Diagnosis and management of urinary bladder paragangliomas: A Sino-American-European retrospective observational study.

OBJECTIVE: Paragangliomas of the urinary bladder (UBPGLs) are rare neuroendocrine tumours and pose a diagnostic and surgical challenge. It remains unclear what factors contribute to a timely presurgical diagnosis. The purpose of this study is to identify factors contributing to missing the diagnosis of UBPGLs before surgery. DESIGN, PATIENTS AND MEASUREMENTS: A total of 73 patients from 11 centres in China, and 51 patients from 6 centres in Europe and 1 center in the United States were included. Clinical, surgical and genetic data were collected and compared in patients diagnosed before versus after surgery. Logistic regression analysis was used to identify clinical factors associated with initiation of presurgical biochemical testing. RESULTS: Among all patients, only 47.6% were diagnosed before surgery. These patients were younger (34.0 vs. 54.0 years, p < .001), had larger tumours (2.9 vs. 1.8 cm, p < .001), and more had a SDHB pathogenic variant (54.7% vs. 11.9%, p < .001) than those diagnosed after surgery. Patients with presurgical diagnosis presented with more micturition spells (39.7% vs. 15.9%, p = .003), hypertension (50.0% vs. 31.7%, p = .041) and catecholamine-related symptoms (37.9% vs. 17.5%, p = .012). Multivariable logistic analysis revealed that presence of younger age (<35 years, odds ratio [OR] = 6.47, p = .013), micturition spells (OR = 6.79, p = .007), hypertension (OR = 3.98, p = .011), and sweating (OR = 41.72, p = .013) increased the probability of initiating presurgical biochemical testing. CONCLUSIONS: Most patients with UBPGL are diagnosed after surgery. Young age, hypertension, micturition spells and sweating are clues in assisting to initiate early biochemical testing and thus may establish a timely presurgical diagnosis.

REGISTRI: Regorafenib in first-line of KIT/PDGFRA wild type metastatic GIST: a collaborative Spanish (GEIS), Italian (ISG) and French Sarcoma Group (FSG) phase II trial.

BACKGROUND: Approximately 15% of adult GIST patients harbor tumors that are wild-type for KIT and PDGFRα genes (KP-wtGIST). These tumors usually have SDH deficiencies, exhibit a more indolent behavior and are resistant to imatinib. Underlying oncogenic mechanisms in KP-wtGIST include overexpression of HIF1α high IGFR signaling through the MAPK pathway or BRAF activating mutation, among others. As regorafenib inhibits these signaling pathways, it was hypothesized that it could be more active as upfront therapy in advanced KP-wtGIST. METHODS: Adult patients with advanced KP-wtGIST after central confirmation by NGS, naïve of systemic treatment for advanced disease, were included in this international phase II trial. Eligible patients received regorafenib 160 mg per day for 21 days every 28 days. The primary endpoint was disease control rate (DCR), according to RECIST 1.1 at 12 weeks by central radiological assessment. RESULTS: From May 2016 to October 2020, 30 patients were identified as KP-wtGIST by Sanger sequencing and 16 were confirmed by central molecular screening with NGS. Finally, 15 were enrolled and received regorafenib. The study was prematurely closed due to the low accrual worsened by COVID outbreak. The DCR at 12 weeks was 86.7% by central assessment. A subset of 60% experienced some tumor shrinkage, with partial responses and stabilization observed in 13% and 87% respectively, by central assessment. SDH-deficient GIST showed better clinical outcome than other KP-wtGIST. CONCLUSIONS: Regorafenib activity in KP-wtGIST compares favorably with other tyrosine kinase inhibitors, especially in the SDH-deficient GIST subset and it should be taken into consideration as upfront therapy of advanced KP-wtGIST. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02638766.

Deubiquitinase USP9X loss sensitizes renal cancer cells to mTOR inhibition.

Mammalian target of rapamycin (mTOR) is a central regulator of mammalian metabolism and physiology. Aberrant hyperactivation of the mTOR pathway promotes tumor growth and metastasis, and can also promote tumor resistance to chemotherapy and cancer drugs; this makes mTOR an attractive cancer therapeutic target. mTOR inhibitors have been approved to treat cancer; however, the mechanisms underlying drug sensitivity remain poorly understood. Here, whole exome sequencing of three chromophobe renal cell carcinoma (chRCC) patients with exceptional mTOR inhibitor sensitivity revealed that all three patients shared somatic mutations in the deubiquitinase gene USP9X. The clonal characteristics of the mutations, which were amassed by studying multiple patients' primary and metastatic samples from various years, together with the low USP9X mutation rate in unselected chRCC series, reinforced a causal link between USP9X and mTOR inhibitor sensitivity. Rapamycin treatment of USP9X-depleted HeLa and renal cancer 786-O cells, along with the pharmacological inhibition of USP9X, confirmed that this protein plays a role in patients' sensitivity to mTOR inhibitors. USP9X was not found to exert a direct effect on mTORC1, but subsequent ubiquitylome analyses identified p62 as a direct USP9X target. Increased p62 ubiquitination and the augmented rapamycin effect upon bortezomib treatment, together with the results of p62 and LC3 immunofluorescence assays, suggested that dysregulated autophagy in USP9X-depleted cells can have a synergistic effect with mTOR inhibitors. In summary, we show that USP9X constitutes a potential novel marker of sensitivity to mTOR inhibitors in chRCC patients, and represents a clinical strategy for increasing the sensitivity to these drugs.

International initiative for a curated SDHB variant database improving the diagnosis of hereditary paraganglioma and pheochromocytoma.

BACKGROUND: SDHB is one of the major genes predisposing to paraganglioma/pheochromocytoma (PPGL). Identifying pathogenic SDHB variants in patients with PPGL is essential to the management of patients and relatives due to the increased risk of recurrences, metastases and the emergence of non-PPGL tumours. In this context, the 'NGS and PPGL (NGSnPPGL) Study Group' initiated an international effort to collect, annotate and classify SDHB variants and to provide an accurate, expert-curated and freely available SDHB variant database. METHODS: A total of 223 distinct SDHB variants from 737 patients were collected worldwide. Using multiple criteria, each variant was first classified according to a 5-tier grouping based on American College of Medical Genetics and NGSnPPGL standardised recommendations and was then manually reviewed by a panel of experts in the field. RESULTS: This multistep process resulted in 23 benign/likely benign, 149 pathogenic/likely pathogenic variants and 51 variants of unknown significance (VUS). Expert curation reduced by half the number of variants initially classified as VUS. Variant classifications are publicly accessible via the Leiden Open Variation Database system (https://databases.lovd.nl/shared/genes/SDHB). CONCLUSION: This international initiative by a panel of experts allowed us to establish a consensus classification for 223 SDHB variants that should be used as a routine tool by geneticists in charge of PPGL laboratory diagnosis. This accurate classification of SDHB genetic variants will help to clarify the diagnosis of hereditary PPGL and to improve the clinical care of patients and relatives with PPGL.

Recurrent Germline DLST Mutations in Individuals with Multiple Pheochromocytomas and Paragangliomas.

Pheochromocytomas and paragangliomas (PPGLs) provide some of the clearest genetic evidence for the critical role of metabolism in the tumorigenesis process. Approximately 40% of PPGLs are caused by driver germline mutations in 16 known susceptibility genes, and approximately half of these genes encode members of the tricarboxylic acid (TCA) cycle. Taking as a starting point the involvement of the TCA cycle in PPGL development, we aimed to identify unreported mutations that occurred in genes involved in this key metabolic pathway and that could explain the phenotypes of additional individuals who lack mutations in known susceptibility genes. To accomplish this, we applied a targeted sequencing of 37 TCA-cycle-related genes to DNA from 104 PPGL-affected individuals with no mutations in the major known predisposing genes. We also performed omics-based analyses, TCA-related metabolite determination, and 13C5-glutamate labeling assays. We identified five germline variants affecting DLST in eight unrelated individuals (∼7%); all except one were diagnosed with multiple PPGLs. A recurrent variant, c.1121G>A (p.Gly374Glu), found in four of the eight individuals triggered accumulation of 2-hydroxyglutarate, both in tumors and in a heterologous cell-based assay designed to functionally evaluate DLST variants. p.Gly374Glu-DLST tumors exhibited loss of heterozygosity, and their methylation and expression profiles are similar to those of EPAS1-mutated PPGLs; this similarity suggests a link between DLST disruption and pseudohypoxia. Moreover, we found positive DLST immunostaining exclusively in tumors carrying TCA-cycle or EPAS1 mutations. In summary, this study reveals DLST as a PPGL-susceptibility gene and further strengthens the relevance of the TCA cycle in PPGL development.

Prevalence of pathogenic germline variants in patients with metastatic renal cell carcinoma.

PURPOSE: Germline pathogenic variants are estimated to affect 3-5% of renal cell carcinoma (RCC) patients. However, higher mutational prevalence in non-clear cell RCC (non-ccRCC) and advanced disease has been suggested. METHODS: To clarify the prevalence of pathogenic germline variants in metastatic RCC, we sequenced 29 cancer susceptibility genes in 294 unselected metastatic RCC cases plus 21 patients with clinical hereditary features. In 145 tumors, genes frequently mutated in RCC were sequenced and methylation was assessed in selected cases. RESULTS: Germline variants in RCC predisposition genes (FH, VHL) were detected in 1.4% of the unselected metastatic patients, with higher frequency in non-ccRCC versus ccRCC (6.4% and 0.4%; P = 0.0025) and in younger patients (P = 0.036). Among the 315 studied patients, 14% of non-type 1 papillary cases (4 of 28), all metastatic <1 year after diagnosis, carried a FH germline variant with loss of heterozygosity and tumor genome hypermethylation. Variants in other cancer-associated genes (e.g., MUTYH, BRCA2, CHEK2) occurred in 5.1% of the unselected series, with unclear significance for RCC. CONCLUSION: Our findings confirm a high prevalence of pathogenic germline variants in RCC predisposition genes in metastatic non-ccRCC, and highlight that metastatic patients with papillary type 2 or unconventional histologies compatible with FH would benefit from genetic screening.

The recurrent p.(Pro540Ser) MEN1 genetic variant should be considered nonpathogenic: A case report.

Pheochromocytoma/paraganglioma (Pheo/PGL) associated with pituitary adenoma (PA) is rare in clinical practice, and a common pathogenic mechanism has been suggested owing to the germline pathogenic variants found in some cases. Our aim is to propose a reassignment for a recurrent MEN1 genetic variant found in a 54-year-old male patient with bilateral pheochromocytoma and GH-secreting PA. Pheo/PGL genes study was carried out in DNA samples from Pheo as well as PA and no pathological variants or large deletions were detected. Additionally, a MEN1 gene analysis was performed, and a heterozygous germline variant in exon 10: c.1618C>T; p.(Pro540Ser) was found. No MEN1 gene deletions/duplications were detected. In evaluating a causal relationship between the c.1618C>T MEN1 variant and both tumors, we took into account that missense variants are common pathogenic variants in MEN1, and the population frequency of this variant is too high to be considered pathogenic. His son (aged 38 and carrier) is asymptomatic, and computational analysis showed discrepancies. We propose that this recurrent variant, previously considered as likely pathogenic, subsequently as variant of uncertain significance, and likely benign should now be reclassified as benign.

Metabolomics, machine learning and immunohistochemistry to predict succinate dehydrogenase mutational status in phaeochromocytomas and paragangliomas.

Phaeochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumours with a hereditary background in over one-third of patients. Mutations in succinate dehydrogenase (SDH) genes increase the risk for PPGLs and several other tumours. Mutations in subunit B (SDHB) in particular are a risk factor for metastatic disease, further highlighting the importance of identifying SDHx mutations for patient management. Genetic variants of unknown significance, where implications for the patient and family members are unclear, are a problem for interpretation. For such cases, reliable methods for evaluating protein functionality are required. Immunohistochemistry for SDHB (SDHB-IHC) is the method of choice but does not assess functionality at the enzymatic level. Liquid chromatography-mass spectrometry-based measurements of metabolite precursors and products of enzymatic reactions provide an alternative method. Here, we compare SDHB-IHC with metabolite profiling in 189 tumours from 187 PPGL patients. Besides evaluating succinate:fumarate ratios (SFRs), machine learning algorithms were developed to establish predictive models for interpreting metabolite data. Metabolite profiling showed higher diagnostic specificity compared to SDHB-IHC (99.2% versus 92.5%, p = 0.021), whereas sensitivity was comparable. Application of machine learning algorithms to metabolite profiles improved predictive ability over that of the SFR, in particular for hard-to-interpret cases of head and neck paragangliomas (AUC 0.9821 versus 0.9613, p = 0.044). Importantly, the combination of metabolite profiling with SDHB-IHC has complementary utility, as SDHB-IHC correctly classified all but one of the false negatives from metabolite profiling strategies, while metabolite profiling correctly classified all but one of the false negatives/positives from SDHB-IHC. From 186 tumours with confirmed status of SDHx variant pathogenicity, the combination of the two methods resulted in 185 correct predictions, highlighting the benefits of both strategies for patient management. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Germline mutations in the new E1' cryptic exon of the VHL gene in patients with tumours of von Hippel-Lindau disease spectrum or with paraganglioma.

BACKGROUNDS: The incidence of germline mutations in the newly discovered cryptic exon (E1') of VHL gene in patients with von Hippel-Lindau (VHL) disease and in patients with paraganglioma or pheochromocytoma (PPGL) is not currently known. METHODS: We studied a large international multicentre cohort of 1167 patients with a previous negative genetic testing. Germline DNA from 75 patients with a single tumour of the VHL spectrum ('Single VHL tumour' cohort), 70 patients with multiple tumours of the VHL spectrum ('Multiple VHL tumours' cohort), 76 patients with a VHL disease as described in the literature ('VHL-like' cohort) and 946 patients with a PPGL were screened for E1' genetic variants. RESULTS: Six different genetic variants in E1' were detected in 12 patients. Two were classified as pathogenic, 3 as variants of unknown significance and 1 as benign. The rs139622356 was found in seven unrelated patients but described in only 16 patients out of the 31 390 of the Genome Aggregation Database (p<0.0001) suggesting that this variant might be either a recurrent mutation or a modifier mutation conferring a risk for the development of tumours and cancers of the VHL spectrum. CONCLUSIONS: VHL E1' cryptic exon mutations contribute to 1.32% (1/76) of 'VHL-like' cohort and to 0.11% (1/946) of PPGL cohort and should be screened in patients with clinical suspicion of VHL, and added to panels for Next Generation Sequencing (NGS) diagnostic testing of hereditary PPGL. Our data highlight the importance of studying variants identified in deep intronic sequences, which would have been missed by examining only coding sequences of genes/exomes. These variants will likely be more frequently detected and studied with the upcoming implementation of whole-genome sequencing into clinical practice.

PTEN expression and mutations in TSC1, TSC2 and MTOR are associated with response to rapalogs in patients with renal cell carcinoma.

The mammalian target of rapamycin (mTOR) pathway inhibitors are key drugs for the treatment of many tumor types, however, there are no predictive biomarkers in clinical use. Here, we performed a molecular and immunohistochemical characterization of key mTOR pathway components in a series of 105 renal cell carcinoma patients treated with rapalogs, aimed at identifying markers of treatment response. Mutational analysis in MTOR, TSC1 and TSC2 was performed through targeted next-generation sequencing (NGS), and immunohistochemistry (IHC) was performed for PTEN, pAKT, pS6K1, pS6 and p21. Among patients with NGS data, 11 of 87 (13%) had mTOR pathway mutations (8 in MTOR, 1 in TSC1 and 2 in TSC2). When comparing the molecular data to the response of the patients, we found that partial response was more frequent in cases with mTOR pathway mutations than in those without mutations (odds ratio [OR] = 0.08, 95% confidence interval [CI] = 0.008-0.79, p = 0.030 univariate; p = 0.038 multivariable). Regarding IHC, negative PTEN staining was detected in 58% of the tumors, and it was more frequent in rapalog responder patients (OR = 0.24, 95% CI = 0.065-0.86, p = 0.029 univariate; p = 0.029 multivariable). Mutations and PTEN IHC were not mutually exclusive events and its combination improved response prediction (OR = 0.16, 95% CI = 0.04-0.62, p = 0.008 univariate; p = 0.013 multivariable). The staining of other proteins did not show and association with response and no association with PFS was observed in unselected patients. In conclusion, our findings suggest that mTOR pathway mutations, negative PTEN IHC and their combination are potential markers of rapalog response.

Hsa-miR-139-5p is a prognostic thyroid cancer marker involved in HNRNPF-mediated alternative splicing.

It is critical to identify biomarkers and functional networks associated with aggressive thyroid cancer to anticipate disease progression and facilitate personalized patient management. We performed miRNome sequencing of 46 thyroid tumors enriched with advanced disease patients with a median follow-up of 96 months. MiRNome profiles correlated with tumor-specific histopathological and molecular features, such as stromal cell infiltration and tumor driver mutation. Differential expression analysis revealed a consistent hsa-miR-139-5p downexpression in primary carcinomas from patients with recurrent/metastatic disease compared to disease-free patients, sustained in paired local metastases and validated in publicly available thyroid cancer series. Exogenous expression of hsa-miR-139-5p significantly reduced migration and proliferation of anaplastic thyroid cancer cells. Proteomic analysis indicated RICTOR, SMAD2/3 and HNRNPF as putative hsa-miR-139-5p targets in our cell system. Abundance of HNRNPF mRNA, encoding an alternative splicing factor involved in cryptic exon inclusion/exclusion, inversely correlated with hsa-miR-139-5p expression in human tumors. RNA sequencing analysis revealed 174 splicing events differentially regulated upon HNRNPF repression in our cell system, affecting genes involved in RTK/RAS/MAPK and PI3K/AKT/MTOR signaling cascades among others. These results point at the hsa-miR-139-5p/HNRNPF axis as a novel regulatory mechanism associated with the modulation of major thyroid cancer signaling pathways and tumor virulence.

Molecular characterization of chromophobe renal cell carcinoma reveals mTOR pathway alterations in patients with poor outcome.

Chromophobe renal cell carcinoma (chRCC) is a histologically and molecularly distinct class of rare renal tumor. TCGA studies revealed low mutational burden, with only TP53 and PTEN recurrently mutated, and discovered alterations in TERT promoter and in the electron transport chain Complex I genes. However, knowledge on drug targetable genes is limited and treatments at metastatic stage do not follow a molecular rationale. In a large series of 92 chRCC enriched with metastatic cases, we performed an in-depth characterization of mTOR pathway alterations through targeted NGS and immunohistochemistry (IHC) of phospho-S6, tuberin, and PTEN. Mutations in mitochondria, telomere maintenance and other renal cancer related genes and p53 IHC, were also assessed. The impact on metastasis development and disease specific survival was determined, using TCGA-KICH series (n = 65) for validation. mTOR pathway mutations (MTOR, TSC1, TSC2) were present in 17% of primary tumors, most of them being classified as pathogenic. Mutations were associated with positive IHC staining of phospho-S6 and PTEN (P = 0.009 and P = 0.001, respectively) and with chRCC eosinophilic variant (P = 0.039), supporting a biological relevance of the pathway. mTOR pathway mutations were associated with worse clinical outcomes. Survival analysis gave a hazard ratio of 5.5 (P = 0.027), and this association was confirmed in TCGA-KICH (HR = 10.3, P = 0.006). TP53 mutations were enriched in metastatic cases (P = 0.018), and mutations in telomere maintenance genes showed a trend in the same direction. p53 IHC staining pattern was associated with the underlying TP53 defect, and negative PTEN IHC staining (82% of cases) suggested PTEN loss as a chRCC hallmark. In conclusion, our study provides with novel genomic knowledge in chRCC and identifies novel markers of poor survival. Furthermore, this is the first study showing that mTOR pathway mutations correlate with poor prognosis, and may help to identify patients with increased sensitivity to mTOR inhibitors.

Plasma metanephrines and prospective prediction of tumor location, size and mutation type in patients with pheochromocytoma and paraganglioma.

Objectives: Plasma free metanephrines are commonly used for diagnosis of pheochromocytoma and paraganglioma (PPGLs), but can also provide other information. This multicenter study prospectively examined whether tumor size, location, and mutations could be predicted by these metabolites. Methods: Predictions of tumor location, size, and mutation type, based on measurements of plasma normetanephrine, metanephrine, and methoxytyramine were made without knowledge of disease in 267 patients subsequently determined to have PPGLs. Results: Predictions of adrenal vs. extra-adrenal locations according to increased plasma concentrations of metanephrine and methoxytyramine were correct in 93 and 97% of the respective 136 and 33 patients in who these predictions were possible. Predicted mean tumor diameters correlated positively (p<0.0001) with measured diameters; predictions agreed well for pheochromocytomas but were overestimated for paragangliomas. Considering only patients with mutations, 51 of the 54 (94%) patients with NF1 or RET mutations were correctly predicted with those mutations according to increased plasma metanephrine, whereas no or minimal increase in metanephrine correctly predicted all 71 patients with either VHL or SDHx mutations; furthermore, among the latter group increases in methoxytyramine correctly predicted SDHx mutations in 93% of the 29 cases for this specific prediction. Conclusions: Extents and patterns of increased plasma O-methylated catecholamine metabolites among patients with PPGLs allow predictions of tumor size, adrenal vs. extra-adrenal locations and general types of mutations. Predictions of tumor location are, however, only possible for patients with clearly increased plasma methoxytyramine or metanephrine. Where possible or clinically relevant the predictions are potentially useful for subsequent clinical decision-making.

Concomitant Medications and Risk of Chemotherapy-Induced Peripheral Neuropathy.

BACKGROUND: Peripheral neuropathy is the dose-limiting toxicity of many oncology drugs, including paclitaxel. There is large interindividual variability in the neuropathy, and several risk factors have been proposed; however, many have not been replicated. Here we present a comprehensive study aimed at identifying treatment and physiopathology-related paclitaxel-induced neuropathy risk factors in a large cohort of well-characterized patients. PATIENTS AND METHODS: Analyses included 503 patients with breast or ovarian cancer who received paclitaxel treatment. Paclitaxel dose modifications caused by the neuropathy were extracted from medical records and patients self-reported neuropathy symptoms were collected. Multivariate logistic regression analyses were performed to identify concomitant medications and comorbidities associated with paclitaxel-induced neuropathy. RESULTS: Older patients had higher neuropathy: for each increase of 1 year of age, the risk of dose modifications and grade 3 neuropathy increased 4% and 5%, respectively. Cardiovascular drugs increased the risk of paclitaxel dose reductions (odds ratio [OR], 2.51; p = .006), with a stronger association for beta-adrenergic antagonists. The total number of concomitant medications also showed an association with dose modifications (OR, 1.25; p = .012 for each concomitant drug increase). A dose modification predictive model that included the new identified factors gave an area under the curve of 0.74 (p = 1.07 × 10-10). Preexisting nerve compression syndromes seemed to increase neuropathy risk. CONCLUSION: Baseline characteristics of the patients, including age and concomitant medications, could be used to identify individuals at high risk of neuropathy, personalizing chemotherapy treatment and reducing the risk of severe neuropathy. IMPLICATIONS FOR PRACTICE: Peripheral neuropathy is a common adverse effect of many cancer drugs, including chemotherapeutics, targeted therapies, and immune checkpoint inhibitors. About 40% of survivors of cancer have functional deficits caused by this toxicity, some of them irreversible. Currently, there are no effective treatments to prevent or treat this neuropathy. This study, performed in a large cohort of well-characterized patients homogenously treated with paclitaxel, identified concomitant medications, comorbidities, and demographic factors associated with peripheral neuropathy. These factors could serve to identify patients at high risk of severe neuropathy for whom alternative non-neurotoxic alternatives may be considered.

Metabolome-guided genomics to identify pathogenic variants in isocitrate dehydrogenase, fumarate hydratase, and succinate dehydrogenase genes in pheochromocytoma and paraganglioma.

PURPOSE: Metabolic aberrations have been described in neoplasms with pathogenic variants (PV) in the Krebs cycle genes encoding succinate dehydrogenase (SDH), fumarate hydratase (FH) and isocitrate dehydrogenase (IDH). In turn, accumulation of oncometabolites succinate, fumarate, and 2-hydroxyglutarate can be employed to identify tumors with those PV . Additionally, such metabolic readouts may aid in genetic variant interpretation and improve diagnostics. METHODS: Using liquid chromatography-mass spectrometry, 395 pheochromocytomas and paragangliomas (PPGLs) from 391 patients were screened for metabolites to indicate Krebs cycle aberrations. Multigene panel sequencing was applied to detect driver PV in cases with indicative metabolite profiles but undetermined genetic drivers. RESULTS: Aberrant Krebs cycle metabolomes identified rare cases of PPGLs with germline PV in FH and somatic PV in IDHx and SDHx, including the first case of a somatic IDH2 PV in PPGL. Metabolomics also reliably identified PPGLs with SDHx loss-of-function (LOF) PV. Therefore we utilized tumor metabolite profiles to further classify variants of unknown significance in SDHx, thereby enabling missense variants associated with SDHx LOF to be distinguished from benign variants. CONCLUSION: We propose incorporation of metabolome data into the diagnostics algorithm in PPGLs to guide genetic testing and variant interpretation and to help identify rare cases with PV in FH and IDHx.

Germline ESR2 mutation predisposes to medullary thyroid carcinoma and causes up-regulation of RET expression.

Familial medullary thyroid cancer (MTC) and its precursor, C cell hyperplasia (CCH), is associated with germline RET mutations causing multiple endocrine neoplasia type 2. However, some rare families with apparent MTC/CCH predisposition do not have a detectable RET mutation. To identify novel MTC/CCH predisposition genes we undertook exome resequencing studies in a family with apparent predisposition to MTC/CCH and no identifiable RET mutation. We identified a novel ESR2 frameshift mutation, c.948delT, which segregated with histological diagnosis following thyroid surgery in family members and demonstrated loss of ESR2-encoded ERß expression in the MTC tumour. ERα and ERß form heterodimers binding DNA at specific oestrogen-responsive elements (EREs) to regulate gene transcription. ERß represses ERα-mediated activation of the ERE and the RET promoter contains three EREs. In vitro, we showed that ESR2 c.948delT results in unopposed ERα mediated increased cellular proliferation, activation of the ERE and increased RET expression. In vivo, immunostaining of CCH and MTC using an anti-RET antibody demonstrated increased RET expression. Together these findings identify germline ESR2 mutation as a novel cause of familial MTC/CCH and provide important insights into a novel mechanism causing increased RET expression in tumourigenesis.

Role of MDH2 pathogenic variant in pheochromocytoma and paraganglioma patients.

PURPOSE: MDH2 (malate dehydrogenase 2) has recently been proposed as a novel potential pheochromocytoma/paraganglioma (PPGL) susceptibility gene, but its role in the disease has not been addressed. This study aimed to determine the prevalence of MDH2 pathogenic variants among PPGL patients and determine the associated phenotype. METHODS: Eight hundred thirty patients with PPGLs, negative for the main PPGL driver genes, were included in the study. Interpretation of variants of unknown significance (VUS) was performed using an algorithm based on 20 computational predictions, by implementing cell-based enzymatic and immunofluorescence assays, and/or by using a molecular dynamics simulation approach. RESULTS: Five variants with potential involvement in pathogenicity were identified: three missense (p.Arg104Gly, p.Val160Met and p.Ala256Thr), one in-frame deletion (p.Lys314del), and a splice-site variant (c.429+1G>T). All were germline and those with available biochemical data, corresponded to noradrenergic PPGL. CONCLUSION: This study suggests that MDH2 pathogenic variants may play a role in PPGL susceptibility and that they might be responsible for less than 1% of PPGLs in patients without pathogenic variants in other major PPGL driver genes, a prevalence similar to the one recently described for other PPGL genes. However, more epidemiological data are needed to recommend MDH2 testing in patients negative for other major PPGL genes.

Gain-of-function mutations in DNMT3A in patients with paraganglioma.

PURPOSE: The high percentage of patients carrying germline mutations makes pheochromocytomas/paragangliomas the most heritable of all tumors. However, there are still cases unexplained by mutations in the known genes. We aimed to identify the genetic cause of disease in patients strongly suspected of having hereditary tumors. METHODS: Whole-exome sequencing was applied to the germlines of a parent-proband trio. Genome-wide methylome analysis, RNA-seq, CRISPR/Cas9 gene editing, and targeted sequencing were also performed. RESULTS: We identified a novel de novo germline mutation in DNMT3A, affecting a highly conserved residue located close to the aromatic cage that binds to trimethylated histone H3. DNMT3A-mutated tumors exhibited significant hypermethylation of homeobox-containing genes, suggesting an activating role of the mutation. CRISPR/Cas9-mediated knock-in in HeLa cells led to global changes in methylation, providing evidence of the DNMT3A-altered function. Targeted sequencing revealed subclonal somatic mutations in six additional paragangliomas. Finally, a second germline DNMT3A mutation, also causing global tumor DNA hypermethylation, was found in a patient with a family history of pheochromocytoma. CONCLUSION: Our findings suggest that DNMT3A may be a susceptibility gene for paragangliomas and, if confirmed in future studies, would represent the first example of gain-of-function mutations affecting a DNA methyltransferase gene involved in cancer predisposition.

Advanced sporadic renal epithelioid angiomyolipoma: case report of an extraordinary response to sirolimus linked to TSC2 mutation.

BACKGROUND: Renal epithelioid angiomyolipomas (EAML) are rare tumors with aggressive behavior. EAML can be sporadic or develop within the tuberous sclerosis complex syndrome, where mutations of TSC1 or TSC2 genes (critical negative regulators of mTOR Complex 1) result in an increased activation of mTOR pathway. Optimal EAML treatment, including mTOR inhibitors, remains undetermined. CASE PRESENTATION: Here we present the case of a young adult with a renal EAML that after radical nephrectomy developed metastases, first in liver and then in lumbar vertebrae. After complete surgical resection of these lesions, liver recurrence was detected, this time with incomplete surgical resection. After finding a new liver lesion, systemic treatment with sirolimus started. The patient exhibited a complete and durable response to this drug, being disease free at the time of publication, after 36 months of treatment. Targeted next generation sequencing (NGS) of MTOR, TSC1 and TSC2 genes in the primary tumor, metastasis and blood of the patient, revealed one inactivating TSC2 mutation (c.2739dup; p.K914*) in the tumor cells. Immunohistochemistry revealed decreased TSC2 protein content and increased phospho-S6 in the tumor cells, demonstrating mTOR pathway activation. CONCLUSION: NGS on an EAML patient with an extraordinary response to sirolimus uncovered TSC2 inactivation as the mechanism for the response. This study supports NGS as a useful tool to identify patients sensitive to mTOR inhibitors and supports the treatment of malignant EAML with these drugs.

Biallelic TSC2 Mutations in a Patient With Chromophobe Renal Cell Carcinoma Showing Extraordinary Response to Temsirolimus.

mTOR inhibitors are used to treat renal cell carcinoma (RCC). Treatment response is variable and appears to correlate with genetic alterations that activate mTOR signaling. Recently, everolimus was suggested to be more effective than sunitinib in chromophobe RCC (chRCC), a tumor with frequent mTOR pathway defects. This report presents the genomic and functional characterization of a metastatic chRCC that showed complete response at metastatic sites and 80% reduction in primary tumor size upon temsirolimus treatment. After surgery, the patient remained disease-free for 8 years after temsirolimus therapy. Whole-exome sequencing (WES) revealed 2 somatic variants in TSC2, a critical negative regulator of mTOR: a splicing defect (c.5069-1G>C) and a novel missense variant [c.3200_3201delinsAA; p.(V1067E)]. In vitro functional assessment demonstrated that the V1067E substitution disrupted TSC2 function. Immunohistochemistry in the tumor tissues revealed increased phosphorylated S6 ribosomal protein, indicating mTOR pathway activation. In conclusion, WES revealed TSC2 inactivation as the likely mechanism for this extraordinary response to temsirolimus. These findings support high efficacy of mTOR inhibitors in a subset of patients with chRCC and propose sequencing of mTOR pathway genes to help guide therapy.