Elias, Marjanu Hikmah
(2015)
Molecular genetic and epigenetic mechanisms of primary and secondary resistance to imatinib mesylate treatment in ph chromosome positive chronic myeloid leukemia patients.
Masters thesis, Universiti Sains Malaysia.
Abstract
Imatinib mesylate (IM) is a BCR-ABL targeted tyrosine kinase inhibitor drug
used for frontline therapy in patients with chronic myeloid leukemia (CML). IM is
highly effective and is considered the standard of care in CML management. Even
though IM has become the gold standard frontline treatment of CML, resistance to IM
has emerged as a major problem of concern. Nearly 33% of CML patients on IM therapy
develop resistance which can be either due to BCR-ABL dependent or BCR-ABL
independent mechanisms. BCR-ABL dependent mechanism involves point mutation in
the BCR-ABL tyrosine kinase domain and amplification of the BCR-ABL gene. BCRABL
independent mechanisms include several factors including alteration in
pharmacokinetics of IM with respect to absorption, distribution of metabolism as well as
epigenetic alterations. The present study was undertaken to elucidate the BCR-ABL
dependent mechanism and BCR–ABL independent mechanism involving epigenetic
alterations, in Malaysian CML patients undergoing IM therapy. A total of 205 CML
patients on IM therapy (122 IM resistant and 83 IM good response) were included in this
study. Using denaturing High Performance Liquid Chromatography (dHPLC) followed
by DNA sequencing, 122 IM resistant CML patients were screened for BCR-ABL
mutations. Ninety two IM resistant CML patients who did not show BCR-ABL mutations
(BCR-ABL non-mutated) were investigated for BCR-ABL gene amplification. As part of
epigenetic approach, 175 CML patients comprising of 83 good response and 92 IM
resistant BCR-ABL non-mutated CML patients were subjected to Methylation Specific
High Resolution Melt Analysis (MS-HRM). In BCR-ABL mutation analysis, mutations
were detected in 30/122 patients (24.6%) with two of the CML patients showing double
mutations. Seventeen different types of mutations (T315I, G250E, E255K, E255V,
M351T, Y253H, V289F, E355G, F359V, L387M, H396R, E355A, D276G, A397P and
E281K) including two novel mutations (G251E and N368S) were identified. Since
different mutations confer different levels of resistance, detection as well as
characterization of BCR-ABL mutations is highly relevant in CML patients to guide in
selecting the most suitable IM dosage or changing to other Tyrosine kinase inhibitor
therapy. However, the 92 IM resistant BCR-ABL non-mutated CML patients did not
show amplification of the BCR-ABL gene. With regard to BCR-ABL independent
mechanism, methylation levels of HOXA4 and HOXA5, but not of SOCS1, were found to
be higher in CML patients showing resistance. IM treated CML patients with higher
than 62.5% of HOXA4 and HOXA5 promoter methylation levels were found to be
associated with a higher risk (OR, 4.71; 95% CI, 2.46, 9.03; P<0.001 and OR, 4.26; 95%
CI, 2.22, 8.17; P<0.001, respectively) for developing IM resistance compared to the
optimal response group. Promoter hypermethylation of HOXA4 and HOXA5 genes could
be considered as one of the BCR-ABL independent mechanisms mediating IM resistance
and could be a potential epigenetic biomarker in supplement to the BCR-ABL gene
mutation in predicting IM treatment response among CML patients. In a five-year
survival analysis, the presence of BCR-ABL mutations especially Y253H and E355G
mutations (p-value =0.005 and p-value =0.025 respectively), were found to be associated
with the prognosis and survival of CML patients on IM therapy. However, after
adjusting for other variables in multiple Cox regression analysis, CML stage has
emerged as the only significant prognostic factor (HR: 27.04, p-value <0.001 for BP and
HR: 9.58, p-value <0.001 for AP). The overall results suggest that resistance to IM is not
due to a single or simple mechanism, but is a multi-factorial phenomenon. BCR-ABL
mutations could be considered as molecular marker for predicting the IM response as
well as prognosis of CML patients on IM treatment whereas promoter methylation levels
of HOXA4 and HOXA5 could be considered as epigenetic markers for predicting the
response to IM.
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