Detection of Human Cytomegalovirus DNA in Plasma from Patients with Brain Tumors Using a Sensitive qPCR Assay

Background: Human Cytomegalovirus (HCMV) has been detected in tissue samples from patients with glioblastoma, but its presence in blood has been less studied.

Methods: In this prospective study, we analyzed the levels of HCMV-DNA in 59 plasma samples obtained from patients with brain tumors and from 114 healthy blood donors as controls. Detection was performed using a sensitive qPCR assay.

Results: HCMV-IE DNA was detected in plasma samples from patients with glioblastoma (n = 18), astrocytoma grades II-III (n = 11), and brain metastasis (n = 8). These groups showed significantly lower ΔCT values than healthy donors (p < 0.0001 for glioblastoma and astrocytoma; p = 0.0007 for brain metastases), consistent with higher circulating viral DNA levels than in controls. No significant difference was observed between patients with secondary glioblastoma and healthy donors.

Conclusions: HCMV-DNA is present at higher levels in plasma from patients with glioblastoma, astrocytoma grades II–III, and brain metastases compared with healthy individuals. Quantification of HCMV DNA in plasma by qPCR is a reliable approach for detecting HCMV in brain tumour patients.

The origin of glioblastoma remains uncertain, but Human Cytomegalovirus (HCMV) has been implicated in the development/progression of primary and metastatic brain tumours, including glioblastoma and medulloblastoma [1-6]. Evidence for this association is mixed: several investigations have reported HCMV nucleic acids or proteins in tumour samples [1,7-17], whereas others have not detected viral material in similar specimens [18-25]. The discrepancies in reported HCMV detection may be explained by methodological differences. More sensitive and specific assays are likely required to resolve this paradox [26]. In particular, optimised protocols, such as antigen retrieval methods, are essential for reliable detection of HCMV proteins in tumour tissue, whereas viral proteins are readily identified in specimens from patients with acute infection.

Beyond brain tumours, HCMV antigens have been observed in multiple malignancies, including cancers of the colon, breast, prostate, and ovary [27-30]. Our own work previously demonstrated HCMV in the majority of brain metastases originating from colon and breast cancers [5]. Interestingly, adjacent non-tumour tissue typically remains virus-free, raising the question of whether HCMV contributes directly to tumorigenesis or merely represents a bystander phenomenon. In glioblastoma, we found that lower active viral levels in the tumour at diagnosis correlated with longer patient survival [13], suggesting a potential influence of HCMV on disease progression. If confirmed, the virus may represent a novel target for therapy. Supporting this idea, experimental studies have shown that antiviral agents or HCMV-specific nanobodies can suppress growth of HCMV-positive xenograft tumours, including medulloblastoma and glioblastoma models [4,31]. Antiviral treatment of HCMV or immunotherapy targeting HCMV in glioblastoma patients are promising, with indications of extended survival [32-35], while other treatments and immunotherapy protocols have failed to improve the prognosis for these patients [36]. Ongoing randomized clinical trials are now evaluating anti-HCMV therapy and dendritic cell vaccines encoding viral antigens (NCT04116411, NCT03927222).

HCMV is a highly prevalent herpesvirus, infecting 80-100% of the adult population worldwide. The primary infection is generally asymptomatic or mild and leads to a lifelong latent/persistent infection, from which it can reactivate. Although generally harmless in healthy individuals, reactivation can cause serious disease in immunocompromised patients, such as transplant recipients and those with AIDS. Importantly, reinfection with different viral strains can occur throughout life, and this also emphasize the difficulty in developing a vaccine against this virus.

HCMV establishes latency primarily in CD34 positive bone marrow progenitor cells. Differentiation of monocytes into macrophages or dendritic cells under inflammatory conditions can trigger reactivation [37,38]. Once reactivated, HCMV is capable of spreading to other cells and through different mechanisms manipulates diverse cellular processes, providing it with the capacity to influence tumour biology. HCMV exerts various oncomodulatory roles in tumors [1-3, 39-41] and can in fact establish all the Hallmarks of Cancer. HCMV controls cellular gene expression through direct and epigenetic mechanisms, it causes DNA damage and mutations, controls cell cycle progression and changes cellular metabolism to promote cell growth. HCMV also induces inflammation and angiogenesis, and it controls immune functions to avoid detection and elimination by the immune system [41,42].

Many investigators have found evidence of HCMV proteins in brain tumors [1,10,43], but identifying viral nucleic acids by PCR or sequencing has proven less consistent [18,19,24,26,44,45]. Some groups, however, have demonstrated abundant HCMV DNA or RNA in glioblastoma samples using PCR or in situ hybridization [9,15,39]. Other investigators have also detected HCMV in blood from glioblastoma patients [14,32,46-49]; these studies used whole blood as sample material. To date, however, plasma has not been systematically investigated for HCMV genomes in this population. In the present study, we used quantitative PCR to assess HCMV DNA in plasma samples from patients with brain tumors and from healthy controls.

Clinical samples

Plasma samples were collected at the time of surgery from 59 patients operated for different types of brain tumors at the Karolinska University Hospital (between 2005 and 2009), to be evaluated for PCR analysis. The diagnoses were: glioblastoma WHO grade IV (GBM) (n = 25), astrocytoma grades II-III (n = 18), secondary GBM (n = 4), ganglioglioma (n = 1) and meningioma (n = 1) and brain metastasis (n = 10, primary tumors were ovarian cancer (n = 1), breast cancer (n = 4), malignant melanoma (n = 1), non-small cell lung cancer (n = 4) (Table 1). Blood samples from 114 healthy donors (age range 20-69 years) were obtained during 2011from the blood bank at our hospital. The study was approved by Stockholm´s ethics committee (2005/542-31/1, June 1, 2005, and 2006/755-31-2, July 5, 2006) and by the ethics committee at the Karolinska Institutet (2008/628-31/2, May 22, 2008, 2008/518-31, June 18, 2008 and 2014/2154-32, Dec 16, 2014). Written informed consent was obtained from all subjects involved in this study.

GBM: Glioblastoma WHO grade IV, N: Numbers, F: Female, M: Male, OS: Overall Survival

Glioma patients (grades II-II and glioblastoma) and benign tumor patients, a total of 49 patients, did not receive any treatment before the operation and sample collection. Of the ten patients with brain metastasis, four had not received any treatment other than surgery of the primary tumor in two cases. The other six patients underwent radio-chemotherapy and only one of them also received antibody therapy. In total 53 of 59 patients had not received previous treatments that could potentially have influenced the relationship with HCMV.

qPCR assay

Plasma DNA was isolated using the Qiagen DNA Isolation Kit (Qiagen AB, Kista, Sweden). Concentrations were determined with a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). Each sample was analysed in triplicate using the TaqMan Fast Universal PCR Master Mix (Life Technologies) for detection of HCMV-IE DNA. Amplification was carried out on a 7900HT Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) with primers and probes specific for HCMV-IE. The sequences were as follows: forward primer 5′-GTGACCCATGTGCTTATGACTCTAT-3′, reverse primer 5′-CTCAACATAGTCTGCAGGAACGT-3′, and probe 5′-TTGGTCACGGGTGTCTC-3′ labelled with FAM (Applied Biosystems, custom-made). RNase P (assay ID 4316844; Life Technologies) served as a housekeeping gene for DNA quality and normalization. DNA extracted from mononuclear cells of an HCMV-infected kidney transplant patient, differentiated into macrophages, was included as positive control [50]. Thermal cycling consisted of an initial activation at 95°C for 20 s, followed by 50 cycles of denaturation at 95°C for 1 s and annealing/extension at 60°C for 20 s. Assay sensitivity was one genome copy per reaction, as determined with a vector containing the HCMV IE gene. A Cycle Threshold (CT) value < 37.0 was considered positive. All samples were analysed in triplicates, and assay performance was validated against the internal control vector. Positive control DNA consistently yielded CT values between 30.3 and 31.0 across nine replicates, showing expected pipetting variability. Data were processed with SDS software version 2.4 (Thermo Fisher Scientific, Inc.).

Results were expressed as mean ± Standard Deviation Mean (SDM) and analyzed with Unpaired student t-test. Analysis was performed using Graph Pad Prism software version 9. p < 0.05 was considered significant (****; p < 0.0001 ***; p < 0.001; ** p < 0.01, *). 

Higher HCMV-IE DNA levels are detected in plasma samples obtained from patients with GBM, astrocytoma grades II-III, and brain metastasis compared to healthy donors

59 plasma samples obtained from patients with different types of brain tumors were analyzed for HCMV-IE DNA with a qPCR assay. When 2/3 sample replicates were positive with a CT value < 37.0, the sample was considered positive. HCMV-IE DNA was detected in plasma samples from GBM patients (n = 18), patients with astrocytoma grades II-III (n = 11) and patients with brain metastasis (n = 8) at lower CT values compared to healthy blood donors (p < 0.0001, p < 0.0001 and p = 0.0007, respectively) (Figure 1A). HCMV-IE DNA was detected in plasma samples obtained from 18 of 25 (72%) patients with GBM, in 11 of 18 (61%) patients with Astrocytoma grades II-III, in 8 of 10 (80%) patients with brain metastasis and in 3 of 4 (75%) patients with secondary GBM (Figure 1B). DNA could be extracted from 96 out of 114 plasma samples from healthy blood donors. HCMV IE-DNA was detected in 40 of 96 (42%) plasma samples collected from healthy donors (Figure 1B). The interval of CT values was lower in the positive plasma samples from patients (CT interval; > CT:32.6 < CT:38.5) compared to samples collected from healthy donors (CT interval; > CT:33.7< 41.3).

Figure 1: HCMV IE-DNA using TaqMan PCR in plasma samples obtained from patients with brain tumors compared to healthy blood donors. (A) HCMV-IE DNA was detected in 18 GBM patients, 11 patients with astrocytoma grades II-III and 8 patients with brain metastasis. Delta CT values were lower in patients with brain tumor (p < 0.0001, p < 0.0001 and p = 0.0007, respectively), 2/3 positive CT values < 37 was defined as positive.
(B) HCMV-IE DNA was detected in plasma samples obtained from 18 of 25 (72%) patients with GBM, in 11 of 18 (61%) patients with Astrocytoma grades II-III, in 8 of 10 (80%) patients with brain metastasis and in 3 of 4 (75%) patients with secondary GBM. HCMV IE-DNA was detected in 40 of 96 (42%) plasma samples collected from healthy donors.
GBM- Glioblastoma; Astro- Astrocytoma grades II-III; Met- Metastatic Brain Tumor; Sec-Secondary GBM; HD-Healthy Blood Donors; HCMV- Human Cytomegalovirus; IE- Immediate Early.

5. The CT interval values for positive controls was 30.3-31.0. CT values for one patient with a ganglioglioma and one patient with a meningioma were 36.3 and 37.2, respectively. Delta CT values (∆CT) were calculated by subtracting mean CT of housekeeping gene (RNase P) from mean CT of target gene. Taken together, these observations imply a higher HCMV-DNA presence in plasma of brain tumor patients than in healthy blood donors. The number of patients in secondary glioblastoma (n = 4) and benign tumors (n = 2) were too small for any conclusions to be madeDiscussion

In this study, 59 plasma samples from patients with glioblastoma (n = 25), astrocytoma grades II–III (n = 18), brain metastases (n = 10), secondary GBM (n = 4), and benign brain tumours (n = 2) were analysed by PCR. HCMV-IE DNA was detected in 72% of glioblastoma cases, 61% of astrocytomas, 80% of brain metastases, 75% of secondary GBMs, and both patients with benign tumours. In addition, HCMV DNA was identified in 42% of healthy donors (n = 96). The detection of viral DNA in healthy controls may reflect either reactivation of latent infection or, in some cases, a primary infection. In our earlier study of this donor cohort, 13 individuals were seronegative for HCMV-IgG but still had low levels of HCMV-DNA detectable in blood, consistent with possible primary infection [51]. Across groups, CT values were generally lower in brain tumor patients than in healthy donors. Specifically, ΔCT values were significantly lower in plasma from patients with glioblastoma, astrocytoma grades II–III, and brain metastases (p < 0.0001, p < 0.0001, and p = 0.0007, respectively), indicating higher circulating viral DNA levels. For secondary GBM, ΔCT values also trended lower, though statistical significance was not reached, likely due to the limited sample size (n = 3 of 4). Taken together, these findings support the interpretation that circulating HCMV-DNA is more abundant in patients with brain tumours than in healthy individuals, consistent with higher viral activity in the tumour setting.

Quantification of HCMV DNA in plasma or serum remains widely used in clinical practice for monitoring antiviral therapy and for identifying patients at risk of developing HCMV-related disease. A limitation, however, is that HCMV DNA detected in plasma is often fragmented and may not fully reflect the amount of infectious virus present. Whole blood PCR, on the other hand, may detect latent viral DNA and thereby overestimate active infection risk. Taken together, these data indicate that both plasma and whole blood are valuable sample types for HCMV detection, though each has inherent limitations. Plasma PCR may underestimate the total viral burden, whereas whole blood PCR may exaggerate infection risk. Nonetheless, plasma-based PCR is the most widely adopted method in clinical settings, offering a practical and reliable tool for guiding therapy and monitoring patients vulnerable to HCMV disease.

Many studies have described a high prevalence of HCMV-DNA in tumour tissue from glioblastoma patients [9,14,15, 32,39,46-48], but relatively few studies have examined the presence of HCMV in the blood of brain tumor patients. In our earlier work, we detected HCMV-DNA in 100% of blood cell samples from 42 GBM patients [14]. Mitchell DA, et al. [12], reported viral DNA in whole blood from 80% of 20 glioblastoma patients [32], while Dos Santos CJ, et al. [9], examined peripheral blood from 20 cases and found HCMV-DNA in 55% of samples using nested PCR and in 45% using qPCR In the present study, we analysed plasma samples with a qPCR assay capable of detecting a single copy of HCMV-IE DNA. To our knowledge, this is the first report of the detection of HCMV-DNA in plasma samples obtained from patients with brain tumors. Plasma PCR assays have previously been applied to measure viral load in organ and stem cell transplant recipients [54,58-60], congenital infection [61] and viremic patients [52,57]. Detection of HCMV-DNA in plasma samples has also been used to monitor the viral load in blood of patients with HCMV disease and often exhibit lower sensitivity than when using whole blood as sample material, a difference influenced by assay design and sample quality.

In the present study, we employed a highly sensitive qPCR assay with a detection limit of one viral genome copy per sample. All samples were tested in triplicate, and results were considered positive when at least two of three replicates were positive. Plasma samples were stored in aliquots and not freeze thawed, and internal controls showed a good quality of the DNA. Despite these precautions, whole blood would likely provide higher sensitivity and identify more positive cases. Unfortunately, paired whole blood samples were not available for this study. Future investigations should therefore prioritize direct comparisons between plasma and whole blood in brain tumour patients.

Using the highly sensitive qPCR assay applied in this study, we observed lower CT values for HCMV-IE DNA in plasma from patients with GBM, astrocytoma grades II–III, and brain metastases compared with healthy blood donors, consistent with higher circulating viral DNA levels in brain tumour patients. Notably, HCMV DNA was also detected in 42% of healthy controls, which is a relatively high proportion. This finding likely reflects the natural biology of HCMV, a virus that persists lifelong in a latent state with intermittent episodes of subclinical reactivation. Detection of viral DNA in healthy individuals may therefore represent latent virus, transient reactivation without clinical consequence, or fragmented viral DNA released into the circulation. Another explanation is the very high analytical sensitivity of the qPCR assay, which can identify extremely low DNA levels that do not necessarily indicate the presence of infectious virus. These observations highlight that detection of HCMV DNA, particularly in healthy individuals, should not automatically be interpreted as evidence of active infection. Nevertheless, such findings provide useful information on viral dynamics and suggest that HCMV activity may be more pronounced in patients with brain tumours than in the general population.

Most studies of HCMV-DNA in plasma have used qPCR assays to detect HCMV viremia and to monitor treatment responses. In the present study, the HCMV-DNA levels were close to the detection threshold and and multifold lower than in viremic patients. Even so, HCMV-DNA levels were significantly higher in patients with GBM, astrocytoma grades II–III, and brain metastases compared with healthy donors, supporting the view that HCMV activity is increased in brain tumour patients. In a previous analysis of the same brain tumour cohort, we demonstrated significantly higher HCMV-specific IgG antibody levels in patients than in age-matched healthy blood donors (GBM: p = 0.008; astrocytoma grades II–III: p = 0.003; metastatic brain tumours: p = 0.004) [51]. Moreover, elevated HCMV-specific IgG titers in patients with metastatic brain tumours were associated with longer survival, suggesting that a stronger immune response against HCMV may contribute to improved outcomes [51]. In our most recent analysis, however, HCMV IgG–negative patients showed better prognosis compared to seropositive individuals, again linking HCMV to adverse disease outcome (unpublished data).

These findings raise the possibility that interventions targeting HCMV could improve prognosis in brain tumour patients. Supporting this idea, we previously reported that GBM patients receiving valganciclovir in addition to standard therapy achieved significantly prolonged median overall survival (24.1 months in 102 treated patients vs., 13.3 months in 231 contemporary controls; p < 0.0001) [33]. This treatment approach is currently being evaluated in a randomized controlled trial (NCT04116411). Other groups have also shown that enhancing the immune response against HCMV in a small number of glioblastoma patients is associated with improved survival [34,62]. Taken together, these data suggest that HCMV may contribute to tumour progression in glioblastoma and possibly other brain tumours, and that HCMV-directed therapies could represent a promising strategy to improve outcomes. Such approaches are particularly important given that other medical treatments have so far failed to extend survival in these patients [36].

We found that HCMV-DNA is present at higher levels in plasma of patients with glioblastoma, patients with astrocytoma grades II-III and in patients with brain metastasis compared with healthy blood donors. These results suggest increased HCMV activity in brain tumour patients. Quantification of HCMV-DNA in plasma with the applied qPCR assay proved to be a reliable method for detecting HCMV, even at very low levels, and may serve as a useful tool for monitoring viral activity in this patient group.

1. Cobbs CS, Harkins L, Samanta M, Gillespie GY, Bharara S, King PH, Nabors LB, Cobbs CG, Britt WJ. Human cytomegalovirus infection and expression in human malignant glioma. Cancer Res. 2002 Jun 15;62(12):3347-50. PMID: 12067971.
2. Dziurzynski K, Chang SM, Heimberger AB, Kalejta RF, McGregor Dallas SR, Smit M, Soroceanu L, Cobbs CS; HCMV and Gliomas Symposium. Consensus on the role of human cytomegalovirus in glioblastoma. Neuro Oncol. 2012 Mar;14(3):246-55. doi: 10.1093/neuonc/nor227. Epub 2012 Feb 8. PMID: 22319219; PMCID: PMC3280809.
3. Söderberg-Nauclér C, Johnsen JI. Cytomegalovirus infection in brain tumors: A potential new target for therapy? Oncoimmunology. 2012 Aug 1;1(5):739-740. doi: 10.4161/onci.19441. PMID: 22934266; PMCID: PMC3429578.
4. Baryawno N, Rahbar A, Wolmer-Solberg N, Taher C, Odeberg J, Darabi A, Khan Z, Sveinbjörnsson B, FuskevÅg OM, Segerström L, Nordenskjöld M, Siesjö P, Kogner P, Johnsen JI, Söderberg-Nauclér C. Detection of human cytomegalovirus in medulloblastomas reveals a potential therapeutic target. J Clin Invest. 2011 Oct;121(10):4043-55. doi: 10.1172/JCI57147. Epub 2011 Sep 26. PMID: 21946257; PMCID: PMC3195466.
5. Taher C, Frisk G, Fuentes S, Religa P, Costa H, Assinger A, Vetvik KK, Bukholm IR, Yaiw KC, Smedby KE, Bäcklund M, Söderberg-Naucler C, Rahbar A. High prevalence of human cytomegalovirus in brain metastases of patients with primary breast and colorectal cancers. Transl Oncol. 2014 Dec;7(6):732-40. doi: 10.1016/j.tranon.2014.09.008. PMID: 25500083; PMCID: PMC4311044.
6. Goerig NL, Frey B, Korn K, Fleckenstein B, Überla K, Schmidt MA, Dörfler A, Engelhorn T, Eyüpoglu I, Rühle PF, Putz F, Semrau S, Gaipl US, Fietkau R. Early Mortality of Brain Cancer Patients and its Connection to Cytomegalovirus Reactivation During Radiochemotherapy. Clin Cancer Res. 2020 Jul 1;26(13):3259-3270. doi: 10.1158/1078-0432.CCR-19-3195. Epub 2020 Feb 14. PMID: 32060103.
7. Bhattacharjee B, Renzette N, Kowalik TF. Genetic analysis of cytomegalovirus in malignant gliomas. J Virol. 2012 Jun;86(12):6815-24. doi: 10.1128/JVI.00015-12. Epub 2012 Apr 11. PMID: 22496213; PMCID: PMC3393585.
8. Bianchi E, Roncarati P, Hougrand O, Guérin-El Khourouj V, Boreux R, Kroonen J, Martin D, Robe P, Rogister B, Delvenne P, Deprez M. Human cytomegalovirus and primary intracranial tumours: frequency of tumour infection and lack of correlation with systemic immune anti-viral responses. Neuropathol Appl Neurobiol. 2015 Feb;41(2):e29-40. doi: 10.1111/nan.12172. PMID: 25041908.
9. dos Santos CJ, Stangherlin LM, Figueiredo EG, Corrêa C, Teixeira MJ, da Silva MC. High prevalence of HCMV and viral load in tumor tissues and peripheral blood of glioblastoma multiforme patients. J Med Virol. 2014 Nov;86(11):1953-61. doi: 10.1002/jmv.23820. Epub 2013 Oct 30. PMID: 24173908.
10. Libard S, Popova SN, Amini RM, Kärjä V, Pietiläinen T, Hämäläinen KM, Sundström C, Hesselager G, Bergqvist M, Ekman S, Zetterling M, Smits A, Nilsson P, Pfeifer S, de Ståhl TD, Enblad G, Ponten F, Alafuzoff I. Human cytomegalovirus tegument protein pp65 is detected in all intra- and extra-axial brain tumours independent of the tumour type or grade. PLoS One. 2014 Sep 30;9(9):e108861. doi: 10.1371/journal.pone.0108861. PMID: 25268364; PMCID: PMC4182568.
11. Lucas KG, Bao L, Bruggeman R, Dunham K, Specht C. The detection of CMV pp65 and IE1 in glioblastoma multiforme. J Neurooncol. 2011 Jun;103(2):231-8. doi: 10.1007/s11060-010-0383-6. Epub 2010 Sep 5. PMID: 20820869.
12. Mitchell DA, Xie W, Schmittling R, Learn C, Friedman A, McLendon RE, Sampson JH. Sensitive detection of human cytomegalovirus in tumors and peripheral blood of patients diagnosed with glioblastoma. Neuro Oncol. 2008 Feb;10(1):10-8. doi: 10.1215/15228517-2007-035. Epub 2007 Oct 19. PMID: 17951512; PMCID: PMC2600830.
13. Rahbar A, Orrego A, Peredo I, Dzabic M, Wolmer-Solberg N, Strååt K, Stragliotto G, Söderberg-Nauclér C. Human cytomegalovirus infection levels in glioblastoma multiforme are of prognostic value for survival. J Clin Virol. 2013 May;57(1):36-42. doi: 10.1016/j.jcv.2012.12.018. Epub 2013 Feb 4. PMID: 23391370.
14. Rahbar A, Peredo I, Solberg NW, Taher C, Dzabic M, Xu X, Skarman P, Fornara O, Tammik C, Yaiw K, Wilhelmi V, Assinger A, Stragliotto G, Söderberg-Naucler C. Discordant humoral and cellular immune responses to Cytomegalovirus (CMV) in glioblastoma patients whose tumors are positive for CMV. Oncoimmunology. 2015 Feb 25;4(2):e982391. doi: 10.4161/2162402X.2014.982391. PMID: 25949880; PMCID: PMC4404865.
15. Ranganathan P, Clark PA, Kuo JS, Salamat MS, Kalejta RF. Significant association of multiple human cytomegalovirus genomic Loci with glioblastoma multiforme samples. J Virol. 2012 Jan;86(2):854-64. doi: 10.1128/JVI.06097-11. Epub 2011 Nov 16. PMID: 22090104; PMCID: PMC3255835.
16. Sjöström S, Hjalmars U, Juto P, Wadell G, Hallmans G, Tjönneland A, Halkjaer J, Manjer J, Almquist M, Melin BS. Human immunoglobulin G levels of viruses and associated glioma risk. Cancer Causes Control. 2011 Sep;22(9):1259-66. doi: 10.1007/s10552-011-9799-3. Epub 2011 Jun 30. PMID: 21717196; PMCID: PMC3146711.
17. Stangherlin LM, Castro FL, Medeiros RS, Guerra JM, Kimura LM, Shirata NK, Nonogaki S, Dos Santos CJ, Carlan Silva MC. Human Cytomegalovirus DNA Quantification and Gene Expression in Gliomas of Different Grades. PLoS One. 2016 Jul 26;11(7):e0159604. doi: 10.1371/journal.pone.0159604. PMID: 27458810; PMCID: PMC4961403.
18. Baumgarten P, Michaelis M, Rothweiler F, Starzetz T, Rabenau HF, Berger A, Jennewein L, Braczynski AK, Franz K, Seifert V, Steinbach JP, Allwinn R, Mittelbronn M, Cinatl J Jr. Human cytomegalovirus infection in tumor cells of the nervous system is not detectable with standardized pathologico-virological diagnostics. Neuro Oncol. 2014 Nov;16(11):1469-77. doi: 10.1093/neuonc/nou167. Epub 2014 Aug 25. PMID: 25155358; PMCID: PMC4201076.
19. Cosset É, Petty TJ, Dutoit V, Cordey S, Padioleau I, Otten-Hernandez P, Farinelli L, Kaiser L, Bruyère-Cerdan P, Tirefort D, Amar El-Dusouqui S, Nayernia Z, Krause KH, Zdobnov EM, Dietrich PY, Rigal E, Preynat-Seauve O. Comprehensive metagenomic analysis of glioblastoma reveals absence of known virus despite antiviral-like type I interferon gene response. Int J Cancer. 2014 Sep 15;135(6):1381-9. doi: 10.1002/ijc.28670. Epub 2014 May 8. PMID: 24347514; PMCID: PMC4235296.
20. Garcia-Martinez A, Alenda C, Irles E, Ochoa E, Quintanar T, Rodriguez-Lescure A, Soto JL, Barbera VM. Lack of cytomegalovirus detection in human glioma. Virol J. 2017 Nov 7;14(1):216. doi: 10.1186/s12985-017-0885-3. PMID: 29116009; PMCID: PMC5678593.
21. Holdhoff M, Guner G, Rodriguez FJ, Hicks JL, Zheng Q, Forman MS, Ye X, Grossman SA, Meeker AK, Heaphy CM, Eberhart CG, De Marzo AM, Arav-Boger R. Absence of Cytomegalovirus in Glioblastoma and Other High-grade Gliomas by Real-time PCR, Immunohistochemistry, and In Situ Hybridization. Clin Cancer Res. 2017 Jun 15;23(12):3150-3157. doi: 10.1158/1078-0432.CCR-16-1490. Epub 2016 Dec 29. Erratum in: Clin Cancer Res. 2022 Apr 14;28(8):1737. doi: 10.1158/1078-0432.CCR-22-0565. PMID: 28034905; PMCID: PMC5474132.
22. Lehrer S, Green S, Rosenzweig KE, Rendo A. No circulating human cytomegalovirus in 14 cases of glioblastoma. Neuro Oncol. 2015 Feb;17(2):320. doi: 10.1093/neuonc/nou325. Epub 2014 Dec 1. PMID: 25452391; PMCID: PMC4288528.
23. Poltermann S, Schlehofer B, Steindorf K, Schnitzler P, Geletneky K, Schlehofer JR. Lack of association of herpesviruses with brain tumors. J Neurovirol. 2006 Apr;12(2):90-9. doi: 10.1080/13550280600654573. PMID: 16798670.
24. Priel E, Wohl A, Teperberg M, Nass D, Cohen ZR. Human cytomegalovirus viral load in tumor and peripheral blood samples of patients with malignant gliomas. J Clin Neurosci. 2015 Feb;22(2):326-30. doi: 10.1016/j.jocn.2014.06.099. Epub 2014 Oct 28. PMID: 25443081.
25. Yamashita Y, Ito Y, Isomura H, Takemura N, Okamoto A, Motomura K, Tsujiuchi T, Natsume A, Wakabayashi T, Toyokuni S, Tsurumi T. Lack of presence of the human cytomegalovirus in human glioblastoma. Mod Pathol. 2014 Jul;27(7):922-9. doi: 10.1038/modpathol.2013.219. Epub 2013 Dec 13. PMID: 24336154.
26. Peredo-Harvey I, Rahbar A, Söderberg-Nauclér C. Presence of the Human Cytomegalovirus in Glioblastomas-A Systematic Review. Cancers (Basel). 2021 Oct 9;13(20):5051. doi: 10.3390/cancers13205051. PMID: 34680198; PMCID: PMC8533734.
27. Harkins L, Volk AL, Samanta M, Mikolaenko I, Britt WJ, Bland KI, Cobbs CS. Specific localisation of human cytomegalovirus nucleic acids and proteins in human colorectal cancer. Lancet. 2002 Nov 16;360(9345):1557-63. doi: 10.1016/S0140-6736(02)11524-8. PMID: 12443594.
28. Rådestad AF, Estekizadeh A, Cui HL, Kostopoulou ON, Davoudi B, Hirschberg AL, Carlson J, Rahbar A, Söderberg-Naucler C. Impact of Human Cytomegalovirus Infection and its Immune Response on Survival of Patients with Ovarian Cancer. Transl Oncol. 2018 Dec;11(6):1292-1300. doi: 10.1016/j.tranon.2018.08.003. Epub 2018 Aug 30. PMID: 30172882; PMCID: PMC6121833.
29. Samanta M, Harkins L, Klemm K, Britt WJ, Cobbs CS. High prevalence of human cytomegalovirus in prostatic intraepithelial neoplasia and prostatic carcinoma. J Urol. 2003 Sep;170(3):998-1002. doi: 10.1097/01.ju.0000080263.46164.97. PMID: 12913758.
30. Touma J, Liu Y, Rahbar A, Pantalone MR, Almazan NM, Vetvik K, Söderberg-Nauclér C, Geisler J, Sauer T. Detection of Human Cytomegalovirus Proteins in Paraffin-Embedded Breast Cancer Tissue Specimens-A Novel, Automated Immunohistochemical Staining Protocol. Microorganisms. 2021 May 13;9(5):1059. doi: 10.3390/microorganisms9051059. PMID: 34068349; PMCID: PMC8153275.
31. Wolmer-Solberg N, Baryawno N, Rahbar A, Fuchs D, Odeberg J, Taher C, Wilhelmi V, Milosevic J, Mohammad AA, Martinsson T, Sveinbjörnsson B, Johnsen JI, Kogner P, Söderberg-Nauclér C. Frequent detection of human cytomegalovirus in neuroblastoma: a novel therapeutic target? Int J Cancer. 2013 Nov 15;133(10):2351-61. doi: 10.1002/ijc.28265. Epub 2013 Jul 13. PMID: 23661597.
32. Mitchell D, Archer G, Bigner D, Desjardins A, Friedman A, Friedman H, Lally-Goss D, Herndon J,  Sharon McGehee S,  McLendon R, Perry B, Reardon D, Rich J, Silverman S, Vrendenburgh J,  Sampson J. Efficacy and immunologic effects of RNA-pulsed dendritic cell vaccines targeting human cytomegalovirus antigens in patients with glioblastoma. Cancer Research. 2008.
33. Stragliotto G, Pantalone MR, Rahbar A, Bartek J, Söderberg-Naucler C. Valganciclovir as Add-on to Standard Therapy in Glioblastoma Patients. Clin Cancer Res. 2020 Aug 1;26(15):4031-4039. doi: 10.1158/1078-0432.CCR-20-0369. Epub 2020 May 18. PMID: 32423968.
34. Batich KA, Reap EA, Archer GE, Sanchez-Perez L, Nair SK, Schmittling RJ, Norberg P, Xie W, Herndon JE 2nd, Healy P, McLendon RE, Friedman AH, Friedman HS, Bigner D, Vlahovic G, Mitchell DA, Sampson JH. Long-term Survival in Glioblastoma with Cytomegalovirus pp65-Targeted Vaccination. Clin Cancer Res. 2017 Apr 15;23(8):1898-1909. doi: 10.1158/1078-0432.CCR-16-2057. PMID: 28411277; PMCID: PMC5559300.
35. Liau LM, Ashkan K, Brem S, Campian JL, Trusheim JE, Iwamoto FM, Tran DD, Ansstas G, Cobbs CS, Heth JA, Salacz ME, D'Andre S, Aiken RD, Moshel YA, Nam JY, Pillainayagam CP, Wagner SA, Walter KA, Chaudhary R, Goldlust SA, Lee IY, Bota DA, Elinzano H, Grewal J, Lillehei K, Mikkelsen T, Walbert T, Abram S, Brenner AJ, Ewend MG, Khagi S, Lovick DS, Portnow J, Kim L, Loudon WG, Martinez NL, Thompson RC, Avigan DE, Fink KL, Geoffroy FJ, Giglio P, Gligich O, Krex D, Lindhorst SM, Lutzky J, Meisel HJ, Nadji-Ohl M, Sanchin L, Sloan A, Taylor LP, Wu JK, Dunbar EM, Etame AB, Kesari S, Mathieu D, Piccioni DE, Baskin DS, Lacroix M, May SA, New PZ, Pluard TJ, Toms SA, Tse V, Peak S, Villano JL, Battiste JD, Mulholland PJ, Pearlman ML, Petrecca K, Schulder M, Prins RM, Boynton AL, Bosch ML. Association of Autologous Tumor Lysate-Loaded Dendritic Cell Vaccination With Extension of Survival Among Patients With Newly Diagnosed and Recurrent Glioblastoma: A Phase 3 Prospective Externally Controlled Cohort Trial. JAMA Oncol. 2023 Jan 1;9(1):112-121. doi: 10.1001/jamaoncol.2022.5370. PMID: 36394838; PMCID: PMC9673026.
36. Vanderbeek AM, Rahman R, Fell G, Ventz S, Chen T, Redd R, Parmigiani G, Cloughesy TF, Wen PY, Trippa L, Alexander BM. The clinical trials landscape for glioblastoma: is it adequate to develop new treatments? Neuro Oncol. 2018 Jul 5;20(8):1034-1043. doi: 10.1093/neuonc/noy027. PMID: 29518210; PMCID: PMC6280141.
37. Reeves MB, MacAry PA, Lehner PJ, Sissons JG, Sinclair JH. Latency, chromatin remodeling, and reactivation of human cytomegalovirus in the dendritic cells of healthy carriers. Proc Natl Acad Sci U S A. 2005 Mar 15;102(11):4140-5. doi: 10.1073/pnas.0408994102. Epub 2005 Feb 28. PMID: 15738399; PMCID: PMC554799.
38. Söderberg-Nauclér C, Fish KN, Nelson JA. Reactivation of latent human cytomegalovirus by allogeneic stimulation of blood cells from healthy donors. Cell. 1997 Oct 3;91(1):119-26. doi: 10.1016/s0092-8674(01)80014-3. PMID: 9335340.
39. Matlaf LA, Harkins LE, Bezrookove V, Cobbs CS, Soroceanu L. Cytomegalovirus pp71 protein is expressed in human glioblastoma and promotes pro-angiogenic signaling by activation of stem cell factor. PLoS One. 2013 Jul 5;8(7):e68176. doi: 10.1371/journal.pone.0068176. PMID: 23861869; PMCID: PMC3702580.
40. Michaelis M, Doerr HW, Cinatl J Jr. Oncomodulation by human cytomegalovirus: evidence becomes stronger. Med Microbiol Immunol. 2009 May;198(2):79-81. doi: 10.1007/s00430-009-0107-8. Epub 2009 Feb 7. PMID: 19198878.
41. Söderberg-Nauclér C. Does cytomegalovirus play a causative role in the development of various inflammatory diseases and cancer? J Intern Med. 2006 Mar;259(3):219-46. doi: 10.1111/j.1365-2796.2006.01618.x. PMID: 16476101.
42. Dziurzynski K, Chang SM, Heimberger AB, Kalejta RF, McGregor Dallas SR, Smit M, Soroceanu L, Cobbs CS; HCMV and Gliomas Symposium. Consensus on the role of human cytomegalovirus in glioblastoma. Neuro Oncol. 2012 Mar;14(3):246-55. doi: 10.1093/neuonc/nor227. Epub 2012 Feb 8. PMID: 22319219; PMCID: PMC3280809.
43. Farias KPRA, Moreli ML, Floriano VG, da Costa VG. Evidence based on a meta-analysis of human cytomegalovirus infection in glioma. Arch Virol. 2019 May;164(5):1249-1257. doi: 10.1007/s00705-019-04206-z. Epub 2019 Mar 19. PMID: 30888562.
44. Garcia-Martinez A. Lack of human cytomegalovirus in gliomas. A viral load analysis by quantitative real time-PCR. Laboratory Investigation. 2013.
45. Hashida Y, Taniguchi A, Yawata T, Hosokawa S, Murakami M, Hiroi M, Ueba T, Daibata M. Prevalence of human cytomegalovirus, polyomaviruses, and oncogenic viruses in glioblastoma among Japanese subjects. Infect Agent Cancer. 2015 Jan 27;10:3. doi: 10.1186/1750-9378-10-3. PMID: 25685179; PMCID: PMC4328287.
46. Bahador M, Gras Navarro A, Rahman MA, Dominguez-Valentin M, Sarowar S, Ulvestad E, Njølstad G, Lie SA, Kristoffersen EK, Bratland E, Chekenya M. Increased infiltration and tolerised antigen-specific CD8+ TEM cells in tumor but not peripheral blood have no impact on survival of HCMV+ glioblastoma patients. Oncoimmunology. 2017 Jun 5;6(8):e1336272. doi: 10.1080/2162402X.2017.1336272. PMID: 28919997; PMCID: PMC5593710.
47. Chen TQ, Li ZX, Jiang Y, Xu XD, Gan L, Wang BL. Detection of human cytomegalovirus in tumor tissue and peripheral blood samples of patients with gliomas. Int J Clin Exp Patho. 2017;10(2):1499-1508.
48. Dziurzynski K, Wei J, Qiao W, Hatiboglu MA, Kong LY, Wu A, Wang Y, Cahill D, Levine N, Prabhu S, Rao G, Sawaya R, Heimberger AB. Glioma-associated cytomegalovirus mediates subversion of the monocyte lineage to a tumor propagating phenotype. Clin Cancer Res. 2011 Jul 15;17(14):4642-9. doi: 10.1158/1078-0432.CCR-11-0414. Epub 2011 Apr 13. PMID: 21490182; PMCID: PMC3139801.
49. Ghazi A, Ashoori A, Hanley PJ, Brawley VS, Shaffer DR, Kew Y, Powell SZ, Grossman R, Grada Z, Scheurer ME, Hegde M, Leen AM, Bollard CM, Rooney CM, Heslop HE, Gottschalk S, Ahmed N. Generation of polyclonal CMV-specific T cells for the adoptive immunotherapy of glioblastoma. J Immunother. 2012 Feb-Mar;35(2):159-68. doi: 10.1097/CJI.0b013e318247642f. PMID: 22306904; PMCID: PMC3280423.
50. Ahlfors K, Forsgren M, Ivarsson SA, Harris S, Svanberg L. Congenital cytomegalovirus infection: on the relation between type and time of maternal infection and infant's symptoms. Scand J Infect Dis. 1983;15(2):129-38. doi: 10.3109/inf.1983.15.issue-2.01. PMID: 6308752.
51. Peredo-Harvey I, Bartek J Jr, Ericsson C, Yaiw KC, Nistér M, Rahbar A, Söderberg-Naucler C. Higher Human Cytomegalovirus (HCMV) Specific IgG Antibody Levels in Plasma Samples from Patients with Metastatic Brain Tumors Are Associated with Longer Survival. Medicina (Kaunas). 2023 Jul 5;59(7):1248. doi: 10.3390/medicina59071248. PMID: 37512060; PMCID: PMC10384986.
52. Lisboa LF, Asberg A, Kumar D, Pang X, Hartmann A, Preiksaitis JK, Pescovitz MD, Rollag H, Jardine AG, Humar A. The clinical utility of whole blood versus plasma cytomegalovirus viral load assays for monitoring therapeutic response. Transplantation. 2011 Jan 27;91(2):231-6. doi: 10.1097/TP.0b013e3181ff8719. PMID: 21048530.
53. Ksouri H, Eljed H, Greco A, Lakhal A, Torjman L, Abdelkefi A, Ben Othmen T, Ladeb S, Slim A, Zouari B, Abdeladhim A, Ben Hassen A. Analysis of cytomegalovirus (CMV) viremia using the pp65 antigenemia assay, the amplicor CMV test, and a semi-quantitative polymerase chain reaction test after allogeneic marrow transplantation. Transpl Infect Dis. 2007 Mar;9(1):16-21. doi: 10.1111/j.1399-3062.2006.00171.x. PMID: 17313466.
54. Hebart H, Müller C, Löffler J, Jahn G, Einsele H. Monitoring of CMV infection: a comparison of PCR from whole blood, plasma-PCR, pp65-antigenemia and virus culture in patients after bone marrow transplantation. Bone Marrow Transplant. 1996 May;17(5):861-8. PMID: 8733710.
55. Tang W, Elmore SH, Fan H, Thorne LB, Gulley ML. Cytomegalovirus DNA measurement in blood and plasma using Roche LightCycler CMV quantification reagents. Diagn Mol Pathol. 2008 Sep;17(3):166-73. doi: 10.1097/PDM.0b013e3181599242. PMID: 18382366.
56. Rzepka M, Depka D, Gospodarek-Komkowska E, Bogiel T. Whole Blood versus Plasma Samples-How Does the Type of Specimen Collected for Testing Affect the Monitoring of Cytomegalovirus Viremia? Pathogens. 2022 Nov 19;11(11):1384. doi: 10.3390/pathogens11111384. PMID: 36422636; PMCID: PMC9697577.
57. Kullberg-Lindh C, Olofsson S, Brune M, Lindh M. Comparison of serum and whole blood levels of cytomegalovirus and Epstein-Barr virus DNA. Transpl Infect Dis. 2008 Oct;10(5):308-15. doi: 10.1111/j.1399-3062.2008.00313.x. Epub 2008 May 7. PMID: 18466193.
58. Dioverti MV, Lahr BD, Germer JJ, Yao JD, Gartner ML, Razonable RR. Comparison of Standardized Cytomegalovirus (CMV) Viral Load Thresholds in Whole Blood and Plasma of Solid Organ and Hematopoietic Stem Cell Transplant Recipients with CMV Infection and Disease. Open Forum Infect Dis. 2017 Jul 8;4(3):ofx143. doi: 10.1093/ofid/ofx143. PMID: 28852681; PMCID: PMC5570102.
59. Nitsche A, Steuer N, Schmidt CA, Landt O, Ellerbrok H, Pauli G, Siegert W. Detection of human cytomegalovirus DNA by real-time quantitative PCR. J Clin Microbiol. 2000 Jul;38(7):2734-7. doi: 10.1128/JCM.38.7.2734-2737.2000. PMID: 10878073; PMCID: PMC87012.
60. Razonable RR, Brown RA, Wilson J, Groettum C, Kremers W, Espy M, Smith TF, Paya CV. The clinical use of various blood compartments for cytomegalovirus (CMV) DNA quantitation in transplant recipients with CMV disease. Transplantation. 2002 Mar 27;73(6):968-73. doi: 10.1097/00007890-200203270-00025. PMID: 11923702.
61. Torii Y, Morioka I, Kakei Y, Fujioka K, Kakimoto Y, Takahashi N, Yoshikawa T, Moriuchi H, Oka A, Ito Y. Correlation of cytomegalovirus viral load between whole blood and plasma of congenital cytomegalovirus infection under valganciclovir treatment. BMC Infect Dis. 2023 Jan 19;23(1):31. doi: 10.1186/s12879-023-07995-6. PMID: 36658533; PMCID: PMC9850601.
62. Schuessler A, Smith C, Beagley L, Boyle GM, Rehan S, Matthews K, Jones L, Crough T, Dasari V, Klein K, Smalley A, Alexander H, Walker DG, Khanna R. Autologous T-cell therapy for cytomegalovirus as a consolidative treatment for recurrent glioblastoma. Cancer Res. 2014 Jul 1;74(13):3466-76. doi: 10.1158/0008-5472.CAN-14-0296. Epub 2014 May 4. PMID: 24795429.