BCR-ABL fusion transcripts are found in around 20–30% of adults and in ≤5% of pediatric acute lymphoblastic leukemia (ALL) cases. [1] [2] [3] [4] [5] [6] [7] BCR-ABL is of interest in ALL for two reasons. Firstly, those affected are known to have a poor prognosis under conventional therapy [8] [9] and are therefore considered high risk patients and primarily candidate for intensified therapy regime, including allogeneic transplantations. Secondly, BCR-ABL positive patients can have a better prognosis under treatment regime with ABL tyrosine kinase inhibitors such as imatinib. [10] Commonly three typical BCR-ABL mRNA transcripts e1a2, e13a2, e14a2, are found in about 99% of BCR-ABL positive cases but several atypical ones (e13a3, e14a3, e1a3, e19a2, e6a2) have also been observed. [11] (Figure 1) However, these variants were observed exclusively in chronic myeloid leukemia (CML) cases, and very little is known about atypical transcript in ALL.

Herein, we report such an atypical BCR-ABL mRNA transcripts e6a2 by reverse transcriptase polymerase chain reaction (RT-PCR) in pediatric ALL in Rajiv Gandhi cancer institute and research centre (India).

A five-year-old boy was presented to our hospital with complaint of lymphadenoid and abdominal pain. On examination he was diagnosed with multiple lymph nodes and splenomegaly. His complete blood count examination showed, hemoglobin 9.2 g/dL, total leukocyte count 3600, peripheral smear showed 12% blasts, with lymphoid morphology, 2% polymorphs and platelet count 8000/mm3. No eosinophils, basophils and monocytes were observed in his blood. Bone marrow examination showed 69% blast of lymphoid morphology which was myeloperoxidase (MPO) negative.

Immunophenotyping: This was carried out using flow cytometer (BD FACSCalibur, BD Biosciences, USA) on bone marrow sample. Using CD45 and side scatter gating 73.41% cells were gated. These cells were dim positive to negative for CD45. These were also positive for CD34 (84.37%), CD10 (76.89%), CD19 (81.65%), cyto CD79a (92.21%), cytoCD22 (97.16%), TDT (47.99%), CD123 (85.01%), and HLA-DR (94.75%). Based on immunophenotype features, it was diagnosed as precursor-B-ALL. The sample was analyzed for common ALL translocations by RT-PCR.

RT-PCR: After red blood lyses RNA was isolated using QIAamp RNA Blood Mini Kit Cat. No. 52304. RNA quantity and quality was checked by ultraviolet spectrophotometer and by running on 1.2% MOPS buffer gel. The cDNA synthesis was carried out by RevertAid H Minus First Strand cDNA synthesis Kit (#K1632) by Thermo Scientific. PCR done for BCR-ABL fusion transcript using primers

BCR 12 FP -5’-TGCTGACCAACTCGTGTGTG-3’ (BCR exon 13)

BCR 1 FP- 5’-AACTCGCAACAGTCCTTCGAC-3’ (BCR exon1)

ABL 3 -5’-CCATTCCCCATTGTGATTATAGC-3’ (ABL exon 3)

FISH: Interphase fluorescence in situ hybridization (FISH) done by using Pathvysion dual color dual fusion translocations probe (08L10-001) Kit Abbott Molecular. In which spectrum green was for BCR gene and spectrum orange was for ABL gene.

Sequencing: Amplified product was sequenced with the Big Dye Terminator v1.1 cycle sequencing Kit (Applied Biosystems) and run in an ABI PRISM 310 Genetic Analyzer and results were evaluated.

Results: The RT-PCR showed an atypical large amplicon of 1026bp correspond to e6a2 breakpoint using the primers BCR1 FP and ABL 3 RP, (Figure 2A) Unlike 384 bp products for e1a2 breakpoint by using the same primers. Patient sample also analyzed for other three common ALL translocations (12;21,1;19,4;11) and were found negative.

Fluorescence in situ hybridization showed by using dual color dual fusion probe we found that there is fusion between BCR and ABL gene (Figure 2B) 55 of the 100 interphase nuclei of the bone marrow smear showed fusion of BCR and ABL gene.

Sequencing result showed identical fusion between exon 6 of BCR gene and exon 2 of ABL gene result in an e6a2 fusion transcript were found. (Figure 2C)

Using RT-PCR, we were able to detect atypical e6a2 BCR-ABL transcript in a pediatric patient with precursor-B-ALL, which also be demonstrated by FISH as well as sequencing and no M-BCR rearrangement was seen by RT-PCR. This is an unusual transcript has only been reported in seven patients with myeloid leukemias, 4 with chronic-phase CML, 1 with acute basophilic leukemia. [13] To the best of our knowledge, this patient is the first Indian pediatric BCR-ABL+ precursor-B-ALL with a breakpoint outside M-BCR and the first case to express an e6a2 BCR-ABL. It has been hypothesized that CML patients with e6a2 BCR-ABL transcript may have a worse prognosis possibly because they lack the (GET)/dbl-like domain of BCR. [14] However, there is one study done by Burmeister et al. in which they have shown that an adult ALL patient with BCR-ABL+ (e6a2) breakpoint showed complete remission after induction of allogeneic transplant and still in complete cytogenetic remission on imatinib. [15] Our patient showed good prednisolone response with an absolute blast count of 32 cells/mm³ on day eight of steroid administration. And complete cytogenetic remission on imatinib until 70 days. Still the patient is on imatinib. Till date there is no data on adult and pediatric precursor-B-ALL with BCR-ABL (e6a2) fusion transcript has been reported in India.

Our result suggests that most of the atypical transcripts known from chronic myeloid leukemia can also found in BCR-ABL+ ALL. The e6a2 is the rarest atypical BCR-ABL transcript in pediatric Precursor-B-ALL. Reverse transcriptase polymerase chain reaction based BCR-ABL diagnosis should be design in such a way that it can detect all typical (e13a2, e14a2, e1a2) as well as atypical (e13a3,e14a3, e1a3, e6a2, e19a2) fusion transcripts. Patients with these transcripts also respond to tyrosine kinase inhibitor therapy. A complementary cytogenetic analysis remains mandatory.

The author thanks Dr. Kabir Sachdeva for his valuable assistance for guiding me to publish this case report.

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