Blood-based Screening Research Articles

Use of blood-based biomarkers for early diagnosis and surveillance of colorectal cancer.

Early screening for colorectal cancer (CRC) holds the key to combat and control the increasing global burden of CRC morbidity and mortality. However, the current available screening modalities are severely inadequate because of their high cost and cumbersome preparatory procedures that ultimately lead to a low participation rate. People simply do not like to have colonoscopies. It would be ideal, therefore, to develop an alternative modality based on blood biomarkers as the first line screening test. This will allow for the differentiation of the general population from high risk individuals. Colonoscopy would then become the secondary test, to further screen the high risk segment of the population. This will encourage participation and therefore help to reach the goal of early detection and thereby reduce the anticipated increasing global CRC incidence rate. A blood-based screening test is an appealing alternative as it is non-invasive and poses minimal risk to patients. It is easy to perform, can be repeated at shorter intervals, and therefore would likely lead to a much higher participation rate. This review surveys various blood-based test strategies currently under investigation, discusses the potency of what is available, and assesses how new technology may contribute to future test design.

Blood-Based Testing for Colorectal Cancer Screening

Colorectal cancer (CRC) is the third most common non-skin cancer diagnosed in men and women in the USA and worldwide. While it has been clearly established that screening for CRC, using a variety of methods, is cost effective and has a significant impact on overall survival, screening rates have proven to be sub-optimal. It has been long conjectured that a simple blood-based test, with a specimen drawn at a routine doctor's office visit, would encourage those individuals who have refused or ignored screening recommendations to undergo screening. This article reviews the currently available blood-based screening tests for CRC, including the ColonSentry™ messenger RNA (mRNA) expression panel and the SEPT9 methylated DNA test, and explores newer biomarkers that are near clinical implementation. Also discussed are additional applications for blood-based CRC testing, such as assessing prognosis, disease surveillance, and expansion of screening tests to high-risk populations, such as the estimated 1.4 million individuals in the USA with inflammatory bowel disease.

Aberrant septin 9 DNA methylation in colorectal cancer is restricted to a single CpG island

BackgroundThe septin 9 gene (SEPT9) codes for a GTP-binding protein associated with filamentous structures and cytoskeleton formation. SEPT9 plays a role in multiple cancers as either an oncogene or a tumor suppressor gene. Regulation of SEPT9 expression is complex and not well understood; however, hypermethylation of the gene was recently introduced as a biomarker for early detection of colorectal cancer (CRC) and has been linked to cancer of the breast and of the head and neck. Because the DNA methylation landscape of different regions of SEPT9 is poorly understood in cancer, we analyzed the methylation patterns of this gene in distinct cell populations from normal and diseased colon mucosa.MethodsLaser capture microdissection was performed to obtain homogeneous populations of epithelial and stromal cells from normal, adenomatous, and tumorous colon mucosa. Microdissected samples were analyzed using direct bisulfite sequencing to determine the DNA methylation status of eight regions within and near the SEPT9 gene. Septin-9 protein expression was assessed using immunohistochemistry (IHC).ResultsRegions analyzed in SEPT9 were unmethylated in normal tissue except for a methylation boundary detected downstream of the largest CpG island. In adenoma and tumor tissues, epithelial cells displayed markedly increased DNA methylation levels (>80%, p <0.0001) in only one of the CpG islands investigated. SEPT9 methylation in stromal cells increased in adenomatous and tumor tissues (≤50%, p <0.0001); however, methylation did not increase in stromal cells of normal tissue close to the tumor. IHC data indicated a significant decrease (p <0.01) in Septin-9 protein levels in epithelial cells derived from adenoma and tumor tissues; Septin-9 protein levels in stromal cells were low in all tissues.ConclusionsHypermethylation of SEPT9 in adenoma and CRC specimens is confined to one of several CpG islands of this gene. Tumor-associated aberrant methylation originates in epithelial cells; stromal cells appear to acquire hypermethylation subsequent to epithelial cells, possibly through field effects. The region in SEPT9 with disease-related hypermethylation also contains the CpGs targeted by a novel blood-based screening test (Epi proColon®), providing further support for the clinical relevance of this biomarker.

SILAC‐based quantitative proteomic analysis of gastric cancer secretome

Gastric cancer is a commonly occurring cancer in Asia and one of the leading causes of cancer deaths. However, there is no reliable blood-based screening test for this cancer. Identifying proteins secreted from tumor cells could lead to the discovery of clinically useful biomarkers for early detection of gastric cancer. A SILAC-based quantitative proteomic approach was employed to identify secreted proteins that were differentially expressed between neoplastic and non-neoplastic gastric epithelial cells. Proteins from the secretome were subjected to SDS-PAGE and SCX-based fractionation, followed by mass spectrometric analysis on an LTQ-Orbitrap Velos mass spectrometer. Immunohistochemical labeling was employed to validate a subset of candidates using tissue microarrays. We identified 2205 proteins in the gastric cancer secretome of which 263 proteins were overexpressed greater than fourfold in gastric cancer-derived cell lines as compared to non-neoplastic gastric epithelial cells. Three candidate proteins, proprotein convertase subtilisin/kexin type 9 (PCSK9), lectin mannose binding 2 (LMAN2), and PDGFA-associated protein 1 (PDAP1) were validated by immunohistochemical labeling. We report here the largest cancer secretome described to date. The novel biomarkers identified in the current study are excellent candidates for further testing as early detection biomarkers for gastric adenocarcinoma.

Development of a high-performance, blood-based screening diagnostic to detect early-stage breast cancer.

e22083 Background: Limitations of current screening mammography, particularly in younger women, demonstrate the need for an alternative breast cancer screening strategy. A non-invasive, easily interpreted and low cost test should address this need. Methods: Gene expression microarray analysis was carried out on 128 individual tumor samples representing over 20 tumor types, 86 samples representing 31 diverse normal tissue types, 68 tumor cell lines and 97 diverse normal primary cell cultures. Genes were ranked for elevated expression in either: i) a large number and variety of tumors relative to normal tissues, or ii) in breast tumors. Elevated expression was verified for a subset of genes using qPCR in a set of independent RNA samples. Proteins coded by genes elevated in breast cancer samples were analyzed in a retrospective training set of breast cancer patient sera samples with cancer-free patient and benign pathology controls using ELISA or bead-based detection assay. Results: Based on availability of suitable reagents, 25 candidate biomarkers were assessed in patient sera samples (31-227 patient samples per biomarker) using ELISA or bead-based assays. Individually, the performance of individual markers varied (ROC AUC, 0.51 - 0.88); however, when expression levels of the best performing markers were combined, the multiplex test demonstrated high-sensitivity (&gt;80%) and specificity (&gt;90%) in identifying early-stage breast cancer patients. Conclusions: A multiplex, proteomic-based approach may provide for a high-performance, blood-based screening diagnostic for breast cancer.

Mo1181 Are Colorectal Cancer Screening Participation Rates Better Than We Think? An Analysis of Screening Behaviour Inside and Outside of a Four Year Fecal Occult Blood-Based Screening Program

Mo1181 Are Colorectal Cancer Screening Participation Rates Better Than We Think? An Analysis of Screening Behaviour Inside and Outside of a Four Year Fecal Occult Blood-Based Screening Program

Discovery of early detection markers of pancreatic cancer and clinical application in blood-based screening

Discovery of early detection markers of pancreatic cancer and clinical application in blood-based screening

Blood-based screening for methylation changes in colorectal cancer patients using novel nanotechnologies.

384 Background: Identification of blood-based biomarkers for cancer screening is essential in order to develop novel and minimally invasive methods for colorectal cancer screening. Our lab has successfully applied a novel nanotechnology that allows us to detect and amplify a single tumor DNA fragment in a plasma sample. This DNA is tested for methylation of several genes including TFPI2 which has shown to be highly sensitive and specific for the detection colorectal cancer in stool. Methods: Whole blood was obtained from 18 colorectal cancer patients and plasma was isolated. Plasma was processed using Methylation On Beads nanotechnology (MOB) and bisulfate treated. Methylation status was determined via quantitative PCR method. Results: Two genes, TFPI2 and IGFBP3, were detected with a high sensitivity. TFPI2, demonstrated a methylation frequency of 94.4%, which is concordant with the TFPI2 methylation frequency of 99% in primary colorectal cancer tissues. IGFBP3 showed the methylation frequency of 61.1%, which corresponds with the methylation frequency of 52% in retrospective colorectal cancer tissues in previous studies. Quantification using standard curves indicated a single copy level of DNA found in plasma. Conclusions: Blood-based screening is challenging due to extremely low quantities of circulating DNA in blood. Utilizing a novel nanotechnology that detects DNA at a single copy level, the methylation changes in colorectal cancer were successfully detected in plasmas at similar frequencies as in tissue samples. This study has demonstrated the feasablility and applicability to blood-based screening. Future studies will focus on improving the sensitivity and determining the specificity of this method.

BRCA2 Promoter Hypermethylation in Sporadic Breast Cancer

<i>BRCA2</i> Promoter Hypermethylation in Sporadic Breast Cancer

Abstract LB-382: DKK3, ITIH5 and RASSF1A gene methylation as novel biomarkers for blood-based breast cancer screening: Towards improving early detection of breast cancer

Abstract Aims: For early detection of breast cancer the development of robust blood-based biomarkers that accurately reflect the host tumor is mandatory and thus a growing field of research. The most common alterations in human cancers including breast cancer are changes in the status of DNA methylation, which are therefore quickly emerging as a new pool of potential biomarkers. Thus, we investigated the feasibility of detecting aberrant tumor suppressor gene methylation in cancer cell-derived free circulating DNA in the bloodstream of breast cancer patients. Methods: Using qualitative MSP, we examined the methylation status of seven biologically significant putative tumor suppressor genes, i.e. ITIH5, DKK3, WIF1, RASSF1A, SFRP1, SFRP2 and SFRP5 in DNA extracted from serum. Free circulating DNA was isolated with the QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany). Clinical performance was determined in a training study on 240 serum samples (120 breast cancers, 120 age-matched healthy controls). 20 benign gynaecological disease and 30 colon cancer serum samples were included for additional specificity testing. Results: Based on the training study we could evaluate the top candidate biomarkers with the best values for sensitivity and specificity. A marker panel with DKK3 and ITIH5 detected breast cancer with a sensitivity of 42% (50/120). Specificity of the panel was sufficient with 83%, 100% and 93% in colon cancer samples, benign and healthy control samples, respectively. Control samples revealed unacceptable high methylation rates of SFRP1 and SFRP5 in DNA extracted from colon cancer sera, whereas SFRP2 and WIF1 showed a considerable methylation frequency in sera from healthy controls. The well-established potential biomarker RASSF1A was included as a reference gene. RASSF1A was highly specific in the analysed benign and healthy control samples (100%) why we integrated RASSF1A in our independent test study to improve sensitivity and specificity of the marker panel with DKK3 and ITIH5. Conclusions: The current study suggests that cancer-specific methylation of ITIH5 and DKK3 in serum-derived tumor-borne DNA might be valuable biomarkers for early detection of breast cancer. In the second phase of this project we are currently validating with quantitative MSP ITIH5, DKK3 and RASSF1A as reliable methylation biomarkers in an independent test set consisting of 140 breast cancer serum samples, 140 age-matched healthy controls, 40 benign gynaecological disease and 30 colon cancer serum samples for additional specificity testing. To date, this is the first study investigating the potential of ITIH5 and DKK3 in combination with the well-established biomarker RASSF1A as reliable blood-based methylation biomarkers in a large-scale serum collective of breast cancer and matched control samples. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-382. doi:1538-7445.AM2012-LB-382

Sensitivity of second-generation blood-based methylated Septin9 assay for early-stage colorectal cancer.

419 Background: Early detection of colorectal cancer (CRC) has been shown to decrease mortality, although compliance to CRC screening is low. Availability of a blood-based test is expected to improve CRC screening compliance. Specific detection of CRC using the Septin9 biomarker (mSEPT9) in a large prospective trial of an average-risk CRC screening population exhibited 67% sensitivity for CRC with 88% specificity. Laboratory-developed tests detecting mSEPT9 in plasma are now available in North America and a 2nd generation molecular diagnostic blood test for mSEPT9 is available as a CE-marked kit in Europe. The current research evaluated the clinical performance of the 2nd generation mSEPT9 assay. Methods: Bisulfite-converted DNA (bisDNA) was prepared from 3.5 mL human plasma using the 2ndgeneration plasma DNA preparation kit. Resulting bisDNA was analyzed in triplicate on the ABI7500 Fast Dx (Life Technologies, Inc.) using proprietary HeavyMethyl real-time PCR technology for mSEPT9 and the 2nd generation real-time PCR kit. In a case – control design, plasma from 98 CRC patients (n = 87 stages I - III) and 99 age-matched, colonoscopy-verified normal individuals were processed with the mSEPT9 assay. In addition, plasma from 150 prospectively enrolled average risk individuals scheduled for screening colonoscopy was tested. mSEPT9 was qualitatively analyzed such that any detection of mSEPT9 in a PCR was called “positive”. Results: The revised mSEPT9 assay exhibited 95% sensitivity (95% CI: 89-98%) for CRC. Sensitivity for stage I was 89% (95% CI: 72-96%, n = 27) and sensitivity for stage II was 93% (95% CI: 78-98%, n = 29). The control group was positive at a rate of 16% (95% CI: 10-25%). Specificity of the mSEPT9 assay in the screening cohort was 85% (95% CI: 78-89%). Conclusions: The 2nd generation mSEPT9 assay demonstrated improved sensitivity for CRC without significant impact on specificity. The enhanced design and robustness of the assay will further facilitate its standardized use in routine laboratory settings. Finally, the increased sensitivity of the revised mSEPT9 assay improves the detection of early stage disease and demonstrates the feasibility of a blood-based CRC screening technology.

Sample preference for colorectal cancer screening tests: Blood or stool?

Objective: Despite the high prevalence of CRC and the proven benefits of faecal sampling tests, participation rates in CRC screening are suboptimal. Literature has identified a number of barriers to participation, including faecal aversion. Emerging test technologies suggest blood-based molecular markers might provide an alternative, more acceptable option, for CRC screening tests. We aim to determine preference for blood compared to faeces as the sample for the screening test. Methods: A survey was mailed to 956 South Australians aged 50 to 74 years. Data were collected on sample preference, demographic variables, and ratings of screening test convenience and comfort. Results: The survey yielded a 43% response rate. The majority of participants preferred to provide a blood sample (78% v 22%, p < 0.001). Women were more likely to prefer blood than men (82% vs 74%, p = 0.05). Sample experience influenced preferences, with a significantly higher preference for faeces among participants with experience in faecal sampling (27% vs 17% with no experience, p < 0.05). Participants who preferred to provide a faecal sample rated it significantly more convenient (p < 0.001), more comfortable (p < 0.001), and more acceptable (p < 0.001) than those who preferred blood sampling. Conclusions: Survey participants overwhelmingly indicate a preference for the idea of a blood sample over a faecal sample for CRC screening. Preference was influenced by gender, experience with sampling method and the individual’s perception of sampling convenience, sampling comfort and sample acceptability. Our results suggest population participation rates are likely to improve with blood-based screening tests.

A blood-based screening tool for Alzheimer's disease that spans serum and plasma: findings from TARC and ADNI.

ContextThere is no rapid and cost effective tool that can be implemented as a front-line screening tool for Alzheimer's disease (AD) at the population level.ObjectiveTo generate and cross-validate a blood-based screener for AD that yields acceptable accuracy across both serum and plasma.Design, Setting, ParticipantsAnalysis of serum biomarker proteins were conducted on 197 Alzheimer's disease (AD) participants and 199 control participants from the Texas Alzheimer's Research Consortium (TARC) with further analysis conducted on plasma proteins from 112 AD and 52 control participants from the Alzheimer's Disease Neuroimaging Initiative (ADNI). The full algorithm was derived from a biomarker risk score, clinical lab (glucose, triglycerides, total cholesterol, homocysteine), and demographic (age, gender, education, APOE*E4 status) data.Major Outcome MeasuresAlzheimer's disease.Results11 proteins met our criteria and were utilized for the biomarker risk score. The random forest (RF) biomarker risk score from the TARC serum samples (training set) yielded adequate accuracy in the ADNI plasma sample (training set) (AUC = 0.70, sensitivity (SN) = 0.54 and specificity (SP) = 0.78), which was below that obtained from ADNI cerebral spinal fluid (CSF) analyses (t-tau/Aβ ratio AUC = 0.92). However, the full algorithm yielded excellent accuracy (AUC = 0.88, SN = 0.75, and SP = 0.91). The likelihood ratio of having AD based on a positive test finding (LR+) = 7.03 (SE = 1.17; 95% CI = 4.49–14.47), the likelihood ratio of not having AD based on the algorithm (LR−) = 3.55 (SE = 1.15; 2.22–5.71), and the odds ratio of AD were calculated in the ADNI cohort (OR) = 28.70 (1.55; 95% CI = 11.86–69.47).ConclusionsIt is possible to create a blood-based screening algorithm that works across both serum and plasma that provides a comparable screening accuracy to that obtained from CSF analyses.

Improved blood tests for cancer screening: general or specific?

Diagnosis of cancer at an early stage leads to improved survival. However, most current blood tests detect single biomarkers that are of limited suitability for screening, and existing screening programmes look only for cancers of one particular type. A new approach is needed. Recent developments suggest the possibility of blood-based screening for multiple tumour types. It may be feasible to develop a high-sensitivity general screen for cancer using multiple proteins and nucleic acids present in the blood of cancer patients, based on the biological characteristics of cancer. Positive samples in the general screen would be submitted automatically for secondary screening using tests to help define the likelihood of cancer and provide some indication of its type. Only those at high risk would be referred for further clinical assessment to permit early treatment and mitigate potential overdiagnosis. While the assays required for each step exist, they have not been used in this way. Recent experience of screening for breast, cervical and ovarian cancers suggest that there is likely to be widespread acceptance of such a strategy.

DNA methylation patterns in blood of patients with colorectal cancer and adenomatous colorectal polyps

Colorectal cancer (CRC) screening rates are currently suboptimal. Blood-based screening could improve rates of earlier detection for CRC and adenomatous colorectal polyps. In this study, we evaluated the feasibility of plasma-based detection of early CRC and adenomatous polyps using array-mediated analysis methylation profiling of 56 genes implicated in carcinogenesis. Methylation of 56 genes in patients with Stages I and II CRC (N=30) and those with adenomatous polyps (N=30) were compared with individuals who underwent colonoscopy and were found to have neither adenomatous changes nor CRC. Composite biomarkers were developed for adenomatous polyps and CRC, and their sensitivity and specificity was estimated using five-fold cross validation. Six promoters (CYCD2, HIC1, PAX 5, RASSF1A, RB1 and SRBC) were selected for the biomarker, which differentiated CRC patients and controls with 84% sensitivity and 68% specificity. Three promoters (HIC1, MDG1 and RASSF1A) were selected for the biomarker, which differentiated patients with adenomatous polyps and controls with sensitivity of 55% and specificity of 65%. Methylation profiling of plasma DNA can detect early CRC with significant accuracy and shows promise as a methodology to develop biomarkers for CRC screening.

A Blood-Based Screening Tool for Alzheimer's Disease That Spans Serum and Plasma: Findings from TARC and ADNI

A Blood-Based Screening Tool for Alzheimer's Disease That Spans Serum and Plasma: Findings from TARC and ADNI

A Genetic Strategy for Combined Screening and Localized Imaging of Breast Cancer

Improvements are needed for the early detection of breast cancer, as current imaging methods lack sensitivity to detect small tumors and assess their disease phenotype. To address this issue, the dual reporter adenoviral vector (Ad5/3-Id1-SEAP-Id1-mCherry) was produced with a cancer-specific Id1 promoter driving expression of a blood-based screening reporter (secreted embryonic alkaline phosphatase, SEAP) and a fluorescent imaging reporter (mCherry). This diagnostic system was assessed for its screening potential on breast cancer cell lines of various aggressive phenotypes. Reporter expression was measured and correlated with promoter level expression using Western blot. Adenovirus receptor expression was normalized against reporter expression with luciferase infectivity assays. Ad5/3-Id1-SEAP-Id1-mCherry infected MDA-MB-231 cells combined with uninfected cells were implanted into the mammary fat pad of athymic nude mice to recapitulate low-dose tumor delivery. Id1 driven SEAP expression and mCherry imaging were monitored to validate diagnostic sensitivity and efficacy. Infected breast cancer cell lines displayed SEAP levels in the media that were 10-fold above background by 2days after infection. Ad5/3-Id1-SEAP-Id1-mCherry infected cells (multiplicity of infection = 10) implanted in athymic nude mice demonstrated a 14-fold increase in serum SEAP levels over baseline when as little as 2.5% of the tumor contained infected cells. This robust response was also found for the mCherry reporter, which was clearly visible in tumor xenografts on day2 post implantation. This diagnostic system that combines screening with imaging for early detection and monitoring of breast cancer can be easily extended to other reporters/modalities and cancer-targeting methods. Combining screening with imaging in a genetic, cancer-specific mechanism allows sensitive multi-modal detection and localization of breast cancer.

Prospective Clinical Validation of an Assay for Methylated SEPT9 DNA in Human Plasma as a Colorectal Cancer Screening Tool in Average Risk Men and Women ≥50 Years

Prospective Clinical Validation of an Assay for Methylated SEPT9 DNA in Human Plasma as a Colorectal Cancer Screening Tool in Average Risk Men and Women ≥50 Years

S1136 A New Blood-Based Screening Test for Colorectal Cancer: A Pilot Study

Background: Colonoscopy is considered to be the standard of care for the diagnosis of colorectal cancer. However, population-based studies have reported a subset of patients with cancer who do not undergo colonoscopy. The purpose of this study was to estimate the prevalence and identify the predictors of not having a colonoscopy in the period preceding colorectal cancer diagnosis. Methods: Using the population-based SEER registries, we identified patients aged >= 69 with colorectal cancer diagnosed from 1994-2005. Linked inpatient and outpatientMedicare claimswere used to identify receipt of colonoscopy prior to diagnosis. We divided this group into patients who had did not have colonoscopy within 3 years of diagnosis (Group I) and those who had 1 or more colonoscopies from 6 months prior to 30 days after diagnosis (Group II). Patient, sociodemographic and tumor factors were used to identify predictors of not having colonoscopy in univariate and multivariable logistic regression analysis. Results: We identified 79,032 patients, including 19.6% in Group I and 80.4% in Group II. Among patients in Group I, 31.6% had barium enema, 21.4% had flexible sigmoidoscopy and 57.3% underwent CT scan within 6 months prior to and 30 days after diagnosis. Independent predictors of Group I included age > 85, African American race, non-married, nursing home residence, rural residence, lower comorbidity score, diagnosis before 2000, AJCC Stage II-IV, left sided or rectal tumor site, and emergency presentation. Patients without colonoscopy were also less likely to undergo surgical resection (OR 0.55, CI 0.52-0.59). In a Cox proportional hazards model that adjusted for demographics, stage and treatment, not undergoing colonoscopy was associated with a higher risk of death (HR 1.31, CI 1.28-1.33). Conclusions: In this large, population based analysis, almost 20% of newly diagnosed colorectal cancer patients did not undergo colonoscopy at the time of diagnosis. Although these patients were more likely to be elderly with advanced disease, lack of colonoscopy appears to be an indicator of emergency presentation, less aggressive treatment and poorer prognosis.

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.