Статті в журналах: "Cytokines; T315I mutation; chronic myeloid leukaemia"

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Asari, Kartini, Susan L. Heatley, Teresa Sadras, Tamara M. Leclercq, Stephen Fitter, Chung Hoow Kok, Andrew C. W. Zannettino, Timothy P. Hughes, and Deborah L. White. "In Vitro Modeling of Ph-like ALL Fusions Identifies Novel Kinase-Domain Mutations As Mode of TKI-Resistance - Implications for Targeted Therapy." Blood 128, no. 22 (December 2, 2016): 3957. http://dx.doi.org/10.1182/blood.v128.22.3957.3957.

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Abstract Introduction Treatment-resistant acute lymphoblastic leukemia (ALL) remains a significant clinical issue. Recently, genomic profiling has identified a new subtype of high-risk ALL termed Philadelphia-chromosome-like (Ph-like) ALL, associated with a poor outcome1. Ph-like ALL has a gene expression profile similar to Ph+ (BCR-ABL1+) ALL, characterized by the presence of fusion genes converging on kinase and cytokine signaling pathways. These pathways have been shown to be targetable both in vitro and in case reports by tyrosine kinase inhibitors (TKIs). Despite well-documented efficacy profiles, it is known from TKI-use in chronic myeloid leukemia (CML) andPh+ ALL that resistance is likely, resulting in relapse. Our study aims to model and understand mechanisms of TKI-resistance inPh-like ALL, informing future therapeutic strategies that may avert or overcome resistance, potentially improving patient outcomes. Methods Three Ph-like ALL lines were generated via retroviral-transduction from plasmids of fusion genes identified in patient cohorts (RANBP2-ABL1, SSBP2-CSF1R and PAX5-JAK2, a kind gift from C. Mullighan)2 into Ba/F3 pro-B cells. Transformation was confirmed via growth of cells in the absence of IL-3. Cells were tested for sensitivity to a panel of TKIs (imatinib, dasatinib, ponatinib, ruxolitinib and BMS-911543) via Annexin-V/7-AAD flow-cytometry and western blotting of downstream effector proteins. Drug resistance was generated through exposure of cells to incrementally increasing concentrations of TKIs over a period of 3-6 months, and cell death LD50 determined byAnnexin-V/7-AAD. Sanger sequencing of the 3-prime partner gene of each fusion was performed to identify the emergence of any kinase-domain mutations. Results Ba/F3 Ph-like cells demonstrated sensitivity to TKIs at clinically relevant doses (RANBP2-ABL1: 1 μM imatinib, 5 nM dasatinib & 5 nM ponatinib; SSBP2-CSF1R: 1 μM imatinib, 6 nM dasatinib; PAX5-JAK2: 1 μM ruxolitinib & 2 μM BMS-911543). This correlated with decreased levels of relevant downstream signaling proteins including p-Stat5, p-Erk and p-CrkL. TKI-resistant Ph-like ALL lines were tolerant to a significantly higher concentration of TKIs compared to control (RANBP2-ABL1: 10 μM imatinib, 200 nM dasatinib & 200 nM ponatinib; SSBP2-CSF1R: 10 μM imatinib, 200 nM dasatinib; PAX5-JAK2: 10μMruxolitinib & 10μM BMS-911543; Table 1). Sequencing analysis revealed that Ba/F3 RANBP2-ABL1 imatinib and dasatinib resistant cells acquired the clinically significant ABL1 T315I (c.944C>T) kinase-domain mutation, which was ultimately targetable using the third-generation TKI ponatinib (LD50: 25 nM). An ABL1 E255K (c.763G>A) and c-terminus deletion was discovered in the ponatinib-resistant line. In Ba/F3 SSBP2-CSF1R cells, a novel CSF1R L785M (c.2566C>A) mutation was identified in imatinib-resistant cells whereas a deletion spanning the SSBP2-CSF1R breakpoint was acquired in the dasatinib-resistant line. A JAK2 Y931C (c.3286A>G) point mutation previously associated with resistance to ATP-competitive inhibitors was acquired in Ba/F3 PAX5-JAK2ruxolitinib and BMS-911543 resistant lines. Conclusion In vitro modeling of Ph-like ALL resistance has identified novel kinase domain mutations and deletions that may arise as a result of targeted TKI therapy. In addition, previously identified mutations (T315I and E255K) were also identified. Detection of these mutations is important because alterations in drug-binding regions are known to result in significantly reduced TKI sensitivity, leading clinically to relapse3. This study describes an in vitro platform that can be utilized to inform future clinical approachesincluding the development of rational therapeutic approaches (and/or combination therapies) to avert resistance inPh-like ALL cases treated with rationally targeted therapies. Abbreviations: ABL1 - Abelson tyrosine protein kinase 1 CSF1R - Colony stimulating factor 1 receptor JAK2 - Janus kinase 2 PAX5 - Paired box 5 RANBP2 - RAN-binding protein 2 SSBP2 - Single-stranded DNA binding protein 2 References: 1 Den Boer et al, Lancet Oncology 2009; 10(2):125-34 2 Roberts et al, Cancer Cell 2012; 22(2):153-66 3 Barouch-Bentov & Sauer, Expert Opinion on Investigational Drugs 2011; 20(2);153-208 Disclosures Hughes: Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Australasian Leukaemia and Lymphoma Group (ALLG): Other: Chair of the CML/MPN Disease Group. White:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Honoraria, Research Funding.

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Hughes, Tim, and Giuseppe Saglio. "Expert Opinion on the Treatment of Refractory Chronic Phase Chronic Myeloid Leukaemia." European Oncology & Haematology 13, no. 01 (2017): 17. http://dx.doi.org/10.17925/eoh.2017.13.01.17.

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The development and clinical availability of second-generation tyrosine kinase inhibitors (TKIs) for the treatment of patients who discontinue imatinib therapy has further improved the outlook for patients with chronic phase chronic myeloid leukaemia (CP-CML). There is, however, uncertainty surrounding how best to treat patients after failing second-generation TKIs. A three-section questionnaire was devised by chronic myeloid leukaemia experts to address questions surrounding this issue. Responses were received from 14 out of 34 experts (41.2%). Generally, a reasonable consensus was found among the responses for most issues. There was a complete consensus that ponatinib was suitable for all patients carrying the T315I mutation regardless of the molecular response to prior treatment. There was also complete consensus that allografting is appropriate in any patient who has had blast crises and is back in a second chronic phase. More recommendations for third-line treatment of CP-CML patients are necessary.

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Massaro, Fulvio, Matteo Molica, and Massimo Breccia. "Ponatinib: A Review of Efficacy and Safety." Current Cancer Drug Targets 18, no. 9 (October 17, 2018): 847–56. http://dx.doi.org/10.2174/1568009617666171002142659.

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Ponatinib is a third generation kinase inhibitor designed to overcome the gatekeeper T315I mutation. In different trials this drug showed inhibitory activity against native BCR-ABL1 kinase and several ABL1 mutations. For this reason, ponatinib is currently indicated for the treatment of chronic myeloid leukaemia (CML) in every phase of disease resistant and/or intolerant to dasatinib and nilotinib and for whom imatinib is not indicated anymore or for patients with T315I mutation. The drug is also indicated for Ph+ acute lymphoblastic leukaemia (ALL). Ponatinib was temporarily suspended in 2013 for the occurrence of cardiovascular thrombotic events. Since then, different investigators analyzed baseline characteristics of patient candidates for ponatinib, especially cardiovascular profile, in order to describe general management recommendations in this setting. In this review, clinical trials data about the use of ponatinib in CML and Ph+ ALL patients will be discussed. It will be focused also about the safety and tolerability profile of the drug and future perspectives of employment.

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O’Connor, Lisa M., Stephen Langabeer, Shaun R. McCann, and Eibhlin Conneally. "BCR-ABL Kinase Domain Mutations & SNPs in Imatinib Resistant or Intolerant Chronic Myeloid Leukaemia Patients." Blood 110, no. 11 (November 16, 2007): 2369. http://dx.doi.org/10.1182/blood.v110.11.2369.2369.

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Abstract The Philadelphia chromosome, is formed as a result of a reciprocal translocation between chromosomes 9 and 22 and results in the formation of the hybrid oncoprotein BCR-ABL which is pathognomic of Chronic Myeloid Leukaemia (CML). Imatinib Mesylate (IM), a tyrosine kinase inhibitor that specifically binds BCR-ABL in its inactive conformation and functions as a competitive inhibitor of ATP binding, has revolutionized therapy for patients with CML. However, resistance develops in a significant proportion of cases and is predominantly mediated by single base-pair substitutions within the BCR-ABL kinase domain leading to changes in the amino acid composition and conformational changes in the kinase domain that inhibit IM binding whilst retaining BCR-ABL phosphorylation capacity. Second generation tyrosine kinase inhibitors such as Dasatinib and Nilotinib retain activity in IM-resistant patients due to less stringent binding requirements and represent viable alternatives for IM-resistant or intolerant CML patients. In this study, we undertook to examine the molecular mechanisms underlying IM resistance. A cohort of 33 patients with either primary or acquired resistance (n=31) to IM or intolerance to IM (n=2) was identified by persistently high or increasing levels of BCR-ABL transcripts determined by standardised real-time quantitative PCR. An initial allele-specific PCR screen was used to sensitively detect the clinically significant T315I mutation, which renders patients insensitive to currently available tyrosine kinase inhibitors: four (11.8%) IM resistant/intolerant patients were T315I positive. To further characterise the molecular mechanisms of mutation induced resistance, the BCR-ABL kinase domain was then screened for the presence of a mutation using a sensitive denaturing high performance liquid chromatography (dHPLC) approach. dHPLC can detect a single base pair substitution within the BCR-ABL kinase domain based on hybridization to a non-mutated wild type control. Mutated samples display reduced hybridization capacity to the dHPLC column and elute at an earlier time-point. Sensitivity of dHPLC (0.1–10%) is significantly greater than that of sequencing (15–25%). Following dHPLC analysis, samples showing evidence of mutation were examined by direct sequencing to identify the mutation(s) present. Kinase domain mutations have been identified in 18 of the 33 (55%) patients examined to date and these include p-loop mutations (M244V, G250E, Q252H), IM-binding domain mutations (T315I & F317L), catalytic domain mutations (M351T & E355G), and an activation-loop mutation (L387M). Three previously unreported mutations that may be associated with IM resistance (T267A, L273M, K291Q) were identified. The L273M positive patient also has an M244V mutation and has shown primary resistance to IM & is currently being treated with Nilotinib; the T267A patient has rising BCR-ABL transcripts, while the K291Q patient has had a 3 log reduction of BCR-ABL transcripts following IM dose escalation. In addition to the above mutations, 2 SNPs were identified at E275E and at L248L, which may not be clinically relevant. The identification of clinically significant mutations facilitates selection of alternative approaches to therapy such as IM dose escalation, second generation tyrosine kinase inhibitors or allogeneic stem cell transplantation, if eligible, allowing patient specific approaches to therapy.

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Jørgensen, H. G., and T. L. Holyoake. "Characterization of cancer stem cells in chronic myeloid leukaemia." Biochemical Society Transactions 35, no. 5 (October 25, 2007): 1347–51. http://dx.doi.org/10.1042/bst0351347.

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CML (chronic myeloid leukaemia) is a myeloproliferative disease that originates in an HSC (haemopoietic stem cell) as a result of the t(9;22) translocation, giving rise to the Ph (Philadelphia chromosome) and bcr-abl oncoprotein. The disease starts in CP (chronic phase), but as a result of genomic instability, it progresses over time to accelerated phase and then to BC (blast crisis), becoming increasingly resistant to therapy. bcr-abl is a constitutively active tyrosine kinase that has been targeted by TKIs (tyrosine kinase inhibitors), including IM (imatinib mesylate), nilotinib and dasatinib. We have developed various flow cytometry techniques to enable us to isolate candidate CML stem cells from CP patients at diagnosis that efflux Hoechst dye, express CD34, lack CD38 and are cytokine-non-responsive in culture over periods of up to 12 days in growth factors. These stem cells have been shown to regenerate bcr-abl-positive haemopoiesis in immunocompromised mice upon transplantation. We previously demonstrated that IM was antiproliferative for CML stem cells but did not induce apoptosis. Clinical experience now confirms that IM may not target CML stem cells in vivo with few patients achieving complete molecular remission and relapse occurring rapidly upon drug withdrawal. Our recent efforts have focused on understanding why CML stem cells are resistant to IM and on trying to find novel ways to induce apoptosis of this population. We have shown that CML stem cells express very high levels of functional wild-type bcr-abl; no kinase domain mutations have been detected in the stem cell population. Dasatinib, a more potent multitargeted TKI than IM, inhibits bcr-abl activity more efficiently than IM but still does not induce apoptosis of the stem cell population. Most recently, we have tested a number of novel drug combinations and found that FTIs (farnesyl transferase inhibitors) have activity against CML. BMS-214662 is the most effective of these and induces apoptosis of phenotypically and functionally defined CML stem cells in vitro, as a single agent and in combination with IM or dasatinib. The effect against CML stem cells is selective with little effect on normal stem cells. The drug is also effective against BC CML stem cells and equally effective against wild-type and mutant bcr-abl, including the most resistant mutant T315I. In association with apoptosis, there is activation of caspase 8 and caspase 3, inhibition of the MAPK pathway, IAP-1 (inhibitor of apoptosis protein-1), NF-κB (nuclear factor κB) and iNOS (inducible nitric oxide synthase). Furthermore, BMS-214662 synergizes with MEK1/2 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase 1/2] inhibitors, suggesting a second mechanism other that RAS inhibition for induction of apoptosis. Our intentions are now to explore the activity of BMS-214662 in other cancer stem cell disorders and to move this preclinical work to a clinical trial combining dasatinib with BMS-214662 in CML.

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Khorashad, J. S., N. Thelwell, D. Milojkovic, D. Marin, J. A. Watson, J. M. Goldman, J. F. Apperley, L. Foroni, and A. G. Reid. "A new rapid and sensitive assay for detecting the T315I BCR-ABL kinase domain mutation in chronic myeloid leukaemia." Journal of Clinical Pathology 61, no. 7 (April 22, 2008): 863–65. http://dx.doi.org/10.1136/jcp.2008.056804.

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Gutierrez, J. Valentin Garcia, Jesùs Odriozola, Javier Lopez, Pilar Herrera, Maria Calbacho, Marta Jimenez-Rolando, Maria Tenorio, Anabelle Chinea, and Jose Garcia-Laraña. "Detection of BCR-ABL Mutations in Patients with Chronic Myeloid Leukemia Treated with Imatinib. Experience of One Centre." Blood 114, no. 22 (November 20, 2009): 4280. http://dx.doi.org/10.1182/blood.v114.22.4280.4280.

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Abstract Abstract 4280 Introduction Imatinib (Imatinib) offers outstanding results in the treatment of chronic phase of chronic myeloid leukaemia (CML). However, there are still some patients who do not achieve an optimal response. There are several possible mechanisms both for primary refractoriness and for acquired resistance, but very often the blame falls in the appearance of ABL-kinase-domain (KD) mutations. Different mutations are related to variable degrees of resistance, ranging from the extreme refractoriness of the mutation T315I down to some which appear to have no clinical significance. Aims To define the clinical characteristics and outcome of patients treated in our centre, in which ABL-kinase-domain mutations through direct DNA sequencing have been analysed. Results In 24 of a total 86 patients, KD mutations were looked for. Screening for mutations was undertaken for one of three reasons: at diagnosis in 6 patients, suboptimal response (Leukemia-Net criteria) in 10 cases and Treatment-Failure (LN criteria) in 8 cases. Median time from diagnosis to ABL mutation detection was 38 months. No risk factors at diagnosis for the appearance of KD mutations have been found. Of the 7 mutations, 5 cases corresponded to treatment failures and 2 to patients in so-called suboptimal responses. One patient, after achieving a complete response to Imatinib, developed a T315I KD mutation and died due to blastic crisis despite having received second generation TKI. Another patient in failure (mutation G250E) achieved a molecular response with dasatinib. No mutations were found in 6 patients checked before treatment, but one of these developed a mutation G250E after 18 months, along with the criteria of a suboptimal response and achieved a mayor molecular response with dasatinib. Conclusions KD mutations are found in a proportion of patients in the situation of suboptimal response or failure to IM, more frequently in the latter in our experience, but the clinical significance of some of them is still unclear. On the contrary, DNA sequencing for screening at the moment of diagnosis offers little relevance in chronic phase CML. Disclosures: No relevant conflicts of interest to declare.

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Vráblová, Lucia, Vladimír Divoký, Pavla Kořalková, Kateřina Machová Poláková, Eva Kriegová, Romana Janská, Jan Grohmann, Milena Holzerová, Tomáš Papajík та Edgar Faber. "Deep Molecular Response Achieved with Chemotherapy, Dasatinib and Interferon α in Patients with Lymphoid Blast Crisis of Chronic Myeloid Leukaemia". International Journal of Molecular Sciences 24, № 3 (20 січня 2023): 2050. http://dx.doi.org/10.3390/ijms24032050.

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The treatment outcome in patients with chronic myeloid leukaemia (CML) in blast crisis (BC) is unsatisfactory despite the use of allogeneic stem cell transplantation (ASCT). Moreover, in some patients ASCT is contraindicated, with limited treatment options. We report the case series of two patients with lymphoid BC CML in whom ASCT was not approachable. The first patient developed BC two months after diagnosis in association with dic(7;9)(p11.2;p11.2) and T315I mutation. Blast crisis with central nervous system leukemic involvement and K611N mutation of the SETD2 gene developed abruptly in the second patient five years after ceasing treatment with nilotinib in major molecular response (MMR) at the patient’s request. Both underwent one course of chemotherapy in combination with rituximab and imatinib, followed by dasatinib and interferon α (INFα) treatment in the first and dasatinib alone in the second case. Deep molecular response (DMR; MR 4.0) was achieved within a short time in both cases. It is probable that DMR was caused by a specific immune response to CML cells, described in both agents. The challenging medical condition that prompted these case series, and the subsequent results, suggest a re-visit to the use of a combination of well-known drugs as an area for further investigation.

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Pelz-Ackermann, Oliver, Michael W. Deininger, Michael Cross, Ines Kovacs, Christine Guenther, Knuth Krohn, Song-Yau Wang, Haifa K. Al-Ali, Dietger W. Niederwieser, and Thoralf Lange. "First Results of a New PCR-Based Approach for a Sensitive and Quantitative Monitoring of BCR-ABL Kinase Domain Mutations in Patients with Chronic Myeloid Leukemia." Blood 108, no. 11 (November 1, 2006): 2191. http://dx.doi.org/10.1182/blood.v108.11.2191.2191.

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Abstract Mutations of the ABL kinase domain (KD) are the most frequent cause of acquired resistance to imatinib in patients with chronic myeloid leukaemia (CML), most likely due to selection of mutated clones on imatinib treatment. As new Bcr-Abl inhibitors become available, precise quantification of low level mutation will be required to monitor response. Here we report our results evaluating patients with advanced phase or imatinib resistant CML for KD mutations using a newly developed, sensitive and quantitative Ligation PCR (L-PCR) assay in comparison to direct sequencing. Twenty eight CML patients on imatinib (17 male, 11 female) with a median age of 62 (range 20 to 75) years in blast crisis (n=4), accelerated phase (n=12) or imatinib failure (n= 12) were analysed using both approaches. Sequencing of the ABL KD was performed using forward and reverse primers to ABL exons 4 and 7, while the L-PCR analysis focussed initially on the E255K and T315I mutations. Briefly, pairs of probes specific for either wild type (WT) or mutant BCR-ABL were added to the RT-PCR amplified ABL KD, then ligated under conditions optimized for specificity. Ligated probe pairs were than amplified in a quantitative PCR using universal primers. Quantification was performed using internal cell-in-cell dilutions of BaF3 cell lines expressing wt and mutant BCR/ABL and values were expressed as % BCR-ABLmut/ BCR-ABLWT. In our hands, this assay can detect 0.05 – 0.1% T315I and 0.01–0.05% E255K in a BCR-ABL WT background. The inter-assay variation at the lowest detection level was only 6.7 and 4,7% for the mutations T315I and E255K respectively. Results were scored positive only if two independent runs showed amplification exceeding the lowest controls. All patients were treated with a median imatinib dose of 600 (range 500–800) mg for a median of 10.5 (range 1 to 74) months. Dose reductions due to toxicity were necessary in 8 (29%) patients. Direct sequencing revealed E255K or T315I mutations in three patients, each with more than 20% mutated allele. L-PCR revealed these three patients plus three more with lower levels of mutation (T315I, 0.46% and E255K, 0.16, 0.17%). The patient with 0.46% T315I also showed G250E by sequencing. This patient was subsequently treated with Dasatinib but failed to respond. In twenty two patients negative by L-PCR, sequencing of exons 4 and 7 showed 15 (53%) to be WT, 6 (21%) to have KD mutations F317L, F359V (n=2), H396R, M315T with F359V, Y253F with the K247R polymorphism and one to have an 80 base pair deletion 3′ of the p-loop. In conclusion the L-PCR assay was able to detect T315I and E255K mutations in twice as many patients as did direct sequencing. These low-level mutations would most probably also have been missed by the D-HPLC-based screening. The fact that only L-PCR detected this mutation in a patient who failed to respond to dasatinib implies that the assays generate clinically useful information. mutations. The study is currently being expanded to include further mutations and longitudinal monitoring of a larger cohort of patients.

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Abruzzese, Elisabetta, Gabriella Marfe’, Carla Di Stefano, Malgorzata M. Trawinska, Matteo A. Russo, Romano Silvestri, Giovanni Del Poeta, et al. "Pyrrolo[1,2-B][1,2,5] benzodiazepines (PBTDs) Compounds Induce Apoptosis in Chronic Myeloid Leukaemia Cells from Patients at Onset, Imatinib and Second Generation TK Inhibitors (Dasatinib, Nilotinib) Resistant." Blood 108, no. 11 (November 16, 2006): 1385. http://dx.doi.org/10.1182/blood.v108.11.1385.1385.

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Abstract CML is a clonal myeloproliferative disease in which Bcr-Abl deregulated tyrosine kinase (TK) activity impairs blood cells homeostasis leading to altered growth, homing and apoptosis. Imatinib mesylate (Glivec, Novartis), an ATP competitor molecule, is the gold standard therapy for Bcr-Abl positive diseases, but resistance is increasingly encountered, mainly due to point mutations in the abl kinase portion of the molecule. Second generation TK inhibitors (Dasatinib, Brystol-Myers Squibb and Nilotinib, Novartis) can bind mutated forms of the protein, but T315I mutation appear unresponsive to TK treatments. Apoptosis is a cell suicide mechanism activated to remove redundant, damaged, or infected cells. An essential component of the apoptotic machinery, the caspase family, consists of a group of intracellular cysteine proteases that can be divided into initiator/upstream caspases (2, 8, 9, and 10 caspases), which activate the downstream/effector caspases (3, 6 and 7 caspases). Cleavage of a selected group of substrates by effector caspases is responsible for dismantling of essential cell components, which results in morphological and biochemical changes that characterise apoptotic cell death: cytoskeletal rearrangement, cell membrane blebbing, nuclear condensation and DNA fragmentation. Based on cell line experiments we focused our attention on some members of the pyrrolo[1,2-b][1,2,5]benzothiadiazepines (PBTDs) family to test their potential apoptotic activity in CML. Important apoptotic activity was demonstrated on K562, K562 R-IM, HL -60 ,U937 and Jurkatt cell lines, as evidenced with the concentration and the percentage of the cell death quantified by measuring PI-uptake by flow cytometry, DNA fragmentation, and analyzed by agarose gel electrophoresis generating a characteristic ladder pattern of discontinuous DNA fragments. Three PBTDs coumpounds (RS 678, RS779, RS 2630 RS) were then incubated with peripheral blood and/or bone marrow cells isolated from 30 CML untreated, 25 imatinib-resistant, 10 Dasatinib resistant, and 1 Nilotinib resistant patients. Cells were incubated at the concentration 10μM for,16,24, and 48 hours. Apoptotic DNA fragmentation was tested by agarose gels electrophoresis and was present in all but one patients in a percentage varying from 60 to 85%, compared to 0 to 5% of internal controls. Among the 25 imatinib-resistant patient 23 were tested for abl mutation using D-HPLC. Five patients showed T315I, one F317L, one each M351V, D276G, Y253H, and 2 patients showed 2 mutations (F317l and M315T; and G250G, and F359V). Only one patient Dasatinib reistant carrying M351V mutation showed very low apoptosis (5–10%), while the 5 patients with T315I mutation underwent high apoptotic DNA fragmentation. Apoptosis seem to be triggered by PBTDs through activation of initiator caspases 8 and 9 and effector caspases 3 as eveidenced by western blotting. Although more experiments are needed, these compounds appear promising for a preclinical approach in CML.

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Connor, Lisa M. O’, Stephen Langabeer, Shaun R. McCann, and Eibhlin Conneally. "Mutation Mediated Resistance to the Tyrosine Kinase Inhibitors Imatinib, Dasatinib & Nilotinib in Philadelphia Positive Leukaemia." Blood 112, no. 11 (November 16, 2008): 4245. http://dx.doi.org/10.1182/blood.v112.11.4245.4245.

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Abstract The Philadelphia chromosome is formed as a result of a reciprocal translocation between chromosomes 9 and 22 and results in the formation of the hybrid oncoprotein BCR-ABL. It is observed in over 95% of Chronic Myeloid Leukaemia (CML) and approximately 30% of adult Acute Lymphoblastic Leukaemia (ALL) cases. Imatinib Mesylate (IM), a tyrosine kinase inhibitor that specifically binds BCR-ABL in its inactive conformation has revolutionized therapy for CML and Ph+ ALL. However, resistance develops in a significant proportion of patients and is predominantly mediated by single base-pair substitutions within the BCR-ABL kinase domain leading to changes in the amino acid composition that inhibit IM binding whilst retaining BCR-ABL phosphorylation capacity. Second generation tyrosine kinase inhibitors such as Dasatinib and Nilotinib retain activity in IM-resistant patients due to less stringent binding requirements and represent viable alternatives for IM-resistant patients with a suitable molecular profile. In this study, we undertook to examine the molecular mechanisms underlying IM resistance. A cohort of 40 patients with either primary or acquired resistance or intolerance to IM was identified by persistent high or increasing levels of BCR-ABL transcripts determined by real-time quantitative PCR. An allele-specific PCR screen was used to sensitively detect the clinically significant T315I mutation, which renders patients insensitive to currently available tyrosine kinase inhibitors: five (12.5%) IM resistant/intolerant patients were T315I positive. To further elucidate the molecular mechanisms of mutation induced resistance, the BCR-ABL kinase domain was screened for the presence of a mutation using a sensitive denaturing high performance liquid chromatography (dHPLC) approach. Samples showing evidence of mutation were examined by direct sequencing to identify the mutation(s) present. Kinase domain mutations have been identified in 20 of the 40 (50%) patients examined to date and these include p-loop mutations (M244V, G250E, Q252H), IM-binding domain mutations (T315I), catalytic domain mutations (M351T), an activation-loop mutation (L387M). Three previously unreported mutations were identified in patients with indications of IM resistance (T267A, E275Q) and Nilotinib resistance (L273M). The L273 residue lies adjacent to a region of the BCR-ABL kinase domain bound by Nilotinib. Three patients were found to harbour mutations at two distinct kinase domain residues while one patient harboured mutations at three distinct residues, supporting the theory that patients who develop mutation-mediated resistance to one kinase inhibitor may become resistant to subsequent inhibitors by a similar mechanism. The identification of clinically significant mutations facilitates selection of alternative approaches to therapy such as dose escalation of IM, second generation tyrosine kinase inhibitors or allogeneic stem cell transplant, if eligible, at an early stage in a patient’s disease, tailoring patient specific approaches to therapy.

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Coude, Marie-Magdelaine, Odile Luycx, Marie-Estelle Cariou, Odile Maarek, Hervé Dombret, Jean-Michel Cayuela, and Delphine Rea. "Undetectable molecular residual disease after omacetaxine and nilotinib combination therapy in an imatinib-resistant chronic myeloid leukaemia patient harbouring the BCR-ABL1 T315I gatekeeper mutation." British Journal of Haematology 157, no. 3 (January 9, 2012): 407–10. http://dx.doi.org/10.1111/j.1365-2141.2011.09016.x.

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Kim, Soo-Hyun, Dongho Kim, Dong-Wook Kim, Hyun-Gyung Goh, Se-Eun Jang, Jeong Lee, Wan-Seok Kim, Il-Young Kweon, and Sa-Hee Park. "Analysis of Bcr-Abl kinase domain mutations in Korean chronic myeloid leukaemia patients: poor clinical outcome of P-loop and T315I mutation is disease phase dependent." Hematological Oncology 27, no. 4 (December 2009): 190–97. http://dx.doi.org/10.1002/hon.894.

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Copland, Mhairi, Daniel Slade, Jenny Byrne, Kristian Brock, Hugues De Lavallade, Charles Craddock, Richard Clark, et al. "FLAG-IDA and Ponatinib in Patients with Blast Phase Chronic Myeloid Leukaemia: Results from the Phase I/II UK Trials Acceleration Programme Matchpoint Trial." Blood 134, Supplement_1 (November 13, 2019): 497. http://dx.doi.org/10.1182/blood-2019-125591.

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Background: The outcome of patients with blast phase chronic myeloid leukaemia (CML) remains extremely poor despite the advent of tyrosine kinase inhibitors (TKIs), and the majority of blast phase patients have already failed the currently licensed TKIs during the chronic phase. Currently there is no standard therapy for patients with blast phase CML, but most will receive 2 or 3 courses of chemotherapy or a TKI, followed by stem cell transplantation (alloSCT). In the PACE clinical trial of ponatinib, 23% of patients with blast phase CML achieved major cytogenetic response, with overall survival of 20% at 12 months. To date, there has been no prospective evaluation of salvage chemotherapy in combination with ponatinib. Methods: MATCHPOINT is a seamless Phase I/II trial to determine the optimal dose of ponatinib in combination with conventional chemotherapy (fludarabine, cytarabine, idarubicin and G-CSF; FLAG-IDA) for the treatment of CML in blast phase. An efficient Bayesian design, EffTox, which utilises both efficacy and toxicity to select desirable doses for subsequent patient cohorts, was used. Given the observed patient outcomes and the investigators' prior beliefs, the next cohort's dose is chosen adaptively to optimize the risk-benefit trade-off between efficacy and toxicity. The initial ponatinib dose was 30mg daily, commenced on day 1 of FLAG-IDA chemotherapy. Where possible, ponatinib was given continuously during the remission-induction phase, but ponatinib therapy was interrupted from day 28 of each cycle if haematologic recovery had not occurred. Patients received one or two cycles of FLAG-IDA chemotherapy plus ponatinib at the allocated dose level, and then proceeded to alloSCT followed by ponatinib maintenance. The co-primary outcome measured toxicity (dose-limiting toxicity; DLT) during the first therapy cycle and efficacy (clinical response; haematologic or cytogenetic) after the first cycle. Results: A total of 17 patients were recruited between March 2015 and May 2018. Eight presented with de-novo blast phase CML and 9 had progressed on TKI. Eight patients had additional cytogenetic abnormalities and 3 had BCR-ABL kinase domain mutations (E255K x2 and T315I x1) at study entry. Nine and 8 patients commenced 1 or 2 cycles of FLAG-IDA + ponatinib, respectively. Of these, 16 patients were evaluable for the primary analysis. Treatment-related mortality (TRM) occurred in 3 patients during remission-induction (cardiomyopathy x1, pulmonary haemorrhage x1 and marrow aplasia x1). During cycle 1, 4/16 patients experienced a DLT (raised ALT x1, fulminant cardiomyopathy x1, cerebral sinus vein thrombosis x1, elevated amylase x1). Eleven of 16 (69%) patients achieved a clinical response, defined as either complete haematologic (3 patients) or cytogenetic response (1 minor and 9 major [2 partial and 7 complete]). Five patients achieved major molecular remission after cycle 1. All patients were recommended to be dosed at the 30mg dose, which was defined as the optimal dose for ponatinib with FLAG-IDA. Nine patients proceeded to alloSCT (7 myeloablative and 2 reduced intensity). Four patients developed acute GvHD (grades 2-4) and 3 had CMV reactivation. Four patients re-started ponatinib maintenance post alloSCT, and de-escalated to 15mg if major molecular remission was maintained. Overall 1-year survival was 45.8% (95% CI 26.9-77.7%), estimated using Kaplan Meier. Summary/Conclusion: We report the first prospective trial of ponatinib and conventional chemotherapy for blast phase CML. The EffTox model combined Phase I and II, and delivered efficiency in determining the trial's optimal dose. This innovative statistical model has the potential to be applied in other rare malignancies. We confirm that FLAG-IDA + ponatinib 30mg, is a tolerable combination in blast phase CML, with promising response and survival. Post-transplant ponatinib was well tolerated and no excess toxicity was observed when ponatinib was used both pre- and post alloSCT. The combination of ponatinib and FLAG-IDA represents a potentially important advance in the treatment of blast phase CML, a rare complication which currently has a very poor outcome. Disclosures Copland: Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Honoraria, Speakers Bureau; Cyclacel: Research Funding. Byrne:Ariad/Incyte: Honoraria, Speakers Bureau. Brock:Astrazeneca: Equity Ownership, Honoraria. De Lavallade:Incyte biosciences: Honoraria, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau. Clark:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Ariad/Incyte: Honoraria; Jazz Pharmaceuticals: Honoraria; AbbVie: Honoraria. Milojkovic:Novartis: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau. Yap:Celgene: Honoraria; Faron Pharmaceuticals: Other: Consultancy. OffLabel Disclosure: Ponatinib is a tyrosine kinase inhibitor (TKI). It is licensed to treat: - chronic myeloid leukaemia where the leukaemic cells have gene change (mutation) called T315I - acute lymphoblastic leukaemia that has an abnormal chromosome called the Philadelphia chromosome, or has the T315I mutation

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15

Pelz-Ackermann, Oliver, Michael W. N. Deininger, Michael Cross, Kathrin Wildenberger, Rainer Krahl, Song-Yau Wang, Haifa K. Al-Ali, Dietger W. Niederwieser, and Thoralf Lange. "BCR-ABL Kinase Domain Mutations in the P-Loop and at Imatinib Contact Sites in CML Patients with Imatinib Resistance Are Associated with Higher Maximum Imatinib Doses." Blood 110, no. 11 (November 16, 2007): 2918. http://dx.doi.org/10.1182/blood.v110.11.2918.2918.

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Abstract Objectives: Acquired resistance to imatinib (IM) in patients with chronic myeloid leukaemia (CML) is frequently due to mutations of the BCR-ABL kinase domain (KD). Current thinking holds that this results from the selection of pre-existing mutant clones on IM. As new BCR-ABL inhibitors with differential activity against KD mutant BCR-ABL have become available, precise quantification of key mutations even at a low level is required to adequately monitor responses. Here we report that the occurrence of G250E, Q252H, Y253F/H, E255K/V, T315I, F317L, F359V mutations at the time of IM resistance is independently associated with a high maximum IM dose. Patients and Methods: We have developed highly specific allele-specific ligation-PCR assays (L-PCR) to accurately quantify a panel of frequent KD mutations, including G250E, Q252H, Y253F/H, E255K/V, T315I, F317L, F359V. In limiting dilution experiments the L-PCR assays routinely detect between 0.05 and 0.1% of mutant allele in total BCR-ABL, and their average dynamic range is in the range of 4.5 log. Forty-three patients with imatinib failure were analyzed. The median age was 60 (range 20 – 75) years and the median disease duration 64 (range 3–213) months. Eleven patients were in blast crisis, 20 in accelerated phase and 12 in chronic phase. Results: Patients were treated with chemotherapy (48%) and/or interferon alpha (76%) prior to IM. The median maximal IM dose was 600 (range 500–800) mg and the median duration of total therapy until resistance was 15.5 (range 1 to 75) months. Eighteen patients (42%) had dose reductions due to toxicity. At the time of IM resistance, clonal evolution was present in 21/41 (47%) of the patients. L-PCR identified 50 mutations in 29/43 patients (67%) (table1). One, 2, 3 or 4 different mutations were identified in 14, 11, 2 and 2 patients, respectively. The T315I and E255K/V mutations accounted for 32/50 (64%) of all mutations, while Q252H and Y253H were not detected. Twelve mutations (24%) were confirmed by direct sequencing (DS) and an additional M315T mutation and a K247R polymorphism were detected. Thirty eight (76%) mutations were negative by DS for the corresponding mutation from the L-PCR panel but additional mutations (L248V, M351T, H396Rx2, L298Vx2) were identified. All 38 L-PCR positive mutations were confirmed in a second independent experiment. The mutated clone was significantly smaller in mutations with no confirmation by DS (median 0.2, range 0.05–12.63%) compared to positive by DS (median 40.76, range 13.58–100%, p=0.003, Wilcoxon test). The detection of one or more mutations was significantly more frequent in patients with a maximum IM dose of 800mg (n=15/17, 88%) compared to less than 800mg (14/26, 53%, p= 0.02 Fisher’s exact test). Multivariate analysis (Wald forward) confirmed that a maximum IM peak dose of 800mg is an independent prognostic parameter to detect a mutation from the L-PCR panel at the time of IM resistance. Conclusions:High sensitive testing with L-PCR detects mutations with 4-fold increased frequency compared to direct sequencing.Mutations, including p-loop and T315I, may be selected by exposure to higher drug levels.The predominance of T315I and E255K/V mutations at a low level is consistent with the findings in newly diagnosed patients with Ph+ALL (Pfeifer et al. Blood 2007).

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Cea, Michele, Antonia Cagnetta, Gabriella Cirmena, Anna Garuti, Ilaria Rocco, Claudia Palermo, Ivana Pierri, et al. "Hedgehog Signaling Is Useful as a Novel Molecular Marker for Predicting Relapse and Resistance During Chronic Myeloid Leukemia Treatment." Blood 116, no. 21 (November 19, 2010): 1215. http://dx.doi.org/10.1182/blood.v116.21.1215.1215.

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Abstract Abstract 1215 Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by the expansion of a Leukemic Stem Cell (LSC) clone carrying a Philadelphia translocation, which outgrows the non-malignant haematopoietic stem cells. The tyrosine kinase inhibitors (TKIs) imatinib, nilotinib and dasatinib, are the gold standard for CML treatment since each one shows an impressive rates of complete cytogenetic and molecular response in chronic phase (CP) CML. However the major problem concerning the final efficacy of TKIs therapy is that the majority of responding CP CML patients have detectable BCR-ABL transcripts which might arise from a population of quiescent CML LSC not effectively targeted by TKIs. Therefore the molecular monitoring not always provide a sufficiently precise evaluation of patients to allow the appropriate choice of clinical interventions. Accordingly, it is necessary to monitor the appearance and increase of LSCs to identify and to treat quickly the fundamental responsible for relapse. Thus, we focused Hedgehog (Hh) signalling which has been proved essential for maintenance of cancer stem cells in myeloid leukaemia. Notably recent studies reported that the expansion of BCR-ABL positive leukemic stem cell is dependent on Hh pathway activation. Here, we analyzed the mRNA levels of Smoothened (SMO), a seven-transmembrane domain receptor protein, and Ptch1, a surface receptor regulator of SMO, in 20 CP-CML patients (8 High, 4 Intermediate and 8 Low Sokal Risk respectively) at diagnosis and during the follow-up. Using RT-PCR, in diagnosis setting, we proved that 60% of patients (bone marrow samples) showed Hh signalling significantly activated compared to CD34+ cells from healthy donors. In detail 75% (6/8) of High Sokal Risk CML patients showed an up-regulation of Smo and a down-regulation of Ptch1 mRNA levels, suggestive of active Hh signalling at diagnosis. Conversely Low and Intermediate Sokal Risk CML patients did not show features of Hh activation. Finally we monitored, during the follow-up, the mRNA levels of Smo and Ptch1 together with BCR-ABL to assess the kind of relationship between these parameters. Interestingly we noticed a direct correlation between the increase of BCR-ABL mRNA levels and signs of Hh activity (Smo mRNA increase level and Ptch1 mRNA decrease level). Characteristically, molecular monitoring highlighted that all patients developing resistance to TKIs treatment, showed a tendency to Hh activation (high and low mRNA levels of Patch1 and Smo respectively) few months before the development of Abl KD mutation (10/10). Typically this last behaviour was more evident in patients developing the gatekeeper mutation T315I. Finally, in accordance with published data, we noted that the pharmacological inhibition of Hh signalling impairs the growth of TKIs resistant human CML cell line BaF/T315I, suggesting a novel treatment option. All our data provide molecular evidence for a role of the stemness pathway to predict quickly both the relapse and the TKIs resistance during CML treatment. Therefore, we propose to use Ptch1 and Smo mRNA levels together with BCR-ABL for the molecular monitoring of CP CML. Disclosures: No relevant conflicts of interest to declare.

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Cerveira, N., R. B. Ferreira, S. Bizarro, C. Correira, L. Torres, S. Lisboa, J. Vieira, et al. "PB1922 PONATINIB INDUCES A SUSTAINED DEEP MOLECULAR RESPONSE IN A CHRONIC MYELOID LEUKAEMIA PATIENT WITH AN EARLY RELAPSE WITH A T315I MUTATION FOLLOWING ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION." HemaSphere 3, S1 (June 2019): 874. http://dx.doi.org/10.1097/01.hs9.0000566188.04988.71.

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18

Ibrahim, Amr R., Marco Bua, Jamshid S. Khorshad, Dragana Milojkovic, Lina Eliasson, Richard Szydlo, Letizia Foroni, et al. "Efficacy of Tyrosine Kinase Inhibitors (TKIs) as Third Line Therapy In Patients with Chronic Myeloid Leukaemia In Chronic Phase Who Have Failed Two Prior TKIs." Blood 116, no. 21 (November 19, 2010): 2274. http://dx.doi.org/10.1182/blood.v116.21.2274.2274.

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Abstract Abstract 2274 Patients with CML in chronic phase who have failed imatinib therapy are commonly treated with dasatinib or nilotinib, but a significant proportion fail to respond or relapse in which case they are often treated with the other tyrosine kinase inhibitor (TKI) that they had not yet received. We report here the largest series of CML patients in CP treated with a third line TKI after failing both imatinib and another TKI. We enrolled 26 patients. The median age was 64 years and 54% were male. 20 patients had received dasatinib and 6 nilotinib as second line therapy. All patients were still in first CP at the moment of commencing third line therapy, and none was harboring a T315I mutation. Failure to second line therapy was defined as no CHR at 3 months, no major cytogenetic response (MCyR) at 12 months or loss of a hematological or cytogenetic response. Patients who were unable to continue therapy on account of toxicity were also considered as having failed therapy. The median follow up for the surviving patients after starting third line therapy was 21.5 months (range, 6 – 46.5 months). The 2.5 years (30 months) cumulative incidences of MCyR, CCyR and MMR were 48.2%, 32.4%, 21.1% respectively. Multivariate analysis showed that the achievement of at least MiCyR (<95% Ph-positive) on imatinib (RR=5.6, p=0.03) or on second line therapy (RR=11.8, p=0.006) were the only independent predictors for the achievement of CCyR. When combining both variables we found that patients who had achieved MiCyR on one of the two prior therapies had a significantly better OS and higher probability of achieving cytogenetic response on third line therapy, i.e. the 30 month probability of OS and CCyR were 72.7% vs 20.4% (p=0.03) and of 71.4% vs 0% (p=0.0005) respectively (Figure). During follow up 9 (34.6%) patients died. The probability of OS at 30 months was 46.7%. The achievement of a cytogenetic response on second line and age younger than 64 (possibly reflecting eligibility for transplantation) were the only independent predictors for OS (RR=6.5, p=0.02 and RR=0.13, p= 0.02). Seventeen patients (65%) were classified as intolerant to previous therapies (imatinib or second line TKI). Intolerant patients had a probability of responding to the third line therapy similar to those of the resistant patients, but when this cohort was subdivided according to the type of intolerance we found that 11 patients who had hematologic toxicity with either therapy had a probability of CCyR at 30 months lower than that of the remaining 15 patients (11.1% vs 47.5 %, p=0.03), while the 8 patients with non-hematologic intolerance to the imatinib or to the second line had a probability of 30-month CCyR greater than that of the remaining 18 patients (87.5% vs 5.6%, p<0.001). At 3 months 26 patients remained on follow up, of whom 9 patients had achieved at least MiCyR. These 9 patients had better 30-month probabilities of CCyR and OS than the patients who had failed to achieve MiCyR, namely 88.9% vs 13.3% (p<0.0001), and 87.5% vs. 35.0% (p=0.1). When we excluded the only patient who died of non-leukemia related causes while in CCyR, the probabilities of OS was 100% vs 35.0% (p=0.04) Which patients should be offered third line TKI therapy? Patients who achieved cytogenetic response on first or second line therapy and patients with a history of non-hematologic intolerance to the prior TKI benefited from a third TKI. Patients with primary cytogenetic resistance to two TKIs or with a history of hematologic intolerance should receive an allogeneic stem cell transplant when possible. For patients in this situation who lack a transplant option we would recommend only a short course (3 months) of the third line therapy to identify responders. Non-responders should be offered experimental studies. Disclosures: Marin: Bristol-Myers Squibb: Consultancy; Novartis: Consultancy, Research Funding.

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Kaeda, Jaspal S., Ken I. Mills, Michael G. Kharas, Giuseppe Saglio, Michaela Schwarz, Christian Oberender, Bärbel Pawlaczyk-Peter, et al. "Increased MSI2 expression Is Associated with Aggressive CML and AML." Blood 118, no. 21 (November 18, 2011): 2516. http://dx.doi.org/10.1182/blood.v118.21.2516.2516.

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Abstract Abstract 2516 The events triggering arrested differentiation and a more aggressive disease in chronic myeloid leukaemia (CML) patients are unclear. Dysregulation of MSI2 has been suggested as a causal event in the transformation of chronic phase (CP), a relatively indolent disease phase, to blast crisis (BC), which is usually fatal. The Musashi gene family, regulated by HOXA9, has been shown to control critical cell fate decisions by binding to the 3'untranslated region of target mRNAs, thereby inhibiting translation. This results in dysfunction of the regulatory pathway, leading to hematopoietic stem cell (HSC) proliferation, impaired myeloid differentiation and worse clinical prognosis in CML and AML. In this study we attempt to verify these published results and test the utility of using MSI2 as a prognostic marker in CML and AML. To assess if MSI2 expression levels might be prognostic in CML, we screened 65 heterogeneous patients [median age; 45 years (19–75); M:35; F:30] of whom 54 were in CP and 11 in advanced disease, treated with different modalities. Of these 64 were administered one or more of the tyrosine kinase inhibitors, of these 2 underwent haematopoietic stem cell transplant. One patient was managed with interferon plus cytarabine only. BCR-ABL1 Kinase domain mutations were documented in 8 of the 11 patients in advanced disease, of which 3 were T315I in combination with another mutation and 4 mapped to the P-loop. Of the 30 CP patients screened 10 had KD mutation, of which one was T315I and 2 were within the P-loop. The overall median survival for 11 patients in advanced disease [2 accelerated phase; 9 BC (4 myeloid, 5 lymphoid)] is 3.9 years (1.1–19.0), 7 of the BC had died (median survival 4.0 years). All the CP patients are alive with an overall median survival of 3.54 years (0.46–16.4). In addition we screened 89 diagnostic samples from acute myeloid leukaemia (AML) patients (M:53; F:36) with a median age of 61 years (8–85) and 27 normal control blood donor samples. The MSI2, BCR-ABL1 and GUSß endogenous control gene) mRNA expression were measured by TaqMan real time quantitative polymerase chain reaction (RQ-PCR) in separate assays. The MSI2 and GUSß mRNA were measured in duplicate and BCR-ABL1 in triplicate as were the standards for the three genes assayed. The data were expressed as % ratio of the control gene, only samples with >5500 GUSß copies were included in the data presented here. Msi2 expression was detected in all samples by RQ-PCR but was significantly increased (p=<0.0001) in advanced disease CML patients [median 6.7 (1.3–22.9)], irrespective of lymphoid or myeloid transformation, when compared with CML CP subjects [median 2.2 (0.2–6.3)]. BCR-ABL1 was detectable in all CML samples. The median for BCR-ABL1 copies in the 11 advanced disease patients was 101.4 (0.3–325.8). Remarkably, when patients in CP were classified as less and greater than the 2.16 MSI2 median, the BCR-ABL1 mRNA median values were 19.4 (0.1–1000) and 1.31 (0.02–393) transcripts, respectively, i.e. higher MSI2 expression was associated with lower BCR-ABL1 transcripts (p=0.023). Furthermore, there was no significant difference in MSI2 expression between the normal control population [median 2.16 (1.33–7.53)] and CP patients (p=0.204). The 89 AML samples had a median MSI2 value of 3.67 (0.41–40.17). Outcome data was available for 86 and these were classified into tertiles (low, intermediate and high MSI2). Kaplan-Meier survival analysis revealed a p value of 0.088 when comparing the outcome of patients in the low and high groups and 0.091 between low and intermediate/high. Although this is a small cohort, the difference in outcome was due to only 4 out of 26 (15.4%) patients in the low group who had died with a mean survival of 525 days (182–840); compared to 17 out of 53 (32%) in the intermediate/High group with a mean survival of 52 days (1–120). In summary, our data identified an inverse relationship between MSI2 and BCR-ABL1 expression levels in CP patients. These observations may reflect the finding of lower expression of HOXA family and MSI2 in quiescent CML stem cells compared to normal stem cells. A longitudinal study among the CP patients might indicate how it effects their overall survival. We also provide evidence that increased MSI2 expression correlates with an aggressiveness of CML and early death in AML. Disclosures: No relevant conflicts of interest to declare.

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Benedicte, Deau, Bachy Emmanuel, Raus Nicole, Nicolini Franck-emmanuel, Rea Delphine, Reman Oumedaly, Maillard Natacha, et al. "Impact of Prior Second-Generation Tyrosine Kinase Inhibitors on the Outcome of Hematopoietic Stem Cell Transplantation for Chronic Myeloid Leukemia." Blood 116, no. 21 (November 19, 2010): 2267. http://dx.doi.org/10.1182/blood.v116.21.2267.2267.

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Abstract Abstract 2267 Prior to the introduction of the tyrosine kinase inhibitors (TKI) into clinical practice, the only curative therapy for chronic myeloid leukaemia (CML) was allogeneic hematopoietic stem cell transplantation (HSCT). HSCT is nowadays recommended for patients in accelerated phase, blastic phase or with the T315I mutation and for patients who experience second-line TKIs (TKI2) failure or intolerance. The impact of TKI2 for CML on the results of subsequent HSCT has not yet been clearly established, although preliminary data does not suggest an increase of non relapse mortality (NRM) in patients previously treated with these agents. To assess whether exposure to TKI2s before HSCT adversely affects outcome, we retrospectively analyzed 31 patients with CML reported to the French registry for HSCT between January 2001 and December 2008 who received a first HSCT for imatinib resistant or intolerant CML subsequently treated with either nilotinib or dasatinib or both. The median age at diagnosis was 39.8 years (range, 19–61). At the start of TKI, 15 (48%) patients were in chronic phase, 12 (39%) patients were in accelerated phase and 4 (13%) patients were in blast crisis. For chronic phase patients, 6 (40%) patients were classified as low Sokal risk, 7 (46%) as intermediate risk, and 2 (14%) as high risk. After imatinib failure (among the 27 patients in chronic or accelerated phase at diagnosis), 21 and 6 patients received dasatinib and nilotinib, respectively. Among these patients, 9 patients received a third line therapy, including 5 sequential therapies with both drugs. The best response to the second and third-line treatments with TKI2 was a complete molecular response in 2 patients, a major molecular response in 2 patients, a complete cytogenetic response (CCR) in 7 patients, a complete hematologic response in 14 patients and no response in 2 patients. 14 patients eventually failed TKI2 treatment because of resistance, whereas 8 were considered as intolerant. Of 14 patients who developed resistance to TKI2, 2 had a mutation identified (T315I in both cases). Median interval from diagnosis to HSCT was 19 months (range, 3–151). At time of transplant, 21 patients were in chronic phase, 10 in accelerated phase and none in blast crisis. 19 patients received a graft from an unrelated donor whereas 12 a match related donor. Stem cell source was peripheral blood, bone marrow or cord blood in 20, 8 and 3 patients, respectively. The conditioning regimen was myeloablative in 21 patients combining either TBI and cyclophosphamide (9 patients) or high dose IV busulfan and cyclophosphamide (12 patients), and a RIC for 10 patients. All patients engrafted successfully: median time to neutrophil and platelet recovery was 18 days and 21 days respectively. Grade 2–4 acute graft-versus-host disease (aGVHD) was observed in 11 (37.9%) patients; grade 3–4 aGVHD occurred in 6 (20.6%) patients. Chronic GVHD was observed in 15 (60%) of 25 patients alive after day 100. The median follow-up after HSCT is 27 months (range, 1.2–50.2). At time of analysis, 11 (35%) patients died, 7 (22.5%) from NRM and 4 (12.5%) from progression of disease. NRM was due to infection (3 patients), GVHD (2 patients), post transplant lymphoma disease (1 patient) and unknown cause (1 patient). The 1-year overall survival (OS) was 79.2% (95% CI, 64,3-94,1%) and the estimated 2-year OS was 55,5% (95% CI, 35,0-75,9%). One-year relapse and NRM rates were respectively 10,3% (95% CI, 2.5–24.6%) and 19.1% (95% CI, 6.7–36.2%). In univariate analysis, no variable had a significant impact on outcome among Sokal score, disease phase at diagnosis, Grathwol score, age at HSCT, time from diagnosis to HSCT, or quality of response before HSCT. In a multivariate analysis, only quality of response (at least better than CCR) was significantly associated with a better outcome in terms of OS (p=0.0459, HR=0.17, 95% CI=0.03-0.97). In conclusion, TKI2 prior to HSCT did not result in an increased risk of NRM. However, our observation that patients in cytogenetic or molecular response at time of HSCT have a significant better outcome underscores the importance of a stringent prospective molecular monitoring under TKI2 therapy and the need for prognostic factors of response under TKI2. Such elements could help to better define after initiating TKI2 treatment patients that could really benefit from HSCT and the best timing of the procedure. Disclosures: No relevant conflicts of interest to declare.

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Vannuffel, Pascal, Barbara Cauwelier, Céline De Rop, and Friedel Nollet. "Detection and Monitoring of BCR-ABL1 Kinase Domain Mutations By Next Generation Sequencing." Blood 126, no. 23 (December 3, 2015): 4042. http://dx.doi.org/10.1182/blood.v126.23.4042.4042.

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Abstract Background. Among myeloproliferative diseases, development of chronic myeloid leukaemia (CML) is associated with the emergence of the fusion oncogene BCR-ABL1 resulting from a t(9,22) chromosomal translocation (Philadelphia chromosome). Mutations of the BCR-ABL1 kinase domain constitute a major cause of treatment failure in CML patients receiving tyrosine kinase inhibitor (TKI) treatment. Moreover, the occurrence of cells with multiple mutations is frequently associated with a higher resistance rate to the different TKI (Imatinib, Dasatinib or Nilotinib). So far, the gold standard procedure to detect BCR-ABL1 mutations remains the conventional Sanger Sequencing (SS), endowed with an analytical sensitivity of 10 to 20 %. The recent implementation of Next Generation Sequencing (NGS) allows lowering the sensitivity level and quantitative follow-up of the mutated subclone(s), which probably improves CML patient's treatments management. Aims. In this retrospective study, we evaluate the advantage of NGS approach to i) identify patients harbouring (low level) mutations that have not been not assessed by conventional methods, ii) detect the emergence of mutated clones earlier than SS and iii) monitor evolution of mutations. Methods. Total BCR-ABL1 RNA was transcribed into a long range cDNA covering the kinase, the regulatory, and the SH2/SH3 domains of either p190 or p210 BCR-ABL1 transcripts (exons 4 to 10). From primers designed with the AmpliseqTM Designer Software, a set of 10 amplicons was generated according to the AmpliSeqTM protocol. Bar-coded libraries were sequenced on the Ion Torrent PGM platform and data were analysed with Torrent Suite and NextGene softwares. Serial dilutions of samples harbouring mutations at different levels were used to determine a variant frequency cut-off. Our methodology was applied to a group of 36 patients presenting with poor response to TKI and with no mutation detected by SS and to a set of 100 samples, corresponding to 20 mutated patients, at different time points before the time of mutation identification by SS. Results. From the serial dilutions experiment, the detection limit of the assay was set up to 2 % (R² > 0.997). An overall coverage ranging from 20 000 to 50 000 reads can be achieved for the hotspot mutations when up to 12 samples were tested together on a 316 Ion chip. On the 36 patients tested by NGS versus SS, no one was found to harbour TKI-resistance mutation. NGS successfully detected all mutations identified by SS; mutations were typically detected within 4 months (18/20 patients) and were also detected up to 9 months prior to detection by SS, even in patients with a low abundance of BCR-ABL1 transcripts and in sequencing failure by SS. In 2 patients presenting with up to 3 mutations, evolution of mutations (emergence, expansion or depletion) correlates with clinical data of treatment decisions, i.e. E255K (patient-1) and L248V (patient-2) depletions when switching from Imatinib to Dasatinib, F317L (patient-1), G250E (patient-1) and T315I (patient-2) expansions under Dasatinib and a complete but transient depletion of T315I (patient-2) with the protein synthesis inhibitor homoharringtonine (Omacetaxine). Finally, assessment of the mutation status of one patient with compound mutations following an Illumina protocol on a MiSeq platform had allowed comparison of technologies performances. Conclusions. NGS did not detect mutations in 36 patients poorly responding to TKI with no detectable BCR-ABL1 TK mutation by SS. For 20 patients showing BCR-ABL1 TK mutation by SS, NGS was able to detect the mutation in samples taken up to 9 months prior to the moment when the mutation was observed by SS. Advances in sequencing technologies and further lowering sensitivity levels can contribute even more to earlier detection of mutations and guide an earlier switch of TKI. Quantitative and sensitive monitoring of mutation evolution can also inform the most appropriate and optimized treatment algorithms. A prospective evaluation of the clinical impact of NGS-based BCR-ABL1 mutation detection is ongoing. Disclosures Vannuffel: ARIAD Pharmaceuticals: Research Funding.

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Hayakawa, Fumihiko, Keiki Sugimoto, Shingo Kurahashi, Hironori Matsuyama, Yasuo Harada, Norikazu Hashimoto, Naoto Ohi, et al. "A Novel Direct STAT3 Inhibitor OPB-31121 Induces Tumor-Specific Growth Inhibition In a Wide Range of Hematopoietic Malignancies and Effectively Suppresses the Chemotherapy Resistant Quiescent Cells In Vivo." Blood 116, no. 21 (November 19, 2010): 3277. http://dx.doi.org/10.1182/blood.v116.21.3277.3277.

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Abstract Abstract 3277 Signal Transduction and Activator of Transcription (STAT) proteins are extracellular ligand-responsive transcription factors that mediate a wide range of biological processes such as cell proliferation, apoptosis, differentiation, development, and immune response. Stimulation with cytokines or growth factors results in the tyrosine phosphorylation of STAT proteins via activation of upstream tyrosine kinases like Janus kinase (JAK) family kinases. Activated STAT proteins translocate to the nucleus and regulate gene expression through direct binding to the promoters of responsive genes. STAT3 is widely recognized as being a master regulator of the cellular functions that lead to the cancer phenotype. Constitutive activation of STAT3 is observed in a broad spectrum of human cancers and induces uncontrolled cell proliferation and apoptosis-resistance. It has been identified as a promising target for anti-tumor drug, but to date most of the trials to block STAT-signaling were the inhibition of upstream kinases like JAK family kinases, especially in clinical trials. Here, we report a novel STAT3 inhibitor, OPB-31121, that has no inhibitory effect on kinases including JAK family kinases. OPB-31121 treatment of HEL92.1.7 cells that had constitutive active mutation of JAK2 inhibited phosphorylation of STAT3 without inhibition of JAK2 phosphorylation (Figure A). STAT3 phosphorylation by JAK2 in vitro was also inhibited by OPB-31121 under constant JAK2 autophosphorylation. On the other hand, it did not inhibit dimerization and nuclear translocation of STAT3 once STAT3 was phosphorylated. Also, direct association between OPB-31121 and STAT3 was suggested in vitro. These data implies that one of the mechanisms of OPB-31121 action was the direct inhibition of STAT3 phosphorylation without JAK kinase inhibition. OPB-31121 demonstrated strong growth suppressive effect (IC50 < 10 nM) in cell lines of a wide range of cancer especially hematopoietic malignancies including acute myeloid leukemia (AML) with JAK2 mutation or fms-related tyrosine kinase 3 (FLT3) mutation, chronic myeloid leukemia (CML), and myeloma. It is revealed that STAT3 is constitutively activated by tyrosine kinase signal from oncoprotein or oncogenic autocrine of IL-6 pathway in these cell lines. Of note, OPB-31121 had little growth inhibitory effect on normal hematopoietic cells and hardly affected colony formation of human cord blood cells at 100 nM. We also demonstrated growth suppression or regression of cell lines including HEL92.1.7, KU812 (CML), and TCCy/sr (ALL positive for BCR-ABL with T315I mutation) in NOD/SCID mice (T/C: 1.8 to 39.5 %). For further analyses, we used human leukemia model mouse where clinical samples of human leukemia were transplanted into NOD/SCID/IL2-Rgammac−/− (NOG) mice and could be maintained by serial transplantation. In this system, heterogeneity and hierarchy of differentiation of leukemia cells, if they had, are maintained. OPB-31121 induced significant growth suppression of leukemia cells of BCR-ABL-positive acute lymphoblastic leukemia (ALL), CML-blast crisis (BC), CML-BC with T315I mutation in BCR-ABL, and AML with FLT3/ITD (T/C: 4 to 58 %, Figure B). Furthermore, treatment with cytarabine induced accumulation of quiescent cells that were thought to be relatively resistant to chemotherapy, whereas OPB-31121 did not cause such accumulation, suggesting its effectiveness on quiescent cells (Figure C). We are now investigating the effect of OPB-31121 on leukemia-initiating cells and the results will be shown at the meeting. Taken together, we conclude that OPB-31121 holds promise as a therapeutic agent against a wide range of hematopoietic malignancies. This drug is under phase 1 trial in Hong-Kong, Korea, and the USA. Disclosures: Hayakawa: Otsuka Pharmaceutical Co., Ltd.: Research Funding. Sugimoto:Otsuka Pharmaceutical Co., Ltd.: Employment. Matsuyama:Otsuka Pharmaceutical Co., Ltd.: Employment. Harada:Otsuka Pharmaceutical Co., Ltd.: Employment. Hashimoto:Otsuka Pharmaceutical Co., Ltd.: Employment. Ohi:Otsuka Pharmaceutical Co., Ltd.: Employment. Kodama:Otsuka Pharmaceutical Co., Ltd.: Employment. Sumida:Otsuka Pharmaceutical Co., Ltd.: Employment. Naoe:Chugai pharmaceutical, Zenyaku pharmaceutical, Kyowa-Kirin pharmaceutical, Dainippon-Sumitomo pharmaceutical, Novartis pharmaceutical, Janssen pharmaceutical, Otsuka pharmaceutical: Research Funding.

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23

Cilloni, Daniela, Francesca Messa, Roberto Bernardoni, Francesca Arruga, Monica Pradotto, Enrico Bracco, Cristina Panuzzo, et al. "Identification of Genes Sustaining Bcr-Abl Oncogenic Signalling and CML Progression through a Genetic Tool Based on Human Bcr-Abl Transgenic Drosophila Melanogaster." Blood 112, no. 11 (November 16, 2008): 1091. http://dx.doi.org/10.1182/blood.v112.11.1091.1091.

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Abstract Although the role of Bcr-Abl in the pathogenesis of Chronic Myeloid Leukaemia (CML) is well established, the mechanisms responsible for CML progression are largely unknown. The aims of the study were to perform a genetic screening to identify new genes and pathways leading to CML progression and imatinib resistance and to provide a powerful tool allowing a wide screening of drug libraries. We developed a genetic model based on transgenic human p210 Bcr-Abl Drosophila melanogaster (Dm). We generated two different fly lines expressing either h-p210 wt or carrying the T315I mutation, in a tissue specific manner such as fly eyes or lymph gland, which represents the Dm hematopoietic system. Bcr-Abl expression results into a glazed phenotype of the eyes correlated with the amount of p210 protein. A wide modifier screening was performed using commercially available stocks of Dm carrying small and well characterized chromosome deletions. The resulting progeny was first screened using eye phenotype as read-out system. A first group of flies displayed a more aggressive phenotype since they lack one or more genes encoding for Bcr-Abl negative regulators while a second group displayed a mild phenotype most likely due to the absence of a gene encoding for a gene involved in the oncogenic signalling. Each deletion, responsible for any phenotype change in the progeny, was further analyzed by crossing Bcr-Abl flies with flies carrying the single deletion of each gene included in the identified region. Among the genes identified, PI3K loss of function results into a phenotype improvement thus supporting the tool effectiveness. As control, the cross between Bcr-Abl flies and the constitutively active form of PI3K results not only into a worse phenotype but also into an increased size of the eye, corresponding to an abnormal proliferation process. With this tool we have at now identified a list of genes responsible for a phenotype change including Fax, Dab and Pros which have been more extensively studied in human primary cells collected from patients at diagnosis enrolled in TOPS studies (Cortes, EHA, 2008) and during disease progression, so confirming their involvement in human disease. We found that these genes are downregulated during accelerated phases and blast crisis. Transfection of CML cells with Fax, Dab and Pros reduced proliferation and/or induced apoptosis. By contrast, loss of function of ENA, the CRKL hortologous in Dm did not induce any significant change of the phenotype. In addition, a drug screening was performed by feeding flies with drugs. We set up a rapid method for drug testing based on wt and T315I/Bcr-Abl phenotypes rescue induced by several TKs inhibitors or by combinations. In conclusion, Dm is a rapid genetic tool which allow the selection of a number of genes involved in CML progression and IM resistance. In addition, it allows to identify drugs or combination of drugs active on Bcr-Abl or T315I mutant form. This investigation was conducted by CML Correlative Studies Network (CCSN), TOPS, which is sponsored by Novartis Oncology

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Zhang, Xiaoshuai, Zongru Li, Yazhen Qin, Robert Peter Gale, Xiaojun Huang, and Qian Jiang. "Correlations between Mutations in Cancer-Related Genes, Therapy Responses and Outcomes of the 3 rd Generation Tyrosine Kinase-Inhibitor (TKI) in Persons with Chronic Myeloid Leukemia Failing Prior TKI-Therapy." Blood 138, Supplement 1 (November 5, 2021): 308. http://dx.doi.org/10.1182/blood-2021-150020.

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Abstract Background Most, but not everyone with chronic myeloid leukaemia (CML) responds to imatinib or 2 nd-generation tyrosine kinase-inhibitors (TKIs). Mutations in cancer-related genes and in other than ABL1 may explain variable responses and outcomes to the 3 rd-generation TKIs including ponatinib and olverembatinib. Aim Interrogate correlations between mutations in cancer-related genes and therapy responses and outcomes to 3 rd-generation TKIs. Methods We used deep targeted sequencing for cancer-related mutations and Sanger sequencing for BCR::ABL1 on DNA samples from 167 subjects with CML failing to the prior imatinib and/or 2 nd-generation TKI-therapy and just before receiving a 3 rd-generation TKI. Gene ontology (GO) analysis was used to evaluate functional enrichment in GO terms among mutated genes. Optimal cut-offs for variant allele frequencies (VAFs) of the common mutations were determined by analyzing receiver-operator characteristic (ROC) curves. A Cox multi-variable regression model was used to identify correlations between mutations in cancer-related genes and therapy responses and outcomes of 3 rd-generation TKI-therapy. Results 167 subjects in chronic phase (n = 125) and accelerated phase (n = 42) received ponatinib (n = 28) or olverembatinib (n = 139) therapy. 27 subjects were exposed to imatinib; 79, a 2 nd-generation TKI; 61, imatinib and a 2 nd generation TKI. 142 (85%) subjects had ABL1 mutations including ABL1T315I (n = 116) or others (n = 26). 163 subjects had other cancer-related mutations which were evaluated in epigenetic regulators (n = 150), transcription factors (n = 84), cell signaling (n = 42), tumor suppressors (n = 39), protein kinases (n = 27), chromatin modification (n = 9) and DNA damage repair (n = 3) related-genes according to functional enrichment. The top 10 mutations were ASXL1 (n = 115), RUNX1 (n = 12), KMT2D (n = 12), PHF6 (n = 8), KMT2C (n = 8), IKZF1 (n = 8), STAT5A (n = 8), DNMT3A (n = 7), TET2 (n = 6) and BCOR (n = 6). 20 subjects had high-risk additional chromosomal abnormalities (ACAs). Frequency of BCR::ABL1 mutations was inversely- (p < 0.001) and of cancer-related mutations directly-related (p = 0.009) to increasing exposure to prior TKI therapies. These relationships were especially so for mutations in KMT2C (p = 0.06), DNMT3A (p = 0.09), KDM6A (p = 0.06) and TNFAIP3 (p = 0.08). BCR::ABL1 (82% vs. 95%, p = 0.03), RUNX1 (5% vs. 14%, p = 0.04), KMT2C (3% vs. 10%, p = 0.08) and IKZF1 (3% vs. 10%, p = 0.10) were more common in accelerated phase. With a median follow-up of 34 months (interquartile range [IQR], 12-40 months), 95 and 71 subjects achieved a complete cytogenetic response (CCyR) and major molecular response (MMR). 18 subjects transformed to accelerated (n = 8) or blast (n = 10) phases, 16 died of disease progression (n = 12) or other causes (n = 4). 3-year cumulative incidences of CCyR and MMRwere 65% (95% Confidence Interval [CI], 58, 71%) and 52% (43, 61%). 3-year probabilities of progression-free survival (PFS) and survival were 88% (81, 92%) and 91% (85, 95%). Mutations in tumor suppressor genes were more common in subjects not achieving a CCyR (27% vs. 19%, p = 0.01). In multi-variable analyses ASXL1 mutation with a VAF ≥ 17% and a PHF6 mutation were significantly associated with lower cumulative incidences of CCyR (p < 0.001 and p = 0.032) and MMR (p < 0.001 and p = 0.04). Moreover, subjects with BCR-ABL1T315I mutation had significantly higher cumulative incidences of CCyR (p = 0.07) and MMR (p = 0.04) than those with no BCR-ABL1 mutation and other BCR-ABL1 non-T315I mutation. Increasing age, more Ph 1-chromosome-positive cells, the best prior therapy-response < partial cytogenetic response (PCyR) and more TKI-therapies were associated with poor responses. STAT5A mutation was significantly associated with worse PFS (p = 0.002) and survival (p < 0.001), RUNX1 mutation (p = 0.006), high-risk ACAs (p = 0.07) and accelerated phase (p = 0.002) with worse PFS and increasing age (p = 0.05) and comorbidity(ies) (p = 0.05) with wosre survival. Conclusions ASXL1 mutations with a VAF ≥ 17% and PHF6 mutations were associated with poor responses of the 3 rd-generation TKI-therapy. STAT5A and RUNX1 mutations and high-risk ACAs were also associated with worse outcomes in persons receiving a 3 rd-generation TKI. These data should help physicians select people to receive 3 rd-generation TKIs. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Jiang, Xiaoyan, Hannah Grant, Justin Stebbing, Letizia Foroni, Charles Craddock, Mike Griffiths, Richard E. Clark, et al. "Analysis of BCR-ABL1 Tyrosine Kinase Domain Mutations In Primitive Chronic Myeloid Leukemia Cells Identifies a Unique Mutator Phenotype." Blood 116, no. 21 (November 19, 2010): 3397. http://dx.doi.org/10.1182/blood.v116.21.3397.3397.

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Abstract Abstract 3397 The tyrosine kinase inhibitor (TKI), imatinib mesylate (IM), induces remissions in most chronic phase chronic myeloid leukaemia (CML-CP) patients, but emergence of drug resistance attributable to critical mutations in the BCR-ABL1 TK domain remains a significant clinical problem. We previously demonstrated that primitive (CD34+) CML-CP cells are both intrinsically insensitive to IM and genetically unstable. BCR-ABL1 TK domain mutations are, furthermore, readily detectable in CD34+ CML-CP cells even prior to IM treatment. However, the mechanisms by which they arise are unknown. In this study, we characterized the spectrum of mutations at each of the 3 codon positions in the BCR-ABL1 TK domain of IM-naïve CD34+ cells and used a mathematical model to compare these with those found in the same region of DNA in IM-resistant cells. A total of 460 TK mutations were identified in cells from 15 IM-naïve and from 316 IM-resistant patients. The mutations were non-randomly distributed across the 3 codon positions, with very few mutations at position 3 (1 of 136 mutations, 0.7%, in IM-naïve patients and 31 of 329, 9%, in IM-resistant patients). The remainder showed no bias in their distribution across codon positions 1 and 2 in IM-naïve patient cells but, in IM-resistant cells, a significant positive bias was observed at codon position 2 (p=0.042). Conversely, BCR-ABL1 TK domain sequences from IM-naïve patient cells had significantly more transitions relative to unselected regions of the genome across all codon positions, a significant positive A-to-G mutational bias (A>G, 1.12×10−10) and an under-representation of C-to-T mutations (C>T, 5.34×10−5) when these mutations were compared with unselected regions of the genome. In addition, we observed a T-to-C mutational hotspot (T>C, 1.23×10−4) at codon position 2. IM-naïve cells exhibited a transitional bias at position 2 and overall (1.99×10−4 and 6.19×10−5, respectively; at position 1, P=0.052). We observed a similar pattern of TK codon position mutations at position 1 and overall in sequences derived from IM-resistant cells. Interestingly, in IM-resistant cells, A-to-T (A>T) transversions were over-represented at position 2 and overall (1.41×10−11 and 3.77×10−5). These cells also had a profound transversional bias (1.45×10−4) at codon position 3, suggesting that positive selection occurs at a position where transversions are typically non-synonymous. However, a skewed transition:transversion ratio and transition and transversion frequency at TK codon positions 1 and 2 was observed in both IM-naïve and IM-selected cells with mutations distributed across these 2 codon positions in a significantly uneven fashion. In IM-resistant cells, the frequency of C-to-T mutations (C>T, 3.82×10−11) indicated a bias affecting codon position 2 more often than position 1, and A-to-T transversions occurred significantly more frequently at position 2 compared to position 1 (A>T, 7.45×10−9). This was also the case for T-to-C transitions in TK sequences of IM-naïve cells (T>C, 1.13×10−4). Frequencies of these TK mutations in IM-naïve and IM-resistant cells were also higher than in unselected regions of the genome, providing further evidence that the TK domain mutations in CML-CP cells have a distinct mutational profile. For example, the clinically observed M244V and D276G mutations result from A-to-G transitions and the F359L mutation arises from a T-to-C transition, both of which would be predicted by the activity of a CML-CP mutator. However, the most clinically important mutation, T315I, that confers resistance to most currently available TKIs, is generated by a C-to-T transition, suggesting a high mutational rate generating mutational escape around the principal mutator pattern and profound selection thereafter. Notably, the CML-CP mutational signature is distinct from that of the activation-induced cytidine deaminase (AID)-induced hypermutation reported in CML blast crisis, both in targeting specific codon positions and in the overall mutational pattern. This strongly favors the hypothesis that the genomic instability of primitive CML-CP cells is generated by a different, specific mutational process. The results also suggest that monitoring BCR-ABL mutational hotspots may be clinically useful in anticipating TKI resistance. Disclosures: No relevant conflicts of interest to declare.

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Rossi, Davide, Clara Deambrogi, Tiziana Lischetti, Michaela Cerri, Marianna Spunton, Silvia Rasi, Silvia Franceschetti, et al. "Hypermethylation of SH2-Containing Phosphatase-1 (SHP-1) and Suppressor of Cytokine Signaling-1 and -3 (SOCS-1 and -3) in Philadelphia Negative Chronic Myeloproliferative Disorders (Ph-CMPD)." Blood 108, no. 11 (November 16, 2006): 118. http://dx.doi.org/10.1182/blood.v108.11.118.118.

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Abstract A fraction of Ph- CMPD is characterized by JAK2F617F mutation leading to constitutive JAK-STAT activation. The negative regulators of cytokine signaling SHP-1, SOCS-1 and SOCS-3 have a crucial function in the negative regulation of JAK2 activation/phosphorylation in response to EPO, G-CSF and a subset of cytokines. SHP-1, SOCS-1 and SOCS-3 may be silenced by aberrant DNA methylation in human malignancies. Here we tested chronic phase Ph- CMPD and acute myeloid leukaemia (AML) from Ph- CMPD for aberrant DNA methylation of SHP-1, SOCS-1 and SOCS-3. The study was based on: i) 85 Ph- CMPD, including 35 essential thrombocythemia (ET), 20 polycythemia vera (PV), 15 idiopathic myelofibrosis (IMF), 6 chronic myelomonocytic leukemia (CMML) and 9 Ph- chronic myeloid leukemia (Ph- CML); and on ii) 19 AML from Ph- CMPD, including 4 AML from PV, 10 AML from ET and 5 AML from IMF. Cases were analysed for SHP-1, SOCS-1 and SOCS-3 aberrant methylation by methylation-specific PCR and for JAK2V617F mutation by allele specific PCR. For comparison, 10 samples of normal bone marrow hematopoietic cells were also investigated. Among Ph- CMPD, methylation of SHP-1 occurred in 4/20 (20%) PV and 4/35 (11%) ET, while it was absent in IMF (0/15), CMML (0/6) and Ph- CML (0/9). Methylation of SOCS-1 occurred in 5/20 (25%) PV, 5/35 (14%) ET, 2/15 (13%) IMF and in 1/9 (11%) Ph- CML while it was absent in CMML (0/6). Methylation of SOCS-3 occurred in 11/20 (55%) PV, 13/35 (37%) ET, 4/15 (26%) IMF, 3/6 (50%) CMML and 3/9 (33%) Ph- CML. JAK2V617F mutation was detected in 47/85 (55%) Ph-CMPD, including 17/20 (85%) PV, 18/35 (51%) ET, 12/15 (80%) IMF, 0/6 CMML and 0/9 Ph- CML. SHP-1, SOCS-1 and SOCS-3 methylation was analysed according to JAK2 mutation status in PV, ET and IMF. In this group of patients, SHP-1, SOCS-1 and SOCS-3 methylation occurred in both JAK2 mutated cases (6/47, 13% for SHP-1; 10/47, 21% for SOCS-1 and 18/47, 38% for SOCS-3) and germline cases (2/38, 5% for SHP-1; 2/38, 5% for SOCS-1 and 10/38, 26% for SOCS-3). By combining the results of SHP-1, SOCS-1 and SOCS-3 methylation status, 21/47 (45%) JAK2 mutated cases carried SHP-1 and/or SOCS-1 and/or SOCS-3 methylation as opposed to 12/38 (31%) germline cases. This pattern of SHP-1 and SOCS-1 methylation was conserved also when the analysis was restricted to PV, ET and IMF each as a single group and after stratification for JAK2V617F mutation. Among AML from Ph- CMPD, methylation of SHP-1 occurred in 1/10 (10%) AML from ET, while it was absent in AML from PV and AML from IMF. Methylation of SOCS-1 occurred in 1/4 (25%) AML from PV and 1/10 (10%) AML from ET. One ET patient acquired SHP-1 methylation at transformation to AML. All normal bone marrow samples (n=10) scored negative for SHP-1, SOCS-1 and SOCS-3 methylation. The implications of these results are threefold. First, inactivation by aberrant methylation of SHP-1, SOCS-1 and SOCS-3 is involved in the pathogenesis of Ph- CMPD and is selectively associated with neoplastic hemopiesis. Second, among PV, ET and IMF, methylation of SHP-1, SOCS-1 and SOCS-3 may occur in both JAK2V617F positive and negative cases. Third, the low prevalence of SHP-1 and SOCS-1 methylation in AML from Ph- CMPD suggests that SHP-1 and SOCS-1 silencing is not involved in leukemic transformation.

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Cilloni, Daniela, Francesca Messa, Francesca Arruga, Enrico Bracco, Paolo Nicoli, Emanuela Messa, Stefano Ulisciani, et al. "Development of a Genetic Tool Based on BCR-ABL Transgenic Drosophila Melanogaster for Identification of Genes Involved in CML Progression and Drug Testing." Blood 110, no. 11 (November 16, 2007): 468. http://dx.doi.org/10.1182/blood.v110.11.468.468.

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Abstract Although the role of Bcr-Abl in the pathogenesis of chronic myeloid leukaemia is well established, the molecular mechanisms by which it triggers cellular transformation remains still partially unknown. In addition, the mechanisms responsible for CML progression and the molecules interacting with Bcr-Abl are largely unknown. and many of. In this study, we have developed a novel approach for genetic analysis investigations to identify a number of candidate genes and pathways involved in disease progression and imatinib resistance by using a model of the Drosophila melanogaster. We generated two different stable transgenic fly lines expressing both human p210Bcr-Abl forms (either w.t. or the mutated form T315I) in a tissue specific manner. We have observed that the activation of BCR-ABL led to a particular phenotype in the fly eyes. Transgenic flies will be phenotipically and genotipically characterized carefully by analyzing the eye development. We conducted an extensive genetic screening using both Drosphila transgenic lines overexpressing human Bcr-Abl forms (Bcr-Abl/w.t. and Bcr-Abl/T315I) by making use of P-elements. This technique offers the possibility to randomly insert P-elements into the genome, thus disrupting genomic loci either in correspondence of coding sequences or regulatory elements and altering the gene function. The heterogeneous mutagenized fly population was carefully screened on the basis of the flies phenotype. The first group which we have selected was represented by flies displaying a phenotype even more aggressive than the parental transgenic fly. This kind of flies harbour mutated genes encoding for proteins which enhance the activity of Bcr-Abl, thus being most likely involved in disease progression; A second group was represented by flies displaying a wild-type phenotype. In the latter case the phenotype reversion most likely due to a mutation occurred at a level of a gene encoding for a protein functioning as Bcr-Abl negative regulator. All the data obtained with the use of fly model were confirmed in both cell lines and in primary cells via the overexpression and/or silencing of the genes identified with the Drosophila genetic-screening. Finally we have established and validated a novel tool for drug testing based on the examination of the eye phenotype induced by Bcr-Abl, which can be reverted by the drugs producing a complete block of the kinase activity. With this method we will be able to screen drug libraries to identify molecules able to silence Bcr-AblT315I tyrosine kinase activity. That can be easily accomplished by feeding flies with food previously mixed with the different drug molecules. Molecules showing a good inhibitory activity can be quickly identified because their /ability to revert the abnormal eye phenotype displayed by the transgenic flies. In conclusion, by using a model of the Drosophila melanogaster we have developed a system to identify novel genetic pathways which regulate the development and the progression of CML and we have shown that this novel system can be also used to evaluate the drugs affecting Bcr-Abl regulatory pathway. Moreover, this system does not require a priori knowledge of the function of the genes involved in the disease.

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Copland, Mhairi, Ashley Hamilton, Janet W. Baird, Martin Barow, Elaine K. Allan, Lucy J. Elrick, and Tessa L. Holyoake. "Dasatinib (BMS-354825) Has Increased Activity Against Bcr-Abl Compared to Imatinib in Primary CML Cells In Vitro, but Does Not Eradicate Quiescent CML Stem Cells." Blood 106, no. 11 (November 16, 2005): 695. http://dx.doi.org/10.1182/blood.v106.11.695.695.

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Abstract Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder of the haemopoietic stem cell. It results from acquisition of the Philadelphia chromosome and expression of the oncogenic fusion protein Bcr-Abl. Imatinib mesylate (IM), a rationally designed tyrosine kinase inhibitor of Bcr-Abl, competitively inhibits ATP binding for which conformation of Bcr-Abl is critical. IM induces a complete cytogenetic response in the majority of CML patients in chronic phase, but nearly all patients have detectable disease at the molecular level by quantitative RT-PCR and, therefore, are unlikely to be cured. Dasatinib is a novel, oral, multi-targeted kinase inhibitor that targets Bcr-Abl and Src kinases, and is currently in Phase 2 clinical trials in CML. In vitro, dasatinib has improved (325 fold greater) potency against wild-type Bcr-Abl expressing cells and is capable of binding Bcr-Abl conformations resistant to IM1,2 and it is proposed that dasatinib will eradicate CML in the majority of patients regardless of mutation status (except T315I). To test this, CD34+ primary CML cells were cultured for 6 days in serum free medium supplemented with 5 growth factors. CFSE was used to track cell division. Conditions included: (1) no drug control, (2) continuous IM (5μM; ~IC90 dose), (3) continuous dasatinib (150nM; ~IC90 dose) (4) continuous IM/dasatinib, (5) IM (72hr) then dasatinib (72hr), (6) dasatinib (72hr) then IM (72hr). Crkl phosphorylation status was evaluated by intracellular flow cytometry in CD34+ and CD34+CD38− primary CML cells at 16 and 72 hours as a marker of kinase activity. Both CD34+ and CD34+CD38− cells showed only single copy Bcr-Abl by FISH, but expressed significantly higher Bcr-Abl transcript levels by RT-PCR compared to total mononuclear cells (P=0.031). There was a significant reduction in total viable cells in all treatment arms versus the no drug control (P=0.003). There was accumulation of undivided CFSEmax CD34+ CML cells in all treatments arms relative to the no drug control (P=0.009). There were no significant differences in undivided CFSEmax CD34+ CML cells remaining after 6 days between individual arms, but, collectively, the arms containing IM had significantly greater accumulation of these cells compared to the non-IM containing arms (P=0.045). Total CD34+ cells showed dephosphorylation of Crkl at 16 hours after treatment with IM or dasatinib. However, after 72 hours, the remaining viable CD34+ Bcr-Abl+ cells showed no dephosphorylation of Crkl in response to IM, consistent with enrichment of a resistant population, whereas the dasatinib-treated cells remained dephosphorylated (P=0.01). In the CD34+38− sub-population, there was no Crkl dephosphorylation at 16 or 72 hours with IM, however, dasatinib induced 43% and 50% dephosphorylation at 16 and 72 hours respectively (P=0.009 and P=0.001). Kinase domain mutations were not detected in either the IM or dasatinib-resistant primitive CML cells. These results demonstrate dasatinib is more effective than IM within the stem cell compartment, however, the most primitive quiescent CML cells remain insensitive to both drugs, questioning the relevance of Bcr-Abl as a therapeutic target in these cells.

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29

Ågerstam, Helena, Nils Hansen, Sofia Von Palffy, Carl Sandén, Kristian Reckzeh, Christine Karlsson, Henrik Lilljebjörn, et al. "IL1RAP Antibodies Block IL1-Induced Expansion of Primitive CML Cells and Display Therapeutic Effects in Xenograft Models." Blood 128, no. 22 (December 2, 2016): 1118. http://dx.doi.org/10.1182/blood.v128.22.1118.1118.

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Abstract Chronic myeloid leukemia (CML) is currently treated with tyrosine kinase inhibitors (TKIs) but these do not effectively eliminate the CML stem cells. As a consequence, CML stem cells persist and cause relapse in most patients upon drug discontinuation. Furthermore, no effective therapy exists for the advanced stages of the disease. Thus, there is still a need for novel treatment strategies in CML. We have previously shown that Interleukin 1 receptor accessory protein (IL1RAP), a co-receptor of IL1R1, is highly expressed on primitive CML cells and that a polyclonal IL1RAP antibody can direct natural killer (NK) cells to specifically target and destroy CD34+CD38- CML cells in an in vitro-based antibody dependent cell-mediated cytotoxicity (ADCC) assay (Järås et al, PNAS, 2010). The aim of the present study was to investigate the consequences of IL1RAP expression on primitive CML cells and the in vivo therapeutic efficacy of monoclonal IL1RAP antibodies against CML cells. Primary chronic phase (CP) CD34+ CML cells were cultured in medium supplemented with cytokines known to signal through receptor complexes involving IL1RAP. The addition of IL1 to the cultures resulted in a marked cellular expansion specifically for the primitive CD34+CD38- CML cells. Moreover, the CD34+CD38- cells showed phosphorylation of the downstream mediator of IL1-signaling NFKB. RNA-sequencing confirmed the activation of NFKB and of genes involved in cell cycling, indicating that IL1 stimulation of CD34+CD38- CML cells induced proliferation. Upon addition of an IL1RAP antibody capable of blocking IL1-signaling to the suspension cultures, the IL1-induced expansion and NFKB phosphorylation of CD34+CD38- CML cells was suppressed. Interestingly, both the IL1RAP expression and the response to IL1 as measured by NFKB phosphorylation was retained during TKI treatment of the cells. To assess the in vivo effects of IL1RAP antibodies in CML models, we first engrafted NOD/SCID mice with BCR/ABL1 expressing BV173 cells and treated the mice with the monoclonal IL1RAP antibody mAb81.2. Mice receiving treatment with mAb81.2 displayed a prolonged survival compared to controls, accompanied by reduced levels of leukemic cells in the BM. In vitro studies showed that mAb81.2 lacked a direct effect on cellular expansion or apoptosis. Instead, the IL1RAP antibody could direct NK cells to elicit killing of the leukemic cells, thereby suggesting effector cell mediated mechanisms to be an important in vivo mode-of-action. To validate the in vivo effects on primary CML cells, we next engrafted CP or blast phase (BP) CML cells into immunodeficient mice. Following engraftment of CP CD34+ CML cells into NSG mice and subsequent treatment with mAb81.2, a reduction of human myeloid cells was observed, suggesting that the treatment targeted the leukemic graft. Importantly, mAb81.2 treatment also reduced the levels of candidate CD34+CD38-IL1RAP+ CML stem cells. Finally, BP CML cells were engrafted into NOD/SCID mice that have a more intact effector cell function compared to NSG mice. Following treatment with mAb81.2 a significant reduction of leukemic cells in the BM as well as in the periphery was observed compared to control mice. Importantly, secondary transplantations revealed a therapeutic effect also on the BP CML stem cells. In vitro ADCC assays confirmed that CML BP cells, including a sample with the highly TKI-resistant T315I mutation, could be targeted and killed using mAb81.2. We conclude that IL1RAP antibodies can suppress IL1-induced expansion of primitive CML cells and that in vivo administration of IL1RAP antibodies in CML xenograft models has anti-leukemic effects that extend to the CML stem cells. These results show that an antibody-based therapy against IL1RAP can be used to efficiently target CML stem cells. Disclosures Richter: BMS: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Järås:Cantargia AB: Equity Ownership. Fioretos:Cantargia AB: Equity Ownership.

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Tschiedel, Sabine, Melanie Adler, Tobias Dietrich, Michael Cross, Vladan Vucinic, and Dietger Niederwieser. "Peptides from Nme2 Are Preferentially Expressed By Chronic Phase CML Cells, Recognized By T Cells and Restricted By the Most Frequent HLA-Antigens." Blood 124, no. 21 (December 6, 2014): 4511. http://dx.doi.org/10.1182/blood.v124.21.4511.4511.

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Abstract Nme2 (non metastasis protein 2) is a nucleoside diphosphate kinase that plays multiple roles in signalling and metabolism. We previously identified Nme2 as a tumor antigen in chronic myeloid leukaemia (CML) and found Nme2 protein over-expression to be a universal and specific feature of Bcr/Abl+ cells in chronic-phase of the disease. Nme2 up-regulation in Bcr/Abl+ cells is post-transcriptional and is reversed by TKI treatment. Furthermore, the IL-3 independence induced by Bcr/Abl transfection of Ba/F3 cells is also accompanied by Nme2 over-expression and is reversed by specific miRNA-mediated down-regulation of Nme2, suggesting that Nme2 contributes to Bcr/Abl driven leukemogenesis. Here, we have investigated the potential of Nme2 as a target for CML specific immunotherapy by assessing it's antigenicity in common HLA backgrounds. Peripheral blood mononuclear cells (PBMCs) were obtained from 40 patients with CML and 24 healthy donors who carried the common HLA-A02 (n=21/7); A03 (n=12/8) and A24 (n=8/9) antigens. Together, these represent the most common HLA types in the Caucasian population. Samples were obtained before (n = 13) and after (n = 36) hematopoietic stem cell transplantation (HCT) and Nme2 specific cytotoxic T lymphocytes (CTL) were generated using artificial antigen presenting cells (aAPC) engineered to express both Nme2 and the appropriate HLA-A antigen. PBMC were primed with aAPC and then re-stimulated weekly. Following CD8+ T-cell selection on day 14, IFNγ production in response to Nme2 expressing stimulator cells was evaluated in Elispot assays. In addition Nme2 reactivity was investigated in peripheral blood cells of CML patients by Elispot assays at various time points up to 10 years after HCT. The frequency of Nme2 specific T cells was significantly higher in CML patients pre HCT (39.3 specific T cells/105 CD8+ cells, p=0.004) than in healthy donors (2.17 specific T cells/105 CD8+ cells). A further rise in specific T cell response was detected in CML patients post HCT (142 specific T cells/105 CD8+ cells, p=0.0006), with a significant increase being evident by day +50 after HCT. The subsequent activity varies, with a weakly pronounced second peak occurring around 3 years post-transplant. However, Nme2 specific T cells persisted over the entire observation period with a significant Nme2 specific T cell response still being detectable nearly 10 years post transplant. The highest Nme2 specific T cell activity was found in HLA-A03 positive patients (187 T cells/105 CD8+ cells) and the lowest in the HLA-A02 background (102 T cells/105 CD8+ cells, p=0.00001). Restriction by HLA-A24 was associated with an intermediate activity (141 Nme2 specific T cells/105 CD8+ cells). We identified 8 patients in which an increase of Bcr-Abl transcript level from 0% Bcr-Abl/Abl to a detectable amount (0.28%, range 0.001 to 5.017% Bcr-Abl/Abl) was observed. In all cases, a transient increase in Nme2 reactive T cells (2.7 fold, p=0.007) was associated with re-entry into molecular remission. Furthermore, Nme2 specific T cells were lower before relapse (88 T cells/105 CD8+ cells) than in patients with long lasting molecular remission (186 T cells/105 CD8+ cells, p=0.026).Finally, we successfully generated Nme2 specific CTLs from healthy donors (4/5) as well as from patients with CML who had received HCT (10/10). CTLs showed a mean expansion rate of 23 (healthy donors) to 30 fold (CML patients) and HLA class I restricted Nme2 specific recognition. Partially HLA matched, primary CML cells were targeted by these Nme2 specific CTLs (6/6), regardless of the presence of the T315I Bcr/Abl mutation that bestows resistance to tyrosine kinase inhibitors (TKI). In summary, we show here that Nme2 is a strong inducer of immune response in transplanted CML patients carrying common HLA class I alleles, implying that the immune response to Nme2 may be involved in the GvL effect post HCT. Our results suggest that Nme2 specific CTLs may be of therapeutic relevance in eradicating the residual leukemic cells after therapy with TKI or HCT. Disclosures No relevant conflicts of interest to declare.

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De Lavallade, Hugues, Sophie Jackson, Heather Oakervee, Sebastian Francis, Edward Belsham, Paul Cahalin, Joanne Ewing, et al. "Interim Analysis of a Prospective Multicentre Study Using Next Generation Sequencing for Kinase Domain Mutational Analysis in CML Patients on First or Subsequent TKI Therapy." Blood 134, Supplement_1 (November 13, 2019): 2935. http://dx.doi.org/10.1182/blood-2019-129825.

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Introduction Kinase domain mutations in the BCR-ABL1 gene are associated with resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukaemia (CML). Next-generation Sequencing (NGS) allows detection of low-level kinase domain mutations as well as quantification of Variant Allele Frequency (VAF).We have previously demonstrated that NGS consistently detects early appearance of kinase domain (KD) mutations in CML patients (Kizilors et al. Lancet Haematology 2019)and highlighted the need for regular monitoring for KD mutations.To that end a multicentre prospective non-interventional study of centralised NGS screening to detect KD mutations was launched in the UK and Republic of Ireland to evaluate the use of NGS in clinical practice (ClinicalTrials.gov, number NCT03647215, INCB 84344-401). Methods Patients with CML on first or subsequent TKI therapy who meet the ELN 2013 criteria for warning or failure are eligible for this prospective study. NGS assay was performed on illumnia MiSeq with a single round PCR using our ISO15189 accredited assay as previously described. Results were communicated to the treating haematologist within 10-12 working days. As this is a non-interventional study, clinical intervention was left to the discretion of treating physician. Repeat mutational analysis was/is encouraged until achievement of a sustained MR3/BCR-ABL<0.1%IS. Results Between December 2017 and June 2019, 192 CML patients from 31 institutions were included in this prospective study (median age 56 yrs, 19-91). 184 patients had chronic phase CML, 5 de novo accelerated phase and 2 de novo blastic phase (missing data n=1). 84 (43.8%) patients were on first line TKI (imatinib n=71, nilotinib n=9, dasatinib n=4), while 58 (30.2%) patients were treated with second line TKI (imatinib followed by 2GTKI n= 52, 2GTKI followed by another 2GTKI n=5), 27 (14.1%) patients were treated with 3rdline TKI (including 4/27 patients on ponatinib) and 23 (12.0%) patients were on 4thline (11/23 patients on ponatinib). At the time of study entry, 78 (40.6%) were in MR2 (BCR-ABL 0.1-1%IS), 50 (26.0%) were in MR1 only (BCR-ABL 1-10%IS), and 54 (28.1%) had a BCR-ABL>10%IS. Of note 10 (5.2%) had a BCR-ABL<0.1%IS. Only 93 (48.9%) patients had previous KD mutational analysis performed by Sanger Sequencing (SS) before study entry. 23 of 192 patients (12.0%) were found to have a KD mutation by NGS while in CP after one (n=12), two (n=3) three (n=2) or four (n=6) lines of TKI therapy. The ongoing TKI therapy was imatinib (n=10), dasatinib (n=4), nilotinib (n=2), bosutinib (n=3) and ponatinib (n=5). Incidence of KD mutation was 8/78 (10.2%) in the MR2 group, 8/50 (16%) in the MR1 and 7/54 (12.9%) in patient with BCR-ABL>10%IS. A single mutation was found in 19 patients while two patients had two mutations and two patients had 3 or more mutations. The most frequent mutations found were T315I (n=11), F317L (n=3), G250E (n=3), V299L (n=2) and E459K (n=2). A low-level mutation was found in 8/23 (35%) patients and would otherwise not be detectable by SS. A mutation conferring resistance to the ongoing TKI ('clinically relevant mutations') was found in 16/23 patients (69%). TKI switch was made in at least 7 patients with response obtained in 5/7 patients at last follow up. Update on the remaining 16 patients is currently being collected and interim updated results will be presented. Serial samples from patients tested negative on the first KD mutational analysis were obtained for 27 patients and remained negative on repeat analysis, of whom 7 patients had reduction in BCR-ABL transcript levels in the interim (2 increase and 21 without change). Four patients found with KD mutation(s) also underwent repeat testing for monitoring of VAF showing a reduction in clone size/VAF and BCR-ABL transcript levels in two patients. Conclusions This interim analysis demonstrates the clinical importance of KD mutational analysis using NGS. The high proportion of clinically relevant mutations -ie conferring resistance to the ongoing TKI treatment-highlights the potential clinical impact of early detection of KD mutation by NGS providing guidance for a rationale switch of TKI therapy. KD mutation distribution was similar in patients in MR2 compared to those with higher disease burden suggesting the importance of using NGS while disease burden is low in order to increase the success of early TKI switch. Interim updated results will be presented. Disclosures De Lavallade: BMS: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Incyte biosciences: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau. Jackson:Incyte biosciences: Honoraria, Research Funding, Speakers Bureau. Oakervee:Pfizer: Honoraria; Novartis: Honoraria; Bristol Myers-Squibb: Honoraria. Ewing:Bristol Myers-Squibb: Other: Meeting attendance sponsorship ; Novartis: Honoraria, Other: Meeting attendance sponsorship . Byrne:Ariad/Incyte: Honoraria, Speakers Bureau. Rothwell:Novartis: Honoraria, Other: advisory board; Pfizer: Speakers Bureau; Incyte: Speakers Bureau. Pillai:Celgene: Honoraria. Mehta:Pfizer: Other: Advisory board. Copland:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Cyclacel: Research Funding; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astellas: Honoraria, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Ottmann:Roche: Honoraria; Pfizer: Honoraria; Fusion Pharma: Honoraria; Celgene: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Novartis: Honoraria; Takeda: Honoraria. Radia:Blueprint Medicines: Consultancy; Novartis: Consultancy, Speakers Bureau. Harrington:Bristol-Myers Squibb: Research Funding. Greig:Incyte: Employment. Thompson:Incyte: Employment. Kizilors:Incyte biosciences: Research Funding.

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Traer, Elie, Nathalie Javidi-Sharifi, Anupriya Agarwal, Jennifer B. Dunlap, Isabel English, Jacqueline Martinez, Jeffrey W. Tyner, Melissa Wong, and Brian J. Druker. "FGF2 Mediates Resistance In CML Patients In The Absence Of Kinase Domain Mutations, and Resistance Is Overcome By Ponatinib." Blood 122, no. 21 (November 15, 2013): 3983. http://dx.doi.org/10.1182/blood.v122.21.3983.3983.

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Abstract Background Development of resistance to kinase inhibitors remains a challenge in chronic myeloid leukemia (CML). Kinase domain mutations are a common mechanism of resistance, yet the mechanism of resistance in the absence of mutations remains less clear. Recent evidence suggests that the bone marrow microenvironment provides a sanctuary for leukemia cells, and may be involved in mediating resistance to imatinib – particularly in the absence of BCR-ABL kinase domain mutations. We tested selected cytokines, growth factors, and extracellular matrix proteins expressed by cells in the bone marrow microenvironment for their ability to protect CML cells from imatinib. Results We found that fibroblast growth factor 2 (FGF2) was the most protective protein for the K562 CML cell line when exposed to imatinib. FGF2 was not only capable of promoting growth in short-term culture, but uniquely able to promote long-term resistance in vitro (p<0.0001 by 2-way ANOVA analysis). To analyze the mechanism of resistance, we used siRNA to target the FGF receptors 1-4 and found that only siRNA targeting FGFR3 was able to abrogate the protective effect of FGF2. Phospho-chip and Western blot analysis revealed that FGF2 binds FGFR3, which then signals the downstream kinases Ras, c-RAF, MEK1, and ERK1/2 to promote survival in the presence of imatinib. Inhibition of FGFR3 with the specific FGFR inhibitor PD173074 led to dephosphorylation of this signaling cascade, and restored sensitivity to imatinib of FGF2-mediated resistant K562 cells. Resistance could also be overcome with ponatinib, a multi-kinase inhibitor that targets both BCR-ABL and FGFR, whereas imatinib, nilotinib and dasatinib were all ineffective against FGF2-mediated resistant K562 cells. Although ponatinib was rationally designed to circumvent the BCR-ABL T315I gatekeeper mutation, it was also able to achieve major cytogenetic responses in 62% of patients without detectable kinase domain mutations in the recent PACE trial. We theorized that increased FGF2 may drive resistance in the subset of patients without kinase domain mutations who respond to ponatinib, similar to our in vitro findings. To evaluate this possibility, we identified patients without kinase domain mutations who were responsive to ponatinib and quantified bone marrow FGF2 by immunohistochemistry. In comparison to ponatinib-responsive patients with kinase domain mutations, patients without kinase domain mutations had increased FGF2 in their bone marrow (50.5% versus 36.6%, p=0.033). Moreover, FGF2 in the marrow decreased concurrently with response to ponatinib, further suggesting that FGF2-mediated resistance is interrupted by FGFR inhibition (-15.9% versus 0.8%, when compared to the change in FGF2 of patients with kinase domain mutations, p=0.012). Qualitatively, FGF2 was predominantly localized in supportive stromal cells (consistent with previous reports), supporting a paracrine mechanism of resistance. Furthermore, we also evaluated a single patient without kinase domain mutations who was resistant to ponatinib. In this patient’s marrow, there was no elevation in FGF2 or change in FGF2 with ponatinib treatment. Taken together, inhibition of FGFR appears to be critical for the clinical activity of ponatinib in patients without kinase domain mutations. Conclusions In summary, our data supports a model of resistance in which FGF2 production by the marrow stromal cells promotes resistance to multiple ABL kinase inhibitors without the need for mutation of the ABL kinase domain. Resistance occurs via FGF2 ligand-induced activation of the FGFR3/Ras/MAPK pathway, and can be overcome by concomitant inhibition of ABL and FGFR. In combination with recent clinical data with ponatinib, our data suggest that FGF2-mediated resistance is a major mechanism of resistance in CML patients without kinase domain mutations. These results illustrate the clinical importance of ligand-induced resistance to kinase inhibitors and support an approach of developing rational inhibitor combinations to circumvent resistance, particularly in other kinase-driven malignancies that routinely develop resistance to kinase inhibitors. Disclosures: Tyner: InCyte Corporation: Research Funding. Druker:Novartis, Bristol-Myers Squibb, & ARIAD: Novartis, BMS & ARIAD clin trial funding. OHSU holds contracts; no salary/lab research funds. OHSU & Druker have financial interest in MolecularMD; technology used in some studies licensed to MolecularMD. This conflict reviewed and managed by OHSU. Other.

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Claudiani, Simone, Jane Apperley, Afzal Khan, Richard Szydlo, George Nesr, George Adams, Letizia Foroni, Jamshid S. Khorashad, and Dragana Milojkovic. "Dose Reduction of First and Second Generation TKIs Is Effective in the Maintenance of Major Molecular Response and May Predict Successful Tfr in CML Patients." Blood 132, Supplement 1 (November 29, 2018): 3007. http://dx.doi.org/10.1182/blood-2018-99-119602.

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Abstract Introduction: As patients (pts) diagnosed with chronic myeloid leukaemia (CML) in CP are predicted to have a life expectancy comparable to that of the normal population, clinical concern has focused on the burden of long-term side effects and quality of life. Stopping tyrosine kinase inhibitor (TKI) is possible in a selected group of pts, resulting in a 50% chance of treatment free remission (TFR). Limited data, however, are available on the outcome of TKI dose reduction (DR) in maintaining molecular responses. Methods: We retrospectively analysed the outcome of TKI DR in pts in ≥MR3 treated at our centre from Jan 2000 until May 2015. We defined different low dose groups (LDG), according to the actual TKI dose: for imatinib (IM), 300mg and 200mg; for dasatinib (DAS), 70-80mg, 50mg, 40mg and 20mg; for nilotinib (NIL), 400-450mg, 300mg or ≤200mg; and for bosutinib (BOS), 300mg, 200mg and <200mg. Given the 'real life' setting, pts may have received either 1) multiple DRs of the same TKI or 2) different TKIs at different low doses. For scenario 1): we analysed the dose that maintained ≥MR3 and was used for the longest period of time. In case of loss of molecular response on a lower dose level, the next dose was considered a further 'case' and the patient was analysed more than once. In scenario 2) the patient was analysed once for each TKI received at low dose. MR3 and MR4 were defined conventionally. The molecular recurrence free survival (MRFS) was estimated by Kaplan-Meier. Results: We included 232 pts (IM=83 pts, cases=85; DAS=75, cases=79; NIL=72, cases=73; BOS=32, cases=33), of whom 8 pts were included in two different LDG on the same TKI (because of loss of response on the lower dose: imatinib n=2, dasatinib n=4, nilotinib n=1, bosutinib n=1). 22 and 4 pts received 2 low dose (LD) TKIs and 3 LD TKIs respectively. The total number of cases was 270. Reasons for DR included any degree of adverse event deemed significant by the clinician or pre-emptive DR at the time of introduction of a subsequent TKI due to intolerance to the previous TKI. All IM pts were treated first line, whereas the majority of pts (n=159, 88.8%) received their current 2GTKI as ≥ 2 line. Median follow-up on LD TKI was 25.3 months (1.9-175). Patient characteristics by TKI are shown in Tables 1-4. The 2-year MRFS were: IM, 88.4% (95% CI, 87.7-89.1%), and 92.7% and 77.2% for LDG1 and LDG2 respectively; DAS 92.8% (95% CI, 92.2-93.4%); and 100%, 96.2%, 92.3% and 85.6% for LDG1, LDG2, LDG3 and LDG4 respectively; NIL 93.4% (95% CI, 92.6-94.1%); and 93.3%, 88.9% and 100% for LDG1, LDG2 and LDG3, respectively; BOS 91.7% (95% CI, 90-93%); and 100% for LDG1 and LDG2 and 75% for LDG3 (Figure 1 a,b,c and d). One patient on NIL required DR for grade 3 liver toxicity, progressed to blast crisis after losing MR3 on 300mg daily and died post allo-SCT. One patient, who had achieved only CHR on IM, developed a T315I mutation on 50mg DAS second line while in MR3, having lost MR4, and was changed to ponatinib. One patient on DAS died of an unrelated brain tumour. In each TKI cohort, 59/83 pts (71%) remained on LD IM, 51/75 (68%) pts on LD DAS, 35/72 (48.6%) pts on LD NIL and 30/32 (93.7%) pts on LD BOS. 55 pts stopped LD TKI while in sustained MR4 or greater (IM n=21/83 [25.3%], DAS n=12/75 [16%], NIL n=20/72 [27.8%], BOS n=2/30 [6.6%]) with a 2-year probability of TFR of 79.4% (95% CI, 78.3-80.5%) (compared to 50% at 2 years in EURO-SKI), with a median observation time of 28 months (5-83.7) in non-relapsing pts. TFR in the different cohorts were 85.7%, 62.3%, 80% and 100% for IM, DAS, NIL and BOS respectively. Conclusion: For selected pts in ≥MR3 lowering the TKI dose can improve the tolerability of TKI therapy without impacting responses. The higher rate of TFR observed in our pts than in published stopping studies probably reflects cohorts of pts already shown to maintain deep responses on lower than standard doses of TKI, and mirrors the results of the UK NIHR Destiny study. Disclosures Apperley: Incyte: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau. Milojkovic:Incyte: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau.

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Yeung, David T., Michael Philip Osborn, Deborah L. White, Susan Branford, Michael Kornhauser, Cassandra Slader, Samar Issa, et al. "Early Switch to Nilotinib Does Not Overcome the Adverse Outcome for CML Patients Failing to Achieve Early Molecular Response On Imatinib, Despite Excellent Overall Outcomes in the TIDEL II Trial." Blood 120, no. 21 (November 16, 2012): 3771. http://dx.doi.org/10.1182/blood.v120.21.3771.3771.

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Abstract Abstract 3771 Background: We have previously reported promising results from the TIDEL-II trial, using imatinib (IM) treatment upfront in patients (pts) newly diagnosed with Philadelphia chromosome positive Chronic Myeloid Leukaemia in Chronic Phase (CML-CP), and switching selected pts to nilotinib (NIL) on the basis of failure to achieve time-dependent molecular response (MR). This strategy showed excellent rates of major molecular response (MMR; BCR-ABL ≤0.1% IS) at 12 months (mos) and transformation free survival. Aim: To optimise molecular outcome and survival in treatment naïve CML-CP pts by selective dose escalation of IM for pts with low trough levels and early switching to NIL for pts with poor MR. Methods: TIDEL-II enrolled 210 CML-CP pts across 23 Australasian centres in 2 equal and sequential cohorts. All pts started treatment with IM 600mg/d and dose escalated to IM 800mg/d if IM trough levels were <1000ng/mL. A series of time-dependent MR targets were set: BCR-ABL ≤10%, ≤1% and ≤0.1% (IS) at 3, 6 and 12 mos. Cohort 1 (C1) pts failing to meet these targets dose escalated to IM 800 mg/d. Pts who failed to improve molecular response, or were already on IM 800mg/d, switched to NIL 400mg BID. Pts in cohort 2 (C2) who failed these targets switched to NIL directly. Pts with grade III/IV or persistent grade II toxicity were also allowed to switch from IM to NIL. Results: Median follow up (f/u) for C1 and C2 pts were 42 & 24 mos respectively, and 31 mos for all pts (15–56 mos) – see table 1. The primary end-point, confirmed MMR at 12 mos, was achieved by 64%, with no difference between C1 and C2. This climbed to 75% at 24 mos. At 12 & 24 mos, the proportion of pts with confirmed MR4.5 (BCR-ABL ≤ 0.0032% IS) was 18% and 29% respectively. Six pts progressed to blast crisis (BC) : 4 in their 1st year of treatment, and 1 each in the 2nd and 3rd yrs, resulting in 2 deaths. Four other deaths were recorded, caused by stroke (1), pneumonia (1) and cardiac disease (2); 2 pts had NIL treatment before death. Eighteen mutations had been identified in 11 pts, including 4 pts with the highly resistant mutations T315I or E255K either singly or in combination with others. These were identified in the context of BC (3), loss of MMR (2), lack of MMR by 12 mos (4), and lack of CCR by 6 mos (2). One other pt lost MMR in the absence of a mutation and regained MMR with switching to NIL. Thirty-one pts in C1 switched to NIL: 19 for intolerance and 12 for failure to achieve targets after a trial of IM 800mg/d. Of the latter, with median f/u of 26 mos on NIL, 5/12 reached MMR subsequently. In C2, 44 patients switched to NIL, 12 for intolerance and 32 for failing targets: of the latter, 9 reached MMR with median f/u of 14 mos. In contrast, in the 31 (C1+C2) pts switching for IM-intolerance, all but 2 reached MMR (including 12 patients already in MMR at time of switch). Of the 25 pts with BCR-ABL ≥ 10% at 3mos, 3 pts progressed to BC (1 at 3.5mos), 6 more withdrew from study. Of the remainder, four pts achieved MMR, 9 more achieved BCR-ABL<1% but without MMR. None of these 25 pts have achieved MR4.5. (Table 2). Conclusion: Overall, the TIDEL-II strategy compares well with other upfront studies of CML-CP pts with regard to MR, as well as risk of death and progression to BC. A small proportion of pts experience further falls in BCR-ABL when switching from IM to NIL for failure to achieve deep MR. In the 12% of pts who fail to achieve BCR-ABL ≤10% at 3 mos, there is greater risk of BC and so far no deep MR are seen, despite intensification in kinase inhibition instituted at as early as 3 mos. Alternative approaches are needed both to identify these pts early and protect them from disease transformation. Disclosures: Yeung: Novartis Pharmaceuticals: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. White:Novartis Pharmaceuticals: Research Funding; BMS: Research Funding. Branford:Novartis : Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Ariad : Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cepheid : Consultancy. Slader:Novartis Pharmaceuticals: Employment. Hiwase:CSL Ltd: Research Funding. Schwarer:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ross:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria. Grigg:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Hughes:Novartis Oncology: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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G. Lindström, H. Jonathan, and Ran Friedman. "The effects of combination treatments on drug resistance in chronic myeloid leukaemia: an evaluation of the tyrosine kinase inhibitors axitinib and asciminib." BMC Cancer 20, no. 1 (May 7, 2020). http://dx.doi.org/10.1186/s12885-020-06782-9.

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Abstract Background Chronic myeloid leukaemia is in principle a treatable malignancy but drug resistance is lowering survival. Recent drug discoveries have opened up new options for drug combinations, which is a concept used in other areas for preventing drug resistance. Two of these are (I) Axitinib, which inhibits the T315I mutation of BCR-ABL1, a main source of drug resistance, and (II) Asciminib, which has been developed as an allosteric BCR-ABL1 inhibitor, targeting an entirely different binding site, and as such does not compete for binding with other drugs. These drugs offer new treatment options. Methods We measured the proliferation of KCL-22 cells exposed to imatinib–dasatinib, imatinib–asciminib and dasatinib–asciminib combinations and calculated combination index graphs for each case. Moreover, using the median–effect equation we calculated how much axitinib can reduce the growth advantage of T315I mutant clones in combination with available drugs. In addition, we calculated how much the total drug burden could be reduced by combinations using asciminib and other drugs, and evaluated which mutations such combinations might be sensitive to. Results Asciminib had synergistic interactions with imatinib or dasatinib in KCL-22 cells at high degrees of inhibition. Interestingly, some antagonism between asciminib and the other drugs was present at lower degrees on inhibition. Simulations revealed that asciminib may allow for dose reductions, and its complementary resistance profile could reduce the risk of mutation based resistance. Axitinib, however, had only a minor effect on T315I growth advantage. Conclusions Given how asciminib combinations were synergistic in vitro, our modelling suggests that drug combinations involving asciminib should allow for lower total drug doses, and may result in a reduced spectrum of observed resistance mutations. On the other hand, a combination involving axitinib was not shown to be useful in countering drug resistance.

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Kim, Wan-Seok, Dongho Kim, Dong-Wook Kim, Il-Young Kweon, Soo-Hyun Kim, Hyun-Gyung Goh, Sa-Hee Park, and Jeong Lee. "Dynamic change of T315I BCR-ABL kinase domain mutation in Korean chronic myeloid leukaemia patients during treatment with Abl tyrosine kinase inhibitors." Hematological Oncology, 2009, n/a. http://dx.doi.org/10.1002/hon.918.

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Cerveira, Nuno, Rosa Branca Ferreira, Susana Bizarro, Cecília Correia, Lurdes Torres, Susana Lisboa, Joana Vieira, et al. "Ponatinib induces a sustained deep molecular response in a chronic myeloid leukaemia patient with an early relapse with a T315I mutation following allogeneic hematopoietic stem cell transplantation: a case report." BMC Cancer 18, no. 1 (December 2018). http://dx.doi.org/10.1186/s12885-018-5100-4.

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Статті в журналах з теми "Cytokines; T315I mutation; chronic myeloid leukaemia"

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