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

Our plain-English summary of 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, by Y.Y. Cheng, M.L. Yuen, E.M. Rath, et al (Frontiers in Oncology, 2020).

The study

• Authors: Y.Y. Cheng, M.L. Yuen, E.M. Rath, B. Johnson, L. Zhuang, T.K. Yu, V. Aleksova, A. Linton, S. Kao, C.J. Clarke, B.C. McCaughan, K. Takahashi, K. Lee
• Journal: Frontiers in Oncology
• Published: 2020;10:579327
• Cited by: 16 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Background: 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). 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.

Methods: This 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.

Results: 95% 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.

Conclusion: Our 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.

Copyright © 2020 Cheng, Yuen, Rath, Johnson, Zhuang, Yu, Aleksova, Linton, Kao, Clarke, McCaughan, Takahashi and Lee.

In plain English

This laboratory study tested whether droplet digital PCR (ddPCR), a DNA-counting technique, could detect loss of the CDKN2A and MTAP genes in malignant pleural mesothelioma tumor tissue. The goal was a possible alternative to the established FISH test. The authors used stored surgical tissue from 75 patients. Controls were 12 normal pleural tissue samples and 10 benign reactive mesothelial hyperplasia samples. Not every sample could be tested by every method. By FISH, 79% (44/56) of tumors had complete (homozygous) loss of CDKN2A; by ddPCR, 72% (28/39) did. No benign sample tested by FISH and no normal sample tested by ddPCR showed this loss. ddPCR detected homozygous CDKN2A loss with 72% sensitivity and 100% specificity and agreed with FISH in 92% of these cases. A protein stain for MTAP detected homozygous CDKN2A loss with 64% sensitivity when FISH defined that loss and 68% when ddPCR did; overall in mesothelioma it was 48%. The authors concluded that ddPCR showed good agreement with FISH and could potentially serve as an alternative diagnostic method. Asbestos exposure was not studied.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory diagnostic method-comparison study on stored surgical tumor tissue, comparing ddPCR with FISH and with MTAP immunohistochemistry (IHC)
• Who was studied: 85 patients with malignant pleural mesothelioma were initially included. 75 had enough formalin-fixed paraffin-embedded tissue for analysis: 58 male and 17 female, median age 65 (range 51 to 93), all treated by pleurectomy/decortication. Subtypes were 37 epithelioid, 26 biphasic and 12 sarcomatoid. Controls were 12 normal pleural tissue samples and 10 reactive mesothelial hyperplasia samples. The study was approved at Concord Repatriation General Hospital, Australia. Collection dates were not given.
• Exposure: No asbestos or other exposure was studied. The study examined loss of the CDKN2A and MTAP genes in tumor tissue, measured by ddPCR, FISH and MTAP IHC.
• Main results:
  • By FISH, 79% (44/56) of mesothelioma samples had homozygous deletion of CDKN2A, 18% (10/56) had hemizygous deletion and 3% (2/56) had no deletion. For MTAP, 77% (43/56) had homozygous deletion.
  • By FISH, 53 of 56 mesothelioma cases (95%) had deletion of either or both genes. In 42 of the 44 cases with homozygous CDKN2A loss (95%), MTAP and CDKN2A were homozygously co-deleted.
  • None of the 10 reactive mesothelial hyperplasia samples showed homozygous loss of CDKN2A or MTAP by FISH; 4 showed hemizygous loss.
  • FISH detection of homozygous CDKN2A loss had a sensitivity of 78% and a specificity of 100%. The discussion gives the sensitivity as 79%.
  • By ddPCR, 72% (28/39) of mesothelioma samples had homozygous CDKN2A deletion, 26% (10/39) hemizygous deletion and 2% (1/39) retained CDKN2A.
  • By ddPCR, 58% (25/43) of mesothelioma samples had homozygous MTAP deletion and 42% (18/43) hemizygous deletion.
  • All 12 normal pleural tissue samples showed retention of both CDKN2A and MTAP by ddPCR.
  • ddPCR detection of homozygous CDKN2A loss in mesothelioma had a sensitivity of 72% and a specificity of 100%.
  • By ddPCR, 82% (23/28) of cases showed homozygous co-deletion of MTAP and CDKN2A.
  • ddPCR agreed with FISH in 92% of cases for homozygous CDKN2A loss. The table gives this as 26 of 28 cases.
  • By MTAP IHC, 35 of 72 mesothelioma samples (49%) showed MTAP loss, 36 (50%) showed retained MTAP and 1 (1%) showed partial loss. All 10 reactive mesothelial hyperplasia samples showed retained MTAP staining.
  • Across all mesothelioma cases, MTAP IHC had a sensitivity of 48% and a specificity of 100%.
  • MTAP IHC was also tested as a marker for homozygous CDKN2A loss. It had a sensitivity of 64% and a specificity of 100% when FISH defined that loss, and a sensitivity of 68% and a specificity of 100% when ddPCR defined it.
• The authors' conclusion: The authors concluded that ddPCR showed good concordance with FISH in detecting MTAP and CDKN2A homozygous deletion in mesothelioma. They also concluded it showed similar sensitivity and specificity to FISH in telling mesothelioma apart from reactive mesothelial hyperplasia, and that it could potentially be used as an alternative to FISH. They added that further studies would be needed before it could be used on less invasive samples such as blood.
• Limitations the authors state:
  • Some cases gave different results with ddPCR and FISH. The authors attributed these to counts falling just past a cut-off or to tumor heterogeneity, and called this a potential weakness of the study.
  • ddPCR analyzes the whole tissue section, including non-tumor cells, which could dilute the tumor DNA.
  • Some samples collected more than 10 years before the experiment were compromised and had to be excluded.
  • FISH counts only selected areas of the tumor, so it could still be subject to selection bias.
  • Unlike FISH, ddPCR cannot analyze genetic differences between individual tumor cells.

Topics

Medical terms in this study, explained in our Lexicon: CDKN2A, MTAP, Biomarker, Pleural Mesothelioma, Reactive Mesothelial Hyperplasia, Tumor Suppressor Gene, Fluorescence In Situ Hybridization (FISH).

Related studies

• MTAP immunohistochemistry is an accurate and reproducible surrogate for CDKN2A fluorescence in situ hybridization in diagnosis of malignant pleural mesothelioma — D.B. Chapel, J.J. Schulte, et al. (2020)
• Utility of Methylthioadenosine Phosphorylase Compared With BAP1 Immunohistochemistry, and CDKN2A and NF2 Fluorescence In Situ Hybridization in Separating Reactive Mesothelial Proliferations From Epithelioid Malignant Mesotheliomas — K.B. Berg, S. Dacic, et al. (2018)
• Usefulness of p16/CDKN2A fluorescence in situ hybridization and BAP1 immunohistochemistry for the diagnosis of biphasic mesothelioma — D. Wu, K. Hiroshima, et al. (2017)
• Hemizygous loss of NF2 detected by fluorescence in situ hybridization is useful for the diagnosis of malignant pleural mesothelioma — Y. Kinoshita, M. Hamasaki, et al. (2020)
• Peritoneal malignant mesothelioma (PMM), and primary peritoneal serous carcinoma (PPSC) and reactive mesothelial hyperplasia (RMH) of the peritoneum. Immunohistochemical and fluorescence in situ hybridisation (FISH) analyses — T. Kawai, S. Tominaga, et al. (2016)
• The Use of Immunohistochemistry, Fluorescence in situ Hybridization, and Emerging Epigenetic Markers in the Diagnosis of Malignant Pleural Mesothelioma (MPM): A Review — E. Rozitis, B. Johnson, et al. (2020)

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