RESUMEN
BACKGROUND: Endolymphatic sac tumors (ELSTs) are, with a prevalence of up to 16%, a component of von Hippel-Lindau (VHL) disease. Data from international registries regarding heritable fraction and characteristics, germline VHL mutation frequency, and prevalence are lacking. METHODS: Systematic registration of ELSTs from international centers of otorhinolaryngology and from multidisciplinary VHL centers' registries was performed. Molecular genetic analyses of the VHL gene were offered to all patients. RESULTS: Our population-based registry comprised 93 patients with ELST and 1789 patients with VHL. The prevalence of VHL germline mutations in apparently sporadic ELSTs was 39%. The prevalence of ELSTs in patients with VHL was 3.6%. ELST was the initial manifestation in 32% of patients with VHL-ELST. CONCLUSION: Prevalence of ELST in VHL disease is much lower compared to the literature. VHL-associated ELSTs can be the first presentation of the syndrome and mimic sporadic tumors, thus emphasizing the need of molecular testing in all presentations of ELST. © 2015 Wiley Periodicals, Inc. Head Neck 38: 673-679, 2016.
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Neoplasias del Oído/patología , Saco Endolinfático/patología , Enfermedad de von Hippel-Lindau/complicaciones , Adolescente , Adulto , Anciano , Niño , Femenino , Mutación de Línea Germinal , Humanos , Masculino , Persona de Mediana Edad , Sistema de Registros , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Adulto JovenRESUMEN
Schwannomas are tumors that develop from Schwann cells in the peripheral nerves and commonly arise from the vestibular nerve. Vestibular schwannomas can present unilaterally and sporadically or bilaterally when the tumor is associated with neurofibromatosis Type 2 (NF2) syndrome. The molecular hallmark of the disease is biallelic inactivation of the NF2 gene. The epigenetic signature of schwannomas remains poorly understood and is mostly limited to DNA methylation of the NF2 gene, whose altered expression due to epigenetic factors in this tumor is controversial. In this study, we tested the genomewide DNA methylation pattern of schwannomas to shed light on this epigenetic alteration in these particular tumors. The methodology used includes Infinium Human Methylation 450K BeadChip microarrays in a series of 36 vestibular schwannomas, 4 nonvestibular schwannomas, and 5 healthy nerves. Our results show a trend toward hypomethylation in schwannomas. Furthermore, homeobox (HOX) genes, located at four clusters in the genome, displayed hypomethylation in several CpG sites in the vestibular schwannomas but not in the nonvestibular schwannomas. Several microRNA (miRNA) and protein-coding genes were also found to be hypomethylated at promoter regions and were confirmed as upregulated by expression analysis; including miRNA-21, Met Proto-Oncogene (MET), and PMEPA1. We also detected methylation patterns that might be involved in alternative transcripts of several genes such as NRXN1 or MBP, which would increase the complexity of the methylation and expression patterns. Overall, our results show specific epigenetic signatures in several coding genes and miRNAs that could potentially be used as therapeutic targets.
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Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Genes Homeobox , Neuroma Acústico/genética , Empalme Alternativo , Femenino , Genoma Humano , Humanos , Masculino , MicroARNs/metabolismo , Familia de Multigenes , Proto-Oncogenes MasRESUMEN
Schwannomas and grade I meningiomas are nonmetastatic neoplasms that share the common mutation of gene NF2. They usually appear in neurofibromatosis type 2 patients. Currently, there is no drug treatment available for both tumors, thus the use of wide expression technologies is crucial to identify therapeutic targets. Affymetrix Human Gene 1.0 ST was used to test global gene expression in 22 meningiomas, 31 schwannomas and, as non-tumoral controls, 3 healthy meningeal tissues, 8 non-tumoral nerves and 1 primary Schwann cell culture. A non-stringent P-value cut-off and fold change were used to establish deregulated genes. We identified a subset of genes that were upregulated in meningiomas and schwannomas when compared to their respectively healthy tissues, including PDGFD, CDH1 and SLIT2. Thus, these genes should be thoroughly studied as targets in a possible combined treatment.
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Cadherinas/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Linfocinas/genética , Meningioma/metabolismo , Proteínas del Tejido Nervioso/genética , Neurilemoma/metabolismo , Factor de Crecimiento Derivado de Plaquetas/genética , Antígenos CD , Cadherinas/metabolismo , Estudios de Casos y Controles , Femenino , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Linfocinas/metabolismo , Masculino , Neoplasias Meníngeas , Proteínas del Tejido Nervioso/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transcriptoma , Regulación hacia ArribaRESUMEN
Examining aberrant pathway alterations is one method for understanding the abnormal signals that are involved in tumorigenesis and tumor progression. In the present study, expression arrays were performed on tumor-related genes in meningiomas. The GE Array Q Series HS-006 was used to determine the expression levels of 96 genes that corresponded to six primary biological regulatory pathways in a series of 42 meningiomas, including 32 grade I, four recurrent grade I and six grade II tumors, in addition to three normal tissue controls. Results showed that 25 genes that were primarily associated with apoptosis and angiogenesis functions were downregulated and 13 genes frequently involving DNA damage repair functions were upregulated. In addition to the inactivation of the neurofibromin gene, NF2, which is considered to be an early step in tumorigenesis, variations of other biological regulatory pathways may play a significant role in the development of meningioma.
RESUMEN
BACKGROUND: Vestibular schwannomas are benign tumors that arise from Schwann cells in the VIII cranial pair and usually present NF2 gene mutations and/or loss of heterozygosity on chromosome 22q. Deregulation has also been found in several genes, such as ERBB2 and NRG1. MicroRNAs are non-coding RNAs approximately 21 to 23 nucleotides in length that regulate mRNAs, usually by degradation at the post-transcriptional level. METHODS: We used microarray technology to test the deregulation of miRNAs and other non-coding RNAs present in GeneChip miRNA 1.0 (Affymetrix) over 16 vestibular schwannomas and 3 control-nerves, validating 10 of them by qRT-PCR. FINDINGS: Our results showed the deregulation of 174 miRNAs, including miR-10b, miR-206, miR-183 and miR-204, and the upregulation of miR-431, miR-221, miR-21 and miR-720, among others. The results also showed an aberrant expression of other non-coding RNAs. We also found a general upregulation of the miRNA cluster located at chromosome 14q32. CONCLUSION: Our results suggest that several miRNAs are involved in tumor formation and/or maintenance and that global upregulation of the 14q32 chromosomal site contains miRNAs that may represent a therapeutic target for this neoplasm.
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Cromosomas Humanos Par 14/genética , Perfilación de la Expresión Génica/métodos , MicroARNs/genética , Neuroma Acústico/genética , Adulto , Alelos , Femenino , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , ARN no Traducido/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Vestibular schwannomas are benign neoplasms that arise from the vestibular nerve. The hallmark of these tumors is the biallelic inactivation of neurofibromin 2 (NF2). Transcriptomic alterations, such as the neuregulin 1 (Nrg1)/ErbB2 pathway, have been described in schwannomas. In this study, we performed a whole transcriptome analysis in 31 vestibular schwannomas and 9 control nerves in the Affymetrix Gene 1.0 ST platform, validated by quantitative real-time PCR (qRT-PCR) using TaqMan low density arrays. We performed a mutational analysis of NF2 by PCR/denaturing high-performance liquid chromatography (dHPLC) and multiplex ligation-dependent probe amplification (MLPA), as well as a microsatellite marker analysis of the loss of heterozygosity (LOH) of chromosome 22q. The microarray analysis demonstrated that 1,516 genes were deregulated and 48 of the genes were validated by qRT-PCR. At least 2 genetic hits (allelic loss and/or gene mutation) in NF2 were found in 16 tumors, seven cases showed 1 hit and 8 tumors showed no NF2 alteration. MET and associated genes, such as integrin, alpha 4 (ITGA4)/B6, PLEXNB3/SEMA5 and caveolin-1 (CAV1) showed a clear deregulation in vestibular schwannomas. In addition, androgen receptor (AR) downregulation may denote a hormonal effect or cause in this tumor. Furthermore, the osteopontin gene (SPP1), which is involved in merlin protein degradation, was upregulated, which suggests that this mechanism may also exert a pivotal role in schwannoma merlin depletion. Finally, no major differences were observed among tumors of different size, histological type or NF2 status, which suggests that, at the mRNA level, all schwannomas, regardless of their molecular and clinical characteristics, may share common features that can be used in their treatment.
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Neurofibromina 2/metabolismo , Neuroma Acústico/genética , Neuroma Acústico/metabolismo , Osteopontina/metabolismo , Receptores Androgénicos/metabolismo , Empalme Alternativo , Apoptosis , Proteínas Reguladoras de la Apoptosis/biosíntesis , Caveolina 1/biosíntesis , Caveolina 1/genética , Cromosomas Humanos Par 22/genética , Regulación hacia Abajo , Receptores ErbB/biosíntesis , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Proteínas de Choque Térmico/biosíntesis , Humanos , Integrina alfa4/genética , Pérdida de Heterocigocidad , Masculino , Repeticiones de Microsatélite , Neurregulina-1/genética , Neurregulina-1/metabolismo , Neurofibromina 2/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteopontina/genética , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , ARN Mensajero/genética , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Células de Schwann/patología , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Quinasas p21 Activadas/metabolismoRESUMEN
PURPOSE: Gene expression array analysis is providing key data on the potential candidate genes and biological pathways involved in schwannoma origin and development. In this way we performed expression array studies on tumor-related genes in schwannomas. METHODS: The GE Array Q Series HS-006 (SuperArray, Bethesda, MD, USA) was used to determine the expression levels of 96 genes corresponding to 6 primary biological regulatory pathways in a series of 23 schwannomas. RESULTS: We identified 15 genes down-regulated, primarily corresponding to signal transduction functions, and 26 genes up-regulated, most frequently involving cell adhesion functions. CONCLUSIONS: In addition to the NF2 inactivation (considered as an early step), variations of other biological regulatory pathways might play a key role in schwannoma.
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Perfilación de la Expresión Génica , Neurilemoma/genética , Humanos , Neurilemoma/patología , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
The six major genes involved in hereditary susceptibility for pheochromocytoma (PCC)/paraganglioma (PGL) (RET, VHL, NF1, SDHB, SDHC, and SDHD) have been recently integrated into the same neuronal apoptotic pathway where mutations in any of these genes lead to cell death. In this model, prolyl hydroxylase 3 (EglN3) abrogation plays a pivotal role, but the molecular mechanisms underlying its inactivation are currently unknown. The aim of the study was to decipher specific alterations associated with the different genetic classes of PCCs/PGLs. With this purpose, 84 genetically characterized tumors were analyzed by means of transcriptional profiling. The analysis revealed a hypoxia-inducible factor (HIF)-related signature common to succinate dehydrogenase (SDH) and von Hippel-Lindau (VHL) tumors, that differentiated them from RET and neurofibromatosis type 1 cases. Both canonical HIF-1α and HIF-2α target genes were overexpressed in the SDH/VHL cluster, suggesting that a global HIF deregulation accounts for this common profile. Nevertheless, when we compared VHL tumors with SDHB cases, which often exhibit a malignant behavior, we found that HIF-1α target genes showed a predominant activation in the VHL PCCs. Expression data from 67 HIF target genes was sufficient to cluster SDHB and VHL tumors into two different groups, demonstrating different pseudo-hypoxic signatures. In addition, VHL-mutated tumors showed an unexpected overexpression of EglN3 mRNA that did not lead to significantly different EglN3 protein levels. These findings pave the way for more specific therapeutic approaches for malignant PCCs/PGLs management based on the patient's genetic alteration.
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Muerte Celular/genética , Paraganglioma/genética , Feocromocitoma/genética , Succinato Deshidrogenasa/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Adolescente , Neoplasias de las Glándulas Suprarrenales/genética , Neoplasias de las Glándulas Suprarrenales/metabolismo , Adulto , Anciano , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Niño , Dioxigenasas/genética , Dioxigenasas/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Persona de Mediana Edad , Neoplasias/genética , Paraganglioma/metabolismo , Feocromocitoma/metabolismo , Succinato Deshidrogenasa/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Adulto Joven , Enfermedad de von Hippel-Lindau/genética , Enfermedad de von Hippel-Lindau/metabolismoRESUMEN
The molecular pathology of meningiomas and shwannomas involve the inactivation of the NF2 gene to generate grade I tumors. Genomic losses at 1p and 14q are observed in both neoplasms, although more frequently in meningiomas. The inactivation of unidentified genes located in these regions appears associated with tumor progression in meningiomas, but no clues to its molecular/clinical meaning are available in schwannomas. Recent microarray gene expression studies have demonstrated the existence of molecular subgroups in both entities. In the present study, we correlated the presence of genomic deletions at 1p, 14q, and 22q with the expression patterns of 96 tumor-related genes obtained by cDNA low-density microarrays in a series of 65 tumors including 42 meningiomas and 23 schwannomas. Two expression pattern groups were identified by cDNA mycroarray analysis when compared to the expression pattern in normal control RNA in both meningiomas and schwannomas, each one with patterns similar and different from the normal control. Meningioma and schwannoma subgroups differed in the expression of 38 and 16 genes, respectively. Using MLPA and microsatellites, we identified genomic losses at 1p, 14q, and 22q at nonrandom frequencies (12.5-69%) in meningiomas and schwannomas. Losses at 22q were almost equally frequent in both molecular expression subgroups in both neoplasms. However, deletions at 1p and 14q accumulated in meningiomas with a gene expression pattern different from the normal pattern, whereas the inverse situation occurred in schwannomas. Those anomalies characterized the schwannomas with expression pattern similar to the normal control. These findings suggest that deletions at 1p and 14q enhance the development of an abnormal tumor-related gene expression pattern in meningiomas, but this fact is not corroborated in schwannomas.
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Deleción Cromosómica , Cromosomas Humanos Par 14 , Cromosomas Humanos Par 1 , Perfilación de la Expresión Génica , Genómica , Meningioma/genética , Neurilemoma/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , ADN Complementario/genética , HumanosRESUMEN
BACKGROUND: Head and neck paragangliomas (HNPs) occur as sporadic or familial entities, the latter mostly in association with germline mutations of the SDHB, SDHC, or SDHD (SDHx) genes. Heritable non-SDHx HNP might occur in von Hippel-Lindau disease (VHL, VHL gene), multiple endocrine neoplasia type 2 (MEN2, RET gene), and neurofibromatosis type 1 (NF1, NF1 gene). Reports of non-SDHx HNP presentations are scarce and guidance for genetic testing nonexistent. PATIENTS AND METHODS: An international consortium registered patients with HNPs and performed mutation analyses of the SDHx, VHL, and RET genes. Those with SDHx germline mutations were excluded for purposes of this study. Personal and family histories were evaluated for paraganglial tumors, for the major tumor manifestations, and for family history of VHL, MEN2, or NF1. RESULTS: Twelve patients were found to have hereditary non-SDHx HNPs of a total of 809 HNP and 2084 VHL registrants, 11 in the setting of germline VHL mutations and one of a RET mutation. The prevalence of hereditary HNP is five in 1000 VHL patients and nine in 1000 non-SDHx HNP patients. Comprehensive literature review revealed previous reports of HNPs in five VHL, two MEN2, and one NF1 patient. Overall, 11 here presented HNP cases, and four previously reported VHL-HNPs had lesions characteristic for VHL and/or a positive family history for VHL. CONCLUSIONS: Our observations provide evidence that molecular genetic testing for VHL or RET germline mutations in patients with HNP should be done only if personal and/or family history shows evidence for one of these syndromes.
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Neoplasias de Cabeza y Cuello/diagnóstico , Neoplasia Endocrina Múltiple Tipo 2a/diagnóstico , Paraganglioma/diagnóstico , Enfermedad de von Hippel-Lindau/diagnóstico , Adolescente , Adulto , Análisis Mutacional de ADN , Femenino , Pruebas Genéticas , Neoplasias de Cabeza y Cuello/complicaciones , Neoplasias de Cabeza y Cuello/genética , Humanos , Masculino , Proteínas de la Membrana/genética , Neoplasia Endocrina Múltiple Tipo 2a/complicaciones , Neoplasia Endocrina Múltiple Tipo 2a/genética , Paraganglioma/complicaciones , Paraganglioma/genética , Succinato Deshidrogenasa/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Adulto Joven , Enfermedad de von Hippel-Lindau/complicaciones , Enfermedad de von Hippel-Lindau/genéticaRESUMEN
Identification of the 1p/19q allelic status in gliomas, primarily those with a major oligodendroglial component, has become an excellent molecular complement to tumor histology in order to identify those cases sensitive to chemotherapy. In addition to loss of heterozygosity (LOH), fluorescence in situ hybridization (FISH), or comparative genomic hybridization (CGH), multiplex ligation-dependent probe amplification (MLPA) has been shown to be an alternative methodology to identify deletions of those chromosome arms. We used MLPA to explore the 1p and 19q allelic constitution in a series of 76 gliomas: 41 tumors with a major oligodendroglial component, 34 glioblastomas, and one low-grade astrocytoma. We compared the MLPA findings of the oligodendroglial cases with those previously obtained using LOH in the same samples. Thirty-eight of 41 oligodendrogliomas displayed identical findings by both LOH and MLPA, and losses at either 1p and/or 19q were identified in 12 of 35 (34%) astrocytic tumors. These findings agree with data previously reported comparing MLPA versus FISH or CGH in gliomas and suggest that MLPA can be used in the identification of the 1p/19q allelic deletions on these brain neoplasms.
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Alelos , Neoplasias Encefálicas/genética , Cromosomas Humanos Par 19/genética , Cromosomas Humanos Par 1/genética , Glioblastoma/genética , Pérdida de Heterocigocidad/genética , Oligodendroglioma/genética , Neoplasias Encefálicas/patología , Glioblastoma/patología , Humanos , Técnicas de Amplificación de Ácido Nucleico/métodosRESUMEN
BACKGROUND AND OBJECTIVES: The lesions that involve the paranasal sinuses and the anterior cranial base at the same time are not unusual. These diseases have different features. The aim of this study is to set out the particularities of the non-malignant lesions involving both zones. MATERIAL AND METHODS: Retrospective study of 32 patients between 1986 and 2007 diagnosed with: non-malignant tumours (31.2 %), tumorlike lesions (3.1 %), fibrous-osseous lesions (12.5 %), congenital or acquired malformations (18.7 %) and infection disease (34.3 %). We analyse the diagnostic imaging, the treatment and pathogen mechanism. RESULTS: Only 6 of 43 osteomas involved the paranasal sinuses and anterior cranial fossa (13.04 %): 3 cases have developed meningitis and 1 developed a pneumocephalus. 2 cases are meningiomas: 1 was asymptomatic and the other one caused destruction at subtotal frontal bone. 1 giant hemangioma associated with Klippel-Trenaunay syndrome is treated by combined craniofacial approach. The fibrous-osseous lesions were specifically fibrous dysplasia and affected the ethmoides. The encephalocele were predominating in the malformations group, 2 were diagnosed after repeated meningitis. 11 cases are included by infection: 10 cases caused osteomielitis and the eleventh is a patient with a mucormycosis. Surgery has been used in 84.3 % of the cases: frontal craniotomy 37 %, combined craniofacial approach 18.5 %, subfrontal approach 18.5 %, osteoplastic technique 18.5 %, lateronasal approach 3.7 %, endonasal microscopic resection 3.7 %. CONCLUSIONS: In this study the diagnosis, extension and surgical management were supported in the imaging. A closed separation between the anterior cranial fossa ant the sinus is necessary after the resection. The reconstruction was performed using a pedicled pericranial flap and titanium mesh in most of the cases.
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Enfermedades Óseas/diagnóstico , Enfermedades de los Senos Paranasales/diagnóstico , Base del Cráneo , HumanosRESUMEN
Microarray gene expression profiling is a high-throughput system used to identify differentially expressed genes and regulation patterns, and to discover new tumor markers. As the molecular pathogenesis of meningiomas and schwannomas, characterized by NF2 gene alterations, remains unclear and suitable molecular targets need to be identified, we used low density cDNA microarrays to establish expression patterns of 96 cancer-related genes on 23 schwannomas, 42 meningiomas and 3 normal cerebral meninges. We also performed a mutational analysis of the NF2 gene (PCR, dHPLC, Sequencing and MLPA), a search for 22q LOH and an analysis of gene silencing by promoter hypermethylation (MS-MLPA). Results showed a high frequency of NF2 gene mutations (40%), increased 22q LOH as aggressiveness increased, frequent losses and gains by MLPA in benign meningiomas, and gene expression silencing by hypermethylation. Array analysis showed decreased expression of 7 genes in meningiomas. Unsupervised analyses identified 2 molecular subgroups for both meningiomas and schwannomas showing 38 and 20 differentially expressed genes, respectively, and 19 genes differentially expressed between the two tumor types. These findings provide a molecular subgroup classification for meningiomas and schwannomas with possible implications for clinical practice.
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Neoplasias Meníngeas/genética , Meningioma/genética , Neurilemoma/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Adulto , Anciano , ADN Complementario/genética , ADN de Neoplasias/genética , Femenino , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Neoplasias Meníngeas/clasificación , Meningioma/clasificación , Repeticiones de Microsatélite/genética , Persona de Mediana Edad , Neurilemoma/clasificación , Neurofibromatosis 2/genéticaRESUMEN
Head and neck paragangliomas are usually asymptomatic and benign tumours arising mainly from the carotid body and the vagal, tympanic or jugular glomus. The majority of patients develop sporadic masses, and around 30% of cases harbour germline mutations in one of the succinate dehydrogenase genes: SDHB, SDHC or SDHD. In these hereditary cases, the presence of familial antecedents of the disease, multiplicity/bilaterality, young age at onset, and more recently, presence of gastrointestinal stromal tumours, are main factors to be considered. Here we describe a new mutation (c.256-257insTTT) affecting the SDHC gene in a 60-year-old-patient with a single head and neck paraganglioma, and without familial antecedents of the disease. In silico splice site analysis showed that this variant created a cryptic splice acceptor site and loss of heterozygosity (LOH) supported the pathogenic role of the mutation. Control population analyses did not detect this variant but revealed a novel SDHC polymorphism that exhibited a frequency of 0.3% (3/1020). This latter finding highlights the importance of assessing the clinical relevance of variants of unknown significance by means of analysing sufficient controls. Despite having found a germline mutation in an older, apparently sporadic patient, we consider that the high costs of analysing all susceptibility genes related to the disease support the recommendation of screening for mutations only in patients fulfilling the above criteria.
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Proteínas de la Membrana/genética , Adenoma/genética , Adulto , Femenino , Mutación de Línea Germinal , Humanos , Pérdida de Heterocigocidad , Masculino , Persona de Mediana Edad , Paraganglioma/genética , Neoplasias Hipofisarias/genéticaRESUMEN
Clinical response to Gefitinib (Iressa, ZD1839) has been found to be associated with somatic mutations, primarily of exons 18-21, of the epidermal growth factor receptor gene (EGFR) in non-small cell lung cancer (NSCLC). Evidence of a positive response was also reported recently on a patient with brain metastasis from NSCLC. On the other hand, amplification of EGFR appears to be associated with a poor prognosis. To determine whether EGFR mutations and amplification are involved in the tumorigenesis of brain metastases, we performed polymerase chain reaction/single-strand conformation polymorphism to examine exons 1, 2, and 7-26 of EGFR in a series of 18 brain metastases. The metastases derived from malignant melanoma (three cases), lung carcinoma (six cases), breast carcinoma (three cases), ovarian carcinoma (two cases), and one each from colon, kidney, bladder, and undifferentiated carcinoma. In addition to several sequence polymorphisms, we identified two mutations on E19 consisting of 18-base pair (bp) deletions: 2423-24440del and 2426-2443del. These mutations presented in lesions derived from kidney carcinoma and lung adenocarcinoma. By real-time quantitaive polymerase chain reaction technique, we determined the amplification/overdose status of EGFR by analyzing exons 11 and 25. Amplification (5- to 100-fold) was identified in three tumors, and overdose (low-level gene amplification corresponding to increases of 1- to 5-fold) presented in four additional metastases. These findings suggest that EGFR mutations and polymorphisms are not exclusively present in metastases derived from lung carcinoma. Accordingly, targeting of EGFR to determine molecular alterations of this gene may be useful in the management of patients with brain metastases.
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Neoplasias Encefálicas/secundario , Receptores ErbB/genética , Mutación , Neoplasias/patología , Reacción en Cadena de la Polimerasa/métodos , Adulto , Anciano , Secuencia de Aminoácidos , Secuencia de Bases , Neoplasias Encefálicas/genética , Análisis Mutacional de ADN , ADN de Neoplasias/química , ADN de Neoplasias/genética , Femenino , Dosificación de Gen , Humanos , Masculino , Persona de Mediana Edad , Mutagénesis Insercional , Neoplasias/genética , Polimorfismo Genético , Polimorfismo Conformacional Retorcido-Simple , Eliminación de SecuenciaRESUMEN
We have studied amplification/gene-dosage and sequence variations of the EGFR gene in 41 oligodendroglial tumours graded according to the WHO classification (21 oligodendrogliomas grade II, 13 oligodendrogliomas grade III and 6 oligoastrocytomas grade II-III), using multiplex ligation-dependent probe amplification (MLPA), real-time quantitative PCR, and PCR/SSCP techniques. To determine gene-dose we studied exons 11 (extracellular domain) and 25 (intracellular domain) in the EGFR gene. Overdose (1- to 5-fold increase) was present in exon 11 in 21 of 41 samples (52.5% of cases) and in exon 25, in 7 of 41 samples (17.5% of cases). Gene amplification > 5-fold increase) was present in exon 11, in 17 of 41 samples (42.5% of cases), and in exon 25 in 6 of 41 samples (15% of cases). Three tumours (two grade II oligodendrogliglioma, one mixed oligoastrocytoma) displayed high level amplifications: > 100 gene copies were identified by both real-time quantitative PCR and MLPA analyses. Gene sequence alterations were identified by PCR/SSCP and sequencing in four cases: two missense mutations: G1051A (Ala351Thr) and G2216A (Arg739Hys); one nonsense mutation: C2934T (Asp978Asp); and an 18 bp deletion in position 2423-2441 of E19. These changes were present only in tumoral DNA, not in the corresponding constitutional patients' DNA. We also found four previously unidentified polymorphic variants: G2025A (Ala675Ala), C2233T (Leu745Leu), C2895T (Treo965Treo) and C3168T (Asp1056Asp), and three previously described polymorphic changes: E12+22 Tright curved arrow A, G1748A (Arg583Lys) and A2547T (Glu849Glu). Our findings demonstrate that mutations and amplification/overdose in the EGFR gene are present in low-grade oligodendroglial tumours, and may contribute to the development of these brain neoplasms.