Phase Chronic Myeloid Leukemia Patients Research Articles

The TKI-Free Duration after a First Discontinuation Attempt That Failed in CP CML Patients Is a Predictive Factor of TKI-Free Remission after a Second Attempt

Legros: Incyte Biosciences: Honoraria, Research Funding; BMS: Honoraria; Pfizer: Honoraria, Research Funding; Novartis: Honoraria. Nicolini:Sun Pharma Ltd: Consultancy; Incyte Biosciences: Honoraria, Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau. Etienne:BMS: Honoraria, Speakers Bureau; Incyte Biosciences: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau. Rousselot:Pfizer: Research Funding; Incyte: Research Funding. Rea:Novartis: 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; BMS: Honoraria; Incyte Biosciences: Honoraria. Guerci:INCYTE: Consultancy, Honoraria. Huguet:Incyte Biosciences: Honoraria; Novartis: Honoraria; Servier: Honoraria; Jazz Pharmaceuticals: Honoraria; Amgen: Honoraria; BMS: Honoraria; Pfizer: Honoraria. Coiteux:Novartis: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Incyte Biosciences: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau. Mahon:Novartis: Consultancy, Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Incyte Biosciences: Honoraria, Speakers Bureau.

Accumulation of DNA Damage and Alteration of the DNA Damage Response in Chronic Myeloid Leukemia

The accumulation of DNA damage and the alteration of the DNA damage response (DDR) are critical features of genetic instability that is presumed to be implicated in BCR/ABL1-mediated blastic transformation of chronic myeloid leukemia (CML). The aim of our study was to analyze underlying mechanisms of genetic instability with regard to DNA damage such as DNA double-strand breaks (DSB), DSB repair and DDR signaling during blastic transformation of CML. Immunofluorescence microscopy of γH2AX was performed for quantification of DSB in peripheral blood mononuclear cells (PBMC) of 8 healthy individuals, 24 chronic phase (CP)-CML patients under current/discontinued tyrosine kinase inhibitor (TKI) treatment (21 patients in deep molecular response (DMR), 3 patients in major molecular response (MMR)), 5 CP-CML patients under current/discontinued TKI treatment with loss of MMR, 3 de novo non-treated CP-CML patients and 2 blast phase (BP)-CML patients. In addition, immunofluorescence microscopy of γH2AX/53BP1 was used for semi-quantification of error-prone DSB repair. Furthermore, immunoblotting of p-ATM, p-ATR, p-CHK1, p-CHK2 and p-TP53 was performed in PBMC of CML patients in comparison to PBMC of healthy individuals. Our analysis revealed an increase in numbers of γH2AX foci in PBMC of CP-CML patients under current/discontinued TKI treatment with loss of MMR (1.8 γH2AX foci per PBMC ± 0.4), in PBMC of de novo non-treated CP-CML patients (2.3 γH2AX foci per PBMC ± 0.7) and in PBMC of BP-CML patients (4.9 γH2AX foci per PBMC ± 0.9) as compared to the number of γH2AX foci in PBMC of healthy individuals (1.0 γH2AX foci per PBMC ± 0.1) and in PBMC of CP-CML patients under current/discontinued TKI treatment in DMR/MMR (1.0 γH2AX foci per PBMC ± 0.1) (Figure 1A and B). Analysis of co-localizing γH2AX/53BP1 foci in PBMC suggested progressive activation of error-prone nonhomologous end-joining repair mechanisms during blastic transformation in CML. Signatures of p-ATM, p-ATR, p-CHK1, p-CHK2 and p-TP53 indicated alterations of the DDR. In summary, our data provide evidence for an accumulation of DNA damage in PBMC of CML patients towards BP-CML patients. We hypothesize that ongoing DSB generation, error-prone DSB repair and DDR alterations might be critical mechanisms of blastic transformation in CML. Figure 1 Analysis of γH2AX foci in freshly isolated peripheral blood mononuclear cells (PBMC) of healthy individuals and chronic myeloid leukemia (CML) patients. (A) Exemplary immunofluorescence microscopic images of γH2AX foci (green, Alexa 488) and cell nuclei (blue, DAPI) in PBMC of a healthy individual (HEALTHY#3), a chronic phase CML patient with a deep molecular response to tyrosine kinase inhibitor (CP-CML DMR#16), a de novo non-treated chronic phase CML patient (CP-CML#1) and a blast phase CML patient (BP-CML#2). (B) γH2AX foci levels in PBMC of healthy individuals and in PBMC of CML patients. Figure 1 Disclosures Saussele: Pfizer: Honoraria; Novartis: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Fabarius:Novartis: Research Funding.

Prospective Evaluation of ABL Kinase Domain Mutational Analysis By Next-Generation-Sequencing in Newly Diagnosed CP CML Patients Undergoing First-Line Treatment with Nilotinib Alone or Nilotinib + Pegylated Interferon-α2a in a Prospective Phase III Trial

Prospective Evaluation of ABL Kinase Domain Mutational Analysis By Next-Generation-Sequencing in Newly Diagnosed CP CML Patients Undergoing First-Line Treatment with Nilotinib Alone or Nilotinib + Pegylated Interferon-α2a in a Prospective Phase III Trial

Combination of Nilotinib and Pegylated Interferon Alfa-2B Results in High Rates of MR4.5 at 24 Months - Primary Analysis of the ALLG CML 11 Pinnacle Study

Combination of Nilotinib and Pegylated Interferon Alfa-2B Results in High Rates of MR4.5 at 24 Months - Primary Analysis of the ALLG CML 11 Pinnacle Study

Conventional Real Time Quantitative Polymerase Chain Reaction Method Yields Similar Level of Sensitivity to Digital Droplet Polymerase Chain Reaction for Detection of BCR-ABL p210 Transcripts in Patients with Chronic Phase Chronic Myeloid Leukemia

Introduction: Several studies have investigated the utility of digital droplet polymerase chain reaction (ddPCR) in the detection of p210 BCR-ABL fusion transcripts in chronic myeloid leukemia (CML), and have suggested greater sensitivity compared to real-time quantitative PCR (RT-qPCR). There is limited data regarding the utility and sensitivity of ddPCR in patients with chronic phase CML (CP-CML) who are candidates for tyrosine kinase inhibitor (TKI) discontinuation. We aimed to investigate the capability of ddPCR to detect p210 BCR-ABL transcript levels in CP-CML patients and compare the sensitivity to RT-qPCR. Methods: We analyzed 103 different peripheral blood (n=98) and bone marrow (n=5) samples from 36 patients with CP-CML treated at Moffitt Cancer Center between 2013 and 2018. Samples were from patients who either met clinical criteria for TKI discontinuation or were enrolled in a phase I dose-escalation study evaluating combination therapy with nilotinib plus ruxolitinib who had at least a complete cytogenetic response (CCyR) to TKI therapy with detectable BCR-ABL transcripts by RT-PCR at the time of enrollment(1, 2). BCR-ABL levels were assessed by RT-qPCR at the time of collection with level of transcript reported as % International Standard (%IS). Samples collected from 2013 to 2014 were reported as p210 to ABL and converted to %IS per lab-specific conversion factor. Stored purified RNA samples were obtained retrospectively and ddPCR was performed using the QX-200 Droplet Digital PCR System and QXDx BCR-ABL %IS Kit (Bio-Rad). Droplets were analyzed by QXDx BCR-ABL reporter software to determine p210 transcript level (%IS). Analysis for RNA degradation of stored samples was performed via High Sensitivity RNA ScreenTape (Agilent). Samples that did not pass quality check upon performing ddPCR were excluded from further analysis. Correlation coefficient was calculated with p-value of <0.05 being significant. Results: Of the 36 patients, 28 were in the TKI discontinuation cohort and 10 were enrolled in the clinical trial of nilotinib plus ruxolitinib. Two patients who participated in the clinical trial subsequently also met criteria for TKI discontinuation. In the TKI discontinuation cohort, 46% of patients (n=13) lost major molecular response (MMR) with a median time to loss of MMR of 88 days. We observed a statistically significant correlation between RT-qPCR and ddPCR levels when comparing %IS (correlation coefficient R2=0.42; p<0.0001) as well as calculated molecular response (MR) logarithm values of all available samples (R2=0.67; p <0.0001) (Figure 1). There was a weaker correlation between patients determined to have MMR by RT-qPCR (MR≥3.0) compared to ddPCR (R2=0.55; p <0.0001) (Figure 2). A total of 91 different samples were consistent with MMR when tested by RT-PCR. Of these samples, 85.7% (n=78) were consistent with MMR when tested by ddPCR and the remaining 14.3% (n=13) appeared to have higher detectable levels of p210 transcripts (MR<3.0). When investigating deeper molecular responses, 62 samples were consistent with MR≥4.0 by RT-PCR, and 20.9% (n=13) of these samples did not achieve a deep molecular response (MR<4.0) when tested by ddPCR. Of note, 5 representative samples were also evaluated for potential RNA degradation and 3 were noted to have significant degradation present. Conclusion: Compared to RT-PCR, ddPCR represents a viable method for detection of p210 transcripts for patients with CP-CML. The two methods yield similar efficacy when detecting varying levels of molecular response, however further research is warranted for the ability to detect deeper molecular responses for patients in MMR. Our study is limited by inherent retrospective bias and quality of RNA in stored samples. 1. National Comprehensive Cancer Network. Chronic Myeloid Leukemia (Version 1.2019). 2. Sweet K, Hazlehurst L, Sahakian E, Powers J, Nodzon L, Kayali F, et al. A phase I clinical trial of ruxolitinib in combination with nilotinib in chronic myeloid leukemia patients with molecular evidence of disease. Leuk Res. 2018;74:89-96. Disclosures Talati: Jazz Pharmaceuticals: Honoraria, Speakers Bureau; Daiichi-Sankyo: Honoraria; Astellas: Honoraria, Speakers Bureau; Pfizer: Honoraria; Celgene: Honoraria; Agios: Honoraria. Shelton:Bio-Rad: Employment. Nodzon:Pharmacyclics: Consultancy; Abbvie: Other: Speaker Fees; Genentech: Consultancy, Other: Speaker Fees; Pfizer: Consultancy. Sweet:Stemline: Consultancy; Pfizer: Consultancy; Abbvie: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Jazz: Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Membership on an entity's Board of Directors or advisory committees. Pinilla Ibarz:Novartis: Consultancy; Abbvie: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Consultancy; Janssen: Consultancy, Speakers Bureau; Bayer: Speakers Bureau; TG Therapeutics: Consultancy; Teva: Consultancy; Sanofi: Speakers Bureau.

Preliminary Exploration of Patient Perspectives on Tyrosine Kinase Inhibitors for the Treatment of Chronic-Phase Chronic Myeloid Leukemia

Introduction: Tyrosine kinase inhibitors (TKIs) represent the standard of care for patients with chronic-phase chronic myeloid leukemia (CP-CML). The emergence and availability of second-generation TKIs (dasatinib, nilotinib and bosutinib) has led to increased choice for patients and physicians, with data from randomized clinical trials indicating improved efficacy of such therapies compared to the first-generation TKI (imatinib). Differences in the efficacy of second-generation TKIs are negligible; however, all have distinct dosing instructions and safety profiles. Understanding the patient perspective is invaluable for optimizing healthcare decisions, with interest in information regarding patient preferences growing among a wide variety of stakeholders, including regulatory agencies, reimbursement bodies and prescribers. However, evidence is lacking regarding treatment preferences and priorities for patients with CP-CML. The aim of this study was to explore attributes of currently-licensed TKI therapies that are relevant and important to patients with CP-CML. This research represents the first step in the conduct of a discrete choice experiment (DCE), or conjoint analysis, to explore treatment preferences more broadly among CP-CML patients. Patient-guided design is crucial to ensuring attributes of investigation are meaningful. Hence the qualitative insights will advise and validate attribute and level selection for inclusion in the forthcoming DCE. Methods: In-depth qualitative interviews were conducted with 12 US-based patients with a clinician-confirmed diagnosis of CP-CML. The sample included 10 females and 2 males (median age 46 years) who were diagnosed 2 years (n=3), 4-7 years (n=5), 10-12 years (n=3) and 16.5 years (n=1) prior to interview. Ten patients had received a second-generation TKI. Current treatments were dasatinib (n=8), bosutinib (n=1) and imatinib (n=3). Previous treatments included imatinib (n=6), nilotinib (n=3) and dasatinib (n=1). The interviews utilized concept elicitation techniques to capture attributes of importance in both TKI treatment experience and TKI treatment selection. Transcripts were thematically analyzed using Atlas.ti. Results: Open-ended questioning elicited an array of attributes related to TKI treatments. It was important to all patients to ensure that a CP-CML treatment was effective. Treatment efficacy was conveyed with multiple descriptors; most commonly "BCR-ABL" (n=7) or major molecular response "MMR", polymerase chain reaction "PCR" and "remission" (each n=3). Eight patients discussed the importance of dose frequency, specifically the burden of scheduling self-administration of oral tablets (n=5). Seven patients conveyed the difficulty of taking a TKI with specific meal requirements, most notably when fasting is required prior to and post-administration (n=4). A multitude of important treatment side effects were elicited at investigation into the patient TKI treatment experience. Fatigue was reported by 11 patients (n=10 spontaneous) and considered by most patients (n=8/11) to be directly attributable to treatment versus the CML itself. Four patients considered fatigue an inevitable component of TKI treatment. Nausea was elicited by 7 patients (n=5 spontaneous) and some patients (n=5/9) considered it to be influential in treatment selection. Muscle pain was reported by 6 patients (n=4 spontaneous), while skin rash was reported by 5 patients and bone pain by 4 patients (all spontaneous). Diarrhea was reported by 4 patients (n=3 spontaneous). Additional symptoms reported by ≤2 patients are presented in Figure 1. Spontaneously elicited concepts were ranked by importance. All patients considered efficacy within the top three most important attributes. Six patients considered fatigue to be either of second or third most-importance. The rankings in Figure 2 serve to portray the varied perspectives and experiences of patients on TKI treatments. Conclusions: The TKI treatment experience for CP-CML patients is multifaceted. This type of study - the first that we know of - supplies valuable insight into the patient perspectives of TKI treatment to establish a foundation to better inform relevant decisions in the field. The qualitative findings will provide the input necessary to inform attribute and level selection to characterize treatment profiles ahead of an upcoming patient-preference DCE. Disclosures Mason: Adelphi Values: Employment; Pfizer: Consultancy. Mamolo:Pfizer: Employment, Equity Ownership. Johnson:Pfizer Inc: Consultancy; Adelphi Values Ltd: Employment. Viqueira:Pfizer Inc: Employment, Equity Ownership. Gater:Pfizer Inc: Consultancy; Adelphi Values Ltd: Employment. Russell-Smith:Pfizer: Employment, Equity Ownership. Tatlock:Adelphi Values: Employment. Shah:Pfizer: Employment, Equity Ownership. Cappelleri:Pfizer: Employment, Equity Ownership.

High Throughput Immunophenotyping and Expression Profiling at Single Cell Level Reveal BCR-ABL1 Dependent Surface Markers of Chronic Myeloid Leukemia Stem Cells

BACKGROUND: Bcr-abl1 oncogene targeted treatment with tyrosine kinase inhibitors (TKI) showed an impressive efficacy against proliferating chronic myeloid leukemia (CML) cells. However, rapid relapses in more than half of CML patients after discontinuation of the treatment suggest a presence of quiescent leukemic stem cells inherently resistant to BCR-ABL1 inhibition. Understanding the heterogeneity of CML stem cell compartment is crucial for preventing the treatment failure. Specificity of already established leukemic stem cell (LSC) markers has been tested mainly in bulk CD34+CD38- populations at diagnosis. Phenotypes and molecular signatures of therapy resistant BCR ABL1 positive stem cells is however yet to be established. AIMS: Identification of BCR-ABL1 dependent LSC markers at single cell level by direct comparison their surface and transcript expression with the levels and the presence of BCR-ABL1 transcript at diagnosis and after administration of TKI treatment. METHODS: Total number of 375 cells were obtained from bone marrow and peripheral blood of 4 chronic phase CML patients. Cells were collected prior any treatment and three months after TKI treatment initiation. Normal bone marrow cells and BCR-ABL1 positive K562 cell line were used as controls. Indexed immuno-phenotyping and sorting of CD34+CD38- single cells was performed using a panel of 11 specific surface markers. Collected single cells were lysed and cDNA was enriched for 11 targets using 22 cycle pre-amplification. Expression profiling was carried on SmartChip real-time PCR system (Takara Bio) detecting following genes: BCR-ABL1, CD26, CD25, IL1-Rap, CD56, CD90, CD93, CD69, KI67, and control genes GUS and HPRT. Unsupervised clustering was performed using principal component analysis (PCA). Correlations were measured by Spearman rank method. RESULTS: At diagnosis, majority of BCR-ABL1+ C34+CD38- stem cells co-express IL1-Rap, CD26, and CD69 on their surface (88%, 82%, 78% overlap). Only 56% of BCR-ABL1+ cells positive for aforementioned markers co-express CD25, 28% CD93 and 16% CD56. The expression of these markers could also be detected in 4-11% of BCR-ABL1- cell, although this could be technical inaccuracy caused by the single cell profiling. CD90 marker did not show any correlation with BCR-ABL1 expression. At transcript level the expression of IL-1Rap, CD26, CD25 and CD56 was observed in 62%, 52% 45% and 16% BCR-ABL1+ cells, and up to 7% of BCR-ABL1- cells. CD69 expression was observed in 90% of BCR-ABL+ cells at transcript level, but also in 71% BCR-ABL- cells. BCR-ABL1 independent expression was observed for cKIT. (60% vs. 76 % in positive vs negative). Finally proliferation marker KI67 was expressed only in 6% of the BCR-ABL1+ cells. PCA analysis divided cells into several distinct clusters with BCR-ABL1 as the main contributor, and cKIT, CD69 and CD26, IL-1RAP as other significant factors. Interestingly BCR-ABL1+ cells collected during TKI treatment showed persistent surface expression of IL-1Rap and CD26, while CD56, CD69 and CD93 were only on part of the BCR-ABL1+ cells. CD25 was significantly deregulated during TKI treatment. CONCLUSION: At diagnosis up to 80% of LSC co-express 3 specific surface markers - IL-1RAP, CD26 and CD69. Variable portion of LSC co-express additional markers such are CD25, CD56 and CD93. During TKI treatment the surface expression of majority of markers is decreased, where the best correlated LSC marker is IL-1Rap, followed by CD26 and CD69. CD56 marker seems to persist in the same proportion of cells while CD25 disappears. cKIT is highly expressed in normal BM and HSC from CML patients, but also in some LSC. CD34+CD38- cells show non-proliferating phenotype. Disclosures Mayer: AOP Orphan Pharmaceuticals AG: Research Funding.

The Interaction of Tumor Cells and Myeloid-Derived Suppressor Cells in Chronic Myelogenous Leukemia

Chronic myelogenous leukemia (CML) is a malignant myeloproliferative disease characterized by the formation of the BCR-ABL fusion gene. At present, basic studies of the pathogenesis of relapse after stopping tyrosine kinase inhibitors (TKIs) treatment have mainly concentrated on two main aspects: the leukemia stem cells (LSCs) and the tumor microenvironment. However, whether relapse or non-relapse patients who discontinued TKIs therapy, LSCs are still exist. Among a variety of factors that compose the CML microenvironment, myeloid-derived suppressor cells (MDSC) are considered to be a strong contributor to the immunosuppressive tumor microenvironment. Here, we designed the study to investigate the potential relation between tumor cells and MDSC in CML and find risk factors for relapse after discontinuation. We detected the percentage of MDSC and the BCR-ABL (IS) transcript levels in bone marrow of 50 CML patients in chronic phase at our center. The data indicated that the frequency of MDSC had significant positive correlation with BCR-ABL (IS) transcript levels (Figure 1A). In addition, the counts of MDSC had significant difference at different response stages (Figure 1B), especially the M-MDSC, a subtype of MDSC. The percentage of M-MDSC was significantly higher in patients with newly diagnosed or complete hematological response (CHR) or major molecular response (MMR) compared with those of CML patients obtained complete molecular response (CMR) (Figure 1C). When K562 cells or CD34+ cells were cocultured with M-MDSC at a 1:10 ratio, K562 cells or CD34+ cells proliferated significantly at day 3 (Figure 1D and E). K562 subcutaneous tumor formation in BALB/c node mice confirmed that tumors weight and volume of the coculture group were higher than control. Then, we further investigated whether tumor cells have an impact on MDSC through microvesicles (MV). After adding K562-MV to peripheral blood mononuclear cells (PBMCs) from healthy donors, MDSC counts appeared significantly elevated in the different K562 cells counts group compared to the control group (Figure 1F).To analyze the roles of K562-MV collected before and after TKIs discontinuation on MDSC, we established a TKIs discontinuation model using the K562 cell, which emulates the cessation of TKIs treatment of CML patients in some extent. The results showed that regardless of the Imatinib or Dasatinib treatment, a significant increase was observed in the proportion of MDSC after TKIs treatment cessation compared with the TKIs treatment groups (Figure 1G). Experiments in vivo also proved K562-MV after different treatments promoted the proliferation of MDSC (Figure 1H and I). In conclusion, our study introduces the notion of the role of MDSC as mediators in the cross talk between tumor cells and the microenvironment. MDSC would provide a novel and useful model to predict the relapse of CML by establishing a type of new risk stratification system. MDSC could be also act as a promising target in the relapse of CML. In addition, we found a mutual promotion of proliferation of tumor cells and MDSC, this bidirectional interaction results in a vicious cycle by providing a protective niche against immune attacks. Therapeutic interventions modulating this interaction might accelerate the success of treatment-free remission. Figure 1 Disclosures No relevant conflicts of interest to declare.

A 20-Year Review of Imatinib in Chronic Phase Chronic Myeloid Leukemia Patients after Failure with Interferon Therapy

A 20-Year Review of Imatinib in Chronic Phase Chronic Myeloid Leukemia Patients after Failure with Interferon Therapy

NUP98-HOXA9-Initiates Molecular and Biologic Features of Disease Progression in a Humanized Model of CML

Acute myeloid leukemias (AMLs) are heterogenous diseases often resulting from the acquisition of multiple genetic alterations that deregulate hematopoietic precursor proliferation and block normal differentiation. Chronic myeloid leukemia offers a unique opportunity to identify molecular mechanisms that interfere with normal differentiation in the context of a highly proliferative hematopoietic stem cell clone that produces massive number of functional differentiated myeloid cells due to the presence of a BCR-ABL1 fusion gene. Since the NUP98-HOXA9 (NA9) fusion gene has been identified in some CML blast crisis patients or de novo AML, we asked whether a humanized model of CML progression to AML would result from its lentiviral-mediated introduction into primary CML CD34+ cells from 3 chronic phase CML patients in which >97%, >96%, and 44% of the CD34+ longterm culture-initiating cells were Ph+/BCR-ABL1+. In vitro experiments showed the NA9-transduced cells produced a hugely increased number of granulo-monocytic progenitors in long-term cultures (up to 1000-fold, p=0.03; t-ratio test) and enhanced the serial replating activity of directly clonogenic cells as compared to matched samples of cells transduced with a control vector. In vivo experiments showed that 90% of sublethally irradiated NOD-SCID IL2Rgc-null mice expressing human IL3, GM-CSF and SCF constitutively that were transplanted with these same NA9-transduced cells developed evidence of a progressed myeloproliferative neoplasia. This included tissue infiltrates of eosinophils, basophils and mastocytes, exclusive myeloid differentiation and signs of an imminently fatal leukemia between 8 and 27 weeks post-transplant in 50% of cases, although an excess blast population was not seen in these. RNA-sequencing of cells analyzed just 48 hours after transduction revealed a signature of 53 genes that were more highly expressed in CD34+ CML cells carrying the NA9 fusion gene in comparison to control CML cells. This signature included many genes expressed by hematopoietic stem cells (HSCs), such as 5' HOXA genes, 3' HOXB genes, PBX3, MEIS1,ARID5B, AHR, REL, BMP6, GDF10, SFRP5, PPBP,PLA2G4A suggesting that NA9 induces the expression of a partial HSC program in later CML progenitor types that make up the bulk of the CD34+ CML population. Consistent with this hypothesis, the NA9 signature separated the HSCs, CMPs and GMPs of chronic CML patients as well as those of normal subjects (GSE47927; p=1x10-6, p=2x10-4 respectively). Most genes in the NA9 signature were also found to be over-expressed in CD34+ cells of patients in the accelerated phase and in the blast phase of CML, including ARID5B, AHR, STARD9, TOX, or FOXP1 (p=9.10-8; ANOVA) (GSE4170). The NA9 signature was also significantly enriched in transcripts of genes that are over-expressed in the blasts of AML patients carrying NPM1 mutations or MLL fusions and was predictive of overall survival in the AML cohort of the Cancer Genome Atlas (p=0.02; log-rank test, n=200). This stem cell signature was also associated with an increase in the number of H3K27ac marks (on average 7805 ± 960 peaks for NA9 vs 5888 ± 2739 peaks for control) at 48 hours post-transduction. De novo H3K27ac peaks in NA9+ CD34+ cells were located in proximal and distal enhancers of 993 genes (GREAT parameters TSS ± 2kb from TSS and 100kb max extension). These were significantly enriched genes in the GM-CSF signaling pathway (MySigDB, binomial p-value=2.19x10-8) and that are upregulated in granulocytes and monocytes following LPS exposure. H3K27ac peaks also matched with GATA1, RELA, MEF2A and IKZF1 transcription factor binding sites previously mapped by ChIPSeq experiments (adjusted p-value <0.05). Finally, we identified super-enhancers in 12 genes among which were PBX3, ANGPT1, MBNL1 and PRKACB. Overexpression of the NA9 fusion gene in chronic phase CD34+ CML cells thus appears to reprogram the expression of HSC genes as well as those associated with GM-CSF pathway activation and inflammatory responses via H3K27 acetylation of associated loci leading to a picture of advanced accelerated phase/disease progression but not the complete differentiation arrest seen in terminal blast crisis or frank AML. These findings highlight the multiplicity of biologically important molecular alterations that can result from a single epigenetic perturbation but, nevertheless, are insufficient to create an overt AML phenotype. Disclosures Turhan: novartis: Honoraria, Research Funding; Incyte: Consultancy, Honoraria.

High-Risk Additional Chromosomal Abnormalities in CML Herald Death By Blast Crisis Already at Low Blast Levels

Background. The end phase or metamorphosis is one of the remaining challenges of chronic myeloid leukemia (CML) management. Blast crisis (BC) is a late marker. Earlier diagnosis may improve outcome. The detection of additional chromosomal abnormalities (ACA) at low blast levels might allow earlier treatment when outcome is better. Methods. We made use of 1536 Ph+CML-patients in chronic phase followed in the randomized CML study IV (Hehlmann et al, Leukemia 2017) for a median of 8.6 years. 1510 cytogenetically evaluable patients were analyzed for ACA and blast increase (Flow chart). According to impact on survival ACA were grouped into high-risk (+ 8; +Ph; i(17q); +17; +19 +21; 3q26; 11q23; -7; complex) and low-risk (all other). Prognosis with +8 alone was clearly better than with +8 accompanied by further abnormalities, but still worse than with low-risk ACA. +8 alone was therefore included in the high-risk group. The presence of high- and low-risk ACA was linked to 6 thresholds of blast increase (1%, 5%, 10%, 15%, 20%, and 30%) in a Cox proportional hazards model. Results. 139 patients (9.2%) displayed ACA at any time before BC diagnosis, 88 (5.8%) had high-risk and 51 (3.4%) low-risk ACA. ACA emerged after a median of 17 (0-133) months. 79 patients developed BC. 43 (61%) of 71 cytogenetically evaluable patients with BC had high-risk ACA. 3-year survival after emergence of high-risk ACA was 48%, after emergence of low-risk ACA 92%. At low blast levels (1-15%), high-risk ACA showed an increased hazard to die (ratios: 3.66 in blood; 6.84 in marrow) compared to no ACA in contrast to low-risk ACA. This effect was not observed anymore at blast increases to 20-30% (Figure). 38 patients with high-risk ACA died, 36 with known causes of death which were almost exclusively BC (n=26, 72%) and progression-related transplantation (n=8, 22%). Only 2 patients died of CML-unrelated causes. Conclusions. High-risk ACA herald death by BC already at low blast levels and may help to define CML end phase in a subgroup of patients at an earlier time than is possible with current blast thresholds. Cytogenetic monitoring is indicated when signs of progression surface and response to therapy is unsatisfactory. More intensive therapy may be indicated at emergence of high-risk ACA. Disclosures Hehlmann: Novartis: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Fabarius:Novartis: Research Funding. Krause:Siemens: Research Funding; Takeda: Honoraria; MSD: Honoraria; Gilead: Other: travel; Celgene Corporation: Other: Travel. Baerlocher:Novartis: Research Funding. Burchert:Novartis: Research Funding. Brümmendorf:Novartis: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Ariad: Consultancy; Pfizer: Consultancy, Research Funding; University Hospital of the RWTH Aachen: Employment. Hochhaus:Pfizer: Research Funding; Novartis: Research Funding; BMS: Research Funding; Incyte: Research Funding; MSD: Research Funding. Saussele:BMS: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; Pfizer: Honoraria; Novartis: Honoraria, Research Funding. Baccarani:Novartis: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Takeda: Consultancy.

Efficacy and Safety of Generic Imatinib Compared to Glivec in Chronic Phase - Chronic Myeloid Leukemia-Single Center Retrospective Analysis

Introduction: Glivec, Imatinib (IM), first tyrosine kinase inhibitor (TKI) has proven its efficacy and safety in Chronic Myeloid Leukemia - Chronic Phase (CML CP) in a large number of trials. In developing country like India Generic Imatinib formulations have been used recently as a more cost effective treatment, data on efficacy and safety of these drugs is sparse. Aim: To analyse and compare safety profile, the overall survival (OS) and progression-free survival (PFS) data of CML-CP patients receiving Glivec/Generic IM as first line therapy Method: A single centre retrospective analysis done on CML-CP patients who initiated treatment with either Glivec or Generic IM between July 2012 and July 2019 at Hemato-oncology Care Centre (HOCC) Vadodara India. Total 109 newly diagnosed CML-CP received IM as 400mg daily dose, dose was adjusted if significant cytopenia observed or in paediatric patients. Response assessment was made as per NCCN CML Version 1 2019 guidelines.Molecular monitoring of patients was done through BCR-ABL1 RQPCR, reported on international scale. Major Molecular Response (MMR) was considered when the ratio on international scale was less than 0.1% level or ≥ 3-log reduction in BCR-ABL1 mRNA from the standardized baseline, while Deep Molecular Response-MR4 was considered when the ratio on international scale was less than 0.01%.OS was measured from starting date of IM until to the date of death from any cause while on therapy or last seen.PFS was measured from starting date of IM to transformation to AP or BC or deaths while on therapy. Survival curves were calculated by the Kaplan-Meier method and compared with the log-rank test. Cox regression with Backward Wald Method, Chi-square test and t-test is applied using SPSS software version 21.0. A p-value <0.05 is considered to be statistically significant. Results: A Total 109 patients were analysed, received Glivec (47)43% or Generic IM (62)56%. The mean (SD) age was 42.2 years (13.3 years) in Glivec and 47.5 years (14.9 years) in the Generic IM group (P=0.06). There was male preponderance in both groups (Glivec 55%, Generic IM 62 %). Sokal score stratification in Glivec and Generic groups, was respectively: low risk 43%/40%; intermediate risk 36%/48% and high risk 21%/11% (P=0.08). The median follow‐up period was 36 months (range, 9-84). MMR was 61% and 38% in Glivec and Generic IM respectively(p=0.02), MMR was achieved in 40%,04%,11%,06% of Glivec and 29%,2%,5%,2% of Generic IM group patients, at 1st,2nd,3rd,4th year of treatment respectively(P=0.15).Deep Molecular Response-MR4 was significantly higher 40% in Glivec vs 23% in Generic IM group (P=0.01). Nearly 35% of patients were lost to follow up, 28% and 40% in Glivec and Generic IM group respectively(p=0.27). Reasons for drop out were - cost constraints, noncompliance, lack of understanding, lack of transport. Dose limiting cytopenia was observed in initial 3 months of treatment in 12% and 23 % in Glivec and Generic IM group respectively(P=0.28).The most common non haematological side effects in Glivec and Generic groups were skin hyperpigmentation 19%,17%, abdominal discomfort 16 %,11% ,Facial Puffiness 9%,5%, respectively(P =0.73). In each group 4% patients progressed to AP, managed with second generation TKI in Generic IM group and with increasing dose of Glivec to 600mg daily in Glivec group. Total 5% patients progressed to BC and died, 3% in Glivec vs 2% in Generic IM group. The risk factors for progression to AP or BC were high Sokal score, poor molecular response and dose limiting cytopenia. OS and PFS at 36 months were similar in Glivec and Generic IM (93.6% vs. 96.8% P=0.834) and (89.4% vs. 91.9%, P=0.839) respectively. Conclusion: Both the groups have shown similar OS and PFS at 36 months of median follow up, there was also no difference in the safety profile, meeting Generic IM unmet needs in developing country. However in view of higher MMR and Deep molecular response-MR4 in Glivec group, need long term follow up to determine its impact on outcome especially in reference to treatment free remission. Figure Disclosures No relevant conflicts of interest to declare.

Dasatinib and Nilotinib As Upfront Therapy for Chronic-Phase Chronic Myeloid Leukemia (CML-CP) in Qatar: A Cost Effectiveness Analysis (CEA)

Objectives: Many tyrosine kinase inhibitors (TKIs) are approved for treating the chronic phase of chronic myeloid leukemia (CML-CP). This study aims at identifying the optimal upfront TKI treatment strategy; considering cost-effectiveness for CML patients from Qatar healthcare system perspective. Evaluated strategies included the newer generation TKIs; dasatinib 100 mg once daily and nilotinib 300mg twice daily. We evaluated both treatments considering their molecular and cytogenetic response outcomes at 12 months follow up as well their respective acquisition costs. Methods: A decision tree model was developed linking clinical effectiveness (defined as major molecular response MMR and/or complete cytogenetic response CCyR), utility, and cost data over a 12-months period. The choice of 12 months period is due to the evidence that the degree of short-term molecular and cytogenetic responses at certain follow- up times is correlated with CML prognosis. Patients who achieve a CCyR are shown to have high remission rates as well overall survival (OS) rates. Patients with newly diagnosed CML are adopted from Qatar Cancer Registry.The probability of primary clinical outcome is calculated from DASISON (dasatinib) and ENESTnd (nilotinib) trials. Direct healthcare costs were derived from the national healthcare payer system, whereas ADR data were derived from local incident reporting system. Results: In the first-line treatments of CML patients, with greater MMR (39% nilotinib vs 12% dasatinib) and CCyR (24% nilotinib vs 16% dastinib) response outcomes, and more adverse drug reactions (ADR) than dasatinib (13.3% vs 4%), nilotinib use is more cost effective with annual costs (QAR 232,102) and after 12 months follow up. Despite the lower acquisition annual cost of dasatinib (QAR 217,125), the incremental cost effectiveness ratio (ICER) of nilotinib versus dasatinib per MMR/CCyR achieved was 56,506 QAR per year. There were no cases in both arms that progressed to accelerated or blast phase. At a threshold of 3 times GDP per capita of Qatar and according to WHO recommendation, the nilotinib use is still cost-effective. Limitations: For the surrogate outcome, we only consider the percentage of patients with or without MMR / CCyR response at 12 months. Nevertheless, we do not consider the depth and the duration of the MMR or CCyR. Conclusions: Upfront therapy of CML-CP patients by nilotinib plan appears to be more cost-effective than dasatinib. Although local real-life data for the effectiveness of these drugs are still warranted. The results of this study implying that nilotinib would be advantageous as first-line treatment for CML-CP patients from Qatar healthcare system perspective. Disclosures No relevant conflicts of interest to declare.

BCR-ABL1 p190 in CML: A Minor Breakpoint with a Major Impact

Introduction: The oncoprotein Bcr-Abl has two major isoforms, depending on the breakpoint in BCR gene, p190 and p210. While p210 is the hallmark of chronic myeloid leukemia (CML), p190 occurs in the majority of Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients. p190 occurs as a sole transcript in 1-2% CML patients, associated with distinct features like monocytosis and frequent additional cytogenetic abnormalities (ACA) at diagnosis. It also confers a risk of treatment failure and progression in chronic phase (CP) CML patients. However, the underlying mechanisms are largely unknown. Here we explore the characteristics of p190 and p210 in CML and ALL patients using next generation sequencing, phospho-flowcytometry and high throughput drug testing. Patients and methods: Peripheral blood mononuclear cells (PMNC) were collected at diagnosis from four CP-CML patients harboring p190 isoform from Helsinki University Hospital. Genetic alterations were identified by whole exome sequencing. RNA sequencing was employed to analyze transcriptional profiles of p190 CML (n=3) in contrast to p210 CML patients (n=4). A thorough transcriptional, phosphorylation and drug sensitivity profiling were applied to five p190- and three p210-expressing Ph+ALL patients. Expression alterations were further characterized in two cell line models mimicking BCR-ABL positive leukemia (Ba/F3 and HPCLSK). Phosphorylation profiles were analyzed by flowcytometry and phospho-array (Tyrosine Phosphorylation ProArray, Full Moon Biosystems). For drug sensitivity and resistance testing (DSRT), a custom plate set comprising 75 approved and investigational oncology drugs was used for patient samples and more extensive 528-drugs plates were used for the cell lines. Results: CML patients with p190 had a median age of 72.5 years at the diagnosis (range: 50-80) and all received imatinib as a frontline treatment. Only one patient achieved a fluctuating major molecular response (MMR) by 12 months while the rest of the patients showed primary resistance to treatment and were shifted to a 2nd line TKI, nilotinib (n=2) or proceeded to HSCT (n=1). By exome sequencing we identified 26 variants in p190-CML samples (median per patient=7, range: 2-10), including variants in ASXL1, DNMT3A and KDM4D genes. RNA-sequencing analysis identified 19 and 97 dysregulated genes (Q <0.05) between p190- and p210 in CML and Ph+ ALL cells respectively. In CML, enrichment analysis revealed upregulation of TNF, interferon (IFN), IL1-R and Toll-like receptor (TLR) signaling, TP53-related, cell cycle and apoptosis pathways. Among Ph+ ALL samples, many CML-related genes were upregulated in samples encompassing p210 while IFN-, TP53- and cell cycle-related molecules were upregulated in p190 samples. p190 samples exhibited hyper-phosphorylation of Src kinase compared to p210 samples. DSRT results also revealed increased sensitivity of primary Ph+ ALL-p190 cells to Src-inhibitors (dasatinib and saracatinib), glucocorticoids and MDM2 inhibitors/TP53 activators (SAR405838 and idasanutlin). Regarding cell lines, Ba/F3-p190 showed the upregulation of interferon signaling pathways compared to p210. Src was also hyperphosphorylated in both Ba/F3 and HPCLSK p190 models. In addition to glucocorticoids and Src-inhibitors, compounds blocking the activity of the inhibitors of apoptosis protein (IAP) family were highly effective at reducing the viability of p190 compared to p210 cells in both cell lines. Conclusions: In CML, p190 isoform of BCR-ABL1 is associated with distinct features and should be considered as a high-risk group. Combining clinical, genomic, phosphorylation and drug sensitivity data, we demonstrated that p190 activates specific cancer pathways, notably Src signaling and interferon pathways. Data also suggests that CML patients with p190 could benefit from broad spectrum TKI with Src inhibiting activity or combination of TKI with MDM2- or IAP-inhibitors. Disclosures Heckman: Orion Pharma: Research Funding; Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding. Porkka:Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding. Mustjoki:Novartis: Research Funding; Pfizer: Research Funding; BMS: Honoraria, Research Funding.

Generic Versus Branded Imatinib As Frontline Therapy in Chronic-Phase Chronic Myeloid Leukemia Patients in Italy: A Case-Control Study

Introduction. Imatinib is the most commonly used frontline drug in chronic phase (CP) chronic myeloid leukemia (CML) patients worldwide. In early 2017 a generic formulation of imatinib was introduced in Italy and uniformly replaced branded imatinib (Glivec®), upon requirement of regional health authorities. In the last years various groups reported on the efficacy and safety of generic imatinib with conflicting results, partly related to substandard pharmaceutical quality of some products used in developing countries. In a multicenter cohort of 294 patients treated in Italy with branded imatinib for at least 6 months and then switched to generic imatinib we observed that the majority of patients maintained or improved their molecular response. Here, we analyzed patients who received generic imatinib since diagnosis. Aims. To analyze the rates of molecular responses at 3, 6 and 12 months and of treatment discontinuation in CML patients treated frontline with generic imatinib, compared to a case-matched historical cohort of CML patients who received frontline branded imatinib at our institutions. Methods. We analyzed 31 newly diagnosed CP-CML patients consecutively enrolled in a prospective observational registry between January 2017 and July 2018, treated frontline with generic imatinib 400 mg/day (diverse manufacturers) and evaluable for all the ELN2013 molecular milestones (if not discontinued earlier). They were compared to a retrospective cohort of 31 patients, matched for age, gender, and Sokal risk, diagnosed between 2007 and 2014 and treated with branded imatinib 400 mg/day for at least 24 months before eventual switching to a generic formulation. Definitions of molecular responses were made according to the ELN2013 recommendations. Results. A total of 62 patients were included in the analysis: 31 patients (21 males and 10 females) treated with generic imatinib ("cases") and 31 treated with branded imatinib ("controls"). Median age at diagnosis of the cases was 68 years (range 33-89), Sokal score was low/intermediate/high in 8 (26%), 19 (61%) and 4 (13%) patients, respectively. The controls were matched for gender, age (+/- 4 years, median age 68, range 35-85) and Sokal score. As median follow-up time for the cases was 18.6 months (range 2.2-28.5), controls were censored at 24 months after imatinib start. Optimal molecular response at 3 months was attained in 23/30 (76.7%) cases and in 18/29 (62%) controls (p=0.35); one case died after 2 months of imatinib therapy for a CML-unrelated cause, while 2 controls were molecularly not evaluable. At 6 months, 17/29 (58.6%) cases and 17/30 (56.7%) controls achieved BCR/ABL transcript <1%, respectively (p=1). At 12 months, MMR was attained by 14/30 (46.7%) cases and by 13/29 (44.8%) controls (p=1). Twelve out of 31 patients (38.7%) permanently discontinued generic imatinib due to warning/failure response (n=6), intolerance (n=4) or death while on treatment at 2 and 13 months (meningoencephalitis of unknown origin and cardiovascular event, respectively). Among patients treated with branded imatinib, 12/31 (38.7%) stopped within the 24th month of therapy for resistance (n=8), intolerance (n=3) or death at 16 months (acute renal failure). No patient receiving generic imatinib progressed to advanced phase, while one control developed a blast crisis at 6 months of branded imatinib and deceased shortly after. Estimated overall survival at 24 months in cases and controls was 92.5% and 93.1%, respectively. Conclusions. Our preliminary data suggest an equivalent efficacy of generic imatinib compared to a matched population of historical patients treated with the originator drug in Italy. A continue pharmacovigilance by reporting efficacy and safety outcomes of generic drugs is needed to ensure an optimal management of CML patients. Disclosures Bonifacio: Novartis: Honoraria, Research Funding; Amgen: Honoraria; Incyte: Honoraria; Pfizer: Honoraria. Tiribelli:Pfizer: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Krampera:Novartis: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.

A Combination of CD302 gene Expression and 3-Months BCR-ABL1 Level Predicts Inferior Achievement of Deep Molecular Response in CP-CML Patients Treated with Imatinib

Introduction and Aim. Achievement of deep molecular response (DMR) is the prerequisite for treatment-free remission in chronic phase CML (CP-CML) patients (pts). Pts who fail to achieve early molecular response (BCR-ABL1 > 10% IS) at 3-months (mths), or have high ELTS score at diagnosis have inferior achievement of DMR. We and others have shown that the levels of NK-cell, T-cell, myeloid-derived suppressor cell, and neutrophils in the blood at diagnosis have an impact on DMR achievement. We hypothesized that Cluster of Differentiation (CD) (cell surface marker) gene expression might provide a surrogate marker to characterize immune cell composition. We aimed to identify pts who had a low probability of achieving DMR by 5 years (yrs) by combining 3-mths BCR-ABL1% and CD gene expression. This may enable clinicians to determine whether an individual patient is on a pathway towards DMR and potentially TFR or should be considered for a different therapeutic approach if TFR is the eventual goal. Methods. 119 blood samples from the imatinib-based TIDEL-II trial were subjected to transcriptomic microarray profiling. A total of 357 CD genes classified by the HUGO Gene Nomenclature Committee CD molecular gene group were assessed. We defined DMR as achieving MR4.5 (BCR-ABL1 < 0.0032%) at two consecutive time points. To construct a predictive model, the samples were randomly assigned to discovery and validation cohorts. Recursive partitioning and construction of a regression tree with tenfold cross-validation based on expression of 357 CD genes and 3-mths BCR-ABL1% were used as inputs in the discovery cohort. The performance was assessed based on accuracy of prediction of DMR by 5 yrs. The final model was validated using the independent validation cohort. All the analysis was performed using R statistical software. Results. Clinical variables (age, gender, ELTS, 3-mths BCR-ABL1%, MMR, and MR4.5) were well matched in the discovery (n=60) and independent validation cohort (n=59). The predictive model was constructed using the discovery cohort to reveal two risk groups: poor-risk (PR, 15% achieving MR4.5 at 5 yrs, n=19), and good-risk (GR, 88% achieving MR4.5 at 5 yrs, n=41) groups (Figure 1A-B). This model classified PR group by BCR-ABL1 ≥ 7.5% at 3 mths OR BCR-ABL1 < 7.5% at 3 months with high CD302 gene expression (≥7.9 log2 gene expression; top 15%) at diagnosis. GR group was defined as having both BCR-ABL1 < 7.5% and low CD302 gene expression (<7.9 log2 gene expression). These variables were chosen by the model based on accuracy performance in predicting DMR. CD302 is a C-type lectin receptor involved in cell adhesion and migration. It is expressed in myeloid populations as well as in blasts and leukemic stem cells (LSC) in AML. High expression of CD302 in PR pts may be a surrogate for increased LSC. The model was validated in the independent validation cohort. Pts identified as PR in the validation cohort had significantly lower 5-yrs MR4.5 achievement rate (14%, n=14) compared to those with GR (82%, n=45, p=0.0002, Figure 1C). We asked whether using the more conventional BCR-ABL1 10% cutoff instead of 7.5% in our model would give similar results, but the performance in predicting long-term DMR achievement was inferior: Pts predicted as PR with this criteria had ~2x higher achievement of DMR (e.g. 26% vs 14% using 3-mths BCR-ABL1 10% vs 7.5% cutoff respectively). ELTS score have been associated with the probability of DMR achievement. We compared the performance of ELTS in combination with 3-mths BCR-ABL1% by replacing CD302 gene expression with ELTS. The predictive accuracy was inferior. Pts with 3-mths BCR-ABL1 ≥7.5% OR BCR-ABL1 <7.5% with high/intermediate ELTS (PR-2) had about 3.3-3.5 fold higher DMR achievement rate than the PR group with CD302 in both discovery and validation cohorts (Figure 1D-E). In contrast, pts with 3-mths BCR-ABL1 <7.5% and low ELTS (GR-2) had approximately 1.1-1.2 fold lower DMR achievement rate than the GR group with CD302 in both discovery and validation cohorts (Figure 1D-E). Conclusion. We have constructed a predictive model for DMR achievement for pts who receive optimised frontline imatinib therapy. This model performs better than combining ELTS and 3-mths BCR-ABL1%. We postulate that this predictive model could enable identification of poor risk pts at 3 mths who would benefit from intensified therapeutic approaches to obtain eligibility for TFR and potentially optimal clinical outcome. Disclosures Yeung: Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Pfizer: Honoraria; Amgen: Honoraria. Hughes:Novartis: Other: Advisory Board and Symposia, Research Funding; BMS: Research Funding.

An Updated Safety and Efficacy Results of Phase 1 Study of HQP1351, a Novel 3rd Generation of BCR-ABL Tyrosine Kinase Inhibitor (TKI), in Patients with TKI Resistant Chronic Myeloid Leukemia

Background: HQP1351, a novel, orally active potent 3rd generation TKI, was designed for treatment of patients with chronic myeloid leukemia (CML) resistant to current TKI-therapies including those with T315I mutation. This is an updated report of the safety and efficacy of HQP1351. Methods: It is an open-label, dose escalation and expansion phase 1 trial of HQP1351 which was designed to determine maximum tolerated dose (MTD) and to identify dose-limiting toxicities (DLTs) in patients with CML in the chronic phase (CP) or accelerated phase (AP) resistant or intolerant to ≥ 2 prior TKIs or those with T315I mutation after ≥1 prior TKI. HQP1351 was orally administered once every other day (QOD) in 28-day cycles at 11 dose cohorts ranging from 1mg to 60 mg. The eligible patients received continuous treatments until disease progression to AP or BP, developing intolerant toxicity, consent withdrawal, or death. Blood samples were collected at certain time points on Day 1-2 and Day 27-28 during cycle 1 for PK analyses. Results: From 26 October, 2016 to 27 May, 2019, 101 patients including 87 CP patients and 14 AP patient were enrolled in this study. Seventy-one (70.3%) patients were male. Median duration of follow-up was 12.8 (range, 1.2-31.5) months. Median age was 40 (range, 20-64) years. Median interval from CML diagnosis to starting HQP1351 treatment was 5.8 (range, 0.3-15.2) years. Eighty-three (83.8%) patients received ≥2 prior lines of TKI-therapy. Sixty-two (61.4%) patients harbored T315I mutation. Seventeen patients who initially assigned to the dose escalation cohorts ranging from 1mg to 20 mg moved to 30 mg or higher dose cohorts via intra-patient dose escalation. Fifty-six patients enrolled in the dose expansion part of the trial, including 30mg,40mg and 50mg dose cohorts. Two out of 3 patients at 60mg dose cohort experienced DLT and 50mg QOD was considered as the MTD. During the follow-up period, HQP1351 was well-tolerated in each dose cohort with an exception of 60 mg cohort. All patients experienced ≥1 treatment related adverse events (TRAEs), most of the non-hematologic TRAEs were reported as grade 1 or grade 2. The most common hematologic TRAE of grade 3/4 was thrombocytopenia (49.5%). The incidences of TRAEs tended to be dose-dependent. No death and grade 5 AEs occurred. The incidence of common TRAEs (≥ 10% any grade) are summarized in Table1. HQP1351 showed potent anti-leukemic activities in CML patients. In the 68 evaluable patients with non-CHR at baseline, 63 (92.6%) achieved CHR including 52 out of 55 (94.5%) CP patients and 11 out of 13 (84.6%) AP patients, respectively. In the 95 evaluable patients with non-CCyR at baseline, 56 out of 81 (69.1%) CP patients achieved MCyR including 49 (60.5%) CCyR; and 6 out of 14 (42.9%) AP patients, achieved MCyR including 5 (35.7%) CCyR, respectively. In the 100 evaluable patients, 32 out of 86 (37.2%) CP patients and 5 out of 14 (35.7%) AP patients achieved MMR, respectively. HQP1351 showed highly efficacious in the patients with T315I mutation (Figure A, B). The probability and the depth of response increased with prolonged treatment period (Figure C). As the cut-off date of May 27 2019, 9 patients (5 CP and 4 AP) withdrawn from the study, including progression to AP or BP (n=5), intolerant AEs (n=2), consent withdrawal (n=1), and newly diagnosis of breast cancer (n=1). The progression free survival (PFS) rate at 18-month was 94% in the CP patients and 61% in the AP patients (Figure D). PK analyses indicated an approximately dose proportional increase in exposure (AUC and Cmax) following oral administration of HQP1351 at doses from 1mg to 60 mg. The peak concentration of HQP1351 was reached at 4-8h. The elimination appeared to be linear with a mean terminal T1/2 of 17.5 to 42.5 h. Slight to moderate accumulation was observed on Day 27 following multiple dosing. Reduction of CRkL phosphorylation in PBMCs, a biomarker of BCR-ABL inhibition, has shown to be dose and time dependent in 53 evaluable patients treated with HQP1351. Conclusions: HQP1351 was well tolerated and exhibited significant and durable antitumor activity in the patients with TKI-resistant CML, including those with T315I mutation. Two pivotal studies of HQP1351 in CML-CP and CML-AP patients with T315I mutation are ongoing in China. Disclosures Chen: HealthQuest Pharma Inc.: Employment. Niu:HealthQuest Pharma Inc.: Employment. Men:HealthQuest Pharma Inc.: Employment. Wang:HealthQuest Pharma Inc.: Employment. JI:HealthQuest Pharma Inc.: Employment. Huang:HealthQuest Pharma Inc.: Employment. Yang:Ascentage Pharma Group: Employment. Zhai:Ascentage Pharma Group Inc.: Employment; HealthQuest Pharma Inc.: Employment.

Silent NK/T Cell Reactions Against Dasatinib during Sustained Deep Molecular Response before Discontinuation Are Associated with Longer Treatment-Free Remission: A Multicenter, Single-Arm, Phase 2 Study

Introduction Tyrosine kinase inhibitors (TKIs) markedly enhance the prognosis of chronic myelogenous leukemia (CML), potentially enabling the attainment of deep molecular response (DMR). Subsequently, the discontinuation of TKI became imperative to evade adverse events and financial burden of TKI therapy. In several studies, beginning with the STIM1 (Lancet Oncol 2010;11:1029), nearly 40%-60% of patients with chronic CML who sustained long DMR could discontinue TKI and attain long-term treatment-free remission (TFR). Remarkably, initial dasatinib specifically induces the elevation in lymphocytes, including large granular lymphocytes (LGL), NK cells, and cytotoxic T cells, as well as the decline in regulatory T cells (Tregs) in the early phase of treatment, related to early clinical responses.(Int J Hematol 2014;99:41) Nevertheless, lymphocyte variations by dasatinib during sustained DMR before cessation is an area of growing interest. In the Japanese multicenter prospective D-STOP trial (NCT01627132), we discontinued dasatinib following 2-year consolidation to sustain DMR in chronic CML to assess the TFR rate. We here present the final results of the D-STOP trial, including the peripheral NK/T cell change during dasatinib consolidation associated with successful TFR. Methods: Chronic phase CML patients treated with TKIs who had undetectable BCR-ABL1 mRNA were enrolled. After confirmation of undetectable BCR-ABL1 mRNA (International Scale <0.01%) using a real-time quantitative polymerase chain reaction (RQ-PCR) in the central laboratory, the patient received additional dasatinib treatment for another 2 years as consolidation therapy. Patients who maintained DMR during the consolidation therapy proceeded to discontinue dasatinib. BCR-ABL1 mRNA was monitored every month in year 1 and every 3 months in year 2. Molecular relapse was defined as two successive positive RQ-PCRs for BCR-ABL1 within 1 month. The relapsed patients restarted dasatinib treatment. The primary endpoint was treatment-free survival after 12 months of discontinuation. Lymphocyte subsets were analyzed using flow cytometry during and after the consolidation therapy. Results: Sixty-five patients received consolidation therapy, and 54 discontinued dasatinib treatment after maintenance of DMR for 2 years. Estimated treatment-free remission (TFR) rates at 12 and 36 months were 63.0% [95% confidence interval (CI): 48.7-74.3] and 59.3% (95% CI: 45.0-71.0), respectively.(Figure 1) CD3-CD56+NK, CD16+CD56+NK, CD57+CD56+NK large granular lymphocytes (NK-LGL), CD8+CD4- cytotoxic T, and CD57+CD3+T-LGL cell numbers were relatively elevated throughout 24-month consolidation only in failed patients who molecularly relapsed within 12 months. In successful patients (TFR >12-months), these subsets elevated transiently after 12 months but returned to basal levels after 24-month consolidation. (Figure 2)There were no differences in CD8-CD4+ helper T and Treg. cell numbers during consolidation between 2 groups. Therefore, smaller changes in the NK/T sebsets, particularly NK subset throughout consolidation, exhibited higher TFR rates. TFR rates of those exhibiting elevation in CD3-CD56+NK > 376 cells/mL, CD16+CD56+NK > 241 cells/mL,CD57+CD56+NK-LGL > 242 cells/mL or CD8+CD4- cytotoxic T cells > 212 cells/mL during consolidation compared with others were 26.7% (8.3%-49.6%) versus 78.3% (55.4%-90.3%), HR 0.032 (0.0027-0.38; P = 0.0064) (Figure 3), 31.2% (11.4%-53.6%) versus 85.0% (60.4%-94.9%), HR 0.039 (0.0031-0.48; P = 0.011), 36.8% (16.5%-57.5%) versus 77.3% (53.7%-89.8%), HR 0.21 (0.065-0.69; P = 0.010), or 41.2% (18.6%-62.6%) versus 76.5% (48.8%-84.9%), HR 0.18 (0.019-1.77; P = 0.14), respectively. Conclusion Silent responses of the T/NK subsets to dasatinib throughout consolidation was significant for longer TFR. Elevated NK/T, particularly NK lymphocytes responsive to dasatinib, could be immature cells with little immunosurveillance; their decline subsequently replaced by altered lymphocyte population with less response to dasatinib during sustained DMR might be immunologically significant. Disclosures Kumagai: Pfizer: Honoraria; Otsuka pharmacology: Honoraria; Novartis: Honoraria; Bristol-Myers Squibb: Honoraria. Nakaseko:Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria. Nishiwaki:Novartis: Research Funding. Yoshida:Otsuka: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau. Matsue:Janssen Pharmaceutical K.K.: Honoraria; Novartis Pharma K.K: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Celgene: Honoraria; Ono Pharmaceutical: Honoraria. Morita:Bristol-Myers Squibb: Honoraria. Sakamoto,:Yakult Honsha Co. Ltd: Other: remuneration. Inokuchi:Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria; Celgene: Honoraria; Novartis: Honoraria.

Pioglitazone Did Not Affect PPAR-Γ, STAT5, HIF2α and CITED2 Gene Expression in Chronic Myeloid Leukemia Patients with Deep Molecular Response

Preliminary reports demonstrated that pioglitazone, an antidiabetic drug that is agonist of peroxisome proliferator-activated receptor gamma (PPAR-γ) was able to reduce expression of STAT5 and its downstream targets HIF2α and CITED2, which are key guardians of the quiescence and stemness of chronic myeloid leukemia (CML) leukemia stem cells (LSCs). Leaving quiescence would turn the LSCs more sensitive to imatinib (IM) and cause an erosion of the LSCs. This was demonstrated in vitro and in vivo in CML patients that achieved complete molecular response after pioglitazone use. This was the rational for the design of EDI-PIO trial (Pilot Study of Imatinib Discontinuation in Patients with Chronic Myeloid Leukemia with Deep Molecular Response - Evaluation of Pioglitazone in Treatment-Free Remission) (NCT02852486). In this trial, pioglitazone was given in association with IM, with the aim to pull out the LSCs from the quiescence and sensitizing them to IM effect, increasing treatment-free remission (TFR) rates after treatment interruption. Aims: to evaluate PPAR-γ, STAT5, HIF2α and CITED2 gene expression before and after pioglitazone use in CML patients with criteria for IM discontinuation Patients and methods: EDI-PIO is a prospective, phase II trial. Inclusion criteria: CML in chronic phase, treated with IM for at least 3 years, with stable deep molecular response (MR4.5) for at least 2 years. Patients received pioglitazone 30 mg/day, orally, for 3 months before IM discontinuation. BCR-ABL levels were measured by real-time quantitative PCR monthly in the first year after discontinuation, every two months in the second year, and then every 3 months during the subsequent follow-up. Imatinib was reinitiated at molecular relapse (loss of major molecular response or confirmed loss of MR4.0). Total RNA was extracted from peripheral blood leukocytes, pre and post pioglitazone, and at 3 and 6 months after IM discontinuation. After cDNA synthesis, an aliquot was used for gene expression analysis by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), using specific primers for PPAR-γ, STAT5, HIF2α and CITED2. The relative gene expression was calculated using the equation, 2-ΔΔCT. GAPDH was used as control gene. Statistical analysis was performed using ANOVA. Treatment-free remission (TFR) was calculated from IM discontinuation until molecular relapse, reintroduction of IM by any cause, progression to advanced phases or death to any cause. Results: The study is closed for enrollment. Between June 2016 and January 2019, 32 chronic phase CML patients were recruited, of which 30 patients were included in gene expression analysis. Median age was 55 years at trial initiation; 56.7% were men, 50% low risk Sokal and the median time of IM treatment was 117 months (41-191). The median follow-up time was 20 months. TFR was 60% at 24 months. Eleven patients relapsed and IM was reintroduced, but none presented hematologic relapse or progression to advanced phases. There was no significant difference in STAT5, PPAR-γ, HIF2α and CITED2 expression pre and post pioglitazone, at 3 and 6 months after IM discontinuation. No difference was found in the comparison of the relapsed vs. non-relapsed group. Conclusions: pioglitazone did not affect STAT5, PPAR-γ, HIF2α and CITED2 gene expression in this group of pts with deep molecular response. The ACTIM trial demonstrated a reduction in STAT5 expression in bone marrow cells 6 months after pioglitazone exposure, but pioglitazone was given to pts with MMR, without MR4.0. There was no difference in gene expression in the groups with or without molecular relapse. TFR rates remains similar to those reported in other discontinuation trials. Disclosures Delamain: Novartis: Honoraria. Pagnano:Sandoz: Consultancy; Pint Pharma: Consultancy; Abbvie: Consultancy.

Simultaneous Inhibition of BCR-ABL1 Tyrosine Kinase and PAK1/2 Serine/Threonine Kinases Exerts Synergistic Effect Against Chronic Myeloid Leukemia Cells

Tyrosine kinase inhibitors (TKIs) revolutionized the treatment of BCR-ABL1 tyrosine kinase - positive chronic myeloid leukemia in chronic phase (CML-CP). However, it is unlikely that TKIs will "cure" the disease in majority of patients because CML-CP cells are elusive targets even for the most advanced therapies employing second and third generation of TKIs. Therefore, new treatment modalities are necessary to improve therapeutic outcomes. We showed before that class I p21-activated serine/threonine kinases (PAKs) remained active in TKI-naive and TKI-treated CML-CP leukemia stem and early progenitor cells. The aim of the study was to test whether simultaneous inhibition of signaling pathways activated by BCR-ABL1 and PAK kinases may improve the treatment of CML. Special attention was focused on PAK1 and PAK2, which are expressed in hematopoietic cells and can play an important role in the promotion of CML cells proliferation and survival. PAK kinases were targeted by small molecule inhibitor IPA-3 (inhibitor of PAK1) and shRNA construct for PAK2, BCR-ABL1 kinase was inhibited by imatinib. The studies were carried out using (i) primary CML-CP stem/early progenitor cells and normal hematopoietic counterparts isolated from the bone marrow of newly diagnosed CML-CP patients and healthy donors, respectively, (ii) CML-blast phase cell lines (K562 and KCL-22), and (iii) BCR-ABL1-transformed 32Dcl3 cell line cells. We show here that inhibition of PAK1 or/and PAK2 kinases activity enhanced the effect of IM against CML cells without affecting normal counterparts. We observed that the combined use of IM with IPA-3 increased growth inhibition and apoptosis of leukemia cells. To evaluate the type of drugs interaction median effect analysis method was used. The studies revealed that the type and strength of drug interaction depend on drug concentration. Generally, combination of IM with IPA-3 at the 50% of the cell kill level (EC50) generated synergistic effect. Altogether, we postulate that BCR-ABL1 kinase inhibitor should be combined with PAK1/2 inhibitor to facilitate eradication of CML cells. Disclosures No relevant conflicts of interest to declare.