Giuseppe Stragliotto

Survival in Patients with Glioblastoma Receiving Valganciclovir

A retrospective analysis of selected patients with glioblastoma who received treatment for cytomegalovirus with their anticancer treatment showed surprisingly good 2-year survival. A randomized trial is needed.

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.

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

BackgroundGlioblastoma 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 methodsBrain 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.ResultsHCMV 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.ConclusionIn conclusion, we found that low-grade HCMV infection was strongly associated with long-term survival in GBM patients.

Valganciclovir in patients with glioblastoma.

To the Editor: Söderberg-Nauclér et al. (Sept. 5 issue)1 report that cytomegalovirus (CMV) antigens are detected in more than 99% of human glioblastomas. In their trial, in which the benefit of valganciclovir was evaluated in patients with glioblastoma,2 29% of the 42 participating patients were seronegative for CMV IgG, a result similar to that obtained in larger studies.3 A large proportion of the patients with intratumoral expression of CMV antigens are therefore unlikely to have had CMV infection. In a recent large-scale analysis of transcriptome-sequencing data of viral nucleic acids,4 significant levels of CMV RNA were not detected in human gliomas (glioblastoma multiforme). Out of 22.8 billion sequencing reads from 167 tumors, only 1 sequence corresponded with CMV RNA. These results imply that CMV does not replicate in gliomas and thus that treatment with virusreplication inhibitors, such as valganciclovir, may be futile. Questions regarding the intratumoral expression of CMV antigens in seronegative patients and the apparent lack of intratumoral replication of CMV should be resolved before larger trials of valganciclovir in glioma are initiated.

Human cytomegalovirus immediate early proteins promote degradation of connexin 43 and disrupt gap junction communication: implications for a role in gliomagenesis

A lack of gap junctional intercellular communication (GJIC) is common in cancer. Many oncogenic viruses have been shown to downregulate the junctional protein connexin 43 (Cx43) and reduce GJIC. Human cytomegalovirus (HCMV) is a ubiquitous, species-specific betaherpesvirus that establishes life-long latency after primary infection. It encodes two viral gene products, immediate early (IE) proteins IE1 and IE2, which are crucial in viral replication and pathogenesis of many diseases. Emerging evidence demonstrates that HCMV DNA and proteins are highly prevalent in glioblastoma multiforme (GBM) and in other tumors, but HCMVs role in tumorigenesis remains obscure. In the present study, we examined the effects of HCMV infection on Cx43 expression and GJIC as well as the viral mechanism mediating the effects in human GBM cells and tissue samples. We found that HCMV downregulated Cx43 protein, resulting in disruption of functional GJIC as assayed by fluorescent dye transfer assay. We show that both HCMV-IE72 and IE86 mediate downregulation of Cx43 by silencing RNA targeting either IE72 or IE86 coupled with ganciclovir. This finding was further validated by transfection with expression vectors encoding IE72 or IE86, and we show that viral-mediated Cx43 depletion involved proteasomal degradation. Importantly, we also observed that the Cx43 protein levels and IE staining correlated inversely in 10 human GBM tissue specimens. Thus, HCMV regulates Cx43 expression and GJIC, which may contribute to gliomagenesis.

Cytomegalovirus infection induces a stem cell phenotype in human primary glioblastoma cells: prognostic significance and biological impact

Glioblastoma (GBM) is associated with poor prognosis despite aggressive surgical resection, chemotherapy, and radiation therapy. Unfortunately, this standard therapy does not target glioma cancer stem cells (GCSCs), a subpopulation of GBM cells that can give rise to recurrent tumors. GBMs express human cytomegalovirus (HCMV) proteins, and previously we found that the level of expression of HCMV immediate-early (IE) protein in GBMs is a prognostic factor for poor patient survival. In this study, we investigated the relation between HCMV infection of GBM cells and the presence of GCSCs. Primary GBMs were characterized by their expression of HCMV-IE and GCSCs marker CD133 and by patient survival. The extent to which HCMV infection of primary GBM cells induced a GCSC phenotype was evaluated in vitro. In primary GBMs, a large fraction of CD133-positive cells expressed HCMV-IE, and higher co-expression of these two proteins predicted poor patient survival. Infection of GBM cells with HCMV led to upregulation of CD133 and other GSCS markers (Notch1, Sox2, Oct4, Nestin). HCMV infection also promoted the growth of GBM cells as neurospheres, a behavior typically displayed by GCSCs, and this phenotype was prevented by either chemical inhibition of the Notch1 pathway or by treatment with the anti-viral drug ganciclovir. GBM cells that maintained expression of HCMV-IE failed to differentiate into neuronal or astrocytic phenotypes. Our findings imply that HCMV infection induces phenotypic plasticity of GBM cells to promote GCSC features and may thereby increase the aggressiveness of this tumor.

Early cognitive impairment in a subset of patients with presumed low-grade glioma.

We investigated the presence of cognitive impairment, in adults with presumed low-grade glioma at early stage of disease prior to major treatments, in relation to neurological symptoms and radiological characteristics of the tumour. Sixteen patients were evaluated. A subset of patients was identified with clearly impaired cognition. Patients with cognitive impairment often had large tumours in the left frontal lobe, were relatively young, and most of them were males. We conclude that cognitive dysfunction may be present already at early stage of disease, and that early identification of patients at risk is 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.

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

ABSTRACT 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)-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, IL-17A, transforming growth factor (TGF)-β1, 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.