Short 57 kb CDKN2A FISH probe effectively detects short homozygous deletion of the 9p21 locus in malignant pleural mesothelioma

Abstract

Homozygous deletion (homo-d) of the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene is frequently found in malignant pleural mesothelioma (MPM). Fluorescence in situ hybridization (FISH) is commonly used to detect chromosomal deletion, and sometimes reveals more frequent heterozygous deletion (hetero-d) compared with homo-d. In clinical practice, such CDKN2A FISH results belong to the 'borderline' homo-d rate, which makes it diffcult to defnitively diagnose MPM. Microdeletion, [<200 kilobase (kb)], can induce a 'pseudo' hetero-d signal in FISH assays with long probes owing to redundant probe reactivity. Thus, the present study hypothesized that shorter FISH probes can effectively detect the small deletion status of the CDKN2A gene and increase homo-d rate in MPM, which has high hetero-d and low homo-d status. The present study aimed to evaluate the effectiveness of a shorter CDKN2A FISH probe in diagnosing MPM. CDKN2A FISH with either a 222 kb long probe (L-probe) or a 57 kb short probe (S-probe) was performed in four MPM cases with high hetero-d and low homo-d patterns. Furthermore, immunohistochemistry for methylthioadenosine phosphorylase (MTAP) and quantitative (q)PCR analyses were performed to confrm the microdeletion of the 9p21 locus. The results demonstrated that all four MPM cases retained MTAP protein expression. CDKN2A FISH with L-probe revealed high hetero-d (cases 1-4; 73.3, 37.1, 59.2 and 64.8%, respectively) and low homo-d (cases 1-4; 12.1, 12.4, 25.4 and 22.2%, respectively). CDKN2A FISH with S-probe revealed high homo-d (cases 1-4; 96.8, 90.0, 87.5 and 82.6%, respectively), with low hetero-d (cases 1-4; 0.0, 1.2, 1.2 and 4.3%, respectively). qPCR analysis demonstrated no allele deletions of the MTAP gene and two-allele deletions of the CDKN2A gene in 3/4 cases. Taken together, these results suggest that the S-probe detects the short homo-d of the 9p21 locus more effectively than the L-probe in MPM. This can assist in solving diagnostic diffculties in cases involving high hetero-d with low homo-d.