1. ACRIN EISC VIRGINIA COMMONWEALTH UNIVERSITY AMERICAN COLLEGE OF RADIOLOGY IMAGING NETWORK ACRIN 6688 PHASE II STUDY OF 3'-DEOXY-3'-18F FLUOROTHYMIDINE (FLT) IN INVASIVE BREAST CANCER Agent Name: 3'-deoxy-3'-[F-18] fluorothymidine Agent NSC Number: 750184 IND Number: 71,260

2. ACRIN EISC Protocol Investigators VCU Study ChairVCU Study Co-ChairVCU Study Co-Chair Paul R Jolles, MDHarry D Bear, MD, PhDMichael O Idowu,MD,MPH Dept Radiology Dept of Surgery Dept of Pathology Richmond, VA Richmond, VA Richmond, VA prjolles@vcu.eduprjolles@vcu.edu hdbear@vcu.edumidowu@mcvh-vcu.eduhdbear@vcu.edumidowu@mcvh-vcu.edu ACRIN Study Co-Chair David Mankoff, MD, PhD Lale Kostakoglu, MD, MPH Professor of Radiology Seattle Cancer Care Alliance Mount Sinai School of Medicine Seattle, WA New York, NY 10029 dam@u.washington.edudam@u.washington.edu lale.kostakoglu@mssm.edulale.kostakoglu@mssm.edu VCU Study Statistician ACRIN Study Statistician Donna K McClish, PhD Fenghai Duan, PhD Department of Biostatistics Ctr for Statistical Sciences Richmond, VA 23298 Brown University mcclish@mail2.vcu.edumcclish@mail2.vcu.edu fduan@stat.brown.edufduan@stat.brown.edu

3. ACRIN EISC  FLT is a structural analog of thymidine  Although FLT is not incorporated into DNA, it is trapped in the cell due to phosphorylation by TK  Recently developed disease specific molecular agents induce cell cycle arrest (cytostatic effect) rather than tumor cell death (cytotoxic effect)  Evaluating alterations in DNA metabolism may reflect response to treatment better than alterations in glucose utilization  FLT PET can be used as an imaging probe to assess in vivo cellular proliferation in malignant tumors Buck AK, Methods 2009: 48:205 [F-18] FLT Background

4. ACRIN EISC Buck AK, Methods 2009: 48:205 Preliminary Studies

5. ACRIN EISC aggressiv e lymphoma Low grade lymphoma Ki-67 labeling index: >90% Ki-67 labeling index: < 5% Buck AK, Methods 2009: 48:205 Non-Invasive detection and grading of malignant lymphoma using FLT PET as surrogate marker of tumor proliferation

6. ACRIN EISC Blood The box plot shows the mean percent error (horizontal line within the box), the 25th and 75th percentiles (bottom and top of box, respectively), and the range (bottom and top horizontal bars on vertical whiskers). Shields, AF, Clin Cancer Res. 2008;14:4463 Kenny, EJNMMI 34:1339, 2007 Reproducibility of [18F]FLT Parameters

7. ACRIN EISC Kenny, EJNMMI 34:1339, 2007 Pre Therapy Post Therapy RESPONSE in a patient with grade II lobular ca NO RESPONSE in a patient with grade II IDC 7 dys post- therapy

8. ACRIN EISC Primary Objective:  To correlate the percentage change in SUVs between baseline (FLT-1) and mid-therapy (FLT-3) with pathologic complete response (pCR) to neoadjuvant chemotherapy of the primary tumor in patients with locally advanced breast cancer (LABC) Study Objectives

9. ACRIN EISC Obtain pre-treatment Proliferative Indices Establish Eligibility Baseline Imaging Mid-therapy Imaging Chemotherapy Surgical Resection Chemotherapy Baseline organ function Pathologically confirmed disease Determine primary systemic Rx Ki-67 and mitotic index on bx sample or re-biopsy (if available) 18 FLT PET/CT (FLT-1) 18 FLT PET/CT (FLT-3) 18 FLT PET/CT (FLT-2) Obtain post-treatment Proliferative Indices Pathologic response, Ki-67 and mitotic index, surgical specimens Early therapy Imaging Chemotherapy [F-18] FLT Study Outline

10. ACRIN EISC  Three imaging sessions pre-treatment (FLT-1), after one cycle (FLT-2), at mid-treatment (FLT-3)  FLT-1 PET must be completed within 3 wks prior to beginning chemo  FLT-2 PET must be performed 5-10 dys after initiation of first chemo cycle  FLT-3 must be performed halfway through the therapy protocol and at least 5 dys after completion of the last chemo prior to the mid-point, and prior to the first chemo cycle after the midpoint  For example, in a protocol consisting of 4 cycles of therapy this will be after cycle 2 and before cycle 3 and in a protocol consisting of 6 cycles, this would be after 3 cycles therapy and before cycle 4. In a regimen where a change of chemotherapy is planned, for example a change from doxorubicin-based therapy to taxane-based therapy, the midpoint would occur after the completion of the first type of chemotherapy and before the administration of the second type of chemotherapy. Imaging Protocol Timing of FLT PET Studies

11. ACRIN EISC Secondary Objectives:  evaluate the relationship between FLT-1, FLT-2 and FLT-3 uptake parameters and pCR and residual cancer burden (RCB)  evaluate the relationship between FLT-1, FLT-2 and FLT-3 uptake parameters and non-response (SD or Prog disease)  demonstrate correlation between FLT-1, FLT-2 and FLT-3 uptake parameters and tumor proliferation markers  continue to monitor for potential safety issues and define any physiologic effects associated with [ 18 F] FLT Study Objectives

12. ACRIN EISC Secondary Objectives (cont’d):  evaluate the relationship between FLT-1, and FLT-3 uptake parameters and pCR to neoadjuvant chemotherapy in patients with regional disease in the LNs  compare the changes of FLT-2 and FLT-3 uptake parameters to changes in tm sizes from other serial imaging modalities such as mammograms, MRI, and US, as available  compare changes of FLT-2 and FLT-3 uptake parameters to metabolic changes from FDG-PET, as available Study Objectives

13. ACRIN EISC  Pathologically confirmed breast cancer, determined to be a candidate for neoadjuvant therapy and for surgical resection of residual primary tm after neoadjuvant therapy. This includes all patients with locally advanced breast cancer (Stage IIIB and some IIIA), all patients with Stage IIIC disease (supraclavicular node involvement), and patients for whom neoadjuvant therapy is indicated to make breast conservation surgery feasible. The last group would be expected to have a median tumor diameter of approximately 4 cm  Tumor size >2cm, measured on imaging or estimated by PE  No obvious contraindications for primary  Residual tumor planned to be removed surgically following completion of neoadjuvant therapy  Age >18 Inclusion Criteria

14. ACRIN EISC  ECOG Performance Status ≤ 2 (Karnofsky ≥ 60%)  Normal organ and marrow function, pre-chemotherapy: - leukocytes ≥ 3,000/μl; -absolute neutrophil count ≥ 1,500/μl; -platelets ≥ 100,000/μl; -total bilirubin within N institutional limits; -AST(SGOT)/ALT(SGPT) ≤2.5 times institutional upper limit of N -creatinine within normal institutional limits; -creatinine clearance ≥ 60 mL/min/1.73 m 2 for patients with creatinine levels above institutional normal;  Able to lie still for 1.5 hours  If female, postmenopausal for a min of one year, OR surgically sterile, OR not pregnant, confirmed by ß-HCG blood test, and willing to use adequate contraception  Able to understand and willing to sign a written informed consent document and a HIPAA authorization in accordance with institutional guidelines Inclusion Criteria, con’t

15. ACRIN EISC  Prior treatment (chemo, RT or surgery) to involved breast  Uncontrolled intercurrent illness. Ongoing or active infection, symptomatic congestive heart failure, unstable angina pectoris, cardiac arrhythmia, or psychiatric illness/social situations that would limit compliance with study requirements  Medically unstable  Condition requiring anesthesia for PET scanning;  History of allergic reactions attributed to compounds of similar chemical or biologic composition to F-18 FLT  Pregnant or nursing. Pregnant women are excluded from this study because the effects of [ 18 F]FLT in pregnancy are not known. Breastfeeding should be discontinued if the mother receives [ 18 F]FLT.  Previous malignancy, other than basal cell or squamous cell carcinoma of the skin or in situ ca of the cervix, from which the patient has been disease free for < 5 years  No hormonal therapy is allowed Inclusion Criteria

16. ACRIN EISC  The participant will undergo [ 18 F]FLT injection, immediately after injection a dynamic regional PET/CT imaging will be done for 60 minutes dynamic imaging will be followed by a static whole body image from top of head to upper thigh; 5-7 bed positions  Analyses SUV 30 ∆SUV 60-120 SUV 60 Patlak slope SUV 120 Flux FLT ∆SUV 30-60 k 3 Imaging Protocol Imaging Sessions

17. ACRIN EISC [ 18 F] FLT Parameters Compared To Pre-Rx (FLT-1) parametersKi-67/mit index, biopsy Clinical Response CT Response Pathological Response (pCR and RCB) After one cycle (FLT-2) parameters Clinical Response (absolute values and % change from FLT-1)CT Response Mid-therapy (FLT-3) parametersKi-67/mit index, surgical (absolute values and % change from FLT-1) Clinical Response CT Response Pathological Response (pCR and RCB) Imaging Protocol Imaging Sessions

18. ACRIN EISC Participant Accrual Enrollment Target  54 cases in 18 months Initial Sites: MSSM, UPENN, UW, VCU Site Target: total # of sites ~10 Site enrollment expectations: 60 - 70 percent of what site reported on application Trial enrollment expectations: min 3 patients per month The ACRIN Biostatistics and Data Management Center (BDMC) will monitor participant accrual

19. ACRIN EISC  The presence of any invasive tumor cells will be considered negative for pathologic complete response Following will be performed at the Core Lab at VCU/Dept of Pathology  Ki-67 (MIB-1 antibody) Immunohistochemical staining  Mitotic index  Routine Clinical Histopathology  Calculation of Residual Cancer Burden pCR is a dichotomous, but tm response is a continuous variable with non-response ranging from very small residual tm to resistant tms with progressing disease size of the tumor bed cellularity of residual primary tumor percentage of DCIS component number of positive nodes size of macrometastasis This tool is available at http://www3.mdanderson.org/app/medcalc/index.cfm?pagename=jsconvert3http://www3.mdanderson.org/app/medcalc/index.cfm?pagename=jsconvert3 Therefore, continuous measures of residual cancer burden (RCB) would be expected to be more predictive of clinical outcome than simple dichotomous classification as currently practiced. RCB determined from routine pathologic materials may be a significant predictor of distant relapse-free survival (98). Different parameters will be collected and submitted to the data collection center for calculation of RCB and will include: The Residual Cancer Burden calculation will use clinical information obtained from participating institutions and pathological analysis at VCU. As the calculation of RCB is dependent on the parameters provided by participating institutions, the Core Laboratory at Virginia Commonwealth University will calculate the RCB as long as the parameters needed for the calculation are present in the reports. Tissue Specimen Analysis

20. ACRIN EISC Participants who,  are unable to complete chemotherapy and undego primary tumor surgery will be excluded from the primary analysis. Those who are able to complete the study to midpoint can be included in secondary analysis regardless of outcome  are unable to complete study to midpoint because of therapy toxicity or disease progression will be removed  experience any serious adverse event from the FLT PET imaging procedure as listed in Section 9.0 will be removed from the study  deviate from planned therapy for lack of response or tumor progression will be excluded from primary cohort analysis Criteria for Removal from the Study