ARTICLE DOI: 10.1038/s41467-018-06366-z OPEN Metastatic adrenocortical carcinoma displays higher mutation rate and tumor heterogeneity than primary tumors Sudheer Kumar Gara1, Justin Lack2, Lisa Zhang1, Emerson Harris1, Margaret Cam2 & Electron Kebebew1,3 Adrenocortical cancer (ACC) is a rare cancer with poor prognosis and high mortality due to metastatic disease. All reported genetic alterations have been in primary ACC, and it is 1234567890():,; unknown if there is molecular heterogeneity in ACC. Here, we report the genetic changes associated with metastatic ACC compared to primary ACCs and tumor heterogeneity. We performed whole-exome sequencing of 33 metastatic tumors. The overall mutation rate (per megabase) in metastatic tumors was 2.8-fold higher than primary ACC tumor samples. We found tumor heterogeneity among different metastatic sites in ACC and discovered recurrent mutations in several novel genes. We observed 37–57% overlap in genes that are mutated among different metastatic sites within the same patient. We also identified new therapeutic targets in recurrent and metastatic ACC not previously described in primary ACCs. 1 Endocrine Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. 2 Center for Cancer Research, Collaborative Bioinformatics Resource, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. 3 Department of Surgery and Stanford Cancer Institute, Stanford University, Stanford, CA 94305, USA. Correspondence and requests for materials should be addressed to E.K. (email: [email protected]) NATURE COMMUNICATIONS | (2018) 9:4172 | DOI: 10.1038/s41467-018-06366-z | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-06366-z drenocortical carcinoma (ACC) is a rare malignancy with types including primary ACC from the TCGA to understand our A0.7–2 cases per million per year1,2. The five-year survival findings in the context of not only primary ACC but also other rate for patients with resectable tumors is less than 30%. primary cancer types (Fig. 1c). Next, we tested the idea whether Particularly, patients with metastatic disease have a median sur- primary ACC tumors of the TCGA from patients with metastasis vival of less than 1 year. Unfortunately, most patients with ACC has any effect on mutation burden and found no significant have locally advanced cancer or metastasis at the time of diag- difference in the mutation rate by tumor stage supporting our nosis. Deaths due to ACC are associated with metastatic disease. findings that the mutation burden is higher in metastatic ACC as Our understanding of the pathogenesis of ACC has been compared to primary ACC (Fig. 1d). We also analyzed the rela- greatly improved over the past decade. Molecular studies have tive proportions of the six possible base-pair substitutions in the demonstrated that TP53 inactivating mutations, CTNNB1 acti- transcribed and non-transcribed strands among all the metastatic vating mutations, IGF2 overexpression, damaging mutations in ACC tumors to understand their relevance with other solid ZNRF3, and high-level amplification of TERT are common and tumors including primary ACC tumors. Similar to primary ACC key drivers of ACC3–6. Furthermore, the international con- from the TCGA data and most other solid tumors13,14, metastatic sortium of The Cancer Genome Atlas (TCGA) has identified ACC were characterized by a predominance of C > T transitions additional ACC driver genes including PRKAR1A, RPL22, TERF2, (Fig. 1e). However, we did not notice any significant difference of CCNE1, and NF17. This study further showed that a whole- T > C mutations between transcribed and non-transcribed genome doubling event is a marker for ACC progression and strands in metastatic ACC, unlike in primary ACC tumors prognosis7. Multiple studies have reported that activating and (Fig. 1e, f). inactivating alterations in the TP53/Rb and WNT pathways are We next analyzed genes recurrently mutated in multiple key molecular events in the pathogenesis of ACC4,7. However, all metastatic ACC tumor samples and compared their status in of the above studies are confined to primary ACC, and death primary ACC from the TCGA (Fig. 1g). Similar to primary ACC, from ACC is primarily due to recurrent or metastatic disease. we observed that CTNNB1, DNHD1, and TTN were frequently Despite recent advances in our understanding of ACC, the mutated in metastatic ACC. Nevertheless, we have also identified therapeutic options for ACC are still limited, and treatment with genes such as ENTHD1, HELZ2, PCDH12, SHANK1, and WDR66 combination mitotane-etoposide, doxorubicin, and cisplatin that were more frequently mutated in metastatic ACC but not in results in low response rates8,9. Studies on targeted therapeutic primary tumors (Fig. 1g). Next, we selected the frequently options using IGF-targeting agents or tyrosine kinase inhibitors mutated genes in primary ACC and compared their mutation have not shown good efficacy10–12. It is evident that most of the status in metastatic ACC (Fig. 1h). As expected, the majority of advanced or late-stage cancers or treatment-resistant tumors the genes that are mutated in primary ACC were also mutated in adapt differently and gain additional mutations. Mutations ana- metastatic ACC (Fig. 1h). We also validated 5/5 tested mutations lysis of late-stage cancer tumor sites and treatment-resistant using droplet digital polymerase chain reaction (ddPCR) and 72 tumors has shown biologically informative genomic alterations. of 77 mutations using Sanger sequencing (Supplementary Figs. 1, Given this, it is important to understand the nature of recurrent 2). In addition, we noticed that two metastatic ACCs from the and metastatic ACC to inform candidate genetic changes same patient (Case 1) had a strong hypermutation phenotype involved in cancer progression and to identify therapeutic targets. (Supplementary Table 1), and two additional patients had > /10 Therefore, our study objectives were to analyze the genomic single-nucleotide polymorphisms (SNPs) per megabase compared landscape of metastatic ACC, the nature of different metastatic to a median of 2.63 SNPs per megabase for all metastatic ACCs in tumor sites (tumor heterogeneity), and their commonality and our data set. To determine if these hypermutation phenotypes are differences compared to primary ACC. driven by DNA repair defects, we examined copy number variations (CNVs), somatic SNPs and insertions and deletions (INDELs), and germline SNPs and INDELs for putative loss-of- Results function (LoF) mutations (homozygous deletions; nonsense, non- Metastatic ACC has a higher mutation rate. We performed frameshift INDELs > = 3 amino acids; and frameshift mutations whole-exome sequencing on 33 histologically confirmed meta- in the Wood DNA repair genes)15. For the two tumor samples static ACCs (lung, liver, pancreas, kidney, peritoneum, and other with the highest mutation rates, the only LoF mutation shared tissue sites with matched peripheral blood sample DNA) collected between both tumors is a heterozygous 9-base germline insertion from 14 patients with metastatic ACC. We identified in exon 1of MSH3 (Supplementary Table 2 and Supplementary 15,321 somatic mutations in the coding region of the genome in Fig. 3), which was amplified in copy number and had (allele 33 tumors; 5928 nonsynonymous mutations and 9393 silent frequency > 0.9 in both samples) complete loss of heterozygosity mutations (Supplementary Table 1). The mean somatic mutation (LOH) in both tumor samples. Somatic INDELs overlapping this rate in the coding region of metastatic ACC was 10.17 mutations mutation were also detected for three primary ACCs, and while it per megabase, with 3.93 and 6.24 mutations per megabase for is unclear if any of these samples have LOH and fixation of these nonsynonymous and silent mutations, respectively, (Supple- alterations, one of the three samples (TCGA-PK-A5HB-01) has a mentary Table 1). We compared the overall somatic mutation hypermutation phenotype, possessing the second-highest muta- burden between our cohort of metastatic ACC and the primary tion rate of the 92 primary ACCs available (24). For the ACC ACC tumors from the TCGA, reprocessed through our somatic metastasis with the second-highest mutation rate (Tumor 20; variant calling pipeline (see methods) to eliminate pipeline-driven Supplementary Table 1), no germline LoF events were detected, difference in variant call rates. The ACC metastatic tumors had but multiple somatic LoF events were detected that may be 2.8-fold higher median mutation rate compared to primary ACC contributing to its hypermutation phenotype. We detected a focal (Fig. 1a). Since the mutation rate was compared between primary homozygous deletion of MSH6 (Supplementary Table 2 and ACC from the TCGA cohort to the metastatic cohort, we per- Supplementary Fig. 4), which was not detected in any of the 92 formed the same analysis in one matched primary ACC with a primary ACC tumors available in cBioPortal (Supplementary metastatic lung ACC tumor from the same patient. We found Table 2 and Supplementary Fig. 3). In addition, we detected a that metastatic lung ACC had a threefold higher mutation rate frameshift mutation in ATM that has become homozygous compared to the primary ACC (Fig. 1b). In addition, we com- through an LoH event (Supplementary Table 2 and Supplemen- pared the mutation rate of metastatic ACC with other cancer tary Fig. 4B) and is fixed in the tumor sample (allele