Hereditary BRCA1 Research Articles

Detection of presumed germline pathogenic variants of hereditary breast cancer predisposition genes in circulating tumor DNA: SCRUM-Japan MONSTAR-SCREEN.

1029 Background: Approximately 5-10% of breast cancer are hereditary. Variant allele frequency (VAF) of hereditary breast cancer predisposition genes in circulating tumor DNA (ctDNA) may be useful for detecting presumed germline pathogenic variants. Methods: One hundred and sixty-eight patients with advanced breast cancer (ABC) who underwent ctDNA and tumor tissue sequencing analyses in the SCRUM-Japan MONSTAR-SCREEN, a cancer genome screening project in Japan, from December 2019 to November 2021 were included. The patients were tested and monitored for their genomic alterations by FoundationOne Liquid assay or FoundationOne Liquid CDx assay. The pathogenic variants (PV) of hereditary breast cancer predisposition genes with VAF of 30% or higher in ctDNA were defined as PGPV. The VAF of BRCA1/2 on ctDNA analyses in BRCA1/2 germline pathogenic variant (GPV) carriers and the prevalence of PGPV in five hereditary breast cancer predisposition genes, including ATM, BRCA1, BRCA2, CHEK2, and PALB2, were investigated. Results: From 168 patients with ABC, including 115 Luminal, 32 HER2-positive, and 21 triple negative breast cancer, with a median age of 58 years, 39 PVs in 5 genes were identified with a median VAF of 0.62% (range: 0.1-84.77). ctDNA identified GPV of known BRCA1/2 GPV carriers (1 with BRCA1 and 6 with BRCA2) , with a median VAF of 51.4% (range: 48.2-77.5). The VAF of GPV on ctDNA were higher than 30% in subsequent consecutive samples. Among 161 patients with ABC, excluding 7 known BRCA1/2 GPV carriers, 6 PGPV (1 with BRCA1, 3 with BRCA2, and 2 with PALB2) were detected, with a median VAF of 65.5% (range: 51.2-84.8). Subsequent confirmatory tests were performed for two PGPV, and the variants were confirmed to be of germline origin. Conclusions: VAF on ctDNA analysis can help to easily detect PGPV of hereditary breast cancer predisposition genes. The PGPV detected in ctDNA analysis should be validated by established germline tests, and the results could provide opportunities for targeted therapies, as well as cancer risk assessment of patients and their relatives.

Identification of homologous recombination deficiency (HRD) by RAD51 in a tumor molecular profiling program for precision medicine.

3137 Background: Tumor molecular profiling by panel sequencing helps to identify candidate patients for precision oncology. Mutations in genes of the homologous recombination repair (HRR) pathway confer DNA repair deficiency and sensitivity to DNA damaging agents, such as PARP inhibitors (PARPi) or platinum-based drugs. RAD51 nuclear foci is a biomarker of functional HRR deficiency (HRD). We aimed to incorporate the RAD51 test in an academic molecular profiling program to identify HRD tumors beyond genetic testing. Methods: We included patients with metastatic breast cancer (TNBC, ER+ <60y, 3rd line HER2+ BC), newly diagnosed high-grade epithelial ovarian cancer (HGOC) and metastatic and/or castration-resistant prostate adenocarcinoma (PC) undergoing tumor/germline genetic testing. A customized capture-based targeted sequencing of 450 genes was performed in FFPE tumor samples. RAD51 was manually stained by immunofluorescence and functional HRD was defined as RAD51 score ≤10%. Results: Between January 2021 and January 2022, tumor panel sequencing was performed in 279 tumors. Panel sequencing was informative in 264/279 (95%) samples and the RAD51 test was evaluable in 81/90 (90%). In total, 77 samples were evaluable for both tumor/germline sequencing and RAD51, namely 42 BC, 16 HGOC and 19 PC (Table 1). Functional HRD by RAD51 was observed in 15/77 (20%) cases. Panel sequencing identified 17/77 (22%) cases carrying HRR gene alterations, of which nine were confirmed as HRD by RAD51. In BC, 2/6 tumors with HRD by RAD51 did not carry a germline or tumor mutation in HRR genes. HRD tumor profiling triggered the germline analysis of one hereditary BRCA2 BC patient. Four g BRCA1/2 BC with HRD by RAD51 became HRR proficient (HRP) at PARPi progression. In addition, the RAD51 test identified 2/5 HGOC and 2/4 PC tumors as HRD despite not carrying a tumor HRR gene mutation. Conclusions: RAD51 testing is feasible in an established molecular profiling program and complements gene panel sequencing results by providing evidence of functional HRR status. In particular, the RAD51 test extends the identification of HRD tumors beyond those with HRR gene mutations and captures HRR restoration after PARPi treatment. We aim to expand the analysis of RAD51 to other tumor types.[Table: see text]

Incidence and impact of brain metastasis in patients with hereditary BRCA1 or BRCA2 mutated invasive breast cancer

Patients with hereditary mutations in BRCA1 or BRCA2 (gBRCA1/2) and breast cancer have distinct tumor biology, and encompass a predilection for brain metastasis (BM). We looked into baseline risk of BMs among gBRCA1/2 patients. Patients with gBRCA1/2, stage I-III invasive breast cancer seen between 2000–2017 with parenchymal BMs. Among gBRCA1 with distant breast cancer recurrence, 34 of 76 (44.7%) were diagnosed with brain metastases compared to 7 of 42 (16.7%) patients with gBRCA2. In the comparator group, 65 of 182 (35.7%) noncarrier triple-negative breast cancer (TNBC) and a distant recurrence experienced BM’s. In a competitive risk analysis using death as a competing factor, the cumulative incidence of BMs was similar between gBRCA1 and noncarrier TNBC patients. The time from primary breast cancer diagnosis to detection of BMs was similar between gBRCA1 and noncarrier TNBC patients (2.4 vs 2.2 years). Survival was poor after BMs (7.8 months for gBRCA1 patients vs. 6.2 months for TNBC noncarriers). Brain was a more common site of initial distant recurrence in gBRCA1 patients versus TNBC noncarriers (26.3% vs. 12.1%). Importantly, the presence of BMs, adversely impacted overall survival across groups (HR 1.68 (95% CI 1.12–2.53), hazard ratio for death if a patient had BMs at the time of initial breast cancer recurrence vs. not). In conclusion, breast cancer BMs is common and is similarly frequent among gBRCA1 and noncarrier patients with recurrent TNBC. Our study highlights the importance of improving the prevention and treatment of BMs in patients with TNBC, gBRCA1 carriers, and noncarriers.

Peculiarities of BRCA1/2 testing in patients with advanced HER2-negative breast cancer in the Russian Federation (results of a survey of Russian oncologists)

Hereditary BRCA1 / 2 mutations affect the strategy of surgical treatment in early cancer and systemic treatment in advanced HER2-negative breast cancer. The article presents the results of a survey of Russian oncologists on various aspects of genetic testing for hereditary BRCA1 / 2 mutations in real-world clinical practice. Indications for testing, testing methods, and funding sources were discussed.

Detection of TP53 mutations in plasma of ovarian cancer and breast cancer patients

Background. The analysis of circulating tumor DNA provides wide opportunities for monitoring the results of cancer treatment. Somatic mutations in TP53 gene are present in almost all breast carcinomas developing in hereditary BRCA1 mutation carriers, as well as in the majority of high-grade serous ovarian tumors, which makes it possible to use them for effective monitoring of these diseases.
 The aim of the study was to analyze the content of tumor-specific TP53 mutations in plasma of patients with high-grade serous ovarian cancer (OC) and BRCA1-associated breast cancer (BC).
 Materials and methods. At least one plasma sample was obtained from 10 patients with OC and 7 patients with BRCA1-associated BC. The primary intratumoral status of TP53 gene was determined in the archival tumor material by targeted next generation sequencing. Digital droplet PCR was applied for testing of plasma samples for the presence of tumor-specific TP53 mutations, and in one case, BRAF V600E mutation.
 Results. All 8 plasma samples obtained from OC patients at the time of disease progression, before or during neoadjuvant chemotherapy, were positive for TP53 mutations. In contrast, 8 OC plasma samples obtained during remission, after surgery, or after neoadjuvant chemotherapy did not contain tumor-specific mutations. In breast cancer, circulating tumor DNA was detected in 2 of 4 samples obtained before treatment, and was not detected after the end of therapy or in remission.
 Conclusion. There is a good correlation between the presence of tumor-specific TP53 mutations in circulating DNA and the disease status in OC patients, therefore TP53 is a promising marker for clinical monitoring of ovarian cancer. In breast cancer, circulating tumor DNA is less abundant, therefore TP53 mutations cannot be reliably detected by digital droplet PCR in the plasma of patients with moderate disease burden.

Abstract IA-026: Gaps alone kill BRCA deficient cancer cells

Abstract Defects in DNA repair and/or the protection of stalled DNA replication forks are thought to underlie the chemosensitivity of tumors deficient in the hereditary breast cancer genes, BRCA1 and BRCA2 (BRCA). Challenging this assumption, recent findings indicate that chemotherapies such as cisplatin used to treat BRCA-deficient tumors do not initially cause DNA double-strand-breaks (DSBs). Here, we show that single stranded DNA (ssDNA) replication gaps underlie the hypersensitivity of BRCA-deficient cancer, and we propose that defects in homologous recombination (HR) or fork protection (FP) do not. Specifically, in BRCA-deficient cells, ssDNA gaps develop because replication is not effectively restrained in response to stress. Gap suppression by either restoration of fork restraint or gap filling confers therapy resistance in tissue culture and BRCA patient tumors. In contrast, restored FP and HR can be uncoupled from therapy resistance when gaps are present. Moreover, we find that DSBs are not detected after therapy when apoptosis is inhibited, supporting a framework in which DSBs are not directly induced by genotoxic agents, but rather from cell death nucleases. Together, this data indicate that ssDNA replication gaps underlie the BRCA cancer phenotype, “BRCAness,” and we propose are fundamental to the mechanism-of-action of genotoxic chemotherapies. Citation Format: Sharon B. Cantor, Nicholas Panzarino. Gaps alone kill BRCA deficient cancer cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-026.

Hereditary Breast-Ovarian Cancer and BRCA1 / BRCA2 variants: a single center experience

Hereditary Breast-Ovarian Cancer and BRCA1 / BRCA2 variants: a single center experience

The incidence and impact of brain metastasis in patients with hereditary BRCA1/2 mutated invasive breast cancer in a prospectively followed cohort.

1096 Background: Previous reports suggest the incidence of brain metastasis is higher in patients with hereditary BRCA1 mutations compared to BRCA1 noncarriers among breast cancer patients who develop recurrent disease. PARP inhibitors are now standard therapies for metastatic breast cancer patients with germline BRCA1 or BRCA2 mutations ( gBRCA1/2) based on their efficacy in treating systemic disease. However, as management of systemic disease improves, a concern is that patients with hereditary BRCA mutations may experience higher rates of disease progression in the CNS. We aimed to estimate the incidence of brain metastasis in breast cancer patients with gBRCA1/2 using a prospectively maintained gBRCA database and to assess the impact of brain metastasis on survival. Methods: To determine incidence, we queried a prospectively maintained electronic database that included patients referred to the MDACC genetics department and who underwent gBRCA1/2 testing. We identified patients with stage I-III invasive breast cancer who were treated between 2000-2017 and assessed for disease recurrence and brain metastasis. To expand our cohort for descriptive characteristics (separate from the incidence analysis), we queried the Breast Medical Oncology database for patients with brain metastasis who had undergone BRCA1/2 testing outside the genetics department or at outside institutions. Results: Of 474 patients with Stage I-III breast cancer and gBRCA1, 77 (16.2%) developed distant metastasis (median f/u: 9.1 years). Of these patients, 34/77 (44.2%) developed brain metastasis. In comparison, 42 of 318 (13.2%) of gBRCA2 patients with Stage I-III breast cancer developed distant recurrence (median f/u: 8.4 years), and 7/42 (16.7%) experienced brain metastasis. In gBRCA1 patients with brain metastasis, 45/48 (83.8%) had triple negative disease, and the median time from diagnosis to brain metastasis was 2.45 years. The brain was among the initial sites of disease recurrence in 24/48 (50%) of gBRCA1 patients. For gBRCA1 patients with distantly recurrent disease, median OS from diagnosis was 3.19 years for patients with brain metastasis vs. 5.37 years for patients without brain mets (HR 0.54; 95% CI 0.34 to 0.85; P = 0.0082). Conclusions: Brain metastasis is frequent among breast cancer patients with recurrent disease and hereditary BRCA1 mutations. Development and testing of agents with intracranial activity is critical for improving long-term outcomes in gBRCA1 patients with metastatic breast cancer.

Les cancers héréditaires du sein vus par le pathologiste

Breast cancers occurring in the context of a hereditary mutation of a predisposition gene represent 5 to 10% of all breast cancers, 20 to 25% of which being due to a mutation in the BRCA1 or BRCA2 genes. Authorization to market PARP inhibitors for breast cancer patients with hereditary BRCA1 and 2 mutations has recently been obtained. Given the annual frequency of breast cancer, morphological identification could facilitate the patient care process to limit the search for BRCA1 and 2 mutations to patients whose tumors have very specific characteristics. However, only a few morphological features have been recognized and differ depending on the mutated genes. Breast cancer occurring as part of a mutation in the BRCA1 gene is in 85% of cases of high-grade non-specific type invasive carcinomas with very limited contours, contain numerous lymphocytes in the stroma and are of triple-negative phenotype. Carcinomas associated with mutations in the BRCA2 genes and genes more recently recognized as associated with a risk of development of breast cancer (CHECK2, BMPR1A, BRIP1, PALB2, MUTYH) are most often non-specific invasive carcinomas, although other histological types are possible, grade III, luminal B phenotype. Breast cancer occurring in the context of a constitutional mutation of TP53 occurs in women under 35 years old are of non-specific histological type and with an amplification of HER2 in two thirds of the cases. Those associated with a PTEN mutation are readily of the apocrine type. Finally, very rarely, certain lobular-type breast cancers can occur in the context of a constitutional mutation of the CDH1 gene, which codes for the protein E-cadherin. The morphological and phenotypic characteristics may suggest to the pathologist a carcinoma of the breast occurring in a context of hereditary mutation. However, at the present time the only situations where a morphological sorting makes it possible to accelerate the genetic analysis are those of an invasive carcinoma of non-specific type of triple-negative phenotype in a woman of less than 50 years or that of a diagnosis of HER2 breast cancer amplified in a woman under 31 years of age (Chompret criteria). Family background and personal history are of great importance in the genetic counseling indication decision trees. Unfortunately, to date, no quality antibody has been developed against BRCA1 and 2 to help the pathologist identify hereditary cases. The immunohistochemical analysis of RAD51 could facilitate the identification of tumors possibly sensitive to PARP inhibitors. Progress to identify hereditary cancers is expected thanks to the development of artificial intelligence algorithms from digitized histological slides.

Targeted capture-based NGS is superior to multiplex PCR-based NGS for hereditary BRCA1 and BRCA2 gene analysis in FFPE tumor samples

BackgroundWith the introduction of Olaparib treatment for BRCA-deficient recurrent ovarian cancer, testing for somatic and/or germline mutations in BRCA1/2 genes in tumor tissues became essential for treatment decisions. In most cases only formalin-fixed paraffin-embedded (FFPE) samples, containing fragmented and chemically modified DNA of minor quality, are available. Thus, multiplex PCR-based sequencing is most commonly applied in routine molecular testing, which is predominantly focused on the identification of known hot spot mutations in oncogenes.MethodsWe compared the overall performance of an adjusted targeted capture-based enrichment protocol and a multiplex PCR-based approach for calling of pathogenic SNVs and InDels using DNA extracted from 13 FFPE tissue samples. We further applied both strategies to seven blood samples and five matched FFPE tumor tissues of patients with known germline exon-spanning deletions and gene-wide duplications in BRCA1/2 to evaluate CNV detection based solely on panel NGS data. Finally, we analyzed DNA from FFPE tissues of 11 index patients from families suspected of having hereditary breast and ovarian cancer, of whom no blood samples were available for testing, in order to identify underlying pathogenic germline BRCA1/2 mutations.ResultsThe multiplex PCR-based protocol produced inhomogeneous coverage among targets of each sample and between samples as well as sporadic amplicon drop out, leading to insufficiently or non-covered nucleotides, which subsequently hindered variant detection. This protocol further led to detection of PCR-artifacts that could easily have been misinterpreted as pathogenic mutations. No such limitations were observed by application of an adjusted targeted capture-based protocol, which allowed for CNV calling with 86% sensitivity and 100% specificity. All pathogenic CNVs were confirmed in the five matched FFPE tumor samples from patients carrying known pathogenic germline mutations and we additionally identified somatic loss of the second allele in BRCA1/2. Furthermore we detected pathogenic BRCA1/2 variants in four the eleven FFPE samples from patients of whom no blood was available for analysis.ConclusionsWe demonstrate that an adjusted targeted capture-based enrichment protocol is superior to commonly applied multiplex PCR-based protocols for reliable BRCA1/2 variant detection, including CNV-detection, using FFPE tumor samples.

BRCA1 mRNA expression levels as an predictor of response to neoadjuvant chemotherapy in breast cancer

Background. Most modern studies are limited to the study of only germline mutations of the BRCA1 gene (most often 5382insC). And there are very few works that characterize the different somatic changes in the BRCA1 gene in the tumor, in particular the expression of this gene and its relation to the effect of chemotherapy. Given the linkage of the hereditary BRCA1 mutation with the high efficacy of platinum drugs, it can be assumed that the expression of the BRCA1 gene will be associated with a high sensitivity of the neoplasm to platinum drugs as well. Objective: study of the relationship of BRCA1 gene expression to the efficacy of neoadjuvant chemotherapy (NAC) in breast tumors. Materials and methods. Study included 105 patients with breast cancer T1–4N0–3M0 (IIA–IIIB stage). The diagnosis of breast cancer was verified morphologically. The initial expression of BRCA1 was evaluated in the tumor material. RNA was extracted from biopsy specimens of breast tumor tissue. Expression profiling of the BRCA1 gene was carried out using quantitative real-time polymerase chain reaction. Results. Expression of the BRCA1 gene was evaluated as a result of the study. There was shown its weak correlation with clinico-morphological parameters of patients with breast cancer and parameters of the tumor process. It has been established that an objective response to NAC is associated with a high level of BRCA1 when taxotere is used in monoregime (p = 0.03) and in the CAX (cyclophosphamide, doxorubicin, xeloda) scheme (p = 0.05). Nevertheless, it was shown that, regardless of the regimen used and the number of another NAC courses, high expression of the gene being studied (more than 1.5) in the primary tumor in 70 % of cases generally results in a lack of an objective response to treatment, compared with a low level (less than 0.3) (Fisher test; p = 0.03). Conclusion. The obtained data on the relationship of expression of the BRCA1 gene before treatment may be a predictive marker of the efficacy of NAC in patients with breast cancer.

Abstract 4318: Reversal of BRCA-1 CpG hyperthylation by genistein and (-)-epigallocatechin-3-gallate in human breast cancer cells with activated AhR

Abstract Sporadic breast cancers, which represent the vast majority (∼90%) of breast tumor cases, do not have mutations in the tumor suppressor gene, BRCA-1 but have absent or markedly reduced levels of BRCA-1 similar to those observed in hereditary BRCA-1 tumors. Therefore, understanding the non-mutational mechanisms that contribute to repression of BRCA-1 has important implications for the prevention of both hereditary and sporadic breast tumors. Agonists of the aromatic hydrocarbon receptor (AhR) are ubiquitous in the environment and include dietary compounds, metabolites of fatty acids, industrial pollution, and photoproducts generated in the skin from ultraviolet radiation. In cultured estrogen-receptor (ER)α-positive MCF-7 breast cancer cells, we found that activation of the AhR with the prototype ligand 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) repressed by 50% estradiol-induced BRCA-1 expression. Conversely, the post-treatment with the soy isoflavone genistein and tea (-)-epigallocatechin-3-gallate (EGCG) at physiological doses (1 μM) rescued within 24 to 48 h BRCA-1 expression to levels ∼2.0 to 3.0-fold higher than those measured in cells treated with estradiol alone. Genistein and EGCG reversed BRCA-1 promoter hypermethylation in AhR-activated MCF-7 cells. In ERα-negative and AhR-overexpressing UACC-3199 breast cancer cells, genistein (1 μM) reactivated BRCA-1 expression. The post-treatment of AhR-activated MCF-7 cells with genistein and EGCG antagonized the recruitment of DNA-methyl transferase-1 (DNMT-1) enzyme to the BRCA-1 promoter. These results suggest the involvement of epigenetic mechanisms in the repression of BRCA-1 and reversal effects of genistein and EGCG against breast tumorigenesis associated with activation of the AhR. Citation Format: Ornella I. Selmin, Andreas J. Papoutsis, Donato F. Romagnolo. Reversal of BRCA-1 CpG hyperthylation by genistein and (-)-epigallocatechin-3-gallate in human breast cancer cells with activated AhR. [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 4318.

Abstract P5-06-03: Combination of the PARP inhibitor E7449 with eribulin +/- carboplatin in preclinical models of triple negative breast cancer

Abstract Introduction: In a small neoadjuvant study in patients with triple negative breast cancer (TNBC) the combination of eribulin plus carboplatin was effective, with a pathologic complete response rate of 43% following 4 cycles of treatment. Significant numbers of sporadic TNBC tumors are deficient in DNA repair capacity and share clinical and pathological features with hereditary BRCA1 mutant disease. PARP inhibitors have demonstrated synthetic lethality in cancer cells with defective DNA repair and have therapeutic potential for TNBC. In this study we describe the combination of PARP inhibitor E7449 with eribulin +/- carboplatin in preclinical models of TNBC. Methods: E7449, an orally available PARP inhibitor, was administered in combination with eribulin +/- carboplatin to 4 s.c. xenograft models of TNBC: MDA-MB-436 (BRCA1 mutant, PTEN deficient), MDA-MB-468 (BRCA wild type, PTEN deficient), HCC1806 and MDA-MB-231 (BRCA and PTEN wild type). Results and Discussion: Addition of E7449 to eribulin significantly delayed tumor progression in PTEN deficient MDA-MB-468 xenografts. In the BRCA1 mutant and PTEN deficient MDA-MB-436 xenograft model, combination of E7449 with eribulin enhanced antitumor activity versus eribulin alone. Similar potentiation was observed for carboplatin upon combination with E7449. Treatment of MDA-MB-436 xenografts with the triple combination of E7449 + eribulin + carboplatin was more efficacious than any double combination and was well tolerated at the doses examined. In contrast, no significant combination activity was observed for E7449 plus eribulin in the BRCA and PTEN wild type xenografts HCC1806 and MDA-MB-231, and similarly no potentiation of carboplatin was observed in an MDA-MB-231 xenograft. Notably, combination activity was observed in the BRCA1 mutant (MDA-MB-436) and PTEN deficient (MDA-MB-436 and MDA-MB-468) xenografts and not in the BRCA and PTEN wild-type models (HCC1806 and MDA-MB-231). Data from ongoing studies to evaluate the combination activity of E7449 + eribulin in patient-derived xenograft (PDx) models of TNBC will be presented at the meeting. Potential biomarkers of sensitivity to the combination are under investigation in both cell line xenograft and PDx models and will be described. Conclusion: The addition of E7449 to eribulin +/- carboplatin increased antitumor activity in a subset of TNBC models. Biomarker studies aimed at a better understanding of the underlying cause of sensitivity are underway. The preclinical data support assessment of E7449 + eribulin + carboplatin combination therapy in the current phase I/II clinical trial. Citation Format: Sharon McGonigle, Jiayi Wu, Donna Kolber-Simonds, Natalie C Twine, Jue-lon Shie, Noel Taylor, Sergei Agoulnik, Zoltan Dezso, Shannon McGrath, Mark Matijevic, Shanqin Xu, Galina Kuznetsov, Mary Woodall-Jappe, Kenichi Nomoto. Combination of the PARP inhibitor E7449 with eribulin +/- carboplatin in preclinical models of triple negative breast cancer [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 P5-06-03.

Germline TP53 mutational spectrum in French Canadians with breast cancer.

BackgroundSpecific germline mutations in the hereditary breast-ovarian cancer susceptibility (HBC/HBOC) genes, BRCA1, BRCA2 and PALB2, have been shown to recur in French Canadians of Quebec, Canada, and this has been attributed to common ancestors. Germline TP53 mutation carriers are known to segregate in Li-Fraumeni syndrome families, which feature young age of onset breast cancer. We have reported rare TP53 mutation carriers in French Canadian HBC families, though none recurred possibly due to the limited number of cancer families investigated. Here we describe TP53 germline mutations found in French Canadian cancer families provided from hereditary cancer clinics; investigate 37 new BRCA1 and BRCA2 mutation-negative HBC/HBOC families for the TP53 mutations; and assess the frequency of TP53 mutations in a 1235 French Canadian breast cancer cases not selected for family history of cancer.MethodsTP53 mutation-positive pedigrees from French Canadian cancer families were provided from local hereditary cancer clinics. Bidirectional Sanger sequencing of all protein encoding exons of TP53 was performed using peripheral blood lymphocyte DNA from breast/ovarian cancer probands from 37 HBC/HBOC families of French Canadian descent. Targeted bidirectional Sanger sequencing assay of regions containing the identified TP53 mutations was performed on 1235 French Canadian breast cancer cases not selected for family history cancer.ResultsFive new TP53 mutations were identified in six pedigrees from hereditary cancer clinics. No deleterious mutations were identified in cancer probands from 37 HBC/HBOC families. A targeted mutation screen of the 1235 breast cancer cases identified a c.844C>T [p.Arg282Trp] mutation carrier. This mutation was also found among the six mutation-positive cancer families provided by the local hereditary cancer clinics. The targeted screen also uncovered a new TP53 mutation, c.685T>C [p.Cys229Arg] that was found in two breast cancer cases. All TP53 mutation carriers were among the 656 women with breast cancer diagnosed less than 50 years of age.ConclusionsIn all six new TP53 mutations were identified in French Canadians, where two each occurred in independently ascertained cases/families. Although all newly identified breast cancer mutation carriers reported a family history of cancer, none were consistent with features of Li-Fraumeni syndrome families.

Biomarkers in triple negative breast cancer: A review.

Breast cancer is an intrinsically heterogeneous disease. In the world about 1 million cases of breast cancer are diagnosed annually and more than 170000 are triple-negative. Characteristic feature of triple negative breast cancer (TNBC) is that it lacks expression of oestrogen, progesterone and human epidermal growth factor receptor-2/neu receptors. They comprise 15%-20% of all breast cancers. We did a systematic review of PubMed and conference databases to identify studies published on biomarkers in TNBC. We included studies with biomarkers including: Epidermal growth factor receptor, vascular endothelial growth factor, c-Myc, C-kit and basal cytokeratins, Poly(ADP-ribose) polymerase-1, p53, tyrosinase kinases, m-TOR, heat and shock proteins and TOP-2A in TNBC. We also looked for studies published on synthetic lethality and inhibition of angiogenesis, growth, and survival pathways. TNBC is a complex disease subtype with many subclasses. Majority TNBC have a basal-like molecular phenotype by gene expression profiling. Their clinical and pathologic features overlap with hereditary BRCA1 related breast cancers. Management of these tumours is a challenge to the clinician because of its aggressive behaviour, poor outcome, and absence of targeted therapies. As the complexity of this disease is being simplified over time new targets are also being discovered for the treatment of this disease. There are many biomarkers in TNBC being used in clinical practice. Biomarkers may be useful as prognostic or predictive indicators as well as suggest possible targets for novel therapies. Many targeted agents are being studied for treatment of TNBC.

Abstract B23: Preclinical evaluation of a PARP inhibitor in mice representing genetically different subtypes of pancreatic cancers

Abstract The incredibly short survival of patients diagnosed with pancreatic cancer underscores one of the greatest failures of the medical community: our inability to identify effective treatments for pancreatic ductal adenocarcinoma. Most new compounds in clinical trials fail because of highly toxic side effects in normal tissues and limited efficacy in tumor response. An ideal drug would kill only tumor cells and spare the normal cells. In this regard, poly(ADP-ribose) polymerase (PARP) inhibitors have demonstrated a promising therapeutic potential, particularly in patients with BRCA-deficient tumors. BRCA-deficient cells have an impaired homology-directed repair (HDR) pathway, a mechanism critical for resolving DNA double strand breaks (DSBs). When PARP1 is inhibited, DNA single strand breaks are not repaired and they mature to double strand breaks, which are very toxic for the cell. Hence, inhibition of PARP1 is selectively toxic to cells with an impaired HDR pathway, such as BRCA-deficient tumor cells, but not to normal cells. Because up to 10% of pancreatic cancer patients carry mutations in either BRCA2 or other proteins involved in the HDR pathway, PARP inhibitors may also prove effective in this setting. However, a number of subtleties remain that call this conclusion into question, including poor drug delivery, loss of heterozygosity, and BRCA-independent alteration of the HDR pathway. We designed a preclinical study to evaluate the efficacy of niraparib, a potent PARP inhibitor currently in clinical trials, in three different genetically engineered mouse models representing different pancreatic cancer patient populations. The KPC mice carry mutations in genes that are frequently mutated in pancreatic cancer patients and develop tumors that fully recapitulate the biology of human pancreatic ductal adenocarcinoma. These mice will represent the broad population of patients with spontaneous pancreatic tumors. A second model with heterozygous mutations in BRCA2 (KPCB2+/-) will represent patients with hereditary BRCA2 mutations. A third model with homozygous conditional deletion of BRCA2 (KPCB2f/f) will represent spontaneous tumors that have completely lost HDR functions. We show that BRCA2-deficient KPC tumor cells strongly respond to treatment with niraparib in vitro and in vivo. Niraparib efficiently inhibits PARP activity, increases double strand break formation and induces M-phase cell cycle arrest in tumors of KPCB2f/f mice. In the future, we will use pre- and post-treatment tumor biopsies to determine the molecular mechanisms of response to PARP inhibition. The ultimate goal of this study is to identify genetic determinants of PARP sensitivity and validate our results in a clinical setting. Identification of a biomarker would be an extremely valuable clinical tool to classify pancreatic cancer patients that could respond to the treatment with PARP inhibitors, a tool that is currently not available. Citation Format: Barbara Orelli, Stephen A. Sastra, Carmine F. Palermo, Thomas Ludwig, Kenneth P. Olive. Preclinical evaluation of a PARP inhibitor in mice representing genetically different subtypes of pancreatic cancers. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B23.

Abstract 1554: Identification of BRCA1 and BRCA2 somatic mutations in breast cancer tumors with loss of BRCA1 nuclear expression

Abstract Breast cancer is the first cause of death by cancer in women in Chile as in many countries around the world. Until today, BRCA1 and BRCA2 are the most relevant genes for breast cancer high risk. Moreover, different studies have shown that BRCA1 is under expressed in breast tumors. Our group has found that in a high percentage of hereditary tumors BRCA1 protein have a diminished or null expression, or is mislocalized to the cytoplasm (unpublished data). In this regard, it has become a goal for us to identify if mislocalization and/or loss of expression of BRCA1 and BRCA2 could be related to somatic mutations in the tumors. In this work, we sequenced BRCA1 and BRCA2 in DNA extracted from 14 fresh/frozen tumors. DNA was isolated from each tumor and amplified by PCR in 5 multiplexes (a total of 93 amplicons) considering all exons and intron/exon junctions with approximately 30bp of intronic sequence each side. Multiplexes were then pooled for each patient and processed for sequencing in the GS Junior Roche. All reads were processed with AVA software to detect nucleotide changes in BRCA1 and BRCA2 sequences and analyzed manually to discard misinterpretations. All mutations identified were confirmed by Sanger. From the same tumors we obtained a second section that was formalin fixed and paraffin embedded. This FFPE fragment was cut in 4 μm sections and analyzed by immunohistochemistry and immunofluorescence to assess BRCA1 expression and localization. We found a total of 5 mutations, 3 in BRCA1 and 2 in BRCA2, in 6 tumors. Interestingly, one BRCA1 mutation present in 7 to 20% of reads, was found in 5 tumors. Among those, two tumors presented two different mutations in BRCA2, in addition. The last tumor presented one extra mutation in BRCA1. BRCA1 expression analysis was coherent between immunohistochemistry and immunofluorescence assays. In general 9 tumors showed weak or negative BRCA1 nuclear expression and 5 mainly moderate nuclear staining. In addition 6 tumors presented moderate to strong cytoplasmic expression of the protein, considered abnormal. Considering tumors with weak or negative expression of BRCA1, 40% presents BRCA1 or BRCA2 mutations. The relevance of the absence of BRCA1 expression, or function impairment of BRCA1 and/or BRCA2 due to somatic mutations, relays on the possible treatment with PARP1 inhibitors. Supported by FONDECYT 1120200. Citation Format: Carolina Alvarez, David Wiener, Patricia Gajardo, Wanda Fernandez, Valeria Cornejo, Jorge Gamboa, Pilar Carvallo. Identification of BRCA1 and BRCA2 somatic mutations in breast cancer tumors with loss of BRCA1 nuclear expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1554. doi:10.1158/1538-7445.AM2014-1554

Minor role of BRCA2 mutation (Exon2 and Exon11) in patients with early-onset breast cancer amongst Iranian Azeri-Turkish women.

Breast cancer is the most common cancer in women. Every year, one million new cases are reported worldwide, representing 18% of the total number of cancer in women. Hereditary BRCA1 and BRCA2 mutations account for about 60% of inherited breast cancer and are the only known causes of hereditary breast cancer syndrome. The aim of this study was to determine the frequency of BRCA2 (exon2 and exon11) gene mutation in patients with early-onset breast cancer among Iranian Azeri-Turkish women. We obtained clinical information, family history and peripheral blood from 110 women under the age of 45 with early-onset breast cancer for scanning germline mutations in the exon2 and 11 of BRCA2 genes which comprises over 50% of the gene. Single-strand conformation polymorphism assay was used in order for screening potential mutations on amplified regions followed by direct sequencing analysis to determine the genotypes. Overall, 11 sequence variants were identified in this study group, including four homozygotes and seven heterozygotes silent substitution of c.3807T to C, p.Val1269Val (rs543304). Mutations in BRCA2 were surprisingly infrequent in the early onset breast cancer patients among Iranian Azeri-Turkish women.

Systemic treatment strategies for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is defined by the lack of immunohistochemical expression of the estrogen and progesterone receptors and human epidermal growth factor receptor 2 (EGFR2). Most TNBC has a basal-like molecular phenotype by gene expression profiling and shares clinical and pathological features with hereditary BRCA1 related breast cancers. This review evaluates the activity of available chemotherapy and targeted agents in TNBC. A systematic review of PubMed and conference databases was carried out to identify randomised clinical trials reporting outcomes in women with TNBC treated with chemotherapy and targeted agents. Our review identified TNBC studies of chemotherapy and targeted agents with different mechanisms of action, including induction of synthetic lethality and inhibition of angiogenesis, growth and survival pathways. TNBC is sensitive to taxanes and anthracyclins. Platinum agents are effective in TNBC patients with BRCA1 mutation, either alone or in combination with poly adenosine diphosphate polymerase 1 inhibitors. Combinations of ixabepilone and capecitabine have added to progression-free survival (PFS) without survival benefit in metastatic TNBC. Antiangiogenic agents, tyrosine kinase inhibitors and EGFR inhibitors in combination with chemotherapy produced only modest gains in PFS and had little impact on survival. TNBC subgroups respond differentially to specific targeted agents. In future, the treatment needs to be tailored for a specific patient, depending on the molecular characteristics of their malignancy. TNBC being a chemosensitive entity, combination with targeted agents have not produced substantial improvements in outcomes. Appropriate patient selection with rationale combinations of targeted agents is needed for success.

A Clinically Validated Diagnostic Second-Generation Sequencing Assay for Detection of Hereditary BRCA1 and BRCA2 Mutations

Individuals who inherit mutations in BRCA1 or BRCA2 are predisposed to breast and ovarian cancers. However, identifying mutations in these large genes by conventional dideoxy sequencing in a clinical testing laboratory is both time consuming and costly, and similar challenges exist for other large genes, or sets of genes, with relevance in the clinical setting. Second-generation sequencing technologies have the potential to improve the efficiency and throughput of clinical diagnostic sequencing, once clinically validated methods become available. We have developed a method for detection of variants based on automated small-amplicon PCR followed by sample pooling and sequencing with a second-generation instrument. To demonstrate the suitability of this method for clinical diagnostic sequencing, we analyzed the coding exons and the intron-exon boundaries of BRCA1 and BRCA2 in 91 hereditary breast cancer patient samples. Our method generated high-quality sequence coverage across all targeted regions, with median coverage greater than 4000-fold for each sample in pools of 24. Sensitive and specific automated variant detection, without false-positive or false-negative results, was accomplished with a standard software pipeline using bwa for sequence alignment and samtools for variant detection. We experimentally derived a minimum threshold of 100-fold sequence depth for confident variant detection. The results demonstrate that this method is suitable for sensitive, automatable, high-throughput sequence variant detection in the clinical laboratory.