Lymphotropic Viruses EBV, KSHV and HTLV in Latin America: Epidemiology and Associated Malignancies. A Literature-Based Study by the RIAL-CYTED.

The Epstein-Barr virus (EBV), Kaposi sarcoma herpesvirus (KSHV) and human T-lymphotropic virus (HTLV-1) are lymphomagenic viruses with region-specific induced morbidity. The RIAL-CYTED aims to increase the knowledge of lymphoma in Latin America (LA), and, as such, we systematically analyzed the literature to better understand our risk for virus-induced lymphoma. We observed that high endemicity regions for certain lymphomas, e.g., Mexico and Peru, have a high incidence of EBV-positive lymphomas of T/NK cell origin. Peru also carries the highest frequency of EBV-positive classical Hodgkin lymphoma (HL) and EBV-positive diffuse large B cell lymphoma, not otherwise specified (NOS), than any other LA country. Adult T cell lymphoma is endemic to the North of Brazil and Chile. While only few cases of KSHV-positive lymphomas were found, in spite of the close correlation of Kaposi sarcoma and the prevalence of pathogenic types of KSHV. Both EBV-associated HL and Burkitt lymphoma mainly affect young children, unlike in developed countries, in which adolescents and young adults are the most affected, correlating with an early EBV seroconversion for LA population despite of lack of infectious mononucleosis symptoms. High endemicity of KSHV and HTLV infection was observed among Amerindian populations, with differences between Amazonian and Andean populations.

Analysis of biological and technical variability in gene expression assays from formalin-fixed paraffin-embedded classical Hodgkin lymphomas.

Formalin-fixed paraffin-embedded (FFPE) tissues are invaluable sources of biological material for research and diagnostic purposes. In this study, we aimed to identify biological and technical variability in RT-qPCR TaqMan® assays performed with FFPE-RNA from lymph nodes of classical Hodgkin lymphoma samples. An ANOVA-nested 6-level design was employed to evaluate BCL2, CASP3, IRF4, LYZ and STAT1 gene expression. The most variable genes were CASP3 (low expression) and LYZ (high expression). Total variability decreased after normalization for all genes, except by LYZ. Genes with moderate and low expression were identified and suffered more the effects of the technical manipulation than high-expression genes. Pre-amplification was shown to introduce significant technical variability, which was partially alleviated by lowering to a half the amount of input RNA. Ct and Cy0 quantification methods, based on cycle-threshold and the kinetic of amplification curves, respectively, were compared. Cy0 method resulted in higher quantification values, leading to the decrease of total variability in CASP3 and LYZ genes. The mean individual noise was 0.45 (0.31 to 0.61 SD), indicating a variation of gene expression over ~1.5 folds from one case to another. We showed that total variability in RT-qPCR from FFPE-RNA is not higher than that reported for fresh complex tissues, and identified gene-, and expression level-sources of biological and technical variability, which can allow better strategies for designing RT-qPCR assays from highly degraded and inhibited samples.

Mecanismos genéticos e epigenéticos relacionados com a resposta e a resistência à terapia com inibidores tirosina quinase na leucemia mielóide crônica / [Genetic and epigenetic mechanisms related to response and resistance to tyrosine kinase inhibitor therapy in myeloid leukemia chronic]

O tratamento da leucemia mieloide crônica (LMC) foi radicalmente modificado com a introdução dos inibidores de tirosina quinase (ITK) na prática clínica. Entretanto, a resistência aos ITK é um problema clínico crescente que acomete 35% dos pacientes com LMC em fase crônica (FC). Dentre os mecanismos envolvidos na resistência, se destaca a presença de mutações no domínio quinase (DQ) da proteína BCR-ABL. Porém, estas mutações ocorrem em 30­60% dos pacientes resistentes, evidenciando a heterogenidade dos mecanismos subjacentes ao aparecimento da resistência. Processos epigenéticos, como a metilação do DNA, modificação de histonas e expressão de microRNAs (miRNAs) ainda não foram amplamente estudados em relação à resistência na LMC. Os miRNAs, pequenos RNAs que regulam a expressão gênica, têm surgido recentemente como um mecanismo epigenético importante na fisiologia do câncer. O objetivo principal deste trabalho foi analisar os padrões de resposta de um grupo de pacientes com LMC submetidos à terapia com Imatinibe (IM), visando identificar mecanismos genéticos e epigenéticos relacionados com a resposta e a resistência aos ITK. Para isto, foram estudados 264 pacientes com LMC tratados com IM por um tempo médio de 30 meses (6-123), com 70% dos casos utilizando o inibidor por >12 meses. As taxas de resposta foram de 76% de resposta citogenética completa (RCgC), 63% de resposta molecular maior (RMM) e 29% de resposta molecular completa (RMC) nos pacientes com LMC-FC. Foi identificado um grupo de respondedores tardios, que representam ~30% dos pacientes que não alcançaram uma RCgC e/ou RMM nos tempos ótimos após inicio do IM. Um grupo de 126 pacientes foi selecionado para pesquisa de mutações no DQ. A presença de mutações foi avaliada por sequenciamento direto, por um ensaio de PCR alelo específico (PCR-AS) para detecção da mutação T315I e a quantificação dos clones mutados foi realizada por pirosequenciamento. Dos casos analisados, 23% apresentaram mutações no DQ. As mutações mais frequentes foram T315I (20%) e F359I/V (11,4%). A quantificação realizada por piro-sequenciamento forneceu informações complementares relevantes sobre o papel do tamanho do clone mutado na resposta ou resistência. Em um grupo de 17 pacientes, selecionados de acordo à resposta obtida com IM, foi realizada uma análise do perfil de expressão de miRNAs maduros através de arrays de TLDA (do inglês TaqMan Low Density Array). Foi identificado um grupo de 4 miRNAs diferencialmente expressos ao diagnóstico quando comparados os grupos de pacientes com resposta ao IM vs resistentes ao IM: miR-155, -451, -18b, -125a-5p. Um grupo de 29 miRNAs apresentou um padrão similar de variação da expressão durante o tratamento. Os níveis de expressão dos miR-451 e miR-148a mostraram uma correlação inversa com a carga tumoral aferida pelos níveis do transcrito BCR-ABL. Esta correlação foi mais consistente para o miR-148a. As análises in silico para predição de alvos para o miR148a indicaram 6 alvos potencialmente relacionados com a LMC: os genes E2F3, PIK3R3, IKKα, IKKß, p27Kip e SHP-2. Foi comprovado que a expressão do miR-148a em linhagens celulares de LMC está regulada por metilação do DNA.

The significance of major and stable molecular responses in chronic myeloid leukemia in the tyrosine kinase inhibitor era

Tyrosine kinase inhibitors have changed the management and outcomes of chronic myeloid leukemia patients. Quantitative polymerase chain reaction is used to monitor molecular responses to tyrosine kinase inhibitors. Molecular monitoring represents the most sensitive tool to judge chronic myeloid leukemia disease course and allows early detection of relapse. Evidence of achieving molecular response is important for several reasons: 1. early molecular response is associated with major molecular response rates at 18-24 months; 2. patients achieving major molecular response are less likely to lose their complete cytogenetic response; 3. a durable, stable major molecular response is associated with increased progression-free survival. However, standardization of molecular techniques is still challenging.

Rapid and sensitive allele-specific (AS)-RT-PCR assay for detection of T315I mutation in chronic myeloid leukemia patients treated with tyrosine-kinase inhibitors.

Point mutations in the kinase domain of BCR-ABL were described in 40-90% of patients with chronic myeloid leukemia (CML) resistant to Imatinib. We herein describe the development of a rapid allele-specific (AS)-RT-PCR assay to identify the T315I mutation, which confers full resistance to all available tyrosine-kinase inhibitors (TKI). The mutation status of 65 patients with resistant CML was evaluated, and the T315I was detected in 3/65 (4.6%). Comparisons between sequencing and AS-RT-PCR results, as well as serial dilutions experiments proved that the method is specific and reproducible, with maximum sensitivity of 1 × 10(-3). The developed assay is a convenient and easy tool to be used in research of CML resistance for rapid mutation screening and, together with sequencing, may be included in efficient strategies for early detection of TKI resistance in patients with CML.

The significance of major and stable molecular responses in chronic myeloid leukemia in the tyrosine kinase inhibitor era.

Tyrosine kinase inhibitors have changed the management and outcomes of chronic myeloid leukemia patients. Quantitative polymerase chain reaction is used to monitor molecular responses to tyrosine kinase inhibitors. Molecular monitoring represents the most sensitive tool to judge chronic myeloid leukemia disease course and allows early detection of relapse. Evidence of achieving molecular response is important for several reasons: 1. early molecular response is associated with major molecular response rates at 18-24 months; 2. patients achieving major molecular response are less likely to lose their complete cytogenetic response; 3. a durable, stable major molecular response is associated with increased progression-free survival. However, standardization of molecular techniques is still challenging.

Mutations within the 5' region of FAS/CD95 gene in nodal diffuse large B-cell lymphoma.

CD95 is a cell-surface receptor that mediates apoptosis. A possible association between CD95 mutations and extranodal diffuse large B-cell lymphomas (DLBCL) has been reported. To further elucidate this question, a mutation analysis within the 5' region and exon 9 of CD95 was performed in a series of 66 DLBCL patients, by polymerase chain reaction, single-strand conformational polymorphism, and sequencing in all cases. Four mutations, all within the 5' region, were detected in three cases of primary nodal DLBCL (6.3% of primary DLBCL), probably originated as by-products of the somatic hypermutation process. No CD95 mutations in the two analyzed regions were detected in primary extranodal DLBCL, mediastinal large B-cell lymphoma (MLBCL), and DLBCL arising from indolent low-grade lymphomas. Because of our results, a review of published data with respect to the site of mutations was performed, which suggested a different distribution of mutations in nodal and extranodal DLBCL.