Gene expression profiles of gliomas in formalin-fixed paraffin-embedded material

BACKGROUND: We have recently demonstrated that expression profiling is a more accurate and objective method to classify gliomas than histology. Similar to most expression profiling studies, our experiments were performed using fresh frozen (FF) glioma samples whereas most archival samples are fixed in formalin and embedded in paraffin (FFPE). Identification of the same, expression-based intrinsic subtypes in FFPE-stored samples would enable validation of the prognostic value of these subtypes on these archival samples. In this study, we have therefore determined whether the intrinsic subtypes identified using FF material can be reproduced in FFPE-stored samples. METHODS: We have performed expression profiling on 55 paired FF-FFPE glioma samples using HU133 plus 2.0 arrays (FF) and Exon 1.0 ST arrays (FFPE). The median time in paraffin of the FFPE samples was 14.1 years (range 6.6-26.4 years). RESULTS: In general, the correlation between FF and FFPE expression in a single sample was poor. We then selected the most variable probe sets per gene (n = 17 583), and of these, the 5000 most variable probe sets on FFPE expression profiles. This unsupervised selection resulted in a better concordance (R-2 = 0.54) between expression of FF and FFPE samples. Importantly, this probe set selection resulted in a correct assignment of 87% of FFPE samples into one of seven intrinsic subtypes identified using FF samples. Assignment to the same molecular cluster as the paired FF tissue was not correlated to time in paraffin. CONCLUSION: We are the first to examine a large cohort of paired FF and FFPE samples. We show that expression data from FFPE material can be used to assign samples to intrinsic molecular subtypes identified using FF material. This assignment allows the use of archival material, including material derived from large-randomised clinical trials, to determine the predictive and/or prognostic value of 'intrinsic glioma subtypes' on Exon arrays. This would enable clinicians to provide patients with an objective and accurate diagnosis and prognosis, and a personalised treatment strategy. British Journal of Cancer (2012) 106, 538-545. doi: 10.1038/bjc.2011.547 www.bjcancer.com Published online 20 December 2011 (C) 2012 Cancer Research U

Gene expression profiles of gliomas in formalin-fixed paraffin-embedded material

Background: We have recently demonstrated that expression profiling is a more accurate and objective method to classify gliomas than histology. Similar to most expression profiling studies, our experiments were performed using fresh frozen (FF) glioma samples whereas most archival samples are fixed in formalin and embedded in paraffin (FFPE). Identification of the same, expression-based intrinsic subtypes in FFPE-stored samples would enable validation of the prognostic value of these subtypes on these archival samples. In this study, we have therefore determined whether the intrinsic subtypes identified using FF material can be reproduced in FFPE-stored samples.Methods: We have performed expression profiling on 55 paired FF-FFPE glioma samples using HU133 plus 2.0 arrays (FF) and Exon 1.0 ST arrays (FFPE). The median time in paraffin of the FFPE samples was 14.1 years (range 6.6-26.4 years). Results: In general, the correlation between FF and FFPE expression in a single sample was poor. We then selected the most variable probe sets per gene (n17 583), and of these, the 5000 most variable probe sets on FFPE expre

Immunotherapy in Glioblastoma: Current Shortcomings and Future Perspectives

Glioblastomas are aggressive, fast-growing primary brain tumors. After standard-of-care treatment with radiation in combination with temozolomide, the overall prognosis of newly diagnosed patients remains poor, with a 2-year survival rate of less than 20%. The remarkable survival benefit gained with immunotherapy in several extracranial tumor types spurred a variety of experimental intervention studies in glioblastoma patients. These ranged from immune checkpoint inhibition to vaccinations and adoptive T cell therapies. Unfortunately, almost all clinical outcomes were universally disappointing. In this perspective, we provide an overview of immune interventions performed to date in glioblastoma patients and re-evaluate their performance. We argue that shortcomings of current immune therapies in glioblastoma are related to three major determinants of resistance, namely: low immunogenicity; immune privilege of the central nervous system; and immunosuppressive micro-environment. In this perspective, we propose strategies that are guided by exact shortcomings to sensitize glioblastoma prior to treatment with therapies that enhance numbers and/or activation state of CD8 T cells

Isotype-specific activation of cystic fibrosis transmembrane conductance regulator-chloride channels by cGMP-dependent protein kinase II

Type II cGMP-dependent protein kinase (cGKII) isolated from pig intestinal brush borders and type I alpha cGK (cGKI) purified from bovine lung were compared for their ability to activate the cystic fibrosis transmembrane conductance regulator (CFTR)-Cl- channel in excised, inside-out membrane patches from NIH-3T3 fibroblasts and from a rat intestinal cell line (IEC-CF7) stably expressing recombinant CFTR. In both cell models, in the presence of cGMP and ATP, cGKII was found to mimic the effect of the catalytic subunit of cAMP-dependent protein kinase (cAK) on opening CFTR-Cl-channels, albeit with different kinetics (2-3-min lag time, reduced rate of activation). By contrast, cGKI or a monomeric cGKI catalytic fragment was incapable of opening CFTR-Cl- channels and also failed to potentiate cGKII activation of the channels. The cAK activation but not the cGKII activation was blocked by a cAK inhibitor peptide. The slow activation by cGKII could not be ascribed to counteracting protein phosphatases, since neither calyculin A, a potent inhibitor of phosphatase 1 and 2A, nor ATP gamma S (adenosine 5'-O-(thiotriphosphate)), producing stable thiophosphorylation, was able to enhance the activation kinetics. Channels preactivated by cGKII closed instantaneously upon removal of ATP and kinase but reopened in the presence of ATP alone. Paradoxically, immunoprecipitated CFTR or CF-2, a cloned R domain fragment of CFTR (amino acids 645-835) could be phosphorylated to a similar extent with only minor kinetic differences by both isotypes of cGK. Phosphopeptide maps of CF-2 and CFTR, however, revealed very subtle differences in site-specificity between the cGK isoforms. These results indicate that cGKII, in contrast to cGKI alpha, is a potential activator of chloride transport in CFTR-expressing cell types