Like uveal melanomas, primary leptomeningeal melanocytic neoplasms (LMNs) frequently carry GNAQ and GNA11 mutations. However, it is currently unknown whether these LMNs harbor mutations in BAP1, SF3B1 and/or EIF1AX like uveal melanomas as well. In this study, we used Sanger sequencing for the detection of mutations in SF3B1 (hotspots in exon 14 and 15) and EIF1AX (exon 1 and 2 and flanking intronic regions) in a series of 24 primary LMNs. Additionally, BAP1 immunohistochemistry was used as a surrogate marker for the detection of inactivating mutations in the BAP1 gene.

PSMB5 mutations and upregulation of the β5 subunit of the proteasome represent key determinants of acquired resistance to the proteasome inhibitor bortezomib (BTZ) in leukemic cells in vitro. We here undertook a multi-modality (DNA, mRNA, miRNA) array-based analysis of human CCRF-CEM leukemia cells and BTZ-resistant subclones to determine whether or not complementary mechanisms contribute to BTZ resistance. These studies revealed signatures of markedly reduced expression of proteolytic stress related genes in drug resistant cells over a broad range of BTZ concentrations along with a high upregulation of myristoylated alanine-rich C-kinase substrate (MARCKS) gene expression. MARCKS upregulation was confirmed on protein level and also observed in other BTZ-resistant tumor cell lines as well as in leukemia cells with acquired resistance to other proteasome inhibitors. Moreover, when MARCKS protein expression was demonstrated in specimens derived from therapy-refractory pediatric leukemia patients (n = 44), higher MARCKS protein expression trended (p = 0.073) towards a dismal response to BTZ-containing chemotherapy. Mechanistically, we show a BTZ concentration-dependent association of MARCKS protein levels with the emergence of ubiquitin-containing vesicles in BTZ-resistant CEM cells. These vesicles were found to be extruded and taken up in co-cultures with proteasome-proficient acceptor cells. Consistent with these observations, MARCKS protein associated with ubiquitin-containing vesicles was also more prominent in clinical leukemic specimen with ex vivo BTZ resistance compared to BTZ-sensitive leukemia cells. Collectively, we propose a role for MARCKS in a novel mechanism of BTZ resistance via exocytosis of ubiquitinated proteins in BTZ-resistant cells leading to quenching of proteolytic stress.

Mucinous carcinoma (MC) is found in 10%-15% of colorectal cancer (CRC) patients. It differs from the common adenocarcinoma (AC) in histopathological appearance and clinical behavior.

Cancer is caused by somatic DNA alterations such as gene point mutations, DNA copy number aberrations (CNA) and structural variants (SVs). Genome-wide analyses of SVs in large sample series with well-documented clinical information are still scarce. Consequently, the impact of SVs on carcinogenesis and patient outcome remains poorly understood. This study aimed to perform a systematic analysis of genes that are affected by CNA-associated chromosomal breaks in colorectal cancer (CRC) and to determine the clinical relevance of recurrent breakpoint genes.

MET has gained interest as a therapeutic target for a number of malignancies because of its involvement in tumorigenesis, invasion and metastasis. At present, a number of inhibitors, both antibodies against MET or its ligand hepatocyte growth factor, and small molecule MET tyrosine kinase inhibitors are in clinical trials. We here describe a novel variant of MET that is expressed in 6% of high-grade gliomas. Characterization of this mutation in a glioma cell line revealed that it consists of an intronic deletion, resulting in a splice event connecting an intact splice donor site in exon 6 with the next splice acceptor site being that of exon 9. The encoded protein lacks parts of the extracellular IPT domains 1 and 2, encoded by exons 7 and 8, resulting in a novel pseudo-IPT and is named MET(Δ7-8). MET(Δ7-8) is located predominantly in the cytosol and is constitutively active. The auto-activating nature of MET(Δ7-8), in combination with a lack of transmembrane localization, renders MET(Δ7-8) not targetable using antibodies, although the protein is efficiently deactivated by MET-specific tyrosine kinase inhibitors. Testing of MET-expressing tumors for the presence of this variant may be important for treatment decision making.

Osteosarcoma is an aggressive bone tumor that preferentially develops in adolescents. The tumor is characterized by an abundance of genomic aberrations, which hampers the identification of the driver genes involved in osteosarcoma tumorigenesis. Our study aims to identify these genes by the investigation of focal copy number aberrations (CNAs, <3 Mb). For this purpose, we subjected 26 primary tumors of osteosarcoma patients to high-resolution single nucleotide polymorphism array analyses and identified 139 somatic focal CNAs. Of these, 72 had at least one gene located within or overlapping the focal CNA, with a total of 94 genes. For 84 of these genes, the expression status in 31 osteosarcoma samples was determined by expression microarray analysis. This enabled us to identify the genes of which the over- or underexpression was in more than 35% of cases in accordance to their copy number status (gain or loss). These candidate genes were subsequently validated in an independent set and furthermore corroborated as driver genes by verifying their role in other tumor types. We identified CMTM8 as a new candidate tumor suppressor gene and GPR177 as a new candidate oncogene in osteosarcoma. In osteosarcoma, CMTM8 has been shown to suppress EGFR signaling. In other tumor types, CMTM8 is known to suppress the activity of the oncogenic protein c-Met and GPR177 is known as an overexpressed upstream regulator of the Wnt-pathway. Further studies are needed to determine whether these proteins also exert the latter functions in osteosarcoma tumorigenesis.

Increased copy number alterations (CNAs) indicative of chromosomal instability (CIN) have been associated with poor cancer outcome. Here, we study CNAs as potential biomarkers of bevacizumab (BVZ) response in metastatic colorectal cancer (mCRC). We cluster 409 mCRCs in three subclusters characterized by different degrees of CIN. Tumors belonging to intermediate-to-high instability clusters have improved outcome following chemotherapy plus BVZ versus chemotherapy alone. In contrast, low instability tumors, which amongst others consist of POLE-mutated and microsatellite-instable tumors, derive no further benefit from BVZ. This is confirmed in 81 mCRC tumors from the phase 2 MoMa study involving BVZ. CNA clusters overlap with CRC consensus molecular subtypes (CMS); CMS2/4 xenografts correspond to intermediate-to-high instability clusters and respond to FOLFOX chemotherapy plus mouse avastin (B20), while CMS1/3 xenografts match with low instability clusters and fail to respond. Overall, we identify copy number load as a novel potential predictive biomarker of BVZ combination therapy.

Cognitive deficits occur frequently in diffuse glioma patients, but are limitedly understood. An important marker for survival in these patients is isocitrate dehydrogenase (IDH) mutation (IDH-mut). Patients with IDH-mut glioma have a better prognosis but more often suffer from epilepsy than patients with IDH-wildtype (IDH-wt) glioma, who are generally older and more often have cognitive deficits. We investigated whether global brain functional connectivity differs between patients with IDH-mut and IDH-wt glioma, and whether this measure reflects variations in cognitive functioning in these subpopulations beyond the associated differences in age and presence of epilepsy.

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.

First line chemotherapy is effective in 75 to 80% of patients with metastatic colorectal cancer (mCRC). We studied whether microRNA (miR) expression profiles can predict treatment outcome for first line fluoropyrimidine containing systemic therapy in patients with mCRC.

Skeletal integrity in humans and animals is maintained by daily mechanical loading. It has been widely accepted that osteocytes function as mechanosensors. Many biochemical signaling molecules are involved in the response of osteocytes to mechanical stimulation. The aim of this study was to identify genes involved in the translation of mechanical stimuli into bone formation. The four-point bending model was used to induce a single period of mechanical loading on the right tibia, while the contra lateral left tibia served as control. Six hours after loading, the effects of mechanical loading on gene-expression were determined with microarray analysis. Protein expression of differentially regulated genes was evaluated with immunohistochemistry. Nine genes were found to exhibit a significant differential gene expression in LOAD compared to control. MEPE, Garnl1, V2R2B, and QFG-TN1 olfactory receptor were up-regulated, and creatine kinase (muscle form), fibrinogen-B beta-polypeptide, monoamine oxidase A, troponin-C and kinesin light chain-C were down-regulated. Validation with real-time RT-PCR analysis confirmed the up-regulation of MEPE and the down-regulation of creatine kinase (muscle form) and troponin-C in the loaded tibia. Immunohistochemistry showed that the increase of MEPE protein expression was already detectable six hours after mechanical loading. In conclusion, these genes probably play a role during translation of mechanical stimuli six hours after mechanical loading. The modulation of MEPE expression may indicate a connection between bone mineralization and bone formation after mechanical stimulation.

The disease course of patients with diffuse low-grade glioma is notoriously unpredictable. Temporal and spatially distinct samples may provide insight into the evolution of clinically relevant copy number aberrations (CNAs). The purpose of this study is to identify CNAs that are indicative of aggressive tumor behavior and can thereby complement the prognostically favorable 1p/19q co-deletion.

Chromosomal instability is a hallmark of cancer, but mitotic regulators are rarely mutated in tumors. Mutations in the condensin complexes, which restructure chromosomes to facilitate segregation during mitosis, are significantly enriched in cancer genomes, but experimental evidence implicating condensin dysfunction in tumorigenesis is lacking. We report that mice inheriting missense mutations in a condensin II subunit (Caph2nes) develop T-cell lymphoma. Before tumors develop, we found that the same Caph2 mutation impairs ploidy maintenance to a different extent in different hematopoietic cell types, with ploidy most severely perturbed at the CD4+CD8+ T-cell stage from which tumors initiate. Premalignant CD4+CD8+ T cells show persistent catenations during chromosome segregation, triggering DNA damage in diploid daughter cells and elevated ploidy. Genome sequencing revealed that Caph2 single-mutant tumors are near diploid but carry deletions spanning tumor suppressor genes, whereas P53 inactivation allowed Caph2 mutant cells with whole-chromosome gains and structural rearrangements to form highly aggressive disease. Together, our data challenge the view that mitotic chromosome formation is an invariant process during development and provide evidence that defective mitotic chromosome structure can promote tumorigenesis.

Development of cancer is driven by somatic alterations, including numerical and structural chromosomal aberrations. Currently, several computational methods are available and are widely applied to detect numerical copy number aberrations (CNAs) of chromosomal segments in tumor genomes. However, there is lack of computational methods that systematically detect structural chromosomal aberrations by virtue of the genomic location of CNA-associated chromosomal breaks and identify genes that appear non-randomly affected by chromosomal breakpoints across (large) series of tumor samples. 'GeneBreak' is developed to systematically identify genes recurrently affected by the genomic location of chromosomal CNA-associated breaks by a genome-wide approach, which can be applied to DNA copy number data obtained by array-Comparative Genomic Hybridization (CGH) or by (low-pass) whole genome sequencing (WGS). First, 'GeneBreak' collects the genomic locations of chromosomal CNA-associated breaks that were previously pinpointed by the segmentation algorithm that was applied to obtain CNA profiles. Next, a tailored annotation approach for breakpoint-to-gene mapping is implemented. Finally, dedicated cohort-based statistics is incorporated with correction for covariates that influence the probability to be a breakpoint gene. In addition, multiple testing correction is integrated to reveal recurrent breakpoint events. This easy-to-use algorithm, 'GeneBreak', is implemented in R ( www.cran.r-project.org) and is available from Bioconductor ( www.bioconductor.org/packages/release/bioc/html/GeneBreak.html).

Three-dimensional visualization of the brain vasculature and its interactions with surrounding cells may shed light on diseases where aberrant microvascular organization is involved, including glioblastoma (GBM). Intravital confocal imaging allows 3D visualization of microvascular structures and migration of cells in the brain of mice, however, with limited imaging depth. To enable comprehensive analysis of GBM and the brain microenvironment, in-depth 3D imaging methods are needed. Here, we employed methods for optical tissue clearing prior to 3D microscopy to visualize the brain microvasculature and routes of invasion of GBM cells.

Although rare, mucoepidermoid carcinoma (MEC) is one of the most common malignant salivary gland tumors. The presence of the t(11;19)(q21;p13) translocation in a subset of MECs has raised interest in genomic aberrations in MEC. In the present study we conducted genome-wide copy-number-aberration analysis by micro-array comparative-genomic-hybridization on 27 MEC samples. Low/intermediate-grade MECs had significantly fewer copy-number-aberrations compared to high-grade MECs (low vs high: 3.48 vs 30; p = 0.0025; intermediate vs high: 5.7 vs 34.5; p = 0.036). The translocation-negative MECs contained more copy-number-aberrations than translocation-positive MECs (average amount of aberrations 15.9 vs 2.41; p =0.04). Within all 27 MEC samples, 16p11.2 and several regions on 8q were the most frequently gained regions , while 1q23.3 was the most frequently detected loss. Low/intermediate-grade MEC samples had copy-number-aberrations in chromosomes 1, 12 and 16, while high-grade MECs had a copy-number-aberration in 8p. The most commonly observed copy-number-aberration was the deletion of 3p14.1, which was observed in 4 of the translocation-negative MEC samples. No recurrent copy-number-aberrations were found in translocation-positive MEC samples. Based on these results, we conclude that MECs may be classified as follows: (i) t(11;19)(q21;p13) translocation-positive tumors with no or few chromosomal aberrations and (ii) translocation-negative tumors with multiple chromosomal aberrations.

Diversity in colorectal cancer biology is associated with variable responses to standard chemotherapy. We aimed to identify and validate DNA hypermethylated genes as predictive biomarkers for irinotecan treatment of metastatic CRC patients. Candidate genes were selected from 389 genes involved in DNA Damage Repair by correlation analyses between gene methylation status and drug response in 32 cell lines. A large series of samples (n=818) from two phase III clinical trials was used to evaluate these candidate genes by correlating methylation status to progression-free survival after treatment with first-line single-agent fluorouracil (Capecitabine or 5-fluorouracil) or combination chemotherapy (Capecitabine or 5-fluorouracil plus irinotecan (CAPIRI/FOLFIRI)). In the discovery (n=185) and initial validation set (n=166), patients with methylated Decoy Receptor 1 (DCR1) did not benefit from CAPIRI over Capecitabine treatment (discovery set: HR=1.2 (95%CI 0.7-1.9, p=0.6), validation set: HR=0.9 (95%CI 0.6-1.4, p=0.5)), whereas patients with unmethylated DCR1 did (discovery set: HR=0.4 (95%CI 0.3-0.6, p=0.00001), validation set: HR=0.5 (95%CI 0.3-0.7, p=0.0008)). These results could not be replicated in the external data set (n=467), where a similar effect size was found in patients with methylated and unmethylated DCR1 for FOLFIRI over 5FU treatment (methylated DCR1: HR=0.7 (95%CI 0.5-0.9, p=0.01), unmethylated DCR1: HR=0.8 (95%CI 0.6-1.2, p=0.4)). In conclusion, DCR1 promoter hypermethylation status is a potential predictive biomarker for response to treatment with irinotecan, when combined with capecitabine. This finding could not be replicated in an external validation set, in which irinotecan was combined with 5FU. These results underline the challenge and importance of extensive clinical evaluation of candidate biomarkers in multiple trials.

Colorectal cancer develops in a multi-step manner from normal epithelium, through a pre-malignant lesion (so-called adenoma), into a malignant lesion (carcinoma), which invades surrounding tissues and eventually can spread systemically (metastasis). It is estimated that only about 5% of adenomas do progress to a carcinoma.

Because of the distinct clinical presentation of early and advanced stage ovarian cancer, we aim to clarify whether these disease entities are solely separated by time of diagnosis or whether they arise from distinct molecular events.

With the improvement of therapeutic options for the treatment of breast cancer, the development of brain metastases has become a major limitation to life expectancy in many patients. Therefore, our aim was to identify molecular markers associated with the development of brain metastases in breast cancer.