Bridging Pediatric and Adult Biomarkers in Graft-Versus-Host Disease
Conditions: Graft-Versus-Host Disease
Sponsor: Indiana University

 
 
 
 
 
Official Title: Bridging Pediatric and Adult Biomarkers in Graft-Versus-Host Disease 
 
NCT number: NCT02194439 
 
Document date: 12Jun2015 

1 
 
 
 
Bridging Pediatric and Adult Biomarkers in Graft-Versus-Host Disease 
 
 
Study Chair 
 
Sophie Paczesny, MD, PhD, Indiana University School of Medicine 
 
Study co-Chair 
 
Jamie L. Renbarger, MD, MS, Indiana University School of Medicine 
 
Protocol Team 
 
Lang Li, PhD, Statistician, Indiana University 
 
Christine Duncan, MD, Dana-Farber Cancer Institute and Boston Children's Hospital 
Kenneth Cooke, MD, Johns Hopkins Sidney Kimmel Comprehensive Cancer Center 
Paul Carpenter, MD, Fred Hutchinson Cancer Research Center 
Robert Krance, MD, Texas Children’s Hospital 
 
Kathryn Leung, MD, Texas Children’s Hospital 
 
David Jacobsohn, MD, Children's National Medical Center 
 
Catherine Bollard, MD, PhD, Children’s National Medical Center 
 
 
Sponsored by 
 
 
 
 
National Institutes of Health (NIH), Biomarkers:  Bridging Pediatric and Adult 
Therapeutics (R01) 

2 
 
PROTOCOL SYNOPSIS 
 
Title: Bridging Pediatric and Adult Biomarkers in Graft-Versus-Host Disease  
Study Design 
 
This   study   is   designed   to   collect   longitudinal   biological   samples   from   patients   after 
hematopoietic cell transplantation (HCT) cared for at multiple bone marrow transplant centers to 
validate biomarkers of both acute and chronic GVHD as well as for use in future unspecified 
research. 
 
After informed consent is signed, this study will involve 1) collection of basic HCT data and clinical 
data available in the medical record and 2) providing blood (and saliva in occasional cases) 
samples for processing, storage, DNA extraction, and analysis (including a seven biomarker 
protein panel as well as future unspecified research purposes) 
 
Pediatric and adult patients will be included (all adult patients will be from the Fred Hutchinson 
Cancer Research Center, the Johns Hopkins Sidney Kimmel Comprehensive Cancer Center, 
and the Indiana University Simon Cancer Center). 
 
Primary Objective: 
1.  To confirm that ST2 alone or the seven-biomarker panel measured at initiation of GVHD 
therapy predict a) D180 post-therapy non-relapse mortality; b) D28 post-therapy non-
response, and c) GVHD grade 1-4 onset D180 post-therapy non-relapse mortality. 
 
Secondary Objective: 
1.  To demonstrate that ST2 alone or the seven-biomarker panel measured at day 14 or day 
21 post-HCT (or a combination of these time points) predicts D180 post-HCT non-relapse 
mortality. 
2.  To demonstrate that ST2 alone or the seven-biomarker panel measured at initiation of 
GVHD symptoms/therapy diagnose acute GVHD as compared to other complications 
presenting with similar symptoms (drug rash, CMV, Clostridium enteritis). 
3.  To demonstrate that ST2 alone or the seven-biomarker panel measured at initiation of 
GVHD symptoms/therapy diagnose the severity of acute GVHD at onset and maximum. 
4.  To develop a repository of biospecimens linked to clinical data for future unspecified 
research. 
 
 
Eligibility: 
All patients receiving an allogeneic hematopoietic stem cell transplant, cord blood transplant, 
bone marrow transplant, T cell depleted marrow, donor lymphocyte infusion (DLI), or donor 
cellular infusion (DCI) can be included. 
Patients (or parent/guardian) must have signed the informed consent document and children 
who are at least 7 years of age must sign the assent document.  For subjects who turn 18 years 
of age during participation in this study, participation in the future use portion of the study after 
their 18th  birthday requires that they sign the informed consent statement following turning 18 
years of age. 
 
Accrual objective: Sample size: 430 

3 
 
Accrual period: The estimated accrual period is 24 months 
 
Study duration: Patients will be followed for 12 months following initiation of GVHD therapy or 
12 months post-HCT for non-GVHD patients. 
This study will not require any extra clinic visits beyond those required for routine clinical patient 
care. 
For subjects who consent to the future unspecified use of their biospecimens for research 
purposes, clinical data collected at any time post-transplant as part of routine clinical care may 
also be accessed. 
 
Governance of samples stored for future research: Samples stored for future research can 
only be released for use by investigators once they have received 1) approval from the Pediatric 
HCT Bio-repository Network (PHCTBN) governance committee and 2) IRB approval for a 
hypothesis driven research question. 

4 
 
TABLE OF CONTENTS 
 
1.  BACKGROUND AND RATIONALE 
1.1. 
Acute Graft-versus-Host Disease (GVHD) 
1.2. 
Therapy resistant GVHD 
1.3. 
Acute GVHD biomarker studies 
 
2.  STUDY DESIGN 
2.1. 
Rationale for study design and objectives 
2.2. 
Eligibility 
2.3. 
Accrual objective and period 
2.4. 
Study duration 
 
3.  STUDY ENDPOINTS 
3.1. 
Primary endpoint 
3.2. 
Secondary endpoints 
3.3. 
Clinical and histological correlatives 
3.4. 
Studies of GVHD Biomarkers 
 
4. PATI ENT ENROLLMENT AND EVALUATION 
4.1. 
Enrollment 
4.2. 
Study monitoring 
4.3. 
Clinical data submission 
4.4. 
Sample Processing 
4.5. 
Sample Storage and Tracking 
4.6. 
Sample Shipment 
4.7. 
HSCT Multi-Center Informatics Infrastructure 
4.8. 
Future Use Sample Analysis 
 
5.  GOVERNANCE 
 
6.  DATA AND SAFETY MONITORING PLAN 
 
7. Data Sharing 
 
 
8.  STATISTICAL CONSIDERATION 
 
8.1  Accrual 
 
8.2 Primary and Secondary endpoints 
 
 
9.  APPENDIX A: HUMAN SUBJECTS 
 
10.  APPENDIX B: INFORMED CONSENT 
 
11. APPENDIX C: STATISTICS 
 

5 
 
12. APPENDIX D: GVHD SCORING 
 
13. APPENDIX E: REFERENCES 
 
14. APPENDIX F: LABORATORY PROCEDURES 
 
15. APPENDIX G: CELLULAR BIOMARKER PANEL FOR VIRUS AND LEUKEMIA 
SPECIFICT CELL IMMUNE RECONSTITUTION   

6 
 
1.  BACKGROUND AND RATIONALE 
 
1.1.    Acute Graft-versus-Host Disease (GVHD) 
 
Allogeneic hematopoietic cell transplantation (HCT) is a potential curative therapy for 
cancers   of   the   blood   and   bone   marrow,   including   leukemias   that   represent 
approximately one third of all cancer cases occurring among children and the leading 
cause of cancer deaths in children1. Use of HCT has increased as new techniques have 
allowed for transplantation in patients who previously would not have been considered 
HCT candidates. Approximately 30,000 allogeneic HCTs will be performed worldwide in 
2020; 20% of allogeneic HCT are performed in the pediatrics population. However, the 
efficacy of this procedure has been impeded by frequent and severe graft versus host 
disease (GVHD). The development of GVHD is tightly linked to the beneficial graft 
versus tumor (GVT) effect, the most potent form of immunotherapy. Nowadays, acute 
GVHD is measured by clinical symptoms in three organ systems: the skin, liver and 
gastrointestinal (GI) tract2-4. Acute GVHD typically occurs between two and eight weeks 
after transplant, but may occur later4, and is often clinically indistinguishable from other 
causes of clinical symptoms such as conditioning regimen toxicity, infection, or 
medication. Endoscopic biopsy is often used to confirm the diagnosis2,5, but histologic 
severity on biopsy has not consistently correlated with clinical outcome3,5-7. A non- 
invasive, reliable blood biomarker specific for GVHD with strong prognosis information 
would thus significantly aid in the management of patients with this disorder. 
 
1.2.    Therapy resistant GVHD 
 
In addition, there are currently no therapies that target GVHD biomarkers, and no effort 
has been made to develop drugs specific for GVHD treatment. Thus far, because of the 
potentially overlapping pathophysiology, all drugs used to treat GVHD were originally 
designed for other diseases, particularly autoimmune disorders or loss of tolerance in 
organ transplantation. Some examples include etanercept and infliximab, which are 
anti-tumor necrosis factor (TNF) drugs approved to treat rheumatoid and psoriatic arthritis. 
Another example, mycophenolate mofetil, is an immunosuppressant used extensively in 
organ transplantation and autoimmune diseases. Some targets have been proposed 
based on preclinical models, but few have been implemented clinically, and none are 
based on patient-specific biomarkers. Steroids, which are general immunosuppressors,  
remain  the  first  line  of  treatment  for  patients  presenting  with GVHD  symptoms.  
However,  less  than  half  of  patients  treated  with  the  standard 
therapeutic regimen for acute GVHD (high dose steroids) have a sustained response8, 
and more severe GVHD is even less likely to respond to treatment9. Only the clinical 
response on day 14 is highly predictive of the response on day 28, and the day 28 
response most accurately estimates overall survival (OS)10. Once GVHD occurs, the most 
important predictor of long-term OS is the primary response to first-line treatment; patients 
who respond completely to treatment have low non-relapse mortality (NRM) equivalent to 
patients without GVHD, whereas patients with incomplete or no response have a NRM 
approaching 50%11. Thus, a major challenge for clinicians is to identify which patients will 
respond to current GVHD treatment and to design more efficient treatment regimens. The 
contribution of this study is expected to be significant, because 

7 
 
the identification of steroid-refractory GVHD biomarker panels at symptom onset has 
tremendous potential for impacting our ability to risk stratify patients before initiating 
GVHD treatment. It may also ultimately guide the intensity and duration of treatment 
and minimize the toxicity associated with chronic steroid administration. The ability to 
identify patients who will not respond to traditional treatment and who are at particularly 
high risk for morbidity and mortality could permit tailored treatment plans, such as 
additional immunosuppressive treatments for high-risk patients that may be more effective 
if introduced early. Equally important is the identification of low-risk patients who will 
respond well to treatment. These patients may tolerate a more rapid tapering of steroid 
regimens to reduce long-term toxicity, infections, and a loss of the graft versus leukemia  
effect.  Follow-up  marker  monitoring  in  high-risk  patients  could  also  help decide 
whether to taper the treatment. 
 
1.3.    Acute GVHD biomarker studies 
 
OVERVIEW. A major barrier to graft-versus-host disease (GVHD) research and treatment 
is that the diagnosis and prognosis of GVHD relies almost entirely on clinical symptoms 
(onset and maximum grade). No simple test for diagnosis and response to treatment of 
acute GVHD exists. We performed discovery and validation experiments to identify a 
panel of informative biomarkers with diagnostic and prognostic value for acute 
GVHD12. We recently identified a skin-specific GVHD marker, elafin with significant 
diagnostic and prognostic ability13. Using the same proteomics strategy, we also identified 
a gastro-intestinal specific GVHD marker, regenerating islet-derived 3-alpha with 
prognostic ability14. These studies have been among the first to apply proteomic 
technology to identify biomarkers for GVHD, yet blood tests were still not able to predict 
a patient’s response to treatment. We, therefore, embarked on a novel proteomics 
experiment as a discovery engine and ELISA for validation, we discovered that among a 
panel of seven biomarkers, ST2 (suppression of tumorigenicity 2 or interleukin-33 
receptor) measured at the initiation of GVHD treatment accurately predicted the day 28- 
non-response and the day 180-post treatment non-relapse mortality in a validation set 
of 381 patients, and outperformed all other biomarkers for discriminating responders from 
non-responders of GVHD therapy (Vander Lugt et al. ASH presentation). These seven 
biomarkers have not yet been validated prospectively in samples from different 
institutions. This study addresses 1) the use (at minimum) of the four-biomarker panel 
(interleukin-2-receptor-alpha, 
tumor 
necrosis-factor-receptor-1, 
interleukin-8, 
and 
hepatocyte growth factor)12, and the two newly identified skin-specific marker, elafin13, and  
gastro-intestinal  specific  marker,  regenerating  islet-derived  3-alpha14    for  a) 
diagnosis of acute GVHD, b) association with therapeutic responses and mortality, c) 
prediction of acute GVHD and 2) the use of ST2 for a) association with therapeutic 
responses and mortality, b) prediction of acute GVHD and non-response. Upon 
completion, these studies will likely result in novel biomarker panels that may facilitate the 
therapeutic decision making process for allogeneic HCT patients. 

8 
 
) 
( 
( 
) 
Sensitivity 
( 
) 
PRELIMINARY STUDIES. 
 
Graft-versus-Host Disease Biomarkers. In our 
initial approach to the identification of GVHD 
biomarkers, we screened patient plasma samples 
by  competitive  hybridization  to  arrays  of 
antibodies specific for 120 diverse proteins 
including 
acute 
phase 
reactants, 
cytokines, 
angiogenic factors, tumor markers, leukocyte 
adhesion molecules, metalloproteinases, and 
inhibitors of metalloproteinases.  We used array 
technology during the discovery phase, followed by  
sandwich  enzyme-linked  immunosorbent assay 
(ELISA) to quantitate the concentrations of 
individual proteins. Due to the limited quantity of 
plasma available for each patient, we used a 
sequential  ELISA  protocol  for  eight  potential 
 
 
 
Fig 
1. 
Individual 
Receiver 
Operating 
Characteristic curves (ROC) for IL-2Rα, 
TNFR1, HGF, IL-8 and the composite panel. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
( 
) 
1- Specificity 
biomarkers rather than multiplex techniques to improve detection and quantification of low 
abundance proteins. This approach identified and validated a four-protein biomarker panel 
with high specificity for GVHD diagnosis (Figure 1) and prognostic value that was 
published as a plenary paper in Blood12.  All four of the biomarkers identified with the 
antibody array approach to biomarker screening have been associated with GVHD in 
previous studies15-26. However, due to the size of our study, we provided  the first 
demonstration that these biomarkers 
Fig  2. Intact Protein Analysis System workflow 
are associated with GVHD clinical 
outcome  and  prognosis.  While  the 
antibody biomarker screen can be 
considered internally unbiased because 
each antibody is represented at an equal  
level,  the  inherent  limitation  of this 
approach is introduced by the availability 
and 
selection 
of 
antibodies. 
To 
overcome these limitations and create a 
broader, less biased approach, we 
utilized 
state-of-the-art 
proteomic 
technology that is straightforward, gel- 
free,   and   based   on   high-resolution 
mass spectrometry. This technique 
interrogates       complex      proteomes 
present in samples, such as plasma, by 
matching mass spectra to a sequence 
database for protein identification. To 
develop this approach, we collaborated 
with Samir Hanash at the University of 
Washington and embarked upon a second discovery study using Intact Protein Analysis 
System (IPAS). The primary goal of this new discovery phase was to find biomarkers 
present at the time of GVHD diagnosis that were specific for GVHD target organs. To 

9 
 
assess the validity of  this approach, we  limited our initial search to  the two most 
frequent GVHD targets (skin and GI tract).  We compared plasma pooled from 10 patients 
with skin-specific GVHD to 10 controls in the first IPAS run, and plasma pooled from 10 
patients with GI tract-specific GVHD to 10 controls in a second IPAS run. The criteria for 
inclusion in the discovery samples were 1) patients conditioned with a myeloablative 
regimen and 2) patients who experienced development of acute GVHD (grade II-IV) 
without other severe complications. The controls included patients without GVHD who 
were matched for age, intensity of the conditioning regimen, donor source (related versus 
unrelated), HLA match (matched versus mismatched), and time of sample acquisition. 
The workflow of IPAS is shown in Figure 2 and described here. IPAS analysis 
compared plasma pooled from 10 patients with GVHD versus plasma pooled from 10 
matched patients without GVHD. The pools were individually immunodepleted of the six 
most abundant plasma proteins (Albumin, IgG, IgA, transferrin, haptoglobin, and 
antitrypsin).  Intact proteins were then labeled on cysteine 
residues with acrylamide. The GVHD– pool received the light acrylamide isotope (12C 
acrylamide), and the GVHD+ pool received the heavy 13C-acrylamide isotope.  The two 
pools were mixed together for further analysis, and specimens were then subjected to a 
two-dimensional  protein  fractionation  where  anion-exchange  chromatography 
represents the first dimension of the protein fractionation. A total of eight pools were 
collected from the anion exchange chromatography and then subjected to a second 
dimension of separation by reversed-phase chromatography. Sixty fractions were 
collected during the run, corresponding to a total of 480 fractions for each experiment. 
Then, the 480 individual digested fractions from each reversed-phase run were re- pooled 
for a total of 96 reverse-phase pools and analyzed on a LTQ-FT mass spectrometer. 
Because protein digestion is performed at the last step, just before liquid 
chromatography–tandem mass spectrometry (LC-MS/MS) analysis, the term  “intact” 
protein analysis was used. The acquired spectra were automatically processed by the 
Computational Proteomics Analysis System for the identification of proteins with a false 
discovery rate of < 5%. The result was a reduced complexity of individual fractions 
subjected to analysis and identification of proteins with a range of concentrations spanning 
seven logs. This technique was therefore able to detect very low abundance 
proteins. 
 
For quantitative proteome analysis, we labeled each GVHD+ pool and matched GVHD– 
pool with differential isotopic labeling. Heavy and light isotopes label approximately 35% 
of proteins, and 834 proteins were confidentially quantified based on differential stable 
isotope labeling in the skin GVHD run. Of the 834 proteins, 66 were increased by at 
least a factor of two in GVHD plasma.  To sequentially refine the list of candidate proteins, 
we further selected the proteins based on their relationship to the tissue of origin (Human 
Protein Atlas [http://www.proteinatlas.org/]), information from published literature, 
pathway networks (MetaCore analytical suite version 4.7, GeneGO, Inc., St. Joseph, MI), 
and the availability of a sandwich ELISA. Fourteen candidates were expressed primarily 
in the skin. Elafin emerged as the lead biomarker candidate of skin GVHD at the time of 
clinical diagnosis because it is highly expressed in skin tissue and has been shown to be 
overexpressed in inflamed epidermis in diseases such as psoriasis. Furthermore, elafin 
is an epidermal proteinase inhibitor that is induced by 
TNF-α27, an inflammatory cytokine that activates host antigen presenting cells (APCs), 

10 
 
Elafin pg/ml 
which in turn activate the mature donor T cells that mediate GVHD pathophysiology 4. 
Concentrations of elafin were measured by ELISA in 492 allogeneic HCT recipients. Elafin 
concentrations were significantly higher in plasma from patients with skin-specific GVHD 
than in control samples from patients without GVHD, with GI-specific GVHD, or with non-
GVHD rash. In addition, elafin concentrations may have value not only as a noninvasive 
diagnostic test, but also as a prognostic marker as published recently in 
Science Translational Medicine13. This data provides a proof-of-principle demonstration 
that  biomarkers of  disease-related,  tissue-specific  changes  can be  detected  in  the 
plasma of patients. We used this approach as the basis for extension of the validation of 
plasma GI biomarkers. We identified Regenerating Islet-Derived 3-alpha (REG3α) as a 
biomarker of lower gastrointestinal GVHD and validated it in 2 independent sets totaling 
1014   patients   from   three   different   centers.   We   showed   that   plasma   REG3α 
concentration at the onset of GVHD diarrhea is the best biomarker (i) to differentiate 
GVHD diarrhea from non-GVHD diarrhea; and (ii) to provide important prognostic 
information, including eventual response to GVHD treatment and survival. We also 
showed that the addition of this marker to clinical stage and histologic grade leads to a 
more refined grading system and risk stratification of patients at the time of GVHD 
diagnosis. This was recently published in Blood14. 
 
Further,  we  first  validated  elafin  as  biomarker  of  resistance  to  GVHD  treatment 
(unpublished data).  As shown in Figure 3, we 
performed additional measurements at four 
weeks    post-treatment    and    correlated    the 
 
Fig 3.  Elafin concentrations at onset and 4- 
week post-treatment in CR and No CR 
patients (paired t-test) 
concentrations of elafin with the response to 
GVHD 
treatment. 
Patients 
with 
complete 
response (CR) at four weeks exhibited no rash 
symptoms and had concentrations of elafin in 
the range of patients without GVHD (white bar), 
whereas patients with persistent or progressive 
rash (No-CR) (30% of the population) had 
unchanged 
elafin 
concentrations. 
Likewise, 
measurements     of     TNFR1     concentrations 
10000 
 
8000 
 
6000 
 
4000 
 
2000 
 
0 
p<0.0001  
ns 
 
 
 
 
 
 
 
 
 
 
 
No GVHD 
Onset    CR    Onset No CR 
declined as compared to the onset in patients who 
had a complete resolution of GVHD symptoms 28. 
N 
277 
81 
54 
 
These studies have been among the first to apply proteomic technology to identify 
biomarkers for GVHD, yet blood tests were still not able to predict a patient’s response 
to therapy, particularly at GVHD onset, when risk-stratification is most beneficial. We 
hypothesized that biomarkers discovered with an intact proteomic analysis system (IPAS) 
approach that we have described above will discriminate between therapy responsive 
(resolution or improvement of GVHD by day (D) 28 post-therapy initiation) and  
unresponsive  (absence  of  complete  or  partial  response)  patients  and  predict survival 
in patients receiving GVHD therapy. 

11 
 
We first performed an IPAS comparing pooled plasma taken at D16 ± 5 post-therapy from 
10 responders (R) and 10 non-responders (NR). Ten candidate biomarkers with an NR/R 
ratio of > 1.5 in the IPAS were measurable by ELISA. We therefore measured 
concentrations in the 20 individual plasma aliquots. Five were significantly increased in 
NR vs. R, with an area under the receiver operator characteristic curve of ≥ 0.85 (ST2, 
IL1sRII, MIF, LYVE, and Lipocalin). These were then measured at therapy initiation 
(D0), with 6 previously validated diagnostic biomarkers of GVHD (IL2Rα, TNFR1, HGF, 
IL8, Elafin, a skin-specific marker, and Reg3α, a gut-specific marker) in plasma samples 
from a validation set of 381 patients with acute GVHD grade 1-4 at onset and treated with 
systemic steroids at the University of Michigan from 2000 to 2011. Preliminary analyses 
(not shown) determined that D0 measurements predicted D28 non-response and D180 
OS. HLA match (match vs. mismatched; Odds Ratio (OR) 1.5, p = 0.07), conditioning 
intensity (full vs. reduced; OR 1.7, p = 0.04), and GVHD onset grade (grade 
3-4 vs. grade 1-2; OR 2.2, p = 0.001) predicted day 28 non-response in univariate 
analysis, while age at transplant (≥ 55 years vs. < 55 years), donor (unrelated vs. related), 
and stem cell source (peripheral blood vs. bone marrow/cord) did not. After adjustment 
for the 3 clinical characteristics which predicted D28 response, multivariate analysis of the 
11 protein concentrations showed that 3 predicted D28 response (ST2, p 
= 0.001; IL1sRII, p = 0.07; and IL8, p = 0.03) and 7 predicted post-therapy D180 OS (ST2, 
p = 0.003; IL1sRII, p = 0.07; IL8, p = 0.05; Elafin, p = 0.06; MIF, p = 0.04; TNFR, p = 
0.03; and Reg3α, p = 0.002 in gut-GVHD subset). Using logistic regression, we examined 
the ability of both the 7 biomarkers and ST2 alone to predict for D28 non- response, 
as ST2 was the most significant marker in all previous analyses. ST2 is the IL33 receptor, 
a member of the IL1/ Toll-like receptor superfamily, which promotes a Th2-type     immune     
response     in 
diseases such as arthritis and asthma 
29. A high biomarker value was defined 
as  a  plasma  concentration  greater 
than 50% above the median value of the 
responders' group. A high panel was 
defined as having at least 5 of 7 high  
biomarkers.  Patients  with  high ST2 
levels were 2.6 times more likely not to 
respond to therapy independent of  the  
aforementioned  significant clinical   
characteristics   (p   <   0.001) while 
patients with a high panel were only  1.9  
times  more  likely  not  to 
respond (p = 0.004). Thus, only ST2 
measurement was used for further 
analyses. Because  ST2 concentrations 
correlated 
with 
response, 
we 
hypothesized that ST2 would    predict    
D180    non-relapse 
Fig 4. Risk stratification with ST2 and onset GVHD grade 
for day 180 NRM 
mortality (NRM), independent of GVHD onset grade, the strongest clinical predictor of 
NRM (20% for GVHD grade 1-2 vs. 50% for GVHD grade 3-4, Hazard ratio (HR) 3.0, p 

12 
 
< 0.001). NRM cumulative incidence curves and HR for the 4 risk categories of low ST2 
/ grade 1-2, low ST2 / grade 3-4, high ST2 / grade 1-2, and high ST2 / grade 3-4 are 
shown in Figure 4. Interestingly, patients presenting with high clinical grade and low 
ST2 had a good prognosis, suggesting that ST2 provides important prognostic information 
at initiation of therapy above the clinical grade. 
 
In conclusion, soluble ST2, the form measured by ELISA, is a decoy receptor that drives 
the   Th2   phenotype   toward   Th1,   a   mechanism   by   which   it   may   act   in   the 
pathophysiology of resistant GVHD. ST2 concentrations obtained at initiation of GVHD 
therapy significantly enhance the accuracy of outcome prediction independent of GVHD 
grade. Measurement of ST2 may allow for early identification of patients at risk for 
subsequent non-response and mortality, and may provide a promising target for novel 
therapeutic interventions. 
 
These six diagnostic biomarkers interleukin-2-receptor-alpha, tumor-necrosis-factor- 
receptor-1, interleukin-8, hepatocyte growth factor, elafin, regenerating islet-derived 3- 
alpha, and suppression of tumorigenicity 2 (ST2) have not been validated blindly in 
samples from different institutions. We therefore hypothesized that this seven GVHD 
biomarker panel, measured during the transplant course, would help diagnose GVHD 
from other post-HCT complications, discriminate between therapy-responsive and 
unresponsive patients and predict survival in samples prospectively obtained on a 
multicenter trial. 
 
Pediatric biomarkers.  As shown above, the majority of candidate biomarkers have 
been   studied   in   adult   population.   The   identification   and   adaptation   of   adult 
biomarkers, for those conditions that are similar in children and adults, is an efficient, 
expedient and economical way to advance knowledge in the use of biom arkers in 
pediatrics. Many biomarkers proposed or developed in pediatrics have not been 
subjected to the rigorous evaluation and validation process needed for their use in 
clinical trials. Cross-age studies of biomarkers, whether between children of differ ent 
ages or between children or adolescents and adults, can provide new insights in 
disease, response to treatment and toxic effects. There are a number of factors that may  
affect  the  usefulness  of  potential  biomarkers  and  surrogate  endpoints  in 
pediatrics. The marked difference in the natural history of some pediatric di seases 
when compared to adults must be taken into account when biomarkers that have 
been  developed  for  the  adult  population  are  used  in  pediatrics.  Following  HCT, 
younger age has been associated with lower GVHD incidence and severity as well as 
better response to treatment and therefore better overall outcomes30,31. In addition, 
children are undergoing development and hence are not a homogeneous population. 
The amount or activity of many biomarkers used in pediatrics varies according to age 
and development; examples include beta-2 microglobulin, and host immunologic 
markers such as CD4 counts. Following HCT, one major difference is the developmental 
stage of the thymus. Furthermore, the usual small sample sizes characteristic of 
pediatric trials may also prevent the validation of surrogate endpoints 
thus multicenter trials are necessary. 

13 
IL-2R  (pg/ml) 
ELAFIN (pg/ml) 
REG3a (ng/mL) 
However, the majority of candidate biomarkers that are associated with a specific 
mechanism relevant to disease progression have been studied in adults.  Advances 
in -omics technology provide a plethora of analytical tools to discover and analyze 
mechanism- and disease-specific biomarkers for risk stratification and potential drug 
development. Translation of innovative systems approaches such as genomics, 
proteomics  and  metabolomics  into  a  developmental  framework  has   not  been 
exploited to understand mechanisms of GVHD treatment efficacy and toxicity in children. 
 
Thus, we have further analyzed our previous GVHD biomarkers focusing solely on 
pediatric  patients  as  this  population  represents  approximately  20%  of  our  total 
allogeneic HCT population. These preliminary data are the ground for further validation 
in samples prospectively obtained on a multicenter trial. We determined that there is a 
statistically significant increase in all four proteins included in the panel (Figure 5A, IL- 
2R as a representative), elafin, a skin-specific GVHD marker (Figure 5B), and REG3α, 
a gastro-intestinal (GI) specific GVHD marker (Figure 5C), based on the presence or 
absence of GVHD in both pediatric (age ≤ 21 yr) and adult (age > 21 yr) HCT recipients. 
 
 
Figure 5: A. IL-2Rα concentrations; B.  Elafin concentrations; C.  Reg3α concentrations, in pediatric 
and adult HCT patients with and without acute GVHD. 
A 
B 
C 
 
 
2500 
 
2000 
 
1500 
 
Peds 
Adult 
  p<0.001   
  p<0.001   
 
  
 
 
10000 
 
8000 
 
6000 
 
Peds 
Adult 
  p=0.001   
  p<0.001   
 
 
500 
 
400 
 
300 
Peds 
p = 0.002 
Adult 
p < 0.0001 
 
1000 
 
4000 
  
 
 
200 
 
500 
 
0 
 
 
n=43 
n=46 
n=180 
n=215 
2000 
 
0 
 
 
n=43 
n=46 
n=179 
n=213 
100 
 
0 
 
 
n=79 
n=25 
n=399 
n=187 
 
GVHD 
- 
+ 
- 
+ 
GVHD 
- 
+ 
- 
+ 
GI GVHD - 
+ 
- 
+ 
We also examined the novel biomarker of unresponsiveness to GVHD treatment: 
suppressor of suppression of tumorigenicity 2(ST2) in the pediatric population. 
Interestingly, the clinical response to GVHD treatment is different in children as compared 
to adults because 37% of children who are non-responders at day 28 post- initiation of 
therapy will be alive at day 180 post-therapy whereas all adults who are non- responders 
at day 28 post-initiation of therapy will be dead at day 180 post-therapy suggesting that, 
as opposed to the adult population, in the pediatric population day 28 post-initiation of 
therapy is not a good surrogate for day 180 post-therapy mortality. Not surprisingly, we 
therefore did not observe a significant difference in onset ST2 concentrations between 
responders and non-responders at day 28 post-initiation of therapy (Figure 6A). However, 
we still observe a significant higher onset ST2 concentrations in patients who will die at 
day 180 post-therapy (Figure 6B). 
 
 
Figure 6:   A.   GVHD onset ST2 concentrations in pediatric and adult responders and non- 
responders at day 28 post-therapy; B.  GVHD onset ST2 concentrations in pediatric and adult, alive 
or dead at day 180 post-therapy. 

14 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Therefore, we feel that analysis of biomarkers in pediatric patients and comparison with 
adults is not only appropriate, but essential in order to further our understanding of this 
disease in this vulnerable patient population. Upon completion, these studies will result 
in novel biomarker panels that may facilitate the therapeutic decision-making 
process for allogeneic HCT patients. The ability to identify patients at high risk for 
GVHD early in their transplant and treatment course has important therapeutic 
consequences,  including  more  stringent  monitoring  and/or  preventative  care. 
This analysis may also offer new key insights into the biology of GVHD and identify 
novel pathways and proteins with potential as therapeutic targets. 

15 
 
2.  STUDY DESIGN 
 
2.1.      Rationale for study design and objectives 
 
For this study, blood samples will be collected on a calendar basis: Day -7, day 7, day 
14, day 21, day 28, day 56, day 100, day 180 and day 360 post-HCT, as well as at 
appearance of clinical symptoms suggestive of acute GVHD and that trigger treatment 
with systemic steroids. A saliva sample will also be collected at the day-7 and day +56 
time-points from subjects with a HCT white blood cell count of <400/L to provide an 
additional specimen for DNA extraction.  Samples will be processed at local sites and 
will then be shipped to the Indiana University Simon Cancer Center (the Data and 
Specimen Coordinating Center) from the other five participating Blood and Marrow 
Transplant Programs (Dana-Farber Cancer Institute and Boston Children's Hospital, 
Johns Hopkins Sidney Kimmel Comprehensive Cancer Center, Fred Hutchinson Cancer 
Research Center, Texas Children’s Hospital, and Children's National Medical Center). 
The appropriate samples will be transported to Dr. Paczesny’s laboratory for analysis of 
a seven-protein biomarker panel. The results from previous discovery and validation 
studies suggested that the panel of six biomarkers can confirm the diagnosis of acute 
GVHD in patients at onset of clinical symptoms of acute GVHD and provide prognostic 
information independent of acute GVHD severity. Furthermore, suppression of 
tumorigenicity 2 measured at initiation of therapy can predict day 28-non-response and 
the day 180-post treatment non-relapse mortality in patients with GVHD treated with at 
least systemic corticosteroids. ST2 outperforms all previously identified biomarkers for 
prognostic information. The plasma protein biomarker panel will include at minimum: 
interleukin-2-receptor-alpha, tumor-necrosis-factor-receptor-1, interleukin-8, hepatocyte 
growth  factor,  elafin,  regenerating  islet-derived  3-alpha  and  suppression  of 
tumorigenicity 2. 
 
Primary Objective: The primary objective is to confirm that ST2 alone or the seven- 
biomarker panel measured at initiation of GVHD therapy predict a) D180 post-therapy 
non-relapse mortality; b) D28 post-therapy non-response, and c) GVHD grade 1-4 
onset. 
 
Secondary Objective: 
1.  To demonstrate that ST2 alone or the seven-biomarker panel measured at day 14 
or day 21 post-HCT (or a combination of these time points) predict D180 post- 
HCT non-relapse mortality. 
2.  To  demonstrate  that  ST2  alone  or  the  seven-biomarker  panel  measured  at 
initiation of  GVHD symptoms/therapy diagnose  acute  GVHD as compared  to 
other complications presenting with similar symptoms (drug rash, CMV, Clostridium 
enteritis). 
3.  To  demonstrate  that  ST2  alone  or  the  seven-biomarker  panel  measured  at 
initiation of GVHD symptoms/therapy diagnose the severity of acute GVHD at onset 
and maximum. 
4. To develop a repository of biospecimens linked to clinical data for future unspecified 
research. 

16 
 
 
 
2.2. 
Eligibility 
 
All patients receiving an allogeneic hematopoietic stem cell transplant, cord blood 
transplant, bone marrow transplant, T cell depleted marrow, donor lymphocyte infusion 
(DLI), or donor cellular infusion (DCI) can be included. 
 Patients (or parent/guardian) must have signed the informed consent document 
 
2.3. Accrual objective and period 
 
 Accrual objective: Sample size 430 
 
 Accrual period: The estimated accrual period is 24 months 
 
2.4. 
Study duration: Patients will be followed for 12 months following initiation of 
GVHD therapy or 12 months post-HCT for non-GVHD patients. 
 This study will not require any extra clinic visits beyond those required for routine 
clinical patient care. 
 For subjects who consent to the future unspecified use of their biospecimens for 
research purposes, clinical data collected at any time post-transplant as part of 
routine clinical care may also be accessed. 
 
 
 
3.  STUDY ENDPOINTS 
 
3.1.    Primary endpoint: The primary objective is to confirm that ST2 alone or the 
seven-biomarker panel measured at initiation of GVHD therapy predict a )  
D180 post-therapy non-relapse mortality; b) D28 post-therapy non-response, and c) 
GVHD grade 1-4 onset. 
 
 
 
3.2. 
Secondary endpoints: 
1.  To demonstrate that ST2 alone or the seven-biomarker panel measured at day 
14 or day 21 post-HCT (or a combination of these time points) predict D180 post- 
HCT non-relapse mortality. 
2. To demonstrate that ST2 alone or the seven-biomarker panel measured at initiation 
of GVHD symptoms/therapy diagnose acute GVHD as compared to other 
complications presenting with similar symptoms (drug rash, CMV, Clostridium 
enteritis). 
3. To demonstrate that ST2 alone or the seven-biomarker panel measured at initiation 
of GVHD symptoms/therapy diagnose the severity of acute GVHD at onset and 
maximum. 
 
3.3. 
Clinical and histological correlatives: 
 
 Pre-transplant characteristics 
Age at transplant, gender, race and ethnicity, Disease diagnosis, disease risk 
(CIBMTR classification), intensity of conditioning, conditioning regimen, donor source, 

17 
 
HLA-match, stem cell source, other significant pre-HCT diagnoses and clinical 
characteristics 
 
 GVHD prophylaxis 
 
 Infectious prophylaxis, if any 
 
 Onset Acute GVHD: day of onset, skin stage, liver stage, gastrointestinal stage 
and overall grade. 
 
 Maximum Acute GVHD: day max, skin stage, liver stage, gastrointestinal stage 
and max overall grade. 
 
 Biopsies: 
o - yes - No, 
o location skin, upper GI, lower GI, liver 
o GVHD yes or no, other diagnostic, specify 
o This will help identify the incidence of other complications presenting with 
GVHD symptom that is not GVHD (e.g. infectious diarrhea, drug rash…) 
 
 Systemic therapy 
o Start day 
o Steroids 2 mg/kg/day  Yes 
No 
o Other first line 
 
 Proportion of complete response (CR), Partial response (PR), mixed response, No 
response, and progression at day 28 post-therapy (among surviving patients) 
 
 Incidence of additional therapy of GVHD given at day 28, and day 56 post- 
therapy (including for GVHD flare). 
 
 Non-relapse mortality (NRM) at day 180 post-therapy for GVHD patients and 
NRM at day 180 post-HCT for patients without GVHD. 
 
 Overall survival, GVHD-free survival at day 180 and 360 post-therapy for GVHD 
patients and NRM at day 180 and 360 post-HCT for patients without GVHD. 
 
 Relapse at day 360 post-HCT. 
 
 Incidence of systemic infections related to GVHD treatment. 
 
 Incidence of chronic GVHD at day 180 and day 360. 
 
 For subjects consenting to future unspecified research, data collected at any 
time-point post-transplant as part of routine clinical care may be accessed for future 
studies. 
 
 

18 
 
 
Staging and grading of acute GVHD 
 
In this study we will classify patients by a modified Keystone grading 
schema (see below). 
 
 
Table 1: Acute GVHD Staging 
Stage 
Skin 
Liver 
(bilirubin) 
Lower GI (stool 
output/day) 
Upper GI 
0 
No rash 
 2 mg/dl 
Adult: < 500 ml/day 
Child: < 10 ml/kg/day 
No protracted nausea and 
vomiting 
1 
Maculopapular rash 
< 25% BSA 
2.1-3 mg/dl 
Adult: 500–1000 ml/day 
Child: 10 -19.9 ml/kg/day 
Persistent severe nausea, 
vomiting with a positive 
upper GI biopsy 
2 
Maculopapular rash 
25 – 50% BSA 
3.1-6 mg/dl 
Adult: 1001-1500 ml/day 
Child: 20 – 30 ml/kg/day 
3 
Generalized erythroderma 
(>50% BSA) 
6.1-15 mg/dl 
Adult: >1500 ml/day 
Child: > 30 ml/kg/day 
4 
Generalized erythroderma 
(>50% BSA) plus bullous 
formation and 
desquamation > 5% BSA 
>15 mg/dl 
Severe abdominal pain 
with or without ileus, 
or grossly bloody stool or 
melena (regardless of 
stool volume) 

For GI staging: 
- 
The “adult” stool output values should be used for patients > 50 kg in 
weight. 
- 
Use 3 day averages for GI staging based on stool output. If stool 
and urine are mixed, stool output is estimated to be 50% of total 
stool/urine mix. 
 
Table 2: Overall Acute GVHD Clinical Grade: 
Grade 0: 
No stage 1-4 of any organ 
Grade I: 
Stage 1-2 skin and no liver or gut involvement 
Grade II: 
Stage 3 skin, or Stage 1 liver, or Stage 1 GI 
Grade III: 
 Stage 0-3 skin, with Stage 2-3 liver, or Stage 
2-3 GI Grade IV: 
 
Stage 4 skin, liver or GI

 
Response 
Definitions: 
 
Complete Response (CR) is defined as a GVHD grade of 0 in all evaluable 
organs. For a response to be scored as CR at day 28, the patient must still be in 
CR on that day and have had no intervening additional therapy for an earlier 
progression, PR or NR. 
Partial Response (PR) is defined as improvement in one or more organs involved 
with GVHD symptoms without progression in others. For a response to be scored 
as PR at day 28, the patient must still be in CR on that day and have had no intervening 
additional therapy for an earlier progression, PR or NR. 

19 
 
 
Mixed Response (MR) is defined as improvement in one or more organs with 
deterioration in another organ manifesting with GVHD symptoms or development 
of GVHD symptoms in a new organ. 
 
Progression is defined as deterioration in at least one organ without any 
improvement in others. 
 
No response (NR) is defined as absence of any improvement or progression as 
defined. Patients receiving secondary therapy (including need to re-escalate steroid dose 
to ≥ 2.5 mg/kg/day of prednisone or equivalent 2 mg/kg/day of methylprednisolone will be 
classified as non-responders. 
 
Proportion of complete response (CR), Partial response (PR), mixed response (MR),  
progression,  and  no  response  (NR)  at  day  28  post-therapy  (among surviving 
patients). Scoring of CR, PR, MR, progression, and NR are in comparison to the patient’s 
acute GVHD status on day 0 of systemic therapy for GVHD. 
 
Incidence of additional systemic GVHD therapy given at day 28, and day 56 post- 
therapy (including for GVHD flare). 
 
Flares are defined as any progression of acute GVHD after an initial response (e.g. earlier  
CR  or  PR)  that  requires  re-escalation  of  steroids  or  initiation  of  additional systemic 
therapy for GVHD. 
 
Steroid dose at day 28 and day 56 post-GVHD therapy 
Dose of methylprednisolone will be converted to prednisone by multiplying the 
methylprednisolone dose by 1.25. Prednisone dose for each patient will be converted to 
mg/kg. The cumulative prednisone dose for each patient at day 28 will be calculated by 
adding the doses (end of each week’s dose) for each of the four weeks of treatment, 
divided by the number of days of survival during this interval. The prednisone dose for 
each patient at day 28 and day 56 will be recorded. 
 
Chronic  GVHD  is  defined  per  the  BMT  CTN  manual  of  procedures  chapter  2. 
Symptoms of  chronic GVHD if present will be reported using the GVHD symptom 
record. The incidence of chronic GVHD at 6 and 12 months will be computed. 
 
Non-relapse mortality (NRM) at day 180 post-therapy for GVHD patients and NRM 
at day 180 post-HCT for patients without GVHD will be computed. The events for 
NRM are death due to any cause other than relapse of underlying malignancy. 

20 
 
Overall  survival,  GVHD-free  survival  at  day 180  and  360 post-therapy for  GVHD 
patients and at day 180 and 360 post-HCT for patients without GVHD will be computed. 
 
Relapse at day 360 post-HCT will be computed. 
 
Incidence of systemic infections related to GVHD treatment through day 180 post- 
therapy for GVHD patients and day 180 post-HCT for patients without GVHD will be 
computed. All microbiologically documented infections (bacterial and fungal) or significant 
infections requiring antibiotic/antifungal therapy occurring within 6 months of initiation of 
therapy for GVHD patients or 6 months post-HCT for no-GVHD patients will be reported 
by site of disease, date of onset, and severity. For definitions, see the BMT CTN manual 
of procedures. 
 
Incidence of severe viral infections related to GVHD treatment through day 180 
post-therapy and in no-GVHD patients will  be recorded (EBV-PTLD, CMV viremia, 
HHV6 encephalitis…). 
 
3.4.    Studies of GVHD Biomarkers 
 
If consent for research samples is obtained, blood samples will be collected on Day -7, 
day 7, day 14, day 21, day 28, day 56, day 100, day 180, and day 360 post-HCT, as 
well as at appearance of clinical symptoms suggestive of acute GVHD and that triggers 
systemic steroid treatment.  These samples will be analyzed for the seven-protein 
biomarker panel. Remaining plasma sample aliquots will be stored for future biomarker 
studies. Additional, whole blood collected from the recipient on day 56 post-HCT 
(representing the donor DNA) and day -7 whole blood or saliva DNA (if white blood cells 
count < 400/μL) (representing recipient DNA) will be collected, frozen and stored for future 
genomic studies. Donor chimerism results recorded at the time nearest to each sample 
collection will be noted.  Please note all sample collection days are targets with collection 
of samples on the closest business day to the suggested day being acceptable. 
 
4.  PATIENT ENROLLMENT AND EVALUATION 
 
 
4.1.    Enrollment 
 
Patients will be registered for this trial using a web-based database that will be utilized for 
collection of all clinical and laboratory data. 
 
Follow this procedure: 
1.  An  authorized  user  at  the  clinical  center  completes  the  initial  screening  by 
entering patients’ demographics. 
2.  If the patient signs the informed consent and assent (if applicable), a study 
number is generated. 
3.  A visit schedule based on enrollment date is displayed for printing. 

21 
 
Subjects enrolled when they are less than 18 years of age will be re-
consented at the first office visit following their 18th birthday.  Data and 
specimens collected from these subjects prior to their 18th birthday may 
continue to be used without this additional consent; however, no clinical 
data or specimens from after the 18th birthday may be collected or utilized 
until the re-consenting process has been completed. 
 
 
4.2. 
Study monitoring 
 
The follow-up schedule is outlined in Table 3 (see below). 
 
 
 
 
Table 3: Required assessments 
 
All allogeneic HCT, day pre/post-HCT GVHD   + Day post-
GVHD 
systemic 
therapy
Chimerism 
-7 7 
14 21 28 56 100 180 360 Onset (0) 28 
56 
If Standard of 
Care in Center 
Biopsy of involved tissue 
(as  part  of  routine  clinical 
care, not research) 
 
 
 
 
 
 
 
 
 
 
X 
 
 
 
Acute GVHD evaluation ^ 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
X 
 
Chronic GVHD evaluation ^ 
 
 
 
 
 
 
X 
X 
X 
 
 
 
 
Steroid dose 
 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
X 
 
Other  GVHD  medications, 
doses, 
and 
drug 
levels 
collected as part of clinical 
care 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
Blood Samples* 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
Saliva  DNA 
(if  WBC  < 
400/µL) 
 
X  
 
 
 
 
X 
 
 
 
 
 
 
 
Chimerism (% donor) ‡ ** 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
*Up to day 28, sample collection may be ±4 days from the target day to 
allow for scheduling flexibility, holidays, etc… 
*Day 100,180, and 360, sample collection will be collected at the clinic visit 
closest to the scheduled time point to allow for scheduling flexibility, holidays, 
etc… 
 
*GVHD onset sample will be collected within a 48 hour window of systemic 
treatment initiation if subject is inpatient 
 
‡ Donor chimerism results recorded at the time nearest to each sample 
collection will be noted. 
 
^ Refer to Appendix D for GVHD assessment forms 
** Will only collect data if already being done as standard of care at center 
 
The timing of follow-up visits is based on the date of BMT and then on the 

22 
 
date of onset of GVHD for post-GVHD assessments. 
 
Assessments 
 
- Baseline weight 
- 
For  all,  complete  acute  GVHD  staging  and  grading  information  
including assessment of rash, diarrhea, nausea/vomiting, liver function and 
weight at day 
7, 14, 21, 28, 56, 100, and 180 post-HCT 
- 
If GVHD or GVHD-like symptoms, 
o recommended  biopsy  of  involved  tissue  (this  is  standard  of  care,  
not research) 
o Complete   acute   GVHD   staging   and   grading   information   including 
assessment of rash, diarrhea, nausea/vomiting, liver function and 
weight at onset of symptoms and 28 and 56 days post-systemic GVHD 
therapy 
o Steroids dose weekly until day 56 
o Samples for laboratory studies at day -7, 7, 14, 21, 28, 56,100 and 
180 
post-HCT and at onset of GVHD symptoms, and 28 days post-
systemic 
GVHD therapy 
- 
Chronic GVHD evaluation at day 100, 180 and 360 
- 
Infections requiring therapy up to 360 days post-HCT 
 
 
 
Sample Collection 
 
Samples will be collected at a time when clinical samples are being collected as 
well if possible. That is, this protocol will NOT require an additional procedure or 
for a patient’s central venous line to be accessed at a time when these things are 
not being done as part of routine clinical care. 
 
Blood will be collected as part of this study for use in evaluation of biomarkers of 
HSCT-related toxicities and outcomes and future unspecified research. 
 
 Blood  will be collected using the schedule detailed above: day -7 and 
the following post-transplant days: 7, 14, 21, 28, 56, 100, 180, and 
360. Please note that the above blood draw schedule is a target only. 
Subjects will never be required to come for a clinic visit specifically to 
have 
samples collected for the purposes of this study. As such, sample 
time- points are targets and samples will be collected at the time 
closest to the target time when collection is feasible. In addition, 
during inpatient periods, sample collection will take place on the 
business day closest to 
the day scheduled per protocol (to allow for flexibility around weekends 
and holidays). 
 

23 
 
 Blood will also be collected at onset of acute GVHD and at day 28 
after initiation of steroid therapy. 
 
  In addition, blood will be collected at the onset of other changes in 
status, which may be clinically significant (such as suspected 
sinusoidal obstruction syndrome, respiratory distress, relapse, etc.). 
 
 Blood volume to be collected:  
 
 
Please note cumulative collection volumes at various points in 
 
 
time of participation are summarized in the table below and are 
 
 
detailed for patients >40 kg in text immediately below for clarity. 
 
 Patients > 40 kg: up to 15 mL per time point) 
 
o 9 scheduled collections in year one = 135 mL 
o 2 scheduled collections in patients who develop acute   
 
GVHD = 30 mL 
 
o Maximum additional blood draws at the time of 
developing other complications will be set at four. 
 
 9 scheduled collections + 2 additional 
collections GVHD collections (if applicable) + 
maximum of four additional collections with 
other complications = 15 collections maximum 
= maximum blood draw of 225 mL. 
 
 
 

24 
 
 
 
Table 4: Summary of maximum blood volume to be collected for this study 
 
Patient Size on Enrollment 
 
> 40 kg 
25-40 kg 
10-25 kg 
<10 kg 
Maximum volume of each scheduled blood draw 
15 mL 
10 mL 
7.5 mL 
5 mL 
Maximum volume of blood of each blood draw if 
develops acute GVHD 
15 mL 
10 mL 
7.5 mL 
5 mL 
Maximum volume of blood with each complication 
15 mL 
10 mL 
7.5 mL 
5 mL 
Maximum volume for scheduled blood draws (9 
collections) 
135 mL 
90 mL 
67.5 mL 
45 mL 
Maximum blood volume if develops acute GVHD (2 
collections) 
30 mL 
20 mL 
15 mL 
10 mL 
Maximum blood volume for other complications (4 
collections) 
60 mL 
40mL 
30 mL 
20 mL 
Maximum volume collected in year one (scheduled 
plus collections with complications) 
225 mL 
150 mL 
112.5 
mL 
75mL 
*Cumulative blood volume will be monitored for each subject and individual blood 
draws will be decreased as needed to maintain annual cumulative blood collection of 
<10% of total blood volume. 
 
Also note, for pediatric patients, we expect patient weights to increase over time due to 
normal growth and as such, updated patient weights will be used to re-calculate 
permitted blood volume collections every six months. 
 
 
 
GVHD scoring 
 
Acute GVHD 
A sample Acute GVHD data record for use in this trial is attached below. 

25 
 
 
 
Today’s Clinical Acute GVHD Assessment 
Today’s date   
 
Patient ID   
  
Karnofsky/Lansky   
 
Codes 
 
 
Differential diagnosis 
0 
1 
2 
3 
4 
GVHD  Drug   Cond  Infect  TPN   VOD 
Other 
Rxn 
Reg 
Skin            □ □ □ □ □ % body rash:         _    □    □    □   □ 
Liver           □ □ □ □ □ Current bili:                  □    □    □   □   □  □              Lower 
GI     □ □ □ □  □ Vol:                              □    □    □   □   □                   Upper GI  
□ □                                  □    □    □   □   □                   Systemic 
agents:             □ CSA               □ Tacrolimus                   □ Infliximab         □ Daclizimab 
□ Pentostatin □ Sirolimus 
□ Etanercept 
□ MMF 
□ Prednisone □ Methylprednisolone 
□ Ontak 
□ Other   
 
Current steroid dose: Prednisone                             mg/kg/day;   Methylprednisolone                          mg/kg/day 
Has the steroid dose been increased to ≥ 2.5 mg/kg/day of prednisone (or 2 mg/kg/day methylpred)? 
□YES   □ NO 
If yes, date dose increased                   ; reason for dose increase: □GVHD flare □ Other (specify)                      
Topical agents:  Skin topical steroids: □ YES □ NO; Non-absorbed oral steroids (e.g. Budesonide, Entocort) 
□ YES □ NO 
Chronic GVHD: Does the patient have evidence of chronic GVHD?  □ YES □ NO 
Codes 
Skin 
Liver (bilirubin) 
Lower GI (stool output/day) 
Upper GI 
0 
No rash 
 2 mg/dl 
Adult: < 500 ml/day 
Child: < 10 ml/kg/day 
No protracted nausea and 
vomiting 
1 
Maculopapular rash 
< 25% BSA 
2.1-3 mg/dl 
Adult: 500–1000 ml/day 
Child: 10 -19.9 ml/kg/day 
Persistent severe nausea, 
vomiting with a positive 
upper GI biopsy 
2 
Maculopapular rash 
25 – 50% BSA 
3.1-6 mg/dl 
Adult: 1001-1500 ml/day 
Child: 20 – 30 ml/kg/day 
3 
Generalized erythroderma 
(>50% BSA) 
6.1-15 mg/dl 
Adult: >1500 ml/day 
Child: > 30 ml/kg/day 
4 
Generalized erythroderma 
(>50% BSA) plus bullous 
formation and desquamation 
> 5% BSA 
>15 mg/dl 
Severe abdominal pain with or 
without ileus, or grossly bloody 
stool or melena (regardless of 
stool volume) 
Signature   
 
Date   
 

26 
 
Chronic GVHD 
 
Patients developing symptoms of chronic GVHD will have their symptoms recorded on 
the chronic GVHD scoring form (Appendix D) at the scheduled follow-up visits: 
 
Table 4: Definite and Possible Manifestation of Chronic GVHD* 
Organ system 
Definite manifestations of chronic 
GVHD 
Possible manifestations of chronic 
GVHD 
Skin 
Scleroderma (superficial and fasciitis), 
lichen planus, vitiligo, scarring 
alopecia, hyperkeratosis pilaris, 
contractures from skin immobility, nail 
bed dysplasia 
Eczematoid rash, dry skin, 
maculopapular rash, 
hyperpigmentation, hair loss 
Mucous Membranes 
Lichen planus, non-infectious ulcers, 
corneal erosions, non-infectious 
conjunctivitis 
Xerostomia, keratoconjunctivitis sicca 
GI tract 
Esophageal strictures, steatorrhea 
Anorexia, malabsorption, weight loss, 
diarrhea, abdominal pain 
Liver 
None 
Elevation of alkaline phosphatase, 
transaminitis, cholangitis, 
hyperbilirubinemia 
GU 
Vaginal stricture, lichen planus 
Non-infectious vaginitis, vaginal 
atrophy 
Musculoskeletal/serosa Non-septic arthritis, myositis, 
myasthenia, polyserositis, 
contractures from joint immobilization 
Arthralgia 
Hematologic 
None 
Thrombocytopenia, eosinophilia, 
autoimmune cytopenias 
Lung 
Bronchiolitis obliterans 
Bronchiolitis obliterans with organizing 
pneumonia, interstitial pneumonitis 
*From BMTCTN MOP chapter 2 
 
Pediatric considerations 
 
Children with chronic GVHD can present with skin, GI, eye or systemic disease leading to failure 
to thrive, persistent immunodeficiency, or pulmonary disease. Chronic “eczema’ or dry skin are 
common manifestations of mild chronic GVHD of skin. Liver disease can present as asymptomatic  
hyperbilirubinemia  or  elevated  alkaline  phosphatase.  As  children  can  have marked elevations 
in alkaline phosphatase during growth spurts, children with this laboratory abnormality should 
have a fractionated alkaline phosphatase to confirm that the band is from the liver and not from 
bone before a diagnosis of chronic GVHD is made. Children with chronic GVHD of the upper GI 
tract may have chronic anorexia or poor growth due to malabsorption. Rather than losing weight, 
children may “fall off” their growth curve with decreased gains in height or weight velocity. In 
children < 2 years of age, this may also affect growth of head circumference.  Chronic  GVHD  
may  also  cause  persistent  low  or  intermediate immunodeficiency. In addition to increasing the 
risk of opportunistic infection, this may cause GI dysfunction and lead to malabsorption, low IgG 
(due to protein losing enteropathy), lactose deficiency, and poor growth. Duodenal intubation with 
quantitation of pancreatic enzymes is useful in these children. If pancreatic enzymes are low, 
children may benefit from supplementation of pancreatic enzymes. 
 
See Appendix D for a sample of chronic GVHD data record for use in this trial. 

27 
 
Summary of Required Assessments 
 
 For all subjects 
 
o complete   acute   GVHD   staging   and   grading   information   including 
assessment of rash, diarrhea, nausea/vomiting, liver function and weight 
at day 7, 14, 21, 28, 56, 100, and 180 post-HCT 
 
o Chronic GVHD evaluation at day 100, 180 and 360 post-HCT (please refer 
to Appendix D for GVHD assessment forms) 
 
o Infections requiring therapy up to 360 days post-HCT 
o Baseline weight 
o Blood samples for laboratory studies and future use at day -7, 7, 14, 21, 
28, 56,100,180, and 360 post-HCT 
 
 
 
 For subjects who develop GVHD or GVHD-like symptoms, 
 
o recommended  biopsy  of  involved  tissue  (this  is  standard  of  care,  not 
research) 
 
o Complete   acute   GVHD   staging   and   grading   information   including 
assessment of rash, diarrhea, nausea/vomiting, liver function and weight 
at onset of symptoms and 28 and 56 days post-systemic GVHD therapy 
 
o Steroid dosing information weekly until day 56 after initiation of steroids 
o Blood samples for laboratory studies and future use at onset of GVHD 
symptoms and 28 days post-systemic GVHD therapy initiation 
 
 
 
4.3.    Clinical data submission 
 
4.3.1. Source documents for all required evaluations and assessments should 
be stored at local sites for all GVHD evaluations (both acute and chronic). 
All clinical data required for this study will be input at local sites into the 
web-based database developed specifically for the Pediatric HCT Bio- 
repository Network.  All clinical information should be updated on subjects 
a least monthly through 1-year post-HCT. 
 
4.3.2. Source documents for 10-15% of required assessments and data will be 
requested for submission to IUSCC for data control evaluations. 
 
 
 
4.4.    Sample Processing 
 
After collection, samples will be processed, aliquoted, and frozen utilizing the standard 
operating procedures developed by the Paczesny laboratory and the Indiana CTSI 
Specimen Storage Facility (described below in the section entitled “Storage”). SOPs for 
sample  processing  established  by  the  Paczesny  laboratory  will  be  followed  for  all 

28 
 
processing procedures.   A manual for sample processing for this study will be provided 
to all participating sites. These can also be provided when requested and during audits. 
 
4.5.    Sample Storage and Tracking 
 
A global unique identifier is randomly assigned to each sample and derivative. Samples 
of the same person can only be linked by authorized persons who have access to the 
data  in  the  database.  All  sample  data  will  be  organized  and  stored  in  the 
SpecimenTrack 
 
system 
 
from  
 
Remedy 
 
Informatics 
(http://www.remedyinformatics.com/product/specimentrack/). 
A  series  of  biobanking 
electronic forms will be created to track the collection, inventory, and distribution of 
HSCT biosamples. Since  SpecimenTrack can be readily integrated with the HSCT 
Registry, the samples will always be associated with their clinical, lab, and genotypic 
annotations. Both the HSCT Registry and SpecimenTrack will be deployed in a HIPAA 
aligned  environment and  supported  by staff  trained  in  HIPAA best  practices.  Both 
systems  are  enterprise  level  systems  with  powerful  and  granular  control  of  user 
permissions,  roles,  and  administration.  The  HSCT  systems  will  be  supported  by 
members of the Indiana CTSI informatics team, who are experienced with deploying, 
maintaining, 
and 
providing 
user 
support 
for 
systems 
such 
as  REDCap 
(https://en.wikipedia.org/wiki/Redcap_%28Research_Electronic_Data_Capture%29). 
Both  Remedy  systems,  MosaicBuilder  and  SpecimenTrack,  are  web  applications 
accessible through a web browser using HTTPS, thus allowing both local and distant 
collaborators to use the system in an encrypted and manner. 
 
The Pediatric HCT Bio-repository Network samples will be stored in the Indiana Biobank 
in affiliation with the Indiana CTSI Specimen Storage Facility (SSF).  The Indiana CTSI 
manages a Specimen Storage Facility dedicated to sample storage in accord with 
International  Society  of  Biologic  and  Environmental  Repositories  (ISBER)  Best 
Practices and protocol defined sample parameters.   The SSF is located in the new 
Wells Children’s Clinical Research Center in Riley Children’s Hospital. 
 
The SSF is directed by Lilith Reeves, CSO, Indiana CTSI, and operates under the Indiana 
CTSI Specimen Storage Facility Advisory Committee, Ken Cornetta, Chair. Quality 
Compliance oversight is provided by Anne Kaiser, Cincinnati Children’s Hospital Medical 
Center (CCHMC) Office of Research Compliance and Regulatory Affairs (ORCRA).  The 
approaches and day-to-day activities of the SSF are managed by QA reviewed SOPs 
organized into the following chapters: 
 
 Administration and Quality Oversight: SOP SF-1-1 through SOP SF-1-13. This 
collection of procedures defines maintenance of SOPs, Scope of Services, 
Organization of the SSF, Management of the SSF, Training, Controlled Document 
Management, Safety Quality Management, Deviation Management, Out-of-
Specification 
Management, 
Audit 
Management, 
Commissioning/ 
Validation/ 
Revalidation policies and Housing for Good Laboratory Practices.  The central 

29 
 
approach of the SSF is to provide the sample storage controls for PI owned collections 
rather than to manage the protocols under which the specimens are collected and 
released.  These SOPs control all procedures associated with specimen intake and 
management while the specimens are located in the SSF. 
 
 Facility  Management: 
SOP  SF-2-1  through  SOP  SF-2-4. 
The  four  facility 
systems within the SSF that are critical to ensuring storage compliance with ISBER 
and specimen quality are Mechanical Freezer Storage Room Controls, Liquid 
Nitrogen System  and Freezer Room  Controls, Controlled Access and Alarm 
Management and Response.   These SOPs define procedures for monitoring and 
responding to situations within the facility critical to ensuring sample integrity. 
 Equipment Management:  SOP SF-3-1-SOP SF-3-9.  All equipment in the SSF is 
maintained, monitored, and function verified according to strict specifications included 
in the SOPs for Refrigerators and Freezers, Liquid Nitrogen Freezers, Pipettes, 
Centrifuges, Biological Safety Cabinets, Timers, Thermometers, Personal O2 
monitors, and Emergency Escape Breathing Apparatus to be used in the event of 
a large-scale liquid nitrogen release event.   These SOPs are written to ensure that 
equipment is used, calibrated, and generally maintained to support quality specimen 
management. 
 Specimen Processing: SOP SF Chapter 4. This group of SOPs defines the 
technical steps for supporting storage of the multiple collections according to the 
defined parameters of each. This chapter of SOPs is ever evolving with the over- 
arching SOPS within the chapter being SOP SF-4-1 Managing Requests for Sample 
Support and SF-4-4 Managing Sample Receipt, Log-in and Tracking. 
 These SOPs are available on-line at IndianaCTSI.org/ictsissf/sops or can be 
provided for review by contacting the SSF Director (Lilith Reeves) or Chair of the SSF 
Advisory Committee (Kenneth Cornetta, MD).  The Indiana CTSI SSF is a CTSI 
Designated Core Laboratory signifying that operating policies and charges are 
reviewed at least annually by the CTSI Translational Technology Resources (TTR) 
core oversight committee.  All personnel in the SSF who have access to PHI have 
completed human subjects training. 
 
4.6.    Sample Shipment 
 
Samples should be shipped Monday through Thursday via Fedex overnight delivery 
every six months.  Samples must be shipped on dry ice to the following address: 
 
Pediatric HCT Biorepository Network 
Wells Children’s Clinical Research Center 
Attention: Lindsey Elmore or Jenny Then 
705 Riley Hospital Dr. RI 2606, Room 2641 
Indianapolis, IN 46202-5225 
 
 

30 
 
Bill sample shipment costs to the following Indiana University account number provided. 
Contact the following individuals prior to all shipments: 
Lindsey Elmore, Operations Manager: lhelvaty@iupui.edu 
Jenny Then, Core Coordinator: jeglassb@iu.edu 
 
4.7.    HSCT Multi-Center Informatics Infrastructure 
 
Data obtained during this clinical trial will be collected at six cancer centers: SKCCC, 
DFCI, FHCRC, IUSCC, TXCCC and CNMC.  Since this proposal involves a very large 
amount of data to be collected from a multi-center clinical trial as well as a large amount 
of proteomic data, we have put procedures in place to ensure the integrity of the data 
transmitted and to allow consistent handling of the clinical data at all sites. The clinical 
and  laboratory data  collected  will  be  organized  and  stored  in  the  HSCT  Registry, 
constructed 
using 
the 
MosaicBuilder 
platform 
from 
Remedy 
Informatics 
(http://www.remedyinformatics.com/mosaic-platform/mosaic-builder-edition-for-it- 
professionals/). Data will either be imported directly from the Cerner Electronic Medical 
Record system, exchanged using flat files, or entered manually into the system using 
electronic forms. The Mosaic enterprise level platform is deployed and maintained as a 
service by the Indiana Clinical Translational Sciences Institute, and enables the creation 
of biobanks that are integrated with clinical, lab, genotypic, and phenotypic data. This 
platform is engineered to support complex workflows, and is deployed in a HIPAA 
aligned environment such that the server, database, and application, are located at 
secured locations and supported by professional staff trained in HIPAA best practices. 
The Mosaic platform is also ontologically aware so that the semantic definition of the 
data  can  be  standardized.  This  further  enables  meaningful  data  integration  and 
querying. All of these factors ensure that the HSCT Registry will be a secure, stable, 
robust and extensible research informatics platform for the future. 
 
4.8.    Future Use Sample analysis 
One objective of this protocol is sample and clinical data collection for future unspecified 
research.  It is anticipated that some of the sample analyses may be carried out prior to 
specific research questions or projects using the advanced diagnostic techniques listed 
below; however, the data generated using these analyses will only be released for data 
analysis once there is IRB approval for the investigation of a specific hypothesis-driven 
research question. 
 
The sample analyses that may be carried out as part of this protocol may include, but 
are not limited to: 
 
    Genomics 
    Proteomics 
    Cytokine analyses 
    Flow cytometry 

31 
 
    Metabolomics 
 
These analyses will generally be performed at one of the six participating sites in the 
PHCTBN.  On occasion, the analyses of research samples will involve collaborators 
at other institutions in order to have access to the most technologically advanced or 
cost effective techniques. All samples analyzed at another institution as part of this 
project will maintain the bar code assigned to the subject on enrollment in this study. 
This process allows for use of a unique identifier that the principal investigator of this 
study may use to link the data to other subject information but eliminates the possibility 
of transmission of privileged health information. Samples analyzed at another center 
will generally be entirely consumed by the research procedures. Any residual will be 
destroyed at the end of this project or returned to the Indiana University storage facility. 
 
 
 
5.  GOVERNANCE 
The PHCTBN is managed by the PIs (Paczesny and Renbarger) and the PHCTBN 
Steering Committee (Duncan, Cooke, Carpenter, Jacobsohn, Bollard, Krance, and 
Leung).  In order for samples/information to be shared with researchers, an IRB-approved 
protocol will need to be provided and the Steering Committee in conjunction with the PIs 
will make determinations about allocation of samples/information to researchers. 
 
 
6.  DATA AND SAFETY MONITORING PLAN  
 
A meeting of the Data and Safety Monitoring Board was held on June 1, 2015.  The  Data  
Safety Monitoring Board consisted of DSMB Chair, Stella Davies, MD, Wade Clapp, MD, 
Scott Baker, MD, and Paul Szabolcs, MD.  The board reviewed and discussed the 
protocol.  It was decided that since this is a low-risk, non-interventional study, it does not 
require a DSMB according to the NIH guidelines.  Therefore, the DSMB has been 
eliminated. 
 
Data and Safety will continue to be monitored by the study Principal Investigators. 
 
7.  DATA SHARING 
Indiana University School of Medicine (IUSM) as well as all the participating institutions are 
committed to supporting the NIH Statements on Sharing Research Data and on Availability 
of Research Results: Publications, Intellectual Property Rights, and Sharing Research 
Resources. These Universities have used a variety of means as appropriate and 
expeditious to share data resulting from sponsored projects with colleagues, such as 
depositing data on a secure web-accessible data warehouses or arranging distribution of 
data, reagents, targets, and protocols to other researchers using established mechanisms 
and repositories. IUSM, and all participating institutions are signatory to the Uniform 
Biological Materials Transfer Agreement and will use the simple letter agreement to 
distribute appropriate research materials to the research community. The availability of 
data sharing will be publicized by individual investigators in publications and presentations. 

32 
 
 
The Universities will assure the timely release and sharing of data no later than the 
acceptance for publication of the main findings from final datasets, and will protect the 
rights and privacy of human subjects who participate in NIH-sponsored research by 
redacting all identifiers, and adopting other strategies to minimize risks of unauthorized 
disclosure of personal identifiers in accordance with authorization and consent documents. 
The Universities agree that data sharing is essential for expedited translation of research 
results into knowledge, products, and procedures to improve human health. 
 
In addition, for this proposal, data from future research studies that may include, but are 
not limited to Genomics, Proteomics, and Metabolomics will also be shared through the 
same mechanism. These analyses will generally be performed at one of the six 
participating sites in the Pediatric HCT Bio-repository Network (PHCTBN). On occasion, 
the analyses of research samples will involve collaborators at other institutions in order to 
have access to the most technologically advanced or cost effective techniques. All samples 
analyzed at another institution as part of this project will be maintain the bar code assigned 
to the subject on enrollment in this study. This process allows for use of a unique identifier 
that the principal investigators of this study may use to link the data to other subject 
information but eliminates the possibility of transmission of privileged health information. 
Samples analyzed at another center will generally be entirely consumed by the research 
procedures. Any residual will be destroyed at the end of this project or returned to the 
Indiana University storage facility. 
 
With respect to licensing patented inventions developed through this project, the 
Universities expect to elect title to inventions made with federal funds by their investigators, 
as appropriate under the Bayh-Dole Act, and in accordance with the NIH policy. The 
Universities understand that the NIH encourages the filing of patent applications on unique 
research resources if doing so will aid in the prompt commercialization of diagnostic, 
prognostic or therapeutic products. Since institutional ownership of such inventions may 
be of concern to collaborators, especially those who are the source of proprietary 
biomarkers, reagents, and/or technologies, the Universities will develop agreements with 
third party collaborators that assure them both adequate patent coverage and opportunities 
to license such patent rights, as appropriate. This agreement will be implemented in a 
manner that does not restrict research use by the scientific community, both nonprofit and 
for profit, but promotes and facilitates their active involvement in this project. 
 
Genomic Studies 
DNA from patients might be used in high-throughput genomic studies (including potentially 
genome-wide association studies or sequencing) for a future project.  
We plan to deposit both the phenotype and genotype data obtained from the work 
proposed in this project into the NIH genotype and Phenotype Database that is managed 
by the National Center for Biotechnology Information (NCBI).  The NIH genotype and 

33 
 
Phenotype Database collects well curate data (clinical and genetics). All the data planned 
for submission will be deidentified/HIPAA compliant and has been properly consented with 
IRB approvals.   
 
We also plan to release the data to qualified researchers who wish to collaborate with the 
investigators involved in this proposal. The data will be available through a secure FTP site 
maintained by the University’s IT department, or sent by encrypted physical media via 
trackable mail.  
Sharing Model Organisms 
N/A 
 
 
8.  STATISTICAL CONSIDERATION 
 
7.1.    Accrual 
 
Accrual will be monitored within each center with the expectation that the enrolled 
patient population is representative of the transplanted patient population at each 
center. Representation will be examined by comparing gender, race, ethnicity and age 
distributions. Accrual of minority patients will be expected to be in proportion to the 
number of minorities patients transplanted at each center. 
 
7.2.    Primary and Secondary Endpoints 
 
Appendix C will detail sample size and statistical analysis to be performed 

34 
 
9.  APPENDIX A: HUMAN SUBJECTS 
 
Subject consent: Candidates for the study will be defined as patients receiving an 
allogeneic hematopoietic stem cell, cord blood, or bone marrow transplant, or T cell depleted 
marrow can be included. The principal investigator or his/her designee at each transplant 
center will contact the candidates and enroll them onto the study. The study coordinator 
at each center will provide the patient with information about the purpose of the study and 
obtain consent. The PIs of the study will provide a template of the consent form to each 
center that can be customized according to their local requirements and submit it for 
review by the local Internal Review Board (IRB). Each center must provide evidence of 
IRB approval. 
 
Confidentiality: Confidentiality will be maintained by individual names being masked and 
assigned a patient identifier code. The code relaying the patient’s identity with the ID 
code will be kept separately at the center. The ID code will be transmitted to the center 
that keeps all the clinical data (separate from the center that keeps the biological data). 
 
Participation of women, children, minorities and other populations: Women, children 
and ethnic minorities will be included in this study. 
 
Accrual will be monitored within each center with the expectation that the enrolled 
patient  population  is  representative  of  the  transplanted  patient  population  at  each 
center. Representation will be examined by comparing gender, race, ethnicity and age 
distributions. Accrual of minority patients will be expected to be in proportion to the number 
of minorities patients transplanted at each center. 

35 
 
10.  APPENDIX B: INFORMED CONSENT TO PARTICIPATE IN RESEARCH 
 
 
Informed Consent to Participate in Research 
 
 
Study Title: Bridging Pediatric and Adult Biomarkers in Graft-Versus-Host Disease 
 
The word “you” in this consent form, may mean either you or your child; “we” means the 
doctors and other staff. 
 
 
 
WHAT IS THE PURPOSE OF THIS STUDY? 
 
You are invited to participate in a research study called “Bridging Pediatric and Adult 
Biomarkers in Graft-Versus-Host Disease.”  It is a study to collect specimens and 
information about patients who receive a bone marrow or stem cell transplant or a donor 
lymphocyte infusion (DLI) to help us to better understand graft-versus-host disease and 
to do future research aimed at understanding more about your disease, stem cell 
transplant and complications that people have after their transplants.  You are about to 
undergo a treatment with chemotherapy and/ or radiotherapy followed by infusion of bone 
marrow-replenishing stem cells or donor lymphocyte cells for the treatment of your 
disease. The purpose of this study is to collect data and blood from patients 
undergoing this type of treatment. We will code data by assigning a number to each 
individual patient and then store the data by that number. No mention of patient identities 
is made in analyses of the data or in any publications, which result from such research. 
 
If you decide to join the study, your blood will be collected and stored. These samples 
will be used for measurement of proteins in blood that might help us predict which patients 
will develop GVHD and for future research. 
 
We will enroll a total of 430 children and adults to this study and biobank. 
 
This consent form will tell you about the purpose of the research, its benefits and 
minimal risks, and your right as a participant in the study. Please take your time to make 
your decision. 
 
Everyone who takes part in research at [insert your institution] should know that: 
 
 Being is any research study is voluntary. 
 You may or may not benefit from being in the study. Knowledge we gain from this 
study may benefit others. 
 If you join the study, you can quit at any time. 
 If you decide to quit the study, it will not affect your care. 
 Please ask the study staff questions about anything that you do not understand, 
or if you would like to have more information. 

36 
 
 You can ask questions now or anytime time during the study. 
 Please take the time you need to talk about the study with your doctor, study 
staff, and your family and friends. It is your decision to be in the study. If you 
decide to join, please sign and date the end of the consent form. 
 
This study does not change the therapeutic standard of care and therefore has 
no more than minimal risk for you. 
 
 
 
WHAT WILL PARTICIPATING IN THIS STUDY INVOLVE? 
 
You are being asked to allow collection of data regarding your transplant to be entered 
into our database. We may collect information from your hospital chart on multiple 
occasions.  As  we  collect  this  database  of  information,  it  can  become  a  valuable 
resource to help us in improving the health and outcomes of subjects receiving a 
transplant. One way to do this is to use results that are in the past medical record (such 
as your clinical lab tests, physical exams, vital signs, for example) to investigate how those 
results may be associated with better or worse outcomes or what complications develop. 
At this time, we do not know which results may hold the key to gaining a better 
understanding. This may change as we gain more knowledge.  The study doctors will 
also collect information related to your treatment. This can include any information 
about your disease, your past medical history, your stem cell transplant course, and 
how you do long term.  This information can be collected from your medical records or 
from the NAME OF YOUR INSTITUTION database.  The investigators will keep track of 
what medicines you receive and how well those medicines work for you and whether 
you have side effects from the medicines. 
 
In addition, you are being asked to allow collection of 15 ml (3 tsp or 1 tablespoons) of 
blood (less for children or adults under 40 kg of body weight—see table below) on multiple 
occasions. This blood will be collected at 9 different times before and after your transplant. 
You will have a total of approximately 9 tablespoons collected throughout the study.  Blood 
will be collected specifically for this study to test biomarkers of GVHD and to use for future 
research.  We may use this blood to run certain tests on the blood to use for future 
research aimed at developing a better understanding of stem cell transplant complications 
and outcomes that certain patients have.  Doctors involved in this study may use some 
of these samples to do certain tests on the blood in order to learn more about 
complications and outcomes that people have after getting stem cell transplants, how to 
best predict which patients are at risk for a complication before it happens, and how to 
diagnose certain complications that are sometimes hard to diagnose.  Samples and 
information will only be released to someone doing a study like this if they have approval 
from the Institutional Review Board (IRB) to do the study. 
 
The specific times of the blood draws will be prior to your transplant and at the following 
times after your hematopoietic stem cell transplant: 
 
 Day 7 

37 
 
 Day 14 
 
 Day 21 
 
 Day 28 
 
 Day 56 
 
 Day 100 
 
 Day 180 
 
 Day 360 
 
All of these planned time-points are approximate and will always be coordinated with a 
time when you are having blood drawn as part of your routine clinical care.  Also blood 
samples will be obtained on the occasion of major complications (or events), or of other 
important clinical findings and their follow up after transplant. Examples of major 
complications might be onset of blood stream infection, onset of veno-occlusive disease 
of the liver, onset of graft-versus-host disease (GVHD), placement of a tube into the airway 
to help breathing, or at the time of relapse. If you develop GVHD, we will collect two 
additional blood samples: at the onset of symptoms that require treatment initiation and 
28 days after treatment begins. The reason that it is important to collect blood samples at 
times like that are that sometimes certain things may be changing in your body around 
these times that could cause changes in the levels of certain chemicals, cells, or other 
things that might help us to better understand how to predict or prevent complications 
from happening.  In this study, we can collect samples like this up to six times (including 
the two additional samples for GVHD).  After collecting the samples, we can do any of the 
following: 
 
 Process and freeze the samples to use for GVHD biomarker test and for future 
unspecified use 
 
 Process and do certain tests on the samples.  We would save this information for 
future research questions about stem cell transplant that investigators ask to do 
and have approval to do in the future. It is sometimes important to do tests like this 
and save the information because some important tests have to be done on fresh 
samples and cannot use samples that have been frozen 
 
The amount of blood you will have collected depends on how much you weigh.  The 
table below summarizes the volume of blood we plan to collect depending on your size. 
If you have a central line or port a cath in place as part of your regular care, blood will 
be drawn from your central line or port a cath.  A central line (central venous catheter) is 
a special type of tubing inserted into a large vein in the chest by a surgeon during a 
short operation. The central line is used to give chemotherapy drugs and to withdraw 
small amounts of blood for testing during treatment. 
 
We  will  collect  a  sample  of  your  saliva  by  asking  you  to  spit  into  an  ORAGENE 
collection kit on day -7 and/or day +56 of your transplant if your total white blood counts 

38 
 
< 400/microliter so that we can use the saliva to extract DNA for genetic tests in the 
future if needed. 
 
 
 
 
 
Table 5: Summary of maximum blood volume to be collected for this study 
 
Patient Size on Enrollment 
 
> 40 kg 
25-40 kg 
10-25 kg 
<10 kg 
Maximum volume of each blood draw 
3 tsp 
2 tsp 
1.5 tsp 
1 tsp 
Maximum volume of scheduled blood draws (9 
collections) 
27 tsp 
18 tsp 
14  tsp 
9 tsp 
Maximum volume of blood if develops acute GVHD 
(2 collections) 
6 tsp 
4 tsp 
3 tsp 
2 tsp 
Maximum volume of blood with complication (4 
collections) 
12 tsp 
8 tsp 
6 tsp 
4 tsp 
Maximum volume in year 1 for scheduled blood 
draws (maximum of 15 collections) 
45 tsp 
30 tsp 
22.5 tsp 
15 tsp 
 
 
Additionally, information about you and your disease and stem cell transplant course 
and complications will continue to be collected forever. It will be stored indefinitely and 
only the investigators for this study will have access to it. 
 
You have the choice of allowing us to draw and store your blood samples and to use them 
for future transplant-related research. The samples may be used up or destroyed without 
your consent and the samples may be used after your death without further consent of 
your estate or legal representative. Future use of your stored sample for genetic studies 
will always require the review and approval of the Institutional Review Board. 
 
I have read the description of this part of the study and willingly give my consent to 
participate in the collection of blood samples from me for use in the future to study my 
disease, hematopoietic stem cell transplant, complications and outcomes of 
hematopoietic stem cell transplant, and other complications of treatment. 
 
Yes   
 or No   
 
  
Initials 
 
Weekly Health Evaluation 
We will evaluate your health every week for 8 weeks after your transplant. If you 
develop acute GVHD, we will also evaluate your health every week for 8 weeks once 
you start your GVHD treatment. Each health evaluation will include tests to evaluate your: 
 GVHD stage and grade 
 Ability to do daily activities (performance status) 

39 
 
 Blood  counts,  liver  and  kidney  function,  and  level  of  GVHD  prevention 
medications in your blood (if it applies to you) 
 Development of any infection 
 
These tests and how often they are scheduled are standard care for patients receiving an 
allogeneic HCT and/or with acute GVHD and would be done even if you were not part 
of this study. 
 
Monthly Health Evaluation 
We will evaluate your health at approximately 3, 6 and 12 months after your transplant. 
Each health evaluation will include tests to evaluate your: 
 GVHD stage and grade 
 Ability to do daily activities (performance status) 
 Blood  counts,  liver  and  kidney  function,  and  level  of  GVHD  prevention 
medications in your blood (if it applies to you) 
These evaluations must be done at the transplant center. Other exams and tests may 
be done at the transplant centers or your local doctor’s office at the discretion of your 
transplant doctor. 
 
These tests and how often they are scheduled are standard care for patients receiving an 
allogeneic HCT and/or with acute GVHD and would be done even if you were not part 
of this study. 
 
 
WHAT BENEFITS MAY YOU EXPERIENCE FROM THIS STUDY? 
 
This study will not directly benefit you.  By taking part in this study you may help us to 
improve treatment for hematopoietic stem cell transplant patients in the future. 
 
WHAT RISKS OR DISCOMFORTS ARE INVOLVED? 
 
Blood Draw Side Effects: 
 
Risks  associated  are  slight,  but  some  possible  risks  include:  excessive  bleeding, 
fainting or feeling lightheaded, bruising, and infection (a slight risk any time the skin is 
broken). To decrease the risk, blood will be drawn by trained personnel and will always 
be taken at a time when blood is being drawn for other tests your study doctor has 
ordered.  You will not have to have an IV placed specifically for this study. 
 
Loss of Confidentiality (personal information): 
 
There is a risk of loss of privacy. All the genetic information gathered in this study will be 
used together with information from other subjects. Your name or other personal health 
information will not be used in the information collected from the study or in publications. 
All your personal and medical data will be considered confidential. All of the information 
collected on you will be coded (codes used to be unable to identify the personal health 
information). Only research staff will have access to the samples.  Only the study doctor 
in charge of this study will be able to access the coded information. 

40 
 
This research follows the Genetic Information Nondiscrimination Act (GINA), a federal law 
which generally makes it illegal for health insurance companies, group health plans, and 
most employers to request the genetic information we get from this research and 
discriminate against you based on your genetic information. 
 
 
 
WHAT FINANCIAL COSTS ARE INVOLVED IN PARTICIPATING? 
 
Costs associated with your care, such as laboratory tests, x-rays, scans, clinic visits, 
medications,  and  hospitalizations,  are  all  considered  a  part  of  standard  care  for 
someone with your illness; therefore you will be responsible for these costs. 
 
Lab tests or procedures required for the research, which are not considered a part of 
standard care for your illness, and the costs for these specified tests will be paid for by 
the study. 
 
 
 
ARE THERE ALTERNATIVES TO PARTICIPATION? 
 
You have the option to choose not to participate in this study. 
 
Refusal to participate in this study or stopping participation will not change the medical 
care you will receive. 
 
 
 
WHAT COMPENSATION IS AVAILABLE IF YOU HAVE A COMPLICATION OR ARE 
INJURED BY PARTICIPATION? 
 
In  the  event  of  physical  injury  resulting  from  your  participation  in  this  research, 
necessary medical treatment will be provided to you and billed as part of your medical 
expenses.   Costs not covered by your health care insurer will be your responsibility. 
Also, it is your responsibility to determine the extent of your health care coverage. 
There is no program in place for other monetary compensation for such injuries. However, 
you are not giving up any legal rights or benefits to which you are otherwise entitled. 
 
 
 
WHAT COMPENSATION IS AVAILABLE FOR PARTICIPATION IN THIS STUDY? 
 
There will be no monetary compensation for participation in this study. 
 
 
 
WHO MAY YOU CALL WITH QUESTIONS ABOUT YOUR PARTICIPATION IN THIS 
STUDY? 

41 
 
For  questions  about the  study or  a  research-related  injury,  contact  the  researcher 
XXXX, MD at XXXX.  If you cannot reach the researcher during regular business hours 
(i.e. 8:00AM-5:00PM), please call the Human Subjects office at XXXXX.  After business 
hours, please call XXXX and ask the operator for the Stem Cell Transplant physician on 
call. 
 
In the event of an emergency, please contact your local medical emergency response 
team by dialing 911.  If you need assistance that is not an emergency, you can always 
reach the study doctor by calling the hospital operator at XXXX or ask for the Stem Cell 
Transplant physician on call. 
 
 
 
For questions about your rights as a research participant or to discuss problems, 
complaints or concerns about a research study, or to obtain information, or offer input, 
contact the Human Subjects office at XXXX. 
 
Additional information is available at the National Cancer Institute’s Cancer Information 
Service at 1-800-422-6237 or visit the NCI’s web sites with a computer: 
 
 Cancer Trials: comprehensive clinical trials information 
http://cancertrials.nci.nih.gov 
 Cancer Net: accurate cancer information 
http://cancernet.nci.nih.gov 
 
 
MAY YOU WITHDRAW FROM THE STUDY? 
 
Furthermore, if you decide you no longer want your blood to be used as part of the 
research, you will always have the right to request that samples no longer be used and 
that any remaining blood be destroyed.   If you choose to have your blood destroyed 
please contact your study doctor in writing  insert name and address of site PI. 
 
Your participation in this study is entirely voluntary.  If you choose not to participate in 
this study, this will in no way interfere with your care.  The same high quality medical 
care will be given to you no matter what your decision might be.  Likewise, you may 
discontinue participation in this study at any time without altering the quality of care 
given to you.  You may also seek treatment from another physician of your choice.  New 
information that may relate to your willingness to participate will be provided to you. 
 
Your participation in this study may be terminated by the investigator for any of the 
following reasons: 
 
A.  If you fail to follow the investigator's instructions. 
 
B.  If you experiences a serious and unexpected side effect that may require evaluation. 

42 
 
C.  If the investigator feels it is in the best interest of your health and welfare. 
 
 
 
WILL YOUR STUDY RECORDS BE KEPT CONFIDENTIAL? 
 
 
 
Efforts will be made to keep your personal information confidential.   We cannot guarantee 
absolute confidentiality.   Your personal information may be disclosed if required by law.  
Your identity will be held in confidence in reports in which the study may be published. 
 
Organizations that may inspect and/or copy your research records for quality assurance 
and data analysis include groups such as the study doctor and his/her research 
associates, the Institutional Review Board or its designees, and (as allowed by law) 
state or federal agencies, specifically the Office for Human Research Protections (OHRP) 
and the National Institutes of Health (NIH). 

43 
 
SUBJECT’S STATEMENT OF CONSENT 
 
I/My child have read the description of this study and have also talked it over with 
my/my child's study doctor to my satisfaction.  I willingly give my consent (to allow my 
child) to participate in this study.  I/My child will receive a copy of the consent to keep for 
future reference. 
 
If the subject is 18 years of age or older: 
 
 
 
Subject’s Printed Name:  
 
 
 
 
Subject’s Signature:  
Date:  
 
 
 (must be dated by the subject) 
 
 
 
 
 
 
 
 
If the subject is 17 years of age or younger: 
 
 
 
Printed Name of Parent:  
 
 
 
 
Signature of Parent:  
Date:  
 
 
 
 
 
 
 
This must be completed: 
 
 
 
Printed Name of Person Obtaining Consent:  
 
 
 
 
Signature of Person 
 
 Obtaining Consent:  
Date:  
 

44 
 
 
Health Insurance Portability and Accountability Act 1 (HIPAA1) Authorization to 
use and disclose individual health information for research purposes 
 
 
A. Purpose 
As a research participant, I authorized the principal investigators and the 
researcher’s staff to use and disclose my individual health information for the 
purpose of conducting the research study: 
Title: Bridging Pediatric and Adult Biomarkers in Graft-Versus-Host Disease 
 
B. Individual health information to be used or disclosed 
My  individual  health  information  that  may  be  used  or  disclosed  to  do  this 
research includes: 
 Demographic information (for example: date of birth, sex, weight) 
 Medical history (for example: diagnosis) 
 Findings from physical exams 
 Laboratory test results  obtained at the time of  the work up and after 
treatment (for example: blood tests, biopsy results) 
 
C. Parties who may disclose my individual health information 
The researcher and the researcher’s staff may collect my individual health 
information from: (list hospitals, clinics and providers from which health care 
information can be requested). 
 
D. Parties who may receive or use my individual health information 
The individual health information disclosed by parties listed in item C and 
information disclosed by me during the course of the research may be received 
and used by the following parties: 
 
Principal investigators and researcher’s staff 
Dr. Sophie Paczesny 
Dr. Jamie Renbarger 
Study sponsors 
U.S. government agencies that are responsible for overseeing research  such as 
the Office of Human Research Protection (OHRP) 
 
U.S. government agencies that are responsible for overseeing public health 
concerns such as the Center for Disease Control (CDC) and federal, state and 
local health departments. 
 
E. Right to refuse to sign this authorization 

45 
 
I do not have to sign this authorization. My decision not to sign this authorization 
will not affect my treatment, payment, enrollment in health plans or eligibility for 
benefits. 
 
F.  Right to revoke 
I can change my mind and withdraw this authorization at any time by sending a 
written notice to [the principal investigator at your transplant center] to inform the 
researcher of my decision. If I withdraw this authorization, the researcher may 
only use and disclose the protected health information already collected for this 
research study. No further health information already about me will be collected 
or disclosed to the researcher for this study. Any unused blood will be destroyed 
if you withdraw your permission. 
 
G. Potential for Re-disclosure 
My  individual  health  information  disclosed  under  this  authorization  may  be 
subject to re-disclosure outside the research study and no longer protected. 
Examples include potential disclosures for law enforcement purposes, mandated 
reporting or abuse or neglect, judicial proceedings, health oversight activities and 
public health measures. 
 
H. This authorization does not have an expiration date. 
 
Title: Bridging Pediatric and Adult Biomarkers in Graft-Versus-Host Disease 
 
 
 
I have read and understood this consent form. The nature and purpose of the 
research study has been explained to me. I understand that I will be given a copy 
of this signed Consent Form. 
 
 
 
 
Participant Name 
 
 
 
 
 
Participant Signature                                                                                                            Date 
 
 
 
 
I certify that I have provided a verbal explanation of the details of the research 
study, including the procedures and risks.  I believe the participant has understood 
the information provided. 

46 
 
 
Counseling Physician 
 
 
 
 
 
Counseling Physician Signature 
Date 

47 
 
Assent to participate in research 
 
XXXX University Assent To Participate in Research 
 
Bridging Pediatric and Adult Biomarkers in Graft-Versus-Host Disease 
 
We are doing a research study.  A research study is a special way to learn about 
something. We are doing this research study because we are trying to find out more 
about patients who receive a bone marrow or stem cell transplant or donor 
lymphocyte infusion (DLI) and complications that people have after their transplants. 
We would like to ask you to be in this research study. 
 
Why am I being asked to be in this research study? 
 
You are being asked to be in this research study because you are about to have 
chemotherapy and/or radiation followed by a bone marrow or stem cell transplant or DLI. 
One thing we want to try to do is find a blood test we can use to diagnose acute Graft-
Versus-Host Disease (GVHD). You are invited to join this research because you might 
develop acute GVHD. We want to learn 
if a blood test will predict patients who will have worse acute GVHD following stem 
cell transplant.  Acute GVHD is common in patients who have stem cell transplants. 
It happens when the new cells attack and damage your tissues such as the skin, 
gut and liver. 
 
What will happen during this research study? 
 
We want to tell you about some things that might happen if you are in the study.  This 
study will take place at XX Hospital.  Blood will be collected specifically for this study 
to use for future research about your disease, stem cell transplant, and problems people 
have after stem cell transplant.   The study doctors will also collect information related to 
your treatment. 
 
Blood will be collected specifically for this study at up to 15 different times before and after 
your transplant. You will have up to three tablespoons of blood drawn for this study over 
a 1-year period. Blood samples will always be coordinated with a time when you are 
having blood drawn as part of your routine clinical care.  Also blood samples will be 
obtained on the occasion of major health complications (or events), like a blood infection 
or liver disease, for example. 
 
We will also get information about you and your disease and what happens to you before 
and after your stem cell transplant from your medical record, which is information that 
your doctor’s office and the hospital have about you. 
 
Are there any bad things that might happen during the research study? 
 
Sometimes bad things happen to people who are in research studies.  These bad 
things are called “risks.”  The risks of being in this study might be loss of your personal 
information.  Blood draw risks also include bleeding, fainting or feeling lightheaded, 

48 
 
bruising, and infection. 
Not all of these things may happen to you.  None of them may 
happen. 
 
Are there any good things that might happen during the research study? 
Sometimes good things happen to people who are in research studies. These good 
things are called “benefits.”  You will not receive a benefit from being in this study.  
However, we hope the information helps to collect information that may benefit future 
stem cell transplant treatments. 
 
Will I get money or payment for being in this research study? 
 
You will not get money for being in this research. 
 
Who can I ask if I have any questions? 
 
If you have any questions about this study, you can ask your parents or guardians or 
your doctor.  Also, if you have any questions that you didn’t think of now, you can ask 
later.  You can call Dr. XXX at XXX. 
 
What if I don’t want to be in the study? 
 
If you don’t want to be in this study, you don’t have to.  It’s up to you.  If you say you 
want to be in it and then change your mind, that’s OK.  All you have to do is tell us that 
you don’t want to be in it anymore.  No one will be mad at you or upset with you if you 
don’t want to be in it. 
 
 
 
My choice: 
 
If I write my name on the line below, it means that I agree to be in this research study. 
 
 
 
 
 
 
Subject’s Signature 
Date 
 
 
 
 
 
 
 
 
 
 
 
Subject’s Name 
 
 
 
 

49 
 
 
 
 
 
Signature of person obtaining assent 
Date 
 
 
 
 
 
 
Name of person obtaining assent

50 
 
 
11. APPENDIX C: STATISTICS 
 
Sample Size and Power Calculation. Three clinical endpoints are chosen as the primary 
hypotheses: D180 Post Therapy non-relapse mortality, D28 post-therapy non-response, 
and GVHD grade 1-4 onset.  Seven biomarkers will be tested at either onset of GVHD 
or at day 14 post-HCT (median 14 days prior GVHD) in predicting these clinical endpoints. 
Therefore, the overall type-I error is justified by a fact of 7*3*2 = 42, and the per 
comparison type I error is controlled at 0.05/42 = 0.0012. The effect size is pre- specified 
by the expected sensitivity and specificity of a biomarker, which ranges from 
0.60 to 0.70. The following table present the power of our study (N = 350) for three clinical 
endpoints. It shows that we shall have more than 91% power to test our hypotheses. 
Given the fact that our preliminary data show that one biomarker has a 
85% sensitivity and specificity, our proposed sample size will have sufficient power to 
test our hypothesis. 
 
Response Type and Rate 
Sensitivity and Specificity 
0.60 
0.65 
0.70 
D180 Post Therapy non-relapse mortality (0.26) 
0.91 
>0.99 
>0.99 
D28 post-therapy non-response (0.41) 
0.96 
>0.99 
>0.99 
GVHD grade 1-4 onset (0.50) 
0.98 
>0.99 
>0.99 
 
Our ‘pre-specified’ expected sensitivity and specificity are based on our previous work 
(taking the median as cutoff) (see Table below, Vander Lugt et al. NEJM, 2013) 
 
Sensitivity and specificity of ST2 concentrations at D14 to predict six-month post-HCT NRM 
 
Cutoff 
Sensitivity  
(95% CI) 
Specificity  
(95% CI) 
75thpercentile 
54% (50-59%) 
83% (77-89%) 
Median 
75% (71-79%) 
56% (48-64%) 
25thpercentile 
92% (89-94%) 
29% (22-36%) 
 
Biomarker Panel. We will construct the most informative and clinically useful biomarker 
panel to predict D180 post-therapy non-relapse mortality, D28 post-therapy non- response, 
and GVHD grade 1-4 onset. To this end, we will use standard logistic regression models 
to integrate the seven biomarkers into a multi-protein biomarker panel. This statistical step 
is a stepwise regression analysis to identify an optimal linear combination  of  biomarkers  
for  the  highest predictive  value  for  predicting  the  three clinical outcomes mentioned 
above. We will first use Univariate and multivariate models similar to those performed in 
the preliminary data. We will also adjust the models for the clinical characteristics at GVHD 
onset: intensity of conditioning regimen, donor HLA match, age, and GVHD onset grade. 

51 
 
After we define the optimized biomarker panel, we will use logistic regression to examine 
the ability of either the biomarker panel or ST2 alone (because ST2 was the most 
significant marker in all previous analyses) to predict D28 non responders. A high 
biomarker value will be defined by a plasma concentration greater than 50% above the 
median value in the responder group. A high panel must have at least five high biomarkers. 
We will then evaluate whether the odds ratio (OR) of the high panel vs. low panel and the 
high ST2 vs. low ST2 are significantly greater, meaning that patients are more likely to not 
respond to therapy independent of the significant clinical characteristics described above. 
If ST2 presents a more similar OR than the panel, ST2 measurement will be used for 
further analyses. We will  then hypothesize that the high panel (or high ST2) provides 
more important prognostic information at therapy initiation than the clinical grade. Similar 
to the preliminary data, we will evaluate if the high panel (or high ST2) at initiation of 
treatment can predict the D180 NRM independent of GVHD onset grade. NRM cumulative 
incidence curves and HR for the four risk categories will be evaluated: low panel/grade 
1-2, low panel/grade 3-4, high panel/grade 1-2, and high panel/grade 3-4. We will evaluate 
the NRM and OS, as described in our previous biomarker studies12,13. Briefly, NRM will be 
modeled using the cumulative incidence regression methods described in Fine et al.32, and 
the OS will be modeled using the Cox proportional-hazards model. 
 
Predictive Model. If we validate the proteins as diagnostic biomarkers, we will in this next 
part of the project ask whether this biomarker can be used to predict the future 
development of GVHD before its clinical signs become manifest. This statistical step will 
evaluate the value of the composite panel as a screening test for the risk of developing 
GVHD by computing the positive and negative predictive values. In order to be clinically 
useful, we expect that both values will be > 75%. In this case, we would expect that the 
biomarkers will correlate with subclinical disease. This possibility is likely, given that our 
preliminary data showed that TNFR1 levels in day 7 post- allogeneic transplant plasma 
correlates with eventual severity and incidence of GVHD, and with overall survival
33,34. 
We propose to use our novel repository of samples and the clinical data from SA 1 to 
determine if any of these biomarkers have predictive ability when measured earlier in the 
patient’s course. We are in a unique position to perform this analysis because our bank 
will contain samples that are collected from patients on a weekly basis during the first 
month after transplant. We will measure concentrations of the biomarkers at D7, D14, and 
D21 post- HCT (7 to 21 days prior to the onset of GVHD) and from similar time points in 
control patients, which will permit the generation of a kinetic curve for the individual 
biomarker. 
We will develop a predictive model using Cox proportional hazard model with time 
dependent covariates.  Time  to  GVHD  development  is  a  time  to  event  outcome,  
protein  biomarkers measured at D7, D14, and D21 post HCT are time dependent 
covariates, and treatment/control data and other clinical and demographic variables will 
be time independent covariates. To perform these analyses GVHD onset will be used as 
the event outcome. The control group includes the patients accrued on this protocol who 
do not develop GVHD. Two strategies will be used to model the time dependent 
biomarkers. One strategy is a simple linear interpolation and extrapolation of biomarker 
measurement to days other than day 7, 14, and 21. The second strategy is to model 
biomarker with either the linear trend or quadratic trend. 
Although these two strategies will pose different Cox regression models with time 

52 
 
dependent covariates, they can be implemented into the likelihood model framework 
proposed by Wulfsohn and Tsiatis
35. We will follow this modeling strategy, and implement 
the model fitting in R. Given Dr. Li’s extensive experiences in modeling longitudinal data 
with time dependent covariates
36,37, we are confident that we will be able to generate 
this time-dependent biomarker signature in predicting time to GVHD development. 

53 
 
12. APPENDIX D: GVHD scoring 
 
A sample Acute GVHD data record suggested for use in this trial is attached below. 
 
Today’s Clinical Acute GVHD Assessment 
Today’s date   
 
Patient ID   
  
Karnofsky/Lansky   
 
Codes 
 
 
Differential diagnosis 
0 
1 
2 
3 
4 
GVHD  Drug   Cond  Infect  TPN   VOD 
Other 
Rxn 
Reg 
Skin            □ □ □ □ □ % body rash:         _    □    □    □   □ 
Liver           □ □ □ □ □ Current bili:                  □    □    □   □   □  □              Lower 
GI     □ □ □ □  □ Vol:                              □    □    □   □   □                   Upper GI     
□ □                                  □    □    □   □   □                   Systemic 
agents:             □ CSA               □ Tacrolimus                   □ Infliximab         □ Daclizimab 
□ Pentostatin □ Sirolimus 
□ Etanercept 
□ MMF 
□ Prednisone □ Methylprednisolone 
□ Ontak 
□ Other   
 
Current steroid dose: Prednisone                             mg/kg/day;   Methylprednisolone                          mg/kg/day 
Has the steroid dose been increased to ≥ 2.5 mg/kg/day of prednisone (or 2 mg/kg/day methylpred)? □YES 
□ NO 
If yes, date dose increased                   ; reason for dose increase: □GVHD flare □ Other (specify)                      
Topical agents:  Skin topical steroids: □ YES □ NO; Non-absorbed oral steroids (e.g. Budesonide, Entocort) 
□ YES □ NO 
Chronic GVHD: Does the patient have evidence of chronic GVHD?  □ YES □ NO 
Codes 
Skin 
Liver (bilirubin) 
Lower GI (stool output/day) 
Upper GI 
0 
No rash 
 2 mg/dl 
Adult: < 500 ml/day 
Child: < 10 ml/kg/day 
No protracted nausea and 
vomiting 
1 
Maculopapular rash 
< 25% BSA 
2.1-3 mg/dl 
Adult: 500–1000 ml/day 
Child: 10 -19.9 ml/kg/day 
Persistent severe nausea, 
vomiting with a positive 
upper GI biopsy 
2 
Maculopapular rash 
25 – 50% BSA 
3.1-6 mg/dl 
Adult: 1001-1500 ml/day 
Child: 20 – 30 ml/kg/day 
3 
Generalized erythroderma 
(>50% BSA) 
6.1-15 mg/dl 
Adult: >1500 ml/day 
Child: > 30 ml/kg/day 
4 
Generalized erythroderma 
(>50% BSA) plus bullous 
formation and desquamation 
> 5% BSA 
>15 mg/dl 
Severe abdominal pain with or 
without ileus, or grossly bloody 
stool or melena (regardless of 
stool volume) 
Signature   
 
Date   
 

54 
 
A sample chronic GVHD data record suggested for use in this trial is attached below. 
 
Today’s Clinical Chronic GVHD Assessment 
 
Today’s date   
 
 
Patient ID   
 
 
0 
1 
2 
3 
Skin 
□ No changes 
□ < 18% BSA lichenoid, 
sclerodermatous, or 
ichthyotic involvement 
□ Eczema hypo or 
hyperpigmentation 
□ 18-50% BSA lichenoid, 
sclerodermatous, or 
ichthyotic involvement 
□ 50% BSA involved 
□ Interference with ADLs 
due to impaired mobility 
□ Skin ulceration 
Hair Loss 
□ None 
□ Mild (< 50%) 
□ > 50% 
Joints 
□ No contractures 
□ Arthralgias 
□ Migratory arthritis 
□ Persistent arthritis in 1 or 
2 joints 
□ Mild joint contractures 
(do not affect ADL) 
□ Polyarticular arthritis 
□ Severe joints 
contractures (interfere 
with ADLs) 
Oral 
□ No changes 
□ Symptomatic but no 
change in diet 
□ Able to eat most foods, 
although some dietary 
changes due to oral 
chronic GVHD 
□ Unable to eat most 
foods due to oral chronic 
GVHD 
Ocular 
□ No changes 
□ Dry Eyes but not 
requiring therapy 
□ Keratoconjuctivitis, 
asymptomatic 
□ Dryness of eyes 
requiring artificial tears, 
lachrymal plugging, or 
Schirmer’s < 5 
□ Keratoconjuctivitis, 
symptomatic 
□ Pseudomembranes 
□ Corneal ulcerations 
□ Loss of vision 
Esophagus 
□ No changes 
□ Symptomatic but can eat 
regular diet 
□ Dysphagia or 
odynophagia requiring 
dietary changes 
□ Need for parenteral 
nutrition 
□ Web/stricture 
formation 
Pulmonary 
□ Asymptomatic 
□ 75-90% FEV1/FVC 
□ Asthma 
□ Dyspnea with exertion 
□ 50-74% FEV1/FVC 
□ Desaturation with 
exercise 
□ Dyspnea with normal 
activities 
□ <50% FEV1/FVC 
□ Requires supplemental 
oxygen 
KPS/Lansky 
□ Asymptomatic and 
fully active (KPS 
100%, ECOG 0) 
□ Lansky = 90-100% 
□ Symptomatic and fully 
ambulatory; restricted in 
physical strenuous activity 
(KPS 80-90%, ECOG 1) 
□ Lansky = 70-80% 
□ Symptomatic; 
ambulatory; capable of 
self-care, > 50% of 
waking hours are spent 
out of bed (KPS 60-70%, 
ECOG 2) 
□ Lansky = 50-60% 
□ Symptomatic; limited 
self-care, spends > 50% 
of waking hours in bed, 
but not bedridden (KPS 
40-50%, ECOG 3) 
□ Lansky = < 50% 
GI 
manifestations 
□ None 
□ Anorexia or 
malabsorption ± < 5% 
weight loss, or in children, 
growth deviation of < 5% of 
pre-transplant percentile 
after 1 year post-transplant 
□ Anorexia or 
malabsorption ±  5-10% 
weight loss, or in 
children, growth 
deviation of < 5% of pre- 
transplant percentile after 
1 year post-transplant 
□ Anorexia or 
malabsorption ± > 20% 
weight loss, or in 
children, growth 
deviation of < 5% of pre- 
transplant percentile after 
1 year post-transplant 
Hematologic 
□ Thrombocytopenia 
not attributed to other 
causes > 100K 
□ Thrombocytopenia not 
attributed to other causes > 
75K 
□ Thrombocytopenia not 
attributed to other causes 
> 50K 
□ Thrombocytopenia not 
attributed to other causes 
< 50K 

55 
 
 
Autoimmune 
(e.g. ITP, AHA) 
□ None 
□ Positive laboratory tests, 
clinically not requiring 
therapy 
□ Require < 6 months 
additional 
immunosuppressive 
therapy to control 
symptoms 
□ Require transfusions 
(AHA) or splenectomy 
(ITP) or > 6 months of 
increased 
immunosuppressive 
therapy to control 
symptoms 
Laboratory and clinical variables 
(Please complete for this visit date) 
Platelets    
 X 109 /L 
Total Bilirubin    
 _._  
mg/dl 
Alkaline Phosphatase   
 
 Units/L 
Weight   
._  
 kg 
Specific manifestations 
(Please circle Yes, No, or Not 
applicable) 
Serositis 
Y / N 
Scleroderma 
Y / N 
Myositis 
Y / N 
Steatorrhea 
Y / N 
Fasciitis 
Y / N 
Vaginitis/Vaginal stricture 
Y / N  / NA 
 
 
*ADL = activities of daily living 
 
 
 
 
 
 
Signature   
 
Date   
 

56 
 
13.APPENDIX E: REFERENCES 
 
1. 
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2. 
Cutler C, Antin JH. Manifestation and Treatment of Acute Graft-Versus-Host- 
Disease. In: Appelbaum F, Forman SJ, Negrin RS, Blume KG, eds. Thomas' 
Hematopoietic Cell Transplantation (ed Fourth): Blackwell Publishing Ltd; 2009:1287- 
1303. 
3. 
Mowat A, Socie G. Intestinal Graft-vs.-Host Disease. In: Ferrara JLM, Cooke KR, 
Deeg HJ, eds. Graft-vs-Host Disease (ed 3rd ed.). New York: Marcel Dekker; 2004:279- 
327. 
4. 
Ferrara JL, Levine JE, Reddy P, Holler E. Graft-versus-host disease. Lancet. 
2009;373:1550-1561 PMCID 2735047 
5. 
Shulman HM, Kleiner D, Lee SJ, et al. Histopathologic diagnosis of chronic graft- 
versus-host disease: National Institutes of Health Consensus Development Project on 
Criteria for Clinical Trials in Chronic Graft-versus-Host Disease: II. Pathology Working 
Group Report. Biol Blood Marrow Transpl. 2006;12:31-47 
6. 
Washington K, Jagasia M. Pathology of graft-versus-host disease in the 
gastrointestinal tract. Hum Pathol. 2009;40:909-917 
7. 
Ertault-Daneshpouy M, Leboeuf C, Lemann M, et al. Pericapillary hemorrhage as 
criterion of severe human digestive graft-versus-host disease. Blood. 2004;103:4681- 
4684 
8. 
MacMillan ML, Weisdorf DJ, Wagner JE, et al. Response of 443 patients to 
steroids as primary therapy for acute graft-versus-host disease: comparison of grading 
systems. Biol Blood Marrow Transplant. 2002;8:387-394 
9. 
Cragg L, Blazar BR, Defor T, et al. A randomized trial comparing prednisone with 
antithymocyte globulin/prednisone as an initial systemic therapy for moderately severe 
acute graft-versus-host disease. Biol Blood Marrow Transplant. 2000;6:441-447 
10. 
Levine JE, Logan B, Wu J, et al. Graft-versus-host disease treatment: predictors 
of survival. Biol Blood Marrow Transplant. 2010;16:1693-1699 PMCID PMC2955996 
11. 
Martin P, Schoch G, Fisher L, et al. A retrospective analysis of therapy for acute 
graft-versus-host disease: initial treatment. Blood. 1990;76:1464-1472 
12. 
Paczesny S, Krijanovski OI, Braun TM, et al. A biomarker panel for acute graft- 
versus-host disease. Blood. 2009;113:273-278 PMCID PMC2615645 
13. 
Paczesny S, Braun TM, Levine JE, et al. Elafin is a biomarker of graft-versus- 
host disease of the skin. Science Translational Medicine. 2010;2:13ra12 PMCID 
2895410 
14. 
Ferrara JL, Harris AC, Greenson JK, et al. Regenerating islet-derived 3 alpha is a 
biomarker of gastrointestinal graft-versus-host disease. Blood. 2011 
15.     Foley R, Couban S, Walker I, et al. Monitoring soluble interleukin-2 receptor 
levels in related and unrelated donor allogenic bone marrow transplantation. Bone 
Marrow Transpl. 1998;21:769-773 
16. 
Grimm J, Zeller W, Zander AR. Soluble interleukin-2 receptor serum levels after 
allogeneic bone marrow transplantations as a marker for GVHD. Bone Marrow Transpl. 
1998;21:29-32 

57 
 
17. 
Holler E, Kolb HJ, Moller A, et al. Increased serum levels of tumor necrosis factor 
alpha precede major complications of bone marrow transplantation. Blood. 
1990;75:1011-1016 
18. 
Kayaba H, Hirokawa M, Watanabe A, et al. Serum markers of graft-versus-host 
disease after bone marrow transplantation. J Allergy Clin Immunol. 2000;106:S40-44 
19.     Miyamoto T, Akashi K, Hayashi S, et al. Serum concentration of the soluble 
interleukin-2 receptor for monitoring acute graft-versus-host disease. Bone Marrow 
Transpl. 1996;17:185-190 
20. 
Nakamura H, Komatsu K, Ayaki M, et al. Serum levels of soluble IL-2 receptor, 
IL-12, IL-18, and IFN-gamma in patients with acute graft-versus-host disease after 
allogeneic bone marrow transplantation. J Allergy Clin Immunol. 2000;106:S45-50 
21. 
Shaiegan M, Iravani M, Babaee G, Ghavamzadeh A. Effect of IL-18 and sIL2R 
on aGVHD occurrence after hematopoietic stem cell transplantation in some Iranian 
patients. Transpl Immunol. 2006;15:223-227 
22. 
Visentainer JEL, Lieber SR, Persoli LBL, et al. Serum cytokine levels and acute 
graft-versus-host disease after HLA-identical hematopoietic stem cell transplantation. 
Exp Hematol. 2003;31:1044-1050 
23. 
Okamoto T, Takatsuka H, Fujimori Y, Wada H, Iwasaki T, Kakishita E. Increased 
hepatocyte growth factor in serum in acute graft-versus-host disease. Bone Marrow 
Transpl. 2001;28:197-200 
24.     Or R, Kalinkovich A, Nagler A, et al. Soluble tumor necrosis factor (sTNF) 
receptors: a possible prognostic marker for bone marrow transplantation-related 
complications. Cytokines Mol Ther. 1996;2:243-250 
25. 
Schots R, Kaufman L, Van Riet I, et al. Proinflammatory cytokines and their role 
in the development of major transplant-related complications in the early phase after 
allogeneic bone marrow transplantation. Leukemia. 2003;17:1150-1156 
26. 
Uguccioni M, Meliconi R, Nesci S, et al. Elevated interleukin-8 serum 
concentrations in beta-thalassemia and graft-versus-host disease. Blood. 
1993;81:2252-2256 
27. 
Pfundt R, Wingens M, Bergers M, Zweers M, Frenken M, Schalkwijk J. TNF- 
alpha and serum induce SKALP/elafin gene expression in human keratinocytes by a 
p38 MAP kinase-dependent pathway. Arch Dermatolog Res. 2000;292:180-187 
28. 
Levine JE, Paczesny S, Mineishi S, et al. Etanercept plus methylprednisolone as 
initial therapy for acute graft-versus-host disease. Blood. 2008;111:2470-2475 PMCID 
PMC2361693 
29. 
Kakkar R, Lee RT. The IL-33/ST2 pathway: therapeutic target and novel 
biomarker. Nat Rev Drug Discov. 2008;7:827-840 
30. 
Gale RP, Bortin MM, van Bekkum DW, et al. Risk factors for acute graft-versus- 
host disease. Br J Haematol. 1987;67:397-406 
31. 
Jacobsohn DA. Acute graft-versus-host disease in children. Bone Marrow 
Transpl. 2008;41:215-221 
32. 
Fine JP, Gray RJ. A proportional hazards model for the subdistribution of a 
competing risk. J Am Stat Assoc. 1999;94:496-509 
33. 
Choi SW, Kitko CL, Braun T, et al. Change in plasma tumor necrosis factor 
receptor 1 levels in the first week after myeloablative allogeneic transplantation 

58 
 
correlates with severity and incidence of GVHD and survival. Blood. 2008;112:1539- 
1542 PMCID PMC2515125 
34. 
Kitko CL, Paczesny S, Yanik G, et al. Plasma elevations of tumor necrosis factor- 
receptor-1 at day 7 postallogeneic transplant correlate with graft-versus-host disease 
severity and overall survival in pediatric patients. Biol Blood Marrow Transpl. 
2008;14:759-765 PMCID PMC2577819 
35. 
Wulfsohn MS, Tsiatis AA. A joint model for survival and longitudinal data 
measured with error. Biometrics. 1997;53:330-339 
36. 
Li L, Brown MB, Lee KH, Gupta S. Estimation and inference for a spline- 
enhanced population pharmacokinetic model. Biometrics. 2002;58:601-611 
37. 
Li L, Lin X, Brown MB, Gupta S, Lee KH. A population pharmacokinetic model 
with time-dependent covariates measured with errors. Biometrics. 2004;60:451-460 
 
 

59 
 
14. APPENDIX F: LABORATORY PROCEDURES 
 
Research specimens 
Research samples collected in this protocol will be used for both protocol-defined testing 
as well as future, undefined research supporting the protocol. All research sample aliquots 
will be given unique bar code designations that cannot be linked back to the recipient.   
 
Patient samples will be collected as specified below. 
 
 Peripheral Blood sample on day -7, pre-transplant: Whole blood, Plasma  
 Peripheral Blood samples on day 56 post-transplant: Whole blood (donor DNA) 
 Donor chimerism results will be recorded at the time nearest to the DNA sample 
collection 
 Peripheral Blood samples at day -7, 7, 14, 21, 28, 56, 100, 180 post-transplant, and 
if the patient develops GVHD, two additional blood samples at the onset of symptoms 
that need treatment initiation and 28 days after treatment begins: Plasma 
 
Samples processed into frozen cryovial aliquots will be shipped to the central Laboratory at 
IU every 6 months on dry ice by Fedex or UPS overnight priority. 
Table 6: Collection, processing, and shipping procedures for patient blood samples 
for protocol-defined biomarker testing and for future undefined research 
Time 
points 
Collection 
of sample 
Type of processing and 
storage 
Specimen  Purpose 
of sample 
Shipping specifications 
Day -7 
pre-
transplant 
12 mL 
peripheral 
blood (two 6 
ml fill EDTA 
purple-top 
tube) 
Centrifuge the EDTA containing 
whole blood tube at 1000-1300 
g or 2100 rpm for 10 minutes 
within 60 minutes of collection. 
Remove the separated plasma. 
Aliquot to 1-4 cryovials and 
freeze at - 80º C in a scientific 
grade freezer. 
Plasma 
Future 
Biomarker
studies 
Batch ship every 6 
months, frozen on dry ice 
in a shipping container, 
Fedex overnight priority to 
the Well’s CCRC 
Laboratory at IU. 
3 mL 
peripheral 
blood (one 3 
ml fill EDTA 
lavender-top 
tube)  
Source of 
patient DNA 
Gently mix the tube by gently 
inverting the tube for 60 seconds 
and freeze at - 80º C in a 
scientific grade freezer. 
Whole 
Blood. 
Until 
extracted 
for  
DNA 
Future 
Biomarker
studies 
Batch ship every 6 
months, frozen on dry ice 
in a shipping container, 
Fedex overnight priority to 
the Well’s CCRC 
Laboratory at IU.  
Day 7 
post-
transplant 
12 mL 
peripheral 
blood (two 6 
ml fill EDTA 
purple-top 
tube) 
Centrifuge the EDTA containing 
whole blood tube at 1000-1300 
g or 2100 rpm for 10 minutes 
within 60 minutes of collection. 
Remove the separated plasma. 
Aliquot to 1-4 cryovials and 
freeze at - 80º C in a scientific 
grade freezer. 
Plasma 
Seven 
protein 
biomarker 
panel/ 
Future 
Biomarker 
studies 
Batch ship every 6 
months, frozen on dry ice 
in a shipping container, 
Fedex overnight priority to 
the Well’s CCRC 
Laboratory at IU.  
Day 
14 
post-
transplant 
12 mL 
peripheral 
blood (two 6 
ml fill EDTA 
Ficoll (without diluting the 
blood). Centifuge at 2000 rpm 
at room temperature for 30 
minutes, without brake. Remove 
the separated plasma. Aliquot 
Plasma/ 
PBMCs 
Seven 
protein 
biomarker 
panel/ 
Future 
Batch ship every 6 
months, frozen on dry ice 
in a shipping container, 
Fedex overnight priority to 

60 
 
purple-top 
tube) 
to 1-4 cryovials and freeze at - 
80º C in a scientific grade 
freezer. 
Then collect the “buffy coat” 
(PBMCs), Wash three times 
with cold sterile PBS 1X to 
remove Ficoll (1400 rpm 4C. 10 
min.). Count cells. 
Freeze cells in 1 ml aliquots 
using 90% FBS and 10% 
DMSO: 1-5M/vial, at least 2 
cryotubes up to 4 cryotubes. 
Gradual freezing: PBMCs are 
stored 24 hours (not more) in -
80°C in the ethanol blue box 
(Cryo-safe cooler , to achieve a 
-1°C /min. rate of cooling)  and 
then transfer in liquid nitrogen 
(liquid phase being better than 
gas phase). See details of the 
protocol below 
Biomarker
studies 
the Well’s CCRC 
Laboratory at IU. 
 
Day 
21 
post-
transplant 
12 mL 
peripheral 
blood (two 6 
ml fill EDTA 
purple-top 
tube) 
 
Ficoll; see protocol below.  
 
Plasma/ 
PBMCs 
Seven 
protein 
biomarker 
panel/ 
Future 
Biomarker 
studies 
Batch ship every 6 
months, frozen on dry ice 
in a shipping container, 
Fedex overnight priority to 
the Well’s CCRC 
Laboratory at IU. 
Day 
28 
post-
transplant 
12 mL 
peripheral 
blood (two 6 
ml fill EDTA 
purple-top 
tube) 
Ficoll; see protocol below. 
 
Plasma/ 
PBMCs 
Seven 
protein 
biomarker 
panel/ 
Future 
Biomarker
studies 
Batch ship every 6 
months, frozen on dry ice 
in a shipping container, 
Fedex overnight priority to 
the Well’s CCRC 
Laboratory at IU.  
Onset 
GVHD 
treated 
with 
systemic 
steroids 
12 mL 
peripheral 
blood (two 6 
ml fill EDTA 
purple-top 
tube) 
Ficoll; see protocol below. 
  
 
Plasma/ 
PBMCs 
Seven 
protein 
biomarker 
panel/ 
Future 
Biomarker 
studies 
Batch ship every 6 
months, frozen on dry ice 
in a shipping container, 
Fedex overnight priority to 
the Well’s CCRC 
Laboratory at IU. 
 
28 
days 
post-
treatment 
for GVHD 
12 mL 
peripheral 
blood (two 6 
ml fill EDTA 
purple-top 
tube) 
Ficoll; see protocol below. 
 
Plasma/ 
PBMCs 
Future 
biomarker 
studies 
Batch ship every 6 
months, frozen on dry ice 
in a shipping container, 
Fedex overnight priority to 
the Well’s CCRC 
Laboratory at IU. 
Day 
56-
60 post-
transplant 
12 mL 
peripheral 
blood (two 6 
ml fill EDTA 
purple-top 
tube) 
Ficoll; see protocol below. 
 
Plasma/ 
PBMCs 
Future 
Biomarker
studies 
Batch ship every 6 
months, frozen on dry ice 
in a shipping container, 
Fedex overnight priority to 
the Well’s CCRC 
Laboratory at IU. 
 
3 mL 
peripheral 
blood (one 3 
ml fill EDTA 
lavender-top 
tube)  
Source of 
patient DNA 
Gently mix the tube by gently 
inverting the tube for 60 seconds 
and freeze at - 80º C in a 
scientific grade freezer. 
 
Whole 
Blood. 
Until 
extracted 
for  
DNA  
 
Future 
Biomarker
studies 
Batch ship every 6 
months, frozen on dry ice 
in a shipping container, 
Fedex overnight priority to 
the Well’s CCRC 
Laboratory at IU. 
 

61 
 
Day 100 
post-
transplant 
12 mL 
peripheral 
blood (two 6 
ml fill EDTA 
purple-top 
tube) 
Ficoll; see protocol below. 
 
Plasma/ 
PBMCs 
Future 
Biomarker
studies 
Batch ship every 6 
months, frozen on dry ice 
in a shipping container, 
Fedex overnight priority to 
the Well’s CCRC 
Laboratory at IU. 
 
Day 180 
post-
transplant 
12 mL 
peripheral 
blood (two 6 
ml fill EDTA 
purple-top 
tube) 
Ficoll; see protocol below. 
 
Plasma/ 
PBMCs 
Future 
Biomarker
studies 
Batch ship every 6 
months, frozen on dry ice 
in a provided shipping 
container, Fedex 
overnight priority to the 
Well’s CCRC Laboratory 
at IU. 
 
Day 360 
post-
transplant 
12 mL 
peripheral 
blood (two 6 
ml fill EDTA 
purple-top 
tube) 
Ficoll; see protocol below. 
 
Plasma/ 
PBMCs 
Future 
Biomarker
studies 
Batch ship every 6 
months, frozen on dry ice 
in a shipping container, 
Fedex overnight priority to 
the Well’s CCRC 
Laboratory at IU. 
 
 
 
 
Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs) and Plasma by 
Ficoll separation and cryopreservation Protocol 
Purpose 
This Standard Operating Procedure (SOP) describes procedures for the isolation and 
cryopreservation of Plasma and Peripheral Blood Mononuclear Cells (PBMC) from whole blood. 
 
Scope 
2.1 This procedure is to be utilized for processing blood samples for the isolation, cryopreservation, 
and storage of Plasma and PBMC samples.  
 
Materials and Reagents 
No. 
Name 
Description 
Concentration 
1.0 
Purple top 
vacutainers 
BD # 367863 
 
2.0 
Ficoll or 
lymphoprep 
(sterile) 
Amersham #17544202 (GMP 
grade)   
Use undiluted. 
3.0 
PBS 1X (-
calcium, - 
magnesium) 
(sterile) 
Hyclone SH30256.02 
 
4.0 
Cryotubes 
Orange top for cells, Corning 
 
5.0 
   Cryotubes  
Clear top for plasma 
 
 

62 
 
Procedure 
 
Do not dilute the blood  
 
For blood samples of up to 11 ml, add 5 ml of ficoll to a 15 ml tube. For blood samples over 
11 ml, use 10 ml of ficoll in a 50 ml tube.  
 Layer cells dropwise onto Ficoll.  
 Let the drops hit the side of the tube and settle slowly to avoid disturbing the meniscus. 
 
 
Centifuge – see table below 
 
Multi-Center GVHD Biomarkers and Biobank Centrifuge Instructions 
Parameter 
Setting 
RPM 
2000 
Time 
30 minutes 
Temperature 
Room Temperature (20-24°C) 
Brake 
Off 
Acceleration 
Off 
 
 

63 
 
 
 
Carefully observe the cloudy “buffy coat” at the interface between the layers.  
 
Collect first the plasma and distribute to 1-4 clear top tubes (1.5 to 1.7 ml in each of the 
tubes). Plasma samples are aliquoted without additives into cryovials and stored at -80°C. 
 
Then collect the cloudy “buffy coat” (= PBMCs), avoiding the Ficoll layer, using a 10 mL 
pipet (for 15 mL conical tube) or 25 mL pipet (for 50 mL Conical tube). Place into a clean 
15 ml tube. 
 
Wash once with 15 ml sterile PBS 1X to remove trace of Ficoll (1400 rpm 4C. 10 min.) 
 
Wash PBMCs again with PBS 1X @ 1200 rpm, 4C. 5 min. 
 
Resuspend pelleted cells in PBS and count. 

64 
 
 
Only if necessary (most of the time you don’t need to), lyse residual erythrocytes. 
 
The last wash should be @900 rpm 10 min, to get rid of the platelets and trash). 
 
Freeze cells in 1 ml aliquots using 90% FBS and 10% DMSO: up to 30M/vial (see table 
below), at least 2 orange top cryotubes up to 4 orange top cryotubes. Gradual freezing: 
PBMCs are stored 24 hours (not more) in -80°C in the ethanol blue box (Cryo-safe cooler , 
to achieve a -1°C /min. rate of cooling)  and then transfer in liquid nitrogen (liquid phase 
being better than gas phase). 
 
PBMC Aliquot Instructions 
Number of Cells 
Number of cryotubes 
1 -10 million 
Distribute in 2  
10 - 20 million 
Distribute in 3  
20 - 35 million 
Distribute in 3 
35 million and over 
Distribute in 4 
 
 
References 
- 
University of Michigan BMT laboratory SOP 
- 
Baylor Institute for Immunology laboratory SOP 
- 
HIV/AIDS Network Coordination SOP 
 
 

65 
 
15. APPENDIX G- CELLULAR PROTEIN BIOMARKER PANEL FOR VIRUS AND 
LEUKEMIA SPECIFICT CELL IMMUNE RECONSTITUTION   PROTEIN BIOMARKER 
PANEL 
1. SPECIFIC AIMS 
 
Cellular populations in the peripheral blood may also serve as biomarkers for GVHD (e.g. 
Tregs). Therefore, we anticipate improving the panel by adding biomarkers of virus and 
leukemia specific T cell immune reconstitution that will be generated in collaboration with 
Dr. Bollard.  This integrated proteomic/cellular approach will provide not only candidate 
biomarkers but also insights in the complex pathology of GVHD. 
 
Accumulating evidence suggests that low levels of circulating Tregs following allo-HSCT in 
humans are associated with a higher incidence of GVHD in adults, and a causal link 
between these phenomena is suggested by murine models that show infusion of donor 
Tregs at the time of transplant or shortly thereafter prevents GVHD while maintaining the 
graft-versus-leukemia (GVL) effect. We hypothesize that increased donor-derived 
Tregs detected in the peripheral blood of pediatric patients is associated with a 
decreased risk of GVHD while sparing the cell mediated immune response to virus 
and tumor antigens after allo-HSCT. These hypotheses are formulated from extensive 
preclinical and clinical data and will be tested as follows: 
 
 
Aim 1: To determine whether increased functional Tregs post allogeneic HSCT is 
associated with decreased GVHD without affecting cellular immune responses 
against leukemia and viruses.     
We will evaluate patient peripheral blood samples. We will measure recovery of CD4+ 
CD25+ FoxP3+ T cells as well as other major T-cell subsets using flow cytometry and 
confirm whether these cells are of donor or recipient origin. We will evaluate the ability of 
the CD4+ CD25+ Tregs to suppress alloreactive responses and analyze the mechanism 
should such suppression occur. We will also measure T-cell immune reconstitution to 
leukemia antigens (WT1, PR1, PRAME) and viruses (EBV, CMV and Adenovirus) after allo-
HSCT. We will measure the frequency of virus and leukemia reactive T cells and test their 
functionality in cytokine secretion assays.  
 
2. BACKGROUND AND SIGNIFICANCE 
 
2.1 Regulatory T cells. A subpopulation of T cells that are CD4+ CD25+ plays a crucial 
regulatory role in the immune system. These Tregs control autoimmune processes1 and 
modulate post-transplant alloreactive responses. Tregs constitutively express the 
transcription factor FoxP3, which is a key regulator of Treg development in humans and 
mice.2-4  Intracellular expression of FoxP3 is one of the most specific markers for Tregs,5 
although other markers such as CD4, CD25hi and CD127lo, may also define the most potent 
subpopulations.6 In short-term in vitro assays, Tregs suppress proliferation of both CD4+ 

66 
 
and CD8+ T cells after polyclonal or antigenic stimulation in a cell contact-dependent and 
cytokine-independent manner.7 Moreover, interleukin 2 (IL-2) is essential for the generation 
of Tregs and crucial in maintaining their suppressive function.8 Murine models show that 
high numbers of Tregs infused at the time of allo-HSCT can prevent GVHD while preserving 
the GVL effect.9;10,11 Indeed, if Tregs present in the transplant product are depleted prior 
to infusion into irradiated allogeneic recipient mice, GVHD is significantly accelerated.7  
Additional studies have shown that Tregs infused post-transplant were able to control 
established GVHD.4  
 
Human studies have demonstrated that poor Treg reconstitution post HSCT correlate with 
occurrence of acute or chronic GVHD.12,13 Phenotypic and PCR analyses of FoxP3+ 
regulatory T cells in 57 transplant recipients (27 with active, chronic GVHD and 30 without 
active, chronic GvHD) showed a decreased frequency of Tregs in patients with chronic 
GVHD compared to patients without (p<0.0001), and FoxP3 expression was reduced in 
chronic GVHD patients compared to those without or to controls (p=0.009 and 0.01, 
respectively).14  Furthermore, there is an association between high donor levels of FoxP3+ 
Tregs and a reduced risk of GVHD after HLA-matched allo-HSCT.15 The above data 
support efforts to determine in the pediatric setting whether rapid Treg immune 
reconstitution after allo-HSCT will prevent GVHD.  
 
2.2 Recovery of cellular immunity after allo-HSCT.  Normal immune reconstitution 
following HSCT extends over many months and even years, rendering the patient highly 
susceptible to bacterial, viral, and fungal infections as well as leukemia relapse. Viral 
infections are one of the leading causes of morbidity and mortality among allo-HSCT 
recipients. Reactivation of latent viruses such as Epstein-Barr Virus (EBV) and 
cytomegalovirus (CMV), and infection with community viruses such as adenovirus and RSV 
are frequent and significant causes of morbidity and mortality.16  Virus-specific T-cells are 
a critical protective element after allogeneic transplantation,17 and the most prolonged 
delays in virus-specific immune responses are associated with protocols specifying high 
levels of T-cell depletion and post-transplant immune suppression to prevent GVHD.  
 
Similarly, several groups have looked at the immune reconstitution of tumor-specific T cells 
and their effect on relapse. Along with viral infection, we and others have shown that relapse 
is one of the main causes of failure after allogeneic stem cell transplant. T-cells specific for 
leukemia associated antigens such as the Wilms tumor antigen (WT1) have been shown to 
contribute to a graft-versus-leukemia (GVL) effect after allogeneic stem-cell transplantation 
(HSCT) for acute leukemia. Furthermore, loss of these leukemia specific CD8+ T-cell 
responses has been shown to be associated with relapse.18  We hypothesize that a 
balanced regulatory and virus and leukemia specific T cell recovery prevents GVHD 
without delaying antiviral immune reconstitution.  
 
3 .  PRELIMINARY DATA 
 

67 
 
3.1. Preliminary data. To demonstrate our ability to measure the recovery of Tregs, NK 
cells and antigen-specific T cells, we isolated PBMC from transplant recipients immediately 
before and early (2-6 months) and late (>6 months) after transplantation (Fig. 1A-C). We 
plated the PBMC in a standard IFN- ELISPOT at a concentration of 2x105cells/well, and 
stimulated them with peptide mixtures (pepmixes) spanning the adenovirus hexon and 
penton 
antigens, CMV 
IE1 and pp65 
and 
the EBV 
antigens 
BZLF1, EBNA1, 
EBNA3A, 
EBNA3B 
and 
EBNA3C. 
To 
measure 
general 
T-cell 
function, 
we 
stimulated 
PBMC 
with 
Staphylococcus 
aureus (Staph) 
as a positive 
control, while unstimulated PBMC served as a negative control. Virus specific responses 
are shown in Fig. 1B. While we were able to detect robust specific T-cell reactivity to Staph 
early, we saw little evidence of virus-specific T-cell function in samples collected early (2 
months) post-transplant. However, by 6 months after allo-HSCT, specific reactivity is 
detectable against viral antigens, indicating that patients who do not have GVHD can 
restore viral specific immunity within 3-6 months.19,20 To detect tumor-specific T-cells, we 
stained the PBMC with HLA A2-restricted tetramers as shown in Fig. 1C and D. Detection 
of WT-1- and PRAME-specific T cells in the peripheral blood of patients with lymphoid and 
myeloid malignancies after allo-HSCT strongly correlated with a low relapse rate after 
transplant.18  These preliminary data support our proposal to detect antigen-specific T cells 
as well as NK cells, post-transplant in the peripheral blood of patients after allo-HSCT. 
Hence, these results demonstrate the feasibility of tracking the effects of BMSC infusions 
on post-transplant immune reconstitution in patients with GVHD. 
 
4. RESEARCH PLAN 
 
4.1. Immune reconstitution of T-cells and NK cells Blood will be obtained at the 
designated timepoints (Day +14, +21, +28, +56, +100, +180 and +360 post HSCT as well 
Figure 1. Immunological studies of patients post HSCT. A.) T regs can be 
detected and analyzed post HSCT B.) T cell immunity to viral antigens as 
determined by IFNg ELISPOT in 8 patients. C and D. Identifying CMV- and 
WT-1 specific T cells from peripheral blood of 2 patients by tetramer analysis 

68 
 
as at time of complications such as acute and chronic GVHD onset, start of systemic 
steroids, or other complications). Peripheral Blood Mononuclear Cells (PBMC) will be 
obtained by Ficoll separation. Flow cytometric analyses will be used to quantify T cells 
(CD3+ CD4+ CD8) and T-cell subsets, including naïve and memory T cells, and NK cells. 
Once the T cells and NK cells have recovered to a predefined level (≥ 500 CD3+ cells/μL), 
samples will be frozen for batch analysis in the functional assays described below (4.2 to 
4.5). 
4.2. Are functional Tregs increased in pediatric patients without GVHD?  To determine 
Treg phenotype, we will use the intracellular FoxP3 Treg kit (eBioscience) as well as CD4 
and CD25 fluorescent antibodies (Fig. 1A). Treg induced suppression will be demonstrated 
by effects on thymidine uptake and CSFE assays in mixed lymphocyte reactions. 
Specifically, we will examine the response of T cells to a standard APC (Allogeneic PBMC 
or K562 cells expressing the high affinity IgG receptor CD64) loaded with OKT3. We will 
determine the mechanism for the suppression by measuring cytokine secretion (IL-10 and 
TGF-β). For thymidine uptake assays, PBMC will be separated into CD4+ CD25+ and CD4+ 
CD25- fractions using fluorescent cell-sorting or magnetic bead separation. The CD4+ 
CD25- fraction (responder cells) will be plated into a round-bottom 96-well plate coated with 
anti-CD3 (OKT3) or allogeneic PBMC. The CD4+ CD25+ Treg fraction will be added at a 1:1 
ratio and incubated at 370C. On day 3, each well will be pulsed with 1 Ci of 3H-thymidine. 
After 18 hours of additional incubation, plates are harvested and processed for counting.20  
 
4.3. Do pediatric patients with GVHD have decreased pathogen-specific immune 
reconstitution compared to those without? Ten to 40 mL of blood (depending on the 
patient’s size) will be taken at designated intervals post-HSCT to measure the frequencies 
of virus-specific T cells as well as NK cells. Absolute lymphocyte counts (ALC) will be 
measured to determine both absolute and relative frequencies of NK cells and virus-specific 
T-cells. Functional assays will determine the reconstitution of immunity to pathogens 
frequently detected in patients after allo-HSCT, including EBV, CMV, Aspergillus and 
adenoviruses. 
(i) Capturing viral reactivation. Peripheral blood collected post-HSCT will be used for 
measurement of viral load (adenovirus, EBV and CMV), using PCR. These data will capture 
at each sample timepoint. 
(ii) Immunity to common pathogens. To evaluate the immune response to EBV, CMV, 
Aspergillus and adenovirus, we will measure the precursor frequency of cytotoxic T 
lymphocytes (CTL) specific for these pathogens in ELISPOT assays.   
 
4.4   What is the antileukemic T-cell activity in patients who develop GVHD?  All 

69 
 
patients will be regularly monitored for disease status by our standard clinical management 
techniques, which include (i) morphologic analysis of bone marrow samples to assess 
“conventional” remission status and (ii) minimal residual disease analyses of marrow and 
peripheral blood samples using chromosomal markers. As an additional measure of 
disease recurrence, recipients will be regularly monitored for (iii) the level of donor 
chimerism in myeloid and lymphoid cells in the blood and marrow, as per our standard 
institutional protocols. Hence, in patients with myeloid and lymphoid malignancies, we will 
measure the precursor frequency of CTL specific for the leukemia-associated antigens 
PR3, PRAME and WT-1 using (i) A2-restricted tetramer assays, (ii) ELISPOT and/or 
intracellular cytokine assays using pepmix-pulsed PBMC.20   
 
4.5. Do patients with GVHD have impaired NK cell persistence and function? To 
evaluate NK cell persistence and function post-HSCT, we propose to undertake 
immunophenotypic analyses of clinical samples obtained post-HSCT. NK-cell persistence 
will be defined as an absolute circulating NK cell count of >100 cells/μL after HSCT. To 
address the issue of possible dysfunction of NK cells in patients with GVHD, we propose to 
perform immunophenotypic analyses of clinical PBMC samples at the timepoints indicated 
above. Multiparameter (12-color) flow cytometry will be performed for NK cell receptors to 
evaluate the expression of activating and inhibitory receptors, NK activation and maturation, 
exhaustion (e.g. CD57 and T-bet)21,22 and homing (e.g. CD62L, CCR7, CLA). Effector 
function will be assessed by the 51chromium release assay, CD107a degranulation23,24 and 
cytokine release (by intracellular cytokine assay [IFNγ]), using K562 and primary leukemia 
cells collected and stored from recipients pre-transplant. Plasma samples obtained from 
the recipient before and after HSCT will be assayed for cytokine levels using the BD 
Cytometric Bead Array for Th1/Th2 cytokines and ELISA assays.  
 
4.6. Statistical analysis. The change in general immune reconstitution in patients with 
versus without GVHD will be summarized with descriptive statistics.  After logarithmic 
transformation, if deemed appropriate, we will analyze the difference in changes by a two-
sample t-test. In addition to descriptive statistics, Treg and antigen-specific T-cell immune 
reconstitution over time, as measured by phenotyping assays and functional assays will be 
plotted over time and represented as means ± SD.  
 
4.7. Expected outcomes/potential problems. GVHD is known to be immune suppressive 
and we predict that there will be an increased Treg response in patients without GVHD with 
preservation of their cell mediated immune response. Given recent reports in the literature 
and our own preliminary data, we predict that if there is an increase in Tregs in patients 
without GVHD, that this will not dampen T-cell immune reconstitution against viral or 

70 
 
leukemia antigens. We also expect the immune reconstitution of NK cells to be unaffected 
in patients who do not have GVHD but that NK cells in patients with GVHD are either 
decreased and/or dysfunctional. Although unlikely, we cannot exclude the possibility that 
NK cells will not be detected in the periphery. This could reflect a complete loss of the NK 
cells or perhaps their limited expansion in vivo. It is also possible that T-cells and NK cells 
will home to sites of leukemia, prompting us to look for their presence in the bone marrow 
as well as blood. We do not anticipate any major obstacles with the proposed immune 
reconstitution studies, owing to our expertise at the CNMC in multiparameter flow cytometry 
and T-cell and NK cell functional immunoassays.24-26  However, it is entirely possible that 
smaller patients will have too few lymphocytes in their peripheral blood, especially in the 
early post-HSCT period, to accommodate the proposed testing scheme. If so, we will 
prioritize our tests, requesting additional samples if necessary. Phenotypic analyses, 
specifically for biomarker assays and CD4+ CD25+ FoxP3+ Tregs, will be performed first, 
followed by general phenotyping assays (T-cell subsets, NK cells and B cells), functional 
Treg assays and frequencies of CMV, then EBV and then adenovirus-specific CTL. In 
patients with evidence of viral infection, evaluation of the T-cell response specific to the 
infecting virus will be given priority.  
 
5. REFERENCES 
 
1. 
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