High-risk human papillomavirus (HPV) types cause cervical lesions of varying severity, ranging from transient productive infections to high-grade neoplasia. Disease stratification requires the examination of lesional pathology, and possibly also the detection of biomarkers. P16(INK4a) and MCM are established surrogates of high-risk HPV E6/E7 activity, and can be extensively expressed in high-grade lesions. Here we have combined these two cellular biomarkers with detection of the abundant HPV-encoded E4 protein in order to identify both productive and transforming lesions. This approach has allowed us to distinguish true papillomavirus infections from similar pathologies, and has allowed us to divide the heterogeneous CIN2 category into those that are CIN1-like and express E4, and those that more closely resemble nonproductive CIN3. To achieve this, 530 lesional areas were evaluated according to standard pathology criteria and by using a multiple staining approach that allows us to superimpose biomarker patterns either singly or in combination onto an annotated hematoxylin and eosin (H&E) image. Conventional grading of neoplasia was established by review panel, and compared directly with the composite molecular pathology visualized on the same tissue section. The detection of E4 coincided with the onset of vacuolation, becoming abundant in koilocytes as the MCM marker declined and cells lost their defined nuclear margins as visualized by standard H&E staining. Of the dual marker approaches, p16(INK4a) and E4 appeared most promising, with E4 generally identifying areas of low-grade disease even when p16(INK4a) was present. Extensive p16(INK4a) expression usually coincided with an absence of E4 expression or its focal retention in sporadic cells within the lesion. Our results suggest that a straightforward molecular evaluation of HPV life-cycle deregulation in cervical neoplasia may help improve disease stratification, and that this can be achieved using complementary molecular biomarker pairs such as MCM/E4 or, more promisingly, p16(INK4a)/E4 as an adjunct to conventional pathology.
高危型人乳头瘤病毒（HPV）感染导致了各种严重程度不同的宫颈病变，从一过性活动性感染到高级别肿瘤均与此有关。宫颈病变分层需要做组织病理学检查，并且也可能需要生物标记检测。P16（INK4a）和MCM是评价高危型HPV E6/E7活动性的确定指标，且可以在高级别病变中广泛表达。在这里，我们联合这两个细胞生物标记以及大量HPV编码E4蛋白的检测鉴别了活动性和转化性病变。这种方法帮助我们鉴别了真正的乳头瘤病毒感染与一些相似性病变，并且将异质性CIN2类分为两类：一类为CIN1样且表达E4，另一类与非活动性CIN3更加相似。为了达到这种预期结果，我们根据规范的病理学标准、使用多重染色方法评估了530例的病变区域，这种多重染色方法为单独一种或多种生物标记类型联合复染于标注的HE图像之上。由复审小组确定常规肿瘤分级，然后直接比较同一张组织切片上可见的混合性分子病理学结果。能够检测到E4表达与开始形成空泡同时发生，并且E4在挖空细胞中大量表达，同时MCM标记表达下降且通过标准化HE染色可见细胞失去其明确的细胞核边缘。p16（INK4a）和 E4双重标记显得最有希望，E4一般标记低级别病变区域，即使是p16（INK4a）阳性也是如此。广泛性p16（INK4a）表达经常伴有E4表达缺失或病变内散在细胞的局灶性表达。我们的结果表明，宫颈肿瘤中对HPV异常生活周期进行直接分子评估有助于改善病变分层，同时所使用的互补性分子学生物标记对（如MCM/E4及更有希望的p16（INK4a）/E4）可以作为常规病理学的辅助性诊断方法。