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999 20 Rar [BEST]

Warranty The warranty for new as well as unworn and used items is 24 months from delivery of the goods to the buyer. Claims for damages are excluded from this and are governed by the statutory provisions. Otherwise, the claims of the buyer against the seller for defects are governed by the statutory provisions.

999 20 rar

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JAK2 V617F mutation recently was identified as a pathogenic factor in typical chronic myeloproliferative diseases (CMPD). Some forms of myelodysplastic syndromes (MDS) show a significant overlap with CMPD (classified as MDS/MPD), but the diagnostic assignment may be challenging. We studied blood or bone marrow from 270 patients with MDS, MDS/MPD, and CMPD for the presence of JAK2 V617F mutation using polymerase chain reaction, sequencing, and melting curve analysis. The detection rate of JAK2 V617F mutants for polycythemia vera, chronic idiopathic myelofibrosis, and essential thrombocythemia (n = 103) was similar to the previously reported results. In typical forms of MDS (n = 89) JAK2 V617F mutation was very rare (n = 2). However, a higher prevalence of this mutation was found in patients with MDS/MPD-U (9 of 35). Within this group, most of the patients harboring JAK2 V617F mutation showed features consistent with the provisional MDS/MPD-U entity refractory anemia with ringed sideroblasts and thrombocytosis (RARS-T). Among 9 RARS-T patients, 6 showed the presence of JAK2 V617F mutation, and in 1 patient without mutation, aberrant, positive phospho-STAT5 staining was seen that is typically present in association with JAK2 V617F mutation. In summary, we found that RARS-T reveals a high frequency of JAK2 V617F mutation and likely constitutes another JAK2 mutation-associated form of CMPD.

Biomarkers offer the potential for determining whether the MDS/MPD entities are more appropriately classified separately or have a pathogenesis more closely related to either MDS or CMPD. Unfortunately, cytogenetic analysis does not resolve this issue, since many of the typical chromosome abnormalities associated with MDS, such as trisomy 8, del(13q), and del(20q) also can be found in CMPD.1 Recently, a point mutation in DNA coding for the Jak homology domain 2 (JH2) pseudokinase domain of Janus kinase 2 (JAK2) has been found in a significant proportion of patients with CMPD, including polycythemia vera (PV), essential thrombocythemia (ET), and chronic idiopathic myelofibrosis (CIMF).2-11 This G-to-T transversion at nucleotide 1849 results in substitution of phenylalanine for valine at codon 617 (JAK2 V617F; Gene Bank accession NM_004972), leading to constitutive activation of the JAK2 tyrosine kinase. As shown in transfection experiments using a murine bone marrow transplant model, expression of the JAK2 V617F kinase is responsible for the evolution of an erythrocytosis in recipient mice due to a defective hematopoietic clone.6 Interestingly, JAK2 V617F mutants have been found only rarely in patients with MDS, including refractory anemias and cytopenias.12-14

The relatively close association of the JAK2 V617F mutation with certain CMPD could provide insight into how the MDS/MPD entities are related to either MDS or CMPD. When a large cohort of patients with CMML was examined for the presence of the JAK2 V617F mutation, this defect was found only in a small proportion of patients,12,13,15 suggesting that CMML is less biologically related to CMPD.

We have created a high throughput molecular assay allowing for rapid detection of JAK2 V617F mutants and studied patients with typical MDS and MDS/MPD to determine the frequency of JAK2 V617F mutations in these disorders. We focused on a cohort of patients with RARS-T to determine whether JAK2 V617F mutation status might suggest a pathogenesis more closely related to CMPD than to MDS.

Informed consent was obtained in accordance with the Declaration of Helsinki for sample collection from patients and controls according to protocols approved by the Institutional Review Board of the Cleveland Clinic Foundation. The MDS patient characteristics are presented in Table 1. They were classified according to the WHO1,16,19 and were also assigned an International Prognosis Scoring System score.20 Hematologic controls included cohort of patients with CMPD (Table 1).

DNA was extracted either from fresh bone marrow, peripheral blood, or paraffin-embedded bone marrow aspirate tissue. Total blood was subjected to density centrifugation with Lymphocyte Separation Medium (Mediatech, Manassas, VA). The granulocyte layer was collected, and residual erythrocytes were removed by osmotic lysis using 0.2% NaCl for 30 seconds.21 The Genomic DNA Purification Kit (Gentra Systems, Minneapolis, MN) was used for DNA isolation. When necessary, DNA was obtained from paraffin tissue blocks as follows: cells were lysed in buffer (50 mM Tris [tris(hydroxymethyl)aminomethane]*Cl, 1 mM EDTA [ethylenediaminetetraacetic acid], and 0.5% Tween-20, pH 8.6) following deparaffinization and proteinase K digestion. DNA was extracted with phenol/chloroform and precipitated with sodium acetate/ethanol.

40 ng of DNA was amplified using primers previously published.3 Amplicons were generated in 25 μL reaction volume with 120 nM forward primers and 120 nM reverse primer, 1 PCR buffer without MgCl2, 0.8 mM dNTPs, 1.8 mM MgCl2, and Taq DNA polymerase (Invitrogen, Carlsbad, CA). PCR conditions were: initial denaturation at 94C for 4 minutes, 30 cycles with denaturation at 94C for 30 seconds, annealing at 58C for 30 seconds, and elongation at 72C for 40 seconds.

Images shown in Figures 4 and 5 were obtained via digital microscopy using an Olympus BX51 microscope (Olympus America, Melville, NY) equipped with either a UPlanFl 40/0.75 numeric aperture (NA) or a UPlanFl 100/1.30 NA objective. Images were captured using a Dage-MTI Model DC330E charge-coupled device (CCD) camera (Dage-MTI, Michigan City, IN) attached to the microscope with a U-TV1X-2 video adapter (Olympus America) and a 0.45 camera coupler (Diagnostic Instruments, Sterling Heights, MI). Imaging hardware included a Scion CG-7 RGB color frame grabber (Scion, Frederick, MD), and imaging software included Adobe Photoshop version 7.0 (Adobe Systems, San Jose, CA) and a Scion Series 7 TWAIN module version 2.0. Additional image processing was performed using Photoshop.

Frequency and type of JAK2 V617F mutation within diagnostic categories. (G/G: wild type; G/T or T/T: heterozygous or homozygous for mutation) (left panel). Within WHO MDS/MPD-U, 6 of 9 cases that were positive for JAK2 V617F mutation occurred in RARS-T. When separated from MDS/MPD-U, 67% of RARS-T cases showed a JAK2 V617F mutation, but only 12% of the remaining MDS-MPD-U were positive (right panel).

Previous reports demonstrated that JAK2 V617F mutation is present in a significant proportion of patients with PV (65%-97%), ET (23%-57%), and CIMF (34%-50%).2-6 Subsequent studies in related diseases, including MDS, showed that in typical MDS and CMML,8,12-15 only a low prevalence of JAK2 V617F can be found. We have hypothesized that JAK2 V617F mutation may be present in patients who show features common to typical CMPD and MDS, such as those with MDS/MPD overlap subentities, and we focused our study on this group of patients. The experimental group included 57 patients with proliferative forms of MDS who under the WHO classification system were classified as MDS/MPD overlap syndromes.1,16,19 This group consisted of CMML, JMML, atypical CML, and MDS/MPD-U, which also includes the provisional entity RARS with thrombocytosis (RARS-T) (Table 1). Among these patients, 9 fulfilled the criteria for RARS-T, and 26 belonged to the MDS/MPD-U category. In addition, patients with traditional chronic myeloproliferative diseases and typical MDS patients served as hematologic control groups.

Immunohistochemical detection of p-STAT. Positive staining is present in nuclei of megakaryocytes and erythroid precursors (A) in a patient (Table 3, Patient no. 7) with RARS-T heterozygous for JAK2 V617F mutation (G/T). Negative antibody control (B). Positive staining is present in nuclei of erythroid precursors but absent in megakaryocytes (C) in a patient (Table 3, Patient no. 1) with RARS-T who lacks JAK2 V617F mutation. Negative antibody control (D). Original magnification, 100.

For the purpose of this study, we have devised and cross-validated molecular assays allowing for a sensitive and unambiguous detection of homozygous and heterozygous JAK2 V617F forms. As an initial test we carried out allele-specific PCR (AS-PCR). This method enabled us to distinguish between G/G wild-type and mutated G/T or T/T alleles (Figure 1A); positive result of AS-PCR (2 amplicons: 364 bp, control band; and 203 bp, carrying G/T or T/T mutation) was confirmed by either sequencing JAK2 V617F (exon 14) (Figure 1B) or Light Typer melt analysis (LTPMA; Figure 1C). DNA dilutions showed detection sensitivity of 10% (Figure 2). Both methods allowed for distinction between G/T and T/T JAK2 V617F mutants (Figure 1B-C). 041b061a72


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