Kinetics Of Low-Level Mutant Clones Detected By Subcloning and Sequencing In Tyrosine Kinase Inhibitor Resistant CML

BCR-ABL1 kinase domain (KD) point mutation causes resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML) patients through impaired binding of TKI to the target site. Recent studies have reported that multiple mutations detected in 2-9% of patients with imatinib (IM)-resistant CML were associated with poor response rate and survival outcomes. However, biological characteristics and dynamics of multiple low-level mutations are still not assessed with a quantitative serial follow-up data in the same populations.

The aim of this study was to investigate biological characteristics and dynamics of low-level mutations in the serial samples from the patients carrying multiple mutations using subcloning and sequencing.

Since 2002, 414 CML patients were screened for mutation analysis due to sign of resistance to TKIs including imatinib (IM), nilotinib (NIL), dasatinib (DAS), bosutinib (BOS), radotinib (RAD) or ponatinib (PON) at Seoul St Mary’s Hospital using direct sequencing and allele specific oligonucleotide-polymerase chain reaction (ASO-PCR). Among them, 31 patients carried ≥ 2 BCR-ABL1 kinase domain mutations. We analyzed 137 samples from these 31 patients using subcloning and sequencing (in total, 2737 colonies were sequenced). By cloning and sequencing, two or more missense mutations present in the same clone were defined as compound mutation and co-existence of single missense mutations in the separated clones was defined as polyclonal mutation. Co-existence of single missense mutation and compound mutation harboring two or more missense mutations in the same clone was defined as mixed mutation. Missense mutations detected by direct sequencing are defined as predominant mutations. Missense mutations detected by cloning and sequencing but not by direct sequencing are defined as low-level mutations.

In a total of 2737 colonies from 137 samples, 1596 (58%) colonies harbored ≥ 2 missense mutations with a median 2 (range, 2 – 7) mutations, and 905 (33%) colonies with a single mutation and 236 (9%) colonies with wild type were observed.

In 2737 colonies, 692 different low-level mutations were detected by cloning and sequencing but not by direct sequencing. Among them, M244V, G250E, Y253H, E279K, T315I, F317L, M351T, E355A, F359I, and F359V were detected by direct sequencing in the followed-up samples, and the others remain undetectable by direct sequencing. To address whether these low-level mutations were distinct in the patients with TKI resistance, we applied the cloning and sequencing to samples from 3 healthy controls and 3 patients with optimal response to IM. 38 different mutations were detected healthy controls. 52 different mutations were detected in optimal responders. S349P, N374S, E450G, and S485P were detected in both healthy controls and optimal responders. All mutations detected in optimal responders except 3 missense mutations (V335A, F382V, and A395S) were detected as low-level mutations in the 31 patients’ cohort with TKI resistance. Of 38 different mutations detected in healthy control, 31 mutations except 7 missense mutations (Y264H, M343R, A350V, E373G, G298R, E462K, and T495K) were also detected as low-level mutations in the 31 patients’ cohort.

We showed basic characteristics and dynamics of low-level mutations by subcloning and sequencing. Some of the low-level mutant clones harboring previously known (clinically common) TKI resistant mutations changes to the predominant in the followed-up samples. Except them, most low-level mutant clones were not detected repeatly and did not increase gradually in the serial samples, implying that they may not have clinical significance.

No relevant conflicts of interest to declare.