Diagnosing colorectal carcinoma: clinical and molecular approaches
Authors: J. M. Jessup, H. R. Menck, A. Fremgen, D. P. Winchester
Journal: CA A Cancer Journal for Clinicians (1997)

Colorectal carcinoma is the fourth most prevalent carcinoma and second most frequent cause of death from cancer in the United States, with an estimated 131,200 new cases and 54,900 deaths in 1997.1 Although the death rate from large bowel cancer may be decreasing slightly, it still remains a health risk that consumes national resources and creates considerable personal suffering. Dietary modification may decrease the neoplastic transformation potential of bowel mucosa,2 but the widespread adoption of low fat, high fiber diets will not eliminate totally the risk of large bowel cancer. The consumption of aspirin3, 4 or other nonsteroidal anti-inflammatory drugs5 may also reduce the risk of developing the adenomatous polyps that precede large bowel cancers. However, not all patients benefit from these chemoprevention strategies and some may have deleterious side effects. Recently, fecal occult blood testing (FOBT) and large bowel endoscopy have been found to detect cancers at an earlier stage and decrease the formation of colorectal carcinomas. Also, the recent identification of specific genetic mutations that cause two different types of inherited colorectal carcinoma suggests that in the near future early diagnosis will be possible through blood or stool tests. As a result, considerable enthusiasm now exists that both the rate of earlier diagnosis and the outcome of colorectal carcinoma will be improved. This review first assesses the common patterns of presentation of colorectal carcinoma, then summarizes the current status of clinical diagnostic methodology, and finally outlines the future potential of molecular diagnostic tests. Critical to the successful application of molecular diagnostic tests is an appropriate understanding of the clinical background within which the tests will be used. Initially, this review focuses on the clinical presentation of colorectal carcinoma, then considers the genetic and biologic causes of colorectal carcinoma. Any molecular approach to diagnosis must explain the range of clinical and pathologic features involved in the manifestations of the disease at presentation. Currently, more patients are diagnosed because they develop signs or symptoms of colorectal cancer than are identified in an asymptomatic state. A major goal is to increase the proportion of patients who are diagnosed while they are asymptomatic because these patients present at an earlier stage that is more amenable to cure.6 The clinical and genetic conditions associated with the development of colorectal carcinoma (Table 1) represent the situations in which the clinician should consider the diagnosis of colorectal carcinoma. Although it is difficult to identify the fraction of patients who are symptomatic at diagnosis, analysis of a large number of patients who are diagnosed in hospitals across the country is informative because it demonstrates the distribution of stage at diagnosis. The National Cancer Data Base (NCDB), a national cancer management and outcomes data base that is a joint effort of the American Cancer Society and the American College of Surgeons,7 currently captures more than 50 percent of the estimated new cases of cancer in the United States and is an excellent starting point for an analysis of the patterns of care and outcome for patients with cancer. Data on the interaction of age, ethnicity, site of primary carcinoma, and stage at presentation are presented in Table 2. As reported earlier in a study of colon cancer,8 patients over the age of 70 years are more likely to present with stage I or II disease than are younger patients. This trend is also present in rectal cancer, in which 62 percent of patients over 80 years of age have stage I or II disease compared with 50 percent for those patients who are less than 50 years old and 53 percent for those who are between 50 and 60 years old (Table 2). Ethnicity is another significant factor because African Americans are less likely to present with stage I or II colon or rectal cancer than are non-Hispanic whites (Table 2). Hispanics are also slightly less likely to be diagnosed with stage I or II colorectal carcinoma than are non-Hispanic whites (Table 2). However, Asians have a pattern of stage at diagnosis similar to that of non-Hispanic whites (data not shown), and the sample of Native Americans is too small to draw any conclusions about their stage at presentation. The data in the NCDB do not reveal any significant effects of median income, region of the country, or type of hospital on the stage of disease at diagnosis (data not shown). As a result, these data from the NCDB suggest that age and ethnicity are important factors to consider in the diagnosis of colorectal carcinoma. Age has two contrasting effects on the diagnosis of colorectal carcinoma. Young (less than 40 years old) patients have a worse outcome than middle-aged patients, whereas older (more than 70 years old) patients present with earlier stage of disease. Bacon9 first reported that colorectal carcinoma is diagnosed before age 40 in 2 to 6 percent of all colorectal carcinoma patients. The delay in attributing symptoms to a possible colorectal cancer may contribute to the worse outcome.10, 11 Although several series indicate that there is a worse outlook for patients who develop colorectal carcinoma before the age of 40,12-17 two series18, 19 suggest that younger patients have the same survival as older patients, possibly because the distribution of patients with stage III and IV disease was similar to that of the older patients in their series. The outcome of younger patients may be worse because they present with more advanced disease or disease with a poorer histologic grade of differentiation. Several authors10, 12-16 have found that younger patients present with more nodal or visceral metastases than do older patients, as is the case with the current data from the NcDB (Table 2). However, the data of others13, 14, 17, 20 suggest that the outcome of younger patients is worse than that of older patients, even when matched by stage. The frequency of more advanced disease in younger patients may result from longer delays in diagnosis compared with older patients; however, the frequency of rectal bleeding or abdominal pain in these young patients is similar to that in older patients. Further, Adkins et al16 observed that eight of the 45 patients were found incidentally during evaluation for other problems, which suggests that the potential delay in detecting a cancer from the onset of symptoms was less than three months. Similarly, in other studies18, 20 there was no significant difference in symptom duration between young and old patients. In summary, there is a consensus that the disease presents at a more advanced state in younger patients. Therefore, patients who develop colorectal cancer before age 40 in the absence of a defined genetic syndrome, such as familial adenomatous polyposis or hereditary nonpolyposis colonic cancer, may still have an accelerated rate of mutations that may provide clues to the analysis of the genetic mechanisms that cause sporadic colorectal carcinoma. Older patients with colorectal cancer are defined as older than 75 or 80 years at the time of diagnosis. Comorbid disease and type of presentation may produce a worse outcome. The series of Payne et al,21 Wise et al,22 and Hobler23 suggest that sepsis and complications of surgery may occur more frequently in the elderly population. However, Arnaud et al24 showed that the postoperative five-year survival rate of older patients was similar to that of younger patients. In addition, the three-year23 and five-year21 cancer-specific survivals of patients over the age of 75 were the same as that of younger patients. Interestingly, several authors suggested that more right-sided cancers arise in the elderly than in the slightly younger population.18, 21 The elderly seem to have a slightly increased frequency of emergency operations, since 7.4 percent of older patients required emergency surgery, compared with four percent for patients younger than 75 years.21 Thus, the biologic behavior of cancers is not likely to be more aggressive in older patients than in patients between 40 and 70 years old. Ethnicity may be associated with an increased frequency of advanced cancer at diagnosis in African Americans. Thomas et al25 suggest that inner-city blacks are diagnosed with colorectal carcinoma at an earlier age than white patients, and that this trend is significantly greater in black males than in white males. Boring et al26 have reported that there has been a significant increase in cancer-specific death rates in both black men and women for colorectal cancer compared with white men and women over the last 30 years. In fact, the cancer-specific death rate declined in white men and women, whereas it increased in black men and women by 47 and 16 percent, respectively.26 Interestingly, when incidence rates are stratified by educational level and socioeconomic status, the incidence of cancer of the colon and rectum is higher for whites than for blacks for each stratification.26 As in the current NCDB data, Boring et al26 observed a trend in which the frequency of localized cancer at diagnosis is lower in blacks (30 percent) than in whites (36 percent). Thus, the poorer outcome may be attributable to more limited access to care. Weaver et al,27 reporting their experience over a 10-year period at Meharry Medical College, found that their black patients tended to present with more advanced disease than did those observed by Boring et al.26 Their experience may also reflect limited access to health care systems. A major challenge for improving early diagnosis is to increase the proportion of stage I and II colorectal cancers diagnosed in African Americans. The symptoms and signs of carcinoma of the colon and rectum are rectal bleeding (either gross blood in the stool or a guaiac-positive reaction on digital rectal examination), abdominal pain, change in bowel habits, nausea, vomiting, abdominal distention, weight loss, fatigue, and anemia. Rectal bleeding may be associated with an improved outcome, possibly because it prompts earlier diagnosis. Various authors28-31 have observed that rectal bleeding as a presenting symptom was associated with a better overall survival in univariate analyses, but when rectal bleeding was analyzed in multivariate analysis in which it was corrected for stage and site, it either had no effect29, 31 or became less important as an independent prognostic variable.28 Similarly, Wiggers et al32 found that only 19 percent of patients who presented with rectal bleeding died of disease compared with 33 percent of patients who did not present with rectal bleeding (P=0.017). Cappell and Goldberg33 observed that rectal bleeding was 2.8 times more prevalent at the time of diagnosis in early stage I lesions than in stage IV cancers. Also, Graffner and olsson34 reported that rectal bleeding was more frequently associated with rectal (50 percent) than colon (14 percent) cancer. Thus, rectal bleeding may be associated with early stage lesions (perhaps because early lesions are more vascularized than more advanced lesions) and, as a result, may carry a better prognosis. Thus, blood on the stool on digital rectal examination, on guaiac test, or by history must be further evaluated and not dismissed as caused by hemorrhoids. Abdominal pain is another symptom that may lead to the diagnosis of large bowel cancer. Two types of abdominal pain are caused by cancer of the colon or rectum. The first is cramping or colicky pain associated with complete or partial bowel obstruction. Although uncommon in rectal cancer, it may be a presenting symptom in colonic cancer. This symptom may be best considered as representing the clinical syndromes of obstruction or perforation that are discussed later. Wiggers et al32 identified abdominal pain and change in bowel habits as significant covariates in their multivariate analysis of patients with colorectal carcinomas. Either pelvic pain or tenesmus may be a symptom of a locally advanced rectal cancer and may be caused by involvement of peripheral nerves.35 Such a locally advanced rectal cancer may be successfully treated with a multimodality approach. However, pain is more likely to be associated with a stage III than a stage I lesion and, as a result, may have a worse prognosis. Other complaints, such as nausea, vomiting, anorexia, and weight loss of more than 5 kg, are uncommon at presentation but are more likely to be associated with advanced than early stage cancers.33 Finally, hemorrhoids at presentation may not be associated with survival but may be associated with early stage cancers of the colon.33 These data reinforce the need to evaluate the entire colon when a patient presents with rectal bleeding and hemorrhoids. Anemia secondary to microscopic bleeding (e.g., iron deficiency anemia), especially from a right-sided colonic cancer, may be a bad prognostic factor, particularly when it is associated with fatigue and weight loss because it is associated with a fivefold increased risk of metastatic disease.33 In summary, rectal bleeding may be associated with early stage disease and better survival. Complete or partial bowel obstruction often adversely affects outcome. Constitutional signs and symptoms-such as general malaise, weight loss, and profound anemia-are often associated with advanced disease and reflect the poor outcome of patients who present with visceral metastases. The definitions of both obstruction and perforation are either imprecise or not stated. Much of the literature either assumes that the reader knows what an obstruction is or does not indicate whether obstruction is complete (i.e., total absence of flatus or bowel movements for at least a day) or partial. Similarly, the type of perforation (e.g, free into the peritoneal cavity or contained, occurring through the cancer or proximal to a complete obstruction) is often not well described. The incidence of complete obstruction appears to range between two and 16 percent of newly diagnosed cases of colorectal cancer.20, 26, 36 The location of the primary cancer may affect the probability of developing a complete obstruction. Levien et al37 found that carcinomas of the splenic flexure were more likely to obstruct than were carcinomas at other sites. In contrast, obstruction from rectal cancers appears to be uncommon.38 Fielding et al39 suggest that the proportion of patients with obstruction follows the incidence of cancer at each site, whereas other reports suggest that the left colon36 or the ascending colon38 is the most frequent site of obstruction. The importance of bowel obstruction is supported by its function as an independent covariate in a multivariate analysis of outcome, even with stage included in the analysis. Crucitti et al40 studied 361 patients with colonic or rectal cancer and found that the presence of obstruction was a significant predictor of death from cancer even when stage was included in the analysis (obstructed patients had a 31 percent five-year survival compared with a 72 percent survival for patients who were not obstructed). Similar findings have been reported by others.29, 36, 41, 42 Wolmark et al42 suggested that the development of obstruction added to the poorer prognosis of right-sided colonic cancer, but there was no difference in outcome between right-sided and left-sided colonic cancers in the Gastrointestinal Tumor Study Group colonic adjuvant therapy trials, although in that study the presence of obstruction increased the risk of death from cancer 1.4-fold. Perforation occurs less commonly. Approximately three-quarters of patients operated upon emergently had obstructions, whereas one-quarter had perforations.43 Among the patients with perforations, only one-quarter of perforations were in the prestenotic bowel or the dilated cecum; most were through the tumor. Of the perforations, half were free perforations into the abdominal cavity, and the remainder were perforations into either an abscess cavity or a phlegmon. Interestingly, five percent of patients had perforations on more than one occasion. Perforation appears to be associated with local recurrence of disease, most likely because malignant cells are dispersed throughout the area of infection.28, 41 Perforation increases the risk of death from cancer 3.4-fold.28 These different patterns of presentation change the distribution of pain and must be taken into account by the clinician. Inflammatory bowel disease is a premalignant lesion that causes a predisposition to colorectal carcinoma when the disease enters a chronic, active phase.44 The propensity with which carcinoma develops may be associated with the degree of dysplasia caused by the inflammatory process.45-47 Although chronic ulcerative colitis has long been associated with an increased risk for the development of invasive carcinoma, recent studies have shown that chronic Crohn's disease is also associated with the subsequent development of cancer.48 As a result, among the symptomatic patients it is important to remember that inflammatory bowel disease may lead to bowel cancer. The cumulative incidence of colorectal cancer in inflammatory bowel disease is estimated to be five to 10 percent at 20 years and 12 to 20 percent at 30 years.48 This reinforces the need for surveillance in patients with inflammatory bowel disease to ensure early diagnosis. The pioneering work of Muto, Bussey, and Morson49 revealed that the majority of colorectal carcinomas develop from an adenomatous polyp. This polyp-to-carcinoma sequence is based on several descriptive but supportive findings: adenomatous polyps have a distribution of location similar to that of cancers that arise in the large bowel49, 50 but appear an average of five years earlier than does large bowel cancer,49, 51 many large bowel cancers display remnants of the polyp adjacent to the primary cancer, serial biopsy of polyps that have not been resected reveals a slow process of transformation to an invasive cancer,49, 52 endoscopic removal of adenomatous polyps decreases the risk of developing bowel cancer by 76 to 90 percent,6 and natural history studies of small polyps left in the colon reveal that some polyps less than 0.5 cm in diameter may regress whereas others either remain unchanged or slowly progress to a larger more dysplastic phenotype.53 Cannon-Albright et al54 have estimated that 19 percent of the general population forms adenomatous polyps and, therefore, is at risk to develop colorectal carcinoma. Three recent findings have markedly advanced our understanding of the relationship between the formation of adenomatous polyps and invasive carcinomas. First, the lesion that precedes the development of a polyp is an aberrant crypt focus (ACF) that becomes a microadenoma. The ACF begins as an outpouching of an epithelium-lined sac from the side of the crypt of Lieberkuhn that expands and eventually develops into a separate broad-mouthed crypt.55 Microadenomas are identified by methylene blue dye staining at colonoscopy or in the pathology specimen because their crypt openings to the lumen of the bowel are several times larger than the normal crypt opening.56 Roncucci56 has reported that microadenomas may be dysplastic, whereas others57, 58 have shown that loss of tumor suppressor genes and activation of proto-oncogenes may occur with frequencies similar to these events in polypoid adenomas. ACF and microadenomas are important because these lesions develop into adenomas and carcinomas in animal models of carcinogenesis.59 However, the evidence that ACFs develop into adenomatous polyps is circumstantial: patients with familial adenomatous polyposis (FAP) have more ACFs than do patients with sporadic colorectal carcinomas, who have more ACFs than do controls without cancer.60 Thus, ACF may be an important intermediate marker to aid in the diagnosis of patients with colorectal cancer. The second finding is that not all adenomas are exophytic; they may be flat and difficult to detect on colonoscopy. The Japanese have focused on the flat adenoma61-64 as a precursor lesion to carcinoma; many flat adenomas are ulcerated lesions that may be more aggressive than exophytic cancers.65 These lesions have also been appreciated in European patients.66 Further, flat adenomas may be precursors to the cancers that develop in hereditary nonpolyposis colorectal cancer (HNPCC) because they display the microsatellite instability that is observed in HNPCC and have a proximal large bowel distribution.64 Lynch and his associates have identified several kindreds of patients who display the hereditary flat adenoma syndrome (HFAS) in which colon cancers develop at a later age than in FAP patients, have a proximal colon distribution, and arise in association with flat adenomas.67 However, genetic linkage studies suggest that these kindreds are linked to deletions on chromosome 5q and may be variants of FAP rather than altered presentations of HNPCC, which has prompted the use of the term attenuated familial adenomatous polyposis (AFAP) for this syndrome.68 The third observation is that a fraction of bowel cancers appear to arise without an antecedent polyp. Several reports have suggested that adenocarcinomas of the bowel may arise without polypoid remnants or altered adjacent mucosa.69, 70 Bedenne et al71 demonstrated that ulcerated, infiltrating cancers in the right colon were least likely to be associated with an adenomatous polyp. These and other results suggested to Jass72 that 10 to 30 percent of colorectal cancers may not develop from an antecedent adenomatous polyp but arise de novo from mucosa that appears otherwise normal or has microadenomas. This is important because the current technology for diagnosis in asymptomatic individuals requires that a polyp be removed in order to prevent bowel cancer. As a result, under the present screening guidelines somewhere between 70 and 90 percent of large bowel cancers may be identified and removed before they become frankly invasive. However, under the best of circumstances 10 to 30 percent of large bowel cancers may not develop from a benign polyp and, as a result, may not be detectable or treatable until the lesion is frankly invasive. Current evidence indicates that colorectal carcinoma develops through as many as three related but slightly different molecular pathways. The first and best described molecular pathway involves mutation in a gene called the adenomatous polyposis coli (APC) gene. This mutation causes the appearance of hundreds to thousands of adenomatous polyps in the large bowel and is the cause of the autosomally dominantly inherited FAP and related syndromes.73 The molecular alterations that occur in this pathway largely involve deletions of alleles of tumor suppressor genes, that is, loss of heterozygosity (LOH) in APC, p53, and the deleted in colorectal cancer (DCC) gene74 combined with mutational activation of proto-oncogenes, especially c-Ki-ras (Fig. 1; see also reviews in the literature75, 76). In contrast, mutation of the human homologues of the bacterial mutHLS complex (hMSH2, hMLH1, hPMS1, and hPMS2) produces the DNA mismatch repair defects that appear to be the cause of HNPCC.77-82 The mutations in DNA mismatch repair enzymes lead to genetic instability that is reflected in errors in accurate replication of the repetitive di-, tri-, and tetranucleotide repeats that are scattered throughout the genome.83-86 As a result, the genomic instability identified in HNPCC is commonly referred to as either replication error positive (RER+) or, because microsatellite regions in the genome contain the nucleotide repeats, as microsatellite instability (MIN). Although these replication errors produce mutations in many of the same tumor suppressor genes and proto-oncogenes that are affected in the FAP pathway, there is a significantly lower frequency of LOH in the HNPCC pathway85 (Fig. 2). Genetic alterations in the familial adenomatous polyposis (FAP) type of neoplastic transformation of bowel epitheilum. Activation generally implies a mutational event, although it may merely be an increase in the expression of a normal gene product or the dephosphorylation of a peptide such as c-src APC = adenomatous polyposis coli gene; DCC = deleted in colorectal cancer gene; LOH = loss of heterozygosity. (Reproduced with permission from Jessup et al.76) Genetic alterations in the hereditary nonpolyposis colorectal carcinoma (HNPCC) type of neoplastic transformation of bowel epithelium. Unlike the events in Fig. 1, mutation without loss of heterozygosity occurs in the adenomatous polyposis coli (APC) gene, p53, and c-Ki-ras in this pathway. The passage through the various classes of adenomas may be shortened.(Reproduced with permission from Jessup et al.76) Finally, although many sporadic carcinomas appear to follow either the FAP (with mutation and LOH of APC, p53, and DCC) or the HNPCC (with RER+ phenotype and mutation of APC, p53, and DCC without LOH) pathway, colorectal carcinomas that arise de novo seem to have a slightly different pattern of molecular alterations that may suggest a third, yet to be defined molecular pathway. De novo carcinomas may have rates of mutation in the p53 gene87 similar to those of carcinomas that arise in association with adenomatous polyps but do not appear to contain c-Ki-ras mutations.88 Interestingly, the expression of the intestinal mucin gene product MUC2 seems to be lost in de novo carcinomas but is retained in both mucinous cancers and carcinomas arising from polyps.89 These studies with de novo colorectal carcinomas need to be better developed before the de novo carcinoma is accepted as another molecular pathway for neoplastic transformation (Table 3). Familial adenomatous polyposis syndrome is associated with loss of APC, a tumor suppressor gene on chromosome 5q21.73 Cytogenetic analyses implicated loss of material on the short arm of chromosome 5 (5q) as occurring frequently in patients with familial adenomatous polyposis90 and the APC gene has been unambiguously identified.91-93 The affected gene is the earliest identified primary genetic change in development of adenomas and is present in adenomatous polyps with a diameter of 5 mm.94 The gene is also mutated in sporadic tumors and in other familial syndromes such as Gardner's syndrome.91 The function of APC is not completely understood, but parts of the sequence are homologous with such structural proteins as myosin and keratin.93 some similarities have been noted between APC and the GTPase activating protein (GAP), which regulates the c-ras gene products, suggesting a potential role for this gene in signal transduction processes.94 APC also binds β-catenin, which implicates it in cell adhesion events because the catenins are essential for the function of the intercellular adhesion molecule cadherin.95, 96 In addition, APC binds to microtubules.97 Mutations of APC are generally either point mutations or insertions of mobile genetic elements that lead to chain termination and production of truncated proteins.98, 99 Further studies are required to understand the function of the protein and the significance of the different locations of mutations. For example, Gardner's syndrome is thought to be a variant of the familial adenomatous polyposis syndromes, with distinct pathologic manifestations in which extracolonic tumors develop in addition to adenomatous polyps. Yet identical mutations are found in the classic familial adenomatous polyposis syndromes and Gardner's syndrome, suggesting that the different phenotypes are the result of other genetic alterations. Nevertheless, the location of the mutation may influence the number of adenomatous polyps produced.68, 100-105 The cloning of the APC gene presents powerful diagnostic opportunities, particularly for individuals with a family history of colon cancer. Because this gene is altered in the germline or in all the cells of the body in patients with FAP, screening in the future will involve simply collecting peripheral blood cells to detect APC mutations. However, this blood test will not identify patients who are at risk for developing sporadic bowel adenomas. The recent description of identifying mutations in K-ras by amplifying DNA isolated from stool through the use of the polymerase chain reaction (PCR) technology106 may allow for similar approaches to the analysis of stool from patients at risk for the development of adenomatous polyps. Because 75 percent of adenomatous polyps have a mutation in APC,74 stool may contain sufficient mutant APC DNA to be isolated and identified. Additionally, as the function of the gene product of APC is better understood, important insights are likely to be gained into the molecular basis for the development of adenomas. The HNPCC syndrome represents an alternative pathway for the induction of colorectal carcinomas. As described by Lynch and colleagues,107, 108 this is a syndrome characterized by the development of colon cancer, usually in the proximal colon, in three first-degree relatives in two generations with at least one of the colon cancers occurring in a person younger than 50 years.109 Two variants of the syndrome are recognized: Lynch syndrome I, in which carcinomas are limited to the colon, and Lynch syndrome II, which has extracolonic carcinomas in such sites as the endometrium, breast, biliary tract, and pancreas, as well as in the proximal colon. Each syndrome may have a few adenomatous polyps present when the carcinoma is detected, but the number of polyps is usually less than five,110 and the polyps often appear as the cancer is diagnosed.108 The lack of preneoplastic lesions or associated stigmata (e.g., the osteomas and epidermoid cysts of FAP patients) combined with variable penetrance