应用液滴数字聚合酶链反应检测口咽鳞癌中人乳头瘤病毒 16 型。

Detection of human papillomavirus type 16 in oropharyngeal squamous cell carcinoma using droplet digital polymerase chain reaction.

机构信息

Department of Surgery, Division of Otolaryngology-Head and Neck Surgery University of Alberta, Edmonton, Alberta, Canada.

Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada.

出版信息

Cancer. 2016 May 15;122(10):1544-51. doi: 10.1002/cncr.29976. Epub 2016 Mar 17.

DOI:10.1002/cncr.29976

PMID:26989832

Abstract

BACKGROUND

The incidence of oropharyngeal squamous cell carcinoma caused by oncogenic HPV (HPV-OPSCC) is rising worldwide. HPV-OPSCC is commonly diagnosed by RT-qPCR of HPV-16 E6 and E7 oncoproteins or by cyclin-dependent kinase inhibitor 2A, multiple tumor suppressor 1 (p16) immunohistochemistry (IHC). Droplet digital PCR (ddPCR) has been recently reported as ultra-sensitive and highly precise method of nucleic acid quantification for biomarker analysis. We aimed to validate this method for the detection of HPV-16 E6 and E7 in HPV-OPSCC.

METHODS

Participants were recruited from January 2015-November 2015 at initial presentation to the University of Alberta Head and Neck Oncology Clinic. RNA was extracted, purified and quantified from prospectively collected participant tissues, and ddPCR was performed with fluorescent probes detecting HPV-16 E6 and E7. Results from ddPCR were compared with p16 IHC performed by clinical pathology as standard of care.

RESULTS

Head and neck tissues were prospectively obtained from 68 participants including 29 patients with OPSCC, 29 patients with non-OPSCC and 10 patients without carcinoma. 79.2% of patients with OPSCC were p16 positive. The sensitivity and specificity of ddPCR HPV E6/E7 compared with p16 IHC in OPSCC was 91.3 and 100%, respectively. The amount of target RNA used was ≤1 ng, 20-50 times lower than reported by other for RT-qPCR HPV E6/E7.

CONCLUSIONS

摘要

背景

由致癌 HPV（HPV-OPSCC）引起的口咽鳞状细胞癌的发病率在全球范围内正在上升。HPV-OPSCC 通常通过 HPV-16 E6 和 E7 癌蛋白的 RT-qPCR 或通过细胞周期蛋白依赖性激酶抑制剂 2A、多个肿瘤抑制因子 1（p16）免疫组化（IHC）来诊断。液滴数字 PCR（ddPCR）最近被报道为用于生物标志物分析的核酸定量的超灵敏和高度精确的方法。我们旨在验证该方法用于检测 HPV-OPSCC 中的 HPV-16 E6 和 E7。

方法

参与者于 2015 年 1 月至 2015 年 11 月在艾伯塔大学头颈肿瘤诊所初次就诊时被招募。从前瞻性收集的参与者组织中提取、纯化和定量 RNA，并使用荧光探针进行 ddPCR 检测 HPV-16 E6 和 E7。ddPCR 的结果与临床病理学作为标准护理的 p16 IHC 进行比较。

结果

前瞻性获得了 68 名参与者的头颈部组织，包括 29 名 OPSCC 患者、29 名非 OPSCC 患者和 10 名无癌患者。79.2%的 OPSCC 患者 p16 阳性。ddPCR HPV E6/E7 与 OPSCC 中的 p16 IHC 相比，其敏感性和特异性分别为 91.3%和 100%。目标 RNA 的使用量≤1ng，比其他报告的 RT-qPCR HPV E6/E7 低 20-50 倍。