Human cytomegalovirus infection in tumor cells of the nervous system is not detectable with standardized pathologico-virological diagnostics

Abstract

Background: Experimental findings have suggested that human cytomegalovirus (HCMV) infection of tumor cells may exert oncomodulatory effects that enhance tumor malignancy. However, controversial findings have been published on the presence of HCMV in malignant tumors. Here, we present the first study that systematically investigates HCMV infection in human nervous system tumors by highly sensitive immunohistochemistry in correlation with the HCMV serostatus of the patients.

Methods: Immunohistochemical and quantitative PCR-based methods to detect different HCMV antigens and genomic HCMV DNA were optimized prior to the investigation of pathological samples. Moreover, the pathological results were matched with the HCMV serostatus of the patients.

Results: HCMV immediate-early, late, and pp65 antigens could be detected in single cells from HCMV strain Hi91-infected UKF-NB-4 neuroblastoma cells after 1:1024 dilution with noninfected UKF-NB-4 cells. Genomic HCMV DNA could be detected in copy numbers as low as 430 copies/mL. However, we did not detect HCMV in tumors from a cohort of 123 glioblastoma, medulloblastoma, or neuroblastoma patients. Notably, we detected nonspecifically positive staining in tumor tissues of HCMV seropositive and seronegative glioblastoma patients. The HCMV serostatus of 67 glioblastoma patients matched the general epidemiological prevalence data for Western countries (72% of female and 57% of male glioblastoma patients were HCMV seropositive). Median survival was not significantly different in HCMV seropositive versus seronegative glioblastoma patients.

Conclusions: The prevalence of HCMV-infected tumor cells may be much lower than previously reported based on highly sensitive detection methods.

Keywords: cytomegalovirus; glioma; oncomodulation.

Fig. 1.

Immunocytochemistry (ICC) and immunohistochemistry (IHC) displaying detection limits in HCMV diagnostics. (A) ICC of paraffin-embedded HCMV strain Hi91-infected UKF-NB-4 neuroblastoma cells showing HCMV-IEA positive nuclei in 30%–40% of the cells (arrow). (B) ICC of paraffin-embedded HCMV-infected UKF-NB-4 cells diluted with noninfected cells in a ratio of 1:1. (C) ICC of paraffin-embedded HCMV-infected UKF-NB-4 cells diluted with noninfected cells in a ratio of 1:4. (D) ICC of paraffin-embedded HCMV-infected UKF-NB-4 cells diluted with noninfected cells in a ratio of 1:16. (E) ICC of paraffin-embedded HCMV-infected UKF-NB-4 cells diluted with noninfected cells in a ratio of 1:64. (F) ICC of paraffin-embedded HCMV-infected UKF-NB-4 cells diluted with noninfected cells in a ratio of 1:256. (G) ICC of paraffin-embedded HCMV-infected UKF-NB-4 cells diluted with noninfected cells in a ratio of 1:1024. (H) Cerebellar tissue from a patient with HCMV encephalitis showing atypical HCMV-positive nuclei (arrow) of infected neurons and positive nuclei of surrounding glial cells (arrow-heads). (Scale 100 µm).

Fig. 2.

Tumors of the nervous system are virtually negative for HCMV antigens. (A) Neuroblastoma stained with the DAKO-antibody against HCMV early antiegen (EA) and HCMV immediate-early antigen (IEA) showing only HCMV-negative nuclei. (B) Human medulloblastoma stained with the DAKO antibody against HCMV EA/IEA also being completely negative. (C and D) Two different glioblastoma specimens also being negative for HCMV EA/IEA. (E) Glioblastoma being slightly positive for HCMV in some areas. (F) Specimen of a patient suffering from cerebellar HCMV encephalitis showing HCMV EA/IEA-positive nuclei of neurons and surrounding glial cells. (Scale 100 µm).

Fig. 3.

Increased anti-HCMV antibody concentrations do not lead to stronger staining intensities or frequencies in tumors of the nervous system in either positive or negative cases. Negative glioblastomas were stained for pp65 antigen (A: antibody dilution 1:200; B: antibody dilution 1:5) and immediate early antigen (IEA); (C: antibody dilution 1:50; D: antibody dilution 1:5) using different antibody concentrations. Slightly HCMV-positive glioblastomas were stained for pp65 antigen (E: antibody dilution 1:200; F: antibody dilution 1:5), and IEA (G: antibody dilution 1:50; H: antibody dilution 1:5) using different antibody concentrations (Scale 50 µm).

Fig. 4.

Old intratumoral bleeding and fixation artefacts are potential pitfalls in immunohistochemistry-based HCMV diagnostics. (A) Positive glioblastoma case with gemistocytic cells being positive for HCMV-pp65 antigen and also for (B) HCMV immediate early antigen (IEA). (C) Same glioblastoma case stained without primary antibody also showing a similar positive signal indicating an unspecific reaction. (D and E) Neuroblastoma with hematoidin artefacts appearing like positive tumor cells (arrows). (F) Neuroblastoma with formalin pigments (arrow). (Scale 50 µm).

Fig. 5.

Patient survival is not associated with the HCMV serostatus of glioblastoma patients. (A) Contingency analysis of HCMV serostatus by sex. (B) Kaplan Meyer survival analysis investigating dependence of HCMV serostatus and patient survival. Log-rank P = .362; Wilcoxon P = .125.