The ne plus ultra for deep BCR-ABL sequencing?

In this issue of Blood, Soverini et al demonstrate the benefits of ultra-deep sequencing (UDS) compared with conventional Sanger sequencing in detecting BCR-ABL kinase domain mutations. This might improve tailoring of the treatment for resistant patients with chronic myeloid leukemia and Ph⁺ acute lymphoblastic leukemia (Ph⁺ ALL) in the future.¹ 

Soon after the introduction of the first tyrosine kinase inhibitor (TKI) imatinib 12 years ago, the first reports appeared of escape mechanisms of malignant clones bearing mutations in the BCR-ABL kinase domain of resistant patients.^2,3  These mutations confer clinical resistance by introducing steric changes of the BCR-ABL protein and thereby hinder binding of BCR-ABL inhibitors. More than 100 different mutations have been described so far, all of them verified by conventional Sanger sequencing.⁴  Of these, only 7 mutated amino acid sites remained problematic for selecting a presumably effective “next-line” TKI.^5,6  One of the open clinical questions is why some resistant patients with 1 or more of the 7 mutations do not respond even though rationally selecting treatment according to in vitro and in vivo experiences. European LeukemiaNet recommendations have been published for how best to identify BCR-ABL mutations in case of unsatisfying response or frank failure on TKI treatment.⁷  These conclude that conventional sequencing should be considered the standard methodology due to its wide availability and robustness. Owing to its low sensitivity, in the range of 10% to 15%, more sensitive techniques have been developed, each of them bearing specific drawbacks.⁸ 

In this issue, Soverini et al¹  applied UDS for mutation detection which combines higher sensitivity (∼1%) with universal coverage of the clinically relevant kinase domain of the BCR-ABL fusion gene. UDS performs thousands of parallel sequencing reactions in separate small picoliter units with each of them hosting 1 DNA molecule. Thereby, single minor clones could be dissected and quantitatively classified according to their bearing 1, 2, 3, or more mutations. Furthermore, in cases with 2 mutations detected by conventional sequencing, UDS was capable of revealing the presence of 2 different clones with 1 mutation each (polyclonal mutations) or both mutations in 1 clone (compound mutations). The latter were previously described to increase oncogenic potency.⁹ 

Because subclones were not detectable with Sanger sequencing in 55% of the samples, UDS was able to unravel multiple clonal dynamics in consecutive samples of patients changing treatment due to resistance.

The findings presented here provide highly differentiated insights in the architecture of multiple resistant clones, including as-yet undetectable mutations by conventional sequencing. Hereby, drawn pictures provide, in part, a retrospective explanation for the examined patients not responding to treatment changes. To justify more widespread use of UDS in TKI-resistant chronic myeloid leukemia or Ph⁺ ALL patients, further trials are warranted to evaluate the clinically relevant sensitivity limits as well as to provide evidence that rational treatment changes according to UDS results lead to better disease control.