Improved Diagnosis of the Transition to JAK2V617F Homozygosity: The Key Feature for Predicting the Evolution of Myeloproliferative Neoplasms

Most cases of BCR-ABL1-negative myeloproliferative neoplasms (MPNs), essential thrombocythemia, polycythemia vera and primary myelofibrosis are associated with JAK2V617F mutations. The outcomes of these cases are critically influenced by the transition from JAK2V617F heterozygosity to homozygosity. Therefore, a technique providing an unbiased assessment of the critical allele burden, 50% JAK2V617F, is highly desirable. In this study, we present an approach to assess the JAK2V617F burden from genomic DNA (gDNA) and complementary DNA (cDNA) using one-plus-one template references for allele-specific quantitative-real-time-PCR (qPCR). Plasmidic gDNA and cDNA constructs encompassing one PCR template for JAK2V617F spaced from one template for JAK2Wild Type were constructed by multiple fusion PCR amplifications. Repeated assessments of the 50% JAK2V617F burden within the dynamic range of serial dilutions of gDNA and cDNA constructs resulted in 52.5364.2% and 51.4664.21%, respectively. The mutation-positive cutoff was estimated to be 3.65% (mean +2 standard deviation) using 20 samples from a healthy population. This qPCR approach was compared with the qualitative ARMS-PCR technique and with two standard methods based on qPCR, and highly significant correlations were obtained in all cases. qPCR assays were performed on paired gDNA/cDNA samples from 20 MPN patients, and the JAK2V617F expression showed a significant correlation with the allele burden. Our data demonstrate that the qPCR method using one-plus-one template references provides an improved assessment of the clinically relevant transition of JAK2V617F from heterozygosity to homozygosity.Fil: Gonzalez, Mariana Selena. Consejo Nacional de Invest.cientif.y Tecnicas. Instituto de Medicina Experimental;Fil: de Brasi, Carlos Daniel. Consejo Nacional de Invest.cientif.y Tecnicas. Instituto de Medicina Experimental;Fil: Bianchini, Michele. DTO. DE GENETICA;Fil: Gargallo, Patricia Martha. INST. DE INVEST. HEMATOLOGICAS;Fil: Stanganelli, Carmen Graciela. Academia Nacional de Medicina de Buenos Aires. Instituto de Invest. Hematologicas "mariano R. Castex";Fil: Zalcberg, Ilana. Molecular Biology, Laboratory, Instituto Nacional do Caˆncer, Rio de Janeiro, Brazil;Fil: Larripa, Irene Beatriz. Consejo Nacional de Invest.cientif.y Tecnicas. Instituto de Medicina Experimental

Linfoma burkitt-like em um lactente: relato de caso

Os linfomas não Hodgkin da infância, incluindo os linfomas de Burkitt e Burkitt-like são raros em lactentes. Um caso de linfoma não Hodgkin B em uma lactente de 13 meses de idade é descrito. Ao diagnóstico a paciente apresentava extenso comprometimento abdominal associado à ascite, derrame pleural e síndrome de lise tumoral. A análise imunofenotípica mostrou um fenótipo compatível com células linfóides oriundas do centro germinativo e a origem clonal dessas células foi demonstrada por reação em cadeia da polimerase. Não foi demonstrada associação do linfoma com infecção pelo vírus Epstein-Barr e/ou virus da imunodeficiência adquirida. O caso apresentado enfatiza a necessidade de considerar o diagnóstico de linfoma mesmo em lactentes.Childhood non-Hodgkin's lymphomas, including Burkitt and Burkitt-like, are rarely diagnosed in infants. A case of B-cell lymphoma in a 13-month-old girl with extensive abdominal disease, ascites, pleural effusion, and tumor lysis syndrome is reported. Phenotypic analysis showed a germinal center B-cell phenotype, and a B-cell clonality was confirmed by polymerase chain reaction. There was no evidence of Epstein-Barr and HIV infection. The case herein reported emphasizes the need for considering the diagnosis of lymphoma even in very young children

Epstein-Barr Virus (EBV) detection and typing by PCR: a contribution to diagnostic screening of EBV-positive Burkitt's lymphoma

BACKGROUND: Epstein-Barr virus (EBV) is associated to the etio-pathogenesis of an increasing number of tumors. Detection of EBV in pathology samples is relevant since its high prevalence in some cancers makes the virus a promising target of specific therapies. RNA in situ hybridization (RISH) is the standard diagnostic procedure, while polymerase chain reaction (PCR)-based methods are used for strain (EBV type-1 or 2) distinction. We performed a systematic comparison between RISH and PCR for EBV detection, in a group of childhood B-cell Non-Hodgkin lymphomas (NHL), aiming to validate PCR as a first, rapid method for the diagnosis of EBV-associated B-cell NHL. METHODS: EBV infection was investigated in formalin fixed paraffin-embedded tumor samples of 41 children with B-cell NHL, including 35 Burkitt's lymphoma (BL), from Rio de Janeiro, Brazil, by in situ hybridization of EBV-encoded small RNA (EBER-RISH) and PCR assays based on EBNA2 amplification. RESULTS: EBV genomes were detected in 68% of all NHL. Type 1 and 2 accounted for 80% and 20% of EBV infection, respectively. PCR and RISH were highly concordant (95%), as well as single- and nested-PCR results, allowing the use of a single PCR round for diagnostic purposes. PCR assays showed a sensitivity and specificity of 96% and 100%, respectively, with a detection level of 1 EBV genome in 5,000–10,000 EBV-negative cells, excluding the possibility of detecting low-number EBV-bearing memory cells. CONCLUSION: We describe adequate PCR conditions with similar sensitivity and reliability to RISH, to be used for EBV diagnostic screening in high grade B-NHL, in "at risk" geographic regions

Avaliação de risco em mioloma múltiplo: resultados preliminares do grupo brasileiro de estudos de mieloma

The Durie/Salmon staging system continues to be used worldwide in patients with multiple myeloma. However, in recent years, new systems have been proposed. The International Myeloma Working Group performed a retrospective study with 11,179 patients and proposed an "International Staging System" utilizing serum levels of â2 microglobulin and albumin. In addition, current research has focused on the usefulness of cytogenetic and molecular data as prognostic factors. These data suggest that these parameters are powerful discriminators of a poor prognostic group of myeloma patients. Indeed, these prognostic indexes have been utilized in clinical trials, with interesting and encouraging results.O esquema de Durie / Salmon continua a ser utilizado para estadiar os pacientes com mieloma múltiplo. Recentemente, um novo sistema mais simples e eficaz foi proposto. O "International Myeloma Working Group" realizou um estudo retrospectivo com 11.179 pacientes e a partir destes dados propôs a criação de um "International Staging System (ISS)" utilizando os níveis séricos de ß2 microglobulina e de albumina ao diagnóstico. Além do ISS a pesquisa está voltada para identificar alterações citogenéticas e moleculares que se correlacionem com o prognóstico no mieloma múltiplo. Estes fatores prognósticos têm sido utilizados para estratificar pacientes em ensaios clínicos com resultados promissores

Avaliação de risco em mioloma múltiplo: resultados preliminares do grupo brasileiro de estudos de mieloma

The Durie/Salmon staging system continues to be used worldwide in patients with multiple myeloma. However, in recent years, new systems have been proposed. The International Myeloma Working Group performed a retrospective study with 11,179 patients and proposed an "International Staging System" utilizing serum levels of â2 microglobulin and albumin. In addition, current research has focused on the usefulness of cytogenetic and molecular data as prognostic factors. These data suggest that these parameters are powerful discriminators of a poor prognostic group of myeloma patients. Indeed, these prognostic indexes have been utilized in clinical trials, with interesting and encouraging results30269sem informaçãoO esquema de Durie / Salmon continua a ser utilizado para estadiar os pacientes com mieloma múltiplo. Recentemente, um novo sistema mais simples e eficaz foi proposto. O "International Myeloma Working Group" realizou um estudo retrospectivo com 11.179 pacientes e a partir destes dados propôs a criação de um "International Staging System (ISS)" utilizando os níveis séricos de ß2 microglobulina e de albumina ao diagnóstico. Além do ISS a pesquisa está voltada para identificar alterações citogenéticas e moleculares que se correlacionem com o prognóstico no mieloma múltiplo. Estes fatores prognósticos têm sido utilizados para estratificar pacientes em ensaios clínicos com resultados promissoressem informaçã

Avaliação de risco em mioloma múltiplo: resultados preliminares do grupo brasileiro de estudos de mieloma

The Durie/Salmon staging system continues to be used worldwide in patients with multiple myeloma. However, in recent years, new systems have been proposed. The International Myeloma Working Group performed a retrospective study with 11,179 patients and proposed an "International Staging System" utilizing serum levels of â2 microglobulin and albumin. In addition, current research has focused on the usefulness of cytogenetic and molecular data as prognostic factors. These data suggest that these parameters are powerful discriminators of a poor prognostic group of myeloma patients. Indeed, these prognostic indexes have been utilized in clinical trials, with interesting and encouraging results.O esquema de Durie / Salmon continua a ser utilizado para estadiar os pacientes com mieloma múltiplo. Recentemente, um novo sistema mais simples e eficaz foi proposto. O "International Myeloma Working Group" realizou um estudo retrospectivo com 11.179 pacientes e a partir destes dados propôs a criação de um "International Staging System (ISS)" utilizando os níveis séricos de ß2 microglobulina e de albumina ao diagnóstico. Além do ISS a pesquisa está voltada para identificar alterações citogenéticas e moleculares que se correlacionem com o prognóstico no mieloma múltiplo. Estes fatores prognósticos têm sido utilizados para estratificar pacientes em ensaios clínicos com resultados promissores.30suppl 26

miRNome profiling of clonal stem cells in Ph+ CML

Chronic myeloid leukemia (CML) is a myeloid stem cell neoplasm characterized by an expansion of myeloid progenitor cells and the presence of BCR-ABL1 oncoprotein. Since the introduction of specific BCR-ABL1 tyrosine kinase inhibitors (TKI), overall survival has improved significantly. However, under long-term therapy patients may have residual disease that originates from TKI-resistant leukemic stem cells (LSC). In this work, we analyzed the miRNome of CML LSC, normal hematopoietic stem cells (HSC) obtained from the same CML patients, and stem and progenitor cells obtained from healthy donors (HD) by next-generation sequencing. We detected a global decrease of microRNA levels in LSC and HSC from CML patients, and decreased levels of microRNAs and snoRNAs from a genomic cluster in chromosome 14, suggesting a mechanism of silencing of multiple non-coding RNAs. Surprisingly, HSC from CML patients, despite the absence of BCR-ABL1 expression, showed an altered miRNome. In silico analysis revealed an association between validated microRNAs and multiple metabolic pathways, suggesting that these molecules may be mediators of the previously reported dysregulation of LSC metabolism. This is the first report of the LSC miRNome that distinguishes between BCR-ABL1+ LSC and their BCR-ABL1- counterparts, providing valuable data for future studies.Fil: Ruiz, María Sol. Fundación Cáncer. Centro de Investigaciones Oncológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sánchez, María Belén. Fundación Cáncer. Centro de Investigaciones Oncológicas; ArgentinaFil: Bonecker, Simone. Instituto Nacional de Câncer; BrasilFil: Furtado, Carolina. Instituto Nacional de Câncer; BrasilFil: Koile, Daniel Isaac. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Yankilevich, Patricio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Cranco, Santiago. Instituto Alexander Fleming; ArgentinaFil: Custidiano, María del Rosario. Instituto Alexander Fleming; ArgentinaFil: Freitas, Josefina. Hospital Nacional Profesor Alejandro Posadas; ArgentinaFil: Moiraghi, Beatriz. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Pérez, Mariel Ana. Gobierno de la Provincia de Buenos Aires. Ministerio de Salud. Hospital Interzonal General de Agudos "prof. Dr. Rodolfo Rossi".; ArgentinaFil: Pavlovsky, Carolina. Fundación Para Combatir la Leucemia; ArgentinaFil: Varela, Ana Inés. Gobierno de la Ciudad de Buenos Aires. Hospital General de Agudos "Ramos Mejía"; ArgentinaFil: Ventriglia, Verónica. Hospital Nacional Profesor Alejandro Posadas; ArgentinaFil: Sánchez Ávalos, Julio César Américo. Instituto Alexander Fleming; ArgentinaFil: Larripa, Irene Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Zalcberg, Ilana. Instituto Nacional de Câncer; BrasilFil: Mordoh, Jose. Fundación Cáncer; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Valent, Peter. Medical University of Vienna; AustriaFil: Bianchini, Michele. Fundación Cáncer; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

TET2 mutations as a part of DNA dioxygenase deficiency in myelodysplastic syndromes

Decrease in DNA dioxygenase activity generated by TET2 gene family is crucial in myelodysplastic syndromes (MDS). The general downregulation of 5-hydroxymethylcytosine (5-hmC) argues for a role of DNA demethylation in MDS beyond TET2 mutations, which albeit frequent, do not convey any prognostic significance. We investigated TETs expression to identify factors which can modulate the impact of mutations and thus 5-hmC levels on clinical phenotypes and prognosis of MDS patients. DNA/RNA-sequencing and 5-hmC data were collected from 1665 patients with MDS and 91 controls. Irrespective of mutations, a significant fraction of MDS patients exhibited lower TET2 expression, whereas 5-hmC levels were not uniformly decreased. In searching for factors explaining compensatory mechanisms, we discovered that TET3 was upregulated in MDS and inversely correlated with TET2 expression in wild type cases. Although TET2 was reduced across all age groups, TET3 levels were increased in a likely feedback mechanism induced by TET2 dysfunction. This inverse relationship of TET2 and TET3 expression also corresponded to the expression of L-2-hydroxyglutarate dehydrogenase, involved in agonist/antagonist substrate metabolism. Importantly, elevated TET3 levels influ-enced the clinical phenotype of TET2 deficiency whereby the lack of compensation by TET3 (low TET3 expression) was associated with poor outcomes of TET2 mutant carriers