Ovarian carcinomas with genetic and epigenetic BRCA1 loss have distinct molecular abnormalities - PubMed (original) (raw)

doi: 10.1186/1471-2407-8-17.

Alessandro De Luca, Niki Boyd, Sean Young, Armelle Troussard, Yolanda Ridge, Pardeep Kaurah, Steve E Kalloger, Katherine A Blood, Margaret Smith, Paul T Spellman, Yuker Wang, Dianne M Miller, Doug Horsman, Malek Faham, C Blake Gilks, Joe Gray, David G Huntsman

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Ovarian carcinomas with genetic and epigenetic BRCA1 loss have distinct molecular abnormalities

Joshua Z Press et al. BMC Cancer. 2008.

Abstract

Background: Subclassification of ovarian carcinomas can be used to guide treatment and determine prognosis. Germline and somatic mutations, loss of heterozygosity (LOH), and epigenetic events such as promoter hypermethylation can lead to decreased expression of BRCA1/2 in ovarian cancers. The mechanism of BRCA1/2 loss is a potential method of subclassifying high grade serous carcinomas.

Methods: A consecutive series of 49 ovarian cancers was assessed for mutations status of BRCA1 and BRCA2, LOH at the BRCA1 and BRCA2 loci, methylation of the BRCA1 promoter, BRCA1, BRCA2, PTEN, and PIK3CA transcript levels, PIK3CA gene copy number, and BRCA1, p21, p53, and WT-1 immunohistochemistry.

Results: Eighteen (37%) of the ovarian carcinomas had germline or somatic BRCA1 mutations, or epigenetic loss of BRCA1. All of these tumours were high-grade serous or undifferentiated type. None of the endometrioid (n = 5), clear cell (n = 4), or low grade serous (n = 2) carcinomas showed loss of BRCA1, whereas 47% of the 38 high-grade serous or undifferentiated carcinomas had loss of BRCA1. It was possible to distinguish high grade serous carcinomas with BRCA1 mutations from those with epigenetic BRCA1 loss: tumours with BRCA1 mutations typically had decreased PTEN mRNA levels while those with epigenetic loss of BRCA1 had copy number gain of PIK3CA. Overexpression of p53 with loss of p21 expression occurred significantly more frequently in high grade serous carcinomas with epigenetic loss of BRCA1, compared to high grade serous tumors without loss of BRCA1.

Conclusion: High grade serous carcinomas can be subclassified into three groups: BRCA1 loss (genetic), BRCA1 loss (epigenetic), and no BRCA1 loss. Tumors in these groups show distinct molecular alterations involving the PI3K/AKT and p53 pathways.

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Figures

Figure 1

Figure 1

Assessment of BRCA1 loss (A) Mutation screening showing the abnormal denaturing high performance liquid chromatography profile corresponding to the 1351delAT mutation in tumor 223. The single blue line represents the electropherogram from a normal control, while the purple line represents the abnormal profile formed by the mutated exon 11c in tumor 223. (B) Direct DNA sequencing demonstrating the 185delAG mutation in tumor 283. Only the mutant allele is seen in the tumor because LOH is present. (C-E) Loss of heterozygosity (LOH) analysis using BRCA1-associated microsatellite markers visualized on an ABI Prism 3100 Genetic Analyzer, where LOH is defined as >50% decrease in area under the curve when germline DNA (upper tracing) and tumor DNA (lower tracing) are compared. (C) The lack of LOH in tumor 240 demonstrated using microsatellite marker D17S1185, (D) LOH in tumor 283 demonstrated using microsatellite marker D17S855. (E) Microsatellite instability demonstrated in tumor 156 using microsatellite marker D17S1185. (F, G, H, and I) Methylation analysis of BRCA1 gene using fluorescence-based, quantitative, real-time PCR (TaqMan) using SYBR Green 1 as detection method. Two sets of primers, designed specifically for bisulfite converted DNA, were used: a methylated set for the BRCA1 gene and a reference set (MYOD1) to control for input DNA. Specificity of the reactions for methylated DNA were confirmed separately using human genomic DNA (unmethyated; F) and CpG methylated Jurkat genomic DNA (methylated; G), respectively. H and I show representative examples of results from assessment of BRCA1 loss through promoter hypermethyation. Tumor 178 shows only unmethylated BRCA1 promoter, while tumor 345 shows evidence of BRCA1 promoter hypermethylation.

Figure 2

Figure 2

Summary of BRCA1 abnormalities and associated features: Pathology refers to the tumor histopathology. Serous or Ser = serous carcinoma; Undiff = undifferentiated carcinoma; HG = high-grade; LG = low-grade; Clear cell = clear cell carcinoma; Endo = endometrioid carcinoma; G1 = grade 1; G2 = grade 2; G3 = grade 3. BRCA1 Status: Mut = mutation; G = germline; S = somatic; N = no mutations. LOH = loss of heterozygosity where LOH indicates that loss of heterozygosity is present, NO indicates that loss of heterozygosity is not present, and MSI indicates that microsatellite instability is present in the tumor. Meth refers to BRCA1 promoter hypermethylation. Tumors containing ≥ 4% fully methylated molecules are designated as methylated (M) and are highlighted in orange, whereas tumors containing < 4% are designated as unmethylated (U). RNA refers to relative RNA expression compared to the average RNA expression in all samples, where the average RNA expression = 1.0. Tumors with relative RNA expression <0.7 are highlighted in aqua as showing BRCA1/BRCA2 loss. IHC refers to BRCA1 immunohistochemistry; (+) indicates tumors with > 1% of nuclei stained positive for BRCA1, (-) indicates tumors with <1% of nuclei positive. N/A indicates that the data is not available for technical reasons.

Figure 3

Figure 3

Summary of analysis of high grade (HG) serous/undifferentiated ovarian tumors: MIP copy number results are shown for c-myc and PIK3CA loci. MIP copy number values over 3.0 are highlighted and correspond to amplification. Relative mRNA levels for PIK3CA and PTEN were assessed using qRT-PCR; levels over 1.3 (highlighted in green) are considered elevated and levels below 0.7 (highlighted in aqua) indicate decreased transcript levels. Associated immunohistochemical markers p21, p53, and WT-1 refer to immunohistochemical staining results. Scoring of immunostaining was done as follows: p21: 0 = <5% nuclei positive and 1 = >5% of nuclei positive. p53: 0 = <50% nuclei positive and 1 = >50% of nuclei positive. WT1: 0 = <5% nuclei positive, 1 = 5–50% nuclei positive, and 2 = >50% nuclei positive. N/A indicates that the data is not available for technical reasons.

Figure 4

Figure 4

Correlation between decreased PTEN mRNA levels and amplification at the PIK3CA locus: Relative PTEN mRNA levels as determined by qRT-PCR are plotted along the X-axis and PIK3CA MIP copy number results are plotted along the Y-axis for high grade serous ovarian tumors with BRCA1 mutations (open circles) and high grade serous ovarian tumors with epigenetic loss of BRCA1 (filled triangles). MIP copy number values over 3.0 indicate amplification.

Figure 5

Figure 5

Immunohistochemistry results: Representative immunohistochemistry results for high grade serous ovarian tumors with BRCA1 mutations (tumor #327, top row), with epigenetic loss of BRCA1 (tumor #332, middle row), and without loss of BRCA1 (tumor #372, bottom row). Immunohistochemical staining is shown for BRCA1 (left column), p53 (middle column) and p21 (right column).

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