Inti Peredo

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.

Activation of neural and pluripotent stem cell signatures correlates with increased malignancy in human glioma.

The presence of stem cell characteristics in glioma cells raises the possibility that mechanisms promoting the maintenance and self-renewal of tissue specific stem cells have a similar function in tumor cells. Here we characterized human gliomas of various malignancy grades for the expression of stem cell regulatory proteins. We show that cells in high grade glioma co-express an array of markers defining neural stem cells (NSCs) and that these proteins can fulfill similar functions in tumor cells as in NSCs. However, in contrast to NSCs glioma cells co-express neural proteins together with pluripotent stem cell markers, including the transcription factors Oct4, Sox2, Nanog and Klf4. In line with this finding, in high grade gliomas mesodermal- and endodermal-specific transcription factors were detected together with neural proteins, a combination of lineage markers not normally present in the central nervous system. Persistent presence of pluripotent stem cell traits could only be detected in solid tumors, and observations based on in vitro studies and xenograft transplantations in mice imply that this presence is dependent on the combined activity of intrinsic and extrinsic regulatory cues. Together these results demonstrate a general deregulated expression of neural and pluripotent stem cell traits in malignant human gliomas, and indicate that stem cell regulatory factors may provide significant targets for therapeutic strategies.

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.

Neuronavigation for arteriovenous malformation surgery by intraoperative three-dimensional ultrasound angiography.

OBJECTIVE Neuronavigational devices have traditionally used preoperative imaging with limited possibilities for adjustment to brain shift and intraoperative manipulation of the surgical lesions. We have used an intraoperative imaging and navigation system that uses navigation on intraoperatively acquired three-dimensional ultrasound data, as well as preoperatively acquired magnetic resonance imaging scans and magnetic resonance angiograms. The usefulness of this system for arteriovenous malformation (AVM) surgery was evaluated prospectively. METHODS Nine consecutive patients with Spetzler Grade 1 (n = 3), 2 (n = 3), 3(n = 2) or 4 (n = 1) AVMs underwent operation using this intraoperative imaging and navigation system. The system provides real-time rendering of three-dimensional angiographic data and can visualize such projections in a stereoscopic (virtual reality) manner using special glasses. The experiences with this technology were analyzed and the outcomes assessed. Angiographic reconstructions of three-dimensional images were obtained before and after resection. RESULTS Conventional navigation on the basis of preoperative magnetic resonance angiography was helpful to secure positioning of the bone flap; stereoscopic visualization of the same data represented a powerful means to construct a mental three-dimensional picture of the extent of the AVM and the feeder anatomy even before skin incision. Intraoperative ultrasound corresponded well to the intraoperative findings and allowed confirmation of feeding vessels in surrounding gyri and rapid identification of the perinidal dissection planes, regardless of brain shift. The latter feature was particularly helpful because the intraoperative navigational identification of surgical planes leads to minimal exploration into the nidus or dissection at a greater distance from the malformation. Application of the system was thought to increase surgical confidence. In two patients, postresection ultrasound prompted additional nidus removal. Ultrasound angiography seemed to allow some degree of resection control, although its sensitivity was not thought to be sufficient. All AVMs were radically removed without new permanent morbidity. CONCLUSION The complexities of handling the pathological vessels of AVMs were ameliorated by intraoperative three-dimensional ultrasound and navigation because the three-dimensional outline of the vasculature (feeders, nidus, and draining veins) provided a means to adapt resection strategies, define dissection planes, and interpret intraoperative findings. It is difficult to provide a scientifically valid definition of “added value.” However, in our experience, the added confidence and the improved mental image of the lesion that resulted from this technology improved the quality and flow of surgery.

Optimized protein extraction from cryopreserved brain tissue samples.

Optimal standard conditions for protein extraction and solubilization from frozen tissue samples have been examined. Quantitative differences in specific protein amounts or post-translational modifications underlie many, if not all, disease states. Maximal and standardized extraction and solubilization of protein from diseased or healthy tissue is important to make the whole protein complement available for proteomic analysis, and to make the best use of a precious resource. Minimal degradation of the protein amino acid backbone, or of phosphorylated amino acid side chains, during sample preparation is essential to preserve the analytical utility of the extract. We have investigated parameters of brain tissue disintegration, and of extraction/solubilization temperature, time and volume and have reached 98% extraction of brain tissue, corresponding to about 100 µg protein per mg tissue wet weight, by an SDS-based method: Tissue disintegration in the frozen state, by ball mill grinding followed by extraction and solubilization in 2% SDS for 10 min, at 70°C, in a volume corresponding to ten times the tissue wet weight, with shaking. The treatment with SDS sample buffer can inhibit protease and phosphatase activity. Moreover, endogenous enzymes can be inhibited by incubation at high pH. The resulting protein extracts can be used for both one-dimensional SDS gel-electrophoresis and for two-dimensional isoelectric focusing/SDS electrophoresis. The proposed standard protocol has the potential to find wide application where protein extraction, solubilization, identification and quantitation from cryopreserved clinical samples are desirable.

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.

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.

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.