High Incidence of Somatic BAP1 alterations in sporadic malignant mesothelioma

Abstract

Background: Breast cancer 1-associated protein 1 (BAP1) is a nuclear deubiquitinase that regulates gene expression, transcription, DNA repair, and more. Several findings underscore the apparent driver role of BAP1 in malignant mesothelioma (MM). However, the reported frequency of somatic BAP1 mutations in MM varies considerably, a discrepancy that appeared related to either methodological or ethnical differences across various studies.

Methods: To address this discrepancy, we carried out comprehensive genomic and immunohistochemical (IHC) analyses to detect somatic BAP1 gene alterations in 22 frozen MM biopsies from U.S. MM patients.

Results: By combining Sanger sequencing, multiplex ligation-dependent probe amplification, copy number analysis, and cDNA sequencing, we found alteration of BAP1 in 14 of 22 biopsies (63.6%). No changes in methylation were observed. IHC revealed normal nuclear BAP1 staining in the eight MM containing wild-type BAP1, whereas no nuclear staining was detected in the 14 MM biopsies containing tumor cells with mutated BAP1. Thus, IHC results were in agreement with those obtained by genomic analyses. We then extended IHC analysis to an independent cohort of 70 MM biopsies, of which there was insufficient material to perform molecular studies. IHC revealed loss of BAP1 nuclear staining in 47 of these 70 MM biopsies (67.1%).

Conclusions: Our findings conclusively establish BAP1 as the most commonly mutated gene in MM, regardless of ethnic background or other clinical characteristics. Our data point to IHC as the most accessible and reliable technique to detect BAP1 status in MM biopsies.

Figure 1. Point mutations of BAP1 genes detected by Sanger sequencing

(A) Tumor NYU524. Genomic DNA sequencing shows point mutation (G → T) at the splicing site at the end of intron 14. (B) cDNA sequencing revealed a 70 nt insertion from part of intron 14 (c.1891_1892Ins 70, p.Glu630fsX23). (C) Tumor NYU1024. Genomic sequencing displayed G insertion in exon 14. (c.1762_1763 Ins G) (D) cDNA sequence of the corresponding region, (p.Pro588fsx55).

Figure 2. Detection of BAP1 protein expression in sporadic MM tumor samples by immunohistochemistry

Wild-type BAP1 tumor sample NYU047 shows strong nuclear staining in all tumor cells. All samples carrying alteration of BAP1 gene are shown (Photomicrographs magnification: 400x).

Figure 3. Gross BAP1 gene alterations detected by MLPA and TaqMan copy number assays

MLPA assay is measured at 17 points, one each on 17 exons of BAP1 gene. TaqMan copy number assay is measured at six points on BAP1 gene; one each on exon 2, 3, 6, 12, 14 & 17. (A, B) NYU540 MLPA and TaqMan data (C, D) NYU559 MLPA and TaqMan data (E, F) NYU1419 MLPA and TaqMan data (G, H) NYU207 MLPA and TaqMan data.

Figure 4. Detection of aberrant forms of BAP1 by MLPA and cDNA sequencing

(A) Tumor NYU851. MLPA assay detected deletions of exons 1, 4, and 15. (B) NYU851 cDNA sequencing revealed two aberrant splicing at Exon 8–9 and Exon 11–17 in one allele. (C) MLPA on NYU966 showed deletion of exon 1 and 4. (D) Tumor NYU966 cDNA sequencing revealed a cDNA with skipping of most of exon 12, 13, 14, and 6 bp of exon 15 as well as a wild type cDNA (exons 14 and 15 shown). (E). MLPA on NYU1306 detected triplication, copy number increase to 3 copies, in most exons. (F) NYU1306 cDNA sequencing revealed a deletion of exon 12 and 13. (G). NYU1250 cDNA sequencing revealed a new splicing isoform of BAP1 due to a 121bp deletion in exon 13. (H) Aberrant usage of criptic splicing site AG, found in NYU1250, is shown by arrow on reference DNA sequence at intron 12 and exon 13. Nucleotides in green indicate the exon sequence.

Figure 5. BAP1 promoter and body methylation analysis

(A) Sample NYU047 (B) Sample NYU524. Low BAP1 promoter methylation was detected by pyrosequencing in all sporadic MM samples tested. Pyrosequencing values detected in MM tumor samples are as low as the normal control DNA. Two representative samples are shown, where yellow highlights indicate the percentage of methylation at each site. (C) The Cancer Genome Atlas portal database (https://tcga-data.nci.nih.gov/tcga/) was examined for methylation at BAP1 gene, which was covered by nineteen probes (data generated by use of the Infinium HumanMethylation450 BeadChip).