Precision Diagnostics in Inflammatory Bowel Disease, Cellular Therapy and Transplantation (The PREDICT Trial)
Precision Diagnostics in Inflammatory Bowel Disease, Cellular Therapy and Transplantation (The PREDICT Trial)
The goal of the Precision Diagnosis in Inflammatory Bowel Disease, Cellular Therapies, and
 Transplantation (PREDICT) trial is to apply a systems-biology approach to enable precision
 diagnostics for the key immunologic outcomes for patients with Inflammatory Bowel Disease,
 Cellular Therapeutics and Transplantation. This approach will deepen the understanding of the
 molecular mechanisms driving auto- and allo-immune diseases and serve as a critical platform
 upon which to design evidence-based treatment paradigms for these patients.

 This research study will examine the immunology of auto- and allo-immune gastrointestinal
 disturbances such as Inflammatory Bowel Disease (IBD), Graft-versus-Host Disease (GVHD), and
 Functional Gastrointestinal Disorder (FGID), as well as the immune manifestations after CAR-T
 and other cellular therapeutics. The Investigators seek to use blood and tissue samples in
 order to better understand the mechanisms driving these diseases and their therapies.

 The Investigators further hypothesize that longitudinal systems-based immunologic analysis
 will enable the patient-specific determination of the molecular evolution of IBD, GVHD and
 the response to cellular therapeutics, 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 these patients.
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).

 Hypothesis #3: We hypothesize that we can define the molecular mechanisms, phenotypic and
 functional immunologic characteristics involved in distinct determinants of adoptive cellular
 therapies, including the efficacy, longevity and toxicity associated with cellular therapy.
 Longitudinal characterization of cellular therapeutics and the endogenous immune response
 they elicit using advanced immunologic analysis including flow cytometry, mass spectrometry,
 TCR deep sequencing and single-cell transcriptomics will allow identification and distinction
 of pathways critical for efficacy and toxicity and enable subsequent therapeutic modulation.

 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.

 Specific Aim #3: Identify the molecular and cellular immunologic mechanisms involved in
 determining the clinical response to cellular therapies and distinguish pathways critical to
 a successful anti-tumor response from those involved in adverse effects.

 Objective 1: Characterize the cellular product prior to administration and track its
 distribution, kinetics, persistence and function longitudinally in vivo in the patient's
 peripheral blood and when applicable bone marrow, CSF and other tissues, using qPCR-based
 transgene detection (if applicable), flow cytometry, mass cytometry, TCR deep sequencing and
 whole transcriptome analysis on T cells and other immune cells contained in the cellular
 product.

 Objective 2: Longitudinally interrogate the interplay of the cellular therapy with the
 endogenous immune system and delineate the role of the endogenous immune response in the
 efficacy, persistence and toxicity of cellular therapy, using flow cytometry, mass cytometry,
 TCR deep sequencing, whole transcriptome analysis on endogenous immune cells and analysis of
 soluble factors and antibodies.

 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 cells12,13 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%14-17. 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 disease17.

 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 anorexia12. Histopathological diagnostic criteria for GI GVHD
 includes identification of crypt cell apoptosis, crypt destruction and/or mucosa
 denudation18. 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 absent19 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 tract20. 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 occured18. 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 GVHD21. Clostridium difficile and cytomegalovirus infections can
 also have similar clinical and histopathological presentations22. Use of mycophenolate
 mofetil23 and proton pump inhibitors24 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 GVHD18,25 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 GVHD26-28. 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 GVHD29-31. 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 patients32-35 with the incidence of this
 disease rising in the past 2 decades36 chronic GVHD causes significant mortality, and in
 those patients that survive, it can result in profound effects on quality of life37-40.
 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 application41. 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 inhibitiors (CNIs)42-44 and unfortunately,
 approximately 50% of patients will fail and require second line treatment45,46 with
 failure-free survival at 2 years following second-line therapy being only 25%47. 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 technologies30,48-54. 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.

 Cellular Therapy: Novel adoptive cell therapies are increasingly entering clinical trials and
 are available as FDA approved biologics, thereby providing a therapeutic option for
 previously refractory patients. This broad and exciting field includes chimeric-antigen
 receptor (CAR) T cells directed against a variety of antigens55, T cells genetically modified
 to express TCRs56,57, cytokine-stimulated NK-cells58, as well as tumor vaccine approaches
 with endogenous, irradiated tumor samples59 or immune cells pulsed with tumor antigen.60
 Chimeric antigen-receptor T cells have led to dramatic responses, particularly in patients
 with CD19+ B-cell malignancies61-64, leading to FDA approval of several products. While
 unprecedented clinical remissions can be achieved initially, the approach is frequently
 limited by the durability of the response and CAR T cell persistence with an EFS of 50% at 12
 months after infusion61, tumor escape mechanisms65 and significant toxicity involving
 cytokine release syndrome, neurologic toxicity66. In the context of CAR T cell therapy, key
 factors for successful application have been identified and include incorporation of a
 costimulatory signaling mechanism67,68, association of in vivo CAR T cell expansion with
 response69 and contribution of T cell phenotypic subgroups with capacity to proliferate and
 persist long term70. Additionally, important insights into the roles of IL-6 and IL-1 in
 cytokine release syndrome and neurotoxicity71,72 and the contribution of a pan-T cell
 infiltrate and high cytokine levels in the CNS as mediators of neurotoxicity 73, have been
 gained by our group and others. However, there is a significant unmet need to apply a
 systematic approach interrogating the determinants of a successful response, toxicity,
 interaction with the endogenous immune system and short- and long-term effects of
 interventions aimed at reducing toxicity such as administration of the IL6R-antagonist
 Tocilizumab or IL-1 blockade74.

 In this study we aim to systematically interrogate the characteristics of cell therapy
 products, evolution after administration in vivo and behavior in different compartments such
 as peripheral blood, bone marrow, CNS and if applicable tissues and their interplay with the
 endogenous immune system. Single cell transcriptomics, coupled with TCR-sequencing (when
 applicable BCR sequencing) and phenotypic characterization of transferred and endogenous
 immune cells as well as analysis of secreted immune factors such as cytokines, chemokines and
 antibodies in the plasma will be employed and correlated with clinical responses. This
 unbiased, systematic precision-diagnostic approach will allow identification of critical
 pathways which are common or distinct depending on the nature of the cell therapeutic
 approach, target and disease entities and associated or segregable from undesired toxicities.
 This will critically inform future rational design of cellular therapeutics and inform
 possible therapeutic interventions after cell therapy administration.
Inclusion criteria for HSCT and IBD-FGID cohorts:

 -  Patients must be at least 6 years old

 -  Weigh >/= 10 kg.

 -  Patients eligible for inclusion are those receiving a first allogeneic hematopoietic
 stem cell transplantation (bone marrow, peripheral blood, or cord blood transplant);
 or patients being evaluated for inflammatory bowel disease (new diagnosis or follow up
 of established disease); or patients being evaluated for functional bowel disease (new
 diagnosis or follow up of established disease).

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

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

 Inclusion Criteria for Cellular Therapy Cohort:

 -  Weight >/= 10kg

 -  Patients receiving adoptive cellular therapy

 -  Patient and/or legal guardian must sign written informed consent

 Inclusion Criteria for HCT Related Donor cohort:

 -  Age >6 years of age

 -  Weight >10kg

 Exclusion Criteria

 -  None