Phase Chronic Myeloid Leukemia Patients Research Articles

Heterogeneous BCR-ABL Signal Patterns Identified By Fluorescence in Situ Hybridization Can Predict Leukemic Clonal Evolution and Poor Prognosis in BCR-ABL-Positive Leukemia

BCR-ABL translocation occurs in approximately 90% of chronic myeloid leukemia (CML), 25% of acute lymphoblastic leukemia (ALL) and less than 5% of acute myeloid leukemia (AML) cases that produce BCR-ABL fusion proteins, which are responsible for proliferative signals and leukemogenesis. Although extensive use of tyrosine kinase inhibitors (TKIs) has resulted in high and durable response rates and prolonged survival times in BCR-ABL-positive CML and acute leukemia patients in recent years, relapse and drug resistance are still big challenges for clinicians. Monitoring the quantification of BCR-ABL fusion genes by quantitative PCR, minimal residual diseaseby flow cytometry and ABL kinase mutations by sequencing are effective means of predicting disease relapse and resistance; however, the prognostic impact of BCR-ABL signal modes by fluorescence in situ hybridization (FISH) in leukemia patients remains elusive. In the present study, we analyzed BCR-ABL signal modes using FISH in 243 CML-chronic phase (CML-CP), 17 CML-blast phase (CML-BP) and 52 ALL patients. A total of 52.9% (n=9) of the complex BCR-ABL signal modes (≥ two types of BCR-ABL signal modes) were observed in the CML-BP patients, followed by 30.8% (n=16) in the BCR-ABL-positive ALL patients, and only 2.1% (n=5) in the CML-CP patients. Conversely, typical single BCR-ABL signal modes (single 1R1G2F fusion signals) were observed in 29.4% (n=5) of the CML-BP, 53.8% (n=28) of the BCR-ABL-positive ALL and 73.7% (n=179) of the CML-CP patients. Patients with atypical single BCR-ABL signal modes (no 1R1G2F single fusion signal, such as 1R1G1F or 1R1G3F) were similar, and they accounted for 17.6% (n=3), 15.4% (n=8) and 24.3% (n=59) of the CML-BP, ALL and CML-CP patients, respectively. Additionally, multiple BCR-ABL signal modes presented very complexity and various. In total, twelve BCR-ABL signal modes were observed in this cohort: 1R1G2F, 1R1G1F, 2R1G1F, 1R2G1F, 2R2G1F, 1R2G2F, 1R1G3F, 1G3F, 2G3F, 1G4F, 1R1G4F and 1R4F. More importantly, the development of BCR-ABL signal patterns can indicate leukemic clonal evolution, disease progression and relapse by sequentially monitoring the BCR-ABL modes at different time points of disease progression using FISH. In this study, we observed five clonal evolution modes related to disease progression among our ALL patients. First, patients presented sensitive single clones at disease onset, which disappeared after treatment, whereas primary clones gradually progressed to drug-resistant clones until relapse during treatment. Second, patients presented sensitive single clones during disease onset that disappeared after treatment, whereas new single resistant clones were observed during relapse. Third, patients presented sensitive single clones during disease onset, whereas new and primary resistant subclones simultaneously occurred during relapse. Fourth, patients presented many different subclones during disease onset. Some preponderant subclones were sensitive and disappeared after treatment, whereas other minor subclones were resistant and gradually progressed to preponderant subclones until relapse. Fifth, patients simultaneously presented many different subclones during disease onset. Some minor subclones were sensitive and disappeared after treatment, whereas the preponderant subclones were resistant to TKIs or chemotherapy drugs. All of the BCR-ABL-positive ALL patients received a TKI combined with chemotherapy treatment. Furthermore, we analyzed the impact of complex BCR-ABL signal modes on survival time in the ALL patients. We observed that patients with complex BCR-ABL signal modes had very poor estimated overall survival (OS) times compared with patients with single BCR-ABL signal modes (typical and atypical single BCR-ABL signal modes) (5.0 vs. 15.0 month, P=0.006). Moreover, there were no OS differences between the BCR-ABL-positive ALL patients and the CML-BP patients (13.0 vs.10.0 month, P=0.984). Conclusion: Our data show that complex BCR-ABL signal modes usually occurred in patients with BCR-ABL-positive CML-BP, followed by ALL patients and rare in CML-CP patients. Complex BCR-ABL signal modes with various in forms predicted leukemic clonal evolution and poor prognoses. Monitoring BCR-ABL signal mode changes may be an effective means to provide prognostic guidance and treatment choices for patients with BCR-ABL-positive leukemia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Dasatinib-Induced Lymphocyte Elevation and Imbalance of T Cell Receptor V Beta Gene Expression in Chronic Phase Chronic Myelogenous Leukemia Patients

Introduction: Chronic myelogenous leukemia (CML) is a myeloproliferative neoplasms caused by a reciprocal translocation of chromosomes 9 and 22 [t(9;22)], producing the BCR-ABL fusion gene. Imatinib (Ima), the 1st generation tyrosine kinase inhibitor (TKI), has drastically improved the prognosis of CML compared to previous therapies. Imatinib and the 2nd-generation TKIs dasatinib (Dasa) and nilotinib (Nilo) are generally used for chronic phase CML (CP-CML) as induction chemotherapy. Lymphocyte proliferation and increased large granular lymphocytes (LGLs) are sometimes observed in patients treated with Dasa. These LGLs have been reported to be mainly natural killer (NK) cells. However, there is insufficient data about T-lymphocytes, including CD8 T cells, in CML patients treated with TKIs. We analyzed lymphocyte counts and percentages in CP-CML patients treated with Dasa and other TKIs. We also examined T cell receptor (TCR) V beta gene repertoire in some patients.Methods: The patients in this study were CP-CML patients diagnosed at our hospital from August 2006 to June 2016 and treated with TKIs. The main aims were to examine (1) lymphocyte rates and (2) cytotoxic T lymphocytes (CTLs) and TCR V beta gene repertoire. These analyses were conducted using flow cytometry. All statistical analyses were performed using GraphPad Prism 6 software.Results: Table 1 summarizes data comparing patients treated with Dasa and those treated with other TKIs. Six patients were treated with Dasa only. Seven and 2 patients, respectively, were treated with Ima or Nilo only. Eleven patients were treated with multiple TKIs including Dasa, and 2 patients were treated with multiple TKIs not including Dasa. The median age at first TKI administration was 60 years in patients treated with Dasa and 72 years in patients treated with other TKIs. No significant statistical difference was observed in age at first TKI administration. The median follow-up period of patients treated with TKIs without Dasa was significantly longer than that of patients treated with TKIs including Dasa. This was due to the difference in the timing of approval of Ima (1st generation TKI) and Dasa (2nd generation TKI). There was no significant difference in the best response between the Dasa and non-Dasa groups. There was also no significant difference in overall survival between the 2 groups (Log-rank, P = 0.7283). Pleural effusion was observed in 5 patients in the Dasa group and 1 in the non-Dasa group, without statistical significance. Figure 1 shows lymphocyte elevation in the Dasa group compared to the non-Dasa group with regard to both lymphocyte counts and percentages. Figure 2 shows the time courses of lymphocyte percentages. Counts from 1, 3, 6 and 12 months from the initiation of Dasa (A) and non-Dasa TKIs (B) are indicated. The lymphocyte rates in the non-Dasa group showed no significant change (Figure 2B). However, gradual lymphocyte elevation was clearly seen with the passage of time in Dasa group patients, with statistical significance (Figure 2A). The time courses of lymphocyte counts in 2 cases are shown in Figure 3. Lymphocyte elevations were clearly observed after changing to Dasa from Nilo (Figure 3A) and Ima (Figure 3B). We also analyzed the detailed lymphocyte fraction in some patients. Representative data of a patient treated with Dasa only are shown in Table 2 and Figure 4. NK cells were found in the peripheral blood, as have been seen in previous reports, and, interestingly, CTLs were also detected at all time points (Table 2). It is notable that about 20% of the peripheral blood consisted of CTLs 35 months after the first administration, which was the same percentage as that of NK cells. TCR V beta gene repertoire in the same patient is shown in Figure 4. Oligoclonal CD8 T cell expansion was observed using V beta 13.1, V beta 7.1, and V beta 2 expression. These results suggest that not only NK cells but also CTLs may play an important role in attacking CML blasts in patients treated with Dasa.Conclusions: In CP-CML patients treated with TKIs, lymphocyte elevation was obviously observed only in patients treated with Dasa. CTL analyses and TCR V beta gene analyses suggest that CTLs can be induced along with NK cells in patients treated with Dasa. These results show that Dasa can work in a different way than other TKIs. Clarifying the tumor antigens recognized by these CTLs may bring new insights and connect to new cellular therapies for refractory CML cases. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

E14a2 and e14a2+e13a2 BCR-ABL Transcripts Are Associated with Earlier Cytogenetic and Molecular Response Rates in CML-CP Treated with Imatinib

Background: The prognostic significance of BCR-ABL1 transcripts in chronic myeloid leukemia (CML) is still controversial. Methods: All consecutive CML patients in chronic phase treated with Glivec between January 2010 and December 2011 and generic imatinib between January 2015 and February 2017 from 8 Brazilian centers were analyzed. BCR-ABL1 transcripts were evaluated by multiplex qualitative RT-PCR. Only patients with BCR-ABL transcripts e13a2 and/or e14a2 were included in this analysis. All patients started imatinib less than six months from diagnosis. Study data were collected and managed using REDCap electronic data capture tools. Demographic data were collected at diagnosis: age, gender, blood cell counts, Sokal, Hasford and EUTOS score, cytogenetics and BCR-ABL transcript type. The definition of the responses followed the European Leukemia Net 2013 criteria. Event-free survival (EFS) was measured from starting of treatment until loss of complete hematologic remission, loss of complete cytogenetic response (CCyR), loss of major (MMR), progression to accelerated (AP) or blast phase (BC), or death from any cause at any time after initial therapy. Overall survival (OS) was measured from starting of imatinib until last follow-up or date of death from any cause at any time. Progression-free survival (PFS) was measured from date of imatinib starting to transformation to AP or BC or deaths while on therapy. SPSS 21.0 software (IBM Corp., Armonk, NY, USA) was used for chi-square, t-test, ANOVA, when adequate, considering p-value <0.05 as significant.Results191 CML-CP patients treated with imatinib (121 Glivec and 70 generic imatinib) were analyzed. Patient's characteristics are described in Table 1. The median age of patients was 46 (18-89) years. Sokal score: 52.1% low; 33.8% intermediate and 14.1% high risk. BCR-ABL transcripts: 109 (57%) presented e14a2 transcripts, 74 (38.8%) e13a2 and 8 (4.2%) both transcripts. There was no difference between the groups concerning age, gender, Hasford, EUTOS scores, whereas white blood cell counts at diagnosis was higher in patients with e13a2 transcripts (P=0.009). Higher rates of CCyR at 6 months were observed in e14a2 and both transcripts group, compared to e13a2 (58.4%, 60% and 30%, respectively - P=0.003); e14a2 group had higher rates of BCR-ABL transcripts <10% measured by real-time quantitative polymerase chain reaction (RQ-PCR) at 3 months (82.9%, 85.7% and 59.3%; P=0.007) and BCR-ABL <1% at 6 months (78.4% e14a2; 61.7% e13a2 and 43% both transcripts; P=0.039). The groups presented similar OS (89 %, 87% and 83%, P=0.49) and PFS (88%, 84% and 71%, P=0.08). EFS was inferior in the e13a2 group (45% vs. 65% e14a2 vs. 54% both transcripts; P=0.02).Conclusion: Patients with BCR-ABL transcripts e14a2 presented higher rates of CCyR at 6 months and higher rates of optimal molecular response at 3 and 6 months when compared to e13a2 transcripts, but there was no difference in overall and progression-free survival by 5 years. DisclosuresPagnano:Bristol-Meirs Squibb: Consultancy, Speakers Bureau; Roche: Speakers Bureau; Amgen: Consultancy; Novartis: Consultancy.

Comparison of Molecular Responses between e14a2 and e13a2 BCR-ABL1 Transcripts in Patients with Chronic Myeloid Leukemia in Chronic Phase Treated with Front-Line Second Generation Tyrosine Kinase Inhibitors: Taiwan CML Study

Background and Purpose: e14a2 (b3a2) and e13a2 (b2a2) are the two major BCR-ABL1 transcripts of CML. Our previous study (EHA 2016, abs P609) showed that patients with e13a2 transcripts received imatinib as front-line therapy had a significant higher treatment failure rate at 6 and 12 months, a lower cumulative incidence of MMR by 2 years, a higher frequency of lymphoid crisis, and a trend of inferior progression-free survival (PFS) as compared to those with e14a2 patients. The prognostic relevance of the two transcript subtypes following front-line second generation tyrosine kinase inhibitors (TKIs) therapy remains to be determined. We aimed to compare the molecular responses between e14a2 and e13a2 transcripts and their impact on outcomes of CML patients treated with front-line nilotinib or dasatinib.Patients and Methods: Between March 2008 and April 2017, there were 260 newly diagnosed CML patients in chronic phase with age older than 18 years, received 2nd generation TKIs as front-line therapy. BCR-ABL1 transcripts included 170 e14a2, 67 e13a2, 19 both e14a2 and e13a2, 2 e1a2, and one each of e14a3 and e13a3. The 237 patients carrying either e14a2 or e13a2 transcripts but not both were enrolled in this study, 143 patients received nilotinib and 94 patients received dasatinib. The e14a2 subtype accounted for 76.9% (n = 110) in the nilotinib-treated group and 63.8% (n = 60) in the dasatinib-treated group. The BCR-ABL1 levels expressed as International Scale (IS) were measured by TaqMan RQ-PCR assay with the lab-specific conversion factor every 3 months in a central laboratory.Results: Of the 237 patients with e14a2 or e13a2 transcripts, there was no difference in age, gender and Sokal score between the two transcript subtypes in each treatment group. In the nilotinib-treated group, patients with e14a2 achieving optimal molecular responses based on the ELN definition at 3, 6, and 12 months were significantly higher than those with e13a2 subtype, and had a significantly lower failure rate at 6 months (P < 0.001). In the dasatinib-treated patients, no difference was observed between the two transcript subtypes in the molecular responses at these early landmark time points. The median time to MMR or MR4.5 wasnotdifferent between the two transcript subtypes in both treatment groups. With a median follow-up of 27.5 months in the nilotinib-treated patients, 76% of e14a2 patients achieved MMR compared with 49% of e13a2 patients (P= 0.004) whereas no difference was observed for the frequency of MR4.5 between the two subtypes; 5 patients had disease progression and died. With a median follow-up of 20.8 months in the dasatinib-treated patients, there were no differences in MMR and MR4.5 rates, disease progression occurred in 4 patients and 3 of them died. In patients treated with nilotinib, the cumulative incidence of CCyR by 12 months and MMR by 24 months were significantly lower in e13a2 subtype compared with e14a2 subtype (P= 0.022, Fig. 1A and P= 0.037, Fig. 1B, respectively) but no difference in the cumulative incidence of MR4.5 between e14a2 and e13a2 by 36 months (P= 0.434). There were no difference in the cumulative incidence of CCyR by 12 months (P= 0.658, Fig. 1C), MMR by 24 months (P= 0.735, Fig. 1D) and MR4.5 by 36 months (P= 0.871) between the two transcript subtypes in the dasatinib-treated group. PFS and overall survival (OS) were not different between the two transcript subtypes in both nilotinib- and dasatinib-treated groups. Sokal risk group did not influence molecular responses, PFS or OS with respect to transcript subtypes in both treatment groups. The molecular responses and outcomes of e14a2 and e13a2 CML patients treated with front-line nilotinib and dasatinib are summarized in Table1.Conclusions: The results of Taiwan CML study demonstrated that patients with e13a2 transcripts had a significantly inferior early molecular responses compared with e14a2 when treated with front-line nilotinib. There was no difference in molecular responses between the two transcripts in patients treated with dasatinib. Sokal risk group had no impact on the treatment responses, irrespective of transcript subtypes. [Display omitted] DisclosuresShih:Novartis (Taiwan) Co., Ltd.: Research Funding.

ABCG2 but Not ABCB1 RNA Levels at Diagnosis and during Treatment Correlates with Response to Imatinib and 2G-Tkis Front-Line Therapy in Chronic Phase CML Patients

Introduction. Overexpression of multidrug resistance proteins ABCB1 and ABCG2 confers resistance to anticancer drugs, including tyrosine kinase inhibitors (TKIs). However, little is known about the significance of ABCB1 and ABCG2 expression during TKI treatment for chronic myeloid leukemia (CML) and possible correlation with response to therapy.Aims. To investigate the role of ABCB1 and ABCG2 expression during the first 12 months of treatment with imatinib and 2G-TKIs for chronic phase CML patients and correlation with molecular response.Methods. We analysed expression of ABCB1 and ABCG2 RNA at diagnosis and after 3, 6 and 12 months of TKI therapy in 20 patients with CML, divided in two groups according to response to treatment. Group A (n=10) comprised patients treated with imatinib that displayed optimal response at 3 months (defined as BCR/ABL <10%), 6 months (BCR/ABL < 1%) and at 12 months (BCR/ABL < 0.1%); group B (n=10) included patients treated with imatinib (n=5) or 2G-TKIs (nilotinib in 3 and dasatinib in 2 cases, respectively) who did not meet the criteria for optimal molecular response at all three timepoint or that switched to alternative treatment after at least the 3rd month. RNA levels were expressed using the 2-ΔΔCt method proposed by Livak and Schmittgen, using ABL as normalizing gene and the expression level of the K562 cell line as calibrator, in order to standardize the results.Results. ABCB1 expression at diagnosis was similar in group A (mean±SD = 0.21±0.22) and in group B (0.15±0.20; p=0.48); similarly, expression during treatment was similar in responding and non-responding patients at 3 (0.82±0.32 vs 0.83±0.41; p=0.66) 6 (0.85±0.22 vs 0.90±0.50; p=0.65) and 12 (1.05±0.24 vs 0.77±0.42; p=0.21) months. Conversely, ABCG2 expression at diagnosis was lower in group A (0.85±0.71) compared to group B (2.83±2.84; p=0.06). RNA levels increased in both groups during treatment, but were always lower in patients with optimal response compared to cases without response or treatment failure: at 3 months (5.12±3.31 vs 49.44±82.70; p=0.13), at 6 months (8.23±8.55 vs 12.71±10.04; p=0.32) and at 12 months (6.05±4.87 vs 13.00±9.93; p=0.05).Considering response to therapy, all patients in group A are still in imatinib with sustained optimal response; all patients in group B receiving imatinib switched to alternative therapy (nilotinib = 2, dasatinib = 1, allogeneic SCT for evolution to blast phase = 1) or died for non-CML causes while not in complete cytogenetic response (n=1), while 3/5 patients receiving 2G-TKI changed therapy (nilotinib to dasatinib = 1, dasatinib to ponatinib = 1, nilotinib to allogeneic SCT for aplasia = 1) and 2/5 patients continued the same drug achieving MMR beyond the 12th month.Discussion. With the limits of a small number of patients, our data suggest that high ABCG2 levels at diagnosis seems to be associated with non-optimal response to imatinib and 2G-TKIs, and an increase in ABCG2 during the first months of treatment is a hallmark of poor response. DisclosuresTiribelli:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Incyte: Consultancy, Honoraria.

“Duration of Deep Molecular Response” Has Most Impact on the Success of Cessation of Tyrosine Kinase Inhibitor Treatment in Chronic Myeloid Leukemia - Results from the EURO-SKI Trial

“Duration of Deep Molecular Response” Has Most Impact on the Success of Cessation of Tyrosine Kinase Inhibitor Treatment in Chronic Myeloid Leukemia - Results from the EURO-SKI Trial

Vitamin K3 (menadione) Induces Cytotoxicity in Chronic Myeloid Leukemia Stem Cells By Upregulating DYRK2 through the Inhibition of SIAH2 Ubiquitin Ligase

Leukemic stem cells (LSCs) generated by the transformation of normal hematopoietic stem cells (HSCs) by BCR-ABL are elusive targets that can initiate and sustain leukemia owing to their unique capacity to regenerate themselves during cell division (self-renewal) and differentiate into a bulk of leukemia cells. Although chronic myeloid leukemia (CML) can be successfully managed using tyrosine kinase inhibitors (TKIs), drug discontinuation trials have shown that 30-60% of patients, depending on the drug used for first- and second-line treatment, can remain in treatment-free remission. Thus, discontinuation is still considered experimental and there are not safety guidelines to stop treatment. A better understanding of self-renewal mechanisms in LSCs will support the development of LSC-specific drugs to induce treatment-free remission in chronic-phase patients by eradicating BCR-ABL-positive LSCs. We recently discovered in mouse models that deletion of the Klf4 gene encoding for the transcription factor KLF4 severely abrogated progression of BCR-ABL(p210)-induced CML by impairing survival and self-renewal of LSCs but not normal hematopoietic stem cells (HSCs). Mechanistically, KLF4 represses the Dyrk2 gene and thus loss-of-KLF4 resulted in elevated DYRK2 protein levels, which was associated with p53-mediated apoptosis and inhibition of self-renewal via depletion of c-Myc protein in LSCs and not in normal HSCs. Here, we report that this mechanism is active in bone marrow cells from chronic-phase CML patients and that stabilization of DYRK2 protein by inhibition of the ubiquitin E3 ligase with vitamin K3 (VK3) has the potential therapeutic approach. Treatment of CML cell lines and bone marrow cells from CML patients with VK3 induced dose-dependent cytotoxicity and apoptosis. This was correlated with increased DYRK2 expression, depletion of c-Myc, and activation of p53-mediated apoptosis. Most importantly, VK3 induced DYRK2 expression and abrogated capacity of purified CD34+ CML stem cells, but not normal CD34+ cells, to generate colonies in methylcellulose assays. Cytotoxicity of VK3 largely depended on the expression of DYRK2, the reduced form menadiol inhibited cell viability similarly to menadione (VK3), and the presence of p53 enhanced cytotoxicity by inducing apoptosis in addition to inhibition of self-renewal. VK3 augmented cytotoxicity of Imatinib in CML cell lines and inhibited cell survival in Imatinib-resistant CML cells. The ability to target CML LSCs was confirmed by in vivo administration of VK3 in BCR-ABL induced mouse model followed by transplantation of leukemic bone marrow in secondary recipient mice. Altogether, our results suggest that stabilization of DYRK2 by inhibition of SIAH2 can induce apoptosis in bulk leukemia cells and LSCs by activating p53 and depleting c-Myc. DisclosuresNo relevant conflicts of interest to declare.

Very Early Molecular Response at 1 Month Is a Predictor of the Outcome of Chronic Phase Chronic Myeloid Leukemia Patients Treated with Tyrosine Kinase Inhibitors

Background The early molecular response (EMR) by tyrosine kinase inhibitors (TKIs) is important to predict optimal response and survival in chronic phase chronic myeloid leukemia (CML-CP). However, EMR achievement can be affected by various clinical factors such as dose intensity, adverse events, blood level and risk scores. However, innate intrinsic sensitivity to TKIs of CML clones has not been considered.Aims To define the optimal molecular response at early time point of treatment initiation with minimal dose interruption, we identified the optimal BCR-ABL1 transcript level measured at 4 weeks after TKI initiation and assessed the clinical impact.Methods In this single center study, 258 newly diagnosed CML-CP patients were treated with various TKIs (130 imatinib and 128 second generation TKIs; 80 dasatinib, 33 nilotinib, 13 radotinib, and 2 bosutinib) and BCR-ABL1 transcript level were analyzed at 4 weeks (28 + 3 days) after frontline TKI initiation.All of patients had e13a2 or e14a2 transcript and a receiver operating characteristic (ROC) curve from BCR-ABL1 transcript level on Day+28 was calculated to predict EMR at 3 months and major molecular response (MMR) at specific timepoints. The results of qRT-PCR were analyzed with at least 4.5-log sensitivity in the central laboratory (Leukemia Research Institute, The Catholic University of Korea, Seoul, Korea).Results Median age was 43 years (18-81 years) and 127 patients were male (Table 1). At 4 weeks, the cut-off values of BCR-ABL1 transcript for achieving 12 month-MMR was 40.89% (very early molecular response; VEMR, P < 0.0001) by ROC curve analysis. One hundred thirty four patients achieving VEMR (<40.89% at 4 weeks) had significantly higher 5-year probabilities of Event-free survival (6.4% vs 13.4 %; P=0.021), cumulative incidence of complete cytogenetic response (89.5% vs 76.8 %; P=0.0001), and complete molecular response (25% vs 11 %; P=0.032) than no VEMR patients. Various predictive factors for achieving VEMR including age, sex, risk score, initial WBC, basophils, splenomegaly, baseline BCR-ABL1 transcript level were analyzed and the data will be presented.Conclusions The very early molecular response defined on the basis of the BCR-ABL1 transcript level measured at 4 weeks of TKI treatment is important for the prediction of long-term responses and survivals. As it also allows for the prediction of CMR, VEMR can be used as one of the important tools to select candidates for treatment free remission. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Impact of Body Mass Index on Outcome in Patients with Chronic Myeloid Leukemia in Chronic Phase Treated with Imatinib in First-Line

BackgroundImatinib mesylate (IM) 400 mg daily still represents the gold standard for chronic phase chronic myelogenous leukemia (CP-CML). Currently, the initiation dose of IM is adapted to the body weight only in childhood. There are some concerns regarding the adaptation of IM prescription in overweighted patients that may respond poorly to this tyrosine kinase inhibitor (TKI). However, this has not been extensively studied in the first-line setting. In addition, it is known that IM is able to promote weight gain and further, obesity has been identified as a possible risk factor in several solid cancers and leukemias.AimsIn a retrospective observational monocentric study, we evaluated the impact of the weight and the body mass index (BMI) measured at diagnosis on CP CML patients outcomes, on IM first-line, in terms of survival, responses and kinetics.MethodsWe retrospectively enrolled in this study, newly diagnosed CP CML patients, treated with IM in first-line between 2000 and 2017. Overall survival (OS), progression-free survival (PFS), failure-free survival (FFS, defined as progression to advanced phases death, loss of CHR, CCyR, or MMR, discontinuation of IM for toxicity, primary cytogenetic resistance) were analysed since IM initiation in intention-to-treat. Responses (hematologic, cytogenetic, molecular) were analysed according to standard methods. Molecular results were standardised according to the ELN/Eutos programs since 2003, and were expressed as BCR-ABL (IS) in %. Clinical data were extracted from medical files, weight and height measured for all patients at diagnosis, and later nearly all patients being weighted at each time point. The BMI was categorized into 4 stages according to the OMS classification: underweight (BMI < 18.5), normal weight (18.5 ≤ BMI ≤ 24.9), overweight (25 ≤ BMI ≤ 29.9), and obesity (BMI ≥ 30). Serum Albumin (g/l) was regularly assessed, and the nutritional risk index (NRI) was also calculated.ResultsWe enrolled 107 patients in this study, 62 (58%) males, with a median age at diagnosis of 55.36 (17-83) years. Sokal risk and Hasford scores were low for 32% and 39%, Intermediate for 48% and 52%, and high for 20% and 9% respectively, (2 patients unknown). Eutos score was low for 95% of patients. The median BMI at diagnosis was 24.61 (17-44.3) Kg/m2. The median weight at diagnosis was 69 (48-128) Kg. We analysed the patients in 2 groups: A: low + normal BMI (<24.9) and B: overweighted + obese (>25). The median follow-up was 97.3 (3-17) months. Age at diagnosis, Sex ratio, duration on IM, Sokal, Euro, Eutos risk scores, presence of ACA at diagnosis, menopauses women, IM duration were not different between groups A & B. There was significantly more diabetic patients in group B (3 vs 11, p=0.018). The rates of CCyR at 6 months, MMR at 12 months were not different between the 2 groups (38% in group A vs 48.9% in group B, p=0.388; and 48.94% in group A vs 66.67% in group B, p=0.133). The rate of MR4 (56 [44.09-71.91]% group A vs 67.7 [52.60-80.74]% group B, p=0.817), MR4.5 (48 [33.95-62.05]% group A vs 48.9 [34.05-63.73]% group B, p=0.97) at 5 years were not significantly different.Five patients died, all in group B, from a lymphoid blast crisis in 1 patient, secondary tumours in 3 (lung, colon, prostate cancer + cholangiocarcinoma) and terminal renal insufficiency in 1. The OS rate at last follow-up was significantly better in low/normal BMI 100% months (A) versus 84.7 [71.5-100] % months (B, p=0.022) (Fig 1A), as well as the PFS 100% months versus 86.5 [75.-99.7] % months (p=0.027) (Fig 1B).The FFS rate was not different (p=0.794). Interestingly, 25 (23.4%) patients fulfilling the STIM criteria for Treatment-Free Remission, were 17 in arm A and 8 in arm B. Of note, the BMI regularly increases over time with an increase of 0.045 [0.006-0.084] Kg/m2 per month, and patients may become overweighted over time (42% at diagnosis, 45.8% at latest follow-up).ConclusionThis retrospective analysis on an homogeneous population demonstrates that being overweighted at diagnosis of CP CML, and undergoing IM treatment first-line, selectively impacts on survival. However, the rates of responses do not seem to be altered, and the majority of such patients died from CML unrelated causes, mostly from other cancers. The mechanisms responsible for that are probably complex, multifactorial, and confounding factors may interfere, and finally remain to be deciphered. [Display omitted] DisclosuresLabussiere-Wallet:Novartis: Speakers Bureau. Nicolini:Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Incyte Biosciences: Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; ARIAD: Honoraria, Speakers Bureau.

The Impact of Early Molecular Response at 3 Months on Long-Term Survival Was Enhanced in the CP CML Patients Treated with Second-Generation Tyrosine Kinase Inhibitors

Background: Recent studies have demonstrated that early molecular milestones were able to identify high-risk chronic myeloid leukemia patients treated with frontline imatinib (IM) and second-generation tyrosine kinase inhibitors (2G-TKIs). 2G-TKI treated patients achieved faster and deeper molecular responses compared with IM treated patients. Because CML patients currently have multiple treatment options, the impact of early molecular milestones on long-term outcomes needs to be determined in each agent treated patients. The aim of this study is to evaluate the impact of 3-month early molecular response (EMR) on long-term outcomes of CML patients treated with IM and different 2G-TKIs, as additional information to guide clinical decisions on switching to a different TKI.Methods: 646 new CP CML patients who were treated with frontline IM (n = 362) or 2G TKIs (n = 284) were analyzed. Molecular responses were monitored using qRT-PCR assay with 3 month intervals, and then 6 month intervals after achieving major molecular response (MMR). Main study objectives were to evaluate the long-term outcomes, including failure-free survival (FFS), progression-free survival (PFS), and overall survival, according to 3-month EMR. FFS was measured from the date of treatment start until death, progression to AP or BP, or ELN failure on treatment, whichever came first. PFS and OS collected survivals on patients who were treated with other TKIs after frontline therapy discontinuation.Results: A total of 646 patients were treated with IM (n = 362), dasatinib (DAS; n = 117), nilotinib (NIL; n = 85), and radotinib (RAD; n = 82). The median age was 43 years (range, 11-87). The percentages of patients with low, intermediate, and high Sokal risk scores were 37%, 39% and 22%, respectively, with 2% unknown risk. With a median follow-up of 75.4 (IM; range, 4.4-195.9) and 34.1 (2G-TKIs; range, 5.2-138.4) months, 383 (59.3%; 168 IM and 215 2G-TKIs) patients continue on the frontline therapy and 263 (40.7%) patients were permanently discontinued or changed to other TKIs due to intolerance (72 IM, 26 DAS, 2 NIL, 21 RAD), ELN failure (36 IM, 3 DAS, 2 RAD), progression (3 IM, 1 DAS, 2 NIL, 1 RAD), death (1 IM, 1 DAS, 1 NIL), and others (10 warning, 9 pregnancy, 59 treatment-free remission study, and 13 follow-up loss). In 362 patients treated with IM, patients achieving BCR-ABL1 ≤ 10% at 3 months (n = 270, 74.6%) had a better outcomes in terms of 8-y FFS (91.7% vs 64.4%, P<0.001), 8-y PFS (95.5% vs 84.2, P<0.001), and 8-y OS (97.0% vs 83.2%, P <0.001) compared with those of the patients with BCR-ABL1 >10%. In 284 patients treated with 2G-TKIs, achievement of 3-month EMR (n = 252, 88.7%) was associated with a higher 6-y FFS (95.5% vs 82.1%, P = 0.001), 6-y PFS (97.2% vs 36.0, P<0.001), and 6-y OS (100% vs 66.2%, P <0.001). The prognostic impact of 3-month EMR was observed in both IM and 2G-TKIs treated groups. However, in IM treated group, 3-month EMR failure showed relative risk (RR) of 4.96 (P <0.001) for PFS and 4.54 (P = 0.001) for OS, respectively, whereas in 2G-TKIs treated group, 3-month EMR failure showed RR of 10.71 (P <0.001) for PFS and 26.42 (P = 0.005) for OS, respectively.Conclusions: Our data showed the 3-month EMR provided long-term prognostic information in patients treated with frontline IM and 2G-TKI. However, the prognostic impact of 3-month EMR failure was enhanced in the patients treated with 2-G TKIs. However, further investigations in a larger patient population with longer follow-up are needed. DisclosuresKim:Takeda: 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; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Il-Yang: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Long-Term Outcome in Children and Adolescents with Chronic Myeloid Leukemia in Chronic Phase Treated with High-Dose Imatinib— the Italian Experience

The availability of imatinib (IM) and thereafter of second-generation tyrosine kinase inhibitors (TKI2) has extended therapeutic options for childhood chronic myeloid leukemia (CML) with a favorable influence on disease outcome and overall prognosis. The aim of this report was to evaluate the response to therapy and long-term outcome of children and adolescents with CML in chronic phase (CP) treated with high-dose imatinib (IM). CP-CML patients (pts) aged Between March 2001 and June 2015, 58 pts with a diagnosis of CP-CML (males/females 1.6; median age 114/12 years) were treated with IM at dose of 340 mg/m2/day in 14 Italian pediatric centers. Sixteen (27.5%) had an HLA-matched sibling. Thirty-eight pts (65.5%) received IM for a period >24 months. After 3 months of IM treatment, 83% and 56% of ptsshowed BCR-ABL1 IS ≤10% and ≤1%, respectively. BCR-ABL1 transcript levels ≤0.1% were found in 61% and 69% of pts at 12 and 18 months. At 24 months, 66% and 54% of pts showed BCR-ABL1 levels ≤0.1% and ≤0.01%, respectively. Overall, 91%, 85%, 56% and 39% of pts achieved CCyR, MR3, MR4, MR4.5 and MR5 after a median time of 6.1, 12.5, 16.4 and 19 months, respectively. BCR-ABL1 IS ≤10% and ≤1% after 3 months of treatment did not impact on the overall CCyR, MR3, MR4-MR4.5 and MR5. Treatment with IM was stopped in 29/58 pts (50%) after a median time of 16.7 months (range: 1.4-157) because of failure (58.5%), toxicity or intolerance (14%) and allogeneic hematopoietic stem cell transplant (HSCT) (27.5%). Eighteen pts (62%) received one or more TKI2, 3 pts (10%) α-interferon (IFN), 1 pt conventional chemotherapy (3.5%) and 16 pts (55%) underwent a HSCT (Table 1). Transplants were performed after a median of 12 months: 10 from an identical sibling, 5 from a matched unrelated donor and 1 from an umbilical cord blood. Three early transplanted pts had disease recurrence after 24, 36 and 83 months, respectively; they were rescued with IM ± conventional chemotherapy ± 2nd HSCT (2 pts) or dasatinib (1 pt). Among 29 pts who continued IM, 15 (52%) (1 in MR3, 6 in MR4-MR4.5 and 8 in MR5) started intermittent treatment (IM for 3 weeks a month); thereafter, 5 pts resumed continuous IM and 1 patient shifted to dasatinib because of response loss; 3 adolescents with long-lasting BCR-ABL1 IS In conclusion, this larger cohort of pts with a longer follow-up confirmed our previous results on the high response rates in children and adolescents treated with high-dose IM and the prognostic impact of the transcript levels after 3 months of treatment on the disease outcome. Unfortunately, failures occurred late during treatment with IM. Over time, the efficacy of IM and the availability of TKI2 also in childhood CML has limited the use of HSCT leading to avoid transplants even in children with an identical sibling. Both transplant and TKI did not prevent pregnancies. Although the stop of treatment is not recommended outside of clinical trials, IM or TKI2 can be safely discontinued in some children and adolescents with a durable deep molecular response. Disclosures Saglio: Ariad: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Foa: Roche: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Sandoz: Consultancy, Speakers Bureau; Abbvie: Consultancy, Speakers Bureau.

The Bone Marrow Niche Uses Mir-300 As a Biological Rheostat to Selectively Control Stem Cell-Driven Malignant Hematopoiesis and Innate Anti-Cancer Immunity

Inhibition of protein phosphatase 2A (PP2A) tumor suppressor is essential for chronic myelogenous leukemia (CML) stem cell (LSC) maintenance, disease evolution and innate anti-cancer immunity. In CML, persistence of ABL tyrosine kinase inhibitor (TKI)-resistant quiescent LSCs, disease development and inhibition of natural killer (NK) cell growth and anti-cancer activity depend on cell-autonomous and bone marrow (BM)-generated signals.Here we show that expression profiling of CD34+ BM progenitors from healthy individuals and CML patients in chronic (CP) and blastic (BC) phase identified a discrete number of microRNA (miRs) altered in CML and/or during blastic transformation. Among the miRs downregulated in CML, we focused on miR-300 because it directly inhibited expression of multiple components of the PP2A inhibitory pathway (i.e. Jak2, hnRNPA1, SET) and of other factors essential for LSC maintenance and disease progression (e.g. CCND1/2, b-catenin, Myc, Twist-1), when ectopically expressed in CML but not in normal CD34+ cells.MiR-300 acted as a potent tumor suppressor in leukemic CD34+ progenitors by inducing cell cycle exit and promoting spontaneous and (TKI)-induced apoptosis, whereas no effects were exerted in normal miR-300-transduced CD34+ cells. In CD34+ CML-BC progenitors miR-300 downregulation required the BCR-ABL1 kinase-dependent inhibition of C/EBPb, which was capable of binding and transcriptionally activating miR-300 promoter.Conversely, low O2 levels and mesenchymal stromal cell (MSC)-derived exosomes abnormally increased miR-300 expression in a TKI-insensitive manner to induce and/or preserve LSC quiescence and suppress NK cell growth. Indeed, lentiviral-mediated miR-300 inhibition in MSCs significantly prevented the MSC growth inhibitory activity on CD34+ CML-BC and NK cells. Accordingly, miR-300 levels were found higher in CD56+CD3- NK cells from CML patients at diagnosis compared to healthy individuals.Induction/maintenance of high miR-300 levels in LSCs (CD34+ CFSEMAX and CD34+CD38- cells) required the hypoxia-induced inhibition of BCR-ABL1 activity and induction of C/EBPb expression/activity. However, MSC-derived exosomal miR-300 also contributed to increase miR-300 expression in LSCs.LSCs escaped from the miR-300 pro-apoptotic activity through an autocrine/paracrine mechanism that required the release by CD34+ CML stem/progenitors of TGFb1 that, in turn, induced the expression of TUG1, a lncRNA acting as a miR-300 sponge. In fact, shRNA-mediated TUG1 suppression or Ab-dependent TGFb1 inhibition decreased the quiescent (CFSEMAX) CD34+ LSC number. By contrast, miR-300 inhibition did not alter LSC survival and self-renewal, further supporting a role for TUG1 as a miR-300 sponge. Accordingly, TUG1 was markedly induced in CFSEMAX but not dividing CD34+ CML cells. In fact, low ectopic miR-300 expression induced growth arrest (decreased LTC-ICs and CD34+CD38- CFC/replating activities) without affecting the number of CFSEMAX LSCs.By contrast, high doses of miR-300 (CPG-miR-300; 500nM) but not scramble oligonucleotides impaired LSC survival (LTC-IC) and self-renewal (CFC/replating), induced a marked killing of quiescent LSCs (decreased CD34+ CFSEMAX cells) and dividing CD34+ CML progenitors, and impaired CML-BC engraftment in NRG-SGM3 mice. Notably, the association of high CPG-miR-300 with TUG1 silencing (CPG-anti-TUG1) further reduced CD34+ CFSEMAX LSC number. Notably, TUG1 was highly expressed in normal CD34+ stem/progenitor cells; in fact, neither low nor high miR-300 levels altered their survival and/or self-renewal.Altogether our results indicate that while loss of miR-300 expression is essential for survival/proliferation of leukemic progenitors and, therefore, for development of a full-blown leukemia, increased miR-300 expression is necessary for reduced NK cell number/activity and maintenance of the LSC reservoir. In this scenario, induction of TUG1 may occur to preserve LSC survival after migration into the BM endosteal niche where quiescence is induced by MSCs and low O2 levels through marked induction of intracellular (endogenous and exosomal) miR-300 levels. Thus, disrupting the miR-300/TUG1 balance may represent a potential therapeutic approach for treatment/eradication of LSC-derived leukemias and restoration of innate immunity.This work is supported in part by NHI-NCI R01CA163800-01 DisclosuresGambacorti-Passerini:Pfizer: Consultancy, Honoraria, Research Funding; BMS: Consultancy. Deininger:ARIAD: Consultancy; Incyte: Consultancy; BMS: Consultancy, Research Funding; Celgene: Research Funding; Pfizer: Consultancy; Novartis: Consultancy, Research Funding; Ariad Pharmaceuticals, Bristol Myers Squibb, CTI BioPharma Corp, Gilead, Incyte, Novartis, Pfizer, Celgene, Blue Print, Galena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding. Milojkovic:ARIAD: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Incyte: Honoraria, Speakers Bureau. Apperley:Pfizer: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Therakos: Honoraria; Incyte: Honoraria; Sun Pharma: Honoraria; Novartis: Consultancy, Honoraria, Other: travel, accommodations, expenses , Research Funding, Speakers Bureau; Bristol Myers Squibb: Consultancy, Honoraria, Research Funding.

Imatinib (IM) in Combination with Hydroxyurea Showed Significantly Lower Major Molecular Response Rates at 6 Months but Similar MMR at 18 Months in Patients with CML1st CP Compared to IM Alone. Final Results of the CML2004 Study. NCT 02480608

Introduction: Imatinib (IM) monotherapy remains an important option to treat newly diagnosed patients with chronic myeloid leukemia (CML) in chronic phase (CP). Hydroxyurea (HU) is effective in controlling elevated white blood cell counts and was widely used to treat CML prior to the era of tyrosine kinase inhibitors (TKIs). After combinations of IM and HU showed additive effects on Ph+ cells in vitro the East German Study Group conducted a study in patients with CML first chronic phase.Methodes: Starting in 2002, 20 adult patients with newly diagnosed CP-CML were included in the phase I study to identify the dose of HU in combination with standard dose IM (400mg daily) that would result in mild myelosuppression (white blood cell count 3,000-4,000/mL). Starting dose of HU was 500mg daily increasing by 500mg every 3 weeks to a maximum of 3,000 mg daily. In line with the protocol, 500mg HU was chosen as the starting dose for the randomized phase II study which tested the combination vs. standard dose IM, with the rate of major molecular response (MMR) at 18 months as the primary endpoint.Additional 93 patients were enrolled in the phase II of the study, 5 of whom were excluded, leaving 88 patients to be randomized 2:1 to the IM/HU (n=59) and IM (n=29) arm, respectively. Three patients (2 IM/HU, 1IM) were lost to follow-up. As prospectively designed, IM/HU patients (n=77) from the phase I and II were analyzed in combination with patients from the CML study IV (n=49) selected by propensity score matching. The median age of the 154 patients was 55 years (range 18 - 82). The EUTOS long-term survival prognostic score was available for 141 patients and was high in 8 (5.7%), intermediate in 35 (24.8%) and low in 98 (69.5%), with no significant differences between treatment groups. The median HU dose was 500mg (range 152-3000); the median IM dose was 400 mg (range 145-617mg) in both arms.Results: The 5-year overall survival (OS) was 90.4% and 84,9% in the IM/HU and IM arm respectively (not significant). Similarly, progression-free survival (PFS) at 5 years amounted to 86.7% in the IM/HU and 84.9% in the IM arm.With IM/HU, the probabilities of complete cytogenetic response (CCR) at 6, 12, and 18 months were 54.3, 84.0, and 93.7%, respectively. In the IM arm, the corresponding numbers were 70.4, 84.9, and 83.3% (p=not significant).Molecular Response Rates (MMR) in the two arms were evaluated at 6 months with 21.6 (IM/HU) vs. 41.1% (IM) (p=0.0383) and at 12 months with 41.9 (IM/HU) vs. 58.9% (IM; not significant). The primary endpoint of the study (MMR rate at 18 months) did not significantly differ between the IM/HU (65.8%) and the IM (66.0%) arms.No significant differences were detected in time to event analyses of OS and PFS or in group comparisons between the probabilities of CCR and MMR.The gross numbers of adverse events in general or of adverse events of grade 4 were not different between the two arms. However, cumulative incidences showed an earlier occurrence in the IM/HU than in the IM arm (p= 0.0343, Gray test)Conclusion: Compared to Imatinib only, the combination of Imatinib and HU resulted in a lower MMR rate at 6 months but a similar MMR rate at 18 months. Furthermore, IM/HU was associated with more early adverse events. There was no indication of a beneficial effect in the treatment of CML patients in 1st chronic phase using the combination of IM with HU. DisclosuresLange:Novartis: Honoraria, Research Funding. Al-Ali:Gilead: Consultancy, Honoraria; Alexion: Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Pfirrmann:Novartis: Honoraria; BMS: Honoraria. Hehlmann:Novartis: Research Funding; BMS: Consultancy. Deininger:Incyte: Consultancy; BMS: Consultancy, Research Funding; Ariad Pharmaceuticals, Bristol Myers Squibb, CTI BioPharma Corp, Gilead, Incyte, Novartis, Pfizer, Celgene, Blue Print, Galena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; ARIAD: Consultancy; Gilead: Research Funding; Celgene: Research Funding; Novartis: Consultancy, Research Funding.

First-Line Dasatinib in BCR-ABL1+ Chronic Myeloid Leukemia in Early Chronic Phase: A Gimema Prospective Multicentric Observational Study

Background: Dasatinib (DAS) is a 2nd generation (2nd gen) tyrosine kinase inhibitor (TKI) approved for 1st line treatment of chronic myeloid leukemia (CML) patients based on DASISION study results. Most data on 2nd generation TKIs are from company-sponsored studies, but the patient selection and the high rate of patient switching to other treatments may jeopardize data interpretation. Interestingly, despite significant differences in terms of cytogenetic and molecular response, 2nd gen TKIs did not produce relevant advantages in terms of overall survival (OS). A post-marketing surveillance in large independent trials is extremely important to confirm efficacy and the safety data.Aims: To describe, in the clinical practice, the response to DAS and the events leading to permanent treatment discontinuation.Methods: An italian multicentric prospective observational study of CML patients treated frontline with DAS has been conducted by the GIMEMA CML Working Party. DAS was given at the discretion of investigators, according to prescribing information, without any exclusion criteria, apart from age (<18 years old) and prior treatment with TKIs or interferon. Molecular response was assessed in standardized molecular laboratories (Labnet network): major molecular response (MR3.0 or MMR), BCR-ABL1 < 0.1% IS; MR4.0, BCR-ABL1 < 0.01% IS or undetectable disease with ≥10,000 ABL1 copies; MR4.5, BCR-ABL1 < 0.0032% IS or undetectable disease with ≥32,000 ABL1 copies. Progression was defined as transformation to advanced (accelerated or blast) phase, according to ELN criteria.Results: 147 CML patients in early chronic phase have been enrolled in 27 italian hematologic centers. The median age was 57 years (65 years or older: 34% of patients). High risk patients according to Sokal, Euro and EUTOS score were 28%, 11%, and 13%, respectively. Median follow-up: 12 months. At 3 months, 88% of patients had BCR-ABL1 transcript levels < 10% IS (at the same timeopoint, < 1% in 66% of patients). At 6 months, 81% of patients had BCR-ABL transcript levels < 1%. The MMR rates at 6 and 12 months were 53% and 72%, respectively. The proportion of patients achieving a MR4 or a MR4.5 at least once was 35% and 16%, respectively. At the last contact, the patients still on treatment with DAS were 88%, while 12% permanently interrupted the study drug. The reasons for study discontinuation were: 1% progression in advanced phase, 2% failure, 1% unrelated death, 1% lost to follow-up, 7% adverse event (1% hematologic toxicity, 4% pleural effusion, 1% tachyarrythmia, 1% other non-hematologic adverse event). No pulmonary hypertension or cardiovascular ischemic adverse events were reported. The probability of OS and progression-free survival at 1 year was 98% and 99%, respectively.Conclusion: The median age of patients receiving DAS in our observational trial was close to the median age reported in population-based CML registries, with one third of patients at least 65 years old; consequently, we may argue that, outside of clinical trials, the decision to treat or not to treat patients with DAS is probably not influenced by age. The proportion of high risk patients, as expected, was higher than reported by population-based CML registries. High molecular response rates were observed and the most frequent reason of permanent treatment discontinuation was the occurrence of adverse events (in particular, pleural effusion). DisclosuresCastagnetti:Pfizer: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Gugliotta:Incyte: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Breccia:Novartis: Consultancy; Pfizer: Consultancy; Incyte: Consultancy; Bristol Myers Squibb: Consultancy. Iurlo:Pfizer: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Stagno:Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Incyte: Consultancy, Honoraria. Abruzzese:Novartis: Consultancy; Incyte: Consultancy; BMS: Consultancy; Pfizer: Consultancy. Crugnola:Celgene: Honoraria; BMS: Honoraria; Novartis: Honoraria. Specchia:Pfizer: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Tiribelli:Bristol Myers Squibb: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Bonifacio:Incyte: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees. Galieni:Abbvie: Other: Advisory Board; Takeda: Other: Advisory Board. Soverini:Incyte Biosciences: Consultancy; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy. Foà:Roche: Consultancy, Speakers Bureau; janssen: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Sandoz: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; AbbVie: Consultancy, Speakers Bureau. Martinelli:ARIAD/INCYTE: Consultancy; ROCHE: Consultancy; JOHNSON & JOHNSON: Consultancy; CELGENE: Consultancy; PFIZER: Consultancy; AMGEN: Consultancy. Saglio:Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Ariad: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Incyte: Consultancy, Honoraria. Pane:Novartis: Honoraria, Speakers Bureau. Baccarani:Novartis: Consultancy, Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Incyte ARIAD: Consultancy, Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau. Rosti:Pfizer: Research Funding, Speakers Bureau; Incyte: Research Funding, Speakers Bureau; Bristol Myers Squibb: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau.

Response to Ponatinib Is Not Related to the Drug Plasma Levels in Chronic Phase CML

BackgroundPonatinib is currently licensed for the treatment of patients resistant or intolerant to at least 2 tyrosine kinase inhibitors (TKI) or in patients harboring a T315I ABL mutation, in chronic, accelerated and blast phase chronic myelogenous leukemia (CML) or Ph+ ALL. Surprisingly, very little is known in terms of pharmacokinetics of this drug in CML patients and their impact on outcomes.AimsWe evaluated the impact of Ponatinib plasma concentrations determinations in chronic phase CML (CP CML) patients during treatment, and outcomes.MethodsWe enrolled after informed consent patients in CP CML in 3 cooperative centers, receiving Ponatinib at any dose and measured the plasma concentration of this compound at any time during treatment by UPLC with mass tandem detection. Data were expressed as µg/L and 24H concentrations (C24h, expressed in mg/kg/d). The C24h were estimated according to the average half-life calculated for the entire cohort. After informed consent, basic clinical data, hematologic, cytogenetic and molecular results were captured retrospectively as well as outcome. qRT-PCR results were expressed as BCR-ABL ratios in %, standardised according to the international scale, with at least 32,000 copies of ABL as control.ResultsThirty-seven CP CML patients have been involved in this study, and 50 blood samples were studied. There were 20 males and 17 females of a median age at diagnosis of 44.6 (7.6-82.5) and of 55.7 (25-86) years at ponatinib plasma measurement. Sokal score was low for 18%, intermediate for 12% and high for 70% of patients (9 patients unknown). One patient had a variant Ph, one a cryptic Ph and all the others a classical form of the Ph chromosome. All patients had a “major” BCR transcript except 1 (e19a2).The median BCR-ABL at diagnosis was 66.5 (33-110) % (IS). The median follow-up since diagnosis was 8.65 (2.5-16.7) years, 8.57 (2.48-16.7) years since first TKI initiation, and 4.32 (0.77-6.1) years since Ponatinib initiation. Two patients received Ponatinib as first-line (EPIC trial). Three patients received Ponatinib as second line, 12 as third line and 20 as fourth line therapy, all for resistance to previous lines of TKI. Fourteen patients (38%) were ABL mutated (10 T315I, 1 V379I, 1 Q252H, 1 L298V, 1 E255K). The median dose at Ponatinib initiation was 45 (30-45) mg daily and at Ponatinib plasma measurement at 30 (5-45) mg daily which represented a median C24h of 0.41 (0.05-0.94) mg/kg/day. The blood test was performed after a median of 12.25 (0.5-92.5) hours after last oral dose. Median plasma concentrations of Ponatinib were 30.75 (0-89.1) mg/l. When considering a target therapeutic concentration of 20 mg/l (= 40 nM, Cortes J. et al. NEJM 2013), 39 patients were above this level and 11 below. At plasma determination, 9 patients were in CHR, 1 in minimal CyR, 1 in minor CyR, 7 in partial CyR, 14 were in CCyR, 13 in MMR, 3 in MR4, and 2 in MR5. The median BCR-ABL at that time was 0.15 (0-47.9)% (IS). There was no significant correlation between the response observed and the plasma concentrations measured in patients and their molecular response (Figure 1.), Spearman correlation test: r= 0.134, p=0.34.There was no correlation neither with the onset of vascular events but the blood sampling was not necessarily performed when these events occurred (n=8). However and indeed, this population might be highly selected, as the majority of the patients without severe side effects and responding to treatment are maintained on Ponatinib. At latest follow-up, 4 patients died in this heavily pre-treated population of patients, 2 from post-allogeneic infectious complications, 1 form a stroke, 1 from a metastatic spinocellular carcinoma.ConclusionThe determination of TKI plasma levels is surely useful in screening patients for uncompliance (here one patient), however their relationship to the efficacy or toxicity of such a compound remains to be further assessed in a more homogeneous and larger population of patients. [Display omitted] DisclosuresNicolini:Incyte Biosciences: Honoraria, Speakers Bureau; ARIAD: Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau. Rea:Pfizer: Honoraria; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Incyte: Honoraria.

Long-Term Survival of Patients with Chronic Phase Chronic Myeloid Leukemia Who Received Autologous Stem Cell Transplantation and Further Exposed to Tyrosine Kinase Inhibitors. a Study on Behalf of the French Society of Blood and Marrow Transplantation and the French Group of CML

Background: Prior to the introduction of tyrosine kinase inhibitors (TKI), the median survival of chronic phase chronic myeloid leukemia (CP-CML) patients was approximately 60 months [Roy L et al, Blood 2006] and the standard treatment with interferon-alpha resulted in complete cytogenetic responses in about 30% of the patients [Bonifazi F. et al, Blood 2001]. Peripheral blood autologous stem cell transplantation (Auto-SCT) was first attempted for patients in transformation in order to restore a second CP and was introduced secondarily in CP to try to prolong the response. The principal rationale for autografting in CP resides on the reduction of the tumor burden and the number of leukemic cells at risk of developing blastic transformation, and the possibility of eradicating already mutated cells. In addition, it takes advantage as well of the endogenous mobilization in the circulation of Ph- HSCs. Nevertheless, Auto-SCT alone was not able to maintain a long-term cytogenetic and/or molecular remission. Nowadays, TKIs represent the state-of-the-art therapy for CML and the concept of Auto-SCT has only little interest while long-term follow-up and outcome in this setting are currently unknown.Aims: The aim of our study is to evaluate the long-term outcome of CP-CML patients who received Auto-SCT and who have received TKIs later during their treatment course.Patients: A total of 182 patients were included in this study, 114 (63%) were males and 68 (37%) females. The median age at diagnosis was 45 years (range: 20-66), interferon-alpha was initiated and then all patients received Auto-SCT between years 1989 and 2004 while they remained in CP, 30% of these patients were included in the auto-CML trials and the rest were treated accordingly. The median time between diagnosis and Auto-SCT was 24 months (range: 1-113). Stem cells source was peripheral blood in 172 (95%) of patients, mostly collected at diagnosis, and bone marrow in 10 (5%) patients. Patients were conditioned by Busulfan 4mg/Kg/day 4 days + Melphalan 140 mg/m2 1 day prior to the cells reinfusion. TKIs were introduced later on in these patients, information concerning the context of their use as well as response after treatment is currently under collection from centers and will be reported later during the meeting.Results: After a median follow-up of 14.5 years (range: 2-30), the probability of overall survival (OS) for the whole population from diagnosis at 10, 15 and 20 years is 97% (95% CI: 92-99), 82% (95% CI: 74-88) and 68% (95% CI: 56-77). OS from date of Auto-SCT at 10, 15 and 20 years was 90% (95%CI: 83-94), 78% (95% CI: 69-85) and 63% (95%CI: 51-74) respectively. When we stratify the population according to age at Auto-SCT, patients younger than 45 years had an OS from diagnosis at 10, 15 and 20 years of 100%, 96% and 86% respectively compared to 94%, 72% and 54% respectively for those with 45 years and older, p=0.0015. A total of 33 (18%) patients died, 15 (8%) of them because of disease progression, 2 secondary malignancies, and the rest because of other causes including pneumonia, hemorrhage, renal failure and cirrhosis.Additional data requests have been sent to centers querying about prognosis, molecular responses, treatment and disease details. An extensive analysis will be performed and the study will be updated with more results.Conclusion: We demonstrate here with the preliminary results that Auto-SCT followed by TKIs has induced remarkable rates of long-term survival and raise the question about the place of intensification in the therapeutic strategy in CML. [Display omitted] DisclosuresCharbonnier:Incyte: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau. Coiteux:BMS: Speakers Bureau; Incyte: Speakers Bureau. Rea:Novartis: Consultancy, Honoraria; Incyte: Honoraria; BMS: Consultancy, Honoraria; Pfizer: Honoraria. Delmer:Roche: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Janssen: Honoraria; Abbvie: Consultancy, Honoraria. Mahon:PFIZER: Consultancy, Honoraria; BMS: Consultancy, Honoraria; NOVARTIS PHARMA: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; INCYTE: Honoraria. Nicolini:Incyte Biosciences: Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; ARIAD: Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau. Etienne:BMS: Speakers Bureau; Incyte: Speakers Bureau; Novartis: Consultancy, Research Funding.

Nilotinib/Interferon-α Combination Rapidly Enhances Leukaemia-Associated Antigen-Specific Cytotoxic T-Lymphocyte Immune Responses, Limits Natural Killer Cell Maturation and Triggers B Cell Remodelling

▪Aim: We investigated the immune effects of nilotinib (NIL) combined with pegylated-interferon-α2b (IFN) in a subgroup of newly diagnosed chronic phase Chronic Myeloid Leukemia (CML) patients within the ALLG CML11 PINNACLE trial. NIL was given for the first 3 months (mo), followed by combination with IFN for 21 mo (from mo 4 - 24), then NIL alone from 25 mo.Methods: We assessed 12 patients serially from diagnosis, 2, 3, 5, 7, 10, 16 and 28 mo, including samples at 1, 8 and 15 days post-IFN commencement (median of 8 samples per patient). Multi-parameter flow cytometry was used to characterise effector phenotype and function of innate immune Natural Killer (NK) cells, Regulatory T cells (Treg), Myeloid Derived Suppressor Cells (MDSC), CD4+/CD8+ T cells and B cells. Functional cytotoxic T-lymphocyte (CTL) responses to leukaemia-associated antigens (LAAs) WT1, BMI-1, PR3 and PRAME were assessed by interferon-γ (IFN-γ)/tumour-necrosis factor-α (TNF-α) FLUROSPOT using peptide libraries of 15-mer peptides overlapping by 11 amino acids spanning the entire protein, or HLA-A*0201 specific peptides in HLA-A*0201+ patients.Results: All patients in this cohort had BCR-ABL1 <10% at 3 mo following NIL alone, 91% had optimal BCR-ABL1 responses (<1% and <0.1% at 6 and 12 mo respectively) with NIL/IFN. Cytolytic CD3-CD56dimCD16br NK cells increased after NIL (median; diagnosis 20.6% vs 2 mo 38.6%, p=0.002), the phenotype was more mature (CD57+) with increased CD161, NKp46, and NKp30 receptors. In contrast, the absolute number of CD56dimCD16br NK cells reduced as early as 8 days post-IFN, was lowest at 7 mo (151 cells/μl) on combination treatment vs 2 mo NIL alone (543 cells/μl, p=0.01). NK maturation status also decreased post-IFN and was subsequently restored upon IFN cessation (Table 1). A trend toward increasing immunoregulatory CD56brCD16-/dim NK cells was observed post-IFN (10 mo; 58 cells/μl) vs 2 mo NIL alone (32 cells/μl). NKp30/46 expression increased further on combination therapy, other NK receptors including CD161, KIR2DL2/DL3/DS2 and KIR2DL5 expression and functional degranulation response were unaffected by IFN. Programmed death-1 receptor expression, indicative of immune activation, increased on NK cells post-IFN, but was unchanged in T cells, B cells and monocytes.Low CD62L which is associated with impaired immune function was observed on CD4+/CD8+ T cells at diagnosis. CD62L surface expression increased from 2 mo NIL and remained increased with NIL/IFN combination. CD62L was highest at 28 mo. Naïve/memory T cell analysis showed decreasing CD8+ but not CD4+ effector memory (EM) cells on combination therapy (10 mo; 13.7%) vs diagnosis (23.3%, p=0.04) and 2 mo NIL (23.9%) with no change in central memory (CM). Functional IFN-γ and/or TNF-α LAA-CTL responses were observed in 83% of patients, with peak responses 15 days post-IFN. PRAME-CTLs (predominantly IFN-γ driven) were the most abundant and detected in all responders, with BMI-1- and PR3-CTLs in 50% of responders.CD3-CD19+ B cell numbers remained unchanged however, Transitional B cells (CD24++CD38++) the first cells which migrate to the peripheral blood, rapidly increased as early as 15 days post-IFN (283 cells/μl) vs diagnosis (70 cells/μl, p=0.001) and 2 mo NIL (61 cells/μl, p=0.008). CD27-IgD+ Naïve B cells demonstrated a similar increased pattern of expression. In contrast, CD27+IgD+ non class switched and CD20+CD38dim class switched memory B cells decreased following combination therapy.Suppressor Treg numbers decreased post-NIL vs diagnosis, and increased with IFN. Treg displayed a shift from EM phenotype to CM on NIL, and remained unchanged on combination therapy. T cell immunoreceptor TIGIT+ Tregs representing a highly activated/suppressive phenotype were unchanged at all time points. Predominantly granulocytic (Gr)-MDSC (CD66b+CD15+) were observed at diagnosis, reducing early in patients on NIL alone and remaining low throughout the study period.Conclusion: This first immunological report of NIL/IFN combination in newly diagnosed CML patients identifies rapid and divergent immunomodulatory effects encompassing both the innate and adaptive immune system. IFN alters NK cell phenotype, without affecting effector function, increases specific early B cell subsets, is associated with enhanced LAA-CTL responses and reduces suppressive Gr-MDSC. Correlation of patient immune responses and clinical outcomes is ongoing. [Display omitted] DisclosuresWhite:Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding. Yeung:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Ariad: Honoraria, Research Funding. Hughes:BMS: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Yong:Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Research Funding; Novartis: Honoraria, Research Funding.

Cytogenetic and Molecular Responses after 2 Years of Tyrosine Kinase Inhibitor (TKI) Treatment in Simplicity, an Observational Study of Chronic-Phase Chronic Myeloid Leukemia (CP-CML) Patients (Pts) in Routine Clinical Practice

Abstract Introduction: Data on clinical response and factors associated with response in CP-CML outside clinical trials are limited.SIMPLICITY (NCT01244750) is an ongoing observational study of CP-CML pts in routine clinical practice receiving first-line (1L) imatinib (IM), dasatinib (DAS) or nilotinib (NIL) in the US and Europe. This analysis aims to describe the factors associated with achievement of complete CyR (CCyR) and major MR (MMR). Methods: Clinical response after start of 1L TKI was assessed by CyR (karyotype or FISH) and MR (only tests by PCR on the international scale were included) as determined by the treating physician. Proportions of patients achieving CCyR and MMR between 30 days and either 12 or 24 months (mos) are presented; data at specified time points will be provided at presentation. Logistic regression models were used to identify factors associated with CCyR and MMR by 12 and 24 mos after 1L TKI initiation. Results: By March 03 2017, 1242 pts were enrolled prospectively in SIMPLICITY, receiving IM (n=416), DAS (n=418) or NIL (n=408); 55% were male. The median (interquartile range; min-max) age of pts at initiation of 1L TKI was 57 (46-68; 18-91) years; 35% of pts were treated in Europe, 65% in the US; 53% at academic centers and 47% at community practices. Numbers of pts followed by 3, 6, 12 and 24 months were 1233, 1224, 1195 and 1121, respectively. Of pts followed 12% (n=151), 31% (n=384), 44% (n=522) and 51% (n=567) had documented CyR testing by 3, 6, 12 and 24 mos; 23% (n=285), 55% (n=672), 72% (n=857) and 84% (n=937) of pts had documented MR testing by 3, 6, 12 and 24 mos; and 7% (n=82), 19% (n=231), 32% (n=379) and 42% (n=475) of pts had documented testing by both CyR and MR by 3, 6, 12 and 24 mos. Proportions of pts remaining on their 1L TKI declined with duration of follow-up. Of pts with documented CyR testing, 93% (n=141), 93% (n=356), 89% (n=464) and 85% (n=484) had remained on their 1L TKI by 3, 6, 12 and 24 mos, respectively. Of pts with documented MR testing, 93% (n=266), 91% (n=610), 85% (n=728) and 78% (n=730) had remained on their 1L TKI by 3, 6, 12 and 24 mos, respectively. In this non-controlled study, proportions of pts achieving CCyR were similar for pts tested by 12 and by 24 mos. For both periods, the proportion of pts achieving CCyR was highest for pts on 1L DAS, lower for 1L NIL, and lowest for 1L IM. Proportions of pts achieving MMR were higher for pts tested by 24 mos than by 12 mos. For both periods, the proportion of pts achieving MMR was highest for pts on 1L NIL, lower for 1L DAS, and lowest for 1L IM. Proportions were lower when calculated among pts followed through the period but not necessarily tested (intention-to-treat). Multivariate logistic regression analysis identified factors associated with MMR or CCyR by 12 and 24 mos after 1L TKI initiation. Receiving a second-generation (2G) TKI (i.e. DAS or NIL) as 1L therapy was associated with CCyR by 12 mos (odds ratio [OR]=2.6; p Conclusions: Response monitoring observed in clinical practice is not aligned with ELN/NCCN guidelines, so outcomes for the full SIMPLICITY cohort may differ from presented data on monitored patients. The lack of correlation between a 2G TKI as 1L therapy and achievement of MMR is unexpected, but may result from switches to 2L TKI in IM suboptimal responders or from differences in testing patterns in the individual cohorts; the uncontrolled nature of this study may have also contributed. Early switching of TKI treatment (previously found to be driven by toxicity, not response, and often from IM to 2G TKI) was associated with achieving MMR or CCyR by 12 mos. Not switching TKI, potentially a marker of better tolerance and adherence, was associated with achieving MMR or CCyR by 12 mos and 24 mos. Download : Download high-res image (116KB) Download : Download full-size image Figure . Disclosures Cortes: Teva: Research Funding; ARIAD: Consultancy, Research Funding; Sun Pharma: Research Funding; ImmunoGen: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. Goldberg: Bristol Myers Squibb: Research Funding, Speakers Bureau; Pfizer: Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau; Jazz: Speakers Bureau; Ariad: Speakers Bureau; Celgene: Speakers Bureau; COTA: Employment, Equity Ownership. Hehlmann: Novartis: Research Funding; BMS: Consultancy. Mauro: Bristol-Myers Squibb: Consultancy. Zyczynski: Bristol-Myers Squibb: Employment. Sen: ICON Clinical Research: Employment. Foreman: ICON Clinical Research: Employment. Gajavelli: Bristol-Myers Squibb: Employment. Gambacorti-Passerini: BMS: Consultancy; Pfizer: Consultancy, Honoraria, Research Funding.

Loss of G0S2 in Kinase-Independent TKI Resistance and Blastic Transformation of CML

Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 have turned chronic myeloid leukemia (CML) from a fatal to a chronic condition. Despite impressive results, resistance is a clinical problem, and TKIs do not target the CML leukemic stem cell (LSC) population, which is independent of BCR-ABL1 kinase activity. To identify mechanisms associated with BCR-ABL1 kinase-independent TKI resistance, we compared the transcriptional profile of TKI-naïve CD34+ cells from newly diagnosed CML patients who subsequently either responded (n=41) or did not respond (n=18) to imatinib over a twelve-month period (McWeeney et al. Blood 2010). The transcriptional profile of imatinib resistance in non-responders revealed striking overlap with reported signatures of CML blastic transformation and also implicated pathways involved in LSC survival. These findings suggest that similar biological mechanisms may be driving three different scenarios of BCR-ABL1-independent resistance: 1) primary kinase-independent TKI resistance, 2) transformation of CML from the chronic phase (CP-CML) to the blastic phase (BP-CML), and 3) persistence of residual leukemia in TKI-responsive patients.Among the genes most profoundly downregulated in patients destined to fail imatinib therapy was G0S2 (>3-fold, p<0.02), encoding a small protein that regulates multiple cellular functions, including apoptosis, quiescence, lipid metabolism, and oxidative phosphorylation. We compared G0S2 mRNA expression in CD34+ cells from normal cord blood (n=5) and from patient specimens lacking BCR-ABL1 kinase domain mutations, including newly diagnosed CP-CML (n=6), TKI-resistant CP-CML (n=2), and BP-CML (n=5). G0S2 was 4-fold downregulated in newly diagnosed CP-CML compared to normal cord blood CD34+ cells (p<0.01), and further downregulated by >3-fold in TKI-resistant samples and BP-CML (p<0.01), consistent with published microarray data (McWeeney et al. Blood 2010). Retrospective analysis of survival data for patients whose samples were evaluated in the transcriptional profile revealed that low G0S2 expression correlated with a shorter overall survival (n=35, p=0.036). Consistently, G0S2 mRNA and protein were downregulated in TKI-resistant K562R cells lacking BCR-ABL1 kinase domain mutations compared to TKI-sensitive controls. Ectopic expression of Flag-tagged G0S2 (G0S2-Flag) significantly reduced colony formation and increased apoptosis of TKI-sensitive and TKI-resistant K562 cells, and also CD34+ cells from CP-CML (n=5, p<0.05) and BP-CML (n=3, p<0.05) patients, having a more pronounced effect in BP-CML in the presence of imatinib. G0S2-Flag did not affect colony formation or apoptosis of normal cord blood CD34+ cells (n=2), which already express high levels of G0S2. Importantly, qRT-PCR revealed that G0S2 mRNA was significantly lower in primitive CD34+38- stem cells compared to more committed CD34+38+ progenitor cells in CP-CML (n=6, p<0.01) but not normal cord blood CD34+ cells (n=4, p=ns). These data suggest that loss of G0S2 plays a role not only in primary TKI resistance and blastic transformation of CML, but also in TKI resistance of the CML LSC. Consistently, CFSE staining revealed that G0S2 mRNA levels in CP-CML CD34+ cells are lowest in cell populations with the least number of cell divisions.Altogether, these findings suggest that G0S2 plays a role in regulating leukemic stem and progenitor cell survival and TKI response in vitro, and that restoring G0S2 expression combined with BCR-ABL1 inhibition may be a novel strategy to induce treatment-free remission in CML patients with kinase-independent TKI resistance and BP-CML, or to eradicate the CML LSC population. Future studies will assess the functional role of G0S2 in LSC survival and BCR-ABL1 leukemogenesis in vivo, and determine the mechanism by which G0S2 is downregulated in TKI resistance. DisclosuresDeininger:BMS: Consultancy, Research Funding; Ariad Pharmaceuticals, Bristol Myers Squibb, CTI BioPharma Corp, Gilead, Incyte, Novartis, Pfizer, Celgene, Blue Print, Galena: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy; Celgene: Research Funding; Gilead: Research Funding; ARIAD: Consultancy.

Inhibition of Nucleocytoplasmic Export Enhances Selective Elimination of Leukemic Stem Cells in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a hematopoietic stem cell malignancy driven by BCR-ABL1 tyrosine kinase and effectively managed with tyrosine kinase inhibitors (TKIs) such as imatinib. Reactivation of BCR-ABL1 through mutations in the kinase is a common mechanism of resistance, but fails to explain clinical resistance in a significant proportion of cases, a situation referred to as BCR-ABL1-independent resistance. Additionally, most patients require continuous TKI therapy to avoid recurrence of active leukemia, suggesting that primitive, fully leukemogenic CML stem cells (LSCs) are BCR-ABL1-independent. We have previously demonstrated that BCR-ABL1-independent CML cells from patients with overt resistance require functional nucleocytoplasmic export (NCE) and are sensitive to knockdown of the NCE regulator RAN and selective inhibitors of NCE (SINEs) such as the CRM-1 inhibitor KPT-330 (selinexor, Karyopharm) [Khorashad et al. Blood. 2015;125(11):1772-81]. We hypothesize that reliance on NCE may extend to LSCs, the reservoir for persistent CML. To test this, we performed in vivo and in vitro experiments to evaluate the effects of a prototype SINE, KPT-330, on LSCs (defined as CD34+CD38-) and leukemic progenitor cells (LPCs, CD34+CD38+) isolated from newly diagnosed chronic phase CML patients and normal cord blood (CB).Treatment with KPT-330 (50 nM) reduced colony formation by CML (n=4) LPCs and LSCs to 42% and 55% of controls, compared to 78% and 96% for CB (n=3) (Table 1). When combined with imatinib (2.5 µM), KPT-330 treatment dramatically reduced colony formation by LPCs and LSCs to 8% and 2% of controls, compared to 66% and 43% for CB. Single agent KPT-330 (50 nM) had modest effects on apoptosis in LSCs and LPCs, but the combination increased apoptosis in LPCs and LSCs by 280% and 940% relative to controls, compared to 180% and 270% respectively for CB (Table 1). These data suggest that combining KPT-330 with imatinib has the most profound effects on LSCs, with a large differential compared to normal hematopoietic stem cells.To test the effect of KPT-330 on functionally defined LSCs, CML CD34+ cells were cultured ex vivo with or without KPT-330 (50 nM) and/or imatinib (2.5 µM) for 72 hours, followed by plating in long term culture-initiating cell (LTC-IC) assays (n=5) or injection into NSG mice (n=20, 5 per treatment arm). While no reduction in colonies was seen in LTC-IC experiments with single agent KPT-330 or imatinib, a 64% reduction in LTC-IC colonies was observed with combination treatment (p<0.05). Genotyping of LTC-IC colonies by fluorescent-in-situ-hybridization (FISH) revealed that BCR-ABL1+ colonies were reduced by 11% and 22% relative to controls, when treated with KPT-330 or imatinib respectively, but combination treatment reduced BCR-ABL1+ colonies by 38%, significantly lower than either single agent (p<0.05). Thus far results are available from one xenograft experiment. All treatment groups engrafted with a median of 80% human CD45+ cells in bone marrow and 54% in spleen at 12 weeks after injection. Human CD45+ cells were sorted and genotyped. The median proportion BCR-ABL1+ cells in the control was 40%, and was not significantly reduced by imatinib or KPT-330 alone. However combination treatment reduced BCR-ABL+ cells by 42% (p<0.01).To identify potential CRM1 clients regulated in LSCs, we performed a meta-analysis of published gene expression data on CML vs. normal C34+38- or quiescent cells. Candidate genes with differential expression between CML and normal were cross-referenced against putative CRM1 cargos predicted by the LocNES tool [Xu et al. Bioinformatics. 2015;31(9):1357-65]. Confocal microscopy is ongoing to validate cytoplasmic relocalization upon KPT-330 exposure for several candidates, including CD26, an established marker of LSCs that has recently been shown to enhance quiescence in hematopoietic cells [O'Leary et al. Leukemia. 2017 Apr 21;Epub ahead of print].Our data illustrates that interrupting nucleocytoplasmic export by SINEs preferentially eliminates CML LSCs and enhances their sensitivity to TKIs, suggesting that combinations of TKI and SINEs represent a clinical strategy to target BCR-ABL1 independent resistance, as well as persistent residual CML. Mechanistic studies exploring the effects of nuclear entrapment of certain NCE cargo proteins of biological significance in CML LSCs are ongoing and will be reported. [Display omitted] DisclosuresDeininger:Celgene: Research Funding; Incyte: Consultancy; Novartis: Consultancy, Research Funding; ARIAD: Consultancy; Gilead: Research Funding; Pfizer: Consultancy; BMS: Consultancy, Research Funding; Ariad Pharmaceuticals, Bristol Myers Squibb, CTI BioPharma Corp, Gilead, Incyte, Novartis, Pfizer, Celgene, Blue Print, Galena: Consultancy, Membership on an entity's Board of Directors or advisory committees.