**1. Introduction**

Colorectal carcinoma (CRC) is a common form of cancer that is estimated to be responsible for approximately 694,000 deaths worldwide each year [1]. It is the third most common form of cancer in males and the second in females, with an estimated 1.4 million new cases diagnosed annually. The natural history of progression of adenomatous polyps to CRC has been well described by the adenoma-carcinoma sequence, a stepwise process which recognizes that the majority CRC arises

from adenomatous polyps (**Figure 1**) [2]. Colon and rectal cancer is staged from radiological, histopathological and intraoperative findings with the TNM (tumornodes-metastasis) system [3] or historically with the Dukes staging system [4]. The stage at diagnosis correlates to prognosis; the 5-year survival of patients with stage one disease is 90%, stage two is 71%, stage three 53% and stage four is only 14% [5]. Therefore, diagnosis and treatment of early stage disease is associated with significantly better outcomes than late stage disease.

Screening programs aim to detect asymptomatic patients with early stage disease where there is a conferrable survival benefit. Investigations used for screening require appropriate levels of sensitivity and specificity, this is to ensure adequate probability of disease detection and to reject patients without the disease in question. Fecal immunochemical tests (FIT) stool screening tests suffer from low predictive values for CRC and as such, positive tests can lead to unnecessary investigation with colonoscopy and other modalities. When considering the discovery of a biomarker for clinical use, the test must have both high sensitivity and specificity to capture the appropriate patient cohort without falsely reassuring patients. In addition, it must be specific in early stage disease, where the natural history of the disease can be successfully altered by surgical intervention. Currently, the participation in CRC screening programs is suboptimal, particularly given that early diagnosis and subsequent treatment significantly correlate to improved outcomes. Depending on the country or region, and the screening test offered, participation can be as low as 40% of the target population [10]. In the context of this poor compliance and subsequent effects on patient morbidity and mortality, there has been increased interest in the role of plasma-based biomarkers as a screening tool for the detection of early stage CRC.

Early stage screening for CRC is via stool-based tests or endoscopic or radiological investigations. Stool-based tests include guaiac-based fecal occult blood (FOB) tests or fecal immunochemical tests (FIT) [6, 7]. Other methods include; colonoscopy, computed tomography colonography, flexible sigmoidoscopy or capsule colonoscopy [8]. Currently, FIT testing is the main method of population based screening for average risk patients as it has 83% sensitivity and 93% specificity [9]. However, the FIT test sufferers from low compliance rates [10]. Colonoscopy is also used for screening and diagnosis but it is a procedure associated with risk, with complications estimated to occur between 0.5–2.8 per 1000 procedures and a mortality rate of 0.007% [11]. Patients who participate in screening programs and

#### **Figure 1.**

*Summary of plasma protein biomarkers according to disease stage. (A) Normal mucosa, (B) adenomatous polyp, (C) stage one and two adenocarcinoma (D) and stage three and four adenocarcinoma. Beneath each image representing each clinical stage, corresponding plasma protein biomarkers are listed. Each biomarker is placed corresponding to the earliest point where it has been identified. Images (A–C) are 40× objective lens. Image (D) 12.5× objective lens, hematoxylin-eosin staining. After deparaffinization and rehydration of colon sections were counterstained with hematoxylin (Gill's formulation, vector laboratories, California, USA). Imaging was performed on an Axioplan-2 microscope (Carl Zeiss AG, Oberkochen, Germany).*

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**Table 1.**

Ionization • Surface-enhanced laser desorption/ ionization

• Matrix-assisted laser desorption ionization

*Overview of common MS analytical technologies.*

*Finding Needles in Haystacks: The Use of Quantitative Proteomics for the Early Detection…*

**Analyzer Technology Advantages Disadvantages References**

Clinical mass spectra Good reproducibility Lower cost Efficient conversion of precursor to product Preferential for targeted analysis

High sensitivity High resolution Multi-stage MS Compact mass analyzer

Fast Can be used for pulsed ionization methods High ion transmission Good mass range

High resolution Accurate-mass detection Good for non-targeted analysis

High-throughput

Fast Minimal fragmentation required Rapid identification Good sensitivity for low abundant proteins

Poorer resolution Peak heights represented as a function of mass Limited application for pulsed ionization

Poor quantitation Poor dynamic range Affected by space charge effects and ion molecule reactions

Collision energy not well defined Many parameters which can affect the

quality

Requires pulsed ionization method or ion beam switching Analyzers used can have limited dynamic range Limited precursor selectivity

Decay of coherent ion packets

Poorer reproducibility Requires small sample volume Detection limits at attomole level Limited to detection of proteins of relatively low molecular mass Dependent of change in expression

profiles

[129, 130]

[129, 130]

[129, 130]

[129, 130]

[131, 132]

methods

*DOI: http://dx.doi.org/10.5772/intechopen.80942*

cylindric magnets to filter ions based on the mass to charge ratio (m/z).

of radio frequency and direct current (DC) electrical fields which allow ions to be trapped for analysis.

different ion velocities allowing separation based on mass.

between electrodes which results in orbiting ions. The ions oscillate at various frequencies which enable mass-tocharge measurement.

Uses a laser energy absorbing matrix to create ions from molecules.

Quadruple Parallel

Ion trap Combination

Time of flight Utilizes

Orbitrap Utilizes DC


*Finding Needles in Haystacks: The Use of Quantitative Proteomics for the Early Detection… DOI: http://dx.doi.org/10.5772/intechopen.80942*

#### **Table 1.**

*Overview of common MS analytical technologies.*

*Advances in the Molecular Understanding of Colorectal Cancer*

cantly better outcomes than late stage disease.

detection of early stage CRC.

from adenomatous polyps (**Figure 1**) [2]. Colon and rectal cancer is staged from radiological, histopathological and intraoperative findings with the TNM (tumornodes-metastasis) system [3] or historically with the Dukes staging system [4]. The stage at diagnosis correlates to prognosis; the 5-year survival of patients with stage one disease is 90%, stage two is 71%, stage three 53% and stage four is only 14% [5]. Therefore, diagnosis and treatment of early stage disease is associated with signifi-

Screening programs aim to detect asymptomatic patients with early stage disease

Early stage screening for CRC is via stool-based tests or endoscopic or radiological investigations. Stool-based tests include guaiac-based fecal occult blood (FOB) tests or fecal immunochemical tests (FIT) [6, 7]. Other methods include; colonoscopy, computed tomography colonography, flexible sigmoidoscopy or capsule colonoscopy [8]. Currently, FIT testing is the main method of population based screening for average risk patients as it has 83% sensitivity and 93% specificity [9]. However, the FIT test sufferers from low compliance rates [10]. Colonoscopy is also used for screening and diagnosis but it is a procedure associated with risk, with complications estimated to occur between 0.5–2.8 per 1000 procedures and a mortality rate of 0.007% [11]. Patients who participate in screening programs and

*Summary of plasma protein biomarkers according to disease stage. (A) Normal mucosa, (B) adenomatous polyp, (C) stage one and two adenocarcinoma (D) and stage three and four adenocarcinoma. Beneath each image representing each clinical stage, corresponding plasma protein biomarkers are listed. Each biomarker is placed corresponding to the earliest point where it has been identified. Images (A–C) are 40× objective lens. Image (D) 12.5× objective lens, hematoxylin-eosin staining. After deparaffinization and rehydration of colon sections were counterstained with hematoxylin (Gill's formulation, vector laboratories, California, USA). Imaging was performed on an Axioplan-2 microscope (Carl Zeiss AG, Oberkochen, Germany).*

where there is a conferrable survival benefit. Investigations used for screening require appropriate levels of sensitivity and specificity, this is to ensure adequate probability of disease detection and to reject patients without the disease in question. Fecal immunochemical tests (FIT) stool screening tests suffer from low predictive values for CRC and as such, positive tests can lead to unnecessary investigation with colonoscopy and other modalities. When considering the discovery of a biomarker for clinical use, the test must have both high sensitivity and specificity to capture the appropriate patient cohort without falsely reassuring patients. In addition, it must be specific in early stage disease, where the natural history of the disease can be successfully altered by surgical intervention. Currently, the participation in CRC screening programs is suboptimal, particularly given that early diagnosis and subsequent treatment significantly correlate to improved outcomes. Depending on the country or region, and the screening test offered, participation can be as low as 40% of the target population [10]. In the context of this poor compliance and subsequent effects on patient morbidity and mortality, there has been increased interest in the role of plasma-based biomarkers as a screening tool for the

**130**

**Figure 1.**

undertake colonoscopy examination have an estimated 90% decreased incidence of colon cancer than those who do not [12]. Early detection of polypoid disease and subsequent removal of polyps therein prevents progression to CRC [8].

Over the last 2 decades, unprecedented technological advancement in proteinbased mass spectrometry (proteomics) has radically changed the landscape of biomarker research [13] (**Table 1**). This has facilitated the characterization of complex cellular proteomes [14–19], research that has identified hundreds of over and under expressed proteins in carcinoma patients using tumor tissue, histological sections, plasma or fecal samples when compared to matched normal tissues [20–24]. Despite this, with the exception of Carcinoembryonic antigen (CEA) and Cancer antigen 19-9 (CA 19-9) [25], no new protein biomarkers have made it into routine clinical practice [21, 26, 27]. In this book chapter, we have sought to present an overview of the diagnostic and prognostic protein biomarkers of early stage CRC to aid in the development of accommodating future screening tools that will continue to increase the rate at which early stage CRC is diagnosed and treated. We also review the use of contemporary proteomic approaches to address many of the long-standing challenges in the field of human CRC plasma proteomics and speculate on the future clinical applications of these technologies (**Figure 2**).

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*Finding Needles in Haystacks: The Use of Quantitative Proteomics for the Early Detection…*

**2. Proteomic technologies for the identification of plasma proteins of** 

The use of blood or plasma for screening or diagnosis of CRC is the most attractive non-invasive material available for the identification of clinically relevant protein biomarkers. Most commonly, candidate protein biomarkers of early stage CRC are identified using MS-based proteomics techniques. Below we list the limitations and advantages of the most common sample preparation and proteomics techniques specifically to identify candidate biomarkers in the plasma of early stage CRC. These techniques face a number of limiting factors, which have reduced the utility of proteins revealed by proteomics. Indeed, factors including the extreme dynamic range of proteins within plasma [28], the variability in collection and processing methods [21], preanalytical and analytic processes [29], and the inherent heterogeneity of patient samples [30], have all hindered uniform consent for which

biomarkers are the most relevant for use in the setting of early stage disease. As a small number of highly abundant proteins such as; albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, fibrinogen, comprise 90% of the human plasma proteome [31], therefore little capacity is left for the identification of lower abundance proteins to be used as early stage markers of CRC [32]. Researchers have thus turned to immunodepletion strategies to enrich for low abundant proteins, resulting in a 25% increase in identified proteins and 4-fold increased enrichment of non-targeted plasma proteins using peptide isoelectric focusing (IEF), followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) [31]. These pioneering studies have paved the way for high-resolution LC-MS/MS studies employed on depleted samples, routinely affording researchers with the capacity to identify 100 s if not 1000 s of plasma proteins during the course of a proteomics

In the context of proteogenomic approaches to biomarker discovery [33], recent studies have also made some progress in reducing variability during collection and processing, revealing the suitability of human plasma proteins for qualitative and quantitative proteomic analysis after collection and storage for up to 48 hours at room temperature in cell free DNA-optimized blood collection tubes [21]. These tubes have been developed to overcome some of the issues that delays in processing time, temperature, and handling contribute to the deterioration of non-protein– based biomarkers [34] and now protein biomarkers [21]. Although not yet in widespread use, future studies may show that these cell stabilization tubes reduce plasma contamination by proteins originating from blood cells during collection and storage, thus increasing the reproducibility of proteomics-based biomarker

Two-dimensional electrophoresis (2DE) coupled to mass spectrometry is a very accurate and sensitive method of large-scale protein separation using human CRC tissue [35]. The application of this preparative platform, which facilitates the resolution of protein mixtures on the basis of proteins isoelectric point and molecular weight has been extensively employed using CRC tissue [36–38]. These techniques can be combined with any analytical MS platform to identify changes in protein abundance between samples. Results of these studies are most commonly validated using orthogonal immunological-based techniques using plasma including; ELISA, flow cytometry, immunoblotting. Recently, two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) was employed on early and late stage CRC plasma samples, identifying apolipoprotein A1 (APOA1) as a potential marker

*DOI: http://dx.doi.org/10.5772/intechopen.80942*

**early stage CRC**

investigation [21] (**Figure 2**).

discovery projects (**Figure 2**).

**2.1 Gel-based separation platforms**

#### **Figure 2.**

*Overview, and advantages and disadvantages of using gel-free quantitative proteomic approaches for the identification of plasma protein biomarkers of early stage colorectal carcinoma.*

*Finding Needles in Haystacks: The Use of Quantitative Proteomics for the Early Detection… DOI: http://dx.doi.org/10.5772/intechopen.80942*
