Myelodysplastic Syndrome/Myeloproliferative Neoplasm with Ring Sideroblasts and Thrombocytosis with Cooccurrent SF3B1 and MPL Ge

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Myelodysplastic Syndrome/Myeloproliferative Neoplasm with Ring Sideroblasts and Thrombocytosis with Cooccurrent SF3B1 and MPL

Gene Mutations: A Case Report and Brief Review Downloaded from https://academic.oup.com/labmed/article/51/3/315/5681672 by guest on 01 October 2021 of the Literature Chang-Hun Park, MD,1 Jae Won Yun, MD, PhD2 Hyun-Young Kim, MD,2,3 Ki-O Lee, AS,4 Sun-Hee Kim, MD, PhD2 Hee-Jin Kim, MD, PhD 2*

Laboratory Medicine 2020;51:315-319

DOI: 10.1093/labmed/lmz076

ABSTRACT Background: Myelodysplastic syndrome/myeloproliferative neoplasm mutations in MPN disease (JAK2 V617F/exon 12, CALR gene exon 9, with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T) is a new and MPL gene exon 10 mutations). disease entity in the current WHO classification. Genetically, 60%–90% of cases have mutations in SF3B1, strongly associated with RS, and more Results: The molecular study revealed SF3B1 K666T and MPL W515R than half of them cooccur with JAK2 V617F. This report describes the mutations, while BCR-ABL1 or JAK2 V617F/exon 12 and CALR rare case of MDS/MPN-RS-T with SF3B1 mutation cooccurring with an mutations were all negative. MPL mutation.

Methods: We report a 79-year-old man who was referred because Conclusion: This is a rare case of concomitant SF3B1 and MPL of generalized edema. Peripheral blood testing showed macrocytic mutations in MDS/MPN-RS-T. anemia and thrombocytosis, and bone marrow analysis demonstrated dyserythropoiesis with RS and increased megakaryocytes. A molecular Keywords: myelodysplastic syndrome, myeloproliferative neoplasm, ring

study was performed to detect SF3B1 mutations and recurrent sideroblasts, thrombocytosis, SF3B1, MPL

Myelodysplastic syndrome/myeloproliferative neoplasm and thrombocytosis (RARS-T), is a new disease entity in the with ring sideroblasts and thrombocytosis (MDS/MPN- current WHO classification of hematologic malignancies.1,2 RS-T), formerly referred to as refractory anemia with RS Hematologically, MDS/MPN-RS-T has features of both MDS and MPN, having dysplasia with increased ring sideroblasts Abbreviations: (≥15%) and thrombocytosis (platelet count, ≥450 × 109/L).1 MDS/MPN-RS-T, myelodysplastic syndrome/myeloproliferative neoplasm It is a rare disease and has a relatively indolent course with ring sideroblasts and thrombocytosis; RARS-T, refractory anemia with RS and thrombocytosis; CBCs, complete blood counts; Hb, hemoglobin; MCV, compared to other MDS/MPN diseases, such as chronic mean corpuscular volume; WBC, white blood cell; FISH, fluorescence in situ myelomonocytic leuekemia and atypical chronic myeloid hybridization; ET, essential thrombasthenia; PMF, primary myelofibrosis; leukemia.3 W515, tryptophan 515; AML, acute myeloid leukemia; BM, bone marrow.

1Department of Laboratory Medicine & Genetics, Samsung Changwon Genetically, mutations in the SF3B1 gene are the most fre- Hospital, Sungkyunkwan University School of Medicine, Changwon, quent molecular defect in MDS/MPN-RS-T (60%~90%).1,4,5 2 Korea, Department of Laboratory Medicine & Genetics, Samsung Medical The SF3B1 gene encodes subunit 1 of the splicing factor Center, Sungkyunkwan University School of Medicine, Seoul, Korea, 3 3b protein, and the SF3B1 mutant has a strong genotype– Department of Laboratory Medicine, Gyeongsang National University Hospital, Jinju, Korea, 4Samsung Biomedical Research Institute, Samsung phenotype correlation with the presence of RS. On the other Medical Center, Seoul, Korea. hand, genes mutated in MPN diseases are also commonly affected, with the JAK2 V617F mutation detected in ~60% of *To whom correspondence should be addressed. [email protected] cases. Approximately half of patients with MDS/MPN-RS-T

have concomitant SF3B1 and JAK2 V617F mutations.4 Unlike JAK2 V617F, other MPN mutations, such as MPL and CALR mutations, are infrequent in MDS/MPN-RS-T.6,7

In this article, we report the rare case of concomitant SF3B1 and MPL mutations in a patient with MDS/MPN-RS-T; also, clinical and molecular findings are discussed. Downloaded from https://academic.oup.com/labmed/article/51/3/315/5681672 by guest on 01 October 2021

Clinical History

A 79-year-old man was referred to our hospital because of generalized edema. He had a past medical history of benign prostatic hyperplasia. Mild splenomegaly was detected on abdominal computed tomography.

Laboratory Information

The peripheral blood smear showed macrocytic anemia with anisopoikilocytosis (teardrop cells, elliptocytes, and acanthocytes), left-shifted neutrophils with eosinophilia, and thrombocytosis. Complete blood counts (CBCs) were hemoglobin (Hb) of 116 g/L, with an increased mean corpuscular volume (MCV) 101.8 fL, white blood cell (WBC) count of 7.74 × 109/L (myelocytes, 2%; band, 5%; neutrophils, 44%; monocytes, 10%; lymphocytes, 11%; and eosinophils, 29%), and platelets of 795 × 109/L. The bone marrow study demonstrated dyserythropoiesis, increased eosinophil precursors, and increased megakaryocytes (Images 1A and 1B). Blasts were less than 5%. Iron stain revealed ring sideroblasts up to 24% (Image 1C). The cellularity was normal for age (~20%), and there was no evidence of fibrosis. A chromosome study revealed 46,XY,i(14)(q10)[10]/46,XY[10]. Fluorescence in situ hybridization (FISH) analyses for FGFR1 (dual-color break-apart probe; MetaSystems, Altlussheim, Germany), PDGFRB (dual-color break-apart probe; Kreatech Diagnostics, Leica Biosystems, IL, USA), FIP1L1-PDGFRA (LSI tricolor prob; Vysis, Abbott Laboratories, Abbott Park, IL, USA), Image 1 and BCR/ABL1 (LSI dual-color translocation probe; Vysis) Bone marrow cytomorphology in the patient with myelodysplasic were performed for eosinophilia and increased eosino- syndrome/myeloproliferative neoplasm with ring sideroblasts philic precursors in bone marrow, which showed normal and thrombocytosis. Bone marrow aspirate smear showed (A) signals. megakaryocytic hyperplasia (Wright Giemsa, ×400) and (B) dyserythropoiesis with multinuclearity and increased eosinophil A molecular genetic study was performed on DNA extracted precursor (Wright Giemsa, ×1000). (C) Iron stain revealed frequent from bone marrow aspirate specimens to detect SF3B1 ring sideroblasts (Prussian blue, ×1000).

316 Lab Medicine 2020;51;315–319 www.labmedicine.com DOI: 10.1093/labmed/lmz076 Case Study

gene mutations and recurrent gene mutations in MPN diseases (JAK2 V617F/exon 12 mutations, CALR gene exon 9 mutations, and MPL gene exon 10 mutations). PCR was performed on a thermal cycler (Applied Biosystems, Foster City, CA, USA), and direct sequencing analyses were performed by using the BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems) on the ABI 3100 genetic analyzer (Applied Biosystems). The primers for the experiments were designed by the authors (available upon Downloaded from https://academic.oup.com/labmed/article/51/3/315/5681672 by guest on 01 October 2021 request). In addition, reverse transcription-PCR was performed to detect BCR-ABL rearrangement.

As a result, we detected a c.1997A>C (p.K666T) mutation in the SF3B1 gene and a c.1543T>A (p.W515R) mutation in the MPL gene. Both mutations are previously reported, rare mutations. BCR-ABL rearrangement was negative, and JAK2 V617F/exon 12 mutations and CALR gene exon 9 mutations were also all negative. In order to confirm the somatic nature of the SF3B1 K666T and MPL W515R mutations, we Figure 1 additionally performed targeted sequencing analyses using Detection of SF3B1 c.1997A>C (p.K666T) mutation and MPL the DNA extracted from the patient’s buccal swab speci- c.1544G>T (p.W515R) mutation in the bone marrow specimen from mens and found negative results (Figure 1). Collectively, the the patient. The mutations were negative in the DNA extracted patient was diagnosed as having MDS/MPN-RS-T. from buccal swab specimen of the patient, confirming the somatic nature.

Patient Follow-Up that the patient’s bone marrow cells had a somatic SF3B1 K666T mutation and an MPL W515R mutation, and chromo- The patient received treatment with anagrelide and some analyses showed isochromosome 14q. The workup hydroxyurea. The patient is currently on follow-up for for the possibility of a concomitant clonal hematologic dis- 5 months from the diagnosis. During the 1 week of the initial order associated with eosinophilia in the patient, including anagrelide treatment, the hemoglobin level decreased from FISH for FGFR1, PDGFRA, PDGFRB, and BCR/ABL1 106 g/L to 87 g/L but shortly recovered to 103 g/L without rearrangement, were all negative. According to a study transfusion (Figure 2A). The platelet count decreased from involving 92 patients with MDS/MPN-RS-T, mutations in 828 × 109/L to 578 × 109/L after the initial week of anagrelide the spliceosome components were the most frequent, with treatment and then increased to 720 × 109/L; it then fell to the SF3B1 mutations in 90%, followed by SRSF2 mutations in reference range after combination therapy with hydroxyurea 6.7%, U2AF1 mutations in 5.3%, and ZRSR2 mutations in (Figure 2B). After 142 days from the start of anagrelide 2.7%.4 The second common group of gene mutations was treatment, the patient had to stop anagrelide therapy due to those in the signaling pathway genes, JAK2 V617F (57%), non-ST segment elevation myocardial infarction. MPL (2.7%), and CBL (4%).4 Common mutations in SF3B1 are K700E (49%), H662Q (17%), R625L (7%), K666N (5%), K666R (5%), G742D (5%), E622D (5%), K666E (2%), K666T (2%), and E622Q (2%).5 Thus, the mutation identified in our patient, K666T, is a previously reported but rare mutation Discussion and Laboratory Role of SF3B1. The JAK2 V617F mutation commonly occurs in Diagnosis in association with SF3B1 mutations in MDS/MPN-RS-T, while MPL mutations are rare (2.7%).4 The MPL gene en- In this report, we describe a patient with MDS/MPN-RS-T codes thrombopoietin receptor and is the major regulator of with a rare genetic profile. Molecular genetic tests revealed megakaryopoiesis. Mutations in the exon 10 (juxtamembrane

www.labmedicine.com Lab Medicine 2020;51;315–319 317 DOI: 10.1093/labmed/lmz076 Case Study

A that maintains the protein in an inactive form when without 125 cytokine, is the mutation hotspot, and substitution with other 115 amino acids (typically W515L or W515K) leads to consti- 110 tutive activation of the protein and the MPN phenotype.8,9 105 Other rare mutations in W515 are W515N and W515R, and 100 our patient had W515R. A review of the literature showed 3 95 cases of MDS/MPN-RS-T with an MPL mutation, all of which 90 were W515L (Table 1).

Hemoglobin (g/L) 85 80 The SF3B1 gene mutation status was not described in Downloaded from https://academic.oup.com/labmed/article/51/3/315/5681672 by guest on 01 October 2021 Anagrelide 2 mg plus hydroxyurea 500mg 75 those cases. Thus, our patient represents the rare case 03160 91 121 of MDS/MPN-RS-T with concomitant SF3B1 K666T and BM study Days After Visit MPL W515R mutations, both of which are rare. Clinical and at Our Hospital hematologic features of MDS/MPN-RS-T with MPL mutation B are limited. Based on the data from our case and other 3 900 cases, the median age of patients at diagnosis was 71 years )

/L 800 9 (range, 63–82 years). The median value of hemoglobin was 700 115 g/L (range, 76–133 g/L), and the median platelet count 9 9 600 was 900 × 10 /L (range, 795–1222 × 10 /L). WBC counts were normal. According to Patnaik et al., 82 cases with 500 MDS/MPN-RS-T had a median Hb level of 95 g/L (range, 400 56–105 g/L), median WBC count of 8.2 × 109/L (range, 300 1.9–27 × 109/L), and median platelet count of 582 × 109/L Platelet Counts (× 10 Anagrelide 2 mg plus hydroxyurea 500mg 200 (range, 454–1798 × 109/L), with a median age at diagnosis of 03160 91 121 72 years (range, 48–93 years).5 Thus, MDS/MPN-RS-T with BM study Days After Visit at Our Hospital the MPL mutation seems to have milder anemia and higher platelet counts than does the overall MDS/MPN-RS-T. C 16 Cytogenetically, approximately 80% of patients with MDS/ 14 MPN-RS-T do not have detectable clonal cytogenetic 12 abnormalities.4,5,13 The cytogenetic aberration detected 10 in our patient, isochromosome 14q, has not previously 8 been reported in patients with MDS/MPN-RS-T. According 6 to the Mitelman database,14 the majority of patients with 4 Hemoglobin (g/L) isochromosome 14q presented with MDS, followed by 2 Anagrelide 2 mg plus hydroxyurea 500mg acute myeloid leukemia (AML). 0 03160 91 121 The frequency of thrombotic events in MDS/MPN-RS-T is BM study Days After Visit at Our Hospital thought to be similar to that of ET but more frequent than MDS-RS.15 Sixty percent of patients with MDS/MPN-RS-T Figure 2 with an SF3B1 mutation had thrombotic events; whereas Clinical course of the patient. Changes in (A) hemoglobin levels, (B) there were no thrombotic events in patients without an platelet counts, and (C) white blood cell counts during the treatment SF3B1 mutation.16 According to Patnai, et al., patients course and follow-up are shown. x axis, course in days; BM, bone marrow. with MDS/MPN-RS-T (especially those with SF3B1 mutations) are divided into 2 categories, low risk (0 factors) and high risk (1 or 2 factors), based on age of >60 years and a domain) are reported in 3%–5% of essential thrombasthenia prior thrombotic event.17 The high-risk group is indicated (ET) and primary myelofibrosis (PMF), disease entities clas- for cytoreductive therapy with hydroxyurea. However, the sified in MPN characterized by predominantly increased cytoreductive therapy in MDS/MPN-RS-T might exacer- megakaryopoiesis. Tryptophan 515 (W515), the amino acid bate anemia. Clinically, the most common disease-related

318 Lab Medicine 2020;51;315–319 www.labmedicine.com DOI: 10.1093/labmed/lmz076 Case Study

Table 1. Hematological Features of Cases of MDS/MPN-RS-T with MPL Mutation Patient Sex Age at Dx Hb MCV Leukocytes Platelets Mutation in: Chromosome Reference no. (y) (g/L) (fL) (×109/L) (×109/L) Study SF3B1 MPL 1 F 63 76 109 8.2 834 Unknown W515L del(13q) 10 2 M 63 133 ? 6.4 1222 Unknown W515L Normal 11 3 F 82 114 90 8.70 966 Unknown W515L Unknown 12 4 M 79 116 101.8 7.74 795 K666T W515R i(14)(q10) The present case

M, male; F, female; Dx, diagnosis; Hb, hemoglobin; MCV, mean corpuscular volume. Downloaded from https://academic.oup.com/labmed/article/51/3/315/5681672 by guest on 01 October 2021

manifestation of MDS/MPN-RS-T is anemia. Our patient was mutations in SF3B1 or other spliceosome genes accompanied by JAK2V617F and ASXL1 mutations. Haematologica. at high risk for thrombosis due to his old age (>60 years), and 2015;100(4):e125–e127. he had mild anemia. For those reasons, he received combin- 5. Patnaik MM, Lasho TL, Finke CM, et al. Predictors of survival ation treatment with anagrelide and hydroxyurea to prevent in refractory anemia with ring sideroblasts and thrombocytosis (RARS-T) and the role of next-generation sequencing. Am J Hematol. anemia and thrombosis. The most common side effects of 2016;91(5):492–498. anagrelide are headache, nausea, diarrhea, edema, palpita- 6. Broséus J, Lippert E, Harutyunyan AS, et al. Low rate of calreticulin tions, and an increased number of myocardial contractions.18 mutations in refractory anaemia with ring sideroblasts and marked thrombocytosis. Leukemia. 2014;28(6):1374–1376. Potential cardiovascular adverse effects include vasospasm 7. Klampfl T, Gisslinger H, Harutyunyan AS, et al. Somatic mutations of the coronary arteries, congestive heart failure, arrhythmia, of calreticulin in myeloproliferative neoplasms. N Engl J Med. or acute coronary syndrome.18 In addition, myocarditis, silent 2013;369(25):2379–2390. myocardial infarction, and myocardial ischemia have been re- 8. Pardanani AD, Levine RL, Lasho T, et al. MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients. ported in young patients with ET and without cardiovascular Blood. 2006;108(10):3472–3476. risk factors following the administration of anagrelide.18,19 9. Pikman Y, Lee BH, Mercher T, et al. MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia.PLoS Med. 2006;3(7):e270. 10. Hao L, Sen S, Sugumar D. An MPL W515L mutation in refractory anemia with ringed sideroblasts associated with marked thrombocytosis: A case report. Oncol Lett. 2015;9(2):749–751. Conclusion 11. Schnittger S, Bacher U, Haferlach C, et al. Detection of an MPLW515 mutation in a case with features of both essential thrombocythemia We detected concomitant SF3B1 K666T and MPL W515R and refractory anemia with ringed sideroblasts and thrombocytosis. Leukemia. 2008;22(2):453–455. mutations in a patient with MDS/MPN-RS-T, which is a rare 12. Schmitt-Graeff AH, Teo SS, Olschewski M, et al. JAK2V617F case in the literature. Albeit based on a limited number of mutation status identifies subtypes of refractory anemia with ringed cases, we speculated that MDS/MPN-RS-T with SF3B1 sideroblasts associated with marked thrombocytosis. Haematologica. 2008;93(1):34–40. and MPL mutations might have milder anemia and higher 13. Patnaik MM, Tefferi A. Refractory anemia with ring sideroblasts and platelet counts than would the overall MDS/MPN-RS-T RARS with thrombocytosis. Am J Hematol. 2015;90(6):549–559. population. LM 14. Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer (2019). Bethesda, MD: Cancer Genome Anatomy Project, National Cancer Institute; 2019. http://cgap.nci.nih.gov/Chromosomes/Mitelman. 15. Broseus J, Florensa L, Zipperer E, et al. Clinical features and course of refractory anemia with ring sideroblasts associated with marked thrombocytosis. Haematologica. 2012;97(7):1036–1041. 16. Visconte V, Makishima H, Jankowska A, et al. SF3B1, a splicing factor References is frequently mutated in refractory anemia with ring sideroblasts. Leukemia. 2012;26(3):542–545. 1. Swerdlow SH, Campo E, Harris NL, et al, ed. WHO classification 17. Patnaik MM, Tefferi A. Refractory anemia with ring sideroblasts (RARS) of tumours of haematopoietic and lymphoid tissues. Lyon, France: and RARS with thrombocytosis (RARS-T): 2017 update on diagnosis, International Agency for Research on Cancer; 2017. risk-stratification, and management. Am J Hematol. 2017;92(3):297– 2. Arber DA, Orazi A, Hasserjian R, et al. The 2016 revision to the World 310. Health Organization classification of myeloid neoplasms and acute 18. Mazzucconi MG, Redi R, Bernasconi S, et al. A long-term study of leukemia. Blood. 2016;127(20):2391–2405. young patients with essential thrombocythemia treated with anagrelide. 3. Talati C, Padron E. An exercise in extrapolation: clinical management Haematologica. 2004;89(11):1306–1313. of atypical CML, MDS/MPN-unclassifiable, and MDS/MPN-RS-T. Curr 19. Lim YH, Lee YY, Kim JH, et al. Development of acute myocardial Hematol Malig Rep. 2016;11(6):425–433. infarction in a young female patient with essential thrombocythemia 4. Jeromin S, Haferlach T, Weissmann S, et al. Refractory anemia treated with anagrelide: a case report. Korean J Hematol. with ring sideroblasts and marked thrombocytosis cases harbor 2010;45(2):136–138.

www.labmedicine.com Lab Medicine 2020;51;315–319 319 DOI: 10.1093/labmed/lmz076