Months Of Nilotinib Treatment Research Articles

Efficacy and safety of nilotinib in chronic myeloid leukaemia patients who failed to achieve a treatment-free remission period after imatinib discontinuation: Results of the French Nilo post-STIM study.

Molecular recurrence (MRec) occurs in about half of all patients with chronic myeloid leukaemia (CML) who discontinue tyrosine kinase inhibitors (TKI) in sustained deep molecular response. A second TKI discontinuation has been attempted in some patients who regain the discontinuation criteria after resuming treatment. Nilotinib treatment affords faster and deeper molecular responses than imatinib as first-line therapy. We prospectively evaluated the efficacy and safety of nilotinib (300 mg twice daily) in chronic-phase CML patients who experienced MRec, after imatinib discontinuation and analysed the probability of TFR after a new attempt in patients treated for 2 years with sustained MR4.5 for at least 1 year. A total of 31 patients were included in the study between 2013 and 2018. Seven (23%) patients experienced serious adverse events after a median of 2 months of nilotinib treatment leading to discontinuation of treatment. One patient was excluded from the study for convenience. Among the 23 patients treated for 2 years with nilotinib, 22 maintained their molecular response for at least 1 year (median: 22 months) and stopped nilotinib. The TFR rates at 24 and 48 months after nilotinib discontinuation were 59.1% (95% confidence interval [CI]: 41.7%-83.7%) and 42.1% (95% CI: 25%-71%) respectively (NCT #01774630).

P50-3 Treatment-free remission in e19a2 BCR-ABL1 chronic myeloid leukemia

Chronic myeloid leukemia (CML) has become a chronic disease, for which the chronic phase is manageable with tyrosine kinase inhibitor (TKI) therapy. Several TKI discontinuation studies in patients with durable deep molecular response demonstrate that stopping TKI therapy is feasible, and this situation is called treatment-free remission (TFR). In present, The NCCN guidelines and the ESMO guidelines provide guidelines for TKI discontinuation. In these guidelines, quantifiable BCR-ABL1 transcript and monitoring using international scale (IS) are involved in the criteria of TKI discontinuation. Because IS can quantify only e13a2 or e14a2 transcripts, other transcript variants does not match the TFR criteria. Here we describe an e19a2-positive CML patient who maintain TFR. A 77-year old woman was diagnosed e19a2-positive CML, and achieved deep molecular response by nilotinib. Since 66 months of nilotinib treatment, e19a2 transcript has been undetected. Since 84 months of nilotinib treatment, she has refused subsequent medication for CML and therefore careful monitoring by QT-PCR has been done. By 40 months since nilotinib discontinuation, she has been well without any sign of withdrawal syndrome and e19a2 transcript has been undetected. In literature search, there is no report of the e19a2-positive CML who obtained TFR. As in the case we presented here, TFR may be achievable in patients with transcript variants other than e13a2 or e14a2. The problem to be solved is the standardization of transcript quantification. Both the ELN guidelines and the NCCN guidelines are created on the premise that they are monitored by the IS method which can only use for e13a2 or e14a2 transcript, meaning there is no treatment guidelines for other variants. Establishing standardized quantification of variant transcripts based on global clinical trials may contribute to prepare the treatment guideline including TFR as similar to e13a2 or e14a2 transcript.

Gene Expression Profiling and Cellularity of Bone Marrow CD34+/Lin- Cells of Patients with Chronic-Phase Chronic Myeloid Leukemia at Diagnosis Vs. 12 Months of First-Line Nilotinib Treatment

Introduction: Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder with heterogeneous biological and clinical features. The biomolecular mechanisms of CML response to tyrosine-kinase inhibitors (TKI) are not fully defined. Despite the TKI nilotinib being a very effective drug for the treatment of CML, drug resistance can emerge. In the contest of the REL-PhilosoPhi34 study (EudraCT: 2012-005062-34) on behalf of the Rete Ematologica Lombarda, we undertook gene expression profiling (GEP) of selected bone marrow (BM) CD34+/lin- cells of 30 patients with CP-CML at diagnosis vs. the same patients after 12 months of nilotinib treatment to investigate molecular signatures characterizing both conditions.Methods: MNCs as well as CD34+/lin- cell counts of 30 CML patients were measured at diagnosis and after 3 and 6 months of nilotinib treatment, respectively.We performed GEP of BM CD34+/lin- cells of 30 patients at diagnosis vs. the same patients after 12 months of nilotinib treatment to investigate the gene expression changes induced by nilotinib treatment using Affymetrix GeneChip HTA 2.0.Results: The study demonstrated that the number of the BM CD34+/lin- cells dramatically decreased between the diagnosis and after 3 as well as 6 months of nilotinib treatment (Tab.1). We noticed that the BM CD34+/lin- cells slightly increased between 6 and 12 months of nilotinib treatment (Fig.1).FISH analysis detected CD34+/lin- Ph+ cells in 30 CML patients at diagnosis. No positive Ph+ nuclei were detected on CD34+/lin- cells after 12 months of treatment.GEP identified 264 statistically significant differentially expressed genes at diagnosis vs. 12 months of nilotinib. Functional enrichment analysis revealed groups of genes belonging to 14 pathways differentially active during nilotinib treatment.CD34+/lin- cells after 12 months of nilotinib treatment showed altered expression of genes involved in metabolic processes: AGPAT4, LPCAT3, MBOAT2, ELOVL6, GDPD1, APOC1, ACSM3, PLD4, GLIPR1, SPNS3 (lipid metabolism), HK1, PDK3, UGGT1 (glycaemia), PRKAR2A, PRKAR2B, PTPN1, MARCKS, CAB39, RXFP1, SORT1, TBC1D4 (insulin resistance), CR1, HBB, HBBP1, HBD, HBG2, SERPINE2, KEL, F13A1 (coagulation cascade), and TLN1, FCGR2A, ITGA2B (platelet activation). Several pathways involved in cell proliferation (CSF2RB, CSF1, FLT3), RAS signaling pathway(RAB31, RASA3, RRAS2, ANGPT2), drug transporters(ABCC5), cell adhesion and cytoskeleton(CDH2, ICAM4, SELL, TEK), B cell differentiation(CD40LG, BLNK, JAK3) were up regulated whereas sphingolipid metabolism (SGMS2 and SGPP1) and apoptosis (CCNG1, GAS2, SPTA1, CDH) were down regulated in CML CD34+/lin- cells at diagnosis, respectively.Conclusions: GEP analyses of CD34+/lin- cells of CP-CML patients at diagnosis vs. 12 months of nilotinib treatment demonstrated that several pathways were significantly differently regulated.From our results, we can speculate that nilotinib might interfere with biologic mechanisms (lipid and glycemic metabolism, insulin resistance, coagulation cascade and platelet activation) that are relevant in CML patients treated with TKI as previously determined by several studies in literature.The alteration of RAS signaling pathway , drug transporters, cell adhesion, cytoskeleton, B cell differentiation, sphingolipid metabolism and apoptosis in CML CD34+/lin- cells at diagnosis may underlie the increased cell proliferation that is characteristic of CML and may play a significant role in recognizing resistance mechanisms of leukemic stem cells, such as stem cell-niche interactions, cell cycle, survival, self-renewal, and metabolism.GEP studies on a larger cohort of CML patients at diagnosis vs. 12 months of nilotinib are ongoing. We believe that BM CD34+/lin- cells from CML patients at diagnosis and after nilotinib treatment harbor differences in certain biologic and genetic properties, that may predict how well they will respond to nilotinib treatment and we will test this hypothesis. [Display omitted] DisclosuresRossi:Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. Orlandi:Novartis: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Incyte: Speakers Bureau. Iurlo:Novartis: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria.

Preliminary Experience of Imatinib after Nilotinib in the First Line Treatment of Chronic Myeloid Leukemia

Background Chronic myeloid leukaemia (CML) is a clonal myeloproliferative disorder, hallmarked by the BCR-ABL fusion gene which encodes for a constitutively activated BCR/ABL tyrosine-kinase, effectively treated with Tyrosine-Kinase Inhibitors (TKIs). The first line TKI Imatinib (IM) can obtain a high cumulative CCyR; compared to IM, deeper and faster molecular responses can be obtained in newly diagnosed CML patients with the second generation TKIs, Nilotinib (NIL) and Dasatinib. Despite the three TKIs have been evaluable as first line therapy for a long time, our experience largely concern the second generation TKIs after IM but not evidence are reported regarding IM after second generation TKIs.On behalf of the Rete Ematologica Lombarda (REL) we designed the PhilosoPhi34 study (EudraCT: 2012-005062-34). This study was divided into three consecutive phases. An initial recruitment phase, a “core” phase of treatment lasting for 12 months (mos) and an “observational” phase lasting for an additional period of 24 mos. NIL 300 mg BID standard dose was administered for one year (“core” phase). At the end of this “core” phase of the trial, during the “observational” period, treatment options were up to the Investigator's choice (i.e. any TKI approved as first line treatment could be used), based on individual patient evaluation.Furthermore, fluctuations of BCR/ABL ratio are known, in particular during the two first years of CML treatment, characterized by a higher risk of disease progression or relapse.Aims Despite this consideration, our purpose is to verify time courses of Molecular Response (MR) IS in the IM treated patients, during the “observational” phase of the PhilosoPhi34 study.Methods We evaluate the PhilosoPhi34 database. MR IS is reported at 3, 6 and 12 mos of NIL treatment (“core” phase) and every 6 mos in the “observational” phase. Until now, the “observational” phase is ongoing and the database is unlocked.Results Among the 74 reported pts who started the “observational” phase, with at minimum a CCyR, 62 pts maintained NIL, 10 pts switched to IM and 2 pts switched to other TKI. Other two pts switched to IM after 18 and 24 months of NIL treatment (i.e. +6 mos and +12 mos of the “observational” phase, respectively). Among the 62 pts that maintained NIL, 33 showed a MR ≥ 4.0 IS and 20 showed a MR 3.0 IS. In the NIL group 15/62 pts showed an increase of the BCR/ABL ratio after 6 mos of the “observational” phase, but only 5/33 (15%) pts lost MR4.0 IS and 1/20 (5%) pts transient lost MR3.0 IS. Overall, 1/62 pts failed at 26 mos of treatment (+14 mos of “observational” phase) harbouring a resistant mutation. All the other NIL treated pts, despite molecular fluctuations, maintained or improved MR over time. Among the 10 pts that switched to IM after 12 mos of NIL treatment, 6 were in MR ≥ 4.0 IS and 4 in MR3.0 IS. Of them, 7/10 pts showed an increase of the BCR/ABL ratio but only 1/6 (16,6%) pts lost MR4.0 IS and 1/4 (25%) pts lost MR3.0 (Table1). No pts failed treatment. Of note, the probability to lose MR≥ 4.0 IS or MR 3.0 IS between NIL treated pts vs IM treated pts proved not significant with the Fisher's test (p: 0.43 and p: 0.28, respectively).Conclusion As expected, our preliminary data show fluctuation of BCR/ABL ratio and of MR IS. IM treated pts present initial molecular fluctuations statistically similar to the fluctuation showed by NIL treated pts evaluated at the same time point. Despite fluctuations, MR IS is maintained or improved, over time. More data are necessary to confirm these exploratory results. Pointing out the possible adverse events occurring during long-term NIL treatment and the percentage of CML pts with vascular or metabolic disease at diagnosis, we consider a short-term treatment with NIL before IM a new possible strategy for CML cure. [Display omitted] DisclosuresOrlandi:Novartis: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Incyte: Speakers Bureau. Iurlo:Pfizer: Consultancy, Honoraria; Bristol Myers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Rossi:Sanofi: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.

Progressive Down Regulation of JAK-STAT, Cell Cycle, and ABC Transporter Genes in CD34+/Lin- Cells of Chronic-Phase Chronic Myeloid Leukemia (CP-CML) Patients at Diagnosis Vs. 12 Months of Nilotinib Treatment Vs. Healthy Subjects

In the PhilosoPhi34 study (EudraCT: 2012-005062-34) on 87 CP-CML patients, we analyzed the gene expression profiling (GEP) of bone marrow (BM) CD34+/lin- cells of 79 patients with chronic-phase chronic myeloid leukemia (CP-CML) patients at diagnosis and after 12 months of nilotinib treatment (Pungolino et al. AM J Hematol. 2018). FISH analyses identified CD34+/lin- Ph+ cells in all 79 CML-CP patients at diagnosis. 78/79 patients achieved at least a complete cytogenetic response after 12 months of nilotinib whereas 1/79 patients relapsed at 12 months. No Ph+ nuclei were detected in 78/79 patients at 12 months. We have demonstrated that genes involved in the JAK-STAT signaling pathway, the cell cycle, and the ATP-binding cassette (ABC) transporters were significantly over expressed in patients at diagnosis compared to 12 months of nilotinib treatment (Trojani et al, PLoS One. 2019). In this preliminary study, we isolated BM CD34+/lin- cells from 9 healthy donors (CTRLs). We investigated the gene expression profiling of 79 CP-CML patients at diagnosis vs. the same patients after 12 months of nilotinib treatment (12 months) vs. 9 CTRLs using Affymetrix HTA 2.0. Data was preprocessed and normalized using RMA and ComBat. Selection of differentially expressed genes (DEg) was performed at diagnosis vs. 12 months of nilotinib vs. CTRLs, using Statistical Analysis for Microarrays (SAM) on 3 groups and a Benjamini Hochberg false discovey rate threshold of 5%, followed, for significance comparisons, by a pair-wise SAM test. We focused on 34 genes of the cell cycle and mitosis, 6 genes belonging to the JAK-STAT signaling pathway, and the ABC transporter gene ABCD3 which were significantly under expressed at diagnosis vs. 12 months of nilotinib vs. CRTLs (Tab.1). In the cell cycle, we found that ORC2, ORC4, ORC5, MCM6, and HDAC2 (G1 phase) were progressively significantly down regulated at diagnosis vs. 12 months vs. CTRLs. We noticed HDAC2 which showed the high fold changes of 2.89 and 4.29 in the comparison between 12 months vs. CTRLs and between diagnosis vs. CTRLs, respectively. This gene plays a crucial role in CML, as HDAC inhibitors treatment represent an effective strategy to target leukemic stem cells in CP-CML patients receiving tyrosine kinase inhibitors. CCNA2, CDK7, CDC6, DBF4 (S phase), WEE1, PRKDC, ATM, MDM2, CCNB1 (G2 phase), and TTK, MAD2L1, BUB1, BUB3, ANAPC4, CDC27, SMC3, YWHAE, and YWHAZ (M phase) were progressively down regulated at diagnosis vs. 12 months vs. CTRLs. Among them, SMC3, YWHAE and YWHAZ showed the following high fold changes in the comparison between 12 months vs. CTRLs and between diagnosis vs. CTRLs: 2.31 and 3.15, 2.59 and 3.94, 2.75 and 4.15, respectively. Notably, the proto-oncogene MDM2 which promotes the development of tumors by targeting p53, was progressively up regulated in CTRLs vs. 12 months vs. diagnosis. In the mitosis, we detected that 10 genes playing a crucial role in mitotic chromosome organization, were progressively under expressed at diagnosis vs. 12 months vs. CTRLs (Tab.1). Notably, CLAPS2, ZW10 and ANLN (hematopoietic cell differentiation) regulate the exit from mitosis. NDC80, SMC4, ZNF207, and NEK2 take part in the mitotic sister chromatid segregation whereas CENPE and SMC2 are mitotic cell cycle check points. In the JAK-STAT signaling pathway, SOS1, PIK3CA, IL7, JAK2, STAM, and PTPN11 were progressively up regulated in CTRLs vs. 12 months vs. diagnosis (Tab.1). ABCD3, encoding a protein which pumped drugs out of the cells, was progressively under expressed at diagnosis vs. 12 months vs. CRTLs as shown in Tab.1. In conclusion, we found progressive gene expression changes in BM CD34+/lin- cells of 79 CP-CML patients at diagnosis vs. 12 months of nilotinib vs. the normal cell counterparts of 9 CTRLs in the cell cycle, JAK-STAT, and the ABC transporter ABCD3. FISH analyses demonstrated that the BM CD34+/lin- cells of 78/79 patients after 12 months of nilotinib were Ph-negative. Despite, our GEP results suggested that BM CD34+/lin- cells after 12 months of nilotinib treatment and the normal cell counterparts differed mostly in the expression of genes regulating the cell cycle and the JAK-STAT signaling pathway. Disclosures Rossi: Daiichi-Sankyo: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria. Elena:Novartis: Consultancy; Pfizer: Consultancy. Iurlo:Pfizer: Other: Speaker Honoraria; Incyte: Other: Speaker Honoraria; Novartis: Other: Speaker Honoraria.

Nilotinib Deregulates Cell Cycle Checkpoints, ABC Transporters Genes and JAK-STAT Signaling Pathway of CD34+/Lin- Cells in Chronic-Phase Chronic Myeloid Leukemia (CP-CML) Patients after 12 Months of Treatment

IntroductionChronic myeloid leukemia (CML) is a stem cell disease characterized by the constitutive activity of the oncoprotein BCR-ABL that activates multiple signal transduction pathways. Tyrosine-kinase inhibitor (TKI) nilotinib successfully inhibits the activation and the proliferative function of BCR-ABL in patients with CP-CML. Despite the success of nilotinib, some patients become refractory suggesting the presence of a population of Philadelphia positive (Ph+) quiescent stem cells escaping the drug activity. Thus, the molecular mechanisms underlying CML remain poorly understood.In this study, we enrolled 87 CP-CML patients (Pungolino et. al. Am J Hematol. 2018). Samples were collected on the behalf of the Rete Ematologica Lombarda (REL) the PhilosoPhi34 study (EudraCT: 2012-005062-34), which included 15 centers from Italy. We undertook gene expression profiling (GEP) of selected bone marrow (BM) CD34+/lin- cells of 80 patients at diagnosis vs. the same patients after 12 months of nilotinib to investigate gene expression changes induced by the treatment.MethodsWe isolated CD34+/lin- cells from BM samples in 87 patients at diagnosis whereas the same cells were also selected from 80/87 patients after 3, 6 and 12 months of nilotinib (Trojani et. al. Cancer Biomark. 2017). BM mononuclear cells (MNCs) as well as BM CD34+/lin- cells of all 80 CML patients were counted at diagnosis and during the treatment with nilotinib (at 3, 6, and 12 months, respectively). Standard FISH tested isolated BM CD34/lin- cells for the 87 patients at diagnosis, and for 80/87 patients after 3, 6 and 12 months of nilotinib treatment, respectively.Therefore, we performed GEP analyses of selected BM CD34+/lin- cells of 80/87 patients at diagnosis vs. the same patients after 12 months of nilotinib treatment. Then, we executed bioinformatic preprocessing and correction for batch effects on raw microarray data. Finally, we conducted differential expression analysis and significantly perturbed genes were subjected to functional clustering.ResultsWe observed a wide variability of the number of BM MNCs as well as the number of the BM CD34+/lin- cells among the 80 CP-CML patients at diagnosis and after 3, 6 and 12 months of nilotinib for each patient (Table 1). Figure 1 showed that the number of the BM CD34+/lin- cells dramatically decreased between the diagnosis and after 3 as well as 6 months of nilotinib. We noticed that the BM CD34+/lin- cells slightly increased between 6 and 12 months of nilotinib which might be caused by the gradual repopulation of the normal CD34+/lin- cells in the bone marrow as FISH results suggested.FISH analysis detected CD34+/lin- Ph+ cells in 87 CP-CML patients at diagnosis. No positive Ph+ nuclei were detected on CD34+/lin- cells of 79/80 patients after 12 months of treatment (to categorize a sample as negative, at least 200 nuclei were examined). All of these 79 patients achieved at least complete cytogenetic response. 1/80 patient relapsed at 12 months. We conducted GEP analyses on 78 subjects because, due to experimental issues, two patients were not considered for differential expression analyses, as the microarray CEL files of the 12 months' samples were corrupted and missed probe intensities for most of the probes.GEP analyses determined 2,959 significantly differently expressed probes between diagnosis and after 12 months of nilotinib treatment. Functional clustering identified some pathways significantly enriched between diagnosis and 12 months of nilotinib. Among these pathways, we found that ABCC4, ABCC5, and ABCD3 genes associated with ATP-binding cassette (ABC) transporters were up regulated at diagnosis. GEP results highlighted that 26 genes belonging to cell cycle, mitosis, DNA damage and repair were over expressed at diagnosis. Moreover, GEP data demonstrated that JAK-STAT signaling pathway was deregulated: JAK2, IL7, STAM, PIK3CA, PTPN11, RAF1, SOS1 were over expressed whereas IL22RA1 was under expressed at diagnosis vs. 12 months of nilotinib, respectively.ConclusionsIn summary, we reported that BM CD34+/lin- cells from CP-CML patients after 12 months of nilotinib were characterized by changes of expression of genes involved in cell cycle checkpoints and mitosis, ABC transporters genes that pump drugs outside form the cells, and JAK-STAT signaling pathway genes responsible for the proliferation, differentiation and cell survival in CML. DisclosuresRossi:Teva: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees.

Impact of Treatment Free Remission (TFR) with Nilotinib in 2nd Line for Chronic Myeloid Leukemia on Savings That May Fund All BCR-ABL Tests in the Brazilian Public Healthcare System during and after Nilotinib Treatment

Objectives: To estimate the savings and opportunity costs of nilotinib considering treatment free remission after 2nd line treatment of chronic myeloid leukemia (CML) with Nilotinib to fund BCR-ABL molecular test under the Brazilian public healthcare system (SUS) perspectiveMaterials and methods: A budget impact analysis was developed to estimate the savings for the public health system with TFR eligible patients. ENESTop (Mahon FX et al., 2018), an ongoing, single-arm, phase 2 study is the first trial specifically evaluating treatment-free remission (TFR; ie, stopping tyrosine kinase inhibitor [TKI] treatment without a loss of response) in patients with chronic myeloid leukemia in chronic phase (CML-CP) who achieved a sustained deep molecular response after switching from imatinib to nilotinib. In this trial, patients using nilotinib, who had 4.5 log molecular response after at least 24 months of Nilotinib treatment after switching from imatinib, entered into 12 months of consolidation phase and if they maintain MR 4.5 log response (77%), treatment was discontinued as planned in study protocol (57.9% TFR rate at 48 weeks - primary endpoint).A systematic review of the literature was developed over the efficacy of the use of nilotinib in 2nd line for patients diagnosed with CML. Efficacy data used to input the economic model is informed in a published randomized controlled clinical trial (ENESTcmr trial, Hughes TP et al., 2015), a 48 month, open-label, randomized, phase 3 study designed to investigate whether patients with complete cytogenetic response but persistent minimal residual disease on long-term imatinib ( ≥ 2 years prior imatinib) could achieve MR 4.5 log response by switching from imatinib to Nilotinib 400 mg BID. In this study, 33%, 42%, 46% and 52% of patients achieved 4.5 log molecular response by 12, 24, 36 and 48 months of nilotinib treatment after switching from imatinib. The total annual cost in 2017 with nilotinib was extracted from an official Brazilian database (http://paineldeprecos.planejamento.gov.br).Results: Considering that MR 4.5 log response patients will start consolidation in the first year, the economic impact will reduce in BRL 7,2M in the second year year, reaching a saving of BRL 11,4M in the fifth year. The total accumulated saving for the Brazilian public system (SUS) in 5-years was over BRL 38,1M. We also evaluated the necessary investments on BCR-ABL monitoring tests for all CML patients in the public market, as this test is not reimbursed by Brazilian public healthcare system (SUS). We estimated a budget impact of BRL 4.5M per year considering an average of 2 monitoring tests per patient, thus in 5 years an investment of 22,5M in BCR-ABL would be necessary.Discussion: The deep molecular response (MR 4.5 log) in may lead to patient discontinuation in those patients achieving 4.5 log molecular response after at least 24 months of nilotinib treatment and maintaining this response after 12 months. Furthermore, the possibility of TFR with nilotinib will save resources in health sytems allowing to invest in other needs. As demonstrated in this study, savings generated by nilotinib in 2nd line considering the possibility of TFR could allow the incorporation of BCR-ABL test for all CML patients in the public health system (SUS), which is essential in the current management of CML.Conclusion: Nilotinib in 2nd line treatment may generate savings for the public healthcare system (SUS) that could fund the BCR-ABL test for all CML patients. DisclosuresVivona:Novartis: Employment. Leite:Novartis: Employment. Matsuo:Novartis: Employment. Teich:Novartis: Consultancy.

Safety and Efficacy of a Switch to Nilotinib in Patients with CML-CP Showing MMR to Imatinib: Results of a Multicenter Phase II Trial (NILSw Trial)

Introduction:Tyrosine-kinase inhibitor (TKI) treatment generally induces a good response and shows long-term efficacy in patients with chronic myeloid leukemia in chronic phase (CML-CP). The ENESTnd trial demonstrated that nilotinib was superior to imatinib in terms of inducing rapid and deep responses and preventing transformation to acute phase/blast crisis in patients with untreated CML-CP. The optimal goal of CML-CP treatment is to cure the patient, such that they remain tumor free without continuing TKI treatment. The STIM trial showed that continuous complete molecular response (CMR) is essential for discontinuing TKI, while a major molecular response (MMR) is insufficient.Aim:Here we evaluated the efficacy and safety of a change to nilotinib in Japanese patients who achieved MMR with continuous detectable BCR-ABL1 transcript levels after imatinib treatment.Patients and methods:Patients with CML-CP who had achieved MMR but not CMR (MR4.5) after over 18 months of imatinib therapy were switched to treatment with nilotinib 400 mg twice daily for up to 24 months. BCR-ABL1 transcript levels were assessed every three months. The primary endpoint was the accumulative rate of CMR (MR4.5) up to 24 months after initiating nilotinib.Results:Between Feb. 2012 and Jan 2014, 53 patients were enrolled, of whom 39 met the eligibility criteria. We evaluated a total of 38 patients with a median age of 57.5 years (range, 22-76 years). Of these 38 patients, 27 completed 24 months of nilotinib treatment. The remaining 11 discontinued nilotinib due to retraction of consent (3 patients), MMR loss (1 patient), intolerance (3 patients), or adverse events (5 patients). Twenty patients (52.6%, 90% CI: 33.3-61.8%) achieved CMR (MR4.5). The accumulative incidences of achieving CMR (MR4.5) by 3, 6, 9, 12, 15, 18, 21, and 24 months were 21.1%, 34.2%, 42.1%, 47.4%, 47.4%, 47.4%, 47.4%, and 52.6%, respectively. None of the patients transformed to acute phase/blast crisis. Neither the periods of achieving CCyR nor MMR correlated to the incidence of achieving CMR (MR4.5) after switching from imatinib to nilotinib. The adverse events were consistent with those found in other nilotinib studies: grade 3/4 adverse events included anemia (5.3%), neutropenia (2.6%), thrombocytopenia (2.6%), lipase increase (5.3%), hypoglycemia (5.3%), hypophosphatemia (39.5%), ascites (2.6%), AST, ALT increase (2.6%), pulmonary edema (2.6%), muscle pain (2.6%), nausea (2.6%), skin rash (2.6%), dyspnea (2.6%), and hyponatremia (2.6%). Cardiovascular events included atrial fibrillation (G2), chest tightness and dyspnea (G1), myocardial infarction (G2), and heart failure (G3), which occurred in one patient each and the latter three complications led to nilotinib discontinuation.Discussion and Conclusion:This NILSw trial showed that nilotinib rapidly induced CMR in 52.6% of patients who had achieved MMR with continuous detectable BCR-ABL1 levels after long-term imatinib therapy. The safety concerns associated with nilotinib were close to those expected. Cardiovascular events during nilotinib treatment must be managed. Switching to nilotinib 400 mg twice daily may represent an alternative treatment strategy for inducing a deeper response (CMR) in relatively imatinib-resistant CML-CP patients. DisclosuresShibayama:Novartis Pharma: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria, Research Funding, Speakers Bureau; Takeda: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Ono Pharmaceutical: Speakers Bureau. Kawaguchi:Novartis: Honoraria. Kuroda:Janssen: Honoraria; Astra Zeneca: Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Nakamae:Mochida Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Novartis Pharma KK: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Other: travel/accommodation/meeting expenses, Research Funding. Matsumura:Bristol-Myers Squibb Company: Honoraria; Novartis Pharma K.K: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Consultancy, Honoraria; Pfizer Japan Inc.: Honoraria. Sunami:Takeda: Research Funding; Celgene: Honoraria, Research Funding; Novartis: Research Funding; Bristol-Myers Squibb K.K.: Research Funding; Daiichi Sankyo: Research Funding; Sanofi: Research Funding; Janssen Pharmaceutical: Research Funding; Ono Pharmaceutical: Research Funding. Akashi:Chugai Pharmaceutical Co., Ltd.: Research Funding; Kyowa Hakko Kirin: Consultancy, Research Funding; Shionogi & Co., Ltd: Research Funding; Asahi Kasei Pharma Corporation: Research Funding; Astellas Pharma: Research Funding; Bristol Meyers Squibb: Research Funding; Sunitomo Dainippon Pharma: Consultancy; Celgene: Research Funding. Kanakura:Bristol Myers: Research Funding; Alexionpharma: Research Funding; Nippon Shinyaku: Research Funding; Astellas: Research Funding; Eisai: Research Funding; Pfizer: Research Funding; Chugai Pharmaceutical: Research Funding; Shionogi: Research Funding; Kyowa Hakko Kirin: Research Funding; Toyama Chemical: Research Funding; Fujimotoseiyaku: Research Funding.

REL-Protocol PhilosoPhi34: An Open Label, Single Arm, Phase II Study of Nilotinib 300 Mg BID in Newly Diagnosed Chronic Phase Chronic Myeloid Leukaemia Patients, to Study the Disappearance of CD34+/Lin-Ph+ Cells from Bone Marrow during Treatment. Preliminary Data

Background. Chronic Myeloid Leukaemia (CML) can be effectively treated with the first generation Tyrosine Kinase Inhibitor (TKI) Imatinib, and more effectively with the second generation TKI, like Nilotinib. However, despite the deeper and faster responses induced by nilotinib in a large proportion of patients, the possible eradication of the pathological stem cells is not yet clearly elucidated. In fact, in vitro data suggest that quiescent stem cells are not sensitive to Bcr/Abl inhibition (Corbin AS, et al 2011; Hamilton A, et al 2012). A preliminary in-vivo study (Defina M, et al 2012) shows that in patients in CCyR even after short-term of nilotinib therapy, residual leukemic progenitors are rarely detected.Methods. On behalf of Rete Ematologica Lombarda (REL), Italy, we designed a single arm prospectic study, PhilosoPhi34 (EudraCT: 2012-005062-34), with the aim to investigate the efficacy of nilotinib 300 mg BID in obtaining the disappearance of Bone Marrow (BM) leukemic stem cells (CD34+/lin-Ph+) in newly diagnosed CP-CML.Primary objective of the study: to enumerate the BM CD34+/lin-Ph+ cells at the end of 6 months of treatment.Secondary objectives: to enumerate the BM CD34+/lin-Ph+ cells at 3 and 12 months; to assess the percentage of patients showing MR ≤10% IS at 3 months and MR ≤1% IS at 6 months and the MMR IS and MR4.5 IS by 3, 6 and 12 months of treatment.BM blood samples (range of 5-20 ml) were collected at diagnosis and after 3, 6 and 12 months of nilotinib treatment. BM mononuclear cells were purified by density gradient centrifugation and then CD34+/lin- cells were isolated using Diamond CD34 Isolation Kit (Miltenyi Biotec). The purity of CD34+/lin- cells was about 97% as determined by flow cytometry. BM CD34+/lin- cells were counted and a range of 100,000-800,000 has been noted at diagnosis.After the treatment we observed that the number of CD34+/lin- cells dramatically decreased after 3 (1,000-600,000), 6 (1,000-260,000) and 12 months (100-130,000). In particular, CD34+/lin- cells were even less than 1000 at 12 month of treatment.In order to verify the disappearance of leukemic stem cells, isolated CD34+/lin- cells were tested by standard FISH (i.e. to categorize a sample as negative at least 200 nucleus were examined).From April 2013 and June 2015 we enrolled 87 pts, as for protocol. We report here the preliminary results.Results. Of 56 patients in CCyR after 6 months of treatment, FISH performed on BM CD34+/lin- cells nuclei was evaluable in 51 cases (5 negative cases were excluded because of less than 200 nucleus were analysed). In 4 out of 51 patients (7.8%), Ph+ nuclei were detected. The Sokal score of these 4 patients was 1 low, 2 intermediate and 1 high risk with a ratio (positive nuclei/total nuclei) of 295/300, 1/200, 2/92, 3/300, respectively. Among 58 patients tested at 3 months and 44 tested at 12 months of treatment, the number of evaluable patients was 48 and 37, respectively; 8/48 (16.6%) and 0/37 (0%) patients showed Ph+ nuclei. Only 2 out of 8 positive patients had a high Sokal score.Regarding efficacy of treatment, Table 1 summarizes the MRs IS observed after 3, 6 and 12 months of treatment in 71, 57 and 41 patients, respectively.Conclusion. Data of this prospective study confirms that nilotinib 300 mg BID, rapidly and progressively induces the clearance of BM CD34+/lin-Ph+ cells in CP-CML patients. In particular, on CD34+/lin- cells, after 6 months of treatment, only 7.8% of patients showed positive nuclei. On 37 patients after 12 months of treatment, no positive nuclei were detected. So far, the kinetic of reduction of such cells seems not influenced by Sokal score.According to international studies, PhilosoPhi34 shows a very high efficacy of Nilotinib to induce MRs in CP-CML patients, at the standard time points.Table 1Molecular Response (MR) in CP-CML patients treated with Nilotinib 300mg BID from diagnosis.MR IS3 months6 months12 months≤10%67/71 94%57/57 100%40/41 97.50%≤1%57/71 80%55/57 96.50%40/41 97.50%≤0.1%17/71 24%41/57 72%35/41 85%≤0.01%3/71 4%19/57 33%20/41 48.70%MR4.5(UD)2/71 2.80%10 (6)/57 17.5% (10.5%)15 (9)/41 36.5% (22%)UD: undetectableWe acknowledge all REL Colleagues for their collaboration and Novartis SpA for the partial financial support to the study. DisclosuresNo relevant conflicts of interest to declare.

Quantification of BCR-ABL with Digital PCR Results in a Significantly Lower Rate of Deep Molecular Responses When Compared to RT-qPCR in CML Patients Treated in the ENEST1st Trial

Quantification of BCR-ABL with Digital PCR Results in a Significantly Lower Rate of Deep Molecular Responses When Compared to RT-qPCR in CML Patients Treated in the ENEST1st Trial

Identification of New microRNA Biomarkers and Candidate Target Genes in Primitive CML Cells Using Global Comparative RNA analyses

Imatinib mesylate (IM) and other ABL tyrosine kinase inhibitors (TKIs) have had a major impact on early phase CML patient outcomes. However, they are rarely curative and initial and acquired TKI resistance remain challenges. This is attributed to the finding that chronic phase CML stem cells are innately more resistant to IM and other TKIs than the bulk of the more mature cells they generate. To identify differentially expressed and new miRNAs in CD34+ CML stem/progenitor cells that could be potential biomarkers and therapeutic targets, we used Illumina Deep Sequencing to obtain absolute miRNA expression profiles of highly purified CD34+ cells obtained at diagnosis from three CML patients who were classified retrospectively, after IM therapy, as IM-responders and three as IM-nonresponders. CD34+ cells isolated from five normal bone marrow (NBM) samples were similarly analyzed as controls. Bioconductor DESeq analysis revealed 63 differentially expressed miRNAs in the CD34+ cells from CML and NBM samples (P<0.05). Interestingly, 12 of these were differentially expressed in CD34+ cells from the IM-responders and nonresponders. Most of the 63 differentially expressed miRNAs identified were present at reduced levels in the CD34+ CML cells as compared to NBM, but 17 were increased. In addition, 34 novel miRNAs were identified in the CD34+ CML stem/progenitor cells. We next validated sequencing data in CD34+ cells from IM-responders (n=12), IM-nonresponders (n=10) and normal individuals (n=11) using a high-throughput quantitative microfluidic device. These studies confirmed the differential expression in CD34+ CML cells of 32 of the 63 identified miRNAs (P<0.05), including an increased level of oncomirs miR-155 and miR-17-92, and a decreased level of the tumor suppressors, miR-145, miR-151, and miR-452. Importantly, we detected significant changes in some of these miRNAs in CD34+ cells from CML patients after three months of nilotinib (NL) treatment (23 normalized after three months of NL treatment, whereas 10 showed little change). To further correlate miRNA profiles with corresponding mRNA expression changes, and to identify potential target genes, RNA-seq was performed on the same RNA samples. Bioconductor RmiR analysis was performed to match miRNA target genes whose expression was inversely correlated with the expression of the deregulated miRNAs based on three of six prediction algorithms (mirBase, TargetScan, miRanda, tarBase, mirTarget2, and PicTar). We identified 1,210 differentially expressed mRNAs that are predicted targets of the deregulated miRNAs in the comparison of CML and NBM data. Strikingly, only seven differentially expressed mRNAs were predicted targets of the deregulated miRNAs identified from a comparison of the IM-responders and nonresponders. Most of these are predicted to have roles in regulation of the cell cycle, MAPK signaling and TGF-beta signaling pathways by DAVID Bioinformatics Resources, which clusters predicted target genes to known KEGG pathways. Thus, aberrant, differentially expressed miRNAs and target genes identified in primitive CML stem/progenitor cells may serve as useful biomarkers to predict clinical response of CML patients to TKI therapy, and may ultimately lead to identification of potential therapeutic targets for improved treatment of CML patients. DisclosuresNo relevant conflicts of interest to declare.

Microarray of Bone Marrow CD34+/Lin- Cells from Patients with Chronic Myeloid Leukemia (CML)

This study aimed to determine the gene expression profiling (GEP) of bone marrow (BM) CD34+/lin- cells of CML patients at diagnosis and after nilotinib treatment.Microarray was performed on 30 CML patients at diagnosis as well as 8 patients after 12 months of nilotinib treatment using the latest generation Affymetrix GeneChip HTA 2.0 to further investigate genomic complexity.Bone marrow blood (in a range of 5-20 ml) samples were collected from patients at diagnosis and after 3, 6 and 12 months of nilotinib treatment. BM mononuclear cells were purified by density gradient centrifugation at 800 rpm for 20 min and soon after CD34+/lin- cells were isolated using Diamond CD34 Isolation Kit (Miltenyi Biotec). The purity of CD34+/lin-cells was about 97% as determined by flow citometry and the cells were counted at diagnosis and after the treatment with nilotinib (at 3, 6, and 12 months). BM CD34+/lin- cells of 30 patients at diagnosis and after 3 and 6 months of nilotinib were resuspended in RNAlater (Ambion, Life Technologies) and stored at -20°C until RNA extraction was performed. Total RNA was extracted from BM CD34+/lin- cells using MagMAX 96 Total RNA Isolation Kit (Ambion).BM CD34+/lin- cells were counted and a range of 100,000-800,000 has been noted at diagnosis.cRNA was prepared according to OvationPico WTA System V2 kit followed by Encore Biotin Module Kit (NuGEN). Ultimately, cRNA was hybridized to HTA 2.0 and signals were scanned by Affymetrix GeneChip Scanner 3000 following the manufacturer’s instructions.We observed that the number of CD34+/lin- cells dramatically decreased at 3 months (1,000-600,000), after 6 months (1,000-260,000) and after 12 months (100-130,000). In particular, CD34+/lin- cells were even less than 1000 after 12 months of nilotinib.If the number of CD34+/lin- cells after 12 months of nilotinib was less than 1000, we directly resuspended the cells in Prelude direct Lysis module (NuGEN) and microarray experiments were performed without RNA extraction. In this case, cRNA was prepared using Ovation One Direct System kit followed by Encore Biotin Module Kit (Nugen) and finally hybridized to the HTA 2.0.Microarray experiments were performed on CD34+/lin- cells of 30 CML patients at diagnosis with HTA 2.0.Moreover, we performed microarray experiments on CD34+/lin- cells of 8 CML patients at diagnosis vs. 8 CML patients after 12 months of nilotinib using HTA 2.0.Data was preprocessed and normalized using Robust Multi-array Average (RMA) algorithm. The Significant Analysis of Microarrays (SAM) was used to identify genes with statistically significant changes in expression in CML patients at diagnosis and at 12 months of treatment. P-values were corrected for multiple testing using false discovery rate.In conclusion, as far as we know, this is the first study which has isolated BM CD34+/lin-cells from CML patients at diagnosis and after nilotinib treatment.We observed a wide variety of the number of CD34+/lin- cells in 30 CML patients at diagnosis as well as after 3, 6 and 12 months of nilotinib.In particular, CD34+/lin- cells of CML patients at diagnosis were over 100.000. RNA was extracted and cRNA was prepared with OvationPico WTA System V2 kit followed by Encore Biotin Module Kit (NuGEN) and finally hybridized to HTA 2.0.On the other hand, the number of CD34+/lin- cells of CML patients after 12 months of nilotinib markedly decreased as low as 100 cells. In this case, we decided to avoid RNA extraction from the cells and we directly prepared cRNA using Ovation One Direct System kit and Encore Biotin Module Kit (Nugen) followed by the hybridization to the HTA 2.0 Array.To increase the statistical power in biomarker identification, we combined samples pre-processed with different experimental protocols (NuGEN) using ComBat, an empirical Bayes framework that adjusts data for batch effects and is robust to outliers in small datasets. Principal component analysis applied to the expression data before and after ComBat adjustment shows that the adopted normalization procedure effectively removes the systematic bias introduced by different protocols. DisclosuresNo relevant conflicts of interest to declare.

Prognostic Value of Multi-Drug Resistance 1 Gene (MDR1) Expression in Newly Diagnosed Patients with Chronic Myeloid Leukemia on Nilotinib Treatment—a Subanalysis of the ENEST1st Study

Introduction MDR1 gene expression has recently been reported to be associated with clinical outcome in imatinib-resistant patients with chronic myeloid leukemia (CML) on second-line nilotinib treatment (Agrawal et al., Leukemia 2014). High MDR1 expression was predictive for achieving deeper cytogenetic and molecular responses as well as duration of chronic phase. We thus prospectively evaluated the value of MDR1 expression analysis in the first-line situation within a large European, multicenter trial of CML patients under treatment with nilotinib.Methods 305 patients enrolled on the ENEST1st study (Evaluating Nilotinib Efficacy and Safety in clinical Trials as First-Line Treatment, NCT01061177) consented to participate in this substudy. Of these, 225 patients were evaluable due to sample availability and presence of typical e14a2 and/or e13a2 BCR-ABL1 transcripts. The median age was 52 (range 18-83), 39% were female. Prior to therapy, the expression of MDR1, BCR-ABL1, and GUSB were determined using a serial dilution of a plasmid constructs harboring MDR1 and GUSB and BCR-ABL1 and GUSB sequences. Ratios MDR1/GUSB and BCR-ABL1/GUSB were calculated. ROC curves were established using initial MDR1 and BCR-ABL1 expression levels aiming to achieve the primary endpoint of MR4 (defined as BCR-ABL1 ≤ 0.01% on the International Scale [BCR-ABL1IS] or undetectable BCR-ABL1 in cDNA with ≥ 10,000 ABL1 transcripts) at 18 months or to achieve a MMR (≤ 0.1% BCR-ABL1IS) at 9 months. Mann-Whitney tests have been applied using a significance level of 0.05.Results At 9 months, 161 patients (72%) achieved MMR and at 18 months 111 patients (49%) a MR4. The median ratio MDR1/GUSB among all evaluable patients was 11.4% (range, 0.6-318.1), the median ratio BCR-ABL1/GUSB was 22.2% (range 0.09-198.7). Initial BCR-ABL1/GUSB levels did not predict MMR or MR4 at 9 or 18 months. However, initial ratios MDR1/GUSB were predictive for the achievement of MMR at 9 months (best cut-off 3.8%, sensitivity 88.8%, specificity 26.6%, AUC 0.598, p=0.022) and for reaching MR4 at 18 months (best cut-off 7.90%, sensitivity 72.1%, specificity 51.8%, AUC 0.624, p=0.001).Conclusions High MDR1 expression levels in newly diagnosed CML patients appear to be associated with deeper molecular responses within the first 18 months of nilotinib treatment. These data merit further exploration and if validated would justify the investigation of frontline CML therapy guided by baseline MDR1 expression levels. DisclosuresDietz:Novartis: Research Funding. Hanfstein:Bristol-Myers Squibb: Honoraria; Novartis: Research Funding. Rousselot:Novartis: Research Funding. Lange:Novartis: Consultancy, Honoraria, Research Funding. Maier:Novartis: Research Funding. Foroni:Novartis: Research Funding. Gerrard:Novartis: Research Funding. Talmaci:Novartis: Research Funding. Janssen:Novartis: Research Funding. Frank:Novartis: Employment. Saussele:Pfizer: Honoraria, Travel, Travel Other; Bristol-Myers Squibb: Honoraria, Research Funding, Travel, Travel Other; Novartis: Honoraria, Research Funding, Travel Other. Giles:Novartis: Honoraria, Research Funding. Hochhaus:Novartis: Research Funding. Müller:Novartis: Honoraria, Research Funding.

Leukemic Stem Cell Quantification Is Of Prognostic Value In Newly Diagnosed Patients In Chronic Phase Chronic Myeloid Leukemia (CML-CP) Receiving Nilotinib Therapy: Results From The ENEST1st Stem Cell Substudy

BackgroundLeukemic stem cells (LSCs) are considered to be very important therapeutic targets in CML, as it has been shown both in vitro and in vivo that tyrosine kinase inhibitor (TKI) therapy is not able to eradicate these entirely. We previously reported that LSC burden at diagnosis has significant prognostic impact on therapy outcome in imatinib and dasatinib treated CML patients. The ENEST1st study (NCT01061177) is focused on examining the role of first-line nilotinib therapy in patients in CML-CP. This translational, multinational, multicenter ENEST1st substudy addresses the predictive value of the stem cell profile in these patients. Patients and methodsBone marrow (BM) and/or peripheral blood (PB) samples were collected from newly diagnosed CML-CP patients before and after 1 and 3 months of nilotinib treatment. The LSC burden was analyzed by FISH and flow cytometry (FC). With the FISH method, CD34+ cells were pre-selected with paramagnetic beads after which they were fractionated into CD38+ (upper 80%, “progenitor”) and CD38- (lowest 5%, “stem cell”) pools using flow sorting. The proportion of Ph+ cells was assayed by counting 1,000 cells with interphase FISH using specific BCR-ABL1 probes. In the FC method, the stem cell compartment was defined as the lowest 1% of CD34+CD38- cells. LSCs were distinguished from normal hematopoietic stem cells (nHSCs) by either higher light scatter properties, and/or aberrant expression of CD7, CD11b and CD56, and/or higher CD45 and CD90 expression. Patient samples were assigned to as “residual nHSCs present” or “no residual nHSC present” at diagnosis and as “residual LSCs present” or “no residual LSCs present” at 1 and 3 months. Altogether, 48 patients from 6 European countries were investigated. ResultsBy FISH analysis at diagnosis, the proportion of BCR-ABL+ cells in the stem cell compartment varied markedly in individual patients (1%-100%) and was lower (85%) than in progenitor (96%) or whole BM fractions (96%, p<0.05). This LSC burden correlated significantly with high WBC count (r=0.44, p=0.008), hemoglobin (r=-0.51, p=0.002), spleen size (r=0.51, p=0.002) and PB (r=0.57, p=0.0002) and BM (r=0.55, p=0.0005) blast percentage. Weak correlation was also found with Sokal score (r=0.37, p=0.03). By FC method, similar to FISH, the range of LSCs was 0%-100% with a median of 73%. Forty percent of patients had detectable residual nHSC at diagnosis. These patients had significantly lower Sokal score (p=0.001), smaller spleen size (p=0.006), lower platelet count (p=0.04) and lower PB blast percentage (p=0.001). With both methods no prognostic relationship existed between LSC burden and eosinophil/basophil counts. Interestingly, by FISH method, the LSC burden at diagnosis correlated also with BCR-ABL transcript levels according to the international scale at 3 (r=0.39, p=0.03), 9 (r=0.41, p=0.01) and 18 months (r=0.42, p=0.02). Borderline significant associations were also found at 6 (p=0.06) and 12 (p=0.07) months. All patients lacking major molecular remission at 12 or 18 months, had >80% of BCR-ABL+ cells in the stem cell fraction at diagnosis. When LSCs were analyzed by the FC method, patients with residual nHSCs at diagnosis had significantly lower BCR-ABL levels at 3 (0.27 vs 1.74, p=0.025) and 6 (0.13 vs 1.21, p=0.03) months, but this was not reflected in lower BCR-ABL levels at 12 or 18 months. During nilotinib therapy, the proportion of BCR-ABL+ cells by FISH decreased rapidly both the in progenitor and stem cell compartments. At 3 months the median LSC percentage was 0.28%, and it did not differ significantly from the values detected either in the progenitor fraction (0.34%) or whole BM (0.29%). Similarly, only a few patients had detectable LSCs left during follow-up time-points with the FC method. ConclusionsLSC burden at diagnosis reflects the biology of the disease in newly diagnosed CML-CP patients. It also carries a prognostic value in first-line treated nilotinib patients, and a substantial number of patients not meeting the optimal response criteria at the 12-month time-point have high LSC burden at diagnosis. Furthermore, the presence of residual nHSCs at diagnosis is predictive for molecular response at 3 and 6 months. Nilotinib therapy markedly decreases LSC pool during the first 3 months of treatment. Noteworthy, the reduction is as potent in the LSC compartment as it is in more mature progenitor cell or whole BM fractions. Disclosures:Richter:Novartis: Consultancy, Research Funding, Speakers Bureau; Bristol Myers Squibb: Consultancy, Speakers Bureau. Fioretos:Novartis: unrestricted research grant Other. Giles:Novartis: Consultancy, Research Funding. Hochhaus:Novartis: Consultancy, Honoraria, Research Funding, Travel Other; BMS: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria. Ossenkoppele:Novartis: Consultancy, Research Funding, Speakers Bureau; Bristol Myers Squibb: Consultancy, Speakers Bureau. Porkka:Novartis: Consultancy, Research Funding, Speakers Bureau; BMS: Consultancy, Research Funding, Speakers Bureau. Wolf:Novartis: Honoraria, Research Funding; Pfizer: Honoraria; Bristol-Meyers Squibb: Honoraria. Janssen:Novartis: Consultancy, Research Funding. Mustjoki:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau.

Mechanism of impaired glucose metabolism during nilotinib therapy in patients with chronic myelogenous leukemia

Hyperglycemia represents frequent adverse event reported in chronic myelogenous leukemia (CML) patients treated with nilotinib. In order to determine the major mechanism of glucose metabolism impairment, we performed a metabolic analysis using an oral glucose tolerance test as well as assessment of incretins and adipokines at baseline and after 3 months of nilotinib treatment in patients with CML. We proved that rapid insulin resistance, compensatory hyperinsulinaemia, and hypoadiponectinaemia develop after initiation of nilotinib therapy, which clarifies not only the mechanism of impaired glucose metabolism, but also explains the fast development of dyslipidaemia and peripheral artery occlusion in nilotinib-treated CML patients.

Nilotinib exacerbates diabetes mellitus by decreasing secretion of endogenous insulin

We report a 74-year-old female with chronic myelogenous leukemia (CML) in accelerated phase with pre-existing severe type 2 diabetes (T2D) and hemorrhagic gastric ulcers who was successfully treated with nilotinib. We first considered second-generation tyrosine kinase inhibitors for the treatment of this patient, as they elicit a superior response compared with imatinib. We next selected nilotinib, rather than dasatinib, since the increased risk of bleeding associated with dasatinib represented a greater risk of fatality than aggravation of T2D with nilotinib. After improvement of hemorrhagic gastric ulcers and T2D with exogenous insulin therapy, we began nilotinib administration; insulin dose was increased to maintain her glucose levels whereas urine C-peptide level decreased. Conversely, when nilotinib was discontinued due to liver dysfunction, the dosage of injected insulin was decreased and urine C-peptide levels increased. After re-starting nilotinib, the required dose of insulin gradually increased again, and urine C-peptide level decreased, indicating that nilotinib may have impaired secretion of endogenous insulin. The patient obtained a complete cytogenetic response after 3months of nilotinib treatment. Her T2D has since been well controlled by insulin therapy. To our knowledge, this is the first report that nilotinib treatment for patients with severe T2D may induce a reversible decrease in endogenous insulin secretion, although the precise underlying mechanisms remain unknown. We highly recommend sufficient screening and early intervention with exogenous insulin therapy for diabetic CML patients who receive nilotinib.

Analysis of Bone Marrow Microenviroment Factors As Early Markers of Response in Patients with Newly Diagnosed Bcr-Abl Positive CML in Chronic Phase Treated with Nilotinib.

AbstractAbstract 2795In the era of molecular target therapy, whereas Imatinib has shown an overall survival rate of 85% and an estimated event-free survival of 55% in a 8 year result update of IRIS trial, several in vitro data have confirmed that Ph+ stem cells housed within BM niches are resistant to TKI treatments. Second generation TKI Nilotinib has been designed with improved target specificity over Imatinib. Its efficacy in the treatment of patients with CML who are resistant to or intolerant to Imatinib led to the registration of a clinical trial CAMN107EIC01, a phase IIIb, multicentre, open-label study applying Nilotinib in the treatment of newly diagnosed CML. The aim of the current study (CAMN107EIC01 sub-study N10) is to define BM microenvironment markers that nurture and determine leukemic stem cell fate in the BM niches of Nilotinib treated patients. We enrolled 37 patients involving 21 Italian centers, from whom written informed consent has been obtained for sub-study N10. Patients have been monitored by Real Time RT-PCR (RT-qPCR) for the expression of the fusion mRNA BCR-ABL, as specified by the core protocol. Major Molecular Response (MMR) is defined as detectable disease ≤0.1% BCR-ABL according to the international scale (IS). Plasma and mononuclear cells have been collected from BM and PB samples of the enrolled patients at the screening visit (V0) and after 3 months of treatment (V4). We purified total RNA from BM and PB mononuclear cells of the enrolled patients to screen by RT-qPCR the expression of 10 genes (ARF, cKIT, CXCR4, FLT3, LIF, NANOg, PML, PRAME, SET and TIE), involved in the regulation of the stemness and survival signaling of hematopoietic stem cells. RT-qPCR results were normalized by the expression of ABL mRNA (Normalized mRNA copy Number: NCN). Moreover, we evaluated by multiplex ELISA assay BioPlex, the BM plasma concentration level of 20 cytokines (IL1a, IL1b, IL3, IL6, IL7, IL8, IL10, IL12, IL15, G-CSF, M-CSF, SCF, SDF1, TRAIL, HGF, PDGFbb, GM-CSF, MIP-1a, TNFa and VEGF), known to be key factors in the interaction of Ph+ stem cells to BM microenvironment. The interim analysis of MMR until the 12th month is available in 26 out of 37 enrolled patients. We observed that MMR is achieved during the first 12 months of treatments in 17 out of 26 (65%) patients. Molecular analysis showed that the expression of two genes involved in the regulation of stem cell pluripotency (NANOg) and cytokine signaling (SET) were significantly down-regulated at V0 in PB mononuclear cells of patients achieving MMR in comparison to cells from non-responding patients (4vs21 NANOg NCN, p=0.05; 8vs32 SET NCN, p=0.05). These data were also confirmed on BM patient’s sample. Moreover, we investigated the concentration level of the above-mentioned soluble factors in BM plasma samples of all 37 enrolled patients at both V0 and V4. We observed that the BM plasma level of several cytokines produced by leukemic cells significantly decreased during the first 3 months of Nilotinib treatment: IL3 (54vs3ng/ml; p=0.02), M-CSF (56vs12 ng/ml; p=0.005), SCF (170vs104 ng/ml; p=0.007), HGF (7565vs705 ng/ml; p<10-5), IL10 (67vs28 ng/ml; p=0.02), IL12 (167vs73 ng/ml; p=0.03) and VEGF (1326vs404 ng/ml; p= 0.0003). Whereas the plasma level of a stroma-derived cytokine SDF1, playing an important role in regulating the retention of hematopoietic progenitor cells within the BM microenvironment, was significantly increased at V4 respect to V0 (171vs390 ng/ml; p= 0.0004). Also, we observed that SCF and HGF concentration levels were significantly lower in the plasma of patients at V4 that achieved MMR compared to those in non- responding patients (71vs134 ng/ml, p=0.02; 421vs737 ng/ml, p=0.01; respectively). These data indicate that expression analysis of genes involved in cell pluripotency (NANOg) and cell signaling (SET), as well as measurement of the plasma concentration level of several soluble factors produced by leukemic progenitor cells and stroma cells (SCF and HGF) may assist in the early prediction of molecular response in patients treated with Nilotinib. Moreover, further studies need to be conducted to evaluate the functional role of stroma secreted cytokines, such as SDF1 and VEGF, in the regulation of TKI responsiveness in these subset of CML patients, since it may be plausible that the same factors that modulate hematopoiesis promote also leukemogenesis, enhance blast survival and make them resistant to treatment within the BM microenvironment. Disclosures:Paratore:Novartsi: Employment. Galimberti:Novartis: Employment. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Multicenter Phase II Clinical Trial of Nilotinib for Patients with Imatinib-Resistant or Intolerant CML From the East Japan CML Study Group (EJCML) Trial: Evaluation of Molecular Responses by the BCR-ABL1 Mutational Status and Plasma Trough Concentration of Nilotinib

AbstractAbstract 1360 Introduction:The tyrosine kinase inhibitor (TKI) imatinib is used as the first-line therapy for newly diagnosed chronic myeloid leukemia (CML). However, some patients fail to respond or become intolerant to imatinib. Nilotinib is a second-generation TKI with higher selectivity and more potent inhibitory effects on BCR-ABL than imatinib. Several studies have shown hematologic and cytogenetic responses to nilotinib in patients with imatinib-resistant or intolerant CML. Purpose:To investigate the safety and efficacy of nilotinib for patients with imatinib-resistant or intolerant, chronic (CP)- or accelerated (AP)-phase CML from the East Japan CML Study Group (EJCML) trial by evaluating molecular responses in terms of the BCR-ABL1 mutational status and plasma trough concentration of nilotinib. Methods:In this multicenter phase II clinical trial, nilotinib (400 mg bid) was administered orally for one year and the molecular responses were monitored by means of the international scale of quantitative PCR (IS-PCR). BCR-ABL1 mutations were analyzed by direct sequencing at the baseline and 12 months or at the time of the event for discontinuation of the treatment (i.e., progressive disease, insufficient effects, or severe adverse events). The plasma trough concentration of nilotinib was measured by high-performance liquid chromatography 3 months after nilotinib administration. Results:From March 2009 through February 2011, 51 patients were registered in this study, and data of 49 patients whose molecular responses were evaluated by the IS-PCR were analyzed (imatinib-resistant CML = 33, imatinib-intolerant CML = 16; CP CML = 46, AP CML = 3). The median follow-up period was 12.0 months (range = 0.1–13.3 months). At 6 and 12 months, the major molecular response (MMR; ≤0.1% IS) rates were 52.5% and 67.6%, respectively, and the complete cytogenetic response (CCyR)-equivalent (≤1.0% IS) rates were 75.0% and 85.3%, respectively. Five types of BCR-ABL1 mutations (M244V, F317L, N358D, F359V, and E459K) were detected in 6 patients (12.2%) at the baseline, but the M244V, N358D, and E459K mutations disappeared after the nilotinib treatment. Acquired BCR-ABL1 mutations (Y253H, I418V, and exon 8/9 35bp insertion) were detected in 3 patients (8.6%) at 12 months or at the time of the event; these patients did not achieve a CCyR or an MMR. No patients showed an acquired mutation of T315I. Most patients except 11 subjects (22.4%) still received the treatment. The reasons for discontinuation were progressive disease in one patient with an F317L mutation, insufficient effects in one patient without any mutation, and adverse events in 9 patients (thrombocytopenia in 5 patients, hyperbilirubinemia in 2 patients, headache in one patient, and heart disease in one patient). Among 30 patients without BCR-ABL1 mutations, the plasma trough concentration of nilotinib was significantly higher in 21 patients with an MMR than in those without an MMR by 12 months (median = 1255.1 ng/mL vs. 372.8 ng/mL, P = 0.0012 by Mann–Whitney U-test; see the figure). The concentration of 761 ng/mL was significantly associated with an MMR by 12 months in a receiver-operating characteristic (ROC) curve analysis of the best sensitivity (76.2%) and specificity (77.8%). Conclusion:The patients with imatinib-resistant or intolerant, CP or AP CML, even those having BCR-ABL1 mutations M244V, N358D, and E459K, achieved an MMR by 12 months of nilotinib treatment. The plasma trough concentration of the drug was related to the MMR by 12 months, and the plasma threshold of nilotinib should be set above 761 ng/mL. These findings suggest that nilotinib shows good efficacy and tolerability in Japanese patients with imatinib-resistant or intolerant, CP or AP CML. (ClicalTrials.gov, UMIN ID 000002201) [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Evaluation of residual CD34+Ph+ progenitor cells in chronic myeloid leukemia patients who have complete cytogenetic response during first‐line nilotinib therapy

Compared with imatinib, nilotinib is a potent breakpoint cluster region/v-abl Abelson murine leukemia viral oncogene (bcr-abl) kinase inhibitor, and it induces higher rate and rapid complete cytogenetic response (CCyR), yet no clinical data are available regarding its efficacy against chronic myeloid leukemia (CML) stem cells. Earlier studies demonstrated that clusters of differentiation 34-positive, Philadelphia chromosome-positive (CD34(+) Ph(+) ) cells are detectable in about 45% of patients with CML, despite being on long-term imatinib therapy and having achieved sustained CCyR. CD34(+) cells from bone marrow of de novo CML patients in the chronic phase (n = 24) treated with nilotinib (median duration of therapy, 22 months) were isolated and scored for BCR-ABL by fluorescent in situ hybridization (FISH) analysis. Similar analysis was also performed in 5 de novo CML chronic phase patients who achieved CCyR within 3 months of nilotinib therapy. FISH evaluation of a median of 100 CD34(+) nuclei per patient revealed that only 1 of 20 (5%) evaluable patients showed residual Ph(+) progenitor cells. In this patient, just 1 of 140 (0.7%) CD34(+) interphase nuclei was found to be positive for BCR-ABL. Surprisingly, no CD34(+) Ph(+) cells were found even in those 5 patients evaluated after 3 months of nilotinib treatment. This study assessed for the first time the persistence of CD34(+) Ph(+) cells during nilotinib first-line treatment. Preliminary results showed that in patients in CCyR, even after short-term nilotinib therapy, residual leukemic progenitors are very rarely detected compared with imatinib-treated CCyR patients. It is yet to be determined if these findings will have an impact in the path to a cure of CML with tyrosine kinase inhibitors.

Successful Peripheral Blood Stem Cells Collection in Imatinib Pretreated and Nilotinib-Treated Chronic Myeloid Leukemia Patient

We report a case of a successful mobilization and harvest of the peripheral blood stem cells (PBSCs) in imatinib-pretreated and nilotinib treated 52-year-old woman diagnosed with Philadelphia chromosome-positive and BCR-ABL (b2a2) positive chronic phase CML in 2/2002. She failed interferon-alfa and imatinib treatment. She achieved her first complete molecular remission after 16 months of nilotinib treatment and later on was mobilized with filgrastim at a dose of 10 ug/kg/day applied subcutaneously once daily. The total number of 2.98 × 106 CD34+ cells/kg was harvested on the fourth day of the mobilization. The autologous graft of the stem cells was cryopreserved and tested for the residual disease: the FISH revealed negative results and the RT-PCR was positive (BCR-ABL/ABL ratio 0,0017 in RQ-PCR). To our knowledge, this is the first report of successful PBSC harvest in a patient significantly pretreated with imatinib and nilotinib.