Abstract B64: High-sensitivity detection of M351T, F317L, and F311C BCR-ABL kinase domain mutation in CML patients treated with imatinib and dasatinib
Authors: Mir Ab , Sazawal Sudha , Chobey Rekha , Bohra Bijender , Sharma Bharti , Kumar Ravi , Chopra Anita , Mishra Pravas , Mahapatra Manuranjan , Saxena Renu 
Published: May 2012

Abstract
B64

Background
the kinase domain (KD) of BCR-ABL are the leading cause of acquired imatinib resistance .

Objectives
(1) To study the High-sensitivity detection of M351T, F317L and F311C BCR-ABL kinase domain mutation in Chronic Myeloid Leukemia patients treated with TKIs (Imatinib and dasatinib ) .
(2) To evaluate the effect of imatinib dose esculation in the CML patients carrying M351T mutations.

Methods
A total of 100 CML patients were selected who were treated with Imatinib at 400mg/day from 2 to 4 years .They were diagnosed by RT-PCR for BCR-ABL transcripts. Early screening for the M351T, F317L and F311C mutations was performed by allele specific-oligonucleotide-PCR (ASO-PCR). The primer sets designed were similar to those used previously.

Results
We evaluated 100 CML patients for M351T, F317L and F311C ABL mutations by ASO-PCR after three years of Imatinib initiation .Patients were categorized into three groups.
Group A: All 100 CML patients were treated with imatinib at conventional dose of 400mg/day and were screened for M351T mutation after three years of Imatinib initiation . (40%) 40/100 were positive for M351T mutation consequently 20/40 were treated with high dose imatinib at 600 to 800 or 1000 mg/day . After 11 months of dose escalation, 15/20 lost M351T mutation but remaining five who resist M351T mutation, developed a more fatal mutation called gate keeper mutation T315I. 2/5 died , three progressed to advanced disease. The development of T315I mutation was significantly associated with a greater likelihood of subsequent progression to accelerated phase/blast crisis (P&lt; .0002) and shorter survival (P&lt; .001).
Group B: All 100 CML patients were screened for F311C mutation after three years of Imatinib initiation and (10%) 10/100 were positive. After 10 months ,4/10 developed a more fatal mutation in P-loop region (Y253H and E255N) and consequently 2/4 died and one progressed to advanced disease . The presence of F311C may provide an advantage to develop p-loop mutations which are associated with poor prognosis .
Group C: It included 12 imatinib resistant CML patients treated with dasatinib at 70 to 100mg/day .At the initiation of dasatinib ,No one was positive for F317L and T315I mutation. After 10 months of dasatinib treatment, all were screened for F317L/ T315I mutation by ASO-PCR ,4/12 were positive for F317L mutation and no one was positive for T315I mutation .After 6 months of mutation detection 2/4 progressed to blast crisis, and 1/4 died .

Significance
We screen for KD mutations with high sensitivity ASO-PCR as the screening strategy. ASO-PCR has been reported to achieve a sensitivity of up to 1 copy of mutant allele in 104 copies of wild-type allele .

Conclusions
Our results showed that irrespective of others risk factors for resistance, monitoring for emerging mutations at any stage of the gleevec and dasatinib therapy may play a role in detecting patients with worse prognosis, for whom the revision of the therapeutic strategy should be considered . ASO-PCR proved to be a very economical, sensitive and rapid technique for detection of M351T, F317L and F311C ABL mutation and is more sensitive than mutation detection by sequencing. The detection of M351T, F317L and F311C -ABL mutation at any stage has prognostic significance.
Citation Information: Cancer Prev Res 2008;1(7 Suppl):B64.