BRCA1 Loss Of Heterozygosity Research Articles

1879PD - Primary ovarian carcinomas arising in BRCA1 mutation carriers contain a small fraction of BRCA1-proficient cells, which rapidly repopulate tumor mass during neoadjuvant chemotherapy but become outgrown by BRCA1-deficient clones during platinum-free intervals

BackgroundSomatic loss of the wild-type allele is a key event in the pathogenesis of BRCA1-driven carcinomas. BRCA1 deficiency renders pronounced sensitivity of these tumors to platinum compounds and PARP inhibitors. MethodsWe analyzed BRCA1 loss of heterozygosity (LOH) in serial ovarian cancer (OC) samples, which were obtained from BRCA1 germ-line mutation carriers before neoadjuvant chemotherapy (NACT), at surgery and at disease relapse. Results26/36 (72%) OC had BRCA1 LOH before NACT. 15 (58%) of these 26 tumors showed retention of the normal BRCA1 allele after NACT. The analysis of linked SNPs and FISH assay strongly indicated, that the restoration of BRCA1 function is caused not by the second mutation, but by the selection of pre-existing BRCA1-proficient cells. Tumor relapses were available in 7 patients with BRCA1 LOH in the chemonaive tumor and/or BRCA-proficiency in the residual post-NACT neoplasm; 6 (86%) of these relapses had BRCA1 LOH thus resembling the primary tumor. Secondary open reading frame (ORF) restoring BRCA1 mutation was detected in 1 recurrent OC. Serial samples retained same TP53 mutation during the treatment course. Whole exome sequencing revealed that chemonaive, post-NACT and relapsed tumors had both shared and individual mutations. Conclusions1) BRCA1 LOH is not the first event in the pathogenesis of BRCA1-driven cancer: gain of TP53 mutation probably precedes BRCA1 inactivation in order to prevent apoptosis; 2) Chemonaïve BRCA1-driven tumors contain a small fraction of BRCA1-proficient cells, which rapidly repopulate the tumor lump during the first weeks of therapy; 3) Change of BRCA1 status during NACT may call to reconsider the existing approaches to adjuvant therapy, as the residual post-NACT tumor masses are likely to be platinum-resistant; 4) The balance between BRCA1-deficient and BRCA1-proficient cells is a subject of fluctuations, depending whether therapy is applied or not; 5) Clinical trials on BRCA1-driven OCs need to address the issue of intratumoral heterogeneity of BRCA1 status. Legal entity responsible for the studyThe authors. FundingRussian Science Foundation (grant 19-15-00168). DisclosureAll authors have declared no conflicts of interest.

998PD - Analysis of tumour samples from SOLO1: Frequency of BRCA specific loss of heterozygosity (LOH) and progression-free survival (PFS) according to homologous recombination repair deficiency (HRD)-LOH score

BackgroundIn SOLO1 (NCT01844986; GOG-3004), maintenance olaparib significantly improved PFS vs placebo (HR 0.30; 95% CI 0.23–0.41; Moore et al. NEJM 2018) in patients (pts) with newly diagnosed advanced ovarian cancer and a BRCA1 and/or BRCA2 mutation (BRCAm), who were in clinical complete or partial response after first line treatment, which includes surgery and platinum-based chemotherapy. We analyzed BRCA LOH and PFS by genome-wide HRD-LOH score in SOLO1. MethodsArchival diagnostic tumour samples from 341/391 pts from SOLO1 were analysed using the Foundation Medicine F1CDx clinical trial assay. Tumour BRCA1 and BRCA2 LOH was determined using the SGZ-computational method (Sun et al. 2018). HRD-LOH score (genomic instability scar) was also generated and compared with PFS using a Cox Proportional Hazards model. ResultsOf evaluable tumours, 99% (275/277) had BRCA1 or BRCA2 LOH, including 2 pts with a somatic BRCAm. Two germline BRCA2m tumours lacked LOH. 283/341 (83.0%) tumours sequenced at FMI were evaluable for HRDLOH score. Using established cut-offs of 14 and 16 (Swisher et al. 2017); 84% (237/283) and 77% (218/283), respectively, would be considered HRD-LOH high. BRCAm pts with HRD-LOH scores <14 or<16 derived similar benefit with olaparib compared to those with high scores (Table).Table: 998PDTable: 998PDProgression-free survivalHRD-LOH score subgroupTreatment armNNumber (%) eventsHR95% CIHRD-LOH score <14Olaparib Placebo27 199 (33.3) 14 (73.7)0.200.08, 0.45HRD-LOH score ≥14Olaparib Placebo165 7263 (38.2) 56 (77.8)0.320.22, 0.46HRD-LOH score <16Olaparib Placebo43 2218 (41.9) 16 (72.7)0.290.15, 0.58HRD-LOH score ≥16Olaparib Placebo149 6954 (36.2) 54 (78.3)0.290.20, 0.43 ConclusionsAs surgery is part of first-line treatment, platinum sensitivity cannot be determined in all pts. However, our results show that in patients with BRCAm tumours, BRCAm LOH is almost universal in ovarian cancer in the first-line setting. The majority of BRCAm tumours also have high HRD-LOH scores at diagnosis, with significant olaparib benefit demonstrated in pts with both high and low HRD-LOH scores. Assessing BRCA LOH and HRD-LOH scores does not discriminate the extent of olaparib benefit in newly diagnosed BRCAm advanced OC. Clinical trial identificationNCT01844986. Editorial acknowledgementEmma Robinson, PhD, from Mudskipper Business Ltd, funded by AstraZeneca and Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, U.S.A. (MSD). Legal entity responsible for the studyAstraZeneca and Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, U.S.A. (MSD). FundingAstraZeneca and Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, U.S.A. (MSD). DisclosureC. Gourley: Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): AstraZeneca; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Tesaro; Honoraria (self), Advisory / Consultancy, Research grant / Funding (self): Nucana; Honoraria (self), Advisory / Consultancy: Roche; Honoraria (self), Advisory / Consultancy: Clovis Oncology; Honoraria (self), Advisory / Consultancy: Foundation One; Honoraria (self), Advisory / Consultancy: MSD; Honoraria (self), Advisory / Consultancy: Sierra Oncology; Honoraria (self), Advisory / Consultancy: Novartis; Honoraria (self), Advisory / Consultancy: Aprea. J.S. Brown: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. Z. Lai: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. P. Lao-Sirieix: Shareholder / Stockholder / Stock options, Full / Part-time employment, Spouse / Financial dependant, Spouse full-time employee: AstraZeneca; Licensing / Royalties, Named inventor on patent licensed to Medtronic. Not related to the topic of the abstract: Medtronic. C.E. Elks: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. H. McGarvey: Full / Part-time employment: AstraZeneca. T. French: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. T. Milenkova: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. R. Bloomfield: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. P. Rowe: Full / Part-time employment: AstraZeneca. D. Hodgson: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. J.C. Barrett: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca. K. Moore: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Financial conflict of interest with AstraZeneca for 2018 related to speaking engagements following SOLO-1 and travel: AstraZeneca; Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution): Tesaro; Honoraria (self): Prime Oncology; Honoraria (self): Research to Practice; Honoraria (self): PER; Advisory / Consultancy: Advaxis; Advisory / Consultancy: Immunogen; Advisory / Consultancy, Research grant / Funding (self), Research grant / Funding (institution): Clovis; Advisory / Consultancy, Research grant / Funding (self): Genentech/Roche; Advisory / Consultancy, Research grant / Funding (institution): Pfizer; Advisory / Consultancy, Research grant / Funding (institution): Merck; Advisory / Consultancy: Janssen; Advisory / Consultancy: Oncomed; Advisory / Consultancy: Samumed; Advisory / Consultancy: Aravive; Advisory / Consultancy: VBL Therapeutics; Advisory / Consultancy: Eisai; Research grant / Funding (self): Lilly; Research grant / Funding (self): PTC Therapeutics; Research grant / Funding (institution): LEAP, AbbVie, Boehringer Ingelheim, Regeneron. P. DiSilvestro: Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Tesaro. E.A. Harrington: Shareholder / Stockholder / Stock options, Full / Part-time employment: AstraZeneca.

Abstract 435: Cell-free circulating tumor DNA (ctDNA) detects somatic copy number loss in homologous recombination repair genes

Abstract Background: In addition to BRCA1/2 germline and somatic inactivating mutations, loss of heterozygosity (LOH) and biallellic somatic copy number loss are associated with BRCA1/2 loss of function. Patients with cancers harboring these somatic features may benefit from treatment with PARP inhibitors. ctDNA analysis has proven a viable alternative to tumor tissue genotyping, especially in tissue- or time-limited clinical scenarios. However, ctDNA assessment of LOH and biallelic copy number loss is challenging given that ctDNA is diluted by cell-free leukocytic DNA making it difficult to confidently call these genomic events. We developed a method to identify somatic biallelic copy number loss in ctDNA using targeted sequencing of cell-free (cfDNA) across a wide range of cancer types. Methods: A novel statistical model was developed using coverage profiles and single nucleotide polymorphism (SNP) allelic frequencies estimated from plasma samples to determine the presence of LOH and biallellic copy number loss in BRCA1 or BRCA2. For each gene of interest, the model uses observed coverage and fragment size distribution, together with allele frequency of germline SNPs. The model was applied to plasma samples from 28,000 patients with advanced solid tumors sequenced using a 73-gene next generation sequencing ctDNA panel (Guardant360®, Guardant Health, Redwood City, CA). Results: The model was analytically validated using in-silico simulations in order to assess both the limit of blank and limit of detection. This method shows 95% sensitivity in detecting LOH and bi-allellic copy number loss for samples with a maximum somatic variant allele frequency as low as 9%. Sensitivity is mainly driven by the panel size and the depth of coverage of the gene of interest. The model was then applied to the 28,199 patient cohort. BRCA1 and BRCA2 LOH was observed in 2.4% (134/5568) and 7.4% (415/5568) of classic homologous recombination deficient (HRD) cancers including breast, ovarian, prostate, and pancreas. BRCA1 and BRCA2biallelic copy number loss was observed in 0.3% (19/5568) and 0.6% (31/5568) of this same group of HRD cancers. Discussion: In this cohort of 5,568 patients with classic HRD associated cancers, somatic LOH and biallelic copy number loss was detected in BRCA1 in 2.7% of samples and in BRCA2 in 8.0% of samples, which is aligned with previously reported tissue prevalence. BRCA1/2 somatic LOH and biallelic copy number loss can accurately be detected in ctDNA utilizing likelihood-based scores based on coverage and germline SNPs allele frequencies. The ability to identify this therapeutically targetable genomic alteration through a non-invasive ctDNA assessment has significant clinical implications. Citation Format: Catalin Barbacioru, Victoria M. Raymond, Marcin Sikora, Elena Helman, Sante Gnerre, Stephen Fairclough, Darya Chudova, Richard B. Lanman, AmirAli Talasaz. Cell-free circulating tumor DNA (ctDNA) detects somatic copy number loss in homologous recombination repair genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 435.

Abstract P2-06-03: The BRCA1 promoter methylation is one of the mechanisms of th BRCA1 dysfunction of triple-negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is typically associated with aggressive tumor phenotype and poor prognosis. TNBC is also considered highly heterogeneous disease. A better understanding of molecular and histopathological features of TNBC is of great importance, in order to develop a new therapeutic strategy and to improve the prognosis of TNBC. TNBC has many similarities with basal-like breast cancer, and is also associated with BRCAness. The major role of BRCA1 is to respond to DNA damage by participating in cellular pathways for DNA repair, mRNA transcription, cell cycle regulation, and protein ubiquitination. BRCA1 function loss leads to impaired homologous recombination mediated DNA repair. A loss of BRCA1 may thus be a biomarker of responsiveness to DNA damaging agents such as PARP inhibitor and cis/carboplatin. Germline mutations in BRCA1, however, account for approximately 5% of breast cancer cases, somatic mutations in BRCA1 rarely occur. Instead, lower than normal expression of BRCA1 is reported to be an important contributing factor in sporadic tumors. The BRCA1 promoter methylation may thus play an important role in the BRCA1 function loss in sporadic breast cancer. Aim: To evaluate the clinical importance of BRCA1 promoter methylation in breast cancer. Materials and Methods: Specimens were obtained from 71 TNBC and 163 non-TNBC patients who underwent surgery without neoadjuvant therapy in our department between 1990 and 2011. BRCA1 promoter methylation was investigated by using combined bisulfate and restriction analysis (COBRA). The BRCA1 mRNA expression was evaluated by quantitive RT-PCR. The BRCA1 protein level was assessed by immnohistochemistry. Loss of heterozygosity(LOH) at the BRCA1 locus was analyzed with microsatellite markers (D17S855 and D17S579) using our microsatellite analysis system. Copy number variations(CNVs) were analysed by SNP-CGH array (Illumina, HumanOmni2.5-8). Results: We found 12 patients with BRCA1 promoter methylation and all of them were TNBC (p&amp;lt;0.0001). BRCA1 promoter methylation was associated with lymphovessel invasion (p = 0.009), high nuclear grade (p = 0.03), low BRCA1 mRNA expression (p = 0.002) and low BRCA1 protein expression (p&amp;lt;0.0001). CNVs were observed more frequently in TNBC(p = 0.012). High CNVs significantly correlated with BRCA1 LOH (p = 0.0025), but not with BRCA1 promoter methylation among TNBC patients. Conclusions: The BRCA1 promoter methylation is considered to be a specific feature of aggressive phenotype of TNBC and to be one of the mechanisms of BRCA1 dysfunction in breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-06-03.

Loss of Heterozygosity at BRCA1 Locus Is Significantly Associated with Aggressiveness and Poor Prognosis in Breast Cancer

BRCA1 and BRCA2 are two major tumor suppressor genes for hereditary breast and ovarian cancer. In sporadic breast cancer, although somatic mutations of these genes are rare, loss of heterozygosity (LOH) at BRCA1 and BRCA2 loci is common. LOH at BRCA1 and BRCA2 loci were investigated in 202 Japanese invasive breast cancer patients. The relationships between LOH at these gene loci and clinicopathologic characteristics were analyzed. Among 166 informative cases for both BRCA1 and BRCA2 loci, 69 (41.6%) and 52 (31.3%) tumors revealed LOH at BRCA1 and BRCA2 loci, respectively. LOH at BRCA1 LOH or BRCA2 locus was associated with higher nuclear grade (P < 0.0001, P = 0.0187). LOH at BRCA1 locus was associated with estrogen receptor and progesterone receptor negativity (P = 0.001 and P = 0.015) and significantly shorter disease-free survival (P < 0.0001), distant metastasis-free survival (P < 0.0001), and overall survival (P < 0.0001). In contrast, LOH at BRCA2 locus had no associations with estrogen receptor or progesterone receptor status and prognosis. LOH at BRCA1 locus was independently associated with poor prognosis in terms of disease-free survival (hazard ratio 3.08, 95% confidence interval [CI] 1.58-6.18, P = 0.0009), distant metastasis-free survival (hazard ratio 5.18, 95% CI 2.35-12.19, P < 0.0001), and overall survival (hazard ratio 4.97, 95% CI 1.84-15.1, P = 0.0013). LOH at BRCA1 locus could be an independent prognostic biomarker useful in identifying a subgroup of patients with poor prognosis.

Methylation not a frequent “second hit” in tumors with germline BRCA mutations

Mutations in tumor suppressor genes BRCA1 and BRCA2 confer an increased lifetime risk of breast and ovarian cancer. Loss of heterozygosity (LOH) of the wildtype allele has been observed in approximately 80% of tumors from BRCA1 carriers and 70% of tumors from BRCA2 carriers and accounts for the majority of the "second-hits" occurring in BRCA-related tumors. Few sporadic tumors have been reported to have mutations in BRCA. Some sporadic tumors do show LOH of BRCA1 and BRCA2. BRCA1 promoter methylation has also been observed in sporadic ovarian and breast tumors; however, BRCA2 promoter methylation has not been reported in sporadic tumors. The relationship between BRCA LOH and BRCA promoter methylation has not been well characterized in tumors from BRCA germline mutation carriers. The goal of this study was to determine if BRCA1 and BRCA2 promoter hypermethylation serves as a "second-hit" in tumors from mutation carriers that do not show LOH. We studied 38 tumors from BRCA1 carriers and 23 tumors from BRCA2 carriers for LOH. To determine if BRCA1 and BRCA2 promoter hypermethylation serves as a "second-hit" in tumors with germline mutations, we tested 15 tumors lacking LOH and nine tumors with LOH for BRCA1 or BRCA2 promoter methylation. We identified seven BRCA1 tumors and nine BRCA2 tumors lacking LOH. Of these, only one tumor with a BRCA2 mutation showed promoter methylation. These data indicate that promoter methylation is a not a frequent "second-hit" in tumors from BRCA1 or BRCA2 carriers.

Expression of BRCA1 protein in benign, borderline, and malignant epithelial ovarian neoplasms and its relationship to methylation and allelic loss of the BRCA1 gene

BRCA1 is a putative tumour suppressor gene responsible for a hereditary ovarian cancer syndrome. To clarify the possible involvement of BRCA1 in the development of sporadic ovarian neoplasms, this study analysed the immunohistochemical expression of BRCA1 protein in normal ovarian surface epithelium and 119 epithelial ovarian tumours (19 benign, 24 borderline, and 76 malignant tumours). Loss of heterozygosity (LOH) of BRCA1 was examined using three microsatellite markers to analyse the relationship between BRCA1 expression and alterations of the BRCA1 gene. Methylation of the BRCA1 promoter was also analysed by methylation-specific PCR. In ovarian carcinomas showing heterogeneous expression of BRCA1 protein in the same tumour, LOH and methylation status were analysed using microdissection techniques. Finally, the relationship of BRCA1 expression or its genetic alteration to clinicopathological parameters and patient survival was analysed. Ovarian surface epithelial cells expressed BRCA1 protein. Decreased expression of BRCA1 was found in 16% of benign tumours, 38% of borderline tumours, and 72% of carcinomas. LOH of BRCA1 was demonstrated in no benign tumours, 15% of borderline tumours, and 66% of carcinomas. Methylation of BRCA1 was not detected in benign or borderline tumours, but was present in 31% of carcinomas. Reduced expression of BRCA1 correlated with the presence of gene methylation. The frequency of BRCA1 methylation and LOH was higher in serous carcinomas than in other types. In one of the three serous carcinomas that showed heterogeneous expression of BRCA1, BRCA1-positive borderline-like tumour cells were LOH-positive and methylation-negative, whereas adjacent BRCA1-negative carcinoma cells were LOH-positive and methylation-positive. The prognosis of carcinoma patients did not correlate with BRCA1 expression or genetic status. These findings suggest that reduced expression of BRCA1 protein along with genetic and epigenetic changes of the BRCA1 gene play an important role in the development of sporadic ovarian carcinomas, particularly those of serous histology.

Loss of heterozygosity of p53, BRCA1, VHL, and estrogen receptor genes in breast carcinoma: correlation with related protein products and morphologic features.

Correlation of loss of heterozygosity (LOH) of p53, BRCA1, VHL, and estrogen receptor (ER) genes with the expression of related protein products and morphologic features predictive of aggressive biologic behavior was investigated to determine the significance of LOH in these genes. DNA from 35 formalin-fixed, paraffin-embedded breast carcinomas was obtained by microdissection of histologic sections. LOH was determined by polymerase chain reaction (PCR) using primers TP53, D3S1038, D17S855, and ESR for p53, VHL, BRCA1, and ER genes, respectively. p53, ER, and progesterone receptor (PR) protein expression was evaluated by immunohistochemistry. Morphologic evaluation included histologic type, and histologic and nuclear grades. TP53 LOH was identified in 13 (52%), BRCA1 LOH in 3 (17%), VHL LOH in 1 (4%), and ER LOH in 4 (21%) of 25, 17, 24, and 19 informative cases, respectively. p53 and ER protein expression was identified in 20 (57%) and 25 (71%) cases, respectively. TP53 LOH directly correlated with both high histologic and nuclear grade (P<0.01). BRCA1, VHL, and ER LOH was not frequent enough for correlation to morphologic features. Although 4 of 4 ER and 7 of 13 p53 LOH cases expressed related proteins, LOH did not correlate with protein expression. TP53 LOH may be an event contributing to aggressive biologic behavior since it is strongly associated with high histologic and nuclear grade. Missense or nonsense mutations may explain the absence of detectable p53 protein in 6 of 13 cases with p53 LOH. All 4 ER LOH cases expressed ER protein. BRCA1 and VHL LOH is infrequent in sporadic breast carcinoma.

Biophenotypes and survival of BRCA1 and TP53 deleted breast cancer in young women

The aim of this study was to examine the loss of heterozygosity (LOH) of BRCA1 (17q21) and TP53 (17p13.1) in early-onset breast cancer patients; to correlate biopathological characteristics with molecular alterations; and to investigate the survival of LOH-related cancers. BRCA1 and TP53 LOH were evaluated in 78 early-onset breast cancers (< or = 40 years, Group 1) and 80 patients with age > 55 years (Group 2). Cases were characterized for multiple biological markers (ER, PR, proliferation index (PI), NEU and p53). LOH was carried out on microdissected paraffin embedded tissues; microsatellites D17S855 (BRCA1) and D17S786 (TP53) were amplified by fluorescent PCR and analyzed by an automated DNA sequencer. Early-onset breast cancers showed a higher frequency of ductal histotype (89.7% vs. 56.3% p < 0.001), node-positive (53.8% vs. 38.7%), larger size (p = 0.017), higher mitotic rate (p = 0.025), higher nuclear and final grade (p = 0.01 and p = 0.001, respectively). D17S855 LOH was 32.8% in group 1 vs. 21% in group 2; D17S786 LOH was 50.7% vs. 31.3% (p = 0.03), respectively. BRCA1 LOH was correlated with higher PI (p = 0.032) and higher p53 expression (p < 0.001) in group 1 and with higher NEU expression (p = 0.028) in group 2. TP53 LOH was correlated with p53 overexpression (p = 0.03) in group 1. A worse clinical outcome in early-onset LOH related cancers emerged from follow-up data: TP53 and BRCA1 LOH were associated with a shorter relapse free interval (RFI) (p = 0.03) and a poorer overall survival (OS) (p = 0.04), respectively. This study underlines different biological profiles in the two age groups investigated, probably reflecting different mechanisms of carcinogenesis. In accordance with adverse histopathological features in early-onset patients, LOH-related cancers have an unfavorable prognosis.

Association between loss of heterozygosity of BRCA1 and BRCA2 and morphological attributes of sporadic breast cancer

Germline mutations in the breast cancer–associated genes BRCA1 and BRCA2 confer a lifetime risk of malignancy. Distinctive morphological features have been attributed to these familial tumours; however, in sporadic breast cancer, the inter-relationship between loss of heterozygosity (LOH) of these loci and tumour morphology remains to be fully elucidated. We studied a series of 120 sporadic breast carcinomas using microsatellite markers to identify LOH of BRCA1, BRCA2, p53 and PTEN. The associations between loss at each of the loci were examined and related to tumour morphology. LOH of the 4 loci did not occur independently; there were highly significant associations between LOH of BRCA1 and both BRCA2 (p < 0.001) and p53 (p < 0.001). LOH at all 4 loci was significantly associated with a high degree of nuclear pleomorphism. Tumours with LOH of BRCA1 also had high mitotic indices, few tubules and a paucity of DCIS, all of which are morphological features similar to those described for familial cases. Following Bonferroni's correction for multiple tests, we found that the tumours with LOH of BRCA1 were still significantly associated with a high mitotic index (p = 0.0006) and a high degree of nuclear pleomorphism (p = 0.001). Int. J. Cancer 88:204–208, 2000. © 2000 Wiley-Liss, Inc.

Association between loss of heterozygosity ofBRCA1 andBRCA2 and morphological attributes of sporadic breast cancer

Germline mutations in the breast cancer-associated genes BRCA1 and BRCA2 confer a lifetime risk of malignancy. Distinctive morphological features have been attributed to these familial tumours; however, in sporadic breast cancer, the inter-relationship between loss of heterozygosity (LOH) of these loci and tumour morphology remains to be fully elucidated. We studied a series of 120 sporadic breast carcinomas using microsatellite markers to identify LOH of BRCA1, BRCA2, p53 and PTEN. The associations between loss at each of the loci were examined and related to tumour morphology. LOH of the 4 loci did not occur independently; there were highly significant associations between LOH of BRCA1 and both BRCA2 (p < 0.001) and p53 (p < 0.001). LOH at all 4 loci was significantly associated with a high degree of nuclear pleomorphism. Tumours with LOH of BRCA1 also had high mitotic indices, few tubules and a paucity of DCIS, all of which are morphological features similar to those described for familial cases. Following Bonferroni's correction for multiple tests, we found that the tumours with LOH of BRCA1 were still significantly associated with a high mitotic index (p = 0.0006) and a high degree of nuclear pleomorphism (p = 0.001).

Loss of heterozygosity of BRCA1, TP53 and TCRD markers analysed in sporadic endometrial cancer

Genetic alterations of tumour suppressor genes, for which loss of heterozygosity (LOH) is one mechanism of gene inactivation, are important steps in the development of endometrial cancer. To investigate the clinical relevance of LOH of BRCA1 (17q21), TP53 (17p13) and TCRD (14q11) in endometrial cancer, polymerase chain reaction (PCR)-based fluorescent DNA technology for the detection of microsatellite polymorphisms was applied. One hundred and thirteen archival endometrial cancer samples with matched normal tissues were examined. Allele loss at three loci were correlated with age, tumour size, lymph node status, metastases, stage, histological types, grade, expression of oestrogen receptor (ER) and progesterone receptor (PgR), family history of cancer, previous history of cancer or precursor lesions, and previous history of hormone replacement therapy (HRT). LOH for BRCA1 was detected in 18.1%, of TP53 in 26.9%, and of TCRD in 26.3% of informative cases. LOH of BRCA1 correlated with medium grade, positive ER status, and family history of cancer; LOH of TP53 correlated with younger age, high grade, positive PgR status, and with tumours from patients without HRT; LOH of TCRD correlated only with family history of cancer. LOH at all three loci correlated only with grade and positive family history. Allele loss of one of the three tumour suppressor loci did not correlate with disease-free survival (DFS), but LOH of BRCA1 correlated significantly with decreased overall survival (OS). The latter, together with the correlation of LOH of BRCA1 locus with steroid hormone receptor expression, might give a hint to the potential involvement of the co-localised 17β-hydroxysteroid dehydrogenase (HSD) gene in the development of endometrial cancer.

Loss of heterozygosity of BRCA1, BRCA2 and ATM genes in sporadic invasive ductal breast carcinoma.

The present study was undertaken to analyse the loss of heterozygosity (LOH) of the three genes, BRCA1, BRCA2 and ATM, and their correlation to clinicopathological parameters in sporadic breast cancer. We studied 59 sets of invasive ductal carcinoma, compared to matched normal control DNA. Microsatellite markers intragenic to BRCA1 (D17S1323, D17S1322, D17S855), BRCA2 (D13S1699, D13S1701, D13S1695) and ATM (D11S2179) were simultaneously used. In addition, one marker telomeric to BRCA2 (D13S1694) and four markers flanking ATM were analysed (D11S1816, D11S1819, D11S1294, D11S1818). Thirty-one per cent of the informative cases showed loss of heterozygosity for the BRCA1 gene, 22.8% for BRCA2 gene and 40% for ATM. LOH of BRCA1 correlated with high grade tumors (p=0.0005) and negative hormone receptors (p=0.01). LOH of ATM correlated with higher grade (p=0.03) and a younger age at diagnosis (p=0.03) in our set of tumors. No correlations were detected between BRCA2 LOH and any of the analysed clinicopathological parameters. However, a correlation was detected between allelic loss of the D13S1694 marker, telomeric to BRCA2, and larger tumor sizes and negative estrogen receptors, favoring the hypothesis of the presence of another putative tumor suppressor gene, telomeric to BRCA2, in the 13q12-q14 region. Only 11 tumors had LOH at more than one of the three genes, most of them (6/11) associated LOH of BRCA1 and ATM. One tumor only combined loss of the three genes BRCA1, BRCA2 and ATM.

Clinical impact of detection of loss of heterozygosity of BRCA1 and BRCA2 markers in sporadic breast cancer.

The development of familial and sporadic breast cancer is based on genetic alterations of tumour-suppressor genes, for which loss of heterozygosity (LOH) is one mechanism of gene inactivation. To investigate LOH of BRCA1 (17q21) and BRCA2 (13-q12-13) in sporadic breast cancer, polymerase chain reaction (PCR)-based fluorescent DNA technology for detection of microsatellite polymorphisms was applied. A total of 137 breast cancer and 15 benign breast specimens with matched normal tissue were examined. Fluorescent-labelled PCR products were analysed in an automated DNA sequencer (ALFTM Pharmacia). Losses at both loci were correlated with different histological types, age, tumour size, lymph node status, grading and steroid hormone receptor expression, [SHR: oestrogen receptor (ER), progesterone receptor (PgR)]. For BRCA1 (D17S855, THRA1, D17S579) losses could be detected in invasive ductal carcinoma (IDC; n = 108) in 32-38%, invasive lobular carcinoma (ILC; n = 19) in 21-42% depending on the marker applied, but not in benign breast tumours (n = 15). Losses of BRCA1 markers correlated with larger tumour size, higher grade, and PgR expression. For BRCA2 (D13S260, D13S267, D13S171) losses could be detected in 108 IDCs in 30-38%, in 19 ILCs in 17-39% depending on the marker applied, but not in benign breast tumours. Losses of BRCA2 markers correlated only with higher grade. Microsatellite analyses combined with detection of fluorescent-labelled PCR products by an automated laser DNA sequencer can be used for routine determination of LOH. In sporadic breast cancer, LOH of BRCA1 of BRCA2 does not add decisive prognostic value as stated for familial breast cancer.