The performance of BRCA1 immunohistochemistry for detecting germline, somatic, and epigenetic BRCA1 loss in high-grade serous ovarian cancer.

BACKGROUND: BRCA1 expression can be lost by a variety of mechanisms including germline or somatic mutation and promotor hypermethylation. Given the potential importance of BRCA1 loss as a predictive and prognostic biomarker in high-grade serous ovarian cancer, we sought to evaluate the utility of BRCA1 immunohistochemistry (IHC) in screening for BRCA1 loss by germline, somatic, and epigenetic mechanisms. PATIENTS AND METHODS: Patients with advanced high-grade serous ovarian cancer who had previously undergone germline BRCA1 testing were identified. Samples from each tumor were stained for BRCA1 and reviewed independently by two pathologists blinded to BRCA status. Tumors with abnormal BRCA1 IHC and wild-type germline testing underwent further evaluation for somatic BRCA1 mutations and promoter hypermethylation. McNemar's test was used to determine the association of BRCA1 IHC with germline BRCA1 mutations and BRCA1 loss through any mechanism. Kaplan-Meier methods were used to estimate overall survival (OS), and the log-rank test was used to assess differences between groups. RESULTS: Inter-rater reliability between the two pathologists on BRCA IHC interpretation was very good (kappa coefficient 0.865, P = 0.16; McNemar's test). BRCA1 IHC was abnormal in 36% (48/135) of cases. When compared with germline BRCA1 status, BRCA1 IHC had a high negative predictive value (95.4%) but a low positive predictive value (PPV, 52.1%). When accounting for promoter hypermethylation and somatic mutations as alternative methods of BRCA1 loss, the PPV rose to 87.5%. Five-year OS rate was 49.6% [95% confidence interval (CI) 26.3% to 69.3%] for patients with germline BRCA1 mutations, 50.4% (95% CI 27.5% to 69.5%) for germline wild-type BRCA1 and abnormal IHC, and 52.1% (95% CI 38.4% to 64.2%) for germline wild-type BRCA1 and normal IHC (P = 0.92). CONCLUSIONS: BRCA1 IHC interpretation was a highly reproducible and accurate modality for detecting germline, somatic, or epigenetic mechanisms of BRCA1 loss. These results support further development of BRCA1 IHC as a potential biomarker for BRCA1 loss in high-grade serous ovarian cancer.

Molecular analysis of the INK4A and INK4B gene loci in human breast cancer cell lines and primary carcinomas.

The INK4A and INK4B loci are located at 9p21 and have been implicated in the tumorigenesis of various human malignancies. The INK4A gene encodes two cell cycle regulators, p16(INK4A) and ARF, while INK4B encodes p15(INK4B). Previously, we have shown that the p16(INK4) tumor suppressor was not mutated or deleted in primary breast carcinomas. However, primary and metastatic breast carcinomas exhibited a relative hypomethylation of p16(INK4A), which is associated with expression, compared to normal breast tissue. The present study was conducted to determine if inactivation of p15(INK4B) and INK4A exon 1beta (ARF) are common events in breast carcinoma. Mutational analysis was performed by PCR-SSCP, and mRNA expression was evaluated by RT-PCR. Methylation-specific PCR was used to determine the methylation status of the p15(INK4B) promoter. Our results demonstrate that the p15(INK4B) gene was altered in 3 (21%) of the 14 breast cell lines; one had a silent mutation and two had homozygous deletion of the gene. None of the cell lines showed methylation of p15(INK4B). Two (14%) cell lines had homozygous deletion of INK4A exon 1beta. All normal and malignant breast tissue samples were wild-type and non-methylated for p15(INK4B) and wild-type for exon 1beta. Our results show that these structurally and functionally related genes are not invariably affected together, and the most frequently observed alteration at the INK4A and INK4B loci in breast carcinoma appears to be p16(INK4A) hypomethylation.

Genetic alterations of the p14ARF -hdm2-p53 regulatory pathway in breast carcinoma.

TP53 is the most commonly mutated tumor suppressor gene in human cancers. The amplification and overexpression of HDM2 plays a role in tumorigenesis via inactivation of p53-dependent cell cycle arrest. p14ARF, an alternate transcript of the INK4A tumor suppressor locus, prevents hdm2-induced transcriptional silencing of p53 by binding hdm2. The role of this p14ARF-hdm2-p53 regulatory pathway in breast carcinoma is unknown. We hypothesized that p14ARF mutations and HDM2 gene amplification may be alternative mechanisms of p53 inactivation in breast cancer. Mutational analysis of TP53 (exons 5-9) and exon 1beta of pl4ARF was performed by PCR-SSCP and putative mutations were confirmed by sequencing. p14ARF mRNA expression was evaluated by RT-PCR and the presence of HDM2 gene amplification by differential PCR. Among the cell lines, 7/14 (50%) harbored TP53 mutations and 2/14 (14%) had a deletion ofp14ARF exon 1beta with no detectable p14ARF mRNA. None demonstrated HDM2 gene amplification. TP53 mutations were identified in 7/36 (19%) breast tumors and HDM2 amplification in 2/30 (7%) tumors. All the tumors contained an intact p14ARF exon 1beta with corresponding expression of the mRNA. Alterations in the various components of this regulatory pathway were identified in nine (64%) cell lines and 25% of the 36 breast cancers with TP53 mutation being the predominant aberration. Although p14ARF mutations and HDM2 gene amplification appear to be uncommon events in breast carcinoma, deregulation of this pathway may occur via alternative mechanisms in breast carcinogenesis.

Expression of E2F-1 and E2F-4 is reduced in primary and metastatic breast carcinomas.

The E2F family of transcription factors can induce both cell proliferation and apoptosis. Whether they function as oncogenes or tumor suppressors appears to be tissue specific. Their role in breast carcinogenesis remains unclear. We found a decreased expression of E2F-1 and E2F-4 in 70% (7/10) of primary breast carcinomas and in all (10/10) metastatic nodal tissues when compared with the corresponding normal breast tissue. No tumor-specific mutation was detected, but polymorphisms were identified in E2F-1 exon 5 and in the polyserine tract of E2F-4. The presence of polymorphisms did not correlate with E2F expression. Among the 12 human breast cancer cell lines, one contained a missense mutation in E2F-1 exon 2. Five (42%) cell lines overexpressed E2F-1, while three (25%) expressed low levels of the protein. Our results suggest that not only are the E2Fs likely to function as tumor suppressors in breast cancer, but also that their down-regulation may be important in the development of metastases.

In microdissected ductal carcinoma in situ, HER-2/neu amplification, but not p53 mutation, is associated with high nuclear grade and comedo histology.

BACKGROUND: HER-2/neu and p53 are two molecular markers that have been the focus of investigation in patients with invasive breast carcinoma. However, most of the published data have relied on immunohistochemical detection of the proteins as a surrogate marker of the underlying genetic alterations, a detection method that often gives variable results due to technical factors. In addition, there are limited data documenting HER-2/neu amplification and p53 mutations in the various histologic subtypes of ductal carcinoma in situ (DCIS). The authors evaluated a series of microdissected, pure DCIS lesions comprising a spectrum of morphologic subtypes (comedo, micropapillary, papillary, cribriform, and solid) and their corresponding normal breast tissue for genetic aberrations in HER-2/neu and p53. METHODS: HER-2/neu amplification was determined by differential polymerase chain reaction, and p53 mutations were identified by single-strand conformation polymorphism analysis. RESULTS: HER-2/neu amplification was identified in 12 of 30 DCIS samples (40%), and p53 mutations were identified in 6 of 30 DCIS samples (20%). The genetic alterations were not present in any of the normal breast tissue samples. HER-2/neu amplification occurred predominantly in the comedo subtype (69% vs. 18% of the noncomedo subtype; P = 0.008) and in lesions of high nuclear grade (63% vs. 14% of low grade; P = 0.01). There was no difference in the frequency of p53 mutations among the subtypes or between low grade and high grade lesions. No correlation between the presence of the two genetic alterations was observed. CONCLUSIONS: The presence of HER-2/neu amplification, but not p53 mutations, correlates with histologic subtype and nuclear grade. The relatively frequent occurrence of HER-2/neu amplification and p53 mutations in DCIS tissue and their absence in normal breast tissue suggest that these genetic aberrations are important early in breast duct carcinogenesis.

Hypomethylation and increased gene expression of p16INK4a in primary and metastatic breast carcinoma as compared to normal breast tissue.

Controversy continues to surround the role of p16INK4a in cell cycle control and carcinogenesis. Mutations, deletions and changes in methylation patterns of p16INK4a have been proposed as mechanisms leading to abnormal expression of the gene. We show here that primary and metastatic breast carcinomas demonstrate hypomethylation of p16INK4a which is associated with expression of p16INK4a mRNA, as compared to normal breast tissue which demonstrates a relative hypermethylation of p16INK4a associated with the absence of p16INK4a expression. These data suggest that methylation and lack of expression of p16INK4a is not a central mechanism in the development of breast carcinoma, but rather that the gene is functioning and expressed in breast carcinoma more frequently than in normal breast tissue. The role of p16INK4a is much more complex than has been previously hypothesized.