important information about revlimid rems
how to fill a revlimid

1. Healthcare provider (HCP) instructs patient to complete patient survey  
| 2.   | HCP completes survey   |
|------|------------------------|  
3. HCP completes patient prescription form 4. HCP obtains Revlimid REMS® authorization number 5. HCP provides authorization number on patient prescription form 6. HCP faxes form, including prescription  
| 7.   | HCP advises patient that a representative from a Revlimid REMS® contract pharmacy will contact them   |
|------|-------------------------------------------------------------------------------------------------------|
| 8.   | Revlimid REMS® contract pharmacy conducts patient education                                           |
| 9.   | Revlimid REMS®contract pharmacy calls for confirmation number                                         |
| 10.  | Revlimid REMS®contract pharmacy ships REVLIMID® with the FDA-approved MEDICATION GUIDE                |
appendix f
human subjects 1. subject consent

A conference will be held with the patient, donor and family to discuss this study and alternative treatments available for the treatment of multiple myeloma. The conference will be conducted by the Principal Investigator or other designated physician. Potential risks associated with the study interventions should be discussed as objectively as possible. Consent will be obtained using an IRB-approved consent. The BMT CTN will provide a template of the consent form to each center. Each center will customize the template according to their local requirements and submit it for review by the local Internal Review Board (IRB). The DCC will verify the adequacy of the consent forms prior to submission to the IRB. Each center must provide evidence of IRB approval. .
appendix f
2. confidentiality

Confidentiality will be maintained by individual names being masked and assigned a patient identifier code. The code relaying the patient's identity with the ID code will be kept separately at the center. The ID code will be transmitted to the network.
appendix f
3. participation of women and

Women and ethnic minorities and other populations will be included in this study. Accrual of women and minorities at each center will be monitored to determine whether their rates of enrollment are reflective of the distribution of potentially eligible women and minorities expected from data reported to the CIBMTR and from published data on incidence of MM in these groups. Centers will be notified if their rates differ significantly from those expected and asked to develop appropriate recruitment strategies.
appendix g karnofsky performance status
3. participation of women and

| Index                      | Specific Criteria                                                              | General                                                                                                               |
|----------------------------|--------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------|
| 100                        | Normal, no complaints, no  evidence of disease.                                |                                                                                                                       |
| 90                         | Able to carry on normal activity,                                              | Able to carry on normal activity; no special                                                                          |
| minor signs or symptoms of | care needed.                                                                   |                                                                                                                       |
| disease.                   |                                                                                |                                                                                                                       |
| 80                         | Normal activity with effort, some  signs or symptoms of disease.               |                                                                                                                       |
| 70                         | Care for self, unable to carry on  normal activity or to do work.              |                                                                                                                       |
| 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 work, able to live at home and care  for most personal needs, varying amount of  assistance needed.         |
| 30                         | Severely disabled, hospitalization  indicated, but death not  imminent.        |                                                                                                                       |
| 20                         | Very sick, hospitalization  necessary, active supportive  treatment necessary. |                                                                                                                       |
| 10                         | Moribund                                                                       |                                                                                                                       |
| 0                          | Dead                                                                           | Unable to care for self, requires institutional  or hospital care or equivalent, disease may be  rapidly progressing. |
appendix h new york heart
3. participation of women and

The following table presents the NYHA classification of cardiac disease.  
| Class   | Functional Capacity                                                                                                                                                                                                                                           | Objective Assessment                                              |
|---------|---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------|
| I       | Patients with cardiac disease but without resulting limitations of  physical activity. Ordinary physical activity does not cause undue  fatigue, palpitation, dyspnea, or anginal pain.                                                                       | No objective evidence  of cardiovascular  disease.                |
| II      | Patients with cardiac disease resulting in slight limitation of physical  activity. They are comfortable at rest. Ordinary physical activity  results in fatigue, palpitation, dyspnea, or anginal pain.                                                      | Objective evidence of  minimal  cardiovascular disease.           |
| III     | Patients with cardiac disease resulting in marked limitation of  physical activity. They are comfortable at rest. Less than ordinary  activity causes fatigue, palpitation, dyspnea, or anginal pain.                                                         | Objective evidence of  moderately severe  cardiovascular disease. |
| IV      | Patients with cardiac disease resulting in inability to carry on any  physical activity without discomfort. Symptoms of heart failure or  the anginal syndrome may be present even at rest. If any physical  activity is undertaken, discomfort is increased. | Objective evidence of  severe cardiovascular  disease.            |  
Version 4.0 dated *July 24, 2018*
appendix i adverse events
i.1. adverse event reporting

Adverse events (AEs) will be collected on calendar-driven forms and event-driven forms in AdvantageEDC.. The calendar-driven forms are those that appear in the AdvantageEDC Forms grid for each enrolled patient at designated timepoints (e.g. Day 28 post transplant) throughout the course of the study. Completion of calendar-driven forms is expected by the target date for the given assessment period. Calendar Driven forms for the BMT CTN 1401 study are as follows:
- Toxicity Form: this form documents all expected toxicities for the BMT CTN 1401 study; each toxicity is also assigned a grade, based on the NCI CTCAE Version 4.0.  
- Follow Up Status Form: this form documents the status of each patient at various intervals on the study.
Event-driven forms must be completed when a certain event triggers the appearance of the form in the AdvantageEDC Forms Grid. Most often the event-driven form is triggered by information entered on the Follow-up Status Form. Event-driven forms for the BMT CTN 1401 study are as follows:
- **Re-Admission/Hospitalization Form**: this form documents all hospital admissions, *including* the admission for transplant for this study.  
- **Infection Form**: this form documents infections from Day 0 (date of transplant) through the 1-year post-transplant follow-up period.  
- **Progression/Relapse Form**: the form captures detailed information associated with progression or relapse of the primary disease. DO NOT report progression or relapse as an Unexpected, Grade 35 Adverse Event.  
- **Death Form**: this form documents the death of a patient from the time of study enrollment and randomization through the 1-year post-transplant follow-up period.  
- Adverse **Event Forms**: this series of forms captures details on adverse events that are *both* unexpected and grades 3-5, based on the NCI CTCAE Version 4.0, **regardless of attribution** to any of the study interventions. These forms are also used to collect information on any SAE event required by the additional adverse event reporting requirements. These events require expedited reporting and will be reviewed by the Medical Monitor associated with the BMT CTN Data and Coordinating Center (DCC) within 2 business days of receiving the summary of the adverse event from the transplant center. If the Medical Monitor requires additional information to make his/her assessment, the transplant center will have 4 business days to respond to the request for additional information.
appendix i adverse events
i.2 reporting expected toxicities

Expected toxicities for all patients enrolled on BMT CTN 1401 will be collected on the BMT CTN 1401 Calendar-Driven Toxicity Form and some of the event-driven forms (Infection, Readmission/Hospitalization, Progression/Relapse, Graft Failure and Death forms). Any grade 4 expected event not collected on the calendar-driven toxicity or specified event- driven form must be reported through the expedited AE reporting system in AdvantageEDC.
appendix i adverse events
i.3 reporting unexpected grade 3-5

All Unexpected, Grade 3-5 Adverse Events should be reported for every patient enrolled on the study from the time of randomization until 3 **years post maintenance initiation**.
appendix i adverse events
i.4 reporting secondary malignancies

All secondary primary malignancies (SPMs), excluding non-melanoma skin cancers, experienced by participants from the time of enrollment until 3 years post maintenance initiation will be reported using the **Adverse Event forms (AE1-AE6) in AdvantageEDC and** must be reported within three business days of knowledge of the event. The Event Description of the Adverse Event forms should include histologic type. I.5 Reporting Adverse Events from enrollment until randomization No adverse event reporting is required from the time of enrollment until randomization. All patient deaths, SPMs, and progressions must still be reported during this time.
appendix i adverse events
i.6 reporting adverse events after

Additional Adverse Event reporting applies to patients who receive lenalidomide. Refer to Chapter 4 for additional instructions for reporting of Second Primary Malignancies and other special events of interest. Determination of the expectedness of adverse events should be differentiated between the initial transplant, maintenance, or vaccine therapy at the discretion of the investigator. For example, oral mucositis would be an expected risk associated with transplantation, but the investigator should assess the expectedness for oral mucositis during maintenance with or without vaccine therapy.
appendix i adverse events
table i-1 adverse events for

| Adverse Event                        | Collection Type   | Collection Form2   |
|--------------------------------------|-------------------|--------------------|
| AUDITORY DISORDERS Hearing loss      | Calendar-Driven   | Toxicity           |
| BLOOD AND LYMPHATIC DISORDERS Anemia | Calendar-Driven   | Toxicity           |  
Version 4.0 dated *July 24, 2018*  
| Adverse Event                                    | Collection Type   | Collection Form2   |
|--------------------------------------------------|-------------------|--------------------|
| Disseminated intravascular  coagulation3         | Event-Driven      | Adverse Event Form |
| Febrile neutropenia5                             | Event-Driven      | Adverse Event Form |
| Lymphopenia                                      | Calendar-Drive    | Toxicity           |
| Neutropenia                                      | Calendar-Driven   | Toxicity           |
| Thrombocytopenia                                 | Calendar-Driven   | Toxicity           |
| Thrombotic thrombocytopenic  purpura/ Thrombotic | Calendar-Driven   | Toxicity           |
| microangiopathy CARDIAC DISORDERS Asystole3      | Event-Driven      | Adverse Event Form |
| Atrial fibrillation3                             | Event-Driven      | Adverse Event Form |
| Atrial flutter3                                  | Event-Driven      | Adverse Event Form |
| Atrioventricular block3                          | Event-Driven      | Adverse Event Form |
| Left ventricular systolic dysfunction            | Calendar-Driven   | Toxicity           |
| Myocardial infarction3                           | Event-Driven      | Adverse Event Form |
| New or worsening heart failure3                  | Event-Driven      | Adverse Event Form |
| Pericardial effusion3                            | Event-Driven      | Adverse Event Form |
| Pericarditis3                                    | Event-Driven      | Adverse Event Form |
| Sinus bradycardia3                               | Event-Driven      | Adverse Event Form |
| Ventricular arrhythmia                           | Calendar-Driven   | Toxicity           |
| Ventricular tachycardia3                         | Event-Driven      | Adverse Event Form |
| ENDOCRINE DISORDERS Hyperthyroidism3             | Event-Driven      | Adverse Event Form |
| Hypothyroidism                                   | Calendar-Driven   | Toxicity           |
| GASTROINTESTINAL DISORDERS Abdominal pain        | Calendar-Driven   | Toxicity           |
| Anorexia                                         | Calendar-Driven   | Toxicity           |
| Constipation                                     | Calendar-Driven   | Toxicity           |
| Diarrhea                                         | Calendar-Driven   | Toxicity           |
| Diverticulitis3                                  | Event-Driven      | Adverse Event Form |
| Dry mouth                                        | Calendar-Driven   | Toxicity           |
| Dysgeusia (taste alteration)                     | Calendar-Driven   | Toxicity           |
| Dyspepsia (heartburn)                            | Calendar-Driven   | Toxicity           |
| GI perforation3                                  | Event-Driven      | Adverse Event Form |
| Intestinal obstruction3                          | Event-Driven      | Adverse Event Form |
| Ischemic bowel3                                  | Event-Driven      | Adverse Event Form |
| Nausea                                           | Calendar-Driven   | Toxicity           |
| Oral mucositis                                   | Calendar-Driven   | Toxicity           |
| Vomiting                                         | Calendar-Driven   | Toxicity           |  
I-4  
Version 4.0 dated *July 24, 2018*  
| Adverse Event                                                                  | Collection Type   | Collection Form2   |
|--------------------------------------------------------------------------------|-------------------|--------------------|
| GENERAL DISORDERS Chest pain- non-cardiac                                      | Calendar-Driven   | Toxicity           |
| Chills                                                                         | Calendar-Driven   | Toxicity           |
| Edema- generalized                                                             | Calendar-Driven   | Toxicity           |
| Fatigue                                                                        | Calendar-Driven   | Toxicity           |
| Fever                                                                          | Calendar-Driven   | Toxicity           |
| Sudden death3                                                                  | Event-Driven      | Adverse Event Form |
| HEPATOBILIARY/PANCREAS DISORDERS Abnormal liver function tests Calendar-Driven | Toxicity          |                    |
| Hepatitis3                                                                     | Event-Driven      | Adverse Event Form |
| Liver failure3                                                                 | Event-Driven      | Adverse Event Form |
| Pancreatitis3                                                                  | Event-Driven      | Adverse Event Form |
| HEMORRHAGIC DISORDERS Intracranial3                                            | Event-Driven      | Adverse Event Form |
| Gastrointestinal3                                                              | Event-Driven      | Adverse Event Form |
| Genitourinary                                                                  | Calendar-Driven   | Toxicity           |
| Pulmonary/Upper respiratory3                                                   | Event-Driven      | Adverse Event Form |
| IMMUNE SYSTEM DISORDERS Allergic reaction3                                     | Event-Driven      | Adverse Event Form |
| Anaphylaxis (swelling of the skin  and/or swelling of the face or              | Event-Driven      | Adverse Event Form |
| throat)3 INFECTIONS Infections/ Sepsis                                         | Event-Driven      | Infection          |
| INVESTIGATIONS Increased ALT > 3.0 x ULN4                                      | Event-Driven      | Adverse Event Form |
| Increased AST > 3.0 x ULN4                                                     | Event-Driven      | Adverse Event Form |
| Increased bilirubin > 3.0 x ULN4                                               | Event-Driven      | Adverse Event Form |
| Increased creatinine                                                           | Calendar-Driven   | Toxicity           |
| Prolongation of QTc interval4                                                  | Event-Driven      | Adverse Event Form |
| Weight loss                                                                    | Calendar-Driven   | Toxicity           |
| METABOLISM AND NUTRITION DISORDERS Dehydration Calendar-Driven                 | Toxicity          |                    |
| Hypercalcemia                                                                  | Calendar-Driven   | Toxicity           |
| Hyperglycemia                                                                  | Calendar-Driven   | Toxicity           |
| Hypoglycemia                                                                   | Calendar-Driven   | Toxicity           |
| Hypokalemia                                                                    | Calendar-Driven   | Toxicity           |
| Hyponatremia                                                                   | Calendar-Driven   | Toxicity           |
| Tumor lysis syndrome3                                                          | Event-Driven      | Adverse Event Form |
| MUSCULOSKELETAL AND TISSUE DISORDERS Arthralgia Calendar-Driven                | Toxicity          |                    |  
Version 4.0 dated *July 24, 2018*  
| Adverse Event                                                                                     | Collection Type    | Collection Form2   |
|---------------------------------------------------------------------------------------------------|--------------------|--------------------|
| Myalgia                                                                                           | Calendar-Driven    | Toxicity           |
| Muscle weakness (generalized or  specific area)                                                   | Calendar-Driven    | Toxicity           |
| Musculoskeletal pain                                                                              | Calendar-Drive     | Toxicity           |
| NERVOUS SYSTEM DISORDERS Anxiety                                                                  | Calendar-Driven    | Toxicity           |
| Ataxia                                                                                            | Calendar-Driver    | Toxicity           |
| Confusion                                                                                         | Calendar-Driven    | Toxicity           |
| Cranial palsy3                                                                                    | Event-Driven       | Adverse Event Form |
| Depression                                                                                        | Calendar-Driven    | Toxicity           |
| Depressed level of consciousness3                                                                 | Event-Driven       | Adverse Event Form |
| Dizziness                                                                                         | Calendar-Driven    | Toxicity           |
| Edema cerebral3                                                                                   | Event-Driven       | Adverse Event Form |
| Encephalopathy3                                                                                   | Event-Driven       | Adverse Event Form |
| Headache                                                                                          | Calendar-Driven    | Toxicity           |
| Insomnia                                                                                          | Calendar-Driven    | Toxicity           |
| Neuralgia3                                                                                        | Event-Driven       | Adverse Event Form |
| Neuropathy3                                                                                       | Event-Driven       | Adverse Event Form |
| Reversible posterior  leukoencephalopathy syndrome                                                | Event-Driven       | Adverse Event Form |
| (PRES)3 Seizure3                                                                                  | Event-Driven       | Adverse Event Form |
| Severe muscle weakness/paralysis3                                                                 | Event-Driven       | Adverse Event Form |
| Somnolence                                                                                        | Calendar-Driven    | Toxicity           |
| Spinal cord compression (nonmalignant)3                                                                                                   | Event-Driven       | Adverse Event Form |
| Stroke                                                                                            | Calendar-Driver    | Toxicity           |
| Syncope (fainting)3                                                                               | Event-Driven       | Adverse Event Form |
| Tremor                                                                                            | Calendar-Drive     | Toxicity           |
| OCULAR/VISUAL DISORDERS Blurred vision3                                                           | Event-Driven       | Adverse Event Form |
| Conjunctivitis                                                                                    | Calendar-Driven    | Toxicity           |
| Sudden loss of vision3                                                                            | Event-Driven       | Adverse Event Form |
| RENAL DISORDERS Cystitis Non-infective                                                            | Calendar-Driven    | Toxicity           |
| Acute kidney injury3                                                                              | Event-Driven       | Adverse Event Form |
| Chronic kidney disease3                                                                           | Event-Driven       | Adverse Event Form |
| RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS Adult respiratory distress syndrome3 Event-Driven | Adverse Event Form |                    |
| Bronchitis                                                                                        | Calendar-Driven    | Infection          |
| Bronchopulmonary hemorrhage3                                                                      | Event-Driven       | Adverse Event Form |  
I-6  
Version 4.0 dated *July 24, 2018*  
| Adverse Event                                                                                                 | Collection Type   | Collection Form2   |
|---------------------------------------------------------------------------------------------------------------|-------------------|--------------------|
| Cough                                                                                                         | Calendar-Driven   | Toxicity           |
| Dyspnea                                                                                                       | Calendar-Driven   | Toxicity           |
| Hypoxia                                                                                                       | Calendar-Driven   | Toxicity           |
| Pleural effusion (non-malignant)3                                                                             | Event-Driven      | Adverse Event Form |
| Pneumonia                                                                                                     | Calendar-Driven   | Infection          |
| Pneumonitis3                                                                                                  | Event-Driven      | Adverse Event Form |
| Pulmonary Hypertension3                                                                                       | Event-Driven      | Adverse Event Form |
| Sinusitis                                                                                                     | Calendar-Driven   | Toxicity           |
| Sore throat                                                                                                   | Calendar-Driven   | Toxicity           |
| SKIN AND SUBCUTANEOUS TISSUE DISORDERS Dry skin Calendar-Driven                                               | Toxicity          |                    |
| Erythema multiforme3                                                                                          | Event-Driven      | Adverse Event Form |
| Pruritis                                                                                                      | Calendar-Driven   | Toxicity           |
| Pyoderma gangrenosum3                                                                                         | Event-Driven      | Adverse Event Form |
| Rash                                                                                                          | Calendar-Driven   | Toxicity           |
| Sweet's syndrome (acute  neutrophilic dermatosis)3                                                            | Event-Driven      | Adverse Event Form |
| VASCULAR DISORDERS Capillary leak syndrome3                                                                   | Event-Driven      | Adverse Event Form |
| Edema                                                                                                         | Calendar-Driven   | Toxicity           |
| Hypertension                                                                                                  | Calendar-Drive    | Toxicity           |
| Hypotension                                                                                                   | Calendar-Driven   | Toxicity           |
| Thromboembolic event (DVT,  pulmonary emboli)3                                                                | Event-Driven      | Adverse Event Form |
| Vasculitis3                                                                                                   | Event-Driven      | Adverse Event Form |
| OTHER Pregnancy3                                                                                              | Event-Driven      | Adverse Event Form |
| Secondary malignancies6                                                                                       | Event-Driven      | Adverse Event Form |
| Other unexpected grade 3-5 AE                                                                                 | Event-Driven      | Adverse Event Form |
| 1 Last dose of lenalidomide refers to the last dose prior to permanent discontinuation of study lenalidomide. |                   |                    |  
Version 4.0 dated *July 24, 2018*
vcan proteolysis investigation of a
appendix j

Immune Biomarker In Myeloma  
Version 1.0 Principal Investigator Fotis Asimakopoulos, MD, PhD
Co-Principal Investigator Binod Dhakal, MD, MS
BMT CTN 1401 Study Co-Chairs and Protocol Officer David Avigan, MD, Nina Shah, MD, David Chung, MD, Marcelo Pasquini, MD, MS
J-2
chapter 1
1 background and rationale 1.1

Myeloma tumor cells are critically dependent on their interactions with their cellular and noncellular microenvironment (the extracellular matrix) in both bone-marrow and extramedullary niches (1-5). We have recently focused on the unique interplay between extracellular matrix, myeloid and stromal cells in fine-tuning the inflammatory and immune microenvironment of the myeloma niche (6). Myeloma progression is characterized by accumulation of the large matrix proteoglycan, versican (VCAN) (7). VCAN contributes to myeloma homeostasis through the activation of tumor-infiltrating myeloid cells [and resultant production of trophic cytokines, such as IL-1β and IL-6) but also by generating immune privilege (through interference with the functionality of antigen-presenting dendritic cells (DC)] (8). By contrast, the regulated proteolysis of VCAN by ADAMTS proteases generates fragments (VCAN-matrikines, including versikine)
with opposing, *immunostimulatory* activities (9). Thus VCAN proteolysis intensity (and the resultant balance between parent, tolerogenic VCAN and its immunogenic fragments) may help determine the immunogenicity of the tumor microenvironment. Because efficacy of immunotherapies depends on prior cancer-immunity "set points" (10), the VCAN proteolysis status may provide a novel predictive immune biomarker. With regard to the relevance of the VCAN pathway to BMT CTN 1401's scientific premise, we hypothesize that the more immunogenic marrow microenvironment generated through VCAN proteolysis will potentiate the primary effector response generated through CTN1401 vaccination.  
Specifically, we have shown that versikine enhances Batf3-DC differentiation and maturation (IL12 production)(6, 9, 11). Batf3-DC are a key DC subset that is essential for tumor antigen crosspresentation (12) and for efficacy of immunotherapies such as checkpoint inhibition (13).  
Intratumoral Batf3-DC are also critical for the efferent arm of the immune response: Gajewski has shown that Batf3-DC in the tumor microenvironment are responsible for T-cell effector recruitment through chemokine networks involving CXCL9 and CXCL10 (14). Thus, active VCAN proteolysis will be predicted to potentiate effector responses post-ASCT (15). Improved infiltration of primary effector cells and enhanced abundance and activation of (versikinepromoted) Batf3-DC at the tumor site (or, residual disease site) will result in secondary, in situ vaccination effects that will amplify the primary effector response. Therefore, patients with active VCAN proteolysis in the bone marrow are predicted to be more likely to activate a beneficial cancer immunity cycle (16), to generate more robust effector responses and experience better clinical outcomes. Consistent with this hypothesis, at ASH 2017 we presented data that corroborated a correlation between bone marrow VCAN proteolysis and increased CD8+ infiltration and activation postASCT for myeloma (Pagenkopf et al., ASH abstract \#1756, ASH Annual Meeting 2017). Our findings on the immunoregulatory role of VCAN in myeloma were independently corroborated by Bruno Paiva's group: they demonstrated that VCAN-producing macrophages expand postautologous SCT and may be associated with MRD persistence through tolerogenic actions of intact VCAN (17).
chapter 2
2 study design and procedures

This study is a laboratory ancillary study that will investigate the versican proteolysis status on randomized patients for BMT CTN 1401. The study will examine samples provided from 10 patients on the Lenalidomide/Vaccine/GM-CSF arm and 10 patients on either the Lenalidomide alone or the Lenalidomide/GM-CSF arms
chapter 2
2.2 specific study aims

To investigate VCAN proteolysis as a predictive biomarker for dendritic cell (DC) vaccine-based immunotherapy post- ASCT for myeloma. The study will correlate versican proteolysis status with study-mandated immunological endpoints
(myeloma-specific T cells). It is hypothesized that versican proteolysis-*predominant* status will predict those patients that meet or exceed the primary immunologic endpoint criteria (expansion of myeloma-specific T cells). Additionally, the study will correlate versican proteolysis status with the primary and relevant secondary clinical endpoints. It is hypothesized that versican proteolysis-*predominant* status will correlate with improved clinical outcomes.
chapter 2
2.3 patient eligibility

Patients must meet specified eligibility criteria outlined in section 2.3.1 and 2.3.2 to be registered on the study. All questions regarding eligibility criteria should be directed to the protocol coordinator at 301-251-1161. 2.3.1 Patient Inclusion Criteria  
1. Patients successfully enrolled in Segment A of BMT CTN 1401 2. Patients eligible for randomization per Patient Eligibility Criteria for Randomization in Chapter 2 of BMT CTN 1401 3. Patients with an available core biopsy at time of randomization 2.3.2 Patient Exclusion Criteria 1. Patients unable or unwilling to provide informed consent for participation on the ancillary study
chapter 2
2.4 study procedures

Patients will be consented at the time of randomization. Patients who agree to participate in the ancillary study will provide permission for the BMT CTN DCC to obtain a portion of the pre-randomization clinical bone marrow biopsy that was collected for the BMT CTN 1401 parent trial. Following randomization, 4 slides of paraffin-embedded, decalcified bone marrow tissue will be prepared from the core biopsy sample at the clinical site and shipped to University of Wisconsin for analysis. No additional samples are required for this study.
chapter 2
2.5 patients for whom adequate

Shipment of the samples will be tracked in GlobalTrace and on the Specimen Acquisition Form in AdvantageEDC for all required samples. Detailed procedures regarding specimen collection schedules and data entry will be found in the BMT CTN 1401 Research Sample Information Guide and the BMT CTN 1401 Forms Guide.
chapter 2
2.6 participant risks

Risks of the BMT CTN 1401 procedures are described in the parent clinical trial protocol. There are no additional risks associated with this ancillary study.
chapter 2
2.7 study analysis

This is a pilot study of a small initial group and all analyses are exploratory. Results from this pilot study will inform plans for expansion into larger cohorts.
chapter 2
ancillary study references

1. Kawano Y, Moschetta M, Manier S, Glavey S, Gorgun GT, Roccaro AM, Anderson KC, Ghobrial IM.  
Targeting the bone marrow microenvironment in multiple myeloma. Immunol Rev. 2015;263(1):160-72.  
Epub 2014/12/17. doi: 10.1111/imr.12233. PubMed PMID: 25510276.  
2. Lemaire M, Deleu S, De Bruyne E, Van Valckenborgh E, Menu E, Vanderkerken K. The microenvironment and molecular biology of the multiple myeloma tumor. Adv Cancer Res. 2011;110:1942. Epub 2011/06/28. doi: B978-0-12-386469-7.00002-5 [pii]10.1016/B978-0-12-386469-7.00002-5. PubMed PMID: 21704227.  
3. Mitsiades CS, McMillin DW, Klippel S, Hideshima T, Chauhan D, Richardson PG, Munshi NC, Anderson KC. The role of the bone marrow microenvironment in the pathophysiology of myeloma and its significance in the development of more effective therapies. Hematol Oncol Clin North Am.  
2007;21(6):1007-34, vii-viii. Epub 2007/11/13. doi: S0889-8588(07)00115-3 [pii] 10.1016/j.hoc.2007.08.007. PubMed PMID: 17996586.  
4. Podar K, Chauhan D, Anderson KC. Bone marrow microenvironment and the identification of new targets for myeloma therapy. Leukemia. 2009;23(1):10-24. Epub 2008/10/10. doi: leu2008259 [pii] 10.1038/leu.2008.259. PubMed PMID: 18843284; PMCID: 3418600.  
5. Ribatti D, Nico B, Vacca A. Importance of the bone marrow microenvironment in inducing the angiogenic response in multiple myeloma. Oncogene. 2006;25(31):4257-66. Epub 2006/03/07. doi: 1209456
[pii]10.1038/sj.onc.1209456. PubMed PMID: 16518413.  
6. Asimakopoulos F, Hope C, Johnson MG, Pagenkopf A, Gromek K, Nagel B. Extracellular matrix and the myeloid-in-myeloma compartment: balancing tolerogenic and immunogenic inflammation in the myeloma niche. J Leukoc Biol. 2017. doi: 10.1189/jlb.3MR1116-468R. PubMed PMID: 28254840.  
7. Hebron E, Hope C, Kim J, Jensen JL, Flanagan C, Bhatia N, Maroulakou I, Mitsiades C, Miyamoto S,
Callander N, Hematti P, Asimakopoulos F. MAP3K8 kinase regulates myeloma growth by cellautonomous and non-autonomous mechanisms involving myeloma-associated monocytes/macrophages. Br J Haematol. 2013;160(6):779-84. Epub 2012/12/21. doi: 10.1111/bjh.12175. PubMed PMID: 23252623; PMCID: 3594344.  
8. Tang M, Diao J, Gu H, Khatri I, Zhao J, Cattral MS. Toll-like Receptor 2 Activation Promotes Tumor Dendritic Cell Dysfunction by Regulating IL-6 and IL-10 Receptor Signaling. Cell Rep. 2015;13(12):285164. doi: 10.1016/j.celrep.2015.11.053. PubMed PMID: 26711349.  
9. Hope C, Foulcer S, Jagodinsky J, Chen SX, Jensen JL, Patel S, Leith C, Maroulakou I, Callander N,
Miyamoto S, Hematti P, Apte SS, Asimakopoulos F. Immunoregulatory roles of versican proteolysis in the myeloma microenvironment. Blood. 2016;128(5):680-5. doi: 10.1182/blood-2016-03-705780. PubMed PMID: 27259980.  
10. Chen DS, Mellman I. Elements of cancer immunity and the cancer-immune set point. Nature.  
2017;541(7637):321-30. doi: 10.1038/nature21349. PubMed PMID: 28102259.  
11. Hope C, Emmerich PB, Papadas A, Pagenkopf A, Matkowskyj KA, Van De Hey DR, Payne SN, Clipson L, Callander NS, Hematti P, Miyamoto S, Johnson MG, Deming DA, Asimakopoulos F. Versican-Derived Matrikines Regulate Batf3-Dendritic Cell Differentiation and Promote T Cell Infiltration in Colorectal Cancer. J Immunol. 2017;199(5):1933-41. doi: 10.4049/jimmunol.1700529. PubMed PMID: 28754680; PMCID: PMC5568487.  
12. Vanpouille-Box C, Galluzzi L. CD103(+) cells at the forefront of anticancer immunity. Oncoimmunology.  
2017;6(11):e1356154. doi: 10.1080/2162402X.2017.1356154. PubMed PMID: 29147613; PMCID: PMC5674943.  
13. Sanchez-Paulete AR, Cueto FJ, Martinez-Lopez M, Labiano S, Morales-Kastresana A, Rodriguez-Ruiz ME, Jure-Kunkel M, Azpilikueta A, Aznar MA, Quetglas JI, Sancho D, Melero I. Cancer Immunotherapy with Immunomodulatory Anti-CD137 and Anti-PD-1 Monoclonal Antibodies Requires BATF3-Dependent Dendritic Cells. Cancer Discov. 2016;6(1):71-9. doi: 10.1158/2159-8290.CD-15-0510. PubMed PMID: 26493961; PMCID: PMC5036540.  
14. Spranger S, Dai D, Horton B, Gajewski TF. Tumor-Residing Batf3 Dendritic Cells Are Required for Effector T Cell Trafficking and Adoptive T Cell Therapy. Cancer Cell. 2017;31(5):711-23 e4. doi: 10.1016/j.ccell.2017.04.003. PubMed PMID: 28486109.  
15. Chung DJ, Pronschinske KB, Shyer JA, Sharma S, Leung S, Curran SA, Lesokhin AM, Devlin SM, Giralt SA, Young JW. T-cell Exhaustion in Multiple Myeloma Relapse after Autotransplant: Optimal Timing of  
Version 4.0 dated July 24, 2018 Immunotherapy. Cancer Immunol Res. 2016;4(1):61-71. doi: 10.1158/2326-6066.CIR-15-0055. PubMed PMID: 26464015; PMCID: PMC4703436.  
16. Chen DS, Mellman I. Oncology meets immunology: the cancer-immunity cycle. Immunity. 2013;39(1):110. doi: 10.1016/j.immuni.2013.07.012. PubMed PMID: 23890059.  
17. Arana P, Zabaleta A, Lasa M, Maiso P, Alignani D, Jelinek T, Segura V, van den Bossche W, Teodosio C,
Damasceno D, Rodriguez-Otero P, Prosper F, Mateos M-V, Lahuerta JJ, Bladé J, van Dongen JJM, San Miguel J, Orfao A, Paiva B. High-Throughput Characterization and New Insight into the Role of Tumor Associated Macrophages (TAMs) in Multiple Myeloma (MM). Blood. 2016;128(22):482-.  
18. Hope C, Emmerich PB, Papadas A, Pagenkopf A, Matkowskyj KA, Van De Hey DR, Payne SN, Clipson L, Callander NS, Hematti P, Miyamoto S, Johnson MG, Deming DA, Asimakopoulos F. Versican-Derived Matrikines Regulate Batf3-Dendritic Cell Differentiation and Promote T Cell Infiltration in Colorectal Cancer. J Immunol. 2017. doi: 10.4049/jimmunol.1700529. PubMed PMID: 28754680.  
J-7
appendix k t cell/myeloma genomic
ancillary study references

Characterization of the T cell immune response and  
  
myeloma genomic signature as predictors of immunologic and clinical response Version 1.0 Principal Investigator David Avigan, MD
Co-Principal Investigator Jacalyn Rosenblatt, MD
BMT CTN 1401 Study Co-Chairs and Protocol Officer David Avigan, MD, Nina Shah, MD, David Chung, MD, Marcelo Pasquini, MD, MS
appendix k t cell/myeloma genomic
protocol synopsis

Characterization of the T cell immune response and myeloma genomic signature as predictors of immunologic and clinical response Study Design: This is a laboratory ancillary study to the BMT CTN 1401 parent clinical trial. The study will further characterize the T cell response to vaccination with respect to targeting of neoantigens, assessment of T cell clonality, and characterization of the immune landscape via CYTOF analysis. In addition, genomic characterization of immunoregulatory pathways in the myeloma cells at baseline and presence of immunoregulatory noncoding RNAs will be correlated with immunologic response. Samples will consist of additional bone marrow and peripheral blood sampling at the time of collection for other indications outlined in the parent protocol.  
Primary Objective: Determine the impact of therapy on the expansion of T cells  
recognizing tumor associated neo-antigens and compare  
response between patients randomized to the Vaccine/GMCSF/Lenalidomide arm and patients randomized to the  
Lenalidomide/GM-CSF or Lenalidomide Alone arms.  
Secondary Objective: Correlate of neoantigen T cell response with expansion of myeloma specific T cell and achievement and maintenance of complete remission at 1 year post-transplant, characterize T cell clonality and TCR identity following therapy, assess measures of the immune landscape and correlated with expansion of myeloma specific T cell and achievement and maintenance of complete remission at 1 year post-transplant, characterize myeloma cell expression of immunoregulatory pathways by genomic analysis and correlate with expansion of myeloma specific T cell and achievement and maintenance of complete remission at 1 year post-transplant, to characterize the presence of immunoregulatory noncoding RNAs and correlate with expansion of myeloma specific T cell and achievement and maintenance of complete remission at 1 year post-transplant Eligibility Criteria: Eligible patients are patients enrolled on BMT CTN 1401 that are eligible to proceed to randomization within the BMT CTN 1401 protocol specified window. Patients must also have consented to provide optional future research samples and have provided a bone marrow aspirate for future research at time of enrollment on BMT CTN 1401. Patients must sign the additional research sample consent prior to enrollment on this study.  
- **Randomization: Additional** 4mL Bone Marrow Aspirate sample, collected at the time of this required bone marrow aspirate being performed as part of the primary 1401 trial.  
- **Prior to Cycles 1, 2, and 4 of Maintenance: Additional**
10mL Peripheral Blood sample, collected at the time of these required research blood collections are being performed as part of the primary 1401 trial.  
- **Prior to Cycle 9 of Maintenance: Additional** 4mL Bone Marrow Aspirate sample, collated at the time of this required bone marrow aspirate being performed as part of the primary 1401 trial.  
- **At time of Disease Progression:** 5mL Bone Marrow Aspirate collection and 40mL Peripheral Blood collection Additionally, a portion of the research bone marrow aspirate sample provided to the protocoldefined lab and/or BMT CTN Biorepository at time of initial enrollment on to BMT CTN 1401 will be used for this correlative laboratory study.  
Accrual Objective: 60 patients randomized on BMT CTN 1401 (30 randomized to Vaccine/Lenalidomide/GM-CSF and 30 randomized to either Lenalidomide/GM-CSF or Lenalidomide). The estimated accrual period is 2 years  
Treatment Description: Patients will be consented at time of randomization on the BMT  
CTN 1401 parent trial. Eligible patients will undergo sample collections at the following time points:  
| Additionally, a portion of the research bone marrow aspirate sample provided to the protocoldefined lab and/or BMT CTN Biorepository at time of initial  laboratory study.  Accrual Objective: 60 patients randomized on BMT CTN 1401 (30 randomized to  accrual period is 2 years Accrual Period: The estimated accrual period is 2 years.  Study Duration: The estimated study duration is 5 years.   |
|---|
chapter 1
1. background and rationale 1.1.

Despite the advances in biologic therapy for Multiple Myeloma (MM), curative outcomes remain elusive and patients ultimately experience life threatening disease progression. Tumor heterogeneity results in significant plasticity in response to therapy and the emergence of resistant clones[1,2]. In a paradigm shift for cancer therapeutics, cellular immunotherapy has emerged as a highly promising strategy with the potential to eradicate diverse tumor clones and provide ongoing surveillance against disease relapse. Two critical areas of investigation in cellular immunotherapy are the development of chimeric antigen receptor (CAR) T cells and cancer vaccines. One vital area of investigation is the development of tumor vaccines to elicit the expansion of tumor specific effector cells, maintain regulatory mechanism of host immunity to minimize off target effects, and develop long term memory response to protect against relapse. Vaccines targeting individual antigens have been developed, but clinical efficacy may be limited by the inability to target the diversity of tumor clones and the development of antigen negative variants as a means of resistance[22, 23]. The group of Avigan and Rosenblatt has developed a promising cancer vaccine in which patient derived tumor cells are fused with autologous dendritic cells (DCs), presenting a broad array of antigens that capture the heterogeneity of the tumor clonal populations. In diverse animal models, vaccination with DC/tumor fusions results in eradication of established metastatic disease[24-26]. In phase I clinical studies of patients with hematological malignancies and solid tumors, vaccination with DC/tumor fusions was well tolerated, induced the expansion of tumor specific T cells, and resulted in disease regression or stabilization in patients with advanced disease[27-30]. In a recently reported trial, vaccination of patients with acute leukemia with DC/AML fusions following chemotherapy induced durable expansion in the peripheral blood and marrow of tumor reactive lymphocytes as defined by recognition of whole tumor cells and shared tumor antigens. The role of neoantigens in driving this response is also being explored. Remarkably, 71% of vaccinated patients demonstrating ongoing remission at nearly 5 years of follow up despite a median age of 63. These results are in stark contrast to previously reported outcomes where 4-year survival of patients over the age of 55 approximates 10%. The DC/tumor vaccine platform has been studied as a strategy to induce tumor specific immunity and disease response in patients with myeloma. In a phase I trial of patients with advanced MM (median of 4 prior lines of therapy) vaccination with DC/MM fusions was feasible, was not associated with clinical evidence of autoimmunity or cytokine release syndrome, induced the 10 fold expansion of CD4 and CD8 myeloma specific T cells, and resulted in prolonged disease stabilization in nearly 70% of patients. In a subsequent study, vaccination of patients with MM following autologous transplantation induced the persistent expansion of myeloma specific T cells and was associated with the near doubling of patients achieving complete remission between day 100 and 1 year post-transplant in the absence of other maintenance therapy. Of note, based on its immunologic potency and efficacy in targeting post-transplant residual disease in a post-transplant phase II study in MM, the fusion vaccine was chosen for a multicenter randomized trial (CTN 1401). Patients undergoing autologous transplantation are randomized in the post-transplant period in a 2:1:1 pattern to one of three arms A) vaccination with DC/MM fusions and GM-CSF
in conjunction with lenalidomide maintenance; B) lenalidomice maintenance and GM-CSF; c) lenalidomide maintenance alone. Patients undergo tumor cell collection at time of initial presenation, standard induction, stem cell mobilization and collection, and high dose melphalan with stem cell rescue. Patients undergo randomization day 56-80 post-transplant. Patients assigned to the vaccine arm undergo leukapheresis, DC generation, and vaccine production. Patients begin maintenance therapy day 90-100 post-transplant. The primary aim of the study is to compare the vaccine and nonvaccine arms with respect to patients in CR ata 1 year. Secondary aims of the study include assessment of PFS at 2 years, and post-transplant conversion from PR to CR. Immunologic endpoints include an assessment of the impact of therapy on presence of myeloma specific T cells in the peripheral blood and bone marrow. Serial samples of peripheral blood and bone marrow are conducted to provide material or this analysis. 17 leading cancer centers who have been trained and validated for vaccine production as a part of an FDA approved protocol. This study provides an important precedent demonstrating feasibility and the potential for expanding the fusion vaccine platform to a multicenter setting to definitively demonstrate vaccine efficacy. A vital aspect of the design of immunotherapy clinical trials is the development of effective platforms to further characterize the nature of the immunologic response, establish assays that reflect the effect of therapy on the immunologic milieu, and most, importantly, determine those measures that most closely correlate with clinical efficacy. In addition, the identification of biomarkers predictive of immune responsiveness is critical to apply these therapies to the appropriate patient subsets. Personalized cellular therapy offers a unique platform to assess the relative contribution of immunologic response to shared as compared to neo-antigens as determinative of clinical outcome. While mutational burden has been defined as predictive of response to checkpoint inhibition in solid tumor settings, the relevance of neo-antigen burden and associated immunologic response has not been well defined for myeloma or in patients following vaccine therapy. In the present accessory study, we will examine the impact of therapy on the functional and phenotypic assessment on the T cell repertoire including clonal nature of the T cell response, the relative role of shared vs. neoantigens, the functional competence of tumor-specific T cells, and the durability of T cell expansion. In addition, we will characterize landscape of the tumor microenvironment to identify tumor- and immune-related cell subsets (effector, antigen presenting, and accessory cell populations, MM subpopulations) associating with response or resistance to the different immunotherapies. We will utilize whole genome sequencing (WGS) and RNA seq analysis to further interrogate the impact of tumor immunobiology, patterns of T cell clonality, identification of T cell receptors, and characterization of the immune repertoire on response. In this context, utilizing a platform of 60 microRNAs that impact immune presentation and regulation, including miR200c and miR34a, we will determine if levels are predictive of immune responsiveness.
chapter 1
chapter 2 2. study design

The study is a laboratory ancillary study that will further characterize the nature of the T cell response to vaccination and provide for genomic characterization of immunoregulatory aspects of the tumor and microenvironment. These assessments will occur with tumor cells acquired at time of study entry, immune cells obtained from bone marrow prior to maintenance therapy and cycle 9; T cells obtained from peripheral blood prior to cycles 1, 2, 4 and at time of disease progression (if applicable); and characterization of the immune landscape from peripheral blood obtained prior to cycle 5 of maintenance. Each of the blood and bone marrow samples are being obtained at an already designated time of bone marrow aspiration or peripheral blood draw in the parent 1401 protocol.
chapter 1
2.2. hypothesis and specific objectives

Evidence of tumor specific immunity as manifested by response to neo-antigens, oligoclonal expansion and emergence of tumor directed TCR will be more common with the vaccinated cohort and will correlate with expansion of myeloma specific T cells in the blood and achievement and maintenance of complete response at 1 year post-transplant. Similarly, genomic analysis of myeloma cells and tumor microenvironment will demonstrate a correlation between presence of immune activation pathways and microRNA and better response to vaccination.
chapter 1
2.2.2. primary objective

Determine the impact of therapy on the expansion of T cells recognizing tumor associated neoantigens and compare response between the Vaccine/GM-CSF/Lenalidomide arm and patients randomized to the Lenalidomide/GM-CSF or Lenalidomide Alone arms.
chapter 1
2.2.3. secondary objectives

Correlate of neoantigen T cell response with expansion of myeloma specific T cell and achievement and maintenance of complete remission at 1 year post-transplant Characterize T cell clonality and TCR identity following therapy Assess measures of the immune landscape and correlated with expansion of myeloma specific T cell and achievement and maintenance of complete remission at 1 year post-transplant Characterize myeloma cell expression of immunoregulatory pathways by genomic analysis and correlate with expansion of myeloma specific T cell and achievement and maintenance of complete remission at 1 year post-transplant Characterize the presence of immunoregulatory noncoding RNAs and correlate with expansion of myeloma specific T cell and achievement and maintenance of complete remission at 1 year posttransplant
chapter 1
2.3. patient eligibility

Patients must meet specified eligibility criteria outlined in section 2.3.1 and 2.3.2 to be registered on the study. All questions regarding eligibility criteria should be directed to the protocol coordinator at 301-251-1161. 2.3.1. Patient Inclusion Criteria  
1. Patients successfully enrolled in Segment A of BMT CTN 1401 2. Patients eligible for randomization per Patient Eligibility Criteria for Randomization in Chapter 2 of BMT CTN 1401 3. Patients with and available bone marrow aspirate material remaining at the BIDMC
protocol-defined laboratory and/or BMT CTN Biorepository that was collected at the time of initial enrollment on BMT CTN 1401 2.3.2. Patient Exclusion Criteria 1. Patients unable or unwilling to undergo bone marrow aspiration or peripheral blood sampling 2. Patients unable or unwilling to provide informed consent for participation on the ancillary study
chapter 1
2.4. study procedures

Patients who agree to participate in the ancillary study will provide additional blood and bone marrow samples during the course of the parent clinical trial as described below. For additional information on collection and shipment of the required samples, refer to the BMT CTN 1401 Research Sample Information Guide.
chapter 1
2.4.1. peripheral blood collection

Prior to cycles 1, 2, and 4 of maintenance, 10 mL of peripheral blood will be analyzed for Characterization of peripheral blood T cells with respect to clonality, TCR analysis, and neoantigen recognition and plasma analysis for immunoregulatory microRNA signature. This sample is collected in addition to the correlative study samples collected at these time points for the BMT CTN 1401 parent trial.  
At the time of progression, 40 mL of peripheral blood will be analyzed for characterization of marrow infiltrating T cells with respect to clonality, TCR analysis quantifying T cell subsets, and neoantigen recognition and plasma analysis for immunoregulatory microRNA signature. This sample is an additional collection time point for the ancillary study only. Samples at time of disease progression should only be collected on patients who have not yet completed their follow up on the BMT CTN 1401 parent trial.
chapter 1
2.4.2. bone marrow aspirate collection

At time of initial tumor collection 4 mL of bone marrow aspirate are stored in the BMT CTN Bioprepository for patients that consent to provide samples for future research. These samples will be obtained from the biorepository for genomic analysis of tumor cells for immune regulatory signature and determination of neoantigen targets. No additional samples are required at this timepoint. At time of randomization & prior to cycle 9 of maintenance 4 mL of bone marrow aspirate will be collected for characterization of marrow infiltrating T cells with respect to clonality, TCR analysis quantifying T cell subsets, and neoantigen recognition, assessment of density of antigen expression by myeloma cells and subsets of immunoregulatory cells and, plasma analysis for immunoregulatory microRNA signature. This sample is collected in addition to the correlative study samples collected at these time points for the BMT CTN 1401 parent trial.  
At the time of disease progression 5 *mL of bone marrow aspirate will be collected for g*enomic analysis of tumor cells for immune regulatory signature and determination of neoantigen targets, characterization of marrow infiltrating T cells with respect to clonality, TCR analysis quantifying T cell subsets, and neoantigen recognition, plasma analysis for immunoregulatory microRNA signature. This sample is an additional collection time point for the ancillary study only. Samples at time of disease progression should only be collected on patients who have not yet completed their follow up on the BMT CTN 1401 parent trial.
chapter 1
2.5. participant risks

Risks of the BMT CTN 1401 procedures are described in the parent clinical trial protocol. This ancillary study involves additional sampling of bone marrow and peripheral blood at the time these procedures are performed for other indications. Risks of additional sampling include bruising and pain at the site of sample acquisition.
chapter 3
3. study endpoints 3.1. ancillary

Bone marrow mononuclear cells will be isolated from aspirate samples obtained at enrollment to the parent study (1401) by ficoll density centrifugation and segregated into CD38+ plasma cell fraction and CD38- nonmalignant cells. Whole Genome Sequencing (WGS) and RNA sequencing will be performed on malignant plasma cell and normal cell fractions tumor samples to characterize the tumor associated mutational burden. Putative neoantigens will be identified based on the computational algorithms established by Dr. Bhasin. Putative antigens arising from mutational events are further evaluated by algorithms predicting proteasome cleavage, transport through TAP transporter and MHC affinity. The peptides with a proteasome cleavage site at their C terminal but not in the center and with high/moderate TAP binding affinity will be used for MHC binding analysis. The MHC binding analysis will be performed using HLAPred (combination of Propred/Propred1) and IEDB consensus prediction methods. The peptides that are predicted high/moderate binders by both IEDB (IC50 <500nM) and HLAPred (Score > Threshold 3%)
algorithms will be used for CTL epitope prediction using CTLPred. Candidate immunogenic peptides will be synthesized and used as antigens for pulsing autologous antigen presenting cells and testing their immunogenic potential *in vitro* using standard T cell assays. Peptides will be pulsed onto PBMCs collected prior to cycle 1, 2, and 4 of maintenance. The prevalence of antigen specific T cells will be quantified by measuring the percentage of CD4 and CD8 T cells that express IFNγ by intracellular FACS analysis. The potential role of antigenic drift in tumor immune escape will be assessed with respect to presence of the identified neo-antigens in samples obtained at disease progression. Immune response to neo-antigens and shared antigens will be compared between patients undergoing vaccination and lenalidomide maintenance alone, and who do and do not achieve CR following vaccination to correlate response with clinical outcome.  
3.1.2. Assessment of T cell clonality and function T cells will be isolated at baseline and following treatment and will be subjected to RNA seq to identify patterns of clonality as previously described. We will perform a computational algorithm to extract of TCR repertoires from bulk RNA-Seq data for quantitative analysis. Single cell analysis of cytokine expression will be assessed to further characterize the functional status of T cells.
chapter 3
3.1.3. characterization of the immune

The immunologic landscape in the peripheral blood and bone marrow will be interrogated for the bone marrow and peripheral blood sample drawn prior to cycle 1 maintenance therapy and prior to cycle 9 of maintenance. We will use a quantitative flow cytometry based analysis (QiFiKIT, Agilent) to determine the actual antigen density on the surface of MM cells. We will assess specific immune and tumor related cellular parameters. Effector cells will be characterized including CD4+ and CD8+ α/β T cells, γ/δ T cells, NK (CD3-CD56+) cells and B cells (CD19). Effector T cells will be further analyzed for the presence and temporal changes in naïve (Tn: CD45RA+,CCR7+) effector memory (Tem: CD45RACCR7+) central memory (Tcm; CD45RACCR7-) and terminally differentiated effector T cells (TEMRA; CD45RA+CCR7-) T cell activation markers including HLA-DR, CD25, CD69, CD38, CD137, Granzyme B. Regulatory cells including Tregulatory
(CD25+CD25+CD127dimFoxp3+) B regulatory cells (CD19+CD38+CD24+IL-10+) and Myeloid derived Suppressor Cells (MDSC; CD11b+CD33+ CD38+ CD14+ or CD15+) will be quantified as well as expression levels of immune checkpoint receptors PD-1, LAG3, TIM-3, CTLA-4, BTLA, TIGIT, CD47 and GITR. MM cells will be isolated from the bone marrow samples by their typical markers CD138, CD38, CD56 and analyzed for the potential markers for immune escape (PDL-1 PDL-2 (ligand PD-1), GAL-9 (ligand TIM3), HVEM ( ligand BTLA), Sirp1a (ligand CD47) and CD155 (ligand TIGIT)) and immune resistance (survivin, Xiap1, MCL-1 and NOTCH). 3.1.4. Genomic analysis to assess myeloma associated immunoregulatory pathways and clonal evolution Malignant plasma cells will be isolated from bone marrow aspirate samples obtained at time of enrollment in the parent 1401 study as previously described. Expression patterns of a previously identified panel of immune regulatory pathways will be determined. Similarly clonal evolution as evidenced by mutational pattern will be determined in patients demonstrating evidence of disease relapse. 3.1.5. Noncoding RNAs as predictors of response Noncoding RNAs such as microRNAs (miRNA) are critical mediators of oncogenesis and are now being investigated as immunoregulatory molecules. We have demonstrated that miR200c and miR34a modulate tumor expression of PDL1 and recruitment of MDSCs into the bone marrow microenvironment, respectively. Using a panel of 60 miRNA families identified by the Slack lab as having immunoregulatory properties, we will examine how their pattern of expression at baseline is predictive of subsequent immune and clinical response and how alterations in expression correlated with disease progression and immune escape. Serum obtained from bone marrow and peripheral blood samples will be assessed using the Abcam Firefly platform to quantify levels of the target miRNA families.
chapter 3
3.2. definition of disease status

Patient disease status will be assessed per Chapter 3 of the BMT CTN 1401 parent trial.
chapter 3
3.2.2. disease assessment comparison

The parent trial will assess the rates of VGPR or better (VGPR, nCR, CR and sCR) responses at specific time points in all arms. The ancillary study will perform immunologic and genomic analysis to further characterize the immunologic response, immune landscape, and immunoregulatory features of the myeloma cells at baseline. These findings will be compared between patients randomized to the Vaccine/GM-CSF/Lenalidomide arm and patients randomized to the Lenalidomide/GM-CSF or Lenalidomide Alone arms and will be correlated with achievement of CR, maintenance of CR at 1 year and PFS. 3.2.3. Progression-Free and Overall Survival The event for PFS and OS are defined in the BMT CTN 1401 parent clinical, and the same definition is used for the ancillary study.
chapter 3
chapter 4 4. patient enrollment

Patients meeting eligibility criteria defined in Chapter 2 will be consented prior to Randomization on BMT CTN 1401. Patients that are eligible will proceed with randomization on the BMT CTN 1401 parent trial. The Segment B enrollment form will include a question regarding participation on this ancillary study. A separate ancillary study enrollment form is not required. Once the patient is randomized on the BMT CTN 1401 parent trial, a schedule of assessments will be generated which will include any required data collection for the ancillary study.
chapter 3
4.2. patient evaluations and assessments

Sample collection will be tracked in GlobalTrace and on the Specimen Acquisition Form in AdvantageEDC for all required samples. Detailed procedures regarding specimen collection schedules and data entry will be found in the BMT CTN 1401 Research Sample Information Guide and the BMT CTN 1401 Forms Guide.
chapter 3
4.2.2. adverse event reporting

Any adverse events that may occur as a result of a bone marrow aspiration or peripheral blood draw performed as a part of the Ancillary study will be captured through the mechanism specified in the parent trial.  
4.2.3. Follow Up Schedule  
| 4.2.3: FOLLOW UP SCHEDULE                                                                               |                                     |
|---------------------------------------------------------------------------------------------------------|-------------------------------------|
| Study Visit                                                                                             | Target Day                          |
| Tumor Cell                                                                                              | No additional sample needed at this |
| Collection1                                                                                             | timepoint2                          |
| Randomization1                                                                                          | ±14 days from date of randomization |
| Start of Maintenance1                                                                                   | ≤ 33 days prior to initiation of    |
| Cycle 1                                                                                                 | maintenance                         |
| Cycle 21                                                                                                | ≤ 3 days prior to Day 1 of Cycle 2  |
| Cycle 41                                                                                                | ≤ 3 days prior to Day 1 of Cycle 4  |
| Cycle 91                                                                                                | ≤ 3 days prior to Day 1 of Cycle 9  |
| Disease Progression4                                                                                    | ≤ 14 days after date of progression |
| 1 Corresponds to existing BMT CTN 1401 Study Visit 2 Sample will be obtained from BMT CTN Biorepository |                                     |  
4.2.4. Schedule of Assessments The sample collection schedule for this ancillary study is outlined below and in Table 4.3.4.  
1. Bone Marrow Aspirate: A bone marrow aspirate will be collected and shipped to the BIDMC Lab as outlined below:
a. Prior to randomization (4mL) b. Prior to Cycle 9 of maintenance (4mL) c. At the time of disease progression (5mL)
2. Peripheral Blood: A peripheral blood sample will be collected and shipped to the BIDMC Lab as outlined below:
a. Prior to cycles 1, 2, and 4 of maintenance (10mL) b. At time of disease progression (40mL)  
| TABLE 4.2.4: Sample Collection Time Points Tumor Cell  Collection (At  Enrollment) Cycles of maintenance                   | Disease   |       |       |             |
|----------------------------------------------------------------------------------------------------------------------------|-----------|-------|-------|-------------|
| 1                                                                                                                          | 2         | 4     | 9     | Progression |
| Pre                                                                                                                            |           |       |       |             |
| Randomization                                                                                                              |           |       |       |             |
| Sample Collection                                                                                                          |           |       |       |             |
| Bone Marrow Aspirate                                                                                                       | X1        | X     | X     | X           |
| Total Marrow Volume (mL)                                                                                                   | 4mL       | 4mL   | 4mL   | 5mL         |
| Peripheral Blood                                                                                                           | X         | X     | X     | X           |
| Total Blood Volume (mL)                                                                                                    | 10 mL     | 10 mL | 10 mL | 40mL        |
| 1 No additional sample required. Sample will be obtained from remaining material at BIDMC lab and/or BMT CTN Biorepository |           |       |       |             |
chapter 5
5. statistical considerations 5.1. study

Determine the impact of therapy on the expansion of T cells recognizing tumor associated neoantigens and compare response between patients randomized to the Vaccine/GMCSF/Lenalidomide arm and patients randomized to the Lenalidomide/GM-CSF or Lenalidomide Alone arms.
chapter 5
5.1.2. secondary objectives

Correlate of neoantigen T cell response with expansion of myeloma specific T cell and achievement and maintenance of complete remission at 1 year post-transplant Characterize T cell clonality and TCR identity following therapy Assess measures of the immune landscape and correlated with expansion of myeloma specific T cell and achievement and maintenance of complete remission at 1 year post-transplant Characterize myeloma cell expression of immunoregulatory pathways by genomic analysis and correlate with expansion of myeloma specific T cell and achievement and maintenance of complete remission at 1 year post-transplant Characterize the presence of immunoregulatory noncoding RNAs and correlate with expansion of myeloma specific T cell and achievement and maintenance of complete remission at 1 year posttransplant
chapter 5
5.2. sample size and power

Accrual to this study is limited to patients participating in the BMT CTN 1401 trial. The target accrual for this study is 188 patients. Assuming 30% of patients will not proceed to randomization, we plan for approximately 66 patients randomized to the vaccine arm and 33 randomized to one of two control arms. Based on the prior cooperative group studies, we anticipate approximately 40% of patients will achieve CR and sCR at 1 year. In the prior phase II study of patients undergoing post-transplant vaccination in the absence of lenalidomide maintenance, CR was achieved in 29% and 54% of patients at 100 days and 1 year post-transplant respectively. Given that we anticipate that lenalidomide maintenance will enhance vaccine efficacy, we will consider the experimental arm of vaccine/GM-CSF + maintenance promising if the CR rate at 1 year is improved from 40% to 60%. A sample size of 66 patients (132 total) randomized to the vaccine arm and the combined no-vaccine arms will have 85% power to detect this improvement in the CR rate at 1 year from 40% to 60%, using a two-sample comparison of proportions with a one-sided type I error of 10%.
chapter 5
5.3. analysis of the primary

The Ancillary study will compare the expansion of peripheral blood T cells recognizing neoantigens (prior to vaccination to peak treatment levels) in patients receiving the DC/MM fusion vaccine as compared to standard maintenance patients. We will quantify neo-antigen specific T cells by determining expression of IFNg in response to exposure to neoantigen peptides in samples obtained prior to and following study therapy. We hypothesize that vaccination will result in the significant expansion of these T cells in contrast to patients receiving nonvaccine maintenance therapy. Of note, lenalidomide may amplify the presence of the T cell population somewhat via nonspecific immune stimulation of tumor specific populations that are observed during posttransplant lymphopoietic reconstitution.  
The primary endpoint of log 10 peak change in tumor reactive T cells from baseline will be compared across the 3 groups using analysis of variance, and if significant then two-sample t-tests will be conducted for each pairwise comparison to determine whether the vaccine and nonvaccine groups are different from one another. A secondary analysis of peak change in tumor reactive T cells will be done by comparing the proportions of patients who experience >10-fold increase in IFNγ expression, using a chi-squared test. Myeloma reactive T- cell response profiles over time will be compared between the treatment groups using linear mixed models for repeated measures data. Peak immune response as well as most recent immune response will be compared between those in CR and those not in CR at each assessment, using a two-sample t-test, to assess the relationship between immune response and clinical response. Peak and most recent immune response will also be considered as time-dependent covariates in a Cox proportional hazards model to assess their impact on progression-free survival. We will assess whether there is a 2 fold difference in the percentage of the vaccine and nonvaccine groups achieving a minimum 10-fold increase of T-cell recognizing neoantigens, keeping power = 85%, alpha = 0.10, using 2-sided Fisher's exact test. For instance, if 3 out of 15 pts (20%) in non-vaccine group have at least 10-fold increase of T-cell recognizing neoantigens", then with 85% power and at alpha = 0.10 we can say vaccine group will have 66% of patients that have "at least 10-fold increase of T-cell recognizing neoantigens" and detecting a difference of about 46% (20% vs 66%) between non-vaccine group (n1 = 15) and vaccine group (n2 = 30) .  
| (post / pre >= 2) of T-cell recognizing neoantigens in non-vaccine group.  S1 S2 S3 S4 S5 S6 S7   | S8         |        |        |        |        |        |        |        |
|---------------------------------------------------------------------------------------------------|------------|--------|--------|--------|--------|--------|--------|--------|
| Vaccine/GMCSF/Lenalidomide                                                                                                   | 31%        | 46%    | 57%    | 66%    | 73%    | 78%    | 84%    | 89%    |
| Non-Vaccine                                                                                       | d          | =      |        |        |        |        |        |        |
| Group1                                                                                            | 0/15  (0%) | d=1/15 | d=2/15 | d=3/15 | d=4/15 | d=5/15 | d=6/15 | d=7/15 |
| (7%)                                                                                              | (13%)      | (20%)  | (27%)  | (33%)  | (40%)  | (47%)  |        |        |
| 1 Includes patents randomized to both the Lenalidomide/GM-CSF arm and the Lenalidomide Alone arm. |            |        |        |        |        |        |        |        |
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5.4. analysis of the secondary

Additional exploratory analyses will be conducted in a similar fashion to examine a number of secondary immunologic endpoints, including: 1) clonality assessment and TCR identification of T cells, 2) T cell cytokine expression 3) immune landscape analysis 4) genomic analysis of tumor immunoregulatory pathways 5) patterns of immunoregulatory microRNA expression. In each case, log transformations will be considered to induce normality, and if still non-normal then nonparametric tests will be used. Profiles of these secondary immunologic endpoints will be described at each time point for each group using summary statistics and compared between the vaccine and non-vaccine groups using mixed models for repeated measures data. The correlation between the immune environment measures and the myeloma specific T-cell response will be assessed and reported using Spearman rank correlation at each time point. Correlation between immune parameters and clinical outcome as measured by achievement and maintenance of CR at 1 year and PFS will be similarly determined. After the PFS and CR data is analyzed in the parent trial, they will be available for analysis in this ancillary study.
chapter 5
ancillary study refernces

1. Bolli, N., et al., *Heterogeneity of genomic evolution and mutational profiles in multiple myeloma.* Nat Commun, 2014. 5: p. 299 2. Melchor, L., et al., Single-cell genetic analysis reveals the composition of initiating clones and phylogenetic patterns of branching and parallel evolution in myeloma. Leukemia, 2014. 28(8): p. 1705-15.  
3. Uttenthal, B., et al., Wilms' Tumour 1 (WT1) peptide vaccination in patients with acute myeloid leukaemia induces short-lived WT1-specific immune responses. Br J Haematol, 2014. 164(3): p. 366-75.  
4. Kuball, J., et al., Pitfalls of vaccinations with WT1-, Proteinase3- and MUC1-derived peptides in combination with MontanideISA51 *and CpG7909.* Cancer Immunol Immunother, 2011. 60(2): p. 161-71.  
5. Gong, J., et al., Activation of antitumor cytotoxic T lymphocytes by fusions of human dendritic cells and breast carcinoma cells. Proc Natl Acad Sci U S A, 2000. 97(6): p. 2715-8.  
6. Avigan, D., *Dendritic cell-tumor fusion vaccines for renal cell carcinoma.* Clin Cancer Res, 2004. 10(18 Pt 2): p. 6347S-52S.  
7. Rosenblatt J et al. Individualized vaccination of AML patients in remission is associated with induction of antileukemia immunity and prolonged remissions. Sci Transl Med. 2016 Dec 8. Rosenblatt, J., et al., *Vaccination with dendritic cell/tumor fusion cells results in cellular and humoral* antitumor immune responses in patients with multiple myeloma. Blood. 117(2): p. 393-402. 19 9. Rosenblatt, J., et al., *Vaccination with dendritic cell/tumor fusions following autologous stem cell* transplant induces immunologic and clinical responses in multiple myeloma patients. Clin Cancer Res, 2013. 19(13): p. 3640-8.  
Version 4.0 dated July 24, 2018
chapter 5
appendix l

REFERENCES  
1 Ratta, M. *et al.* Dendritic cells are functionally defective in multiple myeloma: the role of interleukin-6. *Blood* 100, 230-237 (2002).  
2 Sharma, A., Khan, R., Joshi, S., Kumar, L. & Sharma, M. Dysregulation in T helper 1/T helper 2 cytokine ratios in patients with multiple myeloma. *Leuk Lymphoma* 51, 920-927, doi:10.3109/10428191003699563 (2010).  
3 Joshua, D. E., Brown, R. D., Ho, P. J. & Gibson, J. Regulatory T cells and multiple myeloma.  
Clin Lymphoma Myeloma 8, 283-286, doi:10.3816/CLM.2008.n.039 (2008).  
4 Beyer, M. *et al.* In vivo peripheral expansion of naive CD4+CD25high FoxP3+ regulatory T
cells in patients with multiple myeloma. *Blood* 107, 3940-3949, doi:10.1182/blood-2005-093671 (2006).  
5 Prabhala, R. H. *et al.* Elevated IL-17 produced by TH17 cells promotes myeloma cell growth and inhibits immune function in multiple myeloma. *Blood* 115, 5385-5392, doi:10.1182/blood2009-10-246660 (2010).  
6 Nielsen, H. *et al.* Immune dysfunction in multiple myeloma. Reduced natural killer cell activity and increased levels of soluble interleukin-2 receptors. *APMIS* 99, 340-346 (1991).  
7 Avigan, D. *et al.* Fusion cell vaccination of patients with metastatic breast and renal cancer induces immunological and clinical responses. *Clin Cancer Res* 10, 4699-4708 (2004).  
8 Gong, J. *et al.* Activation of antitumor cytotoxic T lymphocytes by fusions of human dendritic cells and breast carcinoma cells. *Proc Natl Acad Sci U S A* 97, 2715-2718. (2000).  
9 Gong, J., Chen, D., Kashiwaba, M. & Kufe, D. Induction of antitumor activity by immunization with fusions of dendritic and carcinoma cells. *Nat Med* 3, 558-561. (1997).  
10 Vasir, B. *et al.* Fusion of dendritic cells with multiple myeloma cells results in maturation and enhanced antigen presentation. *Br J Haematol* 129, 687-700 (2005).  
11 Gong, J. *et al.* Reversal of tolerance to human MUC1 antigen in MUC1 transgenic mice immunized with fusions of dendritic and carcinoma cells. *Proc Natl Acad Sci U S A* 95, 62796283. (1998).  
12 Gong, J. *et al.* Immunization against murine multiple myeloma with fusions of dendritic and plasmacytoma cells is potentiated by interleukin 12. *Blood* 99, 2512-2517. (2002).  
13 Rosenblatt, J. *et al.* Vaccination with dendritic cell/tumor fusion cells results in cellular and humoral antitumor immune responses in patients with multiple myeloma. *Blood* 117, 393-402, doi:10.1182/blood-2010-04-277137 (2011).  
14 Rosenblatt, J. *et al.* Vaccination with dendritic cell/tumor fusions following autologous stem cell transplant induces immunologic and clinical responses in multiple myeloma patients. Clin Cancer Res 19, 3640-3648, doi:10.1158/1078-0432.CCR-13-0282 (2013).  
15 McCarthy, P. L. *et al.* Lenalidomide after stem-cell transplantation for multiple myeloma. N
Engl J Med 366, 1770-1781, doi:10.1056/NEJMoa1114083 (2012).  
16 Luptakova, K. *et al.* Lenalidomide enhances anti-myeloma cellular immunity. *Cancer* Immunol Immunother 62, 39-49, doi:10.1007/s00262-012-1308-3 (2013).  
17 Davies, F.E. *et al,* Thalidomide and immunomodulatory derivative augment natural killer cell cytotoxicity in multiple myeloma. *Blood,* 210-216 (2001)
18 WAI, L.E. *et al,* Distinct roles for the NK cell-activating receptors in mediating interactions with dendritic cells and tumor cells. J *Immunol,* 222-229, doi: 10.4049/jimmunol.1002597 (2010).  
19 BGM Durie *et al.* International uniform response criteria for multiple myeloma. *Leukemia*
(2006) 1-7.