In 2007, Jeremy Jass proposed a molecular classification of colorectal cancer including KRAS, BRAF, Mismatch Repair, CIMP and MGMT Status. Since then, many prognostic and predictive studies have been published on this topic, mainly focusing on one single molecular marker. The aim of a potential e-book would be to summarize the knowledge in 2012 from a multidiscipli-nary point of view that can potentially be used as a manual by CRC researchers in every field.

University of Bern

Frontiers in Oncology

Gastrointestinal Cancers

Towards a molecular classification of colorectal cancer

In colorectal cancer (CRC), an internationally standardized molecular classification has not been implemented yet. Nevertheless, there are different pathogenetic aspects that could form the basis for a future molecular classification in CRC (<a href="#B1">1</a>).In the classic model, CRC is divided into two major pathways. The first is the chromosomal instability pathway including 85% of all CRCs. This pathway is based on the adenoma–carcinoma sequence, which is defined by consecutive mutations in the APC, KRAS, and TP53 gene and deletion of the chromosome 18q (<a href="#B2">2</a>). An example of an autosomal dominantly inherited disease, which belongs to the chromosomal instability pathway group, is the familial adenomatous polyposis syndrome (FAP). The second, namely the microsatellite instability pathway (15% of all CRCs) is defined by a mismatch-repair deficiency, which leads to a genomic instability. The microsatellite instability status can be sporadic (12%) or hereditary (3%, Lynch syndrome-associated CRC, HNPCC). Sporadic cases can be differentiated from hereditary cases by using the Amsterdam/Revised Bethesda criteria.In 2007, Jeremy Jass proposed a molecular classification based on clinical, morphological, and molecular parameters, which included five subgroups (<a href="#B3">3</a>).Group 1 (12% of all CRCs): chromosomally stable, MLH1 methylated, MSI-H, BRAF mutated, CpG island methylator phenotype (CIMP) high status; group 2 (8% of all CRCs): chromosomally stable, partially MLH1 methylated, microsatellite stable (MSS, MSI-L), BRAF mutated, CIMP high; group 3 (20% of all CRCs): chromosomally instable, MGMT methylated, MSS/MSI-L, KRAS mutated, CIMP low; group 4 (57% of all CRCs): chromosomally instable, MSS/MSI-L, CIMP negative; group 5 (3% of all CRCs): chromosomally stable, MSI-H, CIMP negative, BRAF wild type.Following up on this last classification, in 2010, Barbara Leggett and Vicki Whitehall published a pathogenetic overview of sporadic CRC including three pathways (<a href="#B4">4</a>).The serrated pathway: MSI-H, CIMP high, BRAF mutated (corresponds to the Jass group 1); the alternative pathway: MSS, CIMP low, KRAS mutated (corresponds to the Jass group 3; the traditional pathway: MSS, CIMP negative, BRAT wild type, KRAS wild type (corresponds to the Jass group 4).In 2012, the Cancer Genome Atlas Network published a promising approach for a future molecular classification based on different pathways (<a href="#B5">5</a>).Group 1 (wnt and TGF-beta pathway): proliferation, stem cell, and progenitor phenotype; group 2 (PIK3CA and RTK&#x2013;RAS pathway): proliferation, cell survival, translation; group 3 (p53 pathway): proliferation, cell survival. This approach may elucidatepromising target molecules for wnt pathway inhibitors or proteins of the PIK3CA and RTK&#x2013;RAS pathway such as IGF2, IGFR, ERBB2, ERBB3, MEK, AKT, and MTOR.Recently, a new proposal for a molecular classification of CRC was published by a research group from Oxford based on 906 stage II and III CRCs (<a href="#B6">6</a>).Group 1: MSI-H and/or BRAF mutated; group 2: chromosomally instable and/or TP53 mutated with KRAS and PIK3Ca wild type status; group 3: chromosomally instable, KRAS and/or PIK3CA mutated; TP53 wild type status; group 4: chromosomally stable, KRAS and/or PIK3CA mutated; TP53 wild type status; group 5: NRAS mutated; group 6: no mutations; group 7: other.All these proposals for a CRC molecular classification have in common that some molecular features such as KRAS, BRAF, microsatellite status, and CIMP are often included. Nevertheless, it has to be kept in mind that molecular markers can be prognostic (i.e., BRAF), predictive (i.e., RAS), or both (i.e., microsatellite status). Therefore, the main aim of the research topic “Toward a molecular classification of colorectal cancer” was to include articles that focus on the role of already established (<a href="#B7">7</a>–<a href="#B13">13</a>) or potentially novel and promising molecular biomarkers such as telomere length (<a href="#B14">14</a>) or microRNAs (<a href="#B15">15</a>) and additionally to give an overview on the molecular pathology of CRC.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Towards a Molecular Classification of Colorectal Cancer

Telomere biology is central to the maintenance of genomic stability and telomeric dysfunction is thought to be an early stage in carcinogenesis. Reports of telomere lengths and their ascribed colorectal cancer (CRC) risks have been discordant, with both very short and very long telomeres implicated. Nevertheless, telomeres appear to play a very central role in cancer initiation. Telomere length changes also appear to impact disease burden, progression, and overall survival. This review covers contemporary views on telomere biology and CRC risk, with a brief overview of analytical methods employed in telomere measurement. We conclude with arguments in favor of including telomere assessment in the molecular profiling of CRCs.

Telomere biology is central to the maintenance of genomic stability and telomeric dysfunction is thought to be an early stage in carcinogenesis. Reports of telomere lengths and their ascribed colorectal cancer (CRC) risks have been discordant, with both very short and very long telomeres implicated. Nevertheless, telomeres appear to play a very central role in cancer initiation. Telomere length changes also appear to impact disease burden, progression, and overall survival. This review covers contemporary views on telomere biology and CRC risk, with a brief overview of analytical methods employed in telomere measurement. We conclude with arguments in favor of including telomere assessment in the molecular profiling of CRCs.

Toward a Molecular Classification of Colorectal Cancer: The Role of Telomere Length

Robust biomarkers that can precisely stratify patients according to treatment needs are in great demand. The literature is inconclusive for most reported prognostic markers for colorectal cancer (CRC). Hence, adequately reported studies in large representative series are necessary to determine their clinical potential. We investigated the prognostic value of three Wnt signaling-associated proteins, β-catenin, E-cadherin, and SOX9, in a population-representative single-hospital series of 1290 Norwegian CRC patients by performing immunohistochemical analyses of each marker using the tissue microarray technology. Loss of membranous or cytosolic β-catenin and loss of cytosolic E-cadherin protein expression were significantly associated with reduced 5-year survival in 903 patients who underwent major resection (722 evaluable tissue cores) independently of standard clinicopathological high-risk parameters. Pre-specified subgroup analyses demonstrated particular effect for stage IV patients for β-catenin membrane staining (P = 0.018; formal interaction test P = 0.025). Among those who underwent complete resection (714 patients, 568 evaluable), 5-year time-to-recurrence analyses were performed, and stage II patients with loss of cytosolic E-cadherin were identified as an independent high-risk subgroup (P = 0.020, formal interaction test was not significant). Nuclear β-catenin and SOX9 protein, regardless of intracellular location, were not associated with prognosis. In conclusion, the protein expression level of membranous or cytosolic β-catenin and E-cadherin predicts CRC patient subgroups with inferior prognosis.

Robust biomarkers that can precisely stratify patients according to treatment needs are in great demand. The literature is inconclusive for most reported prognostic markers for colorectal cancer (CRC). Hence, adequately reported studies in large representative series are necessary to determine their clinical potential. We investigated the prognostic value of three Wnt signaling-associated proteins, β-catenin, E-cadherin, and SOX9, in a population-representative single-hospital series of 1290 Norwegian CRC patients by performing immunohistochemical analyses of each marker using the tissue microarray technology. Loss of membranous or cytosolic β-catenin and loss of cytosolic E-cadherin protein expression were significantly associated with reduced 5-year survival in 903 patients who underwent major resection (722 evaluable tissue cores) independently of standard clinicopathological high-risk parameters. Pre-specified subgroup analyses demonstrated particular effect for stage IV patients for β-catenin membrane staining (P = 0.018; formal interaction test P = 0.025). Among those who underwent complete resection (714 patients, 568 evaluable), 5-year time-to-recurrence analyses were performed, and stage II patients with loss of cytosolic E-cadherin were identified as an independent high-risk subgroup (P = 0.020, formal interaction test was not significant). Nuclear β-catenin and SOX9 protein, regardless of intracellular location, were not associated with prognosis. In conclusion, the protein expression level of membranous or cytosolic β-catenin and E-cadherin predicts CRC patient subgroups with inferior prognosis.

Prognostic Significance of β-Catenin, E-Cadherin, and SOX9 in Colorectal Cancer: Results from a Large Population-Representative Series

PIK3CA, the catalytic subunit of PI3K, is mutated in many different tumors, including colorectal cancer (CRC). Mutations of PIK3CA have been reported in 10–20% of CRC, about 80% of mutations found in two hot spots in exon 9 and exon 20. In RAS wild-type CRC, PIK3CA mutations have been associated with a worse clinical outcome and with a negative prediction of a response to targeted therapy by anti-EGFR monoclonal antibodies. However, these findings have not been confirmed in all studies and subsequent more detailed analysis has revealed that these effects may be restricted to mutations in Exon 20. Finally, mutations in PIK3CA may be the long sought biomarker for successful adjuvant therapy with aspirin in patients with CRC. Therefore, PIK3CA mutations appear to be a promising predictive biomarker; however, further data are needed to conclusively define the impact of somatic mutations in the PIK3CA gene for the management of patients with CRC.

PIK3CA, the catalytic subunit of PI3K, is mutated in many different tumors, including colorectal cancer (CRC). Mutations of PIK3CA have been reported in 10&#x2013;20% of CRC, about 80% of mutations found in two hot spots in exon 9 and exon 20. In RAS wild-type CRC, PIK3CA mutations have been associated with a worse clinical outcome and with a negative prediction of a response to targeted therapy by anti-EGFR monoclonal antibodies. However, these findings have not been confirmed in all studies and subsequent more detailed analysis has revealed that these effects may be restricted to mutations in Exon 20. Finally, mutations in PIK3CA may be the long sought biomarker for successful adjuvant therapy with aspirin in patients with CRC. Therefore, PIK3CA mutations appear to be a promising predictive biomarker; however, further data are needed to conclusively define the impact of somatic mutations in the PIK3CA gene for the management of patients with CRC.

PIK3CA in Colorectal Cancer

Phosphatase and TENsin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor gene located at chromosome 10q23.31, encoding for a 403-amino acid protein that possesses both lipid and protein phosphatase activities. The main function of PTEN is to block the PI3K pathway by dephosphorylating phosphatidylinositol (PI) 3,4,5-triphosphate to PI-4,5-bisphosphate thus counteracting PI3K function. PTEN inactivation is a frequent event in many cancer types and can occur through various genetic alterations including point mutations, large chromosomal deletions, and epigenetic mechanisms. In colorectal cancer (CRC) PTEN is altered through mixed genetic/epigenetic mechanisms (typically: mutations and promoter hypermethylation or 10q23 LOH and promoter hypermethylation), which lead to the biallelic inactivation of the protein in 20–30% of cases. The role of PTEN as a prognostic and predictive factor in CRC has been addressed by relatively few works. This review is focused on the report and on the discussion of the studies investigating these aspects. Overall, at the moment, there are conflicting results and, therefore it has not been clarified whether PTEN might play a prognostic role in CRC. The same is valid also for the predictive role, leading to the fact that PTEN evaluation cannot be used in routinely diagnosis for the early identification of patients who might be addressed to the treatment with EGFR-targeted therapies, at odds with other genetic alterations belonging to EGFR-downstream pathways. The reason of discordant results may be attributable to several issues: (1) the size of the analyzed cohort, (2) patients inclusion criteria, (3) the methods of assessing PTEN alteration. In particular, there are no standardized methods to evaluate this marker, especially for immunohistochemistry, a technique suffering of intra and inter-observer variability due to the semi-quantitative character of such an analysis. In conclusion, much work, especially in large and homogeneous cohorts of cases from different laboratories, has to be done before the establishment of PTEN as prognostic or predictive marker in CRC.

Phosphatase and TENsin homolog deleted on chromosome 10 (PTEN) is a tumor suppressor gene located at chromosome 10q23.31, encoding for a 403-amino acid protein that possesses both lipid and protein phosphatase activities. The main function of PTEN is to block the PI3K pathway by dephosphorylating phosphatidylinositol (PI) 3,4,5-triphosphate to PI-4,5-bisphosphate thus counteracting PI3K function. PTEN inactivation is a frequent event in many cancer types and can occur through various genetic alterations including point mutations, large chromosomal deletions, and epigenetic mechanisms. In colorectal cancer (CRC) PTEN is altered through mixed genetic/epigenetic mechanisms (typically: mutations and promoter hypermethylation or 10q23 LOH and promoter hypermethylation), which lead to the biallelic inactivation of the protein in 20&#x2013;30% of cases. The role of PTEN as a prognostic and predictive factor in CRC has been addressed by relatively few works. This review is focused on the report and on the discussion of the studies investigating these aspects. Overall, at the moment, there are conflicting results and, therefore it has not been clarified whether PTEN might play a prognostic role in CRC. The same is valid also for the predictive role, leading to the fact that PTEN evaluation cannot be used in routinely diagnosis for the early identification of patients who might be addressed to the treatment with EGFR-targeted therapies, at odds with other genetic alterations belonging to EGFR-downstream pathways. The reason of discordant results may be attributable to several issues: (1) the size of the analyzed cohort, (2) patients inclusion criteria, (3) the methods of assessing PTEN alteration. In particular, there are no standardized methods to evaluate this marker, especially for immunohistochemistry, a technique suffering of intra and inter-observer variability due to the semi-quantitative character of such an analysis. In conclusion, much work, especially in large and homogeneous cohorts of cases from different laboratories, has to be done before the establishment of PTEN as prognostic or predictive marker in CRC.

Functions and Regulation of the PTEN Gene in Colorectal Cancer

Different genetic aberrations of BRAF have been reported in various malignancies. BRAF is member of the RAS/RAF/MEK/ERK pathway and constitutive activity of this pathway can lead to increased cellular growth, invasion, and metastasis. The most common activating BRAF mutation in colorectal cancer is the V600E mutation, which is present in 5–15% of all tumors, and up to 80% of tumors with high microsatellite instability (MSI) harbor this mutation. BRAF mutation is associated with proximal location, higher age, female gender, MSI-H, high grade, and mucinous histology, and is a marker of poor prognosis in colorectal cancer. The role of BRAF mutation as a predictive marker in respect of EGFR targeted treatments is controversial. BRAF V600 selective inhibitors have been approved for the treatment of V600 mutation positive metastatic melanoma, but the response rates in colorectal cancer are poor. This might be due to innate resistance mechanisms of colorectal cancers against the treatment solely targeting BRAF. To overcome resistance the combination of treatments, simultaneous inhibition of BRAF and MEK or PI3K/mTOR, might emerge as a successful therapeutic concept.

Different genetic aberrations of BRAF have been reported in various malignancies. BRAF is member of the RAS/RAF/MEK/ERK pathway and constitutive activity of this pathway can lead to increased cellular growth, invasion, and metastasis. The most common activating BRAF mutation in colorectal cancer is the V600E mutation, which is present in 5–15% of all tumors, and up to 80% of tumors with high microsatellite instability (MSI) harbor this mutation. BRAF mutation is associated with proximal location, higher age, female gender, MSI-H, high grade, and mucinous histology, and is a marker of poor prognosis in colorectal cancer. The role of BRAF mutation as a predictive marker in respect of EGFR targeted treatments is controversial. BRAF V600 selective inhibitors have been approved for the treatment of V600 mutation positive metastatic melanoma, but the response rates in colorectal cancer are poor. This might be due to innate resistance mechanisms of colorectal cancers against the treatment solely targeting BRAF. To overcome resistance the combination of treatments, simultaneous inhibition of BRAF and MEK or PI3K/mTOR, might emerge as a successful therapeutic concept.

Toward a Molecular Classification of Colorectal Cancer: The Role of BRAF

Microsatellite instability (MSI) is the molecular hallmark of DNA mismatch repair deficiency. Since its initial description in colorectal cancer (CRC) in 1993 and its association with Lynch syndrome, the most common inherited cancer predisposition world-wide, accumulating evidence suggests that MSI status may also be of concrete prognostic and predictive value in the management of sporadic CRC. This mini review aims at providing a concise survey of the molecular basis and the multifaceted role(s) of MSI status in today’s clinical practice.

Microsatellite instability (MSI) is the molecular hallmark of DNA mismatch repair deficiency. Since its initial description in colorectal cancer (CRC) in 1993 and its association with Lynch syndrome, the most common inherited cancer predisposition world-wide, accumulating evidence suggests that MSI status may also be of concrete prognostic and predictive value in the management of sporadic CRC. This mini review aims at providing a concise survey of the molecular basis and the multifaceted role(s) of MSI status in today&#x2019;s clinical practice.

Toward a Molecular Classification of Colorectal Cancer: The Role of Microsatellite Instability Status

MicroRNA-135b and Its Circuitry Networks as Potential Therapeutic Targets in Colon Cancer

O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair enzyme with the ability to protect cells from DNA mutations by removing alkyl groups from the O6 position of guanine. Colon mucosa is exposed to the direct effects of environmental carcinogens and therefore maintaining a proficient DNA repair system is very important to stay protected against DNA mutagenesis. Loss of MGMT expression is almost exclusively associated with methylation of CpG islands in the MGMT gene promoter region which is found in approximately 40% of colorectal cancers. The role of MGMT loss in colorectal tumorigenesis is complex but numerous studies have documented methylation of this gene even in the normal appearing mucosa as well as in aberrant crypt foci, suggesting that MGMT methylation can be regarded as an early event or “field defect” in colon cancer neoplasia. The focus of this perspective is the role of MGMT in different pathways of colorectal carcinogenesis as well as the implication of this molecule in treatment decisions in colorectal cancer patients.

O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair enzyme with the ability to protect cells from DNA mutations by removing alkyl groups from the O6 position of guanine. Colon mucosa is exposed to the direct effects of environmental carcinogens and therefore maintaining a proficient DNA repair system is very important to stay protected against DNA mutagenesis. Loss of MGMT expression is almost exclusively associated with methylation of CpG islands in the MGMT gene promoter region which is found in approximately 40% of colorectal cancers. The role of MGMT loss in colorectal tumorigenesis is complex but numerous studies have documented methylation of this gene even in the normal appearing mucosa as well as in aberrant crypt foci, suggesting that MGMT methylation can be regarded as an early event or “field defect” in colon cancer neoplasia. The focus of this perspective is the role of MGMT in different pathways of colorectal carcinogenesis as well as the implication of this molecule in treatment decisions in colorectal cancer patients.

Toward a Molecular Classification of Colorectal Cancer: The Role of MGMT

Background: Approximately 20% of all colorectal cancers are hypothesized to arise from the “serrated pathway” characterized by mutation in BRAF, high-level CpG Island Methylator Phenotype, and microsatellite instability/mismatch repair (MMR)-deficiency. MMR-deficient cancers show frequent losses of Cdx2, a homeodomain transcription factor. Here, we determine the predictive value of Cdx2 expression for MMR-deficiency and investigate changes in expression between primary cancers and matched lymph node metastases.Methods: Immunohistochemistry for Cdx2, Mlh1, Msh2, Msh6, and Pms2 was performed on whole tissue sections from 201 patients with primary colorectal cancer and 59 cases of matched lymph node metastases. Receiver operating characteristic curve analysis and Area under the Curve (AUC) were investigated; association of Cdx2 with clinicopathological features and patient survival was carried out.Results: Loss of Cdx2 expression was associated with higher tumor grade (p = 0.0002), advanced pT (p = 0.0166), and perineural invasion (p = 0.0228). Cdx2 loss was an unfavorable prognostic factor in univariate (p = 0.0145) and multivariate [p = 0.0427; HR (95% CI): 0.58 (0.34–0.98)] analysis. The accuracy (AUC) for discriminating MMR-proficient and – deficient cancers was 87% [OR (95% CI): 0.96 (0.95–0.98); p < 0.0001]. Specificity and negative predictive value for MMR-deficiency was 99.1 and 96.3%. One hundred and seventy-four patients had MMR-proficient cancers, of which 60 (34.5%) showed Cdx2 loss. Cdx2 loss in metastases was related to MMR-deficiency (p < 0.0001). There was no difference in expression between primary tumors and matched metastases.Conclusion: Loss of Cdx2 is a sensitive and specific predictor of MMR-deficiency, but is not limited to these tumors, suggesting that events “upstream” of the development of microsatellite instability may impact Cdx2 expression.

Background: Approximately 20% of all colorectal cancers are hypothesized to arise from the “serrated pathway” characterized by mutation in BRAF, high-level CpG Island Methylator Phenotype, and microsatellite instability/mismatch repair (MMR)-deficiency. MMR-deficient cancers show frequent losses of Cdx2, a homeodomain transcription factor. Here, we determine the predictive value of Cdx2 expression for MMR-deficiency and investigate changes in expression between primary cancers and matched lymph node metastases.Methods: Immunohistochemistry for Cdx2, Mlh1, Msh2, Msh6, and Pms2 was performed on whole tissue sections from 201 patients with primary colorectal cancer and 59 cases of matched lymph node metastases. Receiver operating characteristic curve analysis and Area under the Curve (AUC) were investigated; association of Cdx2 with clinicopathological features and patient survival was carried out.Results: Loss of Cdx2 expression was associated with higher tumor grade (p = 0.0002), advanced pT (p = 0.0166), and perineural invasion (p = 0.0228). Cdx2 loss was an unfavorable prognostic factor in univariate (p = 0.0145) and multivariate [p = 0.0427; HR (95% CI): 0.58 (0.34–0.98)] analysis. The accuracy (AUC) for discriminating MMR-proficient and – deficient cancers was 87% [OR (95% CI): 0.96 (0.95–0.98); p &lt; 0.0001]. Specificity and negative predictive value for MMR-deficiency was 99.1 and 96.3%. One hundred and seventy-four patients had MMR-proficient cancers, of which 60 (34.5%) showed Cdx2 loss. Cdx2 loss in metastases was related to MMR-deficiency (p &lt; 0.0001). There was no difference in expression between primary tumors and matched metastases.Conclusion: Loss of Cdx2 is a sensitive and specific predictor of MMR-deficiency, but is not limited to these tumors, suggesting that events “upstream” of the development of microsatellite instability may impact Cdx2 expression.