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High Incidence of Somatic BAP1 Alterations in Sporadic Malignant Mesothelioma

      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.

      Key Words

      Malignant mesothelioma (MM) is an aggressive tumor that arises from the mesothelial cells that form the lining of the pleural, pericardial, and peritoneal cavities.
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      Germline BAP1 mutations predispose to malignant mesothelioma.
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      renal cell carcinoma,
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      cholangiocarcinoma,
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      basal cell carcinoma,
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      and possibly more.
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      Germline BAP1 mutations predispose to malignant mesothelioma.
      ,
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      BAP1 is a nuclear deubiquitinase, which belongs to the ubiquitin C-terminal hydrolase family.
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      Among ubiquitin C-terminal hydrolase family members, BAP1 is unique because of its long C-terminal tail, which contains two nuclear localization signals (NLS1 at 656–661 and NLS2 at 717–722).
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      BRCA1-associated protein-1 is a tumor suppressor that requires deubiquitinating activity and nuclear localization.
      It has been postulated that BAP1 functions as a tumor suppressor,
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      possibly through three or more mechanisms, all requiring the nuclear localization of the BAP1 protein: (1) as component of the Polycomb repressive deubiquitinase complex, BAP1 deubiquitinates histone H2A, leading to transcriptional activation of genes that regulates cell growth
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      ; (2) BAP1 acts as a transcription coregulator, associating with host cell factor-1, YY1, and E2F1 to induce transcription of genes involved in cell cycle regulation
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      The ubiquitin carboxyl hydrolase BAP1 forms a ternary complex with YY1 and HCF-1 and is a critical regulator of gene expression.
      ; and (3) BAP1 contributes to the process of DNA repair.
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      Germline mutations in BAP1 impair its function in DNA double-strand break repair.
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      Tumor suppressor and deubiquitinase BAP1 promotes DNA double-strand break repair.
      Whether BAP1 also has some presently unknown cytoplasmic activity remains unknown.
      Somatic BAP1 mutations are found in sporadic MM (i.e., MMs that occur in individuals that do not carry germline BAP1 mutations), as well as in other malignancies, although the frequency of BAP1 mutations varies widely across different tumor types. Pena-Llopis et al.
      • Peña-Llopis S
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      • Liao A
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      BAP1 loss defines a new class of renal cell carcinoma.
      detected somatic BAP1 mutations in 14% of renal cell carcinoma specimens (24 of 176 tumors) using Sanger and whole-genome sequencing, whereas Harbour et al.
      • Harbour JW
      • Onken MD
      • Roberson ED
      • et al.
      Frequent mutation of BAP1 in metastasizing uveal melanomas.
      detected mutations in 84% of metastasizing uveal melanoma biopsies (26 of 31) using next-generation sequencing (NGS). In sporadic MM, by using Sanger sequencing, we found somatic BAP1 mutations in 22% of U.S. Caucasian MM biopsies (4 of 18).
      • Testa JR
      • Cheung M
      • Pei J
      • et al.
      Germline BAP1 mutations predispose to malignant mesothelioma.
      These results are in accordance with the results of Bott et al.
      • Bott M
      • Brevet M
      • Taylor BS
      • et al.
      The nuclear deubiquitinase BAP1 is commonly inactivated by somatic mutations and 3p21.1 losses in malignant pleural mesothelioma.
      and Zauderer et al.,
      • Zauderer MG
      • Bott M
      • McMillan R
      • et al.
      Clinical characteristics of patients with malignant pleural mesothelioma harboring somatic BAP1 mutations.
      who found 23% (12 of 53) and 20% (24 of 121) BAP1 mutations in sporadic U.S. MM, also by Sanger sequencing. We described loss of BAP1 nuclear staining in MM tumor biopsies containing mutated BAP1.
      • Testa JR
      • Cheung M
      • Pei J
      • et al.
      Germline BAP1 mutations predispose to malignant mesothelioma.
      We and Bott et al.
      • Bott M
      • Brevet M
      • Taylor BS
      • et al.
      The nuclear deubiquitinase BAP1 is commonly inactivated by somatic mutations and 3p21.1 losses in malignant pleural mesothelioma.
      also noted loss of BAP1 nuclear staining in some MM biopsies that apparently had wild-type BAP1 according to Sanger sequencing. Arzt el al.
      • Arzt L
      • Quehenberger F
      • Halbwedl I
      • Mairinger T
      • Popper HH
      BAP1 protein is a progression factor in malignant pleural mesothelioma.
      revealed absence of BAP1 nuclear staining in 60% of 123 MM biopsies in a study that was based exclusively on immunohistochemistry (IHC). Yoshikawa et al.
      • Yoshikawa Y
      • Sato A
      • Tsujimura T
      • et al.
      Frequent inactivation of the BAP1 gene in epithelioid-type malignant mesothelioma.
      found that BAP1 was mutated in 61% of cell cultures (14 of 23) derived from pleural fluids of Japanese MM patients. In this study, Yoshikawa et al.
      • Yoshikawa Y
      • Sato A
      • Tsujimura T
      • et al.
      Frequent inactivation of the BAP1 gene in epithelioid-type malignant mesothelioma.
      performed microarray-based comparative genomic hybridization (array CGH), Sanger sequencing, and real-time polymerase chain reaction (PCR) on DNA extracted from tumor cells established in tissue culture, after several subcultures. Instead, our study
      • Testa JR
      • Cheung M
      • Pei J
      • et al.
      Germline BAP1 mutations predispose to malignant mesothelioma.
      and the studies by Bott et al.
      • Bott M
      • Brevet M
      • Taylor BS
      • et al.
      The nuclear deubiquitinase BAP1 is commonly inactivated by somatic mutations and 3p21.1 losses in malignant pleural mesothelioma.
      and Zauderer et al.
      • Zauderer MG
      • Bott M
      • McMillan R
      • et al.
      Clinical characteristics of patients with malignant pleural mesothelioma harboring somatic BAP1 mutations.
      were based on Sanger sequencing of tumor DNA from primary frozen tissue biopsies. The significant difference in frequency of BAP1 mutations reported may due to differences in ethnicities of the patients studied, lack of sensitivity and/or reproducibility of Sanger sequencing or IHC in detecting BAP1 mutations, procedure of tumor cell isolation and accumulation of novel BAP1 mutations by MM cells in tissue culture,
      • Yoshikawa Y
      • Sato A
      • Tsujimura T
      • et al.
      Frequent inactivation of the BAP1 gene in epithelioid-type malignant mesothelioma.
      lack of specificity of IHC, or some other differences in methodology. To investigate the basis of the discrepancies between these studies and to try to conclusively address this issue, we performed multiplex molecular analyses to comprehensively identify all possible genetic alterations of the BAP1 gene in sporadic MM biopsies. We found that BAP1 is mutated in more than 60% of MM specimens.

      MATERIALS AND METHODS

      Specimen Collection

      Twenty-two MM frozen biopsies and matching normal leukocytes were obtained from the New York University (NYU) Langone Medical Center (HIP). MM samples were harvested during surgery and immediately frozen in liquid nitrogen.
      An independent cohort of 70 formalin-fixed paraffin-embedded tissue slides was obtained from the National Mesothelioma Virtual Bank (NMVB) at the University of Pittsburgh. These latter specimens were used exclusively to extend BAP1 IHC analyses, as the amount of tissue available was insufficient to perform molecular studies. Specimens were de-identified before analysis. Sex, ethnicity, asbestos exposure, age at diagnosis, histology, and stage data were collected and are reported in the Supplementary Table 1A (Supplemental Digital Content 1, http://links.lww.com/JTO/A775).
      Germline BAP1 wild-type (WT) DNAs were obtained from healthy volunteers. Written and informed consent was obtained from all patients included in these studies according to the guidelines set forth by the institutional review boards.

      Laser Capture Microdissection

      Laser capture microdissection was performed to enrich tumor cells from frozen tissue specimens for DNA and RNA isolation. The detailed procedure is described in Supplementary material (Supplemental Digital Content 2, http://links.lww.com/JTO/A776).

      DNA and RNA Extraction

      DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) or QiAamp DNA Micro Kit (Qiagen 56304) as per manufacturer's instructions. Total RNA was extracted using mirVana miRNA isolation kit (Ambion, Austin, TX) as per manufacturer's instructions. Sample quality was evaluated by nanodrop. The 260/280 ratio was between 1.66 and 2.07 for DNA samples and between 1.94 and 2.15 for RNA samples.

      Real-Time PCR

      Total RNA was converted to cDNA using ImProm-II Reverse Transcription System (Promega, Madison, WI), following the manufacturer's instructions. A double-strand cDNA library was synthesized using the LD PCR kit (SMART cDNA Library Construction Kit; Clontech Laboratory, Mountain View, CA) according to the manufacturer's instructions.

      DNA and RNA Sequencing Analysis

      Conventional Sanger sequencing was performed as described.
      • Testa JR
      • Cheung M
      • Pei J
      • et al.
      Germline BAP1 mutations predispose to malignant mesothelioma.
      Genomic BAP1 PCR product sizes ranged from 560 to 670 base pairs (bp) with 100 to 150 bp overlap between primer sets. A similar primer design was employed for cDNA sequencing. Sequencing was conducted using the ABI 3730XL DNA Sequencer, at the Advanced Studies in Genomics, Proteomics and Bioinformatics facility at the University of Hawai'i at Manoa.

      Multiplex Ligation-Dependent Probe Amplification Assay

      Multiplex ligation-dependent probe amplification (MLPA) is a reliable technique extensively validated for detection of medium and large gene alterations, which cannot be detected by conventional Sanger sequencing.
      • Cerutti R
      • Sahnane N
      • Carnevali I
      • et al.
      Identification of the first case of germline duplication of BRCA1 exon 13 in an Italian family.
      ,
      • Yamada H
      • Shinmura K
      • Ito H
      • et al.
      Germline alterations in the CDH1 gene in familial gastric cancer in the Japanese population.
      MLPA assay was performed according to the manufacturer's protocol (SALSA MLPA probemix P417-B1 BAP1; MRC-Holland, Amsterdam, The Netherlands). The detailed procedure is described in Supplementary material (Supplemental Digital Content 2, http://links.lww.com/JTO/A776) and Supplementary Figure 3 (Supplemental Digital Content 3, http://links.lww.com/JTO/A777).

      Immunohistochemistry

      IHC analysis of BAP1 protein expression was performed as described using a mouse monoclonal anti-BAP1 antibody (C-4: Santa Cruz Biotechnology, Dallas, TX).
      • Testa JR
      • Cheung M
      • Pei J
      • et al.
      Germline BAP1 mutations predispose to malignant mesothelioma.
      This antibody recognizes the epitope between aa 430 and 739; therefore, it is predicted to detect BAP1 wild-type and mutant forms that retain the NLS. We had previously reported that BAP1 is expressed in normal pleural cells.
      • Carbone M
      • Ferris LK
      • Baumann F
      • et al.
      BAP1 cancer syndrome: malignant mesothelioma, uveal and cutaneous melanoma, and MBAITs.
      We extensively validated this antibody for the detection of nuclear BAP1 on a number of normal human pleural samples—all showing nuclear staining in 100% of pleural cells, as well as on MM-derived cell lines in which BAP1 gene status has been comprehensively characterized in our laboratory. Histologically normal pleura and liver biopsies from autopsies were used as positive and negative controls, respectively, for BAP1 nuclear staining. Liver biopsies were chosen because they were shown to contain negligible amounts of BAP1 (http://www.proteinatlas.org/ENSG00000163930-BAP1/tissue/liver). We confirmed that BAP1 does not stain the nuclei of liver cells.
      Two U.S. board-certified pathologists with extensive experience in MM diagnosis (MC and AP) independently reviewed all IHC results and concurred with their interpretation. The patients’ characteristics of the control materials are not known, and this information cannot be obtained due to Health Insurance Portability and Accountability Act (HIPAA) constraints.

      Promoter Methylation Analysis

      Tumor DNA was modified with sodium bisulfide by using EZ DNA methylation kit (ZYMO research, Orange, CA) according to the manufacturer's protocol. The detailed procedure is described in Supplementary material (Supplemental Digital Content 2, http://links.lww.com/JTO/A776).

      Whole Gene Methylation Analysis

      BAP1 gene body methylation analysis was performed using the Methyl Primer Express Software v1.0 (Applied Biosystems, Grand island, NY), as described in Supplementary material (Supplemental Digital Content 2, http://links.lww.com/JTO/A776).

      TaqMan Copy Number Assay

      TaqMan copy number assay was performed at the Genomics Shared Resource, University of Hawai'i Cancer Center, according to the manufacturer's instructions (Supplementary material, Supplemental Digital Content 2, http://links.lww.com/JTO/A776).

      Statistical Analysis

      We studied the relationship between BAP1 mutation status and age, sex, ethnicity, asbestos exposure, and histology using Fisher's exact test. Epithelioid MMs were compared with all other histological types; MM from patients older than 65 years were compared with all the others; patients exposed to asbestos were compared with all the others. Overall survival was estimated using the Kaplan-Meier method. Patients alive at the end of the study were censored at the time of the last available follow-up. Using the log-rank test, we compared survival between MM patients with altered BAP1 versus patients with wild-type BAP1.

      RESULTS

      Sanger Sequencing and IHC for BAP1

      Sanger sequencing was performed on DNA extracted from laser microdissected tumor cells and from matching germline DNA isolated from blood leukocytes of each of 22 MM patients. Matching germline DNA was found to contain the wild-type BAP1 gene in 22 of 22 samples. Sequencing analysis detected BAP1 alterations in a total of 6 of the 22 MM biopsies tested (27.3%, Table 1), which is in accordance with our previous findings.
      • Testa JR
      • Cheung M
      • Pei J
      • et al.
      Germline BAP1 mutations predispose to malignant mesothelioma.
      The 22 MM biopsies included 15 samples that had been tested previously by Sanger sequencing, whose DNA was re-extracted from a separate vial containing frozen tumor tissue, and seven samples not previously analyzed yet. All 22 samples were analyzed blindly to test for reproducibility. The results obtained using Sanger sequencing were very reproducible as the same four samples that had tested positive for BAP1 mutations, NYU647, NYU658, NYU866, and NYU937 previously,
      • Testa JR
      • Cheung M
      • Pei J
      • et al.
      Germline BAP1 mutations predispose to malignant mesothelioma.
      retested positive and the 11 samples that had tested negative
      • Testa JR
      • Cheung M
      • Pei J
      • et al.
      Germline BAP1 mutations predispose to malignant mesothelioma.
      retested negative (Table 1). These findings indicated that Sanger sequencing of DNA from tumor cells laser microdissected from frozen MM biopsies is very sensitive and specific in detecting at least certain types of BAP1 DNA alterations.
      TABLE 1Summary of Somatic BAP1 Alterations in Sporadic Malignant Mesotheliomas
      Sample ID2011 Nat Gen ID
      Sample ID from Testa et al.5 (Supplementary Table 1A, Supplemental Digital Content 1, http://links.lww.com/JTO/A775).
      Genomic SequencingMLPA AnalysisDNA Copy Number AssaycDNA SequencingBAP1 Nuclear LocalizationPrediction of Amino Acid ChangeComprehensive InterpretationPromoter Methylation
      NYU-047SP-024WTWNLWT(+)Normal1.7%
      NYU-207WTEx 7–13 deletionEx 7–13 deletionEx 7–13 deletion(–)p.Prol47fsX63
      Putative BAP1 mutant form may not be detected by BAP1 antibody.
      Mutant1.3%
      NYU-217SP-019WTWNLWT(+)Normal1.6%
      NYU-269SP-017WTWNLWT(+)Normal1.3%
      NYU-517SP-016WTWNLWT(+)Normal1.7%
      NYU-524Int 14 splicing site (52, 436, 887 G>T)WNL70 nucleotide insertion at Ex 14 and 15(–)p.Glu630fsX23Mutant1.5%
      NYU-540SP-025WTEntire gene deletionEntire BAP1 deletion(–)Mutant1.4%
      NYU-559SP-022WTEx 4–17 deletionEx 4–17 deletion(–)Mutant3.5%
      NYU-647SP-001Ex 11 (52, 439, 219 C deletion)WNLEx 11 C deletion(–)p.Ser341fsX21
      Putative BAP1 mutant form may not be detected by BAP1 antibody.
      Mutant1.8%
      NYU-658SP-018Ex 17 (52, 436, 398–399 CG deletion)WNLEx 17 CG deletion(–)p.Arg699fsX17Mutant1.4%
      NYU-809SP-011WTWNLWT(+)Normal1.5%
      NYU-851SP-012WTEx 1, 4, 15 deletionWTEx 11–17 deletion(–)p.Val335fsx27
      Putative BAP1 mutant form may be detected by BAP1 antibody.
      Mutant1.3%
      NYU-866SP-013Ex 16 (52, 436, 599–627 Entire deletion of other 29-bp deletion) alleleInsertion of Int 16(–)p.Ala683fsX3Mutant1.2%
      NYU-929SP-023WTWNLWT(+)Normal1.1%
      NYU-937SP-015Ex 9 (52, 440, 352 G deletion)
      Point mutations were detected in each allele.
      Ex 9 deletionEx 9 G deletion(–)p.Val234fsX15
      Putative BAP1 mutant form may not be detected by BAP1 antibody.
      Mutant1.7%
      Ex 13 (52, 437, 664 C deletion)
      Point mutations were detected in each allele.
      WNLEx 13 C deletionp.Ile499fsX72
      NYU-966WTEx 1 and 4 deletionWNLEx 12–15 deletion(–)Deletion between 375 and 633Mutant1.8%
      NYU-1017SP-009WTWNLWT(+)Normal1.5%
      NYU-1024SP-026Ex 14 (52, 437, 281- 52, 437, 282 G insertion)
      • Ex 10, 12, 14,
      • 16, and 17 deletion
      Ex 14 G insertion(–)p.Pro588fsx55Mutant2.1%
      NYU-1250WTWNLDeletion in Ex 13(–)p.Tyr418fsx113Mutant1.5%
      NYU-1306WTTriplicationIncrease of Ex 2, 4, 7, 12, 15, and 17Ex 12, 13, 14 deletion(–)p.Lys368fsx33
      Putative BAP1 mutant form may be detected by BAP1 antibody.
      Mutant1.4%
      NYU-1359WTWNLWT(+)Normal1.6%
      NYU-1419WTRandom exon deletionDecrease of Ex 2, 4, 7, 15, and 17WT(–)Mutant1.4%
      None of these point mutations and alterations were detected in germline DNA samples from these patients. Average promoter methylation % is shown. BAP1 protein nuclear expression are represented (+) positive and (–) negative. Triplication is gain of gene copy number (three copies).
      MLPA, multiplex ligation-dependent probe amplification; WT, wild-type sequence; WNL, within normal limit (duploid, two copies); 一, not tested.
      a Sample ID from Testa et al.5 (Supplementary Table 1A, Supplemental Digital Content 1, http://links.lww.com/JTO/A775).
      b Putative BAP1 mutant form may not be detected by BAP1 antibody.
      c Putative BAP1 mutant form may be detected by BAP1 antibody.
      d Point mutations were detected in each allele.
      In addition, we identified two new BAP1 mutations among the seven new MM biopsies, while five tested negative (BAP1 wild type). A point mutation (G → T) was found at the end of intron 14 on the genomic DNA of NYU524 (Fig. 1A). cDNA sequencing of this region revealed a 70 nucleotide insertion of an extra sequence from intron 14 (Fig. 1B). A single G insertion in exon 14 was detected in MM NYU1024 by sequencing both genomic DNA and cDNA (Fig. 1C). This variant encodes a presumed truncated BAP1 protein (642aa) missing the NLS as a result of the frameshift mutation (Fig. 1D). Overall, Sanger sequencing detected BAP1 mutations in 6 of 22 samples (27.3%) all predicted to result in truncated proteins lacking the NLS.
      Figure thumbnail gr1
      FIGURE 1Point 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 nucleotide 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).
      IHC showed complete lack of BAP1 nuclear staining in 100% of tumor cells of these six mutated samples. Unexpectedly, in eight additional biopsies that appeared to contain wild-type BAP1 by Sanger sequencing, no nuclear staining was detected. Overall, 14 of 22 MM biopsies (63.6%) contained tumor cells lacking BAP1 nuclear staining. Instead, the nuclei of the tumor cells of the additional eight MM biopsies carrying wild-type BAP1, as detected by Sanger sequencing, uniformly (all tumor cells) stained for BAP1 (Fig. 2).
      Figure thumbnail gr2
      FIGURE 2Detection of BAP1 protein expression in sporadic malignant mesothelioma 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, 400×).

      MLPA, TaqMan Copy Number Analysis, and cDNA Sequencing Detected a Number of Previously Unidentified Gross Alterations of BAP1 in Somatic MM

      Tumor DNA from NYU540, NYU559, NYU1419, NYU207, NYU851, NYU966, NYU1306, and NYU1250 appeared to contain wild-type BAP1 according to Sanger sequencing, while none of the tumor cells displayed nuclear BAP1 staining (Fig. 2). NYU540 tumor DNA displayed homozygous deletion of the entire BAP1 gene by MLPA analysis (average normalized dosage quotient [NDQ] of all exon = 0.387; Fig. 3A). This deletion was confirmed by TaqMan copy number analysis (Fig. 3B). NYU559 tumor DNA showed a homozygous deletion from exon 4 to exon 17 by MLPA analysis (exons 4–17 average NDQ = 0.267; Fig. 3C, D). MLPA also revealed a homozygous deletion of the BAP1 gene in NYU1419, with the lowest NDQ value of 0.27 for exon 12 (Fig. 3E). These results were confirmed by TaqMan copy number assay (Fig. 3F). A 3919-bp deletion spanning exon 7 to 13 was detected in NYU207 tumor DNA by MLPA analysis and TaqMan copy number assay (NDQ value of 0.407 at exon 12; Fig. 3G, H), which was further confirmed by genomic DNA sequencing using primers specifically designed to detect this deletion (Supplementary Figure, Supplemental Digital Content 4, http://links.lww.com/JTO/A778). cDNA sequencing (c.437_1729del 1293) revealed that this deletion would result in a putative truncated protein (210 aa) lacking the NLS.
      Figure thumbnail gr3
      FIGURE 3Gross 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 exons 2, 3, 6, 12, 14, and 17. NYU540 MLPA and TaqMan data (A, B), NYU559 MLPA and TaqMan data (C, D), NYU1419 MLPA and TaqMan data (E, F), and NYU207 MLPA and TaqMan data (G, H). MLPA = multiplex ligation-dependent probe amplification.
      Three additional MM biopsies (NYU851, NYU966, and NYU1306) appeared to contain wild-type BAP1 by Sanger sequencing, yet they lacked nuclear BAP1 staining (Fig. 2). These three tumors displayed various types of copy number alterations in BAP1 exons by MLPA. As shown in Figure 4, NYU851 tumor DNA showed low NDQ values in exons 1, 4, and 15 (Fig. 4A). Aberrant splicing was found between exon 8 and exon 9 and cDNA sequencing identified the deletion of 1820 nucleotides from 1003 to 2822 (c.1003_2822 del 1820; Fig. 4B). NYU966 showed decreased copy number of exon 1 and exon 4 (Fig. 4C), while cDNA sequencing identified a transcript with deletion of 769 nucleotides from 1128 to 1896 (c.1128_1896del 769; Fig. 4D). An increase in exon copy number was detected in multiple BAP1 exons using MLPA in tumor NYU1306, particularly exons 1 to 3, 7 to 9, and 10 to 17 showing a NDQ value above 1.4 (Fig. 4E). This copy number increase was confirmed by TaqMan Assay (Supplementary Figure, Supplemental Digital Content 5, http://links.lww.com/JTO/A779). Moreover, NYU1306 cDNA sequencing identified a deletion of 791 nucleotides from 1101 to 1891 leading to a putative truncated BAP1 (c.1101_1891 del 791, 400aa; Fig. 4F).
      Figure thumbnail gr4
      FIGURE 4Detection 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 exons 8–9 and exons 11–17 in one allele. C, MLPA on NYU966 showed deletion of exons 1 and 4. D, Tumor NYU966 cDNA sequencing revealed a cDNA with skipping of most of exons 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 three copies, in most exons. F, NYU1306 cDNA sequencing revealed a deletion of exons 12 and 13. G, NYU1250 cDNA sequencing revealed a new splicing isoform of BAP1 due to a 121-bp deletion in exon 13. H, Aberrant usage of cryptic 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. MLPA = multiplex ligation-dependent probe amplification.
      NYU1250 also lacked nuclear BAP1 staining (Fig. 2). However, in this sample, neither Sanger sequencing nor MLPA could identify BAP1 alterations. cDNA sequencing detected a splicing mutant (c.1250_1371 del 122) in which the end of exon 12 was juxtaposed to the middle of exon 13 (Fig. 4G). A cryptic splicing site AG was found at the junction, which may generate a putative BAP1 protein of only 531 amino acids lacking the NLS (p.Tyr418fsx113; Fig. 4H). This splicing variant has not been reported previously (National Center for Biotechnology Information and GeneCards from the Weizmann Institute of Science).
      Three samples showed BAP1 alterations using both, Sanger sequencing and MLPA. NYU937 showed a reduced NDQ of exon 9. Sanger sequencing revealed a point mutation in the region of the MLPA probe for exon 9 (Supplementary Figure, Supplemental Digital Content 3, http://links.lww.com/JTO/A777). MLPA also detected allele deletions in NYU tumor biopsies 866 and 1024 (Supplementary Figure, Supplemental Digital Content 6, http://links.lww.com/JTO/A780), which were shown by Sanger sequencing to contain DNA mutations in the other allele and lacked BAP1 nuclear staining (Figs. 1C, 2). Together these analyses revealed loss of heterozygosity (LOH) for these two samples.
      No deletions were detected by MLPA, or by other techniques, in MM samples NYU047, NYU0217, NYU269, NYU517, NYU809, NYU929, NYU1017, and NYU1359. IHC revealed positive BAP1 nuclear staining in 100% of the tumor cells in these eight biopsies (Fig. 2 and Table 1).

      BAP1 Promoter and Body Methylation Are Not Altered in MM

      We sought to determine whether DNA methylation could be an additional mechanism leading to BAP1 inactivation in sporadic MM. Transcriptional silencing may occur through methylation around the transcriptional start site; therefore, seven CpG loci, located approximately 450-bp upstream of ATG start codon, were used to check the methylation status of BAP1 (Supplementary Figure, Supplemental Digital Content 7, http://links.lww.com/JTO/A781). Bisulfite-converted pyrosequencing revealed 86% to 98% methylation in a human high methylation genomic DNA control (Supplementary Figure, Supplemental Digital Content 7, http://links.lww.com/JTO/A781), whereas 2% to 4% methylation was detected in the low methylation control (Supplementary Figure, Supplemental Digital Content 7, http://links.lww.com/JTO/A781). We analyzed all 22 sporadic MM biopsies and found a consistently low level (1–4%) of methylation in all the samples. Methylation analysis of patient samples NYU047 and N524 is shown as an example (Fig. 5 and Table 1). These results indicate that hypermethylation is not a significant mechanism for BAP1 inactivation in MM. Recently, gene body methylation was found positively correlated with gene expression.
      • Yang X
      • Han H
      • De Carvalho DD
      • Lay FD
      • Jones PA
      • Liang G
      Gene body methylation can alter gene expression and is a therapeutic target in cancer.
      Therefore, we investigated whether BAP1 body methylation may influence BAP1 gene expression. Using The Cancer Genome Atlas (TCGA) database, we analyzed BAP1 methylation in several tumor types, at 19 sites spanning the entire BAP1 promoter, gene, and 3′ untranslated region (Fig. 5C). Four probes located in the body region and 3′ untranslated region were uniformly heavily methylated in all nonmalignant and malignant tissues examined. The remaining 15 probes including all the ones located in the promoter/exon 1 region were completely or nearly completely unmethylated in all tissue samples examined (Fig. 5C and Supplementary Tables, Supplemental Digital Content 8, http://links.lww.com/JTO/A782, and Supplemental Digital Content 9, http://links.lww.com/JTO/A783), support the interpretation that methylation plays no role in the inactivation of the BAP1 gene.
      Figure thumbnail gr5
      FIGURE 5BAP1 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 19 probes (data generated by use of the Infinium HumanMethylation450 BeadChip). MM = malignant mesothelioma.

      Extended IHC Analyses on a Separate MM Cohort

      Our genetic results were very consistent with BAP1 IHC results, as all BAP1 gene alterations detected resulted in loss of BAP1 nuclear staining by IHC. Thus, IHC appeared a reliable method to identify MM biopsies containing mutated BAP1.
      To extend these results, we performed BAP1 IHC on unstained paraffin embedded glass slides from an independent, annotated cohort from the NMVB (Supplementary Table, Supplemental Digital Content 1, http://links.lww.com/JTO/A775) consisting of a total of 70 cases of U.S. MM biopsies, of which we did not have additional material to conduct genetic analyses. This cohort included 63 Caucasian and seven non-Caucasian cases. We found loss of BAP1 nuclear staining in 47 of all 70 MM (67.1%) and in 44 of the 63 Caucasian MM biopsies (69.8%) (Supplementary Table, Supplemental Digital Content 1, http://links.lww.com/JTO/A775).

      Patients’ Demographics and Possible Clinical Correlations

      Overall, the MM patients studied appeared to have received similar treatments as 45% of BAP1-altered and 44% of patients with normal BAP1 had surgery, and 35% of BAP1-altered and 31% of patients with normal BAP1 received chemotherapy (Supplementary Table, Supplemental Digital Content 1, http://links.lww.com/JTO/A775); thus, it is unlikely that treatment influenced some of the results. We did not find significant relationships between frequency of BAP1 alterations and sex, age of diagnosis, ethnicity, or history of asbestos exposure (Supplementary Table, Supplemental Digital Content 1, http://links.lww.com/JTO/A775). Patients with altered BAP1 had similar survivals than those with normal BAP1 (Supplementary Figure, Supplemental Digital Content 10, http://links.lww.com/JTO/A784). This was expected as each cancer contains many genetic alterations and it would be highly unlikely that mutations of a single gene influence prognosis. There was an apparent increased frequency of BAP1 alterations in patients with epithelial type MM versus other histological subtypes. However, the possible significance of all of these clinical correlates is tempered by the small sample size.

      DISCUSSION

      Using an integrated molecular approach that included Sanger sequencing, MLPA, TaqMan copy number analysis, messenger RNA sequencing, and IHC (Supplementary Figure, Supplemental Digital Content 11, http://links.lww.com/JTO/A785), we conclusively demonstrate that the majority (14 of 22, 63.6%) of sporadic MMs contain somatic BAP1 mutations. Our study underscores the need of using distinct technical approaches to identify the nature of the different types of BAP1 mutations possibly causing lack of BAP1 nuclear staining.
      Our results indicate that the use of a single molecular technique is insufficient to detect all types of BAP1 alterations. Although Sanger sequencing is reliable to detect single point mutations, which are various and frequent in MM and other cancer types,
      • Testa JR
      • Cheung M
      • Pei J
      • et al.
      Germline BAP1 mutations predispose to malignant mesothelioma.
      ,
      • Carbone M
      • Ferris LK
      • Baumann F
      • et al.
      BAP1 cancer syndrome: malignant mesothelioma, uveal and cutaneous melanoma, and MBAITs.
      ,
      • Peña-Llopis S
      • Vega-Rubín-de-Celis S
      • Liao A
      • et al.
      BAP1 loss defines a new class of renal cell carcinoma.
      ,
      • Yoshikawa Y
      • Sato A
      • Tsujimura T
      • et al.
      Frequent inactivation of the BAP1 gene in epithelioid-type malignant mesothelioma.
      ,
      • Abdel-Rahman MH
      • Pilarski R
      • Cebulla CM
      • et al.
      Germline BAP1 mutation predisposes to uveal melanoma, lung adenocarcinoma, meningioma, and other cancers.
      ,
      • Njauw CN
      • Kim I
      • Piris A
      • et al.
      Germline BAP1 inactivation is preferentially associated with metastatic ocular melanoma and cutaneous-ocular melanoma families.
      the MLPA assay is much more reliable to detect large deletions.
      • Herman S
      • Varga D
      • Deissler HL
      • Kreienberg R
      • Deissler H
      Medium-sized deletion in the BRCA1 gene: limitations of Sanger sequencing and MLPA analyses.
      We found that Sanger sequencing was very reliable and reproducible in detecting BAP1 point mutations and small deletions in preparations of DNA from laser dissected MM biopsies containing over 80% purity of tumor cells. However, this technique could not detect large DNA deletions. Conversely, MLPA was reliable to detect large exon gains and losses, even under conditions of relatively low sample purity (~50% of tumor cells). In this setting, MLPA is cost-efficient compared with array CGH, fluorescence in situ hybridization, and gene copy number assay.
      • Hömig-Hölzel C
      • Savola S
      Multiplex ligation-dependent probe amplification (MLPA) in tumor diagnostics and prognostics.
      However, MLPA could not detect single-nucleotide changes unless they fall within the MLPA probe target regions. In our study, 6 of 14 mutations were detected using conventional Sanger sequencing. Of these six mutations, five were not detected by MLPA because the mutated regions were small and not covered by the MLPA probe. On the other hand, seven additional aberrant forms of the BAP1 gene were identified by the MLPA assay. Since the regions of exon copy number changes in these seven biopsies are large, Sanger sequencing could not detect these mutations. For accurate detection of deletions of large exons, such as exon 13 of BAP1, several MLPA probes had to be used. Abnormal splicing forms were detected in one MM biopsy—in which both Sanger sequencing and MLPA had not found any alteration—by RNA sequencing. Introns contain splicing enhancers and silencers,
      • Gamazon ER
      • Stranger BE
      Genomics of alternative splicing: evolution, development and pathophysiology.
      • Adamia S
      • Haibe-Kains B
      • Pilarski PM
      • et al.
      A genome-wide aberrant RNA splicing in patients with acute myeloid leukemia identifies novel potential disease markers and therapeutic targets.
      • Brandão RD
      • Tserpelis D
      • Gómez García E
      • Blok MJ
      Detection of exon skipping events in BRCA1 RNA using MLPA kit P002.
      which may partially explain the presence of some aberrant splicing variants in these MM samples.
      Six of 14 MM biopsies had homozygous BAP1 deletions as determined by both genomic analyses and IHC. For the remaining eight additional mutated samples, lack of nuclear staining supports LOH. However, molecular studies could not definitely establish LOH, possibly because traces of contaminant normal cell DNA were present in the samples.
      There was 100% concurrence between detection of some molecular alterations in 14 of 22 biopsies and absence of BAP1 nuclear staining (Table 1 and Fig. 2). Strong nuclear and weaker cytoplasmic BAP1 IHC reliably identified the eight MM biopsies containing wild-type BAP1 from 14 biopsies containing mutated BAP1, whose nuclei did not stain for BAP1. Among the 14 mutated biopsies, seven contained tumor cells lacking BAP1 staining entirely (Fig. 2). Cytoplasmic BAP1 tumor cell staining was detected in seven biopsies with genetically mutated BAP1 (Fig. 2). Cytoplasmic staining can be expected when mutations result in a truncated BAP1 protein lacking the NLS or bearing mutations in the catalytic subunit that prevent BAP1 auto-deubiquitination which is required for nuclear localization.
      • Mashtalir N
      • Daou S
      • Barbour H
      • et al.
      Autodeubiquitination protects the tumor suppressor BAP1 from cytoplasmic sequestration mediated by the atypical ubiquitin ligase UBE2O.
      All mutated forms of BAP1 detected here and in previous publications fall in this category and, therefore, are expected to lead to similar biological effects, as they all cause complete BAP1 protein loss or BAP1 isoforms sequestered in the cytoplasm and thus loss of BAP1 nuclear activity.
      These results suggest that IHC may be the most reliable and accessible method to identify MM biopsies that harbor BAP1 genetic alterations, while much more expensive and time-consuming genetic approaches can be used to (1) confirm the IHC findings, (2) detect heterozygosity, and (3) understand the mechanisms leading to loss of BAP1 expression.
      To this date, all BAP1 activities have been related to nuclear localization.
      • Jiao Y
      • Pawlik TM
      • Anders RA
      • et al.
      Exome sequencing identifies frequent inactivating mutations in BAP1, ARID1A and PBRM1 in intrahepatic cholangiocarcinomas.
      ,
      • Jensen DE
      • Proctor M
      • Marquis ST
      • et al.
      BAP1: a novel ubiquitin hydrolase which binds to the BRCA1 RING finger and enhances BRCA1-mediated cell growth suppression.
      ,
      • Ventii KH
      • Devi NS
      • Friedrich KL
      • et al.
      BRCA1-associated protein-1 is a tumor suppressor that requires deubiquitinating activity and nuclear localization.
      Analysis of a separate cohort of MM biopsies from the NMVB revealed lack of BAP1 nuclear staining in 47 of 70 biopsies (67.1%), confirming the high frequency of BAP1 mutations in U.S. MMs. Of note, in this cohort, six specimens contained a mixed tumor cell population, some with and some without BAP1 nuclear staining, possibly a result related to intratumoral heterogeneity and/or polyclonality.
      • Gerlinger M
      • Rowan AJ
      • Horswell S
      • et al.
      Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.
      ,
      • Comertpay S
      • Pastorino S
      • Tanji M
      • et al.
      Evaluation of clonal origin of malignant mesothelioma.
      Promoter methylation is a common mechanism of tumor suppressor gene inactivation in several MM and other tumor types.
      • Vandermeers F
      • Neelature Sriramareddy S
      • Costa C
      • Hubaux R
      • Cosse JP
      • Willems L
      The role of epigenetics in malignant pleural mesothelioma.
      ,
      • Christensen BC
      • Houseman EA
      • Godleski JJ
      • et al.
      Epigenetic profiles distinguish pleural mesothelioma from normal pleura and predict lung asbestos burden and clinical outcome.
      For example, Ras association (RalGDS/AF-6) domain family member 1 (RASSF1) and E-cadherin promoter hypermethylation was detected in MM patients.
      • Christensen BC
      • Godleski JJ
      • Marsit CJ
      • et al.
      Asbestos exposure predicts cell cycle control gene promoter methylation in pleural mesothelioma.
      ,
      • Fischer JR
      • Ohnmacht U
      • Rieger N
      • et al.
      Promoter methylation of RASSF1A, RARbeta and DAPK predict poor prognosis of patients with malignant mesothelioma.
      High expression of DNA methyltransferases (DNMT1) was detected in pleural MM.
      • Amatori S
      • Papalini F
      • Lazzarini R
      • et al.
      Decitabine, differently from DNMT1 silencing, exerts its antiproliferative activity through p21 upregulation in malignant pleural mesothelioma (MPM) cells.
      However, we observed no differences in methylation of the BAP1 promoter or gene body. Similarly, no BAP1 methylation alterations were found in clear cell renal carcinoma.
      • Ibragimova I
      • Maradeo ME
      • Dulaimi E
      • Cairns P
      Aberrant promoter hypermethylation of PBRM1, BAP1, SETD2, KDM6A and other chromatin-modifying genes is absent or rare in clear cell RCC.
      Whole-body methylation was also examined, considering the increasing volume of publications documenting a role of body methylation in gene expression.
      • Yang X
      • Han H
      • De Carvalho DD
      • Lay FD
      • Jones PA
      • Liang G
      Gene body methylation can alter gene expression and is a therapeutic target in cancer.
      While methylation data on MM are not available yet within the TCGA database, the data collected from multiple other tumor types indicate that methylation plays no role in the inactivation of the BAP1 gene. In summary, DNA methylation does not seem to be a mechanism that contributes to BAP1 inactivation in sporadic MM.
      Although the information of demographics, histology, and survival is presented, we are not elaborating about possible clinical correlation with BAP1 status as the relatively small size of this cohort may bias interpretation.
      In summary, our results demonstrate that: (1) BAP1 inactivation in MM is achieved primarily by DNA mutation and changes in exon copy number; (2) different types of mutations lead to BAP1 lacking nuclear localization as shown by absence of BAP1 nuclear staining in all of the mutated samples and; (3) ethnicity does not seem to influence the frequency of BAP1 alterations.
      Previously cyclin-dependent kinase inhibitor 2A/2B (CDKN2A/2B) and neuriofibromin 2 (NF2) have been considered the most commonly mutated tumor suppressor genes in MM as they are found mutated in 30% to 50% and 35% to 40% of human MM biopsies, respectively.
      • Hirao T
      • Bueno R
      • Chen CJ
      • Gordon GJ
      • Heilig E
      • Kelsey KT
      Alterations of the p16(INK4) locus in human malignant mesothelial tumors.
      ,
      • Ladanyi M
      • Zauderer MG
      • Krug LM
      • et al.
      New strategies in pleural mesothelioma: BAP1 and NF2 as novel targets for therapeutic development and risk assessment.
      However, the high percentage of BAP1 mutations that we found in sporadic MM (>60%) now identifies BAP1 as the most commonly mutated gene in this malignancy.
      Our findings are supported by two recent NGS studies of the MM genome by Guo et al.
      • Guo G
      • Chmielecki J
      • Goparaju C
      • et al.
      Whole-exome sequencing reveals frequent genetic alterations in BAP1, NF2, CDKN2A, and CUL1 in malignant pleural mesothelioma.
      and Lo Iacono et al.
      • Lo Iacono M
      • Monica V
      • Righi L
      • et al.
      Targeted next-generation sequencing of cancer genes in advanced stage malignant pleural mesothelioma: a retrospective study.
      These two NGS studies revealed that various inactivating mutations occur randomly and are rarely shared among MM biopsies, with the exception of BAP1 that was found mutated in 41%
      • Guo G
      • Chmielecki J
      • Goparaju C
      • et al.
      Whole-exome sequencing reveals frequent genetic alterations in BAP1, NF2, CDKN2A, and CUL1 in malignant pleural mesothelioma.
      and 58%
      • Lo Iacono M
      • Monica V
      • Righi L
      • et al.
      Targeted next-generation sequencing of cancer genes in advanced stage malignant pleural mesothelioma: a retrospective study.
      of MMs, respectively, and to a lesser extent NF2, CDKN2A
      • Guo G
      • Chmielecki J
      • Goparaju C
      • et al.
      Whole-exome sequencing reveals frequent genetic alterations in BAP1, NF2, CDKN2A, and CUL1 in malignant pleural mesothelioma.
      ,
      • Lo Iacono M
      • Monica V
      • Righi L
      • et al.
      Targeted next-generation sequencing of cancer genes in advanced stage malignant pleural mesothelioma: a retrospective study.
      , and possibly CUL1.
      • Guo G
      • Chmielecki J
      • Goparaju C
      • et al.
      Whole-exome sequencing reveals frequent genetic alterations in BAP1, NF2, CDKN2A, and CUL1 in malignant pleural mesothelioma.
      Lo Iacono et al.
      • Lo Iacono M
      • Monica V
      • Righi L
      • et al.
      Targeted next-generation sequencing of cancer genes in advanced stage malignant pleural mesothelioma: a retrospective study.
      also performed IHC studies and found that 52% of 116 MM biopsies from 116 patients stained for nuclear BAP1, an indication of normal BAP1 activity, whereas 48% did not, an indication of mutated BAP1.
      • Lo Iacono M
      • Monica V
      • Righi L
      • et al.
      Targeted next-generation sequencing of cancer genes in advanced stage malignant pleural mesothelioma: a retrospective study.
      BAP1 staining correlated with presence/absence of DNA mutations (p = 0.001)—of note in the same study NF2 staining did not—independently supporting our findings that IHC is a reliable technology to identify BAP1 mutations.
      • Lo Iacono M
      • Monica V
      • Righi L
      • et al.
      Targeted next-generation sequencing of cancer genes in advanced stage malignant pleural mesothelioma: a retrospective study.
      Guo et al.
      • Guo G
      • Chmielecki J
      • Goparaju C
      • et al.
      Whole-exome sequencing reveals frequent genetic alterations in BAP1, NF2, CDKN2A, and CUL1 in malignant pleural mesothelioma.
      performed whole-exome sequencing of 22 MM biopsies from 22 MM patients and found 490 mutated genes of which 447 were mutated only in one biopsy. BAP1 alterations were the most frequent mutations as they were detected in 41% of the 22 biopsies they studied, followed by CDKN2A and NF2. Six of these 22 MM biopsies were also included in our study (NYU269, NYU517, NYU647, NYU658, NYU929, and NYU937), allowing a direct comparison of the sensitivity of the technology used to detect BAP1 mutations. In accordance with our results, Guo et al.
      • Guo G
      • Chmielecki J
      • Goparaju C
      • et al.
      Whole-exome sequencing reveals frequent genetic alterations in BAP1, NF2, CDKN2A, and CUL1 in malignant pleural mesothelioma.
      found that samples NYU269, NYU517, and NYU929 contained wild-type BAP1. They also detected the identical BAP1 mutation in sample NYU937 (p.Ile499fsX15). However, although we detected homozygous mutations in samples NYU647 and NYU658 by Sanger sequencing—further confirmed by lack of nuclear staining by IHC—Guo et al.
      • Guo G
      • Chmielecki J
      • Goparaju C
      • et al.
      Whole-exome sequencing reveals frequent genetic alterations in BAP1, NF2, CDKN2A, and CUL1 in malignant pleural mesothelioma.
      did not (Table 1). This discrepancy may be due to the limitations of the NGS approach, which can produce base substitution errors, and to the fact that differently from Guo et al.,
      • Guo G
      • Chmielecki J
      • Goparaju C
      • et al.
      Whole-exome sequencing reveals frequent genetic alterations in BAP1, NF2, CDKN2A, and CUL1 in malignant pleural mesothelioma.
      we laser microdissected tumor cells from the biopsy, thus decreasing/eliminating the background of contaminating normal cells that contain wild-type BAP1. Alternatively, this discrepancy may be due to intratumor heterogeneity
      • Gerlinger M
      • Rowan AJ
      • Horswell S
      • et al.
      Intratumor heterogeneity and branched evolution revealed by multiregion sequencing.
      and/or polyclonality.
      • Comertpay S
      • Pastorino S
      • Tanji M
      • et al.
      Evaluation of clonal origin of malignant mesothelioma.
      Our results and those from these two recent NGS MM studies underscore the apparent driver role of BAP1 in MMs and point at BAP1 as a potentially useful therapeutic target. In addition, somatic mutations in BAP1 are present in other malignancies
      • Carbone M
      • Yang H
      • Pass HI
      • Krausz T
      • Testa JR
      • Gaudino G
      BAP1 and cancer.
      ; thus, therapies to restore BAP1 activity are of potential relevance to many cancer patients.

      ACKNOWLEDGMENTS

      We thank Mr. Hiroyasu Nishioka for helping our sequencing work during his internship. We also thank Ms. Min-Ae Song at the University of Hawaii Cancer Center Genomics Shared Resource and Dr. Shaobin Hou and Dr. Xuehua Wan at the Advanced Studies in Genomics, Proteomics and Bioinformatics of UH Manoa. We thank Waqas Amin, at the University of Pittsburg, for his precious help in the process of collecting the NMVB cohort specimens and all related clinical information.

      SUPPLEMENTARY MATERIAL

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      Linked Article

      • Genetic Basis of Mesothelioma—More Than Asbestos Exposure
        Journal of Thoracic OncologyVol. 11Issue 2
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          A combination of pathogenic organisms, environmental carcinogen, and genetic predisposition can contribute to carcinogenesis. No causative viruses for mesothelioma have been identified to date, and even though mesothelioma has been attributed largely to asbestos exposure, the genetic basis underlying the disease has lately received attention. Such interest is warranted because asbestos alone cannot explain the varying incidence of mesothelioma among patients with comparable exposure, and most diseases have a multifactorial etiology.
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