Clinical trial NCT01814813
Conditions: Recurrent Glioblastoma Recurrent Adult Brain Tumor Gliosarcoma
Sponsor: Alliance for Clinical Trials in Oncology
Start date: 2013-06-19; Completion date: 2023-05-22
Enrollment: 90 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This randomized phase II trial studies how well giving vaccine therapy with or without bevacizumab works in treating patients with recurrent glioblastoma multiforme that can be removed by surgery. Vaccines consisting of heat shock protein-peptide complexes made from a person's own tumor tissue may help the body build an effective immune response to kill tumor cells that may remain after surgery. Monoclonal antibodies, such as bevacizumab, can block tumor growth in different ways. Some block the ability of tumor cells to grow and spread. Others find tumor cells and help kill them. It is not yet known whether giving vaccine therapy is more effective with or without bevacizumab in treating glioblastoma multiforme.

Detailed description:
The purpose of this study is to compare the effects of a vaccine with bevacizumab versus bevacizumab alone on a patient's brain tumor. The vaccine is called heat shock protein peptide complex 96 (HSPPC-96). HSPPC-96 is experimental. Specifically, HSPPC-96 is a protein that may work to help the body have a response against remaining brain tumor cells. Bevacizumab has been approved by the Food and Drug administration for treating brain tumors that grow back. In this study, patients will either get HSPPC-96 vaccine at the same time as bevacizumab, HSPPC vaccine first and then bevacizumab if the tumor comes back, or bevacizumab alone. The use of HSPPC-96 and bevacizumab is investigational.

The primary objective of the study is to determine whether there is an overall survival advantage of HSPPC-96 administered with bevacizumab, given concomitantly or at the point of progression, in comparison with bevacizumab alone in patients with surgically resectable recurrent glioblastoma multiforme.

The secondary objectives are:

1. to evaluate the safety and tolerability of HSPPC-96 with bevacizumab
2. to evaluate the progression free survival of HSPPC-96 with bevacizumab, given concomitantly or at the point of progression.

Patients must undergo surgery within 28 days from pre-registration. There must be confirmation of adequacy of tissue for vaccine manufacture, tumor tissue submitted to Agenus, confirmation of ≥ 90% resection by central radiology review and vaccine manufacture of at least six vials. Patients will be randomized to one of three treatment arms. Please see the "Arms" section for more details.

Patients will be monitored approximately 5 years post-surgery.

Arms: arm 1: HSPPC-96 0.4mL intradermal on days 1 and 8 of cycles 1 and 2, then on day 1 of each cycle, up to a maximum of 12 doses (10 cycles), plus bevacizumab 10 mg/kg intravenous (IV) on day 1 of each cycle, until progression. HSPPC-96 should be administered at least 60 minutes prior to starting bevacizumab infusion. (1 cycle=14 days)

Note: If HSPPC-96 treatment has ended but there is no evidence of disease progression, the patient should continue to receive bevacizumab at the specified dose until progression. arm 2: HSPPC-96 0.4mL intradermal on days 1 and 8 of cycles 1 and 2, then on day 1 of each cycle, up to a maximum of 12 doses (10 cycles). At progression: bevacizumab 10mg/kg intravenous (IV) on day 1 of each cycle, until further progression. (1 cycle = 14 days)

NOTE: It is possible that HSPPC-96 vaccination may end prior to evidence of progression. In this instance it is important to wait until there is confirmed evidence of progression before initiating treatment with bevacizumab.

Upon confirmation of progression the patient should initiate bevacizumab within 7-42 days from the last dose of vaccine. arm 3: Bevacizumab 10mg/kg intravenous (IV) on day 1 of each cycle, until progression. (1 cycle = 14 days)
Interventions: intervention 1: HSPPC-96 intervention 2: bevacizumab
Intervention descriptions: intervention 1: intradermal infusion intervention 2: intravenous

Protocol documents:
Prot_SAP_000.pdf: 
A071101 
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Version Date: 11/09/2018 
Update #13 
 
ALLIANCE FOR CLINICAL TRIALS IN ONCOLOGY 
 
ALLIANCE A071101 
 
A PHASE II RANDOMIZED TRIAL COMPARING THE EFFICACY OF HEAT SHOCK PROTEIN-PEPTIDE 
COMPLEX-96 (HSPPC-96) (NSC #725085, ALLIANCE IND #15380) VACCINE GIVEN WITH 
BEVACIZUMAB VERSUS BEVACIZUMAB ALONE IN THE TREATMENT OF SURGICALLY RESECTABLE 
RECURRENT GLIOBLASTOMA MULTIFORME (GBM) 
 
Investigational Agent: Autologous Heat Shock Protein Peptide Complex-96 (HSPPC-96)  
(NSC #725085, Alliance IND# 15380) supplied and distributed by Agenus Inc. 
Commercial Agent: Bevacizumab 
ClinicalTrials.gov Identifier: NCT01814813 
 
 
Study Chairs 
 Ian Parney, MD, PhD 
Mayo Clinic 
Orin Bloch, MD 
Northwestern University 
 
 
 
 
 
 
Imaging Co-chair 
Neuro-oncology Co-chair 
Neuro-oncology Committee Chair 
 
 
 
 
 
 
 
 
 
 
 
Correlative Study  
Co-chair 
Community Oncology  
Co-chair 
Correlative Committee Chair 
 
 
 
 
 
  
 
 
 
 
  
 
 
 
 
 
Primary Statistician 
Secondary Statistician 
RTOG Surgical Co-Chair 
RTOG Neuro-onc Co-Chair 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
Protocol Coordinator 
 
 
 
 
 
Participating Groups:  
ALLIANCE / Alliance for Clinical Trials in Oncology (lead) 
NRG / NRG Oncology 
 


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Alliance Central Protocol Operations Office 
 
 
 
Alliance Statistical Data Center 
 
 
 
Expedited Adverse Event Reporting 
https://eapps-ctep.nci.nih.gov/ctepaers/ 
Medidata Rave iMedidata portal 
https://login.imedidata.com 
OPEN (Oncology Patient Enrollment Network) 
https://open.ctsu.org 
Protocol Resources:  
 
A071101 Data Manager 
 
  
 
 
Alliance Biorepository at Mayo Clinic 
Paraffin-embedded tissue: 
 
 
 
  
  
 
Non-paraffin biospecimens: 
 
 
 
 
 
Alliance A071101 Pharmacy Contact 
 
 
 
 
A071101 Nursing Contact 
 
 
 
IROC 
 
 
 
 
 
 
 
Protocol-related questions may be directed as follows: 
Questions 
Contact (via email) 
Questions regarding patient eligibility, treatment, and dose 
modification: 
Study Chair, Nursing Contact, Protocol Coordinator, or Data 
Manager 
Questions related to data submission, RAVE or patient 
follow-up: 
Data Manager  
Questions regarding the protocol document and model 
informed consent: 
Protocol Coordinator 
Questions related to IRB  review 
Regulatory Affairs Manager:
 
Questions regarding CTEP-AERS reporting: 
Alliance Pharmacovigilance Inbox: 
 
For a copy of the Investigator Brochure (IB): 
 
The pharmacogenomic component of this study is conducted as part of the NIH Pharmacogenomics Research Network, which is funded through a 
separate U01 mechanism (see http://www.nigms.nih.gov/pharmacogenomics/research_net.html) for details 
 
 
 
 


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CANCER TRIALS SUPPORT UNIT (CTSU) ADDRESS AND CONTACT INFORMATION 
CONTACT INFORMATION 
 
For regulatory 
requirements: 
For patient enrollments: 
For  study data submission: 
Regulatory documentation 
must be submitted to the 
CTSU via the Regulatory 
Submission Portal. 
Regulatory Submission Portal: 
(Sign in at www.ctsu.org,            
and select the Regulatory 
Submission sub-tab under the 
Regulatory tab.)  
 
Institutions with patients 
waiting that are unable to use 
the Portal should alert the 
CTSU Regulatory Office 
immediately at 
 to receive further 
instruction and support. 
 
Contact the CTSU Regulatory 
Help Desk at 
 
for regulatory assistance. 
Please refer to the patient 
enrollment section of the 
protocol for instructions on 
using the Oncology Patient 
Enrollment Network 
(OPEN) which can be 
accessed at 
https://www.ctsu.org/OPEN
_SYSTEM/ or 
https://OPEN.ctsu.org. 
 
Contact the CTSU Help 
Desk with any OPEN-
related questions at 
. 
 
 
Data collection for this study will 
be done exclusively through 
Medidata Rave.  Please see the data 
submission section of the protocol 
for further instructions. 
 
Do not submit study data or forms 
to CTSU Data Operations. Do not 
copy the CTSU on data 
submissions.   
The most current version of the study protocol and all supporting documents must be downloaded 
from the protocol-specific Web page of the CTSU Member Web site located at https://www.ctsu.org. 
Access to the CTSU members’ website is managed through the Cancer Therapy and Evaluation 
Program - Identity and Access Management (CTEP-IAM) registration system and requires user log 
on with CTEP-IAM username and password. Permission to view and download this protocol and its 
supporting documents is restricted and is based on person and site roster assignment housed in the 
CTSU RSS.  
For clinical questions (i.e.,  patient eligibility or treatment-related) see Protocol Contacts Page 2 
For non-clinical questions (i.e., questions unrelated to patient eligibility, treatment, or clinical 
data submission contact the CTSU Help Desk by phone or e-mail:  
CTSU General Information Line – 
, or
. All calls and 
correspondence will be triaged to the appropriate CTSU representative.  
The CTSU Web site is located at https://www.ctsu.org. 
 


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A071101 
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TABLE OF CONTENTS 
 
SECTION 
 
 
 
 
 
 
 
 
 
 
PAGE 
1.0 
INTRODUCTION ............................................................................................................................. 7 
1.1 
Rationale for Selected Approach and Trial Design ......................................................................... 7 
1.2  Study Rationale ............................................................................................................................. 10 
1.3 
Relevant Data ................................................................................................................................ 10 
1.4 
Inclusion of Women and Minorities ............................................................................................. 12 
2.0 
OBJECTIVES ................................................................................................................................. 12 
2.1 
Primary Objective ......................................................................................................................... 12 
2.2 
Secondary Objectives .................................................................................................................... 13 
2.3 
Correlative Science Objectives ..................................................................................................... 13 
3.0 
ON-STUDY GUIDELINES ............................................................................................................ 13 
4.0 
ELIGIBILITY CRITERIA ............................................................................................................ 13 
4.1 
Pre-registration (Pre-Surgery) Eligibility Criteria ........................................................................ 14 
4.2 
Registration (Post-Surgery) Eligibility Criteria ............................................................................ 15 
5.0 
PRE-REGISTRATION, REGISTRATION/RANDOMIZATION, AND 
STRATIFICATION .................................................................................................................................. 16 
5.1 
CTEP Investigator Registration Procedures .................................................................................. 16 
5.2 
Pre-registration .............................................................................................................................. 18 
5.3 
Registration/Randomization .......................................................................................................... 19 
5.4 
Stratification .................................................................................................................................. 20 
5.5 
Registration to Sub-studies ........................................................................................................... 20 
5.6 
Surgical Quality Assurance ........................................................................................................... 21 
6.0 
DATA AND SPECIMEN SUBMISSION AND CENTRAL RADIOLOGY REVIEW ............ 21 
6.1 
Data Submission ........................................................................................................................... 21 
6.2 
Tumor Tissue Submission for Vaccine Manufacture .................................................................... 21 
6.3 
Blood Submission for Immune Monitoring .................................................................................. 22 
6.4 
Tissue Submission for Biomarker Analysis .................................................................................. 24 
6.5 
Biospecimen Submission Summary .............................................................................................. 26 
6.6 
Specimen Registration and Tracking ............................................................................................ 27 
6.7 
MRI/CT Imaging........................................................................................................................... 28 
7.0 
REQUIRED DATA ......................................................................................................................... 31 
8.0 
TREATMENT PLAN ..................................................................................................................... 33 
8.1 
Arm 1, HSPPC-96 + concomitant bevacizumab ........................................................................... 33 
8.2 
Arm 2, HSPPC-96 with bevacizumab at progression ................................................................... 33 
8.3 
Arm 3, Bevacizumab..................................................................................................................... 34 
9.0 
DOSE MODIFICATIONS AND MANAGEMENT OF TOXICITY ......................................... 34 
9.1 
Hypersensitivity and/or Infusion Reactions .................................................................................. 34 
9.2 
Injection Site Reactions ................................................................................................................ 34 
9.3 
Hypertension ................................................................................................................................. 35 
9.4 
Hemorrhage ................................................................................................................................... 35 
9.5 
Venous Thromboembolic Events .................................................................................................. 35 
9.6 
Arterial Thromboembolic Events .................................................................................................. 36 
9.7 
Renal and Urinary Disorders:  Proteinuria .................................................................................... 36 
9.8 
Fistula, Perforation involving any Organ, Bowel Obstruction, and Wound Dehiscence .............. 36 
9.9 
Other Non-Hematologic Toxicity at Least Possibly Related to HSPCC-96 ................................. 36 
9.10 Dose Modifications for Obese Patients ......................................................................................... 36 


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10.0 CORRELATIVE SUB-STUDY ..................................................................................................... 37 
10.1 Immune Response Study ............................................................................................................... 37 
10.2 Tissue Marker Study ..................................................................................................................... 38 
11.0 DRUG FORMULATION, AVAILABILITY, AND PREPARATION ...................................... 39 
11.1 Qualified personnel ....................................................................................................................... 39 
11.2 Unused agents ............................................................................................................................... 39 
11.3 Bevacizumab dose rounding ......................................................................................................... 39 
11.4 Weight changes ............................................................................................................................. 39 
11.5 Bevacizumab (rhuMAb VEGF, Avastin®) (NSC #704865) ......................................................... 39 
11.6  Heat Shock Protein-Peptide Complex-96 Vaccine (HSPPC-96)(NSC #725085) (Alliance  IND # 
15380) ..................................................................................................................................................... 42 
12.0 ANCILLARY THERAPY .............................................................................................................. 44 
12.1 Supportive Care............................................................................................................................. 44 
12.2 Hormones or Other Agents ........................................................................................................... 44 
12.3 Palliative RT ................................................................................................................................. 44 
12.4 Growth Factors .............................................................................................................................. 44 
12.5 Surgery .......................................................................................................................................... 45 
13.0 CRITERIA FOR PROGRESSION ............................................................................................... 45 
14.0 REMOVAL OF PATIENTS FROM PROTOCOL THERAPY ................................................. 45 
14.1 Duration of Treatment ................................................................................................................... 45 
14.2 Extraordinary Medical Circumstances: ......................................................................................... 46 
15.0 STATISTICAL CONSIDERATIONS .......................................................................................... 46 
15.1  Study overview ............................................................................................................................. 46 
15.2 Objectives ..................................................................................................................................... 46 
15.3 Endpoints ...................................................................................................................................... 46 
15.4  Sample Size Derivation ................................................................................................................. 47 
15.5  Accrual and study duration ........................................................................................................... 47 
15.6  Interim analysis ............................................................................................................................. 47 
15.7 Analysis Plan for Primary and Secondary Endpoints ................................................................... 47 
15.8  Descriptive Factors ....................................................................................................................... 49 
16.0 ADVERSE EVENT REPORTING................................................................................................ 50 
16.1  Expedited Adverse Event Reporting ............................................................................................. 50 
16.2  Expedited Reporting Requirements for Adverse Events that Occur on Studies under an IND 
within 30 Days of the Last Treatment 1 .................................................................................................. 51 
16.3 Comprehensive Adverse Event and Potential Risks List (CAEPR) for Bevacizumab (rhuMAb 
VEGF, NSC 704865) .............................................................................................................................. 53 
16.4 Routine adverse event reporting.................................................................................................... 58 
17.0 REFERENCES ................................................................................................................................ 59 
APPENDIX I: UPC (URINE PROTEIN TO CREATININE) RATIO ................................................ 62 
APPENDIX II: KARNOFSKY PERFORMANCE STATUS ............................................................... 63 
APPENDIX III: VACCINE TISSUE PROCUREMENT AND SHIPPING INSTRUCTIONS ........ 64 
APPENDIX IV: WHOLE BLOOD PBMC PROCESSING AT SITES* ............................................. 67 
APPENDIX V: A071101 VACCINE SCHEMA ..................................................................................... 70 
APPENDIX VI: VACCINE RETURN INSTRUCTIONS .................................................................... 71 


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1.0 
INTRODUCTION 
1.1 
Rationale for Selected Approach and Trial Design 
Primary malignant brain tumors are uniformly fatal, and the 5-year survival rate for the highest 
grade of malignant glial neoplasm, GBM, is now less than 4% [1]. Improvements in 
conventional treatment modalities have provided some progress, however median survival 
remains at just over one year from initial diagnosis for patients treated at tertiary care centers 
[2]. Currently approved therapy for a newly diagnosed GBM patient in the United States 
includes maximal surgical resection followed by radiation and temozolomide [1]. Upon 
recurrence there are few approved options and these include surgical implantation of 
chemotherapy bearing wafers [3] (polifeprosan 20 with carmustine implant, Gliadel® Wafer) 
and systemic administration of the anti-angiogenic agent bevacizumab, which has shown a 
partial response rate of 20% in one trial, and 26% in another [4, 5].  Each of these therapies has 
shown modest improvement in survival of recurrent GBM patients, with notable treatment 
related toxicities including wound breakdown after surgical resection [6].  
There is an unmet medical need for highly specific and non-toxic adjuvant therapy to treat 
recurrent GBM patients undergoing surgical resection. Immunotherapy is an appealing method 
to specifically target tumor cells in glioma patients, while minimizing adverse treatment effects 
[7]. There is evidence of immune mediated processes involved in GBM: antigens in the CNS 
lead to production of cytotoxic T cells and antibody response. Also, antigen specific T cells are 
seen in CNS tumors, and microglia and macrophages act as antigen presenting cells in the CNS. 
CNS tumors themselves cause immunosuppression, as evidenced by low lymphocyte counts, 
low antigen and mitogen responses, and T cells with impaired function. Any immunotherapy 
undertaken must overcome this. One way to overcome this is to resect tumor, as this decreases 
steroid requirement and the decrease in tumor bulk helps to downregulate the 
immunosuppression caused by the tumor [7].  
Several methods have been used successfully to evoke anti-tumor immunity in GBM patients, 
with evidence of peripheral and site-specific immune responses. Prins et al investigated the 
utility of immunotherapy for intracranial tumors in a mouse model. The anti-TRP vaccine 
successfully induced CD8+ T cell response, and this was demonstrated with bioluminescent 
imaging to occur within the CNS. The vaccine was also shown to decrease tumor size and 
increase survival. Wheeler et al treated newly diagnosed patients (after surgery and radiation) 
with either chemotherapy, autologous antigen pulsed dendritic cell vaccine, or vaccine and 
chemotherapy. The groups were not matched in that the chemotherapy only group had less full 
resections. The median overall survival was 15.9 months in chemotherapy, 17.9 months in 
vaccine, and 26 months in both. Follow-up work by this same group also found increased 
survival time in immune responders versus nonimmune responders, on the order of 1.8 versus 
1.2 years. A single arm trial by Liau et al looked at 12 patients with newly diagnosed or recurrent 
GBM, treated with dendritic cell tumor peptide vaccine, and found a median overall survival of 
2 years. A phase II trial with matched cohort by Sampson et al looked at a peptide vaccine to 
EGFRvIII. Patients had newly diagnosed disease and minimal residual disease (>95% resected). 
With the 18 vaccine patients, the median progression free survival was 14.2 months, and the 
matched cohort was 6.3 months. The median overall survival was 26 months in the vaccine 
cohort, and 15 months in the non-vaccine group, for a hazard ratio of 5.1.  [8-12].  Overall, it 
can be said that to date there has been data showing potential benefit for the use of 
immunotherapy in GBM. 
Heat Shock Protein-Peptide Complex-96 (HSPPC-96) consists of the heat shock protein 
glycoprotein-96 (HSP gp-96) and a wide array of chaperoned proteins, including autologous 
antigenic peptides. Heat shock proteins (HSP) are molecules that respond to cellular stress and 


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counteract abnormal protein folding. They are known to modulate immune responses, especially 
the HSP gp-96. In a stressful environment, such as a tumor, HSPs are upregulated and highly 
expressed on tumor cells. This protects the tumor and leads to resistance to therapy. HSP 
expression is associated with cellular proliferation, apoptosis evasion, tissue invasion, 
metastasis, and angiogenesis.  
HSPPC-96 immunization works mechanistically by interacting with antigen presenting cells 
(APCs) via specific receptors, including CD91 [13,14]. The highly specific nature of the 
interaction between HSPPC-96 and APCs is a significant advantage over other cancer vaccine 
approaches; and has been shown to facilitate robust CD4+ and CD8+ T-cell immune responses. 
There are multiple mechanisms by which the vaccine works. The first is the following: The 
autologous antigens in the vaccine bind to the chaperone proteins (HSP) with noncovalent 
bonds. Macrophages transfer the antigens from HSP to antigen presenting cells, via receptor- 
mediated endocytosis (with receptors such as CD91). The antigens are then expressed via MHC 
class I on antigen presenting cells (APCs), interacting with naïve T cells, which are activated 
into specific CD8 T cells. 
Another mechanism of the vaccine is an innate immunity pathway. In this mechanism, HSP-Ag 
is internalized into the APC, which activates the APC. This leads to the following: 
• 
Activated APCs express co-stimulatory molecules (CD80, CD86, and CD40) 
and express MHC class II, which interacts with T cells and produces tumor 
specific CD4 cells. 
• 
Activated APCs cause cytokine release (IL-1b, IL-12, IL-6, TNF-alph, 
GMCSF, chemokines, and nitric oxide). IL-12 stimulates NK cells, which 
enhance the antitumor activity of T cells. 
• 
The interaction between HSP-antigen and APCs causes dendritic cell 
maturation, which stimulates NK cells. 
A final mechanism of the vaccine is that exposure to Gp96 leads to translocation of NFkB into 
the APC nucleus, which then regulates cancer cell growth, progression, and apoptosis. 
 
The advantage of the HSPPC-96 vaccine over others is the following: 
• 
Multifactorial mechanism: as described above, the vaccine works by multiple 
pathways to exert its effect, rather than 1 pathway. 
• 
The vaccine is individualized, and does not require identification of 
immunogenic antigens required by other vaccines. 
• 
Multiple antigens are used, which decreases the chance of immune evasion that 
occurs with a single antigen vaccine. 
 
The downside of the HSPCC-96 vaccine is that: 
• 
A large amount of tissue is required 
• 
High purity criteria 
• 
There is difficulty designing immunomonitoring due to the fact that there is not 
a typical antigenic characteristic of GBM. 


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• 
Autoimmune reactions are possible due to the non-specificity of the vaccine 
(although these have not been noted in trials to date) [15,16].  
 
Clinical grade HSPPC-96 can be easily purified from solid tumor and has been safely tested in 
hundreds of patients with melanoma and renal cell cancer. The toxicity encountered in renal cell 
carcinoma in prior trials is the following: injection site erythema (50%), injection site induration 
(48%), back pain (12%), headache (12%), fatigue (10%), nasopharyngitis (9%), incision site 
complication (9%), arthralgia (9%), hypertension (9%), urinary tract infection (8%), nausea 
(7%), diarrhea (6%), asthenia (6%), influenza (6%), constipation (5%), pyrexia (5%), and 
dizziness (5%). In melanoma, toxicity seen was pyrexia (8%), fatigue (6%), and nausea (5%). 
Between the trials there were 3 serious adverse events, 2 were thyroid dysfunction, and 1 was 
cellulitis [17-20].  
A single center single arm Phase 1 clinical trial and a multicenter single arm phase 2 clinical 
trial for recurrent glioblastoma patients treated with HSPPC-96 have been completed.  
Collectively these studies have shown that: 1) HSPPC-96 vaccine made from recurrent GBM 
tumor is safe and well tolerated, 2) HSPPC-96 vaccine extends survival in recurrent GBM 
patients over historical controls, and 3) HSPPC-96 is immunogenic in recurrent GBM patients, 
inducing both an innate and adaptive immune response. Specifically, in the phase I trial, testing 
was performed pre and post vaccine on all patients. Eleven of 12 patients were shown to have a 
significant immune response, which was measured by testing peripheral blood lymphocytes 
(PBLs). PPBLs were tested for phenotype, reactivity to vaccine, IFN gamma production, and 
proliferation. Vaccine was well tolerated with no serious adverse events and the only toxicity 
noted was injection site erythema or induration. The median survival in responders was 47 
weeks, compared to 16 weeks in the non-responder. In the phase II trial, 33 patients were treated. 
Again, there were no serious adverse events attributed to vaccine. Toxicities reported were 
injection site reaction in 12 patients, and fatigue in 9 patients. Eleven patients were still alive at 
time of analysis, with median survival of 333 days for the evaluable population. Also, all patients 
studied showed immune response, based on CD8 IFN gamma production [22, 33].  
 Based on these positive preliminary findings, a large randomized trial, incorporating the current 
best available treatment (bevacizumab), in recurrent GBM is warranted.  Furthermore, as 
described below, there is a theoretical scientific basis for potential synergies between 
bevacizumab and a specific active immunotherapy such as HSPPC-96. 
There are at least two mechanisms by which bevacizumab and HSPPC-96 may work 
synergistically. The first is based on the transient normalization of tumor vasculature achieved 
with a variety of anti-angiogenic agents like bevacizumab, which in turn facilitates infiltration 
of lymphocytes into the tumor. Lymphocytes activated by HSPPC-96 vaccination are thus 
expected to more readily target tumor cells. The second mechanism of potential synergy is based 
on the observation that excessive vascular endothelial growth factor (VEGF) can inhibit the 
differentiation and maturation of dendritic cells (DCs). Such immature DCs are less potent 
stimulators of T cells [24]. Thus, VEGF neutralization by bevacizumab is expected to restore 
the capacity of DCs to optimally process and present HSP associated antigens to T cells. 
 
Published data from both human (sipuleucel-T) and animal studies  
One publication reports the combination of bevacizumab and a cancer vaccine in the clinical 
setting. The vaccine was sipuleucel-T, which was tested in a Phase 1 study with recurrent 
prostate cancer. The agents were administered concurrently. Peripheral blood mononuclear cell 
(PBMC) proliferation and IFN ELISPOT assays were conducted for evaluation of immune 


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Update #13 
response before and at various times after therapy. In all patients evaluated for immune response, 
an increase in response over baseline was detected. Whether the responses were indicative of an 
additive or synergistic effect of bevacizumab and sipuleucel-T could not be determined since 
patients receiving single agent therapy were not included in the study [25]. In animal models 
several studies have reported a synergistic effect of anti-angiogenic agents targeting VEGF or 
VEGF receptors in combination with cancer vaccines. The tumor types tested include 
melanoma, colon cancer, squamous cell carcinoma, breast cancer and fibrosarcoma. Several 
positive effects of the combined therapy were reported, including: (a) prolonged survival, (b) 
tumor shrinkage, (c) reduction in tumor infiltrating regulatory T cells, (d) reduction in tumor 
infiltrating immature DCs and increase in infiltrating mature DCs, (e) increased tumor 
infiltration of CD4+ and CD8+ effector lymphocytes as well as NK cells and (f) increase in 
cytotoxic T-lymphocyte (CTL) response to tumor antigen. It is of note that enhanced tumor 
lymphocyte infiltration was observed despite a reduction in density of tumor vasculature [26, 
27].  
Study Design 
In the present study, patients must have ≥90% resection to be included, which is consistent with 
populations studied in previous trials of HSPPC-96 in recurrent GBM. It is believed that the 
optimal setting for studying cancer vaccine efficacy is in patients who have the least amount of 
residual tumor burden at the time of vaccination.  HSPPC-96 can be easily purified from human 
tissue through a series of biochemical steps.  Surgically resected tumor is homogenized and then 
run through columns that purify the HSPPC-96, eventually concentrating it in sufficient 
quantities to facilitate biological efficacy.  After purification, HSPPC-96 undergoes a series of 
quality tests, which includes confirmation of biological activity through an in vitro antigen 
presentation assay, prior to shipping for clinical use. 
1.2  
Study Rationale 
This trial is important because there are currently no approved adjuvant treatments in recurrent 
GBM that significantly extend survival. This trial is designed to provide sound evidence towards 
determining whether an autologous active immunotherapy, HSPPC-96, used as an adjuvant 
treatment to surgery and in combination (either concomitantly post-surgery or serially at the 
point of progression) with the best available and approved therapy, bevacizumab, in recurrent 
GBM can extend overall survival.  Since this trial includes an arm of bevacizumab alone, this 
affords the opportunity to also better characterize the effect of bevacizumab on overall survival 
in a randomized, controlled setting, which remains an important open clinical question.    
Beyond the primary goal of demonstrating an impact on overall survival, this trial will also 
advance the biological understanding of a vital area of cancer research.  The use of cancer 
vaccines in combination with other immune-based, targeted agents has been an area of 
increasing focus but clinical efforts to undertake combination trials have been limited to date.  
As such, this trial provides the opportunity to advance the understanding of cancer vaccines and 
combination therapy in a meaningful clinical setting.   
In addition, positive findings in recurrent GBM would likely have implications for utility of 
HSPPC-96 in surgically resectable newly diagnosed GBM.  From a biological perspective, 
positive findings could also open additional avenues of research with HSPPC-96 and 
bevacizumab in other cancer indications.   
1.3 
Relevant Data 
The relevant data, which supports the use of the experimental and control arms in the setting of 
recurrent GBM are provided in the summary table below. Please note that the first 2 studies are 
discussed above and full detail is provided therein.  


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Update #13 
 
Experimental Arm:  HSPPC-96 
Population 
Study Design 
Key Results 
Supporting 
publications/ 
abstracts/posters 
Resectable, 
recurrent GBM 
Phase 1, single-arm, 
single center trial to 
evaluate safety and 
immunogenicity 
12 evaluable patients enrolled 
No toxicities attributable to 
HSPPC-96 
92% of patients showed a 
peripheral immune response 
Median OS of 42 weeks 
Crane et. al., Clinical 
Cancer Research, 2012 
Aug 7. [Epub ahead of 
print]http://www.ncbi.n
lm.nih.gov/pubmed/228
72572 [22] 
Resectable 
recurrent GBM 
Phase 2, single-arm, 
multi-center trial to 
evaluate 6-month 
survival rate, overall 
survival and 
immunogenicity 
30 evaluable patients enrolled 
6-month survival of 93% 
Median OS of 47.6 weeks 
All patients showed a peripheral 
immune response 
Parsa et al., J Clin 
Oncol 29: 2011 (suppl; 
abstr 2565)  
http://www.asco.org/A
SCOv2/Meetings/Abstr
acts?&vmview=abst_de
tail_view&confID=102
&abstractID=85010 
[23] 
 
Resectable, 
newly 
diagnosed GBM 
Phase 2, single-arm, 
multi-center trial to 
evaluate overall 
survival and safety 
100% enrolled, 46 patients treated, 
11 deaths reported thus far; 3 
patients to date have reached end of 
study (2 years) without progression 
 Abstract accepted for 
oral presentation at the 
2013 AANS annual 
meeting 
Control Arm: bevacizumab 
Recurrent GBM 
Phase 2, randomized, 
multi center to 
evaluate 6-month PFS 
and OR rates 
25.9% median response rate (per 
label) 
Duration of response of 4.2 months 
(per label) 
6-month PFS of 29% (per Friedman 
et al) 
Median OS of 31 weeks (per 
Friedman et al) 
Approved labeling 
http://www.gene.com/g
ene/products/informatio
n/pdf/avastin-
prescribing.pdf  
Friedman et al., J Clin 
Oncol 27:4733-4740 
http://www.ncbi.nlm.ni
h.gov/pubmed/1972092
7 
Importantly, both the phase I and phase II trials used entry criteria of > 90% resection to generate 
a minimum of 7 grams of tissue to generate 4 more doses of 25 micrograms of HSPPC-96 
vaccine. In the phase I rGBM trial, 12 patients received a median of 5.5 doses with a range of 
4-22, with 3 patients (25%) receiving only 4 doses [46]. Importantly, 11/12 patients generated 
an immune response to HSPPC-96, confirming the immunologic effect of 4 doses. In the phase 
II rGBM trial of 41 patients, the median number of vaccines administered was 6, with a range 
of 1-15 [47]. Based on prior phase I and II trial designs and for consistency, a 4 dose minimum 
for trial entry is required, with the expectation that most patients will have more than that.  The 
4 dose minimum was also used in a ND GBM trial as a criterion for treatment [48].  
 
A 4 dose minimum will increase the number of eligible patients as the number of vials required 
for quality assurance purposes increases with the increased number of vials required for entry.  
While seven grams is felt to be optimal, patients where at least 7 grams of tissue resected could 
be sufficient for vaccine generation (depending on the quality of the tissue), but both clinician 
and patient would need to be advised that vaccine generation might not occur.  


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1.4 
Inclusion of Women and Minorities   
This study will be available to all eligible patients regardless of race, gender, or ethnic group. 
There is no information currently available regarding differential agent effects of this regimen 
in subsets defined by race, gender, or ethnicity, and there is no reason to expect such differences 
to exist. Therefore, although the planned analyses will, as always, look for differences in 
treatment effect based on racial and gender groupings, the sample size is not increased in order 
to provide additional power for such subset analyses. A total of 165 patients may be enrolled. 
Based on prior studies involving similar disease sites, we expect about 13% of patients will be 
classified as minorities by race and about 40% of patients to be women. Expected sizes of racial 
by gender subsets are shown in the following table: 
DOMESTIC PLANNED ENROLLMENT 
Racial Categories 
Ethnic Categories 
Total 
Not Hispanic or 
Latino 
Hispanic or Latino 
Female 
Male 
Female 
Male 
American Indian/ Alaska Native 
  1 
  2 
 
 
    3 
Asian 
  2 
  2 
 
 
    4 
Native Hawaiian or Other Pacific 
Islander 
 
 
 
 
 
Black or African American 
  6 
  8 
 
 
  14 
White 
49 
74 
9 
12 
144 
More Than One Race 
 
 
 
 
 
Total 
58 
86 
9 
12 
165 
 
2.0 
OBJECTIVES  
2.1 
Primary Objective 
To determine whether there is an overall survival advantage of HSPPC-96 administered with 
bevacizumab, given concomitantly or at the point of progression, in comparison with 
bevacizumab alone in patients with surgically resectable recurrent GBM. 


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2.2 
Secondary Objectives 
2.2.1 
To evaluate the safety and tolerability of HSPPC-96 with bevacizumab. 
2.2.2 
To evaluate the progression free survival of HSPPC-96 with bevacizumab, given 
concomitantly or at the point of progression. 
2.3 
Correlative Science Objectives 
2.3.1 To evaluate whether patients who demonstrate an immune response to HSPPC-96 will 
have an improved survival outcome in comparison to those patients who do not 
show immune response.  
2.3.2 To explore the expression of B7-H1 protein expression and PI3K pathway activation 
(which are associated with immunoresistance) at the tissue level before treatment 
and correlate with therapeutic response.  
2.3.3 To explore whether T-cell infiltrate of tumor at baseline correlates with response to 
vaccine.   
 
3.0 
ON-STUDY GUIDELINES 
This clinical trial can fulfill its objectives only if patients appropriate for this trial are enrolled. All 
relevant medical and other considerations should be taken into account when deciding whether this 
protocol is appropriate for a particular patient. Physicians should consider the risks and benefits of 
any therapy, and therefore only enroll patients for whom this treatment is appropriate. Although they 
will not be considered formal eligibility (exclusion) criteria, physicians should recognize that the 
following may seriously increase the risk to the patient entering this protocol: 
• 
Psychiatric illness, which would prevent the patient from giving informed consent. 
• 
Medical condition such as uncontrolled infection (including HIV), which in the opinion of the 
treating physician would make this protocol unreasonably hazardous for the patient. 
• 
Patients with a “currently active” second malignancy other than non-melanoma skin cancers. 
Patients are not considered to have a “currently active” malignancy if they have completed 
therapy and are free of disease for ≥ 3 years.  
• 
Women and men of reproductive potential should agree to use an appropriate method of birth 
control during treatment and for 6 months after the last treatment in this study due to the 
teratogenic potential of the therapy utilized in this trial. Appropriate methods of birth control 
include abstinence, oral contraceptives, implantable hormonal contraceptives or double barrier 
method (diaphragm plus condom) 
• 
Life expectancy of <8 weeks. 
4.0 
ELIGIBILITY CRITERIA    
All questions regarding eligibility criteria should be directed to the Alliance Study Chair. Please note 
that the Study Chair cannot grant waivers to eligibility requirements.  


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Update #13 
4.1 
Pre-registration (Pre-Surgery) Eligibility Criteria  
4.1.1 
Histologic documentation:  Prior histologic diagnosis of GBM at first occurrence.  
4.1.2 
Stage: First or second recurrence of GBM or gliosarcoma considered to be 
surgically resectable.  
4.1.3 
Prior Treatment  
• 
No radiotherapy within 90 days prior to pre-registration.  
• 
No prior treatment with any anti-angiogenic agent targeting the VEGF pathway including 
but not limited to bevacizumab, cediranib, vandetanib, sunitinib, pazopanib, aflibercept or 
sorafenib. 
• 
No prior treatment with HSPPC-96 or other investigational immunotherapy. 
• 
Must have received prior treatment with radiotherapy and temozolomide for histologically 
confirmed GBM at initial diagnosis. 
• 
No tumor directed therapy for most recent progression.  
• 
No prior Gliadel® wafers. 
4.1.4   No clinically significant cardiovascular disease 
• 
Patients with a history of hypertension must be well controlled (<150/90) on a 
regimen of antihypertensive therapy.  
• 
History of arterial thrombotic events within the past 6 months, including transient 
ischemic attack (TIA), cerebrovascular accident (CVA), peripheral arterial 
thrombus, unstable angina or angina requiring surgical or medial intervention in the 
past 6 months, or myocardial infarction (MI).  Patients with clinically significant 
peripheral artery disease (i.e., claudication on less than one block), significant 
vascular disease (i.e., aortic aneurysm, history of aortic dissection) are not eligible. 
• 
Patients who have had a deep vein thrombosis or pulmonary embolus within the 
past 6 months are eligible if they are on stable therapeutic anticoagulation 
• 
No current New York Heart Association classification II, III or IV congestive heart 
failure  
4.1.5 
No significant bleeding within the past 6 months; no bleeding diathesis or 
coagulopathy  
4.1.6 
No history of abdominal fistula, gastrointestinal perforation, or intra-abdominal 
abscess within past 12 months  
4.1.7 
No evidence of any systemic autoimmune disease (e.g. Hashimoto’s thyroiditis) 
and/or any history of primary or secondary immunodeficiency, and no 


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immunosuppressant therapy (with the exception of dexamethasone as noted below) 
for any reason 
4.1.8 
Age ≥ 18 years of age  
4.1.9 
Karnofsky functional status rating ≥70 
4.1.10 No more than 16 mg dexamethasone (or equivalent) per day 
4.1.11 Non-pregnant and non-nursing 
4.2 
Registration (Post-Surgery) Eligibility Criteria  
4.2.1   Pre-registration eligibility criteria continue to be met 
4.2.2 
Histologic documentation:  confirmed histological diagnosis of recurrent GBM or 
gliosarcoma. 
4.2.3  ≥ 90% surgical resection of recurrent GBM confirmed by central radiology review 
by MRI with or without gadolinium per institutional guidelines.  A CT scan is 
allowable in place of MRI only in situations where an MRI is contraindicated (e.g., 
patient has a heart pacemaker, metallic devices in the eye, brain or spine, severe 
claustrophobia).   
4.2.4   Five grams of resected tumor available for vaccine manufacture as determined by 
institutional pathologist. Seven grams of tumor is preferred (see Section 6.5, 
footnote 1).  
4.2.5  Availability of ≥ 4 clinical vials of HSPPC-96  
4.2.6 
Required Initial Laboratory Values:  
Granulocytes 
≥1,500/µL 
Platelet count 
≥100,000/µL 
 
  
Total Bilirubin 
UPC ratio  
OR 
Urine protein 
Calculated creatinine 
clearance 
SGOT/SGPT(AST/ALT) 
≤ 2.0 x ULN 
<1 
 
≤ 1+ 
≥45 ml/min 
≤2.5 x ULN 
 
4.2.7 
No serious, non-healing wounds or ulcers 
4.2.8   
At least 7 days since any minor surgery such as port placement 
4.2.9 
No major surgical procedures, open biopsy or significant traumatic injury ≤28 days 
prior to registration or anticipation of need for elective or planned major surgical 


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Update #13 
procedure during the study. Core biopsy or other minor surgical procedures ≤7 
days prior to registration.   
4.2.10 No active or recent hemoptysis (≥½ teaspoon of bright red blood per episode) ≤30 
days prior to registration. 
4.2.11 No new bleeding on D28 (+/-3) MRI (or CT if MRI is contraindicated). 
4.2.12 No clinical deterioration at the time of registration/randomization. 
4.2.13 If a second surgery is needed for completion of resection, this should be within 30 
days from the first surgery. 
5.0 
PRE-REGISTRATION, REGISTRATION/RANDOMIZATION, AND STRATIFICATION  
5.1 
CTEP Investigator Registration Procedures 
Food and Drug Administration (FDA) regulations and National Cancer Institute (NCI) policy 
require all individuals contributing to NCI-sponsored trials to register and to renew their 
registration annually.  To register, all individuals must obtain a Cancer Therapy Evaluation 
Program 
(CTEP) 
Identity 
and 
Access 
Management 
(IAM) 
account 
(https://ctepcore.nci.nih.gov/iam).  In addition, persons with a registration type of Investigator 
(IVR), Non-Physician Investigator (NPIVR), or Associate Plus (AP) (i.e., clinical site staff 
requiring write access to OPEN, RAVE, or TRIAD or acting as a primary site contact) must 
complete their annual registration using CTEP’s web-based Registration and Credential 
Repository (RCR) (https://ctepcore.nci.nih.gov/rcr). Documentation requirements per 
registration type are outlined in the table below. 
Documentation Required 
IVR 
NPIVR 
AP 
A 
FDA Form 1572 
 
 
 
Financial Disclosure Form 
 
 
 
NCI 
Biosketch 
(education, 
training, 
employment, license, and certification) 
 
 
 
HSP/GCP training 
 
 
 
Agent Shipment Form (if applicable) 
 
 
 
CV (optional) 
 
 
 
 
An active CTEP-IAM user account and appropriate RCR registration is required to access all 
CTEP and CTSU (Cancer Trials Support Unit) websites and applications.  In addition, IVRs and 
NPIVRs must list all clinical practice sites and IRBs covering their practice sites on the FDA 
Form 1572 in RCR to allow the following: 
• 
Added to a site roster 
• 
Assigned the treating, credit, consenting, or drug shipment (IVR only) tasks in OPEN 
• 
Act as the site-protocol PI on the IRB approval 
 


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Additional 
information 
can 
be 
found 
on 
the 
CTEP 
website 
at 
https://ctep.cancer.gov/investigatorResources/default.htm. For questions, please contact the 
RCR Help Desk by email at 
. 
5.2 CTSU Site Registration Procedures 
This study is supported by the NCI Cancer Trials Support Unit (CTSU). 
IRB Approval:   
Each investigator or group of investigators at a clinical site must obtain IRB approval for this 
protocol and submit IRB approval and supporting documentation to the CTSU Regulatory 
Office before they can be approved to enroll patients.  
Assignment of site registration status in the CTSU Regulatory Support System (RSS) uses 
extensive data to make a determination of whether a site has fulfilled all regulatory criteria 
including but not limited to:  
• 
an active Federal Wide Assurance (FWA) number, 
• 
an active roster affiliation with the Lead Network or a participating organization,  
• 
a valid IRB approval, and  
• 
compliance with all protocol specific requirements. 
In addition, the site-protocol Principal Investigator (PI) must meet the following criteria: 
• 
Active registration status 
• 
The IRB number of the site IRB of record listed on their Form FDA 1572 
• 
An active status on a participating roster at the registering site. 
Sites participating on the NCI CIRB initiative and accepting CIRB approval for the study are 
not required to submit separate IRB approval documentation to the CTSU Regulatory Office. 
For sites using the CIRB, IRB approval information is received from the CIRB and applied to 
the RSS in an automated process. Signatory Institutions must submit a Study Specific Worksheet 
for Local Context (SSW) to the CIRB via IRBManager to indicate their intention to open the 
study locally.  The CIRB’s approval of the SSW is then communicated to the CTSU Regulatory 
Office. In order for the SSW approval to be processed, the Signatory Institution must inform the 
CTSU which CIRB-approved institutions aligned with the Signatory Institution are participating 
in a given study. 
5.2.1  Downloading Site Registration Materials 
Site registration forms may be downloaded from the A071801 protocol page located on the 
CTSU members’ website. Permission to view and download this protocol and its 
supporting documents is restricted and is based on person and site roster assignment housed 
in the CTSU RSS. 
• 
Go to https://www.ctsu.org and log in to the members’ area using your CTEP-IAM 
username and password. 
• 
Click on the Protocols tab in the upper left of your screen. 
• 
Either enter the protocol # in the search field at the top of the protocol tree, or, 
• 
Click on the By Lead Organization folder to expand. 
• 
Click on the Alliance link to expand, then select trial protocol # A071801. 


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Update #13 
• 
Click on LPO Documents, select the Site Registration documents link, and 
download and complete the forms provided. 
5.2.2 
Requirements for A071101 Site Registration 
• 
IRB approval (local IRB documentation, an IRB-signed CTSU IRB Certification 
Form, 
Protocol 
of 
Human 
Subjects 
Assurance 
Identification/IRB 
Certification/Declaration of Exemption Form, or combination is accepted).  
5.2.3  Checking Your Site’s Registration Status 
You can verify your site registration status on the members’ section of the CTSU website.  
Check the status of your site’s registration packets by querying the RSS site registration 
status page of the members’ section of the CTSU website.  (Note: Sites will not receive 
formal notification of regulatory approval from the CTSU Regulatory Office.) 
• 
Go to https://www.ctsu.org and log in to the members’ area using your CTEP-IAM 
username and password. 
• 
Click on the Regulatory tab  
• 
Click on the Site Registration tab. 
• 
Enter your 5-character CTEP Institution Code and click on Go. 
Note: The status given only reflects compliance with IRB documentation and institutional 
compliance with protocol-specific requirements outlined by the Lead Network. It does not 
reflect compliance with protocol requirements for individuals participating on the protocol 
or the enrolling investigator’s status with the NCI or their affiliated networks. 
5.2.4 
Submitting Regulatory Requirements 
Submit required forms and documents to the CTSU Regulatory Office via the Regulatory 
Submission Portal, where they will be entered and tracked in the CTSU RSS.  
Regulatory Submission Portal: www.ctsu.org (members’ area)   Regulatory Tab   
Regulatory Submission Portal 
When applicable, original documents should be mailed to: 
 
 
 
Institutions with patients waiting that are unable to use the Portal should alert the CTSU 
Regulatory Office immediately at 
 in order to receive further instruction 
and support. 
5.3 Pre-registration  
5.3.1 
Pre-registration Requirements 
• 
Informed Consent: the patient must be aware of the neoplastic nature of his/her 
disease and willingly consent after being informed of the procedure to be followed, 
the experimental nature of the therapy, alternatives, potential benefits, side effects, 
risks, and discomforts. Human protection committee approval of this protocol and 
a consent form is required. 
• 
All patients must have signed a HIPAA authorization form if applicable.  


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5.3.2 
Patient Pre-registration through OPEN 
Patient enrollment will be facilitated using the Oncology Patient Enrollment Network 
(OPEN).  OPEN is a web-based registration system available on a 24/7 basis.  To access 
OPEN, the site user must have an active CTEP-IAM account (check at < https://eapps-
ctep.nci.nih.gov/iam/index.jsp >) and a 'Registrar' role on either the LPO or participating 
organization roster. 
All site staff will use OPEN to enroll patients to this study.  It is integrated with the CTSU 
Enterprise System for regulatory and roster data.  OPEN can be accessed at 
https://open.ctsu.org or from the OPEN tab on the CTSU members’ side of the website at 
https://www.ctsu.org. 
Prior to accessing OPEN, site staff should verify the following: 
• 
All eligibility criteria have been met within the protocol stated timeframes.  
• 
All patients have signed an appropriate consent form and HIPAA authorization 
form (if applicable). 
Note:  The OPEN system will provide the site with a printable confirmation of registration 
and treatment information.   Please print this confirmation for your records. 
Further instructional information is provided on the OPEN tab of the CTSU members’ side 
of the CTSU website at https://www.ctsu.org or at https://open.ctsu.org.  For any additional 
questions contact the CTSU Help Desk at 
 or 
. 
5.4 Registration/Randomization  
5.4.1 
Registration Procedures  
• 
Confirmation must be sent from site to Agenus informing Agenus that tumor 
material for a patient is being shipped to attempt vaccine manufacture. The Tissue 
Procurement Form (TPF) must be completed and faxed to Agenus as per the 
instructions on the TPF. Sites can obtain the study-specific TPF from the CTSU 
website.  
• 
Bulk tumor tissue must be sent from site to Agenus for vaccine manufacture per 
instructions in Appendix III. The completed Tissue Procurement Form (TPF) must 
be included with the tissue sent to Agenus.  
• 
Notification must be sent to Agenus that ≥ 90% surgical resection of recurrent GBM 
has been confirmed by the Imaging and Radiation Oncology Core central radiology 
review. 
5.4.2 
Registration Requirements 
Receipt by site of the designated Vaccine Production Notification Form from Agenus, 
which will inform the site of the status of the HSPPC-96 vaccine (i.e., successfully 
produced or not successfully produced) and, if successfully produced, the number of 
available vials that will be shipped along with the anticipated shipping date. The Vaccine 
Production Notification Form will be sent by Agenus to the site generally within 14 days 
of receiving the tumor material for processing. There have to be at least 4 clinical vials 
successfully produced for the patient to be considered eligible. If the vaccine was 
successfully produced, but the patient is not eligible to participate, the Vaccine Order Form 
MUST be faxed to Agenus.  The site should check “No, please do not ship vaccine (Arm 
3 or other reason) and identify in the comments section that the patient is a screen failure. 


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5.4.3 
Patient Registration/Randomization through OPEN  
Patients should be randomized 48 to 72 hours after the 28 (+/- 3) day scan with no bleed. 
After randomization, sites must complete the Vaccine Order Form and fax or email it to 
Agenus in order to have vaccine shipped (see Section 11.6 for ordering vaccine). Sites can 
obtain the study specific Vaccine Order Form from the CTSU website. 
The site staff will enter the patient ID number obtained at pre-registration and the patient’s 
age stratum (<55 vs. ≥55 years old) and KPS stratum (70 vs. 80-100) into the OPEN 
registration system to obtain a treatment assignment. Once the randomization is complete, 
note the patient’s treatment assignment in your records. 
OPEN Access Requirements 
 
Patient enrollment will be facilitated using the Oncology Patient Enrollment Network 
(OPEN).  OPEN is a web-based registration system available on a 24/7 basis.  To access 
OPEN, the site user must have an active CTEP-IAM account (check at < https://eapps-
ctep.nci.nih.gov/iam/index.jsp >) and a 'Registrar' role on either the LPO or participating 
organization roster. 
All site staff will use OPEN to enroll patients to this study.  It is integrated with the CTSU 
Enterprise System for regulatory and roster data.  OPEN can be accessed at 
https://open.ctsu.org or from the OPEN tab on the CTSU members’ side of the website at 
https://www.ctsu.org. 
Prior to accessing OPEN, site staff should verify the following: 
• 
All eligibility criteria have been met within the protocol stated timeframes.  
• 
All patients have signed an appropriate consent form and HIPAA authorization 
form (if applicable). 
Note:  The OPEN system will provide the site with a printable confirmation of registration 
and treatment information.   Please print this confirmation for your records. 
Further instructional information is provided on the OPEN tab of the CTSU members’ side 
of the CTSU website at https://www.ctsu.org or at https://open.ctsu.org.  For any additional 
questions contact the CTSU Help Desk at 
. 
5.5 
Stratification 
Patients will be stratified by 
●  Age group: <55 vs. ≥55 years old 
●  KPS group: 70 vs. 80-100 
●  Vials produced: Four or 5 vials of HSPPC-96 vs. 6 or more vials of HSPPC-96 
5.6 
Registration to Sub-studies  
Registration to the correlative science sub-study will be done at the same time as registration to 
the treatment study. Registration to the treatment trial and the sub-study will not be 
completed if eligibility requirements are not met.  
There is one sub-study within A071101: A071101-ST1: Correlative Science in A071101. This 
study must be offered to all patients enrolled on A071101 (although patients may opt to not 
participate). This sub-study does not require separate IRB approval.  


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If a patient answers “yes” to model consent question #1, they have consented to participate in 
the sub-study described in Section 10.1. The patient should be registered to A071101-ST1 at the 
same time they are registered to the treatment trial (A071101). Samples should be submitted per 
Section 6.   
5.7 
Surgical Quality Assurance   
It is recommended that the neurosurgeon has performed at least 20 brain tumor resections over 
the last 12 months.  
Note: The neurosurgeon does not need to hold a Cooperative Group membership. 
 
6.0 
DATA AND SPECIMEN SUBMISSION AND CENTRAL RADIOLOGY REVIEW 
Data collection for this study will be done exclusively through the Medidata Rave clinical data 
management system.  Access to the trial in Rave is granted through the iMedidata application to all 
persons with the appropriate roles assigned in Regulatory Support System (RSS).  To access Rave 
via iMedidata, the site user must have an active CTEP-IAM account (check at < https://eapps-
ctep.nci.nih.gov/iam/index.jsp >) and the appropriate Rave role (Rave CRA, Read-Only, Site 
Investigator) on either the LPO or participating organization roster at the enrolling site. 
Upon initial site registration approval for the study in RSS, all persons with Rave roles assigned on 
the appropriate roster will be sent a study invitation e-mail from iMedidata.  To accept the invitation, 
site users must log into the Select Login (https://login.imedidata.com/selectlogin) using their CTEP-
IAM user name and password, and click on the “accept” link in the upper right-corner of the 
iMedidata page.  Please note, site users will not be able to access the study in Rave until all required 
Medidata and study specific trainings are completed.  Trainings will be in the form of electronic 
learnings (eLearnings), and can be accessed by clicking on the link in the upper right pane of the 
iMedidata screen. 
Users that have not previously activated their iMedidata/Rave account at the time of initial site 
registration approval for the study in RSS will also receive a separate invitation from iMedidata to 
activate their account.  Account activation instructions are located on the CTSU website, Rave tab 
under the Rave resource materials (Medidata Account Activation and Study Invitation Acceptance).  
Additional information on iMedidata/Rave is available on the CTSU members’ website under the 
Rave tab at www.ctsu.org/RAVE/ or by contacting the CTSU Help Desk at 
 or by e-
mail at 
.   
6.1 
Data Submission   
This study will use Medidata Rave for remote data capture (RDC) of all study data. Access the 
RAVE system through the iMedidata portal at https://login.imedidata.com. 
For questions regarding forms completion and submission, please contact the Data Manager 
listed on the Protocol Resources page of the protocol. 
6.2 
Tumor Tissue Submission for Vaccine Manufacture 
The manufacturer of HSPCC-96 may be available for site training. Institutions may contact the 
Alliance at 
 to express their interest in this optional training session 
with Agenus. Agenus will provide training for techniques in tissue procurement, tissue 
processing, packaging and shipping of tissue of vaccine manufacture.  
See Appendix III for detailed instructions for handling, preparation, and submission of tumor 
tissue for HSPPC-96 vaccine manufacture.  
The Tissue Procurement Form (TPF) must be included with the tissue sent to Agenus. 


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6.2.1 
Kits for shipping tissue will be supplied by Agenus and should be ordered at the 
time of IRB submission because they will be needed at time of surgery. Kits are 
ordered by contacting Agenus via e-mail at: 
. 
The dimensions of the kits are 17" x 15" x 15". Please order one or two kits at a time, based 
on your anticipated use, and please allow 3-4 days for shipping. 
 
NOTE: Questions about the protocol and model consent MUST be directed to the 
Protocol Coordinator listed on the title page. For other questions, additional 
resources are identified on page 2.  
 
6.3 
Blood Submission for Immune Monitoring   
For patients who consent to participate, blood samples will be used for the immune response 
analysis described in the correlative study section.  
Only previously designated sites will process the blood at their institution and send PBMCs to 
Mayo BAP for banking.  All other sites will send whole blood to the Mayo BAP for processing 
and banking. 
Collection time points are described below according to treatment arm:  
Arm 1 (HSPPC-96 + concomitant bevacizumab): blood will be drawn prior to first dose of 
vaccine /bevacizumab  (can be drawn on same day of treatment or up to 14 days prior), at the 
vaccine dose #5/bevacizumab dose#3 prior to vaccine administration, at the vaccine dose 
#7/bevacizumab dose #5 prior to vaccine administration, 2 weeks following the final dose of 
vaccine, and 2 weeks following the final dose of bevacizumab.   
Arm 2 (HSPPC-96 followed by bevacizumab at progression): blood will be drawn prior to first 
dose of vaccine  (can be drawn on same day as treatment or up to 14 days prior) at the vaccine 
dose #5 prior to vaccine administration, at the vaccine dose #7 prior to vaccine administration, 
2 weeks following the final dose of vaccine, and 2 weeks following the final dose of 
bevacizumab.   
Arm 3 (bevacizumab alone): blood will be drawn prior to first dose of bevacizumab  (can be 
drawn on same day as treatment or up to 14 days prior), prior to the bevacizumab at dose #3, 
prior to the bevacizumab at dose #5, and 2 weeks following the final dose of bevacizumab.  
Approximately 50 ml of blood will be drawn at the above described time points.  
6.3.1 Blood submission for previously designated sites sending PBMCs 
This applies only to the following previously designated site that will be processing the 
blood at their institution and sending PBMCs to the Mayo BAP for banking: 
 
. 
Blood collected for immunological studies will be processed according to the Appendix 
IV, Whole Blood PBMC Processing at Sites, “Isolation of Peripheral Blood Mononuclear 
Cells (PBMCs) from Human Blood Samples.” These specimens will be batch shipped on 
dry ice to the Mayo BAP for banking. 
Label samples with the following identification: 
1) Procurement date and time 
2) Alliance patient number 


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3) Alliance study number (A071101) 
4) PBMC  
6.3.2  Blood submission for sites sending whole blood   
For sites collecting whole blood and shipping to the Mayo BAP for processing and 
banking: 
Collect approximately 50 ml of peripheral venous blood divided evenly into five 10.0 ml 
green top (sodium heparin) tubes. The sodium heparin tubes should be inverted several 
times to mix and refrigerated immediately until shipped on cold pack by overnight mail to 
the Alliance BAP at Mayo. The samples should be shipped the same day that the blood is 
drawn. 
Label samples with the following identification: 
1) Procurement date and time 
2) Alliance patient number 
3) Alliance study number (A071101) 
4) Whole blood  
Note: Shipment must not involve weekends and holidays. 
6.3.3 
Shipping PBMCs on dry ice or whole blood on cold packs 
For sites batch shipping PBMCs (see Appendix IV) samples should be sent overnight on 
dry ice. For sites shipping whole blood, samples should be sent overnight on cold packs 
(see Section 6.3.2). 
Please be sure to use a method of shipping that is secure and traceable. Extreme heat 
precautions should be taken when necessary. Ship only on Monday-Thursday by overnight 
service to assure receipt. If shipping on Friday, FedEx must be used and the air bill must 
be marked “For Saturday delivery.”  Send the FedEx tracking number to the Biospecimen 
Resource Manager so arrangements can be made to stabilize the samples over the weekend. 
Do not ship specimens on Saturdays. Do not collect specimens the day before a holiday. 
Ship samples to the following address: 
  
 
 
 
 
For questions about blood submission contact: 
 
 
 
 
 


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6.4 
Tissue Submission for Biomarker Analysis  
Submit the following specimens and forms: 
A representative formalin fixed, paraffin-embedded (FFPE) block of tissue from:   
• 
prior histologic diagnosis of GBM at initial diagnosis 
• 
total resection for recurrence  
• 
if possible, from post-progression biopsy 
• 
Specimen Submission: Tissue 
• 
Surgical Pathology Reports from the initial diagnosis and recurrence 
• 
BioMS shipping manifest 
NOTE:  If submission of diagnostic blocks is prohibitive and a non-diagnostic block of 
comparable tumor tissue is available, please prepare a second FFPE specimen to submit for 
biomarker analysis.  
Tissue specimens should be sent to the Alliance Biorepository at Mayo Clinic Pathology 
Coordinator: 
 
 
 
 
 
Use FedEx priority overnight delivery for all shipment. Extreme heat precautions should be 
taken when necessary. Ship only on Monday-Thursday overnight service to assure receipt.  
6.4.1 
Surgical guidelines 
• 
Surgically remove a sample of at least 5 mm3. 
• 
Immerse fresh, surgically removed tumor tissue in 4-10% neutral buffered formalin.  
• 
Immerse tissue as quickly as possible following surgical resection.  
• 
Minimize or avoid necrotic tissue or non-tumor tissues. 
6.4.2 Pathology processing guidelines 
• 
Use a maximum fixation time of 24 hours (longer fixation times lead to over-
fixation and more severe nucleic acid fragmentation). 
• 
Thoroughly dehydrate samples prior to embedding. 
• 
Embed in low melting temperature paraffin.  
For questions pertaining to paraffin-embedded tissue pathology contact: 
 
 
 
 


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6.4.3 Tumor slide preparation guidelines 
If due to institutional policy a block cannot be sent, utilize microfacing of the blocks to submit 
the following: 
• 
15-25 slides containing a minimum of 10 mm2 primary tumor tissue in total, cut at 
a thickness of at least 10µm  
• 
12 slides cut at a thickness of at least 10µm from patient’s tumor both at initial 
diagnosis and recurrent disease resection 
When slides are sent, the following guidelines should be followed: 
• 
Cut new sections from paraffin embedded blocks; do not send precut paraffin 
sections. Cut sections as close as possible to the shipment and processing date (RNA 
degrades significantly faster in the sections that in the block) 
• 
Standard uncharged glass slides should be used to mount the sections. Examples 
standard glass slides: # 4430 Super UP-RITE frosted micro slides, Richard-Allen 
Scientific or equivalent. 
• 
Cut 15-25 x 10-micron unstained tumor tissue sections mounted on regular slides 
for RNA isolation. Each slide must have as much tumor tissue as possible on the 
slide. 
• 
For H&E staining/ tumor visualization, one 5-micron paraffin-embedded section 
must be cut from the same region of the tumor tissue block and mounted on a 
charged slide.  
• 
Do not bake or place cover slips on the unstained slides 
• 
Do not stick adhesive labels on the slides 
• 
Label slides with patient ID number, accession number and order of section (i.e. 1-
25), and thickness of section for 5 or 10 µm. 
The goal of the Alliance Biorepository at Mayo Clinic is to provide investigators with 
quality histology sections for their research while maintaining the integrity of the tissue. 
All paraffin blocks that are to be stored at the Alliance Biorepository at Mayo Clinic will 
be stored at 4ºC to minimize degradation of cellular antigens. For these reasons it is 
preferred that the Alliance Biorepository at Mayo Clinic bank the block until the study 
investigator requests thin sections. Please contact the Alliance Biorepository at Mayo 
Clinic if additional assurances with your hospital pathology department are required.


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6.5 
Biospecimen Submission Summary 
Samples 
A071101 Sample Collection for Arm 1 and Arm 2 
Pre-
registration at 
Surgery 
Pre-
treatment
(prior to 
vaccine 
dose #1)  
Vaccine 
dose #5 
Vaccine 
dose #7 
2 weeks 
after final 
vaccine 
dose 
2 weeks after 
final 
bevacizumab 
dose 
Progression 
Ship to: 
Tumor 
tissue: 5-
7 grams* 
X 
 
 
 
 
 
 
Agenus 
Tumor 
block** 
 
X 
 
 
 
 
X+ 
Biorepo
sitory at 
Mayo 
Whole 
blood (5 
x 10ml 
green top 
tubes) or 
PBMCs 
(isolated 
from 
blood 
collected 
in (5) 
10ml 
green top 
tubes) 
 
X++ 
X++ 
X++ 
X 
X 
 
Mayo 
BAP 
*   Mandatory for vaccine manufacture. Successful manufacture of vaccine is dependent on receipt of viable tissue 
that is processed, packaged, and shipped correctly, and also dependent on sufficient quantity. Tissue must be 
non-necrotic, and must have viable tumor. Depending on the quality of the specimen shipped, it may be possible 
that 5 grams of tumor would be sufficient to manufacture the minimum 4 vials of vaccine that are required for 
treatment on this protocol. However, it is in the best interest of the patients care and potential for good outcomes, 
along with the science of the trial and correlative work on specimens, that maximum tissue is sent and good 
quality tissue is sent. Therefore, the goal with every patient is that the largest quantity of high quality tissue is 
sent for vaccine manufacture. The only tissue utilized at the site should be to confirm that the patient indeed 
has recurrent tumor, as every additional amount of tissue sent will make the difference as to whether the patient 
will be eligible to continue on the trial, and also how many vials of vaccine can be manufactured. 
** Archival paraffin-embedded tissue from prior histologic diagnosis of GBM at initial diagnosis and paraffin block 
tissue from total resection for recurrence. 
+  From post-progression biopsy, if possible. 
++ Blood drawn prior to vaccine administration.  


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*   Mandatory for vaccine manufacture. Successful manufacture of vaccine is dependent on receipt of viable tissue 
that is processed, packaged, and shipped correctly, and also dependent on sufficient quantity. Tissue must be 
non-necrotic, and must have viable tumor. Depending on the quality of the specimen shipped, it may be possible 
that 5 grams of tumor would be sufficient to manufacture the minimum 4 vials of vaccine that are required for 
treatment on this protocol. However, it is in the best interest of the patients care and potential for good outcomes, 
along with the science of the trial and correlative work on specimens, that maximum tissue is sent and good 
quality tissue is sent. Therefore, the goal with every patient is that the largest quantity of high quality tissue is 
sent for vaccine manufacture. The only tissue utilized at the site should be to confirm that the patient indeed 
has recurrent tumor, as every additional amount of tissue sent will make the difference as to whether the patient 
will be eligible to continue on the trial, and also how many vials of vaccine can be manufactured. 
** Archival paraffin-embedded tissue from prior histologic diagnosis of GBM at initial diagnosis and paraffin-
embedded tissue from total resection for recurrence 
+  From post-progression biopsy, if possible. 
++ Blood drawn prior to bevacizumab administration. 
6.6 
Specimen Registration and Tracking 
USE OF THE ALLIANCE BIOSPECIMEN MANAGEMENT SYSTEM (BioMS) IS 
MANDATORY AND ALL SPECIMENS MUST BE LOGGED AND SHIPPED VIA THIS 
SYSTEM. 
BioMS is a web-based system for logging and tracking all biospecimens collected on Alliance 
trials.  
Authorized 
individuals 
may 
access 
BioMS 
at 
the 
following 
URL: http://bioms.allianceforclinicaltrialsinoncology.org/ using most standard web browsers 
(Safari, Firefox, Internet Explorer).  For information on using the BioMS system, please refer 
to the ‘Help’ links on the BioMS web page to access the on-line user manual, FAQs, and training 
videos.  To report technical problems, such as login issues or application errors, please contact: 
.  For assistance in using the application or questions or problems related to 
specific specimen logging, please contact: 
. 
After logging collected specimens in BioMS, the system will create a shipping manifest.  This 
shipping manifest must be printed and placed in the shipment container with the specimens. 
Samples 
A071101 Sample Collection for Arm 3 
Pre-
registration 
at Surgery 
Pre-treatment 
(prior to 
bevacizumab 
dose #1) 
Bevacizumab 
dose #3 
Bevacizumab 
dose #5 
2 weeks 
after final 
bevacizumab 
dose 
Progression 
Ship to: 
Tumor 
tissue: 5-
7grams* 
X 
 
 
 
 
 
Agenus 
Tumor 
block** 
 
X 
 
 
 
X+ 
Biorepo
sitory  
at Mayo 
Whole 
blood (5x 
10ml green 
top tubes) 
or PBMCs 
(isolated 
from blood 
collected 
in (5) 10ml 
green top 
tubes)  
 
X 
X++ 
X++ 
X 
 
Mayo 
BAP 


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6.7 
MRI/CT Imaging  
MRI (or CT only if MRI is contraindicated) imaging is to be completed on the following 
schedule (see Section 7.0):  
• 
Within 28 days prior to surgery 
• 
Within 48 hours post-surgery   
• 
At 28 (+/- 3) days post-surgery 
• 
Every 8 weeks (+/- 14 days) from registration until progression (all arms) and every 
8 weeks (+/- 14 days) until 2nd progression (Arm 2) 
Note: Scans that do not show progression (per sites’ local reads) must be submitted to the IROC 
for banking and quality check (submit within 30 days of scan acquisition). Central review will 
not be performed on these images. Scans that do show progression should be submitted for real 
time review in addition to storage and QC/QA purposes. (Please see time frames below.) 
For patients on Arms 1 and 3, protocol treatment should not be discontinued until central 
radiology review has confirmed progression.  Patients on Arm 2 should not proceed to 
bevacizumab until their first progression has been confirmed by central radiology review or 
discontinue bevacizumab treatment until second progression is confirmed by central radiology 
review. 
The 28 (+/-3) day scan must be submitted to the Imaging and Radiation Oncology Core in real 
time, but central review will not be performed. 
6.7.1 
Remote Central Review  
MRI (or CT) imaging data sets of pre- and post-surgery will be concurrently reviewed by 
the A071101 central review panel within no more than 72 hours after the post-surgery 
data receipt. Confirmation of ≥90% resection for this protocol will be based on the 
centralized review results and NOT on local assessment. 
MRI (or CT) imaging data sets providing evidence of radiographic disease progression as 
determined by the study site investigator will be concurrently reviewed by the A071101 
central review panel within no more than 72 hours after receipt of the scans.  
The Imaging and Radiation Oncology Core (IROC) will contact the A071101 central 
review panel, within 24 hours (except weekends and holidays) after post-surgery 
images being received, for scheduling a real-time remote review.  
The IROC notifies the participating site, Alliance Data Manager, and Agenus of the central 
review result within 24 hours after receiving the result from the central review panel. 
If there are any uncertainties or the participating site disagrees with the first central review 
result, a second review by either another central reviewer or an adjudicator (
 
) will be performed. This process may take additional 24-48 hours turn-
around time, and such review result will be used as the final decision for the outcome 
determinations. The Alliance A071101 trial committee renders final decisions of A071101 
patient inclusion or exclusion. Progression is defined in Section 13. 
For any such related questions, participating sites may directly contact the IROC instead 
of the central reviewer(s), via either the trial email at 
 or 
call at 
 
6.7.2 Data Archiving, Storage, and Submission 
MRI (or CT only if MRI is contraindicated) MUST be completed within 28 days pre-
surgery and within 48 hours post-surgery. The complete imaging data sets must be 


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submitted to the Imaging and Radiation Oncology Core (IROC) in digital DICOM format, 
within no more than 5 business days (pre-surgery) and within 24-48 hours (post-
surgery) upon the image acquisition is completed. BMP files, JPEG files, or hard copies 
(films) are not acceptable. 
The Adjunctive Data Form and the Central Review form—Resection or Central 
Review Form--Progression must be submitted to theIROC together with the entire MRI 
DICOM data sets.   Sites can obtain the study specific forms from the CTSU website. 
De-identify the patient data using institutional procedures to remove patient name and 
medical record number while preserving the patient ID number and protocol number of 
the Alliance trial. The de-identified digital images may be burned to a CD or transferred to 
a PC based system for further electronic data transfer purposes. 
Data should be electronically transferred to the Imaging and Radiation Oncology Core 
Lab by 1) Web Transfer or 2) FTP transfer: 
1) Web Transfer: 
Any PCs with Internet access and web browser (e.g., Internet Explorer, Mozilla Firefox) 
can be used to transfer DICOM images and other required files to the IROC through 
website upload.imagingcorelab.com.  The standard Web Transfer information will be 
provided separately through the specific trial e-mail 
 per 
the request by participating sites before their first data submission. 
2) FTP Transfer:  
Any FTP software can be used to initiate access to the secure FTP Server of the IROC The 
standard FTP access information will be provided separately through the specific trial e-
mail 
 per the request by participating sites before their 
first data submission. 
Send an e-mail notification to inform the Imaging and Radiation Oncology Core Lab at the 
specific trial email of the data submission once the data transfer is completed. 
3) Shipment/Mail (not recommended): 
If the above electronic data transfers cannot be achieved, the de-identified images in 
DICOM format can be burned to a CD, labeled with info of patient ID, study date, 
baseline/follow-ups on the CD cover, and mailed to the IROC via over-night shipment at: 
Imaging and Radiation Oncology Core Department of Radiology 
 
 
 
 
 
 
The IROC will notify site within 24 hours of the data receipt as well as within 72 hours of 
the quality check report via the specific trial email  
 


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Any questions or problems about the data transfer to the Imaging and Radiation Oncology 
Core Lab, email the IROC at the specific trial email, or call the IROC IT group at 
 for help. 
 
 


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7.0 
REQUIRED DATA   
Pre-Study Intervals  
To be completed within 28 DAYS before surgery (pre-registration): 
- History and physical including vital signs 
- Neurological examination 
- Performance status 
- MRI (or CT only if MRI is contraindicated) of brain with or without gadolinium per institutional 
guidelines 
 
To be completed DAY OF SURGERY: 
Collection of tissue for vaccine manufacture 
 Collection of tissue for future biomarker analyses as available per institutional standards  
To be completed within 48 HOURS following surgery:  
 MRI (or CT only if MRI is contraindicated) of brain with or without gadolinium per institutional 
guidelines to evaluate percentage of tumor resected 
To be completed 28 (+/- 3) days POST SURGERY: 
MRI (or CT only if MRI is contraindicated) of brain with or without gadolinium per institutional 
guidelines to evaluate localized bleeding prior to initiating treatment 
To be completed within 16 days prior to registration:  
Any baseline exams to evaluate eligibility  
Blood work 
Serum pregnancy test, if applicable


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Tests & Observations 
Pre-
registration 
Registration 
Day 1 each 
cycle *** 
Post-treatment 
follow up  
History * 
X 
X 
X 
 
Vital Signs1 
 
X 
X 
 
Weight2 * 
 
X 
X 
 
Physical Examination* 
X 
X 
X 
 
Neurological Examination* 
X 
X 
X 
 
Performance Status 
X 
 
X 
 
Serum Pregnancy Test3 
X  
X 
 
 
Injection Site Assessment 
 
 
X5 
 
Solicited Adverse 
Events/Abnormalities 
X 
 
X 
 
Laboratory Studies 
 
 
 
 
Clinical Lab Tests4 ** 
 
X 
X 
 
Urine protein 
 
X 
X6 
 
PT/PTT 
 
X 
X7 
 
Staging and Scans 
 
 
 
 
MRI 
X8 
X8 
X9 
 
Histological Confirmation of recurrent 
GBM 
 
X 
 
 
Survival/Disease Status 
Follow up/Event Monitoring 
 
 
 
 
X10 
Adverse Events: Other11 
 
 
X 
 
Sub-study 
 
 
 
 
PBMC 
See Sections 6.3 and 6.5 
Tissue Collection for Biomarkers 
See Sections 6.4 and 6.5 
1 
Vital signs to include pulse, temperature, blood pressure and respiratory rate to be completed before each 
dose of vaccine and 60 minutes (+/- 5 minutes) post each dose of vaccine. Vital signs to include pulse, 
temperature, blood pressure, and respiratory rate prior to every dose of bevacizumab. 
2 
It is not necessary to change the doses of bevacizumab due to changes in weight unless the calculated 
dose changes by ≥10%. 
3 
For women of childbearing potential only, to be completed at pre-registration and ≤ 16 days prior to 
beginning treatment 
4 
Includes:  
• 
Albumin, alkaline phosphatase, aspartate amino transferase (AST), alanine amino transferase 
(ALT), bilirubin [total], glucose, creatinine,  
• 
Hematology [red blood cell count, platelets, hematocrit, hemoglobin, white blood cell (WBC) 
count, plus WBC differential with absolute counts for neutrophils, eosinophils, basophils, 
lymphocytes, monocytes, and all labs must meet eligibility criteria.]  
• Labs do not need to be repeated at the first administration if completed within 28 days prior to 
administration 
5  
30 to 60 minutes after vaccine administration 
6 
Urine protein within 48 hours of day 1 of bevacizumab cycles only. 
7 
PT/PTT required at registration for all patients; repeat required only in patients on therapeutic doses of 
warfarin 
8 
MRI (or CT only if MRI is contraindicated) completed within 28 days prior to surgery, within 48 hours 
post-surgery, and 28 (+/- 3) days post-surgery. The post-surgical scan at 28 (+/- 3) days is to evaluate 
the patient for their localized bleeding status prior to initiating treatment across all arms. See Section 6.7 
for submission of images to IROC.  
9 
MRI (or CT only if MRI is contraindicated) completed every 8 weeks (4 cycles) from registration until 
progression (all arms) and every 8 weeks (4 cycles) until 2nd progression (Arm 2) 
10 
Every 8 weeks (+/- 14 days) until 18 months from surgery, then every 6 months (+/- 14 days) up to a 
maximum of 5 years. Event monitoring can include, but is not limited to 1) a phone call to the patient, 
2) contact with the last known local medical doctor, or 3) medical record review from a recent clinic 
visit (within about 2 weeks of the event monitoring time point. 


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11 
Grade 1 and 2 with attribution of at least possibly related and all grade 3, 4, and 5 adverse events are to 
be reported. 
* 
To be completed on the timepoints specified above or (-3) days. 
** 
To be collected on the timepoints specified above or (-1) day. 
***       For patients in Arm 2, this colum must be followed while receiving bevacizumab also. 
 
 
8.0 
TREATMENT PLAN 
Note: Kits for shipping tissue will be supplied by Agenus and should be ordered at time of IRB 
submission because they will be needed at time of surgery 
Surgery must be performed within 28 days from pre-registration.  
NOTE: Sites will be followed for appropriate tissue handling. 
 
Protocol treatment is to begin within 35 (+/-7) days   post-surgery. Questions regarding treatment 
should be directed to the Alliance Study Chair. 
Patients will be randomized 1:1:1 to one of three arms.  
It is acceptable for individual therapy doses to be delivered ≤ 24 hour (business day) window before 
and after the protocol defined date for a scheduled dose. In addition, patients are permitted to have a 
new cycle of therapy delayed for up to 5 days for major life events without this being considered a 
protocol violation; documentation to justify this delay should be provided.  
Although not required, patients may take acetaminophen prior to vaccine administration.  
NOTE: Once HSPPC-96 is drawn up into the syringe, the vaccine is stable for up to two hours at 
ambient temperature if immediate administration is not possible. 
8.1 
Arm 1, HSPPC-96 + concomitant bevacizumab  
(1 cycle=14 days)  
HSPPC-96 0.4mL intradermal (see Section 11.6 for details on intradermal administration) on 
days 1 and 8 of cycles 1 and 2, then on day 1 of each cycle, up to a maximum of 12 doses (10 
cycles), plus bevacizumab 10 mg/kg i.v. on day 1 of each cycle, until progression.   
HSPPC-96 should be administered at least 60 minutes prior to starting bevacizumab infusion. 
Note:  If HSPPC-96 treatment has ended but there is no evidence of disease progression, the 
patient should continue to receive bevacizumab at the specified dose until progression. 
8.2 
Arm 2, HSPPC-96 with bevacizumab at progression 
(1 cycle=14 days)   
HSPPC-96 0.4mL intradermal (see Section 11.6 for details on intradermal administration) on 
days 1 and 8 of cycles 1 and 2, then on day 1 of each cycle, up to a maximum of 12 doses (10 
cycles).  
At progression: bevacizumab 10mg/kg i.v. on day 1 of each cycle, until further progression.   
NOTE:  It is possible that HSPPC-96 vaccination may end prior to evidence of progression.  In 
this instance it is important to wait until there is confirmed evidence of progression before 
initiating treatment with bevacizumab.  
Upon confirmation of progression the patient should initiate bevacizumab within 7 to 42 days 
from the last dose of vaccine.  


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8.3 
Arm 3, Bevacizumab    
(1 cycle = 14 days) 
Bevacizumab 10mg/kg i.v. on day 1 of each cycle, until progression. 
9.0 
DOSE MODIFICATIONS AND MANAGEMENT OF TOXICITY 
There are no dose reductions for bevacizumab. Bevacizumab may be delayed or discontinued for 
toxicity as described below. 
9.1 
Hypersensitivity and/or Infusion Reactions 
Allergic or Infusion-Related Reactions 
• 
The initial bevacizumab dose should be administered over a minimum of 90 
minutes. If no hypersensitivity or infusion reactions occur, the second dose should 
be administered over a minimum of 60 minutes. If no hypersensitivity or infusion 
reactions occur with the second dose, the third and subsequent doses should be 
administered over a minimum of 30 minutes. 
• 
For grade 1 or 2 hypersensitivity reactions during bevacizumab infusion, interrupt 
bevacizumab until symptoms resolve to < grade 1, then resume bevacizumab at 
the shortest infusion rate tolerated. Manage symptoms according to institutional 
procedures. Subsequent infusion rates should be titrated as stated above. Consider 
premedication with antihistamines for subsequent doses. 
• 
Subjects who experience clinically evident bronchospasm (regardless of grade) 
should discontinue bevacizumab. Manage symptoms according to institutional 
procedures. 
For grade 3 or 4 allergic reactions considered related to bevacizumab, discontinue bevacizumab. 
Manage symptoms according to institutional procedures. Continue HSPPC-96. 
For grade 1 or 2 reactions considered related to HSPCC-96, manage symptoms according to 
institutional procedures. Continue on study. 
For grade 3 or 4 allergic reactions considered related to HSPCC-96, discontinue HSPCC-96.  
Manage symptoms according to institutional procedures. Continue bevacizumab. 
For grade 1 or 2 infusion-related reaction or cytokine release syndrome as characterized by back 
pain, headache, rigors, erythema, vasodilation, considered related to bevacizumab, decrease 
infusion rate to 50% of the previous rate. Manage symptoms according to institutional 
procedures. When symptoms improve to < grade 1, the infusion rate may be increased in 50% 
increments every 30 minutes. Subsequent infusions can be started at the planned rate. 
For grade 3 or 4 infusion-related reaction or cytokine release syndrome considered related to 
bevacizumab, discontinue bevacizumab. Manage symptoms according to institutional 
procedures. Continue HSPPC-96. 
For anaphylaxis considered related to bevacizumab, discontinue bevacizumab. Manage 
anaphylaxis according to institutional procedures.  Continue HSPPC-96. 
For anaphylaxis considered related to HSPPC-96, discontinue HSPPC-96.  Manage anaphylaxis 
according to institutional procedures. Continue bevacizumab. 
9.2 
Injection Site Reactions 
For grade 1 or 2 injection site reactions, continue on study; make sure to rotate injection sites as 
instructed in Administration section. Manage symptoms according to institutional procedures. 


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For grade 3 or 4 injection site reactions, discontinue HSPCC-96.  Continue bevacizumab. 
9.3 
Hypertension 
 
For grade 1 to 3 hypertension controlled with medication to <150/90 mmHg, continue 
bevacizumab.  
For persistent or symptomatic hypertension despite antihypertensive therapy, delay 
bevacizumab for up to 6 weeks until BP <150/90 mmHg. Continue HSPPC-96. Once 
bevacizumab is held 6 weeks, patient goes off protocol in Arms 2 and 3.  In Arm 1, the patient 
continues the vaccine, stops the bevacizumab, and continues on trial. 
For grade 4 hypertension, discontinue bevacizumab. Continue HSPCC-96. Once bevacizumab 
is held 6 weeks, patient goes off protocol in Arms 2 and 3.  In Arm 1, the patient continues the 
vaccine, stops the bevacizumab, and continues on trial. 
There are no dose modifications for hypertension for HSPPC-96. 
For signs and symptoms suggestive of reversible posterior leukoencephalopathy syndrome 
(RPLS) such as confusion, headache, seizures, and cortical blindness, hold bevacizumab for up 
to 6 weeks. Suspected RPLS should be investigated with MRI as described in Section 11.5. If 
diagnosis of RPLS is confirmed, bevacizumab should be permanently discontinued. If RPLS is 
ruled out via MRI, and signs and symptoms attributed to another cause, may resume 
bevacizumab. If bevacizumab is held or permanently discontinued for RPLS, continue HSPPC-
96. 
9.4 
Hemorrhage 
For grade 1 or 2 bleeding, restart bevacizumab once resolved to < grade 1. Once bevacizumab 
is held 6 weeks, patient goes off protocol in Arms 2 and 3.  In Arm 1, the patient continues the 
vaccine, stops the bevacizumab, and continues on trial. 
For grade 3 or 4 bleeding: Discontinue bevacizumab. Continue HSPPC-96. Once bevacizumab 
is held 6 weeks, patient goes off protocol in Arms 2 and 3.  In Arm 1, the patient continues the 
vaccine, stops the bevacizumab, and continues on trial. 
There are no dose modifications for hemorrhage (non-CNS) for HSPPC-96.  
9.5 
Venous Thromboembolic Events  
Grade 1, 2, 3 or asymptomatic grade 4: Delay bevacizumab. If the planned duration of full-
dose anticoagulation is <2 weeks, protocol treatment should be held until the full-dose 
anticoagulation period is over. If the planned duration of full-dose anticoagulation is >2 weeks, 
protocol treatment may be resumed during the period of full-dose anticoagulation if all the 
following criteria are met:  
• 
The patient must have an in-range INR (usually between 2 and 3) on a stable dose 
of warfarin (or other anticoagulant) prior to restarting therapy; 
• 
The patient must not have had a grade 3 or 4 hemorrhagic event while on study; and 
For recurrent/worsening venous thromboembolic events after resumption of treatment: 
Discontinue bevacizumab.  
For symptomatic grade 4: Discontinue bevacizumab. 
Once bevacizumab is held 6 weeks, patient goes off protocol in Arms 2 and 3.  In Arm 1, the 
patient continues the vaccine, stops the bevacizumab, and continues on trial. 


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9.6 
Arterial Thromboembolic Events 
Arterial thrombosis may include angina, myocardial infarction, stroke (CNS cerebrovascular 
ischemia), or other arterial thrombotic event. In the event of new or worsening grade 2 or any 
grade 3, or 4 arterial thrombotic event, treatment with bevacizumab should be discontinued.  
There are no dose modifications for arterial thromboembolic events for HSPPC-96. 
9.7 
Renal and Urinary Disorders:  Proteinuria 
See Appendix I for information regarding the calculation of UPC (urine protein to creatinine) 
ratio. 
For proteinuria of ≥2+: Confirm total urine protein with a 24-hour urine collection or urine 
protein to creatinine (UPC) ratio. For 2+ proteinuria, give scheduled bevacizumab while 
awaiting the results of the 24-hour collection or UPC ratio. For >2+ proteinuria, delay 
bevacizumab while awaiting results of the 24-hour urine collection or UPC ratio. Continue 
HSPPC-96. 
If monitoring urine protein with UPC, no confirmation is necessary.  
For urine protein 2-3.4 g/24 hours or UPC ratio 2-3.4: Delay bevacizumab until urine protein 
improves to <2g/24 hours or UPC <2. If bevacizumab is delayed more than 6 weeks due to 
proteinuria, discontinue bevacizumab. Continue HSPPC-96. 
For urine protein ≥ 3.5 g/24 hours or UPC ratio ≥3.5: Discontinue bevacizumab. Continue 
HSPPC-96. 
There are no dose modifications for proteinuria for HSPCC-96. 
9.8 
Fistula, Perforation involving any Organ, Bowel Obstruction, and Wound Dehiscence  
• 
For any grade perforation of any organ, GI leak, or any fistula: Discontinue 
bevacizumab. Continue HSPCC-96. 
• 
For any grade bowel obstruction requiring medical intervention: Hold 
bevacizumab therapy until complete resolution. Continue HSPCC-96. If surgery is 
required, the patient may restart bevacizumab after full recovery from surgery and 
after consultation with the study chair. 
• 
For wound dehiscence requiring medical or surgical intervention: Discontinue 
bevacizumab. Continue HSPCC-96. 
Once bevacizumab is held 6 weeks, patient goes off protocol in Arms 2 and 3.  In Arm 1, the 
patient continues the vaccine, stops the bevacizumab, and continues on trial. 
9.9 
Other Non-Hematologic Toxicity at Least Possibly Related to HSPCC-96 
• 
For other grade 1 or 2 non-hematologic toxicity, continue on protocol once 
toxicity is resolved to < grade 1. 
• 
For other grade 3 or 4 non-hematologic toxicity considered at least possibly 
related to HSPCC-96, discontinue HSPCC-96.  Continue bevacizumab. 
9.10 
Dose Modifications for Obese Patients 
There is no clearly documented adverse impact of treatment of obese patients when dosing is 
performed according to actual body weight. Therefore, all dosing is to be determined solely by 
actual weight without any modification unless explicitly described in the protocol. This will 
eliminate the risk of calculation error and the possible introduction of variability in dose 


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administration. Failure to use actual body weight in the calculation of drug dosages will be 
considered a major protocol deviation. Physicians who are uncomfortable with calculating doses 
based on actual body weight should recognize that doing otherwise would be a protocol violation 
10.0 CORRELATIVE SUB-STUDY  
The correlative science study must be offered to patients enrolled on A071101. 
A071101-ST1 will evaluate immune response and tissue markers in patients with resectable 
glioblastoma multiforme (GBM) receiving HSPPC-96. 
10.1 
Immune Response Study   
10.1.1 Background 
The impetus to treat GBM patients with immunotherapy has a clinical and scientific basis. 
Several groups have described a correlation between HIV-mediated immunosuppression 
and intracranial glial tumors [21, 22] [28, 29]. Immunosuppression in transplant recipients 
has also been implicated in the development of intracranial glioma [30]. In contrast, long-
term remission of malignant brain tumors secondary to postoperative infection has 
generated the hypothesis that a heightened immune status can confer some protection 
against intracranial tumor [31, 32]. Consistent with this hypothesis is the epidemiological 
observation that a significant allergy history lowers an individual’s lifetime risk for 
developing an intracranial glioma [33, 34]. Successful active immunotherapy for patients 
with glioma will require development of a specific peptide or polyvalent glioma vaccine. 
Serologic analysis of antigens using recombinant complementary DNA expression cloning 
(SEREX) has identified several tumor-associated antigens in other human cancers [35-42]. 
SEREX has also identified antigens capable of generating an immune response in patients 
with malignant glioma  [43, 44]. An antibody response against one of these antigens, PHF3, 
is associated with an increase in survival. These results represent the first concrete evidence 
that immune responses generated against CNS tumor antigens are potentially protective. 
Moreover, recent phase I and II trials evaluating the immunogenicity of HSPPC-96 in 
vaccinated GBM patients have shown the increase of IFNg secreting T cells and NK cells 
post-vaccination. (Section 1.3) 
10.1.2 Objectives 
The objective is to demonstrate whether patients with an immune response to HSPPC-96 
will have an improved survival outcome in comparison to those patients who do not. 
10.1.3 Methods 
A panel of assays will be performed to characterize the immune response generated to 
HSPPC-96 given in combination with bevacizumab.  This includes the following tests:  
phenotyping of PBMC surface markers, intracellular cytokine staining to detect 
proinflammatory cytokines at the protein level, qPCR to detect cytokines at the mRNA 
level and CFSE dilution to assay T cell proliferation. Re-stimulation assays will be 
performed using autologous vaccine on blood isolated before and after vaccination.  
Recombinant gp96 will be used as a control [22]. The expectation is that patients who 
demonstrate a robust immune response will live longer than those who do not. 
10.1.4 Statistical Design  
This exploratory endpoint is to examine the relationship between immune response to 
HSPPC-96 and overall survival.  The sample size for this correlative study is driven by the 
feasibility of obtaining high quality PBMCs for assessing immunological parameters.   


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Based on the prior clinical experience with HSPPC-96 in GBM it is likely that at least 50% 
of patients will be eligible based on the quality of the PBMCs obtained. We expect the 
relationship between immune response and survival will be similar between Arms 1 and 2, 
thus the analysis will be conducted with two arms pooled together.  The expected accrual 
rate for the whole study is about 11 patients per month (Arms 1:2:3 = 1:1:1) according to 
Section 15.5. Therefore, it can be assumed that an average of 3-4 patients per month (50% 
of patients enrolled in Arms 1 and 2) will be enrolled into this correlative study. Survival 
curves will be estimated for immune responders vs. non-responders separately using 
Kaplan-Meier methods and be compared between the two groups using log-rank tests. We 
expect to observe about 100 events (death) by the time of analyzing the primary endpoint 
within Arms 1 and 2 according to Section 15.4. With a total of roughly 48 events, (assume 
patients enrolled in Arms 1 and 2 who participate in this correlative study provides 50% of 
expected events), we have 80% power in detecting a hazard ratio of 1.85 assuming a 1-
sided type I error rate of 0.1.  
10.2 
Tissue Marker Study 
10.2.1 Background 
It has been published that over expression of B7H1 secondary to activation of PI3 kinase 
results in immunoresistant mechanisms at the local tumor site.  It is therefore postulated 
that patients having low expression levels of B7H1 and PI3 kinase may have a better 
immune response and potentially a better overall response as indicated by improved overall 
survival outcomes versus those patients who have high levels of these markers 
10.2.2 Objectives 
The objective of this study is to explore the expression of B7-H1 protein expression and 
P13K pathway activation at the tissue level before treatment and at recurrence and correlate 
it with the extent of immune infiltration.  We hope to determine that patients with lower 
expression of B7H1 and less PI3 kinase activation at baseline develop a stronger immune 
response post-therapy and show a longer survival. Tissue blocks obtained at the time of 
surgery will be prepared and stained for expression of B7-H1 protein as well as 
downstream markers of PI3K activation (i.e. p-Akt and p-S6K).  The extent of staining will 
be graded and then compared to clinical endpoints. The expectation is that the extent of 
B7-H1 expression and extent of PI3K activation will correlate inversely with clinical 
outcome on the vaccine arms of the study, with no effect on the non- vaccine arm. [45] In 
addition tissue blocks will be stained for T-cell markers (CD3, CD8, CD4) and assessed 
for extent of infiltration, which will be and compared to clinical outcome.  For those 
patients who undergo biopsy or resection after vaccination at time of progression, tissue 
will be collected and analyzed for T-cell infiltrate, and compared to originally tumor tissue 
that was resected at time of surgery for enrollment. 
10.2.3 Methods 
Immunohistochemistry will be performed using B7-H1 specific antibodies as well as 
antibodies specific for phospho-Akt and phosphor-S6Kinase. B7-H1 is an immunoresistant 
protein that is regulated post-transcriptionally by the PI(3)K pathway.  Activation of PI3K 
can be measured by staining for 2 downstream targets phospho-Akt and phosphor-
S6Kinase. 
 10.2.4 Statistical Design 
There is no formal statistical design for this correlative study as it is highly exploratory.  
The endpoint is to explore a potential correlation between B7H1 and PI3 kinase expression 


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levels in baseline tumor tissue, corresponding immune response and overall survival.  
Although tumor tissue samples should be taken on all patients, exploration of this 
correlative measure requires that patients undergo immune response assessments.  
Therefore, approximately 50% of patients enrolled in Arms 1 and 2 (148*50%=74 patients) 
should constitute the sample size for this study.  The monthly accrual rate is the same as 
noted for the immune response assessment. To explore the correlation between biomarkers 
expression levels at baseline with respect to immune response, the alteration in biomarkers 
throughout the study will be summarized descriptively and graphically to evaluate how 
these values changed after the vaccine. Baseline expression levels will be compared 
between responder vs. non-responders using two-sample t-tests (or Wilcoxon rank sum 
tests as needed) as well as side-by-side boxplots. Assume 40% of patients are responders, 
a sample size of 74 will provide us at least 80% power to detect an effect size of 0.67 with 
a two-sided type I error rate of 0.05. We will also explore dichotomized versions of the 
biomarkers and assess their relationship to response using Fisher exact test as well as bar 
graphs as appropriate. We will have at least 80% power to detect a difference of 35% (25% 
vs. 60%) assuming a response rate of 40%, and a two-sided type I error rate of 0.05. We 
will also explore the impact of these biomarkers in the univariate and multivariable setting 
on response using logistic regression model. Cox proportional hazard models will be used 
to explore the relationship between survival and baseline biomarker values. Power 
estimation will be similar to that specified in Section 10.1.4.  
 11.0 DRUG FORMULATION, AVAILABILITY, AND PREPARATION 
11.1 
Qualified personnel 
Personnel who are familiar with procedures that minimize undue exposure to themselves and to 
the environment should undertake the preparation, handling, and safe disposal of 
chemotherapeutic agents in a self-contained, protective environment. 
11.2 
Unused agents 
Discard unused portions of injectable chemotherapeutic agents that do not contain a 
bacteriostatic agent or are prepared with unpreserved diluents (i.e., Sterile Water for Injection 
USP or 0.9% Sodium Chloride for Injection USP) within eight hours of vial entry to minimize 
the risk of bacterial contamination.   
11.3 
Bevacizumab dose rounding 
The total administered dose of bevacizumab may be rounded up or down within a range of 5% 
of the actual calculated dose. 
11.4 
Weight changes 
It is not necessary to change the doses of bevacizumab due to changes in weight unless the 
calculated dose changes by ≥10%. 
11.5 
Bevacizumab (rhuMAb VEGF, Avastin®) (NSC #704865) 
Bevacizumab is a recombinant humanized anti-VEGF monoclonal antibody, consisting of 93% 
human and 7% murine amino acid sequences. The agent is composed of human IgG framework 
and murine antigen-binding complementarity-determining regions. Bevacizumab blocks the 
binding of vascular endothelial growth factor (VEGF) to its receptors resulting in inhibition of 
angiogenesis. 
Availability 
Commercially available. 


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Bevacizumab is supplied as a clear to slightly opalescent, sterile liquid for parenteral 
administration, in 4 mL and 16 mL vials, at a concentration of 25 mg/ml. Each vial also contains 
phosphate, trehalose, polysorbate 20, and Sterile Water for Injection USP. 
Storage and Stability 
Intact vials of bevacizumab should be stored in a refrigerator (2° to 8°C) and should remain 
refrigerated until just prior to use. Do not freeze. Do not shake. The sterile single use vials 
contain no antibacterial preservatives; therefore, vials should be discarded eight hours after 
initial entry. Solutions diluted for infusion may be stored in the refrigerator for up to 8 hours. 
Preparation 
Vials contain no preservative and are intended for single use only. The calculated dose should 
be diluted in 100 mL of 0.9% Sodium Chloride for Injection. Once diluted in 0.9% Sodium 
Chloride for Injection, the bevacizumab solution must be administered within 8 hours. 
Administration 
Bevacizumab is administered as an intravenous infusion. The initial dose should be administered 
over a minimum of 90 minutes. If no adverse reactions occur after the initial dose, the second 
dose should be administered over a minimum of 60 minutes. If no adverse reactions occur after 
the second dose, all subsequent doses should be administered over a minimum of 30 minutes. If 
infusion-related adverse reactions occur, all subsequent infusions should be administered over 
the shortest period that was well tolerated. 
Toxicities 
Hypertension: Hypertension is commonly seen with bevacizumab and oral medications have 
been used to manage the hypertension when indicated. Grade 4 and 5 hypertensive events are 
rare. Clinical sequelae of hypertension are rare but have included hypertensive crisis, 
hypertensive encephalopathy, and reversible posterior leukoencephalopathy syndrome (RPLS). 
RPLS may include signs and symptoms of headache, altered mental function, seizures, and 
visual disturbances/ cortical blindness and requires treatment, which should include control of 
hypertension, management of specific symptoms, and discontinuation of bevacizumab. 
Reversible posterior leukoencephalopathy syndrome (RPLS) or similar leukoencephalopathy 
syndrome: RPLS or clinical syndromes related to vasogenic edema of the white matter have 
been recently reported in association with bevacizumab therapy. These syndromes have been 
seen in < 1% of patients to date. Clinical presentations are variable and may include altered 
mental status, seizure and cortical visual deficit. HTN is a common risk factor and was present 
in most (though not all) patients on bevacizumab who developed RPLS. MRI scans are key to 
diagnosis and typically demonstrate vasogenic edema (hyperintensity in T2 and FLAIR images 
and hypointensity in T1 images) predominantly in the white matter of the posterior parietal and 
occipital lobes; less frequently, the anterior distributions and the gray matter may also be 
involved. RPLS should be in the differential diagnosis in patients presenting with unexplained 
mental status change, visual disturbance, seizure or other CNS findings. RPLS is potentially 
reversible, but timely correction of the underlying causes, including control of BP and 
interruption of the offending drug, is important in order to prevent progression to irreversible 
tissue damage. 
Neutropenia: When combined with chemotherapy, bevacizumab is reported to increase the risk 
of neutropenia over that of chemotherapy alone.  


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Proteinuria: Proteinuria ranging from asymptomatic abnormal urinalysis to nephrotic syndrome 
has been described in 10% or more of patients receiving bevacizumab. See Section 9.7 for dose 
modifications for proteinuria.  
Thromboembolic events: Both venous and arterial thromboembolic (TE) events, ranging in 
severity from catheter-associated phlebitis to fatal, have been reported in patients treated with 
bevacizumab in the colorectal cancer (CRC) trials and, to a lesser extent, in patients treated with 
bevacizumab in NSCLC and breast cancer trials. In the phase III pivotal trial in metastatic CRC, 
there was a slightly higher rate of venous TE events that was not statistically significant in 
patients treated with bevacizumab plus chemotherapy compared with chemotherapy alone (19% 
vs. 16%). There was also a higher rate of arterial TE events (3% vs. 1%) such as myocardial 
infarction, transient ischemia attack, cerebrovascular accident/stroke and angina/unstable 
angina. A pooled analysis of the rate of arterial TE events from 5 randomized studies (1745 
patients) showed that treatment with chemotherapy plus bevacizumab increased the risk of 
having an arterial TE event compared with chemotherapy alone (3.8% vs. 1.7%, respectively). 
Furthermore, subjects with certain baseline characteristics (age ≥ 65 years and/or a history of a 
prior arterial TE event) may be at higher risk of experiencing such an event.  
Aspirin is a standard therapy for primary and secondary prophylaxis of arterial thromboembolic 
events in patients at high risk of such events, and the use of aspirin ≤ 325 mg daily was allowed 
in the five randomized studies discussed above. Use of aspirin was assessed routinely as a 
baseline or concomitant medication in these trials, though safety analyses specifically regarding 
aspirin use were not preplanned. Due to the relatively small numbers of aspirin users and arterial 
thromboembolic events, retrospective analyses of the ability of aspirin to affect the risk of such 
events were inconclusive. However, similar retrospective analyses suggested that the use of up 
to 325 mg of aspirin daily does not increase the risk of grade 1-2 or grade 3-4 bleeding events, 
and similar data with respect to metastatic colorectal cancer patients were presented at ASCO 
2005. Further analyses of the effects of concomitant use of bevacizumab and aspirin in 
colorectal and other tumor types are ongoing. 
Gastrointestinal perforation: Patients with metastatic carcinoma may be at increased risk for the 
development of gastrointestinal perforation when treated with bevacizumab and chemotherapy. 
Bevacizumab should be permanently discontinued in patients who develop gastrointestinal 
perforation. A causal association of intra-abdominal inflammatory process and gastrointestinal 
perforation to bevacizumab has not been established. Nevertheless, caution should be exercised 
when treating patients with intra-abdominal inflammatory processes with bevacizumab. 
Gastrointestinal perforation has been reported in trials in non-colorectal cancer populations (e.g., 
ovarian, renal cell, pancreas, and breast) and may be higher in incidence in some tumor types. 
Wound healing complications: Wound-healing complications such as wound dehiscence have 
been reported in patients receiving bevacizumab. The appropriate interval from discontinuation 
of bevacizumab to subsequent elective surgery required to reduce the risk of impaired wound 
healing has not been determined. Decision on such an interval should take into consideration 
the half-life of bevacizumab (approximately 20 days). It is generally recommended that 
bevacizumab should be discontinued at least 4-8 weeks prior to major elective surgery. In 
addition, bevacizumab should not be restarted until at least 4 weeks after major surgery provided 
that the wound has adequately healed. 
Hemorrhage: Overall, grade 3 and 4 bleeding events were observed in 4.0% of 1132 patients 
treated with bevacizumab in a pooled database from eight phase I, II, and III clinical trials in 
multiple tumor types. The hemorrhagic events that have been observed in bevacizumab clinical 
studies were predominantly tumor-associated hemorrhage (see below) and minor 
mucocutaneous hemorrhage.  


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Tumor-associated hemorrhage was observed in phase I and phase II bevacizumab studies. Six 
serious events, of which 4 had fatal outcome, were observed in a phase II trial of patients with 
non-small cell lung cancer receiving bevacizumab. These events occurred suddenly and 
presented as major or massive hemoptysis in patients with either squamous cell histology and/or 
tumors located in the center of the chest in close proximity to major blood vessels. In five of 
these cases, these hemorrhages were preceded by cavitation and/or necrosis of the tumor. 
Tumor-associated hemorrhage was also seen rarely in other tumor types and locations, including 
central nervous system (CNS) bleeding in a patient with hepatoma with occult CNS metastases 
and continuous oozing of blood from a thigh sarcoma with necrosis.  
Across all bevacizumab clinical trials, mucocutaneous hemorrhage has been seen in 20%-40% 
of patients treated with bevacizumab. These were most commonly grade 1 epistaxis that lasted 
less than 5 minutes, resolved without medical intervention and did not require any changes in 
bevacizumab treatment regimen. There have also been less common events of minor 
mucocutaneous hemorrhage in other locations, such as gingival bleeding and vaginal bleeding.  
Congestive heart failure: CHF has been reported in bevacizumab clinical trials and may be 
increased in incidence in patients with prior exposure to anthracyclines or prior irradiation to 
the chest wall. In a phase III trial (AVF2119g) of capecitabine with or without bevacizumab for 
metastatic breast cancer, 7 subjects (3.1%) who received capecitabine plus bevacizumab 
developed clinically significant CHF compared with 2 subjects (0.9%) treated with capecitabine 
alone; of note, all subjects in this trial had had prior anthracycline treatment. In addition, 2 
subjects had a left ventricular ejection fraction < 50% at baseline and 2 others had prior left 
chest wall irradiation. A recently published phase II study in subjects with refractory acute 
myelogenous leukemia reported 5 cases of cardiac dysfunction (CHF or decreases to <40% in 
left ventricular ejection fraction) of 48 subjects treated with sequential cytarabine, mitoxantrone, 
and bevacizumab. All but one of these subjects had significant prior exposure to anthracyclines 
as well. Other studies are ongoing in this patient population. 
Osteonecrosis of the jaw: There are several reports in the literature suggesting that bevacizumab 
increases the likelihood of osteonecrosis of the jaw in patients receiving bisphosphonates, and 
that bevacizumab may cause osteonecrosis of the jaw in patients not receiving bisphosphonates. 
This effect is thought to be related to inhibition of angiogenesis. 
For a comprehensive list of adverse events and potential risks (CAEPR), see Section 16.3. See 
the most current version of the package insert for additional information. 
11.6 
 Heat Shock Protein-Peptide Complex-96 Vaccine (HSPPC-96)(NSC #725085) (Alliance  
IND # 15380) 
See the Investigator Brochure for additional information.   
To obtain a copy of the IB, contact 
. 
HSPPC-96 is an autologous tumor-derived vaccine under clinical investigation for the treatment 
of a variety of cancer types. It is composed of the 96-kDa heat shock protein gp96 in complex 
with autologous tumor-derived peptides. The gp96 in HSPPC-96 is a highly conserved, 
abundant, nonpolymorphic stress protein found in every cell type of the body. gp96 isolated 
from normal or tumor tissues is found in complex with a diverse repertoire of peptides, which 
are specific to the tissue of origin. Published studies in mouse tumor models have shown that 
HSPPC-96 confers protective immunity only to the tumor from which it is derived and not to 
antigenically distinct tumors. The specific immunogenicity of the HSP preparations can be 
attributed to the unique repertoire of antigenic peptides that exists in different cancers.   


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When injected into the host, HSPPC-96 interacts with antigen presenting cells (APCs) such as 
macrophages, dendritic cells or Langerhans cells, which take up HSPPCs via HSP receptors, 
including CD91. Once internalized by APCs, the peptides chaperoned by the HSPs are 
transferred to major histocompatibility complex (MHC) class I and II molecules in intracellular 
compartments and subsequently expressed at the cell surface.  The cells then migrate to lymph 
nodes, where the complexes are processed so that the peptides chaperoned by the HSPs are re-
presented to naive T cells. The T cells recognize the peptides and, as a result, are stimulated by 
them. Vaccination with HSPPCs thus elicits both a CD8+ and CD4+ T-cell response targeting 
potentially all relevant tumor antigens. The interaction of HSPs with receptors on APCs also 
leads to activation of various components of innate immunity, including cytokine and 
chemokine release by macrophage and dendritic cells, maturation of dendritic cells and 
activation of natural killer cells, as shown in both murine and human systems. Tumor immunity 
is largely mediated by T cells and the ability of HSPPC-96 to stimulate both T-cell arms to 
recognize a large variety of tumor antigens, coupled with the ability to activate innate immune 
responses, uniquely positions the product among other cancer vaccine strategies. 
Availability 
HSPPC-96 will be prepared by Agenus, Inc. and shipped directly to sites within approximately 
2 weeks of Agenus’ receipt of tumor tissue for patients randomized to Arms #1 and #2. HSPPC-
96 vaccine, although produced for patients randomized to Arm #3, will not be shipped to sites. 
Vaccine produced for patients randomized to Arm #3 will be used for quality evaluation and 
will not be available for clinical use. 
HSPPC-96 is supplied in a single-use vial as a clear, colorless solution. Each vial contains 25 
µg of HSPPC-96 in a solution of 9% sucrose-potassium phosphate for intradermal (ID) 
injection. The total volume of each vial of HSPPC-96 is 0.47 mL. The total volume that should 
be administered is 0.4 mL. 
Ordering HSPPC-96 Vaccine from Agenus 
After randomization, the site must complete the Vaccine Order Form and send it back to Agenus 
either by fax (781-674-4260) or by email as a scanned copy to A071101@Agenusbio.com 
indicating whether or not vaccine should be shipped.   
The site should check “Yes, please ship vaccine” for patients randomized to study Arms #1 or 
#2 and “No, please do not ship” for patients randomized to study Arm #3. Additionally, in rare 
instances where vaccine has been produced, but the patient has failed other post-surgical 
eligibility criteria check “No, please do not ship” on the form and identify in the comments 
section that the patient is a screen failure.  
HSPPC-96 Vaccine Label 
Each vial of vaccine vial is labeled with the batch number, patient study number, patient 
initials, and patient date of birth (DOB).  
 
Storage and Stability 
Following successful production of HSPPC-96 vaccine at Agenus, vials are shipped (for those 
patients randomized to Arms #1 and #2) to the clinical site on dry ice and must be stored at -
80°C  ± 20°C until administration to the patient.  Keep the shipping kit until the patient has 
completed vaccine therapy, for return of any unused or expired vaccine. 
Any unused or expired vaccine remaining after a patient has completed vaccine treatment should 
be returned to Agenus. See Appendix VI  


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NOTE: Once HSPPC-96 is drawn up into the syringe, the vaccine is stable for up to two hours 
at ambient temperature if immediate administration is not possible. 
Preparation  
Withdraw the vial from the freezer and roll gently between two fingers until it is completely 
thawed.  Withdraw 0.4 mL, using a TB or insulin syringe. 
Administration 
The route of administration is intradermal injection. Using a 25 g needle, inject into 1 site or 
into 2 adjacent sites (0.2 mL each) a few centimeters apart. Appropriate sites for vaccination 
include the anterior deltoid regions, subclavicular region bilaterally, and medial inguinal regions 
of the upper thighs. Do not administer HSPPC-96 to areas distal to lymph node basins that have 
been resected or in areas just distal to a surgical scar. Rotate the injection sites so injections are 
not repeated at the same site at 2 consecutive administrations and utilize all potential sites for 
the patient before repeating injections at a previously used injection site. Syringes with slip-tip 
detachable needles or luer hubs with greater than 0.1 mL dead space should not be utilized. See 
instructions and diagram on the CTSU website. 
Toxicities 
 
Local injection-site reactions can occur and are usually of minimal extent and mild severity. If 
a local injection-site reaction requires medical attention, the investigator can provide local 
therapy as needed. Occasional systemic toxicities have been reported that have been mild in 
severity. The most frequently reported events include injection site reactions of erythema and 
induration, fatigue, nausea, pyrexia, headache, back pain, arthralgia, constipation, and asthenia.  
For a complete list of events, refer to the IB. 
12.0 ANCILLARY THERAPY   
12.1 
Supportive Care 
Patients should receive full supportive care, including anti-seizure medications, transfusions of 
blood and blood products, antibiotics, antiemetics, etc., when appropriate. The reason(s) for 
treatment, dosage, and the dates of treatment should be recorded on treatment form. 
12.2 
Hormones or Other Agents 
Treatment with hormones or other chemotherapeutic agents may not be administered except for: 
no more than 16 mg dexamethasone (or equivalent) to control symptoms of cerebral edema and 
mass effect and should be discontinued as soon as possible; hormones administered for non-
disease-related conditions (e.g., insulin for diabetes); and intermittent use of dexamethasone as 
an antiemetic.  
12.3 
Palliative RT 
Palliative radiation therapy may not be administered. 
12.4 
Growth Factors 
The following guidelines are applicable unless otherwise specified in the protocol: 
12.4.1 Epoetin (EPO) 
Use of epoetin in this protocol is prohibited. 


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12.4.2 Filgrastim (G-CSF) and sargramostim (GM-CSF) 
Filgrastim (G-CSF), pegfilgrastim and sargramostim (GM-CSF) treatment may not be 
used. 
12.5 
Surgery 
Patients who require surgery during protocol treatment may proceed as such, unless the surgery 
involves resection of GBM. Bevacizumab and/or HSPCC-96 should be held prior to and after 
surgery, at the discretion of the treating physician, for a maximum of 6 weeks.  
If the patient requires an interruption of > 6 weeks, then they will be removed from protocol 
therapy.  
13.0 CRITERIA FOR PROGRESSION  
Patients are followed every 8 weeks until progression.  Standard criteria for response do not apply as 
complete surgical resections of tumor tissue are being performed.  Further, in considering the 
purported mechanism of action of HSPPC-96 it may be difficult to differentiate true progression from 
pseudoprogression (treatment effect); therefore, the definition of progression provided below takes 
this specific protocol setting and the investigational therapy being tested into account.   
In Arm 2, these criteria for progression serve as a trigger to stop HSPPC-96 and start patients on 
bevacizumab.  Determination of subsequent progression after initiation of bevacizumab post-
progression on HSPPC-96 alone should follow the same set of criteria. 
Progressive disease post-baseline is defined as one or more of the following: 
1. New contrast-enhancing lesion outside of radiation field on decreasing, stable, or increasing doses 
of corticosteroids.  
2. Increase by > 50% (modified from >25% according to published RANO criteria) enhancement 
from the first post-surgical scan, or a subsequent scan with smaller tumor size, and the scan 8 weeks 
or later on stable or increasing doses of corticosteroids.  
3. Clinical deterioration not attributable to concurrent medication or comorbid conditions is sufficient 
to declare progression on current treatment.  
4. For patients receiving bevacizumab therapy, significant increase in T2/FLAIR non-enhancing 
lesion may also be considered progressive disease. The increased T2/FLAIR must have occurred with 
the patient on stable or increasing doses of corticosteroids compared with baseline scan or best 
response after initiation of therapy and not a result of comorbid events (e.g., ischemic injury, 
infection, seizures, postoperative changes, or other treatment effects). 
14.0 REMOVAL OF PATIENTS FROM PROTOCOL THERAPY   
14.1 
Duration of Treatment 
14.1.1 Free from Evidence of Disease:  
Continue treatment at the protocol specified doses and regimens as directed based on the 
randomization arms until the appearance of disease progression per Section 13. 
14.1.2 Disease Progression:  
Evidence of disease progression as per Section 13 will be cause for either removal from 
protocol therapy or trigger a patient to stop one therapy and transition to another depending 
upon the randomization arm.  See below for specific details.  Document details including 
tumor measurements on the appropriate form. 


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Patients randomized to Arms 1 or 3 (HSPPC-96+ concomitant bevacizumab (Arm 1) or 
bevacizumab alone (Arm 3)) will be removed from protocol therapy at the point of disease 
progression. 
Patients randomized to Arm 2 (HSPPC-96 followed by bevacizumab at progression) will 
be removed from protocol therapy with HSPPC-96, if they still had remaining vaccinations 
available at the point of progression, and will begin treatment with bevacizumab until 
further progression. 
14.2 
Extraordinary Medical Circumstances:  
If, at any time the constraints of this protocol are detrimental to the patient's health and/or the 
patient no longer wishes to continue protocol therapy, protocol therapy shall be discontinued. In 
this event: 
• 
Notify the Study Chair. 
• 
Document the reason(s) for discontinuation of therapy.  
• 
Follow the patient for survival until death.  
15.0 STATISTICAL CONSIDERATIONS 
15.1  Study overview  
This study will be a randomized phase II trial in patients with surgically resectable recurrent 
GBM. The study includes three arms: Arm 1 - HSPPC-96 with concomitant bevacizumab, Arm 
2 – HSPPC–96 followed by bevacizumab at progression, and Arm 3 – bevacizumab alone (the 
control arm). A dynamic allocation procedure will be used to allocate an equal number of 
patients to different arms (1:1:1). This procedure will balance the marginal distributions of the 
stratification factors between arms. The stratification factors that will be used are age (< 55 vs. 
≥55 years old), KPS (70 vs. 80-100) and the number of HSPPC-96 vials produced for the patient 
(4 or 5 vs. 6 or more). 
15.2 
Objectives 
15.2.1 Primary Objective 
To assess whether HSPPC-96 administered in combination with bevacizumab (given either 
concomitantly with HSPPC-96 or at the point of progression) improves survival overall 
compared to bevacizumab alone in patients with surgically resectable recurrent GBM  
15.2.2 Secondary Objectives  
• 
To evaluate the safety and tolerability of HSPPC-96 when given in combination with 
bevacizumab 
• 
To evaluate progression free survival of HSPPC-96 when given in combination with 
bevacizumab (given either concomitantly with HSPPC-96 or at the point of progression) 
• 
To select an arm with better overall survival between two HSPPC-96 plus bevacizumab 
arms given the combined arm proved to extend the survival as compared to the bevacizumab 
alone arm. 
15.3 
Endpoints 
15.3.1 Primary Endpoint  
The primary endpoint is overall survival (OS), which is defined as the date from study 
registration to the date of death, due to any cause.  


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15.3.2 Secondary Endpoint  
Time to progression free survival: which is defined as the date from study registration to 
the date of first observation of disease progression or death due to any cause (whichever 
comes first). 
15.4  Sample Size Derivation 
The primary objective of this study is to demonstrate that patients with recurrent GBM 
randomized to HSPPC-96 plus bevacizumab arms – either received concomitantly (Arm 1) or 
given at the time of progression (Arm 2) - have improved overall survival as compared to 
patients who were randomized to bevacizumab alone arm (Arm 3). In other words, Arms 1 and 
2 will be combined and then compared to Arm 3 to explore the additive effect of HSPPC–96. 
Based on previous reports, the median OS for recurrent GMB patients treated on bevacizumab 
alone is about 9 months. A sample size of 165 patients (55 per arm) will yield an 85% power to 
detect a 36% decrease in the hazard rate using a one-sided logrank test with a false positive rate 
of 0.1. This is equivalent to a 56% increase in the median survival time from 9 months to 14 
months. Sample size calculation is computed based on an accrual rate of 5 patients per month, 
and a minimum of 12 months follow up period. The required number of events will be 129. 
15.5  Accrual and study duration 
The study will be opened to all sites within The Alliance for Clinical Trials in Oncology (the 
Alliance) through the CTSU mechanism. We anticipate enrolling about 5 eligible recurrent 
GBM patients per month, and the accrual period for the study will be about 33 months and the 
total study duration will be about 45 months. We anticipate pre-registering 254 patients to reach 
a maximum accrual of 165 patients required for the analysis. 
15.6  Interim analysis 
One interim futility analysis will be applied when 50% of events (65deaths) have occurred 
(approximately 2 years after the start of the trial under null hypothesis). Rho family (rho = 2) β 
spending function was used to determine the boundary for the futility interim look. If the hazard 
ratio (experimental arm vs. control armassociated with the treatment covariate in the Cox 
proportional hazards model (adjusted for stratification factors) is> 1.026, the alternative 
hypothesis will be rejected and we will conclude there is little chance of demonstrating the 
combined arm is superior to the control arm and we may recommend terminating the trial.  
15.7 
Analysis Plan for Primary and Secondary Endpoints 
15.7.1 Primary Endpoint 
 
Efficacy analysis will be based on the intention-to-treat principle with all eligible patients 
belonging to the treatment arm to which they were randomized. Patients who have not died 
at the primary analysis time will be censored at the time of last known alive. The 
distribution of overall survival for combined and the control arms will be estimated using 
the Kaplan-Meier method. The hazard ratios and median survivals will be estimated with 
their 95% confidence intervals. The Cox proportional hazards model will be used to assess 
whether the distribution of overall survival times differ with respect to treatment regimen 
having adjusted for the stratification factors (age, KPS and number of vials produced). The 
corresponding p-value associated with the treatment covariate will be compared to the 
nominal p-value to make the conclusion regarding the primary efficacy comparison.  
If the primary efficacy analysis indicates that the combined arm extend the overall survival 
significantly as compared to the bevacizumab alone arm, two HSPPC-96 plus bevacizumab 
arms will be compared to each other to select an arm for following up phase III study using 


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the pick-the-winner method. By the time the primary analyses were completed, we expect 
to observe about 82 events (deaths) between the two experimental arms, which will provide 
about 80% likelihood of selecting the better arm if one arm is superior to the other by 60% 
in median OS (11 vs. 17.6 months), at one-sided significance level of 0.1. 
15.7.2 Secondary Endpoints 
• 
Progression free survival: progression free survival (PFS) will be estimated using 
the Kaplan-Meier method. The hazard ratios and median PFSs will be estimated 
with their 95% confidence intervals, and the distribution of PFS will be compared 
between arms using Cox models.  
• 
Adverse events: a secondary objective of the trial is to further characterize the 
safety and tolerability of HSPPC-96 in combination with bevacizumab. As per NCI 
CTCAE version 5.0, the phrase “treatment-related adverse event” is defined as an 
adverse event that is classified as either “possibly,” “probably,” or “definitely 
related” to study treatment. The maximum grade for each type of treatment-related 
adverse event will be recorded for each patient, and frequency tables for each arm 
will be reviewed to determine patterns. In addition, the number and percentage of 
patients in the treatment group who discontinued HSPPC-96 treatment due to an 
adverse event will be presented, together with the number and percentage of patients 
who died due to an adverse event. In addition, we will review all adverse event data 
that is graded as 3, 4, or 5 and classified as either “unrelated” or “unlikely to be 
related” to study treatment in the event of an actual relationship developing. 
Adverse events and treatment-related adverse events will be evaluated using all 
patients who have received any study treatment as well as summarizing those who 
have been included in the efficacy analyses. Treatment-related adverse events will 
be tabulated for each arm and will be compared among arms using Chi-Square or 
Fisher’s Exact tests. 
 
15.7.3 Adverse Event Stopping Rule 
The stopping rule specified below is based on the knowledge available at study 
development.  We note that the Adverse Event Stopping Rule may be adjusted in the event 
of and in consideration of newly acquired information regarding the adverse event profile 
of the treatment(s) under investigation.  With each arm, a specific interim toxicity analysis 
will be performed once at least 10 evaluable patients have been followed for 3 months 
(accrual will not stop during this three-month time period).  Accrual will be temporarily 
suspended if at any time we observe events considered at least possibly related to study 
treatment (i.e. an adverse event with attribute specified as  “possible”, “probable”, or 
“definite”) that satisfy either of the following: 
• 
If 2 or more patients in the first 10 evaluable patients (or 20% or more 
after first 10 patients have been accrued) experience a grade 4 or higher 
non-hematologic adverse event. 
• 
If 2 or more patients in the first 10 evaluable patients (or more than 20% 
after 10 patients have been accrued) experience a grade 3 or higher CNS 
bleed. 
 
We note that we will review grade 4 and 5 adverse events deemed “unrelated” or “unlikely 
to be related”, to verify their attribution and to monitor the emergence of a previously 
unrecognized treatment-related adverse event. 


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The study team may choose to suspend accrual because of unexpected adverse event 
profiles that have not crossed the specified rule below.  CTCAE v5.0 will be used to 
determine grading for these stopping rules. 
15.7.4 Monitoring 
15.7.4.1  
This study will be monitored by the Clinical Data Update System (CDUS) 
version 3.0. An abbreviated report containing cumulative CDUS data will be submitted 
quarterly to CTEP by electronic means. Reports are due January 31, April 30, July 31, and 
October 31. 
15.7.4.2  
In addition to the regular and frequent assessment of the Adverse Event 
Stopping Rule, this study will also be reviewed by the study chair and study statistician to 
monitor the study for evidence of severe adverse effects and feasibility problems in 
conjunction with production of the annual Alliance Study Summary reports to identify any 
problems with accrual, toxicity, and endpoints. These study summary reports will be 
available to members at www.allianceforclinicaltrialsinoncology.org. 
15.7.4.3 The principal investigator and the study statistician will review the study at least 
twice a year to identify accrual, adverse event/safety data, and any endpoint 
problems that might be developing.  The Alliance Data Safety Monitoring Board 
(DSMB) is responsible for reviewing accrual and safety data for this trial at least 
twice a year, based on reports provided by the study statistician. 
15.7.4.4 In addition to the above monitoring, there will be regularly scheduled (monthly) 
calls with the Study Chairs(s), Statistician(s) andAlliance Executive Officer to 
discuss toxicity reports. 
15.8  Descriptive Factors 
These factors relate to how any subset analysis will be performed. 
15.8.1  
Prior nitrosoureas:  Yes vs. No 
15.8.2  
Prior temozolomide: Yes or No 
15.8.3  
Interval since end of RT (months) 
15.8.4  
Corticosteroid therapy at study entry:  Yes vs. No 
15.8.5 Extent of primary resection:  None vs. biopsy vs. subtotal resection vs. gross total 
resection. 
15.8.6  
Side of primary resection: Right vs. left vs. midline vs. bilateral 
15.8.7 Histologic type of primary tumor: oligodendroglioma vs. oligoastrocytoma  vs. 
astrocytoma 
15.8.8  
Histologic grade of primary tumor: 2 vs. 3 vs. 4 
15.8.9  Number of prior chemotherapy regimens: 0 vs. 1 vs. 2 
15.8.10 Family history of brain tumor:  Yes vs. No  
 
 
If yes, check all that apply: 
_____ Father 
_____ Mother 
_____ Brother/Sister   


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_____ Child 
  
  
_____ Other (list: ______________________________) 
 
16.0 ADVERSE EVENT REPORTING 
16.1  Expedited Adverse Event Reporting 
Investigators are required by Federal Regulations to report serious adverse events as defined 
below. Alliance investigators are required to notify the Alliance Protocol Operations Program, 
the Study Chair, and their Institutional Review Board if a patient has an adverse event requiring 
expedited reporting.  All such events must be reported in an expedited manner using the CTEP 
Adverse Event Reporting System (CTEP-AERS). The descriptions and grading scales found in 
the NCI Common Terminology Criteria for Adverse Events (CTCAE) version 5.0 will be 
utilized for expedited AE reporting beginning April 1, 2018. All treatment areas should have 
access to a copy of the CTEP version 5.0 of CTCAE. A copy of the CTCAE version 5.0 can be 
downloaded 
from 
the 
CTEP 
website 
at: 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm. In the rare event 
when Internet connectivity is disrupted, the report should be submitted as soon as possible once 
connectivity is restored. An electronic report MUST be submitted upon re-establishment of 
Internet connection.  
The Alliance requires investigators to route all expedited adverse event reports through the 
Alliance Protocol Operations Program for Alliance coordinated studies.  Be sure to read this 
entire protocol section, as requirements are described in both the table and bullet points 
following the table.  Note that the table and the Additional Instructions or Exclusions are 
protocol-specific, and in the case of a conflict, the Additional Instructions or Exclusions 
supersede the table.  Most exclusions cover “expected” events that the sponsor would not be 
required to report to the FDA in an expedited manner. 


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16.2 
 Expedited Reporting Requirements for Adverse Events that Occur on Studies under an 
IND within 30 Days of the Last Treatment 1 
FDA REPORTING REQUIREMENTS FOR SERIOUS ADVERSE EVENTS (21 CFR Part 312) 
NOTE:  Investigators MUST immediately report to the sponsor (NCI) ANY Serious Adverse Events, whether or not 
they are considered related to the investigational agent(s)/intervention (21 CFR 312.64) 
 An adverse event is considered serious if it results in ANY of the following outcomes:   
1) Death 
2) A life-threatening adverse event  
3) An adverse event that results in inpatient hospitalization or prolongation of existing hospitalization for ≥ 24 
hours  
4) A persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions  
5) A congenital anomaly/birth defect.  
6) Important Medical Events (IME) that may not result in death, be life threatening, or require hospitalization 
may be considered serious when, based upon medical judgment, they may jeopardize the patient or subject 
and may require medical or surgical intervention to prevent one of the outcomes listed in this definition. 
(FDA, 21 CFR 312.32; ICH E2A and ICH E6). 
 
ALL SERIOUS adverse events that meet the above criteria MUST be immediately reported via CTEP-AERS 
within the timeframes detailed in the table below. 
Hospitalization 
Grade 1 
Timeframes 
Grade 2 
Timeframes 
Grade 3 
Timeframes 
Grade 4 & 5 
Timeframes 
Resulting in 
Hospitalization  
≥ 24 hrs 
10 Calendar Days 
24-Hour; 5 Calendar 
Days 
Not resulting in 
Hospitalization  
≥ 24 hrs 
Not required 
10 Calendar Days 
NOTE:  Protocol specific exceptions to expedited reporting of serious adverse events are found in the 
Specific Protocol Exceptions to Expedited Reporting (SPEER) portion of the CAEPR, and in the 
bullets below. 
Expedited AE reporting timelines are defined as: 
o “24-Hour; 5 Calendar Days” - The AE must initially be reported via CTEP-AERS within 24 hours 
of learning of the AE, followed by a complete expedited report within 5 calendar days of the 
initial 24-hour report. 
o “10 Calendar Days” - A complete expedited report on the AE must be submitted within 10 calendar 
days of learning of the AE. 
1Serious adverse events that occur more than 30 days after the last treatment require reporting as 
follows:  
Expedited 24-hour notification followed by complete report within 5 calendar days for: 
• 
All Grade 4, and Grade 5 AEs that are at least possibly related to treatment 
Expedited 10 calendar day reports for: 
• 
Grade 2 adverse events resulting in hospitalization or prolongation of hospitalization and that 
are at least possibly related to treatment  
• 
Grade 3 adverse events that are at least possibly related to treatment 
 


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Additional Instructions or Exclusion to CTEP-AERS Expedited Reporting Requirements for 
A071101: 
• 
All adverse events reported via CTEP-AERS (i.e., serious adverse events) should also be 
forwarded to your local IRB. 
• 
A071101 uses a drug under an Alliance IND. The reporting requirements above should be 
followed for all agents (any arm) in this trial. 
• 
Treatment expected adverse events include those listed in Sections 11.5 and 11.6, in the 
Investigator’s Brochure for HSPCC-96, and in the CAEPR for bevacizumab. Note that the 
ASAEL column of the CAEPR has been replaced with the Specific Protocol Exceptions to 
Expedited Reporting (SPEER) column. The SPEER includes “expected” severity grades in 
addition to event terms. Events listed in the SPEER only require expedited reporting if the 
severity grade is above the grade noted in the SPEER.  
• 
Grade 3 hypertension, once controlled, does not require continued reporting via CTEP-
AERS. 
• 
Grade 3 proteinuria does not require reporting via CTEP-AERS. 
• 
All new malignancies should be reported through CTEP-AERS whether or not they are 
thought to be related to either previous or current treatment. All new malignancies should 
be reported including solid tumors (including non-melanoma skin malignancies), 
hematologic malignancies, myelodysplastic syndrome (MDS)/acute myelogenous leukemia 
(AML), and in situ tumors. In CTCAE version 5.0, the new malignancies (both second and 
secondary) may be reported as one of the following (1) Leukemia secondary to oncology 
chemotherapy, (2) Myelodysplastic syndrome, (3) Treatment-related secondary 
malignancy, or (4) Neoplasm other, malignant (grade 3 or 4). Whenever possible, the CTEP-
AERS reports for new malignancies should include, tumor pathology, history of prior 
tumors, prior treatment/current treatment including duration, any associated risk factors or 
evidence regarding how long the new malignancy may have been present, when and how 
the new malignancy was detected, molecular characterization or cytogenetics of the original 
tumor (if available) and of any new tumor, and new malignancy treatment and outcome, if 
available.  
• 
Patients who become pregnant on study risk intrauterine exposure of the fetus to agents, 
which may be teratogenic. For this reason, pregnancy occurring on study or within 6 
months following the last dose of study therapy should be reported in an expedited manner 
via CTEP-AERS as “Pregnancy, puerperium and perinatal conditions - Other 
(Pregnancy) under the Pregnancy, puerperium and perinatal conditions SOC and 
reported as Grade 3. 
• 
Pregnancy should be followed up until the outcome of the pregnancy is known at intervals 
deemed appropriate by the investigator and the outcome reported via CTEP-AERS. 
• 
All Adverse Events must be reported in routine study data submissions. AEs reported 
through CTEP-AERS must also be reported in routine study data submissions.


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16.3 
Comprehensive Adverse Event and Potential Risks List (CAEPR) for Bevacizumab 
(rhuMAb VEGF, NSC 704865) 
The Comprehensive Adverse Events and Potential Risks list (CAEPR) provides a single list of 
reported and/or potential adverse events (AE) associated with an agent using a uniform 
presentation of events by body system. In addition to the comprehensive list, a subset, the 
Specific Protocol Exceptions to Expedited Reporting (SPEER), appears in a separate column 
and is identified with bold and italicized text. This subset of AEs (SPEER) is a list of events that 
are protocol specific exceptions to expedited reporting to NCI (except as noted below).  Refer 
to 
the 
'CTEP, 
NCI 
Guidelines: 
Adverse 
Event 
Reporting 
Requirements' 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/docs/aeguidelines.pdf  for 
further clarification. Frequency is provided based on 3540 patients. Below is the CAEPR for 
Bevacizumab (rhuMAb VEGF). 
NOTE: Report AEs on the SPEER ONLY IF they exceed the grade noted in parentheses 
next to the AE in the SPEER.  If this CAEPR is part of a combination protocol using 
multiple investigational agents and has an AE listed on different SPEERs, use the lower of 
the grades to determine if expedited reporting is required. 
 
Version 2.5, May 2, 20181 
 
 Adverse Events with Possible  
 Relationship to Bevacizumab (rhuMAb VEGF) 
 (CTCAE 5.0 Term) 
[n= 3540] 
 
 
 Specific Protocol Exceptions to 
Expedited Reporting (SPEER)  
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious (<3%) 
  
BLOOD AND LYMPHATIC SYSTEM DISORDERS 
  
 
Anemia 
 
 Anemia (Gr 3) 
 
Febrile neutropenia 
 
 Febrile neutropenia (Gr 3) 
 
 
Hemolytic uremic syndrome 
  
CARDIAC DISORDERS 
  
 
Cardiac disorders - Other 
(supraventricular arrhythmias)2  
 
 Cardiac disorders - Other 
(supraventricular arrhythmias)2 (Gr 3) 
 
 
Chest pain – cardiac3 
  
 
 
Heart failure 
  
 
 
Left ventricular systolic 
dysfunction 
  
 
 
Myocardial infarction3 
  
 
 
Ventricular arrhythmia 
  
 
 
Ventricular fibrillation 
  
GASTROINTESTINAL DISORDERS 
  
 
Abdominal pain 
 
 Abdominal pain (Gr 3)  
 
Colitis 
 
 Colitis (Gr 3) 
 
Constipation 
 
 Constipation (Gr 3) 
 
Diarrhea 
 
 Diarrhea (Gr 3) 
 
Dyspepsia 
 
 Dyspepsia (Gr 2) 
 
 
Gastrointestinal fistula4 
  
 
Gastrointestinal hemorrhage5 
 
 Gastrointestinal hemorrhage5 (Gr 2) 
 
Gastrointestinal obstruction6 
 
  
 
 
Gastrointestinal perforation7 
  
 
 
Gastrointestinal ulcer8 
  
 
Ileus 
 
  


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 Adverse Events with Possible  
 Relationship to Bevacizumab (rhuMAb VEGF) 
 (CTCAE 5.0 Term) 
[n= 3540] 
 
 
 Specific Protocol Exceptions to 
Expedited Reporting (SPEER)  
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious (<3%) 
  
 
Mucositis oral 
 
 Mucositis oral (Gr 3) 
 
Nausea 
 
 Nausea (Gr 3) 
 
Vomiting 
 
 Vomiting (Gr 3) 
GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS 
  
 
Fatigue 
 
 Fatigue (Gr 3) 
 
Non-cardiac chest pain 
 
 Non-cardiac chest pain (Gr 3) 
 
Pain 
 
 Pain (Gr 3) 
HEPATOBILIARY DISORDERS 
  
 
 
Gallbladder perforation 
  
IMMUNE SYSTEM DISORDERS 
  
 
Allergic reaction 
 
 Allergic reaction (Gr 2) 
 
 
Anaphylaxis 
  
INFECTIONS AND INFESTATIONS 
  
 
Infection9 
 
 Infection9 (Gr 3) 
 
 
Infections and infestations - 
Other (necrotizing fascitis) 
  
 
Infections and infestations - Other 
(peri-rectal abscess) 
 
  
INJURY, POISONING AND PROCEDURAL COMPLICATIONS 
  
 
Infusion related reaction 
 
 Infusion related reaction (Gr 2) 
 
 
Injury, poisoning and procedural 
complications - Other 
(anastomotic leak)10  
  
 
Wound complication 
 
 Wound complication (Gr 2) 
 
Wound dehiscence 
 
 Wound dehiscence (Gr 2) 
INVESTIGATIONS 
  
 
Alanine aminotransferase increased  
 Alanine aminotransferase increased 
(Gr 3) 
 
Alkaline phosphatase increased 
 
 Alkaline phosphatase increased (Gr 3) 
 
Aspartate aminotransferase increased  
 Aspartate aminotransferase increased 
(Gr 3) 
 
Blood bilirubin increased 
 
 Blood bilirubin increased  
(Gr 2) 
 
Creatinine increased 
 
  
Neutrophil count decreased  
 
 Neutrophil count decreased (Gr 3) 
 
Platelet count decreased 
 
 Platelet count decreased  
(Gr 4) 
 
Weight loss 
 
 Weight loss (Gr 3) 
 
White blood cell decreased 
 
 White blood cell decreased (Gr 3) 
METABOLISM AND NUTRITION DISORDERS 
  
 
Anorexia 
 
 Anorexia (Gr 3) 
 
Dehydration 
 
 Dehydration (Gr 3) 
 
Hyperglycemia 
 
  
 
Hypokalemia 
 
  
 
Hyponatremia 
 
  
MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS 
  
 
Arthralgia 
 
 Arthralgia (Gr 3) 
 
 
Avascular necrosis11 
  


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 Adverse Events with Possible  
 Relationship to Bevacizumab (rhuMAb VEGF) 
 (CTCAE 5.0 Term) 
[n= 3540] 
 
 
 Specific Protocol Exceptions to 
Expedited Reporting (SPEER)  
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious (<3%) 
  
 
Generalized muscle weakness 
 
  
 
Musculoskeletal and connective 
tissue disorder - Other (bone 
metaphyseal dysplasia)12  
 
  
 
Myalgia 
 
 Myalgia (Gr 3) 
 
Osteonecrosis of jaw13 
 
  
NERVOUS SYSTEM DISORDERS 
  
 
Dizziness 
 
 Dizziness (Gr 2) 
 
Headache 
 
 Headache (Gr 3) 
 
 
Intracranial hemorrhage 
  
 
 
Ischemia cerebrovascular 
  
 
Peripheral sensory neuropathy14 
 
  
 
 
Reversible posterior 
leukoencephalopathy syndrome 
  
 
Syncope 
 
  
RENAL AND URINARY DISORDERS 
  
 
 
Acute kidney injury 
  
 
Hematuria 
 
 Hematuria (Gr 3) 
 
 
Nephrotic syndrome 
  
 
Proteinuria 
 
 Proteinuria (Gr 2) 
 
 
Urinary fistula 
  
REPRODUCTIVE SYSTEM AND BREAST DISORDERS 
  
Reproductive system and 
breast disorders - Other 
(ovarian failure)15  
 
 
  
 
 
Vaginal fistula 
  
 
Vaginal hemorrhage 
 
 Vaginal hemorrhage (Gr 3) 
RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS 
  
 
Allergic rhinitis 
 
 Allergic rhinitis (Gr 2) 
 
 
Bronchopleural fistula 
  
 
 
Bronchopulmonary hemorrhage   
 
Cough 
 
 Cough (Gr 3) 
 
Dyspnea 
 
 Dyspnea (Gr 2) 
 
Epistaxis 
 
 Epistaxis (Gr 3) 
 
Hoarseness 
 
 Hoarseness (Gr 3) 
 
 
Pulmonary hypertension 
  
 
 
Respiratory, thoracic and 
mediastinal disorders - Other 
(nasal-septal perforation) 
  
 
 
Respiratory, thoracic and 
mediastinal disorders - Other 
(tracheo-esophageal fistula) 
  
SKIN AND SUBCUTANEOUS TISSUE DISORDERS 
  
 
Dry skin 
 
  
 
Erythroderma 
 
  
 
 
Palmar-plantar 
erythrodysesthesia syndrome 
  
 
Pruritus 
 
 Pruritus (Gr 2) 
 
Rash maculo-papular 
 
 Rash maculo-papular (Gr 2) 


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 Adverse Events with Possible  
 Relationship to Bevacizumab (rhuMAb VEGF) 
 (CTCAE 5.0 Term) 
[n= 3540] 
 
 
 Specific Protocol Exceptions to 
Expedited Reporting (SPEER)  
Likely (>20%) 
Less Likely (<=20%) 
Rare but Serious (<3%) 
  
 
Urticaria 
 
 Urticaria (Gr 2) 
VASCULAR DISORDERS 
  
 
 
Arterial thromboembolism3, 16  
  
Hypertension 
 
 
 Hypertension (Gr 3) 
 
Thromboembolic event 
 
 Thromboembolic event (Gr 3) 
 
1This table will be updated as the toxicity profile of the agent is revised.  Updates will be distributed to all Principal 
Investigators at the time of revision.  The current version can be obtained by contacting 
PIO@CTEP.NCI.NIH.GOV.  Your name, the name of the investigator, the protocol and the agent should be 
included in the e-mail. 
 
2Supraventricular arrhythmias may include supraventricular tachycardia, atrial fibrillation, and atrial flutter. 
 
3The risks of arterial thrombosis such as cardiac or CNS ischemia are increased in elderly patients and in patients 
with a history of diabetes. 
 
4Gastrointestinal fistula may include: Anal fistula, Colonic fistula, Duodenal fistula, Esophageal fistula, Gastric 
fistula, Gastrointestinal fistula, Rectal fistula, and other sites under the GASTROINTESTINAL DISORDERS SOC. 
 
5Gastrointestinal hemorrhage may include: Colonic hemorrhage, Duodenal hemorrhage, Esophageal hemorrhage, 
Esophageal varices hemorrhage, Gastric hemorrhage, Hemorrhoidal hemorrhage, Intra-abdominal hemorrhage, Oral 
hemorrhage, Rectal hemorrhage, and other sites under the GASTROINTESTINAL DISORDERS SOC. 
 
6Gastrointestinal obstruction may include: Colonic obstruction, Duodenal obstruction, Esophageal obstruction, Ileal 
obstruction, Jejunal obstruction, Rectal obstruction, Small intestinal obstruction, and other sites under the 
GASTROINTESTINAL DISORDERS SOC. 
 
7Gastrointestinal perforation may include: Colonic perforation, Duodenal perforation, Esophageal perforation, 
Gastric perforation, Jejunal perforation, Rectal perforation, and Small intestinal perforation. 
 
8Gastrointestinal ulcer may include: Duodenal ulcer, Esophageal ulcer, Gastric ulcer, and other sites under the 
GASTROINTESTINAL DISORDERS SOC. 
 
9Infection may include any of the 75 infection sites under the INFECTIONS AND INFESTATIONS SOC. 
 
10Anastomotic leak may include Gastric anastomotic leak; Gastrointestinal anastomotic leak; Large intestinal 
anastomotic leak; Rectal anastomotic leak; Small intestinal anastomotic leak; Urostomy leak; Vaginal anastomotic 
leak. 
 
11There have been reports of non-mandibular osteonecrosis (avascular necrosis) in patients under the age of 18 
treated with bevacizumab. 
 
12Metaphyseal dysplasia was observed in young patients who still have active epiphyseal growth plates. 
 
13Cases of osteonecrosis of the jaw (ONJ) have been reported in cancer patients in association with bevacizumab 
treatment, the majority of whom had received prior or concomitant treatment with i.v. bisphosphonates. 
 
14Increased rate of peripheral sensory neuropathy has been observed in trials combining bevacizumab and 
chemotherapy compared to chemotherapy alone.  


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15Ovarian failure, defined as amenorrhea lasting 3 or more months with follicle-stimulating hormone (FSH) 
elevation (≥30 mIU/mL), was increased in patients receiving adjuvant bevacizumab plus mFOLFOX compared to 
mFOLFOX alone (34% vs. 2%).  After discontinuation of bevacizumab, resumption of menses and an FSH level 
<30 mIU/mL was demonstrated in 22% (7/32) of these women.  Long term effects of bevacizumab exposure on 
fertility are unknown. 
 
16Arterial thromboembolic event includes visceral arterial ischemia, peripheral arterial ischemia, heart attack, and 
stroke. 
 
Adverse events reported on bevacizumab (rhuMAb VEGF) trials, but for which there is insufficient evidence 
to suggest that there was a reasonable possibility that bevacizumab (rhuMAb VEGF) caused the adverse 
event: 
 
BLOOD AND LYMPHATIC SYSTEM DISORDERS - Bone marrow hypocellular; Disseminated intravascular 
coagulation; Hemolysis; Thrombotic thrombocytopenic purpura 
CARDIAC DISORDERS - Atrioventricular block complete; Atrioventricular block first degree; Cardiac arrest; 
Myocarditis; Pericardial effusion; Restrictive cardiomyopathy; Right ventricular dysfunction 
EAR AND LABYRINTH DISORDERS - Ear and labyrinth disorders - Other (tympanic membrane perforation); 
Hearing impaired; Tinnitus; Vertigo 
ENDOCRINE DISORDERS - Hyperthyroidism; Hypothyroidism 
EYE DISORDERS - Blurred vision; Cataract; Dry eye; Extraocular muscle paresis; Eye disorders - Other 
(blindness); Eye disorders - Other (conjunctival hemorrhage); Eye disorders - Other (corneal epithelial defect); Eye 
disorders - Other (ischemic CRVO); Eye disorders - Other (macular pucker); Eye disorders - Other (transient 
increased IOP > or =30 mm Hg); Eye pain; Floaters; Keratitis; Optic nerve disorder; Photophobia; Retinal 
detachment; Retinal tear; Retinopathy; Vitreous hemorrhage; Watering eyes 
GASTROINTESTINAL DISORDERS - Ascites; Cheilitis; Colonic stenosis; Dry mouth; Dysphagia; 
Enterocolitis; Esophageal pain; Esophageal stenosis; Flatulence; Gastrointestinal disorders - Other (peritonitis); Oral 
pain; Pancreatitis; Proctitis; Rectal mucositis; Rectal stenosis; Typhlitis 
GENERAL DISORDERS AND ADMINISTRATION SITE CONDITIONS - Death NOS; Edema face; Edema 
limbs; Edema trunk; Facial pain; Fever; Flu like symptoms; Gait disturbance; Injection site reaction; Localized 
edema; Multi-organ failure; Sudden death NOS 
HEPATOBILIARY DISORDERS - Cholecystitis; Gallbladder necrosis; Gallbladder obstruction; Hepatic failure; 
Hepatic necrosis 
INJURY, POISONING AND PROCEDURAL COMPLICATIONS - Arterial injury; Bruising; Burn; Dermatitis 
radiation; Fracture 
INVESTIGATIONS - Activated partial thromboplastin time prolonged; Blood antidiuretic hormone abnormal; 
CD4 lymphocytes decreased; CPK increased; Carbon monoxide diffusing capacity decreased; Electrocardiogram 
QT corrected interval prolonged; Forced expiratory volume decreased; GGT increased; INR increased; Lipase 
increased; Lymphocyte count decreased; Serum amylase increased; Weight gain 
METABOLISM AND NUTRITION DISORDERS - Acidosis; Hypercalcemia; Hyperkalemia; 
Hypermagnesemia; Hypernatremia; Hypertriglyceridemia; Hyperuricemia; Hypoalbuminemia; Hypocalcemia; 
Hypomagnesemia; Hypophosphatemia 
MUSCULOSKELETAL AND CONNECTIVE TISSUE DISORDERS - Arthritis; Back pain; Bone pain; Chest 
wall pain; Fibrosis deep connective tissue; Head soft tissue necrosis; Joint effusion; Muscle weakness lower limb; 
Muscle weakness upper limb; Musculoskeletal and connective tissue disorder - Other (polymyalgia rheumatica); 
Neck pain; Osteonecrosis; Pain in extremity; Pelvic soft tissue necrosis; Soft tissue necrosis lower limb 
NEOPLASMS BENIGN, MALIGNANT AND UNSPECIFIED (INCL CYSTS AND POLYPS) - Tumor pain 
NERVOUS SYSTEM DISORDERS - Arachnoiditis; Ataxia; Central nervous system necrosis; Cerebrospinal fluid 
leakage; Cognitive disturbance; Depressed level of consciousness; Dysesthesia; Dysgeusia; Dysphasia; 
Encephalopathy; Extrapyramidal disorder; Facial nerve disorder; Hydrocephalus; Leukoencephalopathy; Memory 
impairment; Myasthenia gravis; Nervous system disorders - Other (increased intracranial pressure); Paresthesia; 
Peripheral motor neuropathy; Pyramidal tract syndrome; Seizure; Somnolence; Tremor; Vasovagal reaction 
PSYCHIATRIC DISORDERS - Agitation; Anxiety; Confusion; Depression; Insomnia; Libido decreased; 
Psychosis 


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RENAL AND URINARY DISORDERS - Bladder spasm; Chronic kidney disease; Cystitis noninfective; Dysuria; 
Renal and urinary disorders - Other (ureterolithiasis); Renal hemorrhage; Urinary frequency; Urinary incontinence; 
Urinary retention; Urinary tract obstruction; Urinary tract pain 
REPRODUCTIVE SYSTEM AND BREAST DISORDERS - Breast pain; Erectile dysfunction; Irregular 
menstruation; Pelvic pain; Vaginal discharge 
RESPIRATORY, THORACIC AND MEDIASTINAL DISORDERS - Adult respiratory distress syndrome; 
Atelectasis; Hypoxia; Nasal congestion; Pulmonary fibrosis; Respiratory failure; Respiratory, thoracic and 
mediastinal disorders - Other (dry nares); Respiratory, thoracic and mediastinal disorders - Other (pulmonary 
infarction) 
SKIN AND SUBCUTANEOUS TISSUE DISORDERS - Alopecia; Hyperhidrosis; Nail loss; Pain of skin; 
Photosensitivity; Purpura; Rash acneiform; Skin and subcutaneous tissue disorders - Other (diabetic foot ulcer); Skin 
and subcutaneous tissue disorders - Other (skin breakdown/ decubitus ulcer); Skin hyperpigmentation; Skin 
induration; Skin ulceration; Stevens-Johnson syndrome 
VASCULAR DISORDERS - Flushing; Hot flashes; Hypotension; Lymphocele; Phlebitis; Vasculitis 
 
Note: Bevacizumab (rhuMAb VEGF) in combination with other agents could cause an exacerbation of any adverse 
event currently known to be caused by the other agent, or the combination may result in events never previously 
associated with either agent. 
 
16.4 
Routine adverse event reporting 
Adverse event data collection and reporting, which are required as part of every clinical trial are 
done to ensure the safety of patients enrolled in the studies as well as those who will enroll in 
future studies using similar agents. Adverse events are reported in a routine manner at scheduled 
times according to the study calendar in Section 7.0.  
Solicited Adverse Events: The following adverse events are considered "expected" and their 
presence/absence should be solicited, and severity graded, at baseline and for each cycle of 
treatment as noted below: 
Allergic reaction (see CTCAE Immune system disorders; not at baseline) 
Infusion related reaction (see CTCAE General disorders and administration site conditions; not 
at baseline) 
Injection site reaction (see CTCAE General disorders and administration site conditions; not at 
baseline) 
Hypertension (see CTCAE Vascular disorders) 
Thromboembolic event, venous or arterial (see CTCAE Vascular disorders) 
Proteinuria (see CTCAE Renal and urinary disorders) 
Wound dehiscence (see CTCAE Injury, poisoning and procedural complications) 
 
 


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17.0 REFERENCES 
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Brem, H., et al., Placebo-controlled trial of safety and efficacy of intraoperative controlled 
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Friedman, H.S., et al., Bevacizumab alone and in combination with irinotecan in recurrent 
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5. 
Cohen, M.H., et al., FDA drug approval summary: bevacizumab (Avastin) as treatment of 
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Clark, A.J., et al., Impact of bevacizumab chemotherapy on craniotomy wound healing. J 
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7. 
Heimberger, A.B. and J.H. Sampson, Immunotherapy coming of age: what will it take to make it 
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Wheeler, C.J., et al., Clinical responsiveness of glioblastoma multiforme to chemotherapy after 
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Liau, L.M., et al., Dendritic cell vaccination in glioblastoma patients induces systemic and 
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Sampson, J.H., et al., Immunologic escape after prolonged progression-free survival with 
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Han, S.J., et al., Biologic principles of immunotherapy for malignant gliomas. Neurosurg Clin N 
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Prins, R.M., et al., Central nervous system tumor immunity generated by a recombinant listeria 
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Binder, R.J. and P.K. Srivastava, Essential role of CD91 in re-presentation of gp96-chaperoned 
peptides. Proc Natl Acad Sci U S A, 2004. 101(16): p. 6128-33. 
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Binder, R.J., D.K. Han, and P.K. Srivastava, CD91: a receptor for heat shock protein gp96. Nat 
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15. 
Srivastava, P.K., Immunotherapy of human cancer: lessons from mice. Nat Immunol, 2000. 1(5): 
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16. 
Yang, I., S. Fang, and A.T. Parsa, Heat shock proteins in glioblastomas. Neurosurg Clin N Am, 
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17. 
Lennerz, V., et al., The response of autologous T cells to a human melanoma is dominated by 
mutated neoantigens. Proc Natl Acad Sci U S A, 2005. 102(44): p. 16013-8. 
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Belli, F., et al., Vaccination of metastatic melanoma patients with autologous tumor-derived heat 
shock protein gp96-peptide complexes: clinical and immunologic findings. J Clin Oncol, 2002. 
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Liyanage, U.K., et al., Increased prevalence of regulatory T cells (Treg) is induced by pancreas 
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Wood, C., et al., An adjuvant autologous therapeutic vaccine (HSPPC-96; vitespen) versus 
observation alone for patients at high risk of recurrence after nephrectomy for renal cell 
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21. 
Testori, A., et al., Phase III comparison of Vitespen, an autologous tumor-derived heat shock 
protein gp96 peptide complex vaccine, with physician's choice of treatment for stage IV 
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22.  
Crane, C., et al., Individual patient-specific immunity against high-grade glioma after vaccination 
with autologous tumor derived peptides bound to the 96 kd chaperone protein. Clin Canc Res, 
2012. 19(1), p. 1-10. 
23.  
Parsa, A., et al., Autologous heat shock protein vaccine (HSPPC-96) for patients with recurrent 
glioblastoma: Results of a phase II multicenter clinical trial with immunological assessments. J 
Clin Oncol 2011. 29(supp), Abstract 2565.  
24. 
Johnson, B., et al., Physiology and therapeutics of vascular endothelial growth factor in tumor 
immunosuppression. Curr Mol Med 2009. 9(6), p. 702-9.  
25.  
Rini, B., et al., Combination immunotherapy with prostatic acid phosphatase pulsed antigen-
presenting cells (Provenge) plus bevacizumab in patients with serologic progression of prostate 
cancer after definitive local therapy. Cancer, 2006. 107(1), p. 67-74. 
26.  
Shrimali, R., et al., Antiangiogenic agents can increase lymphocyte infiltration into tumor and 
enhance the effectiveness of adoptive immunotherapy of cancer. Cancer Res. 2010. 70(15), p. 
6171-80. 
27. 
Huang Y., et al., Vascular normalizing doses of antiangiogenic treatment reprogram the 
immunosuppressive tumor microenvironment and enhance immunotherapy. Proc Nat Acad Sci 
2012. 109(43), 17561-66. 
28. 
Blumenthal, D.T., et al., Primary intracranial neoplasms in patients with HIV. Neurology, 1999. 
52(8): p. 1648-51. 
29. 
Cedeno-Laurent, F. and J.R. Trujillo, Gliomas and brain lymphomas in HIV-1/AIDS patients: 
reflections from a 20-year follow up in Mexico and Brazil. Microbiology Research, 2011. 2(1): p. 
41-44. 
30. 
Schiff, D., et al., Gliomas arising in organ transplant recipients: an unrecognized complication of 
transplantation? Neurology, 2001. 57(8): p. 1486-8. 
31. 
Bohman, L.E., et al., The survival impact of postoperative infection in patients with glioblastoma 
multiforme. Neurosurgery, 2009. 64(5): p. 828-34; discussion 834-5. 
32. 
De Bonis, P., et al., Postoperative infection may influence survival in patients with glioblastoma: 
simply a myth? Neurosurgery, 2011. 69(4): p. 864-8; discussion 868-9. 
33. 
Chen, C., et al., Allergy and risk of glioma: a meta-analysis. Eur J Neurol, 2011. 18(3): p. 387-95. 
34. 
Lachance, D.H., et al., Associations of high-grade glioma with glioma risk alleles and histories of 
allergy and smoking. Am J Epidemiol, 2011. 174(5): p. 574-81. 
35. 
Cooper, C.D., et al., Identification and characterization of peripheral T-cell lymphoma-associated 
SEREX antigens. PLoS One, 2011. 6(8): p. e23916. 
36. 
Kagaya, A., et al., Identification of a novel SEREX antigen family, ECSA, in esophageal 
squamous cell carcinoma. Proteome Sci, 2011. 9(1): p. 31. 
37. 
Song, M.H., et al., Identification of BCP-20 (FBXO39) as a cancer/testis antigen from colon 
cancer patients by SEREX. Biochem Biophys Res Commun, 2011. 408(2): p. 195-201. 
38. 
Heller, A., et al., Immunogenicity of SEREX-identified antigens and disease outcome in 
pancreatic cancer. Cancer Immunol Immunother, 2010. 59(9): p. 1389-400. 
39. 
Kiyamova, R., et al., Identification of tumor-associated antigens from medullary breast carcinoma 
by a modified SEREX approach. Mol Biotechnol, 2010. 46(2): p. 105-12. 
40. 
Shimada, H., et al., Identification of Makorin 1 as a novel SEREX antigen of esophageal 
squamous cell carcinoma. BMC Cancer, 2009. 9: p. 232. 
41. 
Kalnina, Z., et al., Molecular characterisation and expression analysis of SEREX-defined antigen 
NUCB2 in gastric epithelium, gastritis and gastric cancer. Eur J Histochem, 2009. 53(1): p. 7-18. 
42. 
Wang, K., et al., Identification of tumor-associated antigens by using SEREX in hepatocellular 
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43. 
Okada, H., et al., Immunization with an antigen identified by cytokine tumor vaccine-assisted 
SEREX (CAS) suppressed growth of the rat 9L glioma in vivo. Cancer Res, 2001. 61(6): p. 2625-
31. 
44. 
Pallasch, C.P., et al., Autoantibodies against GLEA2 and PHF3 in glioblastoma: tumor-associated 
autoantibodies correlated with prolonged survival. Int J Cancer, 2005. 117(3): p. 456-9. 
45. 
Yang, I., et al., Immune cell infiltrate differences in pilocytic astrocytoma and glioblastoma: 
evidence of distinct immunological microenvironments that reflect tumor biology. J Neurosurg, 
2011. 115(3): p. 505-11. 
46.         Crane C, Seunggu J, and Ahn B. Individual patient-specific immunity against high-grade glioma 
after vaccination with autologous tumor derived peptides bound to the 96 KD chaperone protein. 
Clin Cancer Res 2013, 19: 205-214. 
47.         Bloch O, Carne C, and Fuks Y. Heat-shock protein peptide complex-96 vaccination for recurrent 
glioblastoma: a phase II, single-arm trial. Neuro-Oncology, 2014. 16(2): p. 274-279.  
48.         Bloch O, Raizer J, and Lim M.  Newly diagnosed glioblastoma patients treated with an 
autologous heat shock protein peptide vaccine: PD-L1 expression and response to therapy. J Clin 
Oncol 33, 2015 (supply; abstr 2011). 
 
 
 
 
 
 
 
 
 
 
 
 
 


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APPENDIX I: UPC (URINE PROTEIN TO CREATININE) RATIO 
 
 
The UPC (urine protein to creatinine) ratio directly correlates with the grams of protein found in a 24 
hr urine. The UPC ratio can be used in the place of a 24-hour urine. 
 
Procedure for Obtaining a Urine Protein/Creatinine Ratio: 
 
1. Obtain at least 4 mL of a random urine sample in a sterile container (does not have to be a 24-hour 
urine sample). 
2. Determine protein concentration (mg/dL). 
3. Determine creatinine concentration (mg/dL). 
4. Divide #2 by #3 above: 
 
 
UPC Ratio = 
 
Protein Concentration (mg/dL) 
Creatinine Concentration (mg/dL) 
 
 
 
 


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APPENDIX II: KARNOFSKY PERFORMANCE STATUS 
 
 
 Performance status will be graded according to the following scale: 
 Karnofsky 100 Normal; no complaints; no evidence of disease 
Karnofsky 90 Able to carry on normal activity; minor signs or symptoms of disease 
 Karnofsky 80   Normal activity with effort; some sign or symptoms of disease 
 Karnofsky 70 Cares for self; unable to carry on normal activity or do active work 
 Karnofsky 60  Requires occasional assistance, but is able to care for most personal needs 
 Karnofsky 50 Requires considerable assistance and frequent medical care 
 Karnofsky 40 Disabled; requires special care and assistance 
 Karnofsky 30 Severely disabled; hospitalization is indicated, although death not imminent     
 
 Karnofsky 20 Very sick; hospitalization necessary; active support treatment is necessary 
 Karnofsky 10 Moribund; fatal processes progressing rapidly 
 Karnofsky 0 
Dead 
 
 


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APPENDIX III: VACCINE TISSUE PROCUREMENT AND SHIPPING INSTRUCTIONS 
 
Tissue procurement kits provided by Agenus include: 
50-mL nonpyrogenic tubes and patient information labels 
Absorbent paper 
Saf-T pouches 
Pressure vessels (with foam insert) 
Cardboard pieces (for box support and top of dry ice) 
Original invoice letter (for international shipments only; must be reproduced on study center 
letterhead) 
Shipping box and all shipping/address labels 
Courier documents 
Sites must provide the dry ice (8kg). 
Specimens not shipped to Agenus immediately after procurement must be frozen and stored at 
-80°C ± 20°C. The specimen should not arrive on the weekend or a holiday unless this has been 
agreed to in advance by Agenus. Please call Agenus’ Clinical Affairs at 
 with any 
questions. 
If the tissue will not be shipped immediately, it should be placed into an -80° (+/- 20°) C freezer until 
ready to pack and ship. Do this within a maximum of 30 minutes of removal. Ship as soon as possible. 
Please be careful not to allow thawing when the tissue is packed. 
For tumor tissue stored at sites prior to shipping to Agenus (i.e. overnight or for weekends/holidays), 
the freezer does not need to be sterile but it is recommended that it should be monitored for 
temperature (24/7) with alarms or other mechanism for identifying when temperature goes out of 
specification.  The freezer or its location should have controlled access (locked with specified group 
members having access). The freezer should also be on a preventative maintenance program and 
ideally be hooked up to a back-up power system. 
 
In the OR, wrap the tumor tissue in a sterile cloth or towel, and place in a basin, on ice (not dry ice). 
Ideally, this should be an en bloc resection.  Aspiration contents from a cavitronic aspirator (e.g. 
CUSA) are not acceptable as vaccine manufacturing success with these materials is limited. 
Transport the specimen immediately to the pathology/frozen section suite. 
Share the following steps with the surgical pathologist, along with the tissue procurement kit:
 a. 
Use basic sterile technique.  Care should be taken to avoid any possible cross-
contamination with tissues or fluids from other patients. 
b. 
As small a section of tumor as possible should be kept for routine pathologic studies and 
diagnosis. Tissue for routine pathologic diagnosis should be processed according to institutional 
standards. 
c. 
The pathologist should assess viability of the tumor.  
d. 
When there is necrosis and/or cystic degeneration in the tumor specimen, the necrotic tissue 
and/or cystic component should be removed and should not be sent to Agenus. 
e. 
All tumor tissue should be sent for HSPPC-96® vaccine preparation. A minimum of 5 grams 
of fresh, non-necrotic tumor tissue is sent (7 grams of tissue is preferable).  
 


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The tumor procurement and shipping procedure is as follows: 
 
1. 
The pathologist should section the tissue into 1-2 cm2 sections. The sections should then 
be placed carefully, maintaining sterility, into the 50-cc nonpyrogenic tubes. Each tube 
should be filled no more than ½ to ⅔ full, to allow for expansion of tissue during freezing. 
2. 
The tube(s) must be properly labeled, using only a regular ballpoint pen, with the study 
number (A071101), patient ID number, patient initials (last, first, middle), date of birth 
(e.g., 02/JAN/1901), date of resection, and name of the institution. A completed label must 
be affixed onto each tube. If there is no middle initial, a dash (-) should be included on the 
forms and specimen labels. 
 
Tumor Shipping Checklist: 
Each tube should be placed in a plastic Saf-T pouch, and the Saf-T pouch sealed closed. 
Wrap the tubes in bubble wrap or place in the foam insert. 
The piece of absorbent paper should be placed in the bottom of the orange screw-topped container 
(pressure vessel), and the bundle of tubes placed on top of the paper. Dry ice should NOT be placed 
inside the pressure vessel. 
Place some dry ice in the bottom of the insulated shipper box. 
The pressure vessel container should be placed above the dry ice, in the cardboard support, and more 
dry ice packed surrounding the container. 
Shipping container MUST be filled with at least 8kg of dry ice. Dry ice should be added to about 
10 cm from the top of the box, a second cardboard insert placed above the ice, and the foam plug 
placed into the box. If an insufficient amount of dry ice is placed in the box, and the tissue is not 
frozen on arrival, it cannot be processed and the patient must be withdrawn from the study. 
Complete the Tissue Procurement Form. All the required information must be completed. The form 
should be faxed to Agenus at 
 so that the shipment is expected and can be traced by air 
bill number if it does not arrive. A copy of the form should also be kept for your records. 
The original Tissue Procurement Form should be placed on top of the foam plug and the cardboard 
flaps closed and securely taped shut.  
The following labels should be over the appropriate areas: 
Dry ice (#9) with weight 
Shipping address/content label 
“Shipped from” label 
 
 
 
 
 
 
 
 
 
 
Side of box 
_______ 
_______ 
Dry Ice Label  
“Shipped 
from” label 
Shipping Address/ 
Content 
9 


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Update #13 
Contact courier to arrange for a pick-up, if this was not done in advance.  
Attach the courier air bill to the box. The shipment should be sent to: 
  
 
  
 
  
 
  
 
  
 
  
 
 
 
Below is a list of some common issues that have been encountered with completing the 
Tissue Procurement Form (TPF). Please take great care in filing out this form correctly, 
as any such issues may lead to delays in vaccine production and potential rejection of 
the material if the correct identity cannot be confirmed. 
• 
Patient study number does not match – tube labels vs. procurement 
form  
 
• 
Patient date of birth does not match – tube labels vs. procurement form 
 
• 
Patient initials do not match – tube labels vs. procurement form (i.e., 
ABC ≠ A-C) 
 
• 
Patient study number, date of birth or patient initials are illegible on 
procurement form or tube labels 
 
• 
Tube labels used are not the same as those issued by Agenus 
 
• 
Tubes are not labeled at all, or information is recorded directly onto 
glass of tube and is then smeared upon receipt 
 
• 
Pen or marker used to record information on tube label is not indelible 
ink, and information is then smeared upon receipt 
 
• 
Tubes for more than 1 patient are placed into one pressure vessel 
 
• 
Initials and date of correction are not recorded when cross-outs are 
made on tube labels or Tissue Procurement Form 
 
 
 


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APPENDIX IV: WHOLE BLOOD PBMC PROCESSING AT SITES* 
 
*For previously designated sites only 
 
Isolation of Peripheral Blood Mononuclear Cells (PBMCs) 
from Human Blood Samples 
 
 
1.0 
Suggested Equipment/Materials 
 
Materials 
Manufacturer/Distributor 
Bench-top centrifuge  
The Beckman Coulter Allegra 6R or equivalent1 
(Refrigerated and Room temperature) 
Pipettes 
Rainin or equivalent1 
Dulbecco's Phosphate-Buffered Saline (D-
PBS) (1X) 
GIBCO Cat# 14190-144 or equivalent1 
Ficoll-Hypaque (Histopaque®-1077)  
Sigma-Aldrich Cat# 10771-500ml or equivalent1 
Fetal bovine serum (FBS) 
 
DMSO (Dimethyl Sulphoxide) 
 
-80 oC freezer 
REVCO or equivalent1 
Liquid Nitrogen freezer 
CRYOSAFE or equivalent1 
Sterile 50 mL polypropylene conical tubes 
Becton Dickinson Cat# 352070 or equivalent1 
Sterile T-75 Costar Tissue Culture Flasks 
Sigma-Aldrich Cat# 430641 or equivalent1 
Cryotubes, polypropylene 2.0 ml 
Sigma-Aldrich Cat# 430488 or equivalent1 
Nalgene, Cryo 1°C “Mr. Frosty” Freezing 
Containers  
VWR Cat# 55710-200 or equivalent1 
Serological Pipettes 
5 ml 
10 ml 
25 ml 
Sigma-Aldrich Cat# CLS 4487 or equivalent1 
Sigma-Aldrich Cat# CLS 4488 or equivalent1 
Sigma-Aldrich Cat# CLS 4489 or equivalent1 
1Equivalent means the same quality instrument, or the same quality or grade of material. 
 
 


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2.0 
Blood Separation Procedure 
 
It is suggested that these operations be performed in an ISO Class 5 Cabinet using standard 
aseptic technique. 
Blood (green-top tubes) MUST be kept at room temperature (20-25oC). 
The laboratory must process the blood within 8 hours from the time it is drawn. 
 
2.1 
PBMC Separation 
 
2.1.1 
Whole Blood Separation from Green-top Tubes (Sodium Heparin) 
Five 10 ml Green-top tubes will yield ~50 ml of blood. 
1. Invert Green-top tubes 10 times to re-suspend blood cells in plasma. 
2. Using a 10 ml pipette, pool the blood from all Green-top tubes into a T-75 sterile flask 
containing 65 ml of sterile D-PBS and gently mix the contents by swirling the flask. 
3. Place 12 ml of Ficoll-Hypaque (at room temp) into each of two 50 ml sterile conical tubes. 
4. Using a 25 ml pipette, carefully layer 37 ml of diluted blood on top of Ficoll into each 50 ml 
sterile conical tube. 
5. Centrifuge tubes for 20 minutes at 2000 rpm (900 x g) at 25°C. DO NOT use the centrifuge 
brake.  
*If only one centrifuge is available, then the centrifuge needs to be refrigerated (4oC) following 
this step for the rest of the procedure 
6. With a 10 ml pipette, remove and discard the upper layer (plasma and platelets; yellowish in 
color). 
7. Remove the PBMC interface (white in color) with 10 ml pipette and place each PBMC 
interface into a fresh 50 ml tube (total of 4 tubes). 
8. Fill the tubes up to 50 ml with cold D-PBS and centrifuge for 8 min at 1500 rpm (500 x g) at 
4°C. Use the centrifuge brake. 
9. Discard the clear D-PBS on top of the cell pellet by gently inverting tubes. 
10. Using a 5 ml pipette, re-suspend cell pellet in 5 ml cold D-PBS and gently pipette up and 
down 2-3 times to make a single cell suspension. 
11. Pool the cell suspensions into one 50 ml sterile conical tubes. 
12. Fill the tubes up to 50 ml with cold D-PBS and centrifuge for 8 min at 1300 rpm (400 x g) at 
4°C. Use the centrifuge brake. 
13. Discard the clear D-PBS on top of the cell pellet by gently inverting tubes. 
14. Pool the cells into one tube.  
 
2.2 
Freezing PBMCs 
At the time of use, FBS will be thawed in a water bath at 37oC. Freezing solution will be prepared 
at the site by mixing 45 ml of FBS and 5 ml DMSO. It is suggested that the mixture be prepared 
at the time of blood processing, prior to the freezing step. Freezing solution should be placed on 
ice and kept cold up to the moment of use.  


A071101 
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Version Date: 11/09/2018 
Update #13 
FBS and left over freezing solution can be stored at -20oC, and DMSO stored at room 
temperature. The freezing solution can be frozen again after being thawed and remaining solution 
can be used for another time point or another patient’s sample, whichever comes first. 
1. Thaw/prepare required volume of freezing solution (FBS + 10% DMSO). 
2. Label cryo-tubes (vials) with site ID, patient ID/number, and date. Always use a 
sharpie/permanent marker on the labels, not regular pens or other markers.  
3. Keep freezing solution and the necessary vials on ice at all times during the procedure (in trays, 
placed in a laboratory bucket filled with ice). 
4. Discard the clear D-PBS on top of the cell pellet by gently inverting tubes one at a time. Keep the 
rest of the tubes on ice. 
1. 
Re-suspend cell pellet with 8 ml of freezing solution. 
2. 
Aliquot 1 ml per vial (8 vials). 
3. 
Immediately place vials in -80°C freezer in a “Mr. Frosty” or equivalent slow-cooling container.  
4. 
A) For samples that will be stored at the site and batch-shipped greater than 48 hours after 
collection, the frozen PBMC vials must be transferred from the -80° C freezer into a liquid 
nitrogen freezer or a -140° freezer 24-48 hours after they have been placed in the -80° C freezer. 
They cannot be allowed to stay at  -80° C longer than 48 hours.  Each patient should have a 
separate storage box in the liquid nitrogen freezer.  When they are to be shipped, they should be 
transferred directly from the liquid nitrogen freezer or a -140° freezer to dry ice and shipped. 
B) For samples that are being shipped within 24-48 hours of being first placed in -80° freezer in a 
“Mr. Frosty” or equivalent slow-cooling container, they can be transferred from the -80° freezer 
to dry ice and shipped; at the site that receives them, they are to be transferred from dry ice to 
liquid nitrogen freezer or -140° freezer. They cannot be allowed to stay at  -80° C longer than 48 
hours.  
Note for both 4A and 4B: Use dry ice to transfer vials if necessary to maintain the temperature 
of the frozen vials. 
5. 
For sites which process and temporarily store PBMCs on site and batch ship, the freezer does not 
need to be sterile but it is recommended that it should be monitored for temperature (24/7) with 
alarms or other mechanism for identifying when temperature goes out of specification.  The 
freezer or its location should have controlled access (locked with specified group members having 
access). The freezer should also be on a preventative maintenance program and ideally be hooked 
up to a back-up power system. 
 
 
 
 
 
 
 
 
 
 


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A071101 
71 
Version Date: 11/09/2018 
Update #13 
APPENDIX VI: VACCINE RETURN INSTRUCTIONS 
I. 
Any unused or expired vaccine remaining after a patient has completed vaccine treatment, 
should be returned to Agenus according to the following instructions. 
II. 
Please contact the Agenus Clinical Team in advance (i.e. minimum of 1 day) to inform 
Agenus when you plan to return unused or expired vaccine. 
 
 
 
 
III. 
Agenus will provide a courier air-way bill for this shipment. 
IV. 
Use the qualified HSPPC-96 shipping kit that was received with the original shipment of 
vaccine from Agenus.  The shipping kit should have the following materials, which came as 
part of the vaccine shipment from Agenus: 
• 
Outer cardboard shipping box 
• 
Insulated liner and foam plug 
• 
Pressure vessel container (with foam inserts) 
• 
Cardboard pieces (2: 1 for upright positioning of base of pressure vessel and 1 for 
placement on top of pressure vessel surrounded with dry ice) 
• 
Absorbent paper (on bottom of pressure vessel) 
V. 
Place vials to be returned inside of the pressure vessel, inserted upright in the holes of the 
foam inserts.  Screw the lid shut on the pressure vessel.  Multiple patients’ vaccines may be 
returned in one pressure vessel. 
VI. 
Place a layer of dry ice on the bottom of the insulated liner that is within the outer cardboard 
shipping box.  The pressure vessel container should then be placed above this layer of dry ice, 
with the base of the pressure vessel inserted into the cardboard support.  Additional dry ice is 
then packed around the closed pressure vessel.  The shipping container must be filled with at 
least 8kg of dry ice (i.e. level with the top of the pressure vessel). A second cardboard insert 
should be placed above the dry ice, and the foam plug placed into the box.  The box is then 
sealed with packing tape.   
VII. 
The following labels should be placed on the exterior of the outer cardboard shipping box: 
o Dry ice (#9) with weight (8 kg) 
o Shipping address/content label (Who the package is shipping to)  
o Shipped from label  (Where the package is shipping from) 
VIII. 
Contact the courier to arrange for a pick-up, if a standard pick up time is not established for 
your site or if this was not done in advance. 
IX. 
Attach the courier air bill to the top of the box. The shipment should be sent overnight to: