Human Cytomegalovirus Nucleic Acids Research Articles

Presence of the Human Cytomegalovirus in Glioblastomas-A Systematic Review.

Simple SummaryWhether the human cytomegalovirus (HCMV) is present in samples obtained from patients with glioblastoma (GBM) has been a matter under debate during the last two decades. Many investigators have demonstrated the presence of HCMV proteins and nucleic acids in GBM tumors, while some have not been able to detect it. It is important to evaluate current data and resolve these issues to clarify the possible role of the HCMV in GBM tumorigenesis and if this virus can serve as a potential target of therapy for these patients. In the present systematic review, we aim to review published research studies with a focus to identify differences and similarities in methods used for the detection of the HCMV in GBM samples found to be positive or negative for HCMV. Our data suggest that the HCMV is highly prevalent in glioblastomas and that optimized immunohistochemistry techniques are required to detect it.Glioblastoma is a malignant brain tumor with a dismal prognosis. The standard treatment has not changed in the past 15 years as clinical trials of new treatment protocols have failed. A high prevalence of the human cytomegalovirus (HCMV) in glioblastomas was first reported in 2002. The virus was found only in the tumor and not in the surrounding healthy brain tissue. Many groups have confirmed the presence of the HCMV in glioblastomas, but others could not. To resolve this discrepancy, we systematically reviewed 645 articles identified in different databases. Of these, 81 studies included results from 247 analyses of 9444 clinical samples (7024 tumor samples and 2420 blood samples) by different techniques, and 81 articles included 191 studies that identified the HCMV in 2529 tumor samples (36% of all tumor samples). HCMV proteins were often detected, whereas HCMV nucleic acids were not reliably detected by PCR methods. Optimized immunohistochemical techniques identified the virus in 1391 (84,2%) of 1653 samples. These data suggest that the HCMV is highly prevalent in glioblastomas and that optimized immunohistochemistry techniques are required to detect it.

Human cytomegalovirus infection enhances cell proliferation, migration and upregulation of EMT markers in colorectal cancer-derived stem cell-like cells.

Increasing evidence suggests a link between persistent human cytomegalovirus (HCMV) infection and cancer. Although the role of HCMV in cancer is still elusive, recent studies revealed the presence of HCMV nucleic acids and proteins in different cancer types such as glioblastoma, colorectal, breast, and prostate cancers, and neuroblastoma. Although HCMV may not be directly associated with the neoplastic transformation, the presence of HCMV DNA in the tumorous tissue has been associated with altered clinical outcomes in cancer patients. However, the mechanisms involved in the association between colorectal cancer (CRC) and HCMV are unclear. In this study, we investigated the influence of HCMV infection on CRC or their derived cells. Proliferation and migration assays revealed a high infection efficiency in CRC-derived HT29 and SW480 'stem-like' cells. After 24, 48 and 72 h of HCMV infection, both HT29 and SW480 parental and stem-like cells showed a significant increase in cell proliferation and viability (p<0.0001). Moreover, HCMV infection promoted cell migration. These results demonstrate a significant phenotypic alteration in the CRC cell line upon HCMV infection. Using epithelial to mesenchymal transition (EMT) assays, we demonstrated that the EMT markers and driver genes were upregulated during the virus infection. The WNT signaling pathway, which is associated with the proliferation and migration of CRC cells, was upregulated (6-fold) in HCMV-infected cells as compared to the non-infected cells at day 7 from infection.

Positive Expression of Human Cytomegalovirus Phosphoprotein 65 in Atherosclerosis.

Previous studies showed that human cytomegalovirus (HCMV) is associated with atherosclerosis. However, local vascular atherosclerosis related HCMV infection and protein expression remain unclear. This study aimed to assess the relationship between HCMV infection and atherosclerosis. Formalin-fixed, paraffin-embedded peripheral artery specimens were obtained from 15 patients with atherosclerosis undergoing vascular surgery from 2008 to 2010 at Zhongnan Hospital, Wuhan University. Pathological analyses were carried out after hematoxylin and eosin (H&E) and Masson trichrome staining. In situ hybridization and immunohistochemistry with two different monoclonal antibodies were employed to detect HCMV nucleic acids and proteins, respectively. H&E and Masson trichrome staining showed homogeneous extracellular matrix in femoral artery, while smooth muscle fibers were interlaced with collagen fibers; in carotid artery, inflammatory cell infiltration, foam cell vascular change, cholesterol crystals, and layered collagen fibers were observed. In situ hybridization showed no expression of HCMV nucleic acids in all 15 cases. Immunohistochemical staining for protein immediate-early protein (IE1 72) was negative in all cases, while phosphoprotein 65 (pp65) expression was detected in 14 cases. A high rate of positive pp65 signals was found in patients with atherosclerosis, suggesting that local HCMV infection may be associated with the pathogenesis of atherosclerosis. Further studies on this relationship are warranted.

High Prevalence of Human Cytomegalovirus in Brain Metastases of Patients with Primary Breast and Colorectal Cancers

BACKGROUND: Brain metastases (BMs) develop by largely unknown mechanisms and cause major morbidity and mortality in patients with solid tumors. Human cytomegalovirus (HCMV) is frequently detected in tumor tissue from patients with different cancers. Here, we aimed to determine the prevalence and potential prognostic role of HCMV in BMs. METHODS: We obtained archived samples of BMs from 41 patients with breast cancer and 37 with colorectal cancer and paired primary tumor tissues from 13 and 12 patients in each respective group. In addition, primary breast cancer tissues from 15 patients were included. HCMV proteins were detected with an immunohistochemical technique and Western blot. HCMV nucleic acids were detected with TaqMan polymerase chain reaction (PCR) assay. RESULTS: HCMV proteins were abundantly expressed in 99% of BM specimens, and in 12 of 13 (92%) paired primary breast cancer specimens. All 12 paired colon cancer samples were positive for HCMV proteins. Protein staining was mainly confined to neoplastic cells. Western blot analysis detected an HCMV-IE reactive protein in 53% of breast cancer specimens, and PCR detected the presence of HCMV DNA and transcripts in 92% and 80% of samples, respectively. Patients with high-level expression of HCMV-IE proteins in their tumors had a shorter time to tumor progression and shorter overall survival. CONCLUSIONS: The prevalence of HCMV proteins and nucleic acids is very high in primary and metastatic tumors and may drive the development of metastatic brain tumors; therefore, this virus may represent a potential therapeutic target in metastatic cancer.

Detection of human cytomegalovirus in glioma tumor tissues

A brain tumor that develops from glial cells is called a glioma. About half of all primary brain tumors form from glial cells. Gliomas are divided into subgroups, depending on the origin of the glial cells. Detection of human cytomegalovirus in tumor tissues of glioma patient was performed for the first time in Iran. These data show an association between human cytomegalovirus (HCMV) and malignant gliomas and suggest that HCMV may play an active role in glioma pathogenesis. Cytomegalovirus surgical biopsy specimens of glioma tissues (n = 28) and non-tumor brain tissues (n = 8) were obtained. Total DNA of specimens was extracted, and then, extracted DNA was evaluated by polymerase chain reaction for evidence of HCMV nucleic acids. Here, we show that a high percentage of malignant gliomas are infected by HCMV. Seventy-five percent of grade 4 glioma, 57 % of grade 3 glioma, and 33 % of grade 2 glioma were HCMV positive. All specimens of grade 1 glioma and control tissues were HCMV negative. The relationship between the grade of glioma and the presence of HCMV is significant (P value = 0.035). Fisher exact test was used for statistical analysis.

Sensitive detection of human cytomegalovirus in tumors and peripheral blood of patients diagnosed with glioblastoma

Human cytomegalovirus (HCMV) has been described to be associated with several human malignancies, though the frequency of detection remains controversial. It is unclear whether HCMV plays an active role in malignant tumor progression or becomes reactivated under pathologic conditions that result in chronic inflammation or immunosuppression. In this study, we report on the investigation of detecting HCMV in the tumors and peripheral blood of patients with newly diagnosed glioblastoma multiforme (GBM). Using immunohistochemistry, in situ hybridization, and polymerase chain reaction amplification of viral DNA, the detection of HCMV was investigated in tumor and blood specimens from patients with GBM as well as in the peripheral blood of normal volunteers and patients undergoing craniotomy for diagnoses other than GBM. We found that a high percentage (>90%) of GBM tumors, not surrounding normal brain, are associated with HCMV nucleic acids and proteins. Furthermore, a significant proportion of patients (80%) with newly diagnosed GBM have detectable HCMV DNA in their peripheral blood, while sero-positive normal donors and other surgical patients did not exhibit detectable virus, suggesting either a systemic reactivation of HCMV within patients with GBM or shedding of viral DNA from infected tumor cells into the periphery. These results confirm the association of HCMV with malignant gliomas and demonstrate that subclinical HCMV viremia (presence of viral DNA in blood without clinical symptoms of infection) is a previously unrecognized disease spectrum in patients with GBM.

High Prevalence of Human Cytomegalovirus in Prostatic Intraepithelial Neoplasia and Prostatic Carcinoma

Recent epidemiological data indicate that a history of increased exposure to sexually transmitted diseases is associated with an increased risk of prostate cancer. Human cytomegalovirus (HCMV) is a member of the herpesvirus family, is sexually transmitted in adults and can persistently infect prostatic epithelium in non-immunocompromised hosts. Based on increased awareness of the oncogenic potential of this virus, we decided to reexplore the issue of whether HCMV might be involved in prostate cancer pathogenesis. Paraffin embedded biopsy specimens from 22 randomly selected patients with prostatic intraepithelial neoplasia (PIN) lesions and prostatic carcinoma were analyzed by immunohistochemistry, in situ hybridization, polymerase chain reaction and DNA sequencing to detect HCMV nucleic acids and determine whether HCMV gene products were specifically associated with neoplastic cells. We detected HCMV proteins and/or nucleic acids in all 22 of the 22 preneoplastic and neoplastic prostate lesions evaluated. HCMV proteins were specifically and often highly expressed in basal cell hyperplasia and PIN lesions, and to a lesser degree in carcinoma cells. To our knowledge these data demonstrate for the first time the specific localization of HCMV nucleic acids and proteins in a high percent of PIN and prostate carcinoma lesions, and raise the possibility that HCMV might contribute to the natural history of prostatic cancer.

Specific localisation of human cytomegalovirus nucleic acids and proteins in human colorectal cancer

Colorectal cancer is the second most frequent cause of death from cancer in the USA, and most tumours arise sporadically with no clear cause or genetic predisposition. Human cytomegalovirus is a beta-herpesvirus that is endemic in the human population and can cause life-threatening disease in immunosuppressed adults. In vitro, human cytomegalovirus can transform cells and dysregulate many cellular pathways relevant to colon adenocarcinoma pathogenesis, especially those affecting the cell cycle, mutagenesis, apoptosis, angiogenesis, and cyclo-oxygenase-2 (COX-2) expression. We aimed to assess whether gene products of human cytomegalovirus could be detected in colorectal cancers. We obtained formalin-fixed, paraffin-embedded pathological specimens of colorectal polyps, adenocarcinomas, and adjacent normal mucosa from 29 patients. To detect human cytomegalovirus proteins and nucleic acids, we used immunohistochemistry with two different monoclonal antibodies, in-situ hybridisation, and PCR with DNA sequencing. Human cytomegalovirus proteins IE1-72 and pp65 were detected in a tumour cell-specific pattern in 14 (82%) of 17 and seven (78%) of nine colorectal polyps, respectively, and 12 (80%) of 15 and 11 (92%) of 12 adenocarcinomas, respectively, but not in adjacent non-neoplastic colon biopsy samples from the same patients (none of seven and none of two, respectively). Human cytomegalovirus infection of colon-cancer cells (Caco-2) in vitro resulted in specific induction of Bcl-2 and cyclo-oxygenase-2 proteins, both of which are thought to contribute to progression of colon cancer. Human cytomegalovirus nucleic acids and proteins can be found that specifically localise to neoplastic cells in human colorectal polyps and adenocarcinomas, and virus infection can induce important oncogenic pathways in colon-cancer cells.

Fibroblasts, epithelial cells, endothelial cells and smooth muscle cells are major targets of human cytomegalovirus infection in lung and gastrointestinal tissues

High titre replication of human cytomegalovirus (HCMV) in cell culture is restricted to primary human fibroblasts. During acute infection in vivo, HCMV nucleic acids and antigens have been found in various organs. Using only morphological criteria, inconsistent data have been reported about the cell types that can be infected by HCMV. In particular, the role of fibroblasts in organ infections has remained unclear. To define accurately the target cells of HCMV in vivo, tissue sections from lung and gastrointestinal tract of patients suffering from acute HCMV infection were investigated using immunohistochemical double-labelling analyses. Monoclonal antibodies with defined specificity against immediate early (IE), early (E) and late (L) viral antigens and antibodies directed against cell marker proteins were employed to identify infected cells. The results demonstrated that a broad spectrum of cells was infected by HCMV in vivo. Consistent with their susceptibility in culture, fibroblasts formed a major population of HCMV-infected cells. In contrast, haemopoietic cells were only infrequently stained with virus-specific antibodies. Fibroblasts, epithelial cells, endothelial cells, smooth muscle cells and macrophages appeared to be permissive for HCMV replication. Contrary to this, polymorphonuclear cells showed only IE gene expression, indicating that these cells were abortively infected. The analysis of the distribution of infected cells in tissue supported the hypothesis that endothelial cells and monocytes/macrophages may play a crucial role in the haematogenous spread of HCMV; in contrast, fibroblasts, smooth muscle cells and epithelial cells may form the cell populations important for the multiplication and spread of the virus in infected tissues.

Detection of latent human cytomegalovirus in organ tissue and the correlation with serological status.

The presence of human cytomegalovirus (HCMV) genome in spleen tissue was studied by using DNA hybridization techniques in seropositive and seronegative organ donors without clinical or laboratory confirmed HCMV infection. The serum samples of these patients were screened by latex agglutination test (LA) and enzyme linked immuno sorbent assay (ELISA) for the presence of HCMV antibodies, and confirmed by immunoblotting technique (IB). For the detection of HCMV sequences in spleen tissue dot blot DNA hybridization (DBH) using probes derived from immediate-early and late regions (ES and BH fragment respectively) of the HCMV genome were used. Samples positive in DBH were further tested by in situ DNA hybridization (ISH) using the ES probe. The number of spleen tissue specimens positive for HCMV nucleic acids indicated that HCMV may be present in human beings, even without serological evidence.

Detection of latent human cytomegalovirus in organ tissue and the correlation with serological status

The presence of human cytomegalovirus (HCMV) genome in spleen tissue was studied by using DNA hybridization techniques in seropositive and seronegative organ donors without clinical or laboratory confirmed HCMV infection. The serum samples of these patients were screened by latex agglutination test (LA) and enzyme linked immuno sorbent assay (ELISA) for the presence of HCMV antibodies, and confirmed by immunoblotting technique (IB). For the detection of HCMV sequences in spleen tissue dot blot DNA hybridization (DBH) using probes derived from immediate-early and late regions (ES and BH fragment respectively) of the HCMV genome were used. Samples positive in DBH were further tested by in situ DNA hybridization (ISH) using the ES probe. The number of spleen tissue specimens positive for HCMV nucleic acids indicated that HCMV may be present in human beings, even without serological evidence.

Human cytomegalovirus nucleic acids in tissues from congenitally infected infants

In situ nucleic acid hybridization methods were used to study autopsy tissues from 2 infants with clinical or pathologic evidence of congenital cytomegalovirus infections: a premature infant with multiple congenital malformations and inclusion bodies in several organs and an infant with culture-proved symptomatic congenital cytomegalovirus infection. Cytomegalovirus nucleic acids were detected in lung, kidney, adrenal, thyroid, skin, pancreas, and anterior pituitary tissues. In the second infant, hybridization studies of neural tissues failed to reveal cytomegalovirus nucleic acids despite neuropathologic abnormalities consisting of extensive necrotizing ependymitis and periventricular cavitation.

Examination of brains of AIDS cases for human immunodeficiency virus and human cytomegalovirus nucleic acids.

The role of direct virus infection as a determining factor in acquired immunodeficiency syndrome (AIDS) dementia was investigated using in situ hybridisation for human immunodeficiency virus (HIV) and human cytomegalovirus (HCMV). Four of the five AIDS dementia patients in this series demonstrated HIV infected cells distributed in widely different parts of the brain, but only one case showed HCMV infected cells. The greater abundance of HIV was in subcortical white matter in nodular areas consisting of monocyte/macrophage infiltrates. The cells were occasionally arranged as a multinucleated syncitium. In two cases, a few large cells with the appearance of neurons were positive for HIV hybridisation. By appropriate treatment with ribonuclease, it was shown that hybridisation was primarily to HIV RNA. HCMV infected cells were observed in small numbers in only one of the positive cases, suggesting that HCMV is not a determining factor in AIDS dementia. HCMV positive cells were located in the grey matter, with an appearance suggestive of neurons. Cells expressing the MHC-class II antigen HLA-DR, a marker of reactive microglia and macrophages, were observed to be extensive in affected brain sections in the one case examined. These cells were present in greater number than HIV infected cells. In this case, extensive numbers of HIV infected cells were noticed along the peripheral margin of the substantia innominata. This could indicate infection in this case of a critical brain region from the cerebrospinal fluid.