NY-ESO-1- and survivin-specific T-cell responses in the peripheral blood from patients with glioma.

The prognosis for patients with glioblastoma is grim. Ex vivo expanded tumor-associated antigen (TAA)-reactive T-cells from patients with glioma may represent a viable source for anticancer-directed cellular therapies. Immunohistochemistry was used to test the survivin (n = 40 samples) and NY-ESO-1 (n = 38 samples) protein expression in tumor specimens. T-cells from peripheral blood were stimulated with TAAs (synthetic peptides) in IL-2 and IL-7, or using a combination of IL-2, IL-15 and IL-21. CD4+ and CD8+ T-cells were tested for antigen-specific proliferation by flow cytometry, and IFN-γ production was tested by ELISA. Twenty-eight out of 38 cancer specimens exhibited NY-ESO-1 protein expression, 2/38 showed a strong universal (4+) NY-ESO-1 staining, and 9/40 cancer lesions exhibited a strong (4+) staining for survivin. We could detect antigen-specific IFN-γ responses in 25% blood samples for NY-ESO-1 and 30% for survivin. NY-ESO-1-expanded T-cells recognized naturally processed and presented epitopes. NY-ESO-1 or survivin expression in glioma represents viable targets for anticancer-directed T-cells for the biological therapy of patients with glioma.

Effects of valganciclovir as an add-on therapy in patients with cytomegalovirus-positive glioblastoma: a randomized, double-blind, hypothesis-generating study.

Cytomegalovirus is highly prevalent in glioblastomas. In 2006, we initiated a randomized, double-blind, placebo-controlled, hypothesis-generating study to examine the safety and potential efficacy of Valganciclovir as an add-on therapy for glioblastoma. Forty-two glioblastoma patients were randomized in double-blind fashion to receive Valganciclovir or placebo in addition to standard therapy for 6 months. Magnetic resonance images were obtained before and immediately and 3 and 6 months after surgery to evaluate treatment efficacy by measuring contrast enhancing tumor volume (primary end point). Survival data were analyzed for patients and controls in explorative analyses to aid the design of future randomized trials. Trends but no significant differences were observed in tumor volumes in Valganciclovir and placebo patients at 3 (3.58 vs. 7.44 cm3, respectively, p = 0.2881) and 6 (3.31 vs. 13.75 cm3, p = 0.2120) months. Median overall survival (OS) was similar in both groups (17.9 vs. 17.4 months, p = 0.430). Patients could take Valganciclovir for compassionate use after the study phase. Explorative analyses showed an OS of 24.1 months (95% CI, 17.4-40.3) in patients receiving >6 months of Valganciclovir (Val > 6M) versus 13.1 months (95% CI, 7.9-17.7, p < 0.0001) in patients receiving Valganciclovir for 0 or <6 months, and 13.7 months (95% CI, 6.9-17.3, p = 0.0031) in contemporary controls. OS at 4 years was 27.3% in Val>6M patients versus 5.9% in controls (p = 0.0466). Prolonged OS in Val>6M patients suggest that future randomized trials are warranted and should evaluate whether continuous antiviral treatment can improve outcome in glioblastoma patients.

Two-year survival of low-grade and high-grade glioma patients using data from the Swedish Cancer Registry.

BACKGROUND: Swedish health care legislation requires equal, high-quality health care for all inhabitants, while regional differences of medical availability and treatment potentially allow for different outcomes. This study was undertaken to evaluate whether glioma survival differed between the Stockholm region and the other Swedish regions since the Stockholm region has easier mean access to regional care and had started a specialized neuro-oncology service for all inhabitants of the region. MATERIAL AND METHODS: The Swedish Cancer Registry was searched for all gliomas in the neuroepithelial tissue, aged 16-79 years, and diagnosed between 1996 and the end of 2001. Survival analysis was performed using the Kaplan-Meier method. Survival rates from the Stockholm Regional Cancer Registry area was compared to the other areas in Sweden combined. RESULTS: For high-grade glioma, the 2-year survival was 25% in Stockholm and 14% in the other areas. For low-grade glioma, the 2-year survival was 82% and 72%, respectively. The largest 2-year survival difference was detected for glioblastoma patients aged 16-54 years, with 27% survival in the Stockholm area compared to 12% in the other areas. CONCLUSION: We cannot rule out all possible bias in our study, but results indicated higher 2-year survival for patients with glioma in the Stockholm region than in other regions of Sweden. These data are incompatible with the legislation of equal health care.

Cytokine Networks and Survivin Peptide-Specific Cellular Immune Responses Predict Improved Survival in Patients With Glioblastoma Multiforme.

PURPOSE: We investigated serum cytokine and T-cell responses directed against tumour-associated antigens (TAAs) in association with survival of patients with glioblastoma multiforme (GBM). PATIENTS AND METHODS: Peripheral blood from 205 treatment-naïve patients with glioma (GBM = 145; non-GBM = 60) was obtained on the day of surgery to measure (i) circulating T-cells reacting to viral antigens and TAAs, in the presence or absence of cytokine conditioning with IL-2/IL-15/IL-21 or IL-2/IL-7, and (ii) serum cytokine levels (IL-4, IL-5, IL-6, TNF-α, IFN-γ and IL-17A). Patients were followed-up for at least 1000 days post-surgery. Survivin protein and gene expression in resected GBM tumour tissue were confirmed by immunohistochemistry and real-time polymerase chain reaction, respectively. Antigen-specific T-cell responses were gauged by ICS (intracellular cytokine production). Associations between patient survival and immunological reactivity patterns were analysed using univariate and multivariate statistics. RESULTS: Approximately 2% of patients with GBM and 18% of patients with non-GBM glioma, were alive beyond 1000 days of surgery. Univariate analysis indicated that the combination of three cytokines (IL-4/IL-5/IL-6, p = .0022; IFN-γ/TNF-α/IL-17A, p = .0083) but not a 'partial' combination of these cytokines, the IFN-γ immune response to EBV-EBNA-1 (p < .0001) as well as T-cell responses to the survivin97-111 peptide (p = .0152) correlated with longer survival among patients with GBM. Multivariate analysis identified survivin97-111-directed IFN-γ production with IL-2/IL-15/IL-21 conditioning (p = .024), and the combined presence of serum IFN-γ/TNF-α/IL-17a (p = .003) as independent predictors of survival. CONCLUSION: Serum cytokine patterns and lymphocyte reactivity to survivin97-111, particularly with IL-2, IL-15 and IL-21 conditioning may be instrumental in predicting survival among patients with GBM. This has implications for clinical follow-up of patients with GBM and the targeted development of immunotherapy for patients with CNS tumours.

Correction: Identification of neoepitopes recognized by tumor-infiltrating lymphocytes (TILs) from patients with glioma.

[This corrects the article DOI: 10.18632/oncotarget.24955.].

Identification of neoepitopes recognized by tumor-infiltrating lymphocytes (TILs) from patients with glioma.

Neoepitope-specific T-cell responses have been shown to induce durable clinical responses in patients with advanced cancers. We explored the recognition patterns of tumor-infiltrating T lymphocytes (TILs) from patients with glioblastoma multiforme (GBM), the most fatal form of tumors of the central nervous system. Whole-genome sequencing was used for generating DNA sequences representing the entire spectrum of 'private' somatic mutations in GBM tumors from five patients, followed by 15-mer peptide prediction and subsequent peptide synthesis. For each mutated peptide sequence, the wildtype sequence was also synthesized and individually co-cultured with autologous GBM TILs, which had been expanded in vitro with a combination of interleukin (IL)-2, IL-15 and IL-21. After seven days of culture, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α) and/or IL-17A production was measured by ELISA in culture supernatants, and used as an epitope-specific immune response readout. Mutated peptides that induced a strong cytokine response were considered to contain legitimate neoepitopes. TILs from 5/5 patients with GBM exhibited specific immune reactivity profiles to the nominal target peptides, defined by IFN-γ and/or TNF-α production, as well as IL-17A. Neoepitopes, defined by mutated peptides inducing IFN-γ and/or TNF-α production without or only minimal reactivity to the wildtype sequences, were found for each individual patient. CD8+ TILs dominated the patients' responses to private neoepitopes. The present study shows that neoepitope-specific TIL reactivity constitutes an important arm of anti-tumor immune responses in patients with GBM, and thus a powerful tool for developing next-generation personalized immunotherapies.

Mesothelin as a novel biomarker and immunotherapeutic target in human glioblastoma.

Glioblastoma multiforme (GBM) presents the most malignant form of glioma, with a 5-year survival rate below 3% despite standard therapy. Novel immune-based therapies in improving treatment outcomes in GBM are therefore warranted. Several molecularly defined targets have been identified mediating anti-GBM cellular immune responses. Mesothelin is a tumor-associated antigen (TAA) which is expressed in several solid tumors with different histology. Here, we report the immunological significance of mesothelin in human malignant glioma. Expression of mature, surface-bound mesothelin protein was found to bein human GBM defined by immunofluorescence microscopy, and on freshly isolated, single cell suspension of GBM tumor cells and GBM tumor cell lines, determined by based on flow cytometric analysis. Peripheral blood (PB) from patients with GBM, stimulated with mesothelin peptides and IL-2, IL-15 and IL-21, exhibited increased antigen-specific IFN-γ and TNF-α production. Anti-mesothelin directed T-cell responses could also be detected in tumor - infiltrating lymphocytes (TIL) isolated from GBM speciments. Furthermore, T cells cultured in the presence of IL-2, IL-15 and IL-21 displayed enhanced mesothelin-specific CD4+ and CD8+ subset proliferation, based on ELISA and flow cytometric readouts. Mesothelin-specific IgG antibodies as well as (shed) mature mesothelin protein were detected in plasma samples from patients with GBM by indirect ELISA. Finally yet importantly, we identified distinct immune recognition hotspots within the mature mesothelin component, defined by peptide-specific IFN-γ responses from peripheral T-cells from patients with GBM. Mesothelin may therefore qualify as a viable target for immunotherapeutic approaches for patients with GBM.

Tumor-infiltrating lymphocytes (TILs) from patients with glioma.

Tumor-infiltrating lymphocytes (TILs) may represent a viable source of T cells for the biological treatment of patients with gliomas. Glioma tissue was obtained from 16 patients, tumor cell lines were established, and TILs were expanded in 16/16 cases using a combination of IL-2/IL-15/IL-21. Intracellular cytokine staining (ICS, IL-2, IL-17, TNFα and IFNγ production) as well as a cytotoxicity assay was used to detect TIL reactivity against autologous tumor cells or shared tumor-associated antigens (TAAs; i.e., NY-ESO-1, Survivin or EGFRvIII). TILs were analyzed by flow cytometry, including T-cell receptor (TCR) Vß family composition, exhaustion/activation and T-cell differentiation markers (CD45RA/CCR7). IL-2/IL-15/IL-21 expanded TILs exhibited a mixture of CD4+, CD8+, as well as CD3+ CD4-CD8- T cells with a predominant central memory CD45RA-CCR7+ phenotype. TIL showed low frequencies of T cells testing positive for PD-1, TIM-3 and CTLA-4. LAG3 tested positive in up to 30% of CD8+ TIL, with low (1.25%) frequencies in CD4+ T cells. TIL cultures exhibited preferential usage of Vß families and recognition of autologous tumor cells defined by cytokine production and cytotoxicity. IL-2/IL-15/IL-21 expanded TILs represent a viable source for the cellular therapy of patients with gliomas.

Mesothelin-specific Immune Responses Predict Survival of Patients With Brain Metastasis.

BACKGROUND: Patients with advanced malignancies, e.g. lung cancer, ovarian cancer or melanoma, frequently present with brain metastases. Clinical presentation and disease progression of cancer is in part shaped by the interaction of the immune system with malignant cells. Antigen-targeted immune responses have been implicated in the prolonged survival of patients with cancer. This includes the tumor-associated antigen (TAA) mature mesothelin, a 40kDa cell surface-bound antigen that is overexpressed in several malignancies including lung ovarian and pancreatic cancer. We examined in an observational, prospective study the survival of patients with brain metastases in association with clinical parameters and cellular immune responses to molecularly defined TAAs or viral (control) target antigens. METHODS: Immune cells in peripheral blood obtained from thirty-six patients with brain metastases were tested for cytokine production in response to a broad panel of defined viral and TAA target antigens, including full-length mesothelin. Incubation of immune cells with antigenic targets was carried out in i) medium alone, (ii) in a cytokine cocktail of interleukin (IL)-2/IL-15/IL-21, or (iii) IL-2/IL-7. Supernatants were tested for interferon gamma (IFN-γ) production, after which univariate and multivariate analyses (Cox stepwise regression model) were performed to identify independent clinical and immunological factors associated with patient survival. Patients were followed-up for at least 500days after surgery or until death. FINDINGS: Univariate analysis identified age, gender, radiotherapy and mutational load as clinical parameters affecting survival of patients with brain metastases. Cox multivariate analysis showed that radiotherapy (P=0·004), age (P=0·029) and IFN-γ responses to mature mesothelin, conditioned by IL-2/IL-7 (P=0·045) were independent predictors of the survival of patients from surgery up to follow-up or death. INTERPRETATION: This is the first evidence that immune responses to mesothelin serve as a marker of increased overall survival in patients with brain metastases, regardless of the primary tumor origin. Analyses of immunological markers could potentially serve as prognostic markers in patients with brain metastases and help to select patients in need for adjunct, immunological, treatment strategies.

Enhanced neutrophil activity is associated with shorter time to tumor progression in glioblastoma patients.

Glioblastoma multiforme (GBM) is a highly malignant tumor with a poor outcome that is often positive for human cytomegalovirus (HCMV). GBM patients often have excessive numbers of neutrophils and macrophages near and within the tumor. Here, we characterized the cytokine patterns in the blood of GBM patients with and without Valganciclovir treatment. Furthermore, we determined whether neutrophil activation is related to HCMV status and patient outcome. Blood samples for analyses of cytokines and growth factors were collected from 42 GBM patients at the time of diagnosis (n = 42) and at weeks 12 and 24 after surgery. Blood neutrophils of 28 GBM patients were examined for CD11b expression. The levels of pro- and anti-inflammatory cytokines and chemokines-including interleukin (IL)-1ß, IL-2, IL-6, IL-8, IL-10, IL-12p70, IL-17A, transforming growth factor (TGF)-ß1, interferon-γ, interferon-α, tumor necrosis factor α, and monocyte chemoattractant protein (MCP)-1were analyzed with a bead-based flow cytometry assay. During the first six months after surgery, neutrophil activity was increased in 12 patients and was unchanged or decreased in 16. Patients with increased neutrophil activity had enhanced IL-12p70, high grade HCMV and a shorter time to tumor progression (TTP) than patients without or decreased neutrophil activity (median TTP; 5.4 vs. 12 months, 95% confidence interval; 1.6-10 vs. 0.1-0.6, hazard ratio = 3 vs. 0.4, p = 0.004). The levels of IL-12p70 were significantly decreased in Valganciclovir treated patients (n = 22, T 12W vs. T 24W, p = 0.03). In conclusion, our findings suggest that neutrophil activation is an early sign of tumor progression in GBM patients.

Poor survival in glioblastoma patients is associated with early signs of immunosenescence in the CD4 T-cell compartment after surgery.

Patients with glioblastoma multiforme (GBM) are immunosuppressed and have a broad range of immunological defects in both innate and adaptive immune responses. GBMs are frequently infected with human cytomegalovirus (HCMV), a virus capable of causing immunosuppression. In 42 HCMV-positive GBM patients in a clinical trial (VIGAS), we investigated T-cell phenotypes in the blood and assessed their relation to survival. Blood was collected before and 3, 12, and 24 weeks after surgery, and the frequency of T-cell subsets was compared with that in 26 age-matched healthy controls. GBM patients had lower levels of CD3 cells than the controls, but had significantly higher levels of CD4+CD28- T cells before and 3 and 12 weeks after surgery and increased levels of CD4+CD57+ and CD4+CD57+CD28+ T cells at all-time points. These T-cell subsets were associated with both immunosenescence and HCMV infection. GBM patients also had higher levels of γδ T cells at all-times after surgery and lower levels of CD4+CD25+ cells before and 3 weeks after surgery than healthy controls. Overall survival was significantly shorter in patients with higher levels of CD4+CD28- T cells (p = 0.025), CD4+CD57+ T (p = 0.025) cells, and CD4+CD28-CD57+CD28- T cells (p < 0.0004) at 3 weeks after surgery. Our findings indicate that signs of immunosenescence in the CD4+ compartment are associated with poor prognosis in patients with HCMV-positive GBMs and may reflect the HCMV activity in their tumors.

Ganciclovir concentrations in the cerebral extracellular space after valganciclovir treatment; a case study.

Nearly all glioblastomas (GBMs), brain tumours with very poor prognosis, are infected with human cytomegalovirus (CMV). The anti-CMV drug valganciclovir (VGCV) has shown promise as a treatment option for patients with GBM, but its penetration into the central nervous system (CNS) is unknown. Here we describe a patient with GMB receiving VGCV in whom an intracerebral microdialysis catheter was implanted and ganciclovir (GCV) concentrations in brain extracellular fluid (BECF) and serum were monitored. GCV was rapidly absorbed. Cmax values (at 3 h) in serum and BECF were 19.6 and 10.2 µmol/L, T½ values were 3.2 and 4.5 h, and plasma and BECF AUC0-∞ values were 90.7 and 75.9 µmol h/L, respectively. Thus, VGCV treatment results in significant intracerebral levels of GCV that may be sufficient for therapeutic effects. Further studies of this drug in patients with GBM are warranted.

Discordant humoral and cellular immune responses to Cytomegalovirus (CMV) in glioblastoma patients whose tumors are positive for CMV.

Background. Glioblastoma (GBM) is the most common malignant brain tumor in adults and is nearly always fatal. Emerging evidence suggests that human Cytomegalovirus (HCMV) is present in 90-100% of GBMs and that add-on antiviral treatment for HCMV show promise to improve survival. Methods. In a randomized, placebo-controlled trial of valganciclovir in 42 GBM patients, blood samples were collected for analyses of HCMV DNA, RNA, reactivity against HCMV peptides, IgG, and IgM at baseline and at 3, 12, and 24 weeks of treatment. Results. All 42 tumors were positive for HCMV protein. All patients examined had at least one blood sample positive for HCMV DNA, 63% were HCMV RNA positive, and 21% were IgM positive. However, 29% of GBM patients were IgG negative for HCMV. Five of these samples were positive in an enzyme-linked immunosorbent assay (ELISA) that used antigens derived from a clinical isolate. Blood T cells from 11 of 13 (85%) HCMV IgG-negative GBM patients reacted against HCMV peptides. Valganciclovir did not affect IgG titers, DNA, or RNA levels of the HCMV immediate early (HCMV IE) gene in blood. Conclusion. In GBM patients, HCMV activity is higher than in healthy controls and serology is a poor test to define previous or active HCMV infection in these patients.

Increased survival using delayed gamma knife radiosurgery for recurrent high-grade glioma: a feasibility study.

OBJECTIVE: The current study retrospectively assessed delayed gamma knife radiosurgery (GKRS) in the management of high-grade glioma recurrences. METHODS: A total of 55 consecutive patients with high-grade glioma comprising 68 World Health Organization (WHO) III and WHO IV were treated with GKRS for local recurrences between 2001 and 2007. All patients had undergone microsurgery and radiochemotherapy, considered as standard therapy for high-grade glioma. Complete follow-up was available in all patients; median follow-up was 17.2 months (2.5-114.2 months). Median tumor volume was 5.2 mL, prescription dose was 20 Gy (14-22 Gy), and median max dose was 45 Gy (30-77.3 Gy). RESULTS: The patients with WHO III tumors showed a median survival of 49.6 months with and a 2-year survival of 90%. After GKRS of the recurrences, these patients showed a median survival of 24.2 months and a 2-year survival of 50%. The patients with WHO IV tumors had a median survival of 24.5 months with a 2-year survival of 51.4%. After the recurrence was treated with GKRS, the median survival was 11.3 months and a 2-year survival: 22.9% for the WHO IV patients. CONCLUSION: The current study shows a survival benefit for high-grade glioma recurrences when GKRS was administered after standard therapy. This is a relevant improvement compared with earlier studies that had had not been able to provide a beneficial effect timing radiosurgery in close vicinity to EBRT.

Human cytomegalovirus particles directly suppress CD4 T-lymphocyte activation and proliferation.

CD4 T cells are important regulators of the immune system and are vital for mounting a strong immune response against viral infections. Human cytomegalovirus (HCMV) is known to be a strong modulator of the innate as well as adaptive immune responses. In this study, we found that HCMV directly inhibited proliferation of CD4 T cells and rendered them unresponsive to immunological stimuli. This effect was not observed when CD4 T cells were treated with herpes simplex virus-1/2 or measles virus. When stimulated with phytohemagglutinin, concanavalin A, or phorbol myristate acetate, HCMV-treated T cells were unable to proliferate, revealing an ability of HCMV to inhibit CD4 T cell response. Furthermore, HCMV also prevented proliferation of leukemic T-cell lines. HCMV-treated CD4 T cells expressed the activation markers CD45RO and CD69, were not apoptotic and produced decreased levels of the cytokines IL-4, IFN-γ and TNF-α, compared to untreated controls. The inhibitory effect of HCMV on CD4 T cell proliferation was not mediated by HCMV gH, gB or other immunogenic glycoproteins, since intravenous immunoglobulins or gB- or gH-specific neutralizing antibodies did not prevent the suppression of T-cell proliferation. Our observations show that HCMV inhibits CD4 T cell function with potential clinical consequences for both humoral and cell-mediated immune responses.

Human cytomegalovirus infection levels in glioblastoma multiforme are of prognostic value for survival.

BACKGROUND: Patients with glioblastoma multiforme (GBM) generally live 12-15 months after diagnosis, despite maximal surgical resection, adjuvant radiotherapy, and chemotherapy. HCMV has been detected in 90-100% of GBMs. We recently found that low grade HCMV infection in GBM tumours was highly associated with survival over 18 months (case-control study). Here, we sought to determine whether low-grade HCMV infection in GBMs is associated with prolonged survival in a consecutive patient cohort, analysed retrospectively. STUDY DESIGN: Tumour samples from 75 consecutive GBM patients treated surgically at Karolinska University Hospital in 2004-2005 were examined by immunohistochemistry (IHC) and in situ hybridization for HCMV proteins and DNA, respectively. Tumours were graded 1-4, depending on the percentage of positive cells by IHC. Low-grade HCMV was defined as grade 1 (< 25% of HCMV infected tumour cells). Time to tumour progression (TTP) and survival data were analysed with Cox regression and Kaplan-Meier models. RESULTS: HCMV infection was detected in 74 of 75 tumours (99%). In patients with low-grade HCMV infection, median survival was 20 months longer than in patients with high-grade infections (P = 0.036, HR: 2.2), and TTP was 8 months longer (P = 0.1, HR: 1.8). Two-year survival was much higher in patients with low-grade HCMV infection (63.6% vs. 17.2%, P = 0.003). CONCLUSION: The longer survival in patients whose tumours had low-grade HCMV infection suggests that the level of HCMV infection in GBMs has a prognostic value and that HCMV may contribute to the pathogenesis of GBM.

Low levels of Human Cytomegalovirus Infection in Glioblastoma multiforme associates with patient survival; -a case-control study.

BACKGROUND: Glioblastoma multiforme (GBM) represent the most aggressive brain tumor with a median overall survival of about 12-15 months. Over 90% of GBM tumors have recently been shown to be infected with human cytomegalovirus (HCMV). In this case-control study, we evaluated whether there was an association between the grade of HCMV infection and long-term survival (> 18 months) in GBM patients. MATERIAL AND METHODS: Brain tumor tissue sections from consecutive GBMs patients who survived more than 18 months (n = 40), and an equal number of GBM patients, matched to date of diagnosis and surgery, operated at Karolinska University Hospital in 2000-2005 were selected. HCMV infection grade was determined by estimation of the number of HCMV positive cells (scored negative or grade 1-4) in tumor tissue specimens. Using Chi-Square test and logistic regression analysis, we analyzed whether there was an association between long-term survival and HCMV low-grade infection or other clinical parameters known to be associated with prolonged survival of GBM patients; age under 50 years, radical surgery or low recursive partition analysis (RPA) subclass. RESULTS: HCMV infection was detected in tumor samples from 79 of 80 patients (99%). Among patients surviving > 18 months, HCMV infection grade 1 in the GBM tumor was predominant. A low grade HCMV infection was found in 19 patients, of these 16 survived > 18 months. Thus, 16 of 40 (40%) GBM patients who lived > 18 months had low-grade HCMV infection while only 3 of 40 (8%) GBM patients who lived < 18 months did (p .0006, Chi-Square test). Multiple logistic regression analyses yielded an odds ratio estimate of 6.604 with 95% confidence interval (1.36-32.1) (p .019) for low grade HCMV after adjustment for RPA class III and IV, radical surgery, age and gamma knife treatment. CONCLUSION: In conclusion, we found that low-grade HCMV infection was strongly associated with long-term survival in GBM patients.