SNPs in the Promoter of the Gene Encoding Transmembrane Transporter SLC22A4 (hOCTN1) Are Significantly Associated with an Alteration of Gene Expression and with Resistance to the Imatinib Treatment in Chronic Myeloid Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2463-2463
Author(s):  
Monika Jaruskova ◽  
Rajna Hercog ◽  
Nikola Curik ◽  
Vladimir Benes ◽  
Hana Klamova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Myeloid Leukemia ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Kinase Domain ◽  
Imatinib Treatment ◽  
First Line ◽  
Promoter Regions ◽  
Optimal Response

Abstract Introduction: Apart from the mutations in the kinase domain of BCR-ABL, other important mechanisms of resistance to the first line imatinib (IM) therapy in chronic myeloid leukemia (CML) are pharmacokinetics factors, especially an intracellular concentration of IM. ATP Binding Cassette (ABC, ensuring efflux) and Solute Carrier (SLC, ensuring uptake) super families are responsible for transportation several drugs including IM. Gene expression and activity of the SLC and ABC transporters may be affected by polymorphisms in their promoter regions and may notably contribute to the treatment failure. Objectives: The aim of this study was to identify polymorphisms in the promoter regions of the selected SLC and ABC transporter encoding genes and evaluate their association with the response to the first line IM therapy in CML patients in chronic phase. Material and Methods: The patient cohort consists of 40 CML patients with optimal response and 40 resistant patients (without mutations in the BCR-ABL kinase domain) to the IM in the first line with 24 months follow-up from the therapy initiation. All 80 CML patients were taking a standard dose of IM and in none of them were evidence of non-compliance. The promoter regions (~1000bp) of selected ABC (n=4) and SLC (n=15) genes with the annotated function of drug transportation were amplified. The next generation sequencing was applied for ultra-wide sequencing of altogether 1419 amplicons (454 GS Junior, Roche AppliedScience; MiSeq Series, Illumina). The obtained sequences were analyzed and evaluated with the focus on the presence of single nucleotide polymorphisms (SNPs) using NextGENe software (Softgenetics). The Fisher´s exact binomial test of goodness of fit was used to evaluate haplotype frequency distribution among patient cohort. The expression analysis of a selected gene was analyzed using RT-qPCR (TaqMan® Assays) in total leukocytes of peripheral blood with GUS as a housekeeping gene. Results: Among 1419 evaluated sequences we identified 96 SNPs (2-12 SNPs per one promoter) of which nine was not yet annotated. We identified 2 SNPs, rs460089 (C/G) and rs460271 (C/G), with uniform alleles co-segregations in the promoter of the gene SLC22A4 when GG haplotypes were significantly more frequent in resistant patients (P<.05). Of all resistant patients, 69% carried GG haplotypes, 17% CG and 14% CC. The frequency of haplotypes among responding patients was 31% of GG, 53% CG and 16% CC. As SLC22A4 is imatinib transporter (He L et al. Hum Genomics 2009), we measured the level of the gene expression associated with both co-segregated SNPs. Among all patients analyzed regardless of the response to IM, the GG haplotypes showed a significantly lower expression of SLC22A4 in comparison to CG and CC haplotypes (P<.05). Even greater significant differences in the expression were found when comparing GG haplotypes within patients resistant to IM in comparison to CG haplotypes within patients with optimal response (P<.01). Conclusion: In this work, we found a significantly decreased gene expression of SLC22A4 in total leukocytes of peripheral blood that was associated with GG haplotypes of 2 SNPs in the SLC22A4 promoter, where GG haplotypes were significantly more frequent in CML patients resistant to the first line imatinib treatment in comparison to the patients with an optimal response. We assume that GG haplotypes have altered activity of SLC22A4 promoter resulting in the reduced hOCTN1 expression and thus in lower intracellular IM concentration that may be insufficient for optimal response. The experiments on the SLC22A4 promoter activity alterations associated with GG haplotypes are ongoing. We believe that screening for rs460089 (C/G) and rs460271 (C/G) SNPs among patients with newly diagnosed CML could be an important prognostic genetic factor helping to select a tyrosine kinase inhibitor that is not dependent on the active transportation through the cell membrane. This work was supported by the Ministry of Health of Czech Republic, grant IGA MZ CR NT/13899 and Charles University in Prague, project GA UK/177815. Disclosures Klamova: Bristol Myers-Squibb: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Machova Polakova:Bristol Myers-Squibb: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Evaluation Of hOCT1expression In Patients With Chronic Myeloid Leukemia (CML) Treated With Imatinib In First Line

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4041-4041
Author(s):  
Cintia Do Couto Mascarenhas ◽  
Maria Helena Almeida ◽  
Eliana C M Miranda ◽  
Bruna Virgilio ◽  
Marcia Torresan Delamain ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Chronic Phase ◽  
Taqman Probe ◽  
Molecular Response ◽  
Imatinib Treatment ◽  
First Line ◽  
Transcript Levels

Abstract Introduction The majority of chronic myeloid leukemia (CML) patients (pts) in chronic phase (CP), present satisfactory response to imatinib treatment. However, 25-30% of these pts exhibit suboptimal response or treatment failure. The probability of achieving optimal response may be related with several factors. The human organic cation transporter 1 (hOCT1, SLC22A1), an influx transporter, is responsible for the uptake of imatinib into chronic myeloid leukemia (CML) cells The aim of this study was to analyze hOCT-1 levels at diagnosis of CML patients and correlate with cytogenetics and molecular responses. Methods hOCT-1 expression was evaluated in 58 newly diagnosed CML pts. Pts were treated with imatinib 400-600mg in first line. Samples were collected from peripheral blood at diagnosis and RNA was obtained from total leucocytes. For cDNA synthesis, 3 ug of RNA was used. hOCT-1 expression was evaluated by real-time PCR with TaqMan probe SLC22A1 (Applied Biosystems) and endogenous GAPDH control. The results were analyzed using 2-ΔΔCT. Cytogenetic analysis was performed at diagnosis, 3, 6, 12 and 18 months after starting therapy and then every 12-24 months thereafter if CCR was achieved. BCR-ABL transcripts were measured in peripheral blood at 3-month intervals using quantitative RT-PCR (RQ-PCR). Results were expressed as BCR-ABL/ABL ratio, with conversion to the international scale (IS). Major molecular response (MMR) was defined as a transcript level ≤ 0.1%. Results 58 CML pts, 60% male, median age of 46 years (19-87) were evaluated, 71% in chronic phase (CP), 21% in accelerated phase (AP) and 5% in blast crisis (BC). The mean and median of hOCT-1 transcript levels in the total group was 2.03 and 0.961 respectively (0.008–19.039) and CP pts was 1.86 and 1.00 (0.008-10.34).The median duration of imatinib treatment was 27 months (1-109) and 96.6% achieved complete hematological response, 79.3% complete cytogenetic response and 69% major or complete molecular response. The regression analysis showed correlation between higher transcript levels of hOCT-1 and BCR-ABL transcripts<10%) at 3 months analysis (p<0.0001). Albeit, there was no influence of the hOCT-1 transcript levels at diagnosis in the achievement of cytogenetic and molecular response at 24 months of treatment. Conclusions In this report, we found that high hOCT-1 expression was predictive of BCR-ABL transcripts<10% at 3 months, although we did not find correlation between hOCT-1 levels at diagnosis and the achievement of molecular response at 24 months, studies show that there is correlation between BCR-ABL log reduction in the first months of treatment and the achievement of molecular response. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Evaluation of Peroxiredoxins (PRDX1, PRDX2 and PRDX6) Expression in Patients with Chronic Myeloid Leukemia (CML) Treated with Imatinib in First Line

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5545-5545
Author(s):  
Cintia Mascarenhas ◽  
Lara Woldmar ◽  
Maria Helena Almeida ◽  
Rosangela Vieira Andrade ◽  
Anderson Ferreira Cunha ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Chronic Myeloid Leukemia ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Blood Donors ◽  
Chronic Phase ◽  
Molecular Response ◽  
Imatinib Treatment ◽  
Cdna Synthesis ◽  
First Line

Abstract Introduction: Satisfactory response is present for the majority of chronic myeloid leukemia (CML) patients (pts) in chronic phase (CP) treated with tyrosine kinase inhibitors (ITK) . However, some pts exhibit suboptimal response or treatment failure. The probability of achieving optimal response may be related with several factors. The oxidative stress modulation is tightly related with the physiopathology of various hematologic diseases and can cause cell death, apoptosis and necrosis. Peroxiredoxins (Prdx) are a family of multifunctional antioxidant thioredoxin-dependent peroxidases that protect cells against oxidative stress and modulate signaling cell proliferation pathways and may influence the metabolism of ITKs.The aim of this study was to analyze PRDX1, PRDX2 and PRDX6 levels of CML pts and correlate with cytogenetics and molecular responses. Methods: PRDX1, PRDX2 and PRDX6 expression was evaluated in 20 blood donors, 18 newly diagnosed CML pts and 22 previously treated pts. Pts were treated with imatinib 400-600mg in first line. Samples were collected from peripheral blood at diagnosis or during treatment and RNA samples were submitted to the synthesis of complementary DNA (cDNA) using the kit RevertAid™ HMinus First Strand cDNA Synthesis Kit (Fermentas, Life Sciences). For cDNA synthesis, 3 ug of RNA was used and peroxiredoxins expression was evaluated by real-time PCR with Syber Green (Applied Biosystems) and endogenous (β-Actina and GAPDH) controls. The results were analyzed using 2-ΔΔCT. Statistical analysis were made by using Mann Withney’s T test. Cytogenetic analysis was performed at diagnosis, 3, 6, 12 and 18 months after starting therapy and then every 12-24 months thereafter if CCR was achieved. BCR-ABL transcripts were measured in peripheral blood at 3-month intervals using quantitative RQ-PCR. Results were expressed as BCR-ABL/ABL ratio, with conversion to the international scale (IS). Major molecular response (MMR) was defined as a transcript level ≤ 0.1% (IS). Results: 40 CML pts, 55% male, median age of 53 years (23-84) were evaluated, 60% in chronic phase (CP), 30% in accelerated phase (AP) and 10% in blast crisis (BC). The mean of PRDX transcript levels in the total group was (PRDX1: 0.006 and 10.10 / PRDX2: 0.002 and 16.26 / PRDX6: 0.003 and 49.97) respectively (PRDX1: 1.2 / PRDX2: 0.9 / PRDX6: 15.36). The results showed that there are a significantly difference (p<0.05) in the PRDX gene expression between pts and blood donors. All PRDX expression was reduced in responsive patients, and increase expression in pts resistant to TKI. The median duration of imatinib treatment was 29 months (1-104) and 97% achieved complete hematological response, 75% complete cytogenetic response and 65% major or complete molecular response. The analysis showed that higher levels of PRDX were maybe correlated with a no reduction of BCR-ABL transcripts (p<0.05). As well as, there was may influence of the PRDX levels at diagnosis in the response at 24 months of treatment. Conclusion:Is known that that the increase of ROS in CML leads to an increase of DNA damage, triggering genomic instability and resulting in accumulation of mutations and chromosomal aberrations, and contribute to the mechanism of acquisition of resistance to TKI inhibitors. The decrease of Peroxiredoxins expression observed in the responsive group, could contribute to this process, since the detoxification of these species are compromising and the effects caused by oxidative stress are even more drastic, leading to mutations that could be followed by TKi resistance. The relation between Prdx and CML not yet been elucidated. Disclosures No relevant conflicts of interest to declare.

scholarly journals BCR-ABL1 genomic DNA PCR response kinetics during first-line imatinib treatment of chronic myeloid leukemia

Haematologica ◽  
2018 ◽  
Vol 103 (12) ◽  
pp. 2026-2032 ◽  
Author(s):  
Ilaria S. Pagani ◽  
Phuong Dang ◽  
Ivar O. Kommers ◽  
Jarrad M. Goyne ◽  
Mario Nicola ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Genomic Dna ◽  
Myeloid Leukemia ◽  
Imatinib Treatment ◽  
First Line ◽  
Dna Pcr

scholarly journals Denaturing-HPLC-Based Assay for Detection of ABL Mutations in Chronic Myeloid Leukemia Patients Resistant to Imatinib

2004 ◽  
Vol 50 (7) ◽  
pp. 1205-1213 ◽  
Author(s):  
Simona Soverini ◽  
Giovanni Martinelli ◽  
Marilina Amabile ◽  
Angela Poerio ◽  
Michele Bianchini ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Large Scale ◽  
Kinase Inhibitor ◽  
Direct Sequencing ◽  
Point Mutations ◽  
Kinase Domain ◽  
Imatinib Treatment ◽  
Abl Tyrosine Kinase ◽  
Reverse Transcription Pcr

Abstract Background: Despite the efficacy of the BCR-ABL tyrosine kinase inhibitor Imatinib mesylate for the treatment of chronic myeloid leukemia (CML), resistance has been observed in a proportion of cases, especially those with advanced stages of the disease. Point mutations within the ABL kinase domain are emerging as the most frequent mechanism for reactivation of kinase activity within the leukemic clone. Methods: We developed a denaturing-HPLC (D-HPLC)-based assay for screening for ABL point mutations. For each sample, two partially overlapping fragments of 393 and 482 bp corresponding to the kinase domain were amplified by nested reverse transcription-PCR and analyzed under selected temperature and acetonitrile gradient conditions. Fifty-one bone marrow and/or peripheral blood specimens from 27 CML patients who showed cytogenetic resistance to Imatinib were screened in parallel by D-HPLC and by direct sequencing. Results: In 12 of 27 (44%) patients, D-HPLC showed an abnormal elution profile suggesting the presence of a nucleotide change. Direct sequencing confirmed the presence of a point mutation in all cases. Conversely, all samples scored as wild type by D-HPLC showed no evidence of mutations by direct sequencing. In two cases, novel amino acid substitutions at codons already known for being hot-spots of mutation were identified (F311I and E355D). Conclusions: The proposed D-HPLC-based assay is highly specific and at least as sensitive as sequencing; with respect to the latter, it provides a much faster and less expensive semiautomated system for mutational screening. It may therefore potentially be a valuable tool for regular, large-scale testing of patients undergoing Imatinib treatment.

Specific Drug Transporter Genotypes Are Significantly Associated with Increased Rates of Major and Complete Molecular Responses In Newly Diagnosed Chronic Myeloid Leukemia Patients Treated with Imatinib – A TOPS Correlative Substudy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 670-670
Author(s):  
Simona Soverini ◽  
Sabrina Angelini ◽  
Eleonora Turrini ◽  
Matt Burnett ◽  
Gloria Ravegnini ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Cumulative Incidence ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Phase Iii ◽  
Molecular Response ◽  
Newly Diagnosed ◽  
First Line ◽  
Favorable Alleles ◽  
Summary Measures

Abstract Abstract 670 The availability of multiple options for chronic myeloid leukemia (CML) treatment is not paralleled by the availability of biological predictors of outcome allowing to identify patients (pts) who are more likely to benefit from dasatinib or nilotinib rather than imatinib (IM). Pharmacogenetics has proven a potential source of biomarkers given the known influence of polymorphisms in key genes encoding drug transporters and metabolizing enzymes on drug delivery – hence effectiveness. In CML, only two studies had so far explored this field, but both were conducted in heterogeneous populations including pts at different stages of disease, not all receiving IM first-line. We thus aimed to investigate a panel of 20 single nucleotide polymorphisms (SNPs) in ABCB1, ABCG2, SLC22A1, OATP1A2, OCTN1, CYP3A4 and CYP3A5 genes that can be hypothesized to influence IM transport and metabolism in 189 newly diagnosed CML pts enrolled in the TOPS phase III trial (Cortes et al, J Clin Oncol 2010). Pts selection was exclusively based on availability of written informed consent and sufficient amount of archived material. Median age was 46 years; male to female ratio was 103 to 86; 156 (83%) pts were Caucasian and 23 (12%) were Asian; low, intermediate and high Sokal risk pts were 84 (44.4%), 65 (34.4%) and 40 (21.2%), respectively. Baseline demographic/clinical features did not differ significantly from those of the overall population. Treatment outcomes (complete cytogenetic response [CCyR]; major molecular response [MMR] and complete molecular response [CMR]) were compared according to i) each candidate genotype ii) summary measures based on combinations of SNPs in the same gene and iii) summary measures based on combinations of SNPs in functionally related genes (uptake; efflux). CC genotype in OCTN1 had a favorable impact on the achievement of MMR at 12 months (MMR@12m; P = 0.03). With respect to the summary measures, combination of SNPs in the SLC22A1 gene was significantly correlated with MMR@12m (P = 0.03). When considering summary measures of uptake and efflux, the former was found to be associated with both MMR@12m and CMR@12m (P = 0.003 and P = 0.01, respectively). A separate analysis limited to Caucasian pts (n=156) yielded similar results (Table 1). In addition, the analysis in the Caucasian subgroup evidenced a significant association between the CC genotype in ABCB1 rs60023214 and MMR@12m (P = 0.005) (Table 1). Cumulative incidence plots based on the Kaplan-Meier method were also analyzed in the overall population and in Caucasians, with comparable results. Representative plots are shown in Figure 1. There was evidence for difference among MMR cumulative incidence curves for 2 single SNPs and 2 score measures. Presence of the major allele in OCTN1 (CC) and of the minor allele in CYP3A4 rs2740574 (GG) were associated with increased MMR rate (P = 0.028 and P = 0.042, respectively, in the overall population and P = 0.027 and P = 0.038, respectively, in Caucasians). Similarly, an increase in the number of favorable alleles in the SLC22A1 gene was associated with increased MMR rate (P = 0.030 and P = 0.043 in the overall population and in Caucasians, respectively). In addition, the combination of favorable alleles in the genes involved in IM uptake was associated with increased rates of both MMR and CMR (P = 0.004 and P = 0.015, respectively, in the overall population and P = 0.005 and P = 0.009, respectively, in Caucasians). Our results suggest that SNP genotyping might be helpful in selecting pts who are more likely to benefit from first-line use of more potent inhibitors. Further assessment of the SNPs here identified in larger series of pts is warranted. Supported by Novartis Oncology, Clinical Development, TOPS Correlative Studies Network Disclosures: Hughes: Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad: Honoraria. White:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding. Saglio:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Rosti:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Hatfield:Novartis: Employment. Martinelli:Novartis: Consultancy, Honoraria; BMS: Honoraria; Pfizer: Consultancy.

Baseline Characteristics Of Patients With Chronic Myeloid Leukemia In a Prospective Observational Study (SIMPLICITY)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4026-4026 ◽  
Author(s):  
Jorge E. Cortes ◽  
Rüdiger Hehlmann ◽  
Carlo Gambacorti-Passerini ◽  
Stuart Goldberg ◽  
H. Jean Khoury ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Clinical Practice ◽  
Chronic Myeloid Leukemia ◽  
Clinical Research ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Chronic Phase ◽  
First Line ◽  
Historical Cohort ◽  
Prospective Cohorts

Abstract Background Oral BCR-ABL tyrosine kinase inhibitors (TKIs), including imatinib (IM), dasatinib (DAS) and nilotinib (NIL), have improved survival in chronic-phase chronic myeloid leukemia (CP-CML). Few data are available that compare TKIs in daily clinical practice across multiple regions. Methods SIMPLICITY is an ongoing observational cohort study of adult patients with newly diagnosed CP-CML receiving first-line treatment with IM, DAS or NIL in the USA and Europe (Eu) outside of clinical trials (NCT01244750). The primary objective is to assess effectiveness of these TKIs in clinical practice. The study includes three ‘prospective’ cohorts of patients treated with IM, DAS or NIL since 2010 (the study opened after first-line approval of all three TKIs) and a ‘historical’ cohort treated with IM since 2008. Preliminary baseline demographics are presented for prospective cohorts. Results 860 prospective patients (Eu: 32%, USA: 68%) were enrolled through June 20, 2013, receiving IM (n=399), DAS (n=229) or NIL (n=232). Median age at initiation of first-line TKI was 56 years, with significant differences in pairwise comparisons between DAS and IM and NIL and IM (Table). Demographics were consistent across cohorts. Only 30% of patients had Hasford or Sokal scores recorded. ECOG performance status (PS) was available in 54% of patients. The number of baseline comorbidities per patient (mean: 3.2 + 2.7) was balanced across cohorts; 51% of patients presented with ≥3 comorbidities. Patients in the IM cohort had a higher prevalence of gastrointestinal comorbidities (P=.006 and .007 for DAS vs IM and NIL vs IM, respectively), and the NIL cohort had a higher prevalence of musculoskeletal comorbidities than the DAS cohort (P=.015). The proportions of patients with cardiovascular comorbidities were 38%, 36% and 42% in the DAS, NIL and IM cohorts, respectively, consisting primarily of hypertension (31%) and hyperlipidemia (17%) (P>.05 across cohorts). Coronary artery disease was present in 9%, cardiac arrhythmias in 6%, myocardial infarction in 3% and peripheral arterial disease in 2% of patients. The proportion of patients with diabetes was 10%. Clinicians reported effectiveness as the most common reason for TKI selection; familiarity and cost were also cited as reasons for IM selection (P<.001 vs DAS and NIL). Comorbidities were not drivers of TKI selection in this analysis. Conclusions This is the first report from the prospective cohorts of SIMPLICITY. Demographics were consistent across cohorts. Overall, the SIMPLICITY population is older with potentially more comorbidities than patients enrolled in first-line clinical trials with restrictive inclusion criteria (NEJM 2003 348 994; NEJM 2010 362 2260; NEJM 2010 362 2251). Initial TKI selection does not appear to be driven by baseline comorbidity, rather by perceived effectiveness, cost and familiarity. Hasford/Sokal scores were not recorded in the majority of patients prior to starting first-line TKI therapy. Outcomes data are being collected across cohorts that will inform about a multi-region population treated outside clinical trials. Disclosures: Cortes: Ariad: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Teva: Consultancy, Honoraria, Research Funding. Hehlmann:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding. Gambacorti-Passerini:Bristol-Myers Squibb: Consultancy; Pfizer: Honoraria, Research Funding. Goldberg:Bristol-Myers Squibb: Honoraria, Research Funding, Speakers Bureau; Novartis Oncology: Honoraria, Research Funding, Speakers Bureau; Ariad: Honoraria, Research Funding, Speakers Bureau. Khoury:Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Ariad: Honoraria; Teva: Honoraria. Mauro:Novartis Oncology: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Speakers Bureau. Michallet:Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; MSD: Consultancy, Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding. Paquette:Ariad: Consultancy; Incyte: Consultancy, Honoraria; Novartis: Consultancy. Foreman:ICON Clinical Research: Employment, My employer ICON Clinical Research receives research funding from pharmaceutical companies including manufacturers of CML drugs Other. Mohamed:Bristol-Myers Squibb: Employment. Zyczynski:Bristol-Myers Squibb: Employment. Hirji:Bristol-Myers Squibb: Employment. Davis:Bristol-Myers Squibb: Employment.

Acquisition Of Compound BCR-ABL1 Alleles As A Mechanism Of Resistance To Ponatinib In Chronic Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 853-853
Author(s):  
Don L Gibbons ◽  
Sabrina Pricl ◽  
Paola Posocco ◽  
Erik Laurini ◽  
Maurizio Fermeglia ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
In Silico ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Significant Proportion ◽  
Chronic Phase ◽  
Cytogenetic Response ◽  
Kinase Domain ◽  
The Impact ◽  
In Cells

Abstract BACKGROUND Ponatinib targets the inactive conformation of the ABL1 kinase and avoids interacting with the side chain of the mutated 315 residue. In vitro, ponatinib inhibits all single-point BCR-ABL1 mutations. Yet, a significant proportion of patients with chronic myeloid leukemia in chronic phase (CML–CP) do not respond to ponatinib and a subset loses their response during the course of treatment. The mechanisms of resistance to ponatinib are currently not well characterized. OBJECTIVE To determine the impact of compound BCR-ABL1 mutations (polymutants) on the activity of ponatinib. METHODS BCR-ABL1 mutational status was determined in 70 pts with CML-CP post imatinib failure and during dasatinib therapy by DNA expansion of specific clones followed by DNA sequencing of ≥10 clones. Free energy of binding (DGbind) for the unmutated and all mutant BCR-ABL1 kinase/inhibitor complexes were obtained using Molecular Mechanics/Poisson-Boltzmann Surface Area (MM-PBSA) methodology. Single and polymutant BCR-ABL1 alleles obtained by direct mutagenesis and their expression was forced into Ba/F3 cells by electroporation by the pMX/eGFP-BCR-ABL1 expression vector using the Amaxa System. RESULTS After imatinib failure, 125 ABL1 kinase domain mutations at 113 amino acid positions were detected in 61/70 (87%) pts, including 38 (54%) with mutations in ≥20% of sequenced clones. Mutations conferring resistance to >1µM imatinib were detected in 30 (43%) pts. Polymutant BCR-ABL1 alleles were detected in 29/70 (41%) pts. These patients received dasatinib for a median of 19 mos (range, 2-52), during which dasatinib-resistant mutations were detected in 10/32 (31%) assessable cases (5 with T315I). Polymutants were present in 16/32 (50%) pts (all of them dead in blast phase). The proportion of clones carrying unmutated BCR-ABL1 was markedly lower in patients who only achieved a minor or no cytogenetic response compared to those achieving a major cytogenetic response (p=0.0001), suggesting exhaustion of unmutated clones and expansion of mutant (and polymutant) clones linked to clinical dasatinib resistance. Then, we performed 3D structural analyses to determine the thermodynamic impact of 21 BCR-ABL1 mutants (11 single and 10 double mutants) in the ability of ponatinib to bind the kinase domain (Table). Most single mutants did not result in high ponatinib resistance (except for E255K, IC50=8.8nM; DGbind=-10.99±0.01). However, the association of any 2 of 3 point mutants (T315I, F317L, V299L) in a dual polymutant produced highly resistant BCR-ABL1 proteins that exhibited fold change values from 19 to 40, compared to the unmutated protein, with T315I/F359V displaying the highest resistance (IC50=61nM; DGbind=-10.23±0.03 kcal/mol), unveiling a mechanism of escape to ponatinib. In Ba/F3-based assays, ponatinib (but not imatinib or dasatinib) was active against Ba/F3-BCR-ABL1T315I cells. Polymutants exhibited very high ponatinib resistance (10-fold higher than that of cells carrying BCR-ABL1T315I). As predicted in silico, BCR-ABL1T315I/F359V was the most resistant polymutant tested. Cell growth inhibition was coupled with CrkL and STAT5 phosphorylation inhibition. Ponatinib, while suppressing STAT5 phosphorylation, could not suppress CrkL phosphorylation in cells expressing the BCR-ABL1T315I/F359V polymutant kinase, even at 100 nM (50-fold the IC50 required to inhibit BCR-ABL1T315I). CONCLUSIONS Polymutants are very frequent in pt samples after TKI failure (particularly after sequential TKI therapy) and tend to induce high ponatinib resistance. Our in silico platform predicted very accurately TKI sensitivity in cells carrying different BCR-ABL1 mutations, which makes it clinically applicable for matching specific mutations to the most effective TKI. Some polymutants require ponatinib concentrations not clinically reachable, thus representing a mechanism of escape to ponatinib therapy through selection and expansion of refractory clones. Disclosures: Talpaz: ariad: Research Funding. Cortes:Ariad: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Teva: Consultancy, Honoraria, Research Funding. Quintas-Cardama:ariad: Consultancy.

A Two-Fold Rise of BCR-ABL Transcript Levels Advises BCR-ABL Mutation Analysis in Imatinib-Treated Chronic Myeloid Leukemia (CML) - an Analysis of the Randomized CML-Study IV

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3138-3138
Author(s):  
Benjamin Hanfstein ◽  
Niklas Westhoff ◽  
Rüdiger Hehlmann ◽  
Susanne Saussele ◽  
Michael Lauseker ◽  
...  
Keyword(s):  
Mutation Analysis ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Kinase Domain ◽  
Positive Clone ◽  
Transcript Levels ◽  
Optimal Response ◽  
Abl Kinase ◽  
Equity Ownership ◽  
Kinase Domain Mutations

Abstract Introduction: The clonal selection of a mutant BCR-ABL positive clone can be observed in about one of two patients with imatinib-resistant chronic myeloid leukemia (CML). The early detection of BCR-ABL kinase domain mutations is crucial, since it allows to change the tyrosine kinase inhibitor (TKI) regimen in a timely manner and may therefore prevent disease progression and the accumulation of further genetic lesions. European LeukemiaNet (ELN) recommendations suggest a mutation analysis if optimal response criteria are not achieved at 3, 6, 12 or 18 months, or whenever a loss of optimal response occurs (Soverini et al., Blood 2011). Several attempts have been made to derive this indication from a specific increase of BCR-ABL levels. Here we report on the correlation of a rise in BCR-ABL transcript levels and the prevalence of BCR-ABL kinase domain mutations in imatinib-treated patients of the CML-Study IV. Methods: A total of 1,173 patients were enrolled until 2009 and randomized to one of four imatinib-based treatment arms. BCR-ABLIS of 988 patients was determined in 7,876 samples by quantitative RT-PCR in the central laboratory (median sample number per patient: 8.4, range 1-37; median follow up: 34 months, range 0-86), representing the eligible patients for the study. Thereby, the estimated intra-laboratory variance is assumed to be about 20%. A first rise of BCR-ABLIS to at least two-fold and >0.1% between two samples of a patient's molecular course defined a sample suspected of bearing a mutant BCR-ABL positive clone. A mutation analysis was performed on this critical sample by direct sequencing of ABL exons 4 to 10. Results: A critical rise in BCR-ABLIS was observed in 231 of 988 patients (23%) after a median of 15.2 months on treatment (range 2.8-59.4). In the corresponding sample 33 mutant clones could be detected in 31 patients (13%). Thereby a steeper rise of BCR-ABLIS was correlated with a higher incidence of BCR-ABL mutations in the respective group (table). A total of 18 different mutations could be detected, the most frequent were: M244V, n=7 (21%); E255K, n=4 (12%); T315I, n=3 (9%); L248V, G250E, L387M and F486S, n=2 (6%), respectively. Mutations occur in a substantial proportion (8%) of patients with an only 2 to 3-fold rise of BCR-ABLIS transcript levels (table). Therefore, the most sensitive cut-off should be applied and mutation analysis may be triggered by a doubling of BCR-ABL transcripts at levels >0.1% IS. Conclusion: BCR-ABL kinase domain mutations occur already in a substantial proportion of patients with a doubling of BCR-ABL transcript levels, which should determine mutation analysis. Table 1. Rise of BCR-ABL expression Patients (n) Patients with BCR-ABL mutations (n) Patients with BCR-ABL mutations (%) Inter-sample interval(median, days) 2 to 3-fold 72 6 8.3 98 3 to 5-fold 50 3 6.0 100 5 to 10-fold 39 4 10.3 99 10 to 100-fold 49 10 20.4 98 > 100-fold 21 8 38.1 125 > 2-fold (total) 231 31 13.4 101 Disclosures Hanfstein: Novartis: Research Funding; Bristol-Myers Squibb: Honoraria. Hehlmann:Novartis: Research Funding; Bristol-Myers Squibb: Research Funding. Saussele:Novartis: Honoraria, Research Funding, Travel Other; Bristol-Myers Squibb: Honoraria, Research Funding, Travel, Travel Other; Pfizer: Honoraria, Travel, Travel Other. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Neubauer:MedUpdate: Honoraria, Speakers Bureau. Kneba:Novartis: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Pfirrmann:Novartis: Consultancy; Bristol-Myers Squibb: Honoraria. Hochhaus:Pfizer: Consultancy, Research Funding; ARIAD: Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Müller:Novartis: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding.

Two-Year Consolidation By Nilotinib Is Associated with Successful Treatment Free Remission in Chronic Myeloid Leukemia with MR4.5: Subgroup Analysis from STAT2 Trial in Japan

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1889-1889 ◽  
Author(s):  
Naoto Takahashi ◽  
Chiaki Nakaseko ◽  
Kaichi Nishiwaki ◽  
Hisashi Wakita
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Subgroup Analysis ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Second Generation ◽  
Second Line ◽  
First Line ◽  
Prior Therapy ◽  
Line Therapy ◽  
Treatment Free Remission

Abstract Background Nilotinib (NIL) is a second-generation tyrosine kinase inhibitor (TKI) that exhibits significant efficacy as first- or second-line treatment in patients with chronic myeloid leukemia (CML). Superior rates of deeper molecular responses (DMR) were achieved with NIL vs. imatinib (IM) in patients newly diagnosed with CML in chronic phase (CML-CP) in the ENESTnd trial. In addition, the ENESTcmr study demonstrated that switching to NIL after a minimum of 2 years on IM led to increased rates of DMR vs. remaining on IM. Switching to NIL treatment for 2 years safely led to MR4,5 (BCR-ABLIS…0.0032%) in 47.5% of patients with major molecular response (MMR) on long-term IM therapy in our STAT1 trial. Recently, treatment free remission (TFR) was proposed as one of the goals in CML treatment. Indeed, prospective trials suggest that IM therapy may be safely and successfully discontinued in 40% of CML patients with MR4.5. STAT2 is the first study to evaluate the efficacy of two-year consolidation by NIL for successful TFR in patients with CML-CP who had achieved MR4.5. Before enrolling in STAT2, some patients were treated by not only IM but also NIL because of MMR but no MR4.5 after IM therapy, and some patients changed over from STAT1 to STAT2. Here, we present the results of the subgroup analysis from STAT2 based on the prior treatments at the time of entry into the study. Methods In the STAT2 trial, patients who achieved MR4.5 on IM front line therapy (subgroup 1; SG1) or NIL second line therapy after IM therapy (subgroup 2; SG2) were eligible and NIL was given twice daily at the dose of 600 mg/day for 2 years in consolidation phase. The primary endpoint of STAT2 was the proportion of patients with successful TFR, defined as no confirmed loss of MR4.5 (2 consecutive IS RQ-PCR tests), within the first 12 months of TFR phase. Thirty-five institutions in STAT study group participated. The study was conducted in accordance with the principles of the Declaration of Helsinki. Informed consent was signed by all patients according to institutional guidelines. The study was approved by all institutional review boards and registered with UMIN-CTR (000005904). Results Between July 2011 and December 2012, 96 patients were enrolled in STAT2. Among 96 patients, 50 patients were treated by IM first line only as prior therapy (SG1). On the other hand, 40 patients were treated by IM first line and NIL second line including 21 patients who changed over from STAT1 to STAT2 because they achieved MR4.5 (SG2). Six patients were excluded in this analysis because second generation TKIs were taken as a first line therapy. Among patients treated by NIL for 2 years in this study, 40/50 (80%; 95% CI, 68.4%-88.7%) in SG1 and 33/40 (82.5%; 95% CI, 69.6%-91.5%) in SG2 entered the TFR phase, respectively. The median age was 54.5 years in SG1 and 56.0 years in SG2. The ratio of men to women was 26:14 in SG1 and 18:15 in SG2. The total duration of TKI treatment was 110 months for the SG1 with a median of 86 months of IM, and 24 months of NIL, and 93 months in SG2 with a median of 62 months of IM, and 31 months of NIL,, respectively. All patients achieved MR4.5 at the time of entry into the study and the median time to MR4.5 was 47 months in SG1 and 60 months in SG2.The proportion of patients who maintained TFR at 12 months after stopping NIL was similar across the 2 subgroups: 25/40 (62.5%; 95% CI, 48.3%-77.3%) in SG1, and 23/33 (69.7%; 95% CI, 54.0%-82.5%) in SG2. The Kaplan-Meier (KM) analysis of TFR survival showed that in the 2 subgroups, the majority of events occurred within the first 6 months after stopping NIL (Figure 1). There were no significant differences between these 2 subgroups. Conclusion After two-year consolidation by NIL of CML-CP patients who achieved MR4.5, the TFR rate was 67.9% (90%CI: 58.2% to 76.6%) at 12 months in the STAT2 trial. In the present analysis looking at the prior TKI exposure, the TFR rate was similar in patients treated with IM first line only or who switched from IM to NIL before entering the study, despite the fact that the treatment duration of switched patients was slightly shorter. These findings suggest that two-year consolidation by NIL is associated with successful TFR in CML with MR4.5 that was achieved with IM alone or after switching to NIL. Figure Kaplan-Meiercurve of TFR survival in the 2 subgroups based onthe prior treatmentsbefore two-year consolidation by NIL, IM first line only as prior therapy (subgroup1) and IM first line and NIL second line (subgroup2). Figure. Kaplan-Meiercurve of TFR survival in the 2 subgroups based onthe prior treatmentsbefore two-year consolidation by NIL, IM first line only as prior therapy (subgroup1) and IM first line and NIL second line (subgroup2). Disclosures Takahashi: PFIZER: Honoraria, Research Funding; BMS: Honoraria; NOVARTIS PHARMA: Honoraria, Research Funding. Nakaseko:BMS: Honoraria, Research Funding; PFIZER: Honoraria, Research Funding; NOVARTIS: Honoraria. Nishiwaki:Novartis PHARMA: Research Funding.

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