EARLY DETECTION AND DYNAMICS OF MUTATED CLONES IN CHRONIC MYELOID LEUKEMIA PATIENTS WITH SUBOPTIMAL RESPONSE

FERRI, CRISTIAN; BIANCHINI, MICHELE; ICARDI, GUSTAVO; BELLI, CAROLINA; BENGIÓ, RAQUEL; LARRIPA, IRENE

Baltimore

Conferencia; International Conference Chronic Myeloid Leukemia: Biology and Therapy.; 2012

European School of Haematology

The introduction of the tyrosine kinase inhibitors (TKI) has greatly improved the treatment of chronic myeloid leukemia (CML). However in a small number of cases resistance to treatment is observed. The most frequently identified mechanism of acquired resistance to TKI is the presence of mutations in the BCR-ABL1 kinase domain. This phenomenon correlates with disease progression. The aim of this work was to investigate the early detection of mutated clones in cases with suboptimal molecular response (BCR-ABL1 transcripts > 0.1% after 18 months of treatment). The study was conducted on peripheral blood from 32 CML patients that showed lack or loss of Major Molecular Response (MMR) during TKI treatment. The early screening of BCR-ABL1 mutations was performed by High Resolution Melting (HRM) methodology; the identification and subsequent quantification of mutant clones were carried out by quantitative real time Amplification Refractory Mutation System (ARMS-qPCR). The analysis by HRM indicated us the presence of a nucleotide variation in 11 cases (11/32, 34.4%); while by automatic sequencing only 2 cases (6.2%) resulted positive. By ARMS-qPCR we could report the following mutations: T315I (3 cases), F317L (3 cases), V299L (4 cases) and G250E (1 case). This analysis allowed the monitoring of mutated clones from a low concentration (0.1%). The retrospective study using archival RNA samples from mutated cases allowed us to analyze the kinetics of both total BCR-ABL1 transcripts and mutant clones. Imatinib treated cases harboring T315I or G250E mutations showed growing mutant clones with persistent BCR-ABL1 transcripts. On the contrary, cases harboring F317L (1 case) and V299L (3 cases) mutations showed decreasing clones with favorable response to Nilotinib treatment with a reduction in BCR-ABL1 transcripts. In the remaining 3 cases a longer follow up is necessary to draw conclusions. These results show that our method is highly sensitive and allows an early detection and follow up of emerging or residual clones that eventually increase or decrease depending of the TKI treatment.