Conditions: Castration-Resistant Prostate Carcinoma Metastatic Malignant Neoplasm in the Bone Metastatic Malignant Neoplasm in the Soft Tissues Metastatic Prostate Adenocarcinoma Recurrent Prostate Carcinoma Stage III Prostate Adenocarcinoma AJCC v7 Stage IV Prostate Adenocarcinoma AJCC v7
Allocation: NA | Intervention model: SINGLE_GROUP
Enrollment: 36
Healthy volunteers: no

Brief summary:
This phase II trial studies genetic and molecular mechanisms in assessing response in patients with prostate cancer receiving enzalutamide therapy. Androgens can cause the growth of prostate cancer cells. Antihormone therapy, such as enzalutamide, may lessen the amount of androgens made by the body. Studying samples of tissue and blood in the laboratory from patients with prostate cancer may help doctors better understand castration-resistant prostate cancer. It may also help doctors make improvements in prostate cancer treatment.

Detailed description:
PRIMARY OBJECTIVE:

I. To assess the correlations between baseline molecular features and pathways and prostate-specific antigen (PSA) change (\</\>= 50% decline) at 12 weeks versus (vs.) baseline.

SECONDARY OBJECTIVES:

I. To measure PSA change at 12 weeks and at each study visit vs. baseline after enzalutamide treatment.

II. To measure objective response after enzalutamide treatment. III. To assess the correlations between the baseline molecular features and pathways and progression-free survival, disease-specific survival, and overall survival.

IV. To assess the correlations between the baseline molecular features and pathways and time to PSA progression.

V. To identify molecular features and cellular pathways present in tumors from men with metastatic castrate-resistant prostate cancer (CRPC) that are progressing despite enzalutamide treatment.

VI. To explore correlation between baseline molecular features and pathways and changes in circulating tumor cells (CTCs) counts.

VII. To explore correlation between baseline molecular features and pathways and objective response.

VIII. To assess the correlations between the baseline molecular features and pathways and degree of PSA decline at 12 weeks and maximal PSA decline observed while on study.

IX. To assess the correlations between the baseline molecular features and time on treatment.

EXPLORATORY OBJECTIVES:

I. To assess correlations between cell-free deoxyribonucleic acid (DNA) (cfDNA) molecular features from blood and molecular features and pathways from the biopsy samples.

II. To assess correlations between cfDNA molecular features and endpoints in the primary and secondary objectives listed above.

III. To assess correlations between cell-free DNA and tumor molecular features and changes in PSA after discontinuing enzalutamide.

IV. To explore correlations with baseline molecular features and tissue histology.

V. To explore correlations with baseline tissue histology and PSA change, time to PSA progression, time on treatment, progression-free survival, and overall survival.

OUTLINE:

Patients receive enzalutamide orally (PO) once daily (QD) in the absence of disease progression or unacceptable toxicity. Patients undergo a tumor biopsy of a metastatic site at study entry (prior to initiation of enzalutamide) and after the time of progression. Patients may continue treatment beyond progression at the investigator's discretion.

After completion of study treatment, patients are followed up at 2-3 or 6 weeks and then every 12 weeks thereafter.

Arms:
arm 1: Patients receive enzalutamide PO QD in the absence of disease progression or unacceptable toxicity. Patients undergo a tumor biopsy of a metastatic site at study entry (prior to initiation of enzalutamide) and after the time of progression. Per the investigator, patients may continue treatment beyond progression.

Interventions: intervention 1: Enzalutamide
Intervention descriptions: intervention 1: Given PO

Protocol document:
Prot_SAP_000.pdf: 
ClinicalTrials.gov Identifier: NCT02099864 
Oregon Health & Science University 
OHSU Knight Cancer Institute 
OHSU eIRB Protocol #: 10241 
 
TITLE:   Molecular Mechanisms Underlying Tumor Progression Despite Enzalutamide Treatment  
 
Coordinating Center:  
 
 
The OHSU Knight Cancer Institute  
 
 
 
 
 
 
 
 
 
Oregon Health & Science University   
 
Participating Sites: 
 
 
 
Oregon Health & Science University 
 
 
 
 
 
 
 
 
 
University of California, San Francisco 
 
Principal Investigator:  
Tomasz M. Beer, MD  
Oregon Health & Science University  
3303 SW Bond Ave.  
Portland, OR 97239 
(503) 494-6594 
beert@ohsu.edu  
 
OHSU Co-Investigators: 
Christopher Ryan, MD 
 
 
Julie Graff, MD 
 
 
Jeremy Cetnar, MD  
 
Collaborators: 
 
Joshi Alumkal, MD 
 
 
Jim Korkola, PhD 
 
 
George V Thomas, MD 
 
 
Laura Heiser, PhD 
 
UCSF Co-Investigator: 
Eric Small, MD 
 
 
University of California, San Francisco 
 
 
415-353-9865 
 
 
esmall@medicine.ucsf.edu 
 
Statistician: 
 
Yiyi Chen, PhD 
Oregon Health & Science University  
3181 SW Sam Jackson Park Road 
Portland, OR 97239 
 
 
503-346-0790 
 
 
chenyiy@ohsu.edu 
 
Original Protocol Date: 
September 23, 2013  
Protocol Revision Dates: 
September 17, 2014 
 
 
March 20, 2015 
 
 
August 29, 2016 
 
 
January 6, 2017 
 
 
February 28, 2019 
 
 
June 25, 2019 
Current Version Date: 
March 2, 2020 
 
 


Protocol version: 02March2020 
1 
SCHEMA 
 
 
 
 
 
TABLE OF CONTENTS 
 
1.0 
OBJECTIVES ___________________________________________________________________ 2 
2.0 
BACKGROUND ________________________________________________________________ 3 
3.0 
STUDY POPULATION ___________________________________________________________ 6 
4.0 
REGISTRATION PROCEDURES___________________________________________________ 9 
5.0 
TREATMENT PLAN ____________________________________________________________ 10 
6.0 
DOSING DELAYS/DOSE MODIFICATIONS ________________________________________ 12 
7.0 
ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS ______________________ 12 
8.0 
PHARMACEUTICAL INFORMATION _____________________________________________ 17 
9.0 
BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES ____________________________ 21 
10.0 
STUDY PROCEDURES AND SCHEDULE OF EVENTS ______________________________ 25 
11.0 
MEASUREMENT OF EFFECT ____________________________________________________ 28 
12.0 
DATA REPORTING/REGULATORY REQUIREMENTS ______________________________ 32 
13.0 
STATISTICAL CONSIDERATIONS _______________________________________________ 35 
REFERENCES _______________________________________________________________________ 39 
APPENDIX A ________________________________________________________________________ 41 
APPENDIX B ________________________________________________________________________ 42 
APPENDIX C ________________________________________________________________________ 45 
APPENDIX D ________________________________________________________________________ 47 
APPENDIX E ________________________________________________________________________ 48 
APPENDIX F ________________________________________________________________________ 49 
APPENDIX G ________________________________________________________________________ 50 
 
 


Protocol version: 02March2020 
2 
1.0 
OBJECTIVES 
 
1.1 
Primary Objective 
 
1.1.1 
To assess the correlations between baseline molecular features and pathways and 
PSA change (</> 50% decline) at 12 weeks vs. baseline. 
 
 
1.2 
Secondary Objectives 
 
1.2.1 
To measure PSA change at 12 Weeks and at each study visit vs. baseline after 
enzalutamide treatment. 
 
1.2.2 
To measure objective response defined in Section 11.1.1 after enzalutamide 
treatment. 
 
1.2.3 
To assess the correlations between the baseline molecular features and pathways 
and Progression-Free Survival (defined as time from Day 1 of study drug treatment 
to date of radiographic progression or clinical progression- See Sec 5.3), Disease-
Specific Survival (defined as the time from Day 1 of study drug to date of death 
from prostate cancer), and Overall Survival (defined as time from Day 1 of study 
drug treatment to date of death from any cause). 
 
1.2.4 
To assess the correlations between the baseline molecular features and pathways 
and time to PSA progression. 
 
1.2.5 
To identify molecular features and cellular pathways present in tumors from men 
with metastatic CRPC that are progressing despite Enzalutamide treatment. 
 
1.2.6 
To explore correlation between baseline molecular features and pathways and 
changes in Circulating Tumor Cells (CTCs) counts defined in Sec 11.3.1. 
 
1.2.7 
To explore correlation between baseline molecular features and pathways and 
objective response defined in Section 11.1.1.  
 
1.2.8 
To assess the correlations between the baseline molecular features and pathways 
and degree of PSA decline at 12 weeks and maximal PSA decline observed while 
on study. 
 
1.2.9 
To assess the correlations between the baseline molecular features and time on 
treatment. 
 
 
 
1.3 
Exploratory Objectives 
 
1.3.1 
To assess correlations between cell-free DNA (cfDNA) molecular features from 
blood and molecular features and pathways from the biopsy samples. 
1.3.2 
To assess correlations between cfDNA molecular features and endpoints in the 
primary and secondary objectives listed above.  
1.3.3 
To assess correlations between cell-free DNA and tumor molecular features and 
changes in PSA after discontinuing enzalutamide. 
1.3.4  To explore correlations with baseline molecular features and tissue histology. 


Protocol version: 02March2020 
3 
1.3.5 
To explore correlations with baseline tissue histology and PSA change, time to 
PSA progression, time on treatment, progression-free survival, and overall survival. 
 
 
2.0 
BACKGROUND 
 
2.1 
Study Disease 
 
Worldwide, prostate cancer ranks third in cancer incidence and sixth in cancer mortality in 
men. Prostate cancer growth is dependent on androgens, and depleting or blocking 
androgen action has been a mainstay of treatment for over 6 decades. Hormonal therapies 
include gonadotropin-releasing hormone (GnRH) analogues, androgen receptor antagonists, 
ketoconazole, and estrogenic compounds. Tumors that progress despite castrate levels of 
testosterone in the blood are considered castration-resistant. Despite the early sensitivity of 
these tumors to hormonal strategies, castration-resistant progression generally represents a 
transition to the lethal variant of the illness, and most patients ultimately succumb to this 
disease. The median survival of castration-resistant disease is currently approximately 12 
months.1 
 
Results of clinical investigations and studies on the molecular profiles of these progressing 
tumors show that the androgen receptor (AR) remains functional and that the tumors should 
respond to strategies directed at the androgen receptor signaling axis. Overexpression of the 
AR has been documented in upwards of 50% of castration-resistant prostate cancer (CRPC) 
specimens and is believed to contribute to tumor progression.2 In addition, currently 
approved AR antagonists have the potential to agonize or stimulate androgen receptor 
signaling in the setting of AR overexpression, therefore exacerbating or accelerating 
castration-resistant tumor growth. The decline in serum levels of prostate-specific antigen 
(PSA) seen upon discontinuation of these agents is consistent with the agonist effects 
(“anti-androgen withdrawal syndrome”). 
 
In clinical practice, treatment of advanced prostate cancer is therefore limited by the 
development of resistance to anti-androgen therapies. Most patients receive two or more 
hormonal manipulations and are then offered chemotherapy as they continue to progress. A 
randomized trial in metastatic castration-resistant prostate cancer comparing docetaxel 
administered every three weeks vs. docetaxel weekly, vs. mitoxantrone has shown a modest 
survival benefit for docetaxel every 3 weeks, but this response is not durable.1 Recently, 
cabazitaxel has been approved for patients who progress on docetaxel on the basis of an 
open-label Phase 3 study demonstrating a 2.4 month overall survival benefit for men 
treated with cabazitaxel and prednisone as compared to those treated with mitoxantrone and 
prednisone.3 Because many of these resistant tumors continue to overexpress androgen 
receptors, second generation anti-androgens that are more potent and that are pure 
antagonists may be effective in patients who have failed docetaxel treatment.  
 
As the central role of the AR in prostate cancer progression became apparent, so did the 
need for novel strategies to more effectively target the AR. MDV-3100 is the first fruit of 
this labor and has recently been shown to substantially improve survival in patients with 
castration resistant prostate cancer (CRPC).4-6  
 
This result provides proof of principle for the importance of targeting AR in prostate cancer 
therapy and produces an urgent need to clarify mechanisms that account for  response or 
eventual resistance to enzalutamide, which are largely unknown. A thorough mechanistic 


Protocol version: 02March2020 
4 
understanding of the molecular basis for enzalutamide’s clinical performance will be an 
enormous asset in optimizing the use of the drug. Such understanding can yield rationally 
designed strategies to expand and extend the utility of the drug. Strategies to overcome 
resistance through novel interventions that interrupt emerging mechanisms of resistance are 
a particularly compelling long-term outcome. Equally compelling are strategies to extend 
initial response by optimizing or maximizing those factors that drive response.  
 
The purpose of this study is to determine mechanisms by which CRPC tumors resist 
treatment with the new anti-androgen enzalutamide.4,5  This study provides a tremendous 
opportunity to leverage the molecular analytic resources of a recently funded Stand Up to 
Cancer (SU2C)/Prostate Cancer Foundation (PCF)/American Association for Cancer 
Research (AACR) Dream Team grant.  This Dream Team intends to deploy a broad array 
of analytical strategies to comprehensively clarify the molecular basis of treatment 
resistance in prostate cancer.  A cartoon summarizing the major analytic efforts is shown 
below: 
 
 
 
2.2 
Study Agent(s) 
 
Enzalutamide is a novel small molecule AR antagonist selected for its activity 
against prostate cancer cells with overexpressed androgen receptor.5 Enzalutamide binds 
more tightly to the AR than does bicalutamide. Unlike bicalutamide, Enzalutamide also 
inhibits AR function by blocking nuclear translocation of the androgen receptor and 
deoxyribonucleic acid (DNA) binding. Enzalutamide has no known agonist activity when 
the androgen receptor is overexpressed.  Enzalutamide reduces androgen receptor-
dependent PSA release in bicalutamide-resistant prostate cancer cells. 
 
In a mouse xenograft model of castration-resistant prostate cancer using an androgen 
receptor overexpresssing cell line,  Enzalutamide treatment resulted in a dose-dependent 
reduction in tumor volume (p < 0.05 and p < 0.01 for mid- and high-dose groups vs. 


Protocol version: 02March2020 
5 
vehicle, respectively).5  Enzalutamide treatment decreased tumor volume, resulting in 
unmeasurable tumors in 1/7 animals in the low-dose group and 3/7 animals in the high-dose 
group. As expected, bicalutamide had little effect on tumor growth.5 
 
In addition to the human AR, the targets for which measurable binding was 
detected included the human progesterone receptor with a 50% inhibitory concentration 
(IC50) of 10–25 μM and the rat gamma amino butyric acid-gated chloride channel 
(IC50 = 2.6 μM; Ki = 2.1 μM [1.0 μg/mL]). Binding of  Enzalutamide at 25 μM to the 
human progesterone receptor was too weak to derive a inhibition constant (Ki) value. No 
significant binding was detected with the remaining 70 receptors. 
 
The tolerability, pharmacokinetics (PK), and antitumor activity of  Enzalutamide were 
studied in a multi-center, open-label, dose-escalation study of  Enzalutamide in 140 patients 
with castration-resistant prostate cancer.4 Patients were treated with  Enzalutamide at doses 
of 30–600 mg/day until disease progression or intolerable side effects developed. 
 
The antitumor activity of  Enzalutamide was assessed by post-therapy changes in PSA, soft 
tissue and osseous disease, and circulating tumor cell (CTC) count. PSA declines of ≥ 50% 
from baseline were observed in 62% of chemotherapy-naïve and 51% of post-
chemotherapy patients.4 At the time of the analyses, the median time to PSA progression 
was not yet reached for chemotherapy-naïve patients and was 186 days for post-
chemotherapy patients.4 
 
Among the chemotherapy-naïve patients, there was evidence of radiographic control (no 
progression) in 80% of patients with evaluable soft tissue disease and 63% of patients with 
bone lesions.4 Among the post-chemotherapy patients, there was evidence of radiographic 
control in 65% of patients with evaluable soft tissue disease and 51% of patients with bone 
lesions.4 The median time to radiographic progression was not yet reached for 
chemotherapy-naïve patients and was 201 days for post-chemotherapy patients.4 
Enumeration of CTCs demonstrated that 91% of patients with favorable pretreatment 
counts (i.e., < 5 CTCs/7.5 mL of blood) maintained favorable post-treatment counts, while 
49% of patients converted from unfavorable pretreatment counts (i.e., ≥ 5 CTCs/7.5 mL of 
blood) to favorable post-treatment counts. 
 
At the highest dose of 600 mg/day, two of three subjects had dose-limiting toxicities 
(seizure, rash, respectively).4 One witnessed seizure at 360 mg/day and a possible seizure at 
480 mg/day were also reported. No deaths and no other drug-related serious adverse events 
were reported. Fatigue was the most frequently reported adverse event, with dose-
dependent increases of Grade 3 fatigue (2% at 150, 10% at 240, 21% at 360, and 20% at 
480 mg/day groups). The dose of 240 mg/day was defined as the maximum tolerated dose.  
 
Enzalutamide was absorbed rapidly after oral administration, with maximum plasma 
concentration (Cmax) occurring approximately 30 minutes to 4 hours after dosing. The t1/2 
in patients was approximately 1 week (range 3 to 13 days) and did not appear to be affected 
by the dose size.  Enzalutamide plasma concentrations exhibited a low degree of inter- and 
intra-subject variability and increased linearly with dose. The PK remained linear with 
time, and there was no evidence of inhibition or autoinduction of metabolism during 
chronic administration. In accordance with a 1 week t1/2, it took approximately 1 month to 
reach steady state. The daily fluctuation in steady-state plasma concentrations (i.e., the 
difference between Cmax and minimum plasma concentration [Cmin]) was low, and PK 
profiles approximated a constant infusion. At 160 mg/day, mean plasma concentrations 
fluctuated between 12 μg/mL (Cmin) and 15 μg/mL (Cmax). 


Protocol version: 02March2020 
6 
 
 
 
2.3 
Study and Dose Rationale 
 
 Androgen receptor (AR) signaling is the principal molecular signaling driver of prostate 
cancer progression and dissemination.2,7 As a consequence, therapies that reduce androgen 
receptor signaling, principally by reducing systemic ligand production, constitute the 
mainstay of systemic therapy for advanced prostate cancer.4,8,9 Both pre-clinical 
experiments and clinical observations have clarified that resistance to primary hormonal 
treatments involves androgen-signaling related mechanisms in the majority of patients.2 A 
number of such mechanisms have been proposed; they range from somatic receptor 
mutations of the receptor, receptor splice variants, to a variety of up-stream alterations 
ranging from persistent endocrine ligand production, to paracrine or even autocrine ligand 
production, alterations in receptor cofactors, ligand-independent receptor activation, and 
others.10-19 As the central role of the AR in prostate cancer progression became apparent, so 
did the need for novel strategies to more effectively target the AR. MDV-3100 is the first 
fruit of this labor and has recently been shown to substantially improve survival in patients 
with castration resistant prostate cancer (CRPC).4-6  
 
This result provides proof of principle for the importance of targeting AR in prostate cancer 
therapy and produces an urgent need to clarify the mechanisms of response and resistance 
to MDV-3100, which are largely unknown. A thorough mechanistic understanding of the 
molecular basis for MDV-3100’s clinical performance will be an enormous asset in 
optimizing the use of the drug. Such understanding can yield rationally designed strategies 
to expand and extend the utility of the drug. Strategies to overcome resistance through 
novel interventions that interrupt emerging mechanisms of resistance are a particularly 
compelling long-term outcome. Equally compelling are strategies to extend initial response 
by optimizing or maximizing those factors that drive response.  
 
For our study, we will treat patients with 160mg PO QD of Enzalutamide. This is the FDA-
approved dose used in the recent randomized, placebo-controlled phase III study that 
demonstrated a five month improvement in overall survival.6 This dose was well-tolerated.6 
 
3.0 
STUDY POPULATION 
3.1 
Inclusion Criteria 
 
3.1.1 
Histologically or cytologically confirmed adenocarcinoma of the prostate without 
pure small cell carcinoma. Patients without histologically confirmed 
adenocarcinoma may be eligible if both the treating physician and the study PI 
agree that the patient’s history is unambiguously indicative of advanced 
adenocarcinoma.  
 
3.1.2 
Ongoing androgen deprivation therapy with a GnRH analogue or orchiectomy (i.e., 
surgical or medical castration). Patients who have not had an orchiectomy must 
maintain effective GnRH-analogue therapy for the duration of the trial. 
 
3.1.3 
Radiographic evidence of regional or distant metastases with suspected tumor in an 
area that is safe to biopsy 
 
3.1.4 
Willingness to undergo a tumor biopsy at baseline and at disease progression 
 
3.1.5 
Serum testosterone level < 50 ng/dL at Screening  


Protocol version: 02March2020 
7 
 
3.1.6 
Progressive disease by PSA or imaging in the setting of medical or surgical 
castration. Disease progression for study entry is defined as one or more of the 
following three criteria: 
 
PSA evidence for progressive prostate cancer which consists of a PSA level of at 
least 2 ng/ml which has risen on at least 2 successive occasions, at least 1 week 
apart (#2 & #3a in figure below). If the confirmatory PSA value is less (#3b) than 
the screening (PSA #2) value, then an additional PSA value (PSA #4) greater than 
#2 will be required to document progression of ≥ 1 week  
 
 
 
 
Soft tissue disease progression defined by RECIST 1.1 
 
 
Bone disease progression defined by two or more new lesions on bone scan 
 
3.1.7 
Patient’s physician has already recommended enzalutamide for treatment of 
progression 
 
3.1.8 
ECOG performance status of 0–2 
 
  
 
3.1.9 
Willing and able to give informed consent 
 
  
 
3.1.10 Estimated life expectancy ≥ 6 months 
 
3.1.11 Subjects who have partners of childbearing potential must be willing to use a 
method of birth control with adequate barrier protection as determined to be 
acceptable by the principal investigator and sponsor during the study and for 1 
week after last study drug administration. 
 
3.1.12 A minimum of 4 weeks elapsed off of anti-androgen therapy prior to enrollment for 
flutamide and 6 weeks for bicalutamide and nilutamide without evidence of an anti-
androgen withdrawal response. Patients who NEVER HAD A PSA decline with 
the most recent anti-androgen therapy or in whom the response to the most recent 
anti-androgen was for < 3 months require only a 2 week washout period prior to 
            PSA values to be obtained ≥ 1 week apart 


Protocol version: 02March2020 
8 
first dose of study drug. 
  
3.1.13 A minimum of 4 weeks from prior systemic anti-cancer therapies or 3 weeks for 
radiation treatment prior to enrollment is required.  
 
3.2 
Exclusion Criteria 
 
3.2. 1 Severe, concurrent disease, infection, or co-morbidity that, in the judgment of the 
investigator, would make the patient inappropriate for enrollment 
 
3.2.2 
 Previous treatment with docetaxel for metastatic prostate cancer 
 
3.2.3 
Known metastases in the brain or active epidural disease (NOTE: patients with 
treated epidural disease are allowed) 
 
3.2.4 
Laboratory Values as follows:  
 
Absolute neutrophil count < 1,000/μL,  
 
Platelet count < 75,000/μL, 
 
Hemoglobin < 9 g/dL at the Screening visit; (NOTE: subject may not have 
received any growth factors or blood transfusions within seven days of the 
hematologic laboratory values obtained at the Screening visit). 
 
Total bilirubin (TBL), alanine aminotransferase (ALT) or aspartate 
aminotransferase (AST) > 2.5 times the upper limit of normal at the Screening 
visit. 
 
Creatinine (Cr) > 2 mg/dL at the Screening visit. 
 
PT or INR and a PTT > 1.5 times the upper limit of normal 
 
3.2.5 
Previous treatment with an agent that blocks adrenal androgen synthesis (e.g.. 
abiraterone acetate, TAK-700, TOK-001, ketoconazole) or second generation 
androgen receptor (AR) antagonists (e.g., BMS 641988, ARN-509,TOK-001) 
 
3.2.6 
Systemic corticosteroids greater than the equivalent of 10 mg of prednisone per day 
within 4 weeks of study drug administration are prohibited. 
 
3.2.7 
Structurally unstable bone lesions suggesting impending fracture 
 
3.2.8 
Previous treatment with Enzalutamide (MDV3100)  
 
3.2.9 
Medical contraindications to stopping aspirin, Coumadin or other anticoagulants  
prior to image-guided tumor biopsies Follow institutional guidelines when 
determining drugs to avoid and length of washout (OHSU guidelines can be found 
in Appendix H) 
 
3.2.10 Plans to initiate treatment with an investigational agent during the study 
 
3.2.11 History of seizure or condition that may predispose to seizure. Also, history of loss 
of consciousness or transient ischemic attack within 12 months of Day 1 visit. 
 
3.2.12 Concomitant use of the strong CYP2C8 inhibitors gemfibrozil or trimethoprim 
[Bactrim]) 
 


Protocol version: 02March2020 
9 
3.2.13 History of known malabsorption syndrome or prior surgery(ies) that may lead to 
malabsorption. 
 
3.2.14 Use of herbal products that may have hormonal anti-prostate cancer activity and/or 
are known to decrease PSA levels (e.g., saw palmetto) within 4 weeks of study 
drug administration (Day 1). 
 
3.2.15     Use of the following drugs within 4 weeks of study drug administration: 
 5 α-reductase inhibitors (finasteride, dutasteride),Estrogens, Cyproterone acetate, biologic, 
or other agents with anti-tumor activity against prostate cancer, and androgens 
(testosterone, dihydroepiandrosterone [DHEA], etc.)  
 
3.2.16 A second active malignancy except adequately treated non-melanoma skin cancer 
or other non-invasive or in situ neoplasm 
 
4.0 
REGISTRATION PROCEDURES 
4.1 
Subject Registration 
 
4.1.1 
Local registration 
 
Registration will include the following: 
o A completed Subject Enrollment Form   
o A completed Eligibility Checklist signed by the investigator 
o Complete source documentation for each eligibility criterion 
o Signed  copies of the most recently IRB-approved, informed consent form and 
HIPPA authorization 
 
Registrations from all consented subjects must be entered into the Knight Clinical 
Research Management System (CRMS). 
 
4.1.2 
Multicenter Registration 
The OHSU coordinating center study team will manage subject registration. 
Investigators at participating sites will identify eligible subjects and send screening 
materials with source documents that support eligibility to OHSU in real time and 
in accordance with study protocol.  Designated Knight clinical staff must review 
and verify eligibility before the participating site may enroll and treat its subject. 
The OHSU coordinating center team will verify completeness of documents, enter 
registration information into the Knight CRMS, and assign a study 
number/identifier.  The coordinating center will send an email to the participating 
site indicating whether or not the subject is eligible, verify registration, and assign a 
participant number/identifier. 
 
Registration will include the following: 
o A completed Subject Enrollment Form  
o A completed Eligibility Checklist signed by the investigator 
o Complete source documentation for each eligibility criterion 
o Signed  copies of the most recently IRB-approved, informed consent form and 
HIPPA authorization 
 
Each site must maintain a screening log of all subjects who sign consent, including 
screen failures and those who withdraw consent. The log must also document 


Protocol version: 02March2020 
10 
reason for screen failure. 
 
This log will be submitted to the coordinating center on a regular basis. 
Participating sites are required to retain, in a confidential manner, sufficient 
information on each subject so that the subject may be contacted should the need 
arise. 
 
5.0 
TREATMENT PLAN 
5.1 
Enzalutamide 
 
All patients will receive Enzalutamide 160 mg (four 40 mg capsules) administered orally 
once daily.  Enzalutamide can be taken with or without food.  Capsules are to be swallowed 
whole. Patients will record daily drug administration in a drug diary for tracking purposes 
(see Appendix F for diary.) Treatment adjustments will be at the discretion of the treating 
physician and are not a part of this study. See Section 6.0.  
 
5.2 
Tumor Biopsy at Study Entry and at Disease Progression 
 
All subjects will undergo a tumor biopsy of a metastatic site at study entry (prior to 
initiation of Enzalutamide) and after the time of progression. NOTE: Every effort should be 
made to biopsy NEW lesions if possible. Every effort should be made to perform the 
progression biopsy prior to discontinuation of Enzalutamide treatment. 
 
The tumor biopsies for this study will be collected and shipped via the established 
procedures described below from the University of California, San Francisco, Stand Up to 
Cancer biopsy protocol CC#125519 (OHSU protocol #9204) -Radiologically Guided 
Biopsies Of Metastatic Castration Resistant Prostate Cancer To Identify Adaptive 
Mechanisms of Resistance. 
 
All subjects participating in this protocol will also be enrolled in the study referenced 
above. A separate consent will be obtained for this. 
 
In all subjects, an image-guided (CT or ultrasound) core bone or soft tissue biopsy will be 
performed (please see Appendix B for more details about this procedure). Patients will be 
consented to the appropriate procedure prior to biopsy. All screening imaging for patients 
will be reviewed for eligibility/feasibility of the tumor biopsy. Lesions will be chosen based 
upon the strength of the evidence suggesting the presence of metastasis and with the goal of 
minimizing patient risk. New soft tissue or bone lesions of existing lesions with 
documented radiologic progression should be prioritized for biopsy. If the Radiologist in 
charge of the procedure cannot identify a lesion amenable for biopsy, the patient will be 
considered a screening failure. 
 
The biopsies will be performed in an interventional radiology suite with radiological 
guidance (typically CT or MRI) in accordance with the standard operating procedure in 
Appendix B and institutional standards. CT or MRI will confirm designated lesions 
immediately prior to biopsy. Once the target lesion(s) identified, six (6) to eight (8) biopsies 
will be performed. Preferably, a 16 gauge BonoptyTM needle or biopsy needle with an 
equivalent 16g bore will be used to biopsy the metastatic lesion. If the lesion is a bone 
metastasis, the Bonopty needle will be passed through the cortical bone and into the target 
lesion. Optimal results are obtained when the biopsies are performed on medulary bone 
directly adjacent to blastic lesion. Soft tissue biopsies should be taken so that a core of 


Protocol version: 02March2020 
11 
approximately 10 to 20 mm in length is obtained. Core biopsies will be extracted: 2 will be 
placed in neutral-buffered formalin and 2 to 4 will be immediately frozen on a pre-frozen 
bed of OCT (Optimal Cutting Temperature compound used for frozen sections), covered 
with additional OCT, and kept on dry ice or at -80 C (see Appendix B). 
 
Please see Appendix B for the handling of these tissues. 
 
5.3 
Definition of Progression for Biopsy upon Progression  
 
Radiographic progression: 
a) Soft tissue progression: by RECIST v1.1 
i. Progression at the first scheduled reassessment at Week 12 by RECIST 1.1 
must be confirmed by a second scan performed 6 or more weeks later. 
Confirmatory scans should show progressively worsening disease compared to 
the Week 12 scan.  
ii. Progressive soft tissue disease on CT or MRI per RECIST 1.1 seen for the first 
time after Week 12 does not require confirmation. 
and/or 
 
b) Bone scan progression: The appearance of ≥ 2 new lesions that are confirmed. 
i. New lesions at the first scheduled reassessment at Week 12 must be confirmed 
by a second bone scan performed 6 or more weeks later. The confirmatory 
bone scan should show > 2 additional new lesions compared to the Week 12 
scans.  
ii. New lesions compared to week 12 that are detected for the first time after the 
Week 12 reassessment should be confirmed by a second assessment performed 
6 or more weeks later. Confirmatory scans should show > 2 new lesions 
compared to the prior reassessment. 
 
c) If the investigator’s clinical assessment based on patient symptoms, laboratory data, 
and radiographs suggests the patient may still be clinically benefitting the patient may 
continue on treatment beyond protocol defined radiographic progression.. Every 
attempt should be made to obtain a biopsy when the decision is made to discontinue 
Enzalutamide. 
 
Clinical progression 
d) Clinical progression at investigator’s discretion.  If clinical progression is the only 
trigger for biopsy, tumor assessment by CT of the chest/abdomen/pelvis and Bone scan 
should be obtained prior to biopsy. (The CT and Bone scan does not need to be 
repeated if scans were done within 30 days.)  
 
Note that PSA progression alone does not meet criteria for the progression-triggered 
biopsy. However, if the investigator plans to change therapy due to PSA progression a 
biopsy may be obtained.  
 
Every attempt should be made to obtain the progression biopsy. If in the opinion of the 
investigator the subject’s progression biopsy cannot be performed the reason will be 
documented and the subject will continue to be followed per protocol.  
 
5.4 
General Concomitant Medication and Supportive Care Guidelines 
 
Patients should receive supportive care. This includes antibiotics, anti-emetics, pain 


Protocol version: 02March2020 
12 
medications, and bone targeted therapy. Growth factors (GCSF or Erythropoietin) or 
transfusion with blood products are allowed as long as these cytopenias are not felt to be 
related to Enzalutamide. Please also see section 8.2.  
 
 
5.5 
Banking of Specimens for Potential Future Research  
 
Specimens collected and any serum/plasma will be banked for future research under the 
“Master Protocol for Cancer Research Specimen Bank and Database” (OHSU IRB 2816), 
and will be used to search for biomarkers of response and resistance to therapy.  Future 
research may include genetic research. 
 
 
 
5.6 
Duration of Treatment 
Subjects will remain on Enzalutamide treatment until after progression-triggered biopsy, 
unacceptable toxicity (including any seizures), any adverse event that is intolerable to the 
subject and cannot be ameliorated by the use of adequate medical intervention or that in the 
opinion of the physician would lead to undue risk to the subject if dosing continued, 
withdrawal of consent or if the physician feels it is in the best interest of the subject to 
discontinue therapy. 
 
5.7 
Duration of Follow-Up 
Subjects who have an adverse event(s) related to study treatment (biopsy or Enzalutamide) 
will be followed until resolution or stabilization of the adverse event. Subjects who 
discontinue Enzalutamide treatment prior to confirmed radiographic progression will be 
followed for progression free survival and overall survival.  All subjects will be followed 
for overall survival. See Section 10.3 
 
6.0 
DOSING DELAYS/DOSE MODIFICATIONS 
Dose Delays 
Subjects requiring > 28 consecutive days of drug interruption will meet criteria for study treatment 
discontinuation. 
 
Dose Modifications 
If a patient experiences a seizure, permanently discontinue study drug treatment. 
 
For < Grade 3 AEs that are not seizures that are probably or definitely related to 
enzalutamide, a dose reduction to 120mg is allowed. A further dose reduction to 80mg for 
< Grade 3 AEs that are not seizures but that are persistent despite 120mg dose reduction is 
allowed.If a patient experiences a ≥ Grade 3AE that is not a seizure and is probably or definitely 
related to study treatment, withhold dosing until symptoms improve to Grade 1 or baseline. Once 
the adverse event resolves to Grade 1 or baseline, the study drug should be dose reduced to 120mg 
if treatment is resumed. A further dose reduction to 80mg is allowed if symptoms recur.  
 
 
7.0 
ADVERSE EVENTS:  LIST AND REPORTING REQUIREMENTS 
7.1 
Adverse Events and Potential Risks List(s)  
 
Enzalutamide 
The most common adverse drug reactions (≥ 5%) reported in patients receiving 
Enzalutamide in the randomized clinical trial were asthenia/fatigue, back pain, diarrhea, 
arthralgia, hot flush, peripheral edema, musculoskeletal pain, headache, upper respiratory 
infection, muscular weakness, dizziness, insomnia, lower respiratory infection, spinal cord 


Protocol version: 02March2020 
13 
compression and cauda equina syndrome, hematuria, paresthesia, anxiety, and 
hypertension. Grade 3 and higher adverse reactions were reported among 47% of 
Enzalutamide-treated patients and 53% of placebo-treated patients. Discontinuations due to 
adverse events were reported for 16% of Enzalutamide-treated patients and 18% of 
placebo-treated patients. The most common adverse reaction leading to treatment 
discontinuation was seizure, which occurred in 0.9% of the Enzalutamide-treated patients 
compared to none (0%) of the placebo-treated patients.  
 
Table below shows adverse reactions reported in the randomized clinical trial that occurred 
at a ≥ 2% absolute increase in frequency in the Enzalutamide arm compared to the placebo 
arm. 
 
 


Protocol version: 02March2020 
14 
 
XTANDI 
N = 800 
Placebo 
N = 399 
 
Grade 1-4 
(%) 
Grade 3-4 
(%) 
Grade 1-4 
(%) 
Grade 3-4 
(%) 
General Disorders 
Asthenic Conditionsa 
50.6 
9.0 
44.4 
9.3 
Peripheral Edema 
15.4 
1.0 
13.3 
0.8 
Musculoskeletal And Connective Tissue Disorders 
Back Pain 
26.4 
5.3 
24.3 
4.0 
Arthralgia 
20.5 
2.5 
17.3 
1.8 
Musculoskeletal Pain 
15.0 
1.3 
11.5 
0.3 
Muscular Weakness 
9.8 
1.5 
6.8 
1.8 
Musculoskeletal Stiffness 
2.6 
0.3 
0.3 
0.0 
Gastrointestinal Disorders 
Diarrhea 
21.8 
1.1 
17.5 
0.3 
Vascular Disorders 
Hot Flush 
20.3 
0.0 
10.3 
0.0 
Hypertension 
6.4 
2.1 
2.8 
1.3 
Nervous System Disorders 
Headache 
12.1 
0.9 
5.5 
0.0 
Dizzinessb 
9.5 
0.5 
7.5 
0.5 
Spinal Cord Compression and 
Cauda Equina Syndrome 
7.4 
6.6 
4.5 
3.8 
Parasthesia 
6.6 
0.0 
4.5 
0.0 
Mental Impairment Disordersc 
4.3 
0.3 
1.8 
0.0 
Hypoesthesia 
4.0 
0.3 
1.8 
0.0 
Infections and Infestations 
 
 
 
 
Upper Respiratory Tract 
Infectiond 
10.9 
0.0 
6.5 
0.3 
Lower Respiratory Tract And 
Lung Infectione 
8.5 
2.4 
4.8 
1.3 
Psychiatric Disorders 
Insomnia 
8.8 
0.0 
6.0 
0.5 
Anxiety 
6.5 
0.3 
4.0 
0.0 
Renal And Urinary Disorders 
Hematuria 
6.9 
1.8 
4.5 
1.0 
Pollakiuria 
4.8 
0.0 
2.5 
0.0 
Injury, Poisoning And Procedural Complications 
Fall 
4.6 
0.3 
1.3 
0.0 
Non-pathologic Fractures 
4.0 
1.4 
0.8 
0.3 
Skin and Subcutaneous Tissue Disorders 
Pruritus 
3.8 
0.0 
1.3 
0.0 
Dry Skin 
3.5 
0.0 
1.3 
0.0 
Respiratory Disorders 
Epistaxis 
3.3 
0.1 
1.3 
0.3 
a  Includes asthenia and fatigue. 
b  Includes dizziness and vertigo. 
c  Includes amnesia, memory impairment, cognitive disorder, and disturbance in attention. 
d  Includes nasopharyngitis, upper respiratory tract infection, sinusitis, rhinitis, pharyngitis, and laryngitis. 
e  Includes pneumonia, lower respiratory tract infection, bronchitis, and lung infection. 
 
Seizure 


Protocol version: 02March2020 
15 
In the randomized clinical trial, 7 of 800 (0.9%) patients treated with Enzalutamide 160 mg 
once daily experienced a seizure. No seizures occurred in patients treated with placebo. 
Seizures occurred from 31 to 603 days after initiation of Enzalutamide. Patients 
experiencing seizure were permanently discontinued from therapy and all seizures resolved. 
There is no clinical trial experience re-administering Enzalutamide to patients who 
experienced seizures. 
 
The safety of Enzalutamide in patients with predisposing factors for seizure is not known 
because these patients were excluded from the trial. These exclusion criteria included a 
history of seizure, underlying brain injury with loss of consciousness, transient ischemic 
attack within the past 12 months, cerebral vascular accident, brain metastases, brain 
arteriovenous malformation or the use of concomitant medications that may lower the 
seizure threshold. 
 
Because of the risk of seizure associated with Enzalutamide use, patients should be advised 
of the risk of engaging in any activity where sudden loss of consciousness could cause 
serious harm to themselves or others. 
 
Risks of Biopsy include: bleeding, pain, damage to adjacent organs, and infection.  
 
7.2 
Adverse Event Characteristics  
Adverse events will be collected and attribution will be designated for any untoward 
clinical experience, including but not limited to adverse events related to biopsies and 
associated sedation, venipuncture, as well as Enzalutamide 
 
CTCAE term (AE description) and grade:  The descriptions and grading scales found in 
the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 
will be utilized for AE reporting, with the exception of lab abnormalities which will be 
managed as described below.  All appropriate treatment areas should have access to a copy 
of the CTCAE version 4.0.  A copy of the CTCAE version 4.0 can be downloaded from the 
CTEP web site 
http://ctep.cancer.gov/protocolDevelopment/electronic_applications/ctc.htm.   
 
Attribution of the AE: Unrelated, unlikely, possibly, probably, definitely 
 
Laboratory Abnormalities 
An abnormal lab result should be reported as an adverse event and graded per CTCAE if 
the test result is deemed Clinically Significant (CS) by the responsible or treating 
physician. Clinical significance is based on clinical judgement and individual patient 
situation, at the discretion of the physician. Examples of a test result that may be considered 
CS are described below. This list is not exhaustive and a result that is similar to an example 
below may not be considered CS if the treating physician determines it to be Not Clinically 
Significant (NCS):  
 
Test result is associated with accompanying symptoms, and/or  
 
Test result requires additional diagnostic testing or medical/surgical intervention, 
and/or  
 
Test result leads to a change in study dosing outside of protocol-stipulated dose 
adjustments or discontinuation from the study, significant additional concomitant 
drug treatment, or therapy, and/or  
 
Test result is considered to be an adverse event by the Investigator  
 
 


Protocol version: 02March2020 
16 
7.3 
OHSU IRB Reporting of Unanticipated Problems and Adverse Events 
Unanticipated Problems (UP) and Adverse Events (AE) will be reported to OHSU IRB 
according to the policies, procedures and guidelines posted on the OHSU IRB web site 
http://www.ohsu.edu/research/rda/irb/policies.shtml. 
 
Fatal and life-threatening events must be reported to OHSU IRB within 7 calendar days 
after the PI learns of the event. If any of these require a change (as determined by the PI or 
the IRB) to the protocol or consent form, the PI will make those changes promptly and 
submit the revised documents to the OHSU IRB. 
 
All other UP reports will be submitted to OHSU IRB no later than 15 calendar days of 
notification of the event.  If the event requires changes as determined by  the PI or the IRB) 
to the protocol or consent form, the PI will make the changes promptly and submit the 
revised documents to the IRB.  UP and AE reports are submitted through OHSU e-IRB and 
will be reviewed by OHSU IRB.   
 
7.4 
Central Reporting of Adverse Events for Multicenter Studies  
The SAE/UP reporting for multicenter investigator initiated clinical trials  will follow the 
guidelines outlined in the OHSU Knight Cancer Institute Multi-Center Investigator 
Initiated Trials Coordinating Center Operations Manual.  
 
A participating site must report an SAE to the to the institution’s local IRB for action as 
required, as well as to the OHSU coordinating center study team by fax or email within 24 
hours of learning of the event.  
 
The OHSU coordinating center study team will review and submit SAEs to the FDA, 
OHSU IRB, and any other required contacts as required by the Knight Data Safety 
Monitoring Plan. The principal investigator at the Coordinating Center is responsible for 
distributing IND and/or IDE Action Letters or Safety Reports, as applicable, to 
participating institutions for review and submission to their institution’s local IRB. 
 
7.5 
MedWatch/SAE Reporting  
 
An AE is considered “serious” if, in the view of either the investigator or sponsor, it results 
in any of the following outcomes:  
 
 
Results in death, 
 
Is life threatening (an AE is considered “life-threatening” if, in the view of either the 
investigator or sponsor, its occurrence places the subject at immediate risk of death. It 
does not include an AE that, had it occurred in a more severe form, might have caused 
death.), 
 
Results in persistent or significant disability/incapacity or substantial disruption of the 
ability to conduct normal life functions,  
 
Results in congenital anomaly, or birth defect, 
 
Requires inpatient hospitalization or leads to prolongation of hospitalization 
(hospitalization for treatment/observation/examination caused by AE is to be 
considered as serious), 
 
Other medically important events. 
 
Within 24 hours of awareness of a serious adverse event, whether or not related to the study 
drug, the Investigator will complete and submit a Medwatch 3500A Form to FDA, 


Protocol version: 02March2020 
17 
containing all required information (reference 21 CFR 312.32).  The Investigator will 
submit a copy of this MedWatch 3500A form to Astellas by either e-mail or fax, within the 
same timeframe.. If submission of this SAE to FDA or Astellas or is not possible within 24 
hours, the Investigator’s local drug safety contact (IRB, etc.) should be informed by phone. 
 
The SAE documentation, including the Medwatch 3500A Form and available source 
records should be emailed or faxed to (See Appendix D for the fax cover sheet): 
 
Astellas Pharma Global Development – United States 
Email: Safety-us@us.astellas.com 
Fax number: (847) 317-1241 
 
The following minimum information is required: 
 
Study number/IIT regulatory identifier 
 
Subject number, sex and age 
 
The date of report 
 
A description of the SAE (event, seriousness of the event) 
 
Causal relationship to the study drug 
 
 
8.0 
PHARMACEUTICAL INFORMATION 
 
8.1 
Enzalutamide 
 
Product description: Enzalutamide (MDV3100) will be prescribed at 160mg per day. 
Enzalutamide will be provided as liquid-filled soft gelatin capsules for oral administration. 
Each capsule contains 40 mg of Enzalutamide as a solution in caprylocaproyl 
polyoxylglycerides.  
 
Route of administration: Oral, daily.  
 
8.1.1 
Laboratory Abnormalities  
In the randomized clinical trial, Grade 1-4 neutropenia occurred in 15% of patients 
on Enzalutamide (1% Grade 3-4) and in 6% of patients on placebo (no Grade 3-4). 
The incidence of Grade 1-4 thrombocytopenia was similar in both arms; 0.5% of 
patients on Enzalutamide and 1% on placebo experienced Grade 3-4 
thrombocytopenia. Grade 1-4 elevations in ALT occurred in 10% of patients on 
Enzalutamide (0.3% Grade 3-4) and 18% of patients on placebo (0.5% Grade 3-4). 
Grade 1-4 elevations in bilirubin occurred in 3% of patients on Enzalutamide and 
2% of patients on placebo. 
 
8.1.2 
Infections  
In the randomized clinical trial, 1.0% of patients treated with Enzalutamide 
compared to 0.3% of patients on placebo died from infections or sepsis. Infection-
related serious adverse events were reported in approximately 6% of the patients on 
both treatment arms. 
 
8.1.3 
Falls and Fall-related Injuries 
In the randomized clinical trial, falls or injuries related to falls occurred in 4.6% of 
patients treated with Enzalutamide compared to 1.3% of patients on placebo. Falls 
were not associated with loss of consciousness or seizure. Fall-related injuries were 
more severe in patients treated with Enzalutamide and included non-pathologic 
fractures, joint injuries, and hematomas. 


Protocol version: 02March2020 
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8.1.4 
Hallucinations 
In the randomized clinical trial, 1.6% of patients treated with Enzalutamide were 
reported to have Grade 1 or 2 hallucinations compared to 0.3% of patients on 
placebo. Of the patients with hallucinations, the majority were on opioid-
containing medications at the time of the event. Hallucinations were visual, tactile, 
or undefined. 
 
8.1.5 
Pregnancy 
Enzalutamide can cause fetal harm when administered to a pregnant woman based 
on its mechanism of action. While there are no human or animal data on the use of 
Enzalutamide in pregnancy and Enzalutamide is not indicated for use in women, it 
is important to know that maternal use of an androgen receptor inhibitor could 
affect development of the fetus. Enzalutamide is contraindicated in women who are 
or may become pregnant while receiving the drug. If this drug is used during 
pregnancy, or if the patient becomes pregnant while taking this drug, apprise the 
patient of the potential hazard to the fetus and the potential risk for pregnancy loss. 
Advise females of reproductive potential to avoid becoming pregnant during 
treatment with Enzalutamide. 
 
It is recommended that women who are pregnant or who may be pregnant should 
wear gloves if they need to touch or handle Enzalutamide capsules.  
 
8.2 
Prior and Concomitant Therapy  
 
8.2.1 
Drugs that Inhibit or Induce CYP2C8 
Concomitant use of the strong CYP2C8 inhibitors gemfibrozil or trimethoprim 
[Bactrim]) is excluded in this protocol.  
 
8.2.2 
Drugs that Inhibit or Induce CYP Enzymes 
APPENDIX E provides a list of potent CYP enzyme inhibitors and inducers that 
may have a theoretical concern of potential drug-drug interactions with MDV3100. 
In vitro drug metabolism studies suggest that MDV3100 may have the potential to 
induce CYP3A4 and to inhibit CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, 
CYP2D6, and CYP3A4/5; therefore, concomitant medications that are substrates of 
any of these enzymes should be used with caution, and relevant monitoring should 
be considered, especially for substrates known to cause seizure, because the 
possibility of drug-drug interactions cannot be fully excluded. Since the 
metabolism of MDV3100 is not known, caution should be taken for the 
concomitant use of strong inhibitors and inducers of CYP enzymes and alternative 
products used when available. 
 
Co-administration of a strong CYP3A4 inhibitor (itraconazole) increased the 
composite AUC of enzalutamide plus Ndesmethyl enzalutamide by 1.3 fold in 
healthy volunteers  
 
The effects of CYP3A4 inducers on the pharmacokinetics of enzalutamide have not 
been evaluated in vivo. Co-administration of Enzalutamide with strong CYP3A4 
inducers (e.g., carbamazepine, phenobarbital, phenytoin, rifabutin, rifampin, 
rifapentine) may decrease the plasma exposure of Enzalutamide and should be 
avoided if possible. Selection of a concomitant medication with no or minimal 
CYP3A4 induction potential is recommended. Moderate CYP3A4 inducers (e.g., 


Protocol version: 02March2020 
19 
bosentan, efavirenz, etravirine, modafinil, nafcillin) and St. John’s Wort may also 
reduce the plasma exposure of Enzalutamide and should be avoided if possible  
 
8.2.3 
Effect of Enzalutamide on Drug Metabolizing Enzymes 
Enzalutamide is a strong CYP3A4 inducer and a moderate CYP2C9 and CYP2C19 
inducer in humans. At steady state, Enzalutamide reduced the plasma exposure to 
midazolam (CYP3A4 substrate), warfarin (CYP2C9 substrate), and omeprazole 
(CYP2C19 substrate). Concomitant use of Enzalutamide with narrow therapeutic 
index drugs that are metabolized by CYP3A4 (e.g., alfentanil, cyclosporine, 
dihydroergotamine, ergotamine, fentanyl, pimozide, quinidine, sirolimus and 
tacrolimus), CYP2C9 (e.g., phenytoin, warfarin) and CYP2C19 (e.g., S-
mephenytoin) should be avoided, as Enzalutamide may decrease their exposure. If 
co-administration with warfarin cannot be avoided, conduct additional INR 
monitoring. 
 
8.2.4 
Effect of Other drugs on Enzalutamide  
In a drug-drug interaction trial in healthy volunteers, a single 160 mg oral dose of 
Enzalutamide was administered alone or after multiple oral doses of gemfibrozil 
(strong CYP2C8 inhibitor). Gemfibrozil increased the AUC0-inf of Enzalutamide 
plus N-desmethyl enzalutamide by 2.2-fold with minimal effect on Cmax. The 
results are summarized in Figure 1.  
 
NOTE: Gemfibrozil and trimethoprim (Bactrim) are prohibited during study 
treatment.  
 
In a drug-drug interaction trial in healthy volunteers, a single 160 mg oral dose of 
Enzalutamide was administered alone or after multiple oral doses of itraconazole 
(strong CYP3A4 inhibitor). Itraconazole increased the AUC0-inf of Enzalutamide 
plus N-desmethyl enzalutamide by 1.3-fold with no effect on Cmax. The results are 
summarized in Figure 1. 
 
The effects of CYP2C8 and CYP3A4 inducers on the exposure of Enzalutamide 
have not been evaluated in vivo. 
 
8.2.5 
Effect of Enzalutamide on Other Drugs  
In an in vivo phenotypic cocktail drug-drug interaction trial in patients with 
castration-resistant prostate cancer, a single oral dose of the CYP probe substrate 
cocktail (for CYP2C8, CYP2C9, CYP2C19, and CYP3A4) was administered 
before and concomitantly with Enzalutamide (following at least 55 days of dosing 
at 160 mg daily). The results are summarized in Figure 2. Results showed that in 
vivo, at steady state, Enzalutamide is a strong CYP3A4 inducer and a moderate 
CYP2C9 and CYP2C19 inducer. Enzalutamide did not cause clinically meaningful 
changes in exposure to the CYP2C8 substrate. 
 


Protocol version: 02March2020 
20 
 
 
In vitro, enzalutamide, N-desmethyl enzalutamide, and the major inactive 
carboxylic acid metabolite caused direct inhibition of multiple CYP enzymes 
including CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5; 
however, subsequent clinical data showed that Enzalutamide is an inducer of 
CYP2C9, CYP2C19, and CYP3A4 and had no clinically meaningful effect on 
CYP2C8 (see Figure 2). In vitro, enzalutamide caused time-dependent inhibition of 
CYP1A2. 
 
In vitro studies showed that enzalutamide caused induction of CYP3A4 and that 
enzalutamide is not expected to induce CYP1A2 at therapeutically relevant 
concentrations. In vitro, enzalutamide, N-desmethyl enzalutamide, and the major 
inactive carboxylic acid metabolite are not substrates for human P-glycoprotein. In 
vitro, enzalutamide and N-desmethyl enzalutamide are inhibitors of human P-
glycoprotein, while the major inactive carboxylic acid metabolite is not. 
 
8.2.6  The following medications are prohibited within 4 weeks of study drug 
administration (Day 1):  
 
Flutamide (Patients who never had a PSA decline with the most recent anti-
androgen therapy or in whom response to the most recent anti-androgen was for <3 
months require only a 2 week washout.) 
 
Bicalutamide or nilutamide (6 weeks washout required for these two agents. 
Patients who never had a PSA decline with the most recent anti-androgen therapy 
or in whom response to the most recent anti-androgen was for <3 months require 
only a 2 week washout.)  
 
5 α-reductase inhibitors (finasteride, dutasteride) 
 
Estrogens  


Protocol version: 02March2020 
21 
 
Cyproterone acetate  
 
Biologic, or other agents with anti-tumor activity against prostate cancer  
 
Systemic glucocorticoids greater than the equivalent of 10 mg per day of 
prednisone  
 
Herbal medications that have known hormonal anti-prostate cancer activity and/or 
are known to decrease PSA levels (i.e., saw palmetto) 
 
Androgens (testosterone, dihydroepiandrosterone [DHEA], etc.)  
 
8.2.7 
Medications that may lower the seizure threshold should be used with caution 
 
APPENDIX G provides a partial list of medications that may lower the seizure 
threshold.  
 
9.0 
BIOMARKER, CORRELATIVE, AND SPECIAL STUDIES 
Gene expression analysis 
 
Biopsy Samples  
 
Radiology-guided biopsies will be handled and processed by the study investigators and their 
assigned staff. Biopsies will be collected in the radiology suite. As biopsies are performed, two will 
be placed in 10% NB formalin solution for eventual decalcification (if required) and paraffin 
embedding and four to six biopsies will be placed immediately on pre-frozen pallets of OCT. The 
placement of biopsies on previously frozen OCT rapidly freezes the tissue and immobilizes the 
biopsy. 
 
The frozen sample will be labeled with the patient’s unique, coded number and taken directly or 
shipped by next day Air on dry ice to the laboratory of Dr. Phillip Febbo at UCSF (See Appendix 
B for shipping details).  
 
Samples have to be shipped Monday through Thursday, and Saturday delivery is not allowed. 
Avoid shipping the Thursday prior to a UCSF holiday without prior approval by the study chair.  
 
Frozen tumor biopsies will be cryosectioned and a slide stained with Hematoxylin and Eosin 
(H&E) for histological evaluation. If cancer is present, additional slides will be made and tissue 
subjected to laser capture microdissection for RNA and DNA isolation. 
 
The two biopsies placed in NB formalin will be shipped to the Thomas laboratory at OHSU for 
processing and further work up (Appendix B for shipping details). Paraffin embedded biopsies will 
undergo surface decal (using Immunocal, from Decal Chemical Corp). Hematoxylin and Eosin 
(H&E) staining will be performed to confirm the presence of prostate cancer and document the 
extent of involvement. FFPE samples will be paraffin-embedded and stored at 4° C in airtight 
containers to minimize oxidation of proteins within the blocks.  
 
Unstained sections will be cut and subsequently used for immunohistochemical staining. DNA will 
be extracted from the FFPE tissues (from unstained slides or tissue cores or LCM depending on the 


Protocol version: 02March2020 
22 
amount to tumor present) for downstream sequencing on the Ion Torrent panel. We anticipate that 
240 of the 300 samples (80%) will demonstrate histologically identified prostate cancer.  
 
The H&E stained slides for both the frozen and the paraffin-embedded biopsies will be sent to the 
Huang lab at UCLA for digital scanning and central pathological review to determine the presence 
or absence of tumor and the approximate percentage of tumor in each biopsy. 
 
Determination of AR Activity 
 
Preparation of RNA and DNA  
 
Frozen biopsies will be processed for RNA analysis using laser capture microdissection and 
RNA amplification using adaptations of previously published methods 20. Briefly, 8 micro 
frozen sections are obtained from each biopsy specimen and stained with H&E. Sections 
will be loaded into the Aperio Digital Imaging system and reviewed by the Dream Team 
Molecular Pathologists (Drs. Thomas and Huang) and Dr. Febbo to establish the presence 
or absence of prostate cancer and identify the biopsies most likely to provide sufficient 
tumor for RNA analysis. Multiple sequential 8 micron biopsies are performed prior to rapid 
H&E staining and dehydration with sequential immersion in xylene. The air-dried slides are 
put into a LCM system and prostate cancer cells are identified and collected. Cell material 
selected for analysis is dissolved in RNA or DNA isolation buffer and RNA and DNA are 
isolated separately using standard commercially available kits. RNA and DNA are 
quantified using the RiboGreen or PicoGreen kits (Invitrogen, Inc) and RNA quality is 
assessed with an Agilent 2100 Bioanalyzer using the RNA 6000 Pico Kit (Agilent, Inc). 
Samples with sufficient RNA and RNA with clear 18S and 28S bands present on the 
bioanalyzer are amplified and labeled with biotin using the NuGen WT Ovation kit (NuGen 
Inc). Amplified and labeled target is again quantified and 2.5 micrograms of product are 
sent to Expression Analysis, Inc for hybridization to Affymetrix U133 Plus 2.0 arrays. 
Similarly, DNA is collected from separate but adjacent frozen sections and isolated. DNA 
is quantified using PicoGreen quantification.  
 
For RNA analysis to progress and to apply the AR signature, quality control parameters 
have been set for RNA and microarray data. RNA samples will be considered of sufficient 
quality or quantity if they have un-detectable 18S and 28S peaks on the Agilent 
Bioanalyzer or a RIN < 3 AND sufficient RNA to detect an 18S peak. If RNA quantity is 
below that assessable by the Agilent Bioanalyzer or of questionable quality but sufficient 
for RNA amplification based upon the RiboGreen results, the specimen will be processed 
for microarray analysis and quality assessment of the microarray data will be used to 
determine technical success. Samples resulting in insufficient probe creation (an 
insufficient amount for hybridization) following RNA amplification with NuGen WT 
Ovation, will be re-extracted once and RNA amplification repeated. If two RNA 
amplifications failed, the sample will be considered a technical failure. Once samples are 
hybridized, array-based exclusion criteria include standard QC measures for Affymetrix 
platforms (e.g. %present, 3'/5' ratios), and outliers in relation to the normalization dataset, 
as determined by principal components analysis (PCA). 
 
Application of the AR Signature  
 
The predictor has been derived from the LNCaP cell line data using established 
methodologies 21. Briefly, prior to statistical modeling, LNCaP cell line gene expression 
data is filtered to exclude probe sets with minimal variation (lowest 10% of genes based 
upon standard deviation of expression across the 10 samples used to create signature). 


Protocol version: 02March2020 
23 
When predicting the AR activity of tumor samples, gene selection and identification is 
based on the training data from the LNCaP cell line, and then metagene values are 
computed using the principal components of the expression data. Bayesian fitting of binary 
probit regression models to the training data then permits an assessment of the relevance of 
the metagene signatures in within-sample classification 22, and estimation and uncertainty 
assessments for the binary regression weights mapping metagenes to probabilities. Given 
the LNCaP training set of expression vectors (of values across metagenes) representing two 
biological states (AR active and or inactive), a binary probit regression model, of predictive 
probabilities for each of the two states (high vs. low) for each case is estimated using 
Bayesian methods 21,22 producing estimated relative probabilities – and associated measures 
of uncertainty – of AR activity across the prostate cancer samples. 
 
Determination of Adaptive Pathway Activity  
 
Adaptive pathway activity will be determined using both immunohistochemistry and integrative 
pathway analysis with PARADIGM. Histological sections from the fixed, paraffin-embedded 
biopsies containing tumor will be used for ICH. RNA and DNA from the image-guided biopsies 
will be processed for RNA-sequencing and array comparative genome hybridization (aCGH) which 
will be used by PARADIGM to determine pathway activity. 
 
Immunohistochemistry for selected Adaptive Pathways  
 
Four immunohistochemcial markers will be performed to determine the activity of select 
adaptive pathways including: (1) nuclear AR staining 23\24; (2) PTEN protein 25,26; (3) 
phosphorylated hsp2727; and (4) N-cadherin 28. IHC quantification will be performed with 
automated digital image analysis algorithms to rigorously and systematically measure 
staining intensities, using the inFORM image analysis software (CRi/Caliper Life Sciences, 
Hopkinton, MA) 29. Established qualitative assessments will only be used if staining 
characteristics preclude automated image analysis, and will be performed by both 
pathologists while masked to associated clinical or molecular data. 
 
aCGH Analysis  
 
aCGH analysis will be performed on tumor biopsies and CTCs. Briefly, 0.5 ug of each 
genomic DNA will be fluorescently labeled by following the NimbleGen enzymatic 
labeling protocol, which employs Cy3 and Cy5 labeled random nanomers (TriLink 
Biotechnologies), a heat fragmentation step at 98° C for 10 minutes, and amplification with 
Klenow fragment 5’-3’exo- (NEB). Five micrograms of each Cy5-labeled sample will be 
co-hybridized with 5 ug of Cy3-labeled human male reference DNA (Promega) on Agilent 
SurePrint G3 Human Catalog CGH 4x180K (Part No. G4449A) following the hybridization 
and washing conditions from the Agilent Oligonucleotide Array-Based CGH for Genomic 
DNA Analysis Protocol v6.2. Arrays will be scanned with the Agilent DNA Microarray 
Scanner, and quantified with Feature Extraction 10.5.1.1. CGH processed signal will then 
be uploaded into Biodiscovery Nexus software, where the quality will be assessed and data 
visualized and analyzed for copy number variation to be used for pathway analysis and to 
determine if tumor biopsies and CTCs in the same patient share genetic events. 
 
RNA-sequencing  
 
We will apply a newly developed strategy to perform RNA sequencing from minute 
amounts of total RNA 30. LCM collected material will be lysed with 5 μl Prelude Lysis 
Buffer (NuGen, San Carlos, CA); subjected to cDNA synthesis and amplification without 


Protocol version: 02March2020 
24 
transfer (NuGen, Ovation RNA-seq with some modifications to the standard protocol); 
selected for size and processed using the mixed cDNA standard library preparation 
performed using TruSeq protocol (Illumina, San Diego, CA). Paired-end sequencing will 
be performed on the Illumina HiSeq 2000. RNA-seq is generating very high quality data 
and may replace RNA microarrays for the determination of AR signature activity. 
 
Tumor DNA-sequencing  
 
We will use next generation sequencing to detect potentially actionable mutations with 
patient specimens as well as perform whole exome sequencing to identify activating 
mutations and other genetic events. Genomic DNA from individuals will be extracted from 
collected blood samples (germ line) and tumor biopsies; sonicated to approximately 50 - 
200 bp fragments; and used to make a library for paired-end sequencing (Illumina) or for 
sequencing within the OHSU CLIA laboratory (Ion Torrent sequencing). using established 
protocols 31.  
 
We will use whole genome sequencing or Sanger sequencing of cfDNA from blood and 
DNA from the tumor biopsy specimens. This will enable us to identify molecular features 
(mutations, including but not limited to the AR, copy number alterations, and gene fusions) 
that may predict resistance or early progression despite enzalutamide treatment. 
 
As RNA- and DNA-sequencing can provide similar molecular information as RNA 
microarrays and aCGH, respectively, it is our expectation that sequence-based analysis will 
likely displace array-based analysis during the study. The molecular platform deemed to 
provide the best data for pathway analysis (AR and adaptive pathways) will be used by 
investigators. 
 
Integrative Pathway Analysis to Associate Adaptive Pathways  
Pathway analysis will be both hypotheses driven based upon previously identified adaptive 
pathways as well as unbiased (using pathway analyses to associate biological mechanisms 
to drug resistance). RNA-seq and aCGH data (as well as any additional molecular analyses) 
will be imported into MedBook. Using the molecular data generated from patient samples, 
we will use a software program called PARADIGM for integrative pathway-based analysis. 
 
Adaptive Pathway Activity Analysis  
PARADIGM analysis is well suited to the detection of significant alteration in adaptive 
pathways, and additional key known cancer pathways. The current SuperPathway database 
contains over 16,000 features covering 5,500 proteins (roughly 25% of the genome), which 
are heavily cancer-oriented since the construction of the database was built from NCI’s 
Pathway Interaction Database (PID), Reactome, and BioCarta. Future additions will come 
from KEGG and WikiPathways. Using clinical and molecular data in MedBook, our team 
will apply PARADIGM analysis to determine which adaptive pathways are relatively 
active in each individual biopsied. 
 
Unbiased Pathway Analysis  
In order to identify novel mechanisms underlying eventual disease progression or 
resistance, we will expand our pathway database to include predicted interactions by 
mining functional genomics resources. We have developed the Differential Pathway 
Signature Analysis (DPS) to integrate pathway analysis with clinical response or gene 
mutation. The DPS method is built on many tools: PARADIGM 32, Significance Analysis 
of Microarrays (SAM) 33, Cluster 34, and Java Treeview. PARADIGM is used to integrate 
gene expression and DNA-based events (copy number variation, fusion, mutation etc) into 


Protocol version: 02March2020 
25 
a combined pathway score. These scores are called IPLs (inferred pathway levels). 
PARADIGM inferred pathway features are preferable over gene expression since 
PARADIGM integrates knowledge from both RNA-seq and aCGH.  
 
PARADIGMs output, a relative measure of pathway activities within and across samples as 
continuous variables, will be used to determine the distribution of pathway activity and the 
association with clinical features. 
 
10.0 
STUDY PROCEDURES AND SCHEDULE OF EVENTS 
10.1 
Screening/ Visit 
 
Medical history 
 
ECOG performance status 
 
A complete physical exam including vitals (blood pressure, heart rate, respiration rate, 
temperature and weight)  
 
Laboratory assessments (hematology, chemistry, testosterone, PSA, PT/INR, APTT 
and LDH  
 
Scans (CT scan chest/abdomen/pelvis (preferably with contrast) or MRI (if CT scan is 
contraindicated) and bone scan) 
 
Hematology, PT/INR, and APTT are to be conducted no more than 14 days prior to biopsy.  
The remaining screening evaluations are to be conducted within 35 days of enrollment (day 
of pre-treatment biopsy). Biopsy must be no more than10 days before starting 
Enzalutamide treatment.  
 
 
10.2 
Study Visits 
10.2.1 Enrollment / Baseline biopsy:  
o After eligibility is confirmed and all screening procedures complete per 10.1, 
subject will be registered for the study according to registration procedures 
outlined in Section 4.0.  
o All eligible subjects will undergo a biopsy of a metastatic lesion as described in 
Section 5.2 and Appendices B and C. 
 
10.2.2 Day 1 (start daily dosing of Enzalutamide) pre-dose:  
o ECOG performance status 
o A complete physical exam including vitals  
o Laboratory assessments (hematology, chemistry, and PSA) 
 
10.2.3 Week 6:  
o ECOG performance status 
o A complete physical exam including vitals  
o Laboratory assessments (hematology, chemistry, and PSA) 
 
10.2.4 Week 12:  
o ECOG performance status 
o A complete physical exam including vitals  
o Laboratory assessments (hematology, chemistry, and PSA) 
o Tumor evaluation (CT scan chest/abdomen/pelvis (or MRI) and bone scan)  
 
10.2.5 Every subsequent 12 weeks: 
o ECOG performance status 
o A complete physical exam including vitals  


Protocol version: 02March2020 
26 
o Laboratory assessments (hematology, chemistry, and PSA)  
o Tumor evaluation to be performed every 12 weeks or whenever disease 
progression is suspected based on the investigator’s clinical assessment of 
patient symptoms and laboratory data (CT scan chest/abdomen/pelvis (or MRI) 
and bone scan) 
 
10.2.6 At Progression (See 5.3 for definition):  
o ECOG performance status 
o A complete physical exam including vitals  
o Laboratory assessments (hematology, chemistry, PT/INR, APTT, PSA and 
Investigational samples) 
o Tumor evaluation (CT scan chest/abdomen/pelvis (or MRI) and bone scan) 
within 30 days prior to biopsy. 
o All subjects will undergo a biopsy of a metastatic lesion as described in Section 
5.2 and Appendix B. 
o Patients should have a PSA drawn on the day of taking their last dose of 
enzalutamide (e.g. day of biopsy for those undergoing a progression biopsy 
procedure or day of progression visit when enzalutamide is discontinued.) 
 
10.3 
Follow-up 
After discontinuing enzalutamide, a follow-up PSA should be drawn prior to initiating the 
next therapy (ideally, 2-3 weeks after discontinuing enzalutamide treatment) in those 
patients who are agreeable. In those who have not initiated a new treatment of their prostate 
cancer (radiation, local surgery, or systemic therapy) by 6 weeks, a PSA should be drawn 
then in patients who are agreeable. 
 
After termination of study participation, subjects will be followed using these guidelines:  
10.3.1. If a patient discontinues Enzalutamide treatment for any reason other than 
Radiographic Progression or Clinical Progression as defined in section 5.2, the patient will 
continue to be followed until criteria for Radiographic or Clinical Progression is met. 
Tumor evaluations should follow the same schedule as on study (every 12 weeks).  The 
patient will also be followed for stabilization or resolution of any study-related toxicities.  
 
After progression, all patients will be followed by telephone, doctor visit, or medical record 
review every 12 weeks for survival status.  


Protocol version: 02March2020 
27 
10.4 
Schedule of Events 
 
Screening 
Enrollment 
Day 1 
(Start 
Enzalutamide) 
Week 
6 
Week 
12 
Every 12 
Weeks 
At 
Progression 
or Time 
Recommend
ed for Drug 
to be 
Discontinued 
(See 5.3) 
Follow up 
lab work  
(see 10.3) 
Follow up: 
Every 12 Weeks 
 
Within 35 
days of 
Enrollment 
Within 10 
days of Day 
1 
 
+ 3 Days 
+ 8 Days 
+ 8 Days 
+ 8 Days 
 
+ 8 Days 
Enzalutamide 
(provided) 
 
 
X 
 
X 
X 
 
 
 
Biopsy 
 
X 
 
 
 
 
Xd 
 
 
Informed consent 
X 
 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
Concurrent 
medications 
X 
 
X 
X 
X 
X 
 
 
 
Physical exam 
X 
 
X 
X 
X 
X 
X 
 
 
Vital signs 
X 
 
X 
X 
X 
X 
X 
 
 
Performance status 
X 
 
X 
X 
X 
X 
X 
 
 
Hematologyf 
Xi 
 
X 
X 
X 
X 
X 
 
 
Chemistryf 
X 
 
X 
X 
X 
X 
X 
 
 
PT/INR & APTT 
Xi 
 
 
 
 
 
Xd 
 
 
PSA 
X 
 
X 
X 
X 
X 
Xe 
Xh 
 
Testosterone 
X 
 
 
 
 
 
 
 
 
LDHg 
X 
 
 
 
 
 
 
 
 
Investigational 
samples for 
bankinga 
 
 
 
 
 
 
X 
 
 
Adverse event 
evaluation 
 
X 
X 
X 
X 
X 
X 
 
 
Radiologic 
evaluation 
Xc 
 
 
 
X 
Xb 
Xd 
 
 
Telephone contact 
for Vital Status 
 
 
 
 
 
 
 
 
X 
 
a:  See Appendix C for collection and processing instructions 
b Radiologic evaluations will be done every 12 weeks or when progression is suspected 
c: Scans must be performed within 30 days of the baseline biopsy 
d: Progression biopsy, Progression PT/INR & APTT, and Radiologic Evaluations must be done within 30 days of Progression or time 
recommended for drug to be discontinued 
e. Patients should have a PSA drawn on the day of taking their last dose of enzalutamide (e.g. day of biopsy for those undergoing a progression 
biopsy procedure or day of progression visit when enzalutamide is discontinued. 
f. Chemistry: Na, K, Cl, CO2, BUN, Creat, Ca, Glu, Alb, Alk Phos, T. Bili, AST, T. Protein, ALT  
Hematology :  WBC count with differential, platelet count, hemoglobin, and hematocrit 
g: An LDH may be drawn at any point prior to study drug administration (i.e screening period and on day 1) 
h. After discontinuing enzalutamide, a follow-up PSA should be drawn prior to initiating the next therapy (ideally, 2-3 weeks after 
discontinuing enzalutamide treatment) in those patients who are agreeable. In those who have not initiated a new treatment of their prostate 
cancer (radiation, local surgery, or systemic therapy) by 6 weeks, a PSA should be drawn then in patients who are agreeable. 
i.  Hematology and PT/INR and APTT should be performed no more than 14 days prior to biopsy 


Protocol version: 02March2020 
28 
11.0 
MEASUREMENT OF EFFECT 
11.1 
PSA Response PSA Changes 
 
11.1.1 PSA Response: A ≥ 50% reduction at 12 weeks after the initiation of therapy vs. 
baseline. Baseline PSA will be defined as the measurement obtained immediately 
prior to initiation of Enzalutamide on Day 1 of study.   
 
11.1.2 Waterfall plots will be used to report the percentage change in PSA from baseline 
vs. 12 weeks after starting Enzalutamide treatment for each patient. Waterfall plots 
will also be used to report the maximal PSA decline at any point on study for each 
patient.  Baseline PSA will be defined as the measurement obtained immediately 
prior to initiation of Enzalutamide on Day 1 of study.   
 
11.1.3 PSA Progression (per PSAWG2 guidelines)35:  
 
11.1.3.1 
If PSA has declined from baseline, progression is defined as time from 
start of therapy to first PSA increase that is ≥ 25% and ≥ 2 ng/mL above 
the nadir, and which is confirmed by a second value 3 or more weeks 
later 
 
11.1.3.2 
If there is no decline from baseline, progression is defined as PSA 
increase ≥ 25% and ≥2 ng/mL after 12 weeks, which is confirmed by a 
second value 3 or more weeks later. 
 
Nadir will be defined as the lowest PSA value that was confirmed by a 
second equal or lower measurement. (Thus the nadir PSA is the second 
lowest PSA value measured.)  
 
11.1.4 Stable PSA: Does not meet criteria for response or progression.  
 
11.2 
Antitumor Effect – Solid Tumors 
 
For the purposes of this study, subjects should be re-evaluated for response every 12 weeks.   
 
Objective radiographic response and progression will be evaluated in this study using the 
new international criteria proposed by the revised Response Evaluation Criteria in Solid 
Tumors (RECIST) guideline (version 1.1) [Eur J Ca 45:228-247, 2009].  Changes in the 
largest diameter (unidimensional measurement) of the tumor lesions and the shortest 
diameter in the case of malignant lymph nodes are used in the RECIST criteria. 
 
11.2.1 Definitions 
 
Evaluable for toxicity:  Any subject, who undergoes a biopsy, whether or not he 
receives subsequent study drug treatment, will be analyzed for toxicity related to 
the biopsy. 
 
Evaluable for objective response:  Only those subjects who have measurable 
disease present at baseline, have received at least one cycle of therapy, and have 
had their disease re-evaluated will be considered evaluable for response.  These 
subjects will have their response classified according to the definitions stated 
below.  (Note:  Subjects who exhibit objective disease progression prior to the end 
of cycle 1 will also be considered evaluable.) 


Protocol version: 02March2020 
29 
 
Evaluable Non-Target Disease Response:  Subjects who have lesions present at 
baseline that are evaluable but do not meet the definitions of measurable disease, 
have received at least one cycle of therapy, and have had their disease re-evaluated 
will be considered evaluable for non-target disease response.  The response 
assessment is based on the presence, absence, or unequivocal progression of the 
lesions.  
 
11.2.2 Disease Parameters 
 
Measurable disease:  Measurable lesions are defined as those that can be accurately 
measured in at least one dimension (longest diameter to be recorded for non-nodal 
lesions and short axis for nodal lesions) as >20 mm by chest x-ray, as >10 mm with 
CT scan, or >10 mm with calipers by clinical exam.  All tumor measurements must 
be recorded in millimeters (or decimal fractions of centimeters). 
Note:  Tumor lesions that are situated in a previously irradiated area might or might 
not be considered measurable.  
 
Malignant lymph nodes:  To be considered pathologically enlarged and 
measurable, a lymph node must be >15 mm in short axis when assessed by CT scan 
(CT scan slice thickness recommended to be no greater than 5 mm).  At baseline 
and in follow-up, only the short axis will be measured and followed. 
 
Non-measurable disease:  All other lesions (or sites of disease), including small 
lesions (longest diameter <10 mm or pathological lymph nodes with ≥ 10 to <15 
mm short axis), are considered non-measurable disease.  Bone lesions, 
leptomeningeal disease, ascites, pleural/pericardial effusions, lymphangitis 
cutis/pulmonitis, inflammatory breast disease, and abdominal masses (not followed 
by CT or MRI), are considered non-measurable. 
 
Target lesions:  All measurable lesions up to a maximum of 2 lesions per organ and 5 
lesions in total, representative of all involved organs, should be identified as target 
lesions and recorded and measured at baseline.  Target lesions should be selected on 
the basis of their size (lesions with the longest diameter), be representative of all 
involved organs but, in addition, should be those that lend themselves to reproducible 
repeated measurements.  It may be the case that, on occasion, the largest lesion does 
not lend itself to reproducible measurement in which circumstance the next largest 
lesion which can be measured reproducibly should be selected.  A sum of the 
diameters (longest for non-nodal lesions, short axis for nodal lesions) for all target 
lesions will be calculated and reported as the baseline sum diameters.  If lymph nodes 
are to be included in the sum, then only the short axis is added into the sum.  The 
baseline sum diameters will be used as reference to further characterize any objective 
tumor regression in the measurable dimension of the disease. 
 
Non-target lesions:  All other lesions (or sites of disease) including any measurable 
lesions over and above the 5 target lesions should be identified as non-target lesions 
and should also be recorded at baseline.  Measurements of these lesions are not 
required, but the presence, absence, or in rare cases unequivocal progression of each 
should be noted throughout follow-up.  
 
11.2.3 Methods for Evaluation of Measurable Disease 
 


Protocol version: 02March2020 
30 
All measurements should be taken and recorded in metric notation using a ruler or 
calipers.  All baseline evaluations should be performed as closely as possible to the 
beginning of treatment and never more than 4 weeks before the beginning of the 
treatment. 
 
The same method of assessment and the same technique should be used to 
characterize each identified and reported lesion at baseline and during follow-up. 
Imaging-based evaluation is preferred to evaluation by clinical examination unless 
the lesion(s) being followed cannot be imaged but are assessable by clinical exam. 
Conventional CT and MRI:  This guideline has defined measurability of lesions on 
CT scan based on the assumption that CT slice thickness is 5 mm or less.  If CT scans 
have slice thickness greater than 5 mm, the minimum size for a measurable lesion 
should be twice the slice thickness.  MRI is also acceptable in certain situations (e.g. 
for body scans).   
 
Use of MRI remains a complex issue.  MRI has excellent contrast, spatial, and 
temporal resolution; however, there are many image acquisition variables involved in 
MRI, which greatly impact image quality, lesion conspicuity, and measurement.  
Furthermore, the availability of MRI is variable globally.  As with CT, if an MRI is 
performed, the technical specifications of the scanning sequences used should be 
optimized for the evaluation of the type and site of disease.  Furthermore, as with CT, 
the modality used at follow-up should be the same as was used at baseline and the 
lesions should be measured/assessed on the same pulse sequence.  It is beyond the 
scope of the RECIST guidelines to prescribe specific MRI pulse sequence parameters 
for all scanners, body parts, and diseases.  Ideally, the same type of scanner should be 
used and the image acquisition protocol should be followed as closely as possible to 
prior scans.  Body scans should be performed with breath-hold scanning techniques, 
if possible. 
 
11.2.4 Radiographic Response Criteria 
 
11.2.4.1 Evaluation of Target Lesions 
 
Complete Response (CR): 
Disappearance of all target lesions.  Any 
pathological lymph nodes (whether target or non-
target) must have reduction in short axis to <10 mm. 
Partial Response (PR): 
At least a 30% decrease in the sum of the diameters 
of target lesions, taking as reference the baseline 
sum diameters. 
Progressive Disease (PD): 
At least a 20% increase in the sum of the diameters 
of target lesions, taking as reference the smallest 
sum on study (this includes the baseline sum if that 
is the smallest on study). In addition to the relative 
increase of 20%, the sum must also demonstrate an 
absolute increase of at least 5 mm.  (Note:  the 
appearance of one or more new lesions is also 
considered progression). 
Stable Disease (SD): 
Neither sufficient shrinkage to qualify for PR nor 
sufficient increase to qualify for PD, taking as 
reference the smallest sum diameters while on study. 
 
11.2.4.2 Evaluation of Non-Target Lesions 


Protocol version: 02March2020 
31 
 
Complete Response (CR): 
Disappearance of all non-target lesions and 
normalization of tumor marker level.  All lymph 
nodes must be non-pathological in size (<10 mm 
short axis). 
Note:  If tumor markers are initially above the 
upper normal limit, they must normalize for a 
subject to be considered in complete clinical 
response. 
Non-CR/Non-PD: 
Persistence of one or more non-target lesion(s) 
and/or maintenance of tumor marker level above 
the normal limits. 
Progressive Disease (PD): 
Appearance of one or more new lesions and/or 
unequivocal progression of existing non-target 
lesions.  Unequivocal progression should not 
normally trump target lesion status.  It must be 
representative of overall disease status change, not 
a single lesion increase.  
 
Although a clear progression of non-target lesions only is exceptional, the 
opinion of the treating physician should prevail in such circumstances, and 
the progression status should be confirmed at a later time by the review 
panel (or principal investigator). 
 
11.2.4.3 Evaluation of Best Overall Radiographic Response 
 
The best overall response is the best response recorded from the start of the 
treatment until disease progression/recurrence (taking as reference for 
progressive disease the smallest measurements recorded since the treatment 
started).  The subject's best response assignment will depend on the 
achievement of both measurement and confirmation criteria. 
 
For Subjects with Measurable Disease (i.e., Target Disease) 
Target Lesions 
Non-Target Lesions 
New 
Lesions 
Overall 
Response 
Best Overall Response when 
Confirmation is Required* 
CR 
CR 
No 
CR 
>4 wks. Confirmation** 
CR 
Non-CR/Non-PD 
No 
PR 
>4 wks. Confirmation** 
CR 
Not evaluated 
No 
PR 
PR 
Non-CR/Non-PD/not evaluated 
No 
PR 
SD 
Non-CR/Non-PD/not evaluated 
No 
SD 
documented at least once >4 wks. 
from baseline** 
PD 
Any 
Yes or No 
PD 
no prior SD, PR or CR 
Any 
PD*** 
Yes or No 
PD 
Any 
Any 
Yes 
PD 
* 
See RECIST 1.1 manuscript for further details on what is evidence of a new lesion. 
** 
Only for non-randomized trials with response as primary endpoint. 
*** 
In exceptional circumstances, unequivocal progression in non-target lesions may be accepted as disease progression. 
Note: 
Subjects with a global deterioration of health status requiring discontinuation of treatment without objective evidence of 
disease progression at that time should be reported as “symptomatic deterioration.”  Every effort should be made to 
document the objective progression even after discontinuation of treatment. 
 
 


Protocol version: 02March2020 
32 
For Subjects with Non-Measurable Disease (i.e., Non-Target Disease) 
Non-Target Lesions 
New Lesions 
Overall Response 
CR 
No 
CR 
Non-CR/non-PD 
No 
Non-CR/non-PD* 
Not all evaluated 
No 
not evaluated 
Unequivocal PD 
Yes or No 
PD 
Any 
Yes 
PD 
* 
‘Non-CR/non-PD’ is preferred over ‘stable disease’ for non-target disease since SD is increasingly used as an 
endpoint for assessment of efficacy in some trials so to assign this category when no lesions can be measured is 
not advised 
 
 
 
 
 
11.2.5 Bone Scan Interpretation  
Bone scan progression is defined as the appearance of ≥ 2 unequivocally new 
lesions. For the first scheduled assessment only, a confirmatory scan performed 6 
or more weeks later must show a minimum of 2 or more additional new lesions. If 
changes on bone scan are thought to be due to flare or due to another cause, i.e, 
injury, they will not be considered progression.  Bone scans should be interpreted 
in the context of the entire clinical status of the patient.  The date of progression is 
the date of the first scan that shows the change.  
 
11.2.6 Progression-Free Survival 
 
Progression-free survival is defined as time from Day 1 of study drug treatment to 
date of first documented radiographic or clinical progression (see Sec 5.3).. 
 
 
 
 
 
11.2.7 Overall Survival 
Time to death of all causes will be calculated from Day 1 to the date of death from 
any cause.  
 
 
 
 
 
11.2.8 Disease-Specific Survival 
Time to death from prostate cancer will be calculated from Day 1 to the date of 
death from prostate cancer if this is the cause of death.  
  
12.0 
DATA REPORTING/REGULATORY REQUIREMENTS 
12.1 
Protocol Review  
 
The protocol and informed consent form for this study at OHSU must be reviewed and 
approved in writing by the OHSU Knight Cancer Institute (CI) Clinical Research  Review 
Committee (CRRC) and the appropriate Institutional Review Board (IRB) prior to any 
subject being consented on this study. 
 
All sites must have IRB approval of protocol by the IRB of record before consenting any 
subjects. 
 
12.2 
Informed Consent  
 
Written informed consent will be obtained from all subjects, or the legally authorized 
representative of the subject, participating in this trial, as stated in the Informed Consent 
section of the case of Federal Regulations, Title 21, Part 50.  If a subject’s signature cannot 
be obtained, and for all subjects under the age of 18, the investigator must ensure that the 


Protocol version: 02March2020 
33 
informed consent is signed by the subject’s legally authorized representative. 
Documentation of the consent process and a copy of the signed consent shall be maintained 
in the subject’s medical record.  
 
All sites must have IRB approval of ICF by the IRB of records before consenting any 
subjects. 
 
12.3 
Changes to Protocol  
 
Any modification of this protocol must be documented in the form of a protocol revision or 
amendment signed by the principal investigator and approved by the CRRC and IRB, 
before the revision or amendment may be implemented.  The only circumstance in which 
the amendment may be initiated without regulatory approval is for a change necessary to 
eliminate an apparent and immediate hazard to the subject.  In that event, the investigator 
must notify the CRRC and IRB in writing within 10 working days after the implementation. 
Investigators holding the IND must notify FDA of substantive changes to the protocol. 
 
A participating site must submit proposed changes to protocol to the OHSU Coordinating 
Center for review and endorsement before participating site may implement changes.  
 
12.4 
Maintenance of Records  
 
If the investigator relocates or for any reason withdraws from the study, the study records 
must be transferred to an agreed upon designee, such as another institution, another 
investigator, or to OHSU Knight Cancer Institute Clinical Trials Office.  Records must be 
maintained according to sponsor or FDA requirements. 
 
12.5 
OHSU IRB Reporting of Unanticipated Problems and Adverse Events 
 
Adverse event lists, guidelines, and instructions for AE reporting can be found in Section 
7.0 (Adverse Events: List and Reporting Requirements). 
 
12.6 
OHSU Knight Cancer Institute Data and Safety Monitoring Plan 
 
In addition to complete study and pharmacy files, complete records must be maintained on 
each subject treated on this protocol.  OHSU Knight Cancer Institute, through the auditing 
function of the Knight Clinical Trials Office, is responsible for ensuring that all member 
investigators and affiliate investigators conduct clinical research studies in compliance with 
local IRB standards, FDA regulations and NIH policies and in accordance with the Data 
and Safety Monitoring Plan policies and procedures 
http://ozone.ohsu.edu/cancer/sharedres/kctoresdocs.cfm  
Locally initiated studies will be audited by OHSU Knight CI Auditor.  Newly approved 
studies may be audited any time after enrollment has been initiated.  Each OHSU Knight 
approved treatment protocol will be audited on an annual basis in accordance with the Knight 
Data and Safety Monitoring Plan.  
  
It is the responsibility of each participating site’s principal investigator to ensure that the 
study isconducted in compliance with local IRB standards, FDA regulations, and NIH 
policies. It is also the responsibility of each site’s principal investigator to ensure that quality 
assurance audits at their site are conducted according to their institution’s policies and 
procedures.  The quality assurance audit process provides assurance that reported data 
accurately reflects the data in the primary subject record. 


Protocol version: 02March2020 
34 
 
12.7 
Inclusion of Women, Minorities and Children 
 
12.8.1 Inclusion of Women and Minorities 
No OHSU Knight Cancer Institute study will focus on any particular racial or 
ethnic subset. No subject will be excluded from the study on the basis of racial or 
ethnic origin. Male and minority volunteers will be recruited for this study from the 
general population and 100% men will be studied.  
 
The racial and ethnic composition of the study should represent that of the state of 
Oregon (see Table 1). If the prevalence of the disease being studied is consistent 
across race, ethnicity and gender, then these figures can be used to calculate 
projected enrollments in Table 2. If the disease being studied does not affect both 
genders or all races and ethnicities equally (e.g., cervical cancer only affects women 
and Black males are more likely than White males to have prostate cancer), then this 
information should be taken into account when calculating the projected enrollment. 
The OHSU General Clinical Research Center has links to various sources of 
statistics on their webpage at www.ohsu.edu/gcrc. If a different source is used in 
calculating projected enrollments, that source should be cited below Table 2. 
 
Table 1: Population Demographics - Oregon (%) 
 
Source:   U.S. Census Bureau, 2010    *Totals may not equal 100 due to rounding. 
 
 
Ethnic Category 
Sex/Gender 
Females 
Males 
Total 
Hispanic or Latino 
 
 
11.7 
Not Hispanic or Latino 
 
 
88.3 
Ethnic Category: Total of all subjects* 
 
 
100* 
Racial Category 
 
American Indian or Alaskan Native 
 
 
1.4 
Asian 
 
 
3.7 
Black or African American 
 
 
1.8 
Native Hawaiian or other Pacific Islander 
 
 
0.3 
White 
 
 
83.6 
More than one race 
 
 
3.8 
Unknown/Other 
 
 
5.3 
Racial Category: Total of all subjects* 
 
 
100* 
TOTALS 
50.4 
49.6 
100* 


Protocol version: 02March2020 
35 
Table 2: Projected Accrual for the Present Study  
 
Ethnic Category 
Sex/Gender 
Females 
Males 
Unknown 
Total 
Hispanic or Latino 
0 
4 
0 
 
Not Hispanic or Latino 
0 
32 
0 
 
Unknown 
0 
0 
0 
 
Ethnic Category: Total of all subjects* 
0 
36 
0 
36* 
Racial Category 
 
American Indian or Alaskan Native 
0 
0-1 
0 
 
Asian 
0 
0-2 
0 
 
Black or African American 
0 
0-1 
0 
 
Native Hawaiian or other Pacific Islander 
0 
0-1 
0 
 
White 
0 
30 
0 
 
More than one race 
0 
0-2 
0 
 
Unknown 
0 
0-2 
0 
 
Racial Category: Total of all subjects* 
0 
36 
0 
36* 
Source:   Adapted from U.S. Census Bureau, 2010    *Totals may not equal 100 due to rounding. 
 
12.8.2 Inclusion of Children 
This protocol does not include children for the following reason: Prostate cancer 
does not affect children under the age of 18.  
 
13.0 
STATISTICAL CONSIDERATIONS 
 
13.1 
Study Design 
 
This is a prospective, single-arm multi-center study to identify molecular markers that are 
predictive of the degree and duration of disease control or overall survival with Enzalutamide 
treatment in men with metastatic CRPC.  We expect to enroll 36 patients over the period of 
three years, with an approximate of 12 patients per year.  All subjects will be treated with 
Enzalutamide and followed on protocol until disease progression or intolerance. Two tumor 
biopsies may be conducted: the first biopsy at baseline before Enzalutamide treatment for all 
enrolled patients, followed by the second biopsy at progression.  
 
 
13.2 
Primary and Secondary Objectives and Endpoints 
 
 
 
Primary Objective and Primary Endpoint 
To assess the correlations between baseline molecular features and pathways and PSA 
change (</> 50% decline) at 12 weeks vs. baseline. 
 
Secondary Objectives 
The secondary objectives are:  
 
To measure PSA change at 12 Weeks and at each study visit vs. baseline after enzalutamide 


Protocol version: 02March2020 
36 
treatment.  
 
To measure objective response defined in Section 11.1.1 after enzalutamide treatment 
 
 
 
 
To assess the correlations between the baseline molecular features and pathways and 
Progression-Free Survival (defined as time from Day 1 of study drug treatment to date of 
first documented radiographic progression or clinical progression- See Sec 5.3), Disease-
Specific Survival (defined as the time from Day 1 of study drug to date of death from 
prostate cancer), and Overall Survival (defined as time from Day 1 of study drug treatment 
to date of death from any cause). 
 
 
 
 
 
 
 
To assess the correlations between the baseline molecular features and pathways and time 
to PSA progression (see Sec 11.1). 
 
To identify molecular features and cellular pathways present in tumors from men with 
metasatic CRPC that are progressing despite Enzalutamide treatment. 
 
To explore correlation between baseline molecular features and pathways and changes in 
Circulating Tumor Cells (CTCs) counts defined in Sec 11.3.1. 
 
 
 
To explore correlation between baseline molecular features and pathways and objective 
response defined in Section 11.1.1.  
 
To assess the correlations between the baseline molecular features and pathways and 
degree of PSA decline at 12 weeks and maximal PSA decline observed while on study. 
 
To assess the correlations between the baseline molecular features and time on treatment. 
 
 
 
 
 
13.3 
Analysis Populations 
 
For the primary endpoint, evaluable patients will include those patients for whom data from 
genomic studies and IHC tests are available from the pre-treatment biopsy samples and 
who have completed at least 12 weeks of therapy or who have confirmed disease 
progression prior to 12 weeks of therapy. For the secondary endpoint of time to PSA 
progression, evaluable patients will include those for whom PSA data are available at 
follow-up timepoints on study. For the secondary endpoint of mechanisms at disease 
progression/drug discontinuation, evaluable patients will include those patients for whom 
data from genomic studies and IHC tests are available from both the pre-treatment and 
post-treatment biopsy samples. Any patient who undergoes a biopsy, whether or not he 
receives subsequent study drug treatment, will be analyzed for safety. 
 
 
13.4 
Statistical Analysis Plan 
 
Analysis for Primary Objective 
 
The proportion of patients with or without a >50% decline in PSA values at 12 weeks will 
be reported with 95% exact confidence interval  
 


Protocol version: 02March2020 
37 
Simple Logistic regression model will be used to evaluate the association between PSA 
responseand each of the potential molecular biomarker predictors, including (gene 
expression signatures, copy number alterations, mutations, IPLs (integrated pathway levels 
and IHC measurements). All continuous variables with skewed distributions will be log 
transformed and centered before incorporation. Based on the logistic regression model, a 
receiver-operating characteristic (ROC) curve will be generated and the area under the curve 
(AUC) will be calculated with its 95% confidence interval.  The ROC curve will also be used 
to help determine an optimal cutoff for each molecular predictor for a most accurate 
prediction of PSA response..  
Depending on the actual number of patients who are Enzalutamide-responsive or resistant, 
a multivariable logistic model may be developed, starting with all covariates that have a p-
value < 0.25 from the Simple Logistic regression model.  The model selection will be based 
on the Akaiki Information Criteria (AIC), and the c-index will be reported with the final 
model. If the number of patients who are Enzalutamide responsive or resistant is not big 
enough (less than 10 for each 1 degree of freedom in the model) for the multivariate 
modeling, we will leave the multivariate modeling procedure for future studies.  
 
Analysis for Secondary Objectives 
 
We will use Random Forests classification to identify molecular features and pathways 
present in patients with disease progression or who discontinue Enzalutamide treatment.  
Analyses will be conducted in R using the randomForest library.  Forests will be created 
with at least 10,001 trees (odd number ensures fully deterministic model) and otherwise 
default settings.  We will perform 50 replicates with five-fold CV and step of 0.9 on a log 
scale. The number of predictors with minimum mean CV error across the replicates will be 
chosen as the optimal predictor number.  We will assess performance by ROC AUC 
reported values for Random Forests internal out-of-bag (OOB) testing. The OOB testing is 
based on the fact that each tree in the forest is built on a random 2/3 subset of patients and 
the remaining 1/3 used as test set for that tree. 
 
The association between molecular predictors and survival outcomes (e.g., PFS, DSS and 
OS) and time on treatment will be assessed using cox regression model. In addition, 
Kaplan-Meier plots will be used to graphically illustrate the survival distributions for PFS, 
DSS and OS across the strata of categorical molecular predictors. Logistic regression model 
will be used to assess the association between molecular predictors and response rate. 
Linear regression model will be used to assess the association for changes in CTC counts 
from baseline and maximal PSA observed while on study.  
 
 
13.5 
Sample Size and Power 
 
Our main goal is to identify molecular features and pathways that would allow us predict 
which tumors will have durable disease control with Enzalutamide treatment..  Traditional 
power calculations cannot be used to determine the number of samples (ie, patients) 
required because the assumptions of independence and normality do not hold.37  An 
alternative approach is to model this problem as a learning curve in which the classification 
error is characterized as an inverse power-law38: error = an- + b, where error is the 
expected error rate given n training samples, a is the learning rate,  is the decay rate and b 
is the Bayes error, or minimum error achievable.  This approach has been applied to several 
cancer classification problems, and estimates for the variables can be found in the 
literature.38  For our purposes, we have used the average values from 7 cancer datasets to 
estimate a,  and b.  If we let a = 0.736,  = 0.65 and b = 0.001, we can estimate the error 
for various sample sizes.  We estimate that we can achieve an error rate of ~0.08 with 30 


Protocol version: 02March2020 
38 
samples. Our prior work with metastatic biopsy collection in collaboration with Dr. Philip 
Febbo demonstrated that approximately 80% of patients’ biopsies had sufficient material to 
perform gene expression profiling. Therefore, 36 patients should be sufficient to obtain 
approximately 30 patients with informative samples.  
 
The Knight Cancer Institute Biostatistics Shared Resource will support these analytic 
methods both at the study design stage and during the data analysis stage of the project. 


Protocol version: 02March2020 
39 
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Taplin ME. Drug insight: role of the androgen receptor in the development and progression of 
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de Bono JS, Logothetis CJ, Molina A, et al. Abiraterone and increased survival in metastatic 
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Hu R, Dunn TA, Wei S, et al. Ligand-independent androgen receptor variants derived from splicing 
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Sun S, Sprenger CC, Vessella RL, et al. Castration resistance in human prostate cancer is conferred 
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Carver BS, Chapinski C, Wongvipat J, et al. Reciprocal feedback regulation of PI3K and androgen 
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Mulholland DJ, Tran LM, Li Y, et al. Cell autonomous role of PTEN in regulating castration-
resistant prostate cancer growth. Cancer Cell;19:792-804. 
15. 
Gottlieb B, Beitel LK, Nadarajah A, Paliouras M, Trifiro M. The androgen receptor gene mutations 
database: 2012 update. Human mutation 2012;33:887-94. 
16. 
Linja MJ, Savinainen KJ, Saramaki OR, Tammela TL, Vessella RL, Visakorpi T. Amplification 
and overexpression of androgen receptor gene in hormone-refractory prostate cancer. Cancer Res 
2001;61:3550-5. 
17. 
Visakorpi T, Hyytinen E, Koivisto P, et al. In vivo amplification of the androgen receptor gene and 
progression of human prostate cancer. Nat Genet 1995;9:401-6. 
18. 
Ueda T, Bruchovsky N, Sadar MD. Activation of the androgen receptor N-terminal domain by 
interleukin-6 via MAPK and STAT3 signal transduction pathways. J Biol Chem 2002;277:7076-85. 
19. 
Chang KH, Li R, Papari-Zareei M, et al. Dihydrotestosterone synthesis bypasses testosterone to 
drive castration-resistant prostate cancer. Proc Natl Acad Sci U S A 2011;108:13728-33. 
20. 
Febbo PG, Thorner A, Rubin MA, et al. Application of oligonucleotide microarrays to assess the 
biological effects of neoadjuvant imatinib mesylate treatment for localized prostate cancer. Clin 
Cancer Res 2006;12:152-8. 
21. 
Bild AH, Yao G, Chang JT, et al. Oncogenic pathway signatures in human cancers as a guide to 
targeted therapies. Nature 2006;439:353-7. 


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22. 
Pittman J, Huang E, Nevins J, Wang Q, West M. Bayesian analysis of binary prediction tree models 
for retrospectively sampled outcomes. Biostatistics 2004;5:587-601. 
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Memarzadeh S, Cai H, Janzen DM, et al. Role of autonomous androgen receptor signaling in 
prostate cancer initiation is dichotomous and depends on the oncogenic signal. Proc Natl Acad Sci 
U S A 2011;108:7962-7. 
24. 
Thomas C, Zoubeidi A, Kuruma H, et al. Transcription factor Stat5 knockdown enhances androgen 
receptor degradation and delays castration-resistant prostate cancer progression in vivo. Molecular 
cancer therapeutics 2011;10:347-59. 
25. 
Wang S, Gao J, Lei Q, et al. Prostate-specific deletion of the murine Pten tumor suppressor gene 
leads to metastatic prostate cancer. Cancer Cell 2003;4:209-21. 
26. 
Thomas GV, Horvath S, Smith BL, et al. Antibody-based profiling of the phosphoinositide 3-kinase 
pathway in clinical prostate cancer. Clin Cancer Res 2004;10:8351-6. 
27. 
Lamoureux F, Thomas C, Yin MJ, et al. Clusterin inhibition using OGX-011 synergistically 
enhances Hsp90 inhibitor activity by suppressing the heat shock response in castrate-resistant 
prostate cancer. Cancer Res 2011;71:5838-49. 
28. 
Tanaka H, Kono E, Tran CP, et al. Monoclonal antibody targeting of N-cadherin inhibits prostate 
cancer growth, metastasis and castration resistance. Nat Med 2010;16:1414-20. 
29. 
Suwaki N, Vanhecke E, Atkins KM, et al. A HIF-regulated VHL-PTP1B-Src signaling axis 
identifies a therapeutic target in renal cell carcinoma. Sci Transl Med 2011;3:85ra47. 
30. 
Tariq MA, Kim HJ, Jejelowo O, Pourmand N. Whole-transcriptome RNAseq analysis from minute 
amount of total RNA. Nucleic acids research 2011;39:e120. 
31. 
Tewhey R, Warner JB, Nakano M, et al. Microdroplet-based PCR enrichment for large-scale 
targeted sequencing. Nature biotechnology 2009;27:1025-31. 
32. 
Vaske CJ, Benz SC, Sanborn JZ, et al. Inference of patient-specific pathway activities from multi-
dimensional cancer genomics data using PARADIGM. Bioinformatics 2010;26:i237-45. 
33. 
Efron B, Tibshirani, R. On testing the significance of sets of genes. Ann Appl Stat 2007;1. 
34. 
Eisen MB, Spellman PT, Brown PO, Botstein D. Cluster analysis and display of genome-wide 
expression patterns. Proc Natl Acad Sci U S A 1998;95:14863-8. 
35. 
Scher HI, Halabi S, Tannock I, et al. Design and end points of clinical trials for patients with 
progressive prostate cancer and castrate levels of testosterone: recommendations of the Prostate 
Cancer Clinical Trials Working Group. Journal of clinical oncology : official journal of the 
American Society of Clinical Oncology 2008;26:1148-59. 
36. 
Danila DC, Fleisher M, Scher HI. Circulating tumor cells as biomarkers in prostate cancer. Clin 
Cancer Res 2011;17:3903-12. 
37. 
Pusztai L, Hess KR. Clinical trial design for microarray predictive marker discovery and 
assessment. Annals of oncology : official journal of the European Society for Medical Oncology / 
ESMO 2004;15:1731-7. 
38. 
Mukherjee S, Tamayo P, Rogers S, et al. Estimating dataset size requirements for classifying DNA 
microarray data. Journal of computational biology : a journal of computational molecular cell 
biology 2003;10:119-42. 
 
 


Protocol version: 02March2020 
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APPENDIX A 
ECOG PERFORMANCE STATUS* 
Grade ECOG 
0 
Fully active, able to carry on all pre-disease performance without restriction 
1 
Restricted in physically strenuous activity but ambulatory and able to carry out work of a light or 
sedentary nature, e.g., light house work, office work 
2 
Ambulatory and capable of all selfcare but unable to carry out any work activities. Up and about 
more than 50% of waking hours 
3 
Capable of only limited selfcare, confined to bed or chair more than 50% of waking hours 
4 
Completely disabled. Cannot carry on any selfcare. Totally confined to bed or chair 
5 
Dead 
 
* As published in Am. J. Clin. Oncol.: 
Oken, M.M., Creech, R.H., Tormey, D.C., Horton, J., Davis, T.E., McFadden, E.T., Carbone, P.P.: 
Toxicity And Response Criteria Of The Eastern Cooperative Oncology Group. Am J Clin Oncol 
5:649-655, 1982. 
 
 
 
 


Protocol version: 02March2020 
42 
APPENDIX B 
BIOPSY PROCEDURE 
 
The tumor biopsies for this study will be collected and shipped via the University of 
California, San Francisco, protocol CC#125519 (OHSU protocol #9204) -Radiologically 
Guided Biopsies Of Metastatic Castration Resistant Prostate Cancer To Identify Adaptive 
Mechanisms of Resistance. 
 
Critical to the application of the AR signature, is the collection of prostate cancer tissue for genomic 
analysis. Due to the nature of metastatic prostate cancer, most biopsies will be from bone. Bone biopsies 
present unique challenges with respect to the collection and processing. However, regardless of collection 
technique, rapid freezing of the biopsy material is critical to subsequent success. As part of the trial, we will 
provide kits to each site enrolling patients so as to facilitate a standardized collection of tissue.  
 
Patients are positioned by radiology and conscious sedation may be administered to improve patient 
comfort. This is based on the judgment of the interventional radiologist and may be restricted in some 
centers due to the availability of supportive services.  
 
During the image-guided biopsy procedure we recommend the following general rules when choosing a 
metastatic site and a specific location within the metastatic tumor for biopsy:  
 
1. If the patient has bone only disease, the pelvis is the preferred site for biopsy.  
2. Any biopsy kit capable of obtaining core biopsies from bone is acceptable. The Bonopty Coaxial 
biopsy system with eccentric drill (http://www.vasocare.co.kr/product04-
5.html).http://www.vasocare.co.kr/product04-5.html) is a recommended system. Internal diameter 
is 1.3mm. This is the smallest for collection of usable material.  
3. In the bone, extremely blastic lesions seldom yield usable material. Yields are greater if biopsies are 
performed in marrow of abnormal signal intensity directly adjacent to blastic lesions. Often, we 
pass the needle tangential to the blastic lesion in the marrow space for our most successful 
collections.  
4. In soft tissue, do not biopsy from regions of metastatic lesions that appear necrotic on CT or MRI 
due to extremely poor yield.  
5. It is important that excessive compressive force is not required or used to expel the biopsy from the 
biopsy needle. This obscures cellular morphology. If it is difficult to expel the biopsy from the core 
needle, alternative biopsy kits should be used.  
 
Please follow the directions below when processing the biopsy material:  
 
Required Materials  
 
Included in Kit:  
• Disposable cryo-molds 15x15x5mm  
• Sakura Tissue-Tek OCT compound  
• Metal plate or tray  
• Sarstedt black permanent marker  
• Tissue-Tek Mega-Cassettes  
• Styrofoam cooler (part of shipment box, a new box will be returned once the biopsies arrive at UCSF)  
• Container of neutral buffered formalin (2 provided, keep extra, an additional vial will be included with 
the new box)  


Protocol version: 02March2020 
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*Keep all remaining materials after procedure is completed for use on future biopsies unless otherwise 
noted  
 
Not Included (should be available on site)  
• Dry Ice (pellet form, smaller pellet size works best)  
• Syringe needle (core manipulation; ≤19g)  
• Forceps (fine toothed)  
 
Protocol for Biopsies  
1. Prepare a cold working surface by filling the supplied styrofoam cooler container roughly half full 
of dry ice. Shake the container to form a fairly even layer.  
 
2. Place the metal sheet or tray into the cooler and allow it to cool below -10°C. Temperatures below 
this point will freeze OCT compound quickly, facilitating the block formation. Having a flat 
metallic surface is also important.  
 
3. Prior to heading to the collection, place several cryomolds on the cold metal surface allowing them 
to cool down prior to the biopsy.  
 
4. Label 5 tissue cassettes, then place on dry ice to pre-chill before use during the procedure.  
 
5. Take the cooler setup to the location of the biopsy and be sure to have easy access to it during the 
procedure. Be sure to take the formalin vial in the zip-lock bag as well.  
 
6. Once the radiologist says a core is about ready, fill one of the cryomold trays roughly half-full of 
OCT so that the compound is partially frozen by the time the core is added. The OCT compound 
will appear opaque as it solidifies.  
 
7. Transfer a core to the partially solidified OCT layer in the cryomold. The best method of tissue 
transfer from the biopsy needle to the pallet will depend on the type of needle used. The core biopsy 
can be directly placed into the cryomold of OCT, but care must be taken to ensure the core is lying 
as close to flat as possible as it freezes. Bonopty and other hollow core needles will yield a 
cylindrical core that is ejected via a plunger needle. The pressure created by the plunger directly 
onto the core can unpredictably eject the core from the needle. If the core cannot be placed directly 
on the OCT, it can be ejected onto a gauze pad and transferred immediately onto the OCT so as to 
minimize time for RNA degradation and protein de-phosphorylation. This helps to maintain needle 
sterility and allows for more careful manipulation of the core away from the sterile procedure cart. 
Manipulation of the core from gauze pad to cryomold is done with a syringe needle.  
 
8. Positioning of the core is very important. Be sure to place the core flat down on the center of the 
OCT layer. The tissue can freeze on contact if the OCT layer has solidified, thus preventing 
manipulation once the tissue is on the OCT surface. Adding the core to OCT prior to solidification 
is ideal since the tissue and OCT will then freeze at the same time, resulting in better sectioning 
later. Layers frozen in different stages tend to cause separation during sectioning.  
9. Immediately cover the core with OCT, working in a slow circular motion around the core such that 
OCT fills in the sides, then surrounds the top. Contact of the liquid OCT with the tissue core is 
important to preserve the tissue and facilitate in cryo sectioning later.  
 
10. nce the OCT becomes opaque in each cryomold, the tissue block is ready to be transferred to the 
labeled cold cassette.  
 


Protocol version: 02March2020 
44 
11. In addition to collecting fresh-frozen biopsy cores, 2 core must be collected for paraffin embedding. 
This core should be placed in the container of neutral buffered formalin. Close the jar tightly, and 
return to the zip-lock bag.  
 
12. Package the biopsies into the provided insulated box. The cold cryo cassettes should be placed in 
dry ice in the insulated section of the box, and the formalin vial contained within the zip-lock bag 
should be placed outside of the insulated region of the box. The appropriate spot for the formalin 
vial will be labeled.  
 
13. Include with the shipment, the sample shipment log (Appendix K) that should include comments 
regarding core quality (long cylindrical core, bone shards, mostly blood clot, etc), the quantity of 
cores and any irregularities in the freezing process. The technician performing the collection should 
be sure to note their unique patient ID, the biopsy date, and the time of the procedure as well.  
 
14. Prior to any shipments, the study coordinator and designated laboratory staff at UCSF and OHSU 
must be notified. Notification should come in the form of email at least 2 – 3 days or, if possible, a 
week prior to an anticipated shipment. The email should consist of the date of scheduled biopsy, 
unique patient ID, participating site name and site contact; this should be sent to 
phillip.febbo@ucsf.edu and adam.foye@ucsf.edu and thomasge@ohsu.edu.  
 
15. Mail by Express Overnight delivery to the address specified on the provided shipping labels. Unless 
special arrangements are made, do not ship on Fridays, Saturdays, or Sundays.  
 


Protocol version: 02March2020 
45 
APPENDIX C 
Optional, Investigational Sample Collection, Processing, and Storage 
 
Investigational serum samples will be drawn using venipuncture at each time point dictated by the schedule 
of events (see 10.4). All supplies listed in this appendix, with the exception of the 2ml cryovials supplied by 
OHSU, are to be obtained locally by the sub-site. 
 
Draw 10ml whole blood into an EDTA (lavender top) vacutainer tube. Process blood as soon as possible. It 
is recommended that the resulting plasma is frozen within one (1) hour of blood draw, and that no more 
than two (2) hours elapse from collection to freezing. 
 
Centrifuge whole blood at 1,000g for 10 minutes at room temp. Using a polyethylene transfer pipet, transfer 
at least 1ml of the resulting plasma into each of the four (4) provided Cryovials. Be sure that the cells from 
the buffy coat are not removed with the plasma phase. Cap and label each Cryovial with patient study ID, 
type of sample (plasma), date of collection, and time point (e.g. day 1, week 12, off-study, etc.). 
 
Freeze and store the samples in an upright position at -80°C until shipping to OHSU on dry ice. Samples 
may be stored on site and batch shipped to OHSU. Please alert the OHSU study coordinator listed below of 
any planed shipments to ensure proper receipt.  
 
Follow these instructions to ensure proper transport/receipt of samples: 
 
 
Samples may only be shipped Monday through Thursday via overnight delivery. Do not ship 
samples on Fridays as OHSU is unable to receive packages on Saturdays.  
 
Place the frozen samples and some absorbent material in a plastic biohazard bag and seal the bag. 
 
Samples must be shipped in a Styrofoam insulated shipping container. 
 
Place enough dry ice to cover the bottom of the Styrofoam container, place bagged samples on top 
of dry ice, then cover with 7 to 10 pounds of dry ice. 
 
To ensure timely delivery, the package MUST be properly labeled to indicate the presence of dry 
ice and biological specimens. Consult a local shipping authority if unsure how to properly label. 
 
INVESTIGATIONAL SAMPLES SHOULD BE SENT TO: 
 
SPECIMEN MANAGER 
 
 
OREGON HEALTH & SCIENCE UNIVERSITY 
 
3303 SW Bond Avenue, mailcode: CH14R 
 
PORTLAND, OR 97239 
CONTACT INFORMATION: 
PHONE:  (503) 494-4396 
FAX:  (503) 494-6197 
 
OHSU ONLY 
In addition to the 10ml blood collected by all sites, the OHSU site only will collect an additional 30ml of 
blood in EDTA (lavender top) vacutainer tubes. Centrifuge blood at 1000g for 10 minutes at room temp. 
Transfer the plasma layer (~5ml per tube) to 1.5ml Eppendorf tubes in 1ml aliquots and centrifuge at 
15,000g for 10 minutes at room temp. Transfer the 1ml plasma supernatants to labelled Cryovials. Dislodge 
the remaining buffy coat pellets from the Eppendorf tubes and combined them in one labeled Cryovial. 
Store all samples at -80C. 


Protocol version: 02March2020 
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Protocol version: 02March2020 
47 
APPENDIX D 
 
 
 
 
SAE REPORT COVER SHEET 
 
 
 
IIT Investigator’s Name:  
         
 
(First, Last) 
 
IIT Study Site Information 
 
Institution Name:  
 
      
 
City, State: 
 
      
 
IIT Study Number: 
 
IIT000800 
 
IIT Protocol Title: 
 
      
 
# of pages (including cover page) 
 
      
 
Please return this cover page along with the MEDWATCH form. 
 
 
 


Protocol version: 02March2020 
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APPENDIX E 
 
Below is a partial list of potent CYP inhibitors and inducers.  
 
Potent CYP Inhibitors and Inducers  
CYP 
Inhibitors  
CYP Inducers  
amiodarone  
atazanavir  
clarithromycin  
disulfirum  
fluconazole  
fluoxetine  
fluvoxamine  
fluvoxamine  
gemfibrozil  
indinavir  
itraconazole  
ketoconazole  
moclobemide  
nefazodone  
nelfinavir  
omeprazole  
paroxetine  
quinidine  
ritonavir  
saquinavir  
telithromycin  
carbamazepine  
Rifampin  
 
 
 


Protocol version: 02March2020 
49 
APPENDIX F 
 
Drug Diary 
 
Study: Molecular Mechanisms Underlying Tumor Progression Despite Enzalutamide Treatment  
IRB#: XXXX 
Once the day 1 date is entered, the remaining dates will auto-populate in this form. 
 
Last, First Name: 
MRN: 
Study Period: Week X-X 
Sunday 
 
Monday 
Tuesday 
Wednesday 
Thursday 
Friday 
Saturday 
 
 
 
Enter date 
Enter date 
Enter date 
Enter date 
 
 
 
Week 
Enzalutamide: 
___ 160mg 
Enzalutamide: 
___ 160mg 
Enzalutamide: 
___ 160mg 
Enzalutamide: 
___ 160mg 
Enter date 
Enter date 
Enter date 
Enter date 
Enter date 
Enter date 
Enter date 
Enzalutamide: 
___ 160mg 
Enzalutamide: 
___ 160mg 
Enzalutamide: 
___ 160mg 
Enzalutamide: 
___ 160mg 
Enzalutamide: 
___ 160mg 
Enzalutamide: 
___ 160mg 
Enzalutamide: 
___ 160mg 
Enter date 
Enter date 
Enter date 
Enter date 
Enter date 
Enter date 
Enter date 
Enzalutamide: 
___ 160mg 
Enzalutamide: 
___ 160mg 
Enzalutamide: 
___ 160mg 
Enzalutamide: 
___ 160mg 
Enzalutamide: 
___ 160mg 
Enzalutamide: 
___ 160mg 
Enzalutamide: 
___ 160mg 
Enter date 
Enter date 
Enter date 
Enter date 
Enter date 
Enter date 
Enter date 
Enzalutamide: 
___ 160mg 
Enzalutamide: 
___ 160mg 
Enzalutamide: 
___ 160mg 
Enzalutamide: 
___ 160mg 
Enzalutamide: 
___ 160mg 
Enzalutamide: 
___ 160mg 
Enzalutamide: 
___ 160mg 
Enter date 
Enter date 
Enter date 
 
 
 
 
Enzalutamide: 
___ 160mg 
Enzalutamide: 
___ 160mg 
Enzalutamide: 
___ 160mg 
 
 
 
 
* 
Drug Accountability: Please sign the calendar to indicate you have taken the above medication as instructed and return the signed 
calendar at the next study visit.  Please note any missed doses and the reason missed on the specific date. 
Patient Signature: _________________________________________  
Date: ______________________________________  
 
 
 


Protocol version: 02March2020 
50 
APPENDIX G 
Partial list of medications which lower the seizure threshold. These medicine are not prohibited but should 
be used with caution. 
 
abobotulinumtoxinA 
HALOPERIDOL 
PIMOZIDE 
AMINOPHYLLINE 
HYDROXYZINE 
PROCAINE 
AMITRIPTYLINE 
IMIPENEM 
PROCARBAZINE 
AMPHETAMINE 
IMIPRAMINE 
PROCHLORPERAZINE 
ATROPINE 
IPRATROPIUM BROMIDE PROCYCLIDINE 
BACLOFEN 
ISOCARBOXAZID 
PROMETHAZINE 
BENZTROPINE 
MESYLATE 
ISONIAZID 
PROPANTHELINE BROMIDE 
BIPERIDEN 
LIDOCAINE 
PROPOFOL 
BUPIVACAINE 
LINDANE 
PROPOXYPHENE 
BUPROPION 
LITHIUM 
PROTRIPTYLINE 
CHLORAMBUCIL 
LOXAPINE SUCCINATE 
RASAGILINE MESYLATE 
CHLORPROMAZINE 
MEFENAMIC ACID 
RISPERIDONE 
CITALOPRAM 
HYDROBROMIDE 
MEPENZOLATE 
BROMIDE 
SCOPOLAMINE 
CLOMIPRAMINE 
MEPERIDINE 
SELEGILINE 
CLOZAPINE 
MEROPENEM 
SERTRALINE 
CYCLOSPORINE 
METHSCOPOLAMINE 
BROMIDE 
SOLIFENACIN SUCCINATE 
DARIFENACIN 
HYDROBROMIDE 
METHYLPHENIDATE 
SUMATRIPTAN 
DESIPRAMINE 
METRONIDAZOLE 
THEOPHYLLINE 
DICYCLOMINE 
NALIDIXIC ACID 
THIETHYLPERAZINE 
DORIPENEM 
NEFAZODONE 
THIORIDAZINE 
DOXEPIN 
NORTRIPTYLINE 
TIOTROPIUM BROMIDE 
MONOHYDRATE 
DULOXETINE 
ORPHENADRINE 
CITRATE 
TOLTERODINE 
ERTAPENEM SODIUM 
OXYBUTYNIN 
TRAMADOL 
ESCITALOPRAM 
OXALATE 
OXYTOCIN 
TRANYLCYPROMINE SULFATE 
FENTANYL 
PAROXETINE 
TRIFLUOPERAZINE 
FLUOXETINE 
PENICILLIN V 
POTASSIUM 
TRIHEXYPHENIDYL 
FLUPHENAZINE 
PENTAZOCINE 
LACTATE 
TRIMIPRAMINE MALEATE 
FLUVOXAMINE 
MALEATE 
PERPHENAZINE 
TROSPIUM CHLORIDE 
GLYCOPYRROLATE 
PHENELZINE SULFATE 
 
 
 
 


Protocol version: 02March2020 
51 
 
 
 
Appendix H  
  
OHSU guidelines for drugs to avoid pre-biopsy  
 
 
 
 
 
 
 
 
 
 
 
 
Drugs to avoid pre-biopsy 
Length of washout pre-biopsy 
COUMADIN 
Withhold for at least 5 days prior to biopsy; 
consider bridge with heparin; restart > 24 
hours after biopsy 
CLOPIDOGREL 
Withhold for at least 7 days prior to biopsy 
PRASUGREL 
Withhold for at least 7 days prior to biopsy 
TICARGRELOR 
Withhold for at least 5 days prior to biopsy 
FRACTIONATED HEPARIN 
Withhold  for at least 2-4 hours prior to biopsy 
DABIGATRAN (PRADAXA) 
Withhold for at least 2 days prior to biopsy if 
eGFR >50 or for at least 3  days prior to 
biopsy if eGFR <50; restart > 24 hours after 
biopsy 
RIVAROXABAN (XARELTO) 
Withhold for at least 24 hours prior to biopsy; 
restart > 24 hours after biopsy 
APIXABAN (ELIQUIS) 
Withhold for at least 24 hours before biopsy; 
restart > 24 hours after biopsy 
FONDAPARINUX (ARIXTRA) 
Withhold for at least 3 days prior to biopsy 
TICLOPIDINE (TICLID) 
Withhold for at least 7 days prior to biopsy 
ASPIRIN 
Withhold for at least 5 days prior to biopsy 
DOCLOFENAC (VOLTERAN) 
Withhold for at least 24 hours prior to biopsy 
IBUPROFEN 
Withhold for at least 24 hours prior to biopsy 
INDOMETHACIN 
Withhold for at least 24 hours prior to biopsy 
NAPROXEN 
Withhold for at least 4 days prior to biopsy 
PIROXICAM 
Withhold for at least  11 days prior to biopsy 
AGGRENOX 
Withhold for at least 5 days prior to biopsy