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5.2 clinimacstm system

The mechanism of action of the CliniMACS Cell Selection System is based on magneticactivated cell sorting (MACS). The CliniMACS device is a powerful tool for the isolation of many cell types from heterogeneous cell mixtures, (e.g. apheresis products). These can then be separated in a magnetic field using an immunomagnetic label specific for the cell type of interest, such as CD3+ human T cells.  
The cells to be isolated are specifically labeled with super-paramagnetic particles by an antibody directed toward a cell surface antigen. After magnetic labeling, the cells are separated using a high-gradient magnetic separation column as described below. The magnetically labeled cells are retained in the magnetized column while the unlabeled cells flow through the column for collection. The retained cells are eluted by removing the magnetic field from the column, washing the cells out and collecting them in a separate container from the unlabeled cells.  
The super-paramagnetic particles are small in size (about 50 nm in diameter) and are composed of iron oxide/hydroxide and dextran conjugated to monoclonal antibodies. These magnetic particles form a stable colloidal solution and do not precipitate or aggregate in magnetic fields. The antibody conjugated beads used in this system are highly specific (e.g. CD3+ cells via OKT3 conjugated beads). High-gradient MACS technology has been shown to achieve rapid and highly specific depletion or enrichments of large numbers of target cells from BM, cord blood, and normal peripheral blood mononuclear cells.  
The CliniMACS device incorporates a strong permanent magnet and a separation column with a ferromagnetic matrix to separate the cells labeled with the magnetic particles. The high-gradient system allows the application of strong magnetic forces and a rapid demagnetization. Small ferromagnetic structures, such as the column matrix, placed in a magnetic field concentrate this homogenous field and thereby produce high magnetic gradients. In their immediate proximity, the ferromagnetic structures generate magnetic forces 10,000-fold greater than in the absence of those structures enabling the retention of magnetically labeled cells. After removing the column from the magnet, the rapid demagnetization of the column matrix allows the release of retained cells.  
The CliniMACS device is comprised of a computer controlled instrument incorporating a strong permanent magnet, a closed-system sterile tubing set containing columns with a coated ferromagnetic matrix and a paramagnetic, cell specific, labeling reagent. The instrument will separate the cells in a fully automated process yielding a cell population highly depleted of CD3+ cells. The CliniMACS device is not licensed by the FDA and therefore is investigational.  
The CliniMACS device has separate programs that allow cell selection procedures optimized for either depletion (e.g. CD3+ or CD45RA+) or selection of a target cell population (e.g. CD34+ or CD56+ cells). The basic mechanism is the same for either application; target cells are "tagged" with super-paramagnetic particles and eventually separated from the unlabeled cells using the CliniMACS device as described above. The desired target cells can either be infused or discarded appropriately.
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6.0 required observations and evaluations

All pre/peri/posttransplant and long-term follow-up evaluations for these participants will be carried out as outlined in Appendix D, and guided by the Standard Operating Procedures (SOPs) of the St. Jude Children's Research Hospital, Department of BMTCT, for recipients of allogeneic stem cell transplantation. Copies of these SOPs and ongoing updates can be found at the following site: http://home.web.stjude.org/bone_marrow/clinicalHome.shtml.  
Furthermore, to accommodate the research studies, flexibility in the date is allowed without a deviation from protocol. The degree of flexibility in the timing is also provided in Appendix E.
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6.2 long-term follow-up evaluations

In general, recipients of allogeneic HCT at St. Jude are seen at least annually until 10 years posttransplant in the Department of BMTCT outpatient clinic. For the purpose of this study, research participants will be followed to year 1 post-transplantation. All study participants will be eligible for enrollment in the institutional long-term follow-up protocol for children and young adults who have received stem cell transplantation at St. Jude Children's Research Hospital (BMTFU protocol).
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6.3 evaluation for chimerism and

Evaluation for chimerism and engraftment will be performed on bone marrow or peripheral blood samples according to the timelines noted in Appendix E. Bone marrow chimerism studies will be conducted on or about the following time points: day +21, day +100, and year one posttransplant. However, for research participants who have less than 100% donor chimerism at or about day +21 posttransplant, a repeat bone marrow study (to include chimerism) may be performed approximately one week after the initial procedure.  
The time to neutrophil and platelet engraftment will be recorded. Neutrophil engraftment will be defined as the first of 3 consecutive days of an absolute neutrophil count (ANC) greater than or equal to 500/mm3. Time to platelet engraftment will be designated as the time to platelet count exceeding 20,000/mm3 and 50,000/mm3 without a platelet transfusion in the preceding seven days.  
Chimerism studies using peripheral blood will be obtained on an approximate weekly basis according to the evaluation schedule noted in Appendix E. Additional bone marrow and/or peripheral blood chimerism studies may be performed throughout the course of this study when clinically indicated. Chimerism studies derived from bone marrow may be used in lieu of a specified peripheral blood sample if a bone marrow sample is available. In the event of graft failure/rejection, subsequent chimerism studies may be held as they would not be clinically indicated at that time.  
If there is an initial decrease in donor chimerism to less than 90% at any time on peripheral blood studies, a bone marrow examination will subsequently be performed  
Protocol document date: 09/06/2018 within approximately two weeks to confirm this initial decline. In addition, chimerism analysis may be performed in subsets of lymphocytes, granulocytes, and monocytes for research participants with increasing host chimerism until the research participant attains 100% donor chimerism. Chimerism studies will be performed in the St. Jude Department of Pathology using standard DNA techniques [i.e. VNTR (variable number tandem repeat)] and/or FISH (fluorescent in-situ hybridization) analysis. Chimerism studies will be reported in the database as donor percentages.
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6.4 evaluation for immune reconstitution

Research participants will have immune reconstitution studies of lymphocyte subsets
(i.e. cells, B cells, and NK cells). These studies may be performed more often than what is outlined in Appendix E depending on the clinical status of the research participant.  
Immune analysis as described (Mnemonic listed in 6.4.1 through 6.4.4) will be performed according to the schedule outlined in Appendix D until the immune parameters recover to normal level or donor pattern:
6.4.1 Lymphocyte subsets study: Flow cytometry enumeration.  
6.4.2 VBETA/TREC Research: Thymic output and T-cell repertoire. 6.4.3 Lymphocyte Phenotypes Research: T-cell and NK-cell number and function.  
6.4.4 Quantitative immunoglobulins: IgG, IgM, and IgA levels. 6.4.5 IR-Phenotype: Immune reconstitution of memory and naïve T cells will be investigated in depth in the Youngblood Lab. This may include phenotypic subset characterization, functional correlates, and analysis of the epigenetic signatures of these populations.  
6.4.6 T-Function: T-cell function will be investigated in depth in the Thomas Lab. This may include antigen-specific T-lymphocyte response to viral infections, such as herpes viruses (CMV, HSV and VZV) and respiratory viruses (influenza and RSV).
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6.5 general viral surveillance

Serial PCR testing for CMV, adenovirus, and EBV will be done weekly to day 100 and then on an as needed basis. In research participants with progressive or active infection, samples may be obtained more frequently (St. Jude test mnemonic BMTPCR).
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6.6 minimal residual disease evaluation

Minimal residual disease (MRD) assays in peripheral blood and/or BM by immunologic and molecular methods will be performed for those research participants who have had this test performed during prior therapy for their disease at St. Jude only or those for who samples of diseased marrow were available to identify a leukemic marker for MRD testing. BM (3ml) and/or peripheral blood (5-10 ml) will be obtained from the participants at pre-transplant evaluation, at or about day +21, +100 and at the annual evaluation. MRD assays may be performed more frequently in participants with increasing host chimerism. We will apply immunologic and molecular methods as previously described.54-55 Tests will be performed in the appropriate St. Jude laboratories.
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6.7 research tests on haploidentical

Donors will be offered the option for participation in research studies of immune reconstitution. These tests will be obtained after consent and preferably prior to growth factor administration. Lymphocyte subset analysis of the donor appears to allow for the prediction of the reconstitution of the lymphocyte subsets in the research participant after transplantation. Data in larger donor/research participant pairs will help to verify these observations. A list of these optional research studies are noted in Appendix D and detailed below:
6.7.1 Lymphocyte subsets study Flow cytometry enumeration.  
6.7.2 VBETA/TREC Research Thymic output and T-cell repertoire.  
6.7.3 Lymphocyte Phenotypes Research Lymphocyte number and function. 6.7.4 IR-PHENOTYPE, and T-FUNCTION: In depth characterization of donor lymphocytes
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7.0 evaluation criteria 7.1 adverse

Adverse event (AE) monitoring for on-study research participants will be assessed using the NCI Common terminology Criteria for Adverse Events Version 3.0. The specific criteria for adverse event monitoring are noted Section 9.0 and in Appendix D.
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7.2 gvhd diagnosis and grading

Acute and chronic GVHD will be evaluated, staged, and graded using the criteria found in Appendix B and C respectively.  
Appendix B contains the COG stem cell committee consensus guidelines for establishing organ stage and overall grade of acute GVHD. In the initial 26 patients treated on this protocol prior to amendment 2.0, there were 3 patients initially diagnosed with hyperimmune response syndrome, or similar. This was due to the development of GVHD-like symptoms in some patients that were atypical of acute GVHD syndromes seen in conventional T-cellreplete transplantation. Other patients developed symptoms that were somewhat atypical, but also had a high degree of overlap with acute GVHD. Comprehensive review of the patients transplanted on HAPNK1 prior to amendment 2 was performed by institutional monitors, at least 3 external transplant physicians, and the PI. All patients previously noted to have hyperimmune response syndrome (or similar) were re-coded to acute GVHD. One additional patient was determined to have significant GVHD by audit consensus, giving 4 of the first 26 patients with acute GVHD
≥ grade III. Although we and others96 have noted that GVHD syndromes appear to be biologically different in Naïve T-cell depleted transplantation such as this, with amendment 2.0 any GVHD-like syndrome will be coded as GVHD until such time that a more specific syndrome is fully characterized. The COG stem cell committee consensus guidelines for establishing organ stage and overall grade of acute GVHD has been adopted as the standard GVHD diagnostic guidelines for the Department of BMTCT, and will be applied to patients on this protocol (see Appendix B). In addition, acute GVHD will be assessed at least once a week for the first 100 days per BMTCT SOP 20.01.  
Other disorders that involve donor immune dysregulation, that are distinct from GVHD
are known to occur in transplant recipients. Interstitial pneumonitis syndrome (IPS) and bronchiolitis obliterans organizing pneumonia (BOOP) are two relatively common examples. These disorders are often associated with acute GVHD, but are not considered to be acute GVHD, and are often treated with steroids. These and similar disorders will be captured as AE's and will not be considered to be acute GVHD. The risk of non-GVHD disorders of immune dysregulation in naïve T-cell depleted transplantation is unknown. In the 26 patients transplanted on this study prior to amendment 2, no patient has been diagnosed with IPS or BOOP. However, 3 patients have been diagnosed with toxic epidermal necrolysis (TEN) (n=2) or Steven-Johnson syndrome (SJS) (n=1). TEN and SJS are considered to be variants of a disease continuum that is often triggered by viral infection or drug exposure, and is mediated by cytotoxic T cells.97 All three patients experienced resolution of their TEN/SJS with brief immunosuppressive therapy.98 It can be difficult due to overlapping symptoms, but given that therapeutic recommendations are different, it is important to distinguish such diseases from acute GVHD if possible. Any toxicity that fits criteria for a well described disorder of immune dysregulation (such as the examples above) will be recorded no matter the CTC CAE grade. This allows a full accounting of the incidence of these disorders in naïve T-cell depleted transplantation.  
Appendix C contains a summary of the NIH consensus development project on criteria for clinical trials in chronic GVHD.99 This table will be used for staging/grading of chronic GVHD.
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7.3 performance status

Performance status will be assessed by Karnofsky/Lansky Performance Scores found in Appendix A.
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7.4 hematologic recovery

Posttransplant hematologic recovery will be determined using the engraftment criteria as follows:
7.4.1 Neutrophil engraftment will be defined as the first of 3 consecutive tests performed on different days of an ANC ≥ 500/mm3 with evidence of donor cell engraftment.  
7.4.2 Platelet engraftment will be defined as the first of 3 consecutive tests performed on different days of a platelet count ≥ 20,000/mm3 with no platelet transfusions in the preceding 7 days.
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7.5 graft failure

Primary graft failure will be defined as an ANC never meeting or exceeding 500/mm3 for 3 consecutive tests performed on different days and no evidence of donor chimerism (<5%) before day +42 post-HCT.  
Secondary graft failure will be defined as a decline in ANC to<500/mm3 with a decline in donor chimerism to <5% in research participants with prior engraftment.
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7.6 chimerism

Mixed hematopoietic chimerism will be defined as between 10% and 95% donor chimerism in the absence of immunosuppressive therapy.
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8.0 off-study criteria 8.1 recipient

Recipient research participants will remain on study until one of the following occurs: 8.1.1 Withdrawal from protocol. 8.1.2 Death.  
8.1.3 Donor unable to provide the HSC required for intended recipient to undergo the primary HCT procedure (refer to donor off-study criteria).  
8.1.4 Unable to be contacted and/or effectively monitored by the Principal Investigator
(PI) and/or designees for follow-up (lost to follow-up).  
8.1.5 One year after HSC infusion (i.e. has completed the year +1 post-primary transplant evaluation) and enrollment onto the BMTFU protocol.  
8.1.6 Development of a significant change in health status at any point of therapy which would render receipt of the transplantation procedure or continuation in the study medically unsafe or not in the participant's best interest.  
8.1.7 Requires additional chemotherapy for morphologically confirmed disease relapse
(generally >5%).
Epigenetic or targeted therapy and low dose lymphodepleting chemotherapy with DLI, even if molecularly detectible disease is present, would not trigger this offstudy criteria.
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8.2 additional notes

Recipient research participants enrolled on this study will remain on study for monitoring if one of the following occurs:
8.2.1 Experiences graft failure/rejection.  
8.2.2 Noncompliance with protocol medications/administrations and/or required followup evaluations.  
8.2.3 Positive pregnancy test post-HSC infusion.  
8.2.4 Recipient requires an additional HSC infusion, and unable to receive these cells due to donor issues.
8.3 **Donor Criteria**
Donor research participants will remain on study until one of the following occurs:  
8.3.1 Withdrawal from protocol. Donor research participants may withdraw their consent to participate at any time. Physician may withdraw donor at any time that continuation in the study is deemed medically unsafe or no longer in the donor's best interest.  
8.3.2 Day of transplant recipient or transplant donor death, whichever occurs first.  
8.3.3 Development of a change in health status, including positive pregnancy test or a clinically significant risk for or positive testing for communicable disease, which in the opinion of the PI, would render the donor medically ineligible to serve (or continue to serve) as a therapeutic cell donor.  
8.3.4 Unable to be contacted and/or effectively monitored by PI and/or designees for follow-up as judged by the PI (including non-compliance and lost to follow-up).  
8.3.5 Once the PI has determined that the transplant recipient does/will not require an additional infusion(s) of the donor's cells for the purpose of this protocol, the donor will be taken off study seven days post final cell collection procedure.
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9.0 reporting criteria and continuing

The principal investigator is responsible for promptly reporting to the IRB any adverse events that are unexpected/unanticipated, serious, and that may represent potential harm or increased risk to research participants. When an unexpected death occurs, the PI should report it to the Director of Human Subject's Protection immediately, by phone: (901) 5954359, cell: (901) 336-2894, fax: (901) 595-4361, or e-mail: hsp-1@stjude.org.  
A reportable event entry into TRACKS should follow within 48 hours of notification of the event.  
Serious, unexpected, and related or possibly related events must be reported within 10 business days of notification of the event. At the same time, the investigator will notify the study sponsor and/or the FDA, as appropriate. All other SAEs, including expected death, and all captured AEs will be reported to the IRB at the time of the continuing reviews, with the following exceptions:  
- Any grade III-IV infusion reactions will be reported as soon as possible but every effort should be made to assure reporting is no more than within 10 business days of the event.  
- Any episodes of overall grade III or IV acute GVHD in participants will be reported to the IRB as soon as possible but no more than within 10 business days of the PI's confirmation of the diagnosis/grade of the event.
For this research study, recipient participants will be followed for all NCI Grade III-V
adverse events from the start of conditioning and throughout the first year post HCT, regardless of their relationship to the treatment given. In addition, clinically significant NCI Grade I-II adverse events that are judged to be related/possibly related may be collected per the discretion and judgment of the PI. Examples of "clinically significant Grade I-II adverse events could include, but are not limited to: events meeting criteria for SAE, infections requiring oral systemic therapy, VOD or hemorrhagic cystitis. GVHD events will be recorded on an ongoing basis regardless of stage or grade using the criteria defined in Appendix B, and will not be graded according to NCI criteria.  
With regard to the haploidentical donor participants, they will be followed for all SAEs and any clinically significant AEs, per the judgment of the PI, that are deemed related to the mobilization and/or apheresis procedure from the time of apheresis of NK cells, on through the initiation of mobilization growth factors, until 7-days post last day of the final apheresis procedure. If the transplant recipient requires a second HSC infusion, meaning that the donor is required to undergo the mobilization and apheresis procedure again, collection of this donor safety data will restart upon the initiation of the subsequent mobilization procedure and continue until 7-days post the last day of this apheresis procedure. Timelines for reporting of these donor events to the institutional and federal governing agencies will be according to the same timelines utilized for the recipients. A listing of the captured donor safety data will be provided in a separate table from the transplant recipients within each respective continuing review report.  
The following definitions apply with respect to reporting adverse experiences:
Serious adverse event - any adverse event temporally associated with the participant's participation in research that meets any of the following criteria:
- results in death;
- is life-threatening (places the participant at immediate risk of death from the event as it occurred);
- requires inpatient hospitalization or prolongation of existing hospitalization
- results in a persistent or significant disability/incapacity;
- results in a congenital anomaly/birth defect; or
- any other adverse event that, based upon appropriate medical judgment, may jeopardize the participant's health and may require medical or surgical intervention to prevent one of the above outcomes.  
Unexpected adverse event - any adverse event meeting any of the following criteria:
- an event for which the specificity or severity is not consistent with the protocol related documents, including the applicable investigator brochure, IRB approved consent form, IND/IDE application or any of the product labeling or package inserts;
- an event for which the observed rate of occurrence is significantly increased above what is expected or credible baseline rate for comparison;
- an event for which the occurrence is not consistent with the expected natural progression of any underlying disease, disorder, or condition of the participant(s) experiencing the adverse event and the participant's predisposing risk factor profile for the adverse event.  
The principal investigator is responsible for reviewing the aggregate toxicity reports and reporting to the IRB if the frequency or severity of serious toxicities exceed those expected as defined in the protocol or based on clinical experience or the published literature. Any proposed changes in the consent form or research procedures resulting from the report are to be prepared by the study team and submitted with the report to the IRB for approval.
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9.2 reporting to st. jude

Continuing review reports will be sent to the Institutional Biosafety Committee (IBC) on at least an annual basis using the most current version of the continuing review form found on the IBC website. The safety reports, sent to the IRB for both the donors and stem cell recipients, will be simultaneously forwarded to the IBC. Therefore, reporting for safety events to this committee will be according to the same timelines as reporting to the IRB. This includes notification of achievement of MTD (if/when applicable). As per the direction of the IBC, only those protocol revisions and amendments directly related to the CliniMACS processing and related reagent(s) will require review and consideration by the IBC. Other revisions/amendments will be noted in the IBC continuing review report.
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9.3 reporting to fda (21cfrss312.32

The FDA will be notified in writing (IDE safety report) of any serious and unexpected AE associated with an investigational treatment or device; or any results from laboratory animal tests that suggest a significant risk for human subjects including reports of mutagenicity, teratogenicity, or carcinogenicity.  
Each notification to the FDA should be made as soon as possible and no later than 15 calendar days after the sponsor's initial receipt of the information. The FDA may require additional data to be submitted. In each written IND safety report, the sponsor shall identify all safety reports previously filed with the IND concerning a similar adverse experience, and shall analyze the significance of the adverse experience in light of the previous, similar reports where applicable.  
The sponsor shall also notify the FDA by telephone or by facsimile transmission of any unexpected fatal or life-threatening experience associated with the use of the investigational device as soon as possible but no later than 7 calendar days after the sponsor's initial receipt of the information. Any grade III-IV infusion reactions will be reported as soon as possible but every effort should be made to assure reporting is no more than within 7 business days of the event. Follow-up information to a safety report must be submitted as soon as the relevant information is available.  
If the results of further investigation show an AE that was not initially determined to be reportable should later be deemed reportable, the sponsor shall inform the FDA of the event in a written safety report as soon as possible, but no later than 15 calendar days after the determination is made. Results of the investigation of other safety information shall be submitted, as appropriate, in an information amendment or annual report. Continuing review reports, which will include the up-to-date clinical and safety data, will be submitted to the FDA at least annually.
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9.4 reporting to st. jude

Copies of all correspondence to the St. Jude IRB, including SAE reports are provided to the St. Jude Regulatory Affairs Office. All FDA related correspondence and reporting will be conducted through the Regulatory Affairs Office. Adverse event reporting and annual reporting will be in accord with the FDA Title 21 CFR312.32 and Title 21 CFR312.33, respectively. The Regulatory Affairs Office can be reached at 901-595-2347 (secondary contact: St. Jude Vice President of Clinical Trials Administration 901-5952876).
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9.5 continuing review reports

Continuing review reports of protocol progress and summaries of adverse events will be filed with the St. Jude IRB, and IBC at least annually.
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9.6 data submission to miltenyi

Clinical and safety related data will be provided to Miltenyi Biotec, the manufacturer of the CliniMACS system. Data will include but is not limited to the transplant research participant's age and diagnosis, donor product(s) related information including donor type, the stem cell mobilization, selection, and infusion procedure. Outcome data including lymphohematopoietic reconstitution, immunological response, disease response and transplant complications will be shared with Miltenyi Biotec. Representatives from Miltenyi Biotec will be able to review the participant's (donor and transplant research recipient) laboratory and medical record for data verification purposes. Copies of all reports to the institutional and governing regulatory bodies will also be accessible upon request. In the event that the protocol is placed on a clinical hold by the PI or governing regulatory authorities, representatives from Miltenyi Biotec will be notified as soon as possible.
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9.7 reporting to the center

The Transplant Program at St. Jude is required by the federal government to report transplant information to the Center for International Blood and Marrow Transplant Research (CIBMTR). The CIBMTR is a research partnership of the International Bone Marrow Transplant Registry, the National Marrow Donor Program (NMDP), and the Foundation for the Accreditation of Cellular Therapy (FACT). This organization is responsible for the collection and maintenance of a standardized data warehouse registry of autologous and all allogeneic (related and unrelated donor) transplants performed in the United States.  
The Office of General Counsel, U.S. Department of Health and Human Services, had deemed the CIBMTR not a covered entity under the Privacy Rule (45 CFR 164.512), 45 CFR Parts 160 and 164, and the Health Insurance Portability and Accountability Act (HIPPA) of 1996. For this reason, the submission and disclosure of certain protected health information (PHI), including that required for CIBMTR, is allowable without the individual's authorization (i.e. consent is waived) when such disclosure is made to public health authorities authorized by law for the purpose of preventing or controlling disease, injury, or disability.  
Data resulting from this transplant procedure will be sent for general registry purposes to comply with the federal government requirements. This information for both donor and recipient is submitted using a unique participant identification number. The information submitted for haploidentical recipients is less extensive than recipients of other donor products. For this reason, variables submitted may include but are not limited to the transplant recipient's date of birth, country/state of current residence, diagnosis, basic lympho-hematopoietic reconstitution (e.g. date of ANC and platelet engraftment), postHCT disease status, and basic AEs (e.g. GVHD- yes or no), survival status, date/cause of death.
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10.0 statistical considerations 10.1 statistical

This study is designed as a phase II study. The primary objective of this study is to evaluate the rate of successful engraftment at day +42. Successful engraftment for the purposes of this objective will be patients who do not experience graft failure as defined in section 7.5.  
As detailed in section 2, the use of alternative donors such as haploidentical donors and unrelated umbilical cord blood (UCB) are frequently needed and largely successful. However, the use of these alternative donors is typically complicated with additional problems, particularly poor hematopoietic recovery and graft failure.100 Handgretinger et al found that improved engraftment was obtained in haploidentical donor transplantation with the use of a megadose (high donor CD34+ cell) T-cell depleted graft and administration of OKT3 instead of ATG.26 This combination has since been the foundation of haploidentical donor transplantation at this institution. However, OKT3 is no longer clinically available. This protocol includes a novel preparative regimen using TLI instead of anti-T-cell antibody to facilitate engraftment. Therefore, the primary objective of this study is to assess donor engraftment, ensuring that this novel regimen facilitates donor engraftment at an acceptable rate.  
The majority of published experience with alternative (HLA-mismatched) donor transplantation in children is with umbilical cord blood grafts. The COBALT study included 191 children with hematologic malignancies who received UCB transplantation with TBI based (1350Gy) myeloablative conditioning.101 The cumulative incidence of neutrophil engraftment by day 42 was 80%. The New York Blood Center published outcomes on 1061 patients (78% pediatric) with hematologic malignancies who received myeloablative UCB transplantation with units from their bank.102 The cumulative incidence of neutrophil engraftment was 74% by day 77. The CIBMTR published a comparison of UCB transplantation with HLA-matched unrelated donor transplantation, in which 503 children with acute leukemia received umbilical cord blood  
Protocol document date: 09/06/2018 transplantation.103 They confirmed that although the leukemia-free survival was similar, the rate of neutrophil engraftment (and TRM) were significantly worse with HLAmismatched UCB grafts than with HLA-matched bone marrow grafts. For UCB
recipients, the best engraftment was obtained in the rare 6/6 matched UCB recipients with 85% of the 35 patients achieving neutrophil recovery at day +42. In addition, recipients who received a single antigen mismatch UCB unit with an appropriately high cell dose (n=154) had a neutrophil engraftment rate of 80% at day +42. Recipients of lower cell dose or two antigen mismatch units fared worse.  
Given the data from the paragraph above, we consider a rate of successful engraftment of less than 80% by day +42 to be unacceptable for alternative donor transplantation. The goal of our study is to develop a novel conditioning regimen to facilitate successful engraftment of haploidentical donor grafts at a rate of ≥91%. We do not anticipate censoring during the 42 day time period and we can approximate the rate of successful engraftment at day +42 using a Binomial distribution. (However, patients lost for follow up will be counted as a failure to engraft in order to keep the validity of Binomial distribution approximation). Therefore, in this study, we propose to test the null hypothesis H0: P≤0.80 versus H1: P>0.80, where P is the proportion of research participants successfully engrafted at day +42 posttransplant. With type I error of 10% and type II error of 20%, Simon's two stage optimum design powered at alternative successful engraftment rate P1=0.91 requires 21 evaluable patients at the first stage and 49 evaluable patients in total76. The stopping rules are provided in Table 1, with the understanding that stopping the trial early would be suggestive of the proposed transplant strategy not being an effective treatment option for this group of patients. The interpretation is that if we observe 17 or fewer participants successfully engrafted at day +42 in the first 21 participants, then we would stop the trial for lack of efficacy. However, if we observe 18 or more patients engrafted successfully in the first 21 participants at day +42 in stage one, then 28 more patients will be enrolled in stage two. If we observe 43 or more participants successfully engrafted at day +42 upon completion of the trial, then we conclude that the true rate of successful engraftment is at least 80% and our novel regimen will be proposed for further development and phase III clinical trial.  
Table 1: Stopping rules for lack of efficacy based on the Simon's 2-stage optimum design  
(unacceptable low rate of successful engraftment at day +42)  
Accept H0 if the number of research participants engrafted  
| Accept H0 if the number of research participants engrafted                                                                                                                                                   |      |            |          |        |         |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|------|------------|----------|--------|---------|
| P0                                                                                                                                                                                                           | P1   | (≤ r1/n1 ) | (≤ r/n ) | EN(P0) | PET(P0) |
| 0.80                                                                                                                                                                                                         | 0.91 | 17/21      | 42/49    | 31     | 0.63    |
| Note: r1 and r denote the number of patients successfully engrafted at day +42; EN(P0) denotes the expected sample size under P0;  PET(P0) denotes the probability of early termination at stage I under P0. |      |            |          |        |         |  
All participants who receive the prescribed transplant will be evaluable for the primary outcome (engraftment at day +42). In addition, any patient who starts the conditioning regimen but stops prior to receiving the graft will count as a failure, if the reason for stopping therapy is toxicity from the conditioning regimen. Any patient who dies from toxicity after engraftment but before day +42 will count as a successful engraftment. Any patient who dies prior to engraftment will count as a graft failure. Any patient who rejects the initial graft but is successfully engrafted with another graft will still count as a graft failure.  
Participants who enroll but do not initiate the treatment (receive conditioning and infusions) due to withdrawal of participant, health status change to make the treatment not to the participant's best medical interest, withdrawal of donor participant, etc. will be replaced.  
After the study is finished, for the first primary objective, the rate of engraftment at day
+42 and its 95% Blyth-Still-Casella confidence interval will be estimated based on the binomial approximation.  
As of 1/31/17, 42 patients have received transplantation on this study, 39 had successful engraftment, 1 patient died prior to engraftment, and 2 patients experienced graft failure but were successfully grafted with a progenitor cell boost from the same donor. This indicates that the primary objective is likely to be satisfied (42 successful engraftment) prior to completion of original protocol enrollment goal of 49 recipients. To fulfill a critical patient need, Amendment 4.0 expands the intended enrollment beyond the primary objective enrollment goal. Because this protocol provides specialized T-cell depletion and graft manipulation techniques, a therapy that is only available under the protocol IDE, future patients would not be able to potentially benefit from this therapy once the protocol completes. Preliminary analysis (data censored 10/31/16) show the 1 year EFS (events: death, relapse) in patients with non-refractory (e.g. <5% blasts) hematologic malignancy receiving first allogeneic HCT from a TCD haploidentical donor to be 86.6% (SE:8.2%) on this study compared to 55.6% (SE:8.1%) on the previous two trials (HAPREF, HIFLEX). Therefore, future patients could benefit from this therapy with the expanded enrollment goal of 75 evaluable patients. With past accrual at 12 to 15 patients per year, the study should be extended approximately 2 years. This provides the time necessary to develop and implement a replacement therapeutic protocol that builds upon the progress of this study.  
Secondary benefits to the expanded enrollment on this study include increased precision of the clinical outcome estimates for the primary and secondary objectives, and the ability to compare these outcomes based on whether the donor is KIR matched or mismatched. Tertiary benefits to the expanded enrollment include increased depth of the biologic characterization of immune reconstitution, particularly of donor memory Tcells. Enhanced understanding of memory T-cell function has the potential to lead to the development of new successful therapeutics.  
In addition to the stopping rules based on successful engraftment, we will closely monitor the trial for early excessive toxicities in terms of severe acute GvHD (aGvHD), and therapy related death. Therapy related death is any death in remission and related to protocol therapy. Therapy related death will be monitored for 100 days from the date of transplantation for application of the stopping rules. Most acute GVHD is expected to occ ur i n t h e first 1 0 0 d a ys, h o we v er ac ute G V H D ma y occ ur after Da y 1 0 0, a n d t h e G V H D s y n dr o mes i n naï ve T cell de plete d tra ns pla ntati o n ma y differ bi ol o gicall y fr o m G V H D i n c o n ve nti o nal T cell r e plete s etti n gs. T heref ore a n y ac ute G V H D ≥ gra d e III  
will c o u nt a gai nst t he st o p pi n g r ule, e v e n if it occ urs after 1 0 0 d a ys. T o xicities sec o n dar y t o n o n pr ot oc ol t hera p y f or p osttra ns pla nt persiste nt or rec urr e nt disease will n ot c o u nt t o war ds t he t o xicit y st o p pi n g r ules. T o xicities t hat are sec o n d ar y t o p ost-tra ns pla nt t hera p y t hat is defi ne d i n t he pr ot oc ol (s uc h as escalati n g D LI as descri be d i n secti o n 4. 6) will c o u nt t o war ds t he st o p pi n g r ules. T h e saf et y e n d p oi nts will be m o nit ore d i n de pe n de ntl y a n d a c o nsi derati o n t o st o p pi n g t he trial w o ul d be gi ve n if t here is e vi de nce s u g gesti n g t hat t he r ate of gr a des III I V a G V H D is great er t ha n 2 0 %, or t he rate of tra ns pla nt relat e d deat h is greater t ha n 1 0 %. T he n, t he pla n ne d i nteri m e val uati o n ti me p oi nts a n d st o p pi n g r ules, usi n g gr o u p se q ue ntial desi g n base d o n P oc oc k st o p pi n g b o u n daries after c o nsi deri n g fi ve i nteri m a nal yses ( p val ue t hres h ol d = 0. 0 1 5 8) t o e ns ure a n o verall t y pe o n e err or of 0. 0 5. 1 0 4- 1 0 6 , f or eac h of t he t w o e n d p oi nts are pr o vi de d i n
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t a bles 2 a

After t his a me n d m e nt 4, if t he st o p pi n g r ule f or m o nit ori n g t he e n graft me nt is n ot met i n t he first 4 9 e val ua bl e partici pa nts e nr olle d, t he n after 4 9 e v al ua ble partici pa nts e nr olle d o n t he st u d y, we will n ot m o nit or t h e e n graft me nt a n d will esti mate t he e n graft me nt rate usi n g all e val ua ble partici pa nts e nr olle d o n t he st u d y after t he st u d y is fi nis he d.  
H o we ver, we will c o nti n ue t o cl osel y m o nit or e xcess t o xicit y of gra d e III I V a G V H D  
a n d T R M wit hi n t he first 1 0 0 da ys. W e will c o n d uct t hree m ore i nteri m a nal yses w he n 6 0, 7 0 a n d 7 5 e val ua ble patie nts are e nr olle d o n t he st u d y f or m o nit ori n g t hese t o xicities usi n g t he ne w m o nit ori n g r ule ( Ta bles 2 3) bas e d o n a bi n o mial distri b uti o n a n d a p val ue t hres h ol d of 0. 0 5.  
| T a ble 2. St o p pi n g R ules f or T o xicities B ase d o n Gr a des III- I V a G V H D  # of Resear c h Partici pa nts E nr olle d # Gra des III I V a G V H D O bser ve d    6  2 1 9  3 1 1 2  4 1 1 5  4 9 1 7  6 0 1 8  7 0 2 1  7 5 2 2  Base d o n a bi n o mial distri b uti o n wit h a p- val ue t hres h ol d of 0. 0 5 a d de d o n a me n d me nt 4. 0.   |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| first 1 0 0 d a ys P ost-t r a ns pl a nt  # of Resear c h Partici pa nts  E nr olle d                                | # T hera p y Relat e d Deat h O bser ve d    5   |
|-----------------------------------------------------------------------------------------------------------------------|--------------------------------------------------|
| 2 1                                                                                                                   | 6                                                |
| 3 1                                                                                                                   | 8                                                |
| 4 1                                                                                                                   | 1 0                                              |
| 4 9                                                                                                                   | 1 1                                              |
| 6 0                                                                                                                   | 1 1                                              |
| 7 0                                                                                                                   | 1 2                                              |
| 7 5                                                                                                                   | 1 3                                              |
| Base d o n a bi n o mial distri b uti o n wit h a p- val ue t hres h ol d of 0. 0 5 a d de d o n a me n d me nt 4. 0. |                                                  |  
B ase d o n t he a b o ve T a ble 2 , if si x gra des III I V a G V H D occ ur i n t he first ele ve n e val ua ble res ear c h p artici pa nts treate d, t he n a c o nsi derati o n will be gi v e n t o te m p oraril y st o p pi n g t he trial a n d a me n di n g t he st u d y t o i ncl u de a d diti o nal or alter nati v e p har mac ol o gic G V H D pr o p h yla xis. Si milarl y, fr o m Ta ble 4 if we o bser ve fi ve deat hs relate d t o t h era p y wit hin t he first 1 0 0 d a ys after tra ns pl a ntati o n i n t he first ele v e n e val ua ble res ear c h partici pa nts treate d, t he n a c o nsi derati o n will be gi v e n t o te m p oraril y st o p pi n g t he trial a n d a me n di n g t he st u d y a p pr o priatel y. Li ke wise, if ei g htee n or m or e t he gra d e 3 4 a G v H D wit hi n first 1 0 0 da ys p ost tra ns pla ntati o n occ ur i n t he first 6 0 e val ua ble res ear c h parti ci pa nts treate d, a c o nsi derati o n will be gi ve n t o te m p oraril y st o p pi n g t he trial a n d a me n di n g t he st u d y t o i ncl u de a d diti o nal or alter nati v e p har mac ol o gic G V H D pr o p h yla xis. Si milarl y, if ele ve n or m ore T R M wit hi n first 1 0 0 da ys p ost tra ns pla ntati o n occ ur i n t he first 6 0 e val ua ble res ear c h partici pa nts treate d, t he n a c o nsi derati o n will be gi ve n t o te m p oraril y st o p pi n g t he trial a n d a me n di n g t he st u d y a p pr o priatel y.  
Starti n g wit h A me n d me nt 3. 0, patie nts wit h a KI R matc he d ha pl oi de ntical d o n or are eli gi ble f or t his t hera p y, w hereas bef ore 3. 0, pati e nts c o ul d o nl y recei ve tra ns pla ntati o n fr o m a KI R mis matc he d ha pl oi de ntical d o n or. W hile t he r ate of d o n or e n graft me nt, r ate of ac ut e G v H D, a n d rat e of t hera p y relate d deat h are u nli kel y t o b e r elate d t o KI R  
matc hi n g, wit h t his ne w p o p ulati o n bei n g a d de d wit h a me n d me nt 3. 0, t he st o p pi n g r ules f or effi cac y ( Ta ble 1) a n d t o xicit y ( Ta ble 2 a n d 3) will be a p plie d t o all reci pie nts, t o KI R matc he d d o n or reci pie nts i n de pe n de ntl y, a n d t o KI R mis matc he d d o n or reci pie nts i n de pe n de ntl y.  
Pri or t o a me n d me nt 5. 0, as of date A pril 4, 2 0 1 8, preli mi nar y a nal ysis s h o we d a si g nifica ntl y hi g her rate of G V H D gra d e III I V i n patie nts w h o receive d mara vir o c ( 4 of 5 = 80%) compared to those patients who did not receive maraviroc (12 of 53 = 25%) (p = 0.02). Because the use of maraviroc in these 5 patients treated on amendment 3.0 was a substantive change in therapy that affected the risk of GVHD in those recipients, those 5 patients will be excluded from analysis of the primary objective and the stopping rules above. For the secondary and exploratory objectives as detailed below, the groups (with and without maraviroc) will be analyzed separately and together.
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10.2 statistical analysis for secondary

The estimate of cumulative incidence of relapse will be estimated using Kalbfleisch-Prentice method. Death is the competing risk event. The analysis will be implemented using SAS macro (bmacro252-Excel2007\cin) available in the St. Jude Department of Biostatistics. The Kaplan-Meier estimate of OS and EFS with relapse, death due to any cause and graft failure as events along with their standard errors will be calculated using the SAS macro (bmacro251- Excel2007\kme) available in the Department of Biostatistics at St. Jude, where OS = min (date of last follow-up, date of death) - date of HCT and all participants surviving after 1 year post-transplant will be considered as censored, and EFS = *min* (date of last follow-up, date of relapse, date of graft failure, date of death due to any cause) - date of transplant, and all participants surviving at the time of analysis without events will be censored. The analysis for this objective will be performed when the last evaluable participant has been followed for one-year post transplant.
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10.2.2 estimate the incidence and

The cumulative incidence of acute and chronic GVHD will be estimated using Kalbfleisch-Prentice method. Death is the competing risk event. The SAS macro (bmacro252-Excel2007\cin) available in the Department of Biostatistics at St. Jude will be used for such analysis. The severity of acute GVHD and chronic GVHD will be described. The analysis for this objective will be performed when the last evaluable participant has been followed for 100 days post transplant.  
10.2.3 Estimate the rate of transplant related mortality in the first 100 days after transplantation The cumulative incidence of transplant related mortality will be estimated using the same method as used in evaluating Objective 10.2.2. Deaths before day 100 because of other reasons are the competing risk events. The analysis for this objective will be performed when the last evaluable participant has been followed for 100 days post transplant.
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10.3 analysis for exploratory objectives

The final results of these exploratory objectives are expected to be available when the last evaluable participant has been followed for one-year post transplant.  
10.3.1 Assess the relationship between pre-transplant MRD with transplant outcomes.  
The relationship of pre-transplant MRD and transplant outcomes will be examined through Cox proportional hazard model or generalized linear model. The model can also be used to adjust for other confounding factors such as patient's age at transplant.  
10.3.2 Record immune reconstitution parameters, including chimerism analysis, quantitative lymphocyte subsets, T-cell receptor excision circle (TREC) analysis,
V-beta spectratyping, and phenotype and functional analysis of reconstituted lymphocyte subsets.  
All immune reconstitution measures will be descriptively analyzed.  
10.3.3 The use of CD45RA-depleted DLI for recipeints who have severe viral infections, disease recurrence or progression, or poor immune reconstitution will be recorded, including dose given, date given, and indication for use. To assess efficacy, the resolution of viral infection, resolution of molecular or frank disease, or the correction of severe lymphopenia will be noted. All adverse events that are at least possibly related to CD45RA-depleted DLI and any episode of GVHD will be recorded.
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11.0 data acquisition and quality

A member of the study team will confirm potential participant eligibility as defined in Section 3.1-3.2, complete and sign the 'Participant Eligibility Checklist'. The study team will enter the eligibility checklist information into the Patient Protocol Manager (PPM) system. Eligibility will be reviewed, and a research participant-specific consent form and assent document (where applicable) will be generated. The complete signed consent/assent form(s) must be faxed to the CPDMO at 595-6265 to complete the enrollment process.  
The CPDMO is staffed 7:30 am-5:00 pm CST, Monday through Friday. A staff member is on call Saturday, Sunday, and holidays from 8:00 am to 5:00 pm. Enrollments may be requested during weekends or holidays by calling the CPDMO "On Call" cell phone (901-413-8591) or referencing the "On Call Schedule" on the intranet).
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11.2 data submission

The St. Jude Cancer Center Clinical Research Assistants and Associates assigned to the Department of BMTCT will assure protocol compliance, and conduct all clinical and safety data collection. Data will be entered into an institutional database. The PI will be responsible for review of case report forms for accuracy and completeness prior to entry into the secure departmental database.
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11.3 quality assurance monitoring

This protocol will be monitored for safety and data as per the St. Jude Data and Safety Monitoring Plan for Clinical Trials approved by the NCI in 2010. The Central Protocol and Data Monitoring Office (CPDMO) will verify 100% of the informed consent documentation on all participants and verify 100% of St. Jude participants' eligibility status. The study team will meet at appropriate intervals to review case histories and data quality summaries on all participants. The St. Jude Clinical Research Monitor will assess protocol and regulatory compliance as well as the accuracy and completeness of all data points for the first two participants enrolled, then 15% of study enrollees (and their corresponding donors) every six months. The protocol will be tracked continuously for the accrual of donors and recipients. All AE and SAE reports will be reviewed by the study Principal Investigator for type, grade, attribution, duration, timeliness and appropriateness on all study participants and their corresponding donors. All SAE reports will be reviewed by the monitor every 6 months.  
Protocol compliance monitoring will include participant status, eligibility, the informed consent process, demographics, staging, study objectives, subgroup assignment, treatments, evaluations, responses, participant protocol status, off-study, and off-therapy criteria. The Monitor will generate a formal report which is shared with the Principal Investigator (PI), study team and the Internal Monitoring Committee (IMC). Monitoring may be conducted more frequently if deemed necessary by the CPDMO or the IMC. Continuing reviews by the IRB and CT-SRC will occur at least annually. In addition, SAE reports in TRACKS
(Total Research and Knowledge System) are reviewed in a timely manner by the IRB. The Regulatory Affairs Office will assist the PI in reporting to the FDA and other external oversight agencies, as necessary.  
This plan is supplemented by a focused audit plan as submitted to the FDA on 9/21/15.  
Please see appendix G for details.
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12.0 obtaining informed consent

The ongoing informed consent process will be carried out per the policies and procedures put forth in the St. Jude Investigator's Handbook for Clinical Research (http://home.web.stjude. org/ clinical research/administration/doc/handbook.pdf). The PI or physician sub-investigator will conduct the signature authorization portion of the consent process. Authorization for the recipient procedure will be conducted in the presence of an independent witness such as the St. Jude Ombudsperson/Research Participant Advocate or designee, a professional staff member from the Department of Nursing, or Social Work.
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13.0 references

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cells without graft-versus-host disease. Blood 103:1534-41, 2004 65. Lu SY, Liu KY, Liu DH, et al: High frequencies of CD62L naive regulatory T cells in allografts are associated with a low risk of acute graft-versus-host disease following unmanipulated allogeneic haematopoietic stem cell transplantation. Clin Exp Immunol 165:26477, 2011 66. Crucitti L, Crocchiolo R, Toffalori C, et al: Incidence, risk factors and clinical outcome of leukemia relapses with loss of the mismatched HLA after partially incompatible hematopoietic stem cell transplantation. Leukemia 29:1143-52, 2015 67. Chen YB, Spitzer TR: Current status of reduced-intensity allogeneic stem cell transplantation using alternative donors. Leukemia 22:31-41, 2008 68. Aversa F, Terenzi A, Felicini R, et al: Haploidentical stem cell transplantation for acute leukemia. Int J Hematol 76 Suppl 1:165-8, 2002 69. Aversa F, Velardi A, Tabilio A, et al: Haploidentical stem cell transplantation in leukemia. Blood Rev 15:111-9, 2001 70. Benaim E, Hale G, Horwitz E, et al: Reduced-intensity conditioning (RIC) in haploidentical transplantation. Blood 104:590a, 2004 71. Ortin M, Raj R, Kinning E, et al: Partially matched related donor peripheral blood progenitor cell transplantation in paediatric patients adding fludarabine and antilymphocyte gamma-globulin. Bone Marrow Transplant 30:359-66, 2002 72. O'Donnell PV, Luznik L, Jones RJ, et al: Nonmyeloablative bone marrow transplantation from partially HLA-mismatched related donors using posttransplantation cyclophosphamide. Biol Blood Marrow Transplant 8:377-86, 2002 73. Rizzieri DA, Koh LP, Long GD, et al: Partially matched, nonmyeloablative allogeneic transplantation: clinical outcomes and immune reconstitution. J Clin Oncol 25:690-7, 2007 74. Ogawa H, Ikegame K, Yoshihara S, et al: Unmanipulated HLA 2-3 antigenmismatched (haploidentical) stem cell transplantation using nonmyeloablative conditioning. Biol Blood Marrow Transplant 12:1073-84, 2006 75. Kohrt H, Lowsky R: Total lymphoid irradiation for graft-versus-host disease protection. Curr Opin Oncol 21 Suppl 1:S23-6, 2009 76. Kohrt HE, Turnbull BB, Heydari K, et al: TLI and ATG conditioning with low risk of graft-versus-host disease retains antitumor reactions after allogeneic hematopoietic cell transplantation from related and unrelated donors. Blood 114:1099-109, 2009 77. Lowsky R, Takahashi T, Liu YP, et al: Protective conditioning for acute graftversushost disease. N Engl J Med 353:1321-31, 2005 78. Heinzelmann F, Lang PJ, Ottinger H, et al: Immunosuppressive total lymphoid irradiation-based reconditioning regimens enable engraftment after graft rejection or graft failure in patients treated with allogeneic hematopoietic stem cell transplantation. Int J Radiat Oncol Biol Phys 70:523-8, 2008 79. Palma J, Salas L, Carrion F, et al: Haploidentical stem cell transplantation for children with high-risk leukemia. Pediatr Blood Cancer 80. Lewalle P, Triffet A, Delforge A, et al: Donor lymphocyte infusions in adult haploidentical transplant: a dose finding study. Bone Marrow Transplant 31:39-44, 2003 81. Morton AJ, Gooley T, Hansen JA, et al: Association between pretransplant interferon-alpha and outcome after unrelated donor marrow transplantation for chronic myelogenous leukemia in chronic phase. Blood 92:394-401, 1998 82. Rizzieri DA, Storms R, Chen DF, et al: Natural killer cell-enriched donor lymphocyte infusions from A 3-6/6 HLA matched family member following nonmyeloablative allogeneic stem cell transplantation. Biol Blood Marrow Transplant 16:1107-14 83. Miller JS, Soignier Y, Panoskaltsis-Mortari A, et al: Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood 105:3051-7, 2005 84. Karre K: Immunology. A perfect mismatch. Science 295:2029-31, 2002 85. Triplett BM, Horwitz EM, Iyengar R, et al: Effects of activating NK cell receptor expression and NK cell reconstitution on the outcomes of unrelated donor hematopoietic cell transplantation for hematologic malignancies. Leukemia 23:127887, 2009 86. Savani BN, Mielke S, Adams S, et al: Rapid natural killer cell recovery determines outcome after T-cell-depleted HLA-identical stem cell transplantation in patients with myeloid leukemias but not with acute lymphoblastic leukemia. Leukemia 21:2145-52, 2007 87. Stroncek DF, Clay ME, Smith J, et al: Changes in blood counts after the administration of granulocyte-colony-stimulating factor and the collection of peripheral blood stem cells from healthy donors. Transfusion 36:596-600, 1996 88. Trzonkowski P, Zaucha JM, Mysliwska J, et al: Differences in kinetics of donor lymphoid cells in response to G-CSF administration may affect the incidence and severity of acute GvHD in respective HLA-identical sibling recipients. Med Oncol 21:81-94, 2004 89. Coustan-Smith E, Behm FG, Sanchez J, et al: Immunological detection of minimal residual disease in children with acute lymphoblastic leukaemia. Lancet 351:550-4, 1998 90. Coustan-Smith E, Sancho J, Behm FG, et al: Prognostic importance of measuring early clearance of leukemic cells by flow cytometry in childhood acute lymphoblastic leukemia. Blood 100:52-8, 2002 91. Coustan-Smith E, Sancho J, Hancock ML, et al: Clinical importance of minimal residual disease in childhood acute lymphoblastic leukemia. Blood 96:2691-6, 2000 92. Nucifora G, Larson RA, Rowley JD: Persistence of the 8;21 translocation in patients with acute myeloid leukemia type M2 in long-term remission. Blood 82:712-5, 1993 93. Roberts WM, Estrov Z, Ouspenskaia MV, et al: Measurement of residual leukemia during remission in childhood acute lymphoblastic leukemia. N Engl J Med 336:31723, 1997 94. Knechtli CJ, Goulden NJ, Hancock JP, et al: Minimal residual disease status as a predictor of relapse after allogeneic bone marrow transplantation for children with acute lymphoblastic leukaemia. Br J Haematol 102:860-71, 1998 95. Langebrake C, Creutzig U, Dworzak M, et al: Residual disease monitoring in childhood acute myeloid leukemia by multiparameter flow cytometry: the MRDAML-BFM Study Group. J Clin Oncol 24:3686-92, 2006 96. Bleakley M, Heimfeld S, Loeb KR, et al: Outcomes of acute leukemia patients transplanted with naive T cell-depleted stem cell grafts. J Clin Invest, 2015 97. Nassif A, Bensussan A, Boumsell L, et al: Toxic epidermal necrolysis: effector cells are drug-specific cytotoxic T cells. J Allergy Clin Immunol 114:1209-15, 2004 98. Scott-Lang V, Tidman M, McKay D: Toxic epidermal necrolysis in a child successfully treated with infliximab. Pediatr Dermatol 31:532-4, 2014 99. Filipovich AH, Weisdorf D, Pavletic S, et al: National Institutes of Health consensus development project on criteria for clinical trials in chronic graft-versus-host disease: I. Diagnosis and staging working group report. Biol Blood Marrow Transplant 11:945-56, 2005 100. Ballen KK, Koreth J, Chen YB, et al: Selection of optimal alternative graft source:
mismatched unrelated donor, umbilical cord blood, or haploidentical transplant. Blood 119:1972-80 101. Kurtzberg J, Prasad VK, Carter SL, et al: Results of the Cord Blood Transplantation Study (COBLT): clinical outcomes of unrelated donor umbilical cord blood transplantation in pediatric patients with hematologic malignancies. Blood 112:431827, 2008 102. Barker JN, Scaradavou A, Stevens CE: Combined effect of total nucleated cell dose and HLA match on transplantation outcome in 1061 cord blood recipients with hematologic malignancies. Blood 115:1843-9, 2010 103. Eapen M, Rubinstein P, Zhang MJ, et al: Outcomes of transplantation of unrelated donor umbilical cord blood and bone marrow in children with acute leukaemia: a comparison study. Lancet 369:1947-54, 2007 104. Blyth CR, Still HA: Binomial Confidence-Intervals. Journal of the American Statistical Association 78:108-116, 1983 105. Blyth CR: Approximate Binomial Confidence-Limits. Journal of the American Statistical Association 81:843-855, 1986 106. Casella G, R.L. B: Statistical Inference. Belmont, CA, Wadsworth&Brooks/Cole, 1990  
|       | KARNOFSKY PERFORMANCE STATUS SCALE  ≥ 16 YEARS OLD                              |
|-------|---------------------------------------------------------------------------------|
| Score | General Description                                                             |
| 100   | Normal. No complaints. No evidence of disease.                                  |
| 90    | Able to carry on normal activity. Minor signs or symptoms of disease.           |
| 80    | Normal activity with effort. Some signs or symptoms of disease.                 |
| 70    | Care of self. Unable to carry out normal activity or to do active work.         |
| 60    | Requires occasional assistance, but is able to care for most of his needs.      |
| 50    | Requires considerable assistance and frequent medical care.                     |
| 40    | Disabled. Requires special care and assistance.                                 |
| 30    | Severely disabled. Hospitalization is indicated although death is not imminent. |
| 20    | Hospitalization necessary, very sick, active support treatment necessary.       |
| 10    | Moribund. Fatal processes progressing rapidly.                                  |
| 0     | Dead.                                                                           |
|       |                                                                                 |  
| LANSKY PERFORMANCE STATUS SCALE  < 16 YEARS OLD   |                                                                                                                        |
|---------------------------------------------------|------------------------------------------------------------------------------------------------------------------------|
| Score                                             | General Description                                                                                                    |
| 100                                               | Fully active, normal                                                                                                   |
| 90                                                | Minor restrictions in physically strenuous activity                                                                    |
| 80                                                | Active, but tires more quickly                                                                                         |
| 70                                                | Both greater restriction of and less time spent in play activity                                                       |
| 60                                                | Up and around, but minimal active play; keeps busy with quieter activities                                             |
| 50                                                | Gets dressed but lies around much of the day, no active play but able to participate in all  quiet play and activities |
| 40                                                | Mostly in bed; participates in quiet activities                                                                        |
| 30                                                | In bed; needs assistance even for quiet play                                                                           |
| 20                                                | Often sleeping; play entirely limited to very passive activities                                                       |
| 10                                                | No play; does not get out of bed                                                                                       |
| 0                                                 | Unresponsive                                                                                                           |
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cog stem cell committee consensus

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.
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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 treating physician, a patient experiences this syndrome, details of the event will be recorded in the medical record.  
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  
Protocol document date: 09/06/2018  
APPENDIX B  
| 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 <                                                                                | 2-3 mg/dL         | Adult: 500-999 mL/day Child:                                                                         |             |                                |
| 25% BSA                                                          | 10-19.9 mL/kg/day.  Or persistent nausea, vomiting, or  anorexia, with a positive upper GI  biopsy. |                   |                                                                                                      |             |                                |
| 2                                                                | Maculopapular                                                                                       | rash              | 25-                                                                                                  | 3.1-6 mg/dL | Adult: 1000-1500 mL/day Child: |
| 50% BSA                                                          | 20-30 mL/kg/day                                                                                     |                   |                                                                                                      |             |                                |
| 3                                                                | Maculopapular rash >                                                                                | 6.1-15 mg/dL      | Adult: > 1500 mL/day Child:                                                                          |             |                                |
| 50% BSA                                                          | > 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). |             |                                |  
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 3.2 below). 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 I liver involvement, or Stage l 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                                                                                                                       | Assume no liver GVHD, unless proven by biopsy   |
|------------------------------------------------------------------------------------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------|
| Day 0-35 No Skin/GI                                                                | GVHD  If NO other etiology identified,                                                                                                                                                |                                                                                                                            |                                                 |
| Day 36-100                                                                         | 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                                                                | Worsening bilirubin level                                                                                                                                                             |                                                                                                                            |                                                 |
| present                                                                            | (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).                                                                                                                                                                                                                                                                                                                                                                                                                                                          |
| Skin and/or GI GVHD                                                | Liver GVHD is staged according to the Glucksberg criteria. The                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       |
| worsening                                                          | elevated bill 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 TIP).  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. |
|                                                                    |                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      |  
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  
| Establishing GI GVHD with new onset diarrhea and no skin or liver GVHD                               |                                                                                                                                        |                                                                                                                                                                                                |
|------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------------------------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|
| No skin/liver GVHD                                                                                   | Assume no GI GVHD, unless proven by biopsy                                                                                             |                                                                                                                                                                                                |
| Day 0 through engraftment  No skin/liver GVHD                                                        | NO other etiology of diarrhea  identified:                                                                                             |                                                                                                                                                                                                |
| engraftment through Day 100                                                                          | 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  Worsening diarrhea (includes |                                                                                                                                        |                                                                                                                                                                                                |
| Skin and/or liver GVHD  present                                                                      | 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:  
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  
>100 d Yes No  
| Table 4 Acute GVHD, Chronic GVHD, and Overlap Syndrome  Category Time of Symptoms  Presence of Acute   | Presence of Chronic   |               |     |
|--------------------------------------------------------------------------------------------------------|-----------------------|---------------|-----|
| after HCT or DLI                                                                                       | GVHD features         | GVHD features |     |
| Acute GVHD  Classic acute GVHD                                                                         | <100 d                | Yes           | No  |
| Persistent, recurrent,                                                                                 | >100 d                | Yes           | No  |
| or late-onset acute  GVHDChronic GVHD Classic chronic GVHD                                             | No time limit         | No            | Yes |
| Overlap syndrome                                                                                       | No time limit         | Yes           | Yes |  
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
ind/ide
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.
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2.0 assessment 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. 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 preexisting 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 nonGVHD liver disease or unless a liver biopsy or autopsy specimen is negative.  
Prior acute GVHD in liver with new onset of a disease process that exacerbates preexisting 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.  
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
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3.1 assessing for the presence

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:  
Persistent anorexia, nausea or vomiting in the absence of other known causes of these symptoms will be considered stage I 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.
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3.2 staging of the gastrointestinal

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).
appendix c criteria for grading
3.2 staging of the gastrointestinal

| Score 0                                                                                                                                                                                                                                                                                  | Score 1              | Score 2             | Score 3   |
|------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------|---------------------|-----------|
| Performance Score: ________ KPS ECOG LPS                                                                                                                                                                                                                                                 | Asymptomatic         |                     |           |
| and fully active  (ECOG 0: KPS  or LPS 100%)                                                                                                                                                                                                                                             | Symptomatic, fully   |                     |           |
| ambulatory, restricted  only in physically  strenuous activity (ECOG  1, KPS or LPS 80-90%)                                                                                                                                                                                              | Symtomatic,          |                     |           |
| ambulatory, capable  of self-care, >50% of  waking hours out of  bed (ECOG 2, KPS  or LPS 60-70%)                                                                                                                                                                                        | Symptomatic limited  |                     |           |
| self-care, >50% of  waking hours in bed  (ECOG 3-4, KPS or  LPS <60%)                                                                                                                                                                                                                    |                      |                     |           |
| SKIN Clinical features: Maculopapular rash Lichen planus-like features Papuloquamous lesions or  ichthyosis Hyperpigmentation Hypopigmentaion Keratosis pilaris Erythema Erythroderma Poikiloderma Sclerotic features Pruritus  Hair involvement Nail involvement % BSA Involved _______ | No symptoms          | <18% BSA with       |           |
| disease signs but  NOsclerotic features                                                                                                                                                                                                                                                  | 19-50% BSA OR        |                     |           |
| involvement with  superficial sclerotic  features "not  hidebound" (able to  pinch)                                                                                                                                                                                                      | >50% BSA ORdeep      |                     |           |
| sclerotic features  "hidebound" (unable to  pinch) OR impaired  mobility, ulceration or  severe pruritus                                                                                                                                                                                 |                      |                     |           |
| MOUTH                                                                                                                                                                                                                                                                                    | No symptoms          | Mild symptoms with  |           |
| disease signs but  NOTlimiting oral intake  significantly                                                                                                                                                                                                                                | Moderate             |                     |           |
| symptoms with  disease signs  WITHpartial  limitation of oral  intake                                                                                                                                                                                                                    | Severe symptoms      |                     |           |
| with disease signs on  examination WITH major limitation of oral  intake                                                                                                                                                                                                                 |                      |                     |           |
| EYES                                                                                                                                                                                                                                                                                     | Severe dry eye       |                     |           |
| symptoms significantly  affecting ADL (special  eyeware to relieve pain )  ORunable to work  because of ocular  symptomsOR loss of  vision cause by  keratoconjunctivitissicca                                                                                                           |                      |                     |           |
| GI Tract                                                                                                                                                                                                                                                                                 | No symptoms          | Symptoms such as    |           |
| dysphagia, anorexia,  nausea, vomiting,  abdominal pain or  diarrhea without  significant weight loss  (<5%)                                                                                                                                                                             |                      |                     |           |
| No symptoms                                                                                                                                                                                                                                                                              | Mild dry eye         |                     |           |
| symptoms not affecting  ADL (requiring eyedrops)  <3 x per day) OR  asymptomatic signs of  keratoconjunctivitissiccca                                                                                                                                                                    | Moderate dry eye     |                     |           |
| symptoms partially  affecting ADL  (requiring drops>3x  per day or punctual  plugs),  WITHOUTvision  impairment                                                                                                                                                                          |                      |                     |           |
| Mean tear test (mm): >10 6-10 ≥5 Not done                                                                                                                                                                                                                                                | Symptoms             |                     |           |
| associated with  significant weight loss >  15%, requires  nutritional supplement  for most calorie needs  OResophageal dilation                                                                                                                                                         |                      |                     |           |
| LIVER                                                                                                                                                                                                                                                                                    | Normal LFT           | Elevated Bilirubin, |           |
| AP*, AST or ALT <2 x  ULN                                                                                                                                                                                                                                                                | Symptoms             |                     |           |
| associated with mild  to moderate weight  loss) 5-15%) Bilirubin >3  mg/dl or Bilirubin,  enzymes 2-3 x ULN                                                                                                                                                                              | Bilirubin or enzymes |                     |           |
| > 5 x ULN                                                                                                                                                                                                                                                                                |                      |                     |           |
appendix c (continued)
3.2 staging of the gastrointestinal

| CRITERIA FOR GRADING CHRONIC GVHD GRADE SCORE 0 SCORE 1 SCORE 2                                                                                                                                                                                                                                                                                                                                                                                                                                   | SCORE 3                                                                          |                        |
|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------------------------------------------------------------------------------|------------------------|
| Lungs†                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | No symptoms                                                                      | Mild symptoms          |
| (shortness of breath after  climbing one flight of  steps)                                                                                                                                                                                                                                                                                                                                                                                                                                        | Moderate                                                                         |                        |
| symptoms (shortness  of breath after  walking on flat  ground)                                                                                                                                                                                                                                                                                                                                                                                                                                    | Severe symptoms  (shortness of breath  at rest; requiring 02                     |                        |
| FEV1 ___________ DLCO __________                                                                                                                                                                                                                                                                                                                                                                                                                                                                  | FEV1 >80%                                                                        |                        |
| OR LFS=2                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          | FEV1 60-79%                                                                      |                        |
| OR LFS 3-5                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | FEV1 40-59%                                                                      |                        |
| OR LFS 6-9                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | FEV1 ≥39% OR                                                                     |                        |
| LFS 10-12                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         |                                                                                  |                        |
| JOINTS AND                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        | No symptoms                                                                      | Mild tightness of arms |
| FASCIA                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            | or legs, normal or mild  decreased range of  motion (ROM) AND not  affecting ADL | Tightness of arms      |
| or legs OR joint  contractures,  erythema thought  due to fasciitis,  moderate decrease  ROM AND mild to  moderate limitation  of ADL                                                                                                                                                                                                                                                                                                                                                             | Contractures                                                                     |                        |
| WITH significant  decrease or ROM  AND significant  limitation of ADL  (unable to tie shoes,  button shirts, dress  self etc.) Symptomatic  WITH advance  signs (stricture, labial  agglutination or  severe ulcerations  AND severe pain  with coitus or  inability it insert  vaginal speculum                                                                                                                                                                                                  |                                                                                  |                        |
| Other indicators, clinical manifestations or complications related to chronic GVHD (check all that apply and assign a score to its  severity (0-3) based on its functional impact where applicable (none = 0, mild = 1, moderate =2, severe = 3. GENITAL TRACT No symptoms Symptomatic with  mild signs on exam AND no effect on coitus and  minimal discomfort with  gynecologic exam Symptomatic  with moderate signs  on exam AND with  mild dyspareunia or  discomfort with  gynecologic exam |                                                                                  |                        |  
Symptomatic  
  
WITH advance  
signs (stricture, labial agglutination or severe ulcerations  
AND severe pain  
with coitus or inability it insert vaginal speculum  
Other indicators, clinical manifestations or complications related to chronic GVHD (check all that apply and assign a score to its severity (0-3) based on its functional impact where applicable (none = 0, mild = 1, moderate =2, severe = 3. Esophageal stricture or web ____ Pericarial Effusion ____ Pleural Effusion(s) ____ Ascites (serositis) ____ Nephrotic syndrome ____ Peripheral Neuropathy ____ Myasthenia Gravis ____ Cardiomyopathy ____ Eosinophilia > 500/µl ____ Polymyositis ____ Cardiac conduction defects ____ Coronary artery involvement ____ Platelets <100,000/µl ____ Progressive Onset ____ Other: Specify: ________________________________________________________________________________ Organ scoring of chronic GVHD. * AP may be elevated in growing children, and not reflective of liver dysfunction. † Pulmonary scoring should be performed using both the symptom and pulmonary function testing (PFT) scale whenever possible. When discrepancy exists between pulmonary symptoms or PFT scores, the higher value should be used for final scoring. Scoring using the  
Lung Function Score (LFS) is preferred, but if DLCO is not available, grading using FEV1 should be used. The LFS is a global  
assessment of lung function after the diagnosis of bronchiolitis obliterans has already been established. The percent predicted FEV1 and DLCO (adjusted for hematocrit but not alveolar volume) should be converted to a numeric score as follows: >80% = 1; 70-79% = 2; 6069% = 3; 50-59% = 4; 40-49% = 5; <40% = 6. The LFS = FEV1 score + DLCO score, with a possible range of 2-12. GVHD indicates graft versus host disease, ECOG, Eastern Cooperative Oncology Group, KPS, Karnofsky Performance Status; LPS, Lansky Performance Status; BSA, body surface area; ADL, activities of daily living; LFTs, liver function tests; AP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ULN, upper limit of normal.
appendix c (continued)
appendix c (continued) criteria for

| Final Grade   | Number of   | Maximum Organ  Score**   | Lung Score   |
|---------------|-------------|--------------------------|--------------|
| Organs/Sites* |             |                          |              |
| Mild          | 1 - 2       | 1                        | 0            |
| 3 or more     | 1           |                          |              |
| Moderate      | 1           |                          |              |
| At least 1    | 2           |                          |              |
| Severe        | At least 1  | 3                        | 2 - 3        |
|               |             |                          |              |  
*Determined by adding the total number of organs receiving score > 0 using Figure 1, Appendix B.  
**Defined as the maximum score given to any organ system amongst all organs scored using Figure 1, Appendix B.
appendix c (continued)
appendix d

The St. Jude Department of BMTCT Clinical Research Office standard operating procedure for the documenting and reporting of adverse (SOP 10 Documenting and Reporting of Adverse Events) will provide guidance on the evaluation, collection and reporting of adverse events for this clinical trial. The current version of this document, as well as ongoing updates, can be located at the following website: http://home.stjude.org/bmt/Pages/policies-research.aspx
appendix e
appendix d

Recommended testing and evaluation schedule  
| STANDARD OF  CARE STUDIES    | SAMPLE   | VOLUME                  | PRE   | MONTH 1                            | MONTH 2                 | MONTH 3   | MONTH 6   | MONTH 12   |
|------------------------------|----------|-------------------------|-------|------------------------------------|-------------------------|-----------|-----------|------------|
| Pregnancy Test               | PB       | 2 ml                    | X     | As clinically indicated            |                         |           |           |            |
| Physical Exam                | N/A      | N/A                     | X     | Weekly                             | X                       | X         |           |            |
| CBC with diff.               | PB       | 0.5-2 ml                | X     | Daily until engrafted, then weekly | X                       | X         |           |            |
| Chemistry                    | PB       | 0.25-2ml                | X     | Weekly                             | X                       | X         |           |            |
| Viral surveillance  (BMTPCR) | PB       | 4 ml                    | X     | Weekly                             | As clinically indicated |           |           |            |
| PB                           | 1-2 ml   | Weekly upon engraftment | X     | X                                  |                         |           |           |            |
| Chimerism                    | BM       | 2 ml                    | X     | X                                  | X                       |           |           |            |
| Disease Status  Evaluation   | N/A      | N/A                     | X     | X                                  | X                       | X         |           |            |
| MRD Bone  Marrow             | BM       | 3 ml                    | X     | X                                  | X                       | X         |           |            |
| Lymphocyte  Subset Study     | PB       | 2.5-4 ml                | X     | X                                  | X                       | X         | X         | X          |
| Quantitative                 |          |                         |       |                                    |                         |           |           |            |
| Immunoglobulins              | PB       | 2 ml                    | X     | X                                  | X                       |           |           |            |  
- The information derived from or noted on the physical examinations, standard tests, and other assessments that comprise standard of care for recipients are not required to be transcribed onto case report forms and/or entered into the database. In reference to section 6.1 Evaluations, the above-indicated follow-up regimen for these evaluations is guided by the SOPs of the Department of BMTCT, for recipients of allogeneic stem cell transplantation. As these evaluations are considered standard clinical care (nonresearch), variations in frequency (more or less frequent) of these evaluations can occur due to the participant's current clinical condition and will not be noted as protocol deviations.  
- Disease status evaluations/BM testing results obtained prior to enrollment may be used for the baseline/pre-infusion assessments.  
- Additional chimerism testing may be required as clinically indicated and described in Section 6.3. - Lymphocyte subset studies may be omitted without variance when the absolute lymphocyte count (ALC) is zero.  
- In the event of graft failure/rejection, the post failure/rejection bone marrow, chimerism and several applicable immune studies would not be clinically indicated and these studies may be held.  
APPENDIX E (continued)
Immune reconstitution testing and evaluation schedule for RECIPIENT  
| Immune reconstitution testing and evaluation schedule for RECIPIENT   |        |        |     |         |         |         |         |          |
|-----------------------------------------------------------------------|--------|--------|-----|---------|---------|---------|---------|----------|
| RESEARCH STUDIES                                                      | SAMPLE | VOLUME | PRE | MONTH 1 | MONTH 2 | MONTH 3 | MONTH 6 | MONTH 12 |
| VBETA/TREC  RESEARCH                                                  | PB     | 17 mL  | X   | X       | X       | X       |         |          |
| LYMPHOCYTE  PHENOTYPES                                                | PB     | 17 mL  | X   | X       | X       | X       | X       | X        |
| RESEARCH                                                              |        |        |     |         |         |         |         |          |
| IR-PHENOTYPE  (Youngblood Lab)                                        | PB     | 5 mL   | X   | X       | X       | X       | X       | X        |
| T-FUNCTION                                                            | PB     | 5 mL   | X   | X       | X       | X       | X       | X        |
| (Thomas Lab)                                                          | BM     | 1 mL   | X   | X       | X       |         |         |          |  
- VBETA/TREC Research and Lymphocyte Phenotypes Research results will be maintained in the Immune Monitoring Core Lab.  
- For RESEARCH studies, the posted volumes are the minimum volumes required to perform the respective protocol evaluations.  
- The PRE-transplant bone marrow for T-FUNCTION are optional tests that may be omitted at the discretion of the PI or co-investigator.
appendix e
appendix e (continued) research testing

Prior to initial stem cell collection procedure:  
| OPTIONAL research immune studies testing schedule   |                                                                                                                        |
|-----------------------------------------------------|------------------------------------------------------------------------------------------------------------------------|
| Evaluation                                          | Volume Requirement                                                                                                     |
| Flow cytometry enumeration                          | Lymphocyte Subset Study = 4 mL                                                                                         |
| Thymic output and T cell repertoire                 | VBETA/TREC Research = 17 mL                                                                                            |
| Donor baseline lymphocyte number and function       | Lymphocyte Phenotypes Research = 17 mL                                                                                 |
| Donor baseline immune function (Youngblood Lab)     | IR-PHENOTYPE = 5mL                                                                                                     |
| Donor baseline immune function (Thomas Lab)         | T-FUNCTION = 5mL                                                                                                       |
| -                                                   | All donor research testing to be collected prior to growth factor administration. These optional research tests may be |  
- All donor research testing to be collected prior to growth factor administration. These optional research tests may be collected at separate times.  
- Research testing results will be maintained in a secured database in the respective co-investigator laboratory database.
appendix e
appendix e (continued) research study

Several laboratory tests can only be processed on weekdays; therefore, if the scheduled evaluation falls on a weekend, or during a holiday period, an adjustment in the follow-up visit is expected and would not be noted as a protocol variation. Additionally, in order to accommodate such logistical constraints, evaluation/collection dates of all protocol assessments (required and optional research), may be performed within a reasonable window of the intended date following the guidelines provided in the table below:  
| If the Planned Evaluation  Time Point is:   | Window               |
|---------------------------------------------|----------------------|
| Weekly                                      | + 3 Days             |
| Month 1                                     | Week 2 to Week 6     |
| Month 2                                     | Week 7 to Week 11    |
| Month 3                                     | Week 12 to Month 4   |
| Month 6                                     | Month 5 to Month 7   |
| Month 9                                     | Month 8 to Month 10  |
| Month 12                                    | Month 10 to Month 14 |
|                                             |                      |
appendix e
appendix f

The St. Jude Department of BMTCT Clinical SOPs for standard of care for all allogeneic stem cell infusion recipients and stem cell donors will provide guidance on the evaluation, ongoing clinical care and follow up for this clinical trial. The current versions of these SOPs, as well as ongoing updates, of these documents can be located at the following website:
http://home.web.stjude.org /bone_marrow/clinicalHome.shtml.
appendix e
appendix g

HAPNK1 Audit Plan with Amendment 2.0  
I. All new enrollments will monitored and audited by a designated experienced monitor from our CPDMO (i.e., Protocol Office). Following our institutional process, each new participant data will be reviewed as follows:  
1.On the same day of enrollment the CPMDO staff will review the completed eligibility checklist and informed consent document for completeness and accuracy (100% of enrollments).  
2.In support of compliance with Eligibility and Informed Consent document and process there will be verification of supporting source documents early post enrollment, within 5 days (100% of enrollments).  
3.For the initial six subjects enrolled on Amendment 2.0 (or later protocol versions until 6 new participants are enrolled post activation of Amendment 2.0) the CPMDO monitor will evaluate the following components in each patient within 10 working days of the participant reaching the day +100 milestone:  
a. compliance with treatment including protocol directed GVHD prophylaxis b. adverse events and grade 3-4 toxicities including those contributing to the protocol stopping rules c. assessment and coding of acute GVHD d. evaluation of and reporting of deaths on study (including treatment related mortality, TRM)
e. status of participant at day +100 f. documentation of engraftment and, g. review of source data elements related to assessments for GHVD (for example, pathology biopsy results).  
4.In each case the monitor will compare patient data in the protocol database with source documents. The monitor will utilize the defined criteria as indicated in the HAPNK1 latest protocol version for consent, eligibility, and toxicity assessments and grading, including the newly incorporated COG GVHD Guidelines.  
II. A patient specific audit summary will be generated with findings from each of the components. Results of the patient-specific audit will be provided to the study Principal Investigator (PI) for comment and independently to the VP Clinical Trials Administration. In case of discrepancies related to under reporting of GVHD and TRM, those cases will be interpreted as assigned by the monitor in the context of operationalizing the stopping rules, and will remain as such (i.e. coded as GVHD) until the opinion of an external medical reviewer has been obtained.
III. A final consensus audit summary on the first six enrollments on Amendment 2.0 will be generated and provided to the PI, to the St. Jude Clinical Director. the VP Clinical Trials, St. Jude IRB, as well as to the FDA  
IV. On a monthly basis the study PI, the study CRA, and the Director of Clinical Trial Operations in the BMT service will meet to review ongoing data collection for each enrollment for accuracy, completeness, and timeliness.  
V. If the focused audits of the first six subjects enrolled on Amendment 2.0 reveal no major compliance concerns, the study will continue with an every six month review cycle as required by our Institutional Data Safety Monitoring Plan (with a modification that every other new enrollment will have data audits). In addition to the review elements noted above other components that occur after day +100 (chronic GVHD, other AEs, deaths, status, regulatory documents, etc.) will be reviewed. An overall study monitoring report will generated every six months, and will be reviewed independently at such time by the Institutional Monitoring Committee.