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Australian Studies Suggest BCR-ABL1 Compound Mutations in CML Patients May be PCR Artifacts

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NEW YORK (GenomeWeb) — Using PCR-based methods to detect mutations in the BCR-ABL1 kinase domain of chronic myeloid leukemia patients has been established as a critical tool for monitoring CML patient response to tyrosine kinase inhibitor therapy.

However, according to recent studies from Australian researchers, when PCR assays reveal the presence of a specific kind of mutation — so-called compound mutations, where more than one mutation occurs within the same BCR-ABL1 molecule — it may in fact be an artifact caused by PCR-mediated recombination.

As such, the researchers claim, clinicians must take care that PCR artifacts are not leading to inaccurate assessments of BCR-ABL1 compound mutations, which could have "dire consequences" for patients. In addition, the investigators assert that new techniques are needed to more reliably detect compound mutations — an endeavor that they are now undertaking with the development of a more precise and sensitive assay, corresponding author Wendy Parker, a researcher in the School of Pharmacy and Medical Science at University of South Australia, told PCR Insider in an email this week.

Parker, who also holds an appointment at the Centre for Cancer Biology at Australian lab network SA Pathology in Adelaide, said that over the last decade her lab has developed accurate and sensitive tests to monitor response to TKI therapy for CML patients.

"While the clinical impact of individual mutations on response to certain therapies is now well-established and there are several sensitive tests for their accurate detection, the impact of compound mutations is more elusive," Parker said. This, she added, is primarily because compound mutations are indistinguishable from multiple polyclonal mutations using standard diagnostic techniques such as Sanger sequencing, allele-specific PCR, and mass spectrometry.

"However, recent in vitro and in silico evidence suggests that BCR-ABL1 compound mutations likely represent an emerging resistance mechanism with significant clinical importance, and therefore other techniques are required to enable assessment of their impact on patient outcome," Parker added. This is an especially salient point considering that this resistance may affect new therapies such as the third-generation TKI ponatinib (marketed by Ariad Pharmaceuticals as Iclusig) and combination TKI therapies being prepped for clinical trials, she said.

In the past few years, several important studies have been published regarding the analysis of BCR-ABL1 compound mutations, including two in the journal Blood that used either nested PCR followed by cloning and Sanger sequencing, or next-generation sequencing; and one in the European Journal of Cancer that used NGS.

Considering the importance to their own work, Parker and colleagues at the University of Southern Australia, SA Pathology, and the University of Adelaide decided to take a closer look at these recent studies, all of which found a high incidence of compound mutations in imatinib-resistant CML patients with multiple BCR-ABL1 kinase domain mutations.

As the Australian researchers outlined in a letter to Blood published earlier this month, in most of these reports the same mutations were surprisingly found both as compound mutations and individual mutations in the same patient, which suggested that the same nucleotide substitution occurred independently multiple times in an individual patient — a highly unlikely scenario, physiologically speaking.

"When we closely examined the results of the BCR-ABL1 compound mutation studies published by others we were astonished by the vast clonal complexity reported," Parker said. "While this could be due to unknown interesting biological phenomena, it may also be explained by technological artifact. Before delving into determining the biological mechanism underlying these findings, we sought to establish whether it may indeed be due to artifact."

To test this hypothesis — which was also supported by extensive evidence that PCR frequently mediates recombination between highly similar templates, generating chimeric amplicons with sequences from more than one allele — Parker and colleagues replicated published procedures using mock polyclonal mutant samples created by mixing mutant BCR-ABL1 plasmids or patient samples.

They found that when plasmids were PCR amplified singly, and their amplicons mixed, sequencing of individual clones revealed kinase domain mutations that largely resembled those present in either of the starting plasmids. However, when the plasmids were mixed prior to PCR amplification, a large proportion of the resultant clones had kinase domain mutations that originated from both of the starting plasmids, suggesting that recombination had occurred during amplification.

The researchers repeated these experiments using seven mixtures of five different plasmids and found that 20 percent to 67 percent of clones showed evidence of artificial recombination resulting in compound mutations that were not present in the starting material, compared with 3 percent in control samples.

They also examined three mock samples created by mixing cDNA from eight patients, each with one kinase domain mutation detected by Sanger sequencing and mass spectrometry. They were able to detect artificial compound mutations in clones of all mixtures, including a compound mutation predicted to confer resistance to ponatinib. Further, additional mutations not present in patient samples were detected in some clones, which suggested that inaccurate nucleotide incorporation by the DNA polymerase also contributes to artifacts.

The upshot, the researchers claimed, is that precautions need to be taken by clinicians interpreting results generated by current PCR-based techniques. They offered up one possible precaution in their letter: using a single-round PCR with reduced cycle number, which they showed generated fewer PCR recombination artifacts, as opposed to nested PCR.

"There have also been several other publications detailing ways to reduce PCR artifacts … such as reducing the initial template concentration, keeping the number of PCR cycles low, using a two-step PCR protocol so that extension occurs at the optimal temperature for primer binding, increasing extension time, removing the final extension step, increasing the primer concentration, testing different polymerase enzymes such as processivity-enhanced polymerases, and pooling several individual PCRs," Parker said. Finally, she added, technical duplication or single-molecule PCR approaches using limiting dilutions or microfluidics may be a remedy.

In the meantime, the group plans to continue its studies and is currently developing a "precise and sensitive assay for detecting BCR-ABL1 compound mutations in CML patients, which may be applicable to other cancers," Parker said.

Without such newer, more sensitive assays, "inaccurate classification of compound mutations may impede discovery of genuine drug resistance mechanisms, and could have dire consequences for patients," she added. "Patients could miss out on life-saving therapy, or conversely they could have rapid disease progression and be at risk of serious side effects from non-effective therapy at great cost to the health system."

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