Characterization of the molecular pathology of colorectal cancer (CRC) will be included in the CRC Project in Indiana in order to connect the conventional histologic examination with the evolving molecular signature profiles in CRC and OMIC analysis. Characterization of the molecular pathology will contribute to the understanding of the molecular subsets that exist in the CRC population of Indiana and provide a foundation for assessing the resulting effectiveness of biomarkers whose characteristics differ among molecular subsets.

Molecular Pathways. Five overlapping molecular pathways of tumor initiation and progression are currently known for colorectal carcinogenesis: the chromosomal instability pathway (CIN); the microsatellite instability pathway with high levels of instability (MSI-H); the microsatellite instability pathway with low levels of instability (MSI-L); the hMYH pathway (MYH); and the CpG island methylation pathway/phenotype (CIMP). Three of these pathways (CIN, MSI-H, and CIMP) are involved in the pathogenesis of the vast majority of CRC, and these pathways are characterized in this project.

Analysis Workflow

Routine formalin-fixed paraffin-embedded tissue blocks or unstained slides from blocks will obtained from patients consented at IUSCC. Blocks or unstained slides of tumor and of non-neoplastic control tissue, usually colorectal mucosa, will be shipped under environmentally controlled conditions to Dr. Hamilton’s laboratory at The University of Texas M. D. Anderson Cancer Center in Houston. Sections will be prepared for scalpel microdissection of malignant epithelium for DNA extraction or for immunohistochemistry on positively charged slides, as in previous studies.

The CRC will be evaluated for microsatellite instability status, methylation status, mutation of KRAS and BRAF, and expression of p53 gene product as a surrogate for P53 gene mutation that has about 80% accuracy in CRC. The methods are established in Dr. Hamilton’s laboratory and have been used in previously published studies, summarized in Table 1

P53 overexpression Mutation surrogate immunohistochemistry with antibody D07 Soliman et al., 2001***
*Pyrosequencing: Purified PCR products were sequenced on an ABI Prism 3730 DNA Analyzer with the ABI Prism Big Dye Terminator Cycle Sequencing Kit version 1.1 (Applied Biosystems).
** http://clincancerres.aacrjournals.org/content/14/9/2560.long
*** http://www.nature.com/bjc/journal/v85/n7/abs/6691838a.html

CRC will be classified as CIMP1, CIMP2, CIMP-negative, or HNPCC based on the criterion is given below. The algorithm for classification will consider MSI status based upon the marker panel and expression of mismatch repair gene products, methylation status, BRAF mutation, KRAS mutation, and p53 expression in that order of priority to first identify CIMP1 and HNPCC tumors, followed by CIMP2 and CIMP-negative tumors.

Description of Analysis Results Data

Additional Background

Chromosomal instability pathway (CIN). The vast majority (approximately 80% in Western countries) of colorectal carcinomas develop through the chromosomal instability pathway (CIN) in the conventional adenoma-adenocarcinoma sequence. The hallmark of these CRC is that progression involves structural chromosomal alterations that are reflected in altered total DNA content, cytogenetic aneuploidy, and numerous allelic losses and gains on molecular analyses. These CRC usually have inactivation of the adenomatous polyposis coli (APC) gene on chromosome 5q as an initiating event. Familial adenomatous polyposis with germline APC mutation is the inherited form of CRC in this pathway. Point mutations of the KRAS proto-oncogene and the p53 gene are frequent in CIN CRC with frequencies that are influenced by methylation status [Shen et al. 2007; Suehiro et al. 2008], as described below and summarized in Table 1. KRAS proto-oncogene mutation occurs predominantly in CIN tumors with extensive hypermethylation, and p53 mutation in CIN CRC that lack extensive hypermethylation [Shen et al. 2007; Suehiro et al. 2008].

Microsatellite instability pathway with high levels of instability (MSI-H). CRC with high levels of microsatellite instability (MSI-H) comprise about 15% of cases in Western countries. CRC in this pathway are characterized at the molecular level by numerous nucleotide substitutions and by insertion/deletion mutations in repeated nucleotide sequences (microsatellites). MSI-H CRC result from inactivation of both alleles of a nucleotide mismatch repair gene. These tumors usually have normal total DNA content, relatively normal cytogenetic karyotype, and infrequent allelic imbalances on molecular analyses. Germline mutation of the hMSH2 or hMLH1 mismatch repair gene usually leads to MSI-H CRC in hereditary non-polyposis colorectal cancer syndrome (HNPCC, Lynch syndrome, Warthin-Lynch syndrome), while most sporadic MSI-H CRC result from transcriptional silencing of the hMLH1 mismatch repair gene by promoter hypermethylation, as described below. MSI-H CRC have distinctive clinical-pathologic features. These include right-sided location, poor differentiation, unusual histologic types (mucinous, medullary and signet-ring cell histology), absence of “dirty” necrosis, expansile growth pattern, numerous tumor-infiltrating lymphocytes, and prominent peri-tumoral lymphoid nodules, term Crohn’s-like lymphoid response. MSI-H tumors are identified with a consensus panel of mono- and dinucleotide repeat markers that have altered allelic size in more than 40% of the markers. Serrated adenomas are one of the important precursors to MSI-H CRC [Liang et al. 2008]. Point mutation of the BRAF gene in the RAS/RAF pathway is relatively common in sporadic MSI-H CRC due to hMLH1 hypermethylation [Shen et al. 2007; Suehiro et al. 2008] but does not occur in MSI-H CRC of HNPCC patients. This striking difference in frequencies provides an important marker that assists in distinguishing between sporadic and inherited MSI-H CRC and will be used in our study.

Microsatellite instability pathway with low levels of instability (MSI-L). CRC with low levels of microsatellite instability (MSI-L) are heterogeneous. These tumors have altered allelic size in a minority of markers in the consensus MSI panel. The molecular mechanisms responsible for this small subset of tumors are poorly understood, and the clinical-pathologic characteristics are not yet well defined [Kets et al. 2006]. In most reported studies, this small subset is included with microsatellite-stable CRC that lack change in allelic size in the consensus panel of markers.

hMYH pathway (MYH). CRC that have developed through the hMYH pathway have been described relatively recently. These infrequent inherited CRC have mutations in both copies of the hMYH base excision repair gene due to inheritance of one mutated gene from each parent, resulting in bi-allelic alterations. Progression of these tumors is characterized by high frequency of G-to-T transversion mutations and by allelic loss on chromosome 18q, but the tumors usually has neither chromosomal instability nor microsatellite instability. Understanding the small number of CRC that develop along this pathway is in evolution [Lindor 2009].

CpG island methylation pathway/phenotype (CIMP). About a third of CRC in Western countries develop with the CpG island methylation pathway/phenotype (CIMP). CpG islands are 0.5 to 2 kilobase regions rich in cytosine-guanosine dinucleotide repeats that are present in the 5` region of approximately half of all human genes. Methylation of cytosine residues within CpG islands of promoter regions and proximal exons is associated with loss of gene expression by repression of transcription and altered chromosomal structure. This epigenetic pathway is important in physiologic conditions such as X chromosome inactivation to form the Barr body, genetic imprinting, and aging as well as in neoplasia. In CRC with CIMP, transcriptional inactivation by hypermethylation often occurs in numerous genes that are not usually methylated in non-neoplastic colorectal mucosa. Hypermethylation is often concordant among numerous genes, and this concordance defines the CIMP.

Because of hypermethylation of hMLH1, many CRC that develop due to CIMP also have MSI-H, whereas CRC with extensive hypermethylation that does not involve hMLH1 have chromosomal instability. Clinical-pathologic features of MSI-H/CIMP tumors are similar to those in the MSI-H pathway, as described above, and these tumors have a high frequency of BRAF mutation (Table 1). Microsatellite-stable CRC with CIMP also have characteristic clinical-pathologic findings included right-sided location, poor differentiation, and cribriform gland architecture, whereas corkscrew/serrated gland architecture is not seen in this molecular subtype [Chirieac et al. 2005]. This subset accounts for the majority of CRC that have a KRAS mutation [Shen et al. 2007; Suehiro et al. 2008].

Molecular Categorization of CRC As described above, epigenetic and genetic abnormalities involving the CIN, MSI-H, CIMP, RAS/RAF, and P53 pathways that are important in CRC are not random. Instead, these abnormalities are associated with each other within defined molecular subtypes [Shen et al. 2007; Suehiro et al. 2008], as shown in Figure 1 and summarized in Table 2 below. These findings permit molecular classification of CRC based upon molecular characteristics.

• super-administrator

Researchers should cite this work as follows:

• Stanley R Hamilton; Ann Christine Catlin (2011), "Hamilton Molecular Characterization Laboratory," http://ccehub.org/resources/388.

  BibTex | EndNote

1. molecular characterization