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Acute promyelocytic leukaemia (APL) is a special subtype of acute myeloid leukaemia (AML) which features promyelocytic leukaemia cells, prominent coagulopathy, PML-RARA fusion generated by t(15;17)(q24;q21), and high cure rate in response to all-trans retinoic acid (ATRA) plus arsenic trioxide (ATO) and/or chemotherapy.
Moreover, rearrangement involving RARB and RARG, the other two members of the retinoic acid receptor (RARs) family, has been reported in a group of APL-like leukaemia (APLL) patients, such as TBLR1-RARB, NUP98-RARG, CPSF6-RARG, PML-RARG, NPM1-RARG-NPM1, and HNRNPC-RARG.
Herein, we describe a new case of CPSF6-RARG-positive APLL carrying two fusion transcripts and report a novel fusion variant joining CPSF6 exon 5 to RARG exon 4. Although ATRA was initiated without delay, the patient died early of intracranial haemorrhage. To date, eight cases with CPSF6-RARG/RARG-CPSF6 have been reported, presenting APL morphology and immunophenotype, bleeding diathesis, unresponsiveness to ATRA and arsenic, high mortality rate, and poor prognosis.
This emphasises the clinical importance of identifying this specific fusion gene in APLL without RARA rearrangement.
This study was approved by the Institutional Review Board of the Second Xiangya Hospital, Xiangya School of Medicine, Central South University. Informed consent was obtained from the patient's parents in accordance with the guidelines mentioned in the Declaration of Helsinki.
A 22-year-old previously healthy male presented with a 2-week history of headache, fever and ecchymosis. The haemogram revealed white blood cell (WBC) count of 37.58×109/L, with 94% abnormal promyelocytes, haemoglobin level of 90 g/L, and platelet count of 63×109/L. Biochemical tests showed slightly elevated levels of liver enzymes and markedly increased serum lactate dehydrogenase level of 1424 U/L (reference 120–250 U/L). Coagulopathy was present with a prothrombin time of 16.1 s (reference 10–13 s), fibrinogen levels 1.01 g/L (reference 2–4 g/L), fibrinogen/fibrin degradation product levels 80 mg/L (reference 0–5 mg/L), D-dimer levels 11.81 mg/L (reference 0–0.5 mg/L), and normal activated partial thromboplastin time. Bone marrow (BM) smear showed hypercellularity with 95% hypergranular promyelocytes that featured irregular nuclei, abundant cytoplasmic coarse granules, and strong reactivity to myeloperoxidase cytochemical staining (Fig. 1A,B) in line with APL. Auer rods were absent. The leukaemia cells were positive for CD13, CD33, CD64, CD9, CD117 (partial), and negative for CD34, CD38, HLA-DR, CD56, CD11b, CD14, CD15, and other T or B lymphoid-related markers (Fig. 2). ATRA was initiated on suspicion of APL for 9 days without improvement of coagulopathy. Fluorescence in situ hybridisation (FISH) using a Vysis dual-fusion PML/RARA probe (Fig. 1C) and reverse transcription-polymerase chain reaction (RT-PCR) testing for PML-RARA returned negative results on day 3, and standard ‘7+3’ chemotherapy with idarubicin (10 mg/m2) and cytarabine (100 mg/m2) was administered on day 4. Unfortunately, he experienced worsening headache and then lost consciousness on day 9. Intracranial haemorrhage was confirmed by computed tomography and he died the next day despite resuscitation and supportive care in intensive care.
Fig. 1Histopathological and molecular characterisation of the patient with CPSF6-RARG-positive AML. (A) Wright-Giemsa staining of BM smear shows hypergranular promyelocytes with irregular nuclei, abundant cytoplasmic coarse granules. Auer rods were absent. (B) Myeloperoxidase staining showed strong positivity. (C) Interphase FISH using PML-RARA dual-colour, dual-fusion translocation probes showed no fusion signal. (D) G-banding karyotype revealed a normal karyotype 46, XY [20]. (E) Electrophoresis of RT-PCR products from the patient showed two bands (270 bp and 885 bp) in the bone marrow sample. The BM sample from a PML-RARA-positive APL patient was used as a control. M, DNA marker; P, patient; C, PML-RARA-positive APL patient. (F) Sanger sequencing showed the junction site sequence of the long transcript (transcript 1) that fuses CPSF6 exon 5 to RARG exon 1, and the short transcript (transcript 2) that joins CPSF6 exon 5 to RARG exon 4. (G) Schematic diagram of CPSF6, RARG, CPSF6-RARG fusion proteins. The breakpoint is indicated by red arrows (CPSF6 at amino acid 231 and RARG at 5′UTR or amino acid 61). DBD, DNA-binding domain; LBD, ligand-binding domain; PRD, proline-rich domain; RRM, RNA recognition motif; RSLD, arginine/serine (RS)-like domain; UTR, untranslated region; CPSF6-RARG-L, the long CPSF6-RARG fusion protein; CPSF6-RARG-S, the short CPSF6-RARG fusion protein.
Fig. 2Immunophenotype of the patient with CPSF6-RARG-positive AML. Dot plots of flow cytometry data of bone marrow aspiration sample. Red cluster shows the blast population.
Subsequent chromosome G-banding analysis revealed a karyotype of 46, XY [20] (Fig. 1D). Multiplex RT-PCR showed the absence of 43 leukaemia-related fusion genes, including PLZF-RARA, NUMA1-RARA, STAT5B-RARA, PAKARIA-RARA, FIP1L1-RARA, and NPM1-RARA. Targeted sequencing identified WT1 p.Ser386ArgfsTer62 mutation [variant allele frequency (VAF) 16.3%], WT1 p.Arg385GlufsTer5 (VAF 14%), and NRAS Thr58Ile (VAF 46%). Given the absence of RARA rearrangement, we suspected RARG or RARB fusion as reported in patients with APL features.
RT-PCR using BM samples was performed with the forward primer 5′-TGCAGTCCAGGAAAACTACAC-3′ at CPSF6 exon 4 and reverse primer 5′- ATGGCTTGTAGACCCGAGGA-3′ at RARG exon 5. Two bands were visualised on electrophoresis (Fig. 1E). Sanger sequencing revealed that the long transcript encompassed CPSF6 exon 5 and RARG exon 1 (Fig. 1F, upper panel), as reported by Zhang et al.
The short transcript connects CPSF6 exon 5 to RARG exon 4 (Fig. 1F, lower panel), which is novel. The two transcripts likely resulted from alternative splicing. Both fusions are in-frame and are predicted to encode 828 amino acid and 624 amino acid chimeras, respectively (Fig. 1G).
Including the patient described herein, seven CPSF6-RARG-positive cases and one patient with RARG-CPSF6 have been reported.
Clinical and biological data of these patients are presented in Table 1. Due to the lack of epidemiological data the incidence of this rare AML remains to be determined. The median age for adult patients was 43.6 years (range 22–65 years), and only one 5-year-old paediatric patient has been reported. All cases were east Asian except the RARG-CPSF6-positive patient who was Caucasian. A similar biased race ratio has also been observed in NUP98-RARG-positive APLL.
At presentation, median WBC and platelet counts were 10.9×109/L (range 0.81–37.58) and 65.4×109/L (range 8–229), compared to median WBC 1.5×109/L and platelets 25.5×109/L in PML-RARA-positive APL patients.
Of the seven patients with available coagulation data, six presented with hypofibrinogenaemia. All these patients displayed hypergranular AML-M3 morphological features. The immunophenotype showed a homogenous expression of CD33 and CD13, and negative or partial expression of CD34 and HLA-DR in accordance with APL. Genetically, CPSF6 and RARG are located closely on 12q15 and 12q13 respectively, and as a result, CPSF6-RARG fusion is difficult to detect using conventional karyotyping. In agreement with this, 12q abnormality was not identified in any of the eight patients. Molecularly, the breakpoint of CPSF6 was located at exon 4 or 5, and the breakpoint of RARG varied at exon 1, 2, 3, or 4, generating various fusion transcript variants. Nonetheless the fusion chimera reserves the RNA-recognition motif of CPSF6 and the DNA-binding domain and ligand-binding domain of RARG. The mutation landscape of CPSF6-RARG-positive AML differed from classic APL in that WT1 was the most common co-mutated gene (5/8=62.5%), followed by NRAS/KRAS (3/8=37.5%), FLT3 (1/8=12.5%), DNMT3A (1/8=12.5%), EZH2 (1/8=12.5%), etc. The enriched WT1 mutations in CPSF6-RARG/RARG-CPSF6 AML suggest its involvement in leukaemogenesis.
Table 1Clinical and biological data of CPSF6-RARG-positive AML patients
As summarised in Table 1, all patients were treated with ATRA, and four patients were also treated with arsenic. Among them, six patients were re-evaluated by peripheral blood or bone marrow morphology/flow cytometry during or at the end of induction therapy. Importantly, no signs of differentiation of blast cells were achieved, indicating that CPSF6-RARA-positive AML is unresponsive to ATRA. The four patients receiving intravenous ATO or oral arsenic Realgar-indigo naturalis formula (RIF) had no response, suggesting arsenic resistance. It is worth noting that four patients succumbed to severe haemorrhage events during induction or re-induction, reminiscent of APL in the pre-ATRA era. Close monitoring and aggressive supportive care should be provided to avoid fatal bleeding events. Re-induction with AML-like approaches achieved complete remission (CR) in Patient 2, adopting ‘7+3’ regimen with daunorubicin plus cytarabine (DA). Homoharringtonine plus cytarabine (HA) chemotherapy achieved CR in Patients 5 and 6 who failed previous anthracycline plus cytarabine chemotherapy. Patient 2 achieved long-term leukaemia-free survival for more than 6 years after consolidation with two courses of high dose cytarabine and two courses of DA, while Patients 5 and 6 relapsed and died at 11 months and 32 months, respectively. No-one underwent allogeneic haemopoietic stem cell transplantation (allo-HSCT). The low 2-year and 5-year overall survival rates suggest that allo-HSCT should be performed in first CR for CPSF6-RARG-positive AML patients.
In summary, we report a new case of CPSF6-RARG-positive AML resembling APL and a novel CPSF6-RARG variant that fuses CPSF6 exon 5 to RARG exon 4. Reviewing the literature, CPSF6-RARG-positive AML resembles APL, but is unresponsive to ATRA and ATO. Switching to AML-like approaches with aggressive supportive care is recommended. Accurate identification of this rare subtype of AML is essential to guide therapeutic decisions. Further laboratory and clinical investigations are in urgent need.
Acknowledgements
The authors thank the patients and their families for their contribution to this study. The authors thank Profs Suning Chen and Zhanglin Zhang for updating follow-up information of their patients. We thank Prof Honghu Zhu for reviewing the manuscript and providing constructive advice.
Conflicts of interest and sources of funding
This work was supported by grants from the National Natural Science Foundation of China (Grant No. 81700168 and No. 81900170), Changsha Municipal Natural Science Foundation (Grant No. kq2014234), and Natural Science Foundation of Hunan Province (Grant No. 2021JJ30937). The authors declare they have no competing financial interests and no conflicts of interest to disclose.
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