Optimal Molecular Methods in Detecting p190 (BCR-ABL) Fusion Variants in Hematologic Malignancies: A Case Report and Review of the Literature

Abstract

Patients with BCR-ABL1 positive hematologic malignancies and Philadelphia-like B-lymphoblastic leukemia (B-ALL) are potential candidates for targeted therapy with tyrosine kinase inhibitors (TKI). Before TKIs, patients with B-ALL had a much worse prognosis and current treatments with targeted TKI therapy have improved outcomes. Thus, the detection of BCR-ABL1 is crucial and a false negative BCR-ABL1 result may adversely affect patient care. We report a case of a 76-year-old male with a new diagnosis of B-ALL who was initially found to be BCR-ABL1 negative by quantitative polymerase chain reaction (PCR). A concurrent qualitative PCR was performed which detected a positive BCR-ABL1 result that was confirmed by a next generation sequencing (NGS) based assay and identified as the rare fusion variant e1a3 of p190(BCR-ABL). Based on this result, the patient was placed on dasatinib as a targeted therapy. In the era of molecular diagnostic medicine and targeted therapy, it is essential to have an understanding of the limitations of molecular assays and to follow a comprehensive diagnostic approach in order to detect common abnormalities and rare variants. Incorporating NGS methods in an algorithmic manner into the standard diagnostic PCR-based approach for BCR-ABL1 will aid in minimizing false negative results.

Figure 1

B-Lymphoblastic leukemia. (a) Peripheral blood (Wright-Giemsa, 100x) showing a circulating large sized blast. (b) Bone marrow biopsy (hematoxylin & eosin, 50x) showing fibrotic marrow with small area of immature mononuclear cells. No areas of normal hematopoiesis were seen.

Figure 2

BCR-ABL1 qualitative RT-PCR amplification curves. Lightcycler amplification curves showing the cycle number of the crossing point (CP), the point where the reaction's fluorescence reaches the maximum of the second derivative of the amplification curve corresponding to the point where the acceleration of the fluorescence signal is at its maximum. The CP is 29 cycles for the positive control (purple), 35 cycles for the patient's peripheral blood specimen (grey), and no amplification for the negative control (no DNA) (green). Thus, the BCR-ABL1 fusion transcript was detected from the patient's peripheral blood.

Figure 3

Sequence alignments between e1a2 (p190) and the variant e1a3 (p190) on a portion of the BCR-ABL1 sequence. Example of specific primers [6] (green and blue) used to detect e1a2 with the quantitative RT-PCR method. Notice that the exon a2 sequence (blue) is lacking in the e1a3 transcript, represented by the dotted line. The lack of this primer binding site sequence results in a negative BCR-ABL1 p190 result with the p190 quantitative PCR test.