HCV inter-subtype 1a/1b recombinant detected by complete-genome next-generation sequencing.

Next-generation sequencing (NGS) provides a practical approach to HCV complete-genome sequencing, detecting low-frequency variants and allowing analysis of viral genetic diversity (quasispecies) in the sample, and so far, it is very useful for identifying preexisting drug-resistant mutants and emerging escape mutations, as well as detecting viral recombinants containing genomic regions from different genotypes and subtypes. The aim of this study was to analyze the complete coding region of hepatitis C virus (HCV) genotype 1 (subtypes 1a and 1b) from patients with chronic infection who were direct-acting antiviral (DAA) naïve. Next-generation sequencing (Ion Torrent™ PGM) was used to determine the sequence of the complete coding region of 100 HCV-monoinfected DAA-naïve patients (51 and 49 subtypes 1a and 1b, respectively). We report the first description of nearly complete HCV genome sequences of subtype 1a and 1b isolates from a large population of Brazilian patients with chronic hepatitis C, and HCV-1a grouped in two different clades. Using this methodology, an inter-subtype 1a/1b recombinant was identified in this study.

A Novel Assay for the Identification of NOTCH1 PEST Domain Mutations in Chronic Lymphocytic Leukemia.

Aims. To develop a fast and robust DNA-based assay to detect insertions and deletions mutations in exon 34 that encodes the PEST domain of NOTCH1 in order to evaluate patients with chronic lymphocytic leukemia (CLL). Methods. We designed a multiplexed allele-specific polymerase chain reaction (PCR) combined with a fragment analysis assay to detect specifically the mutation c.7544_7545delCT and possibly other insertions and deletions in exon 34 of NOTCH1. Results. We evaluated our assay in peripheral blood samples from two cohorts of patients with CLL. The frequency of NOTCH1 mutations was 8.4% in the first cohort of 71 unselected CLL patients. We then evaluated a second cohort of 26 CLL patients with known cytogenetic abnormalities that were enriched for patients with trisomy 12. NOTCH1 mutations were detected in 43.7% of the patients with trisomy 12. Conclusions. We have developed a fast and robust assay combining allele-specific PCR and fragment analysis able to detect NOTCH1 PEST domain insertions and deletions.

Improving the diagnosis of meningitis due to enterovirus and herpes simplex virus I and II in a tertiary care hospital.

BACKGROUND: Enterovirus and herpes simplex viruses are common causes of lymphocytic meningitis. The purpose of this study was to analyse the impact of the use molecular testing for Enteroviruses and Herpes simplex viruses I and II in all suspected cases of viral meningitis. METHODS: From November 18, 2008 to November 17, 2009 (phase II, intervention), all patients admitted with suspected viral meningitis (with pleocytosis) had a CSF sample tested using a nucleic acid amplification test (NAAT). Data collected during this period were compared to those from the previous one-year period, i.e. November 18, 2007 to November 17, 2008 (phase I, observational), when such tests were available but not routinely used. RESULTS: In total, 2,536 CSF samples were assessed, of which 1,264 were from phase I, and 1,272 from phase II. Of this total, a NAAT for Enterovirus was ordered in 123 cases during phase I (9.7% of the total phase I sample) and in 221 cases in phase II (17.4% of the total phase II sample). From these, Enterovirus was confirmed in 35 (28.5%, 35/123) patients during phase I and 71 (32.1%, 71/221) patients during phase II (p = 0.107). The rate of diagnosis of meningitis by HSV I and II did not differ between the groups (13 patients, 6.5% in phase I and 13, 4.7% in phase II) (p = 1.0), from 200 cases in phase I and 274 cases in phase II. CONCLUSIONS: The number of cases diagnosed with enteroviral meningitis increased during the course of this study, leading us to believe that the strategy of performing NAAT for Enterovirus on every CSF sample with pleocytosis is fully justified.

Detecção de mutações no gene KIT em leucemia mieloide aguda / The detection of KIT mutations in acute myeloid leukemia

OBJETIVO: Descrever a metodologia para detecção de mutações nos éxons 8 e 17 do gene KIT em pacientes portadores de leucemia mieloide aguda, para implementação desse teste no laboratório clínico do Hospital Israelita Albert Einstein. MÉTODOS: Extração do DNA genômico de 54 amostras de sangue periférico ou medula óssea de pacientes com leucemia mieloide aguda para amplificação, por reação em cadeia da polimerase, sequenciamento e análise de fragmentos. RESULTADOS: Dentre as amostras analisadas, quatro apresentaram mutação no éxon 8, duas no éxon 17 e uma amostra apresentou mutação nos dois éxons. CONCLUSÃO: A pesquisa de mutação nos éxons 8 e 17 do gene KIT foi padronizada com sucesso e o teste está em processo de inclusão no menu de exames do laboratório clínico do Hospital Israelita Albert Einstein.

OBJECTIVE: This study describes a new method used in the clinical laboratory at Hospital Israelita Albert Einstein to detect mutations in exons 8 and 17 of the KIT gene in patients with acute myeloid leukemia. METHODS: Genomic DNA extraction was performed on 54 samples of peripheral blood or bone marrow from patients with acute myeloid leukemia. The extracted DNA was amplified by polymerase chain reaction and sequenced, and the fragments were analyzed. RESULTS: Within the analyzed samples, we detected four mutations in exon 8, two mutations in exon 17, and mutations or a double mutation in one sample. CONCLUSION: The tests detecting mutations in exon 8 and 17 on the KIT gene were successfully standardized. The test is now included among the routine diagnostics employed for patients at Hospital Israelita Albert Einstein clinical laboratory.

The detection of KIT mutations in acute myeloid leukemia.

OBJECTIVE: This study describes a new method used in the clinical laboratory at Hospital Israelita Albert Einstein to detect mutations in exons 8 and 17 of the KIT gene in patients with acute myeloid leukemia. METHODS: Genomic DNA extraction was performed on 54 samples of peripheral blood or bone marrow from patients with acute myeloid leukemia. The extracted DNA was amplified by polymerase chain reaction and sequenced, and the fragments were analyzed. RESULTS: Within the analyzed samples, we detected four mutations in exon 8, two mutations in exon 17, and mutations or a double mutation in one sample. CONCLUSION: The tests detecting mutations in exon 8 and 17 on the KIT gene were successfully standardized. The test is now included among the routine diagnostics employed for patients at Hospital Israelita Albert Einstein clinical laboratory.

Prognostication of soft tissue sarcomas based on chromosome 17q gene and protein status: evaluation of TOP2A, HER-2/neu, and survivin.

BACKGROUND: Topoisomerase 2 alpha (TOP2A), HER-2/neu, and survivin are genes that lie on chromosome 17 and correlate with the prognosis and prediction of target-driven therapy against tumors. In a previous study, we showed that TOP2A transcripts levels were significantly higher in soft tissue sarcomas (STS) than in benign tumors and desmoid-type fibromatoses (FM). Because these genes have been insufficiently examined in STS, we aimed to identify alterations in TOP2A and HER-2 expression by fluorescent in situ hybridization and immunohistochemistry, as well as that of survivin, and correlate them with clinicopathologic findings to assess their prognostic value. METHODS: Eighteen FM and 244 STS were included. Fluorescent in situ hybridization and immunohistochemistry were performed on a tissue microarray. RESULTS: TOP2A and survivin were more highly expressed in sarcomas than in FM. TOP2A was an independent predictor of an unfavorable prognosis; it was combined with formerly established prognostic factors (primarily histologic grade and tumor size at diagnosis) to create a prognostic index that evaluated overall survival. Gene amplification/polysomy (13%) did not correlate with protein overexpression. Survivin and HER-2 expression were not associated with patient outcomes. CONCLUSIONS: These findings might become valuable in the management of patients with STS and possibly in the prospective evaluation of responses to new target-driven therapies.

Identification of genes associated with local aggressiveness and metastatic behavior in soft tissue tumors.

Soft tissue tumors represent a group of neoplasia with different histologic and biological presentations varying from benign, locally confined to very aggressive and metastatic tumors. The molecular mechanisms responsible for such differences are still unknown. The understanding of these molecular alterations mechanism will be critical to discriminate patients who need systemic treatment from those that can be treated only locally and could also guide the development of new drugs' against this tumors. Using 102 tumor samples representing a large spectrum of these tumors, we performed expression profiling and defined differentially expression genes that are likely to be involved in tumors that are locally aggressive and in tumors with metastatic potential. We described a set of 12 genes (SNRPD3, MEGF9, SPTAN-1, AFAP1L2, ENDOD1, SERPIN5, ZWINTAS, TOP2A, UBE2C, ABCF1, MCM2, and ARL6IP5) showing opposite expression when these two conditions were compared. These genes are mainly related to cell-cell and cell-extracellular matrix interactions and cell proliferation and might represent helpful tools for a more precise classification and diagnosis as well as potential drug targets.

Análise do perfil de expressão gênica de lesões melanocíticas / Analysis of gene expression profile of melanocytic lesions

O melanoma cutâneo surge da transformação maligna dos melanócitos. Embora esse tumor seja curável por cirurgia quando o diagnóstico é precoce, o prognóstico de melanoma avançado é baixo em função do alto potencial metastático e falha ao tratamento clínico. O modelo de progressão tumoral sugere uma seqüência de passos: nevo comum, nevo displásico, melanoma primário em fase de crescimento radial, melanoma em fase de crescimento vertical, e melanoma metastático. ... Utilizando a técnica de microarranjos de cDNA, fizemos uma análise sistemática dos genes expressos por 23 amostras de nevos (intradérmico e composto) e 18 melanomas. Nossos resultados mostraram diferenças substanciais na expressão gênica global entre nevos e melanomas, visto que as amostras puderam ser separadas por métodos de agrupamento não-supervisionado. Ainda, identificamos 510 trios classificadores capazes de distinguir amostras com 100% de precisão. Treze módulos funcionais mostraram alteração com significância estatística entre nevo e melanoma. Dentre eles, módulos correspondendo a genes envolvidos com junção intercelular, comunicação e adesão celular reforçam a importância da comunicação celular de melanócitos em seu microambiente. Diversos genes destes módulos foram estudados. Desmocolina 3 e claudina 4, com expressão diminuída em lesões neoplásicas foram validados por RT-PCR. Kalikreína 7 e membros da família das claudinas (claudinas 7 e 11) foram avaliadas por imuno-histoquímica e tiveram expressão diminuída em melanomas. Em resumo, nossos dados apontam diferenças moleculares entre nevo benigno e melanoma primário, particularmente em módulos de adesão e comunicação celular que poderão melhorar o entendimento da biologia dos nevos e melanomas.

Cutaneous melanoma arises from the malignant transformation of melanocytes. Although melanoma is curable by surgery when diagnosis occurs during its early stages, the prognosis of advanced melanoma is poor due to the high metastatic potential and failure to clinical treatment. A sequence of steps for tumor progression has been proposed: commom nevi, dysplastic nevi, RGP (radial growth phase) and VGP (vertical growth phase) primary melanomas, and metastatic melanomas. Nevi are benign lesions composed by melanocytes with focal proliferation, organized in nests and temporally restricted in their growth.They are both precursors and markers for melanoma. Thus, by establishing the molecular profile of nevi, and comparing it to those of melanomas, we may be able to both improve our knowledge on the biology of nevi and identify genes involved in the early steps of tumor progression. We performed a systematic analysis of genes expressed by 23 nevi samples (intradermal and compound) and 18 melanoma samples using cDNA microarray technology. Our results showed substantial differences in global gene expression between nevi and melanomas, as samples were precisely grouped by non-supervised clustering methods. Moreover, we identified 510 molecular classifiers able to distinguish samples with 100% efficiency. Thirteen functional modules showed alterations with statistical significance between nevi and melanoma. Among them were the modules corresponding to genes involved with intercellular junction, cell communication and cell adhesion, highlighting the importance of cellular communication of melanocytes in their microenvironment. Several genes from these modules were further studied. Desmocollin 3 and Claudin 4 were investigated by RT-PCR, and were downregulated in neoplastic lesions. Kalikrein 7 and members of the claudin family (claudin 7 and 11) had decreased expression at the protein level, as observed using immunohistochemistry. In summary, our data pointed to molecular differences between benign nevus and primary melanoma, particularly in modules of cell adhesion and cell communication that could help in the better understanding of the biology of nevi and melanomas. Financial support: CNPq and CEPID/FAPESP (AU)