Targeting TET2 Mutations in Myelodysplastic Syndromes and Acute Myeloid Leukemia (AML) With Azacitidine and Ascorbic Acid
Phase: PHASE2
Conditions: Myelodysplastic Syndromes, Myeloproliferative Neoplasm, Acute Myeloid Leukemia
Sponsor: Case Comprehensive Cancer Center

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STUDY NUMBER:  CASE 1917 
 
ClinicalTrials.gov Number: NCT03397173 
 
Protocol Date: 05/24/2018 
 
STUDY TITLE:  Targeting TET2 mutations in Myelodysplastic Syndromes and Acute Myeloid 
Leukemia with Azacitidine and Ascorbic Acid 
 
 
 
 
PRINCIPAL INVESTIGATOR: Aziz Nazha, MD 
Taussig Cancer Institute, Cleveland Clinic  
Tel: 216-445-0320 
 
 
 
 
E-mail: nazhaa@ccf.org 
 
CO-PI:  
 
 
       Jaroslaw Maciejewski, M.D., Ph.D. 
Taussig Cancer Institute R/40, Cleveland Clinic  
10201 Carnegie Ave, Cleveland, Ohio  
Tel 216-445-5962; Fax 216-636-2495  
E-mail: maciejj@ccf.org 
 
CO-PI:  
 
 
       Mikkael A Sekeres, MD, MS 
Taussig Cancer Institute, Cleveland Clinic  
10201 Carnegie Ave, Cleveland, Ohio  
Tel 216-445-5962; Fax 216-636-2495  
E-mail: sekerem@ccf.org 
 
CO-PI:  
 
 
       Benjamin Tomlinson, MD 
  
 
 
  
       University Hospital Cleveland Medical Center 
 
 
 
 
       Tel: 216-286-9185 
 
 
 
 
       E-mail: benjamin.tomlinson@uhhospitals.org 
 
SPONSOR:  
 
 
Case Comprehensive Cancer Center  
 
 
 
 
 
 
SUPPORT/FUNDING: 
 
Department of Translational Hematology and Oncology 
 
SUPPLIED AGENTS: 
 
Azacitidine, Ascorbic Acid 

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AMENDMENT SUMMARY: 
Original Protocol: 10/13/2017 
 
Amendment 1, Protocol Version 2: 11/20/2017 
 
Amendment 2, Protocol Version 3: 05/24/2018 
 
PROTOCOL SUMMARY 
 
Protocol 
Number/Title 
Case 1917: Targeting TET2 mutations in Myelodysplastic 
Syndromes and Acute Myeloid Leukemia with Azacitidine and 
Ascorbic Acid 
Study Phase 
II 
Brief 
Background/Rationale 
 
 
 
Ten eleven translocation 2 (TET2) mutations are among the most 
common genomic alterations in myeloid malignancies. Somatic 
mutations in TET2 can be identified in 25-30% of patients with 
myelodysplastic syndromes (MDS) and 50-60% of patients with 
chronic myelomonocytic leukemia (CMML), an overlapping 
MDS/myeloprolifrative neoplasm (MPN) disorder, and 15-20% 
of patients with acute myeloid leukemia (AML). Moreover, 
somatic mutations in TET2 gene were identified in healthy 
individuals. The presence of these mutations increases with age 
and can increase the risk of these individuals to develop 
hematologic malignancies and all-cause mortality. Because 
synthetic lethal approaches cannot be applied for the therapy of 
TET2MUT and because TET2 gene is very pleomorphic, 
identification of targeted therapies for patients with TET2 
mutations remains challenging.  
 
The dioxygenase TET2 forms hydroxyl radicals that hydroxylate 
methyl groups on DNA to remove them. Thus, indirectly TET2 
functions as a DNA demethylase that ultimately affects gene 
expression. Consequently, mutations in TET2 result in increased 
methylation of tumor suppressor genes and also possibly cytidine 
deamination leading to C>T transitions at CpG sites. To split O2, 
TET2 needs 3 electrons, 2 from a-ketoglutarate (aKG) and 1 from 
ascorbic acid. Ascorbic acid is therefore an activator of TET2.  
Indeed, in preliminary results with purified TET2, ascorbic acid 
increased the enzymatic activity of mutant TET2 indicating that 
ascorbic acid supplementation may increase the efficiency of 
dioxygenation but this increase was only seen in cells with 
heterozygous mutations. Further, in vitro studies have shown that 
high dose ascorbic acid with plasma concentration of 1000 
μmol/L is toxic to some cancer cells but not normal cells.  
Although trials with oral ascorbic acid in mid 1970s did not show 
any benefit of it in cancer patients, recent trials have shown that 
the addition of high dose IV ascorbic acid to chemotherapy is 
well tolerated, nontoxic, and may improve responses to therapy 
in some patients and can also improve symptoms.  

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Azacitidine (AZA), decitabine (DAC), and lenalidomide 
are the only FDA approved drugs in MDS. Treatment with AZA 
and DAC improves cytopenias and prolongs survival in patients 
with MDS, though only 30-35% of patients respond to therapy. 
Studies have shown that MDS patients with TET2 mutations have 
a higher response rate to AZA. In a study of 82 patients with 
MDS and oligoblastic AML (20-30% BM blasts) treated with 
AZA, TET2 mutations were found in 15% of the patients. 
Patients with TET2 mutations had a higher response to AZA 
compared to wild type patients (82% vs 45%, p = 0.04, 
respectively). In a larger retrospective study, samples from 213 
patients with MDS were sequenced prior to their treatment with 
hypomethylating agents (HMAs) for the presence of 40 recurrent 
gene mutations. TET2 mutations with variant allelic frequency 
(VAF) >10% were associated with a significantly higher overall 
response rate (ORR) compared with WT (60% vs. 43%; OR, 
1.99; 95%CI, 1.05-3.80, P=0.036). Further, mice with 
transplanted TET2-null hematopoietic cells exhibit decrease in 
white blood counts when they are treated with AZA compared to 
vehicle. In another in vitro study, adding vitamin C at 
physiological levels to low doses azacitidine showed synergistic 
inhibition of cancer-cell proliferation and increased apoptosis. 
This synergistic effect is likely the result of both passive DNA 
demethylation by DNMTi and active conversion of 5-
methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) by 
TET enzymes at LTR regions of endogenous retrovirus, because 
vitamin C acts as a cofactor for TET proteins. In addition, TET2 
knockout reduces the synergy between the two compounds. 
Taken together, we hypothesize that the combination of standard 
dose AZA with oral dose of ascorbic acid will improve overall 
response rate (ORR) in MDS and AML patients with TET2 
mutations. 
Primary Objective  
Primary Endpoint(s) 
To estimate the overall response rate (ORR) of the combination 
of standard dose azacitidine and oral dose of ascorbic acid in 
patients with MDS, MDS/MPN overlap, and AML with TET2 
mutations 
Secondary 
Objective(s) 
Secondary Endpoint(s) 
1. The safety profile of the combination in the targeted 
patient population 
2. Response duration  
3. Overall survival of the treated population (compared to 
matched historical cohort of patients treated with single 
agent Azacitidine) 
4. The identification of biomarkers that predict response to 

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the combination 
Correlative 
Objective(s) 
 
Correlative Endpoint(s) 
Identify gnomonic biomarkers for response. Evaluate methylation 
profile and after treatment to determine its relation with response.  
Sample Size 
28 
Disease 
sites/Conditions 
Myelodysplastic Syndromes (MDS), MDS/MPN syndromes, and 
AML 
Interventions 
Azacitidine 75 mg/m2, SC / IV day (1-7) every 28 days cycle 
 
Ascorbic acid 1 g PO  every day (daily) for every 28 days cycle 
 
 
 
 
 
ABBREVIATIONS  
 
CCCC 
Case Comprehensive Cancer Center 
CRF 
Case Report Form 
DCRU 
Dahm’s Clinical Research Unit 
DSTC 
Data Safety and Toxicity Committee 
FDA 
Food and Drug Administration 
ICF 
Informed Consent Form 
IRB 
Institutional Review Board 
PRMC 
Protocol Review and Monitoring Committee 
SOC 
Standard of Care 
CCF 
Cleveland Clinic Foundation 
UH 
University Hospitals 
MDS 
Myelodysplastic Syndromes 
CMML 
Chronic Myelomonocytic Leukemia 
MPN 
Myeloprolifrative Neoplasm 
AML 
Acute Myeloid Leukemia 
DNA 
Deoxyribonucleic acid 
AZA 
Azacitidine 
DAC 
Decitabine 
FDA 
The Food and Drug Administration 
HMAs 
Hypomethylating Agents 
VAF 
Variant Allelic Frequency 
ORR 
Overall Response Rate 
TET2 
Ten eleven translocation 2 
5mC 
5-methylcytosine 
5hmC 
5-hydroxymethylcytosine 
SC 
Subcutaneous 
CALGB Cancer and Leukemia Group B 
NCI 
National Cancer Institute 
AA 
Ascorbic Acid 

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PO 
Orally 
Sp 
Specificity protein 
SVCT-2 Sodium -dependent Ascorbic Acid transporter 2 
EGCG 
Epigallocatechin-3-gallate 
ETP  
Extended Treatment Period 
EOT 
End of Treatment 
CR 
Complete Remission 
PR 
Partial Remission 
HI 
Hematologic Improvement 
IWG 
International Working Group 
WHO 
World Health Organization 
APL 
Acute promyelocytic leukemia 
CNS 
Central Nervous System 
PI 
Principal Investigator 
WBC 
White Blood Cell 
CSF 
Colony-Stimulating Factor 
CTCAE  Common Terminology Criteria for Adverse Events 
AE 
Adverse Event 
SAE 
Serious Adverse Event 
Co-I 
Co-Investigator 
DSMP 
Data and Safety Monitoring Plan 
PRBC 
Packed Red Blood Cell 
ALT 
Alanine transaminase 
AST 
Aspartate aminotransferase 
ESA 
erythropoiesis-stimulating agent 
ECOG 
Eastern Cooperative Oncology Group 
RBC 
Red Blood Cells 
CRi 
Complete Response (CR) with  
incomplete hematologic recovery 
CRp 
Complete Response (CR) with  
incomplete platelets 
IEC 
Independent Ethics Committee 
GCP 
Good Clinical Practice 
ICH 
International Conference on Harmonization 
HIPAA 
Health Insurance Portability and  
Accountability Act of 1996 
OR 
Odds Ratio 
CI 
Confidence Intervals 
EOS 
End of Study 
BUN 
Blood Urea Nitrogen 
MCV 
Mean Corpuscular Volume 
MCHC 
Mean Cell Hemoglobin Concentration 
TP 
Treating Physician 

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TABLE OF CONTENTS 
 
1.0  
INTRODUCTION  
 
1.1 
Background   
1.2  
Name/description of Azacitidine 
1.3  
Name/description of High dose Ascorbic Acid 
1.4  
Rationale   
1.5 
Background and rationale of correlative studies 
 
2.0 
OBJECTIVES 
2.1 
Primary Objective 
 
2.2  
Secondary Objective(s) 
2.3 
Exploratory Objectives(s) 
2.4 
Correlative Objective(s) 
 
3.0 
STUDY DESIGN 
3.1 
Study design 
3.2 
Number of Subjects 
3.3 
Replacement of Subjects 
3.4 
Expected Duration of Treatment and Subject Participation 
 
 
4.0  
SUBJECT SELECTION 
4.1 
Inclusion Criteria 
4.2  
Exclusion Criteria 
4.3 
Inclusion of Women and Minorities 
 
5.0 
REGISTRATION  
 
 
6.0  
TREATMENT PLAN 
6.1 
Treatment Regimen Overview  
6.2 
Phase I Dose Escalation  
6.3  
Definition of Dose Limiting Toxicity 
6.4 
General Concomitant Medications and Supportive Care Guidelines 
6.5 
Criteria for Removal from Study 
6.6 
Duration of Follow-Up 
 
7.0 
DOSE DELAYS / DOSE MODIFICATIONS 
 
8.0 
ADVERSE EVENTS AND POTENTIAL RISKS 
8.1  
Agent Adverse events 
8.2 
Definitions 
8.3 
Serious Adverse Event Report Form 
8.4 
Reporting Procedures for Serious Adverse Event  
8.5 
Serious Adverse Events and OnCoreTM    
8.6 
Data Safety Toxicity Committee 
8.7 
Data and Safety Monitoring Plan 

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9.0 
PHARMACEUTICAL INFORMATION  
9.1 
Investigational Agent(s) 
 
9.2 
Commercial Agent(s) 
 
10.0 
EXPLORATORY/CORRELATIVE  
10.1  Collection of Samples 
  
 
11.0 
STUDY PARAMETERS AND CALENDAR 
11.1 
Study Parameters 
11.2 
Calendar 
 
12.0 
MEASUREMENT OF EFFECT choose appropriate measurement of effect; i.e. 
RECIST (solid tumors) / hematologic diseases / other response measurement 
 
13.0 
RECORDS TO BE KEPT/REGULATORY CONSIDERATIONS 
13.1 
Data Reporting 
13.2 
Regulatory Considerations  
 
 
14.0 
STATISTICAL CONSIDERATIONS 
 
REFERENCES 
 
  
APPENDICES  
The investigator may choose from the following commonly used appendices and modify per 
protocol specifications. The investigator must supply additional appendices as needed including 
questionnaires and surveys.  
 
 
 
APPENDIX I: ECOG / Karnofsky Performance Status Criteria  
 
 
APPENDIX II: Cycle 1 Subject Diary (pill, injectable, etc.) 
 
 
APPENDIX III: Cycle 2+ Subject Diary (pill, injectable, etc.) 
 
 
 
APPENDIX IV: Study Design Time Point Definitions 
 
APPENDIX V: Response Criteria for extended treatment 
 
APPENDIX VI: Clinical Laboratory Parameters 
 
APPENDIX VII: WHO Classification 
 
APPENDIX VIII: Protocol Amendment Summaries 
 
 

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1.0 
Introduction  
 
1.1 
Background of Study Disease  
 
Myelodysplastic syndromes (MDS) are clonal hematopoietic stem-cell disorders characterized 
by ineffective hematopoiesis, peripheral-blood cytopenias, and increased tendency to progress to 
acute myeloid leukemia (AML) 1. The median age of patients with MDS is approximately 70 
years2. This patient population is frequently affected by other comorbid conditions, a factor that 
often influences treatment decisions3. Treatment of MDS is based on prognostic factors that 
predict survival and progression to AML. The most widely used prognostic system for 
therapeutic decision-making is still the International Prognostic Scoring System3. This system 
stratifies patients into the following four groups: low, intermediate-1, intermediate-2, and high 
risk. Risk is based on number of cytopenias, percentage of bone marrow blasts, and karyotype. 
Low risk and intermediate-1 risk are usually grouped together as lower-risk disease, whereas 
intermediate-2 risk and high risk are grouped together as higher-risk disease3. Recently, genome 
sequencing technologies such as whole genome sequencing, whole exome sequencing, and next-
generation targeted deep sequencing have identified several recurrent somatic mutations that 
play an important role in the disease pathophysiology and prognosis4, 5.  
The discovery of new somatic mutations and the newly attained ability to use mutational 
screens as a diagnostic tool opened new opportunities for the development of new targeted 
therapies. Ten eleven translocation 2 (TET2) gene mutations are among the most common 
genomic alterations in myeloid malignancies4, 5. Somatic mutations in TET2 can be identified in 
25-30% of patients with myelodysplastic syndromes (MDS), 50-60% of patients with chronic 
myelomonocytic leukemia (CMML) an overlapping MDS/myeloprolifrative neoplasm (MPN) 
disorder, and 15-20% of patients with AML4-6. The TET2 gene is very polymorphic, with 
hundreds of single nucleotide polymorphisms (SNPs) of unknown clinical impact, but with some 
variants that may be pathogenically important. Similarly, somatic mutations affect all portions of 
the gene, and can be missense or truncating, homo-, hemi- and heterozygous. While the majority 
of TET2 mutations are ancestral, they can also be subclonal, implicating the clonal architecture in 
the consequences of TET2 lesions. Further, somatic mutations in TET2 gene were identified in 
healthy individuals7, 8. The presence of these mutations increases with age and can increase their 

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risk of developing hematologic malignancies and death7, 8. TET2 protein is one of the three 
proteins in TET family that are conserved dioxygenases that catalyzes the conversion of 5-
methyl-cytosine (5-mc) to 5-hydroxymethyl-cytosine (5-hmc) and promotes DNA methylation. 
Dioxygenases function of TET2 enzyme requires a-ketoglutarate (αKG), oxygen, and Fe (II) 
which is enhanced with the presence of ascorbic acid. Ascorbic acid interact with the catalytic 
domain of TET2 enzymes which may promote their folding and recycling of Fe (II) 10. Further, in 
mouse models, ascorbic acid can promote rapid and global increase in 5-hmc followed by DNA 
demethylation for many promoter genes, a change that are TET2 dependent10. Ascorbic acid is 
therefore an activator of TET2. Indeed, in preliminary results with purified TET2, ascorbic acid 
increased the enzymatic activity of WT TET2 and partially overcome the effects of the mixture 
of WT and mutant TET2 (TET2mut) indicating that ascorbic acid supplementation may increase 
the efficiency of dioxygenation and ultimately TET2 activity.  
TET2 belongs to a family of Fe2+ and α-ketoglutarate (αKG) dependent TET (TET1, 
TET2 and TET3) DNA-dioxygenases that catalyze the oxidation of 5-methylcytosine (5mC) to 
5-hydroxymethylcytosine (5hmC), this to 5-formylcytosine (5fC), and this to 5-carboxylcytosine 
(5caC). Ascorbic acid (Vitamin C) is a known enhancer of TET2 activity. We and others have 
shown that Vitmine C enhances the basal TET activity. Higher TET activity results in DNA 
demethylation either actively, by base excision repair of fC and caC, or passively, via replication, 
due to DNA methyltransferase’s inability to recognize/read hmC. The resulting DNA 
demethylation increases gene transcription of context dependent differentiation promoting genes. 
To access the level of TET2, we screened several cell lines with different TET2 genotype using 
Cosmic (https://cancer.sanger.ac.uk/cell_lines/cbrowse/all) and cancer cell encyclopedia 
(https://portals.broadinstitute.org/ccle) see Table 1. To confirm the expression level or protein in 
each cells we grew them to 70-80% confluence in indicated cell culture conditions. The cells 
were harvested and western blot analysis were performed on the nuclear fraction utilizing highly 
specific TET2 antibodies. The data is presented in Fig. 1 A and quantification in Fig. 1B. CMK, 
a TET2+/+ cell line established from the peripheral blood of a 10-month-old boy with down's 
syndrome and acute megakaryocytic leukemia (AML M7) at relapse, showed highest level of 
TET2 while HEL cells have a moderate level of TET2 and carries a mono allelic missense 
mutation in catalytic domain and SIGM5 a TET2-/- cell established from the bone marrow of a 

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63-year-old man with acute myeloid leukemia of monocytic origin (AML FAB M5a) at 
diagnosis shows no TET2 protein expression in western blot analysis.  To ascertain the role of 
ascorbic acid (Vitamin C) in epigenetic modifications, TET2 mutant and wild type MDS and 
MDS/AML we used these three cells. CMK, HEL and SIGM5 cells were propagated as 
described previously. Cells were treated at 300,000 cells/ml (sub confluent) for 12 hours with 
100 µM of  Vitamin C and the genomic DNA were extracted as described previously and 100 ng 
of DNA was spotted on PVDF membrane and cross-linked using UV crosslinking, blocked using 
BSA and probed with 5hmC and 5mC specific antibodies respectively. The spot analysis was 
performed on a Biorad ChemiDoc™ MP Imaging System. The data presented in Fig 2 is 
representative of three independent experiments. Consistent with the TET2 status, the ratio of 
5hmC/5mC was maximum in CMK followed by HEL and SIGM5. Thus we conclude that the 
effect of vitamin C will be similar for mono- and bi-allelic mutant MDS. Based on this data that 
was generated in our lab, we wanted to amend the protocol to include patients with any TET2 
mutations.   
 
 
 
Figure 1A: Western blot analysis results/ Figure 1B: Western blot analysis quantification 
 

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Figure 2: Results of three independent TET2 status experiments 
Azacitidine, decitabine, and lenalidomide are the only FDA approved drugs in MDS. 
Treatment with AZA and DAC improves cytopenias and prolongs survival in patients with MDS, 
though only 30-40% of patients respond to therapy3. Azacitidine was approved in USA based on 
phase III clinical trial that compared azacitidine to best supportive care. Overall response rate to 
azacitidine was 34% with significant delay in AML transformation but no overall survival 
benefit. In a phase III clinical trial conducted in Europe, azacitidine was compared to 
conventional care arm that included best supportive care, low dose cytarabine, and intensive 
chemotherapy11. The median OS for patients treated with azacitidine was 24.5 months compared 
to 15 months for patients treated with best supportive care (HR .58, p = .00001) 11. 
Studies have shown that MDS patients with TET2 mutations have a higher response rate 
to AZA. In a study of 82 patients with MDS and oligoblastic AML (20-30% BM blasts) treated 
with AZA, TET2 mutations were found in 15% of the patients. Patients with TET2 mutations had 
a higher response to AZA compared to wild type patients (82% vs 45%, p = 0.04, respectively) 
12. In a larger retrospective study, samples from 213 patients with MDS were sequenced prior to 
their treatment with hypomethylating agents (HMAs) for the presence of 40 recurrent gene 
mutations13. TET2 mutations with variant allelic frequency (VAF) >10% were associated with a 
significantly higher overall response rate (ORR) compared with WT (60% vs. 43%; OR, 1.99; 

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95%CI, 1.05-3.80, P=0.036) 13. Further, mice with transplanted TET2-null hematopoietic cells 
exhibit decrease in white blood counts when they are treated with AZA compared to vehicle.  
Recent in vitro study have shown that the addition of Ascorbic Acid at physiological 
levels to low doses of azacitidine is synergistic and can increase the inhibition of cancer-cell 
proliferation and increased apoptosis14. This synergistic was related to both passive DNA 
demethylation by DNMTi and active conversion of 5-methylcytosine (5mC) to 5-
hydroxymethylcytosine (5hmC) by TET2 enzymes14. More importantly, TET2 knockout reduces 
the synergy between the two compounds. Further, evidence to suggest that treatment with 
multiagent combination can be beneficial and feasible in patients with MDS/MPN syndromes 
was also shown in a study of the combination of (thalidomide, arsenic trioxide, dexamethasone, 
and ascorbic acid [TADA] 15. With a median on-study follow-up of 5.7 months, 21 patients 
(75%) completed the entire 12-week course of therapy, and 6 patients (29%) responded to 
TADA 15. With a median extended follow-up of 24.1 months for 15 evaluable patients, the 
median progression-free survival was 14.4 months, and the median overall survival was 21.4 
months. 
Taken together, the combination of standard dose AZA with ascorbic acid represent a 
rational, combination that may improve overall response rate (ORR) in MDS and AML patients 
with TET2 mutations.  
 
1.2 Name and Description of Azacitidine 
 
Azacitidine, a ring analog of the pyrimidine nucleoside cytidine, has effects on cell 
differentiation, gene expression, and DNA synthesis and metabolism. Since the early 1970s, 
azacitidine has been investigated in the US for the treatment of acute leukemia. Clinical trials 
have focused primarily on patients with disease refractory to conventional chemotherapy. These 
investigations indicated azacitidine has activity in the treatment of acute myeloid leukemia 
(AML). Clinical trials subsequently have been conducted to evaluate the effects of azacitidine in 
a variety of other malignant and hematologic disorders, including solid tumors, 
hemoglobinopathies (thalassemia and sickle cell anemia), and myelodysplastic syndromes 
(MDS).  
Toxicology studies have been conducted in mice, rats, dogs, and Rhesus monkeys. Most 

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of the studies were performed during the 1970s and early 1980s according to existing guidelines 
and standards in place during that period. The nonclinical studies identified the bone marrow, 
liver, kidneys, and lymphoid tissues (spleen, lymph nodes) as the main target organs of toxicity. 
In single-dose studies, the lethal dose of azacitidine after intravenous (IV) administration in 
mice, rats, and dogs was approximately 250 mg/m2. Repeated daily dosing appears to increase 
the toxicity of azacitidine. The genotoxicity of azacitidine is consistent with that of other 
nucleoside analogs that interact with nucleic acids. Likewise, similar to other agents with 
cytostatic properties, azacitidine was embryotoxic and reduced the reproductive performance in 
mice and rats. It is important to note that animal study data is superseded in many respects by the 
extensive clinical safety data collected in the last two decades.  
Limited azacitidine pharmacokinetic data are currently available. Based on plasma 
concentrations of total radioactivity (which represent parent drug plus circulating metabolites), 
azacitidine is rapidly absorbed when given subcutaneously (SC), with maximum plasma 
concentrations found 0.5 to 2 hours after dosing. Azacitidine and/or its by-products are rapidly 
cleared by the kidneys. The half-lives and percent radioactivity recovered in urine are similar for 
the IV and SC routes of administration.  
The effects of renal or hepatic impairment, gender, age, and race on the pharmacokinetics 
of azacitidine have not been studied. In 1985, the Cancer and Leukemia Group B (CALGB) 
study investigators began clinical trials with azacitidine in MDS patients under the auspices of 
the National Cancer Institute (NCI). Results from the three studies conducted by the CALGB 
(Protocols 8421, 8921, and 9221) have been published. The first two CALGB studies (Protocol 
8421 and Protocol 8921) were uncontrolled Phase 2 investigations. The most recent CALGB 
study (Protocol 9221) was a Phase 3 investigation that compared azacitidine to supportive care 
alone. The azacitidine dose investigated in the CALGB studies was 75 mg/m2/day for 7 days, 
repeated on a 28-day cycle. Azacitidine was administered by continuous IV infusion in the first 
study (Protocol 8421), and by SC injection in the two studies that followed. The dose was 
adjusted based on toxicity and clinical response. In the Phase 3 investigation (Protocol 9221), 
azacitidine produced higher response rates than supportive care alone. In addition, azacitidine 
prolonged the time to transformation to AML or death.  

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The efficacy of azacitidine to treat MDS also was evaluated in 7 open-label studies 
conducted outside the CALGB protocols. The dosage regimen used in 6 of these studies was 75 
mg/m2 given daily for 7 days every 3-4 weeks by SC injection in 4 studies, SC or IV in 1 study, 
and the route was not specified in the last study. The dosage regimen used in the seventh study 
was 5-35 mg/m2/day given by continuous IV infusion for 14 days. The lowest response rate was 
found in this seventh study, which suggests the mechanism of 5-azacitidine’s activity requires 
repeated administration of a minimally effective dose to achieve improvement in hematologic 
parameters.  
As with other antimetabolites, bone marrow suppression (leukopenia, thrombocytopenia) 
is a common adverse event associated with azacitidine. However, myelosuppression generally 
occurs more often and with greater severity at doses higher than those used to treat MDS. 
Gastrointestinal toxicity (i.e., nausea, vomiting, and diarrhea) can limit the dose of azacitidine in 
any patient population. Infrequent adverse effects include neuromuscular aches, generalized 
weakness, renal tubular acidosis, and liver enzyme abnormalities. Erythema and burning at the 
injection site can occur following SC administration, which usually resolves within 24-72 hours.  
1.3 Name and Description of Ascorbic Acid 
 
Ascorbic acid is a water-soluble vitamin. Ascorbic acid has few strictly pharmacological actions. 
Administration in amounts greatly in excess of physiologic requirements causes no demonstrable 
effects. The vitamin is an essential coenzyme for collagen formation, tissue repair and synthesis 
of lipids and proteins. It acts both as a reducing agent and as an antioxidant and is necessary for 
many physiologic functions, e.g., metabolism of iron and folic acid, resistance to infection, and 
preservation of blood vessel integrity. Signs and symptoms of early AA deficiency include 
malaise, irritability, and arthralgia, hyperkeratosis of hair follicles, nosebleed, and petechial 
hemorrhages. Prolonged deficiency leads to clinical scurvy.  
Ascorbic acid is normally present in both plasma and cells. The absorbed vitamin is 
ubiquitous in all body tissues 16. The highest concentrations are found in glandular tissue, the 
lowest in muscle and stored fat. Ascorbic acid is partially destroyed and partially excreted by the 
body. There is a renal threshold for AA; the vitamin is excreted by the kidney in large amounts 

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only when the plasma concentration exceeds this threshold, which is approximately 1.4 mg/100 
mL16. When the body is saturated with ascorbic acid, the plasma concentration will be about the 
same as that of the renal threshold; if further amounts are then administered, most of it escapes 
into the urine. When body tissues are not saturated and plasma concentration is low, 
administration of ascorbic acid results in little or no renal excretion16.  
A major route of metabolism of ascorbic acid involves its conversion to urinary oxalate, 
presumably through intermediate formation of its oxidized product, dehydro-ascorbic acid.  In a 
study of healthy volunteers at the NIH, an in-hospital depletion-repletion study was conducted. 
Seven healthy volunteers were hospitalized for 4-6 months and consumed a diet containing <5 
mg of AA daily16. Steady-state plasma and tissue concentrations were determined at seven daily 
doses of vitamin C from 30 to 2500 mg. AA steady-state plasma concentrations as a function of 
dose displayed sigmoid kinetics. Complete plasma saturation with AA occurred at 1000 mg PO 
daily16.   
Numerous studies have demonstrated that pharmacological doses of ascorbic acid (0.1–
100 mM) decrease cell proliferation in a variety of cancer cell lines 17, 22. Specifically, decreases 
in cell proliferation after ascorbic acid treatment have been reported for prostate, pancreatic, 
hepatocellular, colon, mesothelioma, and neuroblastoma cell lines. The potential mechanisms 
through which treatment with high-dose ascorbic acid may exert its effects on cancer cells have 
been extensively investigated. Several studies have demonstrated that the in vitro direct cytotoxic 
effect of ascorbic acid on various types of cancer cells is mediated through a chemical reaction 
that generates hydrogen peroxide 17,22. Treating colon cancer cells with 2 mM to 3 mM of 
ascorbic acid resulted in down regulation of specificity protein (Sp) transcription factors and Sp-
regulated genes involved in cancer progression. One study suggested that Ascorbic Acid-
mediated prostate cancer cell death may occur through activation of an autophagy pathway. 
Differences in chemosensitivity to Ascorbic Acid treatment in breast cancer cell lines may 
depend on expression of the sodium -dependent Ascorbic Acid transporter 2 (SVCT-2) 22. 
Research has suggested that pharmacological doses of ascorbic acid enhance the effects of 
arsenic trioxide on ovarian cancer cells gemcitabine on pancreatic cancer cells, and combination 
treatment of gemcitabine and epigallocatechin-3-gallate (EGCG) on mesothelioma cells.  

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Ascorbic Acid has also been investigated in clinical trials. One study reported three case 
reports of cancer patients who received IV ascorbic acid as their main therapy 18. During ascorbic 
acid therapy, the patients used additional treatments, including vitamins, minerals, and 
botanicals. According to the authors, the cases were reviewed in accordance with the NCI Best 
Case Series guidelines. Histopathologic examination suggested poor prognoses for these patients, 
but they had long survival times after being treated with IV ascorbic acid 18. Ascorbic Acid was 
given at doses ranging from 15 g to 65 g, initially once or twice a week for several months; two 
patients then received it less frequently for 1 to 4 years. Two studies demonstrated that IV 
ascorbic acid treatment resulted in improved quality of life and decreases in cancer-related side 
effects in cancer patients 18. Studies have shown that ascorbic acid can be safely administered to 
healthy volunteers or cancer patients at doses up to 1.5 g/kg and with screening to eliminate 
treating individuals with risk factors for toxicity (e.g., glucose-6-phosphate dehydrogenase 
deficiency, renal diseases, or urolithiasis). These studies have also found that plasma 
concentrations of ascorbic acid are higher with IV administration than with oral administration 
and are maintained for more than 4 hours 18.  
Ascorbic Acid was also investigated in combination with other cancer treatments. A 
phase I study published in 2012 examined the safety and efficacy of combining IV ascorbic acid 
with gemcitabine and erlotinib in stage IV pancreatic cancer patients 19. Fourteen subjects 
entered the study and planned to receive IV gemcitabine (1,000 mg /m2 over 30 minutes, once a 
week for 7 weeks), oral erlotinib (100 mg daily for 8 weeks), and IV ascorbic acid (50 
g/infusion, 75 g/infusion, or 100 g/infusion 3 times per week for 8 weeks). Minimal adverse 
effects were reported for ascorbic acid treatment19. Five subjects received fewer than 18 of the 
planned 24 ascorbic acid infusions and thus did not have follow-up imaging to assess response. 
Three of those patients had clinically determined progressive disease 19. All of the other nine 
patients had repeat imaging to assess tumor size, and each met the criteria for having stable 
disease. A 2013 phase I clinical study evaluated the safety of combining pharmacological 
ascorbic acid with gemcitabine in treating stage IV pancreatic cancer patients 20. During each 4-
week cycle, patients received gemcitabine weekly for 3 weeks (1,000 mg/m2 over 30 minutes) 
and twice weekly ascorbic acid infusions for 4 weeks (15 g over 30 minutes during the first 
week, followed by weekly escalations in dose until plasma levels reached at least 350 mg/dL [20 
mM]) 20. Among nine patients, mean progression-free survival was 26 weeks and overall 

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survival was 12 months 20. The combination treatment was well tolerated, and no significant 
adverse events were reported. In 2014, a phase I/IIA clinical trial evaluated the toxicities of 
combining IV ascorbic acid with carboplatin and paclitaxel in stage III /IV ovarian cancer. 
Twenty-seven patients were randomly assigned to receive either chemotherapy alone or 
chemotherapy and IV ascorbic acid concurrently 21. Chemotherapy was given for 6 months, and 
IV ascorbic acid was given for 12 months. The addition of IV ascorbic acid was associated with 
reduced chemotherapy-related toxicities. Trials of high-dose IV ascorbic acid with other drugs 
are ongoing 21. A number of studies have included IV ascorbic acid treatment (1,000 mg) with 
arsenic trioxide regimens, with mixed results. The combination therapies were well tolerated and 
suggested beneficial effects in multiple myeloma patients, although the specific contribution of 
ascorbic acid could not be determined. However, similar combination regimens resulted in 
severe side effects and disease progression in patients with acute myeloid leukemia, refractory 
metastatic colorectal cancer, and metastatic melanoma 21. 
 
2.0  
Objectives 
 
2.1       Primary Objective  
 
To estimate the overall response rate (ORR) of the combination of standard dose azacitidine and 
ascorbic acid in patients with MDS, AML, and MDS/MPN overlap with TET2 mutations. 
 
2.2       Secondary Objective(s) 
 
To evaluate the following: 
1. The safety profile of the combination of azacitidine and ascorbic acid in the targeted 
patient population. 
2. The response duration for study patients.  
3. The overall survival of the treated population (compared to matched historical cohort of 
patients treated with single agent Azacitidine) 
4. The identification of genomic biomarkers that may predict response to the combination 
treatment. 

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5. Evaluation of changes in methylation prior and during therapy  
 
3.0 
Study Design 
 
This is an open-label, phase II study that will be conducted at Cleveland Clinic, Taussig Cancer 
Institute.  
Azacitidine will be administered intravenously or subcutaneously at a fixed dose of 
75mg/m2/day for 7 consecutive days, (allowing for weekends, and holidays) of each 28-day 
cycle. Ascorbic acid will be administered orally daily at 1 g/day three days prior to start 
azacitidine and then continues daily for a total of 28 days of each 28 day cycle.  
3.1 
Number of Subjects 
 
Approximately 28 patients will be enrolled in this trial.  
 
3.2  
Expected Duration of Treatment and Subject Participation 
 
Upon signing consent, patients will be enrolled onto the study. Patients will undergo a screening 
period. The screening period is defined as the time period between the signing of the consent to 
the day the patient receives the first dose of study drug. During this period the patient will be 
assessed to determine if the patient meets all inclusion and exclusion criteria to be eligible for the 
study (Appendix IV). 
 
The treatment period is defined as the time between first day of study drug dosage (Day -
3) to C6D28. At this time the patient will be assessed and eligible to move into the Extended 
Treatment Period (ETP). The extended treatment period is defined as C7D1 to the patient meets 
EOT criteria (Section 6: Table 1; Appendix IV) 
 
 
Study treatment is defined as the treatment period and if applicable the extended 
treatment period (Appendix IV). Study drug will be continued during the duration of the study 
treatment unless patients exhibit any EOT criteria (Section 6: Table 1; Appendix IV).   
 

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Treatment should be continued for at least 6 cycles in the absence of toxicities that 
require discontinuation and/or any EOT criteria (Section 6: Table 1; Appendix IV). After 6 
cycles of treatment, a bone marrow biopsy and aspiration will be obtained to evaluate response. 
If patient has achieved either of the below response criteria (Appendix IV) based on their disease 
diagnosis patient will be eligible continue onto the extended treatment period (ETP). The patient 
is eligible to remain on treatment during the ETP until disease progression or patient meets EOT 
criteria (Section 6: Table 1; Appendix IV). 
  
Response Criteria for extended treatment eligibility 
 complete remission, partial remission, or hematologic improvement (CR/PR/HI) per IWG 
2006 criteria for patients with MDS 
 (CR/PR/marrow CR) per IWG 2003 criteria for patients with AML 
 If the response is stable disease and the investigator determines the patient is gaining 
clinical benefit, patient may continue on treatment until progression. 
 
 
At the point in which the patient meets the any EOT criteria (Section 6) the patient will 
enter into the follow up period to follow for survival status. The patient will be followed for up 
to 1 year and/or death, which ever were to occur first. End of study (EOS) is defined as the time 
the patient completes 1 year of survival follow up and/or patient’s death, whichever were to 
occur first. 
 
4.1 
Subject Selection 
 
4.2 Inclusion Criteria 
 
 
Subjects must meet all of the following inclusion criteria to be eligible for enrollment:  
 
4.2.1 Patients must have a confirmed TET2 mutations identified by next generation targeted 
deep sequencing. 
 

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4.2.2 Patients must have MDS, or MDS/MPN overlap defined by 2016 WHO criteria. Both 
newly diagnosed or previously treated MDS or MDS/MPN patients are eligible as long as 
the patient has never received prior treatment with azacitidine or decitabine. 
 
4.2.3 Patients with Leukemic/blast phase transformation MPN. 
 
4.2.4 Patient with AML according to 2016 WHO criteria (excluding APL [AML-M3]). 
 
4.2.4.1 Newly diagnosed patients who are ineligible or declined to receive intensive 
chemotherapy after discussion of risks and benefits of that approach or patients with 
primary refractory/relapsed AML. 
 
4.2.4.2 Patients with active central nervous system (CNS) leukemia eligible at the discretion of 
treating physician. 
 
4.2.4.3 Relapse/Refractory is defined as at least 1 course of treatment for AML excluding any 
patients treated with azacitidine or decitabine. 
 
4.2.4.3.1 Patients should be off any prior treatment or line of therapy for 2 weeks prior to start 
study with the exception of hydrea (Hydroxyurea). 
 
4.2.5 Prior therapy with hydroxyurea, biological or targeted therapy (e.g. flt3 inhibitors, other 
kinase inhibitors) or hematopoietic growth factors is allowed.  
 
4.2.6 Age >18 years. 
 
4.2.7 ECOG performance status < 3. 
 
4.2.8 Patients must have normal organ and marrow function as defined at the discretion of the 
treating physician and PI. 

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4.2.9 Women of childbearing potential must have a negative serum or urine pregnancy     test 
within 10-14 days prior to enrollment. 
 
4.2.10 Patients must have the ability to understand and the willingness to sign a written 
informed consent document. 
 
4.2.11 Patient must be willing to comply with all aspects of the protocol including completing 
the drug diary. 
 
4.2.12 Patient must discontinue any and all use of multivitamin and/or vitamin c medication 24 
hours before first dose of Ascorbic Acid. 
 
4.2 Exclusion Criteria 
 
The presence of any of the following will exclude a subject from study enrollment: 
 
4.2.1 Any prior treatment with azacitidine or decitabine. 
 
4.2.2 Patients diagnosed with APL, AML-M3. 
 
4.2.3 Patients receiving other active treatment for their myeloid malignancy including 
investigational agents with the exception of hydrea for WBC control.  
 
4.2.4 Nursing or pregnant women. 
 
4.2.5 History of allergic reactions to either azacitidine or ascorbic acid. 
 
4.2.6 Patients with uncontrolled intercurrent illness including, but not limited to ongoing or 
active infection, symptomatic congestive heart failure, unstable angina pectoris, cardiac 
arrhythmia, or psychiatric illness/social situations that would limit compliance with study 
requirements.  

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4.2.7 Patients with higher risk of bleeding (deemed by the treating physician) or on 
anticoagulation. 
 
4.2.8 Patients who are unwilling or unable to comply with all study requirements. 
 
4.3 
Inclusion of Women and Minorities  
 
Men, women and members of all races and ethnic groups are eligible and encouraged to 
participate in this trial. 
 
5.0 Registration 
 
All subjects who have been consented are to be registered in the OnCore™ Database. For those 
subjects who are consented, but not enrolled, the reason for exclusion be recorded. 
 
All subjects will be registered through Cleveland Clinic and will be provided a study number by 
contacting the study coordinator once the patient signs the consent. 
 
 
6.0  
Treatment Plan 
 
6.1 
Treatment Regimen Overview 
 
All azacitidine courses will be given at Cleveland Clinic Taussig Cancer Institute or an affiliate 
hospital approved to treat patients. 
 
6.1.1 Azacitidine 
 

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Azacitidine will be administered intravenously or subcutaneously at a fixed dose of 
75mg/m2/day for 7 consecutive days, allowing interruptions for weekends and holidays within 
each 28-day cycle. No dose modifications will be permitted during the treatment period. 
 
6.1.2 Ascorbic Acid 
 
Ascorbic acid will be administered orally daily at 1 g/day three days prior to start azacitidine and 
then continues daily for a total of 28 days of each 28 day cycle. No dose modifications will be 
permitted during the treatment period. 
 
6.2 Treatment Duration 
 
The length of the treatment will vary per patient. The typical treatment period will be 6 months 
for the course of the required treatment. If the patient is eligible to continue treatment during the 
ETP, the patient is able to receive treatment until the patient meets one or more of the EOT 
criteria. 
 
6.3 
General Concomitant Medications and Supportive Care Guidelines 
 
Patients should be off any prior treatment or line of therapy for 2 weeks prior to start study with 
the exception of hydrea.  
Necessary supportive measures for optimal medical care will be given throughout the 
treatment as determined by the treating physician and the patient’s medical need. No 
concomitant chemotherapy, immunotherapy, or therapy with monoclonal antibodies will be 
allowed during the study with the exception of hydroxyurea or corticosteroids for control of 
blood counts. 
Use of a colony-stimulating factor or combinations thereof (e.g. G-CSF, GM-CSF, or 
erythropoietin) are at the discretion of the treating physician and is permitted if judged in the 
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Prophylactic antibiotics, antifungals, and antiviral agents (e.g. levofloxacin, itraconazole, 
valacyclovir, etc.) are recommended; however, the use of these or other drugs will be left to the 
treating physician’s discretion. 
 
6.4 
Criteria for Removal from Study Treatment (EOT) 
 
In the absence of treatment delays due to adverse events, treatment may continue for 6 cycles or 
be extended after 6 cycles unless l one of the following criteria applies: 
 
End of Treatment Criteria 
 Disease progression defined as no response after 6 months of treatment or loss of 
response during treatment as defined in section 12 
 Intercurrent illness that prevents further administration of treatment 
 The investigator and/or treating  physician considers it to be in the best interest of the 
subject to permanently discontinue study treatment 
o This includes if the treatment is deemed to be of excessive toxicity to the 
patient by the treating physician 
 Patient decision to withdraw from treatment (partial consent) or from the study (full 
consent) 
 Pregnancy during the course of the study for a child-bearing participant 
 Death 
 Unacceptable adverse events defined as following: 
o Unacceptable treatment related toxicity, NCI CTC AE version 4.0 Grade 3 or 4 
that fails to recover to baseline or < Grade 3 in the absence of treatment within 
4 weeks 
o Any toxicity or other issue that causes a delay of study drug administration by 
more than 4 weeks  
 
Table 1: End of Treatment Criteria 
 
6.5 
Duration of Follow Up 
 
The clinical course of each adverse event will be followed until resolution, stabilization, or until 
it has been determined that the study treatment or participation is not the cause.  
Serious adverse events that are still ongoing at the end of the study period will necessitate 
follow-up to determine the final outcome. Any serious adverse event that occurs after the study 
period and is considered to be possibly related to the study treatment or study participation will 
be recorded and reported immediately. 

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Following completion of active treatment (EOT) and while still on study (Follow up), 
patients will be followed for survival as of standard of care for up to 1 year. This follow up can 
either be done as a visit to the study doctor’s clinic or via telephone call to the patient.  
 
7.0  
Dose Delays/Dose Modifications 
 
Patients will receive a course of their treatment every 4weeks, but not exceeding more 8 weeks 
between treatment courses (4 weeks of treatment + a window of 4 weeks between cycles). 
Patients should receive their second course of therapy without interruption, regardless of their 
degree of myelosuppression. After the first two courses of therapy, the interval between 
subsequent cycles of Azacitidine therapy could be prolonged (with a maximum of 4 weeks 
prolongation between each 28 day cycle) at the discretion of the investigator. During any dose 
delays of Azacitidine, Ascorpic Acid therapy should not be delayed. Subsequent cycles can be 
given prior to baseline peripheral blood count recovery if considered to be in the best interest for 
the patient and after discussion with the principal investigator and the discussion documented in 
the patient’s medical record.  
No dose modification to azacitidine or ascorbic acid can occur during the treatment 
course.  
 
8.0 
Potential Risks and Adverse events 
 
8.1 
Potential Risks 
 
8.1.1 Azacitidine 
 
 
COMMON, SOME MAY BE SERIOUS 
In 100 people receiving azacitidine more than 20 and up to 100 may have: 

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COMMON, SOME MAY BE SERIOUS 
In 100 people receiving azacitidine more than 20 and up to 100 may have: 
 Fever 
 Fatigue 
 Headache 
 Nausea 
 Vomiting 
 Diarrhea 
 Constipation 
 Loss of appetite 
 Bruising 
 Low blood cell counts 
 Weakness 
 Joint pain 
 Shivering 
 Cough 
 Difficulty Breathing 
 
OCCASIONAL, SOME MAY BE SERIOUS 
In 100 people receiving azacitidine from 4 to 20 may have: 
 Chest pain 
 Pale skin 
 Swelling (such as in arms and legs) 
 Abnormal heart sound 
 Fast heart beat 
 Low blood pressure 
 High blood pressure 
 Fainting 
 Dizziness 
 Anxiety 
 Depression 
 Difficulty sleeping 
 Lack of energy 
 Numbness 
 Pain 
 Skin hives and /or rash 
 Dry skin and /or itching 
 Sweating 
 Low blood levels of potassium 
 Weight loss 
 Abdominal pain, tenderness, and/or swelling 
 Mouth blisters 
 Upset stomach 
 Hemorrhoid bleeding 
 Difficulty swallowing 
 Sore or painful throat 

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OCCASIONAL, SOME MAY BE SERIOUS 
In 100 people receiving azacitidine from 4 to 20 may have: 
 Difficult and/or painful urination 
 Blood in urine 
 Pain 
 Stuffy and/or runny nose 
 Wheezing 
 Lymph node swelling 
 Infection 
 Hair loss 
 
RARE, AND SERIOUS 
In 100 people receiving azacitidine 3 or fewer may have: 
 Irregular heartbeat 
 Heart failure 
 Stoppage of heart and lung function 
 Bleeding in and/or around the brain 
 Seizure 
 Skin condition with fever and skin lesions 
 Abnormal Blood acid/base balance (possible organ damage) 
 Dehydration 
 Inflammation of the gallbladder 
 Terry or coffee ground-like blood in the stool 
 Enlarged spleen 
 Liver failure 
 Kidney failure, even requiring dialysis 
 Build-up of bodily waste products in the blood 
 Coughing up blood 
 Lung inflammation (possible difficulty breathing) 
 Allergic reaction, which may be life-threatening 
 Body-wide inflammation 
 Breakdown products of the cancer cells entering the blood stream 
 
Azacitidine may cause you to develop another type of cancer such as leukemia, a type of blood 
cancer 
 
 
 
 
 
 
 
 
 
 
8.1.2 Ascorbic Acid 

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COMMON, SOME MAY BE SERIOUS 
In 100 people receiving ascorbic acid more than 20 and up to 100 may have: 
 Fatigue 
 Headache 
 Nausea 
 Vomiting 
 Diarrhea 
 Constipation 
 Loss of appetite 
 Bruising 
 Low blood cell counts 
 Weakness 
 Joint pain 
 Shivering 
 Cough 
 Difficulty Breathing 
 
OCCASIONAL, SOME MAY BE SERIOUS 
In 100 people receiving ascorbic acid from 4 to 20 may have: 
 Pale skin 
 Swelling (such as in arms and legs) 
 Abnormal heart sound 
 Fast heart beat 
 Low blood pressure 
 High blood pressure 
 Fainting 
 Dizziness 
 Anxiety 
 Depression 
 Lack of energy 
 Numbness 
 Pain 
 Skin hives and /or rash 
 Dry skin and /or itching 
 Sweating 
 
RARE, AND SERIOUS 
In 100 people receiving ascorbic acid 3 or fewer may have: 
 Kidney stones 
 Kidney failure 
 Bleeding that could be life threatening 
 Loss of blood due to hemolysis (a process where red cell gets destructed) 
 
 

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8.2 
Definitions  
 
8.2.1      Adverse Event   
 
An adverse event (AE) is any unfavorable or unintended event, physical or psychological, 
associated with a research study, which causes harm or injury to a research participant as a result 
of the participant’s involvement in a research study. The event can include abnormal laboratory 
findings, symptoms, or disease associated with the research study. The event does not necessarily 
have to have a causal relationship with the research, any risk associated with the research, the 
research intervention, or the research assessments.  
Adverse events may be the result of the interventions and interactions used in the research; 
the collection of identifiable private information in the research; an underlying disease, disorder, 
or condition of the subject; and/or other circumstances unrelated to the research or any 
underlying disease, disorder, or condition of the subject.  
Abnormal laboratory results will be assessed in real time by the physicians. Treatment for 
any clinically significant values will be completed in real documented in the physician’s note. 
Documentation for the assessment and treatment will be provided in the physician’s note as 
source documentation.  
Clinical staff (i.e. nurses, clinical research coordinators) can be delegated to evaluate and 
assess all adverse events, with the exception of determining relationship to study treatment. 
These assessments will require documented review by either the treating physician or PI. 
 
8.2.2   Serious Adverse Events  
 
A serious adverse event (SAE) is any adverse experience occurring at any dose that results in 
any of the following outcomes:  
 Results in death.  
 Is a life-threatening adverse experience.  The term life-threatening in the definition 
of serious refers to an adverse event in which the subject was at risk of death at the 
time of the event.  It does not refer to an adverse event which hypothetically might 
have caused death if it were more severe.  

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 Requires inpatient hospitalization or prolongation of existing hospitalization.  
Any adverse event leading to hospitalization or prolongation of hospitalization will be 
considered as Serious, UNLESS at least one of the following expectations is met: 
o The admission results in a hospital stay of less than 24 hours OR 
o The admission is pre-planned (e.g., elective or scheduled surgery arranged 
prior to the start of the study) OR 
o The admission is not associated with an adverse event (e.g., social 
hospitalization for purposes of respite care. 
However it should be noted that invasive treatment during any hospitalization may fulfill 
the criteria of “medically important” and as such may be reportable as a serious adverse 
event dependent on clinical judgment.  In addition where local regulatory authorities 
specifically require a more stringent definition, the local regulation takes precedent.  
 Results in persistent or significant disability/incapacity.  The definition of 
disability is a substantial disruption of a person’s ability to conduct normal life’s 
functions. 
 Is a congenital anomaly/birth defect.  
 Is an important medical event.  Important medical events that may not result death, 
be life-threatening, or require hospitalization may be considered a serious adverse 
experience when, based upon appropriate medical judgment, they may jeopardize the 
subject and may require medical or surgical intervention to prevent one of the 
outcomes listed in this definition.  Examples of such medical events include allergic 
bronchospasm requiring intensive treatment in an emergency room or at home, blood 
disease or disorders, or convulsions that do not result in inpatient hospitalization, or 
the development of drug dependency or drug abuse. The development of a new 
cancer is always considered an important medical event. 
 
8.2.3 Adverse Event Evaluation  
 
The investigator or designee is responsible for ensuring that all adverse events (both serious and 
non-serious) observed by the clinical team or reported by the subject, which occur after the 

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subject has signed the informed consent are fully recorded in the subject’s medical records. 
Source documentation must be available to support all adverse events.  
 
The investigator or sub-investigator (treating physician if applicable) or delegated staff will 
provide the following for all adverse events (both serious and non-serious): 
 Event term (as per CTCAE 4.1) 
 Description of the event 
 Date of onset and resolution 
 Expectedness of the toxicity 
 Grade of toxicity  
 Attribution of relatedness to the investigational agent- (this must be assigned by an 
investigator, sub-investigator, or treating physician) 
 Action taken as a result of the event, including but not limited to; no changes, dose 
interrupted, reduced, discontinued, etc. or action taken with regard to the event, i.e. no 
action, received conmed or other intervention, etc. 
 Outcome of event 
An expected adverse event is an event previously known or anticipated to result from 
participation in the research study or any underlying disease, disorder, or condition of the 
subject. The event is usually listed in the Investigator Brochure, consent form or research 
protocol.  
An unexpected adverse event is an adverse event not previously known or anticipated to result 
from the research study or any underlying disease, disorder, or condition of the subject.  
Attribution is the relationship between an adverse event or serious adverse event and the study 
drug.  Attribution of any adverse event can only be assessed by the PI and/or Co-I. Attribution 
will be assigned as follows: 
 Definite – The AE is clearly related to the study drug. 
 Probable – The AE is likely related to the study drug. 
 Possible – The AE may be related to the study drug. 
 Unlikely – The AE is doubtfully related to the study drug. 
 Unrelated – The AE is clearly NOT related to the study drug. 
 

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Protocol must specify if attribution is required for individual components of the treatment 
regimen or the treatment regimen as a whole. 
 
8.3  
SAE Report Form 
 
SAEs will be recorded on the FDA Form 3500A (MedWatch) and Study SAE coversheet but 
should only be reported as instructed below.  The electronic FDA SAE reporting forms should 
not be used.   
 
8.4  
Reporting Procedures for Serious Adverse Events 
 
For the purposes of safety reporting, all adverse events will be reported that occur during 
treatment and through 30 days after the final dose of study drug. Adverse events, both serious 
and non-serious, and deaths that occur during this period will be recorded in the source 
documents. All SAEs should be monitored until they are resolved or are clearly determined to be 
due to a subject’s stable or chronic condition or intercurrent illness(es). Related AEs will be 
followed until resolution to baseline or grade 1 or stabilization.  
 
8.4.1 SAE Reporting Requirements 
 
 Participating investigators (all sites) must report all serious adverse events to the Lead 
Site Principal Investigator (e.g. Sponsor-Investigator) within 24 hours of discovery or 
notification of the event. The participating investigator must also provide follow-up 
information on the SAE until final resolution.  
 The Lead Site Principal Investigator will review the SAE and report the event to the 
FDA, external collaborator(s), and IRB as applicable. 
 It is the Sponsor-Investigator’s responsibility (e.g. lead site PI) to ensure that ALL 
serious adverse events that occur on the study (e.g. ALL SAEs that occur at each 
enrolling institution) are reported to all participating sites. 
 
Institutional Review Board Reporting Requirements: 

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 Investigative sites will report adverse events to their respective IRB according to the local 
IRB’s policies and procedures in reporting adverse events.  
 
8.5 
SAEs and OnCore 
 
 All SAEs will be entered into OnCore.   
 A copy of the SAE form(s) submitted to the sponsor-investigator is also uploaded into 
OnCore. 
8.6  
Data Safety and Toxicity Committee 
 
It is the responsibility of each site PI to ensure that ALL SAEs occurring on this trial (internal or 
external) are reported to the Case Comprehensive Cancer Center’s Data and Safety Toxicity 
Committee. This submission is simultaneous with their submission to the sponsor and/or other 
regulatory bodies.  
  
8.7 
Data and Safety Monitoring Plan (DSMP) 
 
This protocol will adhere to the policies of the Case Comprehensive Cancer Center Data and 
Safety Monitoring Plan in accordance with NCI guidelines.  
 
9.0 
PHARMACEUTICAL INFORMATION  
 
A list of the adverse events and potential risks associated with the investigational or commercial 
agents administered in this study can be found in Section 8.1. 
 
 
9.1 Investigational Agents 
 
9.1.1  Name of Agent 
Azacitidine 
 
Other Names:   
Vidaza 

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Product description: Azacitidine is FDA approved and commercially available.  
 
Preparation for Intravenous Administration: Reconstitute the appropriate number of 
azacitidine vials to achieve the desired dose. Reconstitute each vial with 10 mL sterile water for 
injection. Vigorously shake or roll the  vial until all solids are dissolved. The resulting solution 
will contain azacitidine 10mg/mL. The solution should be clear. Parenteral drug product should 
be inspected visually for particulate matter and discoloration prior to administration, whenever 
solution and container permit.  
Withdraw the required amount of azacitidine solution to deliver the desired dose and inject into a 
50-100 mL infusion bag of either 0.9% Sodium Chloride Infusion or Lactated Ringer’s Infusion.  
Azacitidine is incompatible with 5% Dextrose solutions, Hespan, or solutions that contain 
bicarbonate. These solutions have the potential to increase the rate of degradation of azacitidine 
and should therefore be avoided.  
Intravenous Administration: Azacitidine solution is administered intravenously. Administer 
the total dose over a period of 10-40 minutes. The administration must be completed within 1 
hour of reconstitution of the azacitidine vial.  
Solution Stability: Azacitidine reconstituted for intravenous administration may be stored at 
25°C (77°F), but administration must be completed within 1 hour of reconstitution. If 
reconstituted azacitidine comes into contact with the skin, immediately and thoroughly wash 
with soap and water. If it comes into contact with mucous membranes, flush thoroughly with 
water.  Reconstituted azacitidine may be stored for up to 1 hour at 25°C (77°F) or for up to 8 
hours between 2 and 8°C (36 and 46°F). The azacitidine suspension is single-use and does not 
contain any preservatives. Unused portions of each vial should be discarded properly. Do not 
save any unused portions for later administration. It is recommended that 5-azacitidine in 
solution is administered as soon as possible.  
Storage: Store reconstituted vials at 25o C (77o F); excursions permitted to 15o-30o C (59o-86o 
F) (See USP Controlled Room Temperature). There is no need to protect azacitidine from 

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exposure to light.  
Preparation for Subcutaneous Administration: Reconstitute VIDAZA aseptically with 4 mL 
sterile water for injection. Inject the diluent slowly into the vial. Vigorously shake or roll the vial 
until a uniform suspension is achieved. The suspension will be cloudy. The resulting suspension 
will contain azacitidine 25 mg/mL. Do not filter the suspension after reconstitution. Doing so 
could remove the active substance. 
Preparation for Immediate Subcutaneous Administration: Doses greater than 4 mL should 
be divided equally into 2 syringes. The product may be held at room temperature for up to 1 
hour, but must be administered within 1 hour after reconstitution. 
Preparation for Delayed Subcutaneous Administration: The reconstituted product may be 
kept in the vial or drawn into a syringe. Doses greater than 4 mL should be divided equally into 2 
syringes. The product must be refrigerated immediately. When VIDAZA is reconstituted using 
water for injection that has not been refrigerated, the reconstituted product may be held under 
refrigerated conditions (2ºC - 8ºC, 36ºF - 46ºF) for up to 8 hours. When VIDAZA is 
reconstituted using refrigerated (2ºC - 8ºC, 36ºF - 46ºF) water for injection, the reconstituted 
product may be stored under refrigerated conditions (2ºC - 8ºC, 36ºF - 46ºF) for up to 22 hours. 
After removal from refrigerated conditions, the suspension may be allowed to equilibrate to 
room temperature for up to 30 minutes prior to administration. 
9.1.2  Name of Agent 
Ascorbic Acid 
 
Other Names:   
Vitamin C, 1000 mg   
 
Product description: Ascorbic Acid is a tablet of 1000 mg Patient is to take one tablet once a 
day.   
Product Description 
USP-Verified Dietary Supplement. Recommended by pharmacists. Antioxidant - Helps boost the 
immune system. Vitamin C helps neutralize free radicals and helps iron get absorbed in the body. 
UPS has tested and verified ingredients, potency and manufacturing process. USP sets official 

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standards for dietary supplements. No artificial colors. No artificial flavors. No preservatives. No 
chemical solvents, yeast or gluten. (These statements have not been evaluated by the Food and 
Drug Administration. This product is not intended to diagnose, treat, cure or prevent any 
disease).  
Brand: Nature Made 
This Nature Made product provides vitamin C, a versatile and essential vitamin critical for the 
proper functioning of our immune system. In addition, vitamin C helps manufacture certain 
nerve-transmitting substances and hormones while helping absorb and utilize other nutrients, 
such as vitamin E and iron. Vitamin C also makes collagen, which holds connective tissues 
together in skin, bone, teeth, and other parts of the body.  
Vitamin C is an important and powerful antioxidant that works in the aqueous (water) 
environments of the body, such as the lungs and lens of the eye. Its primary antioxidant partners 
are vitamin E and the carotenes (such as beta-carotene), but it also works with the antioxidant 
enzymes. Vitamin C regenerates oxidized vitamin E and restores the antioxidant potential of 
vitamin E in the body.  
Vitamin C is recommended as part of a daily regimen for women's and men's health, longevity, 
heart strength, and immunity.  
 
No artificial colors  
 
No artificial flavors  
 
No preservatives  
 
No chemical solvents, yeast or gluten. Recommended By Pharmacists.  
 
USP Standards plus Nature Made's Pure Ingredients guarantee optimum quality. 
Guaranteed purity, potency and dissolution.  
Safety Warning 
Do not use if imprinted seal under cap is broken or missing. 

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Ingredients 
Cellulose Gel, Hypromellose, Croscarmellose Sodium, Stearic Acid, Magnesium Stearate, 
Silicon Dioxide, Polyethylene Glycol, Vitamin C 
Directions 
Suggested Use: Take one tablet daily with a meal. Keep bottle tightly closed. Store in a cool, dry 
place, out of reach of children. 
10.0 
EXPLORATORY or CORRELATIVE STUDIES 
 
A blood sample will be obtained for a next generation targeted deep sequencing genomic panel 
of 170 gene mutations prior to start treatment. Quantification of 5hmc and 5mc in patient’s 
derived mononuclear cells will be obtained during the screening period and on C6D15 to 
evaluate for changes in methylation. We will use the ELISA, dot-blot and mass spec assay to 
measure the level of 5hmC, a signature for TET2 activity in HSPCs and 5mC an indicator of the 
lack of TET2 activity in patient derived PBMCs (Peripheral blood mononuclear cells) and bone 
marrow cells. For this purpose total genomic DNA from whole cell nuclear lysates (PBMCs or 
Bone marrow) or from the nuclear lysate of CD34+ cells will be extracted and used for ELISA 
assay using a known standard curve. The mass spec will be performed on the samples after 
digesting with NaOH (Ref: Nature 468, 839–843) and separated and quantified by LCMS-MS 
(Ref: Mo Cell; Volume 64, Issue 5, 1 December 2016, Pages 913–925) utilizing X-bridge 
reverse phase column. Recombinant TET2 protein will be used to prepare the standard reaction 
mixture for 5hmC determination and comparison to the patient samples.  
 
All samples obtained will shipped to and stored at The Cleveland Clinic. Samples will 
remain at the Cleveland Clinic to be used for research purposes for this study as well as for 
future research projects approved by the IRB. 
 
10.1 Collection of Samples 
 

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10 ML of whole blood or bone marrow aspirate whichever available will be collected in extra lab 
tube. Details and instructions about the collection, processing, and shipping will be explain in the 
Lab Manual. All samples should be delivered and/or shipped to: 
 
Attn: Elise Lemelle, Cassie Hirsch, and/or Amy Graham 
 
Principal Research Technologists 
Translational Hematology and Oncology  
Cleveland Clinic, LC – NE6 – 250 
9620 Carnegie Ave. 
Cleveland, OH 44195 
 Phone: (216) 444-0256 
 
11.0  STUDY PARAMETERS AND CALENDAR  
 
11.1 
Study Parameters 
 
11.1.1 Screening Evaluation (Consent signing to Day -4) & Baseline period (Day -17 to day -3) 
 
1. A complete history and physical examination within 30 days prior to study entry.   
2. A whole blood sample (10 ML) that will be used for next generation targeted deep 
sequencing of genes that are common in myeloid malignancies   
3. Hematology exam (Appendix VI) 
4. Serum Chemistry (Appendix VI) 
5. 
Bone marrow biopsy and aspiration with conventional cytogenetic analysis within 30 
days prior to start the study. 10 ML of whole blood or bone marrow aspirate whichever 
available will be collected in extra lab tube. 
6. 
Documentation of transfusion frequency (PRBC and platelets only) prior to first cycle 
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7. 
Documentation of all medication including multivitamins and other over the counter 
medications 
8. 
Dates of multivitamin/vitamin C discontinuation must be documented and occur before 
first dose of study drug. Pre-study laboratory tests must be obtained within 14 days of 
registration.  
9. 
MDS Response Assessment, assessed by the treating physician 
Treatment Period (Day -3 to C6D28) 
1. Hematology exams (Appendix VI) at the start of each cycle (day 1) and the last day of 
cycle 6 (C6D28)  
2. Serum Chemistry (Appendix VI), including Creatinine, bilirubin, ALT or AST at the start 
of each cycle (day 1) 
3. Bone marrow aspirate and/or biopsy to confirm response, to be performed at the end of 
cycle 6. 10 ML of whole blood or bone marrow aspirate whichever available will be 
collected in extra lab tube. All bone marrows samples are to be stored at Cleveland Clinic  
4. Conventional cytogenetics to be performed at the end of cycle 6 
5. Documentation of frequency of transfusions (only PRBC and Platelets) prior to start of 
each new cycle. 
6. Documentation in the medical record of any grade 2 or higher adverse events at each 
visit. For guidelines on SAE Reporting and AE entry for Case Report  
7. Documentation of any prior administration of growth factors and ESA prior to start of 
each cycle  
8. A 10 ML blood sample that will be used for next generation targeted deep sequencing of 
genes that are common in myeloid malignancies  

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9. Physical exam and ECOG performance status occurring at the end of each cycle 
10. Vital signs occulting at each visit 
11. Pregnancy test if applicable 
12. MDS Response Assessment, assessed by the treating physician occurring at the end of 
cycle 6 or EOT, whichever occurs first. 
13. Methylation panel only occurring in Cycle 1 
14. Azacitidine will be administered intravenously or subcutaneously at a fixed dose of 
75mg/m2/day for 7 consecutive days, (allowing interruptions for weekends, and holidays, 
of each 28-day cycle). 
15. Ascorbic acid will be administered orally daily at 1 g/day three days prior to start 
azacitidine and then continues daily for a total of 28 days of each 28 days cycle. 
Extended Treatment Period (Cycles 7+) 
1. Documentation of frequency of transfusions (only PRBC and Platelets) prior to start of 
each new cycle 
2. Documentation in the medical record of any grade 3 or higher adverse events at each 
visit. For guidelines on SAE Reporting and AE entry for Case Report  
3. Physical exams and ECOG performance status occurring at the end of each cycle 
4. Vital Signs at each visit 
5. Hematology exams (Appendix VI) at the start of each cycle (day 1)  
6. Serum Chemistry (Appendix VI), including Creatinine, bilirubin, ALT or AST at the start 
of each cycle (day 1).  
7.  Azacitidine will be administered intravenously or subcutaneously at a fixed dose of 
75mg/m2/day for 7 consecutive days, (allowing interruptions for weekends, and holidays) 

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of each 28-day cycle 
8. Ascorbic acid will be administered orally daily at 1 g/day for a total of 28 days of each 28 
days cycle. 
End of Treatment (EOT)   
1. Documentation of frequency of transfusions (only PRBC and Platelets) prior to start of 
each new cycle 
2. Documentation in the medical record of any grade 3 or higher adverse events at each 
visit. For guidelines on SAE Reporting and AE entry for Case Report  
3. ECOG performance  
4. Hematology exams (Appendix VI)  
5. Serum Chemistry (Appendix VI), including Creatinine, bilirubin, ALT or AST  
16. Optional bone marrow aspirate and/or biopsy to confirm response, to be performed at the 
end of cycle 6. All bone marrows samples are to be stored at Cleveland Clinic  

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11.2 
Calendar  
 
Period 
Screening 
Period 
(Day-17 to 
Day -3) 
Treatment Period 
Extended 
Treatment 
Period 
Cycles 
End Of 
Treat-
ment 
(EOT) 
Follow 
upk 
 
 
Cycle 1 
Cycle 2 
Cycle 3 
Cycle 4 
Cycle 5 
Cycle 6 
 
Day 
 
-3 
1 
7c 
14n 
1 
7c 
1 
7c 
1 
7c 
1 
7c 
1 
7c 
28g 
1 
7c 
 
 
Administration of Azacitidine 
 
 
X 
Xb 
 
X 
Xb 
X 
Xb 
X 
Xb 
X 
Xb 
X 
Xb 
 
X 
Xb 
 
 
Ascorbic Acid 
Dispensement 
 
Xa 
 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
 
X 
 
 
 
disease history and prior therapies 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Record RBC/ platelets transfusion 
X 
X 
 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
 
 
 
History and physical examdm 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
 
Xj 
Vital signsd 
X 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Xj 
ECOG performance statusem 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
Hematologyd m 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Xj 
Serum Chemistryd m 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
Pregnancy test 
X 
 
 
X 
 
Disease response assessmentf 
X 
 
 
X 
X 
X 
Bone marrow biopsy and 
aspirationL 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Xh 
 
 
Xi 
 
Mutational analysis blood draw/L 
Xl 
 
 
X 
 
Methylation profile blood drawL 
X 
 
X 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Drug Diary Accountability 
 
X 
 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
 
 
 
Survival Follow up 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Xk 
Record adverse eventsm 
 
 X  
 
 

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A: For Cycle 1 only, patient will begin treatment with Ascorbic Acid 3 days prior to initial treatment of Azacitidine 
B: Azacitidine administration has a +/- 3 day window to account for breaks in treatment for weekends, and holidays 
C: Day 7 has a +/- 3 day window to account for breaks in treatment for weekends, holidays, and institutional standards 
D: See Appendix VI for required tests 
 
E: See Appendix I for performance status grading 
F: See Section for 12.0 for MDS/AML response assessment criteria 
G: If patient is eligible for the extended treatment period, and moving forward on the next cycle without a dosing delay, the C6D28 
visit can serve as the visit of C7D1. If the patient is not eligible for the extended treatment period the patient’s C6D28 visit can serve 
as the EOT visit. 
H: The cycle 6 bone marrow biopsy can occur up to 14 days (C6D14) prior to the C6D28 visit. 
I: EOT bone marrow biopsy is optional per discretion of the treating physician 
J: These tests in follow up are optional pending discretion of the treating physician 
K: Survival Follow Up will occur bimonthly (60 days +/- 7 days) for a duration of 1 year from patient’s EOT visit. 
L: 10 ML of whole blood or bone marrow aspirate whichever available will be collected in extra lab tube for these tests. 
M: If these procedures require a physician visit, these can occur 3 calendar days prior to the day 1 of that cycle (window of – 3 days). 
N: Cycle 1 Day 14 has a +/- 1 day window to account if the day falls on a holiday or weekend. 
 
 
 
 

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12.0  MEASUREMENT OF EFFECT  
The response criteria recommended by the MDS International Working Group 2006. Responses 
must be verified, documented, and signed at cycle 6 in the local medical record by the local PI. 
(Patients are considered non evaluable if they have received less than 2 courses of treatment, 
unless there is clear disease progression.)  
Definitions: 
Responses for MDS patients: 
Complete Response (CR): Bone marrow: ≤ 5% myeloblasts with normal maturation of all cell 
lines; persistent dysplasia will be noted. Peripheral blood: Hgb ≥ 11 g/dL Platelets ≥ 100 × 
109/L Neutrophils ≥ 1.0 × 109/L, Blasts 0%  
Partial response (PR): All CR criteria if abnormal before treatment except: bone marrow blasts 
decreased by ≥ 50% over pretreatment but still > 5%  
Marrow CR: Blasts ≤ 5% and decrease by ≥ 50% from baseline (baseline blasts should be 
above 5% to be eligible for marrow CR)   
Hematologic Improvement (HI): (responses must last at least 8 weeks) Erythroid response 
[HI-E; (if pretreatment Hgb < 11 g/dL)]; Hgb increase by ≥ 1.5 g/dL; Relevant reduction of units 
of RBC transfusions by an absolute number of at least 4 RBC transfusions/8 weeks compared 
with the pretreatment transfusion number in the previous 8 weeks. Only RBC transfusions given 
for an Hgb of ≤ 9.0 g/dL pretreatment will count in the RBC transfusion response evaluation.  
Platelet response [HI-P; ((if pretreatment Platelet count < 100 x 109 /L)]: Absolute increase of ≥ 
30 × 109/L for patients starting with > 20 × 109/L platelets, or increase from < 20 × 109/L to > 
20 × 109/L and by at least 100%.  
Neutrophil response [HI-N; (pretreatment, ANC < 1.0 × 109/L)]: At least 100% increase and an 
absolute increase > 0.5 × 109/L  

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Progression/Relapse after hematological improvement: ≥1 of the following: ≥50% decrement 
from maximum response levels in granulocytes or platelets; ↓ in Hgb by ≥1.5 g/dL; transfusion 
dependence  
Stable Disease: Not satisfying criteria for Complete Remission, Partial Response, Marrow CR, 
Hematologic Improvement or Progression/Relapse.  
Responses for AML patients: 
Complete Response (CR): Bone marrow: ≤ 5% myeloblasts with normal maturation of all cell 
lines, ANC >/= 1.0 X 109/L, platelet count >/= 100 X 109/L, no detectable Auer rods and no 
extramedulary leukemia. 
Complete Response (CR) with incomplete hematologic recovery (CRi): Responses as in CR 
but ANC < 1.0 X 109/L. 
Complete Response (CR) with incomplete platelets recovery (CRp): Responses as in CR but 
platelets < 100 X 109/L. 
Partial response (PR): All CR criteria if abnormal before treatment except: bone marrow blasts 
decreased by ≥ 50% over pretreatment but still > 5%. 
 
13.0 
DATA REPORTING / REGULATORY CONSIDERATIONS  
 
Adverse event lists, guidelines, and instructions for AE reporting can be found in Section 8.0 
(Adverse Events: List and Reporting Requirements). 
 
13.1 
Data Reporting  
The OnCore™ Database will be utilized, as required by the Case Comprehensive Cancer Center, 
to provide data collection for both accrual entry and trial data management. OnCore™ is a 
Clinical Trials Management System housed on secure servers maintained at Case Western 
Reserve University. OnCore™. Access to data through OnCore™ is restricted by user accounts 
and assigned roles. Once logged into the OnCore™ system with a user ID and password, 

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OnCore™ defines roles for each user which limits access to appropriate data. User information 
and password can be obtained by contacting the OnCore™ Administrator at OnCore-
registration@case.edu. OnCore™ is designed with the capability for study setup, activation, 
tracking, reporting, data monitoring and review, and eligibility verification. This study will 
utilize electronic Case Report Form completion in the OnCore™ database. A calendar of events 
and required forms are available in OnCore™. 
 
13.2 
Regulatory Considerations 
The study will be conducted in compliance with ICH guidelines and with all applicable federal 
(including 21 CFR parts 56 & 50), state or local laws. 
13.2.1 Written Informed consent 
Provision of written informed consent must be obtained prior to any study-related procedures. 
The Principal Investigator will ensure that the subject is given full and adequate oral and written 
information about the nature, purpose, possible risks and benefits of the study as well as the 
subject’s financial responsibility. Subjects must also be notified that they are free to discontinue 
from the study at any time. The subject should be given the opportunity to ask questions and be 
allowed time to consider the information provided.  
The original, signed written Informed Consent Form must be kept with the Research Chart in 
conformance with the institution’s standard operating procedures. A copy of the signed written 
Informed Consent Form must be given to the subject. Additionally, documentation of the 
consenting process should be located in the research chart. 
13.2.2 Subject Data Protection 
In accordance with the Health Information Portability and Accountability Act (HIPAA), a 
subject must sign an authorization to release medical information to the sponsor and/or allow the 
sponsor, a regulatory authority, or Institutional Review Board access to subject’s medical 
information that includes all hospital records relevant to the study, including subjects’ medical 
history.  
 
13.2.3  Retention of records 

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The Principal Investigator of The Case Comprehensive Cancer Center supervises the retention of 
all documentation of adverse events, records of study drug receipt and dispensation, and all IRB 
correspondence for as long as needed to comply with local, national and international 
regulations. No records will be destroyed until the Principal Investigator confirms destruction is 
permitted.  
 
13.2.4  Audits and inspections  
Authorized representatives of the sponsor, a regulatory authority, an Independent Ethics 
Committee (IEC) or an Institutional Review Board (IRB) may visit the site to perform audits or 
inspections, including source data verification. The purpose of an audit or inspection is to 
systematically and independently examine all study-related activities and documents to 
determine whether these activities were conducted, and data were recorded, analysed, and 
accurately reported according to the protocol, Good Clinical Practice (GCP), guidelines of the 
International Conference on Harmonization (ICH), and any applicable regulatory requirements.  
For multi-center studies, participating sites must inform the sponsor-investigator of pending 
audits.  
 
14.0    STATISTICAL CONSIDERATIONS  
 
Definition of primary outcome/endpoint: A binary indicator of overall response, defined as 
achieving CR, PR, or HI) per 2006 IWG criteria for MDS and MDS/MPN overlap and (CR, 
CRp, CRi, or PR) per 2003 IWG criteria for AML after 6 cycles of treatment 
Definition of secondary outcomes/endpoints:  
1.Incidence of adverse events of the combination defined by CTCAE 4.1 criteria.  
Toxicity will be any drug related Grade 3 or 4 toxicity.   
2.Response duration to the combination recorded from start of treatment to progression 
3.Overall survival measured from start of treatment to death or last follow up  
Sample size justification and study design:  The study sample size was determined using 
Minimax Simon's two-stage design (Simon, 1989. The null hypothesis that the true response rate 
is 0.3 will be tested against a one-sided alternative that the true response rate is 0.5. In the first 
stage, 12 patients will be accrued. If there are 3 or fewer responses, the study will be stopped. 
Otherwise, 16 additional patients will be accrued for a total of 28. The null hypothesis will be 

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rejected if 11 or more responses are observed in 28 patients. This design yields a type I error rate 
of 0.1 and a power of 0.8 when the true response rate is 0.5. 
Analysis plan: Descriptive statistics will be used for the primary endpoint and secondary 
endpoints 1,2, and 3. Overall survival will be estimated with Kaplan Meier method and will be 
compared to the survival of a historical cohort of MDS patients treated at our institution with 
single agent Azacitidine between 2006-2012 using a one-sample log rank test.   A Cochran-
Mantel-Haenszel test will be used to test for differences in response rate by mutational status 
controlling for treatment. Unadjusted and adjusted logistic regression models were used to 
predict response to therapy. Models will be adjusted for covariates including age, sex, and other 
prognostic factors. The odds ratio (OR) and 95% confidence intervals (CI) will be estimated for 
the risk group (mutated) and compared with the reference group (WT). 
  

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REFERENCES 
 
 
REFERENCES 
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2. Sekeres MA. Epidemiology, natural history, and practice patterns of patients with 
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3. Garcia-Manero G. Prognosis of myelodysplastic syndromes. Hematology / the Education 
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4. Bejar R, Stevenson K, Abdel-Wahab O, et al. Clinical effect of point mutations in 
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5. Haferlach T, Nagata Y, Grossmann V, et al. Landscape of genetic lesions in 944 patients 
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7. Genovese G, Kahler AK, Handsaker RE, et al: Clonal hematopoiesis and blood-cancer 
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11. Fenaux P, Mufti GJ, Hellstrom-Lindberg E, et al. Efficacy of azacitidine compared with 
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Leukemia 25:1147-52, 2011 
13. Bejar R, Lord A, Stevenson K, et al. TET2 mutations predict response to 
hypomethylating agents in myelodysplastic syndrome patients. Blood 2014;124:2705-12. 
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15. Bejanyan N, Tiu RV, Raza A, et al. A phase 2 trial of combination therapy with 
thalidomide, arsenic trioxide, dexamethasone, and ascorbic acid (TADA) in patients with 
overlap myelodysplastic/myeloproliferative neoplasms (MDS/MPN) or primary 
myelofibrosis (PMF). Cancer 2012; 118(16): 3968-76. 
16. Levine M, Conry-Cantilena C, Wang Y, et al. Vitamin C pharmacokinetics in healthy 
volunteers: evidence for a recommended dietary allowance. Proceedings of the National 
Academy of Sciences of the United States of America 1996; 93(8): 3704-9. 
17. Cameron E, Pauling L. Supplemental ascorbate in the supportive treatment of cancer: 
Prolongation of survival times in terminal human cancer. Proceedings of the National 
Academy of Sciences of the United States of America 1976; 73(10): 3685-9. 
18. Padayatty SJ, Riordan HD, Hewitt SM, Katz A, Hoffer LJ, Levine M. Intravenously 
administered vitamin C as cancer therapy: three cases. CMAJ : Canadian Medical 
Association journal = journal de l'Association medicale canadienne 2006; 174(7): 937-
42. 
19. Monti DA, Mitchell E, Bazzan AJ, et al. Phase I evaluation of intravenous ascorbic acid 
in combination with gemcitabine and erlotinib in patients with metastatic pancreatic 
cancer. PloS one 2012; 7(1): e29794. 
20. Welsh JL, Wagner BA, van't Erve TJ, et al. Pharmacological ascorbate with gemcitabine 
for the control of metastatic and node-positive pancreatic cancer (PACMAN): results 
from a phase I clinical trial. Cancer chemotherapy and pharmacology 2013; 71(3): 765-
75. 

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21. Ma Y, Chapman J, Levine M, Polireddy K, Drisko J, Chen Q. High-dose parenteral 
ascorbate enhanced chemosensitivity of ovarian cancer and reduced toxicity of 
chemotherapy. Science translational medicine 2014; 6(222): 222ra18. 
22. Chen Q, Espey MG, Krishna MC, et al: Pharmacologic ascorbic acid concentrations 
selectively kill cancer cells: action as a pro-drug to deliver hydrogen peroxide to tissues. 
Proc Natl Acad Sci U S A 102:13604-9, 2005. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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APPENDIX I 
 
PERFORMANCE STATUS CRITERIA 
 
ECOG Performance Status Scale 
Karnofsky Performance Scale 
Grade 
Description 
Percent 
Description 
 
0 
 
Normal activity. Full active, able to 
carry on all pre-disease performance 
without restriction.  
 
100 
 
Normal, no complaints, no 
evidence of disease. 
 
90 
Able to carry on normal activity; 
minor signs or symptoms of 
disease. 
 
1 
Symptoms, but ambulatory. Restricted 
in physically strenuous activity, but 
ambulatory and able to carry out work 
of a light or sedentary nature (e.g., 
light housework, office work). 
 
80 
Normal activity with effort; some 
signs or symptoms of disease. 
 
70 
Cares for self, unable to carry on 
normal activity or to do active 
work.  
 
2 
In bed < 50% of the time.  
Ambulatory and capable of all self-
care, but unable to carry out any work 
activities. Up and about more than 
50% if waking hours. 
 
60 
Requires occasional assistance, 
but is able to care for most of 
his/her needs. 
 
50 
Requires considerable assistance 
and frequent medical care. 
 
3 
In bed > 50% of the time.  Capable of 
only limited self-care, confined to bed 
or chair more than 50% of waking 
hours. 
 
40 
 
Disabled, requires special care 
and assistance. 
 
30 
 
Severely disabled, hospitalization 
indicated.  Death not imminent. 
 
4 
100% bedridden.  Completely 
disabled.  Cannot carry on any self-
care. Totally confined to bed or chair. 
 
20 
Very sick, hospitalization 
indicated. Death not imminent.  
 
10 
Moribund, fatal processes 
progressing rapidly. 
5 
Dead. 
0 
Dead 
 
 
 
 
 
 
 
 
 
 
 
 
 

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APPENDIX II 
 
CYCLE 1 SUBJECT PILL DIARY 
 
Subject Name ________________________Protocol #________________ Subject Study ID __________  
Cycle #: _______    
       Month #: _______                                  
INSTRUCTIONS FOR THE SUBJECT: 
   1.  You will take 1 tablet of 1000 mg of Ascorbic Acid (Vitamin C) pills each day. Take the tablets with food 
   2.  Record the date, the number of tablets you took, and what time you took them. 
   3.  If you have any comments please record them in the “Comments” column below. 
   4.  Please bring your pill bottle and this form to your physician when you come for your next appointment.   
   5.  Please sign your name at the bottom of the diary. 
Date 
Day 
Time   
# of 1000 mg Ascorbic Acid 
(Vitamin C) pills and time 
taken 
Comments 
 
-3 
 
 
 
 
-2 
 
 
 
 
-1 
 
 
 
 
1 
 
 
 
 
2 
 
 
 
 
3 
 
 
 
 
4 
 
 
 
 
5 
 
 
 
 
6 
 
 
 
 
7 
 
 
 
 
8 
 
 
 
 
9 
 
 
 
 
10 
 
 
 
 
11 
 
 
 
 
12 
 
 
 
 
13 
 
 
 
 
14 
 
 
 
 
15 
 
 
 
 
16 
 
 
 
 
17 
 
 
 
 
18 
 
 
 
 
19 
 
 
 
 
20 
 
 
 
 
21 
 
 
 
 
22 
 
 
 
 
23 
 
 
 
 
24 
 
 
 
 
25 
 
 
 
 
26 
 
 
 
 
27 
 
 
 
 
28 
 
 
 
Subject’s Signature: ________________________________________ Date: ___________ 
 
 

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APPENDIX III 
 
 CYCLE 2 + SUBJECT PILL DIARY 
 
Subject Name ________________________Protocol #________________ Subject Study ID __________  
Cycle #: _______    
       Month #: _______                                  
INSTRUCTIONS FOR THE SUBJECT: 
   1.  You will take 1 tablet of 1000 mg of Ascorbic Acid (Vitamin C) pills each day. Take the tablets with food 
   2.  Record the date, the number of tablets you took, and what time you took them. 
   3.  If you have any comments please record them in the “Comments” column below. 
   4.  Please bring your pill bottle and this form to your physician when you come for your next appointment.   
   5.  Please sign your name at the bottom of the diary. 
Date 
Day 
Time   
# of 1000 mg Ascorbic Acid 
(Vitamin C) pills and time 
taken 
Comments 
 
1 
 
 
 
 
2 
 
 
 
 
3 
 
 
 
 
4 
 
 
 
 
5 
 
 
 
 
6 
 
 
 
 
7 
 
 
 
 
8 
 
 
 
 
9 
 
 
 
 
10 
 
 
 
 
11 
 
 
 
 
12 
 
 
 
 
13 
 
 
 
 
14 
 
 
 
 
15 
 
 
 
 
16 
 
 
 
 
17 
 
 
 
 
18 
 
 
 
 
19 
 
 
 
 
20 
 
 
 
 
21 
 
 
 
 
22 
 
 
 
 
23 
 
 
 
 
24 
 
 
 
 
25 
 
 
 
 
26 
 
 
 
 
27 
 
 
 
 
28 
 
 
 
Subject’s Signature: ________________________________________ Date: ___________ 

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APPENDIX IV: STUDY DESIGN TIME POINT DEFINTIONS 
 
Study Time Points 
Definitions 
Screening 
Screening will occur from the time of the 
consent until day -4 
Baseline/Enrollment 
Days -17b to Day -3c 
Treatment Period 
Day 1c to C6D28 or patient meets end of 
treatment criteria before completing 6 cycles 
Extended Treatment Period (ETP) 
The extended treatment period is defined as 
C7D1 to the patient meets EOT criteria 
Study Treatment 
The treatment period and if applicable the 
extended treatment period 
End of Treatment (EOT) 
The day patient meets the defined EOT criteria 
(Table 1) and discontinues study treatment. 
Follow up 
Patient will be followed  bimonthly (60 days 
+/- 7 days) for a duration of 1 year after study 
End of Study (EOS) 
The time the patient completes 1 year of 
survival follow up and/or patient’s death, 
whichever were to occur first. 
A: Day 1 is defined as first dose of Azacitidine 
B: Day -17 is defined as 14 days prior to first dose of study Drug (Day -3) 
C: Day -3 is defined as day of Study Drug  
 
  
APPENDIX V: RESPONSE CRITERIA FOR EXTENDED TREATMENT 
 
Response Criteria for extended treatment eligibility 
 complete remission, partial remission, or hematologic improvement (CR/PR/HI) per IWG 
2006 criteria for patients with MDS 
 (CR/PR/marrow CR) per IWG 2003 criteria for patients with AML 
 If the response is stable disease and the investigator determines the patient is gaining 
clinical benefit, patient may continue on treatment until progression. 
 
 
 
 
 
 
 
 
 
 
 

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APPENDIX VI: CLINICAL LABORATORY PARAMETERS 
 
Hematology 
Serum Chemistry Vitals 
Physical Exam 
Bone Marrow Biopsy & 
Aspiration 
Red blood cell count  Sodium  
Temp 
Height(only at 
C1D1) 
Marrow blast percent 
Hemoglobin  
Potassium  
Pulse 
Weight 
Marrow Cellularity 
Hematocrit  
Chloride  
Resp 
 
Auer Rods 
White blood cell 
count  
Bicarbonate  
Blood 
Pressure 
 
Dysplasia 
Neutrophil 
BUN  
SpO2* 
 
Cytogenetics 
Platelets  
Glucose (non-
fasting)  
 
 
 
Blasts percent  
Albumin  
 
 
 
MCV 
AST  
 
 
 
MCHC 
ALT  
 
 
 
Eosinophils  
Serum creatinine 
 
 
 
Basophils  
Protein 
 
 
 
Monocytes  
 Calcium 
 
 
 
Lymphocyte 
Bilirubin 
 
 
 
 
Alkaline 
phosphate 
 
 
 
 This test should only occur at Screening visit 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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APPENDIX VII: WHO classification of myeloid neoplasms and acute leukemia 
Arber et al, 2016 
 
WHO myeloid neoplasm and acute leukemia classification 
Myeloproliferative neoplasms (MPN) 
 Chronic myeloid leukemia (CML), BCR-ABL1+ 
 Chronic neutrophilic leukemia (CNL) 
 Polycythemia vera (PV) 
 Primary myelofibrosis (PMF) 
  PMF, prefibrotic/early stage 
  PMF, overt fibrotic stage 
 Essential thrombocythemia (ET) 
 Chronic eosinophilic leukemia, not otherwise specified (NOS) 
 MPN, unclassifiable 
Mastocytosis 
Myeloid/lymphoid neoplasms with eosinophilia and rearrangement 
of PDGFRA, PDGFRB, orFGFR1, or with PCM1-JAK2 
 Myeloid/lymphoid neoplasms with PDGFRA rearrangement 
 Myeloid/lymphoid neoplasms with PDGFRB rearrangement 
 Myeloid/lymphoid neoplasms with FGFR1 rearrangement 
 Provisional entity: Myeloid/lymphoid neoplasms with PCM1-JAK2 
Myelodysplastic/myeloproliferative neoplasms (MDS/MPN) 
 Chronic myelomonocytic leukemia (CMML) 
 Atypical chronic myeloid leukemia (aCML), BCR-ABL1− 
 Juvenile myelomonocytic leukemia (JMML) 
 MDS/MPN with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T) 

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WHO myeloid neoplasm and acute leukemia classification 
 MDS/MPN, unclassifiable 
Myelodysplastic syndromes (MDS) 
 MDS with single lineage dysplasia 
 MDS with ring sideroblasts (MDS-RS) 
  MDS-RS and single lineage dysplasia 
  MDS-RS and multilineage dysplasia 
 MDS with multilineage dysplasia 
 MDS with excess blasts 
 MDS with isolated del(5q) 
 MDS, unclassifiable 
 Provisional entity: Refractory cytopenia of childhood 
Myeloid neoplasms with germ line predisposition 
Acute myeloid leukemia (AML) and related neoplasms 
 AML with recurrent genetic abnormalities 
  AML with t(8;21)(q22;q22.1);RUNX1-RUNX1T1 
  AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22);CBFB-MYH11 
  APL with PML-RARA 
  AML with t(9;11)(p21.3;q23.3);MLLT3-KMT2A 
  AML with t(6;9)(p23;q34.1);DEK-NUP214 
  AML with inv(3)(q21.3q26.2) or t(3;3)(q21.3;q26.2); GATA2, MECOM 
  AML (megakaryoblastic) with t(1;22)(p13.3;q13.3);RBM15-MKL1 
  Provisional entity: AML with BCR-ABL1 
  AML with mutated NPM1 
  AML with biallelic mutations of CEBPA 

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WHO myeloid neoplasm and acute leukemia classification 
  Provisional entity: AML with mutated RUNX1 
 AML with myelodysplasia-related changes 
 Therapy-related myeloid neoplasms 
 AML, NOS 
  AML with minimal differentiation 
  AML without maturation 
  AML with maturation 
  Acute myelomonocytic leukemia 
  Acute monoblastic/monocytic leukemia 
  Pure erythroid leukemia 
  Acute megakaryoblastic leukemia 
  Acute basophilic leukemia 
  Acute panmyelosis with myelofibrosis 
 Myeloid sarcoma 
 Myeloid proliferations related to Down syndrome 
  Transient abnormal myelopoiesis (TAM) 
  Myeloid leukemia associated with Down syndrome 
Blastic plasmacytoid dendritic cell neoplasm 
 
 
 
 
 
 
 
 
 
 
 

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APPENDIX VIII: PROTOCOL AMENDMENT SUMMARIES  
 
Amendment Number/Version Number/Date: Amendment 1, protocol version 2, 11/20/2017 
Section #/page 
Description of change (s) 
Preparation for 
Intravenous 
Administration, pg 32 
Corrected Chloride Injection or Lactated Ringer’s Injection to Chloride 
Infusion or Lactated Ringer’s Infusion. 
Investigational 
Agents, pg 32 
Added option to deliver Azacitidine to patients subcutaneously due to the 
national IV bag shortage. 
Investigational 
Agents, pg 33 
Corrected the dosages amount for the ascorbic acid as well as the amount of 
tablets. 
Screen test procedures 
Removed Vitamin C level testing 
CLINICAL 
LABORATORY 
PARAMETERS, pg 
53 
Clarified that SpO2 testing should only occur at screening visit 
Appendix IV, pg 54 
Clarified days to align with study calendar 
Study Calendar 
Provided a 3 day window for each day 1 visit requiring a physician visit 
 
Amendment Number/Version Number/Date: Amendment 2, protocol version 3, 05/24/2018 
Section #/page 
Description of change (s) 
Clinical Trials.gov 
number, pg 1 
Added Clinical Trials.gov Number 
Protocol Date and 
Protocol version 
number 
Updated the protocol date and version number throughout the entire 
protocol. 
Eligibility Criteria, pg 
20  
Updated the eligibility criteria from heterozygous TET2 mutations, to 
include all TET2 mutations 
Background of Study 
Disease, pg 10-11 
Updated rationale as to why the eligibility criteria is changing from 
heterozygous TET2 mutations, to include all TET2 mutations 
Study Calendar, pg 
43/44 
Adding Cycle 1 Day 14 has a +/- 1 day window to account if the day 
falls on a holiday or weekend.