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

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.

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Dasatinib (BMS-354825) targets an earlier progenitor population than imatinib in primary CML but does not eliminate the quiescent fraction

Blood ◽

10.1182/blood-2005-07-2947 ◽

2006 ◽

Vol 107 (11) ◽

pp. 4532-4539 ◽

Cited By ~ 458

Author(s):

Mhairi Copland ◽

Ashley Hamilton ◽

Lucy J. Elrick ◽

Janet W. Baird ◽

Elaine K. Allan ◽

...

Keyword(s):

Myeloid Leukemia ◽

Kinase Inhibitor ◽

Single Copy ◽

Kinase Domain ◽

Significant Inhibition ◽

Cell Compartment ◽

Transcript Levels ◽

Abl Kinase ◽

Stem Cell Compartment ◽

Kinase Domain Mutations

AbstractDasatinib (BMS-354825), a novel dual SRC/BCR-ABL kinase inhibitor, exhibits greater potency than imatinib mesylate (IM) and inhibits the majority of kinase mutations in IM-resistant chronic myeloid leukemia (CML). We have previously demonstrated that IM reversibly blocks proliferation but does not induce apoptosis of primitive CML cells. Here, we have attempted to overcome this resistance with dasatinib. Primitive IM-resistant CML cells showed only single-copy BCR-ABL but expressed significantly higher BCR-ABL transcript levels and BCR-ABL protein compared with more mature CML cells (P = .031). In addition, CrKL phosphorylation was higher in the primitive CD34+CD38– than in the total CD34+ population (P = .002). In total CD34+ CML cells, IM inhibited phosphorylation of CrKL at 16 but not 72 hours, consistent with enrichment of an IM-resistant primitive population. CD34+CD38– CML cells proved resistant to IM-induced inhibition of CrKL phosphorylation and apoptosis, whereas dasatinib led to significant inhibition of CrKL phosphorylation. Kinase domain mutations were not detectable in either IM or dasatinib-resistant primitive CML cells. These data confirm that dasatinib is more effective than IM within the CML stem cell compartment; however, the most primitive quiescent CML cells appear to be inherently resistant to both drugs.

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Clinical Activity of Bosutinib by Mutational Status In Patients with Previously Treated Philadelphia Chromosome–positive Leukemias.

Blood ◽

10.1182/blood.v116.21.3434.3434 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3434-3434 ◽

Cited By ~ 2

Author(s):

Carlo Gambacorti-Passerini ◽

H. Jean Khoury ◽

Helio Pinczowski ◽

Tamas Masszi ◽

Dong-Wook Kim ◽

...

Keyword(s):

Research Funding ◽

Kinase Domain ◽

Second Line ◽

Employment Equity ◽

Abl Kinase ◽

T315i Mutation ◽

Equity Ownership ◽

Kinase Domain Mutations ◽

Third Line ◽

Line Setting

Abstract Abstract 3434 Bosutinib (SKI-606) is a dual Src/Abl tyrosine kinase inhibitor (TKI) with minimal inhibitory activity against PDGFR or c-kit. Previous reports of this open-label, phase 1/2 study have demonstrated the efficacy and safety of oral daily treatment with 500 mg bosutinib in adult patients with Philadelphia chromosome–positive (Ph+) chronic phase (CP) chronic myeloid leukemia (CML) following resistance or intolerance to imatinib (second-line setting; Cortes JE, et al. ASCO 2010. Abstract #6502) and dasatinib or nilotinib (third-line setting; Khoury HJ, et al. ASCO 2010. Abstract #6514), or with Ph+ advanced leukemias (ie, accelerated or blast phase CML, acute lymphocytic leukemia) following imatinib failure in the second- and third-line settings (Gambacorti-Passerini C, et al. ASCO 2010. Abstract #6509). Failure of therapy with imatinib and other TKIs may result from mutations in the Abl kinase domain that affect drug binding. Therefore, the current analysis compared the rates of hematologic and cytogenetic responses to 500 mg bosutinib in patients with and without Bcr-Abl kinase domain mutations. Of the 570 patients treated, 53% were male and the median age was 53 years (range, 18–91). Median duration of follow-up was 24.1 months (range, 0.6–53.8) for patients with CP CML in the second-line setting, 22.6 months (range, 0.3–47.3) for patients with CP CML in the third-line setting, and 12.1 months (range, <0.1-45.5) for patients with advanced leukemias. Among the 222 (39%) patients with baseline sequencing analyses across all study cohorts, 119 (54%) patients had 27 unique Bcr-Abl mutations (Table). Patients with and without mutations had similar rates of complete hematologic response (CHR; 78% vs 82%, respectively) and major cytogenetic response (MCyR; 56% vs 53%, respectively). Comparable rates of response to bosutinib were observed across P-loop and non–P -loop Bcr-Abl kinase domain mutations, except for the T315I mutation. In particular, although a small number of patients was evaluable for individual mutations, hematologic and cytogenetic responses were observed with mutations such as F317L, F359V/I/C, and E255K/V, which are commonly associated with resistance following sequential treatment with multiple TKI therapies. Although response rates among patients with the T315I mutation were lower, 2 of 6 (33%) evaluable patients achieved a CHR and 1 of 7 (14%) patients achieved a MCyR. Among patients with mutations, the rate of CHR was higher for those with CP CML in the second-line (95%) and third-line (83%) settings than for those with advanced leukemias (46%). The rate of MCyR was highest among patients with CP CML in the second-line setting (73%). In conclusion, bosutinib was associated with substantial clinical activity in multiple patient populations independent of the presence or absence of Bcr-Abl kinase domain mutations and across all types of mutations, with the exception of the T315I mutation. Response n/n evaluablea (%) Bcr-Abl kinase domain mutation type n CHR MCyR Overall population Any mutation 119 35/45 (78) 44/79 (56) P-loop 35 9/11 (82) 12/21 (57) L248V 5 2/2 (100) 2/4 (50) G250E 11 2/2 (100) 3/5 (60) Y253F 1 1/1 (100) 0/1 Y253H 9 2/3 (67) 4/6 (67) E255K 6 2/3 (67) 2/4 (50) E255V 3 0 1/1 (100) Non–P-loop 84 26/34 (76) 32/58 (55) M244V 6 3/3 (100) 3/5 (60) K263E 1 0 1/1 (100) L273M 1 1/1 (100) 1/1 (100) V299L 1 0 0 F311L 1 0 1/1 (100) T315I 19 2/6 (33) 1/7 (14) F317L 16 4/5 (80) 4/11 (36) G321R 1 1/1 (100) 0 N331S 1 0 1/1 (100) M351T 11 3/4 (75) 6/9 (67) E355G 1 1/1 (100) 1/1 (100) E355G/M244V 1 1/1 (100) 0/1 F359C 3 0 1/2 (50) F359I 4 3/3 (100) 3/3 (100) F359V 8 6/6 (100) 4/7 (57) L384P 1 0/1 0/1 H396P 2 1/1 (100) 2/2 (100) H396R 1 0 0 I432T 1 0 0/1 E453K 1 0 1/1 (100) F486S 3 0/1 2/3 (67) No mutation 103 42/51 (82) 38/72 (53) Second-line CP CML population Any mutation 46 19/20 (95) 29/40 (73) Third-line CP CML population Any mutation 26 10/12 (83) 5/17(29) Advanced leukemia population Any mutation 47 6/13 (46) 10/22 (45) a Evaluable patients had a baseline and post-baseline hematologic or cytogenetic assessments, respectively. Patients who had experienced early progression or death before having a post-baseline assessment were also evaluable. Disclosures: Gambacorti-Passerini: Pfizer Inc: Research Funding. Khoury:BMS, Novartis: Honoraria. Kim:BMS, Novartis, Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Martinelli:BMS: Consultancy, Honoraria; Genzyme: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Honoraria, Research Funding; GlaxoSmithKline: Honoraria. Kelly:Pfizer: Employment, Equity Ownership. Besson:Pfizer: Employment, Equity Ownership. McMullan:Pfizer: Employment, Equity Ownership. Brummendorf:Pfizer: Membership on an entity's Board of Directors or advisory committees. Cortes:Pfizer Inc: Consultancy, Research Funding.

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Validation of the New European LeukemiaNet (ELN) Recommendations for Bcr-Abl Kinase Domain Mutation Analysis In Chronic Myeloid Leukemia: An Analysis of the GIMEMA CML Working Party Studies

Blood ◽

10.1182/blood.v118.21.112.112 ◽

2011 ◽

Vol 118 (21) ◽

pp. 112-112

Author(s):

Simona Soverini ◽

Alessandra Gnani ◽

Caterina De Benedittis ◽

Fausto Castagnetti ◽

Gabriele Gugliotta ◽

...

Keyword(s):

Board Of Directors ◽

Mutation Analysis ◽

Myeloid Leukemia ◽

Research Funding ◽

Chronic Phase ◽

Kinase Domain ◽

Newly Diagnosed ◽

Advisory Committees ◽

Abl Kinase ◽

Mutation Analyses

Abstract Abstract 112 BCR-ABL kinase domain (KD) mutation analysis may be an useful tool for physicians and is being performed in a growing number of laboratories. Recommendations aimed to rationalize the use of mutation testing in chronic myeloid leukemia (CML) have recently (Blood 2011) been compiled by a panel of experts appointed by European LeukemiaNet (ELN) – including specific recommendations as to when mutation analysis should be performed. They came from the expert opinion of the panel members whenever published data were insufficient or contradictory. In order to provide further data to validate or refine these recommendations, we have analyzed the GIMEMA CML WP database recording the results of mutation analyses performed in CML pts (n=1301) receiving imatinib and/or 2nd generation TKIs between January 2004 and July 2011. At dagnosis, mutation analysis was recognized to be useful in the few pts who present in accelerated phase or blast crisis (BC), while it was not recommended in chronic phase (CP) pts. Interrogating our database, we could retrieve 58 mutation analyses in newly diagnosed pts in CP and 12 in newly diagnosed pts in BC. Imatinib-resistant mutations were detected in 0 and 2 pts, respectively. In pts receiving 1st-line imatinib, mutation analysis was recommended both in case of failure and in case of suboptimal response. We have analyzed 399 chronic phase (CP) CML pts receiving first-line imatinib because they were found to meet one of the criteria for failure or suboptimal response. Overall, 45/166 (27.1%) failures were found to be positive for one or more BCR-ABL KD mutations. In particular, mutations were detected in 3/16 (18.8%) pts with less than CHR at 3 months, 1/9 (11.1%) pts with no CyR at 6 months, 4/24 (16.7%) pts with less than PCyR at 12 months, 6/36 (16.7%) pts with less than CCyR at 18 months, 15/49 (30.6%) pts who lost CCyR and 16/32 (50%) pts who lost CHR. More interestingly, only 11/233 (4.7%) suboptimal responders we analyzed were positive for mutations. Among ‘cytogenetic' suboptimal responders, mutations were detected in 1/15 (6.7%) pts with no CyR at 3 months, 1/20 (5.0%) pts with less than PCyR at 6 months, 5/51 (8.2%) pts with less than CCyR at 12 months. Among ‘molecular' suboptimal responders, mutations were detected in 0/52 pts with less than MMR (but having achieved CCyR) at 18 months and in 4/95 (4.2%) pts who lost MMR (but not CCyR). Which rise in Bcr-Abl transcript level should trigger a mutation analysis was the most difficult issue to provide recommendations upon, given the lack of convincing and reproducible data in the literature. It was finally agreed to recommend mutation analysis only in case of MMR loss. In 159 of the CP pts we have analyzed, mutation analysis was specifically requested because of a transcript increase at a single RQ-PCR assessment: 29 pts had less than 1-log increase and 41 pts had a 1-log increase or more – but with no loss of MMR. None of these pts was found to have mutations. Another 36 pts had less than 1-log increase and 53 had a 1-log increase or more, leading to loss of MMR. Mutations were detected in 1 (2.8%) and 3 (5.7%) pts, respectively. In pts receiving dasatinib or nilotinib as 2nd-line agents, mutation analysis was recommended at baseline and then in case of failure according to the provisional definitions proposed by Baccarani et al (J Clin Oncol 2009). Nineteen among the pts we analyzed met these criteria; overall, mutations were detected in 11 (57.8%), including 5/7 pts with no CyR at 3 months, 6/9 pts with minimal CyR at 6 months, 1/4 pts with less than PCyR at 12 months. In addition, newly acquired mutations were detected in 93/131 (71%) pts who lost a previously achieved HR or CyR. We also tested 19 pts who met the provisional definitions for suboptimal response to dasatinib or nilotinib 2nd-line. Mutations were detected in 4/19 pts (21%), including 2/5 pts with minor CyR at 3 months, 1/7 pts with PCyR at 6 months, 1/7 pts with less than MMR at 12 months. Our data indicate that: a) pts harbouring mutations can more frequently be found among cytogenetic suboptimal responders than among molecular suboptimal responders; b) any Bcr-Abl transcript increase that is not associated with MMR loss shouldn't indeed trigger a mutation analysis; c) although definitions of response to dasatinib or nilotinib 2nd-line are still provisional and might soon be refined, not only failures but also suboptimal responses are frequently associated with mutations. Supported by AIL, AIRC, PRIN and FIRB. Disclosures: Soverini: Novartis: Consultancy; ARIAD: Consultancy; Bristol-Myers Squibb: Consultancy. Rosti:Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Research Funding; Novartis: Honoraria; Bristol Myers Squibb: Honoraria. Baccarani:Pfizer Oncology: Consultancy; Novartis: Consultancy; BMS: Consultancy; Ariad: Consultancy; Novartis: Research Funding; Pfizer Oncology: Honoraria; Novartis: Honoraria; BMS: Honoraria; Ariad: Honoraria; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees. Martinelli:Bristol-Myers Squibb: Consultancy; Novartis: Consultancy, Research Funding.

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BCR-ABL kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors: recommendations from an expert panel on behalf of European LeukemiaNet

Blood ◽

10.1182/blood-2010-12-326405 ◽

2011 ◽

Vol 118 (5) ◽

pp. 1208-1215 ◽

Cited By ~ 332

Author(s):

Simona Soverini ◽

Andreas Hochhaus ◽

Franck E. Nicolini ◽

Franz Gruber ◽

Thoralf Lange ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Tyrosine Kinase Inhibitors ◽

Mutation Analysis ◽

Myeloid Leukemia ◽

Kinase Inhibitors ◽

Direct Sequencing ◽

Chronic Phase ◽

Kinase Domain ◽

Abl Kinase

AbstractMutations in the Bcr-Abl kinase domain may cause, or contribute to, resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia patients. Recommendations aimed to rationalize the use of BCR-ABL mutation testing in chronic myeloid leukemia have been compiled by a panel of experts appointed by the European LeukemiaNet (ELN) and European Treatment and Outcome Study and are here reported. Based on a critical review of the literature and, whenever necessary, on panelists' experience, key issues were identified and discussed concerning: (1) when to perform mutation analysis, (2) how to perform it, and (3) how to translate results into clinical practice. In chronic phase patients receiving imatinib first-line, mutation analysis is recommended only in case of failure or suboptimal response according to the ELN criteria. In imatinib-resistant patients receiving an alternative TKI, mutation analysis is recommended in case of hematologic or cytogenetic failure as provisionally defined by the ELN. The recommended methodology is direct sequencing, although it may be preceded by screening with other techniques, such as denaturing-high performance liquid chromatography. In all the cases outlined within this abstract, a positive result is an indication for therapeutic change. Some specific mutations weigh on TKI selection.

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Detection of BCR-ABL Kinase Domain Mutations in Chronic Myeloid Leukemia Patients

Clinical Lymphoma Myeloma & Leukemia ◽

10.1016/j.clml.2016.07.087 ◽

2016 ◽

Vol 16 ◽

pp. S60

Author(s):

Nancy Escobar ◽

Mariana Herrera ◽

Luisa Rosales ◽

Silvana Torselli ◽

Julio Caceres ◽

...

Keyword(s):

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Kinase Domain ◽

Abl Kinase ◽

Kinase Domain Mutations

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PB1920 BCR-ABL GENE ABL KINASE DOMAIN MUTATIONS IN IMATINIB RESISTANT CHRONIC MYELOID LEUKEMIA PATIENTS FROM AZERBAIJAN

HemaSphere ◽

10.1097/01.hs9.0000566180.59245.28 ◽

2019 ◽

Vol 3 (S1) ◽

pp. 873-874

Author(s):

C. Asadov ◽

A. Hasanova ◽

A. Shirinova ◽

N. Karimova ◽

Z. Alimirzoeva

Keyword(s):

Chronic Myeloid Leukemia ◽

Myeloid Leukemia ◽

Kinase Domain ◽

Abl Kinase ◽

Imatinib Resistant ◽

Kinase Domain Mutations

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Low Level BCR-ABL Mutations Below the Detection Limit of Current Standard Screening Techniques Occur Predominantly in the CD34+ Progenitor Cell Compartment in Chronic Phase CML Patients At Diagnosis. A Substudy of the ENEST1st Trial

Blood ◽

10.1182/blood.v120.21.1671.1671 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1671-1671

Author(s):

Jacqueline Maier ◽

Karoline Schubert ◽

Michael Cross ◽

Sabine Leiblein ◽

Kathrin Wildenberger ◽

...

Keyword(s):

Detection Limit ◽

Research Funding ◽

Progenitor Cell ◽

Chronic Phase ◽

Kinase Domain ◽

Cell Compartment ◽

Screening Techniques ◽

Abl Kinase ◽

Cd34 Cells ◽

Kinase Domain Mutations

Abstract Abstract 1671 The presence of BCR-ABL kinase domain mutations below the detection limit of conventional screening techniques (low level mutations, LLM) predicts outcome of subsequent therapy in patients with imatinib resistance (Parker et. al JCO 2011 and Blood 2012). We have further evaluated LLM in the context of the ENEST1st trial, which addresses the frequency of complete molecular responses after 18 months on nilotinib 300mg BID (NI) in newly diagnosed patients with chronic myeloid leukemia (CML) in chronic phase (CP). Here, we have investigated the incidence of detectable LLM in the CD34+ progenitor cell compartment in comparison to total white cells (TWBC). Sixty nine ENEST1st study patients with CP CML provided 10ml of peripheral blood or 2ml bone marrow after written informed consent. CD34+ selection was carried out by MACS® (Miltenyi Biotec) and the CD34+ purity was subsequently determined by fluorescent activated cell sorting (FACS). The results were compared to those derived from stored TWBC from 23 of the same patients and a further 16 patients at diagnosis. Aliquots of 105 CD34+ or at least 106 TWBC were used for RNA extraction, cDNA synthesis and BCR-ABL amplification followed by Ligation PCR (L-PCR) for mutations T315I, Y253H, E255K/V, and F359V. This method has previously been shown to achieve a dynamic detection range of 100% to <0.1% mutant allele (3–3.5 log). No patients showed BCR-ABL kinase domain mutations detected by Sanger sequencing spanning ABL exons 4–9. Forty five of 69 patients (65%) with 105 CD34+ cells and a documented CD34+ purity of >50% were available for BCR-ABL amplification. Amplification was successful from 36 (52%) of these CD34+ samples and from 38 of the 39 (97%) TWBC samples. A total of 180 L-PCR assays of CD34+ cells identified 29 (16%) mutations (T315Ix12, Y253Hx7, E255Kx8/Vx1 and F359Vx1) in CD34+ cells from 21/36 patients (58%). In comparison, 190 assays of TWBC identified 10 (5%) mutations (T315Ix3, Y253Hx6, E255Vx1, p=0.0005) in 8/38 patients (21%, p=0.001 Fishers exact test). Significantly more T315I (33%) and E255K (22%) mutations were observed in CD34+ cells than in TWBC (8%, p=0.007 and 0% p= 0.003 respectively). The quantitative levels of all mutant alleles were median 0.135 (range 0.06–0.535) and 0.1 (range 0.04-0, 25) BCR-ABLmutant/ BCR-ABLunmutated for mutations in CD34+ cells and TWBC, respectively and were not significantly different. Where both CD34+ and TWBC were available from the same patient (n=23), 11 patients showed a total of 18 mutations in the CD34+ fraction but only one of these mutations was confirmed in TWBC. One additional mutation was detectable in the TWBC. The remaining 12 patients with no detectable mutation in the CD34+ fraction showed 3 mutations (2x Y253H, T315I) in 2 patients in TWBC only. In conclusion, LLM with either no (T315I) or intermediate (Y253H, E255K/V, F359V) sensitivity to nilotinib are detectable in CP CML patients at a frequency of 21% in the TWBC but with a significantly higher frequency of 58% in the enriched CD34+ progenitor cell compartment. Longterm patient follow up on the ENEST1st and ENESTobserve studies will allow analysis of the relationship between LLM and clinical outcomes on nilotinib. Disclosures: Hochhaus: Novartis, BMS, MSD, Ariad, Pfizer: Consultancy Other, Honoraria, Research Funding. Frank:Novartis: Employment. Lange:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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