P-loop mutations (Y253H and E255N), their clinical significance and prevalence in Imatinib mesylate resistance CML patients.
Authors: AB Mir, sazawal, Rekha Chobey, Bharti s, Bijender Bohra, choudry, Ravi k, Rathi, Reyaz Mir, Muzafer Pandit, Mishra, Saxena
Journal: Clinical Cancer Research (2008)

B5    Objectives:  (1) To study the incidence of P-loop mutations (Y253H and E255N) by ASO-PCR in newly diagnosed CML patients.  (2) To study the prevalence of P-loop mutations (Y253H and E255N) in relapsed CML patients .   Methods:   Two hundred consecutive patients who presented with a diagnosis of CML had blood examined by RT-PCR for BCR-ABL transcripts. They were started on treatment with imatinib. Early screening for the T315I mutation was performed by allele specific-oligonucleotide-PCR (ASO-PCR). The primer sets designed were similar to those used by Ho-Young Kang et al for P-loop mutations (Y253H and E255N) . The sequences of forward and reverse primers were Y253H N-GCTGGGCGGGGGCCAG & Y253H M-GCTGGGCGGGGGCCAGC and E255N-GCGGGGGCCAGTACGGGG & E255M-GCGGGGGCCAGTACGGGGT.The sensitivity of ASO-PCR is superior to that of direct sequencing as it could detect one mutant allele in 100~100,000 wild type sequences. Mutated and wild-type alleles were specifically amplified in a 50 ul and mixture 100 ng of gDNA .      Results:  All patients were monitored for hematologic and molecular responses, time to progression, survival and toxicity. Results: Patients were divided into two groups on the basis of theeir original presentation. Group A included 190 CP-CML patients who started imatinib at 400mg/day. After a median follow-up of 36 months or 3 years (range 3 to 5 years 60 (31.6%) 30/190 patients had developed molecular relapse and hematologic progression.  P-loop mutations were detected about (66%) {20/30 (Y253H-18/30 and E255N 19/30} of these patients. 15/30 responded to high dose Imatinib for some time but 17 progressed to advanced stage and 5 died.  Group B included 10 patients in advanced phase (AP or BC) at presentation. All were treated with imatinib at 600 to 800 mg/day. After 6 months of treatment 8 (80%) had evidence of P-loop mutation and 2 died. In total 20% to 24% (30+8) 200 of relapsed CML patients were positive for Y253H/E255N mutation ; those originally in chronic phase progressed rapidly to advanced phase.  No Y253H/ E255N T315I mutations were detected in patients in early-CP. Survival and time-to-progression curves were obtained from Kaplan-Meier method. There was a significant difference in the survival rate of patients with or without T315I mutations. Patients with T315I mutations had short survival. Side-effects of imatinib included anemia (n=60), thrombocytopenia (n=40 ) and hypopigmentation of skin (n=20 ).   Conclusions:  Our findings suggest that the early detection of P-loop mutations  mutations proved helpful in clinical management of therapeutic decisions.  ASO-PCR is a very economical, sensitive and rapid technique for detection of P-loop mutations  mutations and is more sensitive than mutation detection by sequencing.  The speed, simplicity, and sensitivity of ASO-PCR assays will be useful for routine monitoring of mutations, especially for frequently identified mutations.  The detection of P-loop mutations  was associated with imatinib resistance and poor prognosis.