Positive Chronic Myeloid Leukemia Patients Research Articles

The study of the impact of additional chromosomal aberrations and c-MYC and BCR::ABL1 genes amplification on CML patient’s characteristics: relation to haematological parameters and patient outcome

BackgroundChronic myeloid leukaemia is characterised by genetic instability which results in additional cytogenetic aberrations that have been linked to progression to advanced phase. Genomic study linked amplified genes in the form of c-MYC and/or the rare BCR::ABL1 genes amplification to chronic myeloid leukaemia. The effect of these genes’ amplification on patients’ characteristics and disease progression still needs further study.This cross-sectional study aimed to investigate the frequency of additional chromosomal aberrations in addition to c-MYC and BCR::ABL1 genes amplification in chronic myeloid leukaemia patients and their impact on patient’s characteristics, disease progression, and level of remission. The study included cytogenetic analysis of 49 Philadelphia positive chronic myeloid leukaemia patients and investigation of c-MYC and BCR::ABL1 genes amplification by fluorescence in situ hybridization.ResultsPatients with additional chromosomal aberrations represented 36.7% and had significantly lower platelet count (P = 0.003) and higher blast count (P = 0.008). The acquisition of additional chromosomal aberrations was significantly higher in chronic myeloid leukaemia patients with advanced stages (P = 0.014). Follow-up of the patients for 6 months revealed significant higher frequency of additional chromosomal aberrations in patients with failure of remission (P < 0.0001). A highly significant association between cases with failure of molecular remission (P = 0.001) and co-existing additional chromosomal aberrations.Amplification of the c-MYC gene was detected in 6 cases. The cases with c-MYC amplification showed significantly higher peripheral blood and bone marrow blasts (P = 0.029 and P = 0.008, respectively) and significantly lower platelet count (P = 0.044). Amplification of c-MYC was significantly associated with additional chromosomal aberrations (P = 0.011). Molecular remission was not achieved in any of the instances with c-MYC amplification. A highly significant association between c-MYC amplification and poor patient outcome was detected (P = 0.002). BCR::ABL1 amplification was detected in three cases, and ABL amplification was detected in four cases. Patients with BCR::ABL1 amplification showed significantly higher blast count. BCR::ABL1 amplification was significantly associated with disease progression and failure of molecular remission (P = 0.002).ConclusionAdditional chromosomal aberrations, c-MYC amplification, and BCR:ABL1 amplification in chronic myeloid leukaemia stratify patients with disease progression, which may lead to better interventions and improved outcome in the future chronic myeloid leukaemia patients.

BCR-ABL Gene Transcript Types of Patients with Chronic Myelogenous Leukemia in Yogyakarta, Indonesia.

Background:The aim of this study was analyzing the BCR-ABL transcript types of patients with chronic myeloid leukemia (CML) in Dr Sardjito General Hospital, Yogyakarta, Indonesia. This study is very relevant because the data concerning BCR-ABL gene transcript types is very limited in Indonesia. Furthermore, it is important for patient’s management, particularly in defining the tyrosine kinase inhibitors (TKIs) therapy and monitoring after therapy. The introduction of TKIs has become a major advance in the management of patients with CML, especially in the chronic phase (CML-CP), in which most patients are diagnosed. Methods:One hundred eighty five (185) of 370 recruited patients were included in this study (2010–2014). RNA samples were isolated from mononuclear cells of peripheral blood of the subjects taken at primary diagnosis. Detection of BCR-ABL gene transcript types was done using multiplex reverse transcriptase PCR (multiplex RT-PCR) and/or nested PCR following the cDNA synthesis. When the first PCR set failed to amplify the BCR-ABL gene, RT-conventional PCR and/or nested PCR would be applied. The proportion of each transcript type was calculated among the BCR-ABL positive CML patients. Results:Approximately 99% (183/185) of CML patients are BCR-ABL positive, with the most common type is major b3a2 (136/183; 74.3%), followed by major b2a2 (41/183; 22.4%). Two samples (1.1%) showed co-expression of b3a2 and b2a2; 1 sample showed co-expression of b3a2 and fragment at 500bp; and 3 samples showed uncommon fragments. Conclusion:Ninety nine percent (99%) of CML patients in Yogyakarta, Indonesia are BCR-ABL positive, with 74.3% have b3a2 transcript, 22.4% have b2a2 trascript, 1.1% have co-expression of b3a2 and b2a2 transcript, and the rest (2.2%) have uncommon bands that still need to be confirmed.

Nilotinib Vs Nilotinib Plus Pegylated Interferon α (Peg-IFN) Induction and Nilotinib or Peg-IFN Maintenance Therapy for Newly Diagnosed BCR-ABL1 Positive Chronic Myeloid Leukemia Patients in Chronic Phase (TIGER Study): The Addition of Peg-IFN Is Associated with Higher Rates of Deep Molecular Response

Introduction: The TIGER (CML V)-study* (NCT01657604) is a multicenter, randomized phase III trial to evaluate efficacy and tolerability of nilotinib (NIL) 2*300mg/d monotherapy vs NIL 2*300mg/d + pegylated interferon α2b (Peg-IFN) 30-50μg/week as first line therapy for chronic myeloid leukemia (CML) patients (pts) in chronic phase and discontinuation of therapy after Peg-IFN maintenance (Figure). Methods: In August 2012, recruitment started with a pilot phase, aiming to validate the recommended dose of Peg-IFN. 25 pilot phase patients were treated with the combination of NIL 2*300 mg daily and Peg-IFN (30-50μg/week according to tolerability and commenced after ≥6 weeks NIL monotherapy). During the main phase of the study, 692 newly diagnosed pts were randomized between NIL 2*300 mg/d and NIL/Peg-IFN combination according to the outcome of the pilot phase. Results: Within 5 years, a total of 717 pts (429 male; median age 51 years, range 18-85; 12.9% EUTOS high risk) were recruited from 109 sites in Germany, Switzerland, and the Czech Republic. 702 pts with typical BCR-ABL1 transcripts (97.9%) were eligible for molecular follow-up assessments according to the international scale (IS). Fifteen pts (2.1%) expressed atypical BCR-ABL1 transcripts. 692 pts were randomized after EUTOS risk stratification to receive NIL monotherapy (n=353) or NIL/PEG-IFN combination therapy (n=339). Median observation time since recruitment was 41 months. Up to now, 477 pts concluded the induction phase by achieving a confirmed major molecular response, MMR (BCR-ABL1 transcript levels ≤0.1% IS, which qualified for entering the maintenance phase of the study using NIL or Peg-IFN monotherapy. During the maintenance phase, 199 pts achieved or sustained MR4 (BCR-ABL1 ≤0.01% IS) for at least one year and then discontinued all therapy. While the rate of MMR at 12 and 18 mo - the first primary endpoint of the study - was not different between the treatment arms, adding Peg-IFN to upfront NIL significantly improved rates of MR4 and MR4.5, BCR-ABL1 ≤0.0032% IS) (Table). In competing risk analysis, median time to MMR was 5.7 vs 5.4 mo, to MR4 20.9 vs 12.5 mo, and to MR4.5 33.8 vs 23.2 mo for NIL vs NIL/Peg-IFN, respectively. After NIL discontinuation, during Peg-IFN maintenance therapy, rate of molecular recurrence (BCR-ABL1 &amp;gt;1% IS) after 18 mo was 28%. From 199 pts who discontinued all therapy, 63 experienced a molecular relapse (BCR-ABL1 &amp;gt;0.1%). Relapse free survival by 18 mo after treatment discontinuation was 61% in the total cohort. By protocol, it is too early to assign relapse rates to the randomized treatment arm. Frequencies of adverse events after 24 mo of therapy were 90 and 92% (grade 1-5) and 36 and 42% (grade 3-5) for NIL vs NIL/Peg-IFN, respectively. Adverse events of special interest (all grades) were fatigue in 34.6 vs 40.4%, thrombocytopenia in 13.3 vs 18.9% and elevation of the alanin aminotransferase (ALAT) in 11.0 vs 18.9% of pts in the NIL vs NIL/Peg-IFN arms, respectively. Fifteen pts (2.1%) progressed to accelerated phase or blast crisis; 22 pts (3.1%) received an allogeneic stem cell transplantation, 10 of them after disease progression. In total, 22 pts (3.1%) died, 16 during the induction phase, 4 in the maintenance phase and 2 in treatment free remission. Four deaths were related to CML, 3 to vascular complications. Conclusions: This per protocol interim analysis demonstrates feasibility of the first-line treatment with NIL 2*300 mg/d combined with PEG-IFN 30-50 μg/week. Peg-IFN, when added upfront to NIL further increases the rates of MR4 and MR4.5, which may translate into higher rates oftreatment free remission. *The study is being conducted on behalf of the German CML Study Group, the Swiss Group for Clinical Cancer Research (SAKK) and the East German Study Group on Hematology and Oncology (OSHO). Disclosures Hochhaus: Pfizer: Research Funding; Novartis: Research Funding; BMS: Research Funding; Incyte: Research Funding; MSD: Research Funding. Burchert:Novartis: Research Funding. Saussele:Novartis: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria. Baerlocher:Novartis: Research Funding. Brümmendorf:Janssen: Consultancy; Ariad: Consultancy; University Hospital of the RWTH Aachen: Employment; Pfizer: Consultancy, Research Funding; Merck: Consultancy; Novartis: Consultancy, Research Funding. La Rosée:Novartis: Research Funding; Bristol-Myers-Squibb: Consultancy, Other: Travel support, Speakers Bureau. Heim:Novartis: Research Funding. Krause:Siemens: Research Funding; Takeda: Honoraria; MSD: Honoraria; Gilead: Other: travel; Celgene Corporation: Other: Travel. le Coutre:Bristol-Myers Squibb: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau. Niederwieser:Daichii: Speakers Bureau; Cellectis: Consultancy. Lange:Novartis: Research Funding. Fabarius:Novartis: Research Funding. Hänel:Novartis: Honoraria; Amgen: Honoraria; Takeda: Other: advisory board; Celgene: Other: advisory board; Roche: Honoraria. Stegelmann:Novartis: Consultancy, Honoraria; Incyte: Consultancy, Honoraria. Mayer:AOP Orphan Pharmaceuticals AG: Research Funding. Hasford:Novartis: Research Funding. Hehlmann:Novartis: Research Funding. Ernst:Novartis: Research Funding. OffLabel Disclosure: Combination of Nilotinib and PEG-IFN alpha is being tested is off-label and being tested in the TIGER study.

Ten Years of Treating Chronic Myeloid Leukemia in Rural Rwanda: Feasible Approaches for Low-Resource Settings

Objective: To describe the characteristics and outcomes of chronic myeloid leukemia (CML) patients treated through the Glivec Patient Assistance Program (GIPAP) in Rwanda. Methods: We retrospectively reviewed all BCR-ABL positive CML patients treated with imatinib at two rural hospitals between 2009 and 2018. Clinical data were analyzed using descriptive statistics, Kaplan-Meier method, and proportional hazards regression. Results: One hundred twenty-four patients were included; median age 35 (range: 9-81), 60% male, 88% without comorbidities. All patients presented with symptomatic disease, median duration 11 months (IQR: 4-24) and median WBC count 225.5 x 109/L (IQR: 117.7-308.5). At diagnosis, 71% were in chronic phase. On imatinib, 91% achieved complete hematologic remission (CHR); 67% within 90 days. Of those who achieved CHR, 51 (45%) lost it at some point and, of which, 36 (71%) regained CHR. At four years, overall survival was 79% (95% CI:69%-86%) for the entire cohort; 93% (95% CI: 79%-98%) for 53 patients who remained in CHR, 89% (95% CI: 69%-96%) for 34 patients who lost but regained CHR, 19% (95% CI: 1%-54%) for 12 patients who lost and never regained CHR. Never achieving CHR was associated with increased mortality (HR: 40.89; 95% CI: 9.88-169.32; p&lt;0.001) as was losing CHR without regaining it (HR 15.73; 95% CI: 4.14-59.81; p&lt;0.001) compared to never losing CHR. Conclusions: Here we demonstrate the feasibility of coupling molecular diagnostics with chronic targeted therapy in a rural low-resource setting through health systems strengthening and international collaboration. Our CML patients are younger, slower to achieve CHR with imatinib, and have higher rates of primary and early hematologic resistance than historical cohorts, suggesting biological differences that warrant further investigation. It is uncertain what role non-adherence to medication influences these data. Without molecular response assessment, hematologic response is an accurate marker of disease control and survival. Our imatinib failure rates highlight the need for access to resistance testing and second generation tyrosine kinase inhibitors, as well as adherence rates. Disclosures No relevant conflicts of interest to declare.

PS1193 CHARACTERISTICS AND OUTCOMES OF THE TREATMENT PATIENTS WITH T315I MUTATION CML

Background:Resistance to tyrosine kinase inhibitors (TKI) in patients with chronic myeloid leukemia (CML) is frequently caused by point mutations in the BCR‐ABL kinase domain, including the gatekeeper mutant T315I, which confers a high degree of resistance to all currently approved tyrosine kinase inhibitors except ponatinib.Aims:To evaluate the results of different treatments in CML patients with mutation T315I.Methods:Retrospective analysis of 79 BCR‐ABLT315I ‐positive CML patients (pts) was done. Allogeneic bone marrow transplantation (allo‐HSCT) was made in 20 pts, 59 pts received only pharmacological therapy (41 pts received TKI as monotherapy or in combination with other drugs, 10‐ α‐IF, other 8 pts received hydroxyurea or chemotherapy). At the time of allo‐HSCT 6 pts were in CP 1, 7‐ CP≥2, 6 pts had AP and 3 pts were in BC. Median (Me) age at the time of mutation detected was 42 years (13–75) (38 years in HSCT‐group). In allo‐HSCT group 13 pts had unrelated donors, 13(65%) pts received more than 2 lines TKIs before HSCT. The number of points on EBMT scale: 3–4 points ‐ 16 pts, 5–7 points ‐ 6 pts. Conditioning regimen in 13 (70%) pts had reduced intensity. Me time to HSCT after T315I detection was 7 months (0–72).Results:Me follow‐up time after T315I detection was 21 months (1–100). 5‐years OS in whole group was 42%. According to multivariate analysis only CML phase at the time of mutation detection significantly affect to survival in whole group. All pts in BC (n = 7), 3 in HSCT group and 4 in non‐HSCT group, died within first year after T315I indication wherein Me survival time was 1,3 month. 5‐years OS in non‐HSCT group (n = 59) was 42% with Me survival time 2,8 years. 5‐years OS after allo‐HSCT (n = 20) was 37% with Me survival time 5 months. However, 7 pts live more than 2 years after allo‐HSCT, 2 ‐ more than 6 years. All living patients after allo‐HSCT are in deep molecular response. Pts in the 2nd CP have OS 47% (CI 95% 29–53%), p = 0.46. There was no significant difference in 5‐years OS between TKI (n = 41) and non‐TKI (n = 18) pharmacological therapy (non‐HSCT) groups (42% and 47% respectively, p = 0,53). In the group that received α‐interferon in the combination with other drugs (n = 10), the 5‐year OS is higher than in the group that did not receive interferon therapy (n = 49), 72% vs 45%, respectively (p = 0.21).Summary/Conclusion:Detection of T315I mutation in TKI‐resistant patients is extremely unfavorable factor for survival, and it is a great reason for switching to ponatinib or other new potential investigated drugs if possible. Allo‐HSCT is an effective method of treatment for this group of patients in case of good selection taking into consideration transplant risk, especially for patients in CP ≥2. The administration of pharmacological therapy in the 1st CP is an effective method of treatment, which allows to control the disease for a long time.

Novel mutations in the kinase domain of BCR-ABL gene causing imatinib resistance in chronic myeloid leukemia patients

Mutations in the drug binding region of BCR-ABL lead to imatinib resistance during the management of chronic myeloid leukemia (CML). In our study, 62 Philadelphia positive (Ph+) CML patients showing conspicuous expression of BCR-ABL gene were treated with imatinib. At the end of 3 months, 21/62 (33.87%) patients did not obtain complete hematological response (CHR) and also showed no significant decrease in BCR-ABL gene expression. In all the imatinib-resistant patients BCR-ABL gene was PCR amplified and sequenced. The sequence analysis showed four novel missense mutations p.(Leu301Ile), p.(Tyr320His), p.(Glu373Asp), p.(Asp381Asn) and six already reported mutations p.(Val256Gly), p.(Thr315Ile), p.(Gly250Glu), p.(Tyr253His), p.(Phe317Leu), p.(Met351Thr) which contributed in the formation of inactive enzyme and also two novel frameshift mutations p.(Glu281*) and p.(Tyr393*), which resulted in truncated protein formation. Further, the structural analysis revealed all these mutations affected P-loop, gatekeeper, catalytic and activation loop domain regions of the enzyme causing poor imatinib binding in the ATP region. The primary intention of the study was to find out the mutations in the BCR-ABL gene causing imatinib resistance. This study highlights the need for BCR-ABL gene sequence analysis to detect the mutations in CML patients in order to properly guide the therapy.

Aberrant DNA methylation at HOXA4 and HOXA5 genes are associated with resistance to imatinib mesylate among chronic myeloid leukemia patients.

Imatinib mesylate is a molecularly targeted tyrosine kinase inhibitor drug. It is effectively used in the treatment of chronic myeloid leukemia (CML) patients. However, development of resistance to imatinib mesylate as a result of BCR-ABL dependent and BCR-ABL independent mechanisms has emerged as a daunting problem in the management of CML patients. Between these mechanisms, BCR-ABL independent mechanisms are still not robustly understood. To investigate the correlation of HOXA4 and HOXA5 promoter DNA hypermethylation with imatinib resistance among CML patients. Samples from 175 Philadelphia positive CML patients (83 good response and 92 BCR-ABL non-mutated imatinib resistant patients) were subjected to Methylation Specific High Resolution Melt Analysis for methylation levels quantification of the HOXA4 and HOXA5 promoter regions. Receiver operating characteristic curve analysis was done to elucidate the optimal methylation cut-off point followed by multiple logistic regression analysis. Log-Rank analysis was done to measure the overall survival difference between CML groups. The optimal methylation cut-off point was found to be at 62.5% for both HOXA4 and HOXA5. Chronic myeloid leukemia patients with ≥63% HOXA4 and HOXA5 methylation level were shown to have 3.78 and 3.95 times the odds, respectively, to acquire resistance to imatinib. However, overall survival of CML patients that have ≤62% and≥63% methylation levels of HOXA4 and HOXA5 genes were found to be not significant (P-value=0.126 for HOXA4; P-value=0.217 for HOXA5). Hypermethylation of the HOXA4 and HOXA5 promoter is correlated with imatinib resistance and with further investigation, it could be a potential epigenetic biomarker in supplement to the BCR-ABL gene mutation in predicting imatinib treatment response among CML patients but could not be considered as a prognostic marker.

P210 and P190 BCR-ABL fusion transcripts variants frequencies among Philadelphia chromosome-positive chronic myeloid leukemia in Sudan

Breakpoint cluster region-abelson (BCR-ABL) leukemic fusion gene types in chronic myeloid leukemia (CML) correlate with the disease clinical course and outcome. There are variations in the reports of previous studies about the frequencies and distribution of BCR-ABL transcripts in chronic myelogenous leukaemia among Sudanese patients. This research aims to determine the frequencies of BCR-ABL fusion transcript variants in Sudan. One hundred (informed consent) Philadelphia positive chronic myeloid leukaemia patients, in chronic phase, were enrolled in this study. EDTA anticoagulated peripheral blood samples were collected from each participant, RNA was extracted from mononuclear cells by (TRIzol) reagent. BCR-ABL transcripts were detected by qRT-PCR technique with specific primers forP190 and P210 BCR-ABL transcript variants. The typical p210 BCR-ABL transcripts (b3a2 or b2a2) were detected in all patients (100%) the b3a2 transcript was detected in 96/100 (96%) and the b2a2 transcript was detected in 4/100 (4%).co-expression of p210/p190 (b2a3/e1a2) was detected in 6/100 (6%). p190 variant was not detected independently.

Long-term patient-reported outcomes from an open-label safety and efficacy study of bosutinib in Philadelphia chromosome-positive chronic myeloid leukemia patients resistant or intolerant to prior therapy.

BACKGROUNDHealth‐related quality of life (HRQOL) in patients with chronic‐phase chronic myeloid leukemia (CML) is important because of the requirement for long‐term treatment. This study assessed HRQOL in bosutinib‐treated patients with Philadelphia chromosome–positive CML and resistance or intolerance to 1 (chronic‐phase second‐line [CP2L]) or more (chronic‐phase third‐line [CP3L]) tyrosine kinase inhibitors who had 264 weeks or more of follow‐up (ClinicalTrials.gov identifier NCT00261846).METHODSPatient‐reported HRQOL was assessed with the EuroQol 5‐Dimensions Questionnaire (EQ‐5D) and the Functional Assessment of Cancer Therapy–Leukemia (FACT‐Leu).RESULTSIn total, 284 and 119 patients composed the CP2L and CP3L cohorts, respectively. At treatment completion, more than 50% of the patients in the CP2L and CP3L cohorts completed the EQ‐5D and FACT‐Leu assessments. The EQ‐5D and EQ‐5D visual analog scale scores were stable in both cohorts throughout treatment. The mean FACT‐Leu scores were generally stable over time but were lower in magnitude in the CP3L cohort versus the CP2L cohort. The FACT‐Leu scale scores of a subset of patients with chronic diarrhea (CP2L, n = 101; CP3L, n = 30) were similar to the scores of the larger cohorts. Minimally important differences (MIDs) from baseline for the FACT‐Leu scale scores were observed for the following: emotional well‐being (EWB), Functional Assessment of Cancer Therapy–General (FACT‐G) Total, FACT‐Leu Total, and Functional Assessment of Cancer Therapy Trial Outcome Index (FACT‐TOI) in the CP2L cohort and FACT‐Leu Total in the CP3L cohort. Among patients with chronic diarrhea, MIDs were observed for EWB, FACT‐G Total, FACT‐Leu Total, and FACT‐TOI in the CP2L cohort and for EWB, FACT‐G Total, and FACT‐Leu Total in the CP3L cohort.CONCLUSIONSHRQOL was maintained with long‐term bosutinib treatment for patients with CP2L and CP3L CML. Cancer 2018;124:587‐95. © 2017 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

Prognostic significance of micro RNA 150 marker in BCR-ABL positive chronic myeloid leukaemia patients on imatinib mesylate

Background: Micro-RNAs control gene expression by destabilizing targeted transcripts and inhibiting their translation. In chronic myeloid leukaemia (CML), abnormal expressions of miRNAs have been described. The current treatment for newly diagnosed cases of CML is imatinib mesylate which produces rapid haematological responses. It is currently impossible to predict whether a patient will develop resistance to imatinib mesylate. This makes identification of predictors of resistance to imatinib an important goal in management of patients with CML. MicroRNA expression patterns can be used to predict outcome which can be remission or relapse. This study therefore, was set to assess the possible use of microRNA 150 for prognostication.Methods: Fifty peripheral blood samples previously collected from CML patients who were being treated with imatinib mesylate and stored in the refrigerator at +4°C were analyzed for the expression of microRNAs 150. Total RNA was extracted from guanidium isothiocynate (GITC) lysate of the blood samples using RNeasy mini spin column. The total RNA was converted to complimentary DNA by random hexamer priming using Murine Moloney Leukaemia Virus Reverse Transcriptase. Real time Multiplex PCR was used for detecting Breakpoint Cluster Region-Abelson Murine Leukaemia (BCR-ABL) transcript type.Results: The patients’ samples showed an expression of miRNA-150. Correlation of BCR-ABL ratio with miRNA-150 was done and the Spearman correlation coefficient (Rho) between BCR-ABL1 and miRNA-150 was 0.442 (p = 0.001; CI = 0.18-0.65) showing that there was a positive correlation between BCR-ABL1 and miRNA-150. The coefficient of determination was 20% (CI = 3-42%), which implies that about 20% of BCR-ABL1 ratio could be accounted for by the miRNA-150 values.Conclusions: Therefore, once patients who are on imatinib achieve molecular remission of the CML, the miRNA-150 can be useful in predicting outcome which could be relapse or complete molecular remission but is weak at diagnosis in predicting such outcome.

The use of Imatinib resistance mutation analysis to direct therapy in Philadelphia chromosome/BCR-ABL1 positive chronic myeloid leukaemia patients failing Imatinib treatment, in Patan Hospital, Nepal.

Philadelphia chromosome/BCR-ABL1 positive chronic myeloid leukaemia (CML) can be successfully treated with Glivec (Imatinib), which is available free of cost through the Glivec International Patient Assistance programme (GIPAP) to patients with proven CML without means to pay for the drug. We review the acquired mutations in the tyrosine kinase encoded by the BCR-ABL1 gene underlying Glivec failure or resistance in a cohort of 388 imatinib-treated CML patients (149 Female and 239 male) registered between February 2003 and June 2016 in Nepal. Forty-five patients (11 female 34 male) were studied; 18 different BCR-ABL1 mutations were seen in 33 patients. P-loop mutation, Kinase domain and A-loop mutations were seen in 9, 16 and 4 patients respectively. Other mutations were seen in five patients. A T315I mutation was the most common mutation, followed by F359V and M244V. Sixteen mutations showed intermediate activity to complete resistance to Glivec. Among the 45 patients evaluated for BCR-ABL1 mutations, 4 were lost to follow-up, 14 died and 27 are still alive. Among the surviving patients, 16 are receiving Nilotinib, 5 Dasatinib and 3 Ponatinib, while 3 patients were referred to India, one of who received allogenic bone marrow transplantation. Understanding the spectrum of further acquired mutations in BCR-ABL1 may help to choose more specific targeted tyrosine kinase inhibitors that can be provided by GIPAP.

The Comparison of Efficacy Between Generic and Branded Imatinib in Achievement of Overall Survival and Cytogenetic Responses in CML Patients in Bosnia and Herzegovina

Introduction Imatinib mesylate (Glivec, Novartis) is the first tyrosine kinase inhibitor (TKI) targeting the BCR-ABL1 fusion protein responsible for the pathogenesis of chronic myeloid leukemia (CML). Low cost generic alternatives to imatinib are an integral part of cost effective healthcare strategies for developing countries. However, the use of generics has been associated with different clinical outcomes. In this study, we compared outcomes of two groups of patients who received Glivec as first-line therapy (Group 1) to patients who received generic imatinib as first-line therapy (Group 2) in Bosnia and Herzegovina.Material and methods This was a multicenter retrospective cohort study of BCR-ABL1 positive CML patients (n = 53) in the Federation of Bosnia and Herzegovina between 1 June 2005 and 31 March 2016. Glivec was used from 01 June 2005 until 30 September 2013, when all patients had to switch to generics, which was mandated by the Federal Solidarity Fund that allocates targeted cancer therapies. The following generic imatinib was available: Anzovip (Zdravlje, Actavis) from 09/2013 to 09/2014, Meaxin (Krka) from 09/2014 to 12/2015, and Plivatinib (Pliva) from 12/2015. Patient data was collected from the database of the Federal Solidarity Fund, a subsidiary of the Federal Health Insurance Agency. Branded and generic imatinib was administered orally at dosage of 400 mg/day. Patients who were switched to nilotinib received orally 400 mg/day. Patients on Glivec included in this study started therapy from 0-6 months from time of diagnosis, while patients who started with generics did not wait for therapy. Patient variables that were collected included age, gender, town, date of diagnosis, date of start of therapy, monthly TKI dosage, adverse side effects, progression, lethal outcome, prognostic factors and diagnostic parameters, including cytogenetics and molecular testing. In September 2013, Glivec stopped being available in Bosnia and all CML patients were switched to generic therapy Anzovip. Median duration of each therapy is given in Table 1.Results We compared patients on Glivec as first-line therapy (Group 1, n=26) to patients on first-line generic imatinib (Group 2, n=27) with the follow-up period of at least three years for each group. When we compare Groups 1 and 2 using intention to treat analysis, Kaplan-Meier estimated rate of overall survival at 24 months of therapy was 88% vs. 68%, respectively (p=0.14), while 69% vs. 70% achieved CCyR (p=0.12), respectively. In Group 1, 27% (7/26) patients switched to nilotinib (treatment failure in 2 patients and side effects in 5 patients), 54% (14/26) patients switched to generics because Glivec was no longer available, and 19% (5/26) patients stopped therapy (2 patients stopped therapy and 3 patients died). Of the 7 patients who switched to nilotinib, 71% (5/7) achieved CCyR, 29% (2/7) achieved MMR and none died. Of 19 patients who stayed on imatinib, 68% (13/19) achieved CCyR, 63% (12/19) achieved MMR and 3/19 (16%) died. Of the 54% (14/26) patients who were switched from branded imatinib to generic imatinib, one patient (7%) lost complete cytogenetic response.Regarding Group 2, 52% (14/27) of patients switched to nilotinib due to treatment failure (n=8) and side effects (n=6), while 48% (13/27) of patients stayed on generics. Of patients who switched to nilotinib, 43% (6/14) achieved CCyR and 15% (2/14) achieved MMR. Of the patients who stayed on generic imatinib, 100% (13/13) achieved CCyR and 85% (11/13) achieved MMR.Conclusion Our results suggest that there was no obvious difference in the treatment efficacy between generic and branded imatinib. At 3 years, there was no significant difference in the overall suvival and achievement of CCyR between first-line Glivec and first-line generic imatinib (p=0.14, and p=0.12, respectively).* Median duration of therapy on generic imatinib [Display omitted] DisclosuresRadich:Bristol-MyersSquibb: Consultancy; Pfizer: Consultancy; ARIAD: Consultancy; TwinStrand: Consultancy; Novartis: Consultancy, Other: laboratory contract.

Gene expression profile of circulating CD34+ cells and granulocytes in chronic myeloid leukemia

PurposeWe compared the gene expression profile of peripheral blood CD34+ cells and granulocytes in subjects with chronic myeloid leukemia (CML), with the accent on signaling pathways affected by BCR–ABL oncogene. MethodsThe microarray analyses have been performed in circulating CD34+ cells and granulocytes from peripheral blood of 7 subjects with CML and 7 healthy donors. All studied BCR–ABL positive CML patients were in chronic phase, with a mean value of 2012±SD of CD34+cells/μl in peripheral blood. ResultsThe gene expression profile was more prominent in CML CD34+ cells (3553 genes) compared to granulocytes (2701 genes). The 41 and 39 genes were significantly upregulated in CML CD34+ cells (HINT1, TXN, SERBP1) and granulocytes, respectively. BCR–ABL oncogene activated PI3K/AKT and MAPK signaling through significant upregulation of PTPN11, CDK4/6, and MYC and reduction of E2F1, KRAS, and NFKBIA gene expression in CD34+ cells. Among genes linked to the inhibition of cellular proliferation by BCR–ABL inhibitor Imatinib, the FOS and STAT1 demonstrated significantly decreased expression in CML. ConclusionThe presence of BCR–ABL fusion gene doubled the expression quantity of genes involved in the regulation of cell cycle, proliferation and apoptosis of CD34+ cells. These results determined the modified genes in PI3K/AKT and MAPK signaling of CML subjects.

Down-regulation of miR-199b associated with imatinib drug resistance in 9q34.1 deleted BCR/ABL positive CML patients

Chronic myeloid leukemia (CML) occurs due to t(9,22) (q34;q11) and molecularly BCR/ABL gene fusion. About 15-18% Philadelphia positive CML patients have gene deletions around the translocation breakpoints on 9q34.1. The microRNAs (miRNAs), namely miR-219-2 and miR-199b, centromeric to the ABL1 gene are frequently lost in CML patients. We have designed a study to determine miR-219-2 and miR-199b expression levels which would help to understand the prognosis of imatinib therapy. A total of 150 CML patients were analyzed to identify 9q deletion. Fluorescent in-situ hybridization (FISH) was performed using BCR/ABL dual color, dual fusion probe to study the signal pattern and BAC probes for miR-199b and miR-219-2 (RP11-339B21 and RP11-395P17) to study the miRNA deletions. The expression level of miRNA was analyzed by real-time polymerase chain reaction (RT-PCR). FISH analysis revealed 9q34.1 deletion in 34 (23%) CML patients. The deletions were not detected using BAC probes for miRNAs in 9q deleted patients. The expression analysis showed down-regulation of miR-199b and miR-219-2 in the 9q deleted patients (34 CML) as compared to a pool of patients without deletion. However, miR-199b (9q34.11) was significantly (p=0.001) down-regulated compared to miR-219-2. The follow-up study showed that the miR-199b was found to be strongly associated with imatinib resistance, as 44.11% patients showed resistance to imatinib therapy. Hence, the deletion in 9q34.1 region (ABL) plays an important role in disease pathogenesis. Eventually, miRNAs can provide new therapeutic strategies and can be used as a prognostic indicator.

Phase I study of NRC-an-019, a tyrosine kinase inhibitor, in imatinib-resistant chronic myeloid leukemia (CML) in an Indian tertiary care hospital.

7080 Background: NRC-AN-019 is an orally administered tyrosine kinase inhibitor (TKI) of the Bcr-Abl protein-tyrosine kinase. The drug was given to patients as a ready-to-use liquid formulation. Methods: The primary objectives of the Phase-I study were to estimate the Dose Limiting Toxicity (DLT) and Maximum Tolerated Dose (MTD) of NRC-AN-019 and also to establish the safe dose for Phase II clinical trial. The secondary objectives were to study the pharmacokinetic properties and antileukemic activity of NRC-AN-019. Philadelphia positive CML patients in all phases, in the age group of 18-65 years were eligible. They had to be resistant or intolerant to Imatinib, with ECOG ≤ 2. Dosing was initiated at 50 mg/day and dose escalation was done in increments of 50 mg per each additional cohort. The maximum dose administered was 450 mg/day. The protocol-specific study duration was 30 days; patients continued to receive the study drug subsequently based on Investigator’s decision. Results: A total of 30 patients were enrolled (3 at 50 mg, 4 at 100 mg, 3 at 150 mg, 3 at 200 mg, 4 at 250 mg, 4 at 300 mg, 3 at 350 mg, 3 at 400 mg, and 3 at 450 mg). DLT was not observed and MTD was not reached. The recommended Phase II-A dose is 300 mg/day, with a scope for escalation up to 450 mg/day. The maximum plasma concentration of NRC-AN-019 was 9,342 ng/mL and AUC was 1,262,191 ng.hr/mL. Common adverse events were skin rash, nausea, and vomiting. Hematological response was observed in 11 patients, cytogenetic response in 7 patients and molecular response in 5 patients. The maximum duration a patient has been on NRC-AN-019 is 863 days. Conclusions: Based on the Phase-I data, NRC-AN-019 showed considerable safety and response; it could be a potential treatment option for imatinib-resistant CML. Clinical trial information: CTRI/2009/091/000204.

Crude and Age-Adjusted Ph+/Bcr-Abl+ Chronic Myeloid Leukemia Incidence Rate in St. Petersburg and Leningrad Region Between 2006–2011

AbstractAbstract 4432 Background:The incidence of chronic myeloid leukemia (CML), reported from some population based registries, varies significantly. CML is known as age-dependent disease, so population age structure may strongly influent on the data. For international comparisons several systems for age-standardization are using in epidemiological studies. We conducted our retrospective study to reveal differences in CML incidence rates on the basis of calculation – crude or age-adjusted according to different population standards in St. Petersburg and Leningrad region. Methods:In 2005 the database of Ph- and/or bcr-abl- positive CML patients (pts) was conducted in St. Petersburg and Leningrad region. Since then the data from all newly diagnosed CML patients were included prospectively on population basis. The database was updated at least bi-annually. The data were obtained from hematologists, as general practitioners and private physicians are not licensed to treat oncohematological disorders. The data were double checked from the list of Imatinib distribution (the only drug reimbursed for first line treatment). To calculate crude CML incidence rate we use the data of the general census of the population in Russia in 2010 (the whole population of our region is 6596434 with population in age 15 and above 5821133). For age-adjusted CML incidence rate we use three of currently existing standards: The Segi (“World”), The Scandinavian (“European”) and the WHO standard (based on world average population between 2000–2025). Results:There are 258 (242 in chronic, 9 in accelerated and 7 in blastic phases) CML adult (15 years and above) pts, registered during 2006–2011. The median age is 53 years (48,5 and 55,5 years for men and women respectively). Sokal score was evaluable in 209 pts. It is low in 37%, intermediate in 35% and high in 28% pts. The crude CML incidence rate is slightly higher in men than in women with ratio 1,2:1. Mean annual crude CML incidence rate was 0,65 per 100 000 whole population of Saint Petersburg and Leningrad region, but it was 0,74 in adult population (15 years old and above). Mean annual CML incidence rates in the same age groups were slightly higher in all three standardized systems: 0,94 in Segi, 0,84 in Scandinavian and 0,88 in WHO standard populations. CML incidence rates in all age groups are presented in the table 1. CML incidence rate was lowest in young pts. It was unexpectedly very low in senior pts.CML incidence rates nearly for all age groups were slightly higher in St. Petersburg than in the Leningrad region. The majority of pts (98%) were treated with Imatinib (93% first or second line) or other tyrosine kinase inhibitors (5% first line-in international clinical trials, 18% after Imatinib failure or intolerance). Stem cell transplantation was performed only in 8/258 (3%) pts. Only 25235 (7,5%) evaluable pts progressed from chronic to advanced phases. Only 29/258 (11%) pts dead mostly due to CML (21 CML related deaths were reported). Estimated 5 years overall survival is 91,5%. Mean annual overall CML pts death rate was 1,9% (mean annual death rate between 2006–2010 in whole population of our region was 1,6%). Mean pts accumulated very fast - annual CML prevalence increasing rate between 2005–2011 was more than 14% (Picture 1). Conclusions:CML incidence both crude and age-adjusted in our population based registry is nearly the same in young and middle age, but much lower in senior (65 years and above) pts groups in comparison with published data from other registries which probably represents peculiarities of health system rather than real incidence. In the tyrosine kinase inhibitors era CML patients death rate is very low (nearly the same as in whole population) and CML pts is accumulated very fast in our region.Tab. 1Mean annual crude and age adjusted CML incidence rate in St. Petersburg and Leningrad region between 2006–2011.Age groups (years)CML incidence rateCrudeBy Segi (“World”)By Scandinavian (“European”)By WHO world standard15–190,250,140,180,1520–240,290,320,360,3125–290,380,410,470,4130–340,700,930,790,7335–390,450,550,470,4640–440,921,010,870,9245–490,730,930,790,9250–541,021,671,191,5555–591,102,021,351,7860–641,302,081,672,2465–691,171,180,881,2070–740,962,401,602,1775–790,792,271,141,5080–840,773,541,771,9485+0,401,010,510,80 [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Efficacy and Safety of Radotinib in Chronic Phase Chronic Myeloid Leukemia Patients with Resistance or Intolerance to BCR-ABL Tyrosine Kinase Inhibitors: Radotinib Phase 2 Clinical Trial

AbstractAbstract ▪695▪This icon denotes a clinically relevant abstract BackgroundRadotinib is a novel, selective Bcr-Abl tyrosine kinase inhibitor (TKI) developed by IL-YANG Pharm, South Korea. Radotinib showed a good efficacy and safety profile to chronic myeloid leukemia (CML) in preclinical and phase 1 clinical studies. To investigate the clinical efficacy and safety of radotinib 400 mg twice daily, data from CML patients treated during phase 2 clinical trial are reported. MethodsPhiladelphia chromosome (Ph+)-positive chronic phase CML (CP-CML) patients who failed or were intolerable to TKIs (imatinib and/or dasatinib and/or nilotinib) were enrolled between July 2009 and November 2011. Patients were treated with radotinib 400 mg twice daily for 12 cycles (1 cycle=4 weeks). The primary end point was an achievement of major cytogenetic response (MCyR, Ph+£35%) by 12 months. Safety parameters were also analyzed. ResultsA total of 77 CP CML patients (18 years of age or over) were enrolled from 12 sites in Korea, India, and Thailand. This analysis includes data from last enrolled patient who received at least 3 months of radotinib therapy. The median age of patients was 47 (range; 24–76) years, and 65 (84.4%) were imatinib-resistant and 12 (15.6%) were imatinib-intolerant. Four patients also had intolerance to dasatinib. With a median follow-up of 10.6 months, treatment with radotinib is ongoing in 46 patients (59.7%) and 31 patients (40.3%) discontinued the treatment including two deaths (2.6%). However, there were no CML-related deaths. Median duration of radotinib exposure was 296 (8–798) days. Overall MCyR rate was 63.6%, including 35 patients (45.4%) complete cytogenetic response and 14 patients (18.2%) partial cytogenetic response. The median time to MCyR was 2.8 months (85 days) and the median duration of MCyR was 315 (range; 5–726) days. Of patients achieving complete cytogenetic response, 37% (13/35) achieved major molecular response. Within follow-up durations, 44 patients (57.1%) required dose interruption and 41 patients (53.3%) had dose reduction. Most common grade 3/4 hematologic and laboratory adverse events (AEs) were thrombocytopenia (27.3%), neutropenia (10.4%), anemia (6.5%), and hyperbilirubinemia (31.2%). Common non-hematologic AEs were rash (29.8%), fatigue (14.3%), nausea/vomiting (14.3%), headache (13.0%), and pruritus (11.7%). The majority of AEs were easily manageable with temporal dose interruption and/or reductions. In all patients with CP-CML treated with second-line radotinib, estimated progression-free survival and overall survival rate at 12months was 84.9% (95% CI, 72.7–92.0%) and 97.4% (95% CI, 89.9–99.3% ), respectively. ConclusionRadotinib phase 2 trial confirmed the efficacy and safety of radotinib 400 mg twice daily in patients with CP-CML after failure to TKIs. Most of the AEs occurred in the early treatment period, were tolerable, and were easily controlled by dose interruption or reduction.TableSummary of patients' characteristics, efficacy, and safety profile.Total patientsN=77Age47 (24-76) yearsGendermale54 (70.1%)female23 (29.9%)Disease status at screeningImatinib resistance65 (84.4%)Resistance42 (54.5%)Resistance + intolerance20 (26.0%)Resistance + intolerance + dasatinib intolerance3 (3.9%)Imatinib intolerance12 (15.6%)Intolerance11 (14.3%)Intolerance + dasatinib intolerance1 (1.3%)Cytogenetic responseComplete cytogenetic response35 (45.5%)Partial cytogenetic response14 (18.2%)Minor cytogenetic response4 (5.2%)Minimal cytogenetic response7 (9.1%)No cytogenetic response14 (18.2%)Not applicable3 (3.9%)Dose modificationDose interruption44 (57.1%)Dose reduction41 (53.3%)Grade 3/4 hematologic adverse eventsThrombocytopenia21 (27.3%)Neutropenia8 (10.4%)Anemia5 (6.5%)The most common grade 3/4 laboratory adverse eventHyperbilirubinemia24 (31.2%)Common non-hematologic adverse events (> 10%)Rash23 (29.8%)Fatigue11 (14.3%)Nausea/vomiting11 (14.3%)Headache10 (13%)Pruritus9 (11.7%) Disclosures:Off Label Use: Radotinib, new BCR/ABL tyrosine kinase inhibitor, treatment for CML. Lee:IL-YANG Pharm.: Employment. Park:IL-YANG Pharm.: Employment. Woo:IL-YANG Pharm.: Employment. Kim:IL-YANG Pharm.: Employment. Lee:IL-YANG Pharm.: Employment. Cho:IL-YANG Pharm.: Employment. Shin:IL-YANG Pharm.: Employment. Kim:IL-YANG Pharm.: Employment. Kim:IL-YANG Pharm.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Impact of additional cytogenetic aberrations at diagnosis on prognosis of CML: long-term observation of 1151 patients from the randomized CML Study IV

AbstractThe prognostic relevance of additional cytogenetic findings at diagnosis of chronic myeloid leukemia (CML) is unclear. The impact of additional cytogenetic findings at diagnosis on time to complete cytogenetic (CCR) and major molecular remission (MMR) and progression-free (PFS) and overall survival (OS) was analyzed using data from 1151 Philadelphia chromosome–positive (Ph+) CML patients randomized to the German CML Study IV. At diagnosis, 1003 of 1151 patients (87%) had standard t(9;22)(q34;q11) only, 69 patients (6.0%) had variant t(v;22), and 79 (6.9%) additional cytogenetic aberrations (ACAs). Of these, 38 patients (3.3%) lacked the Y chromosome (−Y) and 41 patients (3.6%) had ACAs except −Y; 16 of these (1.4%) were major route (second Philadelphia [Ph] chromosome, trisomy 8, isochromosome 17q, or trisomy 19) and 25 minor route (all other) ACAs. After a median observation time of 5.3 years for patients with t(9;22), t(v;22), −Y, minor- and major-route ACAs, the 5-year PFS was 90%, 81%, 88%, 96%, and 50%, and the 5-year OS was 92%, 87%, 91%, 96%, and 53%, respectively. In patients with major-route ACAs, the times to CCR and MMR were longer and PFS and OS were shorter (P < .001) than in patients with standard t(9;22). We conclude that major-route ACAs at diagnosis are associated with a negative impact on survival and signify progression to the accelerated phase and blast crisis.

Regulation of hTERT by BCR-ABL at multiple levels in K562 cells.

BackgroundThe cytogenetic characteristic of Chronic Myeloid Leukemia (CML) is the formation of the Philadelphia chromosome gene product, BCR-ABL. Given that BCR-ABL is the specific target of Gleevec in CML treatment, we investigated the regulation of the catalytic component of telomerase, hTERT, by BCR-ABL at multiple levels in K562 cells.MethodsMolecular techniques such as over expression, knockdown, real-time PCR, immunoprecipitation, western blotting, reporter assay, confocal microscopy, telomerase assays and microarray were used to suggest that hTERT expression and activity is modulated by BCR-ABL at multiple levels.ResultsOur results suggest that BCR-ABL plays an important role in regulating hTERT in K562 (BCR-ABL positive human leukemia) cells. When Gleevec inhibited the tyrosine kinase activity of BCR-ABL, phosphorylation of hTERT was downregulated, therefore suggesting a positive correlation between BCR-ABL and hTERT. Gleevec treatment inhibited hTERT at mRNA level and significantly reduced telomerase activity (TA) in K562 cells, but not in HL60 or Jurkat cells (BCR-ABL negative cells). We also demonstrated that the transcription factor STAT5a plays a critical role in hTERT gene regulation in K562 cells. Knockdown of STAT5a, but not STAT5b, resulted in a marked downregulation of hTERT mRNA level, TA and hTERT protein level in K562 cells. Furthermore, translocation of hTERT from nucleoli to nucleoplasm was observed in K562 cells induced by Gleevec.ConclusionsOur data reveal that BCR-ABL can regulate TA at multiple levels, including transcription, post-translational level, and proper localization. Thus, suppression of cell growth and induction of apoptosis by Gleevec treatment may be partially due to TA inhibition. Additionally, we have identified STAT5a as critical mediator of the hTERT gene expression in BCR-ABL positive CML cells, suggesting that targeting STAT5a may be a promising therapeutic strategy for BCR-ABL positive CML patients.

Absolute Quantification of Mutant T315I BCR-ABL Transcripts At 6 Months Identifies Imatinib Resistant CML Patients with a Low Probability to Achieve MMR After 12 Months on Second Line Tyrosine Kinase Therapy

Abstract 1679The BCR-ABL T315I mutation is associated with resistance against imatinib (IM), nilotinib (NI) and dasatinib (DA) in patients with chronic myeloid leukemia (CML). Although this mutation can be detected by different techniques and at different levels of the mutated clone, the prognostic significance of the absolute amount of the mutated allele is widely unknown. We therefore sought to determine absolute T315I allele levels after 6 months on second line NI or DA therapy to identify a cut off value for predicting the achievement of major molecular response after 12 months (MMR12; ≤0.1% BCR-ABL transcripts at international scale).Forty randomly selected BCR-ABL positive CML patients (n=24 male, median age 64, range 39–78 years) in chronic (n=31), accelerated (n=7), or blastic phase (n=2) with failure to IM were treated with DA 140 mg/day (n=20) or NI 800 mg/day (n=20) and analyzed by denaturing high-performance liquid chromatography (D-HPLC) and high-sensitive allele-specific ligation PCR (L-PCR) for quantification of mutated T315I BCR-ABL allele normalized by beta-glucuronidase (GUS) expression. This method was previously shown to obtain a dynamical detection range of 100% to <0.1% mutant T315I allele (3–3.5 log). The levels of the BCR-ABL T315I alleles at 6 months were correlated with the achievement of a MMR12 on second line treatment. The L-PCR investigator was blinded for the MMR12 results.On second line therapy, MMR12 was achieved by 12 (20%) patients. After 6 months, 37 (10 with MMR12) patients were available for D-HPLC assessment and showed a T315I mutation in 3 (8%) patients. None of these patients achieved MMR12. In comparison, 35 evaluable patients (8 with MMR12) showed >10−5 BCR-ABLT315I/GUS (n=10, 20%), <10−5 (n=8, 22%) or no (n=17, 48%) T315I mutation by L-PCR. All patients with >10−5 BCR-ABLT315I/GUS did not achieve MMR12, all patients with <10−5 developed MMR12 (p<0.0001). This pre-defined value of >10−5 BCR-ABLT315I/GUS as a negative predictor for MMR12 was applied to a new second independent group of 40 IM resistant patients (n=17 male, median age 60, range 32–79 years) all in first chronic phase, MMR12 n=9, 22.5%) equally distributed to NI and DA. In this group 12 (30%) patients showed >10−5 BCR-ABLT315I/GUS after 6 months and all but one did not achieve MMR12. In contrast, 5 patients with <10−5 showed MMR12, 3 patients did not (p=0.0181).In conclusion, absolute quantification of the mutant T315I allele is helpful to identify patients with a low chance of MMR12 on second line nilotinib or dasatinib treatment. Therefore, relevant mutations should be quantified into clinical trial protocols to identify certain amounts with prognostic significance of the mutated clones for a specific mutations on a given TKI treatment in CML. Disclosures:Lange:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hochhaus:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.