Background

The PI3K pathway is the most commonly activated signaling pathway in human cancer. The loss of PTEN further contributes to the tumorigenic impact of the active PI3K pathway, and have direct effect on prognosis of breast cancer. Although many small molecules are used in clinic to target the PI3K pathway, only minority of breast cancer patients respond to these drugs. This study identified the role of a deubiquitinating enzyme, USP10 in the regulation of PI3K pathway in breast cancer.

Methods

A genome wide RNAi screen targeting all known deubiquitinating enzymes was performed and level of phospho-AKT was assessed by western blotting. Significant hits of the screen were validated and mechanism of action in PTEN-mediated regulation of PI3K pathway and resistance to PI3K inhibitors in breast cancer has been investigated.

Results

We identified USP10 as a critical regulator of PI3K pathway. MAGI1 has been recently identified as PTEN interacting protein, and along with MEK1 functions to promote PTEN recruitment to the plasma membrane. We found USP10 as a part of complex between MEK1, MAGI1 and PTEN. Functionally, we demonstrated that USP10 stabilizes ITCH which is an E3 ligase for MEK1 resulting in degradation of MEK1 and decreased PTEN plasma membrane localization. We showed that downregulation of USP10 decreases activation of AKT, and results in decreased colony forming ability of breast cancer cells. Furthermore, expression analysis of USP10 revealed significantly higher levels of USP10 in breast cancer patients and its expression was correlated with the tumor progression and poorer overall survival in the breast cancer patients. Interestingly, we showed the upregulation of USP10 protein level in PI3K inhibitor resistant breast cancer cells as compared to their parental control and further depletion of USP10 resensitized these cells to PI3K inhibitors. In addition, positive correlation between USP10 and PTEN protein levels was observed in the PDX model of breast cancer patients who progressed after receiving PI3K inhibitor.

Conclusions

Overall, our results identify overexpression of USP10 as a potential mechanism for loss of PTEN functionality and PI3K pathway in breast cancer patients, and its possible involvement in resistance to PI3K inhibitors.

Legal entity responsible for the study

National University of Singapore

Funding

National University of Singapore

Disclosure

All authors have declared no conflicts of interest.

Background

A phase I clinical trial of Pevonedistat (MLN4924), a NEDD8-activating enzyme inhibitor, in AML and MDS showed modest clinical activity as a single agent. A phase II trial with Pevonedistat plus Azacytidine versus single-agent Aza is ongoing. As yet, there is no data regarding activity or dysfunction of the NEDDylation pathway in MDS. We examined NEDDylation pathway protein expression and its prognostic relevance in MDS.

Methods

On a tissue microarray (TMA) with bone marrow biopsies from 119 MDS patients, 40 AML pts, and 11 normal controls, we established immunohistochemistry (IHC) for NEDD8, NAE1 and UBE2M. Semi-quantitative analysis was done according to Remmele-Stegner immunoreactive score (IRS) (0 to 12 points). Clinical follow-up was available for 116 pts.

Results

Of 96 evaluable MDS pts, 46 (39.7%) showed an abnormal karyotype. Ten patients who received disease-modifying treatment prior to biopsy were excluded from survival analysis. On IHC, 100% of 40 AMLs and 11 normal controls were clearly positive (IRS>4) for all three NEDDylation pathway proteins. MDS patients showed a different pattern. While NEDD 8 was positive in the majority (60.6%), NAE1 and UBE2M were expressed in only 30.8% and 29.4%, respectively. For each protein, expression was not correlated with MDS type (WHO 2016), IPSS-R risk group, overall survival or AML transformation. Patients with strong expression of all three proteins survived longer (51 vs. 26 months), but the difference did not reach statistical significance (p = 0.10). Low expression of NAE1 and UBE2M significantly correlated with the presence of karyotype anomalies (p = 0.01 and p = 0.02, respectively).

Conclusions

In contrast to AML, expression of NEDD8 conjugation pathway proteins was low in a substantial proportion of MDS patients. The significant association between low or absent expression of NAE1 and UBE2M, and the presence of karyotype anomalies, suggests that low NEDDylation pathway activity may contribute to chromosomal instability. Nevertheless, since NEDD8 increases DNMT3b-dependent DNA methylation, NEDDylation pathway inhibition may be useful to augment treatment with hypomethylating drugs or counteract resistance to this therapy.

Legal entity responsible for the study

Universitätsklinikum Düsseldorf

Funding

Takeda Pharmaceutical Company

Disclosure

F. Majidi, N. Gattermann: The current research project is funded by Takeda Pharmaceutical Company. All other authors have declared no conflicts of interest.

Background

Ovarian cancer is the leading cause of death from gynaecologic malignancy. The platinum-based chemotherapy remains the standard initial treatment for ovarian cancer patients. Platinum resistance and recurrence is a formidable problem that impacts clinical outcomes in ovarian cancer patients. XRCC1-Ligase III heterodimer is a key player in DNA base excision repair (BER), single strand break repair (SSBR) and alternative non-homologous end joining (alt-NHEJ) pathway for double strand breaks (DSBs) Our objective was to evaluate if XRCC1-ligase III expressions could predict platinum and clinical outcome in epithelial ovarian cancers.

Methods

Investigation of the expression of XRCC1 and Ligase III in ovarian epithelial cancer was carried out on tissue microarrays of 525 consecutive ovarian epithelial cancer cases treated at Nottingham University Hospitals (NUH) between 1997 and 2010 and their expression was correlated to clinicopathological outcomes as well as to recurrence free survival (RFS) and ovarian cancer specific survival (OCSS).

Results

High nuclear XRCC1 expression was significantly associated with serous cystadenocarcinomas (p = 0.0000), higher FIGO stage at presentation (p = 0.001), residual tumour following surgery (p = 0.038), measurable disease before chemotherapy (p = 0.002) and platinum resistance (p = 0.002). High cytoplasmic ligase III expression was significantly associated with higher FIGO stage (p = 0.002), higher histology grade (p = 0.028), residual tumour following surgery (p = 0.001), measurable disease before chemotherapy (p = 0.006) and platinum resistance (p = 0.025). High nuclear staining was significantly associated with serous cystadenocarcinomas (p = 0.017). High XRCC1 was significantly positively highly correlated with high nuclear LIG3 (p < 0.0000). High XRCC1 and LIG III protein expressions were correlated with poor outcome.

Conclusions

XRCC1-Ligase III heterodimer is a promising predictive biomarker of platinum response in epithelial ovarian cancer.

Legal entity responsible for the study

This work has been approved by Nottingham Biobank Committee

Funding

Has not received any funding

Disclosure

All authors have declared no conflicts of interest.

Background

NOX66 is under development to enhance standard chemotherapy and radiotherapy. The target of idronoxil (the active constituent of NOX66), ENOX2, is an optimal candidate anti-cancer drug due to its tumour-cell specific expression. Inhibition of ENOX2 leads to a disruption of the TMEP, causing a cascade of intracellular actions leading to direct cytotoxicity and inhibition of DNA repair pathways. Three Phase 1 studies are underway to observe safety and efficacy signals - investigating multiple dose levels of NOX66 as monotherapy and in combination with carboplatin, palliative dose EBRT, and ¹⁷⁷Lu-PSMA (a theranostic for treatment of prostate cancer). Data has previously been presented showing NOX66 400mg to be well tolerated as monotherapy and in combination with carboplatin. Here we present data for patients receiving 800mg NOX66.

Methods

Nineteen patients with end stage metastatic cancer (breast, lung, prostate and ovarian) were enrolled between March and September 2017 across four centres in Georgia. Patients were allocated to one of three treatment cohorts: Cohort 1 (n = 8) receives 400mg NOX66 daily, Cohort 2 (n = 8) and 3 (n = 3) receive 800mg daily. Each patient receives NOX66 for up to seven months in the following regimen: - Part A: Monotherapy - 14 consecutive days of NOX66 treatment followed by 7 Days rest (Cohorts 1 and 2 only); - Part B: Carboplatin AUC4 - 3 x 28 Day cycles; NOX66 Days 1-7, Carboplatin Day 2; - Part C: Carboplatin AUC6 - 3 x 28 Day cycles; NOX66 Days 1-7, Carboplatin Day 2. Efficacy is measured by change in from baseline in radiological scans (using RECIST v 1.0 criteria) at End of Cycle 3 and Cycle 6. Adverse Events are monitored from enrolment until 30 days post Cycle 6.

Results

Data presented is for Cohort 2, Part A and B (Cohort 1 presented at ESMO2017). Of the 8 patients treated with 800mg NOX66, 7 completed monotherapy (one withdrawal prior to dosing). No AEs related to NOX66 were reported. Following three cycles of combination with carboplatin AUC4, 1 patient reported partial response, 4 stable disease and 1 disease progression. No AEs related to NOX66 have been reported.

Conclusions

NOX66 at 800mg is well tolerated as monotherapy and in combination with low dose carboplatin. Efficacy signals support further investigation of NOX66 in combination with standard chemotherapy.

Clinical trial identification

Protocol NOX66-001A; NCT02941523

Legal entity responsible for the study

Noxopharm

Funding

Noxopharm

Disclosure

G. Kelly: Employee, board member and significant shareholder of the sponsor company, Noxopharm Limited. I. Minns: Employee of the sponsor company, Noxopharm Limited.

Background

Glioblastoma multiforme (GBM) is the most aggressive form of malignant primary brain tumors in adults with a survival of only 12-15 months. We previously showed that ZR2002, a chimeric aminoquinazoline designed to possess mixed EGFR tyrosine kinase (TK) inhibitory and DNA targeting properties exhibits potent activity against GBM established cell lines and brain tumor stem cells in vitro.

Methods

We analyzed the in vivo plasma and brain pharmacokinetics (PK) of ZR2002 using various doses and via different routes in CD-1 mice. Mice were sacrificed at 15 min, 30 min, 1 hr, 3 hr, 8 hr, 24 hr after drug administration to quantify the amount of ZR2002 in plasma and brain homogenate of mice using RB10 as an internal standard and HPLC-MS/MS method. For the in vivo efficacy of ZR2002, U87/EGFRvIII cells stably transduced to express luciferase were injected into the right corpus striatum of the brains of 4–6 week-old Nu/Nu nude mice. We monitored tumor growth by luciferase bioluminescence imaging and mice were randomized to receive ZR2002 or vehicle control.

Results

Doses at 12.5 mg/kg IV, and 50 mg/kg PO ZR2002 did not cause any observable toxicity up to 24 h or in a subsequent experiment testing higher doses up to 150mg/kg PO compared to vehicle control with longer time follow-up over three weeks following drug administration. ZR2002 was detectable in the brain homogenate of mice at 3.58 µg/g or 2.76 µg/g for PO/50 mg/kg/3 hr (t max) or IV/12.5 mg/kg/5 min (t max), respectively. Our efficacy study showed that ZR2002 significantly improved the survival of intracranial U87/EGFRvIII tumor-bearing mice, compared to the control group (p value <0.0005).

Conclusions

We conclude that ZR2002 is able to cross the blood brain barrier and exhibits anti-tumor activity in U87/EGFRvIII orthotopic tumor mouse model, suggesting that this combi-molecule should be further developed in pre-clinical studies as a new treatment strategy in GBM.

Legal entity responsible for the study

McGill University

Funding

This research is partly funded by the Canadian Cancer Society grant #70217 (PK study) and Cancer Research Society grant#22716 (Efficacy Study)

Disclosure

All authors have declared no conflicts of interest.

Background

The poly(ADP-ribose) polymerase inhibitors (PARPi) are used in a wide range of human solid tumours but a limited evidence is reported in rhabdomyosarcoma (RMS), the most frequent soft-tissue sarcoma in children and adolescents. Our study evaluated the effects of Olaparib, a specific PARP1/2 inhibitor, and AZD2461, a newly synthetized PARP1/2/3 inhibitor, in RMS cells both as single-agent and in combination with ionizing radiation (IR).

Methods

Cell viability was monitored by trypan blue exclusion dye assays. Cell cycle progression and apoptosis were measured by flow cytometry, and alterations of specific molecular markers were investigated by Real Time PCR, Western blotting and immunofluorescence experiments. Irradiations were carried out at a dose rate of 2 Gy (190 UM/min) or 4 Gy (380 UM/min). Radiosensitivity was investigated by clonogenic assays.

Results

Olaparib (1.5 and 5 μM) or AZD2461 (5 and 10 μM) treatments dose-dependently reduced RH30 and RD cell growth by arresting cell cycle at G2/M phase. A significant modulation in the expression/activation and subcellular localization of specific cell cycle regulators, such as cyclin B1, cyclin D1, cdc2, cdc25C and p21, was observed in both Olaparib- and AZD2461-treated cells in comparison to mocked controls. Phosphorylation of H2AX (γH2AX), a specific marker of DNA damage, was significantly and persistently induced by Olaparib and AZD2461 exposure, this leading to cellular death as assessed by BCL2 down-regulation, caspase-3 activation and cytoplasmic vacuolization. PARPi treatment was also able to induce autophagy in addition to apoptosis, as confirmed by the concurrent LC3-II cleavage and p62 reduction. Interestingly, both PARP inhibitors strongly enhanced the effects of IR by blocking DNA damage repair, increasing G2/M arrest and drastically reducing the colony formation capacity of RMS-cotreated cells.

Conclusions

Our findings suggest that combined exposure to PARP inhibitors and IR might have therapeutic benefits in the treatment of RMS tumours compared with single-agent exposure, since stronger cytotoxic effects are induced, and compensatory survival mechanisms are prevented.

Legal entity responsible for the study

Department of Paediatrics - “Sapienza” University of Rome, Rome, Italy

Funding

Io, domani … Associazione per la lotta ai tumori infantili Onlus; Associazione Fabrizio Procaccini Onlus.

Disclosure

All authors have declared no conflicts of interest.

Background

Sphingosine kinases (SphKs) are enzymes catalyzing the formation of a bioactive lipid messenger, sphingosine-1-phosphate (S1P) through phosphorylation of sphingosine. So far, two isoforms of SphK have been described: SphK1, which has cytosolic distribution, and SphK2, known to locate predominantly in the nucleus. Overactivation of SphKs is involved in tumor growth and progression through regulation of cell proliferation and apoptosis.

Methods

We studied the expression, the intracellular localization and the activity of SphK1 and SphK2 in non-small cell lung cancer (NSCLC) cell lines and in cancer patients.

Results

In NSCLC cells, Sphk1 was found in the cytosol, while Sphk2 showed a nuclear localization. Surprisingly, we observed SphK1 expression also in the nuclear compartment. In several of the tested cell lines, the expression of SphK1 was even higher in the nucleus compared to the cytosol. Consistently, in a NSCLC tissue microarray, SphK1 nuclear expression was detected in a wide number of samples. Based on these data, we hypothesize the presence of a nuclear import signal in the SphK1 sequence, probably recognized by Karyopherin beta2. We found, in the aminoacidic sequence of SphK1, a R/K/H-X(5)-P-Y C-terminal motif within a structurally disordered and positively charged regions of about 30 amino acids. In order to dissect the role of nuclear SphK1, we used different tools able to modulate nuclear import/export. Moreover, specific inhibitors of SphKs were tested. Among them, fingolimod (FTY720), an oral drug initially defined as a S1P receptors antagonist but also acting as a SphK1 inhibitor, is effective in NSCLC cell lines: a reduction of cell density of about 50% is obtained with a drug concentration of about 2.5 µM. Interestingly, fingolimod seems to be more effective in cells with a nuclear/cytosolic ratio of SphK1 expression > 1.

Conclusions

We demonstrate for the first time that SphK1 has a nuclear localization in NSCLC and we are now investigating its biological role in this context. Moreover, we propose nuclear SphK1 as a novel druggable target in human NSCLC cells. Since fingolimod is a clinically available drug, our results may suggest SphK1 targeting as a therapeutic opportunity to be tested in NSCLC patients.

Legal entity responsible for the study

Prof. Roberto Bianco

Funding

Has not received any funding

Disclosure

All authors have declared no conflicts of interest.

Background

Despite intensive multimodal therapy, more than 50% of children with high-risk neuroblastoma eventually succumb to the disease. MYC signaling is a predominant driver of high-risk neuroblastoma, caused either by amplification of MYCN or by activation of cMYC. The bromodomain and extra-terminal (BET) domain-containing protein BRD4 was reported to cooperate with MYCN in the epigenetic regulation of super-enhancer driven genes in neuroblastoma. The BET inhibitors JQ1 and OTX015 were shown to repress BET/MYCN-mediated transcriptional control and antitumoral efficacy in MYCN-driven neuroblastoma. The potent BET inhibitor TEN-010, a derivative of JQ1, is currently in clinical trials for various MYC-driven adult tumors. Its efficacy against neuroblastoma is yet to be established.

Methods

In a preclinical setup, we investigated the antitumoral activity of TEN-010, OTX015 and JQ1 in neuroblastoma cell lines (n = 15) using ATP detection to assess cell viability 72h after treatment. To increase the therapeutic efficacy and decrease the risk of resistance formation, we subsequently turned towards combination therapies. We combined TEN-010 with conventional chemotherapeutics (e.g. etoposide) and with substances interfering with key pathways in neuroblastoma (e.g. volasertib).

Results

Seven cell lines were highly sensitive to TEN-010 with IC50 values ranging 85nM to 632nM. Five cell lines showed an intermediate response (IC50: 1.5-4.8µM). Six of the seven highly sensitive lines displayed a decrease in viability by more than 75% at 1µM. Highest sensitivity was observed in cells with high MYCN/MYC activity, whereas TEN-010 showed a weaker effect in MYCN-non-amplified and low c-myc expressing cell lines. While OTX015 appears effective at lower concentrations, our data indicate a higher specificity, and thus potentially a larger therapeutic window, for TEN-010. First results of combinatorial approaches indicate synergistic effects lowering the IC50s into the low nanomolar range.

Conclusions

Our results support the notion that BET proteins have crucial functions in MYC/MYCN-driven neuroblastoma and suggest BET inhibition as an effective treatment option that may complement current standard therapy.

Legal entity responsible for the study

N/A

Funding

German Cancer Consortium (DKTK), Innovative Medicines Initiative 2

Disclosure

All authors have declared no conflicts of interest.

Background

Biomarkers CD44 and CD24 are routinely used to identify breast cancer stem cells (BCSCs). BCSCs are chemotherapy resistant and bear high tumorigenesis and metastatic capabilities. Wnt/β-catenin signaling is involved in maintaining CSCs and thus is responsible for recurrence and poor prognosis. Role of Wnt receptor LRP6 in breast cancer promotion and progression is well known. We hypothesized that interactions between cancer cells, macrophages, and endothelial cells induce cancer stemness via activation of Wnt/-β catenin pathway, and blocking that pathway will reduce primary and metastatic tumors.

Methods

Breast cancer cells were co-cultured with macrophages and endothelial cells with or without Wnt inhibitors. BCSC markers were quantified by flowocytometry. CD44+/CD24- cells were FACS sorted and applied to 3D cultures with or without Wnt inhibitors and/or Doxorubicin. In our in vitro studies we utilized two types of Wnt inhibitors, a small molecule LPR6 tyrosine kinase inhibitor Salinomycin and a function blocking monoclonal antibody (2F1)^A. We tested the efficacy of 2F1 in two mouse models in combination with Doxorubicin. One model used SCID mice bearing allografts of MDA-MB-231 cells. The second used FvB mice bearing syngeneic Met-1 tumors.

Results

Co-culture of breast cancer cells with macrophages and/or endothelial cells, significantly increases cells expressing BCSC markers, while inhibition of Wnt receptor significantly reduced them. The CD4+/24- cells are found highly resistant to Doxorubicin both in 2D and 3D cultures. In our in vivo experiments the 2F1-Doxorubicin combination significantly reduced primary tumors and lung and bone marrow metastasis as well as improved animal viability.

Conclusions

Inhibition of Wnt pathway significantly reduces the induction of BCSCs in vitro. In vivo both primary tumor and metastatic events were drastically reduced in two mouse models when treated with 2F1 and Doxorubicin. This study introduces LRP6 blocking antibody 2F1 as a promising antitumor agent that can be further developed for breast cancer targeted therapy in patients whose tumors express higher copy numbers of LRP6. Reference: A. Hu Y1, Chen Y, et al. Invest Ophthalmol Vis Sci. 2013 Jan 7;54(1):141-54.

Clinical trial identification

Sumanta Goswami^1,2, Gargi Bandopadhyaya¹, Justin Stein¹, Srinjoy Goswami¹, John S. Condeelis² and Maja H. Oktay^2,3

¹Department of Biology, Yeshiva University, New York, NY 10033. ²Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461 ³Department of Pathology Montefiore Medical Center, Bronx, NY 10467

Legal entity responsible for the study

Sumanta Goswami

Funding

Has not received any funding

Disclosure

All authors have declared no conflicts of interest.

Background

Most targeted therapies in cancer have reached approval based on clinical studies performed in unselected patients. Small subsets of patients present exceptional responses (ER), which could be driven by a low level of genomic alterations in genes identified as causally implicated in cancer.

Methods

Adult patients with advanced solid cancers (breast, lung, colorectal, ovarian and kidney cancers and melanoma) having presented an ER to an approved antineoplastic targeted therapy will be included. ER is defined using the definition chosen by the NCI which combines the three criteria: - complete or partial response, - lasting > 6 months, - and not expected in > 10% of the patients in this drug – organ situation ER will be reviewed each month by the Response Confirmation Committee composed of the study coordinators and at least one expert of each organ. The primary objective is to identify whether tumors characterized by a low level of genomic alterations are associated with ER. A low level of genomic alteration is defined by the presence of less than the 5th quantile of genomic alterations to be expected in the given tumor type. For each tumor type, it is desired to test the null hypothesis H0: π = 0.05 against the one-sided alternative hypothesis π > 0.05. For each of six cohorts, a sample size of 44 patients is necessary to achieve 80% power at π = 15 with a one-sided level 5% test.

Results

As of December 2017, 147 patients were screened in 31 French centers. Forty-two patients were included in the study (19 patients with breast cancer, 11 patients with kidney carcinoma, 5 with melanoma, 4 with lung cancer, 2 with colorectal cancer and 1 ovarian cancer). The 5 most frequent drugs were: sunitinib, everolimus, bevacizumab, trastuzumab and pazopanib. The EXPRESS study is still recruiting. Study completion date is estimated to be in August 2019.

Conclusions

The identification of molecular traits associated with ER might serve the development of predictive classifiers for precision medicine. This study also represents a unique opportunity to better understand cancer biology.

Clinical trial identification

NCT02701907

Legal entity responsible for the study

UNICANCER R&D

Funding

Institut National du Cancer (INCA)

Disclosure

All authors have declared no conflicts of interest.

Background

Increasing evidence shows that microRNAs (miRNA), a family of small non-coding RNAs, play a pivotal role in regulating mRNA translation. Recently, some miRNAs have been shown to be involved in regulating cancer stem cell (CSC) properties. Because CSCs are highly resistant to conventional chemotherapy and radiation therapy, and heavy ion radiotherapy is effective in treating those of chemo-radioresistant cancers, in this study we attempt to explore new molecular mechanisms of CSC targeted therapies by carbon ion beam alone or in combination with a miRNA200c mimic.

Methods

Human pancreatic CSCs (CD44+/ESA+) sorted from hum pancreatic cancer cells PK45 and PANC1 were treated with carbon ion beam, X-ray irradiation alone or in combination with a miRNA200c mimic, followed by cell viability assay, colony and spheroid formation ability assay, caspase 3/7 activity assay, and real-time PCR analysis of apoptosis and autophagy-related gene expression were also performed.

Results

The colony, spheroid formation assays confirmed that a subpopulation of CD44+/ESA+ have exactly CSC properties compared with CD44-/ESA- cells in pancreatic cancer cells. Carbon ion beam combined with a miRNA200c mimic significantly decreased CSC viability. The colony, spheroid formation ability of CD44+/ESA+ cells was significantly inhibited by carbon ion beam combined with miRNA200c compared with X-ray irradiation, carbon ion beam alone. Apoptosis analysis showed that caspase activity of 3/7 was significantly enhanced by carbon ion beam in combination with a miRNA200c mimic. Real-time PCR analysis showed that some of apoptosis-related and autophagy-related genes were significantly induced by carbon ion beam alone or in combination with a miRNA200c mimic in pancreatic cancer cells.

Conclusions

Taken together, because the carbon ion beams have a well-defined range with well-localized energy called “spread out Bragg peak (SOBP)”, and release enormous energy at the end of their range, carbon ion beams therefore can kill radioresistant CSCs more than photon beams, and combination with a miRNA200c mimic further enhances those actions, based on the present data.

Legal entity responsible for the study

National Institutes for Quantum and Radiological Science and Technology

Disclosure

All authors have declared no conflicts of interest.

Background

Cancer cells rewire metabolic pathways to enable their uncontrolled proliferation through a phenomenon called the Warburg effect. Cancer cells adopt this metabolic diversion through epigenetic modifications such as chromatin remodeling and histone acetylation (H3k27ac). Such inheritable modifications influence DNA accessibility to regulate gene expression. One such epigenetic modifier, WD40 protein, is shown to be involved in regulation of transcription, and protein modifications. Its dysfunction might influence the development of diseases such as cancer and metabolic diseases. Our lab has discovered a WD40 containing protein which we refer to as WD40 that serves as an oncogenic driver in breast cancer cells. Based on preliminary findings, we propose that WD40 functions as an oncogenic driver, which plays a critical role in epigenetic reprogramming in genes that drive the Warburg effect and that promote the enrichment of CD44^high/CD24^low population.

Methods

/Results: We determined endogenous levels of WD40 in different breast cancer cells and found that WD40 is markedly elevated in breast cancer cell lines.

Results

Methods/Results (Cont): WD40 knockdown growth curve assays revealed that breast cancer cells depend on WD40 for their growth. Further, recombinant WD40 expression in normal mammary epithelial cells initiates a transcriptional program that is coupled to the emergence of both tumor spheres and strong tumor-promoting properties in vivo (NOD-SCID). Transcriptomic and metabolomic analysis revealed that WD40 orchestrates dramatic reprogramming of the glycolytic infrastructure of breast cancer stem cells and promotes enrichment of CD44^high/CD24^low population. Furthermore, our ChIP assay results indicate that WD40 increases H3k27ac signature on promoter/HRE regions of its key target genes to facilitate their transcription.

Conclusions

Taken together, breast cancer cells have elevated WD40 and depend on WD40 for survival. Recombinant WD40 expression results in tumor formation in NOD-SCID mice. WD40 initiates aerobic glycolysis and promotes enrichment of CD44^high/CD24^low phenotype. This knowledge can be used to design novel therapeutic strategies to reverse epigenetic modifications in cancer.

Legal entity responsible for the study

University of Toronto

Funding

Connaught Fund

Disclosure

The author has declared no conflicts of interest.

Background

NOTCH signalling is a key development pathway whose aberrant activation is known to play a role in multiple human cancers. When the NOTCH pathway is activated by genetic lesions (over expression of ligands/receptors, GOF mutations in receptors, chromosomal translocations), it becomes a major oncogenic driver for NOTCH-dependent cancers and resistance to standard of care treatment.

Methods

So far, two therapeutic approaches have been attempted to block NOTCH signalling in the clinics: (i) using monoclonal antibodies (mAbs) against NOTCH ligands/receptors and (ii) small molecule gamma-secretase inhibitors (GSIs). Clinical activity of these agents was observed, but exposures were usually limited due to various toxicities. To inhibit pan-NOTCH signalling in human tumors independently of the mechanisms of NOTCH activation, and most downstream of the pathway, we have previously reported the discovery of a new class of small molecules protein-protein interaction (PPI) inhibitors able to target assembly of the NOTCH transcription complex, and downregulate expression of NOTCH target genes (e.g. c-MYC, CCND). Here we report the pharmacological characterization of CB-103, a first-in-class oral small molecule, PPI inhibitor of the NOTCH transcription complex.

Results

In vitro studies demonstrated that CB-103 triggers a dose-dependent downregulation in NOTCH signalling with a unique mechanism of action. CB-103 was active in a subset of cancer cell lines from various malignancies, including different solid tumour indications, lymphomas and leukaemias. Moreover, CB-103 demonstrated anti-tumour efficacy in the Triple-Negative Breast Cancer HCC1187 model, resistant to GSIs due to NOTCH2 chromosomal translocation, and in multiple in vivo models of NOTCH-driven T-ALL (cell lines and patients derived xenograft), as single agent or in combination with various anticancer drugs.

Conclusions

Safety pharmacology and toxicology studies have been completed and revealed an excellent non-clinical safety profile of CB-103. Clinical development of CB-103 with a first-in-human Phase I/IIA clinical study in solid tumours and haematological malignancies has been started in Spain, Netherlands and Switzerland.

Clinical trial identification

EUDRACT #2017‐001491‐35

Legal entity responsible for the study

Cellestia Biotech AG Hochbergerstrasse 60C CH-4057 Basel Switzerland

Funding

Cellestia Biotech AG.

Disclosure

D. Weber: Employee (CMO) and shareholder of Cellestia. R. Lehal: Employee (CSO) and shareholder of Cellestia. J-P. Bourquin: Member of Medical Advisory Board Cellestia. M. Bauer: Employee (CEO) and shareholder of Cellestia. M. Murone: Employee (COO) and shareholder Cellestia. F. Radtke: Chairman of the Board and shareholder of Cellestia. All other authors have declared no conflicts of interest.

Background

The Hedgehog (Hh) signaling pathway is an evolutionarily conserved pathway of signal transmission which plays a significant role in the normal embryonic development of invertebrates and vertebrates. In the adult organism, Hh signaling pathway is mostly inactive or poorly active while its hyperactivation is associated with carcinogenesis. Binding of the Hh ligands, Sonic Hedgehog (SHh), Indian Hedgehog (IHh) and Desert Hedgehog (DHh) along with PTCH protein activates Hh signaling resulting in increased activity of the GLI transcription factors that activate targeted genes. The status of Hh pathway components in serous ovarian carcinomas is poorly understood.

Methods

Formalin-fixed paraffin-embedded samples of 11 low-grade (LGSC), 40 high-grade serous ovarian carcinomas (HGSC) and 7 normal/benign ovarian tissues (controls) were used for this study. SHh and IHh protein expression was explored using immunohistochemistry. DNA methylation pattern of SHh and IHh gene was analyzed by methylation-specific PCR (MSP).

Results

SHh and IHh expression was significantly higher in both LGSC (p < 0.001 and p = 0.011, respectively) and HGSC (p < 0.001 and p = 0.003, respectively) compared with normal/benign ovarian tissues. There was no statistically significant difference in SHh and IHh protein expression between LGSC and HGSC. SHh and IHh DNA methylation was observed in HGSC [(2/40, 5%) and (1/40, 2.5%), respectively]. In all normal ovarian tissues no methylation was detected.

Conclusions

A significant proportion of serous ovarian carcinomas exhibits increased SHh and IHh protein expression, which indicates that these Hedgehog signaling pathway components may be actively involved in pathogenesis of serous ovarian carcinomas. Therefore, SHh and IHh might serve as potential therapeutic targets for serous ovarian carcinomas. Low methylation levels of the respective genes in HGSC and absence of methylation in LGSC and normal ovarian tissues indicate alternative mechanisms of SHh and IHh activation. Further clinical studies should confirm the clinical and therapeutic relevance of the observed Hh alterations.

Legal entity responsible for the study

School of Medicine, University of Zagreb

Funding

School of Medicine, University of Zagreb

Disclosure

All authors have declared no conflicts of interest.

Background

Pancreatic ductal adenocarnoma (PDAC) is the fourth leading cause of cancer related death in men and women.¹⁵ Approximately 60-70% of PDACs arise in the head of the pancreas. Since the early diagnosis of head of the pancreas tumors is difficult, patients are frequently at an advanced stage at the time of diagnosis and have extremely short survival. Furthermore, the mechanism of pathogenesis in PDAC is not completely understood and there are currently no effective therapies. Recent studies demonstrated that miRNAs play critical roles in various types of tumors including PDAC and thereby has high diagnostic value for screening and great clinical value for cancer therapy. The aim of the present study was to determine the expression profile of miRNAs in head of pancreas and examine its association with prognosis.

Methods

A total of 56 patients who underwent pancreaticoduodenectomy for head of pancreas were analyzed for 12 different miRNAs by RT-PCR.

Results

miR-21 and miR-10b were 11.78-fold (P = 0.004) and 9.62-fold (P = 0.021) higher, and the miR-143 expression was 5.49-fold lower (P = 0.0412) in tumor tissues compared with normal tissues. The over expression of miR-21 was related with lymphatic invasion (P = 0.0203). Moreover, high miR-21 expression was an independent poor prognostic factor for overall survival (P = 0.0242).

Conclusions

The elevated expression of miR-21 may be involved in the progression of head of pancreas tumors and may therefore be considered a prognostic factor for patients with poor prognosis.

Legal entity responsible for the study

Ekrem Kaya

Funding

Has not received any funding

Disclosure

All authors have declared no conflicts of interest.

Background

Breast cancer is a complex and heterogeneous disease. Luminal B breast cancers molecular subtype are estrogen receptor-positive (ER+), highly proliferative, resistant to standard therapies and have a poor prognosis. Although they express hormone receptors, their metastatic risk and resistance to hormone therapy and to conventional chemotherapy demand to develop appropriate therapies. Studies have suggested that RAB 25 (Ras-related protein 25) a member of Rab small GTPase family, is involved in Luminal B breast cancer pathogenesis. To better understand this subtype we compared RAB25 DNA copy number aberrations (CNAs) and expression level in luminal B tumors with those observed in breast cancers of the other molecular subtypes.

Methods

To further define molecular alterations associated with the luminal B subtype we studied, with high-Troughput molecular analysis (CGHarray), copy number aberrations and RAB25 gene expression (DNA microarray) deregulation in primary breast cancer samples.

Results

RAB25 gene is not targeted by genomic alterations in a high proportion of breast cancers. RAB25 mRNA expression and RAB 25 protein level was deregulated in breast cancers. Comparison with clinical features between breast tumors with and without RAB 25 deregulation showed that RAB25 high expression was associated with the expression of estrogen receptor (ER), high expression of Ki67 proliferative index and luminal B breast cancer molecular subtypes.

Conclusions

we have identified RAB25 mRNA and protein expression deregulated in luminal B breast cancer molecular subtype suggesting RAB25 as a potential therapeutic target in this aggressive subtype and for which no targeted therapy exists at yet. A better understanding of the involvement of RAB25 in luminal B breast cancer might explain the role in the development and/or hormone resistance of this aggressive subtype.

Legal entity responsible for the study

IPC-Molecular Oncology

Funding

Has not received any funding

Disclosure

The author has declared no conflicts of interest.

Background

Ovarian cancer is associated with the highest mortality rate among gynaecologic malignancies. There is a need to refine classification of ovarian cancer and identify novel targets. The mammalian target of rapamycin (mTOR) pathway has a crucial role in the regulation of translation of specific proteins associated with ovarian cancer progression. The major downstream effectors of mTOR are eukaryotic initiation factor 4E-binding protein 1 (4EBP1) and ribosomal protein S6 kinase (p70S6K). We aimed to investigate the biological significance of this pathway in ovarian cancer.

Methods

Investigation of mTORC1, 4EBP1 and p70S6K protein expression, was carried out on tissue microarrays of 195 consecutive ovarian epithelial cancers treated at Nottingham University Hospitals (NUH) between 2000 and 2007. Clinicopathological and outcome data were collected.

Results

High cytoplasmic expression of both 4EBP1 and p70S6K was associated with serous type carcinoma (p = 0.005 and p = 0.02 respectively). High expression of 4EBP1 was seen more frequently in cases treated with early debulking (p = 0.005). Univariate outcome analysis showed an inverse association between 4EBP1 expression and overall survival (p = 0.005) and development of local recurrence (p = 0.005). P70S6K showed inverse association with local recurrence (p = 0.002). High cytoplasmic expression of mTORC1 was inversely associated local recurrence (p = 0.032) and borderline significance with poor overall survival (p = 0.079).

Conclusions

mTORC1 and its downstream effectors, 4EBP1 and p70S6K positive expression predicts local recurrence and poor survival in ovarian cancer patients. Therefore, this could be targeted as a potential pathway that could improve patients’ survival and reduce tumour recurrence.

Legal entity responsible for the study

N/A

Funding

Has not received any funding

Disclosure

All authors have declared no conflicts of interest.

Background

Telomeres play a vital role in maintaining the integrity of the genome. Mammalian telomeres are certainly transcribed into telomeric repeat-containing RNA (TERRA). This Long non-coding RNA participates in the regulation of telomere length and telomerase activity. As reported, lncRNAs play important roles in gastric cancer progression. Telomerase and its major catalytic subunit (hTERT) are upregulated in most cancers, including gastric cancer. RNA interference (RNAi) has been proven to be a powerful tool for gene knockdown and hold good promise for the treatment of human diseases including cancer. In this study, we investigated the effect of hTERT repression on the TERRA expression and telomere length in multiple passages.

Methods

AGS gastric cancer cell line was treated with hTERT FlexiTube siRNA and Hiperfect transfection reagent. Cell viability was examined by MTT assay. A DAPI staining method was used to analysis cell cycle by Flow Cytometry. Real-time PCR was used to evaluate the expression level of hTERT and TERRA and assessment of telomere length.

Results

The MTT assay showed that increasing the exposure time to 48 hours decreased cell viability below the 50% viability mark. The flow cytometry cell cycle analysis showed a significant increasing in the number of cells in G1 phase and decreasing in the number of cells in S phase. The results of the qRT-PCR analysis demonstrated that siRNA transfection decreased the hTERT expression, although has no significant effect on TERRA expression in early passages. However, upregulation of TERRA expression in the passage of 20 compared to the control cells has been shown. Also, telomere length measurement in each passage was decreased after hTERT siRNA treatment.

Conclusions

The significant downregulation of hTERT mRNA inhibited the cell viability of AGS cells and cell cycle arrest. This study provides the fact that downregulation of hTERT expression had no effect on TERRA expression level in early passages of AGS cell line. Also, showed a direct link between decreasing in telomere length and downregulation of hTERT expression.

Legal entity responsible for the study

Ardabil University of Medical Sciences

Funding

Ardabil University of Medical Sciences

Disclosure

All authors have declared no conflicts of interest.

Background

Telomerase is in charge of telomere extending and is triggered in around 90% of cancers. hTERT is the controlling subunit of telomerase and plays a critical role in the activation of telomerase. The mechanism through which hTERT regulate the invasion and metastasis of cancer is unclear. miRNAs can regulate the expression of hTERT. It was previously reported that miR-1266 can target hTERT in gastric cancer. In this study, we have made the first report of miR-1266-5p role on the hTERT expression, telomerase activity, and biological functions, including cell proliferation and cell cycle in AGS, MCF7, A375, and HepG2 cell lines.

Methods

AGS, MCF7, A375, and HepG2 cell lines were transfected with miR-1266-5p mimic and inhibitor reagents. The Expression levels of miR-1266-5p, hTERT, and transfection efficiency were analyzed by Taqman qRT-PCR. The cell proliferation and cell cycle changes were detected by MTT Calorimetric Assay and flow cytometry, respectively. Also, Quantitative TRAP Assay was used to detect telomerase activity.

Results

The expression of miR-1266-5p significantly was increased after transfection by mimic compared to control cells. While its expression was decreased by the inhibitor. Upregulated miR-1266-5p significantly decreased cell growth, although inhibitor promoted cell proliferation. This finding was confirmed by cell cycle analysis, as upregulation of miR-1266-5p induced cells cycle arrest at the transition of G1 to S phase and led to G0/G1 entry, while the downregulation of miR-1266-5p promoted cell growth and led to G2/M entry. Concordantly, the overexpression of miR-1266-5p resulted in down-regulated hTERT expression and also suppressed telomerase activity.

Conclusions

Taken together, the findings showed that miR-1266-5p acts as hTERT and telomerase activity suppressor. miR-1266-5p could also decrease cell proliferation and induce cell cycle arrest, while its inhibitor eliminates miR-1266-5p effects. Thus, upregulation of miR-1266-5p may be considered as a novel therapeutic target in cancer.

Legal entity responsible for the study

Ardabil University of Medical Sciences

Funding

Ardabil University of Medical Sciences

Disclosure

All authors have declared no conflicts of interest.

Background

The regulation of microRNA (miRNA) biogenesis, function and degradation is regulated by a range of mechanisms involving RNA-binding-proteins and protein-RNA interactions. The potential contribution of regulatory miRNAs to the expression of these RNA interactor proteins that could control other miRNAs expression is still unclear.

Methods

Here, we demonstrate a circuit of miRNAs regulation between oncogenic and tumor suppressor miRNAs in a chemotherapy-resistant ovarian cancer model.

Results

We identified and characterized miR-25-3p and miR-15a-5p as negative regulators of RNA-binding protein hnRNPA1 expression that is required for the processing of miR-18a-3p, an inhibitor of the K-RAS oncogene. The inhibition of miR-25-3p and miR-15a-5p decreased the proliferation, motility, invasiveness and angiogenesis potential and also increases apoptosis when combined with docetaxel. Alteration of this regulatory circuit causes poor overall-survival outcome in ovarian cancer patients.

Conclusions

These results highlight miR-25-3p, miR-15a-5p as key regulators of miR-18a-3p expression and its downstream target K-RAS, through direct modulation of hnRNPA1 expression. Our results demonstrate the therapeutic potential of inhibiting miR-25-3p, miR-15a-5p and the use of miR-18a-3p/KRAS ratio, as a prominent outcome prognostic factor.

Legal entity responsible for the study

MD Anderson Cancer Center

Funding

National Institutes of Health, USA

Disclosure

All authors have declared no conflicts of interest.

Background

Astrocytomas are the most common primary brain tumors that are on the basis of their histology, molecular characteristics and prognosis classified into 4 different malignancy grades. As one of the key oncogenic pathways in human malignancies that has been associated to many human cancers the Wnt signaling pathway has also been implicated in gliomagenesis. In the present study we aimed to identify the status of SFRP1 promoter hypermethylation in different malignancy grades of astrocytoma and assess its parallel expression levels in search for their connection with clinical data.

Methods

Promoter hypermethylation of critical molecular component of Wnt signaling – SFRP1 was studied in 24 astrocytomas of different malignancy grades by using methylation-specific PCR (MSP). In order to examine the consequence of hypermethylation, the SFRP1 protein was investigated by immunohistochemistry and analyzed by quantitative stereological analysis.

Results

SFRP1 promoter hypermethylation was found in 32% of astrocytomas. Hypermethylation of SFRP1 promoter was progressively rising in higher astrocytoma grades with the highest significant distribution in glioblastoma (P = 0.042). The expression of SFRP1 protein showed 45.8% of cases with weak or lack of expression, 25% with moderate and 29.2% with strong expression levels. Our study showed that cases with methylated SFRP1 promoter expressed significantly less SFRP1 protein than unmethylated ones (P = 0.031). Furthermore, we have shown that the levels of beta-catenin, pathways indicator of oncogenic activity, were elevated with often nuclear localization. Statistical analysis showed that glioblastoma samples with unmethylated SFRP1 promoter had significantly less beta-catenin (P = 0.033). The activation of Wnt signaling was further confirmed by the expression of its transcriptional activators. Strong expression of LEF1 was significantly associated to higher grades (P = 0.006).

Conclusions

Our findings showed that SFRP1 acts as an antagonist in the evolution of astrocytoma which will contribute to better understanding of astrocytoma molecular profile and offer methylation biomarker of progression.

Legal entity responsible for the study

School of Medicine, University of Zagreb

Funding

Croatian Science Foundation (grant number 6625)

Disclosure

All authors have declared no conflicts of interest.

Background

RAS mutations promote resistance to anti-EGFR targeted agents in colorectal cancers (CRCs) by disabling RAS intrinsic GTPase activity, therefore increasing an EGFR-independent activation of PI3K/AKT and MAPK pathways. However, up to 25% of CRC patients are refractory to EGFR inhibitors even in absence of the above- mentioned mutations. It is, then, crucial to identify alternate routes of kinase pathway activation that sustain the constitutive or acquired resistant phenotype. p21-Activated Kinase (PAK) family proteins play an important role in the context of cancer progression, and are related to Ras pathway. Therefore, we focused on the involvement of PAK signaling pathway in the onset of cetuximab resistance.

Methods

We used different human colorectal cancer models, with or without KRAS/NRAS mutations. We also generated SW48 cells stably transfected with different K-RAS mutation. We tested the effects of PAK inhibition by PF-3758309 both in vitro and in vivo.

Results

All cell lines are sensitive to the PAK inhibitor PF-3758309, as shown by the inhibition of cell proliferation and cell viability, in both RAS wild type and RAS mutated cell lines. Upon PF-3758309 treatment we found an inhibition of the phosphorylation of different signal transducers downstream to PAKs, such as MEK1 (Ser298) and beta-Catenin (Ser675). Moreover, we found that PF-3758309 enhances EGFR phosphorylation in RAS mutated cells, an effect probably mediated by a beta-Catenin-dependent regulation of the DEP1 phosphatase. Based on these data, a combination of PF-3758309 with cetuximab is now under evaluation, in vitro and in vivo.

Conclusions

We demonstrated that PAK inhibition is effective in CRC cell lines, independently from the RAS mutational profile. Moreover, we hypothesize a PAK-dependent regulation of EGFR phosphorylation, mediated by beta-Catenin and DEP1. Based on our preliminary data, we plan to further investigate the role of PAK signaling in the onset of cetuximab resistance both in vitro and in vivo. by a beta-Catenin-dependent regulation of the DEP1 phosphatase. Based on these data, a combination of PF-3758309 with cetuximab is now under evaluation, in vitro and in vivo.

Legal entity responsible for the study

Prof. Roberto Bianco

Funding

Has not received any funding

Disclosure

All authors have declared no conflicts of interest.

Background

Estrogen drives cellular proliferation and survival in estrogen receptor-positive (ER+) breast cancer. Exposure to endogenous or exogenous estrogen is a well-established cause of breast cancer and target of endocrine therapies such as antiestrogens and aromatase inhibitors. However, their efficacy is limited by intrinsic or acquired endocrine resistance which remains a significant clinical challenge. A third of patients given the antiestrogen therapy tamoxifen for 5 years develop recurrence and metastasis within 15 years. Gene expression studies of endocrine resistance suggest the dysregulation of epigenetic enzymes has an important role in survival signaling and cellular proliferation in acquired endocrine resistance. The development of epigenetic modifier inhibitors offers the promise of dynamically targeting mediators of acquired resistance that may be exploited as biomarkers and therapeutic targets to improve patient prognosis.

Methods

The in vitro work was performed using endocrine-resistant and endocrine-sensitive breast cancer cell lines. Proliferation was measured by changes in cellular confluency over time and viability determined by MTT assay. In vivo studies were investigated using a mouse xenograft model.

Results

In this study, we identified a histone methyltransferase that is regulated epigenetically and when targeted in combination with endocrine therapy it significantly reduces the proliferation and the viability of resistant cells in vitro, and it leads to a significant reduction in tumour growth in a mouse xenograft model.

Conclusions

Tamoxifen and histone methyltransferase inhibitor reduce proliferation and viability of tamoxifen-resistant and sensitive breast cancer cell lines. Histone methyltransferase inhibitor re-sensitises resistant breast cancer and causes tumour regression in a mouse xenograft model. Therefore, tamoxifen-resistant ER+ breast cancer can be re-sensitised by epigenetic therapy.

Legal entity responsible for the study

QIMR Berghofer

Funding

QIMR Berghofer

Disclosure

All authors have declared no conflicts of interest.

Background

Obesity and High body mass index are associated with a higher risk of developing renal cell carcinoma (RCC). Leptin is a peptide hormone produced predominantly by adipocytes that is elevated in obese individuals. The main function of leptin is to regulate body weight and appetite. Leptin may play a role in carcinogenesis of many cancers including RCC. We aimed to investigate the role of leptin Receptor gene (A/G) polymorphism (rs1137101) in RCC.

Methods

This study was carried out by cooperation between Clinical Oncology & Nuclear Medicine, Medical Biochemistry and Internal Medicine Departments, Faculty of Medicine, Menoufia University in the period from May 2015 to April 2017. It was conducted on 123 individuals classified into; group I: included 73 patients with histological diagnosis of RCC and group II: 50 healthy control subjects. For RCC patients Staging was done according to the American Joint Committee on Cancer: the 7th edition, stage IV patients received SUTENT® (sunitinib malate) as first line treatment. Genotyping of the Gln223Arg (rs1137101) A/G polymorphism was measured by Real-Time PCR and compared between both groups and correlated with different patients, disease characteristics and survival.

Results

This study included 66 males and 57 females, the mean age 58.65 + 8.62. The results of this study revealed that there was a significant statistical difference as regards genotype frequency of LEPR gene A/G polymorphism between the two studied groups, with the highest frequency of GG genotype among RCC patients (67.1%), while AA genotype had the highest frequency in the control group (60%); (p < 0.001). In RCC patients with GG genotype is associated with more advanced stage (stage III and IV) (46.9%) compared to GA & AA genotypes (12.5%), and higher nuclear grade G3 (28.5%) compared to GA, AA genotypes (4.2%). GG genotype has worse survival versus GA and AA genotypes; p value <0.001.

Conclusions

Leptin Receptor gene (A/G) polymorphism (rs1137101) might be a candidate risk factor for developing RCC. In RCC patients with GG genotype is associated with more advanced stage and higher nuclear grade and shorter survival compared with patients with GA & AA genotypes.

Legal entity responsible for the study

Menoufia University

Funding

Has not received any funding

Disclosure

All authors have declared no conflicts of interest.

Background

Response to inflammatory stimuli is under a tight cellular regulation, including post-transcriptional mechanisms, to ensure the accurate response of cells to various environmental stresses. The aim of this study was to investigate the post-transcriptional regulatory effect of NaBt, a well-known HDAC inhibitor, on COX-2 mRNA in colon cancer cell lines via AU rich elements (ARE) in the 3’UTR.

Methods

Caco-2 and HT-29 cells were treated with NaBt and mRNA and protein expressions were determined by qPCR and Western blot, respectively. COX-2 enzymatic activity was determined by Prostaglandin E2 (PGE2) ELISA assay. mRNA stability was assessed by an Actinomycin D (ActD) chase assay.

Results

Treatment of both cell lines at short (0-6h) and long (48h) time points with NaBt strongly reduced COX-2 mRNA and protein expression. This was accompanied by a strong suppression of stabilizing ARE-binding proteins at 48h. Interestingly, PGE₂ levels remained unchanged most likely due to the robust induction of COX-1 by NaBt. The ActD chase assay indicated that in the absence of new mRNA synthesis, NaBt induced stability of COX-2 mRNA in Caco-2 cells while it enhanced COX-2 mRNA decay in HT-29 cells. In the same experimental setup in Caco-2 cells, NaBt and NaBt+ActD together enhanced phosphorylation of the stress-related protein kinase MAPKAPK2 (MK2) compared to ActD alone, which can induce mRNA stabilization through nucleo-cytoplasmic shuttling of AREBPs like CUGBP2 and HuR. In HT-29 cells, NaBt and NaBt+ActD treatments decreased p-MK2, but increased the phosphorylation of Chk2, which in turn modulates the nucleo-cytoplasmic shuttling of HuR to facilitate COX2 mRNA degradation. Mild reduction in p-Chk2 was observed in Caco-2 cells in the presence of NaBt.

Conclusions

These findings suggest that NaBt can regulate mRNA stability of COX-2 through post-transcriptional mechanisms and in a contextual manner, dependent on upstream signaling. Future studies will indicate how NaBt can affect the dichotomy in Chk2/MK2 activation in the presence of cellular stress.

Background

Chronic atrophied gastritis is the most important and independent risk factor for gastric adenocarcinoma, i.e., precancerous condition, especially in cases of intestinal metaplasia development.

Methods

Aiming the early detection of gastric cancer authors had conducted molecular-genetic researches in 60 patients with chronic atrophied gastritis. The objects of research were gastric mucosal layer taken from 3 points and blood samples. Polymerase chain reaction was used to determine the areas od DNA methylation in APC, E-cadherin, hMLHI and TINP3 genes.

Results

Total hypomethylation was identified in 10 (16,6%) patients, in other 50 cases areas of DNA methylation were not found. Comparison of mucosal samples with blood samples revealed that the rate of concordance was in 7 cases (70%). In cases where we identified methylations in 3 and 4 genes in 2 patients was found stomach cancer.

Conclusions

In cases of identification of DNA areas methylation patients are recommended to undergo endoscopic monitoring every 6 months. In cases of absence of methylation endoscopy should be performed once a year.

Legal entity responsible for the study

National cancer Research Centre MoH Republic of Uzbekistan, Centre of High Technologies Academy of Sciences Republic of Uzbekistan

Funding

Has not received any funding

Disclosure

The author has declared no conflicts of interest.

Background

Ovarian cancer is the fifth leading cause of cancer death in women and the most lethal gynecologic malignancy with a rate of survival of 40%. Aberrant GAS6-AXL signaling pathways are associated with many human diseases, including bone diseases, immune-suppression, fibrosis, cancer progression and metastasis. Experimental evidences demonstrated that patients expressing high levels of Axl shows shorter over-survival than patients expressing low levels of Axl in epithelial ovarian cancer. Therefore, there is the need to find novel strategies for silencing and blocking this signaling pathway and the dissemination of ovarian cancer.

Methods

In this study we hypothesized that dithiophosphate modified AXL-aptamers lead long-lasting bio-disponibility and high stability, resulting in decreased migration, and proliferation. We synthetized and screened a library of 17 aptamers that differ in the position of the modification inside the sequence.

Results

Among these 17 dithiophosphate aptamers, we selected the aptamers that showed better effect on the downregulation of p-Axl. The analysis of p-Axl in ovarian cancer cell lines a specific and strong binding when compared with scramble control. In vitro cell viability, growth, and migration, as well as in vivo therapeutic effectiveness in murine xenograft models, were also assessed following the inhibition of p-Axl in ovarian cancer. The experimental validation identifies significant inhibition of p-AXL, clonogenic ability, and migration. Furthermore, treatment with modified AXL-aptamers lead long-lasting bio-disponibility, high stability and inhibited growth of SKOV3ip1.

Conclusions

These findings identify dithiophosphate modified AXL-aptamers as potential therapeutic tool to target GAS6-AXL signaling pathway in ovarian cancers expressing high levels of this oncogenic protein.

Legal entity responsible for the study

Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA

Funding

National Institute of Health

Disclosure

All authors have declared no conflicts of interest.

Background

Btk is non-receptor tyrosine kinases involved in the activation of signaling pathways responsible for maturation and viability of the cells. It plays an important role in the development of B-cell tumors, activating antiapoptotic pathways. Btk has previously been reported to be overexpressed in prostate cancer which correlated with cancer grades. Colorectal cancer is among the five most frequent causes for cancer-related deaths in Europe. This kind of cancer often accompanied by anemia which is treated with erythropoietin supplement. The aim of this study was to assess the effects of combination therapy with erythropoiethin beta (Epo) and LFM-A13 (Btk inhibitor) on colorectal carcinoma cells both in in vitro and in animal models.

Methods

DLD-1 and HT-29 human colon adenocarcinoma cells were cutured in medium with Epo and LFM-A13. Cell proliferation was measured with an automated cell counter. Expression of Btk by Western Blotting and Akt mRNA by RT-PCR, and its protein was assessed and confocal microscopy, respectively. Analysis of apoptosis by flow-cytometry was also carried out. Nude mice were inoculated with adenocarcinoma cells and treated with Epo and LFM-A13.

Results

Herein, we found that simultaneous use of Epo and LFM-A13 exert an additive inhibitory effect on colon cancer cell growth. Featured therapeutic scheme resulted in effective cell killing, accompanied by attenuation of Btk, Akt signaling pathway and increased of apoptosis. This combination is also effective in vivo studies, where the combined administration of Epo and LFM-A13 significantly reduces the rate of growth of tumor cells and leads to the complete regression of the colorectal cancer cells.

Conclusions

Results of this study show that that adding Epo significantly enhances the antitumor activity of LFM-A13. The results of our study indicate the potential use of a combination of Epo and LFM- A13 as an effective therapeutic approach for colorectal cancer.

Legal entity responsible for the study

Justyna Magdalena Hermanowicz

Funding

National Science Centre, Poland No. DEC-2017/01/X/NZ5/00362

Disclosure

All authors have declared no conflicts of interest.

Background

Due to the adverse side effects of current cancer chemotherapeutics development of targeted anti-cancer medications is under intensive focus. In the present study a fusion protein consisting of the catalytic and translocation domains of diphtheria toxin fused to BR2, a cancer specific cell penetrating peptide, was produced as a new targeted anti-cancer agent and its cytotoxic effects on various cell lines including MCF-7 and HeLa (as cancerous), and HEK293 and HUVEC (as normal) cell lines was evaluated.

Methods

DT386-BR2 protein was expressed in E. coli cells followed by protein purification using affinity chromatography and authenticated by SDS-PAGE and Western blotting. Next, the purified protein was subjected to evaluation of its cyto-lethal effects on the mentioned cell lines at various concentrations. DT386 and BR2 alone were also produced and used as negative controls. In this regard, MTT assay followed by flow cytometry were used for cytotoxicity assessment and prediction of induced cell death mechanism.

Results

SDS-PAGE and Western blotting confirmed expression of DT386-BR2 fusion protein by revealing a band of about 47kDa. In case of the cancerous cell lines, i.e. HeLa and MCF-7 cells, significant reduction in survival percent was shown when compared to the controls. However, no significant cytotoxicity was observed in case of the normal cell lines, HUVEC and HEK293 cells. In addition, DT386 and BR2 alone did not show any significant cytolethal effects. The IC50 of DT386-BR2 for HeLa and MCF-7 was about 0.55 and 2.08 μg/ml, respectively. Furthermore, flow cytometry revealed dose dependent apoptosis induction by DT386-BR2 after 12 hrs.

Conclusions

This chimeric protein showed promising cancer cell specific cytotoxicity and might be considered as a new drug candidate for treatment of some types of cancer. Further studies regarding non-specific cytotoxicity and in vivo animal and preclinical studies of this protein are undergoing.

Legal entity responsible for the study

Isfahan University of Medical Sciences

Funding

Research Deputy of Isfahan University of Medical Sciences

Disclosure

All authors have declared no conflicts of interest.

Background

Besides significant progress in the field of cancer therapy, breast cancer remains the major ongoing health problem among the women worldwide because of the side effects of available drugs. This problem can be overcome by loading herbal extract into the polymeric nanoparticles that will aid in site-specific drug delivery and increase in the retention time of drug, thereby improving therapeutic efficacy.

Methods

In the present study, the anti-cancer activity of nanoparticles loaded with extract of Artemisia absinthium (ANPs) was evaluated in breast cancer cell lines (MCF-7 and MDA MB-231). Identification of the plant was done by employing HPTLC. NIPAAM-VP-AA nanoparticles (NPs) were synthesized by the free radical mechanism. Cellular uptake study was performed by confocal microscopy. The size of NPs was determined by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The cytotoxicity of ANPs was evaluated by apoptotic assays like MTT assay, AnnexinV/FITC apoptosis assay, propidium iodide (PI) staining, DNA degradation assay and DAPI nuclear staining.

Results

The active compounds of the plant viz. artemisinin, artemisinic acid, and alpha-thujone showed the Rf value of 0.52 ± 0.02, 0.32 ± 0.02 and 0.48 ± 0.01, respectively. Among the different plant parts used for extract preparation, the whole plant extract displayed maximum cytotoxicity with least IC₅₀ value (≈300µg/µL for both cell lines). Therefore, whole plant extract was loaded into the polymeric NPs. Using DLS and TEM the average particle size of synthesized NPs was found to be 130 nm and 80 nm, respectively. Confocal imaging of rhodamine B tagged NPs showed significant uptake of NPs by the cells. The apoptotic assays showed a significant apoptosis in extract loaded NPs in comparison to extract alone whereas void nanoparticles do not significantly affect the cell survival.

Conclusions

The results obtained reflect the significant efficacy of extract loaded NPs in inducing apoptosis in breast cancer cells even at a very low drug concentration used. Thus, the synthesized ANPs can be used for further in vivo studies and clinical research which may prove to be of immense therapeutic utility in breast cancer treatment.

Legal entity responsible for the study

Dr. Saima Wajid

Funding

Has not received any funding

Disclosure

All authors have declared no conflicts of interest.

Background

NSCLC patients undergoing platin chemotherapy often develop resistance within 9-12 months and drug-resistance is the primary reason for the low median survival of 10-14 months among stage IV patients. Therefore, resistance is the fundamental therapeutic limitation in the later part of the progressive disease. Researchers showed that suppressed Caspase-3 is one of the primary reasons for failure to induce apoptosis by the cell during cisplatin resistance. Recently, AXL and TWEAK/FN14 pathways have been shown to be upregulated in cisplatin-resistant cells causing activation of EMT and survival pathways. Therefore, we examined whether targeted dual inhibition of AXL and FN14 successfully reverses the cisplatin resistance in NSCLC.

Methods

We tested cisplatin-resistant EGFR mutant H1975 and KRAS mutant A549 cells following knockdown of AXL and FN14. We used gelatin nanoparticles conjugated(gelNP) with AXL and FN14 siRNAs and cetuximab as a targeting agent. We compared efficacy of gelNP with small molecule AXL and FN14 pharmacological inhibitors. Mice (n = 27) were divided into four groups and treated with cisplatin, AXL/FN14 inhibitors, and gelNP along with controls. We investigated cellular toxicity(IC₅₀) using MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye assay and mechanism using western blot. Cytochrome-C induced apoptosis was detected using fluorescence microscopy.

Results

There was a marked decrease in average tumor volume, both in pharmacological and genetic inhibitor (gelNP) treated groups. IC₅₀ values decreased significantly in dual inhibition (<200%). Mutual regulation of AXL and FN14 were observed, presumed through src-RhoA-ROCK pathways. Although in-vitro AXL suppression showed increase in EGFR, dual inhibition reversed the trend. During dual inhibition decreased p-src, p-50, p-AKT, and survivin, and increased FHIT, cleaved caspase 3 and cleaved PARP levels were observed. Cytochrome c release further confirmed intrinsic apoptosis.

Conclusions

Our results show that targeted dual inhibition of AXL and TWEAK/FN14 using targeted therapy by gelNP can reverse cisplatin resistance by inducing higher caspase 3 cleavage through FHIT upregulation that can enhance survival of NSCLC patients undergoing chemotherapy.

Legal entity responsible for the study

University of Missouri-Columbia, USA

Funding

Michael J and Sharon R Bukstein Endowment funds

Disclosure

All authors have declared no conflicts of interest.

Background

Certain chemotherapy and radiotherapy regimens induce Immunogenic Cell Death (ICD) in tumor cells, contributing to the control of tumors by T cells. Markers of ICD includes HMGB-1 and ATP, expression of phosphatidylserine and Calreticulin (CRT) on the surface of apoptotic cells. ICD promotes the maturation of dendritic cells (DC) and the further activation of CTLs. This work was oriented to the implementation of an in vitro screening system of tumor cells, to induce ICD for their possible use in the immunotherapy of cancer in the future.

Methods

Tumor lines (REH, CRL2338 (Her2/neu +++) and KATO III) were treated in vitro with various agents used in antitumor therapy (Carboplatin, Gemcitabine, Doxorubicin, Oxaliplatin, Cyclophosphamide, and Paclitaxel). We established the in vitro conditions (time and concentration) of the treatments to characterize the type of death induced by these agents.

Results

ICD induction in the tumor cells were examined by the capacity of treated tumor cells to elicit the maturation of DC and expansion of activated CTLs. The treatment with different agents evidenced various degree of susceptibility of these cells to ICD inducers. These variations were reflected by different activation profiles of Caspases 3,7 and 8. In some tumor cell types, we detected an alteration of the mitochondrial membrane potential status and alteration of the cell cycle. Finally, we observed endoplasmic reticulum stress; and expression of Annexin V, CRT, HMGB-1 and ATP and others like ROS, NF-kB, COX-2, PPAR-y. The evaluation of immune recognition of treated cells showed an important role in the activation of the innate and adaptive response. This was evidenced by the stimulation of DCs such as phagocytosis, homing, production of Il-12 and cross-presentation to CD8+ T cells as well as activation of CD8 T lymphocytes specific for tumor antigens.

Conclusions

In summary our results led us to conclude that induction of ICD in vitro in tumor cells emerges as a useful tool for the selection of tumor cells as possible vaccines against different tumors that efficiently stimulate the functions of APCs and the activation of antitumor CTLs.

Legal entity responsible for the study

Universidad Nacional de Colombia

Funding

Universidad Nacional de Colombia y Fundación Salud de los Andes

Disclosure

All authors have declared no conflicts of interest.

Background

Ovarian cancer (OC) is the second most common gynecologic malignancy, but its mortality ranks the highest in the world. Pim1, belongs to a group of constitutively activated serine/threonine kinases, has been reported in many types of cancer. Little is known about Pim1 in OC.

Methods

The protein expression of Pim1 was verified from the human protein atlas (www.proteinatlas.org), as well as ovarian cancer cell lines, and its association with survival were then analysed by bioinformatic analysis. CCK-8 assay and colony formation were used to measure cell proliferation. In order to evaluate the mechanism of Pim1 in HGSOC, Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) and Lactate analysis were performed to find that Pim1 maintains Warburg effect via c-Myc-glycolysis signaling axis. In Vivo subcutaneous xenograft inoculation was also performed to certify its role.

Results

Silencing/overexpressing of Pim1 suppressed/promoted OC cells proliferation in vitro. Pim1 significantly influenced glycolysis by OC cells and was associated with p-c-myc protein levels and subsequent c-myc regulated key enzymes (such as PGK1, LDHA) in the glycolytic pathway. Pim1-knockdown also inhibited ovarian tumor growth in vivo. Moreover, Pim1 inhibitor SMI4a exerted chemosensitizing effects on cisplatin. Pim1 was also overexpressed in ovarian cancer and correlated with the poor overall survival of patients by bioinformatics analysis.

Conclusions

Together, these results suggest that Pim1 contributes to OC proliferation and glycolysis through p-c-myc axis, which may serve as a potential target for OC patients.

Legal entity responsible for the study

Fudan University Shanghai Cancer Center

Funding

Has not received any funding

Disclosure

All authors have declared no conflicts of interest.

Background

In recent years, tryptophan degradation has received increasing attention as a potent immunosuppressive mechanism involved in the maintenance of immunological tolerance in both tumor draining lymph nodes and tumors. Indoleamine 2,3-dioxygenase 1 (IDO1) is a tryptophan metabolizing enzyme and its mechanisms of action as an immune regulator involves tryptophan deprivation, production of immunosuppressive metabolites (kynurenines), and activation of signaling events through binding of tyrosine phosphatases (SHPs). IDO1 is chronically activated in many cancer patients and its expression and enzyme activity correlate with a poor prognosis in patients with various cancers. In the past, IDO1 inhibition was mostly achieved using the racemic mixture of 1-D,L-methyltryptophan (1-MT). As it became apparent that IDO1 inhibition may be a promising target for cancer therapy, the individual stereoisomers of 1-MT were investigated in more details. 1-L-MT was shown to more effectively inhibit IDO1 in enzyme assays and in cancer cell lines. However, 1-D-MT showed superior anti-tumor activity in mouse models and was therefore chosen for clinical trials. 1-D-MT is currently being tested in phase II clinical trials (indoximod) as an adjunct to conventional chemotherapy, although its immunostimulatory mechanism is still unknown. We here investigated whether 1-D-MT could interfere with IDO1 signaling rather than catalytic activity.

Methods

The enzymatic activity of IDO1 was measured in vitro in terms of the ability of both purified protein or stably transfected cells to metabolize tryptophan to kynurenine. In these cells we also evaluated IDO1 protein turnover and its ability to bind SHPs through both FRET analysis and immunoprecipitation.

Results

1-D-MT did not inhibit IDO1 catalytic activity, but affected IDO1-SHPs binding.

Conclusions

Our data indicated that 1-D-MT anticancer activity may rely on the inhibition of IDO1 signaling but not catalytic activity.

Legal entity responsible for the study

University of Perugia

Funding

This work was supported by the European Research Council (338954- DIDO to U. Grohmann).

Disclosure

All authors have declared no conflicts of interest.

Background

Indoleamine 2,3-dioxygenase (IDO1), a tryptophan catabolizing enzyme, is recognized as an authentic regulator of immunity in several physiopathologic conditions. In cancer, IDO1 can either be expressed directly by the tumor cells themselves, or induced indirectly in host antigen presenting cells by the tumor and its expression has been associated with a worse clinical outcome in a variety of cancers, such as melanoma, ovarian cancer, and colorectal cancer. This consideration has driven to the definition of IDO1 as an investigational immune checkpoint target and to the development of several IDO1 inhibitors, some of which have entered clinical evaluation. Its mechanisms of action as an immune regulator, is composite and involves tryptophan deprivation and production of immunosuppressive metabolites (kynurenines). We recently demonstrated that IDO1 also acts as a signal-transducing molecule, independently of its enzymic function. In particular, in a microenvironment dominated by TGF-β, we found that IDO1 is involved in intracellular signaling events responsible for the self-amplification and maintenance of a stably regulatory phenotype in plasmacytoid dendritic cells (pDCs), a DC subset. In the literature, IDO1 has been described as a protein with a cytoplasmic localization. However, no thorough analysis of modifications of this localization in different conditions has been performed so far.

Methods

We investigated the intra-cellular localization of IDO1 by means of confocal microscopy and western blot analysis of sucrose isopycnic gradient.

Results

Besides confirming the main cytoplasmic localization of the protein IDO1, we detected the presence of a significant amount of IDO1 in subcellular structures, corresponding to early-endosomes, especially when it acts as a signal-transducing molecule.

Conclusions

Thanks to these data, it is possible to assume the enzyme IDO1 has not exclusively a cytoplasmic localization, but there could be a balance between its localization in the cytosol and early endosomes, possibly related to two distinct IDO1-mediated (catalysis vs. signaling) tolerogenic mechanisms.

Legal entity responsible for the study

University of Perugia

Funding

This work was supported by the European Research Council (338954- DIDO to U. Grohmann)

Disclosure

All authors have declared no conflicts of interest.

Background

Cancer chemoprevention by dietary phytochemicals is particularly attractive for their potential low toxicity and for their ability to modulate a plethora of signal transduction pathways in biological processes associated with cancer. Olive oil, a major component of the Mediterranean diet, is an abundant source of phenolic compounds. Olive oil production is associated with the generation of waste material, termed ‘olive mill wastewaters’ (OMWW), that have been reported to be enriched in polyphenols. Here we investigated whether the use of purified extracts from OMWW (A009) might be effective in exerting chemopreventive activities in three different prostate cancer (PCa) cell lines (PC-3, DU-145, LNCap) in vitro.

Methods

The SANIST platform, based on the Surface-Activated Chemical Ionization/Electrospray Ionization mass spectrometry (SACI/ESI-MS) was used to determine polyphenol content A009. Chemopreventive activity of A009 was tested by proliferation assays and functional study for cell adhesion, migration and invasion. Molecular and biochemical studies were performed to investigate signalling involved in chemopreventive properties of A009, by real-time PCR and western blotting.

Results

Mass spectrometry analysis revealed that hydroxytyrosol (HyT) was the major component of A009 our extracts and used as a reference compound to test A009 chemopreventive properties in vitro. A009 significantly reduced PCa cell viability up to 96 hours in all cell lines investigated, in a similar manner that HyT. A009 inhibited PCa cell adhesion, migration, invasion and sprouting. Molecularly, we found the PCa cell line treatments with the A009 purified extracts resulted in inhibition of the IL-6/STAT3 pathway, along with reduced secretion of immune suppressive (IL-10) and pro-angiogenic (CXCL8, CXCL12) factors.

Conclusions

Our results suggest that A009 extracts show promising chemopreventive properties on PCa in vitro and support the idea of repositioning a waste-derived material for nutraceutical employment, with environmental and industrial cost management benefits.

Legal entity responsible for the study

N/A

Funding

Azienda Agricola Fattoria La Vialla, Castiglinfibocchi, Arezzo, Italia

Disclosure

All authors have declared no conflicts of interest.

Background

Cancer chemotherapy is still hampered by clinical failures due to multi-drug resistance (MDR) of tumor cells. In the present study, we have investigated the cytotoxicity of 20 methanol extracts from 10 medicinal plants against the sensitive leukemia CCRF-CEM cells. The most cytotoxic extracts were then further tested on a panel of 8 human cancer cell lines, including various MDR phenotypes.

Methods

The cytotoxicity of the 20 methanol extracts from 10 Cameroonian medicinal plants was determined using a resazurin reduction assay. Meanwhile, flow cytometry was used to measure cell cycle, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS).

Results

In the preliminary assay using CCRF-CEM cells, 12 extracts from five plants displayed IC50 values below 80 μg/mL, namely Albizia adianthifolia, Alchornea cordifolia, Alchornea laxiflora, Pennisetum purpureum, and Spathodea campanulata. the four best extracts were from two plants: Albizia adianthifolia roots (AAR) and bark (AAB) as well as Alchornea cordifolia leaves (ACL) and bark (ACB) had respective IC50 values of 0.98 μg/mL, 1.45 μg/mL, 8.02 μg/mL and 12.57 μg/mL in CCRF-CEM cells. They were further tested in 8 other cell lines as well as in normal AML12 hepatocytes. IC50 values ranging from 2.71 μg/mL (towards glioblastoma U87MG.ΔEGFR cells) to 10.30 μg/mL (towards breast adenocarcinoma MDA-MB-231-BCRP cells) for AAB, from 3.43 μg/mL (towards U87MG cells) to 10.77 μg/mL (towards colon carcinoma HCT116 (p53−/−) cells) for AAR and from 0.11 μg/mL (towards CCRF-CEM cells) to 108 μg/mL (towards leukemia CEM/ADR5000 cells) for doxorubicin (as control drug) were obtained. ACL and ACB extracts displayed selective activities. AAR and ACL extracts induced apoptosis in CCRF-CEM cells, through caspases activation and loss of MMP, while apoptotic cell death was mediated by MMP diruption and increase ROS production for ACL.

Conclusions

Some of the tested plants namely Albizia adianthifolia, Alchornea cordifolia, Alchornea laxiflora, Pennisetum purpureum, Spathodea campanulata represent a potential source of novel anticancer drugs. Especially, Albizia adianthifolia and Alchornea cordifolia revealed considerable cytotoxic activities that could be exploited to develop phytomedicines to fight cancers including MDR phenotypes.

Legal entity responsible for the study

Ministry of Scientific Research and Innovation (MINRESI) University of Yaounde I

Funding

Has not received any funding

Disclosure

The author has declared no conflicts of interest.

Background

Terpenoids and their potential analogues have attracted recent attention to their anti-cancer activity with lower adverse effects. Acetyl-11-keto-boswellic acid (AKBA) is a derivative of boswellic acid that exerts anti-cancer properties against different types of cancer cells. AKBA is known to induce apoptosis via activation of caspase 8 and also induction of epigenetic pathways via regulation of histone deacetylase gene expression. However, molecular targets and pathways are not identified. In this study we implemented in-depth non-labelled proteomic profiling of MCF-7 breast cancer cells treated with AKBA.

Methods

Breast cancer cell line MCF7 was treated at IC50 dose of AKBA. Protein lysates from treated and untreated cell lines were purified and digested with trypsin. Isolated peptides were subjected to non-labelled proteomic profiling using Orbitrap lC-MS using service facility. Proteomic signal ratios were normalized using variance stabilizing normalization and corrected for false detection ratio.

Results

A total of 137 proteins were significantly up-regulated or downregulated. A set of mitochondrial proteins were downregulated and set of autophagy pathway proteins were up-regulated. Further confirmation of mitophagy pathway was studied using western blot of the SQTRM, PLINK, and PINK-1 proteins. Similarly, mitochondrial contents were measured to confirm mitochondrial degradation. To assess if mitochondrial loss is due to the autophagy pathway, we performed dual staining for LC3 and mitochondrial markers.

Conclusions

Our results show for first time that AKBA induced apoptosis via mitophagy pathway which explains previously known AKBA induction of caspase-8 pathway. AKBA provides a novel therapeutic agent that is capable of induction of apoptosis in cancer cells with less toxicity to normal cells.

Legal entity responsible for the study

Sultan Qaboos University

Funding

The Oman Research Council

Disclosure

All authors have declared no conflicts of interest.

Background

Pancreatic cancer is the fourth leading cause of cancer death, with an overall 5-year survival rate of <10%. These statistics have not changed in almost 50 years. Chemotherapy-resistance is a major barrier for successful therapy of pancreatic duct adeno carcinoma (PDAC). Resistance may arise de novo or is acquired upon intensified chemotherapeutic regimens. Hence, a better characterization of the underlying mechanisms and the identification of new drug targets are a therapeutic necessity.

Methods

Primary pancreatic cancer cells were isolated from CKP (Ptf1a+/Cre, Kras+/LSL-G12D, P53loxP/loxP) mouse and cultured with increasing concentrations of gemcitabine to induce gemcitabine-resistant cell lines. The resistance status was determined by dose-response experiments, calculating the half maximal inhibitory concentration (IC50) of gemcitabine using cell titer assays. Alterations in the Pi3k-akt pathway were identified by genome-wide analyses and verified by Western blot.

Results

Our data indicate that the pi3k-akt pathway is over-activated in gemcitabine-resistant cells. Accordingly, the gemcitabine-resistant cells were more sensitive to pi3k inhibitors than gemcitabine-naïve cells cultured in parallel. Besides the pi3k pathway, several key genes controlling energy metabolism and proliferation were also significantly altered in the gemcitabine-resistant cells as compared with controls.

Conclusions

Gemcitabine treatment might have unwanted side effects by triggering the pi3k-akt pathway in treatment-induced resistance. In order to prevent or overcome this development, a parallel application of pi3k-akt inhibitors along or subsequent to gemcitabine plus abraxane may constitute a meaningful adjuvant treatment strategy. This hypothesis needs to be tested in further detail.

Background

Cancer burden as a worldwide health issue arises from its increasing incidence together with depletion of efficacious treatment modalities. The current available treatments for cancer are surgery, radiotherapy and chemotherapy. The pharmaceutical industry is adopting a new paradigm shift towards a newer class of peptide-based drug that is expected to overcome the drawbacks of the conventional cancer therapeutics by offering more selectivity, easier synthesis, a wider safety profile and a lower cost of manufacture. The interest in peptides as anticancer agents began in the last few decades owing to their intrinsic properties, such as cationicity and small size, enabling them to compete as an efficacious anticancer agent.

Methods

In this study, we used data from the publicly available databases of anticancer peptides (ACPs) and the Red Sea metganomic database, created during AUC/KAUST Red Sea microbiome project. The project was done in two phases; in silico analysis followed by in vitro validation of the computational results. In silico analysis of our metagenome database resulted in a set of peptide hits that share similar composition to ACPs. One potential ACP hit was submitted for further computational prediction of its structure and function. Then, its sequence was chemically synthesized for subsequent in vitro functional assessment through cytotoxicity (MTT) assay, apoptosis/necrosis detection assay (Annexin/PI assay) and RNA expression analysis of Caspase 3. We used HepG2, U2OS, MCF7 and Hela as model cancer cell lines for testing its cytotoxicity.

Results

The peptide showed evident, yet variable, dose dependent cytotoxicity in all tested cell lines. Membranolysis and Apoptosis could be concluded as possible mechanisms of action from the results of annexin assay and RT-PCR.

Conclusions

Our results, despite being consistent and in line with each other, more investigative techniques should be done for elucidation of the molecular mechanism of action of our anticancer peptide lead.

Legal entity responsible for the study

American University in Cairo

Funding

American University in Cairo

Disclosure

All authors have declared no conflicts of interest.

Background

Triple-negative breast cancer (TNBC) is an aggressive disease, with poor therapeutic alternatives, whose incidence and mortality seem to be increased by smoking habit. In line with our previous evidence (Di Giacomo et al. Food Chem Toxicol 2018,111:393), we evaluated the antitumor properties of the natural sesquiterpene β-caryophyllene (CRY) in triple negative breast cancer as a possible novel targeted therapeutic strategy. Particularly, its effect on the molecular pathways of STAT3 and IL-8, and the involvement of cannabinoid and β2-adrenergic systems, whose control on cell proliferation has been reported, were studied.

Methods

Cytotoxicity of CRY was evaluated in triple negative MDA-MB-468 breast cancer cells, also after pre-treatment with CB1-, CB2- and β2-adrenergic receptor antagonists (AM281, AM630 and ICI 118.551). To study the ability of CRY to interfere with the pro-carcinogenic effects of tobacco smoke, cells were treated with a condensed sample of cigarette smoke (CSC). The levels of STAT3/ERP57, IL-8 and ROS were measured. At last, we looked at the expression of BIRC5, MMP2 and TPX2 genes, known to regulate cancer progression and metastatization.

Results

CRY was found able to significantly inhibit the growth of MDA-MB-468 cells: the effect was slightly reduced by AM630, while a remarkable inhibition by ICI 118.551 was displayed. CRY also inhibited the activation of STAT3 pathway and the pro-oxidant effects of CSC. IL-8 level resulted significantly reduced by CSC, while the basal level was restored by CRY. Furthermore, a remarkable inhibition of CSC-induced cell migration and BIRC5, MMP2 and TPX2 gene expression was found.

Conclusions

Data obtained highlight antiproliferative properties of CRY in triple negative breast cancer, with a possible β2-adrenergic system contribution. CRY acts partly by a CB2- activation, although other CB1/CB2 independent mechanisms appear involved. CRY also interferes with smoking-induced progression and metastatization of breast cancer cells: this effect can be approached as a chemopreventive strategy in breast oncologic patients. Present results encourage further studies on CRY as chemopreventive agent or in targeted supporting therapies for breast cancer.

Legal entity responsible for the study

Department of Physiology and Pharmacology, Sapienza University of Rome

Funding

Sapienza University of Rome (University Projects 2016 and 2017)

Disclosure

All authors have declared no conflicts of interest.

Background

Breast cancer (BC) is the most commonly diagnosed cancer in women worldwide. The progression, metastasis and drug resistance of breast cancer cells were suggested to be driven by a special population within the tumor called cancer stem cells (CSC). Targeting CSC is extremely challenging due to the high expression levels of ATP-binding cassette (ABC) transporters which facilitates the multi drug resistance (MDR) capacity of CSC. The aim of this work was to test whether Energy restriction mimetic agents (ERMAs) as OSU-CG5 can counteract cancer multidrug resistance by limiting the availability of energy in CSC. In addition to exploit the mechanistic synergy between OSU-CG5 and a conventional chemotherapeutic agent as Doxorubicin.

Methods

Doxorubicin-resistant breast cancer cells (MCF-7 and MDA-231 cell lines) were generated by the exposure to increasing concentrations of doxorubicin. The degree of resistance was assessed by MTT cell viability assay and immunofluorescence. In addition, the alteration in the expression of ABC transporter proteins was determined by Western blot. The induction of CSC was assessed by flow cytometry looking at the expression of breast CSC markers CD44 and CD24. The antitumor effect of OSU-CG5 and/or doxorubicin, in addition to the mechanistic synergy between them was determined by MTT assay, caspase3/7 assay and Western blotting.

Results

The generated doxorubicin-resistant breast cancer cells (MCF-7 and MDA-231 cell lines) showed less sensitivity to doxorubicin and an increase in breast cancer CD44⁺/CD24^low cells, in addition to increased expression of ABC proteins. This stem cell enriched population was declined and the anticancer effect of doxorubicin was significantly synergized by its combination with OSU-CG5, suggesting that ERMAs preferentially inhibit stem/progenitors in breast cancer cells.

Conclusions

The results suggested that targeting breast CSC population by ERMAs could be a rational strategy to minimize their multi-drug resistance, and the combination with classical chemotherapeutic agent as doxorubicin may represent a clinically relevant strategy for cancer treatment that ultimately lead to new approaches to improve the survival of cancer patients.

Legal entity responsible for the study

University of Sharjah

Funding

Al Jalila Foundation, United Arab Emirates (Grant number AJF201610)

Disclosure

All authors have declared no conflicts of interest.

Background

Paclitaxel (PTX) is an essential component of the first-line treatment in breast cancer (BC). However, the conventional PTX formulation currently used has been associated with many dose-limiting toxicities. mTOR inhibitors, such as everolimus (EVER), were found to increase sensitivity to PTX in BC. Importantly, administering PTX and EVER at a synergistic ratio could allow for reducing the dose and toxicities of PTX. BC tumors co-expressing HER2 and EGFR were shown to have poor prognosis and reduced survival compared to other BC subtypes. Therefore, this research aims to develop a dual-targeted polymeric nanoparticle (NP) formulation encapsulating PTX and EVER at the synergistic molar ratio to improve cytotoxicity and cellular uptake in BC cells co-expressing HER2 and EGFR.

Methods

The combination of PTX and EVER was evaluated in BC cells and the optimal synergistic ratio was defined. PTX+EVER-loaded NP formulation was successfully prepared. Antibody Fab fragments specific to HER2 and EGFR were prepared by digestion of Trastuzumab (TmAb) and Panitumumab (PmAb), respectively. The cytotoxicity and cellular uptake of untargted-NPs, TmAb(Fab)-NPs, and TmAb(Fab)-PmAb(Fab)-NPs were studies in SKBR3 (HER2 +++/EFGR ++) and MCF-7 (HER2-/EGFR+) BC cells.

Results

The optimal synergistic ratio of PTX and EVER combination was defined at 1:0.5, exhibiting synergy in six BC cell lines. PTX+EVER-loaded NPs were spherical with less than 100 nm in diameter, had a total drug loading of 9% (wt%), and exhibited sustained drug release in vitro under physiological conditions for 168 h. A significant increase in the cyototoxicity and cellular uptake was observed for TmAb(Fab)-PmAb(Fab)-NPs relative to TmAb(Fab)-NPs and untargeted-NPs in SKBR3, while this increase was insignificant in MCF-7.

Conclusions

Co-encapsulation of PTX and EVER in HER2-EGFR targeted polymeric NPs can lead to a significant increase in the cytotoxicity and cellular uptake in BC cells co-expressing HER2 and EGFR relative to untargeted and HER2-targeted NPs. These data show high potential for a dual-targeted NP formulation of PTX and EVER combination to improve tumor growth inhibition and reduce PTX dose-limiting toxicities in BC tumors co-expressing HER2 and EGFR in vivo.

Legal entity responsible for the study

Leslie Dan Faculty of Pharmacy, University of Toronto

Funding

Canadian Institutes of Health Research (CIHR)

Disclosure

All authors have declared no conflicts of interest.

Background

Vascular targeted photodynamic therapy (VTP) is based on in-situ photosensitization of a circulating drug leading to intravascular reactive oxygen species (ROS) generation and the subsequent tumors’ blood vessel occlusion that lead to the tumor necrosis. We have developed a series of bacteriochlorophyll derivatives, among them WST11 (TOOKAD®-Soluble) a water-soluble agent that has just been granted approval by the European community (EMA) for the treatment of early/intermediate prostate cancer. Application to other cancers, specifically to pancreatic ductal adenocarcinoma (PDAC) is being evaluated and may require adjustments of the treatment conditions. The following work presents the first pre-clinical results using WST11-VTP to treat syngeneic PDAC in mice.

Methods

We utilize KPC cells derived from genetically engineered mouse model of spontaneous pancreatic adenocarcinoma to induce orthotopic PDAC tumor in C57B mice. Cells are injected in the mouse pancreas and are treated 7 days post-implantation. WST11 is injected i.v. by constant rate infusion followed by illumination of the exposed tumor at 755nm. Tumor growth is monitored using a Vevo 3100 US imaging apparatus. In parallel experiments, a window exposing the pancreas blood vasculature through a glass coverslip is positioned in a few mice. This allows live monitoring of the new angiogenic blood vessels during treatment using a fluorescent microscope and speckle imaging techniques.

Results

WST11-VTP of orthotopic KPC tumors was tested using different drug/light doses. Two doses produced 75-80% tumor necrosis with minimal toxicity, namely 5/7 mg/kg WST11 combined with 100/120mW light. One month post-VTP, the surviving mice were sacrificed histopathological evaluations revealed dramatic tumor shrinkage and necrosis in the treated animals. Moreover, the combination of VTP with Gemcitabine seemed to further improve the treatment success.

Conclusions

WST11-VTP showed good safety/efficacy profile while applied to orthotopic KPC tumors in syngeneic mice. The combination with Gemcitabine seem to be beneficial and is being further evaluated. These results might in the future rationalize early clinical trials in PDAC patients.

Legal entity responsible for the study

Weizmann Institute of Science

Funding

Robert and Yaddle Sklare Professiorail Chair in Biochemistry and the Wade Thompson Family foundation.

Disclosure

All authors have declared no conflicts of interest.

Background

Increased levels of Prolactin (Prl) receptors have been found in endocrine dependent cancers e.g. breast and prostate cancer as well as in ovarian cancer and glioblastomas. Prl stimulates cell growth by activating the intracellular JAK-STAT pathway. A new line of research in the last years suggest that human cytomegalovirus (CMV) infection can also contribute to several human malignancies including glioblastoma brain tumor and ovarian cancer. A supportive study, showed that CMV DNA was present in 50% of fresh ovarian carcinoma tissue samples (39 samples). It was suggested that human CMV is able to create a more malignant phenotype of tumor cells by the action of its regulatory proteins and non-coding RNA, which will affect their proliferation, invasion of other tissues, survival and other cellular properties. Furthermore, prolactin receptor expression and circulating prolactin levels have been shown to be higher among women with ovarian cancer vs. benign-condition or healthy control.

Methods

Our preliminary studies showed that an experimental CMV infection of glioblastoma and ovarian cancer cell lines activates Prl and Prl signals. We have generated a high affinity Prl receptor antagonist that can be recombinantly produced in E-coli. This antagonist, prevents dimerization of the receptor and consists of around 200 amino acids (similar molecular weight as Prl).

Results

Using cell-based assays in glioblastoma cell lines we found that Prl receptor antagonists reduce STAT activation resulting in cellular growth retardation. An interesting (unpublished) finding is that the Prl system appear to be correlated to the activity of CMV virus in glioblastoma and ovarian cancer.

Conclusions

This might open a new therapeutic angel of combining anti-viral treatment and PRLRA along with anti-cancer agents.

Legal entity responsible for the study

Sultan Qaboos University

Funding

Has not received any funding

Disclosure

All authors have declared no conflicts of interest.

Background

Gene expression profiles in spheroid cultivated cells are more similar to natural tumors, than profiles of the same cells in monolayer culture. Tumor spheroids are heterogeneous cellular aggregates that, when greater than 500 μm diameter, are frequently characterized by hypoxic regions and necrotic centers. Architecture of three-dimensionally (3D) propagated cells is very similar to avascular tumor areas. The gradient of diffusion in cell aggregates leads to reduced proliferation rates and increased drug resistance. The purpose of the work was to conduct a comparative study between 3D and monolayer growth systems of MCF-7 cells, and prove the value of spheroid model.

Methods

As experimental model was used adhesion line of breast adenocarcinoma MCF-7. Cells in 2D and 3D culture were incubated during 5 days under conditions of starvation. The number of live cells was evaluated using MTT-colorimetric assay. Apoptotic index was assessed by flow cytometry.

Results

MCF-7 cells growth parameters differ significantly in 2D and 3D growth systems. Cells in 2D system are more sensitive to serum starvation then 3D cultures. Cell viability increases dramatically in 3D system. The level of apoptotic and necrotic cells for 2D growth in serum starvation conditions (39.2±7.3% and 33.5±2.8% respectively) were twice increased in comparison with conditions of complete culture medium (19.0±1.3% and 11.4±1.7% respectively), whereas incomplete medium have no detectible effects on 3D cultured cells. However, the 3D cells percentage in G₀/G₁ phase of the cell cycle was increased in 1.6 times in serum free conditions, whereas it was not changed in complete medium that can indicate similarity to natural tumors.

Conclusions

Therefore, the 3D growth system has been proposed as an adequate and valuable model to study tumor growth and response to therapeutic substances.

Legal entity responsible for the study

Taras Shevchenko National University of Kyiv, ESC “Institute of Biology and Medicine”

Funding

Has not received any funding

Disclosure

All authors have declared no conflicts of interest.

Background

Increasing the target delivery of generated dendritic cells (DCs) to the lymphoid tissue of the recipient can significantly improve the efficiency of immunotherapy. This can be achieved by using iron oxide nanoparticles under the action of a magnetic field – a magnetic nanocomplex (MNC).

Methods

60 CBA mice were used for experimental study. As a model of an experimental tumor, sarcoma 37 (Sa 37) was used. 9x10⁵ cells/mouse of Sa 37 cells were injected into the hip. As a source of DCs syngeneic mice splenocytes were used. Mechanically modified lyophilized Sa 37 cells (0.05 mg/ml) and MNC of Fe₂O₃ nanoparticles (8x10⁻¹² g/cell, Sigma-Aldrich) were used for DCs loading. After the introduction of DC, animals were exposed to the magnetic field for 1 hour. The control group of mice was transfected only with Sa 37. DC vaccine therapy was started on the 7th day after tumor transplantation (TT). DC vaccine was injected intradermally 3 times every 3 days. Volume of the primary tumor was determined with three days interval starting from the 10th day after TT. At the 25th day after TT, tumor mass, amount of leukocytes, leukocyte formula of the peripheral blood and absolute number of cells in the lymphoid organs of animals were determined. The expression levels of GAPDH, VEGF-α, IL-10, TGF-β, FOXP3, and Hsp70 genes in tumor cells were analyzed using a quantitative PCR method.

Results

DCs loaded with MNC didn't have significant effect on the hematological and immunological parameters in animals with Sa 37. However, a significant antitumor effect was observed. In DC+MNC group reduction in tumor volume was observed comparing with control (p = 0.022) and DC monotherapy (p = 0.0005). Also, the tumor mass in the DC+MNC group was lower in relation to control (p = 0.028). Probably, inhibition of the tumor growth occurred due to the reduction of the S-phase cell number by 1.6 times. The combined effect of DCs and MNC significantly reduced expression levels of FoxP3, VEGF-α, IL-10 mRNA in tumor cells. FoxP3 expression level decreased by 1.92 times, VEGF-α by 2.94 times (p = 0.0074) and IL-10 (p = 0.0048) compared with the control.

Conclusions

Intradermal introduction of DC-based vaccine loaded with MNC was accompanied by a pronounced antitumor effect in mice with Sa 37.

Legal entity responsible for the study

National Cancer Institute of Ukraine

Funding

Has not received any funding

Disclosure

All authors have declared no conflicts of interest.

Background

E-cadherin (CDH1), a cell adhesion glycoprotein is widely associated with breast cancer where it is involved in tumor growth, invasion and metastasis. Down regulation of its gene is hall mark for epithelial-mesenchymal transition, which is an essential process for tumor metastasis. While various pathways are associated with E-cadherin, one of the prominent signaling pathways is via epidermal growth factor reception (EGFR), which is found to be over expressed. Silencing the over expressed EGFR gene by using a specific short-interfering RNA (siRNA), or inducing the expression of CDH1 gene with the help of a plasmid could improve management of malignant cancers by disturbing cancer cell interactions with adjacent cells and extracellular matrix; however, tumor-targeted delivery of these complexes is a major challenge in cancer therapy. This could be overcome by using pH-sensitive carbonate apatite nanoparticles that can enhance the intracellular delivery and release of the bound DNA or siRNA from endosomes to cytosol, after being transported to the tumor.

Methods

Carbonate apatite nanoparticles were employed to deliver CDH1 plasmid and EGFR siRNA individually as well as in combination into human and murine breast cancer cell lines and also into breast tumors induced in mammary pads of 6-8 weeks old Balb/c mice. While MTT assay was performed for cell viability in vitro, tumor regression study was performed in vivo. For tumor regression, two doses of intravenous (IV) treatments were given. Further, protein expression of treated and untreated in vitro samples was confirmed by Western Blot analysis.

Results

Delivery of carbonate apatite nanoparticles with electrostatically associated plasmid or siRNA was found to reduce the tumor burden in mice. The synergistic effect for combined delivery of plasmid CDH1 and EGFR siRNA led to further reduction in the tumor growth. Western blot analysis confirmed the suppression of downstream signaling pathways following delivery of CDH1 gene and EGFR siRNA.

Conclusions

Simultaneous delivery of plasmid CDH1/EGFR siRNA with carbonate apatite nanoparticles could be a promising option for effectively treating breast cancer.

Legal entity responsible for the study

Monash University Malaysia

Funding

FRGS/1/2016/STG05/MUSM/02/3 granted by Ministry of Higher Education, Malaysia

Disclosure

All authors have declared no conflicts of interest.

Background

Since 2007, oncologists have been using the GeneSearchTM Breast Lymph Node (BLN) Assay for the detection of metastatic breast cancer. This test detects the presence of Mammaglobin-1 (MGB1) expression in lymph nodes of breast cancer patients. Although MGB1 is overexpressed in 90% of breast cancers, its aberrant expression and function in breast cancer tissue is still misunderstood. We have recently reported the first evidence for MGB1 as potent induction of breast cancer malignancy and disease progression. More precisely, loss of MGB1 expression correlates with a decrease of cell proliferation, spheroid formation, migration, and invasion capacities of breast cancer cells. Concomitantly, we also show that MGB1 expression activates pro-malignant signaling cascades leading to epithelial to mesenchymal transitioning (EMT). More interestingly, we have also discovered new MGB1 gene products that result from transcript editing. The objective of this research is to elucidate the role of MGB1 gene products in breast cancer, we set out to investigate and characterize MGB1 isoforms in breast cancer cell processes.

Methods

MGB1 isoforms were identified by 3’and 5’-RACE assays and next generation sequencing using the minion platform. The novel MGB1 gene products were subsequently cloned.

Results

In total, we have identified 7 different MGB1 product variants. We strongly believe that each MGB1 product has a specific cellular role, which leads to breast cancer malignancy and disease progression. To perform loss and gain of function experiments, we are genetic engineering breast cancer models that specifically code and express individual MGB1 variants with and without tracking epitope tags.

Conclusions

This study will elucidate the molecular and cellular mechanism leading to breast cancer progression. MGB1 variants may provide new avenues for therapeutic or prognostic strategies.

Legal entity responsible for the study

N/A

Funding

Mitacs Globalink, Baxter and Alma Ricard, Canadian Institute of Health Research, Beatrice Hunter Cancer Research Institute, New Brunswick Health Research Institute, O'Brien Foundation, Canadian Breast Cancer Foundation, Atlantic Canada Opportunities Agency, Université de Moncton Campus Moncton

Disclosure

All authors have declared no conflicts of interest.

Background

Osteosarcoma is the most common bone tumour and affects younger patients. The combination of chemotherapy with aggressive surgical resection results in survival rates achieving 60%-70% in patients with localized disease. Unfortunately, 30% of osteosarcoma patients present metastatic disease at diagnosis and only 20-30% will become long-term survivors. Therefore, finding new drugs to treat these patients is a research priority. Preclinical and clinical studies suggest involvement of ErbB network aberrations in the aetiology of osteosarcomas. The present study assessed the effect of Afatinib, an irreversible ErbB family blocker, in osteosarcoma cell lines.

Methods

Cell viability was assessed in osteosarcoma cell lines HOS, SJSA-1, Saos-2, U-2OS and MNNG using Afatinib at increasing concentration (0, 0.5, 1, 1.5, 2, 2.5, 3 and 5 μM). Motility was measure by scratch assay and invasion was performed using transwell chamber. Western blot was used to evaluate EGFR/HER2 pathway.

Results

In cell viability assays Afatinib displayed micro Molar (μM) anti-proliferative activity in all cell lines tested; IC₅₀ values (2.03 +/- 0.35) for SJSA-1, (1.62 +/- 0.38) for U-2OS, (1.8 +/- 0.24) for HOS, (2.08 +/- 0.48) for Saos-2 and (1.8 +/- 0.37) for MNNG cell lines. Motility scratch assays revealed that Afatinib is able, in a statistically significant and dose dependent manner, to reduce the width of the scratch after 24 hrs in all cell lines (p < 0.01). The scratch line width decreased by +/-45 and +/-28 microns when treated with 1 μM or 2 μM, respectively. In the Transwell cell invasion assay, Afatinib, at different concentrations was found to inhibit the invasion behavior of the non-metastatic cell line HOS and the metastatic cell line MNNG in a dose-dependent manner (p < 0.05). The number of invasive osteosarcoma cells in Afatinib treated groups was gradually reduced as the concentration of Afatinib was increased. The inhibitory effect of Afatinib on EGFR and HER2 pathway was evaluated in HOS osteosarcoma line. Afatinib showed a significant reduction in EGFR and HER2 phosphorylation and downstream signalling (p < 0.01).

Conclusions

Afatinib shows anti proliferative activity and decreases the migration capacity of osteosarcoma tumours cells lines.

Legal entity responsible for the study

Boehringer Ingelheim RCV GmbH & Co KG, Mexico

Funding

Boehringer Ingelheim RCV GmbH & Co KG, Mexico

Disclosure

M. Cruz Ramos: For this work Boehringer Ingelheim provided afatinib drug for in vitro experiments and partial financial. F. Solca: Works at Pharmacology and Translational Research, Boehringer Ingelheim RCV GmbH & Co KG. All other authors have declared no conflicts of interest.

Background

Adriamycin (ADR) is a potent anticancer drug widely used to treat a variety of human neoplasms. However, its clinical use is hampered because of severe side effects such as cardiotoxicity and heart failure. ADR-induced cardiomyopathy (AIC) has been reported to be caused by myocardial damage and dysfunction through oxidative stress, DNA damage, and inflammatory responses. Nevertheless, the remedy for ADR cardiomyopathy is still not developed. We describe the effect of NAD⁺/NADH modulation by NAD(P)H quinone oxidoreductase 1 (NQO1) enzymatic action on AIC.

Methods

AIC was established by intraperitoneal injection of adriamycin in C57BL/6 wild-type and NQO1 knockout mice. Before and after exposure to ADR, the mice were orally administered dunnione, a substrate of NQO1. Cardiac biomarker levels in the plasma, cardiac dysfunction, oxidative biomarkers, and mRNA and protein levels of pro-inflammatory mediators were determined to compare the cardiac toxicity of each experimental group.

Results

All biomarkers of Cardiac damage and oxidative stress, and mRNA levels of pro-inflammatory cytokines, including cardiac dysfunction were significantly increased in ADR-treated mice. However, this increase was significantly reduced by dunnione in wild-type, but not in NQO1 knockout mice. In addition, a decrease in SIRT1 activity due to a decrease in the NAD⁺/NADH ratio by PARP-1 hyperactivation was associated with ADR-induced cardiotoxicity through increased nuclear factor (NF)-κB p65 and p53 acetylation, whereas an increase in NAD⁺/NADH ratio by NQO1 enzymatic action using dunnione as a substrate recovered SIRT1 activity and subsequently deacetylated NF-κB p65 and p53, thereby attenuating ADR-induced cardiotoxicity.

Conclusions

Dunnione has a cardioprotective effect against ADR-induced cardiomyopathy through NQO1 enzymatic action. Thus, modulation of NAD⁺/NADH by NQO1 may be a novel therapeutic approach to prevent chemotherapy-associated heart failure, including AIC.

Legal entity responsible for the study

N/A

Funding

National Fund

Disclosure

All authors have declared no conflicts of interest.

Background

Targeted delivery of therapeutic agent meets with hurdle of finding target ligand or receptor those are expressed tumor cells in homogenous patterns but not on normal cells. Apoptosis is an event that is programmed in all cells exposed to specific stimuli and this phenotypic event does not vary in patient to patient. In attempt to overcome limitations of conventional targeted therapy, radiation-induced apoptosis-targeted chemotherapy (RIATC) was suggested in our previous study. Caspase-3 cleavable docetaxel prodrug is treated following local administration of low dose radiation in tumor site which induces apoptosis. After initial activation of the prodrug, in-situ amplification of prodrug from expressed caspase-3 boost further activation of the prodrug.

Methods

We designed EMC-KGDEVD-PABC-DCX containing albumin binding maleimide moiety, and docetaxel linked to a caspase-3 cleavable peptide moiety (DEVD). To upregulate caspase-3 expression in the tumor site, we used radiation to induce apoptosis in target tumor site. EMC-KGDEVD-PABC-DCX combined with multiple dose radiation was administered in breast tumor bearing balb/c nu mice. Toxicities profile of EMC-KGDEVD-PABC-DCX were evaluated.

Results

EMC-KGDEVD-PABC-DCX was activated in the tumor site and free docetaxel was relased out to exert cytotoxic effects on neighboring tumor cells, which repetitively induced the expression of caspase-3. We have achieved complete response of 60% and 80% for 1 mg/kg and 3 mg/kg of EMC-KGDEVD-DCX combined with radiotherapy. No severe toxicities were observed in our treatment doses.

Conclusions

Upon activation of the prodrug, free docetaxel can be released out and exert its cytotoxic effect to bystander cancer cells by stabilising microtubule assembly. Therefore, with our novel therapy using peptide-docetaxel conjugate, outstanding tumor killing effect can be exhibited selectively in tumor site with reducing side effects to normal tissues.

Legal entity responsible for the study

Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, and College of Medicine or College of Pharmacy, Seoul National University

Funding

Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, and College of Medicine or College of Pharmacy, Seoul National University

Disclosure

All authors have declared no conflicts of interest.

Background

In this study, we addressed antiproliferative effects of trastuzumab (Herceptin^®) and nimotuzumab (Theraloc^®) in a combined application with EGF on MCF-7 cells. The epidermal growth factor (EGF) receptors have been found to be implicated in the ontology and maintenance of tumor tissues, which has fostered the discovery and development of molecularly targeted anti-EGF-receptor therapies. However, the accessibility of chemotherapeutic preparations and humanized antibodies to tumor cells in the G0 phase of the cell cycle is lower than to the cells of theproliferative pool. Given this, antitumor efficiency can potentially be enhanced by synchronizing cells in the proliferative pool. To explore this opportunity, we added EGF to tumor cells in combination with nimotuzumab and trastuzumab (antibodies against EGF-R typeI and II, respectively).

Methods

We added aforementioned compounds to MCF-7 cells in the exponential and stationary phases of growth. Cytotoxic/cytostatic effects and cell survival were assessed by the MTT assay, cytofluorimetry and counting cells stained with trypan blue.

Results

The cytotoxic/cytostatic effect of nimotuzumab in combination with EGF was 56% relative to control, whereas nimotuzumab alone resulted in 45%. In the stationary phase of growth, application of nimotuzumab+EGF did not reveal any effects. Trastuzumab did not produce any antiproliferative effects in combination with EGF during exponential growth, but this combination led to a 60% cytotoxic/cytostatic effect in the stationary phase. Application of trastuzumab alone resulted in a 17% effect during exponential growth and 23% in the stationary phase of MCF-7 cells growth. Trastuzumab in combination with EGF can inhibit cell migratory activity. This combination leads to twice decreased migration area for 2 D growth mode, and 20% for spheroid mode. Trastuzumab also promotes cells attachment to substrate under the serum starvation conditions. Index of adhesion increases in 1,5 times. The combination of trastuzumab and EGF usage promote twice increased adhesion. Nimotuzumab in combination with EGF doesn’t demonstrate any detectible influence on MCF-7 cells adhesion or migration. All the data were statistically significant at p < 0.05.

Conclusions

Utilization humanized antibodies against the EGF receptor in complex with EGF may be considered as a potentially efficient antitumor combination.

Legal entity responsible for the study

Taras Shevchenko National University of Kyiv

Funding

Has not received any funding

Disclosure

All authors have declared no conflicts of interest.

Background

The advent of different methods for 3-dimensional (3D) cell culture has allowed scientists to address some of the limitations of conventional methods. 3D cell culture systems attempt to mimic the in vivo tumour microenvironment accurately allowing more physiological studies of cancer cell response to treatment. A major component of the tumour microenvironment which has been overlooked during past years of drug discovery and cancer studies is the interstitial fluid pressure (IFP). The aim of this project is to develop novel methods for studying breast cancer cell behaviour in terms of invasion and drug responses in a more physiologically relevant environment.

Methods

The MDA-MB231 breast cancer cell line presents characteristics of the triple negative breast cancer subtype. In the 3D system MDA-MB231 cells were seeded in a dense 80 mg/ml collagen scaffold surrounded by stromal collagen to form an artificial cancer mass (ACM) enabling the invasive properties of cancer cells to be studied. The morphology/metabolic activity of the cells in 2D, 3D, static and dynamic conditions were evaluated using an Alamar Blue assay and confocal microscopy.

Results

The cells showed lower metabolic activity in 3D compared to 2D. Whereas, so far, gene expression levels for epidermal mesenchymal (EMT) markers has demonstrated that the dynamic environment results in altered expression of snail, vimentin and cadherin but does not appear to affect MMP14 which is involved in collagen type I degradation. Furthermore, the initial results, showed that cell grown in 3D ACM which were exposed to ECM (collagen) produced more vimentin protein than the cells cultured as 2D monolayers .

Conclusions

The results obtained in this study have shown that the proliferation rate and drug responsiveness of cancer cell lines varies according to the different microenvironments in which the cells are cultured. Applying flow and the implementation of IFP affects the expression levels of EMT related markers. The next step will be to evaluate this further in the context of protein expression levels and cellular invasion.

Background

Prostate cancer (PC) is one of the leading cause of cancer related deaths in men. PC progression and recurrence following initial treatments possesses mortality threat amongst these patients. Cell cycle re-entry of quiescent cancer cells has been suggested for cancer progression and recurrence. The slow progression of PC allows a window of opportunity for intervention through diet. An inverse association of flavonoids intake and PC development has been demonstrated in epidemiological studies. We hypothesized that citrus peel that is rich in various bioactive compounds including flavonoids may impede cell cycle re-entry by quiescent PC cells.

Methods

Actively proliferating prostate cancer cells (PC-3) were induced into quiescence by contact-inhibition. The cells were released from quiescence by diluting the cells to lower density in the presence of citrus peel extract.

Results

Our study revealed that the experimental quiescent PC-3 progressed from G0/G1 to S phase following release from quiescence. Compared with the 20% reduction of G0/G1 cells upon cell cycle re-entry, only 3% reduction of G0/G1 cells was noted in the presence of citrus peel extract at 48 hours. In parallel, there was a significant decreased in DNA synthesis in PC-3 cells treated with the extract compared to the control as evaluated by EdU incorporation assay. The cell death was not observed in PC-3 cells when tested using Annexin V-FITC assay. Moreover, the compound responsible for inhibiting the cell cycle re-entry was isolated and identified using chromatographic method. Relevant analysis to validate the compound activity is underway.

Conclusions

This study suggests that citrus peel extract is able to inhibit the cell cycle re-entry of PC cells. The recovery and utilization of bioactive compounds from orange peel waste will open an avenue for developing affordable fortifying food products with potential to reduce the risk of cancer recurrence.

Legal entity responsible for the study

The University of Sydney

Funding

The University of Sydney and LangTech International Pty Ltd.

Disclosure

All authors have declared no conflicts of interest.

Background

Radiotherapy is one of the commonly used approaches to treat cancer. The research trend and breakthroughs in radiotherapy is focusing on the high linear energy transfer (LET) radiation like the alpha particles. Thus, the alpha particles based method is an important option of radiotherapy development, and Targeted Alpha Therapy (TAT) is the most important application of using alpha particles in the past decade. This paper highlights the research work done on computational modelling of TAT at Canadian Nuclear Laboratories (CNL).

Methods

Mathematical model is developed to study the blood flow through a two-dimensional vasculature embedded in a solid tumour or a normal cell. A mesoscale modeling (with a length scale of 1 µm – 1mm and a time scale of 1 ns - 1 µs) is conducted in order to gain an understanding of dynamic alpha-immuno-conjugate (AIC)-attached blood flow interaction in vasculature and its influence on AIC motion in the main flow direction and the perturbations/instabilities of the AIC trajectories in the transverse direction. Microdosimetic modeling of TAT using Monte Carlo toolkit to calculate the amounts of the energy deposited in a simplified DNA model of cancer cells and evaluate the absorbed background dose for normal cells. Coupled the computational fluid dynamic (CFD) simulation with the Monte Carlo prediction to evaluate the efficacy of the AIC during transport process of killing tumour cells and the toxicity to the surrounding normal cells. Post-processing large amount of data calculated by high fidelity CFD simulation and microdosimetric analysis and help review the efficacy of the TAT treatment that is invisible in experiment in pre-clinical stage using a powerful visualization tool.

Results

A coupled model based on the Geant4 Monte Carlo micro-dosimetry technique and Computational Fluid Dynamics analysis was established. The transient drug delivery process and background dose to the cells along the pathway were investigated. A mesoscale numerical simulation in a simple 2D capillary was performed to determine the transient toxicity of the Alpha-Immuno-Conjugate to the DNA of a targeted cell.

Conclusions

The paper demonstrates the feasibility of coupling CFD simulations and microdosimetic modeling to evaluate the efficacy of the TAT realistically and accurately.

Legal entity responsible for the study

Canadian Nuclear Laboratories (CNL)

Funding

Has not received any funding

Disclosure

The author has declared no conflicts of interest.

Background

Determining the influence of molecular alterations on pharmacological responses in cancer cell lines from the omic perspective is the logical first step towards making oncology treatment for patients more effective, specific, and targeted. Our established CellMiner (and in development CellMinerCDB (https://discover.nci.nih.gov/cellminercdb/) web-applications provide high-quality, clean, and numerically extensive molecular and pharmacological data for these purposes.

Methods

The CellMiner multiple databases described are described in detail at https://discover.nci.nih.gov/cellminer/datasetMetadata.do. The CellMinerCDB databases and website features are described in detail at: https://discover.nci.nih.gov/cellminercdb/.

Results

CellMiner provides extensive drug and molecular data for the NCI-60 cancerous cell lines, with activity profiles generated by the Developmental Therapeutics Program (). These databases each contain ∼1000 cell lines, and so provide a broader spectrum of cancer types as well as greater numbers within cancer types, and also have overlap with the NCI-60 (cell lines).

Conclusions

The CellMiner/CellMinerCDB web applications empower researchers to take advantage of the individual strengths of these databases in an integrated fashion. Together, they advance the potential of cancer cell line pharmacogenomic data to lay the foundation for, focus and provide validation for both experimental and clinical studies.

Clinical trial identification

none

Legal entity responsible for the study

The National Cancer Institute (NCI).

Funding

Has not received any funding

Disclosure

All authors have declared no conflicts of interest.

Background

Inflammatory reactions play an important role in all stages of tumor development. A shift to the mesenchymal phenotype causes an increase of migratory capacity of tumor cells. Epithelial-mesenchymal transition (EMT) can also be caused by local inflammation. Searching for factors that can inhibit the transition of the cell population from the epithelial to the mesenchymal phenotype is very important for antitumor therapy. Interferon alfa (IFNα-2b) can be such a factor that has a direct effect on proliferation, differentiation and migration of tumor cells. The aim of this study was to compare the effect of IFNα-2b on the expression of EMT markers and on the basic cellular processes on 2D and 3D growth models.

Methods

2D cell culture was cultured in standard conditions with DMEM nutrient medium (Sigma, USA). The initial cell density was 2 × 104 cells/cm². Concentrations of IFNα-2b were 103, 104 and 105 M/mL. For the initial generation of spheroids to DMEM nutrient medium 2% carboxymethylcellulose was added (Bio-Rad, USA). The spheroid culture was maintained for 7 days on an orbital shaker (PSU-10i, Biosan, Latvia). Every 24 h cells were counted in suspension and adhesion fractions using the routine method. Cell viability was evaluated by MTT assay. The Stemi2000 software AxioVisionRed 4.7 was used for image processing. The volume of spheroids was calculated by Bjerkvig formula. Markers were detected by applying IHC method with primary monoclonal antibodies: Ck (clone AE1/AE3, Dako, USA), vim (Clone V9, Dako, USA), EpCAM (Sigma, USA).

Results

The change of the cell growth type caused a change of the expression of some EMT markers (CKs, EpCAM, Vim). IFNα-2b had a cytotoxic effect on tumor cells, and decreased the migration ability. IFNα-2b caused an increasing of Ck and EpCAM expression by 50.5% and 47.8%, respectively, compared with control in 2D cell culture. In 3D cell culture these increases were 33% and 34%, respectively, compared with the control. Expression of vim significantly showed no difference compared with the control.

Conclusions

IFNα-2b stimulated the differentiation and inhibited a migrational ability of tumor cells on early stages of breast cancer development.

Legal entity responsible for the study

Department of Biotechnical Problems of Diagnostics, Institute for Problems of Cryobiology and Cryomedicine, NAS of Ukraine

Disclosure

All authors have declared no conflicts of interest.

Background

With increasing clinical demands for MEK inhibitors in cancer treatment, overcoming the resistance to MEK inhibitors is becoming an important issue. Especially, the activation of PI3K-Akt pathway is well known to impede the sensitivity to MEK inhibitors. Indeed, a number of early-phase clinical studies, which test the combination efficacy of MEK and PI3K dual inhibition, have been performed; however, the results of these studies remain unsatisfactory. Thus, feasible combination therapies with MEK inhibitors are required to inhibit PI3K-Akt signaling. Mavalonate pathway is not only essential for cholesterol synthesis but also crucial for cell survival with prenylation of small GTPases, which are the upstreams of PI3K-Akt pathway. We thus hypothesized that the blockage of mevalonate pathway using the cholesterol-lowering drugs statins could enhance the efficacy of MEK inhibitors.

Methods

We used two MEK inhibitors: trametinib and CH5126766 (a dual RAF-MEK inhibitor under the phase 2 trials) and two mevalonate pathway blockers: fluvastatin and simvastatin against three cancer cell lines: human breast cancer MDA-MB-231, human melanoma SK-MEL28 and human non-small cell lung cancer A549. Cell growth was analyzed by WST-8 assays and colony formation assays. Apoptotic cells were quantified by flow cytometry. The activation of Akt and the expressions of apoptotic molecules were evaluated by western blotting.

Results

The combined treatment of MEK inhibitors with statins induced significant increases of apoptosis in all cell lines compared to each single treatment. Mechanistically, single treatments of MEK inhibitors increased the phosphorylated Akt, which was suppressed by the addition of statins. Furthermore, the supplementation of mevalonate negated the combinatorial apoptosis.

Conclusions

The present study indicated that the inhibition of the mevalonate pathway suppressed the activation of Akt, resulting in the induction of MEK inhibitor-mediated apoptosis in cancer cells. We propose that statins can be feasible sensitizers for MEK inhibitors. While in vivo studies are needed, the clinical study should be positively considered to examine whether the co-targeting the mevalonate pathway could enhance the efficacy of MEK inhibitors.

Legal entity responsible for the study

Kyoto Prefectural University of Medicine

Funding

Has not received any funding

Disclosure

T. Taguchi: Research expenses from Eisai, Takeda, Daiichisankyo, Fuji film RI pharma, Chugai. All other authors have declared no conflicts of interest.

Background

The therapeutic use of magnetic field effects on intramembranous radical pair mechanisms, a promising new application to liposomal drug-delivery systems (DDSs), will depend to a large degree on technological improvements in the membrane structure of liposomes equipped with various ions and properties. Our previous reports already provided valuable findings about the potential of liposomal DDSs with magnetic controls. In the present research, we used flutamide (FM) as a radical pair-forming drug to investigate whether magnetic fields can control the drug release through changes in the physical properties of membranes.

Methods

For the quantitative analysis of FM-corresponding escape-radicals, we utilized the rate of the disappearance of FM, and the appearance of a cleavage product derived from a FM-corresponding cage product. The relative yields of the escape-radical with magnetic field effects Φ_ER were determined referring to the reported method, based on the following relation:

Φ_ER = Φ_–FM – Φ_UP – Φ_CP

(1)

where Φ_–FM is the FM-photodegradation yield, Φ_UP is the yield related to the radical pair-unrelated photoreaction product, and Φ_CP is the yields related to the radical pair-corresponding cleavage product.

Results

Judging from the overall result of our liposomes prepared in this research, the escape-radical releases obtained using a magnetic field of 0.25 T had not gone as completely as it could have gone. Such differences in the field dependence, we believe, are results of the liposomal membrane packing and the influence of free radicals generated from a cage product derived from a FM-corresponding singlet radical pair, independently of the escaping process of a FM-corresponding triplet radical pair.

Conclusions

We assumed the possibility that the precision of liposomal drug releases with magnetic controls may be obtained by having no bis-allyl proton in its membrane structures. In addition, it may be supposed that at least 0.1 T is needed for detectable magnetic field effects.

Background

Elderly cancer patients aged 65 or more are generally frail compared with younger patients. They are considered to be the age group of less fitting for clinical trials, especially investigating feasibility. For the past decade, the types of the agents have been changing and the number of drug classes has been increasing. The aim of our study was to investigate the impact by age on dose-limiting toxicities (DLT) in each type of agents.

Methods

Retrospective analysis was implemented on patients who participated in phase 1 oncology trials between 1995 and 2016 in our institution. DLT was defined in each trial protocol. The rate of DLT was compared between younger patients (< 65 year) and elderly patients (≥ 65 year) for cytotoxic agents and molecular targeted agents.

Results

In this period, 973 patients underwent 214 enrolments in trials of cytotoxic agents and 996 enrolments for molecular targeted agents. For cytotoxic agents, 165 enrolments with 23 DLT events (13.9%) occurred in the younger group and 49 enrolments with 13 DLTs (26.5%) in the elderly group (p = 0.05). With molecular targeted agents, 728 enrolments with 57 DLTs (7.8%) occurred in the younger group and 268 enrolments with 31 DLTs (11.6%) in elderly group (p = 0.08).

Conclusions

In phase 1 trials, enrolment of elderly patients should be carefully discussed especially in trials of cytotoxic agents. With molecular targeted agents, age groups exert less impact on DLT.

Legal entity responsible for the study

National Cancer Center Hospital

Funding

N/A

Disclosure

S. Iwasa: Grants and personal fees from Eli Lily, personal fees from Taiho, grants and personal fees from Chugai, grants from Novartis, grants from Merck-Serono, grants from Daiichi-Sankyo, grants from Bristol-Myers Squibb, from null, outside the submitted work. Y. Fujiwara: Grants from AbbVie, grants and personal fees from AstraZeneca, grants from Chugai, grants from Daiichi-Sankyo, grants from Eisai, grants from Eli Lilly, grants from Incyte, grants from Merck Serono, grants and personal fees from MSD, grants from Novartis, personal fees from Taiho, grants and personal fees from BMS, personal fees from Ono, outside the submitted work. T. Shimizu: Research expenses from Bristol-Myers Squibb, Daiichi-Sankyo, PharmaMar, FivePrime, 3D Medicine, Takeda-Milleniam, personal fees (lecture fees) from Chugai Pharmaceutical CO., LTD., Taiho Pharmaceutical Co., Ltd., Ono Pharmaceutical Co., Ltd., Ono Pharma Taiwan Co., Ltd., Boehringer Ingelheim, outside the submitted work. N. Yamamoto: Grant from Bristol-Myers Squibb, Chugai, Taiho, Eisai, Lilly, Quintiles, Astellas, Novartis, Daiichi-Sankyo, Pfizer, Boehringer ingelheim, Kyowahakko Kirin, Bayer, Ono Pharmaceutical Co, Ltd and Takeda, other from Chugai., Ono Pharmaceutical Co., Ltd., AstraZeneca, Pfizer, Lilly and Bristol-Myers Squibb outside the submitted work. All other authors have declared no conflicts of interest.

Background

The fundamental principle of precision oncology is the correspondence of molecular, genomic and clinical data with the underlying mechanisms of specific therapeutic agents in order to provide more effective and rational cancer treatment strategies.

Methods

Retrospective study that describes the demographic aspects in a series of 28 consecutive cases of patients of different types of neoplasia screened to Phase I trials, where used a panel of 52 genes Oncomine Focus Assay Panel (ONCOMINE), done through NGS with the simultaneous analysis of DNA and RNA, being able to assess mutations, hotspots, variations in single nucleotide (SNV), indels, variations in the number of copies (CNVs) and gene fusions with a low requirement for sample quantity, and for use in paraffined samples (FFPE).

Results

The median age was 61 years, with variation from 19 to 81 years-15 men 56.57%, and 13 women 43.46%. The clinical diagnoses of the patients, being the majority of Lung Cancer, Colon Cancer, and Pancreatic Cancer, with 25%, 17.86%, and 10%,71% respectively. More than 75% of the patients were already on lines 3 and 4 of their treatments. Among the patients, 42.86% have been newly biopsied, to do this test. Adenocarcinoma has been the most frequently present histological subtype. The average time for availability of the test reports is 20 days, with variation from 9 to 39 days. One patient did not have enough material to carry out the test, and in all the others good DNA amplification was obtained. RNA amplification has not been obtained in 3 cases. In 60.71% of the cases in this sample, potential molecular targets were detected, however, of the patients screened for Phase 1 trials, only one was enrolled in a trial with a drug targeted to the alteration presented. Multiple types of mutations were assessed.10 patients didn't have any relevant alterations.

Conclusions

Our analysis shows the feasibility of using genomic tests in clinical practice. In the future, most patients will harbor pharmacologically treatable genomic alterations, a result consistent with our observation. The current trials in immunotherapy may explain the low recruitment in targeted drug trials There is an urgent need for new trial designs to address this new reality.

Legal entity responsible for the study

Start Madrid at HM Sanchinarro Hospital

Funding

Has not received any funding

Disclosure

All authors have declared no conflicts of interest.

Background

Several mechanisms have been described to explain the emergence of acquired resistance to MAPK inhibitors. Using tumor DNA sequencing, genetic alterations activating the MAPK and the PI3K/AKT pathways have been associated to emergence of resistance mechanisms. Studies focusing on targeted mRNA analysis have associated gene expression alterations to resistance. Nevertheless, resistance to BRAF inhibitors (BRAFi) remained unexplained for nearly half of melanomas highlighting the need of an approach to exhaustively characterize resistance mechanisms to such therapies.

Methods

Tumor samples at baseline and relapse were collected from 20 patients with a BRAF^V600 mutated metastatic melanoma. Relapse and best response were assessed using RECIST criteria. After DNA and mRNA extraction, copy number variations (CNV) and mRNA expression were evaluated in a targeted panel of 41 genes with a validated/suggested role in BRAFi resistance using pyrosequencing, Sanger sequencing and quantitative PCR. Differences of mutational gene status, CNVs and mRNA expression were analyzed by comparing relapse to baseline specimens.

Results

At least one acquired resistance mechanism was observed for each patient. Alterations of MAPK pathway (BRAF, MAP3K8…), tyrosine kinase receptors (TKR) (KIT, ERBB2…), apoptosis (PTEN, BCL2…) and cell cycle (CDK4, RB1…) related genes were observed in 16, 15, 17 and 20 patients respectively. Five patients had multiple relapse samples (Total= 15) allowing the assessment of tumor heterogeneity. Alterations of MAPK pathway, TKR and apoptosis related genes were thus retrieved in 13/15 samples and cell cycle pathway was altered in all.

Conclusions

Using a comprehensive approach combining a panel of genes mutational analysis, CNV and mRNA expression, our study allowed the characterization of at least one resistance mechanism in all relapse tumors. We showed that while there was heterogeneity in the individual genetic alterations, these overlapped when analyzing them within their signaling pathways. This work enlarges the identification of resistance mechanisms to BRAFi and can be combined with those emerging for immunotherapy to improve the clinical management of patients.

Legal entity responsible for the study

APHP

Funding

Institut National du Cancer

Disclosure

M. Battistella: Consulting role for Histalim, Bristol-Myers Squibb, and Innate Pharma. S. Dalle: Principal investigator in studies conducted by Roche-Genentech and Novartis. C. Lebbe: Declares honoraria from Roche, advisory roles at Roche, GSK, Novartis, BMS, MSD, and Amgen and travel accommodation provided by Roche. S. Mourah: Declares a consulting role at Roche, Janssen and Novartis. All other authors have declared no conflicts of interest.

Background

The proper selection of patients in phase I studies is crucial in order to guarantee not only a safer approach to patients but also to obtain reliable results. Recently, Gustave Roussy Immune Score (GRIm-score) predicted overall survival for patients involved in phase I immune-oncology (IO) trials in a more accurate way when compared to other classical scores. The goal of our study is to validate the GRIm-score in our institution.

Methods

We retrospectively studied 171 patients (pts) treated in phase IO trials at our institution between January 2012 and August 2017. Variables such as haemoglobin, neutrophil-to-lymphocyte ratio (NLR), albumin level, LDH, gender, number of previous lines and tumour type were assessed. GRIm score was calculated as follows: LDH > ULN (+1), Albumin < 35 g/dl (+1), NLR>6 (+1). According to GRIm score patients were stratified into low risk (0-1) or high risk (2-3). Overall survival (OS) and OS at 90 days were studied.

Results

A total of 171 pts (M/F: 115/56) were evaluated. The median age was 59.53 (95%CI 57.68-61.38) Mean number of previous lines were 2.25 (95%CI 2.03-2.48). The most frequent tumour histologies were colorectal carcinoma (28.24%), non-small cell lung cancer (15.29%), renal carcinoma? (11.18%) and melanoma (8.24%). Patients with 0 – 1 risk factors were classified as low-risk (n = 114) whereas patients with 2-3 risk factors were considered high-risk (n = 57). Low-risk patients presented an OS of 13.8 months (95% CI 8.51-20.44) whereas high-risk patients presented an OS of 3.02 months (95% CI 2.3-4.3, HR = 2.66; 95% CI1.76-4.03, p < 0.001). When predicting overall survival at 90 days, we found that patients with low-risk GRIm score punctuation were more prone to be alive at 90 days when compared to patients with high-risk score (OR = 6.61; 95% CI 3.06-14.29, p value <0.001).

Conclusions

We have externally validated the GRIm score in an unselected population. Our data suggests that GRIm score predicts overall survival in patients enrolled in phase I IO trials and may be useful when selecting patients. The correct selection of the patients to be enrolled in clinical trials can help to improve the quality of the trials and improve toxicity profile and survival outcomes.

Legal entity responsible for the study

Clinica Universidad de Navarra

Funding

Has not received any funding

Disclosure

I. Melero: Consultant for Roche, BMS, Lilly, Biotech, Alligator and TurkTurk; Research grant from Roche -Genentech BMS, Alligator. All other authors have declared no conflicts of interest.

Background

Genetic biomarker-driven trials are powerful ways to test targeted drugs, but are often complicated by the rarity of the biomarker-positive population. “Umbrella” trials circumvent this issue by testing multiple hypotheses to maximize accrual. However, allocation strategy (ie which drug to administer first, in case multiple actionable mutations coexist) greatly affects sample size and should be carefully planned based on relative mutation frequencies. Inadequate planning results in lack of statistical power and inconclusive trials. The bigger the trial, the higher the chance of conflicting drug allocation. Biomarker-based trial design may be facilitated by leveraging data from public sequencing projects, but no specific computational tool is available.

Methods

We developed the package Precision Trial Designer (PTD) in the R programming language. We used TCGA data to show its potential in a simulated 10-arm imaginary trial on multiple cancers, based on genetic alterations suggested by the 2017 Molecular Analyses for Personalised Therapy (MAP) conference. We validated PTD predictions versus real data from the SHIVA trial (Lancet Oncol 2015). Finally, we deployed PTD as an open access, web-based app using the Shiny-R Studio platform.

Results

PTD estimates parameters useful for trial design, most importantly 1) the fraction of patients with ≥ 1 actionable alteration 2) the frequency matrix of mutation co-occurrence 3) the number of patients needed to molecularly screen (NNMS) for a given design (time-to-event or proportion-based) 4) the allocation rule that maximizes patient accrual (systematically assigning patients to the drug associated with the rarer mutation). In the MAP imaginary trial, PTD “optimal” design reduces NNMS by up to 71.8% (3.5x) vs non-optimal designs. In SHIVA, the fraction of patients with actionable alteration was correctly predicted (33.51% vs 32.92%, within 95% confidence interval), as well as allocation to specific treatment groups and statistical power.

Conclusions

PTD correctly estimates crucial parameters to overcome issues in designing precision oncology trials. Its availability as an open-access web app creates a useful resource for the community of clinical researchers in the field of targeted therapy.

Legal entity responsible for the study

European Institute of Oncology

Funding

AIRC-15988, ANR-10-EQPX-03, Italian Ministry of Health RF-2013-02357231

Disclosure

All authors have declared no conflicts of interest.

Background

Radiation induces DNA damage, leads to cell cycle arrest and cell death. We investigated the effect of adjuvant radiation therapy (RT) on systemic innate and adaptive immune cells in female breast cancer (BC) patients by assessing circulating white blood cells (WBCs) and its subpopulations.

Methods

Peripheral blood cell numbers from BC patients (n = 114, median age 64) who received adjuvant RT (50 Gy) at Dept. Oncology, Ryhov hospital, Sweden, were analyzed. Blood (30 ml) was obtained from BC patients before and after adjuvant RT. The number of total WBCs and its subpopulations were analyzed by Sysmex XE5000 instrument. The phenotype of ex vivo fresh peripheral white blood cell subpopulation were analyzed by BD FACSCanto II flow cytometer and BDFACSDiva software program. Paired T-test was used for comparison between the patients before and after adjuvant RT, Significant p-values <0.05.

Results

1. After adjuvant RT, the number of WBCs, neutrophils and lymphocytes were significantly decreased 2. Neutrophil to lymphocyte ratio (NLR) is suggested to be a biomarker for poor prognostic and short survival time for cancer patient. Interestingly, the NLR was significantly increased after treatment, p-values = <0.001. 3. As for total lymphocytes, adjuvant RT decreased the numbers in all T-cell subpopulations studied (CD3+, CD4+, CD8+, and CD3 + 56 + [NKT cells]) and in CD56 + (NK cells).

Conclusions

Our results indicated that adjuvant RT of BC patients induces systemic alterations in number of innate and adaptive immune cells. This might be due to the bystander or distant immunosuppression from adjuvant radiation. Alternative, adaptive and innate immune response cells are redistributed from circulation to the radiation site. The significance of these alteration on clinical outcome need further investigation.

Legal entity responsible for the study

Regional Ethical Review Board of Linköping

Funding

Clinical Cancer Research in Jönköping and Futurum, Ryhov Hospital, Jönkoping Sweden

Disclosure

All authors have declared no conflicts of interest.

Background

To improve efficiency and safety of phase I trials, various new designs have been proposed. We reported that rule-based design and model-based design were similar profile in efficiency and safety (Shimomura A, et al. TAT 2017). The limitation of the study was retrospective single institutional study. Then, we investigated the literature or presentation based analysis to compare model-based design with rule-based design of phase I trials.

Methods

Published article or presentation of phase I trials which conducted at our institute between Dec 1996 and Dec 2016 were reviewed. Trials which met to the following criteria were excluded; combination trials, trials without dose escalation, trials with expansion cohort only, uncompleted trials and dose-finding trials (to assess safety and pharmacokinetic profiles in two or three doses up to the maximum tolerated dose (MTD) previously determined in the West). Efficiency and safety were assessed in two design groups; rule-based design (conventional 3 + 3 design and accelerated titration design) and model-based design (CRM using escalation with overdose control). Number of patients who treated in MTD and around MTD (from under 20% to over 20% of MTD) was assessed to evaluate efficiency. Number of patients who treated over MTD or developed dose limiting toxicity (DLT) was assessed to evaluate safety.

Results

Of 47 trials, 35 trials have published. A total of 670 patients were included to the analysis. Five-hundred sixty (83.6%) and 110 (16.4%) were assigned to rule-based and model-based trials, respectively. 183 (32.7%) patients in rule-based trials and 17 (15.5%) patients in model-based trials were treated in MTD (p=.0001). 256 (41.6%) patients in rule-based trials and 23 (20.9%) patients in model-based trials were treated around MTD (p=.0013). Number of patients who treated over MTD was 63 (11.3%) in rule-based design and 8 (7.3%) in model-based design (p=.196). Frequency of DLT was 74 (13.2%) in rule-based design and 7 (6.4%) in model-based design (p=.0308).

Conclusions

Trials with rule-based design tend to treat more number of patients not only at both MTD/around MTD but also with DLTs than model-based design equipping trials.

Legal entity responsible for the study

Akihiko Shimomura

Funding

Has not received any funding

Disclosure

S. Iwasa: Grants and personal fees from Eli Lily, personal fees from Taiho, grants and personal fees from Chugai, grants from Novartis, grants from Merck-Serono, grants from Daiichi-Sankyo, grants from Bristol-Myers Squibb, from null, outside the submitted work. Y. Fujiwara: Grants from AbbVie, grants and personal fees from AstraZeneca, grants from Chugai, grants from Daiichi-Sankyo, grants from Eisai, grants from Eli Lilly, grants from Incyte, grants from Merck Serono, grants and personal fees from MSD, grants from Novartis, personal fees from Taiho, grants and personal fees from BMS, personal fees from ONO, outside the submitted work. T. Shimizu: Research expenses from Bristol-Myers Squibb, Daiichi-Sankyo, PharmaMar, FivePrime, 3D Medicine, Takeda-Milleniam, personal fees (lecture fees) from Chugai Pharmaceutical Co., Ltd., Taiho Pharmaceutical Co., Ltd., Ono Pharmaceutical Co., Ltd., Ono Pharma Taiwan Co., Ltd., Boehringer Ingelheim, outside the submitted work. N. Yamamoto: Reports grant from Bristol-Myers Squibb, Chugai, Taiho, Eisai, Lilly, Quintiles, Astellas, Novartis, Daiichi-Sankyo, Pfizer, Boehringer ingelheim, Kyowahakko. Kirin, Bayer, ONO PHARMACEUTICAL Co. Ltd., and Takeda, other from Chugai., Ono Pharmaceutical Co., Ltd., AstraZeneca, Pfizer, Lilly and Bristol-Myers Squibb outside the submitted work. All other authors have declared no conflicts of interest.

Background

Lenvatinib (LenvimaTM, LVT), an oral tyrosine kinase inhibitor, has shown efficacy in iodine-131–refractory thyroid cancer with a median progression free survival (PFS) in the LVT 24 mg daily arm of 18.3 months compared to 3.6 months in the placebo arm. Yet, with a 97.3% incidence of all grades treatment-related adverse effects, dose interruption or dose reduction occur at 82.4% and 67.8%, respectively. The objective of this study was to assess real-life tolerance and LVT plasma exposure after dose reduction in thyroid cancer patients.

Methods

This monocentric prospective observational study included metastatic differentiated thyroid cancer patients treated with LVT in monotherapy according a signed informed consent. Toxicity evaluation was performed monthly after lenvatinib initiation according CTCAE guidelines. Biological parameters, concomitant treatment, morphological tumoral response were also recorded. Blood samples were collected monthly. LVT plasma concentrations were quantified using a routine LC-MS/MS method. A previously published population pharmacokinetic model of LVT was used to compute the individual PK parameters AUC_0-24h and C_trough with NONMEM software (version 7.2). PK parameters in patients experiencing or not adverse event were compared using Wilcoxon test.

Results

LVT Dose reduction occurred in the 13 included patients during the month following LVT initiation (20mg QD n = 1; 14mg QD n = 6; 10mg QD n = 6). Over the 45 observations, median AUC_0-24h reached 1797.2 [984.9 to 3169.8] ng.h.mL⁻¹. LVT treatment was generally well-tolerated as grade 3 HTA events were reported in only 4 patients and did not lead to treatment discontinuation. To date, treatment duration ranged from 3 to 15 months. Grade 1 and 2 diarrhea, hypertension, asthenia occurred during the follow-up, but only HTA appears to be associated with plasma concentration (p = 0.79, p = 0.033 and p = 0.39 respectively).

Conclusions

LVT was well tolerated and no treatment discontinuation was needed. Despite systematic dose reduction, plasma exposure was consistent with the expected exposure at the initial dose. Further exploration is needed to assess treatment duration and tumoral response in real-life treated patients.

Legal entity responsible for the study

Assistance Publique des Hôpitaux de Paris

Funding

Has not received any funding

Disclosure

C. Chougnet: Has a conflict of interest with EISAI. All other authors have declared no conflicts of interest.

Background

Temozolomide (TMZ) is a standard treatment for melanoma and glioblastoma and it has shown limited but encouraging activity in patients with metastatic colorectal cancer (mCRC). In multiple cancer types, tumor expression of the DNA repair protein O⁶-methylguanine-DNA methyltransferase (MGMT) is a marker of poor response to TMZ. MGMT promoter methylation is associated with loss of MGMT expression and response to TMZ. We hypothesized that mCRC patients whose tumors expressed quantities of MGMT protein below a pre-defined cutoff would have better outcomes on TMZ than patients with MGMT expression above the cutoff. To test our hypothesis, we assessed MGMT by mass spectrometry in the tumor samples of patients with refractory mCRC and MGMT promoter methylation who had received TMZ.

Methods

Archived formalin-fixed, paraffin-embedded tissue sections were obtained from 24 patients from two phase 2 trials of TMZ. A pathologist marked the tumor areas, which were then microdissected and solubilized. In each tumor sample, multiple protein biomarkers including MGMT were quantified with selected reaction monitoring mass spectrometry. An MGMT cutoff of 200 amol/ug was based on the limit of quantitation from a concentration curve. The Mantel-Cox log-rank and the Fisher’s exact tests were used for survival comparisons.

Results

MGMT protein was detected in 13 of 24 (54.2%) colorectal tumor samples (range: 229.3-784.8 amol/µg). The overall response rate was 29%. Patients with MGMT protein levels below a cutoff of 200 amol/ug (n = 11) had a notably higher response rate than patients with MGMT levels above the cutoff (64% vs. 0%; p = 0.001 Fisher’s test). Also a longer progression-free survival was observed (4.3 vs. 1.6 months, HR = 0.36, 95% CI = 0.13-1.10, p = 0.054). Results for overall survival were consistent but not statistically significant (8.9 vs 6.9 months, HR = 0.55, p = 0.221).

Conclusions

Patients with mCRC whose tumors expressed low or undetectable levels of MGMT protein had better outcomes following TMZ treatment than their counterparts. Quantitative proteomic analysis of MGMT could potentially be used to select CRC patients for TMZ treatment. The results of validation studies are forthcoming.

Legal entity responsible for the study

NantOmics, LLC

Funding

Has not received any funding

Disclosure

F. Cecchi: Employee at NantOmics

Background

First line standard treatment for advanced HER2-positive breast cancer is trastuzumab/pertuzumab/docetaxel (HPT). HPT therapy has shown significant response rates and prolonged overall survival compared with single anti-HER2 antibody containing therapy. However, some patients with HER2-overexpressing breast cancer show primary resistance to HPT therapy. There is no predictive marker to identify response to HPT therapy. Therefore, we explored the combination of microRNA (miRNA) that is associated with response to HPT therapy.

Methods

HER2-positive advanced breast cancer patients who were treated in our institute from October 2013 to August 2016 were enrolled in this study. Serum samples were obtained just before initiation of HPT therapy. A comprehensive quantitative expression analysis of miRNA was performed using the by DNA chip “3D-Gene^® (Toray Industries Inc.)” Clinicopathological information was obtained from electronic medical record, retrospectively. miRNA was explored, which was associated with complete response or stable disease as defined by RECIST ver 1.1.

Results

Among 36 patients, 32 samples from each patient were available to evaluate. Median age was 54-year-old. 14 patients (44%) were hormone receptor-positive. Sixteen out of 32 (50%) patients had metastatic disease at diagnosis and the rest was recurrent. Overall response rates were the following: complete response (n = 7, 21.9%), partial response (n = 19, 59.4%), stable disease (n = 6, 18.8%). miR-A, miR-B and miR-C were candidates to predict complete response. Sensitivity for each miRNA was 63.6%, 72.7%, 63.6%, respectively, and specificity was 94.7%, 89.5% and 94.7%. The area under the curve (AUC) was 0.77, 0.86, 0.83. With regard to prediction of stable disease, miR-X, miR-Y and miR-Z were candidate. Sensitivity for each miRNA was 80%, 80% and 100%, and specificity was 88%, 80% and 64%, respectively. The area under the curve (AUC) was 0.81, 0.82 and 0.86.

Conclusions

We identified candidate miRNAs which may predict response to HPT therapy. Further validation cohort was needed.

Legal entity responsible for the study

National Cancer Center Research Institute

Funding

Japan Agency for Medical Research and Development

Disclosure

All authors have declared no conflicts of interest.

Background

KRAS and microsatellite instability (MSI) status are well established markers in personalized therapy of colorectal cancer (CRC). The immune checkpoint inhibitor Programmed Death 1 (PD1) and its ligand (PD-L1) have been reported in recent studies as promising targets for cancer therapy. However, there is still a debatable result in the correlation between PD1/PD-L1 expression and KRAS and MSI status as the essential CRC biomarker. Therefore, this study aims to characterize molecular profiles of KRAS, MSI, and TP53 in their association with PD-L1 expression in an Indonesian CRC patient cohort.

Methods

We screened formalin-fixed paraffin-embedded (FFPE) samples from 88 CRC patients for three biomarkers (KRAS, TP53 and PD-L1) and their MSI status. MSI status was defined by the MMR protein expression (MSH2, MLH1, PMS2, MSH6) using monoclonal antibodies by immunohistochemistry (IHC) methods. IHC was also used to detect PD-L1 and TP53 protein expression. High resolution melting PCR along with Sanger sequencing were carried out to detect KRAS mutation status. All the data was statistically analyzed using Fischer exact test.

Results

Only 12.6% (16 out of 88) of sample showed PD-L1 expression. Statistical analysis showed the association between MSI with PD-L1 expression (p = 0.0001). The study showed no association between PD-L1 expression with TP53 (p = 0.9164) and KRAS status (p = 0.3749). Despite that, PD-L1 expression still tended to be higher in patients with wild type KRAS (82.3%/17.7%; wt/mut) and TP53 (62.5%/37.5%; wt/mut).

Conclusions

PD-L1 expression was associated with patient MSI status in this Indonesian cohort. Defining a patient’s MSI status could be suggested as an important step in a screening process for CRC treatment while using anti PD1/PD-L1 targeted therapy.

Legal entity responsible for the study

Institutional Review Board, Stem Cell and Cancer Institute, Jakarta

Funding

PT. Kalbe Farma Tbk., Jakarta, Indonesia

Disclosure

All authors have declared no conflicts of interest.

Background

Cobas^® 4800 BRAF V600 Mutation Test is a CE-marked and FDA-approved in vitro diagnostic assay used to detect metastatic melanoma patients eligible for treatment with BRAF inhibitors. The aim of the study is to compare the performance of two additional commercial kits to assess BRAF mutations in this context

Methods

A total of 247 Metastatic melanoma specimens were tested for BRAF V600 mutations at three laboratories (Vall d´Hebron, 12 de Octubre and Virgen Macarena Hospitals) with the COBAS® BRAF V600 V1 (Roche), Idylla BRAF (Biocartis) and COBAS® BRAF/NRAS V2 (Roche) kits. Overall (OPA) Positive (PPA) and Negative (NPA) percent agreements were determined between the cobas V1 test and the other assays. We also analyzed the sensitivity and specificity of each assay used in this study

Results

BRAF mutations were detected in 35.2% (87/247), 44.5% (110/247) and 42.9% (106/247) of samples by COBAS^® BRAF V600 V1, Idylla and COBAS V2 kits respectively. Similar ratios of invalid results were observed in the 3 different technologies (1.2%, 1.6% and 1.6% respectively). Comparing COBAS V1 with other methods we obtained OPA 90%, PPA 79.6% and NPA 99.2% by Idylla and OPA 90%, PPA 80.3% and NPA 98.5% by COBAS V2 test. Finally, Idylla vs COBAS V2 showed OPA 96%, PPA 95.4% and NPA 97%. Sensitivity and Specificity for each assay in this global comparison were also calculated showing [82.8-100] by COBAS V1, [99-97.8] by Idylla and [98.1-97.8] by COBAS V2. Additionally, COBAS V2 test detected other BRAF non-V600 mutations (2 K601E and 1 G466A/V or G469A/R/V) and also NRAS mutations (2 NRAS G12X, 2 NRAS G13X and 41NRAS Q61X).

Conclusions

Both methodologies COBAS V2 and Idylla have a good performance to detect BRAF V600 mutations in metastatic melanoma samples with a high concordance between them. Both techniques compared with COBAS V1 detect more mutant BRAF V600 cases with High Sensitivity and Specificity. Finally, COBAS V2 detects more mutation out of V600 in BRAF and NRAS genes that allow managing those patients for potential different therapies.

Legal entity responsible for the study

Vall d´Hebron Institute Research

Funding

Has not received any funding

Disclosure

F. Garcia Verdes-Montenegro and M. Ulrech: Employee of Roche-Farma. All authors have declared no conflicts of interest.

Background

The chemokine receptor CXCR5 is selectively expressed on B cells and it is involved in lymphocyte homing and the development of normal lymphoid tissue. Its principle ligand is CXCL13 or B lymphocyte chemoattractant (BLC). Three polymorphisms in CXCR5 gene, rs148351692C/G, rs6421571C/T, and rs78440425G/A, have been identified in CXCR5 gene. The aim of the work is to study the detection and to assess the impact of genetic polymorphisms of CXCR5 on the susceptibility and response to therapy of non-Hodgkin lymphoma (NHL).

Methods

Fifty patients with NHL as well as fifty healthy control subjects were included in this study. CXCR5 rs148351692C/G, rs6421571C/T, and rs78440425G/A genotypes were determined by PCR-RFLP.

Results

Our study revealed that both CXCR5 rs148351692C/G, rs6421571C/T gene polymorphisms are associated with increased risk of developing NHL with OR = 29.57 (95% CI = 8.96 - 97.56) and 4.45 (95% CI = 1.68 - 11.8) respectively, while CXCR5 rs78440425G/A showed no statistical significant difference between NHL patients and the control group regarding the risk of developing NHL. Moreover, double and the triple combined gene polymorphisms is associated with about 129 fold and 108 fold respectively increased risk of developing NHL.

Conclusions

CXCR5 rs148351692C/G, rs6421571C/T, and rs78440425G/A gene polymorphisms could be involved in the patho-physiology and development of NHL. They may be useful as predictive molecular markers for prognosis and disease outcome in NHL patients.

Legal entity responsible for the study

Cairo University

Funding

Has not received any funding

Disclosure

All authors have declared no conflicts of interest.

Background

Growth factor receptor bound protein 7 (GRB7) is a member of Grb family, and it is an adaptor protein. GRB7 has five functional domains, participated in many different signaling pathway.Human epidermal growth factor receptor 2 (HER2) is classified as a subtype in breast cancer. Studies until now have indicated that GRB7 and HER2 are highly correlated.

Methods

To further confirm the relationship between GRB7 and HER2, GRB7 mRNA and protein levels were analyzed in different breast cancer cell lines and clinical patients tissue (N = 213).

Results

We found that GRB7 highly expressed in HER2 positive breast cancer cell lines and HER2 positive patients’ tissues. GRB7 definitely existed in cytosol and nucleus in SKBR3 breast cancer cell, but GRB7 could not regulate HER2 gene expression directly. In addition, we constructed the knocked down GRB7 plasmid and overexpression GRB7 plasmid. We proved that HER2 would decrease when we knocked down GRB7; HER2 protein level would not change when we overexpressed GRB7.Utilizing a nature compound named curcumin to treat SKBR3 cells decreased GRB7 protein expression. Further analysis using cycloheximide to identify the possible degradation mechanism of GRB7, and the consequence indicated that GRB7 protein was directed to the path of hydrolysis, rather than being inhibited during the synthesis.

Conclusions

By inhibiting GRB7 and can decrease the excessive expression of HER2. These results imply that GRB7 could be a potential molecule for therapy and prognosis prediction in breast cancer patients.

Legal entity responsible for the study

N/A

Funding

This study was supported by the Ministry of Science and Technology, Taiwan (MOST 106-2320-B-038-061 -MY3).

Disclosure

The author has declared no conflicts of interest.

Background

Read-through of genetic nonsense mutations has been proven effective in mice models and for some human clinical conditions and can lead to the expression of a full-length protein. Based on initial work that showed aminoglycoside and macrolide antibiotics can read-through adenomatous polyposis coli (APC) nonsense mutations, we have initiated a clinical trial for erythromycin treatment in familial polyposis (FAP) patients that result from nonsense mutations in the APC gene.

Methods

A prospective, open label interventional study with oral erythromycin 250mg BID for 4 months. The study included a baseline and post-treatment colonoscopies at 4 and 12 months, in which a polyp count and measurements were conducted. Polyps were analyzed for Wnt target genes expression. Repeated measures ANOVA was used for the comparison of polyp number and size across repeated measurements. Annual rate of changes in polyp number and size were compared pre and post intervention using the dependent samples t test.

Results

We recruited 9 patients, 5 completed 12 months of follow up. Polyp number at baseline, after 4 and 12 months were 37.0±31.7, 33.6±38.6 and 30.8±36.7, respectively (0.099 between subject effect) and polyp maximal size 7.0±1.4, 6.3±2.1 and 4.1±1.0 (<0.001 between subject effect). Mean pre and post treatment (4 months) values for 9 patients were 73.7±13.9 and 36.3±23.0 for KI67; 38.1±72.6 and 16.2±24.3 for C-MYC; 16.8±23.9 and 6.1±7.0 for AXIN; 2.6±6.0 to 0.08±0.06 for CYCLIN D. The individual rate of polyp number and maximal size change according to the change between 1 year pre-study to study baseline colonoscopies was compared with the rate between baseline and 12 months post study results. Polyp number change was 33.8±77.7% before and -38.7±23.5% after (p = 0.125) and for polyp maximal size: 33.8±77.7% and -38.7±23.5% (p = 0.125).

Conclusions

In this pilot study, initial results point to molecular and clinical effects of erythromycin, which indicate that APC read-through is feasible in FAP and requires further study.

Clinical trial identification

Open label cohort study

Legal entity responsible for the study

Tel Aviv Sourasky Medical Center

Funding

Rising Tide, Gateway

Disclosure

All authors have declared no conflicts of interest.

Background

Bladder cancer is a heterogeneous malignancy with wide scale of clinical manifestation. Different chromosomal aberrations have been already identified in bladder tumors. Older age, Asian ancestry and a positive family history of bladder cancer have long been recognized as important risk factors. The aim of the present investigation was to study the major chromosomal aberrations (CA) like deletion, translocation, inversion and mosaic in bladder cancer patients of Tamil Nadu, Southern India.

Methods

A total of 62 blood samples were collected from various hospitals in Tamil Nadu, Southern India. Equal numbers of normal healthy subjects were chosen after signing a consent form. Volunteers provided blood samples (5 ml) to establish leukocyte cultures. Cytogenetic studies were performed by using Giemsa-banding technique and finally the results were ensured by using fluorescence in situ hybridization (FISH). Fluorescence in situ hybridization (FISH) has been used to identify the target genes for some of these chromosomal alterations.

Results

Showed frequent CA in chromosomes 1, 8, 9, 10, 11, 13, and 14. By conventional cytogenetics, predominant changes included gain of chromosome 8, loss of Y, deletions of 9q and l0q, and the appearance of double minutes. In the present investigation, major CA like deletion, translocation, inversion and mosaic were identified in experimental subjects. In comparison with experimental subjects, the control subjects exhibited very low levels of major CA (P < 0.05).

Conclusions

In the present study, the high frequency of centromeric rearrangements indicates a potential role for mitotic irregularities associated with the centromere in bladder cancer tumorigenesis. Identification of chromosome alterations may be helpful in understanding the molecular basis of the disease in better manner.

Legal entity responsible for the study

Annamalai University, Tamil Nadu, India

Funding

Has not received any funding

Disclosure

All authors have declared no conflicts of interest.

Background

Pain control in oral and neck carcinoma patients treated with chemoradiation are a challenge to manage, achieving a pain control and avoiding the complication of pain killers is as more important as pain control. In this study locally advanced squamous cell carcinoma treated with concurrent chemo radiation involving inj cisplatin for all patients and few patients were given inj nimotuzumab along with inj cisplatin. Controlling pain for moderate- severe level with tramadol without antiemetics is not tolerated by most of the patients, adding a drug for a drug induced complication (emesis) is a burden for patients, hence drug with least complication is always better.

Methods

Three groups (40 patients in each group) of locally advanced oral and neck squamous cell carcinoma treated with concurrent chemoradiation (with inj.cispaltin +/- inj.nimotuzumab) were taken. For pain control Group-A had tapentadol 50mg twice daily if pain persisted dose were increased up 200mg with 6 hours interval, Group-B had tramadol plus paracetamol with antiemetics, Group-C had tramadol plus paracetamol without antiemetics. On 3^rd and 4^th weeks of treatment patients had grade III and grade IV severe pain and pain score was assessed with facial expression as per WHO criteria.

Results

In group-A, of 40 patients 36 (90%) had good pain relief with pain score0-1 of which 31 patients showed pain relief with 50mg twice daily and 4 had dose escalation of 50mg four times daily, 1 patient showed skin allergy with good pain control, 4 patients were switched to tab. morphine 10gm every 4^th hour. In group-B, 35 (87.5%) patients had good pain relief with pain score 0-1, but 5 patients did not tolerate due to emesis, sedation in spite of good pain relief, 5 patients were switched to tab. morphine 10mg every 4^th hourly. In group-C only 7 (17.5%)patients tolerated with pain relief, rest did not tolerate emesis and sedation and were given symptomatic care.

Conclusions

Tapentadol statistically had similar pain control as that of tramadol with paracetamol, but was superiorly tolerated by the patients without emesis.

Legal entity responsible for the study

CBCC-Oncology Services, Saveetha University Chennai

Funding

Has not received any funding

Disclosure

The author has declared no conflicts of interest.

Background

Breast cancer is the leading cause of cancer-related deaths in women aged 45 and younger in developed countries, and although generally improving, survival rates for young women with breast cancer remain lower than for older women. The aim of the study was to evaluate the percentage and the outcome of women younger than 45 with HER2 positive disease.

Methods

Medical files of 625 women diagnosed with breast cancer between 2007-2015 were retrospectively analysed. There were 134 (21.4%) patients younger than 45 at diagnosis. In this subgroup of patients 32 were diagnosed with HER2 positive disease. The time to tumor progression of young patients with HER2 disease was compared with a matched control group of patients with HER2 disease older than 45. All patients received chemotherapy and trastuzumab. No patents received neoadjuvant trastuzumab (due to lack of reimbursement).

Results

In the group of 32 young women, median age at diagnosis was 36.5 years, stage distribution was 12.5%, IIA, 9.4% in stage IIB, 43.7% stage IIIA, 21.9% stage IIIB,12.5% stage IV. Compare to the rest of the patients, the younger was diagnosed more often with advanced and metastatic disease (p = 0.043). The incidence of HER2 positive disease was similar in our group (23.8%) compare to entire group (26.4%). Ki 67 percentage ranged between 11% and 75% (median was 35%). The median disease-free survival for young group was 65 months were for control was not reached; the 3-year and 5-year disease-free survival were 58% and 50%, respectively compare to 63% and 55% for older women. The 3-year and 5-year overall survival were 78% and 58%, respectively significantly lower than in the matched controlled group P = 0.039.

Conclusions

Our lot of patients diagnosed with HER2 positive disease aged less than 45 years was diagnosed in a much more advanced stage and had a poorer prognosis compared with HER2 positive patients older than 45 years.

Legal entity responsible for the study

N/A

Funding

Has not received any funding

Disclosure

All authors have declared no conflicts of interest.

Background

Expectation of benefit from the antiepidermal growth factor receptor (anti-EGFR) therapeutics is a not yet solved question in metastatic colorectal cancer (mCC). MicroRNAs (miRNAs), which are short non-coding RNA molecules and important regulators of cell signaling pathways crucial for the growth of human cancer cells. Several studies have examined the expression profiles of miRNAs in response to different chemotherapy treatments and found that the expression patterns may be associated with the treatment response. Therefore, our aim was to evaluate the differential expression profiles of miRNAs in mCC treated with anti-EGFR therapeutics (cetuximab, panitumumab).

Methods

Overall 21 patients with wild type KRAS, BRAF, NRAS, PTEN and PI3 CC were included into our analysis. Using real-time PCR, we analyzed the expression levels of 18 different human miRNAs in the twenty-one CC tumor samples. Relationship between data and disease-free survival (DFS) were analysed by using SPSS.

Results

Among evaluated miRNAs, miR-31-5p exhibited the most dramatic difference in expression, with 5.01-fold in cancer tissues compared with the controls (P = 0,01). The expression levels of miR-21 and miR-31-5p were significantly higher, miR-451 was lower in non-responders tissues (P = 0.02, P = 0.03, P = 0.01; respectively). In Kaplan–Meier analysis, a high miR-31-5p expression level was significantly associated with shorter DFS (P = 0.003).

Conclusions

This study indicated that up regulation of miR-31-5p and miR-21; down regulation of miR-451 might serve as a potential indicator for anti-EGFR resistance in mCC patients who are expected to benefit from anti-EGFR therapeutics.

Legal entity responsible for the study

Ozkan Kanat

Funding

Has not received any funding

Disclosure

All authors have declared no conflicts of interest.

Background

Targeted anticancer therapies have been approved by the Food and Drug Administration for use in some men with metastatic castrate-resistant prostate cancer (MCRPC). This study aimed to forecast the first-line MCRPC drug-treatable populations (DTP) by global geographic/economic regions over the period 2017-2027.

Methods

Using country-specific cancer registries, prostate cancer incidence was estimated for 45 countries, representing approximately 90% of world population in 2017. Observed correlations between gross domestic product (GDP), prostate cancer risk, and background survival were used to forecast incidence for lower-income countries under study over the next ten years. Diagnosed incident cases were stratified by Tumor Node Metastasis (TNM) stage and risk categories using National Comprehensive Cancer Network (NCCN) 2013 Guidelines. A GDP-based forecast was applied to TNM stage/NCCN risk at diagnosis based on expected improvements in prostate cancer screening for lower-income countries. Metastatic recurrence-free survival was forecast using a function of historical trends to account for improvements in cancer therapy. First-line MCRPC DTP represents the total number of cases eligible for drug treatment in the first-line MCRPC setting each year. Using estimates of stage- and risk-stratified incident cases, and recurrent cases as described above, annualized estimates of first-line MCRPC DTP were obtained. To estimate incident cases and DTP globally, aggregate estimates for each region were divided by the proportion of countries in that region for which direct estimates were made using the methods described above.

Results

In 2017, there were an estimated 331,000 first-line MCRPC DTP worldwide. The first-line MCRPC DTP worldwide will increase by 30% over the period 2017-2027, with higher growth across lower-income countries (40%), than across high-income countries (14%).

Conclusions

The first-line MCRPC DTP is expected to increase globally, primarily driven by increases in diagnosed incidence in conjunction with growing and aging populations in the lower-income regions.

Legal entity responsible for the study

Decision Resources Group, Burlington, MA, USA

Funding

Has not received any funding

Disclosure

The author has declared no conflicts of interest.

Background

HCC is the third most common cause of cancer related mortality, globally. Although early diagnosis is associated with better prognosis. Sorafenib, an orally administered, small molecule multikinase inhibitor, has proven efficacy in advanced HCC, by targeting cellular signaling pathways that orchestrate tumor angiogenesis and proliferation via Vascular Endothelial Growth Factor Receptor(VEGFR), Platelet-Derived Growth Factor Receptor β (PDGFR-β) and Raf -1. The impact of targeted therapy on the Quality of life of cancer patients is gaining importance as these molecules have markedly improved the Overall survival and Progression free survival.

Methods

Prospective observational study. 28 HCC patients enrolled in the study were treated with T. Sorafenib 400 mg Once Daily by the treating oncologist Patient reported QoL data was collected using Functional Assesment of Cancer Therapy – General (FACT- G) Version 4 questionnaire (Maximum score: 108) further subdivided into 4 domains: Physical well-being (Max score: 28), Social well-being (Max score: 28), Emotional well-being (Max score: 24), Functional well-being (Max score: 28). It is administered prior to treatment initiation, and following therapy, at 3months and 6 months respectively. Attainment of higher FACT-G scores correlate with better quality of life. Mean Overall Survival was also assessed as a secondary objective.

Results

Table: 134P. Baseline Characteristics

Statistical analysis was done using SPSS 18. Descriptive analysis of QoL data thus obtained for the total population is as follows: QoL Mean score: 73.29 ± 23.689 at baseline, 77.61± 21.339 at 3 months and 80.20 ± 24.669 at 6 months. Similarly for the 4 domains of FACT- G: PWB Mean score: 17.89 ± 6.082 at baseline, 19.36 ± 5.532 at 3 months and 20.08 ± 6.422 at 6 months SWB Mean score: 20.79 ± 3.542 at baseline, 21.18 ± 4.010 at 3 months, and 21.40 ± 5.050 at 6 months EWB Mean score: 17.36 ± 7.072 at baseline, 18.507 ± 5.885 at 3 months and 18.84± 6.555 at 6 months FWB Mean score: 17.25 ± 8.536 at baseline, 18.57 ± 7.451 at 3 months, and 19.88 ± 7.677 at 6 months. Mean Overall Survival was 9.563 ± 0.438 months.

Conclusions

Over a period of 6 months the quality of life has improved, with the functional well-being domain showing the most improvement in our scenario. Sorafenib as systemic therapy in advanced HCC has a favourable clinical profile without adversely affecting the quality life of the patient.

Clinical trial identification

IEC No. 07/17/2016/MCT

Legal entity responsible for the study

Government Medical College Thiruvananthapuram

Funding

Has not received any funding

Disclosure

All authors have declared no conflicts of interest.

Background

Axitinib is an oral multikinase inhibitor, which is licenced for use in advanced renal cell carcinoma after failure of prior systemic treatment. We have assessed the efficacy and general tolerability of axitinib in a real world setting and audited our local dose escalation/reduction practice.

Methods

Using our electronic patient records and chemotherapy prescribing systems, 43 patients with advanced renal cell carcinoma on axitinib were identified.

Results

31 patients had clear cell carcinoma, 12 had papillary withFirst 17 females and 26 males between the ages of 37-86 years (median of 69). First line: 5patients within A PREDICT trial. Second line: 33 patients who had sunitinib (n = 17), pazopanib(n = 14), everolimus (n = 1), interferon alfa (n = 1) as first line. 4 patients had axtinib as third line, 1 as fourth line treatment after failure of previous systemic treatment lines. 39 (90.7%) patients were commenced on 5mg, 3(6.9%) on 3mg due to borderline performance status and 1 on 7mg for reasons unknown. 28 (65%) reported fatigue with 18 (41.9%), requiring either treatment breaks or steroid use.20 (46.5%) had hypothyroidism. 14 (32.5%) had grade 2-3 hypertension. 7(16.3%) had grade 1-2 diarrhoea. 2 (4.65%) had grade 2 palmar plantar syndrome. Doses were escalated in 15 cases. In 4 cases from 5mg to 7mg due to disease progression (cycles 4, 9,10) and in 11 cases due to good tolerance. Dose of 10mg was achieved in 2 cases. In 2 cases the doses were reduced back from 7mg to 5mg due to grade 3 fatigue Doses were reduced 13 cases. In 7 cases, dose reduction from 5mg to 3mg due to grade 3 fatigue. (n = 4) and grade 2-3 diarrhoea (n = 3). In 3 cases dose reduction from 5mg to 4mg due to grade 3 fatigue (n = 2) and grade 3 diarrhoea (n = 3). In 15 (34.8%) cases, neither escalation nor reduction was done, only 3 had grade 2 side effects of anorexia, 1 of diarrhoea and 1 had a grade 3 hypertension. The remaining 8 (18.6%) either had grade 1 side effects or no side effects and would have benefited from a dose increase. The average no of axtinib cycles to progression was 10.3 (range 2-34).

Conclusions

Side effects experienced are similar to those listed in the literature. Patients commenced on appropriate doses. Changes currently being made to prescribing system to facilitate appropriate changes to doses.

Legal entity responsible for the study

The Royal Free Hospital Hampstead

Funding

Has not received any funding

Disclosure

All authors have declared no conflicts of interest.

Background

Lung cancer remains the commonest cancer and the most common cause of cancer-associated death worldwide. Use of chemotherapeutic drugs are significant in managing patients with lung cancer, however, due to serious adverse drug reactions (ADRs) these drugs are often underutilized. Our aim was to perform a meta-analysis and systematic review of studies looking at pharmacogenetic drug transporter variants as predictors of ADRs in lung cancer patients undergoing chemotherapy.

Methods

Papers were sourced from Medline, Cochrane Library, CINHL, EMBASE, Web of Knowledge and Scopus. The Cochrane Collaboration Risk of Bias Tool v13 was used to evaluate six types of bias domains for each of the publications reviewed. Where applicable, random effect meta-analysis was used and funnel plots displayed.

Results

We report findings on the ATP-binding cassette superfamily (ABC) members ABCB1, ABCC1, ABCC2, ABCG2, ABCA1, ABCC4 and ABCC5. For influx transporters, findings for the solute carrier organic anion transporter family (SLC, SLCO) SLC29A1, SLC2A3, SLC31A1, SLCO1B1 and SLCO1B3 are included. Significant increased risk of irinotecan-induced neutropenia was noted in NSCLC patients expressing ABCB1 2677G>T/G (P = 0.002) or ABCC2 3972T>T (P = 0.04). ABCG2 421C>A was associated with a significant 2-fold increased risk of gefitinib-induced skin toxicity (P = 0.0009). East-Asian patients treated with irinotecan and expressing the SLCO1B1 -1118G>A variant had a 2 -3 fold significant increased risk of diarrhea and neutropenia (P < 0.05). American patients expressing the SLC19A1 IVS2(4935)G>A variant were associated with pemetrexed/gemcitabine induced grade 3+ leukopenia.

Conclusions

Whilst current data shows promise regarding ABCB1, ABCC2, ABCG2, SLCO1B1 and SLC19A1 pharmacogenetics, many more studies are required that incorporate larger patient numbers and designed to minimize bias before we can begin to utilizes these variants to optimize chemotherapeutic treatment.

Legal entity responsible for the study

Zoulikha Zair

Funding

Has not received any funding

Disclosure

All authors have declared no conflicts of interest.