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ROLE OF GENETIC ABNORMALITIES IN COLO-RECTAL CANCERS
Lt. Col. V. P. Singh
INHS ASVIN1, Colaba, Mumbai-400 005


The molecular genetics of colorectal neoplasia are among the best understood of common human cancers. Several characteristics of the disease itself have contributed lo the understanding colorectal neoplasia. First, colorectal neoplasia is common, so many tumours are available for study. Second, the pathogenesis of colorectal carcinoma is unique in that the malignant tumour in most instances, arises in a preexisting benign tumour, the adenoma, constituting an adenoma adenocarcinoma sequence. Furthermore, patients who have or are at risk of developing colorectal neoplasia have epithelial proliferative and differentiation abnormalities in their grossly normal colorectal mucosa that are thought to be precursors to tumour formation. As a conse-quence, the entire spectrum of subtle early abnormalities to fully malignant and metastatic cancers is available for study through endoscopic biopsy of the large bowel and polypectomy specimens, as well as through surgical resection specimens.
Various abnormal genes have been found in colorectal neoplasms, including oncogenes, which have abnormal stimulatory actions, and tumour suppressor genes, which undergo inactivation resulting in loss of normal inhibitory mechanisms. These molecular genetic abnormalities have been identified by analysis of adenocarcinomas, adenomas of various sizes, and mucosa from the large bowel in patients with various clinical settings.

DNA METHYLATION:
Abnormal DNA methylation throughout the genome appears to play an important role in colorectal neoplasia, Generalised DNA hypomethylation is evident even in small adenomas and specific areas of hypermethylatlon of genes also occurs. Increased expression of DNA methyltranferase, the enzyme responsible for cytosine methylation, is identifiable In the non-neoplastic mucosa of patients with colorectal tumours, as well as In the adenomas and adeno-carcinomas themselves. DNA hypermethylatlon can precede allelic losses and may cause or at least indicate chromosomal areas that are genetically unstable". Consequently. DNA methylation abnormalities may play a key role in the earliest events. This finding is of special interest because of the recognised epithelial proliferative and differentiation abnormalities in the non-neoplastic mucosa of patients who have or are at risk of developing colorectal neoplasia.

ADENOMATOUS POLY-POSISCOLI (APC) GENE:
The mutated APC gene responsible for adenomatous polyposis syndrome is located on the long arm of chromosome. The APC gene appears to play a critical role in early events in the colorectal mucosa of patients with adenomatous polyposis, as evidenced by epithelial proliferative and differentiation abnormalities in the large bowel before the onset of adenoma development in at risk members of pedigrees who have inherited the abnormal gene. The mutated APC gene thus appears to predispose to adenoma development in the large bowel, but also to tumours of other organs in patients with adenomatous polyposis. In addition to being responsible for adenomatous polyposis, alteration of the APC gene appears to play a role in sporadic colo-rectal carcinoma because the locus of the gene on chromosome 5q is often deleted in sporadic carci-nomas and adenomas.

MUTATED IN COLO-RECTAL CARCINOMA (MOC)GENE:
The search for the adenomatous polyposis gene led to the identification of another nearby related gene on chromosome 5q, the MCC gene. The potential inter-relationship of MCC and APC is thus under scrutiny. MCC is included in some 5q allelic deletions that occur in colorectal adenomas and adenocarcinomas, and the gene is mutated in some carcinomas.

RAS GENES:
The ras gene family encodes 21,000-dalton membrane-bound proteins involved in signal transduction. Mutations of K-ras and N-ras are frequent in colorectal neoplasms. Codon 12 of K-ras is most frequently mutated. The prevalence of ras gene mutations is relatively similar in larger-adenomas and carcinomas.

DELETED IN COLO-RECTAL CARCINOMA (DCC) GENE:
Deletion of the DCC gene is frequent in adenocarcinomas of the large bowel and less so in the adenomas in which they arise. Thus DCC deletion appears to be a relatively late event in the adenoma-adenocarcinoma sequence.

P53 GENE:
The p53 gene on the short arm of chromosome 17 encodes a 53,000-dalton nuclear phosphoprotein. The p53 gene is most strinkingly associated with the development of carcinomas in adenomas. Frequent examples of deletion and mutation in a carcinoma, but not in the adenoma in which the cancer arose have been identified, p53 is frequently abnormal in other types of cancer as well". Of note. the p53 mutations in colorectal cancer and adenomas are predominantly transitions (Purinetopurine.pyrimidine to pyrimidine) and occur in many codons, thus arguing against the direct role of a single environmental agent.

PREFERENTIAL SEQU-ENCE OF GENETIC ALTERATIONS:
The preferred sequence of alterations discussed previously is based on the prevalences of the alterations in lesions in various stages of the adenoma-adenocarcinoma sequence. Of note, all of the abnormalities rarely occur in an individual carcinoma. For example, a ras gene mutation occurs in only about 50% of colorectal carcinomas. In addition, the order of alteration is by no means inviolate. Thus accumulation of alterations in both oncogenes and tumour-suppressor genes, rather than a set order of the alterations, appears to be important in the adenoma-adenocarcinoma sequence.

OTHER GENES:
The complexity of the molecular genetic alterations in colorectal neoplasia is evident from the preceding discussion, but even more genes are involved. Abnormalities of the c-myc and c-src oncogenes have been found. The nm23 gene on chromosome 17 is frequently deleted in colorectal carcinomas, Furthermore, allelotyping and molecular genetic analysis suggest that additional suppressor genes altered in colorectal neoplasms may also reside on 1p, 8pand22q.

CLINICAL APPUCATIONS:
The clinical application of the molecular genetics of colorectal neoplasia is in its infancy and additional studies are clearly needed to assess utility. Three areas of potential application are evident currently: risk assessment, screening and diagnosis, and prognostication.

RISK ASSESSMENT:
Risk assessment can now be carried out in offspring of patients with adenomalous polyposis syndrome. Molecular genetic evaluation for identification of patients who have inherited a mutated APC gene from an affected parent allows tailoring of clinical follow-up. This approach can concentrate endoscopic surveillance and patient evaluation on offspring who have inherited the mutated APC gene. Although this application of molecular genetics to risk assessment is important, adenomatous polyposis accounts for less than 1% of all colorectal carcinomas. Thus this advance will not lead to a dramatic reduction in overall mortality from this common human cancer.
Familial aggregation of colorectal carcinoma in the absence of a recognizable syndrome is well known. First degree relatives of patients with colorectal cancer have a two-fold to four-fold higher risk than the general population. Studies in the Mormon population in Utah have suggested that inheritance of susceptibility to colorectal adenoma is an autosomal dominant trait with high frequency in the population. Identification of the gene(s) involved in susceptibility to ordinary colorectal neoplasia would have extraordinary implications for reducing mortality from colorectal carcinoma by improving risk assessment using molecular genetic methods.

SCREENING:
Screening for colorectal carcinoma currently employs fecal occult blood testing by various methodo-logies. This approach is far from ideal because of poor sensitivity, specificity, and predictive values, Application of molecular genetics to identify mutated DNA or gene product shed from colorectal neoplasms may be feasible for screening and diagnosis.

PROGNOSTICATION:
The first widespread clinical application of molecular genetics may be in prognostication for patients with colorectal carcinoma. Metastasis is a complicated, multi-step process. It is unrealistic to believe that such a complicated process could be under the control of a single gene. More likely, multiple genes are needed in the various steps. Nonetheless, some of the identified molecular abnormalities in primary colorectal carcinomas are associated with the presence of distant metastasis at the time of resection and with pool prognosis in patients without Initial evidence of disseminated disease. Deletion of the p53 locus on chromosome 17p, deletion of the DCC locus on chromosome 18q. and high fractional allelic loss are associated with distant metastasis and poor survival. One or more of these alterations may eventually be clinically useful markers for poor prognosis.
The most theoretical clinical application of molecular genetics is therapy. Correction of the molecular genetic abnormalities in tumour cells has great appeal as cancer therapy, but the technical problems to be solved are very substantial. For example, the ability to deliver the normal gene to all tumour cells poses a formidable challenge. Pharmacologic agents that block the effects of oncogenes or mimic the effects of tumour suppressor genes may be more likely to succeed.

SUMMARY
Reported molecular genetic abnormalities involve tumour-suppressor genes that undergo inactivation (for example, APC, MCC, DCC, p53 and possibly genes on chromosomes 8p, 1 p, and 22q) and dominant-acting oncogenes (e.g. ras, src, myc). Multiple clonal genetic abnormalities accumulate during the development of colorectal carcinoma in adenomas. Altered DNA methylation is an early event, and the specific gene alterations occur in a preferential order. Clinical application of molecular genetics in patients who are at risk for or have developed colorectal carcinoma is in its infancy. Patients with predisposition to colorectal carcinoma as a result of inheritance of familial adenomatous polyposis can now be identified by genetic analysis of ten APC gene on chromosome 5q21. In patients who undergo curative resection of colorectaf cancer, deletion of the p53 gene on chromosome 17p. deletion of the DCC gene on 18q. and high fractional allelic loss (fraction of non-acrocentricautosomal arms with deletion) in the primary tumour may indicate increased likelihood of occult disseminated disease and thus poor prognosis. Additional studies are needed to establish the role of molecular genetics in the management of colorectal carcinoma.

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