HEMASPHERE-2020-0140; Total nos of Pages: 4; HEMASPHERE-2020-0140 Powered by EHA Case Report Polycythemia Vera Evolution to Chronic Myelomocytic Leukemia: The Prognostic Value of Next Generation Sequencing Maria Huguet1, Lurdes Zamora1, Isabel Granada1, Elisa Orna1, Alba Mesa1, Gustavo Tapia2, Marc Sorigué1, Marco Antonio Fernández-Sanmartín3, Blanca Xicoy1, José-Tomás Navarro1 Correspondence: José Tomás Navarro (e-mail: jnavarro@iconcologia.net). P olycythemia vera (PV) is a myeloproliferative neoplasm monocytosis. Bone marrow aspiration and biopsy were (MPN) that usually evolves to a myelofibrotic phase performed showing significant dyserythropoiesis and dysgranu- and is characterized by the presence of cytopenias. lopoiesis, as well as megakaryocytes with hyposegmented nuclei. There is also a small incidence of progression to acute An increase in the number of mature monocytes was observed leukemia, preceded or not by a myelodysplastic syndrome. and blasts and promonocytes accounted for 11% of the nucleated However, the evolution of PV to a myelodysplastic/myeloprolif- bone marrow cells. Flow cytometry revealed 5% of myeloid erative disorder, such as chronic myelomonocytic leukemia blasts, which were positive for CD34, CD117, HLA-DR, CD33, (CMML), has been scarcely described. We report a case of CD13, CD4 and CD11b, and mature plus immature monocytes JAK2V617F-positive PV which, 6 years after diagnosis, pre- represented 30% of the cellularity. Cytogenetics showed a sented monocytosis along with dysplasia and genetic abnormali- complex karyotype, and eventual fluorescence in situ hybridiza- ties indicating disease progression to CMML. This case also tion (FISH) analysis confirmed TP53 deletion (Fig. 1). FISH for shows the crucial role of next generation sequencing (NGS) BCR-ABL1, PDGFR-alpha and PDGFR-beta were negative. A techniques in the study of MPNs to identify patients at high risk. targeted NGS myeloid panel (Sophia Genetics, Switzerland) A 58-year-old Caucasian man was diagnosed with PV in 2013. detected the JAK2V617F mutation together with other mutations The JAK2V617F mutation was present with allele burden of (Table 1). Cytoreductive treatment was stopped and close follow- 34.13%. A bone marrow biopsy was performed showing absence up was decided. Thirty days after discontinuation of hydroxy- of reticulinic fibrosis. The karyotype was normal, and no further urea, leucocytosis (WBC 28  109/L) with absolute and relative molecular studies were performed at that time. The patient was monocytosis (7.8  109/L and 28%, respectively) were observed, diagnosed with PV, and therapeutic phlebotomies were started. as well as the presence of 4% of blasts on the peripheral blood In May 2016, treatment with hydroxyurea was initiated because smear, and an increase of serum lactate dehydrogenase (747 U/L, of the appearance of leucocytosis and thrombocytosis. The blood normal up to 240). Flow cytometry performed in peripheral counts remained within normal values under cytoreductive blood showed that 95.16% of all monocytes were CD14+/CD16 treatment for the following years. (classical subtype), suggesting the diagnosis of CMML.1 A bone In September 2019 the patient consulted due to general marrow aspirate showed dyserythropoiesis in 72% of precursors, malaise. Blood counts showed pancytopenia with relative dysgranulopoiesis in 90% and dysmegakaryopoiesis in 30%. Moreover, monocytes were increased (9%), and the sum of blasts 1 and promonocytes again represented 11% of bone marrow Hematology Department, ICO-Hospital Universitari Germans Trias i Pujol, Josep cellularity. Additionally, 65% of ring sideroblasts were observed Carreras Leukaemia Research Institute, Universitat Autònoma de Barcelona, Badalona, Spain on Perls stain, with decreased macrophage iron levels. The blast 2 Pathology Department, Hospital Universitari Germans Trias i Pujol, Universitat population presented the same myeloid phenotype described in Autònoma de Barcelona, Badalona, Spain the previous study. Trephine biopsy showed hypercellular bone 3 Flow Cytometry Facility, Germans Trias i Pujol Research Institute, Campus Can marrow, with an increase of the erythroid precursors and Ruti, Badalona, Spain. This work was supported in part by 2017 SGR288 (GRE) from CERCA dysplastic megakaryocytes. The CD34 population positive by Programme/Generalitat de Catalunya, and by funds from Josep Carreras immunohistochemistry represented 5% to 10% of the bone International Foundation and “La Caixa” Foundation. marrow cellularity. The authors declare no conflicts of interest. These results demonstrated that the disease had undergone Copyright © 2020 the Author(s). Published by Wolters Kluwer Health, Inc. on several changes after 6 years of evolution, such as remarkable behalf of the European Hematology Association. This is an open access article distributed under the terms of the Creative Commons Attribution-Non dysplasia involving the three myeloid lineages, appearance of Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible atypical monocytosis and the presence of an immature popula- to download and share the work provided it is properly cited. The work cannot tion (promonocytes and blasts) representing less than 20% of the be changed in any way or used commercially without permission from the bone marrow cellularity. These findings led to the diagnosis of journal. HemaSphere (2020) 4:5(e466). http://dx.doi.org/10.1097/ CMML. These cytologic changes were associated with the HS9.0000000000000466. acquisition of clonal genetic abnormalities. The karyotype Received: 9 May 2020 / Accepted: 7 July 2020 switched from normal to complex with the acquisition of 1 HEMASPHERE-2020-0140; Total nos of Pages: 4; HEMASPHERE-2020-0140 Huguet et al Polycythemia Vera Evolution to Chronic Myelomocytic Leukemia Figure 1. Karyotype and FISH analysis performed at disease progression. (A) Karyotype in the bone marrow sample: 20 metaphases were analyzed. Nineteen metaphases presented a hypodiploid, complex and monosomic karyotype. The following abnormalities were observed: chromosomes 5, 7, 17 monosomies and an unbalanced translocation between 9q11 and 17q11.2. (B) FISH TP53/CEP17 study in interphase nucleus (XL TP53/17cen probe, MetaSystems). The TP53 gene is located at 17p13 (red signal) and the CEP17 probe is located at the centromere of chromosome 17 (green signal). Only one red signal for the17p13 region is observed, confirming a TP53 deletion. The presence of a single green signal can be explained by the unbalanced translocation between 9q11 and 17q11, and the subsequent loss of one chromosome 17 centromere. TP53 deletion (the absence of TP53 deletion at PV diagnosis was these mutations were already present at the time of PV diagnosis, confirmed by FISH). Furthermore, the NGS study showed the an NGS study was retrospectively performed. Strikingly, the presence of additional mutations (Table 1). To confirm whether same mutations along with a JAK2V617F mutation were already 2 HEMASPHERE-2020-0140; Total nos of Pages: 4; HEMASPHERE-2020-0140 (2020) 4:5 www.hemaspherejournal.com Table 1 Variants detected in next generation sequencing (NGS) targeted gene panel, performed at PV diagnosis (2013) and progression (2019). ∗ Classification Gene Chr. Exon c.DNA Protein Total readings VAF† in 2019 (%) VAF† in 2013 (%) Class 1 JAK2 9 14 c.1849G>T p.(Val617Phe) 5233 91.71 43.68 ∗ Class 1 ASXL1 20 13 c.1934dupG p.(Gly646Trpfs 12) 6563 35.91 10.44 Class 1 TP53 17 5 c.523C>G p.(Arg175Gly) 3325 82.41 13.18 Class 3B(‡) TET2 4 3 c.1285G>A p.(Gly429Arg) 5582 50.21 46.96 ∗ Classification of the variants: Class 1: relevant in the clinical management of myeloid hemopathies. It has been established as a pathogenic variant in myeloid hemopathies and alters an actionable gene. Class 2: it has been established as a pathogenic variant in solid tumors or non-myeloid hemopathies and alters an actionable gene. Class 3: variant not previously described, affects an actionable gene and in silico predictors or classifies mutations as: • Class 3A: likely pathogenic. • Class 3B: uncertain significance (VUS). • Class 3C: likely benign. † VAF: variant allele frequency. ‡ This variant has not been previously described in public databases (either as somatic or germline) and in silico predictors classified it, some as benign and others as pathogenic. In this situation, NGS recommendations suggest to classify these variants as of uncertain significance. Chr = chromosome. present 6 years ago, but with lower variable allele frequencies patients in chronic phase,6,7 it is detected in approximately 45% (VAFs) (Table 1). All the changes observed suggested that we cases of acute leukemia evolved from PV.7,8 were in front of the same disease but evolving into a pre-leukemic Although acute leukemia, preceded or not by a myelodysplastic phase. phase, is widely described as a complication of PV, evolution to To support the hypothesis that the CMML had evolved from CMML seems to be uncommon and has been scarcely reported. JAK2V617F-positive PV, we purified CD14+/CD16 cells In an article published by Holcombe in 1991, 2 cases similar to (classical subtype monocytes, which are increased in CMML) the case described herein were reported, but genetic analyses were from peripheral blood by cell sorting (BD FACSAria II), and not available in those patients.9 Recently, Andrei et al published a Sanger sequencing confirmed the presence of the JAK2V617F and case report describing a CMML transformation from PV ASXL1 mutations (already present at PV diagnosis) in this with acquired mutations other than JAK2.10 In this case they CMML cell subtype. With these results, the patient was confirmed that the CD14+ population carried the JAK2V617F diagnosed with PV that had transformed into CMML type 2. mutation, and they concluded that the CMML was originated Due to the poor prognosis conferred by the cytogenetic and from the PV clone. On the other hand, the JAK2V617F mutation molecular alterations, treatment with intensive chemotherapy is reported to be present in 7.8% of de novo CMML cases,10 followed by an allogeneic bone marrow transplant was especially in proliferative subtypes. considered. Before the treatment was started and only six weeks Absolute monocytosis can be seen in myeloid neoplasms other after the diagnosis of CMML, the disease progressed to acute than CMML.11 In the case of primary myelofibrosis, monocytosis myeloid leukemia. Finally, the patient was treated with has been associated with progression to an accelerated phase.12 Venetoclax and Azacytidine and died of respiratory failure one Regarding PV, monocytosis is a frequent finding with an month later without an identified infectious agent. incidence of nearly 21%.13 The Barraco study suggested that Polycythemia vera is a myeloproliferative neoplasm character- PV patients with monocytosis present a specific phenotype ized by polyglobulia, bone marrow erythroid hyperplasia and the consisting in older age (>60 years) and a higher frequency of presence of a JAK2 mutation.2 The median survival of patients other mutations (TET2 and SRSF2).13 Furthermore, monocytosis with PV is approximately 14 years, being up to 24 years in in PV cases seems to have a negative impact on OS, identifying a younger patients.2 The main PV-related complications with an subgroup of patients with a biologically more aggressive disease impact on overall survival (OS) are thrombotic events and that share some genetic features with CMML.13 progression to myelofibrosis or acute myeloid leukemia.3 At 10 In the case described here, additional mutations were detected years, the probability of progression to acute leukemia is 2.3% to at PV diagnosis, and two (ASXL1, TP53) have been reported to 14.4%, and it is often preceded by myelodysplasia.4 In fact, it has confer poor prognosis in several myeloid neoplasms.6,7 Striking- been observed that dyserythropoiesis acquisition in general, and ly, the evolution to CMML -with the subsequent progression to especially the presence of ring sideroblasts, are markers of acute leukemia- was associated with a significant increase of the progression in PV.5 VAF of all the mutations, including the two with poor prognosis. Although only 20% of patients with PV present cytogenetic However, the TET2 VAF detected in the 2013 and 2019 studies abnormalities at diagnosis,4 patients may acquire karyotype would suggest that it may be a typical polymorphism for this alterations during the course of the disease.4 Multiple studies patient, but since the patient died before germline tissue could be have associated the acquisition of a complex karyotype with an obtained, we were unable to verify the true origin of this variant. increased risk of progression to blast phase,3,6 as occurred in the These findings highlight the crucial role that NGS techniques present case. currently have in the diagnosis and follow-up of MPN, being able Regarding molecular findings, recent retrospective studies have to identify patients with higher risk of evolution to a leukemic reported that approximately 30% of JAK2-positive PV cases phase and, consequently, the use of NGS should be considered present concomitant mutations other than JAK2 (most frequently and extended to routine practice.7,14 In fact, a recently study by TET2 and ASXL1).7 The presence of ASXL1, SRSF2, and IDH2 Tefferi et al demonstrated the utility of integrating genetic mutations has been associated with a lower OS and an increased information in the new survival scores for MPN.14 risk of progression to fibrosis or blast phase.2,7,8 On the other In summary, we present an uncommon case of transformation hand, although the TP53 mutation is observed in only 1% of PV of PV to CMML with the same genetic alterations in the cells of 3 HEMASPHERE-2020-0140; Total nos of Pages: 4; HEMASPHERE-2020-0140 Huguet et al Polycythemia Vera Evolution to Chronic Myelomocytic Leukemia the 2 populations of the disease (indicating a common origin). of patients with an increased risk of progression to blast phase. Ann Diagn Pathol. 2019;40:45–48. The aggressive character of the disease and the fatal outcomes 6. Hidalgo-Lopez JE, Carballo-Zarate A, Verstovsek S, et al. Bone marrow reported suggest that the appearance of monocytosis in PV cases, findings in blast phase of polycythemia vera. Ann Hematol. 2018; as described in primary myelofibrosis, might be considered as a 97:425–434. sign of evolution to an accelerated or preleukemic phase. This 7. Tefferi A, Lasho TL, Guglielmelli P, et al. Targeted deep sequencing in case also highlights the usefulness of performing NGS studies at polycythemia vera and essential thrombocythemia. Blood Adv. 2016;1:21–30. diagnosis of MPN and considering some genetic alterations in 8. Yogarajah M, Tefferi A. Leukemic transformation in myeloproliferative upcoming prognostic scores for PV, as suggested in recent neoplasms: a literature review on risk, characteristics and outcome. studies.14 Mayo Clin Proc. 2017;92:1118–1128. 9. 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