CPSF6-RARG-positive acute myeloid leukaemia resembles acute promyelocytic leukaemia but is insensitive to retinoic acid and arsenic trioxide

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.1Sanz M.A. Fenaux P. Tallman M.S. et al.Management of acute promyelocytic leukemia: updated recommendations from an expert panel of the European LeukemiaNet.Blood. 2019; 133: 1630-1643Crossref PubMed Scopus (287) Google Scholar In <5% of morphologically defined APL, PML-RARA is absent. In addition to GTF2I-RARA reported by our group,2Li J. Zhong H.Y. Zhang Y. et al.GTF2I-RARA is a novel fusion transcript in a t(7;17) variant of acute promyelocytic leukaemia with clinical resistance to retinoic acid.Br J Haematol. 2015; 168: 904-908Crossref PubMed Scopus (33) Google Scholar 16 variant RARA fusion genes have been described in patients with different sensitivities to ATRA.1Sanz M.A. Fenaux P. Tallman M.S. et al.Management of acute promyelocytic leukemia: updated recommendations from an expert panel of the European LeukemiaNet.Blood. 2019; 133: 1630-1643Crossref PubMed Scopus (287) Google Scholar,3Geoffroy M.C. de Thé H. Classic and variants APLs, as viewed from a therapy response.Cancers (Basel). 2020; 12: 967Crossref Scopus (23) Google Scholar 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.3Geoffroy M.C. de Thé H. Classic and variants APLs, as viewed from a therapy response.Cancers (Basel). 2020; 12: 967Crossref Scopus (23) Google Scholar 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.4Liu T. Wen L. Yuan H. et al.Identification of novel recurrent CPSF6-RARG fusions in acute myeloid leukemia resembling acute promyelocytic leukemia.Blood. 2018; 131: 1870-1873Crossref PubMed Scopus (30) Google Scholar, 5Qin Y.Z. Huang X.J. Zhu H.H. Identification of a novel CPSF6-RARG fusion transcript in acute myeloid leukemia resembling acute promyelocytic leukemia.Leukemia. 2018; 32: 2285-2287Crossref PubMed Scopus (28) Google Scholar, 6Zhao J. Liang J.W. Xue H.L. et al.The genetics and clinical characteristics of children morphologically diagnosed as acute promyelocytic leukemia.Leukemia. 2019; 33: 1387-1399Crossref PubMed Scopus (37) Google Scholar, 7Zhang Z. Jiang M. Borthakur G. et al.Acute myeloid leukemia with a novel CPSF6-RARG variant is sensitive to homoharringtonine and cytarabine chemotherapy.Am J Hematol. 2020; 95: e48-e51Crossref PubMed Scopus (14) Google Scholar, 8Jiang M. Zhou Y.R. Zhan Y. et al.Application of transcriptome sequencing and fusion genes analysis in the diagnosis of myeloid leukemia with normal karyotype. (Chinese.Zhonghua Yi Xue Za Zhi. 2021; 101: 939-944PubMed Google Scholar, 9Han X. Jin C. Zheng G. et al.Acute myeloid leukemia with CPSF6-RARG fusion resembling acute promyelocytic leukemia with extramedullary infiltration.Ther Adv Hematol. 2021; 122040620720976984Crossref Google Scholar, 10Miller C.A. Tricarico C. Skidmore Z.L. et al.A case of acute myeloid leukemia with promyelocytic features characterized by expression of a novel RARG-CPSF6 fusion.Blood Adv. 2018; 2: 1295-1299Crossref PubMed Scopus (19) Google Scholar 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. 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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.3Geoffroy M.C. de Thé H. Classic and variants APLs, as viewed from a therapy response.Cancers (Basel). 2020; 12: 967Crossref Scopus (23) Google Scholar 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.7Zhang Z. Jiang M. Borthakur G. et al.Acute myeloid leukemia with a novel CPSF6-RARG variant is sensitive to homoharringtonine and cytarabine chemotherapy.Am J Hematol. 2020; 95: e48-e51Crossref PubMed Scopus (14) Google Scholar 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.4Liu T. Wen L. Yuan H. et al.Identification of novel recurrent CPSF6-RARG fusions in acute myeloid leukemia resembling acute promyelocytic leukemia.Blood. 2018; 131: 1870-1873Crossref PubMed Scopus (30) Google Scholar,9Han X. Jin C. Zheng G. et al.Acute myeloid leukemia with CPSF6-RARG fusion resembling acute promyelocytic leukemia with extramedullary infiltration.Ther Adv Hematol. 2021; 122040620720976984Crossref Google Scholar 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.11Zhang J. Shen H. Song H. et al.A novel NUP98/RARG gene fusion in pediatric acute myeloid leukemia resembling acute promyelocytic leukemia.J Pediatr Hematol Oncol. 2022; 44: e665-e671Crossref PubMed Scopus (1) Google ScholarAt 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.12Cicconi L. Testi A.M. Montesinos P. et al.Characteristics and outcome of acute myeloid leukemia with uncommon retinoic acid receptor-alpha (RARA) fusion variants.Blood Cancer J. 2021; 11: 167Crossref PubMed Scopus (5) Google Scholar 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 patientsNo.Sex/Age (years)CBCFib (g/L)ImmunophenotypeKaryotypeGeneticsCo-mutationsTreatmentResponse/OutcomeRef1F/48WBC 0.81×109/LHB 4.2 g/LPLT 92×109/L1.67CD13+, CD33+, MPO+, CD9 partial+, CD64 partial+, HLA-DR–, CD117–, CD34–, CD14–, CD11b–92, XXXX[2]CPSF6 E4-RARG E4DNMT3AInduction therapy: ATRA + ATO + daunorubicinATRA + ATO + IAGDecitabineNo CRNo CRDied of intracranial hemorrhage at 3 months32F/51WBC 20.15×109/LHB 6.5 g/LPLT 45×109/L1.66CD13+, CD33+, cMPO+, CD9 partial+, CD34 partial+, HLA-DR–, CD2–, CD7–, CD10–, CD11c–, CD14–, CD38–del(12) (p12)[2]/46, XX[18]CPSF6 E4-RARG E1WT1, KRASInduction therapy: ATRA + daunorubicinNo CR3DACRConsolidation therapy: 2 cycles high dose cytarabine, and 2 cycles DASustained leukaemia free status at 72 months3M/38WBC 1.68×109/LHB 8.0 g/LPLT 79×109/L1.15CD33+, CD13+, MPO+, CD117+, CD123+, CD34+, CD9 partial+, CD64 partial+, CD11b–, HLA-DR–, CD38–, CD56–, CD14–46, XY[20]CPSF6 E4-RARG E2WT1Induction therapy: ATRA + oral arsenic RIFNo CR4MADied of diffuse alveolar haemorrhage on 37th day4M/26WBC 16.4×109/LHB 10.5 g/LPLT 52×109/LCoagulopathyCD33+, CD13+, CD64+, CD117 partial+, HLA-DR weak+, CD34–, CD56–, CD19–, CD2–, CD5–, CD123–, CD14–, CD11b–, TdT–45, X, -Y [10]/45, idem, add(6) (q?13) [2]/46, XY[8]RARG I9-CPSF6 E5WT1, NEAT1, BMPR1A,Induction therapy: ATRA + IANA55M/5WBC 7.65×109/LHB 8.6 g/LPLT 83×109/LNANA46, XY[20]NAASXL2, FLT3Induction therapy: ATRA + ATO + DANo CR6MAENo CR4 cycles high dose cytarabine based chemotherapyNo CR2 cycles HACRRelapsed and died at 11 months6M/55WBC 1.23×109/LNormalMPO+, CD13+, CD33+, CD71 partial+, CD14–, CD19–, CD34–, CD38–, CD64–, CD117–, CD11b–, CD11c–, HLA-DR–, T/B-lymphoid-related markers–46, XY[20]CPSF6 E5-RARG E1NoneInduction therapy: ATRA + ATONo CR7,8HB 7.6 g/LIANo CRPLT 60×109/LHACRConsolidation therapy: HARelapsed and died at 30 months7F/65WBC 1.60×109/LHB 8.9 g/LPLT 49×109/L0.5CD33+, CD13+, CD117+, CD56+, CD34–, HLA-DR–46, XX[20]CPSF6 E4-RARG E4WT1, KRASATRAInduction therapy: HANo CRDied of intracranial hemorrhage at 1 month98M/22WBC 37.58×109/LHB 90 g/LPLT 63×109/L1.0CD13+, CD33+, CD64+, CD9+, CD117–, CD34–, HLA-DR–, CD56–, CD11b–, CD14–, CD15–, CD38–46, XY[20]CPSF6 E5-RARG E1,CPSF6 E5-RARG E4WT1, NRASInduction therapy: ATRA + IADied of intracranial hemorrhage on 10th dayThis caseATO, arsenic trioxide; ATRA, all-trans retinoic acid; CBC, complete blood count; CR, complete remission; DA, daunorubicin and cytarabine; DIC, disseminated intravascular coagulation; E, exon; Fib, fibrinogen; HA, homoharringtonine and cytarabine; I, intron; IA, idarubicin and cytarabine; IAG, idarubicin (6 mg/m2), cytosine arabinoside (10 mg/m2) and granulocyte-colony stimulating factor; MA, mitoxantrone and cytarabine; NA, not available; Ref, reference; RIF, Realgar-indigo naturalis formula. Open table in a new tab ATO, arsenic trioxide; ATRA, all-trans retinoic acid; CBC, complete blood count; CR, complete remission; DA, daunorubicin and cytarabine; DIC, disseminated intravascular coagulation; E, exon; Fib, fibrinogen; HA, homoharringtonine and cytarabine; I, intron; IA, idarubicin and cytarabine; IAG, idarubicin (6 mg/m2), cytosine arabinoside (10 mg/m2) and granulocyte-colony stimulating factor; MA, mitoxantrone and cytarabine; NA, not available; Ref, reference; RIF, Realgar-indigo naturalis formula. 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. 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.