Acute promyelocytic leukemia with tetraploid karyotype and multiple PML-RARA fusion signals

全文

✉ Correspondence to: Hitoshi Ohno, MD, PhD Department of Hematology, Tenri Hospital 200 Mishima, Tenri, Nara 632-8552, Japan e-mail: [email protected]. 120. Pictures at Bedside and Bench. Tenri Medical Bulletin 2020;23(2):120-124 DOI: 10.12936/tenrikiyo.23-018. Acute promyelocytic leukemia with tetraploid karyotype and multiple PML-RARA fusion signals. Mariko Fujii1, Wataru Maruyama1, Chiyuki Kishimori2, Katsuhiro Fukutsuka2, Naoharu Nagai2, Masahiko Hayashida2, Shinichi Kotani1, Futoshi Iioka1, Takashi Akasaka1, Hitoshi Ohno1,2✉. 1Department of Hematology, Tenri Hospital; 2Tenri Institute of Medical Research. Received 2020/8/26; accepted 2020/9/8; released online 2020/12/25. Keywords: acute promyelocytic leukemia, tetraploid karyotype, ider(17)(q10)t(15;17)(q24;q21), FISH, PML-RARA fusion signal. Key Figure. G-banding karyotype obtained from the BM. The karyotype was: 92,XXXX,−5,−6,−9,der(15)t(15;17)(q24;q21)×2,der(17) t(15;17),ider(17)(q10)t(15;17)×2,+2mar. The der(15)t(15;17)(q24;q21), der(17)t(15;17), and ider(17)(q10)t(15;17) chromosomes are indicated by the arrows, arrowhead, and asterisks, respectively.. APL with tetraploid karyotype. 121Tenri Medical Bulletin Vol. 23 No. 2 (2020). A woman in her thirties was admitted to our hospital for severe pain on the right side of the lower abdomen. Computed tomography demonstrated diverticulitis of the ascending colon associated with regional peritonitis. As a blood test disclosed leukocytopenia, she was referred to the hematology depar tment on the next day of admission. Her hemoglobin level was 10.3 g/dL, white cell count was 0.83 × 103/µL, and platelet count was 67 × 103/µL. Her uric acid level was 2.8 mg/dL, total protein level was 5.6 g/dL, albumin level was 3.0 g/dL, lactate dehydrogenase level was 320 U/L, C-reactive protein level was 5.26 mg/dL, and procalcitonin level was 2.9 ng/mL. The prothrombin time was 22.3 sec, activated partial thromboplastin time was 38.3 sec, fibrinogen level was 37 mg/dL, antithrombin III level was 67% (reference, 70–125%), fibrin degradation product level was 260.0 µg/mL, D-dimer level was 83.0 µg/mL, and soluble fibrin monomer complex level was 95.4 µg/mL (reference, < 7 µg/mL).. Examination of the bone marrow (BM) revealed marked hypercellularity with 88.9% leukemia cells. The cells were giant in size with bizarre, cup-shaped, or convoluted nuclei, and contained variable numbers of azurophilic granules in the cytoplasm (Figure 1A). Occasional cells had abundant Auer rods, exhibiting a faggot-cell appearance (Figure 1B). Myeloperoxidase was strongly positive (Figure 1C). Flow cytometry. revealed high forward-scatter (FSC) and side-scatter (SSC) light-intensity values, indicating a large cell size and intracellular complexity, and a surface antigen prof i le of CD2−, CD13+, CD33di m, CD34−, CD56−, CD117dim, and CD123−/dim. The DNA index was 1.96 relative to normal diploid cells (Figure 2).. On G-banding of metaphase spreads obtained from the BM, cells had a tetraploid karyotype with two copies of der(15)t(15;17)(q24;q21), one copy of der(17) t(15;17), and two copies of isochromosome of the long arm of the der(17)t(15;17) [ider(17)(q10)t(15;17)(q24;q21)] (Key Figure), which is a rare additional cytogenetic abnormality in acute promyelocytic leukemia (APL).1. We then performed fluorescence in situ hybridization (FISH) using the PML/RARA dual-color, dual-fusion probe, which demonstrated that the fusion signals were localized on these 3 derivative chromosomes and 2 isochromosomes (Figure 3A). Accordingly, interphase nuclei carried 2 red (PML), 2 green (RARA), and 7 yellow (fusion) hybridization signals (Figure 3B). The PML-RARA mRNA exhibited the long-form configuration and internal tandem duplication of the fms-like tyrosine kinase-3 (FLT3-ITD) was negative (Supplementary Figure S1).. Initial treatment included transfusions of fresh frozen plasma and platelet concentrates, nafamostat mesylate, and antithrombin III to control coagulopathy. Symptoms. Figure 1. Morphology of leukemia cells in the BM. (A) Wright-stained leukemia cells. Three reactive lymphocytes (Ly) are indicated as a size reference for leukemia cells. (B) Giemsa-stained leukemia cells containing bundles of Auer rods. (C) Myeloperoxidase staining of leukemia cells exhibiting strong positivity. Auer rods were not stained. Original magnification, ×100 objective lens.. a b c. Ly. Ly. Ly. Fujii M et al.. 122 Tenri Medical Bulletin Vol. 23 No. 2 (2020). Figure 2. Flow cytometry of leukemia cells in the BM. Gated cells on the SSC/FSC scattergram were subjected to a single-color analysis. Positive cell populations for each antigen are indicated by horizontal bars.. Figure 3. FISH using the Vysis LSI PML/RARA dual-color, dual-fusion translocation probe kit, consisting of red-labeled PML and green- labeled RARA probes (Abbott Laboratories, Abbott Park, IL). (A) FISH of a metaphase spread. G-banding and FISH images captured through triple band-pass, rhodamine, and fluorescein isothiocyanate (FITC) filters are aligned. Hybridization signals on der(15), der(17), and ider(17), in addition to normal pairs of chromosomes 15 and 17, are indicated by arrowheads of their respective colors. (B) FISH applied to the BM smear slide. FISH images captured through DAPI, triple band-pass, rhodamine, and FITC filters are aligned. Hybridization signals on a representative nucleus are indicated by arrowheads of their respective colors (R, red; G, green; Y, yellow).. APL with tetraploid karyotype. 123Tenri Medical Bulletin Vol. 23 No. 2 (2020). and signs related to acute diverticulitis were ameliorated by intravenous antibiotics. On day 3 of admission, we initiated all-trans retinoic acid (ATRA) in combination with idar ubicin.2 She developed dif ferent iat ion syndrome two weeks after the initiation of treatment, which was resolved by dexamethasone. She achieved complete remission (CR) af ter induction therapy (Supplementary Figure S2) with negative amplification of the PML-RARA mRNA, and subsequently completed 3 cycles of consolidation therapy. She is currently undergoing maintenance therapy composed of ATRA, 6-mercaptopurine, and oral methotrexate.3. We described a patient who presented with APL carrying a tetraploid karyotype. As leukemia cells exhibited giant cellular morphology and the patient had localized but significant gastrointestinal tract infection, we initially treated her cautiously. However, the leukemia was rationally stratified into the low- risk category (i.e., white cell count ≤ 10 × 103/µL and platelet count > 40 × 103/µL) and poorer prognosis predictors, although variably reported, including CD56 expression, FLT3-ITD mutation, and short-form PML- RARA mRNA,4 were negative. We therefore treated her according to the protocol designated for low-/ intermediate-risk patients2 and she readily achieved CR.. Tetraploidy can pathophysiologically develop by cell fusion or erroneous cell division, inducing chromosomal instability and potentially promoting chromosomal translocations.5 However, as tetraploid leukemia with a recurrent translocation is rare,6-9 it remains unclear whether the tetraploid karyotype is related to specific clinical features or treatment outcomes. Regarding APL, of 15 reported cases of tetraploid karyotype and duplicate t(15;17)(q24;q21), CR was achieved in 13 (87%) after ATRA-based therapy, suggesting that the treatment outcome of these patients was similar to that of patients with diploid APL.7 In the present case, as the PML-RARA fusion gene is generated on der(15) t(15;17), leukemia cells theoretically carried two copies of PML-RARA (i.e., one copy per chromosomal set), and the remaining 5 fusion signals represented the. reciprocal RARA-PML, whose role in the pathogenesis of APL has not been fully elucidated.1 Thus, although there were multiple copies of the fusion signal, the level of leukemogenic PML-RARA chimeric protein in the cells was likely comparable to that in diploid APL cells, possibly accounting for the favorable treatment course in response to the standard ATRA-based therapy.. REFERENCES. 1.. 2.. 3.. 4.. 5.. 6. 7. 8. 9. M a n o l a K N , K a r a k o s t a M , S a m b a n i C , e t a l . I s o c h r o m o s o m e d e r (17) ( q10) t (15;17) i n a c u t e promyelocytic leukemia resulting in an additional copy of the RARA-PML fusion gene: report of 4 cases and review of the literature. Acta Haematol 2010;123:162-170. Lo-Coco F, Avvisati G, Vignetti M, et al. Front-line treatment of acute promyelocytic leukemia with AIDA induction followed by risk-adapted consolidation for adults younger than 61 years: results of the AIDA-2000 trial of the GIMEMA Group. Blood 2010;116:3171-3179. Sanz MA, Lo Coco F, Martin G, et al. Definition of relapse risk and role of nonanthracycline drugs for consolidation in patients with acute promyelocytic leukemia: a joint study of the PETHEMA and GIMEMA cooperative groups. Blood 2000;96:1247-1253. Lo-Coco F, Cicconi L, Breccia M. Current standard treatment of adult acute promyelocytic leukaemia. Br J Haematol 2016;172:841-854. Storchova Z, Kuffer C. The consequences of tetraploidy and aneuploidy. J Cell Sci 2008;121:3859-3866. Yang R, Jiang M, Zhao J, et al . Identification of chromosomal abnormalities and genomic features in near- triploidy/tetraploidy-acute leukemia by fluorescence in situ hybridization. Cancer Manag Res 2019;11:1559-1567. Dal ia SM, Horna P, Zhang L. Tetraploidy acute promyelocytic leuemia with double t(15;17)/PML-RARA, a case report with review of literature. Int J Clin Exp Pathol 2014;7:5363-5368. Pang CS, Pettenati MJ, Pardee TS. Clinicopathological analysis of near-tetraploidy/tetraploidy acute myeloid leukaemia. J Clin Pathol 2015;68:236-240. Bene MC, Castoldi G, Derolf A, et al. Near-tetraploid acute myeloid leukemias: an EGIL retrospective study of 25 cases. Leukemia 2006;20:725-728.. Fujii M et al.. 124 Tenri Medical Bulletin Vol. 23 No. 2 (2020). ４倍体核型急性前骨髄球性白血病の一例. 藤井麻梨子 1，丸山亙 1，岸森千幸 2，福塚勝弘 2，永井直治 2，林田雅彦 2，小谷慎一 1，. 飯岡大 1，赤坂尚司 1，大野仁嗣 1,2. 1 天理よろづ相談所病院血液内科 2 天理よろづ相談所医学研究所. Supplementary Figure S2. Treatment course for induction therapy. Abbreviations: ATRA, all-trans retinoic acid; CFPM, cefepime; TAZ/PIPC, tazobactam/piperacillin; MEPM, meropenem; VCM, vancomycin; MCFG, micafungin; FFP, fresh frozen plasma, ATIII, antithrombin III; and FDP, fibrin degradation products. The days after admission are shown.. Supplementary Figure S1. Polymerase chain reaction (PCR)-based tests for PML-RARA fusion mRNA and FLT3-ITD mutation. (A) Nested reverse transcriptase (RT)-PCR amplifying the PML-RARA fusion mRNA. The positions of the PCR products are indicated by the green (long-form) and blue (short-form) arrows with each size (bp, base pair). (B) PCR amplifying exons 14 through 15 of the FLT3 gene. Unmutated products of 329 bp in size were generated in this case.