Clinical trial NCT03298087
Conditions: Newly Diagnosed Oligometastatic Prostate Cancer
Keywords: oligometastatic SBRT abiraterone apalutamide
Sponsor: VA Office of Research and Development
Start date: 2018-07-01; Completion date: 2024-03-31
Enrollment: 28 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
This is a trial for patients with newly diagnosed metastatic prostate cancer with 5 or fewer sites of metastases. The trial involves surgery (removal of the prostate) or radiation to the prostate, six months of hormone therapy, and stereotactic body radiotherapy to the sites of metastasis.

Detailed description:
This is a single arm Phase II clinical trial in patients with newly diagnosed M1a,b prostate cancer and 1-5 radiographically visible metastases treated with radical prostatectomy (and post-operative fractionated radiotherapy for pT 3a, pN1, or positive margins) or radiation to the prostate, metastasis directed SBRT, and complete ADT with LHRH analog leuprolide, abiraterone acetate with prednisone, and apalutamide (ARN-509) for a total of six months of systemic therapy. The primary endpoint of our study is the percent of patients achieving a serum PSA of \<0.05 ng/mL six months after recovery of serum testosterone (for patients undergoing radical prostatectomy) or PSA \<nadir+2 (for patients undergoing prostate radiation).

Arms: arm 1: Radical prostatectomy (and post-operative fractionated radiotherapy for pT=3a, pN1, or positive margins), metastasis directed SBRT, and complete ADT with LHRH analog leuprolide, abiraterone acetate with prednisone, and apalutamide (ARN-509) for a total of six months of systemic therapy.
Interventions: intervention 1: radical prostatectomy intervention 2: stereotactic body radiotherapy intervention 3: Leuprolide intervention 4: apalutamide intervention 5: abiraterone
Intervention descriptions: intervention 1: surgical removal of the prostate intervention 2: Highly targeted radiation intervention 3: Lowers serum testosterone intervention 4: antiandrogen intervention 5: Inhibits androgen synthesis

Protocol documents:
Prot_SAP_000.pdf: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 Title: Systemic and tumor-directed 
therapy for oligometastatic prostate 
cancer 
 
 


 
 
 
 
 
 
Technical Title: A Single-Arm, Open-Label, Phase II Study of Systemic and Tumor 
Directed Therapy for Newly Diagnosed Oligometastatic M1 Prostate Cancer 
 
Sponsor: 
VA West Los Angeles Medical Center 
 
Principal Investigator:   
 
Nicholas G. Nickols, M.D., Ph.D. 
Radiation Oncology 
VA West Los Angeles Medical Center 
 
Protocol  
Version 9.0, September 06, 2022 
 
IND 133606 
 
 
 
 
 
 
 
 
 
 
 
Proprietary and Confidential 
The information in this document is considered privileged and confidential, and 
may not be disclosed to others except to the extent necessary to obtain 
Institutional Review Board approval and informed consent, or as required by 
Federal and State laws. Persons to whom this information is disclosed should be 
informed that this information is privileged and confidential and should not be 
further disclosed. 
 
Funding to conduct the trial is provided by Department of Veterans Affairs (VA ORD, 
CSR&D Merit Review Award (CX-16-006), Nickols (PI), 1I01CX001575-01). Drug support 
(apalutamide and abiraterone acetate) is provided by Janssen Scientific Affairs, LLC. 


i 
 
TABLE OF CONTENTS 
Page 
STUDY TEAM ROSTER ............................................................................................................. 2 
PARTICIPATING STUDY SITES .............................................................................................. 4 
List of Abbreviations ................................................................................................................. 9 
2. BACKGROUND AND RATIONALE................................................................................... 11 
3. STUDY DESIGN .................................................................................................................... 13 
4. SELECTION AND ENROLLMENT OF PARTICIPANTS ............................................... 17 
5. STUDY INTERVENTIONS ................................................................................................... 21 
6. STUDY PROCEDURES ....................................................................................................... 37 
7. SAFETY ASSESSMENTS ................................................................................................... 40 
8. INTERVENTION DISCONTINUATION .............................................................................. 49 
9. STATISTICAL CONSIDERATIONS ................................................................................... 49 
10. DATA COLLECTION AND QUALITY ASSURANCE ................................................... 51 
11. PARTICIPANT RIGHTS AND CONFIDENTIALITY ...................................................... 51 
12. FUNDING AND INSURANCE ........................................................................................... 53 
13. PUBLICATION OF RESEARCH FINDINGS .................................................................. 53 
14. SUPPLEMENTS/APPENDICES ....................................................................................... 53 


2 
 
STUDY TEAM ROSTER  
 
Principal Investigator:  
Nicholas Nickols, M.D., Ph.D. 
 
VA West Los Angeles Medical Center 
Radiation Oncology  
11301 Wilshire Blvd 
Los Angeles, CA 90073 
Ph: 310-268-3390 
 
UCLA Radiation Oncology 
BOX 951714, B3-109 CHS 
Los Angeles, CA 90095-1714 
Ph: 310-825-8278   
Fax: 310-206-1260 
 
e-mail: nnickols@mednet.ucla.edu; Nicholas.nickols@va.gov 
 
Co-Investigators: 
 
Matthew Rettig, M.D. 
 
VA West Los Angeles Medical Center 
Hematology-Oncology 
11301 Wilshire Blvd 
Los Angeles, CA 90073 
 
UCLA Institute of Urologic Oncology 
Department of Urology and Division of Hematology and Oncology 
300 Stein Plaza, 3rd Floor 
Los Angeles, CA 90095 
Ph: 310-794-3565 
Fax: 310-794-3512 
 
e-mail: mrettig@mednet.ucla.edu; matthew.rettig@va.gov 
 
 
Isla P. Garraway, M.D, Ph.D. 
 
VA West Los Angeles Medical Center 
Urology 
11301 Wilshire Blvd 
Los Angeles, CA 90073 
 
UCLA Urology 
BOX 951738, 12-145 Factor 
Los Angeles, CA 90095-1738 
e-mail: IGarraway@mednet.ucla.edu; isla.garraway@va.gov 
 


3 
 
Steve P. Lee, M.D., Ph.D. 
 
VA Long Beach Healthcare System 
Radiation Oncology  
5901 E 7th St 
Long Beach, CA 90822 
 
UCLA Radiation Oncology 
BOX 956951, Ste B265 
200 Medical Plaza  
Los Angeles, CA 90095-6951 
e-mail: splee@mednet.ucla.edu; steve.lee5@va.gov 
 
Mark Jordan, M.D. 
 
VA Long Beach Healthcare System 
Urology 
5901 E 7th St 
Long Beach, CA 90822 
Ph: 562-826-5562 
Fax: 562-826-8117 
e-mail: Mark.Jordan2@va.gov 
 
Michael G. Chang, M.D. 
 
Hunter Holmes McGuire VA Medical Center 
Radiation Oncology 
1201 Broad Rock Blvd 
Richmond, VA 23249 
e-mail: michael.chang@vcuhealth.org 
 
 
Bhaumik B. Patel, M.D. 
 
Hunter Holmes McGuire VA Medical Center 
Department of Internal Medicine 
Division of Hematology, Oncology & Palliative Care 
1201 Broad Rock Blvd. 
Richmond, VA 23249 
e-mail: bhaumik.patel@vcuhealth.org 
 


4 
 
 
 
PARTICIPATING STUDY SITES 
 
West LA VA, Los Angeles, CA, United States 
(Nickols, Rettig, Garraway) 
 
Long Beach VA, Long Beach, CA, United States 
(Lee, Jordan) 
 
Hunter Holmes McGuire VA Medical Center, Richmond, VA, United States 
(Chang, Patel) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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PRÉCIS/SUMMARY 
Study Title  
Systemic and tumor-directed therapy for oligometastatic prostate cancer 
Objectives  
Primary Objective:  
The primary objective of this study is to assess the efficacy of combined modality therapy 
of systemic and tumor directed therapy for newly diagnosed (de novo) oligometastatic 
M1a,b prostate cancer.  In this study, androgen deprivation therapy (ADT) is limited to a 
six-month duration. The primary endpoint of our study is the percent of patients achieving 
a serum PSA of <0.05 ng/mL six months after recovery of serum testosterone ≥150 ng/dL 
(i.e., undetectable disease burden off-therapy and after gonadal recovery) if primary 
tumor therapy is radical prostatectomy, and PSA of < nadir + 2 ng/mL six months after 
recovery of serum testosterone ≥150 ng/dL if primary tumor therapy is radiation therapy.  
 
Secondary Objectives: 
The secondary objectives of the study include a number of clinical endpoints enumerated 
below: 
 
•  Time to biochemical progression. 
•  Time to radiographic progression 
•  Time to additional antineoplastic therapy 
•  Prostate cancer specific survival 
• 
Assessment of health related quality of life using the Functional Assessment 
of Cancer Therapy - Prostate (FACT-P) scale 
 
Tissue available for optional correlative analyses: 
Pre-treatment metastatic biopsies 
Radical prostatectomy specimens (prior to systemic or radiotherapy) 
Serial blood collections 
Metastatic biopsies at radiographic progression (recommended) 
Design  
This is a single arm Phase II clinical trial in patients with newly diagnosed M1a,b prostate 
cancer and 1-5 radiographically visible metastases treated with radical prostatectomy 
(and post-operative fractionated radiotherapy for pT≥3a, pN1, or positive margins) or 
radiotherapy (including inclusion of the pelvic lymph nodes), metastasis directed SBRT, 
and complete ADT with LHRH analog leuprolide, abiraterone acetate with prednisone, 
and apalutamide (ARN-509) for a total of six months of systemic therapy. The primary 
endpoint of our study is the percent of patients achieving a serum PSA of <0.05 ng/mL 
six months after recovery of serum testosterone ≥150 ng/dL if primary tumor therapy is 
radical prostatectomy, and PSA of < nadir + 2 ng/mL six months after recovery of serum 
testosterone ≥150 ng/dL if primary tumor therapy is radiation therapy. [Based on prior 
studies, >90% of patients treated with ADT for six months will recover serum testosterone 
to ≥150 ng/dL within six months after cessation of ADT with a median time to recovery of 
three months (1,2)] This primary endpoint was selected to offer a rapid and sensitive 
assessment of treatment efficacy. PSA is a biomarker for disease burden in prostate 
adenocarcinoma and offers a non-invasive and sensitive assessment of disease control 


6 
 
after treatment in the vast majority of patients.  Durable PSA control in the context of 
recovery of gonadal function (testosterone ≥150 ng/dL) after ADT is therefore a surrogate 
for disease control. See Figure 1 for trial schema. 
 
 
Currently, metastatic prostate cancer patients are treated with ADT indefinitely, or 
intermittently. Based on data from prior trials of discontinuous ADT in prostate cancer 
patients with metastatic disease, essentially all patients will relapse with a rising PSA after 
discontinuation of ADT. The current study hypothesis is that 20% of patients will achieve 
a PSA <0.05 ng/mL six months after recovery of testosterone to >150 ng/dL, or PSA of < 
nadir + 2 ng/mL six months after recovery of serum testosterone ≥150 ng/dL if primary 
tumor therapy is radiation therapy. N=25 evaluable patients has 80% power to detect a 
20% response rate at a significance (alpha) of 0.1. Assuming 10% of patients may not 
recover testosterone to ≥150 ng/dL or may drop out, we anticipate a total study N=28. 
 
Apalutamide is a next-generation antiandrogen in ongoing trials in locally advanced, 
castrate resistant, and metastatic prostate cancer (3,4). Apalutamide is FDA approved for 
the treatment of non-metastatic castration resistant prostate cancer and metastatic 
castration sensitive  prostate cancer.  Abiraterone acetate is an androgen synthesis 
inhibitor approved for metastatic castrate resistant prostate cancer (mCRPC) (5) and is 
also FDA approved for high risk metastatic castration sensitive prostate cancer. SBRT 
directed to metastatic foci in prostate cancer achieves local of greater than 95% (9). 
Radiotherapy directed to the primary tumor is also now a standard of care for patients 
with oligometastatic castration sensitive prostate cancer (PMID:30355464). 
 
This trial includes a radiographic directed biopsy of a metastatic lesion prior to initiation 
of therapy unless unsafe to perform, not feasible, or declined by patient. As such, 
evaluable tissues may include metastatic and primary tumor tissue acquired prior to 
initiation of hormone or radiotherapy. Additionally, blood is collected for correlative 


7 
 
analyses. A metastatic biopsy at the time of radiographic progression is recommended 
but not required. 
 
Correlative Objectives, Overview 
 
Background. This trial will yield evaluable tissue from previously untreated prostate 
cancer patients with metastatic disease. We will identify the primary tumor in each patient 
that gave rise to metastasis, and compare these potentially lethal primaries with the other 
tumors that likely did not yield metastases. We will also compare these potentially lethal 
primary tumors to their corresponding metastases whenever possible. In doing so, we 
may (1) identify characteristic features of potentially lethal primary prostate tumors that 
are destined for metastasis, and (2) identify which pathways become activated within 
these potentially lethal primary tumors during or after the metastatic event. Importantly, 
these investigations all use tissue that is acquired at the outset for each patient enrolled 
in our trial, and these investigations do not depend on the clinical outcome of the trial. 
See Figure 2 for overview of the key correlative Aims. 
 
Sources of Tissue. From VA West LA, fresh tissue from the prostatectomy or biopsy 
will be obtained in addition to FFPE tissue from the prostatectomy specimen or initial 
prostate biopsy. Fresh prostate tissue obtained at surgery at VA West LA will be shared 
with Dr Garraway’s study VA #0001____. From the other sites, FFPE tissue from the 
prostatectomy specimen or initial prostate biopsy will be obtained, and fresh tissue will 
not be obtained due to issues of feasibility. 
 
Correlative Aim 1. What genomic and transcriptomic features are present in lethal 
intraprostatic tumors that ultimately give rise to metastatic disease that are absent, or 
different, in those that do not? To answer this question, we aim to conduct whole exome 
deep sequencing (WES), RNA sequencing (RNA-seq), and comparative genomic 
hybridizations (CGH) of the metastatic and intraprostatic tumors of the 28 patients 
enrolled in our Phase II trial. This will enable identification of the “true” primary tumors 
from which the biopsied metastases arose. Then, we will combine the aforementioned 
genomic analyses with RNA-seq data to identify discriminating features of the “true” 
primary lesions that distinguish them from the intraprostatic tumors that did not yield these 
metastatic lesions.  
1.1 
To identify which intraprostatic tumor in each patient gave rise to metastasis. 
1.2 
To discover genomic and transcriptomic features whenever possible specific to the 
intraprostatic tumors that ultimately gave rise to metastasis versus those that did not. 
 
Correlative Aim 2. What genomic and transcriptomic features are present in the 
metastases that are absent, or different, from the corresponding primary tumors from 
which they metastasized? This will involve comparison between the primary tumors and 
their metastases. We may also specifically interrogate selected markers of kinase activity 
(MEK/ERK) and differentiation (EMT). 
2.1 
To discover genomic and transcriptomic features specific to metastatic prostate 
tumors as compared to the primary tumors from which they arose. 
 


8 
 
2.2 
To evaluate MAPK (ERK) activation and markers of epithelial to mesenchymal 
transition (EMT) in metastatic prostate cancer lesions as compared to their corresponding 
primary tumors. 
 
Correlative Aim 3. What genomic and transcriptomic features are present in circulating 
tumor cells and DNA that predict for response? Are there changes in the 
immunophenotype of circulating lymphocytes over the course of treatment? 
2.1 
To evaluate biomarkers of response using circulating tumor cells (CTCs). 
 
2.2 
To evaluate biomarkers of response using circulating tumor DNA (ctDNA). 
 
2.3 
To evaluate immunophenotypes of circulating immune cells. 
 
Interventions and Duration  
 
Screening 
Screening will occur between day -90 and day 0 (day 1 = date of surgery for patients 
undergoing radical prostatectomy as primary tumor therapy; day 1 = date of initiation of 
ADT/ +/-3 days apalutamide + abiraterone + prednisone for patients undergoing 
radiotherapy as primary tumor therapy). Screening evaluation will include a history and 
physical examination, performance status evaluation, laboratory studies, and biopsy of a 
metastatic lesion.  Staging by NaF or PSMA PET-CT or Fluciclovine (Axumin) PSMA 
PET-CT (with diagnostic CT of the chest, abdomen/pelvis) is required. If biopsy is not 
diagnostic, or unsafe to perform, not feasible, or declined, then a secondary imaging 
modality (for example, MRI) must also be consistent with metastatic disease (unless 
PSMA PET-CT or Fluciclovine (Axumin) PSMA PET-CT was used for initial staging). 
 
On Study Interventions 
Patients who undergo radical prostatectomy as primary tumor therapy will undergo radical 
prostatectomy prior to initiation of systemic or radiotherapy. After prostatectomy, patients 
will begin six months of complete ADT with leuprolide, apalutamide, and abiraterone 
acetate. Patients who undergo radiotherapy as primary tumor therapy can undergo the 
radiotherapy anytime while on the six months of complete ADT with leuprolide, 
apalutamide, and abiraterone acetate, provided they will complete the radiotherapy while 
still on the ADT. All patients will undergo SBRT to all visible sites of metastases, unless 
the metastases would be included in pelvic RT volumes (i.e., common iliac M1a lymph 
nodes). The SBRT may be done anytime while the patient is still on ADT. Some patients 
who undergo prostatectomy will receive post-operative pelvic radiotherapy (for ≥pT3a, 
N1, positive margins). The total duration of systemic therapy is six months. Patients will 
be evaluated on treatment every 30 days, and subsequently after systemic therapy is 
complete every three months thereafter. A research biopsy of a metastatic lesion at the 
time of radiographic progression will be encouraged but not required.  
Sample Size and Population  
Study population. Patients to be enrolled will have untreated metastatic prostate cancer 
with 1-5 metastasis (exclusive of pelvic, N1, metastases) identified by NaF PET-CT or 
PSMA PET-CT or Fluciclovine (Axumin) PET-CT (with diagnostic CT of the chest, 


9 
 
abdomen, pelvis) that have undergone biopsy of a metastatic lesion. 
Sample Size. Currently, metastatic prostate cancer patients are treated with ADT 
indefinitely or intermittently. Based on data from prior trials of discontinuous ADT in 
prostate cancer patients with metastatic disease, essentially all patients will relapse with 
a rising PSA after discontinuation of ADT (33,34).  The current study hypothesis is that 
20% of patients will achieve a PSA <0.05 ng/mL six months after recovery of testosterone 
to >150 ng/dL if primary tumor therapy is radical prostatectomy or PSA of < nadir + 2 
ng/mL six months after recovery of serum testosterone ≥150 ng/dL if primary tumor 
therapy is radiation therapy.. A sample size of N=25 evaluable patients has an 80% power 
to test the null hypothesis response rate of 5.5% against a two sided alternative response 
rate of 20% at a significance level (alpha) of 0.1. Assuming 10% of patients may not 
recover testosterone to ≥150 ng/dL or drop out, we anticipate a total study N=28. 
 
List of Abbreviations 
 
AD = androgen dependent 
ADT = androgen deprivation therapy 
AE = adverse event 
ALT = alanine aminotransferase 
AR = androgen receptor 
AST = aspartate aminotransferase 
CBC= complete blood count 
CFR= Code of Federal Regulations 
CRF = case report form 
CRPC = castration resistant prostate cancer (mCRPC, m denotes metastatic) 
CT = computed-tomography 
CTCAE = Common Terminology Criteria for Adverse Events 
ctDNA = circulating tumor DNA 
DEXA = dual energy x-ray absorptiometry 
DiPSC = Differential Pathway Signature Analysis 
DMC = Data Monitoring Committee 
ECOG = Eastern Cooperative Oncology Group 
EKG = electrocardiogram 
EMT = epithelial to mesenchymal transition 
ERK = extracellular signal related kinase (p-ERK = phosphorylated form of ERK) 
FDA = Food and Drug Administration 
FFPE = formalin fixed, paraffin-embedded 
GnRH = gonadotropin-releasing hormone 
IBC= Institutional biosafety committee 
IHC = immunohistochemistry 
IND = investigational new drug 
INR= international normalized ratio 
IRB = institutional review board 
ISPRC = the Internal Scientific Peer Review Committee 
LDH = lactate dehydrogenase 
LFT= liver function test 
LHRH = luteinizing hormone releasing hormone 
LVEF = left ventricular ejection fraction 


10 
 
MAPK = mitogen activated protein kinase 
MEK = mitogen-activated extracellular signal-related kinase 
MRI = magnetic resonance imaging 
NCI = National Cancer Institute 
NIH= National Institute of Health 
NYHA = New York Heart Association 
OCT = optimal cutting temperature freezing compound 
ORC = Office of Regulatory Compliance 
ORSP = Office of Research Subject Protection 
PARADOGM = Pathway Recognition using Data Integration on Genomic Models 
PNBx = prostate needle biopsy 
PSA = prostate specific antigen 
PI= principal investigator 
PI3K = phosphatidylinositol 3-kinase 
PT= prothrombin time 
PTEN = phosphatase and tensin homolog 
RP = radical prostatectomy 
RAS = proto-oncoprotein, guanosine-nucleotide-binding protein/GTPase 
RECIST = Response Evaluation Criteria in Solid Tumors 
SAE = serious adverse event. 
SPORE = Specialized Program of Research Excellence (in prostate cancer) 
SQ = subcutaneous 
SRC = proto-oncoprotein (p-SRC = phosphorylated form of SRC) 
SU2C = Stand Up To Cancer 
TUNEL = assay of apoptosis 
UCLA= University of California, Los Angeles 
WCDT = West Coast Dream Team (SU2C WCDT) 
 
 
 
 
1. STUDY OBJECTIVES 
1.1 Primary Objective 
1.1  
The primary objective of this study is to assess the efficacy of combined 
systemic and tumor directed therapy for newly diagnosed (de novo) 
oligometastatic M1a,b prostate cancer patients with 1-5 metastases 
(exclusive of pelvic nodal N1 metastases).  The primary endpoint of our 
study is the percent of patients achieving a serum PSA of <0.05 ng/mL six 
months after recovery of serum testosterone ≥150 ng/dL if primary tumor 
therapy is radical prostatectomy or PSA of < nadir + 2 ng/mL six months 
after recovery of serum testosterone ≥150 ng/dL if primary tumor therapy 
is radiation therapy. (i.e., undetectable disease burden off-therapy and 
after gonadal recovery). 
1.2 Secondary Objectives 
1.3.1 
Time to biochemical progression. 
1.3.2 
Time to additional antineoplastic therapy  
1.3.3 
Prostate cancer specific survival  


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1.3.4 
Safety and tolerability 
1.3.5 
Assessment of health related quality of life using the Functional 
Assessment of Cancer Therapy - Prostate (FACT-P) scale. 
1.3 Correlative Objectives 
Correlative Aim 1 
1.1 Identification of metastasis-generating intraprostatic tumors. 
1.2 Determination of differences between metastasis-generating intraprostatic 
tumors and other intraprostatic tumors. 
 
Correlative Aim 2 
2.1 Determination of differences between metastatic and metastasis- 
        generating primary tumors. 
2.2 Determination of ERK activation and markers of EMT between metastatic  
        and primary tumors 
 
Correlative Aim 3.  
2.1 
To evaluate biomarkers of response using circulating tumor cells (CTCs). 
2.2 
To evaluate biomarkers of response using circulating tumor DNA (ctDNA). 
2.3 
To evaluate immunophenotypes of circulating immune cells. 
 
2. BACKGROUND AND RATIONALE 
2.1 Background on Condition, Disease, or Other Primary Study Focus 
Metastatic prostate cancer is incurable and treatment of metastatic prostate cancer is 
palliative. The current standard of care is ADT (androgen deprivation therapy) (10); 
however, relapse on ADT is inevitable. Modern cohorts of patients treated with ADT alone 
have a median time to failure and overall survival of 11 and 42 months, respectively (11). 
Patients are maintained on ADT continuously or intermittently, until death. 
2.2 Study Rationale 
New therapeutics that suppress androgen signaling include abiraterone acetate and 
enzalutamide, which interfere with androgen synthesis and androgen receptor-androgen 
binding, respectively, are approved for castrate resistant prostate cancer (5,12,13) and 
these and apalutamide are currently used also for castration sensitive metastatic prostate 
cancer and under evaluation for high-risk localized prostate cancer in combination with 
radiotherapy (RT) (4,14). Stereotactic body RT (SBRT) enables safe and accurate 
delivery of ablative doses of ionizing radiation to metastatic prostate cancer lesions with 
local control exceeding 95% (9). The addition of definitive local therapy to systemic 
therapy in high-risk localized prostate cancer improves survival (15,16). Clinical data also 
support the notion that the addition of local therapy in node positive (17–19) and M1 
patients also favorably impact survival (20). The STAMPEDE trial evaluated the role of 
prostate radiotherapy and lifelong conventional ADT in M1 patients and found improved 
survival of prostate radiotherapy for oligometastatic M1 patients (21). Osseous 
metastases account for 90% of all prostate cancer metastases and bones are the first site 


12 
 
of metastatic spread in more than 80% of patients (22–24). The identification of metastatic 
sites in prostate cancer has greatly improved through use of NaF PET-CT and PSMA 
PET-CT scans (25,26), with reported sensitivity and specificity >95% (27), superior to that 
of conventional technetium bone scans and CT scans (27,28). 
 
With this confluence of treatment and diagnostic advances, the question arises if a 
multimodal approach with aggressive, early treatment of newly diagnosed M1 prostate 
cancer should be attempted with curative intent. 
 
A striking difference in prostate cancer 
specific mortality is noted in newly 
diagnosed M1b patients with 5 or fewer 
visible metastases (exclusive of pelvic 
lymph nodes) versus those with more 
than 5 (Figure 3) (unpublished). This 
retrospective 
data 
supports 
the 
hypothesis that metastatic prostate 
cancer 
patients 
comprise 
a 
heterogeneous group with a divergent 
clinical course. To at least a first 
approximation, clinical course appears 
to be associated with the number of 
metastases. This analysis led our team 
to select patients with newly diagnosed 
metastatic prostate cancer with 5 or 
fewer visible metastases for inclusion in 
this prospective Phase II clinical trial.  
Both localized and metastatic castrate resistant prostate cancers (mCRPCs) are 
characterized by frequent copy number alterations, gene fusions, and rearrangements 
(29,30). Some characteristic differences between primary and mCRPCs have been 
identified, which include a greater frequency of androgen receptor gene amplification or 
pathway alteration (50% vs 90-100%), pten loss or PI3K/AKT activation (50% vs 90-
100%), tp53 mutations (3-20% vs 50%), and MAP Kinase (ERK) pathway activation (30% 
vs 90-100%), for primary and mCRPC, respectively (31).  Elucidation of the critical 
pathways associated with emergence of metastases from the primary prostate cancer 
could identify actionable targets for systemic therapy to be used both early in the disease 
course and in patients with treatment refractory mCRPC. 
Understandably, prior studies assessing metastatic prostate cancer analyzed castrate 
resistant tumors taken from heavily pre-treated patients on long term ADT (30,32).  As 
such, in these studies the initial genomic events driving metastasis may be obscured 
through subsequent genomic and transcriptomic adaptations occurring within tumors 
during systemic therapy. Therefore, our study, which incorporates genomic analysis of 
both primary and metastatic sites pre-treatment, is poised to address the specific question 
of which gene expression pathways, mutations, and copy number alterations are 
associated with early metastases, de novo, independent of therapeutic pressure.  
 
➢ Hypothesis. The combination of aggressive local, metastases directed, and 
systemic therapy is an effective treatment for metastatic prostate cancer patients 
with limited metastatic disease burden.  The correlative analyses will identify key 


13 
 
features unique to intraprostatic tumors that metastasize, the proximal drivers of 
the metastatic event, and biomarkers associated with response to this multimodal 
treatment strategy. 
3. STUDY DESIGN 
This is a single arm Phase II clinical trial in patients with newly diagnosed M1a,b prostate 
cancer and 1-5 radiographically visible metastases treated with primary radical 
prostatectomy (and post-operative fractionated radiotherapy for pT≥3a, pN1, or positive 
margins) or primary radiotherapy (and pelvic lymph node radiotherapy), metastasis 
directed SBRT, and complete ADT with GnRH analog leuprolide, androgen synthesis 
inhibitor abiraterone acetate, and antiandrogen apalutamide for a total of six months of 
systemic therapy. The primary endpoint of our study is the percent of patients achieving 
a serum PSA of <0.05 ng/mL six months after recovery of serum testosterone ≥150 ng/dL 
for patients treated with primary radical prostatectomy and PSA of < nadir + 2 ng/mL six 
months after recovery of serum testosterone ≥150 ng/dL for patients treated with primary 
radiotherapy. 
 
• Type of trial: Phase II, open-label, single-arm. 
• Outcomes: 
o Primary outcome is a sustained undetectable disease burden after 
gonadal recovery: 
▪ The primary endpoint of our study is the percent of patients achieving 
a serum PSA of <0.05 ng/mL six months after recovery of serum 
testosterone ≥150 ng/dL for patients treated with primary radical 
prostatectomy and PSA of < nadir + 2 ng/mL six months after 
recovery of serum testosterone ≥150 ng/dL for patients treated with 
primary radiotherapy. [Based on prior studies, >90% of patients 
treated with ADT for six months will recover serum testosterone to 
≥150 ng/dL within six months after cessation of ADT with a median 
time to recovery of three month (1,2).] This primary endpoint was 
selected to offer a rapid and sensitive assessment of treatment 
efficacy. PSA is a biomarker for disease burden in prostate 
adenocarcinoma 
and 
offers 
a 
non-invasive 
and 
sensitive 
assessment of disease control after treatment in the vast majority of 
patients.  Durable PSA control in the context of recovery of gonadal 
function (testosterone ≥150 ng/dL) after ADT is therefore a surrogate 
for disease control. It is expected that determination of the primary 
endpoint for each patient will occur between 9 and 12 months after 
completion of systemic therapy. 
o Secondary outcomes are: 
▪ Time to biochemical progression 
▪ Time to radiographic progression 
▪ Time to initiation of alternative antineoplastic therapy 
▪ Prostate cancer specific survival  
▪ Assessment of health related quality of life using the Functional 
Assessment of Cancer Therapy - Prostate (FACT-P) scale 
▪ Safety and tolerability 
o Correlative objectives: 
▪ 
Correlative Aim 1 


14 
 
1.1 Identification of the metastasis-generating intraprotatic tumor. 
1.2 Determination of differences between metastasis-generating 
intraprotatic tumors and other intraprostatic tumors 
▪ 
Correlative Aim 2 
2.1 Determination of differences between metastatic and 
metastasis-generating primary tumors. 
2.2 Determination of ERK activation and markers of EMT 
between metastatic and primary tumors 
▪ 
Correlative Aim 3 
3.1 Analysis of ctDNA for predictors of response  
3.2 Analysis of CTC’s for predictors of response. 
3.3 Analysis of changes in circulating immunophenotypes 
 
• Study population: Patients with metastatic prostate cancer with 1-5 sites of 
metastases (exclusive of pelvic nodal N1 metastases) that have not undergone 
prior treatment. Additionally, the patient must have at least 1 metastatic site that 
has been biopsied (unless deemed unsafe to perform, not feasible, or declined by 
patient) prior to enrollment on the study. If biopsy is not diagnostic, or unsafe to 
perform, then a secondary imaging modality (for example, MRI) must also be 
consistent with metastatic disease (unless PSMA PET-CT or Fluciclovine (Axumin) 
PET-CT) was used as initial staging). 
 
• Number of patients: Currently, metastatic prostate cancer patients are treated with 
ADT indefinitely. Based on data from prior trials of discontinuous ADT in prostate 
cancer patients with metastatic disease, essentially all patients will relapse with a 
rising PSA after discontinuation of ADT (33,34). The current study hypothesis is 
that 20% of patients will achieve a PSA <0.05 ng/mL six months after recovery of 
testosterone to >150 ng/dL. N=25 evaluable patients has 80% power to detect a 
20% response rate at a significance (alpha) of 0.1. Assuming 10% of patients may 
not recover testosterone to ≥150 ng/dL or drop out, we anticipate a total study 
N=28. 
 
• Study Sites:  
o West LA VA Medical Center (Los Angeles, California, United States of 
America) 
o Long Beach VA Medical Center (Long Beach, California, United States of 
America) 
o Hunter Holmes McGuire VA Medical Center (Richmond, Virginia, United 
States of America) 
o  Other VA sites may be added 
 
• Duration of study: 
o Entire study: ~48 months. 
o Individual patient enrollment: ~15-18 months (screening period, active 
treatment, follow-up to primary endpoint). 
 
• Interventions: 
o Pre-treatment biopsy of a metastatic lesion during the screening period 
unless not feasible, unsafe, or declined by patient. Biopsy site will be 
determined by safety.  This biopsy, if done, is used both for diagnostic 


15 
 
confirmation of metastatic disease as well as correlative analyses. 
o Biopsy of a metastatic lesion at progression. This biopsy is not required.  
However, it is encouraged. This biopsy is a research biopsy that will provide 
tissue for correlative analyses.  
o Study treatments summary (detailed in Section 5.1) 
 
• Tumor directed Therapy. 
 
o Primary Tumor therapy. Patients may undergo either 
surgery or radiotherapy as primary tumor therapy. The 
decision is via shared decision making between the 
investigators and the patient. Either are acceptable. 
 
o Surgery. Patients who  undergo surgery as primary 
tumor treatment will undergo radical prostatectomy 
(open or robotic assisted) with pelvic node dissection 
by an experienced urologic surgeon. 
▪ Post-operative pelvic RT. Patients found after 
surgery to be stage pT3a or higher, have 
positive margins, or pathologically involved 
pelvic 
nodes, 
will 
receive 
post-operative 
fractionated IMRT delivered to the pelvic nodes 
and prostate bed. This pelvic RT may extend 
after completion of ADT. 
 
 
o Primary tumor radiotherapy. Patients who undergo 
radiotherapy as primary tumor treatment will undergo 
radiotherapy to the prostate, seminal vesicles, and 
pelvic lymph nodes. Allowed dose fractionations 
include: 
▪ Prostate: 40 Gy in 5 fractions or 60 Gy in 20 
fractions or 79.2 Gy in 44 fractions 
▪ SVs:  25-40 Gy in 5 fractions, or 42-43 Gy in 20 
fractions, or 45 Gy in 25 fractions. 
▪ Pelvic LNs: 25 Gy in 5 fractions or 42-43 Gy in 
20 fractions or 45 Gy in 25 fractions. 
 
The selection of dose and fractionation will be 
made at the discretion of the investigator, and 
typically depends on anatomical considerations 
and baseline urinary and bowel function. Gross 
disease may be prescribed a higher dose at the 
investigator’s discretion. 
 
o Metastasis Directed Therapy. 
 
SBRT. 
Patients 
will 
undergo 
SBRT 
to 
all 
radiographically visible sites of M1 metastases within 
two months of initiation of ADT. SBRT offers ablative 


16 
 
dose-escalation to tumor targets with simultaneous 
dose-restraint to normal tissues, which is not possible 
using conventionally radiotherapy. SBRT utilizes high-
precision radiotherapy delivery machines, integrated 
image guidance systems, advanced planning software, 
and a high level of professional and technical 
expertise.  
 
 
▪ Systemic Therapy.  Complete ADT with GnRH analog leuprolide (6 
month depot, 45 mg, once), antiandrogen apalutamide (240 mg PO 
daily), and abiraterone acetate co-administered with prednisone 
(1,000 mg and 10 mg PO daily), will begin immediately after surgery 
for patients who undergo radical prostatectomy for primary tumor 
therapy and at day 1(+/-3 days) for patients who undergo 
radiotherapy as primary tumor therapy, and continue for a total of 6 
months. 
 
o Pre-treatment assessment of eligibility: 
▪ Staging. To ensure sensitive and 
accurate staging, patients must 
undergo NaF PET-CT or PSMA 
PET-CT or Fluciclovine (Axumin) 
PET-CT (with fused diagnostic 
CT of the chest abdomen and 
pelvis), 
and 
have 
1-5 
radiographically 
visible 
metastases 
(pelvic 
nodal 
metastases are not included). At 
least one site with radiographic 
suspicion for metastases will be 
biopsied for both diagnosis and 
for correlative genomic analyses, 
unless deemed to be unsafe to 
perform, or not feasible, or 
declined by patient, 
then a 
secondary imaging modality (for 
example, MRI) must also be 
consistent 
with 
metastatic 
disease (unless PET-CT was or 
Fluciclovine (Axumin) PET-CT used as initial staging). Patients must 
not have visceral metastases, but may have one or more pelvic 
lymph nodes involved, up to a maximum diameter of 2 cm. Metastatic 
sites (exclusive of N1 pelvic nodes) must be amenable to treatment 
via SBRT. 
 
The reported sensitivity and specificity of NaF PET-CT for detection 
of osseous metastases in prostate cancer patients both exceed 
>95% when interpreted by an experienced physician. (The specificity 


17 
 
of this technique derives from thoughtful correlation of PET tracer 
intensity with underlying sclerotic foci on the fused high resolution CT 
scan). In contrast, the sensitivity and specificity of technetium 99 
bone scans in this population are 70%, 57%, respectively. The value 
of using NaF PET-CT for staging in this trial is illustrated in Figure 4, 
where a patient with newly diagnosed metastatic prostate cancer 
underwent both technetium 99 and NaF PET-CT scans prior to 
treatment. The technetium 99 scan revealed a single metastasis to 
the left seventh rib, while the NaF PET CT revealed an additional 
ninth rib metastasis as well as a sacral metastasis. Although not 
widely available at this time in the United States, we anticipate that 
PSMA-PET CT will become available over the next few years. 
Therefore, we also allow for staging by PSMA PET-CT in this trial. In 
addition, fluciclovine (Axumin) PET/CT was recently FDA approved 
for prostate cancer. As such, we also allow staging by fluciclovine 
(Axumin) PET/CT. 
 
▪ Blood tests to assess for adequate organ function - liver, kidney, 
bone marrow, coagulation 
 
▪ Assessment of the PSA and testosterone, total testosterone must be 
>200 ng/dL prior to ADT. (The optimal time to measure total 
testosterone is between 8 and 9 am) 
 
▪ Adequate performance status and labs 
 
• Randomization: This is a non-randomized study. 
 
• Other: All molecular and pathologic analyses germane to the study objectives will 
be performed under direction of the investigators. This process leverages the 
established infrastructure enabled by existing collaborative programs.  
4. SELECTION AND ENROLLMENT OF PARTICIPANTS 
Patient Recruitment 
Patients with metastatic prostate cancer that:  
i. have not received prior therapy for prostate cancer, 
ii. have at least 1 metastatic lesion that has been biopsied (unless deemed to be unsafe, 
not feasible, or declined by patient) will be considered for this trial.   
 
Patients will be recruited from the urology, medical oncology, and radiation oncology 
clinics at the participating centers. This clinical trial is listed on the clinicaltrials.gov 
website. 
4.1 Inclusion Criteria 
Patients must meet all of the following inclusion criteria to participate in this study. 
1. Biopsy confirmed diagnosis of prostate adenocarcinoma (primary small cell 
carcinoma of the prostate is not allowed, however adenocarcinoma with 
neuroendocrine differentiation is allowed) 


18 
 
2. Age ≥18 
3. Presence of 1-5 visible metastases (by NaF PET-CT or PSMA PET-CT or 
Fluciclovine [Axumin] PET-CT including diagnostic CT of the chest, abdomen, 
and pelvis) 
a. At least one metastasis must be M1a-b 
b. Visceral metastases are not allowed 
c. Patients may have any number of pelvic nodal metastases (but largest must 
be <2 cm in shortest diameter) 
d. Metastases must be amenable to treatment with SBRT 
e. Biopsy of one metastasis must be attempted, unless unsafe or not feasible 
to perform, or declined by patient. If biopsy is not diagnostic, or unsafe to 
perform, or declined by patient, then a secondary imaging modality (for 
example, MRI) must also be consistent with metastatic disease (unless 
PSMA PET-CT or Fluciclovine [Axumin] PET-CT was used as initial 
staging). 
4. Patient must be fit to undergo radical prostatectomy or prostate radiotherapy, 
SBRT to all visible sites of metastases, ADT, 
5. Total testosterone >200 ng/dL prior to ADT (optimal time to measure total 
testosterone is between 8 and 9 am) 
6. Adequate performance status (ECOG 0-1) 
7. Clinical laboratory values at screening:  
a. Hemoglobin ≥9.0 g/dL, independent of transfusion and/or growth factors 
within 3 months prior to randomization 
b. Platelet count ≥100,000 x 109/µL independent of transfusion and/or growth 
factors within 3 months prior to randomization 
c. Serum albumin ≥3.0 g/dL 
d. GFR ≥45 mL/min  
e. Serum potassium ≥3.5 mmol/L 
f. Serum total bilirubin ≤1.5 × ULN (Note: In subjects with Gilbert’s syndrome, 
if total bilirubin is >1.5 × ULN, measure direct and indirect bilirubin and if 
direct bilirubin is ≤1.5 × ULN, subject may be eligible) 
g. Aspartate aminotransferase (AST) or alanine aminotransferase (ALT) <2.5 
× ULN 


19 
 
8. Medications known to lower the seizure threshold (see list under prohibited     
medications) must be discontinued or substituted at least 4 weeks prior to study 
entry. 
4.2 Exclusion Criteria 
All candidates meeting any of the following exclusion criteria will be excluded from 
participation in the study. 
1. Any evidence of spinal cord compression (radiological or clinical) 
2. Prior pelvic malignancy 
3. Prior pelvic radiation 
4. Concurrent malignancy aside from superficial skin cancers or superficial 
bladder tumors 
5. Inability to undergo prostatectomy, radiotherapy, or ADT 
6. Primary small cell carcinoma of the prostate (prostate adenocarcinoma with 
neuroendocrine differentiation is allowed) 
7. Inflammatory bowel disease or active collagen vascular disease 
8. History of any of the following: 
a. Seizure or known condition that may pre-dispose to seizure (e.g. prior 
stroke within 1year to randomization, brain arteriovenous malformation, 
Schwannoma, meningioma, or other benign CNS or meningeal disease 
which may require treatment with surgery or radiation therapy) 
b. Severe or unstable angina, myocardial infarction, symptomatic congestive 
heart failure, arterial or venous thromboembolic events (eg, pulmonary 
embolism, cerebrovascular accident including transient ischemic attacks), 
or clinically significant ventricular arrhythmias within 6 months prior to 
randomization  
9. Current evidence of any of the following: 
a. Uncontrolled hypertension 
b. Gastrointestinal disorder affecting absorption 
c. Active infection (eg, human immunodeficiency virus [HIV] or viral hepatitis) 
d. Any chronic medical condition requiring a higher dose of corticosteroid 
than 10 mg prednisone/prednisolone once daily  
e. Any condition that in the opinion of the investigator would preclude 
participation in this study 
f. Concomitant strong CYP3A4 inducers. (If a strong CYP3A4 inducer must 
be co-administered, abiraterone acetate dose frequency will be adjusted).  


20 
 
g. Treatment with CYP2D6 substrates that have a narrow therapeutic index. 
If an alternative treatment cannot be used, a dose reduction of the 
CYP2D6 substrate may be considered. 
h. Baseline severe hepatic impairment (ChildPugh Class B & C) 
i. Concomitant CYP2C8 inhibitors with narrow therapeutic index. (If a 
concomitant CYP2C8 inhibitor with narrow therapeutic index must be co-
administered patients should be monitored closely for signs of toxicity 
related to the CYP2C8 inhibitor with a narrow therapeutic index if used 
concomitantly with abiraterone acetate) 
10. Presence of visceral metastases (i.e., stage M1c) 
4.3 Study Enrollment Procedures 
The PI and co-PIs will ensure that this study is conducted in full compliance with the FDA 
standards for human research as specified in 21 CFR 312. The study will not be initiated 
at a clinical site until the informed consent form has been approved by the clinical site’s 
Institutional Review Board (IRB). All potential revisions of the protocol must be reflected 
in the informed consent form and reviewed by the IRB & Janssen. Separate informed 
consent will be obtained for all surgical procedures and biopsies that may be performed 
as part of the patient’s standard medical management.  
 
Patients will be provided complete informed consent that will be sought in the participating 
clinics by the Study PI’s, one of the designated sub-investigators or by appropriately 
designated investigators at other participating sites. Subjects will have the option of 
having family and/or anyone else they deem appropriate present during the informed 
consent process. The PI and/or co-investigators will discuss the study with patients 
verbally. The patients will be given the informed consent forms and encouraged to take 
the forms home for careful reading and review at each patient’s leisure. After reading the 
informed consent form, the patients will be encouraged to return to the clinic to meet with 
the study investigators to ask any and all questions that the patients may have. The 
investigators will in turn pose questions that verify that the patients understand the nature 
and risks of the study.  
 
Subjects will be informed that they may refuse participation in or withdraw from this study 
at any time without prejudice or any negative effect on subsequent care. In addition, 
subjects will be informed that their health care provider may be one of the investigators 
on this study and a conflict of interest may therefore exist in that the investigator may be 
interested in both the patient’s welfare and in the conduct of the study.  
 
The patient should agree to use a condom (even men with vasectomies) and another 
effective method of birth control if he is having sex with a woman of childbearing 
potential or agrees to use a condom if he is having sex with a woman who is pregnant 
while on study drug and for 3 months following the last dose of study drugs. Must also 
agree not to donate sperm during the study and for 3 months after receiving the last 
dose of study drug. 


21 
 
5. STUDY INTERVENTIONS 
5.1 Interventions, Administration, and Duration 
 
5.1.1. Study Drugs. 
 
Study groups, medications, and dosing: 
 
This is a single-arm study.  All enrolled patients will initiate leuprolide, apalutamide 
and abiraterone acetate beginning immediately (within 0-3 days) after radical 
prostatectomy. Patients will receive a single dose of leuprolide, SQ, 45 mg, a six 
month depot, and start at the recommended dose of apalutamide (ARN-509, 240 
mg PO daily), and abiraterone acetate co-administered with prednisone (1,000 
mg PO and 10 mg PO daily, respectively), by mouth daily, for a duration of six 
months.  Based on the experience from clinical trials with apalutamide and 
abiraterone acetate in prostate cancer these are thought the optimal doses and 
are well tolerated when given on a continuous daily basis.  These drugs will be 
continued for six months. Based on the experience from clinical trials in metastatic 
prostate cancer this is thought the optimal doses and is tolerated with acceptable 
toxicity.   
• 
Leuprolide. Leuprolide (or another GnRH analog) is the standard of care 
for patients with M1 prostate cancer, and expected side effects for a six-
month course include loss of libido, fatigue, and hot flashes. Leuprolide 
has been used in multiple previous and ongoing Phase III trials in 
prostate cancer patients. 
 
• 
Apalutamide. Apalutamide is FDA approved for the treatment of non-
metastatic castration resistant prostate cancer and also metastatic 
castration sensitive prostate cancer. It is thus part of standard of care for 
these patients. It has been evaluated in several previous and ongoing 
trials for local and metastatic prostate cancer and is tolerated in 
combination with ADT, radiotherapy, and surgery. It can be associated 
with fatigue and rare convulsions. 
 
• 
Abiraterone acetate is also part of standard of care for these patients 
and is also approved for mCRPC in combination with prednisone, and is 
well tolerated in combination with ADT, radiotherapy, and surgery. It can 
be 
associated 
with 
mineralocorticoid 
excess, 
hepatotoxicity, 
osteoporosis, and has some drug-drug interactions through inhibition of 
CYP2D6. After discontinuation of abiraterone, prednisone will be 
continued, but tapered. A recommended prednisone taper schedule is to 
decrease the dose by 1 mg/day every two weeks (see Appendix 14.4). 
 
A Material Safety Data Sheet (MSDS) describing the occupational hazards and 
recommended handling precautions for the study drugs (available from Janssen 
for apalutamide and abiraterone acetate) will be provided to site staff if required 
by local laws or will otherwise be available upon request. 
 
Toxicity will be attributed to specific study drugs based on judgment of the treating 


22 
 
physician, or study PI or co-investigators. Toxicity will be judged as likely related, 
possibly related, or unlikely related to the study drug. If likely or possibly related, 
then dose modifications to the study drug in question will be made. In the situation 
where an unexpected toxicity is observed but not clearly attributable to a study 
drug or procedure, then attempts will be made to identify the source of toxicity 
along with treatment and/or palliation of the toxicity. In this situation, dose 
modifications may be made at the treating physician, study PI or co-investigators’ 
discretion. 
 
Apalutamide 
• 
Acquisition: Supplied by Janssen. Will be sent to the sponsor site (West 
LA clinical research pharmacy) and West LA VA will distribute the drug to 
additional sites. The contents of the label will be in accordance with all 
applicable regulatory requirements.   
 
• 
No special preparation of apalutamide is required. Under normal 
conditions of handling and administration, apalutamide is not expected to 
pose significant safety risks to site staff.   
 
• 
Dosing: 240 mg by mouth daily. Apalutamide will be supplied as 60 mg 
tablets to be administered with or without food.  If a subject misses a dose, 
subject may take the dose if the next scheduled dose is at least 12 hours 
later.  If the next scheduled dose is due in less than 12 hours, subject 
should skip the dose and resume dosing the next day at the regular time. 
 
• 
Patients will be supplied with a drug diary.  They will be asked to annotate 
daily when they take the medication and to regard all other medications, 
supplements, they are taking – dose and frequency. 
 
• 
Thyroid stimulating hormone (TSH) will be evaluated throughout the study 
(with T3 and T4 done only if TSH is abnormal) as follows: screening, day 
1, and monthly during treatment. 
• 
Dose modifications for apalutamide are described below. 
• 
Safety Monitoring Thyroid stimulating hormone (TSH) is evaluated 
throughout the study (with T3 and T4 done only if TSH is abnormal) as 
follows: Screening, and every 30 days for the duration of treatment. 
Apalutamide Investigational Product Details 
 
 
Investigational Product 
Product name: 
Apalutamide (formerly ARN-509) 
Dosage form: 
Tablet 
Unit dose strength(s): 
60 mg 
Dosage level(s): 
240 mg 
Route/Administration: 
Oral, continuous once daily dosing  
Dosing instructions: 
Apalutamide may be taken with or without food.  


23 
 
Manufacturer/source of 
procurement: 
Janssen 
 
 
Risks of Apalutamide include: 
Very common (≥ 10% of people experience these)  
• Fatigue  
• Skin rash  
• Joint pain and muscle spasms (Arthralgia) 
• Weight loss 
• Fall  
• Fracture  
• Increased blood pressure (hypertension)  
• Hot flush  
• Diarrhea 
• Decreased appetite**  
 
Common (≥1% -<10% of people experience these):  
• Itching 
• Changes in thyroid function (hypothyroidism)   
• Increases in cholesterol and blood triglycerides 
• Change in experience of taste (dysgeusia) 
• Reduced or blocked blood flow to the heart, including heart attack (ischemic heart 
disease, including myocardial infarction).  
• Reduced or blocked blood flow to the brain, including stroke (ischemic 
cerebrovascular disorders) 
• Alopecia (hair loss) 
 
Uncommon (≥ 0.1% - <1% of people experience these):  
• Seizures 
• Inflammation with the lungs that may lead to permanent damage (Interstitial lung 
disease)* 
 
Rare (≥0.01% - <0.1%) 
 
***Life-threatening rash with blisters and peeling over much of the body (Stevens-
Johnson syndrome/Toxic epidermal necrolysis) 
 
 
***Skin rash with fever, and blood cell abnormalities including increase in white 
blood cells (lymphocytes and eosinophils), a decrease in platelets and potential life 
threatening inflammation of internal organs (Drug Reaction with Eosinophilia and 
Systemic Symptoms (DRESS) 
*This is information provided voluntarily by doctors using apalutamide in routine 
clinical practice where frequency of this event can be estimated from clinical trials 
and is assessed as uncommon (0.1%) 


24 
 
** This information provided voluntarily by doctors using apalutamide in routine 
clinical practice where frequency cannot be estimated; however, frequency of this 
event can be estimated form clinical trials and is assessed as very common. 
***this information provided voluntarily by doctors using apalutamide in routine 
clinical practice where frequency cannot be estimated; however, frequency of this 
event can be estimated from clinical trials and is assessed as rare. 
 
Other information on apalutamide: 
 History of seizures: Seizures have been observed very rarely in subjects 
taking part in apalutamide studies.  Subjects will be confirmed that they have no 
history of seizures and will be checked throughout the study that they are not 
taking other medications that can increase their risk of seizures. 
  Development of rash: More than 1 in 10 patients have developed a rash. Some 
rashes may need medical attention.  The rash may be confined to one area of the 
body or may spread across the body.  Rashes can be painful, blister on or near the 
lips, eyes or genital and peel on areas of skin surface.  Subject’s may be given 
medicines to apply to the skin or take by mouth to help the signs and symptoms of 
rash.  Also the study medication may be temporarily held. In most cases, rashes 
can be treated with topical or oral medications so that subjects can continue 
apalutamide treatment. 
 Difficulty breathing: Scarrring of the inner lining of the lung (Interstitial lung 
disease) has been observed in patients taking apalutamide.  Subject’s will be 
checked for history of lung problems and monitored throughout study for symptoms 
of shortness of breath, breathing difficulty, cough or fever. 
         SAEs to apalutamide are reported to Janssen and Janssen determines respec           
attribution to SAEs. 
 
        Dose reductions or discontinuation of apalutamide will be made due to toxicities 
that are attributed to apalutamide, or not clearly attributable to another drug in this 
study (e.g., abiraterone acetate, leuprolide). 
 
      Dose modifications for toxicity attributed to apalutamide given concurrently with 
abiraterone acetate will be guided by the Investigator Brochure provided by the 
drug manufacturer, as detailed below: 
 
Toxicity* 
Dose of abiraterone 
acetate 
Dose of apalutamide 
Dose of prednisone 
Grade 1 or 2 
No change 
No change 
No change 


25 
 
≥Grade 3 or higher 
 
No change 
Hold until Grade 1 or baseline, resume 
at 180 mg (3 tablets). ** 
 
No change 
First Recurrence 
≥Grade 3 
 
No change 
Hold until Grade 1 or baseline, resume 
at 120 mg (2 tablets). ** 
 
No change 
Second Recurrence 
≥Grade 3 
 
No change 
 
Permanently Discontinue  
 
No change 
First occurrence of 
seizure of any grade 
or any Grade 4  
toxicity of any 
duration*** 
neurotoxicity 
No change 
Permanently Discontinue 
No change 
 
 
 
 
* 
Toxicity that is attributed to apalutamide or not clearly attributed to another drug 
on this study. 
 
** 
Patients who require a treatment interruption > 2 weeks should discontinue 
apalutamide if it is believed to be responsible for the treatment interruption. 
 
*** 
Individual exclusions to this criteria may be allowable for grade 4 emesis or 
clinically insignificant laboratory abnormalities that resolve within two days of optimum 
treatment at the investigators’ discretion. 
 
Rash 
Dose modifications for rash are allowed only for apalutamide and are summarized in 
below table. 
If the skin rash has any component of desquamation, mucosal involvement, or pustules, 
stop dosing with apalutamide, refer to dermatologist for evaluation, and a skin biopsy is 
recommended (in addition to the interventions listed in below Table) If the skin rash is 
Grade 3 or higher, asking the subject to consent to documentation by a photograph and 
further evaluation by a dermatologist should also be considered. 
 


26 
 
5.1.1.1 
Severity 
5.1.1.2 
Intervention 
5.1.1.3 
Grade 1 
• 
Continue apalutamide at current dose 
• 
Initiate dermatological treatmenta 
o Topical steroid cream AND 
o Oral Antihistamines 
• 
Monitor for change in severitya 
5.1.1.4 
Grade 
2 
(or 
symptomatic 
Grade 1)b 
• 
Hold apalutamide for up to 28 days 
• 
Initiate dermatological treatmenta 
o Topical steroid cream AND 
o Oral Antihistamines 
• 
Monitor for change in severitya 
o If rash or related symptoms improve, reinitiate 
apalutamide when rash is Grade≤1. Consider 
dose reduction at a 1 dose level reductionc. 
5.1.1.5 
Grade ≥3d 
• 
Hold apalutamide for up to 28 days 
• 
Initiate dermatological treatmenta 
o Topical steroid cream AND 
o Oral Antihistamines AND 
o Consider short course of oral steroids 
• 
Reassess after 2 weeks (by site staff), and if the rash is 
the same or has worsened, initiate oral steroids (if not 
already done) and refer the subject to a dermatologist 
o Reinitiate apalutamide at a 1 dose level 
reductione when rash is Grade≤1. 
o If the dose reduction will lead to a dose less than 
120mg, the study drug must be stopped 
(discontinued) 
• 
If after 28 days, rash has not resolved to Grade≤1, 
contact PI to discuss further management and possible 
discontinuation of study drug.  
 
Note: Rash may be graded differently according to the type of rash and associated symptoms. 
For example, maculo-papular rash is graded by body surface area covered and not severity of 
the rash. Please consult NCI-CTCAE Version 4.03 for specific grading criteria for other types of 
rash. 
 


27 
 
a Obtain bacterial/viral cultures if infection is suspected 
b Subject presents with other rash related symptoms such as pruritus, stinging, or 
burning 
c 1 dose level reduction = 60mg (1 apalutamide tablet) 
d If there is blistering or mucosal involvement, stop apalutamide dosing immediately and 
contact PI 
e If a subject previously started oral corticosteroids, continue for at least 1 week after 
resumption of reduced dose of apalutamide. If the proposed total oral steroid use will 
exceed 28 days, contact Janssen. 
 
Abiraterone Acetate 
• 
Acquisition: Supplied by Janssen. Will be sent to the sponsor site (West LA 
VA clinical research pharmacy) and West LA VA will distribute the drug to 
additional sites. The contents of the label will be in accordance with all 
applicable regulatory requirements.   
 
• 
Based on its mechanism of action, abiraterone acetate may harm a 
developing fetus. Therefore, women who are pregnant or women who may 
be pregnant should not handle abiraterone acetate without protection e.g., 
gloves.   
 
Dosing: 1000 mg by mouth daily. Abiraterone acetate will be supplied as 250 
mg tablets to be taken without food.   Abiraterone acetate must be taken on 
an empty stomach. No food should be consumed for at least two hours 
before the dose is taken and for at least one hour after the dose of 
abiraterone acetate is taken. Do not crush or chew the tablets.  If a subject 
misses a dose, subject may take the dose if the next scheduled dose is at 
least 12 hours later.  If the next scheduled dose is due in less than 12 hours, 
subject should skip the dose and resume dosing the next day at the regular 
time. Abiraterone acetate is administered with prednisone, 5 mg, PO to be 
taken twice daily. 
 
Patients will be supplied with a drug diary.  They will be asked to annotate 
daily when they take the medication and to regard all other medications, 
supplements, they are taking – dose and frequency. 
 
LFTs will be assessed every 2 weeks for the first three months, and then at 
each subsequent blood draw (monthly) thereafter until abiraterone acetate 
and prednisone are discontinued.  
 
LFTs are also assessed monthly after discontinuation of ADT (leuprolide, 
abiraterone acetate, and apalutamide) for the first six months, and then every 
three months thereafter. 
 
• 
Dose modifications for abiraterone acetate in combination with apalutamide 
are described below.  
 
 
 
 


28 
 
Abiraterone acetate Investigational Product Details 
 
Investigational Product 
Product name: 
Abiraterone acetate 
Dosage form: 
Tablet 
Unit dose strength(s): 
250 mg 
Dosage level(s): 
1000 mg 
Route/Administration: 
Oral, continuous once daily dosing  
Dosing instructions: 
Abiraterone acetate should be taken without food.  
Manufacturer/source of 
procurement: 
Janssen 
 
 
Risks of abiraterone acetate include: 
Frequent (≥ 20%) [May occur in 20 or more patients in 100]  
• hypokalaemia (low blood potassium, a mineral that helps regulate 
heart rate/function, fluid balance in the body and is needed for 
adequate body function) 
• hypertension (high blood pressure) 
Very Common (10% to 19%) [May occur between 10 and 19 patients in 100]  
• edema peripheral (swelling of the legs as a result of the body 
keeping too much fluid) 
Common (5% to 9%) [May occur between 5 to 9 patients in 100] 
• dyspepsia (uncomfortable feeling in upper belly, indigestion)  
• hematuria (presence of blood in the urine) 
• fractures (a break in the bone) 
• alanine aminotransferase increased and/or aspartate 
aminotransferase increased (enzymes in the blood that measure 
the function of the liver). It is common to see increases in liver 
enzymes but this does not mean the liver is not functioning 
normally. Monthly blood tests are done to monitor acceptable 
levles.  
• urinary tract infection 
Less Common (< 5% [May occur in fewer than 5 patients in 100] 
• hypertriglyceridemia (high levels of fats (triglycerides) in the blood)  
• angina pectoris (chest pain) 
• atrial fibrillation (a fast and irregular heartbeat) 
• tachycardia (rapid heartbeats) 
Uncommon (< 1%) [May occur between 1 and 9 patients in 1000]  
• adrenal insufficiency (decreased function of adrenal glands that 
normally help maintain blood pressure, balance minerals and fluid 
in your body)  
• Cardiac failure (heart failure, the heart is unable to supply enough 
blood flow to meet the body’s needs) 


29 
 
• Arrhythmia (changes in the rhythm of the heart) 
• Abnormal ECG with QT prolongation (an abnormal finding on the 
ECG) 
• Bone density decreased (loss of strength of bones) 
• Myopathy (muscle weakness and /or muscle pain) 
Unknown (frequency isn’t determined since data was derived from post-marketing 
experience and there was no report from clinical studies)   
• failure of the liver to function (called acute liver failure) 
• allergic alveolitis (swelling and irritation of the lung)  
• Rhabdomyolysis (breakdown of muscle tissue) 
• Torsades de Pointes (rapid or irregular heart rate which can cause 
sudden cardiac death) 
There is a small chance of severe allergic reaction to the drug which may be life-
threatening. 
Other information on abiraterone acetate: 
• Abiraterone acetate may cause harm to the liver. About 13% of 
patients taking abiraterone acetate have had abnormal blood levels of 
liver enzymes.   Rarely, liver failure can occur. Subject’s liver function 
will be monitored closely by blood tests every two weeks for the first 3 
months of the study and monthly thereafter.  If elevations in the liver 
function tests show abnormalities, the dose of the study medication will 
be adjusted or discontinued. 
• Abiraterone acetate should be used with caution in patients with a 
history of heart disease. Before treatment with abiraterone acetate, 
high blood pressure must be controlled and low potassium must be 
monitored and corrected, when low.  
 
Use With Glucose-lowering Agents   Isolated cases of hypoglycemia have 
been reported when abiraterone acetate and prednisone was administered to 
patients with pre-existing diabetes receiving pioglitazone and repaglinide. 
Blood sugar should be monitored in patients with diabetes.  
SAEs to abiraterone acetate are reported to Janssen and Janssen determines 
respective attribution to SAEs. 
 
Dose modifications for LFT abnormalities attributed to abiraterone acetate given 
concurrently with apalutamide will be guided by the prescribing information 
provided by the drug manufacturer, as detailed below: 
 
Toxicity 
Dose of abiraterone acetate 
Dose of  apalutamide 
Dose of prednisone 


30 
 
Grade 1 or 2 
No change 
No 
change 
No 
change 
 
 
 
 
Grade 3 
Hold until return to baseline 
or to AST or ALT 
≤2.5 x ULN and total bilirubin 
≤1.5 x ULN, resume at 750 
mg (3 tablets) only after 
discussion and agreement 
with medical monitor 
 
 
 
 
Hold until return to 
baseline 
 
 
 
 
No 
change 
 
Recurrence Grade 3 
Hold until return to baseline 
or to AST or ALT 
≤2.5 x ULN and total bilirubin 
≤1.5 x ULN, resume at 500 
mg (2 tablets) only after 
discussion and agreement 
with medical monitor 
 
 
 
 
Hold until return to 
baseline 
 
 
 
 
No 
change 
Grade 4 
 
Discontinue AA treatment 
 
Hold until return to 
baseline 
No change or consider 
tapering if AA 
discontinued 
 
Concurrent elevation of 
AST/ALT > 3x ULN 
with bilirubin >2x ULN 
(unless the concurrent 
elevation is related to 
biliary obstruction or 
other causes unrelated to
study treatment) 
 
Discontinue AA treatment 
 
No change or consider 
tapering if AA 
discontinued 
 
Dose modifications for hypokalemia attributed to abiraterone acetate given 
concurrently with apalutamide will be guided by the prescribing information 
provided by the drug manufacturer, as detailed below: 
 
Toxicity 
Dose of abiraterone acetate 
Dose of apalutamide 
Dose of prednisone 
 
 
 
Grade 1 or 2 
Initiate oral potassium 
supplementation, titrate to 
≥3.5 to ≤5.0 mmol/L, 
maintenance at ≥4.0 mmol/L 
recommended 
 
 
 
No 
change 
 
 
 
No change 
 
 
 
≥Grade 3 
Hold and initiate IV 
potassium and cardiac 
monitoring, resume only after 
discussion and approval by 
the medical 
monitor 
 
 
 
No 
change 
 
No change or consider 
tapering if AA is 
discontinued 
 
Dose modifications for hypertension and edema/fluid retention attributed to 
abiraterone acetate given concurrently with apalutamide will be guided by the 


31 
 
prescribing information provided by the drug manufacturer, as detailed below: 
 
Toxicity 
Dose of abiraterone acetate 
Dose of apalutamide 
Dose of prednisone 
Grade 1 or 2 
No change 
No change 
No change 
 
≥Grade 3 
Hold until Grade 1 or 
baseline, resume at full dose 
 
No 
change
 
No change 
First 
Recurrence 
≥Grade 3 
Hold until Grade 1 or baseline, 
resume at 750 mg (3 tablets) 
 
No change 
 
No change 
Second 
Recurrence 
≥Grade 3 
Hold until Grade 1 or baseline, 
resume at 500 mg (2 tablets) 
 
No change 
 
No change 
Third 
Recurrence 
≥Grade 3 
 
Discontinue 
 
No change 
No change or consider 
tapering if AA is 
discontinued 
 
 
 
5.1.2. Surgery. 
 
Radical Prostatectomy 
Radical prostatectomy with pelvic lymph node dissection will be performed at the 
VA hospital by a urologic oncologist. The prostatectomy may be performed as an 
open or robot-assisted procedure at the discretion of the treating urologist.  
Surgery will be completed after radiographic directed metastatic biopsy but prior 
to systemic or radiotherapy. 
  
 
5.1.3. Radiotherapy 
 
Primary prostate radiotherapy. Patients who undergo radiotherapy as primary 
tumor therapy will undergo radiotherapy directed to the prostate, seminal vesicles, 
and pelvic lymph nodes.  
• 
Allowed dose fractionations include: 
o 
Prostate: 40 Gy in 5 fractions or 60 Gy in 20 fractions or 79.2 Gy in 44 
fractions 
o 
SVs:  25-40 Gy in 5 fractions, or 42-60 Gy in 20 fractions, or 45-29.2 Gy in 
44 fractions. 
o 
Pelvic LNs: 25 Gy in 5 fractions or 42-43 Gy in 20 fractions or 45 Gy in 25 
fractions. 
The selection of dose and fractionation will be made at the discretion of the 
investigator, and typically depends on anatomical considerations and baseline 
urinary and bowel function. Gross disease may be prescribed a higher dose at the 
investigator’s discretion. If a pelvic node is >1.5 cm on the simulation CT scan (RT 
planning scan) or was enlarged or PSMA positive on the staging scan, then the 
enlarged node may be boosted to a higher dose while respecting normal tissue 
dose constraints. This radiation may be done anytime while the patient is on ADT. 
Standard RTOG contouring guidelines are recommended but not required. 
 
 


32 
 
SBRT. SBRT offers ablative dose-escalation to tumor targets with simultaneous 
dose-restraint to normal tissues, which is not possible using conventional 
radiotherapy.  SBRT utilizes high-precision radiotherapy delivery machines, 
integrated image guidance systems, advanced planning software, and a high level 
of professional and technical expertise.   Unlike conventional RT, which is 
delivered in multiple fractions of 1.8-2 Gy (Gy is the unit of measurement for dose 
delivered), SBRT is delivered in one to five fractions of typically 8 Gy or greater. 
Patients will undergo SBRT to all visible sites of metastases within two months of 
initiation of ADT. Prior to SBRT, patients may be re-imaged with a NaF or PSMA 
PET-CT or Fluciclovine (Axumin) PET-CT in the treatment position for RT 
planning. (Patients suspected of having >5 sites of metastases at this time will be 
censored 
from 
primary 
analysis but will be treated per 
protocol 
at 
patients 
and 
investigators discretion and 
the 
tissue 
used 
for 
the 
correlative 
analyses). 
Simulation CT scan will be 
fused to the NaF or PSMA 
PET-CT 
or 
Fluciclovine 
(Axumin) PET-CT. SBRT will 
be delivered in 1 to 5 fractions. 
SBRT 5 fraction regimens 
should deliver a total dose of 
25 to 50 Gy. Acceptable 3 
fraction 
regimens 
should 
deliver a total dose of 27-36 
Gy. Single fraction regimens 
should deliver a dose of at 
least 
18 
Gy. 
Spinal 
metastases may be treated 
with a single fraction of 15 to 
22 Gy, or 3 fractions to a total 
dose of 24-36 Gy, or 5 fractions to a total dose of 30-35 Gy. Dose selection will 
depend on adequate sparing of normal tissue with maximization of dose delivered 
to metastatic sites. A biologically equivalent dose (BED) to tumor of >100 Gy (for 
an alpha-beta ratio of 3) is a goal but is not required (11). Dose constraints per 
NRG (RTOG) B001 are recommended (35). 
 
Figure 5 shows a recent SBRT treatment plan for a patient with biopsy proven de-
novo oligometastatic prostate cancer. Image fusion between the simulation CT 
scan and the NaF PET was used to delineate the target. This patient received a 
single dose of 20 Gy (BED = 153 Gy) to this site of osseous metastasis without 
toxicity. Note the sharp dose fall-off and conformity of the dose delivered to the 
metastatic lesion, made possible by SBRT technique. (Image is taken from the RT 
planning MIM software package.) 
 
Post-operative pelvic RT. Patients found to have pT3a or higher, positive 
margins, or pathologically involved nodes after radical prostatectomy, will receive 
post-operative fractionated IMRT delivered to the pelvic nodes and prostate bed. 


33 
 
The pelvic nodes will receive 45-50.4 Gy in 25-28 fractions and the prostate bed 
will receive 66-72 Gy in 34-40 fractions with daily image guidance. If a pelvic node 
is >1.5 cm on the simulation CT scan (RT planning scan) or was enlarged or PSMA 
positive on the staging scan, then the enlarged node may be boosted to a higher 
dose while respecting normal tissue dose constraints. This pelvic RT can extend 
beyond or be completed after ADT. Standard RTOG contouring guidelines are 
recommended but not required. 
 
Tissue handling Recommendations: 
 
Research Biopsies: 
• A pre-treatment radiographic directed metastatic biopsy for both diagnostic and 
correlative analysis is required for enrollment unless not safe to perform, not 
feasible, or declined. If this biopsy is not safe, not feasible, or not diagnostic of 
metastatic prostate cancer, then secondary imaging modality [for example, MRI] 
must also be consistent with metastatic disease [unless PSMA PET-CT or 
Fluciclovine (Axumin) PET-CT was used as initial staging]. A second biopsy 
obtained at time of progression will be encouraged but is optional.  The biopsy site 
will be determined primarily by safety. Biopsies will be performed by Interventional 
Radiology.  
• Lesions will be chosen based upon the strength of the evidence suggesting the 
presence of metastasis and with the goal of minimizing patient risk. The biopsies 
will be performed in an interventional radiology suite with radiological guidance 
(typically CT or MRI) in accordance with institutional standards. CT or MRI will 
confirm designated lesions immediately prior to biopsy. Once the target lesion is 
identified, up to six 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 approximately 
10 to 20 mm in length is obtained. Core biopsies will be extracted: at least 3 will 
be placed in neutral-buffered formalin and 3 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. 
Recommended procedures for biopsies are included in Appendix 14.4. 
 
Research Blood Samples: 
• Blood samples will be collected during screening, at 1 month, 2 months, at the 
end of systemic treatments, and at each follow-up visit (q30 days for the first 6 
months, then q3 months thereafter) whenever possible. 
• Blood will ideally be collected in 2 x 5 ml Streck Cyto-Chex® BCT tubes and 1 x 
10 mL Cell-Free DNA BCT@ tubes, and 1 x 10 mL Cell-Free RNA BCT@ tubes. 
The blood is used for immunophenotyping, CTC, ctDNA, and ctRNA analyses. 
Blood in these tubes are stable at room temperature for shipping. Upon receipt, 
blood is processed for immunophenotyping, DNA, and RNA isolation. 
 
Recommendation for processing of prostatectomy specimens: 
• After resection, the prostate is sectioned into multiple slices (total number depends 
on the size of the prostate, but usually 5-8). Alternating slices are frozen, while 


34 
 
other slices are fixed in formalin and embedded in paraffin.  
• A GU pathologist will identify distinct loci of prostate adenocarcinoma by 
histopathology and the corresponding (adjacent) frozen and formalin slices will be 
processed 
for 
genomic/transcriptomic 
analyses 
and 
sectioning 
for 
immunohistochemistry, respectively.  
• Each focus of prostate cancer will be laser capture microdissected from frozen 
tumor sections prior to “omics” analyses if necessary.  
• When a single lesion exhibits areas of differing Gleason grade, an area 
representative of each component will be separately microdissected and subjected 
to analysis. 
• For sites aside from VA West LA, FFPE blocks of the primary tumor and initial 
biopsy may be sent. 
• For VA West LA, the fresh (tissue collected after enrollment which has not been 
exposed to fixative; ie: non-FFPE) prostate tissue may also be shared with Dr 
Garraway’s Study VA #0001. 
 
Shipment of tissue: 
• All specimens (blood, frozen biopsies, frozen prostatectomy specimen slices) are 
delivered in person to the laboratory of Dr. Nicholas Nickols at UCLA, if acquired 
at West LA VA. Frozen tissues are packed on dry-ice. Blood is shipped room 
temperature. 
• Formalin fixed biopsies tissue and prostatectomy specimen slices are shipped 
after diagnostic evaluation by the institution. These are also shipped to the 
laboratory of Dr Nicholas Nickols. 
• Samples shipped can be accompanied by a Sample Shipment Log (supplied by 
Sponsor, West LA VA). 
• Emails will be sent to Nicholas.nickols@va.gov / nnickols@mednet.ucla.edu (PI) 
within three working days prior to shipment whenever possible. 
• Samples should be shipped Monday through Thursday and Saturday Delivery is 
not allowed, unless special arrangements are made in advance. 
• Shipping Address: 
Nicholas Nickols MD PhD 
UCLA Radiation Oncology 
BOX 951714, B3-109 CHS 
Los Angeles, CA 90095-1714 
Tissue will be stored in the laboratory of Nicholas Nickols in appropriately secure 
laboratory research areas. 
5.2 Handling of Study Interventions 
Acquisition of study drugs: 
 
• Apalutamide and abiraterone acetate will be provided by Janssen and sent directly 
to West LA VA (West LA VA Clinical Research Pharmacy). Drug will be stored per 
manufacturers’ recommendations in the Clinical Research Pharmacy. West LA VA 
will distribute to other participating centers.  
• Leuprolide will be purchased from commercial suppliers. 
 
Drug accountability: The Clinical Research Pharmacy will maintain an inventory of the 
study drugs, including the lot number, amount shipped, date of shipment, dates/amounts 


35 
 
dispensed, and remaining amounts of drug. A minimum amount of drug will be kept on 
hand at all times (enough drug to treat 5 patients for four weeks).  Unused or expired 
study medication will be destroyed following the institution’s or drug vendor policies and 
procedures. 
5.3. Concomitant Interventions 
5.3.2 Allowed interventions: 
 All except 5.3.3. 
5.3.3 Required Interventions: 
NaF-PET CT or PSMA PET-CT or Fluciclovine (Axumin) PET-CT, (If biopsy 
is not diagnostic, or unsafe to perform, or not feasible, or declined, then a 
secondary imaging modality [for example, MRI] must also be consistent with 
metastatic disease [unless PSMA PET-CT or Fluciclovine (Axumin) PET-
CT was used as initial staging]) as per protocol and for determining 
eligibility. 
Laboratory assessment as per protocol and for determining eligibility. 
Treatments as described in this protocol. 
 
5.3.4 Prohibited Interventions 
• Herbal products that may have hormonal anti-prostate cancer activity 
and/or are known to decrease PSA levels (e.g., saw palmetto), 
 
• Any medication outside the trial agents known to affect serum androgen 
levels or PSA. 
 
• Prohibited Concomitant Medications During Trial. As a class effect, AR 
antagonists have been associated with seizures due to an off-target 
mechanism of action (gamma amino butyric acid chloride channel [GABAA] 
inhibition). Drugs known to lower the seizure threshold or cause seizures 
are prohibited and a representative list is included below:  
• Atypical antipsychotics (e.g. clozapine, olanzapine, risperidone, 
ziprasidone) 
• Bupropion 
• Lithium 
• Meperidine and pethidine 
• Phenothiazine antipsychotics (eg, chlorpromazine, mesoridazine, 
thioridazine) 
• Tricyclic antidepressants (eg, amitriptyline, desipramine, doxepin, 
imipramine, maprotiline, mirtazapine 
• Concomitant 
strong 
CYP3A4 
inducers 
(e.g., 
phenytoin, 
carbamazepine, rifampin, rifabutin, rifapentine, phenobarbital) will be 
avoided during abiraterone acetate treatment. Although there are no 
clinical data with this dose adjustment in patients receiving strong 
CYP3A4 inducers, because of the potential for an interaction, if a strong 
CYP3A4 inducer must be co-administered, increase the abiraterone 


36 
 
acetate dosing frequency to twice a day only during the co-
administration period (e.g., from 1,000 mg once daily to 1,000 mg twice 
a day). Reduce the dose back to the previous dose and frequency, if 
the concomitant strong CYP3A4 inducer is discontinued. 
 
• Restricted Concomitant Medications.  
 
• Apalutamide is metabolized primarily by human CYP3A4, thus co-
administration with strong inhibitors or inducers of CYP3A4 should be 
avoided as much as possible. Apalutamide may also induce CYP3A4; 
therefore, caution should be taken when administered in conjunction 
with CYP3A4 substrates that have a narrow therapeutic index. 
Examples of the strong CYP3A4 inhibitors and inducers include the 
following:  
 
• 
Strong 
CYP3A4 
inhibitors: 
itraconazole, 
clarithromycin, 
erythromycin, 
diltiazem, 
verapamil, 
delavirdine, 
atazanavir, 
indinavir, 
nefazodone, 
nelfinavir, 
ritonavir, 
saquinavir, 
telithromycin, voriconazole, grapefruit juice (or grapefruits); co-
administration with any of these agents may increase apalutamide 
plasma 
concentrations. 
Strong 
CYP 
inducers: 
phenytoin, 
carbamazepine, rifampin, rifabutin, rifapentin, phenobarbital, 
efavirenz, tipranivir, St. John's wort; co-administration with any of 
these agents may decrease apalutamide plasma concentrations.  
The potential for drug-drug interaction between apalutamide and 
warfarin (eg, Coumadin) is unknown at present. If a subject is 
taking warfarin, re-assess PT (prothrombin time)/international 
normalized ratio (INR) as clinically indicated and adjust the dose of 
warfarin accordingly. 
 
• Abiraterone acetate is a substrate of CYP3A4. In a dedicated drug 
interaction trial, co-administration of rifampin, a strong CYP3A4 
inducer, decreased exposure of abiraterone by 55%. Avoid 
concomitant strong CYP3A4 inducers during abiraterone acetate 
treatment. If a strong CYP3A4 inducer must be co-administered, 
increase the abiraterone acetate dosing frequency. In a dedicated drug 
interaction trial, co-administration of ketoconazole, a strong inhibitor of 
CYP3A4, had no clinically meaningful effect on the pharmacokinetics 
of abiraterone acetate. Abiraterone acetate is an inhibitor of the hepatic 
drug-metabolizing enzymes CYP2D6 and CYP2C8. In a CYP2D6 drug-
drug interaction trial, the Cmax and AUC of dextromethorphan 
(CYP2D6 substrate) were increased 2.8- and 2.9-fold, respectively, 
when dextromethorphan was given with abiraterone acetate 1,000 mg 
daily and prednisone 5 mg twice daily. Avoid co-administration of 
abiraterone acetate with substrates of CYP2D6 with a narrow 
therapeutic index (e.g., thioridazine). If alternative treatments cannot be 
used, exercise caution and consider a dose reduction of the 
concomitant CYP2D6 substrate drug. In a CYP2C8 drug-drug 
interaction trial in healthy subjects, the AUC of pioglitazone (CYP2C8 
substrate) was increased by 46% when pioglitazone was given together 


37 
 
with a single dose of 1,000 mg abiraterone acetate. Therefore, patients 
should be monitored closely for signs of toxicity related to a CYP2C8 
substrate with a narrow therapeutic index if used. 
 
5.4 Adherence Assessment 
 
Adherence to study drugs (apalutamide and abiraterone acetate) will be defined as at 
least 80% of treatment intervention pills taken as determined by pill count during follow-
up visits and assessment of drug diaries at scheduled visits. Patients must undergo 
radical prostatectomy and/or all required radiation treatments.  
 
 
6. STUDY PROCEDURES 
6.1  Schedule of Evaluations 
 
                Treatment Visits (q30d) 
 
Follow up (visits are 
q30 d for first 6 M; q3 
M thereafter) 
Visit 
-90 to 0 
Day 1 
Day 
30    
(1 Mo) 
 31-60 
days 
2, 3, 4, 
5, 6 
Month  
Follow up 
Window (days) 
  
1 
+/-7 
  
+/-7 
+/-7 
Informed consent  
x 
  
  
  
  
  
Inclusion/exclusion criteria 
x 
 
 
 
 
 
Demographics 
x 
  
  
  
  
  
Scan used to find out if cancer has spread to the bone 
(with CT of the chest, abdomen and pelvis)  
x 
  
  
  
  
  
Biopsy or MRI if this biopsy is unsafe to perform, not 
feasible or declined  
x 
  
  
  
  
  
Quality of Life Questionnaire 
x 
 
x 
 
x 
x 
Medical assessment of quality of patient life 
x 
  
x 
  
x 
x 
Medical history 
x 
  
x 
x 
x 
x 
Physical Examination 
x 
  
x 
x 
x 
  
Vital signs, including weight 
x 
  
x 
x 
x 
 x 
Current Medications/Drug Diary Review 
x 
  
x 
x  
x 
x 
Blood Draws to check for thyroid and liver function   
x 
  
x 
  
x 
x 
PSA, total testosterone 
x 
  
x 
  
x 
x 
Research Blood 2 
x 
  
x 
  
x 
 x 
Urine collection 
x 
  
x 
  
x 
x 
Surgery (Radical Prostatectomy) if this is chosen 
  
x 
  
  
  
  
SBRT to all sites of metastases 6 
  
  
  
x 
  
  
Pelvic RT (if indicated) 3 
  
  
  
  
X (may extend beyond first 6 months)   
 
Adverse Events 
  
x 
x 
x 
x 
x 


38 
 
1. LFTs are drawn with each blood chemistry. In addition, for the first 3 months LFTs are also drawn q2 
weeks. (After the first 3 months, they are drawn with the scheduled blood chemistries). 
2. Research blood draws: 2 x 5 ml Streck Cyto-Chex BCT@ tubes, 1 x 10 mL Cell-Free DNA BCT@ tube, 
and 1 x 10 mL Cell-Free RNA BCT@ tube. 
3. Pelvic (post operative) RT may be done anytime, and may be completed after systemic therapy. It is done 
only for patients with pT3+, positive margins, or pN1. 
4. Leuprolide, apalutamide, and abiraterone acetate will be initiated on Day 1(+/- 3 days), if primary tumor 
therapy is radiotherapy or after radical prostatectomy, within 0-3 days, if primary tumor therapy is surgery 
5. Apalutamide, abiraterone acetate, and prednisone are stopped 6 months (+/- 7 days) after leuprolide 
injection. Prednisone will be tapered down after discontinuation of study drugs. 
6. SBRT can be done anytime within the first 2 months of ADT, but preferably during the second month. 
 
6.2 Description of Evaluations 
6.3.1 Screening Evaluation 
Patients with metastatic prostate carcinoma that have not undergone prior 
treatment for prostate cancer and have undergone a metastatic biopsy will be 
evaluated for this study unless deemed unsafe or not feasible to perform, or 
declined by patient. Patients must be staged with NaF or PSMA PET-CT or 
Fluciclovine (Axumin) PET-CT. If the biopsy is not diagnostic, declined, or not 
feasible, MRI must be consistent with metastatic prostate cancer. 
 
Consenting Procedure 
Before any screening procedure is performed, informed consent will be obtained. 
One consenting process and a single informed consent form will be used to 
describe both the screening and study procedures. One of the study investigators 
will approach patients who may be eligible for this study from the Urology Clinic, 
Radiation Oncology clinic, or medical oncology clinic within the West LA VA or 
Long Beach VA Health System. At participating sites, consent will be obtained by 
one of the sub-investigators at that site. The nature and purpose of the study will 
be described in detail, the patients will be given the IRB-approved consent form, 
and patients will be encouraged to ask questions and read the consent form at 
their leisure (including taking it home to read it and ask questions at a subsequent 
time). An enrollment note in the electronic medical record will document the 
informed consent process has been appropriately completed.  
 
Screening Assessments  
Screening studies must be completed within 28 days of enrollment as defined by 
initiation of study drug treatment.  
• History and physical. 
• Confirmation of carcinoma of the prostate pathology.  
• Laboratories- blood tests, urine analysis 
• PSA and testosterone assessment 
• Radiographic directed biopsy of metastatic lesion unless deemed unsafe or 
Leuprolide Injection 54 
  
x 
  
  
  
  
Start apalutamide,  abiraterone acetate , prednisone 
daily (6 months) 
  
x 
  
  
  
  
Radiotherapy (prostate, seminal vesicles, pelvic lymph 
nodes) 
 
Done any time during 6 months 
systemic therapy after ADT start 
 


39 
 
not feasible to perform, or declined by patient 
• Imaging studies (NaF or PSMA PET-CT, diagnostic CT scan of the chest, 
abdomen/pelvis). If biopsy is not diagnostic, or unsafe to perform, or not 
feasible, or declined by patient, then a secondary imaging modality (for 
example, MRI) must also be consistent with metastatic disease (unless 
PSMA PET-CT was used as initial staging). 
• Blood samples to determine circulating tumor genetic information and 
immunophenotyping 
• Assessment of performance status (e.g., ability to perform daily activities) 
 
6.3.2 Enrollment, Baseline, and/or Randomization 
 
Enrollment (Day 1) 
Enrollment will occur when patients have met all eligibility criteria and initiate 
treatment, which begins with radical prostatectomy, if primary tumor therapy is 
surgery. Enrollment must occur within 90 days of the initiation of screening, which 
starts at the time that informed consent is signed.  
 
6.3.3 Treatment and Follow-up Visits  
Scheduled treatment visits will be on days 31 (1 month), and then every 30 days 
(monthly), until patient completes the treatment study period (all systemic and 
radiotherapy completed), and then every 30 days for six months, and then every 3 
months thereafter (+/- 7 days). See 6.1 Schedule of Evaluations.  At these visits, 
the following will occur: 
• Review of medical history and physical examination and vital signs. 
• Laboratories- blood tests, urine analysis. 
• Pill count of study drugs, review of drug diary with concomitant medications 
• Adverse events assessment.  
• Laboratory assessments 
 
6.3.4 Completion/Final Evaluation 
Each patient will remain on study until unacceptable toxicity, disease progression, 
or withdrawal of consent, or two years, whichever occurs first.  The patient will 
have a follow up visit 2 to 4 weeks after going off study or as clinically indicated.  
At this visit, the following will occur: 
• Review of medical history and physical, and vital signs  
• Blood and urine tests  
• Review of concomitant medications  
• Review of Adverse Events (AE), or any side effects from the study drugs or 
other interventions 
 


40 
 
7. SAFETY ASSESSMENTS 
7.1 Specification of Safety Parameters 
Toxicities of the study drugs will be monitored with frequent laboratory assessments and 
exams. Safety during radiotherapy will be assessed by standard on treatment visits and 
standard operating procedures for quality assurance of radiotherapy. An individual Case 
Safety Report (ICSR) will be used to document safety events to be reported. 
 
Individual Case Safety Report (ICSR)  
A valid ICSR must contain the four minimum criteria required to meet regulatory 
reporting requirements. 
• an identifiable subject (but not disclosing personal information such as the 
subject’s name, initials or address) 
• an identifiable reporter (investigational site) 
• a Janssen medicinal product 
• an adverse event, outcome, or certain special situations 
The minimum information required is: 
• suspected Janssen medicinal product (doses, indication) 
• date of therapy (start and end date, if available) 
• batch or lot number, if available 
• subject details (subject ID and country) 
• gender 
• age at AE onset 
• reporter ID 
• adverse event detail (AE verbatim in English), onset date, relatedness, 
causality, action taken, outcome, (if available) 
• Janssen protocol ID 
 
Product Quality Complaint (PQC) 
A product quality compliant is defined as any suspicion of a product defect 
related to a potential quality issue during manufacturing, packaging, release 
testing, stability monitoring, dose preparation, storage or distribution of the 
product, or delivery system.  Not all PQCs involve a subject.  Lot and batch 
numbers are of high significance and need to be collected whenever available. 
Examples of PQC include but not limited to: 
• Functional Problem: e.g., altered delivery rate in a controlled release 
product 
• Physical Defect:  e.g. abnormal odor, broken or crushed tablets/capsules 
• Potential Dosing Device Malfunction: e.g.,  autoinjector button not working, 
needle detaching from syringe 
• Suspected Contamination 
• Suspected Counterfeit 
 


41 
 
Hospitalization 
For reports of hospitalization, it is the sign, symptom or diagnosis which led to the 
hospitalization that is the serious event for which details must be provided. 
Any event requiring hospitalization or prolongation of hospitalization that occurs 
during the study must be reported as a serious adverse event, except 
hospitalizations for the following: 
• Hospitalizations not intended to treat an acute illness or adverse event (e.g., 
social reasons such as pending placement in long-term care facility) 
• Surgery or procedure planned before entry into the study. [Note: 
Hospitalizations that were planned before the start of data collection and 
where the underlying condition for which the hospitalization was planned 
has not worsened will not be considered serious adverse events. Any 
adverse event that results in a prolongation of the originally planned 
hospitalization is to be reported as a new serious adverse event.] 
 
Life-Threatening Conditions 
The cause of death of a subject in a study within 30-days of the last dose of 
Apalutamide or Abiraterone acetate, whether or not the event is expected or 
associated with the study drug, is considered a serious adverse event. 
 
Disease progression should not be recorded as an adverse event or serious 
adverse event term; instead, signs and symptoms of clinical sequelae resulting 
from disease progression/lack of efficacy will be reported if they fulfill the serious 
adverse event definition. 
 
Unlisted (Unexpected) Adverse Event/Reference Safety Information 
An adverse event is considered unlisted if the nature or severity is not consistent 
with the applicable product reference safety information. For a medicinal 
product(s) with a marketing authorization, the expectedness of an adverse event 
will be determined by whether or not it is listed in the applicable product 
information.   
For abiraterone acetate, the link to the package insert is: 
http://www.zytiga.com/sites/default/files/pdf/full_product_information.pdf 
 
For apalutamide, the link to the package insert is:  
http://www.janssenlabels.com/package-insert/product-monograph/prescribing-
information/ERLEADA-pi.pdf    
 
 
Special Reporting Situations  
 
Safety events of interest for a Janssen medicinal product that require expediting 
reporting and/or safety evaluation include, but are not limited to: 
• Drug exposure during pregnancy (maternal and paternal) 


42 
 
• Overdose of a Janssen medicinal product  
• Exposure to a Janssen medicinal product from breastfeeding  
• Suspected abuse/misuse of a Janssen medicinal product 
• Inadvertent or accidental exposure to a Janssen medicinal product  
• For Abiraterone acetate only, any failure of expected pharmacological action 
(i.e., lack of effect) of a Janssen medicinal product 
• Medication error involving a Janssen medicinal product (with or without 
patient exposure to the Janssen medicinal product, e.g., name confusion) 
• Suspected transmission of any infectious agent via administration of a 
medicinal product 
• For abiraterone acetate only, unexpected therapeutic or clinical benefit from 
use of a Janssen medicinal product 
These safety events may not meet the definition of an adverse event; however, 
from a Janssen Scientific Affairs perspective, they are treated in the same 
manner as adverse events. Special situations should be recorded on the Adverse 
Event page of the CRF. 
Any special situation that meets the criteria of a serious adverse event should be 
recorded on a Serious Adverse Event Report Form and be reported to Janssen 
Scientific Affairs within 24 hours of becoming aware of the event. 
7.2 Methods and Timing for Assessing, Recording, and Analyzing Safety 
Parameters 
Safety of the study interventions, including study drug treatments, and SBRT, and 
research biopsies will be assessed by frequent monitoring by: 
• History and physical 
• Laboratories 
• Pill counts, drug diary review 
7.3 Adverse Events and Serious Adverse Events 
Adverse Event (AE) 
An adverse event is any untoward medical occurrence in a clinical study subject 
administered a medicinal (investigational or non-investigational) product. An adverse 
event does not necessarily have a causal relationship with the treatment. An adverse 
event can therefore be any unfavorable and unintended sign (including an abnormal 
finding), symptom, or disease temporally associated with the use of a medicinal 
(investigational or non- investigational) product, whether or not related to that 
medicinal (investigational or non-investigational) product. (Definition per International 
Conference on Harmonisation [ICH]). 
This includes any occurrence that is new in onset or aggravated in severity or 
frequency from the baseline condition, or abnormal results of diagnostic procedures, 
including laboratory test abnormalities. 
 


43 
 
Adverse Events of Special Interest. There are no adverse events of special interest 
identified for apalutamide or abiraterone acetate. 
 
Adverse event (AE) monitoring and reporting is a routine part of every clinical trial. An 
adverse event is any adverse change (developing or worsening) from the patient’s 
baseline (pre-treatment) condition, including intercurrent illness, which occurs during 
the course of a clinical study after treatment has started, whether considered related to 
treatment or not.  Adverse event data will be collected at each patient visit.      
 
Adverse reaction: An adverse reaction is defined as any adverse event caused by the 
use of a drug or other intervention.  Adverse reactions are a subset of all suspected 
adverse reactions for which there is reason to conclude that the drug or intervention 
caused the event. 
 
Suspected adverse reaction: A suspected adverse reaction is defined as any adverse 
event for which there is a reasonable possibility that the drug or intervention caused the 
adverse event. For the purposes of IND safety reporting, “reasonable possibility” indicates 
that there is evidence to suggest a causal relationship between the drug or intervention 
and the adverse event.  A suspected adverse reaction implies a lesser degree of certainty 
about causality than an adverse reaction. 
 
Unexpected adverse reaction: An adverse event or suspected adverse reaction is 
considered unexpected if it is not consistent with the risk information described in the 
general investigational plan or elsewhere in the protocol.  
 
A SERIOUS adverse event (SAE) is defined as follows:  
 
Death: Report of any patient's death for any reason should be reported as a serious 
adverse event. 
 
Suspected Transmission Of Any Infectious Agent Via A Medicinal Product: Report 
any transmission of any infectious agent if suspected to be transmitted by a medicinal 
product. 
 
Medically Important Event*: any medically important event should be reported as a 
serious adverse event.  
 
* Medical and scientific judgment should be exercised in deciding whether expedited 
reporting is also appropriate in other situations, such as important medical events that 
may not be immediately life threatening or result in death or hospitalization but may 
jeopardize the subject or may require intervention to prevent one of the other 
outcomes listed in the definition above. These should usually be considered serious. 
 
Life-Threatening: Report if the patient was at substantial risk of dying at the time of the 
adverse event or it is suspected that the use or continued use of the product would result 
in the patient's death.  
 
 
Examples: Pacemaker failure; gastrointestinal hemorrhage; bone marrow suppression; 
infusion pump failure which permits uncontrolled free flow resulting in excessive drug 
dosing.  


44 
 
 
Hospitalization (initial or prolonged): Report if admission to the hospital or prolongation 
of a hospital stay results because of the adverse event.  
 
 
Examples: Anaphylaxis; pseudo-membranous colitis; or bleeding causing or prolonging 
hospitalization.  
 
Disability: Report if the adverse event resulted in a significant, persistent, or permanent 
change, impairment, damage or disruption in the patient's body function/structure, 
physical activities or quality of life.  
 
 
Examples: Stroke due to drug-induced hypercoagulability; peripheral neuropathy.  
 
Congenital Anomaly: Report if there are suspicions that exposure to a medical product 
prior to conception or during pregnancy resulted in an adverse outcome in the child.  
 
 
Examples: Vaginal cancer in female offspring from diethylstilbestrol during pregnancy; 
malformation in the offspring caused by thalidomide.  
 
Medically Significant or Requires Intervention to Prevent Permanent Impairment or 
Damage: Report if you suspect that the use of a medical product may result in a condition, 
which required medical or surgical intervention to preclude permanent impairment or 
damage to a patient. 
 
 
Examples: Acetaminophen overdose-induced hepatic toxicity requiring treatment with 
acetylcysteine to prevent permanent damage; burns from radiation equipment requiring 
drug therapy; breakage of a screw requiring replacement of hardware to prevent poor 
union of a fractured long bone.  
7.4 Reporting Procedures 
All adverse events encountered during the clinical study will be reported on the case 
report form (CRF).  
 
The intensity of clinical adverse events will be graded according to the Common 
Terminology Criteria for Adverse Events v 4.0 (CTCAE) grading system in the toxicity 
categories. The investigator must evaluate and document the adverse event for 
severity, grade it according to the CTCAE v 4.0, judge causal relationship to the study 
drug under study, take appropriate action to care for the patient and to document the 
outcome.  
 
All information recorded in the case report form must be verifiably documented in the 
source (i.e., medical record or physician office chart). 
 
When specific adverse events are not listed in the CTCAE, they will be graded by the 
Investigator as none, mild, moderate or severe according to the following grades and 
definitions: 
 
 
 


45 
 
Grade 0 
No AE (or within normal limits) 
Grade 1 
Mild; asymptomatic or mild symptoms; clinical or diagnostic observations 
only; intervention not indicated 
Grade 2 
Moderate; minimal, local, or noninvasive intervention (e.g., packing, 
cautery) indicated; limiting age-appropriate instrumental activities of daily 
living (ADL) 
Grade 3: 
Severe or medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; 
limiting self-care ADL 
Grade 4: 
Life-threatening consequences; urgent intervention indicated 
Grade 5: 
Death related to AE 
 
When an adverse event occurs, it is the responsibility of the investigator to evaluate and 
record into the source documents, the nature of the symptom, prescribe the appropriate 
remedy and to report the event.  The adverse event must be reported to the IRB, 
pharmaceutical companies sponsoring the study drugs, FDA and any other appropriate 
agencies (e.g., IBC and NIH for gene medicine trials.)      
 
Any clinical adverse event or abnormal event or abnormal laboratory test value that is 
serious (including death or congenital anomaly) occurring during the course of the study 
must be reported to the Institutional Review Board and FDA in writing as soon as is 
practical and in accordance with international and local laws and regulations, with a copy 
forwarded to pharmaceutical companies sponsoring the study drugs.  
 
If the event is Serious and Unexpected, the event should be reported immediately (in 
writing within 10 days, 48 hours for death) by the Principal Investigator to the IRB, Clinical 
Sciences Research and Development (CSR&D) centralized Data Monitoring Committee 
(DMC), pharmaceutical companies sponsoring the study drugs‘ NIH and the VA IBC for 
gene medicine trial and to any other appropriate agency;  
Any clinical adverse event is to be recorded on the case report form, managed medically 
as appropriate, and the event is followed until resolution.  Follow-up report to the initial 
serious adverse event report is required to document final resolution or outcome of the 
event.  
 
Abnormal Laboratory Test Values 
In the event of unexplained abnormal laboratory test values, the tests should be repeated 
immediately and followed up until they have returned to the normal range and/or an 
adequate explanation of the abnormality is found. 
7.5 Follow-up for Adverse Events 
All SAEs occurring during the course of the study will be reported to the Principle 
Investigator and IRB within 24 hours of the knowledge of the occurrence. Where the initial 
report is made verbally or by telephone, a written confirmation within a further 24 hours 
must follow. 
 
Within 24 hours of first awareness of the event (immediately if the event is fatal or life-
threatening), Principal Investigator will report to  Janssen Scientific Affairs, LLC, by 
facsimile any Serious Adverse Event (“SAE,” as defined below) that occurs during the 
SAE reporting period (as defined below) in a Study subject. PI will report such SAEs using 


46 
 
an FDA MEDWATCH form and a Serious Adverse Event Fax Cover Sheet.  SAEs should 
be reported as soon as they are determined to meet the definition, even if complete 
information is not yet available. 
 
The SAEs that are subject to this reporting provision are those that occur from after 
systemic treatment initiation through 30 days after last treatment. 
 
The details regarding SAE reporting to Janssen Scientific Affairs, LLC are as follows:  
 
SAE reporting to Janssen Scientific Affairs, LLC 
 
Within 24 hours of awareness of a serious adverse event, whether or not related to a 
study drug, the Investigator will complete and submit a Medwatch 3500A Form to FDA, 
containing all required information (reference 21 CFR 312.32). The Investigator will 
submit a copy of this MedWatch 3500A form to Janssen Scientific Affairs, LLC by either 
e-mail or fax, within the same timeframe. If submission of this SAE to FDA or Janssen 
Scientific Affairs, LLC 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 Janssen Scientific Affairs, LLC. 
 
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 or intervention 
 
Follow-up information for the event should be sent within promptly (within 7 days) as 
necessary.  
 
Non-Serious AEs.  All non-serious adverse events should be reported to Janssen 
Scientific Affairs according to the timeframe outlined in the Research Funding Agreement 
section entitled Reporting of Data 
 
Pregnancy.  Because the Janssen medicinal product may have an effect on sperm, 
pregnancies in partners of male subjects exposed to a Janssen medicinal product will be 
reported by the principal investigator within 24 hours of their knowledge of the event using 
the Serious Adverse Event Form.  Depending on local legislation this may require prior 
consent of the partner. Follow-up information regarding the outcome of the pregnancy 
and any postnatal sequelae in the infant will be required 
 
Product Quality Complaint (PQC) Reporting.  A PQC may have an impact on the safety 
and efficacy of the product. Timely, accurate, and complete reporting and analysis of PQC 
information from studies are crucial for the protection of patients, investigators, and 
Janssen Scientific Affairs, and are mandated by regulatory agencies worldwide. Janssen 
Scientific Affairs has established procedures in conformity with regulatory requirements 


47 
 
worldwide to ensure appropriate reporting of PQC information. Lot and/or Batch #s shall 
be collected or any reports failure of expected pharmacological action (i.e., lack of effect). 
The product should be quarantined immediately and if possible, take a picture. All initial 
PQCs involving a Janssen medicinal product under study must be reported to Janssen 
Scientific Affairs by the principal investigator within 24 hours after being made aware of 
the event. The Janssen contact will provide additional information/form to be completed.  
If the defect for a Janssen medicinal product under study is combined with either a serious 
adverse event or non-serious adverse event, the principal investigator must report the 
PQC to the Janssen Scientific Affairs according to the serious adverse event reporting 
timelines.  A sample of the suspected product should be maintained for further 
investigation if requested by Janssen Scientific Affairs. 
 
SAE Follow-up 
 
For all SAEs, the investigator must submit follow-up reports to the IRB. Institutional 
Review Board is to be notified regarding the patient’s subsequent course until the SAE 
has subsided, or until the condition stabilizes, the patient dies, or receives alternative 
therapy. 
 
Local reporting for data and safety monitoring for the protocol will require SAE’s to be 
reported to the Office of Research Subject Protection (ORSP using the Adverse Event 
Reporting form and the FDA MEDWATCH SAE reporting form. 
 
On the anniversary date of the IRB approved protocol, the principal investigator will be 
required to report to the IRB, the number of patients entered on the trial, the number of 
patients treated, a summary of all adverse events reported to date using CTCAE version 
4.0 grading, a specific list of serious adverse events requiring immediate reporting, and 
significant literature reporting developments that may affect the safety of participants or 
the ethics of the study. 
 
The IRB will review annual data and safety monitoring reports and make 
recommendations on whether the study should continue unchanged, require 
modification/amendment, or be closed based on unacceptable risk to participants. 
7.6 Safety Monitoring 
Data and Safety Monitoring Plan 
 
Overview.  The VA Clinical Sciences Research and Development (CSR&D) centralized 
Data Monitoring Committee (DMC) will monitor this study.   
 
The CSR&D DMC provides an ongoing independent evaluation of the progress of studies, 
including participant accrual and retention, adverse events monitoring, and analysis plan. 
This is a service that is provided by CSR&D to ensure independent oversight of the safety 
and integrity of this project. No other DMC or Data and Safety Monitoring Board (DSMB) 
review is needed. 
 
The DMC office is located within the Cooperative Studies Program Coordinating Center 
(CSPCC) at the Edward Hines, Jr. VA Hospital in Hines, IL. 
 


48 
 
The DMC Office at Hines then communicates directly with the PI to provide DMC related 
information. 
 
After the DMC has recommended approval for this trial to begin and the first participant 
has been randomized, a progress report will be due in approximately four months and 
then every four months thereafter (April 15, August 15 and December 15).   The PI 
typically will not need to call in for the DMC review after the initial review. 
 
The DMC makes recommendations to the director of CSR&D for endorsement.  The 
recommendations range from approval (unconditionally or with conditions to be 
addressed) to probation to termination. 
 
Following each DMC meeting, the PI will receive meeting minutes containing any action 
items for which a response will be requested typically within 30 days.  Then the process 
starts all over again for the next reporting period.  
 
In addition to the quarterly progress reports, all Serious Adverse Events are to be reported 
to the DMC. 
Level of Risk of a Study 
 
Level of Risk of a Study  
The intensity level of study oversight is determined by the risk category.  Some of the 
factors that are considered when assigning the Level of Risk category include: 
• A biostatistical design and appropriate procedures for proper data management so 
that the information collected can be properly validated. 
• Appropriate Serious Adverse Event reporting procedures must be in place. 
• The study duration must be appropriate and must be based on a realistic rate of 
enrollment. 
• Data collection and data management must be adequate to verify and ensure 
subject eligibility.  
 
Assignment of risk 
Assigning risk ensures that the data and safety monitoring is based on the level of risk 
(low, medium, or high) to ensure that the data and safety monitoring activities are 
appropriate.  Below are some of the criteria used to make a decision regarding the 
assignment of risk: 
• Expected duration of the study based upon the estimated rate of enrollment. 
• Type of study population (e.g., children, geriatric) 
• The procedures used in the trial are commensurate with the degree of risk. 
• Adequate data management systems in place and appropriate case report forms 
• Proper serious adverse event reporting procedures in place 
• Proper biostatistical design and data analysis procedures in place. 
 
 
 
 


49 
 
Level of Risk 
Level 2 
• Compliance Officer meets with PI/Staff prior to study initiation; review regulatory 
requirements and operating system.  Compliance Officer provides real time 
monitoring to determine eligibility prior to enrollment onto the protocol. 
• Real time QA monitoring of the subjects and data collection occurs for all subjects 
entered onto the trial.  
• Comprehensive QA auditing within first year or first 10 subjects enrolled, whichever 
comes first.  Subsequent audit frequency will be annually.  
• Frequency of DSMB Summary Report is typically on a biannual basis or 
approximately every six months. 
8. INTERVENTION DISCONTINUATION 
 
Intervention can be stopped at any time per patient or treating physician choice.  
Study follow up will be discontinued for the following reasons: 
• Unacceptable toxicity 
• Patient no longer under the care of enrolling physician. 
• Withdrawal of informed consent (Subject’s decision to withdraw for any reason) 
In the event of an adverse event, further study interventions may be discontinued as 
judged by the physician based on the severity and nature of the event. 
 
Patients must maintain adherence to study drugs and other treatments ( 80% of 
treatment intervention pills taken as determined by pill count during follow-up visits at 
Days 30, and during scheduled visits until last day of study drug treatment; patients must 
complete all SBRT treatments and undergo prostatectomy. Patients who do not meet 
these criteria will be censored, replaced, and accordingly, additional patients will be 
recruited and enrolled for this purpose.  
9. STATISTICAL CONSIDERATIONS 
9.1 General Design Issues 
This is a single arm Phase II clinical trial in patients with newly diagnosed M1a,b prostate 
cancer and 1-5 radiographically visible metastases treated with radical prostatectomy 
(and post-operative fractionated radiotherapy for pT≥3a, pN1, or positive margins) or 
radiotherapy (including inclusion of the pelvic lymph nodes), metastasis directed SBRT, 
and complete ADT with LHRH analog leuprolide, abiraterone acetate, and apalutamide 
(ARN-509) for a total of six months of systemic therapy. The primary endpoint of our study 
is the percent of patients achieving a serum PSA of <0.05 ng/mL six months after recovery 
of serum testosterone ≥150 ng/dL for patients treated with primary radical prostatectomy 
and PSA of < nadir + 2 ng/mL six months after recovery of serum testosterone ≥150 ng/dL 
for patients treated with primary radiotherapy. [Based on prior studies, >90% of patients 
treated with ADT for six months will recover serum testosterone to ≥150 ng/dL within six 
months after cessation of ADT with a median time to recovery of three month (1,2).] This 
primary endpoint was selected to offer a rapid and sensitive assessment of treatment 


50 
 
efficacy. PSA is a biomarker for disease burden in prostate adenocarcinoma and offers a 
non-invasive and sensitive assessment of disease control after treatment in the vast 
majority of patients.  Durable PSA control in the context of recovery of gonadal function 
(testosterone ≥150 ng/dL) after ADT is therefore a surrogate for disease control. 
9.2 Sample Size  
 Currently, metastatic prostate cancer patients are treated with ADT indefinitely. Based 
on data from prior trials of discontinuous ADT in prostate cancer patients with metastatic 
disease, essentially all patients will relapse with a rising PSA after discontinuation of 
ADT (33,34).  
 
The current study hypothesis is that 20% of patients will achieve a PSA <0.05 ng/mL 
six months after recovery of testosterone to >150 ng/dL. A sample size of N=25 
evaluable patients has an 80% power to test the null hypothesis response rate of 5.5% 
against a two sided alternative response rate of 20% at a significance level (alpha) of 
0.1. Assuming 10% of patients may not recover testosterone to ≥150 ng/dL, we 
anticipate a total study N=28. 
9.3 Outcomes 
9.3.1 Primary outcomes 
The primary endpoint of our study is the percent of patients achieving a serum PSA 
of <0.05 ng/mL six months after recovery of serum testosterone ≥150 ng/dLfor 
patients treated with primary radical prostatectomy and PSA of < nadir + 2 ng/mL 
six months after recovery of serum testosterone ≥150 ng/dL for patients treated 
with primary radiotherapy. PSA and testosterone are checked every 30 days after 
completion of systemic therapy for the first six months and then every three months 
thereafter. 
9.3.2 Secondary outcomes 
▪ Time to biochemical progression 
▪ Time to radiographic progression  
▪ Time to initiation of alternative antineoplastic therapy 
▪ Prostate cancer specific survival  
▪ Assessment of health related quality of life using the Functional Assessment of 
Cancer Therapy - Prostate (FACT-P) scale 
▪ Safety and tolerability 
▪ Correlative analyses (described in appendix) 
9.4 Data Analyses 
Primary outcome.  
The primary endpoint of our study is the percent of patients achieving a serum PSA of 
<0.05 ng/mL six months after recovery of serum testosterone ≥150 ng/dL for patients 
treated with primary radical prostatectomy and PSA of < nadir + 2 ng/mL six months after 
recovery of serum testosterone ≥150 ng/dL for patients treated with primary radiotherapy. 
[Based on prior studies, >90% of patients treated with ADT for six months will recover 
serum testosterone to ≥150 ng/dL within six months after cessation of ADT with a median 
time to recovery of three month (29,30).] This primary endpoint was selected to offer a 
rapid and sensitive assessment of treatment efficacy. PSA is a biomarker for disease 


51 
 
burden in prostate adenocarcinoma and offers a non-invasive and sensitive assessment 
of disease control after treatment in the vast majority of patients.  Durable PSA control in 
the context of recovery of gonadal function (testosterone ≥150 ng/dL) after ADT is 
therefore a surrogate for disease control. It is expected that determination of the primary 
endpoint for each patient will occur between 9 and 12 months after completion of systemic 
therapy. PSA and testosterone are checked at least every 90 days after completion of 
systemic therapy. 
 
Correlative Analyses. 
Described in Section 15. 
10. DATA COLLECTION AND QUALITY ASSURANCE 
10.1 Data Collection Forms 
Data will be collected for each patient, including age and general medical history.  To 
assess for eligibility and primary/secondary outcomes, the following will be performed and 
collected: routine blood tests, urine analysis, PSA and testosterone, NaF PET with 
diagnostic CT scans of the chest/abdomen/pelvis.  Additionally, tissue from the primary 
tumor and metastatic biopsy prior to therapy, blood, and (recommended) biopsy at 
progression are used to perform correlative analysis. 
 
IHC studies will be performed in the Pathology Core of the Prostate Cancer SPORE and 
data maintained on an Excel Spreadsheet. The sequencing studies will be performed and 
only coded data transmitted for analysis. Codes will be maintained on a password-
protected computer in the PI’s office.  
10.2 Data Management 
Access to individual identifiable patient information will be available to the PI, the co-PIs, 
and study staff. Case report forms will be generated to track clinical, laboratory, and 
radiographic data. The study site will maintain records for its patients; data will be 
compiled by the PI and co-PIs for overall analysis. 
10.3 Quality Assurance 
10.3.1 Training 
All staff has and will continue to undergo appropriate training related to 
human subjects.  
10.3.2 Protocol Deviations 
Protocol deviations will be recorded at the site at which they occur and 
documented with a note to chart. Deviations will be reported to the 
coordinating site and sponsor (West LA VA). 
11. PARTICIPANT RIGHTS AND CONFIDENTIALITY 
This study will be conducted in accordance with the ethical principles that have their origin 
in the Declaration of Helsinki as stated in 21 CFR §312.120(c)(4); consistent with GCP 
and all applicable regulatory requirements. The Investigator must comply with the 
applicable regulations in Title 21 of the Code of Federal Regulations (21 CFR §50, §54, 


52 
 
and §312), GCP/ICH guidelines, and all applicable regulatory requirements.  The IRB 
must comply with the regulations in 21 CFR §56 and applicable regulatory requirements. 
11.1 Institutional Review Board (IRB) Review 
 
This protocol and the informed consent document and any subsequent modifications will 
be reviewed and approved by the IRB responsible for oversight of the study.  Subject 
recruitment materials and any other written information to be provided to subjects are also 
reviewed and approved by the IRB before any protocol related procedures are performed 
on any subjects.  The study will also be approved by the local IRB’s at the other sites prior 
to initiation.  
11.2 Informed Consent Forms 
A signed consent form will be obtained from each participant. For participants who cannot 
consent for themselves, such as those with a legal guardian (e.g. person with power of 
attorney), this individual must sign the consent form. The consent form will describe the 
purpose of the study, the procedures to be followed, and the risks and benefits of 
participation. A copy will be given to each participant or legal guardian and this fact will 
be documented in the participant’s record.  
11.3 Participant Confidentiality 
All patient reports and clinical samples will be identified by an assigned coded 
number/letter to maintain patient confidentiality (e.g. sample 1B). The PI will maintain a 
log of patients’ codes, names, and contact information, which will be kept in a locked 
cabinet in his office, which in turn will remain locked in his absence. Every effort will be 
made to keep all documents with patient identifiers under the strictest confidentiality. 
Information that is collected during the study will be stored at the research site:  paper 
copies will be kept in the safe, and computer files will be protected by passwords. 
 
However, by signing the consent form, patients are authorizing the use and disclosure of 
identifiable health information relevant to this study. This information comes from medical 
records and research study-specific information that is obtained for the purposes of this 
study.  
 
Patient records, the research information, and the informed consent forms may be 
inspected by a representative of the California Department of Health, the FDA, the 
National Institutes of Health, the IRB, and Janssen Scientific Affairs, LLC. Thus, because 
of the possibility that information may be released to one of these regulatory institutions, 
absolute confidentiality cannot be guaranteed. Results of this study may be published, 
but the names or identities of subjects will not be revealed, and medical records will 
remain confidential unless the disclosure is required by law.  
 
Research study files and specimens will be kept for up to 10 years after completion of the 
study. Remaining samples and research files will have the sample number removed and 
files will be destroyed as described in Health Authority (i.e. the FDA or EMA) guidelines. 
Specimens (tissue and blood) may be used outside of what is outlined in this protocol, if 
these investigations may lead to improved understanding of cancer biology and 
treatments. 


53 
 
11.4 Study Discontinuation 
The study may be discontinued at any time by the IRB, the NIH, the OHRP, the FDA, or 
other government agencies as part of their duties to ensure that research participants are 
protected. 
 
12. FUNDING AND INSURANCE 
The study sponsor (VA Greater Los Angeles Healthcare System, with drug support from 
Janssen Scientific Affairs, LLC) is funding the non-standard of care interventions. 
13. PUBLICATION OF RESEARCH FINDINGS 
 
Publication of the results of this trial will be governed by the policies and procedures of 
the IRB’s of the participating sites. Patient confidentiality will be maintained in any 
presentation, abstract, or manuscript.  
14. SUPPLEMENTS/APPENDICES 
14.1 A copy of the CTCAE version 4.0 can be downloaded from the CTEP web site 
(http://ctep/cancer.gov). 
14.2 Investigators’ Brochures (see attached). 
14.3 ECOG Performance Status. 
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 self-care but unable to carry out any work activities.  
Up and about more than 50% of waking hours 
3 
Capable of only limited self-care, confined to bed or chair more than 50% of waking hours 
4 
Completely disabled. Cannot carry on any self-care. 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 ClinOncol 5:649-655, 1982. 
 
14.4 Recommended Procedures for Radiographic-Directed Biopsy. 
 
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:  


54 
 
 
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.  
 
The Principal Investigator, Dr. Nickols, should be contacted directly with any 
questions regarding the most appropriate approach to collect biopsy material from 
specific patients enrolled in the trial.  
 
Please follow the directions below when processing the biopsy material:  
 
Required Materials  
• 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)  
• Dry Ice (pellet form, smaller pellet size works best)  
• Syringe needle (core manipulation; ≤19g)  
• Forceps (fine toothed) 
 
Recommended 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.  At the collection site, lay out supplies needed for sample handling.  
3.  Place several cryomolds on the dry ice 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 


55 
 
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.  Once the OCT becomes opaque in each cryomold, the tissue block is ready 
to be transferred to the labeled cold cassette.  
11.  In addition to collecting fresh-frozen biopsy cores, up to 3 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 (provided by sponsor, 
West LA VA) 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 West LA VA 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 Nicholas.nickols@va.gov / 
nnickols@mednet.ucla.edu (study PI).   
15.  Mail by Express Overnight delivery. Unless special arrangements are made, 
do not ship on Fridays, Saturdays, or Sundays. 
 
14.5 Recommended Prednisone Taper after discontinuation of abiraterone 
acetate. 


56 
 
 
Weeks after discontinuing 
abiraterone acetate 
Prednisone dose (mg/day) 
0 
9 
2 
8 
4 
7 
6 
6 
8 
5 
10 
4 
12 
2 
14 
1 
16 
0.5 
18 
0 
15. CORRELATIVE ANALYSES 
Our prospective Phase II trial of prostatectomy, metastasis directed stereotactic body 
radiation therapy (SBRT), and systemic therapy in newly diagnosed M1a,b 
oligometastatic prostate will yield evaluable tissue (metastatic lesions through biopsy and 
the primary malignant prostate by prostatectomy) from previously untreated prostate 
cancer patients with metastatic disease. We will identify the primary tumor in each patient 
that gave rise to the biopsied metastasis, and compare these potentially lethal primaries 
with the other intraprostatic tumors that likely did not yield metastases. We will also 
compare these potentially lethal primary tumors to their corresponding metastases. In 
doing so, we will (1) identify characteristic features of potentially lethal primary prostate 
tumors that are destined for metastasis, and (2) identify which pathways become 
activated within these potentially lethal primary tumors during or after the metastatic 
event. Importantly, these investigations all use tissue that is acquired at the outset for 
each patient enrolled in our trial, and these investigations do not depend on the clinical 
outcome of the trial.  
I. SPECIFIC AIMS FOR CORRELATIVE ANALYSES 
Aim 1. What genomic and transcriptomic features are present in lethal intraprostatic 
tumors that ultimately give rise to metastatic disease that are absent, or different, in those 
intraprostatic tumors that do not? To answer this question, we will conduct whole exome 
deep sequencing (WES), RNA sequencing (RNA-seq), and comparative genomic 
hybridizations (CGH) of the metastatic and intraprostatic tumors of the 28 patients 
enrolled in our Phase II trial. This will enable identification of the “true” primary tumors 
from which the biopsied metastases arose. Then, we will combine the aforementioned 
genomic analyses with RNA-seq data to identify discriminating features of the “true” 
primary lesions that distinguish them from the intraprostatic tumors that did not yield these 
metastatic lesions.  
1.1 To identify the intraprostatic/ primary tumor in each patient gave rise to the 
biopsied metastatic lesion. 
1.2 To discover genomic and transcriptomic features specific to the intraprostatic 
/primary tumors that ultimately gave rise to metastases versus those that did not. 
Aim 2.  What genomic and transcriptomic features are present in the metastases that are 
absent, or different, from the corresponding primary tumors from which they 
metastasized? This will involve comparison between the “true” primary tumors identified 


57 
 
in Aim 1.1 and their metastases. We will also specifically interrogate selected markers of 
kinase activity (MEK/ERK) and differentiation (EMT). 
2.1 To discover genomic and transcriptomic features specific to metastatic prostate 
tumors as compared to the primary tumors from which they arose. 
2.2 To evaluate MAPK (ERK) activation and markers of epithelial to mesenchymal 
transition (EMT) in metastatic prostate cancer lesions as compared to their 
corresponding primary tumors.  
Aim 3.  What genomic and transcriptomic features are present in circulating tumor cells 
and DNA that predict for response? Are there changes in the immunophenotype of 
circulating lymphocytes over the course of treatment? 
3.1 To evaluate biomarkers of response using circulating tumor cells (CTCs). 
3.2 To evaluate biomarkers of response using circulating tumor DNA (ctDNA). 
3.3 To evaluate immunophenotypes of circulating immune cells. 
II. Unique opportunities for translational investigation.  Our trial, which offers the 
opportunity to assess both the untreated malignant prostates as well as the matching 
metastatic tumors in the same homogenous and untreated cohort, thereby lends itself 
to the following provocative correlative studies: 
1. Identification of the intraprostatic lesion from which the metastatic tumors 
arose. It is likely that 80% or more of clinically significant malignant prostates are 
multifocal (36,37). Multiple prior studies lend credence to the notion that the 
multiple tumors within a malignant prostate may arise independently (38–40). A 
recent study specifically assessing the genomic heterogeneity in multifocal 
prostate tumors found minimal overlap in single nucleotide variants and no overlap 
in copy number alterations (CNAs) between the different tumor foci within 
malignant prostates (36), supporting a multi-clonal model for separate tumor foci. 
Another recent study using tumor tissue derived from warm autopsies on 63 
patients with mCRPC revealed limited genomic intra-individual heterogeneity 
between metastases (41), supporting the hypotheses that a single metastatic 
biopsy may give a reasonable portrait of the genomic landscape of metastatic 
disease burden within an individual patient, and that metastases within most 
patients likely arise from a single tumor within the primary. Although notable 
exceptions have been reported (42), most published studies support the notion 
that rare subclones from within a primary tumor initially seed metastasis, which 
then go on to seed additional sites, often metastasis-to-metastasis (43). In Aim 
1.1, we identify the primary lesion that gave rise to the biopsied metastasis in each 
patient in our trial. 
 
2. Discovery of genomic features of potentially lethal primary prostate tumors 
that give rise to metastatic lesions versus those that do not. It has been 
hypothesized that the index lesion (largest lesion with the highest grade) within a 
malignant prostate may be primarily responsible for disease progression (44,45). 
Based on this hypothesis, a number of clinical trials aimed to investigate focal 
therapies (such as HIFU) directed against the index lesion, some with a high failure 
rate (46). A critical question remains: what genomic features characterize the 
potentially lethal primary prostate tumors from the more indolent intraprostatic 
tumors co-existing within the same malignant prostate? Prior studies have traced 
the evolutionary history of prostate tumors over time using genome sequencing. In 


58 
 
one report, surprisingly, the lethal metastatic clone arose from a region of low-
grade tumor within the prostate in the presence of other, larger lesions of higher 
grade (47). In Aim 1.2, we compare the prostate tumors that gave rise to the 
biopsied metastases to those that did not, in order to better define the distinct 
biology of potentially lethal primary prostate cancers. 
 
3. Discovery of pathways directly associated with the emergence of 
metastases. Both localized and metastatic castrate resistant prostate cancers 
(mCRPCs) 
are 
characterized 
by 
frequent 
CNAs, 
gene 
fusions, 
and 
rearrangements (29,30). Some characteristic differences between primary and 
mCRPCs have been identified, which include a greater frequency of androgen 
receptor gene amplification or pathway alteration (50% vs 90-100%), pten loss or 
PI3K/AKT activation (50% vs 90-100%), tp53 mutations (3-20% vs 50%), and MAP 
Kinase (ERK) pathway activation (30% vs 90-100%), for primary and mCRPC, 
respectively (31).  Understandably, prior studies assessing metastatic prostate 
cancer analyzed castrate resistant tumors taken from heavily pre-treated patients 
on long term ADT (30,32), and principally compared them to localized prostate 
tumors in The Cancer Genome Atlas (TCGA) (32).  As such, in these studies the 
initial genomic events driving metastasis may be obscured through subsequent 
genomic and transcriptomic adaptations occurring within tumors during systemic 
therapy. Furthermore, the prostate tumors represented in the TCGA include some 
potentially lethal primary tumors but also a large number of primary tumors that 
would likely never have progressed (48). In our study, we will be able to compare, 
directly, the pathways activated in the metastatic lesions versus the specific 
primary tumor from which these metastases arose. We conduct this analysis in 
Aim 2. 
 
4. Discovery of genomic biomarkers and pathways in patients with 
oligometastatic prostate cancer who have a favorable response to treatment. 
The total duration of active therapy in our trial is a relatively brief 6 months from 
prostatectomy to completion of systemic therapy. Our trial uses a rapid efficacy 
endpoint: PSA <0.05 ng/mL six months after recovery of testosterone to 150 ng/dL. 
We will use this marker of treatment efficacy (responders vs non-responders) to 
perform pattern recognition/discovery of factors that may be characteristic of the 
tumors from patients with a favorable response. This may allow for identification of 
markers characteristic of a favorable response to therapy in oligometastatic 
prostate cancer patients. Although this analysis is not a part of this proposal 
(because not all patients will have a primary endpoint determination within two 
years), we will conduct this analysis in due course. 
 
5. “Liquid biopsy” by detection of circulating tumor cells. We will incorporate 
evaluation of circulating tumor cells (CTCs) detected in enrolled patients pre-
treatment using VortexChip, a technology enabling of detection of CTCs and 
downstream evaluation by next generation sequencing on as few as three CTCs 
per sample.  
 
III. PRELIMINARY DATA 
A. Epithelial to Mesenchymal Transition (EMT) is associated with acquisition of the 
metastatic phenotype.  Our group has a strong interest in understanding the 


59 
 
mechanisms that underlie the development of metastasis. EMT is a key 
developmental process in which epithelial cells acquire a migratory and invasive 
phenotype characterized by the coordinated loss and gain of various epithelial (e.g. 
E-cadherin) and mesenchymal (e.g. N-cadherin and vimentin) markers, respectively. 
In our model systems, we have shown that EMT is a common response of both normal 
and malignant prostates to castration, which in the case of prostate cancer is sufficient 
to cause metastasis (49–51). The expression of an EMT has been validated in gene 
expression datasets of metastatic prostate cancer (49–52). Preclinical studies by our 
groups demonstrate that therapeutic blockade of EMT or EMT-associated genes can 
inhibit metastasis and can synergize with androgen ablation to prevent castration 
resistance (49–51). Numerous studies by our group and others have implicated SRC 
in the development of castration resistance (53,54). We have shown that SRC 
activation can induce castration resistance and also EMT (43,52,54). SRC activation 
was associated with activation of the MAPK pathway, a finding that suggests that 
cross-talk between SRC and the MAPK pathway may be operative in the development 
of castration resistance, EMT, and metastasis. We therefore postulated that 
development of EMT, driven by SRC and MAPK, may underly development of 
metastasis. 
 
B. MAPK: 
a 
potential 
driver 
of 
metastases. Our team, as part of the 
Stand up to Cancer / West Coast 
Dream 
Team 
(SU2C/WCDT), 
investigated transcriptomic differences 
between 
mCRPC 
and 
localized 
prostate 
cancer 
through 
RNA-
sequencing 
of 
laser 
capture 
microdissected tumor tissue obtained 
from metastatic biopsies of patients 
with mCRPC (n=53 in this analysis) as 
compared to localized prostate cancer 
(TCGA, n>600). To identify evidence of 
activation of protein kinase pathways 
from RNA-seq data sets, we employed 
a bioinformatics tool known as Virtual 
Inference 
of 
Protein-activity 
by 
Enriched Regulon analysis (VIPER 
analysis), which infers kinase activity 
based on gene expression changes 
attributable to the kinase itself as well as downstream transcription factors. ERK was 
identified as the most hyperactivated kinase in the mCRPC cohort compared to the 
primary prostate cancer cohort (Figure 6A) and its activation was more than 140-fold 
higher in mCRPC metastases (Figure 6B), although there is some overlap between 
the cohorts at the upper and lower extremes of the TCGA and SU2C cohorts, 
respectively. Additionally, phosphorylated ERK, a marker of MAPK activation, was 
detected in 80% of bone metastases in a separate series of 30 patients with mCRPC. 
These results suggest MAPK activation is a characteristic of mCRPC and has led our 
team to embark on a PCF funded Phase II clinical trial of the MEK inhibitor trametinib 
in patients with mCRPC. (ERK is the phosphorylation target of MEK).  
 


60 
 
However, a slightly different interpretation of the aforementioned data implicating 
MEK/ERK activity in mCRPC, which is consistent with our prior work implicating MAPK 
in the acquisition of EMT, is that the ERK hyperactivation in mCRPC may, in fact, 
constitute an early event required for metastasis, and that the small but noticeable 
overlap in ERK activation between the mCRPC and TCGA cohorts, represents those 
potentially lethal primary tumors (or their subclones) within TCGA that ultimately 
metastasized.   We are currently supplementing this analysis by correlating p-ERK 
staining of primary prostate cancer TMAs with clinical outcomes in our database that 
includes clinical outcomes over the last 25 years (since 1991). 
In fact, there is pre-clinical evidence that MAPK activation may be an early event 
driving prostate cancer metastasis. In an 
autochthonous murine model of localized 
prostate cancer, activation of MAPK induced 
widespread metastases associated with 
epithelial to mesenchymal transitions in the 
absence of androgen suppression (51). In 
this model, pharmacologic inhibition of MEK 
suppressed metastatic growth with minimal 
effect on the primary tumor (51). We 
therefore hypothesize that MAPK activation 
is an early event in prostate cancer 
metastases, which may occur prior to, and 
independent of, androgen suppression. 
(This 
hypothesis 
does 
not 
preclude 
additional crosstalk between the MAPK 
pathway and other pathways associated 
with 
later 
emergence 
of 
castration 
resistance).  
C. Circulating tumor cells (CTCs) represent 
a promising method to obtain genetic 
information about tumors as they evolve 
in response to treatment, as a “liquid 
biopsy.” Many studies have shown the 
promise and potential of analysis of CTCs, 
though many currently available methods 
are 
protracted 
and 
require 
lengthy 
preprocessing 
and 
processing 
steps, 
limiting the viability of collected cells. To 
circumvent these limitations, the ‘Vortex 
Chip’ technology for isolating CTCs using 
microfluidic chips (55,56) (Figure 7). This 
technology is inexpensive, and utilizes 
vorticeal fluid flows to capture cells by size 
alone based on differential trapping and 
without the need for surface labeling. Accordingly, cells that have an epithelial 
phenotype as well as those that have undergone EMT are captured with our Vortex 
Chip. The processing of an 8 mL sample is rapidly completed within ten minutes 
(55,56), and typically achieves CTC purity of >80%. Furthermore, captured CTCs are 


61 
 
viable and can be cultured or sent for downstream analyses such as 
immunohistochemistry or DNA and RNA sequencing.  
 
We have successfully performed CTC isolation and downstream analyses on dozens 
of patients with advanced prostate cancer, including DNA and RNA sequencing on 
individual and pooled CTCs.  Figure 7C demonstrates representative results from 
three CTCs isolated from two distinct patients. RNA sequencing was performed on 
each of these cells and compared to the results of single cell RNA sequencing of three 
individual VCaP cells. Single cell RNA-seq data was mapped on hg19 human genome 
normalized to exonic reads, by length of the gene and by reads per kilobase per million 
mapped exonic reads (RPKM). Clustering was then performed using Pearson's 
correlation with average linkage. The results show clustering between CTCs from 
patient #2 (CTC2-5 and CTC2-7) and CTC from patient #1 clusters separately. VCaP 
cells cluster tightly together.  
 
IV. HYPOTHESES 
• The primary tumors that give rise to metastases may have genomic features that 
are distinguishable from those lesions that do not. 
• Genomic analyses of these de novo metastatic prostate cancers compared 
concurrently to the specific primary tumors from which they arose will identify 
pathways and genomic alterations directly associated with the emergence of 
metastasis. These analyses will not be obscured by adaptive responses to prior 
therapies, or to genomic features of indolent prostate tumors without metastatic 
potential. (These are some limitations of prior studies that have evaluated the 
genomics of metastatic prostate cancer as compared to localized prostate cancer).  
• EMT and MAPK activation are associated with the emergence of metastasis in 
prostate cancer. 
• The tumors in patients with low volume metastatic prostate cancers that have a 
durable response to local, metastases directed, and systemic therapy may have 
biology distinct from the tumors in patients that do not have a durable response to 
this therapy. 
 
V. APPROACH. First, we will identify the primary tumor that gave rise to the biopsied 
metastatic tumor for each patient. Then, we will examine differences between these 
primary tumors that gave rise to metastases versus those that did not (Aim 1). Finally, 
we will examine differences between the metastatic tumors versus their “true” primary 
prostate tumors (Aim 2).   
A. Aim 1.  We will determine the genomic relationship between the metastatic tumors to 
those of the tumors within the malignant prostate to identify the “true” primary tumor 
that gave rise to the metastatic lesion in each patient. This will involve comparative 
genomic hybridizations, whole exome deep sequencing, and RNA-sequencing of the 
laser captured microdissected metastatic and primary tumors. 
1. Rationale.  Primary prostate tumors are frequently multifocal, with malignant 
prostates exhibiting two or more spatially distinct foci of tumor. It has been 
hypothesized that the “index lesion”, defined by size and/or grade, may be 
responsible for disease progression. However, recent investigations call into 
question this hypothesis. Identification of the intraprostatic tumors in our patient 
cohort that gave rise to the biopsied metastases will enable specific 
characterizations of these lethal primary tumors. 


62 
 
2. Recommended Tissue procurement and handling (for both Aims 1 and 2).   
a. Primary tumor tissue. After resection, the prostate is sectioned into multiple 
slices. Alternating slices are kept fresh on ice, while other slices are fixed in 
formalin and embedded in paraffin. A GU pathologist will identify distinct loci of 
prostate adenocarcinoma by histopathology and the corresponding (adjacent) 
frozen and formalin slices will be processed for genomic/transcriptomic 
analyses and sectioning for immunohistochemistry. Each focus of prostate 
cancer will be laser capture microdissected from frozen tumor sections prior to 
“omics” analyses. When a single lesion exhibits areas of differing Gleason 
grade, an area representative of each component will be separately 
microdissected and subjected to analysis. 
b. Metastatic tumor tissue. As part of the SU2C/WCDT, our group has 
experience in conducting radiographically directed biopsies with 68% of 
attempted bone biopsies yielding tissue sufficient for sequencing analysis (with 
an overall success of 78% taking all metastatic sites into account). Cores (n=6-
8) will be freshly frozen (n=4-6) and formalin-fixed for paraffin embedding (n=2). 
Laser capture microdissection of frozen specimens from biopsies will also be 
performed to enrich samples for tumor tissue for sequencing analyses, as is 
standard practice for metastatic biopsies in our SU2C/WCDT cohort. 
3. Experimental.  
a. For Aim 1.1, we will perform unsupervised clustering based on CNAs 
(determined by CGH) of the metastatic and primary tumors for each patient. As 
well, we will perform unsupervised clustering based on somatic mutational 
status in exons, and transcript levels by RNA-sequencing. These analyses are 
expected to identify the intraprostatic primary tumor foci most closely related to 
the metastatic tumor for each patient (i.e. the metastasis generating 
intraprostatic tumor, or “true” primary). 
b. For 
Aim 
1.2, 
genomic 
analyses 
will 
be 
performed 
for 
pattern 
recognition/discovery of factors that may be characteristic of the “true” primary 
tumor versus other non-metastasizing intraprostatic tumors. We will apply 
various bioinformatic tools for comparisons amongst the primary tumors: 
Pathway Recognition using Data Integration on Genomic Models (PARADIGM) 
and Differential Pathway Signature Analysis (DiPSC) (57–59). PARADIGM 
analysis integrates omics level datasets (i.e. mRNA expression, CNAs, and 
mutation status) to determine pathway activity and key signaling nodes. DiPSC 
utilizes a statistical bootstrap and Pearsons Correlation to determine the 
relationship of phenotype (in this case, the potentially lethal “true” primaries 
versus indolent primaries) to these patterns of pathway activation, for 
determination of discriminative networks characteristic of each phenotype. 
These analyses are similar to those used in the context of the SU2C/WCDT 
project and have been developed and applied by Ted Goldstein, PhD (UCSC). 
 
4. Considerations for genomic analyses (for both Aims 1 and 2). 
 
Comparative genomic hybridizations will be performed on the microdissected 
metastatic and primary tumors for each patient and normalized against germline 
DNA (PBMCs) from each individual. The circular binary segmentation algorithm of 


63 
 
the DNAcopy Bioconductor package will be used to segment DNA copy number 
data and identify regions with abnormal copy number. Data Pre-Processing: Our 
pre-processing pipeline for exome and RNA-sequencing involves the following 
steps: Quality control, Mapping reads, Counting reads per feature, and 
Normalization, filtering and correction for batch effects.  Quality control. Quality 
control will be performed on the raw sequence data files from exome capture and 
RNA sequencing experiments, which contain multiple short-read sequences with 
Phred quality score information. Useful tools for QC and pre-processing are 
FastQC and FASTX-Toolkit. FastQC aims to provide a QC report that can spot 
problems which originate either in the sequencer or in the starting library material. 
The QC metrics we will be looking at include: a) Per-base sequence quality; b) 
Per-sequence GC content for possible library contamination; c) Over-representing 
sequence potential adapter contamination; d) Duplicating sequences for 
enrichment bias. Once we have identified the quality issues, a collection of 
command-line tools called FASTX-Toolkit will be used to perform a series of pre-
processing on the FASTA/FASTQ files. Our experience with similar data confirms 
that performing quality control and necessary pre-processing with the raw 
sequence data files will produce better mapping results. Mapping reads. Once the 
raw sequence data files are cleaned and quality control is performed, we will map 
short reads of each sample to the reference genome hg19 by a short read aligner 
TopHat2. TopHat2 aligns exome and RNA-sequencing reads to mammalian-sized 
genomes using the ultra-fast high-throughput short read aligner Bowtie2 as its core 
read-alignment engine. After mapping, a SAM/BAM file will be generated for each 
sample containing the aligned sequence data.  We will use the flagstat command 
in SAMTools to perform quality check on the alignment files. SNV calling. For 
exome reads, VarScan will be utilized to annotate mutations and to generate single 
nucleotide variant (SNV) and indel calls. Counting reads per feature. The final 
step of data preprocessing is to summarize and count the aligned reads by 
genomic features (e.g., genes, transcripts) for each sample. A useful tool for this 
task is HTSeq-count. HTSeq-count counts the reads that align to each exon and 
sum them up for each gene. For dealing with reads that overlap more than one 
feature, we will use the ‘union’ mode setting. Normalization, filtering and 
correction for batch effects. For both exome and RNA sequencing, we will use 
a Bioconductor package ‘edgeR’ to perform normalization, filtering and batch 
effects correction. We will implement Trimmed Mean of M Values (TMM) 
normalization, which estimates scale factors between samples that can be 
incorporated into currently used statistical methods. We will also filter out tags with 
very low counts to reduce the number of features being tested and thus an increase 
in statistical power. For batch-effect correction, our strategy would be to include 
potential batch effects as additive terms when performing statistical modeling and 
testing. We will compute the false discovery rate (FDR) of Benjamini and Hochberg 
to correct for multiple testing. 
B. Aim 2. We will determine genomic and transcriptomic features specific to the 
metastatic prostate cancers as compared to the “true” primary tumors from which they 
arose (identified in Aim 1.1), and also investigate evidence of EMT and MAPK 
activation in these metastatic versus primary tumors. 
1. Rationale.  Prior studies evaluating genomic and transcriptomic features 
characteristic of metastatic prostate cancer have relied upon tumor tissue derived 
from heavily pre-treated patients, potentially obscuring the drivers of the initial 


64 
 
metastatic event, and compared these metastatic cancers to localized prostate 
cancers, many of which would never have yielded metastases. Therefore, in order 
to specifically interrogate the immediate drivers of a metastatic event, one would 
ideally compare metastatic tumors to the specific intraprostatic tumor from which 
these metastases arose. Our own data suggest an EMT may accompany or drive 
metastasis, and that MAPK activation may be a characteristic feature of metastatic 
prostate cancers and potentially primary prostate cancers with high metastatic 
potential. 
2. Experimental. 
a. For Aim 2.1, we will determine genomic and transcriptomic features specific to 
the metastatic prostate cancers versus the “true” primary tumors that gave rise 
to these metastases.  The genomic analyses will be performed for pattern 
recognition/discovery of factors that may be characteristic of metastatic versus 
the primary tumors using the bioinformatic tools described in Aim 1 
(PARADIGM and DiPSC). 
b. For Aim 2.2, we will assess MAPK activity (p-ERK), and EMT markers (N-
cadherin and vimentin) in each metastasis and its corresponding “true” primary 
tumor. IntMax scores (scale of 0-300) = (% of positive tumor cells [scale of 0-
100]) X (intensity of staining based on 0-3 scale [scale of 0-3]) will be measured 
by quantitative image analysis for p-ERK as well as EMT markers (N-cad, and 
vimentin) by immunohistochemistry. Comparisons will be made between 
tissues from metastatic sites versus the primary tumors. Average IntMax will 
be compared using a paired t-test or Wilcoxon signed-rank test (if the IntMax 
scores do not follow a normal distribution). Fifteen evaluable tumors per group 
(metastasis vs primary) provides 90.5% power to detect a mean difference of 
1.5 standard deviations (SDs) between groups, and 78.4% power to detect a 
mean difference of 1.3 SDs. The effect sizes used for this power analysis are 
based on our preliminary data in EMT markers expressed in local versus 
metastatic prostate cancer specimens and from animal model systems. We, in 
fact, expect to have at least 19 evaluable metastatic tumors (total N=28 x 68% 
successful biopsies yielding tissue for analysis = 19 evaluable specimens). 
Therefore, our study is likely sufficiently powered to detect differences in these 
histological markers. 
 
C. Aim 3. Evaluation of CTCs, ctDNA, and immunophenotypes in blood. We will 
incorporate evaluation of circulating tumor cells (CTCs) detected in enrolled patients 
pre-treatment. VortexChip isolates highly purified CTCs by size alone and without 
need for antibody capture. Not relying on detection of epithelial markers, it allows 
capture of circulating tumor cells that have undergone EMT. It also enables 
downstream analyses by next generation sequencing.  
 
1. We have detected CTCs in 80% of patients with metastatic prostate cancer (N=20, 
Figure 3B). In a pilot experiment, we successfully conducted RNA-sequencing in 
three cells from two patients with mCRPC (Figure 3C), and we have shown that 
VortexChip can capture prostate cancer CTCs that have undergone EMT and 
express vimentin and N-cadherin (not shown). 
2. We will obtain blood from the patients at their baseline visit and capture the 
resulting CTCs using Vortex Chip. Samples will be included in the study if at least 


65 
 
5 CTCs are present for analysis. We will perform RNA sequencing and whole 
exome sequencing analysis on up to 20 CTCs isolated from each specimen, with 
a minimum of 5 cells. We will sort CTCs by expression of vimentin and N-cadherin 
to detect CTCs that may have undergone EMT. We will use clustering analysis of 
SNVs in the CTCs, and matched primary and metastatic tumors in each patient in 
an attempt to determine the site of origin of the CTCs. 
3. Circulating nucleic acids (RNA, DNA) will be isolated using the Qiagen Circulating 
Nucleic Acids Kit, and quantified by the Agilent Bioanalyzer High Sensitivity DNA 
Kit. DNA samples will then be whole genome amplified with an equal amount of 
normal reference DNA. 
4. PBMCs will be isolated over Ficoll-gradients and stored frozen until analysis. 
Lymphoid and myeloid subset phenotyping will be assessed by antibody staining 
and multi-color flow cytometry (LSR-Fortessa, BD). Circulating cytokines, PGE2, 
prostate-tumor 
specific 
antibodies, 
HMGB-1 
protein 
and 
the 
tryptophan/kynurenine ratio in plasma will be quantified by ELISA. Tumor-specific 
CD4 and CD8 T cell activities to known HLA-matched, immunodominant, tumor-
associated antigens will be assessed ex vivo by IFNγ-ELISPOT and dextramer 
staining. The tumor antigens of choice will include PSA and PSMA. 
5. We anticipate that most patients in the trial will have detectable CTCs based on 
our prior experience in the detection of CTCs in metastatic prostate cancer 
patients, although a smaller frequency of CTC positivity is possible due to a lower 
expected burden of disease in this oligometastatic patient cohort. We also 
anticipate that the CTCs likely originate from the “true” primary tumors or 
metastatic tumors. However, it is possible that we may trace them to the other 
intraprostatic tumors in one or more patients. We anticipate that changes in 
lymphoid and myeloid subsets may be occur over time, as well as tumor specific 
T cell responses. 
 
 
 


66 
 
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