Clinical trial NCT02538471
Conditions: Metastatic Breast Cancer
Sponsor: Weill Medical College of Cornell University
Start date: 2015-08-10; Completion date: 2019-02-20
Enrollment: 3 participants; Allocation: NA; Intervention model: SINGLE_GROUP

Brief summary:
Patients with metastatic breast cancer receiving at least one single agent chemotherapy and demonstrating stable disease or disease progression at two consecutive clinical/radiological assessments (at an interval of at least 2 weeks).

Transforming growth factor-beta (TGFΒ) blockade will enhance response of irradiated tumors and improve the function of Dendritic and T cells. Patients will receive 300 mg/day of study drug administered via oral drug tablet every day for 14 days on and 14 days off (=28 day cycle). Radiation to a metastatic site will be delivered at a dose of 7.5 Gy, given consecutively on days 1-3-5.

Detailed description:
Transforming growth factor-beta (TGFβ) is a pleiotropic cytokine which belongs to a superfamily of ligands, including bone morphogenetic proteins and activins \[1-5\]. Under normal conditions, members of the TGFβ family maintain homeostasis in many organ systems. In normal and non-cancerous cells, TGFβ limits the growth of epithelial, endothelial, neuronal, and hematopoietic cell lineages through anti-proliferative and apoptotic responses. In addition, TGFβ exerts potent effects that influence immune function, cell proliferation/ functional differentiation, cell adhesion, extracellular matrix production, cell motility, angiogenesis, and cytokine production. TGFβ has been implicated as an important factor in the growth, progression, and metastatic potential of advanced cancers. Although TGFβ has been shown to suppress the growth of epithelial cells in the early stages of tumor development (premalignant conditions), the effect on advanced cancers is more complex \[1, 5-6\]. Increased production of TGFβ has been found in many neoplasms such as breast, prostate, gastric, renal, and epidermal carcinomas, and elevated plasma TGFβ levels in patients have been correlated with advanced disease, metastases, and lower survival rates \[7-13\]. In these later stage cancers, TGFβ induced growth suppression is lost, and instead, TGFβ promotes tumor growth and metastasis.

Eli Lilly has developed and produced a Transforming Growth Factor-beta (TGF-β) receptor type-1 kinase inhibitor. LY2157299 monohydrate (LY2157299) is a small molecule that inhibits the TGF-β receptor type 1 kinase activity. LY2157299 was developed to investigate its activity in patients with glioblastoma where TGF-β has been demonstrated to play a specific role in tumor progression. In addition, LY2157299 was investigated in other patient populations, either as a stand-alone therapy or in combination with standard anti-tumor treatment regimens for indications including hepatocellular carcinoma and pancreatic cancer. Future investigations include indications with likely TGF-β associated pathway activation, such as melanoma, breast and prostate cancer as well as hematologic malignancies.

Arms: arm 1: Study Drug: Enrolled patients will receive 300 mg/day of LY2157299. LY2157299 will be administered as an oral drug tablet.

The study drug will be administered orally on a 28-day cycle (1 cycle=28 days), every 2 weeks, or 14 days on / 14 days off. Blood samples will be obtained at baseline, and weeks 2, 6 and 15 for immune monitoring.

Radiation therapy : Patients will receive Radiation therapy to a metastatic site at a dose of 7.5 Gy, given consecutively on days 1, 3 and 5, during Week 1 of their treatment.
Interventions: intervention 1: Radiation therapy intervention 2: Study Drug
Intervention descriptions: intervention 1: Imaging by PET/CT will be performed at baseline, 5 weeks and 15 weeks. The chosen metastatic sites will receive conformal external beam radiation 7.5 Gy/fraction x 3, to a total of 22.5 Gy over the course of one week. intervention 2: Patients will receive 300 mg/day of study drug administered via oral drug tablet every day for 14 days on and 14 days off (=28 day cycle) .

Protocol documents:
Prot_SAP_000.pdf: 
NCT# NCT02538471 
WCM IRB # 1505016222 
Title: LY2157299 Monohydrate (LY2157299) and 
Radiotherapy in Metastatic Breast Cancer 
Final Approved version date: 04.21.2017  
IRB approval date: May 19th, 2017. 
 
Document History 
 
Amendments  
Version  Version Date  
Protocol Amendment 4  
4.0  
04.21.2017  
Protocol Amendment 3  
3.0  
11.22.2016  
Protocol Amendment 2  
2.1  
07.18.2016  
Protocol Amendment 1  
2.0  
09.29.2015  
Initial Protocol  
1.0  
07.10.2015  


 


 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
           
 
 
 
 
 
     
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
     
 
 
 
 
 
 
 
 
     
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 


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Co-Investigators UCLA: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Study Contact at WCM:   
 
 
 
Sharanya Chandrasekhar 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Participating Sites:  1. New York-Presbyterian Weill Cornell Medicine 
                                       Stich Radiation N-046                                        
                                       525 East 68th street, 
                                       New York, NY – 10065 
 
 
                                   2. David Geffen School of Medicine at UCLA 
                                       10833 Le Conte Avenue 
                                       Los Angeles, CA 90095-1714 
 


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Confidentiality Statement 
 
This document is confidential and is to be distributed for review only to investigators, 
potential investigators, consultants, study staff, and applicable independent ethics 
committees or institutional review boards. The contents of this document shall not be 
disclosed to others without written authorization from WCM. 


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List of Abbreviations 
 
All abbreviations used throughout the protocol must be defined.  
 
1D11 
Murine monoclonal antibody against TGFΒ 
4T1 murine 
model 
murine breast tumor model 
ADL 
Activities of daily living 
AE 
Adverse event 
AE 
Adverse Event  
Akt  
Agammaglobulinemia Tyrosine Kinase) 
ATM 
Ataxia Telangectasia Mutated 
AUC 
Area Under the Curve - a measure of total drug concentration in  
blood plasma over a period of time 
BCC 
Basal Cell Carcinomas 
B-HCG 
Beta Human Chorionic Gonadotrophin 
BUN 
Blood Urea Nitrogen 
CAT 
Cambridge Antibody Technology 
CBC 
Complete blood count 
CHF 
Congestive Heart Failure 
CI 
Confidence interval 
CIN 
Cervical Intraepithelial Neoplasia 
Cmax 
Maximum concentration of drug 
CNS 
Central nervous system 
CR 
Complete response 
CRF 
Case report/Record form 
CTCAE 
Common Terminology Criteria for Adverse Events 
DC  
Dendritic Cells 
DDR 
DNA Damage Response 
DLT 
Dose Limiting Toxicity 
DSMB 
Data Safety Monitoring Board 
DTC 
Disseminated Tumor Cells 
ECG 
Electrocardiogram 
ECM 
Extra Cellular Matrix 
EGFR 
Epidermal Growth Factor Receptor  
ELISA 
Enzyme-Linked Immunosorbent Assay 


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EMT 
Epithelial-Mesenchymal Transdifferentiation 
ER 
Estrogen Receptor 
ERK  
Extracellular Signal-Regulated Kinase 
FSGS 
Focal Segmented Glomerulosclerosis 
GC1008 
Fresolimumab, human IgG4 kappa monoclonal antibody against TGFΒ 
GI 
Gastrointestinal 
Hct 
Hematocrit 
Hgb 
Hemoglobin 
HIV 
Human Immunodeficiency Virus 
HLA-A2 
MHC Class I, A cell surface antigen: Human Leukocyte Antigen serotype within HLA-A "A" 
serotype group  
HPF 
High-power field  
HTN 
Hypertensions 
IL-6 
Interleukin-6 
IPF 
Idiopathic Pulmonary Fibrosis 
IRB 
Institutional Review Board 
irRC 
Immune response criteria 
IV 
Intravenous 
JNK  
c-Jun N-terminal protein Kinase 
KA 
Keratoacanthoma 
Kd 
Dissociation Constant 
LAP 
Latency-Associated Protein 
LY2157299  LY2157299 Monohydrate 
MAPK  
Mitogen-Activated Protein Kinase 
MDA-MB-
231 
human breast cancer cell line 
MHC-I 
Major Histocompatibility Complex class I 
NCI 
National Cancer Institute 
NK 
Natural Killer Lymphocytes 
nM 
Nanomolar 
NOAEL 
No-Observed-Adverse-Effect Level 
OS 
Overall survival  
PBMC 
Peripheral Blood Mononuclear Cells 
PD 
Pharmacodynamic 
PFS 
Progression free survival  


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PI3K  
Phosphoinositide 3-Kinase 
PLT 
Platelet 
PO 
Per Os – orally 
PR 
Partial response 
PSA 
Prostate-Specific Antigen 
PTHrP 
Parathyroid Hormone-related Peptide 
RCC 
(RENCA) 
murine renal cell carcinoma 
RECIST 
Response evaluation criteria in solid tumors 
RT 
Radiation Therapy 
SAE 
Serious adverse event 
SC 
subcutaneous 
SCC 
Squamous Cell Carcinomas 
SD 
Stable disease 
Smad  
Sma- [small body size] and Mad-related protein 
sur1M2 
synthesized analogue of survivin in which a better anchor residue (methionine) 
 replaces the natural threonine at position 2 so as to better bind HLA-A2 
TRII:Fc 
soluble type II TGFΒ receptor:Fc fusion protein 
TBRS 
TGFβ gene Response Signature 
TGFΒ 
Transforming Growth Factor-beta 
TGFΒR 
Transforming Growth Factor-beta Receptor 
TNFR 
Tumor Necrosis Factor Receptor 
T-reg 
Regulatory T cells 
TTP 
Time to progression 
ULN 
Upper limit of normal 
USP 
United States Pharmacopoeia 
CFR 
Code of Federal Regulations 
CRF 
Case Report Form 
DSMB 
Data Safety Monitoring Board 
DSMP 
Data Safety Monitoring Plan 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HRBAF 
Human Research Billing Analysis Form 
ICF 
Informed Consent Form 
IND 
Investigational New Drug 


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IRB 
Institutional Review Board 
PHI 
Protected Health Information 
PI 
Principal Investigator 
REDC 
Research Electronic Data Capture 
SAE 
Serious Adverse Event 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
WCM 
Weill Cornell Medicine 
UAP 
Unanticipated Problem 
HIPAA 
Health Insurance Portability and Accountability Act of 1996 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Document History  
 
Amendments 
Version 
Version Date  
Protocol Amendment 4 
4.0 
04.21.2017 
Protocol Amendment 3 
3.0 
11.22.2016 
Protocol Amendment 2 
2.1 
07.18.2016 
Protocol Amendment 1 
2.0 
09.29.2015 
Initial Protocol 
1.0 
07.10.2015 
 
Summary of changes: Protocol Amendment 4 Version 4.0 dated 04.21.2017 
 
1. Removal of the following cardiotoxicity testing; hsCRP, Cystatin C and BNP. Due to a 
change in WCM processing procedures, hsCRP, cystatin C and BNP testing are no long 
being processed at WCM and are now being sent to an outside lab which takes 7-10 days to 
be resulted.  Due to the delay in receiving the results we are removing these labs so as to 
not delay the enrollment of metastatic breast cancer patients onto this study. 
 
2. Per DSMB recommendation, the Data Safety Monitoring Plan section 12.1 has been 
revised to reflect the study will be reviewed on an annual basis. 
 
 
3. Pre study and On study evaluations have been amended to include the following: 
A. Patients must have an MRI of the brain.  A Brain CT with contrast is also acceptable 
B. Patients must also have a PET/CT or a CT of the  Chest/Abdomen/ Pelvis 
C. Patients can begin two weeks from prior treatment if they are asymptomatic from 
previous therapy 
 
Summary of changes: Protocol Amendment 3 version 3.0 dated 11.22.2016 
 
1. DoD requested changes: Research Monitor responsibilities – Section 12. 
 
2. WCM DSMB recommendation for semi-annual review – letter dated 10.20.2016. 
 
Summary of changes: Amendment 2 dated 07.18.2016 
 
1. Data safety Monitoring board recommendations from 03.24.2016 were added to the 
protocol section 12.1.  
 
 
 
“The WCM DSMB is to perform a data and safety analysis every three months 
(quarterly) following the accrual of the first subject”.  
 
 
Summary of Changes: Amendment 1 09.29.2015 
 
1. Adding Maria Fenton-Kerimian as the Research Nurse 
 


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2. Data Safety Monitoring board review modifications  
 
 


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PROTOCOL SUMMARY 
 
TGFβ blockade will enhance response of irradiated tumors and improve the function of 
Dendritic and T cells. 
 
Full Title: 
 
 
 
LY2157299 Monohydrate (LY2157299) and Radiotherapy in 
Metastatic Breast Cancer 
Short Title:  
 
 
LY2157299 and Radiotherapy in Metastatic Breast Cancer 
Clinical Phase: 
 
 
Phase II 
Principal Investigator: 
Dr. Silvia C. Formenti, M.D.  
Sample Size: 
 
N= 28 (WCM will enroll a total of 18 patients and UCLA will enroll 
10 patients) 
Accrual Ceiling: 
 
35   
Study Population:  
Patients must have biopsy proven metastatic breast cancer and 
have stable disease or progressed after at least one course of 
chemotherapy or hormonal therapy. This study will not enroll 
vulnerable population. Women > 18 years and < 90 years. 
Accrual Period: 
 
3 years  
Study Design: 
 
TGFΒ blockade will enhance response of irradiated tumors and 
improve the function of Dendritic and T cells. Patients will receive 
300 mg/day of study drug administered via oral drug tablet every 
day for 14 days on and 14 days off (=28 day cycle). Radiation to a 
metastatic site will be delivered at a dose of 7.5 Gy, given 
consecutively on days 1-3-5. 
 Study Duration: 
 
Study participation will involve 15 visits over the course of 16 
weeks (approx.4 months). Patients will have an annual follow-up 
up to 5 years.  
Study Agent/  
Intervention Description: LY2157299 will be administered oral drug tablet: 300mg/day. 
Patients will receive 300 mg/day of study drug administered via 
oral drug tablet every day for 14 days on and 14 days off (=28 day 
cycle) .   
Primary Objective:  
1. to assess safety and feasibility of combining TGFβ receptor I 
kinase inhibitor LY2157299 and local radiotherapy  
 
 
2. To determine if treatment with TGFΒ receptor I kinase inhibitor 
LY2157299 and localized RT achieves an abscopal tumor 
regression; 
Secondary Objectives: 
1. to estimate the local response rate of combining TGFΒ receptor 
I kinase inhibitor LY2157299 and local radiotherapy 
 
 
2. To determine if treatment with TGFΒ receptor I kinase inhibitor 
LY2157299 and localized RT enhances tumor-specific immunity in 


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patients with metastatic breast cancer, and if this is associated 
with abscopal tumor regression. 
 
 
3. To determine if treatment with TGFΒ receptor I kinase inhibitor 
LY2157299 and localized RT alters the numbers and function of T-
reg cells in patients with metastatic breast cancer. 
 
Endpoints: 
            Primary Endpoint: A. To assess safety and feasibility of combining 
TGFβ receptor I kinase inhibitor LY2157299 and local radiotherapy  
 
 
B. To determine if treatment with TGFΒ receptor I kinase inhibitor 
LY2157299 and localized RT achieves an abscopal tumor 
regression  
 
 
Secondary Endpoint: A. to estimate the local response rate of 
combining TGFβ receptor I kinase inhibitor LY2157299 and local 
radiotherapy,  
 
 
B. to determine if treatment with TGFβ receptor I kinase inhibitor    
                                                      LY2157299 and localized RT enhances tumor-specific immunity  
     in patients with metastatic breast cancer, and if this is associated 
    with abscopal tumor regression.  
 
          C. to determine if treatment with TGFβ receptor I kinase inhibitor    
                                                  LY2157299 and localized RT alters the numbers and function of  
 T-reg cells in patients with metastatic breast cancer. 


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SCHEMA 
Patients with metastatic breast cancer receiving at least one single agent chemotherapy and 
demonstrating stable disease or disease progression at two consecutive clinical/radiological 
assessments (at an interval of at least 2 weeks). Stable disease is defined as: < 30% decrease in 
the sum of the longest diameter (LD) of target lesions, taking as reference the baseline sum LD or < 
20% increase in the sum of the LD of target lesions without the appearance of one or more new 
lesions. After obtaining informed consent: 
 
Baseline CT and PET scan 
Definition of target and non-target lesions: Non-target lesion is treated by radiation while Target 
lesions are followed to assess possible abscopal effect 
 
WEEK 1-2, DAY 1-14 
 
 
Administration of LY2157299   
 
 
(150 mg BID, = 300 mg/day) 
 
 
WEEK 1, DAYS 1, 3, 5 
 
RT to Non-target lesion #1 
 
7.5 Gy /fraction to a total dose of 22.5 Gy. 
 
  
WEEK 5, (+/-8 days) 
 
 
 
PET/CT evaluation.  
 
 
WEEK 5-6, DAY 1- 14 
 
 
Administration of LY2157299   
 
 
(150 mg BID, = 300 mg/day) 
 
 
WEEK 5, DAYS 1, 3, 5 
 
 
RT to Non-target lesion #2  
 
7.5 Gy /fraction to a total dose of 22.5 Gy. 
 
 
WEEK 9-10, DAY 1-14 
 
 
Administration of LY2157299   
 
 
(150 mg BID, = 300 mg/day) 
 
 
WEEK 13-14, DAY 1-14 
 
 
Administration of LY2157299   
 
 
(150 mg BID, = 300 mg/day) 
 
 
WEEK 15 (+/-8 days) 
 
 
 
PET/CT evaluation 
 
 
Baseline blood samples 
for immunological studies 
Week 2, day 14 blood samples 
for immunological studies 
Week 6, (day 42) blood samples 
for immunological studies 
Week 15 blood samples 
for immunological studies 
Week 9 (+/- 8 days) blood 
samples for pK values 


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TABLE OF CONTENTS 
 
Protocol Summary ............................................................................................... 12 
SCHEMA ............................................................................................................ 14 
1. 
STUDY OBJECTIVES .................................................................................... 18 
1.1. 
Primary Objectives ............................................................................. 18 
1.2. 
Secondary Objectives ........................................................................ 18 
2. 
BACKGROUND .............................................................................................. 18 
2.1 
 TGF β .................................................................................................. 18 
2.1.1 TGF β in Oncology: Rationale for Blocking TGF β signaling 
Therapy ................................................................................................... 20 
2.2 
 Investigational Agent or Device ....................................................... 21 
2.2.1 Pharmacokinetics and Pharmacology ........................................ 21 
2.2.2 Preclinical Studies ........................................................................ 24 
2.2.3   Toxicology Studies ..................................................................... 24 
2.2.4 Clinical Experience with LY2157299 ........................................... 25 
2.3 
 Rationale ............................................................................................ 26 
2.4 RT Dose Rationale .................................................................................. 27 
2.5  
Risk/Benefit Assessment .................................................................. 27 
2.6 
        Correlative Studies Background ............................................... 28 
2.6.1 TGF β gene response signature .................................................. 28 
2.6.2 Circulating levels of TGF β .......................................................... 28 
2.6.3 Tumor-specific immune responses ............................................ 28 
2.6.4   Levels of circulating Tregs ........................................................ 29 
3. 
SUBJECT SELECTION .................................................................................. 29 
3.1 
 Study Population ............................................................................... 29 
3.2 
Inclusion Criteria ................................................................................ 30 
3.3 
Exclusion Criteria ............................................................................... 31 
4 
REGISTRATION PROCEDURES ................................................................... 33 
4.1  
General Guidelines ............................................................................ 33 
4.2  
Patient Registration Process ............................................................ 33 
5.1 Investigational Agent or Device Administration ................................... 34 
5.2 Pretreatment Evaluations ....................................................................... 34 
5.3 On-Study Evaluations ............................................................................. 35 
5.4 General Concomitant Medication and Supportive Care Guidelines ... 37 
5.5 Duration of Therapy ................................................................................ 37 
5.6 Duration of Follow Up ............................................................................. 37 
5.7 Criteria for Removal from Study ............................................................ 37 
5.8 Alternatives ............................................................................................. 38 
5.9 Compensation ......................................................................................... 38 
5.10 Study Calendar ...................................................................................... 38 
6  Pharmaceutical Information ............................................................................ 39 


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6.1 
Investigational Agent ......................................................................... 39 
6.2 
Availability .......................................................................................... 39 
6.3 
Agent Accountability .......................................................................... 40 
7 DOSING Delays/Dose Modifications ................................................................ 40 
8 Adverse Events: List and Reporting Requirements ....................................... 40 
8.1 Adverse Events and Potential Risks List .............................................. 40 
8.1.1 Recording of Adverse Events ...................................................... 41 
8.1.2 Reporting of AE to WCM IRB ....................................................... 41 
8.2 Expedited Adverse Event Reporting ..................................................... 41 
8.3 Reporting Serious Adverse Events ....................................................... 42 
8.4   Routine Adverse Event Reporting Guidelines .................................... 43 
8.5   Safety Profile of LY2157299 ................................................................. 43 
9   Correlatives and Special Studies .................................................................. 44 
9.1 Laboratory Correlatives Studies ............................................................ 44 
9.1.1 TGF β gene response signature .................................................. 44 
9.1.2 Circulating Levels of TGF β ......................................................... 45 
9.1.3 Anti-angiogenic Activity of LY2157299 in vivo ........................... 45 
9.1.4 Tumor specific Immune responses ............................................. 45 
9.1.5 Levels of circulating Tregs .......................................................... 46 
9.2 Collection of Specimen(s) ...................................................................... 46 
9.3 Handling of Specimens .......................................................................... 47 
9.4 Storage of Samples ................................................................................ 47 
9.5 Shipping of specimens ........................................................................... 48 
9.6 Site(s) Performing Correlative Study .................................................... 48 
9.7 Coding of Specimens for privacy protection ........................................ 48 
10 Investigator Resources .................................................................................. 48 
10.1 Qualifications ........................................................................................ 48 
10.2 Location and Use of WCM and UCLA Facilities ................................. 49 
10.3 Conflict of Interest ................................................................................ 49 
11 Measurement of effect .................................................................................... 49 
11.1 Anti-tumor Effect ................................................................................... 50 
11.1.1 Definitions ................................................................................... 50 
11.1.2 Disease Parameters .................................................................... 50 
11.1.3 Methods for Evaluation of Measurable Disease ....................... 51 
11.1.4 Response Criteria ....................................................................... 52 
11.1.5 Response Review ....................................................................... 53 
12 Data Reporting/ Regulatory Considerations ................................................. 53 
12.1 
Monitoring plan .................................................................................. 54 
12.2 
Stopping rules (for the individual patient and for the study as a 
whole) ............................................................................................................ 54 
12.3 
Data Management .............................................................................. 54 
12.4 
Confidentiality .................................................................................... 55 
13 
Statistical Considerations ........................................................................ 56 


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14.1 Endpoints .............................................................................................. 56 
14.1.1 Primary endpoint ...................................................................... 56 
1. 
To assess safety and feasibility of combining TGF β receptor I kinase .. 56 
inhibitor LY2157299 and local radiotherapy ...................................................... 56 
14.1.2 Secondary endpoints ............................................................... 56 
APPENDIX 1     SPECIMEN COLLECTION PROCEDURES......................... 57 
1.2 Plasma, FROZEN (TGFβ1, VEGF) ................................................. 58 
References .................................................................................................... 59 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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1. 
STUDY OBJECTIVES 
 
 
 
1.1. 
Primary Objectives 
 
 
1.1.1 
To assess safety and feasibility of combining TGFβ receptor I kinase  
                                  Inhibitor LY2157299 and local radiotherapy  
 
1.1.2 
To determine if treatment with TGFβ receptor I kinase inhibitor  
 LY2157299 and localized RT achieves an abscopal tumor regression; 
 
1.2. 
Secondary Objectives 
 
 
                    1.2.1     To estimate the local response rate of combining TGFβ 
                                     Receptor I kinase inhibitor LY2157299 and local radiotherapy 
 
 
 
 
    1.2.2      To determine if treatment with TGFβ receptor I kinase inhibitor  
                                    LY2157299 and localized RT enhances tumor-specific immunity in       
                                          patients with metastatic breast cancer, and if this is associated with     
                                           abscopal tumor regression. 
 
 
 
 
    1.2.3. 
To determine if treatment with TGFβ receptor I kinase inhibitor 
LY2157299 and localized RT alters the numbers and function of T-reg 
cells in patients with metastatic breast cancer. 
 
2. 
BACKGROUND 
 
2.1 
TGF β 
 
Transforming growth factor-beta (TGFβ) is a pleiotropic cytokine which belongs to a superfamily of 
ligands, including bone morphogenetic proteins and activins [1-5].  Under normal conditions, 
members of the TGFβ family maintain homeostasis in many organ systems.  In normal and non-
cancerous cells, TGFβ limits the growth of epithelial, endothelial, neuronal, and hematopoietic cell 
lineages through anti-proliferative and apoptotic responses.  In addition, TGFβ exerts potent effects 
that influence immune function, cell proliferation/ functional differentiation, cell adhesion, 
extracellular matrix production, cell motility, angiogenesis, and cytokine production. 
TGFβ exists in 3 isoforms: TGFβ 1, β2, and β3. Each isoform is encoded by distinct, highly 
conserved genes and is a 25 kilodalton homodimeric, disulphide-bonded protein.  TGFβ members 


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are expressed in a tissue-specific and developmentally regulated fashion.  TGFβ 1 is expressed 
most commonly and is found in endothelial, hematopoietic, and connective tissues.  TGFβ 
2 is found primarily in epithelial and neuronal tissues, and TGFβ 3 resides in mesenchymal 
tissues.  In vitro, each has similar activities.  However, data from knockout mice suggest that each 
may be associated with distinct phenotypes. 
TGFβ is secreted by cells in a biologically inactive “latent form” by virtue of its association with 
latency-associated proteins (LAPs).  Much of the TGFβ /LAP “pro-drug” is stored in the extracellular 
matrix as a complex.  However, other notable sites exist, including platelet granules and the surface 
of certain cells such as regulatory T cells.  The mechanism of release of active TGFβ may allow for 
local control.  Activation can occur either under acidic conditions or through the action of proteases 
such as thrombospondin-1, plasmin, and prostate-specific antigen (PSA). 
TGFβ binds to cells via 3 major receptors:  TGFβ RI, TGFβ RII, and TGFβ RIII (transforming growth 
factor-beta receptor type I, type II, and type III).  The binding of TGFβ to receptors occurs in a 
specific sequence and results in a cascade of events leading to the formation of a receptor complex 
and the phosphorylation and activation of TβRI.  The receptor complex has serine/threonine kinase 
activity and can activate the Smad (Sma- [small body size] and Mad related protein) pathway by 
phosphorylation of Smad2 or 3.  A key event is the formation of an activated Smad2(3)/4 complex, 
which is then transported to the nucleus where it induces gene transcription.  This leads to a variety 
of effects on cell differentiation and growth. 
Although TGFβ/TGFβR/Smad is an essential pathway, TGFβ’s influence on cellular activities 
appears to be much more complex.  TGFβ also binds to other receptors, such as endoglin, a cell-
surface glycoprotein associated with proliferation of human endothelial cells and angiogenesis.  
Other signaling pathways, such as ERK (extracellular signal-regulated kinase), JNK (c-Jun N-
terminal protein kinase), MAPK (mitogen-activated protein kinase), PI3K (phosphoinositide 3-
kinase), Rho-kinase, Akt (agammaglobulinemia tyrosine kinase), and GTPases (guanosine 
triphosphatases) may also be involved.  In addition, other receptor/signal pathways may intersect 
with the Smad pathway – including estrogen receptor (ER), androgen receptor (AR), steroid, 
epidermal growth factor receptor (EGFR), and other TGFβ family members such as the activins.  
Because of these interactions, the overall effects of TGFβ cannot always be predicted based on 
examination of any single pathway such as Smad. 
 
 


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2.1.1 
TGF β in Oncology: Rationale for Blocking TGF β signaling Therapy 
 
TGFβ has been implicated as an important factor in the growth, progression, and metastatic 
potential of advanced cancers.  Although TGFβ has been shown to suppress the growth of epithelial 
cells in the early stages of tumor development (premalignant conditions), the effect on advanced 
cancers is more complex [1, 5-6].  Increased production of TGFβ has been found in many 
neoplasms such as breast, prostate, gastric, renal, and epidermal carcinomas, and elevated plasma 
TGFβ levels in patients have been correlated with advanced disease, metastases, and lower 
survival rates [7-13].  In these later stage cancers, TGFβ induced growth suppression is lost, and 
instead, TGFβ promotes tumor growth and metastasis. 
TGFβ can influence many aspects of cell physiology.  In the later stages of tumor development, 
TGFβ acts to promote tumor cell motility, migration, invasiveness, and metastasis through autocrine 
and paracrine effects.  TGFβ has direct actions on tumor cells and can induce an aggressive 
appearing, morphologic cell change to occur [14].  In fact, investigators have found that transformed 
epidermal cells grown in the presence of TGFβ acquire the ability to form spindle cell carcinomas 
when transplanted into animals [14].  These phenotypic changes following exposure to TGFβ are 
referred to as an epithelial-mesenchymal transition (EMT) and are characterized by a “fibroblast-
like”/spindle cell morphology, down regulation of E-cadherin and cytokeratin, loss of cell-cell 
junctions, and remodeling of the cytoskeleton.  As a consequence of these changes, TGFβ causes 
the cells to become more motile and to migrate.  These TGFβ-induced changes have been 
described in many different cancer models [6, 14-16]. 
In addition to the direct effects above, TGFβ can promote tumor growth and progression through 
paracrine functions.  TGFβ alters the tumor microenvironment to fulfill the space and nutrient 
requirements needed for the growth of primary and metastatic cancers.  This remodeling of the 
tumor stroma and environment occurs through the induction of angiogenesis, by increasing ECM 
deposition, and by inducing the production of factors such as parathyroid hormone-related peptide 
(PTHrP), which stimulates bone resorption by increased osteoclastic activity [5, 17-19].  Importantly, 
TGFβ can also deactivate the anti-tumor defenses of the hosts by suppressing the immune system. 
 With broad activity over natural killer (NK) cells, T cells, monocytes/ macrophages, and dendritic 
cells, TGFβ can affect the initiation and stimulation of both primary and secondary immune 
responses as well as suppress anti-tumor effector cells [20-25].  These autocrine and paracrine 
effects combine to make TGFβ a key factor in the promotion of tumor growth and metastasis. 
For these reasons, neutralizing or blocking TGFβ represents a unique method of intervening and 


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disrupting a key promoter of tumor growth and may provide a new therapeutic opportunity.  
Investigators have examined the anti-tumor activity of inhibiting anti-TGFβ soluble receptors.  These 
agents have demonstrated direct anti-tumor activity and have increased the survival of animals 
bearing a wide variety of neoplasms, including breast cancer, prostate cancer, melanoma, hepatic 
cancer, and pancreatic cancer [5, 20, 26-36].  Inhibition of soluble receptors can act through a 
variety of mechanisms including increased apoptosis of primary tumors and the prevention of tumor 
cell migration, ECM deposition, angiogenesis, bone remodeling, and interleukin-6 (IL-6) and PTHrP 
secretion.  Receptor kinase inhibitors can also reverse TGFβ induced immune suppression and can 
activate important NK and T cell mediated anti-tumor responses [20, 22-23, 25, 37].  All of these 
effects contribute to the anti-tumor activity of blocking TGFβ. 
 
2.2 
Investigational Agent or Device 
 
Description of LY2157299 
 
Eli Lilly has developed and produced a Transforming Growth Factor-beta (TGF-β) receptor type-1 
kinase inhibitor. LY2157299 monohydrate (LY2157299) is a small molecule that inhibits the TGF-β 
receptor type 1 kinase activity. LY2157299 mechanism of action is determined by changes in 
phosphorylated SMAD (pSMAD) levels, with subsequent anti-tumor activity demonstrated in 3 in-
vivo tumor animal models: 2 breast cancer (MX1, 4T1) and one non-small cell lung cancer (NSCLC) 
model (CALU6). 
LY2157299 was developed to investigate its activity in patients with glioblastoma where TGF-β has 
been demonstrated to play a specific role in tumor progression. In addition, LY2157299 was 
investigated in other patient populations, either as a stand-alone therapy or in combination with 
standard anti-tumor treatment regimens for indications including hepatocellular carcinoma and 
pancreatic cancer. Future investigations include indications with likely TGF-β associated pathway 
activation, such as melanoma, breast and prostate cancer as well as hematologic malignancies. 
 
2.2.1 
 
 
 


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2.2.2 Preclinical Studies 
 
Pharmacokinetic and toxicology exposure and metabolism studies were conducted in Fischer 
344 rats and beagle dogs utilizing LY2157299. The compound is well absorbed after oral 
administration and is dose linear up to 250 mg/kg in rats and 20 mg/kg in dogs. In rats, exposure 
was higher in females than those in males, but no discernable difference in exposure between 
male/female dogs. LY2157299 is extensively metabolized, with 2 oxidative pathways accounting 
for most of the metabolic activity. The parent compound is the major circulating material. Protein 
binding is not high, 56 – 68% in mouse, rat and dog plasma, 66% in human plasma. In [14C]-
LY2157299, the majority of the radioactivity was present in feces or bile of the rats or dogs, with 
<25% of the dose recovered in urine including <10% of the parent compound. 
2.2.3   Toxicology Studies 
 
Eli Lilly and Company has performed toxicology studies in rodents and dogs.  Details of each 
of these studies can be found in the Investigator Brochure. 
In 6-month long-term nonclinical toxicity studies, daily dosing of LY2157299 resulted in dose-
progressive incidence of vascular degeneration to the base of the aorta in dogs and rats. 
Vascular changes led to transmural aneurysms and mortality in mid-high dose-treated rats. Due 
to the possibility of aneurysm formation, patients with a history of or predisposition of developing 
aneurysms should not receive LY2157299. Subsequent cardiovascular function studies carried 


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out in dogs revealed that LY2157299 can cause a decrease in blood pressure and increases in 
heart rate and arterial pulse pressure, all reversible. There were no compound-attributable 
changes in QT intervals, arrhythmias, or altered waveforms noted. Because of the associated 
cardiac events in animal studies, patients on LY215799 undergo cardiac monitoring, including 
assessment of heart rate, blood pressure and cardiac functions. Regular monitoring of 
biomarkers for cardiac events, including troponin-I, B type natriuretic peptide and high sensitivity 
C-reactive protein is required..TGF- is an important immunomodulator, hence, careful 
monitoring of immune functions and infection rates is important. Because LY2157299 inhibits 
TGF-receptor type-1 kinase, an autoimmune reaction may occur. To date, no clinically 
significant immune reactions have been reported or observed up to doses of 300 mg/day in a 14 
days-on / 14 days-off treatment regimen. 
LY2157299 should not be administered to pregnant patients or all efforts should be made to 
prevent pregnancy due to TGF-’s role in embryogenesis. Literature searches as well as 
treatment with LY2157299 in pregnant rats showed increased resorptions and alterations in fetal 
skeletal morphology at dose equivalents of 300 mg/day in humans. 
LY2157299 does not demonstrate any direct bone marrow toxicity in either animal studies with 
continuous dosing for 6 months, nor in human colony-forming unit studies with human bone 
marrow progenitor cells. There have been 2 cases with drug-related severe thrombocytopenia 
and associated pan-cytopenia observed, but they cannot be explained at this juncture. 
Thromboembolic events are common events in cancer, present in up to 70% of glioblastoma 
cases. Whenever they occurred, embolic events detected during administration of LY2157299 in 
glioblastoma patients were successfully treated with low-molecular heparins, with LY2157299 
not affecting treatment interventions for embolic events. 
2.2.4 Clinical Experience with LY2157299 
To date, approximately 406 patients have been treated with LY2157299, with no definite drug-
related toxicity profile emerging. The potential risks outlined in the Investigator’s Brochure 
provided by Lilly are based upon the assumed mechanism of action for LY2157299 and the 
reported cases where LY2157299-associated toxicity was found to likely be the related cause 
due to lack of other contributing factors. 


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2.3 
Rationale 
 
Five years ago, Drs. Formenti, Demaria and McBride argued for a revival of the notion of combining 
RT and IT in the treatment of cancer [45], the basic concept of this trial. The rationale is based on 
findings that radiation generates “danger” signals in tissues [46] that, under certain conditions, 
enhance immune presentation of tumor antigen liberated from radiation-damaged cells, thus 
working as a vaccine.  This hypothesis stems from the ideas of Matzinger that the immune system is 
more concerned with responding to damage than to “foreignness” [47].  Radiation elicits damage 
signals, by up-regulating pro-inflammatory cytokines that are involved in DC maturation, 
immunomodulatory molecules such as major histocompatibility complex class I (MHC-I), co-
stimulatory and adhesion molecules, death receptors of the tumor necrosis factor receptor (TNFR) 
family, and heat shock proteins (reviewed in [46]). McBride’s lab showed that irradiation of DC 
greatly enhances their ability to cross-present tumor antigen, a relevant immunological pathway 
during RT [48]. 
Increased production of TGFβ has been found in many neoplasms such as breast, prostate, gastric, 
renal, and epidermal carcinomas, and elevated plasma TGFβ levels in patients have been 
correlated with advanced disease, metastases, and lower survival rates [7-13]. TGFβ can deactivate 
the anti-tumor defenses of the host by suppressing the immune system.  With broad activity over 
natural killer (NK) cells, T cells, monocytes/macrophages, and dendritic cells, TGFβ can affect the 
initiation and stimulation of both primary and secondary immune responses as well as suppress anti-
tumor effector cells [20-25]. 
Barcellos-Hoff’s lab and other groups have shown that ionizing radiation induces TGFβ activation in 
vitro and in vivo in normal and cancer cells [49-55], which is at least in part occurring through a 
redox mechanism that acts directly on the secreted latent protein [56-57].  Moreover, TGFβ is 
actually required for execution of the epithelial damage response to radiation [58-59]. Dr. Barcellos-
Hoff’s group made the surprising discovery that either genetic loss or pharmaceutical inhibition of 
TGFβ inhibits ataxia telangiectasia mutated (ATM) kinase activity, thereby impeding the DNA 
damage response (DDR) and as a consequence, increasing radiation cell kill [59].  In addition, 
LY2109761, a similar compound to LY2157299 was evaluated in radiation-induced fibrosis models 
and showed a reversal of TGF-β-associated pro-inflammatory responses. This suggests that small 
molecule inhibitors of the TGF-bRI kinase may have a beneficial impact on TGF-β-mediated 
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Recent studies underscore the growing concern that high TGFβ levels in tumors drive cancer cells 
towards a more aggressive behavior and support their survival, while simultaneously limiting 
immune-responses by the host and, possibly, co-opting normal tissue functions like angiogenesis. 
Together with the recognition that radiation triggers activation of TGFβ, which in turn promotes DNA 
damage repair, and mediates EMT [60] these results suggest a novel therapeutic advantage for 
TGFβ inhibition in the context of radiotherapy. 
 
2.4 RT Dose Rationale  
 
RT to a measurable metastatic site will be delivered at a dose of 7.5 Gy X 3 (22.5 Gy total dose), 
given consecutively on days 1-3-5, by intensity modulated radiotherapy (IMRT) and/or image guided 
radiotherapy (IGRT) techniques, to best spare the normal tissue. The choice of this fractionation 
regimen of RT is based on the NYU preclinical experience of radiation and ipilimumab [61], that 
suggests superiority of the  8 Gy x 3 fractions regimen (for a total dose of 24 Gy) to induce abscopal 
effects in murine cancer models. In humans, the biological equivalent can be calculated by applying 
the linear quadratic model [62] to determine which dose delivered in 3 fractions  would results in 
comparable tumor control to that of the standard regimen of 3 Gy x 10 fractions (total dose 30 Gy) 
commonly used in the clinic to palliate metastatic disease. The α/β is a tissue and effect-specific 
parameter associated with this conversion model, which α/β is taken to be 10 Gy for cancer 
metastases [63-65]. As demonstrated in Table 1, the closest equivalence is achieved by 7.5 Gy x 3, 
which explains the choice for this trial. Importantly a 3 fraction regimen is very user-friendly (only 3 
visits to radiation oncology). Both UCLA and WCM are equipped to provide Intensity Modulated RT 
(IMRT) and, if necessary Image guided RT (IGRT), if the site targeted is close to vital organs. 
 
Table 1. Biologically effective dose (BED) for different fractionation regimens of radiotherapy 
 
8 Gy x 3 
6 Gy x 5 
7 Gy x 3 
7.25 Gy x 3 
7.5 Gy x 3 
3 Gy x 10 
standard dose 
(= 10 Gy) 
43.2 
48 
35.7 
37.5 
39.4 
39 
 
 
2.5  
Risk/Benefit Assessment  
 
 
The currently available information on the toxicology profile of LY2157299 is quite favorable, 
and the dose and fractionation of radiotherapy used in this study is unlikely to be associated 
with severe side effects. Thus the combination represents a viable alternative for metastatic 
breast cancer patients interested in investigational immunotherapy approaches. 


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2.6        Correlative Studies Background 
 
2.6.1 TGF β gene response signature 
 
A well-defined and robust TGFβ gene response signature (TBRS) [66] was strongly associated with 
three specific subsets of human breast cancer [67]. These findings suggest that the TGFβ pathway 
is activated in particular breast cancer subgroups and may predict for response to TGFβ 
neutralization, a concept that will be tested in this study. We plan to assess TGFβ gene response 
signature (TBRS) positivity in primary tumors (from blood cells and formalin-fixed and paraffin-
embedded archival tissue samples) as predictive biomarker of response to LY2157299 plus 
radiation therapy combination therapy. Based on past experience, we assume that approximately 
50% of subjects will have TBRS-positive tumors. Subjects who have TBRS-positive tumor and 
derive clinical benefit will be considered true positive. Sensitivity, specificity, positive and negative 
predictive value will be estimated. 
2.6.2 Circulating levels of TGF β  
 
As a biomarker, many studies have suggested that circulating TGFβ levels provide an index of 
tumor response to RT and may predict normal tissue toxicity. We will monitor circulating levels of 
TGFβ prior to, during and after radiation therapy in patients with metastatic cancer before and 
after LY2157299 treatment. Measuring TGFβ presents several challenges. First, TGFβ is 
produced and circulates in a latent form that is abundant, but its activity is highly regulated and 
short-lived.  Second, the low levels of active TGFβ present in serum can easily be obscured by 
the potentially high background of TGFβ unintentionally released by platelet degranulation in 
specimens. Lastly, most methods require exogenous latent TGFβ activation by acid or heat, 
which compromises the simultaneous detection of other cytokines. These factors contribute to 
erratic measurements by most commonly used assays, including the widely used ELISA. We will 
measure TGF-β1 levels in EDTA and/or CDTA from collected plasma utilizing an ELISA design 
developed by Lilly Research Laboratories. 
2.6.3 Tumor-specific immune responses  
 
Tumor-specific responses will be assessed initially in HLA-A2+ cells that will be re-stimulated ex 
vivo with sur1M2 survivin peptide and additionally, for HER2neu positive tumors, the HER: p369-
377 peptide. Non-HLA-A2 patients will be tested using peptides immunodominant for their 


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known HLA type. Quite a range of known epitopes is available [68-69]. While it is unrealistic to 
expect that all responses will be detected with equal sensitivity as this is influenced by the 
binding avidities, positive longitudinal responses to any peptide with time after treatment would 
indicate significant responses. In a follow-up assay, if sufficient PBMCs are available, the ability 
of LY2157299 plus RT to alter the hierarchy of the response and to determine whether 
responses to all epitopes are equally affected will be tested using HLA-A2+ PBMCs that respond 
to sur1M2 survivin but with 4 distinct survivin peptides [70]. We anticipate that LY2157299 plus 
RT will enhance tumor-specific immune responses and cause epitope spreading in the majority 
of patients. 
             
 
 2.6.4   Levels of circulating Tregs 
 
The levels of circulating Tregs will be evaluated in patients. Tregs are especially relevant for the 
study because of their relative radiation resistance and because of the importance of TGF- in 
their generation and function. Because the composition of PBMCs changes during cancer 
treatment [71], it is important that Treg levels are assessed both as a % of CD4 cells, as a % 
total lymphocytes, % total PBMCs, and as absolute counts.  The assay can be performed at the 
same time as tetramer staining and on the same sample if necessary (7), enabling the 
assessment of lymphocyte representation in one sample and the determination of CD4:CD8 T 
cell ratios. Since Tregs are radiation resistant, if significant differences in response are found 
between patients, efforts could also be made to correlate changes with dose volume histograms 
obtained during RT. It is anticipated that Treg numbers and function will decrease in patients 
receiving LY2157299 plus RT, in particular in those who show enhanced tumor immunity and 
demonstrate abscopal anti-tumor effects. In general, RT increases Treg activity, but it is 
expected that LY2157299 will counter this effect allowing immune activation. 
3. 
SUBJECT SELECTION 
 
3.1 
Study Population 
 
Patients must have biopsy proven metastatic breast cancer and who have stable disease or 
progressed after at least one course of chemotherapy or hormonal therapy. In addition, patients 


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must have at least 3 metastases with at least one measurable metastatic lesion and who meet 
the inclusion and exclusion criteria will be eligible for study participation. 
3.2  
Inclusion Criteria 
  
3.2.1  Biopsy proven breast carcinoma which is persistent and metastatic or     
      recurrent and metastatic. 
3.2.2  Patients must have failed at least one line of chemotherapy for metastatic   
    disease.  
3.2.3  Patients who are HER2+ as defined by ASCO CAP guidelines must have  
    failed all prior therapy known to confer clinical benefit  
3.2.4   Patients must have at least 3 distinct metastatic sites with at least one    
    measurable lesion which is at least 1 cm or larger in largest diameter. 
3.2.5 
At the time of enrollment, patients must be ≥ 4 weeks‡ since all of the 
following treatments (and recovered from the toxicity of prior treatment to  
Grade 1, exclusive of alopecia): 
• 
major surgery; 
• 
radiotherapy; 
• 
chemotherapy (note: must be ≥ 6 weeks since therapy if treated with a 
nitrosourea, mitomycin, or monoclonal antibodies such as 
bevacizumab);  
• 
immunotherapy; 
• 
Biotherapy/targeted therapies. 
 (‡ - Patients can begin two weeks from prior treatment if they are 
asymptomatic from previous therapy) 
3.2.6 
Patient ≥ 18 years of age. 
3.2.7 
Patient life expectancy > 6 months. 
3.2.8 
ECOG status of  0 or 1 
3.2.9 
Adequate organ function including: 
a) Marrow:  Hemoglobin   10.0 g/dL, absolute neutrophil count 
(ANC)  1,500/mm3, and platelets  100,000/mm3. 
b) Hepatic:  Serum total bilirubin  1.5 x upper limit of normal (ULN) 
     (Patients with Gilbert’s Disease may be included if their total bilirubin is  
3.0 mg/dL), alanine aminotransferase (ALT), and aspartate aminotransferase 
(AST)  2.5 x ULN.  If the patient has known liver metastases, an ALT and/or 
AST  5 x ULN are allowed. 
c) Renal: Estimated or measured creatinine clearance  60 mL/min. 
d) Other:  Prothrombin time (PT) and partial thromboplastin time (PTT) < 
ULN. 
3.2.10 Patients must have negative tests (antibody and/or antigen) for hepatitis  


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      viruses B and C unless the result is consistent with prior vaccination or prior  
      infection with full recovery. 
 
3.2.11 Male and female patients of child-producing potential must agree to use   
            effective contraception while enrolled on study and receiving the   
            experimental drug, and for at least 3 months after the last treatment. 
 
 
 
 
 
3.2.12 Ability to understand and the willingness to sign a written informed  
                              consent document. Patients may not be consented by a durable power of   
                              attorney. 
 
 
 
 
 
3.3  
Exclusion Criteria  
 
 
 
3.3.1 Patients diagnosed with another malignancy – unless following curative  
intent therapy, the patient has been disease free for at least 2 years and the 
probability of recurrence of the prior malignancy is  5%.  Patients with 
curatively treated early-stage squamous cell carcinoma of the skin, basal cell 
carcinoma of the skin, or cervical intraepithelial neoplasia (CIN) are eligible 
for this study. 
3.3.2 Concurrent cancer therapy is not permitted. 
3.3.3 Uncontrolled central nervous system (CNS) metastases, meningeal  
          carcinomatosis, malignant seizures, or a disease that either causes or     
          threatens neurologic compromise (e.g., unstable vertebral metastases). 
3.3.4 History of ascites or pleural effusions, unless successfully treated. 
3.3.5 Patients with an organ transplant, including those that have received an  
          allogeneic bone marrow transplant. 
3.3.6 Patients on immunosuppressive therapy including: 
a) Systemic corticosteroid therapy for any reason, including replacement 
                        therapy for hypoadrenalism.  Patients receiving inhaled or topical  
corticosteroids may participate (if therapy is < 5 days and is limited to 
systemic steroids as antiemetics). 
b) Patients receiving cyclosporine A, tacrolimus, or sirolimus are not   
eligible for this  study. 
3.3.7 Use of investigational agents within 4 weeks prior to study enrollment  
          (within 6 weeks if the treatment was with a long-acting agent such as a   
 
 
 monoclonal antibody). 


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3.3.8  Patients with moderate or severe cardiac disease: 
a)  have the presence of cardiac disease, including a myocardial infarction 
within 6 months prior to study entry, unstable angina pectoris, New York 
Heart Association Class III/IV congestive heart failure, or uncontrolled 
hypertension. 
b)  have documented major electrocardiogram (ECG) abnormalities (not 
responding to medical treatments) at the investigator’s discretion (for 
example, symptomatic or sustained atrial or ventricular arrhythmias, 
second- or third-degree atrio ventricular block, complete bundle branch 
block, ventricular hypertrophy, or recent myocardial infarction). 
c)   have major abnormalities documented by echocardiography (ECHO) with 
Doppler (for example, moderate or severe heart valve function defect 
and/or left ventricular ejection fraction <50%, evaluation based on the 
institutional lower limit of normal). For additional details, refer to ECHO 
protocol. 
d) have predisposing conditions that are consistent with development of 
aneurysms of the ascending aorta or aortic stress (for example, family 
history of aneurysms, Marfan-Syndrome, bicuspid aortic valve, evidence 
of damage to the large vessels of the heart documented by computed 
tomography (CT) scan with contrast). 
3.3.9   Troponin I above ULN,  
3.3.10  Patients with a remote history of asthma or active mild asthma may 
            participate. 
3.3.11 Active infection, including unexplained fever (temperature > 38.5oC). 
3.3.12  Systemic autoimmune disease (e.g., systemic lupus erythematosus,  
            active rheumatoid arthritis, Marfan Syndrome, etc.). 
3.3.13 A known allergy to any component of LY2157299. 
3.3.14 Patients who, in the opinion of the Investigator, have significant medical  
            or psychosocial problems that warrant exclusion.  Examples of significant 
            problems include, but are not limited to: 
a) 
Other serious non-malignancy-associated medical conditions that  
          may be expected to limit life expectancy or significantly increase   
          the risk of SAEs. 


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b) 
Any condition, psychiatric, substance abuse, or otherwise, that, in 
the opinion of the Investigator, would preclude informed consent, 
consistent follow-up, or compliance with any aspect of the study 
3.3.15  Pregnant or nursing women, due to the unknown effects of LY2157299  
            on the developing fetus or newborn infant. 
 
4 
REGISTRATION PROCEDURES 
 
 
4.1  
General Guidelines  
 
All eligible patients who are referred to the Radiation Oncology or Medical Oncology Department 
at Weill Cornell Medicine for radiation will be offered the opportunity to participate in this 
experimental protocol. Treatment may commence once all mandatory pretreatment studies are 
documented, informed consent is obtained and the patient is approved according to the eligibility 
checklist. Eligibility will be determined by the investigators of the study. 
4.2  
Patient Registration Process  
 
Before any protocol specific procedures can be carried out, investigators/staff will fully explain 
the details of the protocol, the study procedures and the aspects of patient privacy regarding 
research information. Patients will be provided a comprehensive explanation of the proposed 
treatment including the type of therapy, the rationale for treatment on the protocol, alternative 
treatments that are available, any known adverse events, the investigational nature of the study 
and the potential risks and benefits of the treatment. The informed consent document will meet 
all requirements of the Institutional Review Board (IRB). All subjects/patients are informed in the 
consent that participation or refusal to participate in the research study will not affect any of the 
clinical treatment or services to which they would otherwise be entitled. 
The physicians who may obtain informed consent are listed on the title page of this protocol. The 
informed consent form will be signed by the participant and the registering physician. Once 
signed, a copy will be given to the patient and one will be maintained with the patient’s medical 
record. Once eligibility is confirmed and informed consent is documented, the patient will be 
registered by the study coordinator/data manager. 


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Patients will be centrally registered with the Office of Billing Compliance. To register a 
patient, submit the following documents via the JIRA Registration Process:  
1. Legible copy of the HRBAF 
2. First and last page of signed informed consent form  
    Registration must be completed within 24 hours of the signing of informed consent.  
 
5.  
TREATMENT PLAN 
 
5.1 Investigational Agent or Device Administration 
 
Enrolled patients will receive 300 mg/day of LY2157299. LY2157299 will be administered as 
an oral drug tablet.   
The study drug will be administered orally on a 28-day cycle (1 cycle=28 days), every 2 weeks, 
or 14 days on / 14 days off. Blood samples will be obtained at baseline, and weeks 2, 6 and 15 
for immune monitoring. Imaging by PET/CT or CT(Chest/Abdomen/Pelvis) will be performed at 
baseline, 5 weeks and 15 weeks. The chosen metastatic sites will receive conformal external 
beam radiation 7.5 Gy/fraction x 3, to a total of 22.5 Gy over the course of one week (on 
alternating days): 
• lesion 1 will be irradiated at week 1 (RT starts 1 day after 1st dose of LY2157299), 
• lesion 2 will be irradiated at week 5 (RT starts  after 1stdose of  the second cycle 
of LY2157299) 
5.2 Pretreatment Evaluations  
 
Screening and Baseline Evaluations/Procedures 
These evaluations will be carefully reviewed to determine the patient’s eligibility for the 
study (see study calendar, section 5.10). 
1. Signed, written informed consent:  Consent must be completed prior to 
performing any study-related procedures  
2. Complete physical examination including height, weight and full skin exam. 


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3. Medical history:  Detailed documentation of demographics, disease and treatment 
history with outcomes. 
4. ECOG performance status assessment. 
5. Concurrent medications. 
6. Echo / Doppler. 
7. Hematology:  CBC with differential and platelet count and peripheral blood smear. 
Coagulation (PT/INR, PTT); Serum chemistries:  Electrolytes (sodium, potassium, 
chloride, and bicarbonate), blood urea nitrogen (BUN), creatinine, glucose, and liver 
function tests (aspartate aminotransferase (AST) and/or alanine aminotransferase 
(ALT), alkaline phosphatase (ALP), total bilirubin, lactate dehydrogenase (LDH), and 
Troponin I.. 
8. Tumor measurement at baseline by PET/CT or CT(chest/abdomen/pelvis). 
9. MRI or Brain CT with contrast is required at baseline  
10. Stool fecal occult blood test of 2 consecutive samples: if patients have positive 
fecal occult blood, full GI workup will be conducted and patients will be excluded 
if active bleeding is detected. 
11. LY2157299 Serum pregnancy test for women of childbearing age. 
 
5.3 On-Study Evaluations  
 
(See study calendar, section 5.10) 
1. Physical exam: including complete skin evaluation and evaluation of oral mucosa 
prior to start of each RT cycle and prior to each LY2157299 administration. 
2. Hematology: CBC with differential and platelet count and peripheral blood smear. 
PT/INR,PTT should be evaluated following multiple doses of LY2157299; Serum 
chemistries: Electrolytes (sodium, potassium, chloride, and bicarbonate), BUN, 
creatinine, glucose, and liver function tests (AST, ALT, ALP, total bilirubin, LDH, 
and Troponin I C). These will be carried out prior to start of each RT cycle and 
prior to each cycle of 2 weeks of BID LY2157299 administration. 
3. ECHO/Doppler : Week 8 within 5 days prior to starting week 9 LY2157299 
administration and Week 15 
4. Study Special Tests: TGFβ levels (at wk 1 day -1 and week 15 visit), immune 
evaluation at weeks 1 day -1, 2, 5 and 15 LY2157299 PK (pre- & after completion 
of drug cycle day 14 & at day 42). 
5. Tumor measurement by PET/CT/ CT(chest/abdomen/pelvis) at weeks 5 and 15. 
6. Adverse events evaluated and recorded using the NCI CTCAE version 
4.0throughout the treatment period (weeks 0-15). 
 


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To date, LY2157299 at the planned dose for this study is well tolerated in patients. If a toxicity is 
observed, a dose reduction may be considered based on the aggregate safety information of 
LY2157299, if other plausible reasons for a toxicity have been excluded. 
Specifically, no bone marrow toxicities were detected in nonclinical toxicology studies; 1 toxicity 
was reported in a patient with glioblastoma in whom a Grade 4 thrombocytopenia was observed 
and this toxicity was potentially associated with administration of LY2157299 monotherapy. If a 
similar case occurs, the drug should be discontinued until platelet counts either return to Grade 
1 or baseline.  
If a patient experiences any of the following events that are considered possibly related to 
LY2157299 and not to study disease, LY2157299 will be omitted until the event resolves: 
 
• 
Grade 4 Thrombocytopenia 
• 
Grade 3 thrombocytopenia associated with bleeding 
• 
Grade 4 Anemia 
• 
absolute neutrophil count (ANC) <0.5 x 109/L for longer than 7 days, or ANC <1.0 x 109/L 
      with a single temperature of >101ºF/38.3ºC or a sustained temperature of >100.4ºF/38ºC 
for more than 1 hour, or platelet count <25,000 x 109/L 
• 
Common Terminology Criteria for Adverse Events Grade 3 or 4 non-hematologic toxicity 
Non-hematologic and hematologic toxicity must resolve to CTCAE Grade 0, 1, or baseline (with 
the exception of alopecia, fatigue, skin rash, nausea, vomiting, constipation, or diarrhea that can 
be controlled with treatment) before start of next cycle. If recovery is >29 days (Day 1of the next 
cycle), patient must be withdrawn from study treatment. 
 
If moderate or severe heart valve toxicities are observed, or the patient develops aneurysms of the 
great vessels of the heart, the patient must immediately be discontinued from the treatment and the 
toxicity reported to sponsor within 24 hours (see references on the ECHO assessment based on the 
Guidelines of the American and European Societies of Cardiac Echocardiography). If the patient 
develops new cardiovascular lesions in the chest, an MRI or CT with contrast of the abdomen 
should be performed to evaluate for development of abdominal vascular lesions. Exceptions to this 
rule must be approved by the IRBs and sponsor. 


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5.4 General Concomitant Medication and Supportive Care Guidelines 
 
No concomitant cancer therapies, immunosuppressive agents or anti-coagulation therapies are 
permitted during the study unless specified below.  Inhaled or topical corticosteroids are allowed.  
Use of alternative medications (herbals, botanicals, etc.) is strongly discouraged during the 
entire study period.  In addition, starting new medications other than anti-cancer agents may be 
associated with side effects; therefore, medications should not be started for the first time during or 
around the time of LY2157299 administration. 
 
 
5.5 Duration of Therapy 
 
Patients completing the week 15 visit will have completed the study treatment.  
 
5.6 Duration of Follow Up 
 
Patients will be followed with follow-up visits monthly for the first three months after completing 
therapy then annually for 5 years with physical exam, performance status evaluation, labs and 
Echocardiography and Doppler (see study calendar, section 5.10). 
5.7 Criteria for Removal from Study 
 
Patients may withdraw consent and discontinue participation in the study at any time, without 
prejudice to further treatment. A patient’s participation in the study may also be discontinued at any 
time at the discretion of the Investigator. 
 
The following are reasons why the Investigator may remove a patient from study treatment and 
further follow-up: 
• 
The patient withdraws consent; 
• 
The patient is found to be not eligible after enrollment; 
• 
The patient is non-compliant with study requirements; 
• 
The Study is terminated 
 
The following are reasons why the Investigator or Sponsor may remove a patient from study 
treatment but continue follow-up: 
• 
General or specific changes in the patient’s condition render the patient   
• 
 unacceptable for further treatment per the investigator’s judgment; 


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• 
The patient becomes pregnant during the study (note: patients who become 
pregnant during the study must be followed throughout their pregnancy; it will also 
be important to follow any infants exposed to LY2157299 during fetal 
development); 
• 
The patient suffers an intolerable AE; 
• 
The patient develops progressive disease (progression at week 5 PET/CT or 
CT(chest/abdomen/pelvis) does not warrant study discontinuation if clinically 
stable);  
• 
The patient requires a prohibited medication; 
• 
There is new information to suggest a significant change in risk/benefit for  
          LY2157299;  
 
5.8 Alternatives 
 
Standard of care treatment will be offered to all eligible patients. 
 
5.9 Compensation  
 
Patients will receive no compensation for participating in the trial. 
 
 
 
5.10 Study Calendar  
 
   
Pre-
Stu
dy 
Wk 1 
(Day -
1) 
0 
Wk 
1 
(Day 
+1) 
(+/- 
3day
s) 
Wk 
2 
(+/- 
3 
days) 
Wk 
3 
(+/- 
3 
days) 
Wk 
4 
(+/- 
8 
days) 
Wk 
5 
(+/- 
3 
days) 
Wk 
6 
(+/- 
3 
days) 
Wk 
7 
(+/- 
3day
s) 
Wk 
8 
(+/- 
3 
days) 
Wk 
9 
(+/- 
8 
days) 
Wk 
10 
(+/- 
3 
days) 
Wk 
11 
(+/- 
3 
days) 
Wk 
12 
(+/- 
3 
days) 
Wk 
13 
(+/- 
3 
days) 
Wk 
14 
(+/- 
3 
days) 
Wk 
15 
(+/- 
8 
days
) 
 
Follo
w-up 
Investigational Agent 
LY2157299 a 
 
 
X 
Xa,d  
  
 
X 
Xa,d 
 
 
Xa,d 
Xa,d 
 
 
Xa,d 
Xa,d 
 
 
RT 
 
 
Xd 
 
 
 
Xd 
 
 
 
 
 
 
 
 
 
 
 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
EKGb 
Xb 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Concurrent meds 
X 
X-----------------------------------------------------------------------------------------------------------------------X 
 
B-HCGc 
Xc 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Performance status 
X 
X 
 
X 
 
X 
 
X 
 
 
 
 
 
X 
 
 
Xd 
X 
Physical examd  
(Vital signs) 
Xd 
Xd 
 
Xd 
 
Xd 
 
Xd 
 
Xd 
 
Xd 
 
Xd 
 
 
Xd 
X 
CBC w/diff, plts, Serum 
chemistrye,  
CEA/CA 27.29f 
Xe, f 
Xe 
 
Xe 
Xe 
 
Xf 
Xe 
 
Xe 
Xe 
 
 
Xe 
 
 
 
Xe f. 
 
PT/INR/PTT 
X 
X 
 
X 
X 
 
 
X 
 
X 
X 
 
 
X 
 
 
X 
 
Hepatitis B and C HbSAg, 
HCV Ab (antibody and/or 
antigen) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Stool fecal occult blood test 
(2 consecutive)  
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Adverse event evaluation 
 
X-----------------------------------------------------------------------------------------------------------------------X 
 
PET/CT/CT(CAP) 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
 
X 
 
MRI or Brain CT with 
contrast 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Tumor tissue for TBRS 
signature 
Xg 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Blood draw for VEGF 
 
X 
 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
Blood draw for 
immunological studies 
 
X 
 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
X 
 
Blood draw (pre and post) 
for LY2157299 pK 
 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
 
Blood draw for LY2157299 
cardiotoxicity (Troponin I ) 
X 
X 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
 
 
  X 
Echo/Doppler 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
 
X 
 
a: 
LY2157299  Administration of investigational drug is given 14 days on and 14 off. 
B: 
Before 1st RT & repeated during the study if indicated. 
C: 
Serum pregnancy test (women of childbearing potential). 
D: 
PE must include full vital signs,  skin and oral mucosa exam and prior to each LY2157299 administration and each RT cycle.  
        PE continue monthly for the first 3 months following therapy then yearly for 5 years. 
E: 
Albumin, alkaline phosphatase, total bilirubin, bicarbonate, BUN, calcium, chloride, creatinine, glucose, LDH, phosphorus, potassium, total protein, 
SGOT[AST], SGPT[ALT], sodium 
f.     CEA/CA 27.29 if elevated at baseline will be repeated at weeks 5 and 15. 
g. 
Archival tissue. 
 
 
 
6  PHARMACEUTICAL INFORMATION  
 
6.1 Investigational Agent  
 
LY2157299 is a receptor kinase inhibitor of TGF receptor type I (ALK5).   
Packaging and Labeling 
LY2157299 will be supplied in open-label blister packs or other appropriate packaging of 
film-coated, paracapsule tablets that are light yellow/yellow in appearance. 
Drug Shipment and Storage 
Eli Lilly & Co. will provide the clinical material for this study. 
6.2    Availability 
 


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Full records must be maintained to account for the study drug supplied to the Investigators, 
the disposition of the study drug, and the return or destruction of unused supplies. 
6.3    Agent Accountability 
 
All investigational drugs are stored with the Investigational Pharmacy. A separate binder is 
issued to each study as well as a bin for storage. The bins and binder are labeled with the protocol 
number and drug name. At WCM, the Investigational Pharmacy is located in a separate room within 
the Inpatient Pharmacy. There is limited access, and only the Pharmacists and support personnel 
working in the Research Pharmacy have access. The hours of operation are Monday- Friday from 8-
5pm. At the end of the day the room is locked. The research Pharmacist and staff can open the 
room using magnetic ID badges. Only the Research staff and management can enter the research 
area. Initial shipment of the investigational agent (LY2157299) will be delivered to WCM 
Investigational Pharmacy, which will disburse the agent within the WCM research group and to all 
collaborating institutions. 
At UCLA, the Investigational Pharmacy is located in the Ronald Regan Medical Center. There is 
limited access, and only the Pharmacists and support personnel working in the Research Pharmacy 
have access. The hours of operation are Monday- Friday from 8-5pm. This area is locked at all 
times. 
 
7 DOSING DELAYS/DOSE MODIFICATIONS 
 
LY2157299 will be administered as a flat dose of 300 mg/day, divided in a BID administration 
of 150 mg each, as an oral tablet. 
8 ADVERSE EVENTS: LIST AND REPORTING REQUIREMENTS 
 
8.1 Adverse Events and Potential Risks List 
 
LY2157299 has no reported history of Adverse Events or Serious Adverse Events. 
 
 
CTCAE term (AE description) and grade:  The descriptions and grading scales found 
in the revised NCI Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 
will be utilized for AE reporting.  A copy of the CTCAE version 4.0 can be downloaded from 
the CTEP web site (http://ctep.cancer.gov). 


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Attribution of the AE: 
Definite – The AE is clearly related to the study treatment. 
Probable – The AE is likely related to the study treatment. 
Possible – The AE may be related to the study treatment. 
Unlikely – The AE is doubtfully related to the study treatment. 
Unrelated – The AE is clearly NOT related to the study treatment 
8.1.1 Recording of Adverse Events 
 
All adverse events will be recorded on a patient specific AE log. The AE log will be 
maintained by the research staff and kept in the patient’s research chart.    
8.1.2 Reporting of AE to WCM IRB 
 
All Aes occurring on this study will be reported to the IRB according to the IRB policy, which 
can be accessed via the following link:  
http://researchintegrity.weill.cornell.edu/forms and policies/forms/Immediate Reporting Poli
cy.pdf.  
 
8.2 Expedited Adverse Event Reporting 
 
 
The principal investigator is responsible for monitoring the safety of patients who enroll in the 
study.  All AEs occurring after any administration of the study drug will be followed until 
resolution.  The descriptions and grading scales found in the revised NCI CTCAE version 4.0 
will be used for adverse event reporting.  A copy of the CTCAE version 4.0 can be downloaded 
from the CTEP web site (http://ctep.cancer.gov/reporting/ctc.html). 
A serious adverse event (SAE) is any adverse drug experience that occurs at any dose that 
results in any of the following outcomes: 
• Death. 
• Life-threatening adverse drug experience. 
• Requires inpatient hospitalization or prolongation of existing hospitalization. 


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• For the purpose of this study, hospitalizations for protocol-scheduled procedures, blood 
product transfusions, or for social reasons (i.e., awaiting transport home) will not be 
considered SAEs. 
• Persistent or significant disability/incapacity. 
• A congenital anomaly/birth defect. 
• Important medical events: Defined as AEs that, based upon appropriate medical 
judgment, may jeopardize the patient and may require medical or surgical intervention to 
prevent 1 of the outcomes listed above, even though these events may not be 
immediately life-threatening or result in death or hospitalization. 
All SAEs occurring on this study will be reported to the IRB according to the IRB policy, 
which can be accessed via the following link:  
http://researchintegrity.weill.cornell.edu/forms and policies/forms/Immediate Reporting Poli
cy.pdf.  
 
8.3 Reporting Serious Adverse Events  
 
All new SAEs and Medical Events of Interest occurring during the study or within 45 days of the 
last administration of LY2157299 will be reported as long as the patient has not withdrawn 
consent.  The principal investigators are responsible for reporting SAEs to the IRB and the FDA 
(21 CFR §312.32] or other applicable regulatory authority.  The principal investigator is 
responsible for submitting follow-up reports for all SAEs regarding the patient’s subsequent 
course until the SAE has resolved or until the patient’s condition stabilizes (in the case of 
persistent impairment), or the patient dies. 
Institution and Investigator understand and agree that Investigator and Institution are obligated 
under applicable law and regulations to report any serious and related adverse event, if any that 
occurs during treatment with the Product to the Institution’s IRB/Ethics Committee and to the 
governing regulatory authority in accordance with applicable filing timelines promptly after any 
such event occurs.  Investigator, within 24 hours (US) or one business day (EU) of first 
knowledge of such serious and related adverse event, will notify Lilly’s assigned medical 
Monitor. 
Prior to or at the time of filing any such report with the governing regulatory authority, the 
Investigator will also transmit an information copy of the report as sent to the authorities to Lilly. 


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The Investigator shall make available to Eli Lilly promptly such records as may be necessary and 
pertinent to investigate any such expedited adverse event, if specifically requested by Lilly. 
Furthermore, the Investigator will inform Eli Lilly of the following: 
• 
 any events that result in protocol amendments for safety reasons, as well as any safety 
related regulatory action such as a clinical hold of the Research 
• 
any pregnancies occurring in patients who are exposed to the Product in connection with 
the Research. 
• 
In addition, the Investigator shall notify Lilly within 24 hours (US) or one business day 
(EU) of first knowledge of any Product complaints (communication of dissatisfaction that 
alleges deficiencies related to the identity, quality, durability, effectiveness, safety, 
labeling, purity, stability, and appearance). 
The Investigator will also inform Eli Lilly within 1 business day of becoming aware of any 
actions from any authority that may affect the performance of the Research 
Medical Events of Interest 
For reporting purposes, the Investigator will make note of any medical events of interest that 
occur during the treatment period. 
8.4   Routine Adverse Event Reporting Guidelines 
 
The descriptions and grading scales found in the revised NCI CTCAE version 4.0 will be 
used for adverse event reporting to the institutional IRB, DSMC, and sponsor.  The IRB 
Reportable Events Forms 
(http://researchintegrity.weill.cornell.edu/forms and policies/forms/Immediate Reporting Po
licy.pdf.) should be used for all reportable adverse events. 
8.5   Safety Profile of LY2157299 
 
Mild to Moderate (Related to LY2157299):  
 
Diarrhea (12%), Fatigue (26%), Anemia (14%), Nausea (15%), Vomiting (10%), Abdominal 
pain (14%), Headache (13%), Neutropenia (5%), Anorexia (4%), Pruritus (4%), Constipation 
(3%).  


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Possible Side Effects related to LY2157299: 
 
Neutropenia, Thrombocytopenia, Lymphopenia, Leukopenia, Dysgeusia, Anorexia, Abdominal 
Discomfort, Dyspepsia, Pruritus, Dehydration, Asthenia, elevated liver enzymes, elevated 
creatinine.  
 
Rare but Serious side effects related to LY2157299: 
 
Pneumonia/Lobar Pneumonia, Pulmonary embolism, Deep vein Thrombosis, Sepsis, 
Neutropenia, Ascites, Anemia, Neoplasms, Pancreatitis, Asthenia, Retroperitoneal Hematoma, 
Heart attack, Stroke, Atrial Thrombosis, Heart Failure.  
 
Death resulting from side effects considered related to LY2157299 in less than 1% of subjects 
have been reported.  
 
 
 
9   CORRELATIVES AND SPECIAL STUDIES 
 
9.1 Laboratory Correlatives Studies 
 
Immune-monitoring studies will be conducted at UCLA under the supervision of Dr. McBride as 
well as at WCM under the direction of Dr. Demaria and Mary Helen Barcellos-Hoff. Blood 
samples will be obtained at baseline, and weeks 2, 5 and 15 for immune monitoring. All samples 
obtained for immune-monitoring studies will be processed for assay testing at the respective 
sites of collection. The following analyses will be performed: 
9.1.1 TGF β gene response signature 
 
Assessment of TGFβ gene response signature (TBRS) positivity in primary tumors or 
metastases as predictive biomarker of response to palliative LY2157299 plus radiation 
therapy combination therapy will be conducted.  
We will use the 153-gene TGFβ response signature (TBRS) developed by Massague` and 
colleagues.  TBRS identified TGFβ activity in 38/67 of metastatic lesions extracted from bones, 
lungs and other sites representing the natural metastatic spectrum of human breast cancer 
samples [72].  The resulting data will be processed by the WCM Core to ascertain the degree of 
TBRS in each patient; the study Biostatistician and staff will evaluate the associations with 


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outcomes.  We predict that the presence of TBRS in the primary or metastatic cancer will predict 
tumor response to RT+LY2157299. Based on past experience, we assume that approximately 
50% of subjects will have TBRS-positive tumors. Subjects who have TBRS-positive tumor and 
derive clinical benefit will be considered true positive.  
9.1.2 Circulating Levels of TGF β 
 
Monitoring of circulating levels of TGFβ prior to, during and after radiation therapy in patients 
with metastatic breast cancer before and after LY2157299 treatment.  We recognize that TGFβ 
measurements are complicated by the presence of the neutralizing antibody.  We will develop a 
new ELISA design based on LAP antibodies to overcome this limitation, in collaboration with Dr. 
Daniel Rifkin at NYU. Plasma TGFβ levels will also be measured. 
9.1.3 Anti-angiogenic Activity of LY2157299 in vivo 
 
Exploration of whether LY2157299 has anti-angiogenic activity in vivo by comparing circulating 
VEGF levels in plasma before and after treatment (ELISA). Plasma VEGF levels will be 
measured. 
9.1.4 Tumor specific Immune responses 
 
The most accepted technology for assessing T-cell responses to tumor antigen, namely 
ELISPOT and tetramer assays will be used. ELISPOT assays measure local release of 
cytokines by activated T cells following ex vivo re-stimulation by antigen. IFN production is most 
often used as a surrogate marker of T cell-mediated anti-tumor immunity. ELISPOT can be as 
sensitive as the tetramer assay and can be adapted to measure T cell avidity, although this will 
not be performed in this study, as it is not relevant to its aims. However, ELISPOT will be used to 
measure the hierarchy of epitope-specific responses for survival, since this might increase 
following radiation-induced tumor cell death, and indicate epitope spreading which we believe is 
an important correlate of clinical response [73].  
The MHC tetramer assay uses flow cytometry to enumerate individual CD8 T lymphocytes that 
specifically recognize a peptide antigen of choice. Tetramers specific for CD4 or CD8 T cells are 
available, though MHC-I tetramers only will be used as they are more advanced.  The tetramer 
assay is very able to detect expected increased responses in PBMCs from patients receiving 
RT. A detailed statistical analysis on data obtained with repeated sampling of over 100 patient 


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samples at UCLA showed that if antigen-specific T cells started below the lower limit of detection 
of 0.038%, they could be considered to have been boosted if they had an increase to over 
0.066%, representing the mean + 3 standard deviations (unpublished).  The assay does not 
require ex vivo T cell clone expansion, minimizing possible artifacts.  It is chosen primarily 
because it directly quantifies tumor-specific CD8 T cells. However, it does not assess the 
functional status of cells; which is why both tetramer staining and ELISPOT assays will be 
performed, but with the expectation of a direct correlation. A lack of correlation would indicate 
involvement of divergent mechanisms that would be investigated further, depending on the 
results.  
All 4 longitudinal samples from 5 HLA-A2 patients will be tested at one time using one vial of 
cells, in parallel with PBMCs from controls.  Initially, PBMCs from HLA-A2+ patients, who should 
be 45% of the cohort, will be tested for CD8+ T cells that bind tetramers containing the  
(LMLGEFLKL) survivin peptide, which has a high binding affinity for HLA-A2 [69] and, for 
patients whose tumors are also HER2/neu positive, also the HER:p369-377 peptide [74]. The 
usual controls will be included, including a nonsense tetramer and PBMCs from controls, as 
described previously [71]. If ELISPOT assays indicate utility, non-HLA-A2 patients will be tested 
by tetramer assay using peptides immunodominant for their known HLA type [68-69]. 
9.1.5 Levels of circulating Tregs 
 
Tregs will be detected primarily by staining PBMCs with antibodies for CD25, CD4, and 
intracellular FoxP3 (FACS analysis). Additional markers will be CD127, CD45RA/RO, and 
CTLA4. We include an examination of CTLA-4 because CTLA-4 signaling seems to be required 
for Treg function, possibly through TGF- induction [75-76]. In addition, the NYU team has 
shown in preclinical models that anti-CTLA-4 synergizes with RT. CD45RA/RO and CD127 will 
be included because of recent reports that these mark subsets of Tregs with distinct proliferative 
and migratory properties that are relevant to cancer [77]. CD25, CD4, and intracellular FoxP3 
staining will be performed at the same time as tetramer staining (see above) but another vial of 
each sample will be used for additional evaluation of Treg function, performed as previously 
described [71]. 
9.2 Collection of Specimen(s) 
 


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Peripheral blood for immune monitoring will be obtained from all trial participants at 5 time 
points: 1) baseline, 2) day 14; 3) day 42 infusion and 4) time of disease evaluation, at week 15. 
Blood will be drawn prior to radiation since radiation may increase TGFβ levels as shown in 
animal models. Up to an average of 60 ml of peripheral blood for research purposes will be 
collected at each time point into EDTA Lavender top tubes from consented patients enrolled in 
the trial, for analysis of plasma and PBMCs. 
Tissue biopsy is not mandatory for participation in the trial, but archived tumor samples (slides 
and blocks, where available) will be obtained from consented patients. This tissue may be 
utilized to study TGFβ response expression signatures. 
Sample collection is described in more detail in Appendix 1. 
9.3 Handling of Specimens 
 
PBMCs will be isolated within 24 hours of blood draw. Plasma will be stored for measurements 
of circulating TGF. An aliquot of 2x106 cells will be removed for HLA typing. DNA isolated with 
the QIAamp DNA Blood Mini prep kit (Qiagen) will be submitted to the UCLA Immunogenetics 
Center (UIC) for PCR-based HLA typing that allows accurate and reliable results in resolving 
allele-level differences. The UIC is internationally recognized as a pioneer in HLA typing and 
crossmatch testing for tissue and organ transplants and a World Health Organization reference 
laboratory for HLA typing. Remaining cells will be frozen in 4 x 1 ml aliquots in 10% DMSO in 
human AB serum and plasma in 2.5 ml cryovials.  Samples will be stored initially at -80C and 
then liquid N2 until used. All freezers will have alarm systems to avoid sample loss. Batches of 
PBMCs will be sent on dry ice overnight to UCLA from the other centers and plasma samples 
similarly shipped to WCM, one vial set at a time to prevent complete loss of samples due to 
unlikely shipping delays. All samples will be coded for laboratory analysis.  A master list with the 
patient’s name and an identification number will be kept in a locked cabinet. 
9.4 Storage of Samples 
 
 
Serial blood samples for immune monitoring collected at baseline, and at selected time 
points outlined in section 5.10.  


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All blood and tissue samples will be stored in a -80°C locked freezer in the WCM Bio 
repository for an indefinitely for research purposes only. These specimens will not be linked to 
any clinical data and will be de-identified in the clinical research database - only the data 
manager will have access to the master list with the patient name and an identification number. 
This master list will be secured in a locked cabinet at the WCM.  Only the investigators listed on 
this protocol will have access to these samples as is described in sections 9.1.1-9.1.5. 
 
The storage of your blood and tissue is optional and you may withdraw your consent for the 
banking of these specimens at any time. You may make this request by writing to the Principal 
Investigator Silvia C. Formenti, M.D. at Weill Cornell Medicine, 525 East 68th Street, New York, 
NY 10065. 
9.5 Shipping of specimens 
 
Frozen blood specimens from WCM will be de-identified and shipped to UCLA at -80°C. 
9.6 Site(s) Performing Correlative Study 
 
Correlative studies (as described in sections 9.1.1-9.1.5) will be performed at UCLA. 
 
9.7 Coding of Specimens for privacy protection 
 
All patients enrolled will be given a unique identifier (study ID number). Only the data manager 
will know the code linking patient and study ID number. Patients will be assigned a unique code 
number. All specimens collected will be de-identified and assigned the same unique study 
number of the corresponding patient and will also be marked with the collection time point. 
Clinical information regarding toxicities and response will likewise be stored in a de-identified 
database using only the unique identifier (study ID number). 
 
10 INVESTIGATOR RESOURCES  
 
10.1 Qualifications  
 
 


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At Cornell Dr. Formenti, will be responsible for the accrual and care of study patients. Research 
Nurse will be in charge of study screening, eligibility checklist and will participate in the process 
of acquisition of an informed consent, after the faculty has discussed the trial with the patient.  
Dr. Paul Christos will participate in the design of the study and oversee the statistical analysis 
and interpretation of the data. 
At UCLA Dr. Steinberg, Dr. Steve Lee, Dr. Phillip Beron, Dr. Kupelian, Dr. Glaspy, Dr. 
McCloskey, and Dr. Hurvitz will be responsible for the accrual and care of study patients.  Dr. 
McBride will be responsible for overseeing the laboratory correlative studies. The research 
component at UCLA will be conducted by the Radiation Clinical Oncology Research Team. . 
 
10.2 Location and Use of WCM and UCLA Facilities 
 
At WCM therapy will be administered in the Department of Radiation Oncology at the Stich 
Radiation Center. LY2157299 will be administered at the infusion center at 525 East 68th street, 
Starr 3, New York, NY – 10065. 
At UCLA therapy will be administered in the Department of Radiation Oncology, 200 UCLA 
Medical Plaza, Suite B265, Los Angeles, CA. 90095. LY2157299 will be administered at the 
CTSI infusion center at UCLA Santa Monica, CA, 2825 Santa Monica, Ste. 200 Santa Monica, 
CA 90404. 
10.3 Conflict of Interest 
 
There are no conflicts of interest to declare. 
 11 MEASUREMENT OF EFFECT 
 
Abscopal responses will be assessed at 15 weeks based on [78] (Table 2). This choice of this 
interval is to enable a sufficient length of study to detect abscopal responses. In fact, the 
experience in the clinical trials of Ipilimumab in melanoma demonstrated occasional initial 
progression of disease with appearance of new metastatic sites when compared to the baseline 
imaging assessment. “Flares” at the first PET/CT or CT(Chest/Abdomen/Pelvis) assessment 
were often followed by clinical responses to the antibody. While the mechanism of action of 


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Ipilimumab is different from that of LY2157299, the experience with TGF inhibition in malignant 
glioma reported by Hau et al [79] confirms the lesson from the melanoma trials, i.e. that 
whenever the response is mediated by the immune system the following course is likely to 
manifest: 1) achievement of a clinical and radiological measurable response is slower than that 
of a typical response to cytotoxic therapy (RT or chemotherapy); 2) it can be heralded by an 
initial “flare” or emergence of new metastatic sites and, 3) with time it may be dramatic and 
would have been missed if the drug was discontinued too soon [80] [81]. 
Table 2: Immune response criteria (irRC) 
 
Summary of irRC 
Note:  
New, measurable lesions are incorporated into tumor burden 
CR 
Disappearance of all index and non-index lesions in two consecutive observations not less 
than 4 weeks apart, without the development of any new lesions. 
PR 
≥ 50% decrease in tumor burden compared with baseline in two observations at least 4 
weeks apart 
SD 
50% decrease in tumor burden compared with baseline cannot be established nor 25% 
increase compared with nadir 
PD 
At least 25% increase in tumor burden compared with nadir (at any single time point) in 
two consecutive observations at least 4 weeks apart  
 
11.1 Anti-tumor Effect  
 
11.1.1 Definitions 
 
Patients will be evaluated by PET/CTor CT(chest/abdomen/pelvis) at baseline, week 5 and week 
15. However, systemic anti-tumor response will be by PET/CT or CT(chest/abdomen/pelvis) as 
defined by irRC at 15 weeks (primary endpoint). 
Patients who have received at least one 14 days cycle of the study drug will be followed for 
toxicity (lack of grade 4 toxicity– primary safety end point).Patients will receive a total of 14 
doses of LY2157299 (one dose daily for 14 consecutive days), per cycle. Patients who have 
received any treatment will also be evaluated for abscopal response to the metastatic sites using 
the immunologic response criteria based on the 5 and 15 week PET/CT or 
CT(chest/abdomen/pelvis) scans as outlined above in Table 2.  
11.1.2 Disease Parameters 
 


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Measurable lesions are defined as those that can be accurately measured in at least one 
dimension (longest diameter to be recorded) as > 20 mm with conventional techniques (CT, 
MRI, x-ray) or as > 10 mm with CT scan. All tumor measurements must be recorded in 
millimeters (or decimal fractions of centimeters). 
All other lesions (or sites of disease), including small lesions (longest diameter < 20 mm with 
conventional techniques) are considered non-measurable disease. Bone lesions, 
leptomeningeal disease, ascites, pleural/pericardial effusions, lymphangitis cutis/pulmonis, 
inflammatory breast disease, abdominal masses (not followed by CT or MRI) and cystic lesions 
are all non-measurable. 
11.1.3 Methods for Evaluation of Measurable Disease 
 
All measurable lesions that are not irradiated are identified as target lesions and recorded 
and measured by PET/CT or CT (chest/abdomen/pelvis) at baseline and at weeks 5 and 15. The 
abscopal response based on target lesions is defined in Table 2. 
A sum of the longest diameter (LD) for all target lesions will be calculated and reported as 
the baseline sum LD. The baseline sum LD will be used as reference by which to characterize the 
objective tumor clinical response (definition in Table 2). 
In parallel, assessment at PET scanning will be conducted. After at least a 4 hour fast, blood 
glucose will be checked. Patients will be injected with 10-15 mCi of F-18 FDG (fluoro-deoxy-
glucose) in a resting state. After 45 minutes, whole body PET scans with attenuation correction will 
be performed from the cerebellum to the mid-thigh. Images will be corrected to provide standardized 
uptake values in g/ml. The Full Width Half Maximum of the scanner is approximately 4.6 mm. 
Therefore, we expect to be able to accurately assess lesions > or equal to 1 cm in size. A qualitative 
assessment of lesions will be performed, i.e., visibility of lesion on F-18 FDG PET. In addition, the 
maximum standardized uptake value will be determined for each lesion. Criteria as described by the 
EORTC PET study group will be used to assess response based on standard uptake value 
(SUV) [82]: 
Progressive metabolic disease: 
 
 
Increase in SUV of more than 25% 
 
Visible increase in the extent of FDG uptake > 20% in longest dimension 
 
New metastatic lesions 
 
Stable metabolic disease: 
 
Increase in SUV of less than 25% or decrease of less than 15% 


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No visible increase in the extent of FDG uptake 
Partial metabolic response: 
 
Reduction in SUV between 15% and 25% after one cycle of treatment 
Reduction in SUV of more than 25% after more than one cycle of treatment 
Complete metabolic response: 
 
Complete resolution of FDG uptake 
 
11.1.4 Response Criteria 
 
 
 
11.1.4.1 Evaluation of Target Lesions 
 
                  Criteria for evaluation of response are reported in Table 2. 
                       11.1.4.2 Evaluation of Non-Target Lesions (irradiated Lesions) 
 
Complete Response (CR): 
Disappearance of all non-target lesions and 
normalization of tumor marker level 
Incomplete Response/ 
Stable Disease (SD): 
Persistence of one or more non-target lesion(s) 
or/and maintenance of tumor marker level above 
the normal limits 
Progressive Disease (PD): 
Appearance of one or more new lesions and/or 
unequivocal progression of existing non-target 
lesions 
 
Although a clear progression of “non-target” lesions only is exceptional, in such 
circumstances, the opinion of the treating physician should prevail, and the progression status 
should be confirmed at a later time by the review panel (or study chair). 
Note: If tumor markers are initially above the upper normal limit, they must normalize for a 
patient to be considered in complete clinical response. 
 
                             11.1.4.3. Evaluation of Best Overall Response  
 
The best overall clinical response is the best clinical response recorded from the start of the 
treatment until disease progression/recurrence (taking as reference for progression disease the 
smallest measurements recorded since the treatment started). The patient’s best response 
assignment will depend on the achievement of both measurement and confirmation criteria (see 
section 12.1.4.4 below). 
Notes: Patients with a global deterioration of health status requiring discontinuation of treatment 
without objective evidence of disease progression at that time should be classified as having 


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“symptomatic deterioration”. Every effort should be made to document the objective progression, 
even after discontinuation of treatment. 
In some circumstances, it may be difficult to distinguish residual disease from normal tissue. 
When the evaluation of complete response depends on this determination, it is recommended 
that the residual lesion be investigated (fine needle aspirate/biopsy) before confirming the 
complete response status. 
                         11.1.4.4 Confirmation Criteria 
 
To be assigned a status of PR or CR, changes in tumor measurements must be confirmed by 
repeat assessments that should be performed after a minimum of 4 weeks, no less, after the 
criteria for response are first met.  
11.1.5 Response Review  
 
All responses will be reviewed by an expert(s) independent of the study at the study’s 
completion. 
12 DATA REPORTING/ REGULATORY CONSIDERATIONS 
 
 
The WCM Data and Safety Monitoring Committee (DSMC) is the central monitoring board for 
this study. The UCLA Cancer Institute Data and Safety Monitoring Committee (DSMC) is the 
local monitoring board for UCLA patients. Peter Martin, M.D. is the Co-Chairman of the 
Cancer Clinical Trials at the WCM Data Safety Monitoring Committee. He will also serve in 
the capacity of the Independent Research Monitor.   
The Research Monitor, Peter Martin, MD, is responsible to oversee the safety of the 
research and report observations/findings to the IRB or a designated institutional official. The 
Research Monitor will review all unanticipated problems involving risks to subjects or others 
associated with the protocol and provide an independent report of the event to the IRB. The 
Research Monitor may discuss the research protocol with the investigators; shall have 
authority to stop a research protocol in progress, remove individual human subjects from a 
research protocol, and take whatever steps are necessary to protect the safety and well-
being of human subjects until the IRB can assess the monitor’s report; and shall have the 


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responsibility to promptly report their observations and findings to the IRB or other 
designated official and the HRPO. 
12.1 Monitoring plan 
 
This study will be conducted in accordance with the guidelines in the 2001 NCI approved data 
Safety and Monitoring plan for the WCM Cancer Institute and with the UCLA approved data 
Safety and Monitoring plan for the UCLA Cancer Institute. Reports to the Data Safety and 
Monitoring Committee will include the following information: accruals, targets, responses, 
adverse events and evidence of reporting to appropriate review committees. The WCM Data 
and Safety Monitoring Board (DSMB) will review the IRB approved protocol, the data and safety 
monitoring plan and any stopping guidelines during protocol initiation. The WCM DSMB is to 
perform a data and safety analysis annually (as per DSMB letter dated 04.20.2017) following the 
accrual of the first subject. . The WCM DSMB may also convene as needed if stopping criteria 
are met or other safety issues arise that the Principal Investigator and/or IRB would like the 
WCM DSMB to address. 
As per WCM DSMB recommendations, the safety analysis will be performed annually, at which 
point the protocol and safety analysis should be submitted to the DSMB for review.  
 
12.2 Stopping rules (for the individual patient and for the study as a 
whole) 
 
Patients will be discontinued from study therapy when any clinical adverse event greater 
than Grade 3 or intercurrent illness in the opinion of the Principal Investigator, indicates that 
continued treatment with the study therapy is not in the best interest of the subject. Patients 
will be monitored until the resolution of the adverse event. Based on PI’s discretion, patients 
will resume or will be withdrawn from the study therapy.  
If five or more non-hematological toxicities, Grade 3 and above, are reported over a period 
of 3 months, will result in temporary discontinuation of combination therapy.  
12.3 Data Management  
 


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All patient data will be entered and maintained using,REDCAP, CTSC data management 
system.  These data include clinical data and all patient safety data. The REDCap provides audit 
trails that track creation and modification of records that include user id and timestamp. 
Depending on the database account privileges, the data managers may be able to correct a 
discrepancy or if not, route it to the project data manager at WCM who can take appropriate 
action to correct the problem. Data clarification forms can also be printed out when necessary to 
be sent to the project data manager at JCTO.  
The Nurse practitioner will also be documenting adverse events in EPIC during every follow up 
visit, All Adverse Events are captured in REDCap database and a report will be sent to Data 
safety monitoring board during their quarterly review meetings.  
All key end points will be source verified by a second person at each site and errors will be 
corrected. Once the data is verified and all discrepancies are closed, the data can be 
locked/frozen. Locking and freezing can be done at different granular levels and will follow 
institutional SOPs and any specific requirements for the project. 
Security measures that will be taken in order to protect patient data will include firewall 
technology and database level security which will be achieved by assigning roles and privileges 
to different levels of users and by requiring that the users authenticate themselves using user id 
and password. Additional security for data transfer between remote clients and servers will be 
achieved by using digital certificates/SSL. All data will be backed-up to tape periodically 
according to the Institutional SOPs. All data will be stored for at least 5 years following the 
termination of this study. 
12.4 Confidentiality 
 
The medical, hospital and research records associated with this study are considered 
confidential. Members of the treating team and designated study assistants will have access to 
the records as required to administer treatment and comply with the protocol. Neither the name 
nor any other identifying information for an individual will be used for reporting or publication 
regarding this study. All laboratory and baseline data will be de-identified and transferred via 
secure links to the database at WCM. Patient records will be made available for inspection to 
auditing agencies to satisfy regulatory requirements. Privacy will be maintained throughout the 
consent process and protocol treatment.   


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13 
STATISTICAL CONSIDERATIONS 
 
The characteristics of patients and their disease will be described for the study as a whole and 
by study site  using summary statistics (including means, standard deviations, quantiles) and 
graphical displays (e.g., boxplots) for quantitative variables and frequency distributions and 
proportions for qualitative data. Clinical and immune response rates will be estimated along with 
exact 95% confidence limits. Similarly, for each immunological parameter, summary statistics 
and graphical displays will be provided at baseline, before radiation, at week 5, and at week 15. 
Tests for normality will be used (eg Kolmogorov-Smirnov tests at alpha = 0.05); transformations 
(eg, logarithmic) will be used to meet the normality assumptions. The patterns of change over 
time  for each of the immunologic parameters will be evaluated using mixed effects regression 
models that incorporate time as a fixed effect and subject as a random effect, and take into 
account different numbers of observations for each patient (missing data, [83]). Exploratory 
analyses will also be undertaken to describe the association of these parameters and changes 
in parameters with response recognizing that the numbers of subjects will be small. Additionally, 
the TGFβ gene suppressor signature (see 9.1.1.) will be estimated for each subject and the 
AUC (area under the Receiver Operating Characteristic Curve) will be estimated. The sensitivity 
and specificity of the defined cut point will be also be provided along with 95% confidence 
intervals.   
 
14.1 Endpoints 
 
14.1.1 
Primary endpoint  
 
1. 
To assess safety and feasibility of combining TGF β receptor I kinase    
         inhibitor LY2157299 and local radiotherapy 
2. 
To determine if treatment with TGFβ receptor I kinase inhibitor  
          LY2157299 and localized RT achieves an abscopal tumor regression  
 
14.1.2 
Secondary endpoints 
 
1. 
To estimate the local response rate of combining TGFβ receptor I kinase 
inhibitor LY2157299 and local radiotherapy 
2. 
To determine if treatment with TGFβ receptor I kinase inhibitor LY2157299 and 


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localized RT enhances tumor-specific immunity in patients with metastatic breast 
cancer, and if this is associated with abscopal tumor regression. 
3. 
To determine if treatment with TGFβ receptor I kinase inhibitor LY2157299 and 
localized RT alters the numbers and function of T-reg cells in patients with 
metastatic breast cancer. 
 
A. Analysis Populations 
 
Efficacy analysis will be conducted on all patients who are entered into the study and 
received any treatment. Additional supportive efficacy analyses will be conducted for those 
patients who have completed week 15 visit.  
B. Sample Size 
 
i. Accrual estimates 
 
 
 
Expected number of eligible patients will be 4 per month. 
ii. Sample size justification 
 
In this trial, a total of 28 patients with metastatic breast cancer will be accrued both at WCM and 
at UCLA (18 patients at WCM and 10 patients at UCLA) to treatment with 
radiotherapy + 300 mg/daily LY2157299 for 14 days on / 14 days off, and followed for evaluation 
of abscopal responses based on the immune response criteria at 15 weeks.   With 28 patients 
enrolled in this study, we can test the null hypothesis that the abscopal response rate at 15 
weeks is ≤0.05 versus the alternative that the response rate is 0.19 with α= 0.05 and power of 
80%. If there are 3 or fewer responses in the 28 patients, then the combination will be rejected 
for further study in this single stage design (calculations from PASS 2008, J. Hintze, NCSS, 
Kaysville, Ut).  The trial is designed to allow for up to 56 patients to be enrolled to ensure that 28 
patients will be evaluable at 15 weeks. Response rates will also be estimated for all enrolled 
patients.  
 
 
 APPENDIX 1     SPECIMEN COLLECTION PROCEDURES 
 
NOTE regarding samples for TGF-β assessment: 
 
It is important to follow special steps to reduce the release of TGF-β into the blood sample –
 particularly, using a straight 20 or 21 gauge needle instead of a butterfly, minimizing tourniquet 


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stasis, directly drawing blood into Vacutainer tubes instead of using a manual syringe and transfer, 
and avoiding contact EDTA. In addition, CTAD tubes are sensitive to light, so they should be kept 
inside the kit boxes when not in use. 
The suggested method of phlebotomy is to minimize the breakage of platelets and thus the release 
of TGFβ from platelet granules. The order of blood tubes was designed to avoid potential cross-
contamination of the plasma CTAD tube with any potential anticoagulants, EDTA and heparin, as 
these agents may activate platelets and/or interfere with the tests. 
 
If the sites need to draw their standard hematology and chemistry labs before any research tests, it 
is recommended that: 
 
1. Standard blood tubes containing anticoagulant be drawn first (e.g. CBC, PT/INR, and PTT) 
followed by collection tubes which do not contain anticoagulants (e.g. serum chemistries). 
2. The biomarkers should then be drawn in the order specified below in order to adequately 
separate the anti-coagulant tubes from the CTAD tube. 
 
Blood draws should be performed in the following order in order to minimize cross contamination of 
the TGFβ plasma collection: 
1. SST tube (Gold hemoguard) 
2. CTAD tube (blue top) 
 
 
1.2 Plasma, FROZEN (TGFβ1, VEGF) 
 
Note: CTAD tubes are sensitive to light, so they should be kept inside the kit boxes when not 
in use. 
 
o 
Collect blood into two 4.5 mL blue top Vacutainer® (CTAD) tubes using a 20 or 21-gauge 
straight needle and the following special instructions: 
 
Tourniquet stasis should be minimized. If possible, do not use a tourniquet when 
drawing the sample for this tube. If a tourniquet is needed, minimize the constriction 
strength and try to remove it at the start of the venipuncture 
 
It is recommended that blood is drawn directly into CTAD tubes in order to avoid platelet 
shearing/destruction. Do not use a standard syringe, manual pull, and transfer to tubes 
method to draw blood. 
 
A central line should not be used for blood draws the day of infusion. In the case 
where it is not at all possible to draw the blood peripherally, then the central line must 
have been flushed with 2-5 ml of normal saline to clear any heparin 
o 
Invert tubes 3-4 times and immediately place in ice water bath for no more than 30 
minutes 
o 
Remove from bath and centrifuge whole blood at 2,500 RCF (xg) for 30 minutes at 4°C. 
Turn the centrifuge brake OFF. 


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o 
Immediately after centrifugation, pipette ~0.5 mL plasma into 7-8 cryovials, Use only the 
top 2/3 of the plasma volume. 
o 
Label the cryovials with the Patient Initials, Patient ID #, sample collection date (dd/mmm/yyyy 
format), sample collection time (24 hour clock) and sample type as serum for the specific 
biomarkers. 
Immediately freeze all cryovials in the upright position at or below -70ºC (-20ºC is NOT acceptable) 
in a non-defrosting freezer until shipped. Keep all samples frozen until ready to ship. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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