Displaying One Session

Poster Area (Hall 4) Poster Display session
Date
28.09.2019
Time
12:00 - 13:00
Location
Poster Area (Hall 4)
Poster Display session 1 Poster Display session

Basic Science (ID 6604)

Lecture Time
12:00 - 12:00
Speakers
  • Caroline Dive (Manchester, United Kingdom)
  • Iain M. Hagan (Manchester, United Kingdom)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00
Poster Display session 1 Poster Display session

10P - Development of chimeric antigenic receptor (CAR) against VEGFR2 for solid tumour treatment (ID 1757)

Presentation Number
10P
Lecture Time
12:00 - 12:00
Speakers
  • Li-Shuang Ai (New Taipei city, Taiwan)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Cancer is known to be the second cause of death worldwide. Vascular endothelial growth factors (VEGFs) regulate blood vessel development, the overexpressed of VEGF/VEGFR2 are often associated with angiogenesis, invasion, metastasis and prognosis in cancers. Targeting VEGFR2 by monoclonal antibodies has been associated with a survival benefit across multiple malignancies including lung, gastric and colorectal cancers. In addition to anti-VEGFR2 targeted therapies, immunotherapy including checkpoint blockade, adoptive cell therapy, and chimeric antigen receptor T cell (CART) acts as potential strategies to cure cancer. In this study, we propose VEGFR2-CART as a potential therapeutic strategy for different solid tumors treatment, and its therapeutic efficacy will be further investigated and verified.

Methods

In this study, the anti-VEGFR2 single chain fragment variable (scFv) antibodies were screened by using an automated high throughput phage display system. The binding affinity various CDR sequences to VEGFR2 determined by binding ELISA. We generated CART by transducing human PBMCs with a VEGFR2-CAR 2nd generation lentiviral vector. Cytotoxic ability of the generated CART was assessed by a LDH cytotoxicity assay. To evaluate the biologic effect of VEGFR2-CART cells, a breast xenograft model was performed by injecting HCC1428 cells subcutaneously into the flank of NOD/SCID mice.

Results

Seventy-two VEGFR2 clones with various CDR sequences with the range of binding affinities of 10-8 to 10-10 M were selected. Cell toxicity assays showed that 50∼60% of VEGFR2-overexpressed FS293 cells were killed by VEGFR2-CART cells. To evaluate the biologic effect of VEGFR2-CART cells, a breast xenograft model was performed by injecting HCC1428 cells subcutaneously into the flank of NOD/SCID mice. The results demonstrate that the VEGFR2-CART cells significantly inhibit the growth of HCC1428 due to reduced tumor sizes and weights.

Conclusions

The cytotoxicity assay showed that the VEGFR2-overexpressed FS293 cells were apparently killed by VEGFR2-CART cells. The antitumor effects of the VEGFR2-CART cells have been proved in a murine tumor model, suggesting that targeting VEGFR2 with CART cells can be a novel strategy in cancer immunotherapy.

Legal entity responsible for the study

Development Center for Biotechnology.

Funding

The government of the Republic of China (Taiwan).

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

11P - Triple blockade of EGFR, MEK and PD-L1 as effective antitumor treatment in PD-L1 overexpressing, MEK inhibitor resistant colon cancer cells (ID 4156)

Presentation Number
11P
Lecture Time
12:00 - 12:00
Speakers
  • Nunzia Matrone (Napoli, Italy)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Molecular mechanisms driving acquired resistance to anti-EGFR therapies in metastatic Colorectal Cancer (mCRC) are complex but generally involve the activation of the downstream RAS-RAF-MEK-MAPK pathway.

Methods

In order to understand the mechanism underlying MEK inhibitor (MEKi) resistance, we generated three different MEKi resistant models, two all RAS WT (SW48-MR and LIM1215-MR) and one mutated in RAS (HCT116-MR).

Results

These models showed features related to the gene signature of Colorectal Cancer CMS4 with upregulation of immune pathway as confirmed by Microarray and Western blot analysis. In particular, moving forward the MEKi phenotype we assisted to the loss of epithelial features and acquisition of mesenchymal markers and morphology. Moreover, this change in the morphology is accompanied by up-regulation of PD-L1 expression and activation of EGFR and its downstream pathway, independently of cell line status mutation. To extend these in vitro findings, we performed an in vivo study using MC38 and CT26 MEKi resistant syngeneic models that we have previously generated. Combined treatment of MEKi, EGFR inhibitor (EGFRi) and PD-L1inhibitor (PD-L1i) resulted in a marked inhibition of tumor growth in both MC38–MR and CT26-MR xenograft model.

Conclusions

These results suggest a strategy to potentially overcome immunotherapy resistance in CMS4-like tumors and to improve the efficacy of MEK inhibition by co-treatment with other agents providing an additional therapeutic strategy via modulation of host immune responses.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

12P - EGFR-mediated PD-L1 upregulation in HER2+ breast cancer (BC) cell line models (ID 2949)

Presentation Number
12P
Lecture Time
12:00 - 12:00
Speakers
  • Nicola Gaynor (Dublin, Ireland)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

The tyrosine kinase inhibitors (TKIs) lapatinib (L), a reversible dual EGFR/HER2 inhibitor, and afatinib (A), an irreversible pan-HER inhibitor, are approved for the treatment of HER2+ BC and EGFR mutant non-small cell lung cancer (NSCLC), respectively. Anti-PD-1 and anti-PD-L1 immune checkpoint inhibitors are showing clinical activity in HER2+ BC and NSCLC. Pre-clinical studies in EGFR-mutant NSCLC models suggest that there may be a link between EGFR activity and PD-L1 expression. This study investigates the potential of the EGF signalling pathway to alter PD-L1 expression in parental and L- or A-resistant HER2+ BC cell lines to examine the pre-clinical rationale for the combination of anti-PD-1/PD-L1 agents and TKIs in HER2+ BC.

Methods

Parental (HCC1954-P, SKBR3-P) and TKI-resistant HER2+ breast cancer cell lines (L-resistant HCC1954-L, A-resistant SKBR3-A) were treated with the EGFR ligand EGF (10ng/ml) alone, L (1µM) alone or the combination of EGF and L for 24 hours. Protein lysates were generated. PD-L1 (CST E1L3N), EGFR (ThermoFisher MS665) and pEGFR y1068 (Millipore 07818) expression levels were assessed by Western blot. Densitometry analysis was carried out on triplicate data using Total Lab Quant. A Student’s t test p value of < 0.05 was considered significant.

Results

All cell lines examined showed a significant increase in PD-L1 expression upon EGF treatment (HCC1954-P – 3-fold increase (p = 0.02), HCC1954-L - 2.5 fold increase (p = 0.03), SKBR3-P - 4.4 fold increase (p = 0.03), SKBR3-A - 1.9 fold increase (p = 0.01)). L was capable of blocking EGF-mediated PD-L1 upregulation in the HCC1954-P, SKBR3-P an SKBR3-A cell lines (p = 0.04, p = 0.04 and p = 0.01 respectively). L was not capable of blocking EGF-mediated PD-L1 upregulation in the HC1954-L cell line. For parental and resistant cell line models, pEGFR levels corresponded to PD-L1 expression upon EGF and L treatment.

Conclusions

The results indicate PD-L1 expression can be altered by manipulation of EGFR activity in HER2+ breast cancer cell line models. The development of targeted therapy resistance can interfere with the ability of EGFR to impact PD-L1 levels. Further examination of EGFR and PD-L1 is warranted in HER2+ breast cancer models.

Legal entity responsible for the study

The authors.

Funding

Cancer Clinical Research Trust.

Disclosure

A. Canonici: Research grant/Funding (institution): Boehringer Ingelheim. J. Crown: Full/Part-time employment: OncoMark; Honoraria (self), Advisory/Consultancy, Speaker Bureau/Expert testimony, Research grant/Funding (institution): Eisai; Honoraria (self): Amgen; Honoraria (self), Advisory/Consultancy, Research grant/Funding (institution): Puma Biotechnology; Honoraria (self), Advisory/Consultancy: Seattle Genetics; Honoraria (self), Research grant/Funding (institution): Boehringer Ingelheim; Honoraria (self), Advisory/Consultancy, Speaker Bureau/Expert testimony, Travel/Accommodation/Expenses: Pfizer; Honoraria (self), Advisory/Consultancy: Vertex; Honoraria (self), Speaker Bureau/Expert testimony: Genomic Health; Honoraria (self), Advisory/Consultancy, Research grant/Funding (institution), Travel/Accommodation/Expenses: Roche; Honoraria (self), Travel/Accommodation/Expenses: MSD Oncology; Travel/Accommodation/Expenses: AstraZeneca; Travel/Accommodation/Expenses: Abbvie. D.M. Collins: Research grant/Funding (self): Puma Biotechnology; Research grant/Funding (self): Roche. All other authors have declared no conflicts of interest.

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13P - The impact of cortisol on immune cells and its effect on cancer-immune cells co-culture in a 3D spheroid of ovarian cancer (ID 4270)

Presentation Number
13P
Lecture Time
12:00 - 12:00
Speakers
  • Maysa Al-natsheh (Brighton, United Kingdom)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Ovarian cancer (OC) is one of the deadliest gynaecologic cancers predominantly of the epithelial subtype (EOC). Despite advances in treatments, relapse and dissemination is very likely to occur after remission. The heterogenous nature and complex immunosuppressive environment of the tumour implicate a heterogenous clinical outcome and a key role for the immune system in the disease prognosis. Diagnosis and treatment of cancer is often a traumatic experience associated with mental and physical comorbidities, cortisol is released in response to stress and has a controversial role to either promote or suppress stress depending on its physiological environment. Strong evidence is showing that stress hormones can influence tumour biology. In this study, the impact of cortisol on splenocytes and T cells and immune-cancer interactions were explored to probe a mechanism for its action in OC.

Methods

C57BL/6j mice were either subjected to chronic restraint stress for 2 hours a day over a period of six weeks or left alone in their home cages (no stress group). Splenocytes were then extracted and first examined for DNA damage measured by immunofluorescence using phosphorylated γ-H2AX. Splenocytes were next co-cultured with ovarian spheroids for five days and spheroids size and splenocytes infiltration were compared against no stress group. The impact of acute stress on DNA damage was tested in vitro on T lymphocytes subjected to physiological concentration of cortisol for 2h.

Results

Chronic stress significantly increased DNA damage in mouse splenocytes (p = 0.0027). Ex vivo T lymphocytes exposed to cortisol also showed a significant increase in DNA damage (p = 0.017). Furthermore, a significant increase in the size of spheroids co-cultured with splenocytes from stress group was observed (p = 0.0213) and cortisol inhibited immune cell trafficking into the spheroids.

Conclusions

In conclusion, both chronic stress and acute exposure to cortisol can increase DNA damage in splenocytes and T lymphocytes reducing their infiltration into cancer spheroids. This data may have impact for patient with ovarian cancer experiencing extreme stress.

Legal entity responsible for the study

The author.

Funding

Has not received any funding.

Disclosure

The author has declared no conflicts of interest.

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14P - Application of sonoporation to increase anticancer drug efficacy in 2D and 3D NSCLC cell cultures (ID 1568)

Presentation Number
14P
Lecture Time
12:00 - 12:00
Speakers
  • Vilma Petrikaite (Kaunas, Lithuania)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

In order to improve the efficacy of chemotherapy an increasing attention is given to the drug transport to tumors. Sonoporation is the application of ultrasound (US) to increase cell membrane permeability. It is thought that US induces expansion, contraction, and explosion of microbubbles (MB) thus creating pores in the cell membrane, enhancing drug delivery and efficacy. It is important to investigate this phenomenon not only into monolayer cultured cells but also into cell spheroids that imitate the biological characteristics of tumor better than 2D cell cultures. The aim of our study was to evaluate the influence of US on the efficacy of three anticancer drugs doxorubicin (DOX), 5-fluorouracil (5-FU) and paclitaxel (PTX) into 2D and 3D A549 non-small cell lung cancer cell cultures.

Methods

US pulse repetition frequency of 10 Hz and 1 MHz center frequency were generated with peak negative pressure of 0.5 MPa and 50% duty cycle. SonoVue™ MB were used. The effect of DOX on cell viability was tested by MTT assay. Spheroids were formed using 3D Bioprinting method mixing A549 cells with human fibroblasts. DOX delivery in 2D and 3D cultures was assessed using fluorescence microscopy. DOX toxicity in tumor spheroids was evaluated according to the change of spheroid size.

Results

Separately applied US and MB did not increase DOX cytotoxicity. Meanwhile, the combination of US and MB increased DOX efficacy by approximately 4% when compared to DOX alone. US exposure did not show a positive effect on DOX delivery in 2D cancer cell cultures. On the other hand, US increased DOX delivery in tumor spheroids. 15 sec. of US exposure increased DOX penetration in the edge and middle zones of spheroids from 12 to 60%. 2 min. of US exposure decreased the amount of DOX in these zones. US also increased DOX, 5-FU and PTX toxicity in cancer cell spheroids 8-, 1.2- and 1.5-fold, respectively.

Conclusions

US is a promising physical method to enhance anticancer drug efficacy, especially into 3D cell cultures. However, there is a lack of evidence about its efficacy and further studies are needed.

Legal entity responsible for the study

The authors.

Funding

Lithuanian University of Health Sciences; Kaunas University of Technology.

Disclosure

All authors have declared no conflicts of interest.

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15P - Tr1-like cells in human peripheral blood are part of the T effector memory pool and are preferentially stimulated via CD55 (ID 5400)

Presentation Number
15P
Lecture Time
12:00 - 12:00
Speakers
  • Iniobong A. Charles (Nottingham, United Kingdom)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Type 1 regulatory (Tr1) T cells, like every effector T cells, arise from stimulation of naïve T cell precursor and enter a long term memory pool. While they can adapt their function to specific environmental cues, sometimes called plasticity, their phenotype remains broadly fixed. Unlike natural T regulator cells that emerge from the thymus with a defined phenotype (CD4+CD25hiCD127loFoxP3+), there is uncertainty over the characterisation of inducible Tr1 cells.

Methods

In this study we stimulated human total CD4+ T cells with PMA/ Ionomycin and also demonstrated that these cells respond specifically to costimulation via CD97-CD55. Dual cell surface capture (CSA) was used to assay for IL-10 and IFN-γ and intracellular staining for Tr1 markers.

Results

A small (<5%) population were IL-10+ IL-4-, IFN-γ- and expressed other markers commonly associated with Tr1-like cells. These included; CD49b, LAG-3, CD226, PD-1, CTLA-4, and TIM-3. However they were negative for FoxP3 expression, and expressed Cmaf, which is thought to be responsible for the transcriptional events within this population. Furthermore, we show that these Tr1-like cells form part of the immunological memory and reside predominantly within the effector memory (CD62L- CD45RO+, TEM) pool. Unlike the majority of other studies where Tr1 is generated by chronic stimulation of PBMCs in the presence of recombinant IL-10 for several days, as far as we are aware, this is the first report characterising Tr1 directly ex vivo from human peripheral blood.

Conclusions

We have also demonstrated that these cells respond specifically to costimulation via CD97-CD55 to drive proliferation and maintain the IL-10 single positive, Tr1 phenotype outlined above. This supports the idea that once differentiated from naïve precursors, Tr1-like cells respond to CD55 costimulation to maintain a small (<5%) pool with a committed IL-10+ IL-4-IFN-γ- phenotype.

Legal entity responsible for the study

The University of Nottingham.

Funding

The Commonwealth Scholarships Commission, London.

Disclosure

All authors have declared no conflicts of interest.

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16P - Functional analysis of tumour infiltrating lymphocytes in triple negative breast cancer focusing on granzyme B (ID 5817)

Presentation Number
16P
Lecture Time
12:00 - 12:00
Speakers
  • Hitomi Kawaji (Fukuoka, Japan)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes in breast cancer and has a worse prognosis than others. Tumor infiltrating lymphocytes (TIL) are considered to be a prognostic factor in TNBC. Cytotoxic T cells (CTL) produce cytokines and cytotoxic substances such as perforin and granzyme B, and attack target cells. We have previously shown that combination of PD-L1 expression and TIL is useful as a functional indicator of tumor immune activation. Granzyme B released from CTL induces apoptosis by passing through pores formed by perforin on the cell membrane of target cells. We focused on the cytotoxic substance granzyme B and explored functional of TIL.

Methods

This study included 228 patients with primary TNBC who underwent resection without neoadjuvant chemotherapy at Kyushu University Hospital (Japan), between January 2004 and December 2014. We retrospectively analyzed granzyme B, CD8, PD-L1 expression assessed by immunohistochemistry and TIL in 228 TNBCs. Granzyme B was evaluated in TIL, ≥ 3% was defined as high expression, and < 3% as low. We also explored the correlation between immunologic features on tumors and immune cells and the clinicopathological characteristics of the tumors, their response to chemotherapy and clinical outcome.

Results

Among the 228 tumors, granzyme B expression was classified as high in 106 (46.5%), low in 122 (53.5%). Granzyme B-high is correlated with high levels of TIL (p = 0.004), CD8 expression of T cell (p = 0.016) and PD-L1 of tumor cells (p<.0001). In the TIL-high and granzyme B-high group, it is considered that the anti-tumor immune system is activated, and it tend to be the most favorable prognosis. On the other hand, in the TIL-high and granzyme B-low group, despite lymphocyte migration, it is presumed that they do not recognize the antigen, that is, they are in a state of immune tolerance and thus have a poor prognosis. In the group with granzyme B-high, the patients treated with anthracycline as adjuvant chemotherapy significantly improved their prognosis (p = 0.043). The high expression of granzyme B may be a predictor of postoperative chemotherapy.

Conclusions

Granzyme B is an important substance of cytotoxic pathway and has been possible to be an indicator of TIL activation.

Legal entity responsible for the study

Masafumi Nakamura.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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17P - Aberrant glycolysis associates with inflammatory tumour microenvironment and promotes metastasis in triple negative breast cancer (ID 2287)

Presentation Number
17P
Lecture Time
12:00 - 12:00
Speakers
  • Chengwei Lin (Taipei City, Taiwan)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Triple-negative breast cancer (TNBC) is account for 10∼25% of breast cancer incidence with more aggressive phenotype, metastatic capability, and poorer prognosis than other subtypes. Altered cancer metabolism is an emerging hallmark of cancer. Previous studies reported that TNBC cells exhibit greater aerobic glycolysis than non-TNBC cells; however, the detailed regulatory mechanism is largely unknown.

Methods

The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases were used to analyze expression of glycolytic genes and clinical relevance in TNBC patients. Immunohistochemistry assay was performed to confirm the expression of glycolytic gene in breast cancer tissues. Gain-of-function and loss-of-function studies were conducted in TNBC cell lines. The effect of inflammatory microenvironment was carried out by coculture of macrophages and TNBC cells, and the expression of inflammatory cytokines were performed by real-time PCR.

Results

We compared the differential genes expression in TNBC and non-TNBC by in silico analysis. Interestingly, the expression of glucose transporter 3 (Glut3) is upregulated in TNBC patients, compared to non-TNBC patients. Mechanistically, overexpression of Glut3 regulated the expression of epithelial-mesenchymal transition (EMT) genes and promoted invasiveness and metastasis of TNBC cells. Activation of IL6/STAT3 signaling regulates the expression of Glut3 and glycolytic genes, and coexpression of IL6/Glut3 rendered poorer survival outcome, particularly in TNBC. Moreover, conditioned medium (CM) from Glut3-expressing tumor cells induced macrophage activation and production of pro-inflammatory cytokines, and patients with high Glut3 level associated with inflammaotry signautres.

Conclusions

Upregulation of IL6/STAT3 signaling axis promotes expression of Glut3 and glycolytic genes. Elevation of Glut3 in TNBC induces macrophage activation and production of pro-inflammatory cytokines. Our data show the possible association between glucose metabolism and inflammatory microenvironment in TNBC.

Legal entity responsible for the study

The author.

Funding

Ministry of Science and Technology, Taiwan.

Disclosure

The author has declared no conflicts of interest.

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18P - Anti-cancer effects of differentiation-inducing factor-1 in triple negative breast cancer (ID 735)

Presentation Number
18P
Lecture Time
12:00 - 12:00
Speakers
  • Fumi Tetsuo (Fukuoka, Japan)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Breast cancer is the most common cancer in women and its metastasis markedly exacerbates patients’ prognosis. In particular, triple negative breast cancer (TNBC), which lacks estrogen receptors (ER), progesterone receptors (PR) and human epidermal growth factor receptor 2 (HER2), is an aggressive subtype of breast cancer frequently forming metastatic lesions. Differentiation-inducing factor-1 (DIF-1) identified in Dictyostelium discoideum inhibits cell proliferation and migration of various mammalian cancer cells. However, the effect of DIF-1 on TNBC has not been examined. Here, we investigated whether DIF-1 shows anti-proliferative and anti-metastatic effects on TNBC by in-vivo and in-vitro experiments.

Methods

In in-vivo experiments, we used cancer xenograft model mice in which murine TNBC 4T1/Luc cells (1.0 × 106 cells/mL) were injected into mammary fat pads to evaluate the effects of intragastric administration of DIF-1 (300 mg/kg/day) on the primary tumor growth and lung metastasis. In in-vitro experiments, we carried out assays for cell proliferation, migration and invasion to evaluate the anti-proliferative and anti-metastatic effects of DIF-1. We also conducted Western blotting and real-time RT-PCR to identify the mechanisms for DIF-1’s anti-cancer effects.

Results

In vivo, administration of DIF-1 significantly suppressed the primary tumor growth and lung metastasis without adverse effects such as weight loss and myelosuppression. In vitro, DIF-1 reduced cyclin D1 and c-Myc by inhibiting transcription and promoting degradation of the proteins, which resulted in the suppression of cell proliferation. DIF-1 also suppressed cell migration and invasion and reduced the protein expressions of snail, twist, vimentin and MMP-2, crucial factors in epithelial-mesenchymal transition (EMT).

Conclusions

DIF-1 exerts anti-cancer effects in TNBC by reducing cell cycle accelerators and reversing EMT. Our findings suggest that using DIF-1 as a lead compound could develop a novel anti-cancer agent for TNBC.

Legal entity responsible for the study

The study protocol was approved by the Committee on Ethics of Animal Experiments of Kyushu University. Animal handling and procedures were carried out in compliance with the Guidelines for Animal Experiments, Kyushu University, and the Law (No. 105) and Notification (No. 6) of the Japanese Government.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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19P - The inhibitory effect in oral squamous cell carcinoma cells by knocking down matrix metalloproteinase 9 (ID 2105)

Presentation Number
19P
Lecture Time
12:00 - 12:00
Speakers
  • Xinyan Zhang (Beijing, China)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Extracellular matrix (ECM) is a basic step of tumor invasion and metastasis. Matrix metalloproteinase (MMPs) family is a kind of zinc-ion-dependent endopeptidase, which can degrade almost all protein components in the extracellular matrix, destroy the histological barrier of tumor cell invasion, and play a key role in tumor invasion and metastasis. The role of MMP-9 in tumor invasion and metastasis has attracted increasing attention and is considered as the main proteolytic enzyme in this process. The aim of this study was to investigate the role of MMP-9 in human oral squamous cell carcinoma cells.

Methods

Human oral squamous cell carcinoma (OSCC) cells CAL27 and SCC-15 were cultured in DMEM high glucose medium with 10% FBS. The MMP-9-shRNA Lentiviral clone and control virus was constructed and transfected into OSCC cells. Cell proliferation was detected by MTT assay. Colony formation was evaluated by colony formation assay. Cell migration and invasion was evaluated using the scratch assay and transwell invasion assay. OSCC cells Knockdown with MMP-9-shRNA were injected into zebra fish and OSCC tumor model in zebra fish was established and evaluated.

Results

MMP-9 expression was down regulated significantly in RNA and protein level after transfection. Knockdown of MMP-9 gene by shRNA could inhibit oral squamous cell cancer cells growth and colony formation, and markedly suppress cell migration and invasion in vitro. And also knockdown of MMP-9 gene could significantly inhibit OSCC cells metastasis in zebra fish.

Conclusions

MMP-9 plays an important role in OSCC cells metastasis.

Legal entity responsible for the study

Beijing Stomatological Hospital & School of Stomatology, Capital Medical University.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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20P - Matrix metalloproteinases and their tissue inhibitors genes abnormal DNA methylation in breast cancer (ID 5939)

Presentation Number
20P
Lecture Time
12:00 - 12:00
Speakers
  • Olga Simonova (Moscow, Russian Federation)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Matrix metalloproteinases (MMPs) specifically hydrolyze the extracellular matrix proteins. MMPs are inhibited by tissue inhibitors of MMPs (TIMPs). Changes in the expression levels of MMPs and TIMPs have been described in various cancers, including breast cancer (BC).

Methods

We analyzed methylation status of 11 MMP genes (MMP2, MMP11, MMP14, MMP15, MMP16, MMP17, MMP21, MMP23B, MMP24, MMP25, MMP28) and 4 TIMP genes (TIMP1, TIMP2, TIMP3, TIMP4) in 183 BC samples and 183 matched samples of morphologically normal adjacent tissues, 6 BC cell lines (ZR711, HBL100, HS578T, BT474, T47D, MCF7), and 6 autopsy samples of normal breast tissues by methylation sensitive restriction enzyme digestion PCR (MSRE-PCR).

Results

In our collection of BC samples, cancer related abnormal methylation was observed for the MMP2, MMP23B, MMP24, MMP25, and MMP28 genes of the MMPs family (Table). Other MMP genes under study were nonmethylated both in tumors and in normal tissues, as well as the TIMP genes TIMP2 and TIMP3. The promoter regions of TIMP1, TIMP4, MMP14, and MMP21 were found to be constitutively methylated in breast tissues, no matter cancerous or normal. A multiple correspondence analysis demonstrated that nonmethylated status of the promoter regions of MMP2, MMP23B, MMP24, MMP25, MMP28 is associated with lack of HER2 expression. The methylated status of the MMP23B is associated with a higher level (3+) of HER2 expression. Table: MMP genes differentially methylated in BC.

20P MMP genes differentially methylated in BC

GeneMethylated in BC, %Methylated in normal breast tissuesPresence (+) or absence (–) of methylation in BC cell lines
ZR751MCF7T47DBT474HBL100HS578T
MMP27,7 (14/183)0+++---
MMP23B17 (31/182)0+++-++
MMP2411,9 (20/168)0---++-
MMP2515,4 (28/182)0-++-+-
MMP284,9 (9/183)0+++++-

Conclusions

Taken that HER2-positive BC is a more aggressive disease, one can assume that unsuppressed expression of the MMP2, MMP23B, MMP24, MMP25, MMP28 genes allowed by the absence of abnormal methylation of their promoters is characteristic for less aggressive BCs. Legal entity responsible for the study: Research Centre for Medical Genetics. Funding: The research was carried out within the state assignment of Ministry of Science and Higher Education of the Russian Federation.

Legal entity responsible for the study

Research Centre for Medical Genetics.

Funding

Ministry of Science and Higher Education of the Russian Federation.

Disclosure

All authors have declared no conflicts of interest.

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21P - Uveal melanoma cell lines depend on multiple signaling pathways for survival (ID 2703)

Presentation Number
21P
Lecture Time
12:00 - 12:00
Speakers
  • John J. Park (Sydney, Australia)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Uveal melanoma (UM) is a rare cancer that arises from melanocytes in the uveal tract of the eye. Despite effective treatment for primary UM, > 50% of patients develop metastatic disease. There is currently no effective treatment for metastatic UM and median life expectancy is < 8 months. About 90% of UM are characterised by mutations in the GNAQ or GNA11 GTPases and several signalling cascades downstream of G-protein activation have been identified as potentially targetable. These include the protein kinase C (PKC), mitogen activated protein kinase (MAPK), phosphatidylinositol-3-kinases (PI3K), mammalian target of rapamycin (mTOR), and YES-associated protein (YAP) pathways. Aim to understand the relative contribution of oncogenic signaling pathways in proliferation and survival of UM.

Methods

The response 11 UM cell lines to 6 selective inhibitors was investigated using cell viability assays and cell cycle analyses by flow cytometry. Inhibition of selected pathways was examined using Western analysis of downstream effector proteins. The inhibitors used in this study included PKC inhibitors (AEB071 and LXS196), MEK inhibitor (trametinib), PI3K/mTOR inhibitor (BEZ235), YAP inhibitor (verteporfin) and ARF6 inhibitor (NAV2729).

Results

PKC inhibitors were most effective with 8 GNAQ/11 mutant UM cell lines showing some degree of sensitivity to each of the inhibitors, although sensitivity was usually associated with proliferative arrest rather than cell death. (see Table) Expression levels of pMARCKS and pERK were strongly inhibited by PKC inhibitors, however inhibition of these effector proteins did not reflect the degree of UM cell sensitivity.

21P Summary of UM cell lines to each inhibitor. Combined result of cell viability assay and cell cycle analysis

Cell LineMutationTrametinibBEZ235NAV2729AEB071VerteporfinLXS196
Mel270GNAQsensitivesensitivesensitivesensitivesensitivesensitive
OMM1GNA11resistantsensitiveintermediate sensitivitysensitivesensitivesensitive
92.1GNAQresistantintermediate sensitivitysensitiveintermediate sensitivitysensitiveintermediate sensitivity
Mel202GNAQresistantintermediate sensitivitysensitiveintermediate sensitivitysensitiveintermediate sensitivity
OMM1.3GNAQresistantintermediate sensitivityresistantintermediate sensitivityresistantintermediate sensitivity
OMM1.5GNAQresistantresistantresistantintermediate sensitivityresistantintermediate sensitivity
MP41GNA11resistantresistantresistantintermediate sensitivityresistantintermediate sensitivity
MP46GNAQresistantresistantresistantresistantresistantintermediate sensitivity
MP38GNAQresistantresistantresistantresistantresistantresistant
Mel290Nilresistantintermediate sensitivityresistantresistantresistantresistant
Mel285Nilresistantresistantresistantresistantresistantresistant

Conclusions

The sensitivity of GNAQ/11 mutation UM cell lines to 6 targeted drugs is heterogeneous and no single dominant signalling pathway was identified. This suggest that multiple, independent signal pathways contribute to the survival of UM. Thus, inhibition of any single pathway is unlikely to be effective in the treatment of majority of metastatic UM.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

22P - XAF1 and ZNF313 complex stimulates ER stress-induced apoptosis via direct GRP78 inhibition (ID 4849)

Presentation Number
22P
Lecture Time
12:00 - 12:00
Speakers
  • Sungchan Jang (Seoul, Korea, Republic of)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Endoplasmic reticulum (ER) stress-induced, unfolded protein response (UPR)-mediated apoptosis is a protective mechanism whose failure contributes to malignant tumor progression. XAF1 is a stress-inducible, pro-apoptotic tumor suppressor which is inactivated in many human tumor cells. The molecular signal mechanism explaining its function in controlling ER stress, however, remains largely unclear. In this study, we explored the role of XAF1 in UPR signaling and ER stress-induced apoptosis.

Methods

XAF1 and GRP78 expression were determined by RT-PCR and immunoblot assay. the interplay of XAF1 with GRP78 was defined using immunoprecipitation and in vitro pull-down assays. ER stress was induced using thapsigargin, tunicamycin and brefeldin A. Flow cytometry was used to assess cell cycle progression and apoptosis.

Results

XAF1 expression is upregulated at the transcriptional level in response to ER stress and this activation is abolished by suppression of PERK-Nrf2 but not of IRE1a or ATF6 signaling. This indicates that XAF1 is a target of Nrf2 in transcriptional level and activated by ER stress through the PERK pathway. This XAF1 induction greatly enhances the apoptotic response of tumor cells to ER stress while blockade of XAF1 induction reduces apoptotic sensitivity of tumor cells to ER stress. Intriguingly, XAF1 decreases the protein level of the ER stress sensor, GRP78, and consequently activates UPR signaling. Mechanistically, XAF1 binds directly to GRP78 and stimulates its proteasomal degradation in an E3 ligase ZNF313-dependent manner. XAF1 facilitates ZNF313 interaction with GRP78 and thereby promoting ZNF313-mediated K48 ubiquitination of GRP78. Unlikely wild-type XAF1, mutant XAF1 lacking GRP78- or ZNF313-binding domain shows no activity to promote ER stress-induced apoptosis.

Conclusions

Conclusion: XAF1 sensitizes cells to ER stress and drives apoptotic switch of UPR function away from cell cycle arrest by directly antagonizing GRP78 through the assembly of a ZNF313-mediated destruction complex. Our study identifies first a cell-fate decisions function of XAF1 under ER stress conditions.

Legal entity responsible for the study

Sung-Gil Chi.

Funding

BK.

Disclosure

All authors have declared no conflicts of interest.

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23P - XAF1 assembles a destructive complex to induce BRCA1-mediated apoptosis via suppressing ERa and switching estrogen function (ID 4801)

Presentation Number
23P
Lecture Time
12:00 - 12:00
Speakers
  • Seung-hun Jang (Seoul, Korea, Republic of)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) is a tumor suppressor that is frequently lost or down-regulated by aberrant promoter hypermethylation in multiple human cancers. To explore XAF1’s candidacy for a suppressor in breast tumorigenesis, we investigated its expression status in tumor cell lines and tissues, effect on ER regulation of cell growth, and the molecular basis for its function.

Methods

XAF1 expression was examined by RT-PCR, immunoblot and bisulfite sequencing. Molecular basis for the XAF1 interplay with ERa and BRCA1 was defined using gene transfection, siRNA-mediated depletion, immunoprecipitation, pull-down assays, ubiquitination assay, immunohistochemistry and animal studies.

Results

XAF1 expression was lost or abnormally diminished by promoter hypermethylation in a substantial fraction of cell lines and primary tumors. XAF1 expression shows an inverse correlation with ERα expression. In ERα-expressing cells, restoration of XAF1 expression extremely increased cellular sensitivity to estrogen-induced, ERα-mediated apoptosis. Likewise, in ERα-nonexpressing cells, recovery of ERα restoration led to the recovery of apoptotic response to estrogen in XAF1-dependent fashion. Intriguingly, XAF1 was found to directly bind to and destabilizes ERα and this interaction is crucial for its apoptosis-promoting activity. Mechanistically, XAF1 has the characteristic of binding with E3 ligase, XAF1 interacts with BRCA1 and subsequently stimulates BRCA1 binding to ERα and BRCA1-mediated K48 polyubiquitination of ERα. Using a series of truncated mutants, we determined the domains of XAF1, ERα, and BRCA1 that are required for the assembly formation. Additionally, XAF1 was characterized to be upregulated by estrogen at the transcription level through the p38, JNK, and NF-kB signaling pathway.

Conclusions

XAF1 is promoted by estrogen and its activation directs the apoptotic switch of estrogen function through the assembly of BRCA1-mediated ERα destruction complex. Our study illuminates the mechanistic consequence of epigenetic inactivation of XAF1 in human breast tumorigenesis.

Legal entity responsible for the study

Sung-Gil Chi.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

24P - Cancer associated fibroblasts promote cancer progression via Wnt2 secretion in colorectal cancer (ID 3416)

Presentation Number
24P
Lecture Time
12:00 - 12:00
Speakers
  • Hideaki Karasawa (Sendai, Japan)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Recent studies have shown that the tumor microenvironment plays a significant role in the progression of solid tumors. As an abundant component of the tumor microenvironment, cancer associated fibroblasts (CAFs) have been shown to promote tumorigenesis and cancer aggressiveness, but their molecular characteristics remain poorly understood.

Methods

CAFs and normal fibroblasts (NFs) were isolated from freshly resected specimens from patients with colorectal cancer (CRC). RNA sequence was performed to obtain gene expression profiles of CAFs and NFs. We analyzed the profiles and compared gene expression differences between CAFs and NFs. Moreover, immunohistochemistry, proliferation assay and migration assay were performed for evaluating function of the isolated genes.

Results

RNA sequencing analysis revealed that the gene sets related to the Wnt signaling pathway were highly expressed in CAFs, as well as TGFβ signaling, which is considered to be a regulator of CAFs. Among the components of this pathway, Wnt2 was specifically expressed. Thus, we performed immunohistochemical analysis on Wnt2 in 171 patients who underwent surgery for colorectal adenocarcinoma. Positive staining for Wnt2 was often observed in cancer stroma, although the immunoreactivity was negative or weak in cancer cells. Wnt2 expression in CAFs was significantly correlated with depth of tumor (P < 0.001), lymph node metastasis (P = 0.044), TNM stage (P = 0.010), venous invasion (P < 0.001), liver metastasis (P = 0.045), and recurrence (P = 0.013). Subsequent in vitro analyses revealed that cancer cell invasion and migration were significantly decreased in a conditioned medium from immortalized CAFs transfected with siRNA targeting Wnt2.

Conclusions

The present study showed that Wnt2 expression in CAFs was significantly related to factors leading to tumor progression, such as depth of tumor invasion and lymph node metastasis, as revealed by immunochemical examination. We also demonstrated that Wnt2 protein derived from CAFs induced cancer cell migration and invasion in CRC. These findings suggest that Wnt2 secreted by CAFs is a key role as a critical mediator in CRC progression and is a potential therapeutic target for CRC.

Legal entity responsible for the study

The authors.

Funding

Japan Society for the Promotion of Science.

Disclosure

All authors have declared no conflicts of interest.

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25P - Paired-related homeobox 1 overexpression promotes invasion and metastasis and is a prognostic factor for worse disease-free survival in patients with lung cancer (ID 4273)

Presentation Number
25P
Lecture Time
12:00 - 12:00
Speakers
  • Jung-jyh Hung (Taipei City, Taiwan)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Lung cancer is the main cause of cancer-related death worldwide. Epithelial-mesenchymal transition (EMT) is one of the major molecular mechanisms inducing tumor invasion and metastasis. Paired-related homeobox 1 (Prrx1) is a newly found EMT-inducing transcription factor. The prognostic value of Prrx1 in lung cancer has not been well demonstrated.

Methods

H1299-Prrx1 cell lines were generated by transfecting the pcDNA3.1-Prrx1 plasmid into H1299 cells. The H1299-Prrx1-Twisti cell lines were generated by transfecting the pSUPER-Twisti plasmid into H1299-Prrx1 cells. Immunofluorescence staining was done to demonstrate expression of E-cadherin and vimentin in these cells. Soft agar clonogenicity assay was done in these cell lines to demonstrate anchorage-independent growth. Cell migration and invasiveness assay were done in these cells to demonstrate their invasion and migration ability. In vivo tail vein metastasis assay was done to demonstrate the ability of Prrx1 overexpression in metastasis in vivo. Immunohistochemistry analysis of Prrx1 expression was done in 110 specimens of lung cancer patients.

Results

Prrx1 overexpression increased transformation activity, induced EMT and increased migration/invasiveness of H1299 cells. Prrx1 overexpression also increased metastatic ability in vivo. Prrx1 overexpression upregulated the expression of Twist and its downstream target matrix metalloproteinase 1 (MMP1). EMT phenotypes, increased migration/invasiveness of H1299-Prrx1 cells, and increase MMP1 expression were reversed by siRNA-mediated repression of Twist expression or a phosphatidylinositol (PI) 3-kinase inhibitor. Prrx1 overexpression predicted lower recurrence-free survival in patients with lung adenocarcinoma.

Conclusions

Prrx1 overexpression induces EMT through upregulation of PI 3-kinase/Akt/Twist/MMP1 axis. Prrx1 overexpression also predicts recurrence in lung cancer patients.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

27P - LncRNA-GC1 contributes to gastric cancer chemo-resistance through inhibition of miR-551b-3p and the overexpression of dysbindin (ID 4241)

Presentation Number
27P
Lecture Time
12:00 - 12:00
Speakers
  • Xin Guo (Beijing, China)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Chemo-resistance is one of the important causes of poor prognosis of gastric cancer patients, but the exact mechanism is still not very clear. The regulation of lncRNA and miRNA and its downstream target genes has become in focus of the field.

Methods

In this study, we performed lncRNA and miRNA microarray analysis of chemo-resistant cells (SGC7901/Oxaliplatin). Then, bioinformatics analysis, TaqMan real-time PCR, western blot, luciferase reporter gene assay, RNA co-immunoprecipitation, cell proliferation and apoptosis analysis were used to explicit the lncRNA-miRNA-mRNA pathway in gastric cancer chemo-resistance. Last, nude mice were used to verify the promotion of chemo-resistance in vivo.

Results

In this study, we identified a lncRNA-GC1, which was upregulated in chemo-resistant gastric cancer tissues and cells, and a miRNA-551b-3p, which was downregulated in chemo-resistant gastric cancer tissues and cells. Firstly, miR-551b-3p can directly bind to the non-coding region of dysbindin mRNA, thereby negatively regulating the expression of dysbindin which was involved in chemotherapy resistance in gastric cancer cells. Secondly, lncRNA-GC1 promotes chemoresistance in gastric cancer by competitively binding to miR-551b-3p to facilitate dysbindin expression in vitro and in vivo. Thirdly, the expression levels of lncRNA-GC1 were positively correlated with tumor size (P = 0.002), lymph node invasion (P = 0.001) and poor platinum response (P < 0.001). Kaplan-Meier curves show that patients with high lncRNA-GC1 expression exhibit poorer overall survival compared with those with low lncRNA-GC1 expression. Multivariate analysis indicated that lncRNA-GC1 serves as an independent prognostic factor for patients with gastric cancer (P = 0.001).

Conclusions

LncRNA-GC1-miRNA-551b-3p-dysbindin signaling pathway may serve as a predictor for oxaliplatin response and a potential therapeutic target for gastric cancer.

Legal entity responsible for the study

Guo Xin.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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28P - GLPG 1790, a new selective EPHA2 inhibitor, is active in colorectal cancer cell lines belonging to the CMS4/mesenchymal-like subtype (ID 5388)

Presentation Number
28P
Lecture Time
12:00 - 12:00
Speakers
  • Pietro Paolo Vitiello (Napoli, Italy)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

EPHA2 tyrosine kinase receptor is implicated in tumor progression, stemness phenotype and resistance to treatment in a wide range of cancers. We investigated the effects of GLPG1790, a new selective Eph receptor inhibitor, in colorectal cancer (CRC) across the 4 consensus molecular subtypes (CMS).

Methods

We tested the antiproliferative effect of GLPG 1790 used alone or in combination with either 5-fluorouracil, oxaliplatin or SN38 (the active metabolite of irinotecan) in a panel of 11 CRC cell lines encompassing the 4 consensus molecular subtypes (CMS). Cell cycle analysis was performed in order to understand possible cell cycle perturbation after treatment. Pathway analysis using western blot (WB) was also performed. We then evaluated the expression of stemness genes upon treatment using qRT-PCR.

Results

GLPG 1790 is active in CRC cell lines, with the strongest activity in the cell lines from the CMS4/mesenchymal-like cluster. Combination with chemotherapeutics is not synergistic according to Chou-Talalay model. The selective inhibitor elicits a persistent inactivation of EPHA2 receptor, associated to G0-G1 cell cycle block in the sensitive cell lines. Furthermore, GLPG 1790 is able to decrease the expression of cancer stem cell genes in cell lines belonging to the CMS4 group.

Conclusions

EPHA2 blockade using the selective inhibitor GLPG 1790 has a strong antiproliferative effect in the chemorefractory subgroup of CMS4/mesenchymal-like CRC cell lines, associated to a G0-G1 cell cycle arrest.

The stronger efficacy of GLPG1790 on the mesenchymal-like subtype is probably due to the impairment of cancer cell stemness and induction of cell differentiation after treatment.

Legal entity responsible for the study

The authors.

Funding

Università della Campania "Luigi Vanvitelli"; Galapagos NV (drug supply); Associazione Italiana per la Ricerca sul Cancro (AIRC).

Disclosure

P.P. Vitiello: Travel/Accommodation/Expenses: Amgen; Research grant/Funding (institution): Bayer; Research grant/Funding (institution): Merck; Research grant/Funding (institution): Roche; Research grant/Funding (institution): Servier; Research grant/Funding (institution): Ipsen; Travel/Accommodation/Expenses: BMS; Travel/Accommodation/Expenses: Sanofi. C. Cardone: Research grant/Funding (institution): Amgen; Research grant/Funding (institution): Bayer; Research grant/Funding (institution): Ipsen; Research grant/Funding (institution): Merck; Research grant/Funding (institution): Roche. D. Ciardiello: Travel/Accommodation/Expenses: Sanofi. L. Poliero: Travel/Accommodation/Expenses: BMS. C. Borrelli: Travel/Accommodation/Expenses: BMS. N. Zanaletti: Travel/Accommodation/Expenses: BMS. P. Vitale: Travel/Accommodation/Expenses: BMS. T. Troiani: Honoraria (self), Research grant/Funding (institution): Roche; Honoraria (self), Research grant/Funding (institution): Merck; Honoraria (self), Research grant/Funding (institution): Bayer; Honoraria (self), Travel/Accommodation/Expenses: Amgen; Travel/Accommodation/Expenses: Servier; Travel/Accommodation/Expenses: Sanofi; Travel/Accommodation/Expenses: Novartis. F. Ciardiello: Advisory/Consultancy, Research grant/Funding (institution): Amgen; Advisory/Consultancy, Research grant/Funding (institution): Bayer; Advisory/Consultancy, Research grant/Funding (institution): Roche; Advisory/Consultancy, Research grant/Funding (institution): Merck; Advisory/Consultancy: Servier; Advisory/Consultancy: Pfizer; Research grant/Funding (institution): Ipsen. E. Martinelli: Honoraria (self), Research grant/Funding (institution): Amgen; Honoraria (self), Research grant/Funding (institution): Merck; Honoraria (self), Research grant/Funding (institution): Bayer; Honoraria (self), Research grant/Funding (institution): Roche; Honoraria (self), Honoraria (institution): Servier. All other authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

29P - Characterisation of growth hormone signal transduction in primary melanoma cell lines (ID 5208)

Presentation Number
29P
Lecture Time
12:00 - 12:00
Speakers
  • Karla S. Sousa (Auckland, New Zealand)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Growth hormone (GH) signalling is initiated by binding of GH to the GH receptor (GHR) and activation of Janus kinase 2, which in turn, promotes phosphorylation of STAT5, PI3K/AKT and MAPK. High levels of GHR expression have been reported in melanoma. This project aims to characterise GH signalling in primary melanoma cell lines and identify cell lines which respond to GH and GHR inhibition, with a view to testing in preclinical xenograft studies.

Methods

Over 24 cell lines established at the Auckland Cancer Society Research Centre (ACSRC) in New Zealand were tested for response to GH, prolactin (PRL) and a GHR antagonist, by assessing STAT5 phosphorylation by western blot. mRNA expression was assessed by Nanostring PlexSet. Endogenous protein was assessed by ELISA.

Results

From the panel of melanoma cell lines, 62% responded to GH stimulation and GHR inhibition. mRNA expression analysis demonstrated that the IGF1 receptor and GHR were highly expressed across cell lines. Most of the cell lines expressing GHR mRNA responded to GH. GH1 mRNA was expressed at very low levels in a subset of cell lines, and ELISA analysis confirmed the presence of endogenous GH protein. siRNA-mediated knockdown of the GHR reduced GHR mRNA expression and STAT5 phosphorylation by GH. GH increased cell viability in a subset of melanoma cell lines. A vemurafenib-resistant melanoma cell line and B16/F10 mouse cell line were also sensitive to GH and GHR inhibition. Exon analysis will be performed for GH and related genes.

Conclusions

In this study, we have demonstrated GH stimulates signal transduction and promotes enhance of viability of primary melanoma cell lines, and that GHR blockade prevents this effect, indicating that blocking GHR signalling might be a useful strategy to treat melanoma. The next step is to understand whether GHR inhibition plays a role in regulating tumour growth in vivo.

Editorial acknowledgement

Li Family PhD Scholarship.

Legal entity responsible for the study

Peter Shepherd.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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30P - LAPTM5 protein can regulate TGF-β mediated MAPK and smad signaling pathways in ovarian cancer cell (ID 3156)

Presentation Number
30P
Lecture Time
12:00 - 12:00
Speakers
  • Yan Gao (Beijing, China)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

LAPTM5 (lysosomal-associated protein transmembrane 5) is a protein that is preferentially expressed in immune cells, and it interacts with the Nedd4 family of ubiquitin ligases. Recent studies in T and B cells identified LAPTM5 as a negative regulator of T and B cell receptor levels at the plasma membrane, but a positive modulator of inflammatory signaling pathways and hence cytokine secretion in macrophages. However, the expression and function of LAPTM5 in ovarian cancer remains undefined.

Methods

LAPTM5 expression in ovarian cancer, benign and normal ovarian tissues was examined by immunohistochemistry. The LAPTM5 mRNA and protein level of OV cells were detected by Q-PCR and Western blotting respectively. Cell apoptosis, migration and invasion changes were observed through high-content screening. The proteins expression involved in epithelial-mesenchymal transition (EMT) and TGF-β mediated signaling pathways was verified by Werstern blotting and immunofluorescence. The role of LAPTM5 on tumorigenesis in vivo was detected by the xenograft model.

Results

Our study showed that in human OV cell lines and tissues, LAPTM5 were significantly induced at both mRNA and protein levels. Furthermore, an OV cell model with downregulated LAPTM5 were established, revealing a significantly alteration of apoptosis. Moreover, analysis of the changes of migration and invasion, showed significant reduced LAPTM5 suppressed cell metastasis. Proteins involved in EMT were strongly altered, which plays a central role in cell metastasis. In addition, phosphorylated ERK1/2, p38 and JNK, key members of mitogen-activated protein kinase (MAPK) family, and phosphorylated Smad2 and Smad3 of Smad signaling pathways mediated by TGF-β regulating OV cells EMT, were strongly decreased. And, LAPTM5 knockdown also inhibited tumorigenesis in xenograft model.

Conclusions

Taken together, our results suggested that LAPTM 5 acts as a positive modulator of MAPK and TGFβ/Smad signaling pathways mediated by TGFβ in OV cells.

Legal entity responsible for the study

Beijing Obstetrics and Gynecology Hospital, Capital Medical University.

Funding

The National Natural Science Foundation of China (grants 81672838), Beijing Municipal Science & Technology Commission (No. Z181100001718193).

Disclosure

All authors have declared no conflicts of interest.

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31P - Effects of novel targeted anticancer drugs on cytotoxicity, apoptosis, angiogenesis, EMT, drug resistance and autophagic mechanism (ID 592)

Presentation Number
31P
Lecture Time
12:00 - 12:00
Speakers
  • Seyma Aydinlik (Bursa, Turkey)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Metastatic colon cancer has a survival rate less than %5 despite the use of effective chemotherapeutic regimen. Prognostic and predictive importance of EGFR is still contradictive in colon cancer. Tyrosine kinase inhibitor canertinib was used in this study to block the EGFR receptor. The effect of Palladium (Pd) (II) compound [PdCl(terpy)](sac).2H2O] and canertinib combination treatment was investigated in this study. 5-fluorouracil (5-FU), a chemotherapeutic drug was used as positive control.

Methods

The cytotoxic effects of the combination of Pd (II)+canertinib and the 5-FU+canertinib combination on colon cancer cell line (HCT-15, HT-29) were determined by the SRB test. Apoptosis, the acidic vesiculations, mitochondrial depolarization were determined by flow cytometry, Annexin-V-Cy3/SYTOX, acridine orange, JC-1 staining. Alterations in proteins related to EGFR-associated pathway, EMT, apoptosis/autophagy and drug carrier were evaluated by western blot. Invasion, wound healing and colony forming ability were determined. Tube forming in HUVEC and vessel forming ability was assessed by matrigel and CAM test respectively.

Results

A significant decrease in p-ERK, p-P38, p-EGFR protein levels was observed with EGFR inhibition, while the cell viability was decreased. Cell death was determined as mitochondrial apoptosis. LC3-II, Beclin-1, Atg5 protein levels, and increased acidic vesicles and decreased p-MTOR protein levels were associated with increased autophagy in combination therapy. In addition, increased E-cadherin expression and decreased N-cadherin, fibronectin and vimentin expression related to decrease in invasion, migration, and colony forming ability. ROS expression and DNA damage increased. Significant differences in MDR-1 and MRP-1 protein levels were observed with EGFR inhibition. In addition, the ability to create tubes and vessels has decreased significantly.

Conclusions

EGFR inhibition along with Pd(II) has been shown to increased cytotoxicity, decreased invasion, migration and vessel formation abilities. Furthermore, induced ROS levels and increased autophagic signaling are found to be necessary for mitochondria-dependent apoptosis in colon cancer cells.

Legal entity responsible for the study

Uludag University.

Funding

Scientific Research Projects Foundation (BAP) of Uludag University of Turkey [Project No. (DDP(F)-2017)].

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

32P - Delineating the mechanisms of alpha 1-3 fucosyltransferase FUT11 in ovarian cancer (ID 3235)

Presentation Number
32P
Lecture Time
12:00 - 12:00
Speakers
  • Qi Chen (Beijing, China)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Ovarian cancer is the most lethal gynecological cancer and the biological mechanisms remains unclear. The prevalence of abnormal fucosylation has been reported to be closely related to cancer progression and prognosis. It was reported that only FUT3 and/or FUT6 in the α 1-3/4 fucosyltransferase subfamily were required for TGF-β-mediated epithelial-to-mesenchymal transition (EMT) in colorectal cancer. However, it was also reported that the α 1-6 fucosyltransferase FUT8 facilitated TGF-β binding to receptor and stimulated the EMT in breast cancer. As an α 1-3 fucosyltransferase distinct from the α 1-3/4 and 1-6 fucosyltransferase subfamily, the role of FUT11 in ovarian cancer development is less understood.

Methods

FUT11 was identified as a novel oncogene related with the EMT process in ovarian cancer by bioinformatic analysis and real-time PCR. The transwell chamber assays were used to examine the changes of the invasion and migration ability after FUT11 knocked-down by siRNA treatment in ovarian cancer cells SK-OV-3 and HEY. Western-blot was usd to determine the changes of EMT genes by FUT11 depletion in response to TGF-β 1. Furthermore, expression and location of R-Smads and Co-Smad were analyzed by western blot and immunofluorescence assay, to evaluate the impact of FUT11 depletion on the TGF-β/Smad pathway.

Results

FUT11 was highly expressed in ovarian cancer, and increased FUT11 expression was significantly correlated with poor survival of the ovarian patients. FUT11 depletion impaired the invasion and migration of the ovarian cancer cell lines, through regulation of the TGF-β 1-induced EMT process. These inhibitory effect was due to downregulation of the expression of SMAD2 and SMAD3, as well as prevention of their translocation from cytoplasm to nucleus. Although expression of SMAD4 were slightly affected, their translocation from cytoplasm to nucleus were inhibited by FUT11 depletion.

Conclusions

FUT11 promotes invasion and migration of ovarian cancer through regulation of the TGF-β 1-induced EMT process. FUT11 affects the response to TGF-β 1 by regulation of the expression level of R-Smads and Co-Smad, as well as their translocation from cytoplasm to nucleus.

Legal entity responsible for the study

Beijing Obstetrics and Gynecology Hospital, Capital Medical University.

Funding

The National Natural Science Foundation of China (81502353 & 81672838), Beijing Municipal Science & Technology Commission (No. Z181100001718193), and Beijing Obstetrics and Gynecology Hospital, Capital Medical University (FCYY201713).

Disclosure

All authors have declared no conflicts of interest.

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33P - The tyrosine kinase inhibitor dasatinib blocks tumour growth, invasion and recurrence potential by interrupting the communication between cancer cells and their surrounding microenvironment in triple negative breast cancer (ID 3577)

Presentation Number
33P
Lecture Time
12:00 - 12:00
Speakers
  • Miriam Nuncia (Albacete, Spain)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Triple-negative breast cancer (TNBC) is the most aggressive among breast cancer subtypes, as these tumors frequently develop resistance to the treatment used. External signals provided by the surrounding tumor microenvironment (TM), which in mammary tumors is mainly constituted by adipose tissue (AT), control this resistance. Therefore, therapies that targets not only the cancer bulk but also its surrounding TM may be more effective. Preliminary in vitro and in vivo studies using mesenchymal stem cells from TNBC patient’s AT (MSCTNBC) showed how a conditioned medium (CM) prepared from MSCTNBC (MSCTNBC-CM) promoted tumorigenicity, invasion, and chemoresistance. In the present work, molecular mechanism will be investigated to identify novel druggable targets in TNBC.

Methods

TNBC cells (MDA-MB-231, BT549, and HS578T) were exposed to MSCTNBC-CM. The activation profile of tyrosine kinase receptors (RTKs) was evaluated using a commercial array. The effect of the inhibitors in the absence and presence of MSCTNBC-CM on TNBC cells recurrence potential, invasion, and cell death were evaluated in vitro through clonogenic, matrix invasion, and flow cytometry assays. Impact on tumour growth was evaluated in a MSCTNBC-TNBC preclinical model (BALB-nu mice).

Results

RTKs activation profile in response to MSCTNBC-CM revealed that TM secreted factors activates Src protein family (SFK) in TNBC. The use of the SFK inhibitor Dasatinib, both in vitro and in vivo, showed a marked reduction of invasion and recurrence potential, an induction of cell death, and a lower of tumour growth.

Conclusions

In this study, we describe SFK as mediators in the communication within the tumour adipose niche and provides fundamental information to understand TNBC progression, as well as its behavior in response to chemotherapy. The specific blockade of the SFK signaling pathway with Dasatinib can interrupt this communication and revert TM protective effect, resulting in death and smaller tumour size. Our results open the gate to the development of new strategies targeting TM to treat TNBC patients.

Legal entity responsible for the study

The authors.

Funding

Diputación de Albacete.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

34P - NORE1A induces a feedback termination of TNF signaling by antagonizing TNFR1 through ITCH-mediated destruction complex (ID 4808)

Presentation Number
34P
Lecture Time
12:00 - 12:00
Speakers
  • Jieun Ahn (Seoul, Korea, Republic of)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

NORE1A/RASSF5 is a tumor suppressor that is commonly inactivated in a variety of cancers. NORE1A is frequently down-regulated during tumor development and its inactivation often correlates with up-regulation of Ras activity. Although NORE1A was reported to be activated by NF-kB, its role in NF-kB signaling and the underlying mechanism have not been addressed. In this study, we explored the role for NORE1A in the regulation and function of the TNF-NF-kB pathway.

Methods

NORE1A effect on TNF regulation of inflammation, growth, and apoptosis was examined by flow cytometry, migration and invasion, and apoptosis assays. NORE1A regulation of TNF-NF-kB signaling was analyzed by quantitative RT-PCR, immunoblot, immunoprecipitation, and promoter luciferase assay.

Results

Upon exposure to TNF-a, NORE1A is induced by NF-kB signaling at the level of transcription while its activation suppresses TNF activation of NF-kB, indicating that NORE1A functions as a feedback terminator of TNF-NF-kB signaling. As predicted, NORE1A blocks TNF activation of pro-inflammatory gene transcription, epithelial-to-mesenchymal transition (EMT), invasion and migration. Mechanistically, NORE1A binds directly to TNF receptor I (TNFR1) and ubiquitin E3 ligase ITCH to promote ITCH-mediated K48-linked ubiquitination of TNFR1 and subsequent lysosomal degradation. Additionally, we identified that the PPXY motif of NORE1A interacts directly with ITCH using a series of deletion mutants. This interaction protects BAX from ITCH-mediated ubiquitination and thus activates its apoptotic function. NORE1A interaction with ITCH plays a crucial role for both TNFR1 degradation and BAX stabilization and activation.

Conclusions

In this study, we demonstrate first that NORE1A is a feedback terminator of TNF-NF-kB signaling, which directly antagonizes TNFR1 by facilitating the ITCH-TNFR1 interaction. Our study also shows that NORE1A binding to ITCH releases ITCH from BAX and activates BAX-mediated apoptosis. These data illuminate the mechanistic consequence of NORE1A inactivation in tumorigenesis.

Legal entity responsible for the study

Sung-gil, Chi.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

35P - Hsp90 inhibitors enhance the antitumoral effect of osimertinib and overcome osimertinib resistance in non-small cell cell lung cancer cell models (ID 1294)

Presentation Number
35P
Lecture Time
12:00 - 12:00
Speakers
  • Jordi Codony-Servat (Barcelona, Spain)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

The epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) osimertinib improves therapy for non-small cell lung cancer (NSCLC) patients who possess EGR mutations. However, invariable acquired resistance appears due to several molecular mechanisms. Hsp90 protein clients are involved in these processes.

Methods

Cell viability and colony formation assays were performed in PC9, H1975 and PC9-derived osimertinib-resistant NSCLC cell lines tested with osimertinib plus hsp90 inhibitors, luminespib or ganetespib. To analyze the mechanism of action of these compounds and its combination, western blot analysis was carried out to study the protein expression and activation.

Results

In our laboratory five osimertinib-resistant cell lines were generated from PC9 NSCLC cell line and overexpression or activation of several proteins, such as, AXL, Yap, bcl2, Akt, Stat3 and IGF-1R were detected. Hsp90 inhibitors, Ganetespib and Luminespib, inhibited cell viability and colony formation in H1975, PC9 and PC9-derived osimertinib-resistant cell lines, and the combination of these inhibitors with osimertinib achieved enhanced cell viability reduction. Luminespib downregulated the expression of several proteins involved in osimertinib-resistance and the combination of this compound plus osimertinib caused an important decrease of expression in several of these proteins, such as, Stat3, Yap, Akt, EGFR and Met. In addition, osimertinib activated the phosphorylation of several membrane receptors and downstream molecules, such as, Met, Yap, Stat3, Akt and Src, and its activation was partially inhibited by Luminespib.

Conclusions

We conclude that hsp90 inhibitors and osimertinib exhibit good efficiency in inhibiting cell viability and colony formation, while at the same time, inhibiting expression and activation of proteins involved in osimertinib-resistance. Our preclinical study shows that the combination of hsp90 inhibitors and osimertinib may represent an effective strategy for NSCLC patients with resistance to osimertinib treatment.

Legal entity responsible for the study

Pangaea Oncology.

Funding

Pangaea Oncology.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

36P - Expression of IL-17RA promotes cancer stem-like properties of colorectal cancer cells by Stat3 activation (ID 1559)

Presentation Number
36P
Lecture Time
12:00 - 12:00
Speakers
  • Chih-Yung Yang (Taipei, Taiwan)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Colorectal cancer stem cells (CSCs) serve crucial functions in tumor relapse, metastasis and therapy failure. Interleukin 17 receptor A (IL-17RA) is a potent mediator in the pathogenesis and progression of colorectal cancer. Our previous study showed that IL-17RA could promote angiogenesis, tumor growth and metastasis. In addition, IL-17RA was correlated with CRC recurrence. Recent study showed IL-17RA play a potential role in self-renewal in glioma stem cells. This study aims to evaluate the functional role and mechanism of IL-17RA in colorectal CSCs.

Methods

In this study, IL-17RA stable overexpression cells were used to examine the change of sphere formation, proliferation, CSC markers and epithelial-mesenchymal transition biomarkers and the regulation mechanism of signaling pathway. The specific IL-17RA signaling inhibitors were used to evaluate the function of the IL-17RA signaling affecting the CSC markers, epithelial-mesenchymal transition gene expression and sphere formation.

Results

We observed high IL-17RA expression can significantly promote the self-renewal by increasing stem cell markers CD133, ALDH1, Lgr5 and Sox2 expression and the ability to form tumorsphere in SW480 and SW620 cells. In additional, IL-17RA overexpression markedly increased chemoresistant ability and mesenchymal markers Vimentin, Slug, Snail, Zeb1expression. The STAT3 inhibitor remarkably decreased the CD133 stem cell gene, drug resistance and tumorsphere ability.

Conclusions

IL-17RA can enhance the self-renewal ability via activating the STAT3 pathway. Therefore, we expect that IL-17RA could serves as a prognosis marker and a potential therapeutic target in CRC.

Legal entity responsible for the study

Chih-Yung Yang.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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37P - Adaption of pancreatic cancer cells to AKT1 inhibition induces the acquisition of cancer stem-cell like phenotype through upregulation of mitochondrial functions (ID 1615)

Presentation Number
37P
Lecture Time
12:00 - 12:00
Speakers
  • Hugo Arasanz (Pamplona, Spain)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

AKT/PKB is a protein kinase that plays a key role in pancreatic adenocarcinoma. Three isoforms with a similar structure but non-overlapping functions have been described: AKT1/PKBα, AKT2/PKBβ and AKT3/PKBγ. Our project studies the molecular routes of adaption to single AKT isoform silencing.

Methods

We have individually silenced AKT isoforms using short hairpin RNAs (shRNAs) delivered by lentivirus. A shRNA against an irrelevant gene was used as control. When cells adapted to the modification, high-throughput quantitative proteomics analyses were performed to evaluate the differentially altered molecular routes. Mitochondrial protein expression was determined by Western Blot. Mitochondrial function was evaluated using Agilent SeaHorse XF. Cancer stem-cell like phenotype was determined by CD44 and EpCAM expression. A subsequent silencing of the escape routes discovered was performed.

Results

Only cells adapted to AKT1 silencing, but not AKT2 or AKT3, exhibited a cancer stem-cell like phenotype with a sharp increase in CD44/EpCAM expression compared to the other cell lines or control (p < 0.0001). shAKT1 expressing cells presented a potentiation of mitochondrial functions determined both by quantitative proteomics, and by basal and maximal oxygen consumption rate (p < 0.0001). Double silencing of AKT1 and the mitochondrial protein TFB2M caused a prolonged cell growth arrest.

Conclusions

When exposed to stable AKT1 inhibition, pancreatic adenocarcinoma cells adapt by switching their metabolism from glycolysis to mitochondrial respiratorion, which suggests a reversion of Warburg effect, and adopting cancer stem-cell like phenotype. Cancer stem cells have been proposed as one of the main factors favouring therapy resistance. Targeting mitochondrial metabolism might improve the efficacy of conventional treatments.

Legal entity responsible for the study

Grupo de Inmunomodulación.

Funding

Grupo de Inmunomodulación.

Disclosure

All authors have declared no conflicts of interest.

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38P - Bub3 is phosphorylated by the ataxia-telangiectasia mutated kinase in mitosis and required for activation of the mitotic spindle checkpoint in breast cancer (ID 4793)

Presentation Number
38P
Lecture Time
12:00 - 12:00
Speakers
  • Mingming Xiao (Tianjin, China)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

ATM is an important cell cycle checkpoint kinase. This protein is an controller of cell cycle checkpoint that is required for cell response to DNA damage and for genome stability. The spindle assembly checkpoint (SAC) controls accurate chromosome segregation to maintain genome stability. All kinetochores correctly attach to the spindle, which allow mitosis to proceed. Bub3 is the mainly component of the SAC, in which its kinase activity can stabilize the correct kinetochore–microtubule attachments. Genomic stability is a hallmarks of breast cancer, and the level of Bub3 in breast cancer are higher than that in normal tussue. Understanding how the ATM-Bub3 signal contributes to kinetochore-based signaling may help us to recognize the occurrence of breast cancer.

Methods

Stable Isotope Labeling with Amino Acid in Cell Culture (SILAC) to identify ATM interation protein in breast cancer. Bioinformatics analyze the phosphorylation site of Bub3. Biacore assay and in vitro kinase assay to validate the phosphorylation site. Flow cytometry assay to check the mitotic index. Mutation of the phosphorylation site of bub3 and Co-IP were proformed to identify the interation of Bub3 and Bub1 or ZNF207, and western blot to be used to test the activity of Bub1.

Results

Through Stable Isotope Labeling with Amino Acid in Cell Culture (SILAC), we identified additional mitotic proteins that interact and can be phosphorylated by mitotically activated ATM kinase. We provide both in vitro and in vivo evidence that ATM phosphorylates Bub3 on Serine 135. This phosphorylation event promotes Bub1 activity in the mitotic spindle checkpoint. Further, we find that phosphorylation of Bub3 on Ser135 is required for formation of the Bub3-Bub1 complex, which is essential for Bub1 Ser314 phosphorylation. Mutation of Ser135 to alanine of Bub3 resulted in an enhanced interaction with BuGZ/ZNF207 and reduced interaction with Bub1, which lead to a spindle checkpoint defect.

Conclusions

Our findings highlight the functional significance of ATM-mediated kinetochore protein phosphorylation and elucidate a detailed regulatory mechanism of the SAC in breast cancer.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

39P - The regulation of INK4 locus by long non-coding RNAs (ID 1448)

Presentation Number
39P
Lecture Time
12:00 - 12:00
Speakers
  • Yojiro Kotake (Iizuka, Japan)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

INK4 locus is located on human chromosome 9p21 region and encodes three tumor suppressor genes, p15, p16 and ARF. So far, we revealed that a long non-coding RNA (lncRNA), ANRIL, transcribed from INK4 locus represses the transcription of p15 and p16 genes. ANRIL associates with polycomb protein complexes and recruits them on INK4 locus, leading to the transcriptional repression. The depletion of ANRIL inhibits the proliferation of cancer cells such as non-small cell lung cancer and colorectal cancer, indicating that ANRIL functions to promote cancer cells proliferation. Recently, we found a novel lncRNA induced by oncogenic Ras signal (named LION: lncRNA induced by oncogenic Ras signal). In this study, we showed that LION is involved in the transcriptional regulation of INK4 locus and cell proliferation.

Methods

Human non-small cell lung cancer cell H1299 and colorectal cancer cell HCT116 were transfected with siRNA oligonucleotides against LION. The expression analysis of INK4 locus genes was performed by quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR). Cell cycle analysis was performed by using Muse cell analyzer.

Results

RT-PCR analysis showed that LION is highly expressed in several cancer cells such as non-small cell lung cancer, cervical cancer and colorectal cancer compared with normal lung fibroblasts. Silencing LION by siRNA oligonucleotides inhibits the proliferation of H1299, HCT116 and HeLa cells. Q-RT-PCR analysis showed that silencing LION increases the p15 and p16 mRNA, suggesting that LION is involved in the transcriptional repression of INK4 locus. Cell cycle analysis showed that silencing LION causes G2/M phase arrest in cell cycle, suggesting that LION functions to promote G2/M transition.

Conclusions

LION is involved in the promotion of cancer cells proliferation such as H1299 and HCT116 cells via regulating p15, p16 and other genes related to G2/M phase control.

Legal entity responsible for the study

The authors.

Funding

JSPS KAKENHI.

Disclosure

All authors have declared no conflicts of interest.

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40P - Vascular endothelial growth factor in colorectal cancer pathology, survival and treatment (ID 1858)

Presentation Number
40P
Lecture Time
12:00 - 12:00
Speakers
  • Liz Baker (Stockton on Tees, United Kingdom)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Angiogenesis (the growth of new blood vessels) is important for tumour invasion and metastasis in order for a tumour to grow beyond a few millimetres in size. Vascular endothelial growth factor (VEGF) is thought to be the predominant angiogenic factor in colorectal and other cancers. As a result, numerous studies have looked at VEGF expression in colorectal cancer patients and several therapeutic agents have been trialled that target the VEGF pathway.

Methods

VEGF levels were determined in 100 paired tumour and normal colorectal tissue samples (1998-2001) and 75 pre-operative plasma samples (2000-2002) by ELISA. Tissue (pg/mg protein) and plasma VEGF levels (pg/ml) were correlated with the tumour pathology. Survival analysis was performed for disease-free and overall 15-year survival (Kaplan Meier, p < 0.05). The study had ethics approval.

Results

VEGF levels were significantly up-regulated in colorectal tumour tissue compared to normal tissue. The median VEGF levels were 288 (range, 30-4,156) pg/mg protein for tumour, 38 (0-833) for normal colon and 90 (0-1,262) pg/ml for pre-operative plasma samples. Tumour levels of VEGF correlated with the tumour depth, differentiation and whether the tumour had undergone lymphatic invasion e.g. well differentiated, 230 (79-1,348), moderate 392 (30-4,156) and poorly differentiated 685 (88-2,612)pg/mg protein. Plasma VEGF levels significantly correlated with tumour differentiation and vascular invasion,e.g. 211 (14-889) vascular invasion and 80 (0-785)pg/ml, no vascular invasion. The 15-year survival status for patients with tissue samples was 30% were alive and well, 1% was alive with recurrence and 69% had died. For the 75 plasma patients; 27% patients were alive and well with the remaining 67% having died. VEGF levels in plasma but not tissue samples significantly correlated with both disease-free and overall 15-year survival, with patients with low plasma VEGF levels having a better survival outcome (p < 0.05, Kaplan Meier).

Conclusions

VEGF levels in both colorectal tumour tissue and pre-operative plasma samples correlated with the tumour pathology. However, only pre-operative plasma VEGF levels correlated with disease-free and overall 15-year survival.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

41P - LCSCAF1 maintains cancer stem-like traits by stabilizing c-Myc protein and promotes metastasis and recurrence in lung cancer (ID 5011)

Presentation Number
41P
Lecture Time
12:00 - 12:00
Speakers
  • Tao Guo (Dalian, China)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Aberrantly overexpressed lung cancer stem cell associated factor-X (LCSCAFX) has been implicated in non-small cell lung cancer (NSCLC) development.

Methods

To explore the effect of LCSCAFXon the lung cancer stem-like traitsin vitro and in vivo.

Results

We reported that enforced expression of LCSCAFX promoted NSCLC cells invasion and metastasis, drug resistance and recurrencein vitro and in vivo, whereas depletion of LCSCAFX suppressed metastasis and relapse in NSCLC. Moreover, we found that LCSCAFX expression was elevated in lung cancer stem cells (LCSCs). Indeed, knockdown of LCSCAFX impaired lung cancer stem-like traits. Conversely, ectopic overexpression of LCSCAFX enhanced the self-renewal ability of lung cancer cells in vitro. In addition, elevated LCSCAFX robustly enhanced tumor initiating frequencies, as well as growth rates of NSCLC cells derived tumor xenografts in immunodeficient mice. Furthermore, mechanistic investigations established that LCSCAFX stabilized c-Myc protein by suppressing its ubiquitylation and proteasomal degradation, which is required for lung cancer stem-like properties maintenance. Consistently, immunohistochemical analysis of clinical lung tumor tissues showed that LCSCAFX was elevated in NSCLC tissues and high LCSCAFX expression serve as an independent prognostic marker in patients with NSCLC. Importantly, genetic depletion of LCSCAFX combined chemotherapy dramatically inhibited NSCLC progression in patient-derived xenograft (PDX) model.

Conclusions

These findings demonstrated a previously unappreciated role of LCSCAFX in the regulation of LCSCs, assigning LCSCAFX as a promising therapeutic target for lung cancer treatments.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Targeting androgen receptor and Wnt pathway in endocrine-resistant breast cancer. (ID 3572)

Lecture Time
12:00 - 12:00
Speakers
  • Virginia Figueroa (Buenos Aires, Argentina)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00
Poster Display session 1 Poster Display session

43P - XAF1 enhances temozolomide induced autophagic cell death through AMPK signaling pathway (ID 4955)

Presentation Number
43P
Lecture Time
12:00 - 12:00
Speakers
  • Mingoo Lee (Seoul, Korea, Republic of)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

XIAP-associated factor 1 (XAF1) is a pro-apoptotic tumor suppressor whose expression is inactivated in many human malignancies. To explore the XAF1’s candidacy for a suppressor in the pathogenesis of human glioma, we investigated its expression and function in tumor cell lines and tissues.

Methods

Expression study was performed using quantitative RT-PCR and immunoblot assays. Functional interplay between XAF1 and AMPK was determined by gene transfection, siRNA-mediated depletion.

Results

XIAP-associated factor 1 (XAF1) is a pro-apoptotic tumor suppressor whose expression is inactivated in many human malignancies. In this study, we explored the XAF1’s candidacy for a suppressor in human glioma pathogenesis. XAF1 reduction is more common in high grade tumors versus low grade tumors and tightly associated with aberrant hypermethylation at 7 CpG sites in the 5’ proximal region of the promoter. XAF1 expression decreases proliferation and colony-forming ability of glioma cells while its depletion enhances cellular resistance to genotoxic drugs, such as temozolomide (TMZ), etoposide and cisplatin. The XAF1 promoter is activated in response to TMZ through JNK-IRF-1 signaling and its activation greatly increases cellular response to TMZ-induced cell death. Furthermore, XAF1 promotes autophagic cell death (ACD) by activating AMP-activated protein kinase (AMPK) in a XIAP-independent manner. Both AMPK-activating and ACD-inducing effects of XAF1 are linked to its activity to decrease intracellular ATP level, oxygen consumption, and mitochondrial membrane potential. XAF1 proteins translocate to the mitochondria and the zinc finger (ZF) 6 domain is essential for its mitochondrial distribution. Consistently, a mutant XAF1 lacking the ZF6 fails to decrease ATP level, activate AMPK, and trigger AMPK-mediated autophagic cell death. Collectively, this study demonstrates that epigenetic inactivation of XAF1 contributes to the malignant progression of human glioma by rendering tumor cells a survival advantage via the attenuation of AMPK signaling.

Conclusions

Epigenetic inactivation of XAF1 contributes to the malignant progression of human glioma by rendering tumor cells a survival advantage via the attenuation of AMPK signaling.

Legal entity responsible for the study

The authors.

Funding

National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2018R1D1A1B07041512).

Disclosure

All authors have declared no conflicts of interest.

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44P - The effect of cortisol on methylation patterns in breast cancer cell lines (ID 5616)

Presentation Number
44P
Lecture Time
12:00 - 12:00
Speakers
  • Haya Intabli (Brighton, United Kingdom)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Epigenetic changes are highly responsive to environmental changes, including stress. The release of stress hormones; such as glucocorticoids, in response to stress have been shown to induce epigenetic modifications in neuronal cells. However, the role of glucocorticoids on epigenetic changes underlying important processes such as cell cycle regulation, apoptosis, and proliferation in breast cancer are not yet established. Furthermore, it is not known if cortisol can induce irreversible epigenetic changes on these cellular processes and whether these changes relate to the duration of the stress response. In this study, cortisol-induced epigenetic changes and the potential involvement of DNA methylation was assessed.

Methods

We analyzed the expression levels of maintenance DNA methyltrasferase (DNMT1) in MDA-MB-231, Hs-578T, MCF7, and T47D breast cancer cell lines by real-time PCR. We also used Qiagen Epitect Methyl ll Complete PCR array for Tumour Suppressor genes to analyse the level of methylation in 94 tumour suppressor genes. The methylation level on the Long Interspersed Nuclear Element (LINE-1) was used as surrogate marker for global DNA methylation.

Results

Our results show that cortisol significantly decreased the expression of DNMT1 in the triple negative cells lines MDA-MB-231 (p < 0.005), and Hs-578T (p < 0.05). We also showed that cortisol induced aberrant methylation characterised by loss of methylation on promoter regions of key tumour suppressor genes in MDA-MB-231 cells, such as DAPK1, MGMT, AKT1, CDKN1A, and ABL1 and hypomethylation of the global genome.

Conclusions

Taken together, cortisol induced aberrant methylation patterns which may have important implications on progression of the disease.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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45P - Global and sex-specific epigenome-wide association studies for the identification of the main methylated loci related to smoking in a mediterranean population (ID 4649)

Presentation Number
45P
Lecture Time
12:00 - 12:00
Speakers
  • Judith Begona Ramirez Sabio (Sagunto, Spain)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Tobacco use has been reported to be the main cause of 90% of male and 80% of female lung cancers, as well as a relevant risk factor for other cancers such as oropharynx, larynx, esophagus, stomach, liver, pancreas, kidney, bladder, and colorectum. Smoking directly affects DNA methylation and although several genes have been reported to be differentially methylated in smokers, gender differences and population-specific differences remain to be further investigated. Our aim was to undertake global and sex-specific genome-wide epigenomic studies (EWAS) in a Mediterranean population to identify the main smoking DNA-methylated genes.

Methods

We analyzed 88 participants in the PREDIMED PLUS-Valencia study paired by age and sex (44 males and 44 females). We included current smokers, former and non-smokers according to the WHO classification. We performed genome-wide DNA methylation using Illumina 850K methylation EPIC arrays. Differential methylation (M-values) was statistically analyzed with Partek, including multivariate adjustement.

Results

In the whole sample, the top-ranked differentially methylated CpG sites (blood) were: cg05575921 in the AHRR (aryl hydrocarbon receptor repressor) gene (P = 2.73E-14), cg21566642 (P = 1.51E-12); cg01940273 (P = 2.10E-10); cg03636183 in the F2RL3 (coagulation factor II thrombin receptor-like 3) gene (P = 6.90E-09); cg23576855 in the AHRR (9.05E-08); and cg17739917 in the RARA (retinoic acid receptor, alpha) gene (P = 1.90E-07), replicating previous finding of the hypomethylation in the AHRR and F2RL3 sites in smokers. In the sex-specific analyses, we detected for both men and women similar hypomethylation in the cg05575921-AHRR site, but some heterogeneity for other loci including the BRCA1 gene.

Conclusions

Smoking has consistent effect of on the methylation of the AHRR gene, but some gender differences for other genes have been detected. This may be related with further gender-smoking differences for some cancers.

Clinical trial identification

2011-005398-22.

Legal entity responsible for the study

Instituto de Salud Carlos III (ISCIII), Ministerio de Ciencia, Innovación y Universidades.

Funding

Instituto de Salud Carlos III (ISCIII), Ministerio de Ciencia, Innovación y Universidades.

Disclosure

All authors have declared no conflicts of interest.

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46P - Whole transcriptomics analyses of mimicking circulating tumor cells (CTCs) by single-cell RNA sequencing (scRNAseq) (ID 4984)

Presentation Number
46P
Lecture Time
12:00 - 12:00
Speakers
  • Jessica Garcia (Lyon, France)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Circulating tumor cells (CTCs) are shed by the solid tumor into the bloodstream. Consistently, single-cell sequencing of CTCs is a powerful tool to further decipher tumor spatiotemporal heterogeneity, plasticity and to identify new pharmacological targets influencing clinical outcome and response to treatment. The aim is to validate an original workflow allowing the isolation at the single-cell level of mimicking CTCs without interfering with single-cell RNAseq analysis.

Methods

The advances in microfluidic systems and isolation technologies have resulted in the enriched extraction of mimicking CTCs from healthy blood samples. The ClearCell Fx (Biolidics Limited) was used as a label-free microfluidic system for enrichment of wholly intact CTCs, while the cellenONE F1.4 system (Cellenion) was used to isolate single CTCs. The later allowed high-throughput automated isolation and dispensing of single CTCs in 96-well plates. ScRNA libraries were prepared with the NebNext Single Cell/Low Input kit (New England Biolabs).

Results

The h-TERT-shp53/RAS HMEC breast cancer cell line was used as mimicking-CTC cell line to evaluate transcriptomic changes. We compared the transcriptomic profiles at each major step: (1) directly after trypsinization, (2) after spiking in whole blood and enrichment with the ClearCell Fx device, (3) after isolation with the cellenONE instrument in bulk of 200 cells and at the single-cell level. The preparation of the scRNAseq library was successful in over 90% of cases at the single-cell level and 100% for bulk samples. Minor effects of the enrichment and isolation methodology were observed at the single-cell level on the transcriptomic profiles. The quality of the libraries and the sequencing provided reliable scRNAseq analyses.

Conclusions

This protocol could be further improved by adding a pre-selection of CTCs based on negative sorting, using a fluorescent anti-CD45 immune labelling exclusion. Such knowledge of single-cell biology may lead to the development of specific therapies to limit tumor progression and seeding of tumoral cells into secondary healthy organs by blocking newly identified targets.

Legal entity responsible for the study

Léa Payen-Gay.

Funding

New England Biolabs.

Disclosure

All authors have declared no conflicts of interest.

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47P - Comparison of enzymatic-and bisulfite conversion to map the plasma cell-free methylome in cancer (ID 5926)

Presentation Number
47P
Lecture Time
12:00 - 12:00
Speakers
  • Nicole Lambert (San Diego, United States of America)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Aberrant methylation is pervasive in cancer and a promising source of diagnostic and predictive biomarkers. Bisulfite conversion, the current standard for detecting methylated cytosines, requires higher DNA input requirements due to destruction of DNA, and is problematic for the low cell-free DNA (cfDNA) recovery from plasma. Enzymatic methyl-seq (EM-Seq) uses enzymatic conversion in place of bisulfite. Here, we compare quality metrics and cancer-associated signals of whole-genome sequenced libraries with bisulfite conversion (WGBS) and EM-Seq.

Methods

We collected blood and isolated cfDNA from 7 healthy controls and 15 participants with cancer. Paired libraries from each participant were prepared using WGBS and EM-seq, were sequenced to a median depth of 1.7x, and processed using a custom bioinformatics pipeline.

Results

The quality of data produced by both methods was largely comparable, including highly correlated methylation levels across CpG islands (average Pearson R = 0.97). However, EM-seq performed better on a number of technical metrics including: higher conversion efficiency, increased alignment quality, and coverage. For library pairs where CNAs were detectable (n = 7), the assessed tumor fractions were similar between the two methods, with 6 out of 7 having estimates within 1.15 fold. WGBS libraries showed evidence of DNA damage, including consistently shorter fragments (7.9 ± 2.1 bp shorter mean length in WGBS relative to paired EM-Seq) and the requirement of more amplification cycles (12 vs 8). Like standard whole genome sequencing, control EM-seq libraries had less variability in genome-wide coverage distributions and preserved the canonical cfDNA fragment length distribution.

Conclusions

Our results show that both approaches produce concordant estimates of methylation levels throughout the genome. However, EM-seq demonstrates a number of advantages, including higher performance in key quality metrics. We conclude that EM-Seq is a suitable method for assessing plasma cell-free methylome in a cancer setting and avoiding bisulfite-associated DNA degradation.

Legal entity responsible for the study

Lexent Bio Inc.

Funding

Lexent Bio.

Disclosure

N. Lambert: Full/Part-time employment: Lexent Bio Inc. A. Robertson: Full/Part-time employment: Lexent Bio Inc. R. Srivas: Full/Part-time employment: Lexent Bio Inc. N. Peterman: Full/Part-time employment: Lexent Bio Inc. J. Close: Full/Part-time employment: Lexent Bio Inc. T. Wilson: Full/Part-time employment: Lexent Bio Inc. P. George: Full/Part-time employment: Lexent Bio Inc. H. Wood: Full/Part-time employment: Lexent Bio Inc. B. Wong: Full/Part-time employment: Lexent Bio Inc. A. Tezcan: Full/Part-time employment: Lexent Bio Inc. H. Tezcan: Full/Part-time employment: Lexent Bio Inc.

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Poster Display session 1 Poster Display session

48P - Detection of low mutations in hepatocellular carcinoma by using circulating tumour DNA (ID 5454)

Presentation Number
48P
Lecture Time
12:00 - 12:00
Speakers
  • Esl Kim (Seoul, Korea, Republic of)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Liver cancer (LC) is presumed to be the sixth most common and newly diagnosed cancer and the fifth leading causing of cancer death around the world in 2018. Hepatocellular carcinoma (HCC) is the most frequent liver cancer and the chronic infection with hepatitis B/C virus is the major risk factor of HCC. Unlike tissue biopsy, liquid biopsy is based on collection of a sample in non-invasive and convenient way at multiple time points over the course of disease. For circulating cell free DNA, it can be used for Next Generation Sequencing (NGS) to detect genetic mutations.

Methods

Whole Blood of patients are collected and plasma was separated. The circulating tumor DNA (ctDNA) is extracted from the plasma samples and quantified using Qubit High Sensitive assay and Agilent D1000 assay. The extracted ctDNA is used for molecular barcoded library construction and captured with probe for next generation sequencing. All samples were then analyzed to identify genetic mutations.

Results

Currently, 20 HCC patients were collected and samples were extracted, sequenced and analyzed. Two thirds of the patients were hepatitis B-viral infected and the one third of the patients were Non-B, Non-C HCC. Most patients were staged 3 others were staged 1 or 2 in HCC. The total ctDNA in 2ml plasma of each patietns were ranged from 2.0 ng to 200ng. As total amount of ctDNA were varied, input ctDNA to construct library was differed ranging from 2ng to 50ng. After molecular tagged libraries were constructed, were pooled and captured with probe for next generation sequencing on Nextseq 500/550 300cycles. Data analysis was performed: TP53 mutations were most frequent and the frequency of mutations are ranged from 3% to 40%.

Conclusions

To fully utilize advantages of the liquid biopsy and circulating tumor DNA, detection of low frequency mutations in the cancer is essential. Detection of low frequency mutation in circulating tumor DNA of hepatocellular carcinoma via optimization of circulating tumor DNA Isolation in plasma samples, optimization of library preparation and target capture condition by using molecular barcoding and customized somatic mutation probe, may improve prognosis, diagnosis, prediction and monitoring of therapeutic response and detection of residual tumor burden.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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49P - Variants in the JAK1 and JAK2 genes in the risk and prognosis of patients with cutaneous melanoma (ID 4428)

Presentation Number
49P
Lecture Time
12:00 - 12:00
Speakers
  • Bruna F. Carvalho (Campinas, Brazil)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Cutaneous melanoma (CM) deserves prominence among tumors due to its high mortality. Single nucleotide variants (SNVs) in genes of immune system pathways in T lymphocytes and abnormal melanocytes may favor tumor proliferation and progression. The aim of the present study was to verify whether SNVs JAK1 c.1648 + 1272G>A and c.991-27C>T and JAK2 c.-1132G>T and c.-139G>A alter the risk of CM and prognosis of CM patients.

Methods

We analysed 248 patients with CM seen at diagnosis at the General Hospital and 274 blood donors (controls) from the Haematology and Haematology Centre of the University of Campinas (UNICAMP). Genotypes of SNVs were identified in DNA of blood samples by real time polimerase chain reaction (RT-PCR), and gene expressions were evaluated by quantitative PCR (qPCR). The statistical meaning of diferences between groups was calculated by t test and Mann-Whitney test. The logistic regression was used to obtain odds ratio (ORs) adjusted for age, cutaneous phototype, nevi and sun exposure, considering the 95% confidence interval (CI). The progression free survival and overall survival were obtained using the Kaplan-Meier estimates and differences between curves were analyzed by the log-rank test. The prognostic value of each variable in patients survival was verified through the univariate and multivariate Cox analyses.

Results

Patients with the JAK1 c.991-27CC genotype (1,22 vs. 0,75 UA, P = 0,01) and allele C (1,11 vs. 0,75 UA, P = 0,02) showed higher mRNA than others. The median follow-up of CM patients was 93 months (variation: 5-221 months). In univariate Cox analysis, patients with the JAK1 c.1648 + 1272GG and JAK1 c.991-27CC isolated genotypes and JAK1 c.1648 + 1272GG plus JAK1 c.991-27CC combined genotype had 1,78, 1,71 and 1,82 more chances of presenting progression of disease, respectively. Patients with JAK1 c.1648 + 1272GG plus JAK1 c.991-27CC plus JAK2 c.-1132GG plus JAK2 c.-139GG combined genotype had 2,11 more chance of presenting disease progression than others.

Conclusions

Our data suggest, for the first time, that JAK1 c.1648 + 1272G>A and c.991-27C>T and JAK2 c.-1132G>T and c.-139G>A SNVs of the JAK/STAT intracellular signaling pathway can alter JAK1 expression and prognosis of CM patients.

Legal entity responsible for the study

The authors.

Funding

CAPES, FAPESP.

Disclosure

All authors have declared no conflicts of interest.

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50P - P-Rex1 expression in breast cancer patients (ID 4409)

Presentation Number
50P
Lecture Time
12:00 - 12:00
Speakers
  • Angela Lara Montero (Buenos Aires, Argentina)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Worldwide, Breast cancer (BC) is the leading cause of cancer-related mortality in women. In Argentina, breast cancer is the second leading cause of death in women, with an incidence of 18.47%. Given the importance of breast cancer for public health, and the diversity of patients’ response to current therapies, the identification of new therapeutic targets is crucial. Rac1 is a Rho GTPase widely involved in motility, mitogenesis, transformation, and metastasis. P-Rex1 is a Rac1 activator essential for the migration of breast cancer cells. It has been reported that P-Rex1 is overexpressed in breast cancer of the luminal type and that its silencing inhibits the migration of cancer cells. In order to validate P-Rex1 as a therapeutic target and/or prognostic biomarker, we aimed to analyze its presence in samples of Argentinean patients from the Marie Curie Hospital of Buenos Aires City, and determine its relationship with clinical and histopathological parameters.

Methods

The levels of P-Rex1 were determined both in biopsy material and in tumor tissue (and its corresponding healthy adjacent tissue) from primary surgery of patients not undergoing neoadjuvant therapy. The levels of messenger RNA (mRNA) were analyzed by quantitative PCR and protein by western blot (WB) and immunohistochemistry.

Results

When the mRNA levels of the biopsies classified according to the molecular subtype were compared, elevated P-Rex1 levels were observed not only in those samples classified as ER+/PR+ breast cancer but also in 50% of the samples of triple negative breast cancer (TNBC). These data were validated by an in-silico analysis made from public access databases (Metabric and the GEO repository of NCBI). P-Rex overexpression in TNBC patients has not been previously reported. The aberrant expression of P-Rex1 was detected in all stages of the disease, and a negative correlation between the expression of P-REX1 and the proliferation marker Ki-67 was determined. P-Rex1 levels were significantly higher in tumors compared to normal breast samples, in agreement with previous reports.

Conclusions

These results confirm the potential of P-Rex1 as a therapeutic target not only for the treatment of luminal tumors but also for a certain population of TNBC patients.

Legal entity responsible for the study

Eva Wertheimer.

Funding

Instituto Nacional del Cáncer-Ministerio de Salud-Argentina (INC, Asistencia Financiera IV, 2018).

Disclosure

All authors have declared no conflicts of interest.

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51P - Modulation of risk of cutaneous melanoma patients by variants in STAT3 gene and functional analysis (ID 4185)

Presentation Number
51P
Lecture Time
12:00 - 12:00
Speakers
  • Gabriela V. Gomez (Sao Paulo, Brazil)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

The JAK/STAT signaling pathway supports the development, progression and metastatic potential of cutaneous melanoma (CM). In normal cells the activation of STAT3 is rapid and transient, but in abnormal melanocytes the activation of the protein occurs in a constitutive manner favoring tumor expansion. STAT3 is coded by the polymorphic gene in humans and thus it is possible that normal individuals show inherited differences in pathway functionality. The aim of the study was to evaluate whether STAT3 c.*1671T>C and STAT3 c.-1937C>G variants influence the risk of CM patients and its functional consequences.

Methods

We evaluated 248 MC patients and 274 controls. DNA was analyzed by real-time polymerase chain reaction (PCR) for genotyping. Luciferase assay and gene expression in a genetically modified SK-MEL-28 melanoma cell line to present the STAT3 c.-1937C>G ancestral and variant genotypes were realized in study. Differences between groups were assessed by the Fisher or chi-square test. Comparisons of luciferase and gene expression were performed using t-tests and ANOVA or Mann-Whitney and Kruskal-Wallis tests.

Results

Individuals with STAT3 c.*1671TT and T allele, and STAT3 c.-1937CC genotype had 1.76 (95% CI: 1.08-2.85, P = 0.02), 1.42 (95% CI: 1.03-1.96, P = 0.02) and 1.67 (95% CI: 1.06-2.63, P = 0.02)-fold increased risks of CM than individuals with the other genotypes and allele, respectively. Individuals with TC haplotype of the STAT3 variants were at 1.70 (95% CI: 1.22-2.36, P = 0.001)-fold increased risk of CM than those with other haplotypes. In a genetically modified melanoma cell line to present the distinct genotypes of STAT3 c.-1937C> G, we observed a 30% increase in the promoter activity of the gene and increase of mRNA in cells with the CC genotype compared to those with the GG genotype.

Conclusions

The data present, for the first time, preliminary evidence that inherited abnormalities in the JAK/STAT pathway, related to STAT3 gene, alter the CM risk. These results may contribute to identify individuals at high risk for CM, who deserve special measures for prevention or early diagnosis.

Legal entity responsible for the study

The authors.

Funding

FAPESP and CAPES.

Disclosure

All authors have declared no conflicts of interest.

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52P - Spectrum of pathogenic germline mutations in Chinese lung cancer patients through next-generation sequencing (ID 3909)

Presentation Number
52P
Lecture Time
12:00 - 12:00
Speakers
  • Ying Huang (Chongqing, China)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Lung cancer is currently a leading cause of cancer-associated mortality worldwide. Despite the increasing evidences of cancer-related variants that were associated with lung cancer risk, investigations of genetic factors and their roles in genetic susceptibility to lung cancer were limited.

Methods

Genomic profiling of DNA was performed through next-generation sequencing (NGS) on tissue or liquid biopsy from 3,651 Chinese patients with lung cancer between January 01, 2017 and May 07, 2019 in 3D Medicines database. Patients with germline mutations were identified, and their clinical information were collected.

Results

Of 3,651 patients with lung cancer, 58 (1.59%) were identified to carry one pathogenic germline mutations in 14 potentially cancer predisposition genes, with a frequency of 1.45% in lung adenocarcinoma (N = 2837), 1.96% in squamous cell lung cancer (N = 612), and 3.12% in small cell lung cancer (N = 160), respectively. None has been found in the small subset of adenosquamous lung carcinoma (N = 42). Amongst all, the highest mutation prevalence was found in BRCA2 (0.47%), BRCA1 (0.22%), CHEK2 (0.22%), TP53 (0.19%), and RAD50 (0.11%). Notably, a majority (57.1%) of the detected germline mutations fell in DNA damage repair (DDR) pathways, including BRCA2, BRCA1, CHEK2, RAD50, ATM, ATR, PALB2, and MRE11A. No significant correlation of the germline mutation prevalence and patients’ histology type was observed (P = 0.26).

Conclusions

This is the first systematic study in germline mutations in Chinese patients with sporadic lung cancer. Our study uncovered the mutation spectrum in Chinese lung cancer population and provided valuable clues for the assessment of the genetic susceptibility to lung cancer.

Legal entity responsible for the study

First Affiliated Hospital of Army Military Medical University.

Funding

Has not received any funding.

Disclosure

Y. Zhang: Full/Part-time employment: 3D Medicines Inc. Z. Zhao: Full/Part-time employment: 3D Medicines Inc. S. Cai: Full/Part-time employment: 3D Medicines Inc. All other authors have declared no conflicts of interest.

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53P - Poor prognostic impact of NTRK2 gene variation in esophageal squamous cell carcinoma (ID 3061)

Presentation Number
53P
Lecture Time
12:00 - 12:00
Speakers
  • Ye Chen (Shanghai, China)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies and lack of effective treatment. Gene fusions involving Neurotrophic Tyrosine Receptor Kinase 2 (NTRK2) have been proven across numerous malignancies. Anti-TRK targeted therapies provide opportunity to treat patients with NTRK2 rearranged cancers. Herein, we aimed to determine the variation and potential prognostic impact of NTRK2 in ESCC.

Methods

In 513 ESCCs resected at Zhongshan Hospital, Fudan University, from January 2007 to November 2010, NTRK2 gene variation was analyzed by Fluorescence in situ hybridization (FISH) in tissue microarrays (TMAs). Survival was estimated by Kaplan-Meier method with the log-rank test.

Results

513 ESCC patients included 420 males and 93 females, ranged in age from 34 to 83 years (mean 61.1 year). The median follow-up time was 36 months (range: 3 to 102 months), the 5-year disease-free survival (DFS) and overall survival (OS) rate for all patients was 32.6 % and 33.1%. The translocation rate of NTRK2 in tumor cells was observed in 11.1% (57/513) and 2.34% (12/513) of patients when the cut-off value was set as low (1% ≤ translocation rate < 15%) and high (rate ≥ 15%), respectively. Survival analysis indicated that patients with high-NTRK2 translocation had a trend poorer DFS (P = 0.063) and poorer OS (P = 0.042) compared with those without NTRK2 gene translocation. Furthermore, high-NTRK2 translocation (P = 0.010) had a lower DFS compared patients without NTRK2 translocation after 18 months. Meanwhile, high-NTRK2 translocation group (P = 0.013), and low-NTRK2 translocation group (P = 0.024) had a lower OS compared patients without NTRK2 translocation after 22 months. The gene copy number (GCN) alterations of NTRK2 were observed in 36.1% (185/513), 5.8% (30/513), 1.4% (7/513) when the cut-off value was set as ≥ 2, ≥ 3, ≥ 4, respectively. We found that NTRK2 alteration was related to DFS and OS when GCN ≥ 2, it was associated with poorer 5-year survival (DFS, P = 0.019, OS, P = 0.027).

Conclusions

High-NTRK2 translocation in ESCC was a poor prognostic parameter especially after 18 or 22 months. Increased GCN of NTRK2 also was a poor prognostic indicator. Low-NTRK2 translocation presented in ESCC, indicating a few ESCC patients might be NTRK-targeted candidates.

Legal entity responsible for the study

Zhongshan Hospital, Fudan University.

Funding

National Natural Science Foundation of China (No. 81702372), Shanghai Natural Science Foundation of China (No. 18ZR1406800), Shanghai Municipal Commission of Health and Family Planning, Key-developing disciplines (No. 2015ZB0201), and Shanghai Municipal Commission of Science and Technology (No. 19441904000).

Disclosure

All authors have declared no conflicts of interest.

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54P - Mutation profile of Tibetan lung cancer revealed by whole exome sequencing (ID 4735)

Presentation Number
54P
Lecture Time
12:00 - 12:00
Speakers
  • Xin Wang (Chengdu, China)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Lung cancer incidence and mortality could be influenced by altitude. In Tibet population Lung cancer was reported to be the sixth deadly malignant cancer, compared with the first deadly tumor in mainland of China. This study is conducted to investigate the mutation profile of Tibet non-small cell lung cancer by whole-exome sequencing.

Methods

We enrolled 17 Tibetan lung cancer patients. Composing of seven adenocarcinomas (Tibetan-LUAD) and 10 squamous carcinomas (Tibetan-LUSC). Paired tissues from malignant tumors and normal lymph nodes were subjected to whole exome sequencing by Illumina X TEN platform using paired-end 150X strategy.

Results

Tibetan LUAD harbored less somatic mutations comparing to median number of somatic mutations detected in TCGA. EGFR mutated most frequently in Tibetan LUAD, but TP53 only mutated in two patients, one of which harbored EGFR mutation as well suggesting reduced responsiveness to first line EGFR-TKIs. Mutation burden of Tibetan-LUSC was similar with TCGA LUSC cohort. In Tibetan LUSC, eight patients carried TP53 mutations. Besides, C>A transversion ratio was lower in Tibetan population than in TCGA for both adenocarcinomas and squamous carcinomas. Mutational signature 1 and 4, which were defined in COSMIC to be related with aging and smoking, contributed largely in Tibetan NSCLC. Other mutational signatures related with failure of DNA double-strand break-repair by homologous recombination and AID/APOBEC cytidine deaminases activation were also noticed. In Tibetan LUAD, amplification in 18 cytobands with q value< 0.25 were remarked. Deletions in two cytoband regions (17p13.1 and 19q13.41) were detected and the q value were 0.025 and 0.056 respectively. For Tibetan-LUSC, amplifications in 22 cytoband regions were remarked with q value <0.25 and deletions were detected in eight cytoband regions with q value between 0.001 and 0.25.

Conclusions

We revealed different mutational landscape, including base substitution status, mutational signature, frequently mutated genes, between Tibetan NSCLC, Chinese Han NSCLC and TCGA NSCLC and provide some insights about the possible role of high-altitude environment on NSCLC and uncovered potential candidate genes for further investigation.

Legal entity responsible for the study

Guowei Che.

Funding

Genetron Health (Beijing) Co. Ltd., Beijing, China.

Disclosure

All authors have declared no conflicts of interest.

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55P - Synergistic activity between niraparib and chemotherapy in colorectal cancer: Molecular determinants from a preclinical model (ID 5236)

Presentation Number
55P
Lecture Time
12:00 - 12:00
Speakers
  • Pietro Paolo Vitiello (Napoli, Italy)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Poly-ADP-Ribose Polymerase inhibitors (PARPi) constitute a class of drugs that interfere with DNA damage response and are already available or in current advanced development either alone or in combination with DNA damaging agents such as chemotherapeutics. Some features of colorectal cancer (CRC), like microsatellite instability and MAPK-induced replication stress, make it a good candidate for exploring the combination of PARPi with chemotherapeutics. Previous data have evidenced how PARPi/chemotherapy combinations are effective in various cancers, albeit early clinical trials showed only modest activity in unselected CRC patients.

Methods

We tested the activity of the PARPi niraparib (MK-4827) used alone or in combination with either 5-fluorouracil, oxaliplatin or irinotecan (SN38) in a panel of 12 CRC cell lines with known molecular characteristics. Proliferation, cell cycle and apoptosis assays were performed. A correlation between combination synergism and the following molecular features was obtained: RAS/BRAF mutation, HER2 amplification, microsatellite status, mutational and transcriptomic profiles from the Cancer Cell Line Encyclopedia (CCLE) database. Mice xenografts using the most representative cell lines are ongoing. Patient-derived 3D primary cultures (PDPCs) are being used to confirm the data obtained in vitro.

Results

Niraparib is synergistic with the investigated chemotherapeutics in most of the cell lines explored. The best candidate for combination is SN38, which is synergistic in 9/12 cell lines analysed. MAPK activation, microsatellite instability (MSI) and mutations in genes involved in homologous recombination repair (HRR) are good predictors of synergism. Transcriptomic analysis is ongoing. Mice xenografts and PDPCs models are in progress and will be presented at the Congress.

Conclusions

The combination of niraparib and irinotecan/SN38 is effective in an in vitro model of CRC. MAPK activation, MSI and mutation in HRR-associated genes are predictors of synergism and are currently being validated in in vivo and ex vivo models. These findings could lead to a better patient selection for this combination in CRC.

Legal entity responsible for the study

The authors.

Funding

Università della Campania Luigi Vanvitelli; Associazione Italiana per la Ricerca sul Cancro (AIRC).

Disclosure

P.P. Vitiello: Research grant/Funding (institution), Travel/Accommodation/Expenses: Amgen; Research grant/Funding (institution): Bayer; Research grant/Funding (institution): Ipsen; Research grant/Funding (institution): Merck; Research grant/Funding (institution): Roche; Travel/Accommodation/Expenses: BMS; Travel/Accommodation/Expenses: Sanofi-Genzyme. D. Ciardiello: Travel/Accommodation/Expenses: Sanofi. C. Cardone: Research grant/Funding (institution): Ipsen; Research grant/Funding (institution): Roche; Research grant/Funding (institution): Merck; Research grant/Funding (institution): Amgen; Research grant/Funding (institution): Bayer. G. Martini: Research grant/Funding (self): Amgen. C. Borrelli: Travel/Accommodation/Expenses: BMS. L. Poliero: Travel/Accommodation/Expenses: BMS. V. De Falco: Travel/Accommodation/Expenses: BMS; Travel/Accommodation/Expenses: Novartis. E.F. Giunta: Travel/Accommodation/Expenses: Novartis; Travel/Accommodation/Expenses: BMS. M. Terminiello: Travel/Accommodation/Expenses: BMS. T. Troiani: Research grant/Funding (institution): Roche; Research grant/Funding (institution): Merck; Research grant/Funding (institution): Bayer; Travel/Accommodation/Expenses: Servier; Travel/Accommodation/Expenses: Amgen; Travel/Accommodation/Expenses: Sanofi; Travel/Accommodation/Expenses: Novartis. F. Ciardiello: Advisory/Consultancy, Research grant/Funding (institution): Merck; Advisory/Consultancy, Research grant/Funding (institution): Bayer; Advisory/Consultancy, Research grant/Funding (institution): Amgen; Advisory/Consultancy, Research grant/Funding (institution): Roche; Advisory/Consultancy: Servier; Advisory/Consultancy: Pfizer; Research grant/Funding (institution): Ipsen. E. Martinelli: Honoraria (self), Research grant/Funding (institution): Amgen; Honoraria (self), Research grant/Funding (institution): Bayer; Honoraria (self), Research grant/Funding (institution): Merck; Research grant/Funding (institution): Roche; Honoraria (self), Research grant/Funding (institution): Servier. All other authors have declared no conflicts of interest.

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56P - cRGDfK (cRGD) conjugated pyropheophorbide-a (Pyro), a new tumour photodynamic agent, is highly accumulated and specific in tumour cell killing (ID 4051)

Presentation Number
56P
Lecture Time
12:00 - 12:00
Speakers
  • Fengwei Wang (Tianjin, China)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Pyropheophorbide-a (Pyro) is a highly promising photosensitizer for tumor photodynamic therapy (PDT), while its very limited tumor-accumulation ability seriously restricts its clinically applications. Higher accumulation of photosensitizers are very important for the treatment of deeply seated and larger tumors. Conjugation of Pyro with tumor homing peptide ligands could be a very useful strategy to optimize the physical properties of Pyro. Herein, we reported our studies on the conjugation of Pyro with a cyclic cRGDfK (cRGD) peptide, an integrin binding sequence, with the aim to development a highly tumor specific photosensitizers for PDT application.

Methods

In order to further reduce the non-specific uptake and thus reduce the background distribution of the conjugates in the normal tissues, we opted to add a highly hydrophilic polyethylene glycol (PEG) chain and an extra strongly hydrophilic carboxylic acid group as the linker to avoid the direct connection of strong hydrophobic Pyro macrocycle and cRGD ligand. While hydrophilic modification may affect the PDT activity of photosensitizers via reduction of the membrane attachment. We reported here the synthesis and characterization of these conjugates. Their tumor accumulation ability and photodynamic induced cell killing ability were evaluated through both in vitro cell-base experiment and in vivo distribution and tumor therapy experiments with tumor-bearing mice.

Results

As a result, the synthesized conjugate significantly improved the tumor enrichment and tumor selectivity of Pyro, and abolished the xenograft tumors in the murine model through one time of PDT treatment.

Conclusions

In view of the simple synthesis process and excellent tumor treatment ability, this compound has good clinical application potential.

Legal entity responsible for the study

Tianjin Union Medical Center.

Funding

Nankai University.

Disclosure

All authors have declared no conflicts of interest.

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57P - The expression of MMR, CD133 and the presence of p53 wt predict the response to cabazitaxel in malignant neural tumours cell lines (ID 859)

Presentation Number
57P
Lecture Time
12:00 - 12:00
Speakers
  • Kevin Doello (Granada, Spain)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Cabazitaxel is used in the treatment of metastatic prostate cancer. It is not a substrate of P-glycoprotein, which is involved in cellular detoxification of chemotherapeutic agents and in the expulsion of drugs at blood-brain barrier. Our objectives are to test Cabazitaxel in vitro in malignant neural cell lines such as glioblastoma, anaplastic glioma and neuroblastoma, and to find any predictor of response that allows us to select potential responder patients in glioblastoma multiforme and other neural tumors.

Methods

Cell lines A172 (glioblastoma, p53 wild type), LN229 (glioblastoma, p53 mutated), SF268 (anaplastic glioma, p53 mutated) and SK-N-SH (neuroblastoma, p53 wild type) were cultured in 48-well plates and increasing doses of Cabazitaxel were tested (0.1, 0.5, 1, 5, 10, 25, 50, 100 μM). After 48 hours of treatment, the proliferation values were studied by spectrophotometric assays. Also, gene expression studies of the MMR complex (miss-match repair) were performed by RT-qPCR for the primers of MLH1, MSH2, MSH3, MSH6, PMS2 genes. We also analyzed the expression of CD133 marker by RT-qPCR assays. A PCR study with Bisulfite kit was carried out to detect the promoter methylation of MGMT.

Results

IC50 values at 48 hours were 13.76 μM in SF268, 6.77 μM in SK-N-SH, 2.88 μM in LN 229 and 1.34 in A172. The expression values of MMR complex proteins were significantly higher in line A172 (relative value 1), followed by line LN229 (0.25) and negative in lines SK-N-SH and SF268. The methylation of MGMT promoter was positive in lines A172 and LN229. Finally, the expression of CD133 was higher in A172 (0.6), followed by LN229 (0.4), SK-N-SH (0.2) and SF268 (0). Pearson correlation coefficient for the relationship between CD133 expression and IC50 values is -0.96.

Conclusions

1. Glioblastoma, anaplastic glioma and neuroblastoma cell lines have a high sensitivity to Cabazitaxel in vitro. 2. Cell lines with MGMT methylation and MMR expression (A172 and LN 229) are the most sensitive. Of them, line A172 presents a p53 wild type, and therefore, a greater sensitivity to Cabazitaxel. 3. The expression of CD133 correlates inversely with the values of IC50 to Cabazitaxel, and may also be a predictor of response.

Legal entity responsible for the study

UGR.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

58P - IKS01, a next generation antibody drug conjugate (ADC) designed to be efficacious in tumors with low and moderate levels of folate receptor expression (ID 2497)

Presentation Number
58P
Lecture Time
12:00 - 12:00
Speakers
  • Jenny Thirlway (Newcastle-upon-Tyne, United Kingdom)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Frequent over-expression of FRA in ovarian and non-small-cell lung cancer (NSCLC), and relative lack of expression in normal tissue, make it an attractive therapeutic target. Ovarian cancer is the ninth most common cancer in women and the leading cause of death in gynaecological cancer. Lung cancer remains a leading cause of cancer-related death with NSCLC accounting for 80 % of cases. ADCs are the focus of intense interest as a means to provide selective tumor killing with increased efficacy and less off-target toxicity than standard of care chemotherapies. Clinical evaluation of mirvetuximab soravtansine in solid tumor indications demonstrated that FRA can be successfully targeted by an ADC. However, anti-tumor activity appears to be generally limited to patients whose tumors express high levels of FRA. IKS01 is an ADC comprised of an FRA-targeting antibody conjugated via Iksuda’s PermaLink conjugation technology to Femtogenix’s highly potent FGX2-62 payload. FGX2-62 is a pyrridinobenzodiazepine DNA mono-alkylating agent with pM potency in a range of tumor cell lines. IKS01 has been designed to be sufficiently potent to have target-dependent efficacy even in tumors with low or moderate expression of the target antigen.

Methods

IKS01 was generated with a drug to antibody ratio of 2. Efficacy was evaluated in human cell line derived xenograft models with a range of FRA expression, including the choriocarcinoma Jeg-3 model (high expression), the ovarian adenocarcinoma OV-90 model (low expression) and the lung adenocarcinoma NCI-H2110 model (moderate expression). An FRA-targeting benchmark ADC with a format that has shown clinical efficacy was included for comparison.

Results

IKS01 is highly effective in causing tumor regressions in FRA-expressing tumor models at doses that are well tolerated. IKS01 was significantly more active than benchmark ADC in the high FRA-expressing Jeg-3 model and caused complete or near complete regressions in low/moderate FRA-expressing xenograft models where the benchmark ADC showed limited or no activity.

Conclusions

IKS01 shows marked anti-tumor efficacy in pre-clinical models of ovarian and lung cancer, even in models with low or moderate FRA expression.

Legal entity responsible for the study

Iksuda Therapeutics Ltd.

Funding

Iksuda Therapeutics Ltd.

Disclosure

J. Thirlway: Shareholder/Stockholder/Stock options: Iksuda Therapeutics Ltd. A. Lodge: Shareholder/Stockholder/Stock options: Iksuda Therapeutics Ltd. A. Pelava: Shareholder/Stockholder/Stock options: Iksuda Therapeutics Ltd. D.J. Williamson: Shareholder/Stockholder/Stock options: Iksuda Therapeutics Ltd. D. Carta: Shareholder/Stockholder/Stock options: Iksuda Therapeutics Ltd. M. Al Nakeeb: Shareholder/Stockholder/Stock options: Iksuda Therapeutics Ltd. J. Mysliwy: Shareholder/Stockholder/Stock options: Iksuda Therapeutics Ltd. P.J.M. Jackson: Shareholder/Stockholder/Stock options: Femtogenix Ltd. D.E. Thurston: Shareholder/Stockholder/Stock options: Femtogenix Ltd. R.J. Lutz: Shareholder/Stockholder/Stock options: Iksuda Therapeutics Ltd.

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Poster Display session 1 Poster Display session

59P - Novel non-camptothecin compounds with antiproliferative activities against breast cancer cells (ID 1636)

Presentation Number
59P
Lecture Time
12:00 - 12:00
Speakers
  • Wen-shan Li (Taipei City, Taiwan)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Even though new molecular targeted agents are now being developed, they are still premature in application of clinical use for triple negative breast cancers (TNBCs). Therefore, it is highly demanded to explore the effective antitumor agents against TNBCs. Novel non-camptothecin topoisomerase I inhibitors are discovered and evaluated in this study.

Methods

Human MDA-MB-231 (BCRC-60425), BT-549 (ATCC® HTB-122™) and MCF-7 (BCRC-60436) were used in this study. Top1 assay was determined by gel mobility assay to validate the Top1-mediated DNA cleavages at different concentrations of non-camptothecin compounds. Cell viability analysis (MTT assay), comet assay and flow cytometry analysis were used to evaluate their growth inhibitory activity, DNA damage, and induction of cell arrest. Mechanistic pathways were studied and validated through western blot analysis, flow cytometry and confocal microscopy analysis. Binding interactions between top1 and non-camptothecin compound were analyzed by computational analysis (Discovery Studio).

Results

The IC50 values (growth inhibitory activity) of these non-camptothecin compounds are in the micromolar to nanomolar range (1.8 μM to 190 nM) against MDA-MB-231, BT-549 and MCF-7 cell lines. These compounds significantly inhibited the process in which supercoiled DNA strand transforms into its relaxed state and showed antitumor spectra similar to camptothecin rather than doxorubicin. Comet tails were observed to increase significantly with various doses of compounds in a dose-dependent manner. In addition, their mode of actions were shown to involve G2/M arrest of the cell cycle along with a dose-dependent increase in protein levels of cleaved caspase-3 and cleavage of cPARP. Except apoptosis pathway, non-camptothecin compounds also induce necrosis, and autophagy. Favorable ADMET characteristics of non-camptothecin compounds were observed using ADMET Descriptors.

Conclusions

Our results have provided evidence for therapeutic intervention in the treatment of TNBC using new top1 inhibitors, non-camptothecin compounds.

Legal entity responsible for the study

Wen-Shan Li.

Funding

Ministry of Science and Technology, Taiwan and Academia Sinica, Taiwan.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

60P - Sensitization of estrogen receptor-positive breast cancer cells to tamoxifen by novel epi-oligomycin A (ID 3443)

Presentation Number
60P
Lecture Time
12:00 - 12:00
Speakers
  • Margarita A. Yastrebova (Moscow, Russian Federation)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Breast cancer is the most common cancer in women. Most breast tumors are ER(+)/hormone-dependent, this makes it possible to treat them with tamoxifen and other SERM/SERD drugs. Combination treatment of tamoxifen with novel drugs provides an efficient way for reduction of the drug concentration. The aim of the work was to obtain novel less toxic oligomycin derivatives for combinatorial treatment with tamoxifen.

Methods

Oligomycin A was produced at BIOAN (Moscow, Russia) using Streptomyces avermitilis NIC B62. The oligomycin derivatives were synthesized from oligomycin A. MCF-7 breast cancer cell line and MCF-10A human mammary epithelial cell line were obtained from the ATCC collection. Hormone-resistant subline was developed by long-term treatment of MCF-7 line with tamoxifen. Antiproliferative activity was measured by MTT.

Results

A series of less toxic oligomycin A derivatives, selectively modified at the C-33 position of the side chain, was synthesized, and their activity against hormone-dependent breast cancer cells was tested. Lead compound, a (33S)-diastereomer of oligomycin A (epi-oligomycin A), was chosen, which showed high activity towards MCF-7 breast cancer cells and MCF-7/TR hormone-resistant cells. Low doses of tamoxifen and epi-oligomycin A, which had no significant effects on the growth of MCF-7 cells, were evaluated. In combination, low doses of tamoxifen and epi-oligomycin A caused a significant antiproliferative effects on MCF-7 cells. Moreover, it was possible to return the sensitivity of hormone-resistant subline to tamoxifen by epi-oligomycin A treatment. Non-malignant epithelial cells, MCF-10A, were less sensitive to epi-oligomycin A.

Conclusions

Novel epi-oligomycin A enhances response to tamoxifen, which allows for reduction of the effective drug concentration. Combinatorial strategy with epi-oligomycin A may hold promise in development of therapies against breast tumours, including those with acquired resistance to the hormonal treatment. The research is supported by Russian Science Foundation (chemistry, agreement 15-15-00141) and the Russian Foundation for Basic Research (biology, 18-015-00422).

Legal entity responsible for the study

The authors.

Funding

Russian Science Foundation (chemistry, agreement 15-15-00141) and the Russian Foundation for Basic Research (biology, 18-015-00422).

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

61P - Autophagy inhibition enhances leflunomide-induced cytotoxicity in human bladder cancer cells (ID 840)

Presentation Number
61P
Lecture Time
12:00 - 12:00
Speakers
  • Li Cheng (Wuhu, China)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Dihydroorotate dehydrogenase (DHODH) is one of the essential enzymes in the de novo biosynthesis of pyrimidine and might be a potential therapeutic target for cancer suppress. The anti-proliferative and apoptosis-inducing effects of leflunomide, a potent DHODH blocker, have been demonstrated in multiple human cancers. This study aims to investigate the cytostatic effects of leflunomide on bladder cancer and the involved mechanism.

Methods

Two human bladder cancer cell lines, T24 and 5637, were used in this study. After incubation with varied doses of leflunomide, the cell viability, apoptosis and cell cycle assay were determined with MTS, cell colony assay and flow cytometry. Western blot was used to evaluate the expression changes of cleaved-PARP, proteins involved in Akt/mTOR/P70S6K signaling pathway and cell autophagy pathway. AVO stain assay was performed to detect the autophagosome. Moreover, the cytostatic effects of leflunomide were further investigated after the modulation of cell autophagy with autophagy agonist rapamycin and inhibitor chloroquine.

Results

Our data demonstrated that leflunomide markedly inhibited the growth of both bladder cancer cells via inducing cell apoptosis and cell cycle arrest in S phase in a time- and dose-dependent manner. After leflunomide treatment, the phosphorylation levels of Akt, mTOR and p70S6K proteins in both cells were significantly down-regulated. Furthermore, AVO stain assay revealed the decline of autophagosome under the incubation of leflunomide. Modulation of autophagy with rapamycin and chloroquine observably attenuated and enhanced the cytostatic effects of leflunomide, respectively.

Conclusions

Leflunomide significantly reduced the cell viability of bladder cancer cells via Akt/mTOR/P70S6K signaling pathway. In addition, cell autophagy was demonstrated to be involved, combination leflunomide with autophagy modifier exerted enhanced antitumor effects in bladder cancer, which offered novel ideas for bladder cancer treatment.

Legal entity responsible for the study

The author.

Funding

Has not received any funding.

Disclosure

The author has declared no conflicts of interest.

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Poster Display session 1 Poster Display session

62P - mTOR inhibition in the treatment of resistant breast cancer (ID 1735)

Presentation Number
62P
Lecture Time
12:00 - 12:00
Speakers
  • María J. Rodriguez (Ciudad Autónoma De Buenos Aires, Argentina)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Breast cancer is the malignant neoplasm with the highest incidence and mortality in women worldwide. Approximately 70% of breast tumors express hormone receptors (HR+). Although antiestrogen tamoxifen has been successful for HR+ breast cancer treatment, a significant amount of these tumors eventually develop resistance. In these cases, the addition of CDK4/6 inhibitor palbociclib has shown clinical effectiveness. However, there is still a need to elucidate the mechanisms underlying resistance to these treatments in order to design better combination therapies for each case and improve patient outcomes. The aim of this work was to study these mechanisms using cell variants generated to mimic what occurs in the clinic.

Methods

We developed three cell lines with acquired resistance to tamoxifen (T47D-TR), palbociclib (T47D-PR) and tamoxifen+palbociclib (T47D-TPR), by exposing T47D wild type cells to progressively increasing concentrations of the drugs over a period of about 12 months.

Results

Western blot analysis showed decreased levels of HR, higher activation levels of PI3K/Akt/mTOR pathway, along with greater levels of cell cycle associated proteins in all resistant cell lines. The resistant tumors generated as xenografts in immunosuppressed mice showed more invasive characteristics than those generated from the T47D-wt cell line, despite the lower growth rates showed by all resistant lines. Finally, we analyzed the response to palbociclib and to PI3K/Akt/mTOR inhibitors in the resistant cell lines. We found that all resistant lines were sensitive to BKM-120 (PI3K inhibitor) and to rapamycin (mTOR inhibitor), and the combination with palbociclib showed an additive inhibitory effect in both cases. However, the sensitivity of the resistant cells was greater to rapamycin than to BKM-120.

Conclusions

Taken together, our results support the use of mTOR inhibitors, rather than PI3K inhibitors, in combination with CDK4/6 inhibitors in the treatment of both tamoxifen and palbociclib resistant tumors.

Legal entity responsible for the study

FONCYT-INC-CONICET.

Funding

FONCYT-INC-CONICET.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

63P - Study of photodynamic therapy in vitro (ID 6068)

Presentation Number
63P
Lecture Time
12:00 - 12:00
Speakers
  • Irene Jiménez Munguía (Moscow, Russian Federation)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Photosensitizers (PS) are commonly used in photodynamic therapy to treat skin cancer. PS molecules bind to cell membrane and damage it by singlet oxygen (SO) generated under illumination. In our laboratory, we study in vitro the processes involved in photodynamic therapy on a model bilayer lipid membranes (BLM) by measuring the boundary potential applying the Intramembrane Field Compensation Method (Sokolov and Kuz’min, Biofizika, 25:170, 1980).

Methods

This method allowed to monitor the binding of PS on BLM and damage of target molecules (TM) of SO - di-4-ANEPPS under excitation of PS by light. In present investigation, we studied the adsorption and photodynamic efficiency of new positively charged porphyrins, namely b-imidazolyl substituted porphyrin and it’s Zn(II) and In(III) complexes; and two phosphorus (V) complexes of meso-(p-pyridyl)-triphenylporphyrin bearing hydroxyl and ethoxyl axial ligands. We observed a linear dependence of the boundary potential change on the logarithm of concentrations of each PS.

Results

The photodynamic efficiency of these porphyrins was assessed by determining the rate of oxidation (R) of TM adsorbed either on the same or opposite surface of the BLM where molecules PS were present. The values R for both positions of TM were close indicating that BLM is highly permeable to singlet oxygen. The values R were proportional to surface density of the porphyrin molecules in the membrane.

Conclusions

This investigation indicate that the main factor influencing the photodynamic efficiency of the porphyrins is their adsorption on the BLM. The work was supported by the Russian Foundation for Basic Research (project 19-04-00694) and the Ministry of Education and Science of the Russian Federation in the framework of Increase Competitiveness Program of NUST «MISiS» (№ К4-2017-053).

Legal entity responsible for the study

National University of Science and Technology.

Funding

NUST-MISiS, Russian Science Foundation, Russian Academy of Sciences.

Disclosure

All authors have declared no conflicts of interest.

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64P - The potential of neratinib plus dasatinib in overcoming and preventing neratinib resistance in HER2-positive breast cancer models (ID 3011)

Presentation Number
64P
Lecture Time
12:00 - 12:00
Speakers
  • Neil Conlon (Dublin, Ireland)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Neratinib (NER) is an irreversible pan-HER kinase inhibitor with demonstrated clinical activity for HER2-positive and HER2-mutated breast cancers (BC). Mechanisms of resistance to NER are poorly understood. The Src/Abl inhibitor dasatinib (DAS) has shown ability to overcome resistance to HER2-targeted agents such as lapatinib (LAP) and TRAS in vitro. This pre-clinical study investigated the efficacy of DAS in combination with NER to overcome or prevent NER resistance in BC models.

Methods

Anti-proliferative effects of NER, LAP, TRAS, DAS, and NER plus DAS were assessed in five NER-resistant HER2+ BC cell lines (HCC1954-N, HCC1569-N, EFM192A-N, BT474-N, and SKBR3-N) by acid phosphatase assay. IC50values and Combination index (CI) values were calculated to determine synergy (CI < 0.8) using Calcusyn. Neratinib resistance was defined as an IC50value > 150 nM NER. Apoptosis induction was assessed by Caspase 3/7-Glo assay. Reverse phase protein array was used to determine changes in key signalling pathways in HCC1954-N cells. BC cells were treated twice weekly with NER and/or DAS and crystal violet stained when confluent to examine resistance development.

Results

All NER resistant cell lines examined had significantly reduced response to NER (11-83 fold increase in IC50values versus parental cells), as well as LAP and TRAS, compared to their parental cells.The combination of NER and DAS displayed synergy in all five NER resistant cell lines (CI = 0.1-0.6). NER plus DAS caused a strong induction of apoptosis in the HCC1954-N cells (p = 0.015). NER/DAS treatment caused changes in 23 phospho- or total protein levels, including suppression of Akt, MAPK, Src, p38 and AMPK signalling. NER alone (9 phospho- and 1 total proteins) and DAS alone (3 phospho- and 3 total proteins) altered fewer targets. The addition of DAS to NER prevented the emergence of NER resistance in parental HCC1954 and HCC1569 cells.

Conclusions

This study provides pre-clinical rationale for the combination of NER and DAS in NER-resistant HER2+ BC and shows that this combination warrants further investigation.

Legal entity responsible for the study

The authors.

Funding

Puma Biotechnology.

Disclosure

N. Conlon: Research grant/Funding (institution): Puma Biotechnology. J. Crown: Full/Part-time employment: OncoMark; Honoraria (self), Advisory/Consultancy, Speaker Bureau/Expert testimony, Research grant/Funding (institution): Eisai; Honoraria (self): Amgen; Honoraria (self), Advisory/Consultancy, Research grant/Funding (institution): Puma Biotechnology; Honoraria (self): Seattle Genetics; Honoraria (self), Advisory/Consultancy, Speaker Bureau/Expert testimony, Travel/Accommodation/Expenses: Pfizer; Honoraria (self), Advisory/Consultancy: Vertex; Honoraria (self), Advisory/Consultancy, Research grant/Funding (institution), Travel/Accommodation/Expenses: Roche; Honoraria (self), Travel/Accommodation/Expenses: MSD Oncology; Travel/Accommodation/Expenses: AstraZeneca; Travel/Accommodation/Expenses: Abbvie; Honoraria (self), Advisory/Consultancy, Research grant/Funding (institution): Boehringer Ingelheim; Honoraria (self), Speaker Bureau/Expert testimony: Genomic Health. D.M. Collins: Research grant/Funding (institution): Puma Biotechnology; Research grant/Funding (institution): Roche. All other authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

65P - Novel HDACi, MHY446, induces apoptosis via regulation of mitochondria-endoplasmic reticulum interaction in HCT116 human colorectal cancer cells (ID 2644)

Presentation Number
65P
Lecture Time
12:00 - 12:00
Speakers
  • Nam Deuk Kim (Busan, Korea, Republic of)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Colorectal cancer (CRC) is one of the leading causes of cancer-related death in the world. Despite considerable research and advancement in CRC diagnosis and therapy, CRC is still clinically challenging and becoming the highest incidence cancer worldwide. There is a need to develop novel therapeutic agents for this malignancy.

Methods

MHY446, a novel histone deacetylase inhibitor (HDACi), was synthesized on the backbone of hydroxamic acid derivatives that are one of the HDAC inhibitors. We evaluated cytotoxic effects and underlying molecular mechanisms of actions of MHY446 in HCT116 human colorectal cancer cells.

Results

MHY446-treated HCT116 cells exhibited cell viability inhibition and an increase in the sub-G1 phase populations and late apoptosis. MHY446-induced apoptosis is accompanied by the activation of caspase-8, -9, and -3; subsequent cleavage of poly(ADP-ribose) polymerase; and alteration in the ratio of Bax/Bcl-2 protein expression level. The presence of Z-VAD-FMK, a pan-caspase inhibitor, significantly attenuated the MHY446-induced apoptosis indicating that apoptotic cell death was caspase-dependent cascading through the activation of both intrinsic and extrinsic signaling pathways. Furthermore, MHY446 caused the loss of mitochondria membrane potential and induced the generation of reactive oxygen species (ROS), evidenced by the scavenging of ROS by N-acetyl-L-cysteine (NAC). Additionally, MHY446 promoted the upregulation of the expression level of endoplasmic reticulum (ER) stress-associated proteins such as BIP, PDI, IRE1α, PERK, p-elF2α, and CHOP. NAC effectively reduced the expression of ER stress-related protein levels, suggesting that ROS acts as an upstream signaling molecule in triggering the ER stress pathway.

Conclusions

Taken together, these results demonstrate that MHY446 exerts its anticancer activities through the regulation of ROS-induced ER stress. These results suggest that MHY446 is a promising candidate for the treatment of CRC.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

66P - Dual inhibition of TGF-β and AXL as a novel treatment for colorectal cancer (ID 3085)

Presentation Number
66P
Lecture Time
12:00 - 12:00
Speakers
  • Davide Ciardiello (Napoli, Italy)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

AXL and transforming growth factor β (TGF-β) signalling pathways play a key role in modulating tumour microenvironment by promoting epithelial to mesenchymal transition (EMT), stromal activation and regulating immune response in colorectal cancer (CRC). Here we have evaluated the effect of double blockade of TGF-β and AXL receptors, as an innovative therapeutic strategy for CRC.

Methods

We assessed the correlation of TGF-β and AXL by an in silico analysis of a human CRC gene expression profile database. Afterwards, an evaluation of AXL expression of in a panel of human CRC cell lines by Western Blot (WB) and Real time PCR was performed. The sensitivity of HCT116 and LOVO cells to treatment with galunisertib (LY21209761), a TGF-β R1 inhibitor, and R428, an AXL inhibitor, was assessed by MTT, cell migration, and colony forming assays. Furthermore we generated patient derived 3D spheroid cultures from primary CRC, and tested their susceptibility to galunisertib and R428 by MTT assay. Finally, in vivo experiments were done with HCT116 and LOVO tumour xenografts in immunodeficient mice.

Results

In silico analysis showed an association between high expression levels of AXL and TGF-β R1 in CMS4 CRC. AXL was differently expressed on cell lines, and interestingly it resulted increased in HCT116 and LOVO after TGF-β inhibition, probably representing a mechanism of cancer cell escape. Dual blockade with galunisertib and R428 determined a reduction in cell migration of 50% in HCT116 and 37% in LOVO cells, respectively, and colony formation of approximately 90 % in both cell lines. Moreover, combined treatment displayed a strong synergistic activity in 3D cultures derived from five human CRC samples, resulting in the inhibition of proliferation ranging from 80 to 90%. The in vivo experiments of HCT116 and LOVO subcutaneous xenograft models are currently on going and results will be presented.

Conclusions

Combined TGF-β and AXL inhibition showed a promising activity in preclinical models of CRC and could represent a novel potential therapeutic strategy for patients with CRC.

Legal entity responsible for the study

Università degli studi della Campania "Luigi Vanvitelli".

Funding

ICURE project, Università degli studi della Campania "Luigi Vanvitelli".

Disclosure

D. Melisi: Research grant/Funding (self): Lilly.

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67P - PARP inhibition enhances cisplatin sensitivity in cervical cancer by modulating β-catenin signaling (ID 1314)

Presentation Number
67P
Lecture Time
12:00 - 12:00
Speakers
  • Minakshi Mann (Delhi, India)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Cisplatin (CDDP) is used in the treatment of locally advanced disease (FIGO stage IIB-IVA) as well recurrent/metastatic cervical cancer. However toxic side effects and acquired resistance limits its efficacy. Enhanced DNA repair is one of the mechanisms responsible for acquired cisplatin resistance. Poly(ADP-ribose) polymerase (PARP) inhibitors have been approved for treating BRCA mutated cancers like breast and ovary cancer, however, little is known about the therapeutic efficacy and mechanism of action of PARP inhibitors in non-BRCA cancer like cervical cancer, either as a single agent or in combination with cisplatin.

Methods

Effect of PARP-1 inhibition (PJ34/PARP-1siRNA) was determined in vitro on cell viability, apoptosis, cell cycle progression, proliferation, invasion and metastasis, clonogenecity and β-catenin signaling in cervical cancer cell lines HeLa and SiHa.

Results

Combination of CDDP with PJ34 or PARP-1 siRNA significantly reduced the cell proliferation and induced cell cycle arrest and apoptosis. Also, a significant decrease in cell survival as well as cell invasion and migration was observed as compared with either CDDP/PJ34 alone. The enhanced CDDP sensitivity by PARP-1 inhibition was determined to be due to inhibition of β-catenin signaling as shown by a decrease in MMP-2 activity, MMP-9, c-myc and cyclin-D1 expression upon treatment with PJ34 and PARP-1 siRNA.

Conclusions

Our data provides experimental evidence on the contribution of PARP-1 inhibition in enhancing the cytotoxicity of CDDP in cervical cancer cells. We also present novel findings on the suppression of β-catenin and its downstream signaling components by PARP-1 inhibitor.

#: 5μM of CDDP was used both alone and in combination with PJ34; ##: A gradient of CDDP from 0-15μM was used to evaluate the combined effect of PJ34 and CDDP on IC50 value of CDDP. p: *<0.05, **<0.01. (H): HeLa; (S): SiHa.

Legal entity responsible for the study

The authors.

Funding

All India Institute of Medical Sciences.

Disclosure

All authors have declared no conflicts of interest.

Combined effect of PJ34 and CDDP on cisplatin sensitivity in cervical cancer cell lines

Cancer cell parameterOnly CDDP (10μM)Only PJ34 (10μM)CDDP(10μM) + PJ34(10μM)p value/fold difference
Clonogenic formation fraction#0.23 ± 0.08 (H) 0.31± 0.06 (S)0.67 ± 0.09 (H) 0.65 ± 0.01 (S)0.13 ± 0.05 (H) 0.15 ± 0.07 (S)* (H) * (S)
Fold migration0.71 ± 0.06 (H) 0.75 ± 0.03 (S)0.89 ± 0.07 (H) 0.99 ± 0.08 (S)0.54 ± 0.05 (H) 0.34 ± 0.02 (S)** (H) * (S)
Relative invasion43.4 ± 2.25 (H) 47.9 ± 7.07 (S)66.6 ± 5.94 (H) 70.5 ± 5.74 (S)15.8 ± 2.81 (H) 23.4± 4.13 (S)** (H) ** (S)
IC50 value##8.25μM (H) 10.8μM (S)31.5μM (H) 33.0μM (S)3.6μM (H) 3.4μM (S)2.29 fold 3.18 fold

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68P - Synergistic effect of DSF combined treatment with cisplatin in atypical teratoid/rhabdoid tumors (AT/RT) (ID 2417)

Presentation Number
68P
Lecture Time
12:00 - 12:00
Speakers
  • Seung Ah Choi (Seoul, Korea, Republic of)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

An atypical teratoid/rhabdoid tumor (AT/RT) is an aggressive tumor of the central nervous system that generally occurs in children under three years of age. There is no effective chemotherapy in most AT/RT patients. Within a tumor, small subpopulation cells called cancer stem cells do not respond to or are resistant to conventional anticancer drugs. In previous our studies, we observed the expression of aldehyde dehydrogenase (ALDH), a stem cell marker in AT/RT cells. Based on this, we confirmed the anticancer effect using disulfiram (DSF) known as ALDH inhibitor. Herein, we have investigated the anticancer effect of DSF with cisplatin in order to improve the effectiveness of AT/RT treatment.

Methods

Patient-derived primary cultured cells and established cell lines were utilized in vitro experiments. The combination effects of DSF with cisplatin was confirmed by cell viability, flow cytometry, ELISA and immunofluorescence analysis. The mechanism was identified by western blot analysis. Tumor volumes and survival rates were analyzed via bioluminescence live imaging in an AT/RT orthotopic xenograft mouse model to verify in vivo therapeutic effects.

Results

Our results demonstrate the anticancer effects of the combination of DSF and cisplatin both in vitro and in vivo. The combined treatment significantly inhibited the cell viability and ALDH enzyme activity of all AT/RT cells. The combination of DSF and cisplatin has been shown to modulate C-jun and ATF3 protein expression and activate PARP to induce apoptosis much more effectively. Importantly, the combination of DSF with cisplatin resulted in decreased tumor volume and increased long-term survival rate in AT/RT animal models.

Conclusions

Our study suggests that combination therapy of DSF and cisplatin can be used as a novel treatment strategy for AT/RT, which is difficult to treat with conventional chemotherapy.

Legal entity responsible for the study

The authors.

Funding

Grant from the Seoul National University Hospital Research Fund (04-2018-0280) and a grant from the National Research Foundation (NRF) of Korea (2017R1A2B2008422).

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

69P - Reactive oxygen species induced by OSU-A9 inhibit the growth of duodenal cancer and gastric cancer cells through dephosphorylating intranuclear pyruvate kinase muscle isozyme M2 (ID 1149)

Presentation Number
69P
Lecture Time
12:00 - 12:00
Speakers
  • Li-Yuan Bai (Taichung, Taiwan)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Pyruvate kinase M2 (PKM2) is a key enzyme responsible for the final step of glycolysis. Whether and how the pyruvate kinase M2 is involved in reactive oxygen species (ROS)-mediated cytotoxicity of gastrointestinal cancer is unknown.

Methods

One duodenal cancer cell line AZ521, and two gastric cancer cell lines NUGC and SCM-1 and were treated with OSU-A9 which is known to induce cytotoxicity of acute myeloid leukemia through ROS generation. The in vitro cytotoxic and proapoptotic activities of OSU-A9 was evaluated by the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay and annexin V – propidium iodide staining, respectively. Overexpression experiment was performed by transfection with indicated plasmid using Lipofectamine 2000 according to the manufacturer’s protocol.

Results

OSU-A9 induced a dose- and time-dependent cytotoxicity and apoptosis in duodenal cancer and gastric cancer cells through ROS generation. The IC50 of OSU-A9 at 24 and 48 h for AZ-521, NUGC and SCM-1 were 2.68 and 1.83, 3.34 and 2.81, and 2.71 and 2.36 mM, respectively. Pretreatment with ROS scavenger rescued the cancer cells from apoptosis and concomitant PARP cleavage, implicating a key role of ROS in OSU-A9-induced cell death. Furthermore, OSU-A9-mediated ROS down-regulated pTyr105-PKM2 which occurred in cell nucleus rather than in cytoplasm. Ectopic overexpression of PKM-2 partially overcame the cytotoxicity of OSU-A9, which implied a role of phosphorylated PKM2 beyond glycolysis in survival of duodenal cancer and gastric cancer cells.

Conclusions

This study shows that ROS-mediated intranuclear PKM2 dephosphorylation, in part, contributes to the anticancer activity of OSU-A9 in duodenal cancer and gastric cancer. Differential down-regulation of phosphorylated PKM2 between nucleus and cytoplasm suggests a non-glycolytic role of PKM2 in cell survival response to ROS stress.

Legal entity responsible for the study

The authors.

Funding

The Ministry of Health and Welfare, Taiwan.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

70P - New therapy for intrahepatic cholangiocarcinoma targeted to cancer associated fibroblasts (ID 1862)

Presentation Number
70P
Lecture Time
12:00 - 12:00
Speakers
  • Takahiro Yamanaka (Maebashi, Japan)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

The microenvironment such as cancer associated fibroblasts (CAFs) was reported to be involved in progression of intrahepatic cholangiocarcinoma (ICC). Nintedanib (BIBF1120) is a tyrosine kinase inhibitor of PDGFR, FGFR and VEGFR which is approved for idiopathic pulmonary fibrosis. And nintedanib was reported to suppress liver fibrosis in mouse through the suppression of hepatic stellate cells activation (Ozturk Akcora B, et al. Sci Rep. 2017 Mar 14;7:44545). Therefore, we hypothesized that nintedanib can inhibit the activity of CAFs of ICC. The purpose of this study is to clarify the effect of CAFs on ICC and develop a new therapy for ICC targeted to CAFs.

Methods

CAFs were established from surgically resected ICC tissues. The effect of nintedanib on proliferation and expression of alpha smooth muscle actin (αSMA) of CAFs was examined. The effect of CAF-CM (conditioned medium of CAFs) and nintedanib-CAF-CM (CM of CAFs treated with nintedanib) on proliferation and invasion of ICC cell lines (HuCCT1, RBE) was examined. The humoral factors of CAF-CM and nintedanib-CAF-CM were analyzed using cytokine array and ELISA. The effect of nintedanib and gemsitabine (GEM) treatment on xenograft model co-implanted with HuCCT1 and CAFs was examined in vivo.

Results

Nintedanib(1µM) suppressed the proliferation of CAFs and expression of αSMA in CAFs. CAF-CM significantly promoted the proliferation and invasion of ICC cell lines. But in nintedanib-CAF-CM, all of those cancer promoting effects were cancelled. In cytokine array and ELISA of CAF-CMs, expression of IL-6, IL-8, VEGF, VCAM1, and Osteopontin were suppressed in nintedanib-CAF-CM compared with CAF-CM. HuCCT1 + CAFs xenograft was increased more than HuCCT1 alone in vivo. Combination treatment with nintedanib (30mg/kg) and GEM (50mg/kg) inhibited HuCCT1 + CAFs xenograft growth than nintedanib or GEM alone in vivo. The xenograft treated with nintedanib and GEM showed reduction of both αSMA-positive staining in stroma and proportion of Ki-67 positive ICC cells.

Conclusions

Nintedanib suppressed CAFs activity and the production of cancer-promoting cytokines produced by CAFs. Combination therapy with nintedanib and GEM may be a new promising therapy to overcome refractory ICC.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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71P - Macrophage-cancer cell fusion is mediated by phosphatidylserine-CD36 receptor interaction and induced by ionizing radiation (ID 782)

Presentation Number
71P
Lecture Time
12:00 - 12:00
Speakers
  • Ivan Shabo (Stockholm, Sweden)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Cell fusion (CF) is a normal biological process that plays major role in mammalian development and differentiation. Through CF, somatic cells acquire nuclear reprogramming and epigenetic modifications to form pluripotent hybrid cells, which constitute an efficient process of rapid evolution to generate hybrids with new genetic, phenotypic and functional properties at a rate exceeding that achievable by random mutagenesis. Experimental and clinical evidences indicate that CF occurs in solid tumors and contribute to cancer progression by generating progeny with metastatic features and resistance to oncologic treatments. Phosphatidylserine (PS) is a glycerophospholipid, recognized by CD36 receptor in macrophage to detect apoptotic cells. The PS-CD36 interaction is a crucial step in fusion between monocytes and tumor cells. The aim of this study is to investigate the impact of the PS-CD36 expression in macrophage–cancer cell fusion and in relation to ionizing radiation (IR).

Methods

GFP-labeled breast cancer MCF-7 cells and macrophages (differentiated monocyte cell line THP-1) were used in CF in vitro experiment with IR (figure 1). MCF-7/macrophage hybrids were generated by spontaneous CF, isolated by fluorescence activated cell sorting and confirmed by fluorescence microscopy. We treated the hybrids and their maternal cells (MCF-7 and macrophages) with IR (ɣ-irradiation, 0Gy, 2.5Gy and 5Gy). Anti-CD36 antibody, 40µg and 80µg, was used to block CD36 receptor. CF rate is based on the number of seeded macrophages, since they do not proliferate. The proportion of hybrids = (number of hybrids/number of seeded macrophages in same sample) x100.

Results

IR induces the exposure of PS (dose dependent) on MCF-7 cells, which was not found in macrophages. IR also induces significant expression of CD36 in THP-1 cells. CF was inhibited by blocking of CD36 receptor with anti-CD36 antibodies (figure 2).

Conclusions

IR induces macrophage-cancer cell fusion by stimulating the exposure of PS on MCF-7 cells and the expression of CD36 in macophages. CF and the generation of metastatic hybrids can be prevented by inhibiting the PS-CD36 interaction, a mechanism constituting an essential step in macrophage-cancer CF.

Legal entity responsible for the study

Ivan Shabo, Karolinska Institutet, Stockholm Sweden.

Funding

Swedish Society of Medicine.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

72P - Effects of three products in the prevention and treatment of chemotherapy and radiation therapy-induced oral mucositis (ID 3181)

Presentation Number
72P
Lecture Time
12:00 - 12:00
Speakers
  • Francesca Zannier (Rho, Italy)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Oral pain, odynophagia and dysphagia, weight loss, dehydration, systemic infection, feeding support and hospitalization should be mentioned as the main determinated effect of oral mucositis (OM); this toxicity is often linked to chemo- or chemoradiotherapy. Oral Mucositis can reduce patients’compliance to cancer treatment and decreased their quality of life; it could be also reduce survival caused by discontinuation or dose reduction of anti-neoplasm therapy. The purpose of this study is to evaluate the impact of the three different products on OM outcomes of patients who received radio and chemotherapy in solid cancer.

Methods

From March to June 2018, 60 patients (pts) were randomized by Medical Oncology Unit of Rho - Milan. The pts were divided in 3 arms, belanced for tumor site and treatment, to receive the following medical devices: Gelx (oral spray), Episil (oral solution) and Gelclair (oral gel). Primary aim of the study was evaluation of OM onset, severity, reduction or remission and pain relief with the three devices in study. We analysed data with forms compiled by pts in 2 different steps: T0 (baseline) and follow-up (after 16 w); the clinicians evaluated OM grade in 2 steps: after 4 w and after 8 w. The pts’safety profile form included 10 items concerning difficulty of speaking, feeding, drinking and change of taste.

Results

The data analysis showed a significant difference among the 3 Groups. OM grade of Group 2 after 4 weeks is higher if compared with Group 1 and Group 3 After 4 weeks two cases of grade 4 mucositis has been achieved in Group 2, none in Group 1 and Group 3. Group GELX (Zinco gluconate included) demonstrated an overall better efficacy, in every analyzed safety profile items.

Conclusions

Even if prevention and treatment of OM are not yet clearly defined and recognized in the clinical practice, the study shows how the correct use of anti- mucositis product (accompanied by an appropriate hygienic and dietetic regimen) seems to be effective in the prevention of this pathology, which is the most frequent side effects on patients undergoing chemotherapy.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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73P - Boiling histotripsy-induced mechanical ablation modulates tumour microenvironment by promoting immunogenic cell death of cancers (ID 2433)

Presentation Number
73P
Lecture Time
12:00 - 12:00
Speakers
  • Cheol-Hee Shin (Seoul, Korea, Republic of)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Boiling histotripsy is a promising non-invasive High-Intensity Focused Ultrasound (HIFU) technique that employs HIFU mechanical effects to fractionate solid tumours without causing any signicant thermal damage. It has been suggested that boiling histotripsy may induce a strong immune response due to the absence of denatured antigenic protein at the HIFU focus. However, the underlying immunological mechanisms of this technique are poorly understood. The main objectives of this present study were to (a) investigate the feasibility of employing High-intensity Focused Ultrasound-induced Mechanical Ablation (boiling histotripsy) in the treatment of solid tumors; (b) examine the relationship between the degree of mechanical damage induced by boiling histotripsy and the level of immune activity, and (c) finally to provide a better understanding of the effects of boiling histotripsy on immune response.

Methods

In vitro 3D model system was used to investigate the effects of histotripsy on tumor immune microenvironment. MDA-MB-231 tumor spheroids were made with 10,000 cells for 72hrs and embedded in collagen gels (6mg/ml). Cell Death Pathway array was performed to determine the mechanism of tumor death. Proteome cytokine array was performed to identify antigenic factors that were secreted by damaged solid tumors. Macrophage polarization induced by immunogenic cell death was examined using qPCR array.

Results

Our results showed that cancer cells (MDA-MB-231) underwent immunogenic cell death via TNF-mediated necrosis signaling pathway after the boiling histotripsy exposure. Significant increases in secretions of damage-associated molecular patterns (CRT, HSP70, HMGB-1), pro-inflammatory cytokines (IFN-γ, IL-1α, IL-1β, IL-18) and chemokines (IL-8) which were related to M1 macrophage activation were also observed. Furthermore, the levels of these signaling proteins increased with the degree of mechanical damage induced by the boiling histotripsy.

Conclusions

We demonstrate that histotripsy causes the mechanical destruction of cancer cells and promotes immunogenic cell death, ultimately leading to an anticancer immune response.

Legal entity responsible for the study

The authors.

Funding

Korea Institute of Science and Technology (KIST).

Disclosure

All authors have declared no conflicts of interest.

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74P - The bacterial receptor NOD2 mediates LGR5+ intestinal stem cells protection against irradiation via mitophagy activation (ID 2663)

Presentation Number
74P
Lecture Time
12:00 - 12:00
Speakers
  • Antonin Levy (Villejuif, France)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

The NOD2 agonist muramyl-dipeptide (MDP), a peptidoglycan motif common to all bacteria, supports LGR5+ intestinal stem cells (ISCs) survival through NOD2 activation upon an otherwise lethal oxidative-stress-mediated signal. Yet the underlying protective mechanisms remains unknown.

Methods

Irradiation was used as stressor and primarily murine-derived intestinal organoids as a model system.

Results

MDP induced a strong reduction of total and mitochondrial reactive oxygen species (ROS) within ISCs, which was associated with mitophagy induction. ATG16L1 KO and NOD2 KO organoids did not benefit from the MDP-induced cytoprotection. We showed the MDP-dependent induction of ISC mitophagy upon stress in vivo.

Conclusions

In LGR5+ ISCs, NOD2 allows irradiation-generated ROS clearance through ATG16L1-activated mitophagy.

Legal entity responsible for the study

The authors.

Funding

This work was supported by European Research Council (ERC) advanced grant 339579-DECRYPT to P.J.S and by French National Research Agency (ANR) grant 17-CE14-0022 (i-Stress) to P.S.. A.L. was personally funded by: i) Poste d’Accueil INSERM and ii) Soutien pour la formation à la recherche translationnelle en cancérologie (ITMO Cancer, INCa - Plan Cancer 2014-2019, allocation number: ASC17040JSA).

Disclosure

All authors have declared no conflicts of interest.

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75P - Pulse mode irradiation regimen of PDT results in high progression free and overall survival in mice with model tumour (ID 3213)

Presentation Number
75P
Lecture Time
12:00 - 12:00
Speakers
  • Alexey Bogdanov (Saint-Petersburg, Russian Federation)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Photodynamic therapy (PDT) is one of actively developing modalities of cancer treatment. During PDT procedure systemically injected photosensitizer molecules are activated by laser irradiation in tumor location leading to generation of singlet oxygen which induces cytotoxic effect. In modern clinical practice PDT uses continuous wave irradiation. In such mode fast depletion of molecular (triplet) oxygen in tissue occurs leading to reduction of PDT efficiency. Previously we have shown that photodynamic treatment using pulse mode irradiation regimens could be more efficient in damaging tumor cells than standard PDT due to reoxygenation (Klimenko et. al., Photodiagnosis Photodyn Ther., 2016). In present study we have evaluated specially designed pulse mode PDT regimes on tumor model in vivo.

Methods

PDT pulse mode irradiation regimens were designed using physics and mathematical modelling. BALB/c male mice were subcutaneous injected into right flanks with CT26 murine colon carcinoma cells. At day ten after inoculation mice were randomized into 3 groups with 10 animals in each (control group, PDT at continuous wave irradiation mode – CW group, PDT at pulse irradiation mode – PM group) and PDT was made. The RadachlorinⓇ photosensitizer and «Lahta-Milon» semiconductor laser (662 nm) were used. After treatment the tumor size was estimated every two days. All experiments were performed in accordance with the animal ethics guidelines.

Results

We have designed new pulse mode irradiation regimens of PDT. We have shown that antitumor efficiency of PDT at pulse irradiation mode is much better than one at standard continuous wave irradiation mode. Median survival after day of treatment was 14 days in control group, 16 days in CW group and 28 days in PM group. In PM group 5 animals had total regress of tumor and progression free survival till the end of experiment (100 days after treatment) compared with only 1 animal in CW group. Thus, we can suggest that overall survival should be much higher in PM group.

Conclusions

We have demonstrated that PDT using pulse mode irradiation regimens is much more effective that PDT using standard regimens and leads to higher progression free and overall survival in mice with CT26 colon carcinoma.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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76P - Proton-sensitizing effect of small molecule inhibitor of P300 histone acetyltransferase C646 in human pancreatic cancer cells (ID 3722)

Presentation Number
76P
Lecture Time
12:00 - 12:00
Speakers
  • Sungwon Shin (Seoul, Korea, Republic of)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Histone acetylation plays an important role in regulating the chromatin structure and is tightly regulated by histone acetyltransferases (HAT) and histone deacetylases (HDAC). Recent studies have demonstrated that several chromatin-regulating proteins can modulate cellular responses to other cytotoxic modalities including ionizing radiation and chemotherapeutic drugs. Even though histone acetylation is one of the key mechanisms of epigenetic regulation, relatively little is known about the cancer therapeutic potential of HAT inhibitors. This is in stark contrast to the well-studied effects of HDAC inhibitors. In this study, we investigated the proton beam-sensitizing effect of C646, a selective small molecule inhibitor of p300 histone acetyltransferase in human pancreatic cancer cells.

Methods

Cell viability assay (CCK-8; Dojindo), Clonogenic survival assay, Cell cycle analysis (PI staining). Annexin V-FITC and PI staining, γ-H2AX foci, Western blot, BxPC-3 Xenograft tumor model, TUNEL assay.

Results

AsPC-1, BxPC-3 and Mia-paca2 cells exhibited increase in radiosensitivity when exposed to C646 at all doses of proton beam tested. C646 pre-treatment induce led to increase of sub-G1 population and abolishment of G2/M arrest. Flow cytometry analysis with annexin V staining and Western blot analysis showed that The pre-treatment with C646 significantly enhanced proton-induced apoptotic cell death through the down-regulation of anti-apoptotic molecules. Our in vivo results demonstrate synergistic effects of combination therapy with C646 and proton beam. In BxPC-3 xenograft tumor model, C646 pre-treatment increased proton-induced tumor growth inhibition and apoptotic cell death in tumor tissues. In addition, the combination treatment of C646 with proton beam increased DNA damage and decreased activation of DNA repair pathway compared with proton alone.

Conclusions

Our results suggest that the C646 may increase proton- induced apoptosis through the modulation of various pro- and anti- apoptotic molecules in human pancreatic cancer cells. These results provide proof of principle that the inhibition of histone acetylation by HATis can be exploited to develop new proton-sensitizer.

Legal entity responsible for the study

The authors.

Funding

National Research Foundation of Korea (NRF).

Disclosure

All authors have declared no conflicts of interest.

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77P - Red-blood-cell-membrane-enveloped magnetic nanoclusters as a biomimetic theranostic nanoplatform for bimodal imaging guided cancer photothermal therapy (ID 5805)

Presentation Number
77P
Lecture Time
12:00 - 12:00
Speakers
  • Sheng Wang (Shanghai, China)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Due to their intrinsic thermal and magnetic resonance imaging properties, magnetic nanoparticles (MNCs) have attracted more and more attention in the biomedical field. However, relatively weak photothermal conversion (PTC) efficiency and low tumor homing capacity has hampered its further application in vivo.

Methods

To solve these problems, we modified near-infrared (NIR) light-absorbing materials onto the surface of MNCs to increase PTC efficiency and increase MNCs tumor homing capacity by coated red blood cell (RBC) membranes.

Results

Our data show that after being loaded with NIR cypate molecules, the as-prepared Cyp-MNCs showed remarkably increased NIR absorbance and resultant PTC efficiency compared with the MNCs. By disguising itself, Cyp-MNCs coated with erythrocyte membranes inherit the long circulation characteristics of RBCs to improve the MNCs’ tumor homing capacity. By tracking the NIR fluorescence of cypate under an in vivo fluorescence imaging system, we discovered that such Cyp-MNC@RBCs upon intravenous injection show significantly improved tumor homing capacity compared with bare cypate-loaded MNCs. The antitumor efficiency of biomimetic Cyp-MNC@RBCs was particularly prominent and was superior to biomimetic MNC@RBCs. Additionally, fluorescence and T2-weighted magnetic resonance imaging (MRI) functionalities were all retained attributable to Cyp-MNC@RBCs cores.

Conclusions

Our study would provide a promising procedure for other similarly enhanced photothermal treatments by increasing PTC efficincies and improving tumor-homing capacity.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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78P - Urine cell-free and extracellular vesicle cargo miRNAs as biomarkers for prostate cancer diagnosis (ID 5251)

Presentation Number
78P
Lecture Time
12:00 - 12:00
Speakers
  • Ivan A. Zaporozhchenko (Novosibirsk, Russian Federation)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Prostate cancer (PCa) is one of the major cancers in men, but discrepancy between incidence and mortality calls for a careful approach to screening and diagnosis. Controversy between US Preventive Services Task Force recommendations and the outcomes clinical trials illustrate that the shortcomings of existing biomarkers and high demand in novel approaches for prostate cancer screening. Recent studies have suggested miRNAs as new candidates for cancer biomarkers. In this study, we have investigated the use of miRNAs found in cell-free nucleoprotein complexes and urine extracellular vesicles (EVs) as prostate cancer biomarkers.

Methods

Urine extracellular vesicles were isolated by standard ultracentrifugation protocols. Cell-free supernatant containing nucleoprotein complexes was obtained after 17.000g centrifugation. miRNA from supernatant and urine EVs were isolated as described previously (Lekchnov et al, Anal Biochem, 2016) and profiled using customized miRCURY LNA miRNA qPCR panels (Exiqon Ltd).

Results

Profiling of the expression of 84 miRNAs in paired samples of urine supernatant and urine EVs of 10 healthy donors (HD), 10 PCa patients and 10 patients with benign prostatic hyperplasia (BPH) has identified subsets of miRNAs differentially expressed between the groups. Using ratio-based normalization miRNA pairs with significantly different expression were selected and verified in an independent sample of 15 HD, 15 PCa and 15 BPH patients. To facilitate highly specific PCa detection a stepwise diagnostic algorithm based on miRNA expression in HD group (median ± 2SD cut off values) and hierarchical analysis of analytical systems was proposed (Bryzgunova et al, PLoS One, 2019). Diagnostic panels developed using miRNA profiling data and comprised of 24 miRNAs (17 miRNA ratios) could classify PCa and BPH patients and HD with 100% specificity and 97.5% accuracy. Diagnostic system based on the expression of 5 miRNA pairs in urine (miR-30a: miR-125b, miR-425: miR-331, miR-29b: miR-21, miR-191: miR-200a, miR-331: miR-106b) can be potentially used to identify PCa.

Conclusions

The data shows promise of urine cell-free and vesicle cargo miRNA as prostate cancer biomarkers.

Legal entity responsible for the study

The authors.

Funding

The study was supported by the Russian Science Foundation (no. 16-15-00124). Pavel P. Laktionov acknowledges financial support within the framework of the Russian state funded budget project # АААА-А17-117020210026-2 to the ICBFM SB RAS.

Disclosure

All authors have declared no conflicts of interest.

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79P - Parkin, APEX1 and BCL2L1 tissue expression in southern Brazilian patients with different breast cancer molecular subtypes (ID 3657)

Presentation Number
79P
Lecture Time
12:00 - 12:00
Speakers
  • Bianca B. Cabral (Curitiba, Brazil)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Breast cancer is the most commonly occurring cancer in women and is the leading cause of cancer death in women in Brazil. Despite being widely studied, it is a disease that is not yet fully understood in its complexity. On the other hand, changes in parkin (parkin RBR E3 ubiquitin protein ligase) expression patterns have been described in several diseases, including breast cancer. Nevertheless, the association of these changes with cancer prognostic and predictive factors remains unclear. Also, parkin regulates APEX1 (apurinic/apyrimidinic endodeoxyribonuclease 1), a protein crucial for repair of oxidized DNA damage, and BCL2L1 (BCL2 like 1), an apoptotic regulator. Both proteins were related to breast cancer.

Methods

Immunohistochemical analysis of Parkin, APEX1 and BCL2L1expression were performed in different breast cancer ductal invasive samples from Southern Brazilian patients to characterize their patterns and explore its association with clinical features and disease outcome.Therefore, 112 samples were organized into 15 Tissue Micro Arrays. The samples were classified in four molecular subtypes based on clinicopathological criteria (46 Luminal B, 24 Luminal A, 24 Basal and 18 HER2). These samples were obtained from Hospital Nossa Senhora das Graças (Curitiba, PR, Brazil) patients who were followed from 5 to 18 years.

Results

Parkin, APEX1 and BCL2L1 showed different expression patterns between the subtypes assessed. While in Luminal A and B APEX1 expression was increased, parkin expression was higher among Basal and HER2 subtypes. BCL2L1 expression was increased mainly in HER2 subtype. When regarding to estrogen (ER) and progesterone (PR) receptors, APEX1 was more expressed in ER/PR-positive samples, whereas Parkin and BCL2L1 in negative ones. No such difference was observed considering only HER2. BCL2L1 expression pattern was significantly associated with disease-free survival time independent of molecular subtypes, lymph node status and tumor size (p = 0.020). The higher the expression levels of BCL2L1, the shorter the disease-free survival time.

Conclusions

The expression of BCL2L1 is direclty associated with relapse of ductal invasive breast tumors, independently of other clinical variables.

Legal entity responsible for the study

Pontifícia Universidade Católica do Paraná (PUCPR).

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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80P - Obesity and prognosis in breast cancer (ID 2839)

Presentation Number
80P
Lecture Time
12:00 - 12:00
Speakers
  • Noha Y. Ibrahim (Cairo, Egypt)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Obesity carries a high risk of breast cancer with worse treatment outcome. Different molecular subtype and prognostic factors are linked to high body mass index (BMI) and affects overall and progression-free survival.

Methods

All 950 breast cancer patients presented to kasr Alainy oncology center (NEMROCK) from 2004 - 2014 at Al kasr alainy oncology center (NEMROCK) were followed up with a median period of 4.2years till Dec 2018. The body mass index (BMI) was assessed at diagnosis in 760 female patients. Disease-free survival (DFS) and overall survival (OS) were assessed and compared between three groups: non-obese (BMI <30), obese (BMI 30-34.9) and severely obese (BMI >40).

Results

The mean age was 50.1 years with Obesity in 63.29% of cases (23.82% and 39.47% in obese and severely obese respectively). Significant correlations between non-obese and severely obese with age (52 vs.48 years, p < 0.001), menopausal status (31.3 vs.46.9%, p < 0.001), molecular types (non- luminal; 25 vs. 50% p < 0.011), Her2 status (44.4 vs. 27.2%, p = 0.014) and hormonal therapy (Tamoxifen alone, 44.3 vs. 30.4%, p = 0.001). Mean Overall survival(OS) was significantly better in non-obese groups compared to obese and severely obese (102.5, 80, 88months, P=value 0.019), with no impact on DFS (p = 0.40).In multivariate analysis, lymph node stage (p < 0.001; OR: 1; 95% CI: 0.07-0.46), BMI (p = 0.001; OR: 1; 95% CI: 0.14-0.61), and hormonal treatment tamoxifen alone(p = 0.001; OR: 1; 95%CI: 1.4-16.4) remained significantly associated with OS.

Conclusions

severe obesity (BMI >40) have worse overall survival with no impact on disease free survival.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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81P - SIPA1 is a modulator of HGF induced tumour metastasis via the regulation of tight junctions in lung adenocarcinoma cells (ID 5132)

Presentation Number
81P
Lecture Time
12:00 - 12:00
Speakers
  • Chang Liu (Cardiff, United Kingdom)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Hepatocyte growth factor (HGF) regulates cell motility, proliferation and morphology and has a crucial impact on the ability of tumors to metastasize. Tight Junctions (TJs) are the first barrier that cancer cells must overcome to metastasize. In previous studies, signal induced proliferation associated protein 1 (SIPA1) has been shown to be involved in the metastasis of tumors. Breast and prostate cancer cells exhibit reduced response to HGF after SIPA1 knock down which was concurrent with changes in the regulation of TJs. It appears that SIPA1 influence HGF mediated control of TJs in cancer cells. We aimed to discover the role of SIPA1 in the HGF-mediated regulation of TJs and metastatic spread in lung adenocarcinoma.

Methods

Expression of SIPA1 was measured in human lung tumor tissues (n = 148) together with adjacent background tissue (n = 148). IHC was performed to examine SIPA1 expression within a lung tissue microarray. In vitro cell function assays were carried out after knock down of SIPA1 in the A549 cell line with/without HGF. Related TJs protein expression was analyzed using qPCR and western blotting.

Results

Patients with lung cancer exhibited a higher expression level of SIPA1 in tumor compared with background tissues (n = 148, p = 0.0141). High expression of SIPA1 was associated with advanced T stage of lung cancer (T3 vs T1, p = 0.014) and poor prognosis (p = 0.0021). Moreover, knockdown of SIPA1 reduced the aggressive behavior and enhanced the barrier function of the A549 cells. The SIPA1 knockdown cells showed a decreased response after treatment with HGF in invasion, proliferation and barrier function assays. In addition, receptor of HGF (Met) and several TJ components such as JAM1, Claudin5, Claudin10, Claudin11 and Claudin20 had reduced expression, whilst others such as Claudin1, Claudin19 had increased expression after knockdown of SIPA1.

Conclusions

SIPA1 may act as a targeting molecule and a marker for prognosis in lung adenocarcinoma. It has an effect on pathways associated with aggressive behavior and barrier function in lung cancer cells. HGF induced malignant behavior of cancer cells requires the presence and influence of SIPA1, which can effect changes in cancer cell behavior by interacting with the TJ complex.

Legal entity responsible for the study

Tracey A. Martin - Cardiff China Medical Research Collaborative, School of Medicine, Cardiff University.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

82P - Dose differential modulation of the autophagic behaviour of estrogen expressing breast carcinoma cells (ID 860)

Presentation Number
82P
Lecture Time
12:00 - 12:00
Speakers
  • Mariam A. Fouad (Cairo, Egypt)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Breast cancer is a major public health problem among women. The use of phytoestrogens like Resveratrol (RSV) has been emerged as a promising chemopreventive and chemotherapeutic agent against breast cancer. However, its effect is reported to be variable according to the administered dose. So this study investigated the underlying mechanisms to the dual action of RSV on estrogen expressing type of breast carcinoma cells (MCF-7).

Methods

MCF-7 cells treated with different low and high doses of RSV and the consequent cytotoxicity, cell cycle, oxidative, angiogenic and inflammatory responses of the cells were evaluated. The autophagic behavior of MCF-7 cells was assessed by RSV combination with autophagic inhibitor (cloroquine). The effect of the combination on cell survival, autophaghic markers expression along with acridine orange staining and cell apoptosis was determined.

Results

Compared to the higher concentration, low dose of RSV (10 µg/ml) exhibited significant anti-survival effect accompanied with cell accumulation at G0/G1phase, higher MDA and VEGF secretion. Concerning the higher doses of RSV (30 and 50 µg/ml), more cells accumulated at S and G2/M phases, and significantly higher levels of GSH, COX-2, PGE2 and nitrate secretion were observed. Autophagic inhibition of RSV exhibited significant acidic lysosomal staining, anti-survival and pro-apoptotic effects on breast carcinoma cells. The combination of low RSV concentration (10 µg/ml) with autophagic inhibitor caused significant decrease in Beclin-1, LC3-B, BCl-2 and MCl-1 expressions, while higher doses of RSV (30 and 50 µg/ml) combination induced Beclin-1, BCl-2 and MCl-1 but reduced LC3-B expressions compared with the single treatment of RSV.

Conclusions

The cytotoxic effect of RSV depends on the autophagic response of breast carcinoma cells in relation to the applied dose.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

83P - Synthetic peptide of tumour–associated antigen L6 formulated with polymer-based adjuvant enhances anti-tumour effects in mice (ID 2304)

Presentation Number
83P
Lecture Time
12:00 - 12:00
Speakers
  • Shih-jen Liu (Zhunan, Taiwan)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Tumour associated antigen L6 (TAL6) is a cell surface protein of the transmembrane-4 superfamily (TM4SF) also known as TM4SF1. TAL6 is over-expressed in more than 80 % of human lung, breast, colon and ovarian tumours but not normal tissues. Synthetic peptides are attractive for cancer immunotherapy because of their safety and flexibility. We have identified an B cell epitope of TAL6 using monoclonal antibodies. The synthetic peptide epitope formulated with adjuvants can induce anti-tumour immunity.

Methods

The linear B cell epitope was identified by anti-TAL6 monoclonal antibody using overlapping peptides cover the extracellular domain of TAL6. The synthetic peptide containing B cell epitope was formulated with different adjuvants to immunize mice to determine their immunogenecity. Furthermore, the sera of immunized mice were used to examine the antibody-dependent cellular cytotoxicity (ADCC) against human cancer. Moreover, the anti-tumor effects were evaluated in mouse model.

Results

The anti-TAL6 recognized a minimal linear region at amino acid 121-129. We formulated synthetic peptide (a.a.114-133) and a pan-DR peptide with different adjuvants to immunize mouse and evaluate their immunogenicity. The peptides formulated with an emulsion type nanoparticle (PELC) adjuvant and a toll-like receptor 9 agonist (CpG ODN) (PELC/CpG) induced the greatest ADCC responses in TAL6-expresssed cell lines. The induced anti-tumor immunity inhibited the growth of TAL6-positive cancer cells in mouse.

Conclusions

These data suggested that a peptide containing B cell epitopes of TAL6 formulated with PELC/CpG adjuvant is feasible for cancer immunotherapy.

Legal entity responsible for the study

The authors.

Funding

Ministry of Science Technology, Taiwan.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

85P - Improving detection level of somatic mosaicism in neurofibromatosis type 1 (ID 4419)

Presentation Number
85P
Lecture Time
12:00 - 12:00
Speakers
  • Kristina Karandasheva (Moscow, Russian Federation)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Neurofibromatosis type 1 (NF-1) is a tumor predisposition syndrome resulting from mutations in NF1 antioncogene. Differential diagnosis with genetic testing is essential in NF-1 diagnostics due to its clinical variability. Identifying of pathogenic mutations is challenging because of the gene size and structure, especially for low-level somatic mosaicism. Notably, patients with mosaic NF-1 demonstrate no less severe phenotype than those with classic form. Nowadays, NGS and Sanger sequencing are recognized diagnostics methods and they both are not sensitive enough to mosaic genotypes with small fractions of alternative alleles. We believe that this can be resolved with a special approach to sequencing data analysis.

Methods

In order to re-evaluate previously obtained NGS (Ion AmpliSeq technology) results for 275 probands with clinical diagnosis “NF-1” or “NF, unspecified” for whom germline mutations in NF1 and NF2 genes were not identified, we developed an improved data analysis pipeline to search for somatic mutations, which includes: (1) programmatic pool-based division of NGS reads, (2) elimination of out-of-design aligned reads, (3) exclusion of systematic sequencing errors, (4) variant calling with low stringency parameters (alternative allele frequency, AF ≥ 0.05; read depth, DP ≥ 20). Newly detected mutations were verified using Sanger sequencing and heteroduplex analysis. Sanger results were analysed using our in-house SeqBase software, highly sensitive to mosaic variants.

Results

Application of our NGS data reanalysis algorithm allowed us to detect 12 cases (4.3%) of somatic mosaic mutations among 275 probands otherwise lacking molecular verification of neurofibromatosis diagnosis. The majority of the identified mutations are nonsense or frameshift, with AF ranging from 0.051 to 0.296. Mutations were verified with alternative methods of molecular diagnostics.

Conclusions

Improved sequencing data analysis allows to detect NF-1 cases with mosaic genotype in cases unresolved by conventional analysis pipeline. Yet, success rate is strongly dependent upon the fraction of the mutant allele, thus alternative quantitative methods are required for exhaustive NF-1 molecular diagnosis.

Editorial acknowledgement

Federal State Budgetary Institution "Research Centre for Medical Genetics"

Legal entity responsible for the study

Federal State Budgetary Institution "Research Centre for Medical Genetics".

Funding

The research was carried out within the state assignment of Ministry of Science and Higher Education of the Russian Federation.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

Identification of microRNA expression profiles in tamoxifen-resistant MCF-7 sublines (ID 5266)

Lecture Time
12:00 - 12:00
Speakers
  • Olga E. Andreeva (Moscow, Russian Federation)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00
Poster Display session 1 Poster Display session

87P - Preclinical pharmacokinetic/pharmacodynamic (PK/PD) relationship of ABN401, a highly selective met inhibitor, in gastric and non-small cell lung cancer models (ID 5283)

Presentation Number
87P
Lecture Time
12:00 - 12:00
Speakers
  • JooSeok Kim (Seoul, Korea, Republic of)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

ABN401 is a highly selective best-in-class Met inhibitor that is targeting MET-driven cancers. Met which is also known as a hepatocyte growth factor (HGF) receptor is encoded by the MET gene. MET alterations, including amplification and mutation, and Met overexpression are well known for tumorigenic transformation, tumor growth, angiogenesis, invasion, and metastasis in several cancer types, including gastric and non-small-cell lung cancer (NSCLC). ABN401 shows significant MET signaling inhibition in in vitro and in vivo preclinical studies, especially for cell lines, xenograft, and PDx models which have high MET amplification and/or MET exon 14 deletion.

Methods

In this study, to confirm the pharmacokinetic and pharmocodynamic correlation of ABN401, gastric cancer and NSCLC xenograft models were used. SNU-5, a gastric cancer cell line and EBC-1, a NSCLC cell line both of which has high MET amplification, were used for the xenograft model and dosed at 10 mg/kg and 30 mg/kg of ABN401 by oral administration. Pharmacokinetic parameters were analyzed in both plasma and tumor tissue samples. Pharmacodynamic biomarkers including Met and phospho-Met (T1234/1235) and downstream signaling were analyzed by immunoblotting. In addition, an expression of Met and phospho-Met were analyzed by immunohistochemistry.

Results

The PK results demonstrated that in both gastric and NSCLC xenograft models ABN401 drug was readily distributed to tumor tissues. According to both PD studies, ABN401 showed inhibitory effects of Met and downstream signaling in a time dependent manner for both cancer types. There was also correlated between the PK parameters in plasma and tumor samples and pharmacodynamics studies.

Conclusions

This preclinical PK/PD correlation study of ABN401 provides evidence for human dose predictions and dosing strategy for clinical studies.

Legal entity responsible for the study

The authors.

Funding

Abion Inc.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

88P - Transcription factors of snail family in the regulation of resistance of breast cancer cells to hypoxic conditions (ID 5488)

Presentation Number
88P
Lecture Time
12:00 - 12:00
Speakers
  • Alvina I. Khamidullina (Moscow, Russian Federation)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

In the course of tumor growth, the cells that are far from the blood vessel are exposed to hypoxic conditions. As part of this process transcription factors (TFs) of the Snail family (Snail, Slug, Twist1, and Zeb1) are activated launching the epithelial-mesenchymal transition (EMT) program. Snail family factors are the most studied as stimulators of EMT, promoting tumor progression, migration and invasion of tumor cells. The aim of our research is to study adaptive mechanisms of human breast cancer (BC) cells to hypoxia and the analysis of the role of the TF Snail in this process.

Methods

We used human BC cells MCF-7, MDA-MB-231 and HBL-100 which were cultured in standard DMEM containing 10% fetal calf serum at 37oC and 5% СО2. For hypoxia modeling, the cells were cultured in a CO2-incubator maintaining O2 concentration at 1%.

Results

Knockdown of the Snail by small interfering RNA (siRNA) leads to increased sensitivity of breast cancer cells to hypoxia, due to increased blocking of cells in the S-phase of cell cycle. Cells were more resistant to hypoxia at a lower density, where Snail expression was increased, but further hyperexpression of Snail had not lead to increased resistance to hypoxia. Knockdown of Snail leads to an compensatory increase of Slug`s expression, while the knockdown of Twist1 and Zeb1 leads to decreased Slug expression. Knockdown of Twist1 decreases cell proliferation both in hypoxia and normoxia, through activation of cell cycle inhibitor p21WAF1, specifically in the G2/M phase, in p53-wild type HBL-100 cells. We used a chemical inhibitor of p53-Snail interaction GN25, to examine if blocking of this interaction could lead to increased sensitivity in hypoxia. We have shown that this compound has a greater cytotoxic effect in normoxia than hypoxia, due to increased Snail activity.

Conclusions

Modulation of Snail-associated signaling pathways is a perspective target for increase of cancer cell tolerance to chronic hypoxia, and inhibition of metastatic process in breast cancer cells.

Legal entity responsible for the study

The authors.

Funding

Grant no. 14.W03.31.0020 between the Ministry of Science and Education of the Russian Federation and Institute of Gene Biology, Russian Academy of Sciences.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

Tumour biology and pathology (ID 6603)

Lecture Time
12:00 - 12:00
Speakers
  • Thomas Helleday (Stockholm, Sweden)
  • Therese Sorlie (Oslo, Norway)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00
Poster Display session 1 Poster Display session

1987P - Metastasis is impaired by endothelial-specific Dll4 loss-of-function through inhibition of epithelial-to-mesenchymal transition and reduction of cancer stem cells and circulating tumour cells (ID 5417)

Presentation Number
1987P
Lecture Time
12:00 - 12:00
Speakers
  • Liliana Mendonça (Vila Verde, Portugal)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Systemic inhibition of Dll4 results in a vast reduction of cancer metastasis. Whether this effect is endothelial-mediated remains to be clarified. Therefore, we proposed to analyze the impact of endothelial Dll4 loss-of-function on metastasis induction on three early steps of the metastatic process, regulation of epithelial-to-mesenchymal transition (EMT), cancer stem cell (CSC) frequency and circulating tumour cell (CTC) number.

Methods

Lewis Lung Carcinoma (LLC) cells or LLC- green fluorescent protein marked were used to model mouse tumour metastasis in vivo, by subcutaneous transplantation into endothelial-specific Dll4 loss-of-function mice.

Results

We observed that endothelial-specific Dll4 loss-of-function is responsible for the tumour vascular regression that leads to the reduction of tumour burden. It induces an increase in tumoural blood vessel density, but the neovessels are poorly perfused, with increased leakage and reduced perivascular maturation. Unexpectedly, although hypoxia was increased in the tumour, the number and burden of macro-metastases was significantly reduced. This is likely to be a consequence of the observed reduction in both EMT and CSC numbers caused by the endothelial-specific Dll4 loss-of-function. This multifactorial context may explain the concomitantly observed two-fold reduction of the circulating tumour cell count. Furthermore, our gene transcription analysis suggests that endothelial Dll4/Notch function mediates tumour hypoxia-driven increase of EMT. We observed that the downregulation of Dll4/Notch1/Hey1 signalling caused a reduction in Snail-1 and Twist expression, known inducers of EMT.

Conclusions

These findings show that the effect of Dll4 inhibition in suppressing cancer metastasis is, at least, partially endothelial-mediated. This is accomplished through the arrest of the EMT process and reduction of CSC clone selection in the primary tumour, regulated by non-cell-autonomous endothelial signalling. Therefore, endothelial Dll4 may constitute a promising target for the prevention of metastasis.

Legal entity responsible for the study

CIISA - Centro de Investigação Interdisciplinar em Sanidade Animal.

Funding

This work was supported by the Portuguese Foundation for Science and Technology (FCT), grants PTDC/CVT/115703/2009 to AD; PTDC/SAU-ONC/116164/2009 and PTDC/SAU-ONC/121742/2010 to AT. CIISA has provided support through project UID/CVT/00276/2019, funded by FCT. LM is a PhD student supported by a studentship from FCT (SFRH/BD/74229/2010).

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

1988P - Identification of novel and known FGFR gene fusions in Chinese non-small cell lung cancer (ID 5494)

Presentation Number
1988P
Lecture Time
12:00 - 12:00
Speakers
  • Weixin Zhao (Shanghai, China)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Oncogenic fibroblast growth factor receptor (FGFR) gene fusions have been described in diverse tumor histology. FGFR inhibitors including erdafitinib have demonstrated promising results in patients whose tumors harbor FGFR gene fusions or mutations. We hereby assessed the frequency of FGFR rearrangements in the Chinese non-small cell lung cancer (NSCLC) population.

Methods

A total of 10,966 NSCLC patients whose tumor specimen and/or circulating cell-free DNA (cfDNA) submitted for genomic profiling by hybridization capture-based targeted next-generation sequencing (NGS) of exons and introns of cancer related genes were retrospectively reviewed. Patients’ clinical characteristics and treatment history were retrieved from the database for further evaluation.

Results

FGFR fusions retaining the intact kinase domain were identified in 0.11% (12/10966) of NSCLC cases examined, including 9 fibroblast growth factor receptor 3 (FGFR3)-transforming acidic coiled-coil containing protein 3 gene (TACC3) fusion-positive cases that was mostly reported in solid tumors, one fibroblast growth factor receptor 2 (FGFR2)-internexin neuronal intermediate filament protein α gene (INA) fusion that was previously reported in gliomas, one novel fibroblast growth factor receptor 4 (FGFR4)-Rap guanine nucleotide exchange factor like 1 gene (RAPGEFL1) fusion case, and one novel fibroblast growth factor receptor 1 (FGFR1) gene fusion involving the 5’UTR of Solute Carrier Family 20 Member 2 gene (SLC20A2) which may possibly drive the overexpression of FGFR1. Concomitant EGFR mutations or EGFR amplification were observed in 6 patients, four of which were previously treated with EGFR tyrosine kinase inhibitors (TKIs), but disease progressed prior to NGS tests. FGFR fusions may arise as resistance mechanism to EGFR TKI therapies in patients who carried founder EGFR alterations. No other known concurrent driver mutations were detected in the remaining 6 cases.

Conclusions

We hereby report a frequency of FGFR fusions of 0.11% in a large Chinses NSCLC population involving known and novel FGFR gene fusion partners including TACC3, INA, RAPGEFL1, and SLC20A2. Oncogenic FGFR fusions may arise as mechanisms of acquired resistance.

Legal entity responsible for the study

Geneseeq Technology Inc.

Funding

Has not received any funding.

Disclosure

Q. Ou: Full / Part-time employment, NA: Geneseeq Technology Inc. X. Wu: Leadership role, NA: Geneseeq Technology Inc. X. Wang: Full / Part-time employment, NA: Nanjing Geneseeq Technology Inc. Y.W. Shao: Leadership role, NA: Geneseeq Technology Inc. All other authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

1989P - WNT pathway mutations (APC/CTNNB1) and immune checkpoint inhibitors (ICI) response in metastatic non-small cell lung cancer (NSCLC) patients (ID 3412)

Presentation Number
1989P
Lecture Time
12:00 - 12:00
Speakers
  • Francisco Javier Ros Montana (Barcelona, Spain)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

WNT pathway mutations (mut) are uncommon in NSCLC. Recent preclinical studies suggest that APC/CTNNB1 mut induce immune resistance in lung adenocarcinoma but how they may impact on immunotherapy response in the clinics remains unclear. Objective: our aim is to describe the clinical outcomes - Overall survival (OS) and progression-free-survival (PFS) and response rate (RR) - of a metastatic NSCLC cohort with APC/CTNNB1 mut treated with immunotherapy.

Methods

We selected those patients with metastatic NSCLC and APC or CTNNB1 mut detected through Amplicon-Seq (tissue), Foundation (plasma), Guardant (plasma) or exome sequencing (tissue) performed from 2014 to 2018.

Results

Incidence of APC/CTNNB1 mut in NSCLC in our hospital has been 1,26% and 0,7% respectively by Amplicon-Seq (tissue) and 10.4% and 1.04% respectively by exome sequencing (tissue). We identified 27 patients with APC (81%) or CTNNB1 (19%) mutations. The median age of the patients was 59 years (44-74), 72% of them were male and most frequent histology was adenocarcinoma (66%). Fifteen of the patients received ICI, 27% as a first line treatment and 47% as a second line. Interestingly, 1 patient had EGFR mut (exon 19 and 20) and 4 patients had KRAS mut. Median OS of the whole cohort from date of diagnosis was 24.5 months (CI95% 13.7-NA). Median OS was 23.5 months (CI95% 10.4-NA) for those patients who harboured CTNNB1 mut and 57.3 months (CI95% 13.7-NA) for those patients with APC mut (HR 1.8, CI95% 0.32-10, p = 0.5). Median PFS with ICI was 6.6 months (CI95% 0.9-NA). Median PFS was 0.7 months (CI95% 0.5-NA) for those patients with CTNNB1 mut and 8.9 months (CI95% 1.8-NA) with APC mut (HR 6.7, CI95% 1.1-41, p = 0.04). None of the 2 CTNNB1 mut patients responded to ICI, while RR in the APC mut cohort was 30% (4 out of 13).

Conclusions

In our cohort, APC mut did not show to impair clinical outcomes. Few CTNNB1 mut cases hinder conclusive analyses. Correlation with other predictive biomarkers such as tumor mutation burden and PDL1 expression is ongoing.

Legal entity responsible for the study

Vall d´Hebron Institute of Oncology (VHIO).

Funding

Grant from: Carlos III Institute of Health, Madrid, Spain: PI14/01248 and PI17/00938.

Disclosure

P. Iranzo: Advisory / Consultancy, Travel / Accommodation / Expenses: BMS; Advisory / Consultancy, Travel / Accommodation / Expenses: Roche; Advisory / Consultancy, Travel / Accommodation / Expenses: Merck; Advisory / Consultancy, Travel / Accommodation / Expenses: Boehringer; Advisory / Consultancy, Travel / Accommodation / Expenses: MSD; Advisory / Consultancy: Rovi; Advisory / Consultancy: Kyowa Kirin; Advisory / Consultancy, Travel / Accommodation / Expenses: Grunental. A. Callejo: Advisory / Consultancy, Travel / Accommodation / Expenses: BMS; Advisory / Consultancy, Travel / Accommodation / Expenses: Roche; Advisory / Consultancy, Travel / Accommodation / Expenses: Pfizer; Advisory / Consultancy, Travel / Accommodation / Expenses: Boehringer; Advisory / Consultancy, Travel / Accommodation / Expenses: MSD; Advisory / Consultancy: Kyowa kirin; Travel / Accommodation / Expenses: Celgene. N. Pardo: Advisory / Consultancy: BMS; Advisory / Consultancy, Travel / Accommodation / Expenses: Roche; Advisory / Consultancy, Travel / Accommodation / Expenses: Pfizer; Advisory / Consultancy: Boehringer. A. Martinez: Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: BMS; Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Roche; Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: MSD; Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Pfizer; Advisory / Consultancy, Speaker Bureau / Expert testimony, Travel / Accommodation / Expenses: Boehringer. A. Navarro: Advisory / Consultancy, Travel / Accommodation / Expenses: BMS; Advisory / Consultancy, Travel / Accommodation / Expenses: Roche; Advisory / Consultancy, Travel / Accommodation / Expenses: Pfizer; Advisory / Consultancy, Travel / Accommodation / Expenses: Boehringer; Advisory / Consultancy: Oryzon Genomics; Travel / Accommodation / Expenses: MSD. S. Cedres: Advisory / Consultancy: BMS; Advisory / Consultancy, Travel / Accommodation / Expenses: Roche; Advisory / Consultancy, Travel / Accommodation / Expenses: Pfizer ; Advisory / Consultancy, Travel / Accommodation / Expenses: Boehringer; Advisory / Consultancy: MSD; Advisory / Consultancy: Amphera. R. Dienstmann: Advisory / Consultancy, Speaker Bureau / Expert testimony: Roche; Speaker Bureau / Expert testimony: Symphogen; Speaker Bureau / Expert testimony: Ipsen; Speaker Bureau / Expert testimony: Amgen; Speaker Bureau / Expert testimony: Sanofi; Speaker Bureau / Expert testimony: MSD; Speaker Bureau / Expert testimony: Servier; Research grant / Funding (institution): Merck. H.G. Palmer: Research grant / Funding (institution): Bayer; Research grant / Funding (institution): Blueprint; Research grant / Funding (institution): Merus; Research grant / Funding (institution): Cellestia. A. Vivancos: Advisory / Consultancy, Research grant / Funding (institution): SYSMEX; Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy: MERCK; Advisory / Consultancy, Research grant / Funding (institution): Roche; Advisory / Consultancy, Research grant / Funding (institution): BMS; Advisory / Consultancy: Guardant Health; Licensing / Royalties, Technology Transfer DX Field: Ferrer; Research grant / Funding (institution): DEBIO; Research grant / Funding (institution): Cellestia; Research grant / Funding (institution): Chittern. E. Felip: Advisory / Consultancy: Abbvie; Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Blueprint Medicines; Advisory / Consultancy: Boehringer Ingelheim,; Advisory / Consultancy: Bristol-Myers Squibb; Advisory / Consultancy: Celgene; Advisory / Consultancy: Eli Lilly; Advisory / Consultancy: Guardant Health; Advisory / Consultancy: Janssen; Advisory / Consultancy: Medscape; Advisory / Consultancy: Merck KGaA; Advisory / Consultancy: Merck Sharp & Dohme; Advisory / Consultancy: Novartis; Advisory / Consultancy: Pfizer; Advisory / Consultancy: Roche; Advisory / Consultancy: Takeda; Advisory / Consultancy: Touchtime; Research grant / Funding (institution): Fundación Merck Salud; Research grant / Funding (institution): Grant for Oncology Innovation EMD Serono. All other authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

1990P - Leukocytosis as a negative prognostic factor in patients with lung cancer: Which subpopulation of leukocytes is responsible? (ID 1815)

Presentation Number
1990P
Lecture Time
12:00 - 12:00
Speakers
  • Filip Kohutek (Trencin, Slovak Republic)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Leukocytosis is common in patients with malignancies and might be a negative prognostic factor in lung cancer (LC). Leukocytes are, however, a heterogenous population of blood cells. Therefore, subpopulation of leukocytes responsible for this effect should be identified. It is presumed that neutrophilia and lymphocytopenia might be a negative factor in patients with lung cancer.

Methods

Leukocyte, absolute neutrophil, lymphocyte, eosinophil, basophil and thrombocyte count were determined before beginning of treatment in patients with LC. Patients with recent bleeding, elevated CRP or treated with best-supportive care were excluded. The impact of leukocytosis, neutrophilia, lymphocytopenia, eosinophilia and basophilia on progression- free survival (PFS) was determined. Neutrophils to lymphocytes ratio (NLR) at various cut-offs were determined along with platelets to lymphocytes ratio (PLR) and their effect on PFS was assessed. Kaplan- Meier and Cox- regression statistical analysis were used.

Results

200 patients were examined in our retrospective study. Median PFS was 7.41 months. Leukocytosis and neutrophilia significantly worsened PFS in patients with LC [HR 0.5200, (95%CI 0.3684 to 0.7340), P < 0.0001 and HR 0.5556 (95%CI 0.4068 to 0.7589) P = 0.0001]. These results were confirmed in Cox- regression analysis (covariates: age, gender, stage). Lymphocytopenia, basophilia, eosinophilia and thrombocytosis had no impact on survival rates. Elevated NLR (cut- off 3.54) significantly worsened PFS in patients with LC [HR 0.6069 (95%CI 0.4475 to 0.8231), P = 0.0013] even after adjustment to covariates (age, gender, stage). Elevated NLR at lower cut-offs (2.15 and 1.77) and elevated PLR (cut-off 123) did not affect the survival rate.

Conclusions

Neutrophilia appears to be a negative prognostic factor in patients with LC. Impairment in other white blood cell lines does not affect PFS, however, significantly elevated NLR (≥ 3.54) seems to have detrimental effect on survival rates.

Legal entity responsible for the study

Faculty Hospital Trencin, Department of Oncology.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

1991P - Identification of MET gene amplifications using next-generation sequencing in non-small cell lung cancer patients (ID 5022)

Presentation Number
1991P
Lecture Time
12:00 - 12:00
Speakers
  • Sergi Clavé (Barcelona, Spain)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

MET amplifications present a potential therapeutic target in NSCLC and while FISH is conventionally used to asses it, there is no clinically defined cutoff. NGS is becoming routine in molecular diagnostics, and provides a means to assess MET amplification in the context of comprehensive genomic profiling. Here, we assess MET amplifications in NSCLC, in addition to TMB and concomitant driver mutations.

Methods

222 NSCLC cases were screened, and 26 samples were selected based on increased MET gene copy number (GCN) detected by FISH (Cappuzzo score, MET GCN >5). Cases were reclassified to determine amplification versus high polysomy by University of Colorado Cancer Center (UCCC) FISH-criteria. Archival tissue samples were evaluated by PGDx elio™ tissue complete (assay under development; Personal Genome Diagnostics), a 500+ gene NGS panel.

Results

Eight patients were amplified by UCCC FISH-criteria (1 patient high-amplified (MET/CEP7 >5) and 7 patients intermediate-amplified (>2.2 to < 5)). Five of these 8 patients (4 intermediate-amplified; 1 high-amplified) were also found to have MET amplification by NGS (>3 fold cutoff) and were male smokers, with a median age of 57 years, with no concurrent driver alterations. One MET-amplified sample also had a concomitant exon 14 skipping mutation. The 3 discordant cases were highly heterogeneous by FISH with focal MET amplifications. Eighteen patients exhibited high copy number gain by FISH but were negative for MET amplifications according to UCCC FISH-criteria and NGS. Five of these cases had driver mutations (2 = KRAS exon 2 mutations and 3 = EGFR sensitizing mutations). TMB scores in NGS MET-positive were lower than NGS MET-negative cases: 5.9 vs. 12.5 mut/Mb exome equivalent.

Conclusions

NGS and FISH produced similar MET amplification status. The wide range of MET classifications via FISH, together with tumor heterogeneity and lack of clinically relevant thresholds, could contribute to discordance. In most cases negative for MET amplification by NGS, alternative driver mutations and/or higher TMB were identified. These results suggest that, while these technologies provide complementary value, further clinical data is needed to define clinically meaningful MET alterations.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

E. Weingartner: Full / Part-time employment: Personal Genome Diagnostics. G. Cerqueira: Full / Part-time employment: Personal Genome Diagnostics. D. Nichol: Full / Part-time employment: Personal Genome Diagnostics. J. Simmons: Full / Part-time employment: Personal Genome Diagnostics. All other authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

1992P - Prognostic role of CD73 in metastatic non small cell lung cancer according to the presence of driver alterations (ID 4925)

Presentation Number
1992P
Lecture Time
12:00 - 12:00
Speakers
  • Giulia Galli (Milan, Italy)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

CD73 is an enzyme involved in the conversion of extracellular AMP into adenosine. Adenosine inhibits T lymphocytes, contributing to immune escape. CD73 promotes proliferation and migration and has been associated to a negative prognosis in various cancers. Data are limited on its role in metastatic NSCLC, particularly with genetic drivers.

Methods

We collected patients (pts) with EGFR Mutant (EM), ALK Rearranged (AR) and Wild Type (WT) metastatic NSCLC treated outside trials at our Institution between 2010 and 2018. Cases without tissue samples were excluded. CD73 expression was determined by immunohistochemistry (Ab133582 ABCAM). Semiquantitative H score (HS) was calculated multiplying the membrane intensity score (0-3) by the % of stained cells to yield a value of 0–300. Median value of HS was chosen as a cutoff. Overall Survival (OS) was estimated through Kaplan-Meier method. After a gene expression profiling on TCGA NSCLC datasets (n = 488), we divided EM, AR, WT cases according to CD73 median level. Cibersort analysis was performed in these series to profile the immune infiltrate.

Results

Fifty-four EM, 28 AR and 40 WT NSCLCs were identified. Median HS was 0 for EM and WT, 100 for AR cases. No imbalances in CD73 expression emerged according to main clinico-pathological variables. EM pts with a high HS showed a trend towards a worse OS (23.4 vs 39.5 months, p .2237); on the contrary, high HS had a non-significant positive prognostic role in AR (53.3 vs 25.1 months, p .1263) and WT pts (59.6 vs 18.7 months, p .5063). In accordance with our data, we observed some differences among the CD73 high subgroups profiling infiltrate of TCGA cases: WT were enriched in Treg and resting dendritic cells (DC), AR showed an increase in M1 macrophages, EM had a reduction in activated NK cells along with an increase in activated DC. Nanostring is ongoing on our series.

Conclusions

CD73 expression seems to have a different prognostic role for EM, AR and WT metastatic NSCLC. In particular, the trend towards a better outcome for AR cohort is a novel finding, potentially supported by a specific inflammatory infiltrate. Despite the small number of analyzed cases, CD73 role in NSCLC different subgroups warrants further investigation. Nanostring results will be presented.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

G. Lo Russo: Honoraria (self), Travel / Accommodation / Expenses: AstraZeneca; Honoraria (self), Travel / Accommodation / Expenses: MSD International GmbH; Honoraria (self), Travel / Accommodation / Expenses: BMS; Honoraria (self), Travel / Accommodation / Expenses: Eli Lilly. D. Signorelli: Honoraria (self), Travel / Accommodation / Expenses: AstraZeneca; Honoraria (self), Travel / Accommodation / Expenses: MSD International GmbH; Honoraria (self), Travel / Accommodation / Expenses: BMS. F.G.M. de Braud: Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: Amgen; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: AstraZeneca; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: Boehringer-Ingelheim; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: BMS; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: Eli Lilly; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: F. Hoffmann-La Roche; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: Ignyta; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: Merck Sharp and Dohme; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: Merck Serono; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: Novartis; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: Pfizer. M.C. Garassino: Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution): AstraZeneca; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution): MSD International GmbH; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution): BMS; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution): Boehringer Ingelheim Italia S.p.A; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution): Celgene; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution): Eli Lilly; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution): Ignyta; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution): Incyte; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution): Inivata; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution): MedImmune; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution): Novartis; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution): Pfizer; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution): Roche; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self), Research grant / Funding (institution): Takeda; Honoraria (institution), Research grant / Funding (institution): Tiziana; Honoraria (institution), Research grant / Funding (institution): Foundation Medicine; Research grant / Funding (self): AIRC; Honoraria (institution): AIFA; Honoraria (institution): Italian Moh; Honoraria (institution): Transcan. C. Proto: Honoraria (self), Travel / Accommodation / Expenses: MSD International GmbH; Honoraria (institution), Travel / Accommodation / Expenses: BMS; Honoraria (institution), Travel / Accommodation / Expenses: Eli Lilly. All other authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

1993P - JAK-STAT inhibitor overcomes interferon γ-reduced, NK cell-mediated cytotoxicity in non-small-cell lung cancer cells (ID 785)

Presentation Number
1993P
Lecture Time
12:00 - 12:00
Speakers
  • Riki Okita (Kurashiki, Okayama, Japan)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Immune checkpoint inhibitors targeting PD-1/PD-L1 axis have shown promising results in patients with non-small-cell lung cancer (NSCLC); however, the objective response rate is less than 30%. One major PD-L1 inducer, interferon γ (IFNg), has anti-tumour properties and is secreted by T cells and NK cells. IFNg-induced PD-L1 is considered to be a major mechanism that facilitates cancer cell escape from host immunity. Therefore, a better understanding of how IFNg stimulus influences the regulation of PD-L1 expression in cancer cells is warranted.

Methods

NSCLC cell lines: A549, PC-9, and LC2/ad were used for in vitro assays. Comparative analysis between IFNg and EGF was conducted in LC2/ad cells because EGF stimuli also enhanced PD-L1 expression in this cell line. PD-L1 expression was evaluated using flow cytometry while cell signalling pathway analysis was assessed using phospho-receptor tyrosine kinase arrays. To assess the NK cell-mediated cytotoxicity, NK cells were collected from healthy donors. NK killing was evaluated by lactate dehydrogenase release assay. Apoptotic cells were assessed via flow cytometry using Annexin V.

Results

IFNg stimulus significantly upregulated PD-L1 expression in all cell lines. In LC2/ad cells, IFNg activated STAT1 signalling while EGF activated AKT, MAPK, and ribosomal protein S6 (RPS6). Although IFNg-induced PD-L1 was clearly blocked by the JAK-STAT inhibitor, tofacitinib, EGF-induced PD-L1 was not. Both IFNg- and EGF-induced PD-L1 were blocked by PI3K and MAPK inhibitors; however, they were not blocked by the RPS6 inhibitor. Interestingly, IFNg downregulated NK cell-activating ligand expression while upregulating MHC class I molecules, the phenotype of which can theoretically escape NK cells easily. Thus, we confirmed that IFNg stimulus attenuated NK cell-mediated cytotoxicity in LC2/ad cells; however, tofacitinib blocked IFNg-reduced NK killing. We also confirmed that tofacitinib, at concentrations less than 3 μM, did not cause NK cell apoptosis.

Conclusions

Tofacitinib blocks IFNg-induced immunoescape from NK cells in NSCLC cells, thereby suggesting that NK cell therapy combined with tofacitinib might be a promising strategy in overcoming tumour immune escape.

Legal entity responsible for the study

Riki Okita.

Funding

Japan Society for the Promotion of Science (JSPS) Kakenhi Grant (25462189 and 16K10696),.

Disclosure

M. Nakata: Research grant / Funding (institution), The sponsor had no control over the interpretation or presentation of this work.: Kyowa Kirin. All other authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

1994P - Low LATS2 expression is associated with poor prognosis in non small cell lung cancer (ID 2326)

Presentation Number
1994P
Lecture Time
12:00 - 12:00
Speakers
  • Si-hyong Jang (Cheonan, Korea, Republic of)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Large tumor suppressor kinase 2 (LATS2) is one of the core components in the Hippo signaling pathway, and it functions as a tumor suppressor associated with regulating tumor cell proliferation and apoptosis. Dysregulated LATS2 expression has been reported in various human cancers. The purpose of this study is to explore LATS2 expression and its clinicopathological significance in non-small cell lung cancer (NSCLC) and its subtypes.

Methods

We examined LATS2 protein expression by immunohistochemistry in 184 resected NSCLC specimens on tissue microarray. We also reviewed the clinical data and performed a clinicopathological analysis.

Results

Of 184 lung cancer specimens, 40 (21.7%) showed low LATS2 expression. Low LATS2 expression was significantly correlated with disease recurrence (p = 0.047) and low LATS2 expression had a tendency of poor prognostic clinicopathological features including large tumor size, the presence of vascular and lymphatic invasion and distant metastasis. The low LATS2 expression group showed a statistically poorer overall survival (OS) (p = 0.004) and disease-free survival (DFS) (p = 0.014) than did the high expression group in Kaplan-Meier analysis with log-rank test. In multivariate analysis with the Cox proportional hazards model, downregulated LATS2 expression in NSCLC was an independent prognostic factor of poor OS and DFS. Furthermore, we evaluated the prognostic significance of LATS2 expression in two major subtypes of NSCLC, squamous cell carcinoma and adenocarcinoma, using the Kaplan-Meier curves with log-rank test. In both squamous cell carcinoma and adenocarcinoma, low LATS2 expression group showed worse prognosis than high LATS2 expression group (OS (p = 0.144), DFS (p = 0.022) in squamous cell carcinoma and OS (p = 0.045), DFS (p = 0.271) in adenocarcinoma).

Conclusions

In conclusion, we demonstrated that downregulated LATS2 expression may predict aggressive biologic behavior and a worse prognosis in NSCLC and we also suggested the possibility of LATS2 as a therapeutic target in both squamous cell carcinoma and adenocarcinoma of the lung.

Legal entity responsible for the study

The authors.

Funding

Soonchunhyang University Research Fund.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

1995P - Application of ESCAT and OncoKB scales in liquid biopsy (LB) in advanced NSCLC patients (pts): Is it feasible and reliable? (ID 5960)

Presentation Number
1995P
Lecture Time
12:00 - 12:00
Speakers
  • Michael G. McCusker (Baltimore, United States of America)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Next generation sequencing (NGS) of cfDNA in NSCLC is entering in clinical practice. Several commercially available platforms using NGS report wide variety of somatic aberrations. Vendors also include therapeutic suggestions to guide oncologists. In addition, new levels of evidence tools are now available for tissue results, but not still used in LB.

Methods

Advanced NSCLC pts underwent commercial 73-gene cfDNA NGS analysis. ESMO Scale for Clinical Actionability (ESCAT) and OncoKB were used to grade levels of evidence for categorize aberrations and compared with variant allele frequency (VAF), treatment decisions, and vendor suggestions.

Results

77 samples from 73 advanced NSCLC pts (73% adenocarcinoma, 27% squamous cell carcinoma) at the time of diagnosis (49%) or during disease course (51%) were analyzed. Median turnaround time was 8 days (range 5-17), with no genotyping failures. There were 323 unique somatic alternations identified: Sequence mutations (254), amplifications (43), synonymous mutations (23), and fusions (3). Median cfDNA % was 0.7 (range 0.05-49.5); 7 samples had no detectable genetic alterations. Variants of unknown significance were 33% of point mutations. We detected 87 and 88 potentially actionable genetic alterations according to ESCAT (IA-IV) and OncoKB (1-4), respectively, and 26% received a matched targeted drug. Discrepancies between these two tools and vendor suggestions were reported in 4 cases. We performed a subset analysis of 64 samples: Median VAF was 4.37 % (range 0.16-43.05) and a VAF < 1% was reported in 16 samples; 45% of alterations (excluding amplifications) were clonal events (cfDNA% divided by VAF > 0.5). Among EGFR and ALK positive samples median VAF was 7.98% (range 0.29-41.2); 3/17 samples presented a VAF below 1%, with no detrimental effect on treatment response.

Conclusions

The application of ESCAT and OncoKB is feasible in LB. Driver mutations with low VAF are amenable to receive treatment. VAF could be included as complementary tool to grading systems to better understand the significance of aberrations.Discrepancies between vendor therapeutic suggestions and evidence-based grading systems requests caution in the use of information outside the molecular tumor board.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

C.D. Rolfo: Honoraria (self), personal fees : Novartis; Honoraria (self), personal fees : MSD; Non-remunerated activity/ies, non-financial support : OncoDNA; Honoraria (self), personal fees and non-financial support : GuardantHealth. All other authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

1996P - IDH1R132H mutation induces a less aggressive phenotype of glioma cells and affects the radiosensitivity by interacting with Wnt/β-catenin signaling (ID 4855)

Presentation Number
1996P
Lecture Time
12:00 - 12:00
Speakers
  • Xuetao Han (Shijiazhuang, China)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

IDH1 R132H mutant (IDH1R132H) glioma patients have better prognosis compared with IDH1 wild-type (IDH1wt) patients. However, the molecular mechanism is largely unknown. In this study, we investigated the biological behaviors of IDH1R132H and IDH1wt glioma cells, intends to explore whether the IDH1R132H protein could affect radiosensitivity. Additionally, we studied potential cross-talk between IDH1 and Wnt/β-catenin signaling in regulating radioresistance.

Methods

We established stable transfected U87MG and SY5Y cell lines with IDH1R132H or IDH1wt gene. The proliferation, migration and invasion ability were determined respectively by CCK-8, wound healing assay and Transwell assay. The radiosensitivity was detected by colony formation assay after 0, 2, 4, 6, 8, 10Gy radiation. Using Western blot, we assayed changes in β-catenin protein of glioma cells before and after 10 Gy radiation.

Results

The glioma cells were divided into three groups: blank control group, IDH1R132H group and IDH1wt group after stable transfection. CCK-8 assay showed that proliferation was significantly decreased in IDH1R132H group. Wound healing assay and Transwell migration assay both showed that migration capacity of IDH1R132H group was reduced than the other two groups. Transwell invasion assay also showed that IDH1R132H group has the lowest invasion rate. After 10Gy radiation, the proliferation, migration and invasion ability of IDH1R132H group was further lower than the other two groups. Similarly, colony formation assay showed formation rate of IDH1R132H group was reduced than the other groups. Western blot revealed that β-catenin protein was declined in IDH1R132H group. Furthermore, after 10Gy radiation, β-catenin protein increased in all three groups, but it was more obvious in IDH1wt group.

Conclusions

These data suggest that IDH1R132H mutation leads to a less aggressive biological behavior and increase the radiosensitivity. The molecular mechanism may be that IDH1 can activate the Wnt/β-catenin signaling pathway. Radiotherapy in combination with inhibitor of Wnt/β-catenin signaling may be an attractive approach for IDH1wt glioma patients.

Legal entity responsible for the study

The Second Hospital of Hebei Medical University.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

1997P - Impact of Angiopoietin-2 on glioblastoma response to combined chemo-radiotherapy (ID 2641)

Presentation Number
1997P
Lecture Time
12:00 - 12:00
Speakers
  • Charly Helaine (Caen, France)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Glioblastoma (GB) are brain tumors with a poor prognosis despite multimodal treatment combining resection, chemotherapy (CT) and radiotherapy (RT). The rich vascularization of these tumors led to the introduction of anti-angiogenic therapy with most efforts focused on the vascular endothelial growth factor (VEGF). However, the angiopoietins (Ang) have emerged as alternative regulators of angiogenesis. In particular, in GB, Ang2 is up-regulated and stimulates tumor angiogenesis in concert with VEGF but also activates pro-angiogenic functions of macrophages. However, Ang2 functions are context-dependent. Therefore, we sought to elucidate the involvement of Ang2 in the interaction of glioma response to CT and RT, both therapeutic modalities known to alter tumor angiogenesis and inflammation.

Methods

To recapitulate high levels of Ang2 in GB patients, Ang2 was overexpressed in murine glioma cells (GL261-Ang2). Effects of Ang2 were studied on an orthotopic syngenic model of GB (GL261 cells) in response to combined CT/RT. C57bl/6 mice were co-treated with temozolomide (TMZ 10 mg/kg; i.p.) and brain tumors were irradiated with X-rays (4 Gy) at 7, 9 and 11 days post-cell injection. The tumor growth and its microenvironment were followed by MRI and immunohistology analyses.

Results

We showed that, in this model, the chronic overexpression of Ang2 does not modify tumor progression, but leads to a decrease in vessel density (-39±10%, p < 0.001) and to an increase in CD68+ inflammatory cells (+24±8%, p < 0.05), compared with the control tumor group (GL261). Interestingly, when combined with CT/RT, the overexpression of Ang2 in the tumor induces a robust delay in tumor recurrence (>3 months) compared with treated GL261 tumors (18±3 days). In vitro, no difference in the chemo-radiosensitivity of GL261 and GL261-Ang2 cells was noticed, suggesting a paracrine effect of Ang2 on the tumor microenvironment. Accordingly, we showed that Ang2 sensitizes the tumor vasculature to CT/RT and sustains inflammatory cells in the tumor microenvironment until 3 months post-treatment.

Conclusions

These results suggest that Ang2 might influence the therapeutic response of GB by acting on angiogenesis and inflammation.

Legal entity responsible for the study

E. Petit.

Funding

This study was funded by the Région Normandie, the Centre National de la Recherche Scientifique (CNRS), the Université de Caen Normandie (UNICAEN), the European Union-Fonds Européen de Développement Régional (FEDER), ARCHADE, HABIONOR European project, la Fédération pour la Recherche sur le Cerveau par l’opération Rotary «Espoir en tête » (FRC), EdNBise 497 - Normandie Université.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

1998P - The discovery of RNA-aptamers that selectively bind and inhibit glioblastoma stem cells by targeting EphA2 (ID 5743)

Presentation Number
1998P
Lecture Time
12:00 - 12:00
Speakers
  • Alessandra Affinito (Napoli, Italy)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults. Despite progress in surgical and medical neuro-oncology, prognosis for GBM patients remains dismal. It has been demonstrated that the modest benefit of conventional therapies is due to the presence of glioblastoma stem cells (GSCs) that cause tumor relapse and chemoresistance. Thus, the identification of specific ligands for GSCs could be fundamental for GBM improvement in survival.

Methods

Here, using a cell-SELEX approach on human primary GSCs, we generated RNA aptamers –namely, a 40L sequence and A40s, a truncated form– that bind GSCs.

Results

The aptamers were selective for human GSCs, they were able to inhibit stemness, cell growth and migration, and strongly reduced tumor proliferation in vivo. Moreover, 40L and A40s were rapidly internalized upon target binding and, therefore, may serve as selective vehicles for therapeutics. Furthermore, A40s is able to cross the blood brain barrier (BBB). Using several approaches, we have identified the aptamer target in the Ephrin type-A receptor 2 (EphA2) which is overexpressed in GSCs compared to differentiated cells.

Conclusions

Given the role of GSCs in GBM recurrence and therapy resistance, 40L and A40s represent innovative therapeutic and diagnostic tool for GBM.

Legal entity responsible for the study

Gerolama Condorelli.

Funding

Federico II, Naples.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

1999P - Impact of tumor reoxygenation by nanoparticles on tumor associated macrophages (TAMs) (ID 4160)

Presentation Number
1999P
Lecture Time
12:00 - 12:00
Speakers
  • Aurélie E. Ferré (Caen, France)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Glioblastoma (GB), a highly hypoxic brain tumor (Bekaert et al. 2017), is characterized by a massive macrophage (M&Phi;) infiltration (Lapa et al. 2015). Hypoxia triggers a shift to a pro-tumoral M2 phenotype in GB (Leblond et al. 2016). Thus, strategies aiming to reduce hypoxia could promote an anti-tumoral M1 phenotype. Among these reoxygenation strategies, we recently developed a new approach with zeolites nanoparticles. These zeolites are able to carry hyperoxic/hypercapnic gases and release them according to a hypoxic gradient. We have demonstrated that the charge balancing cation changes affinity to the gases but also the ability to track zeolite with MRI. Our objective is to study the reoxygenation efficacy of zeolites specifically in the GB and to evaluate their impact on tumor associated M&Phi; with in vitro and in vivo studies.

Methods

Faujasite zeolites (FAU, ∼20nm of diameter) were used and modified by ion exchange with various cations (Fe, Gd, Cu, Ag). GB model was obtained by orthotopic glioblastoma cells implantation (U251) in nude rats (ONCOModels/Unicaen). 7T MRI (Bruker/Cyceron) was used to follow zeolites after intravenous injection and for oxygen measurement. Murine bone marrow derived M&Phi; were prepared and polarized to M1 and M2 using LPS/IFNg or IL4 as previously described (Leblond et al. 2016). Zeolites were added in M&Phi; medium and their impact on M&Phi; were evaluated by crystal violet dye assay, flow cytometry (Plateau ICORE/Unicaen) and polarization assays.

Results

Our results show that zeolites are able to accumulate and release the carried gases specifically in the brain tumor leading to tumor reoxygenation. Regarding the effect on M&Phi;, our preliminary results show, in vitro, the safety of as-prepared zeolites or Fe, Gd or Cu dopped zeolites on M0, M1 and M2 M&Phi; cultures. Similarly, no alteration of the cell cycle was observed. As a positive control of cell death, the presence of Ag-dopped zeolites dramatically decreased M0 and M1 M&Phi; viability.

Conclusions

Zeolites can deliver oxygen to the brain tumor and may improve the effectiveness of conventional treatments. Zeolites do not exhibit toxicity on primary cultures of M&Phi;. Additional studies are underway to evaluate the effect of zeolites on the polarization of M&Phi;, both in vitro and in vivo.

Editorial acknowledgement

Région Normandie, CNRS, Université de Caen Normandie, Ministère de l'Enseignement Supérieur et de la Recherche, European Union-Fonds Européen de Développement Régional (FEDER), HABIONOR European project, co-funded by the Normandy County Council, the French State in the framework of the interregional development Contract “Vallée de la Seine” 2015-2020, ARCHADE, Fédération pour la Recherche sur le Cerveau (FRC) et INCa (INCA-11699).

Legal entity responsible for the study

The authors.

Funding

Région Normandie, CNRS, Université de Caen Normandie, Ministère de l’Enseignement Supérieur et de la Recherche, European Union-Fonds Européen de Développement Régional (FEDER), HABIONOR European project, co-funded by the Normandy County Council, the French State in the framework of the interregional development Contract “Vallée de la Seine” 2015-2020, ARCHADE, Fédération pour la Recherche sur le Cerveau (FRC) et INCa (INCA-11699).

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

2000P - Prognostic significance of c-Rel/p50 heterodimer in the tumor microenvironment of uveal melanoma (ID 2474)

Presentation Number
2000P
Lecture Time
12:00 - 12:00
Speakers
  • Seema Kashyap (New Delhi, India)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Uveal melanoma is the most common primary intraocular malignancy in adults. Almost half of patients with uveal melanoma may die from metastatic disease and the most frequent site of metastases is the liver. It is considered as an inflammatory phenotype. NFκB plays a connecting link between the inflammation and cancer. c-Rel and p50 are together considered as a heterodimer (c-Rel/p50) which plays an important role in inflammation and cancer progression. Therefore, the aim of the study is to detect c-Rel/p50 heterodimer expression in the tumour microenvironment of uveal melanoma and its prognostic significance.

Methods

Evaluation of c-Rel/p50 heterodimer expression was performed in 75 formalin fixed uveal melanoma tissues by using immunohistochemistry. Transcriptional analysis was performed on 58 fresh tissues by real-time PCR (q-PCR). Co-Immunoprecipitation (Co-IP) was done on 10 representative cases to assess the presence of c-Rel/p50 heterodimer and validation of immunohistochemistry results was performed by western blotting. Results were then correlated with clinicopathological parameters.

Results

Immunoexpression of c-Rel+/p50+ is localized to the cytoplasm in normal choroid whereas both cytoplasmic and nuclear expression was seen in 33.33% cases. At the transcriptional level, upregulation of c-Rel+/p50+ heterodimer was shown in 43.10%. Expression of both cytoplasmic and nuclear c-Rel+/p50+ heterodimer were found to have significant correlation with cases having tumour infiltrating lymphocytes, macrophages (CD68+) and high pigmentation. There was a statistically significant difference in the overall survival of patients with nuclear and cytoplasmic expression of c-Rel+/p50+ heterodimer (p < 0.05).

Conclusions

We conclude that c-Rel+/p50+ heterodimer may be used as a poor prognostic indicator of survival outcome in uveal melanoma. Further translational and validation studies are required to confirm its usage as a therapeutic drug target in metastatic uveal melanoma.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

2001P - Synergistic role of BAP1 and DNA damage response pathway in uveal melanoma and its prognostic significance (ID 1769)

Presentation Number
2001P
Lecture Time
12:00 - 12:00
Speakers
  • JAYANTI JHA (Delhi, India)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Uveal melanoma is the most common primary cancer of the eye mainly arising from choroid which metastasizes to the liver and frequently leads to death. Till date, there is no current study on the correlation between ATM and BAP1 expression in uveal melanoma. Both proteins trigger DNA damage response (DDR) which leads to DNA repair. Literature reveals that ATM modifies the BAP1for the activation of DNA repair which signifies that there is a connecting link between ATM and BAP1. Therefore, the aim of the study is to detect the co-expression of ATM and BAP1 protein in uveal melanoma patients.

Methods

Sixty-nine formalin fixed paraffin embedded choroidal melanoma samples were taken to evaluate the expression of ATM and BAP1 by immunohistochemistry and validated by western blotting. To check BAP1 mutation, Sanger sequencing was done on control and UM samples. Results were then correlated with clinical and histopathological parameters. To determine the prognostic significance, Kaplan–Meier analysis and multivariate analysis by Cox’s Proportional Hazards Model was performed.

Results

There was a male preponderance in our study. Histopathological high-risk factors were identified in 48% cases. Both ATM and BAP1 show loss of nuclear expression in 55% of the cases and this was statistically significant with high pigmentation, LTD >10mm, TIL and cell type (p < 0.05). On multivariate analysis, advanced tumour staging and epithelioid cell type are found to be independent prognostic factors.

Conclusions

Our data suggest that loss of nATM and nBAP1might serve as a potential prognostic marker in the pathogenesis of uveal melanoma leading to increased risk of metastasis. These findings demonstrate the synergistic role of ATM and BAP1 proteins and may have a therapeutic potential in uveal melanoma. However, further studies are required in a larger cohort of patients with longer follow up and translational validation needs to be performed.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

2002P - CXCR4, CCR2 and CCR5 expression in subsets of tumor cells with stem and/or EMT features (ID 5037)

Presentation Number
2002P
Lecture Time
12:00 - 12:00
Speakers
  • Olga E. Savelieva (Tomsk, Russian Federation)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Numerous investigations have been focused on the role of CXCR4, CCR2 and CCR5 in solid tumors, including breast cancer. These chemokine receptors make cancer cells move out of the circulation and traffic into organs with high amounts of chemokines, and thus forming metastases. In this connection, the objective of our stady was to investigate CXCR4, CCR2 and CCR5 expression in subsets of tumor cells with stem and/or EMT features in primary tumor and peripheral blood in breast cancer patients.

Methods

Twenty-three patients with breast cancer, between 29 and 69 years of age, were included in this study. Five-color confocal microscopy was used to investigate tumor cells with stem and/or EMT features, CXCR4, CCR2 and CCR5 expression in primary tumor. Flow cytometry was used to analyze the similar phenotypes of circulating tumor cells (CTC). All patients signed an informed consent for voluntary participation.

Results

In the primary tumor, CXCR4 was expressed on subsets of non-stem and stem-like tumor cells with EMT features. CCR5 was more often expressed by tumor cells with stem and EMT features. The count of such cells in the tumor decreased after neoadjuvant chemotherapy (NACT). CK7+CD45-CD44-CCR2+ and CK7+CD45-CD44-CXCR4+ CTC (without stem features) were detected in breast cancer patients with and without NACT. At the same time, the count of non-stem CXCR4+ CTC was significantly higher in the blood of patients after NACT. CK7+CD45-CD44-N-cadh+CCR5+ CTC (with EMT features) were more frequent in the blood of breast cancer patients after NACT, and its count was significantly higher. It is interesting to note that stem-like CTC didn’t express CCR2 and CCR5. Stem-like CXCR4+ CTC were rarely detected and had no EMT features in patients, regardless of the NACT performance. The count of CCR5+ tumor cells without stem and EMT features correlates with the count of non-stem CCR5+ CTC. The count of CXCR4+ tumor cells with stem and EMT features correlates with the count of non-stem CXCR4+ CTC with EMT features.

Conclusions

Thus, the potency for recruitment into the circulation is found in tumor cells with EMT features, regardless of the stemness status. The study was supported by the Russian Science Foundation (grant 19-75-30016).

Legal entity responsible for the study

The authors.

Funding

Russian Science Foundation.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

2003P - Expression of mutant p53 affects cancer cell sensitivity to topotecan (ID 5729)

Presentation Number
2003P
Lecture Time
12:00 - 12:00
Speakers
  • Rimma Mingaleeva (Kazan, Russian Federation)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Inactivation of tumor suppressor p53 is a common event in tumor progression. In almost 50% of cases p53 inactivation is caused by mutations that primarily affect DNA-binding domain. Oncogenic missense mutation Y220C is the ninth most common for p53 and is annually observed in ∼100,000 new diagnosed cancer cases worldwide. Presence of this mutation disturbs tertiary structure of the p53 DNA-binding domain that further leads to destabilization of the whole protein, its partial denaturation and loss of transcriptional activity. In some cases, p53 mutations result in more aggressive cancer cells and alter drug sensitivity.

Methods

We employ CRISPR-Cas9 gene editing technology to generate p53 knock-out and Y220C mutant MCF-7 cell lines. Quantitative analysis of alterations in intracellular protein levels were performed by immunoblotting, analysis of p53-dependent gene expression by quantitative real-time reverse transcription PCR; cell proliferation and chemotherapy cytotoxicity by MTS test. Statistical analysis was performed using ANOVA and Holm-Sidak method for multiple comparison, p ≤ 0.05 was considered to be statistically significant.

Results

We have confirmed that p53 mutation Y220C leads to p53 inactivation. Proliferation rate of p53 knockout and mutant MCF-7 cell lines was 15% higher than wild type. We have shown statistically significant decrease in topotecan cytotoxicity towards knockout and mutant cells compared to wild-type – 14% and 26%, respectively.

Conclusions

Decline of topotecan cytotoxicity in mutant and knockout cells can be explained by topotecan-mediated induction of p53 that leads to higher levels of cell death in wild-type MCF-7. References Bulatov, E., Zagidullin, A., Valiullina, A., Sayarova, R., Rizvanov, A. (2018). Small molecule modulators of RING-type E3 ligases: MDM and Cullin families as targets. Frontiers in pharmacology, 9, 450.

Legal entity responsible for the study

Albert Rizvanov.

Funding

Grant of the President of the Russian Federation (MK-4253.2018.4).

Disclosure

All authors have declared no conflicts of interest.

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2004P - Breast cancer organoids: A new tool for the prediction of drug penetration and patient outcome (ID 5725)

Presentation Number
2004P
Lecture Time
12:00 - 12:00
Speakers
  • Giuseppina Roscigno (Napoli, Italy)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

One limitation in the breast cancer research field is that there are few in vitro models of breast cancer able to predict drug response and thus clinical patients’ outcome, mainly because the present models do not take account of the tumor heterogeneity. To address this issue, we developed an in vitro 3D organoid culture system using primary human breast cancer tissue that could be used to recapitulate in vivo organs. A major difficulty in the development of such models is to identify in vitro conditions that preserve the breast cancer phenotypes observed in vivo.

Methods

We isolated organoids from breast cancer patients and optimized the organoids growing conditions. By immunofluorescence assay we confirmed the receptor status, and the preservation of epithelial (E-cadherin) or the fibroblasts components included in the tissue microenvironment (TME) (alfa-SMA and FAP). Here we used organoids as mimic of tumor burden for drug penetration studies. In particular, we studied doxorubicin (DOX) penetration and biodistribution, by several means: immunofluorescence studies, caspase assay, and cell viability.

Results

We demonstrated by IF studies, that miR-340, an oncosuppressor miR, was able to modulate DOX penetration. At the same time, miR-340 induced an increase in DOX sensitivity assessed by caspase-3 assay, PARP cleavage, as well as p38 phosphorylation. Breast cancer organoids have also been used to better understand the role of the TME in breast cancer response to therapy. We assessed drug sensitivity in the presence or absence of conditioned media obtained from cancer activated fibroblasts (CAFs). We found that organoids treated with conditioned media exhibit lower level of caspase 3 activation compared to the control upon palbociclib treatment, suggesting the importance role of TME in drug resistance.

Conclusions

Thus, organoids can be considered as a new tool for studying breast cancer and developing personalized medicine approaches and to test possible use of RNA therapeutics.

Legal entity responsible for the study

Gerolama Condorelli.

Funding

AIRC.

Disclosure

All authors have declared no conflicts of interest.

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2005P - Aptamer-mediated exosomes detection for early breast cancer identification (ID 5680)

Presentation Number
2005P
Lecture Time
12:00 - 12:00
Speakers
  • Cristina Quintavalle (Napoli, Italy)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Increasing evidence indicates that the release of exosomes by tumor cells play a key role in tumor progression, drug resistance, immune surveillance escape, angiogenesis, tumor invasion, and metastasis. For this reason, cancer-derived exosomes are emerging as very interesting targets for early diagnosis and therapy in cancer, including breast cancer. Indeed, progresses in developing nucleic acids-based therapeutic compounds, including antisense DNAs, aptamers, short interfering/microRNAs, and short activator RNAs, have attracted great interest as emerging platforms for precise cancer treatment.

Methods

We have recently developed a novel differential SELEX (Systematic Evolution of Ligands by Exponential enrichment) strategy by using exosomes purified from epithelial BC primary cells in the positive selection step and exosome-derived from primary normal epithelial breast cell lines in the negative selection step.

Results

Three aptamers have been shown to be enriched in different families, and their ability to selectively bind BC cells-derived exosomes has been proven. Interestingly, two of them can selectively bind triple negative-derived exosomes, compared to more differentiated BC cells. To optimize the best aptamers, shortened version of aptamers (about 35mer) have been generated and their binding ability has been tested. Moreover, the aptamers showed no binding affinity for lung cancer and glioblastoma-derived exosomes. We also tested by binding assay the ability of one of the identified aptamers, named ex-50sh, to selective recognize exosomes isolated from serum of breast cancer patients. Proteomic analysis of the putative target is in progress. Immunofluorescent experiments showed that the short aptamers can also block the uptake of MDA-231-derived exosomes on MDA-231 cell lines. Moreover, ex-50sh is able to block the EMT transformation induced by MDA-231 exosomes in low malignant MCF7.

Conclusions

Altogether, the selected aptamers will provide new insights in the molecular characterization of breast cancer exosomes and, most importantly, innovative tools for early breast cancer diagnosis.

Legal entity responsible for the study

Gerolama Condorelli.

Funding

University of Naples Federico II.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

2006P - MicroRNA-181c promotes tamoxifen resistance in breast cancer cells via upregulation Akt/mTOR axis (ID 2460)

Presentation Number
2006P
Lecture Time
12:00 - 12:00
Speakers
  • Alexander M. Scherbakov (Moscow, Russian Federation)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

The main purpose of the work was to study the key exosomal factors involved in the development of hormonal resistance of breast cancer cells. The work is based on our previous data, which demonstrated the effect of exosome-mediated transferring of hormonal resistance in in vitro cultured MCF-7 breast cancer cells.

Methods

Estrogen-dependent breast cancer cells MCF-7 and the tamoxifen-resistant subline MCF-7 /T were used as an experimental model. The analysis of exosomal microRNAs was performed by HiSeq2500 and at least 5 million reads per samples were obtained. MicroRNA was extracted from by PureLink RNA Micro Kit; library preparation was carried out with NEBNext® Small RNA Library Prep Set for Illumina®. Transfection of the RNA oligonucleotides was performed using Metafectene PRO (Biontex) to result in the final RNA concentration of 50 nM.

Results

A comparative analysis of exosomal miRNAs of MCF-7 and resistant MCF-7/T cells was carried out. In total, 2588 miRNAs have been identified in the exosomes. Among them, mir-181 family, which is one of the negative regulators of estrogen-dependent growth, was identified as the group of miRs, hyperexpressed in the resistant exosomes. Following this, we analysed the role of mir-181c, one of the main members of miR-181 family, in the regulation of cell growth and hormonal response. Mir-181c transfection was found to induce the estrogen-independent growth and partial tamoxifen resistance of MCF-7 cells. The study of the signaling proteins showed that mir-181c transfection, in contrast to scrambled RNA transfection, caused the increase of the amount of Raptor, phosphorylated forms of mTOR and Akt that correlated with increased AP-1 transcriptional activity.

Conclusions

We have demonstrated the involvement of miR-181c in the development of hormonal resistance of breast cancer cells that allows us to consider mir-181 as the perspective target of the treatment of hormone- independent cancers. The research was supported by the Russian Science Foundation (19-15-00245, miRNA analysis) and RFBR (#18-29-09017, tamoxifen resistance).

Legal entity responsible for the study

The authors.

Funding

Russian Science Foundation, project 19-15-00245.

Disclosure

All authors have declared no conflicts of interest.

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2007P - Spatio-temporal separation of tumor infiltrating CD8+ T-cells and HER2/neu+ tumor cells in tumor-immune milieu of infiltrating ductal carcinoma of the breast (ID 3751)

Presentation Number
2007P
Lecture Time
12:00 - 12:00
Speakers
  • Sandhya Sreedharan (Bangalore, India)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Designated tumor infiltrating lymphocytes (TIL) within tumor microenvironment (TME) comprised of both regulatory cells and effector cells, and their interaction with tumor cells account for the immune escape and immunosurveillance mechanisms respectively. The number and location of CD8+ tumor infiltrating lymphocytes (TILs) are the measures of immune response and carries prognostic significance and predictive potential in the era of immuno-oncology. The present study was an attempt to find the relation between the treatment response and the spatio-temporal separation of tumor infiltrating CD8+ T-cells and HER2/neu+ tumor cells in breast cancer TME.

Methods

In this study, a total of 7 breast cancer patients were enrolled and surgically removed fresh tumor tissues were evaluated for the baseline HER2/neu and CD8 expression by immunohistochemistry (IHC) using dual stain (duplex IHC). The tumor tissues were also tested for the response to HER2 blocker in a novel humanized ex vivo platform that preserves tumour microenvironment using tumour explants, maintained in defined tumour grade-matched matrix support and autologous patient serum.

Results

Tumors responded to HER2 blockade had a majority of the CD8+ T cells (60-80%) concentrated around the HER2/neu+ tumor area (within 50-100µm). While, the non-responding tumors had significantly lesser number of the CD8+T cells (20-40%) proximal to HER2/neu+ zone compared to the responders. The present study indeed delineated a preferential spatial relationship of tumor infiltrating CD8+ cells in HER2/neu+ tumor region especially for the recognition of tumor antigen, cross presentation and tumor cell killing.

Conclusions

Assessment of distribution pattern of effector cells surrounding tumor area had a correlation on the efficacy of HER2 inhibition. It warrants validation in the larger cohort of patient tumors to underpin its clinical relevance for possible immunotherapies like combination of check point inhibitors with HER2/neu blocker and others. Identification of other co-stimulatory and pathway molecules on the tumors not responded to HER2 blocker is being studied to understand the mechanism of resistance.

Legal entity responsible for the study

The authors.

Funding

Mitra RxDx Inc.

Disclosure

All authors have declared no conflicts of interest.

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2008P - Large genomic rearrangements in BRCA1 and BRCA2 genes in the Portuguese population (ID 4664)

Presentation Number
2008P
Lecture Time
12:00 - 12:00
Speakers
  • Joao M. Moreira-Pinto (Loures, Lisboa, Portugal)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

BRCA1 and BRCA2 are the genes most frequently associated with hereditary breast and ovarian cancer (HBOC). Besides point mutations, BRCA1/2 large genomic rearrangements (LGR) have been observed, with different frequencies in specific populations. This study aims to update the frequency of BRCA1 and BRCA2 LGR in our population, as well as to characterize the gene distribution of these genetic events.

Methods

All high risk pts referred to our multidisciplinary program between 2000-2018 that consented on genetic testing were reviewed for BRCA1 and BRCA2 point pathogenic variants (several methodologies throughout the years including Conformation Sensitive Gel Electrophoresis; Conformation Sensitive Capillary Electrophoresis; Sanger Sequencing and Next Generation Sequencing) and LGR with Multiplex Ligation dependent Probe Amplification (MLPA).

Results

3763 of 3901 index pts consented on genetic testing and 473 pts had 476 different pathogenic variants (PV). In 3 cases double germline PV were observed: BRCA1 (c.68_69delAG) and CHEK2 [c.(908 + 1_909-1)_(1095 + 1_1096-1)del]; ATM (c.8264_8268delATAAG) and PALB2 (c.751C>T); BRCA2 (c.156_157insAlu) and CHEK2 (c.593-1G>T). BRCA1 PV were detected in 151 pts with 24 LGR of these (15,9%) being detected by MLPA. The most frequent BRCA1 LGR was c. (4185 + 1_4186-1)_(4357 + 1_4358-1)dup (n = 8). BRCA2 PV were detected in 260 pts with 99 LGR: 92 pts with the Portuguese founder mutation c.156_157insAlu (detected by specific PCR or MLPA) and 7 pts with other LGR (detected by MLPA). The second most frequent BRCA2 LGR was c. (8632 + 1_8633-1)_(8754 + 1_8755-1)dup (n = 5). Nine CHEK2 PV were detected with the SALSA MLPA probemix P045 BRCA2/CHEK2: c.1100delC (8 pts) and c. (908 + 1_909-1)_(1095 + 1_1096-1)del (1pt). The total percentage of BRCA1 and BRCA2 LGR in our cohort was 26%. Excluding the Portuguese founder mutation c.156_157insAlu, the prevalence of LGR was 6,5%.

Conclusions

There is a high prevalence of LGR in the Portuguese population, mostly because of the Portuguese founder mutation c.156_157insAlu. Even if excluding this event, other LRG still account for 6,5% of the BRCA1 and BRCA2 pathogenic variants. These results emphasize the importance of testing LGR in all patients with high risk HBOC.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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2009P - Non-BRCA1/2 hereditary breast and ovarian cancer: Findings from a multidisciplinary program (ID 4611)

Presentation Number
2009P
Lecture Time
12:00 - 12:00
Speakers
  • Ana Monteiro (Lisbon, Portugal)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Non-BRCA1/2 pathogenic variants have increasingly been associated with breast and ovarian cancer (BOC). In this study, we analyze the clinical and molecular characteristics of non-BRCA1/2 BOC identified in our program.

Methods

Index pts without BRCA1/2 pathogenic variants identified between 2004-2018, were counselled for multigene sequencing after multidisciplinary decision. Either the BRCA Hereditary Cancer MASTR Plus (Multiplicom) or the Trusight (Ilumina) were performed in the MiSeq platform (Illumina). Counselling included the possibility of opt out.

Results

From all 4277 index pts tested, 377 non-BRCA1/2 (96,3% breast and 22,6% ovarian cancer) consented on reanalysis and in 31 (8.2%) a pathogenic variant was identified. All but one (with both ATM and PALB2 mutated) pt harbored one pathogenic variant (Table). For 38 pts (10,1%) only selected genes from the panel were studied and 11 (2,9%) opted out of TP53.

2009P

GenePathogenic variant
CHECK2c.1100delC (1) c.319 + 2T>A (6) c.593-1G>T (1)
PALB2c.1192delG (1) c.2257C>T; p.Arg753Ter (2) c.172_175delTTGT; p.Gln60ArgfsTer7 (1) c.751C>T (1)*
RAD51Cc.404G>A; p.C135Y (2) c.709C>T;p.Arg237 (1) c.887_896del10 (1) c.8890_899delTTGTTCCTGC;p.Leu297HisfsTer2 (1)
ATMc.3802delG;p.Val1268Ter (1) c.8264_8268delATAAG (2)* c.8292_8293delTG; p.Ser2764ArgfsTer4 (1)
TP53c.586C>T;p.Arg196Ter (1) c.725G>A (1) c.1010G>A;p.Arg337His (1)
BLMc.298_299delCA; p.Gln100Glufs (1) c.2206dupT; p.Tyr376LeufsTer5 (1)
RAD50c.2516_2517insA;p.Asp840ArgfsTer5 (2)
BRIP1c.3874 + 2T>C (1) c.484C>T; p.Arg162Ter (1)
FAM175Ac.1106dupG;p.Ser370iLefsTer2 (1)

Predictive factors for positive tests were a lower median age of BC diagnosis (37vs41) and a complex phenotype (9.7%vs3.8%). Neither the family male: female ratio (3,2% vs 7,2%) or prostate cancer (19.4%vs21,4%) were predictive, but other cancers were more frequent in hereditary cases (83.9%vs78,8%). All individuals were invited for prospective follow up.

Conclusions

A recurrent CHECK2 event explained 19% of all cases and opt out, as well as incomplete panels, may have underestimated the relevance of the TP53 gene. Younger age at BC diagnosis, complex phenotype and aggregation with other cancers were predictive for positive test. Additional follow up will add to the impact of non-BRCA1/2 tests in clinical practice.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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2010P - Quantitative imaging and characterization of collagen patterns in high grade serous ovarian carcinoma (HGSOC) (ID 5340)

Presentation Number
2010P
Lecture Time
12:00 - 12:00
Speakers
  • Ruby Y. Huang (Taipei City, Taiwan)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

HGSOC is known to demonstrate diverse molecular heterogeneity. It is unclear whether the tumor microenvironments such as the stromal components in the extracellular matrix (ECM) also exist heterogeneity.

Methods

As a proof-of concept study, characterization of collagen patterns was performed on 60 unstained formalin-fixed paraffin embedded (FFPE) HGSOC samples (three sections from each of the 20 patients). Each section with 5 micron thickness and a minimum tumour surface area of 40 mm2 were scanned by using a multiphoton imaging system (Genesis 200, HistoIndex) following deparaffinization. Parameters including Collagen Area Ratio (CAR), Collagen Fiber Density (CFD), Collagen Reticulation Index (CRI), Collagen Fiber Number (CFN), Collagen Fiber Thickness (CFT), and Collagen Fiber Length (CFL) were analysed. Unsupervised hierarchical clustering analysis was performed.

Results

Unsupervised hierarchical clustering of the collagen parameters revealed four distinct patterns in HGSOC samples. G1 tumors consisted of long and thick collagen fibers with high CFT and CFL. G2 tumors were low in all the collagen parameters, suggesting a relatively “clean” stromal without collagen deposition. G3 tumors consisted of dense collagen fibers with high CRI suggestive of extensive cross alignment among the fibers. G4 tumors were high in CFN and low in CRI, CFT and CFL, suggesting a stroma loaded with high amount of thin and short collagen fibers without cross alignment.The collagen patterns were not exclusive for specific organ sites (ovary, fallopian tube, other metastatic sites) except that the collagen pattern from the omental metastases were mainly G2 (10/18; 55.6%) and G4 (6/18; 33.3%).

Conclusions

The stromal component in the ECM of HGSOC can be successfully quantitated by the multi-photon imaging technology on unstained FFPE sections. The collagen component exhibited significant heterogeneity in terms of the number, thickness, length, and reticulation patterns. The contribution of different collagen patterns to clinical outcomes and the correlation with known molecular subtypes in HGSOC warrants further investigation in larger cohorts.

Legal entity responsible for the study

National University of Singapore.

Funding

National Medical Research Council of Singapore.

Disclosure

All authors have declared no conflicts of interest.

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2011P - Semiquantitative assessment of vimentin expression in prostate cancer (PC) (ID 4209)

Presentation Number
2011P
Lecture Time
12:00 - 12:00
Speakers
  • Marina Puchinskaya (Minsk, Belarus)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Vimentin (Vim) is one of the mesenchymal markers, that are often upregulated in cancer cells during epithelial-mesenchymal transition (EMT) – a process endowing tumor cells with invasiveness, pro-metastatic potential and, arguably, cancer stem cells (CSC) properties. It is normally seen in cytoplasm, but cell surface Vim was also detected on cells with CSC characteristics. Vim expression may be used as a surrogate marker of EMT in tumor tissue samples from patients and aid in prognosis evaluation. We aimed to characterize Vim expression in prostate cancer (PCa) using our method of semiquantitative staining assessment.

Methods

Vim expression was assessed in 48 cases of PCa using immunohistochemistry with primary rabbit polyclonal antibodies (ThermoFischer, 1:1000). UnoView Rabbit Detection Kit was used for visualization. Specificity of staining was confirmed by another antibody (mouse monoclonal, DAKO, 1:200) in selected cases. To assess Vim expression semiquantitatively we proposed a weighed staining index (WSI) that takes into account the proportion of stained cells as well as the ratios of staining intensities and can be applied to different cellular compartments staining separately. It combines advantages of simple ad oculus semiquantitative staining evaluation and obtaining numerical characteristics of marker expression.

Results

Median WSI for membranous staining was 5 (0 – 52) (7, if cases without expression were excluded), for cytoplasmic – 20.5 (0 – 64) (22.5, if cases without expression were excluded). In 2 (4.2%) cases marker expression was absent in both membrane and cytoplasm. WSI in PCa was significantly higher for cytoplasmic staining. However, in 3 (6.25%) cases WSI for membranous staining was higher than that for cytoplasmic. Significant correlation was found between WSI for cytoplasmic and membranous staining (Spearman test, r = 0.51, p < 0,05). However, no correlation was found between WSI for both compartments and E-cadherin expression (as assessed by average staining intensity score – a method similar to WSI – using Aperio software previously).

Conclusions

Vim expression as a sign of EMT in the tumor is present in 95.8% cases of PCa and is more prevalent in cytoplasm. WSI is a useful tool to assess Vim (and possibly other markers) expression.

Legal entity responsible for the study

M. Puchinskaya.

Funding

Belarusian Republican Foundation for Fundamental Research.

Disclosure

All authors have declared no conflicts of interest.

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2012P - Heat shock protein 90 chaperones and protein kinase D3 regulates androgen-independent prostate cancer development (ID 3309)

Presentation Number
2012P
Lecture Time
12:00 - 12:00
Speakers
  • Attila Varga (Budapest, Hungary)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Prostate cancer is the second leading cause of cancer related deaths in men worldwide. Heat Shock Protein 90 (Hsp90) is expressed in tumour cells at high levels – 3-5% of total proteins – and regulates the function of oncogenes and other tumour related proteins. Protein kinase D3 (PKD3) has a proven role in the progression of androgen-independent prostate cancer. In the present study we set out to explore the impact of Hsp90 and PKD3, respectively, on prostate cancer growth and their potential interaction.

Methods

We employed the DU145 and PC3 well-characterized androgen-independent prostate cancer cell lines. Cell viability was determined by Trypan Blue exclusion cell counting. Apoptosis analysis was performed by flow cytometry after AnnexinV and propidium-iodide co-staining. Protein levels were detected by western blot and protein-protein interactions were investigated by co-immunoprecipitation.

Results

We found that the clinically used Hsp90 inhibitor ganetespib induced apoptosis and significantly reduced the viability of the androgen-independent DU145 and PC3 cell lines. The pan-PKD inhibitor CRT0066101 also decreased cell viability of the prostate cancer cells in a dose-dependent manner. Further, we demonstrated that ganetespib reduced PKD3 protein level in a concentration-dependent manner and induced its proteasomal degradation. Finally, a co-immunoprecipitation study revealed a physical connection between PKD3 and Hsp90.

Conclusions

We identified and confirmed an Hsp90-PKD3 chaperone client interaction, which may be important in prostate cancer cell survival. Further studies are under way to characterize the biological significance of our findings. Our results contribute to better understand the pathological signalling of androgen-independent prostate cancer cells and to find novel treatment strategies.

Legal entity responsible for the study

MTA-SE Pathobiochemistry Research Group.

Funding

National Research, Development and Innovation Office - Hungary.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

2013P - The SWI/SNF driven reprograming for the AR cistrome is NSD2 dependent (ID 3441)

Presentation Number
2013P
Lecture Time
12:00 - 12:00
Speakers
  • Katia Ruggero (Barcelona, Spain)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Resistance to androgen receptor signaling is arguably the principal hallmark of lethal prostate cancer. Several mechanisms account for this resistance, including mutations in the AR, restoration of signaling downstream of the pharmacological blocking and activation of alternative oncogenic pathways.

Methods

We used the Nkx3.1CreERT2/+; Ptenfloxed/floxed; p53floxed/floxed (NPp53) mice that develop CRPC upon castration and undergo neuroendocrine differentiation with anti-AR treatment to isolate Enzalutamide resistance prostate cancer cells.

Results

Recently, we showed that activation of the chromatin remodeler Nsd2 is required for PCa metastasis and is strongly associated to tumor progression, and that its silencing markedly reduced the metastatic burden and increased survival. Our current data indicates that Nsd2 silencing sensitizes PCa cells to anti-AR treatment. Nsd2 KO using CRISPR/Cas9 resulted in a markedly enhanced efficacy of Enzalutamide and a significant reduction of AR transcriptional activity with either Enzalutamide or Abiraterone using two different reporter assays. Next, To identify Nsd2-dependent AR co-regulators we immunoprecipitated chromatin-bound endogenous AR in NPp53 cells and NPp53-Nsd2KO and performed nano-LC-MS/MS mass spectrometry on the purified peptide mix. In particular, data indicates that AR association with SWI/SNF members is stronger in the absence of Nsd2, suggesting that Nsd2 overexpression might impair AR interaction to the SWI/SNF complex. We next tested whether Nsd2 in fact binds SWI/SNF subunits by co-immunoprecipitation in the NPp53 cells and whether BAF155, BAF170 and BRG1 association to AR increases in the absence of Nsd2. As suspected, there is a remarkable increase in the binding of AR to SWI/SNF subunits BAF155, BAF170 and BRG1 in the absence of Nsd2.

Conclusions

Together, our data suggests that the mechanisms by which Nsd2 overexpression drives aggressive castration resistant and androgen independent PCa may be in part through destabilizing the AR-SWI/SNF interaction and altering the specificity of the AR cistrome. Ongoing work will further elucidate this by analyzing chromatin accessibility data coupled with transcriptomics and ChIPseq data for the AR and the BAF170, BAF155 and Brg1 SWI/SNF subunits.

Legal entity responsible for the study

Alvaro Aytes.

Funding

EAU.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

2014P - IGF1R inhibition affects the survival of HT29 cancer cells by alterations of the TLR9- and autophagy signaling (ID 1659)

Presentation Number
2014P
Lecture Time
12:00 - 12:00
Speakers
  • Györgyi Műzes (Budapest, Hungary)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

In HT29 cells, an interplay between self-DNA-induced TLR9-signaling and autophagy response was found with significant effects on cell survival and kinetic parameters. The IGF/IGF1R system plays a determinant role in the pathogenesis and progression of colorectal cancer. This pathway is upstream of mTORC1, a negative regulator of autophagy. I mammalian systems chronic IGF1R inhibition was shown to attenuate autophagosome formation. The interrelated action of IGF1R inhibition and TLR9/autophagy signaling in cancer cells has not yet been clarified. The present study was designed to assess this complex network using HT29 colon carcinoma cells.

Methods

HT29 cells were incubated for 72 h with genomic (g), hypermethylated (m), and fragmented (f) tumor self-DNAs, and with/without inhibitors of IGF1R (picropodophyllin), autophagy (chloroquine) and TLR9 (ODN2088), respectively. Cell viability was measured by MTT assay. Transcriptional changes of TLR9-signaling, IGF1R, mTORC, Akt, and the autophagy process were assayed by Human v3 miRNA Assay (NanoString). Autophagy proteins were detected by immunocytochemistry, while morphology of apoptosis and autophagy by transmission electron microscopy (TEM).

Results

In case of autophagy g- and f-DNAs caused significant upregulation of Beclin1, Atg16L1, LC3 mRNAs, and downregulation of mTORC, and Akt, verified by immunocytochemistry, as well. IGF1R-inhibition alone altered inversely the autophagy-associated gene- and protein-expressions. Upon combined inhibition of autophagy, TLR9 and/or IGF1R-signaling varying degree of autophagy was detected in all groups of tumor cells according to NanoString and TEM. Incubation with IGF1R inhibitor and with m-DNA no expected suppression of tumor cell survival, induction of apoptotic death, and activation of mitophagy were found. Moreover, IGF1R inhibition affected negatively the cell-protective effect f-DNA on macroautophagy and lipophagy.

Conclusions

Our study provided evidence for an interaction between the inhibited IGF1R and TLR9/autophagy signaling with major influences on survival, proliferation and death of HT29 cells subjected to intact/modified self-DNAs. (Funded by StartUp.).

Legal entity responsible for the study

Ferenc Sipos.

Funding

StartUp.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

2015P - Characterization of atypical dMMR (deficient MisMatch Repair) tumors: A study from a large cohort of 4948 cases (ID 1379)

Presentation Number
2015P
Lecture Time
12:00 - 12:00
Speakers
  • Marion Jaffrelot (Toulouse, France)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

MMR testing is performed to screen Lynch Syndrome, evaluate the prognosis of colorectal cancer (CRC) and predict the efficacy of PD1/PDL1 blockade in all tumor types. Two methods are available: immunohistochemistry (IHC) using antibodies against MMR proteins and molecular biology (MB) for assessing microsatellite instability (MSI). Classically, dMMR tumor corresponds to loss of expression of two proteins (MLH1 and PMS2 or MSH2 and MSH6) associated with MSI. Atypical profiles of dMMR tumors have sporadically been described. The aim of our study was to describe the frequency and characteristics of these atypical cases.

Methods

All MMR testing performed in our center between 2007 and 2017 were checked to select cases with both available IHC and MB. Then, all dMMR cases were reviewed to identify atypical cases which were defined by: isolated loss of expression of one protein, loss of expression of two proteins without MSI, normal expression of the four proteins with MSI, aberrant loss of proteins, or MSI-low. Biological data of atypical cases were controlled and clinical data were collected for each case.

Results

4948 MMR tests were performed, 3800 had both available IHC and MB data, and 585 were dMMR (15 %). Among them, 97 cases were atypical and after biological control, 8 cases were re-classified typical; allowing to finally identify 89 atypical cases: 60 CRC, 10 endometrial carcinoma, 8 digestive non CRC and 11 others types of cancers. A strong correlation with genetic syndromes was observed for those atypical profiles.

2015P

Isolated PMS2 or MSH6 loss n = 53Expression of the four proteins n = 5MSH2/MSH6 or MLH1/PMS2 loss n = 16Aberrant loss of proteins n = 15
MSI433-13
MSI low128-
MSS *9-82
Clinical characteristicsPredominantly CCR Genetic predisposition syndrome (73%)Exclusively CCR or endometrial Genetic predisposition syndrome (≥40%)Predominantly Non CRC (63%)None

(* MSS: microsatellite stability)

Conclusions

Even using controlled IHC and MB, 15% of dMMR tumors have an atypical profile. These atypical cases mainly involve non CRC cancer with a strong prediction for Lynch syndrome. Their therapeutic impact particularly for immunotherapy should be now evaluated.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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2016P - Modulation of TLR9-dependent autophagy response via inhibition of c-Met signaling influences the survival of HT29 cancer cells (ID 1657)

Presentation Number
2016P
Lecture Time
12:00 - 12:00
Speakers
  • Ferenc J. Sipos (Budapest, Hungary)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

In HT29 cells, an interplay between self-DNA-induced TLR9- and autophagy responses was found with remarkable effects on survival and differentiation of tumor cells. c-Met activation is known to drive the progression of colorectal cancer by promoting signaling cascades that mainly result in alterations of cell motility, survival and proliferation. c-Met inhibition was shown to inhibit autophagy. In cancer cells the interrelated role of c-Met inhibition and TLR9/autophagy signaling has not yet been clarified, so we aimed to assess this complex interplay.

Methods

HT29 cells were incubated for 72 h with genomic (g), hypermethylated (m), and fragmented (f) tumor self-DNAs, and with/without inhibitors of c-Met (diisothiocyanatostilbene), autophagy (chloroquine) and TLR9 (ODN2088), respectively. Cell viability was measured by MTT assay. Transcriptional changes of TLR9-signaling, PI3K, CD95, c-Met, Bcl2, cytochrome-c, and the autophagy process were assayed by Human v3 miRNA Assay (NanoString). Autophagy proteins were detected by immunocytochemistry, while morphology of apoptosis and autophagy by transmission electron microscopy (TEM).

Results

Self-DNAs g and f resulted in significant upregulation of Beclin1, Atg16L1, LC3 mRNAs, and downregulation of PI3K, Bcl2, CD95, and cytochrome-c, verified by immunocytochemistry, as well. c-Met inhibition alone altered inversely the autophagy-associated gene- and protein-expressions. In each group of tumor cells using combined inhibition of autophagy, TLR9 and/or c-Met-signaling varying degree of autophagy was observed according to NanoString and TEM. Following combined incubation with c-Met inhibitor and m-DNAs no expected suppression of tumor cell survival and induction of apoptosis and mitophagy were detected. Further, c-Met inhibition changed the cell-protective effect f-DNA on macroautophagy.

Conclusions

Our study provided evidence for an intense crosstalk between the inhibited c-Met canonical and non-canonical signaling pathways, and the TLR9/autophagy response with profound impacts on survival, proliferation and death of HT29 cells subjected to intact/modified self-DNAs.

Legal entity responsible for the study

Ferenc Sipos.

Funding

StartUp.

Disclosure

All authors have declared no conflicts of interest.

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2017P - Positive feedback activation of notch signal by obesity enhances colorectal tumorigenicity (ID 3045)

Presentation Number
2017P
Lecture Time
12:00 - 12:00
Speakers
  • Dake Chu (Xi'an, China)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Investigations revealed that the steep rise of CRC incidence was significantly linked to increased obesity rates. However, the molecular interactions of obesity with CRC are still not well understood. The Notch signaling pathway is critical for many cellular processes. It is also a key player in regulating body energy metabolism. Therefore, we aimed to investigate the potential interaction and mechanism of Notch signal between obesity and CRC tumorigenesis.

Methods

In the present study, we randomly recruited 968 cases of CRC specimens, 262 cases of colorectal intraepithelial neoplasia specimens and 185 cases of normal epithelium specimens. Target protein expression was investigated by immunohistochemistry. C57BL/6J mice were randomly allocated intogroups fed with standard rodent chow, high-fat diet (HFD), and HFD treated with DBZ or DAPT. Potential differentially expressed proteins were screened by iTRAQ, and these identified proteins were then analyzed.

Results

Notch1 intracellular domain and DLL4 was up-regulated in overweight participants compared with normal-weight ones. In overweight participants, Notch1 was increased from normal epithelium, intraepithelial neoplasia to CRC. Obesity was identified at week 5 in mice fed with HFD, which began to lead to upregulation of DLL4 and consequent increased Notch1 activity in colorectal tissues. While mice treated with DBZ and DAPT were almost resistant to HFD-induced obesity then. After 10 weeks, Notch1 activity in mice fed with HFD was significantly up-regulated compared with those fed with standard rodent chow. In addition, glucose tolerance and insulin sensitivity were also ameliorated by DBZ and DAPT treatment, through a PP2A-SHIP2 dependent manner.

Conclusions

Notch signaling activation is linked to obesity in both nonmalignant participants and CRC patients. Obesity induced by HFD can increase Notch activity by DLL4-Notch1 pathway. While inhibition of Notch signaling can attenuate high fat diet–induced obesity by improving insulin resistance.These results indicate that activation of Notch signaling by its positive feedback with obesity could be a molecular bridge that connecting obesity and CRC.

Legal entity responsible for the study

Xian Jiaotong University.

Funding

National Nature Science Foundation of China.

Disclosure

All authors have declared no conflicts of interest.

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2018P - The pathological and functional roles of BRPF1 in hepatocellular carcinoma (ID 2285)

Presentation Number
2018P
Lecture Time
12:00 - 12:00
Speakers
  • Lai Hung Carol Cheng (Hong Kong, Hong Kong PRC)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Bromodomain-containing proteins are important epigenetic regulators that specifically recognize acetylated histones and implicated in regulating gene expression. Bromodomain inhibitors showed a prominent therapeutic effect on metabolic diseases and various types of cancers in clinical trials. In this study, we employed RNA-Seq to interrogate the expression profile of bromodomain-containing proteins in human hepatocellular carcinoma (HCC). We found that Bromodomain and PHD Finger Containing 1 (BRPF1) was among the most significantly overexpressed bromodomain-containing proteins in HCC.

Methods

The expression profile of bromodomain-containing proteins in our HCC patients was analyzed by RNA-Seq. BRPF1 knockout in MHCC97L was accomplished by using lentiviral-based CRISPR gene editing system. Cell proliferation, colony formation, and cell migration assays were performed to assess in vitro function of BRPF1. A nude mice orthotopic liver xenograft model was used to study the role of BRPF1 in HCC tumorigenicity and lung metastasis in vivo. Sphere formation assay and qPCR were performed to study the stemness properties of BRPF1 knockdown cells. Apoptosis and cell cycle arrest assay was performed by flow cytometry. Senescence was detected by B-galactosidase staining, mRNA and protein expression of senescence-associated makers.

Results

Clinically, high BRPF1 expression was associated with poorer survival rates in HCC patients. The upregulation of BRPF1 mRNA in HCC was significantly associated with gene copy gain and gene amplification. ROC analysis indicated that BRPF1 was a potential biomarker for HCC detection. shRNA-mediated knockdown and CRISPR-mediated knockout of BRPF1 suppressed cell proliferation and colony formation in MHCC97L. Knockout of BRPF1 also reduced tumorigenicity in liver orthotopic injection model in nude mice. We found that BRPF1 expression was elevated in CD133+ liver cancer stem cells. Inactivation of BRPF1 also reduced the expression of genes related to cancer stemness and cancer renewal ability in sphere formation. BRPF1 inhibition induced apoptosis, cell cycle arrest as well as cellular senescence.

Conclusions

Our findings suggest that BRPF1 may contribute to HCC development and cancer stemness.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

2019P - Protein tyrosine phosphatase non-receptor type 3 (PTPN3) could be a new therapeutic target for pancreatic cancer (ID 3210)

Presentation Number
2019P
Lecture Time
12:00 - 12:00
Speakers
  • Akio Yamasaki (FUKUOKA, Japan)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Pancreatic cancer (PC) is a refractory disease and we have not an effective therapeutic strategy yet. Therefore, the development of a new therapeutic procedure is urgently required. Recently, protein tyrosine phosphatase (PTP) has been attracted as a new therapeutic target for some types of cancers. However, the biological significance of PTP in PC has never been elucidated yet. In the present study, to develop effective therapeutic strategy for refractory PC, the biological significance of PTP non-receptor type 3 (PTPN3) in PC is investigated.

Methods

Three pancreatic ductal adenocarcinoma cell (PDAC) lines were used as target cells. Inhibition or overexpression of PTPN3 was performed using PTPN3 siRNA/shRNA and plasmid, respectively. Protein expression was analyzed by western blot and immunostaining. Proliferation assay was performed by MTT assay. Migration and invasion were estimated by time-lapse imaging and matrigel invasion assay. Mice xenograft experiments were performed using BALB/c nude mice. Twenty-three surgically resected human PC specimens were used for immunostaining.

Results

1) PTPN3 is highly expressed in PC cells compared to that in normal pancreatic duct cells by immunostaining. 2) Inhibition of PTPN3 significantly decreased migration and invasion in PDAC through inhibition of epithelial mesenchymal transition (EMT) and matrix metalloproteinase (MMP)-2 expression. 3) PTPN3 inhibition significantly decreased proliferation in vitro in PDAC. 4) PTPN3 overexpression led to increased proliferation, migration and invasion in PDAC. 5) Tumor volume in mice injected with PTPN3-inhibited PDAC was significantly lower than that in control mice. The expressions of Ki-67 and VEGF in PTPN3-inhibited PDAC were lower than those in control. 6) Signaling from PTPN3 was through PI3K and MAPK signaling pathways. 7) When intensity of PTPN3 expression was divided into 2 groups; weak and strong, intensity of PTPN3 expression was positively correlated with tumor size (T factor) and clinical staging.

Conclusions

These results suggest that PTPN3 contributes to the induction of malignant phenotypes of pancreatic cancer and that PTPN3 could be a new effective and specific therapeutic target for PC.

Legal entity responsible for the study

Kyushu University Hospital, Cancer Therapy and Research.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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2020P - A Novel bispecific BCMAxCD3 T cell-engaging antibody that treat multiple myeloma (MM) with minimal cytokine secretion (ID 3920)

Presentation Number
2020P
Lecture Time
12:00 - 12:00
Speakers
  • Zhenyu Li (Linyi, China)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

B-cell maturation antigen (BCMA) is a potential therapeutic target in treatment of patients with MM. BCMA is widely expressed at the surface of myeloma cells and induces proliferative signals. BCMA-directed BCMA antibody conjugated with toxin has recently demonstrated outstanding anti-tumor responses and acceptable tolerability in MM treatment, allowing patients to achieve deeper responses with minimally added toxicity.

Methods

We used ELISA and FACS to verify AP163 can be combined with CD3/BCMA protein on the cell surface. We used the reporter gene method to verify that it induced T cell activation and target cell killing. In vivo, efficacy was studied in NCI-H929 and Raji xenograft models in female NPG mice. NPG mice were injected subcutaneously into the right dorsal flank with NCI-H929/Raji cells and human PBMC. Then the mice were treated with AP163 and tumor volume was measured twice/week. Cynomolgus monkeys were mainly used for toxicity testing. Intravenous administration is consistent with clinical administration. Its main toxicological target is the increase of cytokines and a series of adverse reactions.

Results

we constructed AP163 which binds to BCMA-expressing cell lines. AP163 induced both types of cell crosslinking, followed by T cell activation, cytokine production, proliferation and redirected target cell killing, with EC50 values in the picomolar range. The affinities of AP163 with human and cynomolgus CD3 are comparable. In vitro, AP163 only induced minimal cytokine release in the presence of BCMA target. When administered to mice bearing human MM xenografts and human T cells, AP163 eradicated subcutaneous tumors in two xenograft models tested and significantly delayed tumor growth at doses as low as 0.04mg/kg. AP163 displayed a 9-h half-life in cynomolgus and was well tolerated in non-human primates, even at high doses up to 5 mg/kg.

Conclusions

AP163 clears tumor cells in a BCMA+ and T-cell dependent manner in vitro and in vivo. Our preclinical data suggest that AP163 induces less cytokine secretion than a conventional bispecific in vitro, with reduction of tumor cell in vivo. AP163 is highly efficacious, safe and convenient for the treatment of patients with MM.

Legal entity responsible for the study

National Engineering Laboratory of High Level Expression In Mammalian Cells.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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2021P - Quantitative spatial profiling of lymphocyte-activation gene 3 (LAG-3)/major histocompatibility complex class II (MHC II) interaction in gastric and urothelial tumors (ID 2691)

Presentation Number
2021P
Lecture Time
12:00 - 12:00
Speakers
  • Cyrus V. Hedvat (Princeton, United States of America)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

LAG-3, expressed on exhausted T cells, negatively regulates effector T-cell activation and may promote regulatory T-cell activity. MHC II, one of the ligands for LAG-3, is expressed by antigen-presenting cells and is heterogeneously upregulated on tumor cells in a variety of cancers. The LAG-3/MHC II interaction may activate LAG-3 and inhibit antitumor immunity. We performed digital spatial analysis to define the geographic association of LAG-3+ tumor-infiltrating lymphocytes (TILs) with individual MHC II+ and MHC II− tumor cells.

Methods

Bladder and gastric tumor samples (n = 20 each) of varying MHC II+ tumor expression (0–100%) were serially sectioned and stained by IHC for LAG-3, MHC II (human leukocyte antigen-DP, -DQ, and -DR), and Pan-cytokeratin. Slides were scanned via an Aperio AT2 scanner using a 20× objective and whole slide images were digitally aligned and analyzed via HALO software. LAG-3 engagement scores for the density of LAG-3+ TILs (LAG-3-D) and the proportion in close proximity (within 30 µm) to tumor cells (LAG-3-P) were computed for each sample using R software.

Results

MHC II was expressed by at least 1% of tumor cells in 55% of bladder and 70% of gastric samples. LAG-3-D and LAG-3-P within an individual tumor were significantly greater when associated with MHC II+ tumor cells (median [interquartile range] LAG-3-D = 6.53 [1.76, 24.9] cells/mm2; LAG-3-P = 46.7 [30.1, 70.4] % engaged) compared to MHC II− tumor cells (LAG-3-D = 0.616 [0.213, 2.38] cells/mm2 [P < 0.001]; LAG-3-P = 17.5 [6.09, 30.1] % engaged [P < 0.001]). Furthermore, we found significantly lower LAG-3/MHC II− tumor engagement by LAG-3-P in gastric compared to urothelial tumors (P < 0.001).

Conclusions

Digital spatial analysis of tumor cells and TILs in the tumor microenvironment is feasible without multiplex assays and can capture cell–cell relationships in tumors with heterogeneous MHC II staining. These data suggest preferential localization of LAG-3-expressing TILs to MHC II+ tumor cells within a proximity that may allow engagement and activation of LAG-3 and help define the importance of spatial analysis in predictive biomarker development for immunotherapy.

Editorial acknowledgement

Editorial assistance was provided by Kathryn Woods, PhD, of Complete HealthVizion, funded by Bristol-Myers Squibb.

Legal entity responsible for the study

Bristol-Myers Squibb.

Funding

Bristol-Myers Squibb.

Disclosure

C.V. Hedvat: Shareholder / Stockholder / Stock options: Bristol-Myers Squibb; Full / Part-time employment: Bristol-Myers Squibb. G. Lee: Shareholder / Stockholder / Stock options: Bristol-Myers Squibb; Full / Part-time employment: Bristol-Myers Squibb. V. Baxi: Full / Part-time employment: Bristol-Myers Squibb. K. Dziuba: Full / Part-time employment: Bristol-Myers Squibb. D. Locke: Shareholder / Stockholder / Stock options: Bristol-Myers Squibb; Full / Part-time employment: Bristol-Myers Squibb. B. Li: Shareholder / Stockholder / Stock options: Bristol-Myers Squibb; Full / Part-time employment: Bristol-Myers Squibb. R. Edwards: Shareholder / Stockholder / Stock options: Bristol-Myers Squibb; Full / Part-time employment: Bristol-Myers Squibb.

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2023P - Evaluating the prevalence of the expression of PD-L1 in NSCLC specimens with short-duration formalin fixation using IHC 22C3 pharmDx (ID 2182)

Presentation Number
2023P
Lecture Time
12:00 - 12:00
Speakers
  • Keiichi Ota (Fukuoka, Fukuoka, Japan)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Immune checkpoint inhibitors mediated by PD-1 and PD-L1 are promising treatments for various tumors. The PD-L1 expression in tumor cells was found to be correlated with the likelihood of a response to PD-1- or PD-L1-targeted therapy in non-small cell lung cancer (NSCLC). For PD-L1 immunohistochemistry (IHC), 22C3 pharmDx is the only companion diagnostic assay that can identify NSCLC patients suitable for pembrolizumab treatment. Specimens are scored and divided into 3 categories (<1%, 1-49%, ≥50%) based on the tumor proportion score (TPS). The PD-L1 prevalence in NSCLC patients screened for the KEYNOTE-024 trial was 31% with TPS <1%, 39% with TPS 1-49%, and 30% with TPS ≥50%. While specimens used for the IHC 22C3 assay are recommended to be formalin-fixed for 12-72 h, the specimens were fixed in 10% neutral-buffered formalin for less than 6 h in our institution. Thus, we retrospectively assessed the PD-L1 prevalence in NSCLC patients to determine whether the PD-L1 TPS differs according to the duration of formalin fixation.

Methods

We screened consecutive NSCLC patients who underwent tumor biopsy by bronchoscopy between January 2017 and June 2018 at National Hospital Organization Fukuokahigashi Medical Center. In the present study, we only included 70 patients whose tumors were formalin-fixed for less than 6 h and whose PD-L1 expression had been evaluated by IHC with antibodies to human PD-L1 (22C3 pharmDx).

Results

The PD-L1 prevalence in patients with NSCLC in our hospital was 26 (37%) with TPS<1%, 23 (33%) with TPS 1-49%, and 21 (30%) with TPS ≥50%, suggesting that small biopsy samples obtained by bronchoscopy that were formalin-fixed for <6 h are suitable for PD-L1 IHC. We also performed analyzed in 35 surgically-resected NSCLC specimens that had been formalin-fixed for 1-7 days by PD-L1 IHC. The PD-L1 prevalence was 20 (57%) for TPS < 1%, 9 (26%) for TPS 1-49%, and 6 (17%) for TPS ≥50%.

Conclusions

The PD-L1 prevalence in specimens with short-duration formalin fixation was consistent with the results of the KEYNOTE-024 trial. We will present our conclusion regarding these results, including the characteristics of patients and the PD-L1 prevalence in specimens fixed for 12-72 h in our institution, from July 2018 onward.

Legal entity responsible for the study

National Hospital Organization Fukuokahigashi Medical Center.

Funding

JSPS KAKENHI JP18K15927.

Disclosure

All authors have declared no conflicts of interest.

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2024P - [18F]-FDG PET/CT in predicting PD-L1 status in nasopharyngeal carcinoma (ID 5255)

Presentation Number
2024P
Lecture Time
12:00 - 12:00
Speakers
  • Liang Zhao (Xiamen, China)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

PD-1/PD-L1 blockades have shown promising antitumor activity in clinical trials of advanced nasopharyngeal carcinoma (NPC). Being the most wildly used molecular imaging in clinic, 18F-FDG PET/CT provides multiple information of tumor biology. In this study, we investigated the association of PD-L1 expression with 18F-FDG uptake and clinical features in patients with NPC.

Methods

84 patients were initially histopathologically diagnosed with NPC between December 2016 and March 2019. All tissue specimens and PET/CT images were collected before any treatment. The PD-L1 antibody we used in this investigation was SP263 (Ventana, USA). High PD-L1 expression in tumor cells (TC) or tumor-infiltrating immune cells (TIIC) was defined as ≥ 50% of corresponding cells with membranous staining.

Results

The values of tumor SUVmax and TLG in patients with positive TC PD-L1 expression were significantly higher than those of negative TC PD-L1 expression (SUVmax: 10.3±4.1 vs. 6.9±3.2; P < 0.001; TLG: 77.7±64.5 vs. 35.0±20.5; P < 0.001), while the SUVmax values and TLG in patients with positive TIIC PD-L1 expression is lower than those of negative TIIC PD-L1 expression (SUVmax: 7.0±3.4 vs. 9.7±4.1; P = 0.011; TLG: 29.1±14.4 vs. 71.3±61.2; P = 0.001). Univariate analysis showed that the expression of PD-L1 in TC was associated with tumor T stage (P = 0.044) but not with serum Epstein-Barr virus (EBV) load (P = 0.816). On the other hand, the expression of PD-L1 in TIIC was related to both T stage (P = 0.028) and serum EBV load (P = 0.003). In multivariate logistic regression analysis, PD-L1 expression in TC was positively associated with tumor SUVmax (P = 0.003) and TLG (P = 0.001), while PD-L1 expression in TIIC was negatively associated with SUVmax (P = 0.038) and serum EBV load (P = 0.025). Through the ROC curve, the SUVmax cut-off value of 6.7 and the TLG cut-off value of 41.3 can be used to predict the PD-L1 status in TC with an accuracy of 78.6% and 71.4%, while the SUVmax cut-off value of 7.2 can be used to predict the PD-L1 status in TIIC with an accuracy of 72.6%.

Conclusions

18F-FDG uptake with NPC lesions were positively correlated with PD-L1 expression in TC and negatively correlated with PD-L1 expression in TIIC. 18F-FDG PET / CT imaging may be useful for predicting the PD-L1 status in patients with NPC.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

2025P - Expression of PD-L1 in Chinese patients with common cancers (ID 4910)

Presentation Number
2025P
Lecture Time
12:00 - 12:00
Speakers
  • Min Zheng (Guilin, China)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

PD-L1 immunohistochemistry (IHC) data in China was less reported. We systematically investigated PD-L1 IHC data of Chinese patients generated by Origimed.

Methods

PD-L1 was stained using IHC on cancer samples from 2060 Chinese patients in Origimed since 2017. Written informed consent was obtained from each patient. IHC staining was performed on FFPE tissue sections using anti-PD-L1 antibody 28-8. Tumor Proportion Score (TPS) was applied on all the samples. We investigated PD-L1 TPS in lung adenocarcinoma (n = 893), lung squamous carcinoma (n = 172), liver cancer (n = 404), esophageal cancer (n = 186), colorectal cancer (n = 166), pancreatic cancer (n = 136) and gastric cancer (n = 103). We then further applied PD-L1 combined positive score (CPS) on the 103 gastric cancer samples. All the slides were reviewed by the same senior pathologist. 95% confidence interval (CI) was obtained by bootstrap. Information entropy (Shannon’s formula) was measured in natural units.

Results

The highest proportion of PD-L1 TPS > = 1% was observed in lung squamous carcinoma (49%; 95% CI: [43%, 59%]), followed by lung adenocarcinoma (25%; 95% CI: [22%, 28%]), liver cancer (14%; 95% CI: [11%, 18%]), esophageal cancer (12%; 95% CI: [8.1%, 17%]), pancreatic cancer (11%; 95% CI: [5.9%, 16%]), gastric cancer (7%; 95% CI: [1.9%, 12%]) and colorectal cancer (4%; 95% CI: [1.2%, 6.6%]). The proportion of PD-L1 CPS > = 1 for gastric cancer was 17% (95% CI: [9.7%, 24%]). In gastric cancer, information entropy of TPS and CPS was 0.40 and 0.78, respectively.

Conclusions

The proportion of PD-L1 TPS > = 1% in Chinese lung squamous cell carcinoma (49%) was much higher than that in lung adenocarcinoma (25%). PD-L1 CPS contained more information than TPS in gastric cancer. Thus, we recommend applying CPS to gastric cancer in China.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

X. Pan: Full / Part-time employment: Origimed. Y. Dai: Full / Part-time employment: Origimed. D. Chen: Full / Part-time employment: Origimed. K. Wang: Full / Part-time employment: Origimed. X. Dong: Full / Part-time employment: Origimed. All other authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

2026P - The clearance of EGF by tumor-associated macrophages is suppressed by chemotherapeutic agent cisplatin (ID 4227)

Presentation Number
2026P
Lecture Time
12:00 - 12:00
Speakers
  • Irina V. Larionova (Tomsk, Russian Federation)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Tumor-associated macrophages (TAMs) are a major component of innate immunity supporting primary tumor growth and metastasis. Chemotherapy is one of the main treatment strategies for solid tumors, while chemoresistance is the major limitation of the chemotherapy for patients with various types of cancer. A number of evidence indicate that TAMs can accumulate in tumors after chemotherapy and contribute to chemoresistance.

Methods

For the endocytic uptake of EGF we used flow cytometry analysis. Confocal microscopy was used for the analysis of stabilin-1-mediated internalization and endocytic trafficking of EGF in CHO cells and in modeled TAMs differentiated in the presence of conditioned supernatants of breast cancer (MCF-7) and colorectal cancer (Colo206F) cell lines. We performed next-generation sequencing of RNA samples obtained from our modeled TAMs. Validation of sequencing data by real-time PCR was performed for selected genes implicated in the endocytic uptake: DNM3, STX8, DENND1A and EHD1.

Results

For the first time we demonstrated that stabilin-1 ectopically expressed in CHO cells mediates endocytic uptake of EGF, key growth factor stimulating progression of breast and colorectal cancer. In the model of primary human TAMs, we have demonstrated that cisplatin decreases stabilin-1-mediated internalization and endocytic trafficking of EGF, without affecting gene expression of scavenger receptor stabilin-1. Molecular mechanisms of cisplatin effect on TAMs were uncovered using high throughput RNA sequencing. Gene set enrichment analysis identified that cisplatin contributes to defects in endocyting machinery reducing membrane biogenesis and vesicular transport. Significant suppression of DNM3, STX8, DENND1A and EHD1 genes expression by cisplatin was confirmed by RT-PCR.

Conclusions

We suggested that suppression of receptor-mediated clearance of tumor-supportive factors, such as EGF, by chemotherapeutic drugs may potentially lead to the tumor progression and/or relapse as a mechanism of macrophage-mediated chemoresistance. This study was supported by grand RSF №19-15-00151.

Legal entity responsible for the study

National Research Tomsk State University.

Funding

RSF N19-15-00151.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

2027P - VHIO-300 and a thousand and one nights: A tale of precision medicine (ID 5222)

Presentation Number
2027P
Lecture Time
12:00 - 12:00
Speakers
  • Ginevra Caratù (Barcelona, Spain)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Targeted hotspot cancer NGS panels are becoming an integral part of clinical practice for detection of clinically relevant mutations. Tumor mutation burden (TMB) has arisen as a biomarker in the immunotherapy field, challenging established hotspot panels. So far, testing has been steep, limited to whole exome sequencing (WES) or external commercial NGS assays, neither of which are feasible strategies for future local testing. Clinical routine implementation requires development and validation of new, comprehensive tests, able to deal with day-to-day reality in testing labs, such as input DNA quality and amount limitations or turnaround-time restraints.

Methods

We designed a custom NGS assay, VHIO-300, to detect mutations, copy number alterations (CNA), gene fusions, loss-of-heterozygosity and TMB scoring from tumor samples. VHIO-300 is a capture enrichment of over 430 cancer genes, covering enough sequence to allow TMB calculation (≈1.3 Mb), as well as 2 Mb of non-coding genomic regions. Here, we report the analytical and clinical validation procedures. Analytical sensitivity was conducted on positive control DNAs derived from 5 ATCC cell lines comprising 40 SNVs and indels. Clinical samples with WES data were used to determine specificity, sensitivity and robustness. VHIO-300 performance for TMB calling was tested using 50 FFPE-derived tumor specimens compared to scores provided from both WES and Foundation One test.

Results

Panel design was tuned to improve detection of capture-sensitive variants such as indels, that affect relevant genes in cancer like EGFR, BRCA1/2 or KIT, improving analytical sensitivity by two-fold. The VHIO-300 panel achieved 92.5% clinical sensitivity and 99.9% clinical specificity for SNVs and indel calling compared to WES data. CNA achieved 85% sensitivity and 99.6% specificity compared to array comparative genomic hybridization. Several in-silico TMB calculation algorithms were tested with WES data, the most accurate included both synonymous and non-synonymous calls (r2=0.94).

Conclusions

We provide a complete analytical validation of VHIO-300 panel that makes it a convenient choice for clinical laboratories where a comprehensive genome profiling of FFPE tumor samples is required.

Legal entity responsible for the study

Vall d’Hebron Institute of Oncology.

Funding

Bristol-Meyers Squibb.

Disclosure

A. Oaknin: Advisory / Consultancy, Travel / Accommodation / Expenses: AstraZeneca; Advisory / Consultancy, Travel / Accommodation / Expenses: PharmaMar; Advisory / Consultancy: Clovis Oncology; Advisory / Consultancy: Inmunogen ; Advisory / Consultancy: Genmab ; Travel / Accommodation / Expenses: Roche. M. Alsina Maqueda: Honoraria (self), Advisory / Consultancy: Bristol-Myers Squibb; Advisory / Consultancy: MSD; Advisory / Consultancy: Servier; Research grant / Funding (institution): Merck. J. Capdevila: Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self): Bayer; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony: Eisai; Honoraria (self), Advisory / Consultancy: AstraZeneca; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony: Novartis; Honoraria (institution), Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self): Ipsen; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony: Merk; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony: Sanofi; Honoraria (self), Advisory / Consultancy, Speaker Bureau / Expert testimony: Amgen. E. Elez Fernández: Research grant / Funding (institution): Novartis; Honoraria (self), Research grant / Funding (institution): Roche; Research grant / Funding (institution): Bayer; Honoraria (self), Research grant / Funding (self): Merck Sorono; Honoraria (self), Research grant / Funding (self): Sanofi. T. Macarulla Mercade: Advisory / Consultancy: Roche; Advisory / Consultancy: Bayer; Advisory / Consultancy: Sanofi; Advisory / Consultancy: Servier. J. Carles: Advisory / Consultancy, Research grant / Funding (institution): Bristol-Myers Squibb; Advisory / Consultancy, Speaker Bureau / Expert testimony: Bayer; Advisory / Consultancy, Speaker Bureau / Expert testimony: Johnson & Johnson ; Advisory / Consultancy: MSD Oncology; Advisory / Consultancy: Roche; Advisory / Consultancy, Speaker Bureau / Expert testimony: Astellas Pharma; Advisory / Consultancy: AstraZeneca. C. Saura: Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Roche; Advisory / Consultancy: Novartis; Advisory / Consultancy: Pfizer; Advisory / Consultancy: Puma; Advisory / Consultancy: Sanofi. J.A. Rodon: Research grant / Funding (institution): Bayer; Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy: Roche Pharmaceuticals ; Advisory / Consultancy: Eli Lilly; Advisory / Consultancy: Orion Pharmaceuticals; Advisory / Consultancy: Servier Pharmaceuticals; Advisory / Consultancy: Peptomyc. R. Dienstmann: Advisory / Consultancy: Roche; Research grant / Funding (self): merck. P.G. Nuciforo: Advisory / Consultancy: Bayer; Advisory / Consultancy: Novartis; Advisory / Consultancy: MSD. E. Garralda: Speaker Bureau / Expert testimony: Bristol-Myers Squibb; Leadership role: ESMO Women for Oncology; Non-remunerated activity/ies: ESMO. E. Felip: Advisory / Consultancy, Speaker Bureau / Expert testimony: Bristol-Myers Squibb; Advisory / Consultancy, Speaker Bureau / Expert testimony: AstraZeneca; Advisory / Consultancy, Speaker Bureau / Expert testimony: Boehringer Ingelheim; Advisory / Consultancy, Speaker Bureau / Expert testimony: Merck KGaA; Advisory / Consultancy, Speaker Bureau / Expert testimony: Merck Sharp & Dohme; Advisory / Consultancy, Speaker Bureau / Expert testimony: Novartis; Advisory / Consultancy, Speaker Bureau / Expert testimony: Pfizer; Advisory / Consultancy, Speaker Bureau / Expert testimony: Roche. J. Tabernero: Advisory / Consultancy: Foundation Medicine; Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Bayer; Advisory / Consultancy: Genentech; Advisory / Consultancy: Chugai; Advisory / Consultancy: Merck Serono; Advisory / Consultancy: Novartis; Advisory / Consultancy: Hoffmann-La Roche Ltd; Advisory / Consultancy: Pfizer; Advisory / Consultancy: Servier; Advisory / Consultancy: Symphogen; Advisory / Consultancy: Biocartis; Advisory / Consultancy: Roche Diagnostics; Advisory / Consultancy: Boehringer Ingelheim. A. Vivancos: Advisory / Consultancy, Research grant / Funding (institution): Bristol-Myers Squibb; Advisory / Consultancy, Research grant / Funding (institution): Sysmex; Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy, Research grant / Funding (institution): Merck; Advisory / Consultancy: Roche; Advisory / Consultancy: Guardant Health; Research grant / Funding (institution): Debio; Research grant / Funding (institution): Cellestia Biotech. All other authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

2028P - Matched whole-genome sequencing and whole-exome sequencing with circulating tumor DNA (ctDNA) analysis are complementary modalities in clinical practice (ID 5668)

Presentation Number
2028P
Lecture Time
12:00 - 12:00
Speakers
  • Robin Imperial (Kansas City, United States of America)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Concordance between whole genome sequencing (WGS)/whole exome sequencing (WES) and circulating tumor DNA (ctDNA) is not known. Therefore, we studied concordance between matched WGS/WES and ctDNA in various solid tumors. In addition, we carried out an analysis of hotspot and actionable alterations in our data.

Methods

Retrospective analysis of 64 stage III/IV solid tumors (Gastrointestinal n = 20 Breast n = 12 Lung n = 19 Other n = 13) who underwent matched WGS/WES and ctDNA (77 genes) by commercially available platforms. Concordance was determined at patient and gene levels.

Results

Patient concordance between tissue and ctDNA results was 58% (n = 37). Concordance was higher when patients received systemic chemotherapy prior to tissue NGS and ctDNA compared to no chemotherapy (78% n = 21 Vs 43% n = 12, respectively p = 0.01) and positively correlated increasing tumor mutation burden. There was no significant difference in concordance when time interval between tissue NGS and ctDNA analysis was evaluated (55% n = 24 Vs 65% n = 13, <90 days Vs > =90 days respectively p = 0.20). Similarly, no statistical difference in concordance was found when tissue NGS was done on primary or metastatic sites (48% n = 12 Vs 64% n = 25, respectively p = 0.20). TMB positively correlated with the number of ctDNA mutations only in those who received chemotherapy (r(25)=0.74 p < 0.0001) but did not correlate in chemotherapy naïve (r(27)=0.11 p = 0.60). Gene level concordance between tissue and ctDNA was 21% across all tumor types. The most common concordant genes with somatic alterations across all tumors were TP53, KRAS, and PIK3CA. 121 hotspots were identified in tumor NGS and 63 were in ctDNA. Hotspot concordance was 34%. Tissue NGS identified 68 targetable alterations in 31 patients and ctDNA analysis identified 87 in 34 patients. 48 of these were concordant and 59 were discordant (tissue 20, ctDNA 39).

Conclusions

Our analysis noted significant discordance between WGS/WES and ctDNA analysis. TMB and prior chemotherapy may affect concordance between NGS and ctDNA. Tissue NGS and ctDNA are complementary modalities and incorporating ctDNA with tissue NGS can increase the likelihood of capturing additional targetable alterations.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

2029P - Exploring the role of genes associated with familial cancer syndromes on the development of multiple primary tumors (ID 5772)

Presentation Number
2029P
Lecture Time
12:00 - 12:00
Speakers
  • Atanaska V. Mitkova (Sofia, Bulgaria)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Over the past decade an increase in the incidence and severity of multiple primary neoplasias has been observed. The main causes of multiple primary tumors (MPT) are genetic factors, environmental factors, infections with oncogenic viruses, etc. The aim of the current study was to explore the role of genes associated with familial cancers in MPT development.

Methods

The study included 12 MPT patients, of which 6 women with metahronous/synchronous breast and ovarian tumors; and 6 men who developed primary tumors with different localisation: bladder/bile ducts; rectum/pancreas; prostate/colon; prostate/sigma; sigma/stomach; palate/larynx+hypopharynx/tongue, respectively. Seventy five (9/12) of the patients had family history of cancer and 50% (6/12) early onset (<50y). Mutational screening was performed by NGS of a panel of 94 tumor-associated genes on MiSeq platform (Illumina).

Results

A total of 82 variants were found of which 18.3% were evaluated as clinically significant. Among selected variants 33.3% (5/15) were pathogenic, 13.3% (2/15) likely pathogenic and 53.3% (8/15) variants of uncertain significance (VUSs). Pathogenic/likely pathogenic variants were detected in the genes BRCA1 (20%), MLH1 (13.3%), BRCA2 (6.7%) and CDH1 (6.7%) while VUSs in PMS1, GPC3, DIS3L2, PRF1, STK11, DICER1, RET, and MSH6, respectively.

Conclusions

Overall, the genetic cause of MPT was found in 58.3% (7/12) of the patients. Further research is needed to evaluate the functional effect of all VUSs.

Editorial acknowledgement

Grants D-71/03.05.2018/MU-Sofia; KP-06-OPR03/1719.12.2018/NSF; DUNK01-2/2009/NSF, MES Bulgaria.

Legal entity responsible for the study

Medical University of Sofia.

Funding

Medical University Sofia; National Science Fund, Ministry of Education and Science, Bulgaria. Grants D-71/03.05.2018/MU-Sofia; KP-06-OPR03/1719.12.2018/NSF; DUNK01-2/2009/NSF, MES Bulgaria.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

2030P - Doxorubicin resistance: early and advanced tumors can use two different strategies based on initial and profound abnormalities in microRNA expression signature (ID 4784)

Presentation Number
2030P
Lecture Time
12:00 - 12:00
Speakers
  • Volodymyr Halytskiy (Kiev, Ukraine)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Doxorubicin is an anthracycline antibiotic that acts as a DNA intercalating agent, inhibiting the topoisomerase II and inducing the apoptotic cell death, mainly due to the accumulation of double-strand DNA breaks (DSB). This research aims to identify in what way the abnormalities in microRNA (miRNA) expression profile in cancer cells can confer them the resistance to doxorubicin.

Methods

MiRNA targets within gene transcripts were predicted in silico using the TargetScan software.

Results

Binding sites for miRNAs miR-21, miR-96, miR-183 and miR-365, which are usually upregulated in cancer cells, were revealed in transcript of TOP2A gene encoding topoisomerase II-alpha. Transcripts of proapoptotic genes BID, BCL2L11 (BIM), BMF, BAX, BAK1, PMAIP1 (NOXA), and BBC3 (PUMA) as well as transcripts of tumor suppressor genes TP53 and PTEN carry targets for at least one of hyperexpressed miRNAs miR-19, miR-21, miR-23, miR-27, miR-29, miR-155, miR-181, miR-221/222 and miR-375. Downregulation of anti-onco-miRNAs let-7, miR-22, miR-34, miR-101, miR-125, miR-140, miR-143, miR-199, miR-200, miR-203, miR-204 and miR-205 allows overexpression of antiapoptotic genes BCL2, BCL2L1 (Bcl-XL), MCL1 and AKT1. In the same way, downregulation of the anti-onco-miRNAs can lead to overexpression of ABCA1/4/12, ABCB1 (MDR1), ABCB6, ABCC1 (MRP1) and ABCC3/5/8/11 genes encoding the ATP binding cassette (ABC) transporters. Moreover, multiple targets for the both up- and downregulated miRNAs were found in transcripts of XRCC5/6, PRKDC, LIG4, DCLRE1C, NHEJ1 (XLF), RAD50/51/51B/51D/52/54B/54L, MRE11A, NBN (NBS1), GEN1, ATM and ATR genes encoding the key elements of non-homologous end joining and homologous recombination pathways.

Conclusions

Obviously, in case of doxorubicin administration, strategy of early tumors consists in strengthening of miR-21 expression with the purpose to silence TOP2A gene and escape from immediate apoptotic death. Advanced tumors with more profound abnormalities in miRNA signature overexpress antiapoptotic genes as well as genes responsible for DSB repair and drug efflux and, therefore, can do without the TOP2A silencing.

Legal entity responsible for the study

Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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2031P - From tumor transcriptomes to underlying cell type proportions to better predict prognosis and response to treatments (ID 3456)

Presentation Number
2031P
Lecture Time
12:00 - 12:00
Speakers
  • Yuna Blum (Paris, France)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Tumor pathologists classify tumors according to cell-level and tissue-level criteria, using molecular markers in addition to morphological patterns, as reported in WHO tumor classifications. Their work describes to some extent both inter-tumor and intra-tumor heterogeneity, but it is a tedious task with potential reproducibility issues. The molecular subtyping of tumors represents a sometimes parallel and sometimes convergent effort to describe the heterogeneity of tumors. It is automated, which attenuates the limitations of pathological scoring mentioned above, but so far it is poorly adapted to describe intra-tumor heterogeneity.

Methods

We propose a novel method, WISP (Weighted In Silico Pathology), which finely measures intra-tumor heterogeneity by automatically estimating the proportions of cell types present in a bulk tumor sample, these cell types being predefined based on histological or high-throughput molecular criterions.

Results

We illustrate the relevance of our approach in several tumor types including lung cancer, glioblastoma and pancreatic cancer. We show that the cell types that describe tumors for a given cancer type can fairly well recapitulate existing molecular classifications and are very consistent with an independent pathological scoring. We show that our method offers a more standardized and finer-grained solution for describing tumor heterogeneity than either pathological scoring or molecular subtyping. More importantly, we show that the proportion of certain cell types is strongly associated to prognosis and drug response.

Conclusions

This study provides a framework for standardized molecular pathology and fully reshapes the way in which we think about pathology or molecular subtyping. We believe that our results are a proof of concept demonstrating the importance of considering cell types intra-tumor proportions for personalized clinical care.

Legal entity responsible for the study

The authors.

Funding

CIT Program - Ligue Contre le Cancer.

Disclosure

All authors have declared no conflicts of interest.

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2032P - Optimization of automated germline DNA extraction from non-tumoral formalin-fixed paraffin embedded (FFPE) tissues (ID 4976)

Presentation Number
2032P
Lecture Time
12:00 - 12:00
Speakers
  • Omar Youssef (Helsinki, Finland)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Recent studies have identified a complex but definitive role of germline DNA in cancer predisposition. Archived formalin-fixed paraffin-embedded (FFPE) samples from cancer patients are vital for several molecular and genomic studies. For retrospective studies investigating gene mutations, loss of heterozygosity and copy number changes, non-tumoral FFPE samples can be used as a source of germline DNA. The key challenge with FFPE DNA purification is usually due to the fixation process, which causes cross-linking and fragmentation of FFPE DNA. In spite of the development of several techniques for FFPE DNA extraction, automated extraction can provide efficient DNA purification with the least hands-on and the least contamination. We compared two different automated approaches with special focus on DNA yield and quality using the DNA integrity number (DIN) value.

Methods

The study was carried out on 48 non-tumoral FFPE samples from cancer patients. Two FFPE pretreatment methods were used simultaneously: GeneRead DNA FFPE Kit (removes cytosine deamination artifacts from FFPE) and QIAsymphony DSP DNA Kit. After the initial pretreatment, the extraction step was performed using the automated QIAsymphony SP instrument for both methods. Finally, the purified DNA was assessed using TapeStation for measuring the concentration and DIN value.

Results

The median DNA concentration using the GeneRead method was 13.85 ng/ul (1.13-111 ng/ul), while for QIAsymphony DSP the median DNA was 5.3 ng/ul (1.07-156 ng/ul). Of the total 48 FFPE samples, 40 purified by GeneRead and 29 purified by QIAsymphony DSP have DNA concentrations above the functional quantitative range of DIN. The median DIN value was 3.3 and 4.1 for GeneRead and QIAsymphony DSP, respectively.

Conclusions

This study demonstrates that the FFPE pretreatment step has an effect on automated DNA extraction. Highly efficient extraction of FFPE DNA based solely on quantity can be misleading as the DNA may be highly fragmented. The removal of cytosine deamination artifacts from FFPE samples can enhance DNA purification with less degradation. This may also have a crucial influence on the downstream molecular approaches such as DNA sequencing.

Legal entity responsible for the study

The author.

Funding

Has not received any funding.

Disclosure

The author has declared no conflicts of interest.

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Poster Display session 1 Poster Display session

2033P - HIV, HBV and HCV screening practices in oncology: A cross-sectional interregional survey (ID 4242)

Presentation Number
2033P
Lecture Time
12:00 - 12:00
Speakers
  • Isabelle Poizot-Martin (Marseille, France)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

HIV screening is recommended at time of cancer diagnosis. Moreover, HBV screening, is recommended during immunotherapy due to its risk of reactivation. Furthermore, HCV screening, before using immunotherapy is justified by the prevalence of dysimmune disorders during HCV infection. The main objective of this survey was to evaluate screening practices for HIV, HBV and HCV in cancer patients.

Methods

A cross-sectional study carried- out across 16 French regions between 25/10/2018 and 31/12/2018, evaluating performance of HIV, HBV and HCV systematic screening at time of cancer diagnosis and/or before immunotherapy. An electronic questionnaire was sent to participants in multidisciplinary concertation meetings.

Results

The responses of 290 participants (including 101 surgeons, 61 organ specialists, 50 oncologists, 30 radiotherapists, 21 hematologists and 13 general practitioners) were analyzed. Of the 16 regions targeted, 8 of them are represented by 160 participants (55%). A systematic screening for HIV, HBV and HCV at time of cancer diagnosis was reported by 59 (20%), 66 (23%) and 63 (22%) respondents, respectively. A screening on a case by case for HIV, HBV and HCV was reported by 113, 103 and 102 respondents, respectively while 117 respondents stated to never prescribe HIV testing (40%), 121, HBV testing (42%) and 125 HCV testing (43%). Before immunotherapy, 122 respondents stated that they were not concerned and 89 routinely screened for HIV (31%), 97 for HBV (33%) and 94 for HCV (32%). A screening on a case by case for HIV, HBV, and HCV was reported by 38, 36 and 34 respondents, respectively while 40 respondents stated to never prescribe HIV testing (14%), 34, HBV testing (12%) and 39, HCV testing (13%).

Conclusions

This survey highlights the insufficiency of HIV, HBV and HCV systematic screening at time of cancer diagnosis and/or before immunotherapy. There is a need to raise awareness about the importance of systematic screening in health care providers.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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2034P - Genetic landscape of KEAP1 and NFE2L2 mutated cancers from the AACR GENIE database (ID 1267)

Presentation Number
2034P
Lecture Time
12:00 - 12:00
Speakers
  • Mark Zaki (Boston, United States of America)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

NFE2L2 encodes Nrf2, which is activated at times of cellular stress to neutralize reactive oxygen species. KEAP1 acts as a negative regulator of Nrf2. Nrf2 has been implicated in tumorigenesis of many human cancers via the mTOR pathway. The prevalence and genetic landscape of NFE2L2 and KEAP1 mutants remains poorly characterized.

Methods

We extracted data from AACR GENIE 5.0 database, including only subjects with complete demographic, mutational, and CNA data (n = 54,237). We quantified the prevalence of NFE2L2 and KEAP1 mutations, determined location frequency by gene subdomain, copy number alterations, and most frequent co-mutated genes.

Results

We identified 720 NFE2L2 mutations and 1422 KEAP1 mutations. NFE2L2-mutated samples showed a difference in age (61.6 vs 55.9 years, p<.01) but had no gender difference (48.9% vs 49.1% female, p = 0.8). NFE2L2 was most commonly mutated in head and neck (6.0%), anal (5.1%), and endometrial (4.5%) cancer samples. 391 (54.3%) mutations were seen in the Neh2 binding domain. TP53(51%), KMT2D(29.4%), and PIK3CA(26.4%) were most commonly co-mutated. KEAP1-mutated samples showed no difference in age (58.3 vs 59.0 years, p = 0.75) nor gender (40.2% vs 48.4% female, p = 0.31). KEAP1 mutations were most common in non-small cell lung (9.4%), unknown primary (6.9%), and small cell lung (4.0%) cancers. 696 (48.9%) mutations were seen in the KELCH binding domain. TP53 (53.8%), STK11(35.0%), and KRAS (30.9%) were the most common co-mutations. Based on prevalence identified in this study and publicly available epidemiological data, we estimate an annual incidence of nearly 60,000 cancers with NFE2L2 or KEAP1 mutations in the United States alone.

Conclusions

NFE2L2 and KEAP1 mutations in their respective binding domains were found in a wide variety of cancer types. Based on estimated incidence of these mutations, mTOR inhibitors may play an important role in treating a signficant number of cancers.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

2035P - β-arrestin1 is involved in the Ras-induced malignant transformation (ID 878)

Presentation Number
2035P
Lecture Time
12:00 - 12:00
Speakers
  • Takashi Shibano (Stockholm, Sweden)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

IGF-1R, a member of the tyrosine receptor family, is well documented in various experimental models to be an important factor in cell transformation, tumor progression, protection from apoptosis and metastasis. In the absence of IGF-1R, most oncogenes are unable to induce malignant transformation, suggesting that some signaling pathways activated by IGF-1R are essential for transformation. We have recently demonstrated that β-arrestin1 (β-arr1), a key regulator of G Protein Coupled Receptor (GPCR), is involved in IGF-1R signaling. Upon ligand binding, β-arr1 brings Mdm2, an E3 ubiquitin ligase, to the IGF-1R resulting in receptor ubiquitination. β-arr1 also functions as a scaffold to activate the MAPK pathway like GPCR. This study aims to investigate whether the β-arr1 mediated signal of IGF-1R is necessary for malignant transformation.

Methods

Mouse embryonic fibroblasts (MEFs) and a bladder cancer cell line, T24 cells were used as target cells. β-arr1 knockdown was performed by siRNA transfection. Activation of MAPK and PI-3K pathways was analysed by western blot. Cell proliferation was evaluated by PrestoBlue assay. Cellular transformation was estimated by colony formation assay in soft agar.

Results

To investigate whether β-arr1 is important for transformation, we stably transfected WT MEF and β-arr1 deficient MEFs with some oncogenes such as HRasV12, PvMT, and vSrc. In the absence of β-arr1, HRasV12 was not able to induce malignant transformation. Similar sensitivity to absence of β-arrestin1 was not observed with PyMT or vSrc. β-arr1 regulates IGF-1-induced activation of the MAPK and PI-3K pathways, both pathways being less active in the absence of β-arr1. These data suggested that β-arr1 is involved in Ras-mediated malignant transformation. In T24 cells with the same mutation in HRas, β-arr1 knockdown attenuate IGF-1 induced MAPK and PI-3K pathways like MEFs. Consistent with less activation of the downstream, IGF-1 induced cell proliferation and colony formation are decreased in β-arr1 knockdown cells, indicating that β-arr1 is required for malignant phenotypes in T24 cells.

Conclusions

These data suggest that β-arr1-dependent signaling by the IGF-1R regulates mechanisms involved in malignant transformation and progression of cancer cells.

Legal entity responsible for the study

Karolinska institutes, Department of Oncology-Pathology, Leonard Girnita’s group.

Funding

Swedish Research Council, Swedish Cancer Society, The Swedish Childhood Cancer Foundation.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

2036P - Incidence of second cancer among PLWHIV: A retrospective observational study of a series of 601 patients in the French CANCERVIH network (ID 4143)

Presentation Number
2036P
Lecture Time
12:00 - 12:00
Speakers
  • Jean-Philippe Spano (Paris, France)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Cancer incidence is increasing but cancer survival is improving. The life expectancy of PLWHIV also increases with improved HAART, so it is to be expected that PLWHIV are likely to develop a second primitive cancer. It is known that the risk of second cancer is slightly higher after a first cancer and depends on several parameters: the location of the first cancer (the risk is higher after H&N, lung cancer or Hodgkin’s lymphoma) and environmental risk factors such as tobacco or alcohol intake but few studies are available nowadays on this subject. PLWHIV have a high risk of cancer: what for a second cancer incidence?

Methods

The files of 601 patients presented at the multidisciplinary meeting of the CANCERVIH network were reviewed. CANCERVIH is a national "rare cancers" network, accredited by the French National Cancer Institute. For all these patients, the history of cancer, indicated by the referring physicians, was examined. History of cancer that was neither metastasis nor locoregional relapse was considered a first primitive cancer. Cancers developing in the same location were considered as first primitive cancers only if they were considered in complete response at least 5 years before and no relapse was detected in the meantime.

Results

Of the 601 patients reviewed, 72 (12%) had a history of at least one cancer. Fourteen (2,3%) patients had at least 2 previous cancers. For one it was his 7th cancer’s case.

2036P

First cancer (number of patients)Number of secondary cancer(s) (number of patients)
Kaposi (25)1 (23) 2 (2)
NHL (7)1 (5) 2 (2)
Anal canal (6)1 (2) 2 (3) 3 (1)
Prostate (6)1 (4) 2 (1) 6 (1)
Breast (5)1 (5)
Head and Neck (5)1 (4) 4 (1)
Hodgkin lymphoma (4)1 (4)
Kidney (4)1 (3) 2 (1)
Skin melanoma (3)1 (2) 2 (1)
Eye (2)1 (1) 3 (1)
Anal margin (1)1 (1)
Lung (1)1 (1)
Ovary (1)1 (1)
Large intestine (1)2 (1)
Liver (1)1 (1)

Conclusions

In this study, the initial locations at risk of second cancer seems to concern primarily AIDS defining cancer and among the non-AIDS defining cancers, anal cancer appeared the first one ahead breast and head and neck cancers, showing the importance of maintaining permanent immuno-virological control in these patients’ population: such results confirm the need to maintain and emphasize prevention and screening programs in this high cancer risk population including for those with a sustained undetectable HIV viral load and/or immune restoration.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

2037P - A challenging task – Identifying carcinoma of unknown primary (CUP) patients according to ESMO guidelines: The CUPISCO trial experience (ID 5145)

Presentation Number
2037P
Lecture Time
12:00 - 12:00
Speakers
  • Chantal Pauli (Zurich, Switzerland)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

The CUPISCO trial (NCT03498521) is an ongoing, phase II, randomised, multicentre study comparing molecularly-guided therapy with standard platinum-based chemotherapy in newly diagnosed poor-risk CUP patients.

Methods

Eligible patients have poor-risk adeno- or undifferentiated CUP as defined by ESMO 2015 guidelines and tissue for molecular sequencing. Local sites initiate the screening process with potentially eligible patients. Patients then undergo central Eligibility Review (ER), a cooperative effort between a central pathology laboratory, external referent oncologists and each site’s investigator and pathology laboratory to confirm the diagnosis. Patients with favourable prognostic subsets or with a strong suspicion of an existing primary site of origin based on immunohistochemistry (IHC) signature and clinical picture are excluded.

Results

As of 19 March 2019, 157 patients had been screened, of whom 91 (58%) failed screening. Three patients were successfully re-screened. Of the 88 patients who permanently failed screening, 23 were due to technical reasons (e.g. insufficient quality/quantity of tissue for sequencing), 20 for failure to meet inclusion/exclusion criteria not directly related to CUP diagnosis, and 14 for other reasons (e.g. declining health status). A set of 31 patients were not enrolled because the CUP diagnosis could not be confirmed at the IHC level, 19 of those after ER review. Central IHC review results included pathological signatures more typical of specific primary tumours (e.g. prostate cancer or melanoma), or marker combinations typically positive in favourable CUP subsets or rare tumour entities.

Conclusions

Experience with the CUPISCO study has highlighted challenges with standardised screening and diagnostic processes in an international clinical trial and the difficulties inherent in accurate diagnosis of poor-risk CUP. Confirming a CUP diagnosis for a clinical trial with multiple review checkpoints can result in many reasons for screen failures. By sharing this experience, we aim to foster understanding and to improve diagnostic algorithms for CUP.

Clinical trial identification

NCT03498521.

Editorial acknowledgement

Medical writing assistance was provided by Ian Leighton, PhD, Nspm Ltd, Meggen, Switzerland, and supported by F. Hoffmann-La Roche Ltd, Basel, Switzerland.

Legal entity responsible for the study

F. Hoffmann-La Roche Ltd.

Funding

F. Hoffmann-La Roche Ltd.

Disclosure

C. Pauli: Honoraria (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: Roche. T. Bochtler: Honoraria (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: Roche. L. Mileshkin: Travel / Accommodation / Expenses: Roche; Travel / Accommodation / Expenses: Beigene. G. Baciarello: Advisory / Consultancy, Travel / Accommodation / Expenses: Amgen; Advisory / Consultancy, Travel / Accommodation / Expenses: Janssen Oncology; Advisory / Consultancy, Travel / Accommodation / Expenses: Sanofi; Advisory / Consultancy, Travel / Accommodation / Expenses: Astellas-Pharma; Advisory / Consultancy: Roche; Travel / Accommodation / Expenses: AstraZeneca; Travel / Accommodation / Expenses: Ipsen. F. Losa: Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution), Travel / Accommodation / Expenses: Roche; Advisory / Consultancy, Research grant / Funding (institution): Amgen; Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Merck; Advisory / Consultancy, Speaker Bureau / Expert testimony: Sanofi; Advisory / Consultancy: Servier. J.S. Ross: Shareholder / Stockholder / Stock options, Full / Part-time employment: Foundation Medicine Inc. S. Yalcin: Honoraria (self): Roche; Honoraria (self): Sanofi; Honoraria (self): Amgen; Honoraria (self): Novartis; Honoraria (self): Lilly; Honoraria (self): MSD; Honoraria (self): Merck Serono. A. Beringer: Full / Part-time employment: F. Hoffmann-La Roche Ltd.. S. Foser: Full / Part-time employment: F. Hoffmann-La Roche Ltd. J. Scarato: Full / Part-time employment: F. Hoffmann-La Roche Ltd.. M. Mueller-Ohldach: Full / Part-time employment: Hoffmann-La Roche Ltd. H. Moch: Honoraria (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: Roche. A. Krämer: Honoraria (self), Research grant / Funding (institution), Travel / Accommodation / Expenses: Roche. All other authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

2038P - Incidence and outcome of chronic lymphocytic leukemia with deletion 17p: An Indian experience; challenges and opportunities (ID 1737)

Presentation Number
2038P
Lecture Time
12:00 - 12:00
Speakers
  • Ajay Gogia (New Delhi, India)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Deletion 17p (del17p) is a rare genomic aberration found in patients with CLL and its incidence is 5-9% in untreated patients and 30- 50% of relapsed and refractory cases. The presence of del 17p correlates with poor response to chemotherapy and unfavourable outcome. There is a paucity of data regarding incidence and outcome of CLL patients with del 17p from India.

Methods

This prospective study included consecutive treatmentnaïve and relapsed/refractory patients who were diagnosed with CLL and registered at the Department of Medical Oncology, Dr. BRAIRCH, All India Institute of Medical Sciences, New Delhi, between June 2013 and December 2018. Del 17p was assessed by FISH in peripheral blood samples.

Results

Total 220 patients (150 new and 70 relapse/refractory) recruited in this study, del 17 p was found in 18 (12%) patients in new cases and 18 (25.5%) in relapsed/refractory cases. The median age in 36 cases were 57 years (35-80) with male:female ratio was 6:1. As per clinical Rai stage:5 cases were in stage 0 & I, 10 cases were in stage II, 8 cases were in stage III and 13 cases were in stage IV. ZAP-70 was positive in 58%, CD 38 was positive in 45%, CD49d was positive in 66% and 80% of cases were IGVH unmutated. Out of 18 newly diagnosed cases, 11 patients received treatment [ 4 – Ibrutinib, 4 -Bendamustine + Rituximab (BR), 2- Chlorambucil and Prednisolone (CP),1 -Fludarabine, cyclophosphamide and rituximab (FCR)] while 7 patients were kept under observation. The overall response rate was 63.6% and complete response was 4 (36.36%). Out of 18 Relapse/refractory cases, 15 patients were required treatment (4- Ibrutinib, 4- BR, 5 -RCHOP/RCVP, 2 CP] with an overall response rate of 40% and CR rate of 12%. Eighteen patients died (11-disease progression, 4 infections and 3 -other reasons). Eight patients developed Richter’s transformation and died. The median progression free survival and overall survival was 14 months and 24 months respectively.

Conclusions

This is the first study on CLL with del17 p reported from India. Its incidence is 12 % in upfront cases and 25.5% in relapsed /refractory cases. Chemoimmunotherapy has dismal outcome and Ibrutinib was used in a limited number of patients because of financial constraints.

Legal entity responsible for the study

The authors.

Funding

Has not received any funding.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

2039P - Driving solo? Investigation into collaborating mutations in SDH-deficient neoplasia (ID 2596)

Presentation Number
2039P
Lecture Time
12:00 - 12:00
Speakers
  • Jonathan K. Killian (Cambridge, United States of America)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Succinate dehydrogenase (SDH) is a highly conserved component of energy production and metabolism. Genetic or epigenetic inactivation of SDH (dSDH) creates a state of intracellular pseudohypoxia that is uniquely oncogenic for subtypes of GIST, renal cell carcinoma (RCC), paraganglioma/pheochromocytoma (PGL/Pheo), and pituitary adenoma/carcinoma (PAC). dSHD stems from homozygous loss of one of four subunit encoding genes SDHA, SDHB, SDHC, or SDHD (aka, SDHx). dSDH is not targetable, and preclinical models are poorly viable. In the current study we analyze comprehensive genomic profiles (CGP) from a dSDH pan-tumor cohort for potential therapy-enabling non-SDHx genomic alterations and biomarkers (GA).

Methods

231,706 clinical grade CGP were searched for dSDH GIST, RCC, PGL/Pheo, and PAC. GA were determined on 1.1 Mb genome sequence encompassing up to 324 cancer genes. dSDH was further established by histopathology review and analysis of SDHx homozygous loss. Germline and zygosity status of SDHx variants were performed by SGZ algorithm. Non-SDHx GA were assessed for therapeutic actionability.

Results

82 SDH-deficient neoplasms were analyzed (Table). 0 of 38 GIST had highly actionable GA. In one case a subclonal KIT variant was identified in a recurrence of an SDHA-mutant GIST treated with Imatinib for several years. No clonal canonical driver kinase mutations were identified in KIT, PDGFRA, NF1, BRAF, FGFR1 or NTRK1-3. 0 of 36 dSDH PGL/Pheo and 0 of 1 PAC had potential therapeutic options. 2 of 7 dSDH RCC reported potential treatment options for oncogenic variants in EGFR and CDK4. No dSDH tumors had high TMB, microsatellite instability, or LOH score indicative of homologous recombination defect (HRD). Overall, 2 of 82 tumors (2.4%) harbored highly actionable GA.

2039P

82 SDH-deficient tumors
GIST (n = 38)PGL/Pheo (n = 36)RCC (n = 7)PAC (n = 1)
Age (mean)33404944
Sex ratio (M/F)16/2124/124/30/1
SDHA111231
SDHB72130
SDHC4110
SDHD2200
SDHx-WT14000
%SDHx germline696410050
GA/tumor (pathogenic non-SDHx)0.580.892.290
MSI0000
TMB (mut/Mb, mean)1.862.430.9
HRD (LOHscore >16%)0000

Conclusions

This study identifies SDH deficiency as a lone driver of malignancy and without associated actionable GA in > 97% of cases, and underscores the need for novel therapeutic approaches targeting SDH deficiency itself.

Legal entity responsible for the study

Foundation Medicine.

Funding

Foundation Medicine.

Disclosure

J.K. Killian: Full / Part-time employment: Foundation Medicine. D.C. Pavlick: Full / Part-time employment: Foundation Medicine. E.S. Sokol: Full / Part-time employment: Foundation Medicine. M. Montesion: Full / Part-time employment: Foundation Medicine. D.X. Jin: Full / Part-time employment: Foundation Medicine. B. Kaplan: Full / Part-time employment: Foundation Medicine. D. Lin: Full / Part-time employment: Foundation Medicine. J. Vergilio: Full / Part-time employment: Foundation Medicine. J.A. Elvin: Full / Part-time employment: Foundation Medicine. N. Ngo: Full / Part-time employment: Foundation Medicine. E. Severson: Full / Part-time employment: Foundation Medicine. S. Ramkissoon: Full / Part-time employment: Foundation Medicine. D. Duncan: Full / Part-time employment: Foundation Medicine. C. Edgerly: Full / Part-time employment: Foundation Medicine. A. Hemmerich: Full / Part-time employment: Foundation Medicine. G.M. Frampton: Full / Part-time employment: Foundation Medicine. V.A. Miller: Full / Part-time employment: Foundation Medicine. S.M. Ali: Full / Part-time employment: Foundation Medicine. J.S. Ross: Full / Part-time employment: Foundation Medicine. All other authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

2040P - The potential of a novel antiangiogenic VEGFR1-D2 binding peptide in oncology therapeutics (ID 1499)

Presentation Number
2040P
Lecture Time
12:00 - 12:00
Speakers
  • Afsaneh Sadre Momtaz (Rasht, Iran)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Tumor growth depends on pro-angiogenic vascular endothelial growth factors (VEGF-A/-B) via binding to VEGF receptors (VEGFR1 and VEGFR2) that are biomarkers of tumor angiogenesis. Importantly, interfering with this interaction by small molecules impairs tumor growth and angiogenesis.

Methods

A novel peptide (known as VGB3) was synthesized using a solid-phase peptide synthesis (SPPS) procedure on 2-chlorotrityl chloride (2-CTC) resin. A disulfide bridge was formed in the peptide and it was purified using RP-HPLC. Recombinant His-tagged VEGFR1D2 obtained from inclusion bodies and purified on a Ni2+-NTA agarose resin. The equilibrium constant for the binding of VGB3 to VEGFR1-D2 molecule was determined by microscale thermophoresis (MST). The accumulation of the peptide in 4T1 mammary carcinoma tumors (MCTs) was assessed using 2D optical imaging. Anti-angiogenic activity of VGB3 were investigated using MTT, scratch assay, and two- as well as three-dimensional angiogenesis assays. Downstream signaling pathways were assessed by quantitative estimation of protein expression using western blotting and immunohistochemistry. (IHC).

Results

We have indicated that VGB3 is able to block VEGFR1-D2 in human umbilical vein endothelial cells (HUVECs) and 4T1 mammary carcinoma tumor (MCT) cells. This resulted in inhibition in proliferation of HUVEC, 4T1 MCT, and U87 glioblastoma cells and MST results reveals that VGB3 is a high-affinity (Kd=1.96± 0.69 µM, n = 3, P < 0.0001) binder to VEGFR1-D2. Moreover, fluorescence imaging in Balb/c mice demonstrated that fluorescein isothiocyanate (FITC)-VGB3 accumulated in tumors. VEGFA-stimulated proliferation, migration, and 2 and 3D tube formation in HUVECs were strongly inhibited by VGB3 (n = 3, P < 0.0001). In a murine 4T1 MCT model, VGB3 strongly inhibited AKT and ERK1/2 phosphorylation and tumor cell downstream signaling pathways associated with proliferation, migration, and angiogenesis, and led to the increased apoptosis index and suppression of systematic spreading of the tumor compared to PBS-treated controls.

Conclusions

These results indicate that VGB3 may be applicable for antiangiogenic and antitumor therapy.

Editorial acknowledgement

S. Mohsen Asghari and Prof. Matthew Groves

Legal entity responsible for the study

S. Mohsen Asghari and Matthew Groves.

Funding

University of Guilan.

Disclosure

All authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

Developmental therapeutics (ID 6605)

Lecture Time
12:00 - 12:00
Speakers
  • Elena Garralda (Barcelona, Spain)
  • Hendrik-Tobias Arkenau (London, United Kingdom)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00
Poster Display session 1 Poster Display session

456P - First-in-human phase I study of TAS-117, an allosteric AKT inhibitor, in patients with advanced solid tumours (ID 1775)

Presentation Number
456P
Lecture Time
12:00 - 12:00
Speakers
  • Mayu Yunokawa (Tokyo, Japan)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

TAS-117 is a novel highly potent and selective oral allosteric AKT inhibitor. This study investigated the safety, efficacy, pharmacokinetics, pharmacodynamics, and pharmacogenomics profiles of TAS-117 in patients (pts) with advanced solid tumors, for whom no standard treatment remains.

Methods

The primary objective was to evaluate the safety profile of TAS-117, including the identification of the maximum tolerated dose (MTD) and the recommended dose (RD) with regimen (RR) in a 21-day cycle. Dose escalation was assessed in a once-daily repeated dosing regimen (QD), starting at 4 mg/day, with an accelerated titration design. Dose-limiting toxicity (DLT) was evaluated in a first cycle. After RD and RR were determined, pts with endometrial cancer (EC) harboring PIK3CA or AKT gene alterations or ovarian clear cell carcinoma (OCC) were enrolled for further safety evaluation.

Results

TAS-117 was administered QD (n = 12) and in a 4 days on/3 days off regimen (4d/3d) (n = 10). The dose was escalated to 24 mg/day in QD dosing and 32 mg/day in 4d/3d; the MTD was not reached. The DLT was a grade 3 rash maculo-papular at 32 mg/day in 4d/3d dosing. The RD and RR were determined as TAS-117 24 mg/day and 4d/3d. As of 24 Apr 2019, 42 pts (15 with PIK3CA-mutated (mt) EC, 7 with AKT-altered EC, and 20 with OCC) were enrolled, and safety profiles were investigated. The common (≥30%) treatment-related adverse events (TRAEs) were rash maculo-papular (grade 3, observed in 42.5% of pts), stomatitis, hyperglycemia, white blood cell decrease, and neutrophil count decreased. Common TRAEs, especially rash maculo-papular, were manageable with dose reduction, dose interruption, or symptomatic therapy. TAS-117 exposure tended to increase in a dose-dependent matter. In efficacy-evaluable pts, objective responses were observed in 1 pt with PIK3CA-mt EC and 5 pts with OCC. The disease control rate was 61.5% in 13 pts with PIK3CA-mt EC, 80.0% in 5 pts with AKT-altered EC, and 37.5% in 16 pts with OCC.

Conclusions

TAS-117 had a manageable safety profile with clinical antitumor activity in pts with advanced solid tumors. Further investigation of the drug in a combination therapy is in preparation.

Clinical trial identification

JapicCTI-152780.

Legal entity responsible for the study

Taiho Pharmaceutical Co., Ltd.

Funding

Has not received any funding.

Disclosure

S. Takahashi: Honoraria (self), Research grant / Funding (self): Eisai; Honoraria (self): Bristol-Myers-Squibb; Honoraria (self), Research grant / Funding (self): Taiho; Honoraria (self): Bayer; Research grant / Funding (self): MSD; Research grant / Funding (self): Astrazeneka; Research grant / Funding (self): Quintiles; Research grant / Funding (self): IQVIA; Research grant / Funding (self): Daiichi-Sankyo; Research grant / Funding (self): Ono pharmaceutical. D. Aoki: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Taiho Pharmaceutical Co., Ltd.; Honoraria (self), Advisory / Consultancy: AstraZeneca K.K.; Honoraria (self), Research grant / Funding (institution): Chugai Pharmaceutical Co., Ltd.; Honoraria (self), Advisory / Consultancy: MSD K.K.. K. Yonemori: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Eisai; Honoraria (self), Research grant / Funding (institution): Taiho; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Novartis; Honoraria (self), Research grant / Funding (institution): Pfizer; Advisory / Consultancy, Research grant / Funding (self), Research grant / Funding (institution): Ono; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Bayer; Research grant / Funding (institution): Chugai; Research grant / Funding (institution): Daiichi Sankyo; Research grant / Funding (institution): Eli Lilly; Research grant / Funding (institution): ICON Japan; Research grant / Funding (institution): Kissei; Research grant / Funding (institution): Kyowa Hakko Kirin; Research grant / Funding (institution): MSD; Research grant / Funding (institution): Nippon Kayaku; Research grant / Funding (institution): Sanofi; Research grant / Funding (institution): Takeda; Research grant / Funding (institution): 3D MATRIX. H. Hara: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Chugai Pharma; Honoraria (self), Research grant / Funding (institution): Taiho Pharmaceutical; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Merck Serono; Honoraria (self): Yakult Honsha; Honoraria (self), Research grant / Funding (institution): Lilly; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Ono Pharmaceutical; Honoraria (self), Research grant / Funding (institution): Takeda; Honoraria (self): Bristol-Myers Squibb; Advisory / Consultancy, Research grant / Funding (institution): MSD; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Boehringer Ingelheim; Research grant / Funding (institution): Dainippon Sumitomo Pharma; Research grant / Funding (institution): Daiichi Sankyo; Research grant / Funding (institution): Pfizer; Research grant / Funding (institution): LSK BioPharma; Research grant / Funding (institution): Eisai; Research grant / Funding (institution): Incyte. K. Hasegawa: Honoraria (self), Research grant / Funding (self): Daiichi-Sankyo; Honoraria (self): Chugai; Honoraria (self): Nippon Kayaku; Honoraria (self): Bayer; Honoraria (self): AstraZeneca; Advisory / Consultancy: MSD; Research grant / Funding (self): OncoTherapy Science; Research grant / Funding (self): Yakult Honsha. K. Takehara: Advisory / Consultancy, Speaker Bureau / Expert testimony: AstraZeneca; Speaker Bureau / Expert testimony: Kyowa Kirin; Speaker Bureau / Expert testimony: Taiho; Speaker Bureau / Expert testimony: Nippon Kayaku; Speaker Bureau / Expert testimony: Janssen; Speaker Bureau / Expert testimony: Treumo; Speaker Bureau / Expert testimony: Ono; Speaker Bureau / Expert testimony: Daiichi Sankyo; Speaker Bureau / Expert testimony: Chugai Pharma; Speaker Bureau / Expert testimony: Eisai. K. Harano: Honoraria (self): Eisai; Honoraria (self), Advisory / Consultancy: Taiho; Honoraria (self): AstraZeneca; Honoraria (self), Non-remunerated activity/ies: Chugai; Advisory / Consultancy: Takeda. E. Noguchi: Honoraria (self), Research grant / Funding (institution): Pfizer; Honoraria (self), Research grant / Funding (institution): Nippon Kayaku; Advisory / Consultancy, Research grant / Funding (institution): Novartis; Advisory / Consultancy, Research grant / Funding (institution): Chugai; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Bayer; Research grant / Funding (institution): Daiichi Sankyo; Research grant / Funding (institution): Eisai; Research grant / Funding (institution): Eli Lilly; Research grant / Funding (institution): ICON Japan; Research grant / Funding (institution): Kissei; Research grant / Funding (institution): Kyowa Hakko Kirin; Research grant / Funding (institution): MSD; Research grant / Funding (institution): Ono; Research grant / Funding (institution): Sanofi; Research grant / Funding (institution): Taiho; Research grant / Funding (institution): Takeda; Research grant / Funding (institution): 3D MATRIX. T. Doi: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Novartis; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Chugai; Advisory / Consultancy, Research grant / Funding (institution): Merck Serono; Advisory / Consultancy: Otsuka; Advisory / Consultancy, Research grant / Funding (institution): Lilly; Advisory / Consultancy: Amgen; Advisory / Consultancy, Research grant / Funding (institution): Kyowa Hakko Kirin; Advisory / Consultancy, Research grant / Funding (institution): Boehringer Ingelheim; Advisory / Consultancy, Research grant / Funding (institution): Bayer; Advisory / Consultancy: AstraZeneka; Advisory / Consultancy, Research grant / Funding (institution): MSD; Advisory / Consultancy, Research grant / Funding (institution): Daiichisankyo; Advisory / Consultancy, Research grant / Funding (institution): Dainippon Sumitomo; Advisory / Consultancy, Research grant / Funding (institution): Takeda; Advisory / Consultancy, Research grant / Funding (institution): Abbvie; Research grant / Funding (institution): Taiho, Zenyaku Kogyo, Astellas; Research grant / Funding (institution): Janssen, Eisai, Sanofi; Research grant / Funding (institution): NanoCarrier, Quintiles, Pfizer; Research grant / Funding (institution): Bristol-myers; Research grant / Funding (institution): Bristol-Myers-Squibb. All other authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

457P - First-in-human study of ABBV-621 in patients (pts) with previously treated sold tumours: Dose-optimization cohorts (ID 4584)

Presentation Number
457P
Lecture Time
12:00 - 12:00
Speakers
  • Emiliano Calvo (Madrid, Spain)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

ABBV-621, a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor agonist, induces cell death in tumor cells by activation of death receptor 4 and 5. Maximum tolerated dose was not reached in dose escalation (Ratain et al. ASCO 2019; NCT03082209). Herein is reported the analysis of safety and antitumor activity in additional pts enrolled in the dose-optimization cohorts with the goal of identifying the recommended phase 2 dose.

Methods

Eligible pts (≥18 years, relapsed/refractory [R/R] KRAS-mutant colorectal [CRC] or pancreatic cancer, ECOG 0–2, measurable disease, willingness to undergo mandatory biopsies) received ABBV-621 intravenously (1.25–7.5 mg/kg) on day (D) 1, D8, and D15 of a 21-D cycle. Analyzed tumor types were evenly distributed across dose levels (DL).

Results

As of 16 Apr 2019, 48 pts received ≥1 dose of ABBV-621 (CRC, n = 24/pancreatic, n = 24; 1.25 mg/kg, n = 8/8; 3.75 mg/kg, n = 8/8; 7.5 mg/kg, n = 8/8). Median age: 63 years (range, 43–76); 65% male, 73% ≥3 prior treatment (Tx) regimens. Median duration of ABBV-621 exposure was 36 days (range, 1–251) with a median 2 Tx cycles (range, 1–12). Tx-emergent and Tx-related AEs are summarized in the table. Ten pts (20.8%) experienced AEs leading to discontinuation; 4 (9.3%) pts had dose-limiting toxicities possibly associated with ABBV-621 administration: respiratory failure (7.5 mg/kg; Grade 5, the only Tx-related death), increased ALT and AST (1.25, 3.75 mg/kg), toxic hepatitis (1.25 mg/kg), non-cardiac chest pain (1.25 mg/kg), and pleuritic pain (1.25 mg/kg). ALT, alanine aminotransferase; AST, aspartate aminotransferase A partial response was observed in 1 pt with CRC (1.25 mg/kg) and 1 pt with pancreatic cancer (3.75 mg/kg). Stable disease at 12 weeks was the best response in 20 pts (41.7%; CRC, n = 9/pancreatic, n = 11).

457P

AEs, n (%)Related to ABBV-621, n (%)
All grade (≥5 patients)
Grade 3/4
Serious
1.25 mg/kg (n = 16)3.75 mg/kg (n = 16)7.5 mg/kg (n = 16)Total (N = 48)1.25 mg/kg (n = 16)3.75 mg/kg (n = 16)7.5 mg/kg (n = 16)Total (N = 48)1.25 mg/kg (n = 16)3.75 mg/kg (n = 16)7.5 mg/kg (n = 16)Total (N = 48)
Fatigue4 (25.0)3 (18.8)4 (25.5)11 (22.9)00000000
Increased ALT1 (6.3)4 (25.0)5 (31.3)10 (20.8)1 (6.3)1 (6.3)1 (6.3)3 (6.3)01 (6.3)01 (2.1)
Stomatitis1 (6.3)3 (18.8)5 (31.3)9 (18.8)00000000
Increased AST1 (6.3)3 (18.8)4 (25.0)8 (16.7)1 (6.3)1 (6.3)2 (12.5)4 (8.3)01 (6.3)01 (2.1)
Decreased appetite2 (12.5)1 (6.3)4 (25.0)7 (14.6)00000000
Diarrhea1 (6.3)3 (18.8)3 (18.8)7 (14.6)00000000
Nausea2 (12.5)1 (6.3)4 (25.0)7 (14.6)0000001 (6.3)1 (2.1)
Vomiting3 (18.8)1 (6.3)3 (18.8)7 (14.6)0000001 (6.3)1 (2.1)
Dysgeusia2 (12.5)2 (12.5)1 (6.3)5 (10.4)00000000
Pyrexia01 (6.3)4 (25.0)5 (10.4)00000000

Conclusions

Administration of ABBV-621 in pts with R/R CRC and pancreatic cancer shows an acceptable safety profile at all DL and evidence of antitumor activity.

Clinical trial identification

NCT03082209.

Editorial acknowledgement

Medical writing support was provided by Yanci M. Baker, PhD, from Aptitude Health, Atlanta, GA, and funded by AbbVie.

Legal entity responsible for the study

AbbVie Inc.

Funding

AbbVie Inc.

Disclosure

E. Calvo: Honoraria (self): HM Hospitales Group; Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (institution): Novartis; Advisory / Consultancy: Nanobiotix; Advisory / Consultancy: Janssen-Cilag; Advisory / Consultancy: PsiOxus; Advisory / Consultancy: Seattle Genetics; Advisory / Consultancy: EUSA Pharma, Inc.; Advisory / Consultancy: AbbVie; Advisory / Consultancy: Celgene; Advisory / Consultancy, Research grant / Funding (institution): AstraZeneca; Advisory / Consultancy: Guidepoint Global; Advisory / Consultancy, Travel / Accommodation / Expenses: Roche/Genentech; Advisory / Consultancy: Gerson Lehrman Group; Advisory / Consultancy: Pfizer; Advisory / Consultancy: Servier; Advisory / Consultancy: amcure; Leadership role: Foundation INTHEOS; Research grant / Funding (institution): BeiGene; Research grant / Funding (institution), Shareholder / Stockholder / Stock options: START; Shareholder / Stockholder / Stock options: Oncoart Associated; Shareholder / Stockholder / Stock options: International Cancer Consultants. M.J.A. de Jonge: Advisory / Consultancy: Faron Pharmaceutical Ltd. D.W. Rasco: Advisory / Consultancy: Boehringer Ingelheim; Advisory / Consultancy: Lily; Research grant / Funding (institution): Celgene; Research grant / Funding (institution): Millennium; Research grant / Funding (institution): Rexahn; Research grant / Funding (institution): Five Prime Therapeutics; Research grant / Funding (institution): Pharmacyclics; Research grant / Funding (institution), Travel / Accommodation / Expenses: Asana BioSciences; Research grant / Funding (institution): Eisai; Research grant / Funding (institution): Aeglea; Research grant / Funding (institution): Merck; Research grant / Funding (institution): Ascentage; Research grant / Funding (institution): MacroGenics; Research grant / Funding (institution): Apexian; Research grant / Funding (institution): Birdie; Research grant / Funding (institution): AbbVie; Research grant / Funding (institution): Constellation; Research grant / Funding (institution): Syndax; Research grant / Funding (institution): Astex; Research grant / Funding (institution): Compugen; Research grant / Funding (institution): Coordination; Research grant / Funding (institution): GSK; Research grant / Funding (institution): Incyte. V. Moreno: Advisory / Consultancy: Puma Biotechnology; Travel / Accommodation / Expenses: Sanofi; Travel / Accommodation / Expenses: Regeneron. Y. Chang: Shareholder / Stockholder / Stock options, Full / Part-time employment: AbbVie. M. Chiney: Full / Part-time employment, Former employee: AbbVie; Full / Part-time employment, Current employee: Bristol-Myers Squibb. M. Motwani: Shareholder / Stockholder / Stock options, Full / Part-time employment: AbbVie. S. Penugonda: Shareholder / Stockholder / Stock options, Full / Part-time employment: AbbVie. A.M. Petrich: Shareholder / Stockholder / Stock options, Full / Part-time employment: AbbVie. M.J. Ratain: Advisory / Consultancy: Acentage Pharma; Advisory / Consultancy: Cyclacel; Advisory / Consultancy: Aptevo Therapeutics; Advisory / Consultancy: Shionogi; Research grant / Funding (institution): Dicerna; Research grant / Funding (institution): AbbVie; Research grant / Funding (institution): Genentech/Roche; Licensing / Royalties: UGT1A1 genotyping for irinotecan; Licensing / Royalties: Genomic prescribing system; Officer / Board of Directors: Value in Cancer Care Consortium. P. LoRusso: Advisory / Consultancy: Agios; Advisory / Consultancy: AbbVie; Advisory / Consultancy: Genmab; Advisory / Consultancy: Halozyme; Advisory / Consultancy: Roche/Genentech; Advisory / Consultancy: CytomX; Advisory / Consultancy: Five Prime; Advisory / Consultancy: Takeda; Advisory / Consultancy: Sotio; Advisory / Consultancy: Cybrexa; Advisory / Consultancy: Agenus.

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Poster Display session 1 Poster Display session

458P - Safety, efficacy, PK and PD biomarker results of the first-in-human study of mutant isocitrate dehydrogenase 1 (mIDH1) inhibitor BAY 1436032 in patients (pts) with mIDH1 advanced solid tumours (ID 3620)

Presentation Number
458P
Lecture Time
12:00 - 12:00
Speakers
  • Wolfgang Wick (Heidelberg, Germany)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

BAY 1436032 (BAY) inhibits R132X-mIDH1, which aberrantly generates oncometabolite 2-hydroxyglutarate (2-HG). In animal studies, BAY decreased 2-HG levels in mIDH1 tumours and showed brain penetration (brain/plasma ratio ∼0.2). Plasma 2-HG levels are increased in pts with mIDH1 tumours like intrahepatic cholangiocarcinoma (IHCC), but not in mIDH1 gliomas.

Methods

In this ongoing phase I dose-escalation and expansion study, BAY was administered orally (150-1500 mg BID) to pts with advanced mIDH1-R132X solid tumours. 4 expansion cohorts (1500 mg BID) enrolled pts with anaplastic and advanced grade 2 gliomas, glioblastoma (GBM), IHCC and other tumours. Response was assessed by RECIST (solid tumours) and RANO criteria (gliomas). Plasma BAY and 2-HG levels were measured.

Results

29 pts were treated in the escalation part in 5 dose-escalation cohorts and 52 in the expansion part. Pts had glioma (n = 55), IHCC (n = 16), chondrosarcoma (n = 7) and other tumours (n = 3). As of the 8 Nov 2018 preliminary cut-off, the most common all-cause treatment-emergent AEs reported in ≥ 10% of pts were: headache (20%), vomiting, diarrhoea and fatigue (19% each), nausea (16%), ALT increased (12%) and blood bilirubin increased (11%). There was 1 DLT (grade 3 rash maculopapular, 1500 mg BID). MTD was not reached; RP2D was 1500 mg BID. BAY exposure increased broadly dose proportionally from 150-1200 mg, with high variability. Consistent with short half-life, no accumulation was observed. Plasma 2-HG reduction occurred in nearly all non-glioma pts, with up to 98% inhibition. A pt with anaplastic astrocytoma (AA) (1200 mg BID) achieved complete response and is still on treatment (>23 months at cut-off); 2 glioma pts (AA and oligodendroglioma, both 1500 mg BID) had a partial response. 6 additional pts remained on treatment with stable disease at cut-off (3 gliomas, 2 GBM, 1 IHCC; treatment duration 4-11 months). Updated safety, efficacy and PK/PD analyses will be presented.

Conclusions

BAY is well tolerated as anticipated due to specificity for mIDH1. BAY inhibited mIDH1-R132X activity as evidenced by a PD effect in pts with measurable 2-HG, with objective response in 3 pts (all gliomas).

Clinical trial identification

NCT02746081.

Editorial acknowledgement

Medical writing assistance was provided by Laura Valenzo, PhD, of Complete Health Vizion and funded by Bayer AG.

Legal entity responsible for the study

Bayer AG.

Funding

Bayer AG.

Disclosure

W. Wick: Non-remunerated activity/ies, patent: IDH vaccine; Non-remunerated activity/ies, patent: IDH diagnostic; Non-remunerated activity/ies, patent: APG diagnostic. G. Tabatabai: Research grant / Funding (self): Roche Diagnostics; Research grant / Funding (self): Medac; Travel / Accommodation / Expenses: BMS; Travel / Accommodation / Expenses: Novocure; Travel / Accommodation / Expenses: Medac; Speaker Bureau / Expert testimony: Medac; Speaker Bureau / Expert testimony: Novocure; Advisory / Consultancy: AbbVie; Advisory / Consultancy: BMS; Advisory / Consultancy: TIGER NIS (Novocure); Advisory / Consultancy: ON-TRK NIS (Bayer). M. Schuler: Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Boehringer Ingelheim; Advisory / Consultancy: Bristol-Myers Squibb; Advisory / Consultancy: Novartis; Advisory / Consultancy: Roche; Honoraria (self): Abbvie; Honoraria (self): Alexion; Honoraria (self): Boehringer Ingelheim; Honoraria (self): Bristol-Myers Squibb; Honoraria (self): Celgene; Honoraria (self): Lilly; Honoraria (self): MSD; Honoraria (self): Novartis; Honoraria (self): Pierre Fabre; Research grant / Funding (institution): AstraZeneca; Research grant / Funding (institution): Boehringer Ingelheim; Research grant / Funding (institution): Bristol Myers-Squibb; Research grant / Funding (institution): Novartis. K. Rorhberg: Research grant / Funding (self), Non-remunerated activity/ies: Bayer; Research grant / Funding (self), Travel / Accommodation / Expenses, Non-remunerated activity/ies: Roche; Research grant / Funding (self), Non-remunerated activity/ies: Loxo Oncology; Research grant / Funding (self), Non-remunerated activity/ies: Orion Pharma; Research grant / Funding (self), Non-remunerated activity/ies: Pfizer; Research grant / Funding (self), Non-remunerated activity/ies: PUMA; Research grant / Funding (self), Non-remunerated activity/ies: Cantargia; Research grant / Funding (self), Non-remunerated activity/ies: Genmab; Research grant / Funding (self), Non-remunerated activity/ies: Novartis; Research grant / Funding (self), Non-remunerated activity/ies: Incyte; Research grant / Funding (self), Non-remunerated activity/ies: Symphogen; Research grant / Funding (self), Non-remunerated activity/ies: AstraZeneca; Research grant / Funding (self), Non-remunerated activity/ies: Alligator; Research grant / Funding (self), Non-remunerated activity/ies: Merck; Travel / Accommodation / Expenses: Sanofi; Research grant / Funding (self), Non-remunerated activity/ies: BMS. S.P. Chawla: Research grant / Funding (self): Amgen; Research grant / Funding (self): Roche; Research grant / Funding (self): Threshold Pharmaceuticals; Research grant / Funding (self): GlaxoSmithKline; Research grant / Funding (self): Immune Design; Research grant / Funding (self): TRACON Pharma; Research grant / Funding (self): SARC; Research grant / Funding (self): Karyopharma Therapeutics; Research grant / Funding (self): Jansen. F. Janku: Research grant / Funding (self): Novartis; Research grant / Funding (self): Genentech; Research grant / Funding (self): Bayer; Research grant / Funding (self): BioMed Valley Discoveries; Research grant / Funding (self): Astellas; Research grant / Funding (self): Agios; Research grant / Funding (self): Plexxikon; Research grant / Funding (self): Deciphera; Research grant / Funding (self): Piqur; Research grant / Funding (self): Symphogen; Research grant / Funding (self): Bristol-Myers Squibb; Research grant / Funding (self): Asana; Research grant / Funding (self): Upsher-Smith Laboratories; Advisory / Consultancy: Guardant Health; Advisory / Consultancy: IFM Therapeutics; Advisory / Consultancy: Synlogic; Advisory / Consultancy: Deciphera; Advisory / Consultancy: Trovagene; Advisory / Consultancy: Immunomet; Shareholder / Stockholder / Stock options: Trovagene. Y. Narita: Speaker Bureau / Expert testimony, Research grant / Funding (self): Chugai Pharmaceutical; Speaker Bureau / Expert testimony, Research grant / Funding (self): Ono Pharmaceutical; Speaker Bureau / Expert testimony, Research grant / Funding (self): Abbvie; Speaker Bureau / Expert testimony, Research grant / Funding (self): Dainippon-Sumitomo; Speaker Bureau / Expert testimony, Research grant / Funding (self): Daiichi-Sankyo; Speaker Bureau / Expert testimony, Research grant / Funding (self): Eisai; Speaker Bureau / Expert testimony, Research grant / Funding (self): Stella-pharma; Speaker Bureau / Expert testimony, Research grant / Funding (self): Ohtuka; Speaker Bureau / Expert testimony, Research grant / Funding (self): Meiji-seika; Speaker Bureau / Expert testimony, Research grant / Funding (self): SBI pharma. Y. Ando: Honoraria (self), Research grant / Funding (self): Chugai Pharmaceutical Co.,Ltd.; Honoraria (self), Research grant / Funding (self): Kyowa Hakko Kirin Co.,Ltd.; Honoraria (self), Research grant / Funding (self): Nippon Kayaku Co., Ltd.; Honoraria (self), Research grant / Funding (self): Yakult Honsha Co.,Ltd.; Honoraria (self), Research grant / Funding (self): Eli Lilly Japan K.K.; Honoraria (self), Research grant / Funding (self): Mochida Pharmaceutical Co.,Ltd.; Honoraria (self), Research grant / Funding (self): Ono Pharmaceutical Co.,Ltd.; Honoraria (self), Research grant / Funding (self): Taiho Pharmaceutical Co.,Ltd.; Honoraria (self): Novartis Pharma K.K.; Honoraria (self): Merck Serono Co.,Ltd.; Honoraria (self): Bayer Holding Ltd.; Honoraria (self): Bristol-Myers Squibb; Honoraria (self): Otsuka Holdings Co.,Ltd.; Honoraria (self): Sawai Pharmaceutical Co., Ltd; Honoraria (self), Research grant / Funding (self): Daiichi Sankyo Company, Ltd.; Research grant / Funding (self): Eisai Co.,Ltd.; Research grant / Funding (self): Hisamitsu Pharmaceutical Co., Inc.; Research grant / Funding (self): Takeda Pharmaceutical Co.,Ltd.; Honoraria (self): Tsumura & Co.; Honoraria (self): Shionogi & Co.,Ltd; Honoraria (self): Janssen Pharmaceutical K.K.; Honoraria (self): Pharma International Inc. H.J. Lenz: Advisory / Consultancy, Speaker Bureau / Expert testimony, Research grant / Funding (self): Bayer; Advisory / Consultancy, Speaker Bureau / Expert testimony: Roche; Advisory / Consultancy, Speaker Bureau / Expert testimony: Merck KG. M. Ikeda: Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Bayer; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Eisai; Honoraria (self), Advisory / Consultancy: MSD; Research grant / Funding (self): Ono; Advisory / Consultancy, Research grant / Funding (self): ASLAN; Honoraria (self), Research grant / Funding (self): Yakult; Research grant / Funding (self): J-Pharma; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Bristol-Myers Squibb; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Eli Lilly; Honoraria (self), Research grant / Funding (self): Sumitomo Dainippon; Research grant / Funding (self): Kowa; Honoraria (self): Abbott; Honoraria (self): EA Pharma; Advisory / Consultancy, Research grant / Funding (self): Chugai; Honoraria (self), Advisory / Consultancy: Daiichi Sankyo; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Novartis; Honoraria (self), Advisory / Consultancy: Teijin Pharma; Advisory / Consultancy, Research grant / Funding (self): Kyowa Hakko Kirin; Advisory / Consultancy, Research grant / Funding (self): NanoCarrier; Advisory / Consultancy: Shire; Honoraria (self), Research grant / Funding (self): Taiho; Research grant / Funding (self): AstraZeneca; Research grant / Funding (self): Baxalta; Research grant / Funding (self): Merck Serono; Advisory / Consultancy, Research grant / Funding (self): Takara Bio; Research grant / Funding (self): Zeria; Honoraria (self): Nobelpharma; Honoraria (self), Advisory / Consultancy: Otsuka; Honoraria (self): Kaken Pharmaceutical; Honoraria (self): Gilead; Advisory / Consultancy: Astellas Pharma; Research grant / Funding (self): Takeda Pharmaceutical; Advisory / Consultancy: Micron. I. Genvresse: Full / Part-time employment: Bayer AG. C. Rentzsch: Full / Part-time employment: Bayer AG. S. Reschke: Full / Part-time employment: Bayer AG. C. Cyris: Full / Part-time employment: CHRESTOS Concept GmbH & Co. KG. C. Cai: Full / Part-time employment: Bayer HealthCare Pharmaceuticals, Inc. M. Jeffers: Full / Part-time employment: Bayer HealthCare Pharmaceuticals, Inc. C. Peña: Full / Part-time employment: Bayer HealthCare Pharmaceuticals, Inc. O. Bähr: Honoraria (self): Novocure; Honoraria (self): Medac Pharma; Honoraria (self): BMS. All other authors have declared no conflicts of interest.

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Poster Display session 1 Poster Display session

459P - Proof of concept clinical study by US-guided intratumor injection of VCN-01, an oncolytic adenovirus expressing hyaluronidase in patients with pancreatic cancer (ID 5465)

Presentation Number
459P
Lecture Time
12:00 - 12:00
Speakers
  • Manuel Hidalgo (Madrid, Spain)
Session Name
Poster Display session 1
Location
Poster Area (Hall 4), Fira Gran Via, Barcelona, Spain
Date
28.09.2019
Time
12:00 - 13:00

Abstract

Background

Dense stroma is a hallmark of hard-to-treat cancers such as pancreatic adenocarcinoma. VCN-01 oncolytic adenovirus is designed to replicate in cancer cells with dysfuncti