Julius Peter, Siyumbwa Stepfanie N, Moonga Phyllis, Maate Fred, Kaile Trevor, Haynatski Gleb, Minhas Veenu, Snow Jazmine, Peterson Kerstin, Gihozo Patience, Streeter Sam, Kaur Salan, Evans Annika, Gonzalez Daniela, Samwel Kandali, Kang Guobin, West John T, Wood Charles, Angeletti Peter C
Department of Pathology and Microbiology, School of Medicine, Lusaka, Zambia.
University Teaching Hospital, Eye Hospital, Lusaka, Zambia.
Front Oncol. 2022 Apr 14;12:864066. doi: 10.3389/fonc.2022.864066. eCollection 2022.
The etiopathogenesis of ocular surface squamous neoplasia (OSSN) is not fully understood. We assessed the frequency of oncogenic viruses in OSSN by immunohistochemistry (IHC) and polymerase chain reaction (PCR) for human papillomavirus (HPV), Epstein-Barr virus (EBV), Merkel cell polyomavirus (MCPyV), Kaposi sarcoma virus, and adenovirus. Cases from Zambia were prospectively enrolled using a cross-sectional study design between November 2017 and March 2020.
Demographic and clinical data [age, sex, HIV status, antiretroviral therapy (ART) history, CD4 count, plasma viral load] and tumor biopsies were collected from 243 consenting patients. Tumor samples were bisected, and half was used for DNA isolation, while the other half was formalin fixed and paraffin embedded (FFPE) for histopathology analysis. The expressions of latent EBV nuclear antigen 1 (EBNA1), CDKN2A/p16INK4A (p16), and MCPyV large T-antigen (LT) were tested by IHC. Multiplex PCR was used to detect 16 HPV genotypes and four other DNA tumor viruses [Kaposi's sarcoma-associated herpesvirus (KSHV), EBV, MCPyV, and adenovirus]. Relationships between HIV status, viral DNA and protein expression, and tumor grades were determined by statistical analysis.
OSSN tumors from patients were 29.6% preinvasive and 70.4% invasive. Patients presented with unilateral tumors that were 70.4% late stage (T3/T4). OSSN patients were HIV positive (72.8%). IHC on 243 FFPE biopsies resulted in the detection of EBNA1 (EBV), p16 high-risk HPV (HR-HPV), and MCPyV LT expression in 89.0%, 4.9%, and 0.0%, respectively. EBNA1 was expressed in all grades of preinvasive [cornea-conjunctiva intraepithelial neoplasia (CIN)1, 100%; CIN2, 85.7%; CIN3, 95.8%; and carcinoma (CIS), 83.8%] and in invasive (89.2%) OSSN. PCR on 178 samples detected EBV, HR-HPV, and MCPyV in 80.3%, 9.0%, and 13.5% of tumors, respectively. EBV was detected in all grades of preinvasive and invasive OSSN. EBV detection was associated with high HIV viral loads (p = 0.022). HR-HPV was detected in 0.0% CIN1, 0.0% CIN2, 5.6% CIN3, 13.0% CIS, and 7.0% invasive OSSN.
Our findings of EBV DNA and EBNA1 protein in all the grades of preinvasive and especially invasive OSSN are consistent with a potential causal role for EBV in OSSN. A role of HPV in OSSN was not clearly established in this study.
眼表鳞状上皮肿瘤（OSSN）的发病机制尚未完全明确。我们通过免疫组织化学（IHC）和聚合酶链反应（PCR）检测人乳头瘤病毒（HPV）、爱泼斯坦-巴尔病毒（EBV）、默克尔细胞多瘤病毒（MCPyV）、卡波西肉瘤病毒和腺病毒在OSSN中的感染频率。2017年11月至2020年3月期间，采用横断面研究设计对赞比亚的病例进行前瞻性纳入。
收集243例同意参与研究的患者的人口统计学和临床数据[年龄、性别、HIV状态、抗逆转录病毒治疗（ART）史、CD4细胞计数、血浆病毒载量]以及肿瘤活检样本。将肿瘤样本一分为二，一半用于DNA提取，另一半用福尔马林固定、石蜡包埋（FFPE）用于组织病理学分析。通过免疫组织化学检测潜伏性EBV核抗原1（EBNA1）、CDKN2A/p16INK4A（p16）和MCPyV大T抗原（LT）的表达。采用多重PCR检测16种HPV基因型以及其他四种DNA肿瘤病毒[卡波西肉瘤相关疱疹病毒（KSHV）、EBV、MCPyV和腺病毒]。通过统计学分析确定HIV状态、病毒DNA和蛋白表达与肿瘤分级之间的关系。
患者的OSSN肿瘤中29.6%为浸润前病变，70.4%为浸润性病变。患者表现为单侧肿瘤，其中70.4%为晚期（T3/T4）。OSSN患者HIV阳性率为72.8%。对243份FFPE活检样本进行免疫组织化学检测，结果显示EBNA1（EBV）、p16高危型HPV（HR-HPV）和MCPyV LT的表达率分别为89.0%、4.9%和0.0%。EBNA1在所有浸润前病变分级[角膜-结膜上皮内瘤变（CIN）1，100%；CIN2，85.7%；CIN3，95.8%；原位癌（CIS），83.8%]以及浸润性（89.2%）OSSN中均有表达。对178份样本进行PCR检测，结果显示分别在80.3%、9.0%和13.5%的肿瘤中检测到EBV、HR-HPV和MCPyV。在所有浸润前病变和浸润性OSSN分级中均检测到EBV。EBV检测与高HIV病毒载量相关（p = 0.022）。在CIN1中HR-HPV检测率为0.0%，CIN2中为0.0%，CIN3中为5.6%，CIS中为13.0%，浸润性OSSN中为7.0%。
我们在所有浸润前病变尤其是浸润性OSSN分级中均发现EBV DNA和EBNA1蛋白，这与EBV在OSSN中可能的致病作用一致。本研究未明确HPV在OSSN中的作用。
