Novel Illumina‐based next generation sequencing approach with one‐round amplification provides early and reliable detection of BCR‐ABL1 kinase domain mutations in chronic myeloid leukemia

Abstract

The occurrence of mutations in the BCR-ABL1 kinase domain (KD) can lead to treatment resistance in chronic myeloid leukaemia patients. Nowadays, next-generation sequencing (NGS) is an alternative method for the detection of kinase domain mutations, compared to routinely used Sanger sequencing, providing a higher sensitivity of mutation detection. However, in the protocols established so far multiple rounds of amplification limit reliable mutation detection to approximately 5% variant allele frequency. Here, we present a simplified, one-round amplification NGS protocol for the Illumina platform, which offers a robust early detection of BCR-ABL1 KD mutations with a reliable detection limit of 3% variant allele frequency.

Hierarchical distribution of somatic variants in newly diagnosed chronic myeloid leukaemia at diagnosis and early follow‐up

“…This is also consistent with our earlier findings, where retrospectively tracked mutations from treatment failure were detected only in two of seven patients at diagnosis (VAF <3%). 29 In contrast to our present data, earlier studies analysing the CD34 + fraction in prospective CP-CML cohorts showed up to 32% mutation incidence. 16,17 In one of these, our team previously reported 8% BCR-ABL1 KD mutation incidence (four of 50 patients), detected with the GS Junior Roche NGS platform.…”

“…In both studies, mutations could only be detected at progression in 58% (19/33) and 38% (three of eight) patients. This is also consistent with our earlier findings, where retrospectively tracked mutations from treatment failure were detected only in two of seven patients at diagnosis (VAF <3%) 29 . In contrast to our present data, earlier studies analysing the CD34 + fraction in prospective CP‐CML cohorts showed up to 32% mutation incidence 16,17 .…”

“…Total RNA was reversely transcribed and subjected to NGS library preparation as described previously 29 . Sequencing the complementary DNA region corresponding to BCR‐ABL1 KD using the Sanger method was performed as described previously 30 …”

Imatinib Resistance in Chronic Myeloid Leukemia Associated with a D363G BCR::ABL1 Kinase Domain Mutation

“…While Sanger sequencing is the traditional method of detection, this technique lacks sensitivity for the early detection of BCR::ABL1 KDM. More recently, nextgeneration sequencing (NGS) approaches have demonstrated improved limits of detection and are becoming increasingly adopted [2][3][4]. Uncommon and novel mutations continue to be described in TKI-resistant CML and Philadelphia chromosome-positive acute lymphoblastic leukemia patients [5][6][7].…”

Differences in Variants in the Structural Domain of <b><i>BCR-ABL1</i></b> Kinase between Chinese Han and Minority Patients with Chronic Myeloid Leukemia by Sanger Sequencing and Next-Generation Sequencing

“…However, SS has some limitations that may be addressed by next-generation sequencing (NGS) [Soverini et al, 2019]. Studies have shown that NGS has a higher sensitivity for mutation detection and can detect pathogenic variants and copy number changes in tumor cytokines [Deregowska et al, 2020;Romzova et al, 2020]. In particular, it has played an important role in detecting early-onset variants in the structural domain of kinases that are not detected by SS and in engaging in early clinical intervention to improve clinical care for patients [Kizilors et al, 2019].…”