Background

Breast cancer stem cells (BCSCs) are responsible for breast cancer metastasis and treatment failure. Hence, eliminating BCSCs poses possibility to eradicate breast cancer. Recently, studies have been suggested that H3K27me3 is implicated in maintenance of cancer stem cells (CSCs), however, the roles of H3K27me3 in BCSCs remain poorly investigated. Hence, we aimed to explore the functionality of H3K27me3 in BCSCs.

Methods

Here, we firstly determined the global level of H3K27me3 in mammosphere-derived cells. Then, we detected the effect of GSKJ4 in cell viability through CCK8 assay. Next, we tested the impact of GSKJ4 on BCSCs expansions with CD44+CD24- phenotype and ALDH1-positive via flowcytometry. In addition, the impact on self-renewal capacity of BCSCs were also asked using mammosphere formation assay and colony formation assay. Further, western blot and Q-PCR were conducted to explore the effect of GSKJ4 in the expression level of stemness-related markers. Finally, we determined the influence of GSKJ4 on tumorigenicity using a xenograft model and investigated the underlying mechanisms.

Results

We identify H3K27me3 as a negative modulator of stemness of BCSCs and suggest GSKJ4 is a promising drug targeting BCSCs. we show that the H3K27me3 level is decreased in mammosphere-derived BCSCs. In breast cancer cells, we demonstrate that GSKJ4 could markedly inhibit the proliferation. Strikingly, we show that GSKJ4 could effectively suppress BCSCs including its expansion, self-renewal capacity, and the expression of stemness-related markers. Additionally, our xenograft model confirms that GSKJ4 is able to effectively inhibit the tumorigenicity of MDA-MB-231. Mechanistically, the inhibition effect of GSKJ4 in BCSCs is via inhibiting JMJD3 and UTX thus causing increment of H3K27me3 level, which results in suppressing stemness factors including NANOG, SOX2 and OCT4.

Conclusions

Our results provide strong supports that epigenetic modification is associated with maintenance of properties of BCSCs and reveal that GSKJ4 is capable to be a prospective agent targeting BCSCs.

Legal entity responsible for the study

N/A

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Cancer stem cells (CSCs) have been proven to be tumorigenic and may be responsible for the resistance to chemo-radiation therapy, disease recurrence and metastasis. Chemo-radiation therapy modulates oxidative stress in cancer cells, leading to cellular adaption response including modulation of cell survival and antioxidant defense mechanisms. However, the redox status alteration of breast CSCs is not yet clearly understood. The aim of this study was to elaborate the impact of rotenone-modulated oxidative stress on the survival of human breast CSCs (CD24-/CD44+) which might be beneficial to understand the underlying mechanism of chemo-radiation therapy resistance.

Methods

Human breast CSCs (CD24-/CD44+) and non-CSCs (CD24-/CD44-) were treated with rotenone and DMSO (vehicle) for 6 hours, respectively. The effects of rotenone on oxidative stress were assessed by analysing intracellular reactive oxygen species (ROS) level using dihydroethidium assay, as well as mRNA expression and specific activity of MnSOD antioxidant. Finally, cell survival was determined using MTS assay, as well as through analysis of survivin mRNA expression.

Results

Our results showed that rotenone could not modulate the superoxide level of human breast CSCs (CD24-/CD44+), in contrast to that of non-CSCs (CD24-/CD44-). Albeit MnSOD synthesis in human breast CSCs has been excessively enhanced following rotenone treatment, the enzyme activity was still lower than in non-CSCs. Importantly, the cell viability of CSCs was higher than that of non-CSCs, which related to the increase of survivin.

Conclusions

We conclude that human breast CSCs (CD24-/CD44+) could survive better than their counterpart non-CSCs (CD24-/CD44-) when treated with rotenone. This impact might be associated with the increase of antioxidant MnSOD expression and survivin mRNA expression.

Legal entity responsible for the study

Faculty of Medicine, Universitas Indonesia

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Triple-negative breast cancer (TNBC) remains a difficult-to-treat cancer and the biology beneath TNBC is a research interest. Tryptophan-kynurenine metabolism plays an important role in epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs) and immune escape. Previous studies have focused on the expression and function of the first step and the rate-limiting enzyme in tumor cells, whereas the second step catabolic enzyme kynurenine 3-monooxygenase (KMO) was rarely addressed in tumorigenesis. Hence, we sought to investigate of the mechanism and functions of KMO in TNBC carcinogenesis.

Methods

KMO gene alteration and mRNA transcripts were analyzed from the The Cancer Genome Atlas (TCGA) database. MDA-MB-231 and MDA-MB-468 TNBC cell lines were used for in vitro studies. Cell proliferation, colony formation, transwell migration/invasion assays and tumorsphere forming ability were used for functional study. Signal transduction pathways were assessed by Western blot, quantitative real-time PCR and reporter assays. The effect of KMO on tumor growth was tested in nude mice with breast cancer xenografts.

Results

TCGA analysis showed high-frequency of KMO amplification alterations, which was related to poor overall survival in breast cancers. KMO transcripts were up-regulated in the tumor tissues of breast cancers, especially in TNBC. The functional assays showed that ectopic KMO expression promoted tumorigenesis, including cell growth and abilities of colony formation, migration, invasion, and tumorsphere formation. Moreover, western blot analysis revealed expressions of epithelial marker E-cadherin were decreased and mesenchymal markers N-cadherin, and Twist were increased by KMO overexpression in MDA-MB-468 cells. Interestingly, the mRNA and protein levels of pluripotency genes including CD44, Nanog, Oct4, and SOX-2 were also suppressed by KMO knockdown. Data of reporter gene assay showed that the activities of Nanog, Oct4, and SOX-2 promoters were enhanced by KMO overexpression. Furthermore, knockdown KMO decreased the xenografted tumor growth of MDA-MB-468 cells, suggesting its oncogenic role in TNBC.

Conclusions

Our data highlight the novel and critical roles of KMO in TNBC progression and metastasis.

Clinical trial identification

Not applicable

Legal entity responsible for the study

Taipei Veterans General Hospital

Funding

Taipei Veterans General Hospital

Disclosure

All authors have declared no conflicts of interest.

Background

Triple-negative breast cancers (TNBCs) are aggressive and associated with poor prognosis. We have recently demonstrated that PIM1 regulates cell death, tumour growth and chemotherapy response in TNBC. This study aims to further explore the molecular mechanisms by which PIM1 promotes malignant phenotypes in TNBC, in particular cell migration.

Methods

The HumanHT-12 v4 expression array was used to interrogate changes in gene expression upon PIM1 knockdown in TNBC cell lines. Transwell migration assays and time-lapse live-cell imaging were used to study the role of PIM1 in cell migration. To assess the morphology of TNBC cells we stained F-actin with 488-phalloidin. Phospho-kinase arrays were used to elucidate the pathway by which PIM1 may control cell migration.

Results

Gene expression analysis revealed PTPN11 as the most downregulated gene upon PIM1 knockdown in TNBC cell lines. These results were validated by qRTPCR in 3 TNBC cell lines. PTPN11 encodes for the phosphatase SHP2, known to be relevant for the migration of TNBC cells. We therefore studied whether PIM1 was also required for this phenotype in TNBC cell lines. PIM1 knockdown led to a defect on 2D-transwell migration in MDA-MB-231 and SUM159 cells, similar to that observed upon SHP2 knockdown. Interestingly, SHP2 knockdown did not affect short-term cell population growth of TNBC cells, suggesting that PIM1 exerts its role in cell population growth via different mechanisms, as demonstrated previously. Upon PIM1 knockdown, MDA-MB-231 showed lower motility persistence, increased circularity and a reduction of F-actin filaments. To understand the common downstream targets of PIM1 and SHP2 and elucidate the pathway by which PIM1 may control cell migration, we used phospho-kinase arrays. These revealed decreased phosphorylation of PLCg1, FAK and PYK2, proteins involved in cell migration, upon either PIM1 or SHP2 knockdown.

Conclusions

These data suggest that PIM1 regulates cell migration by controlling PTPN11/SHP2 expression and provide further evidence for PIM1 as a target for TNBC therapy, not only to induce apoptosis and prevent tumour growth, but also to prevent TNBC migration.

Clinical trial identification

N/A

Legal entity responsible for the study

King's College London/Breast Cancer Now

Funding

Breast Cancer Now

Disclosure

All authors have declared no conflicts of interest.

Background

Triple negative breast cancer (TNBC) is an aggressive cancer and its prognosis remains poor. Combinational therapies are a promising strategy for enhancing treatment efficacy. Blockade of STAT3 signaling have been shown to enhance the response of cancer cells to conventional chemotherapeutic agents. Here we used a SHP-1 agonist SC-43 to dephosphorylate STAT3, thereby suppressing oncogenic STAT3 signaling, and tested it in combination with docetaxel in TNBC cells.

Methods

TNBC cell lines (HCC-1937, MDA-MB-468, MDA-MB-231) were used for in vitro studies. Cell viability was examined by MTT assay. Combination index was determined using Calcusyn anaysis. Apoptosis was examined by flow cytometry and western blot. Signal transduction pathways in cells were assessed by western blot. In vivo efficacy of SC-43 in combination with docetaxel was tested in nude mice with breast cancer xenografts.

Results

To exam expression of SHP-1 in clinical samples, we analyzed mRNA expression of SHP-1 gene (ptpn6) in a public TNBC dataset (The Cancer Genome Atlas, TCGA). We found that higher SHP-1 mRNA expression is associated with better overall survival in TNBC patients. Sequential combination of docetaxel and SC-43 in vitroshowed enhanced anti-proliferation and apoptosis associated with decreased p-STAT3 and decreased STAT3-downstream effector cyclin D1 in the TNBC cell lines MDA-MB-231, MDA-MB-468 and HCC-1937. Ectopic expression of STAT3 reduced theincreased cytotoxicity induced by the combination therapy. In addition, this sequential combination showed enhanced SHP-1 activity compared to SC-43 alone. Furthermore, siRNA against SHP-1 reduced apoptosis induced by the combination treatment. Moreover, by ectopic expression of SHP-1 mutants that caused SC-43 to lose its SHP-1 agonist capability, the SC-43-induced p-STAT3 signaling inhibition was reduced in the cells subjected to the combination treatment, suggesting SHP-1 plays a crucial role in docetaxel-SC-43-mediated TNBC cell apoptosis, Importantly, combination of docetaxel and SC-43 showed enhanced anti-tumor growth compared to single-agent therapy in MDA-MB-231 xenografted tumor mice.

Conclusions

SHP-1 agonist SC-43 enhanced the anti-tumor effect of docetaxel by SHP-1 dependent STAT3 inhibition in human TNBC cells. We suggest a therapeutic potential of SHP-1 agonist in combination with docetaxel for TNBC.

Legal entity responsible for the study

National Taiwan University Hospital

Funding

NHRI-EX106-10608BI

Disclosure

All authors have declared no conflicts of interest.

Background

Despite the increase in patient survival rates promoted by increased screening and prevention efforts, much faster tumor genome sequencing and developed smart targeted therapies, de novo or acquired chemoresistance remains to be a significant factor for treatment failure in breast cancer therapeutics. Conventional chemotherapy, radiotherapy as well as targeted therapies activate mitochondrial cell death machinery to eliminate cancer cells. BCL‐2 protein family members regulate mitochondrial cell death pathway by controlling mitochondrial outer membrane permeabilization. Neratinib and dacomitinib are potent and irreversible pan‐EGFR inhibitors, which block their autophosphorylation and downstream signaling. Moreover, neratinib and dacomitinib have been shown to activate cell death in HER2‐overexpressing cell lines. The aim of our study was to identify molecular pathways responsible for panHER2 inhibitor resistance in HER2+ breast cancer cells.

Methods

The expression of EGFR and BCL‐2 protein family members was determined by immunoblotting and qPCR. CellTiter‐Glo was used to measure cell viability and AnnexinV/PI staining and flow cytometer was used to evaluate apoptotic response. BH3 profiling was used to determine the apoptotic blocks and mitochondrial cell death priming in breast cancer cells.

Results

Here we showed that increased MCL‐1 and decreased BIM mediate resistance to neratinib in ZR‐75‐30 and SKBR3 cells while increased BCL‐XL and BCL‐2 and decreased BIM promoted neratinib resistance in BT474 cells. Cells were also cross-resistant to dacomitinib. BH3 profiles of HER2+ breast cancer cells efficiently predicted anti‐apoptotic protein dependence and development of resistance to panHER2 inhibitors. Adding specific ERK1/2 inhibitor SCH772984 to neratinib or dacomitinib led to increased apoptotic response in SKBR3 and ZR‐75‐30 cells, but we did not detect a similar response in BT‐474 cells. Reactivation of ERK1/2 was primarily responsible for acquired resistance in SKBR3 and ZR‐75‐30 cells. Intriguingly, both ERK1/2 and Akt/NFkappaB pathways were responsible for neratinib resistance in BT474 cells.

Conclusions

Our results showed that different mitochondrial apoptotic blocks mediated acquired panHER2 resistance in HER2+ breast cancer cell lines as well as highlighted the potential of BH3 profiling assay in prediction of panHER2 resistance in breast cancer cells.

Legal entity responsible for the study

Ozgur Kutuk

Funding

Baskent University

Disclosure

All authors have declared no conflicts of interest.

Background

Breast cancer (BC) is a heterogeneous disease, HER2+ represents between 15-30% of all subtypes. Trastuzumab (T), a monoclonal antibody able to inhibit HER2 activation, has been successfully employed in HER2 amplified tumors both in adjuvant and in metastatic settings, conferring an improvement in DFS, PFS and OS. Despite these results, many patients experience primary or secondary resistance to therapy. The mechanism of resistance is unclear, our aim is to assess AXL, a receptor tyrosine kinases (RTK) implicated in epithelial-to-mesenchymal transition, as potential mechanisms of resistance.

Methods

We used two cell lines to investigate possible mechanisms of primary and secondary resistance to T in HER2+ and hormone receptor negative BC. AU565 sensitive to T (AU565-S), and HCC1954 a primary T-resistant cell line. A third cell line with acquired resistance to T (AU565-R) was generated by treating AU565-S cells with constant dose of T (15mg/mL) for 4 months. Cell viability was estimated by MTT assay. We explored the expression of AXL by Western blot (WB) and quantitative reverse transcriptase PCR (qRT-PCR).

Results

The cell viability analysis at 7 days assay confirmed AU565-S as sensible to T, HCC1954 as primary resistant and the development of a secondary resistance to T (AU565-R) (50% of increased viability from AU565-S). HER2 overexpression in all three cells lines were confirmed by WB and FISH. qRT-PCR indicated an important up-regulation of AXL at mRNA levels in AU565-R and HCC1954 compared to AU565-S (p < 0.05). In the same line, WB analyses showed a significantly increase in AXL protein expression in AU565-R and HCC1954 (2.03 and 7.37 fold, respectively). Finally, a selective AXL inhibitor (TP-0903) has demonstrated reduction of viability in all cell lines and significant restoration of sensitivity to T in AU565-R (p < 0.01).

Conclusions

Our results suggest: 1) AXL could be a potential mechanism of both primary and secondary resistance to T; 2) combination therapy with AXL inhibitor plus T restored T sensitivity in in vitro model with AXL overexpressed. These results merit further study and to explore this RTK as possible therapeutic targets in case of anti-HER2 treatment failure.

Legal entity responsible for the study

Hospital Clinico Universitario of Valencia. Biomedical Research Institute INCLIVA

Funding

None

Disclosure

J.A. Perez Fidalgo: Received fees from AstraZeneca, Ipsen, Novartis, Pfizer and Roche for participation in speaker bureaus. Had travel/accomodation expenses paid/reimbursed by AstraZeneca, Roche and Sandoz. All other authors have declared no conflicts of interest.

Background

Lapatinib is a dual targeting (EGFR/HER2) small molecule tyrosine kinase inhibitor (TKI) approved for the treatment of HER2+ breast cancer. Lapatinib treatment can often lead to acquired resistance and refractory disease. There is no data available on the sensitivity of lapatinib-resistant breast cancer cells to immune cell-mediated cytotoxicity. To investigate the consequences of a lapatinib resistant phenotype on the immune response, we examined T-ADCC and the expression levels of immune-related proteins in a cell line model of lapatinib-resistant HER2+ breast cancer.

Methods

Lapatinib-resistant SKBR3 cells (SKBR3-Lap) were generated by continuous exposure to 250nM lapatinib for 6 months alongside untreated parental cells (SKBR3-Par). FACS-based assays employing peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers were used to measure direct PBMC-mediated cytotoxicity and T-ADCC. Comparative DNA microarray studies (SK-Par vs. SK-LAP) identified differentially expressed immune-related genes which were subsequently examined at the protein level by Western blot.

Results

T-ADCC was 45-50% lower in SKBR3-Lap compared to the SKBR3-Par cell line at the three effector to target cell ratios examined - 1:1 (p = 0.01), 5:1 (p = 0.001) and 10:1 (p = 0.024). Direct immune cell-mediated cytotoxicity was low (<7% at 10:1) for both cell lines. Microarray data identified significant alterations in the growth factor receptor EGFR, the immunosuppressive adenosine receptor A2AR and immune response modulating MHC Class I, HLA-E, and NKTR in SK-LAP compared to SK-Par. Western blots established changes to these targets at the protein level. In addition, protein levels of HER2 were not significantly reduced and programmed death ligand 1 (PD-L1) levels were increased (p = 0.045) in SKBR3-Lap.

Conclusions

Resistance to lapatinib is associated with an attenuated T- ADCC response and an altered profile of immune-related proteins in this HER2+ breast cancer model. Further investigation is warranted to explore if targeting proteins such as A2AR or PD-L1 could play a role in ADCC response in this model.

Legal entity responsible for the study

Cancer Biotherapeutics, National Institute for Cellular Biotechnology, Dublin City University

Funding

Cancer Clinical Research Trust

Disclosure

N. O'Donovan: Research funding from GlaxoSmithKline. J. Crown: Honoraria from Novartis. Research funding from Roche and GlaxoSmithKline. D. Collins: Currently funded by a Roche Postdoctoral Fellowship and has received funding from Roche Products Ireland Ltd. as part of the IRCSET Enterprise Partnership Scheme. All other authors have declared no conflicts of interest.

Disclosure

N. O\'Donovan: Research funding from GlaxoSmithKline. J. Crown: Honoraria from Novartis. Research funding from Roche and GlaxoSmithKline. D. Collins: Currently funded by a Roche Postdoctoral Fellowship and has received funding from Roche Products Ireland Ltd. as part of the IRCSET Enterprise Partnership Scheme. All other authors have declared no conflicts of interest.

Background

Exemestane (Exe) is a third-generation steroidal aromatase inhibitor (AI) that is a standard therapeutic approach for post-menopausal women with estrogen-receptor positive (ER+) breast cancer. Besides its clinical benefit, acquired resistance may develop. Thus, to avoid this drawback it is urgent to find new targets that can improve breast cancer treatment. It is known that 85-95% of the ER+ breast cancers, overexpress androgen receptor (AR), that has a dual function in breast cancer depending on hormonal cell status. It has been described that in AIs-sensitive breast cancer cells this receptor promotes cell death. Several clinical trials are ongoing to study the efficacy of combining AR antagonists, as bicalutamide (CDX), with Exe, but the benefit of targeting AR is not well defined. In that way, this work will investigate the biological significance of AR in Exe-treated breast cancer cells and the effectiveness of targeting AR.

Methods

In ER+ breast cancer cells that overexpress aromatase (MCF-7aro), it was investigated the in vitro effects of the AR antagonist CDX in Exe-treated cells. The cell impact in viability and cell proliferation was studied using MTT assay and flow cytometry, respectively. The cell death was explored by evaluating caspase activities. The expression/activation of AR and the effects on PI3K and MAPK pathways were studied by Western-blot.

Results

Exe induces an overexpression and hyperactivation of AR in MCF-7aro cells. By blocking AR with CDX, it was observed an increase in the reduction of viability and proliferation of Exe-treated cells, when comparing to Exe alone. An increase in activation of caspases-9, -8 and -7 was also observed for the combination. In addition, CDX inhibits the Exe-induced activation of cell proliferation/survival MAPK pathway and caused no effect on PI3K pathway.

Conclusions

This study suggests that, contrary what is described for other AIs, the AR as a pro-survival role in sensitive breast cancer cells treated with Exe and that by targeting AR it is possible to improve the clinical efficacy of Exe, by inhibiting cell proliferation and inducing apoptosis. This work contributes to the understanding of the link between AR and Exe and will highlight new targets to improve breast cancer treatment.

Legal entity responsible for the study

UCIBIO, REQUIMTE, Faculty of Pharmacy, University of Porto

Funding

Fundação para a Ciência e a Tecnologia (FCT): Amaral C. (SFRH/BPD/98304/2013) and Augusto T. (BD/128333/2017) grants

Disclosure

All authors have declared no conflicts of interest.

Background

The phosphatidylinositol 3-kinase (PI3Ks) pathway is commonly altereted in breast cancer patients, but its role is still unclear. Taselisib, a mutant PI3Kα selective inhibitor, and ipatasertib, an AKT inhibitor, are currently under investigation in clinical trials in combination with paclitaxel or hormonal therapies in breast cancer. The aim of this study was to evaluate if PI3K or AKT inhibition can prevent resistance to chemotherapy and potentiate its efficacy.

Methods

The efficacy of combined treatment of ipatasertib and taselisib plus vinorelbine or paclitaxel or eribulin was evaluated in vitro on human breast cancer cells (with different expression profile of hormonal receptors, HER2, and of PI3Kα mutation) on cell survival by using MTT (3,(4,5-dimethylthiazol-2)2,5 difeniltetrazolium bromide) and on cell apoptosis by flow-cytometry analysis. We also investigated the effect of combined treatment on downstream intracellular signaling, by western blot analysis, and on metastatic properties, by migration assays. Finally, we analyzed changes in cell cytoskeleton by immunofluorescence.

Results

A significant synergism of ipatasertib or taselib plus anti-microtubule chemotherapy in terms of anti-proliferative, pro-apoptotic and anti-metastatic effect was observed. The combined treatment completely inhibited the activation of proteins downstream of PI3K and MAPK pathways and affected the expression of survivin. Combined treatments completely disorganized the cytoskeleton in human breast cancer cells, thus suggesting a potential mechanism for this combination.

Conclusions

Targeting PI3K may enhance the efficacy of anti-microtubule drugs in human breast cancer.

Legal entity responsible for the study

Università degli Studi della Campania “Luigi Vanvitelli”, Naples, Italy.

Funding

This work has been supported by Associazione Italiana Per La Ricerca Sul Cancro (AIRC)-Project MFAG 2013-N.14392 and drugs were kindly provided by Genentech (Research proposal nr. OR-214726 for taselisib and nr. OR-214797 for ipatasertib).

Disclosure

All authors have declared no conflicts of interest.

Background

In recent years, increasing evidences showed that several types of dietary approaches restricting food intake, including Short Term Starvation (STS), may exert a protective role against aging and other age-related pathologies as well as cancer. Interestingly, the dietary restriction showed significant anticancer effects able to prevent cancer onset, slow its progression and improve therapy response. Since recent studies showed that miRNAs may modulate sensibility/resistance to antiblastic therapy, the aim of our study was to investigate the STS-induced molecular changes in breast cancer cells treated with doxorubicin, focusing our attention on miRNA expression profile.

Methods

Vitality assays were used to assess the effects of STS on cell proliferation. Using a TaqMan Low Density Array A human microRNA microarray analysis, the expression profile of 377 miRNAs was analyzed in healthy and malignant breast cells, MCF10A and MDA-MB-231 respectively, treated for 24h with 1µM doxorubicin under STS conditions for 48h. In addition, the expression of mRNAs and miRNAs specifically induced by STS was analyzed in MCF-7, MDA-MB-231 and SkBr3 cells using Real-time PCR analyses.

Results

In vitro cell vitality assays showed that STS, in association with doxorubicin treatment, significantly reduces breast cancer cell proliferation and viability, whereas it appears to protect healthy breast cells from chemotherapeutic treatment. Microarray analysis showed that a subset of miRNAs involved in molecular pathways related to drug sensitivity/resistance was found to be differentially expressed in breast cancer cells following the doxorubicin treatment and STS. Finally, expression analysis of hypothetical miRNA gene targets involved in therapy response have confirmed the coherence of our results.

Conclusions

This work establishes, for the first time, an interesting link between anticancer effects of STS and miRNA expression changes in doxorubicin-treated breast cancer cells, suggesting the potential involvement of some miRNAs in molecular pathways mediating the effects of STS in breast cancer.

Clinical trial identification

Not applicable

Legal entity responsible for the study

Department of Surgical, Oncological and Oral Sciences, Section of Medical Oncology, University of Palermo

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

It is well established that primary tumour can modify secondary organs long before the circulating tumor cells reach their metastatic targets. These modifications are mainly exerted through tumor-secreted factors and can act on several cell types to form what is called the pre-metastatic niche. Pre-metastatic endothelial niche play a key role in the extravasation process during metastasis. Here we describe the effect of the mixture of secreted factors by tumor cells over endothelial changes that facilitates tumor cell transendothelial migration.

Methods

Adhesion assay Human umbilical vein endothelial cells (HUVEC) cells were stimulated or not with TNF-α [10ng/ml] or Tumor Secreted Factors (TSFs) [10 µg/ml] from MDA-MB-231 or MCF-7 cell lines. HUVEC monolayers were co-cultured with a U937(³H) cells. After 3h firm attached U937(³H) were lysed and radioactivity was measured. Vascular permeability assay HUVEC monolayers were cultured in Boyden chambers. Cells were stimulated or not with TNF-α [10ng/ml] or TSFs [10 µg/ml] and incubated for 12h. Afterwards a dextran-FITC solution was added for 20 min and the bottom well fluorescence was quantified. Transendothelial migration assay HUVEC monolayers were cultured in Boyden chambers. Cells were stimulated or not with TNF-α [10ng/ml] or TSFs [10 µg/ml] and incubated for 10h. Fluorescent labeled MDA-MB-231 cell suspension 2x10⁴ was added (24h) and the migrant cells were counted under an epifluorescence microscope.

Results

Adhesion assay revealed that HUVEC stimulated with MDA-231 TSFs attached U937 cells 6-fold comparing to the MCF-7 TSFs or control, in a similar way as TNF-α did. The MDA-TSFs were able to increase the HUVEC monolayer permeability exceeded about 30% of the TNF-α induced permeability. A 1.5-fold increase of transendothelial migration cells was observed in HUVEC stimulated with MDA-MB-231 TSFs.

Conclusions

Tumor secreted factors derived from highly metastatic cell line MDA-MB-231 are capable to induce a premetastatic-like endothelial state, increasing the tumor cell transendothelial migration, adhesion to the HUVEC monolayer as well as vascular permeability enhancement.

Clinical trial identification

11-62-2014

Legal entity responsible for the study

Instituto de Investigaciones Biomédicas, UNAM

Funding

Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM), fellowship 509589 from CONACYT.

Disclosure

All authors have declared no conflicts of interest.

Background

In the present study, we attempted to clarify what kind of adhesion molecules and tumor cytotoxic killer activity A-NK cells can express, when cultured for long period in vitro, and then tried experimentally to augment the selective accumulation the A-NK cells into rat mammary tumors, in combination with the prior injection of various kinds of adjuvants into the tumor region. The mechanisms by which the effector cells accumulate in tumor tissue will be discussed.

Methods

1. Animals: Specific pathogen-free (SPF) female rats. 2. Preparation of A-NK cells: A-NK cells were isolated from splenic lymphocytes. 3. Antibodies: Monoclonal antibodies, and Anti adhesion molecule antibodies. 4. Immunohistochemical staining and Flow cytometric analysis. 5. Preparation of mammary tumor bearing rats.

Results

Immunocytochemical and flow-cytometric analysis revealed that most of the A-NK cells strongly expressed lymphocyte-function-associated antigen 1 throughout the incubation. All A-NK cells from 8-150-day cultures, particularly those cultured for 8 days, showed significant cytolytic activity against all targets. Peritumoral injection of various kinds of adjuvant, particularly Freund's complete adjuvant plus bacillus Calmetee-Guerin, resulted in a marked accumulation of A-NK cells in mammary tumor tissues 24 h after injection, and simultaneously in the formation of vessels resembling high-endothelium venules, and expression of the ICAM-1 molecule on the tumor cells in the sites of tumor tissues. When A-NK cells were intravenously administered, significant retardation of tumor growth and prolongation of survival of tumor-bearing rats were observed in the groups that received the prior injection of adjuvants.

Conclusions

These results indicate that the prior injection of proper adjuvant into the peritumoral region is effective for the selective accumulation or infiltration of A-NK cells into the sites of tumor tissues, and results in the marked retardation of tumor growth.

Clinical trial identification

none

Legal entity responsible for the study

School of Rehabilitation Sciences

Funding

None

Disclosure

The author has declared no conflicts of interest.

Background

Expression of nitric oxide synthase (NOS) has been found to correlate with tumour progression in breast cancer, indicating that NO activity may drive malignant growth. Previously we have shown that the stress hormone cortisol acts through a nitric oxide synthase (NOS) mediated pathway to induce production of nitric oxide (NO), and can induce DNA damage in breast cancer.

Methods

Breast cancer cell lines MCF-7 and MDA-MB-231 as well as the mouse mammary tumour cell line 66CL4 were exposed to cortisol and levels of intracellular NO were measured using composite electrochemical sensors. DNA damage was quantified using immunofluorescence and expression of iNOS and metastatic markers VEGF and TWIST were examined using qPCR. An in vivo syngeneic breast cancer model was also used to examine the effect of L-NAME, a NOS inhibitor, on tumour aggressiveness and metastasis in conjunction with daily restraint stress (2hrs) (n = 4/group, repeated in duplicate).

Results

Cortisol significantly increased the expression of iNOS, the generation of NO and DNA damage in breast cancer cells and this was blocked by the NOS inhibitor L-NAME. A significant increase in VEGF and TWIST expression was also observed in response to cortisol. Furthermore, L-NAME also significantly reduced primary tumour growth in stressed mice and reduced the number of metastatic sites/mouse. Tumour microvasculature (as evidenced by CD31 expression) was significantly increased in stressed mice and this was reduced with L-NAME treatment.

Conclusions

We demonstrated that L-NAME, through inhibition of NO signalling, is effective in reducing primary tumour formation and metastatic potential in stressed mice. This data may have impact for patients with breast cancer experiencing extreme stress and further genomic analysis are ongoing.

Legal entity responsible for the study

School of Pharmacy and Biomolecular Sciences, University of Brighton, UK

Funding

Rising star initiative, University of Brighton.

Disclosure

All authors have declared no conflicts of interest.

Background

TP53 gene is the most commonly mutated tumour suppressor in human malignancies. TP53 is mutated in more than 50% of all human cancers, with over 96% of high-grade serous ovarian cancer displaying changes at this locus. Mutations of TP53 gene is associated with malignant transformation and resistance to chemotherapy. In addition, previous studies have shown that ectopic expression of TP53 mutant form in breast cancer cells leads to increased transcription of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR). This enzyme regulates the synthesis of geranylgeraniol which is used to post-translationally modify small GTPase oncogenes. HMGCR is itself considered to be a metabolic oncogene. Statins, which inhibit HMGCR, are potential cancer therapeutics which can cause ovarian cancer (OC) cell apoptosis and regression of xenografts.

Methods

The level of mevalonate pathway (MP) enzymes evaluated in panel of OC cell lines using immunoblotting. in addition, MP enzymes expression were evaluate using qPCR following ectopic expression of wild-type and R248W, R175H, and R273H p53 variants in Skov-3 cells and after inhibition of TP53 expression using siRNA directed to TP53 mRNA in Ovcar-3 cells.

Results

We confirmed that the expression of HMGCR is higher in OC cell lines than in normal epithelial ovarian cells. The level of geranylgeranyl transferase I-β (GGTI-β) and Geranylgeranyl transferase II-β (GGTII-β) was significantly higher in a subset of OC cell lines. The ectopic expression of TP53 variants in Skov-3 cells, which lack endogenous p53 protein, led to significantly increased expression of HMGCR, GGTI-β, GGTII-β and Farnesyltransferase-β (FT-β) enzymes compared to cells transfected with vector. The inhibition of the pre-existing mutations in TP53 encoding R248Q in Ovcar-3 cell line significantly decreased p53 protein and also HMGCR, GGTI-β, GGTII-β and FT-β mRNA.

Conclusions

These data suggest that TP53 mutations play critical role in regulation of the activity of MP enzymes, providing a rationale for the evaluation of the pathway inhibitors such as statins and bisphosphonates in the treatment of OC.

Legal entity responsible for the study

The study was designed by AR and MIA, the experimental work was conducted by MIA and MNA.

Funding

Higher Committee for Education Development in Iraq (MIA ref D-11-296).

Disclosure

All authors have declared no conflicts of interest.

Background

Although less frequent than in older women, breast cancer in very young women (BCVY) (≤35 years old) presents more aggressive and complex biological features. Epigenetic modifications such as miRNA regulation or DNA methylation are reported to play an important role in the onset and progression of cancer. The aim of this work is to identify the epigenetics mechanisms characteristics of BCVY that may be conferring more aggressive features to this group of patients.

Methods

We analysed methylation (Infinium MethylationEPIC BeadChip) from 26 BCVY and 15 samples from women >45 years old. Methylation differences were assessed using Wilcoxon rank sum test. We selected from The Cancer Genome Atlas (TCGA) those genes regulated by significantly different methylated sites and their expression was analysed. MiRNA expression data from TCGA, METABRIC and data previously published from our group was evaluated in a meta-analysis. We then selected those target genes which expression was more affected by miRNA deregulation. Pathway enrichment analysis was performed with most relevant genes from the epigenetic study by Enrichr.

Results

We detected a global hypomethylation profile in BCVY samples and hypermetilation of 502 specific CpG sites exclusive of this group of age. Hypomethylated CpG sites were regulating genes mainly involve in neuronal processes, extracellular matrix and cell communication. Whereas specific hypermethylation was located in genes related to immune system, NOTCH signalling, vesicular trafficking, DNA repair and senescence. MiRNA expression meta-analysis revealed a profile of 22 miRNAs significantly deregulated in BCVY. Pathway enrichment analysis of most affected target genes showed an involvement in neural processes, glucose metabolism, vesicular trafficking, DNA repair, histone and chromatin related proteins, apoptosis, cell cycle, response to DNA damage and senescence.

Conclusions

Our work highlights the presence of epigenomic profile distinctive of BCVY. Both methylation and miRNAs studies points to deregulation of pathways related to neural processes, vesicular trafficking, DNA repair and senescence. All these processes may lead to cancer development and progression, thus genes in these pathways may be potential candidates for further studies.

Legal entity responsible for the study

INCLIVA Research Institute

Funding

None

Disclosure

S. Sanchis: Funded on a FPU predoctoral Fellowship (FPU13/04976) from MINECO, Spanish Government.

G. Ribas: Funded on a Miquel Servet II contract (CPII14-00013) from CarlosIII Health Institute.

M.P. Chilet: Funded by private Patients Fundation LeCado. CIBERONC is an initiative of the Carlos III Health Institute.All other authors have declared no conflicts of interest.

Disclosure

S. Sanchis: Funded on a FPU predoctoral Fellowship (FPU13/04976) from MINECO, Spanish Government.

G. Ribas: Funded on a Miquel Servet II contract (CPII14-00013) from CarlosIII Health Institute.

M.P. Chilet: Funded by private Patients Fundation LeCado. CIBERONC is an initiative of the Carlos III Health Institute.All other authors have declared no conflicts of interest.

Disclosure

S. Sanchis: Funded on a FPU predoctoral Fellowship (FPU13/04976) from MINECO, Spanish Government.

G. Ribas: Funded on a Miquel Servet II contract (CPII14-00013) from CarlosIII Health Institute.

M.P. Chilet: Funded by private Patients Fundation LeCado. CIBERONC is an initiative of the Carlos III Health Institute.All other authors have declared no conflicts of interest.

Background

New diagnostic tools can be useful and give clinical benefits, including diagnosis, prognosis, treatment and monitoring of the disease. A new non-invasive method is the study of liquid biopsies, performed in fluids containing cell-free circulating DNA (cfDNA). Analyses with digital PCR technique (dPCR) allows to establish the levels of cfDNA as well as the absolute quantification of mutant alleles with accuracy. This system scatters the sample among twenty thousand wells (microfluids on the chip), where amplification reactions occur independently and are recorded by a reader.

Methods

Peripheral blood samples were obtained from breast cancer patients and healthy controls. From each sample, the cfDNAs were extracted from plasma using the MagMAX™ Cell Free DNA Isolation Kit and dPCR was done for quantification of samples. We evaluated two genes, PUM1 and RNaseP. For amplification of fragments, master mix 1X (Applied Biosystems) and PCR detection assays of both genes were combined with 1.5 μl of plasma cfDNA, dispersed in the chips and placed in a ProFlex ™ thermocycler (Applied Biosystems) following the program pre-established by the manufacturer, with additional five cycles. Finally, quantification data were obtained with QuantStudio® 3D AnalysisSuite™ Cloud Software. Comparison of DNA concentration in copies per microliter and other statistical calculations were performed with InfoStat 2015.

Results

Significant differences were found in the values of cfDNA between patients and controls for PUM1 (p = 0.0001) and RNase P (p = 0.0003). These results allowed to establish cut-off points between groups at 78,995 and 51,154 copies/uL, respectively. These values can be considered in the classification of groups for further analysis of others samples. Statistical support for the use of markers in diagnosis was also evaluated using the ROC curve that favors the PUM1 marker, with a sensitivity of 75% and a specificity of 95.2%.

Conclusions

Based on the significant differences found between breast cancer patients and controls, cell free DNA is a good biomarker that can be used in the diagnostic of breast cancer. On the other hand, digital PCR has been established as a good tool to check cfDNA levels from plasma of breast cancer patients.

Legal entity responsible for the study

Jose Buleje

Funding

Programa Nacional para la competitividad y Productividad (Innovate Perú)-No.138-PNICP-PIAP-2015, Universidad de San Martin de Porres, Oncosalud - AUNA

Disclosure

All authors have declared no conflicts of interest.

Background

A significant portion of hereditary predisposition to breast cancer (BC) is attributed to yet unknown factors. Russian population is characterized by surprisingly strong founder effect, therefore whole exome sequencing (WES) for a limited number of these genetically homogenous patients has a potential to identify novel BC-predisposing genes.

Methods

WES was performed for 32 Russian BC cases, which demonstrated strong clinical signs of the hereditary disease (family history, BC bilaterality, young onset) and lacked germline mutations in “canonical” BC genes (BRCA1, BRCA2, CHEK2, PALB2, and NBS1/NBN).

Results

Eight patients carried potentially pathogenic mutations in BRCA1 network genes (3 truncations (BLM p.Q548*, RAD51C c.904 + 1G>A, FANCM p.S497fs) and 5 missense mutations (FANCM (p.R100W, p.Q891P), ERCC4 p.R799W, RAD54L p.R394W, RAD50 p.D515G)). The remaining 24 patients were analyzed for the presence of rare non-silent genetic variants; in total, 15437 alleles had ExAC frequency <1%. Use of ACMG-guided bioinformatic pipeline classified these variants for 64 ‘pathogenic/likely-pathogenic’, 12844 of ‘uncertain significance’, and 2529 ‘benign/likely-benign’. Prevalence of 69 top candidates was compared in 640 genetically enriched BC patients, 1200 consecutive BC, 1200 middle-aged healthy females and 460 elderly healthy women. Several likely pathogenic mutations were overrepresented in the BC groups: nonsense PZP p.R680* [9/1845, OR = 13.2]; heterozygous missense LEPREL1 p.P636S [6/1778; OR = 4.4] and ING1 p.P319L [3/1792; OR = 3.9]; homozygous missense BRCA1 p.Q356R [12/1683; OR = 5.5] and EXO1 p.G759E [9/1606, OR = 4.3]. Some potentially pathogenic variants occurred only in the index cases but were absent in other BC patients (rare ExAC alleles: HELLS p.R53C and TP53INP1 p.E27D; newly identified variations: MLH3 p.C1393F, EMSY p.G934* and ATRIP p.R760*).

Conclusions

This study revealed several alleles, which may be associated with increased BC predisposition. However, in contrast to well-known Slavic BRCA founder mutations, newly identified candidates are exceptionally rare and therefore are unlikely to be responsible for a significant share of BC morbidity. Supported by the RSF grant No 16-45-02011

Legal entity responsible for the study

Evgeny N. Imyanitov, Head of the Department of Tumor Biology in the N.N. Petrov Institute of Oncology

Funding

Russian Scientific Fund (grant No 16-45-02011).

Disclosure

All authors have declared no conflicts of interest.

Background

Breast cancer in young women (under 35 years) (BCVY) often presents distinct clinic-pathological features: more aggressive phenotype and worse prognosis than older women. Genomic and molecular alterations play a significant role in breast cancer biology. Due to the low incidence of BCVY (2-5%) these women are underrepresented in most molecular studies. This work presents a comprehensive study of the transcriptome in BCVY, focusing in the search of gene expression biomarkers characteristic of this group of patients.

Methods

We analysed the transcriptome by Clariom™ D (Affymetrix) from 31 BCVY and 11 samples from women >45 years old. Global gene expression was filtered and normalized by RMA method. After initial pre-processing we analysed expression in 3,639 mRNAs, 66,457 lncRNA and 3,271 pre-miRNA and differences were assessed using t-test. We performed a meta-analysis with gene expression data from The Cancer Genome Atlas (TCGA) for validation of results. Pathway enrichment analysis was performed by Enrichr.

Results

showed a specific transcriptomic landscape in BCVY. Clariom D study revealed 134 significant mRNA with p-value< 0.05 that pointed out towards pathways related with olfactory receptors, GPCR signalling, tight junction and cell-cell communication. After meta-analysis with TCGA gene expression data and own data, 43 genes were statistically significant and 15 of those withstood FDR correction (FDR< 0.05). Among those we found PIK3CB, HOXD10, ZNF654, TMEM204, IRX5, PF4, MAGEA2 and TSR2 deregulated in BCVY compared with older women. Pathway enrichment analyses and GO search highlight pathways related to cell-cell communication, cancer processes, chemokine and PI3K signalling pathways, cell differentiation, extracellular matrix, vesicular trafficking, neuronal processes among others.

Conclusions

We find the presence of a distinctive transcriptomic profile of the BCVY samples. Our study points to deregulation of pathways related to cell migration, proliferation and differentiation that promote cancer development and progression. Genes obtained in meta-analyse might be potential target genes for further studies in BCVY which could help to clarify the biological background for the development of the disease in this group of age.

Legal entity responsible for the study

INCLIVA Instituto de investigación

Funding

None

Disclosure

S. Sanchis: Funded on a FPU predoctoral Fellowship (FPU13/04976) from MINECO, Spanish Government. G. Ribas: Funded on a Miquel Servet II contract (CPII14-00013) from CarlosIII Health Institute. M.P. Chilet: Funded by private Patients Fundation LeCado. CIBERONC is an initiative of the Carlos III Health Institute. All other authors have declared no conflicts of interest.

Background

Castration resistant prostate cancer (CRPC) is a rapidly progressing disease state for which there is no cure. The constitutively active androgen receptor (AR) splice variant AR-V7 represents a well-established mechanism of therapeutic resistance and disease progression. This variant lacks the AR C-terminal ligand binding domain and, as such, is not inhibited by androgen deprivation therapy. Designing high-affinity drugs to target the amino terminus of AR and AR-V7 is a major challenge due to the intrinsic disorganized structure of this region. Thus, there is an imperative need to identify novel AR-V7 hub genes in PC that may serve as novel therapeutic targets.

Methods

We performed a highly robust gene expression meta-analysis on PC patient samples. We defined gene modules correlated with PC progression using a Weighted Gene-Co-expression Network Analysis (WGCNA), a powerful systems biology approach. Further, we identified AR-V7 downstream target genes using gene expression profiling and mapped the AR-V7 functional interactome for the first time using a novel high-throughput synthetic genetic array screen in yeast. Finally, we combined the results from our three independent system-level analyses with experimental data to identify hub genes that were upregulated in PC patients, upregulated by AR-V7, and that also functionally interacted with AR-V7.

Results

The identified genes not only included select genes previously linked to PC, such as members of the topoisomerase and cyclin families, but also novel genes that had not been previously linked to PC progression. The identified gene-signature expression correlated with patients’ Gleason score and had a prognostic value that predicted disease free-survival at the time of patient biopsy in large independent cohorts.

Conclusions

In sum, we show here an unbiased integrated system-level analysis of AR-V7 networks, where we combined bioinformatic analysis of patient samples and cell-based approaches to identify new candidate genes in CRPC that may serve as novel prognostic markers and future targeted therapies.

Legal entity responsible for the study

University of Miami Miller School of Medicine

Funding

Sylvester Comprehensive Cancer Center

Disclosure

All authors have declared no conflicts of interest.

Background

Androgen receptor (AR) plays a central role in prostate cancer and continues to be a driver in castration-resistant prostate cancer (CRPC), with increased AR expression in most cases. Approximately half of the men with CRPC respond initially to abiraterone or enzalutamide, but most relapse within 1 to 2 years. Majority of the abiraterone and enzalutamide-resistant tumors have still high AR expression and persistent AR activity. Several precursor steroids, like testosterone (T) and dihydrotestosterone activate AR, are synthesized in adrenal glands and de novo in tumours. CYP11A1 (cytochrome p450scc) is a mitochondrial enzyme catalysing the conversion of cholesterol to pregnenolone (Preg), which is the first rate-liming step in steroid hormone biosynthesis. ODM-208 is a novel, oral, non-steroidal and selective inhibitor of CYP11A1 enzyme and suppresses the synthesis of all steroid hormones and precursors.

Methods

The inhibition of CYP11A1 was measured in vitro by detecting the formation of radiolabelled isocapronic acid in a human adrenal cortex cell line (H295R), and further analysing Preg and T formation by ELISA. Inhibition of the adrenal and testicular hormone production in vivo was tested in the intact male rat assay by analysing plasma concentrations of progesterone (P), corticosterone (C) and T (with LS-MS/MS) after single oral dose of ODM-208. The tumor growth inhibition was studied by using androgen dependent VCaP cells, which were subcutaneously grafted to intact male nude mice. When tumor volumes reached on average 200 mm3, mice were castrated, and after regrowth of the tumors, the oral treatment of ODM-208 was started.

Results

ODM-208 potently inhibits CYP11A1 enzyme and formation of Preg and testosterone with low nM concentrations in vitro. In male rats, clear decreases of P, C and T concentrations can be detected already after single oral administration of ODM-208. In the murine VCaP CRPC xenograft model ODM-208 significantly inhibited tumor growth.

Conclusions

ODM-208 shows promising antitumor activity in preclinical CRPC models and suggests that ODM-208 may have the potential to be an effective treatment in CRPC. Clinical trial in patients with metastatic CRPC is planned to be started in the 2018.

Legal entity responsible for the study

Orion Corporation Orion Pharma Orion Corporation Orion Pharma

Funding

Orion Corporation Orion Pharma

Disclosure

R. Oksala, M. Karimaa, M. Ramela, R. Riikonen, P. Rummakko, G. Wohlfahrt, A. Vuorela, M.V. Mustonen, P. Kallio: Employee: Orion Corporation Orion Pharma. All other authors have declared no conflicts of interest.

Background

Aberrations in the ETS transcription factor family members are a common feature of multiple cancers including prostate cancer (PCa), such as the TMPRSS2:ERG fusion. ELF3, also known as ESE-1, is an epithelial-specific ETS transcription factor involved in regulating cell differentiation in various tissues, however, its role in the prostate is controversial. The aim of this study was to identify the function of ELF3 in normal prostate development and to explore its role in PCa.

Methods

Three model systems were used: prostate cell lines, primary prostate epithelial cells cultured from patient tissue and paraffin-embedded human tissue sections. The function of ELF3 was investigated using knockdown via siRNA transfection and overexpression via lentivirus transduction. Western blots, immunofluorescence and immunohistochemistry were used to measure protein localisation and levels of expression. Other assays measured cell viability, colony forming ability and migration.

Results

ELF3 expression was restricted to the basal layer of the normal prostate epithelium and was not expressed in stroma. Analysis of a prostate tissue microarray indicated that whilst ELF3 is expressed in benign prostate tissue, its expression is lost in low-grade prostate tumours and re-expressed in some more advanced tumours. ELF3 knockdown resulted in decreased migration, cell viability and did not induce stem cell characteristics, whilst promoting basal cell gene expression. ELF3 overexpression increased cell migration. ELF3 was induced in primary prostate epithelial cells following treatment with the clinically approved HDAC inhibitor Vorinostat, which can promote neuroendocrine differentiation.

Conclusions

ELF3 expression correlates with the normal prostate epithelial cell differentiation hierarchy, and may have a role in advanced PCa. Analysis of total gene expression following knockdown of ELF3 will give an indication of transcriptional networks that are regulated by ELF3. In addition, a lentivirus that expresses an ELF3 mutant, which alters its localisation, will be used to assess any cytoplasmic function of ELF3. This comprehensive and clinically relevant approach will allow complete elucidation of the role of ELF3 in prostate cell differentiation and PCa.

Clinical trial identification

N/A

Legal entity responsible for the study

Norman Maitland

Funding

Prostate Cancer UK (PCUK) registered charity

Disclosure

All authors have declared no conflicts of interest.

Background

Inhibition of androgen signalling remains the therapeutic mainstay in castrate-resistant prostate cancer. Retention of active AR signalling or acquisition of splice variants have been reported as mechanisms of resistance to the anti-androgen Enzalutamide. Other non-AR dependent mechanisms of resistance have also emerged including acquisition of a hypoxic microenvironment. We propose treatment-induced hypoxia and the induction of angiogenesis may define a novel mechanism of relapse to Enzalutamide.

Methods

Preclinical experiments were conducted in LNCaP tumors and established human prostate cancer cell lines. Tumour growth, intra-tumoral hypoxia and blood vessel density were measured in vivo. AR expression, activation and target gene expression were measured in vitro. Effects of Enzalutamide on hypoxia-driven, disease-progressing pathways and genes of interest and the role of these genes in resistance to Enzalutamide was investigated.

Results

Enzalutamide promoted persistent hypoxia in LNCaP tumours in vivo, followed by increased blood vessel density and restoration of oxygen tension (>14 days). In vitro, hypoxia increased AR expression and transcriptional activity in LNCaP cells and sustained but did not further potentiate high basal AR and ARv7 activity in 22Rv1 cells. Enzalutamide failed to attenuate the concurrent hypoxia-induced HIF-1 and NF-κB signalling, resulting in up-regulation of disease-progressing genes and pathways. Administration of neutralizing antibodies to two hypoxia-regulated genes, IL-8 and VEGF prolonged Enzalutamide-mediated LNCaP tumour growth control over 28 days in vivo (p < 0.001) and re-sensitised enzalutamide-resistant LNCaP cells in vitro.

Conclusions

Enzalutamide-induced hypoxia upregulates the expression of VEGF and IL-8, whose multi-model signalling effects contribute to microenvironment-promoted resistance in prostate tumours.

Legal entity responsible for the study

David Waugh

Funding

Prostate Cancer UK

Disclosure

J. Worthington: Scientific director at Axis Bioservices. D. Waugh: Consulting/advisory role at Almac Diagnostics, Craigavon, Northern Ireland. All other authors have declared no conflicts of interest.

Background

Cell-free DNA (cfDNA) has been known to be released from tumor cells and evaluated potential biomarkers for therapeutic responses. However, the role of cfDNA in pancreatic cancer has not been well studied. Here we selected KRAS mutation which has been known common over 95% of pancreatic ductal adenocarcinoma (PDA) and evaluated applicability as a prognostic marker through the quantitative analysis of cfDNA and KRAS mutation in the patients with PDA.

Methods

Total of 106 PDA patients were enrolled in the study. The concentration and fraction of KRAS mutation were measured by KRAS screening multiplex droplet digital PCR kit (Biorad, USA) in plasma samples.

Results

KRAS mutation was detected in 97.4% of tissue samples and the correlation with cfDNA was 0.561 with 80.5% positivity. KRAS mutation concentration and fractional abundance showed the association with poor survival in both PFS (P <.001 and P = 0.001) and OS (P = 0.003 and P = 0.006) in the entire stage groups. Specially, the impact for survival of KRAS mutation concentration and fractional abundance was obvious in PFS in resectable group (P = 0.016 and P = 0.02). When we analyzed the receiver operating characteristic (ROC) curve to determine whether KRAS mutation in cfDNA have additive benefits with well-known tumor markers CA19-9, combined with KRAS mutation concentration or KRAS fractional abundance, the value of area under the curve (AUC) was significantly higher than the value calculated as CA19-9 alone.

Conclusions

This study represents that KRAS mutation concentration and fractional abundance in cfDNA could be prognostic marker in pancreatic cancer especially in resectable group.

Legal entity responsible for the study

Sun-Young Kong

Funding

This work was supported by grants from the National Cancer Center of Korea (NCC-1510203).

Disclosure

All authors have declared no conflicts of interest.

Background

AKT/PKB is a protein kinase that plays a key role in cancer, which is expressed as 3 isoforms: AKT1 (PKBα), AKT2 (PKBβ) and AKT3 (PKBγ). Although these isoforms are remarkably similar, there is evidence that each isoform yields specific functions which may vary depending on the cell type. Even so, the underlying molecular pathways specifically regulated by each one of them are unknown.

Methods

To gain insight into the role of each isoform in the biology of human pancreatic adenocarcinoma cells, we have silenced each AKT isoform individually using short hairpin RNAs (shRNAs) delivered by lentiviral transduction. Cells transduced with an unspecific shRNA were used as controls. Then, high-throughput quantitative proteomic analyses were performed to evaluate the differential signaling routes altered by silencing of each AKT isoform.

Results

AKT1 silencing induced the upregulation of 57 proteins and downregulation of 58. AKT2 silencing up-regulated and down-regulated 78 and 101 respectively. AKT3 silencing resulted in the upregulation of 88 and downregulation of 93. The expression levels of 45 proteins were altered exclusively after AKT1 knockdown, while 74 proteins and 89 were specifically altered for AKT2 and AKT3 silencing, respectively. AKT1 silencing up-regulated RNA splicing, GPCR and mTOR pathways, and mitochondrial functions such as the respiratory chain, fatty acid metabolism or mitochondrial DNA synthesis. Pathways related to apoptosis and cell migration were inhibited. AKT2 silencing caused the activation of pathways related to apoptosis, splicing, protein folding and some mitochondrial functions. In contrast, other key metabolic pathways such as nucleic acid synthesis, pentose phosphate pathway, cell adhesion and PI3K signalling were down-modulated. Lastly, AKT3 silencing induced increased splicing and mitochondrial functions, regulation of gene expression and snRNA processing. In this case, the pentose phosphate pathway, cell adhesion, apoptosis, protein synthesis and nucleic acid synthesis were also inhibited.

Conclusions

AKT isoforms have specific functions in pancreatic adenocarcinoma. The individual silencing of each isoform induces a differential alteration of molecular pathways involved in main cellular processes.

Legal entity responsible for the study

D. Escors

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Id1 is an independent prognostic factor in NSCLC-AD. Id1 silencing impairs cell viability and migration of NSCLC-AD cell lines. KRAS is the most frequently mutant gene in NSCLC with no specific therapies clinically available. Here we evaluate Id1 as a potential chemopreventive and therapeutic target in a humanized mouse model of KM NSCLC-AD.

Methods

Several human lung AD cell lines with known mutations (H1792-604, H2009, H358, H1568, H1437, H1703 and H2126) were selected for Id1 silencing using inducible short hairpin RNA (shRNA). Humanized AD xenograft mouse models were generated by subcutaneous injection of H1792-604 and H2009 cell lines (Id1 silenced or Id1 wild type) in flanks of immunodeficient mice. Id1 silencing was activated at the time of tumor cell inoculation (chemoprevention assay) or once the tumors were established (therapeutic assay).

Results

Id1 inhibition was achieved in all selected cell lines compared to their controls. In vivo, in the chemoprevention assay we observed a significant decrease in tumor volume in mice injected with Id1 silenced H1792-604 cells (60% ± 32.39) compared to the control group (356.29% ± 115.32) (p < 0.001). Moreover, mice injected with Id1 silenced H2009 cells never developed tumors compared to control mice (168.35 ± 68.71) (p < 0.001). In the therapeutic assay, the activation of inducible silencing of Id1 in established tumors induced a significant reduction of tumor volume in both xenograft models. Id1 inhibition induced a partial response in 40% of the tumors after injection of H1792-604 cells and in 100% of tumors in H2009 inoculated mice.

Conclusions

These findings encourage further evaluation of Id1 as a potential therapeutic target in KM NSCLC-AD patients.

Legal entity responsible for the study

Clínica Universidad de Navarra

Funding

Clínica Universidad de Navarra

Disclosure

All authors have declared no conflicts of interest.

Background

Platinum-based chemotherapy still represents the standard first-line approach for NSCLC patients, although primary or secondary resistance is frequently observed. Recently the Shh pathway has been associated with resistance to platinum-based chemotherapy in NSCLC. The aim of this work is to investigate whether a combined treatment with cisplatin and the Hedgehog-pathway inhibitor vismodegib could potentiate the anti-tumour effect and to explore possible mechanisms of this synergy.

Methods

Two Human NCSLC cell lines A549 and H460 were treated with single agent Cisplatin, single agent Vismodegib and a combination of the two drugs. MTT cytotoxicity assays were performed and the data were analysed with CompuSyn software. Experiments of apoptosis and cell cycle were done by using flow cytometer. Immunofluorescence with lysoTracker as well as western blot (WB) analysis for the LC3B protein were performed to analyse autophagy.

Results

The CompuSyn analysis showed an important synergistic effect of cisplatin + Vismodegib. Combined treatment induced a significant increase in cellular apoptosis compared with single agent cisplatin. The cell cycle analyses revealed a block in S-phase with the combination treatment. The lysoTracker immunofluorescence assay showed that cisplatin induces an increase of autophagy, while the combination with vismodegib strongly reduces it, finally reverting this effect. These findings were confirmed by WB analysis for LC3B which is significantly increased by single agent cisplatin and reduced by the combined treatment.

Conclusions

Combined treatment with cisplatin and vismodegib has a synergistic effect with an increase in cancer cell apoptosis. Autophagy has been described as a mechanism through which cancer cells escape cisplatin-induced cytotoxicity. Combining cisplatin with vismodegib leads to an inhibition of autophagy, so that it could suggest a new therapeutic approach.

Legal entity responsible for the study

Università della Campania “Luigi Vanvitelli”

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Many studies have found leptin related genes involved in tumorigenesis and have suggested it may play a role in the pathogenesis of lung cancer. Leptin Receptor (LEPR) is expressed in many tissues and cells, including lung mucosal cells. LEPR was reported to be associated with tumor cell proliferation and angiogenesis. Our objective has been to estimate the association between the rs4567312-LEPR gene and lung cancer incidence in a Mediterranean population.

Methods

We analyzed 1094 participants (398 men, 696 women) recruited in the PREDIMED-Valencia Study. Participants were high cardiovascular risk subjects aged 67±6 years at baseline. PREDIMED is a multicenter randomized, controlled trial aimed at assessing the effect of the Mediterranean diet (MedDiet) on cardiovascular prevention (primary outcome). Cancer incidence was a secondary outcome in this trial. Demographic, clinical, life-style, biochemical, and genetic variables were obtained. Subjects were followed-up prospectively from 2003 to 2014 (in the extended-follow-up).

Results

We detected 12 new cases of lung cancer from 2003 to 2014 (1.1% cumulative incidence). Tobacco smoking was strongly associated with lung cancer incidence (91.7% of current or former smokers in lung cancer subjects vs 41.5% in the non-cancer participants (p = 0.001). In the whole population, prevalence of the rs4567312 polymorphism was: 95.5% CC, 4.4% CT and 0.1% TT. We also detected in the whole population an association between this polymorphism and plasma leptin concentrations, 26.9±22.9 ng/mL in CC vs 18.4±16.7 ng/mL in T carriers (p = 0.013). We found a strong association between the rs4567312-LEPR polymorphism and lung cancer risk, being higher in carriers of the T-allele. This association remained statistically significant (OR = 7.61; 95% CI: 1.74-33.37 for T-carriers vs CC) even after adjustment for gender, age, tobacco smoking, dietary intervention group (MedDiet vs control diet) and leptin levels.

Conclusions

T-carriers allele in the rs4567312-LEPR polymorphism presented a higher incidence of lung cancer in this Mediterranean population even after adjustment for tobacco smoking and dietary intervention.

Clinical trial identification

Controlled-trials.com number ISRCTN35739639

Legal entity responsible for the study

Instituto de Salud Carlos III and University of Valencia

Funding

Instituto de Salud Carlos III

Disclosure

All authors have declared no conflicts of interest.

Background

Lung cancer (LC) is causing more than 1.3 million deaths worldwide annually. Early detection of LC is critical for survival but despite recent advancements in LC diagnostics most patients are still diagnosed at advanced stages of the disease. The situation is further complicated by high intratumor heterogeneity and general diversity of lung malignancies. Insights into cancer genetics have kindled interest in molecular cancer diagnostics. One of the lucrative sources of prospective LC biomarkers is cell-free circulating miRNAs. These small non-coding RNAs are frequently deregulated in LC. It is also known that miRNAs can travel in bodily fluids for extended periods of time, shielded from degradation by membrane vesicles or other biopolymers. Recently, specific subsets of miRNAs associated with tumor phenotypes and disease progression have been found circulating in blood of cancer patients and suggested as potential biomarkers for LC.

Methods

In the present study, we have investigated the profiles of circulating miRNAs in blood plasma of LC patients and healthy individuals (HD) in order to identify potential markers for lung cancer diagnostics. Small RNAs were isolated from blood plasma of 20 LC patients and 10 healthy individuals (HD) using protocol reported earlier (Zaporozhchenko et al, Anal Biochem, 2015). Profiles of miRNA expression were obtained using miRCURY LNA miRNA qPCR Panels Plasma/Serum (Exiqon). Ratio based normalization was applied to all miRNA’s with call rate higher than 80%.

Results

Statistical comparison using two-way ANOVA identified 241 ratios (98 individual miRNAs) with significantly different expression between LC patients and HD (p < 0.05 after Benjamini-Hochberg correction). Using LASSO penalization models and manual filtering of miRNAs associated with haemolysis, 7 miRNA ratios were identified as best predictors of cancer. Extended set of miRNAs (n = 19) was selected for further verification in an independent sample of 30 LC patients, 20 HD and 10 patients with hyper- and metaplastic endobronchitis using custom miRCURY LNA miRNA qPCR Pick & Mix Panel.

Conclusions

Based on expression in both data sets 5 ratios containing 7 miRNAs were selected for further validation in an extended cohort of LC and cancer-free individuals.

Legal entity responsible for the study

Laboratory of Molecular Medicine, SB RAS Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russian Federation

Funding

Study has been supported by Russian Foundation for Basic Research (RFBR, grant No. 14-04-01881), BOR grant VI.62.1.4, and Presidium of RAS research program ‘Molecular and Cellular Biology’ No. 6.1.

Disclosure

All authors have declared no conflicts of interest.

Background

DNA methylation is an epigenetic determinant of gene expression. Cytochrome P450 (CYP1A1) is a phase 1 xenobiotic metabolizing enzyme. Glutathione S-transferase P1 (GSTP1) detoxifies metabolites and regulates cellular chemical stress and death. Methylation changes in these genes could play a role in the incidence of cancer. Our aims were to analyze DNA-methylation of selected CpG islands in the CYP1A1 and GSTP1 genes at baseline in subjects who had incident cancer and paired controls, and to determine if DNA methylation in cancer patients changed from baseline to the time close to cancer diagnosis.

Background

DNA methylation is an epigenetic determinant of gene expression. Cytochrome P450 (CYP1A1) is a phase 1 xenobiotic metabolizing enzyme. Glutathione S-transferase P1 (GSTP1) detoxifies metabolites and regulates cellular chemical stress and death. Methylation changes in these genes could play a role in the incidence of cancer. Our aims were to analyze DNA-methylation of selected CpG islands in the CYP1A1 and GSTP1 genes at baseline in subjects who had incident cancer and paired controls, and to determine if DNA methylation in cancer patients changed from baseline to the time close to cancer diagnosis.

Methods

We followed-up 1094 subjects (aged: 67±6years) of the PREDIMED-Valencia study prospectively from 2003 to 2014. Cancer incidence was a secondary outcome in our trial. We analyzed 69 cases of incident cancer (lung, breast and colon) during this follow-up period, and 69 paired controls free of cancer. We analyzed DNA-methylation levels of the CYP1A1 and GSTP1 genes at baseline in both groups. Quantitative DNA methylation analysis was undertaken by MALDI-TOF mass spectrometry. We evaluated methylation levels on chromosome 15 (region: 74726090 -74726460 base pairs from promoter) and chromosome 11 (region: 67583556-67583896 base pairs from promoter).

Results

We detected statistically significant differences in DNA methylation levels at baseline in the CYP1A1 and GSTP1 genes between cancer cases and controls (P=0.008 for the CpG site 26-27 of the CYP1A1 gene and P=0.049 for the CpG 10-11 island at the GSTP1 gene). We detected statistically significant changes in DNA methylation prospectively in cancer patients. DNA methylation at the CpG 4 (CYP1A1 gene) was 0.020±0.034 at baseline vs 0.006±0.013 close to cancer diagnosis (P=0.044). For the GSTP1 gene, methylation of the CpG 34-39 prospectively increased from 0.327±0.046 at baseline to 0.345±0.053 (P=0.014) close to cancer diagnosis.

Conclusions

We have detected differences in DNA-methylation of selected CpG Islands of the CYP1A1 and GSTP1 genes at baseline, between future diagnosed cancer subjects and cancer-free controls. Moreover, in patients subsequently diagnosed with cancer, a change in DNA methylation was observed between baseline and close to the time of cancer diagnosis. This suggests a dynamic influence of DNA methylation that could be modulated for prevention.

Clinical trial identification

Controlled-trials.com number ISRCTN35739639

Legal entity responsible for the study

Spanish Government's Instituto de Salud Carlos III and University of Valencia

Funding

Instituto de Salud Carlos III

Disclosure

All authors have declared no conflicts of interest.

Background

Cancer cells require increased glucose supply to sustain proliferation. One mechanism involves increased expression of glucose transporter (GLUT) genes. But insulin has revealed that protein phosphorylation is another key mechanism in glucose uptake regulation: insulin binding to responsive cells triggers a signalling cascade with phosphorylation of TBC1D4, a negative regulator of endosomal GLUT trafficking, so that more transporters are inserted into the plasma membrane. Previous work from the host lab has identified the family of WNK protein kinases and shown that WNK1 can also phosphorylate TBC1D4 and promote GLUT translocation to the cell surface. Our objective is to understand the contribution of WNK1 to glucose uptake in colorectal cancer cells. Our objective is to understand the contribution of WNK1 to glucose uptake in colorectal cancer cells.

Methods

To characterize the role of WNK1, various colorectal cell lines were first cultivated with different glucose concentrations. Levels of GLUT1 at the cell surface were compared under these conditions and the effect of depleting WNK1 expression by siRNA determined.

Results

For selected conditions, key cell cycle or apoptotic marker proteins were analysed by Western blot and revealed higher apoptotic and cell-cycle arrest phenotypes in WNK1-depleted cells cultured in low glucose medium. In order to dissect key phosphorylation events involved in GLUT1 regulation, mass spectrometry analysis revealed that WNK1 phosphorylates TBC1D4 and the functionally related TBC1D1 at unique Serine residues. The corresponding phospho-mimetic mutants are currently being tested for their ability to increase GLUT1 translocation.

Conclusions

Together, these studies will elucidate the molecular details regulating the translocation of glucose transporters in cancer cells and have the potential to identify novel therapeutic targets.

Legal entity responsible for the study

Peter Jordan

Funding

Fundação para a Ciência e Tecnologia BioISI - Biosystems and Integrative Sciences Institute

Disclosure

All authors have declared no conflicts of interest.

Background

In colon cancer distinct genetic subtypes have been described, one of which involves overexpression of RAC1b, a variant generated by alternative splicing. Aberrant splicing is known to occur in cancer and can be caused by mutation in a gene or splicing factor but also represents a dynamic response to oncogene-induced cellular signaling and in this case it may be pharmacologically targeted. Here we explore how signaling pathways are involved in the deregulation of alternative RAC1b splicing in colorectal tumor cells.

Methods

HT29 colorectal cells represent serrated colorectal tumors with BRAF gene mutation V600E in one allele and RAC1b overexpression. Cells were transfected with shRNA vectors directed against target candidate protein kinase transcripts and their effects on RAC1b levels analyzed 24h later by Western Blot and qRT-PCR. Treatment with kinase inhibitors or anti-inflammatory drugs was performed 24h prior to cell lysis.

Results

Two kinases, SRPK1 and GSK3β, were found required to sustain RAC1b levels and both were shown to act upon the phosphorylation of splicing factor SRSF1, which binds to and promotes the inclusion of the alternative exon in RAC1b. SRPK1 knockdown or pharmacological inhibition reduced SRSF1 phosphorylation decreasing its nuclear translocation and concomitantly RAC1b splicing. The same regulatory pathway was also found to be controlled by GSK3β. Interestingly, GSK3β phosphorylation was identified to serve as target for the anti-inflammatory drug ibuprofen, which inhibits RAC1b overexpression.

Conclusions

Together, our results demonstrate that oncogenic signal transduction pathways deregulate alternative splicing and this may be drug revertable.

Legal entity responsible for the study

Peter Jordan

Funding

Fundação para a Ciência e Tecnologia

Disclosure

All authors have declared no conflicts of interest.

Background

Foreseeing treatment outcome in cancer patients is still a challenge that needs to be addressed. Tumors are complex structures, where the interaction between the tumor cells and the surrounding microenvironment regulates key processes in cancer progression, such as angiogenesis, evasion and modulation of the immune system response, and invasiveness. These interactions confer tumors a high heterogeneity not only inter-patient but also intra-patient. In vitro experimental models have been developed to preserve this heterogeneity present on tumor biopsies by the use of rotary wall and perfused bioreactors. However, the complexity and size of the bioreactors prevent from visual inspection of the sample and the realization of a high-throughput screening. The present work focuses on the combination of microfabrication techniques and microfluidics to downsize classic experimental models. The developed methodology requires only microliter size sample, and allows real time optical inspection.

Methods

A microscopy slide size optically transparent microfluidic bioreactor (µbioreactor) was designed and developed to preserve high cellularity on complex samples through constant perfusion. Colorectal carcinoma (CRC) biopsies were taken after previous patient consent was obtained. CRC biopsies were perfused by cell culture media in the µbioreactor during one week. After perfusion, CRC biopsies were histologically processed, stained and characterized by immunofluorescence.

Results

High cellularity was observed in CRC biopsies after one week of perfusion. Stromal and parenchymal preservation was confirmed by both, histological staining and immunofluorescence.

Conclusions

The use of microfluidic bioreactors can be successfully used to preserve CRC biopsies, maintaining cell heterogeneity while allowing optical inspection. The use of small sample volumes (microliters) allows high throughput screening using regular biopsy samples, a key feature to achieve personalized treatments in cancer.

Legal entity responsible for the study

University of Zaragoza

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

The colorectal cancer is one of the most common cancers in the world being the main cause of death the metastatic spread to the liver. During metastatic progression, a stromal and tumor cell crosstalk mediated by hepatic ICAM-1 and tumor LFA-1 interaction modulate the tumor microenvironment through an inflammatory and immune response. Additionally, a matrix remodeling and angiogenic response is also associated with tumor progression. The main cell type associated to these processes is the fibroblast associated to the tumor. Thus, the aim of this work is to elucidate the effect of ICAM-1/LFA-1 interaction during the angiogenic and desmoplasic response during liver metastatic progression.

Methods

To do so, the effect of ICAM-1/LFA-1 interaction on the tumor progression and recruitment of cancer associated fibroblasts was analyzed by an experimental metastasis assay in vivo and a modified Boyden chamber migration assays in vitro, after either activation of tumor cells with sICAM-1 or blocking of ICAM-1/FA-1 interaction. In addition, the effects of LFA-1 tumor depletion on tumor migratory potential induced by tumor-activated fibroblasts derived factors were analyzed. Also, the effect of the modulation of this pathway on MMPs protein and angiogenic gene expression levels was measured by zymography and qPCR, respectively.

Results

In vivo and in vitro assays showed an increase on fibroblast and tumor cells recruitment after activation of tumor LFA-1 activation by binding with sICAM-1 which was abrogated after blocking of LFA-1. Moreover, the expression levels of MMPS and other proangiogenic factors were decrease after the blockage of ICAM-1/LFA-1 interaction. Also, the collagen deposition was increased after LFA-1 activation and diminished by LFA-1 blocking.

Conclusions

The interaction of ICAM-1 with tumor LFA-1 favors the recruitment of fibroblast within the tumor mediated by a modulation of pro-desmoplasic factors. This favors the remodeling of the tumor stroma and the angiogenic response and promotes tumor metastatic progression. Thus, LFA-1ICAM-1 interaction might be pointed out as a potential target for therapy of the metastatic disease.

Legal entity responsible for the study

Department of Cellular Biology and Histology, University of the Basque Country, School of Medicine and Nursery

Funding

Basque Government

Disclosure

All authors have declared no conflicts of interest.

Background

Metastasis is the main cause of death for most solid tumors. The liver is a metastasis-susceptible organ and represents the first most common site for colorectal cancer (CRC). During tumor progression, the unique hepatic microenvironment suffers a reorganization involving the interaction between the tumor, the different hepatic stromal cells and the extracellular matrix (ECM), which is under an extreme remodeling. Among the receptors involved, discoidin domain receptors (DDR-1 and -2), a class of tyrosine kinase receptors for fibrillar collagen, are emerging as new therapeutic targets in cancer treatment, including colorectal. Aim: We aim to elucidate the implication of DDRs in the prometastatic properties of the CRC cells and stromal cells in the liver.

Methods

First, the expression of DDRs on the stromal cells under tumor activated conditions and on tumor cells in the presence of tumor-activated stromal factors was assessed at protein levels. Second, this was related to the migratory potential under the same conditions. Finally, metalloproteinases expression, known to be induced by DDRs activation and involved in cell migration and ECM remodeling, was determined by western blot and zymography.

Results

DDRs expression was inversely altered in macrophages and fibroblasts after their activation by tumor derived factors. The expression of DDRs on CRC cells was decreased by factors derived from stromal cells. These DDRs deregulated expression was related to changes in the migratory capacity of tumor and stromal cells. Moreover, MMP-9 and MMP-14 expression increased in the stromal cells activated by tumor factors, while TIMP-2 expression was higher in fibroblasts but lower in macrophages. Also, the activation of CRC cells by either fibroblasts or macrophages derived factors induced a differential expression of MMP-2, MMP-9 and MMP-14.

Conclusions

The differential expression of DDRs by cells in the tumor microenvironment could redirect the expression of MMPs inducing the migratory capacity of CRC cells. In conclusion, tumor and stromal cells crosstalk may dysregulate the ECM deposition related to DDRs expression contributing to the extensive stroma remodeling in CRC metastatic diseases.

Legal entity responsible for the study

University of the Basque Country (UPV/EHU)

Funding

University of the Basque Country (UPV/EHU)

Disclosure

All authors have declared no conflicts of interest.

Background

Although apatinib has been demonstrated with potential antitumor activity to multiple solid tumors, the underlying mechanism of apatinib for the treatment of hepatocellular carcinoma (HCC) remains unclear. In the present study, we explore if there is any direct suppression effect of apatinib on HCC cells and its relevant targets.

Methods

To determine the role of apatinib, we investigated its effect on viability, colony formation, apoptosis, migration of 6 HCC cell lines in vitro, and HCC progression in mice model. Using a phospho-receptor tyrosine kinase pathway array with 49 different tyrosine kinases, we screened and verified the tyrosine kinase targets involved in apatinib therapy.

Results

Apatinib treatment significantly inhibited HCC cell viability, proliferation, colony formation, migration, and enhanced cell apoptosis in a concentration-dependent manner (p <0.05). Furthermore, apatinib showed a favorable anti-tumor growth effect (71% of inhibition ratio, p <0.05) in the established human HCC xenograft mice model with good safety. RTK pathway arrays and western blots analysis demonstrated apatinib significantly down-regulated the phosphorylation levels of several tyrosine kinase receptors, especially PDGFR-α and IGF-IR, and inhibited Akt phosphorylation.

Conclusions

These novel data suggested that the apatinib may have a direct anti-HCC effects as a direct multi-target RTK inhibitor of HCC cells and a promising potentiality in HCC clinical therapies.

Legal entity responsible for the study

Xiaojin Li

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

GATA6 is a transcription factor that regulates endoderm differentiation and lineage specification. Dysregulation of GATA6 expression had been reported in cancers of endoderm-derived organs such as the lungs, stomach and pancreas. The aim of this study is to determine the clinical significance of GATA6 in hepatocellular carcinoma (HCC) and characterize its potential functional roles.

Methods

GATA6 mRNA expression was assessed in 74 clinical HCC samples by quantitative polymerase chain reaction (qPCR). Correlation between GATA6 expression and clinicopathological parameters was analyzed. Stable GATA6 knockdown clones were established by a lentiviral-based approach in HCC cell line Huh7, which showed relatively high endogenous GATA6 expression. Functional effects upon GATA6 manipulation were investigated by cell proliferation, migration, invasion and tumorsphere formation assays in vitro.

Results

GATA6 expression was significantly downregulated in HCC tumor tissues when compared with the corresponding non-tumoral liver tissues (p = 0.007). A lower GATA6 expression was correlated with poorer cellular differentiation (p = 0.004). Silencing of GATA6 stimulated HCC cell proliferation, and promoted cell invasive and migratory abilities in vitro. This increase in metastatic capacity was mediated through the activation of epithelial-mesenchymal transition (EMT), as demonstrated by the loss of E-cadherin and gain of vimentin protein expression levels. Suppression of GATA6 augmented the self-renewal ability of HCC cells as demonstrated by the enhanced number and size of tumorspheres formed in both primary and secondary generations. In addition, the expression of various stemness markers such as Nanog, Oct4 and Sox2 were upregulated upon GATA6 silencing.

Conclusions

Our findings suggest that GATA6 is downregulated in HCC which may help to convert HCC cells to a more poorly differentiated state and enhance proliferation, self-renewal ability and metastatic potential.

Legal entity responsible for the study

Department of Pathology, Li Ka Shing Faculty of Medicine, The University of Hong Kong

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

By identifying the mechanism of therapeutic effects of the combination of interferon alpha (IFNα)-2b and 5-fluorouracil (5-FU) on advanced hepatocellular carcinoma (HCC) with portal venous invasion¹, we recently found that 5-FU increased both the expression and secretion of transforming growth factor (TGF)-β in HepG2 cells. In this study, we analyzed the effects of 5-FU on TGF-β-related signaling molecules.

Methods

Hepatoma cells (HepG2 and HuH7) were treated with 5-FU, IFNα-2b, and the combination of 5-FU and IFNα-2b. The total and/or phosphorylated protein levels of TGF-β-related signaling molecules were detected by western blot analysis. Additionally, the effects of above-mentioned treatments on the epithelial–mesenchymal transition (EMT) of the cells were evaluated by performing invasion and migration assays.

Results

With respect to the TGF-β-induced apoptosis signals, 5-FU inhibited not only the phosphorylation of SMAD2, but also reduced the total protein levels of SMAD2, SMAD4, and pINK4b. Conversely, 5-FU stimulated the phosphorylation of TGF-β-induced EMT molecules, such as ERK and JNK, but not p38MAPK. Accordingly, the protein levels of E-cadherin were reduced in the cells treated with 5-FU. On the other hand, IFNα-2b did not affect the levels of TGF-β-induced EMT molecules, whereas the combination of 5-FU and IFNα-2b neutralized the effects of 5-FU on TGF-β-related signaling molecules and restored their protein levels to those observed in the control. Interestingly, the phosphorylated protein levels of SMAD2 and the total protein levels of E-cadherin and p15INK4b increased in 5-FU-stimulated HUH7 cells, but not in HepG2 cells. Furthermore, 5-FU stimulated both cell invasion and migration in HepG2 cells, whereas the combination of 5-FU and IFNα-2b abolished these effects of 5-FU.

Conclusions

Our data suggest that 5-FU induces the EMT of hepatoma cells through TGF-β, and that the higher efficacy of the combination therapy of 5-FU and IFNα-2b results from the inhibition of these effects of TGF-β. The differences observed between HepG2 and HUH7 cells in response to the stimulation with 5-FU indicate that the efficacy of the therapy may differ between patients with hepatitis B (HBV) or C virus (HCV) background.

Legal entity responsible for the study

Iwate Medical University

Funding

Japan Society for the Promotion of Science (JSPS)

Disclosure

All authors have declared no conflicts of interest.

Background

According to Global Cancer Statistics (GCS) Hepatocellular carcinoma (HCC) is the 5^th most common and 2^nd deadliest cancer in the world. The incidence of HCC has increased over the past decades but still an effective therapy has not been developed. Sorafenib, which is the only FDA approved agent, can improve the patient survival just for a few months, therefore liver transplantation is the most efficient way of treatment up to date. In this study, we offer potential drug targets by analyzing the relationship between mutation status and drug treatment response of well-differentiated Huh7 and poorly-differentiated Mahlavu liver cancer cells.

Methods

PI3K/Akt pathway is hyperactive in ∼%40 of HCC. We determined the characterized and determined IC₅₀ values of Sorafenib, PI3Ka and b inhibitors and their combinations. RNAseq experiment were performed with the inhibitor treated cells. The RNAseq data of each cancer cell line (as control) was compared to treated samples. Somatic mutations associated with drug resistance were comparatively identified with RNAseq data workflow wrapped in our laboratory using GATK-MuTect tool (Cibulskis, 2013). The results were further filtrated to obtain the missense mutations. The mutated genes that were identified during the chemical knockdown studies were further analyzed in patient survival data. The functional studies were performed by gene silencing.

Results

SLC39A5, FRG1, PPHLN1 and SRP9 gene mutations were found to be shared among all drug resistant cells. Mutated genes were shown to be associated with cancer perseverance and aggressiveness. In addition, we found an unknown transcript called CTC512. The functional studies demonstrated that once these genes were silenced, the cellular growth was prevented. Gene silencing showed the alteration of the cell cycle progression of drug resistant cells. The affected downstream pathways were further analyzed by western blotting.

Conclusions

Our results indicate potential target genes which are critical to be addressed due to their roles in resistance to drugs in HCC. Once the mutated genes are silenced the cancer progression is prevented. The identified genes can be considered as chemotherapeutical and disease progress targets.

Legal entity responsible for the study

Rengul Cetin Atalay, METU

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

We report a biomimetic delivery platform based on human cytotoxic T-lymphocyte membranes. In this platform, T-lymphocyte membranes were camouflaged to the surface of poly-lactic-co-glycolic acid nanoparticles, with local low-dose irradiation (LDI) as a chemoattractant. These carriers were further anchored with the recombinant protein anti-EGFR-iRGD, improving tumor accumulation, facilitating tumor uptake.

Methods

The T-lymphocyte membrane coating was verified by dynamic light scattering, transmission electron microscopy and confocal laser scanning microscopy. The particle phagocytosis study was performed using a human phagocytic cell line. In vivo NIR fluorescence imaging was performed to monitor the route of nanoparticles. EGFR expression of tumor cells was tested before and after LDI.

Results

This new platform reduced phagocytosis of macrophages by 23.99% (p = 0.002). iRGD-EGFR anchored T-lymphocyte membrane-encapsulated nanoparticles accumulated in tumor site more than unfunctionalized groups, while LDI significantly enhanced the targeting ability. LDI could up-regulate EGFR expression on tumor cells, which was important in the process of localization of iRGD-EGFR anchored T-lymphocyte membrane-encapsulated nanoparticles in tumors.

Conclusions

This new platform included both the long circulation time and tumor sites accumulation ability while LDI could significantly enhance the tumor accumulation ability.

Legal entity responsible for the study

National Natural Science Foundation of China

Funding

National Natural Science Foundation of China

Disclosure

All authors have declared no conflicts of interest.

Background

Peritoneal dissemination often occurs in advanced gastric cancer (GC) patient. However, systemic chemotherapy alone has limited effect on peritoneal metastases. The purpose of this work is to fabricate a regional nanomedicine delivery system with injectable hydrogel, to investigate the sustained drug release, biocompatibility, degradation, and the therapeutic efficacy on advanced GC.

Methods

The injectable hydrogel gelling at body temperature was synthesized by one-step esterification of albumin and polyethylene glycol. The paclitaxel-loaded nanoparticles (PRNP) were prepared by encapsulating the drug in erythrocytes membrane ghost and embedded into the hydrogel (PRNP-Gel). The physical and chemical performances were characterized by dynamic light scattering, electronic microscope and SDS PAGE. The drug loading content, hemocompatibility, degradation, drug release, drug accumulation at tumor site, and anti tumor efficacy was also investigated.

Results

The PRNP-Gel suspension gelated in 15 min after subcutaneous injection. The diameter of PRNP in hydrogel was 133.1±1.6 nm, drug loading efficacy and content were 85.45±1.32% and 22.10±0.25%, respectively. 37.9% of the loaded paclitaxel was released in 196 h in vitro, demonstrating the sustained release properties of PRNP-Gel. No hemolysis was detected within the concentration up to 10 mg/mL, and the PRNP-Gel degraded completely in 200 days after injection. The IC50 of PRNP against MKN45 gastric cancer cells, determined by MTT, was 15.16 ng/mL. In vivo antitumor evaluation found that, the tumor growth inhibition of MKN45 on tumor bearing mice was 64.5% of PRNP-Gel group, which was higher than the PRNP (40.0%, P < 0.05) and Taxol (33.8%, P < 0.05). The average tumor weight was 74.9±40.1 mg, while they were 194.6±90.9 mg and 199.6±73.9 mg in PRNP and Taxol respectively (P < 0.05).

Conclusions

The biocompatible and degradable drug delivery system could release chemotherapeutics in a long-term and sustained manner, exhibited an enhanced drug accumulation at tumor site, resulting in the superior antitumor effect in vitro and in vivo. This work provided a new strategy of therapy for advanced GC.

Legal entity responsible for the study

Hanqing Qian

Funding

National Nature Science Foundation of China

Disclosure

All authors have declared no conflicts of interest.

Background

To investigate the effect of apatinib, a small-molecule tyrosine kinase inhibitor, combined with 5-FU on proliferation, apoptosis and invasiveness of human gastric cancer cells AGS, and provide experimental basis for the treatment of two drugs combination in gastric cancer in clinic.

Methods

The expression of vascular endothelial growth factor receptor 2 (VEGFR2) protein in human umbilical vein endothelial cells (HUVEC) and human gastric cancer cells were assessed by western blotting. 4-methyl-teerazolium (MTT) assay and flow cytometry were used to assess the cytotoxicity and apoptosis effects of the cells in response to control, single apatinib, single 5-FU, and apatinib combined 5-FU groups. Western blotting was used to evaluate the expression of p-Akt, proliferating cell nuclear antigen (PCNA), Caspase-3 and the invasiveness differences of the four groups were detected by wound healing assay and matrix metalloprotein-2 (MMP-2), E-cadherin gene amplification were measured by RT-PCR.

Results

AGS had the expression of VEGFR2. Compared with single drug groups, apatinib combined with 5-FU could significantly suppress the growth, proliferation and induce apoptosis of human gastric cancer cells in time and dose-dependent manners (P<0.05). Western blotting dispalyed p-Akt and PCNA expression decreased after AGS cells treated with apatinb combined 5-FU. Wound-healing assay showed the invasiveness of AGS cells was inhibited and probably through down-regulating MMP-2 and E-cadherin amplification in combined group (P<0.05).

Conclusions

Our study points that apatinib combined 5-FU could inhibit the proliferation of AGS gastric cancer cells by down-regulating the expression of p-Akt. The invasiveness of AGS cancer cell was inhibited by reduced expression of MMP-2 and E-cadherin genes, and provides a theory basis for 5-FU and apatinib combination in clinic with advanced gastric cancer patients who failed to second-line treatment but still had a good performance status.

Legal entity responsible for the study

Bangwei Cao

Funding

National Nature Science Foundation of China

Disclosure

All authors have declared no conflicts of interest.

Background

Eradication of advanced disease remains elusive in the majority of cancers including soft tissue sarcomas (STS) despite advances in our understanding of the molecular mechanisms that drive them. Targeted treatment development to date has largely relied upon data derived from all cells within a tumour sample and/or tumour cell lines. These approaches however, do not account for inherent heterogeneity of cancer cells within a single tumour and is considered an important factor that leads to treatment failure. Understanding intra-tumour heterogeneity is therefore a priority for cancer research and appropriate tumour models with sufficient availability would greatly facilitate the identification of newer targets and factors that lead to treatment resistance. We therefore aimed to develop in vitro models of STS that reflect intra-tumour heterogeneity.

Methods

We obtained tissue from patients having surgery for STS in Sheffield Teaching Hospitals and established primary tissue cultures. Short Tandem Repeat (STR) confirmed the same origin of both clones in both cases. DNA copy number profiling and gene expression microarray analysis were used for molecular characterisation of self-immortalised primary cell lines.

Results

One leiomyosarcoma (Shef-LMS 01) and one myxofibrosarcoma cell line (Shef-MFS 01) established two morphologically-distinct tumour cell types (culture variants) in separate long term cultures. Karyotyping and growth characteristics confirm that both variants in each case are tumour cells and they have remained in culture for over 100 passages. STR profiling confirms that in each case, both clones are derived from the same tumour. DNA copy number analysis with microarray-based comparative genomic hybridisation and gene expressson analysis shows many identical somatic copy number abnormalities (SCNA) between variants, but also numerous genomic and transcriptomic differences.

Conclusions

We believe that these genomic and transcriptomic differences provide clues to clonal evolution in these tumours and may explain the development of resistance to targeted treatment. These cell lines are therefore useful for the identification of novel targets and development of effective therapies for these tumours.

Legal entity responsible for the study

Abdulazeez Salawu

Funding

Weston Park Cancer Charity and Sarcoma UK

Disclosure

All authors have declared no conflicts of interest.

Background

Docosahexaenoic acid (DHA) induces apoptotic cell death through several mechanisms in cancer cells. We have previously demonstrated that DHA triggers apoptosis by increasing reactive oxygen species (ROS) accumulation and the ROS-mediated apoptosis caused by DHA is associated with Nrf2 signaling activation. Here we report that DHA-induced cell death is more susceptible through p62/p-eIF2alpha/NRF2 regulation in LMP-1-expressing nasopharygeal carcinoma (NPC) cells.

Methods

Viability of CNE-LMP1 and HONE-EBV cells was compared with CNE and HONE after DHA treatment by MTT assay. DHA-induced apoptosis was analyzed using the TUNEL assay and Western blot of cleaved form of PARP. Tissue expression of LMP-1 and p62 were observed by immunohistochemistry.

Results

Treatment of four human NPC cells (CNE, CNE-LMP1, HONE, HONE-EBV) with DHA for 24 hr resulted in a dose-dependent inhibition of cell growth. The DHA effect was due to the induction of apoptosis, given that DHA increased the cleaved form of PARP as well as the number of TUNEL-positive cells. The inhibition of CNE-LMP1 and HONE-EBV cells after DHA treatment is more susceptible. compared with CNE and HONE cells without LMP1 by MTT assay. The level of p62 and NRF2 of LMP1-NPC cells were increased after DHA pretreatment cpmpared to control NPC cells. On the other hand, the level of p-eIF2alpha produced reverse result. The activation of Nrf2 signal seems to result from decreased Nrf2 inhibitor, Kelch-like ECH-associated protein 1 (Keap1), because DHA remarkably attenuated Keap1 expression levels. Moreover, silencing Nrf2 by small interfering RNAs inhibited the cytotoxic effect of DHA, indicating that Nrf2 activation plays a positive role in the process of DHA-induced apoptosis. Increased staining for LMP1 and p62 was observed in NPC tissues when compared with the nonneplastic (chronic inflammation) tissues.

Conclusions

These results suggest that differential regulation of p62/p-eIF2alpha/NRF2 contributes to susceptible cell death by DHA in LMP-1-expressing NPC cells. Thus, utilization of DHA may represent a promising therapeutic approach for chemoprevention and treatment of human NPC.

Legal entity responsible for the study

Chungnam National University

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2007-0054932, NRF-2015R1D1A1A01056887) and by the framework of international cooperation program This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2007-0054932, NRF-2015R1D1A1A01056887) and by the framework of international cooperation program managed by National Research Foundation of Korea (2015K2A2A6002008)

Disclosure

All authors have declared no conflicts of interest.

Background

O⁶-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that removes the mutagenic O⁶-alkyl groups from guanines. 1, 3-Bis (2-chloroethyl)-1-nitrosourea (BCNU), a DNA damage reagent, is known to induce cell death of tumors and the ubiquitin dependent proteolysis of MGMT. The present study aims to enhance BCNU cytotoxicity toward cancer cells by modulating MGMT dynamics.

Methods

Human nasopharygeal carcinoma cells, including HONE-1 and TW01, and human colon carcinoma HT-29 cells were used for the BCNU treatments, siRNA knockdown, immunoprecipitation and western blot experiments. The BCNU cytotoxicity was determined using methylene blue assay. Proteins involved in MGMT ubiquitination were confirmed with immunofloresence staining and in vitro protein ubiquitination assays.

Results

We previously identified ubiquitin-conjugating enzyme E2B (UBE2B), a DNA repair enzyme with ubiquitin-conjugating abilities, as a critical regulator of the cell cycle in oral cancer cells. A novel role of UBE2B was further revealed in regulating MGMT dynamics in nasopharygeal carcinoma cells and colon carcinoma cells. Increased colocalization of UBE2B with MGMT was found in BCNU treated cancer cells. Depletion of MGMT or UBE2B in cancer cells resulted in decreased IC50 for BCNU. Lower MGMT expression levels were observed in UBE2B deficient cells. Overexpression of MGMT rescued the UBE2B-depleted cells from the cytotoxic concentrations of BCNU, suggesting that MGMT is a downstream target of UBE2B. The E3 ubiquitin ligase RAD18, that is known as a partner of UBE2B in facilitating PCNA ubiquitination, was analyzed to investigate the mechanism of the UBE2B regulation on MGMT. Interaction of RAD18 and MGMT was observed in cancer cells, and was enhanced under the BCNU treatments. Our results also showed that UBE2B and RAD18 contribute to MGMT ubiquitination under in vitro conditions.

Conclusions

Our study indicated that the UBE2B-RAD18 regulation on MGMT plays an important role in BCNU-induced cancer cell death. Thus, UBE2B inhibition may be considered as a potential strategy for cancer treatment. (This work was supported by Taiwan Ministry of Science and Technology under the grants no. NSC 103-2320-B006-036-MY3).

Legal entity responsible for the study

National Health Research Institutes, Tainan, Taiwan

Funding

Taiwan Ministry of Science and Technology

Disclosure

All authors have declared no conflicts of interest.

Background

Inhibition of EGFR signalling has emerged as a new treatment strategy for oral squamous cell carcinoma (OSCC). Previously, we found that loss of EGFR expression in OSCC was associated with EMT, and might have functional implications with regard to resistance to cetuximab, a monoclonal anti-EGFR antibody. Eribulin (a microtubule inhibitor) reportedly renders breast cancer less aggressive, and less likely to metastasise, by triggering the mesenchymal-to-epithelial (MET) transition. Here, we evaluated whether eribulin-induced MET was associated with re-sensitization of resistant OSCC cell lines to cetuximab.

Methods

In vitro antiproliferative activities were determined in three human OSCC lines (OSC-20, OSC-19 and HOC313) treated with eribulin. These three human OSCC represented different EMT/MET states.

Results

Interestingly, HOC313 cells (mesenchymal phenotype) were highly sensitive to eribulin in comparison with other cell lines, and significantly enhanced the anti-proliferative effect of cetuximab in response to the drug. Eribulin also underwent a MET-associated gene switch that resulted in high EGFR expression in HOC313 cells, and abrogated a TGF-β-induced EMT gene expression signature.

Conclusions

Eribulin-dependent sensitization of OSCC to cetuximab is likely due to induction of MET. Combination therapies based on eribulin and cetuximab have potential as a novel treatment regimen in OSCC.

Legal entity responsible for the study

Kanazawa University

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

The TLR agonist prothymosin α (proTα) pleiotropically stimulates immune cells via generating the appropriate cytokine milieu for their activation. The C-terminal decapeptide proTα(100-109) is considered the immunologically active moiety of proTα, as it restores in vitro the deficient immune responses of cancer patients equally well to proTα. ProTα and proTα(100-109) ligate TLR-4, signal through the TRIF/MyD88-dependent pathways, and promote the maturation of dendritic cells. The latter further stimulate T_H1-type immune responses and prime tumor antigen-reactive T cell functions. We evaluated whether proTα and proTα(100-109) function correspondingly in vivo.

Methods

C57BL/6 mice were subcutaneously inoculated with the syngeneic melanoma B16.F1 cells. Upon palpable tumor formation, mice were intraperitonealy treated with 2 cycles of GM-CSF, proTα(100-109) or proTα, in conjunction with a B16.F1-specific peptide vaccine. Tumor growth and animal survival were monitored. Splenocytes from selected animals were tested for ex vivo cytotoxicity by ⁵¹Cr-release assay and CD107 expression. Excised tumors were analyzed by immunohistochemistry, while serum cytokines were quantified by flow cytometry.

Results

Both peptides therapeutically administered in melanoma-bearing mice in the presence of cancer antigens, retarded tumor growth and prolonged the survival of treated animals by 25 days. Ex vivo analysis of spleen cell cytotoxicity confirmed the in vivo induction of B16.F1-specific and non-specific anti-tumor responses. Tumors of mice treated with proTα/proΤα(100-109) did not show infiltration of smooth muscle fibers and vessels, produced less melanin, presented limited necrotic areas and were characterized by sparse T cell infiltration. Sera from the same animals contained more IFN-γ, whereas the concentration of IL-4 was marginally increased.

Conclusions

Our results show that proTα and proTα(100-109) induce T_H1-biased immune responses in vivo. As both peptides are non-toxic to normal cells, their ability to orchestrate and modulate the desired anti-tumor immune responses in mice, suggests their eventual exploitation as adjuvants in anti-cancer peptide vaccines in humans.

Legal entity responsible for the study

O. Tsitsilonis

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

The accumulation of soluble factors in the tumor microenvironment and their release into the bloodstream lead to systemic effects, resulting in cancer-associated syndromes such as cachexia. Another association observed in the latter scenario is chrono-disruption, which has been related with tumor onset and development. In fact, a positive relationship between master clock integrity and healthiness has already been disclosed; however, it is unclear whether the effects of chronodisruption in cancer are locally or systemically exerted. It is known that lung adenocarcinoma reprograms liver metabolism via a pro-inflammatory response without affecting liver clock genes. Our study evaluated whether a non-metastatic skin tumor can affect clock gene machinery of other organs.

Methods

Eight to sixteen-week old C57BL/6J male mice were inoculated subcutaneously with B16-F10 cells (or PBS, control animals), single housed at 22 ± 2 °C, kept under 12/12h light/dark cycle, and received food and water ad libitum. Two weeks after inoculation mice were euthanized 2 hours after lights on (ZT2) or 2 hours after lights off (ZT14). Skin of control animals and samples of non-tumoral adjacent skin, tumor, liver, lung, and brown adipose tissue (BAT) were collected to evaluate the expression of clock genes (Per1, Per2, Bmal1, and Nr1d1) by qPCR.

Results

No oscillatory profile of clock genes was detected in skin of control animals, tumor adjacent skin, and tumor itself of inoculated animals; Bmal1 expression was reduced in adjacent skin and tumor as compared to skin of control animals. In liver and lung tissue, Per1 and Per2 oscillated in control animals, and tumor inoculation did not affect this oscillatory profile. Temporal oscillation of Bmal1 and Nr1d1 in the liver was lost in tumor-bearing mice. In BAT, Per1 and Bmal1 oscillatory expression was also lost in tumor-bearing mice. In all organs analyzed. Bmal1 transcript was reduced in tumor-bearing mice when compared to control animals.

Conclusions

The presence of a non-metastatic tumor in the skin alters clock machinery in adjacent skin, liver, lungs, and BAT. These data bring new knowledge of how tumor macro-environment affects clock machinery, resulting in a likely chrono-disruption of the organism as a result of a localized tumor in the skin.

Legal entity responsible for the study

University of Sao Paulo (USP) and Sao Paulo Research Foundation (FAPESP)

Funding

Sao Paulo Research Foundation (FAPESP) and National Council of Technological and Scientific Development (CNPq).

Disclosure

All authors have declared no conflicts of interest.

Background

To date little is known about receptor tyrosine kinases (RTK) expression on peripheral blood mononuclear cells (PBMC) and tumor infiltrating lymphocytes (TIL) in cancer patients. The aim of this study was to evaluate expression levels of major RTK: VEGFR1, VEGFR2, PDGFRα, PDGFRβ, FGFR2 in CD45+ population of PBMC and TIL isolated from patients with clear cell renal cell carcinoma (RCC).

Methods

Tumor and blood samples were obtained from 20 patients with RCC immediately after surgical resection of primary tumor. Tumors were harvested into sterile antibiotic-containing RPMI 1640 medium (Gibco). TIL isolation was performed under modified protocol [Baldan et al., 2015]. Isolated TIL and PBMC were prepared for flow cytometry. Cells were double stained with anti-CD45 FITC-conjugated mouse antibody, and with PE-conjugated mouse antibodies to VEGFR1, VEGFR2, PDGFRa, PDGFRb, FGFR2 (all Sony Biotech) and were analyzed on NovoCyte 2000R flow cytometer (ACEA Biosciences). Expression of RTK was evaluated with NovoExpress Software.

Results

Among PBMC 72.1±21.3% cells were CD45-positive. Isolated TIL contain 19.2±5.6% CD45-positive cells. PBMC and TIL express RTK. Expression levels of RTK are summarized in the table.Table:

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Conclusions

To our knowledge, this is first study that showed significant RTK expression on peripheral and RCC-infiltrating lymphocytes in RCC patients. PBMC and TIL have similar RTK expression levels.

Legal entity responsible for the study

Ethical committee, KCRB

Funding

Kidney Cancer Research Bureau

Disclosure

All authors have declared no conflicts of interest.

Background

Bladder cancer affects 430,000 patients and leads to 165,000 deaths annually worldwide. With no major advances in the management of this disease in the last 2 decades, there is an urgent need to identify therapeutic targets with validated biomarkers. Overexpression of Met, a Receptor Tyrosine Kinase, was shown to correlate with poor prognosis in bladder cancer making it an attractive target. Cabozantinib, a Met inhibitor, showed clinical activity in patients with refractory bladder cancer in a clinical trial. However, little is known about how Met exactly signals in bladder cancer and there are no validated biomarkers. This study aims at unravelling Met signalling in bladder cancer.

Methods

Western blots and confocal/low light microscopy were used to assess Met signalling and its role in wound healing in Transitional Cell Carcinoma (TCC) cells. Met expression was assessed by immunohistochemistry in tissue samples (n = 64).

Results

Met is overexpressed in TCC cells and in 78% of invasive bladder cancer tissues. This was associated with a shorter median survival as compared to Low Met levels (12.97 Vs 18.05 months). Stimulation of TCC cells with Met ligand, Hepatocyte Growth Factor (HGF), triggered Met activation and downstream signalling as well as wound healing, all of which were reduced with Met pharmacological inhibitors including Cabozantinib. The PI3K downstream effector AKT was highly activated upon Met activation. Moreover, class I PI3K inhibition with GDC094 significantly inhibited HGF-dependent wound healing. Interestingly, HGF triggered rapid Met endocytosis in TCC cells. Furthermore, pharmacological inhibition of endocytosis reduced Met downstream signalling.

Conclusions

We report that Met is a major target in invasive bladder cancer. Our results further suggest that PI3K may be considered as a co-target of Met to improve patients’ outcome. It may also be developed as a biomarker to help select patients who may respond to Met targeted therapy. Finally, we report for the first time that, upon HGF stimulation, Met gets rapidly endocytosed in TCC cells. Furthermore, inhibiting endocytosis reduced Met dependent signalling. All together, our results open the way for novel strategies to target invasive bladder cancer.

Legal entity responsible for the study

Queen Mary University of London

Funding

Barts Cancer Institute, Queen Mary University of London

Disclosure

T. Powles: Honoraria: AstraZeneca, Bristol-Myers Squibb, Roche and Merck. Research Funding: AstraZeneca, Roche and Merck.

L. Menard: Employee: AstraZeneca.

All other authors have declared no conflicts of interest.

Disclosure

T. Powles: Honoraria: AstraZeneca, Bristol-Myers Squibb, Roche and Merck. Research Funding: AstraZeneca, Roche and Merck.

L. Menard: Employee: AstraZeneca.

All other authors have declared no conflicts of interest.

Disclosure

T. Powles: Honoraria: AstraZeneca, Bristol-Myers Squibb, Roche and Merck. Research Funding: AstraZeneca, Roche and Merck.

L. Menard: Employee: AstraZeneca.

All other authors have declared no conflicts of interest.

Background

Recent studies showed increased interest in telomere biology bladder cancer. These studies were mainly focused on hotspot TERT promoter mutations and their contribution for telomerase activation and clinical outcomes. However, the study of TERT promoter methylation, as an additional deregulatory mechanism of TERT expression, was not studied in this disease. We previously uncovered a region in the TERT promoter region (THOR – TERT hypermethylated oncological region) which is specifically hypermethylated and associated with telomerase activation in cancer tissue. In this study we aim to establish the value of this duet (TERT promoter mutations and THORMeth) in bladder cancer recurrence and progression.

Methods

To explore the impact of TERTpMut and THORMeth on TERT expression and clinical outcomes in UBC we studied two cohorts of UBC patients, 331 FFPE samples. THORMeth status was assessed using quantitative bisulfite pyrosequencing. Sanger Sequencing and ddPCR accessed TERTpMut status. TERT expression was evaluated by ddPCR. Cox proportional hazards models were used to correlate THORMeth and TERTpMut with disease recurrence and progression.

Results

THOR is a diagnostic marker for urothelial bladder cancer. THOR hypermethylated (n = 127, 53.6%) is associated with higher levels TERT expression (p < 0.0001) and is a risk factor for disease recurrence in NMIBC (Log rank p = 0.034) and is crucial for disease progression. TERT promoter mutations are present in all stages and grades (n = 182, 76.8%) and are a risk factor for disease recurrence amongst NMIBC (HR: 3.8, p < 0.0001). Genetic and epigenetic combined signatures in the TERT promoter allow for a clinical bladder cancer classification system based on underlying telomere biology.

Conclusions

THOR is a novel biomarker for UBC, and, as TERTpMut is able to predict disease recurrence in NMIBC. TERTpMut/highTHORMeth, comprises a distinct signature that is associated with disease progression and higher TERT expression. This fact suggests a hypothetical synergism between both mechanisms and highlights the merit of evaluating TERT promoter methylation as other deregulatory mechanism in TERT expression with implications in patients’ outcomes.

Funding

Foundation for Science and Technology, Portugal, Individual Doctoral Grant SFRH/BD/102232/2014.

Disclosure

All authors have declared no conflicts of interest.

Background

Dexamethasone (DXM) is commonly used in the management of glioma patients to treat intracranial edema but patients often suffer from its side effects. The molecular mechanisms of these side effects are poorly studied. DXM seems to affect extracellular matrix (ECM) especially proteoglycans (PGs) known to be a major component of the ECM in brain tissue. The aim of our study was to investigate if the effects of DXM on brain tissue PGs depend on the treatment regimen or DXM dose.

Methods

Effects of different doses and regimens of DXM treatment on the brain ECM were studied using RT-PCR and IHC in the ex vivo model of organotypic brain tissue culture and in vivo experimental animal model. The ex vivo organotypic culture model was chosen instead of in vitro cell culture model as it represents the real 3D structure of the tissue and can be used to study ECM.

Results

The most expressed PGs in rat brain tissue were syndecan-1, glypican-1, decorin, biglycan and lumican. DXM treatment of organotypic hippocampus culture ex vivo led to dose-dependent suppression of brevican, perlecan and biglycan expression and increase in expression of glypican-1, NG2 and versican. In the in vivo experiments, PGs demonstrated age-specific and brain zone-specific expression patterns in normal brain of Wistar rats. The effects of DXM on cortex and hippocampus of the experimental animals were dose- and regimen-dependent. Low-dose DXM treatment led to significant decrease in expression of most PGs in cortex but 3-fold increase in syndecan-1, perlecan and brevican expression in hippocampus. Treatment with high-dose DXM resulted in 2-6-fold increase in most of PGs expression in both brain zones. Long-term treatment led to the most dramatic changes in PGs expression on both mRNA and protein levels, completely changing their expression pattern.

Conclusions

Taken together, obtained data demonstrate an importance of DXM doses/regimens during antiglioma therapy. Long-term treatment and high doses of DXM lead to the most dramatic alteration of PGs composition in brain ECM creating a favorable niche for tumor growth and relapses.

Legal entity responsible for the study

Institute of Molecular Biology and Biophysics

Funding

Russian Science Foundation (RSF grant 16-15-10243).

Disclosure

All authors have declared no conflicts of interest.

Background

If not repaired, DNA double-strand breaks (DSBs) can lead to genomic instability and cell death or neoplastic transformation. The major DSB repair (DSBR) mechanism in higher eukaryotes is non-homologous end-joining (NHEJ). In NHEJ, polynucleotide kinase/phosphatase (PNKP) is the primary enzyme for processing abnormal 5'-OH and 3'-phosphate ends that prevent the final repair step by XRCC4/DNA Ligase IV (Lig IV). This processing step is thought to be mediated by an interaction between the PNKP-FHA domain and CK2-phosphorylated XRCC4 C-terminal tails.

Methods

See below.

Results

Our binding assays show tight binding between XRCC4/Lig IV and PNKP both with and without CK2-phosphorylation of XRCC4. Low-resolution ensemble structures of purified phosphorylated-XRCC4/Lig IV/PNKP ternary complex by small-angle X-ray scattering (SAXS) experiments also suggest a second phosphorylation-independent interaction between the PNKP and XRCC4/Lig IV. Hydrogen-deuterium exchange (HDX) experiments have identified a candidate for this secondary interaction site within a loop in the PNKP phosphatase domain. This site contains the clinically significant PNKP E326K mutation found in the severe form of the hereditary neurological disease MCSZ (microcephaly with early-onset intractable seizures and developmental delay). Activity assays show that the E326K mutation decreases both substrate binding and turnover in PNKP when bound to phosphorylated-XRCC4/Lig IV. Furthermore, UV laser microirradiation in cells show that the E326K mutation also disrupts recruitment of PNKP to DNA lesions.

Conclusions

We have identified a putative secondary interaction site that functionally contributes both to recruitment and catalysis of PNKP in NHEJ. Disruptions to PNKP in this region may result in decreased DNA double-strand break repair in cells and describe a molecular basis of MCSZ. Further, PNKP has other known clinical neurological significance and its presence on chromosome arm 19q has interesting implications in oligodendrogliomas. This interaction surface may prove an interesting target for small-molecule inhibition of DNA strand break repair toward novel radio- and chemo-sensitizing therapies in cancer treatment.

Legal entity responsible for the study

University of Alberta

Funding

Canadian Institutes of Health Research, Alberta Cancer Foundation, Structural Biology of DNA Repair Machines Consortium.

Disclosure

All authors have declared no conflicts of interest.

Background

Neuroblastoma (NB) is the most common extracranial solid cancer in childhood and the most common cancer in infancy in the world. Rottlerin, a naturally occurring polyphenolic compound derived from Mallotus philipinensis, appears to have great potential in cancer therapy because of its effects on proliferation and apoptosis. Genistein is a phytoestrogen and it has been found to inhibit uncontrolled cell growth in several cancers. Recently, we learned that eukaryotic elongation factor-2 kinase (EF2K) is dramatically upregulated in many cancer cells and promotes cell survival and proliferation. Rottlerin and genistein have also showed inhibitory effects on this kinase in other solid tumours like pancreatic cancer. With this is mind, we investigated the effects of rottlerin and genistein in neuroblastoma cells.

Methods

In this study, two human neuroblastoma cancer cell lines (SH-SY5Y and Kelly) were treated with rottlerin and genistein in vitro. Cell proliferation, colony formation and invasion were assessed, and wound-healing tests, western blots (wb), cell cycle and apoptosis analysis by flow cytometry were performed.

Results

Our results showed that rottlerin and genistein treatments caused a significant reduction in cell proliferation, colony formation, and invasion/wound-healing capacity in neuroblastoma cells at concentrations of 5 µM and 30 µM, respectively (p < 0,0001). The combination of these doses also increased the level of inhibition in these analyses (p < 0,0001). Additionally, these drugs also increased the level of apoptosis and caused G1 cell cycle arrest in neuroblastoma cell lines (p < 0,0001). We showed that these treatments markedly inhibit EF2K overexpression. Our wb data suggested that EF2K may enhance tumorigenesis/metastasis through the upregulation of pro-tumorigenic/metastatic pathways in these cells and these agents may produce their anti-proliferative, anti-metastatic and apoptotic effects through EF2K downregulation.

Conclusions

In conclusion, these results indicate that rottlerin and genistein have important effects on neroblastoma cell behaviour and these effects may be caused by downregulation of EF2K.

Legal entity responsible for the study

Mumin Alper Erdogan

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Stress is the pathogenetic basis of many diseases. It leads to decreasing resistance of the organism, including antitumor one. Emotional stress of a cancer patient affects the quality of life and treatment outcomes by decreasing the antitumor resistance. Therefore, correction of emotional state is an urgent task. The purpose of the study was to reveal the influence of stimulation of emotiogenic brain structures on the antitumor resistance of animals with cancer.

Methods

Transplantable solid sarcoma S45 in white outbred male rats weighing 230-250 g was used as a model of tumor growth. Implantation of bipolar stimulating electrodes in the subcortical structures of the brain was performed aseptically by stereotactic coordinates. Electrodes were implanted to nucleus lateralis septi (LS) – “positive emotiogenic structure” and to Globus pallium (GP) – “negative emotiogenic structure”; the data were compared to the values in animals without electrodes (controls). The stimulation was performed daily for 2 months without changing the stimulation current.

Results

Stimulation of emotiogenic brain structures influenced the dynamics of S45 growth, and LS stimulation caused the greatest inhibition of tumor growth.Table:

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Morphological study of the thymus, spleen and lymph nodes of animals with LS showed signs of high functional activity of the organs providing a high level of resistance.

Conclusions

Electrostimulation of LS influences antitumor resistance, significantly improving it. This suggests the expediency of combining specific anticancer drugs and nonspecific effects of psychotropic drugs - anxiolytics in complex treatment of cancer.

Legal entity responsible for the study

Rostov Research Institute of Oncology

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Immunotherapy is currently being explored in many tumour types with encouraging results, but has not yet been evaluated in neuroendocrine tumours (NET). Our aim is to characterise the immune landscape of NET and determine which immune-modulatory pathways control the tumour infiltrating lymphocytes (TILs) in order to develop a rational approach for immunotherapy in this tumour type.

Methods

Peripheral blood and fresh tissue was obtained from consenting patients with NET, and subjected to multicolour flow-cytometry to determine the abundance of CD8+, CD4+FoxP3- effector (CD4eff) and CD4+FoxP3+ regulatory (Treg) T cell subsets and the expression of co-inhibitory and co-stimulatory checkpoint molecules on these subsets. Additionally, matched FFPE tissue was obtained for multiparametric immunohistochemistry to investigate the distribution of the immune infiltrate.

Results

Tissue from 23 NET patients including 19 mid-gut tumours (13: G1 and 6: G2) was analysed. Overall the tumours contained a higher proportion of Tregs compared with peripheral blood (8.5% vs 5%, P = 0.02) and had a CD8:Treg ratio of 18.1:24.3 respectively (P = 0.036). The co-inhibitory molecules CTLA-4 and TIM-3 showed highest expression on Tregs, while PD-1 and LAG-3 expression was similar across all T cell subsets. Co-stimulatory molecules, including ICOS, 41BB and OX-40, were also highest on Tregs, as was the recently identified co-stimulatory receptor TIGIT. Immunohistochemistry revealed that the majority of cases have <1% intratumoural CD4+ and CD8+ T cells but a higher number of peritumoural T cells from all subsets. Where present, T cells were predominantly CD8+ and intratumoural CD163+ macrophages were also identified.

Conclusions

These preliminary results provide novel insight into the immune landscape of NET, and may inform the development of targeted combination immunotherapies. Initial results suggest that checkpoint molecules, such as PD1 and LAG-3, may be potential targets in this tumour type and work is ongoing to further elucidate the immunogenic potential of NET.

Legal entity responsible for the study

University College London

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Despite improvements in treatment for Multiple myeloma (MM) achieved by novel drugs such as proteasome inhibitors (PIs) and immunomodulatory drugs (IMiDs), most patients will ultimately relapse or become refractory to their current treatment. Therefore, it is important to understand the mechanisms of therapeutic resistance in relapsed/refractory MM (RRMM) for improving treatment outcome. Recently, it was reported that serum or plasma of MM patients showed sufficiently stable miRNA signatures with prognostic impacts in MM cohorts, which can be used as minimally invasive markers for predicting and monitoring treatment outcomes. However, the expression patterns and biological implications of miRNAs are still unclear in RRMM patients receiving lenalidomide with dexamethasone (Len-dex).

Methods

We investigated the expression of serum miRNAs by genomewide miRNA array analysis and explored their predictive values in RRMM patients receiving Len-dex.

Results

We explored the associations of miRNAs with treatment outcome of Len-dex treatment and prognosis in 55 RRMMs (25 good responders and 30 poor responders) and built a prediction model for treatment response. Three miRNAs (miR-29c-3p, miR-30c-5p, and miR-331-3p) were found to be significantly down-regulated in poor responders. In survival analysis, lower expression of the three miRNAs was significantly associated with shorter time to progression (TTP) or poorer overall survival (OS). Eight clinical factors were also associated with TTP or OS. By combining the miRNA markers and clinical markers, we designed a a prediction model for response to lenalidomide treatment in RRMM patients. Our model showed better prediction power (AUC=0.855, sensitivity 84%, specificity 76%, and accuracy 81%) than international staging system (ISS) based prediction.

Conclusions

Our results suggest the potential of circulating miRNAs as minimally invasive markers for treatment response and prognosis in RRMM patients.

Legal entity responsible for the study

This study was approved by the Institutional Review Board of The Catholic University of Korea and conducted in accordance with the Declaration of Helsinki.

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

MCL is an aggressive B-cell lymphoma with aberrant expression of several oncogenic effectors and requiring novel anticancer strategies. The nuclear transporter exportin-1 (XPO1) is highly expressed in MCL and is critical for cancer survival and proliferation. mTOR signaling is frequently activated and an important therapeutic target in MCL. In this study, we investigated the antitumor effects and molecular/metabolic changes induced by combined with dual mTOR inhibitor AZD-2014 and XPO1 inhibitor KPT-185 on MCL cells under the hypothesis that mTOR inhibition by AZD-2014 represses KPT-185 inducedupregulation of glycolysis.

Methods

Four MCL cell lines (Jeko-1, X138, JVM-2, and MINO), primary MCL cells, and normal bone marrow samples were utilized. Cell viability was evaluated by MTT assay. Cell cycle and apoptosis were determined by flow cytometric analysis. cDNA array, iTRAQ proteomic, immunoblotting and metabolome analysis using CE-TOF-MS were also performed.

Results

AZD-2014 enhanced KPT-185-induced the inhibition of cell growth and repression of cell viability in MCL cells but not in normal bone marrow cells. Different mTOR inhibitors (AZD-8055 and MLN0128) demonstrated similar effects. AZD-2014+KPT-185 decreased expression of the oncogenic mediator c-Myc andthe translational/transcriptional network regulator HSF1as detected by immunoblotting. iTRAQ proteomic analysis demonstrated thatthe combination caused repression of ribosomal biogenesis. Treatment with either AZD-2014 or KPT-185 depressed phospho-S6.CET-OF-MS metabolite assay showed that AZD-2014+KPT-185inhibited theKrebs cycle, and that AZD-2014 effectively repressed KPT-185-induced upregulation of glycolysis. cDNA array detected downregulation of NOD2which is known to trigger activation of MAP kinases and of NF-kappa-B signaling. Moreover, AZD-2014+KPT-185 activated AMPK, an energy stress marker in a cell type-dependent manner.

Conclusions

Our findings indicated that the combinatorial inhibition of mTOR and XPO1 identifies a novel synthetic lethality mechanism that could be exploited clinically, following satisfactory completion of pre-clinical in vivo studies.

Legal entity responsible for the study

Yoko Tabe

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Immunosuppression is one of the major causes of AML pathogenesis and progression. CD200 and IDO are immunoregulatory factors which overexpressed in some solid tumors and hematological malignancies. Distinct researches have shown CD200 and IDO expression is associated with AML progression. In the current study, we simultaneously examined the expression of these molecules, as the two important factors including in immunosuppression, in the newly diagnosed and relapse AML patients to investigate their correlation with each other.

Methods

48 PBMC samples of newly diagnosed and relapse AML patients were tested and also 32 normal subjects were employed as control. CD200 expression level was examined on cells by Flow cytometry and quantitative real time RT-PCR was used to determine the IDO-1 gene expression. Finally, data were analyzed statistically.

Results

Data showed that CD200 and IDO-1 overexpressed especially in relapsed patients. Comparison between FAB AML subgroups demonstrated no statistical differences in the case of CD200 level but expression of IDO-1 was slightly increased only in M4 subgroup in comparison to M3. Correlation analyses showed strong association between expression of CD200 and IDO-1 in all patients particularly in relapsed AML, whereas no significant correlation was found in normal subjects.

Conclusions

According to the role and overexpression of CD200 and IDO-1 in AML patients and also their two-way correlation with T-regs in disease induction and progression, simultaneous assessment of these parameters are so valuable for more exact prognosis detection. Also inhibition of all these immunoregulatory pathways could be so useful for immunotherapy outcome, especially in relapsed AML.

Legal entity responsible for the study

Tehran University of Medical Sciences

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Acute lymphocytic leukemia (ALL) is the most predominant hematopoietic clonal disorder in children than adult. ALL classifies into two subtypes: B –ALLand T-ALL. The incidence of ALL subtype in urban areas is generally higher than rural areas. In the Western World, the predominant immunophenotype observed in ALL is B-ALL with 60-80% of total case, and T-ALL is only 15-20%. But in case of developing country like India the reverse is true. The objective of the present study is to examine and correlate T-ALL and B-ALL in leukemia patients with respect to socio-demographic factors.

Methods

During May 2015 - April 2017, total 427 ALL patients (male:female::1.9:1), age between 2-60 years attended OPD and IPD of Netaji Subhas Chandra Bose Cancer Research Institute, Kolkata, India. We have collected peripheral blood and/or bone marrow samples for immunophenotyping by FACS after taking written consent from the patients. Each sample is evaluated with a panel of monoclonal antibodies and compared the immunophenotyping data with socio-demographic factors (age, sex, economic and social status etc).

Results

The overall survival of ALL patients (with mean age 13.6 years) in 2 year is 73.6%. In our hospital, the economically weak patients (77.05%) are more abundant than economically sound patients (22.95%). Out of 427 patients, T-ALL (51.28%) is predominantly higher than B-ALL (48.71%). We found that immunophenotyping data is correlated with all the socio demographic data i.e., sex, economic and social status etc. Though disease free survival and event free survival is markedly higher in B-ALL compared to T-ALL, but we found the survival of T-ALL is also increasing.

Conclusions

Our unique findings emphasize that the detection of T-ALL and B-ALL by immunophenotypic analysis for better treatment and outcome of the patients and also trying to correlate the prevalence of T-ALL and B-ALL with economical status and also with other socio-demographic factors in study area.

Legal entity responsible for the study

Netaji Subhas Chandra Bose Cancer Research Institute

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Megakaryocyte hyperplasia is a major characteristic of two myeloproliferative neoplasms (MPNs) known as essential thrombocythemia (ET) and Primary myelofibrosis (PMF). About 35% of ET and PMF patients harbour somatic calreticulin (CALR) mutations. CALR is a calcium (Ca²⁺) buffering protein within the endoplasmic reticulum (ER). Ca²⁺ is an important element for megakaryocyte functions; however the impact of CALR mutations in cellular Ca²⁺ during megakaryocyte hyperplasia remains elusive.

Methods

I-TASSER software was used to study the aminoacid charge using the 3D structure of CALR mutant. Marimo cells and overexpressing CALR mutant HEK293T and DAMI cells were used as cellular models. CALR cellular localization was addressed by flow cytometry and confocal microscopy. Basal Ca²⁺ levels were measured by Fluo-8 staining. Furthermore cells were treated with Fendiline and BTP-2 calcium channel blockers to manipulate cellular Ca²⁺.

Results

The present study shows that CALR mutations change the aminoacid charge of the Ca²⁺ binding region of CALR and that mutant CALR is localized outside the ER, within the cytoplasm and the cellular membrane. These results suggest that CALR mutations could be affecting the Ca²⁺ buffering activity within the ER. Therefore, we further analysed Ca²⁺ basal levels in CALR mutant cells, and our results showed that CALR mutations show lower cellular Ca²⁺levels. These results lead us to think that low intracellular Ca²⁺ levels could be the driving force of megakaryocyte hyperplasia characteristic of ET and PMF. Therefore, we induced low intracellular Ca²⁺ levels in leukemic blast by using Ca²⁺ channel blockers and our results showed that treated cells display an increase of the megakaryocyte marker CD41a in the cell surface, suggesting an induction of megakaryopoiesis in these cells.

Conclusions

These findings elucidate that low intracellular Ca²⁺ caused by CALR mutations could be the driving force of megakaryocyte formation in ET and PMF. This study shows the relevance to understand the role of cellular calcium during megakaryocyte formation and this could unravel the pathogeny of CALR mutant in MPNs.

Legal entity responsible for the study

University of Salford

Funding

University of Salford

Disclosure

All authors have declared no conflicts of interest.

Background

Dendritic cell (DC)-based immunotherapy represents a promising approach for cancer treatment. However, the DC homing rate to the lymphoid tissues is poor, thus hindering the activation of antigen-specific T cells and reducing their antitumor efficacy. Here, we developed an approach based on magnetic nanoparticles (NP) to manipulate DC migration and thus elicit a more robust and efficacious antitumor response in tumor-bearing mice.

Methods

Mouse spleen DCs were loaded with nanocomplexes (NC) consisting of iron oxide NP (8x10⁻¹² g/cell) and lyophilized tumor tissue. To investigate the presence of NP in DCs, the method of Fe2⁺ and Fe3⁺ detection by Prussian Blue Staining was used. DCs were injected intradermally into tumor-bearing mice three times in an amount of 2x10⁵ per mouse at an interval of 3 days starting from day 7 after Lewis lung carcinoma inoculation. One group of mice that received DCs loaded with NC was exposed to a magnetic field for 1 h. The number and volume of metastases and tumor weight were assessed 28 days after tumor inoculation. At the same time, the levels of INF-γ, IL-10, TGF-ß, IL-4, FoxP3 mRNA expression in the spleen and inguinal lymph nodes were determined.

Results

The iron oxide NP showed no toxic effects on the DCs and had no effect on their viability. We found that almost all DCs are able to incorporate magnetic NP after 24h of incubation. Fewest metastases were found in the mice that received DCs loaded with NC and were exposed to a magnetic field: the number of metastases in mice from this group was 1.7 times less than in control mice. It should be noted that the volume of metastatic nodes in the lungs and the mass of the primary tumor were practically the same as in the control mice. The most pronounced decrease in FoxP3 mRNA levels in the lymph nodes, indicating a decrease in the activity of regulatory T cells, was also noted in the mice receiving DCs loaded with nanocomplexes and exposed to a magnetic field. In the mice of this group, a significant decrease in the level of IL-4 in the spleen was detected.

Conclusions

Our results suggest that an approach based on magnetic NC could be a promising strategy for improving the antitumor efficacy of DC-based immunotherapy.

Legal entity responsible for the study

National Cancer Institute of Ministry of Public Health of Ukraine

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

The problem of elaboration of methods for effective mobilization of immune antitumor processes remains urgent. Earlier, it was shown that regression of experimental animal tumors can be achieved under the influence of low-intensive electromagnetic radiations (EMR) and magnetite nanoparticles (NPs) (Garkavi L.H. et al, 1996; 2013).The aim of the study was to determine the possibility of regression of large transplanted tumors of white rats under the influence of low-intensity EMR of millimeter range (EHF) and magnetite NPs.

Methods

In experiments on 123 white outbred male rats (200-300g) with transplanted Pliss lymphosarcoma - low-intensity EMR EHF (42.2 GHz, 10 mW/cm², exposure 15-30 min, low-frequency modulation) and magnetite NPs (10 ± 2 nm) in the form of the magnetic fluid AM-01 (“AM-Cube”, Ekaterinburg) were used. The EMR acted on the animal's head starting 3 days before the tumor was transplanted. NPs were injected into peritumoral zone 2-3 times a week in a single dose of 17.7 mg/kg to animals with already formed tumors. Duration of treatment was 4 weeks. We studied the dynamics of tumor size, histochemical and cytometric changes in tumor tissue. The Wilcoxon test was used to evaluate the results.

Results

When EMR EHF was used, complete regression of tumors with a size of 5-6 cm³ and a partial regression (by 30-40%) of tumors with a size of 10-13 cm³ were noted. The effect was obtained in 33% of the animals. In cases of using of magnetite NPs, tumor regression was observed in 40% of the animals, complete regression of tumors with a size of 5-30 cm³ was observed. Before the regression began, the tumor growth rates did not differ from those in the control group when using as one as the other factor. In addition, the regression was characterized by a rapid rate (5-7 days) and no signs of intoxication in animals. In the peritumoral area considerable macrophage activity and increasing number of cytotoxic T-lymphocytes were noticed.

Conclusions

The timing of the onset of regression, its dynamics and the absence of signs of intoxication during rapid elimination of large tumors indicated the deployment of antigen-presentation processes and specific killing of tumor cells by inducing apoptosis.

Legal entity responsible for the study

Rostov Research Institute of Oncology, Ministry of Public Healthcare of the Russian Federation

Funding

Rostov Research Institute of Oncology

Disclosure

All authors have declared no conflicts of interest.

Background

Colorectal cancer is one of the most prevalent types of cancer worldwide. The GTPase RAC1 and its effector PAK1 have been found overexpressed or hyperactivated in this type of cancers, particularly those with more aggressive and invasive features, which is frequently correlated with resistance to chemotherapeutics and unfavourable clinical prognosis. Previously, we described a new signalling pathway in which activation of RAC1/PAK1 signalling promotes a transcriptional switch between the BCL6 repressor and the STAT5 transcriptional activator at a restricted subset of gene promoters.

Methods

Here we used a novel combinatory ChIP-Seq approach for the genome-wide identification of the BCL6/STAT5-switch target genes.

Results

Ontological enrichment analysis among the identified target genes revealed an overrepresentation of genes involved in DNA damage repair. Using the comet assay as readout for the extent of DNA damage, we show that the activation of RAC1/PAK1 signalling significantly accelerates DNA damage repair through the upregulation of pivotal genes.

Conclusions

This work highlights an additional role for the RAC1/PAK1 signalling axis that may contribute to the chemoresistant phenotype of aggressive colorectal tumours.

Legal entity responsible for the study

Paulo Matos

Funding

Fundação para a Ciência e Tecnologia.

Disclosure

All authors have declared no conflicts of interest.

Background

The metastasis suppressor, N-myc Downstream Regulated Gene-1 (NDRG1) inhibits metastasis in a variety of cancer-types, including cancers of the breast, colon, pancreas and prostate. Its potent anti-oncogenic effects were demonstrated in multiple in vitro and in vivo studies, making it a promising therapeutic target. However, exactly how NDRG1 is regulated in different cancer-types and how different regulatory mechanisms affect NDRG1 function remain to be elucidated. Notably, post-translational modifications (PTMs), phosphorylation and cleavage of NDRG1, have been associated with its function. Therefore, it was crucial to examine whether these PTMs occur universally or selectively in different cancer-types. Further, considering the DNA repair role suggested for nuclear NDRG1, the effects of the above PTMs on nuclear NDRG1 levels was examined.

Methods

DU145 and PC3 prostate cancer cells, PANC-1 pancreatic cancer cells, HT-29 colon cancer cells, HepG2 and Hep3B hepatocellular carcinoma (HCC) cells were utilised. Full-length (FL) and truncated (T) NDRG1 isoforms were detected using a C-terminus directed antibody. The FL isoform was detected using an N-terminus directed antibody. Ser330 or Thr346 phosphorylation (p-NDRG1) was detected using specific antibodies.

Results

For the first time, we demonstrated that phosphorylation and potential cleavage of the NDRG1 protein occurs in all the various cancer cell-types examined. Although the levels varied, both the FL and T NDRG1 and its phosphorylated form were detected in all tumour cells assessed. The FL NDRG1 isoform was predominantly found in the cell nucleus. Ser330 p-NDRG1 was also highly localised in the cell nucleus, while Thr346 p-NDRG1 was mostly cytoplasmic. These cellular distribution patterns were similar in all cancer-types tested.

Conclusions

This study demonstrates for the first time that the NDRG1 protein is phosphorylated and potentially cleaved in diverse cancer cell-types. Further, FL NDRG1 and Ser330 p-NDRG1 were highly localised to the cell nucleus. These results indicate that the N-terminus region and phosphorylation at Ser330 could be crucial for nuclear expression and the well-known anti-metastatic function of NDRG1.

Legal entity responsible for the study

The University of Sydney

Funding

The University of Sydney, Cancer Institute New South Wales National Health and Medical Research Council, Cancer Australia, Cure Cancer Australia Foundation.

Disclosure

All authors have declared no conflicts of interest.

Background

Integrated PET/MR scanners can simultaneously acquire whole body functional PET data together with morphological and functional MR data. Whole-body PET-MRI datasets contain huge amounts of spatially detailed morphological, functional and metabolic information. We propose a method, based on deformable registration to a whole-body atlas, for computer aided detection of lesions in image data from an integrated PET-MRI system.

Methods

Images were acquired using an integrated 3T PET-MRI system (Signa PET/MR, GE Healthcare). Fat and water MR images were collected using a Dixon MR Attenuation Correction (MRAC) sequence (TE 1.67ms, TR 4.05ms, voxel size: 2x2x5.2 mm). Subjects underwent a PET scan after injection of [F18]-FDG (2 MBq/kg) with 3 minutes per bed, with a 100-120 minute interval between injection and scan start. PET reconstruction was performed using GE’s fully 3D Time-of-Flight iterative reconstruction (2 iterations, 28 subsets, standard 5 mm filter, voxel size 3.125x3.125x2.78). Deformable image registration was used to spatially align subjects to a previously created morpphological and functional whole-body imaging atlas (Ekström et al., ISMRM 17), to allow voxel-wise comparisons between the imaged subjects and the atlas. Each voxel in the atlas contains mean and standard deviation of the PET uptake. Utilizing the knowledge that low ADC-values (low diffusion measured by MRI) and high FDG uptake (high metabolism measured by PET) is indicative of malignancy, suspected lesions can be detected by measuring how much the FDG uptake in each voxel deviates from “normality”, as defined by the atlas. This approach generates a voxel-wise “lesion probability map” for the imaged subject. The same registration approach can be used to quantify longitudinal changes in detected lesions, for treatment evaluation.

Results

Lesion probability maps have been generated for patients with manually identified lesions, correctly assigning high values to the regions manually identified as suspected lesions.

Conclusions

The proposed method is promising for lesion detection in whole body PET-MRI images. Future work includes quantitative verification of the accuracy of the detected regions, comparing against manual detection by a radiologist.

Legal entity responsible for the study

Uppsala University

Funding

Antaros Medical

Disclosure

H. Ahlström, J. Kullberg: Co-founder and owner of Antaros Medical.

All other authors have declared no conflicts of interest.

Disclosure

H. Ahlström, J. Kullberg: Co-founder and owner of Antaros Medical.

All other authors have declared no conflicts of interest.

Disclosure

H. Ahlström, J. Kullberg: Co-founder and owner of Antaros Medical.

All other authors have declared no conflicts of interest.

Background

The importance of angiogenesis for solid tumor growth is well recognized and evident by the vast differential of research devoted to the subject for over thirty years. It is a complex process directed by growth factors, receptors, extracellular matrix (ECM)-to-cell and cellto- cell interactions. Tyrosine kinase (TKs) is the protein enzymes catalyze the transfer of a phosphate group from an ATP molecule to a tyrosine residue of the target protein, thus leading to signal transduction. Cytoplasmic TKs such as Src, Abl and Lck have been to date discovered and characterized and found that inhibition signalling pathway. A special Src protein like FAK is a non-receptor protein-tyrosine kinase which have vital role in various cellular function like cytoskeleton reorganization, migration, adhesion, spreading, configuration and destruction of FA, cell protrusions, progression, proliferation and apoptosis. The functional ateration of Src signal may be the reason for cancer and metastasis. So this can predict that Src and their signal inhibition can utilized in cancer therapy. Triazine containing hybrid analogues act as a prime skeleton to inhibit Src family TKs like FAK so in present project we constructed 1,3,5-triazine containing analogues (TCA) as an effective cancer induced angiogensis inhibitor.

Methods

TCA analogues constructed accordingly similarity field positioning and pattern through forge V10. Further more in-silico simulation was done using autodock for most prominent analogues. The analogues derived via multifactorial synthetic protocol. The activity evaluation proceeded via in-vitro assay against MCF-7 (Breast cancer) cell line and further in-ova antiangiogenic potency evaluated against cancer induced chick chorioallantoic membrane.

Results

The newly designed and constructed TCA heterocycles expressed more than 56% of similar field point pattern and intra atomic alignments. The prominent pattern showed by the analogue 8d (chloro-anilino) and 8k (bromo-anilino). In-silico docking on hydrophobic site of Src family protein (PDB: 4BRX) revealed that analogue 8d have binding with CYS502, TR503, GLU506, ILE428, ASN551 while analogue 8k shoed interaction with THR503, GLU506, ILE428, LEU567, ASN551 amino acid residue which was similar like vendatinib. Biological evaluation showed that analogues 8d and 8k have great tendency to inhibit cancer induced angiogenesis with marginal toxicity profile.

Conclusions

We have developed a significant series of anticancer analogues and propound the site of binding on the surface of Src family TKs receptor FAK.

Legal entity responsible for the study

N/A

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Plants have had crucial role in the folklore of ancient cultures for over 5000 years. In addition to the use as food or spices, plants have also been utilized as medicine. Two remaining living traditions, the traditional Indian and Eastern medicine, have contributed most to the current state of knowledge related to medicinal plants. In their folklore, herbal medicines were prepared e.g. as teas or tinctures. These products are used as complementary treatment alongside conventional drugs till today. Another trend begun in the early 19th cent., which involved the isolation of active compounds from plants. This tendency led to the discovery of the analgesic alkaloids morphine and codeine or the cardiac glycoside digitoxin. Recently, a new alkaloid bersavine was isolated from Berberis vulgaris, along berbamine and berberine.

Methods

The dried root bark from Berberis vulgaris was minced and extracted with EtOH. The extract was evaporated, dissolved in HCl and extracted with nonpolar solvent. After the second extraction column chromatography on Al₂O₃ was performed. Subsequently was executed preparative TLC, which reveal a yet unknown alkaloid, later identified by MS and NMR analysis and named bersavine. Effect of bersavine on viability and proliferation was evaluated by WST assay and Trypan blue staining. Next was analysed its impact on cell cycle and apoptosis using the flow cytometry, activity of caspases and Western blot analysis of regulatory proteins was implemented.

Results

Bersavine's effect on cancer cells was first evaluated on panel of 9 different cell lines. Cancer cell lines MOLT-4, Jurkat, HT-29, HeLa and MCF-7 appear to be the most sensitive to the effect of bersavine. Follow-up experiments revealed that bersavine reduced cell viability and proliferation in a dose dependent manner within 24 h of treatment. Moreover, the reduction of cell viability was even more pronounce 48 h following the treatment. The decrease in cell viability was caused by the induction of apoptosis and activation of caspases.

Conclusions

All acquired results suggest that bersavine has very promising activity and it would be worthwhile to subject it to further evaluation.

Legal entity responsible for the study

Charles University, Faculty of Medicine in Hradec Kralove

Funding

Grant Agency of Charles University (Project No. 932616) and SVV-260397/2017.

Disclosure

All authors have declared no conflicts of interest.

Background

Tumor lesions of the sacrum are relatively rare and account for 1-7% of all spinal tumors. Tumors of this localization are usually detected when the tumor reaches a significant size and causes gross neurologic disorders and impaired pelvic organs. Radicality of removal of tumors of the sacrum depends on the involvement of the cauda equina, pelvic organs and vascular structures in the process.

Methods

The study involved 13 pateintd TMA clinic on the basis of the Department of Traumatology, Orthopedics, Neurosurgery with GPH No2 from 2011 to 2016. There were 6 women, 7 men. Age category ranged from 17 to 50 years. 1-stage: Holographic selective angiography by Seldinger's method of small pelvic vessels with subsequent embolization of “feeding” tumor of vessels. In addition, the anatomical features of the arteries are determined taking into account the localization of the tumor process, which is important in the operation. 2-stage: After preoperative embolization, three patients underwent hemisakrumectomy with VS3-VS5, and three patients underwent VS1-VS3 sarcomectomy. In this case, patients were additionally stabilized by TPF systems by Lumbo-Pelviofixation.

Results

In 2 cases during surgery, the tumor was intimately soldered to the roots of the horse tail and their isolation led to traumatization of the horse's tail, resulting in a postoperative delay in urine and stool. These violations were resolved within 2 months. In 4 patients with neurinoma S1, S2, S3 spine, an involuntary resection of these roots was performed. In these cases complications from the pelvic organs were not observed due to the presence of a cross innervation. In 2 patients, because of the duration of the operation, suppuration of the operating wound was noted with subsequent secondary healing. There were no lethal outcomes among 13 patients during follow-up.

Conclusions

The tactics of surgical treatment of sacral tumors, including the preliminary embolization of “feeding” arteries with subsequent radical removal of volume formation, reduces the risk of intraoperative complications, and also allows to remove the tumor totally, which in turn prevents its recurrence.

Legal entity responsible for the study

Traumatology Department

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

The role of stromal cells and the tumor microenvironment has been described to modulate cancer development and tumor drug sensitivity, in part due to the interaction with fibroblasts. Therefore, it is critical to incorporate this feature in our in vitro model and to evaluate its potential impact in early stages of drug development.

Methods

Non Small Cell Lung Cancer (NSCLC) tumor cell aggregates were microencapsulated in alginate capsules, alone or in combination with fibroblasts (immortalized normal and cancer-associated fibroblasts – NFs and CAFs, and human dermal fibroblasts - hDFs), cultured during four weeks; and tumor growth and drug response, both in vitro and in vivo, were evaluated.

Results

Microencapsulation of H1650 and H1437 spheroids in mono- or co-cultures with fibroblasts resulted in viable cultures with tumor aggregate increasing continuously during culture time. However, tumor growth in in vitro co-cultures was dependent on the source of fibroblast and cell line used. When challenged with drugs, co-cultures with fibroblasts in our in vitro 3D model presented, in general, lower sensitivity to therapy after 3 weeks of treatment. H1437+hDFs co-cultures showed less sensitivity to volasertib treatment, with higher DNA concentration (2-fold higher versus mono-cultures) and higher resazurin reduction activity (35% versus 22% in mono-cultures). H1650+NFs co-cultures also demonstrated lower sensitivity to erlotinib and docetaxel treatment, with higher resazurin reduction activity (71% versus 29% in mono-cultures) and higher viable area of aggregates, respectively. Mono and co-cultures can also be injected in mice for the generation of xenografts. Evaluation of tumor growth based on the local of injection, fibroblast source and drug response was compared. In agreement with the in vitro results, only co-culture of H1437+hDFs injected in the lungs significantly enhanced in vivo tumor growth. However, co-culture of H1650 with fibroblasts did not result in altered tumor growth in vivo.

Conclusions

Altogether, we established a 3D model with co-culture of NSCLC tumor cell aggregates and fibroblasts that, depending on the pair used, presented reduced sensitivity to standard of care drugs, better reflecting the clinical observations.

Clinical trial identification

N/A

Legal entity responsible for the study

iBET/ITQB-UNL; AbbVie and Oncotest

Funding

Innovative Medicines Initiative Joint Undertaking (IMI grant agreement n° 115188), resources composed of financial contribution from EU – FP7 and EFPIA companies in kind contribution. iNOVA4Health – UID/Multi/04462/2013, a program financially supported by Fundação para a Ciência e Tecnologia/Ministério da Educação e Ciência, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement is also acknowledged. SA and MFE are recipients of PhD fellowships from FCT (PD/BD/105768/2014 and SFRH/BD/52208/2013, respectively).

Disclosure

All authors have declared no conflicts of interest.

Background

Like BRCA1/2, deleterious mutations in PALB2 underlie deficiencies in homologous recombination-based DNA repair (HRD) and can underlie sensitivity to platinum (Pt) therapies and PARP inhibitors (PARPi). BRCA1/2 reversion mutations have been widely reported to cause therapy resistance. We report analogous reversion mutations in PALB2 for breast, ovarian, and prostate carcinomas (Ca).

Methods

Comprehensive genomic profiling (CGP) of DNA (≥50 ng) extracted from 114,200 samples, both solid tumors and heme malignancies, was performed using hybridization-captured, adaptor ligation-based libraries (mean coverage depth >600X) for up to 315 cancer-related genes. Samples were evaluated for substitutions, indels, copy number changes and rearrangements.

Results

PALB2 mutations were found in ∼1-3% of breast, ovarian and prostate carcinomas. Of 744 samples (0.7% of total) with likely deleterious PALB2 mutations, 7 (0.9%) harbored multiple alterations with at least one predicted to be a reversion. Of these samples, 4 were breast Ca, 1 a prostate acinar adenocarcinoma, 1 a high-grade ovarian serous Ca, and 1 an ovarian carcinosarcoma. For 3 samples the availability of multiple tests indicated the acquisition of the predicted reversion mutation(s) over time. Several reversion mutation types were observed: missense (1), compensatory frameshifts (3), overlapping small indels (2), and a deletion likely to disrupt splicing (1) that may lead to skipping of exon 4 and an in-frame deletion excising a frameshift mutation. Multiple reversion mutations were observed in 2 cases. Only 1/7 cases also showed disruption of BRCA1/2.

Conclusions

In addition to the commonly sequenced genes BRCA1/2, PALB2 mutation can lead to homologous recombination deficiency. As with BRCA, the acquisition of additional mutations predicted to restore at least some PALB2 function and thus potentially confer resistance to therapies dependent on HRD, can be observed in tumors such as breast, ovarian, and prostate carcinomas. CGP is a valuable tool to identify clinically significant, albeit rare, primary PALB2 mutations in BRCA-negative tumors as well as acquired secondary resistance mutations in patients who progress on Pt and PARP inhibitor based therapies.

Legal entity responsible for the study

Foundation Medicine

Funding

Foundation Medicine

Disclosure

L.M. Gay, S. Daniel, J. Suh, S. Ramkissoon, J-A. Vergilio, E. Severson, P.J. Stephens, J.S. Ross, J.A. Elvin: Employee of and stockholder in Foundation Medicine, Inc.

Background

Microsatellite instability (MSI) is a hallmark of mismatch repair (MMR) deficiency and can be attributed to alterations in MMR-related genes including MSH2, MLH1, MSH6, and PMS2. Although alterations in PI3K pathway genes have been reported in MSI-High (MSI-H) colorectal carcinoma (CRC), a comprehensive enrichment analysis of the genomic landscape in MSI-H and MSI-stable (MSS) populations across tumor types is lacking. To better understand the molecular signatures of MSI and investigate new avenues for therapeutic opportunities, we sought to define the genomic landscape of MSI-H tumors across cancer types.

Methods

Comprehensive genomic profiling of 395 cancer-related genes, including MSI status, was performed on ∼70,000 tumors. To identify potential driver alterations enriched in MSI-H tumors, variants in regions likely to be affected by polymerase slippage were excluded.

Results

As expected, alterations in MSH2, MHL1, MSH6, and PMS2 as well as MMR deficiency variants were enriched in MSI-H specimens regardless of tumor type. We confirmed that variants in PI3K genes were enriched in MSI-H tumors in CRC. Importantly, this was observed across all MSI-H tumors, with 57% of pan-solid MSI-H tumors harboring a PI3K pathway variant compared to 24% of MSS tumors. WNT pathway variants were also enriched specifically in MSI-H tumors, except for CRC, in which frequent APC variants in MSS resulted in WNT enrichment in MSS tumors. Together, 84% of MSI-H tumors have at least one PI3K or WNT pathway variant (compared to 48% of MSS samples). Finally, although ERBB2 alterations occur in both MSS and MSI-H tumors, we found that ERBB2 amplifications occur nearly exclusively in MSS tumors, while ERBB2 missense mutations are enriched in MSI-H tumors.

Conclusions

The genomic landscapes of MSI-H and MSS tumors suggest that they acquire alterations in distinct pathways. MSI-H tumors appear to share signaling pathway alterations across diseases, suggesting that MSI-H tumors may be more molecularly similar to one another than they are to MSS tumors of the same disease histology. These data may provide new avenues for exploration of targeted therapies in MSI-H tumors.

Legal entity responsible for the study

Foundation Medicine Inc.

Funding

Foundation Medicine Inc.

Disclosure

S.E. Trabucco: Current employee at Foundation Medicine. S.L. Maund, P.S. Hegde, S-M.A. Huang: Current employee and has ownership interest in Genentech. R. Hartmaier, K. Gowen, KJ. Sun, G.M. Frampton, P.J. Stephens: Current employee and has ownership interest in Foundation Medicine.

Background

There have been reported many gene expression profile which can predict prognosis of early stage breast cancer. We have reported the TP53 signature which can predict dysfunction caused by TP53 gene mutation in transcriptome level, and the status defined by TP53 signature can predict more accurate prognosis of early stage breast cancer compare to the status defined by TP53 DNA sequence. Recently, TP53 signature was reported as robust predictor of early stage breast cancer by meta-analysis (BMC Cancer,2015). The aim of this study is to make clear the molecular feature of poor prognosis group diagnosed by TP53 signature and to make easy and quick diagnostic kit which can be used in clinical situation.

Methods

We have done RNA-seq analysis using Hiseq2500 (Illumina) and reanalyzed TCGA data of breast cancer to make clear molecular feature of poor prognosis group defined by TP53 signature. We made simple diagnostic kit using nCounter (Nanostring technology). Using this simple diagnostic kit, we diagnosed 234 breast cancer sample as TP53 signature mutant or TP53 signature wild, and we proved robust prediction power of TP53 signature for early stage breast cancer. We used RNA-seq data to compare prediction power of TP53 signature to Mammaprint, OncotypeDX, TP53 structural mutation.

Results

TP53 signature mutant group have structural mutation in genes, including BRCA1, BRCA2, Rb1 except for TP53, which function is gene repair. In addition, TP53 signature mutant group shows high expression of PD-L1, high mutation burden and high copy number alternation. Analysis of 190 stage I-II breast cancer patients shows TP53 signature by simple diagnostic kit using nCounter has strong prediction power compare to Mammaprint, OncotypeDX, TP53 structural mutation, and clinical factors.

Conclusions

We have developed TP53 signature as diagnostic system for early stage breast cancer which is useful in clinical situation. Poor prognosis group diagnosed by TP53 signature shows molecular features which overlap good response marker of immune-check point inhibitors.

Legal entity responsible for the study

Japan

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Overexpression of DKK1, a modulator of canonical WNT signaling, is frequently seen in malignant tumors and is often associated with worse survival. Preclinical studies have shown that DKK1 can augment tumorigenesis by promoting angiogenesis as well as enhancing tumor-associated immunosuppression. In this Phase I trial, DKN-01 (a humanized IgG4 monoclonal antibody against DKK1) showed encouraging early efficacy signals when combined with paclitaxel (DP) in EGC pts (Strickler et al., GI ASCO 2017). We performed correlative studies of plasma biomarkers of angiogenesis and inflammation in these pts.

Methods

Blood samples were collected from 34 patients treated with DP at baseline, and then weekly for 4 weeks (w). Plasma biomarkers were measured by multiplexed array for angiogenesis (bFGF, PIGF/PGF, sVEGFR1, sTIE-2, VEGF, VEGF-C, and VEGF-D) and inflammation (IFN-g, TNF-a, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70 and IL-13) (Meso-Scale Discovery) and by ELISA for HGF and SDF-1a (R&D Systems). Biomarker changes were evaluated by Wilcoxon Sign-Rank test, and correlations with response using Kendall’s test values and with survival using two-sided Wald test in the Cox regression.

Results

Six of 34 EGC pts in this subset analysis (18%) had a PR and 11/34 (32%) showed SD with preliminary median PFS and OS of 13.7w and 28.4w, respectively. DP treatment induced significant and sustained increases in plasma IFN-g, IL-8, VEGF-D and decreases in IL-10 (all p < 0.05). Transient increases in bFGF and PlGF and decreases in sTie2 were also observed. IFN-γ, IL-10, and VEGF-D correlated with overall response and change in target lesion size. Finally, OS was poor in pts with high IL-6, IL-8 and TNF-a (HR > 1 all time-points) and prolonged in pts with greater increases in IL-2 and VEGF-D after treatment.

Conclusions

Prospective plasma biomarker analyses showed that DP treatment changed biomarkers of systemic immunity and angiogenesis in EGC pts, and indicated potential associations between inflammation biomarkers and outcomes. These hypothesis-generating results will inform future prospective investigation of these plasma biomarkers as well as paired evaluation of tumor biopsies for this combination regimen.

Clinical trial identification

Clinical trial information: NCT02013154

Legal entity responsible for the study

Leap Therapeutics, Inc.

Funding

Leap Therapeutics, Inc.

Disclosure

D.G. Duda: Consultant fees from Bayer and research funding from Leap Therapeutics, Inc, Merrimack, Bristol-Myers Squibb and Bayer. C. Sirard: Employee of Leap Therapeutics, Inc. (salary and stock options for compensation).

Background

Biomarkers predicting response to a bevacizumab containing therapy in metastatic breast cancer (MBC) are of urgent need. MicroRNAs (miRNAs) are involved in regulation of angiogenesis and development of treatment resistance and could therefore provide predictive information.

Methods

Profiling of 754 miRNAs was performed in FFPE tumor samples from 58 MBC patients who received bevacizumab-containing first-line treatment (learning set). Based on median PFS patients were divided into responders (R) and non-responders (NR). Differentially expressed miRNAs between R and NR were selected and validated in a cohort of 57 patients treated with first-line chemotherapy without bevacizumab (control set), to exclude miRNAs providing prognostic information only. In the learning set a multivariate analysis including clinical and pathological information was performed. MiRNAs significantly associated with PFS were further validated in 203 patients treated within the TANIA phase III trial randomizing between chemotherapy plus bevacizumab and chemotherapy alone for two consecutive treatment lines in patients pretreated with bevacizumab in first-line.

Results

Low expression of five miRNAs (miR-9-5p, miR-20a-5p, miR-21-5p, miR-210-3p, and miR-224-5p) was significantly associated with longer PFS in the learning set. For miR-20a-5p (P = 0.035) and miR-21-5p (P = 0.004) this association remained significant in multivariate analysis. In the control set no correlation between expression of those five miRNAs and PFS was seen. In tumor samples from the TANIA trial, low expression of miR-20a-5p was also significantly associated with longer second-line PFS and longer OS in the bevacizumab arm (HR 0.60, 95%CI 0.37-0.89; P = 0.012 and HR 0.54; 95%CI 0.32-0.83; P = 0.007, respectively) but not in the chemotherapy only arm (HR 0.73, 95%CI 0.48-1.09; P = 0.119 and HR 1.01 95%CI 0.63-1.62; P = 0.964, respectively). For miR-21-5p no significant association with PFS or OS in both treatment arms was observed.

Conclusions

MiR-20a-5p expression in breast cancer tissue showed a promising predictive value for identifying patients deriving greater benefit from bevacizumab-containing therapy.

Legal entity responsible for the study

Richard Greil for the translational research project, Roche for the TANIA trial

Funding

Roche

Disclosure

S.P.P. Gampenrieder, G. Rinnerthaler, A. Egle, R. Greil: Advisory role, speakers’ honoraria, travel grants, research funding (no personal payment) from Roche. C. Monzo Fuentes: Travel grants from Roche. C. Hufnagl, C. Hauser-Kronberger: Travel grants, research funding (no personal payment) from Roche. All other authors have declared no conflicts of interest.

Background

The major histologic subtype of esophageal cancer, esophageal squamous cell carcinoma (ESCC), is one of the most common cancers and has been ranked as the sixth leading cause of cancer-related deaths in the world. Using human genome U133 Plus 2.0 GeneChip, we identified gene down-regulation of Cell adhesion molecule L1 like (CHL1) located at 3p26.3 in ESCC. We analysed its down-regulated expression, biological effects and prognostic significance in ESCC.

Methods

To determine whether the down-regulation of CHL1 was associated with aberrant methylation. Methylation-specific PCR (MSP) was performed in ESCCs and their corresponding non-tumor tissues, as well as ESCC cell lines. Loss of heterozygosity status of CHL1 was evaluated by fluorescence in situ hybridization (FISH). The effects of CHL1 re-expression or knockdown were determined in proliferation, invasion and metastasis assay. CHL1 target genes and related pathways were identified by protein mass spectrometry, co-immunoprecipitation (Co-IP), immunofluorescence (IF) and western-blot. Clinical impact of CHL1 down-regulated expression was assessed in 287 patients with ESCC.

Results

The results showed that down-regulation of CHL1 was significantly associated with allele loss (14/21) and promoter methylation (19/21; P<0.05) in 39 pairs of ESCC and their corresponding non-tumor tissues by using MSP and FISH. Biofunctional investigation of CHL1 revealed that CHL1 significantly decreased cell proliferation, G1-S cell cycle transition, invasion/migration abilities, and promoted xenograft tumor growth as well as lymph node metastasis in vivo. The anti-proliferation effect by CHL1 was mediated through inducing cell cycle arrest at G1/S checkpoint by down-regulation of p21 and p53 and up-regulation of cyclin D1; the inhibiting metastasis role was by suppressing Epithelial-to-Mesenchymal Transition and F-actin formation which was the result of recruitment Merlin to cell surface expression by CHL1. After a median follow-up of 48.23 months, multivariate analysis revealed that patients with CHL1 protein down-expression had a significant decrease in overall survival. Kaplan-Meier survival curves showed that CHL1 down-regulated expression was significantly associated with shorter survival in patients with ESCC.

Conclusions

CHL1 plays a pivotal tumor suppressive role in ESCC; its down-regulated expression is an independent prognostic factor of patient with ESCC.

Legal entity responsible for the study

Henan Cancer Hospital

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Clinicopathological factors predicting for response to Bacillus-Calmette Guerin (BCG) treatment for non-muscle invasive bladder carcinoma (NMIBC) are well defined but there is a paucity of data on genetic factors. Vitamin D has been found to have immunomodulatory effects in pre-clinical bladder cancer studies. Various single nucleotide polymorphisms of the Vitamin D Receptor (VDR) gene has also been found to be associated with response to treatment for mycobacterium. In this study, we evaluated the predictive role of 3 VDR single nucleotide polymorphisms (SNP) in patients with NMIBC in assessing BCG immunotherapy outcome.

Methods

Peripheral blood DNA was prospectively obtained from 140 evaluable EORTC intermediate to high risk NMIBC patients, who underwent post-transurethral resection intravesical regimes of BCG or BCG with interferon alpha. 3 VDR SNPs commonly implicated in susceptibility to tuberculosis infections were evaluated using high resolution melt (HRM) analysis followed by DNA sequencing. Kaplan-Meier together with Log-Rank test and Cox regression methods were used to analyze the data.

Results

Genotype frequencies were similar between the NMIBC patients and controls in accordance to the Hardy Weinberg equilibrium. Mean follow-up time was 91.9 months. Overall mean time to recurrence and progression was 25.8 months and 47.0 months respectively. Kaplan-Meier analysis indicate that individuals carrying the VDR genotype rs1544410 A/G were significantly associated with lower recurrence-free survival rates after BCG therapy (p = 0.007). The VDR rs1544410 “A” allele frequency was found to be higher in patients with bladder cancer recurrences (p = 0.01). No association of VDR genotypes with progression-free survival was found.

Conclusions

Our findings suggest that polymorphisms in the VDR gene correlate with response to BCG therapy in NMIBC patients and further work should be performed to evaluate their utility as predictive markers of response to BCG immunotherapy.

Legal entity responsible for the study

National Healthcare group Domain Specific Review Board

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Chemotherapy (CT) combined with anti-HER2 drugs (H) is the treatment of choice for HER2+ early breast cancer (BC) patients (pts). However HER2+ tumors are clinically and biologically heterogeneous, and treatment response largely varies according to ER status. Predictive biomarkers are urgently needed in this context. We have recently developed a meta-dataset of clinical trials of neoadjuvant CT +/− H (trastuzumab, lapatinib, or both) in HER2+ BC pts annotated for gene expression, hormone receptor status and pathological complete response (pCR) rates. We have shown that a gene-signature (GS) of RB-1 loss-of-function (RBsig) seems to be predictive of response to neoadjuvant CT +/− H in ER+/HER2+ BC pts in this meta-dataset. Here we report the results of additional analyses aimed to evaluate RBsig’s predictive value against 10 previously developed GSs in the subset of ER+/HER2+ BC pts.

Methods

The association of RBsig with pCR was evaluated in comparison with previously described GSs of: low ER signaling, p53 mutation, high PI3K pathway signaling, high expression of HER2 amplicon genes, PAM 50 and 5 immune-related GSs. For each GS, samples were classified as High or Low group using a previously described classifier. Odds Ratio (OR) performance was calculated using the ROCR (v. 1.0) package in R and plotted by forest plot using the survcomp (v. 1.24) package.

Results

RBsig and the HER2 amplicon GS were best at predicting response to neoadjuvant CT +/− H (211 pts; p:0.017). In the subgroup of pts treated with CT alone (n = 94), only the PI3K pathway GS was significantly associated with pCR (p:0.026). In pts treated with CT + H (n = 117) only the HER2 amplicon GS significantly correlated with pCR (p:0.042). RBsig showed a similar trend in both these subgroups (p: 0.078 and 0.104, respectively) The immune GSs and PAM50 were not associated with pCR, independent of treatment received.

Conclusions

RBsig and HER2 amplicon GS are strongly associated with pCR in ER+/HER2+ tumors unselected for treatment. These results support the potential use of such GSs as predictive markers of response to CT +/−H in ER+/HER2+ BC pts. Validation studies are warranted.

Legal entity responsible for the study

Angelo Di Leo

Funding

None

Disclosure

A. Di Leo: Consultant/Advisory Board: Novartis, Pfizer, Lilly. All other authors have declared no conflicts of interest.

Background

Gastric cancer is a common and lethal malignancy, killing over 700,000 people worldwide. Recurrence rates are high even in early stage disease, with 5-year overall survival being < 50% for stage II disease and above. Current guidelines for adjuvant therapy include 5-fluorouracil (5-FU) chemotherapy in combination with radiation. The aim of our study is to develop immunohistochemical biomarkers to predict response to adjuvant chemoradiation in patients with resected gastric cancer.

Methods

A tissue microarray composed of 100 specimens from primary resected gastric cancer cases was constructed. All patients received 5-FU based chemotherapy, and 92% of patients received radiation. Tumors underwent immunohistochemical staining for 11 proteins (HER2, EGFR, p-AKT, PTEN, MTOR, VEGFA, IGF1R, MLH1, MSH2, MSH6, PMS2), and H-scores were calculated. Primary endpoints were progression-free survival (PFS) and overall survival (OS). Survival analysis was performed by Kaplan-Meier method with log-rank test for assessing statistical significance. Multivariate analysis was performed with Cox regression.

Results

Mean follow-up time for the cohort was 39.4 months. Ninety-one of 100 cases had sufficient tissue for biomarker analysis. Interestingly, low expression of MSH2 and MSH6 was significantly associated with shorter PFS, while there was a trend towards worse OS for patients with low MSH2 expression. In multivariate analysis, adjusting for well-characterized prognostic variables, both MSH2 and MSH6 retained their predictive significance. In addition, nuclear expression of the tumor suppressor PTEN was associated with longer OS. No other biomarkers were significantly associated with PFS or OS.

Conclusions

These results indicate that low expression of MSH2 and MSH6 predicts for poorer outcomes in patients with resected gastric adenocarcinoma, independent of other predictive markers.

Legal entity responsible for the study

Hellenic Cooperative Oncology Group

Funding

None

Disclosure

G. Fountzilas: Honoraria: AstraZeneca, Consulting or advisory role: Pfizer, Sanofi, Roche Stock ownership (an immediate family member): ARIAD. All other authors have declared no conflicts of interest.

Background

Breast cancer is the leading cause of cancer-related deaths among women worldwide. Current clinicopathological parameters only partially encompass and predict biological diversity and therefore limit our ability to make informed treatment decisions and predict outcomes. The successful future of oncology will rely on our ability to correctly select patients who would benefit from chemotherapy or benefit from treatment intensification, and spare the rest from unnecessary exposure to toxic and expensive therapies. Tumour biology and prognostic markers may be the key to achieving the above goal. We investigated whether changes in Large scale DNA Organization (LDO) of tumour epithelial nuclei is an indicator of the aggressiveness of the tumour.

Methods

We tested our algorithm on a set of 172 TMA cores samples, coming from 95 breast cancer patients. Thirty five patients died of breast cancer and 60 were still alive 0 years after surgery. This TMA slide was stained with Feulgen-thionin and imaged using an high-resolution Imaging system. Automated segmentation of cell nuclei followed by manual selection of intact, in-focus nuclei resulted in an average of 50 cell nuclei per sample. Approximately 60 features measuring Large-scale DNA organization were calculated.

Results

Forward stepwise Linear Discriminant analysis selected 6 features that, combined linearly, gave the best discrimination between nuclei from alive patients specimens and nuclei from deceased patients specimens. Patient LDO score was defined as the percentage of cell nuclei with a high cell LDO. LDO algorithm correctly classified 82.1% patients, with a specificity of 79% and a sensitivity of 88%. Furthermore, individuals with a high LDO score had a 9x fold increase in relative risk of death. In the multivariate Cox regression model, LDO, Node status and Tumor Grade were all significant predictors of cancer death.

Conclusions

This data suggests that LDO could be used to identify patients more likely to have an aggressive disease and thus select a candidate for more aggressive or novel adjuvant therapies.

Legal entity responsible for the study

Martial Guillaud

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

The molecular mechanisms that determine disease progression and evolution in CLL are not completely known. Telomeres are usually short in CLL and their attrition may contribute to disease evolution. In addition, telomerase activity (TA) levels have also been associated with prognosis and response to treatment. In order to integrate telomere associated variables (TAV) in CLL disease management further studies with robust methodology are required.

Methods

Purified peripheral CD19 (+) B-cells from 19 healthy donors and 42 CLLs in different stages of disease were obtained from the National Bank of DNA (Salamanca, Spain). Samples were tested to determine full telomere length (TL) distribution- including percentage of short telomeres by a high throughput quantitative fluorescence in situ hybridization (HT-Q-FISH) technique. TA by Quantitative Telomere Repeat Amplification Protocol (Q-TRAP) was also quantified. Full statistical analysis of the results in the context of the clinical history of the patients was performed.

Results

Data from the pilot, retrospective study stablished strong correlations between key CLL variables and the severity of the disease. Overall, TL was shorter and TA presented higher values compared to normal age-matched subjects. Interestingly longer TL was observed for all CLL patients with somatic hyper-mutation (SHM) that in turn, was associated with better prognosis. Concomitantly, TA was elevated in those patients with no SHM and was linked to poor response to treatment and negative prognosis. The percentage of short telomeres was significantly higher for Binet C/Rai III and IV cases.

Conclusions

The use of reliable technologies to measure TAV should be integrated during early diagnostic in CLL to enhance the ability to predict disease evolution. This will require larger, prospective, longitudinal clinical studies.

Legal entity responsible for the study

Life Length S.L.

Funding

Life Length S.L.

Disclosure

N. De Pedro, M. Diez, I. Garcia, R. González, B. Garcia, L. Esteban, L. Otero, P. Najarro, J.C. Estrada, J. García: Employee of Life Length. M. Chiesa: Former employee of Life Length.

Background

Bevacizumab-containing therapy improves progression-free survival (PFS) in human epidermal growth factor receptor (HER) 2-negative metastatic breast cancer (mBC), but its use has been questioned due to the lack of benefit in overall survival (OS). To date, biomarkers to predict its positive effect are not available. Interestingly, miRNAs have emerged as regulators of most processes, forming tight interconnected feedback loops with genes under their regulation. Currently, different analyses, such as microarray, are performed in order to identify miRNA biomarkers, using formalin-fixed and paraffin-embedded (FFPE) tissue samples.

Methods

In the present study we recorded clinical data from 57 mBC patients, and selected two (4 + 4) PFS extreme groups for the analysis of the miRnome. Three miRNAs were used for normalization (U6, 191-5p and 103-a-3p). miRNAs for model construction were selected by differential expression between the two groups. Candidates were measured in the remaining 49 cases, and stepwise based Akaike criterion was used for profile generation. Additionally, integrative miRNA and mRNA analyses were done to reveal markers and pathways with potential clinical impact.

Results

We selected two groups of patients with extreme differences on PFS (2.48 ± 1.85 vs 35.43 ±8.03 months) for their miRnome study. The expression profiles of miRNAs in both groups were highly correlated, except for 13 miRNAs where statistical differences arised. These miRNAs were selected as candidates for profile generation on the 49 additional cases, and a combination of five of them (miR-362-3p, miR-150b-5p, miR-671-3p, miR-744-3p and miR-941) was able to accurately discriminate two PFS groups. Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses on miRNA possible target genes revealed interesting pathways to explore in these patients, such as cellular adhesion.

Conclusions

By combining experimental approaches and computational biology, we have identified candidate markers of outcome for bevacizumab-containing therapy. The five miRNAs included in the prognostic profile and cellular adhesion related genes should be explored as potential biomarkers.

Legal entity responsible for the study

Fundación para la Investigación del Hospital Universitario La Paz

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Carcinoma of unknown primary (CUP) accounts for approximately 3% of all malignancies. Identification of common cancer pathway alterations (hallmarks of cancer) in diverse cancer lineages offers a rationale for search for biomarkers of targeted therapies in patients with CUP. Avoiding immune destruction is a more recently recognized common cancer characteristic and biomarkers associated with immune check-point blockade were explored in this study.

Methods

392 cases of CUP were tested with NextSeq platform with a 592-gene panel. Tumor mutational load (TML) was calculated using only somatic nonsynonymous missense mutations; microsatellite instability (MSI) was evaluated with NGS by direct analysis of known MSI loci in the target regions of the sequenced genes. ArcherDx FusionPlex Assay was used to detect gene fusions and 52 gene targets were analyzed for 156 tumors. IHC was used to detect tumor expression of PD-L1 (SP142 antibody) and PD-1 TILs (NAT105 antibody) All tests were done in a CLIA-certified lab.

Results

Average patient’s age was 62.4 years; 52% were female. TML high was seen in 12.2% (48/392) tumors using a cutoff of 17 mutations/Mb. MSI-high was detected in 10/392 (2.6%) of tumors. A total of 70 different genes were found mutated with the incidence ranging from 0.3% to 54%; the most frequent were TP53 (53.5%), KRAS (21.5%) and ARID1A (14.6%). Additional notable targetable mutations include PIK3CA (13.1%), CDKN2A (8.1%), PTEN (4.5%), BRAF (4.1%), ATM (3.3%), NOTCH1 (2.4%) and ERBB2 (1.5%). Targetable gene fusions identified included FGFR2 fusions (N = 2), RET (N = 1), RAF1 (N = 1).Tumors with fusions identified carry a lower TML (average 5.9) than the complete cohort (11.7, p < 0.001) with no MSI-high seen in this subgroup. Tumor expression of PD-L1 was seen in 22.5% (82/365) tumors while PD-1 expression on tumor infiltrating lymphocytes seen in 58.7% (37/63). The highest frequency of gene amplification seen were CCND1 (4.7%), FGF3 (3.4%), FGF4(3.4%), FGF19(3.4%), Her2 (3.1%), MYC (2.9%) and AKT2 (2.4%). Of note, CD274 (PD-L1) was rarely amplified (1.4%).

Conclusions

Using a multiplex testing approach, 28% of CUPs had biomarkers (TML-H, MSI-H and/or PD-L1) of response to the immune check-point blockade were identified, making CUP one of the most likely candidate to benefit from immune checkpoint inhibitors.

Legal entity responsible for the study

Joanne Xiu

Funding

None

Disclosure

J. Xiu, Z. Gatalica: Employee of Caris Life Sciences.

Background

Surgery is the only potentially curative option for patients with pancreatic ductal adenocarcinoma (PDAC), but metastatic relapse remains common. We hypothesized that the expression levels of inflammatory cytokines could predict recurrence of PDAC, thus allowing to select patients who most likely could benefit from surgical resection.

Methods

We prospectively collected plasma at diagnosis from two-hundred eighty-seven patients with pancreatic resectable neoplasms. The expression levels of 23 cytokines were measured in ninety patients with PDAC by using a multiplex analyte profiling assay.

Results

Levels higher than cutoff identified of the T_H2 cytokines interleukin (IL)4, IL5, IL6, of macrophage inflammatory protein (MIP)1a, granulocyte-macrophage colony-stimulating factor (GM-CSF), and monocyte chemoattractant protein (MCP)1, and of IL17a, IFN-γ- induced protein (IP)10, and IL1b were significantly associated with a shorter median OS. In particular, levels of IL4 and IP10 higher than cutoff identified, and level of T_H1 cytokines TNFa and INFg, and of IL9 and IL1Ra lower than cutoff identified were significantly associated with a shorter DFS. In the multivariate analysis, high IP10 was confirmed as negatively associated with OS (HR = 3.097, P=0.014) and IL4 and TNFa remain negatively (HR = 2.75, P=0.002) and positively (HR = 0.224, P=0.049) associated with DFS, respectively. Simultaneous expression of low IL4 and high TNFa identified patients with best prognosis (HR = 0.313, P<0.0001).

Conclusions

We demonstrated that, among a series of cytokines, IL4 is the most significant independent prognostic factor for DFS in resectable PDAC patients, and it could be useful to select patients with high risk of early recurrence who may avoid an unnecessary resection.

Legal entity responsible for the study

University of Verona

Funding

Associazione Italiana per la Ricerca sul Cancro (AIRC)

Disclosure

All authors have declared no conflicts of interest.

Background

Recent data suggest that analysis of tumor mutational burden (TMB), a measure of tumor neo-antigenicity derived from tissue biopsies, has shown clinical utility in predicting outcomes for patients treated with anti-PDL1/PD1 therapies across a range of tumor types. Unfortunately, such analyses require quality tumor tissue that in many cases is not available for patients diagnosed in the metastatic setting. As such, there exists a significant unmet medical need for orthogonal diagnostic approaches that enable the analysis of TMB in patient samples without requiring tumor tissue. Herein, we describe the development of an assay to identify TMB from the circulating tumor DNA derived from blood (bTMB), and the analytical validation (AV) that supports its application in a phase III clinical trial in 1L non-small cell lung cancer comparing the anti-PD-L1 agent, atezolizumab, against standard of care platinum-based doublet chemotherapy (BFAST).

Methods

The bTMB assay delivers a count of somatic base substitutions down to 0.5% allele frequency across 394 genes from as little as 1% tumor content in a cell free DNA (cfDNA) sample. AV focused on establishing accuracy and precision, as well as the limit of circulating tumor DNA required to make precise and reliable bTMB calls. Accuracy of the two different bTMB cutoffs being evaluated in BFAST was established against an orthogonally validated TMB platform. Precision was evaluated by comparing the reproducibility of bTMB calls across replicate samples.

Results

The average PPA, NPA and PPV across both bTMB cutoffs was 95%, 100% and 100%, respectively. The average precision was 96%, with a coefficient of variation of 17% across all replicates. The assay limit of detection was defined as 1% tumor content in at least 20 ng of cfDNA.

Conclusions

We have developed and analytically validated a blood-based assay to determine bTMB with high accuracy and precision from as little as 1% tumor content in 20 ng of cfDNA. Clinical validation of bTMB will be established in a prospective, randomized phase III clinical trial, BFAST, with a primary endpoint of progression free survival.

Legal entity responsible for the study

Foundation Medicine, Inc.

Funding

Genentech, Inc.

Disclosure

D. Fabrizio, C. Malboeuf, D. Lieber, S. Zhong, J. He, E. White, M. Coyne, J. Silterra, T. Brennan, J. Ma, M. Kennedy, D. Lipson, G. Otto: Employee and stockholder of Foundation Medicine. E. Schleifman, S.M. Paul, Y. Li, D.S. Shames, C.A. Cummings, E. Peters, M. Kowanetz: Employee and stockholder at Genentech.

Background

Tumour programmed cell death ligand-1 (PD-L1) expression is a key biomarker in identifying patients who may have an enhanced response to non-small cell lung cancer treatment using anti-PD-1 (e.g. nivolumab and pembrolizumab) or anti-PD-L1 (e.g. atezolizumab and durvalumab). Each treatment is currently used in conjunction with an individual PD-L1 diagnostic immunohistochemistry (IHC) assay and it is unclear whether immunolabelling parameters determined by pathologists are comparable across assays. We extended previous studies (Ratcliffe et al Clin Cancer Res 2017; Ratcliffe et al ASCO-SITC 2017 [abstr 7]) by performing image analysis (IA) with a customised PD-L1 scoring solution to permit a quantitative comparison of the 4 PD-L1 IHC assays.

Methods

We developed an IA scoring algorithm that enabled us to quantify the percentage of positive tumour cells on a whole slide image for 4 PD-L1 assays (Ventana SP263, Ventana SP142, Dako 28-8, Dako 22C3). The analysis was applied to 473 commercially available NSCLC cases (180 cases with SP142). We co-registered the consecutive slides per case and harmonised tumour and exclusion annotations to ensure that readouts of identical areas were compared per case.

Results

In agreement with previous reports, IA results showed concordance between 3 assays, whereas the SP142 assay was discordant. Moreover, high correlation was observed between IA results and pathologist ratings. This correlation could be further improved by matching the information the pathologist received to the same information used in the IA solution: blinding against the assay, scoring on digital scans and masking of non-comparable image regions. The remaining differences represent the differing sensitivity profiles of the assay protocols.

Conclusions

The results of our objective IA suggest differences in sensitivity between the analysed assays. Importantly, despite the observed differences, we confirm previous findings indicating concordance between 22C3, 28-8 and SP263. In addition, our analysis provides a continuous distribution of PD-L1 measurements allowing deeper characterisation of the samples. Tobias Wiestler and Moritz Widmaier are joint first authors.

Clinical trial identification

NA

Legal entity responsible for the study

AstraZeneca PLC

Funding

AstraZeneca

Disclosure

T. Wiestler: Full-time employee Definiens, Stock/shareholder: Definiens/AstraZeneca. M. Widmaier: Full-time employee Definiens. J. Walker, C. Barker, M. Scott: Employee and shareholder AstraZeneca. F. Sekhavati, A. Budco: Employee Definiens. K. Schneider: Full-time employee Definiens AG. K. Steele: Employee MedImmune, shareholder AstraZeneca, Patents/Royalties MedImmune/AstraZeneca. M.C. Rebelatto: Employee AstraZeneca/MedImmune; Stockholder AstraZeneca/MedImmune.

Background

CEA-CD3 TCB (RG7802, RO6958688) is a novel T-cell bispecific antibody targeting CEA on tumor cells and CD3e on T cells. In mouse models, CEA-CD3 TCB displays potent anti-tumor activity, leads to increased intratumoral T cell infiltration and activation and up-regulates the PD-L1/PD-1 pathway.

Methods

Biodistribution was assessed in mice using SPECT/CT. Patient (pt) samples were from 2 dose-escalation studies in CEA-positive solid tumors. Study 1 (S1): single agent weekly (qw) (0.052-600 mg IV; n = 80), and Study 2 (S2): CEA-CD3 TCB qw (5-160 mg IV) plus atezolizumab 1200 mg q3w (n = 46). Analytics: [CEA-CD3 TCB]—bifunctional PK assay; antidrug antibodies—ELISA; immunophenotyping in peripheral blood (PB)—flow cytometry (FCM), in baseline (BSL) and on-treatment (OT; week 7) biopsies by immunohistochemistry and FCM; plasma cytokines—multiplex assay; PD-L1—SP142 assay.

Results

In mice, CEA-CD3 TCB preferentially accumulated in CEA-positive tumors. CEA-CD3 TCB showed near-linear PK in both studies (S1: 35; S2: 28). In pts with matched BSL and OT biopsies, 7/10 CRC pts treated with ≥ 60 mg of CEA-CD3 TCB in S1 had > 2.4-fold increase in CD8 T cells, which did not correlate with RECIST response. In S2, 2/2 CRC pts receiving ≥ 80 mg of CEA-CD3 TCB (with RECIST reduction ≥ 25%), showed > 8-fold increase in CD8/Ki67 T cells. SUVmax decrease (FDG-PET) correlated with BSL levels of CD4-OX40 and CD8-PD1 in S1 and CD8-OX40 in S2. In PB at week 4, a > 4-fold expansion of activated CD8 T cells (HLA-DR/Ki67) but not CD4, was detected in most pts at doses ≥ 60 mg (S1: 24; S2: 9). In most pts, increases in IL-6 were seen after the first TCB infusion and in fewer cases after the second/third infusion in both studies (S1: 62; S2: 33).

Conclusions

On-treatment increases in intratumoral CD8 T cells consistent with the mechanism of action and support that CEA-CD3 TCB is the first tumor-targeted T cell bispecific showing biological activity. The activation level of intratumoral T cells at BSL could be a predictive biomarker of response. In preclinical models, tumor targeting has been demonstrated. Updated data will be presented. Clinical data are reported separately.

Clinical trial identification

NCT02324257

Legal entity responsible for the study

F Hoffmann-La Roche Ltd.

Funding

F Hoffmann-La Roche Ltd.

Disclosure

I. Melero: Advisory board: Bristo-Myers Squibb, Roche-Genentech, AstraZeneca, Lilly, Merck Serono, Bayer, Genmab, Alligator, Bioncotech, Tusk Grants from: Roche-Genetech, Bristo-Myers Squibb, Bioncotech. N.H. Segal: Consulting and research funds from Genentech/Roche. J. Saro: Employee of Roche and stock holder of Roche. A. Marabelle: PI: Roche, Bristo-Myers Squibb, Merck, Pfizer, Lytix pharma, Eisai, AstraZeneca/Medimmune Scientific Consulting: Roche, Pierre Fabre, Onxeo, EISAI, Bayer, Genticel, Rigontec, Daichii Sankyo, Imaxio, Sanofi, BioNTech. J.M. Cleary: Research funding to the institution from Merrimack Pharmaceuticals, Taiho Oncology, Merck, Roche, Abbvie, Precision Biologics, and Bristo-Myers Squibb. H.I. Hurwitz: Honoraria: Roche and Lilly. Consultant: Roche, Bristo-Myers Squibb, Lilly, Novartis, Incyte, TRACON Pharma, Acceleron Pharma, GlaxoSmithKline, OncoMed. Institutional Funding: Roche, GlaxoSmithKline, Novartis, TRACON Pharma, Bristo-Myers Squibb, Regeneron, Lilly, Macrogeneics, NCI. C. Jamois, S. Bouseida, F. Sandoval, V. Karanikas: Roche employee. E. Andersson: RICM participation in the Roche Connect program. M. Bacac: Employed by Roche and own stock options. T. Nayak: Roche stocks. All other authors have declared no conflicts of interest.

Background

It is now demonstrated that gut microbiota composition has a determining influence not only on inflammatory bowel diseases but also more broadly on the immunological status of healthy individuals as well as in patients with cancer. We explored the potential role of baseline gut microbiota in anti-tumor response and in intestinal toxicity of patients with metastatic melanoma treated with anti-CTLA4 mAb ipilimumab. Moreover we explored how the composition of gut microbiota could influence not only local gut-immunity but also distant sites such as anti-tumor immunity.

Methods

Fecal microbiota compositions were prospectively assessed at baseline and before each ipilimumab infusion, using 16S rRNA gene sequencing. Patients were further clustered based on microbiota patterns. Peripheral blood lymphocytes immunophenotypes were studied in parallel.

Results

A distinct baseline gut microbiota composition was associated with both clinical response and colitis. As compared to patients whose baseline microbiota was driven by Bacteroides (Cluster B), patients whose baseline microbiota was enriched with Firmicutes (Cluster A) had longer progression-free survival and overall survival. Most of the baseline colitis-associated phylotypes were related to Firmicutes, whereas no-colitis related phylotypes were assigned to Bacteroides. A low proportion of peripheral blood regulatory T cells was associated with Cluster A, long-term clinical benefit and colitis. Ipilimumab led to a higher Inducible T-cell COStimulator induction on CD4+ T cells and to a higher increase in serum CD25 in patients who belonged to Cluster A.

Conclusions

Baseline gut microbiota enriched with Firmicutes is associated with beneficial clinical response to ipilimumab and more frequent occurrence of ipilimumab-induced colitis.

Clinical trial identification

GOLD study: SC12-018; ID-RCB-2012-A01496-37

Legal entity responsible for the study

Coordination: Franck Carbonnel (AP-HP)

Funding

This study was funded by academic groups only: 1- Gustave Roussy Cancer Campus 2- Fondation Gustave Roussy, 3- Direction Générale de l’Offre de Soins (DGOS; GOLD TRANSLA 12-174), 4- Institut National du Cancer (INCa; GOLD 2012-062 N° Cancéropôle: 2012-1-RT-14-IGR-01) 5- SIRIC SOCRATE (INCa DGOS INSERM 6043) 6- MMO program: ANR-10IBHU-0001.

Disclosure

N. Chaput: Grants from Cytune Pharma. E. Soularue: Medical Board for Novartis. F. Carbonnel: Advisory Board for Vifor, enterome, MSD (oncology), Ferring Lecture fees from Abbvie, Janssen, Mayoly Spindler, Hospira, Pfizer, Takeda Research Grants: Alpha Wassermann, Mayoly Spindler. C. Robert: Occasional consultant for Amgen, MSD, Merck, Novartis, Roche, Bristol-Myers Squibb. All other authors have declared no conflicts of interest.

Background

PD-L1 expression testing is mandatory prior to pembrolizumab prescription in non-small cell lung cancer. Pembrolizumab was made available in the UK through the Early Access to Medicines Scheme (EAMS) in May 2016 and was NICE-approved in December 2016. Our Molecular Pathology Diagnostic Service has been offering PD-L1 testing using Dako’s 22C3 IHC assay in parallel with EGFR and ALK testing. We present here data on the relationships between PD-L1 expression, and EGFR and ALK status in a series of 982 tumours.

Methods

PD-L1 expression testing was performed using the aforementioned assay on the 4800 Dako stainer. EGFR mutation testing was performed using the Therascreen kit on the RGQ platform and using COBAS kit (both screening for exon 19 deletions, L858R, G719X, L861Q, S768I, exon 20 insertions and T790M); ALK translocation was assessed using the D5F3 Ventana antibody on XT platform. PD-L1 was considered positive when more than 1% of tumour cells showed membranous staining.

Results

Of the 982 tumours, 492 were positive for PD-L1 (50.1%), 85 bore EGFR mutations (8.7%) and 14 bore ALK translocations (1.4%). There was no significant difference in PD-L1 expression rate with EGFR mutation status. However, whereas 39.3% tumours with a classical EGFR mutation were PD-L1 positive, 86.4% with a rare EGFR mutation (G719X, L861Q, S768I, exon 20 insertions) were PD-L1 positive (p = 0.0006). PD-L1 positivity rate was 52.3% and 85.7% in ALK non-rearranged and rearranged tumours, respectively (p = 0.01).

Conclusions

Our data show that the presence of classical or of rare EGFR mutations is associated with different degrees of PD-L1 expression, which may have future therapeutic implications. Our data also show that the presence of an ALK translocation is positively associated with PD-L1 expression.

Clinical trial identification

N/A

Legal entity responsible for the study

N/A

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

PD-L1 immunohistochemistry (IHC) correlates only moderately with response to nivolumab treatment. Characterizing PD-1, PD-L1 and T-cell markers in both tumor and stroma might improve the predictive value of tissue IHC as predictive biomarker in this setting.

Methods

From 08-2015 to 12-2016 patients with stage IV NSCLC treated with nivolumab were registered and prospectively followed. A histological tumor biopsy, obtained before start of nivolumab, was required. Tumor PD-L1 expression and immune cell (IC) PD-L1, PD-1, CD3, CD4 and CD8 expression in tumor and stroma was assessed using IHC on serial sections. IC infiltration was scored semi-quantitatively indicating no, very low, low, intermediate, or high infiltration. Presence of CD4+ macrophages in between tumor cells was used to aid assessment of tumor PD-L1 expression. Nivolumab was dosed 3 mg/kg Q2W and response assessment was done by CT every six weeks.

Results

Overall response rate of pts (n = 65) was 23% and quantifiable (≥1%) tumor PD-L1 expression was found in 25% of pts, versus <1% expression in 75% of pts. Univariate analyses showed a significant correlation between the levels of tumor PD-L1 expression (0%, 1-50%, >50%) and associated response rates of 17%, 22% and 57% respectively. Stromal IC expression of PD-L1, CD3, CD4 and CD8 also correlated with response (p < 0.05 for all markers). CD8+ tumor IC infiltration and stromal PD-1+ staining did not correlate with response. In the subgroup of n = 47 pts with negative (<1%) tumor PD-L1 expression, pts with either high CD3 or high PD-L1 stromal IC expression (n = 45) showed a remarkably high response rate of 59% (p = 0.009).

Conclusions

A clear positive correlation was found between PD-L1 expression and response. The distribution of PD-L1 expression was lower compared to historical data. Availability of CD4 IHC that identified PD-L1 positive macrophages is the explanation for lower percentage of tumor PD-L1 positive samples. Stromal PD-L1, CD3, CD4 and CD8 IC expression were all predictive for treatment response. In tumor PD-L1 negative pts, high stromal PD-L1 and/or CD3 IC expression selected pts with a remarkably high response rate.

Legal entity responsible for the study

A. J. de Langen

Funding

VU University Medical Center, Department of Pulmonology

Disclosure

All authors have declared no conflicts of interest.

Background

In the immunotherapy era, a better understanding of heterogeneity in MSI cancers is required. We evaluated gene expression profiles of MSI colorectal (CRC), gastric (GC) and endometrial cancer (EC) samples with our cell-of-origin signature (CRCassigner), which is able to classify samples in differentiated (goblet-like; CMS3), differentiating (transit-amplifying – TA; CMS2) and less differentiated/mesenchymal (stem-like; CMS4 and inflammatory; CMS1) subtypes to identify whether MSI transcriptional heterogeneity exists.

Methods

Normalised RNAseq/microarrays gene expression profiles and microsatellite status were downloaded from TCGA. CRCassigner classification of samples was performed using Pearson correlation. Samples with low classification confidence were classified as “mixed” subtype. Gene selection enrichment analysis (using published immune markers) and differential protein expression analysis (of PDL1 from Cancer Proteome Atlas data) was performed between inflammatory and goblet-like MSI samples.

Results

The majority of MSI-H in all the 3 cancer types expressed the inflammatory profile. While in MSI-H CRC only two subtypes were present (inflammatory - 91% and goblet-like - 9%), 5 subtypes in MSI-H GC (inflammatory - 45%, goblet-like - 24%, stem-like - 21%, TA - 6%, enterocyte - 3%) and 4 subtypes in EC (inflammatory - 36%, stem-like - 36%, goblet-like - 14%, TA - 14%) were present. Inflammatory MSI tumours from all the three cancer types were significantly (p < 0.05) enriched for genes associated with checkpoint inhibition (PDL1), MHC Class I, Type I interferon response and macrophages compared to goblet-like MSI tumours. On the other hand, goblet-like MSI cancer showed enrichment of B-cells.

Conclusions

MSI tumours are heterogeneous and can be stratified by virtue of differentiation states (or cell-of-origin) and different immune phenotypes. With further studies, this heterogeneity may help select MSI cancer patients for immune checkpoint combination therapies.

Legal entity responsible for the study

Anguraj Sadanandam

Funding

NIHR Biomedical Research Centre at the Royal Marsden Hospital and Institute of Cancer Research, London, UK; Cancer Research UK.

Disclosure

A. Sadanandam: Entitled to a share of royalties received by the licensor for a patent patent number PCT/IB2013/060416. Received research funding from Bristol-Myers Squibb for pancreatic cancer. All other authors have declared no conflicts of interest.

Background

Anti-tumor action of the host immune systems and cancer-immune interaction are associated with tumor prognosis. Programmed death ligand-1 (PD-L1) expression was found as an unfavorable prognostic factor in breast cancer in our previous study. Besides, PD-L1 and biomarkers of the host immunity which included Neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR) have been reported in predicting prognosis of biliary tract cancer. However, combination of NLR/PLR and PD-L1 in breast cancer has not been reported. The aim of this study is to evaluate the prognostic role of NLR/PLR and PD-L1 in breast cancer.

Methods

A total of 870 patients with breast cancer treated in Sun Yat-Sen University Cancer Center from 2000 to 2012 with known PD-L1 status were included. Clinicopathological data and pretreatment complete blood count were retrospectively collected. X-tile was used to generate the optimal cut-off value of NLR and PLR. Kaplan-Meier and univariate Cox proportional hazards model analyses were used to compare the survival of patients between different groups.

Results

High PLR group achieved worse result than low PLR group in OS and DFS (5-year OS rate: 82.6% vs 88.8%, p = 0.010; 5-year DFS rate: 78.7% vs 85.6%, p = 0.003). High PLR was associated with shorter DFS (adjusted HR = 1.540, 95%CI: 1.124-2.110, p = 0.007), while high PLR was not an independent factor for OS (adjusted HR = 1.001, 95%CI: 0.999-1.003, p = 0.488). NLR was not associated with patients’ survival outcome. And we found patients with PD-L1 expression and high PLR had the worst prognosis. The 5-year DFS rates were 68.4%, and 85.8% in high PLR+PD-L1 (+) group and low PLR+PD-L1 (-) group respectively (p = 0.002). The 5-year OS rates were 73.4% and 90.1%, respectively (p < 0.001).

Conclusions

High PLR are associated with poor DFS in breast cancer patients. PD-L1 expression combined with high PLR was associated with an aggressive clinical outcome. Further studies are needed to evaluate the predictive value of combination of PD-L1 and peripheral blood immune markers.

Legal entity responsible for the study

Shusen Wang

Funding

National Natural Science Foundation of China (81502302); Science and Technology Program of Guangdong Province (2014A020212384; 2016A020215079)

Disclosure

All authors have declared no conflicts of interest.

Background

Breast cancer subtype (BCS) and lymphovascular invasion (LVI) have both been independently demonstrated as prognostic factors. The objective of this investigation was to evaluate the prognostic power of LVI and lymph node status among BCSs.

Methods

From an institutional database, 2017 women with a histopathologically confirmed diagnosis of breast cancer treated between January 2006 and December 2014 were consecutively selected for participation in this study.

Results

Of the 2017 patients with breast cancer in the BCS groups, the highest OS and RFS rates were observed in luminal A subtype (93.6% vs. 95.1%, respectively) and the lowest were observed in TN subtype (85.3% vs. 83.0%, respectively). There were significant differences in OS according to the LVI status between the luminal A, luminal B and luminal HER2 subtypes. There were also a significant difference in the RFS rate of the luminal A, luminal B, luminal HER2 and HER2 subgroup. Therefore, we inferred that there were stronger links with LVI and BCS with regard to OS and RFS rates. Kaplan-Meier analysis showed that there were significant differences in the OS and RFS rates according to the LVI status among the BCS groups. There were significant differences in OS according to the LVI status in the distribution of the luminal A, luminal B, luminal HER2, and TN subtypes. There were also significant differences in the RFS rates among the luminal B, luminal HER2, and HER2 subtypes. On multivariate analysis, after controlling for age, tumor size was independently associated with LVI and lymph node status among all BCS groups. There were significant differences in OS according to the status of lymph node-negative and LVI-positive in the luminal HER2 subtype, as well as lymph node-positive and LVI-positive in the TN subtype. There were also significant differences in RFS according to the status of lymph node-negative and LVI-positive in the luminal A subgroup.

Conclusions

LVI and lymph node status were important prognostic factor for OS and RFS among all BCSs. In lymph node-negative breast cancer, luminal HER2 had greater predictive value for OS, whereas luminal A displayed greater predictability for RFS. In lymph node-positive breast cancer, the TN subtype had greater predictive value for OS.

Clinical trial identification

nil

Legal entity responsible for the study

Tri-Services General Hospital, National Defense Medical Center

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Immune system plays an important role in tumor surveillance and escape. Recently tumor infiltrating lymphocytes (TILs) have been proposed as a predictive biomarker for clinical outcome and pathological response (pR) after neoadjuvant (neoadj) chemotherapy (CT) in breast cancer. PD-L1 is expressed in about 20% of TNBC, suggesting the possibility of being a therapeutic target for this subtype of cancers. Here we studied the association between PD-L1 expression and pR in TNBC.

Methods

We enrolled 54 pts who had received neoadj CT (EC for 4 cycles followed by Paclitaxel q21 for 4 cycles) between Jan 2008 and Dec 2016 at Policlinico Umberto I and San Giovanni Hospital of Rome. We performed IHC for CD20, CD3, CD4, CD8, CD68, N-CAM and PD-L1 (Ventana SP142 clone) in basal paraffin-embedded biopsies. PD-L1 expression on tumor cells was evaluated both qualitatively (membrane staining intensity 0 to 3+) and quantitatively (% of positive cells.). The percentage of TILs positive for PD-L1 was also recorded. Statistical analysis was performed with T di Student test and χ² test.

Results

We enrolled 54 pts (median age: 50 y; range 28-75) affected by TNBC: 51 ductal (94.4%), 2 metaplastic (3.7%), 1 lobular (1.9%). The clinical stage before neoadj CT was as follow: 12.9% cT1 (7 pts), 72.2% cT2 (39 pts), 3.7% cT3 (2 pts), 1.85% cT4 (1 pt) and 5.5% cTx (3 pts). 23 pts were cN + (42.5%). After neoadj CT 30 pts underwent mastectomy (55%) and 24 conservative surgery (45%). 19 pts (35%) showed pCR. No significant associations were found between pR and cT, cN, age, histotype and KI-67. In 64.8% of basal biopsies (35 pts) PD-L1 was not detected on tumor cells and in 18.5% (10 pts) it was absent in the immune infiltrate. PD-L1 expression was detected in > 25% of tumor cells in 4 pts, all of which showed pCR (p = 0.024). No associations between intensity of membrane staining and pR were detected (p = 0.7). The immune infiltrate was characterized mostly by the presence of CD3+ CD8+. No statistically significant associations between and PD-L1 expression on immune infiltrate were detected.

Conclusions

Basal PD-L1 expression on cancer cells was associated with a better pR in TNBC undergoing neoadj CT. The introduction of anti PD-1/PD-L1 therapy in this setting of pts could lead to interesting results.

Legal entity responsible for the study

Sapienza University of Rome

Funding

None

Disclosure

P. Marchetti: Advisory board and meeting with Pfizer, Roche, Novartis, MSD, Bristol-Myers Squibb, Ipsen, AstraZeneca, Boehringer Ingelheim. All other authors have declared no conflicts of interest.

Background

Assessment of tumor infiltrating lymphocytes (TIL) by pathologists using Hematoxylin-Eosin (H&E), has been described as a prognostic factor in resected NSCLC. We aimed to correlate TIL to the benefit from nivolumab in patients (pts) with treated advanced NSCLC.

Methods

Patients with advanced NSCLC treated with nivolumab, with biopsy available for evaluation, were included between November 2012 and February 2017 in two cancer centers. Patients characteristics and outcome were collected. The percentage of tumor infiltrating lymphocytes in the stroma was evaluated using H&E staining from archival pretreatment tumor tissue samples. Primary endpoint was to correlate TIL density with progression free survival (PFS).

Results

Out of ninety-eight patients included. 60 (61%) pts were male, with median age of 61 years and 85 (89%) were smokers. Sixty three (73%) pts were PS 0-1. Sixty tumors (61%) were adenocarcinoma, 29 (30%) squamous and 9 (10%) other histologies. Among 83 tumors with known molecular profile, 22 (27%) were KRAS mutated 7 (8%) EGFR mutated, 1(1%) ALK positive. The median treatment line was 3 (2-4). The median follow up was 8 months (m)(95%CI[6-19]). The median PFS was 2 m (95%CI[1-5]). The ORR was for 16%. The median TIL density was 5% (2-15). TIL density ≥5% correlated with PFS in univariate and multivariate analysis (HR: 0.48 [0.28-0.82] p = 0.007 and HR:0.31 [0.14-0.68] p = 0.004 respectively). TIL density ≥5% was also associated with better ORR (OR = 3.5, 95%CI [1.06-11.7], p = 0.04).

Conclusions

Pathological assessment of TIL allows an easy evaluation of immune infiltration in NSCLC and independently correlates PFS in NSCLC pts treated with nivolumab. Results from validation cohorts and combination with other morphological and immunohistochemical parameters will be reported.

Legal entity responsible for the study

Ithar Gataa

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Cancer-associated inflammation is a key molecular feature of PC and may affect the clinical course. The aim of this study was to evaluate the prognostic relevance of SIRI based on peripheral neutrophil, monocyte, and lymphocyte counts in metastatic PC and its association with the metastatic site.

Methods

Retrospective analysis of the medical records of patients with pathologically confirmed metastatic PC between January 2011 and December 2016. Patients were classified as having liver metastases (LM) or extrahepatic metastases alone (EM). Associations with overall survival (OS) were analyzed using Cox proportional models.

Results

A total of 37 patients were included (47 men; median age 63). Median TTP was 4 months and median OS was 6 months. 29 patients (78%) had LM and 8 (22%) EM. 33 patients (89%) received CT: 13 (40%) Gemcitabine (GEM) plus Nab-Paclitaxel, 9 (27%) GEM in monotherapy, 7 (21%) GEM plus Erlotinib and 4 (12%) an Oxaliplatine doublet. Mean Ca 19 9 levels in patients with LM were 199349 and with EM 9107. Univariate analysis identified SIRI scores ≥1,9 as significant risk factor for OS. Age, sex and high CA 19,9 levels had no prognostic significance for OS in all groups. Patients with LM showed a higher SIRI than those with EM (p = 0,03). Patients with SIRI scores < 1,9 (55%) compared to those who had SIRI scores ≥1,9 (45%) had a longer OS (p = 0,01). LM were significantly associated with shorter OS (hazard ratio [HR] 2.79; 95% confidence interval [CI] 1.36-5.34; p = 0,002) but not those with EM (HR 1.83; CI 0.71-4.72; p = 0,2). An SIRI ≥1,9 resulted in a shorter OS compared to an SIRI <1,9 (HR 2.13; CI 1.10–4.10; p = 0.024).

Conclusions

SIRI is associated with survival in patients with metastatic PC. In patients with LM, unfavourable SIRI may be associated with higher tumor burden. In our experience, a baseline SIRI ≥1.9 duplicates the risk of mortality and this finding may allow better risk stratification.

Legal entity responsible for the study

Medical Oncology Department, Hospital Universitario de La Princesa

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Endocrine responsiveness in Estrogen Receptor positive (ER+) MBC is based on the level of ER expression on the primary tumor and/or metastatic lesion. In this study, nested in the ET-FES JTC 2011 TRANSCAN project, we used molecular imaging with ¹⁸F-FES to explore intra-patient heterogeneity in ER expression at different metastatic sites and to identify patients who are not likely to benefit from ET.

Methods

ER+ patients at first relapse underwent at baseline a ¹⁸F-FES PET/CT plus a ¹⁸F-fluoro-2-deoxy-D-glucose (FDG) PET/CT. Patients were classified into 4 ¹⁸F-FES/FDG subgroups based on the proportion of FDG avid metastatic tumor load with high ¹⁸F-FES uptake (Gebhart Ann Oncol 2016). Subgroup A was considered positive (100% of concordance); subgroups B and C were considered partially positive or partially negative, with different degrees of ¹⁸F-FES uptake; subgroup D was considered negative (100% of discordance). Patients with global SUV ≥2 received first line ET while those with SUV <2 were randomized to ET or to chemotherapy. HR for progression was calculated comparing patients with concordant ¹⁸F-FES/FDG lesions (group A) with all the other patients.

Results

So far, 80 patients have been enrolled in the ET-FES trial and 79 are included in the present analysis. At baseline evaluation, 53 patien4ts (67.1%) were classified as ¹⁸F-FDG/¹⁸F-FES positive (A); 16 patients (20.3%) showed some degree of intra-patient heterogeneity (11 group B and 5 group C); 10 patients (12.6%) were classified as D with all lesions being ¹⁸F-FES negative. In the 64 patients with a response evaluation available at time of analysis, 26 have shown progression. ET was administered in 40 patients in group A and in 24 patients in groups B + C + D. The use of ET alone in partially positive (B) or partially negative (C) or negative (D) patients was associated with a 79% absolute increase in the risk of progression (HR 1.79, p 0.2) compared to patients in group A.

Conclusions

Pretreatment ER biomarker imaging at different metastatic sites with ¹⁸F-FES PET/CT indicate the presence of a significant intra-patient heterogeneity in MBC and represents a promising tool to select patients who are unlikely to benefit from ET alone.

Clinical trial identification

EUDRACT 2013-000-287-29

Legal entity responsible for the study

E.O. Ospedali Galliera, Genoa, Italy

Funding

TRANSCAN JTC 2011

Disclosure

C. Brambati: Employer Advanced Accelerator Applications. All other authors have declared no conflicts of interest.

Background

Radiomics (RAD) uses advanced image processing techniques to extract a large set of quantitative texture and geometric features from tumor regions of interest, and subject these to a supervised machine learning protocol to train a classifier, which we exploit to develop a predictive signature of response to ICIs. We previously developed a lesion-based predictive RAD classifier of response for recurrent/metastatic squamous cell carcinoma of the head and neck (RM SCCHN) pts to ICIs based on RAD features extracted from their CT images (Prawira, ESMO 2016).

Methods

INSPIRE (NCT02644369) is an investigator-initiated phase II study evaluating biomarkers for pembrolizumab (anti-PD1 monoclonal antibody) in multiple cohorts of pts with advanced solid tumors. The primary endpoint of this project is to validate the previously developed RAD classifier from RM SCCHN pts, with pts from INSPIRE. Texture feature algorithm generation and accuracy determination were as previously described. Cross validation accuracy values were generated for combinations of 3 parameters: fraction, cost, and gamma, yielding a 3 dimensional (3D) accuracy space.

Results

Eighty lesions from 23 pts were available for analysis: median age 59, 22% males. Best response: 12 progressive disease, 3 partial response, 8 stable disease (median duration 18 weeks). Primary site: SCCHN/2, triple negative breast cancer/4, high-grade serous ovarian cancer/11, malignant melanoma/2, other advanced cancers/4. Twentyseven lesions were excluded as RECIST 1.1 responses were not yet available. Fiftythree target lesions were contoured. Per lesion RECIST 1.1 radiological outcome: 17 R, 36 NR. Cross validation in the 3D space yielded a set of ROC curves with an accuracy of 71.4% (AUC 0.41, p = 0.7) with 11.2% sensitivity and 99.9% specificity, where specificity corresponds to the proportion of NR tumors classified correctly, and sensitivity to the proportion of R tumors classified correctly.

Conclusions

Heterogeneous histologies and low pt numbers may account for the negative result in this study, suggesting that RAD may be histology-specific. Further validation in a large independent cohort of RM SCCHN pts treated with pembrolizumab is planned.

Clinical trial identification

INSPIRE (NCT02644369)

Legal entity responsible for the study

Princess Margaret Cancer Centre, Drug Development Program

Funding

Merck

Disclosure

All authors have declared no conflicts of interest.

Background

REG demonstrated efficacy in pre-treated mCRC pts. Lack of predictive biomarkers, potential toxicities and cost/effectiveness concerns highlight the unmet need for better patient selection.

Methods

RAS mutant mCRC pts with biopsiable metastatic deposits were enrolled in this phase II trial. Tissue biopsies (6-12 cores) were obtained at baseline (BL), after 2 months if stable disease (SD) and at disease progression (PD). Dynamic contrast enhanced (DCE) MRI was acquired pre and at day 15 post-treatment. Median values of volume transfer constant (K^trans), enhancing fraction (EF) and their product, KEF [K^trans*EF/100] were generated. Circulating tumour (ct) DNA was collected monthly until PD and tested for clonal RAS mutations by digital droplet PCR. PDOs derived from responders and non-responders pts were implanted orthotopically in the liver of mice and treated with REG for 5 days. Changes in tumour and fractional blood volume (fBV) were monitored by oxygen-enhanced MRI.

Results

mCRC pts (n = 27) with paired MRI scans were analysed; a single target lesion per pt was chosen (25 liver and 2 pelvic metastases). Median KEF decrease was 58.2%. In the 23 analysable pts (4 received ≤1 cycle of treatment due to toxicities),>70% drop in KEF(8/27) was associated with higher disease control rate (DCR) measured by RECIST 1.1 at 2 months (m) (p = 0.05), progression free survival (PFS) [HR = 0.24 (0.07-0.86), p = 0.03], 6-m PFS (43.8% VS 0%) and overall survival (OS) [HR 0.08 (0.01-0.63), p = 0.02]. In all pts with DCR, PFS was found to be 5.6 vs. 4.2 m [HR 0.30 (95% CI 0.06-1.49), p = 0.140) and OS was 15.2 vs. 5.8 m [HR 0.11 (95% CI 0.01-1.06), p = 0.057]. KEF drop correlated with CD-31 reduction in sequential tissue biopsies (p = 0.04). RAS mutant clones decay in ctDNA after 8 weeks of treatment was associated with better PFS [HR 0.21 (0.06 - 0.71), p = 0.01] and OS [HR 0.28 (0.07 - 1.04), p = 0.06]. PDOs xeno-transplants treated with REG compared to controls had significant lower tumour fBV (4.5 VS 10.6, p = 0.03) and lower microvascular density measured by CD31 staining (4.3 VS 8.9, p = 0.02).

Conclusions

Combining DCE-MRI and ctDNA predicts depth and duration of anti-angiogenic response to REG with potential health economic implications.

Clinical trial identification

clinical trials.gov number NCT03010722

Legal entity responsible for the study

The Royal Marsden NHS Foundation Trust

Funding

Bayer Oncology Group

Disclosure

K. Khan: Advisory board for Bayer Oncology Group. D. Cunningham: Research funding from: Roche, Amgen, Celgene, Sanofi, Merck Serono, Novartis, AstraZeneca, Bayer, Merrimack and MedImmune. I. Chau: Advisory roles with Merck Serono, Roche, Sanofi Oncology, Bristol-Myers Squibb, Eli-Lilly, Novartis, Gilead Science. Research funding from Merck-Serono, Novartis, Roche and Sanofi Oncology, and honoraria from Roche, Sanofi-Oncology, Eli-Lilly, Taiho. C. Peckitt: Advisory roles with Sanofi. All other authors have declared no conflicts of interest.

Background

Changes of HER2 status has been reported in circulating tumor cells (CTC) isolated from preclinical models and metastatic breast cancer (MBC) patients. The prospective multicentric phase II “CirCe T-DM1” trial was set up to assess whether HER2-amplified CTC are detectable in HER2-negative MBC and whether these cancers would respond to anti-HER2 therapy.

Methods

HER2-amplified CTC were screened in HER2-negative (HER2-/ER- and HER2-/ER+) MBC patients starting a 3^rd line or 4^th line of systemic therapy. CTC were detected by CellSearch® (Janssen Diagnostics) and FISH was performed on isolated CTCs. HER2-amplification was defined by a HER2/CEP17 ratio ≥2.2. Patients with ≥1 HER2-amplified CTC, measurable disease and adequate organ function were eligible. After stratification according to amplified CTC count (< vs ≥ 3), patients received single agent T-DM1. The primary endpoint of the study was the response rate by RECIST criteria.

Results

From 11/2013 to 08/2016, 155 MBC patients were screened. 11 (9.2%) and 3 patients (2.5%) had 1-2 and ≥3 HER2-amplified CTCs respectively (minimal HER2/CEP17 ratio: 2.5). In the 14 patients with HER2-amplified CTC, the fraction of HER2-amplified CTCs among all detected CTCs was low (median 1.6%, range [0.3%-35.3%]), and presence of HER2-amplified CTCs was not associated with any patients’ characteristics. 11 patients were treated with single agent T-DM1. Partial response was confirmed in one patient with 1 HER2-amplified CTC (among 9 CTC detected); median PFS was 4.9 months (range: 1.8-10.1).

Conclusions

This study shows that CTCs with a true HER2-amplification can be detected in advanced HER2-negative MBC, mostly as a minor CTCs subset. Although one confirmed response was observed in our study, the overall low response rate to specific anti-HER2 therapy does not support the clinical utility of such strategy in that setting.

Clinical trial identification

NCT01975142

Legal entity responsible for the study

Institut Curie

Funding

Roche

Disclosure

All authors have declared no conflicts of interest.

Background

Detection of EGFR mutation in circulating tumor DNA (ctDNA), a powerful blood-based biomarker with multiple clinical applications for lung cancer patient, is technically challenging because it requires sensitivity and specificity. In order to evaluate the performance of the french laboratories performing this assay, we set up a national EQA scheme for circulating tumor DNA.

Methods

Artificial samples were prepared by spiking DNA extracted from control FFPE sections containing specific mutations (Horizon Diagnostics, from 25 to 350 copies/mL of plasma, as determined by digital PCR) in normal plasma (Clinisciences). Aliquots (2 ml) of 10 different samples were sent in dry ice to 43 laboratories. DNA extraction and EGFR testing were performed according to local practice. Laboratories were requested to search for exon 19 deletions, p.L858R, p.G719X and p.T790M mutations. Data were collected on a web questionnaire within one month and compared to the expected results.

Results

We collected 30 complete sets of data. DNA was extracted using the QIAmp® circulating DNA kit (Qiagen; n = 13), the Cobas® cfDNA sample preparation kit (Roche; n = 9) or the Maxwell® system (Promega; n = 7). The most widely used methods were the Cobas® EGFR Mutation Test v2 (Roche; n = 10), digital PCR (n = 8) and NGS (n = 6). Among the 10 labs using the Cobas®, 9 obtained the 10 expected genotypes. This number dropped to 3 (out of 6 labs) for NGS, and 2 (out of 8 labs) for dPCR, because of false negative results, false positive results, and not contributive tests.

Conclusions

Digital PCR and NGS are known to be highly sensitive techniques. However, the results of this EQA suggest that in routine clinical practice, ctDNA analysis requires technical skill or/and validated bioinformatic pipeline to reach high sensitivy and specificity. Under the specific conditions of this scheme, the Cobas® method appeared to be the most robust approach. This external control will allow the laboratories to evaluate their practice and improve their process. A similar approach targeting other genes (BRAF, KRAS and NRAS) is being developed, and additional EQA schemes will be set up at the nationwide level. Supported by a grant from AstraZeneca.

Legal entity responsible for the study

Gen&Tiss

Funding

AstraZeneca

Disclosure

All authors have declared no conflicts of interest.

Background

In a Phase I/II study of Trabedersen (OT-101), a phosphorothioate antisense specific for human TGF- β2 mRNA, patients with advanced pancreatic cancer (PAC) treated in the 2^nd-line and beyond exhibited improved overall survival (OS) when OT-101 was followed with chemotherapy. Here, we examined the association between plasma levels of cyto-/chemokines and OS outcomes to identify potential biomarkers for improved OS.

Methods

37 PAC patients were treated with continuous IV infusion in escalating doses of 2 treatment schedules (7-days-on, 7-days-off and 4-days-on, 10-days-off). Plasma levels of 31 cyto-/chemokines were measured at 8 separate time points over 3 cycles of OT-101 treatment (140 mg/m²/day, 4-days-on, 10-days-off). Standardized maximum levels of individual cyto-/chemokines on Day 2 were subtracted from levels on Day 5 of each cycle of treatment and correlated with log10 transformed OS values. Feedback interactions with PK parameters were also investigated utilizing an ANCOVA model.

Results

A median OS of 14.5 months and 2.6 months was observed for 2^nd-line patients treated with and without subsequent chemotherapies, respectively (p = 0.0009). Increasing difference of IL-8 levels at Day 2 vs Day 5 was positively correlated with OS (R² = 0.58, P = 0.0066). Stratifying for patients with and without chemo, R² increased to 0.99 and 0.77 respectively. Similar results were observed for IL-15, with R² = 0.93 in patients with chemo and R² = 0.50 in those without. ANCOVA models for two PK parameters exhibited significant model fits (F_3,7 = 7.89, P = 0.012 for Simulated Vz Mean; F_3,7 = 8.18, P = 0.011 for Simulated Cl Mean) and the interaction effects resulted in lower p-values for the correlation of OS vs IL-8 levels.

Conclusions

Increasing peak levels of IL-8 and IL-15 response on Day 2 of OT-101 treatment correlated with OS in PAC patients. This correlation with OS was evident regardless of subsequent chemotherapy or not indicating spikes in IL-8 and IL-15 as potential biomarkers for OT-101.

Legal entity responsible for the study

Oncotelic Inc.

Funding

Oncotelic Inc.

Disclosure

L. Hwang, W. Wang, S. Qazi, K. Ng, O. D’cruz, V. Trieu: Employee

Background

The FIRE-3 trial compared 1^st-line therapy of FOLFIRI plus either cetuximab or bevacizumab in 592 KRAS exon 2 wildtype (wt) mCRC patients. The subgroup of extended RAS wt patients consisted of 400 patients. The AADx molecular assay has previously been shown to identify a poor prognosis angiogeneic subgroup across multiple cancer types including colorectal cancer. Both bevacizumab (through inhibition of VEGFR-activation) and cetuximab (through inhibition of EGFR-signaling) would be expected to have anti-angiogenic effects in colorectal cancer. The predictive role of AADx in FOLFIRI plus bevacizumab or cetuximab treated in colorectal cancer patients remains unclear.

Methods

Transcriptional profiling of 501 formalin fixed paraffin embedded pre-treatment samples from the ITT population was performed using the Almac Diagnostics Xcel^TM array. Patients were classified by the AADx assay as ANGIO ON or OFF based on a predefined score. ORRs were compared using Fischeŕs exact test. Progression free survival (PFS) and Overall survival (OS) times were compared using Kaplan-Meier estimation and log-rank tests. Hazard ratios (HR) were estimated according to the Cox proportional hazard method.

Results

The AADX assay was successfully applied to 438 out of 501 specimens available from the study population (n = 752). Of those, 315 had a RAS wt tumor and 123 a RAS mutant. The correlation between RAS status and AADx score with respect to ORR; PFS, and OS were complex (Table). The addition of cetuximab to FOLFIRI was significantly superior to the addition bevacizumab in “ANGIO ON” tumors most likely reflecting the strong link between EGFR-signaling and angiogenesis in colorectal cancer.Table:

120P

Conclusions

Here, we present data demonstrating that it possible to define subgroups in the group of wt mCRC patients within the FIRE-3 trial that responded differently to the addition of cetuximab or bevacizumab.

Clinical trial identification

NCT00433927

Legal entity responsible for the study

Klinikum der Universität München

Funding

ALMAC Inc.

Disclosure

S. Stintzing: Honoraria for talks and advisory board from: Amgen, Bayer, Lilly, Merck, Sanofi, Roche. B. Price, A. McCavigan, S. Walker, P. Harkin, R. Kennedy, L. Knight: ALMAC Diagnostics. V. Heinemann: Honoraria for talks and advisory boards by: Amgen, Servier, Sirtex, Merck, Roche, Bayer, Lilly, Sanofi. All other authors have declared no conflicts of interest.

Background

Angiogenesis inhibitors are widely used for treatment of metastastic colorectal cancer. However, no predictive biomarkers are currently available for patient selection. Besides the tumor-associated endothelial cells, VEGF-signaling inhibitors target CRC cells that express VEGF as well as functional VEGFR1 receptors thereby mediating both paracrine and autocrine VEGF-signaling. The aims of this work is i) to establish the direct influence of aflibercept (Zaltrap®) that inhibits all three VEGFR1 ligands (VEGF-A, VEGF-B and PlGF) on CRC cells, ii) to identify tumor phenotypes associated with resistance to aflibercept in vitro and, iii) to extend these findings to human xenograft models.

Methods

A panel of 12 well-characterized CRC cell lines was used to establish the influence of VEGFR1 stimulation (VEGF-A, VEGF-B, PlGF) and inhibition (aflibercept) on VEGF-mediated tumor cell migration. Expression of VEGF ligands and receptors was determined by qRT-PCR and ELISA assays. The in vivo influence of aflibercept was determined in human xenograft models and complemented by IHC and Western blot analysis.

Results

Aflibercept inhibited the migration of most CRC cells under both normoxia and hypoxia including the highly sensitive HCT-116 cells. In contrast, LS174T cells did not respond to either aflibercept or to purified VEGF ligands. These cells expressed high levels of VEGF ligands and HIF2alpha, even under normoxia. Accordingly, aflibercept showed pronounced in vivo activity toward HCT-116 xenografts with 75% tumor growth inhibition but only 40% tumor growth inhibition toward LS174T tumors, compared to the corresponding vehicle controls. A similar trend was observed for bevacizumab with 41% tumor growth inhibition for HCT-116 and 32% inhibition for LS174T xenografts. IHC analysis of LS174T xenografts confirmed the strong expression of HIF2alpha and VEGF ligands as well as a modest inhibitory effect on the tumor-associated endothelia cells.

Conclusions

We here report that aflibercept has direct antimigratory effects on most CRC cells. Strong expression of HIF-2alpha and VEGF ligands was accompanied by aflibercept resistance in vitro as in vivo.

Legal entity responsible for the study

INSERM U938 and Université Pierre et Marie Curie (UPMC), Sorbonne Universités, Paris, France

Funding

Sanofi-Genzyme

Disclosure

A.K. Larsen: This work was supported in part by Sanofi Genzyme. E. Dochy: Employed by Sanofi-Genzyme. A. de Gramont: This research is in part supported by Sanofi. All other authors have declared no conflicts of interest.

Background

The oral Akt inhibitor IPAT is being evaluated in cancers with a high prevalence of PI3K/Akt pathway activation. In the placebo-controlled randomised phase II LOTUS trial (NCT02162719), adding IPAT to P as first-line therapy for metastatic TNBC improved PFS in unselected patients (stratified hazard ratio [HR] 0.60 [90% CI 0.40–0.91]), with a more pronounced effect in patients with PIK3CA/AKT1/PTEN-altered tumours [Dent, ASCO 2017]. cfDNA sequencing was performed to assess the utility of blood samples for detecting tumour mutations in TNBC.

Methods

Pre-treatment plasma and tumour samples were evaluated for genetic alterations using Foundation Medicine’s FoundationACT® and FoundationOne® next-generation sequencing assays, respectively. Samples from 88 patients were evaluable by cfDNA, 72 of whom also had evaluable tumour samples (52 primary tumour). To test the prognostic effect of cfDNA mutational burden, patients were classified as having high or low variant allele fraction (VAF) in cfDNA using the median as a cutoff.

Results

In 81 patients (92%), at least one mutation was detectable by cfDNA sequencing. Concordance with tissue sequencing was 75%. High VAF was associated with shorter PFS than low VAF in both the IPAT + P arm (HR 2.39 [90% CI 1.19–4.70]) and the placebo + P arm (HR 2.68 [90% CI 1.46–5.11]). High VAF was also associated with the presence of > 1 metastatic site but not tumour volume per RECIST v1.1. In 22 patients (25%), an activating PIK3CA (n = 16) or AKT1 (n = 6) mutation was detected in cfDNA; concordance with tissue sequencing was 100%. PFS improvement with the addition of IPAT to P was more pronounced in patients with detectable PIK3CA/AKT1 mutations (HR 0.15 [90% CI 0.03–0.50]) than in those without a detectable mutation (HR 0.82 [90% CI 0.50–1.31]).

Conclusions

These results highlight the potential role of cfDNA in evaluating patient prognosis as well as identifying genetic markers associated with improved treatment outcomes. Furthermore, they support the occurrence of PIK3CA and AKT1 mutations as early genetic events present in primary tissue samples that are maintained during metastasis.

Clinical trial identification

NCT02162719

Legal entity responsible for the study

F Hoffmann-La Roche Ltd.

Funding

F Hoffmann-La Roche Ltd

Disclosure

M. Wongchenko: Employee of Genentech, Inc. and holds shares in Roche and Ariad Pharmaceuticals. R. Dent: Honoraria for consultancy/advisory boards/speaker engagements from Pfizer, Roche, Eisai, Merck, and AstraZeneca. S-B. Kim: Research funding from Novartis, Sanofi-Aventis, Kyowa-Kirin Inc, and Dongkook Pharma Co., Ltd. C. Saura: Honoraria for consulting/advisory roles from Puma Biotechnology, Pfizer, and Roche and research funding (to her institution) from Genentech and AstraZeneca. M. Oliveira: Honoraria for consulting/advisory roles from Puma Biotechnology and Genentech/Roche and research funding (to the institution) from Genentech and AstraZeneca. J. Baselga: Compensation for a leadership role from Infinity Pharmaceuticals, stock or ownership interest in PMV Pharma, Juno Therapeutics, Infinity Pharmaceuticals, and GRAIL, and honoraria for consulting/advisory role for Eli Lilly, Novartis, and GRAIL. A.V. Kapp, W.Y. Chan, S.M. Singel, D.J. Maslyar, S. Gendreau: Employee of Genentech, Inc. and holds stock in Roche.

Background

Acquired resistance of mCRC patients (pts) to anti-EGFR monoclonal antibodies (mAbs) is frequently due to mutations in RAS/BRAF and EGFR extracellular domain (ECD), and amplifications in MET/ERBB2. Studies suggest that some anti-EGFR mAb refractory pts retain tumor EGFR-dependency potentially targetable by more efficacious agents such as Sym004 – a mixture of two non-overlapping mAb targeting EGFR for degradation. A Phase 2b trial of Sym004 in 254 mCRC pts tissue RAS wt that relapsed on anti-EGFR blockade was recently completed.

Methods

Baseline circulating tumor (ct)DNA profiles (Guardant360, Guardant Health, N = 193 pts) were obtained from blood samples collected from pts in the Sym004 Phase2b trial. Serial blood samples during treatment were analyzed for EGFR-ECD mutation dynamics.

Results

High mutant allele frequency (MAF >20%) of KRAS/NRAS was found in 5% of pts and BRAF V600E and EGFR-ECD mutations in 6.7% and 25% of pts, respectively. At least 1 of these mechanisms of resistance was identified in 32% of the pts. Mutations in KRAS, NRAS, and BRAF V600E, and amplifications of ERBB2 and MET were more likely to co-occur with EGFR-ECD mutations, demonstrating genomic complexity developed as a result of EGFR blockade. Comparative analysis of MAFs of driver genes indicated that EGFR-ECD mutations are subclonal events. The absence of any EGFR-ECD/BRAF/high RAS MAF mutation at baseline was associated with a significantly improved OS in the Sym004 treated pts as compared to control arm with investigator’s choice chemotherapy. Sym004 has previously been demonstrated to retain in vitro efficacy in EGFR-ECD mutated cancer cells, and ctDNA monitoring demonstrated decrease in EGFR-ECD in Sym004 treated pts. This suggests that EGFR-ECD mutated cells are targeted by Sym004, but this activity does not translate into clinically meaningful OS benefit, likely due to other co-occuring resistance mechanisms.

Conclusions

Comprehensive liquid biomarker profiling of 193 mCRC pts captured high intrapatient heterogeneity following anti-EGFR therapy, and identified a highly Sym004 sensitive mCRC pts subset with a RAS/RAF/EGFR-ECD wild type profile. Our data indicates heterogeneous responses of different subclones to targeted agents in mCRC.

Legal entity responsible for the study

Medical Oncology Department, Hospital del Mar, Barcelona, Spain

Funding

Funded by Symphogen

Disclosure

C. Montagut Viladot: Advisory Board for Amgen, Bayer, Merck Serono, Sanofi, Symphogen. T. Tuxen Poulsen, M. Kragh, K. Koefoed, C. Ding, J. Clausell-Tormos, T. Lindsted, M.W. Pedersen, P. Nadler, I.D. Horak: Full time employee at Symphogen. S. Kopetz: Advisory boards for Amgen, Merrimack, Bayer, Sanofi, Array BioPharma, Genentech, MolecularMatch, Symphogen, Guardant Health, EMD Serono, Merck. J. Tabernero: Advisory boards for Amgen, Bayer, Boehringer Ingelheim, Celgene, Chugai, Genentech, Lilly, MSD, Merck Serono, Novartis, Pfizer, Roche, Sanofi, Symphogen, Taiho, and Takeda. All other authors have declared no conflicts of interest.

Background

Non-small cell lung cancer (NSCLC) is the primary cause of cancer-related death, with a 5-year survival rate < 16% mainly because of disseminated disease, even in fully resected early-stage disease. Biomarkers identifying patients at a higher risk of relapse would be useful and circulating microRNAs (miRNAs) are a promising option in this setting.

Methods

We analyzed a case series of 182 patients with resected early stage (IA-IIIA) NSCLC (99 adenocarcinoma [ADC] and 83 squamous cell carcinoma [SCC]). Peripheral blood samples were collected from each patient before surgical resection and serum was obtained after centrifugation and stored at -80 °C until miRNA extraction. A panel of 84 circulating miRNAs was analyzed by Real Time PCR. Data were normalized using an external spike-in (cel-miR-39) and the mean of the two most stable endogenous housekeeping genes chosen separately for ADC and SCC samples. miRNA expression was analyzed in relation to disease-free survival (DFS) by the Cox regression model. Results are reported as hazard ratios (HRs) and 95% confidence intervals (CIs).

Results

In ADC patients, stage was significantly associated with DFS (HR stage II-IIIA vs stage I = 4.94, 95% CI [2.71 - 9.02]). Multiple statistical methods were used to evaluate miRNA expression data. In univariate analysis, two miRNAs (miR-222-3p and miR-22-3p) were significantly associated with DFS (p = 0.033 and p = 0.041, respectively). However, this significance was not maintained after adjusting for multiple testing. In SCC patients, disease stage was significantly related to DFS (HR stage II-IIIA vs stage I = 3.31, 95% CI [1.74 - 6.33]). Five miRNAs (let-7a-5p, miR-126-3p, miR-26a-5p, miR-130b-3p, miR-21-5p) were significantly associated with DFS even after adjusting for multiple testing (False Discovery Rate q-value <0.001).

Conclusions

Pre-surgery circulating levels of let-7a-5p, miR-126-3p, miR-26a-5p, miR-130b-3p and miR-21-5p would appear to be significantly correlated with prognosis in resected early-stage SCC.

Clinical trial identification

Not applicable

Legal entity responsible for the study

IRST-IRCCS

Funding

None

Disclosure

All authors have declared no conflicts of interest.

Background

Plasma cell-free DNA (cfDNA) sampling from patients with advanced cancers offers a minimally invasive source of tumor DNA for molecular testing, including methylation profiling. Tumor sequencing at disease progression offers insights into cancer biology, but is often not done because of logistical and other challenges associated with tumor biopsies.

Methods

Plasma samples were collected from patients with advanced breast, colorectal, non-small cell lung cancer (NSCLC) and melanoma at the time of disease progression and/or on therapy. Up to 30 ng (median 18ng) of plasma cfDNA was tested with the pan-cancer methylation panel to target a total of 10,888 CpG sites using an Illumina HiSeq2500 sequencer to calculate methylation scores. Methylation scores were correlated with cancer types and clinical outcomes.

Results

Of 69 plasma cfDNA samples collected from patients (colorectal cancer, 28; NSCLC, 18, breast cancer, 12; melanoma, 11) at disease progression, methylation scores were consistent with the presence of cancer in 84% (58/69); while in 79% (46/58) of plasma cfDNA samples, methylation scores accurately classified the underlying cancer type. High methylation scores in plasma cfDNA samples collected at disease progression compared to low methylation scores were associated with shorter survival irrespective of the tumor type (3.9 vs 10.4 months, P < 0.001, confirmed on multivariable analysis). In addition, high methylation scores in plasma cfDNA samples collected at progression vs low methylation scores were associated with shorter time to treatment failure (TTF) on systemic therapy (1.6 vs. 2.8 months, P = 0.007). There was a positive correlation between RECIST response (%) to systemic therapy and methylation scores (r 0.32, P = 0.03). Methylations score before therapy (median 87.28) were higher than methylation scores on therapy (median 9.49, P = 0.001).

Conclusions

Methylation scores from plasma cfDNA collected at disease progression from patients with advanced cancers accurately classify underlying cancer types and are associated with survival, TTF and RECIST responses.

Clinical trial identification

MD Anderson Protocols laboratory protocols LAB10-0334 and PA13-0956

Legal entity responsible for the study

MD Anderson Cancer Center

Funding

Sidney Kimmel Foundation for Cancer Research (Filip Janku); Sheikh Khalifa Al Nahyan Ben Zayed Institute for Personalized Cancer Therapy (Filip Janku); National Center for Advancing Translational Sciences (grant no. UL1 TR000371); National Institutes of Health through MD Anderson’s Cancer Center Support Grant (P30 CA016672); Illumina.

Disclosure

L. Liu, J. Toung, R. Vijayaraghavan, R. Zhang, H. Kang: Employee and stock ownership Illumina, Inc. F. Janku: Scientific Advisory Boards: Illumina, Guardant Health; Paid consulting: Trovagene; Stocks: Trovagene. All other authors have declared no conflicts of interest.