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

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., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

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.

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., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest

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., (2015 BMJ Publishing Group Ltd.)

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.

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.

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.

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (Copyright © 2013 UICC.)

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., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

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.

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.

Purpose: Cells in the ependymal region in the adult central nervous system (CNS) have been found to possess neural progenitor cell (NPC) like features including capacity for generating new neurons and glia in response to injury and inflammatory disease. Whether these cells are activated after a peripheral nerve injury has not previously been extensively evaluated., Methods: We investigate the possible activation and effect of NPCs in the ependymal region in the immediate vicinity to the hypoglossal nucleus in the brainstem using two models of injuries, hypoglossal nerve transection and nerve avulsion after which the proliferation, migration and differentiation of ependymal regional NPCs were evaluated., Results: We showed that: (i) immunoreactivity for Sox2 was detected in cells in the ependymal region of the brainstem and that BrdU/Sox2-positive cells were observed after avulsion, but not after transection injury; (ii) avulsion induces re-expression of nestin in the ependymal layer as well as induced NPC migration from the ependymal layer; (iii) the chemokine SDF-1α (a marker for migrating cells) was upregulated ipsilateral to the nerve injury; (iiii) the NPCs migrating differentiated only into GFAP-positive astrocytes in the hypoglossal nucleus., Conclusion: These results suggest that nerve avulsion injury induces in parallel with the retrograde "axon reaction" activation of endogenous NPCs in the ependymal region and further suggest that these cells could be involved in repair and neuroregeneration after injury within the brainstem.

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.

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 microg 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 degrees 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.