1. ACRIN Breast Committee Fall Meeting 2010 6688 PHASE II STUDY OF FLUORINE-18 3'-DEOXY-3'-FLUOROTHYMIDINE (F-18-FLT) IN INVASIVE BREAST CANCER Lale Kostakoglu, MD, MPH David Mankoff, MD, PhD Fenghai Duan, PhD ACRIN Breast Committee

2.  FLT structural analog of thymidine  Although FLT is not incorporated into DNA, it is trapped in the cell through phosphorylation by TK1  FLT PET enables non-invasive imaging and quantification of tm proliferative activity in proportion to DNA synthesis rate  FLT PET can be used as an imaging probe to assess impact of therapy on tm cell proliferation, especially using targeted drugs Buck AK, Methods 2009: 48:205 [F-18] FLT Background

3. Kenny, EJNM 34:1339, 2007 FLT in BREAST CANCER Aim: determine FLT-PET response at 1 wk in BC pts treated with chemo; determine the reproducibility of serial scans  17 discrete lesions in 13 stage II–IV breast ca pts  Imaging prior to and at 1 wk after treatment with chemo  Clinical response assessed 60 dys after initiation of chemo  6 pts significant clinical response at dy 60; these also had a significant reduction in FLT uptake at 1 wk  Decrease in SUV at 1 wk discriminated btw clinical response and SD  FLT response preceded tm size changes SUVmax in CR/PRSUVmax in SD

4. VIRGINIA COMMONWEALTH UNIVERSITY AMERICAN COLLEGE OF RADIOLOGY IMAGING NETWORK ACRIN 6688 (amendment 6) PHASE II STUDY OF 3'-DEOXY-3'-18F FLUOROTHYMIDINE (FLT) IN INVASIVE BREAST CANCER

5. Protocol Investigators VCU Study ChairVCU Study Co-ChairVCU Study Co-Chair Paul R Jolles, MDHarry D Bear, MD, PhDMichael O Idowu, MD 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

6. To correlate the % change in SUVs between baseline (FLT1) and early-therapy (FLT2) with pCR (as a dichotoumous variable) to neoadjuvant chemotherapy of the primary tumor in LABC (Changed to early therapy from mid-therapy) Primary Objective

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

8. evaluate correlation or relationship between, FLT1 and FLT3 uptake parameters and proliferation markers FLT1, FLT2 and FLT3 uptake parameters and pCR of the primary tm and residual cancer burden (RCB) FLT1, FLT2 and FLT3 uptake parameters and non-response of the primary tm (SD or prog) FLT1, FLT2 and FLT3 uptake parameters and pCR in pts with regional disease in the LNs compare changes of, FLT2 and FLT3 uptake parameters to changes in tm sizes from other serial imaging modalities (mammogram, MRI, and US, as available) FLT2 and FLT3 uptake parameters to metabolic changes from FDG PET, as available monitor for potential safety issues and define any physiologic effects associated with FLT administration Secondary Objectives

9.  Three imaging sessions pre-treatment (FLT-1), after one cycle (FLT-2), at completion (FLT-3)  FLT-1 (baseline PET) must be completed within 4 wks prior to chemo initiation  FLT-2 (early PET) must be performed 5-10 dys after initiation of the first chemo cycle  FLT-3 (post therapy PET) will be performed after the completion of chemo and within 3 wks prior to surgery Timing of FLT PET Studies

10.  There is no specific neoadjuvant chemo regimen required for this protocol  Subjects for the study may be recruited from prospective neoadjuvant chemo trials, which may also include targeted agents, such as trastuzumab  However, patients on neoadjuvant protocols using hormonal therapy alone are not eligible Neoadjuvant Therapy

11.  Pathologically confirmed BC, a candidate for neoadjuvant therapy and for surgical resection of residual primary tm after neoadjuvant therapy  Tumor size >2cm, measured on imaging or estimated by PE  No obvious contraindications for primary chemotherapy  Residual tumor planned to be removed surgically following completion of neoadjuvant therapy  Age >18  ECOG Performance Status ≤ 2 (Karnofsky ≥ 60%)  Normal organ and marrow function at 1 st visit: -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; OR creatinine clearance ≥30 mL/min/1.73 m 2 for pts with cr levels above normal; Inclusion Criteria determined to be

12.  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

13.  Prior treatment (any) to the involved breast  Uncontrolled intercurrent illness  Medically unstable  Unable to lie still for 1.5 hrs, requirement of anesthesia  History of allergic reactions attributed to compounds of similar chemical or biologic composition to FLT  Pregnant or nursing or age<18  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  Currently on hormone therapy as a primary therapy (aside from hormonal replacement therapy) Exclusion Criteria

14. Study Calendar <4 wks Pre-Study Pre-therapy (FLT-1) After 1 wk (5-10 dys) (FLT-2) Post-therapy(FLT-3)Surgery FLT PET/CT XXX Informed Consent X DemographicsX Medical Hist. XX HeightXX WeightXXXXX PEX CBCw/diff, Plts X S. Chemistry X AE Evaluation XXX

15. Visit 1: Screening visit -screening assessment will occur to determine eligibility -signed consent form will be obtained prior to study trial -CBC with differential and serum chemistry, and platelets. -If data available in the appropriate time window, they need not be repeated -medical history, demographics, height, weight, and PE -tissue samples/slides from bx will be sent to VCU Pathology Visit 2: FLT PET Imaging Studies (FLT1) -baseline FLT PET scan; within 4 wks prior to chemo Visit 3: FLT PET Imaging Studies (FLT2) -early therapy FLT PET;5-10 dys after the initiation of 1 st cycle Visit 4: FLT PET Imaging Studies (FLT-3) -post therapy FLT PET;after chemo & within 3wks prior to surgery Visit 5: Surgery -After neoadjuvant chemo, surgical resection of residual tm -A portion of residual tm sample/slides will be sent for to VCU Core Path for analysis and proliferation assays within 2 wks post surgery - If no viable tumor remains, a pCR will be documented Study Procedures for pre-study evaluation.

16. 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  The preferred imaging sequence is to obtain dynamic PET imaging first, followed by the torso survey using static PET imaging, however, for patients who are unable to tolerate lying in the scanner for dynamic imaging or for centers where scanner availability is limited, SUVs using static PET imaging starting 60 minutes after injection fulfills the needs of the study Analyses SUV 30 Patlak slope SUV 30-60 Flux FLT SUV 60 k 3 Imaging Sessions

17. FLT Parameters Compared To Pre-Rx (FLT1) parametersKi-67/mit index, biopsy Pre-Rx (FLT1) parametersKi-67/mit index, biopsy Clinical Response Path. Response (pCR and RCB) After one cycle (FL2) parameters Clinical Response After one cycle (FL2) parameters Clinical Response (absolute and % change from FLT1) Response from other imaging modalities (as available) (absolute and % change from FLT1) Response from other imaging modalities (as available) Path Response (pCR and RCB) Post-therapy (FLT3) parametersKi-67/mit index, surg specimen Post-therapy (FLT3) parametersKi-67/mit index, surg specimen (absolute and % change from FLT1) Clinical Response (absolute and % change from FLT1) Clinical Response Response from other imaging modalities (as available) Path. Response (pCR and RCB) Data Analysis

18.  pCR defined as the absence of viable invasive tm at histopathologic exam of post-therapy surgical specimen  This analysis will be performed at the local treating site and reviewed at the central site at VCU  Presence of residual non-invasive cancer (DCIS) in the absence of viable invasive cancer is still considered a pCR  Dichotomous response assessment; pCR vs non pCR  A secondary related measure will also be assessed, the residual cancer burden (RCB) as a more continuous variable which will be used for secondary objectives Pathologic Complete Response http://www3.mdanderson.org/app/medcalc/index.cfm?pagename=jsconvert3

19.  Clinical Response as per routine by the treating physician based upon the % change in anatomic tm size between the pre, early, and post-treatment time points  The assessment of size will be made per routine of the treating physician and will typically be performed by one of the following: PE, mammography, US, or breast MRI  The same method should be used consistently for each patient throughout this study. The categorization of clinical response is categorized as described in Table Response CategoryCriteria Complete Response (CR): Disappearance of the primary tumor Partial Response (PR): At least a 30% decrease in the LD of primary tm, from baseline LD Progressive Disease (PD): At least a 20% increase in the LD of primary tm, taking as reference the smallest LD since treatment started Stable Disease (SD): Neither sufficient shrinkage to qualify for PR nor ncrease to qualify for PD Clinical Response

20. Enrollment Target  54 cases in 18 months Initial Sites: MSSM, UPENN, UW, VCU Site enrollment expectations: 60 - 70 % of what site reported on application Trial enrollment expectations: min 3 pts per mo The ACRIN Biostatistics and Data Management Center (BDMC) will monitor participant accrual Accrual Goals

21. Accrual Plot and Current Accrual Rate Last 3 months: Avg 2 pts/month Last three months: Average 2 pts/month

22. Opened Accrual Participating Institutions and Accrual Status University of Pennsylvania School of Medicine3/4/20101 Washington University Medical School7/28/20100 Thomas Jefferson University Hospital5/4/20102 University of Washington8/18/20101 Virginia Commonwealth Univ. Health System9/14/20097 Scottsdale Medical Imaging, LTD7/28/20100 Milton S. Hershey Medical Center9/21/20100 Excel Diagnostics Imaging Clinics7/28/20101 Mount Sinai Medical Center4/27/20101 Fox Chase Cancer Center8/23/20100 University of Arkansas9/24/20100 Wake Forest University9/24/20100

23. FLT will be purchased from a commercial vendor. The vendor must be authorized within the NCI IND (so far Cardinal and PETNET designated). FLT can only be synthesized on site if the chemistry manufacturing and control procedures are filed within the NCI IND (University of Washington is the inly site). Research Radiopharmaceutical

24.  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

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