1. Recurrent Respiratory Papillomatosis (RRP): Basic Science to Clinical Studies Mark J. Shikowitz, MD Bettie M. Steinberg, PhD Long Island Jewish Medical Center Schneider Children’s Hospital North Shore – LIJ Research Institute Presented at the American Academy of Otolaryngology- Head and Neck Surgery, Orlando FL September 2003

2. Respiratory Papillomas l Benign tumors of airway - larynx > trachea >lungs l Caused by Human Papillomaviruses types 6 and 11 l Frequently recur - can require more than 100 operations l Recurrence due to activation of latent infection l Become malignant in approximately 3% of patients l Irradiation of papillomas increases malignant conversion – 16-fold increased risk * * Lindeberg H, Elbrond O. Malignant tumours in patients with a history of multiple laryngeal papillomas: the significance of irradiation. Clin Otolaryngol. 16:149-51 1991.

3. Appearance of Mild and Severe Larynx Papillomas

4. Demographics of RRP l International Data  incidence 3.8 - 7.0 / million population/ year  prevalence 1/100,000  two peaks - juvenile onset 2-5 years of age  adult onset 20-40 years of age  juvenile 1:1 male/female, adult 2:1 l LIJ Data  total patients treated 254  adult onset: 138 juvenile onset: 116  175 males, 79 females

5. Studies in Laboratory l Signal transduction - control of cell growth and differentiation l Regulation of latent infection l Host immune responses to HPV  T cell functions  Suppression of MHC expression by HPV protein l Efficacy of photodynamic therapy – completed l Efficacy of Celebrex therapy

6. Latency is the Key to This Disease l Latency: presence of viral DNA in clinically and histologically normal respiratory tissue l Source of recurrent laryngeal disease  recurrence not due to “spread of virus” during surgery  all patients have patchy latent infection in larynx, even if in remission for 20+ years l Disease is due to focal activation of latency l Cannot cure disease unless eliminate latency - only control

7. Papillomavirus Life Cycle Normal epithelium squame with virus Latent Infection Papilloma Cancer Viral DNA maintenance Early Viral RNA Late Viral RNA, DNA Infection Co-carcinogen(s) Rare event activation Virus Production (if permissive)

8. Tracheal Papillomatosis and Tracheal Latency l Tracheal disease less frequent than laryngeal disease  35/254 (13.7%) of our patients have tracheal disease l Several possible explanations for low prevalence  Low rate of HPV infection  Low rate of HPV latency  Low rate of activation of latent infection l We have asked which of these explanations might be correct

9. 0 20 40 60 Perent biopsies LarynxTrachea HPV Latency is Equal in Larynx and Trachea

10. Conclusions - Take Home Message l Nearly all patients carry latent tracheal HPV DNA l Therefore, low frequency of tracheal disease not due to infection rate or establishment of latency l Low rate of tracheal disease must be due to tissue- specific factors required for activation of HPV DNA  Permissive tissue for virus is stratified squamous epithelium  Trachea normally ciliated columnar  Must avoid inducing squamous metaplasia (e.g. trach tube, smoking)

11. Design of Photodynamic Therapy Study l Patients with 3 or more surgeries in past year or tracheal involvement eligible l Randomized - two treatment times (6 and 15 months) after enrollment - late group “control” l One dose Foscan  - 0.15 mg/kg l Wash-out time 6 days l Light dose (652 nm) adults: 80-100J, children: 60-80J l Score disease at 3 month intervals for 1+ yr after PDT l Study now ended – PDT improved disease for some patients, but did not prevent long-term recurrence

12. Response to PDT 500 50 100 150 200 250 Controls - PDT treated Months After Enrollment Percent Score at Entry Median and Range 369121518 369121518 21 PDT Results of PDT Study p= 0.006

13. Design New Celebrex Study l Multi-centered study, other sites planned  University of Iowa  University of Alabama, Birmingham  University of Pittsburgh l Patient enrollment requirement: Adults with 3 or more surgeries in past year or tracheal involvement l Randomized - two start times (6 and 12 months) after enrollment - late group “control”, everyone gets Celebrex l Celebrex NOT Standard Dose – must be managed carefully as per study l Score disease at 3 month intervals for total of 2 years

14. Role of Host Immune System in Disease l Simple question - why do patients have recurrent disease? l Approx. 5% of population carries latent HPV in larynx, RRP prevalence is 1/100,000 l Are RRP patients immunocompromised? l Answer: No! Standard tests show no defect in immune system and patients no more likely to have other infections. l Must be specific for HPV

15. Immune Response in RRP Antigen Presenting Cell MHC Class II with viral protein T H 1 Cell T H 2 Cell Humoral Immunity Cell-mediated Immunity Ifn- ,, IL-2 IL-4, IL-10 (MHC Class I) Infected epithelial cell CD 8 T-cell (CD28) B7 T-cell receptor

16. Take Home Message l Many patients have antibodies against HPV l Specific defect in the ability of RRP patients to generate a cell-mediated response to HPV infection - not general immune suppression l This could explain the ability of the virus to activate latent infection repeatedly without developing an effective immune response l Celebrex study will determine whether it improves immune response.