Biophysical Determinants of Photodynamic Therapy and Approaches to Improve Outcome

1. Biophysical Determinants of Photodynamic Therapy and Approaches to Improve Outcome Theresa M. Busch, Ph.D. Department of Radiation Oncology University of Pennsylvania, Philadelphia, PA

2. What is Photodynamic Therapy? • PDT is a directed, light-based method of damaging malignant or otherwise abnormal tissues. Image from Wikipedia

3. hv Oxidation of Organic Substrates Photosensitizer 3O2 1O2 Photosensitizer3 Energy transfer How Does it Work? Type 2 Reaction

4. How Does it Work? • Mechanisms of PDT action • Direct Cell Effects • Direct 1O2-mediated toxicity to tumor cells • Indirect Effects • Vascular damage • During light treatment • Delayed development within several hours after light treatment • Stimulation of host immune responses. • Cell death may occur by apoptosis, necrosis, and/or autophagy

5. PDT Variables • Photosensitizer • Drug type • Dose • Drug-light interval • Light Delivery • Wavelength • Fluence • Fluence rate

6. FDA-Approved Indications (Oncology) Obstructive esophageal cancer* Obstructive endobronchial lung cancer* Microinvasive endobronchial lung cancer Actinic keratosis Barrett’s esophagus/ high grade dysplasia *for palliative intent Clinical Trials Pleural spread of nonsmall cell lung cancer Mesothelioma Intraperitoneal malignant tumors Head and Neck- pre-malignant through advanced disease Brain tumors Skin cancer Prostate cancer What is it used for?

7. Heterogeneity in PDT • Photosensitizer distribution • Tissue optical properties (light distribution) • Microenvironment • Tumor oxygenation • Vascular network

8. Heterogeneity in Photosensitizer Uptake: A Lesson From the Intraperitoneal PDT Clinical Trial • Hahn SM, et al. Clin Cancer Res 12:5464-70, 2006

9. How about light distribution?

10. Light absorption and scattering affects the fluence rate seen by the tissue. Tumor surface 75 mW/cm2 630 nm Normalized fluence rate 3 mm depth Distance (mm)

11. The tumor microenvironment is highly heterogeneous…. • Busch TM, et al. Clin. Cancer Res.10: 4630–4638, 2004

12. …. and PDT exacerbates heterogeneity in hypoxia distribution Control RIF Tumor During PDT 5 mg/kg Photofrin 135 J/cm2, 75 mW/cm2 • Busch TM, et al. Cancer Res.62:, 7273-7279, 2002

13. Heterogeneity AboundsSo what to do? Modify Monitor

14. Approach 1: Modify Light Delivery Rationale: • Lowering PDT fluence rate reduces the rate of photochemical oxygen consumption. • Better maintenance of tumor oxygenation during illumination. • Improves long-term tumor responses • Enhanced direct cell kill • Enhanced vascular shutdown in the treatment field

15. Hypoxia Assay • EF3 and EF5 are nitroimidazole-based drugs that binds to hypoxic cells as an inverse function of oxygen tension. • Detection is by a fluorochrome-conjugated monoclonal antibody. • Fluorescent micrographs are digitally analyzed for binding. Section, Stain for EF3/5 Fluorescence microscopy

16. Hoechst EF3 Labeling of Hypoxia during PDT PDT • RIF murine tumor • EF3 at 52 mg/kg • Treated animals receive Photofrin-PDT at 75 or 38 mW/cm2, 135 J/cm2 • Hoechst 33342 at 1.5 min before tumor excision • Cryosectioning, immunohistochemistry, fluorescence microscopy Hoechst (perfusion) Anti-EF3 Anti-CD31 Hoechst (tissue label)

17. 38 mW/cm2 75 mW/cm2 Fluence rate effects on PDT-created hypoxia EF3 Binding EF3 Binding

18. Low fluence rate reduces intratumor heterogeneity in PDT-created hypoxia

19. Causes of depth-dependent hypoxia during PDT • Light distribution? Tumor surface Normalized fluence rate 3 mm depth Distance (mm)

20. Causes of depth-dependent hypoxia during PDT • Photosensitizer distribution? Photofrin Uptake (ng/mg) S D

21. Causes of depth-dependent hypoxia during PDT • Does not appear to be a result of photochemical oxygen consumption. • How about PDT-induced vascular effects?

22. Getting at heterogeneity in vascular response during PDT sources • Diffuse Correlation Spectroscopy • Measures the temporal correlation of fluctuations in the intensity of transmitted light (785 nm) to provide information on the motion of tissue scatters, e.g. red blood cells • Data used to calculate relative blood flow, i.e. flow normalized to a pre-treatment baseline • Monitoring throughout PDT is facilitated by a non-contact camera probe equipped with optical filters to block the 630 nm treatment light • Separation distance between unique source-detector pairs determines the depth of tissue probed. detectors Distance (mm)

23. Substantial intratumor heterogeneity exists in PDT-created vascular effects • PDT induces an initial increase in blood flow. • PDT leads to significant depth-dependent intratumor heterogeneity in blood flow response during illumination.

24. Intratumor heterogeneity in vascular effects (controls)

25. Lower fluence rate reduces intratumor heterogeneity in relative blood flow during PDT

26. Low fluence rate reduces intratumor heterogeneity in cytotoxic response.

27. Low fluence rate improves long-term tumor response % of animals with tumors <400 mm3

28. Lowering PDT fluence rate improves therapeutic outcome (summary) • Delivering a light dose more slowly provides • Less intra-tumor heterogeneity in PDT-created hypoxia during illumination • Less intra-tumor heterogeneity in vascular responses during illumination • Greater direct cell kill of tumor cells • Better long-term treatment response

29. Heterogeneity AboundsSo what to do? Modify Monitor

30. Monitoring: Rationale • PDT can create significant hypoxia in even vascular-adjacent tumor cells. • Vascular monitoring, including oxygenation and/or blood flow, may be indicative of tumor response.

31. Monitoring: Methods Diffuse optical spectroscopy • Broadband reflectance spectroscopy with a noninvasive probe • Measures tissue optical properties in the range of 600-800 nm • Data used to calculate concentrations of oxyhemoglobin (HbO2) and deoxyhemoglobin (Hb) • Tissue hemoglobin oxygen saturation (SO2 or StO2) = [HbO2]/[HbO2 + Hb] • In mouse tissues SO2 of 50% at pO2 of 40 mmHg • Diffuse correlation spectroscopy with a non-contact probe • Measures temporal fluctuations in transmitted light (785 nm) to provide information on the motion of tissue scatters, e.g. red blood cells • Data used to calculate relative blood flow, i.e. flow normalized to a pre-treatment baseline • Monitoring throughout PDT is facilitated by a non-contact camera probe equipped with optical filters to block the 630 nm treatment light

32. 50 40 30 SO2 (%) 20 10 0 0 h 3 h Before PDT After PDT PDT induces variable changes in tumor hemoglobin oxygen saturation

33. Time-to-400 mm3 (days) Time-to-400 mm3 (days) SO2 after PDT (%) SO2 before PDT(%) Pre- or post-PDT SO2 is not associated with tumor response

34. The PDT-induced change in SO2 in individual tumors is highly predictive of response Relative SO2= SO2 after PDT SO2 before PDT Time-to-400 mm3 (days) Wang H-W, et al. Cancer Res. 64(20):7553-7561, 2004 Relative-SO2

35. The PDT-induced change in blood flow is highly predictive of response Time to a tumor volume of 400 mm3 (days) Slope of decrease in blood flow • Yu G, et al. Clin Cancer Res.11:3543-52, 2005

36. Monitoring (Summary) • Pre-existing tumor SO2 of similarly-sized tumors of the same line can be highly heterogeneous. • PDT-induced changes in SO2 and blood flow can vary from tumor-to-tumor, even for the same PDT treatment conditions. • Individualized measurement of PDT effect on blood flow or blood oxygenation in a given tumor is predictive of long term response in that animal. • Changes associated with better maintenance of tumor oxygen (smaller PDT-induced decreases in SO2 or blood flow) lead to better tumor response. • Diffuse optical spectroscopy, can be readily applied in the clinic and thereby may provide a means for the rapid, individualized assessment of PDT outcome.

37. Conclusions • Both and clinical and preclinical studies indicate that tumors can be characterized by substantial heterogeneity in the essential components of PDT. • MODIFICATION (e.g. light delivery or tumor microenvironment) can be used reduce physiologic, hemodynamic, and cytotoxic heterogeneity. • MONITORING offers potential to optimize treatment through individualized, real-time dosimetry based on hemodynamic responses.

38. PDT at Penn Physicists Timothy Zhu Jarod Finlay Andreea DiMofte Pre-clinical Researchers Theresa Busch Sydney Evans Cameron Koch Stephen Tuttle Keith Cengel Arjun Yodh Xioaman Xing Dermatology Steve Fakharzadeh Surgery Douglas Fraker Joseph Friedberg Scott Cowan Bert O’Malley S. Bruce Malkowicz Ara Chalian Nursing Coordinators Debbie Smith Susan Prendergast Melissa Culligan Medicine Dan Sterman Colin Gilespie Andrew Haas Gregory Ginsberg Laser Specialist/Manager Carmen Rodriguez Biostatistics Rosie Mick Mary Putt Radiation Oncology Eli Glatstein Stephen Hahn Robert Lustig James Metz Harry Quon Neha Vapiwala Keith Cengel Veterinary Medicine Lilly Duda Jolaine Wilson

39. Radiation Oncology Steve Hahn Eli Glatstein Keith Cengel Cameron Koch Sydney Evans Statistics/Image Analysis E. Paul Wileyto Mary Putt Kevin Jenkins Physics and Astronomy Arjun Yodh Xiaoman Xing Guoqiang Yu Hsing-Wen Wang Medical Physics Timothy Zhu Jarod Finlay Ken Wang Carmen Rodriguez Andreea Dimofte Busch lab Elizabeth Rickter Shirron Carter Min Yuan Amanda Maas Grant Support (NIH) R01 CA 85831 P01 CA 87971 Acknowledgements