Assessment of HPV 16, HPV 18, p16 expression in advanced stage laryngeal cancer patients and prognostic significance,

Abstract Introduction: Human papilloma virus is an etiological risk factor for a subset of head and neck squamous cell carcinomas. HPV has been proven to be a powerful prognostic biomarker for oropharyngeal cancer, but its role in the larynx has not been explored in depth. The developmental mechanisms of laryngeal carcinomas are quite complex and controlled by various factors. Smoking and alcohol are most important risk factors. Recent studies indicate that HPV infection also plays an important role in larynx carcinomas. HPV related laryngeal carcinomas especially occur at the supraglottic region of larynx. Objective: We aimed to determine the frequency of HPV/protein16 positivity in patients with laryngeal carcinoma and association of HPV and/or p16 positivity with variables such as age, sex, smoking habits, tumor localization, lymph node metastasis, recurrence and survival in advanced stage laryngeal carcinoma in our study. Methods: This retrospective study included 90 patients with advanced laryngeal carcinoma. The Control group was 10 normal larynx mucosa specimens. The presence of HPV was investigated polyclonally by polymerase chain reaction, and protein16 with immunohistochemical method. In HPV positive cases, the presence of HPV types 16, 18 were evaluated by polymerase chain reaction. Demographic features of patients were noted. Patient survival and association with HPV/protein16 was determined. Results: Polyclonal HPV positivity was detected in 11 (12.2%) of 90 cases. Out of these 11 cases, HPV 16 was positive in 6, HPV 18 in 4, and both HPV 16 and 18 were positive in 1. In 18 (20%) of the cases, p16 was positive. Six of the cases (6.6%) had both HPV and protein16 positivity. In cases where protein16 alone or HPV and protein16 were co-positive, alcohol use was less and the tumor was found more likely to be localized in the supraglottic area. These ratios were statistically significant. Supraglottic localization of tumor was determined to be increased in protein16 positive cases. The correlation between protein16 positivity and supraglottic area location was determined to be statistically significant (p= 0.011). 55.6% of protein16 positive cases was located in the supraglottic region, 33.3% was glottic and 11.1% was transglottic. Although life expectancy over 5 years were numerically higher in HPV and protein16 positive cases, this was not found to be statistically significant. There was no statistically significant relationship between HPV positivity and mean age, differentiation, smoking and alcohol use, tumor progression, lymph node metastasis, localization, recurrence, cause of mortality and treatment methods in our study. The mean follow-up period of our patients was 6.7 years. Conclusion: The close relationship between HPV and oropharyngeal squamous cell carcinoma could not be shown in larynx malignancy in many studies, including our study. Our findings support a limited role of HPV in laryngeal carcinogenesis. Protein16 is not a reliable surrogate for HPV status in laryngeal cancers and is not a predictor of laryngeal cancer survival. Supraglottic localization of tumor was determined to be increased in protein16 positive cases. The correlation between protein16 positivity and supraglottic area location was determined to be statistically significant. There is a need for more populated clinical trials, where neoplastic proliferation is better demonstrated and the accuracy of the results obtained is supported by different techniques.

**引言摘要**：人乳头瘤病毒（Human papilloma virus，HPV）是部分头颈部鳞状细胞癌的致病危险因素。HPV已被证实为口咽癌的强效预后生物标志物，但其在喉部中的作用尚未得到深入探究。喉癌的发生发展机制极为复杂，受多种因素调控，其中吸烟与饮酒是最为关键的危险因素。近期研究表明，HPV感染在喉癌发生中同样发挥重要作用，HPV相关喉癌尤其好发于喉声门上区。

**研究目的**：本研究旨在明确喉癌患者中HPV/蛋白16（protein16）阳性的检出率，并探讨晚期喉癌患者中HPV和/或蛋白16阳性与年龄、性别、吸烟习惯、饮酒史、肿瘤部位、淋巴结转移、复发及生存情况等临床变量的相关性。

**研究方法**：本研究为回顾性研究，共纳入90例晚期喉癌患者，对照组为10例正常喉黏膜标本。采用多克隆聚合酶链反应（polymerase chain reaction，PCR）检测HPV的存在情况，采用免疫组化法检测蛋白16的表达。对于HPV阳性病例，进一步通过聚合酶链反应检测其HPV亚型16、18的存在情况。记录患者的人口学特征，并评估患者的生存情况及其与HPV/蛋白16阳性的相关性。

**研究结果**：90例患者中，11例（12.2%）检出多克隆HPV阳性。在这11例HPV阳性病例中，6例为HPV16阳性，4例为HPV18阳性，1例同时为HPV16与HPV18阳性。18例（20%）患者蛋白16阳性。其中6例（6.6%）患者同时存在HPV与蛋白16阳性。在仅蛋白16阳性或HPV与蛋白16均阳性的病例中，患者饮酒率更低，且肿瘤更倾向于位于声门上区，该差异具有统计学意义。蛋白16阳性病例中，肿瘤声门上区定位的比例显著升高，蛋白16阳性与声门上区定位的相关性具有统计学意义（p=0.011）。蛋白16阳性病例中，55.6%的肿瘤位于声门上区，33.3%位于声门区，11.1%位于跨声门区。尽管HPV与蛋白16阳性患者的5年以上生存率在数值上更高，但该差异未达到统计学意义。本研究中，HPV阳性与患者平均年龄、肿瘤分化程度、吸烟与饮酒史、肿瘤进展、淋巴结转移、肿瘤部位、复发、死亡原因及治疗方式均无统计学意义的相关性。患者的平均随访时间为6.7年。

**研究结论**：包括本研究在内的多项研究均未证实HPV与喉恶性肿瘤之间存在如同口咽鳞状细胞癌那般的紧密关联。本研究结果支持HPV在喉癌发生发展中仅发挥有限作用。蛋白16无法作为喉癌中HPV感染状态的可靠替代标志物，也不能作为喉癌患者生存情况的预测因子。蛋白16阳性病例中，肿瘤声门上区定位的比例显著升高，且该相关性具有统计学意义。未来需要开展更大样本量的临床试验，更好地证实肿瘤增殖情况，并通过多种技术手段验证所得结果的准确性。