22 research outputs found
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Non-p53 Dependent, Leukemia Initiating-Cell Selective, Therapy
Abstract
Abstract 2077
Poster Board II-54
Conventional drug therapy for AML is limited by toxic effects on normal hematopoietic stem cells (nHSC), and dependence on p53/apoptosis pathways that are impaired in malignancy. In hematopoiesis, key transcription factors (TF) determine cell-fate. Here, a difference in nHSC versus leukemia initiating-cell (LIC) TF expression is used to overcome the above limitations.
The DNA methylating enzyme DNA methyl-transferase 1 (DNMT1) is also a component of multi-protein histone methyl-transferase complexes. Accordingly, shRNA mediated depletion of DNMT1 in hematopoietic cells hypomethylated DNA and decreased global H3K27 and H3K9 trimethylation (histone marks associated with transcription repression) by >70%. These epigenetic modifications were reproduced using a clinically relevant method: the cytosine analogue decitabine, added to normal human CD34+ hematopoietic precursor cells at 0.2–0.5uM 2–3X/week, depleted DNMT1, H3K27 and H3K9 trimethylation by >70% and significantly hypomethylated DNA (Illumina CpG Microarray). These decitabine levels did not cause measurable DNA damage (H2AX phosphorylation and Fast Micromethod) or apoptosis (Annexin staining and caspase 3 activity). Therefore, at low levels, decitabine can produce broad chromatin changes that increase TF access to target genes, without causing measurable DNA damage or apoptosis.
The gene-expression/cell-fate consequences of opening chromatin with decitabine likely depend on the pre-existing TF expression pattern. HOXB4 (stem cell TF), CEBPa (lineage-specifying TF), and CEBPe (late differentiation TF) levels were measured by RQ-PCR in CD34+ cells from AML (n=3) versus normal bone marrow (n=3). AML CD34+ expressed >50-fold higher CEBPa, but HOXB4 and CEBPe levels comparable to normal CD34+ cells, a pattern confirmed in microarray gene expression analysis (CD34+ and myeloblasts, AML n=321, normal n=51 (GEO)). Repression of late differentiation TF likely involves chromatin-modification, regardless of underlying cause. Therefore, depleting DNMT1 to open chromatin in AML cells expressing high lineage-specifying TF could resume differentiation and terminate AML self-renewal, while nHSC, with high stem cell TF and little lineage-specifying TF, should continue to self-renew. nHSC and human MLL-AF9 AML cells were treated identically with decitabine for 7 days, then 300,000 each viable MLL-AF9 and nHSC were combined and transplanted into NSG mice (n = 8). Mice that received PBS treated cells died by week 5 (>90% human myeloblasts in bone marrow). Mice that received decitabine treated cells remained healthy until sacrifice for analysis at week 12 (log-rank p = 0.02, no detectable leukemia, >80% normal human hematopoietic cell marrow engraftment). Direct treatment of mice with established MLL-AF9 leukemia with very low dose decitabine 1mg/m2 3X/week extended survival by >20% (log-rank p = 0.04).
Decitabine 0.5uM 2X/week induced morphologic differentiation, but not early apoptosis, in primary patient samples (n=15) and leukemia cell-lines (n=4). Cell-cycle exit by differentiation versus apoptosis may utilize different cyclin dependent kinase inhibitors (CDKN). The THP1 AML cell line contains a homozygous frame-shift mutation in TP53 (p.R174fs*3) and no detectable p53 RNA/protein. THP1 cells were treated with equimolar Ara-C or decitabine. Ara-C weakly upregulated CDKN1A (p21) but not CDKN2B (p15), and produced a transient decrease in cell-counts (D3-5) with recovery and growth similar to control by D7. Decitabine strongly upregulated p15, weakly upregulated p21, and produced gradual but complete and durable abrogation of cell growth by D7.
A 66y patient with transfusion dependent RCMD with 5q-, 15q- and severe comorbidities was treated with metronomic (instead of cycled) very low dose SQ decitabine (0.2mg/kg [7.5mg/m2] 2X/week) to avoid cytotoxicity and sustain differentiation modification. Platelets increased by week 4, hematologic remission occurred by week 8 and cytogenetic remission by week 14 (without significant side-effects).
Rationalizing dose and schedule of decitabine exploits a difference in nHSC and LIC TF expression to selectively terminate LIC self-renewal by a non-p53 dependent differentiation pathway. This approach, distinct from conventional apoptosis-based therapy, could have a very favorable safety profile, with efficacy in MDS/AML with complex cytogenetic abnormalities.
Disclosures:
Off Label Use: Decitabine, to treat myelodysplastic syndrome using a novel dose and schedule. Advani:Cephalon: Research Funding. Saunthararajah:HemaQuest: Consultancy
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Vancomycin-Resistant Enterococcus (VRE) Bacteremia During Acute Myeloid Leukemia (AML) Induction Therapy Is an Independent Predictor of Poor Outcome
Abstract
Abstract 1487
The presence of VRE bacteremia in hematopoietic stem cell transplant recipients portends a worsening clinical course and poor overall survival (Avery et al; BMT, 2005). The impact of VRE bacteremia on outcome in AML patients (pts) receiving induction chemotherapy (IC) has not been established.
We conducted an IRB-approved retrospective study of AML pts who received cytarabine-based IC at Cleveland Clinic between 2000–2008 to determine VRE rates and effect on complete remission (CR) and overall survival (OS). Data on age at AML diagnosis (dx), gender, diabetes, smoking history, history of antecedent hematological disorder, pathologic classification, hematologic parameters at dx and at VRE occurrence, metaphase cytogenetics (per CALGB/Alliance 8461), precedingnon-VRE bacteremias, invasive fungal infection (IFI), time from dx and induction to VRE and number of VRE infections, complete remission (CR) and overall survival (OS) were collected from our AML database.
The association of these factors with VRE bacteremia was assessed using Fisher's exact test, the Cochran-Armitage trend test and Wilcoxon rank sum test. The impact of VRE bacteremia on OS was assessed using a 2:1 matched-pairs analysis based on gender and year of dx (+ 3 years), and factors known to influence outcome: age at dx (+ 5 years), etiology, and cytogenetic risk. The timing of VRE was also accounted for in the matching. Frailty models, which included a term for WBC at dx, were use to assess the impact of VRE bacteremia while taking into account both the impact of WBC and the paired nature of the data.
Of 350 pts evaluable for analysis, the median age at dx was 57 years (range 19–88); 192 (55%) were male; 114 (33%) had secondary AML; median baseline WBC was 10.4K/mL (range, 0.48–550); cytogenetic risk distribution was favorable (14%), intermediate (54%), and unfavorable (32%); 45% were current or former smokers; 17% had a history of diabetes; and 7% had IFI. With the exception of IFI (16% in pts with VRE versus 6% in non-VRE pts, p=.04) there were no significant differences in these factors between the two groups (all p>.08). Of 37 pts (9.8%) who had documented VRE bacteremias during IC, the median interval from the start of IC to VRE infection was 17 days (range, 9–58). The majority (89%) of VRE bacteremias occurred in pts receiving IC between 2005 and 2008 (infection rate of 22%, 33/152) while only 4 infections occurred in 198 pts treated between 2000 and 2004 (infection rate of 2%). One plausible explanation for this epidemiologic shift could be the frequent use of fluoroquinolone prohylaxis to prevent neutropenic fever, which became routine in 2004. The overall CR rate for the cohort was 73%; 70% in VRE pts and 73% in non-VRE pts (p=0.7). Median follow-up was 72.2 months (range 1.1–145.4). Unadjusted median OS for the entire cohort was 12.8 months (95% C.I. 10.6–15.9); 7.1 months (95% C.I. 3.9–16.5) for VRE pts and 13.1 months (95% C.I. 11.2–16.3) for non-VRE pts (p=0.13, Figure 1A). Using the 2:1 matching to adjust for the impact of age, etiology, and cytogenetics, VRE pts had a significantly inferior OS compared to non-VRE pts even after adjusting for WBC at dx (p=0.04 and.80, respectively, Figure 1B). Mutivariableanalyses confirmed this association.
In conclusion, VRE bacteremia in pts undergoing IC for AML is an independent risk factor for worse OS. The routine use of fluoroquinoloneprophylaxis is likely contributing to the increased prevalence of VRE bacteremia. Consideration should therefore be given to escalating VRE appropriate antibiotic care in these patients sooner and in the post-remission setting.
Figure 1. Survival from Diagnosis A. All patients B. VRE cases and matched controls Figure 1. Survival from Diagnosis . / A. All patients . / B. VRE cases and matched controls
Disclosures:
Saunthararajah: Cleveland Clinic Innovation: patent application for oral THU-decitabine., patent application for oral THU-decitabine. Patents & Royalties. Advani:Genzyme: Honoraria, Research Funding; Immunomedics: Research Funding. Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding
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Predictive Factors for Latency Period and a Prognostic Model for Survival in Patients with Therapy-Related AML
Abstract
Abstract 2589
Therapy-related acute myeloid leukemia (t-AML) is an increasingly recognized sequela in patients (pts) receiving chemotherapy or radiotherapy for a primary malignancy or autoimmune disease. Factors that adversely affect treatment response and survival in t-AML pts include poor cytogenetics, type of antecedent disorder (AD) and type of preceding therapy. The goal of this study was to design a comprehensive prognostic model integrating pt disease- and treatment-related characteristics to predict clinical outcome and to assess factors related to the latency period (LP) between the AD and t-AML diagnosis (dx).
We evaluated a retrospective cohort of newly diagnosed t-AML pts treated at Cleveland Clinic from 2001 to 2011. Data on age at initial dx of AD, type of AD, preceding treatment, type of chemotherapy, age at t-AML dx, gender, peripheral blood counts at t-AML dx, peripheral and marrow blasts, pathologic classification, metaphase cytogenetics (per CALGB/Alliance 8461 criteria), LP, complete remission (CR) and overall survival (OS) measured from t-AML dx were collated from our IRB approved AML database. Multivariable log-linear, logistic, and proportional hazards models with step-wise variable selection were used to identify independent predictors of each outcome.
Of 730 patients treated with cytarabine-based induction chemotherapy over the 10-year period, 77 had t-AML. Most (68%) were female; median age at dx of antecedent disorder was 56 years (range, 16–75); median age at t-AML dx was 61 yrs (range, 19–79); and median latency period to t-AML dx was 4.6 yrs (range, 0.5–38.4). Most (71%) had an antecedent solid tumor [breast cancer (44%), prostate (10%); colon (6%), other organ sites (8%); 23% had a prior hematologic malignancy [non-Hodgkin lymphoma (16%), Hodgkin lymphoma (4%), and leukemia (4%)]; and 5% had autoimmune diseases. Previous treatments for AD included radiation (26%), chemotherapy (30%), and chemotherapy and radiation (44%). Of 57 pts previously treated with chemotherapy, 68% received alkylating agents, 65% anthracyclines, 51% mitotic inhibitors (MI) and 30% all three drug classes. Cytogenetic risk distribution at t-AML dx was: favorable (19%), intermediate-risk (52%), and unfavorable (29%). Overall, 48 pts (62%) achieved a CR with induction chemotherapy and median OS was 9.6 months, with 30% surviving >2 years.
Independent prognostic factors of shorter LP were age at AD >55 (p=.001) and prior treatment with MI (p=.001). Median LP for pts aged 55 but no prior MI, and 2.0 years for pts >55 and prior treatment with MI. Age at t-AML (p=.001) was the only independent predictor of CR. Independent predictors for inferior OS were unfavorable cytogenetics (p=.002), antecedent hematologic or autoimmune disease (p=0.007) and platelet counts <25000/μL at the time of t-AML dx (p=0.02). A prognostic model based on these factors categorized t-AML pts into two risk groups based on previous diagnosis type, cytogenetics, and platelet count at t-AML dx (Table 1). This score-based risk stratification used a cutoff of 2 points to categorize pts as favorable or unfavorable. Pts with a favorable profile had an estimated median OS of 28.4 months compared to 5.0 months for pts with an unfavorable profile (p=.0003).
In conclusion, multicomponent prognostic models that integrate well-established disease or treatment-related covariates can be clinically helpful in risk stratifying t-AML pts undergoing induction therapy, identifying those who might benefit from more intensive interventions.
Table 1. Predictive model for OS using cytogenetics, antecedent disease and platelet count at t-AML diagnosis Risk Group Score Patients N Median Survival (Months) Hazard Ratio (95% CI) p Solid tumor (0 points) and favorable cytogenetics (0) OR Solid tumor (0), intermediate cytogenetics (2), and platelets > 25000 (0) OR Favorable 0 or 2 38 28.4 Hematologic/autoimmune (2), favorable cytogenetics (0), and platelets >25000 (0) Unfavorable cytogenetics (3) OR Solid tumor (0), intermediate cytogenetics (2) and platelets 2 37 5.0 2.70 (1.57-4.64) .0003 Hem/autoimmune (2), favorable cytogenetics (0) and platelets <25000 (2) OR Hematologic/autoimmune (2) and intermediate cytogenetics (2)
Disclosures:
No relevant conflicts of interest to declare
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Effective and Non-Cytotoxic p53 Independent Epigenetic-Differentiation Therapy In Xeno-Transplant Models of Human Acute Myeloid Leukemia
Abstract
Abstract 3309
The cytosine analogue decitabine can induce both apoptosis and epigenetic/differentiation effects. Although the regimen commonly used to treat myelodysplastic syndrome has de-escalated doses with an epigenetic mechanism of action in mind, therapy continues to resemble pulse-cycled therapy for apoptosis objectives. This contrasts with the lower dose and one to three times per week schedule of decitabine used for non-cytotoxic epigenetic-differentiation therapy of non-malignant disease. Non-cytotoxic differentiation therapy could have substantial advantages, such as sparing of normal hematopoietic stem cells (HSC), decreased therapy related cytopenia that enables more frequent treatment exposure (a critical consideration with S-phase specific therapy), and a p53-independent mechanism of action. These possibilities were assessed in vitro and in vivo. Concentrations of decitabine that deplete DNMT1 in normal HSC without causing measurable DNA damage or apoptosis were determined. Treatment with equimolar AraC was used as a control. These concentrations of decitabine and AraC (0.5 μM) were used to treat p53 wild-type AML cells produced by retroviral insertion of MLL-AF9 into human CD34+ cells. Unlike AraC, decitabine did not induce apoptosis, but nonetheless terminated AML cell proliferation, accompanied by morphologic changes of differentiation, increased CD14 expression, and late and substantial upregulation of key proteins associated with myeloid cell cycle exit by differentiation, CEBPe and CDKN1B/p27. Decitabine produced an identical effect in p53 null MLL-AF9 leukemia cells (THP1 cells). In contrast, the p53 null cells did not demonstrate apoptosis, differentiation or proliferation inhibition in response to AraC. To determine if the non-cytotoxic differentiation terminated the self-renewal ability of leukemia initiating cells, p53 wild-type MLL-AF9 cells and normal HSC were treated with the identical regimen of decitabine or PBS in vitro then assayed for engraftment ability in NOD/SCID mice. Mice receiving the combination of mock treated normal and mock treated MLL-AF9 cells died of overwhelming leukemia by week 6. Mice receiving the combination of decitabine-treated normal and decitabine-treated MLL-AF9 cells remained healthy and after greater than twice the period of survival of the control group, were documented to have normal human hematopoietic cell engraftment, comparable to that seen in mice receiving normal human CD34+ cells without leukemia cells. To confirm that 0.2 mg/kg of decitabine administered sub-cutaneously on a weekly basis depletes DNMT1 without causing cytotoxicity or severe cytopenia in vivo, NSG mice were treated for 8 weeks. There was no treatment associated cytopenia or bone marrow cell apoptosis although DNMT1 was substantially depleted in bone marrow cells. This decitabine regimen, conventional AraC or vehicle was then used to treat xeno-transplant models of p53 wild-type and p53 null human AML (n=5 per group). In the p53 wild-type model, decitabine treatment was associated with significantly longer median survival than vehicle (>50% increase in survival, median survival 92 versus 61 days, Log-Rank p=0.0188), with one decitabine treated mouse without evidence of disease when the experiment was terminated on day 150. In the p53-null model, decitabine treatment was associated with significantly longer median survival (>20% increase) than AraC and vehicle treated mice (median survival 51, 45, and 42 days respectively, Log-Rank p=0.0004). To complement the above experiment in which AML cell lines were used, a xenotransplant model was established using fresh AML cells from a patient with relapsed treatment refractory AML. These AML cells contained complex chromosome abnormalities. Mice treated with decitabine (n=7) had significantly longer median survival (>100% increase) than AraC or vehicle treated mice (median survival 113, 56, and 50 days respectively, Log-Rank p<0.0001). These observations provide the foundation for AML therapy that is mechanistically distinct and a true alternative to conventional apoptosis-based therapy. This approach to therapy was non-toxic and highly effective in the pre-clinical in vivo models of human AML, as expected from its non-apoptosis based, p53-independent, and normal HSC sparing mechanism of action, and warrants further pre-clinical and clinical study.
Disclosures:
No relevant conflicts of interest to declare