Triple-negative Thrombocythemia and Subsequent Acute Lymphoblastic Leukemia with Additional Somatic Mutations

Abstract

Triple-negative essential thrombocythemia (ET) is a condition in which mutations in JAK2, CALR and MPL are all negative. Transformation to acute myeloid leukemia may occur during the course of ET, while B-acute lymphoblastic leukemia B-(ALL) is rare. We experienced a case diagnosed as B-ALL during the course of triple-negative ET. Notably, cytoreduction was required for the excessive increase in blood cells during the bone marrow recovery period after chemotherapies. Whole exome sequencing identified 17 somatic mutations: 9 were identified in both ET and B-ALL samples, while 8 were specific to B-ALL, suggesting that these 8 might have caused the transformation.

Introduction

Essential thrombocythemia (ET) is a myeloproliferative neoplasm (MPN) in which megakaryocytic cells proliferate autonomously in the bone marrow (BM) due to genetic mutations in hematopoietic stem cells, resulting in a sustainable increase in platelets in the peripheral blood (PB) (1). The recurrent mutations in JAK2 are found in 50-60% of ET cases, CALR in 30%, and MPL in 3% (1). Patients harboring none of these mutations in their BM or PB are diagnosed as triple-negative ET and account for about 12% of ET cases (2).

The time to progression to acute leukemia, known as transformation, ranges from a few months to more than 20 years after the ET diagnosis and the ratio for transformation is estimated to be 0.7-3% at 10 years after the diagnosis (3). According to the JSH-MPN-R18 study, a large-scale retrospective study of ET conducted in Japan, triple-negative-ET accounted for 20.7% of all ET cases. In terms of disease progression, the cumulative 5- and 10-year progression rates for all ET patients were 3.8% and 9.0%, respectively. The rate of progression to acute leukemia was 0.6% and 1.5% at 5 and 10 years, respectively (4). Therefore, the longer the time since the diagnosis, the higher the rate of transformation (5).

Most tumor subtypes of transformation are either myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) and very rarely B-acute lymphoblastic leukemia (ALL) (6). During the typical transformation process from ET to MDS/AML, the ET clones first acquire the IDH1/2, RUNX1, or U2AF1 mutations, and then TP53, NRAS, and BCORL1 mutations accumulate in these clones in the later phase (7,8). However, the genetic events during the transformation process from ET to B-ALL have not been clarified because of the rarity of this entity.

The MPL W515S mutation was reported in a patient harboring both tumors (9). Notably, JAK1/2 inhibitors, which are commonly used for the treatment of MPN, have been reported to increase the incidence of B-cell lymphomas, although the mechanism has not been clarified (10).

We herein report a patient harboring triple-negative ET and subsequently B-ALL. A genetic analysis was conducted to clarify the clonal evolution process of these diseases.

Case Report

A 77-year-old woman was referred to our hospital because of an abnormal PB count in a routine blood test 2 years before (year X-2): white blood cell (WBC) count, 11,000 /μL; hemoglobin concentration (Hb), 10.1 g/dL; and platelet count, 648,000 /μL. Reactive thrombocytosis was excluded based on the symptoms and clinical test findings. She also had no history of diseases that might cause reactive thrombocytosis. The levels of C-reactive protein, ferritin, and immunoglobulin were normal. The values of antinuclear antibodies proteinase 3 (PR3)-antineutrophil cytoplasmic antibody (ANCA) and myeloperoxidase (MPO)-ANCA were under the cut-off.

BM smears showed an increase in megakaryocytes, but those with an abnormal morphology, including separated polynuclear megakaryocytes and small megakaryocytes, accounted for ＜10% [Supplementary material 1A(i)]. There was no marked increase in granulocyte-lineage cells or erythroblasts. Ringed sideroblasts were not seen on iron staining (data not shown). Megakaryocytes had deeply lobulated and hyperlobulated (stag-horn-like) nuclei, while bizarre, highly atypical megakaryocytes and dense clusters of megakaryocytes were not seen in the BM biopsy sample [Figure A (i)]. Fibrotic tissues were not observed by either silver staining [Figure A (ii)] or Masson's Trichrome staining [Figure A (iii)].

Figure.

A: A fibrotic evaluation of a bone marrow biopsy section of ET at the diagnosis. (i) Hematoxylin and Eosin staining, (ii) silver staining, (iii) Masson’s trichrome staining. B: A flow cytometric analysis of B-ALL in peripheral blood. The numbers in the right panels indicate the proportions of each fraction gated by CD45 fluorescence and side-scatter (SSC). C: Clinical course. The white blood cell (WBC) and blast counts (left axis) and platelet (Plt) counts (right axis) during the clinical course. HU: hydroxycarbamide, PSL: prednisolone, DNR+VCR: daunorubicin and vincristine, HD-AraC: high-dose cytarabine. D: Schematic illustration of the clonal evolution of B-ALL from triple-negative ET.

A chromosome analysis by G-Banding showed 46, XX [20/20]. Of note, we routinely examined the V617F mutations in JAK2, exon 9 in CALR, and W515 L and W515K mutations in MPL in routine clinical tests. The JAK2, CALR, and MPL mutations were not detected. We further confirmed that no mutations were found in the JAK2, CALR, or MPL genes by whole-exome sequencing (WES) in the subsequent analysis.

Based on these findings, the patient was diagnosed with triple-negative ET, so she was prescribed low-dose aspirin and hydroxycarbamide [hydroxyurea (HU)] 500 mg/body/day 4 times a week, as she was at high risk for thrombosis due to her age. A blood test at the regular outpatient visit in year X showed a WBC count of 32,000 /μL, with 59% blastoid cells. The Hb level and platelet count were decreased to 8.5 g/dL and 10,000 /μL, respectively. BM aspiration showed a dry tap. Touch preparation of the BM biopsy specimen revealed that 90% of mononuclear cells (MNCs) were blastoid cells, some of which had a slit in the nucleus [Supplementary material 1A(ii)]. MPO staining was negative (data not shown).

A flow cytometric analysis in PB showed that cells with the immunophenotype of CD45^dull+, CD10⁺, CD19⁺, CD34⁺, CD3^-, CD13^-, and CD33^- were aberrantly increased (Figure B). The chromosome analysis by G-Banding could not be obtained. BCR-ABL1 analyzed by fluorescence in situ hybridization was negative. Based on these data, she was diagnosed with B-ALL.

She received remission induction therapy with daunorubicin (DNR; 30 mg/m², days 1-3), vincristine (VCR; 1.3 mg/m², days 1, 8, 15, and 22), and prednisolone (PSL; 60 mg/m², days -7 to 1, days 1-21). A BM examination performed on day 41 showed remission, although giant platelets and dysplastic erythroblasts were still observed [Supplementary material 1A(iii)]. Consolidation therapy with cytarabine (Ara-C, 500 mg/m², days 1-3), etoposide (ETP, 60 mg/m², days 1-3), and dexamethasone (DEX, 40 mg/body, days 1-3) was given. Once the WBC count decreased to 300 /μL, it continuously increased up to 50,000 /μL on day 18. Therefore, HU 500 mg/body/day was administered (Figure C).

WES was performed using genomic DNA extracted from BM MNCs collected at the diagnosis of ET, PB MNCs collected at the diagnosis of B-ALL, and oral mucosa specimens as a reference. Libraries were made using SureSelect XT All Exon Human V7 kits (Agilent, Santa Clara, USA) and then sequenced on the Hiseq X platform (Illumina, San Diego, USA) using the standard 150-bp paired-end protocol (Macrogen, Kyoto, Japan). Sequence alignment as well as mutation calling was performed by the Genomon2 pipeline (https://github.com/Genomon-Project). The median average depth for sequencing was ×205 (×198-×205). A total of 17 somatic mutations were found (Supplementary material 2). Thirteen were missense mutations, while four were splice-site mutations. Mutations in nine genes (MEIS1, NBEAL2, ADAMTSL1, MRGPRE, PLEKHA7, CHD4, TFDP1, PRPF8, and GABRA3) were commonly identified in both ET and B-ALL samples, while those in eight (ULK4, OTOGL, UGGT2, SUGP1, TET2, NAA15, PPFIA1, and SNAP23) were specifically identified in the B-ALL sample (Figure D, Supplementary material 2). The variant allele frequencies (VAFs) of mutations commonly identified in both ET and B-ALL samples were distributed between 0.36-0.49 in ET. Notably, in the B-ALL sample, the VAFs of these mutations also varied from 0.39-0.51, except that that of CHD4, which was relatively high at 0.81. The VAFs of the B-ALL-specific mutations were distributed between 0.31 to 0.58, while that of SUGP1 was relatively low at 0.09.

Discussion

We report a patient who developed B-ALL during the course of ET. The significant increase in the WBC count in the recovery phase of B-ALL treatment indicated that the ET state had not been eradicated. The genetic analysis revealed that nine mutations were identified in both ET and B-ALL, while an additional eight were found in B-ALL only. These data suggest that B-ALL had transformed from the triple-negative ET clone with nine mutations by acquisition of the eight B-ALL-specific mutations (Figure D).

The VAFs of mutations identified in this study were distributed around 40%. These data suggested that most of the mutations existed in almost all of the ET or B-ALL tumor cells, assuming that they were heterozygous. Remarkably, the CHD4 mutation in B-ALL might be loss of heterozygosity, as its VAF in B-ALL was much higher than that in ET. In addition, the SUGP1 mutation only existed in a subclone of B-ALL, as its VAF was much lower than those of the other B-ALL-specific mutations.

Notably, some mutated genes are known to be associated with leukemogenesis. For example, among the genes mutated in both the ET and B-ALL samples, MEIS1 encodes a transcription factor with a homeodomain that belongs to the three amino-acid loop extension (TALE) family (11). A high expression of the MEIS1 gene has been known to play essential roles in leukemogenesis (11). CHD4 belongs to the SNF2/RAD54 helicase family, regulating chromatin organization and nucleosome positioning (12). Somatic mutations in CHD4 have been reported in pediatric AML (13) and POEMS syndrome (14). Mutations in the PRPF8 gene encoding a spliceosomal protein have been reported in a large series of genetic studies in myeloid cancers (15).

ULK4, OTOGL, UGGT2, SUGP1, TET2, NAA15, PPFIA1, and SNAP23 were specifically found in the B-ALL sample. The driver mutations ASXL1, TET2, SRSF2, RUNX1, and TP53 were previously described in other studies on leukemic transformation from MPN, although most of the samples analyzed in those studies might be myeloid leukemia (7,8). A mutation in the TET2 gene encoding an epigenetic regulator was also found in the present B-ALL sample. SUGP1 encoding a splicing factor is mutated in various cancers (16). SUGP1 mutations result in 3' splice site abnormalities, resembling abnormalities caused by mutations in SF3B1, a gene known to be frequently mutated in both myeloid and lymphoid cancers (16). These data suggest that these mutations might be the driver mutations for B-ALL development. Conversely, the roles of ULK4, OTOGL, UGGT2, NAA15, PPFIA1, or SNAP23 mutations in blood cancers are poorly understood.

We described a case of clonal evolution of B-ALL from triple-negative ET. Further attention is warranted if recovery of hematopoiesis is excessive after chemotherapy for ALL; such patients might have hidden ET or other MPNs.

The study was approved by the institutional review board at University of Tsukuba Hospital.

Written informed consent was obtained from the patient.

The data and materials are available upon request.

The authors state that they have no Conflict of Interest (COI).

Financial Support

This work was partially supported by Grants-in-Aid for Scientific Research [KAKENHI: JP21K16261 (T.S), JP21K16262 (Y.S.), JP10832024 to (K.H.), JP21H02945 and JP22K19451 (M.S.-Y)] from the Ministry of Education, Culture, Sports, and Science of Japan; AMED under Grant Number JP20ck0106544 (M.S.-Y.). The work was also supported by the Naito Foundation (M.S.-Y).

Supplementary Material

Supplementary figures

Bone marrow smears during the course

Supplementary Table 1

Mutations found in both ET and B-ALL samples and those only found in B-ALL samples