1. Evolution in Thoracic Oncology: Genomics and Treatment for Early Stage NSCLC David H. Harpole, Jr., M.D. Professor of Surgery Associate Professor of Pathology Vice-chairman, Faculty Affairs Duke University Medical Center

2. Therapy Concerns Before Resection A: What resection is sufficient? - Lobectomy for most patients - Current phase III trials exam sub-lobar resection in higher-risk cohorts B: Who needs therapy? - When is resection alone sufficient? C: What therapy and when? - Before (induction) or after (adjuvant)? - Role for radiotherapy in 2009?

3. Who to Treat: LACE Meta-analysis: Chemotherapy Effect Recommended for stage II-IIIA Not recommended for stage I (A or B)

4. Multivariate Risk ratio p Value Vascular invasion 2.36 0.001 T2 tumor size 1.88 0.002 Visceral pleural + 1.75 0.022 > 15 mitoses / HPF 1.51 0.074 12 24 36 48 60 72 Months 0.0 0.25 0.50 0.75 1.0 Size (cm) (n)5-Year Surv 0 to 215974% 2 to 420760% > 4 4245% p=.0002 Risk Model in Stage I NSCLC: Histopathology Harpole et al., Cancer 1995 Survival (n=410) Who to treat: Tumor Biology

5. Protein Expression (IHC) Stage 1 (n=408) Multivariate Survival AnalysisRisk ratiop Value p531.630.0037 Angiogenesis factor VIII 1.4700.0333 Her2-neu1.4290.0440 CD-441.3990.0500 Rb Rb0.7470.0738 D’Amico et al. J Thor & Cardiovasc Surg, 1999 12 24 36 48 60 72 Months 1.0 0.75 0.50 0.25 Survival Total Markers(n)5-yr.Median 0 to 114077%Not reached 212462%Not reached 3 to 514449%58 months p=0.0001 Who to treat: Tumor Biology

6. Observational Data Observational Data Histopathology (H/E Histopathology (H/E) Molecular Data Molecular Data Protein (IHC) Protein (IHC) DNA / RNA (PCR) DNA / RNA (PCR) Genomic Data Genomic Data Proteomic Data Proteomic Data Microarray Microarray Prognosis based on Tumor Biology

7. Who to treat: Early Stage NSCLC Patients Current Tools for Prognosis Clinical and histopathologic factors Single molecular biomarkers But, the challenge is to provide an individualized patient prognosis Gene expression profiles

8. Current Therapy for Clinical Stage I NSCLC Stage I Stage II and IIIA Adjuvant Chemotherapy (> 30% relapse) Observation (40% relapse) Resection Clinical Stage 1 (45,000 patients in U.S.) Identify Patients at Higher Risk Higher Risk

9. Current Therapy for Clinical Stage I NSCLC Stage I Stage II and IIIA Adjuvant Chemotherapy (> 30% relapse)Observation (40% relapse) Resection Clinical Stage 1 (45,000 patients in U.S.) Develop gene expression profiles that refine risk prediction

10. What is a ‘Genomic Signature’ Phenotype A (Ph A) Phenotype B (Ph B) Ph A Ph B Microarray analysis Probability of Ph A ‘Signature’

11. A Signature of Lung Cancer Recurrence Low Risk High Risk Low Risk > 5yr DFS High Risk < 2yr DFS Measure gene expression in tumors “Metagene” NEJM 2006

12. Metagene Tree Analysis: First 5 of 100 Steps Dead Alive Model has 2100 Genes

13. Early Stage NSCLC Genomic Predictor of Recurrence CALGB 30506 Stage I Lung Cancer Stage I Predicted High Risk Predicted Low Risk NEJM 2006

14. Prediction Models by Pathologic Stage Clinical-Pathology Prediction Model Lung Metagene Predictor 2100 Genes Accuracy > 90%, Sensitivity > 90% For all stages

15. Lung Metagene Predictor by Histology

16. Minnesota + CALGB 9761 Validation Set (n=85) Median F/U 59 mo. Accuracy > 90%, Sensitivity > 90%

17. Additional Metagene Validation Studies ACOSOG Z0030; n=25 Stage 1 ACOSOG Z0030; n=25 Stage 1 Mayo Clinic; Stage 1; n=15 Stage 1 Mayo Clinic; Stage 1; n=15 Stage 1 Washington University; n=71 Stage 1 Washington University; n=71 Stage 1 All together, a total of 4 independent sample sets comprising 195 samples have been used for validation

18. N=143 (73 Stage1A and 70 Stage 1B) Deaths=47 Black (high risk) 31 Red (low risk) 16 Censored=96 p=0.003 NCI Director’s Challenge Blinded Dataset Overall Survival censored at 5 Years 12 24 36 48 60 72 12 24 36 48 60 72Months Survival 1.0.25.75 0.0

19. CALGB 30506: Trial Objectives Hypothesis: A genomics prediction model (LUNG METAGENE SCORE; LMS) provides a method for selecting stage I non-small cell lung cancer (NSCLC) patients for adjuvant chemotherapyHypothesis: A genomics prediction model (LUNG METAGENE SCORE; LMS) provides a method for selecting stage I non-small cell lung cancer (NSCLC) patients for adjuvant chemotherapy Primary ObjectivesPrimary Objectives –To demonstrate a survival advantage for patients randomized to adjuvant chemotherapy compared to an observation arm (present standard of care). Observe a more significant survival advantage for High-score patients treated with chemotherapyObserve a more significant survival advantage for High-score patients treated with chemotherapy –To prospectively validate a prognostic indicator of survival in stage I NSCLC patients identified with LMS who are observed after resection.

20. CALGB 30506: Trial Objectives Secondary ObjectivesSecondary Objectives –Evaluate all other existing gene-based prognostic models –Compare the LMS with best clinico-pathologic model for survival –Validate of gene expression signatures from archived paraffin-embedded tumor samples –Quality of Life / Economics: To assess QOL/Cost in all stage I patients before and periodically after resection for NSCLC.To assess QOL/Cost in all stage I patients before and periodically after resection for NSCLC. To examine the impact of chemotherapy on QOL/Cost for patients in the chemotherapy arm, as compared to patients in the observation arm(s)To examine the impact of chemotherapy on QOL/Cost for patients in the chemotherapy arm, as compared to patients in the observation arm(s)

21. CALGB 30506: Inclusion Criteria Pre-resectionPre-resection –Clinical T2N0 NSCLC –Suspicious tumor on CT size: 2.0 to 6.0 cm (6 cm by CT translates to < 5.0 cm by path)(6 cm by CT translates to < 5.0 cm by path) –Mediastinal node assessment if CT > 1cmCT > 1cm PET suspiciousPET suspicious –Pre-registration after Informed Consent –No other cancer or chemotherapy in 3 years At OperationAt Operation –Verified NSCLC (if no Pre-op Dx) –Resectable by lobectomy (VATS or Open) –No clinically evidence of N2 or N1 + Send frozen sections on nodes prior to enrollSend frozen sections on nodes prior to enroll –Fresh-frozen Tumor to Duke Using simple 5mm Dermal punch biopsyUsing simple 5mm Dermal punch biopsy

22. After the specimen has been released by the pathologist, cut an approximate 1.0 x 1.0 x 0.5 cm block of firm grossly homogeneous lung tumor using a sterile scalpel blade.

23. Using a 5mm dermal punch biopsy, cut three (3) core biopsies of equal size from the tumor. Using a 5mm dermal punch biopsy, cut three (3) core biopsies of equal size from the tumor.

24. The core biopsy may be difficult to extract from the dermal punch. If so, it can be pushed out using a wooden applicator stick inserted into the back end of the punch.

25. Using the forceps, immediately place one tumor biopsy sample per Cryomold onto the frozen layer of OCT. 123456-A 123456-B 123456-C

26. With the cryomold still on the cold plate, place a top layer of OCT onto the tumor specimen. * The sample should be acquired / frozen within 30 minutes.* 123456-A 123456-B 123456-C

27. CALGB 30506: Sample/Information Flow Rejected Rejected Rejected CALGB Site Genome Trials Support Facility H&E Harpole Lab RNA Quality MAW-3 EA: Arrays IGSPCALGB 3 days TISSUE RNA TISSUE RNA DATALMS SCORE PT CANREGISTERPT CANREGISTER REGISTEREDREGISTERED RANDOMIZEDRANDOMIZED 10-14 business days

28. CALGB 30506: Inclusion Criteria: Con’t. After OperationAfter Operation –Pathologic T1N0 and T2N0 (5% dropout) N2 and N1 negativeN2 and N1 negative Tumor >1.70 cm (up to 6.0 cm)Tumor >1.70 cm (up to 6.0 cm) Visceral pleural invasion includedVisceral pleural invasion included –Inadequate tumor sample (10% Dropout) > 50% necrosis> 50% necrosis < 12 µg quality RNA< 12 µg quality RNA Good quality Array data generatedGood quality Array data generated Formal RegistrationFormal Registration

29. CALGB 30506: Statistical Assumptions –Observation arm Survival for 1.7 to 6.0 cm stage I 60% at 5-years Low-score observation survival = 70+%Low-score observation survival = 70+% –(10+% increase) High-score observation survival = <50%High-score observation survival = <50% –(10+% decrease) –Chemotherapy Treatment arm survival 68-70% * (10-15% increase = HR 1.3 to 1.5) –High-score chemotherapy advantage (15+% increase = HR 1.67-2.0)(15+% increase = HR 1.67-2.0) * Based on ANITA and NCI-C JBR-10 30506 is powered based on the chemotherapy treatment study: 85% with a two-sided p<0.05. Patients and treating physicians are Blinded to LMS

30. CALGB 30506 Schema (Stage IA/IB) LM Score 0.55; 40% Adjuvant Chemotherapy Resection T (1.70 to 6.0) N0 Patients + Array Observation Randomize LM Scores Blinded to Investigators, n=1525 (1296 after 15% Ineligibility) Randomize Observation Adjuvant Chemotherapy

31. CALGB 30506: Treatment Plan Assignment to Treatment Arm by StatsAssignment to Treatment Arm by Stats –Observation or Treatment Chemotherapy within 12 weeks of resectionChemotherapy within 12 weeks of resection –Platinum Doublets to mirror ECOG 1505 Cisplatin + DocetaxelCisplatin + Docetaxel Cisplatin + GemcitabineCisplatin + Gemcitabine Cisplatin + VinorelbineCisplatin + Vinorelbine Cisplatin + Pemetrexed - added soonCisplatin + Pemetrexed - added soon This will allow combining these data with the ECOG 1505 chemotherapy-alone arm

32. CALGB 30506: Specimen Timeline Resection to Duke 30506 IGSP BankOvernightResection to Duke 30506 IGSP BankOvernight –H/E Tumor Content / Viability –RNA extraction –Gel-based RNA Quality / DNA Contamination2-3 days RNA to Duke IGSPRNA to Duke IGSP –Agilent RNA array –Probe generation –Affymetrix array –Computational Genomic model7 days Data to CALGB-Duke statsAutomaticData to CALGB-Duke statsAutomatic –Assignment + Randomization2 days Results to Site PI2 daysResults to Site PI2 days Total14 or less days (Post-op visit occurs 14-21 days after discharge)

33. Conclusions 1.CALGB 30506 is NCI-CTEP approved and activated in the CTSU. It has been endorsed by ACOSOG, ECOG, RTOG and Canadian sites. The tissue acquisition, processing, Affymetrix Genechip array generation and data analysis are funded by an NCI RO1. 2.It is hopeful that the use of this genomic prediction model will aid in the appropriate selection of stage 1 patients who will benefit from adjuvant chemotherapy.

34. Enrollment Tips It is known that node + patients have up to 15% increased survival with adjuvant chemotherapyIt is known that node + patients have up to 15% increased survival with adjuvant chemotherapy It might be the case for node-negative tumors, but we need a trial to show that (standard is no chemo)It might be the case for node-negative tumors, but we need a trial to show that (standard is no chemo) Everyone gets an equal chance of observation or chemotherapyEveryone gets an equal chance of observation or chemotherapy Everyone is does not know the score of your tumor except the NCI statisticiansEveryone is does not know the score of your tumor except the NCI statisticians You can pick the pair of drugs with your medical oncologist These are FDA-approved Standard Doses of Standard Drugs for adjuvant therapy in NSCLC. No experimental use. These are covered by insurance and mandated coverage by Medicare/Medicaid.You can pick the pair of drugs with your medical oncologist These are FDA-approved Standard Doses of Standard Drugs for adjuvant therapy in NSCLC. No experimental use. These are covered by insurance and mandated coverage by Medicare/Medicaid.