DNA Analysis of Mutations in the Kinase Domain of BCR-ABL1 By Allele-Specific Digital PCR Is Highly Sensitive and Refines Prediction of Kinetics of Resistant CML Clones
Authors: Polivkova Vaclava , Curik Nikola , Zizkova Hana , Benesova Adela , Burda Pavel , Pecherkova Pavla , Motlova Eliska , Brezinova Jana , Ransdorfova Sarka , Klamova Hana , Hovorkova Lenka , Zuna Jan , Ernst Thomas , Soverini Simona , Machova Katerina 
Published: October 2019

Abstract
Introduction: In chronic myeloid leukemia (CML) resistant to tyrosine kinase inhibitors (TKI), detection of mutations in the BCR-ABL1 kinase domain (KD) is routinely performed on transcript level. To determine the level of BCR-ABL1 KD mutation is important to follow kinetics of resistant CML cells and therapeutically prevent progression. However, the mutation types and levels are not always reliable predictors of subsequent dynamics of mutation-bearing clones and of corresponding clinical consequences (Willis, 2005; Khorashad, 2006; Preuner, 2012). DNA analysis enables more precise quantification of (sub)clonal levels and thus might be more reliable approach to monitor dynamics of BCR-ABL1 KD mutations.
Aim: To study clonal evolution of resistant CML cells using genomic quantification of mutated BCR-ABL1 KD by droplet digital PCR (ddPCR).
Methods: BCR-ABL1 mutation analysis on transcript level was performed using next generation sequencing (NGS) (Nextera XT; Illumina) and on DNA level using allele-specific ddPCR assays detecting T315I, E255K and Y253H (Bio-Rad). The level of genomic BCR-ABL1 mutation was determined as a copy number of mutation divided by a copy number of genomic BCR-ABL1 fusion. Quantification of genomic BCR-ABL1 was performed by ddPCR using patient-specific primers and probes designed to detect individual fusions. ALB (albumin) quantification was used as a control of DNA load/cell numbers. For analyses, mRNA and DNA extracted from KCL-22 cell line resistant to imatinib (IM) and from leukocytes of a patient who developed T315I during TKI therapy were used.
Results: KCL-22 cell line is characterized by 2 Ph chromosomes and by ability to develop resistance by acquisition of BCR-ABL1 mutations early after the exposure to IM. We repeatedly found, that during early cultivation in the presence of IM, BCR-ABL1-T315I transcript increased up to maximum of 50%. Subsequently, after 2 months, BCR-ABL1-E255K transcripts became detectable and increased over time to 100%, while T315I decreased to un-detectable levels. To study the observed kinetics, we isolated 4 clones resistant to 4 µM IM that expressed 1) 50% of T315I, 2) 50% of E255K and 3) 30% of Y253H. In the fourth clone, no BCR-ABL1 mutation was detected, but mutation acquisition was found in KRAS, RUNX1 and ATRX. The levels of mutated BCR-ABL1 transcripts in mutation bearing clones remained stable over time. DNA analyses confirmed the same level of mutated BCR-ABL1 and revealed that in all resistant clones, only 1 Ph chromosome carried the BCR-ABL1 mutation (T315I, E255K or Y253H). Based on quantification of genomic BCR-ABL1 fusion and albumin we found, that the un-mutated BCR-ABL1 fusion was duplicated in Y253H clone, explaining the 30% level of Y253H. To follow a clonal evolution, we mixed the 4 KCL-22 resistant clones and analyzed BCR-ABL1 KD mutations at both mRNA and DNA levels during exposition to IM. We found that T315I clone overgrew other 3 clones in the mixture over time and 1 Ph chromosome remained mutated. These data confirm the T315I mutation being the most resistant; however, the data from the original cell culture, where the 100% E255K clone overgrew the 50% T315I cells, demonstrate, that a less resistant mutation might dominate the culture if present on both Ph chromosomes (as revealed by DNA analysis).
We compared mRNA and DNA approach in 14 samples collected during individualized treatment management of a CML patient, who developed T315I during TKI therapy. The first mutation detection was during warning response preceded by eight samples negative by mRNA-NGS approach; DNA ddPCR analysis reliably detected T315I mutation in 7 of these 8 samples. Six mRNA positive samples were positive by DNA approach, which showed the same level of T315I.
Conclusions: Allele-specific ddPCR together with quantification of BCR-ABL1 genomic fusion represents highly sensitive and reliable method providing fast and precise quantification of BCR-ABL1 mutations. A single DNA analysis is able to uncover clinically relevant events including BCR-ABL1 amplification or additional mutation acquisition, which presumably influence fitness of leukemic cells and clonal evolution during therapeutic interventions. The information provided by DNA mutational analysis may thus refine prediction of mutation kinetics and consequently improve management of progressed CML and Ph+ ALL.
Support GACR 18-18407S, MZCR 00023736, AZV 15-31540, AZV 16-30186A

Disclosures
Klamova: Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Ernst:Novartis: Research Funding. Soverini:Incyte Biosciences: Consultancy; Novartis: Consultancy; Bristol Myers Squibb: Consultancy. Machova:Bristol-Myers Squibb: Consultancy, Other: Educational grant funding; Incyte: Consultancy; Novartis: Consultancy.