Assessment of quantitative polymerase chain reaction for BCR–ABL1 transcripts in chronic myeloid leukaemia: Are improved outcomes in patients with e14a2 transcripts an artefact of technology?

Abstract

Summary The clinical outcome of chronic myeloid leukaemia patients has vastly improved since the introduction of tyrosine kinase inhibitor treatment, with a significant proportion of patients able to achieve treatment‐free remission. However, studies have shown that patients with the e13a2 transcript were less likely to achieve major molecular response compared to those with e14a2 transcripts. Most quantitative polymerase chain reaction (PCR) assays for detection of the BCR–ABL1 fusion gene do not differentiate between the two transcripts and we therefore hypothesised that technical bias linked to the qPCR assay could partially explain the discrepancy in outcomes. We designed an e14a2‐specific assay and identified no difference in results compared to an e13a2 standard assay. We then demonstrated that the commercial e14a2 standards were causing a significant overestimation of the e13a2 transcripts. Finally, we reviewed patient management after the qPCR values were corrected, using our new evaluation. We concluded that despite statistically significant differences in qPCR results, there was no impact on patient management or outcome. We conclude that, at least in our institution, it would be inappropriate to perform separate assays for patients with e13a2 or e14a2.

Impact of BCR::ABL1 transcript type on RT-qPCR amplification performance and molecular response to therapy

“…Although the application of laboratory-specific CFs helps to mitigate against this difference, the increase in variation of the results suggests that CFs may not be fully optimised for the e13a2 transcript. Recent work by Dominy et al [ 31 ] also investigated the effect of transcript-specific standard curves, and our results corroborate and extend their findings. All currently available reference materials for BCR::ABL1 are based on e14a2, which likely accounts for the relative lack of assay optimisation for e13a2.…”

“…This issue is almost certainly not limited to the EAC primer/probe set, but likely affects other assays with similar differences in amplicon sizes between e13a2 and e14a2. It is important to emphasize, however, that the discrepancy is subtle and, although its consequences are apparently detectable in some large studies [ 9 – 11 ], the effect on individual cases is expected to be very small [ 31 ]. Nevertheless, we recommend caution in making clinical decisions based on patient transcript type and stress the need to consider trends in sequential MRD results in addition to the achievement of defined milestones at specific timepoints.…”

BCR::ABL1 transcripts and clinical outcome ‐ interrogating the technique

“…A study by Dominy et al 4 published in this issue, demonstrates that e13a2 transcript levels were indeed overestimated using their molecular assay. BCR :: ABL1 transcript values for e13a2 containing samples were on average 1.38 to 1.95‐fold overestimated.…”

“…The conclusion from Dominy et al 4 was that the systematic overestimation of the e13a2 transcript level using their assay did not sufficiently impact treatment outcome to warrant a redesign of their assay. Guidelines and recommendations rely on a static BCR :: ABL1 value at milestone timepoints for treatment decisions: 10% IS; 1% IS; and 0.1% IS.…”

“…These assessments can be made independently of accurate conversion to the international reporting scale. The method used by Dominy et al 4 was a laboratory developed method rather than one of the widely used commercially available assays. However, the laboratory method used a common set of PCR primers and probes that were developed for method harmonisation by a Europe Against Cancer (EAC) initiative.…”