HHV‐8/KSHV during the development of Kaposi's sarcoma: evaluation by polymerase chain reaction and immunohistochemistry

Abstract

The human gamma-herpes virus-8 (HHV-8) was first described in AIDS-related Kaposi's sarcoma (KS) tumour samples. In this study, we report comparative studies on paraffin-embedded biopsies of AIDS-related KS (AKS) and endemic KS (EKS) with regard to HHV-8 content as evaluated using polymerase chain reaction (PCR) and immunohistochemistry. DNA was extracted either using Chelex-100 or using Qia-gene kit and was evaluated with the help of a semiquantitative PCR assay. The PCR detection of HHV-8 was more sensitive to the Chelex method than to Qia-gene. The threshold for PCR test sensitivity with the help of serial dilution of DNA was at the level of five plasmid ORF-26 regions, and DNA from 25 body cavity-based lymphoma-1 cells. The results expressed as virus load/actin unit showed progressively higher HHV-8 levels in late (nodular) cases, compared to those in early (patch/plaque) stages. Evaluation of HHV-8 DNA levels in tumour tissues, thus, indicates a correlation between virus load and KS stage. Double immunostaining of spindle cells (SC) in KS biopsies for CD34 and HHV-8/latency-associated nuclear antigen (LANA) showed an increase in double-positive SC in the lesions of nodular AKS and EKS cases, compared to that in plaque and patch stages. However, 10-15% of CD34+/LANA- SC cells were observed during the development from patch to nodular cases of AKS and EKS. Our results indicate that PCR analysis is a simple and sensitive diagnostic method for HHV-8 evaluation in KS tissues, processed for conventional histopathology.

Diagnosis and Treatment of Kaposi Sarcoma

“…Failure to demonstrate LANA does not necessarily rule out KS in an appropriate clinicopathological setting [77]. In such cases, LANA-stained sections should be carefully re-assessed for subtle granular staining in KS cell nuclei [76].…”

Asynchronous Progression through the Lytic Cascade and Variations in Intracellular Viral Loads Revealed by High-Throughput Single-Cell Analysis of Kaposi's Sarcoma-Associated Herpesvirus Infection

“…Importantly, KSHV-infected cultures usually exhibit spontaneous lytic replication, where 1 to 5% of cells have entered the lytic gene cascade (39). Assays on pooled samples, such as DNA array, quantitative real-time PCR, and immunoblots, by default, include both latent and lytic subpopulations, thereby obscuring experimental analyses (8,17,32,43). Since gene expression in lytically infected cells is robust and involves a wide range of genes, the coexistence of the two states of infection precludes a precise characterization of the viral transcriptomes (latent and lytic).…”

Variability of HHV8 LNA-1 Immunohistochemical Staining Across the 3 Histologic Stages of HIV-Associated Mucocutaneous Kaposi Sarcoma

“…[12][13][14]19 However, until now, there has not been a large study, which has attempted to address the factors contributing to this variability. This study demonstrates that CD4 count is not predictive of the histologic stage of mucocutaneous KS or the intensity and distribution of HHV8 immunohistochemical staining.…”

“…Although Pantanowitz et al 9 has cautioned against comparing staining across the stages because of the greater number of cells in nodular KS compared with patch/plaque stage, there are earlier studies 10,12,13 that have assessed staining across the different stages.…”