The Role of suPAR Biomarker in Blood Samples of Breast Cancer Patients During and Post Doxorubicin Chemotherapy: Causative vs. Predictor

- Hypothesis 1: Higher suPAR at baseline will predispose to DOX-induced cardiomyopathy to
be observed by histology in mouse models, and tested using LVEF (Left Ventricular
Ejection Fraction) assessment, and surrogate cardiovascular outcome measures as
described in humans.

- Hypothesis 2: suPAR is a marker of DOX-induced cardiomyopathy after exposure to DOX, to
be observed by histology in mouse models, and tested using surrogate cardiovascular
outcome measures as described in humans.

1. Patients meeting outlined eligibility criteria are identified by breast cancer
physicians working in the Rush Cancer Center. After eligibility is verified,
patients are approached by the study coordinators or research fellow to discuss
participation in the trial while patients were being seen by their provider within
the cancer center and given the informed consent form for review. Patients are
contacted approximately one week later to answer questions and plan for baseline
visit if interested in enrolling.

2. After signing the informed consent form, patients are officially enrolled. The
research nurse, study coordinator, or research fellow will conduct research visits
at each of the six study time points outlined in the protocol: pre-chemo, post-2
cycles of doxorubicin, post-4 cycles of doxorubicin, 3 months post-doxorubicin, 6
month post-doxorubicin and 12 months post-doxorubin

3. Each study visit consists of collecting lab work via blood draw as detailed in the
protocol as well as an echocardiogram performed with strain.

4. Results from each test are collected, reviewed by the PI and documented in the
patient's study binder.

5. Preliminary human results have not been analyzed at this time as there is currently
insufficient data collected.

In the laboratory approach of the study, the investigators aimed to establish the role of
suPAR in DOX induced cardiomyopathy. The risk for DOX induced cardiomyopathy is known to
increase with an increase of the accumulative dose. The investigators therefore used two
doses of DOX. One group of mice received weekly injections of DOX at 3mg/kg bodyweight for 6
week (accumulative dose of 18mg/kg: DOX18) whereas another group received an accumulative
dose of 25mg/kg bodyweight (DOX25: 5 weeks, at 5 mg/kg bodyweight). Blood samples were taken
before the first injection, in the middle of treatment and at the end of treatment or at the
day of sacrifice. The bodyweight of the animals was constantly monitored throughout the
study.

At time of sacrifice, the heart weight and tibia length of the mice were quantified and cells
were isolated to determine cellular levels of reactive oxygen species (ROS), contractility
and Ca handling properties.

DOX induced cardiomyopathy can manifest years after treatment. Tissue samples from hearts of
the DOX18 and DOX25 treatment group were obtained and quantitative PCR (Polymerase Chain
Reaction amplification) of the ventricular tissue will help us determine the changes in
protein expression and cellular signaling that underlie these DOX induced changes in cellular
function.

Blood samples obtained from the DOX18 and DOX25 mice will be analyzed for their level of
suPAR to determine changes in suPAR over the duration of treatment and potential correlations
of [suPAR] with the degree of cardiac dysfunction.

The data collected will be analyzed using a descriptive analysis with means and standard
deviations for continuous variables and percentages for categorical variables. A paired
t-test comparing the pre-post difference will be used to compare the differences in the
groups comparing pre to post measures of myocardial damage based on histology for mouse
models, and LVEF (Left Ventricular Ejection Fraction) for human data. Analysis will be
performed using SAS Software.

The expected results from the data collected in this study aim to both gain an understanding
of and describe the relationship of various covariates to primary outcomes.

It is expected to have 50 participants by the study end date. There is a trained team working
on the study, insuring quality of data gathering, recruitment and enrollment of patients.

Inclusion Criteria:

- 18 - 64 years

- Undergoing chemotherapy in the Rush cancer center with a plan for doxorubicin
(Adriamycin®) chemotherapy.

Exclusion Criteria:

- Patients with metastatic breast cancer - complicated chemotherapy regimens, higher
mortality risk

- HER2 positive breast cancer patients planned for trastuzumab therapy

- Patients with baseline cardiomyopathy (reduced LVEF: less than 50%)

- Patients with prior cardiovascular history of myocardial infarction (MI), angina,
Congestive Heart Disease (CHD) death, coronary artery bypass grafting (CABG),
percutaneous coronary intervention (PCI) including percutaneous transluminal coronary
angioplasty (PTCA) and end-stage renal disease (ESRD), atrial fibrillation prior to
cancer diagnosis

- Atrial fibrillation noted on baseline ECG