RESUMO
The presence or absence of estrogen and progesterone steroid hormone receptor expression (ER, PR) is an essential feature of invasive breast cancer and determines prognosis and endocrine treatment decisions. Among the four ER/PR receptor phenotypes, the ER-/PR+ is infrequent, and its clinical relevance has been controversially discussed. Thus, we investigated its clinical significance and gene expression pattern in large datasets. In a retrospective clinical study of 15,747 breast cancer patients, we determined the ER/PR subtype survival probabilities using Kaplan-Meier and Cox regression analyses. From The Cancer Genome Atlas (TCGA) breast cancer dataset, PAM50 expression signature and pathway analyses were performed to test for distinct molecular features. In our cohort, the ER-/PR+ phenotype has been observed at a frequency of 4.1 % and was associated with an improved 10-year survival for stage I cancers compared to the ER+/PR+ reference subtype (median; 95 % CI 88.1 %; 83-93 vs. 84.3 %; 82-86 %, P = 0.024) as was confirmed by multivariate analysis over the entire follow-up (HR 0.59, 95 % CI 0.38-0.92, P = 0.021). This association lacked significance when including all stages. ER-/PR+ patients treated with antihormonal agents (34.5 %) had shorter survival compared to their non-treated counterparts (Log-rank P = 0.0001). PAM50 signatures suggest a distinct configuration for the ER-/PR+ phenotype. This specific phenotype has been further separated by a set of 59 uniquely expressed genes. Our study supports the notion of the existence of an ER-/PR+ phenotype with clinical and molecular features distinct from the large group of ER+/PR+ patients.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/mortalidade , Perfilação da Expressão Gênica , Fenótipo , Receptores de Estrogênio/genética , Receptores de Progesterona/genética , Transcriptoma , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Feminino , Seguimentos , Humanos , Pessoa de Meia-Idade , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Avaliação de Resultados da Assistência ao Paciente , Prognóstico , Análise de Sobrevida , Adulto JovemRESUMO
Tamoxifen is the standard-of-care treatment for estrogen receptor-positive premenopausal breast cancer. We examined tamoxifen metabolism via blood metabolite concentrations and germline variations of CYP3A5, CYP2C9, CYP2C19 and CYP2D6 in 587 premenopausal patients (Asians, Middle Eastern Arabs, Caucasian-UK; median age 39 years) and clinical outcome in 306 patients. N-desmethyltamoxifen (DM-Tam)/(Z)-endoxifen and CYP2D6 phenotype significantly correlated across ethnicities (R(2): 53%, P<10(-77)). CYP2C19 and CYP2C9 correlated with norendoxifen and (Z)-4-hydroxytamoxifen concentrations, respectively (P<0.001). DM-Tam was influenced by body mass index (P<0.001). Improved distant relapse-free survival (DRFS) was associated with decreasing DM-Tam/(Z)-endoxifen (P=0.036) and increasing CYP2D6 activity score (hazard ratio (HR)=0.62; 95% confidence interval (CI), 0.43-0.91; P=0.013). Low (<14 nM) compared with high (>35 nM) endoxifen concentrations were associated with shorter DRFS (univariate P=0.03; multivariate HR=1.94; 95% CI, 1.04-4.14; P=0.064). Our data indicate that endoxifen formation in premenopausal women depends on CYP2D6 irrespective of ethnicity. Low endoxifen concentration/formation and decreased CYP2D6 activity predict shorter DRFS.
Assuntos
Antineoplásicos Hormonais/sangue , Neoplasias da Mama/sangue , Neoplasias da Mama/diagnóstico , Detecção Precoce de Câncer , Pré-Menopausa/sangue , Tamoxifeno/sangue , Adulto , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Estudos de Coortes , Feminino , Humanos , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Prospectivos , Tamoxifeno/uso terapêutico , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: Breast cancer is one of the most common malignancies in women. Genome-wide association studies have identified FGFR2 as a breast cancer susceptibility gene. Common variation in other fibroblast growth factor (FGF) receptors might also modify risk. We tested this hypothesis by studying genotyped single-nucleotide polymorphisms (SNPs) and imputed SNPs in FGFR1, FGFR3, FGFR4 and FGFRL1 in the Breast Cancer Association Consortium. METHODS: Data were combined from 49 studies, including 53 835 cases and 50 156 controls, of which 89 050 (46 450 cases and 42 600 controls) were of European ancestry, 12 893 (6269 cases and 6624 controls) of Asian and 2048 (1116 cases and 932 controls) of African ancestry. Associations with risk of breast cancer, overall and by disease sub-type, were assessed using unconditional logistic regression. RESULTS: Little evidence of association with breast cancer risk was observed for SNPs in the FGF receptor genes. The strongest evidence in European women was for rs743682 in FGFR3; the estimated per-allele odds ratio was 1.05 (95% confidence interval=1.02-1.09, P=0.0020), which is substantially lower than that observed for SNPs in FGFR2. CONCLUSION: Our results suggest that common variants in the other FGF receptors are not associated with risk of breast cancer to the degree observed for FGFR2.
Assuntos
Neoplasias da Mama/genética , Predisposição Genética para Doença , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Estudos de Casos e Controles , Feminino , Variação Genética , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Polimorfismo de Nucleotídeo Único/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 5 de Fator de Crescimento de Fibroblastos/genéticaRESUMO
The International Tamoxifen Pharmacogenomics Consortium was established to address the controversy regarding cytochrome P450 2D6 (CYP2D6) status and clinical outcomes in tamoxifen therapy. We performed a meta-analysis on data from 4,973 tamoxifen-treated patients (12 globally distributed sites). Using strict eligibility requirements (postmenopausal women with estrogen receptor-positive breast cancer, receiving 20 mg/day tamoxifen for 5 years, criterion 1); CYP2D6 poor metabolizer status was associated with poorer invasive disease-free survival (IDFS: hazard ratio = 1.25; 95% confidence interval = 1.06, 1.47; P = 0.009). However, CYP2D6 status was not statistically significant when tamoxifen duration, menopausal status, and annual follow-up were not specified (criterion 2, n = 2,443; P = 0.25) or when no exclusions were applied (criterion 3, n = 4,935; P = 0.38). Although CYP2D6 is a strong predictor of IDFS using strict inclusion criteria, because the results are not robust to inclusion criteria (these were not defined a priori), prospective studies are necessary to fully establish the value of CYP2D6 genotyping in tamoxifen therapy.
Assuntos
Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Citocromo P-450 CYP2D6/genética , Tamoxifeno/uso terapêutico , Idoso , Antineoplásicos Hormonais/farmacocinética , Neoplasias da Mama/genética , Feminino , Variação Genética/genética , Genótipo , Humanos , Menopausa , Pessoa de Meia-Idade , Farmacogenética/métodos , Análise de Sobrevida , Tamoxifeno/farmacocinética , Resultado do TratamentoRESUMO
The therapeutic effect of tamoxifen depends on active metabolites, e.g., cytochrome P450 2D6 (CYP2D6) mediated formation of endoxifen. To test for additional relationships, 236 breast cancer patients were genotyped for CYP2D6, CYP2C9, CYP2B6, CYP2C19, CYP3A5, UGT1A4, UGT2B7, and UGT2B15; also, plasma concentrations of tamoxifen and 22 of its metabolites, including the (E)-, (Z)-, 3-, and 4'-hydroxymetabolites as well as their glucuronides, were quantified using liquid chromatography-tandem mass spectrometry (MS). The activity levels of the metabolites were measured using an estrogen response element reporter assay; the strongest estrogen receptor inhibition was found for (Z)-endoxifen and (Z)-4-hydroxytamoxifen (inhibitory concentration 50 (IC50) 3 and 7 nmol/l, respectively). CYP2D6 genotypes explained 39 and 9% of the variability of steady-state concentrations of (Z)-endoxifen and (Z)-4-hydroxytamoxifen, respectively. Among the poor metabolizers, 93% had (Z)-endoxifen levels below IC90 values, underscoring the role of CYP2D6 deficiency in compromised tamoxifen bioactivation. For other enzymes tested, carriers of reduced-function CYP2C9 (*2, *3) alleles had lower plasma concentrations of active metabolites (P < 0.004), pointing to the role of additional pathways.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Glucuronosiltransferase/genética , Polimorfismo Genético/genética , Receptores de Estrogênio/metabolismo , Tamoxifeno/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Hidrocarboneto de Aril Hidroxilases/sangue , Hidrocarboneto de Aril Hidroxilases/genética , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2D6/sangue , Citocromo P-450 CYP2D6/genética , Sistema Enzimático do Citocromo P-450/sangue , Feminino , Seguimentos , Glucuronosiltransferase/sangue , Humanos , Pessoa de Meia-Idade , Estudos Prospectivos , Tamoxifeno/sangue , Tamoxifeno/químicaRESUMO
Pharmacogenomics follows the concept of individualized medicine via targeted and tailored therapy for each patient. This is aided by current progress in human genome research that provided the basis to link genetic markers with pathogenesis and progression for the definition of new therapy options. To achieve this it will be necessary to show the validity and clinical utility of pharmacogenomic tests prior to release. The implementation of pharmacogenomics into clinical practise is a momentous future challenge that requires the establishment of interdisciplinary networks and professional organizations.
Assuntos
Predisposição Genética para Doença/genética , Testes Genéticos/tendências , Terapia Genética/tendências , Farmacogenética/tendências , Medicina de Precisão/tendências , HumanosRESUMO
BACKGROUND/AIMS: The genetic basis for clear-cell renal carcinomas has been established in familial and many sporadic forms. Whether the latter can be induced by environmental carcinogens remains controversial, with concern over solvents such as trichloroethylene (TCE). To study this putative relationship, we analyzed the VHL gene from a patient with long-term TCE exposure. METHODS: PCR amplification and sequencing of VHL exons 1 - 3 were performed on peripheral blood and tumor tissue. RESULTS: The tumor alone had a previously undescribed mutation in exon 1 of the VHL gene: deletion of a cytidine residue at position 291 relative to the first ATG start codon of the wild-type sequence. This deletion causes a frameshift and predicts an altered protein sequence from position 98 onwards. CONCLUSION: The affected amino acids are in the functionally important beta-domain of the VHL protein that is implicated in substrate binding for ubiquitylation, and we hypothesize the mutation lowers that affinity. There is loss of suppressor function when substrates such as hypoxia-inducible factor have impaired degradation: they accumulate and ultimately cause uncontrolled cell turnover. This association of a proposed occupational cause and occurrence of renal-cell carcinoma emphasizes the availability and use of VHL sequencing for both studying the pathophysiology of malignant transformation and potentially playing a clinical role in genetic counseling or risk assessment.
Assuntos
Carcinoma de Células Renais/genética , Neoplasias Renais/genética , Mutação , Exposição Ocupacional/efeitos adversos , Solventes/efeitos adversos , Tricloroetileno/efeitos adversos , Carcinoma de Células Renais/induzido quimicamente , Carcinoma de Células Renais/diagnóstico , Carcinoma de Células Renais/cirurgia , Humanos , Rim/patologia , Neoplasias Renais/induzido quimicamente , Neoplasias Renais/diagnóstico , Neoplasias Renais/cirurgia , Masculino , Pessoa de Meia-Idade , Nefrectomia , Tomografia Computadorizada por Raios X , Proteína Supressora de Tumor Von Hippel-Lindau/genéticaRESUMO
Different homologues of C4 to C8 perfluoroalkyl carboxylates (PFCAs) and perfluoroalkyl sulfonates (PFASs) were detected in German surface waters, bank filtrates, artificially recharged groundwaters, and drinking waters. If no point sources are located nearby, the typically measured levels are in the low ng/L range. In the presence of point sources, such as a fluorochemical production site, a leaching agricultural fertilizer contaminated with PFCAs and PFASs, or drained PFC containing fire-fighting foams, much higher concentrations in the microg/L range occur. This situation is similar in Germany and other countries.
Assuntos
Alcanossulfonatos/análise , Ácidos Carboxílicos/análise , Fluorocarbonos/análise , Poluentes Químicos da Água/análise , Monitoramento Ambiental , Água Doce , Alemanha , Abastecimento de ÁguaRESUMO
Methyl tert-butylether (MTBE) used as fuel oxygenate poses problems for water suppliers since it is persistent in the aquatic environment and the removal efficiency by conventional water treatment methods (aeration or activated carbon filtration) is rather low. Substitution by other ether compounds such as ethyl tert-butylether (ETBE), tert-amylmethylether (TAME) or di-isopropylether (DIPE) is discussed, however, their environmental behaviour is similar to that of MTBE. Experiments investigating the elimination efficiency of AOP were carried out in tap water and water from Lake Constance. The elimination efficiency for all treatment processes was found to follow the order: MTBE << TAME approximately equal ETBE < DIPE For all compounds under investigation, neither pure ozonation nor UV irradiation yield a considerable concentration decline. Only the formation of highly reactive OH radicals shows a potential for removing the ethers from water. Therefore the addition of H2O2 in equimolar ratio prior to ozone admixing proved to be quite efficient. The application of combined UV/H2O2 showed good results in all cases; the best concentration decline was achieved with UV/ozone. The rate of elimination of the three substitutes for MTBE (ETBE, TAME and DIPE) is higher in all processes; nevertheless, no complete removal could be achieved. Therefore, from the point of view of water suppliers, the use of other ethers as substitute for MTBE is posing the same problems as MTBE.
Assuntos
Ésteres/química , Peróxido de Hidrogênio/química , Oligopeptídeos/química , Oxigênio/química , Ozônio/química , Tosilarginina Metil Éster/química , Água/química , Fontes Geradoras de Energia , Etil-Éteres/química , Éteres Metílicos/química , Minerais/química , Raios UltravioletaRESUMO
Equilibrium and kinetic adsorption of methyl tert-butyl ether (MTBE) onto three coal-based activated carbons, one coconut-based activated carbon, and two zeolites are elucidated in this study. Natural organic matter (NOM) and MTBE competed for the adsorption of activated carbons to different extents. The ideal adsorbed solution theory (IAST) combined with the equivalent background compound (EBC) model can adequately describe the NOM competition and predict the isotherms of MTBE onto the activated carbons. No competitive adsorption was observed for one of the zeolites, mordenite, due to the molecular effect. Besides, the aperture size, and the SiO2/Al2O3 ratio of the zeolite may also play an important role in the adsorption of MTBE from the aqueous phase. The surface diffusion model accurately simulated the transport of MTBE within the adsorbents employed in different water matrices. For all the activated carbons tested, the surface diffusivity of MTBE in natural water was nearly equal to that in deionized water, indicating that no apparently hindering effect occurs. A much slower adsorption kinetic of mordenite in natural water was observed since the opening apertures on mordenite may be appreciably hindered and blocked by NOM.
Assuntos
Adsorção/efeitos dos fármacos , Carvão Vegetal/química , Éteres Metílicos/química , Compostos Orgânicos/farmacologia , Poluentes Químicos da Água/farmacologia , Zeolitas/química , Óxido de Alumínio/química , Silicatos de Alumínio/química , Cinética , Dióxido de Silício/química , Fatores de Tempo , Purificação da Água/métodosRESUMO
Aliphatic and alicyclic amines as well as ethanolamines are extremely polar compounds, frequently found in the environment, and some of them have high toxicity. To address the contamination of selected German surface waters examined and the importance of bank filtration in Eastern Germany, investigations on the behaviour of polar organic nitrogen compounds during water treatment were carried out. Test conditions were designed appropriately for drinking water treatment conditions, and the tests were carried out using model water as well as bank filtrate. Test filter studies of microbial degradation of selected compounds demonstrated the following order of biodegradability: ethanolamine > dimethylamine > pyrrolidine > ethylenediamine. piperidine > diethylamine > morpholine > piperazine > cyclohexylamine. Flocculation tests using iron salts as well as aluminium salts as coagulants showed very low removal rates for the amines. The best results for the removal of the polar organic nitrogen compounds from the water were obtained using ozonation. Based on the reaction-rate constants, the order of degradation by ozone is: piperazine > morpholine > ethylenediamine > piperidine, cyclohexylamine > dimethylamine > ethanolamine > pyrrolidine > diethylamine. Disinfection by chlorine-containing agents under drinking water treatment conditions did not give effective elimination of the selected polar nitrogen compounds.
Assuntos
Aminas/química , Compostos de Nitrogênio/química , Purificação da Água , Abastecimento de Água , Biodegradação Ambiental , Filtração , Floculação , Modelos Teóricos , Microbiologia da ÁguaRESUMO
In order to evaluate the sensitivity and specificity of the recently introduced high-throughput method DHPLC (denaturing high performance liquid chromatography) for mutation screening in the VHL tumor suppressor gene, we subjected DNA from 43 unrelated VHL patients with previously sequenced VHL germline mutations to this method. In addition, 36 genomic DNAs of unrelated individuals suspected of being VHL carriers but with unknown germline status were analyzed by DHPLC and sequencing. The aims of the present study were to compare mutation results obtained by direct sequencing and DHPLC, and a comparison of two different DHPLC systems. The sensitivity of DHPLC was tested with two commercial devices and protocols, i.e., the Varian-Helix system and the Wave Nucleic Acid Fragment Analysis system. Both resolved all but one mutation in exons 2 and 3 of the VHL gene. In contrast, the GC-rich exon 1 showed discrepancies in the rate of mutation detection. Whereas the Varian-Helix system detected 10/15 (67%) of the known mutations, the Wave Nucleic Acid Fragment Analysis system detected 13/14 (93%). All three mutations in samples with unknown mutation status were revealed by both systems raising the mutation detection rate to 72% and 94%, respectively. Cases with different substitutions at the same nucleotide showed different elution profiles, but similar elution profiles could be obtained from different mutations. The Wave Nucleic Acid Fragment Analysis system detected most VHL mutations; however, when a 100% detection rate is needed, sequencing is still required and must therefore be the standard VHL mutation detection procedure. Once a family-specific mutation has been established, DHPLC may be suitable for the rapid and cost-effective determination of VHL carrier status in family members.
Assuntos
Genes Supressores de Tumor/genética , Testes Genéticos/métodos , Mutação em Linhagem Germinativa/genética , Ligases , Proteínas/genética , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Cromatografia Líquida de Alta Pressão , Análise Mutacional de DNA/instrumentação , Análise Mutacional de DNA/métodos , Éxons/genética , Mutação da Fase de Leitura/genética , Sequência Rica em GC/genética , Testes Genéticos/instrumentação , Heterozigoto , Humanos , Mutação de Sentido Incorreto/genética , Desnaturação de Ácido Nucleico , Sítios de Splice de RNA/genética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Temperatura , Proteína Supressora de Tumor Von Hippel-LindauRESUMO
In this paper, analytical methods for the trace-level determination of 60 pharmaceuticals in aqueous samples are presented. The list of compounds amenable to the methods comprises analgesics, antiphlogistics, antirheumatics, beta-blockers, broncholytics, lipid-lowering agents (or their metabolites), antiepileptics, vasodilators, tranquillizers, antineoplastic drugs, iodinated X-ray contrast media, and antibiotics of different kind, mainly sulfonamides, macrolides, and penicillins. All methods are based on automated solid-phase extraction followed by GC-MS (after derivatization of the acid compounds) or HPLC-electrospray ionization MS-MS. After an intense validation, which included the determination of performance data according to the German standard method DIN 32645 (limit of detection, limit of identification, limit of determination), the determination of linearity, recovery, and repeatability and the study of matrix effects, the analytical methods were applied within a monitoring program on the occurrence of pharmaceuticals in groundwaters of Baden-Württemberg. During this monitoring program, it was found that several of the compounds under investigation could be detected in groundwaters and their occurrence could be traced back to an impact of municipal or industrial waste water.
Assuntos
Monitoramento Ambiental , Preparações Farmacêuticas/análise , Poluentes Químicos da Água/análise , Cromatografia Líquida de Alta Pressão , Indústria Farmacêutica , Cromatografia Gasosa-Espectrometria de Massas , Alemanha , Resíduos Industriais , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Hypoxia induces transcription of a range of physiologically important genes including erythropoietin and vascular endothelial growth factor. The transcriptional activation is mediated by the hypoxia-inducible factor-1 (HIF-1), a heterodimeric member of the basic helix-loop-helix PAS family, composed of alpha and beta subunits. HIF-1alpha shares 48 per cent identity with the recently identified HIF-2alpha protein that is also stimulated by hypoxia. In a previous study of hemangioblastomas, the most frequent manifestation of hereditary von Hippel-Lindau disease (VHL), we found elevated levels of vascular endothelial growth factor and HIF-2alpha mRNA in stromal cells of the tumors. Mutations of the VHL tumor suppressor gene are associated with a variety of tumors such as renal clear cell carcinomas (RCC). In this study, we analysed the expression of the hypoxia-inducible factors HIF-1alpha and HIF-2alpha in a range of VHL wildtype and VHL deficient RCC cell lines. In the presence of functional VHL protein, HIF-1alpha mRNA levels are elevated, whereas HIF-2alpha mRNA expression is increased only in cells lacking a functional VHL gene product. On the protein levels, however, in VHL deficient cell lines, both HIF-alpha subunits are constitutively expressed, whereas re-introduction of a functional VHL gene restores the instability of HIF-1alpha and HIF-2alpha proteins under normoxic conditions. Moreover, immunohistochemical analyses of RCCs and hemangioblastomas demonstrate up-regulation of HIF-1alpha and HIF-2alpha in the tumor cells. The data presented here provide evidence for a role of the VHL protein in regulation of angiogenesis and erythropoiesis mediated by the HIF-1alpha and HIF-2alpha proteins.
Assuntos
Carcinoma de Células Renais/metabolismo , Proteínas de Ligação a DNA/biossíntese , Genes Supressores de Tumor/fisiologia , Neoplasias Renais/genética , Ligases , Proteínas Nucleares/biossíntese , Oxigênio/metabolismo , Proteínas/genética , Transativadores/biossíntese , Fatores de Transcrição , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Carcinoma de Células Renais/genética , Cerebelo/metabolismo , Cerebelo/fisiologia , Proteínas de Ligação a DNA/genética , Fatores de Crescimento Endotelial/biossíntese , Fatores de Crescimento Endotelial/genética , Transportador de Glucose Tipo 1 , Hemangioblastoma/genética , Hemangioblastoma/metabolismo , Humanos , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Imuno-Histoquímica , Neoplasias Renais/metabolismo , Linfocinas/biossíntese , Linfocinas/genética , Proteínas de Transporte de Monossacarídeos/biossíntese , Proteínas de Transporte de Monossacarídeos/genética , Mutação , Proteínas Nucleares/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transativadores/genética , Células Tumorais Cultivadas , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Proteína Supressora de Tumor Von Hippel-Lindau , Doença de von Hippel-Lindau/genéticaRESUMO
An ion-pair solid-phase extraction (IPE), ion-pair chromatography (IPC) procedure with fluorescence detection for the quantitative analysis of sulfonated naphthalene-formaldehyde condensates (SNFC) was developed, which provides full resolution of SNFC up to a degree of condensation n = 5 and partial resolution up to n = 15. Liquid chromatography-electrospray ionization-mass spectrometry confirmed that SNFC elute in the order of condensation. Response factors in fluorescence detection proved to be mass-constant, thereby allowing us to determine total SNFC amounts. With this IPC method, the weight- and the number-average molecular weights of these high-volume production chemicals (kiloton per annum), used as synthetic tanning agents, concrete plasticizers, and dispersants, can be determined. Recoveries in IPE range from 73 to 85% in river Rhine water and from 79 to 93% in tap water for n = 2 to n = 7 with limits of detection of 3-8 ng/L for individual homologues from 500 mL of water. The IPE-IPC procedure was applied to samples of secondary industrial effluents, river Rhine water, a river bank filtrate, and a groundwater sample. SNFC up to n = 6 were detected in the treated effluents. Total concentrations ranged from 208 micrograms/L in a secondary treated SNFC production effluent to < 1.4 micrograms/L in groundwater. These first analyses suggest a widespread occurrence of the lower oligomers of SNFC in the aquatic environment.
Assuntos
Formaldeído/análise , Naftalenos/análise , Poluentes Químicos da Água/análise , Cromatografia Líquida , Polímeros/análise , Esgotos/análise , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
To elucidate the role of somatic alterations for renal cancer etiology and prognosis, we analyzed 227 sporadic renal epithelial tumors for mutations and hypermethylations in the von Hippel-Lindau tumor suppressor gene VHL. Tumors were classified according to the recommendations of the Union Internationale Contre le Cancer (UICC) and the American Joint Committee on Cancer (AJCC). Somatic VHL mutations were identified by PCR, single-strand conformation polymorphism analysis, and sequencing, and hypermethylations were identified by restriction enzyme digestion and Southern blotting. Frequencies of VHL alterations were established, and an association with tumor type or tumor type and tumor stage was evaluated. VHL mutations and hypermethylations were identified in 45% of clear cell renal cell carcinomas (CCRCCs) and occasionally (3 of 28) in papillary (chromophilic) renal cell carcinomas (RCCs). Lack of VHL mutations and hypermethylations in chromophobe RCCs and oncocytomas was statistically significant (P = 0.0001 and P = 0.0004, respectively). RCCs carrying VHL alterations showed, in nine cases (12%), mutations at a hot spot involving a thymine repeat (ATT.TTT) in exon 2. Tumor staging was critical to the VHL mutation/hypermethylation detection rate in CCRCCs shown by separate evaluation of patients from medical centers in Munich, Heidelberg, and Mainz. The spectrum of pT1, pT2, and pT3 CCRCCs and the VHL mutation/hypermethylation detection rate varied among these three groups. Altogether, VHL alterations were significantly associated with pT3 CCRCCs (P = 0.009). This is the first evidence of frequent somatic VHL mutations at a particular site within exon 2 and an association of VHL mutations/hypermethylations with a standard prognostic factor.
Assuntos
Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Genes Supressores de Tumor , Neoplasias Renais/genética , Neoplasias Renais/patologia , Ligases , Mutação , Proteínas/genética , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Adenoma Oxífilo/genética , Adenoma Oxífilo/patologia , Substituição de Aminoácidos , Carcinoma de Células Renais/classificação , Metilação de DNA , DNA de Neoplasias/genética , Mutação da Fase de Leitura , Regulação Neoplásica da Expressão Gênica , Humanos , Rim/patologia , Neoplasias Renais/classificação , Estadiamento de Neoplasias , Mutação Puntual , Polimorfismo Conformacional de Fita Simples , Deleção de Sequência , Proteína Supressora de Tumor Von Hippel-LindauRESUMO
Hereditary papillary renal carcinoma (HPRC) is characterized by multiple, bilateral papillary renal carcinomas. Previously, we demonstrated missense mutations in the tyrosine kinase domain of the MET proto-oncogene in HPRC and a subset of sporadic papillary renal carcinomas. In this study, we screened a large panel of sporadic papillary renal carcinomas and various solid tumors for mutations in the MET proto-oncogene. Summarizing these and previous results, mutations of the MET proto-oncogene were detected in 17/129 sporadic papillary renal carcinomas but not in other solid tumors. We detected five novel missense mutations; three of five mutations were located in the ATP-binding region of the tyrosine kinase domain of MET. One novel mutation in MET, V1110I, was located at a codon homologous to an activating mutation in the c-erbB proto-oncogene. These mutations caused constitutive phosphorylation of MET when transfected into NIH3T3 cells. Molecular modeling studies suggest that these activating mutations interfere with the intrasteric mechanism of tyrosine kinase autoinhibition and facilitate transition to the active form of the MET kinase. The low frequency of MET mutations in noninherited papillary renal carcinomas (PRC) suggests that noninherited PRC may develop by a different mechanism than hereditary papillary renal carcinoma.
Assuntos
Carcinoma Papilar/genética , DNA de Neoplasias/genética , Neoplasias Renais/genética , Proteínas de Neoplasias/genética , Mutação Puntual , Proteínas Proto-Oncogênicas c-met/genética , Proto-Oncogenes , Células 3T3/metabolismo , Adenoma/genética , Adenoma/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Transformação Celular Neoplásica/genética , Códon/genética , Análise Mutacional de DNA , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Síndromes Neoplásicas Hereditárias/genética , Fosforilação , Conformação Proteica , Processamento de Proteína Pós-Traducional/genética , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-met/química , Proteínas Proto-Oncogênicas c-met/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
BACKGROUND: The development of renal cell carcinoma (RCC) has been associated with both genetic and environmental factors-with mutations in the von Hippel-Lindau (VHL) tumor suppressor gene for clear-cell RCC specifically and with long-term exposure to high doses of trichloroethylene (TRI), an industrially important solvent, for RCC generally. We investigated whether TRI exposure produces RCC through a specific mutational effect on the VHL gene by analyzing VHL sequences in the RCCs of patients exposed to high, cumulative doses of TRI. METHODS: The level of exposure for each of 44 patients with RCC who had known industrial exposure to TRI was classified according to the duration, frequency, and mode of exposure. Samples of normal and cancerous tissues were microdissected from paraffin-embedded tissue. DNA was isolated from these samples, and somatic VHL mutations were identified by polymerase chain reaction analysis, single-strand conformation polymorphism analysis, DNA sequencing, and restriction enzyme digestion. Control samples included RCC DNA from 107 patients without known TRI exposure and lymphocyte DNA from 97 healthy subjects. RESULTS: RCCs of TRI-exposed patients showed somatic VHL mutations in 33 (75%) of 44 cases. The mutations were frequently multiple and accompanied by loss of heterozygosity, and there was an association between the number of mutations and the severity of TRI exposure. We observed a specific mutational hot spot at VHL nucleotide 454 in the RCCs of 13 (39%) of the patients, and this mutation was present in adjacent non-neoplastic kidney parenchyma in four of these patients. The nucleotide 454 mutation was neither detected in any of the RCCs from patients without TRI exposure nor in any of the healthy subjects. CONCLUSION: Our results suggest that RCC in patients with high, cumulative TRI exposure is associated with a unique mutation pattern in the VHL gene.
Assuntos
Carcinoma de Células Renais/etiologia , Exposição Ambiental/efeitos adversos , Genes Supressores de Tumor/genética , Neoplasias Renais/etiologia , Mutação/efeitos dos fármacos , Solventes/efeitos adversos , Tricloroetileno/efeitos adversos , Doença de von Hippel-Lindau/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Renais/induzido quimicamente , Carcinoma de Células Renais/genética , Estudos de Casos e Controles , DNA de Neoplasias/genética , Feminino , Humanos , Neoplasias Renais/induzido quimicamente , Neoplasias Renais/genética , Perda de Heterozigosidade , Masculino , Pessoa de Meia-IdadeRESUMO
PURPOSE: Renal cell carcinoma occurs as a sporadic tumor but may be part of the autosomal dominant von Hippel-Lindau disease, characterized by retinal and central nervous system hemangioblastoma, pheochromocytoma, pancreatic cysts and renal cell carcinoma. We determine the prevalence of von Hippel-Lindau disease in a series of unselected renal cell carcinoma cases by molecular genetic analysis, and compare sporadic to von Hippel-Lindau renal cell carcinoma with respect to morphology and biology. MATERIALS AND METHODS: We established registers comprising 63 subjects with von Hippel-Lindau renal cell carcinoma, belonging to 30 distinct families (register A), and 460 unselected patients operated on for renal cell carcinoma in an 11-year period (register B). Molecular genetic analysis of the von Hippel-Lindau gene was performed for living patients of register A, representing 80% of von Hippel-Lindau families, and register B, 62% living patients, to identify von Hippel-Lindau germline mutations. In addition, register B was evaluated by a questionnaire (95% response) for familial occurrence of von Hippel-Lindau disease. RESULTS: The prevalence of von Hippel-Lindau renal cell carcinoma was 1.6% in 189 consenting unselected renal cell carcinoma patients. Risk factors for occult germline von Hippel-Lindau gene mutations in register B included familial renal cell carcinoma in 3 of 3 patients (100%), multifocal or bilateral renal cell carcinoma in 1 of 10 (10%) and age younger than 50 years at diagnosis in 1 of 33 (3%). Compared to sporadic von Hippel-Lindau renal cell carcinoma was characterized by an occurrence 25 years earlier, association with renal cysts, multifocal and bilateral tumors, cystic organization and low grade histology, and a better 10-year survival (p < 0.001 each). In von Hippel-Lindau disease metastases occurred only in tumors larger than 7 cm. CONCLUSIONS: von Hippel-Lindau differs from sporadic renal cell carcinoma in morphology and biology. Our data provide arguments for planning surgery for von Hippel-Lindau renal cell carcinoma and should stimulate future investigations.