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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.

Supplementary Fig. S2 shows Immunohistochemical staining of H295R xenograft samples.

Supplementary methods

Adrenocortical carcinoma (ACC) is a rare, but highly aggressive cancer with limited treatment options and poor survival for patients with advanced disease. Improved understanding of transcriptional programs engaged in ACC will help direct rational, targeted therapies. While 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 used independent component analysis and identified a novel Wnt/β-catenin-associated signature in ACC predictive of poor survival. This signature was enriched for the extracellular matrix (ECM), suggesting a potential role for Wnt/β-catenin in regulating the ACC microenvironment. We further investigated the minor fibrillar collagen, collagen XI alpha 1 (COL11A1), and found thatCOL11A1expression 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 expression ofCOL11A1and other ECM components, and decreased viability of cancer cellsin vitro. To investigate the preclinical potential of Wnt/β-catenin inhibitionin vivo, we developed and characterized a novel orthotopic xenograft model utilizing minimally invasive techniques. Treatment with the newly developed Wnt/β-catenin:TBL1 inhibitor Tegavivint significantly reduced tumor growth in this preclinical model. Together, our data supports that 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 further clinical development of Wnt/β-catenin inhibitors in ACC and unveil a novel Wnt/β-catenin-regulated transcriptome.Simple SummaryAdrenocortical carcinoma (ACC) is a rare, often deadly cancer arising from the adrenal gland. Mortality associated with ACC remains unchanged over the last several decades. The rarity of ACC, an incomplete understanding of its molecular basis, and limited availability of pre-clinical models have hampered the development of new effective therapies. The present work aims to address these gaps with a focus on the Wnt/β-catenin cell signaling pathway, which is aberrantly activated in ~40% of ACC tumors. We discover a novel ECM program activated in ACC that is associated with Wnt/β-catenin and poor survival. Wnt/β-catenin inhibition disrupts expression of ECM genes and induces loss of cancer cell viability. To extend these findings, we develop a rapid orthotopic mouse model of ACC and demonstrate that disruption of the Wnt/β-catenin axis with novel small molecule inhibitor Tegavivint is a potential effective therapeutic strategy to reduce ACC tumor burdenin vivo.

SUMMARYProliferating Cell Nuclear Antigen (PCNA) is a sliding clamp protein that coordinates DNA replication with various DNA maintenance events that are critical for human health. Recently, a hypomorphic homozygous serine to isoleucine (S228I) substitution in PCNA was described to underlie a DNA repair disorder known as PCNA-Associated DNA Repair Disorder (PARD). PARD symptoms range from UV sensitivity, neurodegeneration, telangiectasia, and premature aging. We, and others, previously showed that the S228I variant changes the protein binding pocket of PCNA to a conformation that impairs interactions with specific partners. Here, we report a second PCNA substitution (C148S) that also causes PARD. Unlike PCNA-S228I, PCNA-C148S has WT-like structure and affinity towards partners. In contrast, both disease-associated variants possess a thermo-stability defect. Furthermore, patient-derived cells homozygous for theC148Sallele exhibit low levels of chromatin-bound PCNA and display temperature-dependent phenotypes. The stability defect of both PARD variants indicates that PCNA levels are likely an important driver of PARD disease. These results significantly advance our understanding of PARD and will likely stimulate additional work focused on clinical, diagnostic, and therapeutic aspects of this severe disease.

Background Glioblastoma is the most frequent and high-grade adult malignant central nervous system tumor. The prognosis is still poor despite the use of combined therapy involving maximal surgical resection, radiotherapy, and chemotherapy. Metabolic reprogramming currently is recognized as one of the hallmarks of cancer. Glutamine metabolism through glutaminolysis has been associated with tumor cell maintenance and survival, and with antioxidative stress through glutathione (GSH) synthesis. Methods In the present study, we analyzed the glutaminolysis-related gene expression levels in our cohort of 153 astrocytomas of different malignant grades and 22 non-neoplastic brain samples through qRT-PCR. Additionally, we investigated the protein expression profile of the key regulator of glutaminolysis (GLS), glutamate dehydrogenase (GLUD1), and glutamate pyruvate transaminase (GPT2) in these samples. We also investigated the glutathione synthase (GS) protein profile and the GSH levels in different grades of astrocytomas. The differential gene expressions were validated in silico on the TCGA database. Results We found an increase of glutaminase isoform 2 gene (GLSiso2) expression in all grades of astrocytoma compared to non-neoplastic brain tissue, with a gradual expression increment in parallel to malignancy. Genes coding for GLUD1 and GPT2 expression levels varied according to the grade of malignancy, being downregulated in glioblastoma, and upregulated in lower grades of astrocytoma (AGII–AGIII). Significant low GLUD1 and GPT2 protein levels were observed in the mesenchymal subtype of GBM. Conclusions In glioblastoma, particularly in the mesenchymal subtype, the downregulation of both genes and proteins (GLUD1 and GPT2) increases the source of glutamate for GSH synthesis and enhances tumor cell fitness due to increased antioxidative capacity. In contrast, in lower-grade astrocytoma, mainly in those harboring the IDH1 mutation, the gene expression profile indicates that tumor cells might be sensitized to oxidative stress due to reduced GSH synthesis. The measurement of GLUD1 and GPT2 metabolic substrates, ammonia, and alanine, by noninvasive MR spectroscopy, may potentially allow the identification of IDH1mut AGII and AGIII progression towards secondary GBM.

Objectives To analyze the efficiency of a multigenic targeted massively parallel sequencing panel related to endocrine disorders for molecular diagnosis of patients assisted in a tertiary hospital involved in the training of medical faculty. Material and methods Retrospective analysis of the clinical diagnosis and genotype obtained from 272 patients in the Endocrine unit of a tertiary hospital was performed using a custom panel designed with 653 genes, most of them already associated with the phenotype (OMIM) and some candidate genes that englobes developmental, metabolic and adrenal diseases. The enriched DNA libraries were sequenced in NextSeq 500. Variants found were then classified according to ACMG/AMP criteria, with Varsome and InterVar. Results Three runs were performed; the mean coverage depth of the targeted regions in panel sequencing data was 249×, with at least 96.3% of the sequenced bases being covered more than 20-fold. The authors identified 66 LP/P variants (24%) and 27 VUS (10%). Considering the solved cases, 49 have developmental diseases, 12 have metabolic and 5 have adrenal diseases. Conclusion The application of a multigenic panel aids the training of medical faculty in an academic hospital by showing the picture of the molecular pathways behind each disorder. This may be particularly helpful in developmental disease cases. A precise genetic etiology provides an improvement in understanding the disease, guides decisions about prevention or treatment, and allows genetic counseling.

Primary aldosteronism (PA) is the most common form of endocrine hypertension and effects one in 50 adults. PA is characterized by inappropriately elevated aldosterone production via renin-independent mechanisms. Driver somatic mutations for aldosterone excess have been found in approximately 90% of aldosterone-producing adenomas (APAs). Using next-generation sequencing, we identified recurrent in-frame deletions inSLC30A1in five APAs (p.L51_A57del, n=3; p.L49_L55del, n=2).SLC30A1encodes the ubiquitous zinc efflux transporter ZnT1 (zinc transporter 1). The identifiedSLC30A1variants are situated in close proximity of the zincbinding site (H43 and D47) in transmembrane domain II and likely cause abnormal ion transport. PA cases with the uniqueSLC30A1mutations showed male dominance and demonstrated increased aldosterone and 18-oxo-cortisol concentrations. Functional studies of the mutant SLC30A151_57delvariant in a doxycycline-inducible adrenal cell system revealed abnormal Na+conductivity caused by the mutant, which in turn led to the depolarization of the resting membrane potential, and thus to the opening of voltage-gated calcium channels. This resulted in an increase in cytosolic Ca2+activity, which stimulatedCYP11B2mRNA expression and aldosterone production. Collectively, these data implicate the first-in-field zinc transporter mutations as a dominant driver of aldosterone excess in PA.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is the rate-limiting enzyme of the mevalonate pathway, which generates cholesterol and non-sterol compounds such as isoprenoid, which are involved in key steps of tumorigenesis such as cell growth and proliferation. Our aim was to evaluate the role of the mevalonate pathway in adrenocortical tumors (ACTs). Expression pattern of HMGCR, FDFT1, LDLR, SCARB1, StAR, TSPO, CYP11A1, CYP11B1, CYP17A1, CYP21A1, and HSD3B1 genes, involved in the mevalonate pathway and steroidogenesis, was quantified by real-time RT-PCR in 46 ACT [14 adenomas (ACA) and 11 carcinomas (ACC) from adults and 13 ACA and 8 ACC from pediatric patients]. Effects of the mevalonate pathway inhibition on NCI-H295A cell viability was assessed by colorimetric assay. HMGCR was overexpressed in most adult ACT. The expression of TSPO, STAR, CYP11B1, CYP21A1, and HSD3B1 in adult ACC was significantly lower than in ACA (p

Objective: Our aim is to establish genetic diagnosis of congenital generalized lipodystrophy (CGL) using targeted massively parallel sequencing (MPS), also known as next-generation sequencing (NGS). Subjects and methods: Nine unrelated individuals with a clinical diagnosis of CGL were recruited. We used a customized panel to capture genes related to genetic lipodystrophies. DNA libraries were generated, sequenced using the Illumina MiSeq, and bioinformatics analysis was performed. Results: An accurate genetic diagnosis was stated for all nine patients. Four had pathogenic variants in AGPAT2 and three in BSCL2. Three large homozygous deletions in AGPAT2 were identified by copy-number variant analysis. Conclusions: Although we have found allelic variants in only 2 genes related to CGL, the panel was able to identify different variants including deletions that would have been missed by Sanger sequencing. We believe that MPS is a valuable tool for the genetic diagnosis of multi-genes related diseases, including CGL.

Astrocytoma is the most common and aggressive tumor of the central nervous system. Genetic and environmental factors, bacterial infection, and several other factors are known to be involved in gliomagenesis, although the complete underlying molecular mechanism is not fully understood. Tumorigenesis is a multistep process involving initiation, promotion, and progression. We present a human model of malignant astrocyte transformation established by subjecting primary astrocytes from healthy adults to four sequential cycles of forced anchorage impediment (deadhesion). After limiting dilution of the surviving cells obtained after the fourth deadhesion/readhesion cycle, three clones were randomly selected, and exhibited malignant characteristics, including increased proliferation rate and capacity for colony formation, migration, and anchorage-independent growth in soft agar. Functional assay results for these clonal cells, including response to temozolomide, were comparable to U87MG—a human glioblastoma-derived cell lineage—reinforcing malignant cell transformation. RNA-Seq analysis by next-generation sequencing of the transformed clones relative to the primary astrocytes revealed upregulation of genes involved in the PI3K/AKT and Wnt/β-catenin signaling pathways, in addition to upregulation of genes related to epithelial–mesenchymal transition, and downregulation of genes related to aerobic respiration. These findings, at a molecular level, corroborate the change in cell behavior towards mesenchymal-like cell dedifferentiation. This linear progressive model of malignant human astrocyte transformation is unique in that neither genetic manipulation nor treatment with carcinogens are used, representing a promising tool for testing combined therapeutic strategies for glioblastoma patients, and furthering knowledge of astrocytoma transformation and progression.

Lysyl oxidase-like 3 (LOXL3), belonging to the lysyl oxidase family of copper-dependent amino oxidase, is responsible for the crosslinking of collagen and elastin. In tumors, the presence of LOXL3 has been associated with genomic stability, cell proliferation, and metastasis. In silico analysis has shown that glioblastoma was among the tumors with the highest LOXL3 expression levels. Moreover, LOXL3 expression increased with malignancy grade amongst diffusely infiltrative astrocytomas (from grade 2 to 4). In the present work, CRISPR-Cas9 gene editing was used to knock out LOXL3 in the human glioma U87MG cell line to evaluate the role of LOXL3 in glioblastoma. Analyses of protein expression level and genomic mutation mediated by CRISPR-Cas9 by two different sgRNA (denominated sgRNA1 and 2) were performed and the knock out efficiency and genome editing were confirmed. Transcriptome analysis of these cells revealed a downregulation of genes that coded for proteins involved in microtubule organization, cell division, and vesicle trafficking. On the other hand, upregulated genes when LOXL3 was knocked out in U87MG cells were involved in actin cytoskeleton, negative regulation of gene transcription, including chromatin remodeling, and cell-cell adhesion. Functional in vitro assays showed that LOXL3 knocked out cells presented decreased cell proliferation due to longer cell cycles with increased G1 and G2/M checkpoints compared to control cells. Additionally, LOXL3 knocked out U87MG cells presented a higher percentage of cells in the early phase of apoptosis with an increment after temozolomide treatment. Our results suggest an important role of LOXL3 in glioblastoma possibly mediated by cytoskeleton regulation, affecting cell proliferation, cell cycle, and cell death. These results reinforced the importance of LOXL3 in tumors, especially in glioblastoma, highlighting it as a potential therapeutic target. Citation Format: Talita S. Laurentino, Roseli S. Soares, Antonio M. Lerario, Suely K. Marie, Sueli Mieko Oba-Shinjo. LOXL3 knock out affects pathways which involve cytoskeleton regulation, proliferation and apoptosis in glioblastoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2605.

Adrenocortical carcinoma (ACC) is a rare cancer of the adrenal cortex without curative medical therapies. CIMP-high is an aggressive ACC molecular subtype defined by global CpG island hypermethylation with paradoxical activation of adrenal differentiation (driven by master transcription factor SF1) and stemness (driven by β-catenin). We show DNA hypermethylation redistributes histone methyltransferase EZH2 and its mark, H3K27me3. EZH2 inhibition remains lethal to CIMP-high ACC cells, erasing transcriptional programs without altering DNA methylation. We reconcile this phenomenon by discovery of two nuclear complexes, SF1/β-catenin and EZH2/β-catenin, present in physiology and persistent through advanced ACC. We find SF1/β-catenin is a chromatin-bound complex that controls the ACC super-enhancer landscape, while EZH2/β-catenin is restricted to off-chromatin pools. EZH2 inhibition purges SF1/β-catenin from chromatin, sparing EZH2/β-catenin, inducing dedifferentiation and restraining ACC growth in vitro and in vivo. Our studies illustrate how cell-of-origin programs dictate cancer evolution, exposing differentiation as an therapeutic vulnerability. Citation Format: Dipika R. Mohan, Kleiton S. Borges, Isabella Finco, Christopher R. LaPensee, Juilee Rege, Donald W. Little, Tobias Else, Madson Q. Almeida, Derek Dang, James Haggerty-Skeans, Ana Claudia Latronico, Berenice B. Mendonca, Richard J. Auchus, William E. Rainey, Suely K. Marie, Thomas J. Giordano, Sriram Venneti, Maria Candida B. Fragoso, David T. Breault, Antonio M. Lerario, Gary D. Hammer. Epigenetic dedifferentiation as a therapeutic strategy in adrenal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1501.

Objective To perform a prospective genetic investigation using whole exome sequencing of a group of patients with syndromic short stature born small for gestational age of unknown cause. Study design For whole exome sequencing analysis, we selected 44 children born small for gestational age with persistent short stature, and additional features, such as dysmorphic face, major malformation, developmental delay, and/or intellectual disability. Seven patients had negative candidate gene testing based on clinical suspicion and 37 patients had syndromic conditions of unknown etiology. Results Of the 44 patients, 15 (34%) had pathogenic/likely pathogenic variants in genes already associated with growth disturbance: COL2A1 (n = 2), SRCAP (n = 2), AFF4, ACTG1, ANKRD11, BCL11B, BRCA1, CDKN1C, GINS1, INPP5K, KIF11, KMT2A, and POC1A (n = 1 each). Most of the genes found to be deleterious participate in fundamental cellular processes, such as cell replication and DNA repair. Conclusions The rarity and heterogeneity of syndromic short stature make the clinical diagnosis difficult. Whole exome sequencing allows the diagnosis of previously undiagnosed patients with syndromic short stature.

Background Glomerulopathy with fibronectin deposits is an autosomal dominant disease associated with proteinuria, hematuria, hypertension and renal function decline. Forty percent of the cases are caused by mutations in FN1, the gene that encodes fibronectin. Case presentation This report describes two cases of Glomerulopathy with fibronectin deposits, involving a 47-year-old father and a 14-year-old son. The renal biopsies showed glomeruli with endocapillary hypercellularity and large amounts of mesangial and subendothelial eosinophilic deposits. Immunohistochemistry for fibronectin was markedly positive. Whole exome sequencing identified a novel FN1 mutation that leads to an amino-acid deletion in both patients (Ile1988del), a variant that required primary amino-acid sequence analysis for assessment of pathogenicity. Our primary sequence analyses revealed that Ile1988 is very highly conserved among relative sequences and is positioned in a C-terminal FN3 domain containing heparin- and fibulin-1-binding sites. This mutation was predicted as deleterious and molecular mechanics simulations support that it can change the tertiary structure and affect the complex folding and its molecular functionality. Conclusion The current report not only documents the occurrence of two GFND cases in an affected family and deeply characterizes its anatomopathological features but also identifies a novel pathogenic mutation in FN1, analyzes its structural and functional implications, and supports its pathogenicity.

Short stature homeobox (SHOX) haploinsufficiency is a frequent cause of short stature. Despite advances in sequencing technologies, the identification of SHOX mutations continues to be performed using standard methods, including multiplex ligation-dependent probe amplification (MLPA) followed by Sanger sequencing. We designed a targeted panel of genes associated with growth impairment, including SHOX genomic and enhancer regions, to improve the resolution of next-generation sequencing for SHOX analysis. We used two software packages, CONTRA and Nexus Copy Number, in addition to visual analysis to investigate the presence of copy number variants (CNVs). We evaluated 15 patients with previously known SHOX defects, including point mutations, deletions and a duplication, and 77 patients with idiopathic short stature (ISS). The panel was able to confirm all known defects in the validation analysis. During the prospective evaluation, we identified two new partial SHOX deletions (one detected only by visual analysis), including an intragenic deletion not detected by MLPA. Additionally, we were able to determine the breakpoints in four cases. Our results show that the designed panel can be used for the molecular investigation of patients with ISS, and it may even detect CNVs in SHOX and its enhancers, which may be present in a significant fraction of patients.

Context Von Hippel-Lindau (VHL) disease is an autosomal dominant syndrome caused by germline mutations in the VHL gene. Guidelines recommend pheochromocytoma (PHEO) biochemical screening should start at age 5 years. Objective Genotype–phenotype correlations in VHL, focusing on PHEO penetrance in children, were studied. Design We retrospectively evaluated 31 individuals (median age at diagnosis was 26 years) with diagnosed VHL disease. Results PHEO was diagnosed in six children with VHL. A large PHEO (5 cm) was detected in a 4-year-old boy with p.Gly114Ser mutation. PHEO penetrance was 55% starting at age 4 years. VHL missense mutations were identified in 11 of 22 families (50%), frameshift mutations in four (18.2%), stop codon in three (13.6%), splicing site in two (9.1%), and large gene deletion in two (9.1%). The codon 167 (n = 10) was a hotspot for VHL mutations and was significantly associated with PHEO (90% vs. 38%; P = 0.007). PHEOs and pancreatic neuroendocrine tumors (PNETs) were strongly associated with VHL missense mutations compared with other mutations (89.5% vs. 0% and 73.7% vs. 16.7%; P = 0.0001 and 0.002, respectively). In contrast, pancreatic cysts (91.7% vs. 26.3%; P = 0.0001), renal cysts (66.7% vs. 26.3%; P = 0.027), and central nervous system hemangioblastomas (91.7% vs. 47.3%; P = 0.012) were more frequent in VHL with nonmissense mutations. Conclusion VHL missense mutations were highly associated with PHEO and PNETs. Our data support that in children with VHL harboring missense mutations, biochemical screening for PHEO should be initiated at diagnosis.

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.

Aim Congenital hypopituitarism has an incidence of 1:3500–10,000 births and is defined by the impaired production of pituitary hormones. Early diagnosis has an impact on management and genetic counselling. The clinical and genetic heterogeneity of hypopituitarism poses difficulties to select the order of genes to analyse. The objective of our study is to screen hypopituitarism genes (candidate and previously related genes) simultaneously using a target gene panel in patients with congenital hypopituitarism. Methods Screening of 117 subjects with congenital hypopituitarism for pathogenic variants in 26 genes associated with congenital hypopituitarism by massively parallel sequencing using a customized target gene panel. Results We found three novel pathogenic variants in OTX2 c.295C>T:p.Gln99*, GLI2 c.1681G>T:p.Glu561* and GHRHR c.820_821insC:p.Asp274Alafs*113, and the previously reported variants in GHRHR c.57+1G>A and PROP1 [c.301_302delAG];[c.109+1G>A]. Conclusions Our results indicate that a custom-designed panel is an efficient method to screen simultaneously variants of biological and clinical relevance for congenital GH deficiency. A genetic diagnosis was possible in 5 out of 117 (4%) patients of our cohort. We identified three novel pathogenic variants in GHRHR, OTX2 and GLI2 expanding the spectrum of variants associated with congenital hypopituitarism.

Somatic variants in genes that regulate intracellular ion homeostasis have been identified in aldosterone-producing adenomas (APAs). Although the mechanisms leading to increased aldosterone production in APA cells have 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 11 APAs, 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 variants, 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.

Medulloblastomas (MBs) and glioblastomas (GBMs) are high-incidence central nervous system tumors. Different origin sites and changes in the tissue microenvironment have been associated with the onset and progression. Here, we describe differences between the extracellular matrix (ECM) signatures of these tumors. We compared the proteomic profiles of MB and GBM decellularized tumor samples between each other and their normal decellularized brain site counterparts. Our analysis revealed that 19, 28, and 11 ECM proteins were differentially expressed in MBs, GBMs, and in both MBs and GBMs, respectively. Next, we validated key findings by using a protein tissue array with 53 MB and 55 GBM cases and evaluated the clinical relevance of the identified differentially expressed proteins through their analysis on publicly available datasets, 763 MB samples from the GSE50161 and GSE85217 studies, and 115 GBM samples from RNAseq-TCGA. We report a shift toward a denser fibrillary ECM as well as a clear alteration in the glycoprotein signature, which influences the tumor pathophysiology. MS data have been submitted to the PRIDE repository, project accession: PXD023350.

Lysyl oxidase-like 3 (LOXL3), belonging to the lysyl oxidase family, is responsible for the crosslinking in collagen or elastin. The cellular localization of LOXL3 is in the extracellular space by reason of its canonical function. In tumors, the presence of LOXL3 has been associated with genomic stability, cell proliferation, and metastasis. In silico analysis has shown that glioblastoma was among tumors with the highest LOXL3 expression levels. LOXL3 silencing of U87MG cells by siRNA led to the spreading of the tumor cell surface, and the transcriptome analysis of these cells revealed an upregulation of genes coding for extracellular matrix, cell adhesion, and cytoskeleton components, convergent to an increase in cell adhesion and a decrease in cell invasion observed in functional assays. Significant correlations of LOXL3 expression with genes coding for tubulins were observed in the mesenchymal subtype in the TCGA RNA-seq dataset of glioblastoma (GBM). Conversely, genes involved in endocytosis and lysosome formation, along with MAPK-binding proteins related to focal adhesion turnover, were downregulated, which may corroborate the observed decrease in cell viability and increase in the rate of cell death. Invasiveness is a major determinant of the recurrence and poor outcome of GBM patients, and downregulation of LOXL3 may contribute to halting the tumor cell invasion.

ATRX-DAXX-H3.3 chromatin remodeler complex is a well known epigenetic factor responsible for the heterochromatin maintenance and control. ATRX is an important nucleosome controller, especially in tandem repeat regions, and DAXX is a multi-function protein with particular role in histone H3.3 deposition due to its chaperone characteristic. Abnormalities in this complex have been associated with telomere dysfunction and consequently with activation of alternative lengthening of telomeres mechanism, genomic instability, and tumor progression in different types of cancer. However, the characterization of this complex is still incomplete in meningioma. We analyzed ATRX, DAXX and H3.3 expressions and the telomere length in a cohort of meningioma of different malignant grades. We observed ATRX upregulation at gene and protein levels in grade II/III meningiomas. A low variability of telomere length was observed in meningiomas across different ages and malignant grades, in contrast to the shortening of telomere length with aging in normal controls.

Microglia are the tissue-resident macrophages of the CNS. They originate in the yolk sac, colonize the CNS during embryonic development and form a self-sustaining population with limited turnover. A consequence of their relative slow turnover is that microglia can serve as a long-term memory for inflammatory or neurodegenerative events. We characterized the epigenomes and transcriptomes of microglia exposed to different stimuli; an endotoxin challenge (LPS) and genotoxic stress (DNA repair deficiency-induced accelerated aging). Whereas the enrichment of permissive epigenetic marks at enhancer regions explains training (hyperresponsiveness) of primed microglia to LPS challenge, the tolerized response of microglia seems to be regulated by loss of permissive epigenetic marks. Here, we identify that inflammatory stimuli and accelerated aging because of genotoxic stress activate distinct gene networks. These gene networks and associated biological processes are partially overlapping, which is likely driven by specific transcription factor networks, resulting in altered epigenetic signatures and distinct functional (desensitized vs. primed) microglia phenotypes.

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

Whole Exome Sequencing (WES) has been a useful tool to improve molecular diagnosis in hypopituitarism, leading to the discovery of at least 8 new genes in the last 7 years. However, some genes associated with hypopituitarism show low coverage in this methodology, limiting its use for molecular diagnosis. Our objective is to compare three library prepping kits, NimbleGen (Roche), SureSelect (Agilent) and Nextera (Illumina) examining the best performance related to sequencing quality, exon extension coverage (≥98%) and base depth read (≥20x) of 44 genes associated with hypopituitarism and 32 involved in pituitary development. Three different groups composed of 2 HapMap samples (Group 1), 2 Brazilian patients with hypopituitarism and their respective mothers (Group 2) and 109 random Brazilian samples (Group 3) were sequenced in Illumina platform. Group 1 and 3 were performed using all three library prepping kits, while group 2 was performed with NimbleGen and SureSelect. Although all technologies covered the selected genes with similar efficiency regarding poor (less than 20%) and rich (more than 80%) GC areas, SureSelect has shown to reach the most uniform coverage in the selected region with a lower level of duplicate reads, as well as a higher number of identified pathogenic variants.

APOL1 high-risk genotypes (HRG) are associated with increased risk of kidney disease in individuals of African ancestry. We analyzed the effects of APOL1 risk variants on an ethnically diverse Brazilian pediatric nephrotic syndrome (NS) cohort. Multicenter study including 318 NS patients, categorized as progressors to advanced CKD [estimated glomerular filtration rate (eGFR)] 30 mL/min/1.73 m2 through the study). We employed Cox regression with progression time as the outcome and APOL1 genotype as the independent variable. We tested this association in the entire cohort and three subgroups; (1) focal segmental glomerulosclerosis (FSGS), (2) steroid-resistant NS (SRNS), and (3) those who underwent kidney biopsy. Nineteen patients (6%) had an HRG. Of these, 47% were self-reported White. Patients with HRG manifested NS at older ages and presented higher frequencies of FSGS and SRNS. HRG patients progressed to advanced CKD more often than low-risk-genotype (LRG) children in the whole NS cohort (p = 0.001) and the three subgroups. In SRNS and biopsied patients, a single risk variant was associated with trends of higher CKD progression risk. Novel discoveries include a substantial prevalence of HRG among patients self-reported White, worse kidney outcomes in HRG versus LRG children in the FSGS subgroup, and a trend of higher CKD progression risk associated with a single risk variant in the SRNS cohort. These findings suggest APOL1-associated NS extends beyond patients self-reported non-White, the HRG effect is independent of FSGS, and a single risk variant may have a detrimental impact in children with NS.

Objective The transcription factor OTX2is implicated in ocular, craniofacial, and pituitary development. Design We aimed to establish the contribution of OTX2 mutations in congenital hypopituitarism patients with/without eye abnormalities, study functional consequences, and establish OTX2 expression in the human brain, with a view to investigate the mechanism of action. Methods We screened patients from the UK (n = 103), international centres (n = 24), and Brazil (n = 282); 145 were within the septo-optic dysplasia spectrum, and 264 had no eye phenotype. Transactivation ability of OTX2 variants was analysed in murine hypothalamic GT1-7 neurons. In situ hybridization was performed on human embryonic brain sections. Genetically engineered mice were generated with a series of C-terminal OTX2 variants. Results Two chromosomal deletions and six haploinsufficient mutations were identified in individuals with eye abnormalities; an affected relative of one patient harboured the same mutation without an ocular phenotype. OTX2 truncations led to significant transactivation reduction. A missense variant was identified in another patient without eye abnormalities; however, studies revealed it was most likely not causative. In the mouse, truncations proximal to aa219 caused anophthalmia, while distal truncations and the missense variant were tolerated. During human embryogenesis, OTX2 was expressed in the posterior pituitary, retina, ear, thalamus, choroid plexus, and partially in the hypothalamus, but not in the anterior pituitary. Conclusions OTX2 mutations are rarely associated with hypopituitarism in isolation without eye abnormalities, and may be variably penetrant, even within the same pedigree. Our data suggest that the endocrine phenotypes in patients with OTX2 mutations are of hypothalamic origin.

Hypospadias is a common congenital disorder of male genital formation. Children born small for gestational age (SGA) present a high frequency of hypospadias of undetermined etiology. No previous study investigated the molecular etiology of hypospadias in boys born SGA using massively parallel sequencing. Our objective is to report the genetic findings of a cohort of patients born SGA with medium or proximal hypospadias. We identified 46 individuals with this phenotype from a large cohort of 46,XY DSD patients, including 5 individuals with syndromic features. DNA samples from subjects were studied by either whole exome sequencing or target gene panel approach. Three of the syndromic patients have 5 main clinical features of Silver-Russell syndrome (SRS) and were first studied by MLPA. Among the syndromic patients, loss of DNA methylation at the imprinting control region H19/IGF2 was identified in 2 individuals with SRS clinical diagnosis. Two novel pathogenic variants in compound heterozygous state were identified in the CUL7 gene establishing the diagnosis of 3M syndrome in one patient, and a novel homozygous variant in TRIM37 was identified in another boy with Mulibrey nanism phenotype. Among the non-syndromic subjects, 7 rare heterozygous variants were identified in 6 DSD-related genes. However, none of the variants found can explain the phenotype by themselves. In conclusion, a genetic defect that clarifies the etiology of hypospadias was not found in most of the non-syndromic SGA children, supporting the hypothesis that multifactorial causes, new genes, and/or unidentified epigenetic defects may have an influence in this condition.

Context The genetic bases of osteoporosis (OP), a disorder with high heritability, are poorly understood at an individual level. Cases of idiopathic or familial OP have long puzzled clinicians as to whether an actionable genetic cause could be identified. Objective We performed a genetic analysis of 28 cases of idiopathic, severe, or familial osteoporosis using targeted massively parallel sequencing. Design Targeted sequencing of 128 candidate genes was performed using Illumina NextSeq. Variants of interest were confirmed by Sanger sequencing or SNP array. Patients and Setting Thirty-seven patients in an academic tertiary hospital participated (54% male; median age, 44 years; 86% with fractures), corresponding to 28 sporadic or familial cases. Main Outcome Measure The identification of rare stop-gain, indel, splice site, copy-number, or nonsynonymous variants altering protein function. Results Altogether, we identified 28 variants of interest, but only 3 were classified as pathogenic or likely pathogenic variants: COL1A2 p.(Arg708Gln), WNT1 p.(Gly169Asp), and IDUA p.(His82Gln). An association of variants in different genes was found in 21% of cases, including a young woman with severe OP bearing WNT1, PLS3, and NOTCH2 variants. Among genes of uncertain significance analyzed, a potential additional line of evidence has arisen for GWAS candidates GPR68 and NBR1, warranting further studies. Conclusions While we hope that continuing efforts to identify genetic predisposition to OP will lead to improved and personalized care in the future, the likelihood of identifying actionable pathogenic variants in intriguing cases of idiopathic or familial osteoporosis is seemingly low.

Primary ovarian insufficiency (POI) is a heterogeneous disorder associated with several genes. The majority of cases are still unsolved. Our aim was to identify the molecular diagnosis of a Brazilian cohort with POI. Genetic analysis was performed using a customized panel of targeted massively parallel sequencing (TMPS) and the candidate variants were confirmed by Sanger sequencing. Additional copy number variation (CNV) analysis of TMPS samples was performed by CONTRA. Fifty women with POI (29 primary amenorrhea and 21 secondary amenorrhea) of unknown molecular diagnosis were included in this study, which was conducted in a tertiary referral center of clinical endocrinology. A genetic defect was obtained in 70% women with POI using the customized TMPS panel. Twenty-four pathogenic variants and two CNVs were found in 48% of POI women. Of these variants, 16 genes were identified as BMP8B, CPEB1, INSL3, MCM9, GDF9, UBR2, ATM, STAG3, BMP15, BMPR2, DAZL, PRDM1, FSHR, EIF4ENIF1, NOBOX, and GATA4. Moreover, a microdeletion and microduplication in the CPEB1 and SYCE1 genes, respectively, were also identified in two distinct patients. The genetic analysis of eleven patients was classified as variants of uncertain clinical significance whereas this group of patients harbored at least two variants in different genes. Thirteen patients had benign or no rare variants, and therefore the genetic etiology remained unclear. In conclusion, next-generation sequencing (NGS) is a highly effective approach to identify the genetic diagnoses of heterogenous disorders, such as POI. A molecular etiology allowed us to improve the disease knowledge, guide decisions about prevention or treatment, and allow familial counseling avoiding future comorbidities.

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.

Objectives High-throughput sequencing of genomes, exomes, and disease-focused gene panels is becoming increasingly common for molecular diagnostics. However, identifying a single clinically relevant pathogenic variant among thousands of genetic polymorphisms is a challenging task. Publicly available genomic databases are useful resources to filter out common genetic variants present in the population and enable the identification of each disease-causing variant. Based on our experience applying these technologies at Hospital das Clinicas da Faculdade de Medicina da Universidade de Sao Paulo (HCFMUSP), Sao Paulo, Brazil, we recognized that the Brazilian population is not adequately represented in widely available genomic databases. Methods Here, we took advantage of our 5-year experience as a high-throughput sequencing core facility focused on individuals with putative genetic disorders to build a genomic database that may serve as a more accurate reference for our patient population: SELAdb. Results/conclusions Currently, our database comprises a final cohort of 523 unrelated individuals, including patients or family members managed by different clinics of HCFMUSP. We compared SELAdb with other publicly available genomic databases and demonstrated that this population is very heterogeneous, largely resembling Latin American individuals of mixed origin, rather than individuals of pure European ancestry. Interestingly, exclusively through SELAdb, we identified a spectrum of known and potentially novel pathogenic variants in genes associated with highly penetrant Mendelian disorders, illustrating that pathogenic variants circulating in the Brazilian population that is treated in our clinics are underrepresented in other population databases. SELAdb is freely available for public consultation at: http://intranet.fm.usp.br/sela.

CONTEXT: Primary ovarian insufficiency (POI) is a cause of female infertility. However, the genetic etiology of this disorder remains unknown in most patients with POI. OBJECTIVE: To investigate the genetic etiology of idiopathic POI. PATIENTS AND METHODS: We performed whole-exome sequencing of 11 families with idiopathic POI. To gain insights into the potential mechanisms associated with this mutation, we generated two mouse lines via clustered regularly interspaced short palindromic repeats/Cas9 technology. RESULTS: A pathogenic homozygous missense mutation (c.149A>G; p.Asp50Gly) in the POLR3H gene in two unrelated families was identified. Pathogenic mutations in this subunit have not been associated with human disorders. Loss-of-function Polr3h mutation in mice caused early embryonic lethality. Mice with homozygous point mutation (Polr3h(D50G)) were viable but showed delayed pubertal development, characterized by late first estrus or preputial separation. The Polr3h(D50G) female and male mice showed decreased fertility later in life, associated with small litter size and increased time to pregnancy or to impregnate a female. Polr3h(D50G) mice displayed decreased expression of ovarian Foxo3a and lower numbers of primary follicles. CONCLUSION: Our manuscript provides a case of POI caused by missense mutation in POLR3H, expanding the knowledge of molecular pathways of the ovarian function and human infertility. Screening of the POLR3H gene may elucidate POI cases without previously identified genetic causes, supporting approaches of genetic counseling.

Objective Genomic alterations in Hürthle cell carcinomas (HCC) include chromosomal losses, mitochondrial DNA mutations, and changes in the expression profile of the PI3K-AKT-mTOR and Wnt/β-catenin pathways. This study aimed at characterizing the mutational profile of HCC. Methods Next-generation sequencing (NGS) of 40 HCC using a 102-gene panel including, among others, the MAPK, PI3K-AKT-mTOR, Wnt/β-catenin, and Notch pathways. HCC was widely invasive in 57.5%, and lymph node and distant metastases were diagnosed in 5% and 7.5% of cases. During follow-up, 10% of patients presented with persistent/recurrent disease, but there were no cancer-related deaths. Results Genetic alterations were identified in 47.5% of HCC and comprised 190 single-nucleotide variants and 5 insertions/deletions. The Wnt/β-catenin pathway was most frequently affected (30%), followed by MAPK (27.5%) and PI3K-AKT-mTOR (25%). FAT1 and APC were the most frequently mutated genes and present in 17.5%. RAS mutations were present in 12.5% but no BRAF mutation was found. There was no association between the mutational profile and clinicopathological features. Conclusions This series of HCC presents a wide range of mutations in the Wnt/β-catenin, MAPK and PI3K-AKT-mTOR pathways. The recurrent involvement of Wnt/β-catenin pathway, particularly mutations in APC and FAT1, are of particular interest. The data suggest that mutated FAT1 may represent a potential novel driver in HCC tumorigenesis and that the Wnt/β-catenin pathway plays a critical role in this distinct thyroid malignancy.

Background: It is recommended a multidisciplinary approach consisted of clinical, hormonal and genetic workups for diagnosing 46,XY DSD. However, no previous study has quantified how useful is this combined approach. Objectives: To retrospectively review the clinical and genetic findings for diagnosing a large cohort of patients with 46,XY DSD from a single Brazilian center. Methods: 247 non-syndromic 46,XY DSD individuals (159 sporadic and 88 familial cases from 39 families) were studied. Clinical and hormonal data were collected from medical files. Testosterone (T), androstenedione (A) were measured by immunoradiometric or immunofluorimetric assays and dihydrotestosterone (DHT) by RIA after celite chromatography or by liquid chromatography tandem mass spectrometry; T/DHT and T/A ratios were calculated. Analysis of sensitivity (SE), specificity (SP) of T/DHT was performed, being the molecular diagnosis considered the gold standard for diagnosing SRD5A2 deficiency. A T/A>0.8 was considered indicative of 17ß-HSDB3 deficiency. The patients were clinically classified into four subgroups: 1) androgen insensitivity syndrome (AIS), 2) gonadal dysgenesis (GD); 3) defects in androgen synthesis (DAS) and 4) DSD of unknown etiology. Molecular studies were performed by Sanger sequencing and/ or massively parallel sequencing (MPS). Results: The median age at first visit was 14 years (range 0.1 to 59 years). The molecular diagnosis was established in 96.5% of the cases with AIS (n=28/29), in 96% of the subjects with DAS (n=46/48), in 36% of the patients with GD (n=21/57) and in 26.7% (n=15/56) with DSD of unknown etiology. The best cut-off for T/DHT in basal state and hCG stimulated was 12.5 (SE=100%; SP=78.57%) and 24 (SE=87.5%; SP=95.7%) respectively. A T/A Conclusion: Considering the phenotype heterogeneity, pitfalls of the hormonal assessment and number of genes involved, it is reasonable to consider MPS as a first test for diagnosing patients with 46,XY DSD. However, the combination of clinical and molecular diagnosis is more accurate than either strategies alone in diagnosing 46,XY DSD.

Most ovarian steroid cell tumors arise sporadically. However, they can also be observed as a rare manifestation of von Hippel Lindau disease. Here, we present a clinical, pathological and molecular characterization of a steroid cell tumor in a VHL patient. A 14 year old girl with molecularly confirmed diagnosis of VHL developed hirsutism and amenorrhea. Initial clinical hormonal evaluation was notable for elevated 17-OHP of 406ng/dl, androstenedione 275ng/dl, and testosterone 102ng/dl. In order to exclude congenital adrenal hyperplasia as a common cause of hirsutism in adolescents, ACTH stimulation was performed, but no increase in 17-OHP was observed. Anti Muellerian hormone, inhibin (INH) A and INH B were normal. Imaging revealed a bilobed 6cm left adnexal mass. The mass was resected en bloc via a left oophorectomy. Pathological evaluation showed multinodular steroid cell tumor with clear cytoplasm and delicate vascular meshwork. Immunoprofiling demonstrated positivity for inhibin and calretinin; while renal cell carcinoma markers were negative. All laboratory values normalized post-surgery. In addition to clinical measurements pre- and post-surgery, steroid profiles were evaluated by LC-MS/MS. Quantitative RT-PCR analysis showed robust tumor expression of enzymes facilitating the production of androgens, but not estrogens. Further preliminary analysis by exome sequencing confirmed the known germline pathogenic variant in VHL, but no additional obvious somatic driver mutations were identified. Interestingly, the NGS analysis of different specimens from the same tumor revealed multiple different single base pair variants in the VHL gene as a second hit. In summary, hirsutism in VHL patients should raise the suspicion for unusual ovarian tumors. In contrary to the usual theory of a monoclonal expansion after loss of the wt VHL allele, this tumor appeared to be oligoclonal as evidenced by different somatic VHL mutations. This could be either explained by initial parallel occurrence of several clones or that the VHL second hit is not an initial event, but the mutation instead supports tumor expansion following initial steps of tumorigenesis.

The adrenal cortex is an endocrine organ comprised of three histological zones, the outermost zona glomerulosa, the intermediate zona fasciculata, and the innermost zona reticularis. High plasticity of the adrenal gland is supported by pools of stem and progenitor cells that are deployed to sustain physiological and homeostatic demands. In recent decades, exciting new discoveries elucidating the identity, function, and fate of these cell populations have emerged. In this review, we describe paracrine and endocrine signaling loops that are crucial for adrenal biology, focusing on recent studies unpacking the enigmatic nature of adrenal stem and progenitor cell populations.

The adrenal cortex is comprised of distinct concentric zones that produce hormones essential for life - the outermost zona glomerulosa (zG) produces aldosterone, and the innermost zona fasciculata (zF) produces cortisol. Adrenal zonation is maintained by a balance between paracrine (Wnt/β-catenin) and endocrine (ACTH/PKA) signaling. Wnt/β-catenin signaling is maintained in the outer cortex (zG, upper zF) by a gradient of Wnt ligands that diminish centripetally, and PKA signaling is maintained in the inner cortex (zF) by ACTH. Recent studies in vivo suggest that sustained PKA signaling promotes zF proliferation, enabling lineage conversion towards zF by inhibiting Wnt/β-catenin transcriptional programming. While these studies were crucial in elucidating mechanisms supporting adrenal zonation, it remains unclear if PKA-mediated repression of Wnt/β-catenin is carried out at the chromatin level or if it is secondary to ligand-dependent modulation of paracrine signaling as may happen in vivo. To address this question, we utilized the adrenocortical cell line (NCI-H295R), which harbors a mutation in CTNNB1 rendering Wnt/β-catenin signaling constitutively active. We stimulated PKA in NCI-H295R using forskolin, and assessed genome-wide chromatin accessibility by ATAC-seq and transcriptome changes by RNA-seq. Motif analysis of ATAC-seq from baseline NCI-H295R revealed that chromatin accessibility is dominated by transcription factors SF1 (master regulator of the adrenal cortex and steroidogenesis), AP1 (effector of PKA) and LEF1 (effector of Wnt/β-catenin). Following forskolin administration, we observed decreased accessibility in chromatin containing LEF1 binding motifs, and increased accessibility in chromatin bearing AP1 and SF1 motifs, suggesting that PKA activation drives AP1/SF1-dependent transcription and inhibits Wnt/β-catenin-dependent transcription at the chromatin level. Indeed, RNA-seq revealed that forskolin administration decreased the expression of zG and Wnt/β-catenin target genes, while simultaneously increasing expression of AP1/SF1 target genes. Collectively, these data demonstrate that PKA activation leads to profound chromatin remodeling that enables zF identity even in the setting of constitutive Wnt/β-catenin signaling. Ongoing studies are aimed at elucidating how chromatin modifiers and transcriptional machinery coordinate the dynamic regulation of differentiation programs required for adrenocortical homeostasis and zonation.

Corticotroph adenomas (CA) are rare pituitary tumors that impose several challenges in clinical management - CA are difficult to diagnose, often recur, and are associated with high morbidity and mortality. CA are characteristically Tpit-positive and PIT1-negative and comprise ACTH-producing (Cushing’s disease (CD)) and ACTH-silent (AS) classes. The molecular programs contributing to disease pathogenesis in CA are still poorly characterized, largely restricted to the identification of somatic mutations in USP8 in 40-60% of CD adenomas. To more fully characterize the mutational and transcriptional landscape driving both classes of CA, we performed whole-exome sequencing and RNA-seq in 19 CD and 16 AS adenomas. We identified USP8 mutations in 53% of CD (10/19) and 6% of AS (1/16) samples. Strikingly, in 19% of AS tumors (3/16), all exhibiting an unusually aggressive disease course, including two cases with brain metastases, we identified recurrent somatic pathogenic mutations in TP53 and novel loss-of-function mutations in telomere maintenance genes DAXX and ATRX. Furthermore, while all tumors with USP8 mutations (regardless of CD/AS status) exhibited no chromosomal abnormalities as measured by copy-number variation (CNV) and loss of heterozygosity (LOH) analysis, 33% of CD (4/12, including 1 tumor with a DAXX mutation) and 36% of AS (4/11, including all DAXX/ATRX-mutated cases) samples exhibited profound chromosomal instability, characterized by hyperdiploidy, widespread whole-chromosome LOH events, and arm-level breakpoints. Using transcriptome analysis (n=22), we identified three classes of tumors (C1-C3), reflecting these distinct somatic alteration profiles. C1 tumors (n=6) are characterized by chromosomal stability, includes exclusively USP8-mutated CD, and exhibits upregulation of genes involved in metabolic processes and protein acetylation. C2 tumors (n=10) are comprised exclusively of AS (including all TP53- and/or DAXX/ATRX-mutated cases), are characterized by chromosomal instability, and exhibits concordant upregulation of cell cycle programs. Finally, C3 (n=6) contains a mixture of AS and CD cases (including CD without mutations in USP8) and features an expression profile that partly overlap with C1 tumors, but also exhibit higher expression of inflammatory genes. Taken together, our data suggest that CD and AS are distinct molecular subtypes of CA, highlighting the dominant role of USP8 mutations in driving a unique transcriptional program and illustrate for the first time that unlike most cases of CD, AS cases are characterized by profound genomic instability and cell cycle activation, features associated with a more aggressive disease course.

Background: Adrenocortical cancer (ACC) is a rare malignant neoplasia associated with a variable clinical presentation. Pediatric patients generally have a better prognosis when compared to adults. In addition, unlike in adults where ACC which is usually sporadic, 50-80% of pediatric ACC is associated with genetic disorders such as Beckwith-Wiedemann and Li-Fraumeni syndromes. Recently, was showed that 3-5% of adult patients with ACC presented germline variants in DNA mismatch repair genes such as MSH2 and MSH6, the cause of Lynch syndrome (LS). The prevalence of these alterations in pediatric ACC is unknown. We aimed to investigate the prevalence of germline alterations in DNA mismatch repair genes among pediatric and adult patients with adrenocortical tumors (benign and malignant) carriers of the germline TP53 p.R337H mutation. Methods: 35 patients selected (30 pediatric and 5 adult) with functional tumors. ACC was diagnosed in 4 pediatric and in all adult patients. NGS was performed in 35 DNA blood samples by HNPCC MASTR Plus for the identification of SNV in 4 genes (MLH1, MSH2, MSH6, and PMS2) and 3’ UTR of EPCAM. Copy number variation (CNV) analyses were done by Copy Number Targeted Resequencing Analysis (CONTRA) and MLPA. The variants were classified, according to ACMG (American College Medical Genome) by Varsome platform. The protein expression was evaluated by Immunohistochemistry (IHC): MLH1 (clone ES05), MSH2 (FE11), MSH6 (EP49), and PMS2 (EP51). All patients were evaluated for variants in TP53. Results: NGS: 2 children presented 2 pathogenic allelic variants associated with LS (2/30, 6.6%), both patients with benign outcome and follow up of 4 years: 1 deletion in MLH1 (c.1500_1502del) and 1 nonsense in the MSH6 gene (c.328C>T p.Arg110X. CNV: MLPA specific for MLH1/MSH2 showed a normal copy number. ICH: the loss of expression in MLH1/PMS2 was identified in only one case without allelic variants. Discussion: Although our cohort is small, we observed 2 allelic pathogenic variants associated with LS among pediatric with adrenocortical tumors. It is higher than the prevalence of colorectal and endometrial cancer (3.2%) in LS. A personal and family history of LS tumors should be strongly considered for genetic risk assessment in pediatric patients with ACT. If the association with TP53 alteration can influence the tumor’s behavior with early clinical presentation, as seen in hereditary nonpolyposis colorectal cancer, it needs to be investigated. The patients with both alterations must be followed with surveillance, according to the US Multi-Society task force guideline for Lynch syndrome and for Li-Fraumeni syndrome.

Adrenocortical carcinoma (ACC) is a rare, aggressive cancer. Up to 75% of patients develop incurable metastatic disease, highlighting an urgent need for novel medical therapies. We recently identified a rapidly progressive ACC subtype characterized by CpG island hypermethylation (CIMP-high), sustained Wnt/β-catenin signaling, steroidogenic differentiation, and cell cycle activation. CIMP-high status alone accounts for 40% of ACC, but predicts 70% of recurrences and >50% of deaths. Intriguingly, hypermethylated CpG islands in CIMP-high ACC are unmethylated in fetal and adult adrenal cortex, suggesting DNA methylation is supported by cancer-specific mechanisms. We therefore sought to investigate how aberrant epigenetic programming contributes to ACC biology. In embryonic stem cells, the Polycomb repressive complex 2 (PRC2) represses differentiation programs through EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3) deposition in promoter CpG islands free of DNA methylation. Gain or loss of EZH2/PRC2 function prevails in a variety of human cancers, enabling proliferation in a tissue-specific manner. Here, we identify that CIMP-high ACC exhibit high expression of EZH2/H3K27me3, but paradoxically bear DNA hypermethylation in annotated PRC2 target regions. To determine if DNA methylation of PRC2 targets disrupts or is controlled by EZH2, we characterized EZH2’s role in CIMP-high ACC cell line NCI-H295R at baseline and in response to EZH2 inhibition (EZH2i). EZH2-directed IP-MS revealed EZH2 interacts with PRC2 members and DNA methylation-sensitive accessory proteins, but no DNA methyltransferase machinery. ChIP-seq revealed EZH2 and H3K27me3 colocalize in repressive domains genome-wide, but DNA methylation and H3K27me3 are mutually exclusive. EZH2i induced H3K27 demethylation and loss of viability, but with no effect on CIMP-high DNA methylation. These data suggest PRC2 target DNA methylation in CIMP-high ACC is maintained independently of EZH2, enabling EZH2/PRC2 to coordinate alternative programs required for cell survival. We then measured the consequences of EZH2i on the NCI-H295R transcriptome (RNA-seq), EZH2/H3K27me3 deposition genome-wide (ChIP-seq), and chromatin accessibility landscape (ATAC-seq). EZH2i led to global downregulation of cell cycle, Wnt/β-catenin transcriptional programming, and steroidogenic differentiation, partially explained by EZH2i-induced offloading of EZH2 from H3K27me3 domains to accessible promoters genome-wide. Taken together, our studies illustrate how aberrant CpG island hypermethylation in CIMP-high ACC participates in a targetable repressive epigenetic cascade that reinforces oncogenic adrenocortical transcriptional programs. Ultimately, we hope to illuminate novel strategies for tissue-specific disruption of the aberrant epigenetic wiring that defines CIMP-high ACC.