A 48-year-old female presented with a leucoerythroblastic blood film, anemia (hemoglobin 10.9 g/dL), thrombocytopenia (72 x 109/L), normal white cell count, and no splenomegaly. An initial bone marrow (BM) aspirate was hypocellular with increased fibrosis suspected as the underlying cause. The JAK2 V617F and CALR exon nine insertion or deletion mutations were not detected but cytogenetic analysis revealed a karyotype of 46,XX,t(9;22)(q42;q11) in 16 metaphases, trisomy 8 in a further two Ph+ metaphases, and tetrasomy 8 in one further Ph+ metaphase. RT-qPCR analysis with standardized primers and probes did not detect BCR-ABL1 transcripts but further analysis with a qualitative PCR approach detected a single abnormal band which Sanger sequencing demonstrated to be a fusion of BCR exon 6 to ABL1 exon a2 (Figure 1). No mutations were detected in the ABL1 kinase domain. Immunophenotyping of the peripheral blood (PB) demonstrated 12% myeloblasts that were positive for CD7, CD13, CD33, CD34, CD117, and HLADR and negative for CD3, CD4, CD8, CD11c, CD14, CD15, CD19, and CD64. A repeat BM aspirate was hypercellular with 18% myeloblasts morphologically. The corresponding trephine biopsy was hypercellular and had prominent grade three fibrosis with CD34+ blasts accounting for approximately 40% of the total cellularity, consistent with a diagnosis of blast crisis CML with marrow fibrosis.
In order to quantitate e6a2 BCR-ABL1 transcripts, a BCR exon 6 forward primer was used with reverse primer ENR561 and probe ENP541. A standard curve was established by tenfold dilutions of patient presentation PB cDNA (slope -3.538, r2 0.996). Control ABL1 transcripts were detected as previously described. At diagnosis the patients' PB BCR-ABL1/ABL1 transcript level was 78.6%.
An NGS approach was employed to detect additional mutations cooperating with the e6a2 BCR-ABL1 fusion in driving blast crisis in this patient. Amplicon libraries covering 30 commonly mutated genes implicated in myeloid malignancies either covering the entire coding region (CALR, CEBPA, ETV6, EZH2, RUNX1, SH2B3, TET2, TP53, and ZRSR2) or mutational hotspots (ABL1, ASXL1, BRAF, CBL, CSF3R, DNMT3A, FLT3, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NPM1, NRAS, PTPN11, SETBP1, SF3B1, SRSF2, and U2AF1) were generated using 20 ng BM genomic DNA and sequencing performed using Ion AmpliSeq  methodology (Thermo Fisher Scientific, Paisley, UK). Calling of somatic mutations was achieved using an algorithm that excluded synonymous mutations, variants located within intronic or untranslated regions, and those present at a variant allele frequency (VAF) of <5%. A minimum target depth of coverage for variant calls was set at 500x as previously described. A single ASXL1  p.E1102D mutation (c.3306G > T; reference sequence NM_015338.5) was detected with a variant allele frequency of 45.5%.
The patient received one cycle of daunorubicin and cytarabine (DA 3 + 10) with imatinib 400mg daily which was escalated to 600 mg daily achieving hematological and morphological remission and resulting in a PB BCR-ABL1/ABL1 transcript level of 0.06%. She was subsequently treated with a second cycle of chemotherapy (DA 3 + 8) which was complicated by septicemia. Prior to ASCT the PB BCR-ABL1/ABL transcript level was 0.03% (Figure 2). Her imatinib was stopped prior to busulfan and cyclophosphamide conditioning for ASCT from a matched sibling donor that was complicated by mucositis and a coagulase-negative staphylococcus infection during her inpatient stay. After discharge she developed grade four skin graft versus host disease (GVHD) and was treated with high dose steroids subsequently developing CMV reactivation and steroid induced diabetes. Antiviral treatment with valganciclovir caused a pancytopenia requiring treatment with granulocyte colony-stimulating factor. BCR-ABL1 transcripts were not detected by RT-qPCR at day 38 after ASCT with full donor chimerism achieved (100% at day 43 post-ASCT). The patient remains off TKI and generally well at last follow-up. The CMV reactivation has resolved and she continues on steroid taper for GVHD. Continued close RT-qPCR monitoring is planned.