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[Molecular analyses for (early) recognition of hematologic diseases - sense and sensibility for molecular analysis: the art of intelligent decision-making]
No full textDuring the last 10 years several molecular markers have been established as useful tools among the armamentarium of a hematologist. As a consequence, the number of performed hematologic molecular analyses has immensely increased. Often, such tests replace or complement other laboratory methods. Molecular markers can be useful in many ways: they can serve for diagnostics, describe the prognostic profile, predict which types of drugs are indicated, and can be used for the therapeutic monitoring of the patient to indicate an adequate response or predict resistance or relapse of the disease. Many markers fulfill more than one of these aspects. Most important, however, is the right choice of analyses at the right time-points
Coincidence of 5q deletion and the JAK2V617F mutation: report of two patients with overlapping myelodysplastic and myeloproliferative features and review of the literature.
No full textRapid and highly specific screening for NPM1 mutations in acute myeloid leukemia
Get PDFNPM1 mutations, the most frequent molecular alterations in acute myeloid leukemia (AML), have become important for risk stratification and treatment decisions for patients with normal karyotype AML. Rapid screening for NPM1 mutations should be available shortly after diagnosis. Several methods for detecting NPM1 mutations have been described, most of which are technically challenging and require additional laboratory equipment. We developed and validated an assay that allows specific, rapid, and simple screening for NPM1 mutations. FAST PCR spanning exons 8 to 12 of the NPM1 gene was performed on 284 diagnostic AML samples. PCR products were visualized on a 2 % agarose E-gel and verified by direct sequencing. The FAST PCR screening method showed a specificity and sensitivity of 100 %, i.e., all mutated cases were detected, and none of negative cases carried mutations. The limit of detection was at 5-10 % of mutant alleles. We conclude that the FAST PCR assay is a highly specific, rapid (less than 2 h), and sensitive screening method for the detection of NPM1 mutations. Moreover, this method is inexpensive and can easily be integrated in the routine molecular diagnostic work-up of established risk factors in AML using standard laboratory equipment
Hematopoietic and endothelial progenitor cell trafficking in patients with myeloproliferative diseases
No full textBACKGROUND AND OBJECTIVES. The presence of circulating hematopoietic progenitor cells in patients with myeloproliferative diseases (MPD) has been described. However, the exact nature of such progenitor cells has not been specified until now. The aim of this work was to investigate the presence of endothelial precursor cells in the blood of patients with MPD and to assess the role of the endothelial cell lineage in the pathophysiology of this disease. DESIGN AND METHODS. Endothelial progenitor cell marker expression (CD34, prominin (CD133), kinase insert domain receptor (KDR) or vascular endothelial growth factor receptor 2 (VEGFR2), and von Willebrand factor) was assessed in the blood of 53 patients with MPD by quantitative polymerase chain reaction. Clonogenic stem cell assays were performed with progenitor cells and monocytes to assess differentiation towards the endothelial cell lineage. The patients' were divided according to whether they had essential thrombocythemia (ET, n=17), polycythemia vera (PV, n=21) or chronic idiopathic myelofibrosis (CIMF, n=15) and their data compared with data from normal controls (n=16) and patients with secondary thrombo- or erythrocytosis (n=17). RESULTS. Trafficking of CD34-positive cells was increased above the physiological level in 4/17 patients with ET, 5/21 patients with PV and 13/15 patients with CIMF. A subset of patients with CIMF co-expressed the markers CD34, prominin (CD133) and KDR, suggesting the presence of endothelial precursors among the circulating progenitor cells. Clonogenic stem cell assays confirmed differentiation towards both the hematopoietic and the endothelial cell lineage in 5/10 patients with CIMF. Furthermore, the molecular markers trisomy 8 and JAK2 V617F were found in the grown endothelial cells of patients positive for trisomy 8 or JAK2 V617F in the peripheral blood, confirming the common clonal origin of both hematopoietic and endothelial cell lineages. INTERPRETATION AND CONCLUSIONS. Endothelial precursor cells are increased in the blood of a subset of patients with CIMF, and peripheral endothelial cells bear the same molecular markers as hematopoietic cells, suggesting a primary role of pathological endothelial cells in this disease
Imetelstat inhibits growth of megakaryocyte colony-forming units from patients with essential thrombocythemia.
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Progenitor cell trafficking in physiologic conditions and in myeloproliferative diseases: quantification of CD34+ cells by polymerase chain reaction.
No full textBACKGROUND AND OBJECTIVES
Previous studies using flow cytometry have shown that CD34+ cell trafficking is increased in patients with chronic idiopathic myelofibrosis. Few data exist on physiologic CD34 + cell trafficking and the quantification of very low cell ranges requires reliable and sensitive measurement techniques. The aim of this study was to establish a quantitative polymerase chain reaction (PCR) technique for studying CD34+ cell trafficking in physiologic conditions, and in patients with myeloproliferative diseases.
DESIGN AND METHODS
CD34+ cell trafficking was measured in 56 controls [(healthy controls (n=21), patients with ischemic cardiopathy (n=21), patients with secondary thrombocytosis or erythrocytosis (n=14)], and in 37 untreated patients with myeloproliferative diseases diagnosed according to the WHO-criteria [(essential thrombocythemia (n=10), polycythemia vera (n=14) and chronic idiopathic myelofibrosis (n=13)]. Quantitative PCR was used to determine CD34 mRNA expression in peripheral blood samples.
RESULTS
Physiologic CD34 mRNA expression ranges were determined in the healthy control group. Mean CD34 mRNA expression was within the physiologic range in patients with ischemic cardiopathy, secondary thrombocytosis or erythrocytosis, essential thrombocythemia and polycythemia vera (p=0.146), but was significantly increased in patients with chronic idiopathic myelofibrosis (p<0.001). When analyzed individually, 12/13 patients with chronic idiopathic myelofibrosis and 3/14 patients with polycythemia vera showed CD34 mRNA expression above the physiologic range.
INTERPRETATION AND CONCLUSIONS
This is a first report about CD34+ cell trafficking measured by quantitative PCR. Quantitative PCR is a reliable method suitable for the quantification of very low cell populations. Our study confirms the significant increase of CD34+ cell trafficking in patients with chronic idiopathic myelofibrosis, and in a subset of patients with polycythemia vera. Prospective studies are underway to characterize these circulating CD34+ cells and to investigate their role in the pathophysiology of myeloproliferative diseases
Molekulares BCR-ABL1-Monitoring bei CML gemäss neuen ELN-Empfehlungen. Internationale Skala in der Schweiz etabliert
No full textCLLU1 expression distinguishes chronic lymphocytic leukemia from other mature B-cell neoplasms
No full textThe distinction of CLL from other mature B-cell neoplasms, especially from leukemic forms of mantle cell lymphoma or splenic marginal zone lymphoma, can be difficult but has important prognostic and therapeutic implications. We measured CLLU1 (CLL upregulated gene1) mRNA by qPCR and found a highly significant difference between CLL and other lymphoid neoplasms (AUC 0.96, 95%CI 0.93-0.99). Based on our cut-off values we can predict CLL and other mature B-cell neoplasms with high probability (PPV 99% and 94%). Analysis of CLLU1 expression is a rapid and reliable tool that may facilitate the diagnosis of mature B-cell neoplasms especially in inconclusive cases
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