Mutational Status of TET2, JAK2, and MPL in Refractory Anemia with Ringed Sideroblasts Associated with Marked Thrombocytosis.
Authors: Malcovati Luca , Brisci Angela , Pietra Daniela , Porta Matteo G Della , Gallífi Anna , Bruno Francesca , Travaglino Erica , Boggi Sabrina , Rumi Elisa , Passamonti Francesco , Ferrari Maurizio , Invernizzi Rosangela , Cremonesi Laura , Cazzola Mario 
Published: October 2019

Abstract
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.