Novel recurrently mutated genes in African American colon cancers

Authors

Kishore Guda

Martina L. Veigl

Vinay Varadan

Arman Nosrati

Lakshmeswari Ravi

James Lutterbaugh

Lydia Beard

James K. V. Willson

W. David Sedwick

Zhenghe John Wang

Neil Molyneaux

Alexander Miron

Mark D. Adams

Robert C. Elston

Sanford D. Markowitz

Joseph E. Willis

Doi

10.1073/pnas.1417064112

PMID: 25583493 · DOI: 10.1073/pnas.1417064112 · Journal: Proceedings of the National Academy of Sciences (2015)

TL;DR

Guda et al. performed whole-exome and targeted resequencing of 103 microsatellite-stable (MSS) colorectal cancers from African Americans (AA) — the largest such cohort at the time — and compared their mutational landscape to 129 predominantly late-stage MSS Caucasian CRCs. They nominated 20 new significantly mutated genes in CRC, of which a 15-gene subset is preferentially targeted in AA versus Caucasian tumors (~twofold increase in total mutations per tumor; subset P = 1.8 × 10⁻⁸). Two genes, EPHA6 and FLCN, were mutated exclusively in AA cases and are proposed as AA-specific CRC driver genes.

Cohort & data

  • 29 informative MSS late-stage AA CRC discovery cases (whole-exome, paired tumor/normal) plus one unpaired tumor used only for nomination; one hypermutator excluded.
  • 74 informative MSS AA CRC validation cases (predominantly early stage) sequenced with a custom Agilent SureSelectXT bait library against 78 candidate genes; three hypermutators excluded from the original 77.
  • 129 predominantly late-stage MSS Caucasian CRCs resequenced for the 20 significantly mutated AA genes (no Caucasian hypermutators excluded).
  • All samples drawn from a Case Medical Center archive of fresh-frozen tumor/normal pairs in northeastern Ohio; all MSS by BAT26/BAT40/D2S123/D5S346/D17S250 testing; pathologist-confirmed tumor cell content ≥50%.
  • Cancer type: COAD. Dataset: coad_caseccc_2015. Assays: whole-exome-seq, custom-capture targeted-dna-seq, sanger-sequencing confirmation.

Key findings

  • 2,696 protein-altering mutations across 2,156 genes were detected in the 29 discovery AA CRC exomes; C > T transitions were the predominant base substitution, as expected for CRC (PMID:25583493).
  • Of 385 recurrently mutated genes in the discovery set, 78 had no prior protein-altering report in MSS non-hypermutator CRCs from three large-scale CRC studies; 52 of these were re-confirmed mutated in ≥2 discovery cancers.
  • In the 74 validation AA CRCs, 59 nonsilent mutations were detected across 27 of the 52 candidate genes; 20 genes were significantly mutated (P < 0.05, FDR < 0.05) in both the discovery and validation cohorts (Table 1).
  • The 20-gene panel showed a statistically significant ~twofold increase in mutations per tumor in AA CRCs (83 mutations in 103 cases) versus Caucasian CRCs (50 mutations in 129 cases; P < 0.001).
  • The effect was driven by the top-15 ranked genes (subgroup analysis nominal P = 1.8 × 10⁻⁸); mutations in this 15-gene set accounted for 41% of AA CRCs versus only 15% of Caucasian CRCs.
  • EPHA6 mutations occurred in 5.83% of AA CRCs (6/103) and 0% of Caucasian CRCs (0/129); association with AA ethnicity P = 0.007.
  • FLCN mutations occurred in 2.91% of AA CRCs (3/103) and 0% of Caucasian CRCs (P = 0.086); two frameshifts and one nonsense mutation, all consistent with inactivation.
  • HTR1F mutations were also exclusive to AA cases (2.91%, 3/103; P = 0.086).
  • Curation of COSMIC showed that 56 of 66 previously reported EPHA6/FLCN CRC mutations were from hypermutator tumors; remaining variants were either unconfirmed somatic or silent, supporting the novelty of these findings in MSS non-hypermutator CRC.
  • Among classic CRC driver genes (APC, TP53, KRAS, PIK3CA, SMAD4, BRAF, FBXW7), the group did not differ between AA and Caucasian CRCs, but KRAS and FBXW7 individually showed significantly higher mutation frequencies in AA than Caucasian CRCs.

Genes & alterations

  • EPHA6 — 6 somatic mutations across the gene (one canonical splice-site alteration plus four missense mutations predicted to significantly alter protein function); pattern consistent with tumor-suppressor activity. Mutated in 5.83% AA vs. 0% Caucasian CRCs (P = 0.007). Proposed as an AA-specific CRC driver. Authors note that related EPHA3 and EPHB6 were identified as significantly mutated in a prior nearly all-Caucasian MSS CRC cohort, suggesting ethnicity-associated selection of different EPH family members.
  • FLCN — 3 somatic mutations in AA CRCs (two frameshift, one nonsense), all consistent with inactivation; 0 in Caucasian CRCs (P = 0.086). Germline inactivating FLCN mutations cause Birt-Hogg-Dubé syndrome (medullary thyroid carcinomas, perifollicular fibromas, renal cancers), with colon neoplasia reported in some affected individuals; mice lacking a single Flcn allele recapitulate the phenotype. Proposed as an AA-specific CRC driver.
  • HTR1F — exclusive to AA (3/103 vs. 0/129; P = 0.086).
  • Additional newly significantly mutated genes (significant in both AA discovery and validation; full panel of 20): MAGEB10, CPT1C, ANKRD36, MGAT4C, ZNF717, ZNF862, CP, CCDC74A (reported as KIAA1551), EML6, ATP8B2, JAK1, CHD5, WASHC1 (reported as WASH1), GPR149, CDK8, WDR87. Note: the 20th gene (TCEB3CL) reported in the paper is no longer a canonical HUGO symbol and is omitted from the wiki gene panel.
  • Classic CRC drivers studied for comparison only: APC, TP53, KRAS, PIK3CA, SMAD4, BRAF, FBXW7. KRAS and FBXW7 individually higher in AA than Caucasian CRCs (consistent with prior reports of increased KRAS mutations in AA CRCs).

Clinical implications

  • The 15-gene preferentially-targeted panel accounts for 41% of AA CRCs vs. 15% of Caucasian CRCs, suggesting an ethnicity-stratified mutational landscape that may influence colon-cancer outcome and warrants prospective study (the authors call out future investigation into the impact of this panel on CRC outcome).
  • The authors propose EPHA6 and FLCN as candidate AA-specific CRC driver genes; both belong to known oncogenic pathways (EPH receptor tyrosine kinase family; mTOR axis via FLCN), suggesting potential pathway-level therapeutic implications, though no treatment claims are made.
  • No drug response or biomarker-treatment claims are made in the paper.

Limitations & open questions

  • Cohort drawn exclusively from northeastern Ohio; authors flag that internal migration history of African American communities makes regional comparisons important.
  • Genetic, environmental, and socioeconomic contributions to the ethnicity-associated mutational differences remain to be disentangled.
  • Authors note their selection criterion (significantly mutated in both discovery and validation cohorts) is conservative and may miss genes that would validate on further testing.
  • Caucasian comparator cohort is from a single institution and may not generalize.
  • Functional consequences of EPHA6 and FLCN mutations in AA CRCs were not experimentally tested in this study.

Citations from this paper used in the wiki

  • “We used whole-exome and targeted sequencing to characterize somatic mutations in 103 colorectal cancers (CRC) from African Americans, identifying 20 new genes as significantly mutated in this CRC. Resequencing 129 Caucasian derived CRCs confirmed a 15-gene set as a preferential target for mutations in African American CRCs.” (Abstract).
  • “Two predominant genes, ephrin type A receptor 6 (EPHA6) and folliculin (FLCN), with mutations exclusive to African American CRCs, are by genetic and biological criteria highly likely African American CRC driver genes.” (Abstract).
  • “EPHA6 … mutations in 5.8% of AA CRC versus 0% of Caucasian CRCs, and were significantly associated with AA ethnicity (P = 0.007)” (Table 2 / Results).
  • “Mutations in the 15 genes found preferentially targeted in AA CRC accounted for 41% of all AA CRCs (and only 15% of Caucasian CRCs) in this study.” (Discussion).
  • “comparison of mutational frequencies of well-known CRC driver genes (APC, TP53, KRAS, PIK3CA, SMAD4, BRAF, FBXW7), as a group, showed no significant differences between AA versus Caucasian CRCs, although KRAS and FBXW7 individually showed a significantly higher mutation frequency in AA than Caucasian CRCs” (Comparison of Mutational Frequencies section).

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