The Effectiveness of Lactobacillus Plantarum (LBP, IND# 17339) in Preventing Acute Graft-Versus-Host Disease (GvHD) in Children Undergoing Alternative Hematopoietic Progenitor Cell Transplantation (HCT)
Phase: PHASE3
Conditions: Acute Graft Versus Host Disease, Hematopoietic and Lymphatic System Neoplasm, Leukemia, Lymphoma
Sponsor: Children's Oncology Group

 
 
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CHILDREN’S ONCOLOGY GROUP 
 
ACCL1633 
 
The Effectiveness of Lactobacillus plantarum (LBP, 
) in Preventing Acute Graft-versus-Host 
Disease (GvHD) in Children undergoing Alternative Hematopoietic Progenitor Cell Transplantation (HCT) 
 
IND Sponsor for Lactobacillus plantarum: Children’s Oncology Group 
 
A Groupwide Study  
 
 
THIS PROTOCOL IS FOR RESEARCH PURPOSES ONLY, AND SHOULD NOT BE COPIED, REDISTRIBUTED OR USED FOR 
ANY OTHER PURPOSE. MEDICAL AND SCIENTIFIC INFORMATION CONTAINED WITHIN THIS PROTOCOL IS NOT 
INCLUDED TO AUTHORIZE OR FACILITATE THE PRACTICE OF MEDICINE BY ANY PERSON OR ENTITY. RESEARCH 
MEANS A SYSTEMATIC INVESTIGATION, INCLUDING RESEARCH DEVELOPMENT, TESTING AND EVALUATION, 
DESIGNED TO DEVELOP OR CONTRIBUTE TO GENERALIZABLE KNOWLEDGE. THIS PROTOCOL IS THE RESEARCH 
PLAN DEVELOPED BY THE CHILDREN’S ONCOLOGY GROUP TO INVESTIGATE A PARTICULAR STUDY QUESTION OR 
SET OF STUDY QUESTIONS AND SHOULD NOT BE USED TO DIRECT THE PRACTICE OF MEDICINE BY ANY PERSON OR 
TO PROVIDE INDIVIDUALIZED MEDICAL CARE, TREATMENT, OR ADVICE TO ANY PATIENT OR STUDY SUBJECT. THE 
PROCEDURES IN THIS PROTOCOL ARE INTENDED ONLY FOR USE BY CLINICAL ONCOLOGISTS IN CAREFULLY 
STRUCTURED SETTINGS, AND MAY NOT PROVE TO BE MORE EFFECTIVE THAN STANDARD TREATMENT. ANY PERSON 
WHO REQUIRES MEDICAL CARE IS URGED TO CONSULT WITH HIS OR HER PERSONAL PHYSICIAN OR TREATING 
PHYSICIAN OR VISIT THE NEAREST LOCAL HOSPITAL OR HEALTHCARE INSTITUTION. 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
Activated: 
04/30/2018 
Version Date: 
04/19/2021 
Closed: 
04/15/2022 
Amendment # 
2 

 
 
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TABLE OF CONTENTS 
 
SECTION 
PAGE 
STUDY COMMITTEE 
4 
ABSTRACT 
6 
EXPERIMENTAL DESIGN SCHEMA 
7 
1.0 
GOALS AND OBJECTIVES (SCIENTIFIC AIMS) 
8 
1.1 
Primary Aim 
8 
1.2 
Exploratory Aims 
8 
2.0 
BACKGROUND AND HYPOTHESES 
8 
2.1 
Acute graft-versus-host disease (GvHD): Clinical impact 
8 
2.2 
GvHD pathophysiology: Focus on the gastrointestinal tract (GI) 
9 
2.3 
An emerging role for the intestinal microbiota in GI aGvHD: Probiotic therapy 
9 
2.4 
Probiotic Strain 
10 
2.5 
Preliminary Data 
11 
2.6 
Summary 
12 
3.0 
STUDY ENROLLMENT PROCEDURES AND PATIENT ELIGIBILITY 
14 
3.1 
Study Enrollment 
14 
3.2 
Patient Inclusion Eligibility Criteria 
17 
3.3 
Exclusion Criteria 
17 
3.4 
Regulatory Requirements 
18 
4.0 
TREATMENT PLAN 
19 
4.1 
Overview of Treatment Plan 
19 
4.2 
LBP or Placebo Administration 
19 
4.3 
Concomitant Therapy 
21 
5.0 
EMERGENCY UNBLINDING 
21 
6.0 
AGENT INFORMATION 
22 
6.1 
Lactobacillus plantarum (
)         (04/15/2021) 
22 
7.0 
EVALUATIONS/MATERIAL AND DATA TO BE ACCESSIONED 
26 
7.1 
Required Clinical and Laboratory Evaluations 
26 
7.2 
Data Collection 
27 
7.3 
Correlative Studies 
27 
7.4 
Specimen Collection, Processing and Shipping 
31 
8.0 
CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF STUDY 
CRITERIA 
34 
8.1 
Criteria for Removal from Protocol Therapy 
34 
8.2 
Off Study Criteria 
34 
9.0 
STATISTICAL CONSIDERATIONS 
34 
9.1 
Sample Size and Study Duration 
34 
9.2  
Study Design 
34 
9.3 
Data Analytic Plan 
36 
9.4 
Power and Sample Size 
38 

 
 
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9.5 
Interim Monitoring 
39 
9.6 
Gender and Minority Accrual Estimates 
40 
10.0 
EVALUATION CRITERIA 
40 
10.1 
Common Terminology Criteria for Adverse Events (CTCAE) 
40 
10.2 
Acute GVHD 
40 
10.3 
Graft Failure 
41 
11.0 
ADVERSE EVENT REPORTING REQUIREMENTS 
41 
11.1 
Purpose 
41 
11.2 
Expedited Reporting Requirements – Serious Adverse Events (SAEs) 
41 
11.3 
Specific Examples for Expedited Reporting 
42 
11.4 
Reporting Requirements for Specialized AEs 
44 
11.5 
Exceptions to Expedited Reporting 
44 
11.6 
Reporting Requirements - Investigator Responsibility 
44 
11.7 
General Instructions for Expedited Reporting via CTEP-AERS 
45 
11.8 
Reporting Table for Late Phase 2 and Phase 3 Studies – Table A 
46 
11.9 
Protocol Specific Additional Instructions and Reporting Exceptions 
47 
11.10 
Routine Reporting of Adverse Events 
47 
11.11 
Syndrome Reporting 
47 
12.0 
RECORDS AND REPORTING 
47 
12.1 
CDUS 
47 
12.2 
Data Safety and Monitoring Committee 
47 
APPENDIX I: CTEP REGISTRATION PROCEDURES 
49 
APPENDIX II: YOUTH INFORMATION SHEETS 
52 
APPENDIX III: ACUTE GVHD STAGING AND GRADING 
54 
REFERENCES 
61 
 
 

 
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AGENT                               
    
  
Lactobacillus plantarum     
 
  
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
SEE SECTION 7.4 FOR SPECIMEN SHIPPING ADDRESSES 
 
 

 
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The Children's Oncology Group has received a Certificate of Confidentiality from the federal government, 
which will help us protect the privacy of our research subjects. The Certificate protects against the 
involuntary release of information about your subjects collected during the course of our covered studies. 
The researchers involved in the studies cannot be forced to disclose the identity or any information collected 
in the study in any legal proceedings at the federal, state, or local level, regardless of whether they are 
criminal, administrative, or legislative proceedings. However, the subject or the researcher may choose to 
voluntarily disclose the protected information under certain circumstances. For example, if the subject or 
his/her guardian requests the release of information in writing, the Certificate does not protect against that 
voluntary disclosure. Furthermore, federal agencies may review our records under limited circumstances, 
such as a DHHS request for information for an audit or program evaluation or an FDA request under the 
Food, Drug and Cosmetics Act. The Certificate of Confidentiality will not protect against mandatory 
disclosure by the researchers of information on suspected child abuse, reportable communicable diseases, 
and/or possible threat of harm to self or others. 
 
 
ABSTRACT 
 
Despite prophylactic immune suppression, clinically significant (Grade II–IV) acute graft-versus-host 
disease (aGvHD) affects up to 45% of pediatric patients receiving allogeneic hematopoietic cell 
transplantation (alloHCT). As aGvHD is responsible for nearly 20% of deaths following alloHCT, the need 
for better prevention and therapy for aGvHD is readily apparent. Involvement of the gastrointestinal (GI) 
tract in the pathogenesis of aGvHD has been substantiated by the translation of pre-clinical and clinical 
studies. Emerging evidence suggests that perturbations in the microbiota diversity result in aberrant 
systemic immune response as well as pathogen colonization and mucosal invasion, fostering the 
development of aGvHD as well as increasing the risk for subsequent bacteremia with enteric pathogens. 
Pre-clinical studies also suggest that replenishing commensals like Lactobacillus prior to alternative donor 
alloHCT may substantially decrease aGvHD severity and intestinal insult. Our pilot data suggests that 
probiotics are safe to administer prior to and after alternative donor alloHCT in children and adolescents 
(IND#108,977). 
 
The proposed study is a randomized, double-blinded, placebo-controlled intervention trial to determine the 
benefit of probiotic therapy in preventing the development of GI aGvHD in children undergoing initial 
alternative donor alloHCT. Importantly, correlative studies will be performed to elucidate how probiotic 
therapy affects the microbiome. Discovering such benefits of probiotic therapy may have significant impact 
on morbidity, mortality, and cost of care in children and adolescents alternative donor undergoing alloHCT. 
 
 
 

 
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EXPERIMENTAL DESIGN SCHEMA 
 
 
 
 
 
RANDOMIZATION 
Lactobacillus plantarum 
(BLINDED) 
Placebo 
(BLINDED) 
Baseline Evaluation (Initiation of Conditioning)  
Administration of LBP 
or placebo Day 1 of 
Conditioning through 
Day 56 Post alloHCT 
Day 0 (Graft Infusion) Evaluation  
Day 14 Evaluation 
Patients age 2-25 undergoing  
alternative donor alloHCT 
Day 7 Evaluation  
Day 28 Evaluation 
Day 120 Evaluation 
Day 56 Evaluation 
Completion of Study 
Administration of LBP 
or placebo Day 1 of 
Conditioning through 
Day 56 Post alloHCT 

 
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1.0 
GOALS AND OBJECTIVES (SCIENTIFIC AIMS) 
 
1.1 
Primary Aim 
To determine efficacy of orally-administered LBP in preventing the development of GI 
aGvHD in children and adolescents undergoing alternative donor alloHCT. 
 
1.2 
Exploratory Aims 
1.2.1 
To determine whether orally-administered LBP decreases the incidence of Grade 
II–IV aGvHD following alternative donor alloHCT. 
 
1.2.2 
To determine whether LBP administration maintains intestinal integrity as 
measured by mean serum citrulline levels and reduction in mucosal barrier injury 
(MBI) bacteremia. 
 
1.2.3 
To measure the effects of LBP on the intestinal flora phylogenetic composition 
during and after alternative donor alloHCT using 16S rRNA gene deep 
sequencing. 
 
1.2.4 
To measure effects of LBP on intestinal flora function during and after alternative 
donor alloHCT using metagenomic and metabolite profiling. 
 
1.2.5 
To measure proposed immunomodulatory effects of LBP in mean serum levels of 
alloreactive-induced inflammatory cytokines (IL-2, IL-6, IL-12p70, IFN, TNF, 
etc.) in patients receiving LBP compared to placebo.  
 
1.2.6 
To determine whether LBP administration reduces the incidence of Clostridium 
difficile-associated diarrhea in alternative donor HCT patients. 
 
1.2.7 
To determine whether LBP administration reduces hospital days within the first 
120 days post hematopoietic cell transplant (HCT). 
 
1.2.8 
To define the safety of orally administered LBP strains 299 and 299v in alternative 
donor HCT patients as measured by incidence of Lactobacillus plantarum 
bacteremia.  
 
 
2.0 
BACKGROUND AND HYPOTHESES  
 
2.1 
Acute graft-versus-host disease (GvHD): Clinical impact  
Allogeneic HCT (alloHCT) is the infusion of hematopoietic stems cells from another 
person or from another person’s umbilical cord blood into a recipient. Alternative donor 
alloHCT is the infusion of hematopoietic stems cells from a donor who is not an HLA-
matched related family member. Acute GvHD develops during the first 100 days post HCT 
as a result of the donor stem cells recognizing the recipient’s body as a foreign substance 
and subsequently the donor cells attack the recipient’s tissues and organs. The three main 
areas of the body affected by GvHD are the skin, liver and gut. 
 
Despite prophylactic immune suppression, clinically significant (Grade II–IV) aGvHD 
afflicts up to 45% of pediatric patients receiving alternative alloHCT.1 As aGvHD is 

 
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responsible for nearly 20% of deaths following alloHCT,2 the need for better prevention 
and therapy for aGvHD is readily apparent. Preventative regimens for aGvHD incorporate 
immunosuppressive therapy (IST), which largely targets T-cell activation, function and 
cytokine production,3 causing delays in donor-derived immune recovery4-6 that result in 
increased risk for life-threatening infections.7-9 Unfortunately, the rate of aGvHD has 
remained the same for nearly two decades despite the use of current immunosuppressive 
agents. Therefore, new approaches to prevent the development of severe aGvHD are 
critically needed.10,11 
 
2.2 
GvHD pathophysiology: Focus on the gastrointestinal tract (GI)  
The pathophysiology of aGvHD is complex and involves donor T-cell activation by host 
alloantigens and the subsequent secretion of cytotoxic cytokines including TNF and 
IFN.12 In short, experimental and clinical data support the hypothesis that cytokine 
dysregulation associated with aGvHD occurs in three distinct phases.13,14 In Phase I, HCT 
conditioning regimens instigate damage to the GI epithelium, leading to translocation of 
pro-inflammatory factors like endotoxin from the gut lumen into the bloodstream15 and 
activation of host tissues to secrete inflammatory cytokines.16 The resultant inflammatory 
environment activates host antigen-presenting cells (APCs) as well as induces chemokines 
that recruit additional donor leukocytes into host target organs, including skin, liver and 
gut.17 In Phase II, activated host APCs present alloantigens to donor T cells infused within 
the hematopoietic cell graft.18 In addition to antigen presentation, APCs provide co-
stimulation, further enhancing T-cell activation and clonal expansion. Phase III involves T 
cell-mediated apoptosis of epithelial cells, leading to target organ damage and dysfunction, 
further compromising intestinal epithelial integrity and perpetuating inflammation. Thus, 
the breakdown of GI barrier function serves as both a major instigator and propagator of 
the pathophysiology of aGvHD.19,20 
 
The field of proteomics has enabled biomarker discovery and validation for aGvHD21 risk 
stratification. Specifically, cytokines like ST2 (suppression of tumorigenicity 2) and their 
associated receptors like TNFR1 (tumor necrosis factor receptor 1) have been validated as 
early markers of aGvHD onset and severity.22 Furthermore, involvement of the GI tract in 
the pathogenesis of aGvHD has been substantiated by the translation of preclinical 
observations into validated serum biomarkers of GI tract insult. Specifically, aGvHD has 
been shown to lead to histological loss of Paneth cells and intestinal epithelial stem cells. 
Paneth cell destruction results in decreased production of antimicrobial peptides like α-
defensins23 and the release of the intestinal epithelium-derived C-type lectin, regenerating 
islet-derived IIIα (REG IIIα), into the circulation.23 Clinically, Paneth cell loss is associated 
with aGvHD severity and non-relapse mortality.24 Similarly, plasma elevations in REG IIIα 
also associate with aGvHD severity as well as overall response IST used to treat 
aGvHD.25,26 Lastly, strategies to preserve intestinal epithelial stem cells via either 
intracellular signaling activation27 or endogenous IL-2228 reduce aGvHD severity in mouse 
models. The predominant source of IL-22 in the intestine is thought to be RORγt+ innate 
lymphoid cells (ILCs).29 Clinically, activated ILCs have been shown to reduce aGvHD 
severity in acute leukemia patients undergoing alloHCT; as those patients with decreased 
pre-transplant ILC levels experienced more severe subsequent aGvHD.30 
 
2.3 
An emerging role for the intestinal microbiota in GI aGvHD: Probiotic therapy  
Commensal organisms within the GI tract, collectively known as the intestinal microbiota, 
are predominantly comprised of two phyla, Bacteroidetes and Firmicutes, the latter of 
which includes the microbial order Lactobacillales.31 The intestinal microbiota critically 

 
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functions in maintaining the physical, functional, and immunologic barriers within the GI 
tract such that perturbations in microbiota diversity or dysbiosis result in aberrant systemic 
immune response32 as well as pathogen colonization and mucosal invasion.31 For example, 
dysbiosis induced by broad-spectrum antimicrobial therapy causes life-threatening 
infections from Clostridium difficile33 and vancomycin-resistant Enterococcus34 in 
alloHCT recipients. In addition, aGvHD markedly alters the intestinal microbiota in mice 
and in human alloHCT recipients, decreasing intestinal microbial diversity and increasing 
predominance of enteric pathogens.35 As a result, aGvHD increases the risk of bacteremia 
with enteric pathogens resulting from mucosal barrier injury (MBI).36,37 Furthermore, loss 
of microbial diversity is an independent risk factor for mortality following alloHCT. 
Specifically, lower microbial diversity at the time of engraftment significantly associates 
with decrease in overall survival (OS) and increase in transplant-related mortality (TRM), 
particularly from aGvHD and infection.37 
 
Replenishing commensals like Lactobacillus prior to HCT in recipient mice substantially 
decreases aGvHD severity and intestinal insult.35,38 However, such attenuating effects of 
probiotic administration on aGvHD severity was limited to transplant recipients; as donor 
microbiota does not seem to influence alloreactive T-cell function and aGvHD course.39 
Dual effects of probiotics in maintaining intestinal cell function and inhibiting secretion of 
pro-inflammatory cytokines from intestinal mucosa suggest their beneficial role in the 
prevention and attenuation of aGvHD (Figure 1).35,38 Evidence from clinical studies further 
supports the hypothesis that altering the microbiota may affect clinical outcomes. For 
example, the provision of enteral feeding, in contrast to total parenteral nutrition, is 
associated with an increase in survival and reduced rates of aGvHD.40,41 Jenq et al also 
found that poor oral nutrition appears to more directly alter the composition of the 
microbiota compared to the conditioning regimen. It is hypothesized that administration of 
probiotic therapy will restore microbial diversity and maintain epithelial cell integrity, 
which together will preserve immune tolerance and prevent aGvHD. In addition, it is 
hypothesized that probiotic therapy will decrease susceptibility to enteric pathogens by 
maintaining intestinal luminal integrity. 
 
2.4 
Probiotic Strain 
LBP was selected as the ideal single-
species probiotic for several reasons. 
First, LBP is the only probiotic that has 
been studied during the HCT period and 
has an established safety profile when 
used in immunocompromised adults 
and children.42-45 Furthermore, the 
investigators have direct experience 
with its use in the alternative donor 
alloHCT 
setting.45 
Secondly, 
pre-
clinical and clinical studies suggest that 
Lactobacillus may prevent transplant-
related complications. In a murine BMT 
model, Lactobacillus was administered beginning on the first day of conditioning therapy 
and continued for several weeks after transplant. Lactobacillus administration was 
associated with significantly reduced mortality, aGvHD score, and inflammation (lower 
number of inflammatory cells and no evidence of ulcerations or abscesses) as well as less 
translocation of microorganisms into mesenteric lymph nodes.38 In clinical studies 
performed among adults in the post-transplant period, alloHCT patients experienced 

 
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decreases in GvHD-associated histopathology and mortality that were associated with 
Lactobacillus. The protective effect was attributed to the ability of Lactobacillus 
preventing Enterococcus domination in the GI tract.35 Finally, LBP has an inherent ability 
to tolerate many different conditions.46 Its growth requirement for manganese enables it to 
accumulate high intercellular levels of manganese, which provides protection against 
oxygen toxicity by reducing oxygen free radicals attributed to hydrogen peroxide.47 LBP 
also has a high tolerance to low pH thereby surviving passage through acidic conditions 
like the human stomach. 
 
The current study will use the same probiotic, LBP, which was tested in our pilot study. 
LBP is a combination of two genetically similar strains belonging to the LBP species 
[Lp299v (DSM 9843) and Lp299 (DSM 6595)]. In preclinical studies, Lp299v improves 
mucosal barrier function, resulting in reduced permeability and decreased bacterial 
translocation.48,49 Improved gut barrier function associated with Lp299v has been reported 
for patients in intensive care units44 and patients with obstructive jaundice undergoing 
biliary drainage.50 Lp299v also reduces gut permeability and subsequent bacterial 
translocation in methotrexate-induced enterocolitis.51 In the clinical setting, LBP has been 
used in immunocompromised patients, including solid organ transplants,52,53 HIV 
patients42, and children undergoing HCT45 and found to be safe. 
 
Lp299v increases CD25 surface expression on CD4+ and CD8+ cells54 and activates innate 
immunity (CD56+, CD16+ cells), which together exert immunomodulatory properties. 
Lp299v can produce nitric oxide synthase (NOS) and cyclooxygenase-2 (COX-2) via 
down-regulation of nuclear factor kappa B (NFκB) pathway, revealing a partial molecular 
basis for its anti- inflammatory properties, which have been exploited in clinical trials 
involving Crohn’s disease and irritable bowel syndrome. 43,55,56 In a randomized, double-
blind, placebo-controlled cross-over study involving healthy adult volunteers, subjects 
ingested either living or heat-killed LBP (strain WCFS1). Patients consuming live LBP had 
biopsies of the intestinal duodenal mucosa, which revealed altered mucosal gene 
expression patterns and cellular pathways supporting induction of immune tolerance.56 
 
2.5 
Preliminary Data 
 
2.5.1 
Biomarkers of Intestinal Integrity 
Plasma citrulline is a plasma biomarker of intestinal integrity in adults undergoing 
HCT and in pediatric conditions.57-61 To determine the association of plasma 
citrulline and intestinal integrity in children undergoing alternative donor alloHCT, 
we measured plasma citrulline levels in 10 children and adolescents beginning at 
the start of conditioning therapy and then every three to seven days.62 The mean 
citrulline value prior to the initiation of treatment was 20.1 ±7 mol/L. The greatest 
decrease in mean plasma citrulline occurred between the initiation of conditioning 
and the day of hematopoietic graft infusion (Day 0, 7.1 ±4.9 mol/L) and reached 
a maximal mean nadir at Day 7 (6.6 ±5.3 mol/L). Levels did not begin to recover 
to pre-transplant levels until Day 21 (10 ±3.7 mol/L). Significant associations 
were found between plasma citrulline levels and the odds of developing mucositis 
(OR = 0.88, 95% CI=0.79–0.99, p=0.036) and diarrhea (OR=0.70, CI=0.59–0.84, 
p<0.0001). These results lend further support that plasma citrulline is a marker of 
GI integrity following alternative donor alloHCT. Moreover, we have identified a 
therapeutic window in which an intervention may prevent the decline in GI 
integrity as measured by plasma citrulline levels during HCT. 

 
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2.5.2 
Pilot Study of LBP 
A previous pilot study was completed that evaluated the safety and feasibility of 
LBP, the probiotic for the proposed trial, in 31 children and adolescents (mean age 
7.7 ±4.7y) undergoing HCT (IND # 108,977).45 Of the 31 patients, one patient was 
removed due to the inability to tolerate daily LBP dosing, which resulted in 30 
evaluable participants. No patients suffered a serious adverse event (SAE) 
associated with LBP and no cases of LBP bacteremia were observed, surpassing 
the safety stopping criterion (≥1 case of LBP bacteremia among 30 evaluable 
patients). Of note, one patient with severe aplastic anemia developed acute 
appendicitis on Day 28. Pathologic examination of the appendix revealed a 
predominance of gram-negative enteric organisms and only a small number of 
gram-positive 
organisms 
that 
were 
morphologically 
inconsistent 
with 
Lactobacillus (no culture or PCR testing performed). The patient tolerated the 
appendectomy well, and investigators reported that the appendicitis was not related 
to Lactobacillus.  
 
Three patients died prior to Day 100 and none of these deaths were attributed to 
LBP. Causes of death included veno-occlusive disease, idiopathic pneumonia 
syndrome, and multi-organ system failure in one patient, disease progression in the 
second patient, and complications due to aspiration pneumonia in the third patient. 
The observed deaths were within rates observed in the published literature.2 
 
Incidence of non-Lactobacillus bacteremia (20%) and Clostridium difficile-
associated diarrhea (CDAD, 20%) did not exceed published rates in pediatric 
alloHCT recipients.63-66 Microbial pathogens associated with bacteremia or 
fungemia included: Staphylococcus epidermidis (1), Serratia marcescens (1), 
Enterococcus faecium (1), Klebsiella pneumoniae (3), Streptococcus viridans (1), 
and Fusarium species (1). Up to four weekly stool samples were collected from 22 
patients receiving LBP. Of these patients, the majority (96%, 21/22) had at least 
one stool specimen that was positive for Lactobacillus. In this restricted analysis, 
75% stool specimens (77/102) were positive for Lactobacillus, suggesting LBP 
administration colonized patient stool. The pilot study also found LBP 
administration was feasible during alloHCT. LBP was administered successfully 
to 97% (30/31, 95% CI (83%-100%) of enrolled children whom received at least 
50% of the total intended dose of probiotic. Among the 30 evaluable patients, the 
mean and median percent of doses taken were 92 ±14% and 97% (range 50-100%), 
respectively. 
 
Nine patients (9/30, 30%) developed clinically significant (Grade II–IV) aGvHD. 
Five patients (5/9, 56%), experienced Grade II aGvHD, and 4 patients (4/9, 44%) 
experienced Grade III aGvHD. No patient experienced Grade IV aGvHD. Overall 
incidence of GI aGvHD was 23%, which is lower than some published literature.1 
Importantly, the pilot study provides reasonable evidence that LBP administration 
did not increase the incidence of aGvHD and legitimates further investigation into 
defining its effects on GI aGvHD. 
 
2.6 
Summary 
The proposed trial would be the only aGvHD prevention trial that is not cell-or drug-based 
in children and adolescents undergoing initial alternative donor alloHCT. As the majority 
of standard and investigational pharmaceutical therapies for preventing and treating GvHD 

 
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are globally immunosuppressive, these agents increase the risk for infection and potentially 
increase the risk of malignant relapse by delaying or inhibiting donor-derived immunity. 
Therefore, the major potential advantage for probiotic use as aGvHD prophylaxis is 
reduction in GvHD without inducing immunosuppression. 
 
Initiation of probiotic administration in the proposed study (i.e., at the start of conditioning 
and through Day 56) will be instrumental in improving our understanding of the optimal 
timing of probiotic therapy in HCT recipients. We anticipate that the timing of probiotic 
administration will enhance the hypothesized benefits of probiotic therapy, potentially 
fortifying commensals prior to HCT-induced insult and replenishing commensal gradients 
faster after HCT-induced insult. The proposed study will also elucidate the mechanisms of 
probiotic-associated modulation of the intestinal microbiota composition and function that 
likely underlie anticipated clinical effects in reducing incidence of aGvHD. Gut 
decontamination with oral antibiotics is currently the only studied means to modulate the 
intestinal microbiota and has resulted in mixed clinical results.21 The proposed study will 
increase our understanding in how alloHCT affects the microbiome itself as well as how 
probiotic administration might modify intestinal composition and metabolic activity. 
Furthermore, the use of a pure probiotic versus a composite mixture like fecal transplant 
has the ability to attribute proposed clinical benefit to the probiotic strain itself, simplifying 
mechanistic investigation into proposed immunologic effects. Finally, aGvHD presents a 
unique opportunity to study probiotic effects in a very high-risk patient population prior to 
development of inflammation. Taken together, the proposed clinical trial may significantly 
improve survival and quality of life in alloHCT patients and increase scientific 
understanding for how probiotics mediate their effects. 
 
 
 

 
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3.0 
STUDY ENROLLMENT PROCEDURES AND PATIENT ELIGIBILITY 
 
3.1 
Study Enrollment 
 
3.1.1 
Patient Registration 
Prior to enrollment on this study, patients must be assigned a COG patient ID 
number. This number is obtained via the Patient Registry module in OPEN once 
authorization for the release of protected health information (PHI) has been 
obtained. The COG patient ID number is used to identify the patient in all future 
interactions with COG. If you have problems with the registration, please refer to 
the online help. For additional help or information, please contact the CTSU Help 
Desk at 1-888-823-5923 or ctsucontact@westat.com. 
 
In order for an institution to maintain COG membership requirements, every patient 
with a known or suspected neoplasm needs to be offered participation in APEC14B1, 
Project:EveryChild A Registry, Eligibility Screening, Biology and Outcome Study. 
 
A Biopathology Center (BPC) number will be assigned as part of the registration 
process. Each patient will be assigned only one BPC number per COG Patient ID. 
For additional information about the labeling of specimens please refer to the 
Pathology and/or Biology Guidelines in this protocol. 
 
Please see Appendix I for detailed CTEP Registration Procedures for Investigators 
and Associates, and Cancer Trials Support Unit (CTSU) Registration Procedures 
including: how to download site registration documents; requirements for site 
registration, submission of regulatory documents and how to check your site’s 
registration status. 
 
3.1.2 
IRB Approval 
Each investigator or group of investigators at a clinical site must obtain IRB 
approval for this protocol and submit IRB approval and supporting documentation 
to the CTSU Regulatory Office before they can be approved to enroll patients. For 
CTEP and Division of Cancer Prevention (DCP) studies open to the National 
Clinical Trials Network (NCTN) and NCI Community Oncology Research 
Program (NCORP) Research Bases after March 1, 2019, all U.S.-based sites must 
be members of the NCI Central Institutional Review Board (NCI CIRB). In 
addition, U.S.-based sites must accept the NCI CIRB review to activate new 
studies at the site after March 1, 2019. Local IRB review will continue to be 
accepted for studies that are not reviewed by the CIRB, or if the study was 
previously open at the site under the local IRB. International sites should continue 
to submit Research Ethics Board (REB) approval to the CTSU Regulatory Office 
following country-specific regulations.  
 
Sites participating with the NCI CIRB must submit the Study Specific Worksheet 
for Local Context (SSW) to the CIRB using IRBManager to indicate their intent 
to open the study locally. The NCI CIRB’s approval of the SSW is automatically 
communicated to the CTSU Regulatory Office, but sites are required to contact the 
CTSU Regulatory Office at CTSURegPref@ctsu.coccg.org to establish site 
preferences for applying NCI CIRB approvals across their Signatory Network. Site 
preferences can be set at the network or protocol level. Questions about 

 
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establishing site preferences can be addressed to the CTSU Regulatory Office by 
email or calling 1-888-651-CTSU (2878). 
 
Sites using their local IRB or REB, must submit their approval to the CTSU 
Regulatory Office using the Regulatory Submission Portal located in the 
Regulatory section of the CTSU website. Acceptable documentation of local 
IRB/REB approval includes: 
• 
Local IRB documentation; 
• 
IRB-signed CTSU IRB Certification Form; and/or 
• 
Protocol of Human Subjects Assurance Identification/IRB 
Certification/Declaration of Exemption Form. 
 
In addition, the Site-Protocol Principal Investigator (PI) (i.e. the investigator on 
the IRB/REB approval) must meet the following criteria in order for the processing 
of the IRB/REB approval record to be completed: 
• 
Holds an active CTEP status; 
• 
Rostered at the site on the IRB/REB approval (applies to US and Canadian 
sites only) and on at least one participating roster; 
• 
If using NCI CIRB, rostered on the NCI CIRB Signatory record; 
• 
Includes the IRB number of the IRB providing approval in the Form FDA 
1572 in the RCR profile; and 
• 
Holds the appropriate CTEP registration type for the protocol.  
 
Additional Requirements 
Additional requirements to obtain an approved site registration status include: 
• 
An active Federal Wide Assurance (FWA) number; 
• 
An active roster affiliation with the Lead Protocol Organization (LPO) or 
a Participating Organization (PO); and 
• 
Compliance with all protocol-specific requirements (PSRs). 
 
For information about the submission of IRB/REB approval documents and other 
regulatory documents as well as checking the status of study center registration 
packets, please see Appendix I. 
 
Institutions with patients waiting that are unable to use the Portal should alert the 
CTSU Regulatory Office immediately at 1-866-651-2878 in order to receive 
further instruction and support. For general (non-regulatory) questions call the 
CTSU General Helpdesk at: 1-888-823-5923. 
 
Note: Sites participating on the NCI CIRB initiative and accepting CIRB 
approval for the study are not required to submit separate IRB approval 
documentation to the CTSU Regulatory Office for initial, continuing or 
amendment review. 
 
3.1.3 
Study Enrollment 
Patient enrollment will be facilitated using the Oncology Patient Enrollment Network 
(OPEN). OPEN is a web-based registration system available on a 24/7 basis. OPEN is 
integrated with CTSU regulatory and roster data and with the Lead Protocol 
Organization (LPOs) registration/randomization systems or the Theradex Interactive 
Web Response System (IWRS) for retrieval of patient registration/randomization 

 
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assignment. OPEN will populate the patient enrollment data in NCI’s clinical data 
management system, Medidata Rave. 
 
Requirements for OPEN access:   
• 
A valid CTEP-IAM account; 
• 
To perform enrollments or request slot reservations:  Must be on an LPO 
roster, ETCTN corresponding roster, or participating organization roster 
with the role of Registrar. Registrars must hold a minimum of an Associate 
Plus (AP) registration type; 
• 
If a Delegation of Tasks Log (DTL) is required for the study, the registrars 
must hold the OPEN Registrar task on the DTL for the site; and 
• 
Have an approved site registration for the protocol prior to patient 
enrollment. 
 
To assign an Investigator (IVR) or Non-Physician Investigator (NPIVR) as the 
treating, crediting, consenting, drug shipment (IVR only), or receiving investigator 
for a patient transfer in OPEN, the IVR or NPIVR must list the IRB number used 
on the site’s IRB approval on their Form FDA 1572 in RCR. If a DTL is required 
for the study, the IVR or NPIVR must be assigned the appropriate OPEN-related 
tasks on the DTL. 
 
Prior to accessing OPEN, site staff should verify the following: 
• 
Patient has met all eligibility criteria within the protocol stated timeframes; 
and  
• 
All patients have signed an appropriate consent form and HIPAA 
authorization form (if applicable). 
Note: The OPEN system will provide the site with a printable confirmation of 
registration and treatment information. You may print this confirmation for your 
records. 
 
Access OPEN at https://open.ctsu.org or from the OPEN link on the CTSU members’ 
website. Further instructional information is in the OPEN section of the CTSU website 
at https://www.ctsu.org or https://open.ctsu.org. For any additional questions, contact 
the CTSU Help Desk at 1-888-823-5923 or ctsucontact@westat.com. 
 
3.1.4 
Timing 
Patients must be enrolled before taking the first dose of LBP or placebo. Participants 
may be enrolled up to 14 days prior to the planned start of the conditioning regimen 
and LBP or placebo administration. 
 
All clinical and laboratory studies to determine eligibility must be performed 
within 7 days prior to enrollment unless otherwise indicated in the eligibility 
section below. 
 
3.1.5 
Randomization 
Randomization will occur at the time of enrollment. Randomization will take place 
through the OPEN system. Upon enrollment, sites will receive notification of the 
kit number of study product that should be dispensed to the patient. All personnel 
at the site will remain blinded.  
 

 
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3.2 
Patient Inclusion Eligibility Criteria 
Important note: The eligibility criteria listed below are interpreted literally and cannot 
be waived. All clinical and laboratory data required for determining eligibility of a 
patient enrolled on this trial must be available in the patient's medical/research record 
which will serve as the source document for verification at the time of audit.  
 
All Clinical and Laboratory studies, if applicable, must be obtained within 21 days prior 
to start of protocol therapy (repeat if necessary). Protocol therapy must begin within 6 
months of study enrollment. 
 
See Section 7.1 for required studies to be obtained prior to starting protocol therapy. 
 
3.2.1 
Age 
Patient must be ≥ 2 years of age and ≤ 25 years of age at time of enrollment. 
 
3.2.2 
Diagnosis 
Patient must have a diagnosis that is managed with an alternative donor allogeneic 
hematopoietic cell transplant.  
 
3.2.3 
Performance Level  
Patients must have a Lansky (for patients ≤ 16 years of age) or Karnofsky (for 
patients > 16 years of age) performance status score of ≥ 70. Patients who are 
unable to walk because of a chronic underlying condition (such as paralysis), but 
who are up in a wheelchair, will be considered ambulatory for the purpose of 
assessing performance score. See  
https://members.childrensoncologygroup.org/prot/reference_materials.asp under 
Standard Sections for Protocols.  
 
3.2.4 
Hematopoietic Cell Transplant (HCT) 
Patient must be receiving cells from alternative donor defined as one of the following: 
a. Unrelated donor with a complete HLA match or a 1 or 2 HLA mismatch, 
considering only HLA-A, HLA-B, HLA-C, and HLA-DRB1. 
b. Related donor with a 1 or more HLA mismatch (including haplo-identical).  
 
Note: history of HCT or other cellular therapy (e.g., CAR-T cells, donor 
lymphocyte infusions) is permitted. 
 
3.3 
Exclusion Criteria 
 
3.3.1 
Patient plans on receiving stem cells from a matched (8/8) related donor.  
 
3.3.2 
Patient has used a probiotic dietary supplement within 30 days prior to enrollment. 
(Consumption of yogurt products is allowed.) 
 
3.3.3 
Patient has a history of severe GI tract insult including but not limited to previous 
bowel perforation, Grade 4 neutropenic colitis or typhlitis, inflammatory bowel 
syndrome, short small bowel syndrome (Crohn’s disease, ulcerative colitis), 
history of gastrointestinal GVHD, or history of bowel resection.  
 

 
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3.3.4 
Patient has a medical, psychiatric or social issue that would compromise patient 
safety or compliance with protocol therapy, or interfere with consent, study 
participation, follow up, or interpretation of study results. 
 
3.3.5 
Female patients who are pregnant are not eligible. Women of childbearing 
potential require a negative pregnancy test prior to enrollment. 
 
3.3.6 
Patient has diarrhea at the time of enrollment which is Clostridium difficile toxin 
positive. 
 
3.3.7 
Patient is receiving antibiotic therapy for an active bacterial infection. 
 
3.3.8 
Patient is allergic to all third or fourth generation cephalosporins, carbapenems, 
and all aminoglycosides, which are used to empirically treat LBP bacteremia. 
 
3.4 
Regulatory Requirements 
 
3.4.1 
All patients and/or their parents or legal guardians must sign a written informed 
consent. 
 
3.4.2 
All institutional, FDA, and NCI requirements for human studies must be met. 
 
 

 
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4.0 
TREATMENT PLAN 
 
Timing of protocol therapy administration, response assessment studies, and surgical 
interventions are based on schedules derived from the experimental design or on established 
standards of care. Minor unavoidable departures (up to 72 hours) from protocol directed therapy 
and/or disease evaluations (and up to 1 week for surgery) for valid clinical, patient and family 
logistical, or facility, procedure and/or anesthesia scheduling issues are acceptable per COG 
administrative Policy 5.14 (except where explicitly prohibited within the protocol). 
 
4.1 
Overview of Treatment Plan 
This will be a double-blind, randomized, placebo-controlled, intervention study that will 
equally allocate 454 children to either LBP or placebo for approximately two months. All 
patients, patient caregivers, direct healthcare providers, pharmacy and the institutional 
study team will be blinded to the randomization allocation. Sites will receive notification 
of the study product kit number to dispense to the patient via email at OPEN enrollment 
submission. The patient should receive the same study product kit number throughout the 
study. Study participants will receive a course of either LBP or placebo for approximately 
64 days, starting at the initiation of conditioning regimen and ending at Day 56. However, 
participants will be followed until Day 120 to ensure capturing development of aGvHD. 
The table below provides a study overview.  
 
 
Start of 
conditioning 
Day 0 
(Graft 
Infusion) 
Day 7 
Day 14 
Day 28 or 
discharge 
Day 56 
Day 120 
Study Product 
Administration 
(LBP or placebo) 
 
 
 
 
 
 
 
Specimen 
Collection 
-Blood 
-Stool 
-Blood 
-Stool 
-Blood 
-Stool 
-Blood 
-Stool 
-Blood 
-Stool 
-Blood 
-Stool 
-Stool 
 
Please see Section 7.1 for specimen requirements.  
 
4.2 
LBP or Placebo Administration 
 
4.2.1  Administration Schedule 
Dosing will begin on the first day of the transplant conditioning regimen and 
continue through day 56 post HCT. The LBP/Placebo does not necessarily need to 
be given prior to receiving the first dose of the conditioning therapy as long as it 
starts on that first day. The first day of the transplant conditioning regimen is 
defined as the first day of chemotherapy or TBI, excluding any preceding anti 
T-lymphocyte antibody therapy. 
 
4.2.2  Dosing and Administration Guidelines 
Dose: The dose used in the study is 1 x 108 colony forming units (CFU)/kg/day. 
Patients will ingest 1 to 9 mL of reconstituted (mixed solution) LBP or placebo 
once each day. Please refer to the dosing chart below. All doses should be 
calculated based on the body weight at the start of the conditioning regimen. This 
should be the weight performed on the day of admission for HSCT (±72 hours). 
There will be no dose adjustment for weight gain or loss during the course of 
treatment.  

 
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See protocol Section 6.1.5 and the Pharmacy Manual (available on the COG 
protocol web page) for complete and current preparation instructions. The 
LBP/Placebo is supplied in capsules that must be opened and the contents mixed 
into the volume of liquid specified in the Pharmacy Manual. Once mixed, it is 
referred to as the “Mixed Solution” for administration as per the chart below.  
 
Study Product Dosing Chart 
Subject’s Weight (kg)* 
Total Amount of Mixed Solution to be Administered (mL) 
≤ 16 kg 
1 mL 
17-27 kg 
2 mL 
28-38 kg 
3 mL 
39-49 kg 
4 mL 
50-61 kg 
5 mL 
62-72 kg 
6 mL 
73-83 kg 
7 mL 
84-94 kg 
8 mL 
≥ 95 kg 
9 mL 
* Subject’s Weight should be rounded to the nearest whole kilogram and total mixed solution selected 
accordingly. Any decimal < 0.5 round down, any decimal ≥ 0.5 round up. For example: a weight of 
16.5 kg would be rounded up to 17 kg and 2 mL dose selected as per the Dosing Chart above. 
 
• 
Patients may receive LBP or placebo orally or through a nasojejunal, nasogastric 
or gastronomy tube. (Please note, an NJ, NG or G tube should not be placed solely 
for the administration of LBP or placebo.)  
• 
Patient should ingest the dose of LBP or placebo within 30 minutes of mixing. 
• 
LBP or placebo can be taken at any time during the day with or without cold or 
room temperature food.  
• 
Liquids warmer than 98°F (36°C) are not allowed for mixing the product as high 
temperature may kill the bacteria.  
• 
If vomiting occurs within 30 minutes of taking the dose, the dose may be repeated 
once on the same day.  
• 
If a dose is missed, it should be taken immediately and only if there are at least 12 
hours until the next scheduled dose. If there are less than 12 hours until the next 
dose, wait until the next scheduled dose.  
• 
If several doses are missed due to inability to tolerate oral administration (e.g. 
mucositis or nausea/vomiting), therapy should resume as soon as the patient is able 
to tolerate oral administration. Please ensure all missed doses are documented in 
the patient medical record if the patient is in the hospital or patient diary (available 
on the COG protocol web page) if the patient is being treated at home.  
 
Most patients will receive the LPB or placebo in both the inpatient and outpatient 
settings. Adherence to the prescribed regimen will be measured by examination of 
medication administration records and as documented in the electronic medical 
record or other hospital source documentation. Patients will be given a diary to 
document the doses they take at home, which includes instructions for preparation 
and storage.  
 

 
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Adherence will be defined as administration of at least 80% of the prescribed 
doses. In the home setting, medication will be dispensed for a specified time period 
and medication diaries will be completed by families and delivered to the 
institution on a weekly basis at the time of weekly visits. 
 
4.2.3 
Discontinuation due to positive blood culture for LBP 
Patients with a blood culture positive for LBP at any time during the study will 
discontinue the study agent (LBP or placebo) immediately and the patient will be 
removed from protocol therapy. LBP-bacteremia will be considered an 
unexpected severe adverse event and must be reported within 24 hours through 
the CTEP-AERS system. See Section 11.0 for reporting requirements.  
 
For patients who test positive for LBP in the blood, empiric treatment should begin 
with either a third- or fourth-generation cephalosporin or carbapenem. If the clinical 
condition worsens after 24 hours, consideration should be given to adding an 
aminoglycoside. Empiric therapy should continue until sensitivity results are available. 
Prompt consultation with an Infectious Disease Specialist is recommended. 
 
4.3 
Concomitant Therapy 
 
4.3.1 
Supplementation with any additional probiotic dietary supplements is not 
permitted (food containing live cultures like yogurt is allowed) through Day 120.  
 
4.3.2 
Prophylactic use of both broad-spectrum and narrow-spectrum antibiotics is 
permitted. Information regarding antimicrobial therapy (agent, start and stop times, 
indication for use) will be collected throughout the study time period. 
 
 
5.0 
EMERGENCY UNBLINDING 
Only in the event of a serious adverse event wherein the investigator feels that the patient cannot be 
adequately treated without knowing the identity of the study medication may the medication code be 
broken for a particular patient. If emergency un-blinding is required, the patient must stop taking the 
LBP or placebo and be removed from protocol therapy. The treating physician or their designee should 
use the contact information provided below to obtain the patient’s un-blinded treatment information.  
 
Emergency Unblinding Instructions 
 
During normal business hours (9am-5pm Pacific Time) 
Please submit unblinding requests using the procedure below: 
1. Email to BOTH of the following: 
a. ACCL1633_unblinding@childrensoncologygroup.org 
b. Research Coordinator email listed on COG protocol webpage 
 
2. Use email subject line: “ACCL1633 – Requesting treatment unblinding” 
 
3. Provide the following info in the body of the email: 
a. COG Patient ID 
b. Treatment Assignment (Kit Number) 
c. Email address for site investigator 
d. Phone contact for any follow up questions/response to requesting institution 

 
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COG Contact Information 
• 
Study Research Coordinator – Contact information listed on the COG Protocol Webpage 
• 
COG Operations Office main line – (626) 241-1500 
 
After hours 
Please contact the Investigational Drug Service at the Perelman School of Medicine, University of 
Pennsylvania – Emergency pager (800) 670-3151. Be sure to provide a callback number. 
 
When calling, be ready to provide (1) COG patient ID (2) patient’s treatment assignment (kit 
number) and (3) investigator’s callback information.  
 
COG Operations or the Investigational Drug Service located at the Perelman School of Medicine, 
University of Pennsylvania, will contact the site investigator to provide the patient’s unblinded 
treatment information. 
 
Emergency Unblinding CRF 
Once unblinded treatment assignment has been received, complete the Emergency Unblinding CRF 
in iMedidata Rave by accessing the patient’s main page in Rave and using the Add Event dropdown.  
 
 
6.0 
AGENT INFORMATION  
 
6.1 
  
 
 
 
    (04/15/2021) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 

 
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6.1.2 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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6.1.3 
 
 
 
 
 
 
 
 
 
 
 
  
 
6.1.4 
 
  
 
 
 
 
  
 

 
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6.1.5 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
  
 
 
 
  
 
 
 

 
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7.0 
EVALUATIONS/MATERIAL AND DATA TO BE ACCESSIONED 
 
Timing of protocol therapy administration, response assessment studies, and surgical interventions are 
based on schedules derived from the experimental design or on established standards of care. Minor 
unavoidable departures (up to 72 hours) from protocol directed therapy and/or disease evaluations 
(and up to 1 week for surgery) for valid clinical, patient and family logistical, or facility, procedure 
and/or anesthesia scheduling issues are acceptable per COG administrative Policy 5.14 (except where 
explicitly prohibited within the protocol). 
 
7.1 
Required Clinical and Laboratory Evaluations 
 
Studies 
Eligibility$ 
First day of 
conditioning@,& 
Day 0 
(Graft 
Infusion) 
Day  
7 
Day 
14 
Day 28  
or 
Discharge% 
Day 
56 
Day 
120 
History  
X 
----- 
----- 
----- 
----- 
----- 
----- 
----- 
Physical examination, 
evaluation of GvHD 
X 
X 
X 
X 
X 
X 
X 
X 
Performance status 
X 
----- 
----- 
----- 
----- 
----- 
----- 
----- 
Pregnancy test* 
X 
----- 
----- 
----- 
----- 
----- 
----- 
----- 
Screening for Clostridium 
difficile (only if diarrhea at 
enrollment or first day of 
conditioning) 
X 
X& 
----- 
----- 
----- 
----- 
----- 
----- 
Review of LBP 
administration/patient 
diary 
----- 
----- 
X 
X 
X 
X 
X 
 
Blood sample for citrulline 
and cytokine studies  
See Section 7.4.1 
----- 
X^ 
X 
X 
X 
X 
Xa,b 
 
Stool sample for 
correlative studies  
See Section 7.4.2 
----- 
X^ 
X 
X 
X 
X 
Xb 
Xb 
$ All clinical and laboratory studies to determine eligibility must be performed within 7 days prior to 
enrollment/randomization unless otherwise indicated. 
^ Specimen collection on first day of conditioning or anytime within 72 hours before the start of 
conditioning (prior to chemotherapy). See Section 7.4.1 and Section 7.4.2 for overview of procedures and 
a Specimen Collection Manual available on the COG protocol web page with further technical detail. 
@ The first day of the transplant conditioning regimen is defined as the first day of chemotherapy or TBI, 
excluding any preceding anti T-lymphocyte antibody therapy. See Section 4.2.1. 
% Day 28 or day of discharge, whichever comes first.  
* Women of childbearing potential, defined as a female who has had her first menstrual cycle. 
& If the patient has diarrhea on the first day of conditioning and repeat stool culture shows C. diff, then the 
patient is ineligible and must be removed from protocol therapy. Reminder: patient condition(s) prior to 
initiation of protocol therapy must be within parameters required for eligibility. (See Section 8.1) 
a Citrulline sample only. 
b Blood and stool specimens may be collected within 14 days of the designated time point without being 
considered a protocol deviation.  
 
 

 
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7.2 
Data Collection 
The following table represents data that will be collected during the study. 
Studies 
First day of 
conditioning@  
Day 0 
(Graft Infusion) 
Day 
7 
Day 
14 
Day 28 or 
Discharge% 
Day 
56 
Day 
120 
Review of LBP or placebo 
administration # 
X 
X 
X 
X 
X 
X 
----- 
Use of TPN/EN  
(yes or no and days of 
administration) 
X 
X 
X 
X 
X 
X 
X 
Use of antimicrobial agents 
X 
X 
X 
X 
X 
X 
X 
Bloodstream infection 
X 
X 
X 
X 
X 
X 
X 
Clostridium difficile-
associated diarrhea 
X 
X 
X 
X 
X 
X 
X 
@The first day of the transplant conditioning regimen is defined as the first day of chemotherapy or 
TBI, excluding any preceding anti T-lymphocyte antibody therapy. See Section 4.2.1. 
% Day 28 or day of discharge, whichever comes first.  
# Total days product was administered will be collected for each reporting period. See Section 4.2.2.  
 
7.3 
Correlative Studies 
 
7.3.1 
Serum citrulline levels 
Serum specimens will be evaluated for citrulline levels by the laboratory of 
Dr. Regina Santella at CUMC. Serum citrulline will be evaluated from the 
initiation of therapy to Day 56. Given previous experience, it is predicted that 
serum citrulline levels are expected to nadir at Day 7 and not begin to rebound 
until Day 21 in patients receiving placebo. In comparison, it is predicted that 
patients receiving LBP will have a less profound decrease in mean serum citrulline 
at Day 7 and earlier kinetic rebound in mean serum levels, reflecting less mucosal 
insult as hypothesized. Given the proposed effect in maintaining and/or attenuating 
injury to intestinal luminal integrity, it is hypothesized that maintaining higher 
serum citrulline levels in patients receiving LBP will be associated with less GI 
aGvHD and less mucosal barrier insult blood stream infections (MBI BSI). 
 
7.3.2 
Intestinal flora phylogenetic composition 
Evaluation of the intestinal flora will be from the initiation of LBP therapy to Day 
120. DNA from each stool specimen collected will be extracted and purified as we 
have performed previously.34 Phylogenetic profiling using 16S rRNA gene 
sequencing will be performed on the Illumina MiSeq platform with 175 bp paired 
end reads of V4-V5 region amplicons. Sequence data will be compiled and 
processed using mothur74 and QIIME75, screened and filtered for quality,76 then 
classified to the species level77 using the Greengenes reference database.78 
 
7.3.2.1 Evaluating baseline colonization with LBP, loss of LBP during the 
course of hospitalization, re-colonization rate with LBP introduction 
and persistence of re-colonization 
Evidence of baseline colonization with LBP in our experience at MSKCC 
is uncommon. In 230 adult patients admitted for alloHCT, only 24 (10%) 
had detectable levels of LBP as measured by 16S deep sequencing. 
Pediatric patients will have a somewhat higher baseline colonization rate 
with LBP.79 LBP strains isolated from the flora of healthy individuals have 
generally been reported to be antibiotic-sensitive with notable exceptions 

 
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of resistance to streptomycin, tetracycline, levofloxacin, and vancomycin 
being notable exceptions.80 The vast majority of isolates are beta-lactam 
sensitive. Thus, we predict that during the course of alloHCT, patients who 
at baseline are colonized with LBP and require broad-spectrum antibiotic 
therapy for treatment of febrile neutropenia will demonstrate loss of 
commensal LBP. Because patients will continue to be administered LBP 
beyond transplant hospitalization (and after discontinuing antibiotic 
therapy often given during this hospitalization), we also predict that LBP 
introduction will lead to improved recovery of LBP abundance and that 
introduction will lead to persistent colonization. 
 
7.3.2.2 Evaluating the effects of LBP introduction on the abundance of other 
members of the intestinal flora 
LBP species are known to produce lactate via fermentation of a variety of 
sugars.46 Lactate, in turn, can support other strains of bacteria, contributing 
to bacterial diversity. Bacterial strains with demonstrated lactate 
utilization can in turn produce butyrate,81 which can mediate anti-
inflammatory effects through recruitment of regulatory T cells (Tregs).82-
84 Species that can utilize lactate include relatives of Eubacterium halli and 
Anaerostipes caccae.81 LBP species have also been found in animal models 
to mediate suppression of potentially pathogenic commensal bacteria, 
such as E. coli, within the intestinal tract,85 perhaps via promoting 
augmented IgM and IgA responses against these pathobionts. Bacteria 
including E. coli and its relatives from the phylum Proteobacteria have 
been observed to commonly expand and dominate the intestinal tract in 
patients following allo HCT86 and have been found to be associated with 
aggravated GvHD in animal models23 and increased mortality in allo HCT 
patients.37 In a similar manner, introduction of Lactobacillus species have 
also been observed in animal models to suppress the expansion of 
Enterococcus species,35 leading to reduced GvHD. Enterococcal 
expansion, similar to Proteobacterial expansion, has been implicated in 
mediating aggravated GvHD in allo HCT patients.87 Based on these prior 
results reported in the literature, we predict that LBP introduction will 
promote bacterial diversity by supporting the abundance of lactate utilizers 
and preventing domination by Proteobacteria and Enterococci. We will 
quantify microbial diversity using the Shannon diversity index 88 
following closed-reference operational taxonomic unit picking.75 We will 
also perform abundance comparisons evaluating for taxons modulated by 
LBP introduction using linear discriminant analysis (LDA) effect size 
(LEfSe) analysis.89 We will compare relative abundances, determined by 
16S deep sequencing, as well as absolute abundances by utilizing relative 
abundance data combined with quantitative total 16S gene levels using 
validated primers.90 
 
7.3.3 
Evaluating functionality of the intestinal flora 
While isolates with near-identical 16S rRNA gene sequences tend to share many 
traits, they can often have variable gene content and as a result variable properties, 
even strains that are considered to be the same species.91 Thus phylogenetic data 
can at best only provide an approximation of gene content and function. To further 
our understanding of how LBP can potentially reduce the risk of aGvHD in 
pediatric HCT recipients, we propose two targeted discovery approaches designed 

 
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to explore and identify mechanisms by which LBP may mediate a beneficial effect. 
These approaches include whole shotgun metagenomic sequencing and 
commercially available functional metabolite profiling. 
 
7.3.3.1 Evaluating the effects of LBP introduction on intestinal flora function 
via whole shotgun metagenomics sequencing to identify bacterial 
genes and gene pathways associated with modulation of GvHD 
Identifying mechanisms by which bacteria can modulate human 
phenotypes remains a formidable challenge in the microbiota field. One 
promising approach is metagenomic sequencing of whole community 
DNA,91 or “shotgun sequencing.” Whole shotgun metagenomic 
sequencing provides bacterial gene abundance data, which allows 
quantification of the abundance of individual genes and gene pathways. 
Following identification of bacterial DNA sequences, sequences are 
mapped against a reference database, such as the Kyoto Encyclopedia of 
Genes and Genomes (KEGG) database,92 to identify bacterial enzymes in 
metabolic reference pathways. This approach has recently identified 
pathways that are enriched in the flora of patients with Type 2 diabetes 
mellitus,93 obesity,94 and newly-diagnosed Crohn’s Disease.95 
 
We will randomly select a subset of 30 samples from patients with similar 
overall 16S abundance, but 15 samples will have abundant LBP and 15 
additional samples will have undetectable LBP. We will utilize the 
Illumina HiSeq2000 platform, aiming for a mean of 10 gigabases of 101 
bp paired-end reads per sample. For comparative quantification of gene 
content at the individual gene level and the level of KEGG metabolic 
pathways, we will utilize linear discriminate analysis (LDA) effect size 
(LEfSe) analysis89 for biomarker discovery. In addition to comparing 
samples with abundant and reduced LBP levels, we will also examine 
samples from patients who did or did not develop aGvHD. With this 
approach, we hope to identify novel bacterial genes and gene pathways, 
some of which may be LBP-derived, that are associated with modulation 
of aGvHD and may play a role in aggravating or attenuating inflammation. 
As all correlative assays will be performed on the backbone of a COG 
clinical trial, we anticipate high quality and very complete assessment 
laboratory findings. 
 
7.3.3.2 Evaluating the effects of LBP introduction on intestinal flora function 
via functional metabolite profiling 
While whole shotgun metagenomic sequencing provides information 
regarding the functional potential of the intestinal flora, a complementary 
approach is to assay directly for demonstration of substrate utilization and 
metabolite production. By determining bacterial metabolite abundances, 
one can confirm that increased abundance of individual genes and genes 
of metabolic pathways are associated with a corresponding increase in 
enzymatic activity. This method can also identify novel bacterial 
metabolites that are biologically active. One example of a group of 
metabolites that have garnered interest recently is short-chain fatty acids 
(SCFA), which are produced by many bacteria as a byproduct of 
carbohydrate fermentation. SCFA have been found to be important 
modulators of the immune system. In germ-free mice and vancomycin-

 
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treated conventional mice, administration of SCFA (acetate, propionate, 
or butyrate) restored normal numbers of Tregs in the large intestine.84 
GPR43 (a SCFA receptor) has been identified as necessary for mediating 
the anti-inflammatory effects of commensal bacteria96 and is required for 
restoration of large intestinal Tregs by SCFA.84 We will quantitatively 
profile levels of enzymatic substrates and metabolites via UHPLC-MS 
services coupled with an extensive library with >2800 authenticated 
compounds provided by Metabolon (Durham, NC). We will profile 
aliquots of the same stool samples selected for the whole shotgun 
metagenomic sequencing approach detailed above as well as paired serum 
samples collected at the same time as the stool samples. A recent study 
linking intestinal flora with severity of an autism-like syndrome in mouse 
models similarly utilized Metabolon’s services to identify a serum 
metabolite that induced anxiety-like behavior in these mice. Interestingly, 
the levels of this metabolite were modulated by intestinal flora 
composition.97 Using this approach, we will identify novel bacterial 
metabolites, some of which may be LBP-derived, that are associated with 
modulation of aGvHD and may play a role in either aggravating or 
attenuating inflammation. 
 
7.3.4 
Serum cytokines 
To measure proposed immunomodulatory effects of LBP as decreases in mean 
serum levels of alloreactive-induced inflammatory cytokines and of aGvHD-
associated GI insult in patients receiving LBP prophylaxis compared to patients 
receiving placebo. Specifically, TNFR1, ST2 and REG3 will be analyzed given 
their previous validation as early biomarkers for aGvHD initiation and severity.98 
Furthermore, the concentrations of ST2 and REG3α on day 7 post-BMT strongly 
correlate with the development of GVHD-related mortality, steroid-refractory 
GVHD, grade III/IV GVHD and GI GVHD stage 3/4.99 Serial serum biomarker 
levels will be evaluated from the initiation of therapy to Day 30. We expect that 
patients who do not develop GVHD will have lower levels of these biomarkers on 
Days 7 and 14 than patients who develop GVHD. Alloreactive-induced 
inflammation in the early post-transplant period activates recipient antigen-
presenting cells, which, in turn, instruct donor T-cells in the allograft to produce 
cytotoxic soluble factors like TNF and IFN that cause damage to the GI 
epithelium. As maintaining tolerance within the GI tract is an established function 
of the gut microbiota, LBP prophylaxis may induce tolerogenic effects, blunting 
allo-induced inflammation and subsequent tissue damage in transplant recipients. 
Therefore, we hypothesize that LBP-associated maintenance or restoration in the 
microbiota will attenuate serum levels of pro-inflammatory cytokines in the early 
post-transplant period. Specifically, patients receiving LBP prophylaxis will have 
reduced serum levels of inflammatory cytokines compared to control patients. 
 
 

 
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7.4 
Specimen Collection, Processing and Shipping 
 
7.4.1 
Blood Specimens (Citrulline and Cytokines) 
Sample Time 
Points: 
• 
First day of conditioning or anytime within 72 hours before 
the start of conditioning (prior to chemotherapy) 
• 
Day 0 (graft infusion) 
• 
Day 7 
• 
Day 14 
• 
Day 28 or discharge, whichever is first 
• 
Day 56 (citrulline only) 
Sample acquisition can occur within 72 hours before the first day of 
conditioning. Sampling can occur within 24 hours before or after 
designated collection times for D0, D7, D14, D28 without being 
considered a protocol deviation. D56 sampling can occur within 14 
days without being considered a deviation. 
Blood Collection 
Procedure: Draw 6 mL of whole blood in a red top tube (supplied by site). 
Specimen 
Processing: 
1. After collection, allow blood to coagulate at room temperature. 
2. After coagulation (but within 1 hour after collection) centrifuge 
the blood samples at 4°C for 15 minutes at 3000 rpm. 
3. Aliquot the serum (upper layer) into three 1.5 mL cryovials 
(0.5 mL of serum per aliquot). 
4. Store frozen at -80°C (or -70°C) immediately after aliquoting. 
Specimen 
Labeling: 
Freezer-safe labels will be provided in specimen kits and must be 
used as specified in the Specimen Collection Manual. Each cryovial 
must be legibly labeled with: ACCL1633, COG Patient ID, 
Timepoint + Aliquot ID (to track individual vials), and Collection Date.  
Specimen 
Packaging and 
Shipping: 
Samples should remain frozen at -80°C (-70°C is permitted) until ready 
for shipping. Batched shipment after each participant’s completion of 
all specimens is strongly encouraged. Be sure to include enough dry 
ice in the shipment to last 48 hours. Prior to shipment, the lab should 
be notified by e-mail with a copy of Specimen Transmittal Form(s) and 
Specimen Processing Worksheet (with FedEx tracking #) to:   
 
 
 
 
 
 
 
 
Blood should be shipped priority overnight to: 
 
 
 
 
 
 
  
 
 

 
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7.4.2 
Stool Specimens 
Please refer to the Specimen Collection Manual and video on the ACCL1633 page 
on the COG website for detailed instructions. Sites do not require special lab 
certification to process stool samples. However, local personnel collecting or 
processing study specimens should carefully review training references and forms. 
Sample 
Time 
Points: 
• 
First day of conditioning or anytime within 72 hours before the start of 
conditioning (prior to chemotherapy) 
• 
Day 0 (graft infusion) 
• 
Day 7 post-transplant 
• 
Day 14 post-transplant 
• 
Day 28 post-transplant or discharge, whichever is first 
• 
Day 56 post-transplant 
• 
Day 120 post-transplant 
Sample acquisition can occur within 72 hours before the first day of conditioning. 
Sampling can occur within 24h before or after designated collection times for D0, 
D7, D14, D28 without being considered a protocol deviation. If the patient is not 
able to produce a stool sample in the appropriate time frame, obtain sample as soon 
as possible. This will not be considered a protocol deviation. 
For D56 and D120 sample acquisition can occur within 14 days before or after the 
time point without being considered a deviation.  
Supplies 
Required 
for  
Stool 
Collection  
& 
Processing: 
The following stool specimens are required: 
A. 2 weighed Eppendorf vials (metabolomics)  
B. 4 cryovials (1.5 mL) for microbiome sequencing and long term storage  
Materials for Collection: 
A. Stool hats for collection 
B. Aliquot Vials 
- 
2 Eppendorf vials 
- 
4 cryovials  
C. Scale (must weigh in increments of .001 grams) for weighing specimen for 
Eppendorf vials only 
D. Pipette (for watery stool) or scoop and/or orange stick (for solid stool)  
E. Gloves – must be worn at all times when handling stool and the vials. 
F. Mask (optional) 
G.  Black, fine-tip Sharpie pen and Freezer-safe Labels (provided in kits) 
Specimen 
Labeling: 
Reminder: freezer-safe labels will be provided in specimen kits and must be used as 
specified in the Specimen Collection Manual. Each specimen must be legibly 
labeled with: ACCL1633, COG Patient ID#, Timepoint + Aliquot ID, and 
Collection Date.  
For specimens placed in Eppendorf vials, weight before and after filling with stool 
must be documented on the Specimen Transmittal Form. Stool quality should be 
recorded on the Specimen Processing Worksheet. (See the Manual or Worksheet for 
stool quality classification guidelines.) 

 
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Specimen 
Processing: Metabolomics (SCFA, bile acids, amino acids)  
For each sample: 
1. Weigh and record weight of two (2) empty Eppendorf vials separately; indicate 
weight on specimen forms. Indicate one Vial A and the second Vial B. Make sure 
Vial A and Vial B are labeled clearly (e.g. label the 2 vials collected on Start of 
Conditioning as: SOCA, SOCB). 
2. Aliquot using a pipette or scoop and/or orange stick 50-150 mg of sample into each 
vial. Please do NOT exceed 150 mg of stool per aliquot. 
3. Weigh and record weight of Vial A + sample, and Vial B + sample; enter each 
weight on specimen forms, separately. 
4. Confirm that required labels are on each aliquot and that the details (i.e. the vial 
weights) are accurately recorded on the specimen forms. 
5. Store aliquots at -80°C (-70°C is permitted). 
Microbiome Sequencing and Long-Term Storage (4 x 1.5 mL cryovials) 
For each of four (4) aliquots:  
1. Fill the cryovial using a pipette or scoop and/or orange stick with stool.  
2. Tighten the lid back on the vial. 
3. Place label on specimen aliquots and complete the specimen forms. 
4. Store vials at -80°C (-70°C is permitted). 
Specimen 
Packaging 
and 
Shipping: 
Samples should remain frozen at -80°C (or -70°C) until ready for shipping. Batched 
shipment after a participant’s completion of all seven time points is strongly 
encouraged. Be sure to include enough dry ice in the shipment to last 48 hours. Prior 
to shipment, the lab should be notified by e-mailing electronic copies of the Specimen 
Transmittal Form(s) and Specimen Processing Worksheet(s) (with FedEx tracking #) 
to: 
  
 
Single or multiple patient specimens may be bulk shipped to the Microbiome center 
at Columbia University Medical Center. If sending samples on multiple patients, be 
sure each patient’s samples are bagged separately and clearly labeled. All specimen 
shipments must include Specimen Transmittal Form(s) and Specimen Processing 
Worksheet. 
 
 
 
 
 
 
  
  
 
 
 
 
 
 
 
 
   
 
 

 
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8.0 
CRITERIA FOR REMOVAL FROM PROTOCOL THERAPY AND OFF STUDY 
CRITERIA 
 
8.1 
Criteria for Removal from Protocol Therapy 
a. Completion of therapy. 
b. Patient develops a LBP-associated bacteremia. 
c. Patient relapses. 
d. Patient develops another malignancy.  
e. Refusal of further protocol therapy by patient/parent/guardian. 
f. 
Physician determines it is in patient’s best interest. 
g. Patient requires emergency unblinding.  
h. Patient never received transplant.  
i. 
Patient experiences an adverse event requiring removal from protocol therapy.  
j. 
Repeat eligibility studies (if required) or change in patient condition(s) prior to the 
initiation of protocol therapy are outside the parameters required for eligibility (see 
Section 3.2 and Section 3.3). 
k. Patient develops graft failure (see Section 10.3). 
 
Patients who are off protocol therapy are to be followed until they meet the criteria for Off 
Study (see below). Follow-up data will be required unless patient is taken off study. Follow 
up time is defined as the duration of time between patient enrollment and assessment of 
primary outcome (120 days after enrollment). 
 
8.2 
Off Study Criteria 
a. Death. 
b. Lost to follow-up. 
c. Withdrawal of consent for any further data submission. 
d. Patient never received conditioning 
e. Completion of follow-up through Day 120. 
 
 
9.0 
STATISTICAL CONSIDERATIONS 
 
9.1 
Sample Size and Study Duration 
Based on estimates from Center of International Blood and Marrow Transplant Research 
(CIBMTR), approximately 1,400 allogeneic SCTs are performed at COG institutions each 
year with approximately 25% of them (350 per year) meeting the definition of “alternative 
donor” as defined in this study. If 30% of those eligible are enrolled, the accrual rate will 
be about 105 per year. Accounting for start-up time and possible delay of up to 4 months 
between the two stages of the study, we project accrual to the screening stage within 1.2 
years and total accrual duration of 5 years. The sample size of 454 (the first 120 for the 
screening stage and an additional 334 for the confirmatory stage) may be increased to a 
maximum of 500 patients (up to 132 for screening stage and an additional 368 for the 
confirmatory stage) to conservatively account for up to 10% loss due to ineligible or not 
being randomized to a treatment regimen. Evaluable patients are defined as eligible (or 
meeting repeated eligibility criteria) randomized patients. 
 
9.2  
Study Design 
This is a randomized, double-blind intervention trial evaluating the efficacy of LBP in 
preventing GI aGvHD in children and adolescents undergoing alternative donor HCT. 

 
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Patients will be randomized to receive daily LBP or placebo using a 1:1 allocation. 
Randomization will be stratified by stem cell source (cord vs. marrow or PBSC), HLA 
degree of match (complete match vs. 1 mismatch vs. 2 or more mismatches), and GvHD 
prophylaxis (ATG/CAMPATH vs. No ATG/CAMPATH). Haplo patients are those who 
have a related donor with 2 or more HLA mismatches. For stratification, Haplo would be 
included into the stem cell source. AGvHD outcomes will be assessed from day of stem 
cell infusion (Day 0) through Day 120. Accrual will be conducted in two stages: a 
randomized screening stage, and a larger confirmatory stage. The screening stage will 
compare the rate of GI aGvHD between the 2 arms at =0.30 (1-sided). After recruitment 
of 120 patients (up to a maximum of 132 to account for up to 10% loss due to patients 
deemed to be ineligible or not being randomized to a treatment regimen), the evaluation of 
the outcome will be assessed. If the screening stage suggests efficacy (p<0.3), the study 
will proceed to the confirmatory stage (N=454). We will use a modified intention-to-treat 
analytic approach for all evaluable patients. Based on previous CCL studies, we expect the 
inevaluable rate to be very low. 
 
The primary outcome measure is the incidence of Stage 1-4 GI aGvHD from the day of 
stem cell infusion (Day 0) through Day 120.  
 
Endpoint incidence is assumed for evaluable patients who are lost to follow up including 
withdrawal of further data submission and death prior to determination of their outcome 
(stage 1-4 of GI aGvHD and grade II-IV overall GvHD) in the primary analysis. 
 
Patients who never received conditioning are not at risk of aGvHD and will be considered 
invaluable for the primary outcome analysis. Patients who have graft failure are not at risk 
for graft versus host disease, therefore, they will contribute to the primary analysis up to 
date of graft failure. However, they can still be included in the analysis of toxicity data and 
laboratory data. See Section 10.3 for definition of graft failure.  
 
Exploratory endpoints include the incidence of Grade II–IV overall aGvHD, blood stream 
infection (BSI), Clostridium difficile-associated diarrhea and assessment of laboratory 
correlative measures for serum levels of citrulline and GvHD biomarkers and blood/stool 
measures of intestinal flora and function and allo-induced inflammation.  
 
Central Committee Review 
The COG SCT Discipline has created consensus guidelines for GvHD determination that 
are based on modified Glucksberg criteria. These guidelines have been used in COG 
transplant trials for the past decade. We will collect information that includes the highest 
grade of overall GvHD (grades 1-4) as well as the highest stages of organ GvHD (skin, GI 
and liver). For acute GI GvHD, we will collect both stage (1-4) for lower GI GvHD as well 
as presence (yes/no) of upper GI GvHD. Both upper and lower GI GvHD are used to 
determine the stage of GI GvHD, but lower GI GvHD alone will be considered an event. 
Dates of onset and maximal GI GvHD stage and overall grade will be collected. The Study 
Committee will audit the GvHD data (assuring that the reported stages correlate with 
overall grade) for each patient every 3 months. Additional information will be requested 
from the reporting center to review completed GvHD forms if the overall Grade of GvHD 
does not appropriately correspond to the reported stages. This targeted centralized review 
will identify cases most prone to error, will be more efficient than review of all data, and 
will avoid what otherwise would be an enormous hardship for centers. All reviewers will 
be blinded to the patient’s regimen in order to limit any bias in determining the primary 
and exploratory endpoints. The study chair will make the final determination for GvHD 

 
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organ staging and overall grading based on central review and may override staging and 
grading entered by the institution.  
 
9.3 
Data Analytic Plan 
Preliminary Descriptive Analysis: Study variables will be summarized using simple 
descriptive statistics such as count/percentage for categorical variables and mean/standard 
deviation/median/interquartile range for continuous variables. Baseline patient 
demographics and clinical characteristics will be summarized. The two arms will be 
balanced on the factors included in the stratified randomization. Treatment arms will be 
descriptively compared for other potential confounders for possible inclusion in the 
multivariate models. Arm comparison will be performed using simple two-sample tests, 
such as 2 test of proportions for categorical variables and independent t-tests or Wilcoxon 
rank sum tests for continuous variables. Analyses for each of the aims is presented below: 
 
Primary Aim: The primary aim is to determine whether oral LBP prevents the development 
of Stage 1-4 GI aGvHD incidence in children and adolescents undergoing alloHCT. We 
hypothesize that administration of oral LBP will reduce the incidence of Stage 1-4 GI 
aGvHD compared to placebo. As described above, accrual will be conducted in two stages, 
the screening stage and the confirmatory stage. The proportion of 120 centrally reviewed, 
eligible and evaluable randomized patients who received at least 1 dose of treatment having 
Stage 1-4 GI aGvHD incidence from Day 0 through Day 120 will be compared between 
the two arms after the screening stage via two-sample unpooled chi-square test of 
proportions at 1-sided =0.3. If this liberal estimate of efficacy of LBP is demonstrated, 
accrual of the confirmatory stage will continue.  
 
At the end of the confirmatory stage, the proportion of eligible patients having Stage 1-4 
GI aGvHD incidence from Day 0 through Day 120 will be compared between the two arms 
(including patients randomized in both stages) via two-sample unpooled chi-square test of 
proportions at =0.05 (1-sided). We will use a modified intention-to-treat approach for the 
primary analysis.  
 
Secondary analyses will be performed on cumulative incidence of Stage 1-4 GI aGvHD 
using unconditional multivariate logistic regression with covariate adjustment. Covariates, 
to be considered in the statistical models are: use of total body irradiation, duration of broad 
spectrum antibiotic usage, incidence of C. difficile infection, Haplo designation, and 
duration of total parenteral nutrition. We expect few patients to be terminated from aGvHD 
observation prior to Day 120 due to death, loss to follow-up, or consent withdrawal before 
determination of GI aGvHD outcome. Nevertheless, we will also perform secondary 
analyses (time from graft infusion to first Stage 1-4 GI aGvHD onset) via competing risk 
modeling, where graft failure, relapse and death are treated as competing events, generating 
incident curves and comparing treatment arms with the Gray’s test to accommodate 
censoring. Patients without Stage 1-4 GI aGvHD will be censored when they meet criteria 
to be off study. Actuarial analyses will be extended for covariate adjustment using Cox 
proportional hazards regression. If we observe moderate protocol non-compliance with the 
daily LBP, additional analyses, such as as-treated analyses, subset analyses including only 
patients adherent with protocol treatment, or sensitivity analyses, will be performed to 
assess whether treatment adherence influenced the primary outcome. Based on our pilot 
results, we not anticipate a significant adherence problem.  
 
Exploratory Aim 1: This aim is to determine whether administration of LBP prevents the 
development of moderate to severe (Grade II–IV) overall aGvHD. We hypothesize that 

 
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administration of LBP will reduce the incidence of Grade II–IV aGvHD in alloHCT 
recipients compared to placebo. We will use a similar analysis approached as described 
above for the primary aim but using the dichotomous cumulative incidence of Grade II–IV 
aGvHD as the endpoint measure.  
 
Exploratory Aim 2: This exploratory aim is to determine whether LBP administration 
maintains intestinal integrity as measured by serum citrulline levels and reduces the 
incidence of mucosal barrier bloodstream infections (MBIs). We hypothesize that 
administration of LBP will maintain serum citrulline levels in alloHCT patients compared 
to patients receiving placebo. As well, we expect a lower incidence of MBIs in the LBP 
arm compared to placebo.  
 
The citrulline levels at each of the time points will be summarized and described by arm. 
The mean quantitative change in citrulline level from start of conditioning to each of the 
four later time points will be compared between the 2 arms to determine if LBP 
administration maintains citrulline level via two-sample t-test and repeated-measures 
general linear modeling which include adjustments for potential confounders. In such 
analyses, multiple comparison adjustments for the 4 parallel analyses (linear contrasts) will 
be performed with Bonferroni adjustment to maintain an overall fixed  level. Linear 
mixed models, which include data from all time points for longitudinal data analyses with 
adjustment for within-patient correlation, will also be considered.  
 
Likewise, we will compare the incidence of MBIs between two groups looking at the time 
from start of protocol treatment to first infection as the primary endpoint. Patients without 
infection will be censored when they meet criteria to be off study. Cox proportional hazards 
regression will be used to compare the incidence density of infection for the two groups 
adjusted for confounders  (use of total body irradiation, duration of broad spectrum 
antibiotic usage, incidence of C. difficile infection, and duration of total parenteral 
nutrition). Main analyses for this aim will be based on a modified intention-to-treat 
principle (inevaluable patients will be excluded), but additional as-treated analysis, subset 
analysis, or sensitivity analysis may be performed if we observe issues with adherence to 
the randomized treatment on the study.  
 
Exploratory Aims 3 and 4: These aims will examine the effects of LBP introduction on the 
intestinal flora of pediatric alloHCT recipients. The association between LBP 
administration and bacterial genes and pathways, and bacterial metabolites will be 
evaluated with Pearson’s correlation coefficient with possible transformation to 
approximate normality. In addition to confirming the significance, a 95% confidence 
interval will be estimated for each marker. The total length of the interval depends on the 
observed correlation; the length will be 0.53 if the correlation is 0.40 and 0.24 if the 
correlation is 0.8. In addition to correlation, bivariate scatterplots will be used to display 
the association between LBP administration and each marker. Finally, we will perform a 
descriptive analysis examining the association between GvHD outcomes and bacterial 
genes, pathways, and metabolites using Cox regression. Additional regression models will 
adjust for other known GvHD risk factors. 
 
Exploratory Aim 5: This aim will examine the effects of LBP on pro-inflammatory 
cytokines in alloHCT recipients. We hypothesize that LBP-associated maintenance or 
restoration in the microbiota will attenuate serum levels of pro-inflammatory cytokines in 
the early post-transplant period. We will use the same analytic approach as described for 
exploratory Aim 2 above. 

 
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Exploratory Aim 6: This aim will examine the effects of LBP on the incidence (yes/no) of 
Clostridium difficile-associated diarrhea in alternative donor HCT patients. Patients who 
have at least one incidence (positive) of C. diff during any reporting period is considered 
evaluable for this aim. We will compare proportion of C. diff during the study period 
between arms via the chi-square test. 
 
Exploratory Aim 7: This aim will examine whether LBP administration reduces hospital 
days within the first 120 days post hematopoietic cell transplant. Total hospital days over 
the study period is calculated as the duration between the date of admission for conditional 
therapy and the date of discharge (or the study end date). Hospital days will be compared 
between arms by t-test (or Wilcoxon rank-sum test if assumptions for t-test are not met). 
 
Exploratory Aim 8: This aim will examine the safety of orally administered LBP strains 
299 and 299v in alternative donor HCT patients as measured by incidence (yes/no) of 
Lactobacillus plantarum bacteremia. Patients who have at least one incidence (positive) of 
Lactobacillus plantarum during any reporting period is considered evaluable for this aim. 
We will use the same analytic approach as described for exploratory Aim 6 above. 
 
9.4 
Power and Sample Size 
Primary Aim: Review of the literature suggests that the incidence of GI aGvHD ranges 
from 15% to 45% across various studies; therefore a precise estimate of baseline GI 
aGvHD incidence for this study population is not available. The incidence of GI aGvHD 
was 27% in the control arm of the most recent COG trial (ASCT0431), which enrolled a 
patient population where 54% of patients received grafts from matched sibling donors. We 
anticipate a higher rate of GI aGvHD in the control arm of this trial, as we are including 
only alternative donor recipients (refer to eligibility criteria), and therefore project the 
baseline cumulative GI aGvHD incidence through Day 120 to be at least 30%. Success will 
be defined as a 40% relative reduction in the cumulative incidence of GI aGvHD (i.e., 18% 
in LBP arm vs. 30% in placebo arm). A sample size of 120 (60 per arm) for the screening 
stage and 454 (227 per arm) for the confirmatory stage (including patients randomized 
during the screening stage) will provide 85% power to detect an effect size of 40% relative 
reduction at 1-sided =0.3 and 0.05, respectively in a two-sample test of binomial 
proportions. The overall power of the study, which is the probability of establishing 
efficacy in the primary endpoint in both analyses, is lower than the power for each 
component separately and higher than the product of two. Based on simulations, the overall 
power is at 80% in this scenario. The overall type I error for the design is also slightly 
reduced from the type I error of 0.05 for each individual component if they are analyzed 
separately. Based on simulations, the overall type I error is at 0.035.   
 
Exploratory Aims 1 and 2: The sample size for the primary aim drives the overall accrual 
goal and thus the number of subjects available for the exploratory aims. These endpoints 
will be analyzed only at the end of the study, either at the end of the screening stage or 
confirmatory stage. Therefore, the power is estimated for the two situations separately 
based on sample size of 120 (60 per arm) or 454 (227 per arm) without any adjustment for 
power after the confirmatory stage. For Exploratory aim #1, a baseline rate of Grade II–IV 
aGvHD in the range of 30%–50%, the sample size after confirmatory stage is sufficient to 
detect a 15% reduction in aGvHD rate, while the sample size after screening stage is 
sufficient to detect a 20% reduction in aGvHD rate with LBP compared to placebo. Power 
estimation is based on 2-sample test of proportions at =0.05 (2-sided). For Exploratory 
aim #2 on serum citrulline levels, the sample size after confirmatory stage is sufficient to 

 
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detect an effect size of 0.4 standard deviation (SD), assuming we obtain samples from 80% 
or more of the subjects; on the other hand, the sample size after screening stage is sufficient 
to detect an effect size of 0.7 SD if we obtain samples from ≥80% subjects. Power 
estimation is based on two-sample t-test at =0.0125 (2-sided), where  is based on the 
Bonferroni adjustment for the 4 parallel comparisons with an overall =0.05. 
 
9.5 
Interim Monitoring  
Study data will be reviewed by DSMC twice a year during the first 2 years of study 
activation, and once a year thereafter if no concerns are identified.  
 
The study will be conducted in two stages: a randomized screening stage, and a larger 
confirmatory stage. The screening stage will compare the proportion of Stage 1-4 GI 
aGvHD between the 2 arms. The proportion of 120 centrally reviewed, eligible and 
evaluable randomized patients having Stage 1-4 GI aGvHD incidence from Day 0 through 
Day 120 will be compared between the two arms via two-sample test of proportions at 
1-sided =0.3. If the screening stage suggests efficacy (p<0.3), the study will proceed to 
the confirmatory stage (N=454). If efficacy is not established at the screening stage, the 
data will be referred to the COG Data Safety and Monitoring Committee (DSMC) for 
consideration of early termination. 
 
Amendment 1: Haploidentical transplantation in children has increased over the past few 
years. Although single institution studies have demonstrated a remarkably lower acute and 
chronic Graft vs Host Disease incidence and severity, these results have not been 
duplicated in larger multicenter trials. In these multicenter trials, the Grade II-IV Acute 
GvHD rate does not appear to be significantly different from that of other alternative donor 
trials. While we do agree that the rate of chronic GVH appears much less, our current study 
ends at Day + 120 and thus chronic GVH is not measured in this trial. 
 
Therefore, it is presumed to have no impact on the power for the primary aim by having 
haploidentical patients enrolled in this study. The estimated primary outcome rate aGvHD 
and proportion of haploidentical patients will be monitored bi-annually. At each 
monitoring time, by assuming the observed aGvHD rate, proportion of haploidentical 
patients within the control arm, and using the same power simulation (that we’ve used to 
provide power analysis for the protocol), if the estimated power at the end of the study is 
less than 80%, we will adjust the sample size through an Amendment. All observed rates 
will be available only for members of DSMC to review. 
 
 

 
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9.6 
Gender and Minority Accrual Estimates 
 
DOMESTIC PLANNED ENROLLMENT REPORT 
Racial Categories 
Ethnic Categories 
Total 
Hispanic or Latino 
Not Hispanic or Latino 
 
Female 
Male 
Female 
Male 
 
American Indian/ Alaska Native 
0 
0 
0 
0 
0 
Asian 
0 
0 
16 
16 
32 
Native Hawaiian or Other Pacific Islander 
0 
0 
0 
0 
0 
Black or African American 
0 
16 
63 
111 
190 
White 
95 
16 
47 
95 
253 
More Than One Race 
0 
0 
0 
0 
0 
Total 
95 
32 
126 
222 
475 
 
INTERNATIONAL (INCLUDING CANADA) PLANNED ENROLLMENT REPORT 
Racial Categories 
Ethnic Categories 
Total 
Hispanic or Latino 
Not Hispanic or Latino 
 
Female 
Male 
Female 
Male 
 
American Indian/ Alaska Native 
0 
0 
0 
0 
0 
Asian 
0 
0 
1 
1 
2 
Native Hawaiian or Other Pacific Islander 
0 
0 
0 
0 
0 
Black or African American 
0 
1 
3 
6 
10 
White 
5 
1 
2 
5 
13 
More Than One Race 
0 
0 
0 
0 
0 
Total 
5 
2 
6 
12 
25 
 
This distribution was derived from the pilot study data, adjusted for Children’s Oncology 
Group projections. 
 
 
10.0 
EVALUATION CRITERIA 
 
10.1 
Common Terminology Criteria for Adverse Events (CTCAE) 
This study will utilize version 5.0 of the CTCAE of the National Cancer Institute (NCI) for 
toxicity and performance reporting. A copy of the CTCAE version 5.0 can be downloaded 
from the CTEP website 
(http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm). 
Additionally, toxicities are to be reported on the appropriate case report forms. 
 
Please note: ‘CTCAE v5.0’ is understood to represent the most current version of CTCAE 
v5.0 as referenced on the CTEP website (i.e., v5.02 and all subsequent iterations prior to 
version 6.0). 
 
10.2 
Acute GVHD  
See Appendix III protocol specific staging and grading criteria for acute GVHD.  
 

 
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10.3 
Graft Failure 
Primary Graft Failure 
Failure to achieve an ANC ≥ 500/µL after 42 days, determined by three consecutive 
measurements on different days; or < 5% donor cells in blood or bone marrow by Day +42 
(as demonstrated by a chimerism assay), without evidence of JMML.  
 
Secondary Graft Failure 
Initial engraftment followed by severe neutropenia (ANC ≤ 500/µL) that is not caused by 
recurrent leukemia, or < 5% donor cells in the blood or bone marrow (as demonstrated by a 
chimerism assay) without subsequent improvement occurring either spontaneously or after 
growth factor treatment. Improvement is defined as ANC > 500/µL consistently. Severe 
neutropenia with bone marrow cellularity ≥ 25% is not secondary graft failure. 
 
Marrow, UCB or peripheral blood stem cell reinfusion carried out any time after Day 0 
because of inadequate hematopoietic function will be taken as a definitive indication of graft 
failure regardless of AMC values and marrow cellularity. Donor lymphocyte infusion is not 
considered a stem cell reinfusion.  
 
 
11.0 
ADVERSE EVENT REPORTING REQUIREMENTS 
 
11.1 
Purpose 
Adverse event data collection and reporting, which are required as part of every clinical 
trial, are done to ensure the safety of patients enrolled in the studies as well as those who 
will enroll in future studies using similar agents. Certain adverse events must be reported 
in an expedited manner to allow for timelier monitoring of patient safety and care. The 
following sections provide information about expedited reporting. 
 
11.2 
Expedited Reporting Requirements – Serious Adverse Events (SAEs) 
To ensure compliance with these regulations/this guidance, NCI requires that AEs be 
submitted according to the timeframes in the AE reporting tables assigned to the protocol, 
using the CTEP Adverse Event Reporting System (CTEP-AERS). 
 
Any AE that is serious qualifies for expedited reporting. An AE is defined as any 
untoward medical occurrence associated with the use of a drug in humans, whether or not 
considered drug related. A Serious Adverse Event (SAE) is any adverse drug event 
(experience) occurring at any dose that results in ANY of the following outcomes: 
 
1) Death. 
2) A life-threatening adverse drug experience. 
3) An adverse event resulting in inpatient hospitalization or prolongation of existing 
hospitalization (for ≥ 24 hours). This does not include hospitalizations that are part 
of routine medical practice. 
4) A persistent or significant incapacity or substantial disruption of the ability to 
conduct normal life functions. 
5) A congenital anomaly/birth defect. 
6) Important Medical Events (IME) that may not result in death, be life threatening, or 
require hospitalization may be considered a serious adverse drug experience when, 
based upon medical judgment, they may jeopardize the patient or subject and may 

 
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require medical or surgical intervention to prevent one of the outcomes listed in this 
definition. 
 
11.3 
Specific Examples for Expedited Reporting  
 
11.3.1 SAEs Occurring More than 30 Days After Last Dose of Study Drug 
Any Serious Adverse Event that occurs more than 30 days after the last 
administration of the investigational agent/intervention and has an attribution of a 
possible, probable, or definite relationship to the study therapy must be reported 
according to the CTEP-AERS reporting tables in this protocol.  
 
11.3.2 Persistent or Significant Disabilities/Incapacities 
Any AE that results in persistent or significant incapacity or substantial disruption 
of the ability to conduct normal life functions (formerly referred to as disabilities), 
congenital anomalies or birth defects, must be reported via CTEP-AERS if it 
occurs at any time following treatment with an agent under a NCI, COG, or 
industry sponsor IND/IDE since these are considered to be serious AEs. 
 
11.3.3 Death 
 
Reportable Categories of Death 
o Death attributable to a CTCAE term.  
o Death Neonatal: Newborn death occurring during the first 28 days after birth.  
o Sudden Death NOS: A sudden (defined as instant or within one hour of the 
onset of symptoms) or an unobserved cessation of life that cannot be 
attributed to a CTCAE term associated with Grade 5.  
o Death NOS: A cessation of life that cannot be attributed to a CTCAE term 
associated with Grade 5.  
o Death due to progressive disease should be reported as Grade 5 “Disease 
progression” in the system organ class (SOC) “General disorders and 
administration site conditions.” Evidence that the death was a manifestation 
of underlying disease (e.g., radiological changes suggesting tumor growth or 
progression: clinical deterioration associated with a disease process) should 
be submitted.  
 
Any death occurring within 30 days of the last dose, regardless of attribution to 
the investigational agent/intervention requires expedited reporting within 24 hours.  
 
Any death occurring greater than 30 days after the last dose of the investigational 
agent/intervention requires expedited reporting within 24 hours only if it is 
possibly, probably, or definitely related to the investigational agent/intervention. 
 
11.3.4 Secondary Malignancy 
A secondary malignancy is a cancer caused by treatment for a previous 
malignancy (e.g., treatment with investigational agent/intervention, radiation or 
chemotherapy). A metastasis of the initial neoplasm is not considered a secondary 
malignancy.  
 

 
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All secondary malignancies that occur following treatment need to be reported via 
CTEP-AERS. Three options are available to describe the event:  
• 
Leukemia secondary to oncology chemotherapy  
• 
Myelodysplastic syndrome  
• 
Treatment related secondary malignancy  
 
Any malignancy possibly related to cancer treatment (including AML/MDS) must 
also be reported via the routine reporting mechanisms outlined in this protocol. 
 
11.3.5 Second Malignancy  
A second malignancy is one unrelated to the treatment of a prior malignancy (and 
is NOT a metastasis from the initial malignancy). Second malignancies require 
ONLY routine reporting via CDUS unless otherwise specified. 
 
11.3.6 Pregnancy, Pregnancy Loss, and Death Neonatal 
NOTE: When submitting CTEP-AERS reports for “Pregnancy”, “Pregnancy loss”, 
or “Death Neonatal”, the Pregnancy Information Form, available at 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/Pregna
ncyReportForm.pdf, needs to be completed and faxed along with any additional 
medical information to (310) 640-9193. The potential risk of exposure of the fetus 
to the investigational agent(s) or chemotherapy agent(s) should be documented in 
the “Description of Event” section of the CTEP-AERS report. 
 
11.3.6.1 Pregnancy 
Patients who become pregnant on study risk intrauterine exposure of the 
fetus to agents that may be teratogenic. For this reason, pregnancy needs 
to be reported in an expedited manner via CTEP-AERS as Grade 3 
“Pregnancy, puerperium and perinatal conditions - Other (pregnancy)” 
under the Pregnancy, puerperium and perinatal conditions” SOC. 
 
Pregnancy needs to be followed until the outcome is known. If the baby 
is born with a birth defect or anomaly, then a second CTEP-AERS report 
is required. 
 
11.3.6.2 Pregnancy Loss (Fetal Death) 
Pregnancy loss is defined in CTCAE as “Death in utero.” Any 
Pregnancy loss should be reported expeditiously as Grade 4 
“Pregnancy loss” under the “Pregnancy, puerperium and perinatal 
conditions” SOC. Do NOT report a pregnancy loss as a Grade 5 event 
since CTEP-AERS recognizes any Grade 5 event as a patient death. 
 
11.3.6.3 Death Neonatal  
Neonatal death, defined in CTCAE as “Newborn death occurring 
during the first 28 days after birth” should be reported expeditiously as 
Grade 4 “Death neonatal” under the “General disorders and 
administration” SOC when the death is the result of a patient 
pregnancy or pregnancy in partners of men on study. Do NOT report 
a neonatal death resulting from a patient pregnancy or pregnancy in 
partners of men on study as a Grade 5 event since CTEP-AERS 
recognizes any Grade 5 event as a patient death.  

 
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11.4 
Reporting Requirements for Specialized AEs  
 
11.4.1 Baseline AEs 
Although a pertinent positive finding identified on baseline assessment is not an 
AE, when possible it is to be documented as “Course Zero” using CTCAE 
terminology and grade. An expedited AE report is not required if a patient is 
entered on a protocol with a pre-existing condition (e.g., elevated laboratory value, 
diarrhea). The baseline AE must be re-assessed throughout the study and reported 
if it fulfills expedited AE reporting guidelines. 
a. If the pre-existing condition worsens in severity, the investigator must 
reassess the event to determine if an expedited report is required. 
b. If the AE resolves and then recurs, the investigator must re-assess the event 
to determine if an expedited report is required. 
c. No modification in grading is to be made to account for abnormalities existing 
at baseline. 
 
11.4.2 Persistent AEs 
A persistent AE is one that extends continuously, without resolution between 
treatment cycles/courses.  
ROUTINE reporting: The AE must be reported only once unless the grade 
becomes more severe in a subsequent course. If the grade becomes more severe 
the AE must be reported again with the new grade. 
EXPEDITED reporting: The AE must be reported only once unless the grade 
becomes more severe in the same or a subsequent course. 
 
11.4.3 Recurrent AEs 
A recurrent AE is one that occurs and resolves during a cycle/course of therapy 
and then reoccurs in a later cycle/course. 
ROUTINE reporting: An AE that resolves and then recurs during a subsequent 
cycle/course must be reported by the routine procedures. 
EXPEDITED reporting: An AE that resolves and then recurs during a subsequent 
cycle/course does not require CTEP-AERS reporting unless: 
1) The grade increases OR 
2) Hospitalization is associated with the recurring AE. 
 
11.5 
Exceptions to Expedited Reporting 
An expedited report may not be required for a specific protocol where an AE is listed as 
expected. The exception or acceptable reporting procedures will be specified in the 
protocol. The protocol specific guidelines supersede the NCI Adverse Event Reporting 
Guidelines. These special situations are listed under the CTEP-AERS reporting Table A 
for this protocol. 
 
11.6 
Reporting Requirements - Investigator Responsibility 
Clinical investigators in the treating institutions and ultimately the Study Chair have the 
primary responsibility for AE identification, documentation, grading, and assignment of 
attribution to the investigational agent/intervention. It is the responsibility of the treating 
physician to supply the medical documentation needed to support the expedited AE reports 
in a timely manner.  
 

 
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Note: All expedited AEs (reported via CTEP-AERS) must also be reported via routine 
reporting. Routine reporting is accomplished via the Adverse Event (AE) Case Report 
Form (CRF) within the study database.  
 
11.7 
General Instructions for Expedited Reporting via CTEP-AERS  
The reporting methods described below are specific for clinical trials evaluating 
agents for which the IND is held by COG, an investigator, or a pharmaceutical 
company. It is important to note that these procedures differ slightly from those used 
for reporting AEs for clinical trials for which CTEP holds the IND. 
 
The descriptions and grading scales found in the revised NCI Common Terminology 
Criteria for Adverse Events (CTCAE) version 5.0 will be utilized for AE reporting. All 
appropriate treatment areas should have access to a copy of the CTCAE version 5.0. A 
copy of the CTCAE version 5.0 can be downloaded from the CTEP website at: 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm.  
 
An expedited AE report must be submitted electronically via CTEP-AERS at:  
https://eapps-ctep.nci.nih.gov/ctepaers 
• 
Expedited AE reporting timelines are defined as: 
o 24-Hour; 5 Calendar Days - The AE must initially be reported via CTEP-AERS 
within 24 hours of learning of the event, followed by a complete expedited report 
within 5 calendar days of the initial 24-hour report. 
o 7 Calendar Days - A complete expedited report on the AE must be submitted 
within 7 calendar days of the investigator learning of the event.  
• 
Any event that results in a persistent or significant incapacity/substantial disruption of 
the ability to conduct normal life functions, or a congenital anomaly/birth defect, or is 
an IME, which based upon the medical judgment of the investigator may jeopardize 
the patient and require intervention to prevent a serious AE, must be reported via 
CTEP-AERS if the event occurs following investigational agent administration.  
• 
Any death occurring within 30 days of the last dose, regardless of attribution to an 
agent/intervention requires expedited reporting within 24 hours via e-mail to the COG 
CTEP-AERS Coordinator and Study Chair.  
• 
Any death occurring greater than 30 days of the last dose with an attribution of 
possible, probable, or definite to an agent/intervention requires expedited reporting 
within 24 hours via e-mail to the COG CTEP-AERS Coordinator and Study Chair.  
 
CTEP-AERS Medical Reporting includes the following requirements as part of the report: 
1) whether the patient has received at least one dose of an investigational agent on this 
study; 2) the characteristics of the adverse event including the grade (severity), the 
relationship to the study therapy (attribution), and the prior experience (expectedness) of 
the adverse event; 3) the Phase (1, 2, or 3) of the trial; and 4) whether or not hospitalization 
or prolongation of hospitalization was associated with the event.  
 
Fax or email supporting documentation for AEs related to investigational agents to 
COG: Fax # (310) 640-9193; email: COGAERS@childrensoncologygroup.org; Attention: 
COG AERS Coordinator.  
• 
ALWAYS include the ticket number on all faxed documents. 
• 
Use the NCI protocol number and the protocol-specific patient ID provided 
during trial registration on all reports. 

 
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11.8 
Reporting Table for Late Phase 2 and Phase 3 Studies – Table A 
Expedited Reporting Requirements for Adverse Events that Occur on Studies under an 
IND/IDE within 30 Days of the Last Administration of the Investigational 
Agent/Intervention 1 
FDA REPORTING REQUIREMENTS FOR SERIOUS ADVERSE EVENTS (21 CFR Part 312)  
NOTE: Investigators MUST immediately report to the sponsor (COG) ANY Serious Adverse Events, 
whether or not they are considered related to the investigational agent(s)/intervention (21 CFR 312.64)  
An adverse event is considered serious if it results in ANY of the following outcomes:  
1) Death.  
2) A life-threatening adverse event.  
3) Any AE that results in inpatient hospitalization or prolongation of existing hospitalization for 
≥ 24 hours. This does not include hospitalizations that are part of routine medical practice. 
4) A persistent or significant incapacity or substantial disruption of the ability to conduct normal life 
functions. 
5) A congenital anomaly/birth defect.  
6) Important Medical Events (IME) that may not result in death, be life threatening, or require 
hospitalization may be considered serious when, based upon medical judgment, they may jeopardize 
the patient or subject and may require medical or surgical intervention to prevent one of the outcomes 
listed in this definition. (FDA, 21 CFR 312.32; ICH E2A and ICH E6.)  
ALL SERIOUS adverse events that meet the above criteria MUST be immediately reported to the NCI 
via CTEP-AERS within the timeframes detailed in the table below.  
Hospitalization 
Grade 1 
Timeframes 
Grade 2 
Timeframes 
Grade 3 
Timeframes 
Grade 4 & 5 
Timeframes 
Resulting in 
Hospitalization 
≥ 24 hrs 
7 Calendar Days 
 
 
24-Hour Notification 
5 Calendar Days 
Not resulting in 
Hospitalization 
≥ 24 hrs 
Not Required 
7 Calendar Days 
NOTE: Additional Special Situations as Exceptions to Expedited Reporting are listed below.  
 
Expedited AE reporting timelines are defined as:  
“24-Hour; 5 Calendar Days” - The AE must initially be reported via CTEP-AERS within 24 hours of 
learning of the AE, followed by a complete expedited report within 5 calendar days of the initial 24-hour 
notification. 
“7 Calendar Days” - A complete expedited report on the AE must be submitted within 7 calendar days 
of learning of the AE.  
1SAEs that occur more than 30 days after the last administration of investigational agent/intervention 
and have an attribution of possible, probable, or definite require reporting as follows:  
Expedited 24-hour notification followed by complete report within 5 calendar days for:  
• 
All Grade 4, and Grade 5 AEs  
Expedited 7 calendar day reports for:  
• 
Grade 2 adverse events resulting in hospitalization or prolongation of hospitalization  
• 
Grade 3 adverse events  
 
 
 

 
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11.9 
Protocol Specific Additional Instructions and Reporting Exceptions 
• 
LBP-bacteremia will be considered an unexpected severe adverse event. Patients 
with a blood culture positive for LBP will cease to receive the probiotic immediately 
and the patient will be removed from protocol therapy. However, data collection will 
continue on all patients through Day 120, the final data point of the study. A report of 
LBP-bacteremia will initiate a sequence of monitoring events. Centers must report 
LBP-bacteremia within 24 hours of the diagnosis through the CTEP-AERS system. 
• 
Only Grade 3 or 4 Unexpected Serious Adverse Events that are attributable (possibly, 
probably or definitely related) to LBP or the placebo should be reported.  
• 
Any death, regardless of attribution should be reported.  
 
11.10 Routine Reporting of Adverse Events  
Note: The guidelines below are for routine reporting of study specific adverse events on 
the COG case report forms and do not affect the requirements for CTEP-AERS reporting. 
 
Routine reporting is accomplished via the Adverse Event (AE) Case Report Form (CRF) 
within the study database. For this study, routine reporting will include all CTEP-AERS 
reportable events and Grade 3 and higher Adverse Events that are possibly, probably or 
definitely attributable to the study product (LBP or placebo).  
 
11.11 Syndrome Reporting 
Unless otherwise specified in this protocol, syndromes should be reported as a single event 
using the CTCAE term for the composite syndrome, and not as the individual events that 
make up the syndrome. For example, Tumor Lysis Syndrome should be reported under the 
composite definition rather than reporting the component events (hyperkalemia, 
hyperphosphatemia, hypocalcemia, hyperuricemia) separately. 
 
 
12.0 
RECORDS AND REPORTING 
See the Case Report Forms posted on the COG web site with each protocol under “Data 
Collection/Specimens”. A submission schedule is included. 
 
12.1 
CDUS 
This study will be monitored by the Clinical Data Update System (CDUS). Cumulative 
CDUS data will be submitted quarterly to CTEP by electronic means. Reports are due 
January 31, April 30, July 31 and October 31. This is not a responsibility of institutions 
participating in this trial. 
 
12.2 
Data Safety and Monitoring Committee 
To protect the interests of patients and the scientific integrity for all clinical trial research 
by the Children’s Oncology Group, the COG Data and Safety Monitoring Committee 
(DSMC) reviews reports of interim analyses of study toxicity and outcomes prepared by 
the study statistician, in conjunction with the study chair’s report. The DSMC may 
recommend the study be modified or terminated based on these analyses.  
 
Toxicity monitoring is also the responsibility of the study committee and any unexpected 
frequency of serious events on the trial are to be brought to the attention of the DSMC. The 
study statistician is responsible for the monitoring of the interim results and is expected to 

 
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request DSMC review of any protocol issues s/he feels require special review. Any COG 
member may bring specific study concerns to the attention of the DSMC.  
 
The DSMC approves major study modifications proposed by the study committee prior to 
implementation (e.g., termination, dropping an arm based on toxicity results or other trials 
reported, increasing target sample size, etc.). The DSMC determines whether and to whom 
outcome results may be released 
 
 

 
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APPENDIX I: CTEP REGISTRATION PROCEDURES 
 
INVESTIGATOR AND RESEARCH ASSOCIATE REGISTRATION WITH CTEP 
Food and Drug Administration (FDA) regulations and National Cancer Institute (NCI) policy 
require all individuals contributing to NCI-sponsored trials to register and to renew their 
registration annually. To register, all individuals must obtain a Cancer Therapy Evaluation Program 
(CTEP) Identity and Access Management (IAM) account at https://ctepcore.nci.nih.gov/iam. In 
addition, persons with a registration type of Investigator (IVR), Non-Physician Investigator 
(NPIVR), or Associate Plus (AP) must complete their annual registration using CTEP’s web-based 
Registration and Credential Repository (RCR) at https://ctepcore.nci.nih.gov/rcr.    
 
RCR utilizes five person registration types. 
• 
IVR — MD, DO, or international equivalent; 
• 
NPIVR — advanced practice providers (e.g., NP or PA) or graduate level researchers (e.g., PhD); 
• 
AP — clinical site staff (e.g., RN or CRA) with data entry access to CTSU applications such 
as the  Roster Update Management System (RUMS), OPEN, Rave, acting as a primary site 
contact, or with consenting privileges; 
• 
Associate (A) — other clinical site staff involved in the conduct of NCI-sponsored trials; and 
• 
Associate Basic (AB) — individuals (e.g., pharmaceutical company employees) with limited 
access to NCI-supported systems. 
 
RCR requires the following registration documents: 
Documentation Required 
IVR 
NPIVR 
AP 
A 
AB 
FDA Form 1572 
 
 
 
 
 
Financial Disclosure Form 
 
 
 
 
 
NCI Biosketch (education, training, 
employment, license, and certification) 
 
 
 
 
 
GCP training 
 
 
 
 
 
Agent Shipment Form (if applicable) 
 
 
 
 
 
CV (optional) 
 
 
 
 
 
 
An active CTEP-IAM user account and appropriate RCR registration is required to access all CTEP 
and Cancer Trials Support Unit (CTSU) websites and applications. In addition, IVRs and NPIVRs 
must list all clinical practice sites and Institutional Review Boards (IRBs) covering their practice 
sites on the FDA Form 1572 in RCR to allow the following: 
• 
Addition to a site roster; 
• 
Assign the treating, credit, consenting, or drug shipment (IVR only) tasks in OPEN; 
• 
Act as the site-protocol Principal Investigator (PI) on the IRB approval; and 
• 
Assign the Clinical Investigator (CI) role on the Delegation of Tasks Log (DTL).  
 
In addition, all investigators acting as the Site-Protocol PI (investigator listed on the IRB approval), 
consenting/treating/drug shipment investigator in OPEN, or as the CI on the DTL must be rostered 
at the enrolling site with a participating organization.  
 
Additional information is located on the CTEP website at https://ctep.cancer.gov/
investigatorResources/default.htm. For questions, please contact the RCR Help Desk by email at 
RCRHelpDesk@nih.gov. 
 

 
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CTSU REGISTRATION PROCEDURES 
 
This study is supported by the NCI Cancer Trials Support Unit (CTSU).  
 
Protocol Specific Requirements for Site Registration: 
 
• 
IRB approval (For sites not participating via the NCI CIRB; local IRB documentation, an 
IRB-signed CTSU IRB Certification Form, Protocol of Human Subjects Assurance 
Identification/IRB Certification/Declaration of Exemption Form, or combination is 
accepted ) 
 
Submitting Regulatory Documents: 
Submit required forms and documents to the CTSU Regulatory Office using the Regulatory 
Submission Portal on the CTSU website.  
To access the Regulatory Submission Portal log in to the CTSU members’ website, go to the 
Regulatory section and select Regulatory Submission. 
Institutions with patients waiting that are unable to use the Regulatory Submission Portal should 
alert the CTSU Regulatory Office immediately at 1-866-651-2878 in order to receive further 
instruction and support. 
 
Checking Your Site’s Registration Status: 
Site registration status may be verified on the CTSU members’ website.  
• 
Click on Regulatory  at the top of the screen; 
• 
Click on Site Registration; and  
• 
Enter the site’s 5-character CTEP Institution Code and click on Go. 
o Additional filters are available to sort by Protocol, Registration Status, Protocol 
Status, and/or IRB Type. 
 
Note: The status shown only reflects institutional compliance with site registration requirements as 
outlined within the protocol. It does not reflect compliance with protocol requirements for 
individuals participating on the protocol or the enrolling investigator’s status with NCI or their 
affiliated networks. 
 
Data Submission / Data Reporting 
Medidata Rave is a clinical data management system being used for data collection for this trial/study. 
Access to the trial in Rave is controlled through the CTEP-IAM system and role assignments.  
Requirements to access Rave via iMedidata: 
• 
A valid CTEP-IAM account; and  
• 
Assigned a Rave role on the LPO or PO roster at the enrolling site of: Rave CRA, Rave 
Read Only, Rave CRA (LabAdmin), Rave SLA, or Rave Investigator.  
Rave role requirements: 
o Rave CRA or Rave CRA (Lab Admin) role must have a minimum of an Associate 
Plus (AP) registration type; 
o Rave Investigator role must be registered as an Non-Physician Investigator (NPIVR) 
or Investigator (IVR); and 
o Rave Read Only role must have at a minimum an Associates (A) registration type. 
Refer to https://ctep.cancer.gov/investigatorResources/default.htm for registration types and 
documentation required. 
 

 
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Upon initial site registration approval for the study in Regulatory Support System (RSS), all persons 
with Rave roles assigned on the appropriate roster will be sent a study invitation e-mail from 
iMedidata. To accept the invitation, site staff must log in to the Select Login 
(https://login.imedidata.com/selectlogin) using their CTEP-IAM username and password, and click 
on the accept link in the upper right-corner of the iMedidata page. Site staff will not be able to 
access the study in Rave until all required Medidata and study specific trainings are completed. 
Trainings will be in the form of electronic learnings (eLearnings), and can be accessed by clicking 
on the link in the upper right pane of the iMedidata screen. If an eLearning is required and has not 
yet been taken, the link to the eLearning will appear under the study name in iMedidata instead of 
the Rave EDC link; once the successful completion of the eLearning has been recorded, access to 
the study in Rave will be granted, and a Rave EDC link will display under the study name.  
 
Site staff that have not previously activated their iMedidata/Rave account at the time of initial site 
registration approval for the study in RSS will also receive a separate invitation from iMedidata to 
activate their account. Account activation instructions are located on the CTSU website in the Rave 
section under the Rave resource materials (Medidata Account Activation and Study Invitation 
Acceptance). Additional information on iMedidata/Rave is available on the CTSU members’ 
website in the Data Management > Rave section at www.ctsu.org/RAVE/ or by contacting the 
CTSU Help Desk at 1-888-823-5923 or by e-mail at ctsucontact@westat.com.  
 
 

 
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APPENDIX II: YOUTH INFORMATION SHEETS 
 
INFORMATION SHEET REGARDING RESEARCH STUDY – ACCL1633 
(for children from 7 through 12 years of age) 
 
A Study of a Probiotic to Prevent Graft versus Host Disease in Children Receiving a Stem Cell 
Transplant 
 
1. We have been talking with you about a stem cell transplant. One problem from the treatment used 
in stem cell transplants is graft versus host disease (GvHD). GvHD happens when the donor cells 
attack parts of your body causing problems with your stomach, skin and liver. 
 
2. We are asking you to take part in a research study because you are about to have a stem cell 
transplant that may cause GvHD. A research study is when doctors work together to try out new 
ways to help people who are sick. In this study, we are trying to learn more about how to keep 
GvHD from happening.  
 
3. Children in this study will drink water or juice mixed with a probiotic or placebo. Probiotics are 
made up of good bacteria that help keep your stomach healthy. The placebo in this study will be 
made of exactly the same ingredients as the probiotic but will not have the good bacteria mixed in. 
Both the probiotic and placebo will look the same and taste the same. When you join the study, you 
will randomly (like the flip of a coin) be assigned to the probiotic or the placebo. You and your 
doctor will not know which treatment you will get. You will be asked to drink your mixture once 
a day starting on the first day of chemotherapy to get ready for transplant up to 56 days after your 
transplant.  
 
4. As part of this study, the doctors and nurses will collect blood and stool samples to run special tests. 
They will keep track of any GvHD you might have and whether you get any infections.  
 
5. Sometimes good things can happen to people when they are in a research study. These good things 
are called “benefits.” We don’t know for sure if there is any benefit of being part of this study. We 
hope a benefit to you would be to have less GvHD during stem cell transplant. 
 
6. Sometimes bad things can happen to people when they are in a research study. These bad things 
are called “risks.” We do not expect you will have any problems from the probiotic or the placebo. 
It is possible that you could get an infection from the good bacteria found in the probiotic, but we 
have not seen that happen in other children receiving the same treatment as you. Other things may 
happen to you that we don’t yet know about. 
 
7. Your family can choose to be part of this study or not. Your family can also decide to stop being 
in this study at any time once you start. Make sure to ask your doctors any questions that you have. 
 
 

 
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INFORMATION SHEET REGARDING RESEARCH STUDY – ACCL1633 
(for teens from 13 through 17 years of age) 
 
A Study of a Probiotic to Prevent Graft versus Host Disease in Children and Teens Receiving a 
Stem Cell Transplant 
 
1. We have been talking with you about a stem cell transplant. One problem from the treatment used 
in stem cell transplants is graft versus host disease (GvHD). GvHD happens when the donor cells 
attack parts of your body causing problems with your stomach, skin and liver. 
 
2. We are asking you to take part in a research study because you are about to have a stem cell 
transplant that may cause GvHD. A research study is when doctors work together to try out new 
ways to help people who are sick. In this study, we are trying to learn more about how to keep 
GvHD from happening.  
 
3. Children and Teens in this study will drink water or juice mixed with a probiotic or placebo. 
Probiotics are made up of good bacteria that help keep your stomach healthy. The placebo in this 
study will be made of exactly the same ingredients as the probiotic but will not have the good 
bacteria mixed in. Both the probiotic and placebo will look the same and taste the same. When you 
join the study, you will randomly (like the flip of a coin) be assigned to the probiotic or the placebo. 
You and your doctor will not know which treatment you will get. You will be asked to drink your 
mixture once a day starting on the first day of conditioning up to 56 days after your transplant.  
 
4. As part of this study, the doctors and nurses will collect blood and stool samples to run special tests. 
They will keep track of any GvHD you might experience and whether you get any infections.  
 
5. Sometimes good things can happen to people when they are in a research study. These good things 
are called “benefits.” We don’t know for sure if there is any benefit of being part of this study. We 
hope a benefit to you would be to have less GvHD during stem cell transplant. 
 
6. Sometimes bad things can happen to people when they are in a research study. These bad things 
are called “risks.” We do not expect you will have any problems from the probiotic or the placebo. 
It is possible that you could get an infection from the good bacteria found in the probiotic, but we 
have not seen that happen in other teens receiving the same treatment as you. Other things may 
happen to you that we don’t yet know about. 
 
7. Your family can choose to be part of this study or not. Your family can also decide to stop being 
in this study at any time once you start. Make sure to ask your doctors any questions that you have. 
 
 
 

 
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APPENDIX III: ACUTE GVHD STAGING AND GRADING 
Reporting Requirements for Acute GVHD in COG Studies 
In an attempt to standardize reporting of acute GVHD, the COG Stem Cell Transplantation Committee has 
adopted a modification of guidelines that were originally developed at the University of Michigan. 
 
Table 1 outlines standard criteria for GVHD organ staging. However, confounding clinical syndromes (such 
as non-GVHD causes of hyperbilirubinemia) may make staging GVHD in a given organ difficult. In 
addition, timing of organ specific symptoms affects whether that symptom is more or less likely to be true 
GVHD. Please refer to Tables 2 and 3 to assist you in deciding whether to attribute these clinical findings 
to GVHD, especially in situations where a biopsy is not possible. For additional help, please see the text 
which follows the tables. Table 4 reviews the approach to assessing GVHD as acute, chronic, or the overlap 
between the two. 
 
Finally, engraftment syndrome will be reported separately from the GVHD scoring presented below. 
Engraftment Syndrome 
A clinical syndrome of fever, rash, respiratory distress, and diarrhea has been described, just prior 
to engraftment in patients undergoing unrelated cord blood and mismatched transplantation. If, in 
the judgment of the local investigator, a patient experiences this syndrome, details of the event 
should be reported when requested in the study CRFs. 
 
Modified Glucksberg Staging Criteria for Acute Graft versus Host Disease 
 
Table 1 Organ Staging (See tables and text below for details) 
Stage  
Skin  
Liver (bilirubin)  
Gut (stool output/day)  
0  
No GVHD rash  
< 2 mg/dL  
Adult: < 500 mL/day  
Child: < 10 mL/kg/day  
1  
Maculopapular rash  
< 25% BSA  
2-3 mg/dL  
Adult: 500–999 mL/day  
Child: 10 -19.9 mL/kg/day 
Or persistent nausea, 
vomiting, or anorexia, with 
a positive upper GI biopsy.  
2  
Maculopapular rash  
25 – 50% BSA  
3.1-6 mg/dL  
Adult: 1000-1500 mL/day 
Child: 20 – 30 mL/kg/day  
3  
Maculopapular rash 
> 50% BSA  
6.1-15 mg/dL  
Adult: > 1500 mL/day  
Child: > 30 mL/kg/day  
4  
Generalized 
erythroderma plus 
bullous formation and 
desquamation > 5% BSA  
>15 mg/dL  
Severe abdominal pain with 
or without ileus, or grossly 
bloody stool (regardless of 
stool volume).  

 
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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 
presumed to be 50% of total stool/urine mix (see Section 3.2). 
 
For stage 4 GI: the term “severe abdominal pain” will be defined as: 
(a) Pain control requiring institution of opioid use, or an increase in on-going opioid use, PLUS 
(b) Pain that significantly impacts performance status, as determined by the treating MD. 
 
If colon or rectal biopsy is +, but stool output is < 500 mL/day (< 10 mL/kg/day), then consider as 
GI stage 0.  
 
There is no modification of liver staging for other causes of hyperbilirubinemia 
 
Overall Clinical Grade (based on the highest stage obtained): 
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 involvement, 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 involvement 
 
Table 2 Evaluating Liver GVHD in the Absence of Biopsy Confirmation (See Table 3.0 below) 
 
Establishing liver GVHD with no skin or GI GVHD 
No Skin/GI GVHD 
Day 0-35 
Assume no liver GVHD, unless proven by biopsy  
No Skin/GI GVHD  
Day 36-100 
If NO other etiology identified, NO 
improvement with stopping 
hepatotoxic medications/TPN: 
Stage as liver GVHD  
If other etiology identified or 
improves with stopping hepatotoxic 
drugs/TPN: 
Do not stage as liver GVHD  
Establishing liver GVHD with skin or GI GVHD and other cause of hyperbilirubinemia 
Skin and/or GI GVHD 
present  
Worsening bilirubin level (includes 
worsening just prior to onset of skin 
or GI tract GVHD) OR stable 
elevated bilirubin despite resolution 
of non-GVHD cause of increased 
bilirubin:  
Stage as liver GVHD  
Stable or improving bilirubin after 
diagnosis of skin or GI GVHD, 
irrespective of treatment:  
Do not stage as liver GVHD 
Changing liver GVHD stage with other cause of hyperbilirubinemia 
Skin and GI GVHD 
stable, improving, or 
absent  
Liver GVHD staging is carried forward without increase in stage until other 
disease process resolves (e.g., if TTP is diagnosed in the presence of stage 2 
liver GVHD, the liver GVHD stage 2 is carried forward despite rising 
bilirubin level until TTP is resolved. If there is no liver GVHD – stage 0 – 
and new onset TTP, the stage 0 is carried forward until TTP is resolved).  

 
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Skin and/or GI GVHD 
worsening  
Liver GVHD is staged according to the Glucksberg criteria. The 
elevated bili is attributed to GVHD alone. 
 
Thus, when skin or GI GVHD is worsening, there is no downgrading of 
liver GVHD staging for other causes of hyperbilirubinemia. (e.g., if TTP is 
diagnosed in the presence of stage 2 liver GVHD and worsening skin or GI 
GVHD, the liver is staged according to the actual bilirubin level even if 
some of the rise in bilirubin is attributed to TTP). 
 
Similarly, even if there is no liver GVHD at onset of a new process, (such 
as TPN cholestasis), but skin or GI GVHD worsen during that process, then 
liver GVHD is diagnosed and staged according to the height of the 
bilirubin. 
 
There is one exception to this: the diagnosis of TTP, with high LDH and 
unconjugated bilirubin precludes the diagnosis and staging of new liver 
GVHD in the absence of a confirmatory liver biopsy. 
 
 
Table 3 Evaluating GI GVHD in the Absence of Biopsy Confirmation (See Table 4.0 below) 
 
Establishing GI GVHD with new onset diarrhea and no skin or liver GVHD 
No Skin/liver GVHD  
Day 0 through 
engraftment 
Assume no GI GVHD, unless proven by biopsy  
 
No Skin/liver GVHD 
Engraftment through 
day 100  
NO other etiology of diarrhea 
identified: 
Stage as GI GVHD  
Any other etiology of diarrhea 
identified: 
Do not stage as GI GVHD  
Establishing GI GVHD with pre-existing diarrhea and skin or liver GVHD 
Skin and/or liver GVHD 
present  
Worsening diarrhea (includes 
worsening just prior to onset of skin 
or liver GVHD) OR persistent 
diarrhea despite resolution of non-
GVHD cause: 
Stage as GI GVHD  
Improving diarrhea after the 
diagnosis of skin or liver GVHD 
(irrespective of treatment) OR 
persistent diarrhea without resolution 
of underlying non-GVHD cause: 
Do not stage as GI GVHD  
 
 
Differentiating Acute GVHD, Chronic GVHD, and Overlap Syndrome  
There is often confusion differentiating acute from chronic GVHD, especially in the setting of reduced 
intensity transplants, DLI and new prophylactic treatments. The NIH Working Group recently published 
new classifications for GVHD: 
 
 

 
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Table 4 Acute GVHD, Chronic GVHD, and Overlap Syndrome 
Category  
Time of Symptoms 
after HCT or DLI  
Presence of Acute 
GVHD features  
Presence of Chronic 
GVHD features  
Acute GVHD  
 
 
 
Classic acute GVHD  
≤100 d  
Yes  
No  
Persistent, recurrent, or 
late-onset acute GVHD 
>100 d  
Yes  
No  
Chronic GVHD  
 
 
 
Classic chronic GVHD 
No time limit  
No  
Yes  
Overlap syndrome  
No time limit  
Yes  
Yes  
 
• 
Scoring of acute GVHD may need to occur past day 100. In particular, patients should continue to 
be scored for acute GVHD when classic acute GVHD symptoms (maculopapular rash, nausea, 
vomiting, anorexia, profuse diarrhea - particularly if bloody and ileus) persist past day 100 or if 
identical symptoms previously scored as acute GVHD resolve and then recur within 30 days during 
immunosuppression taper but past day 100. 
• 
Those patients being scored as having acute GVHD should NOT have diagnostic or distinctive 
signs of chronic GVHD. 
• 
Patients with both acute and chronic symptoms should be diagnosed as having Overlap 
Syndrome and scored according to their chronic GVHD score.  
 
Further Explanation of Criteria presented in Tables 2 and 3 
 
1.0 Assessment of Skin GVHD  
 
1.1 Presence or Absence of Skin GVHD: Skin GVHD will be considered present if a rash characteristic 
of acute GVHD develops after allogeneic marrow transplantation involving more than 25% of the 
body surface not clearly attributable to causes such as drug administration or infection. The extent of 
the body surface area involved can be estimated by the “Rule of Nines”. In estimating the extent of 
skin GVHD, the area involved is calculated for individual anatomic areas, such as the arm or leg, and 
then the total is derived from a simple summation. Areas that are non-blanching should not be 
considered involved regardless of the overlying color of the rash (red, brown, etc.). Limited 
distribution erythema (with the exception of palms and soles) in the absence of associated rash 
elsewhere on the body will not be considered GVHD.  
 
2.0 Assessment of Liver GVHD  
 
2.1 Assessing for the Presence or Absence of Liver GVHD 
A. Hyperbilirubinemia (total bilirubin > 2.0 mg/dL) in the absence of other signs of acute GVHD in 
the skin or GI tract: 
i) Day 0-35: If hyperbilirubinemia alone is present with no other signs of acute GVHD in other 
organ systems, acute GVHD will not be diagnosed based solely on laboratory abnormalities. 
Acute GVHD will be diagnosed if findings on histopathology studies of liver from a biopsy or 
autopsy are confirmatory. 

 
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ii) Day 35-100: If hyperbilirubinemia (must be conjugated bilirubin) is not improving or is 
exacerbated (especially if serum alkaline phosphatase is increased), in the absence of acute 
GVHD in other organ systems, no other etiologies are identified, and does not improve with 
discontinuation of hepatotoxic drugs, acute GVHD will be diagnosed. However, it is 
distinctly unusual to develop ascites or a coagulopathy in the early stages of acute GVHD of 
the liver alone. In the absence of histopathology studies of liver from a biopsy or autopsy 
specimen, ascites or a coagulopathy secondary to liver dysfunction will be considered to 
indicate the presence of another disease process (e.g. veno-occlusive disease). Recommended 
non-invasive studies to define an etiology for hyperbilirubinemia are: 
a. Imaging of liver (CT or ultrasound) 
b. Hepatitis screen (only if ALT is elevated) 
c. PT 
d. Blood cultures 
e. Review of medication list for potentially hepatotoxic drugs 
f. 
Review of risk factors for viral liver infection (HSV, CMV, VZV, adenovirus, EBV, 
HBV, and HCV) 
g. Hemolysis screen 
 
B. Pre-existing hyperbilirubinemia clearly attributed to an etiology other than acute GVHD in the 
presence of signs of acute GVHD in other organ systems. 
i) If pre-existing non-GVHD liver disease (documented clinically, by lab assessment, or by 
imaging studies) is stable or improving at the onset of signs of acute GVHD in other organs, 
then acute GVHD of the liver will not be considered to be present unless proven by liver 
biopsy or autopsy. 
ii) If hyperbilirubinemia worsens several days before or at the time of onset of signs of acute GVHD 
in other organ systems, GVHD will be considered to be present unless histopathology studies 
of liver are available and negative on a biopsy during that time interval or autopsy results 
exclude GVHD. 
iii) If hyperbilirubinemia persists and is not improving after resolution of a pre-existing non-GVHD 
liver disease process (e.g. localized infection of liver, systemic sepsis, biliary tract obstruction) 
when signs of acute GVHD are present in other organ systems or no other intervening cause 
has been diagnosed, then acute GVHD will be considered to be present in the absence of a new, 
clearly identifiable cause of non-GVHD liver disease or unless a liver biopsy or autopsy 
specimen is negative.  
 
C. Prior acute GVHD in liver with new onset of a disease process that exacerbates pre-existing or 
recently resolved hyperbilirubinemia: 
i) If an etiology other than acute GVHD is clearly identified as causing or exacerbating 
hyperbilirubinemia and acute liver GVHD has been diagnosed and has been stable, improving, or 
resolved, then the liver will not be restaged for acute GVHD until the resolution or stabilizing of 
the concurrent disease process (i.e., the liver stage prior to the onset of the new disease process will 
be carried forward until the new disease process resolves). Example: Acute GVHD of the liver and 
gut is diagnosed on day 20. Treatment of acute GVHD results in falling bilirubin levels to liver 
stage 1. Sepsis or TTP develops with transient worsening of the hyperbilirubinemia. The liver stage 
is not increased, despite a higher bilirubin level, because the cause of worsening hyperbilirubinemia 
is attributed to sepsis or TTP. 

 
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ii) If an etiology other than acute GVHD is clearly identified as causing or exacerbating 
hyperbilirubinemia in the presence of already worsening acute liver GVHD or GVHD of the skin 
or GI tract is simultaneously worsening, then the liver GVHD will be staged according to the actual 
bilirubin level, even though another cause of hyperbilirubinemia is present. 
 
 
3.0 Assessment of GVHD of the Gastrointestinal Tract 
3.1 Assessing for the Presence or Absence of GVHD of the Gastrointestinal Tract 
A. Diarrhea (> 500 mL/day in adults or > 10 mL/kg in pediatric patients) in the absence of other signs 
of acute GVHD in other organ systems 
i) Day 0-engraftment: If diarrhea alone is present without other signs of acute GVHD in other 
organ systems, acute GVHD will not be considered present. Diarrhea will be attributed to acute 
GVHD if histopathology studies of gastrointestinal tract from a biopsy or autopsy are 
diagnostic. 
 
ii) Engraftment-day 100: If diarrhea persists and is not improving, is exacerbated, or develops de 
novo in the absence of acute GVHD in other organ systems, histopathology studies of gut 
biopsies or from autopsy specimens are not available, and no other etiologies are clearly 
identified, acute GVHD will be considered to be the cause. A stool specimen should be 
examined to rule out infectious causes (e.g. rotavirus, adenovirus, and C. difficile toxin). It is 
recommended, if at all possible, that biopsies be obtained for diagnostic purposes. 
B. Pre-existing diarrhea clearly attributed to an etiology other than acute GVHD in the presence of 
signs of acute GVHD in other organ systems: 
i) If pre-existing diarrhea caused by a process other than GVHD has been documented clinically 
or by lab assessment and is stable or improving at the onset of signs of acute GVHD in the skin 
or liver, then acute GVHD of the intestine will not be considered to be present in the absence 
of biopsy confirmation or autopsy report. 
• 
ii) If diarrhea or gastrointestinal symptoms are already present, but worsen significantly at 
the time of onset of signs of acute GVHD in the skin or liver, GVHD will be considered 
present, unless biopsy or autopsy are negative. 
iii) If diarrhea persists after resolution of a pre-existing disease process with signs of acute 
GVHD present in other organ systems, GVHD will be considered present, unless biopsy or 
autopsy are negative. 
C. Prior or present acute GVHD in other organ systems with new onset of diarrhea: 
If diarrhea is clearly attributable to an etiology other than acute GVHD (e.g., infection) and a 
history of acute GVHD exists or acute GVHD is present in other organ systems and is stable, then 
the gastrointestinal tract will not be evaluable for acute GVHD until the resolution or stabilizing of 
the other disease process (e.g., infection) in the absence of biopsy or autopsy confirmation. 
D. Persistent anorexia, nausea or vomiting in the absence of signs of acute GVHD in other organ 
systems:  

 
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Persistent anorexia, nausea or vomiting in the absence of other known causes of these 
symptoms will be considered stage 1 acute GVHD if confirmed by endoscopic biopsy. 
 
If a biopsy is not possible (e.g. secondary to thrombocytopenia) but the clinical findings 
are compatible with acute GVHD, then the patient will be treated and recorded as having 
acute GVHD. 
3.2 Staging of the Gastrointestinal Tract for the Severity of Acute GVHD 
The severity of gastrointestinal tract GVHD will be staged according to modified Glucksberg criteria. 
To minimize errors caused by large day-to-day variation, diarrhea volume is measured as an average 
over 3 days and reported as the volume in milliliters per day. When urinary mixing is noted the stool 
volume will be considered half of the total volume unless nursing staff is able to give a better estimate 
from direct observation. Abdominal cramps are considered significant for staging if the severity results 
in a clinical intervention (e.g. analgesia, fasting, etc.). Blood in the stools is considered significant if 
the blood is visible or hematochezia/ melena is present and not clearly attributed to a cause other than 
GVHD (e.g. epistaxis/ hemorrhoids). 
 
 
 

 
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