Functional CDKN2A assay identifies frequent deleterious alleles misclassified as variants of uncertain significance

Abstract

Pathogenic germline CDKN2A variants are associated with an increased risk of pancreatic ductal adenocarcinoma (PDAC). CDKN2A variants of uncertain significance (VUSs) are reported in up to 4.3% of patients with PDAC and result in significant uncertainty for patients and their family members as an unknown fraction are functionally deleterious, and therefore, likely pathogenic. Functional characterization of CDKN2A VUSs is needed to reclassify variants and inform clinical management. 29 germline CDKN2A VUSs previously reported in patients with PDAC or in ClinVar were evaluated using a validated in vitro cell proliferation assay. 12 of the 29 CDKN2A VUSs were functionally deleterious (11 VUSs) or potentially functionally deleterious (1 VUS) and were reclassified as likely pathogenic variants. Thus, over 40% of CDKN2A VUSs identified in patients with PDAC are functionally deleterious and likely pathogenic. When incorporating VUSs found to be functionally deleterious, and reclassified as likely pathogenic, the prevalence of pathogenic/likely pathogenic CDKN2A in patients with PDAC reported in the published literature is increased to up to 4.1% of patients, depending on family history. Therefore, CDKN2A VUSs may play a significant, unappreciated role in risk of pancreatic cancer. These findings have significant implications for the counselling and care of patients and their relatives.

Data availability

All data generated or analysed during this study are included in the manuscript, supporting files, and as Source data and Source code.

Article and author information

Author details

  1. Hirokazu Kimura

    Department of Pathology, Johns Hopkins University, Baltimore, United States
    Competing interests
    No competing interests declared.
  2. Raymond M Paranal

    Department of Pathology, Johns Hopkins University, Baltimore, United States
    Competing interests
    No competing interests declared.
  3. Neha Nanda

    Department of Pathology, Johns Hopkins University, Baltimore, United States
    Competing interests
    No competing interests declared.
  4. Laura D Wood

    Department of Pathology, Johns Hopkins University, Baltimore, United States
    Competing interests
    No competing interests declared.
  5. James R Eshleman

    Department of Pathology, Johns Hopkins University, Baltimore, United States
    Competing interests
    No competing interests declared.
  6. Ralph H Hruban

    Department of Pathology, Johns Hopkins University, Baltimore, United States
    Competing interests
    Ralph H Hruban, has the right to receive royalty payments from Thrive Earlier Diagnosis for the GNAS in pancreatic cysts invention in a relationship overseen by Johns Hopkins University..
  7. Michael G Goggins

    Department of Pathology, Johns Hopkins University, Baltimore, United States
    Competing interests
    No competing interests declared.
  8. Alison P Klein

    Department of Pathology, Johns Hopkins University, Baltimore, United States
    Competing interests
    No competing interests declared.
  9. Nicholas Jason Roberts

    The Johns Hopkins University, Baltimore, United States
    For correspondence
    nrobert8@jhmi.edu
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8709-0664

Funding

The Sol Goldman Pancreatic Cancer Research Center (Pilot Grant)

  • Nicholas Jason Roberts

National Institutes of Health (S10 OD026859)

  • Nicholas Jason Roberts

The Rolfe Pancreatic Cancer Foundation

  • Nicholas Jason Roberts

National Institutes of Health (P50 CA622924)

  • Alison P Klein
  • Nicholas Jason Roberts

The Japanese Society of Gastroenterology

  • Hirokazu Kimura

The Japan Society for Promotion of Science

  • Hirokazu Kimura

The Joseph C Monastra Foundation

  • Nicholas Jason Roberts

The Geral O Mann Foundation

  • Nicholas Jason Roberts

Art Creates Cures Foundation

  • Nicholas Jason Roberts

Susan Wojcicki and Denis Troper

  • Nicholas Jason Roberts

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Emil Lou, University of Minnesota, United States

Version history

  1. Received: June 9, 2021
  2. Accepted: January 6, 2022
  3. Accepted Manuscript published: January 10, 2022 (version 1)
  4. Version of Record published: February 8, 2022 (version 2)

Copyright

© 2022, Kimura et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,951
    Page views
  • 271
    Downloads
  • 3
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Hirokazu Kimura
  2. Raymond M Paranal
  3. Neha Nanda
  4. Laura D Wood
  5. James R Eshleman
  6. Ralph H Hruban
  7. Michael G Goggins
  8. Alison P Klein
  9. Nicholas Jason Roberts
(2022)
Functional CDKN2A assay identifies frequent deleterious alleles misclassified as variants of uncertain significance
eLife 11:e71137.
https://doi.org/10.7554/eLife.71137

Further reading

    1. Cancer Biology
    Gehad Youssef, Luke Gammon ... Adrian Biddle
    Research Article

    Cancer stem cells (CSCs) undergo epithelial-mesenchymal transition (EMT) to drive metastatic dissemination in experimental cancer models. However, tumour cells undergoing EMT have not been observed disseminating into the tissue surrounding human tumour specimens, leaving the relevance to human cancer uncertain. We have previously identified both EpCAM and CD24 as CSC markers that, alongside the mesenchymal marker Vimentin, identify EMT CSCs in human oral cancer cell lines. This afforded the opportunity to investigate whether the combination of these three markers can identify disseminating EMT CSCs in actual human tumours. Examining disseminating tumour cells in over 12,000 imaging fields from 74 human oral tumours, we see a significant enrichment of EpCAM, CD24 and Vimentin co-stained cells disseminating beyond the tumour body in metastatic specimens. Through training an artificial neural network, these predict metastasis with high accuracy (cross-validated accuracy of 87-89%). In this study, we have observed single disseminating EMT CSCs in human oral cancer specimens, and these are highly predictive of metastatic disease.

    1. Cancer Biology
    2. Medicine
    Dingyu Rao, Hua Lu ... Defa Huang
    Research Article

    Esophageal cancer (EC) is a fatal digestive disease with a poor prognosis and frequent lymphatic metastases. Nevertheless, reliable biomarkers for EC diagnosis are currently unavailable. Accordingly, we have performed a comparative proteomics analysis on cancer and paracancer tissue-derived exosomes from eight pairs of EC patients using label-free quantification proteomics profiling and have analyzed the differentially expressed proteins through bioinformatics. Furthermore, nano-flow cytometry (NanoFCM) was used to validate the candidate proteins from plasma-derived exosomes in 122 EC patients. Of the 803 differentially expressed proteins discovered in cancer and paracancer tissue-derived exosomes, 686 were up-regulated and 117 were down-regulated. Intercellular adhesion molecule-1 (CD54) was identified as an up-regulated candidate for further investigation, and its high expression in cancer tissues of EC patients was validated using immunohistochemistry, real-time quantitative PCR (RT-qPCR), and western blot analyses. In addition, plasma-derived exosome NanoFCM data from 122 EC patients concurred with our proteomic analysis. The receiver operating characteristic (ROC) analysis demonstrated that the AUC, sensitivity, and specificity values for CD54 were 0.702, 66.13%, and 71.31%, respectively, for EC diagnosis. Small interference (si)RNA was employed to silence the CD54 gene in EC cells. A series of assays, including cell counting kit-8, adhesion, wound healing, and Matrigel invasion, were performed to investigate EC viability, adhesive, migratory, and invasive abilities, respectively. The results showed that CD54 promoted EC proliferation, migration, and invasion. Collectively, tissue-derived exosomal proteomics strongly demonstrates that CD54 is a promising biomarker for EC diagnosis and a key molecule for EC development.