Clinical trial NCT03006848
Conditions: Osteosarcoma
Keywords: Osteosarcoma PD-L1 Immunotherapy
Sponsor: St. Jude Children's Research Hospital
Start date: 2017-02-16; Completion date: 2020-03-18
Enrollment: 19 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This clinical trial seeks to determine if avelumab will be effective in facilitating removal of all gross tumor in the event of a relapse of osteosarcoma in pediatric patients. Avelumab will be evaluated using dosing that has previously been determined in adult studies.

Primary Objectives:

* To estimate the response rate to 4 cycles of avelumab in patients with recurrent or progressive osteosarcoma.
* To estimate the 16-week progression free survival of patients with recurrent or progressive osteosarcoma after treatment with avelumab.

Secondary Objective:

* To describe the toxicities associated with the administration of avelumab in patients with recurrent or progressive osteosarcoma.
* To assess the quality of life of patients with recurrent or progressive osteosarcoma undergoing treatment with avelumab, and to explore relationships between clinical factors and patient-reported health-related quality of life (HRQOL) outcomes.

Exploratory Objectives:

* To explore factors associated with response in patients treated with avelumab after recurrent or progressive osteosarcoma (e.g. tumor PD-L1 expression).
* To measure parameters of immune activation including subsets of peripheral blood mononuclear cells (PBMCs) and serum markers of immune activation.
* To evaluate the role of T-cells in immune checkpoint blockade via measures of cell proliferation, co-inhibitory receptor expression on CD8 T cells, T cell repertoire, and epigenetic programming.

Detailed description:
This is a Phase 2 study using a traditional Simon two-stage design. Patients 12 years or greater with recurrent/refractory osteosarcoma will be administered avelumab at a dose of 10 mg/kg intravenously (IV) over 60 minutes on days 1 and 15 of each cycle, with a cycle lasting 28 days.

Patients will receive avelumab every 2 weeks in cycles of 28 days for up to 24 months, or 26 cycles. Progression free survival and response to therapy after 4 cycles of treatment will be assessed. In addition, the toxicity profile of avelumab in this population will be closely monitored.

Arms: arm 1: All participants with recurrent/refractory osteosarcoma who consent to the study.

Interventions: Avelumab and quality of life questionnaires.
Interventions: intervention 1: Avelumab intervention 2: Questionnaires
Intervention descriptions: intervention 1: Patients will be administered avelumab at a dose of 10 mg/kg intravenously (IV) over 60 minutes on days 1 and 15 of each cycle, with a cycle lasting 28 days. Patients will receive avelumab every 2 weeks in cycles of 28 days for up to 24 months, or 26 cycles. intervention 2: To assess quality of life, patients will complete questionnaires at four time points.

Protocol documents:
Prot_SAP_000.pdf: 
OSTPDL1
Revision 1.1, dated: 01-19-2018 
IRB approved: 01-26-2018
Protocol document dated: 01-19-2018
St. Jude
OSTPDL1
Initial version, dated: 07-29-2016. Resubmitted to CT-SRC 09-27-2016. 
Resubmitted to IRB 10-21-2016 and 11-16-2016 (IRB approved: 12-13-2016) 
Activated: January 3, 2017
Amendment 1.0, dated: 04-06-2017 (IRB approved: 05-09-2017)
Activated: June 16, 2017
Revision 1.1, dated 01-19-2018 (IRB approved: 01-26-2018)
Activated: 
A PHASE II TRIAL OF AVELUMAB, A FULLY HUMAN ANTIBODY THAT 
TARGETS CELLS EXPRESSING PD-L1 IN PATIENTS WITH RECURRENT OR 
PROGRESSIVE OSTEOSARCOMA
IND# 132,874 (sponsor: St. Jude)
Principal Investigator
Michael W. Bishop, MD, MS
Department of Oncology
Solid Tumor Division
St. Jude Sub-Investigators
Alberto S. Pappo, MD
Department of Oncology
Solid Tumor Division
Deo Kumar Srivastava, PhD
Guolian Kang, PhD
Zhaohua Lu, PhD
Department of Biostatistics
Jennifer L. Harman, PhD
Department of Psychology
Paul Thomas, PhD
Benjamin Youngblood, PhD
Department of Immunology
Sue Kaste, DO
Michael Doubrovin, MD, PhD
Department of Diagnostic Imaging
St. Jude Children's Research Hospital
262 Danny Thomas Place
Memphis, Tennessee  38105-3678
Telephone:  (901) 595-3300
St. Jude
IRB NUMBER: Pro00006856
IRB APPROVAL DATE: 06/26/2019


OSTPDL1
Revision 1.1, dated: 01-19-2018 
IRB approved: 01-26-2018
Protocol document dated: 01-19-2018
Contents of this document may not be extracted without permission from the Principal 
Investigator
St. Jude
IRB NUMBER: Pro00006856
IRB APPROVAL DATE: 06/26/2019


OSTPDL1
Revision 1.1, dated: 01-19-2018 
IRB approved: 01-26-2018
Protocol document dated: 01-19-2018
List of Collaborating Investigators/Institutions
Contact Information
Leo Mascarenhas, MD, MS
Lee J. Helman, M.D.
Keck School of Medicine
University of Southern California
Children's Hospital Los Angeles
4650 Sunset Blvd 
Mail Stop 57
Los Angeles, CA 90027
Ph: 323-361-6058
lmascarenhas@chla.usc.edu
lhelman@chla.usc.edu
James L. Gulley, M.D., Ph.D.
Center for Cancer Research
National Cancer Institute
Building 10, Room 12N226
Bethesda, MD 20892-1750
Ph: 301-435-2956
gulleyj@mail.nih.gov
Jeffrey Schlom, PhD
Caroline Jochems, MD, PhD
Renee Donahue, PhD
Laboratory of Tumor Immunology and 
Biology
Center for Cancer Research
National Cancer Institute
Building 10, Room 8B09
Bethesda, MD 20892-1750
Ph: 301-496-4343
js141@nih.gov
Sarah B. Whittle, MD
Lisa L. Wang, MD
Nino Rainusso, MD
Texas Children’s Cancer Center
Baylor College of Medicine
1102 Bates Street, Suite 1240.12
Houston, TX
Ph: 832-824-4822
sbwhittl@txch.org
llwang@txch.org 
ncrainuss@txch.org
Filemon Dela Cruz, MD
Paul A. Meyers, MD
Alexander J Chou, MD
Memorial Sloan Kettering Cancer 
Center
1275 York Ave #1408
New York, NY 10065
212-639-5952
delacrf1@mskcc.org
meyersp@mskcc.org
choua@mskcc.org 
St. Jude
IRB NUMBER: Pro00006856
IRB APPROVAL DATE: 06/26/2019


OSTPDL1
Revision 1.1, dated: 01-19-2018 
IRB approved: 01-26-2018
Protocol document dated: 01-19-2018
PROTOCOL SUMMARY
OSTPDL1, A Phase II Trial of Avelumab, a Fully Human Antibody that Targets 
Cells Expressing PDL1 in Patients with Recurrent or Progressive 
Osteosarcoma
Principal Investigator: Michael W. Bishop, MD, MS
IND holder: St. Jude Children’s Research Hospital, IND# 132,874.
Brief background and overview of study: Patients with recurrent or progressive 
osteosarcoma have a poor overall survival rate and are unlikely to achieve cure 
without complete surgical remission. The introduction of new cytotoxic and 
biologic agents has failed to show efficacy in this population. Immunotherapy with 
checkpoint inhibitors has demonstrated dramatic changes in outcomes in several 
adult cancers and may provide greater benefit for tumors with high mutational 
load. The purpose of this phase II study is to assess the efficacy of avelumab for 
the treatment of patients with recurrent or progressive osteosarcoma who have 
measurable residual disease.
Intervention: Single agent avelumab.
Brief outline of treatment plan: Patients will receive avelumab every 2 weeks in 
cycles of 28 days for up to 24 months, or 26 cycles. Progression free survival and 
response to therapy after 4 cycles of treatment will be assessed. In addition, the 
toxicity profile of avelumab in this population will be closely monitored.
Study design: Multi-center phase II study.
Sample size: 32 evaluable patients; 40 total patients.
Data management: Data management and statistical analysis will be provided 
locally by the Comprehensive Cancer Center Developmental Biology Solid Tumors 
Program, and Biostatistics Department at St. Jude Children’s Research Hospital.
Human subjects: The main risk to research participants will be the potential 
toxicities associated with the use of the investigational agent, avelumab. Research 
participants will be informed of toxicities which have been associated with the 
study drug and potential side effects of procedures recommended in this study. 
Adverse events will be monitored, treated, and reported following institutional and 
federal guidelines and regulations.
St. Jude
IRB NUMBER: Pro00006856
IRB APPROVAL DATE: 06/26/2019


OSTPDL1
Revision 1.1, dated: 01-19-2018 
IRB approved: 01-26-2018
Protocol document dated: 01-19-2018
EXPERIMENTAL DESIGN SCHEMA
*Avelumab will be given on Days 1 & 15 of each 28 day cycle
Unresected recurrent or 
progressive osteosarcoma
Study entry
Cycle 1-2
Avelumab
Cycle 3-4
Avelumab
Avelumab*
Continue every 2 weeks 
for up to 24 months, or 
26 cycles
Evaluation every 3 
months
End protocol therapy
Evaluation
Evaluation
Progressive 
disease or 
unacceptable 
toxicity (at any 
time)
Off protocol 
therapy
Progressive 
disease or 
unacceptable 
toxicity
Off protocol 
therapy
Stable 
or 
respon
se
Stable 
or 
respon
se
St. Jude
IRB NUMBER: Pro00006856
IRB APPROVAL DATE: 06/26/2019


OSTPDL1
Revision 1.1, dated: 01-19-2018 
IRB approved: 01-26-2018
Protocol document dated: 01-19-2018
TABLE OF CONTENTS
1.0 OBJECTIVES.....................................................................................1
1.1 Primary Objectives................................................................................1
1.2 Secondary Objective.............................................................................1
1.3 Exploratory Objectives.........................................................................1
2.0 BACKGROUND AND RATIONALE.....................................................1
2.1 Background..........................................................................................1
2.2 Preclinical Studies................................................................................4
2.3 Clinical Studies.....................................................................................6
2.4 Rationale for this Study........................................................................9
2.5 Overview of Proposed Study.................................................................9
2.6 Rationale for Correlative Biology Studies...............................................9
2.7 Rationale for Quality of Life Studies....................................................13
3.0 ELIGIBILITY CRITERIA AND STUDY ENROLLMENT........................14
3.1 Inclusion Criteria................................................................................15
3.2 Exclusion Criteria...............................................................................16
3.3 Recruitment and Screening.................................................................18
3.4 Enrollment at St. Jude.........................................................................18
3.5 Enrollment at Collaborating Sites........................................................18
4.0 TREATMENT PLAN........................................................................18
4.1 Overview of Treatment Plan................................................................18
4.2 Treatment During Cycle 1 and Subsequent Cycles..............................18
5.0 DOSE MODIFICATIONS FOR TOXICITIES.......................................20
5.1 Adverse Drug Reactions Requiring Avelumab Discontinuation or 
Modification................................................................................................20
5.2 Treatment Modification for Symptoms of Infusion-Related Reactions.21
5.3 Management of Immune-Mediated Adverse Reactions........................22
5.4 Autoimmune or Immune System Disorders Affecting Other Organ 
Systems.......................................................................................................28
5.5 Central Venous Catheter.....................................................................28
5.6 Concomitant Therapy.........................................................................28
6.0 DRUG INFORMATION....................................................................28
6.1 Avelumab (MSB0010718C, anti-PD-L1)..............................................28
7.0 REQUIRED EVALUATIONS, TESTS, AND OBSERVATIONS..............29
7.1 Evaluations Before and During Therapy..............................................29
7.2 Extended Safety Follow-up.................................................................31
8.0 CORRELATIVE RESEARCH STUDIES...............................................31
8.1 Tumor Tissue.....................................................................................31
8.2 Immunologic Research – NCI Testing Battery......................................31
8.3 Immunologic Research – St. Jude Testing Battery................................35
9.0 EVALUATION CRITERIA.................................................................37
9.1 Response Definitions and Criteria for Solid Tumors............................37
9.2 Response Criteria for Patients with Solid Tumor and Measurable Disease
40
9.3 Response Criteria for Evaluable Disease..............................................41
9.4 Best Response....................................................................................41
9.5 Duration of Response.........................................................................42
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IRB APPROVAL DATE: 06/26/2019


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9.6 Toxicity Evaluation Criteria.................................................................43
10.0OFF TREATMENT AND OFF STUDY CRITERIA...............................43
10.1 Off Treatment Criteria........................................................................43
10.2 Off Study Criteria...............................................................................43
11.0SAFETY AND ADVERSE EVENT REPORTING REQUIREMENTS........44
11.1 Reporting Adverse Experiences and Deaths to St. Jude IRB..................44
11.2 Reporting to St Jude IRB......................................................................46
11.3 Reporting from St. Jude to Pfizer (Drug Manufacturer)........................46
11.4 Reporting to St. Jude Regulatory Affairs Office and FDA......................47
11.5 Recording Adverse Events and Serious Adverse Events.......................47
11.6 Process for Reporting Adverse Events from and to Collaborating Sites48
12.0DATA COLLECTION, MONITORING AND CONFIDENTIALITY........49
12.1 Data Collection...................................................................................49
12.2 Study Monitoring................................................................................49
12.3 Confidentiality....................................................................................50
13.0STATISTICAL CONSIDERATIONS...................................................50
13.1 Study Design......................................................................................50
13.2 Secondary Objectives.........................................................................52
13.3 Exploratory Objectives.......................................................................52
13.4 Anticipated Completion Dates............................................................54
14.0OBTAINING INFORMED CONSENT.................................................54
14.1 Consent Prior to Research Interventions.............................................54
14.2 Consent at Enrollment........................................................................54
14.3 Consent at Age of Majority.................................................................55
14.4 Consent When English is Not the Primary Language............................55
14.5 Collection of Collaborating Institution Consent Forms........................55
15.0REFERENCES..................................................................................56
APPENDICES
Appendix I:Performance Status Scales/Scores
Appendix II:New York Heart Association Functional Classification
Appendix III: 
Instructions for Collection and Shipping of Research Blood 
Samples to NCI
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1.0 OBJECTIVES
1.1 Primary Objectives
1.1.1To estimate the response rate to 4 cycles of avelumab in patients 
with recurrent or progressive osteosarcoma.
1.1.2To estimate the 16-week progression free survival of patients with 
recurrent or progressive osteosarcoma after treatment with 
avelumab.
1.2 Secondary Objective
1.2.1To describe the toxicities associated with the administration of 
avelumab in patients with recurrent or progressive osteosarcoma.
1.3
Exploratory Objectives
1.3.1To explore factors associated with response in patients treated with 
avelumab after recurrent or progressive osteosarcoma (e.g. tumor 
PD-L1 expression).
1.3.2To measure parameters of immune activation including subsets of 
peripheral blood mononuclear cells (PBMCs) and serum markers of 
immune activation.
1.3.3To evaluate the role of T-cells in immune checkpoint blockade via 
measures of cell proliferation, co-inhibitory receptor expression on 
CD8 T cells, T cell repertoire, and epigenetic programming. 
1.3.4To assess the quality of life of patients with recurrent or 
progressive osteosarcoma undergoing treatment with avelumab, 
and to explore relationships between clinical factors and patient-
reported HRQOL outcomes.
2.0 BACKGROUND AND RATIONALE
2.1 Background 
Osteosarcoma is the most common bone tumor in children and 
adolescents. It is estimated there are approximately 400 new cases of 
pediatric osteosarcoma diagnosed in the United States each year.1 Nearly 
70% of children who are treated with osteosarcoma with current therapies 
that include multimodal chemotherapy with high dose methotrexate, 
doxorubicin, and cisplatin (MAP) and surgery can be cured from their 
disease.2 However, patients who present with metastatic disease or 
relapse continue to fare poorly with estimated survival rates that do not 
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exceed 30%.3-6 Furthermore, for patients who relapse, cure is almost 
impossible if they cannot be deemed surgically free of disease.
3 This 
observation supports the need for identification of active agents that can 
facilitate removal of all gross tumor in the event of a relapse. In a study 
by the German Cooperative Osteosarcoma Study Group, the overall 5-
year survival after a first relapse of osteosarcoma was 23% and the 
survival for patients who did not have a second surgical complete 
response was 0%.
3 In a report from the same group, five year overall and 
event free survival rates for patients who experienced a second or 
subsequent relapses were only 32% and 18% at the time of second 
recurrence and 25% and 0% for those who experienced a third 
recurrence.
7 Other studies have shown similar results. For example, in a 
report of 235 patients treated at the Rizzoli Institute, 5 year post-relapse 
event-free and overall survival were 27% and 28%, respectively.8 
Strategies to improve the outcome of patients with metastatic disease 
and recurrent disease have been largely ineffective. In a study of 43 
patients with osteosarcoma who experienced a first pulmonary 
recurrence and who were treated with inhaled GM-CSF, the 3-year event 
free and overall survival were 7.8 and 35.4% respectively.
9 In another 
study that utilized R1507, a monoclonal antibody against the insulin-like 
growth factor-1 receptor, only 2 of 38 patients with osteosarcoma 
experienced an objective response and the median time to progression 
was 5.7 weeks.10 Recently, an analysis was conducted to assess the 
outcomes for children with recurrent or refractory osteosarcoma who 
were enrolled in a series of phase II studies within the Children’s 
Oncology Group (COG) from 1997 to 2007. These studies included the 
agents topotecan, imatinib, oxaliplatin, ixabepilone, docetaxel, irinotecan 
and rebeccamycin. Radiographic responses were seen in only 3 trials with 
the highest response rate being 11% in patients who were treated with 
topotecan. When pooled across all 7 studies, event free survival for 96 
osteosarcoma subjects with measurable disease was 12% at 4 months 
(95% CI 6.0%-19%).
11 This value is now being used to define a benchmark 
for activity of new compounds in the COG and is being incorporated into 
the design of phase II trials for recurrent osteosarcoma. Using this newly 
adopted endpoint, the COG recently conducted a study of the 
halichondrin B analog eribulin mesylate for patients with metastatic and 
recurrent osteosarcoma. This agent was selected as a result of preclinical 
observations of the Pediatric Preclinical Testing Program of the National 
Cancer Institute (NCI).12 Despite very rapid accrual of 19 patients over a 4 
month period, the efficacy endpoint described above was not met and the 
study is closing. However, the rapid enrollment of patients with recurrent 
osteosarcoma reflect the urgent need to develop novel therapies that 
could be incorporated not only in the relapsed setting but in patients with 
metastatic or newly diagnosed disease, and the willingness of these 
patients and families to participate in investigations of novel agents.
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Other strategies aimed at improving outcome of patients with 
osteosarcoma based on histologic response parameters have also been 
largely unsuccessful. It is well known that the degree of tumor necrosis 
following neoadjuvant chemotherapy is a predictor of clinical outcome in 
childhood osteosarcoma. Thus this parameter has been used as an 
indirect measure of clinical outcome and has been exploited in an 
attempt to improve survival of these patients, via augmentation of post-
operative chemotherapy for patients whose tumors demonstrate an 
inadequate response to pre-operative treatment. This was one of the key 
objectives of the recent EURAMOS trial, an international prospective 
clinical trial encompassing American and European collaborative oncology 
groups, in which patients were stratified after surgery to receive standard 
or modified therapy based on histologic response to upfront treatment 
with MAP chemotherapy. Unfortunately, the results failed to show any 
significant improvement in the outcome of patients who were treated 
with interferon after they achieved a favorable histologic response to MAP 
chemotherapy, or were given additional courses of chemotherapy with 
ifosfamide and etoposide if they experienced a poor histologic 
response.
13,14
Genomic analysis of osteosarcoma has demonstrated that this tumor is 
genomically complex with a large number of somatic non-silent 
mutations averaging 25-32 mutations per case and 1.2 mutations per 
megabase.15,16 In addition, osteosarcoma has a high number of structural 
variations that exceeds the number seen in other pediatric tumors such 
as embryonal rhabdomyosarcoma.15 The majority of osteosarcomas carry 
an aberration in the TP53 pathway, and 50% have hypermutable regions 
known as kataegis.15 Additional recurrent gene alterations found in two 
studies of osteosarcoma include RB1, DLG2, and ATRX. Although 
approximately 24% of osteosarcoma have alterations in the PI3K/mTOR 
pathway characterized by variable alterations of PTEN, or mutations in 
TSC2, NF1, PI3KCA, AKT, or PDPK1,16 no clinical studies targeting this 
pathway in osteosarcoma are currently active.
The use of immunotherapy to treat malignant diseases has been 
advanced in recent years with the use of checkpoint inhibitors, 
particularly with the advent of monoclonal antibodies directed against 
cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and  programmed 
cell death protein 1 (PD-1), both immunomodulatory receptors expressed 
on T cells,17 as well as the ligand for PD-1 (PD-L1) that has been 
identified on a number of tumor types. Dramatic responses and long term 
cures have been documented in patients with various adult malignancies 
including melanoma, non-small cell lung cancer, Hodgkin lymphoma, 
renal cell carcinoma, and colorectal carcinoma.
18-23 Studies using 
checkpoint inhibitors suggest that the response to these agents might be 
in part related to the expression of PD-L1 in tumor tissue; however, the 
combined use of CTLA-4 and PD-1 inhibitors may overcome the lower 
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response rates seen in patients whose tumors had negative PD-L1 
expression and were treated with single agent nivolumab,24 suggesting 
that PD-L1 might not be an optimal surrogate biomarker of response to 
these agents. In addition, it is likely that inducible PD-L1 expression 
appears to be more stringently correlated with PD-1 blockade response 
as a result of adaptive immune resistance.25 More recently, tumor specific 
non-synonymous mutations have been identified as being immunogenic; 
the majority of this “mutanome” is recognized by CD4+ T cells. To this 
effect, increased mutational burden in melanoma has been associated 
with responses to CTLA-4 blockade; however, increased mutational load 
alone may not explain the benefit seen in these patients. The presence of 
specific unique neoepitope signatures that confer higher rates of MHC I 
class binding appears to be essential for achievement of clinical 
response.
26,27 The dramatic response to checkpoint inhibitors in patients 
with mutations in mismatch repair deficient colorectal cancer supports 
the observation that a high mutational load is associated with the number 
of mutation-associated antigens that can trigger an immune response 
against the tumor.19
The high mutational load in osteosarcoma makes this tumor an attractive 
target for immunotherapy. Recent work has demonstrated that human 
metastatic osteosarcoma cells express PD-L1; populations of PD-L1 
positive cells were observed in 12 of 16 metastatic tumors by 
immunofluorescence. The presence of PD-L1 may decrease the activity of 
cytotoxic T lymphocytes (CTLs) expressing PD-1; blockade of PD-1/PD-
L1 improved CTL function in vitro and in vivo. In a K7M2 mouse model of 
osteosarcoma this resulted in decreased tumor burden and prolonged 
survival.28 In addition, in an implantable model of metastatic 
osteosarcoma, T cells infiltrating PD-L1 antibody-resistant tumors up-
regulate additional inhibitory receptors, notably CTLA-4, which impairs 
their ability to mediate tumor rejection. Combinations of α-CTLA-4 and 
α-PD-L1 antibody blockade in the K7M2 mouse model of metastatic 
osteosarcoma produces complete control of tumors in a majority of mice 
as well as immunity to further tumor inoculation.29 In another study an 
RNA-based assay to determine PDL1 expression levels showed 32 of 38 
osteosarcoma tumors expressed PDL1; high levels (3 log or more) of 
PDL1 were expressed in 9 tumor samples. PDL1 expression was positively 
correlated with tumor infiltrating lymphocytes (TILs).30 While data is 
limited on the clinical activity of PD-1 or PD-L1 inhibition in patients with 
osteosarcoma, a recent report from a clinical trial utilizing the Merck PD-
1 inhibitor pembrolizumab in patients with advanced bone and soft tissue 
sarcomas demonstrated a partial response in 1 of 19 enrolled 
osteosarcoma subjects at 8 weeks; limited data on progression free 
survival was available.31
2.2 Preclinical Studies
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2.2.1 Avelumab
Avelumab (also referred to as MSB0010718C) is a fully human IgG-1 
antibody which is directed against PD-L1. Avelumab binds PD-L1 and 
blocks the interaction between PD-L1 and its receptors PD-1 and B7-1. 
The interaction between PD-L1 and PD-1/B7-1 significantly inhibits 
activity of T cells. While PD-L1 can be detected on resting and activated T 
cells, B cells, macrophages, dendritic cells and mast cells, tumor cells 
may also express high levels of PD-L1 on their surface compared with 
normal tissues.32 High levels of PD-L1 expression have been found to be 
associated with disease progression, increased metastasis, poor response 
to treatment, and decreased survival in a number of human cancers.
33 
Blockade of PD-L1 removes the suppressive effects of PD-L1 on anti-
tumor CD8+ T cells, resulting in the restoration of cytotoxic T cell 
response and elimination of tumor cells. Furthermore, as a fully human 
IgG1 monoclonal antibody, avelumab has the potential to facilitate tumor 
cell killing via tumor-directed antibody driven cellular cytotoxicity 
(ADCC).
2.2.2Preclinical Pharmacology Studies
Avelumab functionally enhances T cell activation in vitro and significantly 
inhibits the growth of PD-L1 expressing tumors in vivo. Avelumab binds 
to human PD-L1 with a high affinity of 0.7 nM and competitively blocks 
the interaction of PD-L1 with PD-1. In vitro study results showed that by 
binding to PD-L1, avelumab effectively enhances T cell activation as 
measured by interleukin-2 (IL-2) or interferon-gamma (IFN-γ) 
production. As a monotherapy, avelumab demonstrated anti-tumor 
activity against murine MC38 colon carcinoma tumors characterized by 
high level of PD-L1 expression with an observed dose-dependent trend 
when given every third day for a total of 3 doses. Dosing was determined 
to be 400 µg, approximating to 20 mg/kg. In vivo anti-tumor effects 
were primarily mediated by CD8+ T cells as evidenced by observation 
that in vivo depletion of CD8+ T cells abrogated anti-tumor efficacy. The 
contribution of ADCC as a mechanism of anti-tumor activity was 
demonstrated via use of a deglycosylated version of avelumab to 
abrogate Fc receptor binding, and via systemic depletion of natural killer 
(NK) cells. In both settings, loss of in vivo ADCC potential significantly 
reduced anti-tumor activity.
The combination of avelumab with commonly used cancer treatments, 
such as cytotoxic agents and radiation therapy, resulted in improved 
anti-tumor activity. The most promising combination partners for 
avelumab were gemcitabine or 5-fluorouracil and oxaliplatin, the core 
components of FOLFOX. Radiation therapy was found to be highly 
synergistic with avelumab, capable of causing complete regression of 
established tumors.
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Various immunomonitoring assays were incorporated into the in vivo 
studies. Treatment with avelumab resulted in a consistent increase in the 
percentage of CD8+PD-1+ T cells and an increased frequency of CD8+ T 
cells with an effector memory (TEM) phenotype as determined by flow 
cytometry. These changes correlated with anti-tumor effect. Increases in 
tumor antigen-specific T cell responses, as measured by enzyme-linked 
immunosorbent spot and pentamer immunoassays, were evident 
following treatment with avelumab. These responses were enhanced 
when combined with FOLFOX or radiation. This has led to speculation 
that increases in CD8+PD-1+ T cells, CD8+ TEM cells, and antigen-
specific T cell responses may be leveraged as pharmacodynamic 
biomarkers with translational relevance to the clinical setting
2.2.3Preclinical Pharmacokinetic Studies
Avelumab demonstrated pronounced non-linear pharmacokinetic (PK) 
characteristics in mice and monkeys in single dose studies at doses 
below 20 mg/kg, suggesting a combination of first order catabolic 
clearance and saturable target-mediated clearance. Toxicokinetic data 
from repeated dose toxicity studies in mice, rats and monkeys indicated 
that the PK of avelumab was linear within the dose range of 20 to 
140mg/kg, suggesting that the target mediated clearance could be 
saturated when higher doses are administered. Similar terminal half-lives 
of approximately 60 to 70 hours were observed in toxicity studies in mice 
and monkeys. A PK/pharmacodynamic (PD) study in C57BL/6 mice was 
used to correlate receptor occupancy data of avelumab in blood with drug 
combinations. A plasma concentration of 58.5 µg/mL was calculated as 
required for 95% target occupancy in this model.
Avelumab is immunogenic in mice, rats, and monkeys with a lower 
incidence of anti-drug antibodies (ADAs) at higher doses. The latter is 
probably due to interference of free avelumab with the immunogenicity 
assay (drug interference). In animals, the generated ADAs seem to have 
the potential to increase the clearance of the avelumab. As the fully 
human avelumab represents a foreign protein to the immune system of 
animals, the observed immunogenicity of avelumab in rodents and non-
human primates is not deemed predictive for an immune response to 
avelumab in humans.
2.2.4Preclinical Toxicology Studies
The toxicological profile of avelumab was evaluated in repeat-dose 
toxicity studies of 4-week duration with once weekly IV bolus 
injection/infusion of avelumab in mice, rats, and cynomolgus monkeys. A 
repeat-dose toxicity study with intermittent once weekly IV infusion of 
avelumab over 13 weeks followed by an 8-week recovery period in 
cynomolgus monkeys was also conducted. In addition, in vitro cytokine 
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release assays (CRA) in human and cynomolgus monkey whole blood and 
peripheral blood mononuclear cells (PBMCs) followed by an optimized 
CRA in phyto-hemagglutinin (PHA) pre-stimulated PBMCs from 16 human 
volunteers was completed. Tissue cross reactivity (TCR) studies in normal 
human and cynomolgus monkey tissues have also been performed.
On the basis of the binding affinity data, the cynomolgus monkey and the 
mouse were selected as relevant species for the nonclinical safety testing 
of avelumab. Due to severe hypersensitivity reactions after repeated 
administration of avelumab in mice and the low binding affinity in rats, 
rodent species are not considered appropriate for nonclinical safety 
testing of avelumab and therefore, a single species approach 
(cynomolgus monkey only) is applied.
In cynomolgus monkeys, clinical signs of hypersensitivity have not been 
seen after repeated treatment with avelumab at dose levels of 20, 60, and 
140 mg/kg, respectively, in either the pilot 4-week study or in the pivotal 
13-week IV repeat-dose toxicity study. For both studies, a no observed 
adverse effect level (NOAEL) of 140 mg/kg for systemic toxicity was 
established.
Initial cytokine release assays in human and cynomolgus monkey whole 
blood and PBMCs revealed no clear-cut evidence for release of pro-
inflammatory cytokines. However, a subsequent, optimized CRA 
demonstrated evidence of potential cytokine release in PHA pre-
stimulated PBMCs. However, clinical experience has demonstrated that 
the relative risk of an infusion reaction is low (approximately 2%) and with 
pre-medication, the incidence of infusion reaction drops to less than 1%, 
indicating that the optimized CRA overestimates the risk of cytokine 
release in the clinical setting.
2.3 Clinical Studies
2.3.1 Adult Studies
Clinical data regarding pharmacokinetics and safety for avelumab is 
available from four ongoing clinical trials for the treatment of adult 
metastatic or locally advanced solid tumors, across Phases I, II (EMR 
100070-003), and III. EMR 100070-001 is a Phase I open label multiple 
ascending dose trial with two parts: a dose escalation cohort with a 3+3 
enrollment design to determine the maximum tolerated dose (MTD) of 
avelumab with dose levels of 1, 3, 10 and 20 mg/kg every 2 weeks, and 
an expansion cohort for 16 different tumor subtypes. EMR 100070-002 
is a Phase I trial to investigate the tolerability, safety, pharmacokinetics 
(PKs), biological and clinical activity of avelumab in Japanese patients with 
metastatic or locally advanced solid tumors with an expansion cohort for 
Asian patients with gastric cancer, and was conducted in a similar dose 
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escalation design as EMR 100070-001 with dose levels of 3, 10 and 20 
mg/kg every 2 weeks. EMR 100070-003 is a Phase II, single arm, open-
label trial investigating the activity and safety of avelumab in subjects 
with Merkel cell carcinoma. EMR 100070-004 is a Phase III open-label 
trial of avelumab versus docetaxel in subjects with NSCLC that has 
progressed after platinum-doublet containing therapy.
Data for dose escalation and PKs are available for Phase 1 trials EMR 
100070-001 and EMR 100070-002. For EMR 100070-001, 53 subjects 
were included in the dose escalation cohort. Half-lives were 69, 85, 102 
and 120hours for each dose level, respectively. Cmax and AUC increased 
more than proportionally to dose between 1 to 10 mg/kg, but 
proportionally for doses above 10 mg/kg. Given no significant difference 
between the 10 mg/kg and 20 mg/kg groups, this PK characteristic 
suggests that target mediated drug disposition is involved in the 
clearance of avelumab and a high PD-L1 target occupancy (TO) is likely 
achieved at the trough concentrations for doses of 10 mg/kg. Flow 
cytometry of peripheral blood CD3+ T cells in healthy volunteers 
observed a concentration of 1 µg/mL avelumab required for >95% TO; 
based on trough serum levels observed in EMR 100070-001, all subjects 
receiving 10 mg/kg were projected to reach or exceed >95% TO 
throughout the entire dosing interval. The MTD was not reached. The 
dose escalation cohort was completed and a dose of 10 mg/kg every 2 
weeks was determined to be the biologically optimal dose for further 
evaluation in the expansion portion. Similar results were seen for PKs in 
the Phase I trial EMR 100070-002. C
max and AUC increased proportional 
with dose, and half-lives were 92, 127, and 115 hours for each dose 
level, respectively. 
Safety data are available for 1300 subjects treated in the pooled 
treatment expansion cohorts of EMR 100070-001, 52 subjects in the 
ongoing EMR 100070-002, 88 subjects in the ongoing Phase II trial EMR 
100070-003, and 75 patients treated on the ongoing Phase III trial EMR 
100070-004. For EMR 100070-001, drug-related treatment-emergent 
adverse events (TEAEs; all grades) occurred in 62.5% of patients; the most 
frequent treatment-related TEAEs were fatigue (16.3%), infusion-related 
reaction (IRR) (16.1%), nausea (8.3%), chills (7.8%), diarrhea (6.1%), and 
pyrexia (5.5%). Drug-related TEAEs ≥ grade 3 were reported in 124 
patients (9.5%) in the pooled expansion cohort of EMR 100070-001; the 
most common of these were increased gamma-glutamyl transferase 
(GGT) (0.7%) and IRR (0.7%). Other Grade > 3 treatment-related TEAEs 
included elevated lipase, fatigue, anemia, dyspnea, increased 
transaminases, pneumonitis and autoimmune hepatitis. Potentially 
immune-mediated AEs were observed in 7.6%, including hypothyroidism 
(3.5%), pneumonitis (1%), hyperthyroidism (0.5%), adrenal insufficiency 
and dry eye (0.4%), autoimmune hepatitis and colitis (0.3%), and myositis 
(0.2%). 79 subjects (6.1%) had a drug-related TEAE leading to treatment 
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discontinuation. 25 (1.9%) of these were due to an infusion-related 
reaction; 5 (0.4%) were discontinued for increased GGT levels. Other 
events resulting in discontinuation (each in 3 patients or less) included 
elevated transaminases, increased creatine phosphokinase, increased 
lipase, arthralgia, autoimmune hepatitis, colitis, dyspnea, fatigue, and 
myositis. 142 patients (10.9%) experienced a TEAE leading to death; 5 
(0.4%) of these were determined to be related to treatment. These 
adverse events included radiation pneumonitis, pneumonitis, respiratory 
distress, acute hepatic failure, and autoimmune hepatitis with resultant 
fatal liver failure.
For the 52 subjects with available safety data on EMR 100070-002, 19 
serious TEAEs were experienced in 15 subjects, 14 of which were Grade 3 
or higher. Eleven subjects experienced 13 IRR events, 12 of which were 
deemed non-serious; the lone serious IRR was related to subsequent 
chemotherapy after trial discontinuation and not avelumab. 3 potential 
immune related adverse events included hypothyroidism, 
hyperthyroidism and colitis. 3 subjects had TEAEs resulting in fatal 
outcome, one of which was treatment-related (grade 5 tumor lysis 
syndrome, grade 5 acute kidney injury). For 88 patients enrolled on the 
Phase II trial EMR 100070-003, 78 serious TEAEs were reported in 35 
subjects; 57 Grade > 3 events were reported. Eight treatment-related 
TEAEs were identified in 6 subjects including chondrocalcinosis, 
enterocolitis, synovitis, increased transaminases, tubulointerstitial 
nephritis, hypothyroidism, and IRR; all were grade 2 events. 2 subjects 
had potential immune related adverse events including hypothyroidism 
and grade 2 encephalopathy (not related to treatment). Preliminary safety 
data is available for the Phase III trial EMR 100070-004, with 
approximately 75 patients randomized to receive avelumab and an 
additional 75 receiving docetaxel. Safety data is blinded and assignment 
of events to either drug is not possible. A total of 70 serious TEAEs are 
reported in 39 patients, including 25 Grade > 3 treatment related events. 
Six grade 4 treatment-related serious TEAEs were observed in 4 subjects 
including infusion related reaction, aortic aneurysm, decreased neutrophil 
count, intestinal perforation, peritonitis, and paralytic ileus. Fatal TEAEs 
occurred in 8 subjects, including disease progression (4), COPD/dyspnea, 
general physical health deterioration, and respiratory failure. Two fatal 
serious TEAEs (septic shock, pneumonia) in one subject were considered 
treatment-related.
The safety profile of avelumab is consistent with findings reported for 
other anti-PD-1 or anti-PD-L1 antibodies. Infusion-related reactions 
including hypersensitivity and immune-related AEs/autoimmune 
disorders have been identified as important risks for avelumab. 
Respective risk mitigation measures have been implemented in ongoing 
clinical studies with avelumab.
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Clinical efficacy information of avelumab is based on data from non-
small cell lung cancer (NSCLC) and ovarian cancer expansion cohorts in 
the ongoing Phase I trial EMR 100070-001, and for 20 subjects in the 
gastric cancer expansion cohort of the ongoing Phase I trial EMR 
100070-002. The objective response rate (ORR) for subjects treated in 
the NSCLC cohort was 14.1% (26/184). Nineteen of 26 (73%) responders 
had their first documented response by week 12, but 7 (27%) 
demonstrated response by week 18 or later, suggesting a role for longer 
follow-up duration. The median PFS and OS for the cohort were 11.6 
weeks and 8.4 months, respectively. Clinical activity of avelumab was also 
evaluated retrospectively by tumor PD-L1 expression. An objective 
response was observed in 20 of 122 subjects (16.4%) who were PD-L1 
positive (defined as having at least 1% PD-L1 positive tumor cells) 
compared with 2 of 20 subjects (10%) who were considered PD-L1 
negative (defined as having less than 1% PD-L1 positive tumor cells). A 
longer median PFS (12 vs. 5.9 weeks) and OS (8.9 vs. 4.6 months) were 
both observed in PD-L1 positive compared with PD-L1 negative subjects. 
For the ovarian cancer expansion cohort, the objective response rate 
based on confirmed and unconfirmed responses for treated subjects was 
10.7% (8 of 75 subjects). The median PFS for the ovarian cancer cohort 
was 11.4 weeks. Preliminary efficacy data for gastric cancer patients on 
EMR 100070-002 as of a data cutoff of March 11th 2015 demonstrated 3 
of 20 subjects with confirmed partial responses (PRs), with a median PFS 
of 11.9 weeks. The presence of human anti-human antibodies (HAHAs) 
were also assessed, as this may impact drug efficacy; incidence was low 
with 11 out of 377 subjects with post-treatment samples testing positive 
for the presence of HAHAs.
Currently there are additional clinical trials open or in development for 
avelumab in adults at various levels. These include single agent use or 
combinations with chemotherapies and/or immunotherapies for a variety 
of adult malignancies. Data are not available for these studies.
2.3.2Pediatric Studies
There have been no pediatric phase I or II studies using avelumab that 
have been conducted to date. Therefore this trial will evaluate avelumab 
using a dosage and schedule of administration that has been shown to be 
safe in adults with refractory solid tumors, and we will closely monitor for 
toxicities in all patients. Patients with osteosarcoma are typically 
diagnosed in the second and third decade of life. Several other studies 
have allowed for the use of adult dosing to be extended to patients age 
12 years or greater due to body surface area requirements that are more 
closely aligned with adult patients, including recent COG phase II studies 
specifically targeting patients with recurrent or progressive osteosarcoma 
with agents such as eribulin mesylate and glembatumumab vedotin.
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2.4 Rationale for this Study
This study will evaluate avelumab in patients with osteosarcoma using 
dosing that has previously been determined in adult studies. Review of 
COG phase II trials enrolling osteosarcoma patients reveals that over 80% 
of enrolled patients have been age 12 years or older. Objective response 
using traditional criteria such as the Response Evaluation Criteria in Solid 
Tumors (RECIST) has proven challenging for osteosarcoma, as its dense 
calcified structure limits its ability to demonstrate a radiographic 
representation of drug activity. Using disease control (defined as 
extended stabilization of disease for 4 cycles of therapy or greater) as an 
additional endpoint may allow for the ability to detect a signal of activity. 
Therefore objective response will be evaluated as a primary endpoint, but 
stable disease control will also be monitored.
2.5 Overview of Proposed Study
This is a Phase 2 study using a traditional Simon two-stage design. 
Patients 12 years or greater with recurrent/refractory osteosarcoma will 
be administered avelumab at a dose of 10 mg/kg intravenously (IV) over 
60 minutes on days 1 and 15 of each cycle, with a cycle lasting 28 days. 
Evaluations to assess disease control and response will be completed 
after cycles 2 and 4, and then will be completed every 3 cycles. 
Correlative biologic studies will be obtained and will include measures of 
immune activation, including phenotypic and functional analysis of 
peripheral blood mononuclear cells (PBMCs) and serum analyses of 
cytokines, chemokines, soluble CD-27, soluble CD40L, antibodies, and 
tumor-associated antigens.
In addition to assessment of disease control, response assessments will 
use standard RECIST criteria. Determination of progression will use 
measurements according to RECIST criteria at 16 weeks.
2.6 Rationale for Correlative Biology Studies
As an IgG-1 antibody targeting PD-L1, avelumab may potentially cause 
anti-tumor activity through inhibition of the PD-1/PD-L1 interaction and 
resultant removal of inhibition of the immune system to target tumor 
cells, or may be related to ADCC properties; therefore the mechanism of 
action of avelumab is not fully understood. Therefore, correlative biology 
studies will investigate the activation status of the immune system 
through evaluation of serum markers at baseline and after treatment with 
avelumab. This will include cytokines (IFN-γ, IL-10, IL-12, IL-2, IL-4, 
TGF-β), chemokines, soluble CD27, soluble CD40 ligand, and tumor-
associated antigens. Proliferation of 123 unique subsets of PBMCs will be 
evaluated by flow cytometry at baseline and after exposure to avelumab.
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Increased numbers of mutations have been associated with of tumors 
responsive to immune blockade treatment (including CTLA-4 and PD-1). 
The proposed mechanism of action in immune blockade therapy is the 
activation of host CD4 and CD8 T cell responses against tumor antigens, 
with tumor neoantigens considered to be the primary targets of 
efficacious responses.27,34 In order for a tumor mutation to generate a 
neoantigen, it must occur within a peptide context that can bind to a host 
major histocompatibility complex (MHC). Further, this mutation-bearing 
peptide still needs to be processed and presented efficiently and elicit a 
cognate T cell response. Therefore, it is expected that tumors with high 
numbers of mutations are more likely to generate individual mutations 
that meet all the requirements to be immunogenic neoepitopes. In the 
small number of studies that have been conducted using computationally 
predicted neoepitopes, ~2-10% of the predicted epitopes were confirmed 
to have elicited detectable T cell responses by tetramer staining, with 
some variation likely due to differences in the method of prediction.35
Host T cells play a critical role in controlling many forms of cancer, but 
can progressively lose their anti-tumor functions during prolonged 
exposure to high levels of antigen and inflammation.36 Repression of CD8 
T cell effector functions, commonly referred to as T cell exhaustion, is a 
major obstacle for current therapeutic strategies aimed at utilizing the 
host immune system.37 Acquisition of the functionally exhausted state in 
antigen-specific T cells is controlled by the expression of inhibitory 
receptors.
38,39 These recent advances in our mechanistic understanding 
of T cell exhaustion have led to immune checkpoint blockade therapies 
that reverse the exhausted state by blocking the ligation of inhibitory 
receptors expressed on the surface of tumor-specific CD8 T cells.
40 It is 
now apparent that the development of the fully exhausted phenotype is 
correlated to the successive up-regulation of multiple inhibitory 
receptors, including co-expression of PD-1 with Lag3, 2B4, and 
Tim3.41,42 Simultaneous expression of all four receptors identifies a fully 
exhausted cell, while cells that express a smaller subset of these 
receptors might still retain some function. After checkpoint blockade, the 
loss of inhibitory receptor expression will serve as correlate of rescued 
functionality. 
Studies have also demonstrated that increased PD-1 expression can be 
used as a surrogate marker for cells exposed to antigen, which has 
recently been employed as a strategy to identify tumor-specific CD8 T 
cells43; in this work, all tumor-specific cells expressed PD-1. Few data are 
available regarding the percentage of CD8+ T cells that are PD-1 positive 
in pediatric patients with cancer; between 30 and 70% of CD8+ T cells 
were found to be positive for PD-1 in 27 healthy adult samples44. Based 
on the role PD-1 expression plays in T cell exhaustion, as well as its 
utility in identifying activated tumor-specific CD8 T cells, we will perform 
correlative studies to determine changes in the immunophenotypic 
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profile of PD-1high CD8+ T cells from serial peripheral blood samples 
obtained before and during treatment. We will be measuring the change 
in the percentage of PD-1
high CD8+ T cells in peripheral blood samples 
obtained before and 6 weeks after start of therapy. 
Emerging evidence indicates that immune checkpoint blockade results in 
expansion of a clonal population of tumor-specific cells.45 While this 
expansion is groundbreaking, the oligoclonality of T cells will provide a 
selective pressure against specific antigens and may eventually enhance 
tumor escape. Therefore we must better understand the mechanisms for 
the restricted clonality of rejuvenated CD8 T cells in individuals that 
receive immune checkpoint blockade therapy in order to develop 
strategies the allow for a greater breadth of tumor-specific T cells to 
respond to immune checkpoint blockade.
Repertoire diversity has been established as a key correlate of efficacious 
immune responses against infections in mice and humans.
46,47 This 
includes diversity of epitope targets, as well as repertoire diversity within 
an epitope-specific response. We hypothesize that the generation of 
successful anti-tumor immune responses after checkpoint blockade 
requires the activation of diverse T cell repertoires. Conversely, the 
inability to recruit and reactivate a sufficiently diverse repertoire may be a 
key correlate of treatment failure. The characterization of the anti-tumor 
T cell repertoire has to date been performed by growing T cell lines, or 
using predictive algorithms to detect potential T cell responses based on 
neoepitopes. In the latter case, these are almost exclusively Class I-
restricted responses, though a few reports have demonstrated a robust 
role for anti-tumor Class II-restricted T cells.
48,49 We propose to use an 
unbiased approach to define the T cell repertoire in tumor-associated T 
cells (where tumor tissue is available) or in antigen-exposed (PD-1+) T 
cells from the peripheral blood of tumor patients before and after 
treatment with avelumab. Using single cell techniques, we will 
comprehensively characterize the repertoire of PD1+ cells at baseline and 
following treatment, with a focus on identifying clonotypes that expand 
after treatment. Selected clonotypes that expand following treatment will 
be cloned and expressed in a cell line that will report out T cell receptor 
activation using a Nur77-GFP construct. In patients where tumor tissue or 
tumor sequence is available, we will initially screen the reporter lines 
against the neoantigens present in the tumor, using transgenic antigen 
presenting cells transfected with the appropriate HLA and a synthetic 
construct containing all the identified neoantigens. If positive hits are 
identified, the specific neoantigen(s) will be characterized.
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Figure 1: Differential DNA methylation programming among human naïve and memory CD8 
T cell subsets is coupled to their functional state. (A) FACS based isolation strategy to purify 
naïve and memory CD8 T cells subsets from the peripheral blood of healthy individuals. TN 
= naïve, TEM = effector-memory, TCM = central-memory CD8 T cells. Representative FACS-
based gating strategy to identify the following CD8 T cells: Naïve = CCR7+ CD45RO
- , TEM= 
CCR7- CD45RO
+, and TCM= CCR7
+ CD45RO
+. (B) Bisulfite sequencing analysis of Id2, Prf1 
(Perforin), PD-1, and GzmB (Granzyme B)  regulatory loci from genomic DNA of FACS sorted 
naïve, TEM, and TCM CD8 T cells. Cartoon representation of the promoter region is depicted 
to the left of the representative methylation profiling for each region. Open circles represent 
unmethylated CpG site while closed circles represent methylated CpG. Each horizontal line 
represents a sequenced clone and each number represents a CpG site displayed in the 
cartoon representation of transcriptional start. (C) Graphical summary for the average % CpG 
methylation in Id2, Prf1 (perforin), PD-1, and GzmB (granzyme b) loci. Data represents mean 
+/- SEM (n=2-6 healthy donors).
Several studies have demonstrated that CD8 T cell exhaustion gene 
expression programs can become reinforced and heritably maintained.50-
52 Transcriptional memory is often mediated by epigenetic modification 
to the genome, therefore we hypothesize that acquired exhaustion-
specific epigenetic programs must be erased in order for PD-1 blockade 
to be effective on all exhausted T cells. In support of this hypothesis, we 
have recently determined that conditional deletion of the DNA 
methyltransferase 3a (Dnmt3a cKO) inhibits acquisition of de novo DNA 
methylation programs in antigen-specific CD8 T cells and prevents T cell 
exhaustion during chronic viral infection (Youngblood B, unpublished 
data). Importantly, Dnmt3a deficient antigen-specific CD8 T cells retain 
their ability to mount a polyclonal effector response, and ultimately 
control the chronic pathogen. Moreover, Dnmt3a cKO cells remain 
resistant to T cell exhaustion even when the cKO cells are forced to 
persist in an environment that has artificially high levels of antigen for 
several months. Our data serve as proof of principle that acquired 
epigenetic programs promote T cell exhaustion, and have significant 
implications for generating a greater breadth of long-lived tumor-
specific T cell responses after immune therapy. We have recently 
developed protocols for performing whole-genome methylation analysis 
using low input genomic DNA. We have applied this methodology to 
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determine the epigenetic signature of functional human memory CD8 T 
cells (Figure 1). From our whole-genome analysis we have identified 
differentially methylated regions among the T cell subsets. We have 
developed a PCR based assay to measure the epigenetic signature of 
human memory CD8 T cell subsets (Figure 1B & 1C). These data provide 
insight into the poised state for expression of critical effector molecules 
and inhibitory receptors among the memory CD8 T cell subsets. We 
propose to perform loci-specific and whole genome bisulfite sequencing 
methylation analysis of PD-1+ CD8 T cells from patients before and after 
therapy with avelumab to identify epigenetic programs that are coupled 
to the ability of host T cells to be rejuvenated following PD-1 blockade 
therapy. 
2.7 Rationale for Quality of Life Studies
Cancer and its treatment can cause medical and psychosocial 
complications that adversely affect a patient’s quality of life (physical, 
emotional, social and cognitive functioning) during and after treatment. 
Once complications and their outcomes are specified and measured, 
interventions designed to prevent or diminish the complications can be 
developed, implemented in clinical care, and their effects evaluated. Until 
recently, the majority of studies evaluating quality of life in patients with 
childhood cancer had been conducted in survivorship populations.53-56 
Many of these studies included single time points of assessments, cross-
sectional designs, inclusion of only one or a limited number of quality of 
life domains, or included a heterogeneous sample not specific to any 
diagnosis.57,58 More recently, several studies have emerged implementing 
quality of life measures during active treatment.59-63 The majority of 
reports of quality of life in osteosarcoma patients tend to involve long-
term survivors rather than patients who are actively undergoing 
treatment.
64-66 Two studies included more than a single data point and 
the selected time points varied, ranging from end of treatment to 8 years 
after the end of treatment or at the time of definitive surgery and then 
annually for the next three years.67,68
A number of additional studies exist which include patients with 
osteosarcoma along with a variety of other diagnoses, or include patients 
with both pediatric and adult osteosarcoma; data are reported in group 
fashion and it is impossible to extract scores specific to patients with 
osteosarcoma.69-78 A prospective study of patients with high grade, 
localized osteosarcoma evaluated health-related quality of life (HRQOL) 
and symptom distress at four time points including diagnosis, prior to 
definitive surgery, approximately 2 months post-surgery, and 3 to 6 
months after completion of treatment. Significant improvements in most 
domains and decreases in symptom distress were demonstrated across 
time points during therapy.
63 The international collaborative EURAMOS 
trial for newly diagnosed osteosarcoma patients included self- and 
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parent-reported quality of life measures using the EORTC QLQ-C30 
questionnaire; results have not yet been reported.79 Repeated quality of 
life measurements that are linked to specific clinical indicators may result 
in more clinically useful and interpretable findings. Furthermore, little to 
no data exist examining health-related quality of life measures in 
patients with recurrent osteosarcoma. Better understanding of HRQOL 
outcomes may identify opportunities to improve treatment-related 
morbidities, facilitate decision making, establish effective communication 
strategies and improve satisfaction with care for patients and their 
families.
80,81 In the setting of a disease which has previously 
demonstrated poor prognosis after progression or recurrence, HRQOL 
measures may aid in the identification of reasonable therapeutic 
approaches that allow for optimization of patients’ quality of life in 
balance with treatment goals.
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2.7.1Patient Reported Outcome Measurement Information System 
(PROMIS) – Pediatric and Adult Profiles
The PROMIS is a set of questionnaires utilized to assess patient reported 
outcomes (PROs) consistent with goals established by the National 
Institute of Health (NIH). The PROMIS Pediatric Profile (self-report) is 
validated for use with individuals ages 8 to 17 years, and the PROMIS 
Adult Profile (self-report) is validated for individuals ages 18 years and 
older. Both the PROMIS Pediatric and Adult Profiles include PROs across a 
number of domains including: 1) physical function mobility, 2) anxiety, 3) 
depressive symptoms, 4) fatigue, 5) peer relationships, and 6) pain 
interference. On domains other than the physical function mobility 
domain, respondents rate how often statements describe their 
functioning. For the physical function mobility domain, participants use a 
5-point Likert rating scale to rate how much difficulty is experienced with 
engagement in various activities. Scoring tables are then used to convert 
raw scores for each domain into age-normed T-scores. The PROMIS 
measures have demonstrated strong psychometric properties.82 
Literature is available for its use in pediatric oncology research,83,84 as 
well as in adults with lower extremity bone metastases.85 The PROMIS 
measures have shown known-group validity with ability to distinguish 
subgroups of children with a chronic health condition from other 
conditions as well as general population samples.86,87 Additionally, the 
ability to determine a clinical minimally important difference has been 
established with the PROMIS questionnaires, which has been a 
methodological limitation of other HRQOL instruments.88
Patients will complete the PROMIS Pediatric or Adult Profile (based on 
their age at time of administration) at four time points during the study: 
at diagnosis prior to the start of treatment, at the end of cycle 2 (8 weeks 
+ 1 week), at the end of cycle 4 (16 weeks + 1 week), and at end of 
avelumab therapy. Mixed effect models will be performed to assess 
changes in quality of life domains over time and to assess for impact of 
clinical factors on scores in quality of life domains. 
3.0 ELIGIBILITY CRITERIA AND STUDY ENROLLMENT 
According to institutional and NIH policy, the study will accession 
research participants regardless of gender and ethnic background. 
Institutional experience confirms broad representation in this regard.
All clinical and laboratory studies to determine eligibility must be 
performed within 7 days prior to enrollment unless otherwise indicated. 
Laboratory values used to assess eligibility must be no older than 7 days 
at the start of therapy. If a post-enrollment lab value is outside the limits 
of eligibility, or laboratory values are > 7 days old, then the following 
laboratory evaluations must be re-checked within 48 hours prior to 
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initiating therapy: CBC with differential, bilirubin, ALT (SGPT) and serum 
creatinine. Imaging studies, where applicable, must be obtained within 2 
weeks prior to enrollment.
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3.1 Inclusion Criteria
3.1.1Patients must be > 12 years of age but < 50 years of age at the 
time of enrollment.
3.1.2Patients must have histologic verification of osteosarcoma at initial 
diagnosis or relapse.
3.1.3Patients must have had evidence of having relapsed, progressed or 
become refractory to conventional therapy.
3.1.4Patients must have measurable disease, documented by clinical, 
radiographic or histologic criteria. Disease must be bi-
dimensionally measurable by computed tomography (CT) or 
magnetic resonance imaging (MRI).
3.1.5Patients must have a performance status of ≥ 50 using the 
Karnofsky scale for patients > 16 years of age and the Lansky scale 
for patients ≤ 16 years of age.
3.1.6Patients must have a life expectancy of ≥ 6 weeks.
3.1.7Patients must have fully recovered from the acute toxic effects of 
all prior chemotherapy, immunotherapy, or radiotherapy prior to 
entering this study.
a. Myelosuppressive chemotherapy: must not have received 
within 3 weeks of entry onto this study
b.Biologic (anti-neoplastic agent): at least 7 days since the 
completion of therapy with a biologic agent.
c.Immunotherapies: at least 42 days must have elapsed since a 
prior therapy that included a monoclonal antibody or any 
other type of immunotherapy (e.g. chimeric antigen receptor 
(CAR) T cell therapy).
d. Radiation therapy (RT): ≥ 2 weeks for local palliative RT 
(small port); ≥ 6 months must have elapsed if prior 
craniospinal RT or if ≥ 50% radiation of the pelvis; ≥ 6 weeks 
must have elapsed if other substantial bone marrow (BM) 
radiation.
3.1.8 Organ Function Requirements:
a. Adequate bone marrow function defined as:
· Peripheral absolute neutrophil count (ANC) ≥ 1500/mm
3
· Platelet count ≥ 100,000/mm
3 (transfusion independent)
· Hemoglobin ≥ 9.0 g/dL (may receive RBC transfusions)
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b. Adequate renal function defined as: 
· Creatinine clearance or radioisotope GFR ≥70 
mL/min/1.73m2 OR
· Serum creatinine based on age/gender as follows: 
(threshold creatinine values were derived from the 
Schwartz formula for estimating GFR)89
Age
Maximum creatinine 
(mg/DL)
Male   
Female
12 to < 13 
years
1.2
1.2
13 to <16 
years
1.5
1.4
≥ 16 years
1.7
1.4
c. Adequate liver function defined as:
· Total Bilirubin ≤ 1.5x the institutional upper limit of 
normal (IULN) for age
· ALT (SGPT) and AST (SGOT) < 2.5 x IULN for age (or < 5 x 
IULN for patients with documented metastatic disease to 
the liver)
· Serum albumin > 2 g/dL
d.Serum lipase ≤ upper limit of normal (IULN).
e. Patients must have documented pulse oximetry ≥ 92% on room 
air.
3.1.9Female patients of childbearing potential must have a negative 
serum or urine pregnancy test within 7 days of enrollment.
3.1.10 
Male or female patients who are sexually active and of 
reproductive potential must agree to use an effective contraceptive 
method throughout the study and for at least 60 days after last 
avelumab treatment administration. Abstinence is an acceptable 
form of contraception.
3.1.11 
Patients must not currently be using other investigational 
agents.
3.1.12 
Patients must not currently be using other anti-cancer agent.
3.1.13 
Patients must be able to comply with the safety monitoring of 
the study in the opinion of the investigator.
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3.1.14 
Written, informed consent and assent following Institutional 
Review Board, NCI, FDA and OHRP guidelines.
3.2 Exclusion Criteria
3.2.1 Central nervous system (CNS) metastases.
3.2.2 Current use of immunosuppressive medication, EXCEPT for the 
following: a. intranasal, inhaled, topical steroids, or local steroid 
injection (e.g., intra-articular injection); b. Systemic corticosteroids 
at physiologic doses ≤ 10 mg/day of prednisone or equivalent; c. 
Steroids as premedication for hypersensitivity reactions (e.g., CT 
scan premedication).
3.2.3 Active autoimmune disease that might deteriorate when receiving 
an immuno-stimulatory agent.  Patients with diabetes type I, 
vitiligo, psoriasis, or hypo- or hyperthyroid diseases not requiring 
immunosuppressive treatment are eligible.
3.2.4 Active infection requiring systemic therapy.
3.2.5Known history of testing positive for HIV or known acquired 
immunodeficiency syndrome.
3.2.6 Hepatitis B virus (HBV) or hepatitis C virus (HCV) infection at 
screening (positive HBV surface antigen or HCV RNA if anti-HCV 
antibody screening test positive).
3.2.7Patient who has received vaccination within 4 weeks of the first 
dose of avelumab and while on trials is prohibited except for 
administration of inactivated vaccines.
3.2.8Known prior severe hypersensitivity to investigational product or 
any component in its formulations, including known severe 
hypersensitivity reactions to monoclonal antibodies (NCI CTCAE 
v4.03 Grade ≥ 3).
3.2.9 Clinically significant (i.e., active) cardiovascular disease: cerebral 
vascular accident/stroke (< 6 months prior to enrollment), 
myocardial infarction (< 6 months prior to enrollment), unstable 
angina, congestive heart failure (≥ New York Heart Association 
Classification Class II, see Appendix II), or serious cardiac 
arrhythmia requiring medication.
3.2.10 
Persisting toxicity related to prior therapy (NCI CTCAE v. 4.03 
Grade > 1); however, alopecia, sensory neuropathy Grade  ≤ 2, or 
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other Grade ≤ 2 not constituting a safety risk based on 
investigator’s judgment are acceptable
3.2.11 
Other severe acute or chronic medical conditions including 
colitis, inflammatory bowel disease, pneumonitis, pulmonary 
fibrosis or psychiatric conditions including recent (within the past 
year) or active suicidal ideation or behavior; or laboratory 
abnormalities that may increase the risk associated with study 
participation or study treatment administration or may interfere 
with the interpretation of study results and, in the judgment of the 
investigator, would make the patient inappropriate for entry into 
this study.
3.2.12 
Patients with active diarrhea > CTCAE v4.03 Grade 2.
3.2.13 
Patients who have previously received a prior organ 
transplantation, including allogeneic stem cell transplantation.
3.2.14 
Female patients who are pregnant or actively breastfeeding.
3.2.15 
Patients who have previously received anti-PD1 or anti-PD-
L1 therapy. Patients who have previously received anti-CTLA-4 
therapy (e.g. ipilimumab) are eligible for study.
3.3 Recruitment and Screening
Participants will be recruited by study investigators through their clinical 
practice.
3.4 Enrollment at St. Jude
A member of the study team will confirm potential participant eligibility 
as defined in Section 3.1-3.2, complete and sign the ‘Participant 
Eligibility Checklist’. The study team will enter the eligibility checklist 
information into the Patient Protocol Manager (PPM) system. Eligibility will 
be reviewed, and a research participant-specific consent form and assent 
document (where applicable) will be generated. The complete signed 
consent / assent form(s) must be faxed or emailed to the CPDMO at 595-
6265 to complete the enrollment process. 
The CPDMO is staffed 7:30 am-5:00 pm CST, Monday through Friday. A 
staff member from the St. Jude Cerner Millennium (MILLI) helpline is on 
call Saturday, Sunday, and holidays from 8:00 am to 6:00 pm. If you have 
a prospective research enrollment and need assistance releasing your 
consent, please call the MILLI helpline (901-338-0596) on call number.
3.5 Enrollment at Collaborating Sites
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Collaborating Site research participants should be registered at St. Jude 
within 24 hours of enrollment at the site. The completed Eligibility 
Checklist and entire signed Informed Consent should be faxed to 901-
595-6265. Please call 901-595-2568 if confirmation of the enrollment 
information is needed. The Protocol Eligibility Coordinator will then 
register the research participant in the Patient Protocol Manager (PPM) 
system.
4.0 TREATMENT PLAN
4.1 Overview of Treatment Plan
This is a single arm phase 2 study with a two stage design and an 
endpoint specific to osteosarcoma. Avelumab will be given on days 1 and 
15 of each 4 week cycle. Treatment will be discontinued if there is clear 
evidence of progression with increase in tumor size greater than 20%, or 
with drug related dose limiting toxicity that requires removal from 
therapy, as defined in Section 10.1. Therapy may otherwise continue for a 
maximum duration of 24 months or 26 cycles, whichever occurs first. 
Initial radiographic assessments will take place after cycle 2 and cycle 4 
and will be compared to imaging obtained prior to initiation of therapy. 
For patients who remain on protocol therapy, imaging thereafter will take 
place every 3rd cycle.
4.2 Treatment During Cycle 1 and Subsequent Cycles
One cycle of avelumab treatment is described below. A cycle may be 
repeated every 28 days if the patient has at least stable disease and has 
met laboratory parameters as defined in the eligibility section, and 
detailed below.
4.2.1Premedication
In order to mitigate infusion-related reactions, a premedication with an 
antihistamine and with paracetamol (acetaminophen) 30 to 60 
minutes prior to each dose of avelumab is mandatory (for example, 
25-50 mg diphenhydramine and 500-650 mg paracetamol IV or oral). 
This may be modified based on local treatment standards and guidelines, 
as appropriate.
4.2.2Setting
Avelumab should be administered in a setting that allows for 
immediate access to an intensive care unit or equivalent environment 
and administration of therapy for anaphylaxis, such as the ability to 
implement immediate resuscitation measures.  Steroids 
(dexamethasone 10mg), epinephrine (1:1,000 dilution), allergy 
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medications (IV antihistamines), bronchodilators, or equivalents, and 
oxygen should be available for immediate access.
4.2.3 Observation Period
Following avelumab infusions, patients must be observed for 2 hours 
post-infusion for potential infusion-related reactions.
4.2.4 Avelumab Administration
Prior to avelumab infusion:
Diphenhydramine: IV over 5-15 minutes (per institutional guidelines)
Days: 1 and 15
Dose: 1 mg/kg (MAX 50mg)
Note: Equivalent antihistamine therapy may be considered.
Acetaminophen: By mouth (PO)
Days: 1 and 15
Dose: 10-15 mg/kg (MAX 1000mg)
Note: In the setting of limited PO intake and per institutional guidelines, 
IV acetaminophen may be considered.
Avelumab infusion:
Avelumab: Intravenously over 60 minutes
Days: 1 and 15
Dose: 10 mg/kg/dose
4.3 Criteria for Starting Subsequent Cycles
A cycle may be repeated every 28 days if the patient has again met the 
parameters for organ function requirements as defined in the eligibility 
criteria (Section 3.0), has not met any of the criteria for removal from 
therapy, and has not experienced a dose limiting toxicity. A delay of up 
to 7 days from the scheduled date of the next scheduled treatment dose 
may be allowed.
5.0 DOSE MODIFICATIONS FOR TOXICITIES
5.1
Adverse Drug Reactions Requiring Avelumab Discontinuation or 
Modification
Any Grade 4 ADRs require treatment discontinuation with avelumab except for 
single laboratory values out of normal range that are unlikely related to study 
treatment as assessed by the Investigator, do not have any clinical correlate, and 
resolve within 7 days with adequate medical management.
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Any Grade 3 ADRs require treatment discontinuation with avelumab except for 
any of the following: 
·Transient (≤ 6 hours) Grade 3 flu-like symptoms or fever, which is controlled 
with medical management
·Transient (≤ 24 hours) Grade 3 fatigue, local reactions, headache, nausea, 
emesis that resolves to Grade ≤ 1 
·Single laboratory values out of normal range (excluding Grade ≥ 3 liver function 
test increase) that are unlikely related to study treatment according to the 
Investigator, do not have any clinical correlate, and resolve to Grade ≤ 1 within 
7 days with adequate medical management
·Tumor flare phenomenon defined as local pain, irritation, or rash localized at 
sites of known or suspected tumor
·Grade 3 rash that resolves to Grade ≤ 1 with steroid treatment
·Change in ECOG PS to ≥ 3 that does not resolve to £ 2 within 14 days (infusions 
should not be given on the following cycle, if the ECOG PS is ≥ 3 on the day of 
study drug administration)
Any Grade 2 ADR should be managed as follows:
·If a Grade 2 ADR resolves to Grade ≤ 1 by the last day of the current cycle, 
treatment may continue.
·Upon the second occurrence of the same Grade 2 ADR (except for hormone 
insufficiencies that can be managed by replacement therapy) in the same 
participant, treatment with avelumab has to be permanently discontinued.
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5.2 Treatment Modification for Symptoms of Infusion-Related 
Reactions
In order to mitigate infusion-related reactions, all participants will receive 
pretreatment with H1 blockers and acetaminophen 30 to 60 minutes 
prior to each infusion. A premedication regimen of 1 mg/kg 
diphenhydramine (MAX 50 mg) and 10-15mg/kg acetaminophen (MAX 
1000 mg) is recommended prior to each dose of drug. Participants must 
be observed for 2 hours post-infusion. This regimen may be modified 
based on local treatment standards and guidelines as appropriate.
NCI-CTCAE Grade 
Treatment Modification for Study 
Drug
Grade 1 – mild
Mild transient reaction; infusion 
interruption not indicated; 
intervention not indicated.
Decrease the study drug infusion 
rate by 50% and monitor closely for 
any worsening.
The total infusion time for study 
drug should not exceed 
120 minutes.
Grade 2 – moderate
Therapy or infusion interruption 
indicated but responds promptly to 
symptomatic treatment (for 
example, antihistamines, NSAIDs, 
narcotics, IV fluids); prophylactic 
medications indicated for £ 24 h.
Stop study drug infusion.
Resume infusion at 50% of previous 
rate once infusion-related reaction 
has resolved or decreased to at least 
Grade 1 in severity, and monitor 
closely for any worsening.
Grade 3 or Grade 4 – severe or 
life-threatening
Grade 3: Prolonged (for example, 
not rapidly responsive to 
symptomatic medication and/or 
brief interruption of infusion); 
recurrence of symptoms following 
initial improvement; hospitalization 
indicated for clinical sequelae.
Grade 4: Life-threatening 
consequences; urgent intervention 
indicated.
Stop the study drug infusion 
immediately and disconnect infusion 
tubing from the participant.
Participants have to be withdrawn 
immediately from study drug 
treatment and must not receive any 
further study drug treatment.
·Once the avelumab infusion rate has been decreased by 50% or 
interrupted due to an infusion-related reaction, it must remain 
decreased for all subsequent infusions.
·If the patient has a second infusion-related reaction Grade ≥ 2 on the 
slower infusion rate, the infusion should be stopped and the patient 
should be removed from study treatment.
·If hypersensitivity reaction occurs, the patient must be treated according 
to the best available medical practice. 
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5.2.1Severe Hypersensitivity Reaction
Hypersensitivity reactions must be treated according to best medical 
practices. Patients should also be instructed to report any delayed 
reactions to the investigator immediately:
a. Symptoms: Impaired airway, decreased oxygen saturation (<92%), 
confusion, lethargy, hypotension, pale/clammy skin, cyanosis
b. Management: epinephrine injection, dexamethasone (or equivalent) 
infusion, continuous monitoring, and potential ICU management
Patients who experience an acute severe hypersensitivity reaction should 
no longer receive avelumab therapy.
5.2.2Influenza-Like Symptoms
For prophylaxis of influenza-like symptoms, patients may receive a non-
steroidal anti-inflammatory drug (NSAID) such as ibuprofen 10mg/kg 
(MAX 800 mg) or naproxen sodium 10mg/kg (MAX 500 mg) may be 
administered 2 hours before and 8 hours after the start of each avelumab 
infusion. 
5.2.3Tumor Lysis Syndrome
Because avelumab can induce ADCC, there is a potential risk of tumor 
lysis syndrome. Should this occur, participants should be treated as per 
local guidelines for tumor lysis syndrome including IV fluids, frequent 
monitoring of electrolytes (uric acid, K+, Ca
++, PO
4), and use of 
allopurinol or rasburicase as needed.
5.3
Management of Immune-Mediated Adverse Reactions
Since inhibition of PD-L1 stimulates the immune system, immune-related 
AEs (irAEs) may occur. Treatment of irAEs is mainly dependent upon 
severity (NCI-CTCAE grade):
· Grade 1 to 2: treat symptomatically or with moderate dose steroids, 
more frequent monitoring.
· Grade 1 to 2 (persistent): manage similar to high grade AE (Grade 3 
to 4)
· Grade 3 to 4: treat with high dose corticosteroids
Treatment of gastrointestinal, dermatological, pulmonary, hepatic and 
endocrine irAEs should follow guidelines set forth below.
For Grade 2 toxicities requiring delay of avelumab therapy as listed in the 
tables below, study drug should be held until < Grade 1.  Patients who 
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experience Grade 2 toxicities that do not resolve to < Grade 1 despite 
symptomatic/medical intervention within 14 days should have study drug 
discontinued; in the setting of clinical benefit, investigators may discuss 
continuation of study drug with the PI on a case-by-case basis.
Gastrointestinal irAEs
It is recommended that colitis or enterocolitis of Grade 1 be evaluated for other 
non-immune mediated causes, then monitored closely and treated 
symptomatically without steroids, including a trial of loperamide may be used. For 
Grade ≥ 2 colitis or enterocolitis, recommendations include endoscopy. Even if 
colonoscopy does not reveal gross findings of colitis, biopsies should be 
performed and strong consideration should be given to upper endoscopy and 
biopsies. Patients with gross or biopsy proven colitis or enteritis should receive IV 
steroids (recommend 1 mg/kg methylprednisone daily x 7 days) followed by a 
minimum 30 day taper. In patients with Grade 3 or 4 enterocolitis that does not 
respond to high dose steroids after 7 days, further therapies should be 
administered as clinically indicated in consultation with gastroenterology 
subspecialists.
Concern for immune-mediated liver toxicity may be elicited following hepatic 
enzyme (AST, ALT) elevation of 3-fold over baseline and/or right upper quadrant 
abdominal pain or unexplained nausea or vomiting. Other etiologies for 
transaminitis should be considered and evaluated and may include but are not 
limited to neoplastic, concurrent medications, viral hepatitis, and other toxic 
etiologies. Evaluation for autoimmune etiologies may be evaluated by ANA, pANC, 
and anti-smooth muscle antibody tests as well as hepatology consultation with 
possible biopsy. 
Pancreatitis has rarely been associated with checkpoint inhibitors and should be 
considered in cases of abdominal pain associated with elevations of amylase and 
lipase. Treatment of pancreatitis should be supportive and may include 
consultation with gastroenterology subspecialists. Grade 3 or 4 amylase or lipase 
abnormalities that are not associated with diabetes mellitus, associated liver or 
gallbladder inflammation, or clinical manifestations of pancreatitis and which 
decrease to ≤ Grade 2 within 7 days will not require discontinuation of avelumab.
Any patient experiencing diarrhea (which may be defined as watery stool, or 
increase in the frequency stools above grade 1 with urgency or nocturnal bowel 
movement, or melena or hematochezia) should be further evaluated for etiology 
that should include a search for an infectious etiology, C. Difficile colitis and other 
alternative infections as clinically indicated. Consideration should be given to 
discontinuing medications known to exacerbate colitis.
Severity of Diarrhea / 
Colitis (NCI-CTCAE 
Management
Follow-up
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v4.03)
Grade 1
Diarrhea: < 4 stools/day 
over baseline
Colitis: asymptomatic
Continue avelumab 
therapy
Symptomatic treatment 
(e.g. loperamide)
Close monitoring for 
worsening symptoms
Educate participant to 
report worsening 
immediately
If worsens: treat as Grade 
2 or 3 to 4.
Grade 2
Diarrhea: 4 to 6 
stools/day over baseline; 
IV fluids indicated < 24 
hours, not interfering with 
ADLs
Colitis: abdominal pain, 
blood in stool
Delay avelumab therapy
Symptomatic treatment
If improves to Grade 1 
within 7 days, resume 
avelumab therapy
If persists > 5 to 7 days or 
recur: 0.5-1.0 mg/kg/day 
methylprednisolone (or 
equivalent). 
When symptoms improve 
to Grade 1, taper steroids 
over at least 1 month. 
Consider prophylactic 
antibiotics for 
opportunistic infections, 
and resume avelumab 
therapy per protocol.
If worsens or persists > 3 
to 5 days with oral 
steroids: treat as grade 3 
to 4 
Grade 3 to 4
Diarrhea (Grade 3): ≥ 7 
stools/day over baseline; 
incontinence; IV fluids ≥ 
24 hours; interfering with 
ADLs
Colitis (Grade 3): severe 
abdominal pain, medical 
intervention indicated, 
peritoneal signs
Grade 4: life-threatening, 
perforation
Discontinue avelumab 
therapy per protocol
Start 1.0-2.0 mg/kg/day 
methylprednisolone IV 
(or equivalent)
Add prophylactic 
antibiotics for 
opportunistic infections
Consider endoscopy
If improves:
continue steroids until 
Grade 1, then taper over at 
least 1 month
If persists > 3 to 5 days or 
recurs after improvement:
Add infliximab 5 mg/kg 
unless otherwise 
contraindicated (note: 
infliximab should not be 
used in cases of 
perforation or sepsis)
Dermatological irAEs
Grade of Rash
(NCI-CTCAE v4)
Management
Follow-up
Grade 1 to 2
Covering < 30% body 
surface area
Symptomatic therapy (for 
example, antihistamines, 
topical steroids)
If persists > 1 to 2 weeks 
or recurs:
Consider skin biopsy
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Continue avelumab 
therapy
Delay avelumab therapy
Consider 0.5 to 1.0 
mg/kg/day 
methylprednisolone IV or 
oral equivalent. Once 
improving, taper steroids 
over at least 1 month, 
consider prophylactic 
antibiotics for 
opportunistic infections, 
and resume avelumab 
therapy.
If worsens: 
Treat as Grade 3 to 4
Grade 3 to 4
Covering > 30% body 
surface area; life-
threatening consequences
Delay or discontinue 
avelumab therapy
Consider skin biopsy
Dermatology consult
1.0 to 2.0 mg/kg/day 
methylprednisolone IV or 
IV equivalent
If improves to Grade 1:
Taper steroids over at 
least 1 month and add 
prophylactic antibiotics 
for opportunistic 
infections.
Resume avelumab therapy
Pulmonary irAEs
Grade of Pneumonitis
(NCI-CTCAE v4)
Management
Follow-up
Grade 1
(Radiographic changes 
only)
Consider delay of 
avelumab therapy
Monitor for symptoms 
every 2 to 3 days
Consider Pulmonary and 
Infectious Disease 
consults
Re-image at least every 3 
weeks If worsens:
Treat as Grade 2 or Grade 
3 to 4
Grade 2
(Mild to moderate new 
symptoms)
Delay avelumab therapy
Pulmonary and Infectious 
Disease consults
Monitor symptoms daily, 
consider hospitalization
1 mg/kg/day 
methylprednisolone IV or 
oral equivalent
Consider bronchoscopy, 
lung biopsy
Re-image every 1 to 3 
days
If improves:
When symptoms return to 
near baseline, taper 
steroids over at least 1 
month and then resume 
avelumab therapy and 
consider prophylactic 
antibiotics
If not improving after 2 
weeks or worsening:
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Treat as Grade 3 to 4
Grade 3 or 4
Severe new symptoms; 
new / worsening hypoxia; 
life-threatening
Discontinue avelumab 
therapy
Hospitalize
Pulmonary and ID consults
2 to 4 mg/kg/day 
methylprednisolone IV or 
equivalent
Add prophylactic 
antibiotics for 
opportunistic infections
Consider bronchoscopy, 
lung biopsy
If improves to baseline:
Taper steroids over at 
least 6 weeks
If not improving after 48 
hours or worsening:
Add additional 
immunosuppression 
(e.g., infliximab, 
cyclophosphamide, 
intravenous 
immunoglobulin, or 
mycophenolate mofetil)
Hepatic irAEs
Grade of Liver Test 
Elevation (NCI-CTCAE 
v4)
Management
Follow-up
Grade 1
AST or ALT > IULN to 3.0 
x IULN and/or total 
bilirubin >IULN to 1.5 x 
IULN
Continue avelumab 
therapy
Continue liver function 
monitoring
If worsens:
Treat as Grade 2 or 3-4
Grade 2
AST or ALT > 3.0 to ≤ 5 x 
IULN and/or total bilirubin 
>1.5 to ≤ 3 x IULN
Delay avelumab therapy
Increase frequency of 
monitoring to every 3 
days
If returns to Baseline:
Resume routine 
monitoring, resume 
avelumab therapy
If elevations persist > 5 to 
7 days or worsen:
0.5 to 1 mg/kg/day 
methylprednisolone or 
oral equivalent and when 
LFT returns to Grade 1 or 
baseline, taper steroids 
over at least 1 month, 
consider prophylactic 
antibiotics for 
opportunistic infections, 
and resume avelumab 
therapy
Grade 3 
AST or ALT >5-20 x IULN 
and/or total bilirubin >3- 
10 x IULN
Grade 4 
AST or ALT >20 IULN
Bilirubin >10 IULN
Discontinue avelumab 
therapy
Increase frequency of 
monitoring to every 1-2 
days
1 to 2 mg/kg/day 
methylprednisolone IV or 
If returns to Grade 2:
Taper steroids over at 
least 1 month
If does not improve in > 3 
to 5 days, worsens or 
rebounds:
Add mycophenolate 
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IV equivalent 2.0 
mg/kg/day
Add prophylactic 
antibiotics for 
opportunistic infections
Consult gastroenterologist
Consider obtaining 
MRI/CT scan of liver and 
liver biopsy if clinically 
warranted
mofetil 1 gram (g) twice 
daily
If no response within an 
additional 3 to 5 days, 
consider other 
immunosuppressants per 
local guidelines
Endocrine irAEs
Patients experiencing symptoms such as fatigue, myalgia, impotence, mental 
status changes, constipation, or other symptoms thought to be associated with 
endocrine abnormalities should be evaluated for thyroid, pituitary, or adrenal 
endocrinopathies and an endocrinologist should be consulted. 
Patients with Grade 2 hypothyroidism should be evaluated by an endocrinologist 
for further management. Patients with Grade 2 hypothyroidism adequately 
managed with thyroid hormone replacement may continue on protocol therapy. 
Patients with Grade 3 or greater hypothyroidism will be considered to have had a 
dose-limiting toxicity. These patients should be managed according to table 
below and evaluation by an endocrinologist is recommended for further 
management. Patients who enter the study on thyroid replacement should have 
their medication adjusted to maintain TSH in the normal range.
Endocrine Disorder
Management
Follow-up
Asymptomatic TSH 
abnormality
Continue avelumab therapy
If TSH < 0.5 x LLN, or TSH > 2x IULN, or consistently 
out of range in 2 subsequent measurements: include 
T4 at subsequent cycles as clinically indicated; 
consider endocrinology consult
Symptomatic 
endocrinopathy
Evaluate endocrine 
function
Consider pituitary scan
Symptomatic with 
abnormal lab/pituitary 
scan:
Delay avelumab therapy
1 to 2 mg/kg/day 
methylprednisolone IV or 
oral equivalent
Initiate appropriate 
hormone therapy
No abnormal lab/pituitary 
MRI scan but symptoms 
persist:
If improves (with or 
without hormone 
replacement): 
Taper steroids over at 
least 1 month and 
consider prophylactic 
antibiotics for 
opportunistic infections 
Resume avelumab therapy 
Participants with adrenal 
insufficiency may need to 
continue steroids with 
mineralocorticoid 
component
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Repeat labs in 1 to 3 
weeks
MRI in 1 month
Suspicion of adrenal crisis 
(for example, severe 
dehydration, hypotension, 
shock out of proportion to 
current illness)
Delay or discontinue avelumab therapy 
Rule out sepsis 
Stress dose of IV steroids with mineralocorticoid 
activity 
IV fluids 
Consult endocrinologist 
If adrenal crisis ruled out, then treat as above for 
symptomatic endocrinopathy
Cardiac irAEs
Myocarditis
Management
Follow-up
New onset of cardiac 
signs or symptoms and / 
or new laboratory cardiac 
biomarker elevations (e.g. 
troponin, CK-MB, BNP) or 
cardiac imaging 
abnormalities suggestive 
of myocarditis. 
Withhold avelumab 
therapy
Hospitalize. In the 
presence of life 
threatening cardiac 
decompensation, consider 
transfer to a facility 
experienced in advanced 
heart failure and 
arrhythmia management.
Cardiology consult to 
establish etiology and 
rule-out immune-
mediated myocarditis. 
Guideline based 
supportive treatment as 
appropriate per cardiology 
consult.*
Consider myocardial 
biopsy if recommended 
per cardiology consult.
If symptoms improve and 
immune-mediated 
etiology is ruled out, re-
start avelumab therapy.
If symptoms do not 
improve/worsen, viral 
myocarditis is excluded, 
and immune-mediated 
etiology is suspected or 
confirmed following 
cardiology consult, 
manage as immune-
mediated myocarditis.
Immune-mediated 
myocarditis
Permanently discontinue 
avelumab.
Guideline based 
supportive treatment as 
appropriate per cardiology 
Once improving, taper 
steroids over at least 1 
month and add 
prophylactic antibiotics 
for opportunistic 
infections.
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consult.*
Methylprednisolone 1-2 
mg/kg/day.
If no improvement or 
worsening, consider 
additional 
immunosuppressions (e.g. 
azathioprine, cyclosporine 
A)
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5.4 Autoimmune or Immune System Disorders Affecting Other 
Organ Systems
Patients experiencing symptoms that may be associated with 
autoimmune or immune-mediated adverse events possibly, probably or 
definitely related to protocol therapy should be evaluated and monitored 
closely. These may include but are not limited to pneumonitis, sarcoid-
like granuloma and neurologic events including hypophysitis, 
encephalitis, aseptic meningitis, and cranial neuropathy especially 
seventh cranial nerve (CNVII) palsy. Consideration should be given to 
subspecialty consultation particularly if systemic immune suppression is 
considered.
5.5 Central Venous Catheter
While not required for the purposes of this study, the insertion of a 
central venous catheter/line (CVL) is recommended for all patients for 
intravenous (IV) administration of study drug as well as other supportive 
medications and laboratory blood draws. Placement of the CVL may be 
performed by the treating institution based on local guidelines for best 
medical practice.
5.6 Concomitant Therapy
Concurrent cancer therapy, including chemotherapy, radiation therapy, 
immunotherapy or biologic therapy may NOT be administered to patients 
receiving study drug. If these treatments are administered the patient will 
be removed from protocol therapy. Immunotherapy and 
immunosuppressive drugs (i.e., systemic corticosteroids except for short 
term treatment of allergic reactions or for the treatment of immune-
related adverse events) should not be administered during the study. For 
patients with adrenal insufficiency, physiological dosing of steroids is 
acceptable. Herbal remedies with immunostimulating properties (e.g., 
mistletoe extract) or interference with major organ function (e.g., 
hypericin) should not be used during the study. Vaccines should not be 
administered during treatment, with the exception of administration of 
the inactive influenza vaccine. No other investigational agents may be 
given while the patient is on study.
6.0 DRUG INFORMATION
6.1 Avelumab (MSB0010718C, anti-PD-L1)
Source and pharmacology: Avelumab is a fully human antibody of the 
immunoglobulin (IgG) 1 isotype that specifically targets and blocks PD-
L1.
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Formulation and stability: The product is a sterile, clear and colorless 
solution presented at concentration of 20mg/mL in United States 
Pharmacopeia (USP) type I glass vials closed with a rubber stopper and 
sealed with an aluminum Flip Off© crimp seal closure. Each single use 
20mg/mL vial contains 200mg of avelumab as a preservative-free 
acetate-buffered solution (ph 5.2) containing mannitol and polysorbate 
20 (Tween 20). Only excipients that conform to the current USP are used.
Storage: Avelumab vials must be stored at 2°C to 8°C until use. Vials that 
are stored at room or higher temperature for extended periods of time 
may be subject to degradation. The drug product must not be frozen. 
Rough shaking of solution must be avoided.
Solution preparation: Avelumab must be diluted with 0.9% saline solution 
(sodium chloride injection) supplied in an infusion bag to a total volume 
of 250mL. Prior to preparation of dilution, each vial should be allowed to 
equilibrate to room temperature. For mixing purposes, after dilution of 
avelumab in the infusion bag, the mixture should be gently inverted 10 
times. Vial contents from different lots should not be mixed in the same 
infusion.
Stability: The chemical and physical in-use stability for the infusion 
solution of avelumab in 0.9% saline solution (sodium chloride injection) 
has been demonstrated for a total of 24 hours at room temperature. 
However, from a microbiological point of view, this solution should be 
used immediately and is not intended to be stored unless dilution has 
taken place in a controlled and validation aseptic condition
Supplier: Pfizer will supply avelumab to all participating institutions.
Toxicity: Most commonly reported adverse events are fatigue, influenza-
like illness, pyrexia, lymphopenia. See Investigator’s Brochure for full 
description.
Dosage and route of administration: Avelumab should be administered as 
an intravenous infusion over 60 minutes. Premedication is required; see 
treatment plan sections.
7.0 REQUIRED EVALUATIONS, TESTS, AND OBSERVATIONS
7.1 Evaluations Before and During Therapy
All participants enrolled at St. Jude Children’s Research Hospital should 
be invited to participate in the St. Jude tissue banking protocol (TBANK) 
and pharmacogenetics protocol PGEN5 at the time of study entry. All 
clinical and laboratory studies to determine eligibility must be performed 
within 7 days prior to enrollment unless otherwise indicated. Imaging 
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studies must be obtained within 14 days prior to the start of protocol 
therapy (repeat tumor imaging if necessary).
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Evaluations, Tests and Observations
Studies to be Obtained
Baseline
During 
Cycle 1
Prior to 
Cycle 2
Prior to 
Subsequent 
Cycles
End of 
Therapy
History
X
X
X
X
Physical exam with vital 
signs
X
Weekly
X
X
X
Height, weight, BSA
X
X
X
X
Performance status
X
X
X
X
CBC, diff, platelets1
X
Weekly
X
X
X
Electrolytes, including 
CA++, PO
4, Mg
++1
X
X
X
X
BUN/creatinine1
X
X
X
X
ALT (SGPT), AST and total 
bilirubin1
X
X
X
X
Total protein/albumin
X
X
X
X
Amylase, lipase, CRP
X
X
X
X
Pregnancy test (females)2
X
X
X
X
Free T4 and TSH3
X
X
X
X
Cortisol
X
X
X
X
Tumor disease evaluation4 
X
End of 
cycle 2
End of Cycle 
4, then every 
3 cycles
X
CT Chest5
X
End of 
cycle 2
End of Cycle 
4, then every 
3 months
X
18F-FDG PET/PET-CT or 
bone scan6
X
End of 
cycle 2
End of Cycle 
4, then every 
3 months
X
Pulse oximetry
X
X
with Tumor 
Disease 
Evaluation
X
Quality of Life
X
X7
X
Correlative research studies (see Section 8.0)
Correlative biology 
(baseline and prior to 
cycle 3 only)
X
X
Tumor tissue (if available)
X
1Blood chemistry and hematology assessments: must be performed at baseline, prior to each avelumab dose, at 
end of treatment visit and at 30 days post-treatment safety follow-up.
2Women of childbearing potential require a negative pregnancy test prior to starting treatment and must be 
willing to adhere to effective contraceptive during and for 60 days after the last dose of avelumab. Males who 
are sexually active with women of childbearing potential must be willing to adhere to effective contraception 
during and for 60 days after the last dose of avelumab. Abstinence is an acceptable method of birth control.
3Free T4 and TSH must be performed at baseline and at least every 8 weeks during treatment and at end of 
treatment or 30 days post-treatment safety follow-up (if not performed in the previous 8 weeks)
4 MRI of primary tumor and CT or MRI of all non-primary lesions meeting eligibility criteria. Use the same 
imaging modality for all disease evaluations. AP and lateral radiographs should be obtained of the primary 
tumor (if present) as well as any skeletal metastases.
5CT of the chest is required for all patients to assess for the development of progressive pulmonary disease.
6 
18F-FDG PET/PET-CT or bone scan is required with each disease evaluation. It is recommended to obtain MRI or 
CT scan on any positive bone lesion on 18F-FDG PET/PET-CT or bone scan. Investigators are encouraged to use 
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the same modality used at baseline to follow positive lesions that are identified with 
18F-FDG PET/PET-CT or 
bone scan
7QOL questionnaires will be completed at baseline, at the end of cycle 2 (8 weeks +1week), at the end of cycle 4 
(16 weeks +1week) and at the end of avelumab therapy.
7.2 Extended Safety Follow-up
Given the potential risk for delayed immune-related toxicities, safety 
follow-up must be performed up to 90 days after the last dose of 
avelumab administration.
The extended safety follow-up beyond 30 days after last study drug 
administration may be performed either via a site visit or via a telephone 
call with subsequent site visit requested in case any concerns noted 
during the telephone call.
8.0 CORRELATIVE RESEARCH STUDIES
8.1 Tumor Tissue
All patients enrolling on this protocol require institutional histological 
confirmation of osteosarcoma at the time of original diagnosis. Even 
though biopsy to confirm recurrence is strongly encouraged, histologic 
confirmation of recurrence/relapse is NOT required for inclusion in this 
study. For patients who have available tissue from diagnosis/recurrence, 
submission of tissue for biologic studies is strongly encouraged.
Tumor tissue that becomes available for biological studies will be 
processed into single cell suspensions. Purification of tumor-associated 
lymphocytes will be performed by Percoll gradient centrifugation and 
then frozen in appropriate freezing media or directly sorted for 
lymphocyte analyses (described below).
8.2 Immunologic Research – NCI Testing Battery
All patients 18 years of age and older will have 6 (10ml) green top tubes 
and 2 (7ml) SST and patients less than 18 years of age will have 2 (10ml) 
green top tubes and 1 (7ml) SST. 
See Laboratory Manual for additional information on collection, processing 
and shipment of samples.
Timing: prior to start of therapy and prior to starting Cycle 3.
Blood samples may be used for other research studies, which may include 
phenotypic and functional analysis of immune cell subsets, circulating 
tumor cell evaluation, and analysis for cytokines, chemokines, antibodies, 
tumor-associated antigens and / or other markers.
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The samples will be processed at The Clinical Support Laboratory at the 
Frederick National Laboratory for Cancer Research (see Appendix III): 
Theresa Burks
Leido-Frederick
1050 Boyles Street 
Bldg 469, Room 121 
Frederick MD 21702  
Phone 301-846-5125 or 301-846-1707
On days samples are drawn, Jen Bangh at the Clinical Support Laboratory 
should be notified (phone: (301) 846-5893; fax (301) 846-6536). She 
will arrange same-day courier delivery of the specimens.
The research samples will contain labels on the blood tubes that have the 
patient's initials, date of birth, the assigned protocol, and the date the 
samples were drawn. The transmittal forms accompanying the samples 
also contain the same information.
Once a patient’s treatment schedule has been determined, it should be 
faxed to Caroline Jochems at the Laboratory of Tumor Immunology and 
Biology / NIH (Fax: (301) 496-2756; phone: (301) 402-6274) for 
planning purposes.
Parameters of immune activation
Immunologic testing will include:
· Peripheral blood mononuclear cells (PBMC)
o Phenotypic and functional analysis of 123 immune cell 
subsets by flow cytometry (CD4, CD8, and regulatory T-
lymphocytes, B-lymphocytes, NK cells, NKT cells, dendritic 
cells, myeloid derived suppressor cells and ratios of effector 
to suppressor cells).
· Serum
o Analysis of cytokines (ifn-γ, IL-10, IL-12, IL-2, IL-4, TGF-β, 
etc.), chemokines, soluble CD27, soluble CD40L, antibodies, 
tumor-associated antigens and/or other markers
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Flow-cytometry analysis of PBMC immune cell subsets: 27 markers, 
123 subsets 
1. CD4: Helper T lymphocytes (33 subsets)
2. CD8: Cytotoxic T lymphocytes (30 subsets)
•
Maturation status of T lymphocytes (in CD4 and CD8):
‒
Naïve: CD45RA
+ CCR7
+
‒
Central Memory: CD45RA
- CCR7
+ 
‒
Effector Memory: CD45RA
- CCR7
-
‒
Terminal (EMRA): CD45RA
+ CCR7
-
•
T lymphocyte markers (in total and memory CD4 and 
CD8):
‒
CTLA-4: inhibition
‒
PD-1: activation/inhibition
‒
PD-L1: activation/cross-inhibition
‒
TIM-3: inhibition
‒
41BB: co-stiactivation
‒
ICOS: activation (only on CD4)
3. Tregs: Regulatory T lymphocytes (CD4
+ CD25
+ FoxP3
+ CD127
-) (7 
subsets)
•
CD45RA: Tregs highly expandable in vitro
•
CTLA-4: Treg suppression
•
CD49d: “contaminating” effector lymphocytes (non-Tregs)
•
ICOS: Treg suppression
•
PD-1: activation/inhibition
•
PD-L1: cross-inhibition
4. B lymphocytes: CD19
+ (3 subsets)
•
PD-1: activation/inhibition
•
PD-L1: cross-inhibition
5. NK: Natural killer cells (CD56
+ CD3
-) (20 subsets)
•
CD16
+ CD56
dim : Mature NK, lytic
•
CD16
+ CD56
br: Functional intermediate, lytic, cytokine 
production
•
CD16
- CD56
br: Immature, cytokine production, abundant in 
placenta
•
CD16
- CD56
dim: non-lytic, non-cytokine production
•
TIM-3: activation
•
PD-1: activation/inhibition
•
PD-L1: cross-inhibition
6. NK-T: CD56
+ CD3
+ (4 subsets)
•
TIM-3: activation
•
PD-1: activation/inhibition
•
PD-L1: cross-inhibition
7. cDCs (Conventional DCs): CD3
-CD56
-HLA-DR
+CD1c
+CD303
- (5 
subsets)
8. pDCs (plasmacytoid DCs ): CD3
-CD56
-HLA-DR
+CD1c
-CD303
+ (5 
subsets)
•
Markers of DC activation
‒
CD83: activation 
‒
TIM-3: inhibition
‒
PD-L1: cross-inhibition
‒
PD1: activation/inhibition
9. MDSCs: Myeloid-derived suppressor cells (CD11b
+ HLA-DR
low/- 
CD33
+)  (16 subsets)
•
CD14: Common Myeloid Marker (high in monocytes, dim in 
granulocytes)
•
CD15: Granulocyte marker 
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Samples sent to the Clinical Support Laboratory at NCI
All data associated with patient samples are protected by using a secure 
database. All samples drawn at the NIH Clinical Center will be transported 
to the Clinical Support Laboratory at the Frederick National Laboratory for 
Cancer Research by couriers.
Samples will be tracked and managed by the Central Repository database. 
All samples will be stored in liquid nitrogen. These freezers are located at 
NCI Frederick Central Repository in Frederick, Maryland.
Fisher BioServices manages the NCI Frederick Central Repositories under 
subcontract to Leidos Biomedical, Inc. NCI Frederick Central Repositories 
store, among other things, biological specimens in support of NIH clinical 
studies. All specimens are stored in secure, limited access facilities with 
sufficient security, back-up and emergency support capability and 
monitoring to ensure long-term integrity of the specimens for research.
Specimens are stored in accordance will applicable HHS and FDA 
Protection of Human Subjects Regulations in accordance with Fisher 
BioServices Federal-wide Assurance. Fisher BioServices’ role is limited to 
clinical research databases and repositories containing patient 
specimens. Fisher BioServices neither conducts nor has any vested 
interest in research on human subjects, but does provide services and 
supports the efforts of its customers, many of which are involved in 
research on human subjects. The Fisher BioServices IRB reviews policies 
and procedures for labeling, data collection and storage, access, and 
security. The IRB will review protection of privacy issues prior to 
acceptance of any new work and in the event of change impacting privacy 
issues in existing work.
It is the intent and purpose of Fisher BioServices to accept only de-
identified samples and sample information. To the best of our ability, 
every effort will be made to ensure that protected information is not sent 
electronically or by hard copy or on vial labels.
Sample data are stored in the BioSpecimen Inventory (BSI) System II. This 
inventory tracking system is used to manage the storage and retrieval of 
specimens as well as maintain specimen data. BSI is designed for 
controlled, concurrent access. It provides a real-time, multi-user 
environment for tracking millions of specimens. The system controls how 
and in what order database updates and searches are performed. This 
control prevents deadlocks and race conditions. For security, BSI has user 
password access, 3 types of user access levels, and 36 user permissions 
(levels of access) that can be set to control access to the system 
functions. BSI provides audit tracking for processes that are done to 
specimens including shipping, returning to inventory, aliquoting, 
thawing, additives, and other processes. BSI tracks the ancestry of 
specimens as they are aliquoted, as well as discrepancies and discrepancy 
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resolution for specimens received by the repository. If a specimen goes 
out of the inventory, the system maintains data associated with the 
withdrawal request. Vials are labeled with a unique BSI ID, which is 
printed in both eye-readable and bar-coded format. No patient-specific 
information is encoded in this ID.
Investigators are granted view, input and withdrawal authority only for 
their specimens. They may not view specimen data or access specimens 
for which they have not been authorized. Access to specimen storage is 
confined to repository staff. Visitors to the repositories are escorted by 
repository staff at all times.
NCI Protocol Completion/Sample Destruction
All specimens obtained in the protocol are used as defined in the 
protocol. Any specimens that are remaining at the completion of the 
protocol will be stored in the conditions described above. The study will 
remain open so long as sample or data analysis continues. Samples from 
consenting subjects will be stored until they are no longer of scientific 
value or if a subject withdraws consent for their continued use, at which 
time they will be destroyed. Once primary research objectives for the 
protocol are achieved, intramural researchers can request access to 
remaining samples providing they have an IRB approved protocol and 
patient consent.
The PI will report any loss or destruction of samples to the NCI IRB as 
soon as he is made aware of such loss. The PI will report destroyed 
samples to the IRB if samples become unsalvageable because of 
environmental factors (ex. broken freezer or lack of dry ice in a shipping 
container) or if a patient withdraws consent. Samples will also be 
reported as lost if they are lost in transit between facilities or misplaced 
by a researcher. Freezer problems, lost samples or other problems 
associated with samples will also be reported to the IRB, the NCI Clinical 
Director, and the office of the CCR, NCI.
8.3 Immunologic Research – St. Jude Testing Battery
Three aliquots of peripheral blood mononuclear cells (PBMCs) 
(representing a 10 mL draw in Blue/Black CPT tubes) will be obtained for 
testing for TCR repertoire, T cell phenotype, transcriptional profile, and 
epigenetic landscape. Samples may be frozen for the purposes of 
shipping and handling. Should tumor tissue be available from resection 
of the patient’s primary tumor AND/OR metastatic sites of disease, single 
cell suspensions of tumor cells should also be obtained for analysis.
See Laboratory Manual for additional information on collection, 
processing and shipment of samples
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Timing: prior to start of therapy and prior to cycle 3. Samples should be 
sent via FedEx overnight services to:
Paul G. Thomas, PhD
Integrated Research Center, Room E7054
St. Jude Children’s Research Hospital
262 Danny Thomas Place
Memphis, TN 38105
On the day of sample acquisition, please contact Paul Thomas or Ben 
Youngblood at (901) 595-6749 to arrange for pickup of materials.
Samples will be stained with fluorescently labeled antibodies and sorted 
for single cell analysis (1/3 of the sample) with the remaining sample 
sorted on specific cell phenotypes for transcriptional and epigenetic 
analysis.
Preparation of single cell suspension: Tumor sample (from freshly-
resected surgical specimen) is weighed and sequentially minced into 
small pieces (approx. 3 mm in size) using scissors or scalpel. Tumor 
tissue is digested at 37
oC in RPMI medium supplemented with 5% FBS, 
Collagenase 4, and DNase I. Digested tumors are homogenized by 
pipetting, and filtered through a 70 um cell strainer. The remaining 
undigested pieces are forced to pass through the strainer using a syringe 
plunger. The single cell suspension is washed 3 times with ice cold PBS, 
and RBC are lysed (if needed) using an ammonium chloride isotonic 
solution. The number of live cells is determined using a hemocytometer 
after exclusion of dead cells by Trypan blue dye.
Polyparametric immunophenotypic analysis: Cells are labeled with 
fluorescent conjugated antibodies against the listed human epitopes, and 
subsequently analyzed in a flow cytometer (BD LSRII). We will evaluate 
PD-L1 expression in cell surface of infiltrating leukocytes (CD45+ 
fraction) as well as in CD45- fraction, which primary consists of tumor 
cells. 
Specificity
Fluorophore Clone
CCR7
FITC
G043H7
PD1
PE
EH12.2H7
TIM3
PECY7
F38-2E2
CD45RO
APC
UCHL1 
CD3
APCCY7
SK7
CD8a
PB (421)
RPA-T8
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Samples sent to St. Jude
All data associated with patient samples are protected by using a secure 
database. All samples will be stored in liquid nitrogen. These freezers are 
located at St. Jude in a secure facility. All specimens are stored in secure, 
limited access facilities with sufficient security, back-up and emergency 
support capability and monitoring to ensure long-term integrity of the 
specimens for research. Specimens are stored in accordance will 
applicable HHS and FDA Protection of Human Subjects Regulations in 
accordance with SJCRH Federal-wide Assurance. The St. Jude IRB reviews 
policies and procedures for labeling, data collection and storage, access, 
and security. The IRB will review protection of privacy issues prior to 
acceptance of any new work and in the event of change impacting privacy 
issues in existing work.
It is the intent and purpose of the investigators at St. Jude to accept only de-
identified samples and sample information. To the best of our ability, every 
effort will be made to ensure that protected information is not sent 
electronically or by hard copy or on vial labels.
9.0 EVALUATION CRITERIA
9.1 Response Definitions and Criteria for Solid Tumors
See the table in section 7.1 for the schedule of tumor evaluations. In 
addition to the scheduled scans, a confirmatory scan should be obtained 
28 days following initial documentation of objective response.
Response and progression will be evaluated in this study using the 
revised Response Evaluation Criteria in Solid Tumors (RECIST) guideline 
(version 1.1)
90. Key points are that up to 5 target lesions may be 
identified and that changes in the largest diameter (unidimensional 
CD45
BV605
HI30  
PDL1
BV786
MIH1
7AAD
n/a
(dead cell 
exclusion 
dye)
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measurement) of the tumor lesions but the shortest diameter of 
malignant lymph nodes are used in the RECIST v 1.1 criteria.
9.1.1 Definitions
Evaluable for objective response: Patients who exhibit objective disease 
progression prior to the end of cycle 1 will be considered evaluable for 
response. For all other patients, only those patients who have measurable 
disease present at baseline, have received at least one cycle of therapy, 
and have had their disease re-evaluated will be considered evaluable for 
response.
Evaluable Non-Target Disease Response: Patients who have lesions 
present at baseline that are evaluable but do not meet the definitions of 
measurable disease, have received at least one cycle of therapy, and have 
had their disease re-evaluated will be considered evaluable for non-
target disease. The response assessment is based on the presence, 
absence, or unequivocal progression of the lesions.
9.1.2 Disease Parameters
Measurable disease: Measurable lesions are defined as those that can be 
accurately measured in at least one dimension (longest diameter to be 
recorded) as ≥20 mm by chest x-ray, as ≥10 mm with CT scan, or ≥10 
mm with calipers by clinical exam. All tumor measurements must be 
recorded in millimeters (or decimal fractions of centimeters).
Note: Tumor lesions that are situated in a previously irradiated area 
might or might not be considered measurable. If the investigator thinks it 
appropriate to include them, the conditions under which such lesions 
should be considered must be defined in the database.
Malignant lymph nodes: To be considered pathologically enlarged and 
measurable, a lymph node must be ≥15 mm in short axis when assessed 
by CT scan (CT scan slice thickness no greater than 5 mm). At baseline 
and in follow-up, only the short axis will be measured and followed.
Non-measurable disease: All other lesions (or sites of disease), including 
small lesions (longest diameter <10 mm or pathological lymph nodes 
with ≥ 10 to <15 mm short axis), are considered non-measurable 
disease. Bone lesions, leptomeningeal disease, ascites, pleural/pericardial 
effusions, lymphangitis cutis/pulmonitis, inflammatory breast disease, 
and abdominal masses (not followed by CT or MRI), are considered as 
non-measurable.
Note: Cystic lesions that meet the criteria for radiographically defined 
simple cysts should not be considered as malignant lesions (neither 
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measurable nor non-measurable) since they are, by definition, simple 
cysts. ‘Cystic lesions’ thought to represent cystic metastases can be 
considered as measurable lesions, if they meet the definition of 
measurability described above. However, if non-cystic lesions are present 
in the same patient, these are preferred for selection as target lesions.
Target lesions: All measurable lesions up to a maximum of 2 lesions per 
organ and 5 lesions in total, representative of all involved organs, should 
be identified as target lesions and recorded and measured at baseline. 
Target lesions should be selected on the basis of their size (lesions with 
the longest diameter), be representative of all involved organs, but in 
addition should be those that lend themselves to reproducible repeated 
measurements. It may be the case that, on occasion, the largest lesion 
does not lend itself to reproducible measurement in which circumstance 
the next largest lesion that can be measured reproducibly should be 
selected. A sum of the diameters (longest for non-nodal lesions, short 
axis for nodal lesions) for all target lesions will be calculated and 
reported as the baseline sum diameters. If lymph nodes are to be 
included in the sum, then only the short axis is added into the sum. The 
baseline sum diameters will be used as reference to further characterize 
any objective tumor regression in the measurable dimension of the 
disease.
Non-target lesions: All other lesions (or sites of disease) including any 
measurable lesions over and above the 5 target lesions should be 
identified as non-target lesions and should also be recorded at baseline. 
Measurements of these lesions are not required, but the presence, 
absence, or in rare cases unequivocal progression of each should be 
noted throughout follow-up.
9.1.3 Methods for Evaluation of Measurable Disease
All measurements should be taken and recorded in metric notation using 
a ruler or calipers.  The same method of assessment and the same 
technique should be used to characterize each identified and reported 
lesion at baseline and during follow-up. Imaging-based evaluation is 
preferred to evaluation by clinical examination unless the lesion(s) being 
followed cannot be imaged but are assessable by clinical exam.
Clinical lesions: Clinical lesions will only be considered measurable when 
they are superficial (e.g., skin nodules and palpable lymph nodes) and 
≥10 mm diameter as assessed using calipers (e.g., skin nodules). In the 
case of skin lesions, documentation by color photography, including a 
ruler to estimate the size of the lesion, is recommended.
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Chest x-ray: Lesions on chest x-ray are acceptable as measurable lesions 
when they are clearly defined and surrounded by aerated lung. However, 
CT is preferable.
Conventional CT and MRI: This guideline has defined measurability of 
lesions on CT scan based on the assumption that CT slice thickness is 5 
mm or less. If CT scans have slice thickness greater than 5 mm, the 
minimum size for a measurable lesion should be twice the slice 
thickness. MRI is also acceptable in certain situations (e.g. for body 
scans). Ideally, the same type of scanner should be used and the image 
acquisition protocol should be followed as closely as possible to prior 
scans.
PET-CT: At present, the low dose or attenuation correction CT portion of 
a combined PET-CT is not always of optimal diagnostic CT quality for use 
with RECIST measurements. However, if the site can document that the 
CT performed as part of a PET-CT is of identical diagnostic quality to a 
diagnostic CT (with IV and oral contrast), then the CT portion of the PET-
CT can be used for RECIST measurements and can be used 
interchangeably with conventional CT in accurately measuring cancer 
lesions over time. Note, however, that the PET portion of the CT 
introduces additional data which may bias an investigator if it is not 
routinely or serially performed.
Tumor markers: Tumor markers alone cannot be used to assess 
response. If markers are initially above the upper normal limit, they must 
normalize for a patient to be considered in complete clinical response.
Cytology, histology: These techniques can be used to differentiate 
between partial responses (PR) and complete responses (CR) in rare cases 
(e.g., residual lesions in tumor types, such as germ cell tumors, where 
known residual benign tumors can remain). Cytology should be obtained 
if an effusion appears or worsens during treatment when the measurable 
tumor has met criteria for response or stable disease.
18F-FDG PET/PET-CT: While 18F-FDG PET/PET-CT response assessments 
need additional study, it is sometimes reasonable to incorporate the use 
of 18F-FDG PET/PET-CT scanning to complement CT scanning in 
assessment of progression (particularly possible 'new' disease). 
New lesions on the basis of 18F-FDG PET/PET-CT imaging can be 
identified according to the following algorithm:
a. Negative 18F-FDG PET/PET-CT at baseline, with a positive 18F-FDG 
PET/PET-CT at follow-up is a sign of PD based on a new lesion.
b. No 18F-FDG PET/PET-CT at baseline and a positive 18F-FDG 
PET/PET-CT at follow-up: If the positive 18F-FDG PET/PET-CT at 
follow-up corresponds to a new site of disease confirmed by CT, 
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this is PD. If the positive 
18F-FDG PET/PET-CT at follow-up is not 
confirmed as a new site of disease on CT, additional follow-up CT 
scans are needed to determine if there is truly progression 
occurring at that site (if so, the date of PD will be the date of the 
initial abnormal 
18F-FDG PET/PET-CT scan). If the positive 18F-FDG 
PET/PET-CT at follow-up corresponds to a pre-existing site of 
disease on CT that is not progressing on the basis of the anatomic 
images, this is not PD.
Note: A ‘positive’ 
18F-FDG PET/PET-CT scan lesion means one that is FDG 
avid with an uptake greater than twice that of the surrounding tissue on 
the attenuation corrected image.
9.2 Response Criteria for Patients with Solid Tumor and Measurable 
Disease
9.2.1Evaluation of Target Lesions
Complete response (CR): Disappearance of all target and non-target 
lesions. Any pathological lymph nodes (whether target or non-target) 
must have reduction in short axis to <10 mm. If immunocytology is 
available, no disease must be detected by that methodology. 
Partial response (PR): At least a 30% decrease in the sum of the diameters 
of target lesions, taking as reference the baseline sum diameters.
Progressive disease (PD): At least a 20% increase in the sum of the 
diameters of target lesions, taking as reference the smallest sum on study 
(this includes the baseline sum if that is the smallest on study). In addition 
to the relative increase of 20%, the sum must also demonstrate an absolute 
increase of at least 5 mm. (Note: the appearance of one or more new 
lesions is also considered progressions). Note: in presence of SD or PR in 
target disease but unequivocal progression in non-target or non-
measurable disease, the patient has PD if there is an overall level of 
substantial worsening in non-target disease such that the overall tumor 
burden has increased sufficiently to merit discontinuation of therapy.
Stable disease (SD): Insufficient shrinkage to qualify for PR or insufficient 
increase to qualify for PD, taking as reference the smallest sum diameters 
while on study.
9.2.2Evaluation of Non-Target Lesions
Complete response (CR): Disappearance of all non-target lesions and 
normalization of tumor marker level. All lymph nodes must be non-
pathological in size (<10 mm short axis)
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Note: If tumor markers are initially above the upper normal limit, they 
must normalize for a patient to be considered in complete clinical 
response.
Non-CR/Non-PD: Persistence of one or more non-target lesion(s) and/or 
maintenance of tumor marker level above the normal limits
Progressive disease (PD): Appearance of one or more new lesions and/or 
unequivocal progression of existing non-target lesions. Unequivocal 
progression should not normally trump target lesion status. It must be 
representative of overall disease status change, not a single lesion 
increase.
Overall best response assessment: Each patient will be classified 
according to his “best response” for the purposes of analysis of treatment 
effect. Best response is determined as outlined in Section 9.4 from a 
sequence of overall response assessments.
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9.3 Response Criteria for Evaluable Disease
Note: all participants must have measurable disease, but they can also 
have evaluable disease in addition to measurable disease.
Evaluable disease: The presence of at least one lesion, with no lesion that 
can be accurately measured in at least one dimension. Such lesions may 
be evaluable by nuclear medicine techniques, immunocytochemistry 
techniques, tumor markers or other reliable measures.
Complete response (CR): Disappearance of all evaluable disease.
Partial response (PR): Partial responses cannot be determined in patients 
with evaluable disease.
Stable disease (SD): That which does not qualify as Complete Response 
(CR), Partial Response (PR), or Progressive Disease.
Progressive disease (PD): The appearance of one or more new lesions or 
evidence of laboratory, clinical, or radiographic progression.
Overall best response assessment: Each patient will be classified 
according to his “best response” for the purposes of analysis of treatment 
effect. Best response is determined as outlined in Section 9.4 from a 
sequence of overall response assessments.
9.4 Best Response
9.4.1Evaluation of Best Overall Response
The best overall response is the best response recorded from the start of 
the treatment until disease progression/recurrence (taking as reference 
for progressive disease the smallest measurements recorded since the 
treatment started).  The patient's best response assignment will depend 
on the achievement of both measurement and confirmation criteria.
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Table 1: Patients with Measurable Disease (i.e., Target Disease)
Target Lesions
Non-Target 
Lesions
New Lesions
Overall 
Response
CR
CR
No
CR
CR
Non-CR/Non-PD
No
PR
CR
Not evaluated
No
PR
PR
Non-CR/Non-
PD/not 
evaluated
No
PR
SD
Non-CR/Non-
PD/not 
evaluated
No
SD
PD
Any
Yes or No
PD
Any
PD*
Yes or No
PD
Any 
Any
Yes
PD
* In exceptional circumstances, unequivocal progression in non-target lesions may be accepted as 
disease progression.
Note:  Patients with a global deterioration of health status requiring discontinuation of treatment 
without any objective evidence of disease progression at that time should be reported as 
“symptomatic deterioration.”  Every effort should be made to document the objective progression 
even after discontinuation of treatment.
Table 2: Patients with Non-Measurable Disease (i.e., Non-Target Disease)
Non-Target Lesions
New Lesions
Overall Response
CR
No
CR
Non-CR/Non-PD
No
Non-CR/Non-PD*
Not all evaluated
No
Not evaluated
Unequivocal PD
Yes or No
PD
Any
Yes
PD
*“non-CR/non-PD” is preferred over “stable disease” for non-target disease since SD is 
increasingly used as an endpoint for assessment of efficacy in some trials so to assign this 
category when no lesions can be measured is not advised.
Table 3: Sequences of overall response assessments with corresponding 
best response
1st Assessment
2nd Assessment
Best Response
Progression
Progressive disease
Stable, PR, CR
Progression
Progressive disease
Stable
Stable
Stable
Stable
PR, CR
Stable
Stable
Not done
Not RECIST classifiable
PR
PR
PR
PR
CR
PR
PR, CR
Not done
Not RECIST classifiable
CR
CR
CR
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9.5 Duration of Response
Duration of overall response: The duration of overall response is 
measured from the time measurement criteria are met for CR or PR 
(whichever is first recorded) until the first date that recurrent or 
progressive disease is objectively documented (taking as reference for 
progressive disease the smallest measurements recorded since the 
treatment started).
The duration of overall CR is measured from the time measurement 
criteria are first met for CR until the first date that progressive disease is 
objectively documented.
Duration of stable disease: Stable disease is measured from the start of 
the treatment until the criteria for progression are met, taking as 
reference the smallest measurements recorded since the treatment 
started, including the baseline measurements.
9.6 Toxicity Evaluation Criteria
This study will utilize the CTCAE v4.03 for toxicity and performance 
reporting. A copy of the CTCAE v4.03 can be downloaded from the CTEP 
home page (http://ctep.info.nih.gov). Additionally, the toxicities are to be 
reported on the appropriate data collection forms.
10.0 OFF TREATMENT AND OFF STUDY CRITERIA
A genuine effort must be made to determine the reason(s) why a 
participant fails to return for the necessary visits or is discontinued from 
the trial. Information regarding the reason for not completing the trial 
will be recorded on the appropriate case report forms.
It will be documented whether or not each patient completed the clinical 
study. If for any patient study treatment or observations were 
discontinued, the reason will be recorded on the appropriate case report 
form. Reasons that a patient may discontinue participation in a clinical 
study are listed below. All patients will be followed for survival until they 
meet the criteria for off-study.
10.1 Off Treatment Criteria
· Clinical or radiographic evidence of progressive disease of 
greater than 20% increase baseline target lesions selected 
according to RECIST criteria
· Adverse events/toxicity requiring removal from protocol therapy
· Refusal of further protocol therapy by patient/parent/guardian
· Non-compliance that in the opinion of the investigator does not 
allow for ongoing participation
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· Physician determines it is not in the patient’s best interest to 
continue on therapy
· Repeated eligibility laboratory studies are outside the 
parameters required for eligibility prior to the start of protocol 
therapy
· Study is terminated by the sponsor
· Pregnancy
· Development of a second malignancy
10.2 Off Study Criteria
· Death
· Lost to follow-up
· Patient subsequent enrollment on another study with tumor 
therapeutic intent (e.g. at recurrence) or additional non-protocol 
anti-tumor therapy.
· Withdrawal of consent for any further data submission
· Patient did not receive protocol treatment after study enrollment
· Withdrawal of consent
11.0 SAFETY AND ADVERSE EVENT REPORTING REQUIREMENTS
11.1 Reporting Adverse Experiences and Deaths to St. Jude IRB
Only “unanticipated problems involving risks to participants or others” 
referred to hereafter as “unanticipated problems” are required to be 
reported to the St. Jude IRB promptly, but in no event later than 10 
working days after the investigator first learns of the unanticipated 
problem. Regardless of whether the event is internal or external (for 
example, an IND safety report by the sponsor pursuant to 21 CFR 
312.32), only adverse events that constitute unanticipated problems are 
reportable to the St. Jude IRB. As further described in the definition of 
unanticipated problem, this includes any event that in the PI’s opinion 
was:
· Unexpected (in terms of nature, severity, or frequency) given (1) the 
research procedures that are described in the protocol-related 
documents, such as the IRB-approved research protocol and 
informed consent document, as well as other relevant information 
available about the research; (2) the observed rate of occurrence 
(compared to a credible baseline for comparison); and (3) the 
characteristics of the subject population being studied; and
· Related or possibly related to participation in the research; and
· Serious; or if not serious suggests that the research places subjects 
or others at a greater risk of harm (including physical, 
psychological, economic, or social harm) than was previously 
known or recognized. 
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Unrelated, expected deaths that occur after patient has completed 
protocol treatment do not require reporting to the IRB. Though death is 
“serious”, the event must meet the other two requirements of “related or 
possibly related” and “unexpected/unanticipated” to be considered 
reportable. However, all deaths while on active protocol treatment (or 
within 30 days of last protocol treatment) will be require reporting to the 
IRB.
Deaths meeting reporting requirements are to be reported immediately to 
the St. Jude IRB, but in no event later than 48 hours after the investigator 
first learns of the death.
The following definitions apply with respect to reporting adverse 
experiences: 
Serious adverse event (SAE): Any adverse event temporally associated with 
the subject’s participation in research that meets any of the following 
criteria:
· results in death;
· is life-threatening (places the subject at immediate risk of death 
from the event as it occurred);
· requires inpatient hospitalization or prolongation of existing 
hospitalization;
· results in a persistent or significant disability/incapacity;
· results in a congenital anomaly/birth defect; or any other adverse 
event that, based upon appropriate medical judgment, may 
jeopardize the subject’s health and may require medical or surgical 
intervention to prevent one of the other outcomes listed in this 
definition (examples of such events include:  any substantial 
disruption of the ability to conduct normal life functions, allergic 
bronchospasm requiring intensive treatment in the emergency 
room or at home, blood dyscrasias or convulsions that do not 
result in inpatient hospitalization, or the development of drug 
dependency or drug abuse), a congenital anomaly/birth defect, 
secondary or concurrent cancer, medication overdose, or is any 
medical event which requires treatment to prevent any of the 
medical outcomes previously listed.  
Unexpected adverse event:
· Any adverse event for which the specificity or severity is not 
consistent with the protocol-related documents, including the 
applicable investigator brochure, IRB approved consent form, 
Investigational New Drug (IND) or Investigational Device Exemption 
(IDE) application, or other relevant sources of information, such as 
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product labeling and package inserts; or if it does appear in such 
documents, an event in which the specificity, severity or duration is 
not consistent with the risk information included therein; or 
· The observed rate of occurrence is a clinically significant increase 
in the expected rate (based on a credible baseline rate for 
comparison); or 
· The occurrence is not consistent with the expected natural 
progression of any underlying disease, disorder, or condition of the 
subject(s) experiencing the adverse event and the subject’s 
predisposing risk factor profile for the adverse event. 
Internal events: Events experienced by a research participant enrolled at a 
site under the jurisdiction of St. Jude IRB for either multicenter or single-
center research projects.
External events: Events experienced by participants enrolled at a site 
external to the jurisdiction of the St. Jude Institutional Review Board (IRB) 
or in a study for which St. Jude is not the coordinating center or the IRB of 
record.
Unanticipated problem involving risks to subjects or others: An 
unanticipated problem involving risks to subjects or others is an event, 
which was not expected to occur and which increases the degree of risk 
posed to research participants.  
Such events, in general, meet all of the following criteria: 
· unexpected; 
· related or possibly related to participation in the research; and
· suggests that the research places subjects or others at a greater 
risk of harm (including physical, psychological, economic, or social 
harm) than was previously known or recognized.  An unanticipated 
problem involving risk to subjects or others may exist even when 
actual harm does not occur to any participant.
Consistent with FDA and OHRP guidance on reporting unanticipated 
problems and adverse events to IRBs, the St. Jude IRB does not require 
the submission of external events, for example IND safety reports, nor is 
a summary of such events/reports required; however, if an event giving 
rise to an IND safety or other external event report constitutes an 
“unanticipated problem involving risks to subjects or others” it must be 
reported in accordance with this policy.  In general, to be reportable 
external events need to have implications for the conduct of the study 
(for example, requiring a significant and usually safety-related change in 
the protocol and/or informed consent form).
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Although some adverse events will qualify as unanticipated problems 
involving risks to subjects or others, some will not; and there may be 
other unanticipated problems that go beyond the definitions of serious 
and/or unexpected adverse events.  
Examples of unanticipated problems involving risks to subjects or others 
include:
· Improperly staging a participant’s tumor resulting in the participant 
being assigned to an incorrect arm of the research study;
· The theft of a research computer containing confidential subject 
information (breach of confidentiality); and 
· The contamination of a study drug.  Unanticipated problems 
generally will warrant consideration of substantive changes in the 
research protocol or informed consent process/document or other 
corrective actions in order to protect the safety, welfare, or rights 
of subjects or others. 
The principal investigator has the obligation to report all serious adverse 
events to the FDA and IRB.
11.2 Reporting to St Jude IRB
This is an investigator-initiated study. The principal investigator, Michael 
W. Bishop and St. Jude are conducting the study and acting as the 
sponsor. Therefore, the legal/ethical obligations of the principal 
investigator include both those of a sponsor and those of an investigator.
11.3 Reporting from St. Jude to Pfizer (Drug Manufacturer)
The investigator primary responsibilities in the safety reporting are to 
identify and follow-up on Serious Adverse Events (SAEs) experienced by 
participants in the study and to forward the information to the local 
regulatory authorities and Pfizer, as required by local regulations (for 
regulatory reporting) and IIR agreement (for reporting to Pfizer).
The following reportable events must be submitted to Pfizer within 24 
hours (or immediately for death or life-threatening events) using the 
provided Investigator-Initiated Research Serious Adverse Event Form (IIR 
SAE) with the Pfizer Reportable Events Fax Cover Sheet with each SAE 
submission.
· Adverse Events that are serious AND unexpected
· Exposure during Pregnancy or Breastfeeding (even if not associated 
with an adverse event)
· Occupational exposure (even if not associated with an adverse 
event)
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· Potential drug-induced liver injury (Hy’s Law cases):  These events 
are considered important medical events and should be reported as 
SAEs.
Detailed guidance on the safety reporting is provided in the Safety 
Reporting Reference Manual.
Contact information for submission of reportable events to Pfizer:
Fax: Pfizer U.S. Clinical Trial Department, Fax 1-866-997-8322. 
or
E-mail: CTPGrants@pfizer.com, specifying:
· Protocol
· Research Participant ID
· Site PI
· SAE/Onset
11.4 Reporting to St. Jude Regulatory Affairs Office and FDA
Any unanticipated fatal or unanticipated life-threatening event judged by 
the PI to be at least possibly due to the study treatment, will be reported 
to the FDA by telephone or fax as soon as possible but no later than 
seven calendar days after notification of the event and followed by a 
written safety report as complete as possible within eight additional 
calendar days (i.e. full report 15 calendar days total after notification of 
event).
Unanticipated, non-fatal and non-life-threatening adverse events that 
occur in on-study patients and that are considered due to or possibly due 
to the investigational agent, will be reported to the FDA by written safety 
report as soon as possible but no later than 15 calendar days of the 
notification of the occurrence of the event. Expected SAEs, even 
unexpected fatal SAEs, considered by the PI to be not related to the 
study, will be reported to the FDA in the Annual Review Report along with 
non-serious AEs. All FDA correspondence and reporting will be 
conducted through the St. Jude Office of Regulatory Affairs.
Copies of all correspondence to the St. Jude IRB, including SAE reports, 
are provided to the St. Jude Regulatory Affairs office by the St. Jude study 
team. FDA-related correspondence and reporting will be conducted 
through the Regulatory Affairs office.
11.5 Recording Adverse Events and Serious Adverse Events
Adverse events (AEs) will be evaluated and documented by the clinical 
staff and investigators throughout inpatient hospitalizations and each 
outpatient visit. CRAs are responsible for reviewing documentation 
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related to AEs and entering directly into CRIS protocol-specific database 
for all adverse events grade 3 or higher. Peripheral neuropathy will be 
captured if Grade 2 or higher. The data to be recorded are 1) the event 
description, 2) the NCI CTCAE v4.0 code and grade, 3) the onset date, 4) 
the resolution date (or ongoing if it has not resolved at time of off study), 
4) action taken for event, 5) patient outcome 6) relationship of AE to 
protocol treatment/interventions, 7) if AE was expected or unexpected, 
and 8) comments, if applicable. AEs that are classified as serious, 
unexpected, and at least possibly related will be notated as such in the 
database as “SAEs”. These events will be reported expeditiously to the St. 
Jude IRB within the timeframes as described above.
Cumulative summary of Grade 2 clinically relevant events and all Grades 
3-5 events will be reported as part of the progress reports to IRB at the 
time of continuing review. Specific data entry instructions for AEs and 
other protocol-related data will be documented in protocol-specific data 
entry guidelines, which will be developed and maintained by study team 
and clinical research informatics.
The study team will meet regularly to discuss AEs (and other study 
progress as required by institutional DSMP). The PI will review Adverse 
Event reports generated from the research database, and corrections will 
be made if applicable. Once the information is final the PI will sign and 
date reports, to acknowledge his/her review and approval of the AE as 
entered in the research database.
11.6 Process for Reporting Adverse Events from and to Collaborating 
Sites
Adverse events from collaborating sites will also be reviewed by the PI 
and discussed in study team meetings as described above. SAE reports 
from collaborating sites for AEs that are serious, unexpected, and at least 
possibly related to protocol treatment or interventions will be reported to 
site IRB and the St. Jude IRB within the reporting requirements described 
above. The PI will determine if this is an event that will need to be 
reported expeditiously to all participating sites, considering the following 
criteria:
· Is the AE serious, unexpected, and related or possibly related to 
participation in the research?
· Is the AE expected, but occurring at a significantly higher frequency 
or severity than expected?
· Is this an AE that is unexpected (regardless of severity that may 
alter the IRB’s analysis of the risk versus potential benefit of the 
research and, as a result, warrant consideration of substantive 
changes in the research protocol or informed consent 
process/document?
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With the submission of the “Reportable Event” in St. Jude TRACKS 
application, the PI will indicate if all sites should be notified to report to 
their IRBs, and if the protocol and/or consent should be amended 
(consent will be amended if event is information that should be 
communicated to currently enrolled subjects). Generally, only events that 
warrant an amendment to the protocol and/or consent will be reported 
expeditiously to all sites. However, any event may be reported 
expeditiously to all sites at the discretion of the PI. A cumulative 
summary of Grade 2-5 AEs and expected/unrelated deaths that occur 
more than 30 days after protocol treatment will be reported to all sites 
with study progress report at the time of continuing review. 
For collaborating sites: Serious AND unexpected events are to be 
reported to the St. Jude PI (Dr. Michael Bishop) within 48-72 hours via fax 
or email.
Unexpected deaths must be reported to the St. Jude PI by email or phone 
call to Dr. Michael Bishop within 24 hours of the event.  A written report 
must follow.  The study team should be copied on all correspondence 
regarding the event. Sent report to:
Michael W. Bishop, MD, MS
Department of Oncology, Solid Tumor Division
St. Jude Children’s Research Hospital
262 Danny Thomas Place
Memphis, TN 38105
Phone: 901-595-6407
Email:  Michael.Bishop@stjude.org 
OSTPDL1StudyTeam@stjude.org
12.0 DATA COLLECTION, MONITORING AND CONFIDENTIALITY
12.1 Data Collection 
Electronic case report forms (e-CRFs) will be completed by the clinical 
trials staff from the Cancer Center Comprehensive Center, Developmental 
Therapeutics for Solid Tumor program. Data will be entered from record 
directly into a secure CRIS database, developed and maintained by St. 
Jude Clinical Research Informatics.
Data Management will be supervised by the Director of Clinical Trials 
Management, and Manager of Clinical Research Operations for the 
Developmental Biology for Solid Tumors Program working with Dr. Bishop 
or his designee. All protocol-specific data and all grade 2-5 adverse 
events will be recorded by the clinical research associates into the CRIS 
database, ideally within 2-4 weeks of completion of cycle. All questions 
will be directed to the attending physician and/or PI and reviewed at 
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regularly-scheduled working meetings. The attending physicians (or their 
designees) are responsible for keeping up-to-date roadmaps in the 
patient’s primary SJCRH medical chart.
Regular (at least monthly) summaries of toxicity and protocol events will 
be generated for the PI and the department of Biostatistics to review.
12.2 Study Monitoring
Monitoring of this protocol is considered to be in the high-risk 3 
category (HR-3). The study specific Monitoring Plan is a separate 
document from this protocol. The study team will meet at appropriate 
intervals to review case histories or quality summaries on participants. 
Highlights of the protocol monitoring plan are below:
The Clinical Research Monitor will assess protocol and regulatory 
compliance as well as the accuracy and completeness of all data points 
for the first two participants then 15% of study enrollees every six 
months. Accrual will be tracked continuously for studies that have strata. 
All SAE reports will be monitored for type, grade, attribution, duration, 
timeliness and appropriateness on all study participants semi-annually.
The monitor will also verify 100% of all data points on the first two 
participants and on 15% of cases thereafter. Protocol compliance 
monitoring will include participant status, eligibility, the informed 
consent process, demographics, staging, study objectives, subgroup 
assignment, treatments, evaluations, responses, participant protocol 
status, off-study, and off-therapy criteria. The Monitor will generate a 
formal report which is shared with the Principal Investigator (PI), study 
team and the Internal Monitoring Committee (IMC).  Monitoring may be 
conducted more frequently if deemed necessary by administration, the 
Institutional Review Board (IRB), or the IMC.
Continuing reviews by the IRB and CT-SRC will occur at least annually. In 
addition, SAE reports in TRACKS (Total Research and Knowledge System) 
are reviewed in a timely manner by the IRB/ OHSP.
Source document verification of eligibility for all SJCRH cases will be 
performed within two weeks of completion of enrollment. This will 
include verification of appropriate documentation of consent. Monitoring 
of timeliness of serious adverse event reporting will be done as events 
are reported in TRACKS. 
St. Jude affiliates and domestic collaborating study sites will be 
monitored on-site by a representative of St. Jude at intervals specified in 
the Data and Safety Monitoring Plan.
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12.3 Confidentiality
Data abstracted from the subject’s medical record and entered into CRFs 
will be entered into a secure, password protected, protocol-specific 
database maintained by St. Jude Clinical Research Informatics. The 
database will be stored in secure sites. All participants will be given a 
unique number and that will be their identifier in the trial. That number 
will be recorded on an enrollment log, linking the number to the 
participant’s name, and that log will be stored securely in the trial master 
file. The privacy of the study subject and all confidentialities will be 
handled according to institutional and ICH guidelines. Any publication or 
presentation will not identify individual subjects by name.  
The medical records of study participants may be reviewed by the St. 
Jude IRB, FDA, and clinical research monitors.
13.0 STATISTICAL CONSIDERATIONS
13.1 Study Design
13.1.1 Primary objectives
Primary objective 1: To estimate the response rate to four cycles of 
avelumab in patients with recurrent or progressive osteosarcoma.
Primary objective 2: To estimate the 12-week progression-free survival 
of patients with recurrent or progressive osteosarcoma after treatment 
with avelumab.
Responsible investigators: Michael Bishop, Alberto Pappo
Responsible Biostatisticians: Jianrong Wu
Estimated date for completion of data collection: 12/31/2021
The study is designed by treating RECIST response (CR+PR) after 4-cycle 
treatment of avelumab and the 16-week progression-free survival (PFS) 
as dual binary endpoints using the method proposed by Kocherginsky et 
al (software available upon request).91 For each patient, two outcomes: 
RECIST response (CR+PR) and 16-week PFS will be recorded at the end of 
4-cycle treatment of avelumab, here 16-week PFS is defined as a binary 
endpoint for patients who had response (CR+PR) or stable disease (SD) 
after 4-cycle treatment of avelumab. Patients who fail to be evaluated for 
response or PFS at the end of the 4-cycle will be counted as failure (no 
response and disease progression). The following table summarizes the 
sample size and the decision rule for the interim and final analyses based 
on 10% type I error and 90% power and using the method proposed by 
Kocherginsky et al. The proposed design below (Table 13.1) is used to 
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guard against futility and to facilitate the sample size calculation based 
on the following null and alternative hypotheses: 
H0:  pr≤5% and pnp ≤15%
vs. the alternative
Ha:  pr>20% or pnp >35%
where pr is the true response rate and pnp is the true non-
progression rate.
A total of 32 patients are needed for the study with one interim analysis 
after the first 18 patients had response and PFS evaluation. Accrual will 
be temporarily suspended at the time the interim sample size is reached 
(when the first 18 evaluable patients are enrolled) until the interim rules 
are evaluated. The study will be closed if the thresholds below at stage 1 
are exceeded:
Table 13.1: Bivariate Binomial Two-stage Design
Stage Sample 
Size
Decision rule
1
18
Continue trial if:
# of response ≥ 2 or
# of patients without PD after 4-cycle ≥ 3
2
32
Conclude therapy is promising if:
# of response ≥ 4 or 
# of patients without PD after 4-cycle ≥ 9
This design yields 90% power to detect a true response rate of at least 
20% or a true 16-week progression-free rate (CR+PR+SD) of at least 35%. 
The type I error is estimated as 9.2% if the true response rate is no more 
than 5% and the true 16-week progression-free rate is no more than 
35%, with approximately 45% probability of early stopping in this case.
Patients who are removed from treatment due to toxicity will be also be 
treated as failures (non-response and disease progression). Thus, a total 
of 40 patients will insure that 32 patients are evaluable for the primary 
objectives.
It is anticipated that the study will enroll 10 patients per year, thus the 
study accrual duration will be approximately 4 years and the response 
and 16-week PFS data for all patients will be available in approximate 4.5 
years.
13.2 Secondary Objectives
13.2.1 
To describe the toxicities associated with the administration 
of avelumab in patients with recurrent or progressive osteosarcoma.
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Responsible investigators: Michael Bishop, Alberto Pappo
Responsible Biostatisticians: Jianrong Wu
Estimated date for completion of data collection: 3/31/2023
Target toxicities for avelumab treatment are defined as any grade 3-5 
dyspnea, pneumonitis, infusion-related reactions, or immune related 
adverse events at least possibly attributable to the agent observed 
anytime during the 26-cycle treatment period that a patient is on study 
(including the period between off treatment and off study). Due to a 
limited number of patients, following ad hoc stopping rule will be used to 
stop the trial accrual if 3 or more patients in the first 10 patients had 
target toxicities. Subsequently, the target toxicities will be monitored in a 
continuous fashion. That is at any time after 10th patient, the trial accrual 
will be stopped if the rate of target toxicity exceeds 20%.
All toxicity will be carefully monitored in this trial. Any unexpected grade 
3-4 toxicity will be reported to the IBR for review. Furthermore, in the 
event of a toxic death during the 4-cycle treatment, the accrual will be 
put on hold until a thorough review of the event take place and a 
deliberate decision can be made regarding resuming accrual.
13.3 Exploratory Objectives 
13.3.1 
To explore factors associated with response in patients 
treated with avelumab after recurrent or progressive osteosarcoma (e.g. 
tumor PD-L1 expression).
Responsible investigators: Michael Bishop, Alberto Pappo
Responsible Biostatisticians: Jianrong Wu
Estimated date for completion of data collection: 3/31/2023
Logistic regression analysis will be conducted to explore factors which 
may associate with response.
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13.3.2 
To measure parameters of immune activation including 
subsets of peripheral blood mononuclear cells (PBMCs) and serum 
markers of immune activation.
Responsible investigators: Michael Bishop, Benjamin Youngblood, Paul 
Thomas 
Responsible Biostatisticians: Jianrong Wu, Guolian Kang
Estimated date for completion of data collection: 3/31/2023
Descriptive statistics (mean+/- SE or median, min and max) will be 
provided for the parameters of immune activation including subsets of 
PBMCs and serum markers of immune activation for each time point 
separately and changes between two time points. Box-plot will be 
provided for each time point to visually look at the trend of the 
parameters of immune activation over time of therapy.   
13.3.3 
To evaluate the role of T-cells in immune checkpoint 
blockade via measures of cell proliferation, co-inhibitory receptor 
expression on CD8 T cells, T cell repertoire, and epigenetic 
programming.
Responsible investigators: Michael Bishop, Benjamin Youngblood, Paul 
Thomas
Responsible Biostatisticians: Jianrong Wu, Guolian Kang
Estimated date for completion of data collection: 3/31/2023
For the analysis of T cell repertoire, we will compute statistics (such as 
diversity, entropy, richness) and characterize known receptor specificities 
of the immune repertoire for each time-point at which these studies are 
performed. Comparisons among PD1+ and PD1- and other inhibitory 
receptor-expressing subsets will be assessed for changes in repertoire 
associated with treatment. To assess the proliferation and activation 
phenotypes of memory CD8 T cell subsets will measure the percentage of 
naïve, central-memory (Tcm), effector-memory (Tem), and stem-cell 
(Tscm) memory CD8 T cell populations in the PBMC of treated individuals. 
The percentage of CD8 T cell subsets will be measured prior to treatment 
and following the start of therapy. Additionally, we will purify the 
phenotypically defined memory CD8 T cell subsets and measure subset-
specific DNA methylation programs to determine if therapy results in 
epigenetic changes among the pool of naïve and memory T cell subsets. 
Overall, descriptive statistics (mean +/- SE or median, min and max) will 
be computed for the univariate data for each time point separately and 
changes between two time points and graphic presentations will be given 
for the longitudinal observations to demonstrate the changes across the 
courses of therapy (prior therapy and during therapy).
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13.3.4 To assess the quality of life of patients with recurrent or 
progressive osteosarcoma undergoing treatment with avelumab, and to 
explore relationships between clinical factors and patient-reported 
HRQOL outcomes.
Responsible investigators: Michael Bishop
Responsible Biostatisticians: Jianrong Wu, Zhaohua Lu
Estimated date for completion of data collection: 3/31/2023
Patients will complete the PROMIS Pediatric or Adult Profile (based on 
their age at time of administration) at four time points during the study: 
prior to the start of treatment, at the end of cycle 2 (8 weeks ± 1 week), 
at the end of cycle 4 (16 weeks ± 1 week), and at the end of avelumab 
treatment. Patients less than 18 years old will completed the PROMIS-37 
Profile v.1.1; patients age 18 years and older will complete the PROMIS-
43 Profile v.2.0. Mixed effect models will be used to study the change 
patterns of the T-scores in each quality of life domain over time, and the 
association between clinical factors and the T-scores in each quality of 
life domain. Missing data points will be addressed by multiple imputation 
or full information maximum likelihood methods.
13.4 Anticipated Completion Dates
Anticipated primary completion date: December 2020
Anticipated study completion date: December 2023
14.0 OBTAINING INFORMED CONSENT
14.1 Consent Prior to Research Interventions 
Initially, informed consent for patients enrolling at St. Jude will be sought 
for the institutional banking protocol (TBANK research study), and for 
other procedures as necessary for standard medical care. During the 
screening process for eligibility, informed consent for the St. Jude pre-
screening protocol (SCREEN) or for OSTPDL1 is required before any 
research tests are performed. Collaborating sites will follow local policies 
for screening and consent/assent.
14.2 Consent at Enrollment
The process of informed consent for OSTPDL1 will follow local 
institutional policy. The informed consent process is an ongoing one that 
begins at the time of diagnosis and ends after the completion of therapy. 
Informed consent should be obtained by the attending physician or 
his/her designee, in the presence of at least one non-physician witness. 
Initially, informed consent will be sought for the institutional banking 
protocol (research study), blood transfusion and other procedures as 
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necessary. After the diagnosis of relapsed or refractory solid tumor is 
established, we will invite the patient to participate in the OSTPDL1 
protocol.
Throughout the entire treatment period, participants and their parents 
receive constant education from health professionals at St. Jude and 
collaborating sites, and are encouraged to ask questions regarding 
alternatives and therapy. All families have ready access to chaplains, 
psychologists, social workers, and the St. Jude ombudsperson for 
support, in addition to that provided by the primary physician and other 
clinicians involved in their care. 
We will also obtain verbal assent from children 12 to 14 years old and 
written assent for all participants >14 years of age at St. Jude.  
Collaborating sites will follow institutional guidelines for assent.
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14.3 Consent at Age of Majority
Participants who reach the age of majority while on study will be re-
consented for continued participation on OSTPDL1 at the time of their 
next clinic visit after turning 18 year according to Cancer Center and local 
institutional policy.
14.4 Consent When English is Not the Primary Language
Obtaining consent for non-English speaking participants will follow local 
institutional policy. At St. Jude, when English is not the participant, 
parent, or legally authorized representative’s primary language, the 
investigator or the Social Work department will determine the need for an 
interpreter. This information will be documented in the participant’s 
medical record. Either a certified interpreter or the telephone interpreter’s 
service will be used to translate the consent information. If the short form 
consent is available in the primary language of the participant, parent or 
legally authorized representative, this may be utilized to sign consent 
and joined with a full English consent signed by the research team. The 
process for obtaining an interpreter and for the appropriate use of an 
interpreter is outlined on the SJCRH Interpreter Services website, as well 
as OHSP and CPDMO websites.
14.5 Collection of Collaborating Institution Consent Forms
Signed collaborating institution’s consent forms will be faxed to the St. 
Jude CPDMO Eligibility Office at 901-595-6265. 
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32. Keir ME, Butte MJ, Freeman GJ, et al: PD-1 and its ligands in 
tolerance and immunity. Annu Rev Immunol 26:677-704, 2008
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clinical application. Int Immunol 19:813-24, 2007
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35. van Rooij N, van Buuren MM, Philips D, et al: Tumor exome analysis 
reveals neoantigen-specific T-cell reactivity in an ipilimumab-responsive 
melanoma. J Clin Oncol 31:e439-42, 2013
36. Restifo NP, Dudley ME, Rosenberg SA: Adoptive immunotherapy for 
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39. Wherry EJ, Ha SJ, Kaech SM, et al: Molecular signature of CD8+ T 
cell exhaustion during chronic viral infection. Immunity 27:670-84, 2007
40. Sharma P, Allison JP: The future of immune checkpoint therapy. 
Science 348:56-61, 2015
41. Blackburn SD, Shin H, Haining WN, et al: Coregulation of CD8+ T 
cell exhaustion by multiple inhibitory receptors during chronic viral infection. 
Nat Immunol 10:29-37, 2009
42. Peretz Y, He Z, Shi Y, et al: CD160 and PD-1 co-expression on HIV-
specific CD8 T cells defines a subset with advanced dysfunction. PLoS Pathog 
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43. Gros A, Robbins PF, Yao X, et al: PD-1 identifies the patient-
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44. Duraiswamy J, Ibegbu CC, Masopust D, et al: Phenotype, function, 
and gene expression profiles of programmed death-1(hi) CD8 T cells in healthy 
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46. La Gruta NL, Thomas PG: Interrogating the relationship between 
naive and immune antiviral T cell repertoires. Curr Opin Virol 3:447-51, 2013
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epitopes drive therapeutic immune responses to cancer. Nature 520:692-6, 
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51. Youngblood B, Noto A, Porichis F, et al: Cutting edge: Prolonged 
exposure to HIV reinforces a poised epigenetic program for PD-1 expression in 
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52. Youngblood B, Oestreich KJ, Ha SJ, et al: Chronic virus infection 
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55. Crom DB, Chathaway DK, Tolley EA, et al: Health status and health-
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56. Eiser C, Vance YH, Horne B, et al: The value of the PedsQLTM in 
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57. Bradlyn AS, Harris CV, Spieth LE: Quality of life assessment in 
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58. Varni JW, Burwinkle TM, Katz ER: The PedsQL in pediatric cancer 
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59. Kuhlthau KA, Pulsifer MB, Yeap BY, et al: Prospective study of 
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68. Frances JM, Morris CD, Arkader A, et al: What is quality of life in 
children with bone sarcoma? Clin Orthop Relat Res 459:34-9, 2007
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70. Eiser C, Darlington AS, Stride CB, et al: Quality of life implications 
as a consequence of surgery: limb salvage, primary and secondary amputation. 
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tissue cancer treatment. Clin Orthop Relat Res:74-86, 2004
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73. Nagarajan R, Neglia JP, Clohisy DR, et al: Education, employment, 
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74. Refaat Y, Gunnoe J, Hornicek FJ, et al: Comparison of quality of life 
after amputation or limb salvage. Clin Orthop Relat Res:298-305, 2002
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76. van Riel CA, Meijer-van den Bergh EE, Kemps HL, et al: Self-
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77. Bekkering WP, Vliet Vlieland TP, Koopman HM, et al: Quality of life 
in young patients after bone tumor surgery around the knee joint and 
comparison with healthy controls. Pediatr Blood Cancer 54:738-45, 2010
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78. Stokke J, Sung L, Gupta A, et al: Systematic review and meta-
analysis of objective and subjective quality of life among pediatric, adolescent, 
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79. Whelan JS, Bielack SS, Marina N, et al: EURAMOS-1, an international 
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80. Eiser C, Jenney M: Measuring quality of life. Arch Dis Child 92:348-
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81. Varni JW, Burwinkle TM, Lane MM: Health-related quality of life 
measurement in pediatric clinical practice: an appraisal and precept for future 
research and application. Health Qual Life Outcomes 3:34, 2005
82. Varni JW, Magnus B, Stucky BD, et al: Psychometric properties of the 
PROMIS (R) pediatric scales: precision, stability, and comparison of different 
scoring and administration options. Qual Life Res 23:1233-43, 2014
83. Hinds PS, Nuss SL, Ruccione KS, et al: PROMIS pediatric measures in 
pediatric oncology: valid and clinically feasible indicators of patient-reported 
outcomes. Pediatr Blood Cancer 60:402-8, 2013
84. Dobrozsi S, Yan K, Hoffmann R, et al: Patient-reported health 
status during pediatric cancer treatment. Pediatr Blood Cancer, 2016
85. Janssen SJ, Paulino Pereira NR, Raskin KA, et al: A comparison of 
questionnaires for assessing physical function in patients with lower extremity 
bone metastases. J Surg Oncol 114:691-696, 2016
86. Broderick JE, Schneider S, Junghaenel DU, et al: Validity and 
reliability of patient-reported outcomes measurement information system 
instruments in osteoarthritis. Arthritis Care Res (Hoboken) 65:1625-33, 2013
87. DeWalt DA, Gross HE, Gipson DS, et al: PROMIS((R)) pediatric self-
report scales distinguish subgroups of children within and across six common 
pediatric chronic health conditions. Qual Life Res 24:2195-208, 2015
88. Thissen D, Liu Y, Magnus B, et al: Estimating minimally important 
difference (MID) in PROMIS pediatric measures using the scale-judgment 
method. Qual Life Res 25:13-23, 2016
89. Schwartz GJ, Gauthier B: A simple estimate of glomerular filtration 
rate in adolescent boys. J Pediatr 106:522-6, 1985
90. Eisenhauer EA, Therasse P, Bogaerts J, et al: New response 
evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J 
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91. Kocherginsky M, Cohen EE, Karrison T: Design of Phase II cancer 
trials for evaluation of cytostatic/cytotoxic agents. J Biopharm Stat 19:524-9, 
2009
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APPENDIX I: KARNOFSKY AND LANSKY PERFORMANCE STATUS 
SCALES/SCORES
PERFORMANCE STATUS CRITERIA
Karnofsky and Lansky performance scores are intended to be multiples of 10
ECOG (Zubrod)
Karnofsky
Lansky
Scor
e
Description
Scor
e
Description
Scor
e
Description
100
Normal, no 
complaints, no 
evidence of 
disease
100
Fully active, normal
0
Fully active, able 
to carry on all 
pre-disease 
performance 
without 
restriction
90
Able to carry on 
normal activity, 
minor signs or 
symptoms of 
disease
90
Minor restrictions 
in physically 
strenuous activity
80
Normal activity 
with effort; some 
signs or symptoms 
of disease
80
Active, but tires 
more quickly
1
Restricted in 
physically 
strenuous 
activity by 
ambulatory and 
able to carry out 
work of a light 
or sedentary 
nature, e.g., 
light housework, 
office work
70
Cares for self; 
unable to carry on 
normal activity or 
do active work
70
Both greater 
restriction of and 
less time spent in 
play activity
60
Requires 
occasional 
assistance, but is 
able to care for 
most of his/her 
needs
60
Up and around, but 
minimal active 
play; keeps busy 
with quieter 
activities
2
Ambulatory and 
capable of self-
care but unable 
to carry out any 
work activities; 
up and about 
more than 50% 
of waking hours
50
Requires 
considerable 
assistance and 
frequent medical 
care
50
Gets dressed, but 
lies around much 
of the day; no 
active play, able to 
participate in all 
quiet play and 
activities
40
Disabled, requires 
special care and 
assistance
40
Mostly in bed; 
participates in 
quiet activities
3
Capable of only 
limited self-care, 
confined to bed 
or chair more 
than 50% of 
waking hours
30
Severely disabled, 
hospitalization 
indicated; death 
30
In bed; needs 
assistance even for 
quiet play
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not imminent
20
Very sick, 
hospitalization 
indicated.  Death 
not imminent
20
Often sleeping; 
play entirely 
limited to very 
passive activities
4
Completely 
disabled; cannot 
carry on any 
self-care; totally 
confined to bed 
or chair
10
Moribund, fatal 
processes 
progressing rapidly
10
No play; does not 
get out of bed
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APPENDIX II: NEW YORK HEART ASSOCIATION FUNCTIONAL 
CLASSIFICATION
NYHA 
Class
Symptoms
I
Cardiac disease, but no symptoms and no limitation in ordinary 
physical activity, e.g. no shortness of breath when walking, 
climbing stairs etc.
II
Mild symptoms (mild shortness of breath and/or angina) and 
slight limitation during ordinary activity.
III
Marked limitation in activity due to symptoms, even during less-
than-ordinary activity, e.g. walking short distances (20–100 m).
Comfortable only at rest.
IV
Severe limitations. Experiences symptoms even while at rest. 
Mostly bedbound patients.
The Criteria Committee of the New York Heart Association. (1994). Nomenclature and 
Criteria for Diagnosis of Diseases of the Heart and Great Vessels. (9th ed.). Boston: Little, 
Brown & Co. pp. 253–256.
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APPENDIX III: INSTRUCTIONS FOR COLLECTION AND SHIPPING
OF RESEARCH BLOOD SAMPLES TO NCI
Collection:
PBMCS: green top (Na heparin, 10 mL) tubes, BD Vacutainer Ref #366480
Serum:  red serum separator (8 mL) tubes, Greiner Bio-One Vacutainer Ref 
#455071
*Blood is collected at the following time points: Prior to Cycles 1 and 3.
Shipping:
PBMCs are to be sent on the day of draw; serum is to be frozen and batch-
shipped at intervals. **Please note, PBMCs cannot be shipped within 72 hours 
after a bone scan injection due to the Technetium-99 radioisotope.  If a bone 
scan is scheduled, PBMCs must be drawn prior to bone scan injection.
Materials: 
· Pack blood vials in the EXAKT-PAK shipping kits described below or 
equivalent shipping materials:
For ambient PBMCs:
EXAKT-PAK for Vials Category B D-Pak MD8702V06 (Accommodates 6 vials) 
Includes Inner pack (Ambient) and Insulated Cooler
With 2 cool packs per cooler, part #CP1003, room temperature or slightly 
cooled (not frozen) to keep samples between 18 and 30oC.
For frozen serum:
EXAKT-PAK Frozen Category B D-Pak MD8703V06
Dry ice required (not included)
EXAKT-PAK kits available at:
https://www.exaktpak.com/store/ 
EXAKT Technologies, Inc.
Home office: 7002 N. Broadway Extension
Oklahoma City, OK 73116-9006
405-848-5800
Shipping Address:
· For Research Blood Samples, please FED-EX Overnight to the shipping 
address below:  
Leidos
Biomedical Research
Attn: Ludmila Krymskaya/Theresa Burks
1050 Boyles Street
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Bldg. 469/Room 121
Frederick, MD 21702
Phone 301-846-5125, or 301-846-1707
· Please notify the Frederick laboratory when specimens are being shipped. 
Please email Frederick prior to shipping to notify the lab.
· Emails should be sent to the following individuals:
Ludmila Krymskaya, ludmila.krymskaya@nih.gov
Theresa Burks, burkst@mail.nih.gov
Caroline Jochems, jochemscm@mail.nih.gov
· Additionally, NO specimens should be shipped on Fridays or the day before a 
Federal holiday.
Labeling of Blood Samples:
· Please label research tubes with a coding mechanism. The following 
information should be contained on the label:
o Site 
o Patient enrollment number and initials
o Dose level or arm
o Protocol number
o Date drawn
o Time point – such as ‘baseline’ or ‘prior to cycle 3’
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