Precision Diagnostics in Inflammatory Bowel Disease, Cellular Therapy and Transplantation (The PREDICT Trial)

Hypotheses:

Hypothesis #1: The Investigators hypothesize that they can define the molecular mechanisms
responsible for Inflammatory Bowel Disease (IBD) and gastrointestinal (GI) acute GVHD and
differentiate it from other inflammatory disorders by using advanced immunologic analysis
including flow cytometry, TCR deep sequencing and transcriptomics.

Hypothesis #2: The Investigators further hypothesize that longitudinal systems-based
immunologic analysis will enable the patient-specific determination of the molecular
evolution of IBD as well as acute and chronic GVHD as well post-transplant defects in
protective immunity, and determine which pathways, when perturbed, can cause clinical
disease. The discovery of these pathways will lead to improved diagnostic, prognostic and
treatment approaches, and to personalized therapeutic decision-making for patients undergoing
hematopoietic stem cell transplantation (HCT).

Aims:

Specific Aim #1: To identify the mechanisms specific for IBD and GI acute GVHD and delineate
it from other inflammatory disorders.

Objective 1: Perform flow cytometry, TCR deep sequencing and whole transcriptome analysis on
T cells purified from GI tissue samples taken from patients who undergo endoscopy for
presumed GI GVHD, inflammatory bowel disease (IBD), and functional gastrointestinal disease
(FGID).

Objective 2: Perform flow cytometry, TCR deep sequencing and transcriptome analysis on T
cells from the peripheral blood at the time of endoscopy in patients diagnosed with GI GVHD,
IBD, and FGID.

Specific Aim #2: Characterize the immunologic dysregulation responsible for IBD, acute GVHD,
chronic GVHD and defects in protective immunity in patients undergoing HCT.

Objective 1: Perform longitudinal immune analysis on T cells and B cells purified from
patients with IBD and those undergoing allogeneic HCT. For transplant patients, we will
compare T and B cell immunity in patients who develop acute and chronic GVHD, relapse, and
infectious complications post-transplant and compare to patients without these complications.

Objective 2: Perform microbiome analysis longitudinally in patients with IBD and those
undergoing HCT to determine the impact of microbiome alterations in the development of
post-transplant complications.

Background and Significance:

IBD: Inflammatory bowel disease (IBD) which includes Crohn's Disease (CD) and Ulcerative
Colitis (UC), is a chronic complex gastrointestinal (GI) autoimmune condition that inflicts
1.4 million people in the united states1. The incidence and prevalence of both CD and UC are
increasing over time and encompassing larger areas of the world1,2. In addition, pediatric
IBD comprises 25% of all diagnosed IBD, relegating the child to a lifetime of
gastrointestinal disease and exposure to immunosuppression especially during a period meant
for growth and development. Despite ongoing research into the pathogenesis and genetic
abnormalities, the mechanism behind IBD development and progression is not well understood.
Standard therapies still rely on steroids, other non-specific immunosuppression (such as
methotrexate and azathioprine), and anti-TNF biologics. Although newer therapies such as
agents that block cytokines and leukocyte trafficking are emerging, no universally successful
treatments have been identified. Thus, relapsing forms of IBD continue to lead to systemic
compromise in nutritional absorptive capacity, anemia, and often, to the need for surgical
interventions. Deciphering the mechanisms driving the unique subtypes of IBD (even within UC
and CD) then optimizing treatment based on the underlying systemic dysregulation is a
critical unmet need in the field. While the underlying immune mechanism of IBD remains
undetermined, there is significant data to suggest that IBD may represent an inappropriate
immune response towards self antigens and commensal microbiota in a genetically susceptible
host3. Thus, murine colitis models suggest that mucosal inflammation results from pathologic
T helper- (Th) cell responses, along with regulatory cell defects. These data have emerged
from experiments in IL-2 deficient mice4, IL-10 deficient mice5, TGF-beta6, and TGF-betaGRII
dominant negative transgenic mice7. The pathogenesis also includes an exaggeration in
effector cell responses, which have emerged from experiments in Stat4 transgenic mice8 and
TNFARE mutant mice9. More recently human cytokine analysis suggests that despite clinical
similarities, each subtype of IBD show distinctive cytokine profiles10. Although initial
studies have begun to target specific effector T cell pathways11, the application of target
organ transcriptomics is in its infancy and individual targetable pathways are still elusive.

HCT: Allogeneic HCT is an effective treatment for patients with malignant and non-malignant
hematologic diseases. However, this treatment is complicated with high rates of morbidity and
mortality limiting its broader application. The leading causes of post-transplant morbidity
and mortality include acute and chronic GVHD, relapse and infectious disease. The goal of the
PREDICT trial is to apply a systems approach to understanding the mechanisms driving these
complications, such that evidence-based treatment strategies can be devised.

Acute GVHD: Acute GVHD is mediated by donor-derived allo-reactive T cells becoming activated
and resulting in cytotoxicity against host cells5,6 as well as cytokine-mediated tissue
damage. Moderate to severe acute GVHD can occur in up to 60% of patients undergoing HCT and
the more severe forms have been associated with mortality rates >50%1-4. The most common
sites of the immune-mediated tissue damage are the liver, skin, and gastrointestinal (GI)
tract. GI GVHD occurs in 40-50% of HCT patients and is the major cause of morbidity and
mortality from this disease4.

The diagnosis of GI GVHD is derived from clinical and histopathological findings. GVHD can
occur in both the upper and lower GI tract leading to symptoms of diarrhea, abdominal pain,
nausea, vomiting, and/or anorexia5. Histopathological diagnostic criteria for GI GVHD
includes identification of crypt cell apoptosis, crypt destruction and/or mucosa denudation7.
Unfortunately, the severity of GVHD on histology is poorly correlated with the clinical
course of the disease.

While GI GVHD is a common complication following HCT there remain many barriers to its
consistent and accurate diagnosis. First, diagnosis is dependent on appropriate tissue
sampling. Visible lesions are frequently absent8 and endoscopic findings can be diffuse and
nonspecific. There is also no consensus on the optimal location of the GI tract for biopsies
or number of biopsies needed to secure a diagnosis. There is also frequent discordance
between biopsy specimens from the upper and lower GI tract9. Second, patients presenting
early in the course of GVHD may have subtle histopathological findings that may be missed or
not yet present. At the onset of GVHD few apoptotic cells may be seen and crypt loss and
mucosal damage may yet to have occured7. Lastly, there are also confounding factors that can
lead to the misdiagnosis of GVHD that include conditioning regimen related toxicity,
concomitant infections, and medications which can all cause focal inflammation of the GI
tract. In the first 20 days following a myeloablative conditioning regimen diffuse apoptosis
can be seen mimicking acute GVHD10. Clostridium difficile and cytomegalovirus infections can
also have similar clinical and histopathological presentations11. Use of mycophenolate
mofetil12 and proton pump inhibitors13 have also been found associated with GI tract
apoptosis that can be misdiagnosed as GVHD. All of these factors lead to the high degree of
inter-observer variability in the histological diagnosis of GVHD7,14 and poor correlation
with the clinical observations, illustrating the need for more sensitive and specific methods
of diagnosis.

There have been recent advances in the identification of biomarkers in GVHD that have
diagnostic and prognostic significance. IL-8, IL-2 receptor-alpha, tumor necrosis factor
receptor-1 (TNF-1), hepatocyte growth factor (HGF), elafin, regenerating islet-derived
3-alpha (reg-3alpha), TIM3, IL-6, ST2, B-cell activating factor (BAFF), IL-33, CXCL10, and
CXCL11 have all been found to have utility in predicting the development of GVHD15-17. While
these biomarkers have been identified they have not been extensively validated and are yet to
be clinically adopted as a guide to alter treatment. Moreover, the biomarkers discovered thus
far are often the result of downstream pathway perturbations and discovering the upstream
dysregulation that occurs earlier in the course of the disease may be valuable in developing
diagnostic or prognostic models that could lead to trials aimed at altering the natural
course of the disease.

Our group has previously shown that by using advanced immunologic analysis including flow
cytometry, and whole transcriptome analysis, we can identify previously unrecognized
molecular pathways active in GVHD18-20. We anticipate that by utilizing a systems immunology
approach in patients with acute GVHD we will be able to identify pathways that have
diagnostic and prognostic value. This may enhance our diagnostic capacity and most
importantly, allow us to individualize management of patients based on their specific
immunologic profiles.

Chronic GVHD: CGVHD occurs in 40-60% of transplant patients21-24 with the incidence of this
disease rising in the past 2 decades.25 chronic GVHD causes significant mortality, and in
those patients that survive, it can result in profound effects on quality of life26-29.
Despite the increased frequency of chronic GVHD, accurate diagnosis and evidence-based
therapy is still lacking. Thus, while chronic GVHD biomarkers have been identified there have
yet to be any that qualify for clinical application30. Moreover, these biomarkers often
represent end-stage pathway perturbations and may result from nonspecific inflammation and
tissue damage as well as counter-regulatory mechanisms. In addition to the challenges in
diagnosis, there are significant treatment challenges as well: Thus, treatment of chronic
GVHD has not changed significantly over the past few decades. First line therapy remains
corticosteroids with or without calcineurin inhibitors (CNIs)31-33 and unfortunately,
approximately 50% of patients will fail and require second line treatment34,35 with
failure-free survival at 2 years following second-line therapy being only 25%36. These data
underscore the significant unmet needs in this field, both for molecular diagnostics and
evidence-based treatment paradigms.

Protective Immunity: In addition to the challenges of acute and chronic GVHD, patients
undergoing HCT and those with IBD face other toxicities as well, many of which are related to
dysfunctional immune reconstitution after transplant. However, although the phenomenology of
the many defects in protective immunity (both against infectious pathogens and against
leukemia relapse) is well-documented, the causative molecular mechanisms remain unknown. To
address these questions, our group and others have begun to perform detailed assessments of
immunologic reconstitution after HCT including the application of new T Cell Receptor (TCR)
and B cell Receptor (BCR) deep-sequencing technologies.19,37-43 These technologies allow the
investigation of the breadth and depth of post-transplant immune reconstitution at a level of
molecular detail not previously possible and hold the promise of deepening our understanding
of the impact of infectious pathogens on global immune health and immune reconstitution. The
widespread application of these technologies, and their intersection with detailed assessment
of immune phenotype and function can provide novel insights about the state of immune health
in transplant patients, and holds the promise of identifying patients in need of novel
interventions to improve their post-transplant immune reconstitution.

The goal of the PREDICT trial is to apply a systems-biology approach to enable precision
diagnostics for the key immunologic outcomes post-transplant. This approach will deepen our
understanding of the molecular mechanisms driving the most deadly post-transplant
complications, and serve as a critical platform upon which to design evidence-based treatment
paradigms for transplant patients.

Inclusion criteria for HSCT and IBD-FGID cohorts:

- Patients must be at least 6 years old and weigh >/= 10 kg.

- Patients eligible for inclusion are:

- Patients receiving a first allogeneic hematopoietic stem cell transplantation
(bone marrow, peripheral blood, or cord blood transplant).

- Patients being evaluated for inflammatory bowel disease (new diagnosis or follow
up of established disease).

- Patients being evaluated for functional bowel disease (new diagnosis or follow up
of established disease).

- Patients receiving cellular therapeutics.

- Patients and/or parents or legal guardians must sign a written informed consent.

- IBD/FGID Pediatric patient cohort: Phone consent will be used to obtain second
parent/guardian consent when both parents/guardians are not present at the
initial in-person consent conference.

- Negative pregnancy test within 30 days of enrollment for females of childbearing
potential.

Inclusion Criteria for HCT Related Donor cohort:

- Age >6 years of age

- Weight >10kg

Exclusion Criteria