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Anaplastic thyroid carcinoma is arguably the most lethal human malignancy. It often co-occurs with differentiated thyroid cancers, yet the molecular origins of its aggressivity are unknown. We sequenced tumor DNA from 329 regions of thyroid cancer, including 213 from patients with primary anaplastic thyroid carcinomas. We also whole genome sequenced 9 patients using multi-region sequencing of both differentiated and anaplastic thyroid cancer components. Using these data, we demonstrate thatanaplastic thyroid carcinomas have a higher burden of mutations than other thyroid cancers, with distinct mutational signatures and molecular subtypes. Further, different cancer driver genes are mutated in anaplastic and differentiated thyroid carcinomas, even those arising in a single patient. Finally, we unambiguously demonstrate that anaplastic thyroid carcinomas share a genomic origin with co-occurring differentiated carcinomas and emerge from a common malignant field through acquisition of characteristic clonal driver mutations., Competing Interests: Declaration of interests All authors declare that they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Primary aldosteronism (PA) has been broadly dichotomized into unilateral and bilateral forms. Adrenal vein sampling (AVS) lateralization indices (LI) ≥2 to 4 are the standard-of-care to recommend unilateral adrenalectomy for presumed unilateral PA. We aimed to assess the rates and characteristics of residual PA after AVS-guided adrenalectomy., Methods: We conducted an international, retrospective, cohort study of patients with PA from 7 referral centers who underwent unilateral adrenalectomy based on LI≥4 on baseline and/or cosyntropin-stimulated AVS. Aldosterone synthase (CYP11B2) immunohistochemistry and next generation sequencing were performed on available formalin-fixed paraffin-embedded adrenal tissue., Results: The cohort included 283 patients who underwent AVS-guided adrenalectomy, followed for a median of 326 days postoperatively. Lack of PA cure was observed in 16% of consecutive patients, and in 22 patients with lateralized PA on both baseline and cosyntropin-stimulated AVS. Among patients with residual PA postoperatively, 73% had multiple CYP11B2 positive areas within the resected adrenal tissue (versus 23% in those cured), wherein CACNA1D mutations were most prevalent (63% versus 33% in those cured). In adjusted regression models, independent predictors of postoperative residual PA included Black versus White race (odds ratio, 5.10 [95% CI, 1.45-17.86]), AVS lateralization only at baseline (odds ratio, 8.93 [95% CI 3.00-26.32] versus both at baseline and after cosyntropin stimulation), and CT-AVS disagreement (odds ratio, 2.75 [95% CI, 1.20-6.31])., Conclusions: Multifocal, asymmetrical bilateral PA is relatively common, and it cannot be excluded by robust AVS lateralization. Long-term postoperative monitoring should be routinely pursued, to identify residual PA and afford timely initiation of targeted medical therapy., Competing Interests: Disclosures J. Yang has received project support from DiaSorin Pty Ltd and is supported by a Fellowship from the National Health and Medical Research Council of Australia. A. Vaidya reports consulting fees unrelated to the contents of this work from Corcept Therapeutics, Mineralys, HRA Pharma. A.F. Turcu served as local investigator in a clinical trial conducted by CinCor/AstraZeneca. All other authors declare no conflict of interest.

Adrenocortical carcinoma (ACC) is a rare but highly aggressive cancer with limited treatment options and poor survival for patients with advanced disease. An improved understanding of the transcriptional programs engaged in ACC will help direct rational, targeted therapies. Whereas activating mutations in Wnt/β-catenin signaling are frequently observed, the β-catenin-dependent transcriptional targets that promote tumor progression are poorly understood. To address this question, we analyzed ACC transcriptome data and identified a novel Wnt/β-catenin-associated signature in ACC enriched for the extracellular matrix (ECM) and predictive of poor survival. This suggested an oncogenic role for Wnt/β-catenin in regulating the ACC microenvironment. We further investigated the minor fibrillar collagen, collagen XI alpha 1 (COL11A1), and found that COL11A1 expression originates specifically from cancer cells and is strongly correlated with both Wnt/β-catenin activation and poor patient survival. Inhibition of constitutively active Wnt/β-catenin signaling in the human ACC cell line, NCI-H295R, significantly reduced the expression of COL11A1 and other ECM components and decreased cancer cell viability. To investigate the preclinical potential of Wnt/β-catenin inhibition in the adrenal microenvironment, we developed a minimally invasive orthotopic xenograft model of ACC and demonstrated that treatment with the newly developed Wnt/β-catenin:TBL1 inhibitor Tegavivint significantly reduced tumor growth. Together, our data support that the inhibition of aberrantly active Wnt/β-catenin disrupts transcriptional reprogramming of the microenvironment and reduces ACC growth and survival. Furthermore, this β-catenin-dependent oncogenic program can be therapeutically targeted with a newly developed Wnt/β-catenin inhibitor. These results show promise for the further clinical development of Wnt/β-catenin inhibitors in ACC and unveil a novel Wnt/β-catenin-regulated transcriptome.

Adrenocortical carcinoma (ACC) is a rare cancer in which tissue-specific differentiation is paradoxically associated with dismal outcomes. The differentiated ACC subtype CIMP-high is prevalent, incurable, and routinely fatal. CIMP-high ACC possess abnormal DNA methylation and frequent β-catenin-activating mutations. Here, we demonstrated that ACC differentiation is maintained by a balance between nuclear, tissue-specific β-catenin-containing complexes, and the epigenome. On chromatin, β-catenin bound master adrenal transcription factor SF1 and hijacked the adrenocortical super-enhancer landscape to maintain differentiation in CIMP-high ACC; off chromatin, β-catenin bound histone methyltransferase EZH2. SF1/β-catenin and EZH2/β-catenin complexes present in normal adrenals persisted through all phases of ACC evolution. Pharmacologic EZH2 inhibition in CIMP-high ACC expelled SF1/β-catenin from chromatin and favored EZH2/β-catenin assembly, erasing differentiation and restraining cancer growth in vitro and in vivo. These studies illustrate how tissue-specific programs shape oncogene selection, surreptitiously encoding targetable therapeutic vulnerabilities., Significance: Oncogenic β-catenin can use tissue-specific partners to regulate cellular differentiation programs that can be reversed by epigenetic therapies, identifying epigenetic control of differentiation as a viable target for β-catenin-driven cancers., (©2023 American Association for Cancer Research.)

Aging markedly increases cancer risk, yet our mechanistic understanding of how aging influences cancer initiation is limited. Here we demonstrate that the loss of ZNRF3, an inhibitor of Wnt signaling that is frequently mutated in adrenocortical carcinoma, leads to the induction of cellular senescence that remodels the tissue microenvironment and ultimately permits metastatic adrenal cancer in old animals. The effects are sexually dimorphic, with males exhibiting earlier senescence activation and a greater innate immune response, driven in part by androgens, resulting in high myeloid cell accumulation and lower incidence of malignancy. Conversely, females present a dampened immune response and increased susceptibility to metastatic cancer. Senescence-recruited myeloid cells become depleted as tumors progress, which is recapitulated in patients in whom a low myeloid signature is associated with worse outcomes. Our study uncovers a role for myeloid cells in restraining adrenal cancer with substantial prognostic value and provides a model for interrogating pleiotropic effects of cellular senescence in cancer., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Context: Noninvasive encapsulated follicular variant of papillary thyroid cancer was reclassified as noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) in January 2017. The impact of this nomenclature change at a population level remains unknown., Objective: Examine use of NIFTP across different US regions and populations., Design: Descriptive epidemiology study using SEER-22 data (2000-2019)., Participants: Individuals diagnosed with papillary or follicular thyroid cancer (2000-2019) or NIFTP (2017-2019)., Main Outcome Measures: Annual incidence rates of thyroid cancer by subtype and NIFTP. Using 2018-2019 data, (1) rates of NIFTP at the 17 SEER-22 sites and (2) comparison of demographics for patients diagnosed with NIFTP vs papillary and follicular thyroid cancer., Results: NIFTP comprised 2.2% and 2.6% of cases in 2018 and 2019, respectively. Between 2018 and 2019, large heterogeneity was observed in the regional use of NIFTP diagnosis, with site-specific incidence rates between 0.0% and 6.2% (median 2.8%, interquartile range 1.3-3.6%). A diagnosis of NIFTP (vs papillary and follicular thyroid cancer) in 2018 and 2019 was significantly associated with older age (P = 0.012 and P = 0.009, respectively), Black race (both Ps < 0.001), and non-Hispanic ethnicity (both Ps < 0.001)., Conclusions: Marked variation exists in the use of the NIFTP diagnosis. The recent 2021 coding change that resulted in NIFTP, a tumor with uncertain malignant potential and for which there is no long-term outcome data available, no longer being a reportable diagnosis to SEER will disproportionately affect vulnerable patient groups such as older patients and Black patients, in addition to patients who reside in regions with higher rates of NIFTP diagnoses., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context: Due to its rare incidence, molecular features of primary aldosteronism (PA) in young adults are largely unknown. Recently developed targeted mutational analysis identified aldosterone-driver somatic mutations in aldosterone-producing lesions, including aldosterone-producing adenomas (APAs), aldosterone-producing nodules (APNs), and aldosterone-producing micronodules, formerly known as aldosterone-producing cell clusters., Objective: To investigate histologic and genetic characteristics of lateralized PA in young adults., Methods: Formalin-fixed, paraffin-embedded adrenal tissue sections from 74 young patients with lateralized PA (<35 years old) were used for this study. Immunohistochemistry (IHC) for aldosterone synthase (CYP11B2) was performed to define the histopathologic diagnosis. Somatic mutations in aldosterone-producing lesions were further determined by CYP11B2 IHC-guided DNA sequencing., Results: Based on the CYP11B2 IHC results, histopathologic classification was made as follows: 48 APAs, 20 APNs, 2 multiple aldosterone-producing nodules (MAPN), 1 double APN, 1 APA with MAPN, and 2 nonfunctioning adenomas (NFAs). Of 45 APAs with successful sequencing, 43 (96%) had somatic mutations, with KCNJ5 mutations being the most common genetic cause of young-onset APA (35/45, 78%). Of 18 APNs with successful sequencing, all of them harbored somatic mutations, with CACNA1D mutations being the most frequent genetic alteration in young-onset APN (8/18, 44%). Multiple CYP11B2-expressing lesions in patients with MAPN showed several aldosterone-driver mutations. No somatic mutations were identified in NFAs., Conclusion: APA is the most common histologic feature of lateralized PA in young adults. Somatic KCNJ5 mutations are common in APAs, whereas CACNA1D mutations are often seen in APNs in this young PA population., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context: Somatic gene mutations have been identified in only about half of cortisol-producing adenomas (CPAs). Affected genes include PRKACA, GNAS, PRKAR1A, and CTNNB1., Objective: This work aims to expand our understanding of the prevalence of somatic mutations in CPAs from patients with overt Cushing syndrome (OCS) and "subclinical" mild autonomous cortisol excess (MACE), with an immunohistochemistry (IHC)‒guided targeted amplicon sequencing approach using formalin-fixed paraffin-embedded (FFPE) tissue., Methods: We analyzed FFPE adrenal tissue from 77 patients (n = 12 men, 65 women) with either OCS (n = 32) or MACE (n = 45). Using IHC for 17α-hydroxylase/17,20-lyase (CYP17A1) and 3β-hydroxysteroid dehydrogenase (HSD3B2), we identified 78 CPAs (32 OCS CPAs and 46 MACE CPAs). Genomic DNA was isolated from the FFPE CPAs and subjected to targeted amplicon sequencing for identification of somatic mutations., Results: Somatic mutations were identified in 71.8% (56/78) of the CPAs. While PRKACA was the most frequently mutated gene in OCS CPAs (14/32, 43.8%), somatic genetic aberrations in CTNNB1 occurred in 56.5% (26/46) of the MACE CPAs. Most GNAS mutations were observed in MACE CPAs (5/7, 71.4%). No mutations were observed in PRKAR1A. In addition to the known mutations, we identified one previously unreported mutation in PRKACA. Two patients with MACE harbored 2 adjacent tumors within the same adrenal gland - one patient had 2 CPAs, and the other patient had a CPA and an aldosterone-producing adenoma (identified by IHC for aldosterone synthase)., Conclusion: A comprehensive FFPE IHC-guided gene-targeted sequencing approach identified somatic mutations in 71.8% of the CPAs. OCS CPAs demonstrated a distinct mutation profile compared to MACE CPAs., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Alpha-inhibin expression has been reported in pheochromocytomas and paragangliomas (PPGLs). We analyzed alpha-inhibin immunohistochemistry in 77 PPGLs (37 pheochromocytomas [PCCs] and 40 paragangliomas) and correlated the results with catecholamine profile, tumor size, Ki-67 labeling index, succinate dehydrogenase B subunit and carbonic anhydrase IX (CAIX) staining, and genetic pathogenesis. PPGLs were classified as pseudohypoxic cluster 1 disease with documented VHL mutation or SDHx mutation or biochemical phenotype, whereas NF1-driven and RET-driven PPGLs and those with a mature secretory (adrenergic or mixed adrenergic and noradrenergic) phenotype were classified as cluster 2 disease. The Cancer Genome Atlas data on INHA expression in PPGLs was examined. Alpha-inhibin was positive in 43 PPGLs (56%). Ki-67 labeling indices were 8.07% and 4.43% in inhibin-positive and inhibin-negative PPGLs, respectively (P<0.05). Alpha-inhibin expression did not correlate with tumor size. Alpha-inhibin was expressed in 92% of SDHx-related and 86% of VHL-related PPGLs. CAIX membranous staining was found in 8 of 51 (16%) tumors, including 1 SDHx-related PCC and all 5 VHL-related PCCs. NF1-driven and RET-driven PPGLs were negative for alpha-inhibin and CAIX. Alpha-inhibin was expressed in 77% of PPGLs with a pseudohypoxia signature, and 20% of PPGLs without a pseudohypoxia signature (P<0.05). PPGLs with a mature secretory phenotype were negative for CAIX. The Cancer Genome Atlas data confirmed higher expression of INHA in cluster 1 than in cluster 2 PPGLs. This study identifies alpha-inhibin as a highly sensitive (90.3%) marker for SDHx/VHL-driven pseudohypoxic PPGLs. Although CAIX has low sensitivity, it is the most specific biomarker of VHL-related pathogenesis. While alpha-inhibin cannot replace succinate dehydrogenase B subunit immunohistochemistry for detection of SDHx-related disease, it adds value in prediction of cluster 1 disease. Importantly, these data emphasize that alpha-inhibin is not a specific marker of adrenal cortical differentiation, as it is also expressed in PCCs., Competing Interests: Conflicts of Interest and Source of Funding: The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Diverse subtypes of renal cell carcinomas (RCCs) display a wide spectrum of histomorphologies, proteogenomic alterations, immune cell infiltration patterns, and clinical behavior. Delineating the cells of origin for different RCC subtypes will provide mechanistic insights into their diverse pathobiology. Here, we employed single-cell RNA sequencing (scRNA-seq) to develop benign and malignant renal cell atlases. Using a random forest model trained on this cell atlas, we predicted the putative cell of origin for more than 10 RCC subtypes. scRNA-seq also revealed several attributes of the tumor microenvironment in the most common subtype of kidney cancer, clear cell RCC (ccRCC). We elucidated an active role for tumor epithelia in promoting immune cell infiltration, potentially explaining why ccRCC responds to immune checkpoint inhibitors, despite having a low neoantigen burden. In addition, we characterized an association between high endothelial cell types and lack of response to immunotherapy in ccRCC. Taken together, these single-cell analyses of benign kidney and RCC provide insight into the putative cell of origin for RCC subtypes and highlight the important role of the tumor microenvironment in influencing ccRCC biology and response to therapy., Competing Interests: Competing interest statement: A.A. and P.M. are coauthors on an article [S. M. Esagian et al. BJU International, 10.1111/bju.15324 (2021)]., (Copyright © 2021 the Author(s). Published by PNAS.)

Background: While previous studies indicate that the zonae reticularis (ZR) and glomerulosa (ZG) diminish with aging, little is known about age-related transformations of the zona fasciculata (ZF)., Objectives: To investigate the morphological and functional changes of the adrenal cortex across adulthood, with emphasis on (i) the understudied ZF and (ii) sexual dimorphisms., Methods: We used immunohistochemistry to evaluate the expression of aldosterone synthase (CYP11B2), visinin-like protein 1 (VSNL1), 3β-hydroxysteroid dehydrogenase type II (HSD3B2), 11β-hydroxylase (CYP11B1), and cytochrome b5 type A (CYB5A) in adrenal glands from 60 adults (30 men), aged 18 to 86. Additionally, we employed mass spectrometry to quantify the morning serum concentrations of cortisol, 11-deoxycortisol (11dF), 17α-hydroxyprogesterone, 11-deoxycorticosterone, corticosterone, and androstenedione in 149 pairs of age- and body mass index-matched men and women, age 21 to 95 years., Results: The total cortical area was positively correlated with age (r = 0.34, P = 0.008). Both the total (VSNL1-positive) and functional ZG (CYP11B2-positive) areas declined with aging in men (r = -0.57 and -0.67, P < 0.01), but not in women. The CYB5A-positive area declined with age in both sexes (r = -0.76, P < 0.0001). In contrast, the estimated ZF area correlated positively with age in men (r = 0.59, P = 0.0006) and women (r = 0.49, P = 0.007), while CYP11B1-positive area remained unchanged across ages. Serum cortisol, corticosterone, and 11-deoxycorticosterone levels were stable across ages, while 11dF levels increased slightly with age (r = 0.16, P = 0.007)., Conclusion: Unlike the ZG and ZR, the ZF and the total adrenal cortex areas enlarge with aging. An abrupt decline of the ZG occurs with age in men only, possibly contributing to sexual dimorphism in cardiovascular risk., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Complete resection of adrenal cortical carcinoma (ACC) with or without adjuvant therapy offers the best outcome. Recurrence is common, and in individual cases, the long-term outcome is difficult to predict, making it challenging to personalize treatment options. Current risk stratification approaches are based on clinical and conventional surgical pathology assessment. Rigorous and uniform pathological assessment may improve care for individual patients and facilitate multi-institutional collaborative studies. The International Collaboration on Cancer Reporting (ICCR) convened an expert panel to review ACC pathology reporting. Consensus recommendations were made based on the most recent literature and expert opinion. The data set comprises 23 core (required) items. The core pathological features include the following: diagnosis as per the current World Health Organization classification, specimen integrity, greatest dimension, weight, extent of invasion, architecture, percentage of lipid-rich cells, capsular invasion, lymphatic invasion, vascular invasion, atypical mitotic figures, coagulative necrosis, nuclear grade, mitotic count, Ki-67 proliferative index, margin status, lymph node status, and pathological stage. Tumors were dichotomized into low-grade (<20 mitoses per 10 mm 2 ) and high-grade (>20 mitoses per 10 mm 2 ) ones. Additional noncore elements that may be useful in individual cases included several multifactorial risk assessment systems (Weiss, modified Weiss, Lin-Weiss-Bisceglia, reticulin, Helsinki, and Armed Forces Institute of Pathology scores/algorithms). This data set is now available through the ICCR website with the hope of better standardizing pathological assessment of these relatively rare but important malignancies., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Approximately one-tenth of the general population exhibit adrenal cortical nodules, and the incidence has increased. Afflicted patients display a multifaceted symptomatology-sometimes with rather spectacular features. Given the general infrequency as well as the specific clinical, histological, and molecular considerations characterizing these lesions, adrenal cortical tumors should be investigated by endocrine pathologists in high-volume tertiary centers. Even so, to distinguish specific forms of benign adrenal cortical lesions as well as to pinpoint malignant cases with the highest risk of poor outcome is often challenging using conventional histology alone, and molecular genetics and translational biomarkers are therefore gaining increased attention as a possible discriminator in this context. In general, our understanding of adrenal cortical tumorigenesis has increased tremendously the last decade, not least due to the development of next-generation sequencing techniques. Comprehensive analyses have helped establish the link between benign aldosterone-producing adrenal cortical proliferations and ion channel mutations, as well as mutations in the protein kinase A (PKA) signaling pathway coupled to cortisol-producing adrenal cortical lesions. Moreover, molecular classifications of adrenal cortical tumors have facilitated the distinction of benign from malignant forms, as well as the prognostication of the individual patients with verified adrenal cortical carcinoma, enabling high-resolution diagnostics that is not entirely possible by histology alone. Therefore, combinations of histology, immunohistochemistry, and next-generation multi-omic analyses are all needed in an integrated fashion to properly distinguish malignancy in some cases. Despite significant progress made in the field, current clinical and pathological challenges include the preoperative distinction of non-metastatic low-grade adrenal cortical carcinoma confined to the adrenal gland, adoption of individualized therapeutic algorithms aligned with molecular and histopathologic risk stratification tools, and histological confirmation of functional adrenal cortical disease in the context of multifocal adrenal cortical proliferations. We herein review the histological, genetic, and epigenetic landscapes of benign and malignant adrenal cortical neoplasia from a modern surgical endocrine pathology perspective and highlight key mechanisms of value for diagnostic and prognostic purposes.

Background: Tumor molecular profiling from patients experiencing exceptional responses to systemic therapy may provide insights into cancer biology and improve treatment tailoring. This pilot study evaluates the feasibility of identifying exceptional responders retrospectively, obtaining pre-exceptional response treatment tumor tissues, and analyzing them with state-of-the-art molecular analysis tools to identify potential molecular explanations for responses., Methods: Exceptional response was defined as partial (PR) or complete (CR) response to a systemic treatment with population PR or CR rate less than 10% or an unusually long response (eg, duration >3 times published median). Cases proposed by patients' clinicians were reviewed by clinical and translational experts. Tumor and normal tissue (if possible) were profiled with whole exome sequencing and, if possible, targeted deep sequencing, RNA sequencing, methylation arrays, and immunohistochemistry. Potential germline mutations were tracked for relevance to disease., Results: Cases reflected a variety of tumors and standard and investigational treatments. Of 520 cases, 476 (91.5%) were accepted for further review, and 222 of 476 (46.6%) proposed cases met requirements as exceptional responders. Clinical data were obtained from 168 of 222 cases (75.7%). Tumor was provided from 130 of 168 cases (77.4%). Of 117 of the 130 (90.0%) cases with sufficient nucleic acids, 109 (93.2%) were successfully analyzed; 6 patients had potentially actionable germline mutations., Conclusion: Exceptional responses occur with standard and investigational treatment. Retrospective identification of exceptional responders, accessioning, and sequencing of pretreatment archived tissue is feasible. Data from molecular analyses of tumors, particularly when combining results from patients who received similar treatments, may elucidate molecular bases for exceptional responses., (Published by Oxford University Press 2020. This work is written by US Government employees and is in the public domain in the US.)

Objective: Develop a consensus for the nomenclature and definition of adrenal histopathologic features in unilateral primary aldosteronism (PA)., Context: Unilateral PA is the most common surgically treated form of hypertension. Morphologic examination combined with CYP11B2 (aldosterone synthase) immunostaining reveals diverse histopathologic features of lesions in the resected adrenals., Patients and Methods: Surgically removed adrenals (n = 37) from 90 patients operated from 2015 to 2018 in Munich, Germany, were selected to represent the broad histologic spectrum of unilateral PA. Five pathologists (Group 1 from Germany, Italy, and Japan) evaluated the histopathology of hematoxylin-eosin (HE) and CYP11B2 immunostained sections, and a consensus was established to define the identifiable features. The consensus was subsequently used by 6 additional pathologists (Group 2 from Australia, Brazil, Canada, Japan, United Kingdom, United States) for the assessment of all adrenals with disagreement for histopathologic diagnoses among group 1 pathologists., Results: Consensus was achieved to define histopathologic features associated with PA. Use of CYP11B2 immunostaining resulted in a change of the original HE morphology-driven diagnosis in 5 (14%) of 37 cases. Using the consensus criteria, group 2 pathologists agreed for the evaluation of 11 of the 12 cases of disagreement among group 1 pathologists., Conclusion: The HISTALDO (histopathology of primary aldosteronism) consensus is useful to standardize nomenclature and achieve consistency among pathologists for the histopathologic diagnosis of unilateral PA. CYP11B2 immunohistochemistry should be incorporated into the routine clinical diagnostic workup to localize the likely source of aldosterone production., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Approximately 25% of thyroid nodule fine-needle aspirates (FNAs) have cytology that is indeterminate for malignant disease. Accurate risk stratification of these FNAs with ancillary testing would reduce unnecessary thyroid surgery., Methods: We evaluated the performance of an ancillary multiplatform test (MPTX) that has three diagnostic categories (negative, moderate, and positive). MPTX includes the combination of a mutation panel (ThyGeNEXT®) and a microRNA risk classifier (ThyraMIR®). A blinded, multicenter study was performed using consensus histopathology diagnosis among three pathologists to validate test performance., Results: Unanimous consensus diagnosis was reached in 197 subjects with indeterminate thyroid nodules; 36% had disease. MPTX had 95% sensitivity (95% CI,86%-99%) and 90% specificity (95% CI,84%-95%) for disease in prevalence adjusted nodules with Bethesda III and IV cytology. Negative MPTX results ruledout disease with 97% negative predictive value (NPV; 95% CI,91%-99%) at a 30% disease prevalence, while positive MPTX results ruledin high risk disease with 75% positive predictive value (PPV; 95% CI,60%-86%). Such results are expected in four out of five Bethesda III and IV nodules tested, including RAS positive nodules in which the microRNA classifier was useful in rulingin disease. 90% of mutation panel false positives were due to analytically verified RAS mutations detected in benign adenomas. Moderate MPTX results had a moderate rate of disease (39%, 95% CI,23%-54%), primarily due to RAS mutations, wherein the possibility of disease could not be excluded., Conclusions: Our results emphasize that decisions for surgery should not solely be based on RAS or RAS-like mutations. MPTX informs management decisions while accounting for these challenges., (© 2020 The Authors. Diagnostic Cytopathology published by Wiley Periodicals LLC.)

Introduction: Surgical resection is curative for some patients with early lung squamous cell carcinoma. Staging and clinical factors do not adequately predict recurrence risk. We sought to validate the discriminative performance of proposed prognostic gene expression signatures at a level of rigor sufficient to support clinical use., Methods: The two-stage validation used independent core laboratories, objective quality control standards, locked test parameters, and large multi-institutional specimen and data sets. The first stage validation confirmed a signature's ability to stratify patient survival. The second-stage validation determined which signature(s) optimally improved risk discrimination when added to baseline clinical predictors. Participants were prospectively enrolled in institutional (cohort I) or cooperative group (cohort II) biospecimen and data collection protocols. All cases underwent a central review of clinical, pathologic, and biospecimen parameters using objective criteria to determine final inclusion (cohort I: n = 249; cohort II: n = 234). Primary selection required that a signature significantly predict a 3-year survival after surgical resection in cohort I. Signatures meeting this criterion were further tested in cohort II, comparing risk prediction using baseline risk factors alone versus in combination with the signature., Results: Male sex, advanced age, and higher stage were associated with shorter survival in cohort I and established a baseline clinical model. Of the three signatures validated in cohort I, one signature was validated in cohort II and statistically significantly enhanced the prognosis relative to the baseline model (C-index difference 0.122; p < 0.05)., Conclusions: These results represent the first rigorous validation of a test appropriate to direct adjuvant treatment or clinical trials for patients with lung squamous cell carcinoma., (Copyright © 2020. Published by Elsevier Inc.)

Somatic mutations driving aldosterone production have been identified in approximately 90% of aldosterone-producing adenomas (APAs) using an aldosterone synthase (CYP11B2) immunohistochemistry (IHC)-guided DNA sequencing approach. In the present study, using CYP11B2-guided whole-exome sequencing (WES) and targeted amplicon sequencing, we detected 2 somatic variants in CLCN2 in 2 APAs that were negative for currently known aldosterone-driver mutations. The CLCN2 gene encodes the voltage-gated chloride channel ClC-2. CLCN2 germline variants have previously been shown to cause familial hyperaldosteronism type II. Somatic mutations in CLCN2 were identified in 2 of 115 APAs, resulting in a prevalence of 1.74%. One of the CLCN2 somatic mutations (c.G71A,p.G24D) was identical to a previously described germline variant causing early-onset PA, but was present only as a somatic mutation. The second CLCN2 mutation, which affects the same region of the gene, has not been reported previously (c.64-2&#95;74del). These findings prove that WES of CYP11B2-guided mutation-negative APAs can help determine rarer genetic causes of sporadic PA., (© Endocrine Society 2020.)

Lack of routine fresh or frozen tissue is a barrier to widespread transcriptomic analysis of adrenal cortical tumors and an impediment to translational research in endocrinology and endocrine oncology. Our group has previously pioneered the use of targeted amplicon-based next-generation sequencing for archival formalin-fixed paraffin-embedded (FFPE) adrenal tissue specimens to characterize the spectrum of somatic mutations in various forms of primary aldosteronism. Herein, we developed and validated a novel 194-amplicon targeted next-generation RNA sequencing (RNAseq) assay for transcriptomic analysis of adrenal tumors using clinical-grade FFPE specimens. Targeted RNAseq-derived expression values for 27 adrenal cortical tumors, including aldosterone-producing adenomas (APA; n=8), cortisol-producing adenomas (CPA; n=11), and adrenal cortical carcinomas (ACC; n=8), highlighted known differentially-expressed genes (DEGs; i. e., CYP11B2 , IGF2 , etc.) and tumor type-specific transcriptional modules (i. e., high cell cycle/proliferation transcript expression in ACC, etc.), and a subset of DEGs was validated orthogonally using quantitative reverse transcription PCR (qRT-PCR). Finally, unsupervised hierarchical clustering using a subset of high-confidence DEGs revealed three discrete clusters representing APA, CPA, and ACC tumors with corresponding unique gene expression signatures, suggesting potential clinical utility for a transcriptomic-based approach to tumor classification. Overall, these data support the use of targeted amplicon-based RNAseq for comprehensive transcriptomic profiling of archival FFPE adrenal tumor material and indicate that this approach may facilitate important translational research opportunities for the study of these tumors., Competing Interests: The authors declare that they have no conflict of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

Background: Renal cell carcinomas (RCCs) are a heterogeneous group of neoplasms. Recent sequencing studies revealed various molecular features associated with histologic RCC subtypes, including chromophobe renal cell carcinoma (ChRCC)., Objective: To characterize the gene expression and biomarker signatures associated with ChRCC., Design, Setting, and Participants: We performed integrative analysis on RNA sequencing data available from 1049 RCC specimens from The Cancer Genome Atlas and in-house studies. Our workflow identified genes relatively enriched in ChRCC, including Forkhead box I1 (FOXI1), Rh family C glycoprotein (RHCG), and LINC01187. We assessed the expression pattern of FOXI1 and RHCG protein by immunohistochemistry (IHC) and LINC01187 mRNA by RNA in situ hybridization (RNA-ISH) in whole tissue sections representing a cohort of 197 RCC cases, including both primary and metastatic tumors., Outcome Measurements and Statistical Analysis: The FOXI1 and RHCG IHC staining, as well as the LINC01187 RNA-ISH staining, was evaluated in each case for intensity, pattern, and localization of expression., Results and Limitations: All primary and metastatic classic ChRCCs demonstrated homogeneous positive labeling for FOXI1, RHCG proteins, and LINC01187 transcript. Unclassified RCC with oncocytic features, oncocytoma, and hybrid oncocytic tumor, as well as all but two cases of eosinophilic ChRCC also stained positive. Importantly, metastatic and primary RCC of all other subtypes did not demonstrate any unequivocal staining for FOXI1, RHCG, or LINC01187. In normal kidney, FOXI1, RHCG, and LINC01187 were detected in the distal nephron segment, specifically in intercalated cells. Two cases of eosinophilic ChRCC with focal expression of FOXI1 and LINC01187, and Golgi-like RHCG staining were found to contain MTOR gene mutations upon DNA sequencing., Conclusions: We demonstrate a pipeline for the identification and validation of RCC subtype-specific biomarkers that can aid in the confirmation of cell of origin and may facilitate accurate classification and diagnosis of renal tumors., Patient Summary: FOXI1, RHCG, and LINC01187 are lineage-specific signature genes for chromophobe renal cell carcinoma., (Copyright © 2020 European Association of Urology. Published by Elsevier B.V. All rights reserved.)

Poorly differentiated thyroid carcinomas (PDTC) in young individuals are rare and their clinical and histopathologic features, genetic mechanisms, and outcomes remain largely unknown. Here, we report a detailed characterization of a series of six PDTC in patients ≤21 years old defined by Turin diagnostic criteria studied for mutations and gene fusions characteristic of thyroid cancer using targeted next-generation sequencing (NGS) and whole-exome sequencing (WES). All tumors had solid, insular, or trabecular growth pattern and high mitotic rate, and five out of six tumors showed tumor necrosis. Targeted NGS assay identified somatic mutations in the DICER1 gene in five of six (83%) tumors, all of which were "hotspot" mutations encoding the metal-ion binding sites of the RNase IIIb domain of DICER1. WES was performed in five cases which confirmed all hotspot mutations and detected two tumors with additional inactivating DICER1 alterations. Of these two, one was a germline pathogenic DICER1 variant and the other had loss of heterozygosity for DICER1. No other mutations or gene fusions characteristic of adult well-differentiated thyroid cancer and PDTC (BRAF, RAS, TERT, RET/PTC, and other) were detected. On follow-up, available for five patients, three patients died of disease 8-24 months after diagnosis, whereas two were alive with no disease. The results of our study demonstrate that childhood- and adolescent-onset PDTC are genetically distinct from adult-onset PDTC in that they are strongly associated with DICER1 mutations and may herald DICER1 syndrome in a minority. As such, all young persons with PDTC may benefit from genetic counseling. Furthermore, their clinically aggressive behavior contrasts sharply with the indolent nature of the great majority of thyroid tumors with DICER1 mutations reported to date.

Anaplastic thyroid cancer (ATC) is one of the most aggressive human malignancies, with an average life expectancy of ∼6 months from the time of diagnosis. The genetic and epigenetic changes that underlie this malignancy are incompletely understood. We found that ASH1-like histone lysine methyltransferase (ASH1L) is overexpressed in ATC relative to the much less aggressive and more common differentiated thyroid cancer. This increased expression was due at least in part to reduced levels of microRNA-200b-3p (miR-200b-3p), which represses ASH1L expression, in ATC. Genetic knockout of ASH1L protein expression in ATC cell lines decreased cell growth both in culture and in mouse xenografts. RNA-Seq analysis of ASH1L knockout versus WT ATC cell lines revealed that ASH1L is involved in the regulation of numerous cancer-related genes and gene sets. The pro-oncogenic long noncoding RNA colon cancer-associated transcript 1 (CCAT1) was one of the most highly (approximately 68-fold) down-regulated transcripts in ASH1L knockout cells. Therefore, we investigated CCAT1 as a potential mediator of the growth-inducing activity of ASH1L. Supporting this hypothesis, CCAT1 knockdown in ATC cells decreased their growth rate, and ChIP-Seq data indicated that CCAT1 is likely a direct target of ASH1L's histone methyltransferase activity. These results indicate that ASH1L contributes to the aggressiveness of ATC and suggest that ASH1L, along with its upstream regulator miR-200b-3p and its downstream mediator CCAT1, represents a potential therapeutic target in ATC., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Xu et al.)

Driver somatic mutations for aldosterone excess have been found in ≈90% of aldosterone-producing adenomas (APAs) using an aldosterone synthase (CYP11B2)-guided sequencing approach. In the present study, we identified a novel somatic CACNA1H mutation (c.T4289C, p.I1430T) in an APA without any currently known aldosterone-driver mutations using CYP11B2 immunohistochemistry-guided whole exome sequencing. The CACNA1H gene encodes a voltage-dependent T-type calcium channel alpha-1H subunit. Germline variants in this gene are known as a cause of familial hyperaldosteronism IV. Targeted next-generation sequencing detected identical CACNA1H variants in 2 additional APAs in a cohort of the University of Michigan, resulting in a prevalence of 4% (3/75) in APAs. We tested the functional effect of the variant on adrenal cell aldosterone production and CYP11B2 mRNA expression using the human adrenocortical HAC15 cell line with a doxycycline-inducible CACNA1H I1430T mutation. Doxycycline treatment increased CYP11B2 mRNA levels as well as aldosterone production, supporting a pathological role of the CACNA1H p.I1430T mutation on the development of primary aldosteronism. In conclusion, somatic CACNA1H mutation is a genetic cause of APAs. Although the prevalence of this mutation is low, this study will provide better understanding of molecular mechanism of inappropriate aldosterone production in APAs.

Context: Adrenocortical carcinoma (ACC) is a rare malignancy with high rates of recurrence and poor prognosis. The role of radiotherapy (RT) in localized ACC has been controversial, and RT is not routinely offered., Objective: To evaluate the benefit of adjuvant RT on outcomes in ACC., Design: This is a retrospective propensity-matched analysis., Setting: All patients were seen through the University of Michigan's Endocrine Oncology program, and all those who underwent RT were treated at the University of Michigan., Participants: Of 424 patients with ACC, 78 were selected; 39 patients underwent adjuvant radiation., Intervention: Adjuvant RT to the tumor bed and adjacent lymph nodes., Main Outcomes Measures: Time to local failure, distant failure, or death., Results: Median follow-up time was 4.21 years (95% CI, 2.79 to 4.94). The median radiation dose was 55 Gy (range, 45 to 60). The 3-year overall survival estimate for patients improved from 48.6% for patients without RT (95% CI, 29.7 to 65.2) to 77.7% (95% CI, 56.3 to 89.5) with RT, with a hazard ratio (HR) of 3.59 (95% CI, 1.60 to 8.09; P = 0.002). RT improved local recurrence-free survival (RFS) from 34.2% (95% CI, 18.8 to 50.3) to 59.5% (95% CI, 39.0 to 75.0), with an HR of 2.67 (95% CI, 1.38 to 5.19; P = 0.0035). RT improved all RFS from 18.3% (95% CI, 6.7 to 34.3) to 46.7% (95% CI, 26.9 to 64.3), with an HR 2.59 (95% CI, 1.40 to 4.79; P = 0.0024)., Conclusions: In the largest single institution study to date, adjuvant RT after gross resection of ACC improved local RFS, all RFS, and overall survival in this propensity-matched analysis. Adjuvant RT should be considered a part of multidisciplinary management for patients with ACC., (Copyright © 2019 Endocrine Society.)

Purpose: Adrenocortical carcinoma (ACC) is a rare, aggressive malignancy with few therapies; however, patients with locoregional disease have variable outcomes. The Cancer Genome Atlas project on ACC (ACC-TCGA) identified that cancers of patients with homogeneously rapidly recurrent or fatal disease bear a unique CpG island hypermethylation phenotype, "CIMP-high." We sought to identify a biomarker that faithfully captures this subgroup. Experimental Design: We analyzed ACC-TCGA data to characterize differentially regulated biological processes, and identify a biomarker that is methylated and silenced exclusively in CIMP-high ACC. In an independent cohort of 114 adrenocortical tumors (80 treatment-naive primary ACC, 22 adrenocortical adenomas, and 12 non-naive/nonprimary ACC), we evaluated biomarker methylation by a restriction digest/qPCR-based approach, validated by targeted bisulfite sequencing. We evaluated expression of this biomarker and additional prognostic markers by qPCR., Results: We show that CIMP-high ACC is characterized by upregulation of cell cycle and DNA damage response programs, and identify that hypermethylation and silencing of G0S2 distinguishes this subgroup. We confirmed G0S2 hypermethylation and silencing is exclusive to 40% of ACC, and independently predicts shorter disease-free and overall survival (median 14 and 17 months, respectively). Finally, G0S2 methylation combined with validated molecular markers ( BUB1B-PINK1 ) stratifies ACC into three groups, with uniformly favorable, intermediate, and uniformly dismal outcomes., Conclusions: G0S2 hypermethylation is a hallmark of rapidly recurrent or fatal ACC, amenable to targeted assessment using routine molecular diagnostics. Assessing G0S2 methylation is straightforward, feasible for clinical decision-making, and will enable the direction of efficacious adjuvant therapies for patients with aggressive ACC., (©2019 American Association for Cancer Research.)

Background: Extrathyroidal extension (ETE) by papillary and follicular thyroid carcinoma can be associated with increased risk of tumor recurrence and mortality. In the seventh edition of its Cancer Staging Manual, the American Joint Committee on Cancer (AJCC) defined minimal ETE as the involvement of skeletal muscle (i.e., strap muscles) or perithyroidal soft tissue. The eighth edition of the AJCC Cancer Staging Manual has changed the criteria so that only grossly evident (macroscopic) ETE involving strap muscles (not microscopic ETE involving perithyroidal soft tissue) affects tumor staging. Summary: Concordance of identifying microscopic ETE (as well as extranodal extension by carcinoma metastatic to lymph nodes) was previously evaluated among 11 expert endocrine pathologists. The overall agreement rate was slight when rendering a diagnosis of ETE. Concordance was highest when pathologists assessed the spatial relationship of carcinoma to skeletal muscle. This article discusses the significance of these findings. It also reviews relevant anatomic and developmental considerations related to the boundaries of the thyroid. Conclusions: The results of the concordance study provide additional rationale supporting stringent criteria for diagnosing ETE, as proposed by the eighth edition of the AJCC Cancer Staging Manual. It is expected that these rigid morphologic criteria will potentially reduce interobserver variability and enhance consistency in the diagnosis and staging of thyroid carcinoma.

Somatic variants in genes that regulate intracellular ion homeostasis have been identified in aldosterone-producing adenomas (APA). Although the mechanisms leading to an increased aldosterone production in APA cells has been well studied, the molecular events that cause cell proliferation and tumor formation are poorly understood. In the present study, we have performed whole exome sequencing (WES) to characterize the landscape of somatic alterations in a homogeneous series of APA with pathogenic KCNJ5 variants. In the WES analysis on eleven APA, 84 exonic somatic events were called by 3 different somatic callers. Besides the KCNJ5 gene, only two genes (MED13 and ZNF669) harbored somatic variants in more than one APA. Unlike adrenocortical carcinomas, no chromosomal instability was observed by the somatic copy-number alteration and loss of heterozygosity analyses. The estimated tumor purity ranged from 0.35 to 0.67, suggesting a significant proportion of normal cell infiltration. Based on the results of PureCN analysis, the KCNJ5 variants appear to be clonal. In conclusion, in addition to KCNJ5 somatic pathogenic variant, no significant somatic event that would obviously explain proliferation or tumor growth was observed in our homogeneous cohort of KCNJ5-mutated APA. The molecular mechanisms causing APA growth and tumorigenesis remain to be elucidated.

Somatic mutations have been identified in aldosterone-producing adenomas (APAs) in genes that include KCNJ5, ATP1A1, ATP2B3, and CACNA1D. Based on independent studies, there appears to be racial differences in the prevalence of somatic KCNJ5 mutations, particularly between East Asians and Europeans. Despite the high cardiovascular disease mortality of blacks, there have been no studies focusing on somatic mutations in APAs in this population. In the present study, we investigated genetic characteristics of APAs in blacks using a CYP11B2 (aldosterone synthase) immunohistochemistry-guided next-generation sequencing approach. The adrenal glands with adrenocortical adenomas from 79 black patients with primary aldosteronism were studied. Seventy-three tumors from 69 adrenal glands were confirmed to be APAs by CYP11B2 immunohistochemistry. Sixty-five of 73 APAs (89%) had somatic mutations in aldosterone-driver genes. Somatic CACNA1D mutations were the most prevalent genetic alteration (42%), followed by KCNJ5 (34%), ATP1A1 (8%), and ATP2B3 mutations (4%). CACNA1D mutations were more often observed in APAs from males than those from females (55% versus 29%, P=0.033), whereas KCNJ5 mutations were more prevalent in APAs from females compared with those from males (57% versus 13%, P<0.001). No somatic mutations in aldosterone-driver genes were identified in tumors without CYP11B2 expression. In conclusion, 89% of APAs in blacks harbor aldosterone-driving mutations, and unlike Europeans and East Asians, the most frequently mutated aldosterone-driver gene was CACNA1D. Determination of racial differences in the prevalence of aldosterone-driver gene mutations may facilitate the development of personalized medicines for patients with primary aldosteronism.

Several somatic mutations specific to aldosterone-producing adenomas (APAs) have been described. A small proportion of adrenocortical carcinomas (ACCs) are associated with hyperaldosteronism, either primary aldosteronism or hyperreninemic hyperaldosteronism. However, it is unknown whether they harbor mutations of the same spectrum as APAs. The objective of this study is to describe the clinical phenotype and molecular genotype of ACCs with hyperaldosteronism, particularly the analysis for common APA-associated genetic changes. Patients were identified by retrospective chart review at a specialized referral center and by positive staining for CYP11B2 of tissue microarrays. Twenty-five patients with ACC and hyperaldosteronism were initially identified by retrospective chart review, and tissue for further analysis was available on 13 tumors. Seven patients were identified by positive staining for CYP11B2 in a tissue microarray, of which two were already identified in the initial chart review. Therefore, a total number of 18 patients with a diagnosis of ACC and features of either primary aldosteronism or hyperreninemic hyperaldosteronism were therefore included in the final study. Mutational status for a select list of oncogenes, tumor suppressor genes and genes known to carry mutations in APAs were analyzed by next-generation sequencing. Review of clinical data suggested autonomous aldosterone production in the majority of cases, while for some cases, hyperreninemic hyperaldosteronism was the more likely mechanism. The mutational landscape of ACCs associated with hyperaldosteronism was not different from ACCs with a different hormonal phenotype. None of the ACCs harbored mutations of known APA-associated genes, suggesting an alternative mechanism conferring aldosterone production.

Background: Patterns and prognostic implications of recurrent adrenocortical carcinoma are poorly understood. In this study, we aim to describe temporal and spatial patterns of adrenocortical carcinoma recurrence., Methods: This is a retrospective review of 576 patients with adrenocortical carcinoma evaluated at a single institution. Clinicopathologic and follow-up data were collected longitudinally., Results: A total of 354 patients underwent resection of stage I-III adrenocortical carcinoma. We found that 249 (70%) patients developed disease recurrence. The median recurrence-free interval after primary resection was 11 months. The most common sites of initial recurrence were lung and tumor bed. The shortest time to recurrence was associated with lung or multiple site metastases. We found that 142 of 249 patients developed one or more additional sites of recurrence (median 5 months), most commonly involving the lungs. A total of 20 patients developed a third site of recurrence. We found that 100 patients underwent one or more reoperations or metastasectomies and 79 recurred again after reoperation. Same organ or site recurrence was common after reoperation (67%). Although lung metastases occurred early, recurrences to the peritoneal cavity or to multiple sites were associated with worse survival. Metastasectomy beyond three total operations did not improve overall survival., Conclusion: Survival varies according to site of recurrence and other clinicopathologic factors. Knowledge of patterns of recurrence may assist in anticipating disease course and lead to better informed selection of treatment., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Context: Somatic mutations have been identified in more than half of aldosterone-producing adenomas (APAs) through mutation hotspot sequencing. The underlying pathogenesis of inappropriate aldosterone synthesis in the remaining population is still unknown., Objective: To investigate the prevalence and spectrum of somatic mutations in APAs using an aldosterone synthase (CYP11B2) immunohistochemistry (IHC)‒guided next-generation sequencing (NGS) approach., Methods: Formalin-fixed paraffin-embedded adrenal tissue from white American patients with primary aldosteronism who underwent adrenalectomy at the University of Michigan was used. Genomic DNA was isolated from 75 APAs (identified by CYP11B2 IHC). NGS was performed to identify somatic mutations by sequencing the entire coding region of a panel of genes mutated in APAs., Results: Somatic mutations were identified in 66 of 75 APAs (88%). Of the APAs with somatic mutations, six were smaller than coexisting CYP11B2-negative adrenocortical adenomas. The most frequently mutated gene was KCNJ5 (43%), followed by CACNA1D (21%), ATP1A1 (17%), ATP2B3 (4%), and CTNNB1 (3%). In addition to identification of previously reported mutations, we identified five previously unreported mutations (two in KCNJ5, one in ATP1A1, one in ATP2B3, and one in CACNA1D genes). KCNJ5 mutations were more frequent in women (70% vs 24% in men)., Conclusion: Comprehensive NGS of CYP11B2-expressing adrenal tumors identified somatic mutations in aldosterone-driving genes in 88% of APAs, a higher rate than in previous studies using conventional approaches.

Metastatic medullary thyroid cancer (MTC) is incurable and FDA-approved kinase inhibitors that include oncogenic RET as a target do not result in complete responses. Association studies of human MTCs and murine models suggest that the CDK/RB pathway may be an alternative target. The objective of this study was to determine if CDKs represent therapeutic targets for MTC and to define mechanisms of activity. Using human MTC cells that are either sensitive or resistant to vandetanib, we demonstrate that palbociclib (CDK4/6 inhibitor) is not cytotoxic to MTC cells but that they are highly sensitive to dinaciclib (CDK1/2/5/9 inhibitor) accompanied by reduced CDK9 and RET protein and mRNA levels. CDK9 protein was highly expressed in 83 of 83 human MTCs and array-comparative genomic hybridization had copy number gain in 11 of 30 tumors. RNA sequencing demonstrated that RNA polymerase II-dependent transcription was markedly reduced by dinaciclib. The CDK7 inhibitor THZ1 also demonstrated high potency and reduced RET and CDK9 levels. ChIP-sequencing using H3K27Ac antibody identified a superenhancer in intron 1 of RET. Finally, combined inhibition of dinaciclib with a RET kinase inhibitor was synergistic. In summary, we have identified what we believe is a novel mechanism of RET transcription regulation that potentially can be exploited to improve RET therapeutic targeting.

The classification of human cancers represents one of the cornerstones of modern pathology. Over the last century, surgical pathologists established the current taxonomy of neoplasia using traditional histopathological parameters, which include tumor architecture, cytological features and cellular proliferation. This morphological classification is efficient and robust with high reproducibility and has served patients and health care providers well. The most recent decade has witnessed an explosion of genome-wide molecular genetic and epigenetic data for most cancers, including tumors of endocrine organs. The availability of this expansive multi-dimensional genomic data, collectively termed the cancer genome, has catalyzed a re-examination of the classification of endocrine tumors. Here, recent cancer genome studies of various endocrine tumors, including those of the thyroid, pituitary and adrenal glands, pancreas, small bowel, lung and skin, are presented with special emphasis on how genomic insights are impacting endocrine tumor classification., (© 2018 Society for Endocrinology.)

Careful morphological evaluation forms the basis of the workup of an adrenal cortical neoplasm. However, the adoption of immunohistochemical biomarkers has added tremendous value to enhance diagnostic accuracy. The authors provide a brief review of immunohistochemical biomarkers that have been used in the confirmation of adrenal cortical origin and in the detection of the source of functional adrenal cortical proliferations, as well as diagnostic, predictive, and prognostic biomarkers of adrenal cortical carcinoma. In addition, a brief section on potential novel theranostic biomarkers in the prediction of treatment response to mitotane and other relevant chemotherapeutic agents is also provided. In the era of precision and personalized medical practice, adoption of combined morphology and immunohistochemistry provides a new approach to the diagnostic workup of adrenal cortical neoplasms, reflecting the evolution of clinical responsibility of pathologists.

Context: A subset of thyroid carcinomas expresses an oncogenic paired box 8 (PAX8) and peroxisome proliferator activated receptor γ (PPARγ) fusion protein (PPFP). The PPARγ/PPFP ligand pioglitazone is highly therapeutic in a transgenic mouse model of PPFP thyroid carcinoma, but whether pioglitazone is therapeutic in patients with PPFP thyroid carcinoma is unknown., Case Description: Tumor blocks from 40 patients with progressive thyroid cancer despite standard-of-care therapy were screened for PPFP, and the tumor from only one patient (2.5%) was positive. The patient had a 6.0-cm acetabular soft tissue metastasis from Hürthle cell carcinoma that caused severe pain on weight bearing and had a serum thyroglobulin level of 1974 ng/mL. After 24 weeks of therapy with pioglitazone, the metastatic lesion was 3.9 cm, the thyroglobulin level was 49.4 ng/mL, and the patient was pain-free. Thirteen months after discontinuation of pioglitazone, the metastatic lesion was 3.6 cm, the thyroglobulin level was 4.7 ng/mL, and the patient remained pain-free., Conclusions: Pioglitazone may be therapeutic in patients with PPFP thyroid cancer. However, thyroid cancers that are progressive despite standard-of-care therapy appear to only rarely express PPFP.

The genomic landscape of thyroid cancers that are derived from follicular cells has been substantially elucidated through the coordinated application of high-throughput genomic technologies. Here, I review the common genetic alterations across the spectrum of thyroid neoplasia and present the resulting model of thyroid cancer initiation and progression. This model illustrates the striking correlation between tumor differentiation and overall somatic mutational burden, which also likely explains the highly variable clinical behavior and outcome of patients with thyroid cancers. These advances are yielding critical insights into thyroid cancer pathogenesis, which are being leveraged for the development of new diagnostic tools, prognostic and predictive biomarkers, and novel therapeutic approaches.

A significant portion of paragangliomas (PGL) and pheochromocytomas (PCC) occur in patients with hereditary PGL/PCC syndromes, including those with germline mutations in succinate dehydrogenase (SDHx) subunit genes. Recently, germline fumarate hydratase (FH) mutations have been identified in a subset of PGL/PCC, and patients with hereditary leiomyomatosis and renal cell carcinoma (HLRCC) may have an increased risk of developing PGL/PCC. SDHB immunohistochemistry (IHC) has previously been shown to be useful for identifying SDHx-deficient PGL/PCC, however, FH IHC has never been explored in these tumors. Thus, we characterized SDHB and FH IHC in a large cohort of PGL/PCC patients (n = 41) at our institution who were evaluated for hereditary PGL/PCC syndromes. Overall, there was strong, positive correlation between germline SDHx subunit gene mutation status and SDHB IHC status (r φ = 0.77; P < .0001), with high corresponding sensitivity, specificity, positive predictive value, and negative predictive value (95.0%, 81.8%, 82.6%, and 94.7%, respectively). Although SDHB loss by IHC was highly correlated with germline SDHx gene mutations, its utility in this population was dependent on clinicopathologic context: while all head and neck PGL patients with SDHB-deficient tumors had germline SDHx gene mutations, only a small subset (25.0%) of PCC patients with SDHB-deficient tumors harbored a germline SDHx gene mutation. Finally, although our cohort contained only one HLRCC patient, their tumor was FH-deficient by IHC, and all other PGL/PCC showed retained FH IHC. Thus, in the appropriate clinical setting, SDHB and FH IHC may be useful for identifying PGL/PCC patients for Medical Genetics evaluation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background & Aims: The multiple endocrine neoplasia, type 1 (MEN1) locus encodes the nuclear protein and tumor suppressor menin. MEN1 mutations frequently cause neuroendocrine tumors such as gastrinomas, characterized by their predominant duodenal location and local metastasis at time of diagnosis. Diffuse gastrin cell hyperplasia precedes the appearance of MEN1 gastrinomas, which develop within submucosal Brunner's glands. We investigated how menin regulates expression of the gastrin gene and induces generation of submucosal gastrin-expressing cell hyperplasia., Methods: Primary enteric glial cultures were generated from the VillinCre:Men1 FL/FL :Sst -/- mice or C57BL/6 mice (controls), with or without inhibition of gastric acid by omeprazole. Primary enteric glial cells from C57BL/6 mice were incubated with gastrin and separated into nuclear and cytoplasmic fractions. Cells were incubated with forskolin and H89 to activate or inhibit protein kinase A (a family of enzymes whose activity depends on cellular levels of cyclic AMP). Gastrin was measured in blood, tissue, and cell cultures using an ELISA. Immunoprecipitation with menin or ubiquitin was used to demonstrate post-translational modification of menin. Primary glial cells were incubated with leptomycin b and MG132 to block nuclear export and proteasome activity, respectively. We obtained human duodenal, lymph node, and pancreatic gastrinoma samples, collected from patients who underwent surgery from 1996 through 2007 in the United States or the United Kingdom., Results: Enteric glial cells that stained positive for glial fibrillary acidic protein (GFAP+) expressed gastrin de novo through a mechanism that required PKA. Gastrin-induced nuclear export of menin via cholecystokinin B receptor (CCKBR)-mediated activation of PKA. Once exported from the nucleus, menin was ubiquitinated and degraded by the proteasome. GFAP and other markers of enteric glial cells (eg, p75 and S100B), colocalized with gastrin in human duodenal gastrinomas., Conclusions: MEN1-associated gastrinomas, which develop in the submucosa, might arise from enteric glial cells through hormone-dependent PKA signaling. This pathway disrupts nuclear menin function, leading to hypergastrinemia and associated sequelae., (Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Objective: Correct subtyping of primary aldosteronism (PA) is essential for good surgical outcomes. Adrenal vein sampling (AVS) and/or computed tomography (CT) are used for PA subclassification. Clinical and/or biochemical improvement after surgery, however, is not always achieved in patients with presumed unilateral PA. We aimed to identify the pitfalls in PA subclassification leading to surgical treatment failures., Patients and Design: We retrospectively studied 208 patients who underwent adrenal vein sampling (AVS) for PA subclassification in a tertiary referral centre, between January 2009 and August 2016. Simultaneous bilateral AVS was performed before and after cosyntropin administration. We implemented immunohistochemistry for aldosterone synthase (CYP11B2) and 17α-hydroxylase/17,20 lyase (CYP17A1) in adrenal glands resected from patients without improvement of PA after surgical treatment and from those with limitations in AVS interpretation., Results: Of 55 patients who underwent adrenalectomy, three (5.5%) had no improvement of PA. All three patients underwent partial adrenalectomy to remove a CT-detected nodule present on the same side with AVS lateralization. Immunohistochemistry revealed a CYP11B2-negative nodule in both cases available. All patients who underwent total adrenalectomy based on AVS lateralization benefitted from surgery, including three patients with unilateral unsuccessful AVS and aldosterone suppression in the catheterized side vs inferior vena cava., Conclusions: Radiographically identified adrenal nodules are not always a source of PA, even when ipsilateral with AVS lateralization. These data caution against reliance on imaging findings, either alone or in conjunction with AVS, to guide surgery for PA., (© 2017 John Wiley & Sons Ltd.)