Chronic Myelogenous Leukemia Patients Research Articles

Impact of chromosomal translocation and genomic instability on personalized medicine.

structural exchanges between heterologous chromosomes. We recently reviewed the critical impact of chromosomal translocations on cancer diagnosis, prognosis and treatment [4]. Specific chromosomal translocations are often closely associated with subtypes of leukemia and lymphoma, which has greatly facilitated the diagnosis and stratification of patients for certain types of treatments [4]. A classic example is the t(9;22) translocation, which generates the BCR–ABL fusion product in chronic myelo genous leukemia (CML) patients [5]. The cancer cell-specific BCR–ABL fusion protein is a target of the therapeutic agent STI-571 (Gleevec), which induces remission in the vast majority of CML patients [6]. The efficacy of Gleevec in CML is an early and key success that cancer research has delivered in personalized medicine. The Gleevec story also exemplifies the essential contribution of basic research, which enabled discovery of the molecular mechanisms of leukemo genesis and development of targeted therapies for the disease. Another success of individualized therapy in leukemia is the use of all-trans retinoic acid [7] and arsenic trioxide [8–10] in acute promyelocytic leukemia patients harboring the t(15;17) translocation [11]. The t(15;17) translocation fuses a portion of the PML gene to the RARA gene to encode the PML–RARA fusion protein [12]. Combined therapy of all-trans retinoic acid and arsenic trioxide induces a high rate of remission and survival in the vast majority of acute promyelocytic leukemia patients [13]. Many clinics now routinely screen for these translocations, performing karyotyping and cytogenetic analyses of leukemia or lymphoma cells before a specific treatment is identified. These successful medical practices owe credit to the extensive basic and The concept of personalized medicine The term ‘personalized medicine’, in the context used today, first appeared in a Wall Street Journal article in 1999 (already implicating its potential economic impact), which later appeared in the Oncologist [1]. In 2008, the President’s Council of Advisors on Science and Technology published a report, ‘Priorities for Personalized Medicine’, which provided a comprehensive definition of personalized medicine and highlighted its potential to influence healthcare and economics [101]. In recent years, personalized medicine has gained much wider recognition in the scientific community [2]. An ongoing paradigm shift from standard medicine (one-size-fits-all) to personalized medicine can be attributed to two important factors. First is the increasing incidence of chronic diseases such as cancer or metabolic diseases that tend to display more variation in clinical presentation, rather than acute infectious diseases such as those caused by bacteria. Second are rapid advances in new ‘omics’ technologies, including genomics, proteomics and metabolomics, which enable better characterization of individual variation and profiling of clinical differences at the molecular level. Cancer research has been at the forefront of personalized medicine, largely because of the low treatment response rate in a majority of cancers, the intrinsic heterogeneity of cancers, successful examples of targeted cancer therapy, and recent advances in genomic approaches for cancer pathology [3].

Alantolactone inhibits growth of K562/adriamycin cells by downregulating Bcr/Abl and P‐glycoprotein expression

Alantolactone, a sesquiterpene lactone containing an α-methylene-γ-lactone group, is the active component of Inula helenium (Compositae), a traditional Chinese medicinal herb. It has been reported that alantolactone has the capacity to inhibit tumor cell growth through induction of apoptosis. The purpose of this study was to assess the effects of alantolactone in the adriamycin (ADR)-resistant human erythroleukemia cell line K562/ADR, and provide evidence that it might function as a potent therapeutic agent in chronic myelogenous leukemia (CML) patients with Bcr/Abl and the multidrug-resistance phenotype. Our results showed that alantolactone significantly inhibited K562/ADR cell growth by downregulating Bcr/Abl and P-glycoprotein expression. Alantolactone also induced apoptosis via modulation of protein levels of Bcl-2 family members, caspase activation, poly ADP ribose polymerase cleavage, and cytochrome C release. We also observed that alantolactone induced cell-cycle arrest in the G2/M phase, downregulated cyclin B1 and cyclin-dependent protein kinase 1, and upregulated the cyclin-dependent kinase inhibitor p21. Together, these results demonstrate that alantolactone may be a potent therapeutic agent against CML, and a potential Bcr/Abl inhibitor.

The 2010 Benjamin Franklin Medal in Life Science presented to Peter C. Nowell

Peter Nowell is known for his discovery that a small terminal deletion on chromosome 22 is a consistent feature in leukemic cells from chronic myelogenous leukemia (CML) patients. This was the first direct evidence that a human cancer could be the result of a somatic (chromosomal) mutation, and paved the way for the development of a therapy that now cures 95% of individuals with CML.

Inactivation of P16 (INK4a) Gene by Promoter Hypermethylation is Associated with Disease Progression in Chronic Myelogenous Leukaemia

Background: Chronic Myelogenous Leukemia (CML) has a typical progressive course with transition from a chronic phase to a terminal blast crisis phase. The mechanisms that lead to disease progression remain to be elucidated. Promoter hypermethylation is one of the putative mechanisms underlying the inactivation of negative cell-cycle regulators in haematological malignancies. Therefore, aim of our study was to examine whether the methylation status of P16 (INK4a) gene is a useful biomarker in the development and progression of CML.Material and Methods: The methylation status of p16INK4A gene was evaluated by Methylation Specific Polymerase Chain Reaction (MSP) in 200 CML patients among which, 81 were in CP-CML, 54 in AP-CML and 65 in BC-CML.Results: The p16INK4A gene was hypermethylated in 84 of 200 (42%) of CML patients (P<0.0001). Among the three stages p16 (INK4A) promoter gene was methylated in 26% (CP-CML), 43% (AP-CML and 68% (BCCML) patient (P<0.0001). Methylation was more frequent in blastic and accelerated phase patients than in chronic phase. A significant correlation was found between p16INK4A methylation and loss of Imatinib response. Similarly higher frequency of p16INK4A methylation was reported in CML patients with haematological (P<0.02) and molecular resistances (P<0.04). Significantly higher (p<0.0001) frequency of p16INK4A promoter methylation was reported in patients with thrombocytopenia. However no correlation was found between p16INK4a hypermethylation and other clinic-pathological parameters like age, gender, BCR-ABL transcripts etc.Conclusion: Our results suggest that p16INK4a is a primary target for inactivation by promoter methylation in the disease progression of CML patients and that its detection is useful in the follow up of patients with a high risk of developing CML and resistance to Imatinib therapy.

Inactivation of RIZ1 Gene by Promoter Hypermethylation is Associated with Disease Progression and Resistance to Imatinib in Indian Chronic Myelogenous Leukemia Patients, First Study from India

Background: The epigenetic impact of DNA methylation in chronic myelogenous leukemia (CML) is not completely understood. RIZ1 expression and activity are reduced in many cancers. In CML, blastic transformation is associated with loss of heterozygosity in the region where RIZ1 is located. RIZ1 is a PR domain methyltransferase that methylates histone H3 lysine 9, a modification important for transcriptional repression. In CML blast crisis cell lines RIZ1 represses insulin-like growth factor-1 expression and autocrine signaling. Together these observations suggest that RIZ1 may have a role in the chronic phase to blast crisis transition in CML. Methods: To examine whether promoter methylation is involved in the disease development and progression of CML, we investigated promoter methylation status of RIZ1 gene in 100 chronic myeloid leukemia’s (CML) patients and 50 controls by MSP method. Results: The RIZ1 methylation was studied in 100 CML patients, 9 were cases were methylation positive cases, six of nine were in blastic phase, 2 in chronic phase and one patient in accelerated phase. It was seen that RIZ1 methylation was increased significantly from early to advanced phase. The higher frequency of RIZ1 methylation was reported in haematologically resistant cases (42% vs 2%) and molecularly resistant cases (16.77% vs 1.92%) than the responders. The higher frequency of RIZ1 methylation was found in CML patients who were treated with interferon initially followed by imatinib treatment. Also RIZ1 hypermethylation was associated with faster disease progression p<0.003 than the non methylated cases. No correlation was found between RIZ1 gene methylation with age, thrombocytopenia, types of bcr/abl transcripts of CML patients. Conclusion: We conclude that epigenetic silencing of RIZ1 gene is associated with CML progression and imatinib resistance. Early detection of RIZ1 methylation could be a predictive marker for imatinib resistance and disease progression in CML.

Does HOXA9 Gene Expression in Egyptian Chronic Myelogenous Leukemia Patients Affect Disease Progression? A Retrospective Cohort Study

Objective: Chronic myelogenous leukemia (CML) is a clonal stem cell disease and is consistently associated with the BCR-ABL fusion gene. The chronic phase of the disease tends to pass into an accelerated phase and eventually leads to acute leukemia if left untreated. Oncoproteins necessary for leukemic transformation are both fundamentally and clinically relevant to identify as they might be new molecular targets for the development of specific anti-leukemic drugs. This study is an initial step to define the proportion of HOXA9 gene expression in some Egyptians with chronic-phase CML at diagnosis and to evaluate its relation with BCR-ABL expression and its clinical significance. Materials and Methods: Sixty-two newly diagnosed CML patients (56 in chronic phase, 1 in accelerated phase, and 5 in blastic crises) were enrolled in the study. HOXA9 and BCR-ABL gene expressions were detected by one-step RT-PCR. ABL was chosen as a control gene to calculate HOXA9/ABL and BCR-ABL/ABL ratios from densitometric values of PCR product intensities. Results: HOXA9 expression was encountered in 25/56 (44.6%) of newly diagnosed CML patients in the chronic phase. The median expression was 0.31 (range: 0.08-1.37) in relation to the ABL gene, with a higher frequency of expression in CML patients presenting with splenomegaly (p<0.001), high Sokal score (p<0.001), and BCR-ABL expression from the first round (p=0.004). No association could be detected with other clinical parameters, overall survival, or disease-free survival. Conclusion: HOXA9 expression is closely related to poor prognostic factors, but we could not demonstrate its relationship to patient survival. Conflict of interest:None declared.

Bosutinib in the management of chronic myelogenous leukemia

Bosutinib (SKI-606) is an orally available, once-daily dual Src and Abl kinase inhibitor, approved by the US Food and Drug Administration for the treatment of adults with chronic, accelerated, or blast-phase Philadelphia chromosome-positive chronic myelogenous leukemia who are intolerant of or resistant to first- or second-generation tyrosine kinase inhibitors. Bosutinib effectively overcomes the majority of imatinib-resistance-conferring BCR-ABL mutations except V299L and T315I. In the Bosutinib Efficacy and Safety in chronic myeloid LeukemiA (BELA) trial, bosutinib attained a faster and deeper molecular response than imatinib in newly diagnosed chronic-phase chronic myelogenous leukemia patients. Treatment-emergent adverse events are usually very manageable. Low grade, mostly self-limiting diarrhea represents the most frequently observed toxicity of bosutinib. Anti-diarrheal drugs, antiemetic agents, and/or fluid replacement should be used to treat these patients. The improved hematological toxicity of bosutinib compared with other tyrosine kinase inhibitors has been ascribed to its minimal activity against platelet-derived growth factor receptor and KIT. In this review, we give an overview on the profile of bosutinib, the clinical potential and treatment-emergent adverse events.

Analysis of mutations in the BCR-ABL1 kinase domain, using direct sequencing: detection of the T315I mutation in bone marrow CD34+ cells of a patient with chronic myelogenous leukemia 6 months prior to its emergence in peripheral blood.

It has been shown that the occurrence of the BCR-ABL1 T315I mutation leads to a very poor therapeutic outcome in chronic myelogenous leukemia (CML) patients treated with tyrosine kinase inhibitors. Therefore, early detection of this mutation could potentially lead to early therapeutic intervention and a better prognosis with the ongoing treatment regimen. The detection of BCR-ABL1 kinase domain (KD) mutations was performed by direct sequencing of peripheral blood (PB), total bone marrow (BM), and BM CD34+ cells from a reported CML patient. In this patient, the T315I mutation was detected in BM CD34+ cells 6 months prior to its emergence in PB, suggesting evolution and expansion of the T315I mutation clone, which most likely originated from more primitive CML cells. Our finding reflects the natural development of a T315I mutation within the hematopoietic system of the reported patient and indicates the importance of BCR-ABL1 mutation monitoring in more primitive cell populations. Considering the natural history of T315I development in this reported CML case, we hypothesize that BCR-ABL1 KD mutations may be pre-concentrated in more primitive CML cells, which subsequently expand into the PB. These findings may have future implications for the strategy used for detecting BCR-ABL1 mutations.

A Case with Coexistence of Major and Minor BCR/ABL Fusion Transcript at Lymphoblastic Crisis of Chronic Myelogenous Leukemia in Patients with Major BCR/ ABL Positivity during Chronic Phase

A Case with Coexistence of Major and Minor BCR/ABL Fusion Transcript at Lymphoblastic Crisis of Chronic Myelogenous Leukemia in Patients with Major BCR/ ABL Positivity during Chronic Phase

Complete Clearance of Ph+ Metaphases after 3 Months Is a Very Early Indicator of Good Response to Imatinib as Front-Line Treatment in Chronic Myelogenous Leukemia

Aim: To address the incidence and the prognostic role of a very early standard complete cytogenetic response (CCyR) or all Ph– metaphases (MET–, when <20 cells were evaluable). Methods: We revised 182 chronic phase chronic myelogenous leukemia patients treated with frontline imatinib (IM) at two institutions from June 2002 to June 2011. Results: After 3 months of treatment, 138 patients (75.8%) achieved CCyR/MET– while 44 patients (24.2%) still presented Ph+ metaphases (MET+) (<33%, 24 patients; ≥33%, 20 patients). On univariate analysis, palpable spleen enlargement (p < 0.001), WBC count >100.0 × 10⁹/l at onset (p < 0.001), and male gender (p = 0.019) had a negative impact on achievement of CCyR/MET– at 3 months. Among patients with CCyR/MET– after 3 months, there were 15 failures (10.8%) compared to 21 (47.7%) among patients with MET+ (p < 0.001). The 5-year overall survival was 97.0% in patients CCyR/MET– at 3 months and 91.8% in patients MET+ at 3 months (p = 0.277); the 5-year progression-free survival was 88.2% in patients CCyR/MET– at 3 months and 48.4% in patients MET+ at 3 months (p < 0.001). Conclusions: The achievement of CCyR/MET– at 3 months seems to have prognostic relevance and could be a very early and useful indicator of an excellent response to IM beyond European LeukemiaNet guidelines.

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.

Chronic Myelogenous Leukemia Patients with Highly Nilotinib-Resistant BCR-ABL Mutations Demonstrate Similar Efficacy to Dasatinib, but Have a Higher Likelihood of Developing New Mutations in the Event of Clinical Resistance

AbstractAbstract 3755 Objective:Assess the outcome of imatinib- and/or nilotinib-failure chronic myelogenous leukemia (CML) patients with pre-existing, highly nilotinib-resistant mutations (Y253H, E255K/V and F359V/C), during dasatinib therapy. Methods:Sixty-one CML patients who failed imatinib (n=44) or imatinib and nilotinib (n=17), including 20 in chronic phase (CP), 16 in accelerated phase (AP) and 25 in blast phase (BP), were switched to dasatinib, except for patients with highly dasatinib-resistant mutations (T315I/A, F317L/V/I/C and V299L). Patients were grouped into 3 cohorts: no mutation (n=22), no-specific mutation (any mutation except highly nilotinib-resistant mutations; n=15), or specific mutation (Y253H, E255K/V or F359V/C; n=24), according to baseline BCR-ABL mutation status. Cumulative incidence frequencies of hematological response (HR), complete hematological response (CHR), major cytogenetic response (MCR), complete cytogenetic response (CCR) and major molecular response (MMR) were compared. Probabilities of clinical resistance to dasatinib (according to European LeukemiaNet recommendations), progression-free survival (PFS) and overall survival (OS), and dynamics of BCR-ABL mutations during dasatinib therapy were also assessed. Results:Prior nilotinib was associated with a higher proportion of specific mutation versus no mutation (50.0% vs 13.6%, P=0.001) and non-specific mutation (50.0% vs 13.3%, P=0.02). Patients harboring specific mutation also had a higher frequency of advanced phase CML before dasatinib treatment than patients with no mutation (83.3% vs 50.0%, P=0.008), and a similar frequency to those with non-specific mutation (83.3% vs 66.7%, P=0.266). Response, PFS and OS rates, and follow-up duration were similar among the 3 cohorts by baseline mutation status, as shown in Table. Probability of clinical resistance to dasatinib in patients with specific mutation was not significantly different to those with no mutation (54.2% vs 77.3%, P=0.100) or non-specific mutation (54.2% vs 33.3%, P=0.204) despite that there was a significant difference between the cohort with no mutation and non-specific mutation (77.3% vs 33.3%, P=0.008). Among 32 patients who developed clinical resistance to dasatinib and were assessed for BCR-ABL mutation status, newly detectable mutations were identified in 14 patients and most frequently occurred in those already harboring specific mutation versus those with no mutation (76.9% vs 26.7%, P=0.008) and non-specific mutation (76.9% vs 0%, P=0.015) at baseline. T315I (9/14, 64.3%) was the most common newly acquired BCR-ABL mutation. Harboring specific mutation compared with other mutation states at baseline (76.9% vs 16.7%, P=0.001), and developing hematological resistance rather than cytogenetic resistance during dasatinib therapy (65.0% vs 8.1%, P=0.002), were identified as risk factors associated with the development of new mutations. Conclusions:Imatinib- and/or nilotinib-failure patients with highly nilotinib-resistant BCR-ABL mutations demonstrate similar efficacy to dasatinib as those with no or any other mutation. However, patients with highly nilotinib-resistant mutations had a higher likelihood of developing new mutations at the time of clinical resistance. More extensive studies are needed to assess the significance of various BCR-ABL mutations prior to and during tyrosine kinase inhibitor therapy for CML.Table:Response, survival and clinical resistance to dasatinib by baseline BCR-ABL mutation statusMutation statusP valueNo mutation n=22Non-specific mutation n=15Specific mutation n=24HR, n (%)16 (72.7)14 (93.3)21 (87.5)0.202CHR, n (%)16 (72.7)12 (80.0)19 (79.2)0.832MCR, n (%)a9 (69.2)8 (80.0)12 (70.6)0.826CCR, n (%)a8 (61.5)8 (80.0)10 (58.8)0.511MMR, n (%)a3 (23.1)5 (50.0)4 (23.5)0.2813-year PFS, %84.673.765.20.4693-year OS, %75.680.854.00.798Clinical resistance to dasatinib, n (%)17 (77.3)5 (33.3)13 (54.2)0.027Follow-up (months), median (range)31 (2..C48)24 (2..C48)7 (1..C48)0.277a40 patients were evaluated including 13 with no mutation,10 with non-specific mutation and 17 with specific mutation Disclosures:No relevant conflicts of interest to declare.

CML Patients Show Sperm Alterations At Diagnosis That Are Not Improved On Tyrosine Kinase Inhibitor Treatment

AbstractAbstract 1669Most epidemiologic studies performed in chronic myelogenous leukemia (CML) relate that the disease occurs preferentially in males with a sex ratio of ∼1.2. In addition, CML can be diagnosed in young adults and masculine fertility is a matter of concern, particularly because tyrosine kinase inhibitors (TKI) may impact on spermatogenesis by a selective inhibition of Src kinases, PDGF-R and c-kit. Sperm cryopreservation is recommended by some authors at diagnosis in males that would expect to have children later on. In a retrospective analysis we have analysed the spermograms of 62 chronic phase (CP) and 2 onset blast crisis (BC) CML males referred to our 3 centres between 2001 and 2012, collected at diagnosis before TKI treatment, and we have compared the results obtained to those of 15 healthy volunteer donors from the cryopreservation bank database, after informed consent. In 10 patients we could collect some data for patients being on imatinib mesylate (IM). CML patients had a median age of 31 (16–48) years, significantly younger than that in the control group of healthy donors: 37 (34–45) years (p=0.001). Sokal scores were 24% high, 27% intermediate and 49% low for evaluable patients (13 patients unknown or not available). The median BCR-ABLIS value at diagnosis was 77.65%. Patients had a median duration of 26 (0–38) days of hydroxyurea prior to commencing any TKI and 65% of evaluable patients had HU before TKI. None of the patients got interferon prior to TKI. The semen cryopreservation was performed within a median of 10 (2–102) days after CML diagnosis and after a median abstinence of 5 (0.5–30) days. The median volume of semen obtained in CML patients was 2.95 (0.5–14.9) ml and 3 (1.4–5.3) ml for normal donors (p=0.3). Williams test showed 72 (0–87)% of necrospermia in patients versus 18 (4–32)% in donors (p=0.00003). The median number of spermatozoa obtained was not different in patients [46 (0.03–200) 106/ml] than that in donors [74 (19.2–253) 106/ml] (p=0.24), as well as the number of spermatozoa per ejaculate observed (p=0.49). The motility of spermatozoa at 30 minutes after collection was not different between patients (median = 47.5%) and donors (median = 50%) (p=0.12), however higher numbers of atypical spermatozoa were observed in patients [median = 77.5 (16–100)%] rather than in donors [median = 45% (22–89)%], p=0.008, and the multiple abnormalities index (MAI) was significantly higher in patients [median = 1.99 (1.14–2.7)] than that in donors [median = 1.33 (1.09–1.55)], p=0.00006. There was no correlation between age at diagnosis, Sokal index and the number of spermatozoa per ml obtained (p=0.7 and 0.21 respectively).Ten CP CML patients had spermograms after a median of 1440 (9–1456) days of IM treatment and the results obtained were compared to i) the results of each individual patient at CP diagnosis and ii) to the results of healthy comparators. In comparison to the characteristics observed at diagnosis, the semen volume (median = 3.1 ml), Williams test (median = 65%), the motility at 30 minutes (median = 37.5%) and the MAI (median = 1.71) were not different (p=ns for all), however, the numbers of spermatozoa (median = 14.9 106/ml and = 37.05 ml per ejaculate) collected on IM were significantly lower (p=0.014 and p=0.045 respectively). The different parameters evaluated on IM were compared to those of normal controls and showed significant alterations. The semen volume was not different (p=0.94), neither the motility of spermatozoa (p=0.24), but the Williams test was highly perturbed on IM [median 65 (24–79)% versus 18 (4–32)% in donors] p=0.00003, as well as the numbers of spermatozoa as 106 per ml, collected on IM [median 14.9 (0.67–179)) versus normal [74 (19.2–253)], p=0.0036 or as 106 per ejaculate collected on IM [median 37.5 (2.68–572.8)) versus normal [149 (30–535.3)], p=0.026. Atypical forms were significantly more abundant on IM [median = 80 (68–90)%] versus healthy controls [median = 45% (22–89)], p=0.0058. Finally, the MAI was severely altered on IM [median = 1.71 (1.61–1.98)] versus normal individuals [median = 1.33 (1.09–1.55)], p=0.00013.In conclusion, this work demonstrates the existence of significant sperm alterations in young males with CML at diagnosis of undetermined origin, prior to any treatment. These alterations persist on IM treatment and little is know about the impact of second generation TKI. Thus the most appropriate approach remains a matter of debate in thus setting. Disclosures:Nicolini:Novartis, Bristol Myers-Squibb, Pfizer, ARIAD, and Teva: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Huguet:Novartis, BMS: Speakers Bureau. Michallet:Novartis, Pfizer, Teva, Genzyme, Janssen Cilag, BMS, Merck, Pfizer, Gilead, Alexion: Consultancy, Speakers Bureau. Etienne:Novartis, Pfizer, speaker for Novartis, BMS: Consultancy.

Targeting STAT5 Expression Resulted in Molecular Response Improvement in Patients with Chronic Phase CML Treated with Imatinib

AbstractAbstract 696 Background.We have previously demonstrated that PPAR-γ agonists (such as pioglitazone) negatively regulate the level of Stat5A and Stat5Bgene expression in normal CD34+ bone marrow progenitors (Prost, Le Dantec et al. 2008). Aim.We investigated whether targeting Stat5 expression with pioglitazone may impact clonogenic activity of Chronic Myelogenous Leukemia (CML) cells in vitro and result in molecular response improvement in vivo. Patients and methods.Preliminary in vitro studies tested the ability of pioglitazone to impact viability and clonogenicity of CD34+ primary cells from CML patients (pts). We conducted clonogenic and LTC-IC studies. Cultivated Ph+ -CD34+cells were characterized by facs and analyzed by CSFE assay. BCR-ABL and Stat5 expression were quantified by real time PCR. Control experiments were conducted using lentiviral vectors and siRNA assay for Stat5 and PPAR-g.CML pts were eligible in the ACTIM trial (EudraCT 2009–011675-79) if they were treated by imatinib for at least 2 years with a stable daily dose for at least 3 months and in major molecular response without having achieved CMR4.5 (defined by a BCR-ABL/ABL IS ratio ≤ 0.0032%). After inclusion, pts received imatinib (no dose modification) and started pioglitazone (Actos®) 30 mg/d during 2 months and 45 mg/d thereafter for 12 months. BCR-ABL transcript level was monitored every 3 months during the study period. Primary objective was the proportion of patients achieving a confirmed undetectable level of BCR-ABL transcript. Secondary objectives included cumulative incidence of CMR4.5 and safety. A companion biologic study evaluated imatinib through levels, Stat5 expression in bone marrow at baseline, months 6 and 12 and clonogenic activity of bone marrow mononuclear cells at baseline, months 6 and 12. Results.From the in vitro studies we first demonstrated that pioglitazone at pharmacological doses inhibited cell growth of the Bcr-Abl positive cell line K562 through the activation of the PPAR-g/STAT5 pathway. We next showed that PPAR-g / STAT5 pathway induced a clonogenic defect in CD34+ cells from CML patients. Moreover, the activation of the PPAR-g / STAT5 pathway also induced a clonogenic and a proliferative defect in CML LTC-IC. We then confirmed that imatinib induced a selection of insensitive quiescent CML cells and showed that this effect was abrogated by the activation of the PPAR-g / STAT5 pathway.Twenty seven pts were enrolled in the clinical trial and 24 were evaluable (1 was excluded in CMR4.5, 1 pt was not in MMR and 1 pt had consent withdrawal). Median age was 61.6 years (24.1–79) and median follow-up after inclusion was 13 months (9.8–21). All evaluable pts started pioglitazone as planned. Seven pts (29.2%) discontinued pioglitazone before 12 months, 6 following investigator decision after the warning of the French ministry of health regarding the risk of bladder cancer and 1 after its own decision. No pt discontinued due to adverse events. Discontinuations occurred between month 3 and month 9. Median duration of pioglitazone therapy was 11.2 months (2.6–15.4) median daily dose was 39.9 mg. No interaction was observed between imatinib and pioglitazone in term of through level before (median 850 ng/ml) and after (median 927 ng/ml) pioglitazone initiation (p=ns). Main adverse events were weight gain and worsening fluid retention in 3 pts. Three pts (14%) obtained a confirmed undetectable level of BCR-ABL transcript. The one year cumulative incidence of CMR4.5 was 57%. Stat5 mRNA quantification was significantly diminished in pt samples at M6 and M12 compared to the baseline values and a reduction of the clonogenic potential was also observed in bone marrow cells at M6 and M12. We collected “control pts” with similar characteristic (n=20). None of these pts obtained a confirmed CMR and the cumulative incidence of CMR4.5 in this control group was 27% as compared to 57% in the pioglitazone group (p=0.02). Conclusion.We have extended our in vitro results showing that PPAR-γ agonists resulted in Stat5 down regulation in CML CD34+ cells and preferentially reduced their clonogenic and long term potency in CFCs and LTC-IC assays. We now demonstrated that these effects translate in vivo by the achievement of MMR in more than half of the pts treated with the combination of pioglitazone and imatinib suggesting that it may be possible to target quiescent CML cells in vivo and supporting the concept of stem cell pool erosion. Disclosures:Off Label Use: pioglitazone in CML. Roy:Novartis, BMS: Speakers Bureau. Legros:Novartis, BMS: Research Funding, Speakers Bureau. Coiteux:Novartis, BMS: Speakers Bureau. Mahon:Novartis, BMS: Consultancy, Research Funding.

Osteolineage Connexin 43 Expression Is Required for Stromal Support of Leukemic Stem Cells in Vitro and in Vivo

Abstract 4090Leukemic stem cells (LSC) are believed to be responsible for disease maintenance and/or propagation. A hallmark example is chronic myelogenous leukemia (CML). CML is a lethal malignancy that results from hematopoietic stem cell (HSC) transformation by the oncogene BCR-ABL. BCR-ABL tyrosine kinase inhibitors (TKI) are highly effective drugs to induce disease remission in CML patients and prolong their survival, but do not eliminate the LSC population and are required to be maintained throughout the patient’s life to prevent relapse. Therefore, there is a strong rationale to develop strategies to target residual LSC to enable cessation of TKI treatment without leukemia relapse. Growing evidence indicates that signals from the bone marrow (BM) microenvironment are crucial for the maintenance of LSC and it has been suggested that LSC and HSC may locate in different niches where distinct microenvironment generated signals arise. We have previously demonstrated that Connexin-43 (Cx43) regulates HSC activity by preventing chemotherapy-induced senescence/apoptosis by allowing stromal-mediated ROS scavenging (Taniguchi-Ishikawa E et al., PNAS 2012), and allows homing/engraftment of HSC through stromal mediated trans-stromal migration (Gonzalez-Nieto D et al., Blood 2012). Here, we analyzed whether the microenvironment Cx43 function is required for BCR-ABL LSC maintenance or BCR-ABL LSC are independent of microenvironment Cx43-mediated signals. For this purpose, we utilized binary transgenic mice which express p210-BCR-ABL driven by inducible (Tet-off) expression of Scl in HSC. An enriched fraction of LSC (1,000 Lin−/c-kit+/Sca1+ cells) from Dox-off Scl-tTA × TRE-BCR-ABL (SclTg) mice (CD45.2+) was competitively transplanted with 3×106 CD45.1+ BM cells as previously described by our group (Sengupta A et al., Blood 2010 & Blood 2012) into lethally-irradiated Col1α1-WT (OB/P WT) or Col1α1-Cx43f/f (OB/P Cx43Δ/Δ) mice. HSC/P BM chimera was analyzed at 18 weeks post-transplantation by measuring the BM content of CD45.2+ BM cells, CD45.2+ Lin−/c-kit+/Sca1+ (HSC/multipotential progenitors) and CD45.2+ Lin−/c-kit+/Sca1− (progenitors) BM cells. Chimera of equivalent CD45.1+ populations was used as an internal control of normal HSC engraftment. Interestingly, the content of CD45.2+ or CD45.1+ Lin−/c-kit+/Sca1+ was not significantly modified by the deficiency of Cx43 in the osteolineage microenvironment (average ∼ 0.50% of BM cellularity). However, the content of progenitors from SclTg mice was ∼85% decreased in OB/P Cx43Δ/Δ mice while the content of competitor progenitors was also decreased albeit at a lower level (∼30% reduction), indicating that the hematopoietic ability of LSC and HSC were significantly impaired by the deficiency of Cx43 in the osteolineage microenvironment. To determine whether this defect was exclusively dependent on osteolineage deficient BM stroma, we performed long-term BM cultures of Scl-tTA × TRE-BCR-ABL (or non-Tg control from littermates) LSK cells onto WT or Cx43-deficient irradiated stroma obtained from OB/P Cx43Δ/Δ mice (Gonzalez-Nieto D et al., Blood 2012). At the end of 4 weeks of culture, we observed an average reduction in the number of CFUs of 84% (vs 86% for Non-Tg control HSC) when LSC were cultured on OB/P Cx43-deficient stroma. Altogether, these data indicate that the osteolineage deficiency of Cx43 impairs the hematopoietic activity of both LSC and HSC. BCR-ABL activity in LSC does not confer independence of Cx43-mediated signals from the BM osteolineage microenvironment. Disclosures:No relevant conflicts of interest to declare.

Dasatinib Therapy Affects Bone Homeostasis in Patients with Chronic Myelogenous Leukemia in Chronic Phase Independently of Molecular Response

AbstractAbstract 1682 BackgroundWith the success of targeted oral tyrosine kinase inhibitor (TKI) therapy, long-term/life-long therapy for patients with chronic myelogenous leukemia (CML) can now be envisaged. Dasatinib is a second generation TKI, which in a phase I clinical trial was found to be 100–300 times more potent at inhibiting tyrosine kinases than imatinib. In vitro data suggest that dasatinib therapy also may have a direct effect on bone metabolism by interfering with c-FMS, c-SRC and the PDGF-R pathways (Boufker et al, BMC Cancer 2010, 10:298). In this study, we evaluated the effects of dasatinib monotherapy on bone homeostasis of CML patients treated at our center between 2006 and 2011. The levels of traditional biomarkers of bone health, namely serum bone specific alkaline phosphatase isoenzyme (BAP) and osteoprotegerin (OPG) were measured and were correlated with morphometric assessment of bone marrow biopsy specimens (BM Bx). We also assessed for any correlations between bone homeostasis and the results of conventional cytogenetics and molecular analysis with respect to treatment response. MethodsThe study group included 23 patients with chronic phase CML without evidence of clonal evolution who were treated with dasatinib. The median age was 51 years (range, 25–63) with a male to female ratio of 1.5: 1. Four different treatment groups were assessed (20mg/day, 40 mg/day, 80 mg/day and 100 mg/day). We compared paired BM specimens, obtained at initial diagnosis and 12–51 months (mos) after commencing dasatinib therapy. We performed whole slide imaging (Philips) on routinely stained BM Bx and specimen compartments were quantified using an area pixel count algorithm (Image Pro Plus system Version 6.3, MediaCybernetics, Bethesda, MD, USA) to calculate trabecular bone volume as a percentage area. The morphometric results were correlated with the results of conventional cytogenetics and molecular analysis obtained every 3 mos after enrollment. We also performed competitive inhibition ELISA assays to measure serum levels of OPG (baseline and after 12/18 mos) and BAP (every 4 mos), respectively. ResultsWith a median follow up of 24 mos (range 12–51mo), a significant increase in trabecular bone volume (TBV) was noted (p=0.022, Figure 1); 74% (17 pts) showed an increase in TBV on therapy, with an absolute increase of 5.2% (mean). Twelve of 16 pts (75%) evaluated had a decrease in OPG after 18 mos, which correlated in 90% of pts with an increase in TBV using a linear regression model (p= 0.0017, Figure 2). No significant difference was detected in TBV changes among the four dose groups. There was no significant trend in serum BAP levels, thus no statistical correlation was performed at this time point. All patients treated with dasatinib showed cytogenetic and molecular BCR-ABL1 response. There was no correlation between the degree of TBV change and either cytogenetic or molecular response (Figure 3) Conclusions:Our results indicate that within the therapeutic range for CML patients, dasatinib therapy promotes bone formation. However, there was no correlation between the dose levels studied and degree of bone formation and no correlation between TBV change and either cytogenetic or molecular response. The combination of digital image analysis and bone biomarkers enhances our understanding of bone homeostasis in patients on dasatinib. Further studies in a larger cohort of patients will be helpful to further validate our results.Figure 1: mean and standard deviation of TBV for each visit.Figure 2: correlation between “percent change TBV between visit 1 and visit 2” and 18 months OPG levels (linear regression model R2 =0.292, p= 0.0017)Figure 3: association between “percent change in TBV between visit 1 and visit 2” and “Rate of PCR response change between 3 and 12 months” (Spearman correlation coefficient = −0.05; p =0.81) [Display omitted] [Display omitted] [Display omitted] Disclosures:Cortes:Ariad: research support and consulting, research support and consulting Other; Pfizer: research support and consulting, research support and consulting Other; Novartis: research support and consulting, research support and consulting Other; BMS: research support and consulting Other.

Analysis of Clinical Arterial and Metabolic Parameters in Chronic Phase CML Patients On Nilotinib in a Single Center Cohort

AbstractAbstract ▪3756▪This icon denotes a clinically relevant abstractThe introduction of second generation tyrosine kinase inhibitors (TKI2) in the treatment of chronic phase chronic myelogenous leukemia (CML) has improved the response rates in the second, and now, in the first line setting. Nilotinib, a TKI2 with a restricted kinase inhibition profile, demonstrated higher rates of response over imatinib (IM) as front line therapy with a very good short-term tolerability profile. Even if the proportion of patients (pts) in undetectable disease increases with time, the majority will have to continue this treatment for several yrs to avoid progression. In this perspective, mid- and long-term safety issues are of concern, and recent reports point out the onset of arteriopathies in such pts with a suspected abnormal frequency.We have prospectively analysed our on-nilotinib cohort of CP CML between September 2011 and July 2012 with standard clinical assessments [height, weight, body mass index (BMI), ankle-brachial index (ABI)], and basic routine metabolic laboratory assessments (fasting glucose, total, HDL and LDL cholesterol, HDL cholesterol, LDL cholesterol, triglycerides, glycosylated hemoglobin) and BCR-ABL (IS), to explore and detect at early stages abnormalities that may have some later cardio-vascular consequences for pts on this drug.We analysed a series of 54 CML pts all in CP. The median age at diagnosis was 50 (18–73) and 55 (20–78) yrs at nilotinib initiation. The Sokal score was low for 27%, intermediate for 55%, high for 8%, unknown for 10 pts. At CML diagnosis, 13 pts had hypertension, 4 diabetes mellitus, 2 previous peripheral arterial disease (PAD), 4 coronary artery disease (CAD), and 9 pts had tobacco addiction. Thirty-seven pts had IM first line for a median of 4.2 (0.2–11) yrs, then nilotinib for IM-intolerance (14 pts) or IM-resistance (16 pts), or entering a clinical trial (5 pts) and 2 for unknown reasons for a median of 2.8 (0.5–5.6) yrs. Seventeen pts had nilotinib first line for a median of 1 (0.3–4.7) year. The disease duration ranged from 0.4 to 19.3 yrs (median 5.6). Overall, the median duration of nilotinib was 2.2 (0.3–6.8) yrs. Two pts were in CHR, 11 pts in CCyR, 28 in MMR, 13 in undetectable disease (MR4.5) at assessment. In pts on nilotinib without statins at time of assessment, 62% (n=40) showed high total cholesterol values (>2 g/l), 46% (n=37) had high LDL cholesterol (>1.5 g/l) and 57% (n=37) low HDL cholesterol values (<0.6 g/l). Triglycerides were high (>1.48 g/l) in 18% of pts (n=49). Glycosylated hemoglobin was high (>6%) in 18% of the pts treated (n=38), and glucose (> 5.8 mmol/l) in 20% (n=11). Seven pts (13%) were overweight (BMI>30) and the median BMI was 24.6 (16.7–47). On 54 pts, 25% of pts (n=13) had an abnormal ABI (<1) and true arterial problems were authentified by Doppler ultrasound in 13% (n=7) of pts after a median of 2.6 (0.6–4.4) yrs of nilotinib: carotid atherosclerosis, inflammatory carotiditis, one CAD, 4 PADs, with 5 newly diagnosed on nilotinib. In order to correlate the different parameters together we use the correlation circle strategy (see figure 1 below) resulting from the principle components analysis (PCA), in this circle each arrow represents a studied parameter, and the closest one parameter is to another one, the closest the arrows are. Perpendicular arrows denote no correlation at all. The length of each arrow depends on the weight of each parameter compared to the others within the PCA. [Display omitted] As seen; there is a strong correlation between the duration of nilotinib and the total cholesterol, high LDL cholesterol, arterial problems and age at nilotinib initiation. ABI failed in detecting authentified arterial problems detected by other means. The Pearson correlation test nearly reached statistical significance between nilotinib duration and arterial problems (p=0.07).In conclusion, nilotinib induces clear metabolic disturbances along treatment, particularly for lipids and glucose parameters, which can be responsible for significant arteriopathies, that ABI failed to detect. These results should promote the lipids and glucose parameters follow-up during CML treatment by nilotinib in order to detect disturbances at early times, correct them using strict dietary rules and/or concomitant medications to avoid arterial problems at later time points. Disclosures:Nicolini:Novartis, Bristol Myers-Squibb, Pfizer, ARIAD, and Teva: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Chronic Myelogenous Leukemia in the Era of Tyrosine Kinase Inhibitors: Is Sokal Score Still a Valid Predictor of Prognosis?

AbstractAbstract 4422 Background:In the era when the Sokal score was developed (1984), Tyrosine kinase inhibitors (TKI's) were not available for treatment of Chronic Myelogenous Leukemia (CML). With the treatments available then, the 2-year survival in the low risk group was 90% and in the intermediate and high-risk groups was 65%. Since TKI's were approved for treatment of CML in 2001, overall estimated survival at 5 years has improved to 89%. Aim:To evaluate risk stratification by Sokal score as an indicator of prognosis in patients with CML in the present era of tyrosine kinase inhibitors. Methods:Sokal Score is a prognostic score used to risk stratify patients as low-risk, intermediate-risk and high-risk based on diagnosis based on the spleen size, platelet count, age and blast count. Seventy-eight (78) subjects diagnosed with CML and treated at John H. Stroger Hospital of Cook County between 2000 and 2011 were retrospectively analyzed after institutional review board approval. Ten (10) subjects with blast crisis at diagnosis were excluded from this study. Effect of race, sex, age, white cell count, amount of reticulin fibrosis in bone marrow biopsy, total peripheral blood eosinophil and basophil counts, Lactate Dehydrogenase level, spleen and liver size at diagnosis and, risk stratification by Sokal score on overall survival was evaluated using standard statistical analysis. Results:In our population, 27/68 (39.7%) were more than 45 years old, 30/68 (44.11%) were African American, 20/68 (29.4%) were Hispanic, male to female ratio was 2.4:1. WBC>100,000 was observed in 25/68 (51.4%), 57/68 (83.8%) had LDH>200IU/L, 50/68 (73.5%) had palpable splenomegaly >5cm at the time of diagnosis and 67/68 (99%) of subjects had reticulin fibrosis in bone marrow. Of these, 35/67 (52.2%) had mild, 12/67 (17.9%) had moderate and 20/67 (29.8%) had severe fibrosis. According to Sokal score risk stratification, 22/68 (32.3%) were low risk, 40/68 (58.8%) were intermediate risk and 6/68 (8.8%) were high risk at diagnosis. Five year overall survival (OS) in our population was 90%. On univariate analysis, risk stratification by Sokal score at diagnosis was statistically significant for Overall Survival. Subjects with low risk score had better OS as compared to intermediate and high-risk subjects (P=0.007, HR 11.596, 95%CI 1.946–69.103). Similarly, subjects with intermediate score had better OS compared to high-risk group (P=0.007, HR 0.140, 95%CI 0.020–0.539). Conclusion:Sokal Score is still a valid predictor of prognosis in this new age of tyrosine kinase inhibitors and can still be used for risk stratification at diagnosis for chronic phase CML patients. Disclosures:No relevant conflicts of interest to declare.

Different Strategies for First-Line Treatment of Chronic Myeloid Leukaemia: An Economic Analysis

AbstractAbstract 4696 Purpose:To evaluate the economic outcome of conventional and targeted therapies of patients with chronic myelogenous leukemia (CML). Methods:Two Markov simulation models were designed to measure the cost and quality-adjusted life years (QALYs) for newly diagnosed CML patients whether to be administered conventional therapy or six tyrosine kinase inhibitors (TKIs) based strategies. Clinical and utility data were taken mostly from the literature by a MEDLINE search. Costs were based on local charges. The primary output was reported in terms of incremental cost-effectiveness ratios (ICERs). Sensitivity analyses were performed to examine the robustness of the model output. The impact of patient assistance program (PAP) was assessed. Results:The imatinib plus peginterferon-Á switch 2nd TKIs strategy yielded the most health benefits for newly diagnosed CML patients when compared with the conventional, imatinib switch 2nd TKIs, imatinib switch conventional, imatinib plus IFN-Á switch conventional, nilotinib switch conventional and dasatinib switch conventional strategies (figure 1). Marginal cost-effectiveness was $79,134 or $17,600 per QALY gained relative to conventional strategy without or with PAP (figure 2). Both the nilotinib and dasatinib based strategies were more expensive yet less effective than the alternative strategies and were therefore dominated. Model outputs were sensitive to the cost of TKIs. Conclusion:Imatinib and imatinib plus peginterferon-Á based strategies are potentially more cost-effective and 2nd-generation TKIs may be preferred in the setting of imatinib failure. Patient assistant program in health resource limited setting might notably improve the economic outcome of TKIs based therapies. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Incidence of Late Chronic Anemia in Newly Diagnosed Patients with Chronic Myelogenous Leukemia Responsive to Imatinib

Abstract 3769Anemia has been reported in about 5–8% of newly diagnosed patients with Chronic Myelogenous Leukemia (CML) treated with imatinib in the first 6 months of therapy, and has been generally regarded as transient like neutropenia and thrombocytopenia: in patients responsive to imatinib, however, it is still unclear if the prolonged treatment could induce the appearance of a late chronic anemia. To highlight this issue, we revised 104 CML patients (M/F 54/50, median age at diagnosis 58.0 years, interquartile range 43.3 – 70.2) treated at our Institution with 1st line imatinib therapy for at least 36 months and in stable complete cytogenetic response (CCyR). At the 36th month of imatinib treatment, a late chronic anemia (defined as Hb levels < 11 g/dl for > 6 months) was present in 16/104 patients (15.4%): the anemia was severe (Hb < 10 g/dl) in 10 patients (9.6%) and moderate/mild (Hb ≥ 10 < 11 g/dl) in 6 patients (5.8%). Clinical features at diagnosis of patients who had late chronic anemia at the 36th month compared to the remaining 88 patients without late anemia are shown in the Table:Hb < 11 g/dl at the 36th monthHb ≥ 11 g/dl at the 36th monthpM/F6/1048/400.209Median age (yrs) (IR)68.0 (58.9 – 75.4)56.6 (43.1 – 69.1)0.016Sokal Score: Low3440.002Int839High55Median Hb (g/dl) (IR)11.6 (10.4 – 13.3)12.7 (11.2 – 13.8)0.098Median WBC (× 109/l) (IR)79.0 (33.2 – 136.6)60.5 (31.8 – 112.4)0.423Median PLTS (× 109/l) (IR)624 (299 – 835)412 (296 – 557)0.122All patients with late chronic anemia had a low reticulocyte count and 7/16 a condition of iron deficiency with reduced serum ferritin but no clinical and instrumental sign of chronic blood loss: in this latter group, oral iron supplementation was always ineffective. Four out 16 patients (25%) needed 1 or more red cell transfusions during the follow-up. At landmark analysis from the 36th month of imatinib treatment, there was no significant difference in the cumulative 4-year overall survival for patients with and for those without late chronic anemia (80%, 95% CI 44.8 – 100%, vs 94%, 95%CI 85.8 – 100%, respectively; p=0.068). In conclusion, the occurrence of a late chronic anemia during long-lasting treatment with imatinib has been observed in > 15% of our responsive patients and was severe in about 10% of cases: its occurrence seems more common in elderly patients with higher Sokal score at diagnosis. Late chronic anemia does not seem to affect OS, but the real impact should be evaluated on a large cohort of patients. Disclosures:Tafuri:Sigma Tau Pharmaceuticals: Research Funding.