JAK2 mutations are important criteria for the diagnosis of Philadelphia chromosome-negative myeloproliferative neoplasms. We aimed to assess JAK2 exon 14 and exon 12 mutations by high-resolution melting (HRM) analysis , which allows variation screening. The exon 14 analysis included 163 patients with polycythemia vera , secondary erythrocytoses , essential thrombocythemia , or secondary thrombocytoses, and 126 healthy subjects. The study of exon 12 included 40 JAK2 V617F-negative patients (nine of which had polycythemia vera , and 31 with splanchnic vein thrombosis) and 30 healthy subjects. HRM analyses of JAK2 exons 14 and 12 gave analytical sensitivities near 1% and both intra-and interday coefficients of variation of less than 1%. For HRM analysis of JAK2 exon 14 in polycythemia vera and essential thrombocythemia , clinical sensitivities were 93.5% and 67.9% , clinical specificities were 98.8% and 97.0% , positive predictive values were 93.5% and 79.2% , and negative predictive values were 98.8% and 94.6 , respectively. Correlations were observed between the results from HRM and three commonly used analytical methods. The JAK2 exon 12 HRM results agreed completely with those from sequencing analysis , and the three mutations in exon 12 were detected by both methods. Hence , HRM analysis of exons 14 and 12 in JAK2 shows better diagnostic values than three other routinely used methods against which it was compared. In addition , HRM analysis has the advantage of detecting unknown mutations. High resolution melting (HRM) analysis represents a new generation of mutation scanning technology. HRM molecular methods based on real-time PCR have recently been developed and allow screening of known and unknown mutations, including 1-bp substitutions. Furthermore, melting curve analysis with a high-resolution melting instrument is a simple, high performance, time saving, and low labor-intensive technique that is a sensitive and specific tool for detecting DNA variations.
Several sensitive methods for the detection of JAK2 V617F mutation have been published recently, most of them based on Real Time polymerase chain reaction (PCR). However, only some of them have performed studies of diagnostic validity. This study compares three methods based on Real Time PCR to detect JAK2 V617F mutation: two based on hybridization probes (HP) and peptide nucleic acid probe (PNA) and a third employing allele specific oligonucleotide primers for JAK2 V617F quantification. One hundred forty-nine healthy subjects, 61 essential thrombocythemia (ET), 32 polycythemia vera (PV), 38 secondary thrombocytoses, and 35 secondary erythrocytoses were included. Validity test study for JAK2 617 HP PCR in PV Sensitivity (Se) was 88% and in Specificity (Sp), 100%. In ET, Se was 57% and Sp, 100%. For JAK2 617 PNA PCR in PV, Se was 94% and Sp, 97.8%. In ET, Se was 70% and Sp, 95.7%. In JAK2 V671F allelo-specific-oligonucleotide (ASO) quantitative PCR (qPCR), cutoff point of 1% was established by receiving operating characteristic (ROC) curves. In PV, Se was 93.8% and Sp, 98.5%. In ET, Se was 80% and Sp, 95.9%. Two percent of the healthy subjects were positive by JAK2 617 PNA PCR and 2% by JAK2 617 ASO qPCR. JAK2 V617F mutation was detected in healthy subjects by cloning and sequencing. JAK2 617 HP is an adequate test in differential diagnosis for both erythrocytosis and thrombocytosis. When JAK2 V617F allele burden is low, JAK2 617 ASO qPCR should be performed. Simultaneous determination of JAK2 V617F and PRV-1 overexpression does not improve the diagnostic value of JAK2 V617F tests in MPD.
The aim of this study was to evaluate the biological correlation and prognostic impact of Gata-1, Gata-2, EKLF, and c-MPL transcript level in a group of 41 acute mieloid leukemia (AML) patients. Gata-1 overexpression was related to advanced age and a low percentage of bone marrow blasts and was associated with the expression of CD34 antigen and lymphoid T markers. The negative impact of Gata-1 expression on the probability of achieving complete remission has been confirmed. Gata-2 overexpression was associated with a low percentage of blasts in BM and males. Expression of c-MPL was associated with CD341 AML and M2 FAB AML subtype. A higher expression of EKLF was found in secondary AML versus primary AML. Nevertheless, patients expressing EKLF had a longer overall survival and event free survival than those patients that did not express EKLF. Our study has identified expression of EKLF as a factor with a favorable impact on prognosis in AML. Am. J. Hematol. 84:79-86, 2009. V V C 2008 Wiley-Liss, Inc. IntroductionBlood cells are derived from a small number of pluripotent hematopoietic stem cells (HSCs) endowed with the capacity for self-renewal [1]. Specific hematopoietic transcription factors are crucial for self renewal, choice of cell fate, and cell differentiation during normal hematopoiesis [2]. Acute myeloid leukemia (AML) is characterized by the uncontrolled proliferation of myeloid cells that accumulate at various stages of development, where their further differentiation appears to be blocked. Although disturbance of the expression of transcription factors regulating proliferation, survival, and differentiation of myeloid progenitors is almost certainly a requisite for the pathogenesis of AML, a direct link to myeloid leukemogenesis has been formally demonstrated in only a few transcription factors [2].In AML, disruptions of transcription factors themselves seem to be able to enhance and/or block proliferation of the leukemic cells [2-4] because of either the effect on the factors of fusion proteins generated by chromosomal translocations or mutations in the transcription factors.However, even in the absence of the alterations previously mentioned, deregulation of hematopoietic transcription factors is to be expected in AML cells. Recent studies have provided evidence that differential expression of a limited number of genes, that include certain transcription factors, can be useful to classify AML and are related to variations in patient survival [5][6][7][8].Relevant changes in megakaryocytic and erythroide linage are a feature of M6 and M7 AML, but changes in transcription factors involved in erythroid and megakaryocyte differentiation can also be expected in other AML subtypes. The expression of the transcription factors involved in differentiation of the common bipotent progenitor [9] could affect the clinical course and response to treatment of all types of AML.With the purpose of bringing forward information in the deregulation of normal hematopoetic factors in AML, expression of several transcript...
According to our results, an aberrant methylation pattern does not seem to play a crucial role in MPN pathogenesis; nor does it justify phenotypical differences between PV and ET.
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