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Supplemental Materials, Figures S1-6, Tables S1-4. Fig. S1. Flow diagram for inclusion of patients in the study. Fig. S2. RNA secondary structure analysis of the 5'UTR RCC2 mutation compared to wild type. Fig. S3. Vector construction overview. Fig. S4. Morphological changes in actin fiber patterns following depletion of RCC2 protein in HCT15 cells. Fig. S5. Subgroup analyses of in situ protein expression of RCC2 for a consecutive CRC series. Fig. S6. Representative electropherograms from fragment analysis of the 5'UTR RCC2 mutation. Table S1. Statistics for relevant parameters for the test series (n = 37). Table S2. Statistics for relevant parameters for the validation series (n = 122). Table S3. Statistics for relevant parameters for the validation series (n = 60, R0 patients). Table S4. Primer and PCR details.

Purpose: Colorectal cancer has high incidence and mortality worldwide. Patients with microsatellite instable (MSI) tumors have significantly better prognosis than patients with microsatellite stable (MSS) tumors. Considerable variation in disease outcome remains a challenge within each subgroup, and our purpose was to identify biomarkers that improve prediction of colorectal cancer prognosis.Experimental Design: Mutation analyses of 42 MSI target genes were performed in two independent MSI tumor series (n = 209). Markers that were significantly associated with prognosis in the test series were assessed in the validation series, followed by functional and genetic explorations. The clinical potential was further investigated by immunohistochemistry in a population-based colorectal cancer series (n = 903).Results: We identified the cell-cycle gene regulator of chromosome condensation 2 (RCC2) as a cancer biomarker. We found a mutation in the 5′ UTR region of RCC2 that in univariate and multivariate analyses was significantly associated with improved outcome in the MSI group. This mutation caused reduction of protein expression in dual luciferase gene reporter assays. siRNA knockdown in MSI colon cancer cells (HCT15) caused reduced cell proliferation, cell-cycle arrest, and increased apoptosis. Massive parallel sequencing revealed few RCC2 mutations in MSS tumors. However, weak RCC2 protein expression was significantly associated with poor prognosis, independent of clinical high-risk parameters, and stratifies clinically important patient subgroups with MSS tumors, including elderly patients (>75 years), stage II patients, and those with rectal cancer.Conclusions: Impaired RCC2 affects functional and clinical endpoints of colorectal cancer. High-risk patients with either MSI or MSS tumors can be identified with cost-effective routine RCC2 assays. Clin Cancer Res; 21(16); 3759–70. ©2015 AACR.

Macrophages are a heterogeneous population of cells involved in tissue homeostasis, inflammation, and cancer. Although macrophages are densely distributed throughout the human intestine, our understanding of how gut macrophages maintain tissue homeostasis is limited. Here we show that colonic lamina propria macrophages (LpMs) and muscularis macrophages (MMs) consist of monocyte-like cells that differentiate into multiple transcriptionally distinct subsets. LpMs comprise subsets with proinflammatory properties and subsets with high antigen-presenting and phagocytic capacity. The latter are strategically positioned close to the surface epithelium. Most MMs differentiate along two trajectories: one that upregulates genes associated with immune activation and angiogenesis, and one that upregulates genes associated with neuronal homeostasis. Importantly, MMs are located adjacent to neurons and vessels. Cell–cell interaction and gene network analysis indicated that survival, migration, transcriptional reprogramming, and niche-specific localization of LpMs and MMs are controlled by an extensive interaction with tissue-resident cells and a few key transcription factors.

The prognostic value of CpG island methylator phenotype (CIMP) in colorectal cancer remains unsettled. We aimed to assess the prognostic value of this phenotype analyzing a total of 1126 tumor samples obtained from two Norwegian consecutive colorectal cancer series. CIMP status was determined by analyzing the 5-markers CAGNA1G, IGF2, NEUROG1, RUNX3 and SOCS1 by quantitative methylation specific PCR (qMSP). The effect of CIMP on time to recurrence (TTR) and overall survival (OS) were determined by uni- and multivariate analyses. Subgroup analyses were conducted according to MSI and BRAF mutation status, disease stage, and also age at time of diagnosis (

Background The prognostic impact of KRAS and BRAFV600E mutations in primary colorectal cancer (CRC) varies with microsatellite instability (MSI) status. The gene expression–based consensus molecular subtypes (CMSs) of CRC define molecularly and clinically distinct subgroups, and represent a novel stratification framework in biomarker analysis. We investigated the prognostic value of these mutations within the CMS groups. Patients and methods Totally 1197 primary tumors from a Norwegian series of CRC stage I–IV were analyzed for MSI and mutation status in hotspots in KRAS (codons 12, 13 and 61) and BRAF (codon 600). A subset was analyzed for gene expression and confident CMS classification was obtained for 317 samples. This cohort was expanded with clinical and molecular data, including CMS classification, from 514 patients in the publically available dataset GSE39582. Gene expression signatures associated with KRAS and BRAFV600E mutations were used to evaluate differential impact of mutations on gene expression among the CMS groups. Results BRAF V600E and KRAS mutations were both associated with inferior 5-year overall survival (OS) exclusively in MSS tumors (BRAFV600E mutation versus KRAS/BRAF wild-type: Hazard ratio (HR) 2.85, P

Colorectal cancer (CRC) is a complex cancer disease, and approximately 40% of the surgically cured patients will experience cancer recurrence within 5 years. During recent years, research has shown that CRC treatment should be tailored to the individual patient due to the wide variety of risk factors, genetic factors, and surgical complexity. In this review, we provide an overview of the considerations that are needed to provide an individualized, patient-tailored treatment. We emphasize the need to assess the predictors of CRC, and we summarize the latest research on CRC genetics and immunotherapy. Finally, we provide a summary of the significant variations in the colon and rectal anatomy that is important to consider in an individualized surgical approach. For the individual patient with CRC, a tailored treatment approach is needed in the preoperative, operative, and postoperative phase.

BACKGROUND: Microsatellite instability (MSI) was suggested as a marker for good prognosis in colorectal cancer in 1993 and a systematic review from 2005 and a meta-analysis from 2010 support the initial observation. We here assess the prognostic impact and prevalence of MSI in different stages in a consecutive, population-based series from a single hospital in Oslo, Norway. PATIENTS AND METHODS: Of 1274 patients, 952 underwent major resection of which 805 were included in analyses of MSI prevalence and 613 with complete resection in analyses of outcome. Formalin-fixed tumor tissue was used for PCR-based MSI analyses. RESULTS: The overall prevalence of MSI was 14%, highest in females (19%) and in proximal colon cancer (29%). Five-year relapse-free survival (5-year RFS) was 67% and 55% (P = 0.030) in patients with MSI and MSS tumors, respectively, with the hazard ratio (HR) equal to 1.60 (P = 0.045) in multivariate analysis. The improved outcome was confined to stage II patients who had 5-year RFS of 74% and 56% respectively (P = 0.010), HR = 2.02 (P = 0.040). Examination of 12 or more lymph nodes was significantly associated with proximal tumor location (P < 0.001). CONCLUSIONS: MSI has an independent positive prognostic impact on stage II colorectal cancer patients after complete resection.

Background and Objectives : The aim of the study was to evaluate the incidence of peritoneal carcinomatosis (PC) in a prospectively recorded series of colon cancer patients from a defined cohort and to compare clinicopathological characteristics, survival, and TP53 mutation status in primary tumors from patients with and without PC. Methods : Clinical data from all colon cancer patients admitted in 1993–2006 were registered prospectively (n = 1,124). In a subset of PC patients, DNA was retrieved from tumor tissue and TP53 mutations analyzed and compared to the mutation status in a historical series. Results : In the prospective series 10% of female and 7% of male patients had PC (P = 0.05). The PC patients were younger than those without PC (median 4 years, P = 0.002). The incidence of PC was 10.3% and 6.2% (P = 0.03) in patients with primary tumors in the right and left colon, respectively. TP53 was mutated in 57% of the PC patients as compared to 41% in the series of patients without PC (P = 0.05). Conclusions : The incidence of PC was higher in right-sided colon cancer and among women. PC patients were younger than non-PC patients, and PC was independently associated with TP53 mutation in the primary tumor. J. Surg. Oncol. © 2011 Wiley Periodicals, Inc.

Purpose: Colorectal cancer has high incidence and mortality worldwide. Patients with microsatellite instable (MSI) tumors have significantly better prognosis than patients with microsatellite stable (MSS) tumors. Considerable variation in disease outcome remains a challenge within each subgroup, and our purpose was to identify biomarkers that improve prediction of colorectal cancer prognosis. Experimental Design: Mutation analyses of 42 MSI target genes were performed in two independent MSI tumor series (n = 209). Markers that were significantly associated with prognosis in the test series were assessed in the validation series, followed by functional and genetic explorations. The clinical potential was further investigated by immunohistochemistry in a population-based colorectal cancer series (n = 903). Results: We identified the cell-cycle gene regulator of chromosome condensation 2 (RCC2) as a cancer biomarker. We found a mutation in the 5′ UTR region of RCC2 that in univariate and multivariate analyses was significantly associated with improved outcome in the MSI group. This mutation caused reduction of protein expression in dual luciferase gene reporter assays. siRNA knockdown in MSI colon cancer cells (HCT15) caused reduced cell proliferation, cell-cycle arrest, and increased apoptosis. Massive parallel sequencing revealed few RCC2 mutations in MSS tumors. However, weak RCC2 protein expression was significantly associated with poor prognosis, independent of clinical high-risk parameters, and stratifies clinically important patient subgroups with MSS tumors, including elderly patients (>75 years), stage II patients, and those with rectal cancer. Conclusions: Impaired RCC2 affects functional and clinical endpoints of colorectal cancer. High-risk patients with either MSI or MSS tumors can be identified with cost-effective routine RCC2 assays. Clin Cancer Res; 21(16); 3759–70. ©2015 AACR.

Background: The prognostic impact of an indication of chromosomal instability (CIN) is evaluated in a consecutive series of 952 colorectal cancer patients treated at Aker University Hospital, Norway, during 1993–2003. Microsatellite instability (MSI) in this case series has recently been reported and made it possible to find the co-occurrence and compare the prognostic significance of CIN and MSI. Methods: Data sets for overall survival (OS; n=855) and time to recurrence (TTR; n=579) were studied. To reveal CIN we used automated image cytometry (ICM). Non-diploid histograms were taken as indicative of the presence of CIN. PCR-based measures of MSI in this material have already been described. Results: As with MSI, CIN was found to be an independent predictor of early relapse and death among stage II patients (TTR: n=278: HR 2.19 (95% CI: 1.35–3.55), P=0.002). Of the MSI tumours (16%), 71% were found to be DNA diploid, 21% were DNA tetraploid and 8% were DNA aneuploid. Among microsatellite stable tumours, 24% were DNA diploid, 15% were DNA tetraploid and 61% were DNA aneuploid. Conclusion: For patients presenting with stage II disease, genomic instability as detected by DNA image cytometry has the potential to provide a useful biomarker for relapse and cancer-related death following surgery with curative intent.

The prognostic impact of the number of lymph nodes and ratio in colon cancer is still debated.The aim of this study was to evaluate lymph node harvest in patients with colon cancer over time, and to test the hypotheses that investigation of more lymph nodes, and low lymph node ratio in stage III patients, has positive prognostic impact.This is a prospective, observational study.This study was conducted in a single institution treating all patients with colon cancer in a defined catchment area.All patients admitted in the period 1993 to 2009 (n = 1481) were included.The primary outcomes measured were the number of examined regional lymph nodes according to treatment period, 5-year overall survival and time to recurrence, and univariate (Kaplan-Meier) and multivariate (Cox regression) analyses of prognostic factors.Nine hundred fifty (65%) patients underwent curative resection. Median number of examined lymph nodes increased from 7 to 15 (p0.001), and the proportion of patients with stage III disease increased from 25% to 33% (p = 0.02) during the study period. In patients with stage I to III disease, time to recurrence (proportion of patients without recurrence or death of colon cancer) improved from 65% to 82% during the period (p0.001). An association between lymph node count (8 compared with ≥ 12) and overall survival was found for patients with stage II disease (57% vs 71%, p = 0.004). Hazard ratio for death within 5 years was 0.7 (p = 0.043) when 8 to 11 nodes were examined and 0.6 (p = 0.001) when ≥ 12 nodes were examined (8 reference). In patients with stage III disease, increasing lymph node ratio was associated with reduced overall survival and time to recurrence in uni- and multivariate analyses.This study was limited by the small number of patients in each stage.The number of examined lymph nodes increased in the study period. A stage migration was observed, and time to recurrence improved in patients with stage I to III disease. In patients with stage III disease, lymph node ratio was a stronger prognostic factor than the total number of lymph nodes examined.

Several clinical factors have an impact on prognosis in stage II colorectal cancer (CRC), but as yet they are inadequate for risk assessment. The present study aimed to develop a gene expression classifier for improved risk stratification of patients with stage II CRC.315 CRC samples were included in the study. Gene expression measurements from 207 CRC samples (stage I-IV) from two independent Norwegian clinical series were obtained using Affymetrix exon-level microarrays. Differentially expressed genes between stage I and stage IV samples from the test series were identified and used as input for L1 (lasso) penalised Cox proportional hazards analyses of patients with stage II CRC from the same series. A second validation was performed in 108 stage II CRC samples from other populations (USA and Australia).An optimal 13-gene expression classifier (PIGR, CXCL13, MMP3, TUBA1B, SESN1, AZGP1, KLK6, EPHA7, SEMA3A, DSC3, CXCL10, ENPP3, BNIP3) for prediction of relapse among patients with stage II CRC was developed using a consecutive Norwegian test series from patients treated according to current standard protocols (n=44, p0.001, HR=18.2), and its predictive value was successfully validated for patients with stage II CRC in a second Norwegian CRC series collected two decades previously (n=52, p=0.02, HR=3.6). Further validation of the classifier was obtained in a recent external dataset of patients with stage II CRC from other populations (n=108, p=0.001, HR=6.5). Multivariate Cox regression analyses, including all three sample series and various clinicopathological variables, confirmed the independent prognostic value of the classifier (p≤0.004). The classifier was shown to be specific to stage II CRC and does not provide prognostic stratification of patients with stage III CRC.This study presents the development and validation of a 13-gene expression classifier, ColoGuideEx, for prognosis prediction specific to patients with stage II CRC. The robustness was shown across patient series, populations and different microarray versions.

Background The presence of cancer-specific DNA methylation patterns in epithelial colorectal cells in human feces provides the prospect of a simple, non-invasive screening test for colorectal cancer and its precursor, the adenoma. This study investigates a panel of epigenetic markers for the detection of colorectal cancer and adenomas. Methods Candidate biomarkers were subjected to quantitative methylation analysis in test sets of tissue samples from colorectal cancers, adenomas, and normal colonic mucosa. All findings were verified in independent clinical validation series. A total of 523 human samples were included in the study. Receiver operating characteristic (ROC) curve analysis was used to evaluate the performance of the biomarker panel. Results Promoter hypermethylation of the genes CNRIP1, FBN1, INA, MAL, SNCA, and SPG20 was frequent in both colorectal cancers (65-94%) and adenomas (35-91%), whereas normal mucosa samples were rarely (0-5%) methylated. The combined sensitivity of at least two positives among the six markers was 94% for colorectal cancers and 93% for adenoma samples, with a specificity of 98%. The resulting areas under the ROC curve were 0.984 for cancers and 0.968 for adenomas versus normal mucosa. Conclusions The novel epigenetic marker panel shows very high sensitivity and specificity for both colorectal cancers and adenomas. Our findings suggest this biomarker panel to be highly suitable for early tumor detection.

The incidence of colorectal cancer (CRC) increases with age and early onset indicates an increased likelihood for genetic predisposition for this disease. The somatic genetics of tumor development in relation to patient age remains mostly unknown. We have examined the mutation status of five known cancer critical genes in relation to age at diagnosis, and compared the genomic complexity of tumors from young patients without known CRC syndromes with those from elderly patients. Among 181 CRC patients, stratified by microsatellite instability status, DNA sequence changes were identified in KRAS (32%), BRAF (16%), PIK3CA (4%), PTEN (14%) and TP53 (51%). In patients younger than 50 years (n = 45), PIK3CA mutations were not observed and TP53 mutations were more frequent than in the older age groups. The total gene mutation index was lowest in tumors from the youngest patients. In contrast, the genome complexity, assessed as copy number aberrations, was highest in tumors from the youngest patients. A comparable number of tumors from young (70 years) was quadruple negative for the four predictive gene markers (KRAS-BRAF-PIK3CA-PTEN); however, 16% of young versus only 1% of the old patients had tumor mutations in PTEN/PIK3CA exclusively. This implies that mutation testing for prediction of EGFR treatment response may be restricted to KRAS and BRAF in elderly (>70 years) patients. Distinct genetic differences found in tumors from young and elderly patients, whom are comparable for known clinical and pathological variables, indicate that young patients have a different genetic risk profile for CRC development than older patients.

The aim of the study was to evaluate the incidence of peritoneal carcinomatosis (PC) in a prospectively recorded series of colon cancer patients from a defined cohort and to compare clinicopathological characteristics, survival, and TP53 mutation status in primary tumors from patients with and without PC.Clinical data from all colon cancer patients admitted in 1993-2006 were registered prospectively (n = 1,124). In a subset of PC patients, DNA was retrieved from tumor tissue and TP53 mutations analyzed and compared to the mutation status in a historical series.In the prospective series 10% of female and 7% of male patients had PC (P = 0.05). The PC patients were younger than those without PC (median 4 years, P = 0.002). The incidence of PC was 10.3% and 6.2% (P = 0.03) in patients with primary tumors in the right and left colon, respectively. TP53 was mutated in 57% of the PC patients as compared to 41% in the series of patients without PC (P = 0.05).The incidence of PC was higher in right-sided colon cancer and among women. PC patients were younger than non-PC patients, and PC was independently associated with TP53 mutation in the primary tumor.

Reduced levels of autophagy correlate with tumorigenesis, and several inducers of autophagy have been found to be tumor suppressors. One such autophagic inducer is the Beclin 1 binding protein UVRAG, a positive regulator of the class III PI3K/Vps34 complex. UVRAG has been implicated in the formation and maturation of autophagosomes, as well as in endocytic trafficking and suppression of proliferation and in vivo tumorigenicity. In this study we show that approximately one-third of a large series of colon carcinomas with microsatellite instability (MSI) (n = 102) carry a monoallelic UVRAG mutation, leading to expression of a truncated protein, indicating that this event is involved in tumorigenesis. In order to investigate whether the high incidence of UVRAG mutation in MSI colorectal carcinomas is associated with dysfunctional autophagy we analyzed autophagy levels in several colon cancer cell lines that express wild-type or mutant UVRAG protein. No reduction in autophagy was detected in cell lines expressing mutant UVRAG. Consistent with this, depletion of UVRAG in HEK cells stably expressing GFP-LC3 did not inhibit autophagy, but did decrease epidermal growth factor receptor (EGFR) degradation. Overall our results show that there is no correlation between the presence of the monoallelic UVRAG mutation and inhibition of autophagy. Thus, our data indicate that mechanisms other than autophagy contribute to the tumorigenicity of microsatellite unstable colon carcinomas with monoallelic UVRAG mutation.

Background Estimates suggest that up to 30% of colorectal cancers (CRC) may develop due to an increased genetic risk. The mean age at diagnosis for CRC is about 70 years. Time of disease onset 20 years younger than the mean age is assumed to be indicative of genetic susceptibility. We have compared high resolution tumor genome copy number variation (CNV) (Roche NimbleGen, 385 000 oligo CGH array) in microsatellite stable (MSS) tumors from two age groups, including 23 young at onset patients without known hereditary syndromes and with a median age of 44 years (range: 28-53) and 17 elderly patients with median age 79 years (range: 69-87). Our aim was to identify differences in the tumor genomes between these groups and pinpoint potential susceptibility loci. Integration analysis of CNV and genome wide mRNA expression data, available for the same tumors, was performed to identify a restricted candidate gene list. Results The total fraction of the genome with aberrant copy number, the overall genomic profile and the TP53 mutation spectrum were similar between the two age groups. However, both the number of chromosomal aberrations and the number of breakpoints differed significantly between the groups. Gains of 2q35, 10q21.3-22.1, 10q22.3 and 19q13.2-13.31 and losses from 1p31.3, 1q21.1, 2q21.2, 4p16.1-q28.3, 10p11.1 and 19p12, positions that in total contain more than 500 genes, were found significantly more often in the early onset group as compared to the late onset group. Integration analysis revealed a covariation of DNA copy number at these sites and mRNA expression for 107 of the genes. Seven of these genes, CLC, EIF4E, LTBP4, PLA2G12A, PPAT, RG9MTD2, and ZNF574, had significantly different mRNA expression comparing median expression levels across the transcriptome between the two groups. Conclusions Ten genomic loci, containing more than 500 protein coding genes, are identified as more often altered in tumors from early onset versus late onset CRC. Integration of genome and transcriptome data identifies seven novel candidate genes with the potential to identify an increased risk for CRC.