DIRECT

Primary study endpoint

• PhaseII part: Grade III/IV toxicitywill be scored before each chemotherapeutic gift, after the last cycle and6 months after surgery using NCI-CTCAE v4.03 (see appendix A).
• PhaseIII part: pCR at surgery will bemeasured using Miller and Payne (see appendix B). Study staff evaluatingpCR will be blind to the nature of the intervention.

Secondary study endpoints

• Clinical response: measured by MRI after the last AC or FEC cycle of chemotherapy
• Grade I and II side effects ofchemotherapy: scoredprior to every chemotherapeuticgift according NCI-CTC v 4.03 to quantify toxicity (see Appendix A) and alist of relevant side effects will be filled out (see Appendix C).
• The body’s metabolic and inflammatory response to chemotherapy: every cycle just before start ofchemotherapy measure metabolic parameters (glucose, insulin, insulin-likegrowth factor-1 (IGF-1)) and the inflammatory parameter C-reactive protein(CRP) will be measured. Insulin-like growth factor binding protein 3(IGF-BP3), free thyroxin (FT4), T3 and thyroid-stimulating hormone (TSH) will be measured before the cycles 1, the last AC or FEC cycle and the last docetaxel cycle.
• DNA damage, apoptosis, immunology, tumor/stroma ratio and nutrient sensing pathways in the tumor aftersurgery:

Chemotherapy-induced DNA damage in material of the tumor will be measured using the comet assay and the phosphorylation of H2AX, a protein involved in the DNA damage response.

Caspase-3 (as a measure of apoptosis) will be measured using immunohistochemistry and mTOR, AMPK, sirtuin and IGF-1 pathways will be characterized by Western blot, microarray and polymerase chain reaction. The results of the nutrient sensing pathways of the operation specimen will be compared with the biopsy.

Tumor biology is changing. Not only the tumor itself is important, its relation with the micro-environment determines the aggressive potential. The amount of stroma present in the primary tumor has shown to be an important prognostic parameter. Patients with a high amount of stroma have a worse prognosis whereas patients with no or little stroma show good survival. The Tumor/Stroma Ratio (TSR) can be determined on routine histological sections. Preliminary results also show that stroma has a protective role and that response to therapy differs for both stroma groups. We would like to validate the stroma parameter in this prospective trial. The two stroma groups will be evaluated for outcome and response to therapy. The TSR will be evaluated on HE slides.

• Predictive biomarkers: after surgerymaterial of the tumor will be used to study: hormone receptor percentage,Ki67, immunologic tumor profile and TSR as defined above. The results of the operation specimen will be compared with the biopsy.
• Patient’s quality of life and perceptions:patientswill be asked to fill out EORTC QLC-C30 and EORTC QLQ-BR23 (except the questions about arm and breast symptoms47-53) questionnaire before start of chemotherapy (baseline), before the last AC or FEC cycle and before the last docetaxel cycle and 6 monthsafter surgery. Patients will be asked to fill out a distress thermometer (visualanalogue scale (VAS)) about experienced burden of therapy at baseline, before the last ACor FEC cycle and before the last docetaxel cycle and 6 months after therapy. At baseline and before the last docetaxel cycle patients will beasked to fill out the B-IPQ (two different forms) to measure illness perceptions (see appendix D).
• DFS and OS: disease free survival and overall survival willbe determined after 5 and possibly 10 years according to the national guidelines.
• SNPs: before start therapyan extra blood samples (5ml EDTA tube) will be taken to identify and prospectively validate unique genes that can be used as biomarkers to predict treatment outcome (efficacy and toxicity) after chemotherapy.

Optional secondary study endpoints

• Proteomics andcytokines: before cycle 1 an extra blood sample (10ml gel tube) will be taken to identify protein profiles and cytokines that can be used as biomarkers topredict treatment outcome (efficacy and toxicity) after chemotherapy (see blood sampling 8.6).
• Chemotherapy-induced DNA damage inleukocytes: at the first andthe last AC or FEC chemotherapy cycle, 2 extra blood samples (10 ml heparin tube each) will be taken before chemotherapy (baseline value) and3 hours after start of chemotherapy for quantification ofchemotherapy-induced DNA damage in leukocytes. Chemotherapy induced DNA damage will be measured using the comet assay and the phosphorylation of H2AX, a protein involved in the DNA damage response (see blood sampling8.6).
• Nutrient sensing systems in leukocytes: at the first and the last AC or FEC cycle and before the last docetaxel cycle chemotherapy cycle, 2 extra blood samples (10 ml heparin tube each) will be taken for quantification of nutrient sensing systems in leukocytes before (baseline value) and 3 hours after start of chemotherapy. mTOR, AMPK, sirtuin and IGF-1 pathways will be characterized by Western blot, microarray and polymerase chain reaction (see blood sampling 8.6).