1. Date of download: 6/2/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Immune Response During Therapy With Cisplatin or Radiation for Human Papillomavirus–Related Head and Neck Cancer Arch Otolaryngol Head Neck Surg. 2009;135(11):1137-1146. doi:10.1001/archoto.2009.159 Results of in vitro clonogenic survival assays with increasing radiation doses. A, Human papillomavirus–positive (HPV+) human tumor cells (UMSCC-47 and UPCI-SCC90) are more resistant to radiation than are HPV-negative (HPV−) cells (UMSCC-1, -19, and -84). Cells were irradiated with the indicated dose, and percentage survival was calculated by comparing the number of surviving colonies with the number of cells plated (3 plates per group; the results were averaged across 2 experiments). B, The HPV+ mouse tonsil epithelial cells are more resistant than the HPV− cells. Colony-forming assay of cells was performed as described in Figure 1A. Error bars represent SE. Figure Legend:

2. Date of download: 6/2/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Immune Response During Therapy With Cisplatin or Radiation for Human Papillomavirus–Related Head and Neck Cancer Arch Otolaryngol Head Neck Surg. 2009;135(11):1137-1146. doi:10.1001/archoto.2009.159 Radiation-related clearance of human papillomavirus–positive (HPV+) tumors in mice is enhanced by an intact immune response. Tumor growth curves and survival curves are shown for wild-type C57BL/6 mice (A and B) and C57BL/6 B6.129S7-Rag1 tm1Mom /J (RAG-1) mice (C and D) implanted with HPV+ or HPV negative (HPV−) mouse tonsil epithelial cells. Six mice were used in each group. Tumors were locally irradiated with the indicated amount of radiation 7 days after implantation (XRT). Results of the log-rank test, with α =.01, show that survival of wild-type mice vs RAG-1 mice implanted with HPV+ tumors is significant at 0 Gy (P =.002), 8 Gy (P =.007), 16 Gy (P =.006), and 24 Gy (P =.003) but did not reach significance at 32 Gy (P =.03) due to 1 RAG-1 mouse with tumor clearance. The difference in survival between wild-type mice implanted with HPV+ vs HPV− tumors was significant at 32 Gy (P =.002). Figure Legend:

3. Date of download: 6/2/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Immune Response During Therapy With Cisplatin or Radiation for Human Papillomavirus–Related Head and Neck Cancer Arch Otolaryngol Head Neck Surg. 2009;135(11):1137-1146. doi:10.1001/archoto.2009.159 Growth inhibition of bulky human papillomavirus–positive (HPV+) tumors is enhanced by vaccination with an adenovirus expressing HPV-16 E6 and E7 oncogenes (Ad-E6/E7). The C57BL/6 mice were injected with HPV+ mouse tonsil epithelial cells, and tumors were allowed to grow to 1 cm. Mice were vaccinated with either control (AdEmpty) or Ad-E6/E7 on day 14 after tumor implantation. To determine whether timing of vaccination alters response, a separate group of mice was also vaccinated with Ad-E6/E7 21 days after implantation. Six mice were used in each treatment group. Mice flanks were locally irradiated with 20 Gy of radiation (XRT) on day 28 after tumor implantation. Growth curves (A) and survival curves (B) for the various groups show a slight improvement in survival (P =.003, log-rank test, with α =.01) for the mice receiving vaccination with Ad-E6/E7 14 days before radiation therapy. Figure Legend:

4. Date of download: 6/2/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Immune Response During Therapy With Cisplatin or Radiation for Human Papillomavirus–Related Head and Neck Cancer Arch Otolaryngol Head Neck Surg. 2009;135(11):1137-1146. doi:10.1001/archoto.2009.159 Human papillomavirus–positive (HPV+) mouse tonsil epithelial cells (MTECs) are more resistant to cisplatin than are HPV-negative (HPV−) HPV− MTECs. The MTECs were plated, were allowed to attach, and were treated with cisplatin for 24 hours; colony formation was then assessed. The HPV+ and HPV− MTECs were incubated with escalating doses of cisplatin and were allowed to grow until a 15-cell colony size was achieved. Three plates were used per condition, and the results were averaged across 2 experiments. The percentage of surviving cells that formed colonies were quantified. The HPV+ MTECs are more resistant (approximately 63%) to cisplatin than are the HPV− cells (P <.02, Mann-Whitney test). Error bars represent SE. Figure Legend:

5. Date of download: 6/2/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Immune Response During Therapy With Cisplatin or Radiation for Human Papillomavirus–Related Head and Neck Cancer Arch Otolaryngol Head Neck Surg. 2009;135(11):1137-1146. doi:10.1001/archoto.2009.159 Human papillomavirus–positive (HPV+) tumors are more sensitive to treatment with cisplatin than are HPV-negative (HPV−) tumors in immune-competent mice. Tumor growth rates and survival rates were quantified after the indicated doses of cisplatin in C57BL/6 mice (A and B) C57BL/6 and B6.129S7-Rag1 tm1Mom /J (RAG-1) mice (C). Six mice were used in each group. Mice received 3 weekly intraperitoneal injections of cisplatin, with the first dose starting 1 week after tumor implantation with 1 × 10 6 HPV+ or HPV− mouse tonsil epithelial cells. Difference in survival for HPV+ tumors comparing wild-type vs RAG-1 mice and the difference in survival for HPV+ vs HPV− tumors in wild-type mice was different at 10- and 20-mg/m 2 doses (P <.01, log-rank test, with α =.01). Error bars represent SE. Figure Legend:

6. Date of download: 6/2/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Immune Response During Therapy With Cisplatin or Radiation for Human Papillomavirus–Related Head and Neck Cancer Arch Otolaryngol Head Neck Surg. 2009;135(11):1137-1146. doi:10.1001/archoto.2009.159 Cisplatin response can be partially restored by adoptive transfer of splenocytes into B6.129S7-Rag1 tm1Mom /J (RAG-1) mice. Immune cells from wild-type C57BL/6 mice were adoptively transferred into C57BL/6 RAG-1 mice, human papillomavirus–positive mouse tonsil epithelial cells were implanted, and response to weekly cisplatin was compared with that of naive RAG-1 mice (6 mice per group). Mice receiving adoptive transfer had slower tumor growth and greater partial response than did control RAG-1 mice. Treatment with cisplatin, 20 mg/m 2 every week for 3 weeks, was initiated 1 week after tumor cell implantation (cisplatin treatment). Error bars represent SE. Figure Legend:

7. Date of download: 6/2/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Immune Response During Therapy With Cisplatin or Radiation for Human Papillomavirus–Related Head and Neck Cancer Arch Otolaryngol Head Neck Surg. 2009;135(11):1137-1146. doi:10.1001/archoto.2009.159 Cisplatin response is insufficient to induce clearance of bulky tumors. To compare the response for bulky vs small tumor burdens, C57BL/6 mice were implanted with human papillomavirus–positive mouse tonsil epithelial cells and were treated with cisplatin, 20 mg/m 2 (3 weekly doses), either 7 or 21 days (approximately 1 cm in greatest dimension) after implantation (6 mice per group). Cisplatin treatment of bulky tumors slowed tumor growth but was not sufficient to result in complete remission when tumors were larger. The difference in survival between treatment at day 7 and day 21 was significant (P =.007, log-rank test, with α =.01). Figure Legend:

8. Date of download: 6/2/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Immune Response During Therapy With Cisplatin or Radiation for Human Papillomavirus–Related Head and Neck Cancer Arch Otolaryngol Head Neck Surg. 2009;135(11):1137-1146. doi:10.1001/archoto.2009.159 Clonogenic survival of human papillomavirus–positive (HPV+) and HPV-negative (HPV−) mouse tonsil epithelial cells treated with concurrent radiation and cisplatin in vitro. The cells were split 24 hours after treatment with the indicated doses of radiation and 0.25 μg/mL of cisplatin, and colonies greater than 15 cells were counted at 14 days. Three plates were used per condition, with results averaged from 2 experiments. Surviving fractions of cells were compared with initial numbers of plated cells. No difference was seen in sensitivity to combined radiation and cisplatin use between HPV+ and HPV− mouse tonsil epithelial cells. Error bars represent SE. Figure Legend:

9. Date of download: 6/2/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Immune Response During Therapy With Cisplatin or Radiation for Human Papillomavirus–Related Head and Neck Cancer Arch Otolaryngol Head Neck Surg. 2009;135(11):1137-1146. doi:10.1001/archoto.2009.159 Cisplatin and radiation responses in vivo with and without the adenovirus-expressing human papillomavirus (HPV) 16 E6 and E7 oncogene (Ad-E6/E7) vaccination. A, In vivo response of bulky tumors either untreated (RAG-1 and wild type, no treatment) or treated with 3 doses of concurrent radiation (8 Gy weekly) and cisplatin (3 doses of 20 mg/m 2 weekly) implanted in either wild-type C57BL/6 or C57BL/6 RAG-1 mice (the other 4 groups). The HPV-positive (HPV+) cells were injected in C57BL/6 or C57BL/6 RAG-1 mice and were allowed to grow for 14 days (6 mice per group). In the treatment groups, mice were divided again to be vaccinated with either control AdEmpty or Ad-E6/E7 14 days after tumor implantation (adenovirus treatment). Cisplatin and radiation administration in the treatment groups began on day 21 after implantation of tumor cells. Growth curves (A) and survival curves (B) were calculated for each mouse. Wild-type mice had a significantly better response compared with C57BL/6 RAG-1 mice (P <.01, log-rank test, with α =.01). Wild-type mice vaccinated with Ad-E6/E7 had improved survival and slower growth compared with mice receiving control AdEmpty. C, Luminescent images of HPV+ tumors that co-express luciferase. The groups of the indicated mice are shown after treatment on day 59. Wild-type (C57BL/6) mice treated with Ad-E6/E7 and cisplatin and radiation cleared tumors compared with persistent tumor in RAG-1 mice treated with cisplatin and radiation or C57BL/6 mice treated with AdEmpty and cisplatin and radiation. CRT indicates chemoradiation therapy; XRT, radiation therapy. Figure Legend: