A Single-arm Open Label Biomarker Study of Standard-of-care Radium-223 Chloride for Metastatic Castration-resistant Prostate Cancer
Phase: PHASE2
Conditions: Prostate Cancer, Castration-resistant Prostate Cancer, Castration-resistant Prostate Cancer Metastatic to Bone
Sponsor: Massachusetts General Hospital

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STUDY TITLE: A single-arm open label biomarker study of standard-of-care Radium-223 
chloride for metastatic castration-resistant prostate cancer
Test drug(s):
Radium Ra 223 dichloride  (Radium Ra 223 dichloride, BAY 88-
8223) 
Study purpose:
Exploratory response/biomarker evaluation 
Clinical study phase:
II
Version no.:
Date:
30 July 2019    
Amendment no.:
Amendment: PET/MRI 
Clarification
Date:
N/A    
Bayer Study no.:
ONC-2013-119
Institution Study no.:
14-375
Principal Investigator:
Philip J. Saylor, MD
Massachusetts General Hospital Cancer Center
55 Fruit Street, Yawkey 7E; Boston, MA 02114
psaylor@partners.org; Phone: 617-643-1763, Fax: 617-726-8685
Co-Principal Investigator:
Matthew R. Smith, MD, PhD
Massachusetts General Hospital Cancer Center
55 Fruit Street, Yawkey 7E; Boston, MA 02114
smith.matthew@mgh.harvard.edu; Phone: 617-724-5257
Research Nurse:
Carol Gurski, RN
Data Manager:
To be determined.
Statistician:
Susanna Jacobus, MSc, MBA
Dana Farber Cancer Institute
Email: jacobus@jimmy.harvard.edu; Phone : (617) 582-7927
The study will be conducted in compliance with the protocol, ICH-GCP and any applicable regulatory requirements.
Confidential
The information provided in this document is strictly confidential and is intended solely for the guidance of 
the clinical investigation.  Reproduction or disclosure of this document - whether in part or in full - to 
parties not associated with the clinical investigation, or its use for any other purpose, without the prior 
written consent of the Principal Investigator is not permitted.
Throughout this document, symbols indicating proprietary names (, TM) are not displayed.  Hence, the appearance of 
product names without these symbols does not imply that these names are not protected.
NCT02346526

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Synopsis
[Title]
A single-arm open label biomarker study of standard-of-care Radium-223 
chloride for metastatic castration-resistant prostate cancer
Clinical study phase
II
Study objective(s)
The primary objective of this study is to evaluate the quantitative effect of Ra-
223 on 99mTc-MDP bone scan in men with castration-resistant prostate cancer 
metastatic to bone. Secondary objectives include the description of mean 
percent change in bone scan lesion area by 18-month survival status as well as 
the evaluation of the effects of Ra-223 by measures categorized as follows: 
(A) Imaging biomarkers
(B) Standard and novel circulating tumor cell assays
(C) Circulating biomarkers of the tumor microenvironment
(D) Patient reported pain and quality of life

 
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Name of active ingredient
Dose(s) 
Route of administration
Radium Ra 223 dichloride
50 kBq/kg body weight (55kBq/kg after NIST update)
Intravenous
Indication
Standard-of-care use for castration-resistant prostate cancer metastatic to bone
Diagnosis and main criteria for 
inclusion
This study will prospectively enroll men with castration-resistant prostate 
cancer (CRPC) metastatic to bone. 
Study design
Exhaustive inclusion/exclusion criteria are detailed in the Section 5 of the 
protocol. Schema: 
1.
Screeningassessments include 99mTc MDP bone scan, & blood testing 
2.
Enrollment 
3.
Baseline assessment includes Fluciclovine PET/MRI 
4.
Ra-223, administered monthly for a total of six anticipated doses with 
concurrent protocol-specified correlative imaging and blood testing
5.
Conclusion of study treatment with sixth dose of Ra-223
6.
Follow-up and testing as detailed in experimental protocol
Standard androgen deprivation therapy (ADT) will be continued throughout 
study participation. Men will be treated for 6 months on protocol in the 
absence of progression or intolerable side effects. Men will be evaluated 
according to the table provided in protocol Section 7.1.1. At the end of 6 
months of treatment, Ra-223 will be discontinued. Subsequent therapies will 
be at the discretion of the treating oncologist. 
Type of control
This is a single-arm exploratory biomarker study.
Number of subjects
22
Study Endpoints
Please see Protocol Section 7.13 & 7.14
Plan for statistical analysis
Bone scan response is defined as a ≥ 30% decrease in quantitative bone scan 
lesion area (BSA) from baseline to the end of month 2 of Ra-223 treatment 
(i.e. after 2 of 6 possible Ra-223 doses). In a one-stage design, 22 subjects will 
be enrolled to evaluate the primary endpoint of bone scan response. Assuming 
90% of subjects with adequate data, the evaluable sample size is 20 subjects. 
With 20 subjects, given target power of 90% and 1-sided 5% alpha, a null 
hypothesis of ≤ 10% versus an alternative hypothesis of ≥ 35% in the 
proportion of patients achieving response can be evaluated. If 5 or more 
responses are observed in 20 subjects, this method of response assessment will 
be considered promising. 
Amendment Rationale
This amendment is to clarify that the baseline Fluciclovine PET/MRI should only be 
conducted after a subject is successfully registered to the trial, but prior to starting treatment 
on T1D1. 

 
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Table of Contents
Synopsis......................................................................................................................................2
1.
Introduction .......................................................................................................................11
1.1
Background................................................................................................................11
1.2
Rationale of the study ................................................................................................14
1.3
Radium Ra 223 dichloride .........................................................................................15
1.3.1
Description .........................................................................................................15
Radium Ra 223 dichloride is an alpha particle-emitting radio-pharmaceutical, with a 
half-life ( t1/2 )of 11.4 days.It is a  sterile clear, colorless, isotonic, solution to 
be administered intravenously............................................................................15
1.3.2
NIST ...................................................................................................................15
The quantification of radium-223 radioactivity in Xofigo (radium-223 dichloride;BAY 
88-8223) is based on the primary standardization performed by the US NIST. 
National Institute of Standards and Technology prepares the standard reference 
material (SRM) using an official dial setting (primary standardization) as 
published (2). The NIST SRM is used to calibrate the instruments in production 
and quality control for both the drug substance and drug product. Additionally, 
the NIST SRM is used to prepare theNIST traceable Ra-223 reference materials 
which are then sent to the end-users (e.g., nuclear medicine laboratory 
physicians or technicians) for dial-setting of their dose calibrators, to allow 
verification of the patient dose. ..........................................................................15
In 2014, NIST performed a re-assessment of the primary standardization based on 
preliminary information suggesting a potential discrepancy of approximately 8-
10% between the published NIST primary standardization (2) and results 
obtained by other nationalmetrology institutes (United Kingdom, Germany, 
Japan). After completion of the re-assessment, NIST reported their findings (3) 
and had issued a revised NIST SRM in 2015. The discrepancy in the NIST 
standardization was determined to be −9.5% between activity values obtained 
using the old reference standard relative to the new primary standardization. 
Consequently the current numerical values need to be corrected by approx. + 
10.5%. The current NIST standard for radium-223 dichloride will remain in 
effect until the FDA has fully approved the regulatory variation submitted for 
Xofigo and is anticipated in the 2nd quarter of 2016. .........................................15
The change in the numerical description of the patient’s dose, product strength and 
labeled vial activity does not impact the safety or efficacy of Xofigo. The 
change in the NIST radium-223 standard has no impact on subjects; dose 
subjects are receiving, and will continue to receive...........................................15
Subjects will receive the same actual dose and volume that was studied in Study 15245 
(BC1-06 dosimetry study ) and is associated with the proven safety and efficacy 
of radium-223 dichloride, though the stated nominal radiation dose received is 
being updated to reflect the new standard. The formula for the calculation of the 
volume to be administered has to be changed respectively(see dosing section).
............................................................................................................................15
1.3.3
Mechanism of Action .........................................................................................16

 
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1.3.4
Preclinical...........................................................................................................16
1.3.5
Clinical Experience Summary............................................................................17
2.
Study objectives.................................................................................................................21
3.
Investigator[s] and other study participants ......................................................................21
4.
Study design ......................................................................................................................22
5.
Study population................................................................................................................22
5.1
Eligibility ...................................................................................................................22
5.1.1
Inclusion criteria.................................................................................................22
5.1.2
Exclusion criteria................................................................................................23
5.1.3
Inclusion of Women and Minorities...................................................................24
5.1.4
Excluded therapies and medications, previous and concomitant.......................24
5.2
Withdrawal of subjects from study............................................................................24
5.2.1
Registration Procedures......................................................................................24
5.2.2
Withdrawal .........................................................................................................26
5.2.3
Duration of Follow-up........................................................................................26
5.2.4
Screen Failures/Dropouts ...................................................................................27
5.2.5
Replacement .......................................................................................................27
6.
Treatment...........................................................................................................................27
6.1
Treatments to be administered...................................................................................27
6.1.1
Radium Ra 223 dichloride, 50 kBq/kg body weight, will be administered as a 
bolus intravenous (IV) injection (up to 1 minute) at intervals of every 4 weeks 
for up to 6 cycles (55kBq/kg body weight after implementation of the NIST 
update)................................................................................................................27
6.2
Treatment assignment ................................................................................................27
6.2.1
Radium Ra 223 dichloride..................................................................................27
6.2.2
Dose calibration..................................................................................................29
6.2.3
Dosimetry ...........................................................................................................29
6.2.4
Dose handling.....................................................................................................30
6.2.5
Dose calculation .................................................................................................30
6.2.6
Dose preparation.................................................................................................31
6.2.7
Dose administration............................................................................................31
6.2.8
Dose Modification/Delays..................................................................................31
6.3  Drug logistics and accountability ..................................................................................32
6.3.1 Accountability..........................................................................................................32
6.3.2
Destruction and Return.......................................................................................33
6.4
Treatment compliance................................................................................................33
6.5
Prior and concomitant therapy-..................................................................................33
7.
Procedures and variables ...................................................................................................33
7.1
Schedule of procedures..............................................................................................34
7.1.1
Tabulated overview ............................................................................................35
7.1.2
Timing of assessments........................................................................................36
7.1.3
Medical history...................................................................................................36
7.2
99mTc MDP bone scan: Standard clinical bone scan will be carried out per 
institutional practice...................................................................................................37
7.3
CT a/p: Standard clinical CT of the abdomen and pelvis will be carried out per 
institutional practice...................................................................................................37

 
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7.4
Medication history: Medication history, including any changes in medication use 
since last assessment, will be reviewed. ....................................................................37
7.5
Chemistry panel: Standard comprehensive metabolic panel (including sodium, 
potassium, chloride, bicarbonate, BUN, creatinine, glucose, total protein, albumin, 
AST, ALT, total bilirubin, direct bilirubin, and alkaline phosphatase) will be carried 
out at the MGH clinical laboratory in real time.........................................................37
7.6
CBC with differential: Standard comprehensive metabolic panel (including white 
blood cell count, hemoglobin, hematocrit, platelets, and differential) will be carried 
out at the MGH clinical laboratory in real time.........................................................37
7.7
PSA: Standard PSA assay will be carried out at the MGH clinical laboratory in real 
time. ...........................................................................................................................37
7.8
Fluciclovine PET/MRI: This testing will be carried out only for 10 study 
participants. They will be chosen by investigator discretion in light of their 
willingness and medical eligibility to undergo PET/MRI (e.g. pacemaker is a 
contraindication, claustrophobia is a contraindication). The agent used in this study 
will be the radiolabled agent (fluciclovine) that will be administered prior to 
standard whole body PET imaging which includes skull base to thigh. Fluciclovine 
will be handled by qualified personnel, familiar with procedures that minimize 
undue exposure to themselves and the environment. PET/MRI imaging will be 
carried out per standard/clinical institutional protocol. Thus far, there is limited 
experience using fluciclovine PET/MRI; it is investigational. ..................................37
7.9
CTCs by CellSearch (see Appendix G for detailed instructions): .............................37
At study-designated timepoints, 7.5 mL of whole blood will be collected in a special 
CellSave tube (available from Veridex/Janssen Diagnostics), which is then stored at 
room temperature until processed. It will be sent to the BWH CTC Lab for analysis. 
The BWH Circulating Tumor Cell Laboratory provides circulating tumor cell (CTC) 
services using the Veridex/Janssen Diagnostics CellSearch immunomagnetic 
platform. The lab is located to a CLIA-approved space within the BWH Cytology 
Division (Brigham and Women's Hospital, Medical Research Building, 3rd Floor; 
75 Francis St., Boston, MA 02115). Major equipment includes the CellSearch 
Autoprep, and CellTracks Analyzer II. The CTC Lab provides clinical enumeration 
of CTCs for patients with metastatic prostate cancer using the FDA-cleared 
Veridex/Janssen Diagnostics CellSearch System.  It also functions as the BWH CTC 
Core Lab (http://www.partners.org/researchcores/CTC/CTC_BWH.html) providing 
enumeration and isolation of CTC for research purposes. The current director is 
Alarice Lowe, M.D. (Phone: 617-525-8696, Fax: 617-739-6192, 
Email: aclowe@partners.org). ...................................................................................37
7.10
CTCs by microfluidic chip: (see Appendix B for detailed instructions) ...................38
Two 10 mL samples of peripheral blood will be collected into vacutainer tubes (Becton-
Dickinson) containing the anticoagulant EDTA. Samples will be shipped by U.S. 
Ground courier to the MGH Charlestown Navy Yard laboratories of Drs. Daniel 
Haber and Shyamala Maheswaran. Alternatively, a member of the Haber or 
Maheswaran labs will personally transport the samples from the MGH Cancer 
Center to the MGH Charlestown Navy Yard laboratories. Samples will be processed 
through the experimental platform within 6 hours of collection, according to 
previously established protocols52,53 with modifications subject to current lab 

 
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protocol. Briefly, a 5 mL aliquot of blood will be placed into an air-tight conical 
tube on a rocker assembly, and blood will be pneumatically driven through the 
microfluidic chip at a protocol-specified flow rate. The microfluidic chip will then 
be flushed with phosphate-buffered saline at a protocol specified flow rate to remove 
nonspecifically bound cells. CTCs on the chip will be fixed, permeabilized, and 
stained with antibodies. Staining for PSA, PSMA, DAPI, Ki-67, and other proteins 
will be performed. Captured CTCs will then be identified and enumerated using the 
automated detection system as previously described52. RNA and/or DNA will also 
be isolated from CTCs captured on a separate chip for gene expression analyses. 
Any remaining isolated RNA or DNA will be stored frozen at -70 to -90 degrees C 
for batch global profiling analyses. See Appendix B additional processing 
instructions.................................................................................................................38
7.11
Bone turnover markers (see Appendix H for detailed instructions): .........................38
Sera will be collected from subjects at study-specified timepoints. It will be processed and 
stored as detailed in Appendix H. Serum N-telopeptide concentrations will be 
determined at a Massachusetts General Hospital core laboratory using a competitive 
inhibition enzyme-linked immunosorbent assay (ELISA/EIA) (Osteomark® NTx 
Serum, Alere Scarborough, Inc., Scarborough,ME). The intra-assay and inter-assay 
variability are 4.6% and 6.9%, respectively. Quantitative bone-specific alkaline 
phosphatase (BAP) will be determined at a Massachusetts General Hospital core 
laboratory using an enzyme immunoassay (MicroVueTM BAP EIA; Quidel 
Corporation, San Diego, CA).....................................................................................38
7.12
Blood biomarkers (see Appendix C for detailed instructions): .................................38
Blood samples will be collected at study specific timepoints. For the purposes of this 
testing, at least 10 mL will be collected in a PURPLE top (EDTA plasma) tube. 
After processing, separated peripheral blood cells will be immediately stained using 
fluorescently labeled antibodies and analyzed using an LSR-II flow cytometer 
within the Steele Laboratory's facility. Dr. Dan G. Duda, D.M.D., Ph.D. (Associate 
Professor at MGH/HMS), who has over 12 years of experience in clinical correlative 
studies, supervises the Steele Lab's Core operation. Plasma analysis will be carried 
out for a panel of circulating angiogenic and inflammatory molecules. They include 
vascular endothelial growth factor (VEGF), placental-derived growth factor (PlGF), 
soluble (s)VEGFR1, basic fibroblast growth factor (bFGF), VEGF-C, VEGF-D and 
sTie2 using multiplex (7-plex) and IL-1β, IL-6, IL-8 and TNF-α (4-plex) protein 
array kits from Meso-Scale Discovery (Gaithersburg, MD), and stromal cell-
derived factor 1a (SDF1a), hepatocyte growth factor (HGF), soluble (s)c-MET, and 
s-c-KIT using ELISA from R&D Systems (Minneapolis, MN). Finally, we 
will evaluate biomarkers of tumor hypoxia, by measuring plasma carbonic 
anhydrase IX (CAIX) levels as well as biomarkers of osteoclast and osteoblast 
activity (plasma C-telopeptide and total alkaline phosphatase) using ELISA from 
R&D Systems. Samples will be run in duplicate.......................................................38
7.13
Patient reported outcomes: Key patient reported outcome measures will include:  1. 
Analgesic use as recorded in a medication diary (see Appendix D) 2. Changes in 
quality of life as measured by a validated assessment tool (EQ-5D-3L; see Appendix 
E)  3. Changes in pain and narcotic analgesic use as assessed by 4-item  
questionnaire taken from the MD Anderson Brief Pain Inventory (BPI; see 

 
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Appendix F)54:       A. Pain at its worst in the last 24 hours (BPI question 3)      B. 
Average pain in the last 24 hours (BPI question 5)      C. Effect of pain on general 
activity (BPI question 9A)      D. Effect of pain on sleep (BPI question 9F) ............39
7.14
Efficacy......................................................................................................................39
7.15
Safety .........................................................................................................................40
7.15.1
Adverse events ...................................................................................................40
7.15.2
Pregnancies.........................................................................................................46
7.15.3
Further safety......................................................................................................46
7.16
Other procedures and variables..................................................................................46
7.17
Appropriateness of procedures / measurements ........................................................46
8.
Statistical methods and determination of sample size.......................................................46
9.
Data handling and quality assurance .................................................................................49
9.1
Data recording............................................................................................................49
9.2
Monitoring .................................................................................................................49
9.3
Audit and inspection ..................................................................................................49
9.4
Archiving ...................................................................................................................49
10. Premature termination of the study ...................................................................................50
11. Ethical and legal aspects....................................................................................................50
11.1
Ethical and legal conduct of the study .......................................................................50
11.2
Subject information and consent................................................................................51
11.3
Publication policy ......................................................................................................52
11.3.1
Publications and NIST........................................................................................52
11.4
Confidentiality ...........................................................................................................52
12. Reference list.....................................................................................................................52
Specimen Collection, Handling, Storage:.................................................................................64
Supplies: CellSave Preservative Tubes ....................................................................................64
Procedure:.................................................................................................................................64
Procedure notes: .......................................................................................................................64
Reference: Veridex, LLC,  CellSearch System Training Guide. Page 1-6, section 1-4...........64

 
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List of abbreviations 
AE 
Adverse Event
ALP 
Alkaline Phosphatase
ALSYMPCA 
Alpharadin in Symptomatic Prostate Cancer
ALT 
Alanine Aminotransferase
ANC 
Absolute Neutrophil Count
AST 
Aspartate Aminotransferase
BPI-SF 
Brief Pain Index (Short Form)
BSoC 
Best Standard of Care
CBC 
Complete Blood Count
CRO 
Clinical Research Organization
CRPC 
Castration Resistant Prostate Cancer
CT 
Computed tomography
CTCAE 
Common Terminology Criteria for Adverse Events; version 4.03
DK 
Decay Correction Factor
EBRT 
External Beam Radiation Therapy
ECOG 
Eastern Co-operative Oncology Group
eCRF 
Electronic Case Report Form
EU 
European Union
GCP 
Good Clinical Practice
GCL 
Global Clinical Leader
GMP 
Good Manufacturing Practice
HRPC 
Hormone Resistant Prostate Cancer
IB 
Investigator Brochure
ICF 
Informed Consent Form
ICH 
International Conference on Harmonization
IDMC 
Independent data monitoring committee
IEC 
Independent Ethics Committee
IRB
 Institutional Review Board
IV 
Intravenous
IxRS
 Interactive Voice/Web Response System (IVR/IVRS)
kBq 
Kilobecquerel; SI Unit of Radioactivity
kg 
Kilogram
LHRH 
Luteinizing-Hormone-Releasing Hormone; also known as Gonadotropin-
Releaseing Hormone (GnRH)
mCi 
Millicuries
MedDRA 
Medical Dictionary for Regulatory Activities
mL 
Milliliter
MRI 
Magnetic Resonance Imaging
NCI 
National Cancer Institute
NIST
National Institute od Standards and Technology 
NYHA 
New York Heart Association
OS 
Overall Survival
PS 
Performance Status
PSA 
Prostate Specific Antigen
QoL 
Quality of life
SAE 
Serious Adverse event
SAP 
Statistical Analysis Plan
SAS 
Statistical Analysis Software
SRE 
Skeletal-related Events
SUSAR 
Suspected Unexpected Serious Adverse Reaction
TEAE 
Treatment-emergent Adverse Event
ULN 
Upper Limit of Normal
WHO-DD 
World Health Organization – Drug Dictionary

 
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Definitions of terms
Radium Ra 223 dichloride:  
A targeted alpha particle-emitting pharmaceutical (a 
radiopharmaceutical  drug), is a ready-to-use solution for intravenous 
injection containing the drug substance radium dichloride. The active 
moiety is the alpha particle emitting nuclide Ra-223, present as a 
divalent cation (223Ra2+).
Dose:                                  
Doses are given as kilobecquerel (kBq) per kilogram body weight, with 
the corresponding dose given in millicurie (mCi) per kilogram in 
parenthesis. The term “dose” is used to describe the quantity of 
radioactivity from Radium Ra 223 dichloride administered.
1. Introduction
1.1 Background
Background: Skeletal Morbidity and Ra-223 Activity in CRPC
Bone metastases are present in 80-90% of men with advanced prostate cancer and account for 
much of the considerable disease-related morbidity and mortality. Bone-targeted therapies 
have therefore long been an important potential component of prostate cancer treatment. Until 
recently, bone-targeted therapies had been shown to produce clinical benefits (e.g. prevention 
of skeletal-related events with osteoclast inhibition1, 2 or palliation of bone pain with beta-
emitting radiopharmaceuticals3) but not to improve overall survival. The systemically-
delivered alpha-emitting radiopharmaceutical Ra-223 has recently been shown to 
significantly improve survival as well as prevent skeletal-related events among men with 
CRPC predominantly affecting bone.4-6 These findings led to its 2013 FDA approval. 
Ra-223 Efficacy: Need for Biomarkers
The significant survival advantage demonstrated in phase III study of Ra-223 establishes it as 
an active agent within an expanding group of systemic therapies that improve survival in 
mCRPC. Biomarkers of its activity, however, are lacking. Ra-223 treatment has not been 
shown to dependably improve serum PSA, a biomarker that has correlated with drug activity 
for androgen receptor (AR) targeted therapies (GnRH agonists, androgen biosynthesis 
inhibitors such as abiraterone7, 8, antiandrogens such as enzalutamide9, and taxane 
chemotherapy10, 11) but not for non-AR targeted therapies (immunotherapy with sipuleucel-
T12, radiopharmaceuticals such as Ra-223, and tyrosine kinase inhibitors13-15). In addition, 
Ra-223 has not been shown to produce measurable improvements in CT or bone scan 
evidence of disease. Biomarkers of Ra-223 activity are needed. Useful biomarkers would be 
expected to provide any or all of the following: (1) better clarify Ra-223 mechanism of 
action, (2) improve disease monitoring during Ra-223 therapy, and (3) inform the use of Ra-
223 in sequence or in combination with other agents that are active in CRPC. 

 
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Quantitative Planar 99mTc MDP Bone Scan
Bone scan with Technetium-99m methylene diphosphonate (99mTc MDP) is a widely-used 
and accepted imaging modality in both clinical practice and research related to prostate 
cancer.16 It is an indirect measure of disease activity as its uptake reflects tracer deposition by 
osteoblasts17, 18, a cell type that is highly active in the presence of prostate cancer bone 
metastases. One historical limitation of bone scans is that until recently they have not been 
interpreted in a quantitative way. Guidelines for their use in clinical trials (e.g. Prostate 
Cancer Clinical Trials Working Group 2 (PCWG2)) have recommended only their 
examination for the presence or absence of 2 or more new lesions.16 More recently, computer-
aided quantitative methods for bone scan assessment have been described.19, 20 Computer-
aided quantitative assessments allow for a more rigorous serial assessment of scan evidence 
of bone metastases without the cost or availability barriers that are presented by alternative 
imaging strategies such as sodium fluoride positron emission tomography (PET). We 
hypothesize that serial computer-aided quantitative assessment of 99mTc MDP bone scans will 
reveal Ra-223-induced improvements in skeletal disease burden. 
Pain and Quality of Life with Metastatic CRPC
Bone metastases are the major cause of morbidity and mortality in men with metastatic 
prostate cancer. Beta-emitting radiopharmaceuticals that seek bone (Samarium-153 EDTMP, 
and Strontium-89) are approved to treat bone pain in men with symptomatic bone 
metastases.3 These beta-emitting radiopharmaceuticals have a limited role in the management 
of metastatic prostate cancer, however, because of hematologic toxicity and lack of 
documented benefit on cancer outcomes (survival or disease progression). The effects of the 
alpha-emitting Ra-223 on pain and quality of life are unknown because that information was 
not collected in the pivotal study. Ra-223 may palliate bone pain similar to previously 
available beta-emitting radiopharmaceuticals.3 Alternatively, the more limited depth of 
penetration of Ra-223 (<100 micrometers versus several millimeters for beta emitters) may 
limit the effect of Ra-223 on pain and quality of life. We hypothesize that the known benefits 
of Ra-223 in this population will be accompanied by prospectively-observed improvements in 
patient-reported pain and quality of life. 
Molecular Imaging in Advanced Prostate Cancer 
Functional imaging of neoplasms using PET/CT has become an integral part of evaluation of 
a diverse group of malignancies. 18F-fluoro-deoxyglucose (18F -FDG) is the most widely used 
PET tracer in oncology but has not been useful in prostate cancer due to inadequate 
sensitivity as well as intense PET signal in the bladder due to renal elimination. Anti-1-
amino-3-fluorine 18-fluorocyclobutane-1-carboxylic acid (FACBC; now known as 
fluciclovine) PET is an alternative PET tracer that is an L-leucine analog and has 
demonstrated promising sensitivity and specificity for the detection of prostate cancer.21-23 
Fluciclovine was approved by the FDA in 2016. Since fluciclovine uptake depends on the 
tumor cells themselves and not on physiological response of adjacent bone or lymph tissue, 
detection based on fluciclovine PET imaging is effective for soft tissue, lymphatic, and 
osseous metastases. We hypothesize that quantitative assessment of bone tumor volume as 
assessed by fluciclovine PET will reveal a decrease after 8 weeks of Ra-223 treatment. 

 
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Further, we expect that this study will establish fluciclovine PET as an imaging biomarker of 
Ra-223 activity for potential use in future trials and in clinical practice. 
Circulating Tumor Cells
Detection and isolation of CTCs in peripheral blood is an emerging analytically-valid 
biomarker of prognosis and of response to therapy in men with prostate cancer.24-27 
Enumeration of CTCs at baseline and post-treatment is prognostic of survival, and the 
shedding of cells into the circulation represents an intrinsic property of the tumor that is 
distinct from extent of disease.28, 29 These rare peripherally circulating malignant epithelial 
cells have been identified by several techniques. We propose to use two distinct assays to 
study Ra-223 induced effects on CTCs. First, we will use the only FDA-approved and 
broadly-available assay: separation of the CTCs using immunomagnetic beads conjugated 
with antibody to EpCAM (Veridex CellSearch assay). This assay has been incorporated in 
recent prospective randomized controlled phase III trials of novel agents such as abiraterone 
acetate and enzalutamide. Second, we will use our microfluidic device for CTC isolation30 to 
perform real-time characterization of androgen receptor (AR) signaling activity in CTCs 
(termed the Evans Assay31) as well as analysis of markers of DNA damage and repair (e.g. 
gamma-H2AX). We propose to measure treatment-induced changes in CTC number, 
proliferative index, AR signaling, and DNA damage/repair concurrent with standard and 
experimental imaging assessments. We hypothesize that these studies will further define the 
value of CTCs as a biomarker of Ra-223 activity. 
Bone Turnover Markers
Most bone metastases in men with prostate cancer are characterized by excess bone turnover. 
Number and activity of both osteoblasts and osteoclasts are increased in typical bone 
metastases from prostate cancer. Specific biochemical markers of both osteoblast and 
osteoclast activity are markedly elevated in men with metastatic prostate cancer and levels 
correspond to extent of skeletal involvement.32, 33 In multivariate analyses of data from a large 
prospective clinical trial of men with CRPC and bone metastases, elevated levels of serum 
bone specific alkaline phosphatase (BAP), a specific marker of osteoblast activity, was 
independently associated with shorter overall survival.34 Higher levels of BAP and urinary N-
telopeptide (NTx), a marker of osteoclast activity, are associated with greater risk of skeletal 
morbidity in men with metastatic CRPC. Greater declines in urinary NTx following treatment 
with zoledronic acid are associated with lower risk of skeletal morbidity in men with 
metastatic CRPC.35 We hypothesize that Ra-223 treatment will be associated with reductions 
from baseline in BAP and NTx concurrent with improvement in concurrent assessments by 
standard and experimental imaging studies. 
Biomarkers of Angiogenesis & Inflammation
Biomarkers of inflammation and angiogenesis may substantially improve understanding of 
Ra-223 activity as well as mechanisms of resistance. In particular, SDF1α is a chemokine that 
is highly expressed in the bone marrow and has been shown in preclinical studies to play 
important roles in tumor growth and resistance to therapy. Malignant cells may use SDF1α 
signaling through its receptors CXCR4 and CXCR7 to promote survival and migration. For 
example, CXCR4 and CXCR7 expression has been associated with decreased cell apoptosis 
and increased cell proliferation and migration and new vessel formation, including in prostate 

 
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cancer. In addition, the SDF1α/CXCR4 pathway promotes inflammatory bone marrow 
derived cell (BMDC) recruitment that may facilitate tumor resistance to radiotherapy.36, 37 
BMDCs that infiltrate tumors, also known as the “distant stroma”, are often increased in 
tumors after radiation treatment and have led to the hypothesis that this effect mediates 
resistance.38 Irradiation increases SDF1α expression in both cancer cells and stromal cells.39 
We have also seen an increase in circulating inflammatory factors after hormonal therapy for 
prostate cancer, including increased plasma SDF1α.40 We hypothesize that BMDC infiltration 
after radiation treatment in prostate cancer is mediated by SDF1α, and that SDF1α pathway 
mediates progression after systemically administered radiation for prostate cancer metastatic 
to bone. In exploratory analyses, we will use multiplexed arrays to prospectively measure 
SDF1α as well as other inflammatory and angiogenic biomarkers in plasma at key timepoints 
throughout study-directed therapy.
1.2 Rationale of the study
Radium-223 chloride (Ra-223) is the first bone-seeking radiopharmaceutical to significantly 
improve survival in men with metastatic castration-resistant prostate cancer (mCRPC). 
Despite this clearly-demonstrated benefit in randomized phase III study, the mechanism of 
Ra-223 activity is not fully understood. Further, PSA does not appear to be a reliable 
biomarker of efficacy for therapies such as Ra-223 that do not target the androgen receptor. 
Ra-223 may improve survival through direct anti-tumor effects, through effects on the bone 
microenvironment, or through other mechanisms. We seek to carry out a single arm 
exploratory study that will incorporate comprehensive biomarker studies to clarify its 
mechanism of activity and to establish relevant and clinically-useful biomarkers. Further, we 
will use validated patient reported outcome tools to evaluate the effects of Ra-223 on pain 
and quality of life in the study population. 

 
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1.3 Radium Ra 223 dichloride
1.3.1
Description  
Radium Ra 223 dichloride is an alpha particle-emitting radio-pharmaceutical, with a half-life 
( t1/2 )of 11.4 days.It is a  sterile clear, colorless, isotonic, solution to be administered 
intravenously.                                        
1.3.2
NIST
The quantification of radium-223 radioactivity in Xofigo (radium-223 dichloride;BAY 88-
8223) is based on the primary standardization performed by the US NIST. National Institute 
of Standards and Technology prepares the standard reference material (SRM) using an 
official dial setting (primary standardization) as published (2). The NIST SRM is used to 
calibrate the instruments in production and quality control for both the drug substance and 
drug product. Additionally, the NIST SRM is used to prepare theNIST traceable Ra-223 
reference materials which are then sent to the end-users (e.g., nuclear medicine laboratory 
physicians or technicians) for dial-setting of their dose calibrators, to allow verification of the 
patient dose.
In 2014, NIST performed a re-assessment of the primary standardization based on 
preliminary information suggesting a potential discrepancy of approximately 8-10% between 
the published NIST primary standardization (2) and results obtained by other 
nationalmetrology institutes (United Kingdom, Germany, Japan). After completion of the re-
assessment, NIST reported their findings (3) and had issued a revised NIST SRM in 2015. 
The discrepancy in the NIST standardization was determined to be −9.5% between activity 
values obtained using the old reference standard relative to the new primary standardization. 
Consequently the current numerical values need to be corrected by approx. + 10.5%. The 
current NIST standard for radium-223 dichloride will remain in effect until the FDA has fully 
approved the regulatory variation submitted for Xofigo and is anticipated in the 2nd quarter of 
2016.  
The change in the numerical description of the patient’s dose, product strength and 
labeled vial activity does not impact the safety or efficacy of Xofigo. The change in 
the NIST radium-223 standard has no impact on subjects; dose subjects are receiving, 
and will continue to receive.
Subjects will receive the same actual dose and volume that was studied in Study 15245 
(BC1-06 dosimetry study ) and is associated with the proven safety and efficacy of 

 
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radium-223 dichloride, though the stated nominal radiation dose received is being 
updated to reflect the new standard. The formula for the calculation of the volume to be 
administered has to be changed respectively(see dosing section).
1.3.3
  Mechanism of Action 
The active moiety of Radium Ra 223 dichloride is the alpha particle-emitting isotope 
radium-223 (as radium Ra 223 dichloride), which mimics calcium and forms 
complexes with the bone mineral hydroxyapatite at areas of increased bone turnover, 
such as bone metastases. The high linear energy transfer of alpha emitters (80 
keV/micrometer) leads to a high frequency of double-strand DNA breaks in adjacent 
cells, resulting in an anti-tumor effect on bone metastases. The alpha particle range 
from radium-223 dichloride is less than 100 micrometers (less than 10 cell diameters) 
which limits damage to the surrounding normal tissue.
1.3.4 Preclinical
In single and repeated dose toxicity studies in rats, the main findings were reduced 
body weight (b.w.) gain, hematological changes, reduced serum ALP and microscopic 
findings in the bone marrow (depletion of hematopoietic cells, fibrosis), spleen 
(secondary extra-medullary hematopoiesis) and bone (depletion of osteocytes, 
osteoblasts, osteoclasts, fibro-osseous lesions, disruption/disorganization of the 
physis/growth line). These findings were related to radiation-induced impairment of 
hematopoiesis and a reduction of osteogenesis and occurred beginning in the dose 
range of of 20 kBq/kg (22 kBq/kg after implementation of NIST update) b.w. (0.4 
times the clinically recommended dose).
In dogs, dose-limiting myelotoxicity was observed after a single dose of 450 kBq/kg
(497 kBq/kg after implementation of NIST update), while single doses of 50 kBq/kg
(55 kBq/kg after implementation of NIST update) and 150 kBq/kg (166 kBq/kg after
implementation of NIST update) were considered safe and repeated administration of 
the clinically recommended dose of radium-223 dichloride (50 kBq/kg [55 kBq/kg 
after implementation of NIST update]) at intervals of 4 weeks for 6 months was well 
tolerated without dose-limiting toxicities.
Osteosarcomas, a known effect of bone-seeking radionuclides, were observed at clinically 
relevant doses in rats 7 – 12 months after start of treatment. Osteosarcomas were not 
observed in dog studies. The presence of neoplastic changes, other than osteosarcomas, 
was also reported in the longer term (12 to 15 months) rat toxicity studies. Due to its 
mode of action, and as seen with conventional radiotherapy and other radiotherapeutics, 
radium-223 dichloride may have the potential to induce secondary malignancies. No case 
of osteosarcoma has been reported in clinical studies with Radium Ra 223 dichloride. The 
risk for patients to develop osteosarcomas with exposure to Radium Ra 223 dichloride is 
unknown at present 

 
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Studies on reproductive and developmental toxicity have not been performed. Since 
Radium Ra 223 dichloride binds to bone, the potential risk for toxic effects in the male 
gonads in cancer patients with castration-resistant prostate cancer is very low, but cannot 
be excluded. Studies on the mutagenic and carcinogenic potential of Radium Ra 223 
dichloride have not been performed.
No histological changes were observed in organs involved in the excretion of Radium Ra 
223 dichloride. No significant effects were seen on vital organ systems, i.e. cardiovascular 
(dog), respiratory or central nervous systems (rat), after single dose administration of  up 
to 450 (497 kBq/kg after implementation of NIST update) in dogs, and up to 1000 kBq/kg 
(1100 kBq/kg; after the NIST implementation date) in rats, (9 (dog) to 20 (rat) times the 
clinically recommended dose.
1.3.5 Clinical Experience Summary
The clinical development of Radium Ra 223 dichloride is summarized below41-51. It 
includes phase I and phase II studies in prostate cancer patients with bone metastases. The 
results of these completed studies indicated that safety and tolerability of Radium Ra 223 
dichloride in CRPC/HRPC patients with bone metastases was well tolerated, and that 
there was evidence of dose related efficacy against bone markers and other markers of 
disease. In addition there was an effect on median overall survival in a Phase II (BC1-02) 
placebo-controlled study. These studies enabled the initiation of the Phase III 
ALSYMPCA (ALpharadin in SYMptomatic Prostate CAncer) study.
The clinical safety and efficacy of Radium Ra 223 dichloride have been evaluated in a 
double-blind, randomized, multiple dose, phase III multicenter study (ALSYMPCA) in 
castration-resistant prostate cancer patients with bone metastases. The primary efficacy 
endpoint was Overall Survival (OS). 
At the cut-off date of the pre-planned interim analysis, a total of 809 patients were 
randomized 2:1 to receive Radium Ra 223 dichloride 50 kBq (0.0014 mCi)/kg (55kBq 
(.00149 mCiafter implementation of the NIST update) intravenously every 4 weeks for 6 
cycles (N=541) plus best standard of care or matching placebo plus best standard of care 
(N=268). Best standard of care included e.g. local external beam radiotherapy, 
corticosteroids, antiandrogens, estrogens, estramustine or ketoconazole. 
An updated descriptive analysis of safety and of OS was performed in 921 randomized 
patients prior to implementing crossover (i.e. offering patients in the placebo group to 
receive Radium Ra 223 dichloride treatment).
The results of both, interim and updated analysis, revealed that OS was significantly 
longer in patients treated with Radium Ra 223 dichloride plus best standard of care 
compared to patients treated with placebo plus best standard of care. For the updated 
analysis, an increase in median overall survival of 3.6 months was seen with Radium Ra 
223 dichloride plus best standard of care compared to placebo plus best standard of care 
(HR =0.695 (95% CI 0.581/0.832), median OS 14.9 months versus 11.3 months, 
respectively).

 
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In the ALSYMPCA study, the results of the interim analysis and the updated analysis 
showed also a significant improvement in all main secondary endpoints in the Radium Ra 
223 dichloride arm compared to the placebo arm:
Time to first SRE (defined as time to EBRT, time to first pathological bone 
fracture, time to spinal cord compression and time to surgical intervention) was 
statistically significantly longer in the radium-223 chloride group compared to 
placebo (median number of months=15.6 for radium-223 chloride versus 9.8 
months for placebo (HR=0.658, 95 CI 0.522–0.830, p= 0.00037).
Time to total ALP progression (defined as ≥ 25% increase compared to 
baseline/nadir) was statistically significantly longer in the radium-223 chloride 
group 7.4 months compared to placebo 3.8 months  (HR = 0.167, 95% CI 0.129 – 
0.217; p= <0.00001).
Time to PSA progression (defined as a ≥ 25% increase and an increase in absolute 
value of ≥ 2 ng/mL compared to baseline/nadir ) was also significantly prolonged 
in patients receiving Radium Ra 223 dichloride compared to patients receiving 
placebo (HR = 0.643, 95% CI 0.539,0.768; p = <0.00001)
A total ALP  response (defined as a confirmed ≥ 30% or ≥ 50% reduction 
compared to baseline) at week 12 was observed in higher proportions of subjects 
who were treated with radium-223 chloride group (47%  and 3% respectively) 
compared to those in the placebo (3% and <1% respectively) group. 
Subgroup survival analysis showed a consistent survival benefit for treatment with 
Radium Ra 223 dichloride, independent of total alkaline phosphatase (ALP), current use 
of bisphosphonates, prior use of docetaxel and baseline ECOG status. The results from 
the phase III ALSYMCA study regarding time to external beam radiation therapy (EBRT) 
for pain relief and fewer patients reporting bone pain as an adverse event in the Radium 
Ra 223 dichloride group indicate a positive effect on bone pain. 
The most common adverse reactions (≥ 10%) in patients receiving Radium Ra 223 
dichloride were nausea, diarrhea, vomiting, and peripheral edema (Table 1). Grade 3 and 
4 adverse events were reported among 57% of Radium Ra 223 dichloride-treated patients 
and 63% of placebotreated patients. The most common hematologic laboratory 
abnormalities in Radium Ra 223 dichloride-treated patients (≥ 10%) were anemia, 
lymphocytopenia, leukopenia, thrombocytopenia, and neutropenia (Table 2). Treatment 
discontinuations due to adverse events occurred in 17% of patients who received Radium 
Ra 223 dichloride and 21% of patients who received placebo. 
The most common hematologic laboratory abnormalities leading to discontinuation for 
Radium Ra 223 dichloride were anemia (2%) and thrombocytopenia (2%).
Table 1 shows adverse reactions occurring in ≥ 2% of patients and for which the 
incidence for Radium Ra 223 dichloride exceeds the incidence for placebo.
Table 1 Adverse Reactions in the Randomized Trial

 
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Radium Ra 223 dichloride 
(n=600) 
Placebo (n=301) 
System/Organ 
Class 
Preferred 
Term 
Grades 1-4 % 
Grades 3-4 % 
Grades 1-4 % 
Grades 3-4 % 
Blood and lymphatic system disorders 
Pancytopenia 
2 
1 
0 
0 
Gastrointestinal disorders 
Nausea 
36 
2 
35 
2 
Diarrhea 
25 
2 
15 
2 
Vomiting 
19 
2 
14 
2 
General disorders and administration site conditions 
Peripheral 
edema 
13 
2 
10 
1 
Renal and urinary disorders 
Renal failure 
and impairment 
3 
1 
1 
1 
Laboratory Abnormalities
Table 2 shows hematologic laboratory abnormalities occurring in > 10% of patients and 
for which the incidence for Radium Ra 223 dichloride exceeds the incidence for placebo.
Table 2: Hematologic Laboratory Abnormalities
Laboratory values were obtained at baseline and prior to each 4-week cycle.
As an adverse reaction, grade 3-4 thrombocytopenia was reported in 6% of patients on 
Radium Ra 223 dichloride and in 2% of patients on placebo. Among patients who 
received Radium Ra 223 dichloride, the laboratory abnormality grade 3-4 
thrombocytopenia occurred in 1% of docetaxel naïve patients and in 4% of patients who 
had received prior docetaxel. Grade 3-4 neutropenia occurred in 1% of docetaxel naïve 
patients and in 3% of patients who have received prior docetaxel.
Fluid Status
Dehydration occurred in 3% of patients on Radium Ra 223 dichloride and 1% of patients 
on placebo. Radium Ra 223 dichloride increases adverse reactions such as diarrhea, 
nausea, and vomiting which may result in dehydration. Monitor patients’ oral intake and 
fluid status carefully and promptly treat patients who display signs or symptoms of 
dehydration or hypovolemia.
Injection Site Reactions
Hematologic 
Ra-223 (n=600) 
Placebo (n=301) 
Laboratory 
Grades 1-4 
Grades 3-4 
Grades 1-4 
Grades 3-4 
Abnormalities 
% 
% 
% 
% 
Anemia 
93 
6 
88 
6 
Lymphocytopenia 
72 
20 
53 
7 
Leukopenia 
35 
3 
10 
<1 
Thrombocytopenia 
31 
3 
22 
<1 
Neutropenia 
18 
2 
5 
<1 

 
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Erythema, pain, and edema at the injection site were reported in 1% of patients on 
Radium Ra 223 dichloride.
Secondary malignant neoplasms
No cases of radiation-induced cancer have been reported in reported in clinical trials with 
radium-223 dichloride in follow-up of up to three years. However, the radiation dose 
resulting from therapeutic exposure may result in higher incidence of cancer (e.g. 
sarcomas of the bone, or leukemia), mutations and a potential for development of 
hereditary defects.
Bone Marrow Suppression
In the randomized trial, 2% of patients on the Radium Ra 223 dichloride arm experienced 
bone marrow failure or ongoing pancytopenia compared to no patients treated with 
placebo. There were two deaths due to bone marrow failure and for 7 of 13 patients 
treated with Radium Ra 223 dichloride, bone marrow failure was ongoing at the time of 
death. Among the 13 patients who experienced bone marrow failure, 54% required blood 
transfusions. Four percent (4%) of patients on the Radium Ra 223 dichloride arm and 2% 
on the placebo arm permanently discontinued therapy due to bone marrow suppression.
In the randomized trial, deaths related to vascular hemorrhage in association with 
myelosuppression were observed in 1% of Radium Ra 223 dichloride-treated patients 
compared to 0.3% of patients treated with placebo. The incidence of infection-related 
deaths (2%), serious infections (10%), and febrile neutropenia (<1%) were similar for 
patients treated with Radium Ra 223 dichloride and placebo.
Myelosuppression; notably thrombocytopenia, neutropenia, pancytopenia, and 
leukopenia; has been reported in patients treated with Radium Ra 223 dichloride. In the 
randomized trial, complete blood counts (CBCs) were obtained every 4 weeks prior to 
each dose and the nadir CBCs and times of recovery were not well characterized. In a 
separate single-dose phase 1 study of Radium Ra 223 dichloride, neutrophil and platelet 
count nadirs occurred 2 to 3 weeks after Radium Ra 223 dichloride administration at 
doses that were up to 1 to 5 times the recommended dose, and most patients recovered 
approximately 6 to 8 weeks after administration 
Hematologic evaluation of patients must be performed at baseline and prior to every dose 
of Radium Ra 223 dichloride. Before the first administration of Radium Ra 223 
dichloride, the absolute neutrophil count (ANC) should be ≥ 1.5 x 109/L, the platelet 
count ≥ 100 x 109/L and hemoglobin ≥ 10 g/dL. Before subsequent administrations of 
Radium Ra 223 dichloride, the ANC should be ≥ 1 x 109/L and the plateletcount ≥ 50 x 
109/L. If there is no recovery to these values within 6 to 8 weeks after the last 
administration of Radium Ra 223 dichloride,despite receiving supportive care, further 
treatment with Radium Ra 223 dichloride should be discontinued. Patients with evidence 
of compromised bone marrow reserve should be monitored closely and provided with 
supportive care measures when
clinically indicated. Discontinue Radium Ra 223 dichloride in patients who experience 
life-threatening complications despite supportive care for bone marrow failure.

 
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The safety and efficacy of concomitant chemotherapy with Radium Ra 223 dichloride 
have not been established. Outside of a clinical trial, concomitant use with chemotherapy 
is not recommended due to the potential for additive myelosuppression. If chemotherapy, 
other systemic radioisotopes or hemibody external radiotherapy are administered during 
the treatment period, Radium Ra 223 dichloride should be discontinued.
2. Study objectives
The primary objective of this study is to evaluate the quantitative effect of Ra-223 on 99mTc-
MDP bone scan in men with castration-resistant prostate cancer metastatic to bone. . 
Secondary objectives include the description of mean percent change in bone scan lesion area 
by 18-month survival status as well as the evaluation of the effects of Ra-223 by the 
following measures:  
(A) Imaging biomarkers
I. Concurrent conventional assessments including Computed Tomography scans
II. Baseline assessments including fluciclovine PET (if assigned to PET), 99mTc MDP bone 
scan, & blood testing
(B) Standard and novel circulating tumor cell assays
I. CTC number by FDA-approved assay (Veridex CellSearch)
II. CTC translational biomarkers by microfluidic platform
 (e.g. enumeration, androgen receptor signaling, proliferative index by Ki67 staining)
 (C) Circulating biomarkers of the tumor microenvironment 
I. Bone turnover markers (serum bone specific alkaline phosphatase, N-telopeptide)
II. Plasma biomarkers of inflammation and angiogenesis
 (D) Patient reported pain and quality of life
I. Determine rate of confirmed pain response at week 12 of study treatment 
II. Evaluate change in patient reported quality of life as measured by validated                  
assessment tools
3. Investigator[s] and other study participants
Philip J. Saylor, MD
Massachusetts General Hospital Cancer Center
55 Fruit Street, Yawkey 7E; Boston, MA 02114
psaylor@mgh.harvard.edu; Phone: 617-643-1763, Fax: 617-726-8685
Matthew R. Smith, MD, PhD
Massachusetts General Hospital Cancer Center
55 Fruit Street, Yawkey 7E; Boston, MA 02114
smith.matthew@mgh.harvard.edu; Phone: 617-724-5257

 
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4. Study design
This single-arm exploratory biomarker study will prospectively enroll men with castration-
resistant prostate cancer (CRPC) metastatic to bone. Exhaustive inclusion/exclusion criteria 
are detailed in the Section 5 of the protocol. Schema: 
1. Screening  assessments include 99m TC MDP bone scan & blood testing
2. Enrollment
3. Baseline assessments includes fluciclovine PET (if assigned to PET)
4. Ra-223, administered monthly for a total of six anticipated doses with concurrent 
protocol-specified correlative imaging and blood testing
5. Conclusion of study treatment with sixth dose of Ra-223
6. Follow-up and testing as detailed in experimental protocol
Standard androgen deprivation therapy (ADT) will be continued throughout study 
participation. Men will be treated for 6 months on protocol in the absence of progression or 
intolerable side effects. Men will be evaluated according to the table provided in protocol 
Section 7.1.1. At the end of 6 months of treatment, Ra-223 will be discontinued. Subsequent 
therapies will be at the discretion of the treating oncologist. 
5. Study population
5.1 Eligibility
5.1.1
Inclusion criteria

Male age ≥ 18 years.

Histologically or cytologically confirmed adenocarcinoma of the prostate.

Life expectancy of at least 6 months.

ECOG performance status of zero, one, or two. 

Bone-predominant metastatic CRPC: at least two skeletal metastases on bone 
scan with no lung, liver, and/or brain metastasis (lymph node metastasis is 
allowed). 

Judged by investigator to have progressive disease sufficient to clinically justify 
standard-of-care radium-223 treatment. 

Subjects must be able to understand and be willing to sign the written informed 
consent form. 

All acute toxic effects of any prior treatment have resolved to NCI-CTCAE v4.0 
Grade 1 or less at the time of signing the Informed Consent Form (ICF). 

No intention to use cytotoxic chemotherapy within the next 6 months. 

 
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
Subjects must agree to use adequate contraception beginning at the signing of the 
ICF until at least 6 months after the last dose of study drug. The definition of 
adequate contraception will be based on the judgment of the principal 
investigator.

Acceptable hematology and serum biochemistry screening values:

White Blood Cell Count (WBC) ≥ 3,000/mm3

Absolute Neutrophil Count (ANC) ≥ 1,500/mm3

Platelet (PLT) count ≥ 100,000/mm3

Hemoglobin (HGB) ≥ 9 g/dl (Please note: it is acceptable from 
the standpoint of study eligibility to undergo transfusion in 
order to achieve hemoglobin ≥ 9 g/dl) 

Total bilirubin level ≤ 1.5 x institutional upper limit of normal 
(ULN)

Aspartate aminotransferase (AST) and alanine 
aminotransferase (ALT) ≤ 2.5 x ULN 

Creatinine ≤ 2 x ULN

Albumin > 25 g/L

Willing and able to comply with the protocol, including follow-up visits and 
examinations. 
5.1.2
Exclusion criteria

Treatment with cytotoxic chemotherapy within previous 28 days, or failure to 
recover from AEs due to cytotoxic chemotherapy administered more than 28 days 
previous (however, ongoing neuropathy is permitted). 

Received any investigational compound within 28 days prior to the first dose of 
study drug or planned during the treatment period or follow-up. 

Received systemic therapy with radionuclides (e.g., strontium-89, samarium-153, 
rhenium-186, or rhenium-188, or Radium Ra 223 dichloride) for the treatment of 
bony metastases.

Received previous radiotherapy to approximately >25% of bone marrow.   

Other malignancy treated within the last 3 years (except non-melanoma skin 
cancer or low-grade superficial bladder cancer). 

Visceral metastases as assessed by abdominal or pelvic computed tomography 
(CT) or other imaging modality. 

Presence of brain metastases. 

Lymphadenopathy exceeding 6 cm in short-axis diameter. 

Any size pelvic lymphadenopathy if it is thought to be a contributor to concurrent 
hydronephrosis. 

 
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
Imminent spinal cord compression based on clinical findings and/or magnetic 
resonance imaging (MRI). Treatment should be completed for spinal cord 
compression.

Any other serious illness or medical condition, such as but not limited to:
o
Any infection ≥ National Cancer Institute Common Terminology Criteria 
for Adverse Events (NCI-CTCAE) version 4.03 Grade 2 
o
Cardiac failure New York Heart Association (NYHA) III or IV
o
Crohn’s disease or ulcerative colitis
o
Known bone marrow dysplasia

Fecal incontinence. 

Any condition which, in the investigator’s opinion, makes the subject unsuitable 
for trial participation.
5.1.3
Inclusion of Women and Minorities
Both men of all races and ethnic groups are eligible for this trial. Women are not eligible as 
women can not develop prostate cancer. 
5.1.4
Excluded therapies and medications, previous and concomitant

Concurrent anti-cancer therapy (chemotherapy, radiation therapy, surgery, 
immunotherapy, biologic therapy, or tumor embolization) other than Ra 223 
dichloride and standard methods of androgen deprivation therapy (GnRH agonist, 
GnRH antagonist, or bilateral orchiectomy). 

Prior use of Radium Ra 223 dichloride.

Concurrent use of another investigational drug or device therapy (i.e., outside of 
study treatment) during, or within 4 weeks of trial entry (signing of the informed 
consent form).

Major surgery within 30 days prior to start of study drug.
5.2 Withdrawal of subjects from study
5.2.1 Registration Procedures
General Guidelines for DF/HCC and DF/PCC Institutions
Institutions will register eligible participants with the DF/HCC Quality Assurance Office for 
Clinical Trials (QACT) central registration system. Registrations must occur prior to the 
initiation of protocol therapy. Any participant not registered to the protocol before protocol 
therapy begins will be considered ineligible and registration will be denied. 

 
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An investigator will confirm eligibility criteria and a member of the study team will complete 
the QACT protocol-specific eligibility checklist. 
Following registration, participants may begin protocol therapy. Issues that would cause 
treatment delays should be discussed with the Overall Principal Investigator (PI). If a 
participant does not receive protocol therapy following registration, the participant’s 
registration on the study must be canceled. Notify the QACT Registrar of registration 
cancellations as soon as possible
Registration Process for DF/HCC and DF/PCC Institutions
The QACT registration staff is accessible on Monday through Friday, from 8:00 AM to 5:00 
PM Eastern Standard Time. In emergency situations when a participant must begin protocol 
therapy during off-hours or holidays, call the QACT registration line at 617-632-3761 and 
follow the instructions for registering participants after hours. 
The registration procedures are as follows:

Obtain written informed consent from the participant prior to the performance of any 
protocol specific procedures or assessments. 

Complete the QACT protocol-specific eligibility checklist using the eligibility 
assessment documented in the participant’s medical record and/or research chart. To 
be eligible for registration to the protocol, the participant must meet all inclusion 
and exclusion criterion as described in the protocol and reflected on the 
eligibility checklist.
Reminder: Confirm eligibility for ancillary studies at the same time as eligibility for a 
treatment protocol. Registration to both treatment and ancillary protocols will not be 
completed if eligibility requirements are not met for all studies. 

Fax the eligibility checklist(s) and all pages of the consent form(s) to the QACT at 
617-632-2295. For Phase I protocols, attach participant dose level assignment 
confirmation from the sponsor.

The QACT Registrar will (a) review the eligibility checklist, (b) register the 
participant on the protocol, and (c) randomize the participant when applicable.

An email confirmation of the registration and/or randomization will be sent to the 
Overall PI, study coordinator(s) from the Lead Site, treating investigator and 
registering person immediately following the registration and/or randomization.

 
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5.2.2 Withdrawal
Subjects must be withdrawn from the trial (treatment and procedures) for the following 
reasons: 

Subject withdraws consent from study treatment and study procedures.  A subject 
must be removed from the trial at his own request or at the request of his legally 
acceptable representative.  At any time during the trial and without giving 
reasons, a subject may decline to participate further.  The subject will not suffer 
any disadvantage as a result.

Subject is lost to follow-up.

Death.
Subjects may be withdrawn from the study for the following reasons:

The subject is non-compliant with study drug, trial procedures, or both; including 
the use of anti-cancer therapy not prescribed by the study protocol.

If, in the investigator's opinion, continuation of the trial would be harmful to the 
subject's well-being.

The development of a second cancer (with the exception of non-melanoma skin 
cancer). 

Development of an intercurrent illness or situation which would, in the judgment 
of the investigator, significantly affect assessments of clinical status and trial 
endpoints.

Deterioration of ECOG performance status to 4.

Use of illicit drugs or other substances that may, in the opinion of the 
investigator, have a reasonable chance of contributing to toxicity or otherwise 
skewing trial result.
Any subject removed from the trial will remain under medical supervision until discharge 
or transfer is medically acceptable.
In all cases, the reason for withdrawal must be recorded in the CRF and in the subject's 
medical records. Alternative care options will be discussed with the participant. 
A QACT Treatment Ended/Off Study Form will be filled out when a participant is 
removed from protocol therapy. This form can be found on the QACT website or 
obtained from the QACT registration staff. 
In the event of unusual or life-threatening complications, treating investigators must 
immediately notify the Overall PI, Philip J. Saylor M.D. at 617-724-4000. 
5.2.3 Duration of Follow-up
Participants will be followed for 18 months after removal from protocol therapy or until 
death, whichever occurs first. Participants removed from protocol therapy for unacceptable 
adverse event(s) will be followed until resolution or stabilization of the adverse event. 

 
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5.2.4 Screen Failures/Dropouts
A subject who discontinues study participation prematurely for any reason is defined as a 
“dropout” if the subject has already been registered.
A subject who, for any reason (e.g. failure to satisfy the selection criteria), terminates the 
study before the time point used for the definition of “dropout” (see above) is regarded a 
“screening failure”.
5.2.5 Replacement
No withdrawn subjects will be replaced. 
6. Treatment
6.1 Treatments to be administered
6.1.1 Radium Ra 223 dichloride, 50 kBq/kg body weight, will be administered as a 
bolus intravenous (IV) injection (up to 1 minute) at intervals of every 4 weeks 
for up to 6 cycles (55kBq/kg body weight after implementation of the NIST 
update)
6.2 Treatment assignment
This is an open label, single arm study. A patient number (a unique identification number) 
will be assigned when a subject is evaluated for inclusion into the study. The patients will 
then undergo screening procedures and will enter the trial after successful screening. 
6.2.1 Radium Ra 223 dichloride
The alpha-pharmaceutical Radium Ra 223 dichloride will be administered per standard-
of-care following standard instituational clinical practice. It is a ready-to-use, sterile, 
non-pyrogenic, clear and colorless aqueous solution of Radium Ra 223 dichloride 
(223RaCl2) for IV administration. It should not be diluted or mixed with any solutions. 
Each vial for a single use only.
Radium Ra 223 dichloride is an alpha particle emitter with a physical half-life of 11.4 
days. The product is isotonic and has a pH of 6.0-8.0. The radioactive concentration at 
the reference date is 1000 kBq/mL (1,100 kBq/mL after implementation of NIST 
update). The product has a pre-calibration of 14 days. When administered on a day 
other than the reference day, the volume should be corrected according to the physical 
decay table accompanying each shipment..
Bayer Healthcare LLC will provide Radium Ra 223 dichloride, which will be 
manufactured by Algeta’s contract manufacturer: Institute for Energy Technology, 
Isotope laboratories, Kjeller, Norway. The product is produced according to Good 
Manufacturing Practice (GMP). The product will be delivered in a glass vial, ready-to-
use with a certified activity. Radium Ra 223 dichloride is shipped in a lead container 

 
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and Type A radioactive package according to international transportation guidelines for 
radioactive materials. 
The volume per vial is 6 mL, corresponding to 6 MBq  (6.6 MBq after implementation 
of NIST update) at the reference day. Radium Ra 223 dichloride has a shelf life of 28 
days from production day, when stored at ambient temperature. The shelf life has been 
demonstrated for temperatures from cold storage (2-8°C) up to 40°C. In addition, it has 
been shown that the product quality is not jeopardized upon freezing.
All study drugs will be labeled according to the requirements of local law and 
legislation. For all study drugs, a system of numbering in accordance with all 
requirements of GMP will be used, ensuring that each dose of study drug can be traced 
back to the respective bulkware of the ingredients. 
6.2.1.1 General warning
Radium 223 dichloride should be received, used and administered only by authorized 
persons in designated clinical settings. The receipt, storage, use, transfer and disposal 
Radium 223 dichloride are subject to the regulations and/or appropriate licenses of the 
competent official organization. Radium 223 dichloride should be handled by the user 
in a manner which satisfies both radiation safety and pharmaceutical quality 
requirements. Appropriate aseptic precautions should be taken.
6.2.1.2 Instructions for Use
The administration of Radium Ra 223 dichloride is associated with potential risks for 
other persons (e.g. medical staff, care givers and members of the patient’s family) from 
radiation or contamination from spills of body fluids such as urine, feces, or vomit. 
Therefore, radiation protection precautions must be taken in accordance with national 
and local regulations.
For drug handling
Follow the normal working procedures for the handling of radiopharmaceuticals and 
use universal precautions for handling and administration such as gloves and barrier 
gowns when handling blood and bodily fluids to avoid contamination. In case of 
contact with skin or eyes, the affected area should be flushed immediately with water. 
In the event of spillage of Radium Ra 223 dichloride, the local radiation safety officer 
should be contacted immediately to initiate the necessary measurements and required 
procedures to decontaminate the area. A complexing agent such as 0.01 M ethylene-
diaminetetraacetic acid (EDTA) solution is recommended to remove contamination.
For patient care
Whenever possible, patients should use a toilet and the toilet should be flushed several 
times after each use. When handling bodily fluids, simply wearing gloves and hand 

 
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washing will protect caregivers. Clothing soiled with Radium Ra 223 dichloride or 
patient fecal matter or urine should be washed promptly and separately from other 
clothing.
Radium-223 is primarily an alpha emitter, with a 95.3% fraction of energy emitted as 
alpha-particles. The fraction emitted as beta-particles is 3.6%, and the fraction emitted 
as gamma-radiation is 1.1%. The external radiation exposure associated with handling 
of patient doses is considerably lower in comparison to other radiopharmaceuticals for 
therapeutic purposes as the administered radioactivity will usually be below 8 MBq 
(0.216 mCi). In keeping with the As Low As Reasonably Achievable (ALARA) 
principle, for minimization of radiation exposure, it is recommended to minimize the 
time spent in radiation areas, to maximize the distance to radiation sources, and to use 
adequate shielding. Any unused product or materials used in connection with the 
preparation or administration are to be treated as radioactive waste and should be 
disposed of in accordance with local regulations.The gamma radiation associated with 
the decay of radium-223 and its daughters allows for the radioactivity measurement of 
Radium Ra 223 dichloride and the detection of contamination with standard 
instruments.
6.2.2 Dose calibration
Radium Ra 223 dichloride can be measured in a normal dose calibrator instrument. 
When written approvals for the use of Radium Ra 223 dichloride from the Radiation 
Protection Agency for the specific center have been received by the sponsor, a vial of 
Radium Ra 223 dichloride for technical use will be sent to the study center. Different 
clinical study centers possess dose calibrators from various suppliers; thus, the isotope 
calibration factor may differ from center to center. Consequently, each center must 
perform the Radium Ra 223 dichloride dial setting on their relevant dose calibrator(s). 
For dial setting, the clinical study center will receive a sealed vial containing a Radium 
Ra 223 dichloride solution for calibration only. The vial is identical to the vials used 
for study treatment. The amount of Radium Ra 223 dichloride in the vial will be stated 
on the label. Instructions for the dial setting, including the calibration log form, will be 
enclosed with the dispatch of the calibration sample.
6.2.3 Dosimetry
The absorbed radiation dose calculation was performed based on clinical 
biodistribution data. Calculations of absorbed doses were performed using 
OLINDA/EXM (Organ Level INternal Dose Assessment/EXponential Modeling), a 
software based on the Medical Internal Radiation Dose (MIRD) algorithm, which is 
widely used for established beta and gamma emitting radionuclides. For radium-223, 
which is primarily an alpha emitter, additional assumptions were made for the 
intestine, red marrow and bone/osteogenic cells to provide the best possible absorbed 
dose calculations for Radium Ra 223 dichloride, considering its observed 
biodistribution and specific characteristics.

 
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For an administered activity of 3.65 MBq (0.0987 mCi) (50 kBq (0.00135 mCi) 
(55kBq (.00149 mCi after NIST update) per kg body weight to a 73-kg adult), the 
calculated absorbed doses to the bone (osteogenic cells) is 4.2050 Gy (420.5 rad) and 
to the red marrow is 0.5066 Gy (50.66 rad). The calculated absorbed doses to the main 
excretory organs are 0.0265 Gy (2.65 rad) for the small intestine wall, 0.1180 Gy (11.8 
rad) for the upper large intestine wall and 0.1696 Gy (16.96 rad) for the lower large 
intestine wall. 
The calculated absorbed doses to other organs are low, e.g. heart wall (0.0063 Gy, 0.63 
rad), lung (0.0003 Gy, 0.03 rad), liver (0.0109 Gy, 1.09 rad), kidneys (0.0117 Gy, 1.17 
rad), urinary bladder wall (0.0147 Gy, 1.47 rad), testes (0.0003 Gy, 0.03 rad), and 
spleen (0.0003 Gy, 0.03 rad). 
The hematological adverse drug reactions observed in the clinical studies with  Ra-223  
are much lower in frequency and severity than what could be expected from the 
calculated absorbed doses to the red marrow. This may be related to spatial distribution 
of alpha particle radiation resulting in non-uniform radiation dose to the red marrow.
6.2.4 Dose handling
The Radium Ra 223 dichloride vials must be stored inside their lead container in a 
secure facility. The study drug should be used within 28 days of production. Radium 
Ra 223 dichloride is an alpha-pharmaceutical and should be handled by individuals 
who are qualified by training and experience in the safe handling of radionuclides. One 
dedicated person and a back-up designee will have responsibility as assigned from the 
Primary Investigator for handling and storage of Radium Ra 223 dichloride. All 
administrations of Radium Ra 223 dichloride are based on the certified activity of 
Radium Ra 223 dichloride at the calibration date.
6.2.5 Dose calculation
The dosage of Radium Ra 223 dichloride is 50 kBq/kg body weight (55kBq/kg after 
NIST update).  The patient dose is calculated based on date of injection, a decay 
correction (DK) factor specific to number of days from reference date applied to 
correct for physical decay of radium-223 , and patient weight.  A table with DK 
values according to physical decay of the study medication will be provided with 
every shipment of Radium Ra 223 dichloride.  Radium-223 is an alpha particle emitter 
with a physical t1/2 of 11.4 days. The radioactive concentration at the reference date is 
1,000 kBq/mL (1,100 kBq/mL after implementation of NIST update). 
The volume to be administered for the current dose is calculated as follows:
Body weight (kg)   X dose (50kBQ/kg body weight)a
DK factor X 1000kBq (0.027mCi)/mLb
Data regarding activity should be recorded on the appropriate electronic case report 
form (eCRF) page.

 
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a55 kBq/kg after implementation of NIST update
b1100 kBq (0.0297 mCi)/mL after implementation of NIST update; NIST: National Institute of Standards and
Technology; DK: Decay correction
6.2.6 Dose preparation
Personnel should use appropriate protective clothing and equipment during syringe 
filling and application to prevent contamination with the radioactive solution (medical 
gloves / protective glasses). The individual responsible for study drug preparation will 
draw the correct volume of study drug into a syringe. The size of the syringe should be 
chosen according to the applied volume to reach the required dosing accuracy. Radium 
Ra 223 dichloride should not be diluted or mixed with any solutions. Do not store 
above 40°C (104°F). If the vials have been stored in a refrigerator, they should be left 
at room temperature for 1 hour prior to use, since cold material should not be injected 
in a patient. Store Radium Ra 223 dichloride in the original container or equivalent 
radiation shielding. This preparation is approved for use by persons under license by 
the Nuclear Regulatory Commission or the relevant regulatory authority of an 
Agreement State.
6.2.7 Dose administration
Before administration of study drug, the patient must be well hydrated; the patient 
should be instructed to drink ad libitum. Aseptic technique should be used in the 
administration of Radium Ra 223 dichloride. The syringe should be handed over to the 
individual who will perform the injection. The study medication will be administered 
as a bolus intravenous (IV) injection (up to 1 minute). After administration, the 
equipment used in connection with the preparation and administration of drug is to be 
treated as radioactive waste and should be disposed in accordance with local procedure 
for the handling of radioactive material. 
6.2.8 Dose Modification/Delays
Every effort should be made to administer the full dosing regimen of Radium Ra 223 
dichloride. Adjustment of dose level is not permitted.  
Study visits during the treatment period will occur at protocol-defined intervals (within 
a window of +/- 7 days). 
Dosing delays may be instituted under the following circumstances:
Disease progression: The Investigator should delay cytotoxic chemotherapy, other 
systemic radioisotope, hemibody external radiotherapy or other investigational drug 
until the follow-up period. If such treatments have to be given during the treatment 
period, further study drug administrations must be discontinued. Patients with disease 
progression may continue treatment at the Investigator’s discretion. 
Myelosuppression: Treatment-related changes in hematology parameters may occur.

 
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
If a patient experiences CTCAE v4.03 Grade 3 or 4 neutropenia, 
thrombocytopenia, or anemia the administration of study drug should be 
delayed until recovery to Grade 2 or better. 

If a patient experiences CTCAE v4.03 Grade 3 or 4 neutropenia, 
thrombocytopenia, or anemia lasting > 14 days, further study drug 
administrations must be discontinued. 

Blood transfusion is acceptable. Use of biologic response modifiers, such as G-
CSF or GM-CSF, is allowed in the management of acute toxicity.
Gastrointestinal events: Diarrhea should be treated as per local practice. A further dose 
of study medication should not be given before diarrhea is recovered to CTCAE v4.03 
Grade 2 or baseline levels. Nausea or vomiting should be treated as per local practice.  
A further dose of study medication should not be given before nausea or vomiting is 
recovered to CTCAE v.4.03 Grade 2 or baseline levels.
Spinal Cord Compression: If the patient experiences spinal cord compression during 
the treatment period, the patient should be treated for the event, and may receive 
further study drug administration if adequately recovered. 
Surgical Intervention: If surgery is required, the patient should continue with study 
treatment, if this is considered safe in the treating Investigator’s opinion. The surgeon 
needs to be notified that the patient has been given radioactive drug, and needs to 
follow the guidelines for radioactive protection.  
Non-Pathological Fractures: For traumatic fractures in weight-bearing bones during 
treatment phase, the study drug administration should be delayed for 2-4 weeks from 
the time of fracture. 
Pathological fractures: Pathological fractures may occur as the result of either 
progressive disease or increased physical activity associated with significant pain 
palliation. Pathologic fractures are to be treated in a manner that attempts to maintain 
the best functional status and quality of life. Study treatment may continue as planned.
Any Other Toxicity: Local practice will apply.
6.3  Drug logistics and accountability
All study drugs will be stored at the investigational site in accordance with Good 
Clinical Practice (GCP) and Good Manufacturing Practices (GMP) requirements 
and the instructions given by the clinical supplies department of the Institution 
and will be inaccessible to unauthorized personnel.
6.3.1 Accountability
The investigator, or a responsible party designated by the investigator, must maintain a 
careful record of the inventory and disposition of the agent(s) (investigational or free of 
charge) using the NCI Drug Accountability Record or another comparable drug 
accountability form. (See the CTEP website at http://ctep.cancer.gov/protocolDevelopment  

 
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for the “Policy and Guidelines for Accountability and Storage of Investigational Agents” or 
to obtain a copy of the drug accountability form.)
6.3.2
Destruction and Return
At the end of the study, unused supplies of Ra 223 dichloride and other investigational agents  
should be destroyed appropriately and  according to institutional policies.Destruction will be 
documented in the Drug Accountability Record Form. The certificate of destruction for Ra 
223 dichloride  should be sent to Bayer.
6.4 Treatment compliance
Subject compliance with the treatment and protocol includes willingness to comply 
with all aspects of the protocol, and to have blood collected for all safety evaluations.  
At the discretion of the principal investigator, a subject may be discontinued from the 
trial for non-compliance with follow-up visits or study drug.
6.5 Prior and concomitant therapy-
All medication that is considered necessary for the subject’s welfare, and which is not 
expected to interfere with the evaluation of the study treatment, may be given at the 
discretion of the investigator. All medications (including contrast media) taken within 
2 weeks prior to the start of the study and during the study must be recorded in the 
subject’s source documentation and in the CRF (including start/stop dates, dose 
frequency, route of administration, and indication).
Permitted 

Treatment with non-conventional therapies (e.g., herbs [with the exception of St. 
John’s Wart], acupuncture) and vitamin/mineral supplements is acceptable 
provided that, in the opinion of the investigator, such treatment will not interfere 
with the trial endpoints.

Subjects may receive standard of care for any underlying illness.

In the event of neutropenia, anemia, or thrombocytopenia, subjects may receive 
appropriate supportive care (e.g., transfusion, biologic response modifiers such as 
G-CSF or GM-CSF, prophylactic antibiotics, antifungals and/or antivirals, 
hematopoietic growth factors).

Blood transfusions and erythropoietin are allowed. 

If surgery is required during study drug treatment, the surgeon needs to be notified 
that the patient has been treated with a radioactive product and adequate 
precautions for radioactive protection should be applied during the surgical 
procedure.  The patient should continue with study treatment if considered safe in 
the treating Investigator's opinion.

Concomitant treatments for prostate cancer will be recorded in the CRFs. 
7. Procedures and variables

 
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7.1
Schedule of procedures

 
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7.1.1
Tabulated overview 
Table 1: Treatment
S
B
T1 
D
1
T1 
D
4
T
2
T3
T
4
T
5
T
6
F/
U 
1M
F/
U 
4M
F/U 
18
M
History & physical exam
X
X
X
X
X
X
X
X
X
X
99mTc MDP bone scan
X
X1
X
CT a/p
X
X1,
2
X
Fluciclovine PET/MRI3
X
X1
Medication history
X
X
X
X
X
X
X
X
X
X
Chemistry panel
X
X
X
X
X
X
X
X
CBC with differential
X
X
X
X
X
X
X
X
PSA
X
X
X
X
X
X
X
X
X
CTCs by CellSearch4
X
X
X
CTCs by microfluidic 
chip5
X
X
X
BAP & NTx
X
X
X
X
X
X
X
X
X
X
Blood biomarkers6
X
X
X
X
X
X
X
X
X
X
Patient reported outcomes7
X
X
X
X
X
X
X
X
X
Adverse event reporting
X
X
X
X
X
X
X
Notations: 
Screening studies “S” must be done within 60 days of start of treatment (“T1 D1”). 
Baseline study “B” must be done after a subject is successfully registered to the trial, but 
prior to start of treatment (“T1 D1”).
Treatments T1 through T6 occur at 4 week intervals ± 7 days. 
(1) T3 timepoint scans (bone scan, and CT a/p, and fluciclovine PET/MRI in some) will be 
performed within 10 days prior to the T3 treatment day. 
(2) T3 timepoint CT a/p will be performed only for participants that do not undergo 
fluciclovine PET/MRI.
(3) fluciclovine PET/MRI will be performed at two timepoints each for 10 of the study 
participants. They will be chosen by investigator discretion in light of their willingness and 
medical eligibility to undergo PET/MRI (e.g. pacemaker is a contraindication, claustrophobia 
is a contraindication).  The baseline PET/MRI will only be done after the subject has 
successfully been registered to the trial, but prior to start of treatment (“T1 D1”).  
 (4) CTCs, circulating tumor cells; 7.5 mL of whole blood will be collected in a special 
CellSave tubes as described in Appendix G for BWH core lab testing using the commercially 
available Veridex CellSearch assay; subsequent CTC testing will only be carried out on 
participants who had ≥ 1 detectable CTC on study T1 D1. 
(5) 20 mL of blood will be collected in vacutainer tubes (usually purple top EDTA tubes) for 
Haber/Maheswaran laboratory analysis of circulating tumor cells as detailed in Appendix B; 

 
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subsequent CTC testing will only be carried out on participants who had ≥ 1 detectable CTC 
on study T1 D1. 
(6) Plasma biomarkers; 10 mL of blood will be collected in a purple top (EDTA plasma) tube 
and processed as detailed in Appendix C for batched analysis of inflammatory and angiogenic 
biomarkers. 
(7) Analgesic use and other patient reported outcomes will be collected as detailed in 
Appendices D, E, and F. 
Abbreviations: 
S, screening; B, baseline; 
T1 D1, Ra-223 treatment 1 day 1; 
F/U 1M, follow-up visit 1 month after final Ra-223 treatment; 
F/U 4M, follow-up visit 4 months after final Ra-223 treatment; 
F/U 18M, follow-up visit 18 months after final Ra-223 treatment;
CT a/p, CT of the, abdomen, and pelvis; 
Chemistry panel, includes liver panel also known as LFTs; CBC, complete blood count; 
PSA, serum prostate specific antigen; 
PET, positron emission tomography; 
CTCs, circulating tumor cells; 
BAP, bone-specific alkaline phosphatase; NTx, n-telopeptide.
7.1.2
Timing of assessments
Relevant timing is as detailed above in Section 7.1.1 (notations immediately below table). 
7.1.3
Medical history
Medical history findings (i.e. previous diagnoses, diseases or surgeries) meeting all 
criteria listed below will be collected:
1. Not pertaining to the study indication

Start before signing of the informed consent 

Considered relevant to the study
Detailed instructions on the differentiation between (i) medical history and (ii) adverse 
events can be found in Section 7.15.1.1.

 
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7.2
99mTc MDP bone scan: Standard clinical bone scan will be carried out 
per institutional practice.
7.3
CT a/p: Standard clinical CT of the abdomen and pelvis will be carried out per 
institutional practice. 
7.4
Medication history: Medication history, including any changes in medication use 
since last assessment, will be reviewed. 
7.5
Chemistry panel: Standard comprehensive metabolic panel (including sodium, 
potassium, chloride, bicarbonate, BUN, creatinine, glucose, total protein, 
albumin, AST, ALT, total bilirubin, direct bilirubin, and alkaline phosphatase) 
will be carried out at the MGH clinical laboratory in real time. 
7.6
CBC with differential: Standard comprehensive metabolic panel (including 
white blood cell count, hemoglobin, hematocrit, platelets, and differential) will 
be carried out at the MGH clinical laboratory in real time. 
7.7
PSA: Standard PSA assay will be carried out at the MGH clinical laboratory in 
real time. 
7.8
Fluciclovine PET/MRI: This testing will be carried out only for 10 study 
participants. They will be chosen by investigator discretion in light of their 
willingness and medical eligibility to undergo PET/MRI (e.g. pacemaker is a 
contraindication, claustrophobia is a contraindication). The agent used in this 
study will be the radiolabled agent (fluciclovine) that will be administered prior 
to standard whole body PET imaging which includes skull base to thigh. 
Fluciclovine will be handled by qualified personnel, familiar with procedures 
that minimize undue exposure to themselves and the environment. PET/MRI 
imaging will be carried out per standard/clinical institutional protocol. Thus far, 
there is limited experience using fluciclovine PET/MRI; it is investigational.  
7.9
CTCs by CellSearch (see Appendix G for detailed instructions): 
At study-designated timepoints, 7.5 mL of whole blood will be collected in a 
special CellSave tube (available from Veridex/Janssen Diagnostics), which is 
then stored at room temperature until processed. It will be sent to the BWH CTC 
Lab for analysis. The BWH Circulating Tumor Cell Laboratory provides 
circulating tumor cell (CTC) services using the Veridex/Janssen Diagnostics 
CellSearch immunomagnetic platform. The lab is located to a CLIA-approved 
space within the BWH Cytology Division (Brigham and Women's Hospital, 
Medical Research Building, 3rd Floor; 75 Francis St., Boston, MA 02115). 
Major equipment includes the CellSearch Autoprep, and CellTracks Analyzer 
II. The CTC Lab provides clinical enumeration of CTCs for patients with 
metastatic prostate cancer using the FDA-cleared Veridex/Janssen Diagnostics 
CellSearch System.  It also functions as the BWH CTC Core Lab 

 
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(http://www.partners.org/researchcores/CTC/CTC_BWH.html) providing 
enumeration and isolation of CTC for research purposes. The current director is 
Alarice Lowe, M.D. (Phone: 617-525-8696, Fax: 617-739-6192, 
Email: aclowe@partners.org). 
7.10 CTCs by microfluidic chip: (see Appendix B for detailed instructions) 
Two 10 mL samples of peripheral blood will be collected into vacutainer tubes 
(Becton-Dickinson) containing the anticoagulant EDTA. Samples will be 
shipped by U.S. Ground courier to the MGH Charlestown Navy Yard 
laboratories of Drs. Daniel Haber and Shyamala Maheswaran. Alternatively, a 
member of the Haber or Maheswaran labs will personally transport the samples 
from the MGH Cancer Center to the MGH Charlestown Navy Yard laboratories. 
Samples will be processed through the experimental platform within 6 hours of 
collection, according to previously established protocols52,53 with modifications 
subject to current lab protocol. Briefly, a 5 mL aliquot of blood will be placed 
into an air-tight conical tube on a rocker assembly, and blood will be 
pneumatically driven through the microfluidic chip at a protocol-specified flow 
rate. The microfluidic chip will then be flushed with phosphate-buffered saline at 
a protocol specified flow rate to remove nonspecifically bound cells. CTCs on 
the chip will be fixed, permeabilized, and stained with antibodies. Staining for 
PSA, PSMA, DAPI, Ki-67, and other proteins will be performed. Captured CTCs 
will then be identified and enumerated using the automated detection system as 
previously described52. RNA and/or DNA will also be isolated from CTCs 
captured on a separate chip for gene expression analyses. Any remaining isolated 
RNA or DNA will be stored frozen at -70 to -90 degrees C for batch global 
profiling analyses. See Appendix B additional processing instructions. 
7.11 Bone turnover markers (see Appendix H for detailed instructions): 
Sera will be collected from subjects at study-specified timepoints. It will be 
processed and stored as detailed in Appendix H. Serum N-telopeptide 
concentrations will be determined at a Massachusetts General Hospital core 
laboratory using a competitive inhibition enzyme-linked immunosorbent assay 
(ELISA/EIA) (Osteomark® NTx Serum, Alere Scarborough, Inc., 
Scarborough,ME). The intra-assay and inter-assay variability are 4.6% and 6.9%, 
respectively. Quantitative bone-specific alkaline phosphatase (BAP) will be 
determined at a Massachusetts General Hospital core laboratory using an enzyme 
immunoassay (MicroVueTM BAP EIA; Quidel Corporation, San Diego, CA). 
7.12 Blood biomarkers (see Appendix C for detailed instructions): 
Blood samples will be collected at study specific timepoints. For the purposes of 
this testing, at least 10 mL will be collected in a PURPLE top (EDTA plasma) 
tube. After processing, separated peripheral blood cells will be immediately 

 
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stained using fluorescently labeled antibodies and analyzed using an LSR-II flow 
cytometer within the Steele Laboratory's facility. Dr. Dan G. Duda, D.M.D., 
Ph.D. (Associate Professor at MGH/HMS), who has over 12 years of experience 
in clinical correlative studies, supervises the Steele Lab's Core 
operation. Plasma analysis will be carried out for a panel of circulating 
angiogenic and inflammatory molecules. They include vascular endothelial 
growth factor (VEGF), placental-derived growth factor (PlGF), soluble 
(s)VEGFR1, basic fibroblast growth factor (bFGF), VEGF-C, VEGF-D and 
sTie2 using multiplex (7-plex) and IL-1β, IL-6, IL-8 and TNF-α (4-plex) protein 
array kits from Meso-Scale Discovery (Gaithersburg, MD), and stromal cell-
derived factor 1a (SDF1a), hepatocyte growth factor (HGF), soluble (s)c-MET, 
and s-c-KIT using ELISA from R&D Systems (Minneapolis, MN). Finally, we 
will evaluate biomarkers of tumor hypoxia, by measuring plasma carbonic 
anhydrase IX (CAIX) levels as well as biomarkers of osteoclast and osteoblast 
activity (plasma C-telopeptide and total alkaline phosphatase) using ELISA from 
R&D Systems. Samples will be run in duplicate. 
7.13 Patient reported outcomes: Key patient reported outcome measures will include: 
1. Analgesic use as recorded in a medication diary (see Appendix D)
2. Changes in quality of life as measured by a validated assessment tool (EQ-5D-
3L; see Appendix E) 
3. Changes in pain and narcotic analgesic use as assessed by 4-item  
questionnaire taken from the MD Anderson Brief Pain Inventory (BPI; see 
Appendix F)54: 
     A. Pain at its worst in the last 24 hours (BPI question 3)
     B. Average pain in the last 24 hours (BPI question 5)
     C. Effect of pain on general activity (BPI question 9A)
     D. Effect of pain on sleep (BPI question 9F)
7.14 Efficacy
Primary efficacy variable: The primary objective of this study is to evaluate the efficacy of 
Ra-223 based on bone scan response measured by quantitative analysis of 99mTc-MDP bone 
scan. The bone scan area that we will employ for analysis was developed as a quantitative 
tool to improve the interpretability and clinical relevance of the bone scan. The BSA is a 
method of expressing the tumor burden in bone as a percent of the total skeletal mass. For 
analysis, scan images are intensity normalized, and the lesions are identified and 
segmented by anatomic region-specific intensity thresholding, and area summed (resulting in 
a bone scan area measurement for the skeleton and lesions). This calculated ratio of lesion 
area to bone scan area (calculated from two dimensional planar images) will be used to assess 
percent change with treatment. Bone scan response is defined as a ≥ 30% decrease in 
quantitative bone scan lesion area (BSA) from baseline to the end of month 2 of Ra-223 
treatment (i.e. after the second of six possible Ra-223 doses). 
Secondary variables: Please see statistical methods in protocol Section 8. 

 
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7.15 Safety
The sponsor is responsible to comply with the local regulation and legislation for adverse 
events reporting.
All subjects who receive at least one dose of study treatment will be valid for the safety 
analysis. 
All observations pertinent to the safety of the study treatment will be recorded and included 
in the final report.
All AEs whether considered drug-related or not, will be reported with a diagnosis, start/stop 
dates, action taken, whether treatment was discontinued, any corrective measures taken, 
outcome, and other possible causes.  For all events, the relationship to treatment and the 
intensity of the event will be determined by the investigator.
This trial will use the NCI-CTCAE v4.0 criteria for assessment of toxicity and SAE reporting 
with regard to toxicity grade. 
Safety variables may include but not limited to the following: laboratory changes (complete 
blood counts, electrolytes, chemistry, and coagulation), changes in vital signs (blood pressure, 
heart rate, respiratory rate, and temperature) and ECG and, in some instances, changes in 
chest x-ray images, as produced at the investigator’s discretion (e.g., for evaluation for 
pneumonia).
7.15.1
Adverse events
Investigators should refer to the Safety Information section of the current IB for Ra 223 
dichloride, including the DCSI (development core safety information), for the expected side 
effects of Ra 223 dichloride.  As with any agent, there is always the potential for unexpected 
AEs, including hypersensitivity reactions.  The IB will be updated if any new relevant safety 
data are obtained.
Therapeutic monitoring should be performed following dose selection of Ra 223 dichloride in 
a manner consistent with the local clinical standard of care.  In general, subjects should be 
closely monitored for side effects of all concomitant medications regardless of the path of 
drug elimination.
All concomitant medications must be recorded in the subject’s source documentation.
Subjects must be carefully monitored for AEs.  This monitoring also includes clinical 
laboratory tests.  Adverse events should be assessed in terms of their seriousness, intensity, 
and relationship to the study drug, or other chemotherapy/treatment.

 
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7.15.1.1Definitions
Definition of adverse event (AE)
In a clinical study, an AE is any untoward medical occurrence (i.e. any unfavorable and 
unintended sign [including abnormal laboratory findings], symptom or disease) in a 
patient or clinical investigation subject after providing written informed consent for 
participation in the study.  Therefore, an AE may or may not be temporally or causally 
associated with the use of a medicinal (investigational) product.
A surgical procedure that was planned prior to the start of the study by any physician treating 
the subject should not be recorded as AE (however, the condition for which the surgery is 
required may be an AE if the condition worsens compared to baseline).

Conditions that started before signing of informed consent and for which no symptoms 
or treatment are present until signing of informed consent are recorded as medical 
history (e.g. seasonal allergy without acute complaints).

Conditions that started before signing of informed consent and for which symptoms or 
treatment are present after signing of informed consent, at unchanged intensity, are 
recorded as medical history (e.g. allergic pollinosis).

Conditions that started or deteriorated after signing of informed consent will be 
documented as adverse events.
Definition of serious adverse event (SAE)
An SAE is classified as any untoward medical occurrence that, at any dose, meets any of the 
following criteria (a – f):
a. Results in death.
b. Is life-threatening.
The term ‘life-threatening’ in the definition refers to an event in which the patient 
was at risk of death at the time of the event, it does not refer to an event which 
hypothetically might have caused death if it were more severe.
c. Requires inpatient hospitalization or prolongation of existing hospitalization.
A hospitalization or prolongation of hospitalization will not be regarded as an SAE if 
at least one of the following exceptions is met:
- The admission results in a hospital stay of less than 12 hours.
- The admission is pre-planned. 
(i.e. elective or scheduled surgery arranged prior to the start of the study)
- The admission is not associated with an AE.
(e.g. social hospitalization for purposes of respite care).
However, it should be noted that invasive treatment during any hospitalization may 
fulfill the criterion of ‘medically important’ and as such may be reportable as an SAE 

 
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dependent on clinical judgment.  In addition, where local regulatory authorities 
specifically require a more stringent definition, the local regulation takes precedence.
d. Results in persistent or significant disability / incapacity.
Disability means a substantial disruption of a person’s ability to conduct normal life’s 
functions.
e.
Is a congenital anomaly / birth defect.
f.
Is another medically important serious event as judged by the investigator.
7.15.1.2Classifications for adverse event assessment
All AEs will be assessed and documented by the investigator according to the categories 
detailed below.
7.15.1.2.1 Seriousness
For each AE, the seriousness must be determined according to the criteria given in 
Section 7.15.1.1.
7.15.1.2.2 Intensity
The intensity of the AE is classified according to the CTCAEv4.0.  Grade refers to the 
severity (intensity) of the AE:
CTCAEv4 Grade 1: mild; asymptomatic or mild symptoms; clinical or diagnostic 
observations only; intervention is not indicated.
CTCAEv4 Grade 2: moderate; minimal, local, or noninvasive intervention is indicated; 
limiting to age-appropriate instrumental activities of daily living (ADL; instrumental 
ADL refers to preparing meals, shopping for groceries or clothes, using the telephone, 
managing money, etc).
CTCAEv4 Grade 3: Severe or medically significant but not immediately life threatening; 
hospitalization or prolongation of hospitalization is indicated; disabling; limiting to 
self care ADL (self care ADL refers to bathing, dressing and undressing, feeding self, 
using the toilet, taking medications, and not bedridden).
CTCAEv4 Grade 4: life-threatening consequences; urgent intervention is indicated.
CTCAEv4 Grade 5: death due to an AE.
7.15.1.2.3 Causal relationship 
The assessment of the causal relationship between an AE and the administration of treatment 
is a clinical decision based on all available information.
The assessment is based on the question whether there was a “reasonable causal relationship” 
to the study treatment in question.
Possible answers are “yes” or “no”.
An assessment of “no” would include:

 
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1.
The existence of a clear alternative explanation, e.g. mechanical bleeding at surgical 
site.
or
2. Non-plausibility, e.g. the subject is struck by an automobile when there is no 
indication that the drug caused disorientation that may have caused the event; cancer 
developing a few days after the first drug administration.
An assessment of “yes” indicates that there is a reasonable suspicion that the AE is associated 
with the use of the study treatment.
Factors to be considered in assessing the relationship of the AE to study treatment include:
- The temporal sequence from drug administration:  The event should occur after the drug 
is given.  The length of time from drug exposure to event should be evaluated in the 
clinical context of the event.
- Recovery on drug discontinuation (de-challenge), recurrence on drug re-introduction (re-
challenge):
- Subject’s response after de-challenge or subjects response after re-challenge should be 
considered in the view of the usual clinical course of the event in question.
- Underlying, concomitant, intercurrent diseases:  
Each event should be evaluated in the context of the natural history and course of the 
disease being treated and any other disease the subject may have.
- Concomitant medication or treatment: 
The other drugs the subject is taking or the treatment the subject receives should be 
examined to determine whether any of them may be suspected to cause the event in 
question.
- The pharmacology and pharmacokinetics of the study treatment: 
The pharmacokinetic properties (absorption, distribution, metabolism and excretion) of 
the study treatment, coupled with the individual subject’s pharmacodynamics should be 
considered.
Causal relationship to protocol-required procedure(s)
The assessment of a possible causal relationship between the AE and protocol-required 
procedure(s) is based on the question whether there was a “reasonable causal relationship” to 
protocol-required procedure(s).
Possible answers are “yes” or “no”.
7.15.1.2.4 Action taken with study treatment 
Any action on study treatment to resolve the AE is to be documented using the categories 
listed below.

 
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־Drug withdrawn
־Drug interrupted
־Dose not changed
־Dose increased
־Not applicable
־Unknown
7.15.1.2.5 Other specific treatment(s) of adverse events
־None
־Remedial drug therapy
־Other
7.15.1.2.6 Outcome
The outcome of the AE is to be documented as follows: 
־Recovered/resolved
־Recovering/resolving
־Recovered/resolved with sequelae
־Not recovered/not resolved
־Fatal
־Unknown
7.15.1.3Assessments and documentation of adverse events
7.15.1.4Reporting of serious adverse events
The definition of serious adverse events (SAEs) is given in Section 7.15.1.1.
Each serious adverse event must be followed up until resolution or stabilization, by 
submission of updated reports to the designated person. An isolated laboratory abnormality 
that is assigned grade 4, according to CTC definition, is not reportable as an SAE; unless the 
investigator assesses that the event meets standard ICH criteria for an SAE. CTC grade 4 
baseline laboratory abnormalities that are part of the disease profile should not be reported as 
an SAE, specifically when they are allowed or not excluded by the protocol 
inclusion/exclusion criteria.  
When required, and according to local law and regulations, serious adverse events must be 
reported to the Ethics Committee and Regulatory Authorities. 

 
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All serious adverse events should be reported to Bayer within 24 hours.  In the event of such 
an event, the investigator should refer to the Pharmacovigilance section of the contract for 
reporting procedures.
Requirements for Reporting of Serious Adverse Events: 
All SAEs must be reported to Bayer within 24 hours of the Principal Investigator’s awareness 
and must include the following minimum information: 
1. The name and contact information of the reporter 
2. The name of the study drug(s) 
3. A description of the reported SAE 
4. A patient identified by one or more of the following: 
a. Patient initials 
b. Patient number 
c. Knowledge that a patient who experienced the adverse event exists 
d. Age 
e. Sex 
5. An investigator assessment of study drug causality. For studies with combination therapy, a 
separate causality assessment should be provided for each study drug. 
Additional data which would aid the review and causality assessment of the case include but 
are not limited to: 
The date of onset 
The severity 
The time from administration of study drug(s) to start of the event 
The duration and outcome of the event 
Any possible etiology for the event 
The final diagnosis or syndrome, if known 
The Investigator may report serious adverse drug reactions (SADRs) using either:
An ADEERS form (Adverse Event Expedited Reporting System) available at 
http://ctep.cancer.gov/reporting/adeers.html  
OR
 
 A MedWatch form available at http://www.fda.gov/medwatch/ 
All reports shall be sent electronically to: 
Electronic Mailbox: 
DrugSafety.GPV.US@bayer.com 
Facsimile:  
(973) 709-2185 
Address:
Global Pharmacovigilance - USA 
Mail only:
Bayer HealthCare 
P.O. Box 1000 

 
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Montville, NJ 07045-1000 
Address:  
340 Changebridge Road 
FDX or UPS only  
Pine Brook, NJ  07058 
Reports for all Bayer products can also be phoned in via our Clinical Communications 
Dept: 
Phone: 
1-888-842-2937
7.15.1.5 Expected adverse events
For this study, the applicable reference document is the most current version of the 
investigator’s brochure (IB) / summary of product characteristics.
The expectedness of AEs will be determined by Bayer according to the applicable reference 
document and according to all local regulations.
7.15.2
Pregnancies
The investigator must report to Bayer any pregnancy occurring in a study subject’s partner, 
during the subject’s participation in this study.  The report should be submitted within the 
same timelines as an SAE, although a pregnancy per se is not considered an SAE. For the 
pregnancy of a study subject’s partner, all efforts should be made to obtain information on 
course and outcome, subject to the partner’s consent. For all reports, the forms provided are 
to be used.
7.15.3
Further safety
Progressive disease
If progressive disease leads to signs and symptoms that meet the criteria for an SAE 
(i.e., hospitalization, disability, death, or important medical event), the signs and symptoms 
should be reported as an SAE and not the underlying progressive disease.
Death
If any subject dies during the trial or within 30 days of the end-of-treatment visit, the 
investigator will inform Bayer and record the cause of death in detail (using the SAE Form) 
within 24 hours.
7.16 Other procedures and variables
7.17 Appropriateness of procedures / measurements
The assessments described in the previous sections are widely used and generally recognized 
as reliable, accurate, and relevant for determining the safety and efficacy of therapies in this 
disease.
8. Statistical methods and determination of sample size
Primary Aim

 
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Bone scan response is defined as a ≥ 30% decrease in quantitative bone scan lesion area 
(BSA) from baseline to the end of month 2 of Ra-223 treatment (i.e. after 2 of 6 possible Ra-
223 doses). In a one-stage design, 22 subjects will be enrolled to evaluate the primary 
endpoint of bone scan response. Assuming 90% of subjects with adequate data, the evaluable 
sample size is 20 subjects. With 20 subjects, given target power of 90% and 1-sided 5% 
alpha, a null hypothesis of ≤ 10% versus an alternative hypothesis of ≥ 35% in the proportion 
of patients achieving response can be evaluated. If 5 or more responses are observed in 20 
subjects, this method of response assessment will be considered promising. 
Operating characteristics
True but unknown rate of Bone Scan Response 
Rate
10%
15%
20%
25%
30%
35%
Probability of ≥ 5 
responses in 20 pts
0.04
0.17
0.37
0.59
0.76
0.88
Secondary Aims
A key secondary objective is to evaluate whether quantitative analysis of 99mTc-MDP bone 
scan after 2 months of therapy can identify patients who will achieve long term clinical 
benefit. Towards this end, all subjects will be treated and followed for at least 18 months. 
Survival status at 18 months will be ascertained (i.e. dichotomized as survivor or non-
survivor at 18 months). A two-sample t-test will be used to determine the difference in mean 
percent change in BSA between survivor and non-survivor groups. Reported median survival 
for the study cohort of the ALSYMPCA trial was 14.9 months. Assuming events follow an 
exponential distribution this parallels an 18-month survival rate of 43%. The detectable 
difference will depend on the variability in change in BSA as measured by standard deviation 
(SD). There is 80% power to detect an effect of 1.332 SD given a 2-sided 0.05 alpha and 20 
evaluable patients divided as 9 survivors and 11 non-survivors. 
Bone scans are scheduled for the beginning of month 3/end of month 2 (T3), and one month 
post month 6 (FU1M). Descriptive statistics on absolute and percent change in BSA from 
baseline to these study timepoints will be provided for the overall study cohort and within 
survivor groups. By the end of month 2, bone scan response is expected in 35% of subjects. 
Patients will be cross-classified by bone scan response status at 2 months and survival status 
at 18 months.  Agreement will be quantified using Cohen’s Kappa; however, the sample size 
is too small to produce a realistic confidence interval. Kappa is assessed as follows: <0.00, 
poor; 0.00-0.20, slight; 0.21-0.40, fair; 0.41-0.60, moderate; 0.61-0.80, substantial; 0.81-1.00, 
almost perfect; and 1.00, perfect. Survival time defined as the time from registration to death, 
or if censored to date last known alive will be calculated. Correlation between change in BSA 
after 2 months and survival time will be assessed graphically with scatter plots and using 
Pearson correlation coefficient. Survival distributions will also be estimated using the 
Kaplan-Meier method in a landmark analysis at 2 months. Survival time is defined as the time 
from response classification (at 2 months) to death, or if censored to date last known alive. 
Cox proportional hazards (PH) regression will be used to estimate the bone scan response 
hazard ratio along with 95% confidence limits. 

 
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This study also affords insights into several other potential biomarkers including measures 
related to circulating tumor cells (number, proliferative index, AR signaling, markers of DNA 
damage/repair), bone turnover (BAP, NTx), inflammation and angiogenesis. Fluciclovine 
PET imaging will occur in 10 subjects at baseline and the end of month 2 of treatment. All 
other measures will occur at baseline, during treatment, and post treatment as detailed in the 
required data table. Descriptive statistics will be provided for each measure and timepoint 
along with absolute and percentage change from baseline. Association with overall survival 
and response will be analyzed as exploratory similar to the methods above for bone scan. 
Accrual Targets 
Accrual Targets
Sex/Gender
Ethnic Category
Females
Males
Total
Hispanic or Latino
0
+
1
=
1
Not Hispanic or Latino
0
+
21
=
21
Ethnic Category: Total of all subjects
0
(A1)
+
22
(B1) =
22
(C1)
Racial Category
American Indian or Alaskan Native
0
+
0
=
0
Asian
0
+
1
=
1
Black or African American
0
+
1
=
1
Native Hawaiian or other Pacific 
Islander
0
+
0
=
0
White
0
+
20
=
20
Racial Category: Total of all subjects
0
(A2)
+
22
(B2) =
22
(C2)
(A1 = A2)
(B1 = B2)
(C1 = C2)
Timeline 
We anticipate an average of 2 subjects to be accrued monthly (approximate accrual time: 12 
months). The primary endpoint (assessment of change in total area of bone metastases by 
99mTc MDP bone scan) is assessed 2 months after the start of treatment; the primary analysis 
will take place 14 months after study activation. With an anticipated 12 month enrollment 
period and per-subject 18 months of study-directed treatment and follow-up, the last enrolled 
patient will conclude follow-up 30 months after activation. 
Notable anticipated dates and analyses are as follows: 
T: 0m
Study opens for accrual at MGH
T: 12m
Study accrual completed
T: 14m
Primary analysis
T: 30m
Completion of all study follow-up

 
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9. Data handling and quality assurance
9.1 Data recording
It is the expectation that all data has source documentation available at the site.  The site must 
implement processes to ensure this happens.  
The QACT will collect, manage, and perform quality checks on the data for this study.
Investigative sites within DF/HCC or DF/PCC are responsible for submitting data and/or data 
forms to the QACT according to the schedule set by the QACT. 
9.2 Monitoring
The DF/HCC Data and Safety Monitoring Board (DSMB) will review and monitor study 
progress, toxicity, safety and other data from this study. The board is chaired by a medical 
oncologist from outside of DF/HCC and has external and internal representation. Information 
that raises any questions about participant safety or protocol performance will be addressed 
by the Overall PI, statistician and study team. Should any major concerns arise, the DSMB 
will offer recommendations regarding whether or not to suspend the study.
The DSMB will meet twice a year to review accrual, toxicity, response and reporting 
information. Information to be provided to the DSMB may include: participant accrual; 
treatment regimen information; adverse events and serious adverse events reported by 
category; summary of any deaths on study; audit results; and a summary provided by the 
study team. Other information (e.g. scans, laboratory values) will be provided upon request. 
9.3 Audit and inspection
Inspections by regulatory health authority representatives i.e. FDA and IEC(s)/IRB(s) are 
possible.  The investigator should notify Bayer immediately of any such inspection.
9.4 Archiving
Essential documents shall be archived safely and securely in such a way that ensures that they 
are readily available upon authorities’ request.
Patient (hospital) files will be archived according to local regulations and in accordance with 
the maximum period of time permitted by the hospital, institution or private practice.

 
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10. Premature termination of the study

If risk-benefit ratio becomes unacceptable owing to, for example,
- Safety findings from this study (e.g. SAEs)
- Results of any interim analysis
- Results of parallel clinical studies
- Results of parallel animal studies 
(on e.g. toxicity, teratogenicity, carcinogenicity or reproduction toxicity).

If the study conduct (e.g. recruitment rate; drop-out rate; data quality; protocol 
compliance) does not suggest a proper completion of the trial within a reasonable time 
frame.
The investigator has the right to close his/her center at any time.
For any of the above closures, the following applies:

Closures should occur only after consultation between involved parties.

All affected institutions (e.g. IEC(s)/IRB(s); competent authority(ies); study center; 
head of study center) must be informed as applicable according to local law.

In case of a partial study closure, ongoing subjects, including those in post study 
follow-up, must be taken care of in an ethical manner.
Details for individual subject's withdrawal can be found in Section 5.2.1.
11. Ethical and legal aspects
11.1 Ethical and legal conduct of the study
The procedures set out in this protocol, pertaining to the conduct, evaluation, and 
documentation of this study, are designed to ensure that the investigator abide by Good 
Clinical Practice (GCP) guidelines and under the guiding principles detailed in the 
Declaration of Helsinki.  The study will also be carried out in keeping with applicable local 
law(s) and regulation(s).
Documented approval from appropriate IEC(s)/IRBs will be obtained for all participating 
centers before start of the study, according to GCP, local laws, regulations and organizations.  
When necessary, an extension, amendment or renewal of the EC/IRB approval must be 
obtained and also forwarded to Bayer.
Strict adherence to all specifications laid down in this protocol is required for all aspects of 
study conduct; the investigator may not modify or alter the procedures described in this 
protocol.  
Modifications to the study protocol will not be implemented by the investigator without 
discussion and agreement by Bayer.  However, the investigator may implement a deviation 
from, or a change of, the protocol to eliminate an immediate hazard(s) to the trial subjects 
without prior IEC/IRB/Bayer approval/favorable opinion.  As soon as possible, the 

 
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implemented deviation or change, the reasons for it and if appropriate the proposed protocol 
amendment should be submitted to the IEC/IRB/head of medical institution.  Any deviations 
from the protocol must be explained and documented by the investigator.
The Principal Investigator is responsible for the conduct of the clinical trial at the site in 
accordance with Title 21 of the Code of Federal Regulations and/or the Declaration of 
Helsinki.  The Principal Investigator is responsible for personally overseeing the treatment of 
all study patients.  The Principal Investigator must assure that all study site personnel, 
including sub-investigators and other study staff members, adhere to the study protocol and 
all FDA/GCP/NCI regulations and guidelines regarding clinical trials both during and after 
study completion.
The Principal Investigator at each institution or site will be responsible for assuring that all 
the required data will be collected and properly documented.
11.2 Subject information and consent
Each subject / legal representative or proxy consenter will have ample time and opportunity 
to ask questions and will be informed about the right to withdraw from the study at any time 
without any disadvantage and without having to provide reasons for this decision.
Only if the subject / legal representative or proxy consenter voluntarily agrees to sign the 
informed consent form and has done so, may he/she enter the study.  Additionally, the 
investigator and other information provider (if any) will personally sign and date the form.  
The subject / legal representative or proxy consenter will receive a copy of the signed and 
dated form.
The signed informed consent statement is to remain in the investigator site file or, if locally 
required, in the patient’s note/file of the medical institution.
In the event that informed consent is obtained on the date that baseline study procedures are 
performed, the study record or subject’s clinical record must clearly show that informed 
consent was obtained prior to these procedures.
1. If the patient is not capable of providing a signature, a verbal statement of consent can 
also be given in the presence of an impartial witness (independent of Bayer and the 
investigator).  This is to be documented by a signature from the informing physician as 
well as by a signature from the witness.
The informed consent form and any other written information provided to subjects / legal 
representatives or proxy consenters will be revised whenever important new information 
becomes available that may be relevant to the subject’s consent, or there is an amendment to 
the protocol that necessitates a change to the content of the subject information and / or the 
written informed consent form.  The investigator will inform the subject / legal representative 
or proxy consenter of changes in a timely manner and will ask the subject to confirm his/her 
participation in the study by signing the revised informed consent form.  Any revised written 
informed consent form and written information must receive the IEC/IRB`s approval / 
favorable opinion in advance of use.

 
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11.3 Publication policy
The results should be made public within 24 months of reaching the end of the study. The end 
of the study is the time point at which the last data items are to be reported, or after the 
outcome data are sufficiently mature for analysis, as defined in the section on Sample Size, 
Accrual Rate and Study Duration. If a report is planned to be published in a peer-reviewed 
journal, then that initial release may be an abstract that meets the requirements of the 
International Committee of Medical Journal Editors. A full report of the outcomes should be 
made public no later than three (3) years after the end of the study.
Bayer recognizes the right of the investigator to publish results upon completion of the study.  
However, the investigator must send a draft manuscript of the publication or abstract to Bayer 
at least thirty days in advance of submission in order to obtain approval prior to submission of 
the final version for publication or congress presentation. This will be reviewed promptly and 
approval will not be withheld unreasonably. In case of a difference of opinion between Bayer 
and the investigator(s), the contents of the publication will be discussed in order to find a 
solution which satisfies both parties. All relevant aspects regarding data reporting and 
publication will be part of the contract between Bayer and the investigator/institution
The Principal Investigator will ensure that the information regarding the study be publicly 
available on the internet at www.clinicaltrials.gov.
11.3.1 Publications and NIST
Bayer recommends inclusion of the new NIST standard in abstracts/ publications 
submitted OR pending  publication January 2016 onwards    from IIRs and other non- 
Bayer supported abstracts/publications for consistency. Investigators may choose to 
add a footnote to the publication or within the body of the publication include the new 
NIST standard.
11.4 Confidentiality
All records identifying the subject will be kept confidential and, to the extent permitted by the 
applicable laws and/or regulations, will not be made publicly available.
Should direct access to medical records require a waiver or authorization separate from the 
subject’s statement of informed consent, it is the responsibility of the Investigator to obtain 
such permission in writing from the appropriate individual.
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Schuster DM, Nieh PT, Jani AB, et al. Anti-3-[18F]FACBC PET-CT and 111In-
capromab-pendetide SPECT-CT in Recurrent Prostate Carcinoma: Results of a Prospective 
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Schuster DM, Savir-Baruch B, Nieh PT, et al. Detection of recurrent prostate 
carcinoma with anti-1-amino-3-18F-fluorocyclobutane-1-carboxylic acid PET/CT and 111In-
capromab pendetide SPECT/CT. Radiology 2011;259(3):852-61.
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Schuster DM, Votaw JR, Nieh PT, et al. Initial experience with the radiotracer anti-1-
amino-3-18F-fluorocyclobutane-1-carboxylic acid with PET/CT in prostate carcinoma. J 
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patients with metastatic prostate cancer correlate with disease status. Urology 
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Danila DC, Fleisher M, Scher HI. Circulating tumor cells as biomarkers in prostate 
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progressive, castration-resistant prostate cancer: a reanalysis of IMMC38 trial data. Lancet 
Oncol 2009;10(3):233-9.

 
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of Standards and Technology (NIST)standardization update (agreed on 17 MAR 2015)”
30.
Stott SL, Hsu CH, Tsukrov DI, et al. Isolation of circulating tumor cells using a 
microvortex-generating herringbone-chip. Proc Natl Acad Sci U S A 2010;107(43):18392-7.
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Miyamoto DT, Lee RJ, Stott SL, et al. Androgen receptor signaling in circulating 
tumor cells as a marker of hormonally responsive prostate cancer. Cancer discovery 
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Cancer 2000;82(4):858-64.
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Cook RJ, Coleman R, Brown J, et al. Markers of bone metabolism and survival in 
men with hormone-refractory metastatic prostate cancer. Clin Cancer Res 2006;12(11 Pt 
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Coleman RE, Major P, Lipton A, et al. Predictive value of bone resorption and 
formation markers in cancer patients with bone metastases receiving the bisphosphonate 
zoledronic acid. J Clin Oncol 2005;23(22):4925-35.
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myeloid but not endothelial precursor cells facilitates tumor regrowth after local irradiation. 
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Dawson MR, Duda DG, Fukumura D, Jain RK. VEGFR1-activity-independent 
metastasis formation. Nature 2009;461(7262):E4; discussion E5.
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Kioi M, Vogel H, Schultz G, Hoffman RM, Harsh GR, Brown JM. Inhibition of 
vasculogenesis, but not angiogenesis, prevents the recurrence of glioblastoma after irradiation 
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Chang CC, Lerman OZ, Thanik VD, et al. Dose-dependent effect of radiation on 
angiogenic and angiostatic CXC chemokine expression in human endothelial cells. Cytokine 
2009;48(3):295-302.
40.
Saylor PJ, Kozak KR, Smith MR, et al. Changes in Biomarkers of Inflammation and 
Angiogenesis During Androgen Deprivation Therapy for Prostate Cancer. Oncologist 2012.
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Henriksen G, Breistol K, Bruland OS, Fodstad O, Larsen RH. Significant antitumor 
effect from bone-seeking, alpha-particle-emitting (223)Ra demonstrated in an experimental 
skeletal metastases model. Cancer Res 2002;62(11):3120-5.

 
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45.
Henriksen G, Fisher DR, Roeske JC, Bruland OS, Larsen RH. Targeting of osseous 
sites with alpha-emitting 223Ra: comparison with the beta-emitter 89Sr in mice. J Nucl Med 
2003;44(2):252-9.
46.
Parker C, Heinrich D, O'Sullivan JM, et al. Overall survival benefit and safety profile 
of radium-223 chloride, a first-in-class alpha-pharmaceutical: Results from a phase III 
randomized trial (ALSYMPCA) in patients with castration-resistant prostate cancer (CRPC) 
with bone metastases. J Clin Oncol 2012;30(suppl 5; abstr 8).
47.
Parker C, al. E. Quality of Life (QOL) and updated survival and safety data on 
radium-223 dichloride in patients with castration-resistant prostate cancer (CRPC) with bone 
metastases from the phase 3 ALSYMPCA study. In: ICPU; 2013; 2013.
48.
Sartor AO, al. E. Safety of cytotoxic chemotherapy following radium-223 dichloride 
therapy in the phase 3 ALSYMPCA study in patients with castration-resistant prostate cancer 
(CRPC) with bone metastases. In: ICPU; 2013; 2013.
49.
Vogelzang N, al. E. Updated Analysis of Radium-223 Dichloride Impact on Skeletal-
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With Radium-223 Dichloride in Castration-Resistant Prostate Cancer (CRPC) Patients With 
Bone Metastases. In: ASCO GU; 2013; 2013.
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Heinrich D, al. E. Updated Analysis of Radium-223 Dichloride (Ra-223) Impact on 
Pain, Skeletal-Related Events (SRE), and Survival From the Phase 3 Randomized Trial 
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Metastases. In: EAU; 2013; 2013.
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Stott SL, Lee RJ, Nagrath S, et al. Isolation and Characterization of Circulating Tumor 
Cells from Patients with Localized and Metastatic Prostate Cancer. Sci Transl Med 
2010;2(25):25ra3-ra3.
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Stott SL, Hsu C-H, Tsukrov DI, et al. Isolation of circulating tumor cells using a 
microvortex-generating herringbone-chip. Proc Natl Acad Sci USA 2010;107(43):18392-7.
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of Standards and Technology (NIST)standardization update (agreed on 17 MAR 2015)”
Appendix A: Performance Status Criteria 
ECOG Performance Status Scale
Karnofsky Performance Scale
Grade
Descriptions
Percent
Description
100
Normal, no complaints, no evidence 
of disease.
0
Normal activity.  Fully active, able 
to carry on all pre-disease 
performance without restriction.
90
Able to carry on normal activity; 
minor signs or symptoms of disease.
80
Normal activity with effort; some 
signs or symptoms of disease.
1
Symptoms, but ambulatory.  
Restricted in physically strenuous 
activity, but ambulatory and able 
to carry out work of a light or 
sedentary nature (e.g., light 
housework, office work).
70
Cares for self, unable to carry on 
normal activity or to do active work.
60
Requires occasional assistance, but 
is able to care for most of his/her 
needs.
2
In bed <50% of the time.  
Ambulatory and capable of all 
self-care, but unable to carry out 
any work activities.  Up and about 
more than 50% of waking hours.
50
Requires considerable assistance and 
frequent medical care.
40
Disabled, requires special care and 
assistance.
3
In bed >50% of the time.  Capable 
of only limited self-care, confined 
to bed or chair more than 50% of 
waking hours.
30
Severely disabled, hospitalization 
indicated.  Death not imminent.
20
Very sick, hospitalization indicated. 
Death not imminent.
4
100% bedridden.  Completely 
disabled.  Cannot carry on any 
self-care.  Totally confined to bed 
or chair.
10
Moribund, fatal processes 
progressing rapidly.
5
Dead.
0
Dead.

 
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Appendix B: Standard Operating Procedure for CTC Capture
Materials: Three (3) EpCAM functionalized CTC capture devices; 20 mL of patient’s blood; 
20 mL of PBS, 20 mL of buffer (PBS with 1% bovine serum albumin and 0.01% sodium 
azide), anti-cytokeratin PE (CAM 5.2, BD Biosciences); CD45 FITC (BD Pharmingen); 4’,6-
diamidino-2-phenylindole (DAPI, BD Biosciences); anti-PSA antibody; anti-PSMA antibody; 
Ki67 antibody; PicoPure RNA isolation kit (Arcturus, Sunnyvale, CA); TransPlex Whole 
Transcriptome Amplification Kit (Rubicon Genomics, Ann Arbor, MI), Fluidigm Biomark 
system kit.
Transport of samples: Samples for CTC analyses will be transported from the MGH Cancer 
Center in Boston to the MGH Charlestown Navy Yard laboratories of Drs. Daniel Haber and 
Shyamala Maheswaran. After collection of blood samples, the two vacutainer tubes will be 
de-identified and labels with the subject’s study identification number will be affixed. 
Transport will be performed by U.S. Ground Services, or alternatively, by personal transport 
via a member of the Haber/Maheswaran laboratories. It is expected that the elapsed time 
between blood draw and assay will be under six hours. 
Protocol: Protocol specifics for the samples are subject to ongoing modifications. CTC 
capture, enumeration, and other analysis will be carried out per current Haber/Maheswaran 
lab protocol and will be described in detail in concert with all data reporting. 

 
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Appendix C: Standard Operating Procedure for Blood Biomarkers at the CLIA certified 
Clinical Correlative Studies Core within the Steele Laboratory
Blood will be collected and processed at study specific timepoints as described above. 
Dr. Dan G. Duda, D.M.D., Ph.D. (Associate Professor at MGH/HMS), who has over 13 years 
of experience in clinical correlative studies, supervises the Steele Lab's Core operation. 
1. Collect at least 10 mL of blood in a purple top (EDTA plasma) tube.
2. Maintain sample on wet ice and transport within 1-2 hours of collection to the Steele 
Laboratory facility at MGH main campus (Cox 7).
3. Separate plasma by centrifugation, then aliquot and store at –80ºC until later batched 
analysis.
4. Separated peripheral blood cells (remaining from step <3> above) will be immediately 
stained using fluorescently labeled antibodies and analyzed using an LSR-II flow 
cytometer within the Steele Lab's facility at MGH main campus as previously 
described.55
5. Plasma analysis (samples stored in step <3> above) will later be carried out for a 
panel of circulating angiogenic and inflammatory molecules when all samples have 
been collected at the Steele Lab facility in Charlestown Navy Yard. They include 
protein array kits from Meso-Scale Discovery (Gaithersburg, MD): Human 
ProInflammatory Panel 1 V-PLEX™ Plus (#K15049G-2) – IFN-γ, IL-1β, IL-10, IL-
12 p70, IL-13, IL-2, IL-4, IL-6, IL-8, TNF-α; and Human Angiogenesis Panel 1 V-
PLEX Kit (#K15190D-2) – bFGF, Flt-1, PlGF, Tie-2, VEGF, VEGF-C, VEGF-D, and 
single-analyte ELISA kits from R&D Systems (Minneapolis, MN): for stromal cell-
derived factor 1a (SDF1), hepatocyte growth factor (HGF), soluble (s)c-MET, and s-
c-KIT. Finally, we will evaluate biomarkers of tumor hypoxia, by measuring plasma 
carbonic anhydrase IX (CAIX) levels as well as biomarkers of osteoclast and 
osteoblast activity (plasma C-telopeptide and total alkaline phosphatase) using single-
analyte ELISA kits from R&D Systems. Samples will be run in duplicate.

 
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Appendix D: Opioid/Narcotic Pain Medication Diary
Participant name: ______________
Visit date: ______________
Subject ID: ______________
Visit identifier: ______________
Instructions to study participant: Please complete this diary on the date indicated above as 
“Visit date.” Please enter the number of units you have taken of each pain mediation over the 
prior 24 hour period. If you did not take any listed pain medication, record “NONE” in the “# 
Units Taken” box below. 
Medication Name
(write in name)
Route of 
Administration
(examples: oral, 
patch, other)
Strength per Unit
(example: 10 mg 
tablets)
# Units Taken

 
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Appendix E: EuroQol (EQ)-5D-5L
Under each heading, please check the ONE box that best describes your health TODAY
MOBILITY
I have no problems walking  

I have slight problems walking 

I have moderate problems walking
 

I have severe problems walking
 

I am unable to walk

SELF-CARE
I have no problems washing or dressing myself 

I have slight problems washing or dressing myself 

I have moderate problems washing or dressing myself 

I have severe problems washing or dressing myself 

I am unable to wash or dress myself 

USUAL ACTIVITIES (e.g. work, study, housework, 
family or leisure activities)
I have no problems doing my usual activities 

I have slight problems doing my usual activities 

I have moderate problems doing my usual activities 

I have severe problems doing my usual activities 

I am unable to do my usual activities 

PAIN / DISCOMFORT
I have no pain or discomfort 

I have slight pain or discomfort 

I have moderate pain or discomfort 

I have severe pain or discomfort 

I have extreme pain or discomfort 

ANXIETY / DEPRESSION
I am not anxious or depressed 

I am slightly anxious or depressed 

I am moderately anxious or depressed 

I am severely anxious or depressed 

I am extremely anxious or depressed 


 
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“The values in this protocol have been revised as per the United States (US) National Institute 
of Standards and Technology (NIST)standardization update (agreed on 17 MAR 2015)”

We would like to know how good or bad your health is 
TODAY.

This scale is numbered from 0 to 100.

100 means the best health you can imagine.
0 means the worst health you can imagine.

Mark an X on the scale to indicate how your health is 
TODAY.

Now, please write the number you marked on the scale in the 
box below. 
YOUR HEALTH TODAY = 
10
0
20
30
40
50
60
80
70
90
100
5
15
25
35
45
55
75
65
85
95
The best health      
 you can imagine
The worst health    
 you can imagine

 
30 July 2019
Version: Final
Page:  63 of 65
“The values in this protocol have been revised as per the United States (US) National Institute 
of Standards and Technology (NIST)standardization update (agreed on 17 MAR 2015)”
Appendix F: Brief Pain Inventory
Participant name: ______________
Visit date: ______________
Subject ID: ______________
Visit identifier: ______________
Instructions to study participant: Please complete this diary on the date indicated above as 
“Visit date.”
A. Please rate your pain by circling the one number that best describes your pain at its worst 
in the last 24 hours. 
0         1          2          3          4          5          6          7          8          9         10   
No
Pain as bad as
Pain
you can imagine
B. Please rate your pain by circling the one number that best describes your pain on average. 
0         1          2          3          4          5          6          7          8          9         10   
No
Pain as bad as
Pain
you can imagine
C. Please circle the one number that describes how, during the past 24 hours, pain has 
interfered with your general activity.  
0         1          2          3          4          5          6          7          8          9         10   
Does not
Completely
Interfere 
Interferes 
D. Please circle the one number that describes how, during the past 24 hours, pain has 
interfered with your sleep. 
0         1          2          3          4          5          6          7          8          9         10   
Does not
Completely
Interfere 
Interferes 

 
30 July 2019
Version: Final
Page:  64 of 65
“The values in this protocol have been revised as per the United States (US) National Institute 
of Standards and Technology (NIST)standardization update (agreed on 17 MAR 2015)”
Appendix G: Sample Processing for Veridex CellSearch CTC Analyses
Specimen Collection, Handling, Storage: 

Venipuncture or venous port collection only.

Blood samples must be obtained in CellSave Preservative Tubes. Proprietary 
preservative stabilizes CTCs for up to 96 hours at room temperature.

If multiple tube types are to be drawn, draw the CellSave Tubes last.

Draw 1 CellSave tube of blood (approximately 7.5 mL). 
Supplies: CellSave Preservative Tubes
Procedure: 
1
Fill tube completely – until blood flow stops.
2
Immediately invert gently 8 times.
3
Check for clots.
4
Label tubes with two patient identifiers.
4
Send as soon as possible to Brigham and Women's Hospital, Cytology Division, 
Circulating Tumor Cell Lab, Medical Research Building room 315.
Procedure notes: 

Invert the tube immediately after drawing to avoid clotting. Clotted samples cannot 
be submitted to laboratory.  

Do not rock, vortex or shake samples.  

Store samples at room temperature.  Do not refrigerate.
Reference: Veridex, LLC,  CellSearch System Training Guide. Page 1-6, section 1-4.

 
30 July 2019
Version: Final
Page:  65 of 65
“The values in this protocol have been revised as per the United States (US) National Institute 
of Standards and Technology (NIST)standardization update (agreed on 17 MAR 2015)”
Appendix H: Sample Processing for Bone Turnover Markers
Collection of Specimens: Blood draws will take place at three timepoints (baseline, 3-months, 
and 6-months). Each blood draw will be for 10 mL.
Handling of Specimens: Each sample will be processed for storage as serum as follows: 
1. Label as many 1 mL cryovials as necessary for the serum collected (label to include study 
number, participant identifier, collection time & date, study timepoint, and “serum.” 
2. Allow red top tube to clot for 30 minutes at room temperature. 
3. Spin in a standard clinical centrifuge at ~2500 RPM for 10 minutes at 4 degrees C (or at 
room temperature if done within 2 hours of blood draw). 
4. Aliquot 0.5 mL serum into as many cryovials as are necessary for the sample collected (as 
labeled under item (1)). 
5. Place cryovials into biohazard bag and immediately freeze at -70 to -90 degrees C. 
Storage of Specimens: Cryovials will be frozen and stored at -70 to -90 degrees C.