Randomized Study of Doxorubicin and Cyclophosphamide With or Without Intermittent Sunitinib in the First-line Treatment of Locally Advanced or Metastatic Breast Cancer Patients With Measurable Primary Breast Tumor

This is a single-centre, phase II randomized study of doxorubicin and cyclophosphamide (AC) with or without intermittent sunitinib in patients with measurable primary breast cancer who are receiving pre-operative chemotherapy.

A total of fifty patients with measurable primary tumor will be enrolled over a period of 18-24 months. Eligible patients will be randomized 1:1 to either arm A or arm B. Patients will be stratified according to metastatic status (metastatic vs non-metastatic) and presence or absence of clinical T4 disease.

Arm A (Control arm):

Doxorubicin 60mg/m2 day 1 Cyclophosphamide 600mg/m2 day1, every 3 weeks x 4 cycles

Arm B (Experimental arm):

Days -13 to day 0 (total 14 days) - oral sunitinib 37.5mg daily Cycle 1: day 1 - Cycle 1 AC (60/600mg/m2); days 15-21 - oral sunitinib 37.5mg daily Cycle 2: day 1 - Cycle 2 AC (60/600mg/m2); days 15-21 - oral sunitinib 37.5mg daily Cycle 3: day 1 - Cycle 3 AC (60/600mg/m2); days 15-21 - oral sunitinib 37.5mg daily Cycle 4: day 1 - Cycle 4 AC (60/600mg/m2)

Imaging studies

DCE-MRI

DCE-MRI scan will be performed serially to determine tumor response and change in tumor vascular parameters for each enrolled subject:

Arm A: At baseline (before cycle 1 AC) and once at mid-cycle of cycle 1 (days 12-14). Arm B: At baseline (before sunitinib), after completing at least 12 days of sunitinib and before cycle 1 AC, and during mid-cycle of cycle 1 AC (days 12-14 following cycle 1 AC).

Patient will be evaluated weekly for toxicity assessments and full blood count during cycle 1, and on days 1 and 15 of each subsequent cycle. In addition, patients in Arm B will be evaluated weekly during the first two weeks of sunitinib administration prior to cycle 1 AC.

Special tests Blood sampling

Germline DNA at baseline for pharmacogenetics analysis.

Pharmacokinetic sampling for doxorubicin and cyclophosphamide on day 1, cycle 1 of AC administration.

Pharmacokinetic sampling for sunitinib at baseline and after 7 days and 14 days of sunitinib for subjects in Arm B before the sunitinib dose on that day.

Serial plasma samples for proteomics analysis and for analysis of soluble angiogenic factors, including VEGF-A, VEGF-C, soluble VEGF receptors (sVEGFR-1, sVEGFR-2, sVEGFR-3), placental derived growth factor (PIGF), soluble inter-cellular adhesion molecule 1 (sICAM-1), Tie-2 and stromal derived factor 1a (SDF-1a). Samples will be taken at baseline and every week during cycle 1 AC, followed by every 3 weeks prior to each subsequent cycle of AC. One sample will be taken within 4 weeks of completing 4 cycles of AC, within 4 weeks after surgery, followed by 4-monthly until disease progression. Patients in Arm B will have additional two samples taken on a weekly basis during the first two weeks of sunitinib.

Serial whole blood for gene expression analysis at baseline and 2 weeks after cycle 1 AC for all patients, and after completing at least 12 days of sunitinib and prior to cycle 1 AC for subjects in Arm B.

Serial blood samples for circulating tumor and circulating endothelial cells at baseline and every 3 weeks during the four cycles of AC. Patients in Arm B will have an additional sampling for circulating tumor and circulating endothelial cells after completing at least 12 days of sunitinib and before cycle 1 AC.

Tumor core biopsy

Arm A: Performed at baseline, approximately 3 weekly after cycle 1 AC but before cycle 2 AC, and upon completion of 4 cycles of AC, for a total of 3 tumor core biopsies

Arm B: Performed at baseline, after completing at least 12 days of sunitinib and before cycle 1 AC, approximately 3 MainPrint Page 3 of 23 https://www.b2bresearch.nhg.com.sg/DSRB/DSRBWeb/Protocols/MainPrint.aspx 06/05/2010 weekly after cycle 1 AC but before cycle 2 AC, and upon completion of 4 cycles of AC, for a total of 4 tumor core biopsies.

The final biopsy may be obtained at surgery if the patient is scheduled for lumpectomy or mastectomy. The tumor cores will be stored in liquid nitrogen for subsequent DNA, RNA and protein extraction for biomarker studies including gene expression and proteomics analyses. Three to four samples will be obtained at each time point, and one tumor core at each time point will be stored in formalin and paraffin-embedded for immunohistochemistry analysis of biomarkers.