Frequency of the JAK2V617F Mutation in Platelets from Essential Thrombocythemia (ET) Patients.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4972-4972
Author(s):  
Paula G. Heller ◽  
Paola R. Lev ◽  
Juan P. Salim ◽  
Laura I. Kornblihtt ◽  
Patricia S. Vassallu ◽  
...  
Keyword(s):  
Colony Growth ◽  
Jak2v617f Mutation ◽  
Wild Type ◽  
Rt Pcr ◽  
Myeloid Metaplasia ◽  
Pcr Rflp ◽  
In The Wild ◽  
Allele Specific ◽  
Myelofibrosis With Myeloid Metaplasia

Abstract The JAK2V617F mutation has been recently reported in patients with polycythemia vera (PV) and a proportion of patients with ET and myelofibrosis with myeloid metaplasia. This acquired point mutation constitutively activates JAK2 tyrosine kinase and is believed to underlie growth factor hypersensitivity displayed by hematopoietic progenitors in these disorders. Identification of this genetic abnormality may be useful for diagnostic purposes although its prognostic implication remains to be determined. The prevalence of this mutation in granulocytes from ET patients ranges from 23 to 57% depending on the detection technique used. ET is characterized by predominant involvement of the megakaryocytic lineage and increased platelet production. In order to evaluate whether the frequency of the V617FJAK2 mutation in platelets from ET patients differs from that previously reported in granulocytes, we amplified a fragment of JAK2 cDNA from leukocyte-depleted platelets by RT-PCR followed by digestion with BsaXI restriction endonuclease (PCR-RFLP). The JAK2V617F mutation abolishes a BsaXI restriction site present in the wild-type sequence and generates a different band pattern. In addition, allele-specific RT-PCR (ASPCR) was performed with a primer pair common to both wild-type and mutant alleles and a third primer complementary to the mutant allele. Forty four patients with ET diagnosed according to the PVSG criteria were included, median age 39 years old (14–81), 10 were men; 21 were studied before treatment, 10 during treatment with anagrelide or hydroxyurea and 13 both before and during treatment. Twenty-two (50%) patients were found to harbour the JAK2V617F mutation by both PCR-RFLP and ASPCR, 3 were homozygous. No change in genotype was found in repeated samples from patients studied both before and during treatment. Hemoblobin levels and the frequency of thrombosis were significantly higher in patients with the mutation respect to those without (141 ±14 g/L vs. 129 ± 11 g/L, p=0.009, and 41% vs. 4.5%, p=0.004, respectively). In addition, patients carrying the mutation were significantly older (51 vs. 33 years old, p=0.002) and had lower platelet counts at diagnosis (1.070 x 109/L vs. 1.500 x 109/L, p=0.02). Interestingly, 2 patients harbouring the mutation developed PV on follow-up. In conclusion, the prevalence of the JAK2V617F mutation in platelets from ET patients did not differ from that previously reported in granulocytes. The increase in the frequency of thrombosis found in patients carrying the mutation should be confirmed in a larger study. Besides, the finding of higher hemoglobin levels in this group of patients is of interest since JAK2V617F-positive ET patients have been reported to display PV markers such as endogenous erythroid colony growth and PRV-1 overexpression, suggesting they may have larger involvement of the erythroid lineage. Further studies will help clarify whether the risk of developing PV is higher in this subset of patients.

scholarly journals The Cloning and Molecular Analysis of pawn-B in Paramecium tetraurelia

Genetics ◽  
2000 ◽  
Vol 155 (3) ◽  
pp. 1105-1117 ◽  
Author(s):  
W John Haynes ◽  
Kit-Yin Ling ◽  
Robin R Preston ◽  
Yoshiro Saimi ◽  
Ching Kung
Keyword(s):  
Genomic Library ◽  
Open Reading Frame ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Rt Pcr ◽  
Reading Frame ◽  
Close Proximity ◽  
In The Wild ◽  
Dna Fragment ◽  
Alternative Sites

Abstract Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca2+ current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca2+ current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5′ neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.

JAK2 (V617F) Mutation Status and Neutrophil Apoptotic Resistance in Myelofibrosis with Myeloid Metaplasia (MMM): Correlation and Potential Identification through Phosphorylation Status of STAT3.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3503-3503
Author(s):  
Ruben A. Mesa ◽  
Ayalew Tefferi ◽  
Heather Powell ◽  
Terra Lasho ◽  
David Loegering ◽  
...  
Keyword(s):  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Neutrophil Apoptosis ◽  
Wild Type ◽  
Jak2 Mutation ◽  
Myeloid Metaplasia ◽  
Mutation Status ◽  
Stat3 Phosphorylation ◽  
V617f Mutation ◽  
Myelofibrosis With Myeloid Metaplasia

Abstract Background: We have previously described a resistance to the normal process of apoptosis in neutrophils of patients with myelofibrosis with myeloid metaplasia (MMM) (Blood2003;102:11). Most recently, an activating mutation of JAK2 (V617F) has been described in approximately half of the patients with MMM as well as in variable proportion of patients with other myeloproliferative disorders (MPD). In the current study, we investigated the correlation between JAK2 V617F mutation status and neutrophil apoptosis in MMM. Methods: Neutrophils were isolated by density centrifugation from patients with MMM, other MPDs, and normal controls and assessed for apoptosis at baseline and after 24 hours in culture (IMDM with 20% sterilized fetal calf serum to simulate spontaneous apoptosis). Apoptosis was quantified using three-color flow cytometry using CD45 (to confirm leukocyte presence), annexin V (AN) (marker of apoptosis; detects aberrant externalization of phosphatidylserine during apoptosis), and propidium iodide (PI) (marker of dead cells). Mutation analysis for JAK2 V617F was performed in DNA derived from the isolated neutrophils using genomic DNA amplified by PCR, or extracted from cytogenetic pellets in archived specimens. Apoptotic rates after 24 hours in culture were correlated between patients and controls for both JAK2 mutation status and clinical parameters. Immunoblotting was performed on a subset of patients for correlation of JAK2 mutation status and downstream phosphorylation of the JAK2 target, STAT3, which transcriptionally activates several antiapoptotic genes. Results: Spontaneous neutrophil apoptosis was significantly decreased in MMM patients (n=50; median % apoptotic cells at 41%) compared to both healthy volunteers (n=9; 66%) and patients with other MPD (n=11; 53%) (p=0.002). Resistance to apoptosis in MMM correlated with both anemia (p=0.01) and the presence of the JAK2 V617F mutation (p=0.01). Furthermore, the specific abnormality was more pronounced in patients with homozygous JAK2 V617F; median % apoptotic cells of 47% for patients with wild-type allele (n=22) vs. 39% for heterozygotes (n=23) vs. 22% for homozygotes (n=5; p=0.008). The JAK2 mutation status did not appear dependent on other peripheral blood or clinical features. Neutrophils from 14 MMM patients were assessed simultaneously for both JAK2 mutation and STAT3 phosphorylation status by immunoblotting. Strong expression of phosphorylation of STAT3 was seen in all 3 homozygotes and 4 of 5 heterozygotes, but only 1 of 6 with wild-type allele (p=0.026). Conclusions: Impaired neutrophil apoptosis in patients with MMM correlates with the functional presence of JAK2 V617F in an allele-dose dependent manner and STAT3 phosphorylation. The current observation supports a pathogenetic role for the specific mutation in sustaining clonal myeloproliferation.

Detection of the JAK2 V617F Mutation by Real Time PCR in Granulocytes and Platelets of ET Patients.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4877-4877
Author(s):  
Beatriz Bellosillo ◽  
Eva Gimeno ◽  
Raquel Longaron ◽  
Lourdes Florensa ◽  
Antonio Salar ◽  
...  
Keyword(s):  
Real Time ◽  
Direct Sequencing ◽  
Specific Treatment ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Wild Type ◽  
Rt Pcr ◽  
Jak2 Mutation ◽  
Allele Specific ◽  
V617f Mutation

Abstract Introduction. The JAK2 V617F mutation has been detected in 23%–57% of ET patients by direct sequencing or allele-specific (AS) PCR. It remains unknown, however, if the mutation detected in the granulocyte population, may be equally detected in platelets from these patients. Objective. To compare the detection of the JAK2V617F mutation in granulocytes and platelets from ET patients by real time AS RT-PCR. Patients and methods. Platelets and granulocytes from 50 ET patients from a single institution were studied. Patients were diagnosed according to the WHO criteria. At the time when JAK2 mutation was analyzed 16/50 patients were receiving platelet-lowering therapy ± ASA, 14/50 patients only received ASA and 20/50 received no specific treatment. JAK2 mutation was analyzed by real-time AS RT-PCR with probes specific for the mutated and the wild type form. Results. The V617F JAK2 mutation was detected in 18 out of 50 patients in both granulocytes and platelets by real time AS RT-PCR, and was negative in both cell populations in the remaining 32 patients. In the V617F JAK2 positive cases, the mean Ct(V617FJAK2)/Ct(wild type JAK2) ratio was 1.074±0.062 for granulocytes and 1.038±0.039 for platelets (p=0.048). These values corresponded to a 17.79 ±7.4% of mutated population when granulocytes were analyzed, whereas, a significantly higher percentage of mutated population was observed, 23.45±7.78 %, when platelets were analyzed (p=0.032). Conclusions. The results of V617FJAK2 mutation detection by AS RT-PCR were the same in granulocytes and platelets (either positive or negative). The percentage of clonal population detected in ET patients was significantly higher in platelets than in granulocytes.

Myeloablative Conditioning of Patients with Myelofibrosis with Myeloid Metaplasia Using i.v. Treosulfan and Autologous Peripheral Blood Progenitor Cell Transplantation (PBPCT) with High Doses of CD34+ Cells Results in Hematologic Responses - 32 Months Follow-Up of Three Patients and Short-Term Results of a New Study.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5515-5515
Author(s):  
Stefan Fruehauf ◽  
Eike C. Buss ◽  
Julian Toplay ◽  
Hans H. Kreipe ◽  
Anthony D. Ho
Keyword(s):  
Multicenter Study ◽  
Autologous Transplantation ◽  
Conditioning Regimen ◽  
Allogeneic Transplantation ◽  
High Dose ◽  
Post Transplantation ◽  
Myeloid Metaplasia ◽  
Cd34 Cells ◽  
Myelofibrosis With Myeloid Metaplasia

Abstract Myelofibrosis with myeloid metaplasia (MMM) is a chronic myeloproliferative disorder. Myeloablation with high-dose treosulfan and autologous PBPCT provides a palliative approach. A minimum of 5 x 106 CD34+ cells/kg was collected in all patients after G-CSF-priming (16 μg/kg/d for 4 days). Myeloablation consisted of treosulfan (total dose 42 g/m2). To date we have transplanted 3 patients, all female on an individual basis. We observed a prolonged time to reconstitution of leucocytes > 1/nl post transplantation of 28 days (#1, #2) and 38 days (#3). The observation period post transplantation is 32 months now. Patient #1, who required erythrocyte transfusions twice weekly pretransplant received her last erythrocyte transfusion on day 56; her last Hb-value is 9.5 g/dl. The second patient (#2) recovered to platelet counts higher than pre transplantation (58/nl) at 3 months (143/nl) and had 125/nl at last follow-up. Pat. #3 showed a marked reduction of max. spleen size and a rise in Hb from 9 g/dl to 12 g/dl after 12 months, the last Hb value at 26 months was 7.6 g/dl and she received about 3 erythrocyte transfusions per month again. She then underwent allogeneic transplantation from her sister and is currently alive and well. Three male patients were treated in a new one-armed, multicenter study applying the aforementioned protocol. In two patients we observed again a prolonged reconstitution period of 28 (S1) and 44 (S2) days. In one patient (S1) leukocytosis and thrombocytosis resolved while transfusion dependent anemia persisted. In the second patient (S2) transfusion dependent anemia persisted. He received a backup transplantation but still remains transfusion-dependent. Patient S3 had a critical thrombocytopenia of 16/nl before transplantation and proved to be refractory to thrombocyte transfusions post-transplantation. He developed cerebral hemorrhage and died. The conditioning regimen was well tolerated and despite the prolonged aplasia period neutropenic fever was rare (median 1 day, range 0–6 days). The overall response rate was 60%, the mortality rate was 17% which is both compatible with previous data of autologous transplantation in MMM patients undergoing busulfan conditioning. This multicenter study is continuing to recruit patients (http://leukaemie.krebsinfo.de/kn_home/Studien/studie_101.html).

Responses to Thalidomide Combined with Prednisone Therapy for Myelofibrosis with Myeloid Metaplasia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3623-3623
Author(s):  
Shenxian Qian ◽  
Junfeng Tan ◽  
Pengfei Shi ◽  
Gaqian Gao
Keyword(s):  
Bone Marrow ◽  
Low Dose ◽  
Single Agent ◽  
Drug Effects ◽  
Myeloid Metaplasia ◽  
Baseline Status ◽  
Myelofibrosis With Myeloid Metaplasia ◽  
Sequential Phase

Abstract Myelofibrosis with myeloid metaplasia (MMM) is currently classified as a classic (i.e. not yet molecularly defined) myeloproliferative disorder (MPD), along with essential thrombocythemia (ET) and polycythemia vera (PV). All three MPDs represent stem-cell-derived clonal myeloproliferation that, in the case of MMM, is accompanied by an intense bone marrow stromal reaction that includes collagen fibrosis, osteosclerosis, and angiogenesis. To present the results of a long-term analysis of 2 sequential phase 2 trials of thalidomide (alone or in combination) for palliation of myelofibrosis with myeloid metaplasia (MMM). We analyzed (March 2003 to August 2005) initial and long-term outcomes from 33 patients with symptomatic MMM who had enrolled in either our thalidomide single-agent trial (n=12) or our trial of low-dose thalidomide (50 mg/d) combined with prednisone (n=21). Among the 33 study patients, 18 (54%) showed some improvement in their clinical course. Response rates for specific end points included improvements in anemia (12 of 33 [36%]), thrombocytopenia (8 of 12 [66%]), or splenomegaly (5 of 30 [17%]). The combination of low-dose thalidomide and prednisone, as opposed to single-agent thalidomide, was better tolerated and more efficacious. After a median follow-up of 17 months (range, 9–27 months), 10 of 33 patients (33%) showed an ongoing response, including 8 patients in whom protocol treatment has been discontinued for a median of 21 months (range, 16–27 months). Durable treatment responses were documented for only anemia and thrombocytopenia. Treatment response was not affected by the baseline status of bone marrow fibrosis, angiogenesis, osteosclerosis, cytogenetics. Unusual drug effects, all reversible, included leukocytosis (8 patients) and/or thrombocytosis (6 patients). Thalidomide (alone or combined with prednisone) is an effective first-line treatment of symptomatic anemia or thrombocytopenia in MMM. Thalidomide-based therapy has the potential to produce durable responses in MMM-associated cytopenias, even after discontinuation of the drug.

Comparative Analysis Between Molecular Techniques Used for the Detection of JAK2V617F Mutation in Patients with Chronic Myeloproliferative Disorders

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5178-5178
Author(s):  
Salem H Alshemmari ◽  
Mohmd Edrees ◽  
Marwa Almusailaik
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequence ◽  
Genomic Dna ◽  
Jak2v617f Mutation ◽  
Wild Type ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Arms Pcr ◽  
Polymerase Chain ◽  
Mutant Cells

Abstract Abstract 5178 Several somatic mutations have been known to result in an individual to suffer from one or more classes of MPDS. JAK2V617F mutation is the most common somatic mutation that is known as a major contributor to MPDs. Extraction of Total Genomic DNA from Whole Peripheral Blood Blood samples were collected from each subject in vacutainer tubes containing 1.8mg/ml K3-EDTA. Extraction of total genomic DNA was carried following the protocol of a standard QIAGEN DNA Extraction Kit (QIAGEN, USA). Amplification Refractory Mutation System-Polymerase Chain Reaction (ARMS-PCR) for the Detection of JAK2V617FMutation Amplification Refractory Mutation System-Polymerase Chain Reaction (ARMS-PCR) technique was used in this study to amplify three DNA bands, control (463bp), wild type (229bp), and mutant (279bp) if existent, in which the latter represents cells with JAK2V617F mutation. A 100 ng of DNA template was used for the amplification of the three fragments. HotStart Taq Polymerase Master Mix (Qiagen) was utilized for the amplification JAK2V617FAllele-Specific Real-Time PCR (RT-PCR). Qualitative real-time PCR (RT-PCR) was performed in this study on 100 patients suffering from MPDs, fifty of which were negative and fifty were positive, for the detection of as low as 5% of mutant cells. Standard JAK2 MutaScreen™ Kit (IPSOGEN Cancer Profiler) was used in this procedure, containing JAK2V617F mutant positive control (100%), negative control (0.00%), and a reference sample for the discrimination of negative and very low positive cells. Genomic DNA of MPD samples was diluted in TE buffer (AMBION) to 5.0 ng/μl in concentration. For the amplification of the mutant fragment, TaqMan Universal Master Mix (Applied Biosystems) was added to the mixture of 10x probe/primers and DNA. Polymerase Chain Reaction (PCR) for Direct Sequencing A fragment of 349bp was amplified to include the JAK2 mutational site (V617FG>T) in exon 14. AmpliTaq Gold® PCR Master Mix (Applied Biosystems, USA) was used in this procedure. In this comparative analysis, we diagnosed a total number of 385 MPD patients using three major molecular techniques, direct DNA sequence analysis, ARMS-PCR, and RT-PCR. Out of the 385 patients, 285 were run for DNA sequencing, in which 50 negatives and 50 positives were randomly tested using ARMS-PCR. In comparison, a separate randomized set of 100 (50 negatives & 50 positives) patients that were diagnosed through ARMS-PCR, were also run for RT-PCR for comparative investigation. For the 100 MPD cases that were randomly chosen from the 285 diagnosed set, the 50 positive individuals confirm positivity for JAK2V617F mutation (true positives), whereas 47 were confirmed negative (true negatives) and three patients, in which their somatic cells tested negative using DNA sequencing, were proven positive using ARMS-PCR (false negatives). As shown in Figure (X), well 13–15 display clear 279bp mutant band that represents the presence of JAK2V617F positive cells, whereas the 229bp reflect on the presence of wild type cells. Overall, out of the 100 samples that were run for DNA sequence analysis, misdiagnosis accounts for 3% of the total sample number. On another set of patients, 50 randomly chosen negatives and 50 positives that were assessed using ARMS-PCR were also confirmed for their JAK2V617F somatic mutation through RT-PCR. Results revealed that diagnosis of JAK2V617F mutation utilizing RT-PCR is parallel to the outcome if DNA is tested for positivity using ARMS-PCR. Out of the 100 MPD patients, 50 indicated true negativity, and 50 showed true positivity. Thereby, usage of ARMS-PCR as a diagnostic molecular technique is comparable to RT-PCR. The somatic nature of JAK2V617F mutation has a 3% chance in being misdiagnosed for an MPD when DNA sequencing is implemented over ARMS-PCR based on our results. This is most probably due to the small number of mutant cells that result in a small chromatographic peak on the DNA sequence in response to mutant DNA, hence the false negative diagnosis. Whereas, by utilizing ARMS-PCR as a molecular diagnostic assay, we were able to synthesize mutant DNA of such small number of mutant cells, hence eliminating any chance of misdiagnosis. Intensity of the mutant band displayed on the agarose gel in comparison to the wild type is a reflection of the amount of mutant DNA found in each case. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Linked Linear Amplification for Simultaneous Analysis of the Two Most Common Hemochromatosis Mutations

2003 ◽  
Vol 49 (7) ◽  
pp. 1050-1057 ◽  
Author(s):  
Anthony A Killeen ◽  
John W Breneman ◽  
Arlene R Carillo ◽  
Jason Liu ◽  
Craig S Hixson
Keyword(s):  
Hereditary Hemochromatosis ◽  
Colorimetric Method ◽  
Rflp Analysis ◽  
Wild Type ◽  
Linear Amplification ◽  
Pcr Rflp ◽  
Homozygous Mutant ◽  
Allele Specific ◽  
Novel Method ◽  
The Common

Abstract Background: Two mutations in HFE, G845A (amino acid substitution C282Y) and C187G (H63D), are associated with hereditary hemochromatosis. We developed and validated a novel method, linked linear amplification (LLA), for detection of these two mutations. Methods: Two segments of HFE were amplified by a multiplex LLA reaction that generated biotinylated LLA products. Aliquots of the multiplex LLA reaction were captured in microwells by hybridization to immobilized allele-specific oligonucleotides (ASOs). One wild-type and one mutant ASO represented the DNA sequence at each of the two mutation sites. Hybridization was detected by a streptavidin–horseradish peroxidase-based colorimetric method. Genotypes obtained by LLA and PCR-restriction fragment length polymorphism (PCR-RFLP) methods for 320 individuals were compared. Results: The amplified samples included the following genotypes as determined by PCR-RFLP: wild-type 282 and 63 codons (n = 105), C282Y homozygous mutant (n = 54), C282Y heterozygous (n = 52), H63D homozygous mutant (n = 17), H63D heterozygous (n = 59), and compound H63D and C282Y heterozygous mutant (n = 33). There was complete concordance between the results obtained by LLA and those obtained by PCR-RFLP analysis. The presence of another HFE mutation, A193T (encoding S65C), did not interfere with genotyping at codon 63. Conclusions: LLA provides a reliable method to detect the common mutations in HFE that cause hereditary hemochromatosis.

Cytogenetic Profile, JAK2V617F Mutational Status, and Response to Drug Therapy in Myelofibrosis with Myeloid Metaplasia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2591-2591
Author(s):  
Jacob J. Strand ◽  
Terra L. Lasho ◽  
Susan M. Schwager ◽  
Michelle M. Elliott ◽  
Chin-Yang Li ◽  
...  
Keyword(s):  
Molecular Markers ◽  
Drug Therapy ◽  
Mutation Analysis ◽  
Wild Type Allele ◽  
Wild Type ◽  
Myeloid Metaplasia ◽  
Cytogenetic Abnormalities ◽  
Mutational Status ◽  
Cytogenetic Profile ◽  
Myelofibrosis With Myeloid Metaplasia

Abstract Background: One of the utilities of molecular markers in hematological malignancies is their potential to predict response to drug therapy. Accordingly, we inquired about the effect of both cytogenetic profile and JAK2V617F mutational status on drug therapy response in myelofibrosis with myeloid metaplasia (MMM). Methods: Mutation analysis for JAK2V617 was performed in DNA derived from peripheral blood mononuclear cells, granulocytes, or both. Genomic DNA was amplified by PCR and fluorescent dye chemistry sequencing was performed using the same primers used for amplification (Levin et al. Cancer Cell2005;7:387). Abnormal cytogenetic findings were considered to be either favorable (sole 13q- or 20q- abnormalities) or unfavorable (other abnormalities) (Tefferi et al. BJH2001;113:763). Results: i. Patients and treatment: A total of 69 patients with MMM (median age 62 years, range 38–75; 47 males) received the following drugs as first-line therapy for either anemia or symptomatic splenomegaly; erythropoietin (Epo) alone or in combination with hydroxyurea (n=25), hydroxyurea alone (HU; n=17), interferon alpha (IFN; n=11), combination of thalidomide and prednisone (ThalPred; n=7), androgen preparations (Andro; n=5), and etanercept (n=4). ii. Results of cytogenetic studies and JAK2 mutation screening: Cytogenetic findings were abnormal in 35 patients (51%); favorable in 9 and unfavorable in 26. JAK2V617 mutation analysis revealed wild-type allele in 31 patients (45%) and either heterozygous (n=31) or homozygous (n=7) mutation in the remaining 38 patients (55%). iii. Correlation of response to cytogenetic/molecular markers: Overall, 17 patients (25%) achieved either a major (n=8) or minor (n=9) response in anemia from treatment with one of the aforementioned drugs. Regardless of either JAK2V617 mutational status or cytogenetic profile, treatment-induced responses in anemia were poor for IFN (0%) and HU (12%). In contrast, the best anemia responses were documented for ThalPred (57%), Andro (40%), and Epo-based therapy (32%). Among the 25 patients that received Epo-based therapy, all 3 patients with favorable cytogenetic abnormalities achieved major responses in anemia whereas such responses were seen in none of the 9 patients with unfavorable cytogenetic abnormalities and only 1 of 13 patients with normal cytogenetics (p=0.004). Furthermore, 3 of the 4 major responders were heterozygous for JAK2V617 and one carried the wild-type allele. In contrast, none of the 3 JAK2V617 homozygotes showed any type of response but all 3 also displayed unfavorable cytogenetics. None of the 7 ThalPred-treated patients carried favorable cytogenetics and yet 3 achieved major responses including one with unfavorable cytogenetics. JAK2V617 mutational status was heterozygous in 1 and wild-type in the other two. There were no major responses among the 5 Andro-treated patients despite the presence of favorable cytogenetic abnormalities in 2 patients. In order to investigate the relationship between cytogenetic profile and JAK2V617 mutational status further, we referred to an expanded database of 116 patients and found the incidence of unfavorable cytogenetics to be higher in JAK2V617 homozygotes compared to non-homozygotes (57% vs. 31%; p=0.16) Conclusion: The current study suggests clustering of favorable cytogenetic abnormalities in MMM with anemia response to Epo therapy and unfavorable cytogenetic abnormalities with homozygosity for JAK2V617 mutation.

Incidence and Clinical Profile of JAK2 V617F Mutation in Myelofibrosis with Myeloid Metaplasia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 256-256
Author(s):  
Giovanni Barosi ◽  
Monia Marchetti ◽  
Margherita Massa ◽  
Vittorio Rosti ◽  
Alessandro Vannucchi ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Severity Score ◽  
Jak2 V617f ◽  
Mutant Gene ◽  
Jak2 V617f Mutation ◽  
Homozygous Mutation ◽  
Wild Type ◽  
Myeloid Metaplasia ◽  
V617f Mutation ◽  
Myelofibrosis With Myeloid Metaplasia

Abstract An aberrant tyrosine kinase signalling due to non-receptor tyrosine kinase JAK2 V617F mutation has been highlighted in the pathogenesis of polycythemia vera (PV). In myelofibrosis with myeloid metaplasia (MMM) this mutation has been reported to be present in approximately 50% of the patients and its pathogenetic role is not elucidated. Here we report the results of JAK2 V617F mutation in a retrospective analysis of blood samples from 170 patients with MMM. Search for JAK2 mutation was performed by an allele specific PCR from DNA purified from granulocytes. To evaluate whether the mutation was carried in the homozygous or heterozygous state, digestion of PCR products with BsaXI restriction enzyme was performed. The overall frequency of JAK2 V617F mutation was 60% and homozygosity for the mutation was found in 39.2% of mutant samples. Disease duration was similar in JAK2 mutated and wild type patients. Patients who harboured an homozygous mutation had an higher myeloproliferative severity score (that indexed leukocytosis, thrombocytosis and splenomegaly) than patients who had a wild type or heterozygous genotype. In post-PV MMM, the mutant gene was present in 22/22 (100%) of patients, and the frequency of homozygosity was 59% of the mutated cases. In post-ET MMM (n=13), the mutant gene was present in 46% of the patients, and the frequency of homozygosity was 16% of the mutant samples. In idiopathic MMM (n=135), the incidence of the mutational state was 54.8%, with 35.1% of homozygote mutation. Patients who had received a diagnosis of prefibrotic myelofibrosis (WHO classification) had an incidence of the mutation significantly lower than patients who were diagnosed in the fibrotic stage (6/18, 33% vs 68/112, 60.7%; P=0.001). By considering only patients not receiving cytoreductive or disease modifying agents, patients who had an heterozygote mutation, had a mean Hb value higher than wild type patients (9.4 g/dL vs. 11.4 g/dL; P=0.004). Moreover, patients who had an homozygote mutation had the myeloproliferative severity score higher than both heterozygote and wild type patients (2.26 vs 1.59, P=0.008, and 2.26 vs.1.52, P=0.001, respectively). We conclude that JAK2 V617F mutation is significantly represented in MMM patients. It is a necessary event in the transformation from PV to MMM while not in the transformation from ET to MMM. Patients who harboured an heterozygote state maintained higher Hb values than patients with a wild type genotype, while the homozygote mutation was associated with leukocytosis, thrombocytosis and splenomegaly.

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