Highly Sensitive Mutations Detection in BCR-ABL Positive Leukemia Prior and during Imatinib Treatment.
Authors: Cazzaniga Giovanni , Corradi Barbara , Piazza Rocco , Soverini Simona , Martinelli Giovanni , Gambacorti-Passerini Carlo , Biondi Andrea 
Published: October 2019

Abstract
The most frequently identified mechanism of acquired Imatinib resistance in Ph+ leukemia is BCR-ABL kinase domain point mutations. The observation that 90% of patients with mutations eventually relapsed suggests that harboring any BCR-ABL mutation has prognostic value. Thus, the detection of BCR-ABL mutants prior to and during the course of Imatinib therapy may aid in determining therapeutic strategies. However, current methods for mutation screening have either low sensitivity or are too time consuming and labor intensive to be routinely feasible. In addition, to date approximately 30 different point mutations in the BCR-ABL kinase domain have been isolated from Ph+ patients resistant to Imatinib treatment. This may require a method of mutation detection at large spectrum. We tested the feasibility of using the “SNP mining tool” on the Nanogen microarray device (San Diego, CA) for a rapid and sensitive identification of BCR/ABL mutations. The Nanogen SNP mining tool allows de novo discovery of genetic variations, including SNPs and mutations. The method combines the features of electronically controlled DNA hybridization on open-format microarrays, with mutation detection by a fluorescence-labeled mismatch-binding protein (mutS protein). If the test DNA contains a point mutation when compared to the reference DNA, the heteroduplex will contain a mismatch. This mismatch is detectable by the fluorescent-labeled mutS protein that binds to DNA containing a mismatch at any position of the DNA sequence. Since 500bp DNA fragments can be analyzed simultaneously, the entire ABL tyrosine kinase domain can be monitored by only two overlapping PCR reactions, thus substituting high throughput sequencing as a preliminary screening of point mutations. Electronic addressing of DNA strands to distinct test sites of the chip allows parallel analysis of several individuals or several SNPs/mutations in few individuals. We first performed a preliminary series of experiments on samples known to contain mutations by sequencing of PCR clones. A point mutation in ABL could be identified in a 10 copies dilution of a mutated plasmid. Different point mutations in ABL could be identified in 6 patients in which at least 1 mutated clone was found by PCR, cloning and sequencing. Four patients resistant to Imatinib were tested in 10-fold cDNA serial dilutions. A positive value was found till a 10−4 dilution in 3/4 patients. A sensitivity of 10−3 was found in 1 patient. We also monitored a single CML p210-positive patient in late chronic phase, resistant to IFN and treated with 400mg/die Imatinib. In this patient, always positive by RT-PCR of BCR-ABL, we could demonstrate the presence of the mutation prior to Imatinib and in all follow up time points during Imatinib therapy, since 1 month to 6 months. The follow up of additional Ph+ cases will be analyzed. In conclusion, the sensitivity and the short time required for the analyses of different patients samples, or multiple time points during follow-up, make the Nanogen device as an alternative to DHPLC or cloning, for the rapid identification of BCR/ABL mutations and monitoring of BCR/ABL mutations during Imatinib treatment. Early diagnosis of resistance and prompt treatment with appropriate drug dosing may be essential for increasing the success of cure in Ph+ leukemia.