Anaplastic thyroid carcinoma (ATC) is a frequently lethal malignancy that is often unresponsive to available therapeutic strategies. The tumorigenesis of ATC and its relationship to the widely prevalent well-differentiated thyroid carcinomas are unclear. We have analyzed 22 cases of ATC as well as 4 established ATC cell lines using whole-exome sequencing. A total of 2674 somatic mutations (121/sample) were detected. Ontology analysis revealed that the majority of variants aggregated in the MAPK, ErbB and RAS signaling pathways. Mutations in genes related to malignancy not previously associated with thyroid tumorigenesis were observed, including mTOR, NF1, NF2, MLH1, MLH3, MSH5, MSH6, ERBB2, EIF1AX and USH2A; some of which were recurrent and were investigated in 24 additional ATC cases and 8 ATC cell lines. Somatic mutations in established thyroid cancer genes were detected in 14 of 22 (64%) tumors and included recurrent mutations in BRAF, TP53 and RAS-family genes (6 cases each), as well as PIK3CA (2 cases) and single cases of CDKN1B, CDKN2C, CTNNB1 and RET mutations. BRAF V600E and RAS mutations were mutually exclusive; all ATC cell lines exhibited a combination of mutations in either BRAF and TP53 or NRAS and TP53. A hypermutator phenotype in two cases with >8 times higher mutational burden than the remaining mean was identified; both cases harbored unique somatic mutations in MLH mismatch-repair genes. This first comprehensive exome-wide analysis of the mutational landscape of ATC identifies novel genes potentially associated with ATC tumorigenesis, some of which may be targets for future therapeutic intervention.

Richard Lifton and colleagues identify a recurrent activating mutation in PRKACA, which encodes the catalytic subunit of protein kinase A, in cortisol-producing adrenal tumors. They further show that the mutation results in loss of binding by the regulatory subunit PRKAR1A, leading to increased phosphorylation of downstream targets. Adrenal tumors autonomously producing cortisol cause Cushing's syndrome1,2,3,4. We performed exome sequencing of 25 tumor-normal pairs and identified 2 subgroups. Eight tumors (including three carcinomas) had many somatic copy number variants (CNVs) with frequent deletion of CDC42 and CDKN2A, amplification of 5q31.2 and protein-altering mutations in TP53 and RB1. Seventeen tumors (all adenomas) had no somatic CNVs or TP53 or RB1 mutations. Six of these had known gain-of-function mutations in CTNNB1 (β-catenin)5,6 or GNAS (Gαs)7,8. Six others had somatic mutations in PRKACA (protein kinase A (PKA) catalytic subunit) resulting in a p.Leu206Arg substitution. Further sequencing identified this mutation in 13 of 63 tumors (35% of adenomas with overt Cushing's syndrome). PRKACA, GNAS and CTNNB1 mutations were mutually exclusive. Leu206 directly interacts with the regulatory subunit of PKA, PRKAR1A9,10. Leu206Arg PRKACA loses PRKAR1A binding, increasing the phosphorylation of downstream targets. PKA activity induces cortisol production and cell proliferation11,12,13,14,15, providing a mechanism for tumor development. These findings define distinct mechanisms underlying adrenal cortisol-producing tumors.

Adrenocortical carcinoma (ACC) is a rare and lethal malignancy with a poorly defined etiology, and the molecular genetics of ACC are incompletely understood. To utilize whole-exome sequencing for genetic characterization of the underlying somatic mutations and copy number alterations present in ACC. Screening for somatic mutation events and copy number alterations (CNAs) was performed by comparative analysis of tumors and matched normal samples from 41 patients with ACC. In total, 966 nonsynonymous somatic mutations were detected, including 40 tumors with a mean of 16 mutations per sample and one tumor with 314 mutations. Somatic mutations in ACC-associated genes included TP53 (8/41 tumors, 19.5%) and CTNNB1 (4/41, 9.8%). Genes with potential disease-causing mutations included GNAS, NF2, and RB1, and recurrently mutated genes with unknown roles in tumorigenesis comprised CDC27, SCN7A, and SDK1. Recurrent CNAs included amplification at 5p15.33 including TERT (6/41, 14.6%) and homozygous deletion at 22q12.1 including the Wnt repressors ZNRF3 and KREMEN1 (4/41 9.8% and 3/41, 7.3%, respectively). Somatic mutations in ACC-established genes and recurrent ZNRF3 and TERT loci CNAs were mutually exclusive in the majority of cases. Moreover, gene ontology identified Wnt signaling as the most frequently mutated pathway in ACCs. These findings highlight the importance of Wnt pathway dysregulation in ACC and corroborate the finding of homozygous deletion of Wnt repressors ZNRF3 and KREMEN1. Overall, mutations in either TP53 or CTNNB1 as well as focal CNAs at the ZNRF3 or TERT loci denote mutually exclusive events, suggesting separate mechanisms underlying the development of these tumors.

The telomerase reverse transcriptase gene (TERT) encodes the reverse transcriptase component of the telomerase complex, which is essential for telomere stabilization and cell immortalization. Recent studies have demonstrated a transcriptional activation role for theTERTpromoter mutations C228T and C250T in many human cancers, as well as a role in aggressive disease with potential clinical applications. Although telomerase activation is known in adrenal tumors, the underlying mechanisms are not established. We assessed C228T and C250TTERTmutations by direct Sanger sequencing in tumors of the adrenal gland, and further evaluated potential associations with clinical parameters and telomerase activation. A total of 199 tumors were evaluated, including 34 adrenocortical carcinomas (ACC), 47 adrenocortical adenomas (ACA), 105 pheochromocytomas (PCC; ten malignant and 95 benign), and 13 abdominal paragangliomas (PGL; nine malignant and four benign).TERTexpression levels were determined by quantitative RT-PCR. The C228T mutation was detected in 4/34 ACCs (12%), but not in any ACA (P=0.028). C228T was also observed in one benign PCC and in one metastatic PGL. The C250T mutation was not observed in any case. In the ACC and PGL groups,TERTmutation-positive cases exhibitedTERTexpression, indicating telomerase activation; however, since expression was also revealed inTERTWT cases, this could denote additional mechanisms ofTERTactivation. To conclude, theTERTpromoter mutation C228T is a recurrent event associated withTERTexpression in ACCs, but rarely occurs in PGL and PCC. The involvement of theTERTgene in ACC represents a novel mutated gene in this entity.

Parathyroid carcinoma (PTCA) is an exceptionally rare malignancy, often with a clinical presentation similar to that of benign atypical parathyroid adenoma. Its low incidence portends unclear guidelines for management. Accordingly, thorough examination of clinical and pathologic variables was undertaken to distinguish between PTCA and atypical adenomas. This was a retrospective analysis of a prospective database at a tertiary academic referral center. Between September 2001 and April 2014, 3,643 patients were referred for surgical treatment of PHPT. Of these, 52 harbored aggressive parathyroid tumors: parathyroid carcinomas (n=18) and atypical adenomas (n=34). We analyzed the surgical and clinicopathologic tumor characteristics, and did a statistical analysis. We measured preoperative and intraoperative variables, and postoperative and pathologic outcomes. Parathyroid carcinoma patients present with significantly increased tumor size (3.5 cm vs 2.4 cm, respectively; p=0.002), mean serum calcium (13.0 vs 11.8 mg/dL, respectively; p=0.003) and intact parathyroid hormone (iPTH) levels (489 vs 266 pg/mL, respectively; p=0.04), and a higher incidence of hypercalcemic crisis, compared with patients with atypical adenomas (50% vs 19%, respectively; p=0.072). Parathyroid carcinoma more frequently lacks a distinct capsule (47.1% vs 12.9%, respectively; p=0.03) and adheres to adjacent structures (77.8% vs 20.6%, respectively; p=0.017). Of note, there was no significant difference in loss of parafibromin expression between groups. Clinical distinction between PTCA and atypical adenomas is of critical importance in determining the appropriate extent of resection and follow-up. Loss of parafibromin has not been shown to distinguish between PTCA and atypical adenoma; clearer definition of clinicopathologic criteria for PTCA is warranted and may lead to improved postoperative management.