Characterization of the mutational landscape of anaplastic thyroid cancer via whole-exome sequencing.

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.

Clonal Evolutionary Analysis during HER2 Blockade in HER2-Positive Inflammatory Breast Cancer: A Phase II Open-Label Clinical Trial of Afatinib +/- Vinorelbine.

BACKGROUND: Inflammatory breast cancer (IBC) is a rare, aggressive form of breast cancer associated with HER2 amplification, with high risk of metastasis and an estimated median survival of 2.9 y. We performed an open-label, single-arm phase II clinical trial (ClinicalTrials.gov NCT01325428) to investigate the efficacy and safety of afatinib, an irreversible ErbB family inhibitor, alone and in combination with vinorelbine in patients with HER2-positive IBC. This trial included prospectively planned exome analysis before and after afatinib monotherapy. METHODS AND FINDINGS: HER2-positive IBC patients received afatinib 40 mg daily until progression, and thereafter afatinib 40 mg daily and intravenous vinorelbine 25 mg/m2 weekly. The primary endpoint was clinical benefit; secondary endpoints were objective response (OR), duration of OR, and progression-free survival (PFS). Of 26 patients treated with afatinib monotherapy, clinical benefit was achieved in 9 patients (35%), 0 of 7 trastuzumab-treated patients and 9 of 19 trastuzumab-naïve patients. Following disease progression, 10 patients received afatinib plus vinorelbine, and clinical benefit was achieved in 2 of 4 trastuzumab-treated and 0 of 6 trastuzumab-naïve patients. All patients had treatment-related adverse events (AEs). Whole-exome sequencing of tumour biopsies taken before treatment and following disease progression on afatinib monotherapy was performed to assess the mutational landscape of IBC and evolutionary trajectories during therapy. Compared to a cohort of The Cancer Genome Atlas (TCGA) patients with HER2-positive non-IBC, HER2-positive IBC patients had significantly higher mutational and neoantigenic burden, more frequent gain-of-function TP53 mutations and a recurrent 11q13.5 amplification overlapping PAK1. Planned exploratory analysis revealed that trastuzumab-naïve patients with tumours harbouring somatic activation of PI3K/Akt signalling had significantly shorter PFS compared to those without (p = 0.03). High genomic concordance between biopsies taken before and following afatinib resistance was observed with stable clonal structures in non-responding tumours, and evidence of branched evolution in 8 of 9 tumours analysed. Recruitment to the trial was terminated early following the LUX-Breast 1 trial, which showed that afatinib combined with vinorelbine had similar PFS and OR rates to trastuzumab plus vinorelbine but shorter overall survival (OS), and was less tolerable. The main limitations of this study are that the results should be interpreted with caution given the relatively small patient cohort and the potential for tumour sampling bias between pre- and post-treatment tumour biopsies. CONCLUSIONS: Afatinib, with or without vinorelbine, showed activity in trastuzumab-naïve HER2-positive IBC patients in a planned subgroup analysis. HER2-positive IBC is characterized by frequent TP53 gain-of-function mutations and a high mutational burden. The high mutational load associated with HER2-positive IBC suggests a potential role for checkpoint inhibitor therapy in this disease. TRIAL REGISTRATION: ClinicalTrials.gov NCT01325428.

Increased Levels of Macrophage Inflammatory Proteins Result in Resistance to R5-Tropic HIV-1 in a Subset of Elite Controllers.

UNLABELLED: Elite controllers (ECs) are a rare group of HIV seropositive individuals who are able to control viral replication without antiretroviral therapy. The mechanisms responsible for this phenotype, however, have not been fully elucidated. In this study, we examined CD4(+) T cell resistance to HIV in a cohort of elite controllers and explored transcriptional signatures associated with cellular resistance. We demonstrate that a subgroup of elite controllers possess CD4(+) T cells that are specifically resistant to R5-tropic HIV while remaining fully susceptible to X4-tropic and vesicular stomatitis virus G (VSV-G)-pseudotyped viruses. Transcriptome analysis revealed 17 genes that were differentially regulated in resistant elite controllers relative to healthy controls. Notably, the genes encoding macrophage inflammatory protein 1α (MIP-1α), CCL3 and CCL3L1, were found to be upregulated. The MIP-1α, MIP-1ß, and RANTES chemokines are natural ligands of CCR5 and are known to interfere with HIV replication. For three elite controllers, we observed increased production of MIP-1α and/or MIP-1ß at the protein level. The supernatant from resistant EC cells contained MIP-1α and MIP-1ß and was sufficient to confer R5-tropic resistance to susceptible CD4(+) T cells. Additionally, this effect was reversed by using inhibitory anti-MIP antibodies. These results suggest that the T cells of these particular elite controllers may be naturally resistant to HIV infection by blocking R5-tropic viral entry. IMPORTANCE: HIV is a pandemic health problem, and the majority of seropositive individuals will eventually progress to AIDS unless antiretroviral therapy (ART) is administered. However, rare patients, termed elite controllers, have a natural ability to control HIV infection in the absence of ART, but the mechanisms by which they achieve this phenotype have not been fully explored. This paper identifies one mechanism that may contribute to this natural resistance: some elite controllers have CD4(+) T cells that produce high levels of MIP chemokines, which block R5-tropic HIV entry. This mechanism could potentially be exploited to achieve a therapeutic effect in other HIV-seropositive individuals.

Shifting patterns of genomic variation in the somatic evolution of papillary thyroid carcinoma.

BACKGROUND: Cancer is increasingly understood to arise in the context of dynamically evolving genomes with continuously generated variants subject to selective pressures. Diverse mutations have been identified in papillary thyroid carcinoma (PTC), but unifying theories underlying genomic change are lacking. Applying a framework of somatic evolution, we sought to broaden understanding of the PTC genome through identification of global trends that help explain risk of tumorigenesis. METHODS: Exome sequencing was performed on 53 PTC and matched adjacent non-tumor thyroid tissues (ANT). Single nucleotide substitution (SNS) signatures from each sample pair were divided into three subsets based on their presence in tumor, non-tumor thyroid, or both. Nine matched blood samples were sequenced and SNS signatures intersected with these three subsets. The intersected genomic signatures were used to define branch-points in the evolution of the tumor genome, distinguishing variants present in the tissues' common ancestor cells from those unique to each tissue type and therefore acquired after genomic divergence of the tumor, non-tumor, and blood samples. RESULTS: Single nucleotide substitutions shared by the tumor and the non-tumor thyroid were dominated by C-to-T transitions, whereas those unique to either tissue type were enriched for C-to-A transversions encoding non-synonymous, predicted-deleterious variants. On average, SNSs of matched blood samples were 81 % identical to those shared by tumor and non-tumor thyroid, but only 12.5 % identical to those unique to either tissue. Older age and BRAF mutation were associated with increased SNS burden. CONCLUSIONS: The current study demonstrates novel patterns of genomic change in PTC, supporting a theory of somatic evolution in which the zygote's germline genome undergoes continuous remodeling to produce progressively differentiated, tissue-specific signatures. Late somatic events in thyroid tissue demonstrate shifted mutational spectra compared to earlier polymorphisms. These late events are enriched for predicted-deleterious variants, suggesting a mechanism of genomic instability in PTC tumorigenesis.

Whole-exome sequencing defines the mutational landscape of pheochromocytoma and identifies KMT2D as a recurrently mutated gene.

As subsets of pheochromocytomas (PCCs) lack a defined molecular etiology, we sought to characterize the mutational landscape of PCCs to identify novel gene candidates involved in disease development. A discovery cohort of 15 PCCs wild type for mutations in PCC susceptibility genes underwent whole-exome sequencing, and an additional 83 PCCs served as a verification cohort for targeted sequencing of candidate mutations. A low rate of nonsilent single nucleotide variants (SNVs) was detected (6.1/sample). Somatic HRAS and EPAS1 mutations were observed in one case each, whereas the remaining 13 cases did not exhibit variants in established PCC genes. SNVs aggregated in apoptosis-related pathways, and mutations in COSMIC genes not previously reported in PCCs included ZAN, MITF, WDTC1, and CAMTA1. Two somatic mutations and one constitutional variant in the well-established cancer gene lysine (K)-specific methyltransferase 2D (KMT2D, MLL2) were discovered in one sample each, prompting KMT2D screening using focused exome-sequencing in the verification cohort. An additional 11 PCCs displayed KMT2D variants, of which two were recurrent. In total, missense KMT2D variants were found in 14 (11 somatic, two constitutional, one undetermined) of 99 PCCs (14%). Five cases displayed somatic mutations in the functional FYR/SET domains of KMT2D, constituting 36% of all KMT2D-mutated PCCs. KMT2D expression was upregulated in PCCs compared to normal adrenals, and KMT2D overexpression positively affected cell migration in a PCC cell line. We conclude that KMT2D represents a recurrently mutated gene with potential implication for PCC development.

Novel somatic mutations in primary hyperaldosteronism are related to the clinical, radiological and pathological phenotype.

UNLABELLED: Aldosterone-producing adenomas (APAs) and bilateral adrenal hyperplasia are important causes of secondary hypertension. Somatic mutations in KCNJ5, CACNA1D, ATP1A1, ATP2B3 and CTNNB1 have been described in APAs. OBJECTIVE: To characterize clinical-pathological features in APAs and unilateral adrenal hyperplasia, and correlate them with genotypes. DESIGN: Retrospective study. SUBJECTS AND MEASUREMENTS: Clinical and pathological characteristics of 90 APAs and seven diffusely or focally hyperplastic adrenal glands were reviewed, and samples were examined for mutations in known disease genes by Sanger or exome sequencing. RESULTS: Mutation frequencies were as follows: KCNJ5, 37·1%; CACNA1D, 10·3%; ATP1A1, 8·2%; ATP2B3, 3·1%; and CTNNB1, 2·1%. Previously unidentified mutations included I157K, F154C and two insertions (I150_G151insM and I144_E145insAI) in KCNJ5, all close to the selectivity filter, V426G_V427Q_A428_L433del in ATP2B3 and A39Efs*3 in CTNNB1. Mutations in KCNJ5 were associated with female and other mutations with male gender (P = 0·007). On computed tomography, KCNJ5-mutant tumours displayed significantly greater diameter (P = 0·023), calculated area (P = 0·002) and lower precontrast Hounsfield units (P = 0·0002) vs tumours with mutations in other genes. Accordingly, KCNJ5-mutant tumours were predominantly comprised of lipid-rich fasciculata-like clear cells, whereas other tumours were heterogeneous (P = 5 × 10(-6) vs non-KCNJ5 mutant and P = 0·0003 vs wild-type tumours, respectively). CACNA1D mutations were present in two samples with hyperplasia without adenoma. CONCLUSIONS: KCNJ5-mutant tumours appear to be associated with fasciculata-like clear cell predominant histology and tend to be larger with a characteristic imaging phenotype. Novel somatic KCNJ5 variants likely cause adenomas by loss of potassium selectivity, similar to previously described mutations.

Influence of transformational leadership on innovative work behavior and task performance of individuals: The mediating role of knowledge sharing.

This research tries to investigate the dynamic link between higher education institution (HEIs) transformational leaders (TFL) and follower's outcome innovative work behavior (IWB) and Task Performance) through Knowledge sharing (KNS) in Pakistan. Using quantitative design an adopted construct was used to obtain response from HEIs leaders and employees behavior. The obtained information was analyzed through structural equation modeling (SEM) technique via Smart PLS. Results depict that direct link between University Transformational leadership and employees Innovative work behavior as well as Task Performance. The results further postulate that KNS mediate the relationship between Transformational leadership and employees TSP in the context of HEIs. Surprisingly, KNS could not evident to become a mediating variable to strengthen the relationship between transformational Leadership and employees IWB in the HEIs sector of Pakistan. In addition to enhancing the theoretical comprehension of higher education leadership, the outcomes of this article provide that promoting knowledge sharing culture is valuable asset for both existing and future HEIs leaders in order to promote the culture of innovation and creativity. Although recent studies investigate the role of KNS as a mediator, however the current study use KNS as contemporaneous intervening variable for IWB and Task Performance for the first time. The study also confirms theoretical underpinning of social exchange mechanism in strengthening the relationship between leader member's continuum.

The Impact of Green Practices in Value Chain on Firm Performance in the Context of a Developing Country.

Background: Companies need to go green to remain relevant. Previous studies have confirmed that going green leads to superior performance for companies. However, research of green practices in a value chain requires further attention, especially in identifying the green value chain activities that lead to superior performance. A value chain analysis focuses on identifying competitive advantages of firms through five primary and four support activities. Methods: This study extends from Ong et al. (2019), who developed and validated the instrument for the nine green value chain activities, to also examine their effect on firm performance. The 207 valid responses in this study are collected through a questionnaire survey of the sampling frame consisting of companies in Bursa Malaysia and the Federation of Malaysian Manufacturers Directory. Results: The findings reveal that the companies' green practices in primary value chain activities are higher than in the supporting value chain activities. Technological development is the activity with the lowest green attention among the nine value chain activities. Our multiple regression analysis shows that 25% of the variation in firm performance can be significantly explained by the nine green value chain activities. In terms of the individual green value chain activities, green technology development is the only activity that can positively and significantly explain firm performance. Conclusions: The findings of the study suggest that companies intending to build their green core competence need to engage in green technology development. Companies that go green for the purpose of complying to regulations and fulfilling minimum customers' demands can still embed green practices into their green value chain without compromising their performance.

Computational Methods for Analysis of Tumor Clonality and Evolutionary History.

Cancer is an evolutionary process. Recent advances in sequencing technologies have allowed us to investigate intratumor heterogeneity at the single nucleotide level. Here, we describe computational methods that use sequencing data to identify genetically distinct tumor subclones and reconstruct tumor evolutionary histories.

Chromosome 19 amplification correlates with advanced disease in adrenocortical carcinoma.

BACKGROUND: Familial syndromes with specific genetic drivers account for a subset of adrenocortical carcinomas (ACCs), but the genomic underpinnings of sporadic cases remain poorly understood. Recent advances in copy number variation (CNV) prediction from exome sequencing are facilitating exploration of genomic rearrangements common to these carcinomas. METHODS: ACC and matched, nontumor samples underwent exome sequencing. CNVs were predicted using coverage-depth comparison. Clinicopathologic characteristics of amplification- and deletion-dominant samples were compared and pathway enrichment analysis performed for regions with significant variation. RESULTS: CNVs are distributed broadly across the ACC genome. Individual signatures demonstrate amplification or deletion dominance. Areas of recurrent amplification include chromosomes 5, 12, 19, and 20, whereas chromosomes 1, 10, 18, and 22 are deletion prone. Large-scale amplification of chromosome 19 occurred in 12 of 19 cases (63%), including 6 of 8 amplification-dominant samples (75%) and was associated with stage III/IV disease (P = .002). Genes within this amplified region are overrepresented among the adrenal hyperplasia and steroid biosynthesis pathways (P = 4.2(-5) and 2.5(-5), respectively). CONCLUSION: CNV detection via exome sequencing allows high-resolution cataloging of structural variations in ACC. Large-scale, recurrent amplifications encompassing known adrenal-specific gene pathways correlate with tumor stage. Further functional analysis of individual genes within these regions could provide mechanistic insight into specific drivers underlying pathogenesis and progression of ACC.

Mutational landscape of MCPyV-positive and MCPyV-negative Merkel cell carcinomas with implications for immunotherapy.

Merkel cell carcinoma (MCC) is a rare but highly aggressive cutaneous neuroendocrine carcinoma, associated with the Merkel cell polyomavirus (MCPyV) in 80% of cases. To define the genetic basis of MCCs, we performed exome sequencing of 49 MCCs. We show that MCPyV-negative MCCs have a high mutation burden (median of 1121 somatic single nucleotide variants (SSNVs) per-exome with frequent mutations in RB1 and TP53 and additional damaging mutations in genes in the chromatin modification (ASXL1, MLL2, and MLL3), JNK (MAP3K1 and TRAF7), and DNA-damage pathways (ATM, MSH2, and BRCA1). In contrast, MCPyV-positive MCCs harbor few SSNVs (median of 12.5 SSNVs/tumor) with none in the genes listed above. In both subgroups, there are rare cancer-promoting mutations predicted to activate the PI3K pathway (HRAS, KRAS, PIK3CA, PTEN, and TSC1) and to inactivate the Notch pathway (Notch1 and Notch2). TP53 mutations appear to be clinically relevant in virus-negative MCCs as 37% of these tumors harbor potentially targetable gain-of-function mutations in TP53 at p.R248 and p.P278. Moreover, TP53 mutational status predicts death in early stage MCC (5-year survival in TP53 mutant vs wild-type stage I and II MCCs is 20% vs. 92%, respectively; P = 0.0036). Lastly, we identified the tumor neoantigens in MCPyV-negative and MCPyV-positive MCCs. We found that virus-negative MCCs harbor more tumor neoantigens than melanomas or non-small cell lung cancers (median of 173, 65, and 111 neoantigens/sample, respectively), two cancers for which immune checkpoint blockade can produce durable clinical responses. Collectively, these data support the use of immunotherapies for virus-negative MCCs.

DNA replication stress mediates APOBEC3 family mutagenesis in breast cancer.

BACKGROUND: The APOBEC3 family of cytidine deaminases mutate the cancer genome in a range of cancer types. Although many studies have documented the downstream effects of APOBEC3 activity through next-generation sequencing, less is known about their upstream regulation. In this study, we sought to identify a molecular basis for APOBEC3 expression and activation. RESULTS: HER2 amplification and PTEN loss promote DNA replication stress and APOBEC3B activity in vitro and correlate with APOBEC3 mutagenesis in vivo. HER2-enriched breast carcinomas display evidence of elevated levels of replication stress-associated DNA damage in vivo. Chemical and cytotoxic induction of replication stress, through aphidicolin, gemcitabine, camptothecin or hydroxyurea exposure, activates transcription of APOBEC3B via an ATR/Chk1-dependent pathway in vitro. APOBEC3B activation can be attenuated through repression of oncogenic signalling, small molecule inhibition of receptor tyrosine kinase signalling and alleviation of replication stress through nucleoside supplementation. CONCLUSION: These data link oncogene, loss of tumour suppressor gene and drug-induced replication stress with APOBEC3B activity, providing new insights into how cytidine deaminase-induced mutagenesis might be activated in tumourigenesis and limited therapeutically.

Genetic landscape of metastatic and recurrent head and neck squamous cell carcinoma.

BACKGROUND: Recurrence and/or metastasis occurs in more than half of patients with head and neck squamous cell carcinoma (HNSCC), and these events pose the greatest threats to long-term survival. We set out to identify genetic alterations that underlie recurrent/metastatic HNSCC. METHODS: Whole-exome sequencing (WES) was performed on genomic DNA extracted from fresh-frozen whole blood and patient-matched tumor pairs from 13 HNSCC patients with synchronous lymph node metastases and 10 patients with metachronous recurrent tumors. Mutational concordance within and between tumor pairs was used to analyze the spatiotemporal evolution of HNSCC in individual patients and to identify potential therapeutic targets for functional evaluation. RESULTS: Approximately 86% and 60% of single somatic nucleotide variants (SSNVs) identified in synchronous nodal metastases and metachronous recurrent tumors, respectively, were transmitted from the primary index tumor. Genes that were mutated in more than one metastatic or recurrent tumor, but not in the respective primary tumors, include C17orf104, inositol 1,4,5-trisphosphate receptor, type 3 (ITPR3), and discoidin domain receptor tyrosine kinase 2 (DDR2). Select DDR2 mutations have been shown to confer enhanced sensitivity to SRC-family kinase (SFK) inhibitors in other malignancies. Similarly, HNSCC cell lines harboring endogenous and engineered DDR2 mutations were more sensitive to the SFK inhibitor dasatinib than those with WT DDR2. CONCLUSION: In this WES study of patient-matched tumor pairs in HNSCC, we found synchronous lymph node metastases to be genetically more similar to their paired index primary tumors than metachronous recurrent tumors. This study outlines a compendium of somatic mutations in primary, metastatic, and/or recurrent HNSCC cancers, with potential implications for precision medicine approaches. FUNDING: National Cancer Institute, American Cancer Society, Agency for Science, Technology and Research of Singapore, and Gilead Sciences Inc.

PRKACA mutations in cortisol-producing adenomas and adrenal hyperplasia: a single-center study of 60 cases.

OBJECTIVE: Cortisol excess due to adrenal adenomas or hyperplasia causes Cushing's syndrome. Recent genetic studies have identified a somatic PRKACA(L206R) mutation as a cause of cortisol-producing adenomas. We aimed to compare the clinical features of PRKACA-mutant lesions with those of CTNNB1 mutations, and to search for similar mutations in unilateral hyperplasia or tumors co-secreting aldosterone. DESIGN, PATIENTS, AND METHODS: In this study, 60 patients with cortisol excess who had adrenalectomies at our institution between 1992 and 2013 were assessed, and somatic mutations were determined by Sanger sequencing. A total of 36 patients had overt Cushing's syndrome, the remainder were subclinical: 59 cases were adenomas (three bilateral) and one was classified as hyperplasia. Four tumors had proven co-secretion of aldosterone. RESULTS: Among cortisol-secreting unilateral lesions without evidence of co-secretion (n=52), we identified somatic mutations in PRKACA (L206R) in 23.1%, CTNNB1 (S45P, S45F) in 23.1%, GNAS (R201C) in 5.8%, and CTNNB1+GNAS (S45P, R201H) in 1.9%. PRKACA and GNAS mutations were mutually exclusive. Of the co-secreting tumors, two (50%) had mutations in KCNJ5 (G151R and L168R). The hyperplastic gland showed a PRKACA(L206R) mutation, while patients with bilateral adenomas did not have known somatic mutations. PRKACA-mutant lesions were associated with younger age, overt Cushing's syndrome, and higher cortisol levels vs non-PRKACA-mutant or CTNNB1-mutant lesions. CTNNB1 mutations were more significantly associated with right than left lesions. CONCLUSIONS: PRKACA(L206R) is present not only in adenomas, but also in unilateral hyperplasia and is associated with more severe autonomous cortisol secretion. Bilateral adenomas may be caused by yet-unknown germline mutations.

Whole-exome sequencing characterizes the landscape of somatic mutations and copy number alterations in adrenocortical carcinoma.

CONTEXT: Adrenocortical carcinoma (ACC) is a rare and lethal malignancy with a poorly defined etiology, and the molecular genetics of ACC are incompletely understood. OBJECTIVE: To utilize whole-exome sequencing for genetic characterization of the underlying somatic mutations and copy number alterations present in ACC. DESIGN: 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. RESULTS: 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. CONCLUSIONS: 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.

Genomic landscape of cutaneous T cell lymphoma.

Cutaneous T cell lymphoma (CTCL) is a non-Hodgkin lymphoma of skin-homing T lymphocytes. We performed exome and whole-genome DNA sequencing and RNA sequencing on purified CTCL and matched normal cells. The results implicate mutations in 17 genes in CTCL pathogenesis, including genes involved in T cell activation and apoptosis, NF-κB signaling, chromatin remodeling and DNA damage response. CTCL is distinctive in that somatic copy number variants (SCNVs) comprise 92% of all driver mutations (mean of 11.8 pathogenic SCNVs versus 1.0 somatic single-nucleotide variant per CTCL). These findings have implications for new therapeutics.

Recurrent gain of function mutation in calcium channel CACNA1H causes early-onset hypertension with primary aldosteronism.

Many Mendelian traits are likely unrecognized owing to absence of traditional segregation patterns in families due to causation by de novo mutations, incomplete penetrance, and/or variable expressivity. Genome-level sequencing can overcome these complications. Extreme childhood phenotypes are promising candidates for new Mendelian traits. One example is early onset hypertension, a rare form of a global cause of morbidity and mortality. We performed exome sequencing of 40 unrelated subjects with hypertension due to primary aldosteronism by age 10. Five subjects (12.5%) shared the identical, previously unidentified, heterozygous CACNA1H(M1549V) mutation. Two mutations were demonstrated to be de novo events, and all mutations occurred independently. CACNA1H encodes a voltage-gated calcium channel (CaV3.2) expressed in adrenal glomerulosa. CACNA1H(M1549V) showed drastically impaired channel inactivation and activation at more hyperpolarized potentials, producing increased intracellular Ca(2+), the signal for aldosterone production. This mutation explains disease pathogenesis and provides new insight into mechanisms mediating aldosterone production and hypertension.

Recurrent activating mutation in PRKACA in cortisol-producing adrenal tumors.

Adrenal tumors autonomously producing cortisol cause Cushing's syndrome. 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 (ß-catenin) or GNAS (Gαs). 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, PRKAR1A. Leu206Arg PRKACA loses PRKAR1A binding, increasing the phosphorylation of downstream targets. PKA activity induces cortisol production and cell proliferation, providing a mechanism for tumor development. These findings define distinct mechanisms underlying adrenal cortisol-producing tumors.