Biomarkers of colorectal cancer: recent advances and future challenges.

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Proteomics - Clinical Applications

Biomarkers of colorectal cancer: recent advances and future challenges Caroline Coghlin1, Graeme I Murray2 1

Department of Pathology, Aberdeen Royal Infirmary, NHS Grampian, Aberdeen, UK;

2

Pathology, Division of Applied Medicine, School of Medicine and Dentistry, University of

Aberdeen, Aberdeen, UK.

Address correspondence to: Professor Graeme I Murray Phone: +44(0)1224 553794 Fax: +44(0)1224 663002 Email: [email protected]

Abbreviations: Carcinoembryonic antigen, CEA; chromosomal instability, CIN; circulating cell-free DNA, cfDNA; circulating tumour cell, CTC; colon cancer cancer stem cell, CCSC; colorectal cancer, CRC; CpG island methylator phenotype, CIMP; faecal occult blood, FOB; faecal immunochemical testing, FIT; microRNA, miRNA; microsatellite instability, MSI; reverse phase protein array, RPPA; tumour node metastasis, TNM; 78 kDa glucose-regulated protein precursor, GRP78.

Key words: biomarker, colorectal cancer, diagnosis, immunohistochemistry, predictive, prognosis, proteomics

Word count: 6444

Received: 24-Jul-2014; Revised: 19-Sep-2014; Accepted: 07-Oct-2014 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/prca.201400082. This article is protected by copyright. All rights reserved.


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Abstract Colorectal cancer (CRC) is a common malignancy and it contributes significantly to cancer mortality. Outcomes in colorectal cancer vary between patients and this is due to the complexity of colorectal carcinogenesis. Interactions between tumour cells and their microenvironment, genetic alterations and changes in intracellular signalling networks are just some of the abnormal pathways involved in colorectal cancer development. Recent research has targeted components of all of these systems in order to develop biomarkers to aid in the early diagnosis of CRC and to assist in prognostic stratification. Proteomic analysis of tissue or blood-derived samples from CRC patients has proven to be a valuable technique for the identification of potentially informative biomarkers. Such biomarkers may prove to be clinically applicable and could offer greater patient acceptability when compared to conventional methods such as faecal-based testing. In this article we review the recent advances in the development of protein biomarkers of CRC with an emphasis on biomarkers available in the patient's serum and from tissue-based samples. Future challenges in terms of the development of accurate diagnostic, prognostic and predictive biomarkers of CRC and the importance of validation and patient acceptability are also discussed.

This article is protected by copyright. All rights reserved.


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Introduction Colorectal cancer is one of the commonest cancers in both men and women with an estimated annual incidence of 1 million new cases [1]. It also contributes significantly to cancer-related deaths [2]. The prognosis in colorectal cancer patients is dependent on the stage at which the disease is diagnosed. The 5 year survival for patients with early localised disease may be as high as 90% while patients presenting with distant metastases may have a 5 year survival of less than 10% [2]. Currently, due to a frequent lack of early disease-specific symptoms and a reluctance to seek medical investigation, many colorectal cancer cases present late when the disease is at a relatively advanced stage. Colorectal cancer often appears to develop slowly over years and, in many cases, there is an initial pre-invasive polyp stage. For this reason, unlike some other solid malignancies, colorectal cancer is a good candidate for prevention screening strategies. A meta-analysis of trials based on screening with faecal occult blood (FOB) tests indicated a 25% reduction in colorectal cancer-related mortality [3]. Similarly, recent results of flexible sigmoidoscopy-based screening programmes have shown dramatic reductions in colorectal cancer incidence and mortality [4]. Early detection of colorectal cancer in its localised or preinvasive form is therefore likely to represent the most realistic approach to reducing the number of cancer-related deaths [5]. Current screening strategies that are based around faecal occult blood testing or colonoscopy suffer from poor uptake based on a lack of patient acceptability. An alternative strategy is to develop reliable and specific biomarkers detectable in a more readily accessible medium, such as the peripheral blood, that can accurately and reliably detect colorectal cancer in its earliest stages when treatment options can be maximised [6]. Clinically applicable biomarkers of CRC are not only required for the early detection of the disease, they are also needed for accurate diagnosis, prognostic stratification and surveillance of CRC following treatment [7, 8]. Increasingly, with rapid progress in the development of adjuvant and neoadjuvant therapies and the toxicities associated with these treatments, there is also an urgent need for predictive biomarkers of colorectal cancer [6, 9]. In the clinical setting, biomarkers of CRC may be derived from a patient's tissue (endoscopically derived biopsies), their peripheral blood or a stool sample. Recent research has focussed on diverse biomarkers such as circulating tumour cells (CTC), cell-free DNA (cfDNA) and microRNA (miRNA) but a rich source of potentially important CRC biomarkers is also derived from proteomic analysis of plasma and tissue samples. The aim of This article is protected by copyright. All rights reserved.


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this review is to discuss the recent advances in the development of protein biomarkers for CRC with an emphasis on the discovery and clinical application of protein biomarkers for the early detection, prognosis, response to therapy and post-operative surveillance of colorectal cancer (figure1).

Diagnostic biomarkers in CRC Pathological diagnosis of CRC in formalin fixed tissue derived from colonoscopy procedures is often straight forward based on the site of origin of the lesional tissue and the characteristic histological features of primary CRC. However, accurate identification of CRC in secondary sites often requires detection of protein biomarkers using immunohistochemistry. A panel of immunohistochemical markers can guide appropriate treatment in such cases. CRC cells consistently express cytokeratin 20 and CDX-2 (an intestinal-specific transcription factor) and they are generally negative for cytokeratin 7 [911]. Using technology based on magnetic bead sample preparation, MALDI-TOF MS for mass spectra acquisition and sophisticated software programmes for comparison of data sets, a recent study analysed proteomic profiles of serum samples with the aim of identifying protein biomarkers of early CRC [12]. 35 healthy subjects, 35 patients with advanced colorectal adenomas (classified in terms of morphology and/or size) and 40 patients with established CRC were compared using these techniques. Kininogen-1 was identified as a potentially useful biomarker for both the early detection and for prognostic purposes in CRC. The results were then verified using enzyme-linked immunosorbent assays and immunohistochemistry carried out on tissue samples. Serum levels of kininogen-1 assessed using proteomic techniques could be used to indicate the presence of advanced colorectal adenomas compared to the control group. In addition, measurement of kininogenin-1 levels for diagnosing Dukes stage A and B (stage 1 and stage 2) CRC was found to be more accurate, specific and sensitive than using measurement of serum CEA. The precise role of kininogen-1 in CRC development is not well understood. One other group had previously produced similar results suggesting that kininogen-1 might be useful as a screening biomarker in early CRC [13]. However, at least one other earlier study showed that kininogen-1 was actually present in lower than normal levels in the plasma samples of 118 patients with gastrointestinal cancer [14]. The precise role of kininogen-1 as a protein biomarker of early CRC is therefore yet to be fully established. This article is protected by copyright. All rights reserved.


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Peripheral blood is not the only easily accessible body fluid in which to noninvasively screen for protein biomarkers of early CRC. Using a combination of gene expression and metabolomic mass spectrometry-based proteomic analysis, urine and tissue samples from experimental mice and humans were studied to identify biomarkers of early colorectal carcinogenesis [15]. Thirteen biomarkers of CRC development were initially identified in genetically susceptible mice. These included markers related to nucleic acid metabolism and methylation. Analysing the urine of mice for a panel of these biomarkers enabled the accurate identification of both tumour and adenoma-bearing animals. Analysis of human tumour and non-tumour tissues revealed overlap between some of the biomarkers found in the urine of tumour-bearing mice and malignant human tissue. This work therefore indicates the existence of abnormal metabolic pathways present early in CRC development which could potentially be used for early non-invasive detection of the disease.

Prognostic biomarkers in CRC Outcome in CRC and the choice of treatment strategy is currently guided by staging protocols such as the tumour, node and metastasis (TNM) system. The gold standard for treatment in CRC remains surgical resection but, with the significant risk of progressive disease in stage III or high risk stage II cases, the addition of adjuvant therapies becomes important. Colorectal cancer is a complex and variable disease with at least three major pathways operating within CRC carcinogenesis including chromosomal instability (CIN), microsatellite instability (MSI) and epigenetic alteration through CpG island methylator phenotype (CIMP) [16]. In addition to genetic events occurring within CRC cells, cancer progression is also closely linked to the relationship between tumour cells and their microenvironment [17-20]. Such complexity at the molecular level translates into inevitable variability in terms of clinical outcome, patient presentation and response to chemotherapeutic agents. Therefore, although the TNM system remains useful in guiding prognosis in CRC, many researchers have sought to improve on the accuracy of this framework by developing accurate and reliable biomarkers to predict the clinical outcome in CRC. The development of gene transcript signatures for detecting recurrence and progression in CRC has produced encouraging results although validation studies are still urgently required. ColoGuidePro uses 7 genes to stratify risk of progression in stage III CRC patients [21]. Coloprint™ is an 18-gene test based on the analysis of fresh frozen tissue which aims to improve prognostication in patients with stage II and stage III CRC [22, 23]. This article is protected by copyright. All rights reserved.


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An advantage of these multigene tools is that they attempt to address the problem of heterogeneity in CRC by focussing on multiple target genes. However, although some of these kits are now commercially available none have progressed into routine clinical use. Comparative proteomic analysis of malignant, normal and metastatic colorectal tissue has led to the identification of numerous putative prognostic protein biomarkers. Many such biomarkers have been subsequently validated by immunohistochemical analysis in microarrayed tissue samples which have then been correlated with reference to survival data [24-30]. Prognostically useful protein biomarkers should ideally be readily assessable immunohistochemically in formalin-fixed biopsy fragments from colorectal cancers and they must show clear and reproducible staining patterns which can be consistently correlated with tumour biology and clinically significant outcomes (Table 1). Increased levels of survivin and human telomerase reverse transcriptase, as assessed by immunohistochemistry in colonoscopically-detected adenomas, have been associated with an increased risk of developing metachronous colorectal cancers, with the metachronous colorectal tumours presenting at a mean of 5.6 years from the initial index adenoma [16]. Immunohistochemistry for MLH1 and MSH2 as markers of MSI have been correlated with a relatively good prognosis in early colorectal cancers and with a poor response to some chemotherapy agents [24]. Peng et al used systematic proteomic analysis of CRC tissue in different TNM stages (I-IV) to detect differentially expressed proteins [31]. The aim of this study was to develop molecular staging systems which might supplement, or even replace, conventional TNM-based staging. MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) was used to identify several key proteins involved in intracellular signalling and metabolism including, 78 kDa glucoseregulated protein precursor (GRP78), fructose-bisphosphate aldolase A, carbonic anhydrase I and peptidyl-prolyl cis–trans isomerase A. The patterns of these proteins appeared to be differentially expressed between TNM stages in CRC. The authors concluded that these novel biomarkers could augment TNM staging in CRC and may be particularly useful in risk stratifying stage III and stage IV patients. A study aimed at identifying early changes in the development of CRC compared whole tissue lysates from 28 colorectal adenomas and established CRCs (stages I-IV). This group employed quantitative proteomic analysis using a combination of laser microdissection, non-gel based separation, iTRAQ labelling and MALDI-TOF/TOF MS. A potentially significant protein biomarker, OLFM4 was identified which was shown to be



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increased in adenomas and early stage CRC compared with more advanced disease (stages III-IV) [32]. Lymph node dissemination is known to negatively affect prognosis but there are very few accurate protein biomarkers of CRC metastasis. Recently, three candidate protein biomarkers, FXYD3, S100A11 and GSTM3 were identified as potentially useful markers of regional lymph node metastasis in CRC [33]. This study used a tissue-based proteomic approach employing both MALDI imaging and label-free quantitative proteomics to identify both small and large proteins that were differentially expressed between primary CRC without nodal metastases and those which did have nodal metastases. All three biomarkers were significantly correlated with the presence of nodal metastases in the study cases and these findings were subsequently validated by immunohistochemical staining on an independent primary CRC tissue cohort. Similarly, quantitative proteomic analysis of CRC tissue from lymph node positive and lymph node negative tumours identified MX1 as a protein biomarker for predicting regional metastatic disease [34]. A recent study using iTRAQ technology has shown alterations in NADH dependent oxidative enzymes in colorectal cancers compared with paired normal mucosal samples [35]. Glutathione Stransferase pi expression has also been shown to be both prognostic of outcome and also predictive of response to 5-fluorouracil chemotherapy in Dukes C (stage 3, lymph node positive) colon cancer [36, 37].

Screening biomarkers in CRC If a significant number of cases of CRC develop slowly over many years and include a well-defined pre-malignant polyp stage, then this cancer should represent a good target for effective screening programmes. The aim of such programmes is to detect cancers early and institute potentially curable treatment before the disease can spread [38]. Attempts to diagnose lesions at an early invasive or pre-malignant stage have been made using modalities such as FOB testing, serum carcinoembryonic antigen (CEA) screening and colonoscopy [39]. Each of these methods has its disadvantages. Colonoscopy is invasive and incurs a degree of risk while the other more indirect methods lack sensitivity. The issue of participant acceptability is also extremely important as invasive or faecal-based screening tests inevitably suffer from a degree of poor participant uptake. Guaiac based or immunohistochemical stool testing in colorectal cancer screening has proved useful in the past but false positive results are relatively common and these can result in inappropriate invasive procedures. In recent years multitarget stool DNA tests have been This article is protected by copyright. All rights reserved.


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developed which may represent the most sensitive non-invasive test to date for the early detection of CRC. In a large scale study of 9989 asymptomatic participants faecal immunochemical testing (FIT) was measured against stool DNA analysis with colonoscopy used to determine the tests' accuracy [40]. The DNA test included quantitative molecular assays for KRAS mutations, aberrant NDRG4 and BMP3 methylation and beta actin with the addition of a haemoglobin immunoassay. The stool DNA test had greater sensitivity for the detection of CRC than FIT (92.3% versus 73.8%) and advanced pre-cancerous lesions (42.4% versus 23.8%). However, the stool DNA test produced more false positive results than the FIT test in patients who subsequently had a negative colonoscopy. Although these results are promising, translating this technology into routine clinical practice may require further validation studies and cost effectiveness analysis. In a potential move away from faecal-based or invasive screening modalities, the aim of many recent biomarker studies has been to identify robust plasma-based biomarkers which represent an acceptable form of patient testing. Such biomarkers should ideally be able to accurately identify early invasive or even pre-invasive CRC in a sensitive and specific manner. Several different classes of potential screening biomarker are available for study in the peripheral blood of patients. These include microRNAs, cell-free DNA, circulating tumour cells and proteins biomarkers. miRNAs are small non-coding RNA strands that can post-transcriptionally regulate the expression of multiple target genes. Their measurement in the serum of CRC patients has shown promise for early disease detection and, potentially, for monitoring cancer recurrence after treatment [41, 42] . Levels of the oncogenic miRNA, miRNA-21 were found to be significantly raised in CRC patients and patients with colonic adenomas when compared with healthy controls [43]. Elevated levels of miRNA-21 in both tumour tissue and the patients’ serum were found to be significantly associated with tumour size, the presence of metastases and reduced survival. Although technology is advancing rapidly in this area, extraction of miRNA for analysis in cancer patients' plasma or serum for screening purposes is difficult and this had led to inconsistent results between studies. A standardised laboratory approach may be required for validation of such analysis if it is to translate into clinically accurate and consistent tests. cfDNA which may be released from tumour cells by active secretion, apoptosis or necrosis, has recently been investigated as a source of potentially useful CRC biomarkers [41]. Using quantitative real-time PCR to detect ALU repeats in the plasma or serum of operated or non-operated CRC patients and healthy controls, a recent study showed that This article is protected by copyright. All rights reserved.


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circulating levels of cfDNA were significantly higher in non-operated CRC patients when compared with operated patients and controls [44]. The authors suggested that analysis of cfDNA levels in the peripheral blood of patients could be useful both in the early detection of the disease and in monitoring progression after treatment. Several groups have recently focussed on detecting CTCs. These are malignant cells released from a cancerous lesion into the peripheral bloodstream. Initial attempts to measure and analyse CTCs for the purpose of diagnosing early (stage I) CRC have shown inconsistent results [45]. However, elevated levels of CTCs in the peripheral blood may be a marker of poor prognosis in locally advanced disease or of recurrence after surgery [45, 46]. CTC levels in the peripheral blood may be a marker of disease progression. Their role in metastasis is currently the subject of intense study but their utility as a screening biomarker is yet to be proven [47]. CTCs have not yet been subject to proteomic analysis. The hunt for sensitive and specific protein biomarkers of early CRC in the peripheral blood of screening patients represents the ultimate goal of many proteomics researchers [48, 49]. As yet however, despite a plethora of publications suggesting novel screening protein biomarkers, there are no validated tests that have crossed the boundary from the laboratory into routine clinical practice. A recent proteomic analysis from patients with colorectal adenomas or invasive disease identified a panel of protein biomarkers, including three cytokines and CA19.9 that could be used to identify adenoma-bearing patients from healthy controls or subjects with established CRC [50]. Clearly, a panel of protein biomarkers able to accurately predict the presence of pre-malignant CRC in its adenomatous form from a peripheral blood source would have great clinical application. However, although this study represents a significant step towards this goal it was based on a relatively small sample size and it identified some proteins in its test panel which might be non-specifically raised in nonmalignant diseases [6]. Other studies have focused on plasma-based protein arrays to analyse samples from adenoma-bearing patients, those with developed CRC or controls [51]. However, further analysis and validation is required before such work can be translated into routine screening tests [52]. Research in recent years has indicated that cancer patients with diverse types of solid malignancy produce autoantibodies, detectable in their peripheral blood, against tumour associated antigens. A recent review of published studies on autoantibodies in CRC patients for the early detection of the disease, attempted to summarise their diagnostic utility [53]. Techniques used to screen for peripheral blood autoantibodies included ELISA and immunoblotting. The conclusions drawn from this work, which analysed 67 different studies, This article is protected by copyright. All rights reserved.


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indicated that while single autoantibodies lacked sensitivity when used to screen for early CRC, more promising results had been obtained from using a panel of autoantibody biomarkers. However, further validation will be required before such techniques can be used routinely in clinical practice. Overall recent research into the identification of protein biomarkers in patients with early colorectal cancer has produced some interesting and potentially promising results but to date none of these biomarkers has made the transition from the experimental stage into routine clinical practice. Putative biomarkers require extensive validation studies before they can be considered reliable and accurate and the test employing such biomarkers should be acceptable to the patients and cost effective as a screening tool.

Predictive biomarkers in CRC Surgery is usually the first line of treatment for patients with CRC but there are now increasing options for neoadjuvant and adjuvant therapy. A recent trial of the efficacy of imatinib (a tyrosine kinase inhibitor targeting BCR-Abl) plus panitumumab (an inhibitory anti human epidermal growth factor receptor monoclonal antibody) in patients with metastatic CRC used reverse phase protein array (RPPA) to select activated protein targets of the drug imatinib [54]. Levels of c-kit, PDGFR and c-Abl were assessed in microdissected metastatic tissue using RPPA. Based on the levels of these targets, an experimental arm was selected to receive both imatinib and panitumumab. The control group received panitumumab monotherapy. Pre- and post-treatment biopsies were analysed to assess the effects of the drugs on the protein targets. Although the number of patients in the study was small (only seven), the work described a novel method for selecting patients for personalized treatment based on the activation levels of drug targets in their metastases. Targeted therapy with tyrosine kinase inhibitors is a growing force in the battle against solid tumour metastatic disease. In CRC, dasatinib has been used as an effective inhibitor of the Src family of tyrosine kinases, with significant anti-tumour results. As tyrosine kinase inhibitors can cause side effects and incur considerable expense, biomarkers which can be used to accurately select patients and monitor anti-tumour effects are urgently required. Using global phosphotyrosine proteomics a group recently identified PKCdelta as a marker of responsiveness of Src inhibition in CRC cell lines. This data suggests that the measurement of PKCdelta levels may be a useful biomarker for assessing responses to dasatinib in CRC [55]. However, although these results showed promise in pre-clinical



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models, further validation and confirmatory clinical studies are required before this work can be translated into useful routine practice.

Biomarkers in CRC providing insights into tumour biology The problem of tumour genetic heterogeneity is known to be a barrier to the development of effective targeted therapies. However, heterogeneity at the protein level in tumours has been less well studied. Using MALDI image-guided proteomics a group recently studied the heterogeneity of extracellular and membrane proteins in CRC liver metastasis [56]. Interestingly, all the metastases were found to have a similar zonal pattern of functional protein expression. Such zonal expression of functional proteins within a metastatic deposit was thought to reinforce the need for multiple therapeutic targets to achieve maximum coverage of malignant lesions. The biological role of colorectal cancer cancer stem cells (CCSCs) in malignant recurrence following chemotherapy is controversial, but it has been suggested that they may play a critical role in the development of drug resistance. By performing proteomic analysis on the secretomes of cancer stem cells and their isogenic differentiated counterparts isolated from three different colon tumour cell lines, Emmink's group showed that CCSCs produce proteins involved in survival and in antioxidant networks that could contribute to drug resistance [57]. Over 1000 unique proteins were isolated in the conditioned media of the paired CCSC and differentiated tumour cell cultures, as analysed by mass spectrometry-based proteomics. The proteins isolated from the CCSC media were largely involved in survival pathways but the stem cells were also found to secrete drug metabolising enzymes including aldehyde dehydrogenase 1 and bleomycin hydrolase. This group concluded that a better understanding of the pathways involved in CCSC-mediated drug resistance could help to facilitate targeted therapy in future and could also prevent tumour recurrence after treatment. The development of protein biomarkers in CRC not only provides potentially useful clinical tools but it also represents a rich source of information though which researchers can gain a better understanding of CRC development, progression and metastasis. The phenotypic shift from a colorectal adenoma to an invasive carcinoma is a critical stage in cancer development. By using proteomic analysis to study the chromatin binding fraction within invasive CRC tissue and comparing it to that of non-invasive colorectal adenomatous tissue, Knol's group found significant shifts in the protein translation machinery during the process This article is protected by copyright. All rights reserved.


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of CRC progression [58]. This group found an increase in cell cycle regulators, including Cdk1 and Mapk1 kinases, indicating that advanced CRC tissue contains more actively cycling cells. There were also alterations in the levels of splicing factors between the two tissue groups and significant protein expression changes related to pluripotency and “stemness”. This study found that proteomic analysis of the chromatin binding fraction in malignant and pre-malignant tissues is feasible and informative, despite the inevitable mix of stromal, epithelial and immune cells found in both tissue types. It also indicated that identification of such nuclear proteins involved in the pre-malignant to malignant phenotypic shift in CRC may be useful, both in achieving a better understanding of this complex biological process and in the identification of a future source of clinically applicable protein biomarkers. Extracellular vesicles or exosomes are small membrane bound vesicles that are secreted by a variety of mammalian cell types. Exosomes are now thought to have a significant role in cancer biology, potentially by playing a role in intercellular communication which may enable metastatic progression [59]. These membrane bound messengers can transport not only proteins but nucleic acids, including mRNA and microRNA which have been shown to have a functional effect on recipient cells. Using proteomic analysis of exosome profiles found in human primary and metastatic CRC cell lines, Greening et al identified a range of proteins thought to be fundamental to CRC progression [60]. Metastatic cell-derived exosomes were found to be rich in signal transduction molecules and proteins involved in tumour progression and dissemination such as S100A8, S100A9 and the tyrosine kinase, MET. This work indicates that exosome-derived factors may play a key role in intercellular communication between malignant cells and the supporting cells within their microenvironment. It is postulated that this mode of communication may facilitate tumour progression.

Conclusions Proteomic analysis in CRC has produced a wealth of information in recent years that may lead to the identification of clinically useful protein biomarkers. However, despite the plethora of published studies there are essentially no new validated protein biomarkers of CRC that are currently in everyday clinical use. Diagnostic protein biomarkers used in the histological identification of CRC in its primary or metastatic forms are well documented. Early diagnosis of the disease and early surgical resection still affords the best chance of cure, but accurate and consistent protein biomarkers for use in CRC screening are still lacking. This article is protected by copyright. All rights reserved.


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This is, at least in part, due to a lack of good quality follow-up studies to validate initial results. Inconsistencies including lack of quality assurance and validation between laboratories relating to detection assays also contribute to the problem. As the range of adjuvant therapies used to treat CRC increases, so the need for accurate and consistent clinically applicable predictive biomarkers will rise. This becomes particularly important when the toxicities associated with some of the newer chemotherapeutic agents are taken into account. Protein biomarkers will also be vital in prognostication as the current TNM system is known to have its limitations. Finally, a better understanding of CRC progression and metastasis at the subcellular level is likely to be gained by studying protein biomarkers involved in CRC tumour biology. But no matter how promising an initial biomarker discovery appears to be, the transition from the laboratory into the clinic can only be made after thorough follow up validation studies have been carried out and after the careful development of accurate, cost effective and clinically acceptable tests.

Conflict of interest GIM is a member of the Scientific Advisory Board of Vertebrate Antibodies. CC declares no conflict of interest.



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References [1] Ferlay, J., Shin, H. R., Bray, F., Forman, D. et al., Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008. Int. J. Cancer 2010, 127, 2893-2917.

[2] Jemal, A., Bray, F., Center, M. M., Ferlay, J. et al., Global cancer statistics. CA Cancer. J. Clin. 2011, 61, 69-90.

[3] Hewitson, P., Glasziou, P., Watson, E., Towler, B., Irwig, L., Cochrane systematic review of colorectal cancer screening using the fecal occult blood test (hemoccult): an update. Am. J. Gastroenterol. 2008, 103, 1541-1549.

[4] Elmunzer, B. J., Hayward, R. A., Schoenfeld, P. S., Saini, S. D. et al., Effect of flexible sigmoidoscopy-based screening on incidence and mortality of colorectal cancer: a systematic review and meta-analysis of randomized controlled trials. PLoS Med. 2012, 9, e1001352.

[5] Brenner, H., Kloor, M., Pox, C. P., Colorectal cancer. Lancet 2014, 383, 1490-1502.

[6] Coghlin, C., Murray, G. I., Progress in the identification of plasma biomarkers of colorectal cancer. Proteomics 2013, 13, 2227-2228.

[7] Luo, Y., Wang, L., Wang, J., Developing proteomics-based biomarkers for colorectal neoplasms for clinical practice: opportunities and challenges. Proteomics Clin. Appl. 2013, 7, 30-41.

[8] Martinez-Aguilar, J., Chik, J., Nicholson, J., Semaan, C. et al., Quantitative mass spectrometry for colorectal cancer proteomics. Proteomics Clin. Appl. 2013, 7, 42-54.



Page 15


[9] Coghlin, C., Murray, G. I., Following the protein biomarker trail to colorectal cancer. Colorectal Cancer 2012, 1, 93-96.

[10] Werling, R. W., Yaziji, H., Bacchi, C. E., Gown, A. M., CDX2, a highly sensitive and specific marker of adenocarcinomas of intestinal origin: an immunohistochemical survey of 476 primary and metastatic carcinomas. Am. J. Surg. Pathol. 2003, 27, 303-310.

[11] de Wit, M., Fijneman, R. J., Verheul, H. M., Meijer, G. A., Jimenez, C. R., Proteomics in colorectal cancer translational research: biomarker discovery for clinical applications. Clin. Biochem. 2013, 46, 466-479.

[12] Wang, J., Wang, X., Lin, S., Chen, C. et al., Identification of kininogen-1 as a serum biomarker for the early detection of advanced colorectal adenoma and colorectal cancer. PLoS One 2013, 8, e70519.

[13] Qiu, Y., Patwa, T. H., Xu, L., Shedden, K. et al., Plasma glycoprotein profiling for colorectal cancer biomarker identification by lectin glycoarray and lectin blot. J. Proteome Res. 2008, 7, 1693-1703.

[14] Roeise, O., Sivertsen, S., Ruud, T. E., Bouma, B. N. et al., Studies on components of the contact phase system in patients with advanced gastrointestinal cancer. Cancer 1990, 65, 1355-1359.

[15] Manna, S. K., Tanaka, N., Krausz, K. W., Haznadar, M. et al., Biomarkers of coordinate metabolic reprogramming in colorectal tumors in mice and humans. Gastroenterology 2014, 146, 1313-1324.



Page 16


[16] Soreide, K., Gudlaugsson, E., Skaland, I., Janssen, E. A. et al., Metachronous cancer development in patients with sporadic colorectal adenomas-multivariate risk model with independent and combined value of hTERT and survivin. Int. J. Colorectal Dis. 2008, 23, 389-400.

[17] Comoglio, P. M., Trusolino, L., Cancer: the matrix is now in control. Nat. Med. 2005, 11, 1156-1159.

[18] Sleeman, J. P., Christofori, G., Fodde, R., Collard, J. G. et al., Concepts of metastasis in flux: the stromal progression model. Semin. Cancer Biol. 2012, 22, 174-186.

[19] Coghlin, C., Murray, G. I., Current and emerging concepts in tumour metastasis. J. Pathol. 2010, 222, 1-15.

[20] Coghlin, C., Murray, G. I., The role of gene regulatory networks in promoting cancer progression and metastasis. Future Oncol. 2014, 10, 735-748.

[21] Sveen, A., Agesen, T. H., Nesbakken, A., Meling, G. I. et al., ColoGuidePro: a prognostic 7-gene expression signature for stage III colorectal cancer patients. Clin. Cancer Res. 2012, 18, 6001-6010.

[22] Salazar, R., Roepman, P., Capella, G., Moreno, V. et al., Gene expression signature to improve prognosis prediction of stage II and III colorectal cancer. J. Clin. Oncol. 2011, 29, 17-24.

[23] Maak, M., Simon, I., Nitsche, U., Roepman, P. et al., Independent validation of a prognostic genomic signature (ColoPrint) for patients with stage II colon cancer. Ann. Surg. 2013, 257, 1053-1058.



Page 17


[24] Deschoolmeester, V., Baay, M., Specenier, P., Lardon, F., Vermorken, J. B., A review of the most promising biomarkers in colorectal cancer: one step closer to targeted therapy. Oncologist 2010, 15, 699-731.

[25] Coghlin, C., Carpenter, B., Dundas, S. R., Lawrie, L. C. et al., Characterization and over-expression of chaperonin t-complex proteins in colorectal cancer. J. Pathol. 2006, 210, 351-357.

[26] Murray, G. I., Duncan, M. E., O'Neil, P., Melvin, W. T., Fothergill, J. E., Matrix metalloproteinase-1 is associated with poor prognosis in colorectal cancer. Nat. Med. 1996, 2, 461-462.

[27] Kumarakulasingham, M., Rooney, P. H., Dundas, S. R., Telfer, C. et al., Cytochrome p450 profile of colorectal cancer: identification of markers of prognosis. Clin. Cancer Res. 2005, 11, 3758-3765.

[28] Curran, S., Dundas, S. R., Buxton, J., Leeman, M. F. et al., Matrix metalloproteinase/tissue inhibitors of matrix metalloproteinase phenotype identifies poor prognosis colorectal cancers. Clin. Cancer Res. 2004, 10, 8229-8234.

[29] O'Dwyer, D., Ralton, L. D., O'Shea, A., Murray, G. I., The proteomics of colorectal cancer: identification of a protein signature associated with prognosis. PLoS One 2011, 6, e27718.

[30] Duncan, R., Carpenter, B., Main, L. C., Telfer, C., Murray, G. I., Characterisation and protein expression profiling of annexins in colorectal cancer. Br. J. Cancer 2008, 98, 426-433.



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[31] Peng, Y., Li, X., Wu, M., Yang, J. et al., New prognosis biomarkers identified by dynamic proteomic analysis of colorectal cancer. Mol. Biosyst 2012, 8, 3077-3088.

[32] Besson, D., Pavageau, A. H., Valo, I., Bourreau, A. et al., A quantitative proteomic approach of the different stages of colorectal cancer establishes OLFM4 as a new nonmetastatic tumor marker. Mol. Cell. Proteomics 2011, 10, M111.009712.

[33] Meding, S., Balluff, B., Elsner, M., Schone, C. et al., Tissue-based proteomics reveals FXYD3, S100A11 and GSTM3 as novel markers for regional lymph node metastasis in colon cancer. J. Pathol. 2012, 228, 459-470.

[34] Croner, R. S., Sturzl, M., Rau, T. T., Metodieva, G. et al., Quantitative proteome profiling of lymph node-positive vs. -negative colorectal carcinomas pinpoints MX1 as a marker for lymph node metastasis. Int. J. Cancer 2014, doi: 10.1002/ijc.28929.

[35] Jankova, L., Chan, C., Fung, C. L., Song, X. et al., Proteomic comparison of colorectal tumours and non-neoplastic mucosa from paired patient samples using iTRAQ mass spectrometry. Mol. Biosyst 2011, 7, 2997-3005.

[36] Tan, K. L., Jankova, L., Chan, C., Fung, C. L. et al., Clinicopathological correlates and prognostic significance of glutathione S-transferase Pi expression in 468 patients after potentially curative resection of node-positive colonic cancer. Histopathology 2011, 59, 1057-1070.

[37] Jankova, L., Robertson, G., Chan, C., Tan, K. L. et al., Glutathione S-transferase Pi expression predicts response to adjuvant chemotherapy for stage C colon cancer: a matched historical control study. BMC Cancer 2012, 12, 196-2407-12-196.



Page 19


[38] Carroll, M., Seaman, H., Halloran, S., Tests and investigations for colorectal cancer screening. Clin. Biochem. 2014, 47, 921-939.

[39] van Dam, L., Kuipers, E. J., Steyerberg, E. W., van Leerdam, M. E., de Beaufort, I. D., The price of autonomy: should we offer individuals a choice of colorectal cancer screening strategies? Lancet Oncol. 2013, 14, e38-46.

[40] Imperiale, T. F., Ransohoff, D. F., Itzkowitz, S. H., Levin, T. R. et al., Multitarget stool DNA testing for colorectal-cancer screening. N. Engl. J. Med. 2014, 370, 1287-1297.

[41] Schwarzenbach, H., Hoon, D. S., Pantel, K., Cell-free nucleic acids as biomarkers in cancer patients. Nat. Rev. Cancer. 2011, 11, 426-437.

[42] Yang, I. P., Tsai, H. L., Huang, C. W., Huang, M. Y. et al., The functional significance of microRNA-29c in patients with colorectal cancer: a potential circulating biomarker for predicting early relapse. PLoS One 2013, 8, e66842.

[43] Toiyama, Y., Takahashi, M., Hur, K., Nagasaka, T. et al., Serum miR-21 as a diagnostic and prognostic biomarker in colorectal cancer. J. Natl. Cancer Inst. 2013, 105, 849-859.

[44] da Silva Filho, B. F., Gurgel, A. P., Neto, M. A., de Azevedo, D. A. et al., Circulating cell-free DNA in serum as a biomarker of colorectal cancer. J. Clin. Pathol. 2013, 66, 775-778.

[45] Iinuma, H., Watanabe, T., Mimori, K., Adachi, M. et al., Clinical significance of circulating tumor cells, including cancer stem-like cells, in peripheral blood for recurrence and prognosis in patients with Dukes' stage B and C colorectal cancer. J. Clin. Oncol. 2011, 29, 1547-1555.



Page 20


[46] Galizia, G., Gemei, M., Orditura, M., Romano, C. et al., Postoperative detection of circulating tumor cells predicts tumor recurrence in colorectal cancer patients. J. Gastrointest. Surg. 2013, 17, 1809-1818.

[47] Kin, C., Kidess, E., Poultsides, G. A., Visser, B. C., Jeffrey, S. S., Colorectal cancer diagnostics: biomarkers, cell-free DNA, circulating tumor cells and defining heterogeneous populations by single-cell analysis. Expert Rev. Mol. Diagn. 2013, 13, 581-599.

[48] Reumer, A., Maes, E., Mertens, I., Cho, W. C. et al., Colorectal cancer biomarker discovery and validation using LC-MS/MS-based proteomics in blood: truth or dare? Expert Rev. Proteomics 2014, 11, 449-463.

[49] Uzozie, A., Nanni, P., Staiano, T., Grossmann, J. et al., Sorbitol dehydrogenase overexpression and other aspects of dysregulated protein expression in human precancerous colorectal neoplasms: a quantitative proteomics study. Mol. Cell. Proteomics 2014, 13, 1198-1218.

[50] Choi, J. W., Liu, H., Shin, D. H., Yu, G. I. et al., Proteomic and cytokine plasma biomarkers for predicting progression from colorectal adenoma to carcinoma in human patients. Proteomics 2013, 13, 2361-2374.

[51] Pengjun, Z., Xinyu, W., Feng, G., Xinxin, D. et al., Multiplexed cytokine profiling of serum for detection of colorectal cancer. Future Oncol. 2013, 9, 1017-1027.

[52] Albrethsen, J., Bogebo, R., Moller, C. H., Olsen, J. A. et al., Candidate biomarker verification: Critical examination of a serum protein pattern for human colorectal cancer. Proteomics Clin. Appl. 2012, 6, 182-189.



Page 21


[53] Chen, H., Werner, S., Tao, S., Zoernig, I., Brenner, H., Blood autoantibodies against tumor-associated antigens as biomarkers in early detection of colorectal cancer. Cancer Lett. 2014, 346, 178-187.

[54] Pierobon, M., Silvestri, A., Spira, A., Reeder, A. et al., Pilot phase I/II personalized therapy trial for metastatic colorectal cancer: Evaluating the feasibility of protein pathway activation mapping for stratifying patients to therapy with imatinib and panitumumab. J. Proteome Res. 2014, 13, 2846-2855.

[55] McKinley, E. T., Liu, H., McDonald, W. H., Luo, W. et al., Global phosphotyrosine proteomics identifies PKCdelta as a marker of responsiveness to Src inhibition in colorectal cancer. PLoS One 2013, 8, e80207.

[56] Turtoi, A., Blomme, A., Debois, D., Somja, J. et al., Organized proteomic heterogeneity in colorectal cancer liver metastases and implications for therapies. Hepatology 2014, 59, 924-934.

[57] Emmink, B. L., Verheem, A., Van Houdt, W. J., Steller, E. J. et al., The secretome of colon cancer stem cells contains drug-metabolizing enzymes. J. Proteomics 2013, 91, 84-96.

[58] Knol, J. C., de Wit, M., Albrethsen, J., Piersma, S. R. et al., Proteomics of differential extraction fractions enriched for chromatin-binding proteins from colon adenoma and carcinoma tissues. Biochim. Biophys. Acta 2014, 1844, 1034-1043.

[59] Katsuda, T., Kosaka, N., Ochiya, T., The roles of extracellular vesicles in cancer biology: toward the development of novel cancer biomarkers. Proteomics 2014, 14, 412425.



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[60] Greening, D. W., Kapp, E. A., Ji, H., Speed, T. P., Simpson, R. J., Colon tumour secretopeptidome: insights into endogenous proteolytic cleavage events in the colon tumour microenvironment. Biochim. Biophys. Acta 2013, 1834, 2396-2407.



Page 23

Table 1 Biomarkers and colorectal cancer

Colorectal cancer biomarker

Reference

ColoGuid Pro

21

Coloprint

22, 23

Chaperonin t-complex proteins

25

Matrix metalloproteinases

26, 28

Cytochrome P450

27

14-3-3 beta

29

Aldehyde dehydrogenase 1

29

Annexins

30

GRP78, fructose-bisphosphonate aldolase A, carbonic anhydrase 1 and peptidly-propyl cis-trans isomerase A

31

OLFM4

32

FXYD3, S100A11 and glutathione Stransferase M

33

MX1

34

NADH dependent oxidative enzymes

35

Glutathione S-transferase pi

36, 37




Page 24

Figure 1. Overview of protein biomarkers in colorectal cancer.



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