BRCA1 185delAG Mutation Research Articles

A case\u2013control study of BRCA1 founder mutations 185delAG and 5382insC in a cohort of Egyptian ovarian cancer patients using pyrosequencing technique

BackgroundOvarian cancer (OC) is considered a leading cause of death among women with gynecological malignancies. OC, like breast cancer, shows a familial predisposition to germline mutations in genes BRCA1 or BRCA2, which have proved to play important roles in the incidence and progression of cancers. In Arab countries there are limited data concerning BRCA1 or BRCA2 founder mutations associated with familial ovarian cancer (FOC). Therefore, the aim of this pilot study was to assess two common founder mutations of BRCA1 (185delAG and 5382insC) in a cohort of Egyptian patients with FOC. The study included fifty female patients with FOC and twenty healthy controls. Clinical, laboratory, and pathological findings were assessed as well as response to therapy. Genetic testing for BRCA1 (185delAG and 5382insC) mutations was performed on peripheral blood samples using a short-fragment sequencer (pyrosequencer).ResultsThe BRCA1 185delAG mutation was not observed in either the FOC patients or the controls. However, the carrier frequency of heterozygous BRCA1 5382insC mutation was 8%. All the FOC patients with a BRCA1 5382insC mutation had a positive family history of cancer (p = 0.009). All carriers of the BRCA1 5382insC mutation showed a preliminary good response to chemotherapy. The majority of carrier patients were diagnosed at an advanced stage of the disease with high-grade tumors and distant metastasis (75% of cases).ConclusionThe frequency of the BRCA1 5382insC mutation in FOC patients was 8%. The strong association between the mutation and the positive family history suggests that a wider screening for BRCA1 founder mutations would be valuable in predicting high-risk individuals.

BRCA2 Haploinsufficiency in Telomere Maintenance.

Our previous studies showed an association between monoallelic BRCA2 germline mutations and dysfunctional telomeres in epithelial mammary cell lines and increased risk of breast cancer diagnosis for women with BRCA2 999del5 germline mutation and short telomeres in blood cells. In the current study, we analyzed telomere dysfunction in lymphoid cell lines from five BRCA2 999del5 mutation carriers and three Fanconi Anemia D1 patients by fluorescence in situ hybridization (FISH). Metaphase chromosomes were harvested from ten lymphoid cell lines of different BRCA2 genotype origin and analyzed for telomere loss (TL), multitelomeric signals (MTS), interstitial telomere signals (ITS) and extra chromosomal telomere signals (ECTS). TL, ITS and ECTS were separately found to be significantly increased gradually between the BRCA2+/+, BRCA2+/- and BRCA2-/- lymphoid cell lines. MTS were found to be significantly increased between the BRCA2+/+ and the BRCA2+/- heterozygous (p < 0.0001) and the BRCA2-/- lymphoid cell lines (p < 0.0001) but not between the BRCA2 mutated genotypes. Dysfunctional telomeres were found to be significantly increased in a stepwise manner between the BRCA2 genotypes indicating an effect of BRCA2 haploinsufficiency on telomere maintenance.

Age at diagnosis of cancer in 185delAG BRCA1 mutation carriers of diverse ethnicities: tentative evidence for modifier factors.

Germline pathogenic sequence variants (PSVs) in BRCA1 substantially increase risk for developing breast (BC) and ovarian cancer (OvC). Yet, incomplete penetrance suggests that modifier factors affect phenotypic expression of mutant BRCA1 alleles. Analysis of identical BRCA1 PSV carriers of diverse ethnicities may provide further evidence for modifier factors. Female carriers of the 185delAG BRCA1 PSV identified through high-risk clinics in Israel, and Manchester England from 1998-2018 were eligible. Data were retrieved from patients records and confirmed (in Israel) by cross referencing with the Israeli National Cancer Registry. Overall, 2503 female carriers were included: 1715 (71.4%) Ashkenazi Jews (AJ), 201 (8.3%) Iraqi Jews and 383 (15.9%) of mixed ethnicity. In 102 (4.2%) cases ethnicity could not be ascertained. Of Israeli AJ carriers 649 (37.8%), 256 (14.9%) and 62 (3.6%) were diagnosed with BC, OvC or both cancers, respectively. For the Iraqi Jews these frequencies were 76 (37.8%), 43 (21.4%), and 8 (3.98%), respectively. Age at diagnosis of BC in AJ and Iraqi Jews was 46.7 ± 12.3 years and 52.8 ± 12.2 years, respectively (p = 0.001). For OvC age at diagnosis for AJ was 53.5 ± 10.7 years and for Iraqi Jews 50.1 ± 8.8 years (p = 0.0027). No differences in these parameters were noted between English Jews (n = 110) and non-Jews (n = 32). Age at diagnosis of BC and OvC differs between AJ and Iraqi Jews who carry an identical BRCA1 PSV. This finding supports the existence of modifier factors that may be ethnic specific.

CpG promoter hypo-methylation and up-regulation of microRNA-190b in hormone receptor-positive breast cancer.

Estrogen receptor-positive breast cancer is subdivided into subtypes LuminalA and LuminalB, based on different expression patterns. MicroRNA-190b has been reported to be up-regulated in estrogen receptor-positive breast cancers. In this study we aimed to investigate the role of CpG promoter methylation in regulating miR-190b expression and its impact on clinical presentation and prognosis. DNA methylation analysis for the promotor of microRNA-190b was performed by pyrosequencing 549 primary breast tumors, of which 62 were carriers of the BRCA2999del5 founder mutation, 71 proximal normal breast samples and 16 breast derived cell lines. MicroRNA-190b expression was analysed in 67 primary breast tumors, 14 paired normal breast samples and 16 breast derived cell lines. Tissue microarrays (TMAs) were available for ER (n = 436), PR (n = 436), HER-2 (N = 258) and Ki67 (n = 248). MiR-190b had reduced promoter methylation in estrogen receptor-positive breast cancers (P = 1.02e–12, Median values: ER+ 24.3, ER– 38.26) and miR-190b’s expression was up-regulated in a correlative manner (P = 1.83e–06, Spearman’s rho –0.62). Through breast cancer specific survival analysis, we demonstrated that LuminalA patients exhibiting miR-190b hypo-methylation had better survival than other patients (P = 0.034, HR = 0.29, 95% CI 0.09-0.91). We, furthermore, demonstrated that miR-190b hypo-methylation occurs less frequently in ER+ tumors from BRCA2999del5 mutation carriers than in non-mutated individuals (P = 0.038, Χ2 = 4.32, n = 335). Our results suggest that upregulation of miR-190b may occur through loss of promoter DNA methylation during the development of estrogen-receptor (ER) positive breast cancers, and that miR-190b hypo-methylation leads to increased breast cancer specific survival within the LuminalA- subtype but not LuminalB.

Could the 185delAG BRCA1 mutation be an ancient Jewish mutation?

A predominant mutation within the BRCA1 predisposition gene, 185delAG, has been detected in about 1% of the Ashkenazi population, considered a high-risk group for breast and ovarian cancers. We examined 639 unrelated healthy Jews of Iraqi extraction, a presumed low-risk group, for the existence of this mutation. Three individuals were identified as 185delAG mutation carriers, and haplotype analysis of the Iraqi mutation carriers revealed that 2 of the Iraqis shared a common haplotype with 6 Ashkenazi mutation carriers, and 1 had a haplotype which differed by a single marker. This study suggests that the BRCA1 185delAG mutation also occurs in populations considered at low-risk for breast and ovarian cancers, and that it might have occurred prior to the dispersion of the Jewish people in the Diaspora, at least at the time of Christ.

Abstract 3371: Analysis of telomere length and dysfunction in normal breast tissue from BRCA2 mutation carriers

Abstract The tumor suppressor protein BRCA2 is important for maintaining genomic stability. BRCA2 is essential for homologous recombination repair of DNA double strand breaks and also has a role in protection of stalled replication forks as well as in telomere protection and maintenance. Disruptions in telomere homeostasis can result in excessive telomere shortening and drive chromosome instability, a hallmark of BRCA2-related cancers. Germline mutations in the BRCA2 gene are associated with highly increased risk of breast cancer. A single deleterious founder mutation, BRCA2999del5, has been detected in the gene in the Icelandic population, making it feasible to study the influence of a single mutation at a population level. The BRCA2999del5 mutation is found in 6-7% of female breast cancer patients in Iceland and is associated with complex chromosomal changes in tumor tissue. Furthermore, the BRCA2999del5 mutation is associated with poor prognosis, although mutation carriers seem to differ with respect to age of onset and severity of disease. It is therefore of great interest to identify factors that influence breast cancer risk within the mutation carrier group. The aim of the study was to measure telomere length (TL) and levels of telomere dysfunction in tumor-adjacent normal breast tissue from BRCA2 mutation carriers and non-carriers and investigate their potential relationship with breast cancer risk. The study group consisted of 186 Icelandic breast cancer patients; BRCA2999del5 mutation carriers and sporadic cases, matched with respect to age and year of diagnosis. For TL measurements, paraffin embedded normal breast tissue samples were analyzed by Quantitative Fluorescence in Situ Hybridization. Telomere dysfunction induced foci (TIFs) can be detected where co-localization of telomere and DNA-damage signals occur. For this purpose, the samples were also immunofluorocently stained for the DNA damage repair protein 53BP1. In concordance with previously reported data in the literature, luminal epithelial cells showed the shortest TL and highest number TIFs of the cell types in normal breast tissue. This observation is highly relevant as these are the cells from which most breast cancers originate. No difference was detected in age-adjusted TL between BRCA2 mutation carriers and non-carriers. However, consistent with our recently published data from TL measurements in blood from the same study group, shorter TL is correlated with younger age at breast cancer diagnosis in BRCA2 mutation carriers but not in non-carriers. In conclusion, these results indicate that in BRCA2 mutation carriers, shorter TL in normal breast tissue is associated with earlier breast cancer occurrence. Citation Format: Birna Thorvaldsdottir, Beth A. Cimini, Morgan E. Diolaiti, Katrin Olafsdottir, Jon G. Jonasson, Jorunn E. Eyfjörd. Analysis of telomere length and dysfunction in normal breast tissue from BRCA2 mutation carriers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3371.

Tumor Suppressor Gene Mutations in Iraqi Women with Breast Cancer and Their Relatives

Background: Breast cancer is characterized by various malignant tumors arise from breast tissues,age, genetic factors,diet and environmental factors,reproductive and hormonal factors, some types of benign breast disease andsocio-economic factors are factors correlated to an enlarged risk of breast cancer.Tumor suppressor genes are genes whose loss of roleresults in the elevation of malignancy. Aim of the Study: The present study was conducted to verify the tumor suppressor gene mutations in breast cancer women and their relatives. Material and Methods: Three groups of samples were included. The first consisted of 100 blocks of formalinfixed, paraffinembedded(FFPE) breast cancer tissuesof women(group 1). The second contained blood samples of 46 breast cancerwomen(group 2). The third comprised blood samples of46 apparently healthy women who were relatives to the breast cancer patients of group two(group 3). The ages of patients of samples tissue were 46.78±11.5 years, while for samples of blood patients were 47.34±11.18 years. The ages of the healthy relatives were 40.79±9.84 years. Five tumor suppressor gene mutations were examined.BRCA1 185delAG, BRCA1 5382insC and BRCA2 6174delT mutations were evaluated by mutagenically separated polymerase chain reaction, while CHEK2 1100delC and Tp53 exon 7 mutations were analyzed by RFLP. Results: The amplification of BRCA1gene for185 delAG mutation revealed amplicons of 335 bp for the wild type and 354 bp for the mutant gene, whereas for 5382 insC mutation exhibited 2 amplicons of sizes 271 bp for the wild type and 295 bp for the mutant gene. The analysis of BRCA26174 delT mutation indicated 2 amplicons of 151 bp for the wild type and 171 bp for the mutant gene. Tp53 exon 7 mutation analysis highlighted an amplicon of 125 bp size. Digestion of thisamplicon with the Hae III enzyme resulted in 2 fragments with sizes of 83 bp and 42 bp for the wild type. CHEK2 1100 delC mutation evaluation showed an amplicon of 116 bp size. Digestion of this amplicon with Sca I enzyme exhibited 2 fragments with sizes of 92 bp and 24 bpfor the wild type. Data analysis demonstrated that 27 (27%) out of the 100 enrolled breast cancer patients of tissue samples have mutations in tumor suppressor genes, 11 (23.9%) out of 46enrolled breast cancer patients of blood samples have such mutations, while 8 (17.4%) out of 46 relatives have these mutations. Nine (9%) patients of group1 were indicated to have BRCA1 185delAGmutation, while 4 (8.7%) and 3 (6.5%)women from groups 2 and3 exhibited the same mutationrespectively. BRCA15382insC mutation was identified in 7 (7%), 3 (6.5%) and 1 (2.2%) women of groups 1, 2 and 3 respectively. The BRCA2 6174delT mutation was observed in 5 (6%), 2 (4.3%) and 2 (4.3%)women of groups 1,2 and 3 respectively. Three (3%) patients of group1, 1 (2.2%) and 1 (2.2%) women from groups 2 and 3 were found to have CHEK2 1100delC respectively. Tp53exon 7 mutation was evident in 2 (2%), 1 (2.2%) and 1 (2.2%) women of groups 1, 2 and 3 respectively. No patient had more than one mutation. Conclusions: About one quarter of the investigated breast cancer patients have mutations in their tumor suppressor genes and 17% of their relatives have such mutations. Most of breast cancer patients enrolled in this study are sporadic not familial breast cancer.

Frequency of BRCA1 (185delAG and 5382insC) and BRCA2 (6174delT) mutations in Egyptian women with breast cancer compared to healthy controls

BackgroundThe three founder mutations of BRCA1/2 (185delAG, 5382insC and 6174delT) have been reported to be associated with breast cancer. This work was designed to check for the frequency of these genetic mutations in Egyptian women affected with breast cancer compared to healthy first-degree relatives and unrelated controls. Subjects and methodsThis work is a case control study including 43 women diagnosed with breast cancer. Their genetic data were compared to controls including 63 first degree relatives in addition to 91 healthy unrelated controls from the same locality. DNA deletion or insertion mutations were characterized in blood samples of all participants using the PCR technique. ResultsThe frequency of BRCA1 (185delAG) mutation in BC patients and their first-degree relatives was higher than healthy controls (2.3% and 3.2% vs. 1.1%). However, the other two mutations BRCA1 (5382insC) and BRCA2 (6174delT) showed higher frequencies among healthy controls than BC patients and their first-degree relatives (49.5% vs. 11.6% and 6.3%; 61.5% vs. 11.6% and 14.3%, respectively). Furthermore, BC patients with BRCA1 (185delAG and 5382insC) and BRCA2 (6174delT) mutations showed no significant difference compared to others regarding their clinical and laboratory markers. ConclusionsThis study indicates that BRCA1 (185delAG) mutation might contribute to the incidence of breast cancer among Egyptian women, but not with BRCA1 (5382insC) and BRCA2 (6174delT) mutations. Furthermore, there was no significant association between these three founder mutations and the clinical presentation of BC among Egyptian women.

Abstract AP29: BRCA1 RING DOMAIN–DEFICIENT PROTEINS PROMOTE PARP INHIBITOR AND PLATINUM RESISTANCE

Abstract Although cancers harboring BRCA1 mutations initially respond well to platinum and poly(ADP-ribose)-polymerase inhibitor (PARPi) therapy, resistance invariably arises and is a major clinical problem. The BRCA1185delAG allele is a common inherited mutation located close to the protein translation start site, thought to produce a short peptide devoid of function. In this study, we utilized the SUM1315MO2 cancer cell line that harbors a hemizygous BRCA1185delAG mutation to study PARPi and platinum resistance. SUM1315MO2 cells were initially PARPi and cisplatin sensitive but readily acquired resistance. PARPi and cisplatin resistant clones did not harbor secondary reversion mutations. Rather, increased expression of a RING domain-deficient BRCA1 protein (Rdd-BRCA1) was required for resistance. Translation initiation occurred downstream of the frameshift mutation, likely at the BRCA1-Met-297 codon. In contrast to full-length BRCA1, Rdd-BRCA1 did not require BARD1 interaction for stability. Functionally, Rdd-BRCA1 formed irradiation-induced foci and supported RAD51 foci-formation. Ectopic overexpression of Rdd-BRCA1 promoted partial PARPi and cisplatin resistance in vitro and in vivo. Furthermore, Rdd-BRCA1 protein expression was detectable in recurrent carcinomas from germline BRCA1185delAG mutation carriers. Taken together, these results indicate that RING-deficient BRCA1 proteins are hypomorphic, and when expressed at high enough levels are capable of contributing to PARPi and platinum resistance. Citation Format: Yifan Wang, John J. Krais, Andrea J. Bernhardy, Emmanuelle Nicolas, Kathy Q. Cai, Maria I. Harrell, Hyoung H. Kim, Erin George, Elizabeth M. Swisher, Fiona Simpkins and Neil Johnson. BRCA1 RING DOMAIN–DEFICIENT PROTEINS PROMOTE PARP INHIBITOR AND PLATINUM RESISTANCE [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP29.

RING domain-deficient BRCA1 promotes PARP inhibitor and platinum resistance.

Patients with cancers that harbor breast cancer 1 (BRCA1) mutations initially respond well to platinum and poly(ADP-ribose) polymerase inhibitor (PARPi) therapy; however, resistance invariably arises in these patients and is a major clinical problem. The BRCA1185delAG allele is a common inherited mutation located close to the protein translation start site that is thought to produce a shortened, nonfunctional peptide. In this study, we investigated the mechanisms that lead to PARPi and platinum resistance in the SUM1315MO2 breast cancer cell line, which harbors a hemizygous BRCA1185delAG mutation. SUM1315MO2 cells were initially sensitive to PARPi and cisplatin but readily acquired resistance. PARPi- and cisplatin-resistant clones did not harbor secondary reversion mutations; rather, PARPi and platinum resistance required increased expression of a really interesting gene (RING) domain-deficient BRCA1 protein (Rdd-BRCA1). Initiation of translation occurred downstream of the frameshift mutation, probably at the BRCA1-Met-297 codon. In contrast to full-length BRCA1, Rdd-BRCA1 did not require BRCA1-associated RING domain 1 (BARD1) interaction for stability. Functionally, Rdd-BRCA1 formed irradiation-induced foci and supported RAD51 foci formation. Ectopic overexpression of Rdd-BRCA1 promoted partial PARPi and cisplatin resistance. Furthermore, Rdd-BRCA1 protein expression was detected in recurrent carcinomas from patients who carried germline BRCA1185delAG mutations. Taken together, these results indicate that RING-deficient BRCA1 proteins are hypomorphic and capable of contributing to PARPi and platinum resistance when expressed at high levels.

BRCA1 loses the ring but lords over resistance.

Germline breast cancer 1 (BRCA1) variants are associated with a high risk of breast and ovarian cancers. Many BRCA1-mediated cancers are initially responsive to platinum-based therapy; however, resistance commonly develops. The BRCA1185delAG mutation is common in the Ashkenazi Jewish population and has been thought to result in loss of function due to the introduction of a stop codon in the 5' region of the BRCA1 transcript. Two studies in this issue of the JCI reveal that the BRCA1185delAG mutation results in the production of BRCA1 that lacks the N-terminal really interesting new gene (RING) domain. RING-less BRCA1 was shown to directly mediate chemoresistance, while maintaining some homologous recombination function. These results provide important insight into BRCA1 function and indicate that other truncated proteins could arise through similar alterations in codon usage.

Abstract A26: Telomere length in normal and tumor breast tissue from BRCA2 mutation carriers and sporadic breast cancer cases

Abstract Germline mutations in the BRCA genes are associated with highly increased risk of breast and ovarian cancers and to a lesser extent to prostate and pancreatic cancers. Great number of mutations with variable impact on cancer risk and progression are known in these genes worldwide. A single founder mutation has been detected in each of the BRCA genes in the Icelandic population, making it feasible to study the influence of a single mutation at a population level. The BRCA2999del5 mutation is more frequent and can be found in approximately 6-7% of female breast cancer patients and 40% of male breast cancer patients in Iceland. The mutation leads to a non-functional protein product and is associated with complex chromosomal changes in tumor tissue. Furthermore, the BRCA2999del5 mutation has been associated with poor prognosis, both in breast and prostate cancers, although mutation carriers seem to differ with respect to age of onset and severity of disease. Dysfunctional telomere maintenance can lead to excessive telomere shortening which causes chromosome instability. It is therefore a driving force behind cancer progression and is considered a hallmark of many human cancers. Telomere shortening has been shown to be an early and a common molecular alteration in epithelial cancers, including breast cancers. Previous results from our laboratory, amongst others, have shown that BRCA2 is associated with telomere protection and maintenance. Telomere dysfunction induced foci (TIFs) formation was detected in cell lines derived from heterozygous BRCA2999del5 mutation carriers1. Telomere length has been proposed as a possible predictive factor for cancer risk for various cancers. In the case of breast cancer and BRCA mutation carriers, the results have been conflicting. The aim of the study was to investigate if telomere length in normal and tumor breast tissue is correlated with breast cancer progression and survival in a well-defined group of BRCA2 mutation carriers and sporadic breast cancer cases. The study group consisted of breast cancer patients carrying the BRCA2999del5 Icelandic founder mutation and sporadic cases, matched with respect to age and year of diagnosis. Tumor and normal adjacent tissue samples from these patients were embedded in paraffin and Quantitative Fluorescence in Situ Hybridization method (Q-FISH) was used to carry out telomere length measurements. Telomeres were visualized using a telomeric sequence-specific fluorescence peptide nucleic acid (PNA) probe. A centromere-specific PNA probe was used as an inner control for hybridization efficiency. The results show a clear difference in telomere length of different cell types in normal breast tissue with inner epithelial cells having shorter telomeres than both myoepithelial cells and fibroblasts. These findings support previously reported data in the literature2. The majority of examined tumors have very short telomeres. In mutation carriers, there are indications that less variable telomere length in normal adjacent tissue is associated with reduced breast cancer specific survival. This seems to be more pronounced in the mutation carrier group compared to the sporadic group. Telomere length in adjacent normal tissue appears to be shorter in cases with luminal type than with basal-like tumors.

A84a: Role of BRCA1 dysfunction in sporadic triple-negative breast cancer

Background There is increasing evidence that BRCA1-related DNA-repair defects determine sensitivity to certain agents, such as platinum-based chemotherapy. There is a lot of evidence about a link between TNBC and BRCA1 deficiency. Many clinical characteristics and molecular features are shared by sporadic triple-negative breast cancer and BRCA1-associated breast cancer. The majority of BRCA1-related breast cancers are of basal-like/triple-negative phenotype. Identification of specific markers of BRCA1-dysfunction will be essential to translate an understanding of defective DNA repair into targeted treatments for this poor prognosis subtype. Materials and methods Twenty-two patients with triple-negative breast cancer (TNBC) were treated with neoadjuvant platinum-based chemotherapy followed by surgery. Pathologic treatment response was assessed in correlation with biomarkers of BRCA1-dysfunction. Pathological response was evaluated according to the Chevallier classification (Ch). All patients were screened for germ-line mutations in BRCA1 (185delAG, 5382insC, 4153delA, 4158A/G, C61G) and BRCA2 (6174delT). All samples negative for germ-line mutations in BRCA1 and BRCA2 then submitted to BRCA1-dysfunction screening. Biomarkers of BRCA1-dysfunction included: BRCA1 somatic mutations (C61G, 185delAG, 5382insC), promoter methylation of BRCA1, low BRCA1 mRNA expression, high ID4 mRNA expression, low RAD51 mRNA expression, PTEN (T4721C) mutation, p53 (Arg72Pro, Pro72Pro) mutations. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP), methylation-specific PCR were used for BRCA1-dysfunction analysis. Results Twelve patients (54.5%) achieved a pathological complete response (pCR) (Ch1+ Ch2). Ten patients (45.5%) had a residual disease (Ch3+ Ch4). BRCA1 mRNA expression was absent in 16/22 (72.7%) patients, low in 5/22 (22.7%). 11/21 (52.4%) patients with other than normal BRCA1 mRNA expression achieved a pCR. BRCA1 promoter methylation was detected in 9/22 (40.9%). 6/9 patients with BRCA1 promoter methylation achieved a pCR. RAD51 mRNA levels were low in 14/22 (63.6%), high in 1/22 (4.5%). 8/14 (57.1%) patients with other than normal RAD51 mRNA expression achieved a pCR. High ID4 mRNA levels were determined in 5/22 (22.7%). 3/5 (60%) patients with high ID4 mRNA expression achieved a pCR. p53 (Arg72Pro, Pro72Pro) mutations were identified in 12/22 (54.5%). No patient had PTEN (T4721C) mutation. No statistically significant correlation was found between the BRCA1 mRNA expression, BRCA1 promoter methylation, RAD51 mRNA expression, high ID4 mRNA levels, p53 (Arg72Pro, Pro72Pro) mutations and pCR to neoadjuvant platinum-based chemotherapy ( p > 0.05). Eleven patients had BRCA1 somatic mutations: 6/22 (27.3%) – BRCA1 5382insC, 4/22 (18.2%) – BRCA1 185delAG, 3/22 (13.6%) – BRCA1 C61G. 3/4 patients with BRCA1 185delAG mutation had BRCA1 C61G mutation. BRCA1 LOH was detected in 1/22 (4.5%) patient. All patients with BRCA1 5382insC mutation achieved a pCR ( p = 0.01). All patients with BRCA1 185delAG mutation and BRCA1 C61G mutation had residual disease (0.04 and 0.1, accordingly). Conclusion BRCA1 5382insC somatic mutation is associated with good response to neoadjuvant platinum based chemotherapy ( p = 0.01). BRCA1 185delAG and BRCA1 C61G mutations in the BRCA1 RING domain may predict resistance to neoadjuvant platinum-based chemotherapy (0.04 and 0.1, accordingly).

Abstract POSTER-BIOL-1348: No association between PARK2 and BRCA1-associated gynecologic cancers

Abstract Purpose of study: We sought to identify genomic modifiers of gynecologic cancer penetrance in BRCA1 185delAG mutation carriers. In a pilot study using the Illumina 610K Quad chip to perform a whole genome analysis of 592,532 single nucleotide polymorphisms (SNP), we found significant SNP genotype differences in the PARK2 gene between BRCA1 185delAG mutation carriers who developed gynecologic cancer (cases) compared to those who did not (controls). These SNPS created a haplotype structure that was present at four-times higher frequency in cases than controls (37% vs. 9%, p=0.0009). We hypothesized that PARK2, which has been shown to be mutated in other cancers and to have a tumor suppressive function, plays a role in the biology of BRCA1-associated gynecologic cancer development. Experimental Procedures: To validate a PARK2 risk haplotype in an independent dataset, we isolated germline DNA from 85 BRCA1 185delAG mutation carriers and used TaqMan SNP Genotyping to genotype seven SNPs in PARK2. To determine whether a correlation exits between the PARK2 risk haplotype and PARK2 expression levels in tissue, we quantified PARK2 expression in normal ovaries and ovarian cancer tissue using quantitative real-time PCR (qPCR). To determine the rate of PARK2 mutations in 17 BRCA1-associated ovarian cancers, we used a touchdown PCR protocol to amplify and sequence each PARK2 exon. To identify exon deletions and duplications in the PARK2 gene, we utilized the multiplex ligation-dependent probe amplification (MLPA) technique. Summary of Data: In an independent dataset of 85 BRCA1 185delAG mutation carriers, we found the PARK2 risk haplotype to be present in 35 (17%) of cases. The risk haplotype was present in a higher proportion of cancer cases than benign controls (6/12 or 50% vs. 29/73 or 8%), but this was not significant (p=0.54). We did not find a difference in PARK2 ovarian tissue expression levels in patients with and without the PARK2 risk haplotype. We sequenced the PARK2 gene and performed MPLA analysis from tumor DNA of 17 ovarian cancers from BRCA1 185delAG mutation carriers, but did not identify any deleterious PARK2 mutations or exon deletions and duplications. Conclusions: We did not validate a PARK2 risk haplotype as a modifier of gynecologic cancer penetrance in BRCA1 185delAG mutation carriers. We did not find evidence for PARK2 inactivation in BRCA1-associated gynecologic tumors. Citation Format: Christine Walsh, Hoorig Nassanian, Hasmik Agadjanian, Carl Miller, Sandra Orsulic, Beth Karlan. No association between PARK2 and BRCA1-associated gynecologic cancers [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1348.

Abstract 5467: BRCA1 N-terminal-deficient proteins provide PARP inhibitor and platinum resistance

Abstract Introduction: Tumors harboring BRCA1 mutations initially respond well to platinum and PARP inhibitor therapy; however, resistance invariably arises and is a major clinical problem. The BRCA1 185delAG allele is a common founder mutation located close to the protein translation start site, thought to produce a short peptide devoid of function. Experimental procedures: In this study, we utilized the SUM1315MO2 breast cancer cell line that harbors a BRCA1 185delAG mutation to study mechanisms of PARP inhibitor and platinum resistance. SUM1315MO2 cells were cultured in the presence of increasing concentrations of the PARP inhibitor rucaparib or cisplatin until rucaparib resistant (RR) and cisplatin resistant (CR) clones emerged. Results: DNA sequencing revealed that no BRCA1 gene reversion mutations were present in resistant cells. We next measured BRCA1 protein levels by Western blotting with antibodies specific for the N- and C-terminal domains of BRCA1. As a control, the wild-type BRCA1 protein expressed in MDA-MB-231 cells was detectable with both N- and C-terminal antibodies. In contrast, BRCA1 protein was undetectable in both SUM1315MO2 parental and resistant clones using the N-terminal specific antibody. However, the C-terminal specific antibody identified a more quickly migrating band of low abundance in parental cells, but with elevated expression in both RR and CR clones. We inferred that this protein was devoid of the extreme N-terminal RING domain-containing region that mediates interaction with BARD1. To investigate the functionality of the N-terminal truncated BRCA1 protein (Nt-BRCA1), we measured BRCA1 and RAD51 irradiation-induced focus formation by immunofluorescence. RR and CR clones demonstrated 2.25 - 2.75-fold increase (P = 0.024) in the number of cells with BRCA1 foci compared to parental cells. Similarly, we detected a 2 - 2.85-fold increase (P = 0.0325) in RAD51 foci formation in resistant clones compared to parental cells. Additionally, BRCA1 siRNA treated cells were 12- and 9-fold more sensitive to rucaparib compared to scrambled siRNA-treated control cells (P = 0.0002). Similarly, CR-1 cells treated with BRCA1 siRNAs were 1.56- and 1.7-fold more sensitive to cisplatin (P = 0.0346) compared to scrambled siRNA treated cells. Furthermore, N-terminal truncated BRCA1 proteins were detectable in a primary tumor from a germline BRCA1185delAG mutation carrier. Conclusions: Taken together, these results provide evidence for a novel, mutation location-dependent mechanism of PARP inhibitor and platinum resistance. Citation Format: Yifan Wang, Andrea Bernhardy, Emmanuelle Nicolas, Ryan Winters, Kathy Cai, Kelly Duncan, James Duncan, Maria Harrell, Elizabeth Swisher, Neil Johnson. BRCA1 N-terminal-deficient proteins provide PARP inhibitor and platinum resistance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5467. doi:10.1158/1538-7445.AM2015-5467

BRCA1 185delAG Mutation Enhances Interleukin-1β Expression in Ovarian Surface Epithelial Cells.

Familial history remains the strongest risk factor for developing ovarian cancer (OC) and is associated with germline BRCA1 mutations, such as the 185delAG founder mutation. We sought to determine whether normal human ovarian surface epithelial (OSE) cells expressing the BRCA1 185delAG mutant, BRAT, could promote an inflammatory phenotype by investigating its impact on expression of the proinflammatory cytokine, Interleukin-1β (IL-1β). Cultured OSE cells with and without BRAT were analyzed for differential target gene expression by real-time PCR, western blot, ELISA, luciferase reporter, and siRNA assays. We found that BRAT cells expressed increased cellular and secreted levels of active IL-1β. BRAT-expressing OSE cells exhibited 3-fold enhanced IL-1β mRNA expression, transcriptionally regulated, in part, through CREB sites within the (−1800) to (−900) region of its promoter. In addition to transcriptional regulation, BRAT-mediated IL-1β expression appears dualistic through enhanced inflammasome-mediated caspase-1 cleavage and activation of IL-1β. Further investigation is warranted to elucidate the molecular mechanism(s) of BRAT-mediated IL-1β expression since increased IL-1β expression may represent an early step contributing to OC.

Aurora A is a prognostic marker for breast cancer arising in BRCA2 mutation carriers.

Overexpression of the Aurora A kinase has been shown to have prognostic value in breast cancer. Previously, we showed a significant association between AURKA gene amplification and BRCA2 mutation in breast cancer. The aim of this study was to assess the prognostic impact of Aurora A overexpression on breast cancer arising in BRCA2 mutation carriers. Aurora A expression was evaluated by immunohistochemistry on breast tumour tissue microarrays from 107 BRCA2 999del5 mutation carriers and 284 of sporadic origin. Prognostic value of Aurora A nuclear staining was estimated in relation to clinical markers and adjuvant treatment, using multivariate Cox's proportional hazards ratio regression model. BRCA2 wild‐type allele loss was measured by TaqMan in BRCA2 mutated tumour samples. All statistical tests were two sided. Multivariate analysis of breast cancer‐specific survival, including proliferative markers and treatment, indicated independent prognostic value of Aurora A nuclear staining for BRCA2 mutation carriers (hazards ratio = 7.06; 95% confidence interval = 1.23–40.6; p = 0.028). Poor breast cancer‐specific survival of BRCA2 mutation carriers was found to be significantly associated with combined Aurora A nuclear expression and BRCA2 wild type allele loss in tumours (p < 0.001). Multivariate analysis indicated independent prognostic value of both positive Aurora A nuclear staining (hazards ratio = 10.09; 95% confidence interval = 1.19–85.4, p = 0.034) and BRCA2 wild type allele loss (hazards ratio = 9.63; 95% confidence interval = 1.81–51.0, p = 0.008) for BRCA2 mutation carriers. Aurora A nuclear expression was found to be a significant prognostic marker for BRCA2 mutation carriers, independent of clinical parameters and adjuvant treatment. Our conclusion is that treatment benefits for BRCA2 mutation carriers and sporadic breast cancer patients with Aurora A positive tumours may be enhanced by giving attention to Aurora A targeted treatment.

Ethnic aspects of hereditary breast cancer in the region of Siberia

Ethnic diversity of the population in the region of Siberia suggests the existence of different germline mutations in the BRCA1/2 genes associated with breast and ovarian cancer in different ethnic populations, but spectrum of these mutations has not been studied. Our aim was to evaluate the frequency of the most common mutations BRCA1/2 (BRCA1 5382insC, BRCA1 185delAG, BRCA1 4153delAG, BRCAI T300G, BRCA2 6174delT) in women diagnosed with breast cancer among indigenous people and newcomers living in Siberia. We tested 1281 genomic DNA samples for the presence of BRCA1 5382insC mutation in patients diagnosed with breast cancer considering no family history. 72 patients having hereditary cancer signs were tested for the mutations BRCA1 185delAG, BRCA1 4153delAG, BRCA1 T300G, BRCA2 6174delT. Out of 765 patients of Slavic ethnic group, 27 women (3.5%) were carriers of allele BRCA1 5382insC. The frequencies of mutations in patients with signs of hereditary cancer were: 8.3% in group of young patients (under 40 years), 20.0% in patients with bilateral cancer and 5.7% in patients with family history of breast or ovarian cancers. We tested 516 BC patients residing on the territory of the Buryat-Aginsky district, Republics of Tyva and Altai. Out of them, there were 197 patients among the indigenous population (buryats, tuvinians, altaians), and 319 patients among newcomers (Slavic ethnics). Mutations BRCA1 5382insC were detected only in women from Slavic ethnic groups. The frequency of BRCA1 5382insC mutation was 6% in the group where family history was excluded and 14% in the group of patients with characteristics of family cancer. Allele BRCA1 5382insC was not found in indigenous breast cancer patients, although 59 patients had signs of hereditary cancer. In women from Slavic ethnic group, the BRCA1 185delAG, BRCA1 4153delAG and BRCA1 T300G mutations were detected in 9.1% of cases and were not found in patients among the indigenous population. studies of mutations in the BRCA1 gene in breast cancer patients from Siberia confirmed data on the high frequency of "founder mutation" BRCA1 5382insC in Slavic population and indicate the advisability of further studies to identify the genes responsible for the occurrence of hereditary breast cancer in the indigenous population.

Identification of germ-line 185delAG BRCA1 mutation among breast cancer families in Iran: An experience from northeastern Iran.

e22079 Background: The present study evaluated the prevalence of 185DelAG BRCA1 mutation in the group of Iranian women breast cancer patients, or their relative with family history of breast cancer. Breast cancer is the most common malignancy in women and the leading cause of cancer death in females worldwide. BRCA1 is among the best known cancer susceptibility genes with high penetrance and have multiple cellular functions including critical roles in homologous DNA repair. Identifying gremlin BRCA mutations associated with significant cancer susceptibility has the potential to change all aspects of an individual's care, from screening to cancer treatment. The combination of breast cancer susceptibility alleles together with other risk factors may be important clinically. Methods: Subjects included in this study were thirty nine patients diagnosed with breast cancer and 29 women, from first degree relative of patients. The clinical and demographic details from these individuals were collected from medical records, pathological reports, and when needed and using a detailed questionnaire. Genomic DNA from peripheral blood mononuclear cells was extracted and analyzed by polymerase chain reaction and SSCP methods in order to finding 185DelAG founder mutation. Results: The results indicated that four patients and three healthy relatives had this founder mutation. The identification of these founder mutations facilitates mutation population screening and subsequent management options available to an individual woman including screening or prevention are then dependent on this level of assessed risk. Conclusions: Finally identifying the effective risks regarding breast cancer among Iranian women requires many works. However, a complete BRCA1 gene sequence analysis might be useful, regarding targeted cancer prevention and therapeutics.

Haplotype analysis of the 185delAG BRCA1 mutation in ethnically diverse populations

The 185delAG* BRCA1 mutation is encountered primarily in Jewish Ashkenazi and Iraqi individuals, and sporadically in non-Jews. Previous studies estimated that this is a founder mutation in Jewish mutation carriers that arose before the dispersion of Jews in the Diaspora ~2500 years ago. The aim of this study was to assess the haplotype in ethnically diverse 185delAG* BRCA1 mutation carriers, and to estimate the age at which the mutation arose. Ethnically diverse Jewish and non-Jewish 185delAG*BRCA1 mutation carriers and their relatives were genotyped using 15 microsatellite markers and three SNPs spanning 12.5 MB, encompassing the BRCA1 gene locus. Estimation of mutation age was based on a subset of 11 markers spanning a region of ~5 MB, using a previously developed algorithm applying the maximum likelihood method. Overall, 188 participants (154 carriers and 34 noncarriers) from 115 families were included: Ashkenazi, Iraq, Kuchin-Indians, Syria, Turkey, Iran, Tunisia, Bulgaria, non-Jewish English, non-Jewish Malaysian, and Hispanics. Haplotype analysis indicated that the 185delAG mutation arose 750-1500 years ago. In Ashkenazim, it is a founder mutation that arose 61 generations ago, and with a small group of founder mutations was introduced into the Hispanic population (conversos) ~650 years ago, and into the Iraqi-Jewish community ~450 years ago. The 185delAG mutation in the non-Jewish populations in Malaysia and the UK arose at least twice independently. We conclude that the 185delAG* BRCA1 mutation resides on a common haplotype among Ashkenazi Jews, and arose about 61 generations ago and arose independently at least twice in non-Jews.