ALBERT

Description: The most frequent causes of imatinib resistance are BCR-ABL mutations, usually selected during treatment. Most of them destabilize the inactive conformation necessary for imatinib binding. The T315I mutation, located at the ATP binding pocket is resistant to imatinib and other available kinase inhibitors and is associated with a poor outcome. The aim of this study was to evaluate the frequency of T315I mutation in patients with CML resistant to imatinib and to describe their outcome. Mutation analysis was performed in two institutions, through direct automated sequencing. Total RNA was extracted from peripheral blood or bone marrow and amplification of the kinase domain of ABL from BCR/ABL was performed, using a semi-nested RT-PCR, to cover amino acids 244–486. PCR product was submitted to direct automated sequencing and compared with normal sequences of BCR-ABL gene (M14752, GenBank). A total of 173 resistant imatinib resistant patients were analyzed. BCR-ABL mutations were detected in 47 out of 173 (27%) patients. Among these patients, 14 (30%) presented T315I mutation. The mutation was found in ten patients during imatinib treatment, in one before start of imatinib, in three after dasatinib (2) and nilotinib (1) failure. Three patients have more than one mutation analysis, performed during different time-points of treatment: imatinib resistance, dasatinib resistance (3) and bone marrow transplantation relapse (1). Overall survival from diagnosis was 39,5% and 17,5% from mutation detection, in a median time of 69 and 13 months respectively. After imatinib resistance, eight patients were treated with dasatinib, one with nilotinib and had no hematological response; tree were treated with Hydrea and two were submitted to allogeneic bone marrow transplantation. Five patients died due to disease progression in median time of 19 months after mutation detection. One patient died after bone marrow transplantation, due to GVHD and infection. Eight patients are alive, three in CP, two in BC, two in hematological response and one in complete cytogenetic response after bone marrow transplantation. In conclusion, in this study we confirmed the high frequency of T315I mutation in imatinib resistant patients and their poor prognosis. Most of them started imatinib in a late chronic or advanced phase, with exception of two patients which started imatinib 800mg in early chronic phase, suggesting that there was inhibition of Ph positive sensitive cells and selection of resistant clones.

Description: Abstract 2215 Poster Board II-192 The exact action mechanism of the protein BCR-ABL is unknown, studies in vitro and in animal models showed that the activity of protein tyrosine kinase (TK), by itself, is sufficient to cause the development of CML. Thus, the progression of the disease to accelerated phase or blast crisis may be associated with genomic instability. The use of methods to detect the difference in gene expression between CML and normal granulocytes could bring new insights in the understanding of these complex mechanisms, highlighting new therapeutic targets for the CML treatment. Evaluation of the differential gene expression between CML and normal granulocytes using the Subtractive Suppression Hybridization (SSH) and investigation of the involvement of a set of genes in modulating the development of CML. The SSH libraries were constructed using a pool of granulocytes RNA's extracted from CML patients and from healthy blood donors. Real-Time (RT-PCR) was used to validate the results and evaluation of gene expression in granulocytes and in mononuclear cells in the pool of patients and controls used for the construction of libraries. The expression of the RUNX1 gene, whose expression was the most differential between the libraries, was evaluated in mononuclear cells of patients with CML using RT-PCR. The comparison between granulocytes of CML patients and control granulocytes showed 39 genes exclusively expressed in CML and 169 genes in controls. For validation, we evaluated the expression of genes RUNX1, KPNA6, TOB1, SEPT5, CDC42SE, ITCH, MIER and GP1BA by RT-PCR in the pool of patients and controls used for the construction of libraries. Among these genes, the RUNX1 gene showed substantial difference, and was also studied in mononuclear cells from 20 patients with CML in different stages of the disease and using different types of treatment with TK inhibitors. The expression of this gene was highly altered in patients in advanced phase of disease when compared with patients who are responding to treatment. The genes identified in this study may be related to the development and progression of CML. Among them, the RUNX1 gene was shown to be one of the most promising, since the occurrence of chromosomal translocations in this gene has been associated with different types of acute leukemia – however its association with CML is not yet clear. The results showed a relationship between the expression of this gene and progression of the disease, beyond the identification of several genes that may lead to a better understanding of CML and help in identifying new therapeutic targets. This work was supported by FAPESP. Disclosures: Off Label Use: Development of TKI in the treatment of CML.

Description: Point mutations within the ABL kinase domain are the most frequent mechanism for reactivation of kinase activity of the BCR-ABL gene and have been associated with clinical resistance to tyrosine kinases (TK) inhibitors in CML patients conferring in some of them a poor prognosis. The T315I (Treonine → Isoleucine) is a mutation described in exon 6 of BCR-ABL gene that makes the protein resistant to all kinase inhibitors most currently used for treating CML (imatinib, nilotinib and dasatinib). D-HPLC allows for high throughput mutation screening. This technique is based on heteroduplex formation by PCR products amplified from wild type and mutant alleles. Under optimized denaturing conditions, these heteroduplexes can be distinguished from homoduplex. In this study we screened mutations in exon 6 of BCR-ABL gene in patients treated with kinase inhibitors, in different phases of the disease. We evaluated 85 patients: 9 at diagnosis, 81 in chronic phase, 3 in accelerated phase, one in blast crisis. Thirty four were resistant to imatinib, 10 of them to dasatinib and three had suboptimal response to imatinib. In 9 of 85 (10,5%) samples, D-HPLC showed an abnormal elution profile suggesting the presence of nucleotide changes. Automated sequencing confirmed the presence of two point mutations: T315I (two patients) and F359V (two patients). Five patients requires sequencing confirmation. Patients with T315I mutation failed to imatinib and dasatinib. One of them relapsed after bone marrow transplantation in blast crisis. Patients with F359V mutation were resistant to imatinib. One of them has partial hematological response with dasatinib and the other is in complete molecular response after bone marrow transplantation. D-HPLC seems to be a ship and practical method for routine clinical monitoring for emergence of kinase domain mutations and may be useful for optimizing therapy in CML. Early detection of emerging mutant clones may help in decision-making of alternative treatment.