A Phase II, Open Label Study to Evaluate Denosumab in Patients With ER and/or PR-Positive, HER2-Negative Metastatic Breast Cancer (MBC) With Bone Metastases and Detectable Circulating Tumor Cells (CTCs)
Phase: PHASE2
Conditions: Breast Carcinoma Metastatic in the Bone, Circulating Tumor Cell Count, Estrogen Receptor Positive, HER2/Neu Negative, Progesterone Receptor Positive, Stage IV Breast Cancer
Sponsor: Northwestern University

                                                                 
NU16B09
6.1.17/ Amendment 1
i
A Phase II, Open label Study to evaluate Denosumab in patients with 
ER and/or PR-positive, HER2-negative Metastatic Breast Cancer 
(MBC) with bone metastases and detectable circulating tumor cells 
(CTCs)
Principal Investigator:
Massimo Cristofanilli, MD
                                                    Professor
                                                    Department of Medicine
                                                    Division of Hematology /Oncology
Northwestern University Feinberg School of Medicine
710 N. Fairbanks Ct.
Chicago, IL 60611
312-503-5488
Massimo.cristofanilli@nm.org
Sub-Investigator(s):                  Robert H. Lurie Comprehensive Cancer Center
Northwestern University
Sarika Jain, MD
William Gradishar, MD
                                                    Lisa Flaum, MD
                                                    Cesar Santa-Maria, MD
Zhaomei Mu, MD
                                                                                                       
Biostatistician:
             Alfred Rademaker
             rademaker@northwestern.edu
Study Intervention(s):
Denosumab; AMG162, Xgeva, 
IND Number:
IND Exempt
Funding Source: 
Amgen
Version Date:
6.1.2017
Coordinating Center:
Clinical Trials Office
Robert H. Lurie Comprehensive Cancer Center 
Northwestern University
676 N. St. Clair, Suite 1200
Chicago, IL  60611
http://cancer.northwestern.edu/CRO/index.cfm
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
6.1.17/ Amendment 1
ii
Table of Contents
LIST OF ABBREVIATIONS ............................................................................................................1
STUDY SCHEMA ..........................................................................................................................ii2
1.0
STUDY SUMMARY .............................................................................................................3
1.0
INTRODUCTION – BACKGROUND & RATIONALE..........................................................5
2.0
OBJECTIVES ....................................................................................................................14
3.0
PATIENT ELIGIBILITY......................................................................................................14
4.0
TREATMENT PLAN ..........................................................................................................17
5.0
STUDY PROCEDURES.....................................................................................................21
6.0
ENDPOINT ASSESSMENT...............................................................................................23
7.0
ADVERSE EVENTS ..........................................................................................................24
8.0
DRUG INFORMATION ......................................................................................................29
9.0
CORRELATIVES/SPECIAL STUDIES..............................................................................30
10.0
STATISTICAL CONSIDERATIONS ..................................................................................33
11.0
STUDY MANAGEMENT....................................................................................................35
REFERENCES ..............................................................................................................................38
APPENDICES................................................................................................................................40
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                           
NU16B09
6.1.17/ Amendment 1
1
LIST OF ABBREVIATIONS
AE
Adverse Event
ALT
Alanine Aminotransferase
ALC
Absolute Lymphocyte Count
AST
Aspartate Aminotransferase
ASCO
American Society of Clinical Oncology
BUN
Blood Urea Nitrogen
CBC
Complete Blood Count
CMP
Comprehensive Metabolic Panel
CR
Complete Response
CT
Computed Tomography
CTCAE
Common Terminology Criteria for Adverse Events
CTC
Circulating Tumor Cells
DLT
Dose Limiting Toxicity
DSMB
Data and Safety Monitoring Board
ECOG
Eastern Cooperative Oncology Group
ER
Estrogen Receptor
FOCBP
Female of child bearing potential
H&PE
History & Physical Exam
HER-2
Human Epidermal Growth Factor receptor-2
IV (or iv)
Intravenously
MTD
Maximum Tolerated Dose
MBC
Metastatic Breast Cancer
NCI
National Cancer Institute
ORR
Overall Response Rate or Objective Response Rate
OS
Overall Survival
ONJ
Osteonecrosis of the Jaw
PBMCs
Peripheral Blood Mononuclear Cells
PD
Progressive Disease
PFS
Progression Free Survival
PO (or p.o.)
Per os/by mouth/orally
PR
Partial Response
RANKL
Receptor activator of nuclear factor kappa-B ligand 
SAE
Serious Adverse Event
SD
Stable Disease
SGOT
Serum Glutamic Oxaloacetic Transaminase
SRE
Skeletal-related events
SPGT
Serum Glutamic Pyruvic Transaminase
TNF
Tumor Necrosis factor
TRSAE
Treatment Related Serious Adverse Event
WBC
White Blood Cells
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                           
NU16B09
6.1.17/ Amendment 1
2
STUDY SCHEMA
STAGE IV
Population N=37
Patients with:
Her2/neu negative ER+ and/or PR+ MBC 
Currently on  any line of standard therapy (no investigational 
agents)
≥5 Circulating Tumor Cells (CTCs) (after 2 weeks of standard 
therapy)
With PR or SD on current therapy (clinical or RECIST)
Bone metastases
Evaluation after 3 cycles:
Measure CTCs/radiological & tumor response        
Follow up
Follow-up every 12 weeks (+/-2 weeks) up to 2 
years1
1 Patients may continue standard of care treatment 
not prescribed by study.
Treatment (1 cycle=28 days)
Denosumab 120mg SQ administered Day 1 
every 28 days for 3 cycles (unless patients is 
removed from protocol therapy per section 4.4)
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
3
STUDY SUMMARY
Title
A Phase II, Open label Study to evaluate Denosumab in patients with ER 
and/or PR-positive, HER2-negative Metastatic Breast Cancer (MBC) with 
bone metastases and detectable circulating tumor cells (CTCs).
Short Title
Denosumab therapy for Metastatic Breast Cancer.
Version
 8.15.16
Study Design
A single arm, two-stage Phase II study.
Study Center(s)
Robert H. Lurie Comprehensive Cancer Center of Northwestern 
University (RHLCCC). 
Objectives
Primary Objective

To assess the effect of Denosumab in Her2/neu negative ER+ and/ 
or PR+ metastatic breast cancer patients who are in PR or SD after 
starting systemic therapy, with bone metastases and ≥ 5 CTCs, by 
measuring the fraction of patients with reduction in CTC quantity 
after 3 cycles of denosumab.
Secondary Objectives 

To assess the effect of denosumab on CTCs enumeration 
considered as a continuos variable.

To evaluate median progression-free survival(m-PFS) time.
Sample Size
Maximum accrual limit: 42 patients
Evaluable patients: 37
Diagnosis & Key 
Eligibility Criteria

Patients with a histologically or cytologically confirmed HER-2/neu 
negative breast cancer and pathologic or radiographic evidence of 
bone metastases.  

Must have ≥ 5 CTCs, measured at time of registration.
    (Note: FDA approved commercial testing should be used).

Patients may have either measurable or non-measurable disease 
per RECIST 1.1. 

Patients must have a serum calcium or albumin-adjusted serum 
calcium ≥ 2.0mmol/L (8.0mg/dL) and  2.9 mmol/L (11.5mg/dL) and 
cannot have any pre-existing uncorrected hypocalcemia (serum 
calcium <2.0mmol/L or <8.0mg/dL) at time of registration.

Subjects cannot have prior or current history of osteonecrosis or 
osteomyelitis of the jaw, untreated local gum disease and non-
healed dental or oral surgery at the time of registration.

Subjects cannot have a history of clinically symptomatic brain 
metastases or who required treatment for brain metastases within 4 
weeks of registration. 
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
4
Treatment Plan

Patients will be treated with Denosumab 120mg Subcutaneous at 
Day 1(+/-7 days) of each 28 day cycle for a total of 3 cycles. 

Prior to each dose, patients will be assessed for AEs

Overall response will be assessed after cycle 3. In those with at 
least stable disease, patient will be considered off study treatment 
after 3 cycles (however, patients may continue Denosumab as 
standard therapy) (Note: Patients will still be considered on study 
but in follow-up for purpose of survival endpoint.).
Statistical Methodology
We will conduct a single arm, two-stage Phase II study to assess the 
efficacy of a combination therapy including Denosumab in patients with 
bone metastatic breast cancer and detectable circulating tumor cells 
(CTCs).  The primary outcome is evidence of reduction in the number of 
CTCs. A patient will be defined as a “success” if the number of CTCs at 
end of study treatment is less than the number observed at baseline.  
Patients who do not experience a decrease in CTC count at follow-up or 
are missing follow-up CTC count measurements will be defined as 
“failures”. The new combined treatment would be of interest if the 
proportion (p) of successes is at least 70%. A success proportion of less 
than 50% will be of no interest. We will test the null hypothesis that the 
success proportion is 0.5 versus the alternative that this proportion is 0.7. 
 After testing the combination therapy on 23 patients in the first stage, the 
trial will be terminated if there are 12 or fewer successes. If the trial goes 
on to the second stage, a total of 37 patients will be studied. If the total 
number of successes is less than or equal to 23, the combination therapy 
will be rejected. The type I error for this Simon minimax design (Simon, 
1989) is 4.8% and the power is 80%. The probability of early termination 
under the null hypothesis (i.e., p=0.5) is 66.1%. 
To assess the effect of denosumab on CTCs enumeration considered as 
a continuos variable, the percent change in CTCs from baseline (i.e., 
100*[number of CTCs at follow-up/number of CTCs at baseline] will be 
calculated and compared to zero using either a one sample t-test or a 
Wilcoxon signed rank test.   
To evaluate median progression-free survival (PFS) time for the entire 
cohort, Kaplan-Meier curves will be used.  Cox proportional hazards 
models with time dependent covariates will be used to assess the 
relationship between longitudinal CTC counts (as defined by 2 or more 
CTCs evaluation in the course of treatment) and PFS.
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
5
1.0
INTRODUCTION – BACKGROUND & RATIONALE
1.1
Disease background
Breast Cancer Incidence And Subtypes 
Breast cancer is the most common non-dermatological malignancy in women with an 
estimated 232,670 new diagnoses in 2014; and is the second leading cause of cancer 
death in women with an estimated 40,000 women in the United States succumbing to the 
disease in 2014(1). Breast cancer is a heterogeneous disease comprised of several 
molecular subtypes, which are commonly extrapolated into clinical subtypes based on 
receptor status(2). The specific receptors which are assessed in standard clinical practice 
are the estrogen receptor (ER), progesterone receptor (PR), and human epidermal 
growth factor 2-neu (HER2) receptor. Patients with ER-, PR-, and HER2-negative tumors, 
the so-called triple negative breast cancers (TNBC), biologically tend to display an 
aggressive phenotype, currently do not have targeted therapy options as a standard of 
care, and only have a limited amount of cytotoxic agents available to treat their 
disease(3).
Bone Metastasis in Breast Cancer
Metastatic colonization involves reciprocal interactions between tumor cells and a foreign 
microenvironment. Microenvironments consist of extracellular matrix and normal cells 
such as fibroblasts, endothelial cells and infiltrating inflammatory cells. Products of these 
resident and transient cells include growth factors, chemokines, cytokines and proteases. 
Microenvironments that contain tumor cells are distinct from normal tissues. Differences 
include the presence of hypoxia that drives angiogenesis and invasion, low pH, low 
glucose concentrations, alterations in extracellular matrix proteins and liberation of 
previously bound growth factors. There are differences in the distrubution of site specific 
metastasis in advanced breast cancer. Bone metastasis are the most frequent site of 
recurrence particularly in the luminal subtypes. Moreover, the issue of site-specific 
metastasis has provided the opportunity to investigate the potential role of chemokines, 
particularly CXCR4 and RANKL. 
Understanding of the molecular pathways involved in bone metastases has involved the 
evaluation of key players such as RANKL (also referred to as OPGL, TRANCE or ODF) 
and osteoprotegerin (OPG). Receptor activator of nuclear factor kappa-B ligand (RANKL) 
is a member of the tumour necrosis factor (TNF) family of cytokines that binds to its 
receptor RANK to control osteoclast differentiation, activation and survival. OPG is a 
soluble decoy receptor for RANKL that blocks ligand binding to RANK, thereby 
preventing the signaling required for osteoclast differentiation and activation. RANK is 
also constitutively expressed in normal mammary gland epithelial cells, but RANKL 
expression is induced by sex hormones during pregnancy. Genetically, both RANKL and 
RANK are essential for the development of the lactating mammary gland during 
pregnancy and for lymph node organogenesis in mouse embryos. Furthermore, RANK 
has been shown to be expressed on many different epithelial tissues and epithelial 
tumour cells, and can activate specific downstream signaling pathways. Interestingly, 
RANKL also stimulates migration of primary breast epithelial cells and osteoclasts, 
establishing that RANKL-induced cell migration also occurs in normal, non-transformed 
cells. Importantly, inhibition of RANKL/RANK signaling by OPG in vivo markedly and 
selectively reduces bone metastasis and tumour burden in a melanoma model that does 
not activate osteoclasts. These data supported the evaluation of new molecular 
therapeutics designed to inhibit bone metastases, and these therapeutics have now 
entered clinical testing. Among them, the humanized monoclonal antibody denosumab 
(AMG 162) binds RANKL and inhibits its action. 
Recently a series of investigations have suggested a potential important role of RANKL-
RANK in regulating the metastatic process. Using MMTV-Erbb2 transgenic mice, 
investigators showed that reduced RANK gene dosage is associated with a significant 
decrease in the development of lung metastases. Additional data were generated by 
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
6
evaluation of the most "metastatic" form of breast cancer. Inflammatory breast cancer 
(IBC) is the most aggressive form of breast cancer, associated with poor survival despite 
appropriate multidisciplinary treatment. Patients with IBC have a predictable pattern of 
disease recurrence including soft tissue, bone and CNS disease. There are few cell lines 
and animal models currently available to evaluate the biological charactristics of IBC. 
MARY-X is a human xenograph model of IBC that allowed several important 
observations. MARY-X spheroids expressed embryonal stem cell markers including 
potent transcriptional factors, oct-4, nanog, and sox-2, which are associated with stem 
cell self-renewal and developmental potential. Most importantly, MARY-X spheroids 
expressed a cancer stem cell profile characterized by CD44(+)/CD24(-/low), ALDH1, and 
most uniquely, CD133. Furthermore, we recently showed high expression of RANK and 
RANKL mRNA in MARY-X cells and confirmed in tissue specimens from IBC patients 
suggesting association with stem cell phenotype. These data support a role of this 
pathway in the metastatic process and potentially as a modulator of cancer stem cell 
activity. Moreover, Palafox et al have recently demonstrated that RANKL expression was 
associated with the acqusition of stemness, EMT and capacity to invade and promote 
metastases. High expression of RANKL  in human tumors was associated with estrogen-
receptor negative (ER)-/progesterone-receptor negative (PR-) status and adverse 
prognosis. Furthermore, RANKL has been associated with chemotherapy resistance in 
preclinical models and genetic inactivation of the RANKL-R demonstrated inhibition of 
MPA-induced tumors. 
Circulating Tumor Cells (CTCs) in Metastatic Breast Cancer
The natural history of breast cancer suggests that the disease has the capability to 
develop distant recurrence to specific organs and become a lethal disease. To leave the 
primary site and to soil in the metastatic niche, cancer cells need to disseminate through 
the blood and/or lymphatic system. The “seed and soil” theory, postulated by Paget in 
1889 and revived fully by Hart in 1980, represents a milestone for the study of CTCs in 
metastatic breast cancer (MBC) patients. CTCs might represent the seed necessary for 
cancer dissemination and may be the cells responsible for initiating the metastatic 
process. In 1869, Thomas Ashworth reported that, “Cells identical with those of the 
cancer itself being seen in the blood may tend to throw some light upon the mode of 
origin of multiple tumors existing in the same person.” Only recent advances in detection 
methods have enabled their reproducible identification and further characterization.
Although CTCs provide a link between the primary tumor and metastatic sites, the factors 
involved with CTC survival in the blood circulation and eventual metastasis are not well 
understood. So far, much of what is known about CTCs in MBC patients simply involves 
numbering and prognostic value. Recently, advances in technology have facilitated the 
detection of even very small numbers of CTCs in the peripheral blood of MBC patients. 
Research is currently focused on specifically identifying these CTC subsets and 
characterizing them at the molecular level to ultimately provide a tool that would allow 
tailoring of treatment on an individual basis. Moreover, the biology of the metastatic 
process has led researchers to study the plasticity properties of tumor cells that 
characterize a subset that leaves the primary tumor, invades the blood stream and 
travels to the specific distant organs with a developed metastatic niche. 
CTC identification has been based mostly on the detection of epithelial cell markers, such 
as the epithelial cell adhesion molecule (EpCAM), intracellular cytokeratin expression, 
and nuclei presence (4',6-diamidino-2-phenylindole positivity), in patient blood. 
Identification is further based on the contemporary exclusion of normal blood cells, for 
example by using WBC markers, e.g. CD45. Because CTCs are present in whole blood 
in such low numbers, only 1-10 CTCs per mL of whole blood, detection requires 
enrichment of tumor cells in blood samples. Tumor cells are enriched 
immunomagnetically using ferrofluids coated with antibodies targeting EpCAM. The 
CellSearch® system (Veridex Corporation, Warren, NJ, USA) is the only Food and Drug 
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
7
Administration-cleared CTC detection system and has the most robust clinical data with 
reproducible results across different laboratories. Cristofanilli showed for the first time in 
2004 that the number of CTCs detected by the CellSearch® system before starting a new 
line of treatment is an independent predictor of progression-free survival (PFS) and 
overall survival (OS) in patients with MBC. Patients with ≥5 CTCs per 7.5 ml of peripheral 
blood have significantly inferior PFS (median PFS, 2.7 to 8.2 months) and OS (median 
OS, 10.1 to 21.9 months) compared to patients with <5 CTCs (median PFS, 7 to 12 
months; median OS, 18 to 40.1 months). Moreover, CTC counts at the first follow-up visit 
showed similar prognostic value. This prognostic utility was independently confirmed by 
different groups. These interesting findings suggest that CTC enumeration can be used 
to monitor treatments along with standard imaging modalities. 
CTCs enumeration versus Standard imaging assessment 
In this regard, Budd et al. compared CTC monitoring to bi-dimensional response 
assessment (central review) of 177 patients with MBC. Remarkably, the determination of 
CTCs at baseline and follow-up appeared to have superior prognostic implications in 
patients with measurable MBC compared to standard imaging assessment, particularly in 
patients with more refractory disease. Recently, De Giorgi completed a retrospective 
analysis of 115 MBC patients who started a new line of therapy and who had CTC counts 
and fluoro-deoxyglucose (FDG)-Positron Emission Tomography (PET) scans performed 
at baseline and at 9 to 12 weeks during therapy (midtherapy or time of planned restaging, 
usually around 10-12 weeks). In 102 evaluable patients, the median overall survival time 
was 14 months (range, 1 to > 41 months). Measurement of CTC at time of restaging 
demonstrated that detectable levels correlated with FDG-PET/CT response in 68 (67%) 
evaluable patients. In univariate analysis, midtherapy CTC counts and FDG-PET/CT 
response predicted overall survival (P < .001 and P = .001, respectively). FDG-PET/CT 
predicted overall survival (P = .0086) in 31 (91%) of 34 discordant patients who had 
fewer than five CTCs at midtherapy. Only midtherapy CTC levels remained significant in 
a multivariate analysis (P = .004), further supporting the critical importance of this test in 
the management of MBC.  The correlation between these different monitoring modalities 
in advanced disease was further refined in a series of subsequent analyses from the 
same team of investigators with particular regard to patients with bone metastases. The 
largest study evaluated 195 patients with MBC who were diagnosed with 
relapsed/progressive MBC. These patients underwent FDG–PET/CT scans and provided 
blood samples for CTC analysis. One hundred seventeen (60%) patients had received 
prior treatment of MBC with hormonal therapy (53 cases), chemotherapy with or without 
hormonal therapy (48 cases), or human epidermal growth factor receptor 2 (HER2)-
targeted therapies combined with chemotherapy and/or hormonal therapy (16 cases); 78 
(40%) had newly diagnosed MBC. Interestingly, the analysis demonstrated that among 
the 137 patients with bone metastases at relapse/progression, 83 (61%) had ≥5 CTCs, 
while 54 (39%) had <5 CTCs (P = 0.0122). Higher CTC numbers were detected in 
patients with bone metastases alone and patients with metastases in bone plus other 
sites relative to those with no bone metastases. Moreover, higher CTC numbers were 
detected in the patients with more extensive bone metastases relative to those with one 
or two bone lesions. With regard to the correlation with imaging findings, all but seven of 
the 137 patients with bone metastases had increased FDG uptake within one or more 
lesions. Of these seven cases, four had <5 CTCs; of the remaining three with ≥5 CTCs, 
two also had liver metastases with elevated FDG uptake (CTCs = 143 and 25), while one 
presented with primary tumor with elevated FDG uptake (CTCs = 75). This represented 
the first demonstration of a significant association between CTC detection and bone 
metastases. Moreover, Giuliano et al showed that CTCs decrease to a value of < 5CTCs 
has the most significant impact on PFS and OS but, any decrease can affect prognosis 
compared to patients with CTCs increase suggesting a possible need to evaluate CTCs 
decrease as a continuous variable. Patients with ≥ 5 CTCs consistently demonstrated the 
development of new metastatic sites at time of progression instead of the increase in the 
size of the original recurrent disease, indicating that measurement of CTCs can be a 
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
8
useful tool for evaluation of new anti-metastatic intervention that can affect time to new 
metastasis (TTNM).
1.2
Intervention background & Overview  
Denosumab (AMG 162) is a fully human monoclonal IgG2 antibody to RANKL that binds 
with high affinity and specificity to the soluble and cell membrane-bound forms of human 
RANKL. Denosumab binding prevents activation of RANK and inhibits the formation, 
activation, and survival of osteoclasts. Consequently, bone resorption and cancer-
induced bone destruction are reduced.
1.2.1
Preclinical Studies
 Anti-tumor activity, toxicology, absorption, metabolism, elimination:
Preclinical studies of Denosumab have been performed in mouse models and 
non-human primates. Denosumab has no apparent pharmacologic effect in wild-
type mice or rats. However, administration of the RANKL inhibitor OPG-Fc acts 
as a surrogate for denosumab in mouse models of bone metastases. In mouse 
models of estrogen receptor negative (ER-) human breast cancer, OPG-Fc 
significantly reduced skeletal tumor burden and osteolytic lesions and increased 
survival. Pretreatment of mice with OPG-Fc delayed and reduced the 
development of new bone metastases, osteolytic lesions, circulating markers of 
bone resorption and number of osteoclasts. Similar results have been shown in 
mouse models of ER+ human breast cancer, prostate cancer and non-small cell 
lung cancer treated with OPG-Fc.
Denosumab has been studied in cynomolgus monkeys. Monkeys who received a 
single dose of denosumab had an acute and significant reduction in bone 
resorption as assessed by levels of urine N-telopeptide adjusted for urine 
creatinine (uNTx/Cr). Older monkeys without ovaries who were given long-term 
(16 months) denosumab had significant gains in mass and density of cancellous 
and cortical bone and significant reductions in markers of bone turnover (serum 
C-telopeptide-I [CTX-I], serum bone-specific alkaline phosphatase, and 
uNTx/Cr).
Single-dose pharmacokinetics (PK) and multiple-dose toxicokinetics of 
denosumab following intravenous (IV) or subcutaneous (SC) administration have 
been evaluated in mice, rats, and cynomolgus monkeys. In mice and rats, 
terminal half-life values were 19 and 11 days, respectively. Maximum serum 
concentrations after SC doses of 1 mg/kg occurred at 72 hours post-dose in both 
species. Bioavailability was 86% in mice and 56% in rats.
In cynomolgus monkeys, linear PK was observed at higher doses (1-3 mg/kg). 
These data led to the estimation that denosumab rapidly and potently 
suppresses bone resorption in monkeys with a 50% effective concentration of 
464 ng/mL. In all species, volume of distribution at steady-state indicated lack of 
extensive extravascular distribution. Studies using 125I-labeled denosumab found 
that approximately 76-95% of administered radioactivity was recovered in urine 
and 1-3% was recovered in feces.
Preclinical toxicity studies were conducted in cynomolgus monkeys. No 
toxicologically significant effects were observed in a study of denosumab 
administered to monkeys over the course of a year. No treatment-related effects 
were noted on the cardiovascular or respiratory system. Denosumab showed no 
adverse effects on embryo-fetal development and no maternal toxicity when 
given during organogenesis. Effects observed in mothers and infants were 
consistent with the pharmacological action of denosumab as a monoclonal 
antibody against RANKL and inhibitor of osteoclastic bone resorption.
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
9
1.2.2 
Clinical Studies
Phase I Studies, Pharmacokinetics, Anti-Tumor Activity, Dosing, Toxicology, 
Absorption, Metabolism, Elimination: 
Phase I, placebo-controlled studies in healthy volunteers were conducted to 
evaluate the safety, tolerability, and pharmacodynamic effects of denosumab. 
Two studies of particular interest evaluated denosumab in subjects with cancer-
related bone metastases. In all of the phase I studies, single and multiple doses 
of denosumab were well-tolerated and effectively reduced bone resorption as 
measured by uNTx/Cr and CTX-I. Studies of subjects with renal insufficiency 
indicate that renal impairment does not affect the pharmacokinetics or 
pharmacodynamics of the drug, but patients with chronic kidney disease or end-
stage renal disease appear to be at higher risk of hypocalcemia following 
treatment.
The pharmacodynamic profile of denosumab was consistent across all 
populations studied, including subjects with advanced cancer and bone 
metastases. SC administration of 60mg of denosumab causes a rapid, 
approximately 70%, reduction in bone resorption within 6 hours as measured by 
CTX1, and an 85% reduction by 3 days. The pharmacokinetic profile is not 
affected by age, weight, body mass index, sex, race or renal function. Following 
SC administration, denosumab exhibits dose-dependent, nonlinear PK over a 
wide dose range. The maximum serum concentrations (Cmax) are typically seen 1 
to 4 weeks postdose. After Cmax, serum levels decline over 4 to 5 months with a 
mean half-life of 25 to 30 days. Bioavailability is approximately 60% after SC 
dosing. After repeated doses of 120mg every 4 weeks, steady-state was 
achieved by 6 months.
Phase II studies, studies with same diagnosis as being studied, safety, and 
anticipated adverse events:
Denosumab has been studied in phase II and III trials evaluating its role as a 
potential treatment for postmenopausal osteoporosis or male osteoporosis; bone 
loss due to hormone-ablation therapy in subjects with cancer; rheumatoid 
arthritis; prevention of bone metastases in at-risk populations; prevention of 
skeletal-related events in patients with advanced malignancies involving bone; 
multiple myeloma; giant cell tumor of bone; and hypercalcemia of malignancy. 
Studies evaluating the role of denosumab in treating or preventing bone 
malignancies or metastases are of particular relevance. 
1.2.3  
Rationale for dose selection:
The selection of the denosumab dose and schedule for the phase 3 oncology 
studies was based on safety, pharmacokinetic, and pharmacodynamic data 
obtained from phase 1 and 2 studies. Five SC denosumab dose regimens of 30, 
120, or 180 mg every four weeks (q4w) and 60 or 180 mg every 12 weeks 
(q12w), were compared with an IV bisphosphonate in 255 patients with breast 
cancer and bone metastases. The q12w dosing schedules did not maintain high 
serum concentrations over the entire dosing interval. Ultimately, the 120 mg q4w 
dose was selected as the phase 3 dosing regimen because it was well tolerated 
and achieved maximal suppression of uNTx/Cr over the entire dosing interval in 
most subjects. On November 18, 2010, the FDA approved denosumab (Xgeva™) 
for the prevention of skeletal-related events (SREs) in patients with bone 
metastases from solid tumors. The approval is based on results from three 
international randomized (1:1) double-blind double-dummy trials in patients with 
bone metastases comparing denosumab to zoledronic acid. Trial 20050103 
enrolled 1,901 patients with hormone-refractory prostate cancer, Trial 20050136 
enrolled 2,046 patients with breast cancer, and Trial 20050244 enrolled 1,776 
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
10
patients with advanced multiple myeloma or solid tumors other than breast or 
prostate cancer. In all three trials, patients were randomly assigned to receive 
either 120 mg denosumab subcutaneously every 4 weeks or 4 mg zoledronic 
acid intravenously every 4 weeks (dose-adjusted for reduced renal function). 
Patients with creatinine clearances less than 30 mL/min were excluded, and prior 
intravenous bisphosphonate therapy was not permitted. The primary outcome 
measure in each trial was the determination of non-inferiority in time-to-first SRE 
in patients treated with denosumab as compared to patients treated with 
zoledronic acid. An SRE was defined as a pathologic fracture, radiation therapy 
to bone, surgery to bone, or spinal cord compression due to cancer. Other 
outcome measures included superiority of time-to-first SRE and superiority of 
time-to-first and subsequent SRE. Denosumab therapy resulted in a statistically 
significant delay in the time-to-first SRE and in the time-to-first and subsequent 
SRE compared with zoledronic acid in patients with breast cancer (Trial 
20050136) or hormone-refractory prostate cancer (Trial 20050103). In Trial 
20050244, denosumab was not less effective than zoledronic acid in delaying the 
time-to-first SRE and did not demonstrate superiority. 
1.2.4 
Documented Adverse Events:
The most common adverse reactions in patients receiving denosumab (in at least 
25 percent of patients) were fatigue or asthenia, hypophosphatemia, and nausea. 
The most common serious adverse reaction in patients receiving denosumab 
was dyspnea. The most common adverse reactions resulting in denosumab 
discontinuation were osteonecrosis and hypocalcemia.
                                   Hypocalcemia:
Denosumab can cause severe symptomatic hypocalcemia, and fatal cases have 
been reported from the post-marketing setting based on a recent review of 
cases. All subjects will be advised to take calcium supplements (at least ≥500 mg 
of elemental calcium) and vitamin D (≥400 IU) daily during the study. All subjects 
will be recommended to receive vitamin D and calcium supplementation unless 
documented hypercalcemia (albumin-adjusted serum calcium > 2.9mmol/L 
[11.5mg/dL] or ionized calcium >1.5mmol/L) develops on study. If documented 
hypocalcemia occurs, additional short-term calcium supplementation may be 
necessary necessary.  
Osteonecrosis of the Jaw: 
Osteonecrosis of the jaw (ONJ) can occur in subjects receiving denosumab. The 
risk of ONJ is associated with the length of exposure and risk factors such as 
dental extractions for ONJ. Patients should be monitored for ONJ as part of 
routine clinical practice. Patients should be instructed to maintain good oral 
hygiene and avoid invasive dental procedures, if possible.
A dental examination with appropriate preventive dentistry should be considered 
prior to treatment with denosumab in patients with risk factors for ONJ. Patients 
who are suspected of having or who develop ONJ while on denosumab should 
receive care by a dentist or an oral surgeon. 
A temporary interruption of treatment is strongly recommended for patients who 
develop ONJ during treatment with XGEVA based on individual patient’s 
risk/benefit assessment. For patients in whom invasive dental procedures cannot 
be avoided, the clinical judgment of the treating physician should guide the 
management plan of each patient based on individual benefit/risk assessment.
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
11
In three phase III active-controlled clinical trials in patients with advanced 
malignancies involving bone, ONJ was confirmed in 1.8% of patients in the 
denosumab group treated with 120 mg subcutaneously every 4 weeks. In 
patients receiving zoledronic acid 4mg every 4 weeks (median exposure of 12.0 
months; range 0.1 – 40.5) from the same clinical trials, ONJ was found in 1.3% of 
patients. The trials in patients with breast or prostate cancer included a 
denosumab extension treatment phase (median overall exposure of 14.9 months; 
range 0.1 – 67.2). The patient-year adjusted incidence of confirmed ONJ was 
1.1% during the first year of treatment and 4.1% thereafter. The median time to 
ONJ was 20.6 months (range: 4 - 53).
Atypical femoral fracture:
Atypical femoral fracture has been reported with denosumab. These fractures 
can occur anywhere in the femoral shaft from just below the lesser trochanter to 
above the supracondylar flare and are transverse or short oblique in orientation 
without evidence of communication. Atypical femoral fractures most commonly 
occur with minimal or no trauma to the affected area. They may be bilateral and 
many patients report prodromal pain in the affected area, usually presenting as 
dull, aching thigh pain, weeks to months before a complete fracture occurs. 
During denosumab treatment, patients should be advised to report new or 
unusual thigh, hip, or groin pain. Patients presenting with such symptoms should 
be evaluated for an incomplete femoral fracture, and the contralateral femur 
should also be examined.
Hypersensitivity:
Clinically significant hypersensitivity including anaphylaxis has been reported 
with use of denosumab. Reactions may include hypotension, dyspnea, upper 
airway edema, lip swelling, rash, pruritus, and urticaria. Clinically significant 
hypersensitivity has been identified as a contraindication for treatment with 
denosumab. If an anaphylactic or other clinically significant allergic reaction 
occurs, appropriate therapy will be initiated and denosumab therapy permanently 
discontinued.
Musculoskeletal pain:
A safety assessment was performed on a cumulative review of clinical data and 
study serious adverse event and non-study adverse event reports of 
musculoskeletal pain. Based on this assessment, musculoskeletal pain, including 
severe cases, has been identified as an adverse drug reaction based on data 
from post-marketing setting.
1.3
Study Rationale 
1.3.1   Rationale for study design and endpoints: 
    
The presence of circulating tumor cells (CTCs) in patients with metastatic breast 
cancer (MBC) has been associated with shorter survival time than when CTCs 
are absent. Unlike soluble circulating tumor markers, such as CA15-3 and CA27-
29, the number of CTCs seems not to simply reflect tumor bulk. A detailed 
analysis of tumor burden by the bidimensional sum of the metastatic lesions 
showed limited correlation between CTC levels and radiographic measurement 
of tumor load. However, the detection of CTCs in patients with evidence of 
nonvisceral disease (including metastases in chest wall, lymph nodes, and bone) 
was of higher prognostic significance than in those with visceral disease. In a 
retrospective analysis performed at the University of Texas, M. D. Anderson 
Cancer Center, 195 patients with MBC were evaluated; 103 (53%) had <5 CTCs 
at relapse/progression and 92 (47%) had >5 CTCs. Patients treated with HER2-
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
12
targeted therapies (15 trastuzumab and one lapatinib) had lower CTC counts 
with only one case with >5 CTCs at progression during trastuzumab therapy 
(number of CTCs = 17). Of the 92 patients with >5 CTCs, 83 (90%) presented 
with bone metastases; furthermore, of 50 cases with 21 CTCs, 48 (96%) had 
bone metastases. Of 137 patients with bone metastases at relapse/progression, 
83 (61%) had >5 CTCs, while 54 (39%) had <5 CTCs (P = 0.0122). Higher CTC 
numbers were detected in patients with bone metastases alone and patients with 
metastases in bone plus other sites relative to those with no bone metastases. Of 
the 137 patients with bone metastases, higher CTC numbers were detected in 
the patients with more extensive bone metastases relative to those with one or 
two bone lesions. Moreover, patients with ≥5 CTCs and bone metastases have a 
higher chance of developing additional metastatic disease in distant sites 
compared to a) patients with bone metastases and < 5 CTCs or b) patients with 
only visceral metastases. Moreover, chemotherapy has been shown to minimally 
affect CTC values after first-line therapy (approximately 10-15% decline in 
second -line treatment), suggesting a limited therapeutic benefit of standard 
therapies. Furthermore, all these patients were receiving zoledronic acid as part 
of their medical treatment in consideration of their documented metastatic 
disease to the bone.
In summary, detection of CTCs remains a strong 
prognostic factor in MBC but particularly in patients with bone metastases. Those 
patients appear to have higher levels of circulating epithelial cells in spite of 
continuous treatment with bisphosphonates. These observations raise questions 
about some of the molecular factors that may be responsible for the mobilization 
of cancer cells in those patients, including the role of RANKL. That subset of 
patients with more aggressive disease (bone mets and detectable CTCs) could 
potentially benefit from the development of an agent with CTC-targeting 
properties. 
In summary,we hypothesize that use of the RANKL inhibitor denosumab will 
decrease the number of CTCs measured in patients with MBC and bone 
metastases.
We plan to address the change in CTCs as a continuous variable considering the 
potential benefit associated with any decrease in CTCs but, we will use the 
CTC≥5 as a reference cutoff for maximum benefit. We further hypothesize that 
administration of denosumab will increase the time to progression of disease 
(new site vs. progression of lesions in previous site), which would be an indicator 
of the antimetastatic effect of RANKL inhibition.
1.3.2
Rationale for dose selection:  
We plan to use the standard dosing regimen of 120mg SC q4w, as established in 
phase II studies and used in phase III studies. This dose was well-tolerated without 
significant adverse effects and should pose minimal risk to our patients. (refer 
section 1.2.3.)
1.3.3     Rationale for study patient population:
Beyond the enumeration of CTCs, molecular characterization provides a key to 
demonstrating their cellular origin from primary and metastatic tumor deposits, 
and may also provide clues to their evolution during the course of cancer 
treatment. B. Aktas et al. studied the ER, PR, and HER2 expressions on CTCs 
from 87 MBC patients. Blood samples were analyzed for CTCs with the 
AdnaTest BreastCancer™ (AdnaGen AG, Langenhagen, Germany), which 
enables the immunomagnetic enrichment of tumor cells via epithelial and tumor-
associated antigens. The authors showed that CTCs were ER-negative in 48/62 
(77%) patients with ER-positive tumors, and that 46/53 (87%) patients with PR-
positive tumors did not express PR on CTCs. Primary tumors and CTCs 
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
13
displayed a concordant ER and PR status in only 41% (p=0.260) and 45% 
(p=0.274) of cases, respectively. Moreover, regarding the immunohistochemical 
subtype, most CTCs were triple-negative (39 of 86 patients, 45%) or HER2-
positive (27 of 86 patients, 32%). More recently Nole’ et al reported that the 
detection of CTCs is higher in patients with luminal A and B (HER-2) and 
significantly in patients with bone metastases. The data support the value of 
CTCs for prognostic and biomarker analysis particularly in patients with HER-2 
negative disease and demonstrated ER+ in the primary tumors. Those patients 
have the highest incidence of bone metastases and a higher percentage of 
disease presenting with CTC> 5 making them ideal candidates for studies using 
CTCs as surrogate endpoint of efficacy.
1.3.4 
Rationale for exploratory objectives: 
          
We will evaluate gene expression profiles of circulating tumor cells. We will 
analyze ER and PR expression, of the CTCs. We will also analyze stem cell 
characteristics of these cells by studying epithelial and tumor associated antigens 
associated with epithelial/mesenchymal transition. We will also perform Receptor 
activator of nuclear factor kappa-B ligand (RANKL) analysis.
1.4
Pregnancy and Risk to fetus
Denosumab falls in Pregnancy Category D: 
(http://pi.amgen.com/united_states/xgeva/xgeva_pi.pdf)
Defined as “Drugs which have caused, are suspected to have caused or may be 
expected to cause, an increased incidence of human fetal malformations or irreversible 
damage. These drugs may also have adverse pharmacological effects.”
 
Risk Summary: 
Xgeva can cause fetal harm when administered to a pregnant woman based on findings 
in animals. In utero denosumab exposure in cynomolgus monkeys resulted in increased 
fetal loss, stillbirths, and postnatal mortality, along with evidence of absent lymph nodes, 
abnormal bone growth, and decreased neonatal growth. There are no adequate and well-
controlled studies with Xgeva in pregnant women. Women should be advised not to 
become pregnant when taking Xgeva. 
Clinical Considerations: 
The effects of denosumab on the developing human fetus at the recommended 
therapeutic dose are unknown. The effects of Xgeva are likely to be greater during the 
second and third trimesters of pregnancy. Monoclonal antibodies are transported across 
the placenta in a linear fashion as pregnancy progresses, with the largest amount 
transferred during the third trimester. 
In addition, men enrolled on this study should understand the risks to any sexual partner 
of childbearing potential. The extent to which denosumab is present in seminal fluid is 
unknown. There is potential for fetal exposure to denosumab when a male treated with 
Xgeva has unprotected sexual intercourse with a pregnant partner. Hence, men must 
agree to use adequate contraception (hormonal or barrier method of birth control; 
abstinence) to study entry, for the duration of treatment, and for at least 5 months after 
the completion of treatment.
If the patient becomes pregnant during Xgeva therapy, consider the risks and benefits in 
continuing or discontinuing treatment with Xgeva. 
Animal Data: The effects of denosumab on prenatal development have been studied in 
both cynomolgus monkeys and genetically engineered mice in which RANK ligand 
(RANKL) expression was turned off by gene removal (a “knockout mouse”). In 
cynomolgus monkeys dosed subcutaneously with denosumab throughout pregnancy at a 
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
14
pharmacologically active dose, there was increased fetal loss during gestation, stillbirths, 
and postnatal mortality. Other findings in offspring included absence of axillary, inguinal, 
mandibular, and mesenteric lymph nodes; abnormal bone growth, reduced bone 
strength, reduced hematopoiesis, dental dysplasia, and tooth malalignment; and 
decreased neonatal growth. At birth out to one month of age, infants had measurable 
blood levels of denosumab (22-621% of maternal levels). Following a recovery period 
from birth out to 6 months of age, the effects on bone quality and strength returned to 
normal; there were no adverse effects on tooth eruption, though dental dysplasia was still 
apparent; axillary and inguinal lymph nodes remained absent, while mandibular and 
mesenteric lymph nodes were present, though small; and minimal to moderate 
mineralization in multiple tissues was seen in one recovery animal. There was no 
evidence of maternal harm prior to labor; adverse maternal effects occurred infrequently 
during labor. Maternal mammary gland development was normal. There was no fetal 
NOAEL (no observable adverse effect level) established for this study because only one 
dose of 50 mg/kg was evaluated. 
In RANKL knockout mice, absence of RANKL (the target of denosumab) also caused 
fetal lymph node agenesis and led to postnatal impairment of dentition and bone growth. 
Pregnant RANKL knockout mice showed altered maturation of the maternal mammary 
gland, leading to impaired lactation.
2.0
OBJECTIVES 
2.1
Primary Objectives
To assess the effect of denosumab in  Her2/neu negative ER+ and/ or PR+ metastatic 
breast cancer patients who are in PR or SD after starting systemic therapy with bone 
metastases and ≥ 5 CTCs by measuring the fraction of patients with reduction in CTCs 
after 3 cycles of denosumab.
2.2
Secondary Objectives

To assess the effect of denosumab on CTCs enumeration considered as a 
continuous variable (percent change from baseline) in this population.

To evaluate median Progression-free survival (m-PFS).
2.3
Exploratory objective

CTC enumeration after enrichment. 

To assess the effect on CTC profiling and charaterization of stem cell phenotype 
(CTC-EMT).

To evaluate the type of progressive disease (new site vs. progression of lesions  in 
previous sites).

  To analyze the expression of RANKL.
3.0
PATIENT ELIGIBILITY
The target population for this study is in patients with Her2/neu negative ER+ and/ or PR+ 
Metastatic Breast Cancer (MBC) with bone metastases and detectable circulating tumor cells 
(CTCs). This will be a single-center trial conducted at the Robert H. Lurie Comprehensive Cancer 
Center of Northwestern University. 
A total of 42 patients can be enrolled in order to obtain 37 evaluable subjects needed for this trial. 
Approximately 5 potentially eligible patients are seen per month, and it is anticipated that at least 
2 per month will be accrued. Potential patients may be referred to the Principal Investigator (PI) at 
Northwestern University, Dr. Massimo Cristofanilli (312) 695-0990.
 
Eligibility will be evaluated by the study team according to the following criteria. Eligibility waivers 
are not permitted. Subjects must meet all of the inclusion and none of the exclusion criteria to be 
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
15
registered to the study. Study treatment may not begin until a subject is registered. Please refer 
to Section 11 for complete instructions regarding registration procedures.
3.1
Inclusion Criteria  
3.1.1 
Patients must have histologically or cytologically confirmed ER and/or PR 
positive, HER-2/neu negative metastatic breast cancer. They can be enrolled in 
any line of therapy without investigational agents and should have stable disease 
or a partial response (which can be determined clinically) on current systemic 
treatment. Patients must also have pathologic OR radiographic evidence of bone 
metastases and ≥ 5 CTCs. 
             
(Note: the pathology report that is used by the physician to determine diagnosis, 
will be used to determine patient eligibility. ER and PR status should be available 
at the time of registration).
3.1.2 
Patients may have either measurable or non-measurable within 30 of days of 
registration. (Lesions treated with radiation therapy must not be used as a target 
lesion).
(Note: Per RECIST criteria v. 1.1, Measurable disease is defined as at least one 
lesion that can be accurately measured in at least one dimension. 
Non-measurable disease is defined as All other lesions, including small lesions 
(longest diameter <10mm or pathological lymph nodes with P10 to <15mm short 
axis) as well as truly non-measurable lesions. Lesions considered truly non-
measurable include: leptomeningeal disease, ascites, pleural or pericardial 
effusion, and inflammatory breast disease, lymphangitic involvement of skin or 
lung, abdominal masses /abdominal organomegaly identified by physical exam 
that is not measurable by reproducible imaging techniques.[see Appendix 3]).     
      
3.1.3 
Patients may be enrolled in any line of standard treatment (without investigational 
agents). The start date of current treatment should be at least two 2 weeks or 
more prior to registration.
(Note: Patients will continue to receive the planned active treatment with 
chemotherapy or endocrine therapy (standard of care) and initiate denosumab at 
the recommended dose for this protocol.)
     
3.1.6 
Age > 18 years. Both men and women are eligible for this study.  
3.1.7   ECOG performance status 0- 2 (see Appendix 1).
   
3.1.8
Patients must have normal organ and marrow function as defined below within 
30     days of registration; 

Leukocytes
> 3,000/mcL(without growth factor)

Platelets
> 100,000/mcL (with or without transfusion)

Hemoglobin                            ≥8 (with or without transfusion)

AST&ALT (SGOT&SGPT)
< 2.5  times institutional upper limit of normal   
(For patients with liver metastasis up to ≤5 times of ULN is allowed)

Bilirubin                                   <1.5 ULN  
  
(For patients with liver metastasis up to ≤5 times of ULN is allowed)                                                      

Serum Creatinine
≤1.5 ULN 
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
16

Creatinine clearance
≥ 60 mL/min/1.73 m2 for patients 
with creatinine levels above institutional normal
 
(creatinine clearance should be calculated  per institutional standard)
3.1.9 
Patients must have a serum calcium of ≥2.0mmol/L (8.0mg/dL) or albumin-
adjusted    serum calcium   2.9 mmol/L (11.5mg/dL) within 30 days of 
registration.
(Note: If patients are undergoing treatment for hypocalcemia and the serum 
calcium value at screening is >8.0mg/dl, then the patient will be eligible for this 
study).
     
3.1.10 Females of child-bearing potential (FOCBP) and males with his or her partner    
must agree to use two acceptable methods of effective contraception (see 
Appendix 2), at study entry, for the duration of study participation, and for 5 
months following completion of therapy. Subjects who are surgically sterile (e.g., 
history of bilateral tubal ligation, hysterectomy) or whose sexual partner is sterile 
(e.g., history of vasectomy) are not required to use additional contraceptive 
measures. Should a female patient become pregnant or suspect she is pregnant 
while participating in this study, she should inform her treating physician 
immediately.Likewise, if a male patient impregnates his female partner, he 
should inform the treating physician immediately.
 
NOTE: A FOCBP is any woman (regardless of sexual orientation, having 
undergone a tubal ligation, or remaining celibate by choice) who meets the 
following criteria:

Has not undergone a hysterectomy or bilateral oophorectomy

Has had menses at any time in the preceding 12 consecutive months (and 
therefore has not been naturally postmenopausal for > 12 months)
3.1.11
FOCBP must have a negative serum OR urine pregnancy test ≤ 7 days prior to 
registration.  
3.1.12 Ability to understand and willingness to sign a written informed consent and 
HIPAA consent document prior to registration.
3.1.13   Willingness and ability of subject to comply to study requirements.
3.2
Exclusion Criteria 
3.2.1 
Patients may not be receiving any other investigational agents. A 2 week wash-
out period for investigational agents is required before registration. 
3.2.2 
Patients with clinically symptomatic brain metastases or who required treatment 
for brain metastases within 4 weeks of registration (Stable sequelae acceptable if 
treatment has been completed. These lesions cannot be used as target lesions).
 
3.2.3 
Patients who have a history of allergic reactions attributed to compounds of 
similar   chemical or biologic composition to Denosumab are not eligible (i.e. 
same class of drugs) 
(Note: Prior bisphosphonates are allowed. Patients could have received 
bisphosphonates or be bisphosphonate-naïve. Patients who were previously on 
bisphosphonates can be enrolled in the study, as long as they have a wash-out 
period of 2 weeks prior to registration).
3.2.4 
Patients who are on corticosteroids or immunosuppressant’s are not eligible. A
        
2 week wash-out period for is required before registration. 
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
17
3.2.5 
Patients who have a known additional malignancy that is progressing or requires 
active treatment are not eligible. Patients who have had a prior diagnosis of 
cancer and if it has been < 3 years since their last treatment are also not eligible. 
NOTE: Exceptions include basal cell carcinoma of the skin or squamous cell 
carcinoma of the skin that has undergone potentially curative therapy or in situ 
cervical cancer. 
3.2.6 
Patients who have an uncontrolled intercurrent illness including, but not limited to 
any of the following, are not eligible: 

Hypertension (defined as 160/90 mmHg for 3 consecutive readings 2-5 mins 
apart) that is not controlled on medication.

Symptomatic congestive heart failure.

Unstable angina pectoris.

Psychiatric illness/social situations that would limit compliance with study 
requirements.

Any other illness or condition that the treating investigator feels would interfere 
with study compliance or would compromise the patient’s safety or study 
endpoints.
3.2.7 
Subject is pregnant or breast feeding, or planning to become pregnant within 5 
months after the end of treatment.
3.2.8   Known HIV-positive patients who are on combination antiretroviral therapy.  (This 
is because of the potential for pharmacokinetic interactions with denosumab.)  
3.2.9 
No known prior history or current evidence of osteonecrosis/osteomyelitis of the 
jaw.
3.2.10 No known prior history or current evidence of untreated local gum or oral infection.
3.2.11 No known/planned active dental or jaw condition which requires oral surgery, 
including tooth extraction.
3.2.12   No known non-healed dental/oral surgery, including tooth extraction.
3.2.13 Patients have planned invasive dental procedures during the course of the study.
4.0
TREATMENT PLAN
4.1
Overview
This is a Phase II targeted to enroll a maximum of 42 patients to get 37 evaluable patients 
with confirmed ER and/or PR positive, HER-2/neu negative metastatic breast cancer 
receiving any line of standard therpy, for their disease.  Patients will continue to receive 
the planned active treatment with chemotherapy or endocrine therapy (standard of care) 
and initiate denosumab (XGeva®) at 120 mg SQ every 4 weeks x 3 doses.
Patients will have repeated measurement of CTCs and be re-evaluated by standard 
imaging (CT of metastatic sites or PET/CT)  at study enrollment and again after 3 cycles 
of denosumab. At that time we will determine that fraction of patients with decrease in  
CTCs. In this cohort, a decrease of CTCs during standard treatment is expected in 40-
50% and a normalization of CTCs (< 5 CTCs) is expected only in 10-15% of patients; a 
decrease and a normalization higher than expected from historical cohorts will indicate 
benefit of the current treatment. In the absence of treatment delays due to adverse 
events, treatment may continue for 3 cycles on study or until one of the criteria listed in 
Section 4.4. Patients will be off the treatment phase of the study, but will  continue the 
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
18
same treatment (e.g.chemotherapy with denosumab), as standard of care, under their 
treating physician.  Patients will still be considered on study but in follow-up for purpose 
of survival endpoints.  
4.2
Treatment Administration
All subjects will be treated in cycles of 28 days (4 weeks).  There is a window of +/-7 days 
for scheduling a visit. Treatment will continue until disease progression, unacceptable 
toxicity, patient withdrawal or death.  Missed doses will not be made up.  If treatment 
delays are > 14 days, patients will be taken off study. Patients will still be followed up for 
survival.
All study treatment visits should be scheduled to coincide with standard of care visits or 
treatment visits. Patients do not need to come in separately for research 
treatment/evaluation. They will continue to receive their standrd of care treatment with 
chemotherapy or endocrine therapy, and the study drug Denosumab will be initiated.  
Denosumab will be administered on Day 1 of each 28 day cycle, at the dose of 120mg 
subcutaneously for a total of 3 cycles. There are no required premedications.
Regimen description
Agent
Premeditations, 
precautions
Dose
Route
Schedule
Cycle 
Length
Denosumab
None
120mg
Subcutaneous
Day 1 (+/-7 
days) for 3 
cyclesd
4 weeks (28 
days)
4.2.1
Investigational Agent: Denosumab
Denosumab 120mg to be given Subcutaneous (SQ) in clinic on Day 1 of each     
cycle for a total of 3 cycles. One cycle is defined as 28 days.
Note: Patients will continue to receive the planned active treatment with 
chemotherapy or endocrine therapy (standard of care) and initiate denosumab 
(XGeva®) Patients will be admitted as out patients in the Lurie Cancer Center, 
and drug will be administered by nursing staff. All treatment visits will coincide 
with standard of care visits.
Risks (local) associated with subcutaneous injection of Denosumab

red, dry, or itchy skin

oozing or crusty blisters on skin

peeling skin
4.2.2 
Missed or skipped doses 
Administration of Denosumab may be done in a  +/-7 days window due to 
weather emergencies, missed appointments, holiday or other unplanned events 
not related to toxicity. Missed doses will not be made up.  If treatment delays are 
> 14 days, will be taken off study. Patients will still be followed up for survival.
           
4.2.3   Supportive Care therapy and pre-medications:
          Patients should receive appropriate supportive care therapies and pre-
medications that would otherwise be given with the chemotherapy or endocrine 
therapy regimen they are getting concurrently with Denosumab, including the 
recommended Vitamin D and calcium as outlined below. Patients should also 
receive full supportive care therapies concomitantly during the study including 
transfusions of blood and blood products, antibiotics when appropriate.  Palliative 
radiation therapy is permitted for irradiating  areas of painful bony metastases 
that cannot be managed adequately using systemic or local analgesics, as long 
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
19
as the presence of these osseous metastases was recorded at baseline prior to 
study initiation and there is no definite evidence of objective disease progression 
at the site based on RECIST1.1 criteria (Appendix 3). These sites cannot be 
used as target lesions or as part of the patient’s response.     
4.2.4 
Recommended Supplemental therapy: 
                     It is strongly recommended that all patients receive daily supplements of at least 
500 mg calcium and at least 400 IU of vitamin D, unless documented 
hypercalcemia (albumin-adjusted serum calcium >2.9mmol/L [11.5mg/dL] or 
ionized calcium > 1.5mmol/L [6.0mg/dL] develops on study. 
4.2.5 
Concomitant therapies requiring special attention
The use of erythropoietin, colony stimulating factors (per ASCO guidelines), 
antiemetic agents, nonsteroidal anti-inflammatory drugs (Ibuprofen (400 mg qid) 
can be administered in patients with normal renal function (CrCl >60 mL/min).   
Patients with mild to moderate renal insufficiency (CrCl from 45 to 59 mL/min) 
should avoid taking salicylates or NSAIDs with long elimination half-lives. 
4.2.6
Permitted concomitant medications
Glucocorticosteroids may be used as anti-emetics and megestrol acetate may be 
used for appetite.
4.2.7
Prohibited concomitant medications: 
Use of bisphosphonates are not permitted while participating in this study. 
However, patients who were previously on bisphosphonates can be enrolled in 
the study, as long as they have a wash-out period of 2 weeks prior to registration.
Use of concurrent investigational agents is not permitted.
4.3
Toxicity Management & Dose Delays/Modifications
4.3.1
General Principles 
Dose modifications will not be permitted with denosumab. Concomitant 
chemotherapy or hormonal therapy doses may be modified as per the primary 
treating oncologist.
4.3.2   Specific Toxicities and Dose Delays
Denosumab dosing will be held only for the study treatment-related toxicities 
listed below. Please note that there is a dosing window of +/- 7 days for all other 
delays due to logistical or administrative issues.

The dose of denosumab should be held in the event of hypocalcemia 
< Institutional lower limit of normal (LLN). In the event of clinically significant 
hypocalcemia, correction may be achieved with intravenous calcium repletion.

In the event of a hypersensitivity reaction, subsequent doses of denosumab 
will not be given and the patient will be removed from the study.

The dose of denosumab should be held and permanently discontinued in the 
event of development of Osteonecrosis of the jaw(ONJ)

Any patient who receives at least one dose of study therapy will be evaluable 
for toxicity endpoints. Each patient will be assessed for the development of 
toxicity according to the timeframe referenced in the Schedule of Events table. 
Toxicity will be assessed according to NCl CTCAE version 4.03.
4.4
Duration of Therapy and Criteria for Study Termination
In the absence of treatment delays due to adverse events, study treatment may continue 
for 3 cycles or until one of the following criteria applies:

Disease progression  
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
20

Patients with clinical or radiological evidence of progressive disease per RECIST 1.1 
(Appendix 3)                                                                                    

Inter-current illness that prevents further administration of treatment                  

Prolonged delay of treatment administration related to concurrent illness or toxicity 
would be an indication for study termination. If treatment delays are > 14 days, study 
therapy will be discontinued. 

Adverse Events- Administration of investigational product (denosumab) will be 
withheld for any subject who experiences:

A grade 3 or 4 adverse event per NCl CTCAE version 4.03 related or possibly related 
to investigational product, and/or any AEs in the treating physician’s discretion that 
would affect the safety of the patient.

Any grade of osteonecrosis of the jaw (ONJ). 

Patient becomes pregnant (in case of females).

Patient decides to withdraw from the study.

General or specific changes in the patient’s condition that render the patient 
unacceptable for further treatment in the judgment of the investigator.
4.5
Duration of Follow up
Follow-up beyond the 30-day post-last-dose visit will be done approximately every 12 
weeks (+/-2 weeks) for a maximum of 2 years. These follow-up visits will be done at the 
same time as a standard of care visit. Alternatively, it can also be done by phone calls.
4.6
Removal of Subjects from Study Treatment and/or Study as a Whole
Patients can be taken off the study treatment and/or study as a whole at any time at their 
own request, or they may be withdrawn at the discretion of the investigator for safety, 
behavioral or administrative reasons. The reason(s) for discontinuation must be clearly 
documented on the appropriate eCRF and may include:

Patient voluntarily withdraws from treatment (follow-up permitted).

Patient withdraws consent (no follow-up permitted).

Patient is unable to comply with protocol requirements.(follow-up permitted)

Patient demonstrates disease progression. follow-up permitted

Patient experiences unacceptable toxicity. follow-up permitted

Treating physician determines that continuation on the study would not be in the 
patient’s best interest. follow-up permitted

Patient becomes pregnant. follow-up permitted

Patient develops a second malignancy that requires treatment which would interfere 
with this study. follow-up permitted

Patient becomes lost to follow-up (LTF).  This is defined as 3 attempts, made 1 week 
apart, to contact patient by preferred method of contact.
4.7
Patient Replacement:
            If a patient is enrolled in the study but comes off study before cycle 1 day 1 of 
treatment, the patient may be replaced by the DMC. Request should be made to the 
QA monitor for replacement by DMC.
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
21
5.0
STUDY PROCEDURES
Assessments
Baseline
(-30 days)
W1
(+/-7 days)
W5
(+/-7 
days)
W9
(+/-7 days)
Off study
treatment
(W 13 +/-7 
days)
Follow-up8
Informed Consent1
X
Medical History & 
eligibility
X
Physical Exam2
X
X
X
X
X
X
Concomitant 
Medications
X
X
X
X
X
X
Vitals3
X
X
X
X
X
ECOG5
X
X
X
X
X
CBCw/diff, plts
X
Serum Chemistry4
X
X
X
X
X
AE/Toxicity 
Assessment
X
X
X
X
X
X
Tumor 
measurements7
X7
 X7
 X7
X7
Pregnancy test
(urine or serum11
X
CTC 
Measurement6
 X6
Denosumab 
administration9
X
X
X
Research blood 
tests
  X10
  X10
Survival8
X
X
1 cycle= 4 weeks (28 days)                               
1 Informed consent must be signed within 30 days of registration.  If signature is outside that window the 
patient must sign a new consent.
2A full physical exam will be done as indicated. 
3 Vitals include temperature, blood pressure, pulse rate, and weight. Height will be taken only at baseline.
4Albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, chloride, creatinine, glucose,  
potassium, total protein, SGOT [AST], SGPT [ALT], sodium.
5 See Appendix 1 for ECOG performance status table.
6Standard CTC measurement to be done at baseline.Two CellSave tubes will be used to collect 
approximately 20 ml of peripheral blood. Tubes will be kept at ambient temperature (22-24 degrees C). 
(Note:. This baseline sample will be a standard of care procedure and will be sent to QUEST diagnostics 
through the hospital.) 
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
22
         7Tumor measurements are by CT.  PET-CT can be done as an adjunct per investigator discretion. Tumor 
measurements are required at baseline/screening. It does not need to be repeated unless it is >6 weeks 
from Day 1 of study treatment. It will be repeated after 3 cycles. Documentation (radiologic or clinical) 
must be provided for patients removed from study for progressive disease. After 3 cycles, CT will be done 
per physician discretion as a standard of care procedure. In the follow up phase, patients will have scans 
every 12 weeks (+/- 2 weeks) up to 2 years for PFS data.
8Follow up for PD, resolution of treatment related toxicities, and survival should be every12 weeks (+/- 2 
weeks) up to 2 years (or more frequently if clinically indicated) for PFS data. Survival will be assessed 
during standard clinic visit or by phone call. Note: Any labs or scans relating to a toxicity or disease 
measurement should be conducted at an interval determined by the treating investigator and results 
should be recorded on the applicable long term follow up case report forms.
9 Denosumab administration is standard of care. Doses may be given after completion of study therapy, 
but not as part of the study.
 10 Blood will be collected for correlative studies to analyze the expression of RANKL, and additional CTC 
analyses including enrichment, enumeration and CTC profiling and characterization of stem cell 
phenotypes. Blood samples for these research tests will be collected at baseline and at 3 months, 
coinciding with Denosumab administration.
i)
8ml in 1 10ml Cellsave tube (for RANKL expression). 
ii)
7.0 ml  each in 2 Adna Collect syringes (for CTC enrichment, enumeration and CTC profiling and 
characterization of stem cell phenotypes-)
iii)
20ml in 2 CellSave tubes (for CTC measurement after 3 cycles of Denosumab [wk13];
(Note: Tubes will be kept at ambient temperature [22-24 degrees C])
(Note; these tests will be only be for research purposes and will be conducted at the CTC lab at 
northwestern university [see section 9.0 and the study lab manual for details]).
11Pregnancy test (urine or serum) is only required for FOCBP (refer to section 3.1.10) 
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
23
6.0
ENDPOINT ASSESSMENT
6.1
Definitions
Evaluable for objective response.  Only those patients who have measurable disease 
present at baseline, have received at least one cycle of therapy, and have had their 
disease re-evaluated will be considered evaluable for response. These patients will have 
their response classified according to the definitions stated below.  (Note:  Patients who 
exhibit objective disease progression prior to the end of cycle 1 will also be considered 
evaluable).
Evaluable Non-Target Disease Response.  Patients who have lesions present at baseline 
that are evaluable but do not meet the definitions of measurable disease, have received 
at least one cycle of therapy, and have had their disease re-evaluated will be considered 
evaluable for non-target disease.  The response assessment is based on the presence, 
absence, or unequivocal progression of the lesions. 
The Response Evaluation Criteria in Solid Tumours (RECIST 1.1, Refer Appendix 3) 
criteria will be used for objective tumour response assessment.
Measurable disease is defined as at least one lesion that can be accurately measured in 
at least one dimension.
Non-measurable disease is defined as All other lesions, including small lesions (longest 
diameter <10mm or pathological lymph nodes with P10 to <15mm short axis) as well as 
truly non-measurable lesions. Lesions considered truly non-measurable include: 
leptomeningeal disease, ascites, pleural or pericardial effusion, and inflammatory breast 
disease, lymphangitic involvement of skin or lung, abdominal masses /abdominal 
organomegaly identified by physical exam that is not measurable by reproducible imaging 
techniques 
Assessments will be performed after three cycles of treatment. Once protocol treatment 
has been completed subjects will be assessed every three months or sooner as indicated 
by treating physicians.
6.2
Primary Objectives
To assess the effect of denosumab in in  Her2/neu negative ER+ and/ or PR+  metastatic 
breast cancer patients who are in PR or SD after starting systemic therapy with bone 
metastases and ≥ 5 CTCs (who are on standard systemic therapy) by measuring the 
fraction of patients with reduction in CTCs after 3 cycles of denosumab.
Only those patients who have received at least one cycle of therapy, and have had their 
disease re-evaluated will be considered evaluable for response.
6.3
Secondary Objectives

To assess the effect of denosumab on CTCs enumeration considered as a continuous 
variable (percent change from baseline).

To evaluate median Progression-free (m-PFS) survival  time for the entire cohort.
Only those patients who, have received at least one cycle of therapy, and have had their 
disease re-evaluated will be considered evaluable for response.
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
24
6.4
Exploratory objective

The correlative study outcome will be CTC enumeration after enrichment. 

To assess the effect on CTC profiling and charaterization of stem cell phenotype 
(CTC-EMT).

To evaluate the type of progressive disease (new site vs. progression of lesions  in 
previous site)

To analyze the expression of RANKL.
CTCs are present in whole blood in low numbers (only 1-10 CTCs per mL of whole blood). 
Therefore, enrichment of tumor cells in peripheral blood samples is required for their 
detection and enumeration. The correlative study outcome will be CTC enumeration after 
enrichment. Refer to Section 9.0 for methodology.
Enumeration of CTCs will be every 3 months while patients remain on study. Enumeration 
is standard of care and is ordered as a clinical CTC test. 
             
             To analyze the expression of RANKL, approximately 8ml of blood will be collected in a 10 
ml CellSave tube at the same time as when the CTC sample is collected (at baseline and 
at 3 months, coinciding with Denosumab administration). 
(Note; this test will be only be for research purposes and will be conducted at Dr. 
Zhaomei Mu’s laboratory at northwestern university[see section 5.0 and 9.0 for details]).
7.0
ADVERSE EVENTS
This study will be conducted in compliance with the Data Safety Monitoring Plan (DSMP) of the 
Robert H. Lurie Comprehensive Cancer Center of Northwestern University 
(http://cancer.northwestern.edu/CRO/data/DataandSafetyMonitoringPlanMay2014.pdf).
The level of risk attributed to this study requires high as outlined in the DSMP. In addition, the 
study will abide by all safety reporting regulations, as set forth in the Code of Federal 
Regulations.  
7.1
Adverse Event Monitoring
Adverse event data collection and reporting, which are required as part of every clinical 
trial, are done to ensure the safety of subjects enrolled in the studies as well as those 
who will enroll in future studies using similar agents. Adverse events are reported in a 
routine manner at scheduled times during a trial (see Section 5 for time points). In 
addition, certain adverse events must be reported in an expedited manner to allow for 
optimal monitoring and patient safety and care. 
All patients experiencing an adverse event once consent is signed, regardless of its 
relationship to study drug, will be followed until: 

the adverse event resolves or the symptoms or signs that constitute the adverse 
event return to baseline; 

any abnormal laboratory values have returned to baseline; 

there is a satisfactory explanation other than the study drug for the changes 
observed; or

death.
7.2
Definitions & Descriptions
7.2.1
Adverse Event
An adverse event (AE) is any untoward medical occurrence in a patient receiving 
study treatment and which does not necessarily have a causal relationship with 
this treatment. An AE can therefore be any unfavorable and unintended sign 
(including an abnormal laboratory finding), symptom, or disease temporally 
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
25
associated with the use of an experimental intervention, whether or not related to 
the intervention. 
Recording of AEs should be done in a concise manner using standard, 
acceptable medical terms. In general, AEs are not procedures or measurements, 
but should reflect the reason for the procedure or the diagnosis based on the 
abnormal measurement. Preexisting conditions that worsen in severity or 
frequency during the study should also be recorded (a preexisting condition that 
does not worsen is not an AE). Further, a procedure or surgery is not an AE; 
rather, the event leading to the procedure or surgery is considered an AE.
If a specific medical diagnosis has been made, that diagnosis or syndrome 
should be recorded as the AE whenever possible.  However, a complete 
description of the signs, symptoms and investigations which led to the diagnosis 
should be provided. For example, if clinically significant elevations of liver 
function tests are known to be secondary to hepatitis, “hepatitis” and not 
“elevated liver function tests” should be recorded. If the cause is not known, the 
abnormal test or finding should be recorded as an AE, using appropriate medical 
terminology (e/g/ thrombocytopenia, peripheral edema, QT prolongation).
7.2.2
Severity of AEs
All non-hematologic adverse events will be graded according to the NCI 
Common Terminology Criteria for Adverse Events (CTCAE) version 4.03. The 
CTCAE v4 is available at http://ctep.cancer.gov/reporting/ctc.html
If no CTCAE grading is available, the severity of an AE is graded as follows:

Mild (grade 1): the event causes discomfort without disruption of normal daily 
activities.

Moderate (grade 2): the event causes discomfort that affects normal daily 
activities.

Severe (grade 3): the event makes the patient unable to perform normal daily 
activities or significantly affects his/her clinical status.

Life-threatening (grade 4): the patient was at risk of death at the time of the 
event.

Fatal (grade 5): the event caused death.
7.2.3
Serious Adverse Events (SAEs)
All SAEs, regardless of attribution, occurring from time of signed informed 
consent, through 30 days after the last administration of study drug, must be 
reported upon discovery or occurrence.
An SAE is defined in regulatory terminology as any untoward medical occurrence 
that:

Results in death.
If death results from (progression of) the disease, the disease should be 
reported as event (SAE) itself.

Is life-threatening.
The patient was at risk of death at the time of the event; it does not refer to 
an event that hypothetically might have caused death if it were more severe.

Requires in-patient hospitalization or prolongation of existing 
hospitalization for ≥ 24 hours.

Results in persistent or significant disability or incapacity.

Is a congenital anomaly/birth defect.
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
26

Is an important medical event.
Any event that does not meet the above criteria, but that in the judgment of 
the investigator jeopardizes the patient, may be considered for reporting as a 
serious adverse event. The event may require medical or surgical 
intervention to prevent one of the outcomes listed in the definition of “Serious 
Adverse Event“. 
For example: allergic bronchospasm requiring intensive treatment in an 
emergency room or at home; convulsions that may not result in 
hospitalization; development of drug abuse or drug dependency.
7.2.4
Unanticipated Problems Involving Risks to Subject or Others
A UPIRSO is a type of SAE that includes events that meet ALL of the following 
criteria:

Is unanticipated in terms of nature, severity, or frequency

Places the research subject or others at a different or greater risk of harm

Is deemed to be at least possibly related to participation in the study.   
7.3
Adverse Event Reporting
7.3.1
Routine Reporting
All routine adverse events, such as those that are expected, or are unlikely or 
definitely not related to study participation, are to be reported on the appropriate 
eCRF according to the time intervals noted in the appendices. Routine AEs will 
be reviewed by the Data Monitoring Committee (DMC) according to the study’s 
phase and risk level, as outlined in the DSMP. 
7.3.2
Determining if Expedited Reporting is Required
This includes all events that occur within 30 days of the last dose of protocol 
treatment. Any event that occurs more than 30 days after the last dose of 
treatment and is attributed (possibly, probably, or definitely) to the agent(s) must 
also be reported accordingly.
1)
Identify the type of adverse event using the NCI CTCAE v 4.03.
2)
Grade the adverse event using the NCI CTCAE v 4.03.
3)
Determine whether the adverse event is related to the protocol therapy.  
Attribution categories are as follows:

Definite: AE is clearly related to the study treatment.

Probable: AE is likely related to the study treatment.

Possible: AE may be related to the study treatment.

Unlikely: AE not likely to be related to the study treatment.

Unrelated: AE is clearly NOT related to the study treatment.
4)
Determine the prior experience of the adverse event.
Expected events are those that have been previously identified as 
resulting from administration of the agent. An adverse event is 
considered unexpected, for expedited reporting purposes only, when 
either the type of event or the severity of the event is not listed in:

the current protocol

the drug package insert

the current Investigator’s Brochure
7.3.3
Expedited Reporting of SAEs/Other Events
7.3.3.1 Reporting to the Northwestern University QAM/DMC
All SAEs must be reported to the assigned QAM within 24 hours of 
becoming aware of the event. Completion of the NU CRO SAE Form is 
required.
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
27
The completed form should assess whether or not the event qualifies as 
a UPIRSO. The report should also include:

Protocol description and number(s)

The patient’s identification number

A description of the event, severity, treatment, and outcome (if 
known)

Supportive laboratory results and diagnostics

The hospital discharge summary (if available/applicable)
All SAEs will be reported to, and reviewed by, the DMC at their next 
meeting.
7.3.3.2 Reporting to the Northwestern University IRB
The following information pertains to the responsibilities of the lead site 
(Northwestern University). Additional participating sites should follow 
their local IRB guidelines for reporting to their local IRBs.

Any death of an NU subject that is unanticipated in nature and at 
least possibly related to study participation will be promptly reported 
to the NU IRB within 24 hours of notification. 

Any death of an NU subject that is actively on study treatment 
(regardless of whether or not the event is possibly related to study 
treatment)

Any death of a non-NU subject that is unanticipated and at least 
possibly related and any other UPIRSOs will be reported to the NU 
IRB within 5 working days of notification.

All other deaths of NU subjects not previously reported, other non-
NU subject deaths that were unanticipated and unrelated, and any 
other SAEs that were not previously reported as UPIRSOs will be 
reported to the NU IRB at the time of annual continuing review.
7.3.3.3 Reporting to Amgen
All SUSARs, SAEs, and Aggregated reports should be reported to the 
Amgen as stated in the table below.  The investigator or qualified designee 
must complete the Medwatch form) and send it electronically to Amgen 
contact:
Lorie B. Bruno
Manager, Investigator-Sponsored Studies
Non-Amgen Sponsored Clinical Research (NASCR)
PRA Health Sciences Providing Services to Amgen
Phone:  570-732-4059
Cell:        570-778-8765
Fax:        913-307-5758
Email:   lorieb@amgen.com 
AGGREGRATE REPORTS
Safety Data
Timeframe for submission to Amgen
Annual Safety Report
(e.g. EU Clinical trial Directive[ 
CTD]), Annual safety report, US IND 
Annual report)
Annually
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
28
Other aggregate analyses
(any report containing safety data 
that is generated during the course 
of the study)
At the time of ISS sponsor submission
(Anybody governing research conduct e.g. 
RA, IRB etc.)
Final (End of study report)

Unblinding data for blinded 
studies

Reports of unauthorized use 
of a blinded product
At the time of ISS sponsor 
submission(anybody governing research 
conduct RA, IRB etc.) but not later than one 
calendar year of study completion
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
29
8.0
DRUG INFORMATION
8.1
Agent Denosumab
Denosumab is a human IgG2 monoclonal antibody with high affinity and 
specificity for RANKL. Denosumab has an approximate molecular weight of 147 
kDa and is produced in genetically engineered mammalian (Chinese hamster 
ovary) cells. Denosumab is commercially available and this commercial supply 
will be used for this study.
8.1.1
Other names: AMG162, Xgeva
8.1.2
Classification - human IgG2 monoclonal antibody with high affinity and 
specificity for RANKL. 
8.1.3
Mode of action: Denosumab is a human IgG2 monoclonal antibody with high 
affinity and specificity for RANKL
8.1.4
Storage and stability
Denosumab should be stored protected from light and according to the storage 
and expiration information provided in the package insert.
8.1.5
Protocol dose specifics
Denosumab 120mg to be given subcutaneous (SQ) in clinic on Day 1 of each     
cycle for a total of 3 cycles. One cycle is defined as 28 days.
Note: Patients will continue to receive the planned active treatment with 
chemotherapy or endocrine therapy (standard of care) and initiate denosumab 
(XGeva®) (Refer to section 4.0 for further details).
8.1.6
Preparation
Denosumab is provided as a sterile, colorless to slightly yellow, practically free 
from particles, preservative-free solution intended for subcutaneous injection. 
The vial presentations contain 60 mg/mL denosumab, 17 mM sodium acetate, 
and 4.7% (w/v) sorbitol, at a pH of 5.2, filled to a target deliverable volume of 1.0 
mL; or 70 mg/mL denosumab, 18 mM sodium acetate and 4.6% (w/v) sorbitol, at 
a pH of 5.2, filled to a target deliverable volume of 1.7 mL. The prefilled syringe 
(PFS) drug product contains denosumab at 60 mg/mL, 17 mM sodium acetate, 
4.7% (w/v) sorbitol, and 0.01% (w/v) polysorbate 20, at a pH of 5.2, filled to a 
target deliverable volume of 1.0 mL.
8.1.7
Route of administration for this study
Denosumab will be administered subcutaneously. The use of a 27-gauge needle 
is recommended for administration. A 30-gauge needle should not be used, as 
this may affect the quality of the product.
8.1.8
Incompatibilities: none known 
8.1.9
Availability & Supply 
Denosumab is commercially available and will be administered as standard of care 
drug. The drug will be obtained through prescription from the treating physician. 
The cost of the drug will be billed to the patient’s insurance. 
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
30
8.1.10
Side effects 
                          Risks associated with denosumab:>10%:

hypertension

fatigue

headache

swelling in legs or ankle

lowering of the phosphate levels

lowering of the calcium levels

nausea

decreased appetite

vomiting

constipation or diarrhea

anemia

infection (influenza)

weakness or joint pains

limb pain or back pain

shortness of breath or cough
 
1% to 10%:

chest pain

sciatica

high cholesterol levels 

passing more gas

new secondary cancer

osteonecrosis of the jaw (infection and damage of the jaw bone)

cataract

upper respiratory infection
 
<1% (Limited to important or life-threatening): 

developing antibodies to the drug

infection of the heart

fracture of femur

hypersensitivity/allergy including anaphylaxis

low blood pressure

pancreatitis 
8.1.11
Nursing implications
All research visits will coincide with Standard of Care visits. Vitals will be 
monitored per Standard of Care and Institutional guidelines.
                          
8.1.12
Return and Retention of Study Drug
                         Not applicable 
                         Commercial supply of Denosumab will be used.
9.0
CORRELATIVES/SPECIAL STUDIES
9.1
Enrichment and Enumeration of CTCs 
            CTCs are present in whole blood in low numbers (only 1-10 CTCs per mL of whole 
blood). Therefore, enrichment of tumor cells in peripheral blood samples is required for 
their detection and enumeration.
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
31
9.1.2 
Enrichment and Molecular Characterization of CTCs using Adna Test
AdnaTest BreastCancer and AdnaTest EMT-2/StemCell allow molecular 
characterization of CTCs by combining AdnaTest BreastCancerSelect with 
AdnaTest BreastCancerDetect and AdnaTest EMT-2/StemCellSelect with 
AdnaTest EMT-2/StemCellDetect.  AdnaTest BreastCancerSelect and AdnaTest 
EMT-2/StemCellSelect are highly specific immunomagnetic cell-selection system 
for enriching CTCs from peripheral blood. AdnaTest BreastCancerDetect 
and AdnaTest EMT-2/StemCellDetect allow sensitive analysis of breast cancer-
associated gene and EMT/tumor stem cell-associated gene expression in 
immunomagnetically enriched CTCs by reverse transcription (RT) and PCR 
analysis.
Please see the Laboratory Manual for detailed blood collection, processing and 
shipping instructions.
AdnaTest can be performed using two methods based on the gene expression 
profile. The assay will be performed using reagent kits (QIAGEN) according to 
the manufacturer’s protocols.
Please see the Laboratory Manual for detailed blood collection, processing and 
shipping instructions.
9.1.3
AdnaTest BreastCancerSelect/Adna Test Breast Cancer Detect
Adna Test BreastCancer Select generates a magnetic bead-labeled cell lysate 
that is used for further analysis with the Adna Breast Cancer Detect kit (QIAGEN) 
RT-PCR with the Breast Cancer Detect test generates tumor gene fragments 
GA733-2, Muc-1, Her-2. 
The test is considered positive if a PCR fragment of at least one tumor 
associated transcript is clearly detected.  Peaks with a concentration of > 0.30 
ng/μl are positive.  Peaks that are not detected at the above setting are negative 
(i.e., concentration < 0.15 ng/μl).  Peaks with an intermediate concentration of 
0.15 ng/μl to 0.30 ng/μl are inconclusive and require a re-testing.
Method
Enrichment of CTCs from peripheral blood will be performed using the AdnaTest 
BreastCancerSelect kit (QIAGEN). The kit contains BreastSelect Beads in a 
solution containing sodium azide.  BreastCancerSelect enables the 
immunomagnetic enrichment of tumor cells via epithelial and tumor associated 
antigens.  Antibodies against epithelial and tumor associated antigens are 
conjugated to magnetic beads (Dynabeads) for the labeling of tumor cells in 
peripheral blood.  The labeled cells are extracted by a magnetic particle 
concentrator (AdnaMag-L and MPC-S) and are subsequently lysed. The cell 
lysate is used for further analysis with the AdnaTest BreastCancerDetect.  
AdnaTest BreastCancerDetect is used for the detection of breast cancer-
associated gene expression in immunomagnetically enriched tumor cells by RT-
PCR.  BreastCancerDetect contains oligo (dT)25-coated beads for the isolation of 
mRNA from the lysate of pre-enriched tumor cells.  RT results in cDNA, which is 
the template for tumor cell detection and characterization by multiplex-PCR.  
With the PrimerMix BreastDetect three tumor associated antigens and one 
control gene are amplified.  
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
32
The primers generate fragments of the following sizes: 

GA733-2: 395bp

Muc-1: 293bp

Her-2: 270bp

Actin: 114bp (internal PCR control)  
In addition to the BreastCancerDetect, the ER/PR-Detect is available for the 
PCR-expression analysis of hormone receptor gene for estrogen and 
progesterone in enriched tumor cells.  With the PrimerMix ER/PR-Detect the 
hormone receptor genes for estrogen, progesterone and one control gene, actin, 
are amplified.  
The primers generate fragments of the following sizes: 

ER: 305bp

PR: 270bp

Actin: 119bp (internal PCR control)
Using the AdnaTest ER/PR-Detect, the test is considered positive for ER if a 
peak with a concentration of ≥0.15 ng/μl is detected and positive for PR if a peak 
with a concentration of > 0 ng/μl is detected.  
9.1.4 
AdnaTest EMT-2/Stem CellSelect/ AdnaTest EMT-2/Stem CellDetect: 
AdnaTest EMT-2/Stem CellSelect generates a magnetic bead-labeled cell lysate 
that is used for further analysis with AdnaTest EMT-2/Stem CellDetect. The 
results of the test generate EMT associated gene fragments Akt-2, Twist 1, 
PI3Kα, and Actin (control) , and tumor stem-cell marker ALDH1
Method:
Enrichment of CTCs from peripheral blood will be performed using the AdnaTest 
EMT-2/Stem CellSelect kit (QIAGEN). The kit enables the immunomagnetic 
enrichment of tumor cells via epithelial and tumor associated antigens.  
Antibodies against epithelial and tumor associated antigens are conjugated to 
magnetic beads (Dynabeads) for the labeling of tumor cells in peripheral blood.  
The labeled cells are extracted by a magnetic particle concentrator and are 
subsequently lysed.  The cell lysate is used for further analysis with the AdnaTest 
EMT-2/Stem CellDetect.  
The AdnaTest EMT-2/Stem CellDetect is used for the analysis of EMT-2/Stem 
Cell characteristics in immunomagnetically enriched circulating tumor cells by RT 
and  The kit uses oligo (dT)25-coated beads for the isolation of mRNA from the 
lysate of pre-enriched tumor cells.  RT results in cDNA, which is the template for 
tumor cell detection and characterization by multiplex-PCR.  With the PrimerMix 
EMT-2 three EMT related genes and one control gene are amplified.  The 
PrimerMix Stem Cell amplifies ALDH1, which is accepted as tumor stem-cell 
marker.  The PrimerMix Breast Detect can be used to determine breast cancer 
associated gene expression.  
The different primer-mixes generate the following fragments: 
EMT-2: 

Akt-2: 309bp

Twist 1: 201bp

PI3Kα: 551bp

Actin: 111bp (Internal PCR Control)
Stem Cell: 

ALDH1: 161bp
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
33
9.2 Figure 1. Schematic Overview of Sample Preparation
9.2.1 
Enumeration of CTCs and RANKL analysis:
Samples will be maintained at room temperature and processed within 96 h after 
collection. Enumeration of CTCs and RANKL expression on CTCs will be 
performed on CellSearch System (Janssen Diagnostics, LLC) according to the 
manufacturer’s standard CTC measurement protocol. Briefly, 7.5 mL of whole 
blood  will be processed on the CellSearch system using CellSearch CXC kits 
(Janssen Diagnostics). The CellSearch system consists of a semiautomated 
system (CellPrep) for the preparation of the sample and a CellSpotter Analyzer 
with semi-automated fluorescence-based microscopy system for the identification 
of  CTCs by cellular images. At the same time of CTCs enumeration, expression 
of RANKL on CTCs will be stained by PE (phycoerythrin)-conjugated specific 
RANK antibody (R&D Systems). CTCs will be identified by positive staining for 
cytokeratins (CK) and DAPI (double stranded DNA), and negative staining for 
anti-CD45 (leukocytes marker). RANK-positive CTCs will be identified by positive 
staining for both CK and RANK, and by negative staining for anti-CD45. 
Quantitative results are expressed as the total number of CTCs and RANK-
positive CTCs. 
(Note; this test will be only be for research purposes and will be conducted at Dr.  
Zhaomei Mu’s laboratory at northwestern university [also see section 5.0])
Please see the Laboratory Manual for detailed blood collection, processing and 
shipping instructions.
(Note: left over blood samples from all correlative tests will not be banked for 
future use.)
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
34
For any question related to correlative tests, please contact: 
      
CTC Lab at Northwestern University 
Zhaomei Mu, MD
Research Assistant Professor
Department of Medicine-Hem/Oncology
Feinberg School of Medicine 
710 N. Fairbanks Ct, Olson Rm: 8-523
Chicago, IL 60611; 
Tel: 312-503-5489(primary)
       312-503-3025(secondary)
Email: Zhaomei.mu@northwestern.edu
10.0
STATISTICAL CONSIDERATIONS
10.1
Study Design/Endpoints
We will conduct a single arm, two-stage Phase II study to assess the efficacy of a 
combination therapy including Denosumab in patients with bone metastatic breast cancer 
and detectable circulating tumor cells (CTCs).  The primary outcome is evidence of 
reduction in the number of CTCs. A patient will be defined as a “success” if the number of 
CTCs at follow-up is less than the number observed at baseline.  Patients who do not 
experience a decrease in CTC count at follow-up or are missing follow-up CTC count 
measurements will be defined as “failures”. The new combined treatment would be of 
interest if the proportion (p) of successes is at least 70%. A success proportion of less 
than 50% will be of no interest. We will test the null hypothesis that the success 
proportion is 0.5 versus the alternative that this proportion is 0.7.  After testing the 
combination therapy on 23 patients in the first stage, the trial will be terminated if there 
are 12 or fewer successes. If the trial goes on to the second stage, a total of 37 patients 
will be studied. If the total number of successes is less than or equal to 23, the 
combination therapy will be rejected. The type I error for this Simon minimax design 
(Simon, 1989) is 4.8% and the power is 80%. The probability of early termination under 
the null hypothesis (i.e., p=0.5) is 66.1%. 
Based on data from phase III studies of denosumab, we anticipate that the probability of 
serious adverse events related to these agents including acute reaction, renal failure or 
osteonecrosis of the jaw (if meet criteria for serious adverse event)) will not exceed 0.05. 
In this trial, accrual will be suspended if, at any point among the first 23 patients, four or 
more subjects are observed with a treatment related serious adverse event (TRSAE). 
Assuming that the true probability of a TRSAE is 5%, the probability that we will observe 
at least four subjects with a TRSAE among the first 23 patients is 2.58%. Similarly, 
accrual will be suspended if, at any point in the trial, six or more subjects are observed 
with a TRSAE. If the true TRSAE is 5%, then the probability that the study will be 
declared as too toxic at any point is 2.93%.  Alternatively if the true TRSAE rate is 20%, 
then the probability that the study will be terminated early (i.e., ≤23 subjects accrued) is 
70.4% and the chance of declaring the treatment as too toxic at any point in the study 
exceeds 82%. The population for safety analyses will include all patients who receive at 
least one dose of the combination therapy including denosumab.
10.2
Sample Size/Accrual Rate
We plan to enroll up to 42 patients to obtain 37 evaluable patients.  We anticipate that 
patients will be accrued at a rate of 2-3 per month.
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
35
10.3
Stratification Factors
No stratification is planned.
10.4
Statistical Analysis
For the primary objective, the proportion of successes will be estimates using a bias-
corrected estimator and its confidence interval as described by Porcher and Desseaux 
(2012).
To assess the effect of denosumab on CTCs enumeration considered as a continuos 
variable, the percent change in CTCs from baseline (i.e., 100*[number of CTCs at follow-
up/number of CTCs at baseline] will be calculated and compared to zero using either a 
one sample t-test or a Wilcoxon signed rank test.   
To evaluate median progression-free survival (PFS) time for the entire cohort, Kaplan-
Meier curves will be used.  Cox proportional hazards models with time dependent 
covariates will be used to assess the relationship between longitudinal CTC counts (as 
defined by 2 or more CTCs evaluation in the course of treatment) and PFS.
To detect the antimetastatic effect of denosumab, the first progression will be classified 
as occurring either at  a new site or at a previous site.  A competing risk PFS curve will 
be calculated with new site occurrence as the primary event and previous site as the 
competing event.  Similarly, a competing risk PFS curve will be calculated with previous 
site occurrence as the primary event and new site as the competing event.  These two 
corves will be compared decriptively to determine the recurrence pattern of both types of 
outcomes.  Greater occurrence at pervious sites would indicate an antimetastatic effect.
11.0
STUDY MANAGEMENT
11.1
Institutional Review Board (IRB) Approval and Consent 
It is expected that the IRB will have the proper representation and function in accordance 
with federally mandated regulations. The IRB should approve the consent form and 
protocol.
In obtaining and documenting informed consent, the investigator should comply with the 
applicable regulatory requirement(s), and should adhere to Good Clinical Practice (GCP) 
and to ethical principles that have their origin in the Declaration of Helsinki.
Before recruitment and enrollment onto this study, the patient will be given a full 
explanation of the study and will be given the opportunity to review the consent form. 
Each consent form must include all the relevant elements currently required by the FDA 
Regulations and local or state regulations. Once this essential information has been 
provided to the patient and the investigator is assured that the patient understands the 
implications of participating in the study, the patient will be asked to give consent to 
participate in the study by signing an IRB approved consent form.
Prior to a patient’s participation in the trial, the written informed consent form should be 
signed and personally dated by the patient and by the person who conducted the 
informed consent discussion.
11.2
Amendments
The Principal Investigator will formally initiate all amendments to the protocol and/or 
informed consent. All amendments will be subject to the review and approval of the 
appropriate local, institutional, and governmental regulatory bodies, as well as by 
Janssen Scientific Affairs. Amendments will be distributed by the lead institution 
(Northwestern) to all affiliate sites upon approval by the Northwestern University IRB.
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
36
11.3
Registration Procedures
BEFORE a patient can be treated on study, please complete and submit the following 
items to confirm eligibility and receive a subject identification number: 
• Eligibility eCRF (complete in NOTIS) 
• Eligibility checklist (signed and dated by the treating physician – upload in NOTIS)
 • Signed and dated informed consent document (upload in NOTIS) 
• Pathology Report (upload in NOTIS) 
The QAM will review the registration, register the patient, assign an identification number, 
and send a confirmation of registration to involved personnel. Registration will then be 
complete and the patient may begin study treatment.
11.4
Data Submission
Once a subject is confirmed and registered to the study, eCRFs should be submitted 
according to the detailed data submission guidelines (provided in a separate document).  
Generally, for all phase II patients, data are due at the end of every cycle.  
11.5
Data Management and Monitoring/Auditing
This study will be conducted in compliance with the Data Safety Monitoring Plan (DSMP) 
of the Robert H. Lurie Comprehensive Cancer Center of Northwestern University (please 
refer to Appendices for additional information). The level of risk attributed to this study 
requires high level of monitoring as outlined in the DSMP 
(http://cancer.northwestern.edu/CRO/data/DataandSafetyMonitoringPlanMay2014.pdf) 
The assigned QAM, with oversight from the Data Monitoring Committee, will monitor this 
study in accordance with the study phase and risk level. Please refer to the Appendices 
for additional data submission instructions.
11.6
Adherence to the Protocol
Except for an emergency situation in which proper care for the protection, safety, and 
well-being of the study patient requires alternative treatment, the study shall be 
conducted exactly as described in the approved protocol. 
11.6.1
Emergency Modifications
Investigators may implement a deviation from, or a change of, the protocol to 
eliminate an immediate hazard(s) to trial subjects without prior IRB approval. 
For any such emergency modification implemented, an IRB modification form 
must be completed within 5 business days of making the change, and the QAM 
must be notified within 24 hours of such change. 
11.6.2
Other Protocol Deviations
All other deviations from the protocol must be reported to the assigned QAM 
using the appropriate form.
A protocol deviation is any unplanned variance from an IRB approved protocol 
that: 

Is generally noted or recognized after it occurs.

Has no substantive effect on the risks to research participants.

Has no substantive effect on the scientific integrity of the research plan or the 
value of the data collected.

Did not result from willful or knowing misconduct on the part of the 
investigator(s). 
A protocol deviation may be considered an instance of Reportable New 
Information (RNI) if it:
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
37

Has harmed or increased the risk of harm to one or more research 
participants.

Has damaged the scientific integrity of the data collected for the study.

Results from willful or knowing misconduct on the part of the investigator(s).

Demonstrates serious or continuing noncompliance with federal regulations, 
State laws, or University policies.
11.7
Investigator Obligations
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 PI is responsible for personally overseeing the treatment of all study 
patients. The PI 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, entered onto the appropriate eCRFs, and submitted 
within the study-specific timeframes. Periodically, monitoring visits may be conducted and 
the Principal Investigator will provide access to his/her original records to permit 
verification of proper entry of data. The study may also be subject to routine audits by the 
Audit Committee, as outlined in the DSMP. 
11.8
Publication Policy.  
All potential publications and/or data for potential publications (e.g. manuscripts, 
abstracts, posters, clinicaltrials.gov releases) must be approved in accordance with the 
policies and processes set forth in the Lurie Cancer Center DSMP. For trials that require 
high intensity monitoring, the assigned QAM will prepare a preliminary data summary (to 
be approved by the DMC) no later than 3 months after the study reaches its primary 
completion date (the date that the final subject is examined or receives an intervention for 
the purposes of final data collection for the primary endpoint). If the investigator’s wish to 
obtain DMC-approved data prior to this point (or prior to the point dictated by study 
design), the PI must send a written request for data to the QAM which includes 
justification. If the request is approved, data will be provided no later than 4 weeks after 
this request approval. The data will be presented to the DMC at their next available 
meeting, and a final, DMC-approved dataset will be released along with any DMC 
decisions regarding publication. The investigators are expected to use only DMC-
approved data in future publications. The investigators should submit a copy of the 
manuscript to the biostatistician to confirm that the DMC-approved data are used 
appropriately.  Once the biostatistician gives final approval, the manuscript may be 
submitted to external publishers.
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
38
REFERENCES
AKTAS B, MULLER V, TEWES M, ET AL.  “COMPARISON OF ESTROGEN AND 
PROGESTERONE RECEPTOR STATUS OF CIRCULATING TUMOR CELLS AND THE 
PRIMARY TUMOR IN METASTATIC BREAST CANCER PATIENTS.” GYNECOL ONCOL 
2011;122(2): 356-60.
Amgen. Investigator’s Brochure: Denosumab (AMG 162) . Edition 10.1. July 2011
Ashworth, T. R. "A case of cancer in which cells similar to those in the tumours were seen in the 
blood after death". Australian Medical Journal 1869;14: 146–7.
Bauernhofer T, Zenahlik S, Hofmann G et al. Association of disease progression and poor overall 
survival with detection of circulating tumor cells in peripheral blood of patients with metastatic 
breast cancer. Oncol Rep 2005;13(2):179-184.
Bidard FC, Mathiot C, Degeorges A et al. Clinical value of circulating endothelial cells and 
circulating tumor cells in metastatic breast cancer patients treated first line with bevacizumab and 
chemotherapy. Ann Oncol 2010; 21 (in press).
BUDD GT, CRISTOFANILLI M, ELLIS MJ ET AL. “CIRCULATING TUMOR CELLS VERSUS 
IMAGING—PREDICTING OVERALL SURVIVAL IN METASTATIC BREAST CANCER.” CLIN 
CANCER RES 2006; 12: 6403-6409.
Cristofanilli M, Budd GT, Ellis MJ, et al. "Circulating Tumor Cells, Disease Progression and 
Survival in Metastatic Breast Cancer". NEJM 2004;351 (8): 781–91.
Cristofanilli M, Hayes DF, Budd GT et al. Circulating tumor cells: a novel prognostic factor for 
newly diagnosed metastatic breast cancer. J Clin Oncol 2005;23(7):1420-1430.
Dawood S, Broglio K, Valero V et al. Circulating tumor cells in metastatic breast cancer: from 
prognostic stratification to modification of the staging system? Cancer 2008;113(9):2422-2430.
DE GIORGI U, VALERO V, ROHREN E, ET AL. “CIRCULATING TUMOR CELLS AND 
[18F]FLUORODEOXYGLUCOSE POSITRON EMISSION TOMOGRAPHY/COMPUTED 
TOMOGRAPHY FOR OUTCOME PREDICTION IN METASTATIC BREAST CANCER.” JCO 
2009;27(20): 3303-3311.
DE GIORGI U, VALERO V, ROHREN E, ET AL. “CIRCULATING TUMOR CELLS AND BONE 
METASTASES AS DETECTED BY FDG–PET/CT IN PATIENTS WITH METASTATIC BREAST 
CANCER.” ANN ONCOL 2010; 21(1): 33-39.
FEHM T, MULLER V, AKTAS B ET AL. HER2 STATUS OF CIRCULATING TUMOR CELLS IN 
PATIENTS WITH METASTATIC BREAST CANCER: A PROSPECTIVE, MULTICENTER TRIAL. 
BREAST CANCER RES TREAT 2010;124(2):403-412.
Fizazi, Lipton A, Mariette X, et al. Randomized Phase II Trial of Denosumab in Patients with Bone 
Metastases From Prostate Cancer, Breast Cancer, or Other Neoplasms After Intravenous 
Bisphosphonates. JCO 2009; 27(10):1564-71.
Hart IR, Fidler IJ. Role of organ selectivity in the determination of metastatic patterns of B16 
melanoma. Cancer Res 1980;40:2281–2287
Kennecke H, Yerushalmi R, Woods R et al. Metastatic behavior of breast cancer subtypes. J Clin 
Oncol 2010;28(20):3271-3277.
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
39
Lipton A, Steger GG, Figueroa J et al. Randomized Active-Controlled Phase II Study of 
Denosumab Efficacy and Safety in Patients with Breast Cancer-Related Bone Metastases. JCO 
2007;25(28):4431-37.
Liu MC, Shields PG, Warren RD et al. Circulating tumor cells: a useful predictor of treatment 
efficacy in metastatic breast cancer. J Clin Oncol 2009;27(31):5153-5159.
Munzone E, Botteri E, Sandri MT, et al. Prognostic value of circulating tumor cells according to 
immunohistochemically defined molecular subtypes in advanced breast cancerClin Breast 
Cancer. 2012 Oct;12(5):340-6.
Norton L, Massague J. Is cancer a disease of self-seeding? Nat Med 2006;12(8):875-878. 
Paget S. The distribution of secondary growths in cancer of the breast. Lancet 1889;1:571–573
Palafox M, Ferrer I, Pellegrini P, Vila S, et al. RANK Induces Epithelial–Mesenchymal Transition 
and Stemness in Human Mammary Epithelial Cells and Promotes Tumorigenesis and Metastasis. 
Cancer Res , 2012 72; 2879
 
Riethdorf S, Fritsche H, Muller V et al. Detection of circulating tumor cells in peripheral blood of 
patients with metastatic breast cancer: a validation study of the CellSearch system. Clin Cancer 
Res 2007;13(3):920-928.
SCHRAMEK D, LEIBBRANDT A, SIGL V, ET AL. OSTEOCLAST DIFFERENTIATION FACTOR 
RANKL CONTROLS DEVELOPMENT OF PROGESTIN-DRIVEN MAMMARY CANCER. 
NATURE. 2010 NOV 4; 468(7320):98-102.
Simon, Richard. “Optimal Two-Stage Designs for Phase II Clinical Trials,” Controlled Clinical 
Trials, 1989, Volume 10, pages 1-10.
STOPECK AT, LIPTON A, BODY JJ ET AL. DENOSUMAB COMPARED WITH ZOLEDRONIC 
ACID FOR THE TREATMENT OF BONE METASTASES IN PATIENTS WITH ADVANCED 
BREAST CANCER: A RANDOMIZED, DOUBLE-BLIND STUDY. JCO 2010; 28(35):5132-38.
Eisenhauer EA, Therasse P, Bogaerts J, et al. New response evaluation criteria in solid
tumours: revised RECIST guideline (version 1.1). Eur J Cancer. 2009;45(2):228-47.
 
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
40
APPENDICES
APPENDIX 1 ECOG PERFORMANCE STATUS
Grade
Description
0
Normal activity. Fully active, able to carry on all pre-disease performance 
without restriction.
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).
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.
3
In bed >50% of the time. Capable of only limited self-care, confined to bed 
or chair more than 50% of waking hours.
4
100% bedridden. Completely disabled. Cannot carry on any self-care. 
Totally confined to bed or chair.
5
Dead.
* As published in Am. J. Clin. Oncol.:  Oken, M.M., Creech, R.H., Tormey, D.C., Horton, J., Davis, 
T.E., McFadden, E.T., Carbone, P.P.: Toxicity And Response Criteria Of The Eastern 
Cooperative Oncology Group. Am J Clin Oncol 5:649-655, 1982. The Eastern Cooperative 
Oncology Group, Robert Comis M.D., Group Chair.
APPENDIX 2
Barrier Methods
Intrauterine Device Methods
Hormonal Methods

Male condom plus 
spermicide 

Cap plus 
spermicide 

Diaphragm plus 
spermicide 

Copper T

Progesterone T

Levonorgestrel-relasing

Implants

Hormone shot or 
injection

Combined pill

Minipill

Patch
NOTE: choice of contraception should be discussed with primary treating oncologist to discuss 
the risks and benefits of different modalities of contraception. 
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.

                                                                 
NU16B09
Version Date: 6.1.17/ Amendment 1
41
APPENDIX 3
Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 Criteria for Evaluating 
Response in Solid Tumors
RECIST version 1.1* will be used in this study for assessment of tumor response. 
* As published in the European Journal of Cancer:
E.A. Eisenhauer, P. Therasse, J. Bogaerts, L.H. Schwartz, D. Sargent, R. Ford, J. 
Dancey, S. Arbuck, S. Gwyther, M. Mooney, L. Rubinstein, L. Shankar, L. Dodd, R. 
Kaplan, D. Lacombe, J. Verweij. New response evaluation criteria in solid tumors: Revised 
RECIST guideline (version 1.1). Eur J Cancer. 2009 Jan;45(2):228-47.
Can be accessed at:  
http://ctep.cancer.gov/protocolDevelopment/docs/recist_guideline.pdf
IRB #: STU00203216 Approved by NU IRB for use on or after 7/19/2018 through 7/18/2019.