appendix d
operating characteristics of the modified

| Index                   | Specific Criteria                                                             | General                                                              |
|-------------------------|-------------------------------------------------------------------------------|----------------------------------------------------------------------|
| 100                     | Normal, no complaints, no evidence of                                         | Able to carry on normal activity;                                    |
| disease.                | no special care needed.                                                       |                                                                      |
| 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 for self, unable to carry on normal                                      | Unable to work, able to live at                                      |
| activity or to do work. | home and care for most personal  needs, varying amount of  assistance needed. |                                                                      |
| 60                      | Requires occasional assistance from others but  able to care for most needs.  |                                                                      |
| 50                      | Requires considerable assistance from others  and frequent medical care       |                                                                      |
| 40                      | Disabled, requires special care and assistance.                               | Unable to care for self, requires  institutional or hospital care or |
| 30                      | Severely disabled, hospitalization indicated,                                 | equivalent, disease may be rapidly                                   |
| but death not imminent. | progressing.                                                                  |                                                                      |
| 20                      | Very sick, hospitalization necessary, active  supportive treatment necessary. |                                                                      |
| 10                      | Moribund                                                                      |                                                                      |
| 0                       | Dead                                                                          |                                                                      |
appendix e
operating characteristics of the modified

| Comorbidities                                                                   | Definition                                               | Score   |
|---------------------------------------------------------------------------------|----------------------------------------------------------|---------|
| Migraine/headache                                                               | 0                                                        |         |
| Osteoporosis                                                                    | 0                                                        |         |
| Osteoarthritis                                                                  | 0                                                        |         |
| Hypertension                                                                    | 0                                                        |         |
| Gastrointestinal                                                                | Including inflammatory bowel disease                     | 0       |
| Mild pulmonary                                                                  | DLCo and/or FEV1 >80% or                                 | 0       |
| Dyspnea on moderate activity                                                    |                                                          |         |
| Mild renal                                                                      | Serum creatinine 1.2-2 mg/dl                             | 0       |
| Endocrine                                                                       | 0                                                        |         |
| Bleeding                                                                        | 0                                                        |         |
| Coagulopathy                                                                    | Deep venous thrombosis or pulmonary embolism             | 0       |
| Asthma                                                                          | 0                                                        |         |
| Arrhythmia                                                                      | 1                                                        |         |
| Myocardial                                                                      | Coronary artery disease, congestive HF, history of       | 1       |
| medically documented MI, EF50%                                                 |                                                          |         |
| Mild hepatic                                                                    | Chronic hepatitis, Bilirubin >ULN- 1.5 X ULN, or         | 1       |
| AST/ALT >ULN-2.5XULN                                                            |                                                          |         |
| Cerebro-vascular                                                                | History of transient ischemic attack or cerebro-vascular | 1       |
| accident                                                                        | accident                                                 |         |
| Morbid obesity                                                                  | 1                                                        |         |
| Diabetes                                                                        | Requiring treatment                                      | 1       |
| Depression/anxiety                                                              | 1                                                        |         |
| Infection                                                                       | Requiring continuation of treatment after Day 0          | 1       |
| Rheumatologic                                                                   | SLE, RA, polymyositis, mixed CTD, polymyalgia            | 2       |
| rheumatica                                                                      |                                                          |         |
| Moderate pulmonary                                                              | DLCo and/or FEV1 66-80% or                               | 2       |
| Dyspnea on slight activity                                                      |                                                          |         |
| Peptic ulcer                                                                    | Patients who have required treatment                     | 2       |
| Moderate-severe renal                                                           | Serum creatinine >2 mg/dl, on dialysis, or prior renal   | 2       |
| transplantation                                                                 |                                                          |         |
| Valvular heart disease                                                          | Except mitral valve prolapse                             | 3       |
| Prior solid tumor                                                               | Requiring treatment with chemotherapy                    | 3       |
| Moderate-severe hepatic                                                         | Liver cirrhosis, Bilirubin >1.5 X ULN, or AST/ALT        | 3       |
| >2.5XULN                                                                        |                                                          |         |
| Severe pulmonary                                                                | DLCo and/or FEV1 65% or                                 | 3       |
| Dyspnea at rest or requiring oxygen                                             |                                                          |         |
| Total score is the sum of all comorbidities present at time of transplantation. |                                                          |         |
appendix f 2014 refined disease
operating characteristics of the modified

| patient.                                                                                                                     |            |                 |      |                                                         |                      |      |       |       |
|------------------------------------------------------------------------------------------------------------------------------|------------|-----------------|------|---------------------------------------------------------|----------------------|------|-------|-------|
| Disease                                                                                                                      | Stage      | No, of patients | HR'  | Original DRI    Percentage of patients    New DRI Group | 2-y OS (%)    95% Cl |      |       |       |
| Hodgkin lymphoma CR                                                                                                          | 126        | 0.36            | Int  | 14                                                      | Low                  | 68   | 63-68 |       |
| CLL CR                                                                                                                       | 81         | 0.47            | Low  | Low                                                     |                      |      |       |       |
| Mantie cell lymphoma CR                                                                                                      | 160        | 0.51            | Int  | Low                                                     |                      |      |       |       |
| Indolent NHL CR                                                                                                              | 183        | 0.53            | Low  | Low                                                     |                      |      |       |       |
| AML favorable cytogenetics CR                                                                                                | 190        | 0.64            | Low  | Low                                                     |                      |      |       |       |
| Indolent NHL PR                                                                                                              | 276        | 0.71            | Low  | Low                                                     |                      |      |       |       |
| CLL PR                                                                                                                       | 400        | 0.78            | Low  | Low                                                     |                      |      |       |       |
| CML chronic phase 1/2                                                                                                        | 390        | 0.82            | Low  | Low                                                     |                      |      |       |       |
| CML advanced phase                                                                                                           | 69         | 0.92            | Int  | 2                                                       | Int                  | 51   | 50-52 |       |
| Mantle cell lymphoma PR                                                                                                      | 149        | 0.95            | Int  | Int                                                     |                      |      |       |       |
| Myeloproliferative neoplasm                                                                                                  | Any        | 428             | 0.98 | Int                                                     | Int                  |      |       |       |
| AML intermediate cytogenetics CR                                                                                             | 3611       | Ref             | Int  | Int                                                     |                      |      |       |       |
| ALL CR1                                                                                                                      | 1023       | 1.00            | Int  | Int                                                     |                      |      |       |       |
| T-cell NHL CR                                                                                                                | 171        | 1.00            | Int  | Int                                                     |                      |      |       |       |
| Multiple myeloma CR/VGPR/PR                                                                                                  | 339        | 1.03            | Int  | Int                                                     |                      |      |       |       |
| Aggressive NHL CR                                                                                                            | 181        | 1.05            | Int  | Int                                                     |                      |      |       |       |
| Low-risk MDS adverse cytogenetics                                                                                            | Earlyt     | 13              | 1.06 | High                                                    | Int                  |      |       |       |
| T-cell NHL PR                                                                                                                | 164        | 1.06            | Int  | Int                                                     |                      |      |       |       |
| Low-risk MDS intermediate cytogenetics                                                                                       | Earlyt     | 516             | 1.09 | Int                                                     | Int                  |      |       |       |
| HL PR                                                                                                                        | 25         | 1.09            | Int  | Int                                                     |                      |      |       |       |
| Low-risk MDS intermediate cytogenetics                                                                                       | Advanced t | 235             | 1.18 | Int                                                     | Int                  |      |       |       |
| Indolent NHL                                                                                                                 | Advanced t | 128             | 1.21 | Int                                                     | Int                  |      |       |       |
| CLL                                                                                                                          | Advanced   | 265             | 1.22 | Int                                                     | Int                  |      |       |       |
| High-risk MDS intermediate cytogenetics                                                                                      | Early      | 364             | 1.24 | Int                                                     | Int                  |      |       |       |
| Aggressive NHL PR                                                                                                            | 205        | 1.26            | Int  | Int                                                     |                      |      |       |       |
| T-cell NHL                                                                                                                   | Advanced t | 93              | 1.41 | High                                                    | 20                   | High | 3     | 31-35 |
| AML favorable cytogenetics                                                                                                   | Advanced t | 3               | 1.42 | Int                                                     | High                 |      |       |       |
| HL                                                                                                                           | Advancedt  | 85              | 1.48 | High                                                    | High                 |      |       |       |
| High-risk MDS intermediate cytogenetics                                                                                      | Advanced t | 179             | 1.56 | Int                                                     | High                 |      |       |       |
| High-risk MDS adverse cytogenetics                                                                                           | Early      | 8               | 1.58 | High                                                    | High                 |      |       |       |
| ALL CR2                                                                                                                      | 407        | 1.58            | High |                                                         |                      |      |       |       |
| Int                                                                                                                          |            |                 |      |                                                         |                      |      |       |       |
| AL adverse cytogenetics CR                                                                                                   | 175        | 1.59            | High | High                                                    |                      |      |       |       |
| Mantle cell lymphoma                                                                                                         | Advancedt  | 46              | 1.59 | High                                                    | High                 |      |       |       |
| High-risk MDS adverse cytogenetics                                                                                           | Avanced t  | 3               | 1.59 | Very high                                               | High                 |      |       |       |
| BL CR                                                                                                                        | 23         | 1.65            | NA   | High                                                    |                      |      |       |       |
| Multiple myeloma                                                                                                             | Advanced f | 150             | 1.65 | High                                                    | High                 |      |       |       |
| ALL CR3                                                                                                                      | 61         | 1.70            | Int  | High                                                    |                      |      |       |       |
| Low-risk MDS adverse cytogenetics                                                                                            | Advancedt  | 3               | 1.86 | Very high                                               | High                 |      |       |       |
| AL intermediate cytogenetics                                                                                                 | Advanced   | 1227            | 1.89 | High                                                    | High                 |      |       |       |
| CML blast phase                                                                                                              | 52         | 2.02            | Int  | 4                                                       | Very high            | 2    | 20 27 |       |
| ALL                                                                                                                          | Advanced t | 25              | 2.23 | High                                                    | Very high            |      |       |       |
| Aggressive NHL                                                                                                               | Advanced t | 154             | 2.54 | High                                                    | Very high            |      |       |       |
| AML adverse cytogenetics                                                                                                     | Advanced t | 76              | 2.83 | Very high                                               | Very high            |      |       |       |
| BL‡ PR                                                                                                                       | Advanced ↑ | 12              | 5.21 | NA                                                      | Very high            |      |       |       |
| Int, intermediate.                                                                                                           |            |                 |      |                                                         |                      |      |       |       |
| - Hazard ratio for mortality compared with AML intermediate cytogenetics in CR1.                                             |            |                 |      |                                                         |                      |      |       |       |
| ↑Advanced stage refers to induction failure or active relapse, including stable or progressive disease for NHL, HL, and CLL. |            |                 |      |                                                         |                      |      |       |       |
| #                                                                                                                            |            |                 |      |                                                         |                      |      |       |       |  
Participating sites to use column labeled as "New DRI Group" to report DRI of enrolled
appendix g diagnosis and severity
operating characteristics of the modified

| Skin (Active Erythema Only)                                                                          | Upper GI                                           | Lower Gl (stool output/day)                  |                                                |                                           |
|------------------------------------------------------------------------------------------------------|----------------------------------------------------|----------------------------------------------|------------------------------------------------|-------------------------------------------|
| Stage                                                                                                | Liver                                              |                                              |                                                |                                           |
| (Bilirubin)                                                                                          |                                                    |                                              |                                                |                                           |
| No active (erythematous) GVHD rash                                                                   | <2 mg/dL                                           | No or intermittent nausea,                   | Adult: <500 mL/day or <3 episodes/day          |                                           |
| 0                                                                                                    | vomiting, or anorexia                              | Child: < 10 mL/kg/day or < 4 episodes/day    |                                                |                                           |
| i                                                                                                    | Maculopapular rash                                 | 2-3 mg/dL                                    | Persistent nausea,                             | Adult: 500-999 mL/day or 3-4 episodes/day |
| < 25% BSA                                                                                            | vomiting or anorexia                               | Child: 10-19.9 mL/kg/day or 4-6 episodes/day |                                                |                                           |
| 2                                                                                                    | Maculopapular rash                                 | 3.1-6 mg/dl.                                 | Adult: 1000-1500 mL/day or 5-7 episodes/day    |                                           |
| 25-50% BSA                                                                                           | Child: 20-30 mL/kg/day or 7-10 episodes/day        |                                              |                                                |                                           |
| 3                                                                                                    | Maculopapular rash                                 | 6.1-15 mg/dL                                 | Adult: >1500 mL/day or >7 episodes/day         |                                           |
| >50% BSA                                                                                             | Child: >30 mL/kg/day or >10 episodes/day           |                                              |                                                |                                           |
| A                                                                                                    | Generalized erythroderma                           | > 15 mg/dL                                   | Severe abdominal pain with or without ileus or |                                           |
| (>50% BSA) plus bullous formation                                                                    | grossly bloody stool (regardless of stool volume). |                                              |                                                |                                           |
| and desquamation > 5% BSA                                                                            |                                                    |                                              |                                                |                                           |
| Overall clinical grade (based on most severe target organ involvement):                              |                                                    |                                              |                                                |                                           |
| Grade 0: No stage 1-4 of any organ.                                                                  |                                                    |                                              |                                                |                                           |
| Grade 1: Stage 1-2 skin without liver, upper GI, or lower GI involvement,                            |                                                    |                                              |                                                |                                           |
| Grade II: Stage 3 rash and/or stage 1 liver and/or stage 1 upper GI and/or stage 1 lower GI.         |                                                    |                                              |                                                |                                           |
| Grade III: Stage 2-3 liver and/or stage 2-3 lower GI, with stage 0-3 skin and/or stage 0-1 upper GI. |                                                    |                                              |                                                |                                           |
| Grade IV: Stage 4 skin, liver, or lower GI involvement, with stage 0-1 upper GI.                     |                                                    |                                              |                                                |                                           |
appendix g diagnosis and severity
1. acute gvhd organ staging

| SCORE 0                                                                              | SCORE 1                | SCORE 2                   | SCORE 3             |                        |
|--------------------------------------------------------------------------------------|------------------------|---------------------------|---------------------|------------------------|
| PERFORMANCE                                                                          | ❏ Asymptomatic and     | ❏ Symptomatic,            | □ Symptomatic,      | ❏ Symptomatic,         |
| SCORE:                                                                               | fully active (ECOG     | fully ambulatory,         | ambulatory, capable | limited self-care,     |
| 0; KPS or LPS                                                                        | restricted only in     | of self-care, >50%        | > 50% of waking     |                        |
| 100%)                                                                                | physically             | of waking hours ou        | hours in bed (ECOG  |                        |
| KPS ECOG LPS                                                                         | of bed (ECOG 2,        | 3-4, KPS or LPS           |                     |                        |
| strenuous activity                                                                   |                        |                           |                     |                        |
| (COG 1, KPS                                                                          | KS or LPS 60-          | <60%)                     |                     |                        |
| or LPS 80-90%)                                                                       | 70%)                   |                           |                     |                        |
| SKIN†                                                                                |                        |                           |                     |                        |
| SCORE % BSA                                                                          |                        |                           |                     |                        |
| GVHD features to be scored                                                           | ❏ No BSA               | 1 1-18% BSA               | 1 19-50% BSA        | ❏ >50% BSA             |
| by BSA:                                                                              | involved               |                           |                     |                        |
| Check all that apply:                                                                |                        |                           |                     |                        |
| □ Maculopapular rash/erythema                                                        |                        |                           |                     |                        |
| ❏ Lichen planus-like features                                                        |                        |                           |                     |                        |
| □ Sclerotic features                                                                 |                        |                           |                     |                        |
| ❏ Papulosquamous lesions or                                                          |                        |                           |                     |                        |
| ichthyosis                                                                           |                        |                           |                     |                        |
| 🙂 Keratosis pilaris-like GVHD                                                       |                        |                           |                     |                        |
| SKIN FEATURES                                                                        | Check all that apply:  |                           |                     |                        |
| SCORE:                                                                               | □ No sclerotic         | - Superficial             | □ Deep sclerotic    |                        |
| features                                                                             | sclerotic features     | features                  |                     |                        |
| "not hidebound"                                                                      | □ "Hidebound"          |                           |                     |                        |
| (able to pinch)                                                                      | (unable to pinch)      |                           |                     |                        |
| □ Impaired mobility                                                                  |                        |                           |                     |                        |
| D Ulceration                                                                         |                        |                           |                     |                        |
| Other skin GVHD features (NOT scored by BSA)                                         |                        |                           |                     |                        |
| Check all that apply:                                                                |                        |                           |                     |                        |
| □ Hyperpigmentation                                                                  |                        |                           |                     |                        |
| □ Hypopigmentation                                                                   |                        |                           |                     |                        |
| ❏ Poikiloderma                                                                       |                        |                           |                     |                        |
| □ Severe or generalized pruritus                                                     |                        |                           |                     |                        |
| □ Hair involvement                                                                   |                        |                           |                     |                        |
| □ Nail involvement                                                                   |                        |                           |                     |                        |
| ❏ Abnormality present but explained entirely by non-GVHD documented cause (specify): |                        |                           |                     |                        |
| MOUTH                                                                                | ❏ No symptoms          | ❏ Mild symptoms           | 1 Moderate          | ❏ Severe symptoms with |
| Lichen planus-like                                                                   | with disease signs     | symptoms with             | disease signs on    |                        |
| features present:                                                                    | disease signs with     |                           |                     |                        |
| but not limiting                                                                     | examination with major |                           |                     |                        |
| ♦ Yes                                                                                | partial limitation     | limitation of oral intake |                     |                        |
| oral intake                                                                          |                        |                           |                     |                        |
| 🙂 No                                                                                | significantly          | of oral intake            |                     |                        |
| □ Abnormality present but explained entirely by non-GVHD documented cause (specify): |                        |                           |                     |                        |  
2.
appendix g diagnosis and severity
grading of chronic gvhd (nih

Organ scoring of chronic GVHD. ECOG indicates 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; ULN, normal upper limit. *Weight loss within 3 months. Skin scoring should use both percentage of BSA involved by disease signs and the cutaneous features scales. When a discrepancy exists between the percentage of total body surface (BSA) score and the skin feature score, OR if superficial sclerotic features are present (Score 2), but there is impaired mobility or ulceration (Score 3), the higher level should be used for the final skin scoring. To be completed by specialist or trained medical providers. **Lung scoring should be performed using both the symptoms and FEV1 scores whenever possible. FEV1 should be used in the final lung scoring where there is discrepancy between symptoms and FEV1 scores.  
PROGRESS III - PTCY vs. TAC/MTX Protocol - 1703  
| Version 4.0 dated November 16, 2020                                                  |                            |                          |                          |                       |
|--------------------------------------------------------------------------------------|----------------------------|--------------------------|--------------------------|-----------------------|
| SCORE 0                                                                              | SCORE 1                    | SCORE 2                  | SCORE 3                  |                       |
| E YES                                                                                | □ No symptoms              | ❏ Mild dry eye           | ❏ Moderate dry eye       | ❏ Severe dry eye      |
| symptoms not                                                                         | symptoms partially         | symptoms significantly   |                          |                       |
| Keratoconjunctivitis                                                                 | affecting ADL              | affecting ADL            | affecting ADL (special   |                       |
| sicca (KCS) confirmed                                                                | (requirement of            | (requiring lubricant     | eyeware to relieve pain) |                       |
| by ophthalmologist:                                                                  | lubricant eye              | OR unable to work        |                          |                       |
| eye drops > 3 x per                                                                  |                            |                          |                          |                       |
|                                                                                      | drops < 3 x per            | dy or punctal            | because of ocular        |                       |
| □ No                                                                                 | dy)                        | plugs),                  | symptoms OR loss of      |                       |
| WITHOUT new                                                                          |                            |                          |                          |                       |
| ❏ Not examined                                                                       | vision due to KCS          |                          |                          |                       |
| vision impairment                                                                    |                            |                          |                          |                       |
| due to KCS                                                                           |                            |                          |                          |                       |
| □ Abnormality present but explained entirely by non-GVHD documented cause (specify): |                            |                          |                          |                       |
| GI Tract                                                                             | ❏ No symptoms              | □ Symptoms               | □ Symptoms               | □ Symptoms associated |
| Check all that apply:                                                                | without                    | associated with          | with significant weight  |                       |
| loss* > 15%, requires                                                                |                            |                          |                          |                       |
| ❏ Esophageal web/                                                                    | significant weight         | mild to moderate         |                          |                       |
| loss* (<5%)                                                                          | nutritional supplement for |                          |                          |                       |
| proximal stricture                                                                   | weight loss*               |                          |                          |                       |
| (5-15%) OR                                                                           | most calorie needs OR      |                          |                          |                       |
| or ring                                                                              | moderate diarrhea          | esophageal dilation OR   |                          |                       |
| ❏ Dysphagia                                                                          | without                    | severe diarrhea with     |                          |                       |
| □ Anorexia                                                                           | significant                | significant interference |                          |                       |
| 1 Nausea                                                                             |                            |                          |                          |                       |
|                                                                                      | interference with          | with daily living        |                          |                       |
| dily living                                                                          |                            |                          |                          |                       |
| D Diarrhea                                                                           |                            |                          |                          |                       |
|                                                                                      |                            |                          |                          |                       |
| ❏ Failure to thrive                                                                  |                            |                          |                          |                       |
| □ Abnormality present but explained entirely by non-GVHD documented cause (specify): |                            |                          |                          |                       |
| LIVER                                                                                |                            | □ Normal total           | 1 Elevated total         | 1 Elevated total      |
| bilirubin and                                                                        | bilirubin with ALT         | bilirubin but            | bilirubin > 3 mg/dL      |                       |
| ALT or AP                                                                            | 23 to 5 x ULN or           | <3 mg/dL or              |                          |                       |
| < 3 x ULN                                                                            | AP ≥ 3 x ULN               | ALT > 5 ULN              |                          |                       |
| ❏ Abnormality present but explained entirely by non-GVHD documented cause (specify): |                            |                          |                          |                       |
| LUNGS**                                                                              |                            |                          |                          |                       |
| Symptom score:                                                                       | □ No symptoms              | □ Mild symptoms          | 0 Moderate               | □ Severe symptoms     |
| (hortness of                                                                         | symptoms                   | (shortness of breath at  |                          |                       |
| breath after                                                                         | (shortness of breath       | rest; requiring 02 )     |                          |                       |
| climbing one flight                                                                  | after walking on           |                          |                          |                       |
| of steps)                                                                            | flat ground)               |                          |                          |                       |
| Lung score:                                                                          | ❏ FEV1≥80%                 | □ FEV1 60-79%            | □ FEV1 40-59%            | ❏ FEV1 ≤39%           |
| % FEV1 [                                                                             |                            |                          |                          |                       |
| Pulmonary function tests                                                             |                            |                          |                          |                       |  
❏ Not performed
□ Abnormality present but explained entirely by non-GVHD documented cause (specify):
Version 4.0 dated November 16, 2020  
| SCORE 0                                                                              | SCORE 1                       | SCORE 2                                      | SCORE 3                   |
|--------------------------------------------------------------------------------------|-------------------------------|----------------------------------------------|---------------------------|
| JOINTS AND FASCIA                                                                    | □ No                          | ❏ Mild tightness of                          | ❏ Tightness of arms or    |
| arms or legs,                                                                        | legs OR joint                 | significant decrease of                      |                           |
| symptoms                                                                             |                               |                                              |                           |
| P-ROM score                                                                          | normal or mild                | contractures,                                | ROM AND significant       |
| (see below)                                                                          | decreased range of            | erythema thought                             | limitation of ADL         |
| Shoulder (1-7):                                                                      | motion (ROM)                  | due to fasciitis,                            | (unable to tie shoes,     |
| Elbow (1-7):                                                                         | AND not affecting             | moderate decrease                            | button shirts, dress self |
| Wrist/finger (1-7):                                                                  | ROM AND mild to               |                                              |                           |
| ADL                                                                                  | etc.)                         |                                              |                           |
| Ankle (1-4):                                                                         | moderate limitation           |                                              |                           |
| of ADL                                                                               |                               |                                              |                           |
| ❏ Abnormality present but explained entirely by non-GVHD documented cause (specify): |                               |                                              |                           |
| GENITAL TRACT                                                                        | ❏ No signs → Mild signs + and | ❏ Moderate signs + and → Severe signs + with |                           |
| ( See Supplemental figure 2 )                                                        | females with or               | may have                                     | or without                |
| □ Not examined                                                                       | without discomfort            | symptoms with                                | symptoms                  |
| Currently sexually active                                                            | on exam                       | discomfort on exam                           |                           |
| 0 Yes                                                                                |                               |                                              |                           |
|                                                                                      |                               |                                              |                           |  
❏ Abnormality present but explained entirely by non-GVHD documented cause (specify):  
| Other indicators, clinical features or complications related to chronic GVHD (check all that apply and assign a   |                                       |                         |            |          |
|-------------------------------------------------------------------------------------------------------------------|---------------------------------------|-------------------------|------------|----------|
| score to severity (0-3) based on functional impact where applicable none - 0,mild —1, moderate —2, severe - 3)    |                                       |                         |            |          |
| ❏ Ascites (serositis)                                                                                             | ❏ Myasthenia Gravis                   |                         |            |          |
| □ Pericardial Effusion                                                                                            | 1 Peripheral Neuropathy               | ❏ Eosinophilia > 500/pl |            |          |
| ❏ Pleural Effusion(s)                                                                                             | ❏ Polymyositis                        | ❏ Platelets <100,000/μl |            |          |
| ❏ Nephrotic syndrome                                                                                              | ❏ Weight loss>5%* without GI symptoms | - Others (specify):     |            |          |
| Overall GVHD Severity                                                                                             | ❏ No GVHD                             | ❏ Mild                  | ❏ Moderate | 1 Severe |
| (Opinion of the evaluator)                                                                                        |                                       |                         |            |          |
| Photographic Range of Motion (P-ROM)                                                                              |                                       |                         |            |          |  
  
| Categories of Acute and Chronic GVHD   | Presence  of Acute  GVHD  Features   | Presence of  Chronic  GVHD  Features*   |     |
|----------------------------------------|--------------------------------------|-----------------------------------------|-----|
| Time of  Symptoms  after HCT           |                                      |                                         |     |
| Category Acute GVHD Classic acute GVHD | ≤100 d                               | Yes                                     | No  |
| Late-onset acute GVHD                  | >100 d                               | Yes                                     | No  |
| Chronic GVHD                           | No time                              |                                         |     |
| Classic chronic GVHD                   | limit                                | No                                      | Yes |
| No time                                |                                      |                                         |     |
| Overlap syndrome                       | limit                                | Yes                                     | Yes |
| *As defined in section 4 (below)       |                                      |                                         |     |  
3. **Categories of Acute and Chronic GVHD**  
| 4. Signs and Symptoms of Chronic GVHD²³                                                                                |                                     |                                   |                        |                    |
|------------------------------------------------------------------------------------------------------------------------|-------------------------------------|-----------------------------------|------------------------|--------------------|
| Organ or Site                                                                                                          | Diagnostic (Sufficient to           | Distinctive® (Seen in chronic     | Other Features or      | Common† (Seen with |
| Establish the Diagnosis of                                                                                             | GVHD, but Insufficient Alone        | Unclassified Entities 1           | Both Acute and         |                    |
| chronic GVHD).                                                                                                         | to Establish a Diagnosis)           | chronic GVHD)                     |                        |                    |
| Skin                                                                                                                   | Poikiloderma                        | Depigmentation                    | Sweat impairment       | Erythema           |
| Lichen planus—like                                                                                                     | Papulosquamous lesions              | Ichthyosis                        | Maculopapular rash     |                    |
| Keratosis pilaris                                                                                                      | Pruritus                            |                                   |                        |                    |
| features                                                                                                               |                                     |                                   |                        |                    |
| Sclerotic features Morphea-                                                                                            | Hypopigmentation                    |                                   |                        |                    |
| like features                                                                                                          | Hyperpigmentation                   |                                   |                        |                    |
| Lichen sclerosus—like                                                                                                  |                                     |                                   |                        |                    |
| features                                                                                                               |                                     |                                   |                        |                    |
| Nails                                                                                                                  | Dystrophy                           |                                   |                        |                    |
| Longitudinal ridging,                                                                                                  |                                     |                                   |                        |                    |
| splitting or brittle features                                                                                          |                                     |                                   |                        |                    |
| Onycholysis                                                                                                            |                                     |                                   |                        |                    |
| Pterygium unguis                                                                                                       |                                     |                                   |                        |                    |
| Nail loss (usually symmetric,                                                                                          |                                     |                                   |                        |                    |
| affects most nails)                                                                                                    |                                     |                                   |                        |                    |
| Scalp and                                                                                                              | New onset of scarring or            | Thinning scalp hair, typically    |                        |                    |
| body hair                                                                                                              | nonscarring scalp                   | patchy, coarse or dull            |                        |                    |
| alopecia (after recovery                                                                                               | (not explained by endocrine         |                                   |                        |                    |
| from chemoradiotherapy).                                                                                               | or other causes)                    |                                   |                        |                    |
| Loss of body hair                                                                                                      | Premature gray hair                 |                                   |                        |                    |
| Scaling                                                                                                                |                                     |                                   |                        |                    |
| Mouth                                                                                                                  | Lichen planus—like changes          | Xerostomia                        | Gingivitis             |                    |
| Mucoceles                                                                                                              | Mucositis                           |                                   |                        |                    |
| Mucosal atrophy                                                                                                        | Erythema                            |                                   |                        |                    |
| Ulcers                                                                                                                 | Pain                                |                                   |                        |                    |
| Pseudomembranes                                                                                                        |                                     |                                   |                        |                    |
| Eyes                                                                                                                   | New onset dry, gritty, or           | Photophobia                       |                        |                    |
| painful eyes                                                                                                           | Periorbital hyperpigmentation       |                                   |                        |                    |
| Cicatricial conjunctivitis                                                                                             | Blepharitis (erythema of the        |                                   |                        |                    |
| KCS                                                                                                                    | eyelids with edema)                 |                                   |                        |                    |
| Confluent areas of                                                                                                     |                                     |                                   |                        |                    |
| punctate keratopathy                                                                                                   |                                     |                                   |                        |                    |
| Genitalia                                                                                                              | Lichen planus—like features         | Erosions                          |                        |                    |
| Lichen sclerosus—like features                                                                                         | Fissures                            |                                   |                        |                    |
| Females                                                                                                                | Vaginal scarring or clitoral/labial | Ulcers                            |                        |                    |
| agglutination                                                                                                          |                                     |                                   |                        |                    |
| Males                                                                                                                  | Phimosis or urethral/meatus         |                                   |                        |                    |
| scarring or stenosis.                                                                                                  |                                     |                                   |                        |                    |
| GI Tract                                                                                                               | Esophageal web                      | Exocrine pancreatic insufficiency | Anorexia               |                    |
| Strictures or stenosis in the upper                                                                                    | Nausea                              |                                   |                        |                    |
| to mid third of the esophagus                                                                                          | Vomiting                            |                                   |                        |                    |
| Diarrhea                                                                                                               |                                     |                                   |                        |                    |
| Weight loss                                                                                                            |                                     |                                   |                        |                    |
| Failure to thrive (infants                                                                                             |                                     |                                   |                        |                    |
| and children                                                                                                           |                                     |                                   |                        |                    |
| Liver                                                                                                                  | Total bilirubin, alkaline           |                                   |                        |                    |
| phosphatase > 2 × upper                                                                                                |                                     |                                   |                        |                    |
| limit of normal                                                                                                        |                                     |                                   |                        |                    |
| ALT > 2 × upper limit of                                                                                               |                                     |                                   |                        |                    |
| normal                                                                                                                 |                                     |                                   |                        |                    |
| Lung                                                                                                                   | Bronchiolitis obliterans            | Air trapping and                  | Cryptogenic organizing |                    |
| diagnosed with lung biopsy                                                                                             | bronchiectasis on chest CT          | pneumonia                         |                        |                    |
| BOS                                                                                                                    | Restrictive lung diseasell          |                                   |                        |                    |
| Muscles, fascia,                                                                                                       | Fasciitis                           | Myositis or polymyositis'         | Edema                  |                    |
| Joint stiffness or contractures                                                                                        |                                     |                                   |                        |                    |
| joints                                                                                                                 | Muscle cramps                       |                                   |                        |                    |
| secondary to fasciitis or sclerosis.                                                                                   | Arthralgia or arthritis             |                                   |                        |                    |
| Hematopoietic                                                                                                          | Thrombocytopenia                    |                                   |                        |                    |
| and Immune                                                                                                             | Eosinophilia                        |                                   |                        |                    |
| Lymphopenia                                                                                                            |                                     |                                   |                        |                    |
| Hypo- or hyper-gammaglobulinemia                                                                                       |                                     |                                   |                        |                    |
| Autoantibodies (AIHA, ITP)                                                                                             |                                     |                                   |                        |                    |
| Raynaud's phenomenon                                                                                                   |                                     |                                   |                        |                    |
| Other                                                                                                                  | Pericardial or pleural effusions    |                                   |                        |                    |
| Ascites                                                                                                                |                                     |                                   |                        |                    |
| Peripheral neuropathy                                                                                                  |                                     |                                   |                        |                    |
| Nephrotic syndrome                                                                                                     |                                     |                                   |                        |                    |
| Myasthenia gravis                                                                                                      |                                     |                                   |                        |                    |
| Cardiac conduction abnormality or                                                                                      |                                     |                                   |                        |                    |
| cardiomyopathy                                                                                                         |                                     |                                   |                        |                    |
| ALT indicates alanine aminotransferase; AIHA, autoimmune hemolytic anemia; ITP, idiopathic thrombocytopenic purpura.   |                                     |                                   |                        |                    |
| - In all cases, infection, drug effect, malignancy, or other causes must be excluded.                                  |                                     |                                   |                        |                    |
| ' Can be acknowledged as part of the chronic GVHD manifestations if diagnosis is confirmed.                            |                                     |                                   |                        |                    |
| 8 Common refers to shared features by both acute and chronic GVHD.                                                     |                                     |                                   |                        |                    |
| 1 BOS can be diagnostic for lung chronic CVHD only if distinctive sign or symptom present in another organ (see text). |                                     |                                   |                        |                    |
| - Pulmonary entities under investigation or unclassified.                                                              |                                     |                                   |                        |                    |
| 1 Diagnosis of chronic GVHD requires biopsy.                                                                           |                                     |                                   |                        |                    |
appendix h patient reported outcome
bmt ctn 1703 hemorrhagic cystitis

During the past 7 days, how many days did you see blood in your urine?  
1. No days 2. 1 day 3. 2 days 4. 3-5 days 5. 6-7 days How often did you feel like you needed to empty your bladder right away or else you would have an accident?  
1. Never 2. One time during the past 7 days 3. 2-6 times during the past 7 days 4. Often once a day 5. More than once a day
appendix h patient reported outcome
promis questions

[GISX38] During the past 7 days, how many days did you have loose or watery stools?  
1. No days 2. 1 day 3. 2 days 4. 3-5 days 5. 6-7 days
[GISX42] How often did you feel like you needed to empty your bowels right away or else you would have an accident?  
1. Never 2. One time during the past 7 days 3. 2-6 times during the past 7 days 4. Often once a day 5. More than once a day
[PFA6] Does your health now limit you in bathing or dressing yourself?  
1. Not at all 2. Very little 3. Somewhat 4. Quite a lot 5. Cannot do
[PFA53] Are you able to run errands and shop?  
1. Without any difficulty 2. With a little difficulty 3. With some difficulty 4. With much difficulty 5. Unable to do
[PFA21] Are you able to go up and down stairs at a normal pace?  
1. Without any difficulty 2. With a little difficulty 3. With some difficulty 4. With much difficulty 5. Unable to do PSRPSAT06r1] I am satisfied with my ability to do things for my family.  
1. Not at all 2. A little bit 3. Somewhat 4. Quite a bit 5. Very much
[SRPSAT49r1] I am satisfied with my ability to perform my daily routines.  
1. Not at all 2. A little bit 3. Somewhat 4. Quite a bit 5. Very much
[SRPSAT09r1] I am satisfied with my ability to do the work that is really important to me (include work at home).  
1. Not at all 2. A little bit 3. Somewhat 4. Quite a bit 5. Very much 6.
appendix h patient reported outcome
lee chronic gvhd symptom scale

On a scale of 0 to 4, indicate how much you have been bothered by the following problems in the past month: Skin:
- Abnormal skin color - Rashes
- Thickened skin
- Sores on skin
- Itchy skin Eyes and Mouth:
- Dry eyes
- Need to use eye drops frequently
- Difficulty seeing clearly
- Need to avoid certain foods due to mouth pain - Ulcers in mouth - Receiving nutrition from an intravenous line or feeding tube Breathing:
- Frequent cough
- Colored sputum
- Shortness of breath with exercise
- Shortness of breath at rest
- Need to use oxygen Eating and Digestion:
- Difficulty swallowing solid foods
- Difficult swallowing liquids
- Vomiting
- Weight loss Muscles and Joints:
- Joint and muscle aches
- Limited joint movement
- Muscle cramps - Weak muscles Energy:
- Loss of energy
- Need to sleep more/take naps
- Fevers Mental and Emotional:
- Depression
- Anxiety
- Difficulty sleeping
appendix h patient reported outcome
appendix i references

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12Nash RA, Antin JH, Karanes C, Fay JW, Avalos BR, Yeager AM, et al. Phase 3 study comparing methotrexate and tacrolimus with methotrexate and cyclosporine for prophylaxis of acute graftversus-host disease after marrow transplantation from unrelated donors. Blood. 2000 Sep 15;96(6):2062-8.  
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14Luznik L, Fuchs EJ, Chen AR et al. Post-transplantation high-dose cyclophosphamide (Cy) is effective single agent GVHD prophylaxis that permits prompt immune reconstitution after myeloablative HLA matched related and unrelated bone marrow transplantation (BMT). Biology of Blood and Marrow Transplantation. 2007;13:4.  
15Luznik L, O'Donnell PV, Symons HJ et al. HLA-haploidentical bone marrow transplantation for hematologic malignancies using nonmyeloablative conditioning and high-dose, posttransplantation cyclophosphamide. Biol Blood Marrow Transplant. 2008;14:641-650.  
16Raj, K., Streetly, M., Kazmi, M., Harringon, E., Getzendaner, L. C., Lee, S. J., and O'Donnell, P.  
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17Bolaños-Meade J, Fuchs EJ, Luznik L et al. HLA-haploidentical bone marrow transplantation with posttransplant cyclophosphamide expands the donor pool for patients with sickle cell disease. Blood. 2012;120:4285-4291.  
18Luznik L, Fuchs EJ. High-dose, post-transplantation cyclophosphamide to promote graft-host tolerance after allogeneic hematopoietic stem cell transplantation. Immunol Res. 2010;47:65-77.  
19Ciurea SO, Mulanovich V, Saliba RM et al. Improved early outcomes using a T cell replete graft compared with T cell depleted haploidentical hematopoietic stem cell transplantation. Biol Blood Marrow Transplant. 2012;18:1835-1844.  
20Bashay A, Zhang MJ, McCurdy SR, St. Martin A, Argall T, Anasetti C, et al. Mobilized Peripheral Blood Stem Cells versus Unstimulated Bone Marrow as a Graft Source for T-Cell Replete Haploididentical Donor Transplantation Using Post-Transplant Cyclophosphamide. J Clin Oncol. 2017;35(26):3002-9.  
21Mielcarek M, Furlong T, O'Donnell PV, Storer BE, McCune JS, Storb R, et al.  
Postransplantation cyclophosphamide for prevention of graft-versus-host disease after HLAmatched mobilized blood cell transplantation. Blood. 2016;127(11):1502-8.  
22Harris AC, Young R, Decine S, et al. International, Multicenter Standardization of Acute Graftversus-Host Disease Clinical Data Collection: A Report from the Mount Sinai Acute GVHD
International Consortium. Biology of Blood and Marrow Transplantation 2016;22:4-10.  
23Jagasia MH, Hildegard TG, Arora M, et al. National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graft-versus-Host Disease: I. The 2014 Diagnosis and Staging Working Group Report. Biology of Blood and Marrow Transplantation; Journal of the American Society for Blood and Marrow Transplantation 2015;21:389-401.  
24Armand P, Haesook TK, Logan B, et al. Validation and refinement of the Disease Risk Index for allogeneic stem cell transplantation. Blood 2014;123(23):3664-3671 25Kittelson, J.M. and Emerson, S.S., 1999. A unifying family of group sequential test designs. Biometrics, 55(3), pp.874-882.26Belkaid Y and Hand TW. Role of the microbiota in immunity and inflammation. Cell 2014;157(1):121-41.  
27Andermann TM, Peled JU, Ho C, et al. The microbiome and hematopoietic cell transplantation:
past, present, and future. Biol Blood Marrow Transplant 2018;24(7):1322-40.  
28Holler E, Butzhammer P, Schmid K, et al. Metagenomic analysis of the stool microbiome in patients receiving allogeneic stem cell transplantation: loss of diversity is associated with use of systemic antibiotics and more pronounced in gastrointestinal graft-versus-host disease. Biol Blood Marrow Transplant 2014;20(5):640-5.  
29Weber D, Jenq RR, Peled JU, et al. Microbiota disruption induced by early use of broad spectrum antibiotics is an independent risk factor of outcome after allogeneic stem cell transplantation. Biol Blood Marrow Transplant 2017;23(5):845-852.  
30Jenq RR, Taur Y, Devlin SM, et al. Intestinal blautia is associated with reduced death from graft-versus-host disease. Biol Blood Marrow Transplant 2015;21(8):1373-83.  
31Shono Y, Docampo MD, Peled JU, et al. Intestinal microbiota-related effects on graft-versushost disease. Int J Hematol 2015;101(5):428-37.  
32Peled JU, Devlin SM, Staffas A, et al. Intestinal microbiota and relapse after hematopoietic-cell transplantation. J Clin Oncol 2017;35(15):1650-59.  
33Kashyap PC, Chia N, Nelson H, et al. Microbiome at the Frontier of Personalized Medicine.  
Mayo Clin Proc 2017;92(12):1855-64.  
34Drekonja D, Reich J, Gezahegn S, et al. Fecal microbiota transplantation for clostridium difficile infection: a systematic review. Ann Intern Med 2015;162(9):630-8.  
35Kostic AD, Xavier RJ, and Gevers D. The microbiome in inflammatory bowel disease: current status and the future ahead. Gastroenterology 2014;146(6):1489-99.  
36Wilkinson EM, Ilhan ZE, and Herbst-Kralovetz MM. Microbiota-drug interactions: impact on metabolism and efficacy of therapeutics. Maturitas 2018;112:53-63.  
37Kroemer G and Zitvogel L. Cancer immunotherapy in 2017: the breakthrough of the microbiota. Nat Rev Immunol 2018;18(2):87-88.  
38Ogonek J, Kralj Juric M, Ghimire S, et al. Immune reconstitution after allogeneic hematopoietic stem cell transplantation. Front Immunol 2016;7:507.  
39Suessmuth Y, Mukherjee R, Watkins B, et al. CMV reactivation drives posttransplant T-cell reconstitution and results in defects in the underlying TCRbeta repertoire. Blood 2015;125:383550.  
40van Heijst JW, Ceberio I, Lipuma LB, et al. Quantitative assessment of T cell repertoire recovery after hematopoietic stem cell transplantation. Nature medicine 2013;19:372-7.  
41Perales MA, Goldberg JD, Yuan J, et al. Recombinant human interleukin-7 (CYT107) promotes T-cell recovery after allogeneic stem cell transplantation. Blood 2012;120:4882-91.  
42Meyer EH, Hsu AR, Liliental J, et al. A distinct evolution of the T-cell repertoire categorizes treatment refractory gastrointestinal acute graft-versus-host disease. Blood 2013;121:4955-62.  
43Emerson R, Sherwood A, Desmarais C, Malhotra S, Phippard D, Robins H. Estimating the ratio of CD4+ to CD8+ T cells using high-throughput sequence data. Journal of immunological methods 2013;391:14-21.  
44Wu D, Sherwood A, Fromm JR, et al. High-throughput sequencing detects minimal residual disease in acute T lymphoblastic leukemia. Science translational medicine 2012;4:134ra63.  
45Wu D, Emerson RO, Sherwood A, et al. Detection of minimal residual disease in B
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46Rawstron AC, Fazi C, Agathangelidis A, et al. A complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study. Leukemia 2016;30:92936.  
47Honda K and Littman DR. The microbiota in adaptive immune homeostasis and disease.  
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48Gopalakrishnan V, Spencer CN, Nezi L, et al. Gut microbiome modulates response to anti-PD1 immunotherapy in melanoma patients. Science 2018;359(6371):97-103.  
49Routy B, Le Chatelier E, Derosa L, et al. Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors. Science 2018;359(6371):91-97.  
50Moissev I, Pirogova O, Alyanski A, et al. Graft-versus-Host Disease Prophylaxis in Unrelated Peripheral Blood Stem cell Transplantation with Post-Transplantation Cyclophosphamide, Tacrolimus, and Mycophenolate Mofetil. BBMT 2016;22(6):1037-1042.  
APPENDIX J
Mi-Immune Study Microbiome and Immune Reconstitution in Cellular  
  
Therapies and Hematopoietic Stem Cell Transplantation
(Mi-Immune)
BMT CTN Protocol 1801 Principal Investigators Ami Bhatt, MD, PhD1 Leslie S. Kean MD, PhD18 Miguel-Angel Perales, MD3 John Levine, MD10 Brent Logan, PhD4 Hemant Murthy, MD12 Armin Rashidi, MD, PhD13 Marcie Riches, MD, MS14 Wael Saber, MD, MS4 Karamjeet Sandhu, MD15 Ashley Spahn16 Anthony Sung, MD17  
| Merav Bar, MD2 Brandi Bratrude, PNP, CCRP18 Saurabh Chhabra, MD4 Sung Choi, MD5 William Clark, MD, MS6 Suman R. Das, PhD7 Mary Horowitz, MD, MS4 Rob Jenq, MD8 Rick Jones, MD9   |
|----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
Sponsored by the National Institutes of Health National Heart, Lung, and Blood Institute National Cancer Institute 1Stanford University 2Fred Hutchinson Cancer Research Center 3Memorial Sloan Kettering Cancer Center 4Medical College of Wisconsin 5University of Michigan 6Virginia Commonwealth University 7Vanderbilt University 8MD Anderson Cancer Center 9Johns Hopkins University 10Mount Sinai 11Emmes 12University of Florida  
| 13University of Minnesota 14University of North Carolina 15City of Hope 16National Marrow Donor Program 17Duke University 18Boston Children's Hospital   |
|----------------------------------------------------------------------------------------------------------------------------------------------------------|
chapter 1
1 background and significance

Allogeneic HCT is an effective treatment for patients with malignant and non-malignant hematologic diseases. However, this treatment is complicated by high rates of morbidity and mortality limiting its broader application. The leading causes of post-transplant morbidity and mortality include acute and chronic GVHD, relapse and infectious disease. The goal of the trial is to apply a systems biology approach to understanding the mechanisms driving these complications, such that evidence-based treatment strategies can be devised. Results of this study may also inform the development of accurate and predictive surrogate markers of clinically relevant endpoints that may be informative for future BMT clinical trial design. The sample repository and molecular and clinical databases established through the trial will be made available to the biomedical community and are expected to facilitate studies that will establish the utility of molecular biomarkers for risk assessment, diagnosis and monitoring, and allow more rational treatment strategies to be developed for patients undergoing allogeneic HCT. These studies are also likely to provide mechanistic insights and to identify new therapeutic targets leading to development of more targeted and effective therapies. The goal of this protocol is several-fold: First, it is to test the primary hypothesis that the engraftment stool microbiome diversity (determined by 16s rRNA sequencing analysis of the sample closest after, but within 14 days of neutrophil engraftment) predicts 1-year non-relapse mortality in patients undergoing reduced-intensity allogeneic HCT. Second, it is to perform additional analyses on patient samples to answer key questions concerning: a) the impact of the stool microbiome on transplant outcome as well as the impact of transplant on the microbiome and the associated downstream impact on patient health; and b) reconstitution of the T cell repertoire after transplant. Third, it is to establish a cohort of biologic samples collected prospectively from patients treated on BMT CTN 1703 that will be a shared biospecimen resource for conducting future allogeneic HCT correlative studies. The resulting dataset will become an additional resource generated by the trial and will be made available to the biomedical community. MiImmune is designed to link molecular data and biospecimens with high quality clinical phenotype and outcomes data to identify risk factors for development and severity of key complications after allogeneic HCT. To achieve this goal, patients and donors will be recruited and consent will be obtained at the time that they enroll on BMT CTN protocol 1703. Samples will be collected: (1) from patients and donors pre-transplant; and, (2) from patients post-transplant on a calendar-driven schedule through the first year post-HCT. Shared clinical data will be collected in the context of the primary transplant protocol, BMT CTN 1703, and Mi-Immune-specific data will be collected using Case Report Forms (CRFs). Additional clinical data will be available from data submitted to the Center for International Blood and Marrow Transplant Research (CIBMTR) using the CIBMTR CRFs.
chapter 1
chapter 2 2 study design

In this study, we will collect stool, urine, and blood biospecimens, as well as detailed clinical data on infections and antibiotic exposures in an attempt to understand the microbial and immune recovery predictors of HCT outcomes. This study will form the correlative science arm of the BMT CTN 1703 study (PROGRESS III), which will compare two acute graft-versus-host disease (aGVHD) prophylaxis regimens: tacrolimus/methotrexate (Tac/MTX) versus post-transplant cyclophosphamide/tacrolimus/mycophenolate mofetil (PTCy/Tac/MMF) in the setting of reduced intensity conditioning (RIC) allogeneic peripheral blood stem cell (PBSC) transplantation.
chapter 1
2.2 hypothesis and objectives 2.2.1

The engraftment stool microbiome diversity (determined within 14 days after neutrophil engraftment) predicts 1-year non-relapse mortality.
chapter 1
2.2.2 objectives primary objective

The goal of this protocol is to test the primary hypothesis that the engraftment stool microbiome diversity (determined by 16s rRNA sequencing analysis of the sample closest after, but within 14 days of neutrophil engraftment) predicts 1-year non-relapse mortality in patients undergoing reduced-intensity allogeneic HCT.
chapter 1
secondary objectives microbiome infection and

1) Association of engraftment microbiome diversity with development of aGVHD, cGVHD,
and overall survival (corrected for GVHD prophylaxis).  
2) Association of baseline microbiome diversity with development of aGVHD, cGVHD, and overall survival (corrected for GVHD prophylaxis).  
3) Effect of GVHD prophylaxis on microbiome diversity: Difference in the level of microbiome diversity detected at 6, 12, and 24 months in the 2 arms.
4) Association of microbiome oligodomination at the time of engraftment (>30% of microbiome being a single operational taxonomic unit) with increased subsequent risk of bloodstream infection.  
5) Association of volume of antimicrobial exposure (number and duration of drugs) in first 30 days after transplant with rates of aGVHD, cGVHD, and overall survival.  
6) Association between urine metabolites (such as indoxyl sulfate) at the time of engraftment and rate of acute GVHD.
chapter 1
immune repertoire

1) Effect of GVHD prophylaxis on TCR diversity: Difference in the level of TCR diversity detected at 3, 6, 12 and 24 months in the 2 arms.  
2) Effect of TCR diversity at 3 months, 6 months, 12 months and 24 months on subsequent non-relapse mortality, overall survival, and relapse, using landmark analyses and timedependent covariates.
chapter 1
biorepository

1) To establish a cohort of biologic samples and a linked clinical and molecular dataset from patients treated on BMT CTN 1703. The product of this study will be a shared biospecimen and data resource for conducting future allogeneic HCT mechanistic studies. Examples of future exploratory objectives that can be addressed with this biorepository are the following:
chapter 1
exploratory objectives

1) Effect of antibiotic use (length of exposure, anaerobic sparing vs. not) on level of microbiome diversity at 6, 12, and 24 months.  
2) Effect of aGVHD and/or its treatment on microbiome at 6, 12 and 24 months. 3) Effect of cGVHD and/or its treatment on microbiome at 12 and 24 months. 4) Effect of CMV and BK reactivation on TCR diversity at 3, 6, 12 and 24 months.
5) Effect of patient age on TCR diversity at 3, 6, 12, and 24 months.  
6) Effect of donor age on TCR diversity at 3, 6, 12, and 24 months.  
7) Effect of aGVHD and/or its treatment on TCR diversity at 3, 6, 12 and 24 months. 8) Effect of cGVHD and/or its treatment on TCR diversity at 12 and 24 months.  
9) Correlation of microbiome diversity and TCR diversity at 3, 6, 12, and 24 months.
chapter 1
2.3 eligibility criteria for enrollment

The primary intention of the Mi-Immune protocol is to co-enroll patients on BMT CTN 1703. Patient inclusion and exclusion criteria are those of BMT CTN 1703. Related and unrelated donors must be at least 18 years of age.
chapter 1
2.4 treatment plan

The conditioning, GVHD prophylaxis, and supportive care are described in BMT CTN 1703.
chapter 3
3 study endpoints and definitions

The primary endpoint is non-relapse mortality within one year. This endpoint is defined according to the BMT CTN 1703 protocol. This primary endpoint will be associated with the engraftment stool microbiome diversity (grouped by tertiles) for the primary analysis of this study.
chapter 3
3.2 secondary endpoints 3.2.1 clinical

Standard transplant endpoints and clinical outcomes, with the exception of infections and antimicrobial use, will be collected according to BMT CTN 1703, including long-term follow-up data collection through CIBMTR. The endpoints collected on BMT CTN 1703 include acute GVHD, chronic GVHD, non-relapse mortality, relapse, death and cause of death. In addition, a feasibility endpoint (i.e., proportion of samples successfully collected on schedule), and exploratory analyses of donor/recipient pairs will be included.
chapter 3
3.2.2 infection

Data on infections and antimicrobial agent administration occurring through Day 365 will be collected specifically for patients with Mi-Immune samples collected. The incidence of definite and probable viral, fungal and bacterial infections will be tabulated. The infections will be reported using the BMT CTN Advantage eClinical data reporting system.
chapter 3
3.2.3 correlative endpoints 3.2.3.1 microbiome

The microbiome will be analyzed in two ways:
First, the microbiome taxonomic diversity will be evaluated using 16S ribosomal RNA amplicon sequencing. Data will be generated per standard protocols, as previously described.5 Resultant sequencing data will be quality filtered, adapter sequences will be trimmed, and sequences will be analyzed using the QIIME pipeline.6 Shannon diversity will be calculated, as previously described.7 Second, urinary indoxyl sulfate has been used as a biomarker of microbiome composition.8 We will measure urinary indoxyl sulfate using liquid chromatography followed by mass spectrometry, as previously described.  
9
chapter 3
3.2.3.2 t cell repertoire

T cell repertoire will be analyzed using next generation sequencing on the Adaptive Biotechnologies ImmunoSEQ platform. PBMCs from patients will be collected at 3, 6, 12 and 24 months post HCT. TCR diversity will be assessed using the inverse Simpson's diversity index
(1/Ds), which sums the frequency of each clonotype1.
chapter 4
4 patient evaluation 4.1 pre-transplant

All pre-transplant evaluations occur as dictated by BMT CTN 1703. The data will be captured in the BMT CTN 1703 forms and not re-entered for Mi-Immune. Additional Pre-transplant requirements for Mi-Immune to be collected no sooner than 7 days prior to the start of conditioning regimen:
1. Specimen Collection: Urine, stool, and blood will be collected pre-conditioning (Day -14 to Day -7) and pre-infusion (Day -1 to Day 0). Please note that stool sample collection at pre-conditioning is MANDATORY and urine sample collection is OPTIONAL.  
2. A CBC will be performed pre-conditioning (Day -14 to Day -7) and pre-infusion (Day -1 to Day 0).  
3. Infection data collection at baseline.
chapter 4
4.2 post-transplant evaluations

1. For all enrolled HCT patients, the empty bag or syringe in which the stem cell product was infused will be retrieved by the study team and sent to the Central Reference Laboratory at ambient temperature where it will be washed to obtain donor cells for analysis.  
2. Toxicities associated with specimen collection for Mi-Immune will be collected and reported on days 28, 56, 98, 180, 270, and 365 post-transplant. All other toxicities will be reported for BMT CTN 1703.  
3. Specimen collection (blood, urine, and stool) for microbiome assays will occur weekly starting on day 0 through day 77 (through day 84 for blood), then at day 98, day 180, day 270, 1 year, and 2 years.  
- Weekly stool sample collection for microbiome assays starting Day 0 weekly through Day 28 is MANDATORY.  
- Weekly stool sample collection for microbiome assays starting day 35 through day 77, then at day 98, 180, 270, 1 year, and 2 years are OPTIONAL.  
- Weekly urine sample collection for microbiome assays starting Pre-conditioning through Day 270, then at 1 year, and 2 years are ALL OPTIONAL.  
4. A CBC will be performed weekly from day 0 through day 77 (through day 84 for blood),
then day 98, 180, 270, 365, And 730.  
5. Infection data: This will occur every 14 days from day 0 until day 98, then on day 180, 270, and 365 post-transplant.  
6. Antimicrobial medications: Specific medications including route of administration, start date, and stop date will be collected every 14 days from day 0 to day 98. Medications prior to start of conditioning and on day 180, 270, and 365 post-transplant will be listed with route of administration for the 7 days prior to the form date.  
| Preinfusion  (Day -1 to  0)                               |                            |           |           |           |     |     |     |     |     |     |     |     |    |    |    |    |    |    |    |
|-------------------------------|----------------------------|-----------|-----------|-----------|-----|-----|-----|-----|-----|-----|-----|-----|----|----|----|----|----|----|----|
| Pre                               |                            |           |           |           |     |     |     |     |     |     |     |     |    |    |    |    |    |    |    |
| conditioning  (Day -14 to -7) | 180  + 28                  | 270  + 28 | 365  + 28 | 730  + 60 |     |     |     |     |     |     |     |     |    |    |    |    |    |    |    |
| 0                             | 7                          | 14        | 21        | 28        | 35  | 42  | 49  | 56  | 63  | 70  | 77  | 84  | 98 |    |    |    |    |    |    |
| + 3                           | + 3                        | + 3       | + 3       | + 3       | + 3 | + 3 | + 3 | + 3 | + 3 | + 3 | + 3 | + 7 |    |    |    |    |    |    |    |
| CBC                           | X                          | X         | X         | X         | X   | X   | X   | X   | X   | X   | X   | X   | X  | X  | X  | X  | X  | X  | X  |
| Infections                    | X                          | X         | X         | X         | X   | X   | X   | X   | X   | X   | X   | X   |    |    |    |    |    |    |    |
| Patient  Data                 | Antimicrobial  medications | X         | X         | X         | X   | X   | X   | X   | X   | X   | X   | X   | X  |    |    |    |    |    |    |
| Toxicities                    | X                          | X         | X         | X         | X   | X   |     |     |     |     |     |     |    |    |    |    |    |    |    |
| Peripheral  Blood             | X***                       | X         | X         | X         | X   | X   | X   | X   | X   | X   | X   | X   | X  | X  | X  | X  | X  | X  | X  |
| Patient  Samples              | Urine1                     | X***      | X         | X         | X   | X   | X   | X   | X   | X   | X   | X   | X  | X  | X  | X  | X  | X  | X  |
| Stool2                        | X***                       | X         | X         | X         | X   | X   | X   | X   | X   | X   | X   | X   | X  | X  | X  | X  | X  | X  |    |
| CBC                           | X*                         |           |           |           |     |     |     |     |     |     |     |     |    |    |    |    |    |    |    |
| Peripheral  Blood             | X*                         |           |           |           |     |     |     |     |     |     |     |     |    |    |    |    |    |    |    |
| Related Donor  Samples        | Urine                      | X*        |           |           |     |     |     |     |     |     |     |     |    |    |    |    |    |    |    |
| Stool                         | X*                         |           |           |           |     |     |     |     |     |     |     |     |    |    |    |    |    |    |    |
| Hematopoietic  Stem Cell      | X**                        |           |           |           |     |     |     |     |     |     |     |     |    |    |    |    |    |    |    |
| Product Cells                 |                            |           |           |           |     |     |     |     |     |     |     |     |    |    |    |    |    |    |    |
| Unrelated  Donor  Samples     | Hematopoietic  Stem Cell   | X**       |           |           |     |     |     |     |     |     |     |     |    |    |    |    |    |    |    |
| Product Cells                 |                            |           |           |           |     |     |     |     |     |     |     |     |    |    |    |    |    |    |    |
| *Pre-donation.                |                            |           |           |           |     |     |     |     |     |     |     |     |    |    |    |    |    |    |    |
table 4.2 patient and donor
4.2 post-transplant evaluations

*Pre-donation.  
**Empty bag or syringe in which the stem cell product was infused. ***Samples should be collected Pre-conditioning, pre-antibiotic prophylaxis on Day -14 to Day -7.  
1 Weekly urine sample collection for microbiome assays starting Pre-conditioning through Day 270, then at 1 year, and 2 years are ALL OPTIONAL
2 Weekly stool sample collection for microbiome assays at Pre-conditioning, Pre-infusion/Day 0 weekly through Day 28 is MANDATORY. Weekly stool sample collection for microbiome assays starting day 35 through day 77, then at day 98, 180, 270, 1 year, and 2 years are OPTIONAL. *1703* Version 4.0 dated November 16, *2020*
table 4.2 patient and donor
4.3 collection of research samples

Blood, stool, urine and biopsy research samples will be collected in this study. The majority of study samples will be collected on a calendar driven schedule. Based on historical experience with BMT CTN 1202, we anticipate high compliance (1202 calendar-driven samples were collected at a rate of 96% completeness).
table 4.2 patient and donor
4.3.1 study blood draws

For transplant patients, the blood sample collections will occur at baseline (defined as preconditioning, pre-antibiotic prophylaxis) at day -7 to day -14 prior to start of conditioning. Samples will then be collected weekly from day 0 (pre-infusion) through day 84 (+/- 3 days), then on day +98 +/- 7 days, day +180 +/- 28 days, day +270 +/- 28 days, day +365 +/- 28 days, day +730 +/- 60 days as outlined in Table 4.2. Related donor samples will be drawn prior to the peripheral blood stem cell collection.
table 4.2 patient and donor
4.3.2 stool samples

Stool samples will be collected according to standard operating procedures modified from the NIH
Human Microbiome Project protocols. Briefly, samples will be stored at 4oC within 30 minutes of collection and then will be shipped to NMDP within 24-48 hours. At NMDP, the samples will be aliquoted and stored at -80oC. Samples will be collected with and without preservative solutions to enable downstream DNA, RNA and mass spectrometry (metabolite and metaproteome) analysis. Selected centers with substantial microbiome sampling experience will collect and process samples for additional downstream transcriptome and volatile metabolite sampling, which requires rapid processing. For transplant patients, the stool sample collections will occur at baseline (defined as pre-conditioning, pre-antibiotic prophylaxis) at day -7 to day -14 prior to start of conditioning. Samples will then be collected weekly from day 0 through day 77 (+/- 3 days), then on day +98 +/- 7 days, day +180 +/- 28 days, day +270 +/- 28 days, day +365 +/- 28 days, day +730 +/- 60 days as outlined in Table 4.2.1 (see Laboratory Procedures Table for inpatient and outpatient stool collection schedule). 5-10ml of stool will be obtained at each timepoint and placed into a sterile container. Stool sample will then be placed on ice (4oC) and transferred to the lab.
table 4.2 patient and donor
4.3.3 urine samples

Urine metabolites such as indoxyl sulfate are associated with GVHD and specific intestinal microbiome compositions. Urine collections will be performed according to the standard operating procedures outlined in the Laboratory Procedures Table. For transplant patients, the urine sample collections will occur at baseline (defined as pre-conditioning, pre-antibiotic prophylaxis) at day - 7 to day -14 prior to start of conditioning. Samples will then be collected weekly from day 0 through day 77 (+/- 3 days), then on day +98 +/- 7 days, day +180 +/- 28 days, day +270 +/- 28 days, day +365 +/- 28 days, day +730 +/- 60 days as outlined in Table 4.2. Samples will be placed at 4oC within 30 minutes of collection and for no more than 48 hours prior to aliquoting and deep freeze storage.
chapter 5
5 statistical considerations 5.1 study

This study is designed as a companion study to the BMT CTN 1703 Phase III randomized, open label, multicenter trial to compare PTCy/Tac/MMF versus Tac/MTX for GVHD prophylaxis in patients with controlled malignant disease receiving an allogeneic PBSC transplant after a RIC regimen. Patients enrolled on this study will be asked to provide additional study samples which will be linked with the clinical data collected on the parent protocol, in order to address specific research questions associated with those study samples and to provide banked samples for future studies.
chapter 5
5.1.1 accrual

Accrual will occur in conjunction with the BMT CTN 1703 trial. The target enrollment of the parent trial is 428 patients; we plan to enroll approximately 70%, or n=300 patients, on this companion protocol. It is estimated that 36 months of accrual will be necessary to enroll the targeted sample size. The study will remain open until the last recipient enrolled has been followed for two years post-transplant.
chapter 5
5.1.2 randomization

Patients on the BMT CTN 1703 trial will be randomized at a ratio of 1:1 between the treatment arms; no further randomization will be done on this companion protocol. 5.1.3 Primary Endpoint The primary endpoint for this study is non-relapse mortality within 1 year of transplant. 5.1.4 Primary Objective The primary objective of Mi-Immune is to compare NRM within 1 year between groups defined by engraftment stool microbiome diversity. Tertiles will be used to define 3 approximately equal sized groups.
chapter 5
5.1.5 sample size and power

The primary analysis will be done using a Fine and Gray model comparing the non-relapse mortality within 1 year between the three groups. Non-relapse mortality by 1 year is expected to be approximately 15% overall based on BMT CTN 1203 trial data. Using the method of Latouche (2004), we computed power to detect differences of 20% in the 1 year NRM between any two pairs of groups, using a Bonferroni adjusted type I error of 0.05/3 to account for pairwise comparisons2. The table below shows various comparisons of this difference in NRM over an expected range of NRM for this protocol; the targeted sample size of 300 evaluable patients would have at least 85% power to detect a 20% difference in 1 year NRM between any two microbiome diversity groups. Table: Power to detect 20% differences in 1 year NRM between any two microbiome diversity groups:  
| NRM difference   | Power   |
|------------------|---------|
| 5% vs. 25%       | 99%     |
| 10% vs. 30%      | 93%     |
| 15% vs. 35%      | 85%     |
chapter 5
5.2 interim analysis and pausing

As this is a non-interventional correlative study, no interim analyses will be included specifically as part of this protocol, separate from those used in BMT CTN 1703.
chapter 5
5.3 demographic and baseline characteristics

Demographics and baseline characteristics will be described for all patients and separately for each cohort. Characteristics to be examined are: age, gender, race/ethnicity, performance status, primary disease, disease-specific risk categories, DRI, hematopoietic cell transplant comorbidity index (HCT CI), donor type and HLA matching, donor/recipient CMV status, donor/recipient sex match, donor/recipient ABO match, and conditioning regimen. Between group comparisons will be performed for continuous variables via a Kruskal-Wallis test and for categorical variables, via the chi-square test. Additionally, we will compare the distributions of these characteristics between patients with Mi-Immune samples collected vs. those enrolled on BMT CTN 1703 but who did not provide Mi-Immune samples, to assess whether the Mi-Immune cohort appears representative of the larger trial.
chapter 5
5.4 analysis populations 5.4.1 engraftment

All enrolled patients with an available engraftment stool microbiome diversity measure will be included in this population. The primary analysis as well as secondary analyses focusing on the impact of the engraftment stool microbiome diversity on clinical outcomes will use this population. Outcomes are determined from the time of the engraftment stool sample collection.
chapter 5
5.4.2 baseline microbiome population

All enrolled patients with an available baseline stool microbiome diversity measure will be included in this population. This population will be used for secondary analyses looking at the impact of baseline microbiome diversity on subsequent clinical outcomes. Outcomes will be determined from the time of transplant.
chapter 5
5.4.3 landmark microbiome populations

Various landmark microbiome populations will be used to examine the impact of microbiome diversity measures at that timepoint on subsequent clinical outcomes, or to compare microbiome diversity measures at that timepoint between groups such as randomized GVHD prophylaxis strategy. In each case, patients will be included if they are still at risk for the clinical outcome at the landmark time, and have an available microbiome diversity measure at that timepoint. Clinical outcomes will be assessed starting at that landmark timepoint.
chapter 5
5.4.4 antimicrobial exposure population

All enrolled patients with complete data collection on antimicrobial exposure within the first 30 days and who are still at risk for the clinical outcome at day 30 will be included in this landmark antimicrobial exposure cohort. The antimicrobial exposure population will be used to examine the impact of antimicrobial exposure within 30 days on subsequent clinical outcomes. Clinical outcomes will be assessed starting at 30 days.
chapter 5
5.4.5 landmark tcr diversity populations.

Various landmark TCR diversity populations will be used to examine the impact of TCR diversity measures at that timepoint on subsequent clinical outcomes, or to compare TCR diversity measures at that timepoint between groups such as randomized GVHD prophylaxis strategy. In each case, patients will be included if they are still at risk for the clinical outcome at the landmark time, and have an available TCR diversity measure at that timepoint. Clinical outcomes will be assessed starting at that landmark timepoint.
chapter 5
5.5 analysis plan

5.5.1 Analysis of the Primary Objective: Compare NRM within 1 year between groups defined by engraftment stool microbiome diversity.  
This analysis will use the engraftment stool microbiome cohort. Microbiome diversity will be separated into three groups based on tertiles for the primary analysis. Non-relapse mortality will be summarized for each group using cumulative incidence with relapse as a competing event and compared in a univariate manner between tertiles using Gray's test. A Fine and Gray model will be used to compare microbiome diversity groups after adjustment for baseline covariates; microbiome diversity as well as GVHD prophylaxis received will be forced into the model and stepwise selection will be used to identify additional variables associated with outcome. Interactions between GVHD prophylaxis received and microbiome diversity will be assessed. A secondary analysis will further examine the functional form of the relationship between quantitative values of microbiome diversity and the subdistribution hazard for NRM using splines, and if appropriate, an optimal cutpoint will be determined.
chapter 5
5.5.2 analysis of the secondary

1) Association of engraftment microbiome diversity with development of aGVHD, cGVHD,
and overall survival (corrected for GVHD prophylaxis). This analysis will use the engraftment stool microbiome cohort; diversity will be grouped by tertiles or if a more appropriate classification is identified in the primary analysis, that may be used instead. aGVHD (grade 2-4 or 3-4) and chronic GVHD (any or moderatesevere) will be described in each group using cumulative incidence with death or relapse as a competing event, and compared between groups using Gray's test. Overall survival will be described using the Kaplan-Meier estimator and compared between groups using the log-rank test. Multivariate models will be constructed in a similar manner as for the primary endpoint using Fine and Gray models for aGVHD and cGVHD, and using Cox models for OS.  
2) Association of baseline microbiome diversity with development of aGVHD, cGVHD, and overall survival (corrected for GVHD prophylaxis).
This analysis will use the baseline stool microbiome cohort; diversity at baseline will be separated into three groups based on tertiles for initial analysis. aGVHD (grade 2-4 or 3-4) and chronic GVHD (any or moderate-severe) will be described in each group using cumulative incidence with death or relapse as a competing event, and compared between groups using Gray's test. Overall survival will be described using the Kaplan-Meier estimator and compared between groups using the log-rank test. Multivariate models will be constructed in a similar manner as for the primary endpoint using Fine and Gray models for aGVHD and cGVHD, and using Cox models for OS. We will also consider further examination of the functional form of the relationship between quantitative values of baseline microbiome diversity and the risk of aGVHD, cGVHD, or death using splines, and if appropriate, an optimal cutpoint will be determined.  
3) Effect of GVHD prophylaxis on microbiome diversity: Difference in the level of microbiome diversity detected at 6, 12, and 24 months in the 2 arms.
This analysis will use a series of landmark microbiome populations at each time point. Microbiome diversity will be compared between GVHD prophylaxis groups at each time point using Mann-Whitney tests; randomized GVHD prophylaxis groups will be used rather than GVHD prophylaxis received to maintain the ITT principle, however a secondary analysis using the GVHD prophylaxis received may also be performed. Transformation of diversity will be considered to induce normality. If a normalizing transformation can be identified, analysis of covariance will be used to compare diversity at each time point, after adjustment for baseline diversity and other covariates; stepwise selection will be used to identify additional covariates associated with diversity.  
4) Association of microbiome oligodomination at the time of engraftment (>30% of microbiome being a single operational taxonomic unit) with increased subsequent risk of bloodstream infection.
This analysis will use the engraftment stool microbiome population. The number of bloodstream infections and the number of patients experiencing a bloodstream infection between engraftment and day 100 will be described by presence of microbiome oligodomination group. Prevalence of active bloodstream infection will be described at subsequent visit times by microbiome oligodomination group, and compared using the chisquare test. Additional details on organisms will also be summarized. Bloodstream infection density in the first 100 days will be computed as infection frequencies over 100 patient days at risk. If there are sufficient numbers of bloodstream infection events in the first 100 days, multivariate regression analyses will be performed to examine the association between microbiome oligodomination and infection density, while adjusting for other relevant covariates. Proportional rates/means models will be used to model the infection density, with a robust variance estimator to account for within patient correlation; microbiome oligodomination and GVHD prophylaxis received will be forced into the model, and additional variables will be added using stepwise regression3. Interaction between microbiome oligodomination and GVHD prophylaxis received will be assessed.  
5) Association of volume of antimicrobial exposure (number and duration of drugs) in first 30 days after transplant with rates of aGVHD, cGVHD, and overall survival.
This analysis will use the antimicrobial exposure population. Volume of antimicrobial exposure will be computed by measuring the "daily defined dose" exposure for each individual. This value is calculated on a per antimicrobial basis. Each day that a patient is given a specific antimicrobial agent counts as one daily defined dose. Thus, if a patient receives two different antimicrobials for one day each, the patient has received 2 daily defined doses. Alternatively, if a patient receives one antimicrobial for three days, the patient has received 3 daily defined doses. For combination drugs, such as trimethoprim/sulfamethoxazole, the daily defined doses will be calculated individually for each of the two active antimicrobial components of the combination therapy4. Volume of antimicrobial exposure will be dichotomized at the median. aGVHD (grade 2-4 or 3-4) and chronic GVHD (any or moderate-severe) will be described in each group using cumulative incidence with death or relapse as a competing event, and compared between groups using Gray's test. Overall survival will be described using the Kaplan-Meier estimator and compared between groups using the log-rank test. Multivariate models will be constructed in a similar manner as for the primary endpoint using Fine and Gray models for aGVHD and cGVHD, and using Cox models for OS.  
6) Association between urine metabolites (such as indoxyl sulfate) at the time of engraftment and rate of acute GVHD.
This analysis will use the engraftment stool microbiome cohort; urine metabolites at the time of engraftment will be dichotomized at the median. aGVHD (grade 2-4 or 3-4) will be described in each group using cumulative incidence with death or relapse as a competing event, and compared between groups using Gray's test. Multivariate models will be constructed in a similar manner as for the primary endpoint using Fine and Gray models for aGVHD.
chapter 5
immune repertoire

1) Effect of GVHD prophylaxis on TCR diversity: Difference in the level of TCR diversity detected at 3, 6, 12 and 24 months in the 2 arms. This analysis will use a series of landmark TCR diversity populations at each time point. TCR diversity will be compared between GVHD prophylaxis groups at each time point using Mann-Whitney tests; randomized GVHD prophylaxis groups will be used rather than GVHD prophylaxis received to maintain the ITT principle, however a secondary analysis using the GVHD prophylaxis received may also be performed. Transformation of diversity will be considered to induce normality. If a normalizing transformation can be identified, analysis of covariance will be used to compare diversity at each time point, after adjustment for baseline diversity and other covariates; stepwise selection will be used to identify additional covariates associated with diversity.  
2) Effect of TCR diversity at 3 months, 6 months, 12 months and 24 months on subsequent non-relapse mortality, overall survival, and relapse, using landmark analyses and timedependent covariates
This analysis will use a landmark TCR diversity population at each time point. TCR diversity will be dichotomized as above or below the median at each corresponding landmark time point, and cumulative incidence of non-relapse mortality and relapse will be estimated in each group, with relapse and death in remission respectively as competing risks. Survival will be estimated in each group using Kaplan-Meier method. We will examine the impact of TCR diversity over time as a time-dependent covariate in Cox models for the cause specific hazard for NRM or relapse, or for the hazard of death; GVHD prophylaxis received will be forced into the models, and other covariates will be considered using stepwise variable selection. We will also explore the functional form of the relationship between TCR diversity and outcome using splines and if appropriate will identify an optimal cutpoint.  
*1703* Version 4.0 dated November 16, *2020*  
| Shipping                                                             |                                           |                          |                         |                      |                                                  |    |
|----------------------------------------------------------------------|-------------------------------------------|--------------------------|-------------------------|----------------------|--------------------------------------------------|----|
| Temperature  to BMT CTN  Biorepository                               | Sample Collection  or Shipping  Container |                          |                         |                      |                                                  |    |
| Study Subject                                                        | Research                                  |                          |                         |                      |                                                  |    |
| Sample Type                                                          | Time Points                               | Sample  Quantity         | Processed Research      | Research Sample      | Storage                                          |    |
| Sample Material                                                      | Material Aliquots                         | Temperature              |                         |                      |                                                  |    |
| 5 mL Clot Tube                                                       | Serum                                     | 5 - 0.5 mL aliquots      |                         |                      |                                                  |    |
| 2 mL EDTA Tube                                                       | Whole Blood                               | 2 - 1.0 mL aliquots      | -80⁰ C                  |                      |                                                  |    |
| Pre                                                                      |                                           |                          |                         |                      |                                                  |    |
| conditioning,  Day 14, 28, 98,  180, 365, 730                        | 39 mL                                     | Insulated  Ambient       | Heparin Plasma          | 15 - 0.5 mL aliquots |                                                  |    |
| 32 mL in Sodium  Heparin Tubes                                       | ~3 - 5x106 / 1 mL  aliqouts               | LN                       |                         |                      |                                                  |    |
| PBMC                                                                 |                                           |                          |                         |                      |                                                  |    |
| 5 mL Clot Tube                                                       | Serum                                     | 5 - 0.5 mL aliquots      |                         |                      |                                                  |    |
| 2 mL EDTA Tube                                                       | Whole Blood                               | 2 - 1.0 mL aliquots      | -80⁰ C                  |                      |                                                  |    |
| Peripheral  Blood                                                    | Day 7, 21, 35,                            | 31 mL                    | Insulated               |                      |                                                  |    |
| 42, 49, 56, 63,                                                      | Ambient                                   |                          |                         |                      |                                                  |    |
| 70, 77, 84, 270                                                      | Heparin Plasma                            | 15 - 0.5 mL aliquots     |                         |                      |                                                  |    |
| 24 mL in Sodium  Heparin Tubes                                       | ~2 - 5x106 / 1 mL  aliqouts               | LN                       |                         |                      |                                                  |    |
| PBMC                                                                 |                                           |                          |                         |                      |                                                  |    |
| 5 mL Clot Tube                                                       | Serum                                     | 5 - 0.5 mL aliquots      | -80⁰ C                  |                      |                                                  |    |
| 24 mL in Sodium  Heparin Tubes                                       |                                           |                          |                         |                      |                                                  |    |
| Patient (N = 300)                                                    | Pre-infusion                              | 24 mL                    | Insulated  Ambient      | Heparin Plasma       | 15 - 0.5 mL aliquots ~2 - 5x106 / 1 mL  aliqouts | LN |
| PBMC                                                                 |                                           |                          |                         |                      |                                                  |    |
| Pre                                                                      |                                           |                          |                         |                      |                                                  |    |
| conditioning,  Weekly Day 0  to Day 77, Day  98, 180, 270,  365, 730 |                                           |                          |                         |                      |                                                  |    |
| Urine                                                                | 10-12 mL                                  | 2-8⁰ C                   | Urine - No Preservative | 10 - 1.0 mL aliquots | -80⁰ C                                           |    |
| Sterile Specimen  Container                                          |                                           |                          |                         |                      |                                                  |    |
| Pre                                                                      |                                           |                          |                         |                      |                                                  |    |
| conditioning,  Weekly Day 0  to Day 77, Day  98, 180, 270,  365, 730 | Stool - No Preservative                   | 4 - 1 to 1.5 mL aliquots |                         |                      |                                                  |    |
| Sterile Specimen                                                     |                                           |                          |                         |                      |                                                  |    |
| 10-12 mL                                                             | 2-8⁰ C                                    | Container                |                         |                      |                                                  |    |
| Stool                                                                | -80⁰ C                                    |                          |                         |                      |                                                  |    |
| Stool in RNAlater                                                    | 2 - 1.5 mL processed  aliquots            |                          |                         |                      |                                                  |    |  
*1703* Version 4.0 dated November 16, *2020*  
| Shipping                               |                                                                     |                               |                    |                |                      |                         |                      |        |
|----------------------------------------|---------------------------------------------------------------------|-------------------------------|--------------------|----------------|----------------------|-------------------------|----------------------|--------|
| Temperature  to BMT CTN  Biorepository | Shipping                                                            |                               |                    |                |                      |                         |                      |        |
| Temperature  to Leslie  Kean Lab       | Sample                                                              |                               |                    |                |                      |                         |                      |        |
| Collection or  Shipping  Container     |                                                                     |                               |                    |                |                      |                         |                      |        |
| Study                                  | Sample Type                                                         | Time Points                   | Sample             |                |                      |                         |                      |        |
| Research                               |                                                                     |                               |                    |                |                      |                         |                      |        |
| Subject                                | Quantity                                                            | Processed Research            | Research Sample    | Storage        |                      |                         |                      |        |
| Sample Material                        | Material Aliquots                                                   | Temperature                   |                    |                |                      |                         |                      |        |
| 5 mL Clot Tube                         | Serum                                                               | 5 - 0.5 mL aliquots           |                    |                |                      |                         |                      |        |
| 4 mL EDTA Tube                         | Whole Blood                                                         | 4 - 1.0 mL aliquots           | -80⁰ C             |                |                      |                         |                      |        |
| Peripheral  Blood                      | Pre-donation                                                        | 41 mL                         | Insulated  Ambient | Heparin Plasma | 15 - 0.5 mL aliquots |                         |                      |        |
| 32 mL Sodium  Heparin Tubes            | ~3 - 5x106 / 1 mL  aliqouts                                         | LN                            |                    |                |                      |                         |                      |        |
| PBMC                                   |                                                                     |                               |                    |                |                      |                         |                      |        |
| Related  Donor (N= ~120)               | Urine                                                               | Pre-donation                  | 10-12 mL           | 2-8⁰ C         | Sterile Specimen     | Urine - No Preservative | 10 - 1.0 mL aliquots | -80⁰ C |
| Container                              | Stool - No Preservative                                             | 4 - 1 to 1.5 mL  aliquots     |                    |                |                      |                         |                      |        |
| Stool in RNAlater                      | 2 - 1.5 mL processed                                                | -80⁰ C                        |                    |                |                      |                         |                      |        |
| aliquots                               |                                                                     |                               |                    |                |                      |                         |                      |        |
| Sterile Specimen                       |                                                                     |                               |                    |                |                      |                         |                      |        |
| Stool                                  | Pre-donation                                                        | 10-20 mL                      | 2-8⁰ C             | Container      |                      |                         |                      |        |
| All                                    |                                                                     |                               |                    |                |                      |                         |                      |        |
| Donors  (Will need  protocolspecific  donor  consent)                                        | Empty bag or  syringe in which  the stem cell  product was  infused |                               |                    |                |                      |                         |                      |        |
| Hematopoietic                          | Day 0                                                               | Product Bag                   |                    |                |                      |                         |                      |        |
| Stem Cell                              | or Syringe                                                          | 2-8⁰ C                        |                    |                |                      |                         |                      |        |
| Product Cells                          | Viably Cryopreserved                                                | 2-4 - 10x106 / 1 mL  aliqouts | LN                 |                |                      |                         |                      |        |
| WBC                                    |                                                                     |                               |                    |                |                      |                         |                      |        |
chapter 5
mi-immune references

1Venturi, V., Kedzierska, K., Turner, S.J., Doherty, P.C. & Davenport, M.P. Methods for comparing the diversity of samples of the T cell receptor repertoire. *J. Immunol. Methods* 321, 182–195 (2007).  
2Latouche A, Porcher R, Chevret S (2004). Sample size formula for proportional hazards modelling of competing risks. Statistics in Medicine, 23:3263-3274.  
3D. Lin, L. Wei, I. Yang, Z. Ying. Semiparametric regression for the mean and rate functions of recurrent events. J Royal Statist Soc, 62 (2000), pp. 711-730.  
4"Definition and General Considerations." WHO Collaborating Centre for Drug Statistics Methodology, Norwegian Institute of Public Health, https://www.whocc.no/ddd/definition_and_general_considera/
5Walters W, Hyde ER, Berg-Lyons D, et al. Improved bacterial 16S rRNA gene (V4 and V4-5)
and fungal internal transcribed spacer marker gene primers for microbial community surveys. mSystems 2015;1(1):pii:e00009-15.  
6Kuczynski J, Stombaugh J, Walter WA, et al. Using QIIME to analyze 16s rRNA gene sequences from microbial communities. Curr Protoc Microbiol 2012;Chapter 1.  
7Taur Y, Jenq RR, Perales MA, et al. The effects of intestinal tract bacterial diversity on mortality following allogeneic hematopoietic stem cell transplantation. Blood 2014;124(7):117482.  
8Weber D, Oefner PJ, Hiergeist A, et al. Low urinary indoxyl sulfate levels early after transplantation reflect a disrupted microbiome and are associated with poor outcome. Blood 2015;126(14):1723-8.  
9Zhu W, Stevens AP, Dettmer K, et al. Quantitative profiling of tryptophan metabolites in serum, urine, and cell culture supernatants by liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem 2011;401(10):3249-61.