TERT and its binding protein: overexpression of GABPA/B in high grade gliomas.

Enhanced expression of TERT in gliomas is a result of two hotspot mutations, C228T and C250T, at the promoter region. GA-binding proteins selectively bind at these positions, respectively, causing an activation of the promoter and overexpression of TERT. GABP is a multimeric protein consisting of GABPA and GABPB with its isoforms GABPB1, GABPB1-L, GABPB1-S, GABPB2. In this study, we investigated the mRNA expression and association between TERT and GABPA/B isoforms in tumor samples of different glioma grades. The expression was determined by quantitative real-time PCR and the results were statistically analyzed. We present that TERT is mainly expressed in primary glioblastomas. All GA-binding proteins progress through the glioma grades and have the highest expression levels in secondary glioblastomas. In secondary glioblastomas after chemotherapy, GABPB1 and GABPB1-L are expressed on a lower level than without treatment. In high grades, TERT and GABPA, GAPB1, GABPB1-L, GABPB1-S are upregulated compared to low grades. Between primary and secondary glioblastomas with and without chemotherapy, TERT is elevated in the former while GABPB1 is increased in the secondary glioblastomas. GABPA and GABPB1, GABPB1-L and GABPB1-S positive correlate in primary glioblastomas. The present study confirms the upregulation of TERT in primary glioblastomas while all GABP proteins rise with the malignancy of the gliomas. Further investigations must be made to elucidate the relation between TERT and all GABP proteins as it may play a key role in the gliomagenesis.

Death Receptor 6 (DR6) Is Overexpressed in Astrocytomas.

BACKGROUND/AIM: Death receptor 6 (DR6) is a member of the tumor necrosis factor receptor superfamily. The expression of DR6 is elevated in different kinds of tumors including ovarian, breast cancer and adult sarcoma. In these tumors, the receptor may be handled as a new diagnostic and prognostic marker. Thus, we investigated the expression of DR6 in gliomas. MATERIALS AND METHODS: Tumor and control tissues were extracted during neurosurgery and grouped according to the WHO classification. DR6 expression was investigated in low- and high-grade gliomas PCR (n=70), immunofluorescence staining (n=33) and western blot (n=58). Additional analysis of TCGA-data was performed to assess the general alteration of DR6 in cancer and influence of IDH-mutation on DR6 expression in gliomas. RESULTS: The expression of DR6 was significantly enhanced in gliomas (p<0.05). It showed a trend towards rising expression with increasing malignancy of the tumor. Chemotherapy treatment could have an influence on DR6 expression. CONCLUSION: In our investigation, DR6 acts as a potential suitable diagnostic marker for gliomas.

Heterogeneity of Human Gliomas: Glutathione-S-Transferase Expression Profile During Disease Progression and Under Systemic Therapy.

BACKGROUND/AIM: The glutathione S-transferase pi gene (GSTP1) is a polymorphic gene encoding functionally different Gstp1 isoenzyme proteins. These seem to contribute to xenobiotic metabolism and might also play a role in susceptibility to cancer. The aim of this study was to elucidate the potential role of GSTP1 as a biomarker for disease progression and predictor of chemotherapeutic effect in glioma. MATERIALS AND METHODS: Using quantitative real time PCR and western blotting analysis, a total of 61 astrocytic tumor samples from WHO grade II-IV were investigated. RESULTS: There were no differences in GSTP1 mRNA expression between diffuse astrocytomas, anaplastic astrocytomas, or GBM. No difference was seen between secondary GBM before and after radio-/chemotherapy. CONCLUSION: The expression of GSTP was highly heterogeneous within the surgical specimens. No significant differences in gene and protein expression were detected between the different types of gliomas, suggesting that glioma chemoresistance is probably multifactorial and GSTP1-independent.

Qualitative and Quantitative Analysis of IDH1 Mutation in Progressive Gliomas by Allele-Specific qPCR and Western Blot Analysis.

To date, diagnosis of IDH1 mutation is based on DNA sequencing and immunohistochemistry, methods limited in terms of sensitivity and ease of use. Recently, the diagnosis of IDH1 mutation by real-time polymerase chain reaction was introduced as an alternative method. In this study, real-time polymerase chain reaction was validated as a tool for detection of IDH1 mutation, and expression levels were analyzed for correlation with course of the disease. A total of 113 tumor samples were obtained intraoperatively from 84 patients with glioma having a diagnosis of diffuse glioma (World Health Organization II), anaplastic glioma (World Health Organization III), secondary glioblastoma ± chemotherapy, primary glioblastoma ± chemotherapy (World Health Organization IV). Tumor samples were snap frozen and processed for sectioning and RNA and protein isolation. Presence of IDH1 mutation was determined by DNA sequencing. Hereafter, quantitative expression of IDH1 messenger RNA was assessed using real-time polymerase chain reaction with specific primers for IDH1 mutation and -wt; protein expression was verified by Western Blot analysis and immunohistochemistry. Additionally, 19 samples of low-grade glioma and their consecutive high-grade glioma were analyzed at different time points of the disease. IDH1 mutation was identified in 63% of samples by DNA sequencing. In correlation with the real-time polymerase chain reaction results, a cutoff value was determined. Above this threshold, sensitivity and specificity of real-time polymerase chain reaction in detecting IDH1 mutation were 98% and 94%, respectively. Quantitative analysis revealed that IDH1 mutation expression is upregulated in secondary glioblastoma (mean ± standard error of mean: 3.52 ± 0.55) compared to lower grade glioma (II = 1.54 ± 0.22; III = 1.67 ± 0.23). In contrast, IDH1 wt expression is upregulated in all glioma grades (concentration >0.1) compared to control brain tissue (0.007 ± 0.0016). Western Blot analysis showed a high concordance to both sequencing and real-time polymerase chain reaction results in qualitative analysis of IDH1 mutation status (specificity 100% and sensitivity 100%). Moreover, semiquantitative protein expression analysis also showed higher expression levels of mutated IDH1 in secondary glioblastoma. In our study, real-time polymerase chain reaction and Western Blot analysis were found to be highly efficient methods in detecting IDH1 mutation in glioma samples. As cost-effective and time-saving methods, real-time polymerase chain reaction and Western Blot analysis may therefore play an important role in IDH1 mutation analysis in the future. IDH1 mutation expression level was found to correlate with the course of disease to a certain extent. Yet, clinical factors as recurrent disease or prior radiochemotherapy did not alter IDH1 mutation expression level.

Long-Term Outcome and Health-Related Quality of Life of Elderly Patients After Meningioma Surgery.

OBJECTIVE: The incidence of meningiomas increases with increasing age. Because the median age for the diagnosis is 65 years, the patients' general health condition, comorbidities, and procedural risks will influence the postoperative functional outcomes. The aim of our study was to assess the long-term impairments of health-related quality of life (HRQOL) after meningioma resection in different age groups of elderly patients. METHODS: We analyzed the HRQOL of 133 patients aged >54 years at surgery who had undergone surgical meningioma resection from 2004 to 2010. The median age was 67.3 ± 7.4 years. The average interval between surgery and questionnaire completion was 3.8 ± 2.5 years. Six different 5-year age groups were established. The patients completed the Medical Outcomes Study short-form 36-item questionnaire, and the results were compared with general population values. The Kruskal-Wallis test and Mann-Whitney U test were used for statistical analysis. RESULTS: We found significantly lower levels of physical function, vitality, social role functioning, mental health, and general health perception and significantly greater levels of pain between the older age groups (in particular, 75-79 years) and younger patients aged 55-59 years. The physical component summary showed a steadily and stepwise decline from younger to older patients. However, the most significant differences in HRQOL were not related to age but to comorbidities. CONCLUSION: Our findings suggest that Karnofsky performance scale and American Society of Anesthesiologists scores have a strong effect on long-term HRQOL, especially for older patients after meningioma resection. These data should be a substantial consideration in the preoperative decision-making process.

ACTB and SDHA Are Suitable Endogenous Reference Genes for Gene Expression Studies in Human Astrocytomas Using Quantitative RT-PCR.

BACKGROUND: Quantitative real-time reverse-transcription polymerase chain reaction is frequently used as research tool in experimental oncology. There are some studies of valid endogenous control genes in the field of human glioma research, which, however, only focus on the comparison between normal brain with tumor tissue and malignant transformation toward secondary glioblastomas. Aim of this study was to validate a more general reference gene also suitable for pre- and posttreatment analysis and other evaluations (eg, primary vs secondary glioblastoma). METHODS: This quantitative polymerase chain reaction analysis was performed to test a panel of the 6 most suitable reference genes from other studies representing different physiological pathways (ACTB, GAPDH, POLR2A, RPL13A, SDHA, and TBP) in all common glioma groups, namely: diffuse astrocytoma World Health Organization II, anaplastic astrocytoma World Health Organization III, secondary glioblastoma World Health Organization IV with and without chemotherapy, primary glioblastoma, recurrent glioblastoma, and gliomas before and after radiation. Expression stability was tested during the longitudinal course of the disease in 8 single patients. RESULTS: Evaluation of the expression levels of the 6 target genes showed that ACTB, GAPDH, and RPL13A show higher expression compared to SDHA, POLR2A, and TBP. ACTB, GAPDH, and RPL13A showed different expression levels between astrozytoma grade II and primary glioblastoma. Except for this difference, the candidate genes were not differentially expressed between primary and secondary glioblastomas and between the World Health Organization tumor grades. Furthermore, they remained stable before and after radiotherapy and/or chemotherapy. Therefore, they are adequate references for glioblastoma gene expression studies. The comparison of all tested genes resulted in SDHA and ACTB as most stable reference genes determined by the NormFinder software. Our data revealed lowest intragroup variation in the SDHA, highest in the RPL13A gene. CONCLUSIONS: All tested genes may be recommended as universal reference genes for data normalization in gene expression studies under different treatment regimens both in primary glioblastomas and astrocytomas of different grades (World Health Organization grades II-IV), respectively. In summary, ACTB and SDHA exhibited the best stability values and showed the lowest intergroup expression variability.

Inhibition of the PI3K but not the MEK/ERK pathway sensitizes human glioma cells to alkylating drugs.

BACKGROUND: Intrinsic chemoresistance of glioblastoma (GBM) is frequently owed to activation of the PI3K and MEK/ERK pathways. These signaling cascades are tightly interconnected however the quantitative contribution of both to intrinsic resistance is still not clear. Here, we aimed at determining the activation status of these pathways in human GBM biopsies and cells and investigating the quantitative impact of both pathways to chemoresistance. METHODS: Receptor tyrosine kinase (RTK) pathways in temozolomide (TMZ) treatment naive or TMZ resistant human GBM biopsies and GBM cells were investigated by proteome profiling and immunoblotting of a subset of proteins. Resistance to drugs and RTK pathway inhibitors was assessed by MTT assays. Apoptotic rates were determined by Annexin V staining and DNA damage with comet assays and immunoblotting. RESULTS: We analyzed activation of RTK pathways by proteome profiling of tumor samples of patients which were diagnosed a secondary GBM and underwent surgery and patients which underwent a second surgery after TMZ treatment due to recurrence of the tumor. We observed substantial activation of the PI3K and MEK/ERK pathways in both groups. However, AKT and CREB phosphorylation was reduced in biopsies of resistant tumors while ERK phosphorylation remained unchanged. Subsequent proteome profiling revealed that multiple RTKs and downstream targets are also activated in three GBM cell lines. We then systematically describe a mechanism of resistance of GBM cell lines and human primary GBM cells to the alkylating drugs TMZ and cisplatin. No specific inhibitor of the upstream RTKs sensitized cells to drug treatment. In contrast, we were able to restore sensitivity to TMZ and cisplatin by inhibiting PI3K in all cell lines and in human primary GBM cells. Interestingly, an opposite effect was observed when we inhibited the MEK/ERK signaling cascade with two different inhibitors. CONCLUSIONS: Temozolomide treatment naive and TMZ resistant GBM biopsies show a distinct activation pattern of the MEK/ERK and PI3K signaling cascades indicating a role of these pathways in resistance development. Both pathways are also activated in GBM cell lines, however, only the PI3K pathway seems to play a crucial role in resistance to alkylating agents and might serve as drug target for chemosensitization.

Expression of FAS-L Differs from Primary to Relapsed Low-grade Gliomas and Predicts Progression-free Survival.

BACKGROUND/AIM: The tumor necrosis factor FAS is overexpressed in high-grade gliomas (HGG). Only little is known about FAS or FAS ligand (FAS-L) in low-grade gliomas (LGG). We explored FAS/FAS-L expression in LGG, focusing on differences in primary and relapsed LGG and on its prognostic value. PATIENTS AND METHODS: A total of 133 glioma samples (73 LGG, 60 HGG) were collected. The LGG samples included 15 matched pairs of primary and relapsed tumors. RT-PCR was performed to measure FAS/FAS-L expression, using subunit A, flavoprotein variant (SDHA) as housekeeper. Clinical data included progression free- (PFS) and overall survival (OS). RESULTS: LGG showed significantly lower FAS but higher FAS-L expression than HGG. The FAS-L expression was higher in primary compared to relapsed LGG and had a positive prognostic value concerning PFS (median 45.20 vs. 31.37 months). CONCLUSION: FAS-L could act as a prognostic marker and potential target in primary LGG.

Discordance and Conversion Rates of Progesterone-, Estrogen-, and HER2/neu-Receptor Status in Primary Breast Cancer and Brain Metastasis Mainly Triggered by Hormone Therapy.

BACKGROUND/AIM: Knowing the molecular footprint of tumors is a precondition for personalized medicine. For breast cancer, targeted therapies are frequently based on the molecular status of the tissue gained from the primary tumor operation. However, it is unclear whether metastases in different organs maintain the same status. PATIENTS AND METHODS: We compared the estrogen- (ER), progesterone- (PgR) and HER2/neu receptor status of the primary tumor with brain metastases in a series of 24 consecutive breast cancer patients. RESULTS: 62.5-75% of patients exhibited a constant receptor status between the primary tumor and the brain metastasis, whereas discordance rates of 25-37.5% were found, depending on the receptor. The rate of ER and PgR expression was each 41.6% in the primary tumors and decreased to 12.5% and 16.6% in the brain metastases. In contrast, the rate for Her2+ tumors increased from 41.6% in primary breast cancer to 65.2% in the respective brain metastases. The Ki-67 proliferation index increased significantly from a mean of 21% at the primary tumor site to 60% in brain metastases (p<0.001). All anti-estrogen treated breast tumors lost the estrogen receptor expression in the brain metastases, whereas no Her2/neu conversions occurred after treatment with trastuzumab. CONCLUSION: In summary, receptor conversion is frequent during disease progression. Therefore, the receptor status of the primary tumor is invalid for planning a therapy targeted against brain metastases, especially after hormone-therapy. In these cases, new tissue collection by biopsy or resection is mandatory for the selection of adequate therapeutic targets and accurate decision-making for systemic therapies.

Intracerebral administration of heat-inactivated Staphylococcus epidermidis enhances oncolysis and prolongs survival in a 9L orthotopic gliosarcoma model.

BACKGROUND/AIMS: The association between postoperative infection and prolonged survival in high-grade glioma is still a matter of debate. Previously we demonstrated that the intracerebral (i.c.) injection of heat-inactivated staphylococcal epitopes (HISE) resulted in a well-defined infux of immunocompetent cells across the blood-brain barrier. The present study investigated the potential antitumoral effect of HISE-immunostimulation in an experimental glioma model. METHODS: Wistar rats were intracerebrally implanted with 9L gliosarcoma cells (n=6), 9L cells mixed with HISE (n=12), or phosphate buffered saline (n=4). Tumor growth was measured by serial magnetic resonance imaging (MRI). After death due to the tumor burden, the brains were histopathologically assessed for inflammation and oncolysis. A toxicity assay was performed to quantify potential impairment of HISE on tumor cell growth in vitro. RESULTS: Animals treated by HISE showed a significant increase in average survival and even complete regression of an already established mass in one case. Naïve 9L gliosarcomas failed to recruit significant numbers of systemic immune cells. In contrast, concomitant intracerebral HISE inoculation lead to a oncolysis and a distinct peri- and intratumoral infiltration of macrophages, CD8 and CD4 co-expressing T-lymphocytes in two thirds of the tumor-bearing animals. The toxicity screening showed HISE-mediated oncolysis to be ineffective ex vivo. CONCLUSION: This study describes a novel approach for combatting malignant glioma using inactivated staphylococci as potent immunomodulators. Our results provide an outline for investigating the strategic potential of bacteria as emerging future therapeutics.

A novel experimental in vivo model of cerebral immunomodulation induced by inactivated Staphylococcus epidermidis.

The genesis and appropriate treatment of neuroinflammation in various infectious and non-infectious disorders of the central nervous system is still a matter of debate. We introduce an alternative and simple experimental model for the investigation of the cellular inflammatory response to bacterial antigens by stereotactic intracerebral injection of heat-inactivated Staphylococcus epidermidis (HISE). HISE-injection resulted in well-circumscribed intraparenchymal deposits encompassed by an early micro- and astroglial response and a selective but sustained opening of the blood-brain barrier (BBB). After 24h, the HISE collections were densely infiltrated by granulocytes and few circumjacent macrophages that became the predominating immunocompetent cell type from day 4 on. CD8a+ lymphocytes peaked at day 4, whereas CD4+ and CD20+ lymphocytes increased gradually in number, developing a scattered infiltrate until day 17, indicating the initiation of an adaptive immune response. MHC class II presenting cells were abundantly recruited from day 1 and eventually shaped an increasingly dense accumulation within the lesion. Intracerebral HISE administration provides a controlled, highly reproducible and well defined influx of immunocompetent cells across the BBB leading to a distinct and condensed inflammatory reaction. The technique is straightforward, easily feasible and may significantly enable further investigations of the initiation, maintenance and therapeutic modulation of acute neuroinflammation.

Epigenetic silencing of the candidate tumor suppressor gene PROX1 in sporadic breast cancer.

Extensive hypermethylation and consecutive transcriptional silencing of tumorsuppressor genes have been documented in multiple tumor entities including breast cancer. In a microarray based genome-wide methylation analysis of five sporadic breast carcinomas we identified a hypermethylated CpG island within the first intron of the prospero related homeobox gene 1 (PROX1). We, therefore, investigated CpG island methylation of PROX1 in a series of 33 pairs of primary breast cancer and corresponding normal tissue samples by bisulfite sequencing and COBRA analyses. Seventeen of these (52%) breast cancer samples revealed a significant accumulation of methylated CpG sites along with a significant reduction of PROX1 transcription compared to normal breast tissues of the same patients. Frequent methylation was also observed in brain metastases from primary breast cancer (21/37 = 57% of cases). Secondary, we analysed 38 brain metastases of primary breast carcinomas and detected a significantly reduced expression of PROX1 compared to normal breast tissue (p < 0.001) and primary breast carcinomas (p < 0.05), respectively. Additionally, treatment of breast cancer cell lines with demethylating agents could reactivate PROX1 transcription. In summary, we have identified PROX1 as a novel target gene that is hypermethylated and transcriptionally silenced in primary and metastatic breast cancer.

Metabolic differences between primary and recurrent human brain tumors: a 1H NMR spectroscopic investigation.

High-resolution proton magnetic resonance spectroscopy was performed on tissue specimens from 33 patients with astrocytic tumors (22 astrocytomas, 11 glioblastomas) and 13 patients with meningiomas. For all patients, samples of primary tumors and their first recurrences were examined. Increased anaplasia, with respect to malignant transformation, resulting in a higher malignancy grade, was present in 11 recurrences of 22 astrocytoma patients. Spectroscopic features of tumor types, as determined on samples of the primary occurrences, were in good agreement with previous studies. Compared with the respective primary astrocytomas, characteristic features of glioblastomas were significantly increased concentrations of alanine (Ala) (p = 0.005), increased metabolite ratios of glycine (Gly)/total creatine (tCr) (p = 0.0001) and glutamate (Glu)/glutamine (Gln) (p = 0.004). Meningiomas showed increased Ala (p = 0.02) and metabolite ratios [Gly, total choline (tCho), Ala] over tCr (p = 0.001) relative to astrocytomas, and N-acetylaspartate and myo-inositol were absent. Metabolic changes of an evolving tumor were observed in recurrent astrocytomas: owing to their consecutive assessments, more indicators of malignant degeneration were detected in astrocytoma recurrences (e.g. Gly, p = 0.029; tCho, p = 0.034; Glu, p = 0.015; tCho/tCr, p = 0.001) in contrast to the comparison of primary astrocytomas with primary glioblastomas. The present investigation demonstrated a correlation of the tCho-signal with tumor progression. Significantly elevated concentrations of Ala (p = 0.037) and Glu (p = 0.003) and metabolite ratio tCho/tCr (p = 0.005) were even found in recurrent low-grade astrocytomas with unchanged histopathological grading (n = 11). This may be related to an early stage of malignant transformation, not yet detectable morphologically, and emphasizes the high sensitivity of 1H NMR spectroscopy in elucidating characteristics of brain tumor metabolism.

Diagnostic impact of ornithine decarboxylase in meningiomas.

The objective of this study was to investigate whether activity and protein expression of ornithine decarboxylase (ODC) the metabolism-rate limiting enzyme of the polyamine biosynthesis, which is involved in the regulation of cellular growth and differentiation, reflects the distinct histopathologic characteristics indicative of atypia and anaplasia in meningiomas as well as their impact in recurrences. The authors previously evaluated its value as a critical factor of tumor development in various brain tumors. Among 152 meningiomas, World Health Organisations (WHO) grade I meningiomas (n = 121) exhibited a low ODC activity, while meningiomas WHO grade II (n = 22) and WHO grade III (n = 9), respectively, showed a significantly increased ODC activity. Recurrent WHO grade I meningiomas exhibited the same low enzyme activity and immunohistochemical staining index for ODC positive tumor cells as their primary tumors, whereas raising ODC activity and protein expression in recurrent meningiomas paralleled malignant transformation. These results indicate that ODC reflects aggressive growth and malignization in meningiomas. Especially in recurrent meningiomas, the determination of its activity and protein expression by immunohistochemistry may provide a useful diagnostic tool to recognize malignant progression.