A co-operative evaluation of different methods of detecting BCR-ABL kinase domain mutations in patients with chronic myeloid leukemia on second-line dasatinib or nilotinib therapy after failure of imatinib

Abstract

Background: Various techniques have been employed to detect BCR-ABL kinase domain mutations in patients with chronic myeloid leukemia who are resistant to imatinib. This has led to different reported frequencies of mutations and the finding of a heterogeneous pattern of individual mutations.

Design and methods: We compared direct sequencing alone and in combination with denaturing high-performance liquid chromatography and two high-sensitivity allele-specific oligonucleotide polymerase chain reaction approaches for analysis of BCR-ABL mutations in 200 blinded cDNA samples prior to and during second-line dasatinib or nilotinib therapy in patients with chronic myeloid leukemia in whom imatinib treatment had failed.

Results: One hundred and fourteen mutations were detected by both direct sequencing alone or in combination with high performance liquid chromatography and 13 mutations were additionally detected by the combined technique. Eighty of 83 mutations (96%) within a selected panel of 11 key mutations were confirmed by both allele-specific oligonucleotide polymerase chain reaction techniques and 62 mutations were identified in addition to those detected by combined liquid chromatography and direct sequencing, indicating the presence and a high prevalence of low-level mutations in this cohort of patients. Furthermore, 125 mutations were detected by only one allele-specific oligonucleotide polymerase chain reaction technique. Pre-existing mutations were traceable 4.5 months longer and emerging clones were detectable 3.0 months earlier by allele-specific oligonucleotide polymerase chain reaction than by direct sequencing together with liquid chromatography.

Conclusions: Our results suggest that denaturing high performance liquid chromatography combined with direct sequencing is a reliable screening technique for the detection of BCR-ABL kinase domain mutations. Allele-specific oligonucleotide polymerase chain reaction further increases the number of detected mutations and indicates a high prevalence of mutations at a low level. The clinical impact of such low-level mutations remains uncertain and requires further investigation. Allele-specific oligonucleotide polymerase chain reaction allows detection of defined mutations at a lower level than does denaturing high performance liquid chromatography combined with direct sequencing and may, therefore, provide clinical benefit by permitting early reconsideration of therapeutic strategies.

Figure 1.

Comparison of the number of mutations detected by the four different mutation analysis methods (for a panel of 11 key mutations). In total, 200 blinded cDNA samples prior to and during second-line TKI therapy were simultaneously analyzed by all techniques. Differences between direct sequencing and D-HPLC/DS were not statistically significant. For Y253H, E255K, T315I, and F317L, statistically significant differences were observed between D-HPLC/DS and both ASO methods. Numbers over the bars indicate p values in cases of statistically significant differences, as obtained by Fisher’s exact test.

Figure 2.

(A) Comparison of both ASO PCR methods for high-level mutations (i.e. mutations which were also detected by D-HPLC/DS). Eighty of 83 high-level mutations (96%) were independently detected by both ASO methods. Hatched areas correspond to mutations which were detected by both ASO methods in the same sample. ARMS detected one G250E mutation which was not detected by L-PCR and L-PCR revealed one F317L mutation which was not detected by ARMS. (B) Comparison of both ASO PCR methods for low-level mutations (i.e. mutations which were not detected by D-HPLC/DS). Sixty-two of 187 low-level mutations (33%) were independently detected by both ASO techniques (hatched areas). Forty-eight low-level mutations were detected by ARMS only and 77 by L-PCR only. L-PCR detected statistically significantly more T315I low-level mutations than did ARMS (p<0.0001).

Figure 3.

Evaluation of the diagnostic window for emerging drug-resistant clones and follow-up of pre-existing clones as obtained by ASO PCR versus D-HPLC/DS. A total of 15 mutant clones emerged and 10 pre-existing clones disappeared as detected by D-HPLC/DS over a 12-month period of second-line TKI treatment. Detection of these clones by ASO PCR was compared to that by D-HPLC/DS in serial samples. For individual mutations the respective median differences (months) compared to D-HPLC/DS are illustrated by bars. ASO PCR detected emerging mutations a median of 3.0 months earlier and traced pre-existing mutations 4.5 months longer than D-HPLC/DS.