Acute myeloid leukemia with a novel CPSF6‐RARG variant is sensitive to homoharringtonine and cytarabine chemotherapy

Acute promyelocytic leukemia (APL) is characterized by the PML-RARA fusion gene, which is generated by chromosomal translocation t(15;17) (q22;q21), and can be induced differentiation by all-trans retinoic acid (ATRA) and arsenic.1 However, occasional acute myeloid leukemia (AML) cases with classic morphological, immunophenotypical, and clinical characteristics of APL are identified that do not have the PML-RARA fusion gene, and are resistant to ATRA and arsenic.2 Several reports have shown that some patients with AML resembling APL have retinoic acid receptor beta (RARB) or retinoic acid receptor gamma (RARG) rearrangement.3-5 Patients with CPSF6-RARG, a very rare occult fusion gene by inv(12q13;12q15), are difficult to diagnose; therefore, they are difficult to treat effectively.6-8 In this study, we report a case of AML, morphologically and immunophenotypically resembling APL, and carrying a novel CPSF6-RARG variant that was generated by the fusion of exon 5 of CPSF6, and exon 1 of RARG. Complete remission was achieved after treating the patient with a homoharringtonine (HA) and cytarabine chemotherapy regimen but not with differentiation-inducing therapy or conventional anthracycline plus cytarabine chemotherapy. A 55-year-old previously healthy man was admitted because of fever and pulmonary infection that did not respond to antibiotic treatment for 1 month. Blood tests showed a hemoglobin level of 76 g/L (normal range, 110-150 g/L), white blood cell count of 1.23 × 109/L (normal range, 4-9 × 109/L), and platelet count of 60 × 109/L (normal range, 100-300 × 109). The fibrinogen and D-dimer levels were 2.14 g/L (ref. 2.00-4.00 g/L) and 21.17 μg/mL (normal range, 0.00-0.55 μg/mL), respectively. Prothrombin time and activated partial prothromboplastin time were 13.7 seconds (normal range, 10.5-13.0 seconds) and 38.1 seconds (normal range, 23-35 seconds), respectively. A bone marrow smear revealed hypercellularity, with 93% immature cells that consisted of predominantly abnormal hypergranular promyelocytes with occasional Auer rods (Figure S1A). Moreover, the blasts, including the promyelocytes, were strongly positive for myeloperoxidase (Figure S1B). Flow cytometric immunophenotypic studies demonstrated that the blasts were positive for CD13, CD33, CD117, and CD56 (Figure S1C) but negative for HLA-DR, CD34, CD38, CD15, CD14, CD7, CD2, CD3, CD4, CD8, CD19, CD20, and CD10. The presumptive diagnosis in this patient was APL, and he received ATRA starting on the first day of admission. However, dual-color dual-fusion fluorescence in situ hybridization (DCDF-FISH) with a specific probe for PML and RARA failed to detect the PML-RARA fusion transcript in the bone marrow sample (Figure S1D). A chromosomal analysis using GTG banding indicated a normal karyotype of 46,XY (Figure 1E). Myeloid-related fusion genes (MLL/ELL, MLL/AF17, MLL/AF6, MLL/AF9, MLL/AF10, AML1/ETO, dupMLL, NPM/RARA, PLZF/RARA, PML/RARA, NPM/MLF1, CBFβ/MYH1, DEK/CAN, HOX11, TLS/ERG, and EVI1) were negative by RT-PCR. No hotspot mutations were identified by exon sequencing. To search for the potential fusion gene, we performed RNA sequencing of the bone marrow samples with a HiSeq 2500 system (Illumina, Inc., San Diego, CA, USA) and found 21 fusion genes with a probability of more than 80% using deFuse inspector software (Figure 1A). The sequences of the 21 fusion genes were aligned in the NCBI database (https://blast.ncbi.nlm.nih.gov), and exon 5 of CPSF6 was fused to exon 1 of the RARG fusion gene in frame, which is expected to result in a fusion protein with 828 AMino acids (Figure 1B). To confirm the fusion, we performed RT-PCR using the bone marrow sample and the following primers: forward (at CPSF6 exon 4), 5′-AGACTGGCTTCTACATACTG-3′ and reverse (at RARG exon 2), 5′-AGACTGGCTTCTACATACTG-3′. As expected, a band of approximately 500 bp was visualized by electrophoresis in the bone marrow but not in the NB4 cell line, an APL cell line (Figure 1C). Sanger sequencing revealed that the fusion gene was the fusion of CPSF6 exon 5 with RARG exon 1 (Figure 1D). On the basis of our morphological and immunophenotypical findings, we made a presumptive diagnosis of APL. The patient was treated with 40 mg of ATRA per day for 20 days. Arsenic trioxide (ATO) (10 mg per day) was added starting on day 21. After 1 month of treatment, 74% of the bone marrow cells were promyelocytes. Because the patient demonstrated complete resistance to ATRA and ATO, the treatment was switched to idamycin (10 mg d1-4) and Ara-C (150 mg d1-7) as an induction therapy. The blast cells in the bone marrow were 79% after 20 days of treatment. The patient discontinued therapy and was discharged on September 29, 2018. He was re-admitted on November 6, 2018, and underwent a course of homoharringtonine (4 mg d1-7) and Ara-C (150 mg d1-7) (Figure 1E). He has been in complete remission since December 2018 (Table S1). Because both CPSF6 and RARG genes are located at 12q1, it is difficult to detect the CPSF6-RARG fusion gene using traditional genetic and molecular biological technologies. With the application of second-generation sequencing technology in the clinical diagnosis, four patients were reported with CPSF6-RARG, and one with RARG-CPSF6.6-9 All of the patients were morphologically diagnosed with APL, suggesting that their disease had a similar pathogenesis to that of APL. However, none of the patients with CPSF6-RARG or RARG-CPSF6 experienced a response to ATRA and ATO, suggesting that it is a novel subtype of AML. In previous reports, the fusion sites of CPSF6 were located at the terminal of exon 4 in four patients with CPSF6-RARG, but the fusion sites of RARG varied greatly (1 case at exon 1, 1 at exon 2, and 2 at exon 4).6-8 In our patient, the CPSF6-RARG variant was the longest, and the fusion site was exon 5 of CPSF6 with exon 1 of RARG. However, the role of different transcripts in the pathogenesis and prognosis of AML remains to be determined. Patients with this novel translocation are resistant to ATRA and arsenic, while conventional anthracycline plus cytarabine chemotherapy is equally ineffective.6-8 Homoharringtonine, which was first isolated from cephalotaxus plant alkaloid, inhibits protein translation and induces apoptosis in various cell lines of hematological malignancies in the G1 and G2 phases.10 It has been widely used in the treatment of AML, chronic myeloid leukemia, and myelodysplastic syndrome since the 1970s.11-13 A recent study found that the homoharringtonine chemotherapy regimen is effective in inducing remission in patients with refractory and relapsed AML.14 We treated our patient with the homoharringtonine regimen after the patient failed to experience a response to ATRA, ATO, and conventional idamycin and Ara-C chemotherapy. After one course of treatment, he achieved a complete remission, suggesting that homoharringtonine is a very effective drug for AML with CPSF6-RARG. In summary, we identified a novel CPSF6-RARG transcript in a patient with AML that resembled APL and provided evidence that homoharringtonine is effective as a backbone drug for the treatment of AML with CPSF6-RARG. It would be very interesting to determine whether the same treatment would be effective for AML with other types of RARG rearrangements. This work was supported by the Institutional Research Grant of The University of Texas MD Anderson Cancer Center. The authors declare no competing financial interests. Z.L.Z., M.J., and S.Q.L. performed the experiments. X.B.H., Q.X., D.J., L.F., and H.R.B. performed the clinical analyses. Z.L.Z., G.B., G.T., A.D.B., L.F., H.R.B., and M.J.Y. wrote the manuscript. Figure S1 Morphological, cytochemical, cytometric, and cytogenetic assessment of the patient's AML bone marrow sample. (A) Frequent promyelocytes with hypergranulated cytoplasm and invaginated nuclei are shown (Wright-Giemsa-stained bone marrow smear, × 1000). (B) Myeloperoxidase stain showed strong positivity in AML (×1000). (C) Flow cytometric analysis showed that CD45-dim blasts were gated. The blasts expressed CD33 and CD13, partially expressed CD117 and CD56, and did not express CD34, CD7, CD38, HLA-DR, or other myeloid and lymphoid markers. MON, LYM and DEB are monocytes, lymphocytes, and debris, respectively. (D) Interphase FISH revealed the absence of PML-RARA fusion signals in AML using PML-RARA dual-color, dual-fusion translocation probes. (E) Cytogenetics analysis with G-banding revealed a normal male karyotype 46, XY [20]. Table S1 Laboratory examination and maintenance therapy regimen Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.