ALBERT

Description: The JAK2 (V617F) mutation is found in 95% of patients with polycythemia vera (PV) and in about 60% of those with essential thrombocythemia (ET) or primary myelofibrosis (PMF). In a portion of PV patients, the occurrence of a mitotic recombination of chromosome 9p in a hematopoietic cell that is heterozygous for the mutation generates a subclone of cells that are homozygous for JAK2 (V617F) [N Engl J Med. 2005 Apr 28;352(17):1779–90]. We have previously shown that the vast majority of patients with post-PV myelofibrosis (MF) have high percentages of granulocyte JAK2 (V617F) mutant alleles [Blood. 2008 Apr 1;111(7):3383–7], suggesting that transition from heterozygosity to homozygosity for JAK2 (V617F) is associated with progression to myelofibrosis in PV. Activating mutations of MPL, mainly involving a W515 substitution, have been detected in JAK2 (V617F)-negative patients with ET and PMF. MPL maps on chromosome 1p34, and whether a mitotic recombination of this chromosome occurs and involves a transition from heterozygosity to homozygosity for MPL mutations is currently unclear. At variance with post-PV MF, post-ET MF occurs rarely, with an estimated risk of about 6% at 15 years. We studied 315 patients with myeloproliferative disorders followed at the Department of Hematology Oncology, University of Pavia and Fondazione IRCCS Policlinico San Matteo, Pavia, Italy. This study was approved by the local Ethics Committee. The revised World Health Organization (WHO) criteria were employed for the diagnosis of PV, ET and PMF, whereas the criteria of the International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) were employed for the diagnosis of post-PV MF and post- ET MF. We developed a high-resolution melting-curve (HRM) assay for detecting MPL mutations in granulocyte and T lymphocyte gDNA. The study population included 205 patients with ET [58% of whom carried JAK2 (V617F)], 91 patients with PMF [62% of whom carried JAK2 (V617F)], and 19 patients with post-ET MF [42% of whom carried JAK2 (V617F)]. By means of HRM analysis and direct sequencing, we detected the following MPL mutations in circulating granulocytes but not in T lymphocytes: W515L, W515K, W515A, S505C, and V501A – these latter two being novel mutations. Two patients carried two different MPL mutations concurrently in circulating granulocytes, while another patient carried both JAK2 (V617F) and a MPL mutation. Based on the detection of abnormal melting curve profiles in granulocytes, the prevalence of MPL mutations was found to be 5.4% in all patients with ET [12.6% in JAK2 (V617)-negative subjects], 5.5% in those with PMF [14.3% in JAK2 (V617)-negative subjects], and 21.1% in patients with post-ET MF [36.4% in JAK2 (V617)-negative subjects]. The prevalence of MPL mutations was therefore significantly higher in post-ET MF than in ET (P

Description: About 95% of patients with polycythemia vera (PV) carry the unique V617F mutation in JAK2 exon 14, which encodes a portion of the JH2 auto-inhibitory domain of the Jak2 kinase. Mutations in exon 12 have been recently reported in JAK2 (V617F)-negative patients with PV or idiopathic erythrocytosis. We searched for exon 12 mutations in 168 patients with JAK2 (V617F)-negative myeloproliferative disorders. The 2001 WHO criteria were employed for diagnosis. Of the 168 patients studied, 47 had sporadic PV, 11 had familial PV, 75 had essential thrombocythemia (ET), and 35 had primary myelofibrosis (PM). Seventeen patients with PV, including 15/47 sporadic cases and 2/11 familial cases, were found to carry deletions (n=15) or duplications (n=2) of exon 12 in circulating granulocytes but not in T-lymphocytes. None of the 110 patients with ET or PM was found to be positive. Mutations were detected by sequencing, and were then confirmed by sub-cloning in bacteria in 7/17 cases. Four of the 8 mutations detected were novel, while the most frequent ones were N542–E543del and E543–D544del. Mutations spanned from base 1606 to 1640, and the two duplications modified the rest of the sequence by adding 33 bp. In terms of protein, deletions predicted aminoacid changes spanning from phenylalanine 537 to aspartic acid 544, while duplications predicted changes from phenylalanine 547 onwards within the JH2 pseudokinase domain. Three categories of molecular lesions were identified: those involving a K539L substitution; those involving the E543del; and aminoacid duplications involving a substitution of phenylalanine 547. At clinical onset, 16/17 (94%) patients carrying a JAK2 exon 12 mutation had low serum erythropoietin (Epo) levels, indicating a combination of absolute erythrocytosis and suppressed endogenous Epo production. Moreover, 12/17 patients had erythrocytosis associated with normal white blood cell and platelet counts, i.e., isolated erythrocytosis. This frequency (71%) was significantly higher than that observed in 92 patients diagnosed with JAK2 (V617F)-positive PV at the Department of Hematology, IRCCS Policlinico San Matteo, Pavia, Italy (P12 x 109/L) and/or thrombocytosis (PLT〉400 x 109/L), and only 22% of them had isolated erythrocytosis. Both patients with familial PV carrying an exon 12 mutation had an affected sibling with JAK2 (V617F)-positive PV. While the former showed isolated erythrocytosis, their JAK2 (V617F)-positive siblings had also thrombocytosis. In conclusion: several somatic mutations of JAK2 exon 12 - mostly 6 bp deletions - can be found in patients with a myeloproliferative disorder that is mainly characterized by erythrocytosis associated with low serum Epo levels; a genetic predisposition to acquisition of different JAK2 mutations is inherited in families with myeloproliferative disorders, and the mutation type (exon 12 vs exon 14) contributes to determining their variable clinical phenotype.

Description: Abstract 1862 Refractory anemia with ring sideroblasts (RARS) is a myelodysplastic syndrome (MDS) characterized by isolated anemia, erythroid dysplasia only, less than 5% blasts and 15% or more ring sideroblasts in the bone marrow (2008 WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues). The natural history of RARS is characterized by an initial phase of erythroid hyperplasia and ineffective erythropoiesis, which is usually stable for many years but in a proportion of patients may be followed by a phase of marrow failure, with or without the later emergence of leukemic blasts. Overall, RARS is a benign condition with a median survival of about 9 years (J Clin Oncol. 2005;23:7594-603). Since the vast majority of these patients have no cytogenetic abnormalities, the clonal nature of RARS has been questioned. However, a few studies of X-chromosome inactivation patterns performed in female patients have suggested that RARS derives from clonal proliferation of a multipotent hematopoietic stem cell with the potential for myeloid and lymphoid differentiation. Somatic mutations of TET2 have been recently found in myeloid neoplasms including MDS, where they appear occur early during disease evolution (Nat Genet. 2009;41:838-42), and are currently considered as a reliable clonal marker of these disorders. In this study, we therefore performed a mutation analysis of TET2 in patients with myeloid neoplasms associated with ring sideroblasts. Using direct sequencing, we studied 33 patients with RARS and 28 patients with refractory cytopenia with multilineage dysplasia (RCMD) having 15% or more ring sideroblasts in the bone marrow. Somatic mutations of TET2 were detected in circulating granulocytes from 10 out of 33 (30%) patients with RARS and 10 out of 28 (36%) patients with RCMD and ring sideroblasts. Most of these mutations were novel at the time of this writing. Fourteen patients had a single somatic mutation, and the mutation burden ranged from 10 to 80%. In 9 of these 14 cases, the mutation burden was approximately 50%, consistent with a fully clonal hematopoiesis characterized a single dominant clone that was heterozygous for the mutation. In a female patient with 10% mutant alleles, however, granulocytes carrying mutant TET2 represented only one tenth of clonal granulocytes as determined by X-chromosome inactivation patterns, suggesting the existence of alternative genetic events preceding the TET2 mutation and sustaining clonal dominance. Six patients had multiple somatic mutations of TET2: two mutations in 3 cases, three mutations in 2 cases, and four mutations in the last case. Quantitative evaluation of mutation burden showed concordant values (about 50%) for the multiple mutations in two patients (one with 4 and the other one with 3 somatic mutations of TET2), indicating the existence of a single dominant clone with multiple mutations. In the remaining 4 patients, discordant mutation loads were detected: the dominant mutation was present in about 50% alleles, while the remaining one(s) involved a lower proportion (10-35%) of alleles. These findings are consistent with the initial emergence of a clone of hematopoietic cells carrying a single mutation of TET2 and the subsequent development of subclones that carry additional TET2 mutations and become dominant with time. We also compared gene expression profiles of CD34-positive cells from patients with and without somatic mutations of TET2. While these 2 patient groups both had up-regulation of ALAS2 and down-regulation of ABCB7, distinctive “sideroblastic” features at the molecular level (Blood. 2006;108:337-45), no differentially expressed gene was identified between the 2 groups. These data indicate that somatic mutations of TET2 are unlikely to have a major impact on metabolic pathways at the CD34-positive cell level, and are more consistent with an epigenetic regulation function of TET2. In summary, this study shows that about one third of patients with RARS carry somatic mutations of TET2 in circulating granulocytes, clearly indicating that RARS is a true clonal disorder of hematopoiesis despite it presents as a benign erythroid disorder. In most cases, TET2 mutations appear to cause clonal dominance of hematopoietic stem cells, thus initiating the myelodysplastic process. During the clinical course of the disease subclonal evolution may occur through the acquisition of additional somatic mutations of TET2. Disclosures: No relevant conflicts of interest to declare.

Description: We studied patients with myeloid neoplasm associated with ringed sideroblasts and/or thrombocytosis. The combination of ringed sideroblasts 15% or greater and platelet count of 450 × 109/L or greater was found in 19 subjects fulfilling the diagnostic criteria for refractory anemia with ringed sideroblasts (RARS) associated with marked thrombocytosis (RARS-T), and in 3 patients with primary myelofibrosis. JAK2 and MPL mutations were detected in circulating granulocytes and bone marrow CD34+ cells, but not in T lymphocytes, from 11 of 19 patients with RARS-T. Three patients with RARS, who initially had low to normal platelet counts, progressed to RARS-T, and 2 of them acquired JAK2 (V617F) at this time. In female patients with RARS-T, granulocytes carrying JAK2 (V617F) represented only a fraction of clonal granulocytes as determined by X-chromosome inactivation patterns. RARS and RARS-T patient groups both consistently showed up-regulation of ALAS2 and down-regulation of ABCB7 in CD34+ cells, but several other genes were differentially expressed, including PSIP1 (LEDGF), CXCR4, and CDC2L5. These observations suggest that RARS-T is indeed a myeloid neoplasm with both myelodysplastic and myeloproliferative features at the molecular and clinical levels and that it may develop from RARS through the acquisition of somatic mutations of JAK2, MPL, or other as-yet-unknown genes.

Description: Abstract 418 According to the WHO classification, myelodysplastic/myeloproliferative neoplasms include chronic myelomonocytic leukemia, atypical chronic myeloid leukemia (BCR-ABL1 negative), juvenile myelomonocytic leukemia, and myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN, U). The best characterized of these latter conditions is the provisional entity defined as refractory anemia with ringed sideroblasts (RARS) associated with marked thrombocytosis (RARS-T); up to 60% of RARS-T patients harbor the JAK2 (V617F) mutation. Somatic mutations of TET2 have been recently described in myeloid neoplasms, where they appear to be associated with the amplification of the mutated clone at the early stages of hematopoietic differentiation [N Engl J Med. 2009 May 28;360(22):2355-7]. In order to gain a deeper insight into the pathophysiology of RARS-T, we studied a cohort of 187 patients with myeloid neoplasms and investigated the relationship between ringed sideroblasts, thrombocytosis, and mutational status of TET2, JAK2 and MPL. RARS-T was defined according to the following WHO criteria: i) refractory anemia associated with erythroid dysplasia and ringed sideroblasts ≥ 15%; ii) 〈 5% blasts in the bone marrow; iii) platelet count ≥ 450 × 109/L; iv) presence of large atypical megakaryocytes similar to those observed in BCR/ABL1-negative myeloproliferative neoplasms; v) absence of del(5q), t(3;3)(q21;q26) or inv(3)(q21q26). The combination of ringed sideroblasts ≥ 15% and platelet count ≥ 450 × 109/L was found in 19 subjects fulfilling the diagnostic criteria for RARS-T, while 24 patients had RARS without thrombocytosis. JAK2 and MPL mutations were detected in circulating granulocytes and bone marrow CD34+ cells - but not in T-lymphocytes - from 11 out of 19 (58%) RARS-T patients. Three RARS patients, who initially had low to normal platelet counts, progressed to RARS-T, and two of them acquired JAK2 (V617F) at this time. Somatic mutations of TET2 were found in three of the 15 RARS-T patients studied, and the presence of multiple mutant genes allowed analysis of subclones in two of them. One of these patients carried the following three somatic mutations: TET2 (C1271Y), JAK2 (V617F) and MPL (W515L). Analysis of genomic DNA from circulating granulocytes showed 50% TET2 (C1271Y) mutant alleles but smaller proportions of JAK2 (V617F) and MPL (W515L) mutant alleles (5.8% and 20% respectively). We then analyzed five BFU-E grown from peripheral blood mononuclear cells obtained from this patient. All these five colonies were heterozygous for TET2 (C1271Y), while three of them were heterozygous also for MPL (W515L) and the remaining two were heterozygous also for JAK2 (V617F), clearly indicating that erythroid progenitors carrying JAK2 or MPL mutants belonged to subclones of the dominant TET2 (C1271Y) clone. A woman with the TET2 (S1612LfsX4) mutation (50% granulocyte mutant alleles) and fully clonal hematopoiesis as indicated by X-chromosome inactivation patterns, carried 28% JAK2 (V617F) mutant alleles in circulating granulocytes, indicating that granulocytes harboring JAK2 mutant alleles belonged to a subclone of the initial TET2 (S1612LfsX4) mutant clone. Over a 5-year period, in fact, the initial TET2 mutant clone was completely replaced by the TET2/JAK2 mutant subclone. In other two female patients with RARS-T and no somatic mutation of TET2, granulocytes carrying JAK2 (V617F) represented only a fraction (11 to 22%) of clonal granulocytes as determined by X-chromosome inactivation patterns (96 to 100%). Somatic mutations of TET2 were detected also in a significant proportion of patients with RARS without thrombocytosis, while no JAK2 or MPL mutation was identified in these individuals. These observations suggest that the occurrence of a TET2 mutation may represent the initial event determining clonal dominance of hematopoietic cells both in RARS and RARS-T patients, while the subsequent occurrence of JAK2 and/or MPL mutations likely generates myelodysplastic/myeloproliferative subclones in RARS-T patients. In conclusion, RARS-T is indeed a myeloid neoplasm with both myelodysplastic and myeloproliferative features at the molecular and clinical level, and it may develop from RARS through the acquisition of somatic mutations of JAK2, MPL or other as-yet-unknown genes on the background of clonal hematopoiesis caused by somatic mutations of TET2 or other similar (as-yet-unknown) mutant genes. Disclosures: No relevant conflicts of interest to declare.

Description: We searched for JAK2 exon 12 mutations in patients with JAK2 (V617F)-negative myeloproliferative disorders. Seventeen patients with polycythemia vera (PV), including 15 sporadic cases and 2 familial cases, carried deletions or duplications of exon 12 in circulating granulocytes but not in T lymphocytes. Two of the 8 mutations detected were novel, and the most frequent ones were N542-E543del and E543-D544del. Most patients with PV carrying an exon 12 mutation had isolated erythrocytosis at clinical onset, unlike patients with JAK2 (V617F)-positive PV, most of whom had also elevations in white blood cell and/or platelet counts. Both patients with familial PV carrying an exon 12 mutation had an affected sibling with JAK2 (V617F)-positive PV. Thus, several somatic mutations of JAK2 exon 12 can be found in a myeloproliferative disorder that is mainly characterized by erythrocytosis. Moreover, a genetic predisposition to acquisition of different JAK2 mutations is inherited in families with myeloproliferative disorders.

Description: Key Points Different driver mutations have distinct effects on phenotype of myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms (MDS/MPN). Accounting for driver mutations may allow a classification of these disorders that is considerably relevant for clinical decision-making.