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15.3 sample size

This study is designed as a two-stage phase II trial to estimate OS. Since the goal is to estimate the OS at a long-term time-point (6 months), our design is a generalization of the Simon design. Rather than suspension of the trial for evaluation after stage 1, this design uses an optimal interim analysis for futility without suspension of accrual as proposed by Huang et al17**. The goal of this design minimizes the**
expected sample size under the assumed parameters. Our trial assumes that a 6-month OS of 25% is unacceptably low and **a 6-month OS of 50% is a rate worthy of further study. Both the null and alternative** hypotheses assume that the OS follow an exponential distribution. Given an overall one-sided type I error of 5%, 32 evaluable patients will provide 80% statistical power. When 14 **patients have been enrolled (stage 1), it is expected that the accrual will stop if the 6 month** Kaplan-Meier estimate of OS is less than 30.0%. It is not required that the 14th patient be **followed for 6** months prior to making a decision of whether to continue the trial. Once the 14th patient has been enrolled, the statistician can calculate survival to determine if it falls below the cut-point. If the trial reaches the end of stage II after 32 **evaluable patients, significant clinical activity would require at least** a 6 month Kaplan-Meier estimate of OS of 39.2%. The probability of stopping at the interim analysis under the null hypothesis is 63.5% and 10.9% under the alternative hypothesis. The R package 'Optinterm' with functions 'OptimDes' and 'SimDes' were used to create design parameters. Assuming that 5% of enrolled patients are not evaluable, we expect recruitment of 34 patients.
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accrual

We perform approximately 30 DLIs per year at the collaborating institutions combined. Considering ~50% screen failures and enrollment on competing trials, we expect to complete enrollment in approximately 2 years with an additional year of follow up after the last patient **has been enrolled.**
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15.4 stopping rules toxicity monitoring

Monitoring guidelines are developed to monitor excess toxicity using a continuous monitoring strategy based on an adaptation of Pocock stopping boundaries20**. In the event that a stopping rule is triggered,**
enrollment will be halted and reviewed by the full study committee and if appropriate by the IRB, prior to initiation of re-enrollment. **Additionally, the FDA will be informed if a stopping rule is triggered. The** stopping rule was calculated using the following website: http://cancer.unc.edu/biostatistics/program/ivanova/ContinuosMonitoringForToxicity.aspx
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grade iii-iv agvhd until 3

Stopping rules were developed for excessive grade III-IV aGVHD. Previous results from trials of HMA plus DLI have reported an incidence of ~10% for aGVHD III-IV8,9,18,19**. The goal is to construct a boundary based**
on aGVHD grade III-IV such that the probability of early stopping is at most 5% if the true rate is equal to 10% and our sample size is 34. Given these parameters, the upper stopping boundary for aGVHD grade III-IV is 2 events out of 2 patients, 3 out of 6, 4 out of 10, 5 out of 15, 6 out of 21, 7 out of 27 or 8 out of 33. The probability of early stopping from excessive aGVHD grade III-IV if the true probability is 30% is 86%.
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16 ethical and regulatory considerations

The study will be conducted in accordance with **the appropriate regulatory requirement(s). Essential**
clinical documents will be maintained to demonstrate the validity of the study and the integrity of the data collected. Master files should be established at the beginning of the study, maintained for the duration of the study and retained according to the appropriate regulations.
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16.2 ethical considerations

The study will be conducted in accordance with ethical principles founded in the Declaration of Helsinki.  
The IRB will review all appropriate study documentation in order to safeguard the rights, safety and wellbeing of the patients. The study will only be conducted at sites where IRB approval has been obtained.  
The protocol, informed consent, written information given to the patients, safety updates, annual progress reports, and any revisions to these documents will be provided to the IRB by the investigator.
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16.3 informed consent

All potential study participants will be given a copy of the IRB-approved consent to review. The investigator or designee will explain all aspects of the study in lay language and answer all questions regarding the study. If the participant decides to participate in the study, he/she will be asked to sign and date the consent document. Patients who refuse to participate or who withdraw from the study will be treated without prejudice.
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17 references

1 Schmid C, Labopin M, Nagler A, Niederwieser D, Castagna L, Tabrizi R et al. Treatment, risk factors, and outcome of adults with relapsed AML after reduced intensity conditioning for allogeneic stem cell transplantation. Blood 2012; 119: 1599–606.  
2 Bejanyan N, Weisdorf DJ, Logan BR, Wang H-L, Devine SM, de Lima M et al. Survival of Patients with Acute Myeloid Leukemia Relapsing after Allogeneic Hematopoietic Cell Transplantation: A Center for International Blood and Marrow Transplant Research Study. Biol Blood Marrow Transplant 2015; 21. doi:10.1016/j.bbmt.2014.11.007.  
3 Rashidi A, Weisdorf DJ, Bejanyan N. Treatment of relapsed/refractory acute myeloid leukaemia in adults.  
Br J Haematol 2018. doi:10.1111/bjh.15077.  
4 Guillaume T, Gaugler B, Chevallier P, Delaunay J, Ayari S, Clavert A et al. Escalated lymphodepletion followed by donor lymphocyte infusion can induce a graft-versus-host response without overwhelming toxicity. Bone Marrow Transplant 2012; 47: 1112–7.  
5 Miller JS, Weisdorf DJ, Burns LJ, Slungaard A, Wagner JE, Verneris MR et al. Lymphodepletion followed by donor lymphocyte infusion (DLI) causes significantly more acute graft-versus-host disease than DLI alone. Blood 2007; 110: 2761–3.  
6 Sánchez-Abarca LI, Gutierrez-Cosio S, Santamaría C, Caballero-Velazquez T, Blanco B, Herrero-Sánchez C
et al. Immunomodulatory effect of 5-azacytidine (5-azaC): potential role in the transplantation setting. Blood 2010; 115: 107–21.  
7 Craddock C, Jilani N, Siddique S, Yap C, Khan J, Nagra S et al. Tolerability and Clinical Activity of PostTransplantation Azacitidine in Patients Allografted for Acute Myeloid Leukemia Treated on the RICAZA
Trial. Biol Blood Marrow Transplant 2016; 22: 385–390.  
8 Schroeder T, Rachlis E, Bug G, Stelljes M, Klein S, Steckel NK et al. Treatment of acute myeloid leukemia or myelodysplastic syndrome relapse after allogeneic stem cell transplantation with azacitidine and **donor** lymphocyte infusions--a retrospective multicenter analysis from the German Cooperative Transplant Study Group. Biol Blood Marrow Transplant 2015; 21: 653–60.  
9 Steinmann J, Bertz H, Wäsch R, Marks R, Zeiser R, Bogatyreva L et al. 5-Azacytidine and DLI can induce long-term remissions in AML patients relapsed after allograft. Bone Marrow Transplant 2015; 50: 690–5.  
10 Choi J, Ritchey J, Prior JL, Holt M, Shannon WD, Deych E et al. In vivo administration of hypomethylating agents mitigate graft-versus-host disease without sacrificing graft-versus-leukemia. Blood 2010; 116: 129– 139.  
11 Ghobadi A, Choi J, Fiala MA, Fletcher T, Liu J, Eissenberg LG et al. Phase I study of azacitidine following donor lymphocyte infusion for relapsed acute myeloid leukemia post allogeneic stem cell transplantation. Leuk Res 2016; 49: 1–6.  
12 Welch JS, Petti AA, Miller CA, Fronick CC, O'Laughlin M, Fulton RS et al. TP53 and Decitabine in Acute Myeloid Leukemia and Myelodysplastic Syndromes. N Engl J Med 2016; 375: 2023–2036.  
13 Cany J, Roeven MWH, Hoogstad-van Evert JS, Hobo W, Maas F, Franco Fernandez R et al. Decitabine enhances targeting of AML cells by CD34+ progenitor-derived NK cells in NOD/SCID/IL2Rgnull mice. Blood 2018; 131: 202–214.  
14 Spoerl S, Mathew NR, Bscheider M, Schmitt-Graeff A, Chen S, Mueller T et al. Activity of therapeutic JAK
1/2 blockade in graft-versus-host disease. Blood 2014; 123: 3832–3842.  
15 Carniti C, Gimondi S, Vendramin A, Recordati C, Confalonieri D, Bermema A et al. Pharmacologic Inhibition **of JAK1/JAK2 Signaling Reduces Experimental Murine Acute GVHD While Preserving GVT Effects.** Clin Cancer Res 2015; 21: 3740–3749.  
16 Zeiser R, Burchert A, Lengerke C, Verbeek M, Maas-Bauer K, Metzelder SK et al. Ruxolitinib in corticosteroid-refractory graft-versus-host disease after allogeneic stem cell transplantation: a multicenter survey. Leukemia 2015; 29: 2062–2068.  
17 Huang B, Talukder E, Thomas N. Optimal two-stage phase II designs with long-term endpoints. Stat Biopharm Res 2010; 2: 51–61.  
18 Schroeder T, Czibere A, Platzbecker U, Bug G, Uharek L, Luft T et al. Azacitidine and donor lymphocyte infusions as first salvage therapy for relapse of AML or MDS after allogeneic stem cell transplantation. Leukemia 2013; 27: 1229–1235.  
19 Schroeder T, Rautenberg C, Krüger W, Platzbecker U, Bug G, Steinmann J et al. Treatment of relapsed AML and MDS after allogeneic stem cell transplantation with decitabine and DLI—a retrospective multicenter analysis on behalf of the German Cooperative Transplant Study Group. Ann Hematol 2018; 97:
335–342.  
20 Ivanova, A., Qaqish, B.F., and Schell, M.J. (2005). Continuous toxicity monitoring in phase II trials in oncology. Biometrics 61: 540-545  
| Percentage 100   | Normal, no complaints, no evidence of disease                                 |
|------------------|-------------------------------------------------------------------------------|
| 90               | Able to carry on normal activity; minor signs or symptoms of disease          |
| 80               | Normal activity with effort; some signs or symptoms of disease                |
| 70               | Cares for self; unable to carry on normal activity or do active work          |
| 60               | Requires occasional assistance, but is able to care for most of his/her needs |
| 50               | Requires considerable assistance and frequent medical care                    |
| 40               | Disabled; requires special care and assistance                                |
| 30               | Severely disabled, hospitalization indicated. Death not imminent              |
| 20               | Very sick, hospitalization necessary, active supportive treatment necessary   |
| 10               | Moribund, fatal processes, progressing rapidly                                |
| 0                | Dead                                                                          |
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appendix i - karnofsky performance

REFERENCE Karnofsky DA: Meaningful clinical classification of therapeutic responses to anti-cancer drugs. Editorial: Clin Pharmacol Ther 2:709-712, 1961.
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appendix ii -- gvhd grading

| Consensus Clinical Stage and Grade of Acute GVHD (Glucksberg et al, 1974; Thomas et al, 1975, Przepiorka et al, 1995) Stage Skin Liver Lower Gastrointestinal  Upper Gastrointestinal  Tract Tract 1 Maculopapular rash  Bilirubin 2.0 - 3.0  Diarrhea 500 - 1000  <25% of body surface mg/dl mL/day or 280 - 555  mL/m2  No protracted nausea  and vomiting 2 Maculopapular rash 25- Bilirubin 3.1 - 6.0  Diarrhea 1000 - 1500  50% body surface mg/dl mL/day or 556 - 833  mL/m2  Persistent nausea,  vomiting or anorexia 3 Generalized  Bilirubin 6.1 - 15.0  Diarrhea >1500 mL/day or  erythroderma mg/dl >833 mL/m2  4 Generalized  erythroderma with  bullous formation and  desquamation Bilirubin > 15 mg/dl Severe abdominal pain,  with or without ileus, or  stool with frank blood or  melena   |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|  
| University Of Minnesota Acute GVHD Grading                 |            |             |                |                |
|------------------------------------------------------------|------------|-------------|----------------|----------------|
| Acute GVHD Grade                                           | Skin Stage | Liver Stage | Lower GI Stage | Upper GI Stage |
| I                                                          | 1-2        | 0           | 0              | 0              |
| II                                                         | 3          | 1           | 1              | 1              |
| III                                                        | -          | 2-4         | 2-3            |                |
| IV                                                         | 4          | -           | 4              |                |
| - Each column identifies minimum criteria for organ grade. |            |             |                |                |
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late acute and chronic gvhd

Late acute and chronic GVHD will be assessed using the National Institutes of Health (NIH) Consensus Criteria.  
Patient ID: Date of late acute or chronic GVHD diagnosis (mm/dd/yyy) [_____]/[___]/[__
Onset of chronic GVHD was:
1 Progressive (acute GVHD progressed directly to chronic GVHD)
- Interrupted (acute GVHD resolved, then chronic GVHD developed)
✓ De novo (acute GVHD never developed)
❍ Chronic GVHD flare (symptoms reactivated within 30 days of drug tapering or discontinuation)
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karnofsky performance status

O 100 Normal, no complaints; no evidence of disease O 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 70 Cares for self; unable to carry on normal activity or do active work
- 60 Requires occasional assistance, but is able to care for most of his/her needs 55 40 Disabled; requires special care and assistance
- 30 Severely disabled; hospitalization is indicated. Death not imminent O 20 Very Sick, hospitalization necessary, active supportive treatment necessary O 10 Moribund, fatal processes, progressing rapidly 0 0 Dead
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diagnosis was based on

✔ Histologic evidence / biopsy proven
- Clinical evidence 0 Both ✓ Unknown
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organ/system involvement (check if yes)

✓ Sclerosis of skin Other skin or hair involvement (rash, ulcers, pruritus or itching, dyspigmentation, alopecia, lichenoid skin changes, etc)
O Eyes (xerophthalmia (dry eyes), abnormal Schirmer's test, abnormal slit lamp, corneal erosion /
conjunctivitis, etc)
❍ Mouth (lichenoid changes, mucositis / ulcers, erythema, etc) ✓ Bronchiolitis obliterans
✔ Other lung involvement Organ/System Involvement (check if yes)
O Gastrointestinal tract (esophageal involvement, chronic nausea / vomiting, chronic diarrhea, malabsorption, abdominal pain / cramps, etc)
0 Liver O Genitourinary tract (vaginitis / stricture, etc)
- Musculoskeletal (arthritis, contractures, myositis, myasthenia, etc)
✓ Thrombocytopenia (< 100 x 10^9/L)
- Eosinophilia
✓ Autoantibodies O Other hematologic involvement O Serositis
✔ Weight loss O Other organ involvement from chronic GVHD
Specify other organ:
Ref: Jagasia MH, Greinix HT, Arora M. National Institutes of Health Consensus Development Project on Criteria for Clinical Trials in Chronic Graftversus-Host Disease: The 2014 Diagnosis and Staging Working Group Report. Biol Blood Marrow Transplant. 2015 March; 21(3): 389–401.e1. doi:10.1016/j.bbmt.2014.12.001.