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Prevalence of gains and losses according to histological features

List of genes in MLPA probe mixes

About 5% of colorectal adenomas are estimated to progress to colorectal cancer. However, it is important to identify which adenomas actually carry a high risk of progression, because these serve as intermediate endpoints, for example, in screening programs. In clinical practice, adenomas with a size of ≥10 mm, villous component and/or high-grade dysplasia, called advanced adenomas, are considered high risk, although solid evidence for this classification is lacking. Specific DNA copy number changes are associated with adenoma-to-carcinoma progression. We set out to determine the prevalence of cancer-associated events (CAE) in advanced and nonadvanced adenomas. DNA copy number analysis was performed on archival tissues from three independent series of, in total, 297 adenomas (120 nonadvanced and 177 advanced) using multiplex ligation-dependent probe amplification or low-coverage whole-genome DNA sequencing. Alterations in two or more CAEs were considered to mark adenomas as high risk. Two or more CAEs were overall present in 25% (95% CI, 19.0–31.8) of advanced adenomas; 23% (11/48), 36% (12/33), and 23% (22/96) of the advanced adenomas in series 1, 2, and 3, respectively, and 1.7% (1/58) and 4.8% (3/62) of the nonadvanced adenomas, in series 1 and 2, respectively. The majority of advanced adenomas do not show CAEs, indicating that only a subset of these lesions is to be considered high risk. Nonadvanced adenomas have very low prevalence of CAEs, although those with CAEs should be considered high risk as well. Specific DNA copy number alterations may better reflect the true progression risk than the advanced adenoma phenotype. Cancer Prev Res; 11(7); 403–12. ©2018 AACR.

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Purpose: Early detection of colorectal cancer (CRC) and its precursor lesions is an effective approach to reduce CRC mortality rates. This study aimed to identify novel protein biomarkers for the early diagnosis of CRC.Experimental Design: Proximal fluids are a rich source of candidate biomarkers as they contain high concentrations of tissue-derived proteins. The FabplCre;Apc15lox/+ mouse model represents early-stage development of human sporadic CRC. Proximal fluids were collected from normal colon and colon tumors and subjected to in-depth proteome profiling by tandem mass spectrometry. Carcinoembryonic antigen (CEA) and CHI3L1 human serum protein levels were determined by ELISA.Results: Of the 2,172 proteins identified, quantitative comparison revealed 192 proteins that were significantly (P < 0.05) and abundantly (>5-fold) more excreted by tumors than by controls. Further selection for biomarkers with highest specificity and sensitivity yielded 52 candidates, including S100A9, MCM4, and four other proteins that have been proposed as candidate biomarkers for human CRC screening or surveillance, supporting the validity of our approach. For CHI3L1, we verified that protein levels were significantly increased in sera from patients with adenomas and advanced adenomas compared with control individuals, in contrast to the CRC biomarker CEA.Conclusion: These data show that proximal fluid proteome profiling with a mouse tumor model is a powerful approach to identify candidate biomarkers for early diagnosis of human cancer, exemplified by increased CHI3L1 protein levels in sera from patients with CRC precursor lesions. Clin Cancer Res; 18(9); 2613–24. ©2012 AACR.

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Aim: To assess the potential of biomarker triage testing (BM-TT) in the Dutch colorectal cancer (CRC) screening program. Materials & methods: Using the Adenoma and Serrated pathway to Colorectal CAncer model, we simulated fecal immunochemical test (FIT) 47 -screening and various FIT plus BM-TT screening scenarios in which only individuals with both a positive FIT and BM-TT are referred to colonoscopy. Results: Adding a low polyp sensitivity BM-TT to FIT-screening reduced colonoscopy burden (89–100%) while increasing CRC mortality (27–41%) compared with FIT 47 -screening only. The FIT plus high polyp sensitivity BM-TT scenarios also decreased colonoscopy burden (71–89%) while hardly affecting CRC mortality (FIT 47 0–4% increase, FIT 15 2–7% decrease). Conclusion: Adding a BM-TT to FIT-screening considerably reduces colonoscopy burden, but could also decrease screening effectiveness. Combining FIT 15 with a high polyp sensitivity BM-TT seems most promising.

Screening to detect colorectal cancer (CRC) in an early or premalignant state is an effective method to reduce CRC mortality rates. Current stool‐based screening tests, e.g. fecal immunochemical test (FIT), have a suboptimal sensitivity for colorectal adenomas and difficulty distinguishing adenomas at high risk of progressing to cancer from those at lower risk. We aimed to identify stool protein biomarker panels that can be used for the early detection of high‐risk adenomas and CRC. Proteomics data (LC–MS/MS) were collected on stool samples from adenoma (n = 71) and CRC patients (n = 81) as well as controls (n = 129). Colorectal adenoma tissue samples were characterized by low‐coverage whole‐genome sequencing to determine their risk of progression based on specific DNA copy number changes. Proteomics data were used for logistic regression modeling to establish protein biomarker panels. In total, 15 of the adenomas (15.8%) were defined as high risk of progressing to cancer. A protein panel, consisting of haptoglobin (Hp), LAMP1, SYNE2, and ANXA6, was identified for the detection of high‐risk adenomas (sensitivity of 53% at specificity of 95%). Two panels, one consisting of Hp and LRG1 and one of Hp, LRG1, RBP4, and FN1, were identified for high‐risk adenomas and CRCs detection (sensitivity of 66% and 62%, respectively, at specificity of 95%). Validation of Hp as a biomarker for high‐risk adenomas and CRCs was performed using an antibody‐based assay in FIT samples from a subset of individuals from the discovery series (n = 158) and an independent validation series (n = 795). Hp protein was significantly more abundant in high‐risk adenoma FIT samples compared to controls in the discovery (p = 0.036) and the validation series (p = 9e‐5). We conclude that Hp, LAMP1, SYNE2, LRG1, RBP4, FN1, and ANXA6 may be of value as stool biomarkers for early detection of high‐risk adenomas and CRCs. © 2019 Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

The realtime follow-up of neutrino events is a promising approach to search for astrophysical neutrino sources. It has so far provided compelling evidence for a neutrino point source: the flaring gamma-ray blazar TXS 0506+056 observed in coincidence with the high-energy neutrino IceCube-170922A detected by IceCube. The detection of very-high-energy gamma rays (VHE, $\mathrm{E} > 100\,\mathrm{GeV}$) from this source helped establish the coincidence and constrained the modeling of the blazar emission at the time of the IceCube event. The four major imaging atmospheric Cherenkov telescope arrays (IACTs) - FACT, H.E.S.S., MAGIC, and VERITAS - operate an active follow-up program of target-of-opportunity observations of neutrino alerts sent by IceCube. This program has two main components. One are the observations of known gamma-ray sources around which a cluster of candidate neutrino events has been identified by IceCube (Gamma-ray Follow-Up, GFU). Second one is the follow-up of single high-energy neutrino candidate events of potential astrophysical origin such as IceCube-170922A. GFU has been recently upgraded by IceCube in collaboration with the IACT groups. We present here recent results from the IACT follow-up programs of IceCube neutrino alerts and a description of the upgraded IceCube GFU system., Presented at the 37th International Cosmic Ray Conference (ICRC 2021). See arXiv:2107.06966 for all IceCube contributions. See arXiv:2108.05257 for all H.E.S.S. contributions

Introduction: In colorectal cancer (CRC) surveillance, adenoma characteristics such as size, number of adenomas, dysplasia, and villous components, are used as indicators for risk of developing metachronous cancer and guide the surveillance interval. The current risk groups (e.g., advanced adenomas) cannot optimally distinguish high risk from low risk, which may result in suboptimal surveillance strategies. Specific DNA copy number aberrations are associated with risk of colorectal adenoma-to-carcinoma progression, but these aberrations are present in only a subset of advanced adenomas. Therefore, we hypothesize that specific DNA copy number aberrations may better predict the risk of CRC than advanced adenoma. Aim: To evaluate whether a molecularly-defined high-risk adenoma is a better risk factor for CRC than the presently used advanced adenoma. Materials and methods: DNA copy number profiles were determined, by means of low-coverage whole genome sequencing, on a series of 529 adenomas, selected from a Norwegian adenoma cohort. We retrieved detailed information on adenoma characteristics and whether adenoma patients were subsequently diagnosed with CRC or not. By univariate and multivariate regression analysis we estimated the odds ratio for association between the development of CRC and baseline presence of advanced adenoma versus presence of a molecularly-defined high-risk adenoma, respectively. Results: Molecular high-risk features were observed in 85/267 (32%) of advanced adenomas and in 27/262 (10%) of non-advanced adenomas. The odds ratio for developing metachronous CRC was 3.58 (p=2.84E-8) and 1.90 (p=0.012), when an advanced adenoma or molecular high-risk adenoma was detected at the baseline colonoscopy, respectively. In the multivariate regression analysis only advanced adenoma was as a significant risk factor for CRC. Conclusion: Molecularly-defined high-risk adenomas are associated with an increased risk of CRC. However, the advanced adenoma is a stronger predictor for risk of CRC. Citation Format: Henriette C. Jodal, Eddymurphy U. Akwiwu, Leticia G. Leon, Margriet Lemmens, Pien Delis-van Diemen, Dagmar Klotz, Meike de Wit, Remond Fijneman, Monique van Leerdam, Evelien Dekker, Manon C. W. Spaander, Gerrit A. Meijer, Michael Bretthauer, Magnus Løberg, Veerle M. H. Coupé, Mette Kalager, Beatriz Carvalho. Molecular adenoma features to predict metachronous colorectal cancer risk: A nested-case control study [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A004.

Background The fecal immunochemical test (FIT) is used in colorectal cancer (CRC) screening, yet it leaves room for improvement. Objective To develop a multitarget FIT (mtFIT) with better diagnostic performance than FIT. Design Diagnostic test accuracy study. Setting Colonoscopy-controlled series. Participants Persons (n = 1284) from a screening (n = 1038) and referral (n = 246) population were classified by their most advanced lesion (CRC [n = 47], advanced adenoma [n = 135], advanced serrated polyp [n = 30], nonadvanced adenoma [n = 250], and nonadvanced serrated polyp [n = 53]), along with control participants (n = 769). Measurements Antibody-based assays were developed and applied to leftover FIT material. Classification and regression tree (CART) analysis was applied to biomarker concentrations to identify the optimal combination for detecting advanced neoplasia. Performance of this combination, the mtFIT, was cross-validated using a leave-one-out approach and compared with FIT at equal specificity. Results The CART analysis showed a combination of hemoglobin, calprotectin, and serpin family F member 2-the mtFIT-to have a cross-validated sensitivity for advanced neoplasia of 42.9% (95% CI, 36.2% to 49.9%) versus 37.3% (CI, 30.7% to 44.2%) for FIT (P = 0.025), with equal specificity of 96.6%. In particular, cross-validated sensitivity for advanced adenomas increased from 28.1% (CI, 20.8% to 36.5%) to 37.8% (CI, 29.6% to 46.5%) (P = 0.006). On the basis of these results, early health technology assessment indicated that mtFIT-based screening could be cost-effective compared with FIT. Limitation Study population is enriched with persons from a referral population. Conclusion Compared with FIT, the mtFIT showed better diagnostic accuracy in detecting advanced neoplasia because of an increased detection of advanced adenomas. Moreover, early health technology assessment indicated that these results provide a sound basis to pursue further development of mtFIT as a future test for population-based CRC screening. A prospective screening trial is in preparation. Primary funding source Stand Up to Cancer/Dutch Cancer Society, Dutch Digestive Foundation, and HealthHolland.

The N‐Myc Downstream‐Regulated Gene 4 (NDRG4), a prominent biomarker for colorectal cancer (CRC), is specifically expressed by enteric neurons. Considering that nerves are important members of the tumor microenvironment, we here establish different Ndrg4 knockout (Ndrg4 −/−) CRC models and an indirect co‐culture of primary enteric nervous system (ENS) cells and intestinal organoids to identify whether the ENS, via NDRG4, affects intestinal tumorigenesis. Linking immunostainings and gastrointestinal motility (GI) assays, we show that the absence of Ndrg4 does not trigger any functional or morphological GI abnormalities. However, combining in vivo, in vitro, and quantitative proteomics data, we uncover that Ndrg4 knockdown is associated with enlarged intestinal adenoma development and that organoid growth is boosted by the Ndrg4 −/− ENS cell secretome, which is enriched for Nidogen‐1 (Nid1) and Fibulin‐2 (Fbln2). Moreover, NID1 and FBLN2 are expressed in enteric neurons, enhance migration capacities of CRC cells, and are enriched in human CRC secretomes. Hence, we provide evidence that the ENS, via loss of Ndrg4, is involved in colorectal pathogenesis and that ENS‐derived Nidogen‐1 and Fibulin‐2 enhance colorectal carcinogenesis., Loss of enteric neuronal N‐Myc Downstream‐Regulated Gene 4 (Ndrg4) increases the release of the extracellular matrix proteins, Nidogen‐1 and Fibulin‐2, which accelerates intestinal growth in vitro and promotes colorectal cancer progression in vivo.

Aim: Better stratification of patients with stage II and stage III colon cancer for risk of recurrence is urgently needed. The present study aimed to validate the prognostic value of CDX2 protein expression in colon cancer tissue by routine immunohistochemistry and to evaluate its performance in a head-to-head comparison with tandem mass spectrometry–based proteomics. Patient and methods: CDX2 protein expression was evaluated in 386 stage II and III primary colon cancers by immunohistochemical staining of tissue microarrays and by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis using formalin-fixed paraffin-embedded tissue sections of a matched subset of 23 recurrent and 23 non-recurrent colon cancers. Association between CDX2 expression and disease-specific survival (DSS) was investigated. Results: Low levels of CDX2 protein expression in stage II and III colon cancer as determined by immunohistochemistry was associated with poor DSS (hazard ratio [HR] = 1.97 (95% confidence interval [CI]: 1.26–3.06); p = 0.002). Based on analysis of a selected sample subset, CDX2 prognostic value was more pronounced when detected by LC-MS/MS (HR = 7.56 (95% CI: 2.49–22.95); p < 0.001) compared to detection by immunohistochemistry (HR = 1.60 (95% CI: 0.61–4.22); p = 0.34). Conclusion: This study validated CDX2 protein expression as a prognostic biomarker in stage II and III colon cancer, conform previous publications. CDX2 prognostic value appeared to be underestimated when detected by routine immunohistochemistry, probably due to the semiquantitative and subjective nature of this methodology. Quantitative analysis of CDX2 substantially improved its clinical utility as a prognostic biomarker. Therefore, development of routinely applicable quantitative assays for CDX2 expression is needed to facilitate its clinical implementation.

Exosomes are small extracellular vesicles (~100 nm) that are secreted by all cells and participate in cellular communication. Tumor-derived exosomes play a role in cancer progression by affecting the tumor microenvironment and establishing the pro-metastatic niche. Cancer cells are reported to have an increased release of exosomes, in addition to modulating their cargo. Therefore, blocking exosome secretion by cancer cells could halt cancer progression and be a promising novel drug-target for therapy. Global insight into the functional protein players of this process is key for development of a successful therapeutic strategy. To identify novel drug targets, Mass Spectrometry (MS)-based proteomics was performed on a unique sample set collected from 22 patients, comprising multiple fractions (tissue lysate, soluble secretome and extracellular vesicles) of matched normal and colorectal cancer (CRC) tissues (n=18) and adenomas (n=4). Available CPTAC phospho-proteomic CRC dataset was used to identify phospho-proteins that regulate the potential drug targets. Network clustering was performed using ClusterOne and gene ontology using BinGO (cytoscape). CD63 was measured using confocal microscopy. Exosome release using Coomassie stained gels and TSG101 expression using Western blot. Gene Ontology revealed deregulated pathways in normal versus cancer comparison that were linked to vesicle trafficking. Mainly, the ESCRT-pathway of the exosome biogenesis machinery was down-regulated in cancer while alternative proteins related to endocytosis and exosome release were up-regulated. Targeted data mining to the exosome biogenesis pathway revealed 12 up-regulated proteins (FC Citation Format: Madalena N. Monteiro, Catarina Almeida-Marques, Meike de Wit, Valerie Dusseldorp, Logan Bishop-Currey, Sander R. Piersma, Thang V. Pham, Jaco C. Knol, Hanieh Sadeghi, Gerrit Meijer, Remond J. A. Fijneman, Angelika Hausser, Irene V. Bijnsdorp, Connie R. Jimenez. Exosome pathway inhibition as therapeutic strategy in colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr CC03-01.

Purpose To mimic the perioperative microenvironment where bacterial products get in contact with colorectal cancer (CRC) cells and study its impact on protein release, we exposed six CRC cell lines to lipopolysaccharide (LPS) and investigated the effect on the secretome using in-depth mass spectrometry-based proteomics. Experimental design Cancer cell secretome was harvested in bio-duplicate after LPS treatment, and separated in EV and soluble secretome (SS) fractions. Gel-fractionated proteins were analysed by label-free nano-liquid chromatography coupled to tandem mass spectrometry. NF-κB activation, triggered upon LPS treatment, was evaluated. Results We report a CRC secretome dataset of 5601 proteins. Comparison of all LPS-treated cells with controls revealed 37 proteins with altered abundance in the SS, including RPS25; and 13 in EVs, including HMGB1. Comparing controls and LPS-treated samples per cell line, revealed 564 significant differential proteins with fold-change > 3. The LPS-induced release of RPS25 was validated by western blot. Conclusions and clinical relevance Bacterial endotoxin has minor impact on the global CRC cell line secretome yet it may alter protein release in a cell line-specific manner. This modulation might play a role in orchestrating the development of a permissive environment for CRC liver metastasis, especially through EV-communication. This article is protected by copyright. All rights reserved.

In dieser Dissertation werden Daten aus drei Jahren vom vollständig fertiggestellten IceCube-Detektor benutzt um nach Neutrinos zu suchen, die in Dunkle-Materie-Annihilationen in fünf nahegelegen Zwerggalaxien, der M31 Galaxie und dem Virgo Galaxienhaufen produziert wurden. Um die Messung durchzuführen, wurde zunächst eine Ereignis-Selektion angewandt, die es ermöglicht, die von aus der Atmosphäre stammenden Teilchen dominierte Rate der Ereignisse von zirka 100 Hz auf 0.5 mHz zu reduzieren. Danach wird eine Maximum-Likelihood-Schätzer eingesetzt um zu bestimmen ob es ein Überschuss von Neutrinos aus der Richtung der jeweiligen Quellen gibt, der mit einen Energie-Spektrum übereinstimmt das mann für Dunkle-Materie-Annihilationen erwartet. Für die M31 Galaxie und den Virgo Galaxienhaufen wurde zusätzlich zu dieser Suche nach einer Punktquelle auch eine Suche für ein erweitertes Signal durchgeführt. In allen untersuchten Fällen ist das Ergebnis der Analyse vereinbar mit einer Messung der Hintergrund-Hypothese, und daraus wurden Limits für den über die Geschwindigkeit gemittelten Wirkungsquerschnitt für Dunkle-Materie-Annihilation für verschiedene Endprodukte bestimmt. Für hohe Dunkle-Materie-Massen gibt es ein Überschuss von Neutrinos aus drei der Zwerggalaxien. Dieser Überschuss hat einen globalen p-Wert von 4.9% und ist damit nicht statistisch signifikant. Die Suche für ein erweitertes Signal von der M31 Galaxie und dem Virgo Galaxienhaufen ergab keinen Überschuss. Die Limits auf den über die Geschwindigkeit gemittelten Wirkungsquerschnitt für Dunkle-Materie-Annihilation haben sich im Vergleich zu vorherigen IceCube-Analysen signifikant verbessert, um bis zu einer Größenordnung. Diese ist teilweise auf Grund Verbesserungen für diese Analyse besonders: eine verbesserte Ereignis-Selektion, und für die Analyse ist eine Maximum-Likelihood-Schätzer eingesetzt statt eine Analyse in ein Suchfenster. In this thesis, three years worth of data from the completed IceCube detector is used to search for neutrinos produced in dark matter annihilations in five nearby dwarf galaxies, the M31 galaxy and the Virgo cluster. To do this, an event selection which was developed for this analysis is applied to the data sample to reduce the atmospheric background rate from approximately 100 Hz to less than 0.5 mHz. Then, an unbinned maximum likelihood method is used to determine whether there is an excess of neutrinos from the direction of the considered galaxies or galaxy cluster that has an energy spectrum that matches the spectrum expected from dark matter annihilations. For the M31 galaxy and the Virgo cluster an extended signal with a two-dimensional Gaussian shape and width up to 5 degrees is also considered. In all cases, the results of the analysis are compatible with the background-only hypothesis and limits are set on the velocity-averaged dark matter annihilation cross section for different annihilation channels. For high dark matter masses there is an excess of neutrinos from three of the five dwarf galaxies. This excess has a global p-value of 4.9%, so it is not statistically significant. The search for an extended emission from the direction of the M31 galaxy and the Virgo cluster also did not result in an excess: in both cases the global p-value is larger than 50%. The limits on the velocity-averaged dark matter annihilation cross section have improved significantly (up to an order of magnitude) with respect to the previous IceCube analysis considering these same targets. This is partially due to improvements to this analysis specifically: an improved event selection was used to select the final data sample and an unbinned maximum likelihood method was used for the final analysis instead of a binned analysis method.

Roadmapping in China and in Germany has different connotations, contexts, meanings and cultural backgrounds. In order to understand what is meant by roadmaps — and not to confuse roadmaps with foresight in general — a comparative analysis was jointly performed by the Beijing Research Centre for Science of Science (BRCSS) under the Beijing Academy of Science and Technology and the Fraunhofer Institute for Systems and Innovation Research (ISI). This joint project investigated several cases according to a set of criteria and compared the content. The aim of the project was mutual learning and the results revealed that the differences can be found on different levels like the understanding of the terminology (not only language), cultural understandings, the background of the innovation system and contexts of the roadmaps and the understanding of the objectives. The way the process is performed differed significantly whereas the differences in the processes themselves, the number of participants or the topic fields were rather similar. In the paper, some of the differences are discussed in context and lessons learned are explained.

This chapter focuses on the impact of flows of information through information and communication technologies (ICTs) on the decision-making processes for researchers working in instable, volatile regions. Drawing on the authors’ experiences in Nigeria and Congo-Brazzaville, the paper argues that while ICTs have become important tools for research, they also create new dilemmas for the researcher. These dilemmas emerge from the interface between us as researchers, and constant flows of information about insecurity in our fields that reach us via social media. Paradoxically, in an information rich world, a lack of information can also create such dilemmas. Flows of information – or the lack thereof – are not only data, but also affect us on an emotional level. As such new subjective fields of insecurity emerge for the researcher that produce emotional responses such as anger or disorientation, and that in turn inform decision-making processes, such as an inclination for deeper engagement, or to disengage.

About 5% of colorectal adenomas are estimated to progress to colorectal cancer. However, it is important to identify which adenomas actually carry a high risk of progression, because these serve as intermediate endpoints, for example, in screening programs. In clinical practice, adenomas with a size of ≥10 mm, villous component and/or high-grade dysplasia, called advanced adenomas, are considered high risk, although solid evidence for this classification is lacking. Specific DNA copy number changes are associated with adenoma-to-carcinoma progression. We set out to determine the prevalence of cancer-associated events (CAE) in advanced and nonadvanced adenomas. DNA copy number analysis was performed on archival tissues from three independent series of, in total, 297 adenomas (120 nonadvanced and 177 advanced) using multiplex ligation-dependent probe amplification or low-coverage whole-genome DNA sequencing. Alterations in two or more CAEs were considered to mark adenomas as high risk. Two or more CAEs were overall present in 25% (95% CI, 19.0–31.8) of advanced adenomas; 23% (11/48), 36% (12/33), and 23% (22/96) of the advanced adenomas in series 1, 2, and 3, respectively, and 1.7% (1/58) and 4.8% (3/62) of the nonadvanced adenomas, in series 1 and 2, respectively. The majority of advanced adenomas do not show CAEs, indicating that only a subset of these lesions is to be considered high risk. Nonadvanced adenomas have very low prevalence of CAEs, although those with CAEs should be considered high risk as well. Specific DNA copy number alterations may better reflect the true progression risk than the advanced adenoma phenotype. Cancer Prev Res; 11(7); 403–12. ©2018 AACR.

// Kristiaan Lenos 1, 2 , Jeroen A.C.M. Goos 3 , Ilona M. Vuist 1, 4 , Sjoerd H. den Uil 3, 5 , Pien M. Delis-van Diemen 3, 6 , Eric J.Th. Belt 7 , Hein B.A.C. Stockmann 5 , Herman Bril 8 , Meike de Wit 9, 6 , Beatriz Carvalho 3, 6 , Susan Giblett 10 , Catrin A. Pritchard 10 , Gerrit A. Meijer 3, 6 , Yvette van Kooyk 1 , Remond J.A. Fijneman 3, 6 , Sandra J. van Vliet 1 1 Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands 2 Current address: Laboratory of Experimental Oncology and Radiobiology, Center for Experimental Molecular Medicine, Academic Medical Center, Amsterdam, The Netherlands 3 Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands 4 Current address: Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands 5 Department of Surgery, Spaarne Gasthuis, Haarlem, The Netherlands 6 Current address: Netherlands Cancer Institute, Amsterdam, The Netherlands 7 Department of Surgery, Erasmus Medical Center, Rotterdam, The Netherlands 8 Department of Pathology, Spaarne Gasthuis, Haarlem, The Netherlands 9 Department of Medical Oncology, VU University Medical Center Amsterdam, The Netherlands 10 Department of Biochemistry, University of Leicester, Leicester, UK Correspondence to: Sandra J. van Vliet, e-mail: s.vanvliet@vumc.nl Keywords: colorectal cancer, BRAF, glycosylation, MGL, C-type lectin Received: May 06, 2015 Accepted: June 18, 2015 Published: July 02, 2015 ABSTRACT Colorectal cancer (CRC) is the third most prevalent cancer type worldwide with a mortality rate of approximately 50%. Elevated cell-surface expression of truncated carbohydrate structures such as Tn antigen (GalNAcα-Ser/Thr) is frequently observed during tumor progression. We have previously demonstrated that the C-type lectin macrophage galactose-type lectin (MGL), expressed by human antigen presenting cells, can distinguish healthy tissue from CRC through its specific recognition of Tn antigen. Both MGL binding and oncogenic BRAF mutations have been implicated in establishing an immunosuppressive microenvironment. Here we aimed to evaluate whether MGL ligand expression has prognostic value and whether this was correlated to BRAF V600E mutation status. Using a cohort of 386 colon cancer patients we demonstrate that high MGL binding to stage III tumors is associated with poor disease-free survival, independent of microsatellite instability or adjuvant chemotherapy. In vitro studies using CRC cell lines showed an association between MGL ligand expression and the presence of BRAF V600E . Administration of specific BRAF V600E inhibitors resulted in decreased expression of MGL-binding glycans. Moreover, a positive correlation between induction of BRAF V600E and MGL binding to epithelial cells of the gastrointestinal tract was found in vivo using an inducible BRAF V600E mouse model. We conclude that the BRAF V600E mutation induces MGL ligand expression, thereby providing a direct link between oncogenic transformation and aberrant expression of immunosuppressive glycans. The strong prognostic value of MGL ligands in stage III colon cancer patients, i.e . when tumor cells disseminate to lymph nodes, further supports the putative immune evasive role of MGL ligands in metastatic disease.

Background: The fecal immunochemical test (FIT) for detecting hemoglobin is used widely for noninvasive colorectal cancer (CRC) screening, but its sensitivity leaves room for improvement.Objective: To identify novel protein biomarkers in stool that outperform or complement hemoglobin in detecting CRC and advanced adenomas.Design: Case-control study.Setting: Colonoscopy-controlled referral population from several centers.Participants: 315 stool samples from one series of 12 patients with CRC and 10 persons without colorectal neoplasia (control samples) and a second series of 81 patients with CRC, 40 with advanced adenomas, and 43 with nonadvanced adenomas, as well as 129 persons without colorectal neoplasia (control samples); 72 FIT samples from a third independent series of 14 patients with CRC, 16 with advanced adenomas, and 18 with nonadvanced adenomas, as well as 24 persons without colorectal neoplasia (control samples).Measurements: Stool samples were analyzed by mass spectrometry. Classification and regression tree (CART) analysis and logistic regression analyses were performed to identify protein combinations that differentiated CRC or advanced adenoma from control samples. Antibody-based assays for 4 selected proteins were done on FIT samples.Results: In total, 834 human proteins were identified, 29 of which were statistically significantly enriched in CRC versus control stool samples in both series. Combinations of 4 proteins reached sensitivities of 80% and 45% for detecting CRC and advanced adenomas, respectively, at 95% specificity, which was higher than that of hemoglobin alone (P < 0.001 and P = 0.003, respectively). Selected proteins could be measured in small sample volumes used in FIT-based screening programs and discriminated between CRC and control samples (P < 0.001).Limitation: Lack of availability of antibodies prohibited validation of the top protein combinations in FIT samples.Conclusion: Mass spectrometry of stool samples identified novel candidate protein biomarkers for CRC screening. Several protein combinations outperformed hemoglobin in discriminating CRC or advanced adenoma from control samples. Proof of concept that such proteins can be detected with antibody-based assays in small sample volumes indicates the potential of these biomarkers to be applied in population screening.Primary Funding Source: Center for Translational Molecular Medicine, International Translational Cancer Research Dream Team, Stand Up to Cancer (American Association for Cancer Research and the Dutch Cancer Society), Dutch Digestive Foundation, and VU University Medical Center.

Colorectal cancer (CRC) is a major health problem. Biomarkers associated with molecular changes in cancer cells can aid early detection, diagnosis, prognosis, therapy selection, and disease monitoring. Tumor tissue secretomes are a rich source of candidate biomarkers. To identify CRC protein biomarkers, secretomes of four pairs of human CRC tissue and patient-matched normal colon tissue samples, and secretomes of five CRC cell lines were analyzed by GeLC-MS/MS. Subsequent data analysis was based on label-free spectral counting, Ingenuity Pathway Analysis, Secretome/SignalP, STRING and Cytoscape, resulting in 2703 protein identifications in the tissue secretomes, of which 409 proteins were significantly more present in CRC samples than in controls. Biomarker selection of 76 candidates was based on consistent and abundant over-representation in cancer- compared to control-secretomes, and presumed neoplastic origin. Overlap analysis with previously obtained datasets revealed 21 biomarkers suited for early detection of CRC. Immunohistochemistry confirmed overexpression in CRC of one candidate marker (MCM5). In conclusion, a human reference dataset of 76 candidate biomarkers was identified for which we illustrate that combination with existing pre-clinical datasets allows pre-selection of biomarkers for blood- or stool-based assays to support clinical management of CRC. Further dedicated validation studies are required to demonstrate their clinical applicability.Tissue secretome proteomes are a rich source of candidate biomarkers. Several secretome proteome datasets have been obtained from pre-clinical in vitro and in vivo colorectal cancer (CRC) model systems, yielding promising CRC biomarkers obtained under well-defined experimentally controlled conditions. However, which of these biomarker proteins are actually secreted by human CRC samples was not known. To our knowledge, this is the first study that directly compares secretome proteomes from clinically relevant human CRC tissues to patient-matched normal colon tissues. We identified 76 human CRC protein biomarkers that may facilitate blood-based or stool-based assay development to support clinical management of CRC. Overlap analysis with datasets from well-defined pre-clinical studies helps to determine what clinical application suits these human CRC biomarkers best, i.e. early detection, diagnosis, prognosis, therapy selection, and/or disease monitoring of CRC. This is demonstrated for a CRC mouse model dataset, revealing 21 human CRC biomarkers suited for early detection of CRC.

Colorectal cancer (CRC) is a major cause of cancer-related death in the western world. Screening to detect the disease in an early stage is the most effective approach to tackle this problem. In addition, better diagnostic tools for assessment of prognosis and prediction of response to drug therapy will allow for personalized therapies and better outcomes. Protein biomarkers that reflect tumor biology have the potential to address a wide range of clinical needs. These include diagnostic (screening) biomarkers for early detection, prognostic biomarkers for estimation of disease outcome, predictive biomarkers for adjuvant treatment stratification, and surveillance biomarkers for disease monitoring and treatment response. An important source for the discovery of potential biomarkers comes from mass spectrometry based proteomics research of the biology of CRC development. Here, we review recent colon cancer proteomics studies directed at identification of biomarker proteins. These include studies that use preclinical models (i.e. cell lines or murine tissues) as well as clinical materials (e.g. tissue and stool samples). We separately highlight some studies that focused on identification of cancer stem cell (CSC) related proteins in tumor spheroids, an in vitro model system for investigating CRC treatment response. Recent proteomics studies have generated many new candidate protein biomarkers. However, the lack of follow-up studies that lead to biomarker verification and/or validation remains a limiting factor in the translation of these candidate biomarkers into clinical applications. This is partly due to technological limitations which are bound to diminish with new technologies, including selected reaction monitoring mass spectrometry (SRM-MS). Antibodies are still required, though, both to perform high-throughput validation as well as to develop cost-effective tests for routine use in a clinical setting.

Despite major improvements on the knowledge and clinical management, cancer is still a deadly disease. Novel biomarkers for better cancer detection, diagnosis and treatment prediction are urgently needed. Proteins secreted, shed or leaking from the cancer cell, collectively termed the cancer secretome, are promising biomarkers since they might be detectable in blood or other biofluids. Furthermore, the cancer secretome in part represents the tumor microenvironment that plays a key role in tumor promoting processes such as angiogenesis and invasion. The cancer secretome, sampled as conditioned medium from cell lines, tumor/tissue interstitial fluid or tumor proximal body fluids, can be studied comprehensively by nanoLC-MS/MS-based approaches. Here, we outline the importance of current cancer secretome research and describe the mass spectrometry-based analysis of the secretome. Further, we provide an overview of cancer secretome research with a focus on the three most common cancer types: lung, breast and colorectal cancer. We conclude that the cancer secretome research field is a young, but rapidly evolving research field. Up to now, the focus has mainly been on the discovery of novel promising secreted cancer biomarker proteins. An interesting finding that merits attention is that in cancer unconventional secretion, e.g. via vesicles, seems increased. Refinement of current approaches and methods and progress in clinical validation of the current findings are vital in order to move towards applications in cancer management. This article is part of a Special Issue entitled: An Updated Secretome.

Purpose: Early detection of colorectal cancer (CRC) and its precursor lesions is an effective approach to reduce CRC mortality rates. This study aimed to identify novel protein biomarkers for the early diagnosis of CRC. Experimental Design: Proximal fluids are a rich source of candidate biomarkers as they contain high concentrations of tissue-derived proteins. The FabplCre;Apc15lox/+ mouse model represents early-stage development of human sporadic CRC. Proximal fluids were collected from normal colon and colon tumors and subjected to in-depth proteome profiling by tandem mass spectrometry. Carcinoembryonic antigen (CEA) and CHI3L1 human serum protein levels were determined by ELISA. Results: Of the 2,172 proteins identified, quantitative comparison revealed 192 proteins that were significantly (P < 0.05) and abundantly (>5-fold) more excreted by tumors than by controls. Further selection for biomarkers with highest specificity and sensitivity yielded 52 candidates, including S100A9, MCM4, and four other proteins that have been proposed as candidate biomarkers for human CRC screening or surveillance, supporting the validity of our approach. For CHI3L1, we verified that protein levels were significantly increased in sera from patients with adenomas and advanced adenomas compared with control individuals, in contrast to the CRC biomarker CEA. Conclusion: These data show that proximal fluid proteome profiling with a mouse tumor model is a powerful approach to identify candidate biomarkers for early diagnosis of human cancer, exemplified by increased CHI3L1 protein levels in sera from patients with CRC precursor lesions. Clin Cancer Res; 18(9); 2613–24. ©2012 AACR.

Gain of chromosome 20q is an important factor in the progression from colorectal adenomas to carcinomas. Genes that drive 20q gain are expected to show correlation of mRNA and protein expression levels with 20q DNA copy number status while functionally influencing cancer processes. CSE1L, DIDO1 and RBM39 are located on the 20q amplicon and affect processes such as cell viability and anchorage-independent growth in colorectal cancer. This study aimed to investigate whether CSE1L, DIDO1 and RBM39 may drive 20q amplification.Protein expression levels were examined by immunohistochemical evaluation of tissue microarrays containing a series of colorectal adenoma and carcinoma samples, which were characterized by genome-wide (microarray-based) DNA and mRNA profiling.CSE1L, DIDO1 and RBM39 mRNA expression levels correlated with chromosome 20q DNA copy number status. CSE1L protein expression was not associated with 20q gain, although its expression was increased in carcinomas compared to adenomas. DIDO1 and RBM39 protein expression was quite strong in the majority of tumors irrespective of 20q DNA copy number status.The lack of correlation between protein expression levels and 20q DNA copy number status implies that CSE1L, DIDO1 and RBM39 are merely passengers rather than drivers of chromosome 20q gain in colorectal adenoma-to-carcinoma progression.

Colorectal cancer (CRC) is the second leading cause of cancer death in the western world. The majority of CRCs, which develop from adenoma precursor lesions, show gain of chromosome arm 20q, where BCL2L1 is located. BCL2L1 is an important apoptosis regulating gene that codes for both an anti-apoptotic (Bcl-x(L)) and a pro-apoptotic (Bcl-x(S)) splice variant. The aim of the present study was to investigate whether BCL2L1 contributes to 20q gain-driven colorectal adenoma-to-carcinoma progression. To this end, the functional role of BCL2L1 in cancer-related processes was investigated, and differences in BCL2L1 DNA, mRNA, and protein levels were compared between colorectal adenomas and CRCs, as well as between tumours with and without 20q gain. Down-modulation of BCL2L1 inhibited cell viability and anchorage-independent growth of CRC cells, while invasion was not affected. BCL2L1 DNA copy number and protein expression were increased in CRCs compared to adenomas (p = 0.00005 and p = 0.03, respectively), while mRNA expression was not. Differences in BCL2L1 protein expression were even more pronounced between tumours with and without 20q gain (p = 0.0001). In conclusion, BCL2L1 is functionally involved in several cancer-related processes and its protein expression is associated with 20q gain. This supports a role for 20q gain-dependent expression of BCL2L1 in colorectal adenoma-to-carcinoma progression. However, the absence of a direct correlation between BCL2L1 mRNA and protein expression implies that BCL2L1 protein expression is regulated at the post-transcriptional level by a distinct factor on the 20q amplicon (eg ZNF217, AURKA or miRNAs). Therefore, even though BCL2L1 affects CRC biology in a 20q gain-dependent manner, it is not likely to be a driver of chromosome 20q gain associated adenoma-to-carcinoma progression.

Differences in word associations between monolingual and bilingual speakers of Dutch can reflect differences in how well seemingly familiar words are known. In this (exploratory) study mono-and bilingual, child and adult free word associations were compared. Responses of children and of monolingual speakers were found to be more dispersed across response categories than responses of adults and of L2 speakers, respectively. Log linear analyses show that the distributional patterns of association responses differ among the groups. Age has the largest effect on association responses. Adults give more meaning-related responses than children. Child L1 speakers give more meaning-related responses than child L2 speakers. Form-based and ‘Other’ associations were mostly given by (L2) children. The different findings for mono- and bilingual children and for mono- and bilingual adults show the influence of bilingualism on the development of word associations. The prominent effect of age emphasizes the role of conceptual development in word association behavior, and makes free word association tasks less suitable as an assessment tool for word knowledge.