Kris R. Jatana , M.D . Department of Otolaryngology—Head & Neck Surgery The Ohio State University

1. Significance of Circulating Tumor Cells in Patients with Squamous Cell Carcinoma of the Head and Neck Kris R. Jatana, M.D. Department of Otolaryngology—Head & Neck Surgery The Ohio State University April 29, 2010 American Head and Neck Society Annual Meeting Las Vegas, Nevada

2. Acknowledgements • Department of Chemical and Biomolecular Engineering • Liyang Yang, Ph.D. • PriyaBalasubramanian, M.S. • Jeffrey J. Chalmers, Ph.D. • Department of Otolaryngology – Head and Neck Surgery • Jas C. Lang, Ph.D. • David E. Schuller, M.D. • AmitAgrawal, M.D. • EnverOzer, M.D. • Ted Teknos, M.D. • Courtney A. Jatana, D.D.S. • Elisabeth White, B.A. • This work is supported by National Science Foundation (BES-0124897); the National Cancer Institute (R01 CA97391-01A1); the State of Ohio Third Frontier Program (ODOD 26140000:TECH 07-001). • No Disclosures.

3. NCI-SEER 2009 • 48,010 new cases of oral cavity, pharyngeal, & laryngeal cancer in United States • Estimated 11,260 deaths

4. Research Objective • Develop a prognostic blood test based on detection of circulating tumor cells (CTCs) • Customize treatment • Cancer surveillance

5. Primary tumor Infiltration and invasion Blood vessel Metastases Micrometastases SCCHN Metastasis Pantel K, et al. 1999. Detection and clinical importance of micrometastatic disease.

6. Circulating Tumor Cells • Studies have linked CTCs to poor prognosis • Breast cancer • Colorectal cancer • Prostate cancer • Melanoma • Lung

7. Circulating Tumor Cells (CTC) in the Peripheral Blood? 5 Billion RBCs 7 Million WBCs 295 Million Platelets

8. Cell Immunomagnetic Cell Separation Magnetic labeling Target Antigen Antibody to CD45 Magnetic bead Positive selection of leukocytes

9. Red Cell Lysis Magnetic Separation Magnetic Labeling Cell Tumor Cells Isolated Cytokeratin Immunocytochemistry Study Design Patient Blood Sample

10. Validation of Technique • Blood from healthy humans was spiked with head and neck cancer cell lines • Established reliability and reproducibility of tumor cell detection

11. Circulating Tumor Cell (CTC) Brightfield

12. 0-100 CTCs 68.9% 31.1% 0 CTCs 100-1000 CTCs >1000 CTCs

13. Summary of Results

14. Summary of Results

15. Summary of Results

16. Disease-Free Survival No CTCs p=0.011 CTCs Present

17. Disease-Free Survival No CTCs > 0 to 25 CTCs >25 CTCs

18. DAPI Cytokeratin Vimentin CD44 Combined

19. DAPI Cytokeratin Vimentin CD44 Combined 26 y/o WF with stage II recurrent tongue cancer, 2nd recurrence 2 months after surgery

20. Conclusions • Developed and validated a successful negative selection technique, using immunomagnetic separation, for detection of CTCs in patients with SCCHN. • Evidence of mesenchymal and cancer “stem cell” markers on CTCs. • Clinical follow-up with a mean of 15.2 months has shown a statistically significant improved disease-free survival in those patients with NO detectable CTCs per mL blood.

21. Thank you.

22. References • 1. Suen JY.Management of the N0 neck. Proceedings of the Fourth International Conference of Head and Neck Cancer. The Society of Head and Neck Surgeons and American Society of Head and Neck Surgeons, Toronto, Madison WI: Omnipres, 1996, 557. • 2. Yamamoto E, Miyakawa A, Kohama G. Mode of invasion and lymph node metastasis in squamous cell carcinoma of the oral cavity. Head Neck Surg. 1984; 6: 938-947. • 3. Tankere F, Camproux A, Barry B, et al. Prognostic value of lymph node involvement in oral cancers: a study of 137 cancers. Laryngoscope. 2000; 110: 2061-2065. • 4. Gath, HJ, Brakenhoff, RH. Minimal residual disease in head and neck cancer. Cancer Metastasis Rev. 1999; 18: 109-126. • 5. ShinDM, Lippman SM. Paclitaxel-based chemotherapy for recurrent and/or metastatic head and neck squamous cell carcinoma: current and future directions. Semin. Oncol. 1999; 26: 100-105. • 6. Schantz SP, Harrison LB, Forastiere AA. Tumors of the nasal cavity and paranasal sinuses, nasopharynx, oral cavity and oropharynx.  In: Cancer: Principles and Practice of Oncology 6th edition, editors: Vincent T. Devita, Jr., Samuel Hellman, and Steven A Rosenberg. Lippincott Williams and Wilkins. 2001.p.797-860. • 7. Pantel K, Brakenhoff RH. Dissecting the metastatic cascade. Nat Rev 2004; 4:448-456. • 8. Fidler IJ, Kripke ML. Metastasis results from preexisting variant cells within a malignant tumor. Science. 1977; 197:337-342. • 9. Schmidt-Kittler O, Ragg T, Daskalakis A, et al. From latent disseminated cells to overt metastasis: Genetic analysis of systemic breast cancer progression. PNAS.2003; 100(13): 7737-7742.

23. References Cont’d • 10. Klein CA, Blankenstein TJF, Schmidt-Kittler O, et al. Genetic heterogeneity of single disseminated tumour cells in minimal residual cancer. The Lancet. 2002; 360:683-689. • 11. Klein CA, Seidl S, Petat-Dutter K, et al. Combined transcriptome and genome analysis of singel micrometastatic cells. Nat Biotech. 2002; 20:387-392. • 12. Klein CA, Schmidt-Kittler, O., Schardt, JA, et al. Comparative Genomic Hybridization, Loss of Heterozygosity, and DNA sequence analysis of Single Cells. PNAS.1999; 96:4494-4499. • 13. Braun S, Vogl FD, Naume B, et al. A pooled analysis of bone marrow micrometastasis in breast cancer. Engl J Med. 2005;353(8):793-802. • 14. Mocellin S, Hoon D, Ambrosi A, Nitti D, Rossi CR. The Prognostic Value of Circulating Tumor Cells in Patients with Melanoma: A Systematic Review and Meta-analysis. Clin Cancer Res. 2006; 12:4605-4613. • 15. Naume N, Wiedswang G, Borgen E, et al. Clinical significance of isolated tumor cells inperipheral blood in breast cancer patients three years after diagnosis: comparison between analysis of peripheral blood and bone marrow. Proc. ASCO 2004; 23:844. Abstract #9554. • 16. Cristofanilli M, Budd GT, Ellis MJ, et al. Circulating tumor cells, disease progression and survival in metastatic breast cancer. N. Engl. J. Med. 2004; 351(8):781-791. • 17. Guney K, Yoldas B, Ozbilim G, Derin AT, Sarihan S, Balkan E. Detection of micrometastatic tumor cells in head and neck squamous cell carcinoma. Saudi Med. J. 2007; 28:216-220. • 18. Partridge M, Brkenhoff R, Phillips E, et al. Detection of Rare Disseminated Tumor Cells Identifies Head and Neck Cancer Patients at Risk of Treatment Failure. Clinical Cancer Research, 2003; 9:5287-5294.

24. References Cont’d • 19. Wirtschafter A, Benninger MS, Moss TJ, Umiel T, BlasoffK, Worsham MJ. Micrometatic Tumor Detection in Patients with Head and Neck Cancer. A Preliminary Report. Arch Otoloaryngol Head and Neck Surg Vol. 2002; 128:40-43. • 20. Lara O, Tong X, Zborowski M, Chalmers JJ. Enrichment of Rare Cancer Cells through Depletion of Normal Cells Using Density and Flow-Through, Immunomagnetic Cell Separation. Exp Hematol.2004;32(10):891-904. • 21. Tong X,Yang L, Lang JC. Zborowski M, Chalmers JJ. Application of immunomagnetic cell enrichment in combination with RT-PCR for the detection of rare circulating head and neck tumor cells in human peripheral blood. Cytometry BClin cytom. 2007. Jan. 4. • 22. Riethdorf S, Fritsche H, Muller V, et al. Detection of Circulating Tumor Cells in Peripheral Blood of Patients with Metastatic Breast Cancer: A Validation Study of the CellSearch System. Clin. Cancer Res. 2007; 13(3):920-928. • 23. Coombes RC, Berger U, Mansi J, et al. Prognostic significance of micrometastases in bone marrow in patients with primary breast cancer. J Natl Cancer Inst. 1986; 1:51-53. • 24. Diel IJ, Kaufmann M, Costa SD et al. Micrometastatic breast cancer cells in bone marrow at primary surgery: prognostic value in comparison with nodal status. J Natl Cancer Inst. 1998; 90:1099-1101. • 25. Gerber B, Krause A, Muller H, et al. Simultaneous immunohistochemical detection of tumor cells in lymph nodes and bone marrow aspirates in breast cancer and its correlation with other prognostic factors. J Clin Oncol. 2001; 19:960-971. • 26. Fehm T, Braun S, Muller V, et al. A concept for the standardized detection of disseminated tumor cells in bone marrow from patients with primary breast cancer and its clinical implementation. Cancer. 2006; 107(5):885-92.