Detection of transcriptionally active high-risk HPV in patients with head and neck squamous cell carcinoma as visualized by a novel E6/E7 mRNA in situ hybridization method.
Detection of transcriptionally active high-risk HPV in patients with head and neck squamous cell carcinoma as visualized by a novel E6/E7 mRNA in situ hybridization method.
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DOI：
10.1097/pas.0b013e318265fb2b
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发表时间：
2012-12
期刊：
影响因子：
--
通讯作者：
Westra WH
中科院分区：
文献类型：
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作者：
Bishop JA;Ma XJ;Wang H;Luo Y;Illei PB;Begum S;Taube JM;Koch WM;Westra WH
病毒癌蛋白E6和E7的转录激活证据被认为是临床相关人乳头瘤病毒（HPV）存在的金标准，但E6/E7 mRNA的检测需要RNA提取和PCR扩增-这是一项具有挑战性的技术，仅限于研究实验室。与E6/E7 mRNA互补的RNA原位杂交（ISH）探针的开发允许在常规处理的组织中直接观察病毒转录物，并为临床护理环境中的准确HPV检测打开了大门。使用p16免疫组织化学、HPV DNA ISH和靶向高危HPV E6/E7 mRNA转录物的RNA ISH测定（RNAscope®），检测了包含282例来自不同解剖学亚部位的头颈部鳞状细胞癌（HNSCC）的组织微阵列（TMA）中HPV的存在。E6/E7 mRNA检测还用于检测另外25例口咽癌，其中由于检测结果相互矛盾（即p16阳性，DNA ISH阴性），手术病理报告中记录的HPV状态不确定。用E6/E7 mRNA法检测282例HNSCC中49例（17%）检出HPV，其中口咽癌77例中43例（56%），原发部位不明的转移性HNSCC 3例中2例（67%），鼻腔鼻窦癌7例中2例（29%），头颈部其他部位癌195例中2例（1%）。P16表达与HPV E6/E7 mRNA的存在密切相关：49例HPV阳性肿瘤中有46例显示p16表达，而233例HPV阴性肿瘤中只有22例p16阳性（94%对9%，p <0.0001）。E6/E7 mRNA法与HPV DNA原位杂交法之间的一致率也很高（99%）。对于所选的一组不一致的HNSCC（p16+/HPV DNA-），在25例病例中的21例（84%）中检测到E6/E7转录本的存在。E6/E7 mRNA方法证实了存在转录活性的HPV相关HNSCC，其对口咽部具有强烈的偏好，并且与高水平的p16表达强烈相关。通过RNA ISH检测HPV E6/E7转录本是理想的，因为它证实了整合和转录活性病毒的存在，允许组织中病毒转录本的可视化，并且在临床实验室中进行常规检测在技术上是可行的。
Evidence for transcriptional activation of the viral oncoproteins E6 and E7 is regarded as the gold standard for the presence of clinically relevant human papillomavirus (HPV), but detection of E6/E7 mRNA requires RNA extraction and PCR amplification - a challenging technique that is restricted to the research laboratory. The development of RNA in-situ hybridization (ISH) probes complementary to E6/E7 mRNA permits direct visualization of viral transcripts in routinely processed tissues and has opened the door for accurate HPV detection in the clinical care setting. Tissue microarrays (TMAs) containing 282 head and neck squamous cell carcinomas (HNSCCs) from various anatomic subsites were tested for the presence of HPV using p16 immunohistochemistry, HPV DNA ISH, and a RNA ISH assay (RNAscope®) targeting high risk HPV E6/E7 mRNA transcripts. The E6/E7 mRNA assay was also used to test an additional 25 oropharyngeal carcinomas where HPV status as recorded in the surgical pathology reports was equivocal due to conflicting detection results (i.e. p16 positive, DNA ISH negative). By the E6/E7 mRNA method, HPV was detected in 49 of 282 (17%) HNSCCs including 43 of 77 (56%) carcinomas from the oropharynx, 2 of 3 (67%) metastatic HNSCCs of unknown primary site, 2 of 7 (29%) carcinomas from the sinonasal tract, and 2 of 195 (1%) carcinomas from other head and neck sites. P16 expression was strongly associated with the presence of HPV E6/E7 mRNA: 46 of 49 HPV positive tumors exhibited p16 expression, whereas only 22 of 233 HPV negative tumors were p16 positive (94% versus 9%, p < .0001). There was also a high rate of concordance (99%) between the E6/E7 mRNA method and HPV DNA ISH. For the selected group of discordant HNSCCs (p16+/HPV DNA-), the presence of E6/E7 transcripts was detected in 21 of 25 (84%) cases. The E6/E7 mRNA method confirmed the presence of transcriptionally active HPV-related HNSCC that has a strong predilection for the oropharynx and is strongly associated with high levels of p16 expression. Testing for HPV E6/E7 transcripts by RNA ISH is ideal because it confirms the presence of integrated and transcriptionally active virus, permits visualization of viral transcripts in tissues, and is technically feasible for routine testing in the clinical laboratory.