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1
Chen, Jiaqi, Hongxing Liu, Fang Wang, Yang Zhang, Xue Chen, Daijing Nie, Yu Li, Yincheng Tan, Yuanli Xu, and Xiaoli Ma. "Dynamic Evolution of Ponatinib Resistant BCR-ABL1 T315 and Compound Mutations." Blood 134, Supplement_1 (November 13, 2019): 3796. http://dx.doi.org/10.1182/blood-2019-129579.
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The third-generation tyrosine kinase inhibitor (TKI) ponatinib exhibits activity against all common BCR-ABL1 kinase domain (KD) single mutations, including the highly resistant gatekeeper T315I. However, the drug response is variable and the clinical resistance mutations may still befall with few reports to date. We performed next-generation sequencing (NGS) detection of BCR-ABL1 KD mutations in sequential samples of three BCR-ABL1-positive leukemia patients who developed clinical resistance to ponatinib, to explore the dynamic evolution of ponatinib mutations. Case 1 was diagnosed as chronic myeloid leukemia (CML) chronic phase when he was 29 years old in 2009. His maintenance therapies were imatinib and dasatinib for seven years and then replaced with ponatinib due to the blast crisis of CML and the T315I mutation with variant allele frequency (VAF) of 46%. He did not achieved molecular remission after attempting multiple combined chemotherapy and TKIs including ponatinib, with the T315I mutation persists and eventually increases to VAF of 97%. He was then was medicated with a combined chemotherapy plus dasatinib and ponatinib. But NGS KD mutation investigation showed multiple T315I compound mutations, T315M and T315I mutations six months later. The patients went through salvage allogenic hemopoietic stem cell transplantation (allo-HSCT), and the above polyclonal and compound mutations were still carried after transplantation. Finally, Q252H/T315I (VAF,50%) became the dominant clone, and ponatinib and HQP1351 (domestic TKI designed for T315I) were ineffective for this compound mutation (Figure a). A similar dynamic evolution to the BCR-ABL1 KD mutation of Case1 also occurred in case 2. The 7-year-old boy was diagnosed with BCR-ABL1-positive acute B-lymphocytic leukemia (Ph+B-ALL) in April 2014. NGS results showed that he had D276G (7%), F311I (27%) and F317L (31%) polyclonal mutations in BCR-ABL1 KD after 18 months of imatinib administration which were subsequently treated with ponatinib. After 5 months of treatment with ponatinib, F311I and F317L mutations disappeared, but G250E (5%) and D276G/T315L (4%) compound mutation appeared; subsequent progression to D276G/T315L (32%), G250W/E255V/F311I (4%) and F311I/T315I (58%) polyclonal compound mutations; long induction chemotherapy combined with ponatinib treatment remained unresolved, and finally there was only D276G/T315L compound mutation (VAF, 100%, figure b and d). Notably, a rare mutation T315L (c.943_945delinsCTC/p.T315L) appeared in the BCR-ABL1 KD. D276G is known to be sensitive to various TKIs, so we speculate that ponatinib is ineffective for the T315L mutation. Case 3 was a 46-year-old woman who diagnosed with Ph+B-ALL in July 2018. The Q252H (20%) and T315I (44%) double mutations appeared after oral administration of imatinib for 2 months, and then switched to ponatinib for remission. After 8 months, the bone marrow and peripheral blood samples showed not only the T315I/F359V compound mutation (VAF, 90% and 94%), but also the T315L mutation (VAF, 5% and 6%, c.943_944AC>CT/p.T315L). The clinician combined her BCR-ABL1 KD mutation and condition, ponatinib was discontinued. After 1 month of chemotherapy combined with dasatinib, the patient's condition improved, but the BCR-ABL1 KD mutation progressed to T315L (18%, figure c and e) and T315I/F359V compound mutation (76%). She eventually died from severe pulmonary infection and sepsis. NGS analysis identify KD mutation with sensitivity about 2%, and can also distinguish between compound and polyclonal mutations. All of the ultimately dominant ponatinib resistant mutations (Q252H/T315I, D276G/T315L, and T351I/F359V) in these three cases were T315 compound mutations derived from the T315I or other original mutation with additional mutation event. The T315L/M mutations and compound mutations collaborated by T315 and other KD mutations may confer the major component of ponatinib resistance. The dynamic resistant mutation in these three patients adds to the currently less content compendium of ponatinib clinical resistance. All of the three patients encountered ponatinib toxic side effects and had to discontinue or reduce the dose, which also confer favorable opportunity for the development of drug-resistant mutations. Figure Disclosures No relevant conflicts of interest to declare.
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Manrique, Gonzalo, Roberta Bittencout, Verónica Pérez, Vanesa Sholl, Monica Cappetta, Noel Zubillaga, Rocio Hassan, et al. "Detection of BCR-ABL Kinase Domain Mutations in Chronic Myeloid Leukemia Patients Treated with Tirosin-Kinase Inhibitors." Blood 112, no. 11 (November 16, 2008): 4261. http://dx.doi.org/10.1182/blood.v112.11.4261.4261.
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Abstract Background. Point mutations in the kinase domain (KD) of the BCR-ABL are the most frequent mechanism of drug resistance in CML patients treated with kinase inhibitors (TKI). More than 80 mutations with different frequency and clinical significance have been reported. One of them, the T315I confers resistance to all TKIs available. The detection of mutations in KD allows early identification of high-risk patients and therefore guides clinical therapy decisions. Aim. To assess the mutation status of a group of CML pts resistant to TKI from Uruguay (n=35) and Brazil (n=30). Methods. KD mutation screening was performed by RT-PCR and direct sequencing according to Branford et al. (2002). Additionally, we developed a rapid, specific, sensitive and low cost allele specific (AS)-RT-PCR assay to identify T315I, using Branford’s KD amplification primers in combination with an allele specific primer for the T315I point mutation detection. BCR-ABL transcript levels were also measured by RQ-PCR according to international recommendations. Results and Discussion. RT-PCR and direct sequencing analyses performed in all pts showed the presence of T315l mutation in 3/65 cases. Other 11 showed the alternative mutations Y253H (n=2), E450A, G250E (n=2), E459K (n=2), E450G, F317L (n=2) and E255K; and the remaining 55 showed no mutations in the ABL KD. All 65 samples together with cDNA from 15 non-resistant CML pts and 10 cDNA from non-CML were analyzed by AS-RT-PCR assay for T315l mutation in order to validate the method. T315l was identified in the 3 samples in which the mutation was previously detected by direct sequencing and in 1 pt that had been classified as KD mutation negative. This result was then confirmed by direct sequencing of the AS-PCR product. T315 was neither detected in samples positive for other mutations nor in samples of non-resistant CML and non-CML patients, supporting the specificity of the method. Assessment of the sensitivity of the AS-RT-PCR was performed on serial dilutions experiments using RNA from T315 positive pt into RNA from CML-T315l negative pt, showing that the T315I mutation was detectable to a level of 0.01 % by AS-PCR, while through direct sequencing method the sensitivity was 10–20%. The prevalence of mutations in our study was 15/65 (23%). Conclusions. Our results showed that the AS-RT-PCR described here is a convenient and easy tool to be used in a clinical routine laboratory for rapid screening for BCR-ABL T315. This, together with direct sequencing, constitutes a suitable approach for CML resistance monitoring and therapeutic choice.
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Soverini, Simona, Giovanni Martinelli, Sabrina Colarossi, Alessandra Gnani, Fausto Castagnetti, Gianantonio Rosti, Costanza Bosi, et al. "Mutations at Residues 315 and 317 in the ABL Kinase Domain Are the Main Cause of Resistance to Dasatinib in Philadelphia-Positive (Ph+) Leukemia Patients (pts)." Blood 108, no. 11 (November 16, 2006): 836. http://dx.doi.org/10.1182/blood.v108.11.836.836.
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Abstract Dasatinib (BMS-354825) is a second-generation BCR-ABL inhibitor active against several imatinib-insensitive BCR-ABL mutant forms. We have treated in the phase II program with dasatinib a total of forty-five Ph+ pts who were resistant to or intolerant of imatinib. At the time of writing, twenty pts have failed to respond to or relapsed on dasatinib therapy. In order to assess which pre-existent or emerging ABL kinase domain (KD) mutations are challenging for dasatinib clinical efficacy, we retrospectively analyzed ABL KD sequences before the start of treatment and every month thereafter, until dasatinib discontinuation. Mutation monitoring was done by D-HPLC, followed by sequencing in D-HPLC-positive cases. Eight pts had primary resistance to dasatinib (Table 1). In all cases, a T315I or a F317L mutation was already detectable before the onset of treatment or became detectable after one month. The mutations persisted up to the time of disease progression, which occurred at a median of 1.5 months (range, 1–4) from dasatinib start. Twelve pts had acquired resistance to dasatinib (Table 1). Relapse occurred after a median of 7.5 months (range, 3–15) from dasatinib start. Mutation analysis performed before the onset of treatment showed that five of these pts had a wild-type ABL sequence, while the remaining seven pts had evidence of various imatinib-resistant KD mutations (G250E, Y253H, E255K, D276G, M351T). At the time of relapse, however, most of the original mutant clones had disappeared, whereas mutations at residues 315 (T315I or T315A) and/or 317 (F317L or F317I) had invariably emerged in all but one pt. This pt was found to have developed a novel K356R mutation which is now under characterization. Our results indicate that residues 315 and 317 are mutation hotspots in dasatinib-resistant pts, according to the experimental observation that they are both involved in inhibitor binding. They also provide a proof of principle that novel, inhibitor-specific mutant variants (i.e., T315A, F317I, K356R) may be selected, and raise some concerns about the limitations of single-agent treatment in the long term disease control of advanced phase-CML or Ph+ ALL pts. Supported by European LeukemiaNet, AIL, AIRC, FIRB and PRIN projects. Table 1 Pt Disease Mutation(s) before dasatinib start Best HR Best CgR Months on dasatinib Mutation(s) at relapse NE, not evaluated Primary resistance 1 CML/AP WT NR none 4 T315I 2 CML/AP T315I NR NE 1 T315I 3 CML/myBC T315I NR NE 1 T315I 4 CML/myBC F317L NR none 3 F317L 5 CML/lyBC T315I NR NE 1 T315I 6 Ph+ ALL T315I, M351T, L387M NR NE 2 T315I, M351T, L387M 7 Ph+ ALL T315I NR NE 1 T315I 8 Ph+ ALL F359V NR NE 2 T315I Acquired resistance 9 CP WT CHR minor 15 T315I 10 CML/myBC G250E NEL none 8 F317L 11 CML/lyBC Y253H CHR complete 9 CHR T315I 12 CML/lyBC WT CHR complete 4 T315I 13 CML/lyBC E255K CHR none 3 E255K, T315I 14 CML/lyBC D276G CHR complete 7 T315I 15 CML/lyBC WT CHR partial 9 F317L 16 Ph+ ALL E255K CHR NE 4 T315I 17 Ph+ ALL Y253H CHR complete 13 T315A, F317L, D276G 18 Ph+ ALL M351T CHR complete 13 M351T, F317L 19 Ph+ ALL WT CHR complete 6 F317I 20 Ph+ ALL WT CHR complete 4 K356R
4
Smith, Catherine C., Michael Brown, Jason Chin, Corynn Kasap, Sara Salerno, Lauren E. Damon, Kevin Travers, et al. "Single Molecule Real Time (SMRT™) Sequencing Sensitively Detects Polyclonal and Compound BCR-ABL in Patients Who Relapse on Kinase Inhibitor Therapy,." Blood 118, no. 21 (November 18, 2011): 3752. http://dx.doi.org/10.1182/blood.v118.21.3752.3752.
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Abstract Abstract 3752 Background: Secondary kinase domain (KD) mutations are the most well-recognized mechanism of resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) and other cancers. In some cases, multiple drug resistant KD mutations can coexist in an individual patient (“polyclonality”). Alternatively, more than one mutation can occur in tandem on a single allele (“compound mutations”) following response and relapse to sequentially administered TKI therapy. Distinguishing between these two scenarios can inform the clinical choice of subsequent TKI treatment. There is currently no clinically adaptable methodology that offers the ability to distinguish polyclonal from compound mutations. Due to the size of the BCR-ABL KD where TKI-resistant mutations are detected, next-generation platforms are unable to generate reads of sufficient length to determine if two mutations separated by 500 nt reside on the same allele. Pacific Biosciences RS Single Molecule Real Time (SMRT) circular consensus sequencing technology is a novel third generation deep sequencing technology capable of rapidly and reliably achieving average read lengths of ∼1000bp (Travers et al, 2010) and frequently beyond 3000bp, allowing sequencing of the entire ABL KD on single strand of DNA. We sought to address the ability of SMRT sequencing technology to distinguish polyclonal from compound mutations using clinical samples obtained from patients who have relapsed on BCR-ABL TKI treatment. Results: We analyzed an 863bp area of the BCR-ABL KD in 6 patients who had clinically relapsed on ABL kinase inhibitor therapy. SMRT sequencing detected mutations at a sensitivity of ∼1–2% of the total sequenced population, and successfully distinguished polyclonal from compound BCR-ABL KD mutations in several patient samples. Results were largely consistent with those obtained by PCR subcloning and sequencing, although SMRT sequencing detected additional mutations and/or mutation combinations. In the most complex case, 7 distinct mutation-bearing alleles were detected in an individual patient after sequential relapse on imatinib and dasatinib. Mutant clones contained single and compound mutations combining distinct mutations (Y253H, T315F, T315A, T315I, T319A, E355G). Three distinct substitutions at residue T315 were detected: T315A, T315I and T315F. Notably, these findings are clinically important as the T315A mutation confers resistance to dasatinib but not imatinib, while the T315F and T315I mutations are resistant to all three clinically approved BCR/ABL inhibitors (imatinib, dasatinib, and nilotinib). Phospho-flow analysis for p-Crkl, a direct substrate of BCR-ABL, was conducted following ex vivo exposure of patient cells from the same time point to all three BCR-ABL inhibitors, and demonstrated the existence of distinct populations of cells with varying sensitivity to each drug (i.e. polyclonal drug sensitivity), underscoring the potential clinical importance of distinguishing polyclonal from compound mutations. Additionally, SMRT sequencing routinely detected alleles harboring compound mutations not detectable by conventional direct sequencing. Data analysis of samples from additional patients is ongoing and will be presented. Conclusions: Pacific Biosciences RS SMRT sequencing sensitively detects KD mutations in patient samples and can distinguish TKI-resistant clones containing compound mutations to reveal a complex mutational landscape in an individual patient not detectable by conventional sequencing. SMRT sequencing of the BCR-ABL KD can feasibly be developed into a rapid and economical clinical test with the additional advantages of increased sensitivity and reliability over current methods. Given the growing numbers of patients exposed to multiple TKIs in a sequential manner, the ability to accurately and sensitively characterize drug-resistant alleles promises to further facilitate a personalized approach to patient management. Disclosures: Brown: Pacific Biosciences: Employment. Chin:Pacific Biosciences: Employment. Travers:Pacific Biosciences: Employment. Wang:Pacific Biosciences: Employment. Kasarskis:Pacific Biosciences: Employment, Equity Ownership. Schadt:Pacific Biosciences: Employment, Equity Ownership.
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Shah, Neil P., John M. Nicoll, Susan Branford, Timothy P. Hughes, Ronald L. Paquette, Moshe Talpaz, Claude Nicaise, Fei Huang, and Charles L. Sawyers. "Molecular Analysis of Dasatinib Resistance Mechanisms in CML Patients Identifies Novel BCR-ABL Mutations Predicted To Retain Sensitivity to Imatinib: Rationale for Combination Tyrosine Kinase Inhibitor Therapy." Blood 106, no. 11 (November 16, 2005): 1093. http://dx.doi.org/10.1182/blood.v106.11.1093.1093.
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Abstract Point mutations within the BCR-ABL kinase domain represent the most common mechanism of resistance to imatinib in patients with CML. Preclinical studies have shown that dasatinib (BMS-354825) is effective at inhibiting the kinase activity of imatinib-resistant BCR-ABL mutants with the notable exception of the T315I mutation, which remains highly resistant to imatinib, dasatinib, and AMN107 (Gorre et al, Science 2001; Shah et al, Science 2004; Weisberg et al, Cancer Cell, 2005). Clinical data from Phase I and II studies of dasatinib in CML confirms the in vitro findings. Each of three imatinib-resistant patients bearing the T315I mutation (CP=1; AP=2) did not achieve objective hematologic or cytogenetic responses during treatment with dasatanib on a Phase I study. Additionally, each of two phase II patients with the T315I mutation (CP=1; LBC=1) treated at UCLA showed no evidence of objective response. We have also detected the T315I mutation in each of two cases of acquired resistance in a phase II (LBC =2) study, and in seven of nine patients with acquired resistance to dasatinib in phase I and II studies (CP=1; MBC=3; LBC=2; Ph+ ALL=1). Notably, we detected a novel BCR-ABL mutation, T315A, in one of the two patients who relapsed without a detectable T315I mutation. The patient is a 53 year-old female whose chronic phase CML had progressed to myeloid blast phase while being treated with imatinib. The imatinib-resistant mutation M244V was identified prior to dasatinib treatment. The patient achieved a major hematologic response (<5% blasts with partial recovery of peripheral blood counts) on dasatinib 90 mg orally given twice daily, but relapsed with MBC after six months. Sequence analysis of the BCR-ABL kinase domain at the time of relapse revealed the presence of the imatinib-resistant mutation M244V as well as the novel mutation T315A. This finding is of particular interest because T315A and several other novel BCR-ABL mutations were recently recovered in a saturation mutagenesis study designed to define potential mechanisms of dasatinib resistance. Remarkably, many of these mutations retain sensitivity to imatinib in vitro (Burgess et al, PNAS, 2005). Through periodic molecular monitoring of other dasatinib-treated patients, we have identified a second novel BCR-ABL mutant, F317I, that developed in an imatinib-resistant CP patient after 9 months of treatment. Similar to T315A, F317I was isolated in the saturation mutagenesis screen for dasatinib resistance and is predicted to retain sensitivity to imatinib. Taken together, our findings implicate the T315I mutation as the principle mechanism of resistance to dasatinib, but more importantly, strongly support the use for combination kinase inhibitor therapy in CML to prevent emergence of drug resistant clones. A phase I trial to assess the safety of combining imatinib with dasatinib is planned.
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Deininger, Michael W. N., Michael J. Mauro, Yousif Matloub, Ritwik Sinha, Lynn Ploughman, David Liu, and Jerald Radich. "Prevalence of T315I, Dasatinib-Specific Resistant Mutations (F317L, V299L, and T315A), and Nilotinib-Specific Resistant Mutations (P-loop and F359) at the Time of Imatinib Resistance in Chronic-Phase Chronic Myeloid Leukemia (CP-CML)." Blood 112, no. 11 (November 16, 2008): 3236. http://dx.doi.org/10.1182/blood.v112.11.3236.3236.
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Abstract Mutations in the catalytic domain of ABL kinase (AKD) are a major mechanism of resistance to imatinib. Over 70 mutations in more than 50 amino acid residues have been reported to date. The ‘gatekeeper’ mutation, T315I, which causes complete resistance to all three FDA-approved tyrosine kinase inhibitors (TKIs), was reported to be not uncommon among a heterogeneous set of patients who had failed first-line imatinib therapy. In addition, the F317L, V299L, and T315A mutations were reported to convey a high degree of resistance to dasatinib, and higher frequency mutations within the P-loop (Y253H/F, E255V/K) and F359 mutations were associated with a high degree of resistance to nilotinib. We studied the prevalence of AKD mutations in the START-C phase II trial of dasatinib in patients who have failed imatinib (resistance or intolerance). Baseline mutation data were available for 95 of 99 patients with imatinib intolerance, and 274 of 288 patients with imatinib resistance. Of these patients, 13 (14%) with imatinib intolerance and 136 (50%) with imatinib resistance had AKD mutations. Of the 149 patients with mutations, only 3 (2%) had the T315I mutation. A total of 57 (38%) subjects had mutations in the P-loop (between 248–256): 13 patients with Y253H/F (9%), and 6 patients with E255V/K (4%). Four subjects (3%) had the F317L mutation, and 8 (5%) had F359 mutations. No subjects with V299L or T315A mutations were detected at baseline. The rates of complete cytogenetic response (CCyR) were 52% in patients with any mutation, 69% in patients with Y253H/F, 40% among those with E255V/K, 0% for T315I mutations, 0% for F317L mutations, and 50% for F359 mutations. Patients without mutations achieved a 55% rate of CCyR. These results confirm that select P-loop and F359 mutations are sensitive to dasatinib, while F317L and T315I mutations are resistant to dasatinib treatment. However, the overall incidence of these dasatinib-resistant mutants is low. In contrast, nilotinibresistant mutations in the P-loop (Y253H/F, E255V/K) or at F359 are more common, representing 15% and 5% of all patients with mutations, respectively. Therefore, the likelihood of harboring a nilotinib-resistant mutation at the time of imatinib resistance appears higher than the likelihood of harboring a dasatinib-resistant mutation, and suggests mutation testing may become instrumental for choosing between the various second-line TKI inhibitors to optimize outcomes.
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Kim, Dong-Wook, Dongho Kim, Soo-Hyun Kim, Saengsuree Jootar, Hyun-Gyung Goh, Jeong Lee, Soo-Young Choi, Young-Seok Lee, and Sang-Mi Oh. "Dynamics and Characteristics of BCR-ABL Multiple Mutations In Tyrosine Kinase Inhibitor Resistant Chronic Myeloid Leukemia." Blood 116, no. 21 (November 19, 2010): 3443. http://dx.doi.org/10.1182/blood.v116.21.3443.3443.
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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.
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Deininger, Michael W. N., Jorge E. Cortes, Dong-Wook Kim, Franck E. Nicolini, Moshe Talpaz, Michele Baccarani, Martin C. Müller, et al. "Impact of baseline mutations on response to ponatinib and end of treatment mutation analysis in patients with chronic myeloid leukemia." Journal of Clinical Oncology 31, no. 15_suppl (May 20, 2013): 7001. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.7001.
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7001 Background: BCR-ABL kinase domain mutations frequently cause tyrosine kinase inhibitor (TKI) failure in chronic myeloid leukemia (CML). Ponatinib, a potent oral pan-BCR-ABL TKI, has shown preclinical activity against all single mutants tested, including T315I. The impact of baseline (BL) mutations on response to ponatinib (45 mg once daily) and end of treatment (EOT) mutations in pts discontinuing treatment were evaluated in the phase II PACE trial. Methods: Heavily pretreated chronic phase (CP) CML pts (93% received ≥2 prior TKIs, 60% ≥3) resistant or intolerant to dasatinib or nilotinib (N=203) or with T315I confirmed at BL (N=64) were enrolled. The primary endpt was major cytogenetic response (MCyR). Min follow up at analysis (9 Nov 2012) was 12 mos (median 15 [0.1-25]). Sanger sequencing was done at one central laboratory. Results: At BL, no mutations were detected in 51% of pts, 1 mutation in 39%, and ≥2 mutations in 10%; 26 unique mutations were observed. Responses were observed regardless of BL mutation status. MCyR rates were: 56% overall, 49% in pts with no mutations, 64% 1 mutation, 62% ≥2 mutations; 57% in pts with mutation(s) other than T315I, 74% T315I only, 57% T315I + other mutation(s). Responses were seen against each of the 15 mutations present in >1 pt at BL, including T315I, E255V, F359V, Y253H. 99 pts discontinued, 56 had EOT mutations assessed. 5 pts lost a mutation, 46 had no change, 5 gained mutations (Table). 11 pts lost MCyR (none with T315I); of the 6 discontinuing, 4 had EOT mutations assessed and no changes from BL were seen. Conclusions: Responses to ponatinib were observed regardless of BL mutation status. No single mutation conferring resistance to ponatinib in CP-CML has been observed to date. Data with a minimum follow up of 18 mos, including pts with advanced disease, will be presented. Clinical trial information: NCT01207440. [Table: see text]
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Jabbour, Elias, Hagop Kantarjian, Dan Jones, Srdan Verstovsek, Alessandra Ferrajoli, Farhad Ravandi, Susan O’Brien, and Jorge Cortes. "Characteristics and Outcome of Patients with Chronic Myeloid Leukemia (CML) and T315I Mutation Following Failure of Imatinib Mesylate Therapy." Blood 110, no. 11 (November 16, 2007): 1943. http://dx.doi.org/10.1182/blood.v110.11.1943.1943.
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Abstract Background. T315I is an imatinib pocket binding mutation within the Bcr-Abl kinase domain that is highly resistant, both in vitro and in vivo, to imatinib and to 2nd generation tyrosine kinase inhibitors (TKIs). Several studies have suggested that patients with T315I have a poor outcome. Study Aims. The objectives of this study were to define the clinical characteristics of patients harboring the T315I mutation, and to assess their outcome after imatinib failure. Results. T315I was detected in 27 pts: 20 among a series of 186 pts assayed after imatinib failure (11% of all pts; 21% of all mutations detected) after a median of 37 months (mos) from start of imatinib, and 7 among 23 pts who developed new mutations after a median of 10 mos on therapy with a 2nd generation TKI. Median age was 52 years. Median time from diagnosis to T315I was 41 mos, and the median follow-up from the detection of mutation is 18 mos. At the time of T315I detection, 10 pts were in CP, 9 in AP, and 8 in BP. Fifteen pts (56%) had transformed to accelerated or blast phase at the time of T315I detection. Best response to TKI immediately preceding development of T315I (20 imatinib, 2 nilotinib, 2 dasatinib, 2 bosutinib, 1 INNO-406) was CHR in 13 (48%) and CyR in 9 (33%; complete in 6, partial in 1, minor in 2). The median duration of response was 44 mos. Except for the lack of response to a second TKI (p=0.001), there was no difference in pt characteristics between pts with or without T315I, other mutations, or no mutations. Among the 20 pts with T315I present prior to start of 2nd TKI, 5 responded, all hematologic (3 complete hematologic response -CHR-, 2 partial hematologic response -PHR-, 1 return to chronic phase); in contrast all 5 pts without T315I prior to 2nd TKI, responded (1 major molecular response -MMR-, 2 Minor cytogenetic response -CyR-, 1 CHR, 1 PHR); and among the 2 pts with unknown T315I status at start of 2nd TKI 1 had PHR and 1 complete cytogenetic response -CCyR-. Responses were usually transient but 3 pts had sustained responses for some time despite presence of T315I: 1 pt in AP harboring simultaneously F317L and G250E acquired a T315I mutation 5 mos after the start of nilotinib and achieved MMR that was sustained for 21 mos eventually lost to major CyR. A 2nd pt in AP treated with bosutinib acquired a T315I mutation 6 months after the start of bosutinib, but nonetheless achieved a minor CyR that has been sustained for more than 8 mos. A third patient with Y253H mutation developed T315I 1 mo after therapy with INNO-406 for CML AP; at the last follow-up, 4 months into therapy, he maintained a PHR. 4/14 pts (38%) treated with T315I-directed agents (aurora kinase inhibitors, homoharringtonine) responded. 4 pts received allogeneic stem cell transplant (ASCT) and 2 are alive: 1 in CMR 24+ months after ASCT and 1 in CCyR 9 months after ASCT, wit molecular relapse and recurrence of T315I. 11/27 pts with T315I (40%) died. Patients in CP had better outcome with 87% 2-year survival, compared to 45% in AP and 20% in BP. Survival of patients with T315I was similar to those with other mutations or without mutations (p=0.64). Conclusion. Altough T315I is a mutation highly resistant to conventional BCR-ABL TKI, occasional responses can be observed. Overall survival of patients with T315I mutations is mostly dependent on the stage of the disease.
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Jabbour, Elias, Hagop Kantarjian, Dan Jones, Megan Breeden, Guillermo Garcia-Manero, Susan O'Brien, Farhad Ravandi, Gautam Borthakur, and Jorge Cortes. "Characteristics and outcomes of patients with chronic myeloid leukemia and T315I mutation following failure of imatinib mesylate therapy." Blood 112, no. 1 (July 1, 2008): 53–55. http://dx.doi.org/10.1182/blood-2007-11-123950.
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AbstractChronic myeloid leukemia (CML) with T315I mutation has been reported to have poor prognosis. We analyzed 27 patients with T315I, including 20 who developed T315I after imatinib failure (representing 11% of 186 patients with imatinib failure), and 7 of 23 who developed new mutations after second tyrosine kinase inhibitor (TKI). Median follow-up from mutation detection was 18 months. At the time of T315I detection, 10 were in chronic phase (CP), 9 in accelerated phase, and 8 in blast phase. Except for the lack of response to second TKIs (P = .002), there was no difference in patient characteristics and outcome between patients with T315I and those with other or no mutations. Patients in CP had a 2-year survival rate of 87%. Although the T315I mutation is resistant to currently available TKIs, survival of patients with T315I remains mostly dependent on the stage of the disease, with many CP patients having an indolent course.
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Mitra, Priti, Swati Dasgupta, Chinmay Kumar Basu, Firoj Hossain Gharami, Subrata Mandal, and Ashis Mukhopadhyay. "Study of different mutations in chronic myeloid leukemia in India and their co-relation with drug resistance." Journal of Clinical Oncology 31, no. 15_suppl (May 20, 2013): 7083. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.7083.
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7083 Background: Emergence of ABL point mutations is the most frequent cause for imatinib resistance in CML. Aim of our study is to investigate two potential resistance mechanisms i.e.,mutations of BCR-ABL tyrosine kinase domain (TKD) and Additional Chromosomal Abnormalities during TKI treatment in CML. Methods: Karyotyping and BCR-ABL TKD mutation screening are performed in 100 imatinib resistant CML patients who were on imatinib at the time of loss of hematologic response, cytogenetic or molecular response. Imatinib–Resistance Mutation Analysis (Qualitative) were detected by Nested RTPCR and Sanger’s Sequencing. In 100 cases, 34 received escalated imatinib, 34 nilotinib and another 32 dasatinib. Results: In 100 BCR-ABL positive imatinib, nilotinib and dasatinib resistant cases, 11 different BCR-ABL TKD mutations were detected. Analysis revealed no mutations-43 cases, M351T-12 cases, G250E-10 cases, F317L-8 cases, M244V-5 cases, E255K-4 cases, V379I-4 cases, F359V-3 cases, H396R-3 cases, Y253F-3 cases, E355G-3 cases, T315I-2 cases. 11 novel mutations (F317L, G250E, M244V, Y253F, E255K, M351T, F359V, H396R, V379I, E355G, T315I) conferring imatinib resistance, 10 nilotinib–resistant mutations (M244V, F359V, T315I, E355G, G250E) and 8 dasatinib-resistant mutations (H396R, F317L, H396R, T315I, M351T) were seen in our patient population. T315I was found more frequently in cases on dasatinib than on imatinib therapy. Conclusions: T315I which confers resistance to all TKIs was detected only in 2/100 patients who demonstrated loss of response in our population. As compared with other western studies, incidence of T315I mutation was very low in our study. In addition analysis of mutation patterns at baseline may help in stratifying patients for treatment. For cases with TKI resistance, mutation and ACA screening may play role in identifying patients with poorer prognosis. In our practice if nilotinib–resistant mutation was detected, dasatinib was preferred and for dasatinib-resistant mutation, nilotinib was preferred. We are planning for using bosutinib, panotinib and omacetaxine (SC route) in third line therapy in imatinib resistant different mutation positive chronic myeloid leukemia.
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Mian, Afsar Ali, Hadiqa Raees, Sujjawal Ahmad, Oliver Ottmann, and El-Nasir M. A. Lalani. "Arsenic Trioxide Suppresses Growth of BCR-ABL1 Positive Cells with "Gatekeeper" or Compound Mutation." Blood 138, Supplement 1 (November 5, 2021): 4346. http://dx.doi.org/10.1182/blood-2021-154511.
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Abstract Introduction: Chronic myeloid leukemia (CML) and 30% of adult acute lymphatic leukemia (ALL) are characterized by the Philadelphia chromosome (Ph +), having a (9;22) chromosomal translocation. The BCR-ABL1 fusion protein is the hallmark of Ph + leukemia. BCR-ABL1 is characterized by constitutively activated ABL1 tyrosine kinase activity that determines its transformation potential. Tyrosine kinase inhibitors (TKI) have greatly improved the overall prognosis of these diseases. However, unsatisfactory responses in advanced disease stages, resistance and long-term tolerability of BCR-ABL1 inhibitors represent major clinical problems. The most important resistance mechanism against TKIs is the acquisition of point mutations within the BCR-ABL1 kinase domain that impair drug binding, restoring the oncoprotein's constitutively active tyrosine kinase activity. The selection of leukemic clones driven by BCR-ABL1 harboring point mutations, such as the E255K, Y253F/H (P-loop), H396R (activation loop) or the T315I (gatekeeper). Second- and third generation TKIs such as nilotinib, dasatinib, and ponatinib effectively overcome point mutation-mediated resistance. Ponatinib is the only U.S. Food and Drug Administration approved TKI with activity against all known BCR-ABL1 point mutations, including BCR-ABL1-T315I. However, the emergence of compound mutations (two mutations within the same BCR-ABL1 allele) has been linked to resistance to all approved TKIs, including ponatinib, posing a clinical challenge with limited treatment options. The anti-cancer agent arsenic trioxide (ATO) has been used to treat patients with acute promyelocytic leukemia (APL). APL patients respond very well to ATO therapy and achieve complete remission, possibly through induction of apoptosis and differentiation. In addition, it has been demonstrated that combined treatment of ATO with interferon or nilotinib significantly suppressed cell proliferation. However, the potential effects of ATO on BCR-ABL1 mutations and especially on compound mutation is not apparent. This study aimed to investigate the role of ATO in BCR-ABL1 resistant mutations, including compound mutation in Ph + leukemias. Methods: We undertook preclinical evaluation of ATO and compared it with approved TKIs e.g. imatinib, nilotinib, dasatinib, ponatinib and ABL inhibitor asciminib, in vitro models of CML and primary patient-derived long term cultures (PD-LTC) of Ph + ALL patients with or without mutation. The effects on mutational resistance were investigated in Ba/F3 cells expressing BCR-ABL1 with T315I mutation and T315I-E255K mutation. For non-mutational resistance, we used PD-LTCs from Ph + ALL patients with different levels of non-mutational drug resistance. Cell proliferation was assessed by XTT. Results: ATO efficiently inhibited the growth of all PD-LTCs in cellular assays at dosages of 200-500nM. It also suppressed the growth of Ph + PD-LTC with non- mutational resistance (BV) and the BCR-ABL1-T315I positive PD-LTC (KO) in this dosage range. In all modelsWe treated Ba/F3 cells expressing native BCR-ABL1, BCR-ABL1-T315I mutation and BCR-ABL1-T315I-E255K (compound mutation) with increasing concentrations of imatinib (250, 500 and 1000nM), nilotinib (100, 200 and 400nM), dasatinib (10, 25 and 50nM), ponatinib (10, 50 and 100nM), asciminib) (ABL allosteric inhibitor) (5, 10 and 20nM) and ATO (0.5, 1.0 and 2.0 µM). We found that all the inhibitors significantly inhibited the proliferation of Ba/F3 cells expressing wild type BCR-ABL1 in a dose-dependent manner. In contrast, the growth of Ba/F3 cells expressing BCR-ABL1-T315I was inhibited by increasing concentration of ponatinib, asciminib and ATO. ATO potently inhibited the most challenging mutation (T315I-E255K) with a clinically relevant concentration (IC50 250nM). All approved ABL kinase inhibitors (AKIs) and allosteric inhibitors like asciminib could not inhibit the growth of Ba/F3 cells expressing BCR-ABL1 compound mutation. Conclusions: Our findings indicate that ATO significantly suppressed the proliferation of cells expressing non-mutated BCR-ABL1, single and compound mutated BCR-ABL1. These results support including ATO in treating patients with Ph + leukemias having BCR-ABL1 resistant single or compound mutati Disclosures Ottmann: Novartis: Honoraria; Amgen: Honoraria, Research Funding; Celgene/BMS: Honoraria, Research Funding; Fusion: Honoraria; Incyte: Honoraria, Research Funding.
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Tanaka, Ruriko, Shinya Kimura, Toshiya Hosomi, Mitsuharu Hirai, Rina Nagao, Asumi Yokota, MMSc, Yuri Kamitsuji, et al. "Automated and Rapid Detection of BCR-ABL Kinase Domain Mutations in IM Resistant Patients with Ph+ Leukemias." Blood 114, no. 22 (November 20, 2009): 2590. http://dx.doi.org/10.1182/blood.v114.22.2590.2590.
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Abstract Abstract 2590 Poster Board II-566 Chronic myelogenous leukemia (CML) is caused by a consistent genetic abnormality, termed the Philadelphia chromosome (Ph). It results in the production of BCR-ABL fusion protein, a constitutively active tyrosine kinase. Imatinib mesylate (IM, Gleevec®), the first generation tyrosine kinase inhibitor (TKI), has revolutionized therapy for CML patients. However, resistance for IM develops in a significant proportion of cases, and is predominantly mediated by single point mutations within the BCR-ABL kinase domain. Second generation TKIs such as dasatinib (Sprycel®) and nilotinib (Tasigna®) represent viable alternatives for IM-resistant or intolerant CML patients. Each mutated BCR-ABL has different sensitivity to those TKIs. Thus, it is significantly important to detect early the existence of BCR-ABL mutations and their specificities in treating Ph+ leukemias. We have developed a novel automated method that has high sensitivity to detect a few copies of mutation sequences that are mixed in many copies of normal sequences. This method consists of PCR amplification step and Tm (melting temperature) analysis step that uses a quenching probe. And we have already shown that this system has clinical efficacy in JAK2V617F mutation that is one of the genetic hallmarks of chronic myeloproliferative diseases. (Tanaka R, et al. Leuk Res, 2008). When a whole blood sample or a purified DNA sample reacts with reagents, PCR and Tm analysis automatically processed in the same tube, and whole procedure finishes in approximately 1 hour. The detection of mutation is extremely accurate because the quenching probe is designed perfectly matched for mutated sequence. As Tm value of mutation sequence is higher than that of normal one, it is easy to detect the existence of mutation from the Tm analysis data. We have constructed the probes for 14 mutations concerned for IM-resistance (M244V, G250E, Q252H, Y253F, Y253H, E255V, E255K, T315I, T315A, F317L, M351T, E355G, F359V, and H396R). Considering the clinical significance of T315I mutation, which renders resistance to all currently available TKIs, we refined this method to higher sensitivity for detecting T315I mutation. First, we analyzed the sensitivity of this system on BCR-ABL. In dilution assays using wt and mutated plasmid, the system reliably quantified the mutation in a population containing as few as 3.0% mutant. Moreover, for T315I setting, we successfully detected as few as 0.3% (30 copies from 10,000 copies) mutations by a higher-sensitive assay. Next, we examined the clinical samples. Each sample was also examined by direct sequencing in comparison to our method. Kinase domain mutations were identified in 24 of the 50 (48%) patients. Our automated analysis was enabled to detect mutations in 19 patients, including p-loop mutations (G250E: n=3; E255K: n= 5), IM-binding domain mutations (T315I: n=10), and an activation-loop mutation (H396R: n=1). And all the positive cases (19 of 19) showed a concordance with the result of direct sequencing. On the other hand, 5 cases were detected just by direct sequencing, but all that cases were out of our setting mutations (Q252E, V379I, S417F, E459K). Impressively, in one case, only higher-sensitivity assay could reveal T315I mutation, although it was detected as a wild type both by direct sequence and our usual method. It suggests that the higher-sensitive system could detect low amount of T315I mutation in the earlier stage of disease. In conclusion, sensitivity of our system (3%) is significantly greater than that of direct sequencing (15 – 25%), and results can be obtained within one hour. By the serial monitoring, it is demonstrated the availability of the higher-sensitive analysis (0.3%) to detect T315I mutation. This rapid and accurate detection of clinically significant mutations enables us to contribute to better clinical practice in treating Ph+ leukemia patients, such as in selecting alternative strategies of IM dose escalation, second generation TKIs, or allogeneic stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.
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Okabe, Seiichi, Tetsuzo Tauchi, Shinya Kimura, Taira Maekawa, and Kazuma Ohyashiki. "The Analysis of HDAC Inhibitor, Vorinostat Efficacy against Wild Type and BCR-ABL Mutant Positive Leukemia Cells in Monotherapy and in Combination with a Pan-Aurora Kinase Inhibitor, MK-0457." Blood 112, no. 11 (November 16, 2008): 5025. http://dx.doi.org/10.1182/blood.v112.11.5025.5025.
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Abstract The use of imatinib, an ABL tyrosine kinase inhibitor, has led to a dramatic change in the management of BCR-ABL positive leukemia patients. However, the resistance to imatinib mediated by mutations in the BCR-ABL domain has become a major problem in the treatment. Histone deacetylase (HDAC) inhibitors have been shown to mediate the regulation of gene expression, induce cell growth, cell differentiation and apoptosis of tumor cells. Vorinostat (suberoylamide hydroxamic acid:SAHA) is a hydroxamic acid based polar HDAC inhibitor. Vorinostat have shown efficacy in a wide range of cancers such as cutaneous T-cell lymphoma (CTCL). However, efficacy of vorinostat against the BCR-ABL mutants has fully not known. Here we report on the studies performed against murine Ba/F3 cell line which was transfected wild type (Wt) p210 and p185 BCR-ABL or imatinib resistant BCR-ABL mutants such as G250E, Q252H, Y253F, E255K, M294V, T315I, T315A, F317L, F317V, M351T, H396P and T315I(p185). 48 hours treatment of vorinostat exhibits cell growth inhibition and proapoptotic activity murine Ba/F3 cells ectopically expressing Wt and imatinib resistant BCR-ABL mutants including T315I mutation in a dose dependent manner. IC50 of these cell lines are Wt(720nM), G250E(625nM), Q252H(220nM), Y253F(525nM), E255K(685nM), M294V(785nM), T315I(500nM), T315A(715nM), F317L(560nM), F317V(565nM), M351T(375nM) and H396P(485nM). Aurora kinases play a pivotal role in the regulator of mitotic processes during cell division. MK-0457 is a small molecule inhibitor of the Aurora kinase family and was found to be active against the cells from BCR-ABL positive patients with T315I mutation in clinical trial. Because vorinostat also depleted BCR-ABL, as well as induced apoptosis and sensitized BCR-ABL-expressing leukemia cells, we examined whether vorinostat and MK-0457 enhances the apoptosis in imatinib resistant BCR-ABL-expressing cells. 48 hours treatment of MK-0457 exhibits cell growth inhibition of Ba/F3 cells ectopically expressing Wt and imatinib resistant BCR-ABL mutants including T315I mutation. IC50 of MK-0457 is Wt(215nM), G250E(205nM), Q252H(185nM), Y253F(245nM), E255K(185nM), M294V(238nM), T315I(205nM), T315A(165nM), F317L(200nM), F317V(200nM), M351T(225nM) and H396P(195nM). We examined the intracellular signaling by using these cell lines. We found that caspase 3, and poly (ADPribose) polymerase (PARP) were activated after MK-0457 treatment in a dose dependent manner. Phosphorylation of BCR-ABL and Crk-L which is downstream target of BCR-ABL was reduced after MK-0457 treatment. We found that combination of vorinostat and MK-0457 synergistically cell growth inhibition of Wt and BCR-ABL mutants Ba/F3 cells in 48 hours treatment. Phosphorylation of Crk-L was reduced after vorinostat and MK-0457 treatment. Caspase 3 and PARP activation were also synergistically increased after vorinostat and MK-0457 treatment. We evaluated the activity of MK-0457 and vorinostat in primary BCR-ABL positive acute lymphoblastic leukemia (ALL) cells with the T315I mutation. We found that MK-0457 potently induced cell growth inhibition of primary T315I cells in 48 hours treatment. Moreover, combination of vorinostat and MK-0457 synergistically increased the cell growth inhibition in primary T315I cells. This study demonstrate monotherapy of vorinostat and the combination of vorinostat and MK-0457 are more potent efficacy not only wild type BCR-ABL but also imatinib resistant BCR-ABL mutants cells and represents a promising new strategy for treatment of imatinib resistant BCR-ABL positive leukemias, including those harboring the T315I mutation.
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Shah, Neil P., Brian Skaggs, Susan Branford, Timothy P. Hughes, John M. Nicoll, Ronald L. Paquette, and Charles L. Sawyers. "Sequential Kinase Inhibitor Therapy in CML Patients Can Select for Cells Harboring Compound BCR-ABL Kinase Domain Mutations with Increased Oncogenic Potency: Rationale for Early Combination Therapy of ABL Kinase Inhibitors." Blood 108, no. 11 (November 16, 2006): 751. http://dx.doi.org/10.1182/blood.v108.11.751.751.
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Abstract A critical question in the targeted therapy era relates to whether treatment outcomes will be optimized by sequential or combinatorial use of targeted agents. Selection for CML cells with BCR-ABL kinase domain mutations is the main mechanism responsible for loss of response to imatinib. Dasatinib is an ABL tyrosine kinase inhibitor that has activity against nearly all imatinib-resistant mutations and is approved for the treatment of imatinib-resistant and -intolerant BCR-ABL-associated leukemias. Acquired clinical resistance to sequential use of dasatinib following imatinib failure has been observed. We analyzed the BCR-ABL kinase domain at the time of relapse in 15 patients who lost an initial response to dasatinib, and found evolution of a total of three new mutations at the time of relapse in all cases. The highly resistant BCR-ABL/T315I mutation was detected in 11 cases. The four remaining cases were associated with the evolution of novel mutations (V299L, 3 cases; T315A, 1 case). V299L was also detected in a fourth case that had also evolved T315I. These three dasatinib-resistant mutations were part of a small number of amino acid substitutions previously isolated in a preclinical mutagenesis screen for dasatinib resistance-conferring BCR-ABL mutations. While the T315I mutation is highly resistant to imatinib, V299L and T315A retain sensitivity to imatinib in vitro and have not been previously described in imatinib-resistant cases, raising the potential utility of imatinib rechallenge in select dasaitinib-resistant cases. A significant finding of our studies is the evolution of five unique “compound” mutations (i.e. greater than one mutation on a DNA strand) in the BCR-ABL kinase domain of patients treated sequentially with imatinib and dasatinib. It is noteworthy that although the imatinib-sensitive V299L and T315A mutations evolved in five cases, they were detected in the context of a pre-existing imatinib-resistant mutation in three of these cases, and these cases are therefore unlikely to respond to rechallenge with IM. The T315A mutation was detected in the context of 2 pre-existing IM-resistant mutations (M244V/L364I). Interestingly, in bone marrow transformation assays, the clinically-identified dasatinib-resistant M244V/L364I/T315A mutation was more potently oncogenic than non-mutated BCR-ABL, in contrast to the baseline imatinib resistant M244V/L364I, which like T315A in isolation, was less potent than native BCR-ABL Our studies of CML cases resistant to sequential kinase inhibitor therapy reinforce BCR-ABL kinase domain mutation as the predominant mechanism of resistance to kinase inhibitor therapy, and provide evidence that compound mutations acquired as a result of sequential therapy can not only limit further therapeutic options, but also create more biologically aggressive isoforms of BCR-ABL. Together, these findings provide a strong rationale for early treatment of CML with combinations of kinase inhibitors that have the capacity to collectively prevent selection of resistant kinase domain mutations.
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Cortes, Jorge, Elias Jabbour, Hagop Kantarjian, C. Cameron Yin, Jianqin Shan, Susan O'Brien, Guillermo Garcia-Manero, et al. "Dynamics of BCR-ABL kinase domain mutations in chronic myeloid leukemia after sequential treatment with multiple tyrosine kinase inhibitors." Blood 110, no. 12 (December 1, 2007): 4005–11. http://dx.doi.org/10.1182/blood-2007-03-080838.
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AbstractDasatinib and nilotinib are potent tyrosine kinase inhibitors (TKIs) with activity against many imatinib-resistant chronic myeloid leukemia (CML) clones with BCR-ABL kinase domain (KD) mutations, except T315I. We assessed for changes in the BCR-ABL KD mutation status in 112 patients with persistent CML who received a second-generation TKI after imatinib failure. Sixty-seven different KD mutations were detected before the start of therapy with a second TKI, with T315I seen in 15%. Equal numbers of patients received nilotinib or dasatinib following imatinib, and 18 received 3 TKIs. Response rates were similar for patients with and without mutations, regardless of mutation site except for T315I. Overall, 29 patients (26%) developed new KD mutations after therapy with a second (n = 24) or third (n = 5) TKI, but only 4 (4%) developed T315I. In 73% of cases, the KD mutations that persisted or developed following switch to new TKI were at sites also found in prior in vitro TKI mutagenesis assays. Although there is only a mild increase in mutation frequency with sequential TKI treatment, novel mutations do occur and mutation regression/acquisition/persistence generally reflects the in vitro differential sensitivity predicted for each TKI. In this study, there was no marked increase in development of T315I.
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Jené, Kim. "Therapieoption bei T315I-Mutation." InFo Onkologie 20, no. 2 (March 2017): 60. http://dx.doi.org/10.1007/s15004-017-5644-5.
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Soverini, Simona, Alessandra Gnani, Caterina De Benedittis, Ilaria Iacobucci, Claudia Venturi, Cristina Papayannidis, Mario Luppi, et al. "BCR-ABL kinase domain mutations and resistance in Ph+ acute lymphoblastic leukemia from the imatinib to the second-generation TKI era." Journal of Clinical Oncology 30, no. 15_suppl (May 20, 2012): 6531. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.6531.
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6531 Background: Advent of 2nd-generation TKIs has brought additional treatment options for Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients (pts). To analyze the changes they have determined in mutation frequency and type, we have reviewed the database recording the results of BCR-ABL mutation analyses done in our laboratory from 2004 through 2011. Methods: 781 tests on 258 pts were performed by direct sequencing. Results: 143 pts were analyzed because of imatinib resistance; 101 (71%) had one or more mutations (a single mutation in 91 pts; two mutations in 10 pts). Three mutation types were by far the most frequent: T315I (38 pts, 37%), E255K (19 pts, 18%) and Y253H (19 pts, 18%). Of 84 pts who had developed resistance to 2nd- or 3rd-line therapy with dasatinib, nilotinib or bosutinib after imatinib failure, 65 (77%) were positive for Bcr-Abl mutations; 30 (46%) carried multiple mutations (up to four) and in 19 of them (63%) this was consequence of multiple lines of therapy. The most frequent newly acquired mutation in this setting was the T315I, detected in 35/57 (61%) cases acquiring mutations on dasatinib. Mutation analysis was also performed in 15 resistant pts enrolled in a study of dasatinib as 1st-line treatment of Ph+ ALL; 12 pts were positive, 11 of them had a T315I. Taking advantage of a next-generation sequencer (Roche 454), allowing a high sensitive and quantitative mutation scanning of Bcr-Abl, serially collected samples from 24 selected cases who developed mutations and resistance to one or more TKIs were retrospectively analyzed to study the kinetics of expansion of mutant clones. Results will be presented. Conclusions: Although 2nd generation TKIs are more potent and have much fewer insensitive mutations, long-term disease control remains a problem and the T315I becomes an even tougher enemy. The high genetic instability fosters mutational events anytime during TKI treatment and some mutation types (T315I, Y253H) have been observed to emerge and take over very quickly (from <0.01% to 90% in one-two months). Supported by PRIN, AIL, AIRC, Fondazione CARISBO.
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Goranova-Marinova, Veselina, Alexander Linev, Hristo J. Ivanov, Ivan Zheljazkov, Vily Stoyanova, and Zhanet Grudeva-Popova. "Clinical characteristics, disease evolution and survival in patients with chronic myeloid leukemia, BCR-ABL1 (+) and T315I mutation." Folia Medica 63, no. 5 (October 31, 2021): 670–75. http://dx.doi.org/10.3897/folmed.63.e63366.
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Introduction: The T315I mutation in patients with chronic myeloid leukemia (CML) has been associated with therapeutic resistance and an unfavourable prognosis. Aim: To study the frequency of T315I mutation in patients with CML, BCR-ABL (+), their clinical characteristics, disease evolution, and median survival. Patients and methods: We studied 75 patients with CML and BCR-ABL1 (+). T315I mutation was detected by digital droplet PCR and BCR-ABL1 was analyzed by RT-PCR. A comparative analysis was performed by sex, age, disease phase, risk group, treatment, molecular response (MR), and median survival in T315I (+) and T315I (−) patients. Results: T315I mutation was detected in 11 patients (14.7%). No significant difference was found in the phase, risk group, and first-line therapy. A significantly higher proportion of T315I (+) did not achieve MR >3.5 log: 8 (72.7%) vs. 22 (34.4%) (p=0.023). The lowest mean BCR-ABL1 levels were significantly higher in the CML T315I (+) group compared to the CML T315I (−) group: 12.1±6.0 vs. 3.77±1.28 (p=0.009). The median survival of T315I (+) patients was significantly shorter: 73 months vs. 175 months (p<0.0001, CI 95%). Conclusions: Our data confirm the world experience on the frequency of T315I mutation, including the unfavourable evolution, resistance to TKI treatment and short survival. ddPCR is a highly sensitive method for early detection of genetic mutations which gives the chance for effective treatment.
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Chen, Chen, Na Xu, Wu WanEr, Liu Liang, Xuan Zhou, Huang JiXian, Yin Changxin, Rui Cao, Qifa Liu, and Liu Xiaoli. "The Clinical Characteristics and Efficacy of Chronic Myeloid Leukemia Patients with T315I Mutation." Blood 132, Supplement 1 (November 29, 2018): 5433. http://dx.doi.org/10.1182/blood-2018-99-114113.
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Abstract Background: The application of tyrosine kinase inhibitor (TKIs) has greatly improved the overall survival (OS) and quality of life of chronic myeloid leukemia (CML) patients.However, in the TKIs era, 10% to 25% of CML patients still develop TKIs resistance, and ABL kinase point mutations are the most common reason.Most of the ABL kinase region mutations resistant to imatinib could be alleviated by second generation TKIs, but the T315I mutation resistance to the first and second generation TKIs. Ponatinib is a multi-target tyrosine kinase inhibitor, which belongs to the third generation of TKI inhibitors,and is sensitive for CML or Ph postitive ALL patients with T315I mutation. But,how to apply ponatinib bridging graft or whether ponatinib preventive therapy is needed after transplantation is uncertainty. Methods: 18 CML patients with T315I mutation detected by ABL1 kinase region mutation in Southern Hospital from March 2013 to April 2018 were retrospectively analyzed.G-banding method was used for chromosome analysis and real-time quantitative PCR method was used to detect mutations in ABL1 kinase region by BCR-ABL1 fusion gene Sanger sequencer. Result:18 CML patients with T315I mutation :13 cases chronic phase (CP) ,2 cases in accelerated phase,3 cases in blastcrisis phase(BP); 9 cases in high risk group, 6 cases in middle risk group and 3 cases in low risk grou by Sokal score score system.15 patients by imatinib ,3 patients first-line treatment with dasatinib .In imatinib group, 13 cases conversed to dasatinib because of drug resistance or intolerance, and 5 cases (5 / 13) were converted to ponatinib because of T315I mutation.One case in dasatinib group converted to ponatinib because of T 315 I mutation.A total of 6 patients (6 / 18) were treated with ponatinib. 6 patients (6 / 18) treated by allogeneic hematopoietic stem cell transplantation (Allo-SCT).The median stage of T315I mutation was 12.5 m from the beginning of treatment to the detection of T 315I mutation in 18 patients.At the end of the follow-up, 8 cases died of recurrence and 10 survived: (CMR 2 cases, CHR 1 cases, PR 3 cases, NR 3 cases, 1 cases not regularly followed up, unable to evaluate the disease state), including 6 patients with PO treatment. Conclusion:The point of T315I mutation was detected in patients with CML resistance after long-term sequential therapy frequently. The recurrence rate was still high even if these patients experience allogeneic hematopoietic stem cell transplantation.However,these patients treatment with ponatinib before and after transplantation maybe reduce the recurrence rate and improve prognosis. Key words:Chronic myeloid leukemia;BCR-ABL;T315I;ponatinib. Disclosures No relevant conflicts of interest to declare.
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Nicolini, Franck E., Giovanni Martinelli, Dong-Wook Kim, Andreas Hochhaus, Michael J. Mauro, Jorge Cortes, Charles Chuah, et al. "Epidemiological Study on Survival of Chronic Myeloid Leukemia (CML) and Ph+ Acute Lymphoblastic Leukemia (ALL) Patients with T315I Mutation. Final Analysis." Blood 112, no. 11 (November 16, 2008): 188. http://dx.doi.org/10.1182/blood.v112.11.188.188.
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Abstract The BCR-ABL T315I mutation is one of the major mechanisms of resistance to tyrosine kinase inhibitors (TKIs). Limited data have suggested that patients harboring a T315I mutation have poor outcomes. The objectives of this study were to estimate overall (OS) and progression-free survival (PFS) for CML in chronic (CP), accelerated (AP), or blastic (BP) phase, and Ph+ ALL patients who developed a T315I mutation; and describe the treatment pattern after T315I detection. Methods: This was a retrospective, multicenter observational study. Eligible patients included CML and Ph+ ALL patients who developed T315I mutation between 1999 and 2008. The medical records of 222 patients from 9 countries (France, Italy, Korea, USA, Germany, Singapore, Denmark, UK and Japan) were abstracted, and Kaplan-Meier plots and Cox proportional hazard models were used for survival analysis. Results: Median age at T315I detection was 54 (range, 18–84) years; 57% were male; 75% were Caucasian and 22% were Asian. Before T315I detection, 97% patients received imatinib (25% as a 1st line) and 50% received second generation TKIs. 16% of patients had other mutations detected before T315I detection. The median time between TKI treatment start and T315I detection was 29 months for CP, 15 for AP, 6 for BP, and 9 for Ph+ ALL. After T315I detection, 56% patients received second generation TKIs (30% started after T315I detection), 39% received hydroxyurea (33% started after T315I detection), 35% received imatinib (13% started after T315I detection), 26% received cytarabine, 21% received investigational drugs including 11% MK-0457, 17% underwent stem cell transplantation, and 6% received interferon alpha (5% started after T315I detection). At the time of T315I detection, T315I formed the predominant clone in 87% of patients; 23% had additional mutations detected (11% of these P-loop mutations). OS and PFS from T315I mutation detection are summarized in Table 1. In a preliminary analysis, the following covariates were associated with worse OS in Cox proportional hazard model (adjusted hazard ratio, 95% confidence interval): older age (by median, 2.30, 1.04–5.09) in Ph+ ALL patients, female gender in BP (1.73, 0.96–3.10); worse performance status in Ph+ ALL (1+ vs. 0; 2.18, 1.02–4.68); and detection of T315I by direct sequencing (vs. other methods) in AP (3.03, 0.89–10.29) and Ph+ ALL (2.33, 1.06–5.12). The effect of different treatments on OS will be available at the time of presentation. Conclusion: These results confirm that survival of patients harboring a T315I mutation is dependent on the disease phase at T315I detection. No clear treatment pattern after T315I detection was observed. Age, gender, performance status, and techniques used for T315I detection might be important prognosis factors affecting OS across different phases of CML and Ph+ ALL. Table 1. OS and PFS of CML and Ph+ ALL patients from T315I detection CML CP (N=82) CML AP (N=38) CML BP (N=56) Ph+ ALL (N=46) * Median survival and survival rates were calculated using Kaplan-Meier method. Median follow up time (months) 12.4 15.2 3.0 3.6 Median OS (months) (95% CI) 22.4 (18.2, 48.5) 28.4 (15.9, 49.8) 4.0 (2.0, 5.0) 4.9 (3.4, 7.3) 1-year OS Rate (95% CI) 71% (58–80%) 69% (50–81%) 23% (13–36%) 12% (3–27%) Median PFS (months) (95% CI) 11.5 (9.2, 15.7) 22.2 (9.0, N/A ) 1.8 (1.2, 4.0) 2.5 (1.8, 3.6) 1-year PFS Rate (95% CI) 46% (34–57%) 56% (38–70%) 16% (7–27%) 7% (1–19%)
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Tauchi, Tetsuzo, Seiichiro Katagiri, Seiichi Okabe, Eishi Ashihara, Shinya Kimura, Taira Maekawa, and Kazuma Ohyashiki. "Combined Effects of a Pan-ABL1 Kinase Inhibitor, Ponatinib and Dasatinib Against T315I Mutant Forms of BCR-ABL1: In Vitro and In Vivo Studies." Blood 118, no. 21 (November 18, 2011): 2482. http://dx.doi.org/10.1182/blood.v118.21.2482.2482.
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Abstract Abstract 2482 Ponatinib is a third-generation ABL1 kinase inhibitor optimized using structure-based drug design to bind the inactive, DFG-out conformation of wild-type (WT)-BCR-ABL1 and T315I BCR-ABL1. Ponatinib provides an effective in minimizing resistance, however, certain BCR-ABL1 compound mutations such as Y235H/T315I and E255K/T315I are predicted to be recalcitrant to ponatinib. Combining ponatinib and dasatinib may provide several advantages, including enhanced efficacy and the potential to reduce the emergence of new resistant compound mutations. In the present study, we investigated the combined effects of ponatinib and dasatinib in mutant forms of BCR-ABL1-expressing cells. Co-treatment with ponatinib and dasatinib resulted in significantly more inhibition of growth than treatment with either agent alone in BaF3 cells expressing wt-BCR-ABL1 and BCR-ABL1 mutants (M244V, G250E, Q252H, Y253F, E255K, T315A, T315I, F317L, F317V, M351T, H396P). The observed data from the isobologram indicated the synergistic effect of simultaneous exposure to ponatinib and dasatinib including BaF3 cells expressing T315I. Initially, we performed random mutagenesis for BCR-ABL1 experiments (Blood, 109; 5011, 2007). Single-agent ponatinib completely suppressed outgrowth on resistant clones at 40 nM. As the immediate clinical application of ponatinib is in refractory BCR-ABL1 positive leukemia patients harboring T315I BCR-ABL1 mutation, we next performed resistant screens starting from T315I BCR-ABL1 BaF3 cells to identify BCR-ABL1 compound mutations. Compared with single agents, the combination of ponatinib and dasatinib was more effective at reducing the outgrowth of resistant cell clones. At the highest concentration of ponatinib (2 micro M), the compound mutation narrowed to Y253H/E225K/T315I in single cell by direct sequencing. To assess the mechanism of combination effect between ponatinib and dasatinib on T315I BCR-AB1L compound mutation-expressing cells, we used RNA interference to determine whether reduction of dasatinib off-target molecules such as KHS, Raf-1, B-Raf, ACK-1, FAK, EPHB4 may affect the growth inhibition. BaF3 cells expressing T315I BCR-ABL1 pretreated with Raf-1or EPHB4 siRNA showed enhanced growth inhibition with ponatinib. These results demonstrate that the enhanced growth inhibition by ponatinib and dasatinib in T315I-expressing cells may be mediated by Raf-1 and EPHB4. To assess the in vivo efficacy of ponatinib and dasatinib, athymic nude mice were injected s.c. with BaF3 cells expressing wt-BCR-ABL1 and BCR-ABL1 mutants (M244V, G250E, Q252H, Y253F, E255K, T315A, T315I, F317L, F317V, M351T, H396P). 5 days after injection (average tumor volume, 100 mm3), the mice were randomised into four groups (5 mice per group), with each group receiving either vehicle, ponatinib (30 mg/kg; q.d.), dasatinib (10 mg/kg; q.d.), ponatinib (30 mg/kg; q.d.) + dasatinib (10 mg/kg; q.d.). The ponatinib and dasatinib combination more effectively inhibited tumor growth in mice compared to either vehicle- or ponatinib- or dasatinib-treated mice. Histopathologic analysis of tumor tissue from ponatinib+dasatinib-treated mice demonstrated an increased number of apoptotic cells detected by TUNEL stain. This study provides comparative drug combination profiling analysis of the effect of ponatinib and dasatinib, these two compounds are clinical active for BCR-ABL1-positive leukemia. Both agents commonly target a number of important molecular pathways that regulate cell growth and survival. A single proapoptotic or antiproliferative pathway may not be critical for the therapeutic effects of ABL1 kinase inhibitors. The present findings have important implications for the clinical use of ponatinib and dasatinib as anti-leukemia agent either alone or in combination with other agents. Disclosures: No relevant conflicts of interest to declare.
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Polivkova, Vaclava, Nikola Curik, Hana Zizkova, Adela Benesova, Pavel Burda, Pavla Pecherkova, Eliska Motlova, et al. "DNA Analysis of Mutations in the Kinase Domain of BCR-ABL1 By Allele-Specific Digital PCR Is Highly Sensitive and Refines Prediction of Kinetics of Resistant CML Clones." Blood 132, Supplement 1 (November 29, 2018): 1743. http://dx.doi.org/10.1182/blood-2018-99-119710.
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Abstract Introduction: In chronic myeloid leukemia (CML) resistant to tyrosine kinase inhibitors (TKI), detection of mutations in the BCR-ABL1 kinase domain (KD) is routinely performed on transcript level. To determine the level of BCR-ABL1 KD mutation is important to follow kinetics of resistant CML cells and therapeutically prevent progression. However, the mutation types and levels are not always reliable predictors of subsequent dynamics of mutation-bearing clones and of corresponding clinical consequences (Willis, 2005; Khorashad, 2006; Preuner, 2012). DNA analysis enables more precise quantification of (sub)clonal levels and thus might be more reliable approach to monitor dynamics of BCR-ABL1 KD mutations. Aim: To study clonal evolution of resistant CML cells using genomic quantification of mutated BCR-ABL1 KD by droplet digital PCR (ddPCR). Methods: BCR-ABL1 mutation analysis on transcript level was performed using next generation sequencing (NGS) (Nextera XT; Illumina) and on DNA level using allele-specific ddPCR assays detecting T315I, E255K and Y253H (Bio-Rad). The level of genomic BCR-ABL1 mutation was determined as a copy number of mutation divided by a copy number of genomic BCR-ABL1 fusion. Quantification of genomic BCR-ABL1 was performed by ddPCR using patient-specific primers and probes designed to detect individual fusions. ALB (albumin) quantification was used as a control of DNA load/cell numbers. For analyses, mRNA and DNA extracted from KCL-22 cell line resistant to imatinib (IM) and from leukocytes of a patient who developed T315I during TKI therapy were used. Results: KCL-22 cell line is characterized by 2 Ph chromosomes and by ability to develop resistance by acquisition of BCR-ABL1 mutations early after the exposure to IM. We repeatedly found, that during early cultivation in the presence of IM, BCR-ABL1-T315I transcript increased up to maximum of 50%. Subsequently, after 2 months, BCR-ABL1-E255K transcripts became detectable and increased over time to 100%, while T315I decreased to un-detectable levels. To study the observed kinetics, we isolated 4 clones resistant to 4 µM IM that expressed 1) 50% of T315I, 2) 50% of E255K and 3) 30% of Y253H. In the fourth clone, no BCR-ABL1 mutation was detected, but mutation acquisition was found in KRAS, RUNX1 and ATRX. The levels of mutated BCR-ABL1 transcripts in mutation bearing clones remained stable over time. DNA analyses confirmed the same level of mutated BCR-ABL1 and revealed that in all resistant clones, only 1 Ph chromosome carried the BCR-ABL1 mutation (T315I, E255K or Y253H). Based on quantification of genomic BCR-ABL1 fusion and albumin we found, that the un-mutated BCR-ABL1 fusion was duplicated in Y253H clone, explaining the 30% level of Y253H. To follow a clonal evolution, we mixed the 4 KCL-22 resistant clones and analyzed BCR-ABL1 KD mutations at both mRNA and DNA levels during exposition to IM. We found that T315I clone overgrew other 3 clones in the mixture over time and 1 Ph chromosome remained mutated. These data confirm the T315I mutation being the most resistant; however, the data from the original cell culture, where the 100% E255K clone overgrew the 50% T315I cells, demonstrate, that a less resistant mutation might dominate the culture if present on both Ph chromosomes (as revealed by DNA analysis). We compared mRNA and DNA approach in 14 samples collected during individualized treatment management of a CML patient, who developed T315I during TKI therapy. The first mutation detection was during warning response preceded by eight samples negative by mRNA-NGS approach; DNA ddPCR analysis reliably detected T315I mutation in 7 of these 8 samples. Six mRNA positive samples were positive by DNA approach, which showed the same level of T315I. Conclusions: Allele-specific ddPCR together with quantification of BCR-ABL1 genomic fusion represents highly sensitive and reliable method providing fast and precise quantification of BCR-ABL1 mutations. A single DNA analysis is able to uncover clinically relevant events including BCR-ABL1 amplification or additional mutation acquisition, which presumably influence fitness of leukemic cells and clonal evolution during therapeutic interventions. The information provided by DNA mutational analysis may thus refine prediction of mutation kinetics and consequently improve management of progressed CML and Ph+ ALL. Support GACR 18-18407S, MZCR 00023736, AZV 15-31540, AZV 16-30186A Disclosures Klamova: Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Ernst:Novartis: Research Funding. Soverini:Incyte Biosciences: Consultancy; Novartis: Consultancy; Bristol Myers Squibb: Consultancy. Machova:Bristol-Myers Squibb: Consultancy, Other: Educational grant funding; Incyte: Consultancy; Novartis: Consultancy.
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Palani, Renuka, Richard M. Szydlo, Jane F. Apperley, Gareth Gerrard, Chrissy Giles, Simona Deplano, Alistair Reid, Letizia Foroni, and Dragana Milojkovic. "Clinical Outcome Following Change of Tyrosine Kinase Inhibitor (TKI) According to the Detection of an ABL Kinase Mutation." Blood 124, no. 21 (December 6, 2014): 4557. http://dx.doi.org/10.1182/blood.v124.21.4557.4557.
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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.
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Hughes, Timothy, Giuseppe Saglio, Giovanni Martinelli, Dong-Wook Kim, S. Soverini, Martin Mueller, A. Haque, et al. "Responses and Disease Progression in CML-CP Patients Treated with Nilotinib after Imatinib Failure Appear To Be Affected by the BCR-ABL Mutation Status and Types." Blood 110, no. 11 (November 16, 2007): 320. http://dx.doi.org/10.1182/blood.v110.11.320.320.
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Abstract Nilotinib is a rationally designed 2nd-generation bcr-abl inhibitor. It is ∼30-fold more potent than imatinib against wild-type bcr-abl and active against 32/33 imatinib-resistant bcr-abl mutants in preclinical models. In an open-label phase II study of nilotinib in imatinib-resistant or -intolerant CML-CP patients (pts), we assessed the occurrence of mutations and the efficacy stratified by BCR-ABL mutational status. Prior to therapy, 35 mutations affecting 28 amino acids in the BCR-ABL kinase domain were identified by direct sequencing in 39% (106/270) of the pts analyzed. The incidence of baseline mutation was higher in imatinib-resistant (100/183, 55%) versus imatinib-intolerant pts (6/86, 7%). After 12 months of therapy, complete hematologic response (CHR) was achieved in 85%, major cytogenetic response (MCR) in 60%, and complete cytogenetic response (CCR) in 45% of pts without baseline mutations versus 67, 49 and 29% of pts with mutations. Among patients with baseline mutations, responses were observed broadly in all genotypes identified, but rates of responses differed by the in vitro sensitivity of the mutant clone against nilotinib. Pts with sensitive mutations of ≤100 nM cellular IC50 had the best response rate and were comparable to pts without baseline mutations. Pts with less sensitive mutations (IC50 201–800nM:Y253H, E255K, E255V, F359C) had responses but the response rate were lower then those of the two other groups (IC50 101–200nM and 201–800nM). The nilotinib-resistant T315I mutation (IC50>800nM) was identified at baseline in 5 cases (one pt had a limited response followed by progression). The less sensitive mutations (IC50 201–800nM) and the T315I mutation occurred in 8% and 2% of all pts assessed for baseline mutations, respectively. With a median follow up of 12 months, progression occurred in 15% (25/164) versus 40% (42/106) of pts without and with baseline mutations. Nine of 18 with less sensitive baseline mutations and 3 of 5 with T315I progressed, but the baseline mutation most frequently associated with progression was F359V (7/9). In 67 cases of progression, mutational data at or within 3 months of progression were available in 28 cases. Among the 28 pts, 7 (25%) had no mutation; 9 (32%) had the same baseline mutation (including F359V in 3; Y253F/H in 3; E255K in 1; and T315I in 1). A further 12 (43%) pts showed new emerging mutations at progression, 4 with T315I, 4 E255K, 3 Y253H, and 1 F359C. The other 7 pts with emerging mutations had not progressed. In total 21 pts were found with emerging mutations, 19 (90%) had a different mutation at baseline. In summary, nilotinib responses were observed across a variety of BCR-ABL mutations. Preliminary data suggest that mutational status at baseline and/or the emergence of new mutations may influence disease progression. Less sensitive or resistant mutations represented 10% of the pt population and may be associated with less favorable responses. Longer follow up is required.
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Heinrich, Michael C., Jason R. Morich, Emmanuel J. Beillard, Courtney Fuller, Chad D. Galderisi, and Brian J. Druker. "A Novel, High-Throughput Assay for Detection of ABL T315I Mutations." Blood 108, no. 11 (November 1, 2006): 2334. http://dx.doi.org/10.1182/blood.v108.11.2334.2334.
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Abstract Mutations in the kinase domain of BCR-ABL result in impaired drug binding and are thought to be the leading cause of acquired resistance to the tyrosine kinase inhibitor imatinib (Gleevec®). While imatinib is a highly effective therapy in all stages of chronic myelogenous leukemia (CML), relapse after an initial response is common in patients with advanced disease. The T315I point mutation is one of the most common imatinib-resistant mutations and patients with this mutation are also resistant to two second generation tyrosine kinase inhibitors, dasatinib and nilotinib. Recently, the U.S. FDA approved dasatinib for treatment of imatinib-resistant, Philadelphia chromosome-positive acute and chronic leukemias. Thus, appropriate detection of this mutation is essential to optimal management of patients with imatinib resistance and may be useful for clinical trials of agents that target patients with the T315I mutation. Current methods for mutation detection, such as direct DNA sequencing, are not sensitive enough for detection of point mutations at low levels of BCR-ABL transcript. Conversely, ultrasensitive detection methods such as allele specific PCR (AS-PCR) may be too sensitive and can be plagued by false positive test results. In addition the clinical relevance/significance of mutation detection at ultra sensitive levels ( < 1% mutant) is questionable and not yet known. We developed a novel T315I mutation detection assay, using Fluorescent Resonance Energy Transfer (FRET)-based hybridization probes and melting curve analysis. BCR-ABL amplicons generated from a first round of PCR are amplified using primers flanking the ABL kinase region encoding for codon 315. The resultant amplicon is hybridized with fluorescein-labelled anchor probe and a LC Red 640-labelled T315I mutation specific probe. Wild-type and T315I mutant amplicons are distinguished by melting curve analysis (Roche Light Cycler 480™). Using a series of plasmid and cell line dilutions we determined that the sensitivity of this assay for detection of T315I mutations was 5–10%. Using patient-derived samples we were able to successfully genotype samples containing as few as 20–50 BCR-ABL transcripts. To date, the assay sensitivity and specificity are 100%. The assay is performed in a plate based format (96 or 384 wells) and commercially available software allows automated genotype assignment. This approach is suitable for high-throughput detection of T315I mutations for clinical management of CML patients and/or screening of patients to determine eligibility for clinical studies.
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Aggoune, Djamel, Nathalie Sorel, Marie Laure Bonnet, Jean-Claude Chomel, and Ali G. Turhan. "A Niche-Based Cell Mutagenesis Assay Identifies ABL-Kinase Mutations Appearing in BCR-ABL T315I-Mutated Leukemic Cells Treated with Ponatinib." Blood 120, no. 21 (November 16, 2012): 2436. http://dx.doi.org/10.1182/blood.v120.21.2436.2436.
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Abstract Abstract 2436 The occurrence of ABL-kinase (ABL-K) mutation is a major persisting concern in CML patients treated with tyrosine kinase inhibitor (TKI) therapies. Leukemic stem cell niche can protect the leukemic cells by providing survival and/or quiescence signals but also could favor the occurrence of ABL-kinase mutations. Amongst the ABL-kinase mutations, T315I is one of the most problematic as it induces resistance to all three clinically accepted TKI (Imatinib, Dasatinib, Nilotinib) and has been shown to occur at the level of stem cells (Chomel et al, Leuk lymphoma 2010). Ponatinib (Formerly AP24534) is a multi-targeted TKI which overcomes resistance to T315I mutation as well as to other ABL-kinase mutations. To model the role of the niche in the context of T315I in patients treated with Ponatinib, we designed a niche-based cell mutagenesis assay in the human hematopoietic UT7 cells engineered to express BCR-ABL (UT7-BCR-ABL native cells) and BCR-ABL with T315I (UT7-T315I), via retrovirus mediated gene transfer. Western blot analyses demonstrated that these cells express BCR-ABL and UT7 clones harboring the T315I mutation were resistant to Imatinib, Dasatinib and Nilotinib but sensitive to Ponatinib. We have treated UT7-T315I cells with N-ethyl-N-nitrosourea (ENU, 50microg/ml) for 24 hours and seeded them on previously prepared MS5 stromal cells (plate of 96 wells) in the presence of Ponatinib (30 nM final concentration). As a control, ENU-treated cells were also cultured in the absence of MS5 feeders and Ponatinib-based selection was performed in the same conditions. The same niche-based assay was also performed in the UT7-BCR-ABL native cells and selection process has taken place in the presence of Imatinib (2 microM). Cell medium was changed every week with addition of Ponatinib (UT7-T315I cells) or Imatinib (UT7-BCR-ABL native cells). At week+4, Ponatinib-resistant and IM-resistant wells cultured in the presence (MS5+) or in the absence (MS5-) of the hematopoietic niche were enumerated. In UT7-BCR-ABL native plates, the numbers of IM-resistant outgrowth was identical in MS5+ versus MS5- conditions (51/95 and 54/96, respectively). On the other hand, in UT7-T315I plates, there was a major difference in the numbers of Ponatinib-resistant T315I cells in MS5+ (96/96, 100% survival) as compared to MS5- conditions (65/96, 62 % survival). IM and Ponatinib-resistant clones were amplified and 48 clones resistant to Ponatinib (MS5+ n= 24; MS5- n= 24) and 48 clones resistant to IM (MS5+ n= 24: MS5- n=24) were screened for ABL-K mutations using a denaturing gradient gel electrophoresis assay followed by direct sequencing of the ABL-kinase domain. In the native BCR-ABL mutagenesis assay (with Imatinib), all clones were found mutated, in the presence or absence of MS5 stromal cells. Most of them were mutated in the P-loop region (E255K, G250E, Y253H). In addition, we have found 3 clones harboring the T315I mutation (1 in MS5- and 2 in MS5+ conditions). Concerning T315I clones surviving to Ponatinib, in addition to the T315I mutation, most of the resistant clones harbored an additional P-loop mutation which occrred in the absence or presence of MS5. Moreover, in two clones, two additional mutations were detected in addition to the T315I mutation. Interestingly, when Ponatinib-resistant cells were switched from MS5+ conditions to MS5- conditions, Ponatinib resistance could be abrogated in some but not all cases. In conclusion, our assay shows that the hematopoietic stem cell niche could play a crucial role in conferring resistance to Ponatinib, not only via the occurrence of novel mutations but also by providing survival signals. Preliminary results also suggest that the hematopoietic niche could facilitate the emergence of T315I mutation in cells expressing native BCR-ABL. These results could be important to study the mechanisms of the occurrence and selection of ABL-K mutations in patients treated with TKI including Ponatinib, and to develop niche-targeted therapies to overcome TKI-resistance in CML. Disclosures: Turhan: Novartis, Bristol Myers Squibb: Honoraria, Research Funding.
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Branford, Susan, Junia V. Melo, and Timothy P. Hughes. "Selecting optimal second-line tyrosine kinase inhibitor therapy for chronic myeloid leukemia patients after imatinib failure: does the BCR-ABL mutation status really matter?" Blood 114, no. 27 (December 24, 2009): 5426–35. http://dx.doi.org/10.1182/blood-2009-08-215939.
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Abstract Preclinical studies of BCR-ABL mutation sensitivity to nilotinib or dasatinib suggested that the majority would be sensitive. Correspondingly, the initial clinical trials demonstrated similar response rates for CML patients after imatinib failure, irrespective of the mutation status. However, on closer examination, clinical evidence now indicates that some mutations are less sensitive to nilotinib (Y253H, E255K/V, and F359V/C) or dasatinib (F317L and V299L). T315I is insensitive to both. Novel mutations (F317I/V/C and T315A) are less sensitive/insensitive to dasatinib. We refer to these collectively as second-generation inhibitor (SGI) clinically relevant mutations. By in vitro analysis, other mutations confer a degree of insensitivity; however, clinical evidence is currently insufficient to define them as SGI clinically relevant. Here we examine the mutations that are clearly SGI clinically relevant, those with minimal impact on response, and those for which more data are needed. In our series of patients with mutations at imatinib cessation and/or at nilotinib or dasatinib commencement, 43% had SGI clinically relevant mutations, including 14% with T315I. The frequency of SGI clinically relevant mutations was dependent on the disease phase at imatinib failure. The clinical data suggest that a mutation will often be detectable after imatinib failure for which there is compelling clinical evidence that one SGI should be preferred.
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Kim, Wan-Seok, Il-Young Kweon, Soo Hyun Kim, Hyun Gyung Goh, Jeong Lee, Se Eun Jung, Dongho Kim, et al. "Clinical Characteristics and Outcome of 20 Imatinib Mesylate Resistant Patients with T315I BCR-ABL Mutation." Blood 110, no. 11 (November 16, 2007): 1954. http://dx.doi.org/10.1182/blood.v110.11.1954.1954.
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Abstract Most CML patients are sensitive to imatinib mesylate (IM), however, a small fraction develop resistance, mostly through the emergence onset of BCR-ABL mutation. Dasatinib and nilotinib, novel tyrosine kinase inhibitors (NTKIs), are active against most of IM resistant BCR-ABL kinase domain mutants except T315I. Although T315I mutation has been highly resistant to IM and both NTKIs, precise clinical characteristics and outcome have not been known yet. A total of 81 patients with various phases of CML who were intolerant or were resistant to IM (M:F-52:29, median age: 43 years, range; 12–74 years) were enrolled in this study between May 2005 and Sep 2006. Eighty one patients had received dasatinib and/or nilotinib in phase II or extended access program. At the time of IM failure and every 3 months during NTKIs treatment, mutations were screened by both ASO-PCR and direct sequencing. Clinical characteristics and outcome probabilities were statistically analyzed. T315I was detected in 20 of 31 patients (65%) harboring kinase domain mutations; 9 patients had mutation before NTKIs treatment and 11 patients were developing mutation after NTKIs treatment. Median age was 33 years (range, 19–74 years). Transcripts were Major BCR for all patients and 8 patients had received prior interferon therapy. 6 patients had additional chromosomal abnormalities (ACA) at diagnosis. At the time of T315I emergence, 8 patients were CP, 6 patients were AP, and 6 in BC (4 in myeloid and 2 in lymphoid). Median accumulate dose per day was 398.8 mg/day (range, 205.3–600.0 mg/day). 13 patients were received dasatinib, 5 received nilotinib and 2 received both dasatinib and nilotinib. The best response to NTKIs was complete cytogenetic response (CCyR) in 6 and complete hematologic response (CHR) in 8. Median overall survival (OS) from NTKIs start was 7.0 months for advanced phase and 12.3 months for chronic phase. The 3 year survival rate was 21.5%, with median value was 8.4 months in all patients. With NTKIs therapies [median follow-up, 9.7 months (range, 0.7–27.3 months)], 8 patients are alive (40%); 6 patients are alive with active disease and 2 patients are alive with ongoing response. 10 patients died of disease progression and 2 patients died of pneumonia. Low grade disease phase at discovery of T315I mutation (log-rank P=0.0237) demonstrated significantly favorable outcome but after NTKIs treatment, there was no difference in OS between disease phases (log-rank P=0.271). And there was no difference in OS between T315I mutation and other mutations (P=0.147). However the ACA at diagnosis (P=0.029) and achievement of best CCyR during NTKIs treatment (P=0.085) was different in OS. And there was significant difference in survival between patients with and without ACA and achievement of best CCyR during NTKIs treatment (log-rank P=0.0235), suggesting they have prognostic influences on survival as T315I mutation. In summary, our findings confirm that ACA at diagnosis and achievement of best CCyR during NTKIs treatment are statistically significant prognostic information with respect to survival probability at patients with T315I mutation. However T315I mutation was highly resistant to NTKIs as well as IM, and are associated with poor outcome. As diverse outcomes in patients with T315I mutation have been demonstrated, different strategies such as MK-0457 and/or novel T315I mutation inhibitor should be applied.
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Deininger, Michael W., Jane F. Apperley, Christopher Kevin Arthur, Charles Chuah, Andreas Hochhaus, Hugues De Lavallade, Jeffrey H. Lipton, et al. "Post Hoc Analysis of Responses to Ponatinib in Patients with Chronic-Phase Chronic Myeloid Leukemia (CP-CML) By Baseline BCR-ABL1 Level and Baseline Mutation Status in the Optic Trial." Blood 138, Supplement 1 (November 5, 2021): 307. http://dx.doi.org/10.1182/blood-2021-145995.
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Abstract Introduction: Ponatinib is a third-generation, pan-inhibitory tyrosine kinase inhibitor (TKI) designed to potently inhibit BCR-ABL1 with or without any single resistance mutation, including T315I. Patients (pts) with resistant disease or with T315I BCR-ABL1 mutations respond inadequately to earlier-generation BCR-ABL1 TKIs, leading to poor survival outcomes. OPTIC (Optimizing Ponatinib Treatment in CP-CML, NCT02467270; ongoing) is a phase 2 trial evaluating the safety and efficacy of ponatinib in pts with CP-CML whose disease is resistant to 2 or more TKIs or who have a T315I mutation. Here, we present an in-depth post hoc analysis of OPTIC trial pt responses by baseline disease burden and mutation status. Methods: Pts with CP-CML resistant to ≥2 TKIs or with the BCR-ABL1 T315I mutation were randomized to ponatinib starting doses of 45 mg (cohort A; 45 mg → 15 mg), 30 mg (B; 30 mg → 15 mg), and 15 mg (C) once daily. Doses were reduced to 15 mg after achievement of ≤1% BCR-ABL1 IS in cohorts A and B. Pts could re-escalate to their original starting dose for loss of response. The primary endpoint is ≤1% BCR-ABL1 IS at 12 months; secondary endpoints include cytogenetic and molecular responses and safety outcomes. In this analysis, outcomes are analyzed by baseline T315I mutation status and baseline disease burden in the intent-to-treat (ITT) population. BCR-ABL1 mutations were assessed by Sanger sequencing at a central laboratory (MolecularMD, Portland, OR, USA). Results: 283 pts were randomized (A/B/C: n=94/95/94). At baseline, 84.1% of pts had a high (>10% BCR-ABL1 IS) disease burden; 23.8% had T315I mutation, 17.0% had a mutation other than T315I, and 57.8% had no mutation. The 45 mg →15 mg cohort showed the highest ≤1% BCR-ABL1 IS response rates by 36 months (Figure 1). Subanalysis of pts across the 3 dosing arms showed that pts with T315I mutations had the highest ≤1% BCR-ABL1 IS response rates (60%) by 3 years with the 45 mg → 15 mg dose compared with the other cohorts, with a trend toward higher progression-free survival (PFS) in the 45 mg →15 mg arm (Table 1). Across all 3 cohorts, 97 pts without T315I mutations (ie, no mutation or with mutations other than T315I) achieved ≤1% BCR-ABL1 IS. Median duration of response (mDoR) for pts with a T315I mutation at baseline was 27 months for pts (n=15) in the 45 mg → 15 mg cohort and 12 mo for pts (n=5) in the 30 mg → 15 mg cohort. For pts without T315I mutations, the mDoR was not reached. Across all 3 cohorts, 79% of pts who achieved ≤1% BCR-ABL1IS maintained this response during the study. A total of 25 patients lost responses (Table 2). Of those who lost response, 11 had T315I, 10/11 dose re-escalated; of those who re-escalated, 6/10 regained ≤1% BCR-ABL1IS after dose re-escalation (Table 2). The most common nonhematologic treatment-emergent adverse events (TEAEs) in the ITT population for all cohorts combined were arterial hypertension (28%), headache (18%), and lipase increased (17%). The most common hematologic TEAEs were thrombocytopenia (40%), neutropenia (26%), and anemia (19%). Overall, 6.0% of pts experienced a treatment-emergent arterial occlusive event (TE-AOE); 4.6% experienced a Grade ≥3 TE-AOE. Conclusions: Previous analyses established these largely resistant pts achieved high response rates when treated with ponatinib. Consistent with this, the OPTIC post hoc analysis showed clinical benefit across all 3 dosing regimens regardless of T315I mutation status at baseline; the 45 mg →15 mg cohort showed the highest response rates regardless of baseline disease burden (as assessed by BCR-ABL1 IS levels). Regardless of T315I mutation status, most patients were able to maintain their response after dose reduction to 15 mg/day upon achieving BCR-ABL1IS ≤1%. Compared with patients without a T315I mutation, patients with a T315I mutation at baseline were more likely to lose their response upon dose reduction; however, 60% of responses were regained with dose re-escalation. For pts with T315I mutations, PFS was greater with 45 mg → 15 mg dosing compared with the other arms; for pts without T315I mutations, all 3 doses showed robust PFS and OS outcomes. The data presented further support the benefit of using ponatinib post-2nd generation TKI for pts with resistant disease regardless of baseline T315I mutation status. Figure 1 Figure 1. Disclosures Deininger: Incyte: Consultancy, Honoraria, Research Funding; Fusion Pharma, Medscape, DisperSol: Consultancy; Blueprint Medicines Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding; SPARC, DisperSol, Leukemia & Lymphoma Society: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding; Sangamo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Research Funding. Apperley: Incyte, Pfizer: Honoraria, Research Funding, Speakers Bureau; Bristol Myers Squibb, Novartis: Honoraria, Speakers Bureau. Chuah: Steward Cross: Honoraria; Bristol Myers Squibb: Honoraria, Research Funding; Pfizer: Other: Travel, Research Funding; Novartis, Korea Otsuka Pharmaceutical: Honoraria. Hochhaus: Bristol-Myers Squibb: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Incyte: Research Funding. De Lavallade: Pfizer, Novartis.: Honoraria; Bristol Myers Squibb, Incyte: Honoraria, Research Funding. Lipton: Bristol Myers Squibb, Ariad, Pfizer, Novartis: Consultancy, Research Funding. Lomaia: Novartis: Honoraria; Pfizer: Honoraria; BMS: Honoraria; Pharmstandard: Honoraria. McCloskey: Takeda: Consultancy, Speakers Bureau; Jazz: Consultancy, Speakers Bureau; BMS: Honoraria, Speakers Bureau; COTA: Other: Equity Ownership; Incyte: Speakers Bureau; Amgen: Speakers Bureau; Novartis: Consultancy; Pfizer: Consultancy. Mauro: Bristol Myers Squibb: Consultancy, Research Funding; Takeda: Consultancy; Novartis: Consultancy, Research Funding; Sun Pharma / SPARC: Research Funding; Pfizer: Consultancy. Moiraghi: Novartis, Pfizer, Takeda: Speakers Bureau. Pavlovsky: Novartis, BMS, Pfizer, Takeda: Speakers Bureau. Rosti: Pfizer: Research Funding, Speakers Bureau; Bristol Myers Squibb, Incyte, Novartis: Speakers Bureau. Rousselot: Incyte, Pfizer: Consultancy, Research Funding. Undurraga: AbbVie, Janssen, Novartis, Pfizer, Roche: Other: Advisory Board; Janssen, Novartis, Pfizer: Speakers Bureau. Lu: Takeda: Current Employment. Vorog: Takeda: Current Employment. Cortes: Takeda: Consultancy, Research Funding; Sun Pharma: Consultancy, Research Funding; Bristol Myers Squibb, Daiichi Sankyo, Jazz Pharmaceuticals, Astellas, Novartis, Pfizer, Takeda, BioPath Holdings, Incyte: Consultancy, Research Funding; Bio-Path Holdings, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.
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Kakihana, Kazuhiko, Kazuteru Ohashi, Yuho Najima, Taku Kikuchi, Yasunobu Nagata, Toshimitsu Ueki, Takeshi Kobayashi, et al. "Quantitative Monitoring of T315I BCR-ABL Mutation by the Invader Assay." Blood 110, no. 11 (November 16, 2007): 4543. http://dx.doi.org/10.1182/blood.v110.11.4543.4543.
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Abstract Although imatinib mesylate has a highly inhibitory effect against BCR-ABL kinase, primary and secondary refractoriness have been observed. Point mutation within the kinase domain of BCR-ABL is one of the most important drug-resistant mechanisms. Of 25 reported point mutations, T315I BCR-ABL mutation might be dismal in clinical settings because it mediates clinical resistance to imatinib, nilotinib, and dasatinib, and the only established therapeutic option is hematopoietic stem cell transplantation at this moment. However, there is no information about the kinetics of this mutated clone. Here we developed quantitative Invader assay to monitor T315I BCR-ABL transcript. By using fluorescent resonance energy transfer system, the amount of released 5′ flap is measured as signal intensity, which enables us to calculate the percentage of T315I products with standard curve. Using this assay, we serially monitored T315I BCR-ABL transcript in a CML patient whose BCR-ABL transcript was still detectable (more than 100 copies/microgram RNA) 3 months after starting imatinib therapy. Although we have continued to monitor T315I BCR-ABL transcripts in 13 patients (chronic phase) up to 10 months, there was no patients who appear to be apparently resistant to imatinib due to a given T315I mutant, so far. In contrast, in a case of Ph+ ALL was being treated with chemotherapy including imatinib, we serially monitored both wild type and T315I BCR-ABL transcripts and observed the increased level of T315I transcript during relapse (0%at the time of diagnosis and 54.8% at relapse). Thus, our new strategy could be a useful tool to study the kinetics of the mutant clone and the pharmacokinetics of the drug resistant to T315I mutation.
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Cilloni, Daniela, Enrico Bracco, Sonia Carturan, Valentina Rosso, Valentina Campia, Alessandra Favole, Chiara Calabrese, et al. "Design and Application of a Novel PNA Probe for the Detection At a Single Cell Level of BCR-ABL T315I Mutation in Chronic Myeloid Leukemia Patients." Blood 120, no. 21 (November 16, 2012): 3732. http://dx.doi.org/10.1182/blood.v120.21.3732.3732.
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Abstract Abstract 3732 Background: The BCR-ABL T315I mutation at the gatekeeper residue is frequent in advanced phases of chronic myeloid leukemia (CML) and is one of the main cause of resistance to Tyrosine Kinase Inhibitors (TKI) by disrupting important contact points between the drugs and the enzyme. Although this mutation can be detected by different techniques and at different levels of the mutated clone, the prognostic significance of the absolute amount of the mutated allele is widely unknown. The aim of the study was to develope a novel assay based on peptide nucleic acid (PNA) technology coupled to immuno-fluorescence microscopy (PNA-FISH) for the specific detection at a single cell level of T315I mutation thus improving both the diagnostic resolution and the knowledge on the behaviour of the mutated clone. Methods: We designed a fluorescently-labelled PNA probe, coupled to FISH technology, which allows to distinguish with a high degree of specificity between CD34+ progenitor stem cells harbouring T315I mutation or the wild type form of BCR-ABL. CD34+ cells were enriched from CML patients who were positive for T315I mutation, or from patients who lost T315I mutation after ponatinib treatment. In addition we tested CML patients without T315I mutation as control and BM samples from healthy donors to establish the specificity of the method. CD34+ progenitors cells were enriched by MACS and then cytospun onto slides and hybridized with human species-specific fluorescinated 15 base pairs (bp)-long oligo-PNA, specifically recognizing the BCR-ABL T315I sequence. Slides were analyzed by fluorescence confocal microscopy. Results: We found, with a rather wide variability occurring among patients, a percentage of mutated CD34+ cells ranging from 3 to 90%. In addition these data indicate that fluorescinated BCR-ABL T315I/PNA probe displays a very high specificity towards a single base-pair mismatch. Interestingly, when evaluating the presence of T315I positive cells collected from a patient who lost the mutation (evaluated by sequencing) after ponatinib therapy and acquired T317 mutation a small amount of T315I positive CD34+ cells were still present. This percent did not exceeded 2% of the total CD34+ cell population. Importantly, the lack of positivity detected in CD34 positive cells from CML patient without mutations or in 20 healthy subjects demonstrates a high specificity of this method. Conclusions: BCR/ABL T315I/PNA probe method displays high specificity and reliability in discriminating cell subpopulations harbouring the mutation. In addition, it allows to analyze the CD34+ population at the single cell level and to monitor the behaviour of the clone at the stem/progenitor cell level. This approach allows to monitor longitudinally the evolution of the mutated population over time, the response to specific drugs active against T315I mutants and to characterization the mutated stem/progenitor cell compartment in patients with CML. Disclosures: Saglio: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy.
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Parker, Wendy T., David T. O. Yeung, Alexandra L. Yeoman, Haley K. Altamura, Bronte A. Jamison, Chani R. Field, J. Graeme Hodgson, et al. "The impact of multiple low-level BCR-ABL1 mutations on response to ponatinib." Blood 127, no. 15 (April 14, 2016): 1870–80. http://dx.doi.org/10.1182/blood-2015-09-666214.
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Key Points The association between multiple BCR-ABL1 mutations and inferior response to nilotinib/dasatinib was not seen with ponatinib therapy. However, chronic phase patients with T315I plus additional mutation(s) did have poorer responses to ponatinib than those with T315I only.
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Okabe, Seiichi, Tetsuzo Tauchi, Yuko Tanaka, Juri Sakuta, and Kazuma Ohyashiki. "Anti-Leukemic Activity of Axitinib Against Cells Harboring the BCR-ABL Point Mutation." Blood 126, no. 23 (December 3, 2015): 2769. http://dx.doi.org/10.1182/blood.v126.23.2769.2769.
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Abstract Introduction: The BCR-ABL1 fusion gene is a causative oncogene in chronic myeloid leukemia (CML) and 30-50% of acute lymphoblastic leukemia cases. Although ABL tyrosine kinase inhibitors (ABL TKI) such as imatinib have improved CML treatment, such therapies cannot cure patients with Philadelphia chromosome (Ph)-positive leukemia because of leukemia stem cells. Moreover, some patients develop BCR-ABL point mutations and become resistant to ABL TKI therapy. In particular, the ABL kinase domain mutation T315I is resistant to imatinib and second-generation ABL TKIs (e.g., nilotinib, dasatinib, and bostinib). Accordingly, this mutation is often found in patients with TKI-resistant disease. A third-generation ABL TKI, ponatinib, was recently developed. Ponatinib is a potent oral tyrosine kinase inhibitor that affects both unmutated and mutated BCR-ABL; it is effective against T315I mutant cells and has been approved for TKI-resistant or intolerant CML and Ph-positive ALL patients.Recently, the vascular endothelial growth factor receptor (VEGFR) inhibitor axitinib was found to exhibit anti-leukemic activity against T315I-mutant disease. Axitinib is an orally active and potent TKI of VEGFRs 1, 2, and 3 and inhibits BCR-ABL1, especially the T315I variant, via a distinct binding conformation. Materials and methods: In this study, we investigated whether axitinib could suppress ponatinib-resistant compound mutant cells harboring the T315I mutation and primary samples. Results: A 72 h axitinib treatment inhibited the growth of Ba/F3 T315I cells (Figure 1A). Immunoblot analysis of axitinib-treated cells revealed dose-dependent decreases in BCR-ABL, the downstream molecule CrkL, and ribosomal S6 protein phosphorylation and increases in caspase 3 and Poly (ADP-ribose) polymerase (PARP) activity. Ponatinib and axitinib also induced apoptosis, significantly increased caspase activity, and reduced Akt activity. In contrast, clinically available concentrations of axitinib did not inhibit the growth of ponatinib-resistant Ba/F3 cells. Immunoblot analysis revealed that BCR-ABL, Crk-L, and S6 kinase phosphorylation were not inhibited by axitinib or ponatinib. Similarly, no increase in caspase activity or decrease in Akt activity was observed following axitinib treatment, and neither ponatinib nor axitinib affected apoptosis in these cells. We next evaluated primary T315I-mutant and ponatinib-resistant compound mutant samples. Axitinib potently inhibited the growth of T315I mutant primary cells in a dose-dependent manner. Immunoblot analysis further revealed reduced Crk-L and S6 kinase phosphorylation after axitinib or ponatinib treatment. In contrast, the growth of ponatinib-resistant primary cells was not affected by ponatinib or axitinib. Immunoblotting revealed that neither ponatinib nor axitinib affected the phosphorylation of Crk-L and S6 kinase in ponatinib-resistant cells. Conclusion: In CML, ABL TKI resistance is frequently caused by ABL kinase domain mutations. The T315I mutation is resistant to all ABL TKIs except ponatinib. Although axitinib, which is currently being investigated for efficacy in patients with Ph-positive T315I-mutant leukemia, induced apoptosis in T315I-mutant cells, it was ineffective against cells with a compound mutation including T315I. Current evidence to direct the management of ABL TKI-resistant disease, particularly those harboring T315I and compound mutations, is limited. New molecular-targeted drugs and an understanding of ABL TKI resistance mechanisms are required to apply an appropriate therapeutic approach. Disclosures No relevant conflicts of interest to declare.
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Santos, Fabio P. S., Jorge Cortes, Charles Koller, and Elias Jabbour. "Combination Therapy with Tyrosine Kinase Inhibitors and Agents with Different Mechnisms of Actions Is Effective in a Patient with Chronic Myeloid Leukemia Harboring the T315l BCR - ABL1 Mutation." Blood 114, no. 22 (November 20, 2009): 4282. http://dx.doi.org/10.1182/blood.v114.22.4282.4282.
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Abstract Abstract 4282 Mutations of BCR-ABL1 have been observed in 50% of patients with chronic myeloid leukemia (CML) who develop resistance to imatinib. The gate-keeper mutation T315I is one of the mutations with universal resistance to imatinib and to the second-generation tyrosine kinase inhibitors (TKI) that are approved for the treatment of patients with imatinib failure. The use of new kinase inhibitors with in vitro activity against T315I mutation as well as other agents with different mechanisms of actions is being evaluated in clinical trials. We report the case of a 57-year old man that was diagnosed with CML in 2003. Patient received initial therapy with standard-dose imatinib that was subsequently increased to 800 mg daily. He did achieve a complete cytogenetic response (CCyR) 9 months post dose escalation. He was followed by RT-PCR for BCR-ABL1.. In May, 2007, the patient BCR-ABL1/ABL1 ratio increased to 16.38 but the patient remained in CCyR. BCR-ABL1 sequencing revealed the T315I mutation in 100% of cells (Figure 1). One month later the patient lost CCyR (5% Philadelphia-positive [Ph+] cells) and the BCR-ABL1/ABL1 ratio was 5.08. The patient was started on the T315I specific inhibitor KW-2449 (100 mg orally twice daily for 14 days, every 3 weeks). Patient had a progressive decline in percentage of cells with the T315I mutation (Figure 1). However, at the same time he had an increase in percentage of Ph+ cells. In September, 2007, three months after starting therapy with KW-2449, patient had no cytogenetic response (80% Ph+ cells, PCR for BCR-ABL1 ratio > 100) and the T315I mutation was undetectable. At that time, a new ABL1 sequencing revealed the F359I mutation (no quantification was done). Patient was maintained on KW-2449 for the next 6 months, without significant improvement in cytogenetic response nor BCR-ABL1 ratio, but the clone with the T315I mutation did not reappear. In February, 2008, the patient lost hematologic response and presented with an elevated white blood cell count of 22×109/L. The F359I mutation was still present. Therapy with KW-2449 was stopped and the patient started dasatinib 100 mg/day and Interferon-a 3,000,000 units. Three months later, the patient acheived CCyR with a BCR-ABL1/ABL1 ratio of 0.05. At the last evaluation, 16 months after the start of dasatinib and interferon combination, the patient was maintaining CCyR and major molecular response. In conclusion, this case illustrates the benefit of the use of combination therapy, mainly TKI and agent with different mechanism of action either sequentially (TKI followed by KW-2449) or concomitantly (TKI + interferon) in eradicating resistant disease with T315I clone. Figure 1 Serial Monitoring of Ph+ Cells, T315I Cells and BCR-ABL1/ABL1 Ratio Figure 1. Serial Monitoring of Ph+ Cells, T315I Cells and BCR-ABL1/ABL1 Ratio Disclosures: Cortes: Novartis: Research Funding. Jabbour:Novartis: Speakers Bureau; Bristol Myers Squibb : Speakers Bureau.
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Widyastuti, Reni, Melva Louisa, Ikhwan Rinaldi, Riki Nova, Instiaty Instiaty, and Rizky Priambodo. "Mutation Analysis of ABL1 Gene and its Relation to the Achievement of Major Molecular Response in Indonesian Chronic Myeloid Leukemia Patients." Current Pharmacogenomics and Personalized Medicine 17, no. 1 (April 28, 2020): 48–54. http://dx.doi.org/10.2174/1875692117666190925115852.
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Background: Imatinib mesylate is the first tyrosine kinase inhibitor approved for chronic myeloid leukemia (CML) therapy. Imatinib is an effective drug. However, previous studies have shown that about 20-30% of patients eventually would develop resistance to imatinib. Approximately 40% of imatinib resistance is associated with BCRABL kinase domain mutation. One of the most common and serious variations account for imatinib response is T315I of ABL1 gene. Objective: The study aimed to examine the association of T315I mutation with the ABL1 gene and its relation to major molecular response (MMR) achievement in CML patients. This study also examined other mutations adjacent to T315I, i.e., F311I, F317L, and different possible variations in the ABL1 gene. Methods: This was a cross-sectional study on Indonesian CML patients in chronic phase. We analyzed 120 blood samples from patients in chronic phase who have received imatinib mesylate (IM) for ≥12 months. Results: There were no T315I, F311I, and F317L mutations found in this study. However, we found another variation, which was 36 substitutions from A to G at position 163816 of ABL1 gene (according to NG_012034.1). Conclusions: We found no T315I, F311I, and F317L mutations in this study. Our findings suggest that there might be other factors that influenced the MMR achievement in our study patients. However, there were 36 substitutions from A to G at position 163.816 (according to NG_012034.1) that needed further examination to explore the significance of this mutation in clinical practice.
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Nicolini, Franck E., Michael J. Mauro, Giovanni Martinelli, Dong-Wook Kim, Simona Soverini, Martin C. Müller, Andreas Hochhaus, et al. "Epidemiologic study on survival of chronic myeloid leukemia and Ph+ acute lymphoblastic leukemia patients with BCR-ABL T315I mutation." Blood 114, no. 26 (December 17, 2009): 5271–78. http://dx.doi.org/10.1182/blood-2009-04-219410.
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Abstract The BCR–ABL T315I mutation represents a major mechanism of resistance to tyrosine kinase inhibitors (TKIs). The objectives of this retrospective observational study were to estimate overall and progression-free survival for chronic myeloid leukemia in chronic-phase (CP), accelerated-phase (AP), or blastic-phase (BP) and Philadelphia chromosome—positive (Ph)+ acute lymphoblastic leukemia (ALL) patients with T315I mutation. Medical records of 222 patients from 9 countries were reviewed; data were analyzed using log-rank tests and Cox proportional hazard models. Median age at T315I mutation detection was 54 years; 57% cases were men. Median time between TKI treatment initiation and T315I mutation detection was 29.2, 15.4, 5.8, and 9.1 months, respectively, for CP, AP, BP, and Ph+ ALL patients. After T315I mutation detection, second-generation TKIs were used in 56% of cases, hydroxyurea in 39%, imatinib in 35%, cytarabine in 26%, MK-0457 in 11%, stem cell transplantation in 17%, and interferon-α in 6% of cases. Median overall survival from T315I mutation detection was 22.4, 28.4, 4.0, and 4.9 months, and median progression-free survival was 11.5, 22.2, 1.8, and 2.5 months, respectively, for CP, AP, BP, and Ph+ ALL patients. These results confirm that survival of patients harboring a T315I mutation is dependent on disease phase at the time of mutation detection.
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Zafar, Usva, Mohammed Yusuf, Rikhia Chakraborty, El-Nasir M. A. Lalani, and Afsar Ali Mian. "The ''Gatekeeper'' Mutation T315I in BCR/ABL Confers Additional Oncogenic Activities to Philadelphia Chromosome Positive Leukemia." Blood 134, Supplement_1 (November 13, 2019): 5196. http://dx.doi.org/10.1182/blood-2019-131694.
Full textAbstract:
Chronic myeloid leukemia (CML) and 30% of adult acute lymphatic leukemia (ALL) are characterized by the Philadelphia chromosome (Ph+), having a (9;22) chromosomal translocation. The BCR/ABL fusion protein is the hallmark of Ph+ leukemia. BCR/ABL is characterized by deregulated and constitutively activated ABL tyrosine kinase activity that determines its transformation potential. Tyrosine kinase inhibitors (TKI) have greatly improved the overall prognosis of these diseases, particularly by altering the natural history of chronic phase (CP) CML and preventing the previously inexorable progression to terminal blast crisis (BC). However, unsatisfactory responses in advanced disease stages, resistance and long-term tolerability of BCR/ABL inhibitors represent major clinical problems. The most important mechanism of resistance against TKIs is the selection of leukemic clones driven by BCR/ABL harboring point mutations, such as the E255K, Y253F/H (P-loop), H396R (activation loop) or the T315I (gatekeeper). The "gatekeeper" mutation T315I confers resistance against all approved TKIs, with the only exception of Ponatinib, a multi-target kinase inhibitor. CML and Ph+ ALL, rarely present at diagnosis with a BCR/ABL harboring a resistance mutation to TKI. Resistant clones may be present and only detectable by highly sensitive methods. We have previously shown that the resistance mutations may influence the biology of BCR/ABL and its transformation potential. We therefore hypothesized that the presence of mutations such as the T315I select for a "dormant cell population" which manifests following initial treatments with TKI inhibitors and treatment failure. The aim of this study was to determine whether the ''gatekeeper'' mutation T315I is able to confer biological features to BCR/ABL influencing its leukemogenic potential. We investigated the influence of T315I on the biology of BCR/ABL in CML and Ph+ ALL. We used Ph+ ALL patient derived long term culture (PDLTCs), factor dependent Ba/F3 cells and syngeneic mouse model of BCR/ABL induced CML-like disease. These models allowed the direct comparison of BCR/ABL with BCR/ABL-T315I. We observed significantly slower proliferation of Ba/F3 cells and PDLTCs expressing BCR/ABL-T315I compared to the native BCR/ABL. This was further confirmed by undertaking mitotic index calculations and colony formation assays on both cell types. Furthermore, the induction of a CML-like disease in syngeneic mice was significantly delayed in the presence of T315I (median: BCR/ABL - 27 days; BCR/ABL-T315I - 61 days). We undertook functional studies to determine the putative signaling pathway and found that Ras/Erk1/2 pathway was activated inT315I positive cells. This study may assist towards therapy decisions in patients with CML/Ph+ ALL with a T315I mutation. Disclosures No relevant conflicts of interest to declare.
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Khorashad, Jamshid S., Dragana Milojkovic, Puja Mehta, Mona Anand, Sara Ghorashian, Alistair G. Reid, Valeria De Melo, et al. "In vivo kinetics of kinase domain mutations in CML patients treated with dasatinib after failing imatinib." Blood 111, no. 4 (February 15, 2008): 2378–81. http://dx.doi.org/10.1182/blood-2007-06-096396.
Full textAbstract:
We sought kinase domain (KD) mutations at the start of treatment with dasatinib in 46 chronic myeloid leukemia (CML) patients resistant to or intolerant of imatinib. We identified BCR-ABL mutant subclones in 12 (26%) cases and used pyrosequencing to estimate subsequent changes in their relative size after starting dasatinib. Four patients lost their mutations, which remained undetectable, 3 patients retained the original mutation or lost it only transiently, 3 lost their original mutations but acquired a new mutation (F317L), and 2 developed another mutation (T315I) in addition to the original mutation within the same subclone. This study shows that expansion of a mutant Ph-positive clone that responds initially to a second generation tyrosine kinase inhibitor may be due either to late acquisition of a second mutation in the originally mutated clone, such as the T315I, or to acquisition of a completely new mutant clone, such as F317L.
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Panjarian, Shoghag, Shugui Chen, John Engen, and Thomas Smithgall. "Enhanced SH3:Linker Interaction Suppresses Activating Mutations of the c-Abl Protein-Tyrosine Kinase." Blood 116, no. 21 (November 19, 2010): 1208. http://dx.doi.org/10.1182/blood.v116.21.1208.1208.
Full textAbstract:
Abstract Abstract 1208 Bcr-Abl, the chimeric protein-tyrosine kinase expressed as a result of the Philadelphia chromosome translocation, plays a pivotal role in the initiation and maintenance of chronic myelogenous leukemia (CML). Imatinib (Gleevec) is an ATP-competitive Bcr-Abl inhibitor that selectively kills Bcr-Abl+ CML cells. Despite its clinical success, imatinib is less effective in the advanced stages of CML due to the emergence of drug resistance caused by point mutations in the Abl kinase domain. Second generation Bcr-Abl inhibitors such as dasatinib and nilotinib are active against most imatinib-resistant forms of Bcr-Abl, with the exception of the T315I “gatekeeper” mutant. The Abl gatekeeper residue (Thr315) is located between the ATP-binding site and an adjacent hydrophobic pocket, and forms a key hydrogen bond with imatinib. Additionally, the T315I mutation produces a strong activating effect on the downregulated c-Abl “core,” consisting of the myristoylated N-terminal Ncap, tandem SH3 and SH2 regulatory domains, the SH2-kinase linker, which forms a polyproline type II helix for internal SH3 docking, and the tyrosine kinase domain. Using hydrogen-exchange mass spectrometry, we recently found that the T315I mutation not only induced conformational changes in the Abl kinase domain as expected, but also at a distance in the RT-loop of the SH3 domain. Such changes may allosterically contribute to kinase domain activation by disturbing the negative regulatory influence of SH3:linker interaction. Recently, a new class of allosteric Bcr-Abl inhibitors has been reported that targets the myristate-binding pocket of Abl, which localizes to C-lobe of the kinase domain and away from the active site. Together with our finding that the T315I mutation perturbs SH3:linker interaction, these inhibitors support the existence of an extensive network of allosteric interactions that work together to regulate Abl kinase activity. In this project, we investigated whether enhanced SH3:linker interaction can allosterically reverse the activating effects of the T315I imatinib resistance mutation as well as mutations of the N-terminal myristoylation site and myristic acid binding pocket. We created modified versions of Abl [High Affinity Linker proteins (HALs)] by mutating multiple residues within the SH2-kinase linker to proline, thereby enhancing the SH3 domain binding affinity. Using mammalian cell-based expression assays and immunoblotting with phosphospecific antibodies, we identified five of eleven Abl-HAL proteins that did not exhibit changes in basal kinase activity. The Abl-HAL protein with the greatest enhancement of SH3:linker interaction was then combined with the T315I mutation, a myristoylation-defective mutant, and a myristic acid binding pocket mutation. Remarkably, this HAL substitution completely reversed the activating effect of the myristic acid binding pocket mutation, while substantially suppressing the activity of Abl T315I and the myristoylation-defective mutant. These results indicate that stabilization of SH3:linker interaction allosterically represses Abl activation by a wide variety of mechanisms, and suggests a new approach to allosteric control of Bcr-Abl kinase activity. Disclosures: No relevant conflicts of interest to declare.
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Sandt, Christophe, Olivier Feraud, Marie Laure Bonnet, Rania Khedhir, Marie Claude Meunier, Paul Dumas, Annelise Bennaceur-Griscelli, and Ali G. Turhan. "Infrared Microspectroscopy Allows Direct Identification of Leukemic Cells Expressing T315I-Mutated BCR-ABL Via a Unique Spectral Signature." Blood 124, no. 21 (December 6, 2014): 4510. http://dx.doi.org/10.1182/blood.v124.21.4510.4510.
Full textAbstract:
Abstract The use of tyrosine kinase inhibitor (TKI) therapies has dramatically changed the prognosis of chronic myeloid leukemia (CML) and modified the natural history of the disease. However, the generation of ABL-kinase domain mutations due to the genetic instability of leukemic cells continues to be very significant challenge, especially in advanced phases of the disease. Amongst these mutations, T315I is the most problematic as it reduces the binding of Imatinib, Dasatinib or Nilotinib to their target, leading to a total resistance to all three TKIs. Although the mechanism of the appearance of this mutation is not completely known, it is possible that it induces a signalling pathway not entirely similar to that induced by classical wild-type BCR-ABL. From the mechanistic point of view, the mechanism of resistance of T315I-mutated BCR-ABL to Imatinib is related to the substitution of Isoleucine to Threonine, which normally forms a hydrogen bond with Imatinib, this bond being absolutely required for binding of the drugs to the catalytic site. T315I mutation can be identified by direct sequencing, D-HPLC, allele specific PCR or double gradient gel electrophoresis of the PCR products amplified from the BCR-ABL kinase domain. However these techniques cannot identify directly the leukemic cells expressing T315I-mutated BCR-ABL which are known to co-exist with native BCR-ABL-expressing cells in bone marrow. Infrared microspectroscopy allows the identification of metabolic features of mammalian cells, based on their protein, lipid, amid and nucleic acid contents, generating a spectral signature. In this work we asked whether infrared microspectroscopy can be used to identify metabolic changes occurring in single cells bearing BCR-ABL T315I mutation. For this purpose we have used human (UT7) and murine embryonic stem cells (GS2) expressing either native (N) or T315I-mutated BCR-ABL. In the human UT7 cells expressing N-BCR-ABL, we were able to clearly distinguish at the single cell level, cells expressing BCR-ABL from wild type cells using principal component analysis. However, the presence of T315I mutation induced a clearly different signature, allowing a highly significant separation by the use of spectral signature. To confirm the specificity of this signature, we have used UT7 cells engineered to express either native or T315I-mutated BCR-ABL under the control of Doxycycline (DOX) -sensitive promoters. In this TET-OFF system, the addition of doxycycline to the culture medium inhibits BCR-ABL expression as monitored by Western blots, in approximately 6 days. Using this system, at day 0, UT7 cells expressing N-BCR-ABL and T315I mutated BCR-ABL were clearly distinguishable from parental UT7 cells. Upon inhibition of BCR-ABL expression by addition of Doxycycline, the spectral signature of N-BCR-ABL and T315I-mutated BCR-ABL cells became similar at day 2 and indistinguishable at day 4, but still distinguishable from wild type UT7 cells expressing DOX-inducible GFP. To confirm these results in a different cell context we have used a murine embryonic stem (ES) cell line (GS2) which was transduced with either N-BCR-ABL or T315I-mutated BCR-ABL-expressing lentiviral vectors. BCR-ABL-expressing individual clones grown were analyzed to analyze the spectral signature. In this cell line also, infrared microspectroscopy was able to distinguish ES cells expressing BCR-ABL T315I as compared to N-BCR-ABL. Thus, these results suggest that T315I mutation clearly induces metabolic changes different from N-BCR-ABL in leukemic cells, rendering them identifiable by the analysis of nucleic acid, lipid, amid and sugar contents. This new methodology can now be applied to the identification of primary CML leukemic cells harboring T315I at the single cell level and could be of interest for rapid identification of leukemic cells in a single step. This technique can also be used for rapid screening of novel compounds active against T315I mutation using microfluidic technologies leading to novel drug discoveries. Disclosures Turhan: Bristol Myers Squibb, Novartis: Honoraria, Research Funding.
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Desterke, Christophe, Djamel Aggoune, Marie Laure Bonnet, Nais Prade, Jean-Claude Chomel, Eric Delabesse, and Ali G. Turhan. "T315I-Mutated BCR-ABL Induces a Distinct and Specific Molecular Signature With High Expression Of Zinc Finger (ZNF) Transcription Factors." Blood 122, no. 21 (November 15, 2013): 4899. http://dx.doi.org/10.1182/blood.v122.21.4899.4899.
Full textAbstract:
Abstract Chronic myeloid leukemia (CML) is the paradigm of malignancy treated by targeted therapies by the use of tyrosine kinase inhibitors (TKI), essentially Imatinib, Dasatinib and Nilotinib. Despite their major efficiency, especially as first line therapies, resistance to these drugs develop partly due to genetic instability inherent to CML. BCR-ABL-kinase mutations remain the first cause of resistance, which appears to be due to clonal selection of cells bearing a given mutation under TKI therapies. Amongst these mutations, the “gatekeeper” T315I mutant is a major concern as it confers resistance to all three TKI clinically used and patients with this mutation have a poor prognosis. The inaccessibility of the TKI to the ABL kinase pocket might not be the only “mechanistic” cause of resistance and it has been suggested that T315I-mutated BCR-ABL (Skaggs BJ et al, 2006) could induce a specific phosphoproteome signature. To evaluate this possibility, we decided to determine if a specific gene expression profiling can be associated with T315I-mutated BCR-ABL, as compared to native BCR-ABL. The human hematopoietic cell line UT7 was transfected with retroviral vectors encoding for native BCR-ABL (UT7.11) or BCR-ABL with the T315I mutation (UT7.T315I). The cell lines were characterized by their cell growth, Western blotting and sequencing. UT7.11 cells were sensitive to Imatinib, Dasatinib and Nilotinib as well as to Ponatinib whereas UT7-T315I cells were resistant to all three TKI except for Ponatinib. Affymetrix microarrays were performed in triplicate on each of three groups (UT7, UT7.11, UT7.T315I). The datas were normalized using the dchip software. Bioinformatics analyzes were performed with R software (packages FactoMineR, limma, PAMR) Mev in TM4 software, enrichment analysis with the GSEA software (Broad institute). The principal component analysis (PCA) showed that the overall RNA expression of UT7.T315I was different from that of UT7.11 (native BCR-ABL) and parental UT7. On factorial map, UT7.11 was found more distant from parental UT7 than UT7.T315I. The contrast analysis of the linear model by the algorithm limma between the 3 groups, showed a strong differential signature of UT7.11 as compared to parental UT7 and UT7.T315I (respectively 4792 and 4813 genes). Only 800 genes were found to be differentially expressed between UT7.T315I and parental UT7. In hierarchical clustering, the total signature obtained in limma confirmed a closed profile between parental UT7 and UT7.T315I. Among the results of the limma model, we identified a 286 specific genes signature for UT7.T315I (both different from parental UT7 and UT7.11 and also not regulated between UT7.11 and UT7). This specific list of UT7.T315I was validated with the T315I group sample segregation by different multivariate methods: PCA, hierarchical clustering and non-negative matrix factorization. Among this T315I-specific gene list limma, 34 ZNF family genes were found (11.88%). Predicting class algorithm based on shunkren centroid (PAMR) separated the three group samples with low classification error and a global list of 368 genes: only 75 genes predicted UT7.T315I group and from this list 13 were in the ZNF gene family (13.33%). By the method of gene set enrichment analysis (GSEA), we explored the top 100 ranked genes as upregulated in UT7.T315I by comparing the two other sample groups. This gene set showed a high representation of ZNF family genes (25%). The design of a gene set with ZNF family genes selected showed a positive enrichment of ZNF (NES = +1.35, p-value <0.001) in the UT7.T315I by comparing the two other groups. The majority of these genes is localized in 19q13.41 (ZNF cluster 282). They exhibit C2H2 and Kruppel-associated box (KRAB) domains in their sequence. Interestingly the overexpression of KRAB-ZNF transcription factors has been recently reported in patients with gastrointestinal stromal tumors (GIST) as conferring resistance to Imatinib Mesylate (Rink L., PLOS One 2013). In conclusion, our work revealed for the first time a specific signature of the T315I mutation which includes a strong representation of the ZNF family. The identification of this signature could be interest for future drug screening strategies in advanced phase CML patients progressing under Ponatinib. Current experiments are underway to validate these results by analyzing the expression of ZNF family of genes in primary CML cells with T315I mutation. Disclosures: Turhan: Bristol Myers Squibb, Novartis: Consultancy, Honoraria.
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Mir, A. R., S. Sazawal Sazawal, A. Saxena, and R. Saxena. "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." Journal of Clinical Oncology 27, no. 15_suppl (May 20, 2009): 7061. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.7061.
Full textAbstract:
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.
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Jabbour, Elias, Hagop Kantarjian, Dan Jones, Francis Giles, Gautam Borthakur, Susan O’Brien, and Jorge Cortes. "Clinical Characteristics and Outcome of Patients (pts) with T315I Mutation after Imatinib Failure." Blood 108, no. 11 (November 1, 2006): 2189. http://dx.doi.org/10.1182/blood.v108.11.2189.2189.
Full textAbstract:
Point mutations of the Bcr-Abl kinase domain are the most frequently identified mechanism of resistance in patients (pts) with CML who failed imatinib. T315I is an imatinib pocket binding mutation within the Bcr-Abl kinase domain that is highly resistant to imatinib as well as to the novel tyrosine kinase inhibitors (NTKIs) (eg, dasatinib, nilotinib) both in vitro and in vivo. It has thus been suggested that these pts have a very poor prognosis. To define the clinical characteristics and outcome of these pts, we reviewed all pts with this mutations identified at our institution after failure to imatinib. T315I was detected in 15 of 112 patients (13%) harboring kinase domain mutations. Median age was 49 years (range, 34-66 years). At the time imatinib was started, 12 pts were in chronic (CP) and 3 in accelerated (AP). Ten pts had received prior interferon therapy. The best response to imatinib was major molecular response in 1, major cytogenetic response in 5 (complete in 2), and complete hematologic response in 8. One patient was intolerant to imatinib. The median duration of response was 37 months (range, 10–60 months). T315I was identified after a median of 48 months (range, 10–83 months). At the time the mutation was identified, 9 patients had transformed (7 to AP, 2 to BP). Clonal evolution was observed in only 1 pt. Fourteen patients received subsequent NTKIs; 3 of them received both nilotinib and dasatinib. Among them, 1 patient achieved a partial hematologic response with nilotinib that lasted for 3 months and 1 other had T315I identified while in complete cytogenetic response induced by nilotinib that has been sustained for 16+ months. After a median follow-up of 27 months (range, 3–42 months), 11 pts (73%) are alive: 8 are alive with active disease, and 3 patients are alive with ongoing response: 1 in complete molecular remission following allogeneic stem cell transplantation, 1 in partial cytogenetic response following treatment with aurora kinase inhibtor MK0457, and one in sustained complete cytogenetic response with ongoing treatment with nilotinib. Four patients have died of disease progression. Except for previous treatment with interferon (more frequent in patients harboring the T315I mutation; p=0.024) and the lack of response to the NTKI (p=0.001), there was no difference in patients characteristics and previous response to imatinib compared to patients with other mutations. Similarly, there was no difference in overall survival among patients with T315I mutations or other mutations (p=0.71). In conclusion T315I is a highly resistant mutation to conventional tyrosine kinase inhibitors; however occasional responses can be observed and overall survival may not be as poor as previously reported.
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Hochhaus, Andreas, T. Ernst, P. Erben, M. C. Mueller, M. Emig, S. Kreil, P. La Rosée, et al. "Long Term Observation of CML Patients after Imatinib Resistance Associated with BCR-ABL Mutations." Blood 106, no. 11 (November 16, 2005): 1086. http://dx.doi.org/10.1182/blood.v106.11.1086.1086.
Full textAbstract:
Abstract The major cause of imatinib resistance in CML patients (pts) is clonal selection of cells harboring mutations of the BCR-ABL tyrosine kinase associated with various degrees of insensitivity to the drug. Management and outcome of these pts have been a matter of debate. In particular, retrospective studies suggested a poor prognosis of pts with P-loop mutations, whilst others did not confirm this observation. We sought to determine the outcome of 121 CML pts (73 m, 48 f; median age 61 yrs, range 28–80) with BCR-ABL mutations detected by direct sequencing and associated with hematologic (n=105), cytogenetic (n=14), or molecular (n=2) resistance. Imatinib was commenced between 8/99 and 3/05 after a median of 33.1 mo (range 0–312.3) after diagnosis, the median exposure to the drug was 17.0 mo (0.9–55.4). Resistance occurred after 18.1 mo (0.9–55.4) in chronic phase (CP, n=63), 18.3 mo (5.1–52.8) in accelerated phase (AP, n=33), 11.4 mo (0.9–47.7) in myeloid blast crisis (BC, n=20), and 9.1 mo (1.8–45.6) in lymphoid BC (n=5). Resistance was associated with 41 different mutations leading to a change of 32 amino acids (aa) of the P-loop area (n=38), T315I (n=15), the activation loop (n=11), and other sites (n=74). 15 pts showed 2, 2 pts 3, and one patient 4 mutations in parallel. In addition, resistance was associated with a loss of 27 aa including the P-loop in 2 pts. ABL polymorphisms were detected in 3 pts (T315T, n=1; K247R, n=2). Median time until resistance was 12.1 mo (0.9–52.8) for pts with P-loop mutations, 11.5 mo (2.8–47.7) for T315I, 17.2 mo (5.0–35.5) for activation loop mutations, and 20.4 (2.0–55.4) for others. After hematologic resistance, imatinib therapy was continued for 3.5 mo (0–54.1). Dose was increased in 42 pts up to 800mg/d, which was associated with disease stabilization in 7, hematologic improvement in 4, and cytogenetic or molecular improvement in 2 cases. In 46 pts, imatinib was combined with other drugs, 33 received low dose cytarabine. After imatinib withdrawal, treatment was continued with hydroxyurea, n=53, cytarabine, n=32, and/or other cytostatic drugs. 10 pts underwent allogeneic stem cell transplantation (SCT), 5 of them died. Recently, 2nd generation tyrosine kinase inhibitors were administered to 35 pts (dasatinib, n=19, AMN107, n=15, both drugs, n=1). Censoring pts at the time of SCT or start of alternative tyrosine kinase inhibitors, overall survival after resistance was 29.7 mo (0–52.3). 38 pts died; of these, mutations were located in the P-loop, n=16, at T315, n=4, or at other sites, n=18. Median survival after hematologic resistance was 29.7 mo for pts with P-loop mutations, 13.0 mo for pts with T315I, and 33.5 mo for others (n.s.). Considering CP pts only, median survival in case of P-loop mutations was 35.8 mo and has not been reached for pts with T315I and others (n.s.). In advanced disease, median survival with P-loop mutations was 17.0 mo, for T315I 13.0 mo, and for others 29.5 mo (n.s.). In conclusion, this prospective analysis failed to reveal a significantly impaired survival of pts with P-loop mutations compared to others. There is, however, a trend for a survival disadvantage in pts harboring T315I. Outcome of CML pts with mutations associated with insensitivity to imatinib, in particular P-loop and T315I mutations can be improved by early detection of the mutation and rapid withdrawal of the drug.
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Mian, Afsar, Isabella Haberbosch, Oliver G. Ottmann, and Martin Ruthardt. "Transphosphorylation of Endogenous BCR Mediates the Effect of T315I on the Transformation Potential of BCR/ABL." Blood 124, no. 21 (December 6, 2014): 4523. http://dx.doi.org/10.1182/blood.v124.21.4523.4523.
Full textAbstract:
Abstract Targeting of BCR/ABL by ABL-kinase inhibitors (AKI) such as Imatinib, Nilotinib, or Dasatinib is a proven concept in Philadelphia chromosome positive (Ph+ ) leukemia. In the majority of cases the acquisition of resistance is related to point mutations in BCR/ABL, such as the E255K, Y253F/H (P-loop), H396R (activation loop) or the T315I (gatekeeper). Noteworthy, Ph+ leukemias, both CML and Ph+ ALL, never emerge at diagnosis with a BCR/ABL harboring a resistance mutation even if the clone with the mutation is already existing and detectable by very sensitive methods. This indicates that the mutations confer biological features to the clones unveiled by the treatment. One can hypothesize that the presence of mutations such as the T315I confers a growth disadvantage with respect to native BCR/ABL. We and others have previously shown that the resistance mutations may influence the biology of BCR/ABL regarding its transformation potential. The aim of the study was to determine whether the ‘‘gatekeeper’’ mutation T315I is able not only to induce biochemical modification of BCR/ABL responsible for the to resistance of patients against first and second generation AKI, but also to confer biological features to BCR/ABL influencing its leukemogenic potential. We recently showed that T315I is able to fully restore factor independent growth in Ba/F3 cells of loss of function mutants (LOFM) of BCR/ABL such as that lacking the Y177 (Y177F) or the N-terminal coiled coil oligomerization interface (ΔCC), which was accompanied by a transphosphorylation of endogenous BCR. Based on these findings we systematically investigated the influence of T315I on the biology of BCR/ABL and the role of BCR in this process. As models we used a syngeneic mouse model of BCR/ABL induced CML-like disease, factor dependent 32D and Ba/F3 cells, and Ph+ ALL patient derived long term culture (PD-LTCs). These models allowed the direct comparison of BCR/ABL with BCR/ABL-T315I. Furthermore we took advantage of LOFM such as ΔCC-BCR/ABL and Y177F-BCR/ABL which we studied for their capacity to mediate either transformation potential in Rat-1 fibroblasts (contact inhibition and anchorage-independent growth) and/or factor independent growth in 32D cells in the presence of T315I. The role of the transphosphorylation of BCR was assessed by RNAi against BCR in 32D cells and the Rat-1 cells. Here we show that proliferation of 32D or Ba/F3 cells or the PD-LTCs expressing BCR/ABL-T315I was significantly slower than that of correspondent cells expressing native BCR/ABL. Also the induction of a CML-like disease in syngeneic mice was significantly delayed in the presence of T315I (median: BCR/ABL - 27 days; BCR/ABL-T315I - 61 days). On the other hand T315I was able to restore both transformation potential and factor independent growth of LOFM of BCR/ABL in Rat-1 and 32D cells, respectively. This was accompanied by a transphosphorylation of endogenous BCR at Y177, which led to an activation of Ras/Erk1/2 pathway. This effect of T315I on factor independent growth and transformation mediated by the LOFM and related activation of Ras/Erk1/2 was reverted by targeting the BCR with RNAi. Taken together these data suggest that T315I confer biological features to BCR/ABL which are unveiled only upon treatment or in the presence of LOFM. How these features may influence the destiny of the BCR/ABL-T315I clone in the patient and the role of Ras/Erk1/2 pathway in this process has to be further investigated. Disclosures No relevant conflicts of interest to declare.
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Mishima, Yuko, Yuji Mishima, Yasuhito Terui, Shinya Kimura, and Kiyohiko Hatake. "An Autophagy Inhibitor, Chloroquine, Overcomes An Imatinib-Resistant T315I Mutant of Bcr/Abl, Whose Latent May Exist Before Imatinib Treatment." Blood 114, no. 22 (November 20, 2009): 2183. http://dx.doi.org/10.1182/blood.v114.22.2183.2183.
Full textAbstract:
Abstract Abstract 2183 Poster Board II-160 Background) Imatinib improved the prognosis of CML and Philadelphia chromosome-positive (Ph+) adult acute lymphocytic leukemia (ALL). Despite the stunning efficacy of this agent, a small number of patients develop resistance to imatinib. To overcome these resistant disease, second generation tyrosine kinase inhibitors (TKIs) such as dasatinib, nilotinib have been developed. The efficacy of these TKIs was shown against the various imatinib-resistant cells having gene mutations in Bcr/Abl, but could not inhibit against the gate keeper mutation, T315I. The T315I mutation is often observed in many case of aggressive phase of CML or Ph+ALL. Recently, some new drugs such as aurora kinase inhibitor and HDAC inhibitor, have been considered as the new drug against CML and Ph+ALL. It is known that HDAC inhibitor induced both apoptosis and autophagy in Bcr/Abl positive cells and that the block of autophagy accelerated apoptotic cell death. In recent studies, autophagy has been estimated to one of new target of treatment of Ph+ leukemia cells. Methods) We established two Ph+ ALL cell lines, DPAL and DPAL/T315I, these cell lines from one clinical course of a patient have wild type and T315I mutation in Bcr/Abl, respectively. We analyzed the sequences of genes and the chromosomes of these cell lines. Using these cell lines as well as BaF/3 cell lines stably-transfected with wild type (BaF/3/wild) and mutated Bcr/Abl genes (BaF/3/T315I), we evaluated the effect of chloroquine diphosphate, one of the inhibitors of autophagy on cytotoxicity induced by imatinib or SAHA, one of a HDAC inhibitor. Result) This Ph+ ALL patient had double Ph+ chromosomes with additional chromosomal abnormalities at the first diagnosis. The patients received imatinib combined chemotherapy, then, relapsed after 3 months. T315I mutation was detected without additional chromosomal abnormality at relapse. DPAL was derived from bone marrow before imatinib treatment and DPAL/T315I was derived bone marrow at relapse after imatinib-combined chemotherapy. The DNA sequence analysis of DPAL/T315I cells revealed that one of two Philadelphia chromosomes had T315I mutation on Bcr/Abl. These result indicated that Bcr/Abl positive cells were heterogeneous clones before imatinib treatment in this clinical case. The most dominant populations was wild type Bcr/Abl clone with additional chromosomal abnormalities and one of minor population was T315I-positive cells without additional chromosomal abnormalities. This indicated the imatinib treatment eliminated the wild type Bcr/Abl-positive cells and subsequently T315I-positive cells increased. Other combination chemotherapies with stem cell transplantation could not overcome T315I-positive cells in this clinical course. So, to establish new treatment strategy, we studied about the effect of an autophagy inhibitor, chloroquine, against T315I-positive cells using our established cell lines. The combination treatment of imatinib with chloroquine increased the cell death of DPAL, BaF/3/wild, Ba/F3/T315I cells and inhibited the growth of DPAL/T315I cells. The combination treatment of a HDAC inhibitor, SAHA, with chloroquine induced cell death of not only DPAL cells but also DPAL/T315I cells. The phosphorylation of tyrosine on BCR/ABL did not change by chloroquine. The concentrations of intracellular ATP, assessed by luciferase assay, were decreased by chloroquine treatment. Discussion) From our established cell lines, it was suggested that Bcr/Abl-positive cells were heterogeneous clones in the Ph+ leukemia. Mono-treatment of TKIs against Bcr/Abl might permit the survival of tolerant clones such as T315I. The combination treatment of an autophagy inhibitor with TKIs or HDAC inhibitors were effective against Bcr/Abl-positive cells, even if T315I mutant cells. Our study indicated that autophagy is the protect system in Ph+ leukemia cells and block of autophagy might be one of new strategies for elimination of resistant clone of Bcr/Abl. Disclosures: No relevant conflicts of interest to declare.
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Skaggs, Brian J., Michael R. Burgess, Mercedes E. Gorre, Thomas G. Graeber, and Charles L. Sawyers. "Altered Oncogenic Fitness of Imatinib- and Dasatinib-Resistant BCR-ABL Mutants Is Due to Differential Intrinsic Kinase Activity and Signaling Pathway Selection Defined by Phosphoproteome Profiling." Blood 106, no. 11 (November 16, 2005): 692. http://dx.doi.org/10.1182/blood.v106.11.692.692.
Full textAbstract:
Abstract Point mutations in the kinase domain of BCR-ABL are the most common cause of resistance to imatinib therapy in CML. E255K and T315I are detectable before the initiation of imatinib therapy in a small population of CML patients, suggesting that certain imatinib-resistant BCR-ABL mutants display altered biological fitness relative to wild-type p210 BCR-ABL. To test this hypothesis, we performed primary murine bone marrow transformation assays using BCR-ABL-expressing retroviruses. In a survey of six clinically imatinib-resistant mutations (Y253F, Y253H, E255K, E255V, T315I, M351T) as well as five additional mutations that confer in vitro resistance to the dual SRC/ABL inhibitor dasatinib (L248R, V299L, T315A, F317L, F317V), we identified several that altered oncogenic fitness either positively or negatively relative to p210. To elucidate the molecular basis for altered transformation potential, we examined the kinase activity of p210 versus mutant full-length BCR-ABL. Enzyme kinetics studies with an optimized Abl peptide substrate as well as gel-based in vitro kinase assays illustrated that intrinsic kinase activity does explain some, but not all, of the mutant oncogenic potency phenotypes. The imatinib- and dasatinib-resistant mutant T315I, for instance, was a gain-of-fitness mutant in the bone marrow assay but exhibited a three-fold lower Vmax than p210. In contrast, T315A, a dasatinib-resistant mutation at the T315 gatekeeper residue, exhibits reduced transforming potential relative to wt p210 and is completely unable to phosphorylate Abl substrates in the kinase assays, although it does retain the ability to autophosphorylate. Subsequent kinase assays on immunoprecipitations from stably expressing IL-3-independent Ba/F3 cells illustrated that certain mutants bind and phosphorylate distinct substrates from p210. We further delineated signaling differences between p210 and the mutant panel by utilizing a mass spectrometry-based global phosphorylation profiling technique. Trypsinized proteins from stably expressing p210 and mutant Ba/F3 cells were immunoprecipitated using anti-phosphotyrosine antibodies, then identified and quantified by two dimensional mass spectrometry. Phosphorylation of specific substrates correlates with gain- or loss-of fitness observed in the bone marrow assay. We conclude that certain BCR-ABL kinase domain mutations, in addition to conferring drug resistance, also affect the oncogenic potency of BCR-ABL by altering intrinsic kinase activity as well as downstream targets. Elucidation of alternative signaling pathways activated by these mutants by the combination of biochemical and mass spectrometric methods may provide novel therapeutic targets for imatinib- and dasatinib-resistant CML.
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Khoury, H. Jean, Jorge E. Cortes, Carlo Gambacorti-Passerini, Dong-Wook Kim, Andrey Zaritskey, Andreas Hochhaus, Eric Leip, Nadine Besson, Virginia Kelly, and Tim H. Brümmendorf. "Activity of Bosutinib by Baseline and Emergent Mutation Status in Philadelphia Chromosome–Positive Leukemia Patients with Resistance or Intolerance to Other Tyrosine Kinase Inhibitors." Blood 118, no. 21 (November 18, 2011): 110. http://dx.doi.org/10.1182/blood.v118.21.110.110.
Full textAbstract:
Abstract Abstract 110 Bosutinib is an orally active, Src/Abl tyrosine kinase inhibitor (TKI) that has demonstrated clinical activity and an acceptable safety profile in an open-label, phase 1/2 study of patients (pts) with Philadelphia chromosome–positive (Ph+) leukemia following resistance or intolerance to imatinib. Abl kinase domain mutations may affect drug binding and subsequent TKI efficacy. Responses to bosutinib by Bcr-Abl baseline mutation status and the emergence of mutations during bosutinib treatment in this phase 1/2 study are reported here. A total of 570 pts in 3 cohorts received bosutinib: chronic phase chronic myeloid leukemia (CP CML) following prior imatinib only (CP2L cohort; n = 288); CP CML following prior imatinib plus dasatinib and/or nilotinib (CP3L cohort; n = 118); and advanced leukemia (ADV cohort: accelerated and blast phase CML and acute lymphoblastic leukemia; n = 164) following prior treatment with imatinib only or with other TKIs. For the CP2L, CP3L, and ADV cohorts, the respective median times since diagnosis were 3.6 y (range, 0.1–15.1 y), 6.5 y (range, 0.6–18.3 y), and 3.7 y (range, 0.1–22.1y). Per protocol, Bcr-Abl mutational status was evaluated at baseline (pre-dose) and at the end of treatment (EOT). Mutations were assessed at baseline in 412 pts; of these, 79/212 (37%) CP2L pts, 39/83 (47%) CP3L pts, and 65/117 (56%) ADV pts had ≥1 baseline mutation, and 11 (5%), 9 (11%), and 7 (6%) pts, respectively, had ≥2 baseline mutations. The most common mutations were T315I (n = 31; prior to eligibility exclusion), F359C/I/S/V (n = 23), F317L (n = 21), G250E (n = 19), Y253F/H (n = 16), and M351T (n = 15). Rates of confirmed complete hematologic response (CHR) and major cytogenetic response (MCyR) were generally similar among chronic phase pts (CP2L, CP3L) with and without baseline mutations, although the presence of a mutation negatively impacted response rates in ADV pts (CHR, 17% vs 39%; MCyR, 24% vs 37%; see Table). CHR and/or MCyR were observed broadly across mutations, including those associated with dasatinib (F317L) or nilotinib (Y253F/H, E255K/V, and F359C/I/S/V) resistance, with the exception of the T315I mutation in all cohorts and the F317L and Y253H mutations in the ADV cohort (see Table). Of the 140 pts who were assessed for emergent mutations at treatment completion, a new mutation was identified in 39 (28%) pts. Pts with a new mutation were more likely to have discontinued treatment due to PD/unsatisfactory response (UR; n = 34/39 [87%]) than those without a new mutation (n = 47/101 [47%]). The most common emergent mutations were T315I (n = 14) and V299L (n = 12), and nearly all pts who developed these mutations (93% and 100% of pts, respectively) discontinued treatment due to PD/UR. New mutations were more common in pts with ≥1 baseline mutation (n = 26) versus those without a baseline mutation (n = 13); of those pts, 21/26 (81%) and 13/13 (100%), respectively, discontinued treatment due to PD/UR. Eight pts discontinued treatment due to PD/UR within 6 months of starting bosutinib; all but 1 patient had a T315I or V299L mutation present at baseline or identified at treatment completion. In conclusion, clinical activity to bosutinib was observed in CML pts across baseline Bcr-Abl kinase domain mutations, including those associated with resistance to other second-generation TKIs, except typically for T315I. These results support the observed benefit of bosutinib in pts with Ph+ leukemia following prior treatment with TKIs. Pts in whom new mutations were identified at treatment completion were more likely to have pre-existing mutations and to discontinue treatment due to PD/UR. The most common mutations to emerge during bosutinib therapy were T315I and V299L, some of which may have been present but not detected at baseline.Baseline mutation status, n/n evaluablea (%)IC50 fold increaseCP2L cohortCP3L cohortADV cohortCHRMCyRCHRMCyRCHRMCyRNo mutation120/133 (90)65/121 (54)34/44 (77)15/43 (35)19/49 (39)16/43 (37)≥1 mutation65/78 (83)44/78 (56)26/39 (67)11/35 (31)10/59 (17)13/55 (24) Y253H0.52/22/25/64/61/72/7 F359V0.98/94/90/21/20/20/1 Y253F1.01/10/10000 F317L2.44/43/44/81/70/90/6 F359I2.41/11/12/22/20/21/2 G250E4.35/63/53/60/54/62/7 E255V5.52/21/21/10/10/41/3 E255K9.50/22/30/10/10/41/3 V299L26.1001/20/200 T315I45.42/92/92/70/60/131/13 F359CNA002/21/20/10/1 F359SNA1/11/1001/10/1NA, not available.aTreated pts with adequate baseline response assessment. Disclosures: Cortes: Pfizer Inc: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Gambacorti-Passerini:Pfizer Inc: Honoraria, Research Funding; BMS: Research Funding; Novartis: Honoraria; Biodiversity: Honoraria. Kim:Pfizer Inc: Consultancy, Research Funding; BMS: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Ariad: Research Funding. Hochhaus:Pfizer Inc: Research Funding; BMS: Research Funding; Novartis: Research Funding; Ariad: Research Funding. Leip:Pfizer Inc: Employment. Besson:Pfizer Inc: Employment. Kelly:Pfizer Inc: Employment. Brümmendorf:Pfizer Inc: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Patents & Royalties, Research Funding; BMS: Consultancy, Honoraria.
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Jabbour, Elias, Dan Jones, Hagop Kantarjian, Susan O’Brien, Guillermo Garcia-Manero, Francis Giles, William Wierda, and Jorge Cortes. "Dynamics of BCR-ABL Kinase Domain Mutations in Patients with Chronic Myeloid Leukemia (CML) after Treatment with One, Two or Three Tyrosine Kinase Inhibitors (TKI)." Blood 108, no. 11 (November 16, 2006): 750. http://dx.doi.org/10.1182/blood.v108.11.750.750.
Full textAbstract:
Abstract Dasatinib (D) and nilotinib (N) are potent tyrosine kinase inhibitors (TKIs) with activity against many imatinib (IM) resistant BCR-ABL kinase domain mutants, except T315I. In vitro mutant models have selected specific mutations occurring after incubation with IM, D and N. Therapy with these new TKI may select for patients with T315I or other mutations relatively insensitive to them. We assessed the change in mutation status of the bcr-abl kinase domain (codons 220 to 500) in 113 patients (pts) with CML who received therapy with D and/or N after imatinib failure. Median age was 60 years (range, 21 to 82 years). Seventy-one (63%) pts received prior interferon (IFN). Median time on imatinib was 28 months (range, 2 to 78 months). At the time of imatinib failure, mutations were detected in 46 of 85 (54%) pts who had DNA sequencing. The evolution of mutations after a second TKI was as follows (Table 1). Twenty pts received a third TKI after failing IM and a second TKI. The evolution of mutations in this cohort was as follows (Table 2). Overall, 19 of 101 evaluable pts (19%), cases had new mutations emerge following TKI switch 17 after a 2nd TKI (12 nilotinib, 5 dasatinib), and 2 after a 3rd TKI (2 dasatinib). We analyzed whether these N- and D-associated new mutations were at sites that have been detected following D and N treatment in vitro (Burgess et al, PNAS 2005; Bradeen et al, Blood 2006; Von Bubnoff et al, Blood 2006). Only 14/46 (30%) kinase domain mutations that developed after D (7) or N (7) corresponded with an in vitro-identified site. Only 5 of 134 (4%) mutations identified were T315I (3 after dasatinib, 2 after nilotinib), but the mutation status of these patients was unknown after IM. We conclude that the spectrum of mutations that develops in vivo after TKI switch is broader and includes common imatinib-resistance sites as well. There appears to no marked increase in the incidence of T315I mutation after TKI switch. Table 1. Dynamics of mutations after 2nd TKI Post IM mutation No. Post-2nd TKI Mutation (New + Same + Lost) *1 pt acquired new mutation with persistence of pre-existing mutation, 1 lost 3 mutations and acquired 1, and 1 pt lost 2 mutations. Nilotinib Dasatinib Absent 39 8+NA+NA/21 3+NA+NA/18 Present 46 3+20+3/26 2+16+2*/20 Unknown 28 8/9 13/19 Table 2. Dynamics of mutations after 3rd TKI Post IM mutation No. Post-3nd TKI Mutation (compared to status after 2nd TKI) (New + Same + Lost) Nilotinib Dasatinib Absent 5 0/1 1+NA+NA/4 Present 12 0+1+0/1 2+6+3/11 Unknown 3 1/3 NA