Highly Sensitive Mutations Detection in BCR-ABL Positive Leukemia Prior and during Imatinib Treatment.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1985-1985
Author(s):  
Giovanni Cazzaniga ◽  
Barbara Corradi ◽  
Rocco Piazza ◽  
Simona Soverini ◽  
Giovanni Martinelli ◽  
...  
Keyword(s):  
Point Mutation ◽  
Mutation Detection ◽  
Point Mutations ◽  
Kinase Domain ◽  
Imatinib Therapy ◽  
Imatinib Treatment ◽  
Time Points ◽  
Abl Kinase ◽  
Mining Tool

Abstract The most frequently identified mechanism of acquired Imatinib resistance in Ph+ leukemia is BCR-ABL kinase domain point mutations. The observation that 90% of patients with mutations eventually relapsed suggests that harboring any BCR-ABL mutation has prognostic value. Thus, the detection of BCR-ABL mutants prior to and during the course of Imatinib therapy may aid in determining therapeutic strategies. However, current methods for mutation screening have either low sensitivity or are too time consuming and labor intensive to be routinely feasible. In addition, to date approximately 30 different point mutations in the BCR-ABL kinase domain have been isolated from Ph+ patients resistant to Imatinib treatment. This may require a method of mutation detection at large spectrum. We tested the feasibility of using the “SNP mining tool” on the Nanogen microarray device (San Diego, CA) for a rapid and sensitive identification of BCR/ABL mutations. The Nanogen SNP mining tool allows de novo discovery of genetic variations, including SNPs and mutations. The method combines the features of electronically controlled DNA hybridization on open-format microarrays, with mutation detection by a fluorescence-labeled mismatch-binding protein (mutS protein). If the test DNA contains a point mutation when compared to the reference DNA, the heteroduplex will contain a mismatch. This mismatch is detectable by the fluorescent-labeled mutS protein that binds to DNA containing a mismatch at any position of the DNA sequence. Since 500bp DNA fragments can be analyzed simultaneously, the entire ABL tyrosine kinase domain can be monitored by only two overlapping PCR reactions, thus substituting high throughput sequencing as a preliminary screening of point mutations. Electronic addressing of DNA strands to distinct test sites of the chip allows parallel analysis of several individuals or several SNPs/mutations in few individuals. We first performed a preliminary series of experiments on samples known to contain mutations by sequencing of PCR clones. A point mutation in ABL could be identified in a 10 copies dilution of a mutated plasmid. Different point mutations in ABL could be identified in 6 patients in which at least 1 mutated clone was found by PCR, cloning and sequencing. Four patients resistant to Imatinib were tested in 10-fold cDNA serial dilutions. A positive value was found till a 10−4 dilution in 3/4 patients. A sensitivity of 10−3 was found in 1 patient. We also monitored a single CML p210-positive patient in late chronic phase, resistant to IFN and treated with 400mg/die Imatinib. In this patient, always positive by RT-PCR of BCR-ABL, we could demonstrate the presence of the mutation prior to Imatinib and in all follow up time points during Imatinib therapy, since 1 month to 6 months. The follow up of additional Ph+ cases will be analyzed. In conclusion, the sensitivity and the short time required for the analyses of different patients samples, or multiple time points during follow-up, make the Nanogen device as an alternative to DHPLC or cloning, for the rapid identification of BCR/ABL mutations and monitoring of BCR/ABL mutations during Imatinib treatment. Early diagnosis of resistance and prompt treatment with appropriate drug dosing may be essential for increasing the success of cure in Ph+ leukemia.

Clinical Outcome Following Change of Tyrosine Kinase Inhibitor (TKI) According to the Detection of an ABL Kinase Mutation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4557-4557
Author(s):  
Renuka Palani ◽  
Richard M Szydlo ◽  
Jane F. Apperley ◽  
Gareth Gerrard ◽  
Chrissy Giles ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Mutation Detection ◽  
Point Mutations ◽  
Kinase Domain ◽  
Second Line ◽  
Abl Kinase ◽  
T315i Mutation ◽  
Kinase Mutation

Abstract Introduction Point mutations in the kinase domain of BCR-ABL are the most frequent mechanism of acquired imatinib resistance in patients with chronic myeloid leukemia (CML). Mutation analysis is recommended to guide the selection of appropriate second line therapy in patients with imatinib failure, since some frequently occurring mutations confer clinical resistance to nilotinib and/or dasatinib. To date, more than 80 point mutations have been described following imatinib exposure, but mutations at 7 sites (G250, Y253, E255, T315, M351, F359, and H396) comprise approximately 60% of mutations reported in large series. We retrospectively analysed the impact of 83 ABL kinase mutations (P-loop mutations = 28, T315I mutation = 12 and other mutations = 43) arising in 65 chronic phase (CP) CML patients with imatinib failure. The aim of this study was to define the clinical characteristics of these patients, and to assess their outcome following introduction of second line agents. Methods Between July 2002 and August 2013, 123 CML patients were found to have ABL kinase mutations in our centre. Patients presenting in blast crisis (BC) or accelerated phase (AP), and those who did not require change in therapy (including patients who required imatinib dose escalation) following detection of an ABL kinase mutation were excluded from the analysis. Sixty-five patients in CP who had imatinib failure and detectable ABL kinase mutation, and who required change in therapy were evaluated. Definitions of CML phases, treatment responses and failures were as per definitions of the European LeukaemiaNet. Direct sequencing method was used to detect a range of mutations within the tyrosine kinase domain at the level of ~20% sensitivity and pyrosequencing to detect specific mutations with a sensitivity of ~5%. Results Eighty-three ABL kinase mutations were detected in 65 CP patients at the time of imatinib failure with 35% of patients (23 of 65) harbouring P-loop mutations (including M244V), 18% (12 of 65) with T315I mutation and 46% (30 of 65) with other mutations (catalytic domain, imatinib binding site, activation loop and C-terminal). Composite mutations were present in 10 patients (15%), with 2 patients harbouring both P-loop and T315I mutations. Median time on imatinib therapy was 29.5 months (range, 2-144 months). At the time of mutation detection, 20% of patients (13 of 65) were in CCyR, 54% (35 of 65) in CP, 17% (11 of 65) in AP and 9% (6 of 65) had progressed to BC. Median time from CML diagnosis to mutation detection was 21 months for patients with T315I mutation, 45.5 months for P-loop mutations and 48 months for other mutations. Following mutation detection, patients in CP and AP were treated on second-line agents with dasatinib, nilotinib, bosutinib or ponatinib (based on sensitivity of ABL kinase mutants to ABL kinase inhibitors), and those in BC were treated with chemotherapy +/- TKI. Thirteen patients (20%) underwent allogeneic stem cell transplantation (SCT) for disease control (P-loop = 7, T315I = 5, other = 1). Following the change in TKI therapy, the best response was CMR or MMR in 54% of patients (35 of 65), CCyR in 12% (8 of 65), MCyR in 3% (2 of 65) and CHR in 26% (17 of 65). 5% (3 of 65) progressed to BC. In those who only achieved CHR, MCyR or developed progressive disease despite change in TKI, 6 out of 22 patients were found to have additional mutations during their treatment course, including 5 patients with re-emergence of their original mutation. After a median follow-up of 60 months (range, 3-137 months) from detection of mutation, 12 of the 65 patients (18%) have died, including 6 of 23 (26%) with P-loop mutations, 3 of 12 (25%) with T315I mutation, and 3 of 30 (10%) with other mutations. One patient who died had composite P-loop and T315I mutations. Median overall survival was 250 months for patients harbouring P-loop mutations (P=0.37) and not reached for T315I and other mutations. Conclusion With longer follow-up and the availability of second and third generation TKIs, we have demonstrated that most clinically relevant ABL kinase mutations respond to change in TKI therapy following imatinib failure, with the majority of patients achieving durable cytogenetic and molecular response. This study emphasizes the importance of early detection and characterization of ABL kinase mutations in imatinib resistant patients in order to identify those patients who may benefit from alternative TKI therapy or stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Complete Molecular Response due to nilotinib as III line treatment in a patient with CML and F317L point mutation of the Bcr-Abl kinase domain

2015 ◽  
Vol 5 (5S) ◽  
pp. 21-25
Author(s):  
Maria Iovine ◽  
Giuseppe Monaco ◽  
Mario Troiano ◽  
Antonio Abbadessa
Keyword(s):  
Point Mutation ◽  
Kinase Inhibitors ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Cytogenetic Response ◽  
Kinase Domain ◽  
Molecular Response ◽  
Abl Kinase ◽  
First Line Therapy ◽  
Complete Molecular Response

In 1997, a forty-three years old woman was diagnosed with CML and treated with alfa-Interferon, achieving complete haematological response (CHR). Three years later, patient was switched to hydroxiurea due to thyroid toxicity. For logistic reasons, therapy with imatinib 400 mg/die was initiated only in 2003, obtaining complete cytogenetic response (CCyR) and suboptimal molecular response in twelve months. CCyR and CHR were then lost three years later. Doubling imatinib dose to 800 mg/die gave no positive results. Mutational analysis performed in September 2007 showed F317L point mutation of the Bcr-Abl kinase domain. In October 2007 dasatinib was started and in April 2008 CCyR was reached with suboptimal molecular response. In March 2009 Bcr-Abl transcript progressively increased, and in August 2009 cytogenetic analysis showed loss of CCyR. Therapy with nilotinib 800 mg/die was started, and in October 2009 the patient obtained complete molecular response (CMR). Bcr-Abl kinase-domain point mutations, acquired during first line therapy, are a common cause of resistance to tyrosine kinase inhibitors. While several Bcr-Abl mutations have been identified, involvement of codon 317 has been reported in the literature following treatment with imatinib and dasatinib.

Examination of Point Mutations within the ABL Kinase Domain of Bcr-Abl Fusion Gene in Chinese Patients with Chronic Myeloid Leukemia Who Develop Imatinib Resistance.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4573-4573
Author(s):  
Dongguang Yang ◽  
Ri Zhang ◽  
Honglin Gang ◽  
Jian Gu ◽  
Jian Nong Cen
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Point Mutation ◽  
Mutation Rate ◽  
Myeloid Leukemia ◽  
Fusion Gene ◽  
Point Mutations ◽  
Kinase Domain ◽  
Chinese Patients ◽  
Single Mutation ◽  
Abl Kinase

Abstract Objective: To investigate the point mutations within the ABL kinase domain (KD) of Bcr-Abl fusion gene in Chinese patients with chronic myeloid leukemia who develop IM resistance and investigate the mutation. Method: We collect a total of 17 bone marrow samples obtained from 11 patients who showed hematology resistance(n=7) or cytogenetic refractoriness(n=4). A long semi-nest PCR method was used to amplify the ABL KD of the Bcr-Abl fusion gene. After two cycles of PCR reaction, we get a fragment including 863 bases, purified and sequenced it. Result: The ABL point mutation was detected in 45.45% of patients. The mutation rate of hematology resistance is 57.14% and the mutation rate of cytogenetic refractoriness is 25%. In total, we find three point mutation in all of our patients tested, ie, G250E(2), E255K(1) and T315I(2). In patients who develop hematology resistance, the earliest time to detect mutation is 4 months later after the therapy of IM and the longest time to detect mutation is 9 months later after the therapy of IM. In patients who develop cytogenetic refractoriness, the time to detect mutation is 12 months later after the therapy of IM .For the patients whose samples are available, no single mutation happens before IM thearpy. Conclusion: There is high frequency of point mutations clustered within the adenosine triphosphate-binding region of Bcr-Abl fusion gene in Chinese patients with chronic myeloid leukemia, and the mutation is related to IM therapy. It’s good for patients to switch to another therapeutic strategy when the mutations were detected earlier. Figure Figure

Mutation Analysis of BCR-ABL Tyrosine Kinase Domain In New Chronic Phase-Chronic Myeloid Leukemia Patients with Suboptimal Response or Treatment Failure From Imatinib Treatment.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3441-3441
Author(s):  
Dong-Wook Kim ◽  
Dongho Kim ◽  
Soo-Hyun Kim ◽  
Hyun-Gyung Goh ◽  
Fabrizio Pane ◽  
...  
Keyword(s):  
Treatment Failure ◽  
Board Of Directors ◽  
Kinase Domain ◽  
Imatinib Treatment ◽  
Advisory Committees ◽  
Mutation Status ◽  
Optimal Response ◽  
Failure Group ◽  
Time Points ◽  
Abl Kinase

Abstract Abstract 3441 Resistance to imatinib can occur in chronic myeloid leukemia (CML). Mutations in BCR-ABL kinase domain have been known as the clinically most relevant mechanisms of imatinib resistance. Several studies have shown that 40~80% of the imatinib resistant patients have BCR-ABL kinase domain mutations. However, there has not been much information about mutation status in CML patients with suboptimal response to imatinib. With samples from Tyrosine Kinase Inhibitor Optimization and Selectivity (TOPS) study which investigates efficacy of imatinib 400 mg daily and 800 mg daily with Philadelphia positive CML CP patients, and relationship between various responses and emergence of mutation was investigated by both standard and highly sensitive mutation assays. As a clinical correlative study (CCS) program with samples from TOPS study, we analyzed BCR-ABL mutation from 51 patients with optimal response, suboptimal response or treatment failure to imatinib in order to investigate their mutation status at different time points including diagnosis, 6 months, 12 months and 18 months. In addition, 53 non-clinical trial patients from Seoul St. Mary's hospital were involved in this analysis. All patients were in CP. Suboptimal responders and treatment failures were selected based on European Leukemia Net (ELN) 2009 guideline. CML patients' samples from the TOPS study and Seoul St. Mary's hospital were collected at particular time points after initiation of imatinib treatment and stored as cryopreserved cells or isolated RNAs. Mutations in BCR-ABL kinase domain were analyzed using direct sequencing and allele-specific oligonucleotide (ASO)-PCR (for Y253H, Y253F, G250E, E255K, E255V, T315I, F359V, and M351T). We performed mutation analysis with total 164 samples collected from 104 patients at different time points including diagnosis, 6 months, 12 months and 18 months after initiation of imatinib treatment. Serially collected samples at all time points were not available for all the patients; samples from 31 patients were available at diagnosis, 41 patients at 6 months, 58 patients at 12 months and 34 patients at 18 months (Table 1). We found ten BCR-ABL kinase domain point mutations including G250E, Q252H, Y253H, T315I, F317L, E355G, F359V, F359I and D444Y in 13 patients. In addition, we also found other mutations including 35 base pair insertion between exon 8 and exon 9 of ABL, and deletion of exon 7 of ABL in other 10 patients. No mutation was found from the patients' samples collected at diagnosis. At 6 months, mutation was found 5% (1 of 21), 18% (2 of 11) and 22% (2 of 9) patients in optimal response, suboptimal response and treatment failure group, respectively. ASO-PCR revealed that one patient in optimal response group had T315I. The same mutation status of the patient maintained at 12 months and the patients showed treatment failure at 12 months. At 12 months, mutation portion was 0% (0 of 15), 13% (2 of 15) and 25% (7 of 28) in optimal response, suboptimal response and treatment failure group, respectively. At 18 months, 36% (5 of 14) of suboptimal molecular responders who achieved CCyR, but no MMR showed mutation, and 62% (8 of 13) of failure group who showed less than CCyR had mutations. No particular difference in mutation frequency was found between 400mg group and 800mg group. Patients with suboptimal response or treatment failure showed much higher chance of BCR-ABL point mutation, 35 base pair insertion or exon 7 deletion in comparison with optimal responders, suggesting that mutation screening is important for patients with suboptimal response as well as treatment failure on the basis of ELN guideline. Treatment failure who achieved less than CCyR at 18 months and suboptimal responders who achieved CCyR, but no MMR at 18 months were highly recommended for mutation screening based on these data. Highly sensitive ASO-PCR provided early detection of point mutation in BCR-ABL kinase domain. However, clinical relevance of low level mutant clone, 35 base pair insertion and exon 7 deletion require long-term follow up for better understanding. Table 1. Response Diagnosis 6 months 12 months 18 months Pt with mutation Total patients Pt with mutation Total patients Pt with mutation Total patients Pt with mutation Total patients Optimal response 0 31 1 (5%) 21 0 (0%) 15 0 (0%) 7 Suboptimal response 2 (18%) 11 2 (13%) 15 5 (36%) 14 Failure 2 (22%) 9 7 (25%) 28 8 (62%) 13 Total Patients 31 41 58 34 Disclosures: Pane: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees. Saglio:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hochhaus:Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Outcome of Patients with Philadelphia Positive Chronic Myeloid Leukemia Post Imatinib Failure: Prognostic Significance of ABL Mutation and Additional Clonal Abnormality.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4673-4673 ◽  
Author(s):  
Monika Conchon ◽  
Patricia B Ferreira ◽  
Mafalda MY Novaes ◽  
Luciana Nardinelli ◽  
Mariana Serpa ◽  
...  
Keyword(s):  
Overall Survival ◽  
Chronic Myeloid Leukemia ◽  
Point Mutation ◽  
Myeloid Leukemia ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Fish Analysis ◽  
Imatinib Treatment ◽  
Abl Kinase ◽  
Clinical Resistance

Abstract Abstract 4673 Point mutation within the ABL kinase domain of the BCR-ABL gene has been associated with clinical resistance to imatinib in chronic myeloid leukemia (CML). However, several other mechanisms have been proposed to underlie the development of imatinib resistance such overexpression and amplification of the BCR-ABL gene locus; development of additional chromosomal abnormalities (ACA), activation of BCR-ABL independent mechanisms; increased drug efflux through multidrug resistance gene and binding of imatinib to serum alfa-1 acid glycoprotein. In this regard, we report the outcome of 54 patients Ph+ CML who were resistant to imatinibe and the association with point mutation, ACA and overexpression of BCR-ABL through in situ hybridization (FISH). Patients and Methods Of 54 CML patients, 16 were in early chronic phase (ECP), 24 in late chronic phase (LCP) and 14 in accelerated phase (AP) before imatinib treatment. They were considered imatinib resistant for the following reasons: hematologic resistant or recurrence (7 patients), cytogenetic resistance or recurrence (30 patients) or progression to more advanced phase (17 patients). Mutational analysis was carrying out by direct sequencing and FISH analysis using commercial BCR/ABL t(9;22), dual color, dual fusion probe (Kreatech, Poseidon, The Netherlands). Subsequent therapy was also analyzed, as to whether they underwent to allogeneic SCT, chemotherapy, or received, dasatinib or nilotinib. Results A total of 24 BCR-ABL kinase domain mutations were detected in 54 patients. Two patients had more than 1 mutation. The most frequent mutations were F359V (6/24), T315I (5/24) and F317L (3/24). P-loop mutations accounted for 60% of the remaining amino acid substitutions. Twelve patients were treated with nilotinib 400 mg bid, 50% presented mutation at resistance (3 F359V, 1 Q252H, 1 E355G, and 1 T315I), and all alive but one with T315I mutation died after 1 month. Dasatinib, 100-140 mg daily, was dispensed for 37 patients, mutations were identified in 40%, where 10 patients are still alive (2 T315I, 2 F317L, and 6 others). Allogeneic SCT was performed in 2 patients (1 G250E), and both died. Two patients died (1 F359V) after systemic chemotherapy and 1 patient who received 800 mg of IM is in CCyR after 40 months. There was no difference in overall survival when mutation was present or not at the moment of resistance (log rank, p=0.9). We also compared the karyotype tacked before imatinib treatment with the karyotype performed at the time of resistance; 17 patients developed ACA and were associated with worse overall survival (log rank, p=0,046) when compared with those patients without ACA. FISH analysis did not identify overexpression of BCR-ABL, but 4 cases presented 3 signals for BCR-ABL indeed had an extra Ph chromosome. Conclusions Second-generation TK inhibitors improve overall survival of those patients who presented mutations at resistance. The presence of ACA during imatinib treatment was related with a higher mortality despite the treatment applied and the presence or not of mutations. We also conclude that overexpression of BCR-ABL was not responsible for resistance to imatinib in our study population. Disclosures: No relevant conflicts of interest to declare.

Dynamics and Characteristics of BCR-ABL Multiple Mutations In Tyrosine Kinase Inhibitor Resistant Chronic Myeloid Leukemia.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3443-3443
Author(s):  
Dong-Wook Kim ◽  
Dongho Kim ◽  
Soo-Hyun Kim ◽  
Saengsuree Jootar ◽  
Hyun-Gyung Goh ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Point Mutation ◽  
Clinical Relevance ◽  
Kinase Inhibitors ◽  
Direct Sequencing ◽  
Point Mutations ◽  
Kinase Domain ◽  
Abl Kinase ◽  
Multiple Mutation ◽  
Compound Mutation

Abstract Abstract 3443 BCR-ABL kinase domain (KD) point mutation causes resistance to tyrosine kinase inhibitors (TKI) in CML patients through impaired binding of TKI to the target site. One of the characteristics of patients with BCR-ABL kinase domain point mutations is the fact that some patients have multiple mutations. However there have not been many studies showing that data about clinical relevance or dynamics of multiple mutation during CML treatment. From January 2002 to June 2010 at Seoul St Mary's Hospital, 277 CML patients were screened for mutation analysis due to sign of resistance to tyrosine kinase inhibitors including imatinib, nilotinib, dasatinib or bosutinib. We found that 95 patients have point mutation in BCR-ABL kinase domain through direct sequencing or ASO-PCR. Among them, 17 patients showed multiple mutation containing more than one type of point mutations in BCR-ABL KD. We investigated the patients with multiple mutations to characterize its clinical relevance and dynamics. Once mutation found, follow-up samples from the corresponding patients were collected and analyzed prospectively, or mutation status was analyzed retrospectively with cryopreserved samples if they were available. Status of the patients with multiple mutation is shown in Table 1. In order to investigate whether the multiple mutations are on same clone or on separated clone, we cloned serial samples from the 17 patients. Cloning of cDNA region corresponding to BCR-ABL KD into plasmid was performed and followed by transformation into competent cells, colony formation, plasmid preparation of 20 colonies from each sample, and then direct sequencing. Multiple mutations of 88% patients (15 out of 17) existed compound mutation which means the individual mutant types are located on the same BCR-ABL molecule. In addition of major mutation types which were detectable in direct sequencing analysis, all the patients showed to have minor types of mutations which were found only through BCR-ABL KD cloning and subsequent colony sequencing. To make sure that this minor mutation types were not caused by sequencing error, we also analyzed of 3 patients who showed TKI resistance, but had no BCR-ABL mutation. In addition, samples from 3 normal persons were analyzed with the same method. The frequency of appearance of the minor types of point mutation was reduced in the patient group who showed TKI resistance, but had no BCR-ABL mutation, and then dramatically decreased in the normal person group, indicating that BCR-ABL gene in patients with point mutation are relatively unstable. Analysis of serial samples from a same patient provided evidence of dynamic change of portion of compound mutation. In most case, portion of the clone containing compound mutation was increased as treatment went on, indicating the clone harboring compound mutation can take survival advantage over TKI treatment in comparison of the clone containing individual type of mutation. In addition, some patients showed change in individual mutation type comprising multiple mutation as treatment went on. Currently investigation of clinical relevance of compound mutation and other analyses are being carried on and more results will be provided in detail at the conference. Table 1. Patients Tx at mutation detection (mg) Compound type Compound % 1 Nilotinib400 G250E+T315I 6.7 G250E+D444G 33.3 T315I+D444G 6.7 2 Nilotinib400 M244V+T315I 95.0 3 Dasatinib100 Y253H+T315I 95.0 4 Dasatinib140 T315I+E459K 55.6 5 Dasatinib200 T315I+M351T 66.7 6 Dasatinib100 NCM Dasatinib80 NCM Dasatinib100 M244V+F359V 16.7 7 Bosutinib500 NCM 8 Dasatinib140 T315I+F359C 35.3 9 Imatinib400 E255K+T315I 5.6 10 Dasatinib80 E255V+T315I 90.0 11 Imatinib800 E255K+T315I 10.5 12 Nilotinib800 E255K+T315I 12.5 13 Dasatinib100 F311I+T315I 35.0 F311I+F317Lb 10.0 Imatinib400 F311I+T315I 10.0 F311I+F317La 15.0 F311I+F317Lb 55.0 14 Nilotinib800 Y253H+F359I 5.6 15 Bosutinib500 V299L+E459K 95.0 Nilotinib400 + Dasatinib100 V299L+F359I 5.0 V299L+E459K 55.0 V299L+F317La+E459K 15.0 V299L+F359I+E459K 15.0 V299L+F317La+F359I+E459K 5.0 16 Imatinib600 NCM 17 Imatinib400 NCM NCM: no compound mutation. Disclosures: No relevant conflicts of interest to declare.

Successful treatment of progressive cutaneous mastocytosis with imatinib in a 2-year-old boy carrying a somatic KIT mutation

Blood ◽  
2008 ◽  
Vol 112 (5) ◽  
pp. 1655-1657 ◽  
Author(s):  
Karl M. Hoffmann ◽  
Andrea Moser ◽  
Peter Lohse ◽  
Andreas Winkler ◽  
Barbara Binder ◽  
...  
Keyword(s):  
Germ Line ◽  
Systemic Mastocytosis ◽  
Therapeutic Option ◽  
Kinase Domain ◽  
Imatinib Therapy ◽  
Imatinib Treatment ◽  
Kit Mutation ◽  
Systemic Involvement ◽  
Kit Gene ◽  
Cutaneous Mastocytosis

Abstract Cutaneous mastocytosis (CM) in children is a usually benign skin disorder caused by mast cell proliferation. Progressive disease leading to systemic involvement and fatal outcomes has been described. C-kit receptor mutations have been identified as causative for CM, some of which potentially respond to imatinib treatment as described for patients with systemic mastocytosis. We report successful therapy of progressive CM with imatinib in a 23-month-old boy. KIT gene analysis revealed not only a somatic deletion of codon 419 in exon 8 (c.1255_1257delGAC) which responds to imatinib therapy, but also a novel germ line p. Ser840Asn substitution encoded by exon 18 in the c-kit kinase domain. Family history suggests this exchange does not affect receptor function or cause disease. Imatinib therapy was well tolerated, stopped symptoms and disease progression, and appeared to shorten the course of the disease. Imatinib could possibly represent a novel therapeutic option in patients with progressive CM.

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.

High Sensitive Detection of Imatinib-Resistance Associated Mutations Based on ARMS-PCR.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2943-2943
Author(s):  
Franz X.E. Gruber ◽  
Mikchail Soevershaev ◽  
Marita Olsen ◽  
Bjoern Skogen
Keyword(s):  
Kinase Inhibitors ◽  
Point Mutations ◽  
High Sensitivity ◽  
Kinase Domain ◽  
Single Step ◽  
Sensitive Detection ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Loop Region ◽  
Highly Sensitive Detection

Abstract Background: Point mutations in the Abl kinase domain are associated with resistance against imatinib. Strategies to overcome resistance include dose escalation, combination treatment using imatinib with conventional or other developmental agents or, in the future, imatinib may be replaced by other tyrosine kinase inhibitors which work effectively against mutated clones. Mutational profiling of the BCR-ABL kinase domain will in this scenario become an important analysis as a supplement to BCR-ABL quantitation and may provide the rational basis for therapy, once resistance is diagnosed. Our group reported recently a sensitive, single step PCR assay for quantitation of mutated clones based on the ARMS principle. Aim: We describe an optimized, two step analysis for high sensitivity screening of mutated clones associated with resistance against imatinib targeting all P-Loop mutations, the T315I and M351T. Methods: In a first conventional PCR-reaction a cDNA-region spanning the BCR-ABL breakpoint is amplified resulting in an isolation of the BCR-ABL kinase domain for further analysis. An aliquot is then analysed in a second PCR step, conducted on the real time PCR Taqman platform. Selectivity for the mutated clone is conferred by the amplification refractoriness of non complementary primer 3′-ends (ARMS principle). By introducing potent nucleotide-mismatches in position n-2, selectivity of the assay could be further increased. Even in the P-Loop region, which is known to be a difficult PCR template, misannealing could be reduced to an acceptable level. Results: Assays targeting all P-Loop mutations inclusive the T315I and M351T were tested by analysis of patient samples diluted in normal cDNA and non-mutated BCR-ABL and plasmid dilutions, containing the targeted mutation in a background of wildtype plasmids. Generally a 1:1000 dilution of mutated templates could be detected (sensitivity 0.1%). For some mutations even higher sensitivity could be achieved (0.01%). The level of sensitivity is generally higher than reported for other methods described before. The first PCR step can be conducted in parallel to other PCR-based detection strategies. The second step can be run simultanously to Taqman based BCR-ABL quantitation. This makes the described assay the ideal supplement to general mutation detection approaches like D-HPLC or sequencing strategies. Compared to the single step assay we desribed before, the two step approach increases sensitivity with one or two log factors. Conclusion: The described assay may be suitable for highly sensitive detection of mutated clones in resistant CML patients as a supplement to less sensitive general screening approaches and BCR-ABL quantitation.

Leukemic Stem Cells of Chronic Phase CML Patients Possess Uniquely Elevated BCR-ABL Kinase Activity and Acquire Spontaneous BCR-ABL Kinase Domain Mutations at a High Frequency.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 438-438 ◽  
Author(s):  
Xiaoyan Jiang ◽  
Kyi Min Saw ◽  
Allen Eaves ◽  
Connie Eaves
Keyword(s):  
Stem Cells ◽  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Point Mutations ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Tyrosine Kinase Activity ◽  
Abl Kinase ◽  
Kinase Domain Mutations

Abstract Growing evidence indicates that the therapeutic potential of imatinib mesylate (IM) for the treatment of CML may be limited initially by a relative innate resistance of the leukemic stem cells and eventually by an accumulation of cells with BCR-ABL tyrosine kinase domain mutations. We now show that the amount and tyrosine kinase activity of p210-BCR-ABL in the most primitive and relatively IM-unresponsive lin−CD34+CD38− CML cells is 3 to 10-fold higher than in the majority of the lin−CD34+CD38+ CML progenitors (n=3). These results confirm previous BCR-ABL transcript data and identify elevated p210-BCR-ABL expression to be a likely important factor in the characteristic IM-insensitivity of very primitive CML cells. To determine whether in vivo, CML stem cells also accumulate gene mutations affecting the BCR-ABL kinase domain, cDNAs were prepared from RNA extracts of purified lin−CD34+CD38− cells isolated from 3 chronic phase patients that had not received IM therapy. Bidirectional sequencing of individually cloned cDNAs from these samples revealed BCR-ABL kinase domain mutations in 2 of the 3 patients at frequencies of 10% (1/10), 20% (2*/10,*identical mutations). Incubation of these lin−CD34+CD38− cells in vitro for 2–3 wk ± a high concentration of IM (up to 10 μM, which was sufficient to reduce the tyrosine kinase activity in the input cells by 70±12% and in their 2 wk progeny by 10±5%) selected a subpopulation of more differentiated and completely IM-resistant cells. This was shown in Western blots by the inability of 10 μM IM to reduce either their p210-BCR-ABL tyrosine kinase activity or CrkL phosphorylation and in methylcellulose assays ±5 μM IM. As predicted, IM-selected cells showed a higher frequency of kinase domain mutations (13–20% vs 0–20% of cDNA clones analyzed from 3 wk cells cultured ±IM). Analysis of individual colonies produced from CFCs in the cultured cells showed all (21/21) colonies from IM-selected cells had mutations vs 50% (5/10) in those cultured without IM. The total frequency of mutant cDNAs detected was also increased in the IM-resistant cells (35–55% vs 10–25% mutant cDNAs in selected vs control cells). Interestingly, in most cases, both wild-type and mutant cDNAs were identified in the same colony, indicating de novo generation of mutations in vitro. Overall, >50 different mutations were identified. These included 10 point mutations previously associated with clinical IM resistance (including G250 and T315), another 13 point mutations previously identified in a comprehensive mutational screen, and >20 previously undescribed mutations. Several of the latter affect the critical region of the P loop, the c-helix and the activation loop and would be predicted to confer significant IM resistance. To investigate the possibility that the observed genomic instability of very primitive CML cells might be related to their elevated innate p210-BCR-ABL activity, BCR-ABL transcript levels in individual IM-selected, fully resistant and control (similarly treated but no IM exposure) colonies were compared. This showed that BCR-ABL transcripts were ~20-fold higher (P<0.05) in the resistant colonies (30 assessed from 3 patients). These findings suggest that the increased BCR-ABL expression and activity that uniquely characterizes the most primitive CML cells may contribute not only to their innate insensitivity to IM but also to a deregulation of genomic stability leading to the emergence of IM-resistant mutants and other subclones associated with disease progression.

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