FISH assay development for the detection of p16/CDKN2A deletion in malignant pleural mesothelioma

Our plain-English summary of FISH assay development for the detection of p16/CDKN2A deletion in malignant pleural mesothelioma, by C.T. Chung, C. Santos Gda, D.M. Hwang, et al (Journal of Clinical Pathology, 2010).

The study

• Authors: C.T. Chung, C. Santos Gda, D.M. Hwang, O. Ludkovski, M. Pintilie, J.A. Squire, M.S. Tsao
• Journal: Journal of Clinical Pathology
• Published: 2010;63(7):630-4
• Cited by: 85 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

Aims: To develop a fluorescence in-situ hybridisation (FISH) assay for detecting p16/CDKN2A deletion on paraffin tissue sections for use as an ancillary test to distinguish reactive from malignant mesothelial proliferations.

Method: Dual-colour FISH for p16/CDKN2A and chromosome 9 (CEP-9) was performed on 11 benign mesothelial proliferations and 54 malignant pleural mesothelioma (MPM) cases to establish cut-off values for p16/CDKN2A deletion. A third MYC probe was used to verify cases showing homozygous deletion. Eight equivocal biopsies were used for assay testing.

Results: Cut-off values for p16/CDKN2A deletion were calculated based on FISH signalling patterns obtained from the benign controls (mean percent nuclei plus three standard deviations). Hemizygous deletion was defined as >44% of nuclei showing the hemizygous (one p16/CDKN2A, two CEP-9 signals) or >15% of nuclei showing the monosomy (one p16/CDKN2A, one CEP-9 signal) deletion patterns. None of the benign cases showed a homozygous deletion pattern (no p16/CDKN2A, at least one CEP-9 signal). In the malignant cases, the percentage of nuclei showing homozygous deletion ranged from 1% to 87%. Therefore, the cut-off value for homozygous deletion was defined as >10%. P16/CDKN2A deletion was detected in 61% (33/54) of MPM cases. Among the equivocal biopsies, four showed homozygous and one showed hemizygous p16/CDKN2A deletion. Age over 60 years, asbestos exposure and p16/CDKN2A deletion were associated with a worse prognosis.

Conclusion: Distinction between benign and malignant mesothelial proliferations can be diagnostically challenging. FISH for p16/CDKN2A deletion is a useful test for confirming the diagnosis of MPM.

In plain English

This Toronto study developed a FISH test, which counts gene copies in cells using fluorescent probes, to detect missing copies of the p16/CDKN2A gene in tissue sections. It aims to help tell malignant pleural mesothelioma from benign mesothelial growths. The authors used 11 benign cases to set cut-offs for losing one gene copy. Because none showed loss of both copies, the cut-off for that pattern (over 10% of cells) was set after examining 54 malignant pleural mesotheliomas, allowing for signals lost in slicing. Both copies were lost in 23 of 54 (43%) and one copy only in 10 of 54 (18%): 61% sensitivity and 100% specificity. Seven of eight hard-to-diagnose biopsies, all later confirmed as mesothelioma, could be tested: four showed loss of both copies and one loss of one copy. Looking at one factor at a time, gene loss, age over 60 and previous asbestos exposure (recorded in 50% of patients) were each linked to shorter survival. Two-year survival was 17% with gene loss versus 50% without. The authors concluded it is a clinically relevant confirmatory test for diagnosing malignant pleural mesothelioma.

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: Diagnostic test (assay) development study using archival specimens, with testing on prospectively collected equivocal biopsies and a survival analysis
• Who was studied: 54 archival biopsy and resection specimens of malignant pleural mesothelioma (42 epithelioid, 11 biphasic, 1 sarcomatoid) and 11 reactive mesothelial proliferations as negative controls, plus 8 prospectively collected equivocal biopsies; University Health Network, Toronto, Canada; dates not given
• Exposure: The main factor studied was p16/CDKN2A gene deletion detected by FISH. Previous asbestos exposure was recorded as a clinical variable; no fiber types, products, jobs or doses are given.
• Main results:
  • None of the 11 benign reactive cases showed the homozygous deletion pattern (loss of both p16/CDKN2A copies)
  • Hemizygous deletion cut-offs, set from the benign cases: more than 44% of nuclei with one p16/CDKN2A and one CEP-9 signal, or more than 15% of nuclei with one p16/CDKN2A and two CEP-9 signals
  • The homozygous deletion cut-off was set after examining the malignant cases, at more than 10% of nuclei with no p16/CDKN2A signal and one or two CEP-9 signals, to allow for signal loss from nuclear sectioning
  • Malignant pleural mesothelioma cases: homozygous p16/CDKN2A deletion in 23/54 (43%)
  • Malignant pleural mesothelioma cases: hemizygous-only deletion in 10/54 (18%)
  • p16/CDKN2A deletion had 61% sensitivity and 100% specificity for malignant pleural mesothelioma
  • Equivocal biopsies: 7 of 8 had enough tissue; 4 showed homozygous deletion (mean 49%, range 16 to 75% of nuclei) and 1 showed hemizygous deletion
  • All eight equivocal cases were later confirmed to be malignant pleural mesothelioma
  • Asbestos exposure was documented in 50% of patients and a smoking history in 46%
  • Two-year survival was 50% in patients without p16/CDKN2A deletion versus 17% in patients with the deletion
  • Age over 60 years and previous asbestos exposure were associated with significantly worse survival on univariate analysis. For asbestos exposure the article reports 39% versus 17% survival at 2 years (p = 0.039) without labelling which figure belongs to which group.
  • p16/CDKN2A deletion status was not associated with any of the clinical parameters
• The authors' conclusion: The authors concluded that FISH for p16/CDKN2A deletion on formalin-fixed paraffin-embedded sections is a clinically relevant confirmatory test for diagnosing malignant pleural mesothelioma
• Limitations the authors state:
  • Loss of signals through nuclear truncation in tissue sections can be problematic for analysing deletions
  • A cut-off for homozygous deletion could not be established from the benign controls
  • In two desmoplastic mesothelioma cases, normal and tumour spindle cells were difficult to tell apart under fluorescence microscopy

Topics

The National Library of Medicine indexes this study under subjects our site covers: Mesothelioma.

Medical terms in this study, explained in our Lexicon: Fluorescence In Situ Hybridization (FISH), CDKN2A, Pleural Mesothelioma, Homozygous Deletion, Asbestos Exposure, Prognosis.

Related studies

• Low homozygous/high heterozygous deletion status by p16 FISH correlates with a better prognostic group than high homozygous deletion status in malignant pleural mesothelioma — M. Hamasaki, S. Matsumoto, et al. (2016)
• Fluorescence in situ hybridization (FISH) provides estimates of minute and interstitial BAP1, CDKN2A, and NF2 gene deletions in peritoneal mesothelioma — S. Brich, F. Bozzi, et al. (2020)
• p16 FISH deletion in surface epithelial mesothelial proliferations is predictive of underlying invasive mesothelioma — H. Hwang, C. Tse, et al. (2014)
• CDKN2A and MTAP deletions in peritoneal mesotheliomas are correlated with loss of p16 protein expression and poor survival — A.M. Krasinskas, D.L. Bartlett, et al. (2010)
• Utility of BAP1 Immunohistochemistry and p16 (CDKN2A) FISH in the Diagnosis of Malignant Mesothelioma in Effusion Cytology Specimens — H.C. Hwang, B.S. Sheffield, et al. (2016)
• 9p21 deletion in the diagnosis of malignant mesothelioma, using fluorescence in situ hybridization analysis — M. Takeda, T. Kasai, et al. (2010)

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