Breast Cancer BRCA2 Research Articles

Breast cancer: oophorectomy for BRCA1 ER--negative disease-an open debate.

Risk-reducing salpingo-oophorectomy (RRSO) is a standard intervention in BRCA1 or BRCA2 mutation carriers owing to its associated reduction in mortality related to ovarian and breast cancer. A study has now reported a beneficial impact of adjuvant RRSO in patients with BRCA1 mutations and breast cancer. However, various biases confound these results.

BRCA1 gene variant p.P142H associated with male breast cancer: a two-generation genealogic study and literature review.

Breast cancer occurs rarely in male patient. BRCA1 gene mutation seems to be related to male breast cancer, but its role is not clearly defined. We have identified in a male patient affected by breast cancer the BRCA1 gene variant p.P142H. We performed a literature research using the keywords "male breast cancer", "male breast cancer mutations" and "BRCA" and we reviewed the cases. We found ew other studies regarding BRCA1 variant p.P142H, about female subjects. At the moment, BRCA1 gene variant p.P142H is not certainly classified as neutral or deleterious. Genetic testing for BRCA1 and BRCA2 and PALB2 mutation gene has been performed on our patient. Segregation analysis for this p.P142H BRCA1 variant has been extended to the second generation of the family. Genetic tests revealed a clear inheritance regarding the BRCA1 gene p. P142H variant. Of the eight patients with this specific genetic mutation, four presented breast cancer (bilateral in one case), two female and two male. None of the subjects in the family without the BRCA1 gene variant p. P142H presented breast cancer or other BRCA1 gene mutation-related cancers. Our analysis suggests that the BRCA1 gene variant p.P142H mutation is related with male breast cancer. Starting from these data, it can be inferred that more studies on MBC and its relation with the BRCA1 gene mutation P142H variant must be undertaken to improve prognostic and therapeutic strategies.

Second primary breast cancer in BRCA1 and BRCA2 mutation carriers: 10-year cumulative incidence in the Breast Cancer Family Registry.

BReast CAncer genes 1 and 2 (BRCA1 and BRCA2) mutation carriers diagnosed with breast cancer are at increased risk of developing a second primary breast cancer. Data from high-risk clinics may be subject to different biases which can cause both over and underestimation of this risk. Using data from a large multi-institutional family registry we estimated the 10-year cumulative risk of second primary breast cancer including more complete testing information on family members. We prospectively followed 800 women diagnosed with breast cancer from the Breast Cancer Family Registry (BCFR) who were carriers of a BRCA1 or BRCA2 pathogenic mutation or a variant of unknown clinical significance. In order to limit survival and ascertainment bias, cases were limited to those diagnosed with a first primary breast cancer from 1994 to 2001 and enrolled in the BCFR within 3years after their cancer diagnosis. We excluded women enrolled after being diagnosed with a second breast cancer. We calculated 10-year incidence of second primary breast cancers. The 10-year incidence of a second primary breast cancer was highest in BRCA1 mutation carriers (17%; 95% CI 11-25%), with even higher estimates in those first diagnosed under the age of 40 (21%; 95% CI 13-34%). Lower rates were found in BRCA2 mutation carriers (7%; 95% CI 3-15%) and women with a variant of unknown clinical significance (6%; 95% CI 4-9%). Whereas the cumulative 10-year incidence of second primary breast cancer is high in BRCA1 mutation carriers, the estimates in BRCA2 mutation carriers and women with variants of unknown clinical significance are similar to those reported in women with sporadic breast cancer.

The PARP inhibitor AZD2281 (Olaparib) induces autophagy/mitophagy in BRCA1 and BRCA2 mutant breast cancer cells.

PARP inhibitors are considered promising anti-cancer agents and currently being tested in clinical trials in hereditary breast cancer patients harboring mutations in BRCA1 and BRCA2 genes. In this study, we investigated the antiproliferative effects and mechanism of PARP inhibitors ABT-888 (Veliparib), BSI-201 (Iniparib) and AZD228 (Olaparib) in breast cancer cell lines with BRCA1 or BRCA2 mutations and 9 different BRCA wild-type cell lines with BRCA1 allelic loss. We found that AZD2281 was the most potent in the PARP inhibitors and induces significant growth inhibition (~95%) in BRCA1 mutant (HCC-1937, MDA-MB-436, and SUM-149PT) and BRCA2 mutant (HCC-1428) cell lines. AZD2281 treatment also resulted in growth inhibition ranging from 20 to 50% in cells with BRCA1 allelic loss, including ER(+), HER2/Neu(+) and triple-negative breast cancer (TNBC) cells, but showed no effect in cells without with type BRCA without allelic loss. Knocking down of BRCA1 or BRCA2 in TNBC cells with BRCA1 allelic loss by RNA interference significantly enhanced AZD2281-induced growth inhibition and induced significant autophagy that was associated with mitophagy in cells with BRCA mutations. Inhibition of autophagy by gene knockdown significantly diminished AZD2281-induced mitophagy and apoptosis, indicating that autophagic process mediates some of the downstream effects of PARP inhibitors. In conclusion, our data provide the first evidence of PARP inhibitor AZD2281 autophagy and mitophagy in breast cancer cell lines with BRCA mutations or BRCA-allelic loss. In addition, our results indicate that the patients with BRCA1 allelic loss may also benefit from PARP inhibitor therapy if BRCA is further inhibited.

Prospective evaluation of alcohol consumption and the risk of breast cancer in BRCA1 and BRCA2 mutation carriers.

Given the adverse effect of alcohol in the development of breast cancer among women in the general population, we evaluated whether a similar association exists among women with a BRCA1 or BRCA2 mutation. Information regarding baseline daily alcohol consumption was abstracted from a research questionnaire for 3067 BRCA mutation carriers enrolled in a prospective cohort study. Women were followed biennially until the date of the last follow-up questionnaire, date of breast cancer diagnosis, date of prophylactic bilateral mastectomy, or date of death. Cox proportional hazards models were used to estimate relative risks (RRs) and 95 % confidence intervals (CIs) for invasive breast cancer associated with alcohol consumed at or prior to completion of the baseline questionnaire. After a mean of 5.4 years of follow-up, we observed 259 incident cases of primary invasive breast cancer. Compared with non-users, the adjusted RRs were 1.06 (95 % CI 0.78-1.44) for ever use and 1.08 (0.79-1.47) for current alcohol use. For women in the highest versus lowest quintile of cumulative alcohol consumption, the RR was 0.94 (95 % CI 0.63-1.40; P trend = 0.65). Our findings suggest that alcohol consumption is not a risk factor for breast cancer among women with a BRCA1 or BRCA2 mutation.

Abstract S5-05: Defective stalled replication fork repair and predisposition to hereditary breast cancer

Abstract BRCA1 is a tumor suppressor gene, and germ line BRCA1 mutations increase the risk of breast cancer. While all cells with BRCA1 mutations exhibit a heterozygous BRCA1mut/+ genotype, cancer develops primarily in females, often at young ages and affects almost exclusively the breast and ovaries. Why BRCA1 shows such tissue specificity, and how a normal cell in a BRCA1 mutation carrier (BRCA1mut/+) gives rise to invasive tumor cells are largely unknown. To determine whether BRCA1 heterozygosity in cells confers defect in any of the multiple, known, BRCA1 functions is a potentially valuable step in achieving a better understanding of BRCA1 mutation-driven cancer predisposition. Thus, we have analyzed a collection of primary mammary BRCA1mut/+ epithelial cells and skin fibroblasts obtained from BRCA1 mutation carriers for such functions. We, and others have recently shown that BRCA1 exhibits a new DNA damage repair function – i.e. repair of stalled replication forks (SFR). Stalled forks, when not resolved, lead to mutations, or collapse into double strand breaks (DSBs). Both outcomes result in what is commonly referred to as replication stress (RS), which, when chronic, is a driving force behind cancer development. To determine if SFR is defective in normal/healthy breast cells in BRCA1 mutation carriers, and whether this haploinsufficiency results in the kind of genomic changes that lead to cancer, we have now generated 18 primary fibroblast strains from skin punch biopsies and 10 primary mammary epithelial cell (MECs) strains from prophylactic mastectomies performed on BRCA1 mutation carrying women. This collection includes N=23 different BRCA1 mutations, which, together, span almost the entire BRCA1 gene. BRCA1+/+ control MECs were derived from tissue collected during reduction mammoplasties and control fibroblasts were derived from skin punch biopsies from women with no BRCA1 mutation. Our current data shows that BRCA1mut/+ strains exhibited multiple, normal BRCA1 functions, including the support of homologous recombination- type double strand break repair (HR-DSBR), cell cycle- associated checkpoint functions, centrosome number control, spindle pole formation, Slug expression and satellite RNA suppression. By contrast, nearly all strains were defective in the repair of stalled replication forks and in the suppression of fork collapse, i.e. replication stress. These defects were rescued by reconstituting BRCA1 heterozygous cells with wild-type BRCA1 cDNA, indicating that they are a product of BRCA1 haploinsufficiency. In addition, the development of sufficient replication stalling rendered BRCA1mut/+ cells defective in an otherwise intact BRCA1 function, HR-DSBR. No such ‘conditional’ haploinsufficiency was detected in any of the other non-haploinsufficient functions, noted above. Given the importance of replication stress in cancer development and of an HR defect in breast cancer pathogenesis, these defects, when they develop serially, could contribute to the BRCA1 breast cancer development pathway. Finally, given the important role of BRCA2, another hereditary breast cancer gene, in stalled fork stability, a similar analysis for BRCA2mut/+ cells from BRCA2 mutation carriers is currently underway and will also be reported at the meeting. Citation Format: Shailja Pathania, Sangeeta Bade, Morwenna Le Guillou, Karly Burke, Ying Su, David T Ting, Kornelia Polyak, Andrea L Richardson, Jean Feunteun, Judy E Garber, David M Livingston. Defective stalled replication fork repair and predisposition to hereditary 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 S5-05.

Effect of Oophorectomy on Survival After Breast Cancer in BRCA1 and BRCA2 Mutation Carriers.

Women who carry a germline mutation in either the BRCA1 or BRCA2 gene face a lifetime risk of breast cancer of up to 70%, and once they receive a diagnosis of breast cancer, they face high risks of both second primary breast and ovarian cancers. Preventive bilateral salpingo-oophorectomy is recommended to women with a BRCA mutation at age 35 years or thereafter to prevent breast and ovarian cancer, but it is unclear whether oophorectomy has an impact on survival in women with BRCA-associated breast cancer. To estimate the impact of oophorectomy on survival in women with breast cancer with a BRCA1 or BRCA2 mutation. Retrospective analysis of patients selected by pedigree review of families who received counseling at 1 of 12 participating clinical genetics centers. Patients were 676 women with stage I or II breast cancer and a BRCA1 or BRCA2 mutation who were observed for up to 20 years after receiving a diagnosis between 1975 and 2008. Survival experience was compared for women who did and who did not undergo oophorectomy. In all analyses, the primary end point was death due to breast cancer. Of the 676 women, 345 underwent oophorectomy after the diagnosis of breast cancer and 331 retained both ovaries. The 20-year survival for the entire patient cohort was 77.4%. The adjusted hazard ratio for death attributed to breast cancer in women who underwent oophorectomy was 0.38 (95% CI, 0.19-0.77; P = .007) for BRCA1 carriers and 0.57 (95% CI, 0.23-1.43; P = .23) for BRCA2 carriers. The hazard ratio for breast cancer-specific mortality was 0.76 (95% CI, 0.32-1.78; P = .53) for women with estrogen receptor-positive breast cancer and 0.07 (95% CI, 0.01-0.51; P = .009) for women with estrogen receptor-negative breast cancer. Oophorectomy is associated with a decrease in mortality in women with breast cancer and a BRCA1 mutation. Women with estrogen receptor-negative breast cancer and a BRCA1 mutation should undergo oophorectomy shortly after diagnosis.

Adjuvant Oophorectomy in the Treatment of Early-StageBRCAMutation–Positive Breast Cancer

TheUSPreventiveServicesTaskForce recently releasedasystematic review focused on the risk of breast and ovarian cancer in BRCA1 and BRCA2mutation carriers and the impact of genetic testing and counseling on the patient.1 A metaanalysis of 70 studiesdemonstrated that breast cancer prevalencewas46%to71%by 70 years of age for thosewith BRCA1orBRCA2mutationsandovariancancerprevalencewas 41%to46%forBRCA1and 17%to23%forBRCA2mutationcarriers. Patientswith germlinemutations in theBRCAgene family often choose prophylactic surgery, mastectomy and/or oophorectomy, to reducetheir riskofdevelopingbreastorovariancancer. In this issueofJAMAOncology,Metcalfeetal2 evaluated the impact of oophorectomy on overall survival of patientswhohave already received a diagnosis of breast cancer. Theparticipantswere676patientswith stage I or II breast cancer andBRCA1orBRCA2mutations. Smaller studies have suggested that oophorectomy after a breast cancer diagnosis results in reduced mortality in these individuals at extreme genetic risk. The cohort reported byMetcalfe et al2 did or did nothave their ovaries removedandwereobserved for 20years tomonitor for recurrence. The results provide a validation of the roleof oophorectomy inconveyingbothadisease-free and overall survival benefit for BRCA1mutation carriers. Oophorectomy after the primary diagnosis of breast cancer significantly reducedbreast cancer–specificmortality inwomenwith BRCA1mutations (HR, 0.38 [95% CI, 0.19-0.77]; P = .007) but not in BRCA2mutation carriers (HR, 0.57 [95% CI, 0.23-1.43]; P = .23). In the entire group, oophorectomy was particularly effective for survivalbenefit inwomenwithestrogenreceptor– negativebreast cancer (HR,0.07 [95%CI, 0.01-0.51];P = .009). Ofnote, themean timeelapsedbeforeoophorectomyafter the diagnosis of breast cancerwas 6.1 years. The hazard ratiowas further reduced,however, if the surgerywasperformedwithin 2 years of the breast cancer diagnosis. The data reported here are compelling and suggest that the potential of oophorectomy should become part of the treatment discussion at the time of diagnosis forBRCAmutation carrierswith early-stage breast cancers.

XomAnnotate: Analysis of Heterogeneous and Complex Exome- A Step towards Translational Medicine.

In translational cancer medicine, implicated pathways and the relevant master genes are of focus. Exome's specificity, processing-time, and cost advantage makes it a compelling tool for this purpose. However, analysis of exome lacks reliable combinatory analysis tools and techniques. In this paper we present XomAnnotate – a meta- and functional-analysis software for exome. We compared UnifiedGenotyper, Freebayes, Delly, and Lumpy algorithms that were designed for whole-genome and combined their strengths in XomAnnotate for exome data through meta-analysis to identify comprehensive mutation profile (SNPs/SNVs, short inserts/deletes, and SVs) of patients. The mutation profile is annotated followed by functional analysis through pathway enrichment and network analysis to identify most critical genes and pathways implicated in the disease genesis. The efficacy of the software is verified through MDS and clustering and tested with available 11 familial non-BRCA1/BRCA2 breast cancer exome data. The results showed that the most significantly affected pathways across all samples are cell communication and antigen processing and presentation. ESCO1, HYAL1, RAF1 and PRKCA emerged as the key genes. Network analysis further showed the purine and propanotate metabolism pathways along with RAF1 and PRKCA genes to be master regulators in these patients. Therefore, XomAnnotate is able to use exome data to identify entire mutation landscape, pathways, and the master genes accurately with wide concordance from earlier microarray and whole-genome studies -- making it a suitable biomedical software for using exome in next-generation translational medicine.Availability http://www.iomics.in/research/XomAnnotate

Assessing associations between the AURKA-HMMR-TPX2-TUBG1 functional module and breast cancer risk in BRCA1/2 mutation carriers.

While interplay between BRCA1 and AURKA-RHAMM-TPX2-TUBG1 regulates mammary epithelial polarization, common genetic variation in HMMR (gene product RHAMM) may be associated with risk of breast cancer in BRCA1 mutation carriers. Following on these observations, we further assessed the link between the AURKA-HMMR-TPX2-TUBG1 functional module and risk of breast cancer in BRCA1 or BRCA2 mutation carriers. Forty-one single nucleotide polymorphisms (SNPs) were genotyped in 15,252 BRCA1 and 8,211 BRCA2 mutation carriers and subsequently analyzed using a retrospective likelihood approach. The association of HMMR rs299290 with breast cancer risk in BRCA1 mutation carriers was confirmed: per-allele hazard ratio (HR) = 1.10, 95% confidence interval (CI) 1.04 – 1.15, p = 1.9 x 10−4 (false discovery rate (FDR)-adjusted p = 0.043). Variation in CSTF1, located next to AURKA, was also found to be associated with breast cancer risk in BRCA2 mutation carriers: rs2426618 per-allele HR = 1.10, 95% CI 1.03 – 1.16, p = 0.005 (FDR-adjusted p = 0.045). Assessment of pairwise interactions provided suggestions (FDR-adjusted pinteraction values > 0.05) for deviations from the multiplicative model for rs299290 and CSTF1 rs6064391, and rs299290 and TUBG1 rs11649877 in both BRCA1 and BRCA2 mutation carriers. Following these suggestions, the expression of HMMR and AURKA or TUBG1 in sporadic breast tumors was found to potentially interact, influencing patients’ survival. Together, the results of this study support the hypothesis of a causative link between altered function of AURKA-HMMR-TPX2-TUBG1 and breast carcinogenesis in BRCA1/2 mutation carriers.

Bilateral risk-reduction mastectomy in BRCA1 and BRCA2 mutation carriers: a meta-analysis.

Women with BRCA1 and BRCA2 mutations have substantially elevated risk of developing breast cancer. The aim of this study was to clarify the role of bilateral risk-reducing mastectomy (BRRM) in reducing breast cancer risk in women carriers of BRCA1 and BRCA2 mutations. The Pubmed, MEDLINE and Scopus databases were searched to retrieve articles written in the English language. Two investigators independently extracted the characteristics and results of the selected studies. Only prospective trials with available absolute numbers of breast cancer and death events were included. Pooled hazard ratio (HR) with 95 % confidence interval (CI) was calculated using fixed or random effects model. Meta-analysis of four prospective studies, including 2635 patients, demonstrated a significant risk reduction of breast cancer incidence in BRCA1 and BRCA2 mutation carriers receiving BRRM (HR 0.07; 95 % CI 0.01-0.44; p = 0.004). Among patients without previous risk-reducing salpingo-oophorectomy, a significant benefit was similarly recorded (HR 0.06; 95 % CI 0.01-0.41; p = 0.005). Performing BRRM may lead to highly significant risk reduction of breast cancer in BRCA1 and BRCA2 mutation carriers. These data allow clinicians to discuss more in-depth with patients all the available options in order to design better management strategies.

Radiotherapy in the Treatment of BRCA1 and BRCA2 Mutated Breast Cancer.

Radiotherapy in the Treatment of BRCA1 and BRCA2 Mutated Breast Cancer.

Decreased BECN1 mRNA Expression in Human Breast Cancer is Associated With Estrogen Receptor-Negative Subtypes and Poor Prognosis

Both BRCA1 and Beclin 1 (BECN1) are tumor suppressor genes, which are in close proximity on the human chromosome 17q21 breast cancer tumor susceptibility locus and are often concurrently deleted. However, their importance in sporadic human breast cancer is not known. To interrogate the effects of BECN1 and BRCA1 in breast cancer, we studied their mRNA expression patterns in breast cancer patients from two large datasets: The Cancer Genome Atlas (TCGA) (n=1067) and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) (n=1992). In both datasets, low expression of BECN1 was more common in HER2-enriched and basal-like (mostly triple-negative) breast cancers compared to luminal A/B intrinsic tumor subtypes, and was also strongly associated with TP53 mutations and advanced tumor grade. In contrast, there was no significant association between low BRCA1 expression and HER2-enriched or basal-like subtypes, TP53 mutations or tumor grade. In addition, low expression of BECN1 (but not low BRCA1) was associated with poor prognosis, and BECN1 (but not BRCA1) expression was an independent predictor of survival. These findings suggest that decreased mRNA expression of the autophagy gene BECN1 may contribute to the pathogenesis and progression of HER2-enriched, basal-like, and TP53 mutant breast cancers.

TUSC4 functions as a tumor suppressor by regulating BRCA1 stability.

BRCA1 expression is lost frequently in breast cancers in which it promotes malignant development. In the present study, we performed a global expression analysis of breast cancer cells in which the tumor-suppressor candidate gene TUSC4 was silenced to gain insights into its function. TUSC4 silencing affected genes involved in cell cycle and cell death, which have broad reaching influence on cancer development. Most importantly, we found a cluster pattern of gene-expression profiles in TUSC4-silenced cells that defined a homologous recombination (HR) repair defect signature. Mechanistic investigations indicated that TUSC4 protein could physically interact with the E3 ligase Herc2, which prevents BRCA1 degradation through the ubiquitination pathway. TUSC4 silencing enhanced BRCA1 polyubiquitination, leading to its degradation and a marked reduction in HR repair efficiency. Notably, ectopic expression of TUSC4 suppressed the proliferation, invasion, and colony formation of breast cancer cells in vitro and tumorigenesis in vivo. Furthermore, TUSC4 silencing was sufficient to transform normal mammary epithelial cells and to enhance sensitivity to PARP inhibitors. Our results provide a set of genetic and biologic proofs that TUSC4 functions as a bona fide tumor suppressor by regulating the protein stability and function of BRCA1 in breast cancer.

Mutational landscape of gastric adenocarcinoma in Chinese: Implications for prognosis and therapy

Gastric cancer (GC) is a highly heterogeneous disease. To identify potential clinically actionable therapeutic targets that may inform individualized treatment strategies, we performed whole-exome sequencing on 78 GCs of differing histologies and anatomic locations, as well as whole-genome sequencing on two GC cases, each with three primary tumors and two matching lymph node metastases. The data showed two distinct GC subtypes with either high-clonality (HiC) or low-clonality (LoC). The HiC subtype of intratumoral heterogeneity was associated with older age, TP53 (tumor protein P53) mutation, enriched C > G transition, and significantly shorter survival, whereas the LoC subtype was associated with younger age, ARID1A (AT rich interactive domain 1A) mutation, and significantly longer survival. Phylogenetic tree analysis of whole-genome sequencing data from multiple samples of two patients supported the clonal evolution of GC metastasis and revealed the accumulation of genetic defects that necessitate combination therapeutics. The most recurrently mutated genes, which were validated in a separate cohort of 216 cases by targeted sequencing, were members of the homologous recombination DNA repair, Wnt, and PI3K-ERBB pathways. Notably, the drugable NRG1 (neuregulin-1) and ERBB4 (V-Erb-B2 avian erythroblastic leukemia viral oncogene homolog 4) ligand-receptor pair were mutated in 10% of GC cases. Mutations of the BRCA2 (breast cancer 2, early onset) gene, found in 8% of our cohort and validated in The Cancer Genome Atlas GC cohort, were associated with significantly longer survivals. These data define distinct clinicogenetic forms of GC in the Chinese population that are characterized by specific mutation sets that can be investigated for efficacy of single and combination therapies.

Analysis of PTEN in two BRCA1 and BRCA2 wild-type familial breast cancer patients.

Sir, Our study was designed to sequence the entire exonic regions and intronic junctions of the BRCA1, BRCA2, and PTEN genes with regard to identifying the influence of PTEN mutations in two Iranian families with familial breast cancer and BRCA1/2 wild-type genes [Figure 1]. Figure 1 Pedigrees of the two families with familial breast cancer carrying new germline sequence variants in the PTEN gene: One deletion (del T IVS4-29) and one single-nucleotide substitution (IVS2+65 G>A) Mutations in particular sets of genes are associated with an increased risk of hereditary breast and ovarian cancer (HBOC). These genes are categorized into three groups according to their impact on cancer susceptibility. The first group leading to elevated breast cancer risk (40% up to 85%)[1] with mutations of high penetrance include BCRA1, BCRA2, TP53, PTEN, and STK11.[2] PTEN mutations, as a rare but high penetrance mutation, are considered following the two major known breast cancer susceptibility genes, BRCA1 and BRCA2, in HBOC development.[3] Human genomic PTEN gene locus on chromosome 10q23.3 contains 9 exons encoding a 5.5 kb messenger RNA that has a 403 amino acid open reading frame.[4] Whole gene sequencing was done for three genes in these families. The BRCA1 and BRCA2 in the proband of the families were normal, and no germline abnormality was found in these two genes that are mainly responsible for familial breast cancer. However, two new germline sequence variants were detected in the PTEN gene: One deletion (del T IVS4-29) and one single-nucleotide substitution (IVS2+65 G>A) [Figure ​[Figure2a2a and ​andb].b]. Both new changes found in the PTEN gene were analyzed by the two servers NetGene 2 and alternative splice site predictor. Del T IVS4-29 change was impositioned in NetGene 2 site, the 3’ splice site acceptor changed in the consensus sequences, but did not delete the consensus sequences. The IVS2+65 G>A variation did not cause a new change in splicing site. Figure 2 (a) Heterozygous IVS2+65 G>A of the PTEN gene. (b) Heterozygous del IVS4-29 of the PTEN gene Despite new achievements in this area, many factors leading to breast cancer predisposition are still unknown, and thus need to be identified with further investigations. All the investigations to promote strategies of early cancer diagnosis, which is critical for the survival of affected individuals, might be advantageous. As the main mechanism for activating the PTEN protein is proteasomal degradation,[5] any changes in protein structure influence the integral role of it. The specific variations that we detected may make some mute changes in the protein interactions which may prevent it from performing its task. It can be cleared by the study of protein structure in future investigations. To the best of our knowledge, the study we present here is the first analysis of the PTEN gene in BRCA1 and BRCA2 wild-type breast and/or ovarian cancer in Iranian families. Financial support and sponsorship This work was supported by grants from Iran University of Medical Sciences project number 1021. Conflicts of interest There are no conflicts of interest.

Tumor-associated inflammation as a potential prognostic tool in BRCA1/2-associated breast cancer

The prognosis of BRCA1/2-associated breast cancer partly depends on histologic characteristics. Most of these breast cancers, however, are poorly differentiated. BRCA1-associated cancers are mainly negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Consequently, the use of these histologic features for risk stratification in BRCA1/2 breast cancer is limited. We assessed the prognostic value of additional histologic features, including tumor-associated inflammation and tumor-associated stroma in BRCA1/2 breast cancer patients. From the Rotterdam Family Cancer Clinic database, we collected demographics, tumor characteristics, and follow-up data from female BRCA1/2 breast cancer patients. Tumor samples were centrally reviewed including histologic subtype, differentiation grade, tumor-associated inflammation density, amount of tumor-associated stroma, and intratumor necrosis. The impact of these factors on recurrence-free survival (RFS) was evaluated using univariate and multivariate Cox regression, adjusted for established prognostic features and year of diagnosis. We included 138 BRCA1 and 37 BRCA2 breast cancer patients. Median follow-up after diagnosis was 9.7 years. Independent prognostic factors for RFS were tumor size (hazard ratio [HR], 2.47 for >2 versus ≤2 cm; 95% confidence interval [95% CI], 1.10-5.57), tumor-associated inflammation (HR, 0.18 for moderate/marked versus absent/mild; 95% CI, 0.05-0.61), and intratumor necrosis (HR, 2.60 for presence versus absence; 95% CI, 1.12-6.05). Established prognostic factors as nodal status and differentiation grade were not significantly related to RFS. Subgroup analyses of 138 BRCA1 and 118 triple-negative breast cancer cases showed similar results. Tumor-associated inflammation density was the strongest predictor for RFS in this series of BRCA1/2 breast cancer patients. This provides a potential risk stratification tool that can easily be implemented in routine histologic examination.

Structure and mechanism of action of the BRCA2 breast cancer tumor suppressor.

Mutations in BRCA2 increase susceptibility to breast, ovarian and prostate cancers. The product of human BRCA2, BRCA2 protein, plays a key role in the repair of DNA double strand breaks and interstrand crosslinks by RAD51-mediated homologous recombination. Here, we present a biochemical and structural characterization of full length (3,418 amino acid) BRCA2, alone and in complex with RAD51. We show that BRCA2 facilitates nucleation of RAD51 filaments at multiple sites on single-stranded DNA. Three-dimensional electron microscopy reconstructions revealed that BRCA2 exists as a dimer and that two oppositely-oriented sets of RAD51 molecules bind the dimer. Single stranded DNA binds along the long axis of BRCA2, such that only one set of RAD51 monomers can form a productive complex with DNA and establish filament formation. Our data define the molecular mechanism by which this tumor suppressor facilitates RAD51-mediated homologous recombinational repair.

BRCA1 pathway function in basal-like breast cancer cells.

Sporadic basal-like cancers (BLCs) are a common subtype of breast cancer that share multiple biological properties with BRCA1-mutated breast tumors. Despite being BRCA1(+/+), sporadic BLCs are widely viewed as phenocopies of BRCA1-mutated breast cancers, because they are hypothesized to manifest a BRCA1 functional defect or breakdown of a pathway(s) in which BRCA1 plays a major role. The role of BRCA1 in the repair of double-strand DNA breaks by homologous recombination (HR) is its best understood function and the function most often implicated in BRCA1 breast cancer suppression. Therefore, it is suspected that sporadic BLCs exhibit a defect in HR. To test this hypothesis, multiple DNA damage repair assays focused on several types of repair were performed on a group of cell lines classified as sporadic BLCs and on controls. The sporadic BLC cell lines failed to exhibit an overt HR defect. Rather, they exhibited defects in the repair of stalled replication forks, another BRCA1 function. These results provide insight into why clinical trials of poly(ADP-ribose) polymerase (PARP) inhibitors, which require an HR defect for efficacy, have been unsuccessful in sporadic BLCs, unlike cisplatin, which elicits DNA damage that requires stalled fork repair and has shown efficacy in sporadic BLCs.

Abstract 3812: Targeting BRCA1-deficient breast cancer by inhibition of the DEAD box RNA helicase DDX3X

Abstract Introduction: The DEAD box RNA helicase DDX3X (DDX3) has been demonstrated to have a pro-oncogenic role in breast cancer. Thus, abrogating DDX3 activity was explored as a viable anti-cancer strategy using a small molecule inhibitor of DDX3, referred to as RK-33. RK-33 was found to inhibit non-homologous end joining (NHEJ), a DNA double strand break (DSB) repair mechanism. Therefore, we set out to determine the utility of RK-33 in breast cancers of BRCA1 mutation carriers, as they exhibit deficiencies in homologous recombination, an additional DSB repair mechanism. We hypothesized that BRCA1-deficient breast cancers would be more dependent on NHEJ to maintain genomic stability and thus inhibiting DDX3 activity by RK-33 would be an efficacious treatment strategy. Methods: We evaluated DDX3 protein expression levels by immunohistochemistry in 102 BRCA1 and 29 BRCA2 germline mutation carriers and compared these to those of 345 sporadic breast cancer patients. In addition, DDX3 expression in two BRCA1-deficient (SUM149PT and HCC1937) and two BRCA1-proficient breast cancer cell lines (MCF-7 and MDA-MB-231) was quantified by immunoblotting. Furthermore, we compared the cell killing abilities of RK-33 in these BRCA1 pro- and deficient cell lines, as well as in MCF-7 cells before and after knockdown of BRCA1 by shRNA. Results: High cytoplasmic DDX3 expression is less frequent in BRCA1 (24.5%) and BRCA2 (20.7%) related breast cancer, when compared to sporadic breast cancer (34.2%; OR 0.63; 95% CI 0.38-1.03; P = 0.065). This difference is statistically significant when correcting for higher grade and basal-like subtype in the group of BRCA1 mutation carriers (OR 0.33; 95% CI 0.14-0.77; P = 0.01). The BRCA1 pro- and deficient cell lines expressed similar levels of DDX3 and had a comparable sensitivity to RK-33 with IC-50 values ranging between 2 and 7 uM. Knockdown of BRCA1 in MCF-7 did not result in an increased sensitivity to RK-33. Conclusion: DDX3 expression is lower in breast cancer in BRCA1 mutation carriers, when compared to sporadic breast cancers of similar grade and molecular subtype. BRCA1 pro- and deficient breast cancer cell lines are equally sensitive to inhibition of DDX3 by RK-33. This indicates that abrogating NHEJ activity by RK-33 results in cell death irrespective of additional deficiencies in the DNA repair pathway. Given that RK-33 showed good in vitro efficacy to kill all breast cancer cell lines, additional research is required to evaluate the use of RK-33 as a targeted chemotherapy agent for breast cancer. Citation Format: Marise R. Heerma van Voss, Farhad Vesuna, Guus M. Bol, Paul J. van Diest, Venu Raman. Targeting BRCA1-deficient breast cancer by inhibition of the DEAD box RNA helicase DDX3X. [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 3812. doi:10.1158/1538-7445.AM2014-3812