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.

Yuen Yee Cheng,Ken Takahashi,Candice Julie Clarke,Vesna Aleksova,Man Lee Yuen,Emma M Rath,Brian C Mccaughan,Steven Kao,Anthony Linton,Ben Johnson,Ling Zhuang,Kenneth Lee,Ta-Kun Yu

doi:10.3389/fonc.2020.579327

Abstract

BackgroundThe diagnosis of malignant pleural mesothelioma (MPM) can be difficult, in part due to the difficulty in distinguishing between MPM and reactive mesothelial hyperplasia (RMH). The tumor suppressor gene, CDKN2A, is frequently silenced by epigenetic mechanisms in many cancers; in the case of MPM it is mostly silenced via genomic deletion. Co-deletion of the CDKN2A and methylthioadenosine phosphorylase (MTAP) genes has been researched extensively and discovered to be a highly specific characteristic of MPM. Most studies have used FISH to detect the deletion of CDKN2A and IHC for MTAP as a surrogate for this. In this study, we aim to investigate and validate droplet digital PCR (ddPCR) as an emerging alternative and efficient testing method in diagnosing MPM, by particularly emphasizing on the loss of MTAP and CDKN2A.MethodsThis study included 75 formalin fixed paraffin embedded (FFPE) MPM tissue, and 12 normal pleural tissue and 10 RMH as control. Additionally, primary MPM cell lines and normal pleural samples were used as biomarker detection controls, as established in our previous publication. All FFPE specimens were processed to isolate the DNA, that was subsequently used for ddPCR detection of CDKN2A and MTAP. FFPE samples were also analyzed by fluorescence in situ hybridization (FISH) for CDKN2A and MTAP deletion, and for MTAP IHC expression. Concordance of IHC and ddPCR with FISH were studied in these samples.Results95% and 82% of cases showed co-deletion of both MTAP and CDKN2A when determined by FISH and ddPCR respectively. ddPCR has a sensitivity of 72% and specificity of 100% in detecting CDKN2A homozygous loss in MPM. ddPCR also has a concordance rate of 92% with FISH in detecting homozygous loss of CDKN2A. MTAP IHC was 68% sensitive and 100% specific for detecting CDKN2A homozygous loss in MPM when these losses were determined by ddPCR.ConclusionOur study confirms that MTAP is often co-deleted with CDKN2A in MPM. Our in-house designed ddPCR assays for MTAP and CDKN2A are useful in differentiating MPM from RMH, and is highly concordant with FISH that is currently used in diagnosing MPM. ddPCR detection of these genetic losses can potentially be utilized as an alternative method in the diagnosis of MPM and for the future development of a less-invasive MPM-specific detection technique on MPM tumor tissue DNA.

Highlights

The diagnosis of malignant pleural mesothelioma (MPM) can be difficult, in part due to the difficulty in distinguishing between MPM and reactive mesothelial hyperplasia (RMH)
This present study aims to utilize the Asbestos Disease Research Institute’s (ADRI) extensive MPM collection to re-validate the ability of droplet digital PCR (ddPCR) to detect the loss of CDKN2A, and the prevalence of methylthioadenosine phosphorylase (MTAP) co-deletion in MPM
Some samples that were collected over 10 years prior to our experiment were found to be compromised on the basis that they did not provide sufficient reference copy numbers to differentiate the deletion of CDKN2A or MTAP and were excluded from the experiment

Summary

Introduction

The diagnosis of malignant pleural mesothelioma (MPM) can be difficult, in part due to the difficulty in distinguishing between MPM and reactive mesothelial hyperplasia (RMH). Co-deletion of the CDKN2A and methylthioadenosine phosphorylase (MTAP) genes has been researched extensively and discovered to be a highly specific characteristic of MPM. Most studies have used FISH to detect the deletion of CDKN2A and IHC for MTAP as a surrogate for this. Malignant pleural mesothelioma (MPM) is an aggressive malignancy caused by exposure to asbestos. Most MPM patients are diagnosed at a late stage due to non-specific symptoms presenting at early stages coupled with the long latency period between asbestos exposure and the development of MPM. Delays or errors in diagnosis potentially hinder treatment intervention which can adversely affect the patients’ survival and quality-of-life (QoL). Accurate diagnosis is essential for prognostication and management [5]

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