Human Cytomegalovirus Tegument Protein pp65 Is Detected in All Intra- and Extra-Axial Brain Tumours Independent of the Tumour Type or Grade

Introduction

Human cytomegalovirus (HCMV) has been associated with tumours such as primary intracerebral tumours, neuroblastoma, colorectal cancer, prostate cancer, and non-melanoma skin carcinomas in humans [1]–[6]. Particular interest has been shown for the association between the HCMV protein expression and primary, highly malignant, non-curable brain tumours such as glioblastoma (GBM) [1], [7], [8]. To our knowledge, only a few other types of brain tumours, intra- or extra-axial have been investigated regarding the HCMV protein expression [1], [8]–[12]. Interestingly, no signs of an active infection such as intranuclear inclusions have been observed in these tumours. Meanwhile, the HCMV DNA and RNA have been detected in a subset of samples that have been assessed [1], [7], [13]. Noteworthy, when the HCMV DNA was investigated in a set of GBMs, only 1 out of 80 tumour cells was shown to carry the viral DNA [14].

Recently, it has been suggested that by treating for the HCMV infection, the progression of the primary disease, GBM, is halted even though it is not significant [15]. The high prevalence of the HCMV protein expression, as reported previously in GBM, makes the HCMV an interesting therapeutic target even if only a progression related effect is achieved. Thus, currently there are ongoing studies involving the antiviral therapies as a complementary treatment of subjects with GBM [13], [15], [16].

HCMV is a member of the Betaherpesvirinae subfamily of the Herpesviridae. An HCMV virion contains about 60 virus-encoded proteins and more than 70 host-proteins. Similar to all herpes viruses, the HCMV virion has three basic structural units: capsid, tegument, and envelope. Tegument is the link between the capsid and the envelope. It contains a number of proteins, two of which (pp65 and pp71) are key regulatory proteins that are delivered into the host cell during an infection. pp65 is the most abundant virion protein and has an immunomodulatory role. It is also a major component of the dense bodies – non-infectious viral particles that are assembled during active infection [17], [18]. In contrast, pp71 is a transcriptional transactivator. HCMV can be present in the monocytes during the latent phase of an infection when virions are not produced. During this latent phase, the viral DNA is replicated in close contact with the host DNA using host-cell replication machinery. The presence of the HCMV DNA at this phase can be shown by applying sensitive techniques such as the polymerase chain reaction (PCR). Contrary to this, during an ongoing active infection when the virions are produced, the resulting viral particles are seen as intranuclear inclusions in the affected cells, also called “the owl eyes”[17]–[20].

A primary HCMV infection leads to a life-long viral persistence. The seroprevalence of HCMV in the general population ranges between 50 to 100% [21]–[24]. The HCMV proteins can be detected in the human tissue in various cellular compartments by means of immunohistochemistry (IHC) [18]. Several reports have demonstrated one or more of these proteins in the neoplastic cells in the GBM and other intracerebral tumours (Table 1). Some of the proteins that have been investigated are components of the tegument or envelope of the virion and are expressed during certain phases of the HCMV life circle (immediate early 1 (IE1, 72 kDa), immediate early 76 kDa protein (clone DDG9), early envelope glycoprotein GP48 (clone QB1/42), early/late protein polymerase processivity factor (p52) (clone CCH2) and the late tegument protein pp65) [1], [8], [11], [25].

The proteins produced by the HCMV have been reported to be involved in numerous events, significant for tumour progression. It has been reported that these proteins influence the telomerase activation, angiogenesis, chronic inflammatory environment, immunosuppression, cellular motility and invasion, cell-cycle modulation, and anti-apoptotic effects [21], [26]–[33].

Noteworthy, none or extremely low levels of the HCMV proteins or nucleotides have been reported to be seen in areas of necrosis in tumours, normal brain tissue adjacent to tumour, brain tissue obtained from controls, or subjects suffering from various neurodegenerative diseases [1], [8], [33]. These observations support the notion that the HCMV proteins might be of significance for tumour initiation or progression.

In this study, we have assessed the expression of the HCMV proteins, applying the IHC and tissue microarray (TMA) techniques to a large unselected sample of various intracranial tumours including gliomas, medulloblastomas (MB), primary diffuse large B-cell lymphomas of the central nervous system (CNS DLBCL), and meningiomas. The aim has been to identify the best and most reliable method to assess the expression of the HCMV proteins in surgical samples of tumours and to analyse whether the type of the tumour, grade of the tumour, or the age of the patient influences the obtained expression pattern.

Materials and Methods

The study was carried out on human brain tissue obtained either during surgery (467 subjects) or postmortem (17 subjects). The original sample included material from subjects with intracranial tumours (469 subjects, Table 2), HCMV infection (one case), and brain tissue from control cases without a neurological disease (n = 14). The subjects included had given their consent for the use of the tissue, and the experimental procedures were approved by the local ethical committees in either Finland or Sweden. Some of the originally included cases were lost during the processing; finally, 417 cases remained for the analysis (Figure 1).

Results

Discussion

In this study, we have assessed the expression of the HCMV protein in a large cohort of intra- and extra-axial brain tumours. The cohort in our study included in total 417 tumours of various WHO grades, representing gliomas of various types, MBs, CNS DLBCL, and meningiomas. We noted that the HCMV protein pp65 expression was indeed common in brain tumours and ranged from 65 to 98 percent. This expression seemed to be independent of the tumour type, grade, patient age, and gender.

It is well known that the choice of Ab and sensitive staining protocols are of significance while assessing the HCMV proteins. The previously published results have reported that the HCMV proteins have been seen in 0 to 100% of the assessed tumours [1], [7]–[10], [12], [13], [25], [44]. We tested nine different commercial Abs directed toward different HCMV proteins, including Abs that detect proteins that are expressed at the early stage of the viral cycle to those that are expressed at the later stages. It should be noted that it is difficult to compare our results in detail with those previously published, as previous publications do not always provide details (clone) regarding the used antibodies. In most papers, only the antigen (immediately early/early/late) and the manufacturer producing the Ab are stated. In our study, all of the Abs tested that are commercially available were efficient when assessing the HCMV infected tissue. The hallmark lesions of an HCMV infection, including the inclusions seen in the HE stain, were repeatedly labelled. Thus, all of the Abs applied here can readily be used in the assessment of tissue with an active HCMV infection. In addition, however, labelling of glial cells, neurons, pericytes, erythrocytes, and neuropil was seen to various extents. This labelling seen is difficult to interpret on its own, without inclusions. Thus, the IR as such is not sufficient to verify an active infection. It is noteworthy that some of the tested Abs when applied on the control, uninfected brain tissue, labelled the glial cells, corpora amylacea, pericytes, and red blood cells. Whether these findings are a sign of a latent HCMV infection cannot be ruled out. In general, the choice of the Ab to be used while assessing IR is of great importance. It has previously been shown that applying various Abs to assess the same protein might lead to various results that are interpreted by the assessor in different manners, leading to controversies [45]. Here, to be on the safe side, we excluded all Abs that displayed a strong background staining or stained various cells and cell compartments (nucleus vs. cytoplasm) in the normal brain tissue. This scrutinized approach meant that only one Ab out of the nine tested fulfilled our requirements and was thus chosen. Consequently, this strict approach made us confident that what was detected as IR was indeed the HCMV late protein pp65 in the cells. The specificity of this Ab is also supported by previous report indicating that the Ab #8 recognizes a protein of the expected molecular mass [46].

Our results applying the Ab #8 regarding GBMs and MBs are in line with previously published reports applying the Abs #3, #4, and #6 [1], [7], [9]. In contrast, our results regarding GBM differ in comparison with some of the previously published reports while applying Abs #5, #7, and #8 [9], [12], [13]. This difference, particularly regarding Ab #8, is probably due to the staining protocol applied as has already been discussed in a previous publication [8]. Furthermore, dilution, ARM, and the use of a detection system, parameters that are well known to influence the IHC outcome, are of significance [47], [48]. Here, we carried out a systematic testing applying various dilutions and ARMs to obtain optimal results. Our testing led to the use of heat pretreatment, i.e., pressure cooker with a high pH buffer, as ARM. Others have implemented heat pretreatment with a low pH buffer or enzymatic digestion, ARMs not optimal in our study. Furthermore, the detection system used here, which was polymer based, has high signal amplification (PowerVision, Immunologic) [49]. Others have used different detection systems, including the polymer based [44] or the three-step labelled streptavidin-biotin method [1], [7], [13], [50]. All of these methods are in principle designed to amplify the labelling signal. Thus, due to the different approaches, comparing our results with those previously obtained is not fully possible. Even the thickness of the section has been discussed as being of importance if the staining method used is not sensitive enough [8]. Here, we used the thickness of sections applied in routine diagnostics ranging from 3 to 7 µm, without noting any major differences in the obtained results. The advantage of using thin sections is being able to assess the individual cells rather than the cell masses. A number of additional factors such as fixative, fixation time, and type of embedding medium have been demonstrated in several earlier studies to be of importance for the IHC outcome [51]–[53]. All tumour samples assessed here were, in principle, processed using the fixative and fixation time in a similar way. Although the paraffin used and the dehydration system in Finland and Sweden differed, we did not note any significant influence of these factors on the IHC results.

Thus, based on our observations, the Ab directed toward the HCMV late protein pp65 could readily be used on the formalin fixed tissue, independent of the thickness of the section or the routine handling of the sample (fixative, fixation time, paraffin), whereas one should be cautious regarding the ARM as well as the detection system.

Here, we only assessed the protein expression applying the IHC method. Some previous studies have also carried out the HCMV DNA and/or RNA analysis on the tissue investigated in addition to the IHC analyses [1], [7], [8], [12], [13], [44]. It is worth noting that while applying the DNA and/or RNA –assessment, fewer cases are positive when compared with the IHC-detection of the HCMV protein expression [11], [54]. This quite surprising result might be related to the observation that the HCMV-IR was patchy, that is, unevenly distributed in the tissue section. Thus, while applying different methods in assessing the HCMV, the sampling strategy might significantly influence the obtained results.

Presence of the different HCMV proteins in the gliomas has been analysed earlier in a number of studies [1], [8]–[10], [12], [13], [25], [44], [50]. Several publications, in line with our results, have indicated that the HCMV proteins are indeed frequently present in the GBM [1], [8], [13], [25], [44], [50]. The IR for late protein pp65 in our study was similar to previous reports; however, in addition to the GBMs the pp65 protein was also detected in the low-grade gliomas. Overall, 90% of all the gliomas independent of the WHO grade were HCVM-IR. Furthermore, although not previously reported but based on our results, the molecular subtype of the tumour did not influence the HCMV protein expression.

Medulloblastomas, preferentially seen in children, showed HCMV IR in 65% of the cases. This is in accordance with a previous report describing that 92% of the medulloblastomas expressed early and 73% expressed late proteins [7].

To our knowledge, the analysis of the HCMV in the CNS DLBCL has never been performed previously. In the current study, CNS DLBCL, a high malignant brain tumour, was IR in 76% of the cases. As MBs, these tumours are highly proliferative and subjects with this tumour have an extremely poor prognosis and a short survival time.

Interestingly, contrary to a previous report [1], we noted that 87% of the WHO grade I meningiomas (tumours considered benign with good prognosis) showed the HCMV-IR. In the WHO grade II meningiomas, the percentage was even higher, 90%. The lower HCMV-IR in the WHO grade III meningiomas (75%) could be due to a low number of cases included (n = 8). Our differing results when compared to the previous reports are probably due to selection bias and methodology. In one of the reports, all types of brain tumours were negative, disputing the methods used, and the other study included only nine cases [1], [12]. Thus, our results are in principle unique, indicating that the HCMV protein is expressed in these tumours of low malignancy. It is, however, well known that a meningioma has a high tendency to recur if not extirpated in total, indicating a good “survival” tendency of the tumour cells.

Here, we did not assess systematically the extent of the labelling in each case as has been carried out by other investigators [13]. The labelling, however, was seen in a sample measuring 0.6 to 2 mm in diameter (TMA core), indicating that a dichotomized assessment of the protein does not seem to be influenced by the sampling deficit. However, the distribution of the HCMV-IR might be, as we noted, patchy and uneven. This should be considered while assessing the extent of the HCMV-IR in routine diagnostics. It is common to obtain surgical samples ranging from a few mm (needle biopsy) to several cm (surgical resections); thus, the sampling deficit might be of significance.

We have been unable to assess any potential prognostic aspects related to the expression of the HCMV protein since the operation techniques varied (partial/total resection) and different chemo- and radiotherapy regimens had been applied. However, it is noteworthy that tumours ranging from the WHO grade I to WHO grade IV of various cellular origins and with extremely different proliferation rates (1 up to 90 percent) have all expressed the HCMV protein related to the late stage of the viral cycle.

Signs of an ongoing HCMV infection such as inclusions in the HE or IHC stain were never seen in any of our cases. Thus, we lack proof for an activation of a latent viral infection in this tumour population. What was detected was the expression of the late HCMV protein pp65 known as the tegument protein. This protein can be produced in excess in cells during an active symptomatic/asymptomatic infection and assembled in particles called dense bodies that lack viral DNA. The HCMV seropositivity is high in adults, and it is presumed that most of us have suffered from this infection even unknowingly, asymptomatic. Based on previous studies, the viral DNA can be detected in monocytes whereas the late viral proteins such as pp65 are common in tissue samples from patients with the HCMV infection. Whether all our subjects with the expression of late HCMV protein pp65 in the tumour cells have monocytes harboring the latent HCMV virus is not assessed here. This possibility however cannot be neglected due to the high levels of the HCMV seroprevalence, especially in adults [23], [24], [55]. It is also known that the ability of the HCMV to replicate in the monocytes, which harbor the latent HCMV, is dependent on the state of the cellular differentiation [56], [57]. Interesting to note, however, is that most studies assessing the activation of latent HCMV are carried out in vitro, and in vivo tumour conditions are in general difficult to replicate. Meanwhile, only a few cell types propagate the HCMV in vivo and the GBM cell lines are among them [18].

Some studies have reported that the HCMV proteins are present not only in the brain tumours but also in other types of cancers (skin, breast, colorectal, prostate) [2]–[4], [58]. Based on our results indicating that the late HCMV protein pp65 is present in a wide range of different tumour types within the skull, it emphasizes that further studies assessing the HCMV protein in various pathological conditions are warranted.

Recently, a number of studies have indicated that the anti-HCMV drug treatment can alter the outcome of the GBM in human, animal models, and cell cultures [15], [16], [59]. These drugs, at least two of them, induce apoptosis and thus influence the survival of the tumour cells. Interestingly, one of the drugs is dependent on the viral DNAase (assessed on human material) [15], [59] whereas the other is not (assessed on mouse models and cell cultures) [16]. Surprisingly, both the HCMV-expressing and non-HCMV-expressing tumours, when assessed in an experimental design, seemed to be influenced while using the drug, independent of the the viral DNAase [16].

In conclusion, we systematically analysed the performance of nine commercial HCMV-Abs on the brain tissue samples obtained from a verified HCMV infected patient, from 14 neurologically unimpaired subjects lacking pathology, and on a set of various brain tumours in TMA. The best performing Ab, the late HCMV protein pp65 (clones 2 and 6) was further used to assess the HCMV expression in different extra- and intra-axial brain tumours. This late HCMV protein pp65 was detected in all types of tumours analysed, and the IHC expression did not depend on the patient's age, gender, tumour type, or grade. The labelling pattern observed in the tumours differed from the labelling pattern observed in the tissue with a verified active HCMV infection. No signs of an active HCMV infection were noted; thus, we do not feel confident in using the term “HCMV infection” but rather implement the term “expression of late HCMV protein pp65” in tumour cells, which certainly might be a sign of a latent HCMV infection.

Data Availability

The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper.

Funding Statement

This work was supported by Local Uppsala University Hospital "ALF" grants. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.