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1 Supplementary Materials for A Clinical Trial for Patients with Acute Myeloid Leukemia or Myelodysplastic Syndromes Not Eligible for Standard Clinical Trials Table of Contents Content Page Supplementary Methods Isolation of Human Mononuclear cells 2 Correlative LINE DNA Methylation Analysis 2 Supplementary Figures Figure 1S. LINE DNA Methylation levers during First cycle of Therapy 3 Figure 2S. Kaplan-Meier plots for OS in terms of cytogenetic response 4 Supplementary Tables Table S1. Primer sequences used for pyrosequencing. 5 Table 2S. Univariate analysis for survival at 60-days, OS and EFS 6 Table 3S. Multivariate analysis for survival at 60-days, OS and EFS 7 Table 4S. Individual patient characteristics in the exploratory study 8 Table 5S. Individual patient characteristics in the extension study 10
2 Supplementary Methods Isolation of human mononuclear cells Peripheral blood specimens from patients were drawn into heparin containing tubes. Mononuclear cells were separated using Ficoll-Paque PLUS (GE Healthcare Life Sciences, Pittsburgh, PA) gradient centrifugation. The mononuclear cell - enriched fraction was collected and washed twice with calcium and magnesium-free phosphate-buffered saline (PBS). After centrifugation, PBS was removed, and the samples were immediately frozen at - 80 C. DNA methylation analysis To study the dynamics of DNA hypomethylation during treatment, we sequentially studied LINE methylation, a marker of global DNA methylation. To do so, we used bisulfite pyrosequencing assay. DNA was extracted using standard phenol-chloroform method. Bisulfite modification of DNA was performed by using EpiTect Bisulfite Kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. For pyrosequencing analysis, we performed polymerase chain reactions (PCRs). Bisulfite modified DNA was amplified by PCR with primers shown in Table S1. The degree of methylation was calculated using the PSQ HS 96A 1.2 software (Biotage AB, Uppsala, Sweden). 2
3 Figure 1S. LINE DNA methylation Levels during First Cycle of Therapy. Methylation assays were performed in 19 (63%) patients. Median LINE DNA methylation levels were: 65.06% ( ) on Day 0; 64.58% ( ) on Day 5; 63.26% ( ) on Day 21; and ( ) on Day 28. A significant decrease in LINE methylation levels was identified by Cycle 1 Day 21 compared to baseline (65% vs 63%, p=0.018). Subsequent progressive increase in methylation levels was observed by Cycle 1 Day 28 3
4 Figure 2S. Kaplan-Meier plots for overall survival in terms of cytogenetic response across both treatment arms. 4
5 Table S1. Primer sequences used for pyrosequencing Gene Forward primer Reverse primer Biotin primer Sequencing primer LINE TTTTGAGTTAGG AAAATCAAAAAA AAAATCAAAAAA AGTTAGGTGTG TGTGGGATATA TTCCCTTTC TTCCCTTTC GGATATAGT 5
6 Table 2S. Univariate analysis for survival at 60 days, overall survival (OS) and event-free survival (EFS) Survival at 60 days (Alive/Total=62/79) OS (Event/Total=67/79) EFS (Event/Total=71/79) Covariate OR (95% CI) P-value HR (95% CI) P-value HR (95% CI) P-value Age 0.90 ( 0.83, 0.97 ) ( 1.00, 1.06 ) ( 0.99, 1.04 ) 0.35 Sex Female vs. Male 0.75 ( 0.24, 2.34 ) ( 0.57, 1.63 ) ( 0.71, 1.94 ) 0.55 Diagnosis AML vs. MDS 0.28 ( 0.09, 0.86 ) ( 1.21, 3.24 ) ( 1.19, 3.08 ) 0.01 Complex cytogenetics Yes vs. No 0.37 ( 0.12, 1.13 ) ( 1.58, 4.58 ) < ( 1.09, 2.89 ) 0.02 Treatment Aza+S vs. Aza 2.75 ( 0.92, 8.25 ) ( 0.51, 1.38 ) ( 0.37, 1.00 ) 0.05 Serum creatinine (mg/dl) 2 vs. < ( 0.11, 1.63 ) ( 0.48, 2.13 ) ( 0.68, 2.79 ) 0.37 Total bilirubin (mg/dl) 2 vs. < ( 0.06, 2.49 ) ( 0.35, 2.69 ) ( 0.55, 3.38 ) 0.51 log(hemoglobin) ( 0.19, ) ( 0.05, 2.40 ) ( 0.02, 0.98 ) 0.05 log(platelet count) 1.76 ( 0.98, 3.15 ) ( 0.44, 0.78 ) < ( 0.53, 0.91 ) 0.01 log(wbc) 0.73 ( 0.45, 1.17 ) ( 0.76, 1.24 ) ( 0.90, 1.42 ) 0.29 log(bone marrow blasts) 0.51 ( 0.30, 0.84 ) ( 1.08, 1.66 ) ( 1.00, 1.48 ) 0.06 Survival at 60 days was analyzed using logistic regression model; OS and EFS were analyzed using Cox regression models; OR=Odds Ratio; HR=Hazard Ratio
7 Table 3S. Multivariate analysis for survival at 60 days, overall survival (OS) and event-free survival (EFS) Survival at 60 days (Alive/Total=62/79) OS (Event/Total=67/79) EFS (Event/Total=71/79) Covariate OR (95% CI) P-value HR (95% CI) P-value HR (95% CI) P-value Age 0.91 ( 0.83, 0.99 ) ( 1.00, 1.06 ) ( 0.98, 1.05 ) 0.44 Diagnosis AML vs. MDS 0.48 ( 0.14, 1.66 ) ( 0.94, 2.83 ) ( 1.05, 2.92 ) 0.03 Treatment Aza+S vs. Aza 2.00 ( 0.59, 6.79 ) ( 0.79, 2.38 ) ( 0.43, 1.17 ) 0.17 Complex cytogenetics Yes vs. No 0.31 ( 0.09, 1.08 ) ( 1.32, 3.92 ) ( 1.17, 3.25 ) 0.01 log(platelet count) 0.61 ( 0.44, 0.86 ) Survival at 60 days was analyzed using logistic regression model; OS and EFS were analyzed using Cox regression models; OR=Odds Ratio; HR=Hazard Ratio Forward variable selection was applied with p-value cutoff of Age, diagnosis. treatment and complex cytogenetics were forced to stay in the multivariate model. 7
8 Table 4S. Individual patient characteristics in the exploratory study. Patient Age (years) Diagnosis Inclusion Criteria Time from MDS or AML Diagnosis to Inclusion (days) ECOG PS ACE-27 Number of Cycles 1 50 RAEB Active uterine leiomyosarcoma NR Response Subsequent Evolution and Therapy None. Transfer to hospice for end-oflife care due to progression of solid malignancy 2 76 RAEB Bilirubin 2mg/dL NE None 5.1 Survival (months) Cause of Death 7 Metastatic uterine leiomyosarcoma 3 62 RAEB Metastatic breast cancer CR None 15.9 Unknown 4 71 RCMD Active Chronic lymphocytic leukemia CR 5 62 CMML Bilirubin 2mg/dL NR 6 74 AML Thyroid cancer Creatinine 2mg/dL CR 7 74 RCMD Active ovarian cancer NR Fludarabine and Cytarabine induction due to progression to AML Patient received 1 cycle of therapy with loss of follow-up for subsequent cycles Progressive disease. Enrollment on subsequent clinical trial receiving one cycle of therapy prior to death. None. Transfer to hospice for end-oflife care due to progression of solid malignancy 17.3 Related to MDS-related progressive pancytopenia after 1 month admission in an Complication during consolidation chemotherapy at an 7.8 Disease progression 6.5 Septic shock and multiorgan failure during salvage therapy 2.3 Metastatic ovarian cancer 8 81 AML Cardiac comorbidities (EF <20%) CR None 6.3 Unknown 9 59 RAEB Performance status of CR Allogeneic stem-cell transplantation on an 23.7 Unknown CMML Performance status of CRP None 1.2 Myocardial infarction RAEB Prior colon cancer Prior squamous cell carcinoma of the hypopharynx Coronary artery disease AML Performance status of NR AML Active unresolved infection Performance status of NR None 0.3 Pneumonia Enrollment of Phase II study and subsequent transfer to hospice for end-of-life care due to progressive disease NE None RAEB Relapsed follicular lymphoma NR AML Prior prostate adenocarcinoma Previous cerebrovascular accident RAEB Active breast cancer NR Fludarabine and Cytarabine induction due to progression to AML. Transition to hospice for end-of-life care. 9.4 Unknown 9.2 Unknown Respiratory failure due to probable diffuse alveolar hemorrhage or pneumonia NE None 5.9 Transformation to AML with sepsis and multiorgan failure None. Transfer to hospice for end-oflife care due to progression of solid malignancy 9.8 Progression of breast cancer RAEB Prior Waldeströms Macroglobulinemia CR Allogeneic stem-cell transplantation 10.5 Relapse after allogeneic stem-cell transplantation AML Performance status of CR AML Prior breast ductal carcinoma and glioblastoma multiforme HI/CRi Relapse with enrollment on Phase II study Progressive relapsed /refractory disease. Received several lines of 14.6 Unknown during admission in an 14.2 Pneumonia 8
9 salvaged therapy including fludarabine and cytarabine, 3+7 regimen and decitabine AML Performance status of HI/CRi None 11.9 Unknown AML Cardiac comorbidity NE None 4.4 Acute pulmonary edema RAEB AML Performance status of 3 Creatinine 2mg/dL Bilirubin 2mg/dL Prior diffuse large B cell lymphoma Cardiac comorbidity Bilirubin 2mg/dL Prior folicullar lymphoma NR NR AML Creatinine 2mg/dL CR RAEB Active chronic lymphocytic leukemia NR RA Prior mantle cell lymphoma NR AML Active small cell lung cancer NE AML Prior prostate cancer Cardiac comorbidities RAEB Prior cutaneous T-cell lymphoma PR AML Active colon cancer NR None. Per patient request therapy was discontinued and he was transferred to hospice for end-of-life care Progressive disease. Received salvage therapy with Fludarabine and cytarabine. None. Due to worsening of chronic renal insufficiency and ECOG-PS was transferred to hospice for end-of-lifecare Progression to relapsed/refractory AML. Enrolled on Phase II study (Clofarabine + low-dose cytarabine). Subsequently received Fludarabine and cytarabine Fludarabine and Cytarabine induction due to progression to AML None. Patient was transferred to hospice for end-of-life care due to progression of solid malignancy 0.6 Progression to acute myelogenous leukemia 4.7 Septic shock and multiorgan failure during salvage therapy 21.9 Unknown Unknown during consolidation therapy on an outside institution Respiratory failure due to pneumonia as a complication of induction chemotherapy. 5.6 Disseminated small-cell lung cancer NR None 1.6 Cardiac and disease progression None. Patient was transferred to hospice for end-of-life care None. Patient was transferred to hospice for end-of-life care 29 Unknown 1.1 Unknown NE: Not evaluable due to early death. MDS: Myelodysplastic syndrome. RA: Refractory anemia. RCMD: Refractory cytopenia with multilineage dysplasia. RAEB: Refractory anemia with excess blasts. CMML: Chronic myelomonocytic leukemia. AML: Acute myelogenous leukemia. NR = No response. CR= Complete response. CRi = Complete response with insufficient hematological recovery. CRp = Complete response without platelet recovery. HI = Hematological improvement. 9
10 Table 5S. Individual patient characteristics in the extension study. Pt Age (years) Diagnosis Inclusion Criteria Treatment Arm Time from Diagnosis to Inclusion (Days) PS ACE-27 Number of Cycles Response 1 61 MDS Previous breast cancer A+V CRi 2 81 AML Performance status 3 A ED 3 63 MDS Interstitial pneumonitis A NR 4 59 MDS 5 54 MDS 6 88 AML History of fibromyxoid sarcoma Concurrent metastatic ovarian cancer Concurrent Chronic lymphocytic leukemia A NR A CR Subsequent Evolution and Therapy MUD allogeneic SCT. Postransplant relapse treated with Clofarabine and LDAC. Transfer to palliative care for endof life care None. Died on cycle 1 day 6 of therapy on an Continued azacitidine off protocol at an with loss of follow up Fludarabine and cytarabine with subsequent allogeneic SCT and postransplantation relapse treated on a clinical trial. Transition to palliative care for end-of life care Allogeneic SCT followed with posttransplant azacitidine maintenance Survival (months) Cause of Death 16,2 Unknown 0,2 Unknown 10,6 Unknown 13,7 Unknown 8,6 Pneumonia A+V NR No subsequent therapy 1,7 Unknown 7 80 AML Performance status 3 A Died 8 65 MDS 9 66 AML AML MDS Creatinine 2mg/dL Cardiac and pulmonary comorbidities Creatinine 2mg/dL Performance status 3 Concurrent non- Hodgkin lymphoma Concurrent ovarian carcinoma History of stroke A NR A NE A ED A+V Died AML Bilirubin 2mg/dL A CR AML MDS AML Ineligible for protocol of higher priority due to atrial fibrillation Concurrent malignant mesothelioma Concurrent Thyroid Carcinoma Transformation to AML. Received Fludarabine and cytarabine (BIDFA) induction with no response and multiple. Transition to palliative care for end-of-life care Due to presence of t(8;21) was taken off protocol and treated with FLAG-Ida Hydroxyurea and cytarabine for cytoreduction due to hyperleukocytosis No subsequent therapy. Was admitted at an and expired Relapse with subsequent decitabine therapy followed by BIDFA induction. Transition to hospice for end-of-life care 0,7 Multiorgan failure due to pneumonia related septic shock 6,1 Multiorgan failure due to pneumonia related septic shock 47,5 Alive and in complete remission 0,8 Multiorgan failure due to pneumonia related septic shock 2,0 Unknown 6,0 Unknown A+V ED No subsequent therapy 0,2 Multiorgan failure due to pneumonia related septic shock A+V NR A CR MDS Prostate cancer A CRi AML MDS Bilirubin 2mg/dL Progressive pneumonia Ulcerative colitis HIV Prostate cancer A+V CR A CRp No subsequent therapy. Expired at an Relapse. Received therapy on two subsequent clinical trials. Due to progression of disease transitioned to palliative care for end-of-life care Continued off protocol azacitidine, per patient request, with loss of response and was then treated on a subsequent clinical trial with eventual progression of disease Relapse. Received therapy on subsequent clinical trial with no response Allogeneic SCT with post-transplant relapse treated with off protocol decitabine. Transition to palliative care for 5,6 Unknown 13,7 Unknown 20,4 Unknown 7,1 Multiorgan failure due to pneumonia related septic shock 28,3 Unknown 10
11 19 68 AML AML Anal squamous cell carcinoma History of: MALT lymphoma Neuroendocrine tumor Squamous cell carcinoma Concurrent Multiple Myeloma A+V ED A NR AML Bilirubin 2mg/dL A Died AML AML MDS Concurrent chronic lymphocytic leukemia Coronary Artery Disease Concurrent systemic mastocytosis A+V NR end-of-life care Continued azacitidine off protocol, per patient request, with progressive disease. Transitioned to palliative care for end-oflife care No subsequent therapy. Was admitted on Cycle 1 Day 15 and had a left MCA stroke with subsequent transition to palliative care for end-of-life care Fludarabine and cytarabine (BIDFA) with persistent disease with subsequent decitabine therapy in 0,7 Multiorgan failure due to pneumonia related septic shock 13,5 Unknown 1,0 Unknown 7,6 Unknown A+V Died No subsequent therapy 4,4 Acute myocardial infarction A CRp MDS Performance status 3 A+V CRi MDS MDS AML MDS MDS MDS Concurrent non-small cell lung cancer History of Mantle cell lymphoma Concurrent metastatic sarcoma Concurrent multiple myeloma Prior Burkitt s Lymphoma Cardiac comorbidities (EF <20% and atrial fibrillation) A CR A+V NR A+V NR A+V Died A+V NR A+V Died MDS Recent breast cancer A+V CRp MDS History of Mantle cell lymphoma Severe ataxia Chronic kidney disease A Died AML Coronary artery disease A CR AML Bilirubin 2mg/dL Performance status 3 A CRi MDS Bilirubin 2mg/dL A+V NR MDS AML History of follicular lymphoma Adrenal insufficiency Pulmonary fibrosis A+V NR Progression of disease treated with several clinical trials. Transformation to AML leading to acute renal failure Continued azacitidine single agent with subsequent relapse of disease as AML treated with BIDFA with persistent disease Relapsed disease treated in subsequent clinical trial. Transition to palliative care for end-of-life care Progression of disease with inclusion of subsequent clinical trial Progression of disease treated with off protocol clofarabine and low dose cytarabine with subsequent follow up in No subsequent therapy. Was admitted at an Persistent disease treated on subsequent clinical trial followed by allogeneic SCT No subsequent therapy. Transitioned to palliative care facility for end-of-life care Relapse treated on subsequent clinical trial achieving CR2 No subsequent therapy. Expired at an Persistent disease on supportive care. Diagnosed of breast cancer Persistent disease with transition to supportive care and palliative care A+V ED No subsequent therapy. 0,8 34,2 Acute renal failure 35,7 Unknown 9,5 Unknown 3,7 Acute myocardial infarction 11,1 Massive hemoptysis 1,4 Multiorgan failure due to pneumonia and rectal abscess 4,8 Unknown 1,9 Multiorgan failure due to pneumonia and subdural intracranial hemorrhage 33,3 Alive in CR 1,2 Unknown 35,8 Alive in CR 1,4 Congestive heart failure due to atrial fibrillation 32,3 Alive 7,6 Unknown Respiratory failure due to pneumonia vs diffuse alveolar hemorrhage 11
12 History of breast cancer AML Performance status 3 A CR MDS Creatinine 2mg/dL A+V Died MDS Creatinine 2mg/dL A Died MDS Concurrent chronic lymphocytic leukemia A+V NR MDS Bilirubin 2mg/dL A+V CR AML Neuroendocrine tumor Colon cancer Relapsed refractory disease with multiple lines of therapy No subsequent therapy. Transition to palliative care for end-of-life care No subsequent therapy. Expired on an Transformation to AML treated on several subsequent clinical trials with persistent disease. Transition to palliative care for end-of-life care Allogeneic SCT. Expired due to infectious in CR 16,0 Multiorgan failure due to septic shock 4,5 Pneumonia 1,1 Unknown 10,6 Unknown 12,1 Fungal pneumonia A ED No subsequent therapy 0,6 Multiorgan failure due to septic shock MDS Creatinine 2mg/dL A Died AML Concurrent metastatic neuroendocrine tumor A+V NR MDS Creatinine 2mg/dL A+V CR MDS MDS MDS Concurrent diffuse large B-cell lymphoma Concurrent chronic lymphocytic leukemia Concurrent diffuse large B-cell lymphoma A CR A CRp A+V CRp MDS Performance status 3 A+V CR MDS Performance status 3 A+V CR AML Creatinine 2mg/dL A+V Died MDS AML MDS MDS MDS MDS Ineligible for protocol of higher priority Chronic obstructive pulmonary disease History of myocardial infarction Concurrent refractory mantle cell lymphoma Concurrent chronic lymphocytic leukemia Concurrent metastatic ovarian cancer Ineligible for protocol of higher priority A+V CRp A+V NR A+V CRi A+V CR A+V CRp No subsequent therapy. Expired on an Persistent disease treated with BIDFA. Transition to palliative care for end-of-life care Relapse with subsequent treatment on another clinical trial No subsequent therapy. Expired on an Relapse treated with supportive care and therapy for his CLL. Expired on an Allogeneic SCT. Expired on day +23 of transplant due to infectious No subsequent therapy. Expired on an Relapse treated with multiple subsequent therapies including clinical trials No subsequent therapies. Expired on an Allogeneic SCT. Expired due to posttransplant infectious No subsequent therapy due to refractory disease and multiple infectious. Transition to palliative care for end-of-life care Progression of disease with subsequent supportive care. Transition to palliative care for end-of-life care Relapse with no subsequent therapy due to death at Progression of metastatic solid cancer. Transition to palliative care for end-of-life care 1,5 Unknown 7,4 Unknown 30,1 Alive in CR 5,7 Unknown 10,1 Unknown 5,6 Multiorgan failure due to septic shock 10,3 Unknown 22,4 Pneumonia 3,6 Unknown 8,5 Multiorgan failure due to pneumonia related septic shock 3,1 Multifocal pneumonia 9,4 Unknown 14,1 Unknown 10,0 Unknown A+V CRp Loss of follow up 12,6 Alive MDS Concurrent Non- Hodgkin lymphoma A+V NE Loss of follow up 26,8 Alive MDS Concurrent non- Hodgkin lymphoma A+V NR Persistent disease. Transition to palliative care for end-of-life care 6,2 Unknown AML Concurrent chronic A+V NR No subsequent therapy. Expired on 6,4 Unknown 12
13 lymphocytic leukemia AML Creatinine 2mg/dL A NR AML AML AML AML Polycythemia Vera History of acute myeloid leukemia Concurrent prostate cancer Coronary artery disease with recent CABG Bilateral lung transplantation due to end-stage COPD A+V NR A CR A+V NR A+V PR MDS Creatinine 2mg/dL A+V CRp MDS Concurrent metastatic leiomyosarcoma A+V CRI AML Creatinine 2mg/dL A+V Died MDS Performance status 3 A+V CR MDS AML MDS Creatinine 2mg/dL Pulmonary hypertension Concurrent mantle cell lymphoma Concurrent papillary thyroid carcinoma A+V CR A+V NR A+V NR MDS Creatinine 2mg/dL A+V CRp MDS MDS MDS Concurrent relapsed/refractory chronic lymphocytic leukemia Ineligible for protocol of higher priority due to atrial fibrillation History of squamous cell carcinoma of the penis A+V CRp A+V CR A+V CR AML Pulmonary fibrosis A+V CR Persistent disease. Transition to palliative care for end-of-life care No subsequent therapy. Transition to palliative care for end-of-life care Transition to palliative care for end-of-life care by family and patient decision No subsequent therapy due to unavailable clinical trials. Expired on an No subsequent therapy. Transition to hospice for end-of-life care Continued azacitidine off protocol per patient request. Expired on an outside institution Expired due to during therapy Continued azacitidine off protocol per patient request. Expired at an outside institution Relapsed disease treated on subsequent clinical trial Progression of disease treated on subsequent clinical trial Continued azacitidine of protocol per patient request No subsequent therapy for MDS. Received additional therapy for progressive CLL. Expired at an outside institution Progression of diseases subsequently treated with several clinical trials. Expired due to after transformation Relapsed disease treated with subsequent clinical trial and, due to new progression, off protocol decitabine. Expired at an 2,7 Unknown 0,7 Unknown 5,5 Unknown 7,0 Unknown 4,6 Unknown 1,9 Multiorgan failure due to pneumonia related septic shock 10,9 Unknown 5,5 Multiorgan failure due to pneumonia related septic shock 14,2 Unknown 17,9 Alive with stable disease 3,6 Multiorgan failure due to pneumonia and hemophagocytic syndrome 18,6 Alive with stable disease 18,2 Alive with stable disease 12,2 Unknown 14,5 Intracraneal hemorrhage 18,2 Multiorgan failure due to pneumonia related septic shock 18,0 Pneumonia MDS = Myelodysplastic syndrome. AML = Acute myelogenous leukemia. NR = No response. CR= Complete response. CRi = Complete response with insufficient hematological recovery. CRp = Complete response without platelet recovery. HI = Hematological improvement. 13
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