PEskE: A Phase 0/Surgical Window-of-opportunity Study to Evaluate the Pharmacokinetics and Pharmacodynamics of Evolocumab in Patients With Recurrent High-grade Glioma or Glioblastoma
Phase: EARLY_PHASE1
Conditions: Malignant Glioma, Glioblastoma
Sponsor: Duke University

CONFIDENTIAL 
The information contained in this document is regarded as confidential, and may not be disclosed to another party unless such disclosure is 
required to initiate the study, to conduct study-related activities, or to comply with national, state, or local laws and regulations.  Written 
authorization from the coordinating site and sponsor is required for disclosure otherwise. 
PT-PhIDEE v04.30.14 
 
 
 
 
 
A Phase 0 Study 
 
PEskE: A phase 0/surgical window-of-opportunity study to evaluate the 
pharmacokinetics and pharmacodynamics of Evolocumab in patients with 
recurrent glioma or glioblastoma 
 
 
Sponsor: 
PI – Duke Cancer Institute 
 
 
Funding Source: 
Departmental Funds 
 
 
Protocol Source: 
PI - Duke Cancer Institute 
 
 
Duke IRB#: 
 
Pro000108375 
 
 
Principal Investigator 
Mustafa Khasraw, MBChB, MD, FRCP, 
FRACP 
Room 047 Baker House 
DUMC 3624 
Ph: (919) 684-5301 
Fax: (919) 684-6674 
Mustafa.khasraw@duke.edu 
  
Sub-Investigators 
David Ashley, MBBS, FRACP, PhD 
David.ashley@duke.edu  
 
Annick Desjardins, MD, FRCPC 
Annick.desjardins@duke.edu 
Margaret Johnson, MD 
Margaret.O.Johnson@duke.edu 
 
Daniel Landi, MD 
Daniel.landi@duke.edu 
 
Katherine Peters, MD, PhD 
Katherine.peters@duke.edu 
 
Kirit Singh, MBBS 
Kirit.singh@duke.edu 
 
Statistician 
James E. Herndon, II, PhD 
James.herndon@duke.edu 
Study Coordinator 
Erin Severance 
Erin.Severance@duke.edu 
 
Regulatory Coordinator 
Kate Hogan, MS 
Katherine.hogan@duke.edu 
 
Data Manager 
Eric Lipp, MD 
Eric.lipp@duke.edu 
Original version 1.0: 
20210528 
 
Amended version 1.1: 
20210718 
Revise order of secondary outcomes in Sections 3.1 and 6.  
Amended version 2.0: 
20210831 
Revise Table 4 and Sections 10.1, 10.3, and 10.4 to 
separate the lipid panel from the CMP as lipid panel is 
only required at screening and to make language in 
Section 10.1 consistent with Table 4.  All screening 
tests/procedures should be within 14 days of starting 
evolocumab with the exception of the pregnancy test (see 
also Section 9.1).  MRI has been removed at screening 
from Table 4 and Section 10.1, as disease evaluation is not 
needed for the study endpoints. Physical and neurological 
exams and clinical labs removed from Table 4 and 10.4 as 
they are SOC tests/procedures for pre-op and no study-
specified window is needed.  Clarification regarding 
commercial agent in Section 8.4. 
Amended version 2.1: 
20210927 
Updates to face page of the protocol 
Amended version 3.0 
20211027 
Remove the term “high-grade” from study title and 
throughout protocol.  While the study always included a 

comparator group, we are further defining this group as 
an unblinded, non-randomized, control arm that includes 
patients who consent to participate as a control OR 
specimens requested from the BTBR (see revisions 
throughout Sections 3, 6, 7.1, 10, 11, and 13).  A new 
schema has been added in Section 4 for the control arm.  
Eligibility in Section 9 has been reformatted to indicate 
the criteria that apply to either both arms or treatment 
arms only.  Units for eligible lab values have been 
modified. 
Amended version 4.0 
20220221 
The inclusion criterion for platelets in the treatment arm 
has been revised to allow potential participants who 
received temozolomide in the past year to have lower 
platelets (Section 9.1).  Evolocumab does not have 
myelotoxic side effects, so should not further lower 
platelets. 
Revise injection window prior to surgery from 7-14 days 
to 4-14 days (see revisions in Study Schema in Section 4, 
Table 4, and throughout Sections 7 and 10) to allow for 
subject scheduling flexibility.  
An exploratory objective has been added to assess the 
activity of T cells isolated from tumor specimens (Sections 
6, 7.3, and 13.6). 
 
Amended version 5.0 
20220414 
Biopsy is now allowed as a means to provide tissue in 
addition to surgical resection and debulking. Revised 
inclusion criterion #3 to include biopsy (Section 9.1).   
Revised language to be inclusive of biopsy material 
including reducing minimum amount of tissue collection 
needed to 20 mg from 50 mg. (See revisions throughout 
Sections 3, 6, 7, 9, 10, and 13; Study Schema Figure 1 and 
Figure 2, and Table 4) 
 
 
 

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1 TABLE OF CONTENTS 
1 
TABLE OF CONTENTS ............................................................................................................................................ 3 
2 
LIST OF ABBREVIATIONS ....................................................................................................................................... 7 
3 
PROTOCOL SYNOPSIS AND RESEARCH SUMMARY ............................................................................................... 9 
3.1 
Purpose ........................................................................................................................................................ 9 
3.2 
Background and Significance ....................................................................................................................... 9 
3.3 
Design and Procedure ................................................................................................................................ 10 
3.4 
Selection of Subjects .................................................................................................................................. 10 
3.5 
Duration of Study ....................................................................................................................................... 10 
3.6 
Data Analysis and Statistical Considerations .............................................................................................. 10 
4 
STUDY SCHEMA .................................................................................................................................................. 12 
5 
BACKGROUND AND SIGNIFICANCE .................................................................................................................... 13 
5.1 
Study Disease ............................................................................................................................................. 13 
5.2 
Study Agent ................................................................................................................................................ 13 
5.2.1 
Pre-clinical experience ...................................................................................................................... 13 
5.2.2 
Clinical Experience ............................................................................................................................. 18 
5.3 
Study Purpose/Rationale ........................................................................................................................... 20 
6 
OBJECTIVES AND ENDPOINTS ............................................................................................................................ 21 
7 
INVESTIGATIONAL PLAN ..................................................................................................................................... 22 
7.1 
Study Design ............................................................................................................................................... 22 
7.1.1 
Dose Escalation and Expansion ......................................................................................................... 22 
7.1.2 
Definition of Dose-Limiting Toxicity (DLT) ......................................................................................... 22 
7.1.3 
Dose Modification ............................................................................................................................. 24 
7.1.4 
Safety Considerations ........................................................................................................................ 24 
7.1.5 
Missed Doses ..................................................................................................................................... 24 
7.1.6 
Concomitant Medications ................................................................................................................. 24 
7.1.7 
Study Drug Blinding ........................................................................................................................... 24 
7.2 
Rationale for Selection of Dose, Regimen, and Treatment Duration ......................................................... 24 
7.3 
Rationale for Correlative Studies ............................................................................................................... 25 
7.4 
Definition of Evaluable Subjects, On Study, and End of Study ................................................................... 25 
7.5 
Early Study Termination ............................................................................................................................. 25 
8 
STUDY DRUG ...................................................................................................................................................... 25 
8.1 
Names, Classification, and Mechanism of Action ...................................................................................... 25 
8.2 
Packaging and Labeling .............................................................................................................................. 26 
8.3 
Supply, Receipt, and Storage ..................................................................................................................... 26 
8.4 
Dispensing and Preparation ....................................................................................................................... 26 

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8.5 
Compliance and Accountability .................................................................................................................. 26 
8.6 
Disposal and Destruction ........................................................................................................................... 26 
9 
SUBJECT ELIGIBILITY ........................................................................................................................................... 27 
9.1 
Inclusion Criteria ........................................................................................................................................ 27 
9.2 
Exclusion Criteria ........................................................................................................................................ 27 
10 
SCREENING AND ON-STUDY TESTS AND PROCEDURES ..................................................................................... 27 
10.1 
Screening Examination ............................................................................................................................... 28 
10.2 
Run-In Period ............................................................................................................................................. 29 
10.3 
Treatment Period ....................................................................................................................................... 29 
10.4 
Active Follow-up Period ............................................................................................................................. 29 
10.5 
End of Treatment ....................................................................................................................................... 30 
10.6 
Follow-up Period ........................................................................................................................................ 30 
10.7 
End of Study ............................................................................................................................................... 30 
10.8 
Early Withdrawal of Subject(s) ................................................................................................................... 30 
10.8.1 
Criteria for Early Withdrawal ............................................................................................................. 30 
10.8.2 
Follow-up Requirements for Early Withdrawal ................................................................................. 30 
10.8.3 
Replacement of Early Withdrawal(s) ................................................................................................. 31 
10.9 
Study Assessments ..................................................................................................................................... 31 
10.9.1 
Medical History ................................................................................................................................. 31 
10.9.2 
Physical Exam .................................................................................................................................... 31 
10.9.3 
Correlative Assessments.................................................................................................................... 31 
11 
SAFETY MONITORING AND REPORTING ............................................................................................................ 31 
11.1 
Adverse Events ........................................................................................................................................... 31 
11.1.1 
AEs of Special Interest ....................................................................................................................... 32 
11.1.2 
Reporting of AEs ................................................................................................................................ 32 
11.2 
Serious Adverse Events .............................................................................................................................. 32 
11.2.1 
Reporting of SAEs .............................................................................................................................. 32 
11.3 
Emergency Unblinding of Investigational Treatment ................................................................................ 33 
11.4 
Other Reportable Information ................................................................................................................... 33 
11.5 
Special Warnings and Precautions ............................................................................................................. 33 
11.6 
Safety Oversight Committee (SOC) ............................................................................................................ 33 
11.7 
External Data and Safety Monitoring Board (DSMB) ................................................................................. 33 
12 
QUALITY CONTROL AND QUALITY ASSURANCE ................................................................................................. 33 
12.1 
Monitoring ................................................................................................................................................. 33 
12.2 
Audits ......................................................................................................................................................... 34 
12.3 
Data Management and Processing ............................................................................................................ 35 
12.3.1 
Case Report Forms (CRFs) ................................................................................................................. 35 

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12.3.2 
Data Management Procedures and Data Verification ...................................................................... 35 
12.3.3 
Study Closure ..................................................................................................................................... 35 
13 
STATISTICAL METHODS AND DATA ANALYSIS .................................................................................................... 35 
13.1 
Analysis Sets ............................................................................................................................................... 35 
13.2 
Patient Demographics and Other Baseline Characteristics ........................................................................ 35 
13.3 
Treatments ................................................................................................................................................. 36 
13.4 
Primary Objective ....................................................................................................................................... 36 
13.4.1 
Variable ............................................................................................................................................. 36 
13.4.2 
Statistical Hypothesis, Model, and Method of Analysis .................................................................... 36 
13.4.3 
Handling of missing values, censoring, and discontinuations ........................................................... 36 
13.5 
Secondary Objectives ................................................................................................................................. 36 
13.5.1 
Key Secondary Objectives #1 and #2: Lipid Metabolism and Tumor Cells Expressing MHC-I ........... 36 
13.5.2 
Secondary Objective #3: Serum and Intratumoral Levels of Evolocumab ........................................ 36 
13.6 
Exploratory Objectives ............................................................................................................................... 37 
13.7 
Interim Analysis .......................................................................................................................................... 37 
13.8 
Sample Size Calculation .............................................................................................................................. 37 
14 
ADMINISTRATIVE AND ETHICAL CONSIDERATIONS ........................................................................................... 37 
14.1 
Regulatory and Ethical Compliance ............................................................................................................ 37 
14.2 
DUHS Institutional Review Board and DCI Protocol Review and Monitoring Committee ......................... 37 
14.3 
Informed Consent ...................................................................................................................................... 38 
14.4 
Study Documentation ................................................................................................................................ 38 
14.5 
Privacy, Confidentiality, and Data Storage ................................................................................................. 39 
14.6 
Data and Safety Monitoring ....................................................................................................................... 39 
14.7 
Protocol Amendments ............................................................................................................................... 39 
14.8 
Records Retention ...................................................................................................................................... 39 
15 
REFERENCES ....................................................................................................................................................... 40 
 
 
Table 1. Adverse reactions occurring in ≥ 3% of Evolocumab-treated patients and more frequently than with 
placebo in a 52-week trial ........................................................................................................................................... 19 
Table 2. Adverse reactions occurring in ≥ 1% of Evolocumab-treated patients and more frequently than with 
placebo in pooled 12-week trials ................................................................................................................................. 19 
Table 3. Formulations of Evolocumab ......................................................................................................................... 26 
Table 4. Schedule of Events for Treatment Arm .......................................................................................................... 27 
 
 
Figure 1. Study schema for newly diagnosed or recurrent glioma patients receiving Evolocumab ............................ 12 
Figure 2. Study schema for control patients ................................................................................................................ 12 
Figure 3. PCSK9 depletion attenuates tumor growth in syngeneic mice..................................................................... 14 
Figure 4. PCSK9 knockout combined with anti-PD1 therapy enhances ....................................................................... 15 
Figure 5. Inhibition of PCSK9 overcomes tumor resistance to anti-PD1 therapy ........................................................ 15 
Figure 6. Quantitation of immune effector cells in PCSK9-knockout B16F10 tumors ................................................. 16 

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Figure 7. CD8+ T cell depletion in PCSK9-knockout in B16F10 melanoma models ..................................................... 16 
Figure 8. MHC-1 Expression ......................................................................................................................................... 17 
Figure 9. PCSK9 knockout effect on MHC I localization ............................................................................................... 17 
Figure 10. Proposed mechanism for PCSK9i on increasing cell surface MHC I expression ......................................... 18 

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2 LIST OF ABBREVIATIONS 
AE 
 
Adverse Event 
ALT 
 
Alanine Aminotransferase  
AST 
 
Aspartate Aminotransferase 
BBB 
 
Blood Brain Barrier 
BTBR 
 
Brain Tumor Center Biorepository 
CBC 
 
Complete Blood Count 
CFSE 
 
Carboxyfluorescein Diacetate Succinimidyl Ester 
CMP 
 
Comprehensive Metabolic Panel  
CNS 
 
Central Nervous System 
CR 
 
Complete Response 
CT 
 
Computed Tomography 
CTCAE  
Common Terminology Criteria for Adverse Events  
CTL 
 
Cytotoxic T-Lymphocyte 
DLT 
 
Dose Limiting Toxicity 
DVT 
 
Deep Vein Thrombosis 
DSMB 
 
Data and Safety Monitoring Board  
ECOG 
 
Eastern Cooperative Oncology Group  
FACS 
 
Fluorescence-activated cell sorting  
GO 
 
Gene ontology 
H&P 
 
History & Physical Exam 
HRPP 
 
Human Research Protections Program  
ICI 
 
Immune Checkpoint Inhibition 
IDH 
 
Isocitrate dehydrogenase 
IV 
 
Intravenously 
KPS 
 
Karnofsky Performance Status 
LC-MS/MS 
Liquid chromatography coupled to tandem mass spectrometry  
LDL  
 
Low-density lipoproteins 
MGMT   
O-6-methylguanine-DNA methyltransferase 
MTD 
 
Maximum Tolerated Dose  
NCI 
 
National Cancer Institute 
ORR 
 
Overall Response Rate 
OS 
 
Overall Survival 
PBMCs  
Peripheral Blood Mononuclear Cells 
PCSK9i  
Proprotein convertase subtilisin/kexin type 9 serine protease inhibitor  
PK 
 
Pharmacokinetics 
PD 
 
Pharmacodynamics 
PD1 
 
Programmed Death 1 
PFS 
 
Progression Free Survival 
p.o. 
 
per os/by mouth/orally 
PR 
 
Partial Response 
PRTBTC  
Preston Robert Tisch Brain Tumor Center 
SAE 
 
Serious Adverse Event 
SD 
 
Stable Disease 
SGOT 
 
Serum Glutamic Oxaloacetic Transaminase  
SIL 
 
Stable isotope-labeled 
SPGT 
 
Serum Glutamic Pyruvic Transaminase 
STRING  
Search tool from the retrieval of interactive genes/proteins  
TIL 
 
Tumor Infiltrating Lymphocyte 

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TME 
 
Tumor Microenvironment 
TMZ 
 
Temozolomide 
WBC 
 
White Blood Cells 
WHO 
 
World Health Organization  
XRT 
 
Radiation therapy

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3 PROTOCOL SYNOPSIS AND RESEARCH SUMMARY 
3.1 Purpose 
This Phase 0 surgical window of opportunity trial seeks to evaluate the pharmacokinetic (PK) and 
pharmacodynamic (PD) properties of an FDA-approved proprotein convertase/ kexin type 9 serine 
protease inhibitor (PCSK9i; Evolocumab) in patients with primary and recurrent glioma at the Preston 
Robert Tisch Brain Tumor Center (PRTBTC). Specifically, it will address the following objectives. 
 
Primary Objectives: 
Evaluate whether evolocumab crosses the blood brain barrier (BBB) and is measurable in the resected or 
biopsied tumor specimens of patients with primary and recurrent glioma. 
 
Secondary Objectives: 
1. Among patients treated with evolocumab, assess the association between serum and intratumoral levels 
of evolocumab in the patients treated with evolocumab. 
2. Compare the PDs of evolocumab in treated patients relative to that observed in a control cohort of 
untreated patients. The characterization of the PD properties will include the following: 
a. Lipid metabolism of tumor cells taken from resected or biopsied samples 
b. Analysis of tumor cells expressing MHC-I will be performed by  fluorescence-activated cell sorting 
(FACS) 
 
Hypotheses: 
We hypothesize that: 
1. The PCSK9 inhibitor evolocumab can cross the BBB and accumulate intratumorally 
2. Treatment with evolocumab induces measurable biologic effects when administered to patients with 
glioma. 
3.2 Background and Significance 
Glioma, specifically glioblastoma (GBM), accounts for 46.6% of primary brain tumors, affects approximately 
10,996 people in the US annually 2, and results in a high mortality rate with a median survival ranging from 
16.1 to 20.5 months for patients with newly diagnosed GBM 3-5. Despite aggressive, image-guided tumor 
resection 6, high-dose external beam radiotherapy or brachytherapy, and recent advances in anti-
angiogenic 7 treatments and chemotherapy, patients with GBM typically live less than 15 months from the 
time of diagnosis 8,9. Immunotherapy has revolutionized treatment for several hard-to-treat cancers but 
has failed to make significant improvements in outcomes for patients with glioblastoma 10. This reflects the 
multiple and unique mechanisms of immune evasion and escape in this highly heterogeneous tumor 11. 
Glioblastoma engenders profound local and systemic immunosuppression and is remarkably effective at 
inducing T cell dysfunction, posing a challenge to any immunotherapy-based approach. Through the use of 
a genetic screen, proprotein convertase subtilisin/kexin type 9 serine protease (PCSK9) has been identified 
as important in tumor escape from immune mediated rejection. PCSK9 is a secreted protein that binds to 
the low-density lipoprotein (LDL) receptor redirecting it to the lysosome for degradation rather than 
recycling to the plasma membrane. In preclinical experiments, genetic depletion of PCSK9 in murine tumor 
cells significantly attenuates their abilities to form tumors in a T cell dependent manner. Furthermore, 
PCSK9 depletion overcomes resistance to anti-programmed death 1 (PD1) immune checkpoint therapy 
with a high fraction of mice free of tumors and resistant to later challenges with parental tumor cells. A 
significant increase has also been observed in both the total numbers and diversity of T cells in the PCSK9 
deficient tumors. Importantly, PCSK9 down-regulates MHC-I expression, in a manner similar to its 
regulation of LDL receptor 1. 
 

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Evolocumab is a PCSK9i with a well-established safety profile, is FDA-approved as a lipid-lowering agent, 
and is widely used to treat patients who have elevated levels of LDL 12. To explore the role of PCSK9i in 
glioma, we need first to confirm that Evolocumab crosses the blood brain barrier and achieves 
measurable concentration in human glioma. In this protocol, we will compare levels of Evolocumab in the 
tissue of treated patients to that observed in control subjects after we develop an assay that can measure 
the level of Evolocumab in resected or biopsied brain glioma tissue. This protocol uses a surgical window 
of opportunity approach, collecting tissue from patients undergoing procedures of their primary or 
recurrent glioma to characterize the pharmacokinetic and pharmacodynamics properties of PCSK9i in 
patients with glioma. We will initiate this trial as a first step to assess if it is worthwhile to repurpose 
Evolocumab as an immunotherapeutic agent for treatment of patients with glioma. 
3.3 Design and Procedure 
A maximum of 10 patients with pathologically documented glioma (either newly diagnosed or at first 
progression/ recurrence) will be treated with Evolocumab in this study after providing informed consent. 
Patients will receive 420 mg (the maximum single dose) of Evolocumab subcutaneously into thigh, 
abdomen or upper arm 4-14 days prior to a surgical procedure or biopsy of their tumor. After the 
procedure, leftover tissue not required for histological analysis will be collected, and the level of 
evolocumab will be quantified using liquid chromatography coupled to tandem mass spectrometry (LC- 
MS/MS). At two time points, prior to injection of Evolocumab and at time of surgery or biopsy, peripheral 
blood will be drawn to analyze serum levels of the drug (for comparison to intratumoral). Twenty to one 
hundred milligrams of tumor per patient will be homogenized and normalized using a protein assay 
followed by reduction/alkylation and trypsin digestion. Stable isotope-labelled (SIL) peptides corresponding 
to evolocumab-specific peptides will be spiked into tryptic digests, and LC-MS/MS will be used for targeted 
quantification of the drug-derived and SIL forms of these Evolocumab-specific peptides. Calibration curves 
will be generated by spiking in of Evolocumab into blank matrix (tumor homogenates from the brain 
specimen repository) followed by targeted LC-MS/MS. Data analysis, including normalization to SIL 
standards and fitting of data to the calibration curve will be performed in Skyline as previously described 13. 
If necessary, an immunoprecipitation step using Protein A/G agarose will be utilized to enrich for 
immunoglobulins (including evolocumab) prior to trypsin digestion and LC-MS/MS.  
 
A contemporaneous control arm of 20 patients who undergo craniotomy (without receiving Evolocumab) 
will be consented to participate in this study as control subjects and prospectively observed. Although non-
randomized, these specimens will provide valuable information for exploratory comparison of the 
evolocumab treated and evolocumab untreated tumor specimens and their immune microenvironment.  If 
there are difficulties recruiting prospectively patients to serve as a control group, samples from the Brain 
Tumor Center Biorepository (BTBR) (Pro00007434) will be considered as possible control subjects.  
3.4 Selection of Subjects 
Please refer to Section 9.  
3.5 Duration of Study 
Patients’ active participation in the treatment arm of the study will last approximately four weeks, 
including a two-week screening period, one day for their surgical procedure and two-week safety follow-up 
period. 
 
Control patients’ participation ends after their surgical procedure. 
3.6 Data Analysis and Statistical Considerations 
The primary and secondary objectives are comparisons of two cohorts with respect to PK and PD 
measures: treated and untreated brain tumor specimens. Specifically, tumor specimens will be assessed for 
lipid metabolism, and whether tumor cells express MHC-I. Depending upon the distribution of these 

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outcomes, either a Wilcoxon rank sum test or a two-sample t-test will compare cohorts. Spearman’s 
correlation coefficient will describe the association between serum and intratumoral levels of Evolocumab. 
 
In exploratory analyses, the hypothesis that Evolocumab treatment will lead to global changes in pro-
tumorigenic pathways and the reduction in PSCK9 levels will be assessed. To test this hypothesis, non-
targeted quantitative LC-MS/MS will be performed on resected or biopsied tumor from n=10 treated 
patients versus n=20 untreated patients. Furthermore, if PSCK9 is not among the proteins quantified by 
non-targeted LC-MS/MS, a more sensitive targeted LC-MS/MS analysis will be utilized as described for the 
PK analysis of Evolocumab as described above. Bioinformatics analyses of differentially expressed proteins 
will utilize protein set enrichment, gene ontology (GO) annotations, and search tool from the retrieval of 
interactive genes/proteins (STRING). Adjustments for multiple comparisons will be made. 

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4 STUDY SCHEMA 
 
 
Figure 1. Study schema for newly diagnosed or recurrent glioma patients receiving Evolocumab 
 
Figure 2. Study schema for control patients 
 
 

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5 BACKGROUND AND SIGNIFICANCE 
5.1 Study Disease 
Primary brain tumors are more common than Hodgkin’s disease and account for more human deaths than 
melanoma or other cancers such as bladder or kidney. Glioblastoma (GBM) is the most common primary 
brain tumor, and despite aggressive, computer-guided tumor resection 6, high-dose external beam 
radiation therapy (XRT), and multi-mechanistic chemotherapy delivered at toxic doses, most patients with 
GBM live < 15 months from the time of diagnosis, and patients with recurrent tumors usually survive < 12 
weeks 8,9,14-17). The non-specific nature of standard of care therapy for brain tumors often results in 
incapacitating damage to surrounding normal brain and systemic tissues 18,19. 
 
Immunotherapies have come to the forefront of treatment for many primary tumors, including gliomas. 
Unfortunately, gliomas are immunologically “cold” tumors that do not respond well to traditional 
immunotherapies targeted at checkpoint blockades. An important means by which some glioblastoma cells 
avoid immune-mediated rejection is through the reduced expression of MHC class I molecules, which are 
necessary for the binding of T cell receptors (TCRs) and therefore the tumor cells are ignored by T cells (He 
et al., 2019). However, recent work by Liu et al has demonstrated that antibody based inhibitors of 
proprotein convertase subtilisin/ kexin type 9 (PCSK9) can potentially enhance the efficacy of immune 
checkpoint inhibition by increasing MHC class I molecule expression 1. 
5.2 Study Agent 
Evolocumab is a fully human IgG2 monoclonal antibody PCSK9 inhibitor, currently approved by the FDA for 
use as a lipid lowering agent 20. PCSK9 has important roles in LDL receptor trafficking and determination of 
cardiovascular risk. Evolocumab was studied in a randomized controlled phase III study of 27,564 patients, 
which demonstrated significant reduction in LDL cholesterol and the primary endpoint of cardiovascular 
death, myocardial infarction, stroke, hospitalization for unstable angina, or coronary revascularization, 
resulting in its FDA approval (11.3% vs 9.8% in placebo; HR 0.85, P<0.001) 12. Evolocumab is typically 
administered as a 140-mg subcutaneous injection once every 2 weeks or 420 mg monthly and has been 
shown to have a favorable safety profile, with adverse events overlapping entirely with placebo except for 
injection site reactions 21,22. 
5.2.1 Pre-clinical experience 
5.2.1.1 PCSK9 Knockout Experiments 
Liu et al performed several sets of experiments using immunocompetent syngeneic mice and 
multiple cancer cell lines (B16F10 – melanoma, 4T1 – mammary carcinoma, MC38 – colon 
adenocarcinoma and CT26 – undifferentiated colon carcinoma). By performing CRISPR-cas9 gene 
editing, tumors with PCSK9 knocked out exhibited significantly delayed growth when compared 
to wild-type (data shown in Figure 3). 
 
Although PCSK9 inhibitors were developed to target the low-density lipid receptor (LDLR), and 
cause intracellular degradation, subsequent experiments lowering LDLR demonstrated no anti-
tumor effects or effect on surface expression of major histocompatibility protein class I (MHC I) 
molecules. This indicates that the growth retardation induced by PCSK9 knockout was caused by 
a mechanism separate to that involving the LDLR 23. 
 

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About 1 × 105 vector control and the same number of PCSK9-knockout mouse tumor cells of different types were 
inoculated subcutaneously into syngeneic mice and monitored for tumor formation. Both tumor size and overall 
survival were monitored. a, b, 4T1 breast cancer line, grown in Balb/c mice. n = 9 and 20 mice for control and 
PCSK9-knockout tumor cells, respectively. c, d, B16F10 melanoma line, grown in C57BL/6 mice. n = 12 mice for both 
groups. e, f, CT26 colon cancer line, grown in Balb/c mice. n = 5 mice for both groups. g, h, MC38 colon cancer line, 
grown in C57BL/6 mice. n = 5 mice for both groups. Data shown as mean =/- s.e.m. P values were calculated by two-
way analysis of variance (ANOVA) in a, c, e, g and log- rank test in b, d, f, h. Figure taken from Liu et al in Nature 
2020 1. 
5.2.1.2 PCSK9 knockout combined with anti-PD1 
Further work to evaluate if PCSK9 was a promising target for anti-cancer therapy involved  treating 
syngeneic mice with PCSK9 knock out tumors with a mouse anti-PD1 antibody. Anti-PD1 blocks 
the binding of the immune checkpoint PD1 receptor on T cells with the PD-L1 ligand on tumor 
cells, and therefore increasing T cell activation and subsequently tumor cell death. As described in 
Figure 4, knockout of PCSK9 combined with anti-PD1 resulted in significantly decreased tumor 
volume and greatly enhanced survival in both B16F10 melanoma models and MC38 colon cancer. 
 
Figure 3. PCSK9 depletion attenuates tumor growth in syngeneic mice. 

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Figure 4. PCSK9 knockout combined with anti-PD1 therapy enhances 
5.2.1.3 PCSK9 inhibitors combined with anti-PD1 
 
Figure 5. Inhibition of PCSK9 overcomes tumor resistance to anti-PD1 therapy 
Treatment of MC38 colon cancer with combined anti-PCSK9 antibodies (isotype antibody control, Iso; evolocumab, 
Evo; or alirocumab, Ali) and anti-PD1 antibodies in mice. (a) Experimental protocol; (b) tumour growth curve; (c) 
overall survival. n = 10 mice per group. Shown are combined results from two separate experiments. Data shown as 
mean ± s.e.m. Figure taken from Liu et al in Nature 2020 1. 
 
When considering PCSK9 as an anti-cancer target, clinically approved agents are readily available. 
Evolocumab and Alirocumab are anti-PCSK9 blocking antibodies, approved by the FDA for 
treatment of hypercholesteremia or to reduce cardiovascular risk 20. When evaluated in MC38 
colon cancer tumors, combined with anti-PD1 antibody treatment, significant increases in long-
term survival were seen. This is particularly of note given that anti-PD1 resistant tumors (MC38R) 
also responded to evolocumab, suggesting that PCSK9 plays an important role in the anti-PD1 
resistance pathway (shown in Figure 5). 
a 
b 
c 
(a–c) Treatment of vector control 
and PCSK9-knockout B16F10 
melanoma with an anti-PD1 
antibody in syngeneic mice. (a) 
Experimental protocol; (b) tumor 
growth curve; (c) overall survival. N 
= 5, 5, 5, and 20 mice in the four 
groups, respectively. i.p., intra- 
peritoneal. (d–f) Treatment of vector 
control and PCSK9-knockout MC38 
colon cancer with an anti- PD1 
antibody in syngeneic mice. (d) 
Experimental protocol; e, tumor 
growth curve; (f) overall survival. N 
= 5 mice per group. Data shown as 
mean ± s.e.m. Figure taken from Liu 
et al in Nature 2020 (Liu et al., 
2020). 

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5.2.1.4 Mechanism of anti-tumor effect 
These data appear to demonstrate that anti-PCSK9 antibodies when combined with anti- PD1 can 
enhance survival and overcome anti-PD1 resistance. As mentioned, the mechanism by which this 
occurs appears to be completely separate to the LDLR pathway for which it is currently in clinical 
use. To understand the mechanism by which evolocumab exerts this effect, flow cytometry was 
undertaken to profile immune effector cells present within PCSK9-knockout tumors (shown in 
Figure 6). 
 
(a–e) Quantitative estimate of various immune effector cells per milligram of tumor tissue in vector control and PCSK9-
knockout B16F10 tumors, as determined by flow cytometry. n = 7 tumors per group. NK, natural killer cell. f, Ratio of 
CD8+ cytotoxic T lymphocytes (CTLs) to CD4+ Foxp3+ Treg cells in control and PCSK9-knockout B16F10 tumors. n = 7 
tumors per group. (g, h) Average numbers of tumor- infiltrating IFN-γ+ CD8+ T cells (g) and GZMB+ CD8+ T cells (h) 
per milligram of tumor tissue in control or PCSK9-knockout tumors. n = 7 tumors per group. Figure taken from Liu et a 
in, Nature 2020 1. 
 
These knockout tumors displayed significantly increased number of tumor-infiltrating 
lymphocytes (TILs), with intratumoral IFN- γ+ cytotoxic T lymphocytes (CTLs) exhibiting grater 
response to evolocumab than on treatment with anti-PD1 antibody. Notably, the ability of PCSK9 
knockout to inhibit tumor growth could be abrogated by depletion of CD8+ T cells, in contrast to 
CD4+ depletion which had minimal effect (shown in Figure 7) 1. 
 
 
 
Figure 6. Quantitation of immune effector cells in PCSK9-knockout B16F10 tumors 
a 
b 
Tumor growth (a) and host survival (b) 
from control and PCSK9-knockout B16F10 
tumor cells in C57BL/6 mice depleted of 
CD8+ T cells. n = 6, 5, 5 tumors in the three 
groups. Figure taken from Liu et al in 
Nature 2020 (Liu et al., 2020). 
 
Figure 7. CD8+ T cell depletion in PCSK9-knockout in B16F10 melanoma models 

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c 
a 
b 
 
Figure 8. MHC-1 Expression 
The key effect of CD8+ cytotoxic T cells for PCSK9 knockout anti-tumor effect was shown to be 
correlated with significantly increased expression of MHC I molecules on the surface of tumors in 
which PCSK9 was absent (Figure 8). Notably this effect is absent in LDL receptor knockout 
mice, indicated a separate pathway. This increased expression was also shown to be accompanied 
by an increase in the diversity of T cell receptors, allowing for greater binding potential for CTLs 
and subsequent tumor killing. MHC I expression could be induced by PCSK9 inhibitors, which 
could then be reversed when PCSK9 levels were restored by means of an exogenous protein. 
 
In PCSK9-overexpressing cells, more H2-K1 (mouse MHC class I antigen) was localized in the 
lysosome and less in the plasma membranes (top panels). On the other hand, in PCSK0-knockout 
cells, H2-K1 staining indicated greater localization to the plasma membrane (lower panels) 
Western blot analysis of fractionated cellular lysates confirmed the immunofluorescence staining 
results 1. 
 
By upregulating MHC I expression, greater numbers of intracellular peptides are displayed at the 
cell surface for recognition by CD8+ T cells. These MHC I-peptide complexes exist at the cell 
surface, awaiting recognition before being recycled into the cell membrane. This process begins 
with the formation of endocytic vesicles, which are then sorted by endosomes to be redirected 
to the cell surface or destroyed by lysosomes. Intriguingly, PCSK9 has been shown to interact 
Figure 9. PCSK9 knockout effect on MHC I localization 
MHC-I expression on the surface of  (a) 
Control and PCSK9KO B16F10 cells grown 
in C57BL/6 mice; (b) control and LDLR-KO 
B16F10 cells grown in vitro; (c) LDLR KO 
with or without PCSK9 double knockout 
B16F10 cells grown in vitro. Figure taken 
from Liu et al in Nature 2020 1 

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with MHC I, resulting in its increased trafficking to lysosomes and blocks its return to the cell 
membrane. This results in cells which express PCSK9 having reduced surface levels of MHC I 
which allows them to escape T cell recognition (confocal images shown in Figure 9 and 
mechanism described in Figure 10 1). 
 
 
Figure 10. Proposed mechanism for PCSK9i on increasing cell surface MHC I expression 
As PCSK9 has also been shown to induce degradation of other receptors such as LDLR, CD81 and 
CD36, it may be reasonable to assume that PCSK9 has a key regulatory role in endocytosis of 
membrane receptors and their diversion to lysosomal degradation 23. Therefore, inhibition and 
its resultant upregulation of MHC I may result in greater recognition by CTLs and thus enhanced 
immune effector cell function. 
5.2.2 Clinical Experience 
Standard treatment for hypercholesterolemia relied on diet modification and use of statins until the 
introduction of antibodies, such as evolocumab, that inhibit proprotein convertase subtilisin-kexin type 9 
(PCSK9). Evolocumab is an antibody that binds to PCSK9, where the antibody’s therapeutic effect is to 
reduce LDL-cholesterol and where the therapeutic goal is to reduce risk for heart attack and stroke. 
 
In phase II and III clinical trials (Open Label Study of Long Term Evaluation Against LDL- C [OSLER-1 and 
OSLER-2], respectively) evolocumab decreased LDL cholesterol by 52%, decreased total cholesterol by 
36%, and raised high-density lipoprotein cholesterol by 7% when compared to standard treatment 
(Sabatine et al., 2015). A follow-up study (Further Cardiovascular Outcomes Research with PCSK9 
Inhibition in Patients with Elevated Risk [FOURIER]) was performed to evaluate effects on reducing 
cardiovascular endpoints, such as myocardial infarction, stroke, unstable angina, or need for coronary 
revascularization. The combination of evolocumab plus standard statin therapy significantly reduced the 
risk of cardiovascular death, myocardial infarction, or stroke by 20%, which correlated with decreases in 
LDL levels 12. Evolocumab is currently FDA-approved for treatment of hypercholesterolemia in 
combination with other lipid-lowering medications (e.g., statins, ezetimibe), and is only recommended as 

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single- agent therapy in patients diagnosed with primary hyperlipidemia, which is typically an inherited 
disease. It is given as a 140-mg subcutaneous injection once every 2 weeks or 420 mg monthly 24. 
 
Peak plasma concentrations occur at 3–7 days 21 and evolocumab has a half-life of 11–17 days and like 
other monoclonal antibodies, is eliminated by degradation to short peptides and amino acids. When 
considering adverse reactions, this has been well characterized as part of the FDA approval process. In a 
52- week, double-blind, randomized, placebo-controlled trial, 599 patients received 420 mg of 
evolocumab subcutaneously once monthly. The mean age was 56 years (range: 22 to 75 years), 23% were 
older than 65 years, 52% women, 80% White, 8% Black, 6% Asian; 6% identified as Hispanic ethnicity. 
Adverse reactions reported in at least 3% of evolocumab-treated patients, and more frequently than in 
placebo-treated patients are shown in Table 1. 
 
Table 1. Adverse reactions occurring in ≥ 3% of Evolocumab-treated patients and more frequently than 
with placebo in a 52-week trial 
 
Placebo  (N=302)% 
Evolocumab (N=599)% 
Nasopharyngitis 
9.6 
10.5 
Upper respiratory tract 
infection 
6.3 
9.3 
Influenza 
6.3 
7.5 
Back Pain 
5.6 
6.2 
Injection site reactions 
5.0 
5.7 
Cough 
3.6 
4.5 
Urinary tract infection 
3.6 
4.5 
Sinusitis 
3.0 
4.2 
Headache 
3.6 
4.0 
Myalgia 
3.0 
4.0 
Dizziness 
2.6 
3.7 
Musculoskeletal pain 
3.0 
3.3 
Hypertension 
2.3 
3.2 
Diarrhea 
2.6 
3.0 
Gastroenteritis 
2.0 
3.0 
 
Adverse reactions led to discontinuation of treatment in 2.2% of evolocumab-treated patients and 1% of 
placebo-treated patients. The most common adverse reaction that led to evolocumab treatment 
discontinuation and occurred at a rate greater than placebo was myalgia (0.3% versus 0% for evolocumab 
and placebo, respectively) 24. 
 
In seven pooled 12-week, double-blind, randomized, placebo-controlled trials, 993 patients received 140 
mg of evolocumab subcutaneously every 2 weeks and 1059 patients received 420 mg of evolocumab 
subcutaneously monthly. The mean age was 57 years (range: 18 to 80 years), 29% were older than 65 
years, 49% women, 85% White, 5% Black, 9% Asian; 5% identified as Hispanic ethnicity. Adverse reactions 
reported in at least 1% of evolocumab-treated patients, and more frequently than in placebo-treated 
patients, are shown in Table 2 24. 
 
Table 2. Adverse reactions occurring in ≥ 1% of Evolocumab-treated patients and more frequently than 
with placebo in pooled 12-week trials 
 
Placebo (N=1224) % 
Evolocumab* (N=2052) % 

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Nasopharyngitis 
3.9 
4.0 
Back Pain 
2.2 
2.3 
Upper respiratory tract infection 
2.0 
2.1 
Arthralgia 
1.6 
1.8 
Nausea 
1.2 
1.8 
Fatigue 
1.0 
1.6 
Muscle spasms 
1.2 
1.3 
Urinary tract infection 
1.2 
1.3 
Cough 
0.7 
1.2 
Influenza 
1.1 
1.2 
Confusion 
0.5 
1.0 
*140mg every 2 weeks and 420mg once monthly combined 
5.3 Study Purpose/Rationale 
Despite aggressive, computer-guided tumor resection 6, high-dose external beam radiation therapy (XRT), 
and multi-mechanistic chemotherapy delivered at toxic doses, most patients with glioblastoma (GBM) live 
< 15 months from the time of diagnosis, and patients with recurrent tumors usually survive < 12 weeks 
8,9,14-17. The non-specific nature of standard of care therapy for brain tumors often results in incapacitating 
damage to surrounding normal brain and systemic tissues 18,19. Immunotherapies have come to the 
forefront of treatment for many primary tumors, including gliomas. Unfortunately, gliomas are 
immunologically “cold” tumors that do not respond well to traditional immunotherapies targeted at 
checkpoint blockades. 
 
Recent work by Liu et al has been demonstrated that PCSK9 plays a role in regulating recycling of the major 
histocompatibility class I (MHC-I) and enhances tumor antigen presentation, independent of its role in LDL-
receptor regulation 1. PCSK9 deficient tumors show increased MHC-I expression and CD8+ T-cell infiltration. 
PCSK9 knockout or inhibition synergizes with anti-PD-1 therapy in murine models of melanoma, colorectal 
cancer, and breast cancer. We therefore intend to evaluate if a clinically licensed PCSK9i such as 
Evolocumab can be repurposed as a potential immunotherapeutic for glioma by testing its ability to access 
the intracranial space. 

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6 OBJECTIVES AND ENDPOINTS 
 
Objective 
Endpoint 
Analysis 
Primary 
Evaluate the pharmacokinetics  (PK) of 
evolocumab and whether it can cross the 
blood brain barrier (BBB) to accumulate 
intratumorally in patients with primary and
recurrent glioma or glioblastoma 
Presence of evolocumab detected 
in surgical resection or biopsy 
samples by means of mass 
spectrometry 
See Section 13.4 
Secondary 
Compare lipid metabolism of tumor cells 
taken from resected or biopsied samples 
after dose of evolocumab to resected or 
biopsied samples obtained from s from 
untreated glioma patients.  
Level of lipid metabolism within 
the resected or biopsied tumor as 
assessed via fluorescence- 
activated cell sorting (FACS) 
See Section 13.5.1 
Secondary 
Compare MHC-I expression on tumor cells 
following administration of evolocumab to  
samples from untreated glioma patients 
with tissue 
Extent of MHC-I expression within 
the resected or biopsied tumor, 
as assessed by  fluorescence- 
activated cell sorting (FACS) 
See Section 13.5.1 
Secondary 
Among patients treated with evolocumab, 
describe the strength of the association 
between serum and intratumoral levels of 
evolocumab. Peripheral blood samples to 
be taken before administration of 
evolocumab and at time of surgery or 
biopsy. 
 
Correlation of serum and 
intratumoral levels of 
evolocumab 
See Section 13.5.2. 
Exploratory 
Describe the immune characteristics of 
tumor cells taken from resected or 
biopsied samples 
Sequencing characteristics as 
assessed by multi- omics analysis, 
immune infiltration, and T cell 
profiling 
See Section 13.6 
Exploratory 
Assess the activity of T cells isolated from 
tumor specimens in patients treated with 
or without evolocumab 
Characterization of tumor-
infiltrating lymphocytes (TILs) 
based on flow cytometry  
immunostaining of TILs for 
expression of markers of 
exhaustion (e.g., PD-1, Lag3, 
TIM3) and activation (CD137) 
See Section 13.6 
 
 
 
 

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7 INVESTIGATIONAL PLAN 
7.1 Study Design 
This phase 0/surgical window of opportunity study is being conducted to assess if Evolocumab, a clinically 
approved drug for hypercholesteremia, can cross the blood brain barrier and accumulate within the brain 
tumor of patients with glioma or glioblastoma. Study participants will include newly diagnosed or recurrent 
patients who are due to undergo resection or biopsy of their tumor. Up to 10 patients will be enrolled in 
the treatment arm of this study, while up to 20 patients will serve as controls. 
 
Study treatment arm: Each eligible patient will receive 420 mg of Evolocumab subcutaneously (the 
maximum single dose) 4-14 days prior to surgical resection or biopsy of their tumor. Prior to injection of 
evolocumab (treatment arm only) and at time of surgery surgery, peripheral blood will be drawn to analyze 
serum levels of the drug (for comparison to intratumoral).  
 
Following administration of Evolocumab and surgical procedure, patients in the treatment arm will be 
followed up for two weeks to observe for any evidence of side effects or toxicity. Although the safety 
profile of Evolocumab is well understood, the study will be paused and re-evaluated should any significant 
toxicities occur.  
 
Patients can then proceed to standard of care treatment for their glioma following the trial, with no 
expected impediment caused by the use of Evolocumab.   
 
Control arm: A control group of 20 patients who undergo craniotomy without Evolocumab treatment 
will be prospectively recruited to this study using similar eligibility criteria used to recruit patients to 
receive Evolocumab.  If accrual of these controls is difficult, matched 2:1 (controls: cases) will be 
obtained from the BTBR (Pro00007434).  The control arm will have peripheral blood drawn prior to their 
scheduled day of the surgical procedure or on the day of the surgical procedure.  Patients who consent to 
participate as a control for this study will come off study after tissue is collected for research (if available) 
during their surgical procedure.   
7.1.1 Dose Escalation and Expansion 
The primary objective of this study is to determine whether Evolocumab can cross the blood brain barrier 
and accumulate intra-tumorally. This drug has been approved by the FDA, and the safety data are well 
understood 20. Therefore, the maximum clinically indicated dose for hyperlipidemia (420 mg 
subcutaneously into thigh, abdomen or upper arm) will be used 24. No escalation or expansion of the 
dosage regime is planned. If any evidence of dose-limiting toxicity among patients treated with 
Evolocumab as defined in Section 7.1.2 is observed, the trial will be paused and re-evaluated by the 
investigators. 
7.1.2 Definition of Dose-Limiting Toxicity (DLT) 
Among patients treated with Evolocumab, toxicities will be graded according to the National Cancer 
Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 5 criteria. DLTs will be 
defined as any of the following events that are at least (possibly, probably, or definitely) attributable to 
study treatment (i.e., evolocumab): 
• 
Any Grade 3 or any Grade 4 toxicity (at least possibly, probably, or definitely attributable to study 
treatment [i.e., evolocumab]) that is not reversible (i.e., reversible means the toxicity is trending 
towards improvement and has downgraded to at least Grade 2 or back to baseline) within 2 
weeks, including cerebral edema or worsening neurologic symptoms 

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• 
Any life-threatening event (at least possibly, probably, or definitely attributable to study 
treatment [i.e., evolocumab]) that is not reversible (i.e., reversible means the toxicity is trending 
towards improvement and has downgraded at least to Grade 2 or back to baseline) within 2 
weeks 
• 
Treatment-related death at least possibly, probably, or definitely attributable to study treatment 
(i.e., evolocumab). 
• 
Any grade 2 or higher serious autoimmune toxicities, particularly those affecting vital organs 
(e.g., cardiac, renal, CNS) occurring within 2 weeks of any protocol treatment (i.e., evolocumab). 
 
Exceptions to the above DLT definition include: 
• 
Seizures: Due to the nature of the disease under investigation in this protocol, patients may have 
pre-existing seizures or be susceptible to new seizures as a result of the underlying disease 
process. Although seizures may be defined as Grade 3 or 4 toxicities under NCI CTC, and will be 
reported as such in this protocol, seizures will not be considered an unacceptable adverse event 
if, in the opinion of the Principal Investigator they have not increased in frequency or can be 
attributed to another recognized cause of increasing seizure frequency such as sub- therapeutic 
anti-convulsant levels or biopsy proven tumor progression. 
• 
New neurologic deficits: Due to the nature of the disease under investigation in this protocol, 
patients may develop new neurologic deficits as a result of tumor invasion. A new neurologic 
deficit, which resolves within 2 weeks after initiation of medical therapy, will not be considered 
an unacceptable adverse event. New neurological symptoms will not be an unacceptable adverse 
event if they can be ascribed to tumor progression (e.g. documented with histopathologic 
analyses of biopsy tissue; or; they respond to treatment (e.g., oral steroids, within 2 weeks)). 
• 
Thromboembolism: Due to the high incidence of deep vein thrombosis (DVT) in this patient 
population, patients may have undiagnosed pre-existing DVTs or be susceptible to the 
development of DVTs due to the underlying disease process. Although DVT may be defined as 
Grade 3 or 4 toxicities under NCI CTC, and will be reported as such in this protocol, DVT will not 
be considered unacceptable adverse event in this protocol. 
• 
Syndrome of Inappropriate Antidiuretic Hormone (SIADH): Due to the high incidence of SIADH in 
this patient population, patients may be susceptible to the development of SIADH due to the 
underlying disease process. Although SIADH may be defined as Grade 3 toxicity under NCI CTC, 
and will be reported as such in this protocol, SIADH will not be considered an unacceptable 
adverse event in this protocol unless it is refractory to medical management. 
• 
Muscle Weakness and Weight Gain: Due to the high incidence of muscle weakness and weight 
gain in patients taking steroids, patients may be susceptible to the development of muscle 
weakness or weight gain, which is due to steroids alone. Although muscle weakness may be 
defined as Grade 3 or Grade 4 toxicity and weight gain ≥ 20% may be defined as Grade 3 toxicity 
under NCI CTC, and will be reported as such in this protocol. Muscle weakness or weight gain will 
not be considered an unacceptable adverse event in this protocol if the patient has required 
steroids greater than physiologic doses in the interval between the immunization and the 
development of the toxicity. 
• 
Tumor Progression: Due to the nature of the disease under investigation in this protocol, patients 
may have an increase in pre-existing neurologic deficits or have an onset of new neurologic 
deficits due to tumor progression. Although such neurologic deficits may be defined as 
unacceptable adverse events under NCI CTC, and will be reported as such in this protocol, these 
clinical changes are not an unexpected phenomenon in this disease in the setting of tumor 
growth. As a result, neurologic deficits will not be considered an unacceptable adverse event if 
unequivocal tumor progression can be documented radiographically or histologically. 
• 
Immune Related Exceptions: grade 2 Pneumonitis; grade 2 or 3 colitis or diarrhea; grade 2, 3, or 4 
endocrinopathies (including but not limited to hypophysitis, adrenal insufficiency, 
hyperthyroidism, and type 1 diabetes mellitus). Any of the aforementioned immune-related AEs 
will not be considered unacceptable if the event resolves to Grade 1 after withholding therapy. 

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7.1.3 Dose Modification 
7.1.3.1 Non-hematologic: 
There will be no non-hematologic dose modifications in this study. 
7.1.3.2 Hematologic: 
There will be no hematologic dose modifications in this study. 
7.1.3.3 Other considerations: 
Not applicable 
7.1.4 Safety Considerations 
Evolocumab is not predicted to induce any specific immunologic adverse events given its well described 
safety profile. Although there is potential for immune-genicity, it has been evaluated using an electro-
chemi-luminescent bridging screening immunoassay for the detection of binding anti-drug antibodies. 
 
 
For patients whose sera tested positive in the screening immunoassay, an in vitro biological assay was 
performed to detect neutralizing antibodies. In a pool of placebo- and active-controlled clinical trials, 0.3% 
(48 out of 17,992) of patients treated with at least one dose of evolocumab tested positive for the 
development of binding antibodies. Patients whose sera tested positive for binding antibodies were 
further evaluated for neutralizing antibodies; none of the patients tested positive for neutralizing 
antibodies. There was no evidence that the presence of anti-drug binding antibodies impacted the 
pharmacokinetic profile, clinical response, or safety of Evolocumab 24. Therefore, we do not anticipate any 
immunologic AEs that can affect the patients wellbeing or with standard of care treatment of radiation 
therapy and temozolomide (XRT/TMZ). If any reactions are observed, the study team will pause the study 
and re-evaluate. 
7.1.5 Missed Doses 
Not applicable. 
7.1.6 Concomitant Medications 
Concomitant medications will be managed by the treating physician and recorded at each study visit by 
the study team. 
 
As we wish to monitor immune modulation in both treatment and control groups, corticosteroids should 
be used at the lowest dose possible [less than 0.1 mg/Kg/day (<4 mg of dexamethasone / day)], except for 
the 24 hours prior to surgery or biopsy, to control symptoms of edema and mass effect, and discontinued, 
if possible. Use of corticosteroids should be recorded in the electronic database. 
7.1.7 Study Drug Blinding 
Not applicable. 
7.2 Rationale for Selection of Dose, Regimen, and Treatment Duration 
The primary objective of this study is to determine whether evolocumab can cross the blood brain barrier 
and accumulate intratumorally. This drug has been approved by the FDA, and the safety data are well 
understood 20. 
 
Therefore, the maximum clinically indicated dose for hyperlipidemia (420 mg subcutaneously into thigh, 
abdomen or upper arm) will be used 24. Only a single dose is required 4-14 days prior to administration as 
this is the point at which peak plasma concentrations occur and maximal accumulation in the brain is likely 
to occur, but remaining well within the effective half-life of 11-17 days21,25. This will therefore allow for the 

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greatest chance for detected evolocumab in the brain, should it successfully cross the blood brain barrier. 
No dose escalation is planned. 
7.3 Rationale for Correlative Studies 
To compare the pharmacokinetics (PK) of evolocumab in treated patients, peripheral blood samples will be 
taken to compare serum concentrations of evolocumab to intratumoral concentrations. These are further 
detailed in Section 10.  
 
Tumor-infiltrating lymphocytes (TILs) from tumor or liquid obtained from the surgical field will be 
dissociated into a single cell suspension or fragmented and TILs expanded as one batch following the TILs 
pre-rapid expansion protocol (preREP). The ability to successfully and reliably expand preREP TILs from 
lung cancer and glioblastoma samples has been previously demonstrated in Pro00106078. TILs, in general, 
display a profound degree of exhaustion. Therefore, cells will be stained for expression of surface and 
nuclear markers of exhaustion (PD-1, Lag3, TIM3) and activation (CD137) and differential levels assessed by 
flow cytometry. CD137 is upregulated on T cells during early activation immediately following interaction 
with tumor antigen in the Tumor Microenvironment (TME). We will explore the potential to select and 
expand the non-exhausted CD137+ subset and examine their anti-tumor efficacy in samples from patients 
treated with or without evolocumab. Autologous CD137+ TIL cell product will be co-cultured with 
disassociated CFSE labeled neurospheres for 24 and 48 hours. Cytotoxicity determined by flow cytometry 
of CFSE+ tumor cells will be compared to no TIL exposure. Patient-derived neurospheres will generated in 
vitro based on established protocols 26. 
7.4 Definition of Evaluable Subjects, On Study, and End of Study 
Once a patient has their baseline PK collection (both arms) and receives one dose of Evolocumab 
(treatment arm only), that patient will be considered “on study.” Different definitions for “on study,” “on 
treatment,” “off treatment,” and “off study” may be required in the electronic research record. Rationale 
for taking a patient off protocol treatment will be documented. 
 
Any patient who receives one dose of Evolocumab will be included in safety analyses. Once all data has 
been collected, analyzed, and completely evaluated on all subjects, as defined in the statistical analysis 
plan, and subjects have completed follow up, expired, withdrawn, or been lost to follow up, the study will 
be ended. 
7.5 Early Study Termination 
This study can be terminated at any time for any reason by the PI or Sponsor- Investigator. If this occurs, all 
subjects on study should be notified as soon as possible. Additional procedures and/or follow up should 
occur in accordance with Section 10.7, which describes procedures and process for prematurely withdrawn 
patients. 
8 STUDY DRUG 
8.1 Names, Classification, and Mechanism of Action 
Evolocumab (Repatha®) is a PCSK9 inhibitor and binds to a pro-protein involved in the regulation of LDL 
receptors on liver cells; receptor numbers are increased, which results in increased uptake of LDL-
cholesterol from the blood. Evolocumab is currently indicated 24: 
 
• 
In adults with established cardiovascular disease to reduce the risk of myocardial infarction, stroke, 
and coronary revascularization 
• 
As an adjunct to diet, alone or combined with other low-density lipoprotein cholesterol (LDL-C)-
lowering therapies, in adults with primary hyperlipidemia, including heterozygous familial 
hypercholesterolemia (HeFH), reducing LDL-C 

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• 
As an adjunct to other LDL-C-lowering therapies in patients with homozygous familial 
hypercholesterolemia (HoFH), to reduce LDL-C 
8.2 Packaging and Labeling 
 Please refer to the current evolocumab (REPATHA®) labeling on Drugs@FDA. 
https://www.accessdata.fda.gov/scripts/cder/daf/index.cfm?event=overview.process&ApplNo=125522 
8.3 Supply, Receipt, and Storage 
Evolocumab is available commercially from Amgen and is a clear to opalescent, colorless to pale yellow 
solution supplied as follows: 
 
Table 3. Formulations of Evolocumab 
140 mg/mL single-dose prefilled syringe 
1 pack 
NDC 72511-750-01 
NDC 55513-750-01 
140 mg/mL single-dose prefilled SureClick® autoinjector 
1 pack 
NDC 55513-760-01 
140 mg/mL single-dose prefilled SureClick® autoinjector 
2 pack 
NDC 72511-760-02 
NDC 55513-760-02 
140 mg/mL single-dose prefilled SureClick® autoinjector 
3 pack 
NDC 55513-760-03 
420 mg/3.5 mL single-dose Pushtronex® system (on-body 
infusor with prefilled cartridge) Formulation to be used in this 
study) 
1 pack 
NDC 72511-770-01 
NDC 55513-770-01 
 
Per the package insert: 
The needle cover of the glass single-dose prefilled syringe and the single-dose prefilled autoinjector contain 
dry natural rubber (a derivative of latex). The single-dose on-body infusor with prefilled cartridge is not 
made with natural rubber latex. 
 
Store refrigerated at 2°C to 8°C (36°F to 46°F) in the original carton to protect from light. Do not freeze. Do 
not shake. 
 
For convenience, evolocumab may be kept at room temperature at 68°F to 77°F (20°C to 25°C) in the 
original carton for 30 days. If not used within the 30 days, discard evolocumab. 
8.4 Dispensing and Preparation 
The commercial agent will be purchased from the pharmacy storeroom and managed by ICS.  Evolocumab 
is a sterile, preservative-free, clear to opalescent, colorless to pale yellow solution for subcutaneous use.  
Each single-dose Pushtronex® system (on-body infusor with prefilled cartridge) delivers a 3.5 mL solution 
containing 420 mg Evolocumab, acetate (4.2 mg), polysorbate 80 (0.35 mg), proline (89 mg) in Water for 
Injection, USP. Sodium hydroxide may be used to adjust to a pH of 5.0 24. 
8.5 Compliance and Accountability 
Evolocumab will be delivered from Duke Pharmacy directly to the clinic under the supervision of the 
research nurse, or his/her designee. Evolocumab will be administered according to the approved labeling. 
The patient’s name, Study ID, DOB, and Duke history number will be double verified prior to subcutaneous 
injection as is standard Duke procedure. 
8.6 Disposal and Destruction 
All injection equipment will be disposed of according to standard Duke procedures. Unused medication will 
be returned to Duke pharmacy, if possible, or disposed of as per standard handling of clinical medicines.

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9 SUBJECT ELIGIBILITY 
9.1 Inclusion Criteria 
Both arms: 
1. Adult patients ≥ 18 years old 
2. Newly diagnosed or recurrent glioma or glioblastoma (GBM) (if recurrent, prior pathology report 
indicating glioma) 
3. A clinical indication for gross macroscopic resection, debulking of the glioma, or biopsy, with sufficient 
tumor size that can allow collection of specimens for the required analyses. 
 
Treatment arm only: 
4. Adequate hematologic function within 14 days prior to starting evolocumab defined as follows: 
a. Hemoglobin ≥ 10 g/dL (Note: the use of transfusion or other intervention to achieve Hgb ≥ 10.0 
g/dl is acceptable) 
b. White Blood Cells ≥ 1.5 x 109/L 
c. 
Absolute Neutrophil Count (ANC) ≥ 1.0 x 109/L 
d. Platelets ≥ 100 x 109/L or ≥ 50,000 for patients who received TMZ within the past year 
5. Adequate renal function within 14 days prior to starting evolocumab defined as calculated creatinine 
clearance (CrCL) of ≥ 30 mL/min/1.73m2 by the Cockcroft-Gault formula 
6. Adequate hepatic function within 14 days prior to starting evolocumab defined as follows: 
a. Total bilirubin ≥ 1.5 x institutional upper limit of normal (ULN) (Note:  Patients with known Gilbert 
disease without other clinically significant liver abnormalities are not excluded.) 
b. AST(SGOT) and ALT(SGPT) ≥ 1.5 × ULN 
7. Negative serum pregnancy test (in females of childbearing potential) within 48 hours of starting 
evolocumab.   
9.2 Exclusion Criteria 
Treatment arm only: 
1. Any patient with a history of a serious hypersensitivity reaction to evolocumab or any of the excipients 
in evolocumab 
2. Patients with severe hepatic impairment outside of the range defined in the inclusion criteria within 7 
days of starting evolocumab.  
3. History or evidence of central nervous system bleeding as defined by stroke or intraocular bleed 
(including embolic stroke) not associated with any antitumor surgery within 6 months before 
enrollment 
4. Infection requiring intravenous antibiotics that was completed < 1 week of study enrollment (day 1) 
with the exemption of prophylactic antibiotics for long line insertion or biopsy 
5. Females of reproductive potential and males who are unwilling to practice an acceptable method(s) of 
effective birth control while on study through 1 month (2 half-lives) after receiving the last dose of 
study drug. 
10 SCREENING AND ON-STUDY TESTS AND PROCEDURES 
Table 4. Schedule of Events for Treatment Arm 
Description 
Screening (within 
14 days of starting 
evolocumab) 
Treatment Period 
Active Follow-up Period 

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Pre-Evolocumab 
Screening 
Evolocumab Injection 
Surgical Procedure 
Post-Operative 
Visit 
Day(s) 
 
1 
4-14 
Typically ~2 
weeks following 
surgery 
General Evaluations 
Evolocumab 
Informed Consent 
X1 
 
 
 
Physical Exam 
X 
 
 
X 
Neurologic Exam 
X 
 
 
X 
Adverse Events 
 
Continuous2 
Laboratory Evaluations 
CBC with differential 
X 
 
 
X 
CMP  
X 
 
 
X 
Lipid profile 
X 
 
 
 
Serum 
Pregnancy Test, 
if applicable 
X3 
X3 
 
 
Pharmacokinetic 
blood draws (5 
mL)4 
 
X 
X* 
 
Tumor tissue 
analysis (estimated 
> 20mg available)5 
 
 
X 
 
Treatment 
Evolocumab 
 
X 
 
 
* This blood sample taken at time of surgery or biopsy so that drug level in the blood can be compared to intratumoral 
concentration.  
 
Table 4 shows the schedule of events for the treatment arm of this study, including all study-related general 
evaluations, laboratory evaluations, disease evaluations, and treatments. Of particular importance for this phase 1 
study are the tumor sampling and pharmacokinetic blood draws to investigate evolocumab concentration profiles 
following agent administration.  Patients who consent to participate as a control will only be asked to have the 
following: 
• 
Informed consent on the control consent form 
• 
PK blood draw prior to surgical procedure 
• 
Tumor tissue analysis, if available 
10.1 
Screening Examination 
The initial screening examination will take place at a Duke PRTBTC clinic visit. The patient will sign either 
the main Evolocumab treatment consent form or the control consent form for this study and then undergo 
peripheral blood draw (prior to administration of evolocumab for those in the treatment arm). All patients 
with resectable tumors or tumors amenable to biopsy will complete screening and confirm their eligibility 
per the inclusion/exclusion criteria in Section 9. Screening tests in the treatment arm will consist of a 
physical and neurological exam, CBC with differential, CMP, lipid panel, and Beta HCG quantitative3  (if 
applicable). 
 
                                                                
1 Consent can be obtained within 30 days before starting evolocumab. 
2 Patients in the treatment arm will be followed for ongoing serious adverse events and new onset serious adverse events for 30 days following 
evolocumab injection.  
 
3 Within 48 hours of the first evolocumab injection, if applicable.  Will only be repeated on Day 1 if screening pregnancy test is outside of the 
48-hour window. 
4 Tube type to be determined by the Proteomics Core. 
5 Estimated > 50-100 mg from surgical resection or de-bulking procedure; Estimated > 20 mg from a minimum of 2 cores from biopsy 
 
 

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Once the patient is confirmed eligible, patients in the treatment arm will then receive Evolocumab via 
subcutaneous injection 4-14 days prior to surgical procedure. If the first evolocumab injection does not start 
within two weeks of the screening visit, the CBC with differential, CMP, lipid panel, and clinic visit 
(physical/neuro exam) will need to be repeated. 
 
Patients who consent to participate as a control will not undergo any screening tests or procedures.   
10.2 
Run-In Period 
Not applicable. 
10.3 
Treatment Period 
Day 1: 
• 
Baseline pharmacokinetic (PK_ blood draw (applies to patients who consent to either arm of the 
study) (5 mL drawn). For patients in the treatment arm, PK blood draw should occur before 
Evolocumab injection. For patients in the control arm, this blood draw can occur prior to the 
scheduled day of the surgical procedure or on the day of the surgical procedure.Error! Bookmark not 
defined.  
 
Treatment arm only: 
• 
Beta HCG quantitative3  (if applicable) only if screening pregnancy test is outside of the 48-hour 
window 
• 
Evolocumab injection 
o 
Latex-sensitive patients will be cautioned that the needle cover of the glass single-dose 
prefilled syringe and the single-dose prefilled auto-injector contain dry natural rubber (a 
derivative of latex) that may cause allergic reactions in individuals’ sensitive to latex 
o 
Evolocumab will be allowed to warm to room temperature for at least 30 minutes for the 
prefilled auto-injector or syringe and for at least 45 minutes for the single-dose 
Pushtronex® system (on-body  infusor with prefilled cartridge) if evolocumab has been 
refrigerated. 
o 
Evolocumab will be administered subcutaneously into areas of the abdomen, thigh, or 
upper arm that are not tender, bruised, red, or indurated, avoiding injecting into areas 
with scars or stretch marks. 
o 
The 420 mg dose of Evolocumab can be administered either: 
▪ 
Over 5 minutes by using the single-dose Pushtronex® system (on-body  infusor with 
prefilled cartridge)  
▪ 
By giving 3 injections consecutively within 30 minutes using the single-dose prefilled 
auto-injector or single-dose prefilled syringe. 
• 
Adverse events monitoring 
10.4 
Active Follow-up Period 
Days 4-14 – Surgical resection or biopsy 
• 
Tumor tissue collection (applies to patients who consent to either arm of the study) – this tissue 
will be taken intraoperatively for pharmacokinetic LC-MS analysis to assess for the presence of 
evolocumab. 
o 
Sample will only be taken for analysis if greater than 20 mg of resected or biopsied tissue 
available (excess available beyond that required for histological analysis) 
o 
Sample to be aliquoted into 25 mg sections for resections and 10-20 mg (1-2 cores) for 
biopsies 
 
                                                                
 

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Treatment arm only: 
• 
Pharmacokinetic blood draw (5 mL, drawn at time of surgery or biopsy, i.e., PK draw may occur 
either immediately before or during surgery or biopsy)Error! Bookmark not defined. 
• 
Adverse events monitoring2 
 
About 2 weeks following surgery or biopsy (treatment arm only) 
• 
Physical and neurological exam  
• 
Laboratory evaluations (obtained as part of standard post-operative labs) 
o 
CBC with differential 
o 
CMP  
• 
Adverse events monitoring2 
10.5 
End of Treatment 
Once the baseline PK blood draw is collected (both arms) and the evolocumab injection (treatment arm 
only) is completed, the treatment phase of the study is over and the active follow-up period begins. During 
the active follow-up period (see Section 10.4), patients undergo their standard of care surgical resection or 
biopsy with tissue collection, if available.  Patients in the treatment arm return for post-surgical wound 
care about 14 days post-surgical procedure as needed per standard neurosurgical practice.   
10.6 
Follow-up Period 
Once active follow-up is complete, patients are followed per standard of care.  The study team will review 
medical records of patients enrolled in the treatment arm to collect information for ongoing serious 
adverse events and new onset serious adverse events for up to 30 days following Evolocumab injection.  If 
patients subsequently undergo additional biopsy or resection, a sample of leftover tissue may be 
requested for analysis. 
10.7 
End of Study 
Once all data has been collected, analyzed, and completely evaluated on all subjects as defined in the 
statistical analysis plan and subjects have completed follow up, expired, withdrawn, or been lost to follow 
up, the study will be ended. The study may also be terminated early for any reason by the PI. 
10.8 
Early Withdrawal of Subject(s) 
10.8.1 
Criteria for Early Withdrawal 
Subjects may voluntarily withdraw from the study at any time.  The PI may also withdraw a subject from 
the study at any time based on his/her discretion.  Reasons for PI-initiated withdrawal may include, but is 
not limited to the following: 
 
- 
Adverse events 
- 
Abnormal laboratory values 
- 
Abnormal test procedure results 
- 
Protocol deviation 
- 
Administrative issues 
- 
Disease progression 
- 
Pregnancy 
10.8.2 
Follow-up Requirements for Early Withdrawal 
Patients in the treatment arm will be assessed and followed for adverse event monitoring/safety analysis 
for 30 days following Evolocumab injection. 

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10.8.3 
Replacement of Early Withdrawal(s) 
Patients who are withdrawn by the PI prior to baseline PK collection (both arms) and Evolocumab injection 
(treatment arm) will be replaced.  Patients who do not undergo surgery or biopsy or do not have sufficient tissue 
taken for the pharmacokinetic LC-MS analysis to assess for the presence of evolocumab (treatment arm) will be 
replaced. If there is no enough tissue available for analysis in either cohort, the patients should be replaced. 
10.9 
Study Assessments 
10.9.1 
Medical History 
Medical history will be obtained from the Duke electronic system and from the subject and/or family at 
the screening visit and reviewed at each study visit. This data may include the following: 
• 
All past medical and surgical history 
• 
Current medications 
• 
Changes in physical or neurologic symptoms 
• 
Any adverse events 
10.9.2 
Physical Exam 
Vital signs and physical and neurologic examinations will be assessed for patients in the treatment arm 
and recorded prior to enrollment and at each visit according to Table 4. 
10.9.3 
Correlative Assessments 
The following will be tested from tumor and serum samples (both study arms): 
1. Lipid metabolism and immune characteristics (multi-omics analysis, immune infiltration and T cell 
profiling) of tumor cells taken from resected or biopsied samples 
2. Analysis of tumor cells expressing MHC-I will be performed by fluorescence-activated cell sorting 
(FACS) 
3. Analysis of serum levels of evolocumab, for comparison to intratumoral concentrations 
4. Differential levels of TILs based on flow cytometry after staining of cells for expression of surface 
and nuclear markers of exhaustion (e.g., PD-1, Lag3, TIM3) and activation (CD137) 
11 SAFETY MONITORING AND REPORTING 
The PI is responsible for the identification and documentation of adverse events and serious adverse events, as 
defined below.  Among patients receiving evolocumab and at each study visit, the PI or designee must assess, 
through non-suggestive inquiries of the subject or evaluation of study assessments, whether an AE or SAE has 
occurred. 
11.1 
Adverse Events 
An adverse event (AE) is any untoward medical occurrence in a patient receiving study drug in the 
treatment arm and which does not necessarily have a causal relationship with this treatment.  For this 
protocol, the definition of AE also includes worsening of any pre-existing medical condition.  An AE can 
therefore be any unfavorable and unintended or worsening sign (including an abnormal laboratory 
finding), symptom, or disease temporally associated with the use of Evolocumab, whether or not related to 
use of the Evolocumab.  Abnormal laboratory findings without clinical significance (based on the PI’s 
judgment) should not be recorded as AEs.  But laboratory value changes that require therapy or 
adjustment in prior therapy are considered adverse events. 
 
From the time a patient receives their Evolocumab injection to the End of Study visit (as defined in Section 
10.7), all AEs must be recorded in the subject medical record and adverse events case report form. 
 

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AEs will be assessed according to the CTCAE version 5.0.  If CTCAE grading does not exist for an AE, the 
severity of the AE will be graded as mild (1), moderate (2), severe (3), life-threatening (4), or fatal (5). 
 
Attribution of AEs will be indicated as follows: 
• 
Definite:  The AE is clearly related to the study drug 
• 
Probably:  The AE is likely related to the study drug 
• 
Possible:  The AE may be related to the study drug 
• 
Unlikely:  The AE is doubtfully related to the study drug 
• 
Unrelated:  The AE is clearly NOT related to the study drug 
11.1.1 
AEs of Special Interest 
Special Interest Adverse Events that may occur during the injection procedure include: 
• 
Allergic reaction 
• 
Anaphylaxis 
• 
Pre-syncope 
• 
Syncope 
• 
Vasovagal reaction 
 
Special Interest Adverse Events that may occur after the procedure, but may still be related to injection 
include: 
• 
Injection site complications 
11.1.2 
Reporting of AEs 
A summary of all adverse events (not just those considered related to the study drugs) will be kept. AEs 
will be categorized by organ system, relationship to which treatment, grade of severity, and resolution. 
Weekly review by the PI during weekly Adverse Event meeting of new AEs and the collective AEs will 
occur with the intention of identifying any trends or patterns in toxicity. If significant toxicity is observed 
in the initial 2 patients, or after the first month, the study will be paused, and protocol modification will 
be considered.  If any DLT occurs, as defined in Section 7.1.2, the study will be paused for 
assessment/review to determine if any changes to the protocol are warranted. 
11.2 
Serious Adverse Events 
An AE is considered “serious” if in the opinion of the investigator it is one of the following outcomes: 
 
• 
Fatal 
• 
Life-threatening 
• 
Constitutes a congenital anomaly or birth defect 
• 
A medically significant condition (defined as an event that compromises subject safety or may 
require medical or surgical intervention to prevent one of the three outcomes above). 
• 
Requires inpatient hospitalization or prolongation of existing hospitalization 
• 
Results in persistent or significant incapacity or substantial disruption to conduct normal life 
functions. 
11.2.1 
Reporting of SAEs 
All SAEs should be reported immediately to the PI, Dr. Mustafa Khasraw (Pager: 919-206-0493) or his 
designee (919-684-8111).   
 
All adverse events that are considered serious, unanticipated, and related or possibly related to the 
research (as defined by 21CRF312.32[a]) will be reported to the IRB using the appropriate SAE reporting 
process. 

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11.3 
Emergency Unblinding of Investigational Treatment 
Not applicable. 
11.4 
Other Reportable Information 
Not applicable. 
11.5 
Special Warnings and Precautions 
Not applicable. 
11.6 
Safety Oversight Committee (SOC) 
The Duke Cancer Institute SOC is responsible for annual data and safety monitoring of DUHS sponsor-
investigator phase I and II, therapeutic interventional studies that do not have an independent Data Safety 
Monitoring Board (DSMB). The primary focus of the SOC is review of safety data, toxicities and new 
information that may affect subject safety or efficacy. Annual safety reviews includes but may not be 
limited to review of safety data, enrollment status, stopping rules if applicable, accrual, toxicities, reference 
literature, and interim analyses as provided by the sponsor-investigator.  The SOC in concert with the DCI 
Monitoring Team (see Section 12.1 for Monitoring Team description) oversees the conduct of DUHS 
cancer-related, sponsor-investigator greater-than-minimal-risk intervention studies that do not have an 
external monitoring plan, ensuring subject safety and that the protocol is conducted, recorded and 
reported in accordance with the protocol, standing operating procedures (SOPs), Good Clinical Practice 
(GCP), and applicable regulatory requirements. 
11.7 
External Data and Safety Monitoring Board (DSMB) 
There is no external DSMB for this study. 
12 QUALITY CONTROL AND QUALITY ASSURANCE 
12.1 
Monitoring 
This clinical research study will be monitored both internally by the PI, and institutionally by the Duke 
Cancer Institute (DCI).  In terms of internal review, the PI will continuously monitor and tabulate adverse 
events.  Appropriate reporting to the Duke University Medical Center IRB will be made.  If an unexpected 
frequency of Grade III or IV events occur, depending on their nature, action appropriate to the nature and 
frequency of these adverse events will be taken.  This may require a protocol amendment, dose de-
escalation, or potentially closure of the study.  The PI of this study will also continuously monitor the 
conduct, data, and safety of this study to ensure that: 
• 
Interim analyses occur as scheduled; 
• 
Stopping rules for toxicity and/or response are met; 
• 
Risk/benefit ratio is not altered to the detriment of the subjects; 
• 
Appropriate internal monitoring of AEs and outcomes is done; 
• 
Over-accrual does not occur; 
• 
Under-accrual is addressed with appropriate amendments or actions; 
• 
Data are being appropriately collected in a reasonably timely manner. 
 
DCI Protocol Review and Monitoring systems (PRMS) review of this protocol begins with an initial review by 
the Cancer Protocol Committee (CPC). CPC new protocol review focuses on scientific relevance, study 
design, adequacy of biostatistical input, protocol prioritization, feasibility of completing the study within a 
reasonable time frame and risk assessment of the trial. The PI will abide by CPC assessment of the level of 
risk, which will determine the intensity of subsequent DCI monitoring. CPC also conducts annual scientific 
progress reviews on protocols that are open to enrollment and focus on protocol prioritization, accrual and 
scientific progress. These reviews are conducted at the time of IRB annual renewals and documentation of 
all CPC reviews will be maintained in eIRB/iRIS systems. 

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A determination for the degree of monitoring conducted by the DCI monitoring team is made at the time 
of initial CPC approval to commensurate with the type and level of intervention, phase, endpoints, degree 
of risk, size and complexity of the protocol. A formal, independent monitoring will be conducted by the DCI 
monitoring team according to the risk level and monitoring plan assigned by the CPC until the study is 
closed to enrollment or subjects are no longer receiving study drug or other interventions that are more 
than minimal risk. Additional monitoring may be prompted by findings from monitoring visits, unexpected 
frequency of serious and/or unexpected toxicities, or other concerns.  Monitoring visits may also be 
initiated upon request by DUHS and DCI Leadership, CPC, SOC, a sponsor, an investigator, or the IRB.   
 
The DCI monitoring team reviews the adequacy of informed consent, enrollment of eligible patients, 
implementation of protocol-specified procedures and treatment, adequacy of data collection, and 
appropriateness of adverse event monitoring and reporting. The DCI monitoring team presents final 
monitoring reports to the DCI Safety Oversight Committee (SOC) highlighting safety concerns and 
unresolved issues.  The SOC, at a convened meeting, assigns an overall rating of satisfactory, marginal, or 
unsatisfactory to reflect the overall quality of data, regulatory, consent, eligibility, study conduct and AE 
reporting.  Corrective action plans (CAPs) are developed, implemented, and evaluated as indicated.  The 
SOC will notify the sponsor-investigator and DUHS IRB when significant safety concerns are identified. 
 
The SOC in concert with DCI monitoring team conducts data and safety monitoring for DUHS sponsor-
investigator phase I and II, therapeutic interventional oncology studies that do not have an independent 
DSMB.  These reviews occur at a minimum annually and more frequently for the high risk studies. The SOC 
safety reviews include review of safety data, enrollment status, stopping rules if applicable, accrual, 
toxicities, reference literature, and interim analyses as provided by the sponsor-investigator. The SOC, at a 
convened meeting, assigns a rating of satisfactory when adequate accrual with lack of excessive toxicity is 
present. 
12.2 
Audits 
The Duke Office of Audit, Risk and Compliance (OARC) office may conduct confidential audits to evaluate 
compliance with the protocol and the principles of GCP.  The PI agrees to allow the OARC auditor(s) direct 
access to all relevant documents and to allocate his/her time and the time of the study team to the CTQA 
auditor(s) in order to discuss findings and any relevant issues. 
 
OARC audits are designed to protect the rights and well-being of human research subjects. OARC audits 
may be routine or directed (for cause). Routine audits are selected based upon risk metrics generally 
geared towards high subject enrollment, studies with limited oversight or monitoring, Investigator initiated 
Investigational Drugs or Devices, federally-funded studies, high degree of risk (based upon adverse events, 
type of study, or vulnerable populations), Phase I studies, or studies that involve Medicare populations. 
Directed audits occur at the directive of the IRB or an authorized Institutional Official. 
 
OARC audits examine research studies/clinical trials methodology, processes and systems to assess 
whether the research is conducted according to the protocol approved by the DUHS IRB. The primary 
purpose of the audit/review is to verify that the standards for safety of human subjects in clinical trials and 
the quality of data produced by the clinical trial research are met. The audit/review will serve as a quality 
assurance measure, internal to the institution. Additional goals of such audits are to detect both random 
and systemic errors occurring during the conduct of clinical research and to emphasize “best practices” in 
the research/clinical trials environment. 

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12.3 
Data Management and Processing 
12.3.1 
Case Report Forms (CRFs) 
The electronic CRF (eCRF) will be the primary data collection document for the study.  The eCRFs will be 
updated in a timely manner following acquisition of new source data.  Only approved study staff (e.g., the 
PI, the clinical research coordinator, the data management team, and the assistant research practice 
manager) are permitted to make entries, changes, or corrections in the CRF. 
 
An audit trail will be maintained automatically by the 21 CFR Part 11 compliant electronic CRF 
management system.  Designated personnel will complete user training, as required or appropriate per 
regulations. 
12.3.2 
Data Management Procedures and Data Verification 
Designated personnel using the electronic CRF will have access based on their specific roles in the 
protocol as determined by the PRTBTC Data Manager. 
 
Completeness of entered data will be checked automatically by the eCRF system, and users will be alerted 
to the presence of data inconsistencies.  Additionally, the data management team and the statistical team 
will cross-reference the data to verify accuracy.  Missing or implausible data will be highlighted for the PI 
requiring appropriate responses (i.e. confirmation of data, correction of data, completion or confirmation 
that data is not available, etc.). 
 
The database will be reviewed and discussed prior to database closure, and will be closed only after 
resolution of all remaining queries.  An audit trail will be kept of all subsequent changes to the data. 
12.3.3 
Study Closure 
Following completion of the studies, the PI will be responsible for ensuring the following activities: 
 
• 
Data clarification and/or resolution 
• 
Accounting, reconciliation, and destruction/return of used and unused study drugs 
• 
Review of site study records for completeness 
• 
Shipment of all remaining laboratory samples to the designated laboratories 
13 STATISTICAL METHODS AND DATA ANALYSIS 
All statistical analysis will be performed under the direction of the statistician designated in key personnel.  Any 
data analysis carried out independently by the investigator must be approved by the statistician before publication 
or presentation. 
13.1 
Analysis Sets 
All patients who receive Evolocumab will be included in summaries of adverse events. 
 
PK and PD analyses will include all patients in the treated and untreated groups who have baseline PK 
blood collected and underwent a resection. 
 
Analyses of serum and intratumoral levels of Evolocumab will be conducted among treated and untreated 
patients who were resected or biopsied and had serum collected.  
13.2 
Patient Demographics and Other Baseline Characteristics 
The clinical and sociodemographic characteristics of the patients who are enrolled and treated on this 
study will be summarized by arm.  Frequencies and percentages will be provided for categorical variables; 

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means and standard deviations or medians and interquartile ranges will be provided for continuous 
variables. 
13.3 
Treatments 
A CONSORT diagram will be generated to describe the flow of patients through the study, and will provide 
information about the number of patients who received Evolocumab treatment, the number of control 
patients (either consented on this study as a control or requested from the BTBR), and the number of 
patients who subsequently had a surgical procedure. 
13.4 
Primary Objective 
The primary objective of this study is to assess whether Evolocumab crosses the blood brain barrier (BBB) 
and is measurable in the resected or biopsied tumor specimens of patients with primary and recurrent 
glioma. 
13.4.1 
Variable 
By means of mass spectrometry, the level of Evolocumab within the resected or biopsied brain tumor of a 
patient treated with one dose of Evolocumab will be assessed. 
13.4.2 
Statistical Hypothesis, Model, and Method of Analysis 
The level of Evolocumab within the resected or biopsied tumor will be determined for patients treated 
with Evolocumab, and within a cohort of resected or biopsied control patients who were not treated with 
Evolocumab.  Either a Wilcoxon rank sum test or a two-sample t-test will compare cohorts. 
13.4.3 
Handling of missing values, censoring, and 
discontinuations 
Not applicable. 
13.5 
Secondary Objectives 
This study’s three secondary objectives include a comparison of brain tumor tissues specimens with and 
without Evolocumab with respect to lipid metabolism, and tumor cells expressing MHC-I; and an 
assessment of the relationship between serum levels of evolocumab and intratumoral concentrations. 
13.5.1 
Key Secondary Objectives #1and #2: Lipid Metabolism 
and Tumor Cells Expressing MHC-I 
The first two secondary objectives involve a comparison of the pharmacodynamics (PDs) of Evolocumab in 
treated patients relative to that observed in a control cohort of untreated patients. The characterization 
of the PD properties will include the following: (1) Lipid metabolism of tumor cells taken from resected or 
biopsied samples, and (2) Analysis of tumor cells expressing MHC-I will be performed by fluorescence-
activated cell sorting (FACS).  Analyses similar to those describe for the primary objective will be 
performed. 
13.5.2 
Secondary Objective #3: Serum and Intratumoral Levels 
of Evolocumab 
Among patients treated with evolocumab, the third secondary objective is an assessment of the 
relationship between serum and intratumoral levels of the drug.  These assessments will be obtained at the 
time of surgical procedure. A scatterplot will graphically show the relationship between these two 
measures.  Spearman’s rank correlation will assess their association. 

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13.6 
Exploratory Objectives 
This study has two exploratory objectives: (1) To describe the immune characteristics of tumor cells taken 
from resected or biopsied samples, and (2) To assess the activity of T cells isolated from tumor specimens 
in patients treated with or without evolocumab. 
 
Exploratory Objective #1:  We hypothesize that evolocumab treatment will lead to global changes in pro-
tumorogenic pathways and the reduction in PSCK9 levels and therefore will seek to determine the immune 
characteristics (multi-omics analysis, immune infiltration and T cell profiling) of tumor cells taken from 
resected or biopsied samples. To test this hypothesis, non-targeted quantitative LC-MS/MS will be 
performed on resected or biopsied tumors from n=10 treatment patients versus n=20 untreated control 
specimens. Furthermore, if PSCK9 is not among the proteins quantified by non-targeted LC-MS/MS, a more 
sensitive targeted LC-MS/MS analysis will be utilized as described for the PK analysis of evolocumab above. 
Bioinformatic analyses of differentially expressed proteins will utilize protein set enrichment, gene 
ontology (GO) annotations, and search tool from the retrieval of interactive genes/proteins (STRING).26 
 
Wilcoxon rank-sum test, or a two-sample t-test, will compare groups with respect to these endpoints.  
Given the number of comparisons that may be conducted in these exploratory analyses, adjustment for 
multiple comparisons will be made. 
 
Exploratory Objective #2:  Cells stained for expression of surface and nuclear markers of exhaustion (PD-1, 
Lag3, TIM3) and activation (CD137) will be characterized using flow cytometry. We will explore the 
potential to select and expand the non-exhausted CD137+ subset and examine their anti-tumor efficacy in 
samples from patients treated with or without evolocumab. Cytotoxicity determined by flow cytometry of 
stained tumor cells will be compared to no TIL exposure. Patient-derived neurospheres will generated in 
vitro based on established protocols 26.  Statistical methods described for exploratory objective #1 will be 
used. 
13.7 
Interim Analysis 
Not applicable. 
13.8 
Sample Size Calculation 
The sample size selected was chosen for practical considerations in the setting of a phase 0 study, to allow 
descriptive analysis of the changes of the biologic endpoints in the evolocumab treated (cases) and 
untreated patients (controls). This may provide important biologic insights into the secondary and 
exploratory endpoints. 
14 ADMINISTRATIVE AND ETHICAL CONSIDERATIONS 
14.1 
Regulatory and Ethical Compliance 
This protocol was designed and will be conducted and reported in accordance with the International 
Conference on Harmonization (ICH) Harmonized Tripartite Guidelines for Good Clinical Practice, the 
Declaration of Helsinki, and applicable federal, state, and local regulations. 
14.2 
DUHS Institutional Review Board and DCI Protocol Review and 
Monitoring Committee 
The protocol, informed consent form, advertising material, and additional protocol-related documents 
must be submitted to the DUHS Institutional Review Board (IRB) and DCI Protocol Review and Monitoring 
Committee (PRMC) for review.  The study may be initiated only after the Principal Investigator has received 
written and dated approval from the PRMC and IRB. 
 

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The Principal Investigator must submit and obtain approval from the IRB for all subsequent protocol 
amendments and changes to the informed consent form.  The PRMC should be informed about any 
protocol amendments that potentially affect research design or data analysis (i.e. amendments affecting 
subject population, inclusion/exclusion criteria, agent administration, statistical analysis, etc.). 
 
The Principal Investigator must obtain protocol re-approval from the IRB within 1 year of the most recent 
IRB approval.  The Principal Investigator must also obtain protocol re-approval from the PRMC within 1 
year of the most recent IRB approval, for as long as the protocol remains open to subject enrollment. 
14.3 
Informed Consent 
The informed consent form must be written in a manner that is understandable to the subject population.  
Prior to its use, the informed consent form must be approved by the IRB. 
 
The Principal Investigator or authorized key personnel will discuss with the potential subject the purpose of 
the research, methods, potential risks and benefits, subject concerns, and other study-related matters.  
This discussion will occur in a location that ensures subject privacy and in a manner that minimizes the 
possibility of coercion.  Appropriate accommodations will be made available for potential subjects who 
cannot read or understand English or are visually impaired.  Potential subjects will have the opportunity to 
contact the Principal investigator or authorized key personnel with questions, and will be given as much 
time as needed to make an informed decision about participation in the study. 
 
Before conducting any study-specific procedures, the Principal Investigator must obtain written informed 
consent from the subject or a legally acceptable representative.  The original informed consent form will be 
stored with the subject’s study records, and a copy of the informed consent form will be provided to the 
subject.  The Principal Investigator is responsible for asking the subject whether the subject wishes to 
notify his/her primary care physician about participation in the study.  If the subject agrees to such 
notification, the Principal Investigator will inform the subject’s primary care physician about the subject’s 
participation in the clinical study. 
14.4 
Study Documentation 
Study documentation includes, but is not limited to, source documents, case report forms (CRFs), 
monitoring logs, appointment schedules, study team correspondence with sponsors or regulatory 
bodies/committees, and regulatory documents that can be found in the DCI-mandated “Regulatory 
Binder,” which includes, but is not limited to, protocol and amendments, approved informed consent 
forms, and laboratory certifications. 
 
Source documents are original records that contain source data, which is all information in original records 
of clinical findings, observations, or other activities in a clinical trial necessary for the reconstruction and 
evaluation of the trial.  Source documents include, but are not limited to, hospital records, clinical and 
office charts, laboratory notes, memoranda, subjects’ diaries or evaluation checklists, pharmacy dispensing 
records, recorded data from automated instruments, copies or transcriptions certified after verification as 
being accurate copies, microfiches, photographic negatives, microfilm or magnetic media, x-rays, subject 
files, and records kept at the pharmacy, at the laboratories, and at medico-technical departments involved 
in the clinical trial.  When possible, the original record should be retained as the source document.  
However, a photocopy is acceptable provided that it is a clear, legible, and an exact duplication of the 
original document. 
 
An electronic case report form (CRF) will be the primary data collection document for the study.  The CRFs 
will be updated within two weeks of acquisition of new source data.  Only approved study staff (e.g., the PI, 
the clinical research coordinator, the data management team, and the assistant research practice 
manager) are permitted to make entries, changes, or corrections in the CRF.  The Principal Investigator or 

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authorized key personnel will maintain a record of the changes and corrections.  For eCRFs, an audit trail 
will be maintained by the 21CFR Part 11 compliant electronic CRF management system. 
14.5 
Privacy, Confidentiality, and Data Storage 
The Principal Investigator will ensure that subject privacy and confidentiality of the subject’s data will be 
maintained.  Research Data Security Plans (RDSPs) will be approved by the appropriate institutional Site 
Based Research group. 
 
To protect privacy, every reasonable effort will be made to prevent undue access to subjects during the 
course of the study.  Prospective participants will be consented in an exam room where it is just the 
research staff, the patient and his family, if desired. For all future visits, interactions with research staff 
(study doctor and study coordinators) regarding research activities will take place in a private exam room. 
All research related interactions with the participant will be conducted by qualified research staff who are 
directly involved in the conduct of the research study. 
 
To protect confidentiality, subject files in paper format will be stored in secure cabinets under lock and key 
accessible only by the research staff. Subjects will be identified only by a unique study number and subject 
initials.  Electronic records of subject data will be maintained using a 21 CFR Part 11 compliant dedicated 
database, which is housed in an encrypted and password-protected network server.  Access to electronic 
databases will be limited to those authorized by the PRTBTC Data Manager.  The security and viability of 
the IT infrastructure will be managed by DCI and/or Duke Medicine.   
 
Upon completion of the study, research records will be archived and handled per DUHS HRPP policy.     
 
Subject names or identifiers will not be used in reports, presentations at scientific meetings, or publications 
in scientific journals. 
14.6 
Data and Safety Monitoring 
Data and Safety Monitoring will be performed in accordance with the DCI Data and Safety Monitoring Plan.  
For a more detailed description of the DSMP for this protocol, refer to Section 11 (Sections 11.6 and 11.7 in 
particular) and Section 12. 
14.7 
Protocol Amendments 
All protocol amendments must be initiated by the Principal Investigator and approved by the IRB prior to 
implementation.  IRB approval is not required for protocol changes that occur to protect the safety of a 
subject from an immediate hazard.  However, the Principal Investigator must inform the IRB and all other 
applicable regulatory agencies of such action immediately. 
 
Though not yet required, the PRMC should be informed about any protocol amendments that potentially 
affect research design or data analysis (i.e., amendments affecting subject population, inclusion/exclusion 
criteria, agent administration, etc.). 
14.8 
Records Retention 
The Principal Investigator will maintain study-related records for the longer of a period of at least six years 
after study completion. 

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15 REFERENCES 
 
 
1. 
Liu XJ, Bao XH, Hu MJ, et al: Inhibition of PCSK9 potentiates immune checkpoint therapy for 
cancer. Nature 588:693-+, 2020 
 
2. 
Ostrom QT, Gittleman H, Fulop J, et al: CBTRUS Statistical Report: Primary Brain and Central 
Nervous System Tumors Diagnosed in the United States in 2008-2012. Neuro Oncol 17 Suppl 4:iv1-iv62, 2015 
 
3. 
Chinot OL, Wick W, Mason W, et al: Bevacizumab plus radiotherapy-temozolomide for newly 
diagnosed glioblastoma. N Engl J Med 370:709-722, 2014 
 
4. 
Gilbert MR, Dignam JJ, Armstrong TS, et al: A randomized trial of bevacizumab for newly 
diagnosed glioblastoma. N Engl J Med 370:699-708, 2014 
 
5. 
Stupp R, Taillibert S, Kanner AA, et al: Maintenance Therapy With Tumor-Treating Fields Plus 
Temozolomide vs Temozolomide Alone for Glioblastoma: A Randomized Clinical Trial. JAMA 314:2535-43, 2015 
 
6. 
Kelly PJ: Stereotactic resection and its limitations in glial neoplasms. Stereotact Funct Neurosurg 
59:84-91, 1992 
 
7. 
Vredenburgh JJ, Desjardins A, Herndon JE, 2nd, et al: Bevacizumab plus irinotecan in recurrent 
glioblastoma multiforme. J Clin Oncol 25:4722-9, 2007 
 
8. 
Stupp R, Mason WP, van den Bent MJ, et al: Radiotherapy plus concomitant and adjuvant 
temozolomide for glioblastoma. N Engl J Med 352:987-96, 2005 
 
9. 
Walker MD, Green SB, Byar DP, et al: Randomized comparisons of radiotherapy and nitrosoureas 
for the treatment of malignant glioma after surgery. N Engl J Med 303:1323-9, 1980 
 
10. 
Tan AC, Ashley DM, López GY, et al: Management of glioblastoma: State of the art and future 
directions. CA Cancer J Clin 70:299-312, 2020 
 
11. 
Khasraw M, Reardon DA, Weller M, et al: PD-1 Inhibitors: Do they have a Future in the Treatment 
of Glioblastoma? Clin Cancer Res 26:5287-5296, 2020 
 
12. 
Sabatine MS, Giugliano RP, Keech AC, et al: Evolocumab and Clinical Outcomes in Patients with 
Cardiovascular Disease. New England Journal of Medicine 376:1713-1722, 2017 
 
13. 
Schaller TH, Foster MW, Thompson JW, et al: Pharmacokinetic Analysis of a Novel Human 
EGFRvIII:CD3 Bispecific Antibody in Plasma and Whole Blood Using a High-Resolution Targeted Mass Spectrometry 
Approach. Journal of Proteome Research 18:3032-3041, 2019 
 
14. 
Walker MD, Hunt WE, Mahaley MS, et al: Evaluation of Bcnu and-or Radiotherapy in Treatment 
of Anaplastic Gliomas - Cooperative Clinical-Trial. Journal of Neurosurgery 49:333-343, 1978 
 
15. 
Shapiro WR: Therapy of Adult Malignant Brain-Tumors - What Have the Clinical-Trials Taught Us. 
Seminars in Oncology 13:38-45, 1986 
 
16. 
Salford LG, Brun A, Nirfalk S: 10-Year Survival among Patients with Supratentorial Astrocytomas 
Grade-Iii and Grade-Iv. Journal of Neurosurgery 69:506-509, 1988 
 
17. 
Dinapoli RP, Brown LD, Arusell RM, et al: Phase-Iii Comparative-Evaluation of Pcnu and 
Carmustine Combined with Radiation-Therapy for High-Grade Glioma. Journal of Clinical Oncology 11:1316-1321, 
1993 
 
18. 
Imperato JP, Paleologos NA, Vick NA: Effects of Treatment on Long-Term Survivors with 
Malignant Astrocytomas. Annals of Neurology 28:818-822, 1990 
 
19. 
Hall WA, Fodstad O: Immunotoxins and Central-Nervous-System Neoplasia. Journal of 
Neurosurgery 76:1-12, 1992 
 
20. 
Traynor K: Evolocumab approved for reducing LDL cholesterol. Am J Health Syst Pharm 72:1599-
600, 2015 
 
21. 
Khatib RW, F.: Pharmacology of Medications Used in the Treatment of Atherosclerotic 
Cardiovascular Disease, in Vasan RSS, D. B. (ed): Encyclopedia of Cardiovascular Research and Medicine. Oxford, 
Elsevier, 2018 
 
22. 
Sabatine MS: PCSK9 inhibitors: clinical evidence and implementation. Nat Rev Cardiol 16:155-
165, 2019 

Pro00108375:  Evolocumab in GBM 
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23. 
Liu X, Suo R, Chan CZY, et al: The immune functions of PCSK9: Local and systemic perspectives. J 
Cell Physiol 234:19180-19188, 2019 
 
24. 
Amgen : Repatha (evolocumab) injection, for subcutaneous use: US prescribing information, 
2021 
 
25. 
Kasichayanula S, Grover A, Emery MG, et al: Clinical Pharmacokinetics and Pharmacodynamics of 
Evolocumab, a PCSK9 Inhibitor. Clin Pharmacokinet 57:769-779, 2018 
 
26. 
Hasselbach LA, Irtenkauf SM, Lemke NW, et al: Optimization of high grade glioma cell culture 
from surgical specimens for use in clinically relevant animal models and 3D immunochemistry. J Vis Exp:e51088, 
2014