High-density array-CGH with targeted NGS unmask multiple noncontiguous minute deletions on chromosome 3p21 in mesothelioma

Abstract

We used a custom-made comparative genomic hybridization array (aCGH; average probe interval 254 bp) to screen 33 malignant mesothelioma (MM) biopsies for somatic copy number loss throughout the 3p21 region (10.7 Mb) that harbors 251 genes, including BRCA1 (breast cancer 1)-associated protein 1 (BAP1), the most commonly mutated gene in MM. We identified frequent minute biallelic deletions (<3 kb) in 46 of 251 genes: four were cancer-associated genes: SETD2 (SET domain-containing protein 2) (7 of 33), BAP1 (8 of 33), PBRM1 (polybromo 1) (3 of 33), and SMARCC1 (switch/sucrose nonfermentable-SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily c, member 1) (2 of 33). These four genes were further investigated by targeted next-generation sequencing (tNGS), which revealed sequence-level mutations causing biallelic inactivation. Combined high-density aCGH and tNGS revealed biallelic gene inactivation in SETD2 (9 of 33, 27%), BAP1 (16 of 33, 48%), PBRM1 (5 of 33, 15%), and SMARCC1 (2 of 33, 6%). The incidence of genetic alterations detected is much higher than reported in the literature because minute deletions are not detected by NGS or commercial aCGH. Many of these minute deletions were not contiguous, but rather alternated with segments showing oscillating copy number changes along the 3p21 region. In summary, we found that in MM: (i) multiple minute simultaneous biallelic deletions are frequent in chromosome 3p21, where they occur as distinct events involving multiple genes; (ii) in addition to BAP1, mutations of SETD2, PBRM1, and SMARCC1 are frequent in MM; and (iii) our results suggest that high-density aCGH combined with tNGS provides a more precise estimate of the frequency and types of genes inactivated in human cancer than approaches based exclusively on NGS strategy.

Custom Array Comparative Genomic Hybridization: the Importance of DNA Quality, an Expert Eye, and Variant Validation

“…A small number of other studies mention the presence of false positive and false negative results detected, for instance, by the use of different aberration detection methods [24], or the finding of possible aberration missed at aCGH when reanalyzing the results by Next Generation Sequencing (NGS) [26]. However, in our opinion, the presence of false and positive results is not addressed enough in the results reported in the literature.…”

“…More recently, the growing use of custom arrays, which are based on libraries of validated synthetic probes that can interrogate relevant genomic regions, have further enhanced the resolution capabilities of targeted regions [22,23,24,25,26]. In addition, single nucleotide polymorphism (SNP) arrays have been exploited to search for CNVs: not feasible for single-exon resolution throughout the genome, they nonetheless have the advantage to provide genotypes and detect regions of absence of heterozygosity (AOH) thus also allowing the identification of uniparental isodisomies (UPD) and genetic identity by descent [27].…”

“…Validation on a large number of patient and control samples following aCGH analysis is not practical, and it is not always reported in large screenings [33,34], or only few interesting candidates are validated [26]. …”

BAP1 Loss Predicts Therapeutic Vulnerability in Malignant Peritoneal Mesothelioma

“…As mentioned in the previous section, the mutation analysis also predicted that despite mutation in BAP1 in these three tumors, the entire BAP1 amino-acid chain is still intact and may be functionally active (Additional file 1: Figure S7). Furthermore, we found DNA copy loss of 3p21 locus to include four concomitantly deleted cancer genes -BAP1, SETD2, SMARCC1, and PBRM1, consistent with [20]. Copy-number status of these four genes was significantly correlated with their corresponding mRNA expression (Additional file 1: Figure S12), suggesting that the allelic loss of these genes is associated with decreased transcript levels.…”

Targeting the epigenome in malignant pleural mesothelioma

“…Recent advances in "omics" technologies have enabled comprehensive gene and transcriptome analyses that have provided considerable insight regarding the pathogenesis, prognosis, and treatment of MPM (2,3). Presently, less information is available regarding mechanisms and clinical relevance of epigenetic derangements in MPM (4,5).…”