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CASE REPORT Table of Contents  
Ahead of print publication
Identification of rare atypical BCR-ABL1 transcript: A case report


1 Department of Clinical Laboratory Services and Translational Research, Malabar Cancer Centre, Thalassery, Kerala, India
2 Department of Haematology and Medical Oncology, Malabar Cancer Centre, Thalassery, Kerala, India

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Date of Submission13-Jul-2021
Date of Decision04-Nov-2021
Date of Acceptance05-Nov-2021
Date of Web Publication13-Jan-2023
 

   Abstract 


CML is characterized by the presence of a BCR-ABL1 fusion transcript. Several guidelines have been published for its detection and molecular monitoring. Here, a case is described of chronic myeloid leukemia presenting in the blast phase with a rare variant transcript, with a discussion on possible red flags in its detection and genetic testing and description of the patient's clinical characteristics. This case highlights the pitfalls of using real-time quantitative reverse-transcription polymerase chain reaction (RQ-PCR) for diagnosis of CML, especially when the clinical picture and the test results are discordant.

Keywords: Atypical variant, CML, molecular testing, RQ-PCR


How to cite this URL:
Saha J, Gopinath V, Nair CK, Roshan D. Identification of rare atypical BCR-ABL1 transcript: A case report. Indian J Pathol Microbiol [Epub ahead of print] [cited 2023 Feb 5]. Available from: https://www.ijpmonline.org/preprintarticle.asp?id=367702





   Introduction Top


The BCR-ABL1 fusion gene is the genetic molecular marker of chronic myeloid leukemia (CML).[1] The fusion oncogene translates into an oncogenic protein with constitutive kinase activity. In the majority of patients, the breakpoint region for the BCR gene is located within exons 12 to 16 and the breakpoint region for the ABL gene is located upstream of exon 1b, between 1b and 1a, or between 1a and a2.[2] In CML, e13a2 and e14a2 form the majority, whereas atypical transcripts form about 2% of the total cases.[3] Here, a case is described of CML in blast phase (CML-BP) with a rare variant transcript (e14a3/b3a3), with a discussion on possible red flags in the detection and description of patient's clinical characteristics.


   Case History Top


A 58-year-old male patient complaining of chronic fatigue presented with a white blood cell count of 64.8 × 109/L, hemoglobin of 5.4 g/dL, and platelets of 13 × 109/L. Peripheral blood smear (PBS) examination revealed 85% of WBCs to be blasts. On immunophenotyping, the blasts were positive for CD10, CD19, CD33, CD34, TdT, and HLA-DR and negative for CD14, CD15, CD20, and cCD3, leading to a diagnosis of precursor B-cell acute lymphoblastic leukemia (ALL). The patient had old reports of tests done elsewhere–a complete blood count (CBC) report with raised leucocytes and a PBS report, suggestive of CML. He was tested in another laboratory with real-time quantitative reverse-transcription polymerase chain reaction (RQ-PCR) for BCR-ABL1 transcripts twice, 3 months apart, but both the test reports were negative.

On suspicion of an undiagnosed CML-chronic phase that had progressed to blast crisis, we did reverse transcriptase qualitative multiplex polymerase chain reaction (RT-PCR) using in-house developed primers, described in [Table 1]. Gel electrophoresis of qualitative PCR-amplified product showed a band that was smaller in size than the commoner variants p210 (e14a2 and e13a2) and p190 (e1a2) [Figure 1]a. To confirm that this was indeed an atypical transcript and not a false-positive result, we did fluorescence in situ hybridization (FISH) with BCR-ABL1 dual-color dual-fusion probes (ZytoVision) on the patient's PBS. FISH revealed BCR-ABL1 fusion in 160 cells out of 200 cells examined (nuc ish 1R1G2F[160x], 2R2G[40x]) [Figure 2]. An atypical-sized band in gel electrophoresis and positive FISH analysis pointed toward the presence of an atypical transcript. The quantitative PCR amplified product was purified using AMPure XP magnetic beads-based kit (Beckman Coulter Inc.) and sequenced in 3500 Dx Genetic Analyzer (Applied Biosystems) [Figure 1]b. A nucleotide BLAST tool search of the sequence on NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi) revealed it to be a known atypical variant, e14a3 (b3a3). The patient was started on tyrosine kinase inhibitor (TKI) therapy and chemotherapy. He responded to TKI for the next 2 months. Subsequently, he developed a progressive disease. Resequencing BCR-ABL1 transcript showed the presence of Y253F mutation in the P-loop domain, a known resistance mechanism against TKI therapy.
Table 1: Primers used in multiplex RT-PCR

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Figure 1: (a0Gel electrophoresis of the amplified product. Lane 1 shows an amplified product with e14a3(b3a3) approximate size of 300 base pairs. Lane 2: no amplification for p190. Lane 3: internal control (ABL1). Lanes 4–6: positive control. Lane 4: p210 control. Lane 5: p190 control. Lane 6: internal control. Lanes 7–9: negative control for p210, p190, and internal control. Lane 11: DNA ladder of 100 base pairs. (b) Electropherogram of the amplified atypical BCR-ABL1 product (e14a3/b3a3)

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Figure 2: Image of FISH with dual-colour dual-fusion probes showing a typical 1R1G2F signal pattern. G (green): BCR; R (red): ABL1

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   Discussion Top


The National Comprehensive Cancer Network (NCCN) guidelines recommend doing bone marrow cytogenetics at the initial evaluation to detect Philadelphia chromosome-positive (Ph-positive) cells and document additional chromosomal abnormalities in Ph-positive cells.[4] FISH analysis using dual probes for BCR and ABL1 on PBS is an acceptable method for detecting Ph-positive cells. Double-fusion FISH probes can detect all variant translocations of the Philadelphia chromosome;[4] however, FISH has lower sensitivity than PCR-based methods. RQ-PCR is the most sensitive technique for detecting BCR-ABL1 transcripts; however, it may fail to detect atypical transcripts. If there is a strong clinical suspicion and initial RQ-PCR is negative for BCR-ABL1 transcripts, FISH or cytogenetic analysis should be done. If there is discordance between FISH and RQ-PCR results, qualitative PCR should be done for the confirmation of the presence of atypical transcripts. Qualitative PCR complements FISH because it is more sensitive than FISH; however, it may also fail to generate an amplified product in some rare atypical transcripts where the DNA sequence complementary to the primers is affected.

RQ-PCR can be used for measuring the levels of e13a2, e14a2,[5], and e1a2[4] transcripts using log-reduction from a standardized baseline. However, manufacturers of RQ-PCR kits for BCR-ABL1 may not include primers for the detection of e1a2.

There are two possible explanations of why RQ-PCR was not able to detect atypical transcript (e14a3) in this patient. One is that the breakpoints involved in the atypical transcript caused changes in the DNA sequence complementary to the primers used in the RQ-PCR reaction. The other more plausible explanation is that the primer complementary to the ABL1 region mapped nearby exon 2 and hence was upstream to the breakpoint in the ABL1 gene and not present in the atypical transcript [Figure 3].[6]
Figure 3: Schematic diagram of exons showing breakpoint regions and primer target regions. FP: forward primer, RP: reverse primer, arrows: breakpoint regions for the formation of e14a3 transcript

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In this particular case, the multiplex RT-PCR amplified product was found to have a different size compared to the typical transcript sizes of p210 and p190, indicating that an atypical transcript might be present. It is very important to not consider such results as artifacts/false-positive, especially if the results are not concordant with the clinical picture. Variable-sized bands should always be sequenced to confirm or refute the presence of an atypical transcript. Sequencing our PCR-amplified product revealed it to be a rare variant of the BCR-ABL1 fusion transcript, e14a3, which has been described in both CML and Ph+ ALL cases and was first reported in India in 2013.[3],[7],[8] Data on clinical characteristics and prognostic significance of CML patients harboring this transcript are scarce; five patients evaluated in a study showed variable response to TKI therapy and overall survival ranged from 30 to 112 months.[9]

RQ-PCR is increasingly being used as a diagnostic modality in India and although there have been publications from India identifying RQ-PCR as a “reliable” molecular diagnostic test.[10] This case highlights the pitfall of using only standardized RQ-PCR tests for the detection of BCR-ABL1.

Acknowledgments

The present study was financially supported by the Kerala Biotech Commission of the Kerala State Council for Science Technology and Environment, Government of Kerala.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.]



 
   References Top

1.
Bartram CR, de Klein A, Hagemeijer A, van Agthoven T, Geurts van Kessel A, Bootsma D, et al. Translocation of c-ab1 oncogene correlates with the presence of a Philadelphia chromosome in chronic myelocytic leukaemia. Nature 1983;306:277-80.  Back to cited text no. 1
    
2.
Melo J. The diversity of BCR-ABL fusion proteins and their relationship to leukemia phenotype [editorial; comment]. Blood 1996;88:2375-84.  Back to cited text no. 2
    
3.
Baccarani M, Castagnetti F, Gugliotta G, Rosti G, Soverini S, Albeer A, et al. The proportion of different BCR-ABL1 transcript types in chronic myeloid leukemia. An international overview. Leukemia 2019;33:1173-83.  Back to cited text no. 3
    
4.
Deininger MW, Shah NP, Altman JK, Berman E, Bhatia R, Bhatnagar B, et al. Chronic Myeloid Leukemia, Version 2.2021, NCCN Clinical Practice Guidelines in Oncology. J Natl Compr Canc Netw 2020;18:1385-1415. doi: 10.6004/jnccn.2020.0047.  Back to cited text no. 4
    
5.
Langabeer SE. Standardized molecular monitoring for variant BCR-ABL1 transcripts in chronic myeloid leukemia. Arch Pathol Lab Med 2015;139:969-9.  Back to cited text no. 5
    
6.
Kantarjian HM, Talpaz M, Cortes J, O'Brien S, Faderl S, Thomas D, et al. Quantitative polymerase chain reaction monitoring of BCR-ABL during therapy with imatinib mesylate (STI571; Gleevec) in chronic-phase chronic myelogenous leukemia. Clin Cancer Res 2003;9:160-6.  Back to cited text no. 6
    
7.
Cai H, Yang L, Shen K, Zhang W, Xiong J, Zhang M, et al. A rare e14a3 BCR/ABL fusion transcript in acute lymphoblastic leukemia patient treated with CAR-modified T-cell therapy. Oncol Lett 2018;15:2491-4.  Back to cited text no. 7
    
8.
Vaniawala S, Acharya A, Parekh H, Mukhopadhyaya PN. Rare e14a3 (b3a3) BCR-ABL fusion in chronic myeloid leukemia in India: The threats and challenges in monitoring minimal residual disease (MRD). Anal Cell Pathol 2013;36:85-92.  Back to cited text no. 8
    
9.
Xiaomin G, Yong Z, Jinlan P, Huiying Q, Jiannong C, Yongquan X, et al. Chronic myeloid leukemia with e14a3 BCR-ABL transcript: Analysis of characteristics and prognostic significance. Leuk Lymphoma 2015;56:3343-7.  Back to cited text no. 9
    
10.
Azad NA, Shah ZA, Pandith AA, Rasool R, Jeelani S. Real-time quantitative PCR: A reliable molecular diagnostic and follow-up tool for 'minimal residual disease' assessment in chronic myeloid leukemia. Biosci Rep 2018;38. doi: 10.1042/BSR20180974.  Back to cited text no. 10
    

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Correspondence Address:
Deepak Roshan,
Assistant Professor, Division of Genetics and Cytogenetics, Malabar Cancer Centre, Moozhikkara, Thalassery - 670 103, Kerala
India
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijpm.ijpm_715_21



    Figures

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