Fluorescence in situ hybridization (FISH) provides estimates of minute and interstitial BAP1, CDKN2A, and NF2 gene deletions in peritoneal mesothelioma

The aim of this study was to assess the performance of fluorescence in situ hybridization (FISH) in identifying the copy number profiles of the three key peritoneal mesothelioma tumor suppressor genes BAP1, CDKN2A, and NF2, with particular emphasis on minute homozygous deletions, a copy number abnormality recently unveiled at the 3p21 (BAP1) chromosomal region using high-throughput methods. FISH was performed on 75 formalin-fixed-paraffin-embedded peritoneal mesotheliomas and recognized two types of monoallelic loss (monosomy, and hemizygous deletion) and two types of biallelic loss (canonical homozygous deletion with a complete loss of FISH signal and homozygous deletion with diminished signal). Diminished FISH signals revealed deletions occurring within the genomic region covered by the gene-specific probe and affected all three tumor suppressors. BAP1 homozygous deletions with diminished signal outnumbered canonical homozygous deletions (13 vs 3): conversely, canonical homozygous deletions were prevalent for CDKN2A (2 vs 14). Diminished signal homozygous deletion was the only pattern of biallelic loss observed for NF2 (2 cases). Hemizygous deletion mainly affected BAP1 (21 vs 6), while monosomy was prevalent for CDKN2A (14 vs 7) and particularly for NF2 where it accounts for all monoallelic losses. FISH/immunohistochemistry (BAP1, CDKN2A, and MTAP) correlation showed that all homozygous deletions, including those with diminished signals, resulted in a null BAP1 and CDKN2A immunophenotype but only canonical CDKN2A homozygous deletions resulted in MTAP loss of expression. BAP1 hemizygous deletion, but not monosomy, was also invariably associated with loss of protein expression whereas neither type of CDKN2A monoallelic loss correlated with p16 or MTAP immunohistochemistry. Array comparative genomic hybridization performed on a spontaneously emerging peritoneal mesothelioma cell line provided support for the interpretation of the FISH patterns and allowed us to extend the number of chromatin remodeling factors involved in mesothelioma to SETD7 and PCGF5, two previously unreported genes.