Variant-specific discrepancy when quantitating BCR-ABL1 e13a2 and e14a2 transcripts using the Europe Against Cancer...

Area of Science:

• Molecular diagnostics
• Hematologic oncology
• Quantitative PCR (qPCR)

Background:

• Molecular monitoring of chronic myelogenous leukemia (CML) treatment response relies on BCR-ABL1 quantification.
• The Europe Against Cancer (EAC) qPCR assay is widely used, amplifying both e13a2 and e14a2 BCR-ABL1 transcript variants.
• Distinct amplification curves observed during qPCR suggest potential variant-specific quantification biases.

Purpose of the Study:

• To investigate whether distinct BCR-ABL1 transcript variants (e13a2 vs. e14a2) affect quantitation accuracy using the EAC qPCR assay.
• To compare qPCR results with absolute quantification by droplet digital PCR (ddPCR) across different diagnostic centers.

Main Methods:

• qPCR efficiency was assessed using cell lines with e13a2 or e14a2 variants at three Danish diagnostic centers.
• BCR-ABL1 %IS values from 219 CML patients (e13a2: n=113; e14a2: n=106) were compared between qPCR and ddPCR.
• Bland-Altman analysis was employed to evaluate bias between qPCR and ddPCR quantitation for each variant.

Main Results:

• No significant differences in amplification efficiencies were observed between e13a2 and e14a2 variants.
• Bland-Altman analysis revealed significant average biases between qPCR and ddPCR values, varying by center (4.6-fold, 6.5-fold, 1.8-fold).
• Overall, qPCR %IS values showed a significant 4.7-fold underestimation of the e14a2 variant compared to the e13a2 variant.

Conclusions:

• The EAC qPCR assay demonstrates a potential underestimation of the e14a2 BCR-ABL1 transcript variant relative to the e13a2 variant.
• This discrepancy may impact the accurate molecular monitoring of CML treatment response.
• Further validation and potential assay modifications are warranted to ensure precise quantification of both BCR-ABL1 variants.