BRCA Methylation Testing Identifies a Subset of Ovarian Carcinomas without Germline Variants That Can Benefit from PARP Inhibitor.

Nora Sahnane,Eleonora Di Lauro,Maria Grazia Tibiletti,Sofia Facchi,Fausto Sessa,Giorgio Formenti,Annamaria Salvati,Ileana Carnevali,Domenico Memoli,Jvan Casarin,Francesca Rizzo,Raffaella Bombelli

doi:10.3390/ijms21249708

Abstract

Homologous Recombination Deficiency (HRD) is a frequent feature of high-grade epithelial ovarian carcinoma (EOC), associated with sensitivity to PARP-inhibitors (PARPi). The best characterized causes of HRD in EOCs are germline or somatic mutations in BRCA1 and BRCA2 genes. Although promoter methylation is a well-known mechanism of gene transcriptional repression, few data have been published about BRCA gene methylation in EOCs. In this retrospective study, we quantitatively analyzed by pyrosequencing a selected series of 90 formalin-fixed (FFPE) primary EOCs without BRCA germline mutations. We identified 20/88 (22.7%) EOCs showing BRCA promoter methylation, including 17/88 (19.3%) in BRCA1 and 4/86 (4.6%) in BRCA2 promoters, one of which showing concomitant BRCA1 methylation. Mean methylation levels were 49.6% and 45.8% for BRCA1 and BRCA2, respectively, with methylation levels ≥50% in 10/20 methylated EOCs. Constitutive BRCA methylation was excluded by testing blood-derived DNA. In conclusion, pyrosequencing methylation analysis of BRCA genes is a robust, quantitative and sensitive assay applicable to FFPE samples. Remarkably, a considerable subset of germline BRCA-negative EOCs showed somatic methylation and, likely, HRD. A subpopulation of women with BRCA methylation, even without BRCA mutations, could potentially benefit from PARP-inhibitors; further clinical studies are needed to clarify the predictive role of somatic BRCA methylation of PARP-therapy response.

Highlights

Epithelial ovarian carcinoma (EOC) is the most lethal gynecologic malignancy, and the most frequent cause of cancer-related mortality in women in the world [1]
A promising novel therapy for epithelial ovarian carcinoma (EOC) is based on the inhibition of poly(ADP-ribose) polymerase (PARP), which is synthetically lethal in cancer cells with acquired inactivation of the homologous recombination-mediated repair (HR) pathway [2]
It is supposed that HR deficiency can arise through germline and somatic mutations of a wider set of homologous recombination repair related genes [3,4,5], the well described causes of HR deficiency in EOC are germline or somatic mutations in the BRCA1 and BRCA2 genes that are detected in 12–15% and 5–7% of cases, respectively [6]

Summary

Introduction

Epithelial ovarian carcinoma (EOC) is the most lethal gynecologic malignancy, and the most frequent cause of cancer-related mortality in women in the world [1]. The impact of germline BRCA gene deleterious mutations on PARPi and platinum responses in EOC is well established, the clinical relevance of BRCA promoter methylation is still unknown [7,8,9,10,11]. Mainly due to wide concerns regarding the analytic validity of the published studies, the 2020 ESMO recommendation [16] clearly claimed that currently there isn’t enough evidence to determine the clinical validity of BRCA1 promoter methylation yet, and no datum is available for BRCA2 gene. A new national universal tumor BRCA1/2 workflow was approved [17] to support treatment choice, no strategy is available on the proper handling of BRCA hypermethylated cases with respect to PARPi therapy. We analyzed BRCA1 and BRCA2 promoter methylation in a series of 90 FFPE EOC, selected for the absence of germline BRCA1/2 pathogenetic variants, using pyrosequencing analysis to quantitatively detect BRCA methylation

Results

Patients and Samples

BRCA1 and BRCA2 Gene Promoter Methylation Test