Chronic Phase CML Research Articles

Influence of MDR1 and CYP3A5 genetic polymorphisms on trough levels and therapeutic response of imatinib in newly diagnosed patients with chronic myeloid leukemia

Polymorphisms in genes coding for imatinib transporters and metabolizing enzymes may affect imatinib pharmacokinetics and clinical response. Aim of this study was to assess the influence of polymorphisms in MDR1 and CYP3A5 genes on imatinib trough levels, cytogenetic and molecular response in patients with CML. Newly diagnosed patients with chronic-phase CML started on imatinib therapy were enrolled and followed up prospectively for 24 months. The following single nucleotide polymorphisms were genotyped; C1236T, C3435T, G2677T/A in MDR1 gene and A6986G in CYP3A5 gene. Genotyping was done using PCR-RFLP method and validated by direct gene sequencing. Trough levels of imatinib were measured using LC–MS/MS. Cytogenetic response was assessed by conventional bone-marrow cytogenetics. Molecular response was assessed by qRTPCR using international scale. A total of 173 patients were included, out of which 71 patients were imatinib responders, while 102 were non-responders. Marked inter-individual variability in trough levels of imatinib was seen. Patients with GG genotype for CYP3A5-A6986G (P=0.016) and TT genotype for MDR1-C3435T (P=0.013) polymorphisms had significantly higher trough levels of imatinib. Patients with AA genotype for CYP3A5-A6986G [RR=1.448, 95% CI (1.126, 1.860), P=0.029] and CC genotype for MDR1-C1236T [RR=1.397, 95% CI (1.066, 1.831), P=0.06] &MDR1-C3435T [RR=1.508, 95% CI (1.186, 1.917), P=0.018] polymorphisms were at high risk for failure of imatinib therapy. Patients with CGC haplotype for MDR1 polymorphisms had significantly lower imatinib trough levels and were at a higher risk of imatinib failure [RR=1.547, 95% CI (1.324, 1.808), P<0.001]. GG vs. non-GG genotype for CYP3A5-A6986G [adjusted OR: 0.246; 95% CI (0.116, 0.519); P<0.001] and TT vs. non-TT genotype for MDR1-C1236T [adjusted OR: 0.270; 95% CI (0.110, 0.659); P=0.004] &MDR1-C3435T [adjusted OR: 0.289; 95% CI (0.135, 0.615); P=0.001] polymorphisms were independent factors predicting imatinib response in multivariate analysis. To conclude, MDR1 and CYP3A5 genetic polymorphisms significantly influence plasma trough levels and therapeutic response of imatinib in patients with CML. Genotyping of these polymorphisms could be of value to individualize the therapy and optimize the clinical outcomes.

Gadd45a deficiency accelerates BCR-ABL driven chronic myelogenous leukemia.

The Gadd45a stress sensor gene is a member in the Gadd45 family of genes that includes Gadd45b & Gadd45g. To investigate the effect of GADD45A in the development of CML, syngeneic wild type lethally irradiated mice were reconstituted with either wild type or Gadd45a null myeloid progenitors transduced with a retroviral vector expressing the 210-kD BCR-ABL fusion oncoprotein. Loss of Gadd45a was observed to accelerate BCR-ABL driven CML resulting in the development of a more aggressive disease, a significantly shortened median mice survival time, and increased BCR-ABL expressing leukemic stem/progenitor cells (GFP+Lin- cKit+Sca+). GADD45A deficient progenitors expressing BCR-ABL exhibited increased proliferation and decreased apoptosis relative to WT counterparts, which was associated with enhanced PI3K-AKT-mTOR-4E-BP1 signaling, upregulation of p30C/EBPa expression, and hyper-activation of p38 and Stat5. Furthermore, Gadd45a expression in samples obtained from CML patients was upregulated in more indolent chronic phase CML samples and down regulated in aggressive accelerated phase CML and blast crisis CML. These results provide novel evidence that Gadd45a functions as a suppressor of BCR/ABL driven leukemia and may provide a unique prognostic marker of CML progression.

Early efficacy observation of generic imatinib for treatment of chronic myeloid leukemia in chronic phase

Objective To evaluate the cytogenetic, molecular responses and safety of generic imatinib in newly diagnosed patients with chronic myelogenous leukemia in chronic phase (CML-CP) in 1-year at different stages. Methods From January 2014 to November 2014, 50 CML-CP patients received oral generic imatinib 400 mg/d. The cytogenetic examinations, bcr-abl transcript levels and safety were monitored after 3, 6, 9 and 12 months respectively. Results 46 of 50 patients insisted on oral generic imatinib and followed up 1 year. At 3-month, 52.0 % (26/50) patients reached the complete hematologic responses(CHR) rate, and patients at least achieved minor cytogenetic response (mCyR) and bcr-ablIS≤10 % were 84.0 % (42/50) and 42.0 % (21/50). At 6-month, patients at least achieved part cytogenetic response (PCyR) and bcr-ablIS≤10 % were 73.5 % (36/49) and 59.2 % (29/49). At 12-month, patients achieved complete cytogenetic response (CCyR), bcr-ablIS≤1 % and bcr-ablIS≤0.1 % were 60.9 % (28/46), 63.1 % (29/46) and 45.7 % (21/46). The grade 3 leukopenia, thrombocytopenia and anemia rates were 34 % (17/50), 40 % (20/50) and 30 % (15/50), respectively. No grade 4 hematologic toxicity occurred. The common non-hematologic toxicities included edema [84 % (42/50)], nausea [46 % (40/50)], muscle pain [20 % (10/50)], rash [16 % (8/50)], and impaired liver function [8 % (4/50)]. Conclusion Generic imatinib has a favorable effect in treatment of patients with CML-CP, and without serious adverse reactions. Key words: Leukemia, myeloid, chronic; Cytogenetics; Bcr-abl; Generic imatinib

IL1RAP antibodies block IL-1–induced expansion of candidate CML stem cells and mediate cell killing in xenograft models

AbstractChronic myeloid leukemia (CML) is currently treated with tyrosine kinase inhibitors, but these do not effectively eliminate the CML stem cells. As a consequence, CML stem cells persist and cause relapse in most patients upon drug discontinuation. Furthermore, no effective therapy exists for the advanced stages of the disease. Interleukin-1 receptor accessory protein (IL1RAP; IL1R3) is a coreceptor of interleukin-1 receptor type 1 and has been found upregulated on CML stem cells. Here, we show that primitive (CD34+CD38−) CML cells, in contrast to corresponding normal cells, express a functional interleukin-1 (IL-1) receptor complex and respond with NF-κB activation and marked proliferation in response to IL-1. IL1RAP antibodies that inhibit IL-1 signaling could block these effects. In vivo administration of IL1RAP antibodies in mice transplanted with chronic and blast phase CML cells resulted in therapeutic effects mediated by murine effector cells. These results provide novel insights into the role of IL1RAP in CML and a strong rationale for the development of an IL1RAP antibody therapy to target residual CML stem cells.

Allogeneic Stem Cell Transplantation in First Chronic Phase CML during the Last Decade: Retrospective Analysis of the National SFGM-TC Registry

Background & aimsThe only curative treatment of CML to date, remains allogeneic stem cell transplantation (Allo-SCT) despite some observations of non-detectable disease recurrence after tyrosine kinase inhibitor (TKI) cessation. The scope of allogeneic stem cell transplant for chronic phase (CP-) CML remains debatable and it seems interesting to retrospectively analyse the settings of this procedure in such patients since the introduction of TKI within the therapeutic arsenal of this disease.MethodsWe retrospectively analysed the registry of the Francophone society of stem cell transplantation and cellular therapy (SFGM-TC) from 2002 to 2014, for patients being in CP at diagnosis and at transplant. All data were captured according to thefrench regulations and were collected after signed up informed consent for each patient. All patients were transplanted for different degrees of resistance or severe recurrent intolerance to TKI(s). We segmented the observation period into two parts: 2002-2006 (Imatinib era) and 2006-2014, once second generation TKI were available in our country (TKI2 era). All patients were in CP-CML at diagnosis and first chronic phase at transplant. Second transplants for the same patient were excluded from this analysis.ResultsFrom 2002 to 2014 the proportion of transplants for CML dramatically decreased form 7.1% to <3% of totalallo-SCT performed in the country. Nevertheless, between 2002 and 2014, 191 transplants were performed for CP-CML, 121 during the first period and 70 during the second period. Interestingly, age at transplant is 36 (26-43) for IM period and older, 44 (26-55) years for TKI2 period; with a sex ratio of 1.08 and 1.3 respectively (p=ns). The median interval between diagnosis and transplant was 19 (1.4-197) months for IM period and much longer thereafter[32 (6.6-194) months, p<0.001]. The source of cells varied a lot with 71% of BM, 25.5% PBSC and 3.5% CB for IM period, 37% BM, 56% PBSC and 7% CB for TKI2 period (p<0.001), whereas the proportion of MAC versus RIC remained stable (88.5%/11.5% versus 81%/19%, p=0.262). The use of TBI as a part of the conditioning regimen was drastically reduced during the second period: 37% IM era, 14% TKI2 era (p<001). While ABO match did not differ, the use of unrelated donors largely increased in the second period (66% versus 46%, p=0.015), with less identical sibling donors used (33% versus 52%) in this last period. The proportion of sex match did not differ with a majority of male to male transplants (28% and 37%; p=ns) performed in both groups. The majority of patients wereGratwohlscore 3 in IM period andGratwohlscore 4 in the second period. Overall, the cumulative incidence of grade 2-4 acute GVHD was 32%, 41.3% and 44% at 1,2 and 3 months respectively, and the overall cumulative incidence of chronic GVHD was 26%, 40%, 45%, 50% at 1, 2, 5, and 10 years. The TRM rates were not different between the 2 periods: 22.4%, 23%, 26.65% and 27.8% at 1, 2, 5 and 10 years for IM period and 16.2%, 19.7%, 22.4% and 27.8% at 1, 2, 5 years for TKI2 period (NR 10 year for this period, p=0.508).The overall (OS) and relapse-free survival (RFS) rates according to the two periods are shown in Figure 1, with only a trend in the improvement of OS and RFS in the TKI2 period (log-rank tests, p=0.601 and 0.651 respectively).Gratwohlscore efficiently segregated patients for OS (overall p value = 0.002) and RFS (p=0.007). Multivariate analysis adjusted on OS identified only age (HR=1.02, p=0.05), and a related donor as a favourable variable on outcome (HR=0.53, p=0.031) with no significant influence of age, interval diagnosis-allo-SCT, source of donor cells, and type of conditioning regimen.ConclusionAllo-SCT still remains a curative treatment of CP-CML despite significant toxicities over time and the picture of this procedure in the therapeutic arsenal has dramatically changed over the last decade due to multiple therapeutic options offered now. Despiteallo-SCT of patients with longer diseases histories, probably more co-morbidities, there is an encouraging trend in the improvement of OS and RFS. [Display omitted] [Display omitted] DisclosuresNicolini:BMS: Consultancy, Honoraria; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Coiteux:Novartis, BMS, ARIAD: Speakers Bureau. Michallet:Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Astellas Pharma: Consultancy, Honoraria; MSD: Consultancy, Honoraria; Genzyme: Consultancy, Honoraria. Peffault De Latour:Novartis: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria, Research Funding; Amgen: Research Funding; Pfizer: Consultancy, Honoraria, Research Funding.

Efficacy and Safety of Nilotinib 300 Mg Twice Daily (BD) in Patients with CML in Chronic Phase (CML-CP) Who Are Intolerant to Prior BCR-ABL Tyrosine Kinase Inhibitors (TKIs): Results from the Randomized, Phase IIIb E.N.E.S.Tswift Study

Background : Philadelphia chromosome-positive (Ph+) CML is a myeloproliferative disease characterized by the presence of the abnormal Ph+ in hematopoietic cells. Imatinib, dasatinib and nilotinib are BCR-ABL TKIs commonly used in the treatment of CML-CP. Many patients on BCR-ABL TKI therapy will experience adverse events (AEs). Some of the more common AEs associated with first- and second-generation BCR-ABL TKIs include fluid retention, diarrhea, rash, musculoskeletal pain, nausea, vomiting, muscle cramps, and headache. Some patients will be unable to tolerate these AEs and will discontinue therapy. The current study aimed to assess the efficacy and safety of nilotinib in patients with CML-CP who are responsive to but intolerant of treatment with imatinib or dasatinib. Although the study was stopped early due to low recruitment, here we present results on cross-intolerance and molecular response in the patients who were switched from imatinib or dasatinib to nilotinib. Methods : Eligible adult patients had: Ph+ CML-CP associated with BCR-ABL quantifiable by real-time quantitative reverse transcriptase-polymerase chain reaction (RQ-PCR); received ≥3 months imatinib or dasatinib or both; were Results: The study was stopped early due to low recruitment; 20 patients were enrolled (mean age 53.9 years [range 31-77]; 14 female). 16 patients had received prior imatinib therapy, 4 patients prior dasatinib. Median nilotinib treatment duration was 494 days (mean 480, SD 167.6 days). At screening, 30% of patients were not in MMR, 45% had MMR and 25% had MR4.0. By month 3 and 24 of nilotinib treatment, 55% (11/20) and 65% (13/20) of patients, respectively, achieved at least a 1 log reduction in BCR-ABL levels. 35% (7/20) of patients achieved MR4.5 between baseline and month 3 of nilotinib treatment, and 50% (10/20) achieved MR4.5 at any time up to month 24. The proportion of patients with a molecular response at each visit up to month 15 is shown in the Figure. AEs during prior treatment with imatinib and dasatinib included gastrointestinal events (nausea, vomiting, diarrhea), superficial edema, myalgia, fatigue, rash, and headache, among others. 68% of AEs had resolved by month 3 of nilotinib treatment. Among the 13 evaluable patients on prior imatinib, 7 (54%) had resolution of all AEs during treatment with nilotinib; of 3 evaluable patients on prior dasatinib, 3 (100%) had AE resolution on nilotinib. Grade 3/4 AEs during nilotinib therapy occurred in 3 patients: diabetes mellitus, fatigue, neutropenia, pneumonia, osteoarthritis, and hyperuricemia. Conclusions : Although early termination of the study has not allowed for a robust analysis, these results suggest that nilotinib is effective and well tolerated in most patients intolerant of imatinib or dasatinib. During the first 3 months of switching to nilotinib, 55% of patients had achieved at least a 1 log reduction in BCR-ABL levels, and 35% of patients had achieved MR4.5. The cumulative rate of MR4.5 by 24 months was 50%. Achievement of this endpoint has been linked to favorable long-term outcomes, such as treatment-free remission. Furthermore, the majority of the AEs had resolved by month 3 of nilotinib therapy. This improved tolerance to nilotinib may result in improved treatment adherence. As such, although further study in a larger population is needed for confirmation, these results provide further evidence that nilotinib is a favorable option to establish a molecular response in patients intolerant of imatinib or dasatinib. Disclosures D9Rozario: BMS: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Branford: Qiagen: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding; Ariad: Research Funding; Novartis: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding; Cepheid: Consultancy. Yeung: Ariad: Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding. Anderson: Novartis Pharmaceuticals: Employment. Gervasio: Novartis Pharmaceuticals: Employment. Hughes: Novartis: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity9s Board of Directors or advisory committees, Research Funding.

Patient-Derived Induced Pluripotent Stem Cells Revealed ADAM8/CD156 As a Novel Marker of TKI-Resistant Chronic Myeloid Leukemia Cells

Since the emergence of tyrosine kinase inhibitors (TKIs), long-term survival of patients with chronic myelogenous leukemia (CML) has been improved. However, those TKIs have not fully succeeded in curing CML, mainly due to TKI-resistant CML stem cells. CML stem cells are often difficult to analyze because they represent an extremely minor population of CML cells. To overcome this obstacle, we established integration-free induced pluripotent stem cells (iPSCs) from bone marrow (BM) cells of two patients with CML in chronic phase (CML-CP) and obtained CML pre-hematopoietic progenitor cells (CML-pre-HPCs), immature hematopoietic cells phenotypically defined by CD34+/CD45-/CD43+ cells.In semisolid and liquid cultures, CML-pre-HPCs recapitulated the principal features of CML stem cells, multi-potency and the resistance against imatinib. Gene expression enrichment analysis for CML-pre-HPCs demonstrated that several gene sets, including those related to the maintenance of hematopoietic stem cells were enriched. In addition, we found that a disintegrin and metalloprotease 8 (ADAM8), also known as CD156, was highly enhanced in CML-pre-HPCs and the expression level of ADAM8 was even increased after the treatment of imatinib in vitro. To address the significance of ADAM8 in CMP-CP patient, we evaluated purified ADAM8+ cells by fluorescence-activated cell sorting (FACS) in primary samples.First, FACS analysis found that ADAM8+ cells were enriched more in BM samples of patient with newly diagnosed CML-CP than normal or other types of leukemias among CD34+ fraction. ADAM8+ cells were enriched in CD34+/CD38- fraction compaered to CD34+/38+ fraction in BM of CML-CP patients, indicating that ADMA8+ cells represent immature hematopietic cells. In cell viability assays, ADAM8+/CD38+ CML cells in newly diagnosed CML-CP patient enhibited imatinib-resistance and imatinib-induced apoptosis in vitro was strongly suppressed in ADAM8+ CML cells compared to ADAM8- cells. Even in CD34+/CD38+ fraction, which was previously known as TKI-sensitive fraction, ADAM8+ cells exhibited TKI-resistance in both cell viability and apoptosis assay, indicating that ADAM8 would be a useful marker of TKI-resistant CML cells.Finally, to evaluate the significance of ADAM8 as a marker of TKI-resistant CML cells in vivo, we measured the frequency of CML cells in BM samples of CML-CP patients who had achieved major or complete molecular response (MMR; n = 2 or CMR; n = 1) after the administration of TKIs by limiting dilution analysis. In CML patients with MMR, CML cells remained in ADAM8+ cells at higher frequency in spite of steep decline of CML cells in ADAM8- cells The frequency of CML cells was as high in CD34+/CD38+/ADAM8+ fraction as in CD34+/38- CML stem cell fraction. Even in a patient with CMR, residual CML cells were detected in ADAM8+ population among CD34+/CD38+ fraction, whereas CML cells were undetectable in ADAM8- population.In conclusion, we have established a powerful platform with CML-iPSCs to investigate the pathophysiology of TKI-resistant CML stem cells. Using this platform, we have identified ADAM8 as a novel marker of TKI-resistant CML cells. CD34+/CD38+/ADAM8+ fraction, as well as CD34+/CD38- fraction, was an important population that defines residual CML cells even in CML-CP patients with deep molecular response after the treatment of TKI. ADAM8 would become an attractive candidate of novel therapeutic targets against TKI-resistant CML cells. DisclosuresKataoka:Yakult: Honoraria; Boehringer Ingelheim: Honoraria; Kyowa Hakko Kirin: Honoraria.

In Elderly Patients with CML Second Generation TKIs Are Very Effective and Can be Given Long Term Particularly in the Absence of Comorbidities: A Single Center Experience

INTRODUCTIONSecond and third generation tyrosine kinase inhibitors (TKIs) are available for Philadelphia positive chronic myeloid leukemia (Ph+ CML) treatment, but though median age at diagnosis is 60-65 years, elderly patients are under-represented in clinical trials concerning 2G-TKIs and a very small number of papers has been published with series of CML patients > 65 years treated with 2G-TKIs or ponatinib. AIMwe have reviewed our single-center experience with 2G-TKIs in chronic-phase CML (CP-CML) patients > 65 years, in first or subsequent therapeutic lines. PATIENTS AND METHODSthe patients were 10 females and 13 males; CML diagnosis had been made between 1994 and 2015 at a median age of 67 years (61-77). According to Sokal score, 3 patients (13%) were classified as low, 15 (65%) as intermediate and 5 (22%) as high-risk respectively. B3a2 transcript was detected in 13 (56%), b2a2 in 10 (44%) cases.Overall, 30 therapies with 2G-TKIs were prescribed in 23 patients CP-CML > 65 years old, from 2007 to 2015. At 2G-TKI therapy start the median age was 68 years (65-79). Charlson Comorbidity Index (CCI) score was 0 in 11 patients, 1 in 4 patients, 2 in 4 patients, 3 in 3 patients and 4 in 1 patient.Thirteen of 23 patients received second-generation TKIs (2G-TKIs) up-front (6 nilotinib, 7 dasatinib), 10/23 patients as 2nd line therapy (6 nilotinib, 4 dasatinib) after imatinib failure (8/10) or intolerance (2/10), median time from imatinib start to 2G-TKIs being 18 months (9-120). Five of 23 patients needed a TKI as 3rd therapeutic line (4 dasatinib, 1 bosutinib), 1 patient also as 4th (ponatinib) and 5th line (bosutinib). RESULTSall 23 patients are alive after a median follow up of 45 months (12-259) from diagnosis; median follow up from the first 2G-TKI treatment is 36 months (12-110), in details 37 months (12-110) for patients that received 2G-TKIs up front and 34 months (12-70) for patients that received first 2G-TKI as second line therapy.Seventeen of 23 patients (74%) are still on 2G-TKI therapy, with a median treatment of 37 months (12-110) -table 1-. At the last visit 15/17 patients had MMR or best results (1 MR4.5, 9 MR4, 4 MR3).Six patients of 23 (26 %) stopped definitively 2G-TKI therapy, after a median treatment duration of 31 months (6-68) from the first 2G-TKI (table 2), in all cases for non-hematologic toxicity: 1 recurrent pleural effusion (grade 2), 1 geriatric syndrome, 1 carotid atherosclerotic disease, 1 PAOD, 1 vomiting (grade 3) and 1 pulmonary hypertension. At discontinuation, the disease response was MMR (2 patients), MR4 (3 patients), MR 4.5 (1 patient).Hematologic toxicity occurred during 2G-TKI treatment in 7/23 patients (30%), grade 1-2 in 4, grade 3-4 in 3. Sixteen of 23 (69%) patients experienced non-hematologic toxicity, grade 1-2 in 7 (30%) -5/7 pleural effusion-, and grade 3-4 in 9 (39%) -table 3-.There were no significant differences between patients still on therapy and patients that completely discontinued 2G-TKIs regarding Sokal score risk and b2a2/b3a2 transcripts. Conversely, CCI differed in these 2 categories, because CCI was > 1 in 5/6 (83%) of "discontinued" patients and in 7/17 (41%) of "ongoing" patients. Among the eleven patients with CCI = 0, only 1 discontinued 2G-TKI treatment. CONCLUSIONSOur experience shows that 2G-TKIs are very effective in elderly CP-CML, not only as first line therapy. Non-hematologic toxicity is high and it is the main reason for 2G-TKI discontinuation, that appears to be related to comorbidities. The vast majority of patients without comorbidities remain on 2G-TKI treatment long term. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Correlation of Expression of CD25 in Hematopoietic Stem/Progenitor Cell Fraction of Bone Marrow Cells with Response to Tyrosine Kinase Inhibitors in Chronic Myelogenous Leukemia Patients

Introduction: Tyrosine kinase inhibitors (TKIs) have dramatically improved the prognosis of chronic myelogenous leukemia (CML). The treatment with TKIs continues to improve the depth of response and overall survival of CML patients, but the life-long use of TKI is known to be associated with late complications such as cardiovascular events as well as heavy financial burden, and thus impairs the quality of life. To overcome these issues, many studies evaluating the possibility of TKI discontinuation have been ongoing worldwide. In order to achieve durable treatment-free remission, it is crucial to understand the dynamics of CML-leukemia initiating cells (LICs). We previously reported that CD25 was highly expressed in murine and human CML-LICs (Kobayashi CI et al., Blood, 2014). The aim of this study was to assess whether the proportion of CD25 positive cells in hematopoietic stem/progenitor cell fraction of bone marrow cells in CML patients treated with TKIs is associated with their molecular response and could serve as a novel surrogate marker to stop TKI therapy.Methods: Bone marrow samples were obtained from patients with CML in chronic phase who were diagnosed and have been treated solely with TKIs at Keio University Hospital (Tokyo, Japan). This study was approved by the institutional ethical committee and informed consent was obtained from each patient. Both quantitative and qualitative PCR of BCR-ABL was performed using bone marrow mononuclear cells (BMMNCs). The proportion of CD25 positive cells in bone marrow hematopoietic stem/progenitor cell (HSPC; CD34+CD38-) fraction was evaluated by flow cytometry using FITC-labeled anti-CD34, PE- labeled anti-CD38 and APC-labeled anti-CD25 antibodies. The response to TKIs at the time of evaluation was determined according to the previous report (Yoshida C et al., Int J Clin Oncol, 2012): complete cytogenetic remission (CCyR) defined as Philadelphia chromosome undetectable and quantitative PCR copy numbers >731 among BMMNCs; major molecular remission (MMR) as quantitative PCR copy numbers ≤731, and complete molecular remission (CMR) as undetectable BCR-ABL by quantitative and qualitative PCR.Results: Bone marrow samples obtained from 95 patients were evaluated (median age 53 years old; male/female, 67/28). Analysis was performed prior to TKI exposure in nine patients and under TKI therapy including 2nd generation TKI in 64 patients (imatinib, 22; dasatinib, 33; nilotinib, 9). Remaining 22 patients were treatment free because they enrolled in a clinical trial of TKI discontinuation. The proportion of CD25 positive cells in HSPC fraction significantly decreased in patients with prior TKI exposure relative to patients at diagnosis (n=86; Mean 4.2%, SD 7.0% vs n=9; Mean 22.4%, SD 11.3%, P<0.01). In addition, the proportion of CD25 positive cells in HSPC fraction significantly correlated the level of quantitative PCR (Figure, P<0.0001), and MMR was also dividable from CMR by the proportion of CD25 positive cells (MMR; n=29, Mean 4.6%, SD 4.8% vs CMR; n=49, Mean 2.6%, SD 2.5%, P<0.05). The expression of CD25 was still detected in the majority of patients who achieved CMR, including those who sustained CMR after the discontinuation of TKIs.Conclusion: We confirmed that the expression of CD25 in HSPC fraction of CML patients was significantly correlated with the response to TKI therapy, and may serve as an asset to select patients who are likely to achieve durable treatment-free survival. [Display omitted] DisclosuresKarigane:Celgene: Honoraria. Sakurai:Celgene: Honoraria. Matsuki:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Nippon Shinyaku: Honoraria. Kikuchi:Celgene: Honoraria; Takeda Pharmaceutical Company: Honoraria; Kyowa Hakko Kirin: Honoraria. Mitsuhashi:LSI Medience: Consultancy. Okamoto:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Asahi Kasei Pharma Corp.: Research Funding; Astellas Pharma Inc.: Research Funding; Shionogi & Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Alexion Pharmaceuticals, Inc.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Teijin Pharma Limited: Research Funding; Bristol-Myers Squibb K.K.: Honoraria, Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Pfizer Inc.: Honoraria, Research Funding; JCR Pharmaceuticals Co., Ltd.: Research Funding.

Discontinuation of Dasatinib after Deep Molecular Response for over 2 Years in Patients with Chronic Myelogenous Leukemia and the Unique Profiles of Lymphocyte Subsets for Successful Discontinuation: A Prospective, Multicenter Japanese Trial (D-STOP Trial)

▪IntroductionThe tyrosine kinase inhibitor (TKI), imatinib, dramatically improves the prognosis of chronic myelogenous leukemia (CML) by suppressing the function of BCR-ABL gene. It was shown that imatinib could be discontinued in a proportion of patients with CML who maintained deep molecular response (DMR) for at least 2 years. (Lancet Oncol. 2010; 11:p1029) Treatment with second generation TKI, dasatinib, after imatinib resistance/intolerance could also be discontinued after maintaining DMR for over 1 year in a proportion of patients with CML (Lancet Haematol. 2015; 2:p528). In this Japanese prospective multicenter trial (D-STOP trial by Shimousa Hematology and Kanto CML Study Groups, ClinicalTrials.gov Identifier: NCT01627132), we aimed to discontinue dasatinib in patients with CML who maintained DMR for over 2 years.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 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. 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. Mean age of the patients was 54.2 (25-82) years, and median follow-up period after cessation of dasatinib was 16.2 (7-30) months. Twenty patients relapsed during the observation period. Using Kaplan-Meier analysis, the estimated overall treatment-free survival (TFS) was 62.9% (48.5-74.2) at 12 months (Fig.1). Most relapses occurred within 6 months after discontinuation of dasatinib. All relapsed patients responded again to dasatinib. There was no significant difference either in estimated TFS between males and females or Sokal scores at diagnosis. During the consolidation therapy, the proportion (%) of CD4+CD8−, CD3−CD56+, CD16+CD56+, and CD57+CD56+ cells among total lymphocytes were monitored using flow cytometry in patients who could discontinue dasatinib during the observation periods (group A) and those who relapsed (group B). At the start of the consolidation therapy, there was no significant difference between groups A and B (CD4+CD8−%: 33.6 vs 34.0, p = 0.89, CD3−CD56+%: 24.3 vs 24.1, p = 0.95; CD16+CD56+%: 22.1 vs 23.9, p = 0.57; CD57+CD56+%: 19.7 vs 21.8, p = 0.51, respectively). In group B, the proportion of CD4+CD8− cells gradually decreased, whereas CD3−CD56+, CD16+CD56+, and CD57+CD56+ cells gradually increased during the consolidation therapy. All four types of cells were relatively stable in group A. At the end of the consolidation therapy, there was a significant group difference in the proportion of these subsets (CD4+CD8−%: 29.6 vs 22.2, p = 0.018*; CD3−CD56+%: 25.9 vs 37.3, p < 0.01*; CD16+CD56+%: 23.2 vs 34.4, p < 0.01*; CD57+CD56+%: 21.9 vs 32.1, p < 0.001*; respectively). We concluded that patients with CD4+CD8− cells ≥23.1%, CD3−CD56+ cells ≤40.1%, CD16+CD56+ ≤35.6% or CD57+CD56+ ≤26.6% at the end of the consolidation therapy had significantly higher estimated overall TFS at 12 months than those without each condition (CD4+CD8−%: 84% vs 32%, p < 0.0001* (Fig.2); CD3−CD56+%: 77% vs 25%, p < 0.001* (Fig.3), CD16+CD56+%: 77% vs 25%, p < 0.0001*; CD57+CD56+%; 84% vs 46%, p < 0.01*, respectively). Although increased large granular lymphocytes and NK cells were reported to be associated with high responses to dasatinib (Int J Hematol. 2014; 99:p41), the unique profiles of lymphocyte subsets could predict successful discontinuation of dasatinib.Conclusion:Discontinuation of TKI in patients with chronic phase CML after consolidation therapy with dasatinib for 2 years was feasible with relatively high TFS. The unique profile of lymphocyte subsets might be able to predict successful discontinuation. [Display omitted] [Display omitted] [Display omitted] DisclosuresKumagai:Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau; Pfizer: Speakers Bureau. Nakaseko:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria; Pfizer: Honoraria, Research Funding. Nishiwaki:Novartis: Research Funding. Yoshida:Bristol-Myers Squibb: Honoraria, Speakers Bureau; Otsuka Pharmaceutical: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau. Morita:Bristol-Myers Squibb: Speakers Bureau. Sakamoto:Takeda Pharmaceutical: Consultancy; Yakult: Other: Remuneration. Inokuchi:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria; Celgene: Honoraria; Pfizer: Honoraria.

A Single Center Retrospective Analysis of Adherence to Molecular Monitoring Guidelines in Patients with Chronic Myeloid Leukemia

Introduction In 2016, it is predicted that there will be 8,220 new cases of Chronic Myelogenous Leukemia (CML) in the United States, with an estimated 1,070 deaths from this disease (Siegel RL, et al, CA Cancer J Clin 2016, 66:7-30). Due to the prognostic significance of cytogenetic and molecular response to frontline Tyrosine Kinase Inhibitors (TKIs), expert panels such as the European LeukemiaNet and the National Comprehensive Cancer Network (NCCN) recommend strict guidelines to ensure adequate surveillance of patient's response to TKI therapy (Deininger M, et al, Blood 2009, 114: Abstract 1126). In this retrospective observational study, we studied adherence to current NCCN Guidelines in the management of CML. Methods Using both electronic and archived paper medical records, we identified 26 patients diagnosed with CML from 3/3/2003 to 12/9/2015 who were followed in a single community practice. Information at initial diagnosis regarding age, CML phase, spleen size, and initial TKI therapy selected were obtained. Monitoring parameters including frequency of follow-ups, complete blood counts, chromosomal analysis, molecular testing, and repeat bone marrow aspirates were reviewed. Evaluation for response to treatment with time to Complete Hematologic Response (CHR), time to Major Molecular Response (MMR), and time to Complete Molecular Response (CMR) were noted. Evaluations of treatment adherence and testing for BCR-ABL kinase mutations when milestones were missed or molecular progressions detected were recorded. Reasons for change in therapy and choice of subsequent treatment were also documented. Results The median age at diagnosis of CML was 53, with a male to female ratio of 1.6:1. In this group of 26 patients, only a single patient was diagnosed in the accelerated phase, the rest were in the chronic phase of CML. In 92%, clinical spleen size was logged. However, a single standardized method was not utilized which precluded calculation of Sokal scores for risk classification. In 88.5%, bone marrow aspirates were obtained to establish disease phase and enable bone marrow cytogenetic studies. Out of 26 patients, 16 (61.5%) were initially treated with imatinib, 6 (23%) were treated with dasatinib and 4 (15.4%) were treated with nilotinib. Attempts to achieve superior cytogenetic and deeper molecular response with dasatinib or nilotinib, coupled with lower rates of progression to advanced phases, were most frequently cited as reasons for choosing non-imatinib front line therapy. Only 50% of patients had complete compliance with periodic molecular testing at 3, 6 and 12 months during the first 2 years of diagnosis. The frequency of molecular and cytogenetic response assessments were often less than recommended. Only 2 of the patients studied underwent repeat bone marrow testing, with one enrolled in a clinical trial requiring periodic repeat bone marrow biopsies. The median time to achieve CHR was 27 days, median time to reach MMR was 26.5 weeks and median time to CMR was 7.9 months. Attempts to confirm patient compliance with oral TKI therapy were documented in 22 of the patients (84.6%). Three patients underwent testing for BCR-ABL kinase mutations at the time of molecular progression, however, none tested positive for resistance mutations. No patient had a hematological or cytogenetic relapse, and two patients switched from nilotinib to imatinib because of poor compliance due to toxicity. Conclusion Clinical practice guidelines promote evidence-based recommendations to guide clinicians in the management of CML. TKI therapy has evolved to be the primary treatment for chronic phase CML, and monitoring response to TKI therapy by setting cytogenetic and molecular milestones has improved quality of care and success with treatment. Unfortunately, real-world application of strict time lines for testing may not be feasible in all circumstances. Patient non-compliance with testing and marrow exams, interval illness and hospitalization, and errors in laboratory testing have served as barriers to adherence to NCCN guidelines. Despite a 50% compliance rate, there were no major untoward events (i.e. transformation to accelerated or blast phase) appreciated in this subset of patients. Disclosures No relevant conflicts of interest to declare.

Long-Term Treatment with Imatinib Is Associated with Decreased Estimated Glomerular Filtration Rate and Hemoglobin Level in Patients with Chronic Myelogenous Leukemia

▪Background: Among tyrosine kinase inhibitors (TKIs), imatinib mesylate was the first TKI successfully used for the treatment of chronic myelogenous leukemia (CML) in chronic phase (CP), and a majority of patients still remains on its long-term treatment. Although imatinib has been well tolerated in clinical practice, and the side effect profile has usually been mild to moderate, there are limited data available regarding the long-term TKIs treatment on kidney function and associated complications such as anemia. This study aimed to evaluate the effect of long-term imatinib treatment on estimated glomerular filtration rate (eGFR) and hemoglobin (Hb) level in patients with CML in CP.Patients and methods: By using the institutional database, patients with CML in CP who had been on imatinib as the first and the only TKI for over 5 years at Keio University Hospital (Tokyo, Japan) were selected and retrospectively analyzed. Estimated GFR was calculated by the Modification of Diet in Renal Disease equation for Japanese defined by the Japanese Society of Nephrology. All statistical analyses were performed with EZR, which is a graphical user interface for R.Results: Eighty-two patients were evaluable. The median age at initiating imatinib was 49.5 years (range, 19-76). The median duration of imatinib treatment was 105 months (range, 60-170). During this study period, imatinib was given at a dose of 400mg/day. The dose reduction was indicted in 8 patients but not because of progressing renal impairment. The mean eGFR was 77 ml/min/1.73m2 (range, 38-120), and the value was below 60 ml/min/1.73m2 in 12 patients before initiating imatinib. The mean value significantly decreased to 62 ml/min/1.73m2 (range, 34-98) over the 5 years after imatinib treatment (P<0.001), and the values reached <60 ml/min/1.73m2 in 43 of 82 patients (P<0.001). In an univariate analysis of patients excluding 12 patients with below 60 ml/min/1.73m2, older age and lower eGFR value at the initiation of imatinib were associated with later development of chronic kidney disease (<60 ml/min/1.73m2) (P<0.001 and 0.002, respectively). Mean Hb level at 5 years after starting imatinib was significantly lower as compared with that before (12.9+1.7 g/dl vs. 12.4+1.3 g/dl, P<0.01). The declining rate of eGFR was negatively correlated with those of Hb levels (correlation coefficient -0.249, P<0.05). In 20 patients with low Hb level (9.7+1.2 g/dl) and renal dysfunction, median serum erythropoietin (EPO) level was 31.9 mIU/ml (range, 9.1-119). Furthermore, 11 patients with eGFR<60 ml/min/1.73m2 achieving a durable molecular remission took part in an institutional TKI discontinuation trial. At 1 year after discontinuing imatinib, their mean eGFR values significantly improved (50.0 + 6.5 to 56.0 + 10.2 ml/min/1.73m2, P<0.05) as well as Hb level (12.0 + 1.7 to 14.0 + 1.6 g/dl, P<0.01).Conclusion: Our findings indicated that long-term use of imatinib is frequently associated with reversible but continuous decline in eGFR level, which could lead to anemia partly due to inadequate production of EPO. Although the degree of nephrotoxicity is usually mild, close monitoring of renal function is recommended particularly in older patients with pre-existing renal dysfunction. DisclosuresSakurai:Celgene: Honoraria. Karigane:Celgene: Honoraria. Matsuki:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Nippon Shinyaku: Honoraria. Kikuchi:Celgene: Honoraria; Takeda Pharmaceutical Company: Honoraria; Kyowa Hakko Kirin: Honoraria. Okamoto:Nippon Shinyaku Co., Ltd.: Research Funding; Alexion Pharmaceuticals, Inc.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Asahi Kasei Pharma Corp.: Research Funding; Teijin Pharma Limited: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Shionogi & Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Bristol-Myers Squibb K.K.: Honoraria, Research Funding; Pfizer Inc.: Honoraria, Research Funding; JCR Pharmaceuticals Co., Ltd.: Research Funding.

Outcome of Frontline Treatment with Generic Imatinib in Adult Patients with Chronic Myeloid Leukemia in Algerian Population: A First Multicenter Study

▪Introduction:In a developing country like Algeria, such expensive therapy is not available. Alternative approaches are needed to help these adult. In Algeria 'imatib' (CIPLA-India) was introduced in 2006; but no study has been published yet in the North Africa region regarding response and outcome of this copy in CML patients. The goal of this multicenter study is to characterize newly adult CML in the western region of Algeria and to assess the effectiveness and safety of imatib (IM, copy) as frontline therapy for patients with CML.Patients and Methods:The study was carried out in 7 hematology centers in the western Algeria. Patients, who were diagnosed to be suffering from CML between 1st January, 2007 and 31st December, 2014 were selected for data analysis. All patients received a copy preparation, consisting of the alpha crystal form of imatinib, (IM, copy) at a oral dose of 400 mg daily and monitored for tolerance and side effects while on therapy.Results:Between January 2007 and December 2014, 355 patients with CML were treated with imatib (copy). The median follow- up of the study was 46 months (range: 13-107 months). Complete hematological response (CHR) was seen in 83% of patients within 3 months. According to the Sokal score, 72% patients with low, 78% with intermediate and 69% with high risk disease achieved a CHR in 3 months (p=0.26) and according to the EUTOS score, 81% of patients with low and 70% with high risk disease achieved a CHR in 3 months (p=0.08). The major molecular response (MMR) at six months (M6), M9, M12, M18 and M24 was 21%, 38%, 35%, 51% and 67% respectively and 34% of patients achieved a complete molecular response (CMR). The projected 5-year overall survival (OS) rate was 83%. Side effects of imatib (copy) in this study were similar to those reported previously for the entire imatinib mesylate treatment study and only 8% of patients were intolerant to imatib (copy) and treated with a second generation of BCR-ABL inhibitor.Conclusion:In conclusion, imatib (copy) is effective and safe in treating patients with CML in chronic phase and proves to have a durable outcome. To our knowledge this is the first study reporting the response to imatib (copy) in a Algerian population. DisclosuresNo relevant conflicts of interest to declare.

Young CML Patients Treated Frontline with Imatinib or Second Generation TKIs: Clinical Characteristics and Outcome

Median age of CML patients at diagnosis is reported to be around 66 years. Few data about the characteristics and outcome of patients younger than 40 years are available, and the clinical trials of dasatinib and nilotinib as first line treatment were not stratified by age.We retrospectively analyzed 251 young patients with CML in chronic phase from 12 different Italian Institutions, diagnosed between October 2001 and October 2016. Up to 2011 all patients were treated frontline with imatinib while from January 2011 onwards with imatinib or a second generation TKI (nilotinib or dasatinib), based on clinical judgment. At diagnosis median age was 32,6 years [interquartile range (IQR) 27,6- 36,9]; 143 (57%) were male.Splenomegaly was found in 129 out of 233 evaluable patients, in 29,2% of the cases spleen was palpable >5 cm below the costal margin. The risk score was low in most of the cases (low risk Sokal 71%, low risk Eutos 91,3% vs high risk Sokal 9,8%, high risk Eutos 8,7%). Clinical features of the patients in treatment with different inhibitors are summarized in table 1. There were two main statistically significant differences in the group of patients treated with dasatinib: a higher proportion of high risk (30,8%) according to Eutos score and a lower median Hb level at diagnosis (8,5 gr/dL). These features probably reflected the trend of using dasatinib in patients with a more aggressive disease, as this drug was shown to be more potent since the first in vitro studies.Out of 251 patients 179 were treated with imatinib, 57 (22%) with nilotinib, 15 (5,9%) with dasatinib.Median follow up of the whole cohort was 76,5 months (IQR 41- 116); as expected, the follow up was longer in the imatinib group compared to the nilotinib and dasatinib groups (100 months vs 39,6 and 23,0 respectively).In the whole cohort, the cumulative incidence of Complete Cytogenetic Response (CCyR) and Major Molecular Response (MMolR) were 90,4% and 75,7% respectively; a deep molecular response (negative nested PCR, MR4.0, MR4,5) was achieved by 52.2% of patients, without differences among the 3 groups. Primary resistance occurred in 12,4% of patients, without differences among the 3 groups: secondary resistance occurred in 15.9% of patients, with a higher rate in the imatinib group (19,5%) as compared with nilotinib (7,0%) and dasatinib (6,7%) (p=0.047). Treatment discontinuation due to toxicity was observed in 6.0% of patients, without differences among the 3 groups. Blast transformation occurred in 7 out of 251 patients (2,8%), all in the imatinib group, after a median time from the diagnosis of 31 months (range 4-110). The 4-year cumulative Event-Free Survival (EFS) and Overall Survival (OS) were 72,4% (95%CI 66,7 - 78,5) and 98,1% (95%CI 96,4 - 99,8) respectively, without differences among the 3 groups. All deaths were related to blast transformation in imatinib group.In conclusion, as expected in a younger population, response to treatment and OS were excellent. However, the 4-year EFS was lower than expected, in spite of the presence of patients with predominantly low risk score. Furthermore, while the higher rate of secondary resistance in the imatinib group probably reflects the longer follow-up, it is worth of note that no statistically significant difference was observed between imatinib and 2nd generation TKI groups in terms of OS and EFS. [Display omitted] [Display omitted] DisclosuresBreccia:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Honoraria; Celgene: Honoraria; Ariad: Honoraria; Pfizer: Honoraria.

Downregulation of Mir-146a-5p, Mir-99b-5p, Mir-143-3p, Mir-10a-5p and Mir-151a-3p Associated with PI3K/AKT, p53, NF-Kb, and Fanconi Anemia/BRCA Signaling Pathways Are Observed in Imatinib-Resistant Chronic Myeloid Leukemia Patients without Detectable BCR-ABL kinase Domain Mutations

IntroductionChronic myeloid leukemia (CML) patients who do not achieve landmark responses are considered imatinib (IM)-resistant. IM-resistance can be due to BCR-ABL kinase domain (KD) mutations, although many IM-resistant patients do not harbor KD mutations. The pathogenesis of this phenomenon is unclear. Aberrations in BCR-ABL independent downstream signaling pathways, namely PI3K/AKT, p53, NF-kB and Fanconi anaemia (FA)/BRCA, have been implicated. MicroRNAs (miRNAs) are short non-coding RNAs that control gene expression and are notoriously promiscuous, with one miRNA regulating many mRNAs. In recognition of this, we sought to identify dysregulated miRNAs associated with the above pathways in patients with IM-resistant CML.MethodsEight patients with chronic phase CML who demonstrated primary resistance to IM and tested negative for KD mutations via Sanger sequencing were enrolled. Two healthy volunteers constituted the control group. Peripheral blood samples were taken and miRNA extracted from the white cells. MiRNA profiling was performed using miRNA sequencing (miRNA-seq) of the human microRNAome. MiRNA expression analyses (miEA) were performed on 4 sets of miRNAs associated with 4 different signaling pathways derived from publicly available databases: 237 miRNAs for PI3K/AKT, 223 miRNAs for p53, 221 miRNAs for NF-kB, and 126 miRNAs for FA/BRCA. MiRNA expression levels of the patients were compared with normal controls to obtain the differential expression of the miRNAs. MiRNAs that occur in all 4 pathways were further investigated in silico for their putative gene targets (Tarbase v7.0). Gene targets which were commonly linked with 3 different miRNAs were identified.ResultsMiRNA-seq yielded a total of 790 miRNAs. MiEA showed downregulation of 5 miRNAs in all 4 signaling pathways above: miR-146a-5p, miR-99b-5p, miR-143-3p and miR-10a-5p, miR-151a-3p. MiR-146a-5p and miR-99b-5p were the first and second most downregulated miRNAs in all 4 pathways. Putative gene targets that were commonly modulated by 3 miRNAs were: CDK6 (miR-143-3p, miR-10a-5p, miR-151a-3p), MDM2 (miR-146a-5p, miR-143-3p, miR-10a-5p), PTGS2 (miR-146a-5p, miR-143-3p, miR-10a-5p) and REV3L (miR-146a-5p, miR-143-3p, miR-151a-3p), as shown in Table 1.ConclusionsThe PI3K/AKT pathway is activated in response to IM-exposure to IM-naïve cells, implying a role in IM resistance. As cell cycle modulators, both p53 and NF-kB pathways are regulated within the PI3K/AKT pathway via AKT. FANCD2 is a central FA/BRCA pathway regulator and its transcription is maintained by AKT/mTOR via enhancement of NF-kB activity. The complexity of the interactions above has impeded efforts in identifying a unifying target in counteracting IM resistance. Thus, it is of significance that our results showed 5 differentially expressed miRNAs that consistently featured in the above signaling pathways, 4 of which remarkably influence PI3K/AKT and p53.In our study, PI3K/AKT and p53 pathways are inextricably linked with the same 4 miRNAs (miR-146a-5p, miR-143-3p, miR-10a-5p and miR-151a-3p) targeting the same 2 genes (MDM2 and CDK6) in these 2 pathways. MDM2 is an ubiquitin ligase that regulates the stability of p53a. AKT facilitates the functions of MDM2 to promote p53 ubiquitination. Conversely, inhibition of MDM2 leads to activation of p53 in response to cellular stresses. CDK6 is a cell cycle regulator and its overexpression influences the accumulation of pro-apoptotic p53 proteins, postulating a role in DNA damage response. Both PTGS2 and REV3L genes were commonly targeted by 3 miRNAs, within the NF-kB and FA/BRCA pathways respectively. PTGS2 overexpression has been shown in gastrointestinal adenocarcinomas; it induces carcinoma cell migration and invasion via activation of PI3K/AKT signaling. REV3L is the catalytic subunit of DNA polymerase ζ and plays a central role in DNA damage tolerance and chemoresistance towards DNA damaging agents. Notably, the NF-kB and FA/BRCA pathways were juxtaposed to the PI3K/AKT/p53 system via shared miRNA regulators.This provokes interesting questions regarding IM-resistance: are anti-apoptotic signaling and DNA damage response mechanisms driven simultaneously by these miRNAs? Can these miRNAs be potential biomarkers and therapeutic targets in IM-resistant CML? More studies are needed to further validate our findings. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Comparison of ABL1 and Gusb Reference Genes in the qRT-PCR Analysis of Halving Time and Early Molecular Response to TKI Therapy in Patients with Chronic Myeloid Leukemia

Objectives and background: Early molecular response (EMR) to tyrosine kinase inhibitors (TKI) therapy defined as BCR-ABL1 transcript level ≤ 10% at 3 months is a well-established predictive factor in CML patients. Recently, several study groups presented the concept of BCR-ABL1 decline velocity or "halving time" as a new predictive factor for optimal TKI treatment response. Different reference genes were used in the quantitative RT-PCR (qRT-PCR) analysis to calculate halving time and EMR rate, potentially influencing results of these analyses. The aim of the study was to compare ABL1 and beta-glucuronidase (GUSB) as reference genes used for calculation of halving time and EMR rate in chronic phase CML patients (pts) treated with imatinib 400 mg/d in the clinical settings.Methods: A total of 52 chronic phase CML pts with blood samples collected at baseline and at 3 months of imatinib therapy were investigated. Transcript levels of BCR-ABL1 were determined by qRT-PCR with the use of ABL1 and GUSB as control genes for each patient. Halving time for BCR-ABL1 transcripts was calculated using baseline transcript levels, transcript levels at 3 months of treatment, and number of days between these two points according to methodology reported elswhere1. Using receiver operating characteristic (ROC) analysis, the optimal halving time thresholds for achievement of EMR for each control gene were determined. Optimal thresholds along the ROC curves were calculated using the Youden index.Results: 40 of 52 (76.9%) and 44 of 52 (84.6%) pts achieved EMR with BCR-ABL1 transcripts levels calculated with GUSB and ABL1 as control genes, respectively (p=0.55). In 4 pts there was no BCR-ABL1 transcript reduction from baseline at 3 month using GUSB as a control gene resulting with negative halving times. The GUSB based halving times of these 4 patients were imputed to the longest positive halving time (8530 days) calculated for the patient with the smallest BCR-ABL1 decline. Transcript reduction occurred in all pts when ABL1 was used as a control gene. ROC curve analysis resulted in the optimal halving time thresholds for achievement of EMR of 39 days (area under the curve [AUC ] 0.96, ;95% CI 0.9-1.0, ) and 31 days (AUC 0.95; 95% CI 0.87-1.0, ) for GUSB and ABL1 control gene, respectively.Discussion and Conclusions: Lack of linearity in qRT-PCR analysis observed in blood samples collected in CML patients at diagnosis, with high amount of BCR-ABL1 and wild type ABL1 transcripts could affect the accuracy of early kinetics decline of BCR-ABL1 transcript calculations when ABL1 is used as a reference gene. The analysis with the use of GUSB as a control gene is not biased by this phenomenon. In this study, a non - statistically significant differences in the rate of EMR achieved at 3 months of imatinib therapy and halving time were observed. Analyses performed in our study with ABL1 and GUSB as reference genes suggest that both GUSB and ABL1 are able to identify relevant cut-offs for EMR achievement and could be used for halving time calculation.Reference:Branford S, Yeung DT, Parker WT, et al. Prognosis for patients with CML and >10% BCR-ABL1 after 3 months of imatinib depends on the rate of BCR-ABL1 decline. Blood 2014 Jul 24;124(4):511-8 DisclosuresZawada:Novartis: Speakers Bureau. Czekalska:Novartis: Speakers Bureau. Sacha:Pfizer: Consultancy, Honoraria, Speakers Bureau; Incyte: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Adamed: Consultancy, Honoraria.

Nilotinib As Second-Line Therapy in Patients with Chronic Myeloid Leukemia in Chronic Phase: Thailand Experience

Background: Nilotinib, a second generation tyrosine kinase inhibitor, was proved to have high efficacy on treatment of Philadelphia chromosome positive CML patients who failed or were intolerant to imatinib. Limited data was available on its efficacy and safety in Asian population.Methods: Chronic phase CML patients who have failure, suboptimal response or intolerance to imatinib according to ELN 2009 guideline were treated with nilotinib 400 mg twice daily on 7 centers in Thailand. Prospective data collection for 24 months was performed.Results: There were 106 cases participated in this study, 2 cases with initial T315I mutation were excluded from the study, total 104 cases were analysed. The median age was 46 (16-79) years with a slight male predominance over female at the ratio of 1.4:1 respectively. Twenty five percent received imatinib less than 12 months whilst 20% received imatinib longer than 60 months. The median duration of the prior imatinib treatment was 18 months (2-142 months). Best response to imatinib treatment were major molecular response (MMR) 5.8%, complete cytogenetic response (CCyR) 26%, major cytogenetic response (MCyR) 12.5%, complete hematologic response (CHR) 47%, and no CHR 8.7%. The reasons for nilotinib switching were imatinib failure 65%, imatinib intolerance 28%, imatinib suboptimal response 7%. Sixty-eight percent were completed 24 months follow up. Of those, 32% early discontinued treatment mostly because of unsatisfactory results or adverse events. Two patients died of infection and CNS bleeding during the study period.Evaluation were made every 3 months based on ELN 2009 criteria . Best response to nilotinib treatment included MMR 57%, CCyR 16%, MCyR 6%, CHR 16%, and no CHR 5%. At 3 months, 91%, 35%, and 14% of the patients CHR,CCyR, and MMR, respectively. Achieving CCyR or MMR at 3 months predicted a chance of achieving MMR (P= 0.00001) Those who did not achieve at least CHR at 3 months never achieved MMR, while 86 % of those who achieved CCyR at 3 months achieved MMR and 100% of those achieving MMR at 3 months had sustained MMR throughout the study period (24 months). Imatinib suboptimal response had significantly better outcome as compared to imatinib failure and imatinib intolerance group (P = 0.017). All of suboptimal response cases achieved CCyR , 86% achieved MMR, no early discontinue treatment in this group. While 75% of failure group achieved CCyR, 62% achieved MMR and 62% of intolerance group achieved CCyR, 38% achieved MMR. The reason for poorer response of intolerance group was high rate of early discontinue due to side effects, 17% vs 5% in the imatinib failure group.Initial BCR-ABL mutation analysis was performed on 90 cases, mutations were found on 16 cases, 2 of them were T315I which were excluded from the study. The cases with mutation significantly had poorer response to treatment than those without mutation (P = 0.001). There was one case with initial G250E mutation, who developed T315I mutation after treatment with nilotinib. At 24 months, 1 case progressed to accelerated phase and 3 cases progressed to blastic transformation. The 2-year progression-free survival and 2-year overall survival was 96% and 98%, respectively.Skin rash, musculoskeletal pain, and infection were the three most common non-hematologic adverse events found in 18%, 13%, and 6% of the patients, respectively; however, most of them were grade 1-2, except for 4 cases with grade 3-4 infections .Grade 3-4 hematologic adverse events included thrombocytopenia (12%), neutropenia (11%), anemia (5%), and leucopenia (4%); and most of them were manageable. Although biochemical abnormalities were commonly found, most of them were mild. Grade 3-4 events found were hypophosphatemia (6%), hyperglycemia (4%), and elevated serum lipase (4%). Only 10 cases (9%) permanently discontinued nilotinib due to its adverse effects.Conclusions: Nilotinib, as a second line treatment for Thai patients with chronic phase CML showed excellent efficacy and tolerability. Indication for nilotinib treatment, initial mutation status and depth of response at 3 months after treatment can predict outcomes of the patients.Author contact: KanchanaChansung M.D. Division of Hematology, Department of Medicine, Faculty of Medicine, Khonkaen University, Khonkaen, Thailande-mail: kchansung@gmail.com DisclosuresNo relevant conflicts of interest to declare.

Upfront Imatinib with Selective Early Switching to Nilotinib Leads to Excellent Achievement of Deep Molecular Response in Chronic Phase CML: 5 Year (Final) Analysis of the TIDEL-II Study

Upfront Imatinib with Selective Early Switching to Nilotinib Leads to Excellent Achievement of Deep Molecular Response in Chronic Phase CML: 5 Year (Final) Analysis of the TIDEL-II Study

Imatinib as Second-Line Therapy, Ameliorates or Maintains Molecular Responses Obtained with Ponatinib First-Line in Chronic Phase CML Patients

Background & aimsThe third generation tyrosine kinase inhibitor (TKI) Ponatinib demonstrates extremely potent BCR-ABL inhibitory activity in CML patients in chronic or advanced phase, resistant to 1, 2 or 3 prior TKI (Cortes NEJM, 2013) and it seemed interesting to test this agent in the front-line setting of chronic phase patients. The EPIC trial was a phase III trial set up to compare the molecular outcomes of Imatinib 400 (IM400) versus Ponatinib 45 mg daily at one year. However, this trial has been abrogated at early stages by the FDA despite the impressive molecular efficacy of Ponatinib (Lipton, Lancet Oncol 2016) because of unexpected and unacceptable rates of arterial thrombotic events that occurred in another trial (PACE). A majority of patients in the Ponatinib arm were switched towards IM400, but their outcome is not known. The aim of this study was to analyse the general and molecular outcomes of such patients in our country.MethodsThis is an observational study following the rules and regulations of these studies in our country. Data were monitored and retrospectively collected in the 5 centers involved in the EPIC trial. Molecular analyses were performed locally during ponatinib exposure (in parallel with centralised assessments) and during IM treatment thereafter. Only local results, expressed in % BCR-ABL (IS), were used in this analysis, and all respective molecular biology laboratories have been involved in the Eutos/ELN BCR-ABL accreditation system.ResultsThirteen patients were analysed, 9 males and 4 females, with a median age of 58.5 (range 19.5-69) years at chronic phase CML diagnosis. Sokal scores were low for 4, intermediate for 7, high for 2 patients, Euro score was low for 7, intermediate for 5 and high for 1, Eutos LTS score was low for 10, intermediate for 2 and high for 1. Patients were harbouring a "conventional" Philadelphia chromosome without clonal evolution (20-28 metaphases analysed/patient) at diagnosis. All patients were harbouring a Major BCR-ABL transcript. The median BCR-ABL transcript level was 109 (range 35-440) % at diagnosis. Blood pressure was normal in all patients at diagnosis except one. Ponatinib was initiated in all patients at 45 mg daily after a median of 1.5 (range 0.8-2.75) months after diagnosis. Median Ponatinib duration was 4 (range 0.5-6.3) months. Complete Hematologic Response was obtained in 0.98 (range 0.5-1) month in all patients. At 3 months, median BCR-ABL ratio was 0.19 (range 0.008-2.74) %, all patients being in early molecular response (EMR) and 4 patients being already in major molecular response (MMR). Median Ponatinib dose at cessation was 45 (range 0-45) mg daily. IM was started at 400 mg daily in all patients after a median of 1 (range 1-35) day after Ponatinib cessation and after a median of 4 (range 0.54-6.54) months after Ponatinib initiation.The median BCR-ABL transcripts at 3, 6, 12, 18, 24, 30, 36 and 42 months of Ponatinib (i.e. after a median time of 7, 10, 16, 22, 28, 34, 40 and 46 months of IM) were 0.22 (range 0.01-27)%, 0.08 (0.005-8.99)%, 0.08 (0.01-2.01)%, 0.1 (0.003-1.9)%, 0.03 (0.001-1.61)%, 0.01 (<0.001-0.21)%, 0.01 (0.001-0.21) and 0.01 (0.001-0.02) respectively (see figure 1). On the 10 evaluable patients for molecular analyses, at Ponatinib cessation 40% of patients were >MMR, 40% of patients in MMR, 20% in MR4. At last follow-up, on IM400 10% of patients were in >MMR, 40% in MMR, 20% in MR4, 10% in MR4.5 and 20% in MR5, i. e. 50% in deep molecular response.At last follow-up, all patients were alive, still on IM400 daily for 32 (27.75-37.4) months, except 4 [1 grade 3 asthenia (now on Dasatinib), 2 molecular failure (now on Dasatinib and Bosutinib)], 1 free of treatment]. None of the patients progressed towards advanced phases to date. No thrombotic events occurred in any of the patients on Ponatinib. Three patients developed hypertension on Ponatinib requiring treatment, still ongoing at last follow-up in all.ConclusionThe use of Imatinib after Ponatinib first-line therapy for chronic phase CML patients is safe. Despite a significant tyrosine kinase inhibition inferior activity in vitro, it allows the maintenance or the improvement of molecular responses throughout time and serves as a consolidation therapy. These data might pave the way for the design of future clinical trials using Ponatinib in the front-line setting in conjunction with Ponatinib dose reduction. [Display omitted] DisclosuresNicolini:Ariad, BMS: Consultancy, Speakers Bureau; Novartis: Research Funding, Speakers Bureau. Coiteux:Novartis, BMS, ARIAD: Speakers Bureau. Mouchel:Ariad/Incyte: Employment. Mahon:BMS: Honoraria; Pfizer: Honoraria; Novartis: Honoraria, Research Funding; Ariad: Honoraria. Etienne:novartis: Consultancy, Speakers Bureau; BMS: Speakers Bureau; ARIAD: Speakers Bureau; Pfizer: Speakers Bureau. Guerci:Pfizer: Consultancy; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; ARIAD: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Inhibition of CML Development Following Conditional SIRT1 Deletion in Transgenic BCR-ABL Mice

▪Despite the success of tyrosine kinase inhibitors (TKIs) in treatment of CML, cures remain elusive, as primitive leukemia stem cells (LSC) are retained in patients achieving remission. Previous studies from our group have suggested that Sirtuin 1 (SIRT1) inhibition may represent a novel approach for elimination of LSCs in chronic phase CML. SIRT1 was shown to be overexpressed in CML LSCs, and SIRT1 inhibition using shRNA or a small molecule SIRT1 inhibitor selectively eliminated CML LSCs by increasing p53 acetylation and activity (Li et.al; Cancer Cell 2012). These studies were limited by possible off-target effects and limited duration of in vivo exposure. Here we used a genetic mouse model to definitively delineate the role of SIRT1 in CML development. A model for conditional SIRT1 deletion in hematopoietic stem cells was established by crossing homozygous SIRT1 exon-4 floxed (SIRT1fl/fl) mice with Mx1-Cre mice. To study the requirement of SIRT1 for development of CML, Mx1-cre SIRT1fl/fl mice were crossed with SCL-tTA/BCR-ABL mice, representing a tet-regulated inducible transgenic mouse model of CML, to generate SCL-tTA/BCR-ABL Mx1-Cre SIRT1fl/fl mice (BA Mx1-Cre SIRT1fl/fl). BA SIRT1fl/fl mice lacking Mx1-Cre were used as controls. The mice were maintained on doxycycline until CML induction. Cre mediated deletion of SIRT1 was induced by intraperitoneal pIpC injections (250µg/mouse) administered every other day for a total of 7 doses. SIRT1 knockdown was confirmed by PCR for excised exon-4 and by RT-Q-PCR. Bone marrow (BM) cells from either BA Mx1-Cre SIRT1fl/fl or controls (both CD45.2) were transplanted into irradiated (800 cGy) CD45.1 congenic recipients (2X106 cells/mouse). Cre-mediated deletion of SIRT1 was induced by pIpC injection starting at 4 weeks post-transplant, followed by withdrawal of tetracycline to induce BCR-ABL expression. Serial PB counts and phenotypic evaluation of cell types by flow cytometry (Fig 1 A-B) showed SIRT1 knockdown to have a profound effect on CML development. By 8 weeks after BCR-ABL induction, BA SIRT1fl/fl mice (n=10), showed significantly lower neutrophils (p=0.0003) and Gr-1/Mac-1 positive myeloid cells (p=0.0002) compared to control mice. Subsequently, control mice developed progressive neutrophilic leukocytosis and increasing morbidity from leukemia, whereas BA SIRT1fl/fl mice demonstrated significantly lower WBC counts, without evidence of progressive increase or morbidity (Fig 1 A). This cohort of mice continues to be followed for survival. Another cohort of BA Mx1-Cre SIRT1fl/fl mice was sacrificed at 8 weeks post pIpC injection and BCR-ABL induction to evaluate the effect of SIRT1 knockdown on stem and progenitor populations (n=6 each). SIRT1 deleted mice demonstrated significant reduction in spleen size, weight, cellularity, and myeloid infiltration (Fig 2 A-B), and in myeloid cell expansion in the BM compared to controls (p=0.002). Primitive lineage negative, Sca1 positive, c-Kit negative (LSK) cells and granulocyte-macrophage progenitors (GMP) were significantly reduced in BM and spleen of BA SIRT1 deletedmice compared to control mice, whereas megakaryocyte-erythrocyte progenitors (MEP) were increased (Fig 3 A-B). Long term hematopoietic stem cells (LTHSC) in the BM are reduced following CML development. The percentage and number of LTHSC were significantly increased in SIRT1 deletedmice compared to control mice (Fig 3C-D). We also evaluated the effect of SIRT1 deletion on normal hematopoiesis by studying Mx1-Cre SIRT1fl/fl mice lacking BCR-ABL. SIRT1fl/fl mice without Mx1-Cre were studied as controls. Mx1-Cre SIRT1fl/fl and control mice were treated with pIpC to induce SIRT1 deletion. SIRT1deletedmice did not show significant alteration in blood counts, but demonstrated significantly higher LSK and LTHSC numbers in BM compared to control mice. Upon secondary transfer, recipients of BM from SIRT1deleted mice showed a modest increase in donor cell engraftment at 12 weeks compared to controls (90.8% (83.2-92.2%) vs 83.6% (75.8-86.7%); p=0.001). We conclude that genetic deletion of SIRT1 markedly inhibits all aspects of CML development in transgenic BCR-ABL mice, without impairing normal hematopoiesis. These observations demonstrate a critical role for SIRT1 in leukemia development, and support further evaluation of SIRT1 as a therapeutic target in CML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.