Pilot Study of Stem Cell Transplantation for Children and Young Adults With Refractory Crohn's Disease.
Pilot Study of Stem Cell Transplantation for Children and Young Adults With Refractory Crohn's Disease.
This is a study for people with Crohn's Disease (CD) that are not responsive to standard
 treatment. CD is a chronic disease with an auto-immune component that goes away and relapses
 over the years and causes lifelong impairment of health and quality of life. Regardless of
 the therapy used some patients remain seriously ill with active disease after multiple
 therapeutic options have been exhausted. There is currently no drug that will cure CD. Drug
 treatment is focused on controlling symptoms. Another treatment is to perform surgery but
 again this does not lead to cure and is often linked to infection, short gut syndrome
 problems and psycho-social and cosmetic issues. Therefore, a treatment that does not involve
 surgery or long-term drug treatment may be beneficial especially to young adults.

 Hematopoietic stem cell transplantation (HSCT) has been of value in other auto-immune
 diseases and it is possible that it could be of value in CD. This is a pilot study to
 determine if HSCT is safe and effective for children and young adults with severe CD. For
 this study the stem cells will come from the patient. This is called an autologous
 transplant. The patient will undergo collection and storage of his/her peripheral blood stem
 cells (PBSC). The patient will be given drugs to move (or mobilize) the stem cells from
 his/her bone marrow into his/her blood where they will be collected on a machine called
 apheresis (similar to dialysis.) The cells will be stored and given back to the patient about
 1 month after collection.
Crohn's Disease (CD) is an immunologically mediated chronic illness that has a relapsing and
 remitting course, most commonly presenting in the 2nd or 3rd decade and causing lifelong
 impairment of health and quality of life. Mainstay of clinical treatment for severe disease
 is combination of anti-inflammatory agents like 5-aminosalicyaltes and immunosuppressive
 medications like corticosteroids and newer anti-TNF antibodies like Infliximab. None of the
 drugs are, at present, curative and a relevant subset of patients are refractory to many of
 these pharmacologic approaches.

 Immunoablative treatment followed by autologous stem cell rescue (Autologous HSCT) has been
 tried in this refractory group of patients with successful results. Autologous HSCT is
 effective in this auto-immune setting through the eradication of effector/memory T-cell
 clones due to a direct immuno-ablative effect of drugs used in the preparative regimen; by
 leading to an immune-reset- recovering clones of T- cells from the infused stem cells do not
 mount an auto- immune response and are tolerant to 'self' antigens and, by upregulation of T
 regulatory cells (Treg, CD4+CD25+FOXP3+ or CD8+ FOXP3+) via change in cytokine mileu during
 transplant. Increased population of Tregs restricts the activity of self-reactive effector
 T-cells.

 This pilot study is designed to gain on the success of previously published adult studies of
 autologous HSCT in refractory CD, with the aim to confirm the feasibility, safety and
 efficacy of HSCT and post-transplant cyclophosphamide when given post-HSCT in pediatric
 patients and young adults.

 Since, this combination of immuno-ablative therapy with post-transplant cyclophosphamide has
 not been extensively studied in children with CD, therefore transplant related mortality
 (TRM) and severe toxicity (> grade 3 toxicity by NCI criteria) will be monitored for 100 days
 post-transplant in all the patients and stopping rules will be enforced in case of excessive
 toxicity or TRM (>10%). Clinical assessments will be done for 1 year post-HSCT for disease
 activity, steroid free remission period to evaluate the 'clinical efficacy' of this
 procedure.

 The major aim of this pilot is to generate the preliminary safety data to confirm the
 feasibility and benefit of autologous HSCT in children and young adults with refractory CD.

 Study Design:

 This is a single arm, pilot study designed to estimate the feasibility, safety and benefit of
 immunoablation with hematopoietic stem cell transplantation (HSCT) using autologous stem
 cells followed by administration of post-transplant cyclophosphamide for children and young
 adults with refractory Crohn's disease (CD). The study population is patients with severe and
 refractory Crohn's disease who are ≥10 and ≤ 30 years of age.

 Objectives:

 Primary Objective:

 To determine the feasibility and toxicity of immunoablation with HSCT and post-HSCT infusion
 of cyclophosphamide in children and young adults with refractory Crohn's disease (CD). Death
 (transplant related mortality, TRM) and severe non-hematologic toxicity (≥ grade 3 toxicity;
 NCI Toxicity Criteria version 4.0) within the first 100 days after HSCT will be monitored to
 meet this end-point.

 Secondary Objectives:

 Secondary objectives are evaluations of incidence of HSCT related complications and
 evaluations of response. These include:

 1. Incidence of viral reactivations - CMV and EBV

 2. Incidence of invasive fungal infections

 3. Immune reconstitution after HSCT lymphocyte subpopulations (absolute number of CD3, CD4,
 CD8 and CD19 cells), immunoglobulin levels (IgG) and CD4+ CD25+ CD127+ regulatory T
 cells (Treg) populations on day +30 , +60 , +100, day +180 and yearly post-HSCT.

 4. Evaluate the efficacy and benefit of HSCT in this population at 1 year post HSCT, by
 serial assessments of clinical activity of CD, assessment of disease severity- CDAI
 scores and length of steroid free remissions.

 Hypotheses:

 Primary: The HSCT and post-transplant cyclophosphamide regimen will be well tolerated with
 acceptable toxicity and will lead to <10% transplant related mortality (TRM).

 Background and Rationale:

 Crohn's disease (CD) is a chronic illness that is immunologically mediated, of unknown
 etiology, but probably induced by an exposure to intestinal bacteria or their component
 antigens leading to an excessive T helper type 1 (TH-1) mediated chronic inflammation of the
 gastrointestinal (GI) tract 1. Crohn's disease is a relapsing and remitting disorder most
 commonly presenting in the 2nd or 3rd decade and causing lifelong impairment of health and
 quality of life 2.

 Regardless of the therapy used some patients remain seriously ill with active disease after
 multiple therapeutic options have been exhausted. In addition, patients with refractory
 disease suffer from an inability to eat, frequent nausea, vomiting, diarrhea, malnutrition,
 growth retardation, fistulae formation, abdominal pain and psychological distress, ileostomy
 or colostomy, and multiple surgeries that may lead to short-gut syndrome, severely affecting
 quality of life. Though not well characterized, a distinct excessive mortality exists in this
 group of patients 3,4.

 Patients with refractory CD have a higher mortality rate compared to normal population
 (hazard ratio of 1.73); but deaths are usually related to surgical complications, liver
 disease, infections and intestinal cancer in long term survivors 5. Even though the overall
 mortality is low in this group of patients, they suffer from significant morbidity,
 cortico-steroid side effects and poor quality of life and 50% patients with refractory
 disease will need surgical interventions (ileostomy, colostomy) further affecting the
 complications rate 2,3.

 Many drugs have been used for controlling the symptoms of CD, but no drug is, at present,
 curative and a relevant subset of patients are refractory to many pharmacologic approaches 6.
 Almost all drugs employed in the advanced form of disease, such as immunosuppressives and
 immunomodulators, have potential risks. Mainstay of clinical treatment is combination of
 anti- inflammatory agents like 5-aminosalicyaltes and immunosuppressive medications like
 corticosteroids4. Majority of the patients with severe refractory disease are treated with
 anti- Tumor Necrosis Factor (TNF) antibodies like Infliximab (Remicade), but limited options
 exist for patients who have failed to respond to anti-TNF therapy7. Approximately 40% of
 patients with CD develop intolerable side effects or fail to respond to anti-TNF therapy,
 hence there is a significant population of this subset of patients where options are very
 limited 8.

 Some symptoms may be controlled by prolonged steroids, but there are many known long term
 effects of continued steroid use- muscle loss, osteopenia, cataracts and cushingoid habitus.
 Majority of these patients enroll on other experimental Phase I/II drug trials or resort to
 surgical intervention to control symptoms. Surgical approach (partial gut resection of the
 inflamed segment with ileo-colostomy) is an option, but is associated with infection risk,
 psycho-social and cosmetic issues and increased morbidity due to short gut syndrome,
 especially in young adults. Increased mortality is also associated with surgical
 interventions 2,3.

 Therefore, any intervention that avoids surgery or long term corticosteroids in young adults
 may be beneficial. Given that hematopoietic stem cell transplantation (HSCT) have been of
 some value in other auto-immune diseases characterized by loss of immune tolerance and or a
 TH-1 predominant immune response, it is possible that these procedures could be of value in
 CD 9,10. Autologous transplantation might also be of benefit because clearing the body of
 committed lymphocyte clones might restore the patient to the status quo ante of being
 predisposed to CD but not suffering from it 11.

 Rationale of HSCT for CD

 HSCT may provide benefit in auto-immune disease by:

 1. Eradication of effector/memory T-cell clones due to a direct immuno-ablative effect of
 drugs used in the preparative regimen.

 2. Immune-reset: recovering clones of T-cells from infused stem cells do not mount an
 auto-immune response and are tolerant to 'self' antigens.

 3. Upregulation of T regulatory cells (Treg, CD4+CD25+FOXP3+ or CD8+ FOXP3+) - by change in
 cytokine mileu during transplant. Increased population of Tregs restricts the activity
 of self-reactive effector T-cells.

 4. Engraftment of uncommitted stem cells in the gut mucosa may lead to direct healing of
 mucosa.

 Evidence for the effectiveness of HSCT in Crohn's disease Initial observations were made in
 patients with Crohn's disease and hematologic malignancies. For patients who underwent HSCT
 for their hematologic malignancy they had remission of their Crohn's disease 12.

 Several studies have reported the clinical course of patients with Crohn's disease receiving
 autologous transplantation for another condition. Perhaps the most interesting concerned a
 patient who developed symptoms at the age of 9, who was diagnosed with Crohn's disease 4
 years later and who required substantial treatment over the next 7 years 13. Following
 autologous stem cell transplantation for non-Hodgkin's lymphoma it was reported that there
 was no clinical or laboratory evidence of recurrence of his Crohn's disease in the next 7
 years. Another reported case of Crohn's disease had diffuse pan colitis before
 transplantation and was asymptomatic (but had inflammation) at endoscopy 3 years later 14.
 Another patient who had symptomatic Crohn's disease for 2 years and had undergone surgery was
 free of disease 5 years after transplantation. Systematic colonoscopic evaluation showed
 persisting subclinical inflammation initially with progressive clearance. At Vanderbilt we
 performed an unrelated donor transplant for a patient with AML and Crohn's disease. He is
 currently 10 years post-transplant with no symptoms of Crohn's disease.

 HSCT specifically for Crohn's disease:

 The first report of stem cell transplantation for Crohn's disease concerned 2 patients from
 the Chicago group 15. In both of the initial cases Crohn's disease activity index was more
 than 250 (normal range <150, pathological range 220 - 600) despite treatment with infliximab.
 In both patients, peripheral blood stem cells (PBSC) were mobilized using cyclophosphamide
 and granulocyte colony stimulating factor (G-CSF), enriched ex-vivo by CD 34+ selection.
 Cyclophosphamide and antithyomcyte globulin (ATG) were used for immune conditioning prior to
 transplantation. The first patient was a 22 year old woman with a past history of a right
 hemicolectomy with severe ileocolic Crohn's disease causing intractable diarrhea with 25
 bowel actions per day, fistula and peri-anal sepsis. The second was a 16 year old boy who had
 been unwell for 6 years requiring tube feeding and receiving methotrexate, 6-mercaptopurine
 and 5 amino acyclic acid with Crohn's colitis. In both of these cases, following
 transplantation, diarrhea resolved and the Crohn's disease activity index normalized. In the
 first patient the haemoglobin rose, whereas in the second it remained at a slightly
 sub-normal level. Post-transplantation CRP was within normal limits and the albumin remained
 or became normal. These cases were accompanied by extensive colonoscopic evidence of disease
 activity. Prior to transplantation both cases had areas of severely active Crohn's disease
 with cobble stoning, fissuring and deep ulceration. Following transplantation the bowel
 remained abnormal though changes were much more trivial with superficial erosions only.

 Oyama et al reported on autologous HSCT for 12 patients median age of 27 (15-38) with severe
 refractory Crohn's disease 16. The conditioning regimen consisted of cyclophosphamide (200
 mg/kg) and equine ATG (90 mg/kg). Eleven of 12 patients entered a sustained remission defined
 by a CDAI < 150. After a median follow-up of 18.5 months (range, 7-37 months), only one
 patient has developed a recurrence of active CD, which occurred 15 months after HSCT.

 Hasselbatt et al reported on autologous HSCT 12 patients (ages 24-50) with refractory Crohn's
 disease using high dose cyclophosphamide (200 mg/kg) without ATG 17. PBSC harvest following
 mobilization chemotherapy was successful in 11/12 patients and resulted in a clinical and
 endoscopic improvement in 7/12 patients. Subsequent conditioning and autoPBSCT were performed
 in nine patients and were relatively well tolerated. Among those, five patients achieved a
 clinical and endoscopic remission within 6 months after autoPBSCT. However, relapses occurred
 in 7/9 patients during follow-up, but disease activity could be controlled by low-dose
 corticosteroids and conventional immunosuppressive therapy.

 In 2010 Burt et al reports on autologous HSCT in 24 patients with mean age of 24 years (range
 15-52) with anti-TNF refractory Crohn's disease 18. Stem cells were mobilized from the
 peripheral blood using cyclophosphamide (2.0 g/m2) and G-CSF (10 ug/kg/day), enriched ex vivo
 by CD34 selection, and reinfused after immune suppressive conditioning with cyclophosphamide
 (200 mg/kg) and either equine antithymocyte globulin (ATG, 90 mg/kg) or rabbit ATG (6 mg/kg).
 Eighteen of 24 patients are 5 or more years after transplantation. All patients went into
 remission with a CDAI less than 150. The percentage of clinical relapse-free survival defined
 as the percent free of restarting CD medical therapy after transplantation is 91% at 1 year,
 63% at 2 years, 57% at 3 years, 39% at 4 years, and 19% at 5 years.

 The percentage of patients in remission (CDAI < 150), steroid-free, or medication-free at any
 posttransplantation evaluation interval more than 5 years after transplantation has remained
 at or greater than 70%, 80%, and 6% respectively.

 Regulatory T cells (Tregs) and Auto-immune disease:

 Regulatory T cells (Tregs) are implicated in the suppression of immune responses and in
 maintenance of tolerance 19,20. The percentage of CD4+CD25+FOXP3+ T cells (T regs) is
 significantly decreased in patients with auto-immune diseases like SLE and Type 1 diabetes
 20. Also, transfer of Treg can prevent auto-immune phenotype that develops after Treg
 depletion in animal models.

 During HSCT for auto-immune diseases, an increase in Treg population correlates with
 remission induction. Zhang et.al has shown that HSCT leads to a newly generated population of
 Treg cells that maintain patients in true immunological remission 21.

 Burt et.al, also evaluated the role of Treg cells in some of their patients who underwent
 HSCT for CD. Before HSCT the mean % of Treg cells was significantly low in CD patients (2.62%
 ± 2.01%), compared to normal volunteers (3.86% ± 1.67%). A robust increase in the % of Treg
 was observed after HSCT, but this was not sustained over period of time and may have
 contributed to relapses 18. Hence, there is clinical evidence that a sustained increase in
 Treg population may maintain remission in patients with auto-immune disorder.

 Rationale of the proposed Study:

 This pilot study is designed to estimate the feasibility of HSCT and safety of post-HSCT
 cyclophosphamide infusions for patients with refractory disease who have failed to respond to
 Infliximab/and or other immunosuppressive agents. This is designed as a small pilot study
 with built in 'stopping rules' in case of excessive toxicity or death.

 Rationale for the immuno-ablative regimen:

 Rationale of CY for mobilization: Cyclophosphamide (CY) will be used for mobilization of
 peripheral stem cells and for immuno-ablation prior to HSCT. CY has been traditionally used
 for mobilization in all HSCT for refractory CD.

 Rationale for post-transplant Cyclophosphamide (CY)- Recently, use of post-transplant CY has
 been pioneered by John Hopkins and shown to be a very effective GVHD prophylaxis in patients
 undergoing HSCT for hematologic malignancies, even in the setting of HLA mismatched and
 haploidentical donors. Post-transplant delivery of CY allows for effective deletion of
 proliferating allo-reactive T- cells that cause GVHD without adding to the toxicity of the
 regimens 22. In a recent study by the Hopkins group post-transplant cyclophosphamide was
 successfully used in patients with sickle cell disease undergoing haploidentical bone marrow
 transplant with no toxicities.

 Some studies have performed CD34+ stem cell selections to get rid of all immune-reactive T-
 cells clones and use purified CD34+ infusions for regenerating the immune system 18. The
 problem with this approach is delayed immune-reconstitution post-transplant and a higher
 incidence of infections.

 European trials have used un-manipulated peripheral blood stem cells (PBSCs) as infusions
 (without any T cell depletion or CD34+ selections) for SCT in auto-immune diseases 23. Since,
 peripheral stem cells contain the same T cells clones that are immuno-reactive and are
 infused along with the donor stem cells in non-T cell depleted infusion, this may be one of
 the reasons for relapse in T cell replete HSCT done till date for CD.

 Therefore, to overcome the above-mentioned issues, we propose to use un-manipulated PBSCs,
 but give two doses of CY pre- and two doses post-stem cell infusion to effectively deplete
 the immuno-reactive T cell clones present in the autologous infusion and therefore, decrease
 the incidence of relapse. The total dose of CY remains the same (200 mg/kg), therefore, it
 will not add to the toxicity of the regimen, but may lead to more effective depletion of the
 immune- reactive clones, obviating the need for costly in-vitro T cell depletions/CD34+
 selections.

 Also, CY causes sustained depletion of all T cell subsets, but more profound depletion of
 CD4+ CD25- subsets, resulting in relative enrichment of CD4+CD25+ cells, which we hope to
 expand by using post-transplant cyclophosphamide.

 Rationale for Rabbit-ATG (Thymoglobulin, Genzyme Corp, Cambridge, MA): will also be used for
 effective immune-ablation and for in-vivo T cell depletion post- stem cell infusion. Rabbit
 ATG will be used as this has been shown to promote the expansion of functional Tregs in-vitro
 24. Rabbit ATG was use in the largest study by Burt et al 18.

 (Please note reference numbers can be found on pages 25-26 of the protocol.)
Inclusion Criteria:

 All the subjects considered eligible for the study will be screened and reviewed by the
 Gastroenterology (GI) physicians prior to enrollment (Dedrick Moulton, MD or his designee
 at Vanderbilt Children's Hospital) 2. Age ≥ 10 and < 30 years 3. Disease status:

 1. Confirmed diagnosis of Crohn's Disease: Diagnosis of Crohn's disease that has been
 established based on typical endoscopic/histologic and/or radiological appearances.

 2. Active disease, defined as: Pediatric Crohn's Disease Activity Index (PCDAI) >30 (see
 Appendix I) or Crohn's Disease Activity Index (CDAI) of >250 (see Appendix II) at any
 time within 3 months prior to enrollment and any one of the following- i. Endoscopic
 evidence of active disease confirmed on histology within 3 months prior to enrollment,
 or ii. Clear evidence of active small bowel Crohn's disease on small bowel imaging
 within 3 months prior to enrollment.

 3. Refractory disease- Moderate to severe disease that has been unresponsive to current
 or prior therapy with mercaptopurine and/or azathioprine (thiopurines), methotrexate
 and anti-TNF therapy. Patients should have relapsing disease (i.e. ≥ 1
 exacerbation/year) or corticosteroid dependence despite current or prior thiopurines,
 methotrexate and anti-TNF maintenance therapy or clear demonstration of intolerance or
 toxicity to these drugs. Patients who fail induction therapy with corticosteroids and
 anti-TNF therapy, and are therefore not eligible to receive maintenance therapy with
 thiopurines or methotrexate will also be candidates for enrollment.

 4. Negative stool culture, C. difficile, and negative CMV pcr (in stool or colonic
 biopsy). Patients with CMV colitis will receive a trial of anti-viral treatment and
 only responders will be considered eligible for inclusion.

 5. Patients with a prior ileostomy or colostomy may enter the study. For this group of
 patients', physician's global assessment will be used to assess clinical activity of
 CD, as Pediatric CDAI and CDAI scoring method may not be representative of disease
 activity.

 6. Patients with abscesses are eligible to enroll once the abscesses or any other
 significant infection has resolved.

 Exclusion Criteria:

 1. Pregnancy or unwillingness to use adequate contraception during the study- if a woman
 is of childbearing age.

 2. HIV infection.

 3. Organ function criteria-

 1. Renal: creatinine clearance < 50 ml/min/1.73m2 (measured or estimated).

 2. Cardiac: left ventricular ejection fraction <30% by multigated radionuclide
 angiography (MUGA) or a shortening fraction of < 25% by cardiac echocardiogram.

 3. Pulmonary Function tests: DLCO < 30% or patient on oxygen.

 4. Hepatic: serum bilirubin > 3 mg%; AST and ALT > 3x ULN for the institutional lab.

 4. Uncontrolled Hypertension (using age based criteria) despite at least 2 anti-
 hypertensive agents.

 5. Active Infection or risk thereof-

 1. Current abscess or significant active infection

 2. Perianal infection is not an exclusion criterion, provided there is drainage with
 or without placement of drain.

 3. Abnormal chest x- ray (CXR) consistent with active infection or neoplasm.

 6. Severe diarrhea due to short small bowel; patients believed to have < 700 mm of small
 bowel and diarrhea attributable to this will be excluded.

 7. Patients with toxic megacolon, active bowel obstruction or intestinal perforation.

 8. Unable to collect minimum of 3 x106/kg CD34+ cell dose. These patients will be
 excluded from receiving the preparative regimen.