Whole exome sequencing of adenoid cystic carcinoma

Authors

Philip J. Stephens

Helen R. Davies

Yoshitsugu Mitani

Peter Van Loo

Adam Shlien

Patrick S. Tarpey

Elli Papaemmanuil

Scott M. Lippman

Ultan McDermott

Michael R. Stratton

Peter J. Campbell

Adel K. El-Naggar

P. Andrew Futreal

Doi

10.1172/JCI67201

PMID: 23778141 · DOI: 10.1172/JCI67201 · Journal: J Clin Invest (2013)

TL;DR

Stephens et al. performed whole-exome sequencing on 24 adenoid cystic carcinoma (ACC) tumors with matched normals, plus extension sequencing of SPEN (42 cases) and FGFR2 (25 cases). Beyond the known MYB–NFIB fusion that defines most ACC, they identified mutations in known cancer genes (PIK3CA, ATM, CDKN2A, SF3B1, SUFU, TSC1, CYLD), implicated chromatin-remodeling deregulation in 12/24 cases, found NOTCH1/NOTCH2 mutations in 3 cases, nominated SPEN as a novel ACC cancer gene (truncating mutations in 5 cases, enriched in solid histology), and discovered 3 likely activating FGFR2 mutations that nominate FGFR inhibition (e.g. dovitinib) as a therapeutic avenue.

Cohort & data

  • 24 ACYC cases — 23 pretreatment primary tumors plus 1 local-regional lymph-node metastasis — with matched normal salivary-gland parenchyma PMID:23778141.
  • Histology: roughly equal mixture of cribriform and predominantly solid forms; 19/24 scored positive for MYB activation PMID:23778141.
  • Solution-phase exome capture + next-generation sequencing of the coding exome (whole-exome-seq) with somatic-mutation validation; SNP-array copy-number profiling on the Affymetrix SNP6.0 platform (affymetrix-snp6) analyzed with ASCAT v2.1 PMID:23778141.
  • MYB-NFIB fusion status determined by fusion-transcript sequencing, qRT-PCR, FISH, and 3′ RACE PMID:23778141.
  • Extension cohorts: targeted sanger-sequencing of SPEN across an additional 42 ACC cases and of FGFR2 across an additional 25 ACC cases PMID:23778141.
  • Dataset deposited as acyc_sanger_2013; sequence data in EGA (EGA00001064049); SNP6 data in ArrayExpress E-MTAB-1141; expression data in ArrayExpress E-MTAB-1397 PMID:23778141.

Key findings

  • 312 somatic mutations across 24 exomes (range 2–35 per tumor; mean 13/exome) — lower than most adult solid tumors. Breakdown: 182 missense, 59 silent, 14 nonsense, 45 frameshift, 6 in-frame indels, 6 essential splice site PMID:23778141.
  • Neither histology subtype nor MYB status was a significant predictor of mutation burden (generalized linear model, negative binomial; P = 0.34 for histology, P = 0.28 for MYB) PMID:23778141.
  • Recurrent copy-number losses observed at 1p36, 6q, 9p, and 12q, consistent with prior cytogenetic work; many MYB-positive cases also showed breakpoints/copy-number changes at the MYB and NFIB loci that likely underlie reported 6/9 involvement PMID:23778141.
  • Genes involved in chromatin biology were mutated in 12/24 (50%) cases, including ARID1A, ARID1B, ARID5B, CREBBP, EP300, KDM6A, KDM5A, KMT2C (MLL3), SMARCA2, CHD2, and BRD2 PMID:23778141.
  • SPEN nominated as a new cancer gene in ACC: 6 truncating mutations in 5 of 24 discovery cases (all solid histology), plus 2 additional truncating mutations (p.R1403, p.Q3355) in solid-histology cases on extension of 42 cases. All truncations fall before the SPOC domain, but no consistent LOH was observed — arguing against a simple loss-of-function mechanism PMID:23778141.
  • NOTCH1 missense (p.F1702S) and frameshift (p.Y550fs81) in 2 cases; NOTCH2 compound truncating mutations (p.Q2308fs5, p.E2420*) in a single case (PD3189), which also carried 2 SPEN truncations — together implicating NOTCH-pathway deregulation PMID:23778141.
  • Three likely activating FGFR2 mutations identified across discovery+extension: p.Y376C (identical to Beare-Stevenson cutis gyrata syndrome germline allele and to recurrent endometrial/ovarian carcinoma somatic mutations), an in-frame deletion p.I389_V393>M in the transmembrane domain, and p.K642R in the kinase domain (identical to the Pfeiffer syndrome germline allele) PMID:23778141.

Genes & alterations

  • MYB — activation via t(6;9) MYB-NFIB fusion in 19/24 cases; copy-number breakpoints at MYB locus on SNP6 in many cases PMID:23778141.
  • NFIB — translocation partner of MYB; breakpoints at NFIB locus in MYB-activated cases PMID:23778141.
  • PIK3CA — canonical activating hotspot p.H1047L PMID:23778141.
  • ATM — missense p.R337C in the kinase domain; same residue recurrently mutated (R337S/H/C) in colorectal cancer and B-CLL, supporting oncogenicity PMID:23778141.
  • CDKN2A — truncating frameshift mutation; 3 additional cases had LOH encompassing CDKN2A without nearby NFIB involvement, suggesting a CDKN2A role independent of the fusion event PMID:23778141.
  • SF3B1, SUFU, TSC1, CYLD — truncating mutations in known cancer/tumor-suppressor genes PMID:23778141.
  • NOTCH1 — missense p.F1702S (in the region targeted by T-ALL activating mutations) and frameshift p.Y550fs*81 (upstream of reported truncating activations); functional direction (activating vs tumor-suppressor like HNSCC) not resolvable from mutation pattern alone PMID:23778141.
  • NOTCH2 — compound heterozygous truncations p.Q2308fs5 and p.E2420 (PD3189), reminiscent of activating mutations seen in Hajdu-Cheney syndrome PMID:23778141.
  • SPEN — 8 total truncating mutations (6 in discovery, 2 in extension), all premature stops before the SPOC domain; no clear LOH; co-occurring with NOTCH2 truncations in PD3189 — implicates NOTCH-pathway abrogation PMID:23778141.
  • FGFR2 — p.Y376C, p.I389_V393>M (transmembrane in-frame deletion), and p.K642R (kinase domain); all paralogous to known activating germline craniosynostosis alleles or recurrent somatic activating alleles in endometrial/ovarian cancer PMID:23778141.
  • Chromatin-remodeling genes — ARID1A, ARID1B, ARID5B, KMT2C, KDM6A, KDM5A, CREBBP (including missense p.R1446C in the KAT11 domain), EP300, SMARCA2 (3 somatic missense), CHD2 (truncating), BRD2 (in-frame deletion) — collectively mutated in 12/24 cases PMID:23778141.

Clinical implications

  • The presence of activating FGFR2 mutations in a subset of ACC nominates small-molecule FGFR inhibitors as a therapeutic strategy; the authors specifically highlight the then-open clinical trial of dovitinib (TKI258) in ACC (NCT01524692) and suggest evaluating FGFR2 mutation status in trial participants PMID:23778141.
  • Identification of SPEN and NOTCH1/NOTCH2 mutations supports exploration of NOTCH-pathway targeting as a therapeutic angle in ACC PMID:23778141.
  • Solid-histology ACC, which carries worse prognosis (higher rates of local-regional relapse and distant metastasis), is enriched for SPEN truncations — a possible prognostic and biological subgroup PMID:23778141.

Limitations & open questions

  • Small discovery cohort (24 cases) limits power to detect lower-frequency drivers and to test associations with histology or MYB status PMID:23778141.
  • Functional direction of NOTCH1/NOTCH2 mutations in ACC is not resolvable from mutation pattern alone — they could be activating (per T-ALL/Hajdu-Cheney parallels) or loss-of-function (per HNSCC parallels), and may depend on cooperating mutations PMID:23778141.
  • SPEN truncations are not accompanied by consistent LOH or by loss of transcript expression, so a simple loss-of-function mechanism is unlikely; mechanism remains unresolved PMID:23778141.
  • Predicted therapeutic benefit of FGFR inhibition is inferred from analogous mutations in other tumor types; direct evidence in ACC tumors with these alleles awaits trial readouts PMID:23778141.

Citations from this paper used in the wiki

  • “We have undertaken exome sequencing in a series of 24 ACC to further delineate the genetics of the disease.” (Abstract)
  • “Exome sequencing identified 312 somatic mutations, ranging from 2 in PD3198a to 35 in PD3181a … with a mean of 13 mutations per exome, lower than that reported for most adult solid tumors thus far.” (Results)
  • “Summing over the entire exome series, 12/24 cases have mutations in genes directly involved in chromatin biology.” (Results)
  • “Spen homolog transcriptional regulator (SPEN), an inducible transcriptional regulator on chromosome 1p36, was identified as a cancer gene in ACC with 6 truncating mutations in 5 cases (all of solid histology) in the exome screen.” (Results)
  • “Three of 5 cases had evidence for loss of heterozygosity (LOH). One case (PD3195) was found to have 2 truncating mutations … no discernible LOH.” (Results)
  • “Two mutations, p.Y376C and p.I389_V393>M, were identified in PD3197a and PD3226a. The p.Y376C mutation is identical to that observed in Beare-Stevenson cutis gyrata syndrome.” (Results)
  • “Sequencing of FGFR2 through a further 25 cases identified a p.K642R mutation in the kinase domain identical to that described in Pfeiffer syndrome.” (Results)
  • “Indeed, a trial of one of these, Dovitinib, in ACC has recently opened.” (Discussion)

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