Search

Background Targeted therapy with Tyrosine-Kinase-Inhibitors (TKIs) totally modified the course of treatment of Chronic Myeloid Leukemia (CML). The objectives and the needs of treatment have been modified during the last years and the discontinuation of therapy is now a feasible aim. However, a lot of biological data acquired in the last twenty years, showed that degree and mechanisms of Leukemic Stem Cells (LSCs) clearance during TKI treatment are not clearly established as well as the predictive criteria for a stable and prolonged Treatment Free Remission (TFR). The multicentre, prospective, single-arm PhilosoPhi34 study (EudraCT: 2012-005062-34) was designed by the Rete Ematologica Lombarda (REL), to verify the in-vivo activity and time-course of first-line Nilotinib (NIL) therapy on Bone Marrow (BM) CD34+/lin-Ph+ cells clearance. An exploratory Gene Expression Profiling (GEP) study of CD34+/lin- cells at diagnosis and at 12 months (mos) of treatment, for the first 30 evaluable pts, was included. Preliminary GEP data suggested a correlation between different NFKBIA expression at diagnosis and at 12 mos and the achievement of a deeper Molecular Response (MR) (Pungolino et al, AJH 2018). We report here some results of GEP analysis on all enrolled evaluable pts and their possible correlation with clinical data. Methods BM cells were collected and stored at diagnosis and at 12 mos of treatment. CD34+/lin- cells were purified with a Diamond CD34 Isolation Kit Miltenyi (97% of purity). For GEP analysis we used Affymetrix HG-U133 Plus 2.0 microarray and Genechip platform (Affymetrix) and the Affymetrix GeneChip Scanner 3000. Data was pre-processed and normalized using the Robust Multi-array Average (RMA) algorithm. The Significant Analysis of Microarrays (SAM) was used to identify genes with statistically significant changes in expression. P-values were corrected for multiple testing using false discovery rate, for differentially expressed genes confirmation. We chose to analyse different expression of NFKBIA (the inhibitor of NF-kB onco-gene) in order to confirm the preliminary data reported on the first 30 analysed pts. Pts were monitored according to ELN-recommendation. Biological data were correlated with MR at 3, 12 and 36 mos of therapy. We use Fishers test to compare unbalanced group. Results Out of the 87 enrolled pts, 80 completed the first 12 mos of treatment and 78 (1 failure and 77 CCyR) were evaluable for GEP analysis. We observed 2726 genes symbol differentially expressed of which 1868 are coding genes. Among these, JAK-2 showed a down regulation at 12 mos (p: .024). JAK-2 expression ranged from 2.62 to 4.95 at diagnosis and from 1.48 to 5.58 at 12 mos. Only 26/78 pts increased JAK-2 expression that was > 4 in 1/26 pts, at diagnosis; 2/26 (7.69%) pts showed a H Sokal. Other 52/78 pts decreased JAK-2 expression that was ≥ 4 in 21/52 pts, at diagnosis; 10/52 (19.23%) pts and 6/21 (28,57%) pts showed a H Sokal. Similarly, when we compared low JAK-2 expression (< 3.5) vs vary high expression (≥ 4) 2/21 vs 6/22 pts had H Sokal (9.52% vs 27.27%; p: .0057). Considering the role of JAK-2 and NFKBIA in cell regulation and survival, we evaluated how the combination of their different expression impact on MR (i.e. NFKBIA increased expression/JAK-2 decreased expression vs NFKBIA decreased expression/JAK-2 increased expression). Data are reported in Table 1 and 2. Conclusion GEP analysis showed a down regulation of JAK-2 expression after 12 mos of first line NIL treatment, in 78 early chronic phase CML pts. Data suggest that high expression of JAK-2, at diagnosis, correlate with H Sokal score. However, H Sokal pts with a JAK-2 down regulation, obtain during treatment similar MR compared to L Sokal pts. Additionally, the study confirms our preliminary observation on 30 pts , concerning the role of NKBIA up - regulation in increasing percentage of earlier and deeper MR . The better condition of NFKBIA and JAK-2 expression (up regulation of NFKBIA and down regulation of JAK-2) is associated with a higher percentage of early MR3 and optimal responses over time, despite the higher number of H Sokal pts in this group. A study with NIL as first line treatment combined with low dose of JAK-2 inhibitor and a natural inhibitor of NF-kB (such as curcuma), during the first year of treatment, to increase the deeper MR rate and the probability of TFR is warrented. Disclosures Rossi: Sandoz: Honoraria; Jazz: Membership on an entity's Board of Directors or advisory committees; Celgene: 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; Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria; Teva: Membership on an entity's Board of Directors or advisory committees; Mundipharma: Honoraria; Novartis: Honoraria; Pfizer: 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.

Chronic Myeloid Leukemia is a clonal disorder characterized by the presence of the Ph-chromosome and the BCR-ABL tyrosine-kinase (TK). Target-therapy with Imatinib has greatly improved its outcome. Deeper and faster responses are reported with the second-generation TKI Nilotinib. Sustained responses may enable TKI discontinuation. However, even in a complete molecular response, some patients experience disease recurrence possibly due to persistence of quiescent leukemic CD34+/lin−Ph+ stem cells (LSCs). Degree and mechanisms of LSCs clearance during TKI treatment are not clearly established. The PhilosoPhi34 study was designed to verify the in-vivo activity and timecourse of first-line Nilotinib therapy on BM CD34+/lin−Ph+ cells clearance. Eighty-seven CP-CML patients were enrolled. BM cells were collected and tested for Ph+ residual cells, at diagnosis, 3, 6 and 12 months of treatment. FISH analysis of unstimulated CD34+/lin− cells in CCyR patients were positive in 8/65 (12.3%), 5/71 (7%), 0/69 (0%) evaluable tests, respectively. Per-Protocol analysis response rates were as follows: CCyR 95% at 12 months, MR4.5 31% and 46% at 12 and 36 months, respectively. An exploratory Gene Expression Profiling (GEP) study of CD34+/lin− cells was performed on 30 patients at diagnosis and after, on 79 patients at diagnosis vs 12 months of nilotinib treatment vs 10 healthy subjects. Data demonstrated some genes significantly different expressed: NFKBIA, many cell cycle genes, ABC transporters, JAK-STAT signaling pathway (JAK2). In addition, a correlation between different expression of some genes (JAK2, OLFM4, ICAM1, NFKBIA) among patients at diagnosis and their achievement of an early and deeper MR was observed.

BACKGROUND: 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 are not fully defined. OBJECTIVE: We undertook a gene expression profiling (GEP) study of selected bone marrow (BM) CD34+/lin-cells of chronic-phase CML patients at diagnosis and after 12 months of TKI nilotinib to investigate molecular signatures characterizing both conditions. METHODS:We selected and counted BM CD34+/lin- cells of 30 CML patients at diagnosis and during 3, 6 and 12 months of first-line nilotinib treatment. GEP was performed between CD34+/lin- cells of patients at diagnosis and the same patients after 12 months of nilotinib. RESULTS: The number of BM CD34+/lin- cells dramatically decreased after 3, 6 and 12 months of nilotinib. GEP detected 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. CONCLUSIONS: In conclusion, lipid, glucose and sphingolipid metabolism, insulin resistance, complement and coagulation, platelet activation, cytoscheleton, cell adhesion, transport, B cell differentiation, RAS-signaling pathway, proliferation, growth factors, and apoptosis were significantly deregulated between CML patients at diagnosis and after 12 months of nilotinib.

Introduction Chronic 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. Methods We 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. Results We 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. Conclusions In 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. Disclosures Rossi: 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.

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 1. Molecular Response (MR) in CP-CML patients treated with Nilotinib 300mg BID from diagnosis. MR IS 3 months 6 months 12 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: undetectable We acknowledge all REL Colleagues for their collaboration and Novartis SpA for the partial financial support to the study. Disclosures No relevant conflicts of interest to declare.