William C. Spanos

Immune Response During Therapy With Cisplatin or Radiation for Human Papillomavirus–Related Head and Neck Cancer

BACKGROUND Human papillomavirus (HPV) is the most identifiable cause of head and neck squamous cell cancer (HNSCC). Compared with HPV-negative HNSCC, HPV-positive HNSCC presents at an advanced stage but with significantly better survival. We created a syngeneic mouse model of HPV-positive and HPV-negative HNSCC by transforming mouse primary tonsil epithelial cells with either HPV oncogenes or a nonantigenic RNA interference strategy that affects similar oncogenic pathways. OBJECTIVES To examine the effect of radiation therapy on HPV-positive and HPV-negative tumors in immune-competent and immune-incompetent mice and to examine responses in human cancer cell lines. DESIGN Prospective in vivo murine model. MAIN OUTCOME MEASURES Survival and tumor growth. RESULTS For human and murine transformed cell lines, HPV-positive cells were more resistant to radiation and cisplatin therapy compared with HPV-negative cells. In vivo, HPV-positive tumors were more sensitive to radiation, with complete clearance at 20 Gy, compared with their HPV-negative counterparts, which showed persistent growth. Cisplatin in vivo cleared HPV-positive tumors but not HPV-negative tumors. However, neither radiation or cisplatin therapy cured immune-incompetent mice. Adoptive transfer of wild-type immune cells into immune-incompetent mice restored HPV-positive tumor clearance with cisplatin therapy. CONCLUSIONS The HPV-positive tumors are not more curable based on increased epithelial sensitivity to cisplatin or radiation therapy. Instead, radiation and cisplatin induce an immune response to this antigenic cancer. The implications of these results may lead to novel therapies that enhance tumor eradication for HPV-positive cancers.

The PDZ binding motif of human papillomavirus type 16 E6 induces PTPN13 loss, which allows anchorage-independent growth and synergizes with ras for invasive growth.

ABSTRACT The human papillomavirus (HPV) oncogene E6 has been shown to perform multiple functions (p53 degradation, telomerase activation, etc.) that play a role in oncogenic transformation. Beyond known E6 functions, an undefined mechanism that allows cellular invasion requires the E6 PDZ binding motif (PDZBM). Here, we show that HPV type 16 (HPV16) E6 interacts with and induces loss of a protein tyrosine phosphatase (PTPN13) in a PDZBM-dependent manner. PTPN13 loss induced either by the presence of E6 or by a short hairpin RNA strategy allows for anchorage-independent growth (AIG) and synergy with a known oncogene, Rasv12, resulting in invasive growth in vivo. Restoring PTPN13 expression reverses AIG in cells lacking PTPN13. A genomic analysis of colorectal carcinoma has identified an association between PTPN13 loss-of-function mutations and aberrant Ras signaling. Our findings support this correlation and provide methods for further evaluation of the mechanisms by which PTPN13 loss/Ras expression leads to invasive growth, the results of which will be important for treatment of HPV-related and non-HPV cancer.

Deletion of the PDZ motif of HPV16 E6 preventing immortalization and anchorage-independent growth in human tonsil epithelial cells

Human papillomavirus 16 (HPV16) has been associated with head and neck squamous cell carcinoma (HNSCC) in up to 60% of sampled specimens.

Unilateral vocal fold paralysis in premature infants after ligation of patent ductus arteriosus: Vascular clip versus suture ligature

Objectives: We investigated risk factors associated with unilateral iatrogenic vocal fold paralysis (IVFP) in the context of ligation of patent ductus arteriosus (PDA) and compared the rates of paralysis between vascular clip and suture ligation procedures. Methods: We performed a prospective examination of infants with isolated PDA treated surgically during 1995 to 2005. Statistical significance was determined with a 2-tailed t-test. Results: Of 68 PDA ligations, 13 cases of left-sided IVFP were diagnosed, for an overall incidence of 19%. All cases of IVFP occurred in 55 infants who weighed less than 1 kg at birth. Suture ligature was used in 60% of all PDA ligation patients, and vascular clips in 40%. The incidence of IVFP in patients with vascular clips (19%) was similar to the incidence in those with suture ligature (20%). Hoarseness or stridor was present in 69% of patients with IVFP, compared to 17% of normal controls (p < 0.001). The rate of aspiration was not increased in the IVFP group; however, 15% of the patients with IVFP had episodes of decreased oxygen saturation, versus 7% of infants with normal vocal fold mobility. Conclusions: A hoarse infant with a birth weight of less than 1 kg who has undergone PDA ligation should be examined for unilateral IVFP. Vascular clips and suture ligature are associated with similar rates of IVFP.

Cidofovir incorporation into human keratinocytes with episomal HPV 16 results in nonselective cytotoxicity

Objectives: Recurrent respiratory papillomatosis (RRP) is caused by human papillomavirus (HPV). Surgical excision is the mainstay of treatment; however, medical therapy including cidofovir, a cytosine analog, has been investigated. Human papillomavirus does not encode a viral DNA polymerase, which is the known target of cidofovir in cytomegalovirus infections. Methods: In an effort to better understand the usefulness of cidofovir in the treatment of HPV-related disease, we tested cidofovirs ability to inhibit growth, alter gene expression, and inhibit genome replication. Results: With the use of carbon 14-labeled cidofovir in episomal HPV 16-containing keratinocytes, there was a minimal increase in cidofovir incorporation into episomal DNA versus genomic DNA. Cidofovir decreased the copies of episomal HPV 16 in keratinocytes; however, the copies per cell returned to baseline levels once cidofovir was removed. Expression of a viral oncogene (HPV 16 E6) in transformed keratinocytes with episomal HPV 16 was not decreased by cidofovir. Cytotoxicity in head and neck squamous cell carcinoma lines exposed to cidofovir correlated with cell doubling time, and not with HPV status. Also, tonsil keratinocytes transformed with episomal HPV 16 did not exhibit greater cidofovir-mediated toxicity than did telomerase-transformed keratinocytes. Conclusions: These findings suggest that any potential in vivo benefit of cidofovir therapy results from non–viral-specific cell toxicity at the site of application.

Abstract A55: PD-1 blockade synergizes with cisplatin radiation therapy aiding in clearance of murine HPV+ oropharyngeal carcinoma

Squamous cell carcinomas of the head and neck (SCCHN) are the 6th most common malignancy world-wide with approximately 650,000 diagnoses per year. Human papillomavirus (HPV) causes up to 80% of oropharyngeal (tonsil/base of tongue) SCCHN. Despite presenting at an advanced stage, multiple studies show that HPV+ SCCHN are more curable relative to their HPV- counterparts. Clearance of SCCHN during treatment with cisplatin radiation therapy (CRT) is not only related to direct cytotoxicity, but is also dependent on immune mediated clearance. This immune clearance is strongly dependent on an intact CD4+ and CD8+ cellular response to tumor cells harboring these viral oncogenes. Furthermore, the immune system recognizes and clears HPV + SCCHN only after the start of CRT. The immunologic tolerance to these cancers prior to treatment could be explained by immune checkpoints aimed at preventing unregulated immune activity. The programmed death 1 receptor (PD-1) and its ligand, PD-L1, are one such checkpoint axis. Recent data suggests that the PD-1:PD-L1 pathway may play an important role in immune resistance in head and neck cancer. We evaluated PD-1 in combination with CRT in an immune competent HPV+ SCCHN mouse model. HPV + mouse SCCHN cells were injected subcutaneously into immune competent mice and tumors were treated after growing to palpable size. Using the mouse analog to the human PD-1 blocker, pembrolizumab, we determined that while single-agent therapy does not have significant activity on primary disease or distant metastasis, PD-1 blockade strongly synergizes with CRT. An overall survival of 58% was evident in mice treated with combination PD-1 and CRT versus 8% in mice treated with isotype control (IgG1) in combination with CRT (p = 0.006). These studies provide the impetus for future work investigating less toxic induction of immune responses in combination with blockade of immune checkpoint pathways. Citation Format: Daniel W. Vermeer, Steven Powell, William C. Spanos, John H. Lee. PD-1 blockade synergizes with cisplatin radiation therapy aiding in clearance of murine HPV+ oropharyngeal carcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A55.

Radiation-induced loss of cell surface CD47 enhances immune- mediated clearance of HPV+ cancer

The increasing incidence of human papillomavirus (HPV) related oropharyngeal squamous cell carcinoma (OSSC) demands development of novel therapies. Despite presenting at a more advanced stage, HPV(+) oropharyngeal squamous cell carcinoma (OSCC) have a better prognosis than their HPV(−) counterparts. We have previously demonstrated that clearance of HPV(+) OSCC during treatment with radiation and chemotherapy requires an immune response which is likely responsible for the improved clinical outcomes. To further elucidate the mechanism of immune‐mediated clearance, we asked whether radiation therapy induces tumor cell changes that allow the body to recognize and aid in tumor clearance. Here, we describe a radiation‐induced change in tumor surface protein expression that is critical for immune‐mediated clearance. Radiation therapy decreases surface expression of CD47, a self‐marker. CD47 is frequently overexpressed in head and neck squamous cell carcinoma and radiation induces a decrease of CD47 in a dose‐dependent manner. We show that both in vitro and in vivo tumor cell CD47 protein levels are restored over time after sublethal radiation exposure and that protein levels on adjacent, normal tissues remain unaffected. Furthermore, reduction of tumor cell CD47 increases phagocytosis of these cells by dendritic cells and leads to increased interferon gamma and granzyme production from mixed lymphocytes. Finally, decreasing tumor cell CD47 in combination with standard radiation and chemotherapy results in improved immune‐mediated tumor clearance in vivo. These findings help define an important mechanism of radiation‐related immune clearance and suggest that decreasing CD47 specifically on tumor cells may be a good therapeutic target for HPV related disease.

The in vitro effect of cidofovir on HPV16 versus HPV-HNSCC cell lines

Abstract Problem: Previous studies have suggested that cidofovir selectively inhibits cell proliferation in cervical cancer cell lines that contain human papillomavirus 16 (HPV16), possibly due to inhibition of viral oncogenes. We hypothesize that cidofovir may also selectively alter viral gene expression and cellular proliferation in head and neck cancer cells that contain HPV16. Methods: To test this hypothesis we studied 2 HPV-containing HNSCC cell lines (SCC 47, SCC 90) and 2 non-HPV-containing HNSCC cell lines (SCC 19, SCC 84). We created standard logarithmic dose response curves using the MTT assay. In addition, we examined the expression of viral oncogenes (E6, E7) and their cellular targets (P53, Rb) by quantitative RT-PCR to determine whether any notable response was due to selective cidofovir-induced changes in viral gene expression. Results: Dose response curves demonstrate decreased cell viability at increasing concentrations of cidofovir for all cell lines. There was no apparent difference in the dose response curves between cell lines containing HPV16 and cell lines without HPV16. Selective inhibition of viral oncogenes was not seen. Changes in the expression of P53 and Rb were also similar between cells regardless of HPV16 status. Conclusion: This study demonstrates that nonspecific cellular toxicity of cidofovir in vitro on HNSCC cell lines is independent of HPV16 status. Significance: HPV has recently been implicated in head and neck squamous cell carcinoma. If a chemotherapeutic agent could be used to affect the HPV virus, perhaps this could be used to treat HNSCC. Alternatively, if no effect is found attention could be turned elsewhere. Support: None reported.

Radiation-induced loss of cell surface CD47 enhances immune-mediated clearance of human papillomavirus-positive cancer: Radiation-decreased CD47 improves tumor clearance

The increasing incidence of human papillomavirus (HPV) related oropharyngeal squamous cell carcinoma (OSSC) demands development of novel therapies. Despite presenting at a more advanced stage, HPV(+) oropharyngeal squamous cell carcinoma (OSCC) have a better prognosis than their HPV(−) counterparts. We have previously demonstrated that clearance of HPV(+) OSCC during treatment with radiation and chemotherapy requires an immune response which is likely responsible for the improved clinical outcomes. To further elucidate the mechanism of immune‐mediated clearance, we asked whether radiation therapy induces tumor cell changes that allow the body to recognize and aid in tumor clearance. Here, we describe a radiation‐induced change in tumor surface protein expression that is critical for immune‐mediated clearance. Radiation therapy decreases surface expression of CD47, a self‐marker. CD47 is frequently overexpressed in head and neck squamous cell carcinoma and radiation induces a decrease of CD47 in a dose‐dependent manner. We show that both in vitro and in vivo tumor cell CD47 protein levels are restored over time after sublethal radiation exposure and that protein levels on adjacent, normal tissues remain unaffected. Furthermore, reduction of tumor cell CD47 increases phagocytosis of these cells by dendritic cells and leads to increased interferon gamma and granzyme production from mixed lymphocytes. Finally, decreasing tumor cell CD47 in combination with standard radiation and chemotherapy results in improved immune‐mediated tumor clearance in vivo. These findings help define an important mechanism of radiation‐related immune clearance and suggest that decreasing CD47 specifically on tumor cells may be a good therapeutic target for HPV related disease.

08:20: HPV16 E6 Mediated Invasion through PDZ Binding of PTP-BL

compared with the parental cells. CONCLUSION: Northern blot confirmation of the differentially expressed microRNAs by RT-PCR is ongoing to confirm microarray results. SIGNIFICANCE: In future experiments, the researchers will change the expression of confirmed microRNA in either HEp2/DOC cells or parental cells to determine (1) the role of certain microRNAs in head and neck cancer cell MDR, and (2) the target genes regulated by these microRNAs. Findings will help in understanding MDR molecular mechanisms and development of new approaches to overcome MDR. SUPPORT: VA Advanced Research Career Development Award (PMS).