The mutational landscape of adenoid cystic carcinoma

Abstract

Adenoid cystic carcinomas (ACCs) are among the most enigmatic of human malignancies. These aggressive salivary gland cancers frequently recur and metastasize despite definitive treatment, with no known effective chemotherapy regimen. Here we determined the ACC mutational landscape and report the exome or whole-genome sequences of 60 ACC tumor-normal pairs. These analyses identified a low exonic somatic mutation rate (0.31 non-silent events per megabase) and wide mutational diversity. Notably, we found mutations in genes encoding chromatin-state regulators, such as SMARCA2, CREBBP and KDM6A, suggesting that there is aberrant epigenetic regulation in ACC oncogenesis. Mutations in genes central to the DNA damage response and protein kinase A signaling also implicate these processes. We observed MYB-NFIB translocations and somatic mutations in MYB-associated genes, solidifying the role of these aberrations as critical events in ACC. Lastly, we identified recurrent mutations in the FGF-IGF-PI3K pathway (30% of tumors) that might represent new avenues for therapy. Collectively, our observations establish a molecular foundation for understanding and exploring new treatments for ACC.

Figure 1. Mutational landscape of adenoid cystic carcinoma

(a). Number of validated nonsynonymous somatic mutations per sample across 60 ACC cases. (b). Representative list of recurrent nonsynonymous somatic mutations. Multiple mutations within a given sample in the same gene were only counted once. (c). Pathways affected by driver mutations identified by CHASM. Bonferroni FDR-corrected p-values for pathway enrichment are shown. (d). Location of ACC mutations in key chromatin remodeling genes. HSA, helicase-SANT-associated; Zf-TAZ, TAZ zinc finger; DUF, domain of unknown function; KAT11, histone acetylation protein; ZZ, ZZ-type zinc finger; SNF2_N, SNF2 family N-terminal domain; Helicase_C, Helicase conserved C-terminal domain; TPR, tetratricopeptide repeat; JmjC, JmjC hydroxylase domain; FtsJ, FtsJ-like methyltransferase. (e). Location of ACC mutations in key established cancer genes. PI3K_p85B, PI3K p85-binding domain; PI3K_rbd, PI3K ras-binding domain; PI3Ka, PI3K accessory domain; EGF_Ca, calcium-binding EGF domain; hEGF, human growth factor-like EGF; Ank repeat, ankyrin repeat; DSPc, dual specificity phosphatase, catalytic domain; P53 TAD, P53 transactivation motif; P53_tetramer, P53 tetramerization motif.

Figure 2. Integrated analysis of adenoid cystic carcinoma genetic alterations

Clinical data, validated somatic mutations, validated structural variants, and copy number alterations for 60 ACC cases. Genes highlighted in bold contain significant driver mutations identified by CHASM. Remaining genes contain selected mutations of interest that did not reach significance by CHASM but which are either altered in other malignancies or are integral components of affected pathways. Orange, nonsynonymous somatic mutation (missense, nonsense, splice site, indel); blue, homozygous copy number deletion; red, high-level copy number amplification.

Figure 3. Structural variations and copy number landscape of adenoid cystic carcinoma

(a). Clustered copy number profiles of 60 ACC cases. Clustering revealed 4 sub-clusters of tumors with one each defined by loss of 14q, loss of 12q13, a cluster with two samples containing a large number of copy number alterations, and most tumors in a large group with minimal copy number changes. White, normal (diploid) copy number log-ratio; blue, copy number loss; red, copy number amplification. (b). Circos plots of genetic alterations in 5 ACC cases. Plots depict validated structural genetic variants, DNA copy number alterations, intra- and inter-chromosomal translocations, and sequence alterations. Loss-of-heterozygosity, green track; amplification, red track; copy number loss, blue track; missense mutation, black gene name; nonsense mutation, blue gene name; splice site mutation, orange gene name; inter-chromosomal translocation, red line; deletion, gray line; intra-chromosomal translocation, blue line; insertion, purple line.

Figure 4. Diversity and frequency of ACC genetic changes culminating in aberrant signaling pathways

Alterations are defined by somatic mutations, homozygous deletions, high-level focal amplifications, and structural variants validated by FISH or PCR. Frequencies are expressed as a percentage of all cases. Red background denotes activating alteration, blue background denotes inactivating alteration, white background denotes numerous alterations, and purple background denotes alteration of unclear significance. Interactions based upon IPA or Pathway Commons. (a). Epigenetic modification. (b). DNA damage/checkpoint signaling pathway. (c). MYB/MYC signaling pathway, with fusion denoted by recurrent t(6;9) translocation between MYB and NFIB. (d). FGF/IGF/PI3K signaling pathway. (e). Notch signaling pathway.

Figure 5. Functional consequences of genetic alterations in KDM6A, TP53, PI3K, and FAT4 pathway genes

(a). Overexpression of KDM6A mutants identified in ACC compared to overexpressed wildtype demonstrates increased growth. Experiments performed in triplicate. (b). KDM6A tumor-specific mutants exhibit abrogated H3K27me3 demethylase activity relative to wildtype KDM6A. Representative immunostaining of cells overexpressing wildtype or mutant KDM6A is shown with corresponding quantitation. Arrows represent cells expressing FLAG-tagged KDM6A construct. Experiments performed in triplicate. Scale bars, 20µm. (c). Heatmap shows differential expression of key TP53 transcriptional targets in TP53-pathway altered ACC samples (TP53-mutant or MDM2-amplified) compared to TP53-wildtype ACC samples and normal salivary tissue. (d). PI3K-mutant ACCs demonstrate marked downstream p-AKT and p-PRAS40 immunohistochemistry staining relative to PI3K-wildtype ACCs. Scale bars, 120µm. (e). FAT4 knockdown demonstrates increased growth in human foreskin fibroblasts (HFF-1), immortalized human salivary cells (HSG), and human salivary adenocarcinoma cells (HSY). Experiments performed in triplicate. KD, knockdown. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant (ANOVA). Error bars, ± 1 S.E.M.