scholarly journals Refractory anemia with ringed sideroblasts associated with marked thrombocytosis (RARS-T), another myeloproliferative condition characterized by JAK2 V617F mutation

Blood ◽  
2006 ◽  
Vol 108 (7) ◽  
pp. 2173-2181 ◽  
Author(s):  
Hadrian Szpurka ◽  
Ramon Tiu ◽  
Gurunathan Murugesan ◽  
Samer Aboudola ◽  
Eric D. Hsi ◽  
...  
Keyword(s):  
Melting Curve ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Refractory Anemia ◽  
Melting Curve Analysis ◽  
Jak2 Mutation ◽  
Ringed Sideroblasts ◽  
Polymerase Chain ◽  
V617f Mutation ◽  
Reaction Sequencing

Abstract 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.

Refractory Anemia with Ringed Sideroblasts Associated with Marked Thrombocytosis (RARS-t): A Clinicopathological Entity with Questionable Pathogenesis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5252-5252
Author(s):  
Anupma Nayak ◽  
Abha Goyal ◽  
Judith Brody ◽  
Peihong Hsu ◽  
Steven Savona ◽  
...  
Keyword(s):  
Expression Profiles ◽  
Normal Karyotype ◽  
Jak2 V617f ◽  
Clinicopathological Features ◽  
Jak2 V617f Mutation ◽  
Refractory Anemia ◽  
Simultaneous Occurrence ◽  
Jak2 Mutation ◽  
Ringed Sideroblasts ◽  
V617f Mutation

Abstract Refractory anemia with ringed sideroblasts associated with marked thrombocytosis (RARS-t) is a clinicopathological entity with features of both myelodysplastic syndrome and myeloproliferative disorder. The cases of RARS-t reported in literature are few and do not confirm whether this disease represents a variant of Refractory anemia with ringed sideroblasts (RARS), a variant of Essential Thrombocythemia (ET), or the simultaneous occurrence of two separate disorders (RARS and ET). Because of this ambiguity, RARS-t has been assigned to MDS/MPD- Unclassifiable subcategory as a provisional entity in the WHO (2001) classification. Recent studies have demonstrated association of RARS-t with JAK2 V617F mutation in up to 67% of cases, suggesting its proximity to MPD. In a few studies, presence of JAK2 mutation was related with better prognosis. Based on this evidence, there are chances that this provisional entity may be clubbed with the group of MPD disorders in the revised WHO classification. We present here 4 cases of RARS-t diagnosed within a period of 1 year (2007–2008) at our institution. Clinicopathological features of these cases are mentioned in the attached table (Table-1). Two of these cases were not associated with anemia at the time of presentation, however could be diagnosed as RARS-t based on the presence of dyserythropoetic features and >15% ringed sideroblasts in marrow on iron stains. Other causes of reactive ring sideroblasts and thrombocytosis were ruled out in these cases. 2/4 cases were positive for JAK2 V617F mutation, however no correlation was found between the JAK2 positivity and the clinicopathological features including prognosis. All the 4 patients were given hydroxyurea and pyridoxine therapy. In contrast to other studies, one of our JAK2 positive cases had to be maintained on the transfusion support alone due to the multiple treatment failures. Our data adds to the literature supporting the association of RARS-t with JAK2 mutation. However, we do not agree with the notion that JAK2 positivity renders a better prognosis. Also, lack of any correlation of clinicopathological features in our cases with the JAK2 mutation drives us to think that it might be only a secondary association rather a primary pathogenesis event. Review of the literature reminds us that the pathogenetic events resulting in myelodysplastic component of this overlap syndrome have only been rarely addressed. In future, more studies focusing on the mutations responsible for genesis of ringed sideroblasts such as ALAS2, ABCB7, FECH and gene expression profiles of bone marrow progenitor cells are required for better understanding of this common but under reported entity. Table 1 Case Age Sex Hb. (g/dL) Hct. MCV (fL) RS% Platelet (× 109/L) WBC (× 109/L) JAK2mutation Other cytogenetics Fibrosis Organomegaly Case#1 84 F 12.9 38.9 94.6 >15% 709 8.3 Negative normal karyotype 1+ reticulin Absent Case#2 85 M 10.3 28.3 131 >15% 1072 7.2 Negative normal karyotype 1+ reticulin Absent Case#3 68 M 6.9 25 101.3 >15% 634 5.4 Positive normal karyotype 1+ reticulin Absent Case#4 63 M 13.7 41.5 78.7 >15% 1272 17.7 Positive normal karyotype 1+reticulin Absent

Granulocyte JAK2 (V617F) Mutation Status in Myeloid Neoplasms with Ringed Sideroblasts.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 854-854 ◽  
Author(s):  
Luca Malcovati ◽  
Matteo G. Della Porta ◽  
Daniela Pietra ◽  
Anna Galli ◽  
Erica Travaglino ◽  
...  
Keyword(s):  
Bone Marrow ◽  
X Chromosome ◽  
Jak2 V617f ◽  
X Chromosome Inactivation ◽  
Jak2 V617f Mutation ◽  
Refractory Anemia ◽  
Mutation Status ◽  
Ringed Sideroblasts ◽  
Myeloid Neoplasms ◽  
V617f Mutation

Abstract The most common type of acquired sideroblastic anemia is the myelodysplastic syndrome (MDS) defined as refractory anemia with ringed sideroblasts (RARS). We have previously demonstrated that mitochondrial iron accumulation in this condition is in the form of mitochondrial ferritin (MtF). A gain-of-function mutation of JAK2 is found in most patients with chronic myeloproliferative disorders. A high frequency of this mutation has been also reported in RARS associated with marked thrombocytosis (RARS-T), a provisional entity characterized by marked increase in platelet count, hypercellular marrow with increased megakaryocytes, and ringed sideroblasts. In this study, we investigated the granulocyte JAK2 (V617F) mutation status in 73 patients receiving a diagnosis of myeloid malignancy with ringed sideroblasts at the Division of Hematology, University of Pavia Medical School & IRCCS Policlinico San Matteo Pavia, Italy between 2001 and 2006. According to the WHO classification of the myeloid neoplasms, 23 patients had RARS, 17 had refractory cytopenia with multilineage dysplasia and ringed sideroblasts (RCMD-RS), 16 had refractory anemia with blasts excess, four had MDS with isolated del(5q), and 13 fulfilled the criteria for RARS-T. JAK2 (V617F) mutation status was analyzed on peripheral blood granulocytes through a quantitative real time PCR-based allelic discrimination assay. We compared clinical and biological features of patients with RARS-T with those of patients with refractory anemia or cytopenia with ringed sideroblasts, and found that RARS-T patients had higher neutrophil and platelet counts, lower frequency of cytogenetic abnormalities and higher incidence of the JAK2 (V617F) mutation (P values ranging from <.001 to .02). JAK2 (V617F) was detected in six out of 63 evaluable cases (9.5%), one being diagnosed as MDS with isolated del(5q) and five as RARS-T, resulting in an incidence of mutation in the latter group of 45%. The proportion of mutant alleles ranged between 2.8% and 18.4%, values commonly observed by us in essential thrombocythemia [Blood. 2006 May 1;107(9):3676–82]. Focusing the analysis on RARS-T, a significantly higher hemoglobin level at diagnosis was found in mutated (median value 11.2 g/dL, range 10.1–15.4) compared with non mutated patients (median value 9 g/dL, range 6–9.9) (P=.009). JAK2-positive patients also showed a significantly lower percentage of ringed sideroblasts in the bone marrow (P=.01), and an increased marrow reticulin fibrosis (P=.03). We then evaluated the clonality of hematopoiesis in female patients through analysis of X-chromosome inactivation patterns (XCIPs) in circulating granulocytes and bone marrow CD34-positive cells. Twenty-one out of 23 informative female patients with ringed sideroblasts (91%), as well as 5/6 RARS-T (83%) displayed a clonal pattern of X-chromosome inactivation. These observations suggest that refractory anemia with ringed sideroblasts with marked thrombocytosis is a clonal stem cell disorder significantly associated with the JAK2 (V617F) mutation. This disorder shows both dysplastic and proliferative features, the presence of the mutation being associated with a predominant myeloproliferative phenotype. The recognition of a heterogeneous genetic background in myeloid neoplasms with ringed sideroblasts suggests that different mechanisms might be involved in the induction of mitochondrial ferritin expression in these disorders.

Acquired 1p Uniparental Disomy Is Associated with Biallelic MPL W515L Mutation in RARS-T.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1643-1643
Author(s):  
Hadrian Szpurka ◽  
Lukasz P Gondek ◽  
Sanjay R Mohan ◽  
Eric D Hsi ◽  
Karl S Theil ◽  
...  
Keyword(s):  
Clonal Selection ◽  
Neutral Loss ◽  
Significant Proportion ◽  
Uniparental Disomy ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Refractory Anemia ◽  
Molecular Defect ◽  
Ringed Sideroblasts ◽  
V617f Mutation

Abstract Refractory anemia with ringed sideroblasts and thrombocytosis (RARS-T) has been considered a provisional subtype within the diagnostic entity of myelodysplastic/myeloproliferative diseases (MDS/MPD). Since JAK2 V617F mutation is present in a significant proportion of RARS-T patients (Szpurka et al. Blood, 2006), many investigators consider this entity to be more closely related to classical MPD. However, a significant minority of patients with RARS-T do not display a JAK2 V617F mutation. We have studied a cohort of patients with RARS-T (N=18) characterized by the presence of ringed sideroblasts, reticulin fibrosis and thrombocytosis (>600×109/L), that lack obvious causes of secondary thrombocytosis. While 8/18 patients harbored a JAK2 V617F mutation, a molecular pathogenesis for the remaining patients was unexplained. The successful application of SNP-A to characterize the genomic lesions in MDS prompted us to use this technology to study RARS-T. SNP-A allows detection of copy neutral loss of heterozygosity such as UPD9p which is associated with JAK2 V617F mutation. SNP-A facilitated detection of previously cryptic lesions; 9/18 patients showed an abnormal SNP-A-based karyotype often involving multiple lesions (only 5 of these defects were detected by metaphase cytogenetics). The new lesions seen by SNP-A included gains of chromosome 11p, 20q and 21q; deletion of 2p and various areas of UPD including 1p, 9p, 6p, 2p and 8p. SNP-A allowed identification of seemingly invariant UPD1p in 4/18 patients. As this region includes the Mpl gene, we analyzed patients for the presence of MPL W515L/K mutations which have been described in MPD. We did not find any patients with MPL W515K, however MPL W515L mutation was present in 2/4 RARS-T patients with UPD1p; another patient showed monoallelic MPL W515L variant. In addition, 1 patient with UPD1p harbored both JAK2 V617F and MPL W515L mutations. To further delineate the molecular lesion we analyzed all patients for the presence of abnormal STAT5 activation. An aberrant phospho-STAT5 staining pattern was present in all cases that were positive for either JAK2 V617F or MPL W515L mutations (N=10); unexplained STAT5 activation was found in only 4 cases, pointing towards a molecular defect involving this pathway. In these 4 patients, and in 1 additional with UPD1p who did not harbor MPL W515L mutation, we searched for other genes which might explain the pathogenesis of this disease by potentially causing aberrant activation of STAT5. We sequenced Jak1T478S, Jak1V623A and Ntrk1S677N as well as the transmembrane, juxtamembrane and kinase domain of Tie1, Epha2 and Ephb2 genes, but no mutation was found. In addition, we found a group of phospho-STAT5-negative patients (N=4) that showed typical genetic features of myelodysplasia e.g. del(5), +8 and partial loss of chromosome X; these cases are probably best considered to be of MDS origin rather than MPD. To our knowledge, our work is the first description of biallelic MPL W515L mutation and UPD1p found in RARS-T patients. This data is important for understanding the clonal selection process and pathophysiology of activating mutations in MDS/MPD. Overall, our studies demonstrate that somatic UPD1p is associated with homozygous MPL W515L mutation in MDS/MPD cases. Localizing areas of somatic UPD by SNP-A may help identify candidate genes within the shared regions that are likely targets for mutations.

RARS-T without Thrombocytosis?.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4607-4607
Author(s):  
Christophe Ravoet ◽  
Marie-Agnès Azerad ◽  
Vanessa Delrieux ◽  
Nathalie Meuleman ◽  
Dominique Bron
Keyword(s):  
Bone Marrow ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Refractory Anemia ◽  
Trisomy 8 ◽  
Ringed Sideroblasts ◽  
Platelet Counts ◽  
Normal Platelet ◽  
Transfusion Requirements ◽  
V617f Mutation

Abstract Background: Recently, a provisional entity called “refractory anemia with ringed sideroblasts with thrombocytosis” (RARS-T) has been characterized in the MDS/MPD overlap category of the WHO classification. Diagnostic criteria include RARS features in association with sustained platelet counts > 600 (500) x 109/l and exclusion of cytogenetic abnormalities such as 5q−, t(3,3) and inv(3). Reticulin fibrosis in the bone marrow has been significantly associated with RARS-T. Splenomegaly also seems more frequent than in other RARS subtypes. Among these patients, approximately two thirds present with a JAK2 V617F mutation. On the contrary, JAK2 V617F mutation appears extremely rare in patients with typical RARS (Szpurka H. Blood 2006, Cabello AI. Leuk Research 2005, Malcovati L. ASH 2006, Remacha AF. Haematologica 2006). Case report: In 2001, we observed a 67 years old patient with RARS (22 to 45% ringed sideroblasts). Conventional cytogenetics revealed a trisomy 8 and deletion of chromosome Y. A mild to moderate fibrosis was observed in the bone marrow. At diagnosis, the patient was anaemic but had normal platelet counts which remained strictly normal during the next 6 years. After failure of various treatments, the transfusion requirements increased due to progressive alloimmunization and probably a moderate splenomegaly which had appeared 3 years after the diagnosis (splenic volume evaluated by ultrasonography was completely normal until 2004). In July 2006, the haemoglobin level was 6.1 g/dl despite the transfusion of 11 RBC units in the previous month. In the aim to overcome the suspected hypersplenism, a splenectomy was performed. In the subsequent days following splenectomy, we observed a rapid and persistent increase in platelet count with a peak of 2550 x 109/l. A JAK2 V617F mutation was demonstrated. The normalization and maintenance of platelets was obtained with small doses of anagrelide (0.5mg four doses/week) with RBC transfusion requirements decreasing by 50%. Conclusion: This patient presented most of the main characteristics of RARS-T except thrombocytosis (note that trisomy 8 has been described in association with RARS-T). Our observation suggests that thrombocytosis could be absent in some patients presenting with a very similar pattern of features as described in RARS-T. We postulate that hypersplenism (and possibly other particular biological characteristics) could, in some cases, mask the complete picture of RARS-T.

scholarly journals PCR-HRM for Detecting JAK2V617F Gene Mutation: Is It a Sensitive Assay?

2021 ◽  
Author(s):  
Mitra Rezaei ◽  
Mihan PourAbdollah Toutkaboni ◽  
Babak Salimi ◽  
Sharareh Seifi ◽  
Fatemeh Maryam Sheikholeslami
Keyword(s):  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Sensitive Assay ◽  
Chain Reaction ◽  
Jak2 Mutation ◽  
Pcr Rflp ◽  
Polymerase Chain ◽  
Neoplastic Disorders ◽  
V617f Mutation ◽  
Sensitivity Specificity

Background: A substitution of G to T at nucleotide 1849 in exon 14 of the Janus kinase2 (JAK2) gene is well recognized in myeloproliferative neoplastic disorders (MPNs). Based on WHO guidelines, detection of the mutation is very important to confirm the disease in suspected patients. Methods: Eighty-seven patients with different background diseases were tested for JAK2 V617F mutation by four different methods, including polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), amplification refractory mutation system (ARMS), polymerase chain reaction-high resolution melting (PCR-HRM), and two different commercial kits. Results: The mean age of patients was 53.38±17.43 years, 72.4% were males, and 37.6% were females. JAK2 mutation was detected in 16 patients (18.3%). Of those, 7 (43.75%) suffered from PV, 5 (31.25%) from ET, 3 (18.75%) from PMF, and 1 (6.15%) from unclassified neoplastic disorders. The frequency of JAK2 mutation was 71.4% (5/7) in PV, 80% (4/5) in ET, and 66.7% (2/3) in PMF patients. The sensi- tivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and GE of PCR-HRM for detection of the JAK2 mutation was 86.7%, 100%, 100%, 97.3%, and 97.7%, respectively. While the sensitivity, specificity, PPV, NPV, and GE of PCR-RFLP were 93.3%, 80.5%, 50%, 98.3%, and 82.7%, respectively. On the other hand, the sensitivity, specificity, PPV, NPV, and GE of ARMS assays were evaluated by about 80%, 96%, 100%, 96%, and 96.5%, respectively. Conclusion: This study showed that PCR-HRM was a more sensitive assay to detect the JAK2 V617F mutation than the other assays. So, it can be used as a quick, easy, and effective method for screening the JAK2 V617F mutation in patients with MPNs disorders. PCR-RFLP must accompany it as a gold standard method for confirmation of the mutation of JAK2 V617F.

The JAK2-V617 Mutation Status Identifies Subtypes of Refractory Anemia with Ringed Sideroblasts Associated with Marked Thrombocytosis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2695-2695
Author(s):  
Annette Schmitt-Graeff ◽  
Soon-Siong Teo ◽  
Manfred Olschewski ◽  
Andreas Kirn ◽  
Petra Reinecke ◽  
...  
Keyword(s):  
Jak2 V617f ◽  
Initial Diagnosis ◽  
Jak2 V617f Mutation ◽  
Refractory Anemia ◽  
Mutation Status ◽  
Allelic Ratio ◽  
Positive Group ◽  
Ringed Sideroblasts ◽  
Platelet Counts ◽  
V617f Mutation

Abstract Sideroblastic erythropoiesis is a feature of pure refractory anemia with ringed sideroblasts (RARS), but may also be associated with megakaryocytic proliferation and marked thrombocytosis (RARS-T). Recently, the identification of a JAK2-V617F mutation in small series of patients has suggested that RARS-T is a JAK2 mutation-related disorder. The aim of our study was to correlate the presence of JAK2-V617F with clinical and hematological features in a larger cohort of patients with RARS-T. An allele-specific PCR for JAK2-V617F genotyping was carried out on DNA samples extracted from bone marrow biopsies obtained from 28 patients (mean age 71 years, mean follow-up 72 months). Our assay measures the allelic ratio of the mutated JAK2 allele, i.e. the percentage of JAK2 loci with the mutated dT-nucleotide (%T). We detected the JAK2-V617F mutation in 12/28 patients (43%) with a predominance of the female gender (8/12). In 7 patients, we found %T<50% (range 16 to 43) at first presentation compatible with a heterozygous JAK2-V617F mutation. Since we are analyzing a mixture of cells, we cannot exclude the presence of a small population of cells that are already homozygous for JAK2-V617F. In 4 cases, the mutant allele predominated (%T> 50%), demonstrating cells homozygous for JAK2-V617F at initial diagnosis. In one patient the allelic ratio of mutated JAK2 increased from 0 to 43 and 98 when biopsies obtained 8 and 16 months after the initial diagnosis were analyzed. At the time when JAK2 V617F was negative, the patient fulfilled the criteria of RARS but not yet of RARS-T since the platelet count was < 500 x 10^9L. The acquisition of the JAK2V617 mutation was accompanied by a progression into RARS-T. In another patient, the ratio increased from 19 to 72 within 14 months. In both cases, the increased ratio of mutated JAK2 was associated with rising platelet counts. On histological evaluation, all JAK2-V617F positive cases displayed a megakaryocyte morphology in keeping with a typical myeloproliferative phenotype. In 4 of the negative cases, a predominance of small hypolobated forms indicated myelodysplastic features. With regard to the mutation status, no differences were found for platelet counts, spleen size, marrow fiber score or vascular events. However, V617-positive patients had significantly higher erythrocyte values (p = 0,003), lower MCV (p = 0,0003) and higher leukocyte counts (p = 0,034). The mean survival time of patients in the positive group was 5.14 years versus 6.43 years in the negative group. The mutation-positive patients had a relative death risk which was 82.3% lower than that of the positive group (relative risk of JAK2-V617F positive versus negative 0.177; 95% confidence interval: 0.040–0.789; p = 0,023). Our results suggest that RARS-T may be divided into two biologically distinct subgroups according to the JAK2-V617F mutation status with a more favorable prognosis for the positive disease. An evolution from V617 heterozygosity to homozygosity during the course of RARS-T may occur in a subset of patients.

scholarly journals Analysis of the reannealing- instead of melting-curve in the detection of JAK2 V617F mutation by HRM method

2019 ◽  
Vol Volume 10 ◽  
pp. 235-241
Author(s):  
Alireza Moradabadi ◽  
Ahmad Fatemi ◽  
Ali Noroozi-aghideh
Keyword(s):  
Melting Curve ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
V617f Mutation
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