A group led by researchers from the Children's Cancer Research Institute in Vienna has developed a method using the Roche 454 GS FLX+ to sequence the entire BCR-ABL1 tyrosine kinase domain in order to measure the presence and clonal distribution of mutations in patients with Philadelphia-positive leukemia.
The team published the study in the European Journal of Cancer last month, detailing the performance of this method in a small group of specimens from six patients with chronic myeloid leukemia.
Alexander Kohlmann, who is a member of German leukemia diagnostic lab Münchner Leukämielabor and was not involved in the Austrian study, told Clinical Sequencing News in an email this week that the method is "promising."
"With the availability of a 'long-read GS Junior', as Roche announced to be available soon, it will be very interesting for laboratories in the CML/tyrosine kinase inhibitor space," he added.
According to the Austrian study authors, rapid and sensitive detection of subclones carrying BCR-ABL1 TKD mutations associated with TKI resistance is a growing clinical need in Ph+ leukemias.
Meanwhile, the group wrote, current diagnostic methods, including next-gen sequencing of short fragments, leave room for improvement due to a limited ability to identify compound mutations or to distinguish mutations on the same or different molecules without the addition of potentially costly and laborious steps.
This drove the group to develop its long-range NGS strategy, allowing for coverage of the ABL1 kinase domain in a single long read.
In the study, the researchers tested the method on 11 individual and consecutive samples from six CML patients displaying single, multiple, or no detectable point mutations by established methods including Sanger sequencing, pyrosequencing, and ligation-dependent PCR.
The group described the method, which begins with reverse transcription and PCR amplification of the BCR-ABL1 kinase domain, followed by a specific library preparation protocol and then sequencing on the 454 GS FLX+.
According to the authors, the protocol allowed sequencing coverage of the entire ABL1 kinase domain in a single read, which revealed both single and compound point mutations as well as insertions and deletions in the tested samples. Overall, median coverage of individual target sequences was 38,401 reads, ranging from 20,388 reads to 59,825 reads, the group reported.
Most of the mutations the team detected have been reported previously, the authors wrote. But the group also found new aberrations, including a 540 base pair deletion affecting the whole of exons seven and eight and parts of exons six and nine, as well as two previously unreported compound mutations.
Based on bioinformatic analysis, the researchers set a cutoff point of 1 percent or greater of the entire BCR-ABL1 leukemic clone for subclones to be considered true variants because smaller subclones could not be distinguished from possible artifacts of the amplification and sequencing process.
The team then compared the findings to results using an independent PCR and Sanger sequencing approach in two of the samples to address the possibility of confounding artifacts and establish the accuracy or reliability of the method. These independent measurements confirmed the long-range NGS results the group generated, suggesting the approach is reliable.
According to the study authors, other NGS approaches have been developed to sensitively identify compound mutations in subclones in the BCR-ABL1 kinase domain, notably, a method using four overlapping amplicons for ultra-deep sequencing that was developed by an Italian group and published in Blood earlier this year.
Such approaches may not be readily amenable to screening for compound mutations in the routine clinical setting, the Austrian study authors wrote, due to the current high costs of next-gen sequencing.
The team's LR-NGS approach, meanwhile, offers a reliable and more economic option, the team argued, and thus has the "potential of becoming the method of choice form mutational screening, particularly for [high-throughput] identification of compound mutations in Ph-positive leukemias."