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The molecular pathogenesis and classification of colorectal carcinoma are based on the traditional adenoma–carcinoma sequence in the Vogelstein model, serrated polyp pathway, and MSI.
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The genetic basis for hereditary nonpolyposis colorectal cancer is based on detection of mutations in the MLH1, MSH2, MSH6, PMS2, and EPCAM genes.
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Genetic testing for the Lynch syndrome includes MSI testing, methylator phenotype testing, BRAF mutation testing, and molecular testing for germline mutations in mismatch
Molecular Diagnostics in Colorectal Carcinoma: Advances and Applications for 2018
Section snippets
Key points
Epidemiology
Constitutional (endogenous) as well as environmental (exogenous) factors are associated with the development of colorectal carcinoma. Multiple risk factors have been linked to colorectal carcinoma. Colorectal carcinoma is more common in late-middle-aged and elderly individuals. Men are at a higher risk for developing this malignancy. There is a strong association with a Western type of diet consisting of high-calorie food, rich in animal fat.4
Pathophysiology and molecular genetics
The various molecular alterations described in colorectal carcinoma are enlisted in Box 1.10 The diagrams depict the adenoma–carcinoma sequence and serrated polyp pathway arising from a complex interplay of genetic alterations (Fig. 1, Fig. 2, Fig. 3, Fig. 4).1
Traditional Vogelstein model and APC gene pathway
The traditional model of Vogelstein describes the classic adenoma–carcinoma sequence and accounts for approximately 80% of sporadic colon tumors. The pathogenesis involves mutation of the APC gene early in the neoplastic process.2
Shared by Both Inherited and Sporadic Tumors
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Tendency to occur on right side of colon.
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Medullary carcinoma phenotype.
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Presence of mucinous or signet ring component.
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Presence of tumor infiltrating and peritumoral lymphocytes.
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Crohnlike inflammatory response.
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Pushing tumor borders (Fig. 10; see Tables 4 and 5).32
Quality assurance
The detection of MSI in a tumor by microsatellite analysis requires that the DNA used for the analysis be extracted from a portion of the tumor that contains approximately 40% or more tumor cells. Thus, pathologists should help to identify areas of the tumor for DNA isolation that have at least this minimum content of tumor cells. MSI testing is frequently performed in conjunction with IHC testing for MMR protein expression (ie, MLH1, MSH2, MSH6, and PMS expression). If the results of MMR IHC
Pitfalls
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During IHC evaluation of MSI proteins, an intact expression of all 4 proteins indicates that the tested MMR enzymes are intact.
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It is common for intact staining to be patchy.
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Positive IHC reaction for all 4 proteins does not exclude the Lynch syndrome, because approximately 5% of families may have a missense mutation (especially in MLH1), which can lead to a nonfunctional protein with retained antigenicity.
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Defects in lesser known MMR enzymes may also lead to a similar result, but this situation
Microsatellite instability testing
Frameshift mutations in microsatellites are identified by the amplification of selected microsatellites by PCR and analysis of fragment size by gel electrophoresis or an automated sequencer after extraction of DNA from both normal and tumor tissue (usually formalin-fixed, paraffin-embedded tissue). The sensitivity of the revised panel of MSI testing is at least 90% (Table 8).4, 5, 6, 7
Various fluorescent multiplex PCR-based panels (eg, Promega panel) are used for detection of MSI loci. The
KRAS mutation testing
Mutations in codons 12 and 13 in exon 2 of the coding region of the KRAS gene predict a lack of response to therapy with antibodies targeted to EGFR. The presence of the KRAS gene mutation has been shown to be associated with a lack of a clinical response to therapies targeted at EGFR, such as cetuximab and panitumumab. Although clinical guidelines for KRAS mutational analysis are evolving, provisional recommendations from the American Society for Clinical Oncology are that all patients with
Hereditary Nonpolyposis Colon Cancer
The goal of a genetic workup of families with HNPCC is to identify the underlying germline mutation. Confirmation of the germline mutation allows for the most accurate treatment and follow-up recommendations for the patient, and allows predictive testing to be undertaken in interested family members. The initial approach by most laboratories is to analyze the complete coding sequence of the relevant gene or genes (depending on IHC results), as well as a portion of the intronic regions important
Algorithmic strategies for management of mismatch repair colorectal carcinoma
There is no definitive standardized practice for the triage of colorectal carcinoma for molecular testing. Almost all microsatellite-instable colorectal carcinomas are detected by a combination of MSI and IHC testing. In the presence of deficient MMR, additional loss of protein expression of MSH2/MSH6, MSH6 alone, or PMS2 increases likelihood of the Lynch syndrome. Concomitant incidence of defective MMR, CIMP high, and MLH1 supports the diagnosis of sporadic defective MMR CRC. Detection of a
Molecular investigation of lymph nodes in patients with colon cancer using one-step nucleic acid amplification
A diagnostic system called one-step nucleic acid amplification, has recently been designed to detect cytokeratin 19 messenger RNA as a surrogate for lymph node metastases. In a study by Güler and colleagues,28 analysis of lymph nodes reported negative after standard examination with hematoxylin and eosin resulted in upstaging 2 of 13 patients (15.3%). Compared with histopathology, one-step nucleic acid amplification had a 94.5% sensitivity, 97.6% specificity, and a concordance rate of 97.1%.
Molecular staging individualizing cancer management
GUCY2C is a member of a family of enzyme receptors synthesizing guanosine 3′5′ cyclic monophosphate from guanosine-5′-triphosphate, which is expressed on intestinal epithelial cells but not in extraintestinal tissues. The expression is amplified in colorectal carcinoma compared with normal intestinal tissues. It is identified in all colorectal human tumors independent of anatomic location or grade, but not in extragastrointestinal malignancies. Therefore, it has a potential application in
Novel molecular screening approaches in colorectal cancer
Stool DNA potentially offers improved sensitivity, specificity, and cancer prevention by the detection of adenomas. The basis for stool DNA screening is the identification of genetic alterations in the initiation of a sequenced progression from adenoma to carcinoma, such as mutations in APC, KRAS, DCC, and p53. Key genetic alterations seen in many hereditary forms of CRC correspond with genetic alterations in sporadic CRC, indicating that the somatic occurrence of these genetic alterations
Quantitative Multigene Reverse Transcriptase Polymerase Chain Reaction Assay
Quantitative gene expression assays to assess recurrence risk and benefits from chemotherapy in patients with stages II and III colon cancer have been evaluated and are commercially available. The test provides information on the likelihood of disease recurrence in colon cancer (prognosis) and the likelihood of tumor response to standard chemotherapy regimens (prediction). The Oncotype Dx colon cancer assay evaluates a 12-gene panel consisting of 7 cancer genes and 5 reference genes to
Epigenetic inactivation of endothelin 2 and endothelin 3 in colon cancer
Therapeutic strategies target overexpressed members of the endothelin axis via small molecule inhibitors and receptor antagonists, but this work supports a complementary approach based on the reexpression of endothelin 2 and endothelin 3 as natural antagonists of endothelin 1 in colon cancer.89
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This article has been updated from a version previously published in Clinics in Laboratory Medicine, Volume 33, Issue 4, December 2013.