Co-infection and co-localization of Kaposi sarcoma-associated herpesvirus and Epstein-Barr virus in HIV-associated Kaposi sarcoma: a case report.

Kaposi sarcoma (KS), a multifocal vascular neoplasm frequently observed in HIV-positive individuals, primarily affects the skin, mucous membranes, visceral organs, and lymph nodes. KS is associated primarily with Kaposi sarcoma-associated herpesvirus (KSHV) infection. In this case report, we present a rare occurrence of co-infection and co-localization of KSHV and Epstein-Barr virus (EBV) in KS arising from the conjunctiva, which, to our knowledge, has not been reported previously. Immunohistochemistry (IHC), DNA polymerase chain reaction (PCR), and EBV-encoded RNA in situ hybridization (EBER-ISH) were utilized to demonstrate the presence of KSHV and EBV infection in the ocular KS lesion. Nearly all KSHV-positive cells displayed co-infection with EBV. In addition, the KS lesion revealed co-localization of KSHV Latency-Associated Nuclear Antigen (LANA) and EBV Epstein Barr virus Nuclear Antigen-1 (EBNA1) by multi-colored immunofluorescence staining with different anti-EBNA1 antibodies, indicating the possibility of interactions between these two gamma herpesviruses within the same lesion. Additional study is needed to determine whether EBV co-infection in KS is a common or an opportunistic event that might contribute to KS development and progression.

Yes-associated protein-1 overexpression in ocular surface squamous neoplasia; a potential diagnostic marker and therapeutic target.

Yes-associated protein-1 (YAP-1) is a Hippo system transcription factor, which serves as an oncogene in squamous cell carcinoma, and several solid tumors when the Hippo pathway is dysregulated. Yet, the activity of YAP-1 in ocular surface squamous neoplasia (OSSN) has not been determined. Here, we investigate the relationship between YAP-1 overexpression and OSSN. Using a cross-sectional study design, we recruited 227 OSSN patients from the University Teaching Hospitals in Lusaka, Zambia. Immunohistochemistry was used to assess YAP-1 protein overexpression in tumor tissue relative to surrounding benign squamous epithelium. OSSN patient samples (preinvasive, n = 62, 27% and invasive, n = 165, 73%) were studied. One hundred forty-nine invasive tumors contained adjacent preinvasive tissue, bringing the total number of preinvasive lesions examined to 211 (62 + 149). There was adjacent benign squamous epithelium in 50.2% (114/227) of OSSN samples. Nuclear YAP- 1 was significantly overexpressed in preinvasive (Fisher's (F): p <.0001, Monte Carlo (MC): p <.0001) and invasive (F: p <.0001, MC: p <.0001) OSSN in comparison to adjacent benign squamous epithelium when analyzed for basal keratinocyte positive count, staining intensity, expression pattern, and Immunostaining intensity-distribution index. YAP-1 expression did not differ between preinvasive and invasive OSSN (p >.05), keratinizing and non- keratinizing cancer (p >.05), or between T1/T2 and T3/T4 stages in invasive tumors (p >.05). However, grade 2 and 3 tumors had significantly stronger nucleus YAP-1 overexpression intensity than grade 1 tumors (F: p = .0078, MC: p = .0489). By immunohistochemistry, we identified significant overexpression (upregulation of YAP-1 protein expression) in preinvasive and invasive OSSN lesions compared to neighboring benign squamous epithelium. YAP-1 expression was significantly higher in poorly and moderately differentiated invasive squamous cancer than in well-differentiated carcinomas. Overexpression of YAP-1 within the margin of preinvasive and invasive OSSN, but not in the neighboring normal epithelium, indicates that it plays a role in the development and progression of OSSN.

Epstein-Barr Virus, But Not Human Papillomavirus, Is Associated With Preinvasive and Invasive Ocular Surface Squamous Neoplasias in Zambian Patients.

Background: 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. Methods: 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. Results: 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 in situ (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. Conclusions: 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.

Clinical and Pathologic Presentation of Primary Ocular Surface Tumors among Zambians.

AIM: This study aimed to characterize the clinical and pathologic presentation of ocular surface tumors (OSTs) and to more precisely differentiate the grades of ocular surface squamous neoplasia (OSSN) and benign lesions among Zambians. METHODS: Two-hundred sixty-five Zambian patients presenting with ocular surface growths, suspicious for OSSN, were recruited between November 2017 and November 2019 to a cross-sectional study to investigate their lesions. Sociodemographic data were collected, HIV infection status and vision tests were performed, and lesions were measured and documented. Lesions >2 mm in diameter were excised and sent for pathology analysis. In addition to the biopsies, tears, blood, and buccal swabs were collected. CD4+ T-cell counts were measured by flow cytometry. Lesions were classified according to the WHO guidelines. χ2 and bivariate correlations were used to analyze variable associations and strengths with phi/Cramer's V and correlation coefficients, respectively. Binary logistics was used to adjust for covariance. RESULTS: In this study, 68.3% of the participants were found to be HIV positive. The most frequent diagnoses were invasive OSSN (45.3%), preinvasive OSSN (29.1%), and pterygium (22.6%). Invasive OSSN comprised keratinizing squamous cell carcinoma (SCC) (87.5%), basaloid SCC (3.3%), and spindle cell carcinoma (3.3%). Unusual carcinomas, not described previously, included hybrid SCC (5.0%) and acantholytic SCC (0.8%). Invasive OSSN had advanced tumor (T3/T4) staging (93.3%) at diagnosis. Lymphadenopathy was rare (2.3%), and metastasis was absent. Patients were mostly female (59.2%). Median age was 36 (interquartile ranges 33-41) years (ranges 18-81). Patients with invasive OSSN were more likely to present with pain (p = 0.007), redness (p = 0.034), excessive tearing (p = 0.0001), discharge (p = 0.011), bleeding (p = 0.007), reduced vision (p = 0.0001), fungating lesion (p = 0.001), and blindness (p = 0.005); location at temporal limbus (p = 0.0001), inferior limbus (p = 0.0001), or circumlimbal (p = 0.001); and extension to cornea (p = 0.006) and forniceal palpebral conjunctiva (p = 0.001). Invasive OSSN was associated with any smoking habit and alcohol consumption (p = 0.04 and 0.03, respectively). HIV positivity was strongly associated with OSSN (74.6% OSSN vs. 49.3% benign lesions; p = 0.0001; phi: 0.237 [p = 0.0001]). CONCLUSION: OSTs are very common in Zambia and are strongly associated with HIV coinfection. Patients with OSSN were more likely to be HIV positive than those with pterygia. Despite the commonality of OSTs in sub-Saharan Africa, these cancers have historically been poorly characterized.