Normal Colon Control Research Articles

A distal super-enhancer activates oncogenic ETS2 via recruiting MECOM in inflammatory bowel disease and colorectal cancer

Abnormal activities of distal cis-regulatory elements (CREs) contribute to the initiation and progression of cancer. Gain of super-enhancer (SE), a highly active distal CRE, is essential for the activation of key oncogenes in various cancers. However, the mechanism of action for most tumor-specific SEs still largely remains elusive. Here, we report that a candidate oncogene ETS2 was activated by a distal SE in inflammatory bowel disease (IBD) and colorectal cancer (CRC). The SE physically interacted with the ETS2 promoter and was required for the transcription activation of ETS2. Strikingly, the ETS2-SE activity was dramatically upregulated in both IBD and CRC tissues when compared to normal colon controls and was strongly correlated with the level of ETS2 expression. The tumor-specific activation of ETS2-SE was further validated by increased enhancer RNA transcription from this region in CRC. Intriguingly, a known IBD-risk SNP resides in the ETS2-SE and the genetic variant modulated the level of ETS2 expression through affecting the binding of an oncogenic transcription factor MECOM. Silencing of MECOM induced significant downregulation of ETS2 in CRC cells, and the level of MECOM and ETS2 correlated well with each other in CRC and IBD samples. Functionally, MECOM and ETS2 were both required for maintaining the colony-formation and sphere-formation capacities of CRC cells and MECOM was crucial for promoting migration. Taken together, we uncovered a novel disease-specific SE that distantly drives oncogenic ETS2 expression in IBD and CRC and delineated a mechanistic link between non-coding genetic variation and epigenetic regulation of gene transcription.

P-112 Oleic acid promotes the malignant transformation of Kras-mutant colonic organoids via the expansion of tumorigenic stem cells and abnormal Paneth cells through upregulation of NFATc family

Although the five-year survival rate of colorectal cancer (CRC) is significantly high with localized stage (90%), only 38% of the patients are diagnosed at this stage. Therefore, finding high risk factors of CRC could prevent the disease at the early stage. Obesity and high-fat diet consumption are risk factors of CRC. In detail, oleic acids (OA) were found to accumulate in the adipose tissues of obese patients as well as being the most common long chain fatty acid in dietary lipid. The role of OA in colorectal cancer development is still controversary. On the other hand, organoids, which can be derived from adult tissue stem cells, are the miniatures of organs. Recent works suggest organoids are useful tools to dissect mechanisms involved in cancer development. The current work aims to utilize crypt stem cell-derived organoids to dissect how obesity can contribute to CRC development. Organoids were derived from Lgr5 + -EGFP mice (normal colon control), Lgr5 + -EGFP-creER; LSL-KrasG12D mice (colonic epithelium harbored KrasG12D mutation) and AOM/DSS-induced CRC mice (cancerous colon control). For in-vitro, the impact of OA on the malignant transformation of Kras-mutant colonic organoids was assessed by MTT proliferation assay, 2D transwell migration and invasion assay, irinotecan-resistant test and colony formation assay. The tumor formation in-vivo was induced by subcutaneous engrafting of OA-treated organoids on NOD/SCID mice. RNAseq was done to screen some differential expressed genes when treating OA. The existence of stem cell and abnormal Paneth cell population was demonstrated by immunostaining, flow cytometry and ELISA. OA enhanced the malignant of Kras-mutant colonic organoids through increasing proliferation, migration, invasion, drug resistance and colony formation. Besides, mice which were engrafted with OA-trained Lgr5-Kras organoids possessed larger tumors. The colonic stem cell population which were represented by positive Lgr5-GFP + cells were upregulated in OA-treated group. Interestingly, there is an existence of abnormal Lyz + /CD24 + Paneth cells in OA-treated Kras-mutant organoids. This suggested that OA may promote abnormal differentiation of colonic progenitor cells. Indeed, treatment of OA upregulated ATOH1 and GFI1, which contributed to select between goblet/Paneth and enteroendocrine cell fates. Besides, NFAT family, among which NFATc3 expressed the most, significantly increased in OA-treated organoids. The upregulation of NFATc was induced by inhibition of proteasomal degradation resulting from low expression of the complex RNF123, USP18 and TRIM30A. In addition, inhibition of the nuclear translocalization of NFATc family by VIVIT demonstrated the decline of stemness markers as well as Paneth cell markers. Finally, we showed the role of NFATc, especially NFATc3, in promoting the malignancy of OA-treated Kras-mutant organoids. Our findings suggested the impact of oleic acid on promoting the malignant transformation of Kras-mutant colonic organoids through NFATc-relating pathway. The expansion of tumorigenic stem cells via induction of abnormal Paneth cell, may contribute to the potential of cancer-toward metaplasia in Kras-mutant organoids when exposing to high level of oleic acid. Oleic acid is an unfavorable factor of colorectal cancer. The frequent usage of OA, which may benefit to cardiovascular diseases, may lead to the increased risk of CRC in latent sensitive Kras-mutant individuals.

Neutrophil extracellular traps-associated protein peptidyl arginine deaminase 4 immunohistochemical expression in ulcerative colitis and its association with the prognostic predictors

Neutrophil extracellular traps (NETs) are incriminated in several immune and inflammatory diseases including ulcerative colitis (UC). Analysis of colonic tissues for NETs-related markers in UC carries prognostic and therapeutic implications. This work aims to evaluate the immunohistochemical (IHC) expression of NETs-associated-protein arginine deaminase 4 (PAD4) in colonic biopsies from UC patients in comparison to normal colon (NC). Association between PAD4 expression level and histopathologic grade, patient’s therapeutic response and other clinicopathological prognostic predictors in UC are determined. This cohort study included biopsies from 42 UC patients and 11 NC controls. Clinicopathological data including patient's age at diagnosis, gender, presenting symptoms, anatomical disease extent, extra-intestinal manifestations, type and response to therapy and surgical interventions were recorded and tabulated. Histopathological grading of disease activity and associated epithelial changes were assessed. PAD4 immunostaining was conducted using Horseradish Peroxidase technique and scored semiquantitatively considering intensity and percentage of nuclear staining of lamina propria inflammatory cells. Appropriate statistical tests were applied. Anti-PAD4 was localized mainly in the nuclei of lamina propria infiltrating neutrophils. It was expressed more significantly in UC (95.2 %) compared to NC (p 0.001). Increased PAD4 expression level was significantly associated with increasing histopathologic grade, anatomical disease extent, lacking response to therapy and subjection to radical surgery (p:0.001, = 0.038, 0.046, 0.046 respectively). Age, gender, presenting symptoms, extra-intestinal manifestations and epithelial changes showed insignificant associations. This study characterizes a subset of UC patients with high histopathological grade of activity, pancolonic involvement, strong/moderate PAD4 expression levels and who are unresponsive to routine medical therapeutic regimens rendering them candidates for radical surgery. In conjunction with histopathological grading, IHC evaluation of PAD4 in UC is recommended to guide patient’s selection for targeted therapy using the novel-discovered selective PAD4 inhibitors.

273 Multi-Target DNA Aberrations in Sporadic Colorectal Cancer Tissues Do Not Differ Between Younger and Older Patients

INTRODUCTION: Colorectal cancer (CRC) screening is broadly endorsed for individuals 50–75 years of age. Given recent increases in CRC incidence and mortality for patients <50 years old, the American Cancer Society now recommends initiation of average-risk CRC screening at age 45 (Wolf AMD CA Cancer J Clin 2018). The performance of recommended tests, including the multi-target stool DNA (mt-sDNA; marketed as Cologuard™) test, has been minimally described in populations under 50 years old. To address this knowledge gap, we conducted a retrospective study of DNA markers (KRAS, BMP3, NDRG4) included in the mt-sDNA assay to quantify and compare tissue marker levels in sporadic CRC cases and normal colon controls from patients in 45–49 and 50–64 year-old age groups. Our primary hypothesis was that mt-sDNA marker levels would be statistically similar between cases in both age groups. METHODS: Patient samples were identified from a pathology tissue archive at Mayo Clinic, excluding those with inflammatory bowel disease or familial cancer syndromes. Formalin-fixed, paraffin-embedded tissues were reviewed by a GI pathologist for 45–49 year-old (n = 90) and 50–64 year-old (n = 96) CRC cases; normal colon samples (45–49, n = 76; 50–64, n = 92) were also included for comparison. DNA extracted from core punches was blinded and processed in a randomized order. Quantitative Allele specific Realtime Target and Signal amplification (QuARTS) assays were used to quantify KRAS mutations, and BMP3 and NDRG4 methylation. Marker level in each sample was reported as the QuARTS product of mutant KRAS, methylated BMP3 or NDRG4 relative to the ACTB product in each sample, reported as a percentage. At least 60 patients per group provided sufficient power to demonstrate differences in marker levels of ±25% between case groups and between control groups using the Wilcoxon Rank Sum test. RESULTS: CRC cases in the 45–49 vs 50–64 year-old groups did not differ significantly with respect to sex (P = 0.77) or race/ethnicity (P = 0.06); left-sided CRC was more common in the 45–49 vs 50–64 year-old group (87% vs. 72%; P = 0.02). None of the marker levels were statistically different between cases of the younger vs older groups or between controls of these groups (Figure 1). CONCLUSION: These data support the expectation of no age-related differences in CRC detection by mt-sDNA, as the relevant molecular markers, when they occur, are present at similar levels across age groups 45–49 and 50–64 years.

A novel isoform of IL-33 revealed by screening for transposable element promoted genes in human colorectal cancer.

Remnants of ancient transposable elements (TEs) are abundant in mammalian genomes. These sequences contain multiple regulatory motifs and hence are capable of influencing expression of host genes. TEs are known to be released from epigenetic repression and can become transcriptionally active in cancer. Such activation could also lead to lineage-inappropriate activation of oncogenes, as previously described in lymphomas. However, there are few reports of this mechanism occurring in non-blood cancers. Here, we re-analyzed whole transcriptome data from a large cohort of patients with colon cancer, compared to matched normal colon control samples, to detect genes or transcripts ectopically expressed through activation of TE promoters. Among many such transcripts, we identified six where the affected gene has described role in cancer and where the TE-driven gene mRNA is expressed in primary colon cancer, but not normal matched tissue, and confirmed expression in colon cancer-derived cell lines. We further characterized a TE-gene chimeric transcript involving the Interleukin 33 (IL-33) gene (termed LTR-IL-33), that is ectopically expressed in a subset of colon cancer samples through the use of an endogenous retroviral long terminal repeat (LTR) promoter of the MSTD family. The LTR-IL-33 chimeric transcript encodes a novel shorter isoform of the protein, which is missing the initial N-terminus (including many conserved residues) of Native IL-33. In vitro studies showed that LTR-IL-33 expression is required for optimal CRC cell line growth as 3D colonospheres. Taken together, these data demonstrate the significance of TEs as regulators of aberrant gene expression in colon cancer.

Tu1930 - A Prospective Multi-National Study of the Colorectal Cancer Mucosal Microbiome Reveals Specific Taxonomic Changes Indicative of Disease Stage and Prognosis

Introduction The colorectal cancer (CRC) microbiome is niche specific and individualised. Several putative driver organisms enriched on CRCs have been identified from human studies, but few data exist which properly account for important clinical variables in CRC. In this study, we used a meta-taxonomic approach to demonstrate how the CRC microbiome varies with disease stage, histological markers of prognosis and host molecular phenotypes. Method A prospective study was performed on patients undergoing colonoscopy and elective surgery for CRC at three hospitals in UK and Czech Republic. Tissue was sampled from tumours, adenomas, adjacent normal mucosa and mucosa from healthy colon controls. The V1-2 regions of the 16S rRNA gene were sequenced (Illumina MiSeq); data were processed in Mothur and analysed in Stamp and R. Species assignment was performed with NCBI BLAST for microbial genomes. False discovery rate p value correction accounted for multiple testing. Histological analysis and tumour molecular phenotyping were performed according to Royal College of Pathology guidelines. Results One hundred and ninety six patients were recruited: 158 CRC patients, 24 adenoma patients and 14 normal colon controls (median age 70; range 35–90). Tumours were staged as 6 T0, 4 T1, 23 T2, 97 T3, 27 T4; 99 N0, 40 N1, 27 N2; 6 M1. No significant differences were seen in diversity or taxonomy between the UK and Czech cohorts. Adenoma and healthy colon control samples were taxonomically indistinct. However, CRCs were characterised by reduced Shannon diversity (p Conclusion This large prospective analysis demonstrates that the CRC microbiome is stage-specific and appears to evolve with disease progression. We conclude that oral pathobionts which colonise advanced stage disease relate to markers of tumour prognosis, raising the possibility that they may be directly influencing tumour invasion. Disclosure of Interest None Declared

A Genome-Wide Methylation Approach Identifies a New Hypermethylated Gene Panel in Ulcerative Colitis.

The cause of inflammatory bowel disease (IBD) is still unknown, but there is growing evidence that environmental factors such as epigenetic changes can contribute to the disease etiology. The aim of this study was to identify newly hypermethylated genes in ulcerative colitis (UC) using a genome-wide DNA methylation approach. Using an Infinium HumanMethylation450 BeadChip array, we screened the DNA methylation changes in three normal colon controls and eight UC patients. Using these methylation profiles, 48 probes associated with CpG promoter methylation showed differential hypermethylation between UC patients and normal controls. Technical validations for methylation analyses in a larger series of UC patients (n = 79) were performed by methylation-specific PCR (MSP) and bisulfite sequencing analysis. We finally found that three genes (FAM217B, KIAA1614 and RIBC2) that were significantly elevating the promoter methylation levels in UC compared to normal controls. Interestingly, we confirmed that three genes were transcriptionally silenced in UC patient samples by qRT-PCR, suggesting that their silencing is correlated with the promoter hypermethylation. Pathway analyses were performed using GO and KEGG databases with differentially hypermethylated genes in UC. Our results highlight that aberrant hypermethylation was identified in UC patients which can be a potential biomarker for detecting UC. Moreover, pathway-enriched hypermethylated genes are possibly implicating important cellular function in the pathogenesis of UC. Overall, this study describes a newly hypermethylated gene panel in UC patients and provides new clinical information that can be used for the diagnosis and therapeutic treatment of IBD.

OC-074 The Role of the Fungal Microbiota in the Pathogenesis of De-Novo Paediatric Inflammatory Bowel Disease using next Generation Sequencing

<h3>Introduction</h3> Paediatric Inflammatory bowel disease (IBD) incidence is rising worldwide. Recently the role of the gut microbiota has been recognised as pivotal in disease pathogenesis. IBD microbial studies to date have focused on bacterial diversity assessment in established disease cohorts, with limited studies in treatment naive patients. In contrast to bacteria, the exact role of the colonising fungi and their pathogenic potential has not been fully explored. The aim of the study was to examine candidate fungal triggers at disease onset in children with IBD using pyrosequencing, utilising the Bacteria in Inflammatory bowel disease in Scottish Children Undergoing Investigation before Treatment (BISCUIT) study cohort. <h3>Methods</h3> 128 children undergoing colonoscopy were approached from three Scottish paediatric centres (Aberdeen, Glasgow and Dundee) with 100 ultimately recruited and biopsied; 44 IBD (comprising Crohn’s disease (CD; 29), ulcerative colitis (UC; 13) and IBD-type unspecified (2)), 42 normal colon controls (NCC) and 14 “others”. All IBD patient samples were taken from inflamed tissue. Fungal DNA was amplified on a reduced cohort of 37 recruits (13 CD, 12 UC, 12 NCC) using 18S rDNA primers. Roche 454 Titanium sequencing was conducted by NewGene (Newcastle, UK). Data analysis was performed using QIIME version 1.3.0 workflow. Taxonomy assignment of operational taxonomic units (OTUs) was performed according to ribosomal database project taxonomy. OTU tables were rarefied at 3,000 reads. <h3>Results</h3> Fungal DNA was amplifiable from 7 patient samples, 6 children with a diagnosis of IBD – 4 with CD (BISCUIT1, 31, 62 and 89), 2 children with UC (BISCUIT33 and 104) and 1 NCC (BISCUIT 27). Fungal diversity was assessed in all paediatric samples alongside three adult samples to act as comparison. The adult samples comprised 1 patient with UC (2UC21Aa) and 2 NCC (GH4 and GH9). Phylum level analysis indicated that all fungal sequences belonged to the Ascomycota and Basidiomycota phyla. Control patients contained predominantly Ascomycota sequences ( &gt; 80% of sequences in all patients) whilst 6/7 IBD patients contained exclusively Basidiomycota species. Genus level analysis was undertaken and there was no similarity between fungal profiles from the paediatric and adult samples. <h3>Conclusion</h3> By using robust methodology we have characterised the IBD “fungal microbiota” at diagnosis in children. Based on the current study, it would appear that a distinctly altered fungal species profile is present at IBD disease presentation. Further work should now focus on expanding this study and identifying how to beneficially modify the microbiota using established and novel IBD treatments. <h3>Disclosure of Interest</h3> None Declared

OC-072 The Microaerophilic Microbiota of De-Novo Paediatric Inflammatory Bowel Disease: the Biscuit Study

Introduction Children presenting for the first time with inflammatory bowel disease (IBD) offer a unique opportunity to study etiological agents before the confounders of treatment. Microaerophilic bacteria can exploit the ecological niche of the intestinal epithelium; Helicobacter and Campylobacter are previously implicated in IBD pathogenesis. The aim of the study was to assess these and other microaerophilic bacteria in de-novo paediatric IBD. Methods 100 children undergoing colonoscopy were recruited including 44 treatment naive de-novo IBD patients and 42 with normal colons. Colonic biopsies were subjected to microaerophilic culture with Gram-negative isolates then identified by sequencing. Biopsies were also PCR screened for the specific microaerophilic bacterial groups: Helicobacteraceae, Campylobacteraceae and Sutterella wadsworthensis. Results 129 Gram-negative microaerophilic bacterial isolates were identified from 10 genera. The most frequently cultured was S. wadsworthensis (32 distinct isolates). Unusual Campylobacter were isolated from 8 subjects (including 3 C. concisus, 1 C. curvus, 1 C. lari, 1 C. rectus, 3 C. showae). No Helicobacter were cultured. When comparing IBD vs. normal colon control by PCR the prevalence figures were not significantly different (Helicobacter 11% vs. 12%, p = 1.00; Campylobacter 75% vs. 76%, p = 1.00; S. wadsworthensis 82% vs. 71%, p = 0.312). Conclusion This study offers a comprehensive overview of the microaerophilic microbiota of the paediatric colon including at IBD onset. Campylobacter appear to be surprisingly common, are not more strongly associated with IBD and can be isolated from around 8% of paediatric colonic biopsies. S. wadsworthensis appears to be a common commensal. Helicobacter species are relatively rare in the paediatric colon. Disclosure of Interest None Declared

Tu1704 The Role of the Fungal Microbiota in the Pathogenesis of De-Novo Pediatric Inflammatory Bowel Disease Using Next Generation Sequencing

Introduction Paediatric Inflammatory bowel disease (IBD) incidence is rising worldwide. Recently the role of the gut microbiota has been recognised as pivotal in disease pathogenesis. IBD microbial studies to date have focused on bacterial diversity assessment in established disease cohorts, with limited studies in treatment naive patients. In contrast to bacteria, the exact role of the colonising fungi and their pathogenic potential has not been fully explored. The aim of the study was to examine candidate fungal triggers at disease onset in children with IBD using pyrosequencing, utilising the Bacteria in Inflammatory bowel disease in Scottish Children Undergoing Investigation before Treatment (BISCUIT) study cohort. Methods 128 children undergoing colonoscopy were approached from three Scottish paediatric centres (Aberdeen, Glasgow and Dundee) with 100 ultimately recruited and biopsied; 44 IBD (comprising Crohn’s disease (CD; 29), ulcerative colitis (UC; 13) and IBD-type unspecified (2)), 42 normal colon controls (NCC) and 14 “others”. All IBD patient samples were taken from inflamed tissue. Fungal DNA was amplified on a reduced cohort of 37 recruits (13 CD, 12 UC, 12 NCC) using 18S rDNA primers. Roche 454 Titanium sequencing was conducted by NewGene (Newcastle, UK). Data analysis was performed using QIIME version 1.3.0 workflow. Taxonomy assignment of operational taxonomic units (OTUs) was performed according to ribosomal database project taxonomy. OTU tables were rarefied at 3,000 reads. Results Fungal DNA was amplifiable from 7 patient samples, 6 children with a diagnosis of IBD – 4 with CD (BISCUIT1, 31, 62 and 89), 2 children with UC (BISCUIT33 and 104) and 1 NCC (BISCUIT 27). Fungal diversity was assessed in all paediatric samples alongside three adult samples to act as comparison. The adult samples comprised 1 patient with UC (2UC21Aa) and 2 NCC (GH4 and GH9). Phylum level analysis indicated that all fungal sequences belonged to the Ascomycota and Basidiomycota phyla. Control patients contained predominantly Ascomycota sequences ( > 80% of sequences in all patients) whilst 6/7 IBD patients contained exclusively Basidiomycota species. Genus level analysis was undertaken and there was no similarity between fungal profiles from the paediatric and adult samples. Conclusion By using robust methodology we have characterised the IBD “fungal microbiota” at diagnosis in children. Based on the current study, it would appear that a distinctly altered fungal species profile is present at IBD disease presentation. Further work should now focus on expanding this study and identifying how to beneficially modify the microbiota using established and novel IBD treatments. Disclosure of Interest None Declared

The microaerophilic microbiota of de-novo paediatric inflammatory bowel disease: the BISCUIT study.

IntroductionChildren presenting for the first time with inflammatory bowel disease (IBD) offer a unique opportunity to study aetiological agents before the confounders of treatment. Microaerophilic bacteria can exploit the ecological niche of the intestinal epithelium; Helicobacter and Campylobacter are previously implicated in IBD pathogenesis. We set out to study these and other microaerophilic bacteria in de-novo paediatric IBD.Patients and Methods100 children undergoing colonoscopy were recruited including 44 treatment naïve de-novo IBD patients and 42 with normal colons. Colonic biopsies were subjected to microaerophilic culture with Gram-negative isolates then identified by sequencing. Biopsies were also PCR screened for the specific microaerophilic bacterial groups: Helicobacteraceae, Campylobacteraceae and Sutterella wadsworthensis. Results129 Gram-negative microaerophilic bacterial isolates were identified from 10 genera. The most frequently cultured was S. wadsworthensis (32 distinct isolates). Unusual Campylobacter were isolated from 8 subjects (including 3 C. concisus, 1 C. curvus, 1 C. lari, 1 C. rectus, 3 C. showae). No Helicobacter were cultured. When comparing IBD vs. normal colon control by PCR the prevalence figures were not significantly different (Helicobacter 11% vs. 12%, p = 1.00; Campylobacter 75% vs. 76%, p = 1.00; S. wadsworthensis 82% vs. 71%, p = 0.312).ConclusionsThis study offers a comprehensive overview of the microaerophilic microbiota of the paediatric colon including at IBD onset. Campylobacter appear to be surprisingly common, are not more strongly associated with IBD and can be isolated from around 8% of paediatric colonic biopsies. S. wadsworthensis appears to be a common commensal. Helicobacter species are relatively rare in the paediatric colon.Trial RegistrationThis study is publically registered on the United Kingdom Clinical Research Network Portfolio (9633).

Stool DNA testing for the detection of pancreatic cancer

Pancreatic cancer (PanC) presents at late stage with high mortality. Effective early detection methods are needed. Aberrantly methylated genes are unexplored as markers for noninvasive detection by stool testing. The authors aimed to select discriminant methylated genes and to assess accuracy of these and mutant KRAS in stool to detect PanC. Nine target genes were assayed by real-time methylation-specific polymerase chain reaction (MSP) in bisulfite-treated DNA from microdissected frozen specimens of 24 PanC cases and 30 normal colon controls. Archived stools from 58 PanC cases and 65 controls matched on sex, age, and smoking were analyzed. Target genes from fecal supernatants were enriched by hybrid capture, bisulfite-treated, and assayed by MSP. KRAS mutations were assayed using the QuARTS technique. Areas under the receiver operating characteristics curves (AUCs) for tissue BMP3, NDRG4, EYA4, UCHL1, MDFI, Vimentin, CNTNAP2, SFRP2, and TFPI2 were 0.90, 0.79, 0.78, 0.78, 0.77, 0.77, 0.69, 0.67, and 0.66, respectively. The top 4 markers and mutant KRAS were evaluated in stool. BMP3 was the most discriminant methylation marker in stool. At 90% specificity, methylated BMP3 alone detected 51% of PanCs, mutant KRAS detected 50%, and combination detected 67%. AUCs for methylated BMP3, mutant KRAS, and combination in stool were 0.73, 0.75, and 0.85, respectively. This study demonstrates that stool assay of a methylated gene marker can detect PanC. Among candidate methylated markers discriminant in tissue, BMP3 alone performed well in stool. Combining methylated BMP3 and mutant KRAS increased stool detection over either marker alone.

Abstract 4627: Proximal fluid proteome profiling of human colorectal cancer tissue reveals candidate biomarkers for CRC screening

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer death in the Western world. Detection of CRC at an early stage of disease is associated with a much better prognosis for the patient, and is a realistic approach to reduce CRC mortality rates. Several randomised trials have shown that FOBT screening, ie detection of blood-derived haem in feces, reduces CRC mortality by ∼16%. However, it is commonly recognized that sensitivity and specificity of non-invasive CRC screening tests need to be improved, for which novel biomarkers are urgently needed. Aim: The aim of this study is to identify novel protein biomarkers that can be used for development of a blood-based or stool-based screening test for early diagnosis of CRC. Approach: Tumor “proximal fluids” are a rich source of tumor-derived proteins, comprising proteins that are either secreted, shed by membrane vesicles (exosomes), or externalized due to cell death. Fresh human colon carcinoma tissue and matched normal colon tissue samples were obtained from four patients directly following surgery. Histological evaluation indicated large variation among tumor samples in staging (I, III, and 2x IV) and type (3 adenocarcinomas, 1 mucinous tumor). Tissues were briefly rinsed and incubated in PBS at 37°C for one hour. Proximal fluids of normal and CRC tissues were collected and subjected to in-depth proteome profiling by a GeLC-MS/MS workflow. Quantitative comparisons were based on label-free spectral counting, p-values were calculated using the Mantel Haenszel test, and the single-test threshold for significance was set to p&amp;lt;1e-5 to withstand correction for multiple testing. Ingenuity Pathway Analysis (IPA) was used to support data interpretation. Results: A total of 2817 proteins were identified from proximal fluids of human colon carcinoma and patient-matched normal colon control tissues. Of these, 55 proteins were consistently and significantly (single test p&amp;lt;0.00001) more secreted by CRC samples than by controls. Data integration with a previously obtained mouse colon tumor proximal fluid proteome indicated 26 overlapping candidates, including several minichromosome maintenance MCM proteins that have been proposed in literature as markers for (stool-based) CRC screening. Conclusion &amp; Discussion: We conclude that proximal fluid proteome profiling of human CRC tissue is a powerful strategy to discover novel candidate biomarkers for CRC screening. Despite large variation in tumor stage and type between the four samples analyzed more than 50 candidate CRC biomarkers were identified. Further validation studies are required to investigate whether these candidates are robust biomarkers for CRC screening. Acknowledgments: This research is supported by the Cancer Center Amsterdam. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4627.