What is Imatinib Resistance Testing?

Imatinib is a targeted tyrosine kinase inhibitor for patients suffering from Chronic Myeloid Leukemia (CML)
Patients’ response to Imatinib has been significantly better and with fewer side effects than other available therapies
But even in the best responders, some BCR-ABL1 positive cells usually remain.
Patients who initially responded well to Imatinib have at times relapsed due to the development of resistance caused by changes in the ABL1 kinase domain. This affects Imatinib’s ability of binding to the active site

Prevalence

The diagnostic marker of CML is the Classical Philadelphia chromosome, t(9;22)(q34;q11)
This results in the fusion of the N-terminal region of the BCR gene with the C-terminal kinase domain of the ABL1 gene. The fusion produces an active chimeric protein kinase responsible for leukemogenesis in CML.
CML patients on Imatinib are monitored with quantitative RT-PCR and that patients with a log increase in quantitative BCR-ABL1 transcript levels are recommended to be assayed for the presence of Imatinib-resistance mutations
Over 130 mutations in Imatinib-resistant patients are reported, of which frequent variations attributed to resistance include:
M237I M244V L248V G250E Q252H
Y253F/H E255K/V D276G E279K V299L
F311I/L/V T315I F317L M351T E355G
F359V/C V379I L384M M388L H396R/P
S417Y/T E450G E453K/V/D E459L/K/G F486S
The T315I mutation appears to confer resistance to multiple targeted tyrosine kinase inhibitors, while other mutations may be more responsive to other therapies
Upon understanding the mutation status, the clinician intervenes by increasing the dosage of Imatinib depending on the exact mutation present. Or adding another kinase inhibitor may be effective in controlling BCR-ABL1 levels
Determines the therapeutic strategy for each patient by identifying the mutation status of the ABL1 kinase domain by either:
- Increasing Imatinib dose
- Switching to second-generation inhibitors as Dasatinib, Nilotinib or Ponatinib
- Alternate treatment approaches

MedGenome offers Imatinib Resistance testing by Next Generation Sequencing (NGS)
Imatinib Resistance Test carried out by Next-generation Sequencing (NGS) technology has multiple advantages
- Sanger Sequencing could only detect mutations present above a 20% allele load
- RT-PCR has greater sensitivity, but limited by their short spectrum of mutation detection
Several NGS/Sanger comparison studies reported that Sanger sequencing had misclassified or underestimated kinase domain mutation status in up to 55% of samples, where mutations with 1-15% abundance are present
NGS revealed emerging resistant mutants 2-11 months earlier when compared to Sanger sequencing
Imatinib Resistance Mutation testing by NGS aids in:
- Full characterization of the spectrum of a minor (<20%) mutated variants
- Ability to follow the dynamics of resistant mutations over time
- Reconstruction of the clonal architecture of mutated populations in the case of multiple mutations occurring within the same amplicon.

Assay characteristics	NGS	Sanger sequencing	RT-PCR
Throughput	High	Low	Medium
The sensitivity of mutation detection	>1%	>20%	>5%
Ability to detect novel mutations	Yes	Yes	No
Identification of InDels	Yes	Yes	Yes
Distinguish between compound mutations	Yes	Yes	No
Identification of Polyclonal mutation	Yes	Yes	No
Quantification of mutation burden	Yes	Yes	No