GPA33 (A33) gene codes for a membranous protein that is widely expressed in colorectal cancers, appearing in up to 95% of cases but particularly in well differentiated tumors. (Baptistella, 2016). Upregulation of GPA33 may be a downstream effect of peroxisome proliferator-activated receptor (PPARG) activation, which induces expression of KLF4, a transcription factor that targets GPA33 (Rageul, 2009). Antibodies to GPA33 used in the radiotherapy of colorectal cancers have been proposed as an effective means of treatment for tumors that express the protein (Cheal, 2017).

CK20 (KRT20) is a keratin that is expressed in epithelial cells in colorectal crypts, where levels of this protein have been found to increase gradually from the crypt bottom (where it is absent) to the top (Chan, 2009; Moll, 1990). It is frequently used as a differentiation marker in the colon. Chan et al found that CK20 is positively regulated by CDX1, a homeobox gene involved in promoting intestinal differentiation (Chan, 2009). In colorectal cancer, CK20 typically has strong expression paired with an absence of expressed CK7 (KRT7) (Chan, 2009; Harbaum, 2012) and its expression is often inversely correlated with levels of LGR5 (Shimokawa, 2017). In a study on several hundred primary colorectal tumors, Harbaum et al found that while the majority had high levels of CK20, microsatellite unstable (MSI) tumors tended to have reduced or depleted expression of the protein (Harbaum, 2012). Aggressive, poorly differentiated colorectal tumors and those with high rates of MSI may stain negatively for this marker in immunohistochemistry (Harbaum, 2012; Merlos-Suárez, 2011).

CK7 (KRT7) is a keratin expressed in a number of tissues including in the colon, where its expression is limited to glandular cells (proteinatlas.org). CK7 typically has negative expression in colorectal cancer, and as a marker in IHC antibody panels it is often paired with positive expression of CK20 in support of adenocarcinoma (Harbaum, 2012; Diagnostic Immunohistochemistry p. 521). However, KRT7 expression in colon cancer may vary with the subtype and aggressiveness of the tumor; while high levels of CK20 expression are linked with increased differentiation, conversely in some colorectal tumors higher levels of CK7 and low levels of CK20 have been found and are attributed to poor differentiation (Harbaum, 2012; Harbaum, 2011). Furthermore, microsatellite-stable colorectal cancers with BRAF mutation, a subtype known to be aggressive and have poor prognosis, have been found to express higher levels of CK7 than other typically negative subtypes (Landau, 2014).

Calretinin (CALB2) is a calcium-binding protein involved in calcium signaling. High levels of expression of calretinin in medullary carcinoma of the colon with strong focal staining in IHC have been documented, emphasizing its utility as an immunohistochemistry biomarker for detection of these cancers (Winn, 2008; Lin, 2014).

CDH17 is a cadherin involved in cell adhesion and proliferation and expressed in the glands of the colon, duodenum and small intestine as well as the appendix and gallbladder (Fagerberg, 2014; proteinatlas.org). In a study examining over 700 tumors across multiple tissues including the colon, Panarelli et al found that CDH17 has immunohistochemistry staining patterns in gastrointestinal cancers similar to CDX2 and is a highly specific marker for the intestinal epithelium (Panarelli, 2012). Furthermore, overexpression of this protein has been implicated in metastasis and invasive potential in colorectal cancer (Bartolome, 2014).

Cyclooxygenase 2 (COX-2 / PTGS2) is a protein involved in inflammation, proliferation and cell renewal that is regularly upregulated in colorectal cancer and may play a role in its progression (Liu, 2017; Wang, 2009). Colorectal cancer patients with tumors expressing high levels of COX-2 who are treated with NSAIDs and COXIBs (COX-2 inhibitors) see up to a 50% decrease in their risk of developing colorectal cancer (Wang, 2009). Mutant APC mouse models that undergo inhibition of this protein have also seen a decrease in tumorigenesis (Brown, 2005).

MUC2 is a member of the mucin family of proteins and is a secreted protein that produces protective mucous in the gut (Allen, 1998). MUC2 is implicated in the progression of ulcerative colitis, where it is downregulated (Moehle, 2006), and also in the formation colorectal cancers (Betge, 2016). A MUC2 knockout model in mice demonstrated a propensity for colorectal cancer in its absence, as these mice frequently developed invasive colorectal adenocarcinomas (Velcich, 2002).

CDX2 codes for a homeobox protein involved in intestinal cell differentiation and tumor suppression in the colon (Platet, 2017), and loss of expression of this protein is correlated with colorectal tumorigenesis and poor survival (Platet, 2017; Dalerba, 2016). Platet et al found that higher levels of expression in the colon are correlated with a lack of cellular deformability and an increase in anti-metastatic structural properties (Platet, 2017). Furthermore, loss of expression of this protein may lead to tumor cells attaining stem cell attributes, as described by Lundberg IV et al in their findings that CDX2 downregulation is linked with upregulation of Yamanaka factor SOX2, involved in embryonic development and implicated in the induction of pluripotency (Lundberg, 2016; Takahashi, 2006). They found this to be a frequent phenomenon in MSI-positive (microsatellite unstable) and CIMP (CpG Island Methylator Phenotype) tumors. Jiang et al also found a link between methylation silencing of CDX2 (hypermethylation is a driving factor of CIMP) and colorectal cancer (Jiang, 2016). Downregulation of CDX2 may also be caused by FBXW7, a tumor suppressor with frequent mutations in cancer that manages the degradation of a large number of proteins including the Yamanaka factor Myc (Kumar, 2016; Takeishi, 2014). FBXW7 has been found to negatively regulate CDX2 expression by promoting its degradation together with the serine-threonine kinase GSK3B (Kumar, 2016).

Villin (VIL1) is an actin-binding protein involved in the maintenance of microvilli in epithelial cells and is also involved in the epithelial-mesenchymal transition, and deregulation of this gene may contribute to tumorigenesis (Patnaik, 2016). Loss of expression of villin is seen in poorly differentiated colorectal carcinomas, particularly in microsatellite-unstable (MSI) tumors (Arango, 2012), and is linked to poor survival (Jaudah, 2013).

MOC-31 is a clone of the protein EPCAM (TACSTD1) and is a useful biotarget for metastatic adenocarcinomas, including those originating in the colon. As an immunomarker, MOC-31 antibodies are used to distinguish adenocarcinoma from mesothelial cells with high sensitivity (Morgan, 1999; Kundu, 2011; Hecht, 2006). EPCAM itself is a protein involved in cell adhesion and has been implicated in the progression of a number of cancers (Dai, 2017; Baeuerle, 2007), included a proportion of individuals with Lynch Syndrome who hold inherited deletions in the gene. These deletions lead to silencing of the adjacent repair gene MSH2 via transcriptional read-through, which then causes microsatellite instability and colorectal cancer (as well as other malignancies that also frequently arise from Lynch Syndrome) (Kempers, 2010).

Carcinoembryonic (CEA / mCEA / pCEA) antigen includes a large class of proteins such as CEACAM5 that are biomarkers for colorectal cancer. These cell adhesion proteins have high expression in specifically metastatic cancer, making them suitable targets for immunohistochemistry of late tumors (Bajenova, 2014; Polat, 2014). The degree of expression of CEA is correlated with tumor stage, with particularly pronounced levels in well-differentiated adenocarcinomas relative to early tumors (which are expected to express the same levels as normal tissues) (Vukobrat-Bijedic, 2013).

SATB2 is a transcription factor involved in chromatin remodeling that negatively regulates a number of stem cell markers such as AXIN2, CD44 and NANOG (Li, 2016; Dragomir, 2014). Loss of expression of SATB2 in colorectal cancer has been correlated with an increase in stemness and invasive potential (1). SATB2 may be a useful immunohistochemistry (IHC) marker for primary and metastatic colorectal cancers due to its high specificity (Dragomir, 2014).

Carbohydrate antigen (CA19-9/FUT) is a common metastatic colorectal cancer marker to tetrasaccharide carbohydrate (Sialyl-Lewis A), which has high levels of occurrence alongside CEACAM5 (CEA) expression levels in late tumors (Vukobrat-Bijedic, 2013; Polat, 2014). CA19-9 is an effective marker for circulating tumor cells, as increased levels of CA19-9 in serum coincide with the quantity of circulating tumor cells from colorectal metastases (Zhao, 2017).

TP53 is a tumor suppressor, repair gene and transcriptional regulator, and it is considered the most frequently mutated gene in cancer (Andrysik, 2017). Germline mutations in TP53 lead to Li-Fraumeni syndrome and cancer, including early-onset colorectal cancer (Yurgelun, 2015). In colorectal cancer, the expression of TP53 with gain of function mutations is a regular occurrence and is correlated with an increase in proliferative and invasive attributes in tumors of advanced stage (Andrysik, 2017; Sun, 2016). Furthermore, TP53 is often overexpressed in colorectal dysplasia resulting from ulcerative colitis (Kobayashi, 2017).

BRAF, a MAPK/ERK signaling pathway kinase, is one of the most frequently somatically mutated genes in some cancers, particularly melanoma, thyroid carcinomas, and colorectal cancer (Chang, 2016; Zhang, 2014; Cantwell-Dorris, 2011). BRAF mutation occurs in roughly 10% of colorectal cancers, where detection of the V600E (rs113488022) mutation is useful for subtyping CIMP (CpG Island Methylator Phenotype), particularly when it accompanies the disappearance of the repair protein MLH1 (Cantwell-Dorris, 2011). These microsatellite-unstable (MSI) tumors typically propagate along the sessile serrated adenoma development pathway (Rhee, 2016; Cantwell-Dorris, 2011) and experience epigenetic silencing and loss of function frameshift in a large number of tumor suppressor and cell cycle regulation genes including TGFBR2, BAX, RNF43, CASP5 and KMT2C (MLL3) (Cortes-Ciriano, 2017). This is caused by the hypermethylation-induced silencing of mismatch repair protein MLH1, which occurs subsequent to and may even be caused by BRAF mutation (Fang M, 2014; Cantwell-Dorris, 2011). BRAF V600E is a pro-proliferative mutation, and while the adoption of V600E-mutant BRAF protein alone is not a pathogenic event (this mutation is the source of overproliferation in many benign nevi) (Wu, 2007), when combined with mutations in tumor suppressor genes it leads to carcinogenesis.

PIK3CA is a kinase frequently mutated in cancer, including up to 15% of colorectal cancers. This gene is regularly mutated in proximal cancers alongside KRAS mutation and MGMT silencing (Rosty, 2013), and frequently experiences the same somatic mutations characterized by H1047R, E545K, and E542K (Chang, 2016). The acquisition of somatic mutations in this gene may lower rates of survival in drug-resistant colorectal tumors that also harbor KRAS-mutations (Xu, 2017).

RECQL4 is a RECQ-like helicase involved in aging and DNA repair that forms a complex with TP53 (Qiao, 2016; Croteau, 2012). This protein has mutations, amplified copy numbers and upregulation in a number of cancers including colorectal, breast and gastric cancer (Qiao, 2016; Cancer Genome Atlas Network, 2012; Mo, 2016; Arora, 2016; Lao, 2013; Buffart, 2005; Giannakis, 2016). Amplification of this protein in metastatic colorectal cancer seems to be correlated with other oncogenes on chromosome 8, including Yamanaka factor MYC, SLA, PTK2, PTP4A3, TPD52, MOS (Buffart, 2005), as well as PLEC, EPPK1, RAD21, and PDS5B (Cancer Genome Atlas Network, 2012; Buffart, 2005; Giannakis, 2016). In gastric cancer cells, high levels of expression of RECQL4 have been linked to drug resistance and activation of the multi-drug resistant protein 1 (MDR1 / P-glycoprotein 1 / ABCB1 / CD243) (Mo, 2016). IHC (immunohistochemistry) staining of RECQL4 in colorectal cancer showed this protein localizing to the nucleus (Lao, 2013).

Adenomatous Polyposis Coli (APC) is a gene frequently mutated in colorectal cancer and inherited mutations in this gene are causative for classical familial adenomatous polyposis. This gene is involved in the regulation of cell adhesion through its interaction with CDH1 (e-cadherin) and CTNNB1 (beta-catenin) via the WNT signaling pathway (Markowitz, 2009), and it is considered a tumor suppressor. The loss of functional APC protein (often alongside mutations in TP53 or KRAS) is a frequent event in colorectal cancers and is associated with cellular over-proliferation, chromosomal instability and carcinogenesis (Pino, 2010).

MLH1 is a primary MMR (mismatch repair) DNA repair gene, and loss of function of MLH1 protein leads to microsatellite instability and cancer in the colon and other organs (Papadopoulos, 1994). This can be caused by a “second hit” somatic mutation in MLH1 in patients with inherited heterozygous pathogenic mutations (Ollikainen, 2007), or alternatively through epigenetic silencing via hypermethylation of the MLH1 promoter in CIMP (CpG Island Methylator Phenotype) colorectal tumors (Boland, 2010). Inherited MLH1 mutation is one of the most frequent causes of Lynch Syndrome, and it is a pivotal gene in the mismatch repair functionality of cells (Cohen, 2014). IHC staining for MLH1 is therefore useful for determining the tumor subtype of colorectal cancer patients, as in Lynch Syndrome, Lynch-like cancers and in CIMP cases expression of this protein regularly disappears.

MSH2 is a DNA repair protein involved in mismatch repair and inherited or somatic mutations in this gene are a source of microsatellite instability (MSI) and cancer in the colon. Heterozygous germline mutations in MSH2 are the cause of disease in a large subset of Lynch Syndrome (LS) patients (Cohen, 2014); in LS, somatic mutation of the remaining functional copy of this protein leads to MSI, widespread mutation and LOF in a number of important tumor suppressor and apoptotic genes, and subsequently tumorigenesis throughout the colon (Lynch, 2009).

MSH6 is a DNA repair protein involved specifically in the mismatch repair of mutations that arise from replication and recombination, and somatic and inherited mutations in this gene lead to microsatellite instability and colorectal cancer. MSH6, along with MSH2, MSH3, MLH1, PMS1 and PMS2, is dysfunctional in the germline of many Lynch Syndrome patients (Houlleberghs, 2017; Giglia, 2016), and genetic testing along with immunohistochemistry staining of this protein is useful for diagnosis and subtyping of colorectal tumors (Giglia, 2016).

PMS2 is a DNA mismatch repair protein that is often mutated or downregulated in colorectal cancer. Inherited mutations in PMS2 are the cause of Lynch Syndrome and colorectal cancer in a subset of patients (Rosty, 2016; Cohen, 2014). Knockout of PMS2 results in microsatellite unstable tumors with malignant potential, and IHC staining for this protein can thus be used to subtype colorectal tumors (Worthley, 2005).

AXIN1 is a protein involved in the WNT signaling pathway and acts alongside APC, CK1, AXIN2 and GSK3B in the regulation of B-Catenin (CTNNB1) phosphorylation and degradation (Mazzoni, 2014; Novellasdemunt, 2015). Mutations in this gene are prevalent in microsatellite-unstable (MSI) colorectal cancers, where loss of function of this protein may have a negative activating impact on WNT signaling and B-Catenin management and contribute to tumorigenesis (Novellasdemunt, 2015; Segditsas, 2006).

AXIN2, also known as conductin, is a member of the WNT signaling pathway and maintains the degradation and phosphorylation of B-Catenin (CTNNB1) alongside APC, AXIN1, CK1 and CSK3B in basal colorectal cells (Mazzoni, 2014; Novellasdemunt, 2015; Segditsas, 2006). AXIN2 is somatically mutated in microsatellite-unstable (MSI) colorectal cancers, where loss of function contributes to aberrant activation of WNT signaling and tumorigenesis. Germline mutations in this gene are also associated with a predisposition for colorectal carcinoma (Segditsas, 2006).

TCF7L2 is a member of the WNT signaling pathway and interacts with B-catenin. It is frequently mutated in MSI- (microsatellite instability) positive colorectal tumors, and also forms fusion transcripts with the neighboring VTI1A gene in some colorectal cancers (Nome, 2014). Homozygous germline mutations in this gene (mutations rs12255372 and rs7903146) have been associated with a risk for colorectal cancer as well (Folsom, 2008; Sainz, 2012; Hazra, 2008).