**3. Overview of biomarkers of colorectal adenocarcinoma**

Plasma biomarkers used in clinical practice include Carcinoembryonic antigen and Cancer antigen 19-9, however these investigations have limited use in early diagnosis of CRC [6]. A variety of plasma and histological biomarkers of early and late stage CRC including heat shock proteins, matrix metalloproteinases, complement component proteins, Annexins and S100 proteins are discussed and summarized in **Table 2**.

#### **3.1 Carcinoembryonic antigen**

Carcinoembryonic antigen (CEA) is a cell-surface high molecular weight glycoprotein important for cell adhesion, discovered in 1965 by Gold and Freedman as a component of human colon carcinoma and foetal tissue [62]. The production of CEA typically ceases at birth and it is present in very low concentrations in healthy patients. It can, however, be elevated in CRC, other types of cancer and non-malignant conditions [63]. CEA is one of the most commonly used biomarkers of CRC worldwide, however its sensitivity for the detection of CRC is not good enough to be useful as a diagnostic tool, with plasma elevation >5 μg/L in Dukes type A, B, C and D reportedly 3, 25, 45 and 65% respectively [63, 64] . Limited evidence supports a role of CEA as a marker of CRC prognosis and recurrence; its sensitivity as an indicator of recurrence is estimated to be 80% [65] and post-operative elevation is particularly sensitive for the detection of hepatic and retroperitoneal metastases.

**139**

**Protein** APO1A

Serum samples: Dukes A And B (N = 24), Dukes C and D (N = 24) and healthy controls (N = 26).

Serum protein enrichment and clean up, lysis buffer and labeling, 2D-DIGE.

LS-MS/MS

2419 protein spots detected, 8 proteins up -regulated in early CRC and 2 down regulated. In late stage CRC, 14 proteins up-regulated, 4 proteins were down-regulated.

ELISA: 66 Serum Samples (Early Stage Disease N = 29, Late stage disease N = 19, Healthy controls N = 18). The 5 proteins selected for validation were APO1a, APOe, CFH, SYNJ2, GAL7.

HSP27

Histological samples:

2DGE for 12 CRC samples.

QSTAR Pulsar

HSP27

i hybrid mass

spectrometer

LC-MS/MS

HSP identified, excised and

trypsin digested.

TMA of 404 CRC samples

and 100 controls followed by

IHC analysis.

IHC for the 50 formalin

N/A

HSP40 expressed in 14% of IHC

N/A

samples, 80% of immunoblot

HSP70 in 80% of IHC samples, 60% of

immunoblot

samples using positive

and negative controls.

Immunoblotting

(SDS-PAGE).

Immunoblot for 10 frozen

samples.

HSP60

Histological samples:

Histological samples

MALDI

Approx. 1600 gel protein spots. 17

IMMUNOBLOT demonstrated

[83]

significant overexpression

of HSP60, glutathione-*S*transferase pi, α-enolase, TCP1β. Leukocyte elastase

inhibitor with decreased

expression.

HSP60 overexpression

confirmed with IHC in 20

samples.

proteins with differential expression.

TOF-MS

underwent 2DGE with

internal standards.

ELISA for HSP60 to compare

serum levels.

50 CRC

Serum: 112 CRC and

90 healthy controls

for immunoassay.

HSP40

Histological samples:

50 CRC formalin

fixed, 10 frozen CRC.

404 primary tumors.

**Sample type**

**Workflow**

**MS platform** Nanoacquity UPLC Q-TOF

**Proteins identified**

**Validation set**

**References**

[91]

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

[79]

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

IHC: APO1a the only protein consistently identified.

TMA of 315 CRC in an

[77]

independent cohort.


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

*Advances in the Molecular Understanding of Colorectal Cancer*

peptides present in complex biological samples.

SWATH-MS can be deployed for both discovery and quantitation of all detectable

SWATH-MS also affords the added advantage that it does not rely on prior knowledge of the precursor peptide ions, instead acquiring information in a DIA manner and thus avoiding laborious assay development. The SWATH-MS workflow involves two key steps beginning with the generation of a spectral library (e.g. via conventional LC-MS/MS) through which acquired peptides are identified. During this acquisition mode, the mass spectrometer is programed to step within 2–4 s cycles through a set of precursor acquisition windows covering the mass range accessible by a quadrupole mass analyzer and also that in which most tryptic peptide precursors should fall (400–1200 m/z). During each cycle, the mass spectrometer fragments peptide precursors and records a complete, high accuracy fragment ion spectrum for all precursors that elute on the chromatograph. This is then followed by acquisition of SWATH-MS data for each sample under analysis, interrogation and matching against the spectral library to identify peptides, and finally extraction of specific peptide ions to enable area-under-the-curve quantitation between samples.

The first SWATH-MS study of CRC plasma also simultaneously assessed protein biomarkers from pancreatic cancer, lung cancer, prostate cancer, and ovarian cancer, all from patients diagnosed with early forms of these diseases. This sophisticated study employed sample enrichment and subsequent detection of tissuespecific secreted protein profiles via SWATH-MS. These data were used to generate a digital representation of the proteins from within each plasma sample that could be queried for the presence and quantity of specific peptides using a targeted data analysis [60]. Tumor specific biomarkers were detected for individual cancer types, as well as a common biomarker Thrombospondin-1 (THBS1), which was significantly altered in the blood of four of five carcinomas (CRC, lung, prostate and ovarian) [61]. These ground breaking studies highlight the potential of the new

Plasma biomarkers used in clinical practice include Carcinoembryonic antigen and Cancer antigen 19-9, however these investigations have limited use in early diagnosis of CRC [6]. A variety of plasma and histological biomarkers of early and late stage CRC including heat shock proteins, matrix metalloproteinases, complement component proteins, Annexins and S100 proteins are discussed and summa-

Carcinoembryonic antigen (CEA) is a cell-surface high molecular weight glycoprotein important for cell adhesion, discovered in 1965 by Gold and Freedman as a component of human colon carcinoma and foetal tissue [62]. The production of CEA typically ceases at birth and it is present in very low concentrations in healthy patients. It can, however, be elevated in CRC, other types of cancer and non-malignant conditions [63]. CEA is one of the most commonly used biomarkers of CRC worldwide, however its sensitivity for the detection of CRC is not good enough to be useful as a diagnostic tool, with plasma elevation >5 μg/L in Dukes type A, B, C and D reportedly 3, 25, 45 and 65% respectively [63, 64] . Limited evidence supports a role of CEA as a marker of CRC prognosis and recurrence; its sensitivity as an indicator of recurrence is estimated to be 80% [65] and post-operative elevation is particularly sensitive for the detection of hepatic and retroperitoneal metastases.

generation of analytical MS techniques for the detection of early stage.

**3. Overview of biomarkers of colorectal adenocarcinoma**

**138**

rized in **Table 2**.

**3.1 Carcinoembryonic antigen**


**141**

**Protein** MMP-2 MMP- 1,3,9

Histological samples: 72 CRC with matched normal tissue and serum plasma samples

MMP-7

IHC

N/A

> Grading G0-G3 according to the percentage of strongly stained areas.

76 Histological samples: Normal mucosa (n = 15) or tubular (n = 32), tubulovillous (n = 16), or villous (n = 13) adenomas.

Serum samples: 78 CRC, 38 healthy

Protein concentration

N/A

Significant overexpression of

N/A

MMP-7. MMP-10 and 12 not statically

significant. All associated with

significantly impaired survival.

estimation by commercial

Assay kit. Luminex based

Assay for MMP-7, MMP-10,

MMP-12

MMP-13

TIMP-1

Histological: 94 CRC

with matched healthy

controls

Histological samples:

IHC. Monoclonal antibody

N/A

MMP-13

N/A

[93]

to MMP-13. Detection using

Gelatin Zymography

IMMUNOBLOT assay

N/A

Positive TIMP staining in 53.2%

N/A

[109]

CRC samples, 80.6% of lymph node

metastasis.

Increased levels associated with

decreased disease-free survival and

overall survival

IHC analysis

TMA

249 CRC.

controls.

MMP-12

**Sample type**

**Workflow** ELISA using commercial kits for MMP-1,2,3,9

**MS platform**

N/A

**Proteins identified**

Highly elevated concentrations of MMP-

1, MMP-2, MMP-3 and MMP-9 protein expression in tumor tissue compared with tumor-free tissue (*p* < 0.0001). MMP-2 the only significantly increased in plasma.

MMP-7 identified in cytoplasm of all adenoma cells. Statistically significant difference in degree of overexpression of the three subtypes of colonic adenomas.

N/A

N/A

**Validation set**

**References**

[98]

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

[108]

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

[103]

