Principal Investigator: Charles E. Cox, M.D. Co Investigators: Stefan Glück, M.D.

MINT Title: MINT I Multi-Institutional Neo- adjuvant Therapy MammaPrint Project I Principal Investigator: Charles E. Cox, M.D. University of South Florida, Tampa FL Co Investigators: Stefan Glück, M.D. and Alberto Montero M.D. University of Miami/Sylvester Comprehensive Cancer Center University of Miami, Miller School of Medicine FL Total of 226 patients; up to 10 institutes in the USA 44K gene expression profiling Agendia Inc Central pathology review at Moffitt Cancer Center FL Timelines; Oct Oct 2013

MammaPrint: 70-gene profile prognostic and predictive tumor analysis Will patient benefit from chemotherapy? TargetPrint: Gene expression of ER/PR/HER2 Centralized lab confirmation of receptor status Will patient benefit from hormonal treatment? BluePrint: 80-gene molecular subtyping profile Basal, Luminal, and HER2 subtypes Which therapy works best? TheraPrint: Gene expression of 56 genes Potential markers for prognosis and therapeutic response Potential therapy options saved for the future Breast Cancer Symphony Suite

BluePrint - Identifies molecular subtypes of breast cancer Analysis of Entire Human Genome ~25,000 Genes Prognostic & Predictive Breast Cancer Genes Identified Breast Cancer Basal, Luminal, and HER2-type classification BluePrint Luminal-Type Basal-Type HER2-Type

TargetPrint: quantification of ER/PR/HER2 mRNA levels Analysis of Entire Human Genome ~25,000 Genes Prognostic & Predictive Breast Cancer Genes Identified Breast Cancer Centralized lab confirmation of receptors

IHC microarray Microarray based readout of ER, PR and HER2

TheraPrint - Suggests alternative treatment options in advanced disease Analysis of Entire Human Genome ~25,000 Genes Prognostic & Predictive Breast Cancer Genes Identified Breast Cancer Gene expression analysis for drug sensitivity

TheraPrint: quantitates 56 genes linked to specific drug responses Offers read-out of gene expression for 56 genes Genes might have relevance in breast cancer therapy and prognosis No claims about mRNA level and response can be made Genes Included:

The response to chemotherapy varies in the different molecular subtypes Straver 1 Somlo 2 Hess 3 Total npCRn n n % Luminal-type/MP Low Risk % Luminal-type/MP High Risk % HER2-type % Basal-type % 1. Straver et al. Breast Cancer Res Treat, Somlo et al. ASCO, Hess study as part of Krijgsman et al. In press, 2011

MINT; Study Objectives To determine the predictive power of chemosensitivity of the combination of MammaPrint and BluePrint as measured by pCR. To compare TargetPrint single gene read out of ER, PR and HER2 with local and centralized IHC and/or CISH/FISH assessment of ER, PR and HER2. To identify possible correlations between the TheraPrint Research Gene Panel outcomes and chemo-responsiveness. To identify and/or validate predictive gene expression profiles of clinical response/resistance to chemotherapy. To compare the three BluePrint molecular subtype categories with IHC-based subtype classification.

MINT; Eligibility Criteria Inclusion criteria: Women with histologically proven invasive breast cancer; T2(≥3.5cm)-T4, N0,M0 or T2-4N1M0 DCIS or LCIS are allowed in addition to invasive cancer at T2 or T3 level. Age ≥ 18 years. Measurable disease in two dimensions Adequate bone marrow reserves, adequate renal function, and hepatic function Signed informed consent Exclusion criteria Patients with inflammatory breast cancer. Tumor sample shipped to Agendia with ≤ 30% tumor cells or that fails QA or QC criteria. Patients who have had any prior chemotherapy, radiotherapy, or endocrine therapy for the treatment of breast cancer. Any serious uncontrolled inter current infections, or other serious uncontrolled concomitant disease.

MINT; Nodal staging

Study Design Flowchart Core Needle Biopsies Sample placed in RNA Retain, send to Agendia* Breast Cancer Symphony Suite successful Breast Cancer Symphony Suite not successful Patient ineligible Patient information & informed consent Neo- adjuvant treatment CRF 1 baseline Surgery CRF 2 surgery * (Diagnostic commercial testing for Symphony Breast cancer Suite)Suite FFPE Slides and microscopic worksheets to USF Pathology

Neo-adjuvant therapy For HER2 negative patients: TAC chemotherapy TC chemotherapy Dose Dense AC or FEC100 followed by paclitaxel or docetaxel chemotherapy For HER2 positive patients: TCH chemotherapy Dose adjustments Hematological and non-hematological toxicities should be managed by treating oncologist as per routine clinical practice.

Tissue Collection Tissue should be collected by incisional biopsy (when placing port) or via core needle biopsy. If the tissue is obtained by incisional biopsy then the tissue sample should be no greater than 3 to 4 mm in thickness and between 8 to 10 mm in diameter. Core needle biopsies should be obtained with a 14 gauge or larger needle. ◦ If a 14 gauge needle is used: 5 cores ◦ If a 11 gauge needle is used: 4 cores ◦ If a 9 gauge needle is used: 3 cores

Sending Sample to Agendia A. Remove large specimen tube from kit and open it. B. Place large screw cap with small specimen vial on table and open small specimen vial. C. Place a barcode label from the completed requisition form on to the vial. D. Place the small specimen vial into the large specimen tube and place the tube into the specimen safety bag. E. Place the sealed specimen bag into the shipping kit along with the requisition form. IMPORTANT: ensure that the study sticker is affixed to the requisition form. F. Package the kit into the FedEx shipping pack and attach the pre printed label for shipment to Agendia Inc. G. Please call Fed Ex for pickup.

Central Pathology Review Initial core/incisional biopsy specimens Cold ischemic time (time from collection of specimen to fixation solution) should be restricted to < 1 hour Tissue submitted for histopathological analysis will be fixed in 10% buffered formalin for 6 to 48 hours Send 1 H&E, original ER, PR, and HER2 IHC stains, and 10 unstained sections of representative tumor on positive-charged glass slides for central review (Appendix III). If receptor studies are not available, an additional 10 unstained sections on positive-charged glass slides of representative tumor will be required.

Central Pathology Review Gross specimen processing 1. Lumpectomy/partial mastectomy The specimen is oriented, inked in 6 colors, and sectioned at cm intervals. If gross residual tumor is identified: ◦ Dimensions (width, length, height) are recorded ◦ Distance from all margins if < 0.2 cm is noted, otherwise the closest margin is recorded ◦ The gross residual tumor is entirely submitted in sequential sections (Appendix IV) ◦ Sampling of margins is performed If no-grossly identifiable residual tumor is present: ◦ The dimensions of the biopsy site and surrounding fibrosis/ induration is recorded ◦ The specimen is entirely submitted sequentially (Appendix IV) ◦ Description of margin status is recorded as noted above

Central Pathology Review 2. Total mastectomy/modified radical mastectomy The specimen is oriented and inked accordingly If gross residual tumor is identified: ◦ Dimensions (width, length, height) are recorded ◦ Distance from all margins if < 0.2 cm is noted, otherwise the closest margin is recorded ◦ The gross residual tumor is entirely submitted in sequential sections (Appendix IV) ◦ Sampling of margins is performed If no-grossly identifiable residual tumor is present: ◦ The dimensions of the biopsy site and surrounding fibrosis/ induration is recorded ◦ The area of fibrosis (“tumor bed”), to include biopsy site, is entirely submitted sequentially (Appendix IV) ◦ Description of margin status is recorded as noted above

Central Pathology Review Sentinel lymph node processing Sentinel lymph nodes will be serially sectioned along the long axis at 2-mm intervals. If the lymph node measures 0.5 cm in greatest dimension, it may be bivalve. Specimens are then entirely submitted (Appendix IV).

Clinical Report Form Web based data collection/electronic CRF ◦ CRF1; Baseline information ◦ CRF2; After surgery Relatively easy to complete compared to drug trial minutes for CRF1 and 2

Accessing the Database Accessing the Database ◦ There is one hyperlink to access all the electronic Case Report Forms (CRFs) ◦ You will receive an institution specific site number and password from your Agendia Clinical Research Manager. Institution:

Detailed Data Entry Instructions will be sent to site. Samples will appear in the database if they are found to be eligible. CRF Overview Administrative page

CRF 1: Questions Baseline Patient characteristics Age at diagnosis Ethnicity/ origin Menopausal status Date of biopsy Specimen type Tumor measurements Kind of imaging used Primary tumor size Size largest metastatic lymph node Pathology Date of histologic diagnosis Histopathologic tumor type Histological grade ER, PR and Her2-neu status Vascular invasion TNM Nodal staging Date nodal staging If sentinel lymph node Number of sentinel nodes removed Number of counts Blue dye Histology of SLN: Concordance of nodal histology to primary tumor

CRF 2: Questions Neo-adjuvant treatment Regimen given Was specified number of cycles completed? Any grade 4 or 5 CTC for Adverse Events observed? Surgery information Date breast carcinoma surgery Type of surgery Lymph node assessments Pathology Nodal status ypTN Tumor measurements What kind of imaging has been used ? Primary tumor size Size largest metastatic lymph node Treatment response Lymph node assessments Sentinel Lymph Node Procedure (SLNP) If yes Number of sentinel nodes examined Number of positive sentinel nodes Number of negative sentinel nodes Blue dye Concordance of nodal histology to primary tumor Axillary Lymph Node Dissection (ALND) If yes Number of axillary nodes examined Number of positive axillary nodes Number of negative axillary nodes SLNP and ALND if yes:

Contacts: Jessica Gibson, Clinical Research Manager (619)