Mutational Analysis of Chronic Myeloid Leukemia (CML) Clones Reveals Heightened BCR-ABL1 Genetic Instability and Wild-Type BCR-ABL1 Exhaustion in Patients Failing Sequential Imatinib and Dasatinib Therapy.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1938-1938 ◽  
Author(s):  
Alfonso Quintás-Cardama ◽  
Don L. Gibbons ◽  
Hagop Kantarjian ◽  
Moshe Talpaz ◽  
Nick Donato ◽  
...  
Keyword(s):  
Amino Acid ◽  
Genetic Instability ◽  
Kinase Inhibitors ◽  
Mutational Analysis ◽  
Direct Sequencing ◽  
Cytogenetic Response ◽  
Kinase Domain ◽  
Wild Type ◽  
Abl Kinase ◽  
Tki Resistance

Abstract ABL kinase domain (AKD) mutations are the main mechanism of resistance in patients (pts) with CML who fail tyrosine kinase inhibitors (TKIs) therapy, being found in 20%–40% of cases by direct sequencing (DS). Therefore, many pts fail TKI therapy for unknown reasons. We evaluated the development of AKD mutations among 61 CML pts after imatinib-intolerance (n=10) or -resistance (n=51) enrolled in a phase I study of dasatinib by DS of nested PCR-amplified BCR-ABL1 products as well as by DNA expansion of specific clones (DESC) followed by DNA sequencing of at least 10 clones. Prior to imatinib (400 mg daily in 47, 600 in 13, and 800 in 1), 54 pts were in chronic (CP), 2 in accelerated (AP), and 5 in blastic (BP) phase. At the end of imatinib therapy, 26 pts were in CP, 14 in AP, and 21 in BP. AKD mutations (in ≥1 out of 10 sequenced clones) were detected in 58/61 (95%) pts by DESC (4 pts with wild type [WT] BCR-ABL1) but only in 23/55 (42%) by DS. Overall, 118 AKD mutations at 112 amino acid positions were detected by DESC, of which 77 had never been previously reported. Mutations conferring resistance to >1μM imatinib (M244V, G250E, Q252H, Y253H, E255K/V, F359V, H396R, and T315I) were detected in 20 (34%) pts by DESC, but only in 5 (8%) by DS. Combinations of mutations within the same clone (polymutants) were detected in 33/58 (57%) pts by DESC, with clones expressing 2 (n=41), 3 (n=11), 4 (n=1), or even 5 (n=2) distinct mutations. By contrast, only 1 pt was found to carry 2 different mutations (M244V and M351T) by DS. Dasatinib was subsequently given to 56/61 (92%) pts (53 evaluable for response) for a median of 17 months (range, 1 to 48). DESC available in 15 pts during dasatinib therapy revealed 16 additional mutations (15 amino acid positions), including 5 previously not reported (all in polymutants). Dasatinib-resistant mutations (L248V/R, Q252H, E255K, V299L, T315I/A, and F317L/C/I/S/V) were detected in 7/15 (47%) cases (2 with T315I) by DESC but only in 2/15 (13%) by DS. Of these 15 pts, only 3 (1 CP, 1 AP, and 1 BP) are alive. The proportion of clones harboring WT BCR-ABL1 prior to and during dasatinib therapy decreased significantly (p=0.003), particularly in pts harboring highly dasatinib-resistant mutants. Notably, pts without cytogenetic (CG) response on dasatinib had a lower proportion of WT clones compared with those who achieved at least a partial cytogenetic response (p=0.02). AFTER IMATINIB ON DASATINIB Evaluable Patients No. Clones No. WT Clones (%) Evaluable Patients No. Clones No. WT Clones (%) DASATINIB RESPONSE 53 598 268 (48) 15 130 26 (20) No CG Response 25 242 128 (49) 7 58 5 (9) CG Response Minor 10 79 33 (42) 4 35 9 (26) Partial 6 57 35 (61) 2 18 8 (44) Complete 12 123 46 (37) 2 19 4 (21) In summary, DESC greatly increases the sensitivity of detection of AKD mutants compared to DS and reveals heightened BCR-ABL1 genetic instability among pts failing TKIs, which could explain TKI resistance in pts not carrying resistant mutations by DS. This might be mediated by generation of resistant polymutant clones that perpetuate a “mutator phenotype” leading to WT BCR-ABL1 exhaustion.

Sequencing of Subcloned PCR Products Facilitates Earlier Detection of BCR-ABL1T315I Mutants Compared to Direct Sequencing of the ABL1 Kinase Domain.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1952-1952
Author(s):  
Alfonso Quintás-Cardama ◽  
Don L. Gibbons ◽  
Hagop Kantarjian ◽  
Moshe Talpaz ◽  
Nicholas Donato ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Catalytic Domain ◽  
Direct Sequencing ◽  
Cytogenetic Response ◽  
Median Number ◽  
Kinase Domain ◽  
Therapeutic Implications ◽  
Abl Kinase ◽  
Therapeutic Algorithms ◽  
Pcr Products

Abstract ABL kinase domain mutations represent the most frequent cause of resistance to tyrosine kinase inhibitors (TKIs). The BCR-ABL1T315I mutation affects a highly conserved “gatekeeper” threonine near the ABL catalytic domain, thus causing steric hindrance that precludes ABL TKIs binding. BCR-ABL1T315I retains kinase activity even in the presence of micromolar concentrations of imatinib or dasatinib. Thus, early detection of BCR-ABL1T315I has important prognostic and therapeutic implications. We evaluated the sensitivity of detection of BCR-ABL1T315I in 62 CML pts after failure (n=51) or intolerance (n=11) of imatinib enrolled in a phase I study of dasatinib by direct sequencing (DS) of nested PCR-amplified BCR-ABL1 products as well as by DNA expansion of specific clones (DESC) followed by DNA sequencing of at least 10 clones. Ten (15%) pts were found to carry BCR-ABL1T315I, 4 prior to dasatinib start and 6 during dasatinib therapy. Four pts never responded to imatinib whereas 5 had achieved a complete hematologic response (CHR) and 1 a complete cytogenetic response (CCyR). Imatinib was stopped due to rash (n=1), hematologic resistance (n=4), and progression to accelerated (AP; n=1) or blastic (BP; n=4) phase. Of the 4 pts in whom BCR-ABL1T315I was detected by DESC prior to dasatinib start, 2 are dead and 2 are alive. DS performed on the same samples detected BCR-ABL1T315I only in 2 of them. In pts in whom direct sequencing failed to detect BCR-ABL1T315I, the percentage of clones carrying BCR-ABL1T315I was 10% and 100%, respectively. In one of them, DS detected BCR-ABL1T315I 12 months later, whereas in the other case, DS failed to detect BCR-ABL1T315I in 3 separate occasions. These 2 pts are still alive (1 on hydrea, 1 on bosutinib) but never achieved any cytogenetic response. BCR-ABL1T315I was detected in 6 additional pts after a median time of 5 months (range, 1–6) on dasatinib (dosing ranging from 70 to 140 mg/d): 5 of 5 analyzed by DESC and 1 of 2 determined by DS. Of them, 4 had no response to dasatinib and 2 had transient cytogenetic responses (1 minor, 1 partial) and only one is still alive. In 1 that had no response, for whom paired samples were available, BCR-ABL1T315I was present in 10% of clones but was not detected by DS. Overall, the median number of clones harboring BCR-ABL1T315I was 90% (range, 10%–100%) and the median number of mutants co-expressed with BCR-ABL1T315I was 3 (range, 1–9). DS failed to identify 40 non-BCR-ABL1T315I mutants (including F317L in 1 patient), regardless of the percentage of clones in which they were expressed, except for E355G in 1 patient. Eight pts received dasatinib for more than 3 weeks (median, 5 months; range, 2–13) and were evaluable for response. Six failed to achieve any cytogenetic response and 2 had transient cytogenetic responses (1 minor and 1 partial). Seven (70%) pts died and 3 are alive with no cytogenetic response. In conclusion, DS has a poor sensitivity to detect ABL kinase mutations, particularly when the proportion of mutated clones is low. With the advent of novel T315I inhibitors, prompt detection of this highly-resistant mutation must be prioritized and included in therapeutic algorithms. To maximize the sensitivity of T315I detection, sequencing of subcloned PCR products might be preferable to DS.

Mutational Analysis of Chronic Phase Chronic Myeloid Leukemia (CMLCP) Clones Reveals Heightened BCR-ABL1 Genetic Instability in Patients Failing Sequential Imatinib and Dasatinib Therapy.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2114-2114 ◽  
Author(s):  
Alfonso QuintasCardama ◽  
Don L. Gibbons ◽  
Hagop M. Kantarjian ◽  
Moshe Talpaz ◽  
Nicholas Donato ◽  
...  
Keyword(s):  
Amino Acid ◽  
Kinase Inhibitors ◽  
Mutational Analysis ◽  
Direct Sequencing ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Imatinib Resistant ◽  
High Prevalence ◽  
High Incidence ◽  
Clinical Consequences

Abstract ABL1 kinase domain (AKD) mutations are the most important mechanism of resistance to tyrosine kinase inhibitors (TKIs) in CML. Direct sequencing (DS) techniques detect AKD mutations in 20%–40% of imatinib-resistant pts. Therefore, most pts fail TKI therapy for unknown reasons. We evaluated the incidence and clinical consequences of AKD mutations among 70 pts in CML-CP after imatinib failure (13 intolerant) enrolled in a phase I study of dasatinib. Mutations were studied by DS of nested PCR-amplified BCR-ABL1 products and by DNA expansion of specific clones (DESC) followed by DNA sequencing of ≥10 clones. Patients had received imatinib at 400 mg/d (n=60), 600 mg/d (n=8), or 800 mg/d (n=2). Prior to dasatinib, AKD mutations were detected in 61/70 (87%) pts by DESC, including 38 (54%) with mutations in ≥20% of sequenced clones. Mutations were only detected in 18/38 (47%) by DS. Overall, 125 mutations at 113 amino acid positions were detected by DESC (78 previously unreported). Mutations conferring resistance to >1μM imatinib (M244V, G250E, Q252H, Y253H, E255K/V, F359V, H396R, and T315I) were detected in 30 (43%) pts by DESC, but only in 5 (7%) by DS. Two or more mutations within the same clone (polymutants) were detected in 29/70 (41%) pts by DESC, with clones expressing 2 (n=38), 3 (n=11), 4 (n=1), or even 5 (n=2) distinct mutations. By contrast, only 1 pt was found to carry 2 different mutations (M244V and M351T) by DS. Pts received dasatinib for a median of 19 months (range, 2–52), of whom 68 (97%) are evaluable for response. DESC available in 32 pts during dasatinib therapy revealed 20 additional mutations not present at dasatinib start (19 amino acid positions), including 5 previously not reported (all in polymutant clones). Dasatinib-resistant mutations (L248V/R, Q252H, E255K, V299L, T315I/A, and F317L/C/I/S/V) were detected in 10/32 (31%) cases (5 with T315I) by DESC (but only in 3/16 [19%] by DS). Of these 16 pts 13 died (all in BP) and 3 are alive in CP carrying E255K, T315I, and F317L respectively. The percentage of clones with unmutated BCR-ABL1 before dasatinib decreased significantly compared to those present after a median of 16 wks (range, 4–84) during dasatinib (p=0.001), particularly in pts carrying highly dasatinib-resistant mutants. No differences were seen in the proportion of unmutated BCR-ABL1–expressing clones between pts with no cytogenetic (CG) response and those who achieved a partial (PCyR) or a complete CG (CCyR) response prior to dasatinib therapy. Conversely, DESC during dasatinib therapy showed the proportion of unmutated clones was lower among pts who failed to achieve a CG response compared to those who had a PCyR or CCyR (p=0.0001). AFTER IMATINIB ON DASATINIB No. Evaluable for Response to dasatinib No. clones sequenced No. unmutated Clones (%) No. Evaluable Pts No. clones sequenced No. unmutated Clones (%) DASATINIB RESPONSE 68 680 311 (46) 32 305 94 (30) No CG Response 30 309 149 (48) 13 120 17 (14) CG Response Minor 11 91 32 (35) 5 47 4 (8) Partial 7 69 38 (55) 3 28 13 (46) Complete 20 211 92 (43) 11 110 60 (55) In conclusion, DESC demonstrates a high prevalence of AKD mutations among pts who fail imatinib, revealing heightened BCR-ABL1 genomic instability in this setting. This high incidence of mutations might partly explain TKI resistance in pts found to carry unmutated BCR-ABL1 by DS. The latter might be mediated by generation of resistant polymutant clones that perpetuate a “mutator phenotype” and by exhaustion of clones carrying unmutated BCR-ABL1 alleles.

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.

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 ◽  
2011 ◽  
Vol 118 (5) ◽  
pp. 1208-1215 ◽  
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.

Mutations within BCR-ABL1 295–312 Define a Novel Region Associated with Poor Prognosis in Patients with Chronic Myelogenous Leukemia (CML) Resistant to Imatinib.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1936-1936
Author(s):  
Alfonso Quintás-Cardama ◽  
Don L. Gibbons ◽  
Hagop Kantarjian ◽  
Moshe Talpaz ◽  
Jorge Cortes ◽  
...  
Keyword(s):  
Amino Acid ◽  
Catalytic Domain ◽  
Kinase Inhibitor ◽  
Direct Sequencing ◽  
Amino Acid Position ◽  
Cytogenetic Response ◽  
Kinase Domain ◽  
Myelogenous Leukemia ◽  
Low Frequencies

Abstract ABL kinase domain (AKD) mutations are found in 20%–40% of pts with CML who fail therapy with the tyrosine kinase inhibitor (TKI) imatinib. Except for T315I, resistance conferred by most mutations can be overcome by the 2nd generation TKI dasatinib. In pts failing TKI therapy with no detectable AKD mutations by conventional direct sequencing (DS) of the AKD, other mechanisms of resistance (e.g., BCR-ABL1 amplification, SRC overexpression) have been proposed. However, these mechanisms occur at low frequencies in vivo. To ascertain whether imatinib-resistant pts may harbor mutations not detected by standard DS, we evaluated 61 pts with CML after imatinib failure by DNA expansion of specific clones (DESC) followed by DNA sequencing of at least 10 clones. At this point and prior to start of dasatinib therapy, 26 pts were in CP, 14 in AP, and 21 in BP. A total of 118 distinct AKD mutations at 112 amino acid positions were detected (77 previously unreported) in 58/61 (95%) pts. As previously reported, most mutants mapped to 4 AKD regions: P-loop (16%), catalytic domain (17%), 315–317 region (13%), and activation loop (9%). In addition, mutations were also found to cluster at high frequency to 4 novel AKD regions. One of them spans the residues flanked by amino acid positions 295 and 312 and contained 18% of AKD mutations. In addition to 5 pts who developed the highly dasatinib-resistant mutation V299L, 16/61 (25%) other pts harbored mutations within 295–312 prior to dasatinib start. Eleven (69%) of them never achieved any cytogenetic response on dasatinib and this was associated with significantly worse overall survival than that of patients expressing any other AKD mutation (p=0.02), except for T315I (Figure 1). Structurally, 295–312 mutations can potentially interfere with the N-lobe:helix αC interface. Although the exact energetic consequences of mutations at these residues are difficult to predict, structural analysis appears to indicate that these may hinder the outward torsion of the adjacent helix αC, potentially hampering the transition to the inactive conformation (“αC-Glu In” conformation) to which imatinib binds. Alternatively, the 295–312 region may serve as the structural scaffold for amino acid position 299, a direct dasatinib contact residue. Mutations mapping to 295–312 might distort the topography surrounding 299, thus altering the 299/dasatinib interface. Notably, 5/16 (32%) of these pts carried clones expressing more than 1 mutation within 295–312. All but 1 (80%) of these pts are dead. In summary, the use of techniques with higher sensitivity than conventional DS reveals that AKD mutations are highly prevalent (95%) in pts failing imatinib therapy, which could explain TKI resistance in pts not found to carry resistant mutations by conventional DS. We present evidence supporting the deleterious effect of mutations mapping to the novel 295–312 region. Experiments designed to prove these hypotheses are ongoing. Survival of 295–312 mutants Survival of 295–312 mutants

scholarly journals Molecular monitoring in CML and the prospects for treatment-free remissions

Hematology ◽  
2015 ◽  
Vol 2015 (1) ◽  
pp. 257-263 ◽  
Author(s):  
Michael W. Deininger
Keyword(s):  
Kinase Inhibitors ◽  
Dynamic Range ◽  
Mutational Analysis ◽  
Main Tool ◽  
Cytogenetic Response ◽  
Kinase Domain ◽  
Molecular Response ◽  
Molecular Monitoring ◽  
Landmark Analyses ◽  
Complete Blood Counts

Abstract Monitoring treatment responses in chronic myeloid leukemia (CML) is based on complete blood counts (CBCs) to determine hematologic response, karyotyping of bone marrow metaphase cells to delineate cytogenetic response and quantitative reverse transcription polymerase chain reaction (qPCR) to quantify expression of BCR-ABL1 mRNA (molecular response; MR) in peripheral blood. Fluorescence in situ hybridization (FISH) to identify BCR-ABL1 in interphase nuclei and mutational analysis of the BCR-ABL1 kinase domain (KD) are used in certain clinical circumstances. As most patients treated with tyrosine kinase inhibitors (TKIs) achieve complete cytogenetic responses (CCyRs), qPCR with its increased sensitivity and dynamic range has become the main tool used to monitor CML patients. Landmark analyses of large TKI trials have established MR milestones that identify patients with high risk of failure, are the basis of consensus management guidelines, and have led to a strong push toward qPCR test standardization. Today many laboratories report BCR-ABL1 qPCR results on the international scale (IS), a system based on the conversion of laboratory-specific numerical values to conform to a universal scale. The fact that qPCR is technically demanding and liable to assay variations poses considerable challenges for its routine clinical use. This is important as the prevalence of patients on chronic TKI therapy increases and critical clinical decisions are made based on qPCR results, for example if discontinuation of TKI therapy should be considered. Here we will review the current state of molecular monitoring in CML, focusing on qPCR, the definition of TKI failure and the results of TKI discontinuation studies.

Abnormally Small BCR-ABL Transcripts in CML Patients before and during Imatinib Treatment.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2153-2153 ◽  
Author(s):  
Jamshid S. Khorashad ◽  
Jeffrey H. Lipton ◽  
David Marin ◽  
Dragana Milojkovic ◽  
Nicholas C.P. Cross ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Genetic Instability ◽  
Fusion Gene ◽  
Direct Sequencing ◽  
Pcr Amplification ◽  
Kinase Domain ◽  
Current Evidence ◽  
Imatinib Treatment ◽  
Abl Kinase ◽  
Detection Frequency

Abstract Neoplastic cells bearing fusion genes that express an activated tyrosine kinase may set the scene for accumulation of genetic lesions by dysregulating DNA damage repair mechanisms and causing genetic instability. The observation that the BCR-ABL fusion gene alters pre-mRNA splicing in a variety of other genes including Ikaros and PYK2 supports this hypothesis. However, the only current evidence for acquired genetic change in the BCR-ABL gene itself is limited to finding mutations in the BCR-ABL kinase domain in patients treated with imatinib mesylate (IM). Here we report the observation that some patients with CML have abnormally small BCR-ABL transcripts both before and during treatment. Patients with sub-optimal response to IM are screened for mutations specifically within the BCR-ABL kinase domain by performing nested PCR, thereby excluding amplification of the non-translocated ABL allele. In the first round PCR amplification is performed across the fusion and the amplicons generated are subjected to a second round to yield an expected 863 bp (containing ABL exons 4 through 9 and thus the entire BCR-ABL kinase domain) PCR fragment. Smaller amplicons were observed in 49 (9.9%) of the 494 CML patients investigated. There was marked variation in the mRNA species when the abnormally small amplicons were subjected to direct sequencing; we found exon skipping, intra-exon splicing and insertion of intronic sequences. Similarly, in some cases the open reading frame was maintained whilst in others there were frame shifts leading to premature stop codons. The commonest finding, (22 of the 49 patients) was skipping of ABL exon 7 from codons 362 to 424, which includes the activation loop of the kinase domain. The smaller amplicons persisted even after the first round products had been diluted to 1:160. We also noted that the normal 863 bp fragment was present in some cases but was not detectable in others; its absence could reflect preferential amplification of the smaller transcripts. In a number of cases the same abnormally short amplicons were identified in the same patient studied serially on three or more separate occasions. We subsequently performed a second round of nested PCR with primers designed to amplify across the BCR-ABL junction such that the product included sequences from BCR 13 to ABL exon 9. With these new primers the detection frequency of abnormally small transcripts was increased. Furthermore, we observed the smaller transcripts in all of the 12 patients tested prior to beginning IM therapy. We then sought to determine if the normal ABL allele was involved; in order to avoid amplifying the BCR-ABL kinase domain, we performed a single round of PCR and restricted the analysis to patients in complete cytogenetic remission (CCyR). Only the expected 863 bp amplicon was observed in cDNA samples from 19 CML patients in CCyR whose BCR-ABL/ABL ratios ranged from 0.01 to 0.98. Furthermore, the smaller amplicons were not observed in cDNA samples from 20 normal individuals. We conclude that these abnormalities may result either from aberrant alternative splicing or from spontaneous deletions, or from a combination of both mechanisms. They may be a manifestation of the genetic instability believed to be an integral feature of CML.

Kinase Domain Point Mutations in Ph+ Acute Lymphoblastic Leukemia (ALL) and Lymphoid Blast Crisis of Chronic Myeloid Leukemia (CML) and Their Emergence Following Therapy with Bcr-Abl Kinase Inhibitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1831-1831
Author(s):  
Dan Jones ◽  
Rayjalakshmi Luthra ◽  
Hagop M. Kantarjian ◽  
Megan Breeden ◽  
Susan O’Brien ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Direct Sequencing ◽  
Lymphoblastic Leukemia ◽  
Blast Crisis ◽  
Philadelphia Chromosome ◽  
Point Mutations ◽  
Kinase Domain ◽  
Current Therapy ◽  
Abl Kinase ◽  
T315i Mutation

Abstract Bcr-Abl kinase domain (KD) point mutations are detected in the dominant clone(s) in approximately 45% of CML at the time of disease resistance, developing after an average of 20–35 months of imatinib therapy. However, low numbers of Philadelphia chromosome (Ph)+ tumor cells with KD mutations could be present at earlier timepoints providing a pool of potential resistant subclones. Since current therapy of Ph+ ALL relies on imatinib maintenance therapy, the pattern of Bcr-Abl KD mutations in this tumor is an important and understudied phenomenon. We assessed the frequency and levels of Bcr-Abl KD mutations at different points in ALL, including at diagnosis, upon relapse and following salvage therapy with kinase inhibitors. We performed Bcr-Abl KD mutational analysis by direct sequencing in 25 cases of Ph+ ALL at the time of diagnosis and 25 cases upon disease persistence/relapse. For comparison, we analyzed 22 cases of lymphoid blast crisis of CML (LyBC), most of which transformed following long-term imatinib monotherapy. To track the emergence of mutated clones, we also performed more sensitive analysis for the T315I mutation by pyrosequencing (5% sensitivity) and allele-specific oligonucleotide probe (ASO) PCR (1:500 sensitivity). KD mutations were not seen by direct sequencing in ALL cases at diagnosis. The T315I mutation was also not detected by pyrosequencing (n =25) or ASO-PCR (n = 10) in newly diagnosed ALL. In contrast, Bcr-Abl KD mutations (Y253H in 3, Q252H, T315I, F317L, E355Q, H396R in 1 each) were seen in 8 of 25 (32%) relapsed/persistent ALL, occurring in patients who had been receiving imatinib for a median of 14 months (range 2–26). An additional 3 patients treated with dasatinib or nilotinib for relapse subsequently developed KD mutations (T315I and Y253H, and F317L) after 1, 4 and 9 months of second therapy. KD mutations were seen in 16 of 22 (73%) patients with lymphoid blast crisis, including T315I in 7, E255K and M244V in 2 each, and Y253H, V299L, F311I, E355G, F359V in 1 each. All KD mutations in LyBC developed following imatinib or nilotinib therapy. As with CML, kinase inhibitor therapy particularly in the relapse/salvage setting is the primary risk factor for emergence of Bcr-Abl KD mutations in Ph+ ALL. There is a high frequency of Bcr-Abl KD mutations associated the lymphoid transformation of CML. However, Bcr-Abl KD mutations develop more rapidly in persistent or relapsed Ph+ ALL than in CML and there is a higher frequency of Y253H mutations noted. These findings will likely have consequences for the timing and dosages of imatinib and other kinase inhibitors in maintenance and relapsed ALL regimens.

An Intron-Derived Insertion/Truncation Mutation in the BCR-ABL Kinase Domain in Three CML Patients Undergoing Kinase Inhibitor Therapy.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1953-1953
Author(s):  
Jennifer Laudadio ◽  
Michael W.N. Deininger ◽  
Michael J. Mauro ◽  
Brian J. Druker ◽  
Richard D. Press
Keyword(s):  
Amino Acids ◽  
Drug Resistance ◽  
Amino Acid ◽  
Cdna Clone ◽  
Kinase Inhibitor ◽  
Kinase Domain ◽  
Inhibitor Therapy ◽  
Wild Type ◽  
Abl Kinase ◽  
Truncation Mutation

Abstract Although targeted inhibition of BCR-ABL with imatinib is an effective therapy for patients with chronic myeloid leukemia, a minority acquire mutations in the kinase domain (KD) that cause imatinib resistance. The spectrum of KD mutations thus far discovered, although quite heterogeneous, includes almost exclusively single nucleotide substitutions in key amino acids regulating drug binding or BCR-ABL function. Here, we describe a KD insertion/truncation mutation in 3 CML patients undergoing kinase inhibitor therapy. Two of these patients were being treated with imatinib (for 12 and 17 months), and one with dasatinib (for 13 months after a prior relapse while on imatinib). Suspected drug resistance was assessed by direct DNA sequencing of a BCR-ABL PCR product extending to the end of the kinase domain. Each of these 3 patients had 35 nucleotides from ABL intron 8 inserted at the normal exon 8–9 splice junction, after nucleotide 1423 (amino acid 475) of Genbank cDNA clone NM_005157. In all 3 cases, the mutation was co-expressed with wild type BCR-ABL sequence. The inserted sequence is derived from intron 8, beginning 1151 bp downstream from the normal splice donor site at the end of exon 8. This 35 bp intronic sequence is flanked by excellent consensus splice donor and acceptor sequences, suggesting alternative splicing as the likely mutational mechanism. The insertion creates a premature translational stop codon after 10 intron-encoded amino acids (figure), thus truncating 653 C-terminal amino acids including part of the KD and the entire last exon region - including a proline-rich domain, 3 nuclear localization signals, a DNA-binding domain, an actin-binding domain, and a nuclear export signal. These 3 insertion mutation cases were detected in our diagnostic clinical molecular pathology laboratory after sequencing 174 cases referred to us for suspected kinase inhibitor resistance, 78 of which contained a detectable mutation. The estimated prevalence of the exon 8/9 insertion/truncation mutation is then approximately 1.7% among patients with suspected drug resistance, and this mutation constitutes approximately 3.8% of all mutations. Conclusion: Kinase domain insertions are an alternative (and not entirely uncommon) mutational mechanism in CML patients undergoing kinase inhibitor therapy. The functional significance in terms of kinase activity and drug resistance remains to be addressed. Figure: Amino acid sequence of the C-terminus of the BCR-ABL kinase domain for the wild type and insertion/truncation mutant (with numbering as per GenBank cDNA clone NM_005157). Figure: Amino acid sequence of the C-terminus of the BCR-ABL kinase domain for the wild type and insertion/truncation mutant (with numbering as per GenBank cDNA clone NM_005157).

Outcome of Patients (pts) with Chronic Myeloid Leukemia (CML) After Failure to 2nd and 3rd Tyrosine Kinase Inhibitors (TKIs)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1238-1238
Author(s):  
Liunan Li ◽  
Hagop Kantarjian ◽  
Meng Zhao ◽  
Susan O'Brien, MD ◽  
Elias Jabbour ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Patient Population ◽  
Kinase Inhibitors ◽  
Chronic Phase ◽  
Cytogenetic Response ◽  
Kinase Domain ◽  
Abl Kinase ◽  
Kinase Domain Mutations ◽  
Lost To Follow Up

Abstract Abstract 1238 Background: First and second generation tyrosine kinase inhibitors are effective for most pts with CML in chronic phase. Approximately 80% of pts achieve complete cytogenetic response (CCyR) with imatinib, but nearly 15% of them eventually lose response. With dasatinib, nilotinib or bosutinib, approximately 50% of those who failed imatinib achieve CCyR, and about 15% of them eventually lose response. Using one of these agents after failure to 2 prior TKIs results in CCyR in only about 20%, usually of short duration. Thus, some pts receive and fail therapy with 3rd TKI. No standard therapy is available for these pts. Although their outcome is presumed to be poor, this has not been systematically analyzed. Understanding their outcome is important since new investigational options are being developed to treat this patient population, and understanding their expected outcome is needed to better comprehend the results obtained. Aim: To analyze the outcome of patients who have received and failed 2nd and 3rd TKI. Methods: We reviewed the records of 64 CML pts treated at MD Anderson Cancer Center from 2005–2009 who received treatment with 3 sequential TKIs. The second TKI was bafetinib (INNO-406, 1pt), bosutinib (12 pts), dasatinib (13 pts), and nilotinib (38 pts). Upon failure to 2nd TKI, 27 pts were in chronic (CP), 20 pts in accelerated (AP), 14 pts in blast phase (BP), and 3 pts in 2nd chronic phase, and were started on a 3rd TKI: bafetinib (6 pts), bosutinib (12 pts), dasatinib (35 pts), and nilotinib (11 pts). Results: After a median follow-up of 36 months (mo) (range, 3 – 71), 14 (22%) pts were still on 3rd TKI, including nilotinib (4 pts), dasatinib (7 pts), bafetinib (1 pt), and bosutinib (2 pts). Fifty (78%) pts failed therapy, including 16 pts (1 in 2nd CP, 2 in CP, 4 in AP, and 9 in BP) died during therapy with 3rd TKI. Among the 34 pts alive after 3rd TKI failure, their median age was 59 years (range, 19 to 92), and 17 were female. Their median time from diagnosis of CML was 76 mo (22 to 241). They failed the 3rd TKI after a median of 5.8 mo (range, 0.3 to 45) on therapy, with 27 pts being resistant (1 had minor cytogenetic response, all others 100% Ph+) and 7 pts were intolerant to 3rd TKI. The best response to a 3rd TKI was 1 partial cytogenetic (PCyR), 1 minor and 1 minimal cytogenetic response, 7 complete hematologic responses (CHR), and 24 with no response (NR). Upon failure to 3rd TKI, 16 pts were in CP (4 with BCR-ABL kinase domain mutations including two F359V, and one Y253H and one F317L), 11 in AP (6 with BCR-ABL kinase domain mutations including two G250E, and one each for F317L, F359C, T315I, and F317L), and 7 in BP (3 pts with BCR-ABL kinase domain mutations, two T315I and one V299L). These stages at the end of 3rd TKI represented no stage change in 26 patients, a progression in 5 pts (3 from CP to AP, 1 from CP to BP, 1 from AP to BP), and an improvement in 3 (from AP to CP in 2, from BP to AP in 1). Of those in CP, 16 pts were in complete hematologic responses (CHR). The median Ph+ metaphase after failure to 3rd TKI was 94% (8 to 100). After failure to 3rd TKI, 4 pts received dasatinib, 5 nilotinib, 4 bafetinib, 2 AP24534, 3 omacetaxine + imatinib, 6 single-agent omacetaxine, 4 stem cell transplantation, and 1 each for MK-0457, hydroxyurea, vincristine + deaxmethasone, idarubicin + imatinib + Ara-C, and DCC-2036. One pt was lost to follow-up. Response to subsequent therapy is described in the table 1 . After a median follow-up of 4 mo since failure to 3rd TKI, 15 of 34 pts have died, including 4 of 16 in CP, 5 of 11 in AP, and 6 of 7 in BP. The median survival from failure to 3rd TKI was 25 mo (12 mo for pts in CP, 6 in AP, and 1 in BP). Among patients who are still alive, 12 pts are still receiving the 4th therapy which were 3 pts received nilotinib, 2 dasatinib, 2 homoharringtonine, 2 AP245341, 1 bafetinib, 1 DCC-2036, and 1 hydroxyurea since failure to 3rd TKI, and 6 pts have changed to subsequent therapies after 4th therapy, including 5 pts on AP24534, 1 pt on XL228, and one lost to follow-up. Conclusions: Patients who have failed therapy with 2nd and 3rd TKI have a very poor prognosis with rare responses and a very short expected survival. New therapies that may improve their outcome and prolong their survival are urgently needed for this patient population. Disclosures: No relevant conflicts of interest to declare.

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