Is the presence of HCMV components in CNS tumors a glioma-specific phenomenon?

Tumor tissues were obtained during surgical resections at the Department of Neurosurgery of the First Affiliated Hospital of Zhengzhou University from January 2014 to December 2018. The general data of the patients, including age, sex, date of surgery, extent of resection were collected from the medical records. No patients underwent chemotherapy or radiation prior to surgical therapy. Samples for this research consisted of 65 meningiomas (benign, atypical, and malignant), 45 pituitary adenomas, 20 cavernous hemangiomas, and 30 metastatic carcinomas. The meningiomas were classified into benign (WHO grade I), 57 cases, and atypical/malignant, 8 cases. The pituitary adenomas consisted of 15 cases of growth hormone (GH) pituitary adenoma, 19 prolactin (PRL) pituitary adenomas, and 11 adrenocorticotropic hormone (ACTH) pituitary adenomas. Of these, 12 were invasive pituitary adenomas. Detailed information on the clinical characteristics of the non-glial CNS tumors patients is listed in Table 1.

After surgical resection, samples were divided into two; one part was processed for histopathology studies and the other was transferred into cryotubes and immediately snap-frozen in liquid nitrogen for subsequent molecular analysis. The histological diagnosis was established and verified by two neuropathologists according to the 2016 WHO classification guidelines [13]. For each sample, the percentage of tumor cells was assessed on hematoxylin and eosin-stained frozen sections. Peripheral blood samples were obtained from each patients. This study was approved by the institutional review boards of the hospital and written informed consent was obtained from all patients.

Tumor tissues were paraffin-embedded, and 4 μm sections were analyzed by sensitive immunostaining as described [7, 12]. Briefly, all sections were deparaffinized in xylene and rehydrated through serial dilutions of ethanol. The sections were blocked for endogenous peroxidase activity (3% H₂O₂, 12 min) and incubated with Fc receptor blocker (10 min, 20 °C; Innovex Biosciences, Richmond, CA, USA) before the addition of monoclonal antibodies. Sections were incubated at 4 °C overnight with either anti-IE1–72 (1:40; Abcam, Hong Kong) or anti-pp65 (clones 2 and 6, 1:50, Abcam, Hong Kong). Negative control sections not treated with primary antibody were also included. The slides were then developed with a secondary antibody (Dako Cytomation, Carpinteria, CA). Antigen-antibody complexes were visualized by streptavidin-conjugated horseradish peroxidase (Bio-Maixin, Fu Zhou, KIT-9701) and 3,3′-diaminobenzidine (DAB) as a chromogen, and counterstained with hematoxylin.

Sections were evaluated independently under light microscopy by two investigators blinded to clinical data. The evaluated proportion of positive cells was classified as 0 (0–1%), 1 (2–25%), 2 (26–50%), 3 (51–75%), or 4 (> 75%). For viral antigens, staining intensity was classified as negative, weak, moderate, or strong, with corresponding scores from 0 to 3, referring to the predominant intensity. A combined staining index was determined by multiplying the proportion by the staining intensity. A staining index > 4 was defined as tumors with widespread high IE1–72 or pp65 levels, and a staining index ≤4 as tumors with lower and/or more disseminated levels of staining.

DNA was extracted from tissue or 300 μl of peripheral blood using the Wizard® Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. DNA was quantitated by absorbance at 260 nm. The final DNA extracts were stored at − 80 °C until use.

The detection of HCMV in DNA samples was performed by nested PCR analysis with external and internal primers specific for the HCMV UL55 gene region. External primers (E-1/E-2) amplify a 267 bp fragment whereas internal primers (I-1/I-2) amplify a 146 bp fragment. The primer pairs used and the size of the amplified fragments are shown in Table 2. All samples were tested at least in triplicate. The first round of PCR was carried out in a 25 μl reaction containing 0.25 U of Taq polymerase, 0.5 μl of each external primer (10 mM) and 2 μl of each genomic DNA sample. For the first amplification round, after an initial denaturing step (94 °C, 5 min), 35 cycles of denaturation (94 °C, 30 s), annealing (58 °C, 30 s), extension (72 °C, 30 s) were performed, followed by a final extension step (72 °C, 6 min). Amplified products (2 μl) were then subjected a second round of PCR using the primer pair I-1/I-2; cycling parameters were denaturation (94 °C for 5 min) followed by 40 cycles of amplification (94 °C, 30 s; 59 °C, 30 s; 72 °C, 30 s) followed by a final extension step (72 °C, 5 min). Following second-round amplification, 5 μl PCR products were electrophoresed on 2.0% agarose gel, stained with ethidium bromide, and photographed on an ultraviolet light transilluminator. PCR products were extracted and subjected to direct DNA sequence analysis. During the PCR process, strict precautions were taken to avoid cross-contamination [14].

Immunohistochemical staining was used to detect whether HCMV proteins are expressed in a series of common non-glial CNS tumors including meningioma, pituitary adenoma, cavernous hemangioma, and metastatic carcinoma. We optimized immunohistochemistry conditions for the detection of low levels of HCMV IE1–72 expression. IE1–72 expression was detected in 3.1% (2/65) of meningioma specimens, whereas no IE1–72 protein was detected in atypical/malignant meningioma (Fig. 1a and b). Only a low percentage of IE1–72 immunoreactivity (6.7%, 2/30) was detected in metastatic carcinoma, and we did not detect IE1–72 immunoreactivity in pituitary adenoma or cavernous hemangioma. In HCMV-positive tumors, IE1–72 immunoreactivity was found in the nuclei and the perinuclear cytoplasm (Fig. 1c, d and Fig. 3c, d), and blood vessel endothelial cells within tumors were also HCMV-positive.

To confirm HCMV antigen expression in these samples, HCMV-related protein pp65 was also investigated. We detected the pp65 tegument protein in 4.6% (3/65) of meningioma specimens (Fig. 2c and d), among the 3 pp65 positive specimens, two specimen were also positive for IE1–72 as indicated above; no pp65 expression was detected in atypical/malignant meningioma. HCMV pp65 protein was not detected in pituitary adenoma, cavernous hemangioma, or metastatic carcinoma. Meanwhile, no immunohistochemical staining was detected in negative control sections (Fig. 2a,b and Fig. 3a,b). Our results indicate that HCMV immunoreactivity is only present in a low percentage of common non-glial CNS tumors (Table 1).

To confirm the presence of HCMV in these tumors we performed nested PCR to detect HCMV UL55 sequences in total DNA extracted from all studied tumor samples and control tissues. Samples were considered HCMV-positive when a band of 146 bp was present. HCMV sequences were only detected in the four antigen-positive samples detected earlier, specifically two benign meningiomas and two metastatic tumors. This confirmed in all cases that these products were HCMV sequences. No HCMV DNA was detected in pituitary adenoma or cavernous hemangioma tissues (Fig. 4a and b). Unfortunately, HCMV DNA was not detected in the peripheral blood of the non-glial CNS tumors patients (Additional file 1: Figure S1).