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Background. Approximately 30% of adult ALL patients are characterized by the presence of the Philadelphia (Ph) chromosome, which derives from a reciprocal translocation t(9;22)(q34;q11) and results in a chimeric BCR-ABL oncogene. The prognosis of this subset of patients treated with standard therapies, including multi-agent chemotherapy, Imatinib, and allogeneic stem cell transplantation, is still dismal, due to a high risk of relapse. Dasatinib and Nilotinb are second generation TKIs developed to overcome the problem of resistance to Imatinib in relapsed Ph+ leukemias. Design and Methods. We retrospectively evaluated the single center experience on therapy efficacy of Dasatinib, Nilotinib, and experimental third generation TKIs, administered as second or subsequent line of therapy on 25 relapsed Ph+ adult ALL patients. All patients were previously treated with Imatinib. The median age at time of diagnosis was 50 years (range 18-74), 17 patients were male and 8 female. Ten patients presented a BCR-ABL P190 fusion protein and corresponding fusion transcript, the remaining a BCR-ABL P210. Nineteen patients received Dasatinib, 2 patients Nilotinib and the remaining 4 patients were treated with third generation TKIs. Fourteen patients (56%) were in first relapse, and 7 (28%), 3 (12%) and 1 (4%) were in second, third and fourth relapse, respectively. A mutational analysis was performed in all the patients before TKIs (9 wild type, 16 mutated, including T315I) and at the time of subsequent relapse; gene expression profiling, SNPArray (6.0 Affymetrix chip), and Ikaros deletions were also analyzed. Results. 13 out of 25 patients (52%) obtained a haematological response (HR) (11 patients treated with Dasatinib, 1 patient with Nilotinib and 1 patient with a third generation experimental TKI). 10 patients obtained also a cytogenetic response (CyR) and 6 patients a molecular response (MolR). With a median follow up of 10.8 months (range 2-29 months), median duration of HR, CyR and MolR were 117 days (range 14-385 days); progression free survival were 162 days with Dasatinib and 91 days with Nilotinib. Overall survival was 25.8 months. Interestingly, in 6 out of 9 wild-type patients, treated with Dasatinib, the mutational analysis showed the emergence of T315I or F317I mutation at the time of relapse. Conclusion. Second and third generation TKIs represent a valid approach in relapsed Ph+ adult ALL patients; the subsequent relapse is often associated to the emergence of mutation, conferring resistance to TKIs.

Mutations in the Bcr-Abl kinase domain (KD) are often detected at the time of resistance to tyrosine kinase inhibitor (TKI) therapy in chronic myeloid leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients (pts). It is still unclear whether and in how many pts low level mutations may already be detectable at the time of diagnosis. We therefore analyzed cDNA samples from 22 newly diagnosed pts with Ph+ ALL (n=13) or chronic phase (CP)-CML (n=9) who subsequently received TKI therapy (imatinib, dasatinib or nilotinib). Screening for low level mutations was performed by cloning the Bcr-Abl KD (a.a. 206-524) in a bacterial vector and sequencing 200 independent clones for each pt. All pts had evidence of aberrant KD sequences. Three to twelve different mutations were detected in each pt. Each mutation was present in two to five independent clones. A total of 105 mutations (including 35 silent, 5 nonsense, and 65 missense mutations) were observed. The vast majority (98/105, 93%) of them have never been reported in association with TKI resistance and are likely not to confer any advantage under TKI selective pressure. Interestingly, 93/105 (89%) mutations were transitions: G>A (n=28), A>G (n=23), C>T (n=22), T>C (n=20). Such a high prevalence of transitions (normally occurring 1.4 times more frequently than transversions) suggests that a specific mechanism generating mutations is active in Ph+ cells. One of the nine CP-CML pt received hydroxyurea for 6 months before starting imatinib therapy. In this pt, high-sensitivity mutation screening was performed again immediately before imatinib start and showed further accumulation of mutations. Eight Ph+ ALL pts and three CML pts subsequently relapsed with evidence of mutations, but only one with a mutation (T315I) that was already detectable at diagnosis. The remaining eleven pts are in persistent remission after a follow up ranging from 12 to 36 months, although four of them were harbouring known imatinib-(H396P, D276G, E355G) or dasatinib-(F317L) resistant mutations at low levels. Our observations suggest that: a) Bcr-Abl KD mutations can probably be found at diagnosis in all CP-CML and Ph+ ALL pts; b) mutations seem to arise randomly and most of them are silent or not conferring any growth advantage under the selective pressure of TKIs; c) generation of mutations seems to be linked to Bcr-Abl-driven genetic instability; d) TKI-resistant mutations present at low levels at diagnosis do not always outgrow and lead to relapse, probably because some of them arise in cell clones with limited self-renewal capacity. This warns against high-sensitivity mutation screening of all CML and Ph+ ALL pts before the start of TKI therapy.

By genome-wide analyses of DNA copy number abnormalities in adult BCR-ABL1-positive ALL patients (pts) using single nucleotide polymorphism (SNP) microarrays (Affymetrix 500K and SNP 6.0), we identified a high frequency of genetic alterations of regulators of B lymphoid development (PAX5, EBF1, IKZF1) and cell cycle (CDKN2A, CDKN2B, BTG1). The most frequent somatic copy number alteration was deletion on 7p12 of IKZF1 (68/106 patients, 64%), which encodes the transcription factor Ikaros required for the earliest stages of lymphoid lineage commitment. Here, we characterized and mapped all breakpoints in IKZF1 gene to recognize that two major deletions occur in BCR-ABL1-positive ALL: type A characterized by loss of exons 4-7 (44%) and due to breakpoints occurring in introns 3 and 7; type B characterized by removal of exons 2-7 (19%) and due to a variable pattern of breakpoints in introns 1 and 7. In two pts we had both \#916;2-7 and \#916;4-7 deletions and in one we identified a deletion of all IKZF1 gene and part of GRB10. A variable number of nucleotides (patient-specific) were inserted at the conjunction and maintained with fidelity at the time of relapse. The extent of both deletions correlated with the expression of dominant-negative isoforms with cytoplasmatic localization. The IKZF1 alteration was also identified in B-negative ALL pts (41%) and at the progression of CML to lymphoid blast crisis (66%), but never in myeloid blast crisis CML and chronic phase CML. Known DNA sequences were mapped along the breakpoint cluster regions including heptamer recombination signal sequences recognized by the RAG enzymes during V(D)J recombination and activation-induced cytidine deaminase (AID) consensus motifs (DGYW/WRCH). Gene expression profiling analysis of pts with IKZF1 deletion vs wild-type pts identified a unique signature characterized by a down-regulation of genes involved in pre-B-cell differentiation (e.g. VPREB1, VPREB3, IGLL3, BLK), demonstrating that genomic IKZF1 alterations have a strong impact on trascriptoma and contribute to an impaired B-cell differentiation. Univariate analysis showed that the IKZF1 deletion is a negative prognostic marker influencing the cumulative incidence of relapse (10.1 months for pts with deletion vs 56.1 months for wild-type pts, p=0.0103) and disease-free survival (DFS) (10.1 months vs 32.1 months, respectively, p=0.0229). The negative prognostic impact of the IKZF1 deletion on DFS was also confirmed by multivariate analysis (p=0.0445). In conclusion, deletion of IKZF1 is an important event in the development of BCR-ABL1 B-progenitor ALL which significantly influences clinical outcome.

Purpose: ABL kinase domain mutations have been implicated in the resistance to the BCR-ABL inhibitor imatinib mesylate of Philadelphia-positive (Ph+) leukemia patients. Experimental Design: Using denaturing high-performance liquid chromatography and sequencing, we screened for ABL kinase domain mutations in 370 Ph+ patients with evidence of hematologic or cytogenetic resistance to imatinib. Results: Mutations were found in 127 of 297 (43%) evaluable patients. Mutations were found in 27% of chronic-phase patients (14% treated with imatinib frontline; 31% treated with imatinib post-IFN failure), 52% of accelerated-phase patients, 75% of myeloid blast crisis patients, and 83% of lymphoid blast crisis/Ph+ acute lymphoblastic leukemia (ALL) patients. Mutations were associated in 30% of patients with primary resistance (44% hematologic and 28% cytogenetic) and in 57% of patients with acquired resistance (23% patients who lost cytogenetic response; 55% patients who lost hematologic response; and 87% patients who progressed to accelerated phase/blast crisis). P-loop and T315I mutations were particularly frequent in advanced-phase chronic myeloid leukemia and Ph+ ALL patients, and often accompanied progression from chronic phase to accelerated phase/blast crisis. Conclusions: We conclude that (a) amino acid substitutions at seven residues (M244V, G250E, Y253F/H, E255K/V, T315I, M351T, and F359V) account for 85% of all resistance-associated mutations; (b) the search for mutations is important both in case of imatinib failure and in case of loss of response at the hematologic or cytogenetic level; (c) advanced-phase chronic myeloid leukemia and Ph+ ALL patients have a higher likelihood of developing imatinib-resistant mutations; and (d) the presence of either P-loop or T315I mutations in imatinib-treated patients should warn the clinician to reconsider the therapeutic strategy.

Background. SKI-606 is a novel 4-anilino-3-quinolinecarbonitrile Src and Abl kinase inhibitor. SKI-606 has been shown to be a potent antiproliferative and proapoptotic agent when tested on Bcr-Abl-positive cell lines. The remarkable efficacy of SKI-606 against chronic myeloid leukemia (CML) cells in culture was mirrored by its activity in vivo against CML xenografts: K562 tumors regressed in nude mice when SKI-606 was administered per os once daily over a 5-day period. The crystal structure of the Bcr-Abl kinase domain in complex with SKI-606 has not yet been determined and the mode of binding of this inhibitor is therefore unknown. Moreover, there are currently no published data on the ability of SKI-606 to bind and efficiently inhibit the Bcr-Abl mutants known to confer resistance to imatinib. Aims. In this study, we used a molecular docking approach to a) determine SKI-606 binding mode to the wild-type (wt) form of the Bcr-Abl kinase; b) hypothesize SKI-606 binding mode to the more frequent, clinically relevant Bcr-Abl mutants known not to be inhibited by imatinib; c) predict which novel mutant forms might emerge and interfere with SKI-606 binding. Methods. Modelling of the human Abl kinase was performed with the program Modeller v7.7 (http://salilab.org/modeller) adopting the highly related Mus musculus Abl homologue as a template structure (PDB: 1OPJ, 0.175nm resolution). Chemsketch (http://www.acdlabs.com) was used to build a three-dimensional model of SKI-606. Flexible docking of the ligand to the protein was performed with Autodock v3.0 (http://www.scripps.edu/mb/olson). Results. We first docked SKI-606 on Bcr-Abl with the activation loop in the active (open) and inactive (closed) conformation (the latter is the one to which imatinib binds). According to our results, the interaction between SKI-606 and Bcr-Abl seems to be more stable when the activation loop is in the inactive conformation. The consequent structural study of SKI-606 modeled into wt-Bcr-Abl ATP binding site highlighted the variant residues located within a spherical environment of 0.5nm centered on SKI-606: Y253, T315 and F359 (residues numbered according to ABL exon Ia splice variant). The binding of SKI-606 to the eight Bcr-Abl mutants which are most frequently implicated in clinical resistance to imatinib mesylate was also studied: G250E, Y253H, E255K, T315I, M351T, F359V, H396R. Our results indicated that SKI-606 retains the ability of efficiently binding all the above mentioned Bcr-Abl variants with the exception of the T315I mutant. Finally, we identified six potential residues around SKI-606 that, if mutated, could potentially be able to interfere with the SKI-606/Bcr-Abl interaction: a) the charged residues K271, D381 and H361; b) the hydrophobic/aliphatic residues V299, A380 and M318. Conclusions. Pre-clinical data suggest that SKI-606 is a promising second-generation kinase inhibitor with potent antiproliferative and proapoptotic effects on CML cells. Our docking experiments indicate that SKI-606 may prove effective in imatinibresistant patients since it is expected to retain the ability to bind several Bcr-Abl mutant forms. A phase I trial is about to start in CML and Philadelphia-positive acute lymphoblastic leukemia.

Background SKI-606 is a novel 4-anilino-3-quinolinecarbonitrile Src and Abl kinase inhibitor. SKI-606 has been shown to be a potent antiproliferative and proapoptotic agent when tested on Bcr-Abl-positive cell lines. The remarkable efficacy of SKI-606 against chronic myeloid leukemia (CML) cells in culture was mirrored by its activity in vivo against CML xenografts: K562 tumors regressed in nude mice when SKI-606 was administered per os once daily over a 5-day period. The crystal structure of the Bcr-Abl kinase domain in complex with SKI-606 has not yet been determined and the mode of binding of this inhibitor is therefore unknown. Moreover, there are currently no published data on the ability of SKI-606 to bind and efficiently inhibit the Bcr-Abl mutants known to confer resistance to imatinib. Aims In this study, we used a molecular docking approach to a) determine SKI-606 binding mode to the wild-type (wt) form of the Bcr-Abl kinase; b) hypothesize SKI-606 binding mode to the more frequent, clinically relevant Bcr-Abl mutants known not to be inhibited by imatinib; c) predict which novel mutant forms might emerge and interfere with SKI-606 binding. Methods Modelling of the human Abl kinase was performed with the program Modeller v7.7 (http://salilab.org/modeller) adopting the highly related Mus musculus Abl homologue as a template structure (PDB: 1OPJ, 0.175nm resolution). Chemsketch (http://www.acdlabs.com) was used to build a three-dimensional model of SKI-606. Flexible docking of the ligand to the protein was performed with Autodock v3.0 (http://www.scripps.edu/mb/olson). Results We first docked SKI-606 on Bcr-Abl with the activation loop in the active (open) and inactive (closed) conformation (the latter is the one to which imatinib binds). According to our results, the interaction between SKI-606 and Bcr-Abl seems to be more stable when the activation loop is in the inactive conformation. The consequent structural study of SKI-606 modeled into wt-Bcr-Abl ATP binding site highlighted the variant residues located within a spherical environment of 0.5nm centered on SKI-606: Y253, T315 and F359 (residues numbered according to ABL exon Ia splice variant). The binding of SKI-606 to the eight Bcr-Abl mutants which are most frequently implicated in clinical resistance to imatinib mesylate was also studied: G250E, Y253H, E255K, T315I, M351T, F359V, H396R. Our results indicated that SKI-606 retains the ability of efficiently binding all the above mentioned Bcr-Abl variants with the exception of the T315I mutant. Finally, we identified six potential residues around SKI-606 that, if mutated, could potentially be able to interfere with the SKI-606/Bcr-Abl interaction: a) the charged residues K271, D381 and H361; b) the hydrophobic/aliphatic residues V299, A380 and M318. Conclusions Pre-clinical data suggest that SKI-606 is a promising second-generation kinase inhibitor with potent antiproliferative and proapoptotic effects on CML cells. Our docking experiments indicate that SKI-606 may prove effective in imatinib-resistant patients since it is expected to retain the ability to bind several Bcr-Abl mutant forms. A phase I trial is about to start in CML and Philadelphia-positive acute lymphoblastic leukemia.

Patients with Systemic Mastocytosis (SM) need a highly sensitive diagnostic test for D816V detection of the KIT receptor gene.Along with histology/cytology and flow cytometry evaluation, bone marrow (BM) from 110 consecutive adult patients referred with a suspicion of SM to Multidisciplinary Outpatient Clinic for Mastocytosis in Verona were tested both by Amplification Refractory Mutation System Reverse Transcriptase quantitative real time Polymerase Chain Reaction (ARMS-RT-qPCR) and RT-PCR. +. Restriction Fragment Length Polymorphism (RFLP) followed by Denaturing-High Performance Liquid Chromatography (D-HPLC) and Sanger sequencing.ARMS-RT-qPCR identified D816V mutation in 77 patients, corresponding to 100% of cases showing CD25+ mast cells (MCs) whereas RT-PCR+RFLP/D-HPLC+sequencing revealed D816V mutations in 47 patients.According to the 2008 WHO criteria 75 SM, 1 Cutaneous Mastocytosis (CM), 1 monoclonal MC activation syndrome (MMAS), and 1 SM Associated with Haematologic Non-Mast Cell Disorder (SM-AHNMD) were diagnosed. Seventeen out 75 SM patients (23%) would have not satisfied sufficient WHO criteria on the basis of the sole RT-PCR+RFLP: these patients had significantly lower serum tryptase levels and amount of CD25+ MCs.Therefore, ARMS-RT-qPCR might result particularly useful, in patients that do not fulfil major BM histological criterion, for the recognition of indolent SM with a very low MC burden.

Abstract 2027 Poster Board II-4 Background. The prognosis of Ph+ adult ALL patients treated with standard therapies, including multi-agent chemotherapy, Imatinib, and allogeneic stem cell transplantation, is still dismal, due to a high risk of relapse. Dasatinib and Nilotinb are second generation TKIs developed to overcome the problem of resistance to Imatinib in relapsed Ph+ leukemias. Design and Methods. We retrospectively evaluated the single center experience on therapy efficacy of Dasatinib, Nilotinib, and experimental third generation TKIs, administered as second or subsequent line of therapy on 25 relapsed Ph+ adult ALL patients. All patients were previously treated with Imatinib. The median age at time of diagnosis was 50 years (range 18-74), 17 patients were male and 8 female. Ten patients presented a BCR-ABL P190 fusion protein and corresponding fusion transcript, the remaining a BCR-ABL P210. Nineteen patients received Dasatinib, 2 patients Nilotinib and the remaining 4 patients were treated with third generation TKIs. Fourteen patients (56%) were in first relapse, and 7 (28%), 3 (12%) and 1 (4%) were in second, third and fourth relapse, respectively. A mutational analysis was performed in all the patients before TKIs (9 wild type, 16 mutated, including T315I) and at the time of subsequent relapse; gene expression profiling, SNPArray (6.0 Affymetrix chip), and Ikaros deletions were also analyzed. Results. 13 out of 25 patients (52%) obtained a haematological response (HR) (11 patients treated with Dasatinib, 1 patient with Nilotinib and 1 patient with a third generation experimental TKI). 10 patients obtained also a cytogenetic response (CyR) and 6 patients a molecular response (MolR). With a median follow up of 10.8 months (range 2-29 months), median duration of HR, CyR and MolR were 117 days (range 14-385 days); progression free survival were 162 days with Dasatinib and 91 days with Nilotinib. Overall survival was 25.8 months. Interestingly, in 6 out of 9 wild-type patients, treated with Dasatinib, the mutational analysis showed the emergence of T315I or F317I mutation at the time of relapse. Conclusion. Second and third generation TKIs represent a valid approach in relapsed Ph+ adult ALL patients; the subsequent relapse is often associated to the emergence of mutation, conferring resistance to TKIs. Since TKIs are different and have different efficacy, pharmacokinetic, pharmacodynamic and toxicity profiles, and since are all effective, an alternative experimental option for the treatment of Ph+ ALL could be a combination or a rotation of two or more than two TKIs. Moreover, the addition of new promising targeted molecules, such as Aurora kinase or T315I inhibitors may contribute to overcome the problem of resistance to TKIs. Acknowledgments. Work supported by European LeukemiaNet, FIRB 2006, AIRC, AIL, COFIN, University of Bologna and BolognAIL. Disclosures: No relevant conflicts of interest to declare.