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210 Genetic and epigenetic factors affect RET gene expression in breast cancer cell lines and influence survival in patients
S30
Abstracts
received honoraria from GSK and Roche, and a travel grant from Sandoz. PLP received honoraria from Amgen, Merk Serono, Sanofi and Integragen. LG and PAJ received travel grant from Pfizer. 209 POSTER The sum of gains and losses of genes encoding for protein tyrosine kinase targets predicts response to multi-kinase inhibitor treatment X. Jiang1 , D. Pissaloux1 , C. De La Fouchardiere2 , F. Desseigne2 , Q. Wang1 , M.E. Fondrevelle3 , P. Cassier2 , C. Seigne4 , D. Perol4 , I. Ray-Coquard2 , A. Le Cesne5 , N. Penel6 , O. Tredan2 , J.Y. Blay2 . 1 ´ Centre Leon Berard, Department of Translational Research, Lyon, ´ France; 2 Centre Leon Berard, Department of Medical Oncology, Lyon, 3 ´ France; Centre Leon Berard, Department of Pathobiology, Lyon, France; 4 ´ Centre Leon Berard, Department of Clinical Research, Lyon, France; 5 Gustave Roussy, Department of Medical Oncology, Villejuif, France; 6 Centre Oscar Lambret, Department of Medical Oncology, Lille, France Background: Validated predictive biomarkers for multi-tyrosine kinase inhibitors (MTKI) efficacy are lacking. We hypothesized that interindividual response variability is partially dependent on the somatic DNA copy number alterations (SCNAs), especially those of genes encoding for the protein tyrosine targeted by MTKI (called target genes). Genomic alteration was investigated in MTKI responsive and non responsive patients with different histological subtypes included in the ProfiLER protocol (NCT 01774409). Material and Methods: From 05 mars 2013 to 31 august 2014, 58 patients with advanced cancer treated with one of 7 MTKIs were included in the ProfiLER trial and split into one discovery cohort (n = 13),2 validation cohort (n = 12 and 33). We defined MTKI sensitive as patients who had complete response, partial response, stable disease as best response and resistant as those having progressive disease as best response at 8 weeks. CGH array and mutation sequencing were used to generate molecular profiles. The sum of gains on target genes was termed as tumor target charge (TTC), while the sum of losses were termed tumor target loss (TTL). A prediction algorithm (Called SUMSCAN) based on combination of TTC and TTL were conceived and validated in 2 independent validation cohorts. Results: MTKI sensitive tumor presents a characteristic SCNA profile including high TTC, low TTL, global gained profile, while MTKI resistant tumors presents the opposite (See table). Moreover, PIK3CA hotspot mutations were found only in regorafenib resistant colorectal cancer, but none of the responsive tumor. SUMSCAN favorable patients achieved a longer progression free survival and overall survival (data not shown). Discovery cohort
1st Validation cohort
2nd Validation cohort
Total Age Tumor type
13 63.1 (40.7–75.8) CRC
12 56.0 (41.6–70.5) CRC, STS
Relevant MTKI
Regorafenib
Regorafenib
33 55.9 (24.6–76.1) CRC, STS, HCC, Thyroid differentiated carcinoma RCC Regorafenib, sorafenib, sunitinib, pazopanib, axitinib, vandetanib, cabozantinib
Sensitive vs Resistant (mean, range) Number 6 vs 7 TTC 6.8 (1−14) vs 2.8 (0−7) TTL 1.3 (0−5) vs 2.4 (0−7) SUMSCAN prediction / accuracy
5 vs 7 8.2 (2−14) vs 2.3 (0−5) 1.6 (0−5) vs 3 (0−6) 91.7%
20 vs 13 2.3 (0−9) vs 0.7 (0−3) 0.75 (0−4) vs 1.5 (0−3) 75.8%
Conclusions: The sum of target gene copy number alterations enables to predict for response of cancer patients to MTKI. No conflict of interest. 210 POSTER Genetic and epigenetic factors affect RET gene expression in breast cancer cell lines and influence survival in patients P. Griseri1 , O. Garrone2 , A. Lo Sardo1 , M. Monteverde3 , M. Rusmini1 , F. Tonissi3 , M. Merlano2 , P. Bruzzi4 , I. Ceccherini1 , C. Lo Nigro3 . 1 G. Gaslini Institute, Medical Genetics, Genoa, Italy; 2 S. Croce & Carle Teaching Hospital, Medical Oncology, Cuneo, Italy; 3 S. Croce & Carle Teaching Hospital, Laboratory Cancer Genetics and Translational Oncology, Cuneo, Italy; 4 San Martino IST, Clinical Epidemiology, Genoa, Italy Background: The RET proto-oncogene encodes for a transmembrane tyrosine kinase receptor whose signalling pathway is activated by the binding with one of its ligands (GDNF, neurturin, artemin or persephin) and coreceptors (Gfra1−4). Aberrant RET signaling is oncogenic, as demonstrated by its involvement in different human cancers. In recent years, a large body of evidence has demonstrated that RET is overexpressed in a subset of human breast cancer. In particular, in Estrogen Receptor-positive
(ER+) breast tumors, overexpression of RET resulted to be involved in endocrine resistance. Materials and Methods: In the present study we have investigated the molecular mechanisms (either genomic, transcriptional or post transcriptional) which underlie RET overexpression, and its possible modulation in breast cancer, in two ER+ cell lines, MCF7 and T47D, known to express high e low levels of RET mRNA. Looking for genetic variants responsible of variable RET expression, we have also carried out a pilot association study in 93 ER+ breast cancer patients. Results: We noticed that RET expression is similarly modulated by estrogens in MCF7 and T47D while treatments with TNF, IL-8 and deacetylase inhibitors differently affect RET expression in breast cell lines. After sequencing ER-responding regions and a known enhancer in intron 1 at RET locus, we identified two single nucleotide variants rs12247450 and rs2435357, whose genotype is different between MCF7 and T47D, possibly accounting for the opposite RET expression pattern. In particular, SNP rs2435357 (also known as RET+3 polymorphism) is located at +5kb inside intron 1 and shown to be associated with reduced expression of the gene. T47D cells carry the genotype associated with lower expression of the RET gene and this data can explain both the reduced expression of the RET gene and the different response to Sodium Butyrate. We carried out a pilot study genotyping for this SNP in a cohort of 93 ER + early breast cancer patients. We observed a statistically significant increased OS in patients with one of the variant alleles (CT or TT) of rs2435357 in comparison to those carrying the CC wild type allele. The association was confirmed in multivariate analysis, where nodal status, grading, HER2 status and Ki67 were adjusted for (HR = 0.243, 95%; CI=0.088–0.675; P=0,007). Conclusions: Our data are fully consistent with the observation that RET overexpression is associated to poor prognosis in ER+ breast cancer and strongly candidate this SNP as prognostic factor in ER+ early breast cancer. These findings can be useful to deepen into the role played by RET in breast tumorigenesis and stand RET as a potential candidate molecular target for breast cancer treatment. No conflict of interest. 211 POSTER Screening for the prevalence of EGFR and ALK mutations in lung adenocarcinoma patients in the levant area, a prospective analysis A. Tfayli1 , H. Rafei1 , M. Khalil2 , A. Mina1 , N. Fakhreddin3 , R. Mahfouz4 , F. Farhat5 , H. Rabee6 , S. Hamouri7 , H. Dbouk8 , Z. Salem1 , N. Saghir1 , A. Shamseddine1 , N. Bitar9 , A. Mougharbil10 , J. Makarem11 , W. Daw11 . 1 American University of Beirut Medical Center, Internal Medicine, Beirut, Lebanon; 2 University of Miami Miller School of Medicine, West Palm Beach Regional Campus, Internal Medicine, West Palm Beach, USA; 3 Hammoud Hospital University Medical Center, Pathology, Saida, Lebanon; 4 American University of Beirut Medical Center, Pathology, Beirut, Lebanon; 5 Hammoud Hospital University Medical Center, Internal Medicine, Saida, Lebanon; 6 Koufa Hospital, Internal Medicine, Koufa, Iraq; 7 Jordan University of Science and Technology, Surgery, Amman, Jordan; 8 Jabal Amel Hospital, Internal Medicine, Jabal Amel, Lebanon; 9 Sahel General Hospital, Internal Medicine, Beirut, Lebanon; 10 Makassed General Hospital, Internal Medicine, Beirut, Lebanon; 11 Ain Wazein Hospital, Internal Medicine, Shouf, Lebanon Background: Recent evidence suggests that a significant percentage of lung adenocarcinomas have a driver mutation that is responsible for the development of the tumor and promotion of its growth and spread. Significant variation in the prevalence of these mutations has been documented with race and smoking status being two major factors (more common in Asians and/or non-smokers). To date, no data has prospectively assessed the prevalence of these mutations in a Middle Eastern population. Our study is a multicenter prospective study of formalin fixed paraffin embedded (FFPE) tissues from patients diagnosed with lung adenocarcinoma to assess the prevalence of EGFR and ALK mutations in the Levant Area. Material and Methods: Patients with lung adenocarcinoma were prospectively enrolled as part of a multicenter, multinational study. Tumors were tested for various EGFR mutations in exons 18−21 by PCR and for EML4-ALK translocation by FISH Break-Apart test. Study was open in 10 sites in Lebanon, Jordan and Iraq and was restricted to patients of Middle Eastern origin. Results: 210 patients have been recruited in total. 139 (66.2%) males and 71 females (33.8%) with a mean age of 63.4 years. EGFR testing was done in 205 (97.6%) patients and has been found to be Wild type in 173 (84.4%) patients, mutated in 32 (15.6%). Tissue has been insufficient in 5 (2.4%) of the samples. 46.9% of EGFR positive patients are nonsmoker and 62.5% are females as opposed to 22.4% and 33.8% respectively of the
Abstracts
received honoraria from GSK and Roche, and a travel grant from Sandoz. PLP received honoraria from Amgen, Merk Serono, Sanofi and Integragen. LG and PAJ received travel grant from Pfizer. 209 POSTER The sum of gains and losses of genes encoding for protein tyrosine kinase targets predicts response to multi-kinase inhibitor treatment X. Jiang1 , D. Pissaloux1 , C. De La Fouchardiere2 , F. Desseigne2 , Q. Wang1 , M.E. Fondrevelle3 , P. Cassier2 , C. Seigne4 , D. Perol4 , I. Ray-Coquard2 , A. Le Cesne5 , N. Penel6 , O. Tredan2 , J.Y. Blay2 . 1 ´ Centre Leon Berard, Department of Translational Research, Lyon, ´ France; 2 Centre Leon Berard, Department of Medical Oncology, Lyon, 3 ´ France; Centre Leon Berard, Department of Pathobiology, Lyon, France; 4 ´ Centre Leon Berard, Department of Clinical Research, Lyon, France; 5 Gustave Roussy, Department of Medical Oncology, Villejuif, France; 6 Centre Oscar Lambret, Department of Medical Oncology, Lille, France Background: Validated predictive biomarkers for multi-tyrosine kinase inhibitors (MTKI) efficacy are lacking. We hypothesized that interindividual response variability is partially dependent on the somatic DNA copy number alterations (SCNAs), especially those of genes encoding for the protein tyrosine targeted by MTKI (called target genes). Genomic alteration was investigated in MTKI responsive and non responsive patients with different histological subtypes included in the ProfiLER protocol (NCT 01774409). Material and Methods: From 05 mars 2013 to 31 august 2014, 58 patients with advanced cancer treated with one of 7 MTKIs were included in the ProfiLER trial and split into one discovery cohort (n = 13),2 validation cohort (n = 12 and 33). We defined MTKI sensitive as patients who had complete response, partial response, stable disease as best response and resistant as those having progressive disease as best response at 8 weeks. CGH array and mutation sequencing were used to generate molecular profiles. The sum of gains on target genes was termed as tumor target charge (TTC), while the sum of losses were termed tumor target loss (TTL). A prediction algorithm (Called SUMSCAN) based on combination of TTC and TTL were conceived and validated in 2 independent validation cohorts. Results: MTKI sensitive tumor presents a characteristic SCNA profile including high TTC, low TTL, global gained profile, while MTKI resistant tumors presents the opposite (See table). Moreover, PIK3CA hotspot mutations were found only in regorafenib resistant colorectal cancer, but none of the responsive tumor. SUMSCAN favorable patients achieved a longer progression free survival and overall survival (data not shown). Discovery cohort
1st Validation cohort
2nd Validation cohort
Total Age Tumor type
13 63.1 (40.7–75.8) CRC
12 56.0 (41.6–70.5) CRC, STS
Relevant MTKI
Regorafenib
Regorafenib
33 55.9 (24.6–76.1) CRC, STS, HCC, Thyroid differentiated carcinoma RCC Regorafenib, sorafenib, sunitinib, pazopanib, axitinib, vandetanib, cabozantinib
Sensitive vs Resistant (mean, range) Number 6 vs 7 TTC 6.8 (1−14) vs 2.8 (0−7) TTL 1.3 (0−5) vs 2.4 (0−7) SUMSCAN prediction / accuracy
5 vs 7 8.2 (2−14) vs 2.3 (0−5) 1.6 (0−5) vs 3 (0−6) 91.7%
20 vs 13 2.3 (0−9) vs 0.7 (0−3) 0.75 (0−4) vs 1.5 (0−3) 75.8%
Conclusions: The sum of target gene copy number alterations enables to predict for response of cancer patients to MTKI. No conflict of interest. 210 POSTER Genetic and epigenetic factors affect RET gene expression in breast cancer cell lines and influence survival in patients P. Griseri1 , O. Garrone2 , A. Lo Sardo1 , M. Monteverde3 , M. Rusmini1 , F. Tonissi3 , M. Merlano2 , P. Bruzzi4 , I. Ceccherini1 , C. Lo Nigro3 . 1 G. Gaslini Institute, Medical Genetics, Genoa, Italy; 2 S. Croce & Carle Teaching Hospital, Medical Oncology, Cuneo, Italy; 3 S. Croce & Carle Teaching Hospital, Laboratory Cancer Genetics and Translational Oncology, Cuneo, Italy; 4 San Martino IST, Clinical Epidemiology, Genoa, Italy Background: The RET proto-oncogene encodes for a transmembrane tyrosine kinase receptor whose signalling pathway is activated by the binding with one of its ligands (GDNF, neurturin, artemin or persephin) and coreceptors (Gfra1−4). Aberrant RET signaling is oncogenic, as demonstrated by its involvement in different human cancers. In recent years, a large body of evidence has demonstrated that RET is overexpressed in a subset of human breast cancer. In particular, in Estrogen Receptor-positive
(ER+) breast tumors, overexpression of RET resulted to be involved in endocrine resistance. Materials and Methods: In the present study we have investigated the molecular mechanisms (either genomic, transcriptional or post transcriptional) which underlie RET overexpression, and its possible modulation in breast cancer, in two ER+ cell lines, MCF7 and T47D, known to express high e low levels of RET mRNA. Looking for genetic variants responsible of variable RET expression, we have also carried out a pilot association study in 93 ER+ breast cancer patients. Results: We noticed that RET expression is similarly modulated by estrogens in MCF7 and T47D while treatments with TNF, IL-8 and deacetylase inhibitors differently affect RET expression in breast cell lines. After sequencing ER-responding regions and a known enhancer in intron 1 at RET locus, we identified two single nucleotide variants rs12247450 and rs2435357, whose genotype is different between MCF7 and T47D, possibly accounting for the opposite RET expression pattern. In particular, SNP rs2435357 (also known as RET+3 polymorphism) is located at +5kb inside intron 1 and shown to be associated with reduced expression of the gene. T47D cells carry the genotype associated with lower expression of the RET gene and this data can explain both the reduced expression of the RET gene and the different response to Sodium Butyrate. We carried out a pilot study genotyping for this SNP in a cohort of 93 ER + early breast cancer patients. We observed a statistically significant increased OS in patients with one of the variant alleles (CT or TT) of rs2435357 in comparison to those carrying the CC wild type allele. The association was confirmed in multivariate analysis, where nodal status, grading, HER2 status and Ki67 were adjusted for (HR = 0.243, 95%; CI=0.088–0.675; P=0,007). Conclusions: Our data are fully consistent with the observation that RET overexpression is associated to poor prognosis in ER+ breast cancer and strongly candidate this SNP as prognostic factor in ER+ early breast cancer. These findings can be useful to deepen into the role played by RET in breast tumorigenesis and stand RET as a potential candidate molecular target for breast cancer treatment. No conflict of interest. 211 POSTER Screening for the prevalence of EGFR and ALK mutations in lung adenocarcinoma patients in the levant area, a prospective analysis A. Tfayli1 , H. Rafei1 , M. Khalil2 , A. Mina1 , N. Fakhreddin3 , R. Mahfouz4 , F. Farhat5 , H. Rabee6 , S. Hamouri7 , H. Dbouk8 , Z. Salem1 , N. Saghir1 , A. Shamseddine1 , N. Bitar9 , A. Mougharbil10 , J. Makarem11 , W. Daw11 . 1 American University of Beirut Medical Center, Internal Medicine, Beirut, Lebanon; 2 University of Miami Miller School of Medicine, West Palm Beach Regional Campus, Internal Medicine, West Palm Beach, USA; 3 Hammoud Hospital University Medical Center, Pathology, Saida, Lebanon; 4 American University of Beirut Medical Center, Pathology, Beirut, Lebanon; 5 Hammoud Hospital University Medical Center, Internal Medicine, Saida, Lebanon; 6 Koufa Hospital, Internal Medicine, Koufa, Iraq; 7 Jordan University of Science and Technology, Surgery, Amman, Jordan; 8 Jabal Amel Hospital, Internal Medicine, Jabal Amel, Lebanon; 9 Sahel General Hospital, Internal Medicine, Beirut, Lebanon; 10 Makassed General Hospital, Internal Medicine, Beirut, Lebanon; 11 Ain Wazein Hospital, Internal Medicine, Shouf, Lebanon Background: Recent evidence suggests that a significant percentage of lung adenocarcinomas have a driver mutation that is responsible for the development of the tumor and promotion of its growth and spread. Significant variation in the prevalence of these mutations has been documented with race and smoking status being two major factors (more common in Asians and/or non-smokers). To date, no data has prospectively assessed the prevalence of these mutations in a Middle Eastern population. Our study is a multicenter prospective study of formalin fixed paraffin embedded (FFPE) tissues from patients diagnosed with lung adenocarcinoma to assess the prevalence of EGFR and ALK mutations in the Levant Area. Material and Methods: Patients with lung adenocarcinoma were prospectively enrolled as part of a multicenter, multinational study. Tumors were tested for various EGFR mutations in exons 18−21 by PCR and for EML4-ALK translocation by FISH Break-Apart test. Study was open in 10 sites in Lebanon, Jordan and Iraq and was restricted to patients of Middle Eastern origin. Results: 210 patients have been recruited in total. 139 (66.2%) males and 71 females (33.8%) with a mean age of 63.4 years. EGFR testing was done in 205 (97.6%) patients and has been found to be Wild type in 173 (84.4%) patients, mutated in 32 (15.6%). Tissue has been insufficient in 5 (2.4%) of the samples. 46.9% of EGFR positive patients are nonsmoker and 62.5% are females as opposed to 22.4% and 33.8% respectively of the