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PDF file - 9.8MB, Supplementary Figure S1. TCF3 expression levels in breast cancer cell lines. Supplementary Figure S2. Effects of TCF3 on cell proliferation, apoptosis and tumor growth. Supplementary Figure S3. TCF3 induces changes in the expression of EMT-associated genes. Supplementary Figure S4. Gene expression changes in TCF3-silenced cells. Supplementary Figure S5. Wnt-related gene expression changes in TCF3 silenced cells. Supplementary Figure S6. Inducible Tcf3 transgenic mice. Supplementary Figure S7. Tcf3 inhibition of ductal growth is reversible. Supplementary Figure S8. Tcf3 induces EMT-associated genes in the mouse mammary gland

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Background The prognosis of gastric cancer (GC) is poor with a median overall survival (OS) of less than 12 months in advanced-stage disease. The search for distinct genetic subgroups of GC patients and predictive biomarkers is ongoing. While BRCA1 or BRCA2 germline mutations (gBRCAm) have potential therapeutic implications in ovarian, breast and pancreatic cancers, their significance in GC patients has not been established. Patients and methods A retrospective multi-center data analysis of GC patients with gBRCAm was conducted, detailing the clinical characteristics and disease course in this unique subset of patients. Results Ten GC patients with gBRCAm were identified, six of them with metastatic disease. The median OS of all ten GC patients was 47.5 (13-192) months. Median OS for patients diagnosed with operable disease was 55.5 (13-192) months and of the patients with metastatic disease (calculated from metastatic disease diagnosis) 32 (15-52) months with an exceptional 1-, 2- and 3-year survival rate of 100%, 83.3% and 50%, respectively. Conclusion These preliminary data suggest that gBRCAm in GC patients are associated with a favorable prognosis. Furthermore, gBRCAm might be a predictive biomarker to DNA-damaging agents response in GC patients, similarly to its established role in other malignancies. Further research is needed to confirm our findings.

To study the anticipation phenomenon among hereditary breast cancer patients, by evaluating trends in age at diagnosis and phenotype of breast cancer across two successive generation pairs of BRCA1/2 mutation carriers/non-carriers with breast cancer after reports of an earlier age of diagnosis in successive generations among BRCA1/2 mutation carrier families.A retrospective cohort study. Patient characteristics, pathologic data and survival were compared between mothers and daughters and between carriers and non-carriers.Overall, 126 patients were found, who formed 67 pairs of mothers and daughters diagnosed with breast cancer and genetically tested for BRCA mutations. Age at diagnosis was significantly younger in the daughter versus mother generation, in both groups of BRCA carriers/non-carriers. Tumor characteristics were not different between mothers and daughters. Survival analysis revealed a not significant better outcome for the daughter generation versus the mother generation.Breast cancer appeared to be diagnosed at an earlier age in successive generations among BRCA mutation carriers and non-carriers. The fact that we also observed a downshift at age of diagnosis in non-carrier pairs emphasizes that other factors (environmental, lifestyle, or social) may influence the age at diagnosis.

The likelihood of recurrence in breast cancer patients with hormone receptor-positive (HR-positive) tumors is influenced by clinical, histopathological, and molecular features. Recent studies suggested that activated STAT3 (pSTAT3) might serve as a biomarker of outcome in breast cancer patients. In the present work, we have analyzed the added value of pSTAT3 to OncotypeDx Recurrence Score (RS) in patient prognostication. We have found that patients with low RS (

Introduction Traditionally, women with family history of breast and ovarian cancer, or young age at presentation, are referred to BRCA1\2 mutation testing. The growing information regarding other genes involved in predisposition to breast cancer (regardless of specific family history) and the potential impact of germline alterations on surgical and oncologic management led us to establish a model to perform a quick multigene mutation evaluation among breast cancer patients at our cancer institute. Methods All breast cancer patients that required genetic counselling (according to treating oncologist/surgeon’s judgement) were referred, after thorough explanation and signing informed-consent forms, to a 'genetic coordinator' and 'genetic nurse'. They provided each patient with a second pre-test explanation and ordered wide hereditary cancer gene panel (83-genes, Invitae). Results were given within 2-4 weeks. All patients\results were registered centrally at our center and discussed at routine Onco-genetic Multidisciplinary Board (OMB). A referral for formal genetic counselling is offered to all patients, and rapid track is arranged for patients with positive results. Results During a 12-months pilot period, 130 breast cancer patients were examined via the model, and 109 results were received; 44 patients (41%) had no abnormal findings, 50 patients (45%) had variants of uncertain clinical significance, and 15 patients (14%) were found carriers of pathogenic variants (ATM n=2, BRCA1 n=1, BRCA2 n=2, CHEK2 n=3, MUTYH n=1, PALB2 n=2, RAD50 n=1, RECQL4 n=1, TP53 n=2). Among this group, 3 patients (20%) had immediate panel-driven treatment decisions: Mastectomy to avoid radiotherapy (RAD50 carrier), oophorectomy during lumpectomy (BRCA2 carrier), and bilateral risk-reducing mastectomy instead of lumpectomy (TP53 carrier). Conclusions The creation of an Express-model for broad germline analysis allows to obtain rapid and meaningful results with impact on treatment. We believe that the Express-model should be considered as an integral tool of multimodal care of breast cancer patients. Citation Format: Albert Grinshpun, Einat Carmon, Luna Kaduri, Aviad Zick, Shiri Shkedi, Edna Shalom, Avital Granit, Liza Monas, Tamar Shaldovsky, Tamar Hamburger, Vardiella Meiner, Tamar Peretz. Express model of hereditary multigene mutation analysis in breast cancer patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-14-06.

Introduction: There are several reports of an earlier age of diagnosis in successive generation among BRCA1/2 mutation carriers' families, which may indicate an anticipation phenomenon among hereditary breast cancer (BC) patients. Here, we assess for evidence of anticipation, by evaluating trends in age at diagnosis and phenotype of BC across two successive generations pairs of BRCA1/2 mutation carriers and non-carriers with BC. Methods: The study retrospective cohort was extracted from the oncogenetic database of the Sharett Institute of Oncology (established in 1995), Hadassah Medical Center (N=14,450). Patient characteristics and pathological data were collected from the oncogenetic database, from patients' medical files and pathological reports and compared using independent student's t-test and chi-square tests. Survival analysis was carried out in order to compare survival rates between mothers (M) and daughters (D) and between carriers and non-carriers. Results: The final study cohort includes 126 patients that forms 67 pairs of M and D, both diagnosed with BC and genetically tested for BRCA1/2 mutations. Among M, 35 were BRCA carriers (52%) and 35 were BRCA carriers in the D group (52%). Several patients' characteristics (table 1) were different between M & D. Age at diagnosis of all BRCA carriers was significantly younger in all BRCA carriers vs. wildtype. Moreover, age at diagnosis was significantly younger in D vs. M, in both groups of BRCA carriers and non-carriers. In addition, age at first pregnancy was significantly younger in M vs. D in both groups. Furthermore, tumor phenotype was also different; BRCA carriers had more invasive ductal histology vs. non-carriers, (94% vs. 80%, p=0.024), more triple negative BC, (40% vs. 6%) and more grade 3 BC (60% vs. 18%, P=0.001). Survival analysis revealed significantly better outcomes for D vs. M among BRCA1/2 carriers with triple-negative BC (5-year survival of 91% and 75%, respectively). Conclusions: Breast cancer appeared to be diagnosed at an earlier age in successive generation among BRCA mutation carriers and non-carriers. This observation could be attributed to gene-environment interactions. The fact that we also observed a downshift at age of diagnosis in non-carriers mothers and daughters, emphasizes that other factors (environmental, life style or social) may influence the age at diagnosis. Significantly better survival among second generation of BRCA carriers in triple negative BC can be attributed mainly to better therapy. Further studies are warranted to explore the younger age of the second generation at presentation, and additional means to further improve outcome. Table 1- Patient's CharacteristicsBRCA1/2 carriers BRCA non-carriersParameterMotherDaughterP valueMotherDaughterP valueAge at diagnosis of BC48.2337.38 Citation Format: Aviya Kedmi, Luna Kaduri, Albert Grinshpun, Shiri Shkedi, Vardiella Meiner, Tamar Hamburger, Ofra Maimon, Tamar Peretz. Genetic anticipation among BRCA1/2 mutation carriers with breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-08-12.

Background The prognosis of gastric cancer (GC) is poor with a median overall survival (OS) of less than 12 months in advanced-stage disease. The search for distinct genetic subgroups of GC patients and predictive biomarkers is ongoing. While BRCA1 or BRCA2 germline mutations (gBRCAm) have potential therapeutic implications in ovarian, breast and pancreatic cancers, their significance in GC patients has not been established. Patients and Methods A retrospective multi-center data analysis of GC patients with gBRCAm was conducted, detailing the clinical characteristics and disease course in this unique subset of patients. Results Ten GC patients with gBRCAm were identified, six of them with metastatic disease. The median OS of all ten GC patients was 47.5 (13–192) months. Median OS for patients diagnosed with operable disease was 55.5 (13–192) months and of the patients with metastatic disease (calculated from metastatic disease diagnosis) 32 (15–52) months with an exceptional 1-, 2- and 3-year survival rate of 100%, 83.3% and 50%, respectively. Conclusion These preliminary data suggest that gBRCAm in GC patients are associated with a favorable prognosis. Furthermore, gBRCAm might be a predictive biomarker to DNA-damaging agents response in GC patients, similarly to its established role in other malignancies. Further research is needed to confirm our findings.

Mutations in the BRCA1/2 genes were recently shown to be associated with an increased risk for colorectal cancer. We characterized the largest cohort available of BRCA1/2 mutation carriers with colorectal cancer. We analyzed 32 patients with lower gastrointestinal cancers and germline BRCA1/2 mutations from two large academic hospital registries; 91% of patients were of Ashkenazi ancestry, 78% were women, and 62.5% were carriers of BRCA1 gene mutations. A high percentage of colorectal tumors (34.5%) had a mucinous histology and were located atypically in the left colon. Two patients had anal cancer with unusual histology and an additional patient had mucinous small bowel carcinoma. Gene expression analysis showed significant correlation between the gene signatures of left mucinous colorectal cancer and basal-like breast cancer. Our results imply that Ashkenazi BRCA1/2 mutation carriers with colorectal cancer might have unique characteristics with a high rate of left-sided, mucinous histology colorectal cancer, and possibly anal carcinoma. This report suggests a phenotypic influence of defects in DNA repair genes on colorectal tumors.

Gene expression assays are widely used to predict risk of recurrence in early breast cancer (BC). We report the 21-gene expression assay (Oncotype Dx) recurrence score (RS) distribution of 27 BRCA carriers with estrogen receptor (ER) positive BCs, identified at Hadassah Medical Center, combined with 2 previous studies. Treatment decision and outcomes of the 27 BRCA carriers were compared with an Israeli cohort of 1594 patients published recently. We found Oncotype Dx RS low ( 30) among 12 (21.4%), 23 (41.1%) and 21 (37.5%) of 56 BRCA1 carriers compared with 15 (17.2%), 49 (56.3%) and 23 (26.4%) of 87 BRCA2 carriers (p=0.2). The corresponding distribution in a population of 82,434 women published by Genomic Health was 53.4%, 36.3% and 10.3% for low, intermediate and high RS (p< 0.001 for BRCA1 and BRCA2). Treatment decision regarding chemotherapy according to RS was similar in BRCA1, BRCA2 and the control group. Two of 27 carriers had distant recurrence: a BRCA1 carrier with RS of 18 and a BRCA2 carrier with RS of 22; both have an excellent response to chemotherapy. We found an approximately 3 fold increased rate of high RS among BRCA1 and 2 carriers with ER positive BC compared with the general BC population. These data might indicate that hormone positive BC in BRCA carriers are molecularly unique. The surprisingly good response to chemotherapy in the metastatic setting in 2 patients may suggest that the predictive value of low-intermediate RS in carriers merits further studies. This article is protected by copyright. All rights reserved.

// Rachel Goldberg 1 , Amir Sonnenblick 1 , Esther Hermano 1 , Tamar Hamburger 1 , Amichay Meirovitz 1 , Tamar Peretz 1 , Michael Elkin 1 1 Sharett Institute, Hadassah-Hebrew University Medical Center, Jerusalem 91120, Israel Correspondence to: Michael Elkin, email: melkin@hadassah.org.il Keywords: heparanase, insulin receptor, breast carcinoma, diabetes Received: September 12, 2016 Accepted: December 01, 2016 Published: December 27, 2016 ABSTRACT Recently, growing interest in the potential link between metabolic disorders (i.e., diabetes, obesity, metabolic syndrome) and breast cancer has mounted, including studies which indicate that diabetic/hyperinsulinemic women have a significantly higher risk of bearing breast tumors that are more aggressive and associated with higher death rates. Insulin signaling is regarded as a major contributor to this phenomenon; much less is known about the role of heparan sulfate-degrading enzyme heparanase in the link between metabolic disorders and cancer. In the present study we analyzed clinical samples of breast carcinoma derived from diabetic/non-diabetic patients, and investigated effects of heparanase on insulin signaling in breast carcinoma cell lines, as well as insulin-driven growth of breast tumor cells. We demonstrate that heparanase activity leads to enhanced insulin signaling and activation of downstream tumor-promoting pathways in breast carcinoma cells. In agreement, heparanase enhances insulin-induced proliferation of breast tumor cells in vitro . Moreover, analyzing clinical data from diabetic breast carcinoma patients, we found that concurrent presence of both diabetic state and heparanase in tumor tissue (as opposed to either condition alone) was associated with more aggressive phenotype of breast tumors in the patient cohort analyzed in our study (two-sided Fisher’s exact test; p=0.04). Our findings highlight the emerging role of heparanase in powering effect of hyperinsulinemic state on breast tumorigenesis and imply that heparanase targeting, which is now under intensive development/clinical testing, could be particularly efficient in a growing fraction of breast carcinoma patients suffering from metabolic disorders.

It has been shown that various tRNA synthetases exhibit non-canonical activities unrelated to their original role in translation. We have previously described a signal transduction pathway in which serine 207 phosphorylated lysyl-tRNA synthetase (P-s207 LysRS) is released from the cytoplasmic multi-tRNA synthetase complex (MSC) into the nucleus, where it activates the transcription factor MITF in stimulated cultured mast cells and cardiomyocytes. Here we describe a similar transformation of LysRS due to EGFR signaling activation in human lung cancer. Our data shows that activation of the EGFR results in phosphorylation of LysRS at position serine 207, its release from the MSC and translocation to the nucleus. We then generated a P-s207 LysRS rabbit polyclonalantibody and tested 242 tissue micro-array samples derived from non-small-cell lung cancer patients. Highly positive nuclear staining for P-s207 LysRS was noted in patients with EGFR mutations as compared to WT EGFR patients and was associated with improved mean disease-free survival (DFS). In addition, patients with mutated EGFR and negative lymph node metastases had better DFS when P-s207 LysRS was present in the nucleus. The data presented strongly suggests functional and prognostic significance of P-s207 LysRS in non-small-cell lung cancer.

Hereditary cancer comprises more than 10% of all breast cancer cases. In patients with a family history suggestive of a hereditary component, a mutation is often identified in the high penetrant genesBRCA1andBRCA2. Several founder mutations have been detected in some Jewish communities, yet noBRCA1/2founder mutation had been known in Kurdish Jews. Here, we describe the validation of a 22 hereditary cancer gene panel and aBRCA1mutation found in 4 women from 2 unrelated Kurdish Jewish families utilizing this gene panel.A panel spanning the coding sequences of 22 familial cancer-related genes was planned. Genomic DNA was taken to create libraries using this panel, which were then sequenced using the Ion Torrent PGM. The panel's validity in detecting mutations was tested on 25 samples with previously identified point mutations in theBRCA1,BRCA2,MLH1andPMS2genes; the panel did not test for large deletions or insertions. All previously identified mutations were detected. Next, a different set of 40 cancer patients of Kurdish Jewish descent diagnosed with cancer before the age of 50 years was tested. We identified theBRCA1mutation,c.224_227delAAAG(dbSNP ID rs80357697), in 4 women from 2 unrelated Jewish Kurdish families. The probands were diagnosed with cancer at a young age and had significant family history, suggesting a founder mutation in this population. We suggest testing Kurdish Jewish women with a personal or family history of breast and/ or ovarian cancer for this mutation.

Naama Halpern,1 Yael Goldberg,2 Luna Kadouri,2 Morasha Duvdevani,2 Tamar Hamburger,2 Tamar Peretz,2 Ayala Hubert2 1Institute of Oncology, The Chaim Sheba Medical Center, Tel Hashomer, Israel; 2Sharett Institute of Oncology, Hadassah Medical Center, Hebrew University, Jerusalem, Israel Background: The prognostic and predictive significance of the high-level microsatellite instability (MSI-H) phenotype in various malignancies is unclear. We describe the characteristics, clinical course, and outcomes of patients with MSI-H malignancies treated in a real-life hospital setting.Patients and methods: A retrospective analysis of MSI-H cancer patient files was conducted. We analyzed the genetic data, clinical characteristics, and oncological treatments, including chemotherapy and surgical interventions.Results: Clinical data of 73 MSI-H cancer patients were available. Mean age at diagnosis of first malignancy was 52.3years. Eight patients (11%) had more than four malignancies each. Most patients (76%) had colorectal cancer (CRC). Seventeen patients (23%) had only extracolonic malignancies. Eighteen women (36%) had gynecological malignancy. Nine women (18%) had breast cancer. Mean follow-up was 8.5years. Five-year overall survival and disease-free survival of all MSI-H cancer patients from first malignancy were 86% and 74.6%, respectively. Five-year overall survival rates of stage 2, 3, and 4 MSI-H CRC patients were 89.5%, 58.4%, and 22.9%, respectively.Conclusion: Although the overall prognosis of MSI-H cancer patients is favorable, this advantage may not be maintained in advanced MSI-H CRC patients. Keywords: microsatellite instability, malignancy, treatment, outcome

50-70% of tumors of the so called "triple negative" subtype of breast cancer express EGFR. We hypothesized that addition of anti EGFR to Taxanes will result in increased effectiveness in EGFR expressing tumors. Here we set out to obtain data regarding the safety, tolerability and also the effectivity of the combination of weekly Taxane treatments with Cetuximab -an anti EGFR antibody in this subgroup of breast cancer. 18 triple negative breast cancer patients were treated with weekly Cetuximab and Taxane therapy. Addition of Cetuximab resulted in controllable Dermatologic toxicity in most patients -with grade 3 in two patients. Some impressive results were noted including one CR, one near CR and regression of chemotherapy and radiation resistance skin metastasis. Median TTF -and overall survival -6 and 12 months. Administration of Taxane Cetuximab weekly therapy for triple negative breast cancer patients is feasible. Use of anti EGFR-Taxane combinations should be assessed in larger clinical trials in this patient population perhaps in a similar manner to the lung cancer patients only in those with strong EGFR expression.

Previous studies indicated that BRCA haploinsufficiency was associated with activation of the EGF receptor (EGFR) signaling pathway and increased proliferative activity in mammary epithelial cells of healthy women. We hypothesized that these processes might be reflected in the expression of serologic soluble EGFR (sEGFR) and thymidine kinase 1 (TK1) activity, which signal the initial and final steps of the proliferative pathway, respectively. We found that healthy carriers of BRCA1/2 mutations (n = 80) showed a significantly higher TK1 activity than age-matched controls (P = 0.0003), and TK1 activity was similar in women with BRCA1 and BRCA2 mutations (P = 0.74). The sEGFR concentration was significantly higher in women with BRCA1 than in controls and BRCA2 mutation (P = 0.013 and 0.002, respectively). During follow-up, four of 80 BRCA1/2 mutation carriers developed breast cancer. These women showed a significantly higher TK1 activity and somewhat higher sEGFR concentrations than the other 76 BRCA1/2 carriers (P = 0.04 and 0.09, respectively). All tumors were negative for ovarian hormone receptors, but showed a high EGFR expression. This study was limited by the short-term follow-up (mean, 27 months; range, 5–45), which resulted in a small sample size. Women with BRCA1 and BRCA2 mutations that had undergone risk-reducing bilateral salpingo-oophorectomy (BSO) showed significantly lower sEGFR compared with those without surgery (P = 0.007 and 0.038, respectively). Larger, prospective studies are warranted to investigate whether TK1 and sEGFR measurements may be useful for identifying healthy BRCA1/2 carriers with high risk of developing breast cancer; moreover, sEGFR measurements may serve as effective tools for assessing risk before and after BSO. Cancer Epidemiol Biomarkers Prev; 22(11); 2110–5. ©2013 AACR.

It has been shown that various tRNA synthetases exhibit non-canonical activities unrelated to their original role in translation. We have previously described a signal transduction pathway in which serine 207 phosphorylated lysyl-tRNA synthetase (P-s207 LysRS) is released from the cytoplasmic multi-tRNA synthetase complex (MSC) into the nucleus, where it activates the transcription factor MITF in stimulated cultured mast cells and cardiomyocytes. Here we describe a similar transformation of LysRS due to EGFR signaling activation in human lung cancer. Our data shows that activation of the EGFR results in phosphorylation of LysRS at position serine 207, its release from the MSC and translocation to the nucleus. We then generated a P-s207 LysRS rabbit polyclonalantibody and tested 242 tissue micro-array samples derived from non-small-cell lung cancer patients. Highly positive nuclear staining for P-s207 LysRS was noted in patients with EGFR mutations as compared to WT EGFR patients and was associated with improved mean disease-free survival (DFS). In addition, patients with mutated EGFR and negative lymph node metastases had better DFS when P-s207 LysRS was present in the nucleus. The data presented strongly suggests functional and prognostic significance of P-s207 LysRS in non-small-cell lung cancer.

Regulatory factors controlling stem cell identity and self-renewal are often active in aggressive cancers and are thought to promote their growth and progression. TCF3 (also known as TCF7L1) is a member of the TCF/LEF transcription factor family that is central in regulating epidermal and embryonic stem cell identity. We found that TCF3 is highly expressed in poorly differentiated human breast cancers, preferentially of the basal-like subtype. This suggested that TCF3 is involved in the regulation of breast cancer cell differentiation state and tumorigenicity. Silencing of TCF3 dramatically decreased the ability of breast cancer cells to initiate tumor formation, and led to decreased tumor growth rates. In culture, TCF3 promotes the sphere formation capacity of breast cancer cells and their self-renewal. We found that in contrast to ES cells, where it represses Wnt-pathway target genes, TCF3 promotes the expression of a subset of Wnt-responsive genes in breast cancer cells while repressing another distinct target subset. In the normal mouse mammary gland, Tcf3 is highly expressed in terminal end buds, structures that lead duct development. Primary mammary cells are dependent on Tcf3 for mammosphere formation, and its overexpression in the developing gland disrupts ductal growth. Our results identify TCF3 as a central regulator of tumor growth and initiation, and a novel link between stem cells and cancer. Cancer Res; 72(21); 5613–24. ©2012 AACR.

Variation in penetrance estimates for BRCA1/2 carriers suggests that other environmental and genetic factors may modify cancer risk in carriers. The GSTM1, T1 and P1 isoenzymes are involved in metabolism of environmental carcinogens. The GSTM1 and GSTT1 gene is absent in a substantial proportion of the population. In GSTP1, a single-nucleotide polymorphism that translates to Ile112Val was associated with lower activity. We studied the effect of these polymorphisms on breast cancer (BC) risk in BRCA1/2 carriers. A population of 320 BRCA1/2 carriers were genotyped; of them 262 were carriers of one of the three Ashkenazi founder mutations. Two hundred and eleven were affected with BC (20 also with ovarian cancer (OC)) and 109 were unaffected with BC (39 of them had OC). Risk analyses were conducted using Cox proportional hazard models adjusted for origin (Ashkenazi vs non-Ashkenazi). We found an estimated BC HR of 0.89 (95% CI 0.65-1.12, P=0.25) and 1.11 (95% CI 0.81-1.52, P=0.53) for the null alleles of GSTM1 and GSTT1, respectively. For GSTP1, HR for BC was 1.36 (95% CI 1.02-1.81, P=0.04) for individuals with Ile/Val, and 2.00 (95% CI 1.18-3.38) for carriers of the Val/Val genotype (P=0.01). An HR of 3.20 (95% CI 1.26-8.09, P=0.01), and younger age at BC onset (P=0.2), were found among Val/Val, BRCA2 carriers, but not among BRCA1 carriers. In conclusion, our results indicate significantly elevated risk for BC in carriers of BRCA2 mutations with GSTP1-Val allele with dosage effect, as implicated by higher risk in homozygous Val carriers. The GSTM1- and GSTT1-null allele did not seem to have a major effect.

This paper is a report of response rate (RR) and survival of 34 metastatic melanoma patients who received a dinitrophenyl (DNP)-modified autologous melanoma cell vaccine. In all, 27 patients started the vaccine as a primary treatment for metastatic melanoma and seven started it as an adjuvant, with no evidence of disease at the time, but had developed new metastases. Interleukin-2 (IL-2) was administered in 24 out of the 34 patients: 19 who progressed on vaccine alone and five who had the combination from start. Interleukin-2 was administered in the intravenous, bolus high-dose regimen (seven patients) or as subcutaneous (s.c.) low-dose treatment (17). Overall response for the entire group was 35% (12 patients out of 34), 12% having a complete response (CR) and 23% a partial response (PR). However, only two patients had tumour responses while on the vaccine alone, whereas the other 10 demonstrated objective tumour regression following the combination with IL-2 (two CR, eight PR), lasting for a median duration of 6 months (range 3-50 months). Of the 12 responding patients, 11 attained strong skin reactivity to the s.c. injection of irradiated, unmodified autologous melanoma cells. None of the patients with a negative reactivity experienced any tumour response. Patients with positive skin reactions survived longer (median survival - 54 months). The results suggest enhanced RRs to the combination of IL-2 and autologous melanoma vaccine. Skin reactivity to unmodified autologous melanoma cells may be a predictor of response and improved survival, and therefore a criterion for further pursuing of immunotherapeutic strategies.

Background: Hereditary breast cancer (HBC) comprises more than 10% of all breast cancers (BC). Mutations in the BRCA1/2 genes are found in approximately half of HBC patients. The majority of BRCA1 associated tumors are ER, PR and HER2 negative with basal-like expression pattern. BRCA2 associated tumors are mostly ER positive (ER+). In the present study we aim to further explore clinical and molecular characteristics of BRCA associated BC in 3 different cohorts. Methods: Three different BC databases (DB) were evaluated: (i) Hadassah oncogenetic BC DB (n=4429); (ii) Nick-Zainal et al. BC DB (n=560), and (iii) METABRIC BC DB (n= 1980). We tested for differences in age at diagnosis between BRCA positive (BRCA+) and BRCA negative (BRCA-) patients with either ER+ or ER negative (ER-) tumors. Point mutation analysis was performed in cohorts ii & iii and mRNA differential expression (DEA) and pathway analysis were performed in cohort iii, using Ingenuity Pathway Analysis (IPA). Results: Age (years) at diagnosis for cohorts i, ii,&iii respectively, for ER+ PT:BRCA1-44, NA, 60; for BRCA2-49, 48, 64; for BN – 53, 56, 63. For ER-: BRCA1-42, 42, 47; for BRCA2-48, 52, 49; for BN-49, 54, 56. For cohorts ii&iii, higher frequencies of TP53 and PIK3Ca mutations were found among BRCA+&BRCA-, respectively. DEA was performed between BRCA+&BRCA- in ER- tumors: the major activated pathways involved cancer related processes and were highly significant (up to p=1e-7.5, FDR=1e-4.5). Surprisingly - the most significant pathway was Estrogen Mediated S-phase Entry and the most activated upstream regulator was ERBB2. Similar evaluation in ER+ showed mostly differences in immune related pathways (differences not statistically significant). Conclusions: Younger age at presentation was observed in BRCA1 vs. BRCA2 patients. No age differences were observed between ER+&ER- PT in cohort i&ii, in cohort iii ER- BRCA+ Patients were younger than ER+ BRCA+ (similar age as ER+ BRCA-). BRCA+ show different mutational profile than BRCA-. ER+ BRCA+ and BRCA- show similar genomic characteristics. By contrast, for ER- BRCA+ differs markedly from BRCA-. This might imply that BRCA+ associated tumors consist of two genomically distinct subtypes: (i) ER-, and (ii) ER+. The results may shed light on possible somatic factors which affect the development of BC BRCA+ and carry preventive and therapeutic implications. Citation Format: Peretz TY, Zick A, Luna K, Grinshpun A, Sonneblick A, Iziely B, Hamburger TG, Cohen S, Granit AM, Dvir M, Rosenberg S. ER dependent breast cancer phenotype in BRCA 1/2 carriers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-03-02.

This study evaluates the overall survival and disease free survival of melanoma patients that were treated with an autologous melanoma cell vaccine, administered as a post-operative adjuvant. Included are 43 patients with totally resected metastatic melanoma (28-AJCC stage III, 15-AJCC stage IV), with a median follow up of 34 months (6–62). The treatment consisted of eight doses of a vaccine made of 10–25×106 autologous melanoma cells either released from the surgical specimen or grown in cell cultures. Tumour cells were conjugated with hapten dinitrophenyl, mixed with Bacille Calmette Guérin and irradiated to 110 Gy. Both disease free survival and overall survival were found to be correlated with intensity of evolving delayed type hypersensitivity to subcutaneous injection of unmodified melanoma cells. Patients with a delayed type hypersensitivity reaction of ⩾10 mm had a median disease free survival of 17 months (mean 35 months) and a mean overall survival of 63 months (median not reached). In contrast, patients with a negative or weak delayed type hypersensitivity had a median disease free survival of 9 months (relative risk of recurrence=4.5, P=0.001), and a median overall survival of 16 months (relative risk of death=15, P=0.001). Stage III patients with a positive delayed type hypersensitivity reaction had an improved disease free survival of 16 months and a mean overall survival of 38 months, whereas patients with a negative delayed type hypersensitivity had a median disease free survival of 7 months (relative risk=4.5, P=0.02) and a median overall survival of 16 months (relative risk=9.5, P=0.005). The adjuvant administration of autologous melanoma vaccine was associated with improved disease-free and overall survival to selected patients who successfully attained anti-melanoma reactivity as detected by positive delayed type hypersensitivity reactions to unmodified melanoma cells. British Journal of Cancer (2002) 86, 1534–1539. DOI: 10.1038/sj/bjc/6600251 www.bjcancer.com © 2002 Cancer Research UK

To investigate the prognostic significance of cancer antigen 15-3 (CA15-3) in primary breast cancer (BC).This prospective study included 368 women: 62 patients with benign breast disease (BBD), 159 patients with invasive BC and 88 healthy blood donors (control). The median follow-up was 76 months (range, 43-99 months). Serum CA15-3 was measured with LIAISON® CA15-3® chemilluminescence immunoassay.Significantly high levels of CA15-3 were found in patients with BC compared to controls (p=0.029), but not to the BBD group (p=0.16). Preoperative CA15-3 in patients with BC was significantly associated with tumor size (p=0.003), TNM stage (p=0.005), vascular invasion (p=0.018) and tumor necrosis (p0.05). Increased CA15-3 (30 U/ml) concentrations were more often found in patients with larger tumors (p0.05), advanced stage (p=0.004) and node-positive disease (p=0.007). Patients with normal levels of CA15-3 had better recurrence-free survival (RFS) than those with elevated levels (p0.001). After adjustment for T-stage, grade, tumor necrosis, estrogen receptor (ER) and progesterone receptor (PR) status, CA15-3 remained an important preoperative characteristic with independent impact on RFS (hazard ratio=4.4, 95% confidence interval=1.5-13.1, p=0.007). The independent prognostic contribution of CA15-3, considered on a continuous scale was significant among subgroups of the BC patients with ER/PR-positive (p=0.002), node-positive (p=0.028), node-negative (p=0.003), T1-stage node-negative (p=0.017), luminal-A (p=0.003), luminal-B (p=0.028) and human epidermal growth factor receptor-2 (HER2)/non-luminal disease (p=0.045).Preoperative measurement of CA15-3 allowed identifying high-risk of recurrence for patients with primary BC and might be combined with existing prognostic factors in planning adjuvant treatment.

Purpose: Autologous melanoma cells display a broad variety of tumor antigens and were used for treatment of American Joint Committee on Cancer stages III and IV melanoma as an adjuvant or active therapy. Survival data and immune response were evaluated in vaccinated patients.Experimental Design: Forty-seven patients received 2,4-dinitrophenyl–conjugated autologous melanoma vaccine as an adjuvant (23 patients) or therapy (24 patients). CD4 and CD8 T-cell response in blood sampled before vaccination and after five or eight vaccine doses was evaluated against melanoma cells and autologous peripheral blood mononuclear cells using IFNγ enzyme–linked immunospot. Serum levels of antilivin, an inhibitor of apoptosis, and anti-gp100 IgG were determined.Results: The immunologic effect of the vaccine differed between the two groups of patients. In the adjuvant group, there was a significant increase in CD8 melanoma-reactive T cells (P = 0.035) after vaccination and an increase in antimelanoma CD4 T cells correlating with improved overall survival (P = 0.04). In the therapeutic group, there was no objective tumor regression; antimelanoma T-cell reactivity increased by a small amount, stayed the same, or in some cases decreased. In all patients, a significant increase was noted in CD4 T-cell reactivity against autologous peripheral blood mononuclear cells (P = 0.02), which did not affect survival. Increased antilivin IgG was associated with improved survival. Expression of MHC class II on melanoma cells was vital for the immunogenicity of the vaccine.Conclusion: Autologous melanoma cell vaccine is capable of inducing effective antimelanoma CD4 T-cell activity associated with improved survival. Patients with active metastatic disease generally displayed reduced immune response and gained little from active immunization.

Background A germ line single nucleotide polymorphism (SNP) in the first intron of the gene encoding MDM2 at position 309, an important modulator of p53, has been described. BRCA1/2 mutation have been associated with increased rates of breast cancers with mutated P53. It was shown that the presence of MDM2 309 SNP correlated with younger cancer onset age in individuals with a p53 mutations. The differential effects of this SNP were also linked to estrogen receptor activation. Here we report on our study of 453 Ashkenazi breast cancer patients of whom 180 were positive for the known Ashkenazi BRCA1/2 mutations Methods DNA from breast cancer patients was obtained for analysis of one of the three common BRCA1/2 mutations and MDM2 SNP309. Data regarding cancer onset and death ages was obtained from our database and Statistical analysis was performed using the SPSS® statistical package (SPCC Inc., Chicago, IL), and JMP® software (SAS Institute, Cary, NC). Results The percentage of MDM2 SNP309 in control and BRCA 1/2 population which is similar to that reported for other Jewish Ashkenazi populations at 52.2% for the heterozygotes and 25.0% for MDM2SNP309G/G and 22.8% for MDM2SNP309T/T. There was not a statistical significant difference in median age of disease onset in the different MDM2 SNP309 subgroups of the BRCA1/2 carriers. When we further divided the group into under and above 51 years old ( presumed menopause age) in the BRCA1 positive subset we found that there were less patients of the MDM2SNP309 G/G versus the MDM2SNP309 T/T in the over 51 patient group (p = 0.049). This result has been obtained in a relatively small subgroup and is of borderline statistical significance. Interestingly, in the BRCA1/2 mutation carriers, we found a survival advantage for patients harboring the SNP309 G/G genotype (p = 0.0086) but not for the 272 patients not harbouring this mutations. Conclusion MDM2SNP309G/G main effect on BRCA1/2 positive mutation carriers is linked to its effect on patients survival. Further research is needed in order to understand the reason for this difference.

Background: The risks for cancers other than breast (BC) or ovarian cancer (OC) in BRCA1/2 mutation carriers were elevated in studies of carrier families. However, case control studies did not confirm this observation. Objective: To compare other cancers risks in BRCA1/2 mutation carriers and non-carriers all affected with BC and/or OC. Both groups share risk modifiers of BC/OC, which enable us to assess the role of BRCA1/2 mutations. Methods: 1098 Ashkenazi Jewish women affected with BC and/or OC were ascertained during 1995-2003, molecular testing revealed 229 BRCA1 and 100 BRCA2 carriers and 769 non-carriers. COX proportional hazard models were used to evaluate the risk of other cancers. Analyses were conducted including all other cancers or only those diagnosed after BC/OC diagnosis. Results: The hazard ratio (HR) for any other cancer were 2.6 (95%CI 1.7-4.2, p

Background: Oncotype DX is a 21-gene RT-PCR assay which quantifies the likelihood of breast cancer recurrence and the potential benefit of chemotherapy in patients with early stage, ER positive, Tamoxifen treated breast cancer. Breast cancer in BRCA carriers is considered more aggressive. The aim of our study was to evaluate whether Oncotype Dx recurrence score distribution is different in breast cancer patients with inherited BRCA mutation. Methods: The Oncotype DX assay has been used at Hadassah Medical Center since 2004 on specimens from over 450 patients. We analyzed and compared clinicopathological characteristics and Oncotype Dx recurrence scores of BRCA carriers versus non- BRCA or unknown status of BRCA patients. Results: Ten patients had validated inherited BRCA mutation, five of them are BRCA1 carriers and five BRCA2 carriers. There were no significant differences in the clinicopathological characteristics between the two groups. Oncotype Dx recurrence score distribution between low, intermediate and high risk groups was not significantly different. The mean recurrence score was 18.48 for the non- BRCA or unknown status of BRCA patients and 22.8 for the BRCA carriers patients. This difference was not statistically significant. Conclusion: Estrogen receptor positive breast cancer tumors from BRCA carriers does not display a significantly different Oncotype Dx recurrence score result distribution. These preliminary data suggest Oncotype Dx assay might be used to help tailor treatment in this subset of patients, although further follow up is needed. All patients evaluated for oncotype_DX All evaluated (except BRCA+) n = 456BRCA positiveP valueAgeMean57.4557.60.7 Median5857 Range56 (25-81)34 (42-76) T stageT167.7%70%0.9 T230.7%30% T31.6%0% Tumor sizeMean1.71.360.2 Median1.51.26 Range7.8 (0.2-8)1.3 (0.7-2) GradeGrade 1-278.8%60%0.15 Grade 321.2%40% RSLow50%40%0.57 Intermediate39.8%40% High10.2%20% RSMean18.4822.80.16 Median17.522.5 Range64 (0-64)27 (12-39) Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-05-08.

Variation in the penetrance estimates for BRCA1 and BRCA2 mutation carriers suggests that other genetic polymorphisms may modify the cancer risk in carriers. The RAD51 gene, which participates in homologous recombination double-strand breaks (DSB) repair in the same pathway as the BRCA1 and BRCA2 gene products, is a candidate for such an effect. A single-nucleotide polymorphism (SNP), RAD51-135g-->c, in the 5' untranslated region of the gene has been found to elevate breast cancer (BC) risk among BRCA2 carriers. We genotyped 309 BRCA1/2 mutation carriers, of which 280 were of Ashkenazi origin, 166 noncarrier BC patients and 152 women unaffected with BC (a control group), for the RAD51-135g-->c SNP. Risk analyses were conducted using COX proportional hazard models for the BRCA1/2 carriers and simple logistic regression analysis for the noncarrier case-control population. BRCA2 carriers were also studied using logistic regression and Kaplan-Meier survival analyses. The estimated BC hazard ratio (HR) for RAD51-135c carriers adjusted for origin (Ashkenazi vs non-Ashkenazi) was 1.28 (95% CI 0.85-1.90, P=0.23) for BRCA1/2 carriers, and 2.09 (95% CI 1.04-4.18, P=0.04) when the analysis was restricted to BRCA2 carriers. The median BC age was younger in BCRA2-RAD51-135c carriers (45 (95% CI 36-54) vs 52 years (95% CI 48-56), P=0.05). In a logistic regression analysis, the odds ratio (OR) was 5.49 (95% CI 0.5-58.8, P=0.163). In noncarrier BC cases, carrying RAD51-135c was not associated with BC risk (0.97; 95% CI 0.47-2.00). These results indicate significantly elevated risk for BC in carriers of BRCA2 mutations who also carry a RAD51-135c allele. In BRCA1 carriers and noncarriers, no effect for this SNP was found.

Background Inherited mutations in the breast cancer susceptibility genes (BRCA1 and BRCA2) are associated with a high risk of developing breast (BC) and ovarian cancers (OC) in females of different age and ethnic groups. The spectrum of mutations in these genes varies between populations, with some showing a high frequency of unique mutations. Ashkenazi Jews have a high rate of founder mutations in BRCA1/2, in some other Jewish communities in Israel (Jews who immigrated to Israel from Iraq, Iran and Yemen), other founder mutations had been identified. Still high proportions of Israeli BC cases with strong family history have none of the known mutations at the BRCA1/2 genes. Methods and patients Over 3000 breast cancer patients were evaluated in the oncogentic clinic during 1997-2011. One hundred thirty seven of them underwent full sequencing of the BRCA1/2 genes due to strong family history of breast and/or ovarian cancer or young age at presentation. Clinical and pathological characteristics of these patients were evaluated. Result Sixty seven percent were non Ashkenazi Jews, Mean age at BC onset was 44 (22-77). In 20 patient (15%) BRCA 1(N = 8) or 2 (N = 12) mutations were identified. Three of the carriers had bilateral BC and 5 had OC as second primary .17 were of non Ashkenazi origin. Ninety five percent of the carriers had first degree family history of breast or ovarian cancer. The pathological information was available in half of the carriers. All had high grade (2-3) tumor, 90% of them were HER2 negative and 60% ER positive. Age at presentation had no effect on BRCA1/2 status. BRCAPRO is a predictive model to assess BRCA status and has assessed in 9 carriers, in 6 of them, the probability was >80% and in one - 5%. Conclusions A full sequence of the BRCA1/2 genes was performed in a selected group pf BC patient, who were negative for the common founder mutation in Israel. Fifteen percent were found to carry other BRCA1/2 mutations. We recommend that, patients with the following clinical features: Sephardic origin, first degree family history of BC or OC, high grade tumor, HER-2 negative, should be offered full BRCA1/2 testing. The role of BRCAPRO and other predictive model should be further evaluated. Disclosure All authors have declared no conflicts of interest.

Scope To presents a clinical chemistry test (DiviTum) for analyses of total cell division in human and to show the biochemical character of the s-TK expressed in breast carcinoma (BRCa), and to relate the degree of pathological increase found in pretreatment sera from women with localized BRCa to other clinical parameters as well as to outcome of disease. Methods and patients A standardised assay for s-TK is presented. In principle, the s-TK converts BrdU to its monophosphate which immidiatly is phosphorylated by yeast enzymes to triphosphate, which instantly is immobilised by polymerisation to a DNA-strand by reverse transcriptase using a prA-template bound to the bottom of the 96 well plate (i.e thymidine incorporation). The amount of produced DNA proportional to the s-TK is determined by ELISA using colorimetri. For comparison the Liason TK test was used. Molecular size fractioning of sera used a Superose 6 column. Serum cohorts included healthy women, healthy women with BRCA1/BRCA2 mutations in different loci, women with breast lumps judged as benign, and women with non-metastatic invasive BRCa. Results The pathological s-TK used only ATP as phosphate donor and was confined for all sera to a complex of approx. 700kD. With DiviTum, contrary to Liason, significantly higher s-TK levels, compared to healthy blood donors, were found in patients having benign lumps with atypical hyperplasia and in healthy persons with BRCA1/2 mutations in certain loci. Further, pretreatment s-TK correlated to all parameters indicating aggressivity such as absence of ER/PR receptor, vascularisation, tumor grade and size, triple negativity and BRCA mutation, but not to age, menopausal status, histopathology, HER/neu or nodal status. Univariate analyses showed that pretreatment s-TK (>134Du/L, p 80Du/L p Conclusion The degree of cell division in the BRCA patients, measured as s-TK by DiviTum assay, is an important biomarker of survival and thus useful for staging, prognoses, and therapy monitoring. Disclosure S. Gronowitz: I, Simon Gronowitz am the founder of Biovica International and the inventor of the DiviTum assay for seru thymidine kinase and my family has still shares in the company. All other authors have declared no conflicts of interest.

Abstract #6148 Background Counseling young breast cancer patients regarding the chances of conception following chemotherapy is a subject of great importance. Previous studies have reported a 50-70% rate of amenorrhea following standard breast cancer chemotherapy and very low rates of pregnancy. However a uniform definition of infertility is lacking and as a result many studies reported results observed in pre-menopausal women as a whole without specific analysis and separation according to age. In this retrospective, single institution study, we examined resumption of menses and pregnancy rates in young breast cancer patients who had received chemotherapy. Methods We identified 193 breast cancer patients aged 35 years or younger diagnosed at a single institution between 1996 and 2004. All patients who were treated with adjuvant chemotherapy and had completed at least 1 year of follow-up without evidence of recurrence were included in the study. Results Forty seven patients met the eligibility criteria. The mean time of follow-up was 69 months (range, 12-124 mos.). The mean age at diagnosis was 30 years (range, 19-34 yrs). Chemotherapy consisted of Adriamycin based protocols in 29 cases and CMF (Cyclophosphamide, Methotrexate, 5-Fluorouracil) in 18 cases. GnRH agonists were administered to 28 (61%) of patients for at least two years. All patients had regular menses prior to the initiation of chemotherapy. Three patients continued to receive regular injections with a GnRH agonist beyond two years, one patient underwent oophorectomy, two patients developed amenorrhea whilst 35 patients (35/37 95%) resumed menses subsequent to chemotherapy. Information regarding menstruation was lacking for six patients. Sixteen patients (16/37 43%) gave birth to at least one child after menses resumed at a mean age of 35 years (range, 28-41yrs). Discussion Standard chemotherapy protocols in breast cancer patients under the age of 35 do not preclude fertility preservation and pregnancy. However, not all young breast cancer survivors wish to become pregnant following treatment and therefore the actual rate of fertility is in fact higher than those recorded. Further prospective studies which incorporate documentation regarding patient's desire for future pregnancies need to be undertaken in order to better advise young patients with respect to their individual chances of conceiving following chemotherapy. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6148.

Background The incidence of breast cancer (BC) in Arab women is lower compared to the incidence in the Jewish population in Israel; still, it is the most common malignancy among Arab women. There is a steep rise in breast cancer incidence in the Arab population in Israel over the last 10 years that can be attributed to life style changes. But, the younger age of BC onset in Arab women compared with that of the Jewish population is suggestive of a genetic component in BC occurrence in that population. Methods We studied the family history of 31 women of Palestinian Arab (PA) origin affected with breast (n = 28), ovarian (n = 3) cancer. We used denaturing high performance liquid chromatography (DHPLC) to screen for mutations of BRCA1/2 in 4 women with a personal and family history highly suggestive of genetic predisposition. Results A novel BRCA1 mutation, E1373X in exon 12, was found in a patient affected with ovarian cancer. Four of her family members, 3 BC patients and a healthy individual were consequently also found to carry this mutation. Of the other 27 patients, which were screened for this specific mutation none was found to carry it. Conclusion We found a novel BRCA1 mutation in a family of PA origin with a history highly compatible with BRCA1 phenotype. This mutation was not found in additional 30 PA women affected with BC or OC. Therefore full BRCA1/2 screening should be offered to patients with characteristic family history. The significance of the novel BRCA1 mutation we identified should be studied in larger population. However, it is likely that the E1373X mutation is not a founder frequent mutation in the PA population.