Lab Med Online.  2023 Oct;13(4):370-374. 10.47429/lmo.2023.13.4.370.

A Case of Chronic Myeloid Leukemia with Micro BCR::ABL1 Rearrangement: Precaution in Reverse Transcription PCR to Prevent False Negativity

• Kim SW
• Kim H
• Shin S
• Chung H

• KMID: 2552765
• DOI: http://doi.org/10.47429/lmo.2023.13.4.370

Abstract

We report a patient negative for BCR::ABL1 in qualitative reverse transcription (RT)-PCR but subsequently reported to be positive for t(9;22) (q34;q11.2) in conventional karyotyping. The patient was finally diagnosed with micro-type chronic myeloid leukemia after re-examining RT-PCR and performing targeted RNA sequencing. Through this case, we highlight the risk of false negativity when interpreting RT-PCR to detect microtype fusion. Upon re-examining RT-PCR results, the patient’s internal control band was thicker than others. After extending the electrophoresis run time, a 911-bp internal control band and a target band around the level of 1.0 kb were separated. We confirmed a fusion breakpoint (BCR exon 19 and ABL1 exon 2) by targeted RNA sequencing, and it corresponds to 1,012 bp-sized e19a2 (c3a2) type among four micro-type fusion transcripts that RT-PCR HemaVision ^® kit M6B can detect.

Keyword

Chronic myeloid leukemia; BCR::ABL1; p230; Thrombocytosis; False negative

Figure

• 

  Fig. 1 Presentation of (A) peripheral blood smear (×200 magnification) and (B) a bone marrow aspiration at initial diagnosis (×200 magnification).

• 

  Fig. 2 Qualitative BCR::ABL1 RT-PCR results using a HemaVision® kit (DNA Technology, Aarhus, Denmark). (A) Gel electrophoresis of the M6B lane at initial diagnosis. The index patient’s IC band (yellow arrow) appeared thicker than that of other patients (triangles) without the BCR::ABL1 transcript. (B) Repeated gel electrophoresis with a prolonged run time of up to 50 min revealed a separate band with a larger molecular size (1,012 bp, yellow arrow) than the IC band (911 bp). Abbreviations: L, molecular size ladder (3.0 kb, 2.0 kb, 1.5 kb, 1.0 kb, 800 bp, 600 bp, 500 bp, 400 bp, 300 bp, 200 bp, and 100 bp size from the top); Pt, index patient; NTC, no template control; PC, e13a2 type positive control with 472 bp size.

• 

  Fig. 3 Targeted RNA sequencing analysis (FusionPlex Pan-Heme Panel; ArcherDX, Boulder, CO, USA) confirmed breakpoints in BCR exon 19 and ABL1 exon 2. The fusion reads of BCR::ABL1 numbered 634 (64.63 % of total reads). Breakpoint visualization was performed with the Integrative Genomics Viewer (Broad Institute, Cambridge, MA, USA).

Reference

1. Melo JV. 1996; The diversity of BCR-ABL fusion proteins and their relationship to leukemia phenotype. Blood. 88:2375–84. DOI: 10.1182/blood.V88.7.2375.bloodjournal8872375. PMID: 8839828.

2. Ben-Neriah Y, Daley GQ, Mes-Masson AM, Witte ON, Baltimore D. 1986; The chronic myelogenous leukemia-specific P210 protein is the product of the bcr/abl hybrid gene. Science. 233:212–4. DOI: 10.1126/science.3460176. PMID: 3460176.

3. Ohsaka A, Shiina S, Kobayashi M, Kudo H, Kawaguchi R. 2002; Philadelphia chromosome-positive chronic myeloid leukemia expressing p190(BCR-ABL). Intern Med. 41:1183–7. DOI: 10.2169/internalmedicine.41.1183. PMID: 12521212.

4. Komorowski L, Fidyt K, Patkowska E, Firczuk M. 2020; Philadelphia chromosome-positive leukemia in the lymphoid lineage-similarities and differences with the myeloid lineage and specific vulnerabilities. Int J Mol Sci. 21:5776. DOI: 10.3390/ijms21165776. PMID: 32806528. PMCID: PMC7460962.

5. Ito T, Tanaka H, Tanaka K, Ito K, Kyo T, Dohy H, et al. 2004; Insertion of a genomic fragment of chromosome 19 between BCR intron 19 and ABL intron 1a in a chronic myeloid leukaemia patient with micro-BCR-ABL (e19a2) transcript. Br J Haematol. 126:752–3. DOI: 10.1111/j.1365-2141.2004.05119.x. PMID: 15327531.

6. Mondal BC, Majumdar S, Dasgupta UB, Chaudhuri U, Chakrabarti P, Bhattacharyya S. 2006; e19a2 BCR-ABL fusion transcript in typical chronic myeloid leukaemia: a report of two cases. J Clin Pathol. 59:1102–3. DOI: 10.1136/jcp.2005.029595. PMID: 17021137. PMCID: PMC1861751.

7. Haskovec C, Ponzetto C, Polák J, Maritano D, Zemanová Z, Serra A, et al. 1998; P230 BCR/ABL protein may be associated with an acute leukaemia phenotype. Br J Haematol. 103:1104–8. DOI: 10.1046/j.1365-2141.1998.01098.x. PMID: 9886327.

8. Kim HY, Park S, Kim SH, Kim HJ. 2017; The first report of chronic myelogenous leukemia with e19a2 micro-BCR/ABL1 presenting with massive myelofibrosis. Blood Cells Mol Dis. 65:68–70. DOI: 10.1016/j.bcmd.2017.05.001. PMID: 28571780.

9. Lee J, Kim DS, Lee HS, Choi SI, Cho YG. 2017; Concurrence of e1a2 and e19a2 BCR-ABL1 Ffusion Ttranscripts in a Tyypical Ccase of Cchronic Mmyeloid Lleukemia. Ann Lab Med. 37:74–6. DOI: 10.3343/alm.2017.37.1.74. PMID: 27834071. PMCID: PMC5107623.

10. Kim JH, Lee WM, Ryoo NH, Ha JS, Jeon DS, Kim JR, et al. 2008; A case of atypical CML with micro BCR/ABL rearrangement. Korean J Hematol. 43:184–9. DOI: 10.5045/kjh.2008.43.3.184.

11. Goh HG, Hwang JY, Kim SH, Lee YH, Kim YL, Kim DW. 2006; Comprehensive analysis of BCR-ABL transcript types in Korean CML patients using a newly developed multiplex RT-PCR. Transl Res. 148:249–56. DOI: 10.1016/j.trsl.2006.07.002. PMID: 17145570.

12. Molica M, Abruzzese E, Breccia M. 2020; Prognostic significance of transcript-type BCR-ABL1 in chronic myeloid leukemia. Mediterr J Hematol Infect Dis. 12:e2020062. DOI: 10.4084/mjhid.2020.062. PMID: 32952973. PMCID: PMC7485470.

13. Engvall M, Cahill N, Jonsson BI, Höglund M, Hallböök H, Cavelier L. 2020; Detection of leukemia gene fusions by targeted RNA-sequencing in routine diagnostics. BMC Med Genomics. 13:106. DOI: 10.1186/s12920-020-00739-4. PMID: 32727569. PMCID: PMC7388219.

14. Langabeer SE. 2015; Standardized molecular monitoring for variant BCR-ABL1 transcripts in chronic myeloid leukemia. Arch Pathol Lab Med. 139:969. DOI: 10.5858/arpa.2014-0522-LE. PMID: 26230589.