BRCA1 In Patients Research Articles

Abstract 3922: Prediction of breast cancer progression using nuclear morphometry

Abstract Introduction: The progression of breast cancer (BrCa) involves nuclear morphometric changes, which are used in the pathological diagnosis of breast cancer. Although changes in nuclear morphometry (NM) contribute to histologic alterations observed in breast cancer, an accurate and autonomous quantification of NM changes have remained elusive. Here we created an image analysis macro to quantify changes in nuclear parameters, including size, shape, and DNA content and used these parameters to predict BrCa progression. Materials & Methods: Three tissue microarrays (TMAs) with 140 BrCa cases, stratified by TNM stage were used for this study. The TMAs were generated using 6 mm cores from primary tumors of a Korean cohort from Yengnam University Hospital, Daegu, South Korea. Specimens were obtained from surgical resection between January 1995 and January 2004. Each case was represented by 2 cores on the TMAs. Clinical information were provided by the pathology reports and patients’ medical records. H&E slides were first used for pathological diagnosis of BrCa after which slides were scanned with the Aperio scanner and the image of each core was separated using Aperio ImageScope software. The H&E stained nuclei from the tumor core were quantified using ImagePro® Premier 9.1 software (MediaCybernetics). For each core, data of all regions of interest were pooled and the covariance of each parameter was generated. Data were first analyzed alone and then in combination using multivariate logistic regression (MLR) to predict the aggressive cases or recurrence. For all analysis, p<0.05 was considered statistically significant. Results: Using multivariate logistic regression (MLR) to differentiate aggressive BrCa (TNM T3 & T4) from indolent BrCa (TNM T1 & T2) on the TMAs, our H&E NM model generated an receiver operating characteristic curve-area under the curve (ROC-AUC) of 0.75 with a sensitivity of 50.9% and specificity of 84.3% at the predictive probability cutoff of 0.50. Parameters of nuclei diameter, size, staining intensity, and aspect ratio contributed to the MLR model. In addition, our H&E NM model also showed power in the prediction of recurrence based on the MLR (ROC-AUC = 0.80) with a sensitivity of 25.9% and specificity of 96.3% at the probability cutoff of 0.53. Parameters of nuclei diameter, size, staining intensity, and cluster contributed to the MLR model. The predictive probability (PP) is significantly higher in the aggressive and recurrent cases based on the two MLR models, respectively. Conclusions: We discovered that our automated quantification of tissue nuclear morphometry can be used to accurately predict BrCa aggressiveness and recurrence in women who underwent surgical resection of their primary tumors. Although further confirmatory studies our needed, our data suggests that this tool could provide an accurate predictor of tumor behavior and patient prognosis that could be used to inform therapy for BrCa patients. Citation Format: Neil Carleton, Guangjing Zhu, Linda Resar, Lisa Rooper, Young Kyung Bae, Robert W. Veltri. Prediction of breast cancer progression using nuclear morphometry. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3922.

Abstract 2547: Molecular insights into OGG1 gene, a modifier of cancer risk in BRCA1 and BRCA2 mutations carriers

Abstract Oxidative stress is a source of DNA damage which can lead to genomic instability and telomere shortening. The OGG1 glycosidase plays an important role in the Base Excision Repair (BER) pathway that counteracts the oxidative DNA damage caused by endogenous cell stress. Recently, we have demonstrated that a single nucleotide polymorphism (SNP) in the OGG1 gene can modify cancer risk in carriers of germline mutations in the BRCA1 and BRCA2 genes1. Hence, we aimed to explore the possible role of this variant in DNA damage and telomere shortening to explain the association of this cancer risk variant. We used 2 independent set of samples with a heterogeneous BRCA mutational status. First of all, a familiar breast and ovarian cancer (FBOC) set of 154 BRCA patients (BRCA1, BRCA2 and BRCAX) and 68 controls, were used to measure the mRNA OGG1 expression levels in blood and the leukocyte telomere length (TL). Second, we used a panel of 23 Lymphoblastoid Cell Lines (LCL) from patients harboring germline mutations in BRCA1 and non-carrier controls to validate previous results regarding OGG1 mRNA expression levels and telomere shortening. Additionally we measured gammaH2AX intensity levels in LCL to evaluate the role of the SNP on DNA damage. In the FBOC series, we found that the carriers of the variant had significant decreased expression levels of the OGG1 transcript compared with the non-carriers (p = 0.013). Regarding TL studies, we found that the variant may exert a synergistic effect together with BRCA1/2 mutations on telomere shortening (p<0.0045). TL was not associated to higher cancer risk in our FBOC series, pointing to this phenotype not as the cause of the cancer risk association, but maybe as a hallmark of a higher DNA damage environment when both genetic events are present in the cell. Using the LCL panel we were able to detect OGG1 mRNA down regulation due to the presence of the variant and a significant telomere shortening exclusively in the BRCA1 defective cells harboring the OGG1 SNP after 55 passages. The DNA damage studies, confirmed significant higher intensity levels of gammaH2AX in the damaged LCL harboring the SNP compared to those not harboring it (p<0.0001). These results, may explain the molecular insights behind this modifier variant. Under the prism of a deleterious interaction, in cells harboring mutations in BRCA genes, the presence of the SNP itself, seems to affect OGG1 mRNA down regulation. This could lead to a defective performance of the BER pathway that may lead to genome instability, characterized by higher gammaH2AX intensity in damaged cells and telomere shortening when both genetic events are present, and as consequence, higher cancer risk. 1. Osorio A, et al (2014) PLoS Genet Funding: J.B's laboratory, INNPRONTA 2012, Spanish Ministry of Health (PI12/00070) and (CIBERER). C.B is granted by PI12/00070 M.A.B.'s laboratory, Spanish Ministry of Science and Innovation (SAF2008-05384;2007-A-200950) (TELOMARKER), ERCA (GA#232854) Citation Format: Carlos Benitez-Buelga, Tereza Vaclova, María Sofia Ferreira, Nora Soberon, María Antonia Blasco, Ana Osorio, Javier Benitez. Molecular insights into OGG1 gene, a modifier of cancer risk in BRCA1 and BRCA2 mutations carriers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2547.

Achievements, Investigations in Gynecologic Oncology Explored

Achievements, Investigations in Gynecologic Oncology Explored

Oncotype-DX recurrence score distribution in breast cancer patients with BRCA1/2 mutations.

Oncotype-DX assay has never been validated for BRCA mutation carriers. This study compares the recurrence score (RS) distribution in BRCA-positive breast cancer patients with that of a general population (GP) of patients and reports their outcomes. Eligible patients were BRCA carriers who performed the Oncotype-DX assay. Two sets of databases were cross-linked: BRCA carriers at Rabin Medical Center and Sheba Medical Center with Oncotype-DX tests performed through Clalit Health Services HMO, from 2003 to 2015. Fifty-eight BRCA patients were included (20 BRCA1, 38 BRCA2). The GP included 1020 patients. Compared to the GP, BRCA1 patients were younger, had higher rate of grade three tumors, and higher Ki67. BRCA2 patients had lower PR index, higher rate of grade three tumors, and higher Ki67. Among the GP, 52.9, 37.9, and 9.1% had low, intermediate, and high risk RS, respectively. Corresponding rates were 15, 35, and 50% in BRCA1 patients, and 18.4, 52.6, and 29% in BRCA2 patients. Subgroup analysis revealed a similar RS distribution pattern regardless of the nodal status. Median follow-up was 45months. Four BRCA patients (7%) developed disease recurrence. RS of these patients were in the intermediate and low range. All recurrences occurred in chemo-naïve patients who had not undergone bilateral oophorectomy. This study revealed significantly different RS distributions between BRCA patients and the GP. RS values shifted toward high and intermediate risk categories. This pattern held regardless of the nodal status and was more pronounced in the BRCA1 group.

Highly favorable outcome in BRCA-mutated metastatic breast cancer patients receiving high-dose chemotherapy and autologous hematopoietic stem cell transplantation.

Breast cancer carrying BRCA mutation may be highly sensitive to DNA-damaging agents. We hypothesized a better outcome for BRCA-mutated (BRCA(mut)) metastatic breast cancer (MBC) patients receiving high-dose chemotherapy and autologous hematopoietic stem cell transplantation (HDC AHSCT) versus unaffected BRCA (BRCA wild type; (BRCA(wt))) or patients without documented BRCA mutation (BRCA untested (BRCA(ut))). All female patients treated for MBC with AHSCT at Institut Paoli-Calmettes between 2003 and 2012 were included. BRCA(mut) and BRCA(wt) patients were identified from our institutional genetic database. Overall survival (OS) was the primary end point. A total of 235 patients were included. In all, 15 patients were BRCA(mut), 62 BRCA(wt) and 149 BRCA(ut). In multivariate analyses, the BRCA(mut) status was an independent prognostic factor for OS (hazard ratio (HR): 3.08, 95% confidence interval (CI): 1.10-8.64, P=0.0326) and PFS (HR: 2.52, 95% CI :1.29-4.91, P=0.0069). In this large series of MBC receiving HDC AHSCT, we report a highly favorable survival outcome in the subset of patients with documented germline BRCA mutations.

Abstract S3-01: EndoPredict (EPclin) score for estimating residual distant recurrence (DR) risk in ER+/HER2- breast cancer (br ca) patients treated with 5 years adjuvant endocrine therapy alone: Validation and comparison with the oncotype DX recurrence score (RS)

Abstract Background: RS is widely used for estimating 10-yr risk of DR for br ca patients receiving 5yrs adjuvant endocrine treatment alone. EndoPredict is a prognostic test which unlike RS combines its eight-gene expression signature (EP score) with tumor size and nodal status to provide the EPclin score with a single low/high risk cut-off at 10% risk of DR at 10yrs similar to the low/intermediate risk cut-off with RS. Aims: To determine the prognostic value of EP/EPclin over 10yrs, 0-5yrs and 5-10yrs in ER+/HER2- br ca patients and to compare this with the RS (i) alone (primary objective) and (ii) in addition to the clinical treatment score (CTS [developed in TransATAC]: nodes (N); tumour size; grade; age; tam or AI). Methods: mRNA from 928 ER+/HER2- primary br ca from patients treated with anastrozole or tamoxifen in the ATAC trial on which RS was already available were assessed for the EP/EPclin. Primary end point was DR. Kaplan–Meier and Cox regression analyses were used to determine DR risk for 0-10, 0-5 and 5-10yrs follow-up. Likelihood ratio tests (LR-chi sq) were used to assess the prognostic information provided by EP, EPclin, and RS alone and in combination with CTS. Results: Median follow-up was 9.95 yrs. The table shows the LR-chi sq for the prognostic information provided by each marker alone (univariate) and the information on top of CTS for all patients, and N- and N+ subgroups in 0-10, 0-5 and 5-10yrs. EP and EPclin provided substantially more prognostic information than RS across all three time periods and subgroups, except for N- patients in 0-5yrs. EP and EPclin also provided more information in addition to CTS than RS in 0-10yrs due to better performance of EP and EPclin in 5-10yrs where RS added no significant information to CTS. Using predefined cut-offs, EPclin and RS identified 546 (58.8%) and 573 (61.7%) patients as low risk respectively (HR (95%CI) low vs. non-low risk: 5.9 (3.9-9.1) and 2.7 (1.9-3.8), respectively). The EP score significantly added prognostic information to the RS over 0-10yrs and 5-10yrs in the overall, N- and N+ populations. Cases classified discordantly by EPclin and RS followed the EPclin classification more closely than RS classification. Prognostic information from EP, EPClin, RSLR-chi-sqAll patientsNode-negativeNode-positiveUnivariate0-10yrs0-5; 5-10yrs0-10yrs0-5; 5-10yrs0-10yrs0-5; 5-10yrsEP49.325.7; 23.630.811.9; 15.614.57.9; 6.6EPclin139.380.0; 59.340.017.0; 22.748.332.2; 16.1RS29.126.1; 5.621.318.7; 4.88.08.0; 1.0CTS149.885.0; 64.735.619.0;16.961.635.2; 26.3added to CTS EP16.46.9; 9.815.55.2; 6.68.32.3; 3.4EPClin20.310.5; 9.917.03.6; 9.05.46.4; 2.3RS12.811.8; 2.318.78.1; 1.44.13.7; 0.7LR-chi sq >3.8, p<0.05 Conclusions: EPclin was highly prognostic for DR in all patients, N-, N+, early and late metastasis. EPclin provided more prognostic information than RS in part but not solely because of the integration of the EP score with N and tumour size parameters. The data stress the importance of including clinicopathological factors in deriving an overall estimate of risk. Citation Format: Dowsett M, Sestak I, Buus R, Kronenwett R, Denkert C, Krappmann K, Scheer M, Petry C, Dubsky P, Cuzick J. EndoPredict (EPclin) score for estimating residual distant recurrence (DR) risk in ER+/HER2- breast cancer (br ca) patients treated with 5 years adjuvant endocrine therapy alone: Validation and comparison with the oncotype DX recurrence score (RS). [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S3-01.

Frequency and Significance of Abnormal Pancreatic Imaging in Patients with BRCA1 and BRCA2 Genetic Mutations.

Objective. Pancreatic adenocarcinoma is typically diagnosed in advanced stages resulting in a significant reduction in the number of patients who are candidates for surgical resection. Although the majority of cases are believed to occur sporadically, about 10% show familial clustering and studies have identified an increased frequency of BRCA germline mutations. The role of screening for pancreatic adenocarcinoma in these populations is unclear. Our study aims to identify the abnormal pancreatic imaging findings in BRCA1 and BRCA2 mutation carriers. Methods. A retrospective review of patient medical records with known BRCA1 and BRCA2 mutations was conducted. Data was collected and all available abdominal imaging studies were reviewed. Results. A total of 66 patients were identified, 36 with BRCA1 and 30 with BRCA2 mutations. Only 20/66 (30%) had abdominal imaging (14 BRCA1 and 6 BRCA2 patients). Of those patients with abdominal imaging, abnormal pancreatic imaging findings were detected in 7/20 (35%) cases. Conclusion. Our study shows a high incidence of abnormal pancreatic imaging findings in patients with BRCA genetic mutations (35%). Larger studies are needed to further define the role of pancreatic cancer screening and the significance of abnormal imaging findings in BRCA1 and BRCA2 mutation carriers.

Understanding the role of the kynurenine pathway in human breast cancer immunobiology.

Breast cancer (BrCa) is the leading cause of cancer related death in women. While current diagnostic modalities provide opportunities for early medical intervention, significant proportions of breast tumours escape treatment and metastasize. Gaining increasing recognition as a factor in tumour metastasis is the local immuno-surveillance environment. Following identification of the role played by the enzyme indoleamine dioxygenase 1 (IDO1) in mediating maternal foetal tolerance, the kynurenine pathway (KP) of tryptophan metabolism has emerged as a key metabolic pathway contributing to immune escape. In inflammatory conditions activation of the KP leads to the production of several immune-modulating metabolites including kynurenine, kynurenic acid, 3-hydroxykynurenine, anthranilic acid, 3-hydroxyanthranilic acid, picolinic acid and quinolinic acid. KP over-activation was first described in BrCa patients in the early 1960s. More evidence has since emerged to suggest that the IDO1 is elevated in advanced BrCa patients and is associated with poor prognosis. Further, IDO1 positive breast tumours have a positive correlation with the density of immune suppressive Foxp3+ T regulatory cells and lymph node metastasis. The analysis of clinical microarray data in invasive BrCa compared to normal tissue showed, using two microarray databank (cBioportal and TCGA), that 86.3% and 91.4% BrCa patients have altered KP enzyme expression respectively. Collectively, these data highlight the key roles played by KP activation in BrCa, particularly in basal BrCa subtypes where expression of most KP enzymes was altered. Accordingly, the use of KP enzyme inhibitors in addition to standard chemotherapy regimens may present a viable therapeutic approach.

Magnesium as a diagnostic marker of cancer

Magnesium plays a key role in many essential cellular processes. It is recognized that magnesium deficiency may lead to many disorders like cardiovascular disease, diabetes mellitus, hypertension, myocardial infarction or even cancer. Connection between magnesium concentration in body fluids and cancer occurrence still becomes unclear. Magnesium ions are crucial cofactors for enzymes involved DNA repairing. Thereby magnesium deficiency may result in genomic instability and consequently cause cancer. The aim of our study was to investigate if serum magnesium concentration may be useful biomarker for early detection of prostate and laryngeal cancer and also if serum Mg concentration may be marker of selection for CT of the lung, laryngeal examination, PSA levels or prostate biopsy. Independently we have investigated a prospective study where our aim was to investigate if the serum Mg concentration is a marker of increased breast cancer risk. We performed retrospective investigation in laryngeal cancer patients (n = 123) and prostate cancer groups (n = 166). Each group was compared with healthy controls group, which consists of equal to cases amount of controls. Controls and cases were matched by age, gender, negative supplementation status, negative status of hormonal contraceptives and smoking status. In the prospective study (sera collected before cancer diagnosis) Mg concentration was determined in BRCA (-) women and compared to controls. Mg serum levels ( 20800 µg/l) are associated with 2-fold higher occurrence of laryngeal cancers. Magnesium serum level is not a useful marker for selection of the patients for prostate cancer screening. Finally, magnesium concentration may be a marker for breast cancer risk in BRCA (-) patients but further investigation is needed.

Occult Histopathology and Its Predictors in Contralateral and Bilateral Prophylactic Mastectomies.

The last decade has seen an increasing prevalence of prophylactic mastectomies with decreasing age of patients treated for breast cancer. Data are limited on the prevalence of histopathologic abnormalities in this population. This study aimed to measure the prevalence of histopathologic findings in contralateral prophylactic mastectomy (CPM) and bilateral prophylactic mastectomy (BPM) patients and identify predictors of findings. Our institution's prophylactic mastectomies from 2004 to 2011 were reviewed. Breast specimens with prior malignancies were excluded. Patient factors and pathology reports were collected. Independent predictive factors were identified with univariate and multivariate logistic analysis. A total of 524 specimens in 454 patients were identified. Malignancy was found in 7.0% of CPM and 5.7% of BPM specimens. In CPM patients, ipsilateral lobular carcinoma-in situ [odds ratio (OR) 4.0] and mammogram risk group (OR 2.0) were predictive of malignancy. Age group (OR 1.5), ipsilateral lobular carcinoma-in situ (OR 2.3), and prior bilateral salpingo-oophorectomy (OR 0.3) were predictive of moderate- to high-risk histopathology. Only increasing age group was predictive of increased moderate- to high-risk histopathology in BPM patients (OR 2.3). There were no independent predictors of malignancy in BPM. BRCA status was not predictive in either CPM or BPM. Patients with lobular carcinoma-in situ in the index breast or high-risk mammograms have a higher prevalence of malignancies. Although BRCA patients may benefit from prophylactic mastectomy, the genetic diagnosis does not increase the prevalence of detecting occult pathology. BPM patients can be counseled about relative risk, where occult pathology increases with age.

Effect of BRCA2 mutation on familial breast cancer survival: A systematic review and meta-analysis.

Reports of BRCA2 genetic mutations on the prognosis of familial breast cancer (BC) patients have been contradictory. True difference in survival, if it exists, would have important implications for genetic counseling and in treatment of hereditary BC. The purpose of this study was to compare overall survival rate (OSR) among BRCA2 mutation carriers, non-carriers and sporadic BC patients. We searched the PUBMED and EMBASE databases and retrieved 4529 articles using keywords that included breast cancer, BRCA, prognosis and survival. Nine articles were selected for systematic review and among them 6 were included in our meta-analysis. We used the fixed and random effect models to calculate the summary odds ratio (OR) and corresponding 95% confidence interval (CI). BRCA2 mutation carriers had significantly higher long-term OSR than non-carriers (OR=0.69 [95% CI=0.5-0.95]), while both short-term and long-term OSR of BRCA2 mutation carriers did not differ from those of patients with sporadic disease (OR=1.11 [95% CI=0.74-1.65]; 0.85 [95% CI=0.38-1.94], respectively). For BC-specific survival rate (BCSSR), BRCA2 mutation carriers had a similar BCSSR to the non-carriers (OR=0.61 [95% CI=0.28-1.34]). There was no significant difference in disease-free survival (DFS) between BRCA2 mutation carriers and patients with sporadic disease. Our results suggest that BRCA2 mutation increases long-term OSR in hereditary BC, which reminds us a new prospect of management of the disease.

Alternatively spliced tissue factor synergizes with the estrogen receptor pathway in promoting breast cancer progression

Procoagulant full-length tissue factor (flTF) and its minimally coagulant alternatively spliced isoform (asTF), promote breast cancer (BrCa) progression via different mechanisms. We previously showed that flTF and asTF are expressed by BrCa cells, resulting in autoregulation in a cancer milieu. BrCa cells often express hormone receptors such as the estrogen receptor (ER), leading to the formation of hormone-regulated cell populations. To investigate whether TF isoform-specific and ER-dependent pathways interact in BrCa. Tissue factor isoform-regulated gene sets were assessed using ingenuity pathway analysis. Tissues from a cohort of BrCa patients were divided into ER-positive and ER-negative groups. Associations between TF isoform levels and tumor characteristics were analyzed in these groups. BrCa cells expressing TF isoforms were assessed for proliferation, migration and invivo growth in the presence or absence of estradiol. Ingenuity pathway analysis pointed to similarities between ER- and TF-induced gene expression profiles. In BrCa tissue specimens, asTF expression was associated with grade and stage in ER-positive but not in ER-negative tumors. flTF was only associated with grade in ER-positive tumors. In MCF-7 cells, asTF accelerated proliferation in the presence of estradiol in a β1 integrin-dependent manner. No synergy between asTF and the ER pathway was observed in a migration assay. Estradiol accelerated the growth of asTF-expressing tumors but not control tumors invivo in an orthotopic setting. Tissue factor isoform and estrogen signaling share downstream targets in BrCa; the concomitant presence of asTF and estrogen signaling is required to promote BrCa cell proliferation.

Association of Somatic Mutations of ADAMTS Genes With Chemotherapy Sensitivity and Survival in High-Grade Serous Ovarian Carcinoma.

Chemotherapy response in the majority of patients with ovarian cancer remains unpredictable. To identify novel molecular markers for predicting chemotherapy response in patients with ovarian cancer. Observational study of genomics and clinical data of high-grade serous ovarian cancer cases with genomic and clinical data made public between 2009 and 2014 via the Cancer Genome Atlas project. Chemotherapy response (primary outcome) and overall survival (OS), progression-free survival (PFS), and platinum-free duration (secondary outcome). In 512 patients with ovarian cancer with available whole-exome sequencing data, mutations from 8 members of the ADAMTS family (ADAMTS mutations) with an overall mutation rate of approximately 10.4% were associated with a significantly higher chemotherapy sensitivity (100% for ADAMTS-mutated vs 64% for ADAMTS wild-type cases; P < .001) and longer platinum-free duration (median platinum-free duration, 21.7 months for ADAMTS-mutated vs 10.1 months for ADAMTS wild-type cases; P = .001). Moreover, ADAMTS mutations were associated with significantly better OS (hazard ratio [HR], 0.54 [95% CI, 0.42-0.89]; P = .01 and median OS, 58.0 months for ADAMTS-mutated vs 41.3 months for ADAMTS wild-type cases) and PFS (HR, 0.42 [95% CI, 0.38-0.70]; P < .001 and median PFS, 31.8 for ADAMTS-mutated vs 15.3 months for ADAMTS wild-type cases). After adjustment by BRCA1 or BRCA2 mutation, surgical stage, residual tumor, and patient age, ADAMTS mutations were significantly associated with better OS (HR, 0.53 [95% CI, 0.32-0.87]; P = .01), PFS (HR, 0.40 [95% CI, 0.25-0.62]; P < .001), and platinum-free survival (HR, 0.45 [95% CI, 0.28-0.73]; P = .001). ADAMTS-mutated cases exhibited a distinct mutation spectrum and were significantly associated with tumors with a higher genome-wide mutation rate than ADAMTS wild-type cases across the whole exome (median mutation number per sample, 121 for ADAMTS-mutated vs 69 for ADAMTS wild-type cases; P < .001). ADAMTS mutations may contribute to outcomes in ovarian cancer cases without BRCA1 or BRCA2 mutations and may have important clinical implications.

Abstract IA28: BRCA-mutated pancreas adenocarcinoma: Emerging therapeutic implications

Abstract Germline BRCA mutations, in particular BRCA 2, incur an increased risk of developing pancreas adenocarcinoma. BRCA mutations occur in 5-7% of an unselected pancreas adenocarcinoma population. In patients with Ashkenazi Jewish heritage and pancreas adenocarcinoma the frequency of BRCA mutations may be significantly higher at 12-15%. In patients with familial pancreatic cancer, defined as having at least one first degree relative with pancreas adenocarcinoma, the frequency of BRCA 1 or 2 mutation carriers is estimated at 11-17%. PALB2 (partner and localizer to BRCA2) mutations occur in a smaller fraction of patients with pancreas adenocarcinoma. The PALB2 protein stabilizes the BRCA 2 protein and anchors it to the nucleus, allowing it to carry out its DNA repair function. More recently, PALB2 has been shown to associate with the BRCA 1 protein, providing a linkage between BRCA 1 and 2 in homologous recombination repair of double-stranded DNA breaks. Preclinical data has demonstrated hypersensitivity of BRCA 2 deficient pancreatic cell lines to DNA cross-linking agents mitomycin and cisplatin and more recently to the novel agents, poly (ADP-ribose) polymerases (PARP) inhibitors. We and others have reported increased sensitivity to DNA damaging agents in patients with pancreas adenocarcinoma arising on a background of a known BRCA 1 or 2 germline mutation. Poly ADP ribose polymerases are a family of enzymes, two of which, PARP1 and 2, are key components of the DNA repair mechanism for cells with single-strand breaks and nucleoside base damage. PARP inhibition has particular application in tumors with pre-existing defects in homologous recombination, such as BRCA 1 or 2 deficient cells. Inhibition of PARP in these cells leads to transformation of single-strand breaks into double-strand breaks (DSB), which are cytotoxic in cells, which are unable to repair DSBs by homologous repair. This effect of synthetic lethality provides the therapeutic rationale for development of PARP inhibitors for BRCA 1 or 2 mutation associated pancreas adenocarcinoma. Veliparib (ABT-888) is an orally available, small molecule inhibitor of polyADP-ribose polymerase. Three trials are evaluating the role of role of veliparib in combination with cisplatin-based therapy in patients with BRCA or PALB2 mutated pancreas adenocarcinoma. Data will be presented on the first of these trials, which is a phase IB study designed to determine the recommended phase II dose of veliparib in combination with cisplatin and gemcitabine for patients with a either a known BRCA or PALB2 mutation or a very strong personal or family history of malignancy. Between 02/12 and 10/13, 33 patients were screened and 17 enrolled. Male = 10, Female = 7. Median age = 60 years (range 42- 72). BRCA = 9. Non-BRCA = 8. Four dose levels of veliparib were evaluated; Two dose-limiting toxicities of veliparib were observed at an 80mg daily continuous dosing schedule (neutropenia, thrombocytopenia). Overall grade 3-4 toxicities: Hematologic, fatigue and one fatal bowel perforation tumor/diverticulitis. For N= 9 BRCA patients tumor response: 5 (56%) partial responses and 4 (44%) stable disease. No objective responses were seen in the non-BRCA patients. The recommended phase II dose of Veliparib is 80 mg PO BID day 1-12 q 3 weeks with fixed dose cisplatin and gemcitabine. The triplet combination of cisplatin, gemcitabine and veliparib shows high activity in BRCA-related pancreas adenocarcinoma. A randomized phase II trial evaluating cisplatin/gemcitabine +/- veliparib is underway in BRCA/PALB2-mutated pancreas adenocarcinoma (NCT01585805). In addition to the clinical trials, there are a series of correlative science questions focused around: (1) characterization of the molecular phenotype of BRCA/PALB2 mutated pancreas adenocarcinoma, (2) mechanisms of resistance to platinum and PARP inhibition, (3) evaluation of assays for homologous repair, (4) evaluation of loss of heterozygosity at BRCA 1, 2 loci in tumor, and other considerations.

Survival of patients with structurally-grouped TP53 mutations in ovarian and breast cancers.

The objective of this study was to determine if ovarian cancer patients with a TP53 mutation grouped by location of the mutation within the p53 protein structure exhibit differential survival outcomes. Data from patients with high grade serous ovarian cancer (HGS OvCa) (N = 316) or breast cancer (BrCa) (N = 981) sequenced by The Cancer Genome Atlas (TCGA) was studied by Kaplan-Meier and Cox proportional hazards survival analysis. A TP53 DNA binding domain (BD) missense mutation (MM) occurred in 58.5% (185/316) of HGS OvCas and 16.8% (165/981) of BrCas. Patients with a TP53 DNA BD MM grouped by structural location had significantly different overall survival (OS) and progression free survival (PFS). Median OS (months) of HGS OvCa patients by structural group were: Sheet-loop-helix stabilizers, 31.1; DNA minor groove residue R248, 33.6; Wild-type, 34.2; all other MMs, 44.5; DNA major groove residues, 84.1, and zinc ion coordinating residues, 87.0 (log-rank p = 0.006). PFS of DNA major groove MM cases was longer than TP53 wild-type cases (19.1 versus 10.1 months, log-rank p = 0.038). HGS OvCa and BrCa patients with structurally-grouped TP53 DNA BD MMs have different survival outcomes.

Comparative analysis of 1% lymphazurin versus 1% methylene blue for sentinel lymph node mapping in early stage breast cancer.

e12055 Background: Sentinel lymph node mapping (SLNM) is the standard for staging clinically node negative (-ve LN) breast cancer (BrCa) patients (pts). Lymphazurin (L) has been the most commonly u...

Abstract P5-10-09: Correlation between germline and tumor CYP450 2D6 gene polymorphisms

Abstract Tamoxifen is used for the treatment and prevention of ER (estrogen receptor)-positive BrCa (Breast Cancer). Several studies have shown that genetic variations in the CYP2D6 gene and/or drug-dependent inhibition of CYP2D6 enzyme activity (drug-drug interactions or DDIs) are associated with significant reductions in the circulating levels of endoxifen the active tamoxifen metabolite. These studies demonstrated that changes in CYP2D6 metabolism, as predicted from CYP2D6 genotyping, affect efficacy. Recent negative results with regards to CYP2D6 genotyping from two large studies suggest that testing for CYP2D6 status has no practical clinical value. Tumor DNA was used for genotyping in a large fraction of the BrCa patients in these studies. Genetic bias may result when CYP2D6 genotyping is carried out on the tumor (somatic) genome rather than the host genome (germline DNA) since it is the host genome that determines CYP450 enzyme activity within the liver and GI tract. Also expanded CYP2D6 polymorphism coverage, using a more comprehensive genotyping panel, may improve risk stratification when using CYP2D6 genotyping as a prognosticator in BrCa patients treated with tamoxifen. We hypothesize that BrCa tissues will harbor numerous mutations, due to a mutator phenotype inherent in most cancers, including within the CYP450 family of genes and, specifically, within the CYP2D6 gene. We expect that comparisons between germline DNA isolated from peripheral blood, and mutated DNA isolated from BrCa (somatic DNA) specimens within the same patient will reveal extensive differences in CYP2D6 genotypes. The aim of this study, is to extensively genotype 70 BrCa patients using a retrospective cohort with matched blood and tumor tissue from an existing biobank at UTHealth. We plan to perform genotype to phenotype conversions on each patient, for both germline and somatic DNA. We will look for discrepancies in genotypes and phenotypes and determine the magnitude of genetic bias possible in such cohorts. Method: DNA samples were extracted from matched archived tumor cells, dissected by laser microdissection microscopy and blood. Genotyping was performed by clinically validated Taqman® discrimination assays on the most common alleles for CYP2D6. We also studied the genotype status of these paired samples for CYP2C9, VKORC1, Factor II, Factor V, MTHFR, CYP3A4 and CYP3A5 genes. CYP2D6 gene copy number and gene rearrangement with CYP2D7 pseudogene were also assessed by Taqman copy number assays at 3 three different sites within gene. Genotype-phenotype conversion was performed using an in-house developed, clinically validated genotype-phenotype translator package. Genotype agreement was assessed between the two DNA sources. Results : Noticeable non-concordant results between DNA from breast tumors and blood were observed in the genotyping of polymorphisms in the CYP2D6 gene. However, strong agreement between DNA from breast tumors and blood was detected in the genotyping of polymorphisms in all other studied genes. These results suggest that previous publications refuting the association between CYP2D6 genetic polymorphisms and tamoxifen efficacy could have reached an inaccurate conclusion due to genetic bias and study design. Further research in this biomarker area is needed. Citation Format: Mehdi Dehghani, Anneliese O Gonzalez, Neda Hashemi-Sadraei, Songlin Zhang, Kevin P Rosenblatt. Correlation between germline and tumor CYP450 2D6 gene polymorphisms [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-10-09.

Abstract B54: Non-malignant cells from metastatic niches induce estrogen receptor-positive breast cancer cell resistance to therapy

Abstract Metastatic lesions remain the main cause of mortality in breast cancer (BrCa) and their resistance to systemic therapies has been mainly attributed to mutations in BrCa cells, while the non-mutually exclusive role of the metastatic microenvironment has been largely overlooked. To address the role of the local microenvironment of metastatic sites in conferring drug resistance to BrCa cells, we assembled heterotypic in vitro three-dimensional (3D) tissue cultures comprised of estrogen receptor-positive (ER+) BrCa cells and non-malignant cells from organs frequently targeted by metastatic disease. BrCa cells (MCF7, T47D, and ZR75-1) expressing the firefly luciferase gene were grown in extracellular matrices (collagen type I and Matrigel) alone or with nonmalignant cells from the respective metastatic target tissues, and exposed to clinically achievable concentrations of FDA-approved antineoplastic agents widely used for the treatment of BrCa. Interestingly, BrCa cell response to antiestrogens (hydroxytamoxifen (4-OHT), fulvestrant or raloxifene) in 3D conditions was marked by acinar differentiation of tumor spheroids into structures that resemble normal breast epithelium. Co-culture of BrCa cells with immortalized accessory cells from the bone (BMSCs and osteoblasts); brain (astrocytes); liver (hepatocytes); and lung (fibroblasts) in 3D (but not 2D) conditions, inhibited the antiestrogen-induced acinar differentiation and conferred resistance to 4-OHT, raloxifene, and fulvestrant at clinically relevant doses. The results obtained with immortalized human BMSCs were further confirmed in co-cultures of BrCa cells with primary human BMSCs isolated from bone metastatic site. MCF7 cells injected s.c. in nude mice together with BMSCs (heterotypic xenografts) had decreased response to tamoxifen compared with monotypic xenografts (MCF7 alone). Furthermore, immortalized BMSCs or metastatic patient-derived BMSCs induced MCF7 cell resistance to several chemotherapeutic agents including vinca alkaloids (vincristine, vinblastin and vinorelbin) and taxanes (docetaxel, paclitaxel, cabazitaxel). Notably, primary BMSCs from healthy donors and brain astrocytes isolated from cancer-free neurosurgical specimens did not induce BrCa cell resistance to anti-estrogens or chemotherapeutic agents. BMSCs induced in MCF7 cells downregulation of TGFβ2; upregulation of a transcriptional signature enriched for genes associated with high-grade tumors, including genes involved in IGF1R, IL6 and EGFR superfamily members; NF-kappaB and other antiapoptotic pathways; and elevated c-Myc protein levels. The co-cultures of MCF7 cells with BMSCs had increased sensitivity to inhibitors of c -myc, IGF1R, IL-6 and JAK/STAT pathways when compared to the respective mono-cultures. Our results indicate that local nonmalignant cells present in the metastatic niche can function as “accessories” to the tumor, conferring BrCa cell resistance to antiestrogens and chemotherapeutic agents in 3D tissue cultures and xenograft models. Preclinical modeling of the composition and 3D architecture of metastatic lesions can help elucidate the mechanisms of stroma-induced drug resistance and may reveal new therapeutic targets for refractory BrCa patients with metastatic disease. Citation Format: Eugen Dhimolea, Richard Groen, Culhane Aedin, Mitsiades Nicholas, Polyak Kornelia, Mitsiades Constantine. Non-malignant cells from metastatic niches induce estrogen receptor-positive breast cancer cell resistance to therapy. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B54. doi:10.1158/1538-7445.CHTME14-B54

Mutational profiling of familial male breast cancers reveals similarities with luminal A female breast cancer with rare TP53 mutations.

Background:Male breast cancer (MBC) is still poorly understood with a large proportion arising in families with a history of breast cancer. Genomic studies have focused on germline determinants of MBC risk, with minimal knowledge of somatic changes in these cancers.Methods:Using a TruSeq amplicon cancer panel, this study evaluated 48 familial MBCs (3 BRCA1 germline mutant, 17 BRCA2 germline mutant and 28 BRCAX) for hotspot somatic mutations and copy number changes in 48 common cancer genes.Results:Twelve missense mutations included nine PIK3CA mutations (seven in BRCAX patients), two TP53 mutations (both in BRCA2 patients) and one PTEN mutation. Common gains were seen in GNAS (34.1%) and losses were seen in GNAQ (36.4%), ABL1 (47.7%) and ATM (34.1%). Gains of HRAS (37.5% vs 3%, P=0.006), STK11 (25.0% vs 0%, P=0.01) and SMARCB1 (18.8% vs 0%, P=0.04) and the loss of RB1 (43.8% vs 13%, P=0.03) were specific to BRCA2 tumours.Conclusions:This study is the first to perform high-throughput somatic sequencing on familial MBCs. Overall, PIK3CA mutations are most commonly seen, with fewer TP53 and PTEN mutations, similar to the profile seen in luminal A female breast cancers. Differences in mutation profiles and patterns of gene gains/losses are seen between BRCA2 (associated with TP53/PTEN mutations, loss of RB1 and gain of HRAS, STK11 and SMARCB1) and BRCAX (associated with PIK3CA mutations) tumours, suggesting that BRCA2 and BRCAX MBCs may be distinct and arise from different tumour pathways. This has implications on potential therapies, depending on the BRCA status of MBC patients.

Methylation status and immunohistochemistry of BRCA1 in epithelial ovarian cancer.

Cancer initiation and progression are controlled by genetic and epigenetic events. One epigenetic process which is widely known is DNA methylation, a cause of gene silencing. If a gene is silenced the protein which it encodes will not expressed. 1. Identify the methylation status of BRCA1 in patients with epithelial ovarian cancer (EOC)and assess BRCA1 protein expression in tumor tissue. 2. Examine whether BRCA1 gene methylation and BRCA1 protein are associated with survival of epithelial ovarian cancer patients. The study design was a prospective-cohort study, conducted at Sardjito hospital, Yogyakarta, Indonesia. A total of 69 cases were analyzed in this study. The data showed that the methylation status of BRCA1 in EOC was positive in 89.9%, with clear protein expression of BRCA1 in 31.9%. Methylation status and expression of BRCA1 were not prognosticators of EOC patients. Menarche, CA125 level, clinical stage and residual tumor were independent factors for prognosis.