Study of genetic profile of patients with Philadelphia-negative Myeloproliferative Neoplasms (MPN)

As neoplasias mieloproliferativas (NMPs) Filadelfia negativo como a policitemia vera (PV), trombocitemia essencial (TE) e mielofibrose primária (MFP) são desordens clonais da célula tronco hematopoiética caracterizadas pela produção excessiva de células mielóides diferenciadas. Este fenômeno ocorre devido à uma mutação somática (JAK2V617F) que ativa a via JAK-STAT de transdução de forma constitutiva. Esta mutação é mais frequente na PV, ocorrendo em 95% dos casos, e em 50% dos casos de TE e MFP. Outro defeito genético que ocorre é a mutação no receptor de trombopoetina, MPL. As mutações em MPL podem ser germinativas ou somáticas e menos de 10% dos pacientes com TE e MFP apresentam essa alteração genética. Entretanto, grande parte dos pacientes com TE e MFP que não apresentam mutação em JAK2V617F ou MPL podem apresentar mutações somáticas no gene CALR. Em adição às mutações somáticas que causam mieloproliferação, outras alterações genéticas em genes que funcionam como reguladores epigenéticos são encontrados nas NMPs nos genes TET2, IDH1, IDH2 e ASLX1. Objetivo: Estabelecer um perfil genético em pacientes com NMP através da avaliação de mutações nos genes JAK2, CALR, MPL, IDH1, IDH2, TET2 e ASXL1 assim como estabelecer uma correlação laboratorial destas na PV, TE e MFP. Casuística e Métodos: Foram utilizadas amostras de sangue periférico de 104 pacientes que foram enviadas para o Laboratório de Biologia Tumoral do Serviço de Hematologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo para avaliação diagnóstica. Quinze dos 104 pacientes são de pacientes com PV (14,4%), 20/104 (19,2%) com MFP, 20/104 (19,2%) com TE e 49/104 (47,1%) com outras doenças hemtológicas. Foi feita a avaliação da prevalência de mutações somáticas, seja por sequenciamento ou análise de fragmentos, nos genes JAK2 (exon 12 e Y931C), MPL, IDH1, IDH2, CALR, TET2 e ASXL1. PCR-RFLP foi realizada para identificação de mutações em JAK2V617F. Resultados: A mutação JAK2V617F foi observada em 30 (28,8%) pacientes (12 PV, 11 TE e 7 MFP), a mutação JAK2 exon 12 foi observada em apenas um (0,96%) paciente com PV, mutação JAK2Y931C em 4 (3,8%) pacientes (1 PV, 2 TE e 1 MFP) e 8 (7,7%) pacientes apresentaram mutações em CALR (3 TE e 5 MFP). Mutações nos genes epigenéticos como IDH1 foram observadas em 9 (8,7%) pacientes (2 TE, 2 MFP, 1 SMD, e 4 pacientes com suspeita de NMP), mutações em IDH2 estão presentes em 5 (4,8%) pacientes (2 TE, 1 SMD/leucemia e 4 pacientes com suspeita de NMP), mutações em ASXL1 foram identificadas em 13 (12,5%) pacientes (1 PV, 3 TE, 2 MFP, 3 SMD/leucemias e 4 com suspeita de NMP) e finalmente, mutações em TET2 foram encontradas em 33 (31,7%) pacientes (3 PV, 5 TE, 4 MFP, 8 SMD/leucemias e 13 pacientes com suspeita de NMP). Além disso, no caso da PV, os pacientes que apresentam mutações em JAK2V617F apresentam valores aumentados de plaquetas (mediana de 5,41 x 105/mm3 plaquetas) em relação aos pacientes sem a mutação (mediana de 2,06 x 105/mm3 plaquetas), com diferença estatística (p=0,031). Pacientes do mesmo grupo que apresentam mutações em TET2 apresentam, opostamente aos com mutações em JAK2V617F, menores valores de plaquetas (mediana de 1,75 x105/mm3 plaquetas) em relação aos pacientes sem mutações no gene TET2 (mediana de 5,41 x 105/mm3 plaquetas), com diferença estatística (p=0,048). No caso da MFP, os pacientes que apresentam mutações em JAK2V617F apresentam valores maiores de leucócitos (mediana de 1,09 x104/mm3 leucócitos) do que os pacientes que não apresentam a mutação (mediana de 6,99 x103/mm3 leucócitos) com diferença estatística (p=0,046), já os pacientes que apresentam mutações no gene ASXL1 apresentam valores menores de hemácias (mediana de 2,43 x106/mm3 hemácias) em relação aos pacientes que não apresentam mutação (mediana de 3,71 x106/mm3 hemácias) com diferença estatística (p=0,042). Conclusão: O trabalho permitiu fornecer um perfil genético dos pacientes com NMP estudados. Além disso, é possível observar que algumas mutações epigenéticas podem influenciar em diferenças clínicasMyeloproliferative neoplasms (MPNs) Philadelphia (Ph) chromosome negative, such as polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are clonal disorders of hematopoietic stem cell characterized by increased proliferation of differentiated myeloid cells. This phenomenon occurs due somatic mutation (JAK2V617F) that constitutively stimulates the JAK-STAT signaling pathway. This mutation is more frequent in PV, around 95%, and between 50% in ET and PMF. Other genetic aberration can be observed in the thrombopoietin (TPO) receptor MPL. Mutations in MPL can be in the germline line or somatic and less than 10% of patients with TE or PMF would harbor this genetic alteration. Otherwise, patients with TE or PMF without JAK2V617F or MPL mutation could present somatic mutations in calreticulin (CALR). In addition to somatic mutations that cause myeloproliferation, other genetic alterations that function as epigenetic regulators were identified in genes as TET2, IDH1, IDH2 e ASLX1 in MPN. Objective: Establish genetic profile in patients with diagnosis of PV, ET, and PMF through genetic alterations in the following genes: JAK2, MPL, CALR, IDH1, IDH2, TET2 e ASXL1, and correlate those alterations with demographic characteristic of the study population. Casuistic and Methods: Peripheral blood samples from 104 patients referred to the Tumor Biology Laboratory of the Department of Hematology of Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo for diagnostic investigation were analyzed. Fifteen out 104 samples were from PV patients (14.4%), 20/104 (19.2%) in PMF, 20/104 (19.2%) in ET and 49/104 (47.1%) with other hematologic diseases. Identification of somatic mutations was made, either by direct sequencing or fragment analysis in JAK2 (exon 12 and Y931C), MPL, IDH1, IDH2, CALR, TET2 and ASXL1. PCR-RFLP was performed to identify JAK2V617F mutation. Results: JAK2V617F mutation was observed in 30 (28.8%) patients (12 PV, 11 ET and 7 PMF), JAK2 exon 12 in only one (0.96%) patient with PV, JAK2Y931C in 4 (3.8%) patients (1 PV, 2 ET and 1 PMF), and 8 patients (7.7%) presented CALR mutation (3 ET and 5 PMF). Mutations in the epigenetic genes as IDH1 were observed in 9 (8.7%) patients (2 ET, 2 PMF, 1 MDS and 4 patients with suspected MPN), IDH2 mutations were present in 5 (4.8%) patients (2 ET, 1 MDS/leukemia, and 4 patients with suspected MPN), ASLX1 mutations were identified in 13 (12.5%) patients (1 PV, 3 ET, 2 PMF, 3 MDS/leukemia and 4 with suspected MPN) and finally, TET2 mutations were present in 33 (31.7%) patients (3 PV, 5 ET, 4 PMF, 8 MDS/leukemia, and 13 with suspected MPN). In addition, patients with PV who harbor JAK2V617F have increased platelet counts (median 5.41 x 105/mm3 platelets) compared to those without the mutation (median 2.06 x 105/mm3 platelets, p=0.031). Patients in the same group with TET2 mutation, as opposed to those with JAK2V617F, presented low platelets counts (median of 1.75 x 105/mm3 platelets) compared to those without TET2 mutation (median 5.41 x 105/mm3 platelets, p=0.048). Presence of JAK2V617F in patients diagnosed with PMF have a greater number of leukocytes (median 1.09 x104/mm3 leukocytes) when compared to patients without the mutation (median 6.99 x 103/mm3 leukocytes, p=0.046). Patients with PMF who presented mutations in ASXL1 gene have a lower number of red blood cells (median of 2.43 x 106/mm3) compared to patients without mutations in the same gene (median 3.71 x 106/mm3, p=0.042). Conclusion: The present study allows us to provide a genetic profile of patients with MPN. Furthermore, it is possible to observe that some epigenetic mutations could influence in some clinical difference

Pretherapeutic Expression of the hOCT1 Gene Predicts a Complete Molecular Response to Imatinib Mesylate in Chronic-Phase Chronic Myeloid Leukemia

In this retrospective study we evaluated the pretherapeutic mRNA expression of the hOCT1 (human organic cation transporter 1) gene in patients with chronic-phase (CP) chronic myeloid leukemia (CML) who varied in terms of their response to imatinib (IM). hOCT1 mRNA was quantified by real-time PCR. Patients were classified as expressing either high (n = 44) or low hOCT1 mRNA (n = 44). the complete cytogenetic response rates observed at 6, 12 and 18 months were 47.7, 84.1 and 91%, respectively, in patients with high hOCT1 mRNA and 47.5, 81.8 and 86.3%, respectively, in patients with low hOCT1 transcripts. the major molecular response rates were not significantly different between patients with high and low hOCT1 mRNA after 6 months of therapy (22.7 vs. 9.1%; p = 0.07), but they were significantly different after 12 months (54.5 vs. 31.8%; p = 0.026) and 18 months (77.2 vs. 56.8%; p = 0.034). Complete molecular responses were observed in 5 patients with low and 17 patients with high hOCT1 mRNA (p = 0.003). the 5-year event-free and overall survival analyses revealed no significant differences between the groups. These data imply that knowledge of the pretherapeutic level of hOCT1 could be a useful marker to predict IM therapy outcome in treatment-naive CP CML patients. Copyright (C) 2012 S. Karger AG, BaselMaria Cecilia Souto Vidigal Foundation, São Paulo, BrazilUniv São Paulo, Dept Hematol, Sch Med, Tumor Biol Lab,Fac Med, BR-05403000 São Paulo, BrazilUniv São Paulo, Cytogenet Lab, Fac Med, BR-05403000 São Paulo, BrazilUniv São Paulo, Discipline Hematol, Fac Med, BR-05403000 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Translat Med, São Paulo, BrazilSão Paulo Inst Trop Med, São Paulo, BrazilSão Paulo Inst Canc, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Translat Med, São Paulo, BrazilWeb of Scienc

Comparative study of different methodologies to detect the JAK2 V617F mutation in chronic BCR-ABL1 negative myeloproliferative neoplasms

Objectives: A mutation in the JAK2 gene, V617F, has been identified in several BCR-ABL1 negative myeloproliferative neoplasms (MPN): polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Defining the presence or absence of this mutation is an essential part of clinical diagnostic algorithms and patient management. Here, we aimed to evaluate the performance of three PCR-based assays: Amplification Refractory Mutation System (ARMS), High-Resolution Melting analysis (HRM), and Sanger direct sequencing, and compare their results with those obtained by a PCR restriction fragment polymorphism assay (PCR-RFLP). Design and methods: We used blood samples from 136 patients (PV=20; PMF=20; ET=28, and other MPN suspected cases=68). Results: Comparable results were observed among the four assays in patients with PV, PMF, and MPN suspected cases. In patients with a diagnosis of ET, the JAK2 V617F mutation was detected in 67.8% of them by the PCR-ARMS and PCR-HRM assay and in 64% of them by the conventional Sanger sequence approach. The PCR-ARMS and PCR-HRM assays were 100% concordant. With these tests, only one of the 20 patients with ET and one of the three patients with clinically suspected MPN gave different results compared with those obtained by the PCR-RFLP. Conclusions: Our results have demonstrated that the PCR-ARMS and PCR-HRM assays could detect the JAK2 V617F mutation effectively in MPN patients, but PCR-HRM assays are rapid and the most cost-effective procedures. Keywords: Myeloproliferative, JAK2 V617F, Mutation, Wild type, Screenin