Sensitivity Analysis of the MGMT-STP27 Model and Impact of Genetic and Epigenetic Context to Predict the MGMT Methylation Status in Gliomas and Other Tumors.

The methylation status of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene is an important predictive biomarker for benefit from alkylating agent therapy in glioblastoma. Our model MGMT-STP27 allows prediction of the methylation status of the MGMT promoter using data from the Illumina's Human Methylation BeadChips (HM-27K and HM-450K) that is publically available for many cancer data sets. Here, we investigate the impact of the context of genetic and epigenetic alterations and tumor type on the classification and report on technical aspects, such as robustness of cutoff definition and preprocessing of the data. The association between gene copy number variation, predicted MGMT methylation, and MGMT expression revealed a gene dosage effect on MGMT expression in lower grade glioma (World Health Organization grade II/III) that in contrast to glioblastoma usually carry two copies of chromosome 10 on which MGMT resides (10q26.3). This implies some MGMT expression, potentially conferring residual repair function blunting the therapeutic effect of alkylating agents. A sensitivity analyses corroborated the performance of the original cutoff for various optimization criteria and for most data preprocessing methods. Finally, we propose an R package mgmtstp27 that allows prediction of the methylation status of the MGMT promoter and calculation of appropriate confidence and/or prediction intervals. Overall, MGMT-STP27 is a robust model for MGMT classification that is independent of tumor type and is adapted for single sample prediction

PD-1⁺ Tcf1⁺ CD8⁺ T cells from established chronic infection can form memory while retaining a stableimprint of persistent antigen exposure.

Virus-specific PD1 <sup>+</sup> Tcf1 <sup>+</sup> memory-like CD8 <sup>+</sup> T cells (T <sub>ML</sub> s) maintain the CD8 <sup>+</sup> T cell response during chronic viral infection. However, the fate of these cells following cessation of persistent antigen exposure has been unclear. Here, we find that T <sub>ML</sub> s persist upon transfer into antigen-free hosts and form memory following recall stimulation. Phenotypic, functional, and transcriptome analyses show that T <sub>ML</sub> -derived memory cells resemble those arising in response to acute, resolved infection, but they retain features of chronically stimulated cells, including elevated PD-1 and Tox and reduced cytokine expression. This chronic infection imprint is largely accounted for by constitutive Tox expression. Virus-specific Tcf1 <sup>+</sup> CD8 <sup>+</sup> T cells that persist after clearance of systemic infection also display a chronic infection imprint. Notwithstanding, renewed virus exposure induces a recall response, which controls virus infection in part. Thus, cessation of chronic antigen exposure yields a memory CD8 <sup>+</sup> T cell compartment that reflects prior stimulation

Characterization of the blood-brain barrier in genetically diverse laboratory mouse strains.

Genetic variation in a population has an influence on the manifestation of monogenic as well as multifactorial disorders, with the underlying genetic contribution dependent on several interacting variants. Common laboratory mouse strains used for modelling human disease lack the genetic variability of the human population. Therefore, outcomes of rodent studies show limited relevance to human disease. The functionality of brain vasculature is an important modifier of brain diseases. Importantly, the restrictive interface between blood and brain-the blood-brain barrier (BBB) serves as a major obstacle for the drug delivery into the central nervous system (CNS). Using genetically diverse mouse strains, we aimed to investigate the phenotypic and transcriptomic variation of the healthy BBB in different inbred mouse strains. We investigated the heterogeneity of brain vasculature in recently wild-derived mouse strains (CAST/EiJ, WSB/EiJ, PWK/PhJ) and long-inbred mouse strains (129S1/SvImJ, A/J, C57BL/6J, DBA/2J, NOD/ShiLtJ) using different phenotypic arms. We used immunohistochemistry and confocal laser microscopy followed by quantitative image analysis to determine vascular density and pericyte coverage in two brain regions-cortex and hippocampus. Using a low molecular weight fluorescence tracer, sodium fluorescein and spectrophotometry analysis, we assessed BBB permeability in young and aged mice of selected strains. For further phenotypic characterization of endothelial cells in inbred mouse strains, we performed bulk RNA sequencing of sorted endothelial cells isolated from cortex and hippocampus. Cortical vessel density and pericyte coverage did not differ among the investigated strains, except in the cortex, where PWK/PhJ showed lower vessel density compared to NOD/ShiLtJ, and a higher pericyte coverage than DBA/2J. The vascular density in the hippocampus differed among analyzed strains but not the pericyte coverage. The staining patterns of endothelial arteriovenous zonation markers were similar in different strains. BBB permeability to a small fluorescent tracer, sodium fluorescein, was also similar in different strains, except in the hippocampus where the CAST/EiJ showed higher permeability than NOD/ShiLtJ. Transcriptomic analysis of endothelial cells revealed that sex of the animal was a major determinant of gene expression differences. In addition, the expression level of several genes implicated in endothelial function and BBB biology differed between wild-derived and long-inbred mouse strains. In aged mice of three investigated strains (DBA/2J, A/J, C57BL/6J) vascular density and pericyte coverage did not change-expect for DBA/2J, whereas vascular permeability to sodium fluorescein increased in all three strains. Our analysis shows that although there were no major differences in parenchymal vascular morphology and paracellular BBB permeability for small molecular weight tracer between investigated mouse strains or sexes, transcriptomic differences of brain endothelial cells point to variation in gene expression of the intact BBB. These baseline variances might be confounding factors in pathological conditions that may lead to a differential functional outcome dependent on the sex or genetic polymorphism

Rgtsp: a generalized top scoring pairs package for class prediction.

SUMMARY: A top scoring pair (TSP) classifier consists of a pair of variables whose relative ordering can be used for accurately predicting the class label of a sample. This classification rule has the advantage of being easily interpretable and more robust against technical variations in data, as those due to different microarray platforms. Here we describe a parallel implementation of this classifier which significantly reduces the training time, and a number of extensions, including a multi-class approach, which has the potential of improving the classification performance. AVAILABILITY AND IMPLEMENTATION: Full C++ source code and R package Rgtsp are freely available from http://lausanne.isb-sib.ch/~vpopovic/research/. The implementation relies on existing OpenMP libraries

Prognosis of stage II and III colon cancer treated with adjuvant 5-fluorouracil or FOLFIRI in relation to microsatellite status: results of the PETACC-3 trial†

We examined the effect of MSI status in a trial-based cohort of 1254 stage II and III colon cancer (CC) patients treated with 5-FU/LV or FOLFIRI. We confirm that for stage II CC MSI is strongly prognostic for RFS and OS. No significant differences were found between treatment arms. Adding irinotecan to 5-FU did not benefit MSI-H tumors. RFS was better for MSI-H CC both in the right and left colo

BET inhibitors repress expression of interferon-stimulated genes and synergize with HDAC inhibitors in glioblastoma.

The development of rational combination therapies is key to overcome inherent treatment resistance of glioblastoma (GBM). We aim at identifying new druggable targets by disturbing GBM cells with inhibitors of bromodomain and extra-terminal motif (BET) proteins to reveal cancer-relevant vulnerabilities that may sensitize to a second drug. BET proteins are epigenetic modulators and have been associated with proto-oncogene overexpression in cancer. A GBM-derived sphere-line was treated with the BET inhibitor (BETi) JQ1 over a time-course of 48 hours, followed by RNA-sequencing. Four chromatin marks were investigated by chromatin immunoprecipitation followed by sequencing (ChIP-seq). Signatures of interest were functionally validated in vitro and in orthotopic xenografts. Combination therapies were evaluated for synergistic effects. Cancer-relevant pathways significantly modulated by JQ1 comprised interferon alpha (IFN-α) response genes and response signatures to histone deacetylase inhibitors (HDACi). The IFN-signature was reminiscent of a GBM-derived IFN-signature comprising CD274 (PD-L1). Functional pathway analysis suggested that JQ1 was acting directly on the transcriptional level of IFN-response genes and not via the canonical JAK/STAT pathway. This was in line with JQ1 modulated expression and BRD4 and Pol II occupancy at IFN-signature genes, supporting a direct mechanistic interaction. Finally, we showed that combining HDACi with JQ1 acts synergistically in reducing cell viability of GS-lines. Our approach identified BETi-induced vulnerabilities in cancer-relevant pathways, potentially amenable to synergistic combinatorial therapy, such as combination with HDACi. The direct inhibitory effect of BETi on IFN-responsive genes in GBM cells, including CD274, indicates modulation of the tumor immune landscape and warrants further studies

DNA Topoisomerase I Gene Copy Number and mRNA Expression Assessed as Predictive Biomarkers for Adjuvant Irinotecan in Stage II/III Colon Cancer.

PURPOSE: Prospective-retrospective assessment of theTOP1gene copy number andTOP1mRNA expression as predictive biomarkers for adjuvant irinotecan in stage II/III colon cancer. EXPERIMENTAL DESIGN: Formalin-fixed, paraffin-embedded tissue microarrays were obtained from an adjuvant colon cancer trial (PETACC3) where patients were randomized to 5-fluorouracil/folinic acid with or without additional irinotecan.TOP1copy number status was analyzed by fluorescencein situhybridization (FISH) using aTOP1/CEN20 dual-probe combination.TOP1mRNA data were available from previous analyses. RESULTS: TOP1FISH and follow-up data were obtained from 534 patients.TOP1gain was identified in 27% using a single-probe enumeration strategy (≥4TOP1signals per cell) and in 31% when defined by aTOP1/CEN20 ratio ≥ 1.5. The effect of additional irinotecan was not dependent onTOP1FISH status.TOP1mRNA data were available from 580 patients with stage III disease. Benefit of irinotecan was restricted to patients characterized byTOP1mRNA expression ≥ third quartile (RFS: HRadjusted, 0.59;P= 0.09; OS: HRadjusted, 0.44;P= 0.03). The treatment byTOP1mRNA interaction was not statistically significant, but in exploratory multivariable fractional polynomial interaction analysis, increasingTOP1mRNA values appeared to be associated with increasing benefit of irinotecan. CONCLUSIONS: In contrast to theTOP1copy number, a trend was demonstrated for a predictive property ofTOP1mRNA expression. On the basis ofTOP1mRNA, it might be possible to identify a subgroup of patients where an irinotecan doublet is a clinically relevant option in the adjuvant setting of colon cancer.Clin Cancer Res; 22(7); 1621-31. ©2015 AACR

miR-345 in metastatic colorectal cancer: a non-invasive biomarker for clinical outcome in non-KRAS mutant patients treated with 3rd line cetuximab and irinotecan.

INTRODUCTION: MicroRNAs (miRNAs) have important regulatory functions in cellular processes and have shown promising potential as prognostic markers for disease outcome in patients with cancer. The aim of the present study was to find miRNA expression profiles in whole blood that were prognostic for overall survival (OS) in patients with metastatic colorectal cancer (mCRC) treated with cetuximab and irinotecan. METHODS: From 138 patients with mCRC in 3rd line therapy with cetuximab and irinotecan in a prospective phase II study, 738 pretreatment miRNAs were isolated and profiled from whole blood using the TaqMan MicroRNA Array v2.0. Mutation status of KRAS, BRAF, and PI3KCA was known. RESULTS: After Bonferroni adjustment, 6 miRNAs: (miR-345, miR-143, miR-34a*, miR-628-5p, miR-886-3p and miR-324-3p), were found associated with short OS. miR-345 was the strongest prognostic miRNA, significant in the full cohort and in the non-KRAS mutant population. miR-345, as a continuous variable in the full cohort, resulted in a hazard ratio (HR) of 2.38 per IQR (CI 95%: 1.8-3.1, P-value = 2.86e-07, Bonferroni adjusted, univariable analysis) and a HR = 1.75 per IQR (CI 95%: 1.24-2.48, P-Wald = 1.45e-03) in the multivariable analysis adjusted for gender, age, KRAS, PI3KCA and performance status. miR-345 was prognostic in progression-free survival (PFS) with a HR = 1.63 per IQR (CI 95%: 1.25-2.114, P-Wald = 2.92e-4) in the multivariable analysis. In addition, high miR-345 expression was associated with lack of response to treatment with cetuximab and irinotecan. CONCLUSION: We identified miR-345 in whole blood as a potential biomarker for clinical outcome. MiR-345 was a single prognostic biomarker for both OS and PFS in all patients and also in the non-KRAS mutant population

Long-lasting stem cell-like memory CD8+ T cells with a naïve-like profile upon yellow fever vaccination.

Efficient and persisting immune memory is essential for long-term protection from infectious and malignant diseases. The yellow fever (YF) vaccine is a live attenuated virus that mediates lifelong protection, with recent studies showing that the CD8(+) T cell response is particularly robust. Yet, limited data exist regarding the long-term CD8(+) T cell response, with no studies beyond 5 years after vaccination. We investigated 41 vaccinees, spanning 0.27 to 35 years after vaccination. YF-specific CD8(+) T cells were readily detected in almost all donors (38 of 41), with frequencies decreasing with time. As previously described, effector cells dominated the response early after vaccination. We detected a population of naïve-like YF-specific CD8(+) T cells that was stably maintained for more than 25 years and was capable of self-renewal ex vivo. In-depth analyses of markers and genome-wide mRNA profiling showed that naïve-like YF-specific CD8(+) T cells in vaccinees (i) were distinct from genuine naïve cells in unvaccinated donors, (ii) resembled the recently described stem cell-like memory subset (Tscm), and (iii) among all differentiated subsets, had profiles closest to naïve cells. Our findings reveal that CD8(+) Tscm are efficiently induced by a vaccine in humans, persist for decades, and preserve a naïveness-like profile. These data support YF vaccination as an optimal mechanistic model for the study of long-lasting memory CD8(+) T cells in humans

Caloric dose-responsive genes in blood cells differentiate the metabolic status of obese men.

We have investigated the postprandial transcriptional response of blood cells to increasing caloric doses of a meal challenge to test whether the dynamic response of the human organism to the ingestion of food is dependent on metabolic health. The randomized crossover study included seven normal weight and seven obese men consuming three doses (500/1000/1500 kcal) of a high-fat meal. The blood cell transcriptome was measured before and 2, 4, and 6 h after meal ingestion (168 samples). We applied univariate and multivariate statistics to investigate differentially expressed genes in both study groups. We identified 624 probe sets that were up- or down-regulated after the caloric challenge in a dose-dependent manner. These transcripts were most responsive to the 1500 kcal challenge in the obese group and were associated with postprandial insulin and oxidative phosphorylation. Furthermore, the data revealed a separation of the obese group into individuals whose response was close to the normal weight group and individuals with a transcriptional response indicative of a loss of metabolic flexibility. The molecular signature provided by the postprandial transcriptomic response of blood cells to increasing caloric doses of a high-fat meal challenge may represent a sensitive way to evaluate the qualitative impact of food on human health