PROACTIVE: Prevention of Acute and Chronic GVHD Using TocIlizumab in Combination With Standard GVHD Prophylaxis After allogEneic Transplantation
Phase: PHASE2
Conditions: Hematologic Malignancy
Sponsor: Medical College of Wisconsin

Title of Trial:  PROACTIVE: Phase II Single Center Open Label Study for Prevention of Acute 
and Chronic GVHD Using Tocilizumab in Combination with Standard GVHD Prophylaxis After 
Allogeneic Transplantation 
 
Clinical Trials.gov Number:  NCT03699631 
 
Date of Protocol:  September 19, 2019 

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CLINICAL STUDY PROTOCOL 
 
 
  
 
PROACTIVE: Phase II Single Center Open Label Study for Prevention of Acute 
and Chronic GVHD Using Tocilizumab in Combination with Standard GVHD Prophylaxis 
After Allogeneic Transplantation 
  
 
 
Saurabh Chhabra, MD 
 
 
Version 4, September 19, 2019 
 
 
 
Proprietary and Confidential 
The information in this document is considered privileged and confidential and may not 
be disclosed to others except to the extent necessary to obtain Institutional Review 
Board approval and informed consent, or as required by federal and state laws. Persons 
to whom this information is disclosed should be informed that this information is 
privileged and confidential and that it should not be further disclosed. 
 
 
 
 
 

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PROTOCOL SIGNATURE PAGE 
Protocol No.: 32694 
Version Date: 09/19/2019 
1. I agree to follow this protocol version as approved by the Medical College of Wisconsin 
(MCW) Scientific Review Committee (SRC), Institutional Review Board (IRB) and Data 
Safety Monitoring Committee (DSMC).  
2. I will conduct the study in accordance with applicable IRB requirements, federal regulations, 
and state and local laws to maintain the protection of the rights and welfare of study 
participants.  
3. I certify that I, and the study staff, have received the requisite training to conduct this 
research protocol.  
4. I have read and understand the information in the Investigator’s Brochure (or Manufacturer’s 
Brochure) regarding the risks and potential benefits. I agree to conduct the protocol in 
accordance with Good Clinical Practices (ICH-GCP), the applicable ethical principles, the 
Statement of Investigator (Form FDA 1572) and with local regulatory requirements. In 
accordance with the FDA Modernization Act, I will ensure the registration of the trial on the 
www.clinicaltrials.gov website.  
5. I agree to maintain adequate and accurate records in accordance with IRB policies, Federal, 
state and local laws and regulations. 
 
 
MCW Principal Investigator / Study Chair 
 
 
Printed Name 
 
 
Signature 
 
Date 
 

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Title: PROACTIVE: Phase II Single Center Open Label Study for Prevention of Acute and 
Chronic GVHD Using TocIlizumab in Combination with Standard GVHD Prophylaxis 
After Allogeneic Transplantation 
MCW OnCore® No.: IIT-Chhabra-Proactive 
MCW Protocol No.: 32694  
 
Principal Investigator 
Saurabh Chhabra, MD 
Assistant Professor 
Medical College of Wisconsin 
9200 W. Wisconsin Ave.  
Milwaukee, WI. 53226 
Telephone:  414-805-4600 
Fax:  414-805-0596 
Email: schhabra@mcw.edu 
Biostatistician 
Aniko Szabo, PhD 
Associate Professor 
Division of Biostatistics 
Institute for Health & Society 
8701 Watertown Plank Road 
Milwaukee, Wis. 53226 
Telephone:  414-955-4096 
Fax:  414-955-0176 
Email:  aszabo@mcw.edu 
  
Co-Principal Investigator 
William R. Drobyski, MD 
Professor 
Medical College of Wisconsin 
8701 Watertown Plank Road 
Milwaukee, Wis. 53226 
Telephone:  414-805-4600 
Email: wdrobysk@mcw.edu 
 
 
Co-investigator 
Jennifer M. Knight, MD, MS, FACLP 
Assistant Professor of Psychiatry, Medicine, 
and Microbiology & Immunology 
Divisions of Consultation Liaison Psychiatry 
and Hematology/Oncology 
Medical College of Wisconsin 
8701 Watertown Plank Rd. 
Milwaukee, WI 53226 
Email: jmknight@mcw.edu 
Funding Sponsor:  
William R. Drobyski, MD 
Professor 
Medical College of Wisconsin 
8701 Watertown Plank Road 
Milwaukee, Wis. 53226 
Telephone:  414-805-4600 
Email: wdrobysk@mcw.edu 
Investigational Agent(s):  
Tocilizumab 
OnCore® Contact: 
Rachel Thuemling 
Research Program Coordinator 
MCW Cancer Center 
9200 W. Wisconsin Ave.  
Milwaukee, WI. 53226 
Telephone:   414-805-4376 
Email: rthuemli@mcw.edu 
 
 

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REVISION HISTORY 
Revision history is presented in reverse order so that the information pertaining to the most 
current version of the protocol is presented first in this section. 
Version 4, September 19, 2019 
Version 3, June 27, 2019 
Version 2, November 21, 2018 
Version 1, August 24, 2018 
Initial submission of the protocol.  
 

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PROTOCOL SUMMARY 
Title 
Phase II Single Center Open Label Study for Prevention of Acute 
and Chronic GVHD Using Tocilizumab in Combination with 
Standard GVHD Prophylaxis After Allogeneic Transplantation 
Protocol Number 
32694 
Principal Investigator  
Saurabh Chhabra, MD 
Study Sites 
Froedtert & the Medical College of Wisconsin 
Clinical Trial Phase 
II 
Study Disease 
Hematologic malignancy 
Main Eligibility 
Criteria 
Inclusion Criteria: 
1. 
Age ≥18 years. 
2. 
Patients with any hematologic malignancy for which alloHCT 
is indicated. Patients with acute myeloid leukemia (AML) and 
acute lymphoblastic leukemia (ALL) must be in complete 
remission at the time of alloHCT(<5% blasts in the bone 
marrow, normal maturation of all cellular components in the 
bone marrow and absence of extramedullary disease). 
3. 
Myeloablative conditioning (MAC) regimen, based on 
CIBMTR criteria 
4. 
T cell-replete peripheral blood or bone marrow graft. 
5. 
Patients must have a matched related or unrelated donor (at 
least 6/6 match at HLA-A, -B and -C for related donors and 
at least 8/8 match at HLA-A, -B, -C and -DRB1 for unrelated 
donors). 
6. 
Cardiac function: Left ventricular ejection fraction ≥45% for 
myeloablative conditioning. 
7. 
Estimated creatinine clearance ≥40 mL/minute (using the 
Cockcroft-Gault formula and actual body weight). 
8. 
Pulmonary function: DLCO ≥40% (adjusted for hemoglobin) 
and FEV1 ≥50%. 
9. 
Liver function: total bilirubin <3 x upper limit of normal and 
ALT/AST <5 x upper normal limit. 
10. 
Signed informed consent: Voluntary written consent must be 
given before patient registration and performance of any 
study related procedure not part of standard medical care, 
with the understanding that consent may be withdrawn by the 
patient at any time without prejudice to future medical care. 

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11. 
Female patient: A negative pregnancy test will be required 
for women of child bearing potential. Breast-feeding or 
lactation is not permitted. 
12. 
Planned posttransplant maintenance therapy is allowed. 
Exclusion Criteria: 
1. 
Prior allogeneic HCT. 
2. 
Active CNS involvement with malignancy. 
3. 
Patients receiving cord blood or haploidentical allograft.  
4. 
Patients undergoing in vivo or ex vivo T cell-depleted 
alloHCT. 
5. 
Karnofsky Performance Score <60%.  
6. 
Patients with uncontrolled bacterial, viral or fungal infections 
(currently on treatment and with progression of infectious 
disease or no clinical improvement) at time of enrollment.  
7. 
Active hepatitis B or C virus infection or known human 
immunodeficiency virus (HIV) positive. 
8. 
Prior intolerance or allergy to tocilizumab 
9. 
Use of rituximab, alemtuzumab, anti-thymocyte globulin 
(ATG) or other monoclonal antibody planned as part of 
conditioning regimen for GVHD prophylaxis.  
10. 
History of diverticulitis, Crohn’s disease or ulcerative colitis. 
11. 
History of demyelinating disorder. 
12. 
Any current uncontrolled cardiovascular conditions, including 
uncontrolled ventricular arrhythmias, NYHA class III or IV 
congestive 
heart 
failure, 
uncontrolled 
angina, 
or 
electrocardiographic evidence of active ischemia or active 
conduction system abnormalities. 
13. 
Any serious medical or psychiatric illness that could, in the 
investigator’s 
opinion, 
potentially 
interfere 
with 
the 
completion of treatment according to this protocol. 
Study Rationale 
We earlier hypothesized and demonstrated that tocilizumab could 
attenuate the incidence of aGVHD after MAC and RIC alloHCT, 
using matched sibling or unrelated donor. In this study, we 
hypothesize that longer term IL-6 inhibition through treatment with 
tocilizumab by repeated dosing would mediate a beneficial effect not 
only on the risk of aGVHD, but also on chronic GVHD. This will be 
achieved by administering an additional dose of tocilizumab at Day 
+100 post-alloHCT, besides the pretransplant dose, as done in our 
previous clinical trial, thereby providing total prophylaxis against 
both acute and chronic GVHD. 
Primary Objectives 
1. 
Determine the probability of GVHD/relapse-free survival 
(GRFS) defined as survival without grade III-IV acute GVHD, 

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systemic therapy-requiring chronic GVHD, relapse, or death 
at 12 months after matched related/unrelated donor bone 
marrow or peripheral blood alloHCT using myeloablative 
conditioning (MAC). 
Secondary Objectives 
1. 
Cumulative incidence of mild, moderate and severe chronic 
GVHD (by NIH criteria) and limited or extensive chronic 
GVHD (by conventional criteria).  
2. 
Cumulative incidence of grade II-IV acute GVHD at Days 
+100 and +180. 
3. 
Cumulative incidence of grade III-IV acute GVHD at Days  
           +100 and +180. 
4. 
Incidence of primary and secondary graft failure. 
5. 
Probability of non-relapse mortality post-alloHCT. 
6. 
Cumulative incidence of relapse/progression of the primary  
            malignancy. 
7. 
Probability of PFS post-HCT. 
8. 
Probability of OS post-HCT. 
9. 
T cell and myeloid chimerism kinetics following alloHCT at 
Day +28. 
10.     Immune reconstitution following alloHCT at Day +28, Day 
+100, Day +180, and Day +365. 
11.   Patient-reported Quality of Life assessments at baseline 
(enrollment), Days +28, +100, +180 and +365 post-
transplant.  
12a. Effect of GVHD prophylaxis on gut microbiome 
diversity: Difference in the level of microbiome 
diversity during transplant. 
12b. Association of baseline gut microbiome diversity with 
development of aGVHD, cGVHD, and overall 
survival. 
Endpoints 
Primary endpoint: The primary endpoint of this trial is GRFS. An 
event for this outcome is defined as grade III-IV acute GVHD, 
systemic therapy requiring chronic GVHD, relapse, or death. 
Patients who are alive without GVHD will be censored at the last 
follow-up.  
Secondary endpoints: 
• 
Acute GVHD 
• 
Chronic GVHD 
• 
Hematopoietic Recovery 

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• 
Graft Failure 
• 
Non-Relapse Mortality (NRM) 
• 
Disease Relapse or Progression 
• 
Progression-Free Survival 
• 
Overall Survival 
• 
Incidence of Infections 
• 
Donor Cell Chimerism 
• 
Immune Reconstitution 
• 
Patient-reported Quality of Life 
• 
Microbiome diversity pre-engraftment and evolution during 
transplant 
Study Design 
This is a phase II open-label trial designed to evaluate the efficacy 
of tocilizumab in improving GRFS after allogeneic hematopoietic cell 
transplantation (alloHCT) for hematologic malignancy. 
Study Agent/
Intervention 
Description 
Patients enrolled on the clinical trial will receive tacrolimus initiating 
at Day -1 at doses to maintain therapeutic levels per institutional 
preference and continued until at least Day +90 post-transplant. 
Methotrexate will be administered intravenously and dosed at 15 
mg/m2 Day +1 and 10 mg/m2 Days +3, +6 and +11. Tocilizumab will 
be administered intravenously at a dose of 8 mg/kg on Day -1 and 
at day +100 (+/- 14 days). 
Number of Subjects 
32 
Subject Participation 
Duration 
Patients will be followed for at least 12 months following alloHCT. 
Duration of Follow up 
Patients will be followed indefinitely for survival following alloHCT. 
Estimated Time to 
Complete Enrollment: 
The estimated accrual period is 18 months. 
Statistical 
Methodology: 
 
The sample size was selected to achieve 80% power to detect an 
increase in GRFS at 12 months to 40% compared to a historical 
control value of 20% at a one-sided 5% significance level. Based on 
asymptotic z-test for proportions, 29 patients with a known outcome 
are needed (i.e., known GVHD, relapse, or death status at 12 
months). Accounting for the drop out rate of 10%, a total of 32 
patients will be enrolled on the study. 
Analysis of primary endpoint 
The GRFS will be estimated using the Kaplan-Meier estimator and 
plotted with a 95% confidence band. An event for this outcome is 
defined as grade III-IV acute GVHD, chronic GVHD requiring 

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systemic therapy, relapse or death. Patients who are alive without 
GVHD will be censored at the last follow-up. The 12-month GRFS 
will be compared to the prespecified historical control value of 20% 
using a one-sided z-test. 
Analysis of secondary endpoints 
Demographic and other baseline data, such as disease 
characteristics, as well as outcome measures, will be presented 
overall and separately for URD and MRD patients. Categorical data, 
such as gender, race, etc., will be presented by frequencies and 
percentages. Descriptive summary statistics (e.g., frequency, mean, 
median, range and standard deviation) will be used to present 
numeric data. 
Time-to-event outcomes with and without competing risks will be 
analyzed using Kaplan-Meier and Nelson-Aalen estimates, 
respectively, and presented with 95% confidence intervals. Binary 
outcomes will be analyzed using proportions with 95% confidence 
intervals. 
 
 

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STUDY CALENDAR 
Study Assessments/Testing 
Baseline9 
 
 
Post-Transplant 
Day-1 
Day 0 
Day +1± 
2 
Day+8± 
2 
Day+15± 
2 
Day+21± 
2 
Day+28± 
7  
Day+56± 
7 
Day+100
± 14  
Day+180
± 14  
Day+365
± 28  
History, physical exam, weight and 
height* 
X 
 
X 
X 
 
 
 
X 
X 
X 
X 
X 
Karnofsky performance status and 
HCT-CI* 
X 
 
 
 
 
 
 
 
 
 
 
 
HLA typing (donor and recipient) * 
X 
 
 
 
 
 
 
 
 
 
 
 
Toci dose†  
 
X 
 
 
 
 
 
 
 
 
X 
 
 
Transplant data (documentation of 
conditioning/stem cell infusion) 
 
 
X 
 
 
 
 
 
 
 
 
 
CBC with differential1, 
comprehensive metabolic panel2* 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Infectious disease markers3*
 
 
X 
 
 
 
 
 
 
 
 
 
 
 
LVEF*10 
X 
 
 
 
 
 
 
 
 
 
 
 
DLCO and FEV1 * 
X 
 
 
 
 
 
 
 
 
 
X 
X 
Disease evaluation4* 
X 
 
 
 
 
 
 
 
 
X 
X 
X 
Pregnancy test5* 
X 
 
 
 
 
 
 
 
 
 
 
 

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Study Assessments/Testing 
Baseline9 
 
 
Post-Transplant 
Day-1 
Day 0 
Day +1± 
2 
Day+8± 
2 
Day+15± 
2 
Day+21± 
2 
Day+28± 
7  
Day+56± 
7 
Day+100
± 14  
Day+180
± 14  
Day+365
± 28  
GVHD assessments6* 
 
 
 
 
 
 
 
X 
X 
X 
X 
X 
AE assessments7 
X 
 
 
X 
 
 
 
X 
X 
X 
X 
 
Fasting lipid panel 
 
 
 
 
 
 
 
 
X 
 
X 
 
Chimerism8* 
 
 
 
 
 
 
 
X 
 
 
 
 
CMV NAAT peripheral blood* 
 
 
 
 
 
 
 
X 
X 
X 
 
 
Immune reconstitution* 
 
 
 
 
 
 
 
X 
 
X 
X 
X 
QoL assessments (See Appendix 
3)  
X 
 
 
 
 
 
 
X 
 
X 
X 
X 
Research (stool) sample collection 
 
X11 
 
X 
X 
X 
X 
X12 
*Indicate evaluations are currently performed as standard of care at the MCW BMT program.  
1, 2 CBC with differential, comprehensive metabolic panel will be performed as appropriate during hospitalization for alloHCT and on clinic visits and 
at minimum performed on days indicated on the schedule of events. CBC will be checked weekly until neutrophil and platelet recovery. 
3 Infectious disease titers include: CMV, hepatitis panel (hepA ab, hepB SAb, hepB SAg, hepB Core Ab, hepC Ab), HIV and HTLV I/II antibody. 
4 Evaluation of the malignant disease: for acute leukemia, CML and MDS this includes a bone marrow aspirate and biopsy for morphology, 
cytogenetics/FISH and molecular testing as appropriate. For lymphomas, this includes imaging studies, which will be done according to institutional 
practices, or the same as prior to transplant, for matter of comparison.  
5 Pregnancy test must be performed <30 days before the start of the transplant conditioning for females of childbearing potential and may be 
performed per institutional practices. (Serum or Urine) 
6 GVHD assessments performed per the standard operating procedures at the MCW BMT Program, and at minimum on Day + 28, +56, +100, +180, 
+365. The GVHD assessment will include a review of all abnormalities experienced during the entire assessment period and the highest grade 
for each abnormality (whether attributed to GVHD or not) during the assessment period will be recorded. 
7 The AE assessment will include a review of all toxicities including infections experienced during the entire assessment period and the highest 
grade for each AE during the assessment period will be recorded on the AE forms. 

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8 Chimerism in whole blood fractionated as CD3 and CD33. 
9 All baseline tests/procedures must be performed <30 days before the start of the transplant conditioning unless specified otherwise.  
10 LVEF to be estimated by MUGA or transthoracic echocardiogram.  
11 First stool sample for microbiome studies will be collected after admission in the hospital pre-transplant. Subsequent samples will be collected on 
Day+1±2, Day+7±2, Day+15±2, Day+21±2, and until neutrophil engraftment (last stool sample will be collected after neutrophil engraftment).  
12 Stool sample will also be collected at the time of onset of acute or chronic GVHD.  
† Tocililzumab will be dose at 8 mg/kg (maximum dose of 800 mg) once on the Day -1 approximately 24 hours prior to the estimated time of the 
hematopoietic cell infusion, and subsequently, on Day +100 (+/- 14 days, i.e., Days +86 to +114) post-alloHCT. The weight used to calculate the 
dose will be within 7 days of first dose of tocilizumab. Only if the patient’s weight has changed >10% (over the baseline weight), then the dosage of 
tocilizumab (for the second dose) will be changed depending on the weight checked within 14 days prior to the second dose. 
 
 

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TABLE OF CONTENTS 
PROTOCOL SIGNATURE PAGE ..................................................................................................................... 2 
REVISION HISTORY ........................................................................................................................................ 4 
PROTOCOL SUMMARY .................................................................................................................................. 5 
STUDY CALENDAR ....................................................................................................................................... 10 
TABLE OF CONTENTS .................................................................................................................................. 13 
LIST OF ABBREVIATIONS ............................................................................................................................ 15 
1 BACKGROUND ........................................................................................................................................... 17 
2. HYPOTHESIS AND OBJECTIVES ............................................................................................................. 27 
2.1 PRIMARY OBJECTIVES....................................................................................................... 28 
2.2 SECONDARY OBJECTIVES ................................................................................................. 28 
3 STUDY DESIGN .......................................................................................................................................... 29 
3.1 GENERAL DESCRIPTION .................................................................................................... 29 
3.2 PRIMARY ENDPOINT(S) ..................................................................................................... 29 
3.3 SECONDARY ENDPOINT(S) ................................................................................................ 29 
3.4 PRIMARY COMPLETION ..................................................................................................... 32 
3.5 STUDY COMPLETION ......................................................................................................... 32 
4 PATIENT SELECTION ................................................................................................................................ 32 
4.1 ELIGIBILITY CRITERIA ........................................................................................................ 32 
4.2 INCLUSION CRITERIA ......................................................................................................... 33 
4.3 EXCLUSION CRITERIA........................................................................................................ 34 
5 STUDY ENTRY AND WITHDRAWAL; STUDY PROCEDURES .................................................................. 35 
5.1 STUDY ENTRY PROCEDURES ............................................................................................. 35 
5.2 STUDY PROCEDURES DURING TREATMENT ......................................................................... 36 
5.3  POST-TRANSPLANT .......................................................................................................... 36 
5.4 STUDY WITHDRAWAL PROCEDURES .................................................................................. 38 
6  TREATMENT PLAN ................................................................................................................................... 39 
7  ADVERSE EVENTS: DEFINITIONS AND REPORTING REQUIREMENTS ............................................... 42 
7.1 DEFINITIONS ..................................................................................................................... 42 
7.2 KNOWN AES LIST ............................................................................................................. 46 
7.3 TIME PERIOD AND GRADE OF AE CAPTURE ........................................................................ 47 
7.4 MONITORING AND RECORDING AN ADVERSE EVENT ............................................................ 47 
8  PHARMACEUTICAL INFORMATION......................................................................................................... 51 
8.1  ACTEMRA® (TOCILIZUMAB) ................................................................................................ 51 
9 STATISTICAL CONSIDERATIONS ............................................................................................................. 53 
9.0 STUDY POPULATIONS ....................................................................................................... 53 
9.1 STUDY ENDPOINTS ........................................................................................................... 54 
9.2 STUDY DESIGN ................................................................................................................. 55 
9.3 ACCRUAL RATE ................................................................................................................ 55 
9.4 SAMPLE SIZE JUSTIFICATION ............................................................................................. 55 
9.5 STOPPING RULES ............................................................................................................. 55 
10 DATA AND SAFETY MONITORING PLAN (DSMP) ................................................................................. 56 
10.1 STUDY TEAM .................................................................................................................. 56 

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10.2 QUALITY ASSURANCE ..................................................................................................... 56 
10.3 CLINICAL TRIALS OFFICE ................................................................................................. 56 
10.4 DSMC ........................................................................................................................... 57 
11 REGULATORY COMPLIANCE, ETHICS AND STUDY MANAGEMENT .................................................. 57 
11.1 ETHICAL STANDARD ........................................................................................................ 57 
11.2 REGULATORY COMPLIANCE ............................................................................................. 57 
11.3  PRE-STUDY DOCUMENTATION ......................................................................................... 58 
11.4 INSTITUTIONAL REVIEW BOARD ....................................................................................... 58 
11.5  SUBJECT CONFIDENTIALITY AND ACCESS TO SOURCE DOCUMENTS/DATA ......................... 59 
11.6 PROTECTION OF HUMAN SUBJECTS ................................................................................. 60 
11.7 CHANGES IN THE PROTOCOL ........................................................................................... 60 
11.8 INVESTIGATOR COMPLIANCE ........................................................................................... 60 
12 DATA HANDLING AND RECORD KEEPING ............................................................................................ 61 
12.1 OVERVIEW ..................................................................................................................... 61 
12.2 DATA MANAGEMENT RESPONSIBILITIES............................................................................ 61 
12.3 HANDLING AND DOCUMENTATION OF CLINICAL SUPPLIES .................................................. 62 
12.4 SOURCE DOCUMENTS ..................................................................................................... 62 
12.5 CASE REPORT FORMS .................................................................................................... 63 
12.6 STUDY RECORD RETENTION............................................................................................ 63 
REFERENCES ............................................................................................................................................... 64 
APPENDIX 1. PERFORMANCE STATUS CRITERIA .................................................................................... 72 
APPENDIX 2. GVHD ASSESSMENT ............................................................................................................. 73 
APPENDIX 3: QOL MEASURES .................................................................................................................... 79 
APPENDIX 4. GUT MICROBIOME STUDY: STOOL SAMPLE COLLECTION AND PROCESSING ............. 89 
 
 
 

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LIST OF ABBREVIATIONS 
AE 
aGVHD 
ALL 
alloHCT 
adverse event 
acute graft versus host disease 
acute lymphocytic leukemia 
allogenic hematopoietic cell transplant 
ALT 
AML 
alanine aminotransferase 
acute myeloid leukemia 
ANC 
absolute neutrophil count 
AST 
aspartate aminotransferase 
AUC 
area under the curve 
BUN 
blood urea nitrogen 
CBC 
cGVHD 
complete blood cell (count) 
chronic graft versus host disease 
CR 
complete response 
CRF 
case report form 
CTCAE  
Common Terminology Criteria for Adverse Events 
CTEP 
Cancer Therapy Evaluation Program 
CMV 
cytomegalovirus  
DFS 
disease-free survival 
DLT 
dose-limiting toxicity 
DSM 
Data and Safety Monitoring 
DSMC 
Data and Safety Monitoring Committee 
DSMP 
data and safety monitoring plan 
ECOG 
Eastern Cooperative Oncology Group 
FDA 
Food and Drug Administration 
GCP 
GRFS 
good clinical practice 
graft-versus-host disease and relapse-free survival 
GVHD 
graft-versus-host disease 

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HIV 
HLA 
human immunodeficiency virus 
human leukocyte antigen 
ICH 
International Conference on Harmonization 
IRB 
Institutional Review Board 
IV 
KPS 
intravenous 
Karnofsky Performance Score 
LVEF 
MAC 
left ventricular ejection fraction 
myeloablative conditioning 
MCWCC 
Medical College of Wisconsin Cancer Center 
NAAT 
nucleic acid amplification test 
PBPC 
peripheral blood progenitor cell 
QoL 
quality of life 
SAE 
serious adverse event 
SD 
standard deviation 
SRC 
Scientific Review Committee 
UPIRSO   
unanticipated problems involving risks to subjects or others 
 
 

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1 BACKGROUND 
1.1 Graft-versus-Host Disease 
Allogeneic hematopoietic cell transplantation (alloHCT), a curative therapy for hematologic 
malignancies, acts by mediating immunologic graft-versus-tumor effects against the malignancy. 
Unfortunately, these immune responses can also be directed toward normal host tissues, 
resulting in acute graft-versus-host disease (GVHD). 1 GVHD is classified as either acute or 
chronic GVHD, based on clinical characteristics. 2 According to the clinical definitions, classic 
acute GVHD (aGVHD) develops within 100 days posttransplant with distinctive clinical features 
in the skin, gastrointestinal (GI) tract, or liver. However, manifestations of aGVHD can reoccur, 
persist, or present after Day 100 posttransplant and is then classified as recurrent, persistent or 
late onset aGVHD. Classical chronic GVHD is not limited to a specific organ system and can 
present at any time after transplantation. The diagnosis of chronic GVHD is based on 
consensus criteria for each organ system involved. While some clinical features are diagnostic 
(e.g., bronchiolitis obliterans and sclerotic features of the skin), other manifestations require 
additional clinical and/or histological criteria to be fulfilled. Some patients may also present as 
an overlap syndrome with clinical features of both acute and chronic GVHD (cGVHD). Scoring 
systems have been developed to grade the severity of aGVHD, based on the involvement of 
skin, GI tract and liver: Glucksberg scale (grading aGVHD from I to IV) is most widely used 3 
(see Appendix A). Importantly, aGVHD, although T-cell dependent, is known to be mediated, at 
least in part, by inflammatory cytokines. 4 By contrast, the mechanistic pathway in cGVHD 
involves Th17 differentiation 5 and germinal center B cells. 6 
 
The development of GVHD is a three-step process, each involving different subsets of immune 
cells. 7,8 The first step is activation of the innate immune system; this results in a state of 
systemic inflammation prior to the introduction of donor T cells. The chemotherapy and/or 
radiation used in the conditioning therapy invoke tissue damage with release of proinflammatory 
cytokines, e.g., IL-1, TNF-α, IFN-γ and IL-6. These cytokines perpetuate GVHD through direct 
cytotoxic effects on host tissues, 9-11 activation and/or priming of immune effector cells 12 and 
differentiation of proinflammatory and pathogenic alloantigen-specific T cell populations (i.e., 
TH1 and TH17 cells) from naïve T cell precursors. 4,13,14 Damage to the GI barrier allows 
increased translocation of microorganisms from the gut microbiome, with subsequent increased 
levels of circulating lipopolysaccharide, as well as molecules with pathogen- and damage-
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(APC) secrete proinflammatory cytokines and present host, as well as pathogen peptides on 
their major histocompatibility complex (MHC) molecules. A reduction of pretransplant 
inflammation (e.g., using reduced intensity conditioning (RIC), reduction of gut microbiome 
levels) and the presence of genetic factors associated with reduced inflammatory responses are 
associated with a reduced risk of GVHD and transplant-related mortality (TRM). 16-18 
 
The second step is characterized by alloreactive T cell activation, proliferation and differentiation 
as a response to the presentation of host antigens by host APC in a proinflammatory context. 
Stimulation of specific T helper cell subsets is thought to be important for the initiation of later 
cytotoxic T-cell mediated tissue damage. 7,8 Th1 cells release IFN-γ and express the 
transcription factors STAT4 and STAT1/T-bet. 19,20 This Th1 polarization seems to be important 
for the development of aGVHD, especially in the GI tract. 11 Th2 cells characterized by 
expression of the transcription factor GATA-3 and secretion of anti-inflammatory cytokines IL-4, 
IL-10, and IL-13 21 also may play a role in GVHD pathogenesis. 11,22,23 Th17 cells are 
characterized by the expression of the transcription factor RORγt and IL-17 secretion, 24 and are 
thought to be important for aGVHD severity 25 and early transplant-related severe lung injury. 26 
A majority of early posttransplant circulating TCRαβ+ CD4+ and CD8+ T cells release IL-6 at 
relatively high levels together with classical proinflammatory cytokines, such as IFN-γ and 
TNFα. 27 Finally, T regulatory cells (Tregs) are a Th subset characterized by expression of the 
transcription factor FOXP3 and high IL-2R (CD25) expression. These cells play an important 
regulatory role by actively suppressing immune responses through their release of the anti-
inflammatory cytokines IL-10, TGF-β and IL-35, 28 and through direct interaction with other T cell 
subsets. Tregs are suppressed during GVHD, and resolution of GVHD is associated with 
restoration of the Treg function. 29 The inflammatory environment in GVHD is promoted by the 
absence of an effective Treg response as both a relative and an absolute decline of Tregs in the 
peripheral blood and target tissues have been demonstrated in most studies. 13,30-32 The strong 
association between a proinflammatory milieu and the absence of an effective counter-
regulatory response suggests that the inflammatory environment prevents or inhibits Treg 
reconstitution during GVHD. 
 
The third step of GVHD development is characterized by local action of cytotoxic CD8+ T cells 
triggered by the local release of chemokines during the two first steps. These cells mediate 
direct cytotoxic effects upon target cell recognition, including secretion of perforin/granzyme and 
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through polarization of macrophages toward a proinflammatory (M1) phenotype that further 
increases tissue damage through the release of oxidants and proinflammatory cytokines, 
including TNF-α and IL-6. 33,34 
 
A combination of a calcineurin inhibitor (tacrolimus (Tac) or cyclosporine (CSP)) and 
methotrexate (MTX) has been an accepted standard for prevention of GVHD. Despite 
prophylaxis, the incidence of grade II–IV acute GVHD (aGVHD) is 35 to 50% 35 and the rate of 
chronic GVHD is 40 to 60%. The occurrence of GVHD after alloHCT results in increased 
transplant-related mortality (TRM), which also affects overall survival (OS) 36. The risk increases 
with unrelated or partially HLA-matched donors due to the greater genetic disparity between 
donor and recipient. Thus, new therapeutic treatment strategies are needed for prevention of 
acute, as well as chronic GVHD.  
 
1.2 Interleukin-6 
IL-6, a glycosylated protein with a molecular weight of 21-28 kDa,37 is a pleiotropic cytokine that 
is produced by a variety of cell types, including T cells, B cells, fibroblasts, endothelial cells, 
macrophages, monocytes and keratinocytes. 38 Macrophages and monocytes appear to be the 
main sources of IL-6 during acute inflammation, while T cells seem to be a major contributor 
during chronic inflammation. 39 The systemic (serum/plasma) levels usually range from 1.8 to 14 
pg/ml in healthy individuals. 40,41 During inflammation, a more than 105-fold increase can be 
observed, often correlating with disease severity. 42 IL-6 is essential for maturation, proliferation, 
differentiation and maintenance of B cells/plasma cells and several proinflammatory T-cell 
subsets.  
 
With respect to GVHD biology, IL-6 sits at the crossroads where the fate of naïve T cells to 
become proinflammatory T cells or Tregs is determined. In the presence of IL-6 and 
transforming growth factor-β (TGF-β), naïve T cells differentiate into TH17 cells, whereas in its 
absence, Tregs are induced. 43,44 IL-6 produced by dendritic cells after activation through Toll-
like receptors is, in addition, able to inhibit the suppressive function of Tregs. 45,46 Thus, IL-6 
appears to have a pivotal role in directing the immune response toward an inflammatory 
phenotype and away from a regulatory response. Second, IL-6-signaling is crucial for trafficking 
of immune cells to inflamed tissues and lymphoid organs. This is caused both by altered 
expression of adhesion molecules by endothelial cells and by expression of their ligands by 
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may, therefore, be a risk of combined injury during GVHD (e.g., GVHD-induced immunologic 
damage, pharmacologic toxicity and IL-6 inhibition). Finally, IL-6, together with TNF-α released 
from macrophages, has been reported to directly/independently contribute to tissue damage in 
GVHD. 33  
 
The importance of IL-6 in GVHD is also supported by clinical studies showing increased 
incidence and severity of GVHD in patients with elevated plasma levels of IL-6, 47,48 and in those 
with a recipient or donor IL-6 genotype that results in increased IL-6 production. 49,50 IL-6 also is 
important in regulation of stem cells and tissue regeneration in several organs. 2 This has been 
best demonstrated for hematopoiesis, liver cells, GI mucosa and muscle cells. Impaired IL-6 
function in these organs is associated with reduced regeneration after injury, and IL-6 
dysregulation during chronic inflammation can contribute to organ dysfunction. 
 
Signaling through IL-6 occurs by its binding to a low-affinity IL-6 receptor (IL-6R). 51 IL-6R exists 
both in a membrane-bound (mIL-6R) and a soluble form (sIL-6R). The binding of IL-6 to IL-6R 
induces homodimerization of gp130 cell membrane protein for intra-cellular signal 
transduction.52 mIL-6R is only expressed by a limited number of cell types, including 
hepatocytes, hematopoietic cells (neutrophils, naïve T cells, macrophages) and a subset of 
intestinal epithelial cells. IL-6R signaling can be initiated in these cells through the membrane-
associated complex of IL-6, IL-6R and gp130. This is called classical signaling and is often 
associated with tissue regeneration and anti-inflammatory effects. 2,52 IL-6R can also be shed 
from the membrane, generating a soluble form of the receptor, which can bind IL-6 and induce 
an intracellular response in any tissue expressing gp130 through a process called trans-
signaling. 53,54 Thus, cells that do not express the IL-6R themselves can be IL-6 responsive and 
this seems important for many of the proinflammatory effects of IL-6. 37 sIL-6R is mainly formed 
through cleavage of mIL-6R by a disintegrin and metalloprotease (ADAM) 10 and 17 proteases. 
sIL-6R is constitutively released by liver and hematopoietic cells, but activation of ADAM17 
during inflammation causes a rapid local increase in sIL-6R levels. The trans-signaling pathway 
is highly inflammatory, while classic signaling in cells directly expressing IL-6R is crucial for the 
differentiation of donor T cells down a Th17 pathway, which is capable of mediating GVHD. 37,55 
 
Gp130 is noncovalently associated with the Janus kinases (JAKs) JAK1, JAK2 and TYK2. 
Following receptor ligation, the JAKs are auto-phosphorylated, and they also phosphorylate 
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the tyrosine phosphatase SHP-2. Phosphorylated STAT3 dimerizes and is translocated to the 
nucleus where it acts as a transcription factor. 56 SHP-2 activates the RAS/RAF/MAPK/ERK 
pathway, whereas gp130 activation also leads to activation of the PI3K-AKT pathway together 
with the transcriptional regulator YAP1. Most of the IL-6 effects seem to be STAT3-mediated. 
STAT3 is controlled by a negative feedback mechanism; it induces expression of SOCS 
proteins and activation of the SHP-2 phosphatase. SOCS3 then binds with high affinity to the 
same phosphorylated binding site on gp130 as JAK1/2 and thereby inhibits further intracellular 
signaling. IL-6 signaling is also inhibited by internalization and degradation of the receptor 
complex and internalization of gp130 prevents further signaling. 
 
1.3 IL-6 Blockade and GVHD:  Preclinical Studies 
The role of IL-6 in acute and chronic GVHD has been investigated in several mouse models. 
Givon et al. 57 examined the effect of IL-6 on bone marrow reconstitution after syngeneic and 
alloHCT and found that posttransplant treatment with subcutaneous recombinant IL-6 
significantly supported white blood count reconstitution and improved survival in syngeneic and 
allogenic models transplanted with a low stem cell dose. In contrast, mice receiving IL-6 showed 
increases in both the severity of and mortality from GVHD. Chen et al. 58 observed increased 
systemic IL-6 and IL-6R levels early after both syngeneic and allogeneic transplantation. These 
levels returned to baseline over time in the syngeneic group, whereas IL-6 levels remained high 
in mice developing GVHD. Both IL-6 and IL-6R expression increased in the liver and colon and 
the highest IL-6R mRNA levels were observed in these two organs.  
 
Selective IL-6 knockout in neither recipient nor donor cells were sufficient to protect from GVHD. 
However, GVHD treatment with anti-IL-6 resulted in significantly less weight loss, less 
histopathological evidence of damage to colon, liver and lungs, and significantly increased Treg 
levels in the spleen. The increased Treg levels were not dependent on an intact thymus; rather 
the IL-6 blockade increased peripheral generation of Treg cells and reduced the levels of Th1 
and Th17 cells. Similar results were shown by Noguchi et al.; 59 treatment with an anti-IL-6 
antibody reduced liver enzyme levels and occurrence of organ failure and was associated with 
reduced infiltration of Th1 and Th17 cells, increased Tregs and improved survival. 
 
Tawara et al. 60 investigated the effect of IL-6 derived from donor T cells. Selective IL-6 
knockout in donor/graft T cells was associated with less severe GVHD and prolonged survival. 
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histopathologic severity of GVHD, but the Tregs were not altered. Importantly, the GVT effect 
was maintained despite the reduction in GVHD. The systemic cytokine levels and the levels of 
circulating cells were not altered. Selective ablation of IL-6 in recipient bone marrow cells did not 
reduce the incidence or severity of GVHD.  
 
Organ-specific effects of IL-6 in GVHD have been investigated in mouse models. Varelias et al. 
26 examined the role of IL-6 in idiopathic pulmonary syndrome (IPS) after alloHCT and 
demonstrated that local IL-6 secretion induced Th17 cell differentiation that was necessary for 
disease development. IPS could be prevented by IL-17 knockout or using anti-IL-17 antibody. 
Le Huu et al. 61 investigated the role of IL-6 in a sclerodermatous cGVHD model and observed 
increased IL-6 during disease progression. Treatment with anti-IL-6 antibody prior to 
manifestations of scleroderma significantly decreased the severity, while no reduction was 
observed when IL-6 blockade was started after the onset of cGVHD. Treatment with anti-IL-6 
was associated with a significant increase in the number of splenic Treg cells, whereas the 
expression of IFN-γ, TNFα, IL-6, IL-18, TGF-β1, CCL2, CCL3 and CCL5 in affected skin was 
significantly reduced.  
 
The role of STAT3 in the regulation of activation and differentiation of Tregs and Th17 cells after 
alloHCT has been investigated in several mouse models (i.e., STAT3-knockout mice) and in 
vitro models. Emerging evidence also suggests that activation of STATs in B cells is also 
important for GVHD. 62 Several important observations suggest that targeting of IL-
6/JAK2/STAT3 signaling may be effective in preventing GVHD. 63-67 As has been demonstrated 
in our previous clinical trial, IL-6 blockade using tocilizumab is an effective GVHD preventative 
strategy since IL-6 and STAT3 activation are closely linked to the development of both Th17 
and Treg cells, and since early STAT3 phosphorylation posttransplant seems to precede 
development of GVHD. 66 Currently available clinical data indicate that IL-6 blockade is most 
effective when used as GVHD prophylaxis, whereas manifest GVHD is likely less susceptible to 
the effects of IL-6 blockade. 
 
1.4  Tocilizumab  
Tocilizumab (ActemraTM) is a humanized anti-IL-6R antibody that blocks IL-6 signaling and has 
been FDA approved for the treatment of severe active rheumatoid arthritis. It has been shown to 
have remission-inducing efficacy in patients with moderate-to-severe rheumatoid arthritis, 
systemic juvenile idiopathic arthritis and multicentric Castleman’s disease. 68,69 A pilot phase I/II 

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study in patient with active Crohn’s disease also suggested benefit when administered on every 
two-week basis. 70 Tocilizumab is generally well tolerated in patients with rheumatoid arthritis; 
the most commonly reported side effects being dyslipidemia (21–25%), increased liver 
transaminases (5–6%) and transient decreases in neutrophil counts. 71-73 IL-6 blockade inhibits 
the acute phase response and may thereby mask signs of acute severe infections. Although 
some studies in rheumatoid arthritis have shown a higher rate of infections in patients treated 
with tocilizumab, 71,72 this could not be confirmed in a large multicenter study. 73  
 
1.4.1 Tocilizumab and Treatment of GVHD 
Murine studies of GVHD have shown that treatment with an anti-IL-6R antibody is able to 
significantly reduce GVHD-associated mortality and pathologic damage. 58 In addition to a few 
case reports and abstracts, 2 two published case series report the effects of tocilizumab in the 
treatment of steroid-refractory aGVHD. 74,75 
 
Drobyski et al. 75 published a series of eight patients who received tocilizumab for steroid-
refractory aGVHD. Responses were observed in four of six patients with severe aGVHD that 
had failed to respond to first, and in most cases, second-line therapies. Five patients had grade 
IV GVHD (four GI and one skin), while one patient had grade II GI and one had grade III liver 
GVHD. One of these patients died early after tocilizumab administration and was not evaluable, 
one patient did not respond, three patients were classified as partial responders, and two 
patients were considered complete responders. In one of the patients with partial response, 
tocilizumab was discontinued for the possibility of causing worsening of preexisting 
hyperbilirubinemia. Infections were responsible for the major adverse events associated with 
tocilizumab, with a total of 13 documented infections. Roddy et al. 74 reported the effect of 
tocilizumab in patients with steroid-refractory aGVHD, including seven patients with grade IV 
and two with grade III. Two patients were classified as complete responders, and two patients 
had mixed response with persistence of severe aGVHD in one organ but resolution in other 
organs, and four patients did not respond. In this series, four patients had infectious events with 
two deaths being reported, but no liver toxicity was observed. 
 
1.4.2 Tocilizumab and GVHD prophylaxis 
A phase I/II study conducted by Kennedy et al. assessed if tocilizumab could attenuate the 
incidence of acute GVHD 76 when given as adjunct to the GVHD prophylactic regimen. Eligible 
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total body irradiation (TBI)-based myeloablative (MAC) or RIC using unrelated or sibling donors. 
One intravenous (IV) dose of tocilizumab (8 mg/kg, capped at 800 mg, over 60 mins’ infusion) 
was given the day before alloHCT along with standard GVHD prophylaxis (cyclosporine [5 
mg/kg per day on Days –1 to +1, then 3 mg/kg per day to maintain therapeutic levels (trough 
levels of 140–300 ng/mL) for 100 days plus methotrexate [15 mg/m² on Day 1, then 10 mg/m² 
on Days 3, 6 and 11]). The primary endpoint was incidence of grade 2–4 aGVHD at Day 100, 
assessed and graded, as per the Seattle criteria. Immunological profiles were compared with a 
non-randomized group of patients receiving alloHCT, but not treated with tocilizumab. A total of 
48 patients receiving CSP+MTX as GVHD prophylaxis were enrolled into the study. The 
incidence of grade II–IV aGVHD in patients treated with tocilizumab at Day 100 was 12% (95CI 
5–24), and the incidence of grade III–IV aGVHD was 4% (1–13). Grade II–IV aGVHD involving 
the skin developed in five (10%) patients of 48 treated with tocilizumab, involving GI tract in four 
(8%) patients; there were no reported cases involving the liver. Low incidences of grade II–IV 
aGVHD were noted in patients receiving both TBI-based MAC (12% [95%CI 2–34) and 
fludarabine and melphalan RIC (12% [4–27]). There were no reports of graft failure and immune 
reconstitution was preserved in tocilizumab recipients, but with suppression of known 
pathogenic STAT3-dependent pathways. The addition of tocilizumab appeared generally safe 
with no increase in graft rejections, time to neutrophil engraftment, chimerism posttransplant, or 
early relapse compared with historical controls. Only three patients experienced severe liver 
toxicity during the first month after transplantation. This study showed that inhibition of IL-6 
superimposed on the calcineurin inhibitor-based GVHD prophylaxis is a potential strategy to 
reduce the incidence of acute GVHD without compromising immune reconstitution. 76 
 
A phase II study of tocilizumab was conducted by Drobyski et al. in alloHCT patients using 
matched related/unrelated donor and receiving Tac/MTX for GVHD prophylaxis. 77 Tocilizumab 
was administered IV as a single dose of 8 mg/kg the day before alloHCT. A total of 35 patients 
were enrolled in the study and all received busulfan-based conditioning (Flu/BU4, Flu/BU2 or 
BU/CY). The majority of patients (83%) received peripheral blood grafts. The primary endpoint 
is grade II–IV GVHD during the first 180 days posttransplant. The median follow-up of surviving 
patients was 12 months (range 5–16). The incidence of grades II–IV and III–IV aGVHD at Day 
100 was 14% (95CI 5–30%) and 3% (95CI 0–11%), respectively. In the first 100 days post-
alloHCT, there were no cases of acute GVHD of lower GI tract and only one patient developed 
grade IV aGVHD of skin. Five patients died from relapse between 140 to 270 days 
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(n=1). TRM was 9% (95CI 2–20%) and relapse was 17% (95CI 7–31%) at six months. In 
addition, we performed a matched (1:4) case-control analysis with contemporary controls 
(n=130) from the Center for International Blood and Marrow Transplant Research (CIBMTR) 
from 2000 to 2014 that were identified according to the same eligibility criteria for the trial except 
for the use of Tac/MTX as GVHD prophylaxis and were matched for age, Karnofsky 
Performance Score (KPS), disease and donor type. The incidence of grades II–IV aGVHD at 
Day +180 was significantly lower in the tocilizumab/Tac/MTX cohort when compared with the 
CIBMTR Tac/MTX control population (17% vs. 45%, P=0.001). Corresponding probabilities of 
grade II–IV aGVHD-free survival at six months, which was the primary objective of the study, 
were 69% and 42% (P=0.001 by stratified log rank test). There were no differences in relapse, 
TRM, DFS or OS at six months between the two groups. These two prospective studies thus 
show that tocilizumab has promising activity in preventing aGVHD, particularly lower GI tract 
GVHD and late onset aGVHD.  
 
1.5 Success of allogeneic transplant: relapse versus non-relapse mortality 
AlloHCT used for the treatment of hematologic malignancies is associated with two main risk 
factors for poor outcomes: transplantation-related mortality (also referred to as NRM) and 
mortality from disease relapse (relapse-related mortality). Efforts to mitigate one cause of 
mortality have often compromised the other. For example, efforts to reduce GVHD risk by T-cell 
depletion of the allograft can lower transplantation-related morbidity/mortality, but can also 
increase relapse risk. 78,79 Similarly, efforts at reducing relapse with intensified pre-transplant 
conditioning regimen can lead to increased mortality from organ dysfunction, infections, or 
GVHD. 80 Therefore, clinical trials are needed that evaluate the post-transplant outcomes by 
focusing on both NRM and relapse risk concurrently, rather than having only one as the primary 
objective. In addition, both endpoints NRM and relapse mortality do not reflect non-lethal 
morbidity. Because of these concerns, a composite endpoint such as GRFS would be ideal to 
assess all significant and relevant endpoints in a study evaluating a new clinical strategy with 
the potential to impact outcomes after alloHCT. 81 The events for GRFS include grade III-IV 
acute GVHD, systemic therapy-requiring chronic GVHD, relapse, or death in the first year post-
HCT. GRFS, therefore, represents ideal recovery from HCT (at one year) and a measure of 
cure without ongoing morbidity.  
 
Using a cohort of 628 adult patients treated with tacrolimus and methotrexate as GVHD 
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Transplant Research determined that the 1-year probability of GRFS was 23% (95% confidence 
interval [CI] 20-26). 81 In other words, only approximately one-quarter of patients transplanted 
for hematologic malignancy survived without at least one of these major complications during 
the first 12 months after alloHCT. 
 
We earlier hypothesized and demonstrated that tocilizumab could attenuate the incidence of 
aGVHD after MAC and RIC alloHCT, using matched sibling or unrelated donor. 77 In this study, 
we hypothesize that longer term IL-6 inhibition through treatment with tocilizumab by repeated 
dosing would mediate a beneficial effect not only on the risk of aGVHD, but also on chronic 
GVHD. This will be achieved by administering an additional dose of tocilizumab at Day +100 
post-alloHCT, besides the pretransplant dose, as done in our previous clinical trial, thereby 
providing total prophylaxis against both acute and chronic GVHD. 
 
1.6 Studies of the Microbiome in Transplant Patients 
The microbiome, consisting of a varied community of microbes (bacteria, viruses, fungi, 
microeukaryotes, and sometimes multicellular parasites), exists in niches across the human 
body. The skin, lung, nares, vagina, and gastrointestinal tract are among the most heavily 
colonized, with the largest number of microorganisms inhabiting the colonic lumen. While the 
majority of the over trillion organisms that live within a healthy human colon are nonpathogenic 
members of the phyla Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria, alterations 
in the balance of these microorganisms have been associated with adverse outcomes ranging 
from GVHD and infection to relapse post-HCT. 82 This clinical association between intestinal 
microorganisms and HCT outcomes has been investigated for decades – and has informed the 
still controversial practices of infection prophylaxis, gut decontamination, the “neutropenic diet,” 
and isolation of patients in laminar air flow rooms. Single-institution studies have demonstrated 
that low microbial diversity in the stool after allogeneic HCT is associated with poor survival. 83,84 
Additionally, specific alterations have been associated with increased risk of acute GVHD, 
infectious outcomes and most recently, relapse. 85-87 While these findings are compelling, the 
generalizability of these proposed microbial biomarkers is unclear. This is particularly important 
as there is known geographic variation of the intestinal microbiome and practice variability in 
antibiotic use for prophylaxis and treatment from institution to institution, in part due to different 
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Novel methods in microbiome research are likely to facilitate translational breakthroughs – these 
methods allow (1) detailed taxonomic classification of microorganisms at the strain level, (2) 
metabolic characterization of the small molecules and proteins that a microbial community 
makes, (3) measurement of microbial genomic evolution in clinical time courses, and (4) 
culturing of previously fastidious organisms from the microbiome for in vitro investigation and 
cultivation as potential therapeutic live bacterial clinical interventions. Given recent reports 
suggesting that alterations in the microbiome can impact the efficacy of immunologic therapies, 
it is imperative that the link between the microbiome and transplant outcomes is 
investigated.88,89  
1.6.1 Microbiome and Outcomes after Allogeneic Transplantation using Tocilizumab as 
GVHD prophylaxis 
The study will test the secondary hypothesis that the use of IL-6 blockage (tocilizumab) which 
has been shown to lower gastrointestinal GVHD rates preserves the gut microbiome diversity 
(determined by 16s rRNA sequencing analysis of the sequential stool sample collected pre- and 
post-transplant until neutrophil engraftment). Additional analyses on patient samples will be 
conducted to answer the key question concerning the impact of the gut microbiome on 
transplant outcome. Aliquots preserved additionally will establish a cohort of stool samples 
collected prospectively for future sequencing by shotgun metagenomic sequencing and 
metabolomic analysis. 
2. HYPOTHESIS AND OBJECTIVES 
We earlier hypothesized and demonstrated that tocilizumab could attenuate the incidence of 
aGVHD after MAC and RIC alloHCT, using a matched sibling or an unrelated donor. 90 In this 
study, we hypothesize that longer term IL-6 inhibition through treatment with tocilizumab by 
repeated dosing would mediate a beneficial effect not only on the risk of aGVHD, but also on 
chronic GVHD. This will be achieved by administering an additional dose of tocilizumab at Day 
+100 post-alloHCT, besides the pretransplant dose, as done in our previous clinical trial, 
thereby providing total prophylaxis against both acute and chronic GVHD. 
The study will test the secondary hypothesis that the use of IL-6 blockage (tocilizumab) which 
has been shown to lower gastrointestinal GVHD rates preserves the gut microbiome diversity 
(determined by 16s rRNA sequencing analysis of the sequential stool sample collected pre- and 
post-transplant until neutrophil engraftment). Additional analyses on patient samples will be 
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transplant outcome. Aliquots preserved additionally will establish a cohort of stool samples 
collected prospectively for future sequencing by shotgun metagenomic sequencing and 
metabolomic analysis.  
 
2.1 Primary Objectives 
Determine the probability of GRFS defined as survival without grade III–IV acute GVHD, 
systemic therapy-requiring chronic GVHD, relapse, or death at 12 months after matched 
related/unrelated donor bone marrow or peripheral blood alloHCT, using myeloablative 
conditioning (MAC). 
 
2.2 Secondary Objectives 
 
- Cumulative incidence of mild, moderate and severe chronic GVHD (by NIH criteria) and limited 
or extensive chronic GVHD (by conventional criteria).  
- Cumulative incidence of grade II–IV acute GVHD at Days +100 and +180. 
- Cumulative incidence of grade III–IV acute GVHD at Days +100 and +180. 
- Incidence of primary and secondary graft failure. 
- Probability of non-relapse mortality post-HCT. 
- Cumulative incidence of relapse/progression of the primary malignancy. 
- Probability of PFS post-HCT. 
- Probability of OS post-HCT. 
- T cell and myeloid chimerism kinetics following alloHCT at Day +28. 
- Immune reconstitution following alloHCT at Day +28, Day +100, Day +180, and Day +365. 
- Patient-reported Quality of Life assessments at baseline, Day+28, Day+100, +180 and +365 
post-transplant 
- Effect of GVHD prophylaxis on gut microbiome diversity: Difference in the level of microbiome 
diversity during transplant. 
- Association of baseline gut microbiome diversity with development of aGVHD, cGVHD, and 
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3 STUDY DESIGN 
 
3.1 General Description 
This is a phase II, open-label, single-arm trial designed to evaluate the efficacy of tocilizumab in 
improving the GVHD and relapse-free survival. 
 
3.1.1 Number of Subjects  
Thirty-two (32) patients.  
 
3.2 Primary Endpoint(s) 
The primary endpoint of this trial is GRFS. An event for this outcome is defined as grade III–IV 
acute GVHD, systemic therapy requiring chronic GVHD, relapse or death. Patients who are 
alive without GVHD will be censored at the last follow-up.  
 
3.3 Secondary Endpoint(s) 
3.3.1 Acute GVHD 
Cumulative incidences of grades II–IV and III–IV acute GVHD will be determined at Day +100 
and Day +180 post-HCT. Acute GVHD will be graded according to Appendix 1. The time of 
onset of grade II–IV acute GVHD and time to development of the highest grade until Day +180 
will be recorded. Development of acute GVHD will be considered an event for this endpoint. 
Death but not disease relapse will be considered a competing event. 
 
3.3.2 Chronic GVHD 
The cumulative incidence of chronic GVHD by NIH Consensus criteria will be determined. 
Development of chronic GVHD or death will be considered events for this endpoint. The highest 
grade of chronic GVHD will be recorded. In addition, the time of onset of chronic GVHD and the 
time to highest chronic GVHD grade will be recorded. Death but not disease relapse will be 
considered a competing event. 
 
3.3.3 Hematopoietic Recovery 
Hematopoietic recovery will be assessed according to neutrophil and platelet counts recovery 
after HCT. Neutrophil recovery or engraftment is defined as achieving an absolute neutrophil 
count (ANC) ≥500/mm3 for three consecutive measurements on three different days. The first of 
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death without engraftment. Platelet recovery is defined by either the first day of a sustained 
platelet count >20,000/mm3 for three days with no platelet transfusion in the preceding seven 
days. The first day of sustained platelet count above these thresholds will be designated the day 
of platelet engraftment. 
 
3.3.4 Graft Failure 
Graft failure will be assessed as a secondary endpoint, including primary and secondary graft 
failure. Primary graft failure is defined as no neutrophil recovery to > 500 cells/μL by Day 28 
post-HCT. Secondary graft failure will be assessed according to neutrophil count after initial 
hematologic recovery. Secondary graft failure is defined as initial neutrophil engraftment 
followed by subsequent decline in absolute neutrophil counts <500 cells/μL, unresponsive to 
growth factor therapy, but cannot be explained by disease relapse or drugs. 
 
3.3.5 Nonrelapse Mortality (NRM) 
NRM is defined as death after alloHCT without relapse. The cumulative incidence of NRM will 
be estimated at Day +100 and one year after alloHCT. An event for this endpoint is death 
without evidence of disease progression or relapse. Disease progression or relapse will be 
considered a competing event. 
 
3.3.6 Disease Relapse or Progression 
Relapse is defined by either morphological, cytogenetic or radiologic evidence of the 
pretransplant hematologic malignancy. Institution of any therapy to treat persistent, progressive 
or relapsed disease, including the withdrawal of immunosuppressive therapy or donor 
lymphocyte infusion, will be considered evidence of relapse/progression. NRM will be 
considered a competing event. 
 
3.3.7 Progression-Free Survival 
The event for this endpoint is relapse/progression or death. The time to this event is measured 
from transplant to death or relapse/progression, whichever comes first. Patients who are alive 
and disease-free will be censored at last follow-up. 
 
3.3.8 Overall Survival  
The event for this endpoint is death from any cause. The time to this event is measured from the 
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will be censored at last follow-up. 
 
3.3.9  Incidence of Infections 
The incidence of grade≥3 (CTCAE v5) viral, fungal and bacterial infections until day+180 will be 
determined. The cumulative incidence of CMV viremia post-alloHCT will be described. 
 
3.3.10 Donor Cell Chimerism 
Chimerism will be evaluated using sorted whole blood in CD3+ and CD33+ fractions. Mixed 
chimerism is defined as the presence of donor cells, as a proportion of total cells to be >5% and 
<95%. Full donor chimerism is defined as ≥95% of donor cells. Donor cells of ≤5% will be 
considered as graft rejection. Donor cell chimerism will be assessed for all patients at Day +28 , 
and Day +100 for both CD3+ and CD33+ fractions. The proportion of patients with each level of 
chimerism listed above will be described. CD3+ donor cell chimerism will be used to define the 
donor/recipient chimerism status. 
 
3.3.11  Immune Reconstitution 
Quantitative assessments of peripheral blood CD3, CD4, CD8, CD19 and CD56 positive 
lymphocytes will be done through flow cytometric analysis at Days +28, +100, +180, and +365. 
 
3.3.12 Patient-Reported Quality of Life (PR-QoL) Endpoints 
The QoL endpoints of this study are depressive symptoms, anxiety, fatigue, sleep, and pain at 
baseline, Day +28, Day +100, Day +180, and Day +365 post-transplant.  
• 
Depression: This will be addressed through the General Depression subscale of the 
IDAS and includes 20 questions with a scoring range of 20-100 (mean in community 
dwelling adult of 44.99 and standard deviation of 14.75).18 
• 
Anxiety: Anxiety will be assessed using two subscale items of the IDAS including panic 
(health population mean = 12.58, SD = 5.26) and traumatic intrusions (healthy 
population mean = 7.60, SD = 4.20).18  
• 
Fatigue: The FSI will be utilized to assess fatigue; a score of 3 or greater on items 
assessing fatigue in the past week (average of items 1-3; FSI Composite) indicates 
clinically meaningful fatigue.19, 20 The FSI can also be evaluated using the average rating 
of the degree to which fatigue interfered with some general activities (0-10; FSI 
Interference); participants’ ratings of the number of days in the past week they felt 
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week, on average, they felt fatigued (0-100; FSI Percent).20 Individuals scoring at or 
above the cutoff also report significantly greater scores on these other subscales. 
• 
Sleep: Sleep will be assessed using the PSQI, with a score of >5 considered disturbed 
sleep as adjusted for cancer populations.21, 22 
• 
Pain: The BPI assesses pain intensity as well as pain-related interference in function.23, 
24 BPI Pain Severity score ranges from 0-40 (first four items), and the BPI Pain 
Interference score is a mean of the last 7 items (5a-5g) with a range of 0-10. 
3.3.13 Microbiome 
The gut microbiome taxonomic diversity will be evaluated using 16S ribosomal RNA amplicon 
sequencing. Data will be generated per standard protocols 91. Resultant sequencing data will be 
quality filtered, adapter sequences will be trimmed, and sequences will be analyzed using the 
QIIME pipeline 92. Shannon diversity will be calculated 93. 
 
Guidelines for reporting of Depression and/or Suicidal Ideation: 
Should the patient endorse any thoughts of suicidality or self harm per the IDAS, the study co-
investigator for this portion of the ancillary testing (Dr. Jennifer Knight) will contact them by 
phone and standard of care referrals will be made. Should completion of the study surveys 
prompt participants to want treatment for any of the other symptoms, participants will be offered 
a referral for appropriate care through the Quality of Life Center at the Froedtert Cancer Center. 
 
3.4 Primary Completion 
The study will reach primary completion 18 months from the time the study opens to accrual. 
 
3.5 Study Completion 
The study will reach study completion 30 months from the time the study opens to accrual. 
4 PATIENT SELECTION 
4.1 Eligibility Criteria  
Patients must have baseline evaluations performed prior to the first study drug dose and must 
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about all study aspects, including the study visit schedule and required evaluations and all 
regulatory requirements for informed consent. The written informed consent must be obtained 
from the patient prior to enrollment. The following criteria apply to all patients enrolled onto the 
study, unless otherwise specified.  
 
4.2 Inclusion Criteria 
1. Age ≥18 years. 
2. Patients with any hematologic malignancy for which alloHCT is indicated. Patients with 
acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) must be in 
complete remission at the time of alloHCT (<5% blasts in the bone marrow, normal 
maturation of all cellular components in the bone marrow and absence of extramedullary 
disease). 
3. Myeloablative conditioning (MAC) regimen, based on CIBMTR criteria 94  
4. T cell-replete peripheral blood or bone marrow graft. 
5. Patients must have a matched related or unrelated donor (at least 6/6 match at HLA-A, -
B and -C for related donors and at least 8/8 match at HLA-A, -B, -C and -DRB1 for 
unrelated donors). 
6. Cardiac function: Left ventricular ejection fraction ≥45% for myeloablative conditioning. 
7. Estimated creatinine clearance ≥40 mL/minute (using the Cockcroft-Gault formula and 
actual body weight). 
8. Pulmonary function: DLCO ≥40% (adjusted for hemoglobin) and FEV1 ≥50%.  
9. Liver function: total bilirubin <3 x upper limit of normal and ALT/AST <5 x upper normal 
limit. 
10. Signed informed consent: Voluntary written consent must be given before patient 
registration and performance of any study-related procedure not part of standard medical 
care, with the understanding that consent may be withdrawn by the patient at any time 
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11. Female patient: A negative pregnancy test will be required for women of childbearing 
potential. Breast-feeding or lactation is not permitted. 
12. Planned posttransplant maintenance therapy is allowed. 
 
4.3 Exclusion Criteria 
1. Prior allogeneic HCT. 
2. Active CNS involvement with malignancy. 
3. Patients receiving cord blood or haploidentical allograft.  
4. Patients undergoing in vivo or ex vivo T cell-depleted alloHCT. 
5. Karnofsky Performance Score <60%.  
6. Patients with uncontrolled bacterial, viral or fungal infections (currently on treatment and 
with progression of infectious disease or no clinical improvement) at time of enrollment.  
7. Active hepatitis B or C virus infection or known human immunodeficiency virus (HIV) 
positive. 
8. Prior intolerance or allergy to tocilizumab. 
9. Use of rituximab, alemtuzumab, anti-thymocyte globulin (ATG) or other monoclonal 
antibody planned as part of conditioning regimen for GVHD prophylaxis.  
10. History of diverticulitis, Crohn’s disease or ulcerative colitis. 
11. History of demyelinating disorder. 
12. Any current uncontrolled cardiovascular conditions, including uncontrolled ventricular 
arrhythmias, NYHA class III or IV congestive heart failure, uncontrolled angina, or 
electrocardiographic evidence of active ischemia or active conduction system 
abnormalities. 
13. Any serious medical or psychiatric illness that could, in the investigator’s opinion, 
potentially interfere with the completion of treatment according to this protocol.  

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5 STUDY ENTRY AND WITHDRAWAL; STUDY PROCEDURES 
5.1 Study Entry Procedures 
5.1.1 Required Preregistration Screening Tests and Procedures 
The study-specific assessments are detailed in this section and outlined in the Study Calendar. 
A written, signed informed consent form (ICF) must be obtained before any study-specific 
assessments are initiated. A signed ICF copy will be given to the subject and a copy will be filed 
in the medical record. The original will be kept on file with the study records.  
All patients who are consented will be registered in OnCore®, the MCW Cancer Center Clinical 
Trial Management System. The system is password protected and meets HIPAA requirements. 
 
5.1.2 Registration Process 
Patients will be approached for this study after the decision to proceed with transplant is made 
and a suitable HLA-matched donor is identified. Transplant physicians will evaluate the patient 
eligibility onto this study. All source documents that support eligibility include a signed informed 
consent/HIPAA and signed eligibility checklist. These must be available for review and 
verification.  
At the point of registration, the study staff will register the patient in the electronic database 
(where applicable), including demographic, consent and on-study information. The patient will 
be assigned a unique sequence number for the study. 
Patients will be enrolled on this trial within 30 days preceding the conditioning regimen for 
alloHCT. 
 
5.1.3 Pretreatment Period 
Pretransplant Evaluations 
The following observations must be completed within 30 days before the initiation of the 
conditioning regimen unless otherwise specified. 
• 
History, physical examination, height and weight. 
• 
Karnofsky performance status and HCT-Specific Comorbidity Index score. 
• 
CBC with differential and comprehensive metabolic panel. 
• 
Fasting lipid panel. 
• 
Infectious disease markers: CMV antibody, Hepatitis panel (HepA Ab, HepB SAb, HepB 
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• 
LVEF (may be performed >30 days prior to patient enrollment, ECHO or MUGA).  
• 
Pulmonary function tests, including DLCO and FEV1 (may be performed >30 days prior 
to patient enrollment). 
• 
Disease evaluation of the malignant disease: For acute leukemia, CML and MDS, this 
includes a bone marrow aspirate and biopsy for pathology and cytogenetics. For 
lymphomas, this includes imaging studies, which will be done according to institutional 
practices (may be performed >30 days prior to patient enrollment).  
• 
Pregnancy test per institutional practices for females of childbearing potential. NOTE: 
pregnancy test must be performed <30 days before initiation of the conditioning regimen. 
May be serum or urine. 
• 
Research (stool) sample will be collected pre-transplant after admission to the hospital 
during the (pre-transplant) conditioning period. Research samples will be stored 
temporarily at MCW (Silvia Munoz-Price lab) and shipped in batches to Memorial Sloan 
Kettering Cancer Center, New York, NY (Marcel van den Brink lab) for additional 
downstream transcriptome and volatile metabolite sampling, which requires rapid 
processing. 
 
5.2 Study Procedures during Treatment 
Patients must meet eligibility criteria eligible on Day -1 to be treated.  
5.3  Posttransplant 
5.3.1 Posttransplant evaluations 
Study Visit 
Target Day Posttransplant 
Baseline* 
≤30 days from conditioning 
<4 weeks  
Day+1; Day+8, Day+15, 
Day+21 
4 weeks 
28 ± 7 days (21–35 days) 
8 weeks 
56 ± 7 days (49–63 days) 
100 days 
100 ± 14 days (86–114 days) 
6 months 
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Study Visit 
Target Day Posttransplant 
12 months 
365 ± 28 days (337–393 days) 
*Unless specified otherwise 
The following observations will be made: 
• 
History and physical exam will be conducted on Days +28, +56, +100, +180, +365 post-
HCT. 
• 
History and physical exam and laboratory studies of complete blood count and 
comprehensive metabolic panel will be conducted, starting day+1, at least three time a week 
until neutrophil and platelet recovery.  
• 
Assessment for AEs on Days +28, +56, +100, +180 post-transplant. 
• 
Comprehensive metabolic panel on Days +28, +56, +100, +180, +365 post-transplant. 
• 
Fasting lipid panel on Days +56 and +180 post-transplant. 
• 
Chimerism studies performed at Days +28posttransplant. Chimerism will be evaluated in 
whole blood in fractions including CD3+ and CD33+. 
• 
Disease evaluation of the malignant disease at Days +100, +180 and +365 post-transplant. 
• 
Pulmonary function tests, including DLCO and FEV1 at Days +180 and +365 post-
transplant. 
• 
Immune reconstitution panel will be collected at Days +28, +100, +180, and +365. 
• 
Research (stool) samples will be collected post-transplant on a weekly basis until after 
engraftment and subsequently at the time of onset of acute or chronic GVHD (as shown in 
study calendar). Research samples will be stored temporarily at MCW (Silvia Munoz-Price 
lab) and shipped in batches to Memorial Sloan Kettering Cancer Center (Marcel van den 
Brink lab) for additional downstream transcriptome and volatile metabolite sampling, which 
requires rapid processing. 
The post-transplant investigations will be performed within a window ranging from a week to 
four weeks before and after the target date, as mentioned in the table above (Section 5.3.1). 
  
5.3.2 GVHD Assessment 
Patients will be monitored for development of acute and chronic GVHD, per standard operating 
procedures at the MCW BMT Program, but at minimum on Days +28 (+/-7), +56 (+/-7), +100 
(+/-14), +180 (+/-14), +365 (+/-28) post-HCT. Diagnosis of acute GVHD may not necessitate 
biopsy confirmation in at least one involved organ, but would be preferred. When more than one 
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With liver-only GVHD, biopsy confirmation is strongly recommended. Acute GVHD will be 
assessed by consensus criteria (Appendix 2) 95 and chronic GVHD diagnosis and grading will 
be according to NIH Criteria 96,97. Please see Appendix 2.  
Independent GVHD adjudication is mandated in the study. Acute and chronic GVHD will be 
adjudicated by an independent Faculty Research Committee (FRC)-appointed review panel. 
Protocol PIs will be blinded to the adjudication panel and will not be permitted to grade or modify 
GVHD assessments. GVHD case report forms (CRFs) will be completed by treating 
MDs/NPs/APPs in real time, as indicated in study calendar. FRC adjudication panel will grade 
GVHD, using a calendar-driven approach (Days +100 and +180 for aGVHD and Days +180 and 
+365 for chronic GvHD). 
 
5.3.3 Clinical Grading of Chronic GVHD (Appendix) 
 
• 
None. 
• 
Mild chronic GVHD involves only one or two organs or sites (except the lung: see 
below), with no clinically significant functional impairment (maximum of score 1 in all 
affected organs or sites). 
• 
Moderate chronic GVHD involves: (1) at least one organ or site with clinically significant 
but no major disability (maximum score of 2 in any affected organ or site) or (2) three or 
more organs or sites with no clinically significant functional impairment (maximum score 
of 1 in all affected organs or sites). A lung score of 1 will also be considered moderate 
chronic GVHD. 
• 
Severe chronic GVHD indicates major disability caused by chronic GVHD (score of 3 in 
any organ or site). A lung score of 2 or greater will also be considered severe chronic 
GVHD. 
• 
Presence of limited or extensive chronic GVHD will be also be recorded. 
 
5.4 Study Withdrawal Procedures 
5.4.1 Duration of Therapy 
In the absence of treatment delays due to adverse events, treatment may continue for or until:  
 
Disease progression. 
 
General or specific changes in the patient’s condition render the patient unacceptable for 
further treatment in the investigator’s judgment.  
 
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 
Patient decides to withdraw from the study. 
 
Significant patient noncompliance with protocol. 
 
Unacceptable adverse event(s). 
 
See specifics in 5.5.3. 
 
5.5.2 Patient-Initiated Withdrawal:  A patient may decide to withdraw from the study at any 
time. 
 
5.5.3 Investigator-Initiated Withdrawal:  The investigator will withdraw a patient whenever 
continued participation is no longer in the patient’s best interests. Reasons for withdrawing a 
patient include, but are not limited to, disease progression, the occurrence of an adverse event 
or a concurrent illness, a patient’s request to end participation, a patient’s noncompliance as 
determined by the investigator or simply significant uncertainty on the part of the investigator 
that continued participation is prudent. There may also be administrative reasons to terminate 
participation, such as concern about a patient’s compliance with the prescribed treatment 
regimen.  
5.5.4 Sponsor-Initiated Withdrawal:  Sponsor’s decision to discontinue the study. 
5.5.5 Withdrawal Documentation Procedure:  The reason for study withdrawal and the date 
the patient was removed from the study must be documented in the case report form. 
6  TREATMENT PLAN 
6.1 Conditioning Regimens 
Eligible patients will receive myeloablative conditioning at the discretion of the treating 
physician. 
It is recommended that adjusted ideal body weight be used when calculating conditioning 
regimen chemotherapy doses. One exception is fludarabine, which uses actual body weight. 
Ideal Body Weight (IBW) Formulas: 
Males IBW = 50 kg + 2.3 kg/inch over 5 feet. 
Females IBW = 45.5 kg + 2.3 kg/inch over 5 feet. 
Adjusted Ideal Body Weight Formula: 
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6.2  Hematopoietic Cell Transplantation 
Bone marrow or mobilized peripheral blood graft will be the graft source in this study. 
6.2.1 Peripheral Blood Progenitor Cells (PBPC) Mobilization and Collection 
PBPC mobilization and collection will be done according to standard operating procedures from 
MCW BMT program.  
Target CD34 cell doses are between 5–10 x 106 per kg recipient body weight.  
The transportation of the PBPC product from unrelated donors shall be done in accordance with 
NMDP standards. 
6.2.2 PBPC Infusion 
PBPC grafts will be infused according to standard operating procedures from MCW BMT 
program at Day 0. For recipients of related donor PBPC, whose donors require a third day of 
collection (Day +1), these cells will be infused separately from the Day –1 and Day 0 collections 
on Day +1.  
 
6.3 Tacrolimus/Methotrexate/Tocilizumab 
6.3.1 Tacrolimus 
Tacrolimus will be given per standard operating procedures from the MCW BMT program, The 
dose should be adjusted accordingly to maintain a suggested level of 5–10 ng/mL. The dose of 
tacrolimus may be switched to oral at a 1:3 dose equivalence and rounded to the nearest 0.5 
mg at the discretion of the treating physician. If patients are on medications which alter the 
metabolism of tacrolimus (e.g., azoles), the initial starting dose and subsequent doses should 
be altered, as per institutional practices. Tacrolimus taper can be initiated at a minimum of 90 
days post-HSCT, if there is no evidence of active GVHD. The rate of tapering will be done 
according institutional practices, but patients should be off tacrolimus by Day 180 post-HSCT, if 
there is no evidence of active GVHD. 
 
6.3.2 Methotrexate 
Methotrexate will be administered, per institutional practices.. Dose reduction of MTX due to 
worsening creatinine clearance after initiation of conditioning regimen, high serum levels or 
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6.3.3 Tocilizumab 
Tocilizumab will be administered intravenously (IV) at a dose of 8 mg/kg (maximum dose of 800 
mg) once on the Day -1 approximately 24 hours prior to the estimated time of the hematopoietic 
cell infusion, and subsequently, on Day +100 (+/- 14 days, i.e., Days +86 to +114) post-alloHCT. 
The weight used to calculate the dose will be within 7 days of first dose of tocilizumab. Only if 
the weight has changed >10% (over the baseline weight), then the dosage of tocilizumab (for 
the second dose) will be changed depending on the weight checked within 14 days prior to the 
second dose. The infusion will be administered over 60 minutes through a dedicated IV line and 
must not be administered by IV bolus. Commercially available tocilizumab will be utilized for the 
study. 
 
6.3.3.1 Contraindications to Tocilizumab dosing on Days +100 (range, Day +86 to +114): 
1) Grade 4 (according to CTCAE version 5 hematologic or nonhematologic events (which have 
life-threatening consequences and require urgent treatment). Dosing will be allowed upon 
recovery to grade ≤3. Transfusion to increase platelet and hemoglobin is allowed. Grade 3 
febrile neutropenia and grade 3 increased AST/ALT/bilirubin are also contraindications to 
dosing of tocilizumab; recovery to grade 2 or lower would allow for tocilizumab dosing within 
the time frame of Day +86 through Day +114. 
2) Disease (relapse or progression of the primary malignancy) for which treatment is indicated. 
3) Donor leukocyte infusion (DLI) prior to planned second dose of tocilizumab for any 
indication: infection, disease control or to address mixed chimerism. 
4) Grade III–IV acute GVHD. Patients with grade I–II acute GVHD and on topical or systemic 
corticosteroids therapy (equivalent to prednisone ≤20 mg/d) can receive a second dose of 
tocilizumab. 
5) Active chronic GVHD requiring systemic therapy. 
6) Acute GVHD treated with tocilizumab before Day +100. 
 
6.4 Supportive Care 
All supportive care will be given according to standard operating procedures of the MCW BMT 
program. 
 
6.4.1  Growth Factors 
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6.4.2  Prophylaxis against Infections 
Patients will receive infection prophylaxis according to standard operating procedures of the 
MCW BMT program. 
 
6.5 Participant Risks 
See section 7.2, known AEs. 
 
6.6. Quality of Life (QoL) Assessments  
6.6.1. Instruments 
Participants will complete a battery of self-report surveys at the following time points: baseline, 
Day +28, Day +100, Day +180 and Day +365 post-transplant (total of 5 time points). The self-
report surveys to be completed include: Inventory of Depression and Anxiety Symptoms (IDAS; 
depression and anxiety), Fatigue Symptom Inventory (FSI; fatigue), Pittsburgh Sleep Quality 
Index (PSQI; sleep), and Brief Pain Inventory (BPI; pain). QoL measures will be administered to 
English-speaking patients. All QoL assessments will be completed by patients on a single day, 
during the routine clinic visit associated with the corresponding study time point. QoL at each 
time point will be summarized using simple descriptive statistics (mean, SD). Change in patient-
reported outcomes from enrollment to Day 100, 6 and 12 months will be calculated. The primary 
objective is to compare alteration in QoL symptoms between this cohort receiving two doses of 
tocilizumab and the prior tocilizumab cohort receiving only one dose of tocilizumab. PR-QoL 
outcomes among survivors at each time point will be compared in an initial analysis using two 
sample t-statistics. The missing data pattern of the QoL measurements will be examined using 
graphical techniques and logistic regression models conditional on survival.  
Should patients endorse any thoughts of suicidality or self-harm per the IDAS, study Co-I and 
Psycho-Oncology Medical Director, Dr. Jennifer Knight, will contact them by phone.  Should 
completion of the study surveys prompt participants to want treatment for any of the other 
symptoms, participants will be offered a referral for appropriate care through the Quality of Life 
Center at the Froedtert Hospital Cancer Center. 
7  ADVERSE EVENTS: DEFINITIONS AND REPORTING 
REQUIREMENTS 
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7.1.1 Adverse Event (AE) and Serious Adverse Events (SAE) 
An AE is defined as any abnormal laboratory value(s) or test result(s), even when they do not 
induce clinical signs or symptoms or require therapy’, as per 21 CFR 312.32(a). The investigator 
and his or her team will follow the Medical College of Wisconsin policies related to adverse 
event reporting. This information may be found on the Human Research Protection Program 
website.  
 
Serious AE (SAE) means any untoward medical occurrence that at any dose: 
• 
Death. Results in death. 
• 
Life threatening. Is life threatening (refers to an AE in which the patient was at risk of 
death at the time of the event. It does not refer to an event which hypothetically might 
have caused death if it were more severe). 
• 
Hospitalization. Requires inpatient hospitalization or prolongation of an existing 
hospitalization. 
• 
Disability/incapacity. Results in persistent or significant disability or incapacity. 
(Disability is defined as a substantial disruption of a person’s ability to conduct normal 
life functions). 
• 
Medically important event. This refers to an AE that may not result in death, be 
immediately life threatening, or require hospitalization, but may be considered serious 
when, based on appropriate medical judgment, may jeopardize the patient, require 
medical or surgical intervention to prevent one of the outcomes listed above, or involves 
suspected transmission via a medicinal product of an infectious agent. Examples of such 
medical events include allergic bronchospasm requiring intensive treatment in an 
emergency room or at home, blood dyscrasias or convulsions that do not result in 
inpatient hospitalization, or the development of drug dependency or drug abuse; any 
organism, virus, or infectious particle (e.g., prion protein transmitting Transmissible 
Spongiform Encephalopathy), pathogenic or nonpathogenic, is considered an infectious 
agent.  
Clarification should be made between a serious AE (SAE) and an AE that is considered severe 
in intensity (Grade 3 or 4), because the terms serious and severe are NOT synonymous. The 
general term severe is often used to describe the intensity (severity) of a specific event; the 
event itself, however, may be of relatively minor medical significance (such as a Grade 3 
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criteria described above and is usually associated with events that pose a threat to a patient’s 
life or ability to function. A severe AE (Grade 3 or 4) does not necessarily need to be considered 
serious. For example, a white blood cell count of 1000/mm3 to less than 2000 is considered 
Grade 3 (severe) but may not be considered serious. Seriousness (not intensity) serves as a 
guide for defining regulatory reporting obligations. 
 
7.1.2 Unanticipated Problem Involving Risk to Subject or Other (UPIRSO) 
The investigator and his or her team will follow the Medical College of Wisconsin policies related 
to unanticipated problems involving risks to subjects or others. This information may be found 
on the Human Research Protection Program website.  
 
 
7.1.3 AE Attribution and Grading 
Adverse Event Grading 
Grade 
Description 
0 
No AE (or within normal limits). 
1 
Mild; asymptomatic or mild symptoms; clinical or diagnostic 
observations only; intervention not indicated. 
2 
Moderate; minimal, local or noninvasive intervention (e.g., 
packing cautery) indicated; limiting age-appropriate 
instrumental activities of daily living (ADL). 
3 
Severe or medically significant but not immediately life-
threatening; hospitalization or prolongation of hospitalization 
indicated; disabling; limiting self-care ADL. 
4 
Life-threatening consequences; urgent intervention indicated. 
5 
Death related to AE 
 
 
Adverse Event Attribution 
Attribution is an assessment of the relationship between the AE and the medical intervention. 
 

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Relationship 
Attribution 
Description 
Unrelated to investigational 
agent/intervention 
Unrelated 
The AE is clearly NOT related to the 
intervention 
Unlikely 
The AE is doubtfully related to the 
intervention 
Related to investigational 
agent/intervention 
Possible 
The AE may be related to the 
intervention 
Probable 
The AE is likely related to the 
intervention 
Definite 
The AE is clearly related to the 
intervention 
 
Relationship Assessment:  In-Depth Definitions 
For all collected AEs, the clinician who examines and evaluates the subject will determine the 
adverse event’s causality based on temporal relationship and his/her clinical judgment. The 
degree of certainty about causality will be graded using the categories below: 
Definitely Related: There is clear evidence to suggest a causal relationship, and other possible 
contributing factors can be ruled out. The clinical event, including an abnormal laboratory test 
result, occurs in a plausible time relationship to drug administration and cannot be explained by 
concurrent disease or other drugs or chemicals. The response to withdrawal of the drug 
(dechallenge) should be clinically plausible. The event must be pharmacologically or 
phenomenologically definitive, with use of a satisfactory rechallenge procedure if necessary.  
Probably Related: There is evidence to suggest a causal relationship, and the influence of 
other factors is unlikely. The clinical event, including an abnormal laboratory test result, occurs 
within a reasonable time sequence to administration of the drug, is unlikely to be attributed to 
concurrent disease or other drugs or chemicals, and follows a clinically reasonable response on 
withdrawal (dechallenge). Rechallenge information is not required to fulfill this definition.  
Possibly Related: There is some evidence to suggest a causal relationship (e.g., the event 
occurred within a reasonable time after administration of the trial medication). However, the 
influence of other factors may have contributed to the event (e.g., the subject’s clinical condition, 
other concomitant events). Although an adverse drug event may rate only as “possibly related” 
soon after discovery, it can be flagged as requiring more information and later be upgraded to 
“probably related” or “definitely related”, as appropriate. 
Unlikely: A clinical event, including an abnormal laboratory test result, whose temporal 
relationship to drug administration makes a causal relationship improbable (e.g., the event did 
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drugs or chemicals or underlying disease provides plausible explanations (e.g., the subject’s 
clinical condition, other concomitant treatments).  
Unrelated: The AE is completely independent of study drug administration, and/or evidence 
exists that the event is definitely related to another etiology. There must be an alternative, 
definitive etiology documented by the clinician.  
 
7.2 Known AEs List 
7.2.1 Tacrolimus 
 
Tacrolimus side effects include:  
• 
Cardiovascular: hypertension 
• 
Neurologic: confusion, dizziness, insomnia, seizures, tremors, changes in how clearly 
one can think 
• 
Gastrointestinal: nausea, vomiting 
• 
Hematologic: microangiopathic hemolytic anemia, thrombocytopenia 
• 
Endocrine and metabolic: hypomagnesemia, hypokalemia, hypocalcemia, 
hyperlipidemia 
• 
Miscellaneous: unwanted hair growth, changes in vision, liver problems, reversible renal 
insufficiency, infections and posttransplant lymphoproliferative disorders 
7.2.2  Methotrexate 
The most frequently reported adverse reactions associated with methotrexate use as GVHD 
prophylaxis include: 
• 
Neurologic: fever, dizziness, chills, undue fatigue 
• 
Gastrointestinal: ulcerative stomatitis, nausea, abdominal distress, diarrhea 
• 
Hematologic:  leucopenia, anemia and suppressed hematopoiesis (leading to infection) 
• 
Miscellaneous: abnormal liver tests, kidney failure and pulmonary complications after 
transplantation 
7.2.3  Tocilizumab 
Tocilizumab side effects include: 
• 
Hypersensitivity reactions 

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• 
Respiratory: upper respiratory tract infections, nasopharyngitis, bronchitis 
• 
Gastrointestinal: mouth ulceration, upper abdominal pain, gastritis, gastrointestinal 
perforations, pancreatitis. 
• 
Hepatic: transaminases elevation 
• 
Neurologic: headache, dizziness 
• 
Cardiovascular: hypertension 
• 
Dermatologic: skin rash 
• 
Serious infections (See Section 8.1.5) 
7.3 Time Period and Grade of AE Capture 
Serious adverse events that do not meet the requirement for expedited reporting (not related to 
study treatment or expected) will be reported to the Institutional Review Board (IRB) as part of 
the annual renewal of the protocol.   
 
7.4 Monitoring and Recording an Adverse Event  
Definition. Any clinically relevant deterioration in laboratory assessments or other clinical 
finding is considered an AE.  
Reporting source. AEs may be spontaneously reported by the patient and/or in response to an 
open question from study personnel or revealed by observation, physical examination or other 
diagnostic procedures.  
Prior to the trial. Planned hospital admissions or surgical procedures for an illness or disease 
that existed before the patient was enrolled in the trial are not to be considered AEs unless the 
condition deteriorated in an unexpected manner during the trial (e.g., surgery was performed 
earlier or later than planned).  
Pretreatment events following signed informed consent. For serious pretreatment events, 
the investigator must determine both the intensity of the event and the relationship of the event 
to study procedures.  
Treatment events. For serious AEs, the investigator must determine both the intensity of the 
event and the relationship of the event to study drug administration.  
Not serious AEs. For non-serious AEs, the investigator must determine both the intensity of the 
event and the relationship of the event to study drug administration. 
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All adverse events will be followed with appropriate medical management until Day+180 or until 
they are resolved, if they are related to the study treatment. 
 
7.4.1 Procedure for Reporting Drug Exposure during Pregnancy and Birth Events 
If a woman becomes pregnant, or suspects that she is pregnant, while participating in this study, 
she must inform the investigator immediately and permanently discontinue the study drug. The 
sponsor-investigator must notify the DSMC by email. The pregnancy must be followed for the 
final pregnancy outcome. 
If a female partner of a male patient becomes pregnant during the male patient’s participation in 
this study, the sponsor-investigator must also immediately notify the DSMC by email. Every 
effort should be made to follow the pregnancy for the final pregnancy outcome. 
 
7.4.2 Subject Complaints 
If a complaint is received by anyone on the study staff, it will be discussed with the study staff 
and will be addressed on a case-by-case basis. The PI will be notified of any complaints. 
Complaints will be reported to the IRB if indicated.  
If the subject has questions about his or her rights as a study subject, wants to report any 
problems or complaints, obtain information about the study or offer input, the subject can call 
the Medical College of Wisconsin/Froedtert Hospital research subject advocate at 414-955-
8844. This information is provided to the subject in their consent.  
A product complaint is a verbal, written or electronic expression that implies dissatisfaction 
regarding the identity, strength, purity, quality or stability of a drug product.  Study staff, who 
identify a potential product complaint situation should immediately contact the sponsor and 
report the event. Whenever possible, the associated product should be maintained in 
accordance with the label instructions pending further guidance from a sponsor representative. 
Product complaints in and of themselves are not Reportable Events. If a product complaint 
results in an SAE, an SAE form should be completed.  
 
7.4.3 Routine Reporting Procedures for AEs 
Expedited Reporting Procedures for SAEs, SARs, UPIRSOs and DLTs. 
 

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Since this is an investigator-initiated study, the principal investigator, also referred to as the 
sponsor-investigator, is responsible for reporting serious adverse events (SAEs) to any 
regulatory agency and to the sponsor-investigator’s IRB.  
 
Signs or symptoms reported as adverse events will be graded and recorded by the investigator, 
according to the CTCAE. When possible, signs and symptoms indicating a common underlying 
pathology should be noted as one comprehensive event.  
 
The investigator will assess all adverse events and determine reporting requirements to the 
Medical College of Wisconsin Cancer Center (MCWCC) DSMC and MCW IRB. 
 
Only grade 1 and 2 adverse events related to the study drug and all grade 3, 4 and 5 adverse 
events regardless of attribution will be entered into OnCore®. 
 
Reporting to the Data and Safety Monitoring Committee (DSMC) 
Regardless of the causality, all unexpected grade 3, and all grade 4, and 5 SAEs, as well as any 
others requiring expedited reporting as defined in this protocol must be reported to the MCWCC 
DSMC within five calendar days of study staff’s knowledge. 
Report Method:  The investigator will use email to report SAEs and applicable AE to the DSMC. 
The SAE report must include event term(s), serious criteria, and the sponsor-investigator’s or 
sub-investigator’s determination of both the intensity of the event(s) and the relationship of the 
event(s) to study drug administration. Intensity for each SAE, including any lab abnormalities, 
will be determined by using the NCI CTCAE v5 as a guideline whenever possible.  
The criteria are available online at http://ctep.cancer.gov/reporting/ctc.html. 
 
Reporting to MCW Institutional Review Board (IRB) 
The principal investigator must report events to the MCW IRB within five business days of his 
awareness of the event.  
[Guidance on Adverse Event Reporting to the IRB is available online at MCW IRB Policies and 
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Event Type 
Report Recipients  
PI/Study Chair/ 
Coordinating Center 
Institutional 
Review  
Board 
DSMC 
 
 
FDA (if applicable) 
CTO  
Regulatory 
Office  
Other 
Serious Adverse Event 
ASAP 
5 days (or annual 
CPR)1 
5 days 
7 or 15 days2 
ASAP 
 
Unanticipated Problems Involving Risks to 
Subjects of Others 
ASAP 
5 days (or annual 
CPR)1 
5 days 
7 or 15 days2 
ASAP 
 
Evidence of Causal Relationship between Drug 
and AE 
ASAP 
5 days (or annual 
CPR)1 
5 days 
7 or 15 days2 
ASAP 
 
Dose-Limiting Toxicity 
ASAP 
5 days (or annual 
CPR)1 
5 days 
7 or 15 days2 
ASAP 
 
 
 
 
 
 
 
 
Contacts 
Role 
Name 
Entity/Department 
Institution 
Telephone 
Email 
Sponsor-Investigator 
Saurabh Chhabra, MD 
Hematology/Oncology 
MCW 
414-805-0578 
schhabra@mcw.edu 
 
 
Footnotes 
1 Consult MCW IRB Policies (contact your regulatory representative) 
2  FDA guidelines:  Suspected adverse reaction, Unexpected and Serious = 7 Days; If not = 15 days 

 
 
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8  PHARMACEUTICAL INFORMATION 
8.1  Actemra® (Tocilizumab) 
8.1.1  Product Description 
Single-use vials containing tocilizumab, preservative free, sterile concentrate solutions (20 mg/ml) for IV 
infusion are available in the following sizes:  80 mg, 200 mg and 400 mg. The solution is colorless to pale 
yellow with a pH of approximately 6.5.  
Classification:  Immunomodulatory. 
Tocilizumab is a recombinant humanized interleukin-6 receptor inhibiting monoclonal antibody. Tocilizumab 
binds to both soluble and membrane bound IL-6 receptors and results in the blockade of interleukin-6 
signaling through these receptors. 
Pharmacokinetics:   
Pharmacokinetic studies indicate that tocilizumab undergoes biphasic elimination from the circulation. In 
rheumatoid arthritis patients treated with 4 and 8 mg/kg every four weeks, the central volume of distribution was 
3.5 L and the peripheral volume of distribution was 2.9 L with a volume of distribution at steady state of 6.4 L. 
Tocilizumab dosed at 8 mg/kg resulted in a mean steady state area under the curve (AUC), minimum 
concentration (Cmin) and a maximum concentration (Cmax) of 35 + 15 mg∙hr/ml, 9.74 + 10.5 mcg/ml and 183 
+ 85.6 mcg/ml respectively. Tocilizumab AUC, Cmin and Cmax increased with increasing body weight with an  
86% higher exposure in patients greater than 100 kg. As a result, doses exceeding 800 mg (max dosing weight 
100 kg) per infusion are not recommended. 
 
The total clearance of tocilizumab is concentration dependent and is represented by the both the linear 
clearance and the nonlinear clearance. Upon saturation of the nonlinear clearance pathway, the main 
determining factor is linear clearance. The reported linear clearance in the pharmacokinetic studies is estimated 
to be 12.5 mL/h. The concentration dependent half-life is up to 11 days for the 4 mg/kg dose and up to 13 days 
for the 8 mg/kg dose every four weeks at steady state. 
 
Special populations. Pharmacokinetic analysis in adult rheumatoid arthritis patients did not demonstrate a 
change in kinetics based on age, gender or race. The effects of renal and hepatic impairment have not been 
assessed. 
 
Contraindications: 
Side Effects:  Complete and updated adverse event information is available in the Investigational Drug 
Brochure and/or product package insert.  
Tocilizumab side effects include: 

 
 
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• 
Hypersensitivity reactions 
• 
Respiratory: upper respiratory tract infections, nasopharyngitis, bronchitis 
• 
Gastrointestinal: mouth ulceration, upper abdominal pain, gastritis, gastrointestinal perforations 
• 
Hepatic: transaminases elevation 
• 
Neurologic: headache, dizziness 
• 
Cardiovascular: hypertension 
• 
Dermatologic: skin rash 
• 
Serious infections (See Section 8.1.5) 
 
8.1.2  Solution Preparation  
Using aseptic technique, utilize a 100-ml bag of 0.9% sodium chloride injection USP and withdraw a volume 
equal to the volume of the tocilizumab solution required for the dose. Next, withdraw the calculated volume of 
tocilizumab solution necessary for the dose from the vials and slowly inject the tocilizumab to the infusion bag. 
The final solution volume should be 100ml. Gently invert the IV bag to mix the solution. Inspect the prepared 
IV solution for particulates. 
 
8.1.3  Route of Administration  
The infusion will be administered over 60 minutes through a dedicated IV line and must not be administered 
by IV bolus. 
8.1.4  Storage Requirements 
Vials should be stored under refrigeration at 2°C to 8°C (36°F to 46°F). Do not freeze. Protect the vials from 
light by storage in the original package until time of use. Inspect vials visually for particulates and discoloration 
prior to use and discard if particulates or discoloration is noted. Do not use beyond the expiration date on the 
container, package or prefilled syringe. 
Storage of prepared IV solution:  The solution may be stored under refrigeration or at room temperature for up 
to 24 hours and should be protected from light. 
 
8.1.5 Warnings and Precautions 
Serious Infections. The product information labeling contains a black box warning regarding the risk of 
serious infection. Most patients who developed these infections were taking concomitant 
immunosuppressants, such as methotrexate or corticosteroids. Serious infections leading to hospitalization or 

 
 
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death, including tuberculosis, bacterial, invasive fungal, viral and other opportunistic infections have occurred 
in patients receiving tocilizumab. Viral reactivation and cases of herpes zoster exacerbation were reported in 
clinical trials. If a serious infection develops, tocilizumab should be withheld until the infection is controlled. 
Gastrointestinal Perforations. Events of GI perforation have been reported in clinical trials, primarily as 
complications of diverticulitis. Patients presenting with new onset abdominal symptoms should be evaluated 
promptly. 
Laboratory Parameters. Neutropenia, decreases in platelets, transaminase elevations and increases in lipid 
parameters (total cholesterol, LDL and triglycerides) have been reported in relation to the use of tocilizumab. 
Drug Interactions. Elevated levels of IL-6 and other cytokines have been associated with reduced expression 
of some cytochrome (CYP) P450 enzymes. Tocilizumab, through IL-6 inhibition, has the potential to affect 
expression of multiple CYP enzymes by restoring their activity to a higher level than that in the absence of 
tocilizumab. Monitoring of drugs that are metabolized by CYPs with narrow therapeutic index or where the 
dose is individually adjusted is advised. Caution should be exercised when tocilizumab is coadministered with 
CYP3A4 substrate drugs where decrease in effectiveness is undesirable, e.g., oral contraceptives, 
atorvastatin etc. The effect of tocilizumab on CYP450 enzymes may occur within approximately two weeks of 
starting therapy and persist for several weeks after stopping therapy. 
 
8.1.6 Availability 
This drug is commercially available, but will be paid for by the study.  
 
8.1.7 Investigator and Site Responsibility for Drug Accountability. 
Accountability for the study drug is the responsibility of the principal investigator/designee. The investigator will 
ensure that the drug is used only in accordance with this protocol. Drug accountability records indicating the 
drug’s delivery date to the site, inventory at the site, use by each patient, and disposal of the drug (if applicable) 
will be maintained by the clinical site. Accountability records will include dates, quantities, lot numbers, 
expiration dates (if applicable), and patient numbers. 
9 STATISTICAL CONSIDERATIONS 
9.0 Study Populations 
The Full analysis set (FAS) will consist of all eligible patients enrolled in the study. This population will be used 
for the efficacy analyses. 
The Safety set (SS) will consist of all eligible patients enrolled in the study who received at least one dose of 
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9.1 Study Endpoints 
Analysis of Primary Endpoint 
The primary analysis will be performed on the FAS population using the intention-to-treat principle including all 
patients enrolled in the trial. The GRFS will be estimated, using the Kaplan-Meier estimator and plotted with a 
95% confidence band. An event for this outcome is defined as grade III–IV acute GVHD, chronic GVHD 
requiring systemic therapy, relapse or death. Patients who are alive without GVHD will be censored at the last 
follow-up. The 12-month GRFS will be compared to the prespecified historical control value of 20%, using a 
one-sided z-test. 
 
Analysis of Secondary Endpoints 
Demographic and other baseline data, such as disease characteristics, as well as outcome measures, will be 
presented overall and separately for URD and MRD patients. Categorical data, such as gender, race, etc., will 
be presented by frequencies and percentages. Descriptive summary statistics (e.g., frequency, mean, 
median, range and standard deviation) will be used to present numeric data. 
Time-to-event outcomes with and without competing risks will be analyzed, using Kaplan-Meier and Nelson-
Aalen estimates, respectively, and presented with 95% confidence intervals. Binary outcomes will be 
analyzed, using proportions with 95% confidence intervals. 
Effect of GVHD prophylaxis on microbiome diversity: Difference in the level of microbiome diversity detected 
at pre- and post-transplant. Microbiome diversity will be compared at each time point using Mann-Whitney 
tests. Association of pre-transplant microbiome diversity with development of aGVHD, cGVHD, and overall 
survival (corrected for GVHD prophylaxis): This analysis will use the pre-transplant stool microbiome cohort; 
diversity will be grouped by tertiles or if a more appropriate classification is identified in the analysis, that may 
be used instead. Acute GVHD (grade 2-4 or 3-4) and chronic GVHD (any or moderate-severe) will be described 
in each group using cumulative incidence with death or relapse as a competing event, and compared between 
groups using Gray’s test. Overall survival will be described using the Kaplan-Meier estimator and compared 
between groups using the log-rank test. Multivariate models will be constructed in a similar manner as for the 
primary endpoint using Fine and Gray models for aGVHD and cGVHD, and using Cox models for OS. 
 
Missing data 
Patients who are lost to follow-up will be censored at the day of the last contact with known status as 
appropriate for each outcome (for example, last day known alive for overall survival, and last day known alive 
without relapse or GVHD for GRFS). 
 
 

 
 
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9.2 Study Design 
The study is designed as a phase II, open-label, single-center trial to evaluate a novel GVHD prophylaxis 
regimen, Tac/MTX/tocilizumab. The primary endpoint of the study is GRFS, GVHD/ relapse-free survival at 12 
months posttransplant. 
 
9.3 Accrual Rate 
It is estimated that 18 months of accrual will be adequate to enroll the targeted sample size. 
9.4 Sample Size Justification 
The sample size was selected to achieve 80% power to detect an increase in GRFS at 12 months to 40% 
compared with an historical control value of 20% at a one-sided 5% significance level. Based on asymptotic z-
test for proportions, 29 patients with a known outcome are needed (i.e., known GVHD, relapse or death status 
at 12 months). We will recruit 32 subjects to allow for approximately 10% loss to follow-up by 1 year. Patients 
who have received at least one dose of tocilizumab would still be followed if they are withdrawn from the 
study. 
 
 
9.5 Stopping Rules 
 
Formal statistical toxicity monitoring will be in place for 100-day overall mortality. Based on program 
experience, a 100-day mortality rate <10% is expected. The monitoring boundary was selected to provide a 
10% probability of declaring excessive mortality, if the true probability is 10%. The following table shows the 
number of 100-day mortality events that would trigger a safety review depending on the number of evaluable 
patients. For the purposes of the safety monitoring patients in the safety set who are alive with at least 100 
days of follow-up or have died within 100 days are considered evaluable. 
 
Number of Evaluable 
Patients 
Boundary for excessive 
100-day mortality 
1-2 
- 
3-10 
3+ 
11-15 
4+ 
16-20 
5+ 
21-25 
6+ 
26-29 
7+ 
 
The following table shows the probability of finding excessive 100-day mortality during the study for several 
underlying true values of 100-day mortality rate. 

 
 
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100-day mortality rate 
Probability of crossing 
boundary 
5% 
1.5% 
10% 
11% 
20% 
55% 
25% 
76% 
30% 
89% 
 
10 DATA AND SAFETY MONITORING PLAN (DSMP) 
Data and Safety Management Overview 
The MCWCC DSMC and the MCW IRB will approve protocol-specific DSM plans. A local, investigator-
initiated trial will be required to be continuously monitored by the principal investigator of the study with safety 
and progress reports submitted to the DSMC.  
The DSMP for this study will involve the following entities: 
 
10.1 Study Team 
The study team minimally consists of the principal investigator, the clinical research coordinator, regulatory 
specialist and the study biostatistician. While subjects are on treatment, the principal investigator will meet 
regularly with the research coordinator and the study biostatistician to review study status. This review will 
include but not be limited to reportable SAEs and UPIRSOs and an update of the ongoing study summary that 
describes study progress in terms of the study schema. The appropriateness of further subject enrollment and 
the specific intervention for a next subject enrollment is addressed. All meetings including attendance are 
documented.  
 
10.2 Quality Assurance  
The MCWCC Clinical Trials Office (MCWCC CTO) provides ongoing quality assurance audits. Quality 
Assurance: This protocol was classified as intermediate risk and will be reviewed internally by the MCW 
Cancer Center Clinical Trials Office Quality Assurance Staff according to the current version SOP, 6.5.2 
Internal Quality Assurance Reviews. 
10.3 Clinical Trials Office 
The MCWCC CTO provides administrative assistance and support to the DSMC.  

 
 
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10.4 DSMC 
The MCWCC places the highest priority on ensuring the safety of patients participating in clinical trials. Every 
cancer interventional trial conducted at MCW includes a plan for safety and data monitoring. 
More information can be found related to the MCWCC Data and Safety Monitoring Plan at the MCWCC 
website (Data and Safety Monitoring Plan).  
This study will be reviewed by the MCWCC DSMC. A summary of the MCWCC DSMC activities are as 
follows:  
 
Review the clinical trial for data integrity and safety  
 
Review all unexpected grade 3, and all grade 4, and 5 adverse events, as well as any others requiring 
expedited reporting as defined in this protocol (Grades 4 and 5 events must be reported to the DSMC 
within five calendar days of study staff’s knowledge.)  
Grade ≤4 hematological AEs occurring within the first 28 days post-transplant will be routine reported 
to the DSMC. 
 
Review all DSM reports  
 
Submit a summary of any recommendations related to study conduct  
 
Terminate the study if deemed unsafe for patients  
A copy of the MCWCC Data and Safety Monitoring Plan and membership roster will be maintained in the 
study research file and updated as membership changes. The committee will review reports from the study PI 
twice annually (or more frequently if needed) and provide recommendations on trial continuation, suspension 
or termination as necessary.  
Any available DSMC letters will be submitted to the IRB of record as required. 
11 REGULATORY COMPLIANCE, ETHICS AND STUDY MANAGEMENT 
11.1 Ethical Standard 
This study will be conducted in accordance with the ethical principles that have their origin in the Declaration 
of Helsinki as stated in 21 CFR §312.120(c)(4); consistent with GCP and all applicable regulatory 
requirements. 
 
11.2 Regulatory Compliance 
This study will be conducted in compliance with: 
 
The protocol 
 
Federal regulations, as applicable, including: 21 CFR 50 (Protection of Human Subjects/Informed 
Consent); 21 CFR 56 (Institutional Review Boards) and §312 (Investigational New Drug Application; 
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(Prisoners), and D (Children), GCP/ICH guidelines, and all applicable regulatory requirements. The 
IRB must comply with the regulations in 21 CFR §56 and applicable regulatory requirements. 
11.3  Prestudy Documentation  
Prior to implementing this protocol at MCWCC, the protocol, informed consent form, HIPAA authorization and 
any other information pertaining to participants must be approved by the MCW IRB.  
 
11.4 Institutional Review Board 
The protocol, the proposed informed consent form and all forms of participant information related to the study 
(e.g. advertisements used to recruit participants) will be reviewed and approved by the MCW Institutional 
Review Board. Prior to obtaining approval, the protocol must be approved by the Medical College of 
Wisconsin Cancer Center Scientific Review Committee. The initial protocol and all protocol amendments must 
be approved by the IRB prior to implementation.  
 
Informed Consent Process 
Informed consent is a process that is initiated prior to the individual’s agreeing to participate in the study and 
continues throughout the individual’s study participation. Extensive discussion of risks and possible benefits of 
this therapy will be provided to the subjects and their families. Consent forms describing in detail the study 
interventions/products, study procedures and risks are given to the subject and written documentation of 
informed consent is required prior to starting intervention/administering study product. 
Consent forms will be IRB approved and the subject (and Legally Authorized Representative, if necessary) will 
be asked to read and review the document. Upon reviewing the document, the investigator will explain the 
research study to the subject and answer any questions that may arise. In accordance with 46 CR 46.111, the 
subject will sign and date the informed consent document prior to any procedures being done specifically for 
the study.  
A witness should only sign when required, per FH/MCW IRB policy. If a witness signs the document when not 
required, the study staff should document in the legal medical record (or note to file) the relationship to the 
patient and why a witness signed. (i.e., “Although not required, the subject’s spouse was present during the 
consenting process and signed as the witness.” Or “Although not required, hospital staff was present for 
consenting process and signed as a witness.”) 
The subjects will have the opportunity to discuss the study with their surrogates or think about it prior to 
agreeing to participate. The subjects may withdraw consent at any time throughout the course of the trial.  
A copy of the informed consent document will be given to the subjects for their records. The rights and welfare 
of the subjects will be protected by emphasizing to them that the quality of their medical care will not be 
adversely affected if they decline to participate in this study. If there are changes to the consent form, all 
revisions will be reviewed with study subject at the next appropriate opportunity. Patients that require 
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consent will again be performed. Study subjects will not be reconsented for continuing reviews. The MCWCC 
CTO will follow the MCW/FH IRB’s policy for subjects who demonstrate limited English proficiency or limited 
literacy.  
After the subject’s visit in which the consent is signed, it is documented in the clinic chart that the consent has 
been signed and that all questions have been answered to the subject’s satisfaction after adequate time for 
review of the consent. It is also documented that a copy of the consent is given to the subject. The original 
consent is kept with the subject’s study file, and a copy of the consent is sent to the OCRICC office, which will 
then submit to HIM a copy of the signed consent to be scanned into EPIC, the legal medical record. 
 
11.5  Subject Confidentiality and Access to Source Documents/Data 
Subject confidentiality is strictly held in trust by the sponsor-investigator, participating investigators, and any 
staff, [and the sponsor(s) and their agents]. This confidentiality includes the clinical information relating to 
participating subjects, as well as any genetic or biological testing. 
The study protocol, documentation, data, and all other information generated will be held in strict confidence. 
No information concerning the study, or the data will be released to any unauthorized third party without prior 
written approval of the principal investigator. 
The conditions for maintaining confidentiality of the subjects’ records are required for the life of the data. 
These rules apply equally to any and all MCWCC projects.  
One risk of taking part in a research study is that more people will handle the personal health information 
collected for this study. The study team will make every effort to protect the information and keep it 
confidential, but it is possible that an unauthorized person might see it. Depending on the kind of information 
being collected, it might be used in a way that could embarrass the subject or affect his/her ability to get 
insurance. 
While data are being collected and after all data have been collected but are still in the process of being 
analyzed, the subject’s data/PHI are stored in the locked Clinical Research office in the Clinical Trials Office. 
Databases in which the study subject information is stored and accessed are password protected, allowing for 
limited access by authorized personnel only. Data/PHI kept in the Case Report Forms contain the study 
identifiers, subject initials, date of birth and date of service.  
The principal investigator will allow access to all source data and documents for the purposes of monitoring, 
audits, IRB review and regulatory inspections.  
The study monitor or other authorized representatives of the principal investigator may inspect all documents 
and records required to be maintained by the investigator, including but not limited to, medical records (office, 
clinic, or hospital) and pharmacy records for the subjects in this study. The clinical study site will permit 
access to such records. 
 

 
 
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11.6 Protection of Human Subjects 
11.6.1 Protection from Unnecessary Harm 
Each clinical site is responsible for protecting all subjects involved in human experimentation. This is 
accomplished through the IRB mechanism and the informed consent process. The IRB reviews all proposed 
studies involving human experimentation and ensures that the subject’s rights and welfare are protected and 
that the potential benefits and/or the importance of the knowledge to be gained outweigh the risks to the 
individual. The IRB also reviews the informed consent document associated with each study in order to 
ensure that the consent document accurately and clearly communicates the nature of the research to be done 
and its associated risks and benefits. 
 
11.6.2 Protection of Privacy 
As noted, patients will be informed of the extent to which their confidential health information generated from 
this study may be used for research purposes. Following this discussion, they will be asked to sign informed 
consent documents. The original signed document will become part of the patient’s medical records, and each 
patient will receive a copy of the signed document.  
 
11.7 Changes in the Protocol 
Once the protocol has been approved by the MCW IRB, any changes to the protocol must be documented in 
the form of an amendment. The amendment must be signed by the investigator and approved by IRB prior to 
implementation.  
If it becomes necessary to alter the protocol to eliminate an immediate hazard to patients, an amendment may 
be implemented prior to IRB approval. In this circumstance, however, the Investigator must then notify the IRB 
in writing within five working days after implementation.  
The IRB may provide, if applicable regulatory authority(ies) permit, expedited review and approval/favorable 
opinion for minor change(s) in ongoing studies that have the approval /favorable opinion of the IRB. The 
investigator will submit all protocol modifications to the sponsor and the regulatory authority(ies) in 
accordance with the governing regulations. 
Changes to the protocol may require approval from the sponsor. 
Any departures from the protocol must be fully documented in the source documents. 
 
11.8 Investigator Compliance  
The investigator will conduct the study in compliance with the protocol given approval/favorable opinion by the 
IRB and the appropriate regulatory authority(ies).  
Onsite Audits 
Auditing is essential to ensure that research conducted at the Medical College of Wisconsin (MCW) Cancer 
Center is of the highest quality and meets MCW and regulatory agency standards.  

 
 
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Regulatory authorities, the IRB and/or sponsor may request access to all source documents, data capture 
records and other study documentation for on-site audit or inspection. Direct access to these documents must 
be guaranteed by the investigator, who must provide support at all times for these activities. 
12 DATA HANDLING AND RECORD KEEPING 
12.1 Overview 
Every effort is made to uphold the integrity of the project, the research, the institution, and the researchers 
involved. Data collection guidelines and methodologies are carefully developed before the research begins. 
Investigators focus on the following to ensure data integrity:  well-trained data collectors/recorders to ensure 
consistency and quality, well-designed data collection protocols and ongoing monitoring. In this way, study 
rigor and validity are maintained. Data is protected from physical damage as well as from tampering, loss or 
theft.  
 
12.2 Data Management Responsibilities 
This project’s data management is a multidisciplinary activity that includes investigators, research 
coordinators and nurses, data mangers, support personnel, biostatisticians and database programmers. 
Quality control will be applied to each stage of data handling to ensure that all data are reliable and have been 
processed correctly.  
 
12.2.1 Principal Investigator 
The principal investigator oversees the management of patient records/case report forms and ensures that a) 
complete and accurate data will be obtained and provided to the sponsor; b) patient records are maintained to 
include history, prescribed medication and investigational product(s), measurements, exams, evaluations and 
adverse events; c) corrections are applied to clinical research data according to principles of good research 
practice (i.e., single-line delete, date and initial). He or she will ensure that there is correlation between the 
case report forms and the source documents.  
 
12.2.2 Research Coordinator  
A research coordinator creates, collects, and organizes clinical trial documentation. He or she ensures that 
source documentation and data abstraction and entry are being done at protocol specified time points. 
 
12.2.3 Research Nurse/Medical Staff 
The research nurse and medical staff documents protocol-required care or assessment of the subject’s 
outcomes, adverse events and compliance to study procedures. 
 

 
 
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12.2.4 Biostatistician 
The biostatistician may assist in CRF development (content and design), dataset specifications (annotation of 
CRFs and record layout) and validation. 
 
12.3 Handling and Documentation of Clinical Supplies 
The MCWCC principal investigator will maintain complete records showing the receipt, dispensation, return, or 
other disposition of all investigational drugs. The date, quantity and batch or code number of the drug, and the 
identification of patients to whom study drug has been dispensed by patient number and initials will be 
included. The sponsor-investigator will maintain written records of any disposition of the study drug. 
The principal investigator shall not make the investigational drug available to any individuals other than to 
qualified study patients. Furthermore, the principal investigator will not allow the investigational drug to be 
used in any manner other than that specified in this protocol. 
 
12.4 Source Documents 
Source documents for clinical information (patient history, diagnosis, clinical and diagnostic test reports, etc.) 
are maintained in the patient’s clinical file.  
 
 
All source documents will be written following ALCOA standards:  
 
ALCOA Attribute 
Definition 
Attributable 
Clear who has documented the data. 
Legible 
Readable and signatures identifiable. 
Contemporaneous 
Documented in the correct time frame along with the flow of events. 
If a clinical observation cannot be entered when made, chronology 
should be recorded. Acceptable amount of delay should be defined 
and justified. 
Original  
Original, if not original should be exact copy; the first record made 
by the appropriate person. The investigator should have the original 
source document. 
Accurate 
Accurate, consistent and real representation of facts. 
Enduring 
Long-lasting and durable. 
Available and 
accessible 
Easily available for review by treating physicians and during 
audits/inspections. The documents should be retrievable in 
reasonable time. 
Complete 
Complete until that point in time. 
Consistent 
Demonstrate the required attributes consistently. 
Credible 
Based on real and reliable facts. 
Corroborated 
Data should be backed up by evidence. 
 

 
 
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12.5 Case Report Forms  
The principal investigator and/or his/her designee will prepare and maintain adequate and accurate participant 
case histories with observations and data pertinent to the study. Study-specific Case Report Forms (CRFs) 
will document safety and treatment outcomes for safety monitoring and data analysis. All study data will be 
entered into OnCore® via standardized CRFs, in accordance with the study calendar, using single data entry 
with a secure access account. The Clinical Research Coordinator will complete the CRFs as soon as possible 
upon completion of the study visit; the investigator will review and approve the completed CRFs.  
The information collected on CRFs shall be identical to that appearing in original source documents. Source 
documents will be found in the patient’s medical records maintained by MCWCC personnel. All source 
documentation should be kept in separate research folders for each patient. 
In accordance with federal regulations, the Investigator is responsible for the accuracy and authenticity of all 
clinical and laboratory data entered onto CRFs. The principal investigator will approve all completed CRFs to 
attest that the information contained on the CRFs is true and accurate.  
All source documentation and data will be available for review/monitoring by the MCWCC DSMC and 
regulatory agencies. 
 
12.6 Study Record Retention 
The principal investigator is required to maintain adequate records of the disposition of the drug, including 
dates, quantity and use by subjects, as well as written records of the disposition of the drug when the study 
ends.  
The principal investigator is required to prepare and maintain adequate and accurate case histories that 
record all observations and other data pertinent to the investigation on each individual administered the 
investigational drug or employed as a control in the investigation. Case histories include the case report forms 
and supporting data including, for example, signed and dated consent forms and medical records including, 
for example, progress notes of the physician, the individual's hospital chart(s), and the nurses' notes. The 
case history for each individual shall document that informed consent was obtained prior to participation in the 
study. 
Study documentation includes all CRFs, data correction forms or queries, source documents, sponsor-
investigator correspondence, monitoring logs/letters, and regulatory documents (e.g., protocol and 
amendments, IRB correspondence and approval, signed patient consent forms). 
Source documents include all recordings of observations or notations of clinical activities and all reports and 
records necessary for the evaluation and reconstruction of the clinical research study. 
In accordance with FDA regulations, the investigator shall retain records for a period of two years following the 
date a marketing application is approved for the drug for the indication for which it is being investigated; or, if 
no application is to be filed or if the application is not approved for such indication, until two years after the 
investigation is discontinued and FDA is notified. 

 
 
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Filipovich AH, Weisdorf D, Pavletic S, et al: National Institutes of Health consensus 
development project on criteria for clinical trials in chronic graft-versus-host disease: I. Diagnosis and staging 
working group report. Biol Blood Marrow Transplant 11:945-56, 2005 
 

 
 
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APPENDIX 1. PERFORMANCE STATUS CRITERIA 
ECOG Performance Status Scale 
Karnofsky Performance Scale 
Grade 
Descriptions 
Percent 
Description 
0 
Normal activity 
Fully 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% of 
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 2. GVHD ASSESSMENT 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
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ACUTE GVHD ASSESSMENT 
 
 
 
 
STAGING AND GRADING OF ACUTE GVHD 
Staging 
 
 
 

 
 
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Grading of Acute GVHD 
 
 
 

 
 
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Grading of Chronic GVHD (NIH Criteria) 
 
 

 
 
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APPENDIX 3: QOL MEASURES 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
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QOL MEASURES 
 
Participant # _________ 
Date: _________ 
 
IDAS 
Instructions: Below is a list of feelings, sensations, problems, and experiences that people sometimes have. Read each item to 
determine how well it describes your recent feelings and experiences.  Then select the option that best describes how much 
you have felt or experienced things this way during the past two weeks, including today.   
 
 
Not at all 
A little bit 
Moderately 
Quite a bit 
Extremely 
1. I was proud of myself………………………………………………………………... 
1 
2 
3 
4 
5 
2. I felt exhausted………………………………………………………………………. 
1 
2 
3 
4 
5 
3. I felt depressed………………………………………………………………………. 
1 
2 
3 
4 
5 
4. I felt inadequate……………………………………………………………………… 
1 
2 
3 
4 
5 
5. I slept less than usual………………………………………………………………… 
1 
2 
3 
4 
5 
6. I felt fidgety, restless………………………………………………………………… 
1 
2 
3 
4 
5 
7. I had thoughts of suicide ……………………………………………………….….... 
1 
2 
3 
4 
5 
8. I slept more than usual………………………………………………………………. 
1 
2 
3 
4 
5 
9. I hurt myself purposely ……………………………… ...............................................  1 
2 
3 
4 
5 
10. I slept very poorly………………………………………………………………….. 
1 
2 
3 
4 
5 
11. I blamed myself for things
 
 
1 
2 
3 
4 
5 
12. I had trouble falling asleep
 
 
1 
2 
3 
4 
5 
13. I felt discouraged about things……………………………………………………… 1 
2 
3 
4 
5 
14. I thought about my own death
 
 
1 
2 
3 
4 
5 
15. I thought about hurting myself
 
 
1 
2 
3 
4 
5 
16. I did not have much of an appetite
 
 
1 
2 
3 
4 
5 

 
 
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17. I felt like eating less than usual
 
 
1 
2 
3 
4 
5 
19. I did not feel much like eating……………………………………………………… 
1 
2 
3 
4 
5 
21. I felt optimistic……………………………………………………………………… 1 
2 
3 
4 
5 
23. I felt that I had accomplished a lot
 
 
1 
2 
3 
4 
5 
24. I looked forward to things with enjoyment
 
 
1 
2 
3 
4 
5 
25. I was furious………………………………………………………………………… 1 
2 
3 
4 
5 
26. I felt hopeful about the future
 
 
1 
2 
3 
4 
5 
27. I felt that I had a lot to look forward to
 
 
1 
2 
3 
4 
5 
28. I felt like breaking things
 
  
1 
2 
3 
4 
5 
29. I had disturbing thoughts of something bad that happened to me
 
 
1 
2 
3 
4 
5 
30. Little things made me mad
 
 
1 
2 
3 
4 
5 
31. I felt enraged………………………………………………………………………… 
 
1 
2 
3 
4 
5 
32. I had nightmares that reminded me of something bad that happened
 
 
1 
2 
3 
4 
5 
33. I lost my temper and yelled at people………………………………………………. 
1 
2 
3 
4 
5 
34. I felt like I had a lot of interesting things to do.
 
 
1 
2 
3 
4 
5 
35. I felt like I had a lot of energy
 
 
1 
2 
3 
4 
5 
36. I had memories of something scary that happened
 
 
1 
2 
3 
4 
5 
37. I felt self-conscious knowing that others were watching me
 
 
1 
2 
3 
4 
5 
38. I felt a pain in my chest ……………………………………………………………. 1 
2 
3 
4 
5 
39. I was worried about embarrassing myself socially
 
 
1 
2 
3 
4 
5 
40. I felt dizzy or light headed
 
 
1 
2 
3 
4 
5 
41. I cut or burned myself on purpose
 
 
1 
2 
3 
4 
5 

 
 
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42. I had little interest in my usual hobbies or activities
 
 
1 
2 
3 
4 
5 
43. I thought that the world would be better off without me……………………………. 1 
2 
3 
4 
5 
 
 
44. I felt much worse in the morning than later in the day
 
 
1 
2 
3 
4 
5 
45. I felt drowsy, sleepy………………………………………………………………… 1 
2 
3 
4 
5 
46. I woke up early and could not get back to sleep……………………………………. 1 
2 
3 
4 
5 
47. I had trouble concentrating
 
 
1 
2 
3 
4 
5 
48. I had trouble making up my mind
 
 
1 
2 
3 
4 
5 
49. I talked more slowly than usual
 
 
1 
2 
3 
4 
5 
50. I had trouble waking up in the morning……………………………………………. 
1 
2 
3 
4 
5 
51. I found myself worrying all the time
 
 
1 
2 
3 
4 
5 
52. I woke up frequently during the night
 
 
1 
2 
3 
4 
5 
53. It took a lot of effort for me to get going
 
 
1 
2 
3 
4 
5 
54. I woke up much earlier than usual
 
 
1 
2 
3 
4 
5 
55. I was trembling or shaking………………………………………………………….. 1 
2 
3 
4 
5 
56. I became anxious in a crowded public setting
 
 
1 
2 
3 
4 
5 
57. I felt faint……………………………………………………………………………. 1 
2 
3 
4 
5 
58. I found it difficult to make eye contact with people
 
 
1 
2 
3 
4 
5 
59. My heart was racing or pounding
 
 
1 
2 
3 
4 
5 
60. I got upset thinking about something bad that happened
 
 
1 
2 
3 
4 
5 
61. I found it difficult to talk with people I did not know well
 
 
1 
2 
3 
4 
5 
62. I had a very dry mouth……………………………………………………................ 1 
2 
3 
4 
5 
63. I was short of breath………………………………………………………………… 1 
2 
3 
4 
5 

 
 
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64. I felt like I was choking…………………………………………………………….. 
1 
2 
3 
4 
5 
 
 
 
FSI 
 
Instructions: The following questions ask about fatigue during the past week. For each of the following, circle the one 
number that best indicates how that item applies to you. 
 
1. Rate your level of fatigue on the day you felt most fatigued during the past week. 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
Not at all 
fatigued 
 
 
 
 
 
 
 
 
 
As fatigued as 
I could be 
 
2. Rate your level of fatigue on the day you felt least fatigued during the past week. 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
Not at all 
fatigued 
 
 
 
 
 
 
 
 
 
As fatigued as I 
could be 
 
3. Rate your level of fatigue on the average in the last week. 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
Not at all 
fatigued 
 
 
 
 
 
 
 
 
 
As fatigued as I 
could be 
 
4. Rate your level of fatigue right now. 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
Not at all 
fatigued 
 
 
 
 
 
 
 
 
 
As fatigued as I 
could be 
 
5. Rate how much, in the past week, fatigue interfered with: 
 
No 
interference 
 
Extreme 
interference 
a. Your general level of activity ..........................  0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
b. Your ability to bathe and dress yourself .........  0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
c.  Your normal work activity (includes both 
work outside the home and housework) ........  
 
0 
 
1 
 
2 
 
3 
 
4 
 
5 
 
6 
 
7 
 
8 
 
9 
 
10 
d. Your ability to concentrate .............................  0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
e. Your relations with other people .....................  0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
f. Your enjoyment of life ....................................  0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
g. Your mood ......................................................  0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
 
6. Indicate how many days, in the past week, you felt fatigued for any part of the day: 
0 
1 
2 
3 
4 
5 
6 
7 
 
7. Rate how much of the day, on average, you felt fatigued in the past week 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
None of the 
day 
 
 
 
 
 
 
 
 
 
The entire day 
 

 
 
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PSQI 
 
Instructions: The following questions relate to your usual sleep habits during the past month only. Your answers 
should indicate the most accurate reply for the majority of days and nights in the past month. Please answer all questions. 
 
1. During the past month, what time have you usually gone to bed at night? 
 
BED TIME ___________ 
 
2. During the past month, how long (in minutes) has it usually taken you to fall asleep each night? 
 
NUMBER OF MINUTES ___________ 
 
3. During the past month, what time have you usually gotten up in the morning? 
 
GETTING UP TIME ___________ 
 
4. During the past month, how many hours of actual sleep did you get at night? (This may be 
different than the number of hours you spent in bed.) 
 
HOURS OF SLEEP PER NIGHT ___________ 
 
For each of the remaining questions, check the one best response. Please answer all questions. 
 
5. During the past month, how often have you had trouble sleeping because you . . . 
 
a) Cannot get to sleep within 30 minutes 
 
Not during the 
past month _____  
Less than 
once a week _____  
Once or twice 
a week _____  
Three or more 
times a week _____  
b) Wake up in the middle of the night or early morning 
 
Not during the 
past month _____  
Less than 
once a week _____  
Once or twice 
a week _____  
Three or more 
times a week _____  
c) Have to get up to use the bathroom 
 
Not during the 
past month _____  
Less than 
once a week _____  
Once or twice 
a week _____  
Three or more 
times a week _____  
d) Cannot breathe comfortably 
 

 
 
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Not during the 
past month _____  
Less than 
once a week _____  
Once or twice 
a week _____  
Three or more 
times a week _____  
 
 
 
e) Cough or snore loudly 
 
Not during the 
past month _____  
Less than 
once a week _____  
Once or twice 
a week _____  
Three or more 
times a week _____  
f) Feel too cold 
 
Not during the 
past month _____  
Less than 
once a week _____  
Once or twice 
a week _____  
Three or more 
times a week _____  
g) Feel too hot 
 
Not during the 
past month _____  
Less than 
once a week _____  
Once or twice 
a week _____  
Three or more 
times a week _____  
h) Had bad dreams 
 
Not during the 
past month _____  
Less than 
once a week _____  
Once or twice 
a week _____  
Three or more 
times a week _____  
i) Have pain 
 
Not during the 
past month _____  
Less than 
once a week _____  
Once or twice 
a week _____  
Three or more 
times a week _____  
j) Other reason(s), please describe_________________________________________________ 
__________________________________________________________________________ 
 
How often during the past month have you had trouble sleeping because of this? 
 
Not during the 
past month _____  
Less than 
once a week _____  
Once or twice 
a week _____  
Three or more 
times a week _____  
 
 
 
 
6. During the past month, how would you rate your sleep quality overall? 
 
Very good 
__________ 
Fairly good 
__________ 
Fairly bad 
__________ 
Very bad 
__________ 
 

 
 
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7. During the past month, how often have you taken medicine to help you sleep (prescribed or 
"over the counter")? 
 
Not during the 
past month _____  
Less than 
once a week _____  
Once or twice 
a week _____  
Three or more 
times a week _____  
 
 
8. During the past month, how often have you had trouble staying awake while driving, eating 
meals, or engaging in social activity? 
 
Not during the 
past month _____  
Less than 
once a week _____  
Once or twice 
a week _____  
Three or more 
times a week _____  
9. During the past month, how much of a problem has it been for you to keep up enough enthusiasm to get things 
done? 
No problem at all 
__________ 
Only a very slight problem 
__________ 
Somewhat of a problem 
__________ 
A very big problem 
__________ 
 
 
 

 
 
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BPI 
 
Instructions: The following questions ask about pain during the past week. For each of the following, circle the one 
number that best indicates how that item applies to you. 
 
1. Please rate your pain by circling the number that best describes your pain at its worst in the last week. 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
No 
pain 
 
 
 
 
 
 
 
 
 
Pain as bad as you 
can imagine 
 
2. Please rate your pain by circling the number that best describes your pain at its least in the last week. 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
No 
pain 
 
 
 
 
 
 
 
 
 
Pain as bad as you 
can imagine 
 
3. Please rate your pain by circling the number that best describes your pain on the average in the last week. 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
No 
pain 
 
 
 
 
 
 
 
 
 
Pain as bad as you 
can imagine 
 
4. Please rate your pain by circling the number that best describes your pain right now. 
0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
No 
pain 
 
 
 
 
 
 
 
 
 
Pain as bad as you 
can imagine 
 
5. Circle the number that describes how, during the past week, pain has interfered with your: 
 
Does not 
interfere 
 
Completely 
interferes 
a. General activity ................................................ 0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
b. Mood ................................................................ 0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
c. Walking ability ................................................. 0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
d. Normal work (includes both work outside the 
home and housework) ..................................... 
 
0 
 
1 
 
2 
 
3 
 
4 
 
5 
 
6 
 
7 
 
8 
 
9 
 
10 
e. Relations with other people .............................. 0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
f. Sleep ................................................................. 0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
g. Enjoyment of life ............................................. 0 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
 
 
 
 

 
 
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THESE QUESTIONS NEED ONLY BE ANSWERED ONCE; NO NEED TO ANSWER AT SUBSEQUENT 
STUDY FOLLOW-UP VISITS: 
 
Please indicate below what best represents your household income: 
 
_____ <$10,000 
_____ $10,001-$25,000 
_____ $25,001-$40,000 
_____ $40,001-$55,000 
_____ $55,001-$70,000 
_____ $70,001-$85,000 
_____ $85,001-$100,000 
_____ > $100,000 
   
Please indicate which education level best describes you: 
 
_____ Less than 12 years 
_____ High School 
_____ Trade School 
_____ Some College 
_____ College Graduate 
_____ Post-Graduate Degree  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
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APPENDIX 4. GUT MICROBIOME STUDY: STOOL SAMPLE COLLECTION AND 
PROCESSING 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
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Sample Information Management for Microbiota Studies  
  
Each stool sample will be given a unique identifier.   
 
For example:  
Patient 0001 and has sample A and B. Patient 0002 has sample A.  
Sample IDs: MCW_0001-A, MCW_0001-B, MCW_0002-A  
  
As we acquire, aliquot, and store samples, we will not to try to squeeze additional information 
(metadata), such as date or day of collection or patient initials into the sample ID.  Such 
additional information becomes very difficult to deal with later and statistical software programs 
can’t deal with them well. Please don’t name samples things like “Pt 002 Week 3” or “Pt 002 pre-
transplant” or “Pt JFK (Day -2)”.  If a patient has more than one transplant, day and week 
designations are problematic. Another problem with this sort of a sample-naming scheme leads 
to discrepancies between the target time point (e.g. day 30) and the actual timepoint collected 
(e.g. day 32)  
  
The following approach is recommended:   
Keep the names of samples simple and then keep a separate table of sample metadata, as below:   
 
 
 
 
 
 
 
 
 
 

 
 
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Stool Sample Collection, Processing, and Shipping 
 
Sample Collection  
 
  Suggested collection days:  
                   Inpatient: twice weekly (e.g. Mondays and Thursdays) – once pre-transplant and 
weekly (+/- 2 days) post-transplant until after neutrophil engraftment.  
 
Tube Preparation  
     Please aliquot into sturdy tubes that can withstand cold temperatures2. Standard urine- or 
sputum-collection cups crack at freezing temperatures and should not be used.  
     If labeling tubes by hand, please use an alcohol-proof marker3. Sharpie, etc. will dissolve in 
the ethanol used during DNA extraction.  
     If using printed labels, please ensure that they will not peel, crack, or smear at freezing 
temperatures4, 5.  
  
Aliquoting  
     Aliquoting should be done as soon as possible after sample is received.  
     For solid or semi-solid samples, use a clean disposable spatula6 to transfer stool into 
cryovials2.  
o  Prepare 5 aliquots of ~0.5 mL each.  
o  For more liquid samples, can transfer up to 1 mL material.  
     For liquid samples, use transfer pipette7 to prepare 1.5 mL aliquots of material.  
     All aliquots should be stored at -80 until shipping.  
  
Shipping Address:  
Attn: Annie Slingerland Z-1419  
Memorial Sloan-Kettering Cancer Center  
408 East 69th Street New York, NY 10021 United States  
646-888-2304  

 
 
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Shipping   
Samples should be sent overnight, with enough dry ice to last 2-3 days in the event that delivery is 
delayed.  
Please ship only on Monday, Tuesday, or Wednesday to ensure that sample does not sit on 
loading dock over the weekend.  
 
After Shipping  
     Please email Annie (slingera@mskcc.org) with tracking number and a shipping manifest 
(.xlsx or .csv) detailing  
for each tube:  
o  text of written or printed label  
o  unique sample ID (discussed in “Microbiota Data Collection and Management”)  
o  unique ID for associated patient (discussed in “Microbiota Data Collection and 
Management”)  
o  date of collection (or day of transplant/conditioning)  
o  preservative added, if any  
o  # of aliquots made of sample and how many are included in shipment (e.g. 1 of 5; 2 
shipped)  
o  If sending more than 20 tubes, please include a map of each tube’s location within 
the grid of the box  
  
Questions?  
van den Brink lab tel: 646-888-2317 (ask for Tsoni, Annie, or Melissa)  
Dr. van den Brink's office & assistants: 646-888-2304. 
 
Suggested Materials: 
 
1. Outpatient kit: 
 commode specimen collection system (www.fishersci.com, 02-544-208, $2.75/ea, 60/cs)  
               (specimen container will need a unique specimen number label)  

 
 
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 14 x 9 x 9” corrugated box (www.uline.com, S-4985, $0.66 to $0.94/box, multiples of 
25/cs)  
 8 x 6 x 4.25” foam container (www.uline.com, S-18312, $4.70/ea, 20/cs)  
 (2) cold bricks - 7.5 oz (www.uline.com, S-14294, $0.824/ea, 42/cs)  
 12 x 15 biohazard zip lock bag (www.ourshippingsupplies.com A-LN4099, $0.18/ea, 
1000/cs)  
 14 x 10 x 15.5” paper shopping bag (www.uline.com, S-9661, $0.345 to $0.38/bag, 
multiples of 200/cs)  
outpatient collection directions  
2. Microtubes, 2ML, w/ screw cap (www.fishersci.com, NC0445483, or www.sarstedt.com 
72.694.006,$0.32/ea, 1000/cs)  
3. VWR lab markers, black ink, fine point (www.vwr.com, 52877-310, $6.19/ea, 10/pack)  
4. Cryo-Clear clear labels, for 0.5 ml tubes, 0.94" L x 0.5" W 
(http://www.usascientific.com/labelsonsheets.aspx,   
              9125-0238, $3.15/sheet of 119 stickers, 20 sheets/box)  
5. Tough-Spots, small, colors, for 0.5 ml  tubes, 3/8" diameter 
(http://www.usascientific.com/labelsonsheets.aspx,   
              9185-1008, $2.85/sheet of 192 stickers, 20 sheets/box)  
6. Sterile Disposable Spatula, Double-Ended, with Spoon & Scoop 
(https://us.vwr.com/store/product/4531734/vwr- 
              disposable-polypropylene-spatulas, 89097-816, $1.39/ea, 100/pack)  
7. Disposable Graduated Transfer Pipettes, individually wrapped 
(https://www.fishersci.com/shop/products/fisherbrand  
              -disposable-graduated-transfer-pipettes-5/p-163764, 13-711-20, $0.21/ea, 500/pack)  
8. Feces tube, 101x16.5mm, sterile (https://www.fishersci.com/us/en/healthcare-products.html, 
NC0729971, $0.47/ea, 500/case).