1. Differentiation between cancer and inflammation in lung cancer imaging Chumy Nwogu, MD, PhD, FACS Associate Professor of Oncology & Surgery Roswell Park Cancer Institute University at Buffalo MEDAMI September 7 th, 2014

2. Outline Background Clinical Applications of Molecular Imaging in Lung Cancer Evolution of Radioguided Lung Cancer Imaging Limitations Future Prospects

3. Background Lung Ca – most frequent cause of cancer death worldwide 15% SCLC ; 85% NSCLC LN metastasis – most important prognostic factor in locoregional NSCLC Current staging methods are suboptimal 40% of Stage I (node negative) patients will recur within 2 years Understaging – unrecognized micrometastases

4. Lymph Node Drainage Patterns

5. Background PET-CT is currently the imaging modality of choice for NSCLC clinical staging All resected LNs undergo bisection and H&E staining Conventional pathologic techniques can miss micrometastases Defined as 0.2-2.0 mm sized tumor deposits in LNs IHC and RT-PCR (ultra-staging) are much more sensitive but labor intensive & expensive Need for a tool to select most suspicious LNs for ultra-staging

6. PET-CT

7. Background Incidence of micromets is about 19 - 26% Can be detected by multiple step sections, IHC, RT-PCR Skip metastases also occur There is great value in assessing ALL the regional lymph nodes in patients Higher # of lymph nodes examined and lower lymph node ratio are both associated with better disease specific and overall survival Kim AW. Sem in Thorac & Cardiovasc Surg 2009;21(4):298-308 Nwogu C, et al. Number of Lymph Nodes and Metastatic Lymph Node Ratio are Associated with Survival in Lung Cancer. Ann Thor Surg 2012; 93(5):1614-1619

8. Clinical Applications of Molecular Imaging in Lung Cancer Risk stratification of CT screening detected lung nodules Sentinel lymph node mapping Identification of all malignant thoracic lymphadenopathy Localization of small lung nodules Cytotoxic therapy for lymph node metastases

9. Sentinel Lymph Node Mapping Sentinel LNs – 1 st echelon of LNs draining a tumor Initial reported technique: Isosulfan blue SLN identified in 17 of 36 pts (47%) The other pts showed diffuse staining of the lung Difficulty differentiating blue nodes from anthracotic nodes 5 cases of unexpected N2 were identified Little A.G., et al. J Thorac Cardiovasc Surg 1999;117:220-224

10. Use of Tc-99m Successful migration on Tc-99m in 120 of 148 pts (81%) Lack of Tc-99m migration in 19% SLN identified in 104 of 120 pts (87%) 104 of all 148 pts (70%) SLN harbored cancer in 32% of pts Upstaging occurred in 8 of 104 pts (5.5%) Liptay MJ, et al. Ann Thorac Surg 2000;70:384-389

11. RPCI Preclinical Study Pulmonary Lymphatic Mapping in Dogs Technetium 99m sulfur colloid & Isosulfan blue dye Isosulfan blue dye detected SLN in 50% within 5 minutes Tc 99m detected SLN in 83% within 20 minutes Tc 99m technically easier to use Nwogu CE, Kanter PM, Anderson TM Cancer Invest 2002; 20(7-8):944-947

12. Multicenter Phase II CALGB Study 46 pts accrued in 2 yrs (150 planned) SLN detected in 24 of 39 pts (61.5%) SLN accurate in 20 of 24 pts (83.3%) Overall accuracy of 51.2% (20 of 39 pts) Closed early due to poor accrual, low accuracy and loss of funding. Liptay MJ, D’Amico TA, Nwogu CE, et al. J Thorac Oncol 2009;4:198-202

13. Multicenter Phase II CALGB Study Collaboration challenges b/w some nuclear medicine, surgery & pathology depts State radiation safety obstacles Some surgeons possibly still in their learning curve: 8 surgeons performed an average of 5 cases (1-13)

14. Limited adoption of SLN mapping in NSCLC Perceived difficulty of technique Surgical delay after intra-op isotope injection Extra procedure for injection of Tc99m-tin colloid pre-op Controversial clinical benefit Low morbidity of thoracic lymphadenectomy Nwogu CE. Sem in Thorac & Cardiovasc Surg 2009;21(4):323-326

15. Pilot Gamma Probe Study Radioguided Detection of NSCLC lymph node mets Assessment of all lymph nodes (not just the sentinel LNs) Use of 18F-FDG instead of Tc or blue dye Nwogu C, Fischer G, Tan D, et al. Ann Thorac Surg 2006;82:1815-1820

16. Objectives Measure the feasibility of a hand-held gamma probe to detect FDG in malignant tissue Primary tumor Lymph nodes Explore the ability of this technique to improve lymph node staging in NSCLC

17. Gamma Probe

18. Methods 10 pts enrolled – all had pre-op PET scans Pts had tumors >3cm or enlarged thoracic LNs on CT 10-15 mCi FDG injected on the day of surgery Handheld probe used intraop to assess Primary tumor Regional lymph nodes Lung resection with lymphadenectomy Pathologic assessment H&E Serial sections & IHC of probe +, H&E - LNs

19. Results The i ntra-op handheld gamma probe detected All PET positive lesions 2 cases with FDG avid LNs not seen on PET scan Only the ex-vivo counts were useful 9/55 (16%) of all lymph nodes ultrastaged 4 of 10 cases with micrometastases 3 cases (30%) were upstaged 37% false positive rate

20. Conclusions from Pilot Study Detection of occult LN metastases with an FDG-sensitive intra-op gamma probe is feasible This can improve NSCLC staging Larger study required to determine the accuracy and predictive value of this technique

21. Follow-up Gamma Probe Trial 100 patient trial Stage I and II NSCLC patients Specific Aim 1: Assess the utility of an intra-operative gamma probe in detecting FDG avid LNs Specific Aim 2: Assess the clinical relevance of the gamma probe detected lymph node metastases Supported by an NCI K-23 grant (K23 CA122182)

22. Results Median additional operative time for LN mapping was 5 minutes (Range 0-20) Detection of FDG avid (hot) lymph nodes PET-CT: 6 patients Gamma Probe: 86 patients Median # of LNs harvested/patient – 10 (Range 1-21)

23. PET-CT detection of lymph node metastasis (H&E/IHC) PET-CT resultLN Disease by H&E/IHCNo LN Disease by H&E/IHC Positive82 Negative19323 Total27325 Sensitivity = 30% (95%CI=13.75%, 50.18%) Specificity = 99% (95%CI=97.84%, 99.93%)

24. PET-CT detection of lymph node metastasis (RT-qPCR) PET-CT resultLN Disease by RT-PCR No LN Disease by RT-PCR Positive44 Negative33160 Total37164 Sensitivity = 11% (95%CI=3.03%, 25.42%) Specificity = 98% (95%CI=93.87%, 99.33%)

25. Gamma probe detection of lymph node metastasis (H&E/IHC) Gamma probe resultLN Disease by H&E/IHCNo LN Disease by H&E/IHC Positive (Hot)20155 Negative (Cold)7170 Total27325 Sensitivity = 74% (95%CI=53.72%, 88.89%) Specificity = 52% (95%CI=46.59%, 57.59%)

26. Gamma probe detection of lymph node metastasis (RT-PCR) Gamma probe resultLN Disease by RT-PCR No LN Disease by RT-PCR Positive1482 Negative2382 Total37164 Sensitivity = 38% (95%CI=22.46%, 55.24%) Specificity = 50% (95%CI=42.10%, 57.90%)

27. Comparison based on RT-PCR

28. ROC Curve for gamma probe

29. Agreement b/w PET-CT and Gamma Probe PET-CTGamma Probe Positive Gamma Probe Negative Positive35 Negative179178 Percent Agreement b/w PET-CT & Gamma Probe = 49.6% Kappa statistic (measures non-chance agreement): -0.0109 (-0.041, 0.0192; Poor)

30. Results 18 patients had malignant LNs by H&E IHC detected additional metastatic LNs in 4% of patients RT-PCR detected additional metastatic LNs in 47% of patients Median patient follow-up is 24 months Worse disease free survival in patients with positive LNs by H&E/IHC but not by RT-PCR

31. Discussion 90% of patients were in clinical stage I with relatively small tumors Lower probability of occult LN mets compared to pilot study or the literature Upstaging of 4% of pts by IHC and 47% by RT-PCR Need for adjuvant chemotherapy trial on patients with occult LN metastases

32. Discussion The gamma probe was more sensitive but less specific than PET-CT in predicting malignant LN disease The poor specificity of the gamma probe limits its clinical utility Inability to differentiate between malignancy and inflammation Strong need for better radioisotopes (other than FDG)

33. Thoracic Radioguided Node Mapping Challenges High background radioactivity Aerosolization of Tc-99m Cardiac FDG uptake Primary tumor ‘shine-through’ effect Intrapulmonary lymph nodes Need for minimally invasive probes Mediastinoscopy Thoracoscopy Robotic surgery

34. Non-radioactive Tracers Peritumoral injection of various non- radioactive tracers for SLN mapping have been investigated Ferumoxides (magnetite) detected with a sterilizable magnetometer Near-Infrared Fluorescent Quantum Dots or Indocyanine Green (ICG) Oncolytic herpes virus with a green fluorescent protein (GFP) transgene

35. Near Infrared Fluorescence Imaging NIR light is invisible Wavelength 700-1000 nm Special imaging system required Contrast Agents Indocyanine Green (ICG) Quantum Dots (QDs) – fluorescent semiconductor nanocrystals

36. NIR Fluorescence Imaging

37. Soltez EG, et al. Ann Thorac Surg 2005; 79:269-277

38. 18 F-FDG Versus 18 F-fluoro-L- tyrosine PET Protein synthesis and amino acid transport are enhanced in most tumor cells but affected less in inflammatory conditions 22 patients: 11 NSCLC, 10 lymphomas, 2 head & neck carcinomas 18 F-TYR PET only visualized 67% of the lesions identified by 18 F-FDG PET Tumor SUV 5.2 vs. 2.5; tumor: muscle ratio 7.4 vs. 2.7; tumor:mediastinum activity ratio 3 vs. 1.4 - all favored 18 F-FDG PET Conclusion: 18 F-TYR PET not superior to 18 F-FDG PET Hustin R., et al. J Nucl Med 2003; 44(4):533-9

39. 18 F-FLT PET 3’-deoxy-3’-[ 18 F]fluorothymidine PET Most promising nucleoside metabolic tracer 28 women with suspicious breast findings on conventional imaging (mammography & ultrasonography) Whole body and regional breast PET performed Same Sensitivity (92.3%) but better specificity (66.7% vs. 52.4%) with the regional PET Wang J., et al Ann Nucl Med 2014 Aug 20 [Epub ahead of print]

40. 18 F-FLT PET/CT imaging in inflammation Wister rabbit inflammation model 8 rabbits with tibial Staph aureus abscesses 8 rabbits with tibial sterile granulomas After 4 weeks, 18 F-FLT PET/CT - SUV MAX 5.76±0.25 vs. 1.15±0.32 (P<0.01) Conclusion: 18 F-FLT is not a tumor specific tracer Tan Y., et al Exp Ther Med 2014; 8(1):69-72

41. Therapeutic SLN Imaging Targeted SLN Imaging Liposomes Dendrimers Quantum Dots Chemical & biologic cytotoxic agents Attenuated viruses Genetically modified bacteria

42. Future Prospects Reliable, reproducible lymph node mapping techniques Sensitive, affordable path methods for micrometastasis detection Elimination of interference from the heart, major blood vessels or central airway More specific PET radioisotopes 124 I in preclinical studies Heptamethine cyanine based 64 Cu-PET Probe PC 1001

43. Future Prospects Easy detection of peri-tumoral LN radioactivity Integration with other molecular lung cancer staging techniques Coupling therapeutic with diagnostic agents 124 I used for both PET imaging & Photodynamic therapy (PDT) Improvement in NSCLC staging, therapy and outcomes

44. Collaborators Todd Demmy, MD Sai Yendamuri, MD Mary Reid, PhD Alex Adjei, MD, PhD Alan Hutson, PhD Ravi Pandey, PhD Gal Shafirstein, PhD Richard Cheney, MD Dominic Lamonica, MD Paul Bogner, MD Elizabeth Repasky, PhD

45. Thanks Questions?