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1.
Horm Metab Res ; 2020 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-32521546

RESUMO

Most pancreatic neuroendocrine neoplasms (pNEN) occur sporadically but they can also occur as part of multiple endocrine neoplasia type 1 (MEN1). MAX was originally described as an inherited pheochromocytoma-paraganglioma risk gene, but also has recently been implicated in pituitary tumorigenesis. Here we describe the first case of a pNEN associated with an inherited MAX gene deletion in a family with endocrine tumors. The patient was a male carrier of an intragenic exon 3 deletion inherited from his father who had recurrent pheochromocytomas and a macroprolactinoma. The patient underwent screening and hormonal studies but no pheochromocytoma-paraganglioma, pituitary or renal tumors were identified. However, abdominal magnetic resonance imaging (MRI) identified a 1 cm lesion in body of the pancreas. The lesion was hyperintense on T2-weighted signal, and there was hyperfixation of the tumor on 68Ga-DOTANOC PET-CT images. No biochemical evidence of pancreatic hormone excess was identified. Following a guided biopsy, a pathological diagnosis of a low grade pNEN was made and immunohistochemistry showed loss of MAX nuclear staining. Genetic analysis of the tumor tissue indicated copy number neutral loss of heterozygosity consistent with uniparental disomy. This is the first reported case of a MAX deletion associated pNEN and strengthens the argument that MAX may represent an inheritable multiple endocrine neoplasia risk gene. Further analysis of germline and somatic MAX mutations/deletions in large cohorts of unexplained NEN cases could help clarify the potential role of MAX in NEN etiology.

2.
J Pathol ; 251(4): 378-387, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32462735

RESUMO

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.

3.
J Am Acad Dermatol ; 2019 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-31870915

RESUMO

BACKGROUND: Metastatic basal cell carcinoma (mBCC) is a very rare entity, and diagnosis can be challenging. Therapeutic options are limited, and response to targeted therapy is poor. OBJECTIVE: To demonstrate a clonal relationship between BCCs and their metastases and to explore which hedgehog pathway-related mutations are involved in mBCC. METHODS: Genetic analysis was conducted in 10 primary BCCs and their metastases. Genes relevant for BCC development were analyzed in tumor and metastasis material with small molecule molecular inversion probes (smMIPs) for PTCH1, PTCH2, SMO, SUFU, GLI2, and TP53 or with targeted next generation sequencing of the same genes and CDKN2A, CDKN2B, CIC, DAXX, DDX3X, FUBP1, NF1, NF2, PTEN, SETD2, TRAF7, and the TERT promoter. RESULTS: In 8 of 10 patients, identical gene mutations could be demonstrated in the primary tumors and their metastases. A broad spectrum of mutations was found. Four patients had SMO mutations in their tumor or metastasis, or both. All SMO mutations found were known to cause resistance to targeted therapy with vismodegib. LIMITATIONS: In 2 patients there was insufficient qualitative DNA available for genetic analysis. CONCLUSIONS: Molecular testing can help to identify the origin of a BCC metastasis and may be of prognostic and therapeutic value.

4.
Artigo em Inglês | MEDLINE | ID: mdl-31379752

RESUMO

Background: Adrenocortical carcinoma (ACC) is a rare tumor entity with restricted therapeutic opportunities. HSP90 (Heat Shock Protein 90) chaperone activity is fundamental for cell survival and contributes to different oncogenic signaling pathways. Indeed, agents targeting HSP90 function have shown therapeutic efficacy in several cancer types. We have examined the expression of HSP90 in different adrenal tumors and evaluated the use of HSP90 inhibitors in vitro as possible therapy for ACC. Methods: Immunohistochemical expression of HSP90 isoforms was investigated in different adrenocortical tumors and associated with clinical features. Additionally, a panel of N-terminal (17-allylamino-17-demethoxygeldanamycin (17-AAG), luminespib, and ganetespib) and C-terminal (novobiocin and silibinin) HSP90 inhibitors were tested on various ACC cell lines. Results: Within adrenocortical tumors, ACC samples exhibited the highest expression of HSP90ß. Within a cohort of ACC patients, HSP90ß expression levels were inversely correlated with recurrence-free and overall survival. In functional assays, among five different compounds tested luminespib and ganetespib induced a significant decrease in cell viability in single as well as in combined treatments with compounds of the clinically used EDP-M scheme (etoposide, doxorubicin, cisplatin, mitotane). Inhibition of cell viability correlated furthermore with a decrease in proliferation, in cell migration and an increase in apoptosis. Moreover, analysis of cancer pathways indicated a modulation of the ERK1/2-and AKT-pathways by luminespib and ganetespib treatment. Conclusions: Our findings emphasize HSP90 as a marker with prognostic impact and promising target with N-terminal HSP90 inhibitors as drugs with potential therapeutic efficacy toward ACC.

5.
Theranostics ; 9(17): 4946-4958, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31410193

RESUMO

Rationale: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors that present variable outcomes. To date, no effective therapies or reliable prognostic markers are available for patients who develop metastatic PPGL (mPPGL). Our aim was to discover robust prognostic markers validated through in vitro models, and define specific therapeutic options according to tumor genomic features. Methods: We analyzed three PPGL miRNome datasets (n=443), validated candidate markers and assessed them in serum samples (n=36) to find a metastatic miRNA signature. An integrative study of miRNome, transcriptome and proteome was performed to find miRNA targets, which were further characterized in vitro. Results: A signature of six miRNAs (miR-21-3p, miR-183-5p, miR-182-5p, miR-96-5p, miR-551b-3p, and miR-202-5p) was associated with metastatic risk and time to progression. A higher expression of five of these miRNAs was also detected in PPGL patients' liquid biopsies compared with controls. The combined expression of miR-21-3p/miR-183-5p showed the best power to predict metastasis (AUC=0.804, P=4.67·10-18), and was found associated in vitro with pro-metastatic features, such as neuroendocrine-mesenchymal transition phenotype, and increased cell migration rate. A pan-cancer multi-omic integrative study correlated miR-21-3p levels with TSC2 expression, mTOR pathway activation, and a predictive signature for mTOR inhibitor-sensitivity in PPGLs and other cancers. Likewise, we demonstrated in vitro a TSC2 repression and an enhanced rapamycin sensitivity upon miR-21-3p expression. Conclusions: Our findings support the assessment of miR-21-3p/miR-183-5p, in tumors and liquid biopsies, as biomarkers for risk stratification to improve the PPGL patients' management. We propose miR-21-3p to select mPPGL patients who may benefit from mTOR inhibitors.

6.
JAMA Oncol ; 2019 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-31294750

RESUMO

Importance: The risk stratification of adrenocortical carcinoma (ACC) based on tumor proliferation index and stage is limited. Adjuvant therapy after surgery is recommended for most patients. Pan-genomic studies have identified distinct molecular groups closely associated with outcome. Objective: To compare the molecular classification for prognostic assessment of ACC with other known prognostic factors. Design, Setting, and Participants: In this retrospective biomarker analysis, ACC tumor samples from 368 patients who had undergone surgical tumor removal were collected from March 1, 2005, to September 30, 2015 (144 in the training cohort and 224 in the validation cohort) at 21 referral centers with a median follow-up of 35 months (interquartile range, 18-74 months). Data were analyzed from March 2016 to March 2018. Exposures: Meta-analysis of pan-genomic studies (transcriptome, methylome, chromosome alteration, and mutational profiles) was performed on the training cohort. Targeted biomarker analysis, including targeted gene expression (BUB1B and PINK1), targeted methylation (PAX5, GSTP1, PYCARD, and PAX6), and targeted next-generation sequencing, was performed on the training and validation cohorts. Main Outcomes and Measures: Disease-free survival. Cox proportional hazards regression and C indexes were used to assess the prognostic value of each model. Results: Of the 368 patients (mean [SD] age, 49 [16] years), 144 were in the training cohort (100 [69.4%] female) and 224 were in the validation cohort (142 [63.4%] female). In the training cohort, pan-genomic measures classified ACC into 3 molecular groups (A1, A2, and A3-B), with 5-year survival of 9% for group A1, 45% for group A2, and 82% for group A3-B (log-rank P < .001). Molecular class was an independent prognostic factor of recurrence in stage I to III ACC after complete surgery (hazard ratio, 55.91; 95% CI, 8.55-365.40; P < .001). The combination of European Network for the Study of Adrenal Tumors (ENSAT) stage, tumor proliferation index, and molecular class provided the most discriminant prognostic model (C index, 0.88). In the validation cohort, the molecular classification, determined by targeted biomarker measures, was confirmed as an independent prognostic factor of recurrence (hazard ratio, 5.96 [95% CI, 1.81-19.58], P = .003 for the targeted classifier combining expression, methylation, and chromosome alterations; and 2.61 [95% CI, 1.31-5.19], P = .006 for the targeted classifier combining methylation, chromosome alterations, and mutational profile). The prognostic value of the molecular markers was limited for patients with stage IV ACC. Conclusions and Relevance: The findings suggest that in localized ACC, targeted classifiers may be used as independent markers of recurrence. The determination of molecular class may improve individual prognostic assessment and thus may spare unnecessary adjuvant treatment.

7.
Eur J Endocrinol ; 181(1): 45-53, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31067510

RESUMO

Objectives: Inoperable or metastatic paragangliomas (PGLs) and malignant pheochromocytomas (PCCs) are rare tumours with limited options for systemic treatment. Aim of this study was to assess the safety and efficacy of the radiolabelled somatostatin analogue (177LutetiumDOTA0-Tyr3)octreotate (177Lu-DOTATATE) for the treatment of PGLs and PCCs. Methods: Patients with histologically proven inoperable or malignant PGLs and PCCs treated with 177Lu-DOTATATE at our centre were retrospectively analysed. Patients were treated with up to four cycles of 177Lu-DOTATATE with an intended dose of 7.4 Gb per cycle. Response was assessed with use of RECIST 1.1. Results: Thirty patients were included: 17 with parasympathetic, 10 with sympathetic PGLs and 3 with PCCs. Grade 3/4 subacute haematotoxicity occurred in 6 (20%) of patients. A reversible subacute adverse event due to cardiac failure following possible catecholamine release occurred in two patients. Best tumour response was partial response in 7 (23%) and stable disease in 20 (67%), whereas 3 (10%) patients had progressive disease. In 20 patients with baseline disease progression, tumour control was observed in 17 (85%); the median progression-free survival was 91 months in patients with parasympathetic PGLs, 13 months in patients with sympathetic PGLs and 10 months in patients with metastatic PCCs. Conclusion: This study suggests that PRRT with 177Lu-DOTATATE is a safe and effective treatment option for patients with inoperable or malignant PGL and PCC.


Assuntos
Neoplasias das Glândulas Suprarrenais/radioterapia , Octreotida/análogos & derivados , Compostos Organometálicos/uso terapêutico , Paraganglioma/radioterapia , Feocromocitoma/radioterapia , Radioisótopos/uso terapêutico , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Octreotida/uso terapêutico , Doses de Radiação , Receptores de Peptídeos/efeitos da radiação , Estudos Retrospectivos , Resultado do Tratamento
8.
Cancers (Basel) ; 11(5)2019 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-31052272

RESUMO

8658860258318000Recently, genetic alterations in the genes encoding succinate dehydrogenase subunit B and D (SDHB and SDHD) were identified in pet dogs that presented with spontaneously arising pheochromocytomas (PCC) and paragangliomas (PGL; together PPGL), suggesting dogs might be an interesting comparative model for the study of human PPGL. To study whether canine PPGL resembled human PPGL, we investigated a series of 50 canine PPGLs by immunohistochemistry to determine the expression of synaptophysin (SYP), tyrosine hydroxylase (TH) and succinate dehydrogenase subunit A (SDHA) and B (SDHB). In parallel, 25 canine PPGLs were screened for mutations in SDHB and SDHD by Sanger sequencing. To detect large chromosomal alterations, single nucleotide polymorphism (SNP) arrays were performed for 11 PPGLs, including cases for which fresh frozen tissue was available. The immunohistochemical markers stained positive in the majority of canine PPGLs. Genetic screening of the canine tumors revealed the previously described variants in four cases; SDHB p.Arg38Gln (n = 1) and SDHD p.Lys122Arg (n = 3). Furthermore, the SNP arrays revealed large chromosomal alterations of which the loss of chromosome 5, partly homologous to human chromosome 1p and chromosome 11, was the most frequent finding (100% of the six cases with chromosomal alterations). In conclusion, canine and human PPGLs show similar genomic alterations, suggestive of common interspecies PPGL-related pathways.

9.
Virchows Arch ; 474(6): 673-680, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30888490

RESUMO

Next-generation sequencing (NGS) panel analysis on DNA from formalin-fixed paraffin-embedded (FFPE) tissue is increasingly used to also identify actionable copy number gains (gene amplifications) in addition to sequence variants. While guidelines for the reporting of sequence variants are available, guidance with respect to reporting copy number gains from gene-panel NGS data is limited. Here, we report on Dutch consensus recommendations obtained in the context of the national Predictive Analysis for THerapy (PATH) project, which aims to optimize and harmonize routine diagnostics in molecular pathology. We briefly discuss two common approaches to detect gene copy number gains from NGS data, i.e., the relative coverage and B-allele frequencies. In addition, we provide recommendations for reporting gene copy gains for clinical purposes. In addition to general QC metrics associated with NGS in routine diagnostics, it is recommended to include clinically relevant quantitative parameters of copy number gains in the clinical report, such as (i) relative coverage and estimated copy numbers in neoplastic cells, (ii) statistical scores to show significance (e.g., z-scores), and (iii) the sensitivity of the assay and restrictions of NGS-based detection of copy number gains. Collectively, this information can guide clinical and analytical decisions such as the reliable detection of high-level gene amplifications and the requirement for additional in situ assays in case of borderline results or limited sensitivity.


Assuntos
Variações do Número de Cópias de DNA/fisiologia , Dosagem de Genes/genética , Testes Genéticos , Sequenciamento de Nucleotídeos em Larga Escala , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutação/genética , Patologia Molecular/métodos , Análise de Sequência de DNA/métodos
10.
Cancer ; 125(8): 1258-1266, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30644531

RESUMO

BACKGROUND: Somatic mutations in hypoxia-inducible factor 2α (HIF2A) are associated with polycythemia-paraganglioma syndrome. Specifically, the classic presentation of female patients with recurrent paragangliomas (PGLs), polycythemia (at birth or in early childhood), and duodenal somatostatinomas has been described. Studies have demonstrated that somatic HIF2A mutations occur as postzygotic events and some to be associated with somatic mosaicism affecting hematopoietic and other tissue precursors. This phenomenon could explain the development of early onset of polycythemia in the absence of erythropoietin-secreting tumors. METHODS: Correlation analysis was performed between mosaicism of HIF2A mutant patients and clinical presentations. RESULTS: Somatic HIF2A mutations (p.A530V, p.P531S, and p.D539N) were identified in DNA extracted from PGLs of 3 patients. No somatic mosaicism was detected through deep sequencing of blood genomic DNA. Compared with classic syndrome, both polycythemia and PGL in all 3 patients developed at an advanced age with polycythemia at age 30, 30, and 17 years and PGLs at age 34, 30, and 55 years, respectively. Somatostatinomas were not detected, and 2 patients had ophthalmic findings. The biochemical phenotype in all 3 patients was noradrenergic with 18 F-fluorodopa PET/CT as the most sensitive imaging modality. All patients demonstrated multiplicity, and none developed metastatic disease. CONCLUSION: These findings suggest that newer techniques need to be developed to detect somatic mosaicism in patients with this syndrome. Absence of HIF2A mosaicism in patients with somatic HIF2A mutations supports association with late onset of the disease, milder clinical phenotype, and an improved prognosis compared with patients who have HIF2A mosaicism.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Paraganglioma/classificação , Mutação Puntual , Policitemia/classificação , Adolescente , Adulto , Idade de Início , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Pessoa de Meia-Idade , Mosaicismo , Paraganglioma/diagnóstico por imagem , Paraganglioma/genética , Policitemia/diagnóstico por imagem , Policitemia/genética , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Análise de Sequência de DNA
11.
Genet Med ; 20(12): 1652-1662, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30008476

RESUMO

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.


Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Malato Desidrogenase/genética , Paraganglioma/genética , Feocromocitoma/genética , Neoplasias das Glândulas Suprarrenais/patologia , Adulto , Feminino , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Paraganglioma/patologia , Feocromocitoma/patologia , Isoformas de Proteínas
12.
Eur J Endocrinol ; 179(2): 125-133, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29875288

RESUMO

BACKGROUND/AIM: PET with 6-[18F]fluor-l-3,4-dihydroxyphenylalanine (18F-FDOPA) has been shown to be a useful imaging tool with a high sensitivity for the visualization of neuroendocrine tumors (NETs). 18F-FDOPA uptake in tumors other than NETs has been suggested previously, but data on this phenomenon are limited. We therefore studied the non-physiological, false-positive uptake of 18F-FDOPA in a large population of patients with a NET or with a high clinical suspicion of harboring a NET. PATIENTS AND METHODS: Retrospective single-center study among adult patients in whom 18F-FDOPA PET scintigraphy was performed between January 2004 and December 2014. The original scan report was compared with the original pathology report corresponding with the 18F-FDOPA PET-positive lesion. In case this was inconsistent with the diagnosis of a NET, both the scan and the pathology slides were reassessed. Specimens of these non-NET tissues were immunohistochemically stained for AADC. RESULTS: 1070 18F-FDOPA PET scans from 705 patients were evaluated. Focal or multiple 18F-FDOPA-avid lesions were described in 709 18F-FDOPA PET scans (66%). Histology of these 18F-FDOPA PET-positive lesions was present in 508 (72%) cases. In seven cases, the histopathology was not compatible with NET but showed squamous cell carcinoma of the cervix, multiple myeloma (two cases), hepatocellular carcinoma, Schwannoma, adrenocortical carcinoma and a skeletal myxoid chondrosarcoma, with positive immunohistochemical staining for AADC in 67%. CONCLUSIONS: Pathological uptake of 18F-FDOPA does not always indicate the presence of a NET. The possibility of 18F-FDOPA uptake by tumor types other than NETs, although rare, should be considered.


Assuntos
Di-Hidroxifenilalanina/análogos & derivados , Tumores Neuroendócrinos/diagnóstico por imagem , Neoplasias das Glândulas Suprarrenais/diagnóstico por imagem , Neoplasias das Glândulas Suprarrenais/metabolismo , Neoplasias das Glândulas Suprarrenais/patologia , Adulto , Idoso , Di-Hidroxifenilalanina/administração & dosagem , Di-Hidroxifenilalanina/metabolismo , Di-Hidroxifenilalanina/farmacocinética , Reações Falso-Positivas , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Intestinais/diagnóstico por imagem , Neoplasias Intestinais/metabolismo , Neoplasias Intestinais/patologia , Masculino , Pessoa de Meia-Idade , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/patologia , Neoplasias Pancreáticas/diagnóstico por imagem , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Paraganglioma/diagnóstico por imagem , Paraganglioma/metabolismo , Paraganglioma/patologia , Feocromocitoma/diagnóstico por imagem , Feocromocitoma/metabolismo , Feocromocitoma/patologia , Tomografia por Emissão de Pósitrons , Cintilografia , Estudos Retrospectivos , Neoplasias Gástricas/diagnóstico por imagem , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Distribuição Tecidual
13.
Genet Med ; 20(12): 1644-1651, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29740169

RESUMO

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.


Assuntos
Neoplasias das Glândulas Suprarrenais/genética , DNA (Citosina-5-)-Metiltransferases/genética , Paraganglioma/genética , Feocromocitoma/genética , Neoplasias das Glândulas Suprarrenais/patologia , Adulto , Sistemas CRISPR-Cas/genética , Metilação de DNA , Feminino , Mutação com Ganho de Função , Predisposição Genética para Doença , Genótipo , Mutação em Linhagem Germinativa/genética , Humanos , Masculino , Paraganglioma/patologia , Feocromocitoma/patologia , Sequenciamento Completo do Exoma
14.
J Clin Endocrinol Metab ; 103(1): 46-55, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-28938490

RESUMO

Context: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine, usually benign, tumors. Currently, the only reliable criterion of malignancy is the presence of metastases. Objective: The aim of this study was to identify genes associated with malignancy in PPGLs. Design: Transcriptomic profiling was performed on 40 benign and 11 malignant PPGLs. Genes showing a significantly different expression between benign and malignant PPGLs with a ratio ≥4 were confirmed and tested in an independent series by quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemistry was performed for the validated genes on 109 benign and 32 malignant PPGLs. Functional assays were performed with hPheo1 cells. Setting: This study was conducted at the Department of Pathology of the Erasmus MC University Medical Center Rotterdam Human Molecular Genetics laboratory of the de Duve Institute, University of Louvain. Patients: PPGL samples from 179 patients, diagnosed between 1972 and 2015, were included. Main outcome measures: Associations between gene expression and malignancy were tested using supervised clustering approaches. Results: Ten differentially expressed genes were selected based on messenger RNA (mRNA) expression array data. Contactin 4 (CNTN4) was overexpressed in malignant vs benign tumors [4.62-fold; false discovery rate (FDR), 0.001]. Overexpression at the mRNA level was confirmed using qRT-PCR (2.90-fold, P = 0.02; validation set: 4.26-fold, P = 0.005). Consistent findings were obtained in The Cancer Genome Atlas cohort (2.7-fold; FDR, 0.02). CNTN4 protein was more frequently expressed in malignant than in benign PPGLs by immunohistochemistry (58% vs 17%; P = 0.002). Survival after 7 days of culture under starvation conditions was significantly enhanced in hPheo1 cells transfected with CNTN4 complementary DNA. Conclusion: CNTN4 expression is consistently associated with malignant behavior in PPGLs.


Assuntos
Neoplasias das Glândulas Suprarrenais/diagnóstico , Biomarcadores Tumorais/metabolismo , Contactinas/metabolismo , Paraganglioma/diagnóstico , Feocromocitoma/diagnóstico , RNA Mensageiro/metabolismo , Neoplasias das Glândulas Suprarrenais/genética , Neoplasias das Glândulas Suprarrenais/metabolismo , Biomarcadores Tumorais/genética , Contactinas/genética , Perfilação da Expressão Gênica , Humanos , Paraganglioma/genética , Paraganglioma/metabolismo , Feocromocitoma/genética , Feocromocitoma/metabolismo , Prognóstico , RNA Mensageiro/genética
15.
J Clin Endocrinol Metab ; 102(10): 3611-3615, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28973495

RESUMO

Context: The disease course of adrenocortical carcinoma (ACC) patients is heterogeneous. A marker for prognosis and treatment response would facilitate choices for diagnosis and therapy. In other cancer types, circulating cell-free tumor DNA predicted tumor dynamics. Case Descriptions: The present pilot study included six patients. Next-generation sequencing (NGS) showed mutations in three ACC cases. From these patients, blood was drawn before (1 to 2 weeks) and after surgery and cell-free circulating DNA (cfDNA) was isolated. Tumor-specific mutations were found in the cfDNA of one of the three patients, with metastasized ACC at diagnosis. NGS of the tumor showed an NRAS mutation (c.182A>G:p.Q61R) in 78%, a TP53 mutation (c.856G>A:p.E286K) in 60%, and a TERT gene mutation (1295250C>T) in 28% of the reads. The preoperative cfDNA showed the same mutations at a frequency of 64%, 32%, and 2%, respectively. The postoperative cfDNA showed the same mutations but at lower frequencies (52%, 16%, and 3%, respectively). The patient was postoperatively treated with mitotane and chemotherapy. No mutations were detected in the corresponding leukocyte DNA or in the cfDNA from the two other patients. Conclusions: To the best of our knowledge, we report for the first time mutations occurring at high levels in cfDNA collected before and after surgery from one of three patients, after previous identification in the tumor. However, in the cfDNA from two patients with known mutations, we were unable to reliably detect mutations in the cfDNA. Our results indicate that mutation detection in cfDNA can vary among ACC patients, and other approaches might be required to detect the tumor response and monitor progressive disease.


Assuntos
Neoplasias do Córtex Suprarrenal/genética , Carcinoma Adrenocortical/genética , DNA de Neoplasias/análise , Células Neoplásicas Circulantes/metabolismo , Neoplasias do Córtex Suprarrenal/patologia , Carcinoma Adrenocortical/patologia , Idoso , Análise Mutacional de DNA/métodos , DNA de Neoplasias/metabolismo , Feminino , GTP Fosfo-Hidrolases/genética , Genes p53 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Células Neoplásicas Circulantes/patologia , Projetos Piloto , Telomerase/genética , beta Catenina/genética
16.
J Clin Endocrinol Metab ; 102(9): 3491-3498, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28911143

RESUMO

Context: Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with overall poor prognosis. The Ki67 labeling index (LI) has a major prognostic role in localized ACC after complete resection, but its estimates may suffer from considerable intra- and interobserver variability. VAV2 overexpression induced by increased Steroidogenic Factor-1 dosage is an essential factor driving ACC tumor cell invasion. Objective: To assess the prognostic role of VAV2 expression in ACC by investigation of a large cohort of patients. Design, Setting, and Participants: A total of 171 ACC cases (157 primary tumors, six local recurrences, eight metastases) from seven European Network for the Study of Adrenal Tumors centers were studied. Outcome Measurements: H-scores were generated to quantify VAV2 expression. VAV2 expression was divided into two categories: low (H-score, <2) and high (H-score, ≥2). The Ki67 LI retrieved from patients' pathology records was also categorized into low (<20%) and high (≥20%). Clinical and immunohistochemical markers were correlated with progression-free survival (PFS) and overall survival (OS). Results: VAV2 expression and Ki67 LI were significantly correlated with each other and with PFS and OS. Heterogeneity of VAV2 expression inside the same tumor was very low. Combined assessment of VAV2 expression and Ki67 LI improved patient stratification to low-risk and high-risk groups. Conclusion: Combined assessment of Ki67 LI and VAV2 expression improves prognostic prediction in ACC.


Assuntos
Neoplasias do Córtex Suprarrenal/sangue , Neoplasias do Córtex Suprarrenal/mortalidade , Carcinoma Adrenocortical/sangue , Carcinoma Adrenocortical/mortalidade , Biomarcadores Tumorais/sangue , Proteínas Proto-Oncogênicas c-vav/metabolismo , Neoplasias do Córtex Suprarrenal/terapia , Carcinoma Adrenocortical/terapia , Adulto , Idoso , Análise de Variância , Biópsia por Agulha , Estudos de Coortes , Terapia Combinada , Intervalo Livre de Doença , Europa (Continente) , Feminino , Humanos , Imuno-Histoquímica , Internacionalidade , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/patologia , Valor Preditivo dos Testes , Prognóstico , Proteínas Proto-Oncogênicas c-vav/sangue , Análise de Sobrevida , Resultado do Tratamento
17.
J Mol Diagn ; 19(4): 575-588, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28552549

RESUMO

Genetic diagnosis is recommended for all pheochromocytoma and paraganglioma (PPGL) cases, as driver mutations are identified in approximately 80% of the cases. As the list of related genes expands, genetic diagnosis becomes more time-consuming, and targeted next-generation sequencing (NGS) has emerged as a cost-effective tool. This study aimed to optimize targeted NGS in PPGL genetic diagnostics. A workflow based on two customized targeted NGS assays was validated to study the 18 main PPGL genes in germline and frozen tumor DNA, with one of them specifically directed toward formalin-fixed paraffin-embedded tissue. The series involved 453 unrelated PPGL patients, of whom 30 had known mutations and were used as controls. Partial screening using Sanger had been performed in 275 patients. NGS results were complemented with the study of gross deletions. NGS assay showed a sensitivity ≥99.4%, regardless of DNA source. We identified 45 variants of unknown significance and 89 pathogenic mutations, the latter being germline in 29 (7.2%) and somatic in 58 (31.7%) of the 183 tumors studied. In 37 patients previously studied by Sanger sequencing, the causal mutation could be identified. We demonstrated that both assays are an efficient and accurate alternative to conventional sequencing. Their application facilitates the study of minor PPGL genes, and enables genetic diagnoses in patients with incongruent or missing clinical data, who would otherwise be missed.


Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Paraganglioma/genética , Feocromocitoma/genética , Neoplasias das Glândulas Suprarrenais/diagnóstico , Adulto , Análise Mutacional de DNA/métodos , Feminino , Variação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Paraganglioma/diagnóstico , Feocromocitoma/diagnóstico
18.
Cancer Cell ; 31(2): 181-193, 2017 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-28162975

RESUMO

We report a comprehensive molecular characterization of pheochromocytomas and paragangliomas (PCCs/PGLs), a rare tumor type. Multi-platform integration revealed that PCCs/PGLs are driven by diverse alterations affecting multiple genes and pathways. Pathogenic germline mutations occurred in eight PCC/PGL susceptibility genes. We identified CSDE1 as a somatically mutated driver gene, complementing four known drivers (HRAS, RET, EPAS1, and NF1). We also discovered fusion genes in PCCs/PGLs, involving MAML3, BRAF, NGFR, and NF1. Integrated analysis classified PCCs/PGLs into four molecularly defined groups: a kinase signaling subtype, a pseudohypoxia subtype, a Wnt-altered subtype, driven by MAML3 and CSDE1, and a cortical admixture subtype. Correlates of metastatic PCCs/PGLs included the MAML3 fusion gene. This integrated molecular characterization provides a comprehensive foundation for developing PCC/PGL precision medicine.


Assuntos
Paraganglioma/genética , Feocromocitoma/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Ligação a DNA/genética , Feminino , Fusão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas Nucleares/genética , Paraganglioma/etiologia , Feocromocitoma/etiologia , Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , Proteínas Proto-Oncogênicas c-ret/genética , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética
19.
Neuroendocrinology ; 105(4): 384-393, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28122379

RESUMO

AIM: We aimed at exploring the activation pattern of the mTOR pathway in sporadic and hereditary pheochromocytomas (PCCs) and paragangliomas (PGLs). METHODS: A total of 178 PCCs and 44 PGLs, already characterized for the presence of germline mutations in VHL, RET, NF1, MAX, SDHA, SDHB, SDHC, and SDHD as well as somatic mutations in VHL, RET, H-RAS, and MAX, were included in 5 tissue microarrays and tested using immunohistochemistry for mTOR and Rictor as well as the phosphorylated forms of mTOR, p70S6K, AMPK, AKT, 4EBP1, S6, and Raptor. RESULTS: The positive correlation among most of the molecules investigated proved the functional activation of the mTOR pathway in PCCs/PGLs. Total mTOR, p-S6K and p-S6, and mTORC1-associated molecules p-Raptor and p-AMPK were all significantly overexpressed in PGLs rather than in PCCs, and in the head and neck rather than in abdominal locations. None of the markers, except for the low expression of p-mTOR, was associated with malignancy. Cluster 1 PCCs/PGLs had higher total mTOR, p-Raptor, and p-S6 expression than cluster 2 PCCs/PGLs. In contrast, p-mTOR and mTORC2-associated molecule Rictor were significantly overexpressed in cluster 2 tumors. Within cluster 1, molecules active in the mTORC1 complex were significantly overexpressed in SDHX- as compared to VHL-mutated tumors. CONCLUSION: In summary, the mTOR pathway is activated in a high proportion of PCCs/PGLs, with a preferential overactivation of the mTORC1 complex in PGLs of the head and neck and/or harboring SDHX mutations.


Assuntos
Complexos Multienzimáticos/genética , Mutação/genética , Tumores Neuroendócrinos/genética , Paraganglioma/genética , Paraganglioma/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Neoplasias das Glândulas Suprarrenais/genética , Neoplasias das Glândulas Suprarrenais/metabolismo , Adulto , Idoso , Europa (Continente) , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Complexos Multienzimáticos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Tumores Neuroendócrinos/metabolismo , Feocromocitoma/genética , Feocromocitoma/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Estatística como Assunto , Análise Serial de Tecidos , Adulto Jovem
20.
Oncotarget ; 8(9): 14525-14536, 2017 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-28099933

RESUMO

Germline mutations in the succinate dehydrogenase (SDHA, SDHB, SDHC, SDHD, SDHAF2) or Von Hippel-Lindau (VHL) genes cause hereditary paraganglioma/pheochromocytoma. While SDHB (1p36) and VHL (3p25) are associated with autosomal dominant disease, SDHD (11q23) and SDHAF2 (11q13) show a remarkable parent-of-origin effect whereby tumor formation is almost completely dependent on paternal transmission of the mutant allele. Loss of the entire maternal copy of chromosome 11 occurs frequently in SDHD-linked tumors, and has been suggested to be the basis for this typical inheritance pattern.Using fluorescent in situ hybridization, microsatellite marker and SNP array analysis, we demonstrate that loss of the entire copy of chromosome 11 is also frequent in SDHAF2-related PGLs, occurring in 89% of tumors. Analysis of two imprinted differentially methylated regions (DMR) in 11p15, H19-DMR and KvDMR, showed that this loss always affected the maternal copy of chromosome 11. Likewise, loss of maternal chromosome 11p15 was demonstrated in 85% of SDHD and 75% of VHL-related PGLs/PCCs. By contrast, both copies of chromosome 11 were found to be retained in 62% of SDHB-mutated PGLs/PCCs, while only 31% showed loss of maternal chromosome 11p15. Genome-wide copy number analysis revealed frequent loss of 1p in SDHB mutant tumors and show greater genomic instability compared to SDHD and SDHAF2.These results show that loss of the entire copy of maternal chromosome 11 is a highly specific and statistically significant event in SDHAF2, SDHD and VHL-related PGLs/PCCs, but is less significant in SDHB-mutated tumors, suggesting that these tumors have a distinct genetic etiology.


Assuntos
Cromossomos Humanos Par 11/genética , Mutação em Linhagem Germinativa/genética , Proteínas Mitocondriais/genética , Paraganglioma/genética , Succinato Desidrogenase/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Alelos , Feminino , Humanos , Perda de Heterozigosidade
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