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Germline Exome Sequencing for Men with Testicular Germ Cell Tumor Reveals Coding Defects in Chromosomal Segregation and Protein-Targeting Genes - Beyond the Abstract

Testicular germ cell tumor (TGCT) is the most common cancer of young white men and results in the most years of life lost among all adult cancers.1 Although TGCT survival is excellent, morbidity is substantial, making early detection of TGCT an ongoing need.2 To date, no gene with germline variation conferring high-penetrance risk (like BRCA1) has been identified.3

In an approach focusing on DNA-repair genes, we and others previously identified CHEK2 in which coding variations were associated with moderately increased TGCT risk.4 Growing evidence suggests that there is no single major high-penetrance TGCT-predisposition gene,3 making it increasingly likely a multigenic etiology underpins the high heritability observed in TGCT.5

In the current study, germline DNA was obtained through TGCT family history studies at the US National Cancer Institute6 and the University of Pennsylvania.7 High risk for TGCT-predisposing germline variation was defined as either bilateral disease or having at least one affected 1st to 3rd degree relative with TGCT. As controls, we used ethnicity-matched cancer-free males from the Penn Medicine BioBank (PMBB)4 with >80% European ancestry (determined genetically), aged 60-90 years, male by both electronic health record identification and sex chromosome genotyping quality control, and absence of ICD9/10 for presence/history of cancer, benign neoplasm, and family history of cancer. Variant association and gene burden testing were conducted using Rvtests and EPACTS. Multiple testing correction was applied, with p<0.05 indicating a nominal association. Four pathways selected for previously demonstrated or hypothesized associations with TGCT—ciliary genes, sex- and germ-cell development, ABC (ATP-binding cassette) transporters, and known cancer-predisposition genes—were assessed for increased phenotype association using a gene burden analysis. Network analysis was completed using the STRING database.

The cohort consisted of 293 men with high risk TGCT from 228 unique families and 3157 cancer-free controls. The pedigrees showed TGCT only (64%), bilateral TCGT (26%), or both (8.6%) with the ages at diagnosis reflecting the expected distribution. Tumors that were found in affected men were distributed equally, 39% seminoma, 39% non-seminoma, and 22% unspecified.

After multiple testing corrections, loss-of-function variants in ten genes, including NIN and QRSL1 were identified from gene burden association testing. We recapitulated our previous findings associating CHEK2 with TGCT predisposition, and we also identified a signal associated with CFTR (the gene underlying cystic fibrosis), which may become statistically significant in larger studies.4 We identified an association with a specific variant in PIM1, a proto-oncogene. There were no statistically significant associations with the sex- and germ-cell development pathways, or evidence of associations with the regions previously identified by GWAS. However, when considering all significant coding variation together with genes associated with TGCT by GWAS, there were associations with three major pathways: mitosis/cell cycle, co-translational protein targeting, and sex differentiation. Since these pathways overlap with those identified by TGCT GWAS, it suggests an important nexus between pathways affected by both coding and non-coding predisposition to TGCT.8

In summary, to our knowledge, this exome sequencing study of 293 men with familial or bilateral TGCT and 3,157 cancer-free controls is the largest of its kind. We identified germline coding alterations associated with TGCT using a gene-agnostic approach. These findings support the model of a polygenic etiology for familial TGCT which differs from other cancer types (like colo-rectal and breast), in that it includes no high-penetrance high-risk variants. A polygenic etiology may imply a role in cancer-predisposition polygenic risk score (PRS). PRSs are under active investigation for other cancers including breast and prostate. Lastly, this study also reveals new candidate genes for further scientific mechanistic studies (for example, CFTR, PIM1, and CRBN), to better understand the etiology of TGCT or provide targets for future personalized clinical treatment.

Written by: Douglas Stewart, MD, Senior Investigator, Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Rockville, MD

References:

  1. Cancer Statistics Review, 1975-2017 - SEER Statistics.
  2. Chovanec M, Lauritsen J, Bandak M, et al. Late adverse effects and quality of life in survivors of testicular germ cell tumour. Nature Reviews Urology 2021 18:4. 2021;18(4):227-245.
  3. Litchfield K, Loveday C, Levy M, et al. Large-scale Sequencing of Testicular Germ Cell Tumour (TGCT) Cases Excludes Major TGCT Predisposition Gene. Eur Urol. 2018;73(6):828-831.
  4. Aldubayan SH, Pyle LC, Gamulin M, et al. Association of Inherited Pathogenic Variants in Checkpoint Kinase 2 (CHEK2) with Susceptibility to Testicular Germ Cell Tumors. JAMA Oncol. 2019;5(4).
  5. Loveday C, Law P, Litchfield K, et al. Large-scale Analysis Demonstrates Familial Testicular Cancer to have Polygenic Aetiology. Eur Urol. 2018;74(3):248-252.
  6. Greene MH, Mai PL, Loud JT, et al. Familial testicular germ cell tumors (FTGCT) - overview of a multidisciplinary etiologic study. Andrology. 2015;3(1):47-58.
  7. Kanetsky PA, Mitra N, Vardhanabhuti S, et al. A second independent locus within DMRT1 is associated with testicular germ cell tumor susceptibility. Hum Mol Genet. 2011;20(15):3109-3117.
  8. Pluta J, Pyle LC, Nead KT, et al. Identification of 22 susceptibility loci associated with testicular germ cell tumors. Nat Commun. 2021;12(1).
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