RESUMO
OBJECTIVES: Molecular diagnosis for targeted therapies has been improved significantly in non-small-cell lung cancer (NSCLC) patients in recent years. Here we report on the prevalence of rare fusions in NSCLC and dissect their genomic architecture and potential clinical implications. MATERIALS AND METHODS: Overall, n = 5554 NSCLC patients underwent next-generation sequencing (NGS) for combined detection of oncogenic mutations and fusions either at primary diagnosis (n = 5246) or after therapy resistance (n = 308). Panels of different sizes were employed with closed amplicon-based, or open assays, i.e. anchored multiplex PCR (AMP) and hybrid capture-based, for detection of translocations, including "rare" fusions, defined as those beyond ALK, ROS1, RET and <0.5 % frequency in NSCLC. RESULTS: Rare fusions involving EGFR, MET, HER2, BRAF and other potentially actionable oncogenes were detected in 0.5% (n = 26) of therapy-naive and 2% (n = 6) TKI-treated tumors. Detection was increased using open assays and/or larger panels, especially those covering >25 genes, by approximately 1-2% (p = 0.001 for both). Patient characteristics (age, gender, smoking, TP53 co-mutations (56%), or mean tumor mutational burden (TMB) (4.8 mut/Mb)) showed no association with presence of rare fusions. Non-functional alterations, i.e. out-of-frame or lacking kinase domains, comprised one-third of detected rare fusions and were significantly associated with simultaneous presence of classical oncogenic drivers, e.g. EGFR or KRAS mutations (p < 0.001), or use of larger panels (frequency of non-functional among the detected rare fusions 57% for 25+ gene- vs. 12% for smaller panels, p < 0.001). As many rare fusions were identified before availability of targeted therapy, mean survival for therapy-naïve patients was 23.8 months, comparable with wild-type tumors. CONCLUSION: Approximately 1-2% of advanced NSCLC harbor rare fusions, which are potentially actionable and may support diagnosis. Routine adoption of broad NGS assays capable to identify exact fusion points and potentially retained protein domains can increase the yield of therapeutically relevant molecular information in advanced NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/patologia , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Genômica , Mutação , Receptores ErbB/genéticaRESUMO
BACKGROUND: Cholangiocarcinoma (CCA) is a primary malignancy of the biliary tract with a dismal prognosis. Recently, several actionable genetic aberrations were identified with significant enrichment in intrahepatic CCA, including FGFR2 gene fusions with a prevalence of 10-15%. Recent clinical data demonstrate that these fusions are druggable in a second-line setting in advanced/metastatic disease and the efficacy in earlier lines of therapy is being evaluated in ongoing clinical trials. This scenario warrants standardised molecular profiling of these tumours. METHODS: A detailed analysis of the original genetic data from the FIGHT-202 trial, on which the approval of Pemigatinib was based, was conducted. RESULTS: Comparing different detection approaches and displaying representative cases, we described the genetic landscape and architecture of FGFR2 fusions in iCCA and show biological and technical aspects to be considered for their detection. We elaborated parameters, including a suggestion for annotation, that should be stated in a molecular diagnostic FGFR2 report to allow a complete understanding of the analysis performed and the information provided. CONCLUSION: This study provides a detailed presentation and dissection of the technical and biological aspects regarding FGFR2 fusion detection, which aims to support molecular pathologists, pathologists and clinicians in diagnostics, reporting of the results and decision-making.
Assuntos
Neoplasias dos Ductos Biliares , Colangiocarcinoma , Neoplasias dos Ductos Biliares/tratamento farmacológico , Ductos Biliares Intra-Hepáticos/patologia , Colangiocarcinoma/tratamento farmacológico , Genômica , Humanos , Técnicas de Diagnóstico Molecular , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genéticaRESUMO
Alectinib is a standard initial treatment for patients with advanced anaplastic lymphoma kinase (ALK) rearranged non-small-cell lung cancer (NSCLC). The current study analyzed a prospective cohort of 24 consecutive alectinib-treated patients and controls in order to comprehensively characterize longitudinal erythrocyte changes under treatment with ALK inhibitors. Upon starting alectinib, all examined patients developed reticulocytosis and abnormal erythrocyte morphology with anisocytosis and a predominance of acanthocytes (64% of red blood cells on average, range 36−100%) in the peripheral blood smear within approximately 2 weeks. Changes were accompanied by a gradual reduction in Eosin-5-maleimide (EMA) binding, which became pathologic (<80% of cells) within 1−2 months in all cases, mimicking an abortive form of hereditary spherocytosis. The latter could be ruled out in 3/3 of analyzed cases by normal sequencing results for the ANK1, EPB42, SLC4A1, SPTA1, or SBTB genes. The direct Coombs test was also negative in 11/11 tested cases. Besides, anemia, increased LDH, and increased bilirubin were noted in a fraction of patients only, ranging between 42 and 68%. Furthermore, haptoglobin decreases were infrequent, occurring in approximately 1/3 of cases only, and mild, with an average value of 0.93 g/L within the normal range of 0.3−2 g/dL, suggesting that hemolysis occurred predominantly in the extravascular compartment, likely due to splenic trapping of the deformed erythrocytes. These changes showed no association with progression-free survival under alectinib or molecular features, i.e., ALK fusion variant or TP53 status of the disease, and resolved upon a switch to an alternative ALK inhibitor. Thus, alectinib induces mild, reversible erythrocyte changes in practically all treated patients, whose most sensitive signs are aberrant red cell morphology in the peripheral smear, a pathologic EMA test, and reactive reticulocytosis. Frank hemolytic anemia is rare, but mild subclinical hemolysis is very frequent and poses differential-diagnostic problems. Alectinib can be continued under the regular control of hemolysis parameters, but the risk of long-term complications, such as cholelithiasis due to increased serum bilirubin in most patients, remains unclear at present.
RESUMO
Modern concepts in precision cancer medicine are based on increasingly complex genomic analyses and require standardized criteria for the functional evaluation and reporting of detected genomic alterations in order to assess their clinical relevance. In this article, we propose and address the necessary steps in systematic variant evaluation consisting of bioinformatic analysis, functional annotation and clinical interpretation, focusing on the latter two aspects. We discuss the role and clinical application of current variant classification systems and point out their scope and limitations. Finally, we highlight the significance of the molecular tumor board as a platform for clinical decision-making based on genomic analyses.
Assuntos
Neoplasias , Medicina de Precisão , Biologia Computacional , Genômica , Humanos , Neoplasias/genéticaRESUMO
OBJECTIVES: Implementation of tyrosine kinase inhibitors (TKI) and other targeted therapies was a main advance in thoracic oncology with survival gains ranging from several months to years for non-small-cell lung cancer (NSCLC) patients. High-throughput comprehensive molecular profiling is of key importance to identify patients that can potentially benefit from these novel treatments. MATERIAL AND METHODS: Next-generation sequencing (NGS) was performed on 4500 consecutive formalin-fixed, paraffin-embedded specimens of advanced NSCLC (nâ¯=â¯4172 patients) after automated extraction of DNA and RNA for parallel detection of mutations and gene fusions, respectively. RESULTS AND CONCLUSION: Besides the 24.9 % (nâ¯=â¯1040) of cases eligible for approved targeted therapies based on the presence of canonical alterations in EGFR exons 18-21, BRAF, ROS1, ALK, NTRK, and RET, an additional nâ¯=â¯1260 patients (30.2 %) displayed rare or non-canonical mutations in EGFR (nâ¯=â¯748), BRAF (nâ¯=â¯135), ERBB2 (nâ¯=â¯30), KIT (nâ¯=â¯32), PIK3CA (nâ¯=â¯221), and CTNNB1 (nâ¯=â¯94), for which targeted therapies could also be potentially effective. A systematic literature search in conjunction with in silico evaluation identified nâ¯=â¯232 (5.5 %) patients, for which a trial of targeted treatment would be warranted according to available evidence (NCT level 1, i.e. published data showing efficacy in the same tumor entity). In conclusion, a sizeable fraction of NSCLC patients harbors rare or non-canonical alterations that may be associated with clinical benefit from currently available targeted drugs. Systematic identification and individualized management of these cases can expand applicability of precision oncology in NSCLC and extend clinical gain from established molecular targets. These results can also inform clinical trials.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Mutação , Medicina de Precisão , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas/genéticaRESUMO
Medulloblastoma is among the most common malignant brain tumors in children. Recent studies have identified at least four subgroups of the disease that differ in terms of molecular characteristics and patient outcomes. Despite this heterogeneity, most patients with medulloblastoma receive similar therapies, including surgery, radiation, and intensive chemotherapy. Although these treatments prolong survival, many patients still die from the disease and survivors suffer severe long-term side effects from therapy. We hypothesize that each patient with medulloblastoma is sensitive to different therapies and that tailoring therapy based on the molecular and cellular characteristics of patients' tumors will improve outcomes. To test this, we assembled a panel of orthotopic patient-derived xenografts (PDX) and subjected them to DNA sequencing, gene expression profiling, and high-throughput drug screening. Analysis of DNA sequencing revealed that most medulloblastomas do not have actionable mutations that point to effective therapies. In contrast, gene expression and drug response data provided valuable information about potential therapies for every tumor. For example, drug screening demonstrated that actinomycin D, which is used for treatment of sarcoma but rarely for medulloblastoma, was active against PDXs representing Group 3 medulloblastoma, the most aggressive form of the disease. Functional analysis of tumor cells was successfully used in a clinical setting to identify more treatment options than sequencing alone. These studies suggest that it should be possible to move away from a one-size-fits-all approach and begin to treat each patient with therapies that are effective against their specific tumor. SIGNIFICANCE: These findings show that high-throughput drug screening identifies therapies for medulloblastoma that cannot be predicted by genomic or transcriptomic analysis.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Cerebelares/tratamento farmacológico , Meduloblastoma/tratamento farmacológico , Medicina de Precisão/métodos , Animais , Linhagem Celular Tumoral , Neoplasias Cerebelares/genética , Criança , Dactinomicina/farmacologia , Regulação Neoplásica da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Masculino , Meduloblastoma/genética , Camundongos Endogâmicos NOD , Mutação , Polimorfismo de Nucleotídeo Único , Sequenciamento do Exoma , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Brain tumors are the leading cause of cancer-related death in children. Genomic studies have provided insights into molecular subgroups and oncogenic drivers of pediatric brain tumors that may lead to novel therapeutic strategies. To evaluate new treatments, better preclinical models adequately reflecting the biological heterogeneity are needed. Through the Children's Oncology Group ACNS02B3 study, we have generated and comprehensively characterized 30 patient-derived orthotopic xenograft models and seven cell lines representing 14 molecular subgroups of pediatric brain tumors. Patient-derived orthotopic xenograft models were found to be representative of the human tumors they were derived from in terms of histology, immunohistochemistry, gene expression, DNA methylation, copy number, and mutational profiles. In vivo drug sensitivity of targeted therapeutics was associated with distinct molecular tumor subgroups and specific genetic alterations. These models and their molecular characterization provide an unprecedented resource for the cancer community to study key oncogenic drivers and to evaluate novel treatment strategies.
Assuntos
Bancos de Espécimes Biológicos , Neoplasias Encefálicas/patologia , Imuno-Histoquímica , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Linhagem Celular Tumoral , Criança , Pré-Escolar , Metilação de DNA/genética , Feminino , Genômica , Humanos , Masculino , Camundongos , Mutação , PediatriaRESUMO
Medulloblastoma (MB) is a highly malignant pediatric brain tumor. Despite aggressive therapy, many patients succumb to the disease, and survivors experience severe side effects from treatment. MYC-driven MB has a particularly poor prognosis and would greatly benefit from more effective therapies. We used an animal model of MYC-driven MB to screen for drugs that decrease viability of tumor cells. Among the most effective compounds were histone deacetylase inhibitors (HDACIs). HDACIs potently inhibit survival of MYC-driven MB cells in vitro, in part by inducing expression of the FOXO1 tumor suppressor gene. HDACIs also synergize with phosphatidylinositol 3-kinase inhibitors to inhibit tumor growth in vivo. These studies identify an effective combination therapy for the most aggressive form of MB.
Assuntos
Proliferação de Células/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Meduloblastoma/tratamento farmacológico , Meduloblastoma/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/metabolismo , Genes Supressores de Tumor/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Pilocytic astrocytoma (PA) is the most common brain tumor in children but is rare in adults, and hence poorly studied in this age group. We investigated 222 PA and report increased aneuploidy in older patients. Aneuploid genomes were identified in 45% of adult compared with 17% of pediatric PA. Gains were non-random, favoring chromosomes 5, 7, 6 and 11 in order of frequency, and preferentially affecting non-cerebellar PA and tumors with BRAF V600E mutations and not with KIAA1549-BRAF fusions or FGFR1 mutations. Aneuploid PA differentially expressed genes involved in CNS development, the unfolded protein response, and regulators of genomic stability and the cell cycle (MDM2, PLK2),whose correlated programs were overexpressed specifically in aneuploid PA compared to other glial tumors. Thus, convergence of pathways affecting the cell cycle and genomic stability may favor aneuploidy in PA, possibly representing an additional molecular driver in older patients with this brain tumor.
Assuntos
Aneuploidia , Astrocitoma/classificação , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/classificação , Adulto , Fatores Etários , Astrocitoma/genética , Astrocitoma/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Criança , Estudos de Coortes , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Mutação/genética , Estadiamento de Neoplasias , Prognóstico , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemRESUMO
Medulloblastoma is a highly malignant paediatric brain tumour currently treated with a combination of surgery, radiation and chemotherapy, posing a considerable burden of toxicity to the developing child. Genomics has illuminated the extensive intertumoral heterogeneity of medulloblastoma, identifying four distinct molecular subgroups. Group 3 and group 4 subgroup medulloblastomas account for most paediatric cases; yet, oncogenic drivers for these subtypes remain largely unidentified. Here we describe a series of prevalent, highly disparate genomic structural variants, restricted to groups 3 and 4, resulting in specific and mutually exclusive activation of the growth factor independent 1 family proto-oncogenes, GFI1 and GFI1B. Somatic structural variants juxtapose GFI1 or GFI1B coding sequences proximal to active enhancer elements, including super-enhancers, instigating oncogenic activity. Our results, supported by evidence from mouse models, identify GFI1 and GFI1B as prominent medulloblastoma oncogenes and implicate 'enhancer hijacking' as an efficient mechanism driving oncogene activation in a childhood cancer.
Assuntos
Proteínas de Ligação a DNA/genética , Elementos Facilitadores Genéticos/genética , Variação Estrutural do Genoma/genética , Meduloblastoma/genética , Oncogenes/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Animais , Criança , Cromossomos Humanos Par 9/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Meduloblastoma/classificação , Meduloblastoma/patologia , Camundongos , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Smoothened (SMO) inhibitors recently entered clinical trials for sonic-hedgehog-driven medulloblastoma (SHH-MB). Clinical response is highly variable. To understand the mechanism(s) of primary resistance and identify pathways cooperating with aberrant SHH signaling, we sequenced and profiled a large cohort of SHH-MBs (n = 133). SHH pathway mutations involved PTCH1 (across all age groups), SUFU (infants, including germline), and SMO (adults). Children >3 years old harbored an excess of downstream MYCN and GLI2 amplifications and frequent TP53 mutations, often in the germline, all of which were rare in infants and adults. Functional assays in different SHH-MB xenograft models demonstrated that SHH-MBs harboring a PTCH1 mutation were responsive to SMO inhibition, whereas tumors harboring an SUFU mutation or MYCN amplification were primarily resistant.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Hedgehog/genética , Meduloblastoma/genética , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Adolescente , Adulto , Animais , Sequência de Bases , Compostos de Bifenilo/uso terapêutico , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/genética , Criança , Pré-Escolar , RNA Helicases DEAD-box/genética , Variações do Número de Cópias de DNA/genética , Feminino , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Fatores de Transcrição Kruppel-Like/genética , Masculino , Meduloblastoma/tratamento farmacológico , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Dados de Sequência Molecular , Proteína Proto-Oncogênica N-Myc , Transplante de Neoplasias , Proteínas Nucleares/genética , Proteínas Oncogênicas/genética , Receptores Patched , Receptor Patched-1 , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piridinas/uso terapêutico , Receptores de Superfície Celular/genética , Proteínas Repressoras/genética , Transdução de Sinais/genética , Receptor Smoothened , Telomerase/genética , Proteína Supressora de Tumor p53/genética , Adulto Jovem , Proteína Gli2 com Dedos de ZincoRESUMO
Two recurrent mutations, K27M and G34R/V, within histone variant H3.3 were recently identified in â¼50% of pHGGs. Both mutations define clinically and biologically distinct subgroups of pHGGs. Here, we provide further insight about the dominant-negative effect of K27M mutant H3.3, leading to a global reduction of the repressive histone mark H3K27me3. We demonstrate that this is caused by aberrant recruitment of the PRC2 complex to K27M mutant H3.3 and enzymatic inhibition of the H3K27me3-establishing methyltransferase EZH2. By performing chromatin immunoprecipitation followed by next-generation sequencing and whole-genome bisulfite sequencing in primary pHGGs, we show that reduced H3K27me3 levels and DNA hypomethylation act in concert to activate gene expression in K27M mutant pHGGs.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias do Tronco Encefálico/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Histonas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias do Tronco Encefálico/metabolismo , Linhagem Celular Tumoral , Criança , Epigênese Genética , Genes Dominantes , Glioblastoma/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Metilação , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Proteínas de Neoplasias , Complexo Repressor Polycomb 2/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Fatores de Transcrição , Transcrição GênicaRESUMO
Pilocytic astrocytoma, the most common childhood brain tumor, is typically associated with mitogen-activated protein kinase (MAPK) pathway alterations. Surgically inaccessible midline tumors are therapeutically challenging, showing sustained tendency for progression and often becoming a chronic disease with substantial morbidities. Here we describe whole-genome sequencing of 96 pilocytic astrocytomas, with matched RNA sequencing (n = 73), conducted by the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. We identified recurrent activating mutations in FGFR1 and PTPN11 and new NTRK2 fusion genes in non-cerebellar tumors. New BRAF-activating changes were also observed. MAPK pathway alterations affected all tumors analyzed, with no other significant mutations identified, indicating that pilocytic astrocytoma is predominantly a single-pathway disease. Notably, we identified the same FGFR1 mutations in a subset of H3F3A-mutated pediatric glioblastoma with additional alterations in the NF1 gene. Our findings thus identify new potential therapeutic targets in distinct subsets of pilocytic astrocytoma and childhood glioblastoma.
Assuntos
Astrocitoma/genética , Neoplasias Encefálicas/genética , Mutação , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor trkB/genética , Animais , Astrocitoma/metabolismo , Sequência de Bases , Neoplasias Encefálicas/metabolismo , Linhagem Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Pontos de Quebra do Cromossomo , Cromossomos Humanos Par 6 , Cromossomos Humanos Par 9 , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Modelos Moleculares , Proteínas de Fusão Oncogênica/química , Proteínas de Fusão Oncogênica/genética , Conformação Proteica , Proteínas Proto-Oncogênicas B-raf/química , Proteínas Proto-Oncogênicas B-raf/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor trkB/metabolismoRESUMO
Medulloblastoma is an aggressively growing tumour, arising in the cerebellum or medulla/brain stem. It is the most common malignant brain tumour in children, and shows tremendous biological and clinical heterogeneity. Despite recent treatment advances, approximately 40% of children experience tumour recurrence, and 30% will die from their disease. Those who survive often have a significantly reduced quality of life. Four tumour subgroups with distinct clinical, biological and genetic profiles are currently identified. WNT tumours, showing activated wingless pathway signalling, carry a favourable prognosis under current treatment regimens. SHH tumours show hedgehog pathway activation, and have an intermediate prognosis. Group 3 and 4 tumours are molecularly less well characterized, and also present the greatest clinical challenges. The full repertoire of genetic events driving this distinction, however, remains unclear. Here we describe an integrative deep-sequencing analysis of 125 tumour-normal pairs, conducted as part of the International Cancer Genome Consortium (ICGC) PedBrain Tumor Project. Tetraploidy was identified as a frequent early event in Group 3 and 4 tumours, and a positive correlation between patient age and mutation rate was observed. Several recurrent mutations were identified, both in known medulloblastoma-related genes (CTNNB1, PTCH1, MLL2, SMARCA4) and in genes not previously linked to this tumour (DDX3X, CTDNEP1, KDM6A, TBR1), often in subgroup-specific patterns. RNA sequencing confirmed these alterations, and revealed the expression of what are, to our knowledge, the first medulloblastoma fusion genes identified. Chromatin modifiers were frequently altered across all subgroups. These findings enhance our understanding of the genomic complexity and heterogeneity underlying medulloblastoma, and provide several potential targets for new therapeutics, especially for Group 3 and 4 patients.
Assuntos
Neoplasias Cerebelares/genética , Genoma Humano/genética , Meduloblastoma/genética , Envelhecimento/genética , Sequência de Aminoácidos , Transformação Celular Neoplásica , Neoplasias Cerebelares/classificação , Neoplasias Cerebelares/diagnóstico , Neoplasias Cerebelares/patologia , Criança , Cromatina/metabolismo , Cromossomos Humanos/genética , RNA Helicases DEAD-box/genética , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Genômica , Proteínas Hedgehog/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Histona Desmetilases/genética , Humanos , Meduloblastoma/classificação , Meduloblastoma/diagnóstico , Meduloblastoma/patologia , Metilação , Mutação/genética , Taxa de Mutação , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteínas de Fusão Oncogênica/genética , Receptores Patched , Receptor Patched-1 , Fosfoproteínas Fosfatases/genética , Poliploidia , Receptores de Superfície Celular/genética , Análise de Sequência de RNA , Transdução de Sinais , Proteínas com Domínio T/genética , Fatores de Transcrição/genética , Proteínas Wnt/metabolismo , beta Catenina/genéticaRESUMO
Genomic rearrangements are thought to occur progressively during tumor development. Recent findings, however, suggest an alternative mechanism, involving massive chromosome rearrangements in a one-step catastrophic event termed chromothripsis. We report the whole-genome sequencing-based analysis of a Sonic-Hedgehog medulloblastoma (SHH-MB) brain tumor from a patient with a germline TP53 mutation (Li-Fraumeni syndrome), uncovering massive, complex chromosome rearrangements. Integrating TP53 status with microarray and deep sequencing-based DNA rearrangement data in additional patients reveals a striking association between TP53 mutation and chromothripsis in SHH-MBs. Analysis of additional tumor entities substantiates a link between TP53 mutation and chromothripsis, and indicates a context-specific role for p53 in catastrophic DNA rearrangements. Among these, we observed a strong association between somatic TP53 mutations and chromothripsis in acute myeloid leukemia. These findings connect p53 status and chromothripsis in specific tumor types, providing a genetic basis for understanding particularly aggressive subtypes of cancer.
Assuntos
Neoplasias Encefálicas/genética , Rearranjo Gênico , Meduloblastoma/genética , Proteína Supressora de Tumor p53/genética , Animais , Criança , Aberrações Cromossômicas , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Modelos Animais de Doenças , Humanos , Leucemia Mieloide Aguda/genética , Síndrome de Li-Fraumeni/fisiopatologia , Camundongos , Pessoa de Meia-IdadeRESUMO
PURPOSE: Integrated genomics approaches have revealed at least four distinct biologic variants of medulloblastoma: WNT (wingless), SHH (sonic hedgehog), group C, and group D. Because of the remarkable clinical heterogeneity of group D tumors and the dismal prognosis of group C patients, it is vital to identify molecular biomarkers that will allow early and effective treatment stratification in these non-WNT/non-SHH tumors. PATIENTS AND METHODS: We combined transcriptome and DNA copy-number analyses for 64 primary medulloblastomas. Bioinformatic tools were used to discover marker genes of molecular variants. Differentially expressed transcripts were evaluated for prognostic value in the screening cohort. The prognostic power of follistatin-like 5 (FSTL5) immunopositivity was tested for 235 nonoverlapping medulloblastoma samples on two independent tissue microarrays. RESULTS: Comprehensive analyses of transcriptomic and genetic alterations delineate four distinct variants of medulloblastoma. Stable subgroup separation was achieved by using the 300 transcripts that varied the most. Distinct expression patterns of FSTL5 in each molecular subgroup were confirmed by quantitative real-time polymerase chain reaction. Immunopositivity of FSTL5 identified a large cohort of patients (84 of 235 patients; 36%) at high risk for relapse and death. Importantly, more than 50% of non-WNT/non-SHH tumors displayed FSTL5 negativity, delineating a large patient cohort with a good prognosis who would otherwise be considered intermediate or high-risk on the basis of current molecular subgrouping. CONCLUSION: FSTL5 expression denoted a dismal prognosis both within and across medulloblastoma subgroups. The addition of FSTL5 immunohistochemistry to existing molecular stratification schemes constitutes a reliable and cost-effective tool for prognostication in future clinical trials of medulloblastoma.
Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Proteínas Relacionadas à Folistatina/genética , Meduloblastoma/genética , Meduloblastoma/patologia , Biomarcadores Tumorais/biossíntese , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/metabolismo , Criança , Estudos de Coortes , Intervalo Livre de Doença , Proteínas Relacionadas à Folistatina/biossíntese , Perfilação da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Meduloblastoma/metabolismo , Análise em Microsséries , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Análise de SobrevidaRESUMO
Activation of the MAPK signaling pathway has been shown to be a unifying molecular feature in pilocytic astrocytoma (PA). Genetically, tandem duplications at chromosome 7q34 resulting in KIAA1549-BRAF fusion genes constitute the most common mechanism identified to date. To elucidate alternative mechanisms of aberrant MAPK activation in PA, we screened 125 primary tumors for RAF fusion genes and mutations in KRAS, NRAS, HRAS, PTPN11, BRAF and RAF1. Using microarray-based comparative genomic hybridization (aCGH), we identified in three cases an interstitial deletion of ~2.5 Mb as a novel recurrent mechanism forming BRAF gene fusions with FAM131B, a currently uncharacterized gene on chromosome 7q34. This deletion removes the BRAF N-terminal inhibitory domains, giving a constitutively active BRAF kinase. Functional characterization of the novel FAM131B-BRAF fusion demonstrated constitutive MEK phosphorylation potential and transforming activity in vitro. In addition, our study confirmed previously reported BRAF and RAF1 fusion variants in 72% (90/125) of PA. Mutations in BRAF (8/125), KRAS (2/125) and NF1 (4/125) and the rare RAF1 gene fusions (2/125) were mutually exclusive with BRAF rearrangements, with the exception of two cases in our series that concomitantly harbored more than one hit in the MAPK pathway. In summary, our findings further underline the fundamental role of RAF kinase fusion products as a tumor-specific marker and an ideally suited drug target for PA.
Assuntos
Astrocitoma/genética , Neoplasias Encefálicas/genética , Sistema de Sinalização das MAP Quinases/genética , Mutação/genética , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Pré-Escolar , Cromossomos Humanos Par 7/genética , Hibridização Genômica Comparativa , Feminino , Seguimentos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Lactente , Masculino , Camundongos , Células NIH 3T3 , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação/genética , Deleção de Sequência , Estatísticas não Paramétricas , Transfecção/métodosRESUMO
Pilocytic astrocytoma (PA) is emerging as a tumor entity with dysregulated Ras/Raf/MEK/ERK signaling. Common genetic lesions observed in PA, which are linked to aberrant ERK pathway activity, include either NF1 inactivation, KRAS or BRAF gain-of-function mutations. To investigate the mutation spectrum within the proto-oncogene encoding the Ser/Thr-kinase B-Raf in more detail, we analyzed 64 primary tumor samples from children with PA including two patients with neurofibromatosis type 1 (NF1). The well-known BRAF(V600E) mutation was found in 6/64 (9.38%) of our samples. For the first time, we report concomitant presence of a somatic BRAF(V600E) mutation in an NF1 patient indicating that more than one Ras/ERK pathway component can be affected in PA. Furthermore, 2/64 (3.13%) of our samples carried a 3-bp insertion in BRAF resulting in the duplication of threonine 599. This conserved residue is located within the activation segment and, if phosphorylated in a Ras-dependent manner, plays a key role in Raf activation. Here, we demonstrate that this mutant (B-Raf(insT) ) and another B-Raf mutant, which carries two additional threonine residues at this position, display an in vitro kinase activity and cellular MEK/ERK activation potential comparable to those of B-Raf(V600E) . Notably, replacement of threonines by valine residues had similar effects on B-Raf activity, suggesting that the distortion of the peptide backbone by additional amino acids rather than the insertion of additional, potential phosphorylation sites destabilizes the inactive conformation of the kinase domain. We also demonstrate that B-Raf(insT) and B-Raf(V600E) , but not B-Raf(wt) , provoke drastic morphological alterations in human astrocytes.
Assuntos
Astrocitoma/genética , Predisposição Genética para Doença , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Adolescente , Adulto , Alelos , Animais , Astrocitoma/metabolismo , Transformação Celular Neoplásica/genética , Criança , Pré-Escolar , Éxons , Feminino , Genótipo , Humanos , Sistema de Sinalização das MAP Quinases , Masculino , Camundongos , Mutagênese Insercional , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas B-raf/metabolismo , Adulto JovemRESUMO
Autosomal recessive cerebellar ataxia with ocular apraxia type 2 (AOA2) is a neurodegenerative disorder characterised by early onset cerebellar ataxia, sensory-motor neuropathy and frequently increased levels of alpha-fetoprotein. We describe a male patient with a phenotype highly suggestive of AOA2, but only one point mutation found by sequencing of the SETX gene. Further analysis revealed a large out-of-frame tandem duplication, encompassing exons 7, 8, 9 and 10. This duplication event occurred obviously by unequal homologous recombination between AluY sequences. Gross SETX deletions or duplications might be an underestimated cause of AOA2.