Molecular Monitoring in Adult Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia with the Variant e13a3 BCR-ABL1 Fusion
Monitoring BCR-ABL1 transcript levels in patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is a widely adopted method to assess response to therapy. However, a small minority of Ph+ ALL patients express variant BCR-ABL1 transcript types, usually due to splicing of alternative BCR or ABL1 exons. Whether patients expressing these rare, variant BCR-ABL1 transcripts have a distinct phenotype or response to therapy is not known due to the limited number of reported cases. Here, we report the presenting features of Ph+ ALL in a young adult with a variant e13a3 BCR-ABL1 fusion. Molecular monitoring reflected the disease response from diagnosis through allogeneic stem cell transplantation which resulted in undetectable e13a3 BCR-ABL1 transcripts. This case highlights the value of molecular monitoring in Ph+ ALL patients with variant BCR-ABL1 transcripts and the requirement for standardization of such assays.
Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) in adults is an aggressive disease that responds poorly to conventional chemotherapy. Despite improvements in survival with the addition of tyrosine kinase inhibitors (TKI) to chemotherapy, hematopoietic allogeneic stem cell transplantation (ASCT) remains the only curative option in those eligible patients . Molecular monitoring of BCR-ABL1 transcripts is a valuable tool in assessing individual patient response to chemotherapy and ASCT [2–4]. The most common BCR-ABL1 transcripts in Ph+ ALL are the e1a2, e13a2, and e14a2 fusions ; however, approximately 5% of adult patients express variant BCR-ABL1 transcripts . Of these variants, the e13a3 (b2a3) BCR-ABL1 type is extremely rare with scant information regarding optimal therapeutic approach [7, 8]. Characterisation of these rare BCR-ABL1 variants also affords the selection of appropriate primer/probe combinations for reverse-transcriptase quantitative PCR (RT-qPCR) assessment of residual disease. The presentation and clinical course of a patient with e13a3 BCR-ABL1 Ph+ ALL is reported.
2. Case Report
A 23-year-old male presented with chest pain and dyspnoea, a hemoglobin count of 7.4 g/dL, platelet count of <10 × 109/L, and white cell count of 11.6 × 109/L. Bone marrow (BM) biopsy and aspirate demonstrated a 95% infiltration of lymphoblasts (Figure 1). Immunophenotyping of the BM aspirate showed lymphoblasts were CD10-, CD19-, CD20-, CD34-, TdT-, and HLA-DR-positive. Cytogenetic analysis revealed a complex clone in 10 cells analysed by G-banding containing various structural and numerical abnormalities including a derivative chromosome 22 from a translocation between the long arms of chromosomes 9 and 22. The composite karyotype was 44–48, XY, +X, t(1; 14)(p32; q32), −3, −6, add(6)(p21), add(7)(p21), add(8)(p21), −10, add(12)(p13), +16, add(19)(p13), der(22)t(9; 22)(q34; q11.2), +1∼2mar, inc [cp10]. Interphase FISH analysis showed the presence of BCR-ABL1 rearrangement in 64/100 cells analysed. Standardised RT-PCR and Sanger sequencing demonstrated e13a3 BCR-ABL1 transcripts  (Figure 2). E13a3 BCR-ABL1 transcripts lack ABL1 exon a2, thus prohibiting the use of a standardised primer/probe combination for e13a2/e14a2 BCR-ABL1 qPCR . A modified BCR-ABL1 qPCR assay was therefore adopted utilising BCR forward primer ENF501F2  with ABL1 reverse primer ENR1063 and ABL1 probe ENP1043, the latter both complementary to ABL1 exon a3 sequence . The BCR-ABL1 standard curve was constructed using serial dilutions of an e14a3 BCR-ABL1 plasmid over a five log range (100% to 0.001% BCR-ABL1 transcripts) with ABL1 as the reference gene as previously described . Best practice guidelines for BCR-ABL1 qPCR and data interpretation were followed . Presentation e13a3 BCR-ABL1 transcripts were of a high level (BCR-ABL1/ABL1 75.7%) consistent with a diagnosis of pre-pre-B cell Ph+ ALL.
The patient commenced induction treatment with rituximab, dexamethasone, vincristine, and daunorubicin, with imatinib 400 mg oral daily started on day 15. After phase-one induction, the bone marrow aspirate demonstrated a complete morphological response with residual disease detected by immunophenotyping and RT-qPCR (BCR-ABL1/ABL1 8.11%). After phase-two induction, the bone marrow BCR-ABL1/ABL1 level had fallen to 0.053%. Following high-dose methotrexate, the pre-ASCT BCR-ABL1 level was 0.034%. The patient proceeded to ASCT from an unrelated donor after cyclophosphamide and total body irradiation conditioning and recommenced continued imatinib maintenance. BCR-ABL1 transcripts were not detected in the peripheral blood at one, two, and three and a half months post-ASCT (Figure 3). Continued close molecular monitoring is planned.
E13a3 BCR-ABL1 transcripts lack ABL1 exon a2 that encodes part of SH3 domain thought to contribute to leukemogenesis by inhibition of the kinase domain and by STAT5 activation . In chronic myeloid leukemia patients, this transcript results in an indolent and TKI-responsive form of disease [14–16]; however, its prognostic significance in adult Ph+ ALL patients remains unknown due to the limited number of annotated cases. While the possibility exists of lymphoid blast crisis in chronic myeloid leukemia (CML), this transformation in e13a3 BCR-ABL1 CML is rare . In the absence of basophilia, thrombocytosis, and splenomegaly, this case likely represents de novo Ph+ ALL. Additional chromosomal abnormalities and complex karyotypes are frequently observed in Ph+ ALL, as witnessed in this case. There is some suggestion that in Ph+ ALL, additional cytogenetic abnormalities are associated with a shorter overall survival and might therefore be used for stratification purposes . In the post-ASCT setting, monitoring BCR-ABL1 transcript levels is an essential component of Ph+ ALL patient management  with effective standardisation of RT-qPCR assays for this and other variant BCR-ABL1 fusion transcripts required .
Reporting of further cases would enable identification of any phenotypic characteristics of e13a3 BCR-ABL1 Ph+ ALL and help establishing optimal treatment strategies for patients with this rare genotype.
Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this paper.
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