Refractory Anemia With Ring Sideroblasts Associated With Marked Thrombocytosis Research Articles

Molecular Analyses of MDS/MPN Overlap Entities According to WHO Classification Reveal a Distinct Molecular Pattern for MDS/MPN, Unclassifiable

Introduction: The World Health Organization (WHO) classification defines myelodysplastic/myeloproliferative neoplasms (MDS/MPN) based on clinical, morphologic, and laboratory findings that show features of MDS and characteristics more consistent with MPN. This category includes atypical chronic myeloid leukemia (aCML), chronic myelomonocytic leukemia (CMML), MDS/MPN, unclassifiable (MDS/MPN, U), and refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T). In recent years RARS-T, CMML, and also aCML were deciphered by several molecular studies, while MDS/MPN, U cases warrant closer investigations.Aim: To comprehensively investigate mutations in 17 genes known to be mutated in aCML, CMML, MDS/MPN, U, and RARS-T and to define entity specific mutation patterns in comparison to cytogenetic and clinical data.Patients and Methods: We investigated 179 patients diagnosed by cytomorphology, immunophenotyping and genetic studies following WHO criteria: 35 patients were diagnosed as aCML, 58 as CMML, 39 as MDS/MPN, U, and 47 as RARS-T. All patients underwent mutation analyses by a gene panel containing: ASXL1, TET2, DNMT3A, SRSF2, SF3B1, U2AF1, JAK2, CALR, MPL, NRAS, KRAS, CBL, BRAF, CSF3R, RUNX1, SETBP1, and NPM1. Gene mutations were analyzed by Sanger sequencing, next generation sequencing, melting curve analyses, or gene scan. Cytogenetics was available in 172/179 cases and was grouped as normal karyotype (n=128, 74%) or aberrant karyotype (n=44, 26%).Results: In the total cohort the most frequently mutated gene was ASXL1 (41%), followed by TET2 (40%), and the spliceosomal genes SF3B1 (31%) and SRSF2 (30%). Also frequently mutated were JAK2 (21%), NRAS (15%), RUNX1 (12%), and CBL (12%). All other investigated genes showed mutation frequencies below 10%.There were no significant differences between the 4 entities regarding frequencies of aberrant karyotypes (14-37%) and no correlation of the number of molecular mutations (0-6/patient) with any specific karyotype.Addressing the mutation patterns of these 4 entities showed that ASXL1 and TET2 are frequently mutated in all entities (19-60% and 26-53%, respectively), although significant differences between the entities exist (see figure): ASXL1 is less frequently mutated in RARS-T (19%) in comparison to aCML (60%; p<0.001) and CMML (52%; p=0.001), TET2 is more often mutated in CMML (53%) in comparison to MDS/MPN, U (26%; p=0.007) and RARS-T (32%; p=0.031). SRSF2 is more frequently mutated in CMML (53%) than in RARS-T (9%; p<0.001) and MDS/MPN, U (15%; p<0.001), SF3B1 is more often mutated in RARS-T (92%) than in all other entities (aCML: 11%, CMML: 5%, MDS/MPN, U: 13%; for all p<0.001). One important difference between aCML and CMML versus MDS/MPN, U and RARS-T was reflected by two different signaling pathways: i) JAK2/CALR/MPL (JAK/STAT pathway) were significantly more often affected in MDS/MPN, U (33%) and RARS-T (53%), (aCML: 9%, CMML: 7%; p<0.001). ii) NRAS/KRAS/CBL (RAS pathway) were more often mutated in aCML (37%) and CMML (52%), (MDS/MPN, U: 5%, RARS-T: 9%; p<0.001).The MDS/MPN, U cohort included most patients with no mutation in any analyzed gene (11/39, 28%) in contrast to aCML (2/23, 6%), CMML (5/58, 9%), and RARS-T (0/47, 0%). Furthermore all these MDS/MPN, U patients with no gene mutation had a normal karyotype. Looking at co-ocurrences of gene mutations in MDS/MPN, U revealed that SRSF2 and TET2 mutations occur together more frequently (4/10 vs. 2/29 in TET2wt; p=0.028). Of notice, in MDS/MPN, U U2AF1 (18%) was the most frequently mutated spliceosomal gene which was only rarely mutated in the other entities (5%, p=0.015).Conclusions: 1) ASXL1 and TET2 are the most frequently mutated genes found overall in MDS/MPN overlap. 2) SF3B1 mutations are specific for RARS-T. 3) SRSF2 is most frequently mutated in CMML, but also in aCML. 4) MDS/MPN, U is affected by mutations in all spliceosomal genes. 5) The JAK/STAT pathway is more often affected in MDS/MPN, U and RARS-T. 6) The RAS pathway is more often affected in aCML and CMML. 7) MDS/MPN, U shows a specific molecular pattern with characteristics reflecting a mixture of all other MDS/MPN entities.Red: gene mutation, orange: gene mutations combined, light grey: no mutation/normal karyotype, black: aberrant karyotype, white: not analyzed. [Display omitted] DisclosuresMeggendorfer:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Jeromin:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

I walk the other line: myelodysplastic/myeloproliferative neoplasm overlap syndromes.

Patients with the myelodysplastic syndromes/myeloproliferative neoplasm (MDS/MPN) overlap, including chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia (aCML), MDS/MPN-unclassifiable (MDS/MPN-U), and refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T), often present with findings of both dysplasia and marrow proliferation, occupying the border region of two seemingly divergent camps. Historically, these disorders which have been lumped with either MDS or MPN have represented a minority, or been excluded all together, from the development of prognostic models and clinical trials. Therefore, Food and Drug Administration approved therapies specifically for overlap subtypes are lacking. More recently, the revolution in molecular genetics has led to discovery of mutations enacting pathways that result in the distinct biology and presentation of the overlap syndromes. Additionally, these recurrent genetic lesions have a prognostic value and are potential therapeutic targets, which might ultimately improve patient outcomes by reducing disease-related symptoms and complications and by prolonging survival.

Polycythemia vera and essential thrombocythemia: 2013 update on diagnosis, risk‐stratification, and management

Polycythemia Vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms (MPN) primarily characterized by erythrocytosis and thrombocytosis, respectively. Other disease features include leukocytosis, splenomegaly, thrombohemorrhagic complications, vasomotor disturbances, pruritus and a small risk of disease progression into acute myeloid leukemia or myelofibrosis. Almost all patients with PV harbor a JAK2 mutation. When PV is suspected, the presence of a JAK2 mutation highly suggests the diagnosis and its absence, combined with normal or increased serum erythropoietin level, excludes the diagnosis. Differential diagnosis of ET should include reactive thrombocytosis, chronic myeloid leukemia, prefibrotic myelofibrosis and RARS-T (refractory anemia with ring sideroblasts associated with marked thrombocytosis). A JAK2 mutation is found in 50-70% of patients with ET, myelofibrosis or RARS-T and is capable of distinguishing reactive from clonal thrombocytosis. Current risk stratification in PV and ET is designed to estimate the likelihood of thrombotic complications: high-risk is defined by the presence of age >60 years or presence of thrombosis history; low-risk is defined by the absence of both of these two risk factors. Recent data considers JAK2V617F and cardiovascular (CV) risk factors as additional risk factors for thrombosis. Presence of extreme thrombocytosis (platelet count >1,000 × 10(9) /L) might be associated with acquired von Willebrand syndrome (AvWS) and, therefore, risk of bleeding. Risk factors for shortened survival in both PV and ET include advanced age, leukocytosis, and history of thrombosis. Survival is near-normal in ET and reasonably long in PV. The 10-year risk of leukemic/fibrotic transformation is <1%/1% in ET and <3%/10% in PV. In contrast, the risk of thrombosis exceeds 20%. The main goal of therapy is therefore to prevent thrombohemorrhagic complications. In low risk patients, this is effectively and safely accomplished by the use of low-dose aspirin in both PV and ET and phlebotomy (hematocrit target of <45%) in PV. In high risk patients, treatment with hydroxyurea is additionally recommended, although not mandated in older patients without JAK2V617F or CV risk factors. Treatment with busulfan or interferon-α is usually effective in hydroxyurea failures. Screening for clinically significant AvWS is recommended before administrating aspirin in the presence of extreme thrombocytosis.

Refractory Anemia with Ring Sideroblasts Associated with Marked Thrombocytosis Complicated by Massive Splenomegaly Treated with Lenalidomide Resulting in Resolution of Splenomegaly but Severe and Prolonged Pancytopenia

Refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T) is a hematological malignancy that combines features of both a myeloproliferative and myelodysplastic disorder. There have been recent reports of the successful treatment of anemia in 2 patients with RARS-T with lenalidomide. Here we report the successful treatment of massive splenomegaly in a patient with a long history of RARS-T resulting in complete resolution of splenomegaly, but with prolonged severe cytopenias. We also report the acquisition of the t(3;12)(q26;p13) translocation previously described in cases of myelodysplasia and the potential for transformation to myelofibrosis.

Clonal Analysis of SF3B1, JAK2 and MPL in Refractory Anemia with Ring Sideroblasts Associated with Marked Thrombocytosis

Abstract 172Somatic mutations of the RNA splicing machinery have been recently identified in patients with myelodysplastic syndrome (MDS). In particular, a strong association has been found between SF3B1 mutation and the MDS subtype defined as refractory anemia with ring sideroblasts (RARS). Similarly, within myelodysplastic/myeloproliferative neoplasms (MDS/MPN) a high prevalence of SF3B1 mutations has been reported in the provisional entity defined as refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T). These findings strongly support a causal relationship between SF3B1 mutations and ring sideroblasts. Interestingly, a high proportion of RARS-T patients also harbor JAK2 and/or MPL mutations. The available evidence suggests that somatic mutations of SF3B1 might be an early pathogenetic event determining myelodysplastic features, and that subsequent occurrence of JAK2 and/or MPL mutations may cause the myeloproliferative phenotype.In this work, we studied the mutation status of SF3B1, JAK2 and MPL in circulating granulocytes and bone marrow cells from RARS-T patients. We also studied the in vitro growth of hematopoietic progenitors (BFU-E, CFU-GM), and genotyped individual colonies to examine the mutation status of the above genes. The coding exons of SF3B1 were screened using massively parallel pyrosequencing. A real time PCR-based allelic discrimination assay was used for the detection of JAK2 (V617F), while Sanger sequencing was employed for JAK2 exon 12 and MPL exon 10 mutation analysis.Twenty-eight patients affected with RARS-T were assessed for SF3B1, JAK2 and MPL exon 10 mutation status. Eighteen patients (64%) showed somatically acquired mutation of SF3B1. The median mutant allele burden was 43%, consistent with the presence in the majority of patients of clonal hematopoiesis characterized by a dominant clone carrying a heterozygous SF3B1 mutation. Fourteen patients carried the JAK2 (V617F) mutation (median allele burden 6.5%, range 0.4–29.5%), while one had a JAK2 exon 12 mutation. In 13 cases, the JAK2 mutation was detected at the time of diagnosis, whereas in 2 patients, who had a typical RARS phenotype and were negative for JAK2 mutations at clinical onset, JAK2 (V617F) was detected 18 and 32 months after diagnosis, respectively, and concomitantly with a progressive increase in platelet count. Four patients, two of whom were JAK2 (V617F)-positive, carried the MPL (W515L) mutation (median allele burden 27.5%, range 25–50%).Concomitant mutations of SF3B1 and JAK2 or MPL were observed in 8 cases. Seven patients carried an SF3B1 mutation and JAK2 (V617F), while one carried SF3B1 (K700E), JAK2 (V617F), and MPL (W515L). In all these cases, the SF3B1 mutant allele burden was higher than that of JAK2 or MPL, indicating the existence of an SF3B1-mutated dominant clone with minority JAK2- or MPL-mutated clones.We genotyped individual colonies from peripheral blood in 2 patients with concomitant mutations. In a patient with granulocyte SF3B1 and JAK2 mutant allele burdens equal to 45% and 8%, respectively, SF3B1 (H662Q) was detected in 9 of 11 colonies, three of which also carried JAK2 (V617F); the remaining two colonies had wild type SF3B1 and JAK2. These data are consistent with the existence of a dominant hematopoietic clone carrying the SF3B1 mutation and the subsequent emergence of a JAK2-mutated subclone. The other patient, who was initially SF3B1- mutated and JAK2 wild type, at the time of colony assay had a mutant allele burden equal to 50% and 1% for SF3B1 (K700E) and JAK2 (V617F), respectively. Forty-three of 45 colonies were heterozygous for SF3B1 (K700E) and wild type for JAK2. The opposite pattern was observed in the remaining 2 colonies, which carried just JAK2 (V617F). These data indicate the coexistence of two distinct clones, a dominant one carrying the SF3B1 mutation and a minority one carrying JAK2 (V617F).In summary, these observations suggest that the occurrence of an SF3B1 mutation represents an early event in patients with RARS-T, likely causing mitochondrial iron overload, ring sideroblasts, ineffective erythropoiesis and anemia, typical myelodysplastic features. The subsequent occurrence of a somatic mutation of JAK2 or MPL involves the emergence of minority clones and the acquisition of myeloproliferative features. JAK2- mutated clones may emerge as subclones of the dominant SF3B1-mutated clone or as independent clones. Disclosures:No relevant conflicts of interest to declare.

The Recently Suggested MDS/MPN Subgroup “Refractory Anemia with Ring Sideroblasts Associated with Marked Thrombocytosis (RARS-T)” Demonstrates Unique Clinical, Cytogenetic, and Molecular Aspects: A Comprehensive Analysis of 57 Cases Strictly Defined According to WHO Criteria

AbstractAbstract 3081 Introduction:In the new WHO 2008 classification, “refractory anemia with ring sideroblasts associated with marked thrombocytosis” (RARS-T) represents a provisional entity defined by platelets ≥450 ×109/l (being lowered from 600 ×109/l; WHO 2001), proliferation of large megakaryocytes, bone marrow (BM) blasts <5%, and ring sideroblasts ≥15% of nucleated erythropoiesis. The separation of RARS-T from other myeloproliferative/myelodysplastic neoplasms is still under debate. Patients:To further characterize this subtype and to evaluate whether its separate position in the WHO classification is justified from biologic/genetic aspects, we analyzed 57 patients with a diagnosis of RARS-T (strictly defined according to WHO 2008 criteria) for peripheral blood parameters, BM morphology, cyto-/molecular genetics, and clinical profiles. The study cohort consisted of 34 females and 23 males (median age, 76 years, range, 51–92 yrs; 52 de novo; 5 therapy-associated). At the time of analysis, all pts were at diagnosis or therapy naïve. Patients with a sole del(5q) or >5% of blasts were excluded according to WHO criteria. Methods:All BM samples underwent May Giemsa Gruenwald and iron stainings. Chromosomal banding analysis (and FISH if needed) were performed in 56/57 cases. PCR was done for the following markers: JAK2V617F (investigated: n=47), MPLW515 (n=46), NRAS (n=24), TET2 mutations (TET2mut, n=14), MLL-PTD (n=13), FLT3-ITD (n=12), and CBL (n=16). Result:Median WBC count was 7.9 ×109/l (range, 3.1–60.0 ×109/l), median hemoglobin (Hb) level was 10 g/dl (range, 6–13 g/dl), and median platelet count was 572 ×109/l (range, 454-1, 737 ×109/l). The median ring sideroblast count was 60% (range, 18–92%). Karyotypes (KT) were as follows: normal KT: n=52 (52/56; 93%); +8: n=2; -Y: n=1. The most frequent mutation was the JAK2V617F (18/47; 38%); an MPLW515 mutation was detected in 3/46 (7%). From the 46 pts being analyzed both for the JAK2 as for the MPLmut, 21 (45.6%) were observed with one of both markers; there was no coincidence of the JAK2 and the MPL mutations. Furtheron, 5/14 (36%) had a TET2 mutation. Coincidences of molecular markers were observed in 3 pts who had a JAK2V617F and a TET2mut in parallel (TET2mut: 3/10; 30% in JAK2mut pts; vs. 2/4 in JAK2 wildtype pts; n.s.). No patient had a JAK2V617F and MPLW515 in parallel. There was no mutation of the NRAS, MLL-PTD, FLT3, or CBL genes in pts investigated for these markers. A positive JAK2V617F mutated status correlated significantly with higher platelets (p=0.038; T-test), whereas no significant correlations were observed for the respective medians taken as thresholds for leukocytes (≥7.9 ×109/l vs. <7.9 ×109/l vs.), Hb (≥10.0 g/dl vs. <10.0 g/dl), or ring sideroblast percentages (≥60% vs. <60%). All 3 pts with MPLW515mut had platelets ≥600×109/l. Cytogenetic aberrations were independent from the JAK2mut status (normal karyotype: 17/45 JAK2mut; 38%; vs. aberrant KT: 1/2 JAK2mut; n.s.) and the MPLmut status (normal KT: 3/44 MPLmut; 7%; vs. aberrant KT: 0/2; n.s.). Higher WBC count (≥7.9 ×109/l) was correlated to a higher Hb level (≥10 g/dl) (p=0.47) and to higher platelets (≥600 ×109/l) (p=0.011). The patients with RARS-T had a favorable outcome with 84.6% being alive at 2.5 years. Conclusion:Investigation of 57 patients strictly fulfilling the criteria of the WHO 2008 classification was able to confirm the unique profile of RARS-T in all aspects: patients with the RARS-T had a normal karyotype (>90% of all cases), had no prognostically adverse cytogenetic alterations, and frequently showed mutations of the JAK2 (V617F) or MPL (W515K/L) genes (45.6% in total). The molecular profile was even more homogeneous in RARS-T cases with ≥600 ×109/l platelets (the WHO threshold from 2001) due to significantly higher proportions of JAK2V617F positive cases when compared to cases with platelets between 450 and <600 ×109/l. However, from clinical aspects, patients with RARS-T had a favorable outcome in our study independent of the molecular state or the number of platelets. These data support to include RARS-T as definite subtype in the next edition of the WHO classification. The frequent occurrence of TET2 mutations in our cohort has to be noted for future diagnostic and classification approaches. Therefore, in cases suspicious for RARS-T but without evidence of a JAK2V617F, molecular screening should be performed including analysis for alterations of the TET2 and MPL genes. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Diagnosing myelodysplastic/myeloproliferative neoplasms: laboratory testing strategies to exclude other disorders

The 2008 World Health Organization classification of myeloid neoplasms includes the diagnostic category, myelodysplastic/myeloproliferative neoplasms (MDS/MPN), which encompasses those rare clonal myeloid proliferations that at initial presentation, show overlapping myeloproliferative and myelodysplastic features, making classification as either a myelodysplastic syndrome (MDS) or myeloproliferative neoplasm (MPN) problematic. There are four main subcategories, chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia, BCR-ABL1-negative (aCML), juvenile myelomonocytic leukemia (JMML), and myelodysplastic/myeloproliferative neoplasm, unclassifiable (MDS/MPN-U), which also includes the provisional entity, refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T). Notably, the morphological features typical of MDS/MPNs are not specific and can be seen in other myeloid neoplasms at presentation or as part of disease progression or transformation. This review presents a laboratory approach to diagnosing MDS/MPNs in adults that allows for the exclusion of other disorders that may be otherwise indistinguishable. Ancillary studies including cytochemistry, immunohistochemistry, flow cytometry, and genetic testing are discussed. The most appropriate classification of myeloid neoplasms presenting with hybrid myelodysplastic/myeloproliferative features requires a comprehensive clinical and laboratory assessment with careful integration of the morphological, immunophenotypic, genetic, and clinical characteristics.

Mutations of JAK2 and TET2, but not CBL are detectable in a high portion of patients with refractory anemia with ring sideroblasts and thrombocytosis

Mutations of JAK2 and TET2, but not CBL are detectable in a high portion of patients with refractory anemia with ring sideroblasts and thrombocytosis