Genomic Deletion of BAP1 and CDKN2A Are Useful Markers for Quality Control of Malignant Pleural Mesothelioma (MPM) Primary Cultures

Abstract

Malignant pleural mesothelioma (MPM) is a deadly cancer that is caused by asbestos exposure and that has limited treatment options. The current standard of MPM diagnosis requires the testing of multiple immunohistochemical (IHC) markers on formalin-fixed paraffin-embedded tissue to differentiate MPM from other lung malignancies. To date, no single biomarker exists for definitive diagnosis of MPM due to the lack of specificity and sensitivity; therefore, there is ongoing research and development in order to identify alternative biomarkers for this purpose. In this study, we utilized primary MPM cell lines and tested the expression of clinically used biomarker panels, including CK8/18, Calretinin, CK 5/6, CD141, HBME-1, WT-1, D2-40, EMA, CEA, TAG72, BG8, CD15, TTF-1, BAP1, and Ber-Ep4. The genomic alteration of CDNK2A and BAP1 is common in MPM and has potential diagnostic value. Changes in CDKN2A and BAP1 genomic expression were confirmed in MPM samples in the current study using Fluorescence In situ Hybridization (FISH) analysis or copy number variation (CNV) analysis with digital droplet PCR (ddPCR). To determine whether MPM tissue and cell lines were comparable in terms of molecular alterations, IHC marker expression was analyzed in both sample types. The percentage of MPM biomarker levels showed variation between original tissue and matched cells established in culture. Genomic deletions of BAP1 and CDKN2A, however, showed consistent levels between the two. The data from this study suggest that genomic deletion analysis may provide more accurate biomarker options for MPM diagnosis.

CDKN2A and MTAP Are Useful Biomarkers Detectable by Droplet Digital PCR in Malignant Pleural Mesothelioma: A Potential Alternative Method in Diagnosis Compared to Fluorescence In Situ Hybridisation

“…All samples obtained from previously diagnosed MPM patients were reviewed by an experienced pathologist (KL) with MPM-specific IHC markers before the experiments were carried out. Immunohistochemical analysis of MTAP (Abnova, H00004507-M01, 2G4 clone; 1/300 dilution) in MPM tissue samples was performed as described previously ( 7 ). Briefly, MPM cells and MPM tissue were processed for formalin fixed paraffin embedded (FFPE) block and were sectioned at 4 μ m thickness for IHC analysis.…”

“…Recently BAP1 and MTAP IHC have been used for this purpose with reasonably good sensitivity and specificity. However for equivocal cases, FISH for homozygous loss of CDKN2A ( 7 – 9 ) is often used to further differentiate between the two. We have previously shown that BAP1 and CDKN2A loss are frequent in MPM ( 7 ).…”

“…However for equivocal cases, FISH for homozygous loss of CDKN2A ( 7 – 9 ) is often used to further differentiate between the two. We have previously shown that BAP1 and CDKN2A loss are frequent in MPM ( 7 ). Hwang et al , also indicated that the loss of CDKN2A by FISH and BAP1 by IHC are specific biomarkers for MPM diagnosis ( 10 , 11 ).…”

“…ddPCR provided higher sensitivity and precision in molecular diagnostics for pathogens such as hepatitis B virus ( 15 ), human immunodeficiency virus (HIV) ( 16 ), chlamydia trachomatis ( 17 ) and chromosomally integrated human herpes virus 6 ( 18 ). We have previously reported ddPCR to be a useful method in analyzing and detecting the loss of CDKN2A in MPM ( 7 ). This present study aims to utilize the Asbestos Disease Research Institute’s (ADRI) extensive MPM collection to re-validate the ability of ddPCR to detect the loss of CDKN2A, and the prevalence of MTAP co-deletion in MPM.…”

Special Issue on Mechanisms of Mesothelioma Heterogeneity: Highlights and Open Questions

“…A general feature of a tumor is that it comprises tumor cells and stroma containing immune cells, fibroblasts, matrix and blood vessels. Therefore, it is not surprising that in this special issue, the mechanisms of mesothelioma heterogeneity have been addressed extensively at the level of tumoral cells, highlighting differences in genetic alterations [ 1 , 2 , 3 , 4 , 5 ] or temporal differences during tumor progression [ 2 ].…”