Use of Direct Sequence PCR for ABl Kinase Mutations in Patients with CML Blast Crisis, Treated Prior to the Availability of Imatinib Therapy.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2002-2002
Author(s):  
Sara Ghorashian ◽  
Anna Babb ◽  
Jamshid Khorasan ◽  
Jaspal Kaeda ◽  
David Marin ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Mutational Analysis ◽  
Fusion Gene ◽  
Limit Of Detection ◽  
Blast Crisis ◽  
Kinase Domain ◽  
Imatinib Therapy ◽  
Abl Kinase ◽  
Major Teaching Hospital ◽  
Major Teaching

Abstract We identified all patients who developed blast crisis before the advent of imatinib therapy and in whom stored marrow samples were available from a database of CML patients in a major teaching hospital. mRNA was extracted and cDNA synthesized successfully in 20 of 22 cases. The cDNA was subjected to real time PCR for quantification of BCR-ABL transcripts. The ABL allele not involved in the t(9;22) translocation was excluded from mutational analysis by subjecting the cDNA to nested PCR. This was achieved by amplifying from exons 13 and 10 of the BCR and ABL moieties of the BCR-ABL fusion gene using primers B2A and JAMR, respectively. The resulting amplicon was then subjected to nested PCR using primers, NTPB+ and NTPE-, sited within exons 4 and 10 of the ABL gene. The nested PCR yielded an 863 bp fragment in length containing the entire BCR-ABL kinase domain. To test for successful amplification an aliquot of the PCR products were electrophoresed through 2.0% agarose gel. In all cases, a single amplicon was observed and the PCR reaction subjected to magnetic purification. The purified amplicon were then sequenced by Sanger’s dideoxy chain termination reaction using Big-Dye ABI 310 sequencer (Applied Biosystems, Foster City, USA). In each case, the sequence obtained was compared with the published ABL type 1a sequence, Genbank M14752, using Blast 2 software. No mutations were found in any patient. This suggests the absence of a mutated kinase in a proportion of greater than approximately 30% of the total BCR-ABL kinase, due to the limit of detection of the technique. Our results suggest that the predominant form of BCR-ABL in the setting of blast crisis (in the absence of imatinib) is wild type. This goes against theories that kinase mutations confer an intrinsic ‘gain of function’ or a proliferative advantage to a CML clone. Instead, it is likely that mutant forms of BCR-ABL which are relatively resistant to imatinib binding gain prominence through positive selection with the presence of imatinib within the cell milieu.

Early Detection of Imminent Hematologic Relapse Due to BCR-ABL Kinase Domain Mutations in CML Patients on Imatinib Therapy by D-HPLC.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1088-1088
Author(s):  
Thomas Ernst ◽  
Philipp Erben ◽  
Thomas Schenk ◽  
Martin C. Mueller ◽  
Michael Emig ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Direct Sequencing ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Imatinib Therapy ◽  
Tyrosine Kinase Domain ◽  
Abl Kinase ◽  
Loop Region ◽  
Kinase Domain Mutations

Abstract Mutations of the BCR-ABL tyrosine kinase domain constitute the major cause of resistance in chronic myeloid leukemia (CML) patients (pts) on imatinib monotherapy. Sensitivity of conventional sequencing may allow the detection of a proportion of 10% mutated cells. We sought to improve the diagnostic armamentarium to screen for mutations prior to frank relapse. A total of 95 pts (chronic phase, CP, n=47; accelerated phase, AP, n=27; myeloid blast crisis, BC, n=19; lymphoid BC, n=2) who relapsed during imatinib therapy were screened for BCR-ABL kinase domain mutations applying denaturing high performance liquid chromatography (D-HPLC) and direct sequencing. Mutations were detectable in 47/79 (59%) pts with hematologic relapse and in 8/16 (50%) pts with cytogenetic relapse. 22 different point mutations affecting 18 amino acids and a novel deletion of 81bp of ABL exon 4 mapping to the P-loop region of the kinase domain were observed. To investigate the dynamics of the mutated clones D-HPLC was applied to 453 cDNA samples tracking back from relapse towards start of imatinib therapy. D-HPLC was optimized to detect 0.1–0.5% BaF3BCR-ABL cells harboring various mutations in a background of unmutated cells. Hematologic relapse occurred after a median of 12.9 mo (range 0.9–44.2) of imatinib therapy. However, BCR-ABL mutations became first detectable by D-HPLC at a median of 5.8 mo (range 0.0–30.5) after commencing imatinib (p<0.0001). Nine pts (18%; CP, n=4; AP, n=3, myeloid and lymphoid BC, n=1 each) showed evidence for BCR-ABL mutations even prior to imatinib therapy (T315I, n=4; M351T, n=3; M244V, Y253H, n=1 each). The median interval between first detection of the mutation and relapse was 5.6 mo in CP, 8.1 mo in AP, and 2.4 mo in myeloid BC. P-loop mutations were revealed at a median of 2.8 mo, activation loop mutations 2.9 mo, and T315I 6.3 mo prior to relapse. Cytogenetic relapse occured at a median of 19.2 mo (range 10.3–36.3) after start of imatinib therapy. BCR-ABL mutations became first detectable at a median time of 15.8 mo (range 0.0–26.4) after commencing imatinib (difference n.s., p=0.061). In two pts BCR-ABL mutations (M244V in AP, L324Q in CP) were observed in a small clone prior to imatinib therapy. Twenty-five CP pts in continuous complete cytogenetic remission were screened for BCR-ABL mutations to determine the predictive value of minor clones harboring mutations for consecutive relapse. No mutation was found in any of these pts. We conclude that (i) D-HPLC is a reliable and sensitive method to screen for BCR-ABL mutations before and during therapy with tyrosine kinase inhibitors. (ii) The observation of BCR-ABL mutations during imatinib therapy is predictive for relapse. (iii) Mutations may be detectable several months before hematologic relapse, and (iv) the sensitive detection of small mutated clones could provide clinical benefit by triggering early therapeutic interventions, which should be demonstrated in prospective clinical trials.

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.

Several Bcr-Abl kinase domain mutants associated with imatinib mesylate resistance remain sensitive to imatinib

Blood ◽  
2003 ◽  
Vol 101 (11) ◽  
pp. 4611-4614 ◽  
Author(s):  
Amie S. Corbin ◽  
Paul La Rosée ◽  
Eric P. Stoffregen ◽  
Brian J. Druker ◽  
Michael W. Deininger
Keyword(s):  
Imatinib Mesylate ◽  
Kinase Inhibitor ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Myelogenous Leukemia ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Cellular Assays ◽  
Kinase Domain Mutations

Abstract Imatinib mesylate is a selective Bcr-Abl kinase inhibitor, effective in the treatment of chronic myelogenous leukemia. Most patients in chronic phase maintain durable responses; however, many in blast crisis fail to respond, or relapse quickly. Kinase domain mutations are the most commonly identified mechanism associated with relapse. Many of these mutations decrease the sensitivity of the Abl kinase to imatinib, thus accounting for resistance to imatinib. The role of other mutations in the emergence of resistance has not been established. Using biochemical and cellular assays, we analyzed the sensitivity of several mutants (Met244Val, Phe311Leu, Phe317Leu, Glu355Gly, Phe359Val, Val379Ile, Leu387Met, and His396Pro/Arg) to imatinib mesylate to better understand their role in mediating resistance.While some Abl mutations lead to imatinib resistance, many others are significantly, and some fully, inhibited. This study highlights the need for biochemical and biologic characterization, before a resistant phenotype can be ascribed to a mutant.

Properties of CD34+ CML Cells That Predict Clinical Response to Tyrosine Kinase Inhibitors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 324-324
Author(s):  
Xiaoyan Jiang ◽  
Donna Forrest ◽  
Franck Nicolini ◽  
Karen Lambie ◽  
Kyi Min Saw ◽  
...  
Keyword(s):  
Stem Cells ◽  
Tyrosine Kinase ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Abl Kinase ◽  
Cd34 Cells ◽  
Clinical Responses ◽  
Kinase Domain Mutations

Abstract Imatinib (IM) treatment causes remission in a majority of patients with chronic myeloid leukemia (CML) but relapses remain a problem. The frequent presence in relapsing cells of BCR-ABL kinase domain mutations suggests that their prior but undetected acquisition by rare CML stem cells may be a major contributor to IM treatment failures. We have recently demonstrated that enriched populations of CML stem cells (lin−CD34+CD38− cells) are relatively insensitive to IM and possess multiple unique features that would be expected to promote both innate and acquired mechanisms of resistance to BCR-ABL-targeted therapeutics. These include elevated BCR-ABL expression and tyrosine kinase activity, increased expression of ABCB1/MDR1 and ABCG2, decreased expression of OCT1, and a high degree of genetic instability, as demonstrated by a rapid accumulation of BCR-ABL mutations in vitro. To determine whether these parameters may be predictive of clinical responses to IM, immunomagnetically selected CD34+ stem/progenitor cells from 18 chronic phase CML patients’ samples obtained prior to IM therapy were evaluated and the results compared with subsequent clinical responses. Direct sequencing of transcripts cloned from extracts of freshly isolated CD34+ cells (10 clones/sample) detected a high frequency of pre-existing BCR-ABL kinase mutations in the CD34+ cells from 12 of 12 patients regardless of their subsequent IM responses (20–80%). Interestingly, a higher incidence of BCR-ABL kinase domain mutations was found in 5 IM-nonresponders (33–80% of transcripts showed ≥1 BCR-ABL kinase domain mutation) as compared to 5 IM-responders (values of 20-30%, P<0.02). A higher frequency of BCR-ABL kinase domain mutations was also detected in extracts of colonies generated from assays of cells harvested from 3-week suspension cultures initiated with the same starting CD34+ CML cells (21–68% vs 10–43%). A high incidence of BCR-ABL kinase domain mutations was also documented in freshly isolated or cultured CD34+ cells from 2 patients who developed sudden blast crisis (50–63% and 17–83%). Overall, 38 different mutations were identified from freshly isolated CD34+ CML cells and >50 additional mutations were identified in the progeny of CD34+ CML cells cultured ± IM. These included 15 point mutations frequently associated with clinical IM resistance (including G250, Q252, E255, T315, M351, F359 and H396) and >40 mutations not previously described. Furthermore, freshly isolated CD34+ cells from IM-nonresponders (including the 2 patients who developed blast crisis, n=10) showed a greater resistance to IM in vitro (∼2 fold, P< 0.001 with 5 μM and P<0.02 with 10 μM IM) as compared to CD34+ cells from IM-responders (n=8) in the presence of 5 and 10 μM IM, as determined by colony-forming cell (CFC) assays. Although more IM-resistant CFCs were obtained in the presence of IM from 3-week cultures initiated with CD34+ cells from the same IM-nonresponders than from IM responders, these latter differences were not significantly different (P= 0.28). These results suggest that the CD34+ leukemic cells from individual chronic phase CML patients harbor differences in their biologic properties that are predictive of how they will respond to IM therapy and that assessment of these differences may form the basis of rapid, practical and quantitative tests to assist in optimized patient management.

Screening of Mutations in BCR-ABL Kinase Domain in Chronic Myeloid Leukemia (CML) Patients Treated with Kinase Inhibitors by Denaturing High-Performance Liquid Chromatography (D-HPLC).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4580-4580
Author(s):  
Cintia C. Mascarenhas ◽  
Anderson F. Cunha ◽  
Katia B.B. Pagnano ◽  
Rosana A. Silveira ◽  
Fernando F. Costa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
Marrow Transplantation ◽  
Kinase Inhibitors ◽  
Blast Crisis ◽  
Point Mutations ◽  
Kinase Domain ◽  
Molecular Response ◽  
Abl Kinase ◽  
Clinical Resistance

Abstract Point mutations within the ABL kinase domain are the most frequent mechanism for reactivation of kinase activity of the BCR-ABL gene and have been associated with clinical resistance to tyrosine kinases (TK) inhibitors in CML patients conferring in some of them a poor prognosis. The T315I (Treonine → Isoleucine) is a mutation described in exon 6 of BCR-ABL gene that makes the protein resistant to all kinase inhibitors most currently used for treating CML (imatinib, nilotinib and dasatinib). D-HPLC allows for high throughput mutation screening. This technique is based on heteroduplex formation by PCR products amplified from wild type and mutant alleles. Under optimized denaturing conditions, these heteroduplexes can be distinguished from homoduplex. In this study we screened mutations in exon 6 of BCR-ABL gene in patients treated with kinase inhibitors, in different phases of the disease. We evaluated 85 patients: 9 at diagnosis, 81 in chronic phase, 3 in accelerated phase, one in blast crisis. Thirty four were resistant to imatinib, 10 of them to dasatinib and three had suboptimal response to imatinib. In 9 of 85 (10,5%) samples, D-HPLC showed an abnormal elution profile suggesting the presence of nucleotide changes. Automated sequencing confirmed the presence of two point mutations: T315I (two patients) and F359V (two patients). Five patients requires sequencing confirmation. Patients with T315I mutation failed to imatinib and dasatinib. One of them relapsed after bone marrow transplantation in blast crisis. Patients with F359V mutation were resistant to imatinib. One of them has partial hematological response with dasatinib and the other is in complete molecular response after bone marrow transplantation. D-HPLC seems to be a ship and practical method for routine clinical monitoring for emergence of kinase domain mutations and may be useful for optimizing therapy in CML. Early detection of emerging mutant clones may help in decision-making of alternative treatment.

High-sensitivity detection of M351T, F317L, and F311C BCR-ABL kinase domain mutation in chronic myeloid leukemia patients treated with novel tyrosine kinase inhibitors (TKIs) imatinib and dasatinib

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 7061-7061
Author(s):  
A. R. Mir ◽  
S. Sazawal Sazawal ◽  
A. Saxena ◽  
R. Saxena
Keyword(s):  
Mutation Detection ◽  
Advanced Disease ◽  
Blast Crisis ◽  
Prognostic Significance ◽  
Kinase Domain ◽  
High Dose ◽  
Disease Group ◽  
Abl Kinase ◽  
Kinase Domain Mutation ◽  
T315i Mutation

7061 Objectives: (1) To study the detection of M351T, F317L, and F311C ABL kinase domain mutation in CML patients treated with TKIs (imatinib and dasatinib). (2) To evaluate the effect of imatinib dose esculation in the CML patients carrying M351T mutations. Methods: One hundred CML patients were treated with imatinib at 400 mg/day from 2 to 4 years .They were diagnosed by RT-PCR for BCR-ABL transcripts. Early screening for the M351T, F317L, and F311C mutations was performed by allele specific-oligonucleotide-PCR (ASO-PCR). Results: We evaluated 100 CML patients for kinase domain mutation by ASO-PCR after three years of imatinib initiation. Patients were categorized into three groups. Group A All 100 CML patients were treated with imatinib at conventional dose of 400 mg/day and were screened for M351T mutation after three years of imatinib initiation. (40%) 40/100 were positive for M351T mutation consequently 20/40 were treated with high dose imatinib at 600 to 800 or 1,000 mg/day. After 11 months of dose escalation, 15/20 lost M351T mutation but remaining five who resist M351T mutation, developed a more fatal mutation called gate keeper mutation T315I. 2/5 died, three progressed to advanced disease. Group B All 100 CML patients were screened for F311C mutation after three years of imatinib initiation and (10%) 10/100 were positive. After 10 months, 4/10 developed a more fatal mutation in 315 and consequently 2/4 died and one progressed to advanced disease. Group C It included 12 imatinib-resistant CML patients treated with dasatinib at 70 to 100 mg/day. At the initiation of dasatinib, no one was positive for F317L and T315I mutation. After 10 months of dasatinib treatment, all were screened for F317L/ T315I mutation by ASO-PCR, 4/12 were positive for F317L mutation and no one was positive for T315I mutation. After 6 months of mutation detection 2/4 progressed to blast crisis, and 1/4 died. Conclusions: ASO-PCR proved to be a very economical, sensitive, and rapid technique for detection of KD mutations M351T, F317L, and F311C ABL mutation and is more sensitive than mutation detection by sequencing. The detection of M351T, F317L, and F311C -ABL mutation at any stage has prognostic significance. No significant financial relationships to disclose.

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.

Pathobiology of Lymphoid and Myeloid Blast Crisis and Management Issues

Hematology ◽  
2005 ◽  
Vol 2005 (1) ◽  
pp. 188-194 ◽  
Author(s):  
Robert L. Ilaria
Keyword(s):  
Kinase Inhibitors ◽  
Early Stage ◽  
Blast Crisis ◽  
Clonal Evolution ◽  
Philadelphia Chromosome ◽  
Kinase Domain ◽  
Early Stage Disease ◽  
Abl Kinase ◽  
Road Map ◽  
Blastic Phase

Abstract Despite recent improvements in the treatment of early-stage disease, the blastic phase of chronic myeloid leukemia (CML) remains a therapeutic challenge. For imatinib-naïve patients, imatinib provided encouraging hematologic and cytogenetic benefits; however, the vast majority of CML blast crisis cases today arise in patients already on imatinib-based therapy. Clonal evolution and duplication of the Philadelphia chromosome continue to be associated with blastic phase transformation, but recent studies have identified BCR/ABL kinase domain mutations in 30%–40% of blast crisis patients. This implies that BCR-ABL–targeted therapy might have influenced the molecular road map to blastic transformation. In this review, we will examine the effect of imatinib on primitive CML progenitors and how this might influence the pathophysiology of blast crisis. A rational framework for deciding how best to integrate stem cell transplantation, traditional chemotherapy, imatinib, and other BCR-ABL kinase inhibitors in the care of blast crisis patients will also be discussed.

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.

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