Colonic adenocarcinoma is the third commonest malignant neoplasia in societies with Western type life style as diet rich in red meat and fat, lacking in vegetables, fruit and fiber is implicated in colonic carcinogenesis. The colorectal adenocarcinoma has distinct genetics, heterogeneity at histological and molecular level, growth characteristics and precursor lesions which identify high risk groups amenable to surveillance programs.

The adenocarcinoma, familial (adenomatous polyposis or hereditary non-polyposis colorectal (HNPCC cancer), sporadic or associated with chronic inflammatory bowel disease, especially ulcerative colitis follows a multi-step genetic pathway with cumulative genetic events that lead to oncogene upregulation and tumor suppressor gene inactivation. The alterations at DNA level cause a gradual change of the cellular phenotype with the neoplastic cells acquiring invasive and metastatic properties and full expression of lethal carcinoma^1,2.

An acceptable model of colonic carcinogenesis is that of Fearon and Volgestein which explains several aspects of the adenoma-carcinoma sequence and it is based on the following genetic events^3-5. Hypomethylation of DNA results from carcinogenic factors and allows chromosomal irregularities to occur⁶. The normal epithelial cell may enter a proliferative state prone to mutational events in the APC and K-ras genes and the concomitant alterations of both genes are important for adenoma formation. The evolution of early (small) adenoma to intermediate and late adenoma requires additional mutations of the DCC gene (deleted colon cancer) located on chromosome 18q22. As the product of the DCC gene is a cell adhesion molecule, probably functioning as a growth signal receiver allowing progression of the disease, an important phenomenon related to full expression of malignant phenotype with invasive and metastatic capacity are the mutational changes of the p53 tumor supressor gene. In the genetic pathway to malignancy important abnormalities occur in at least four DNA mismatch repair (MMR) genes with inadequate repair of mutations and DNA replication errors at microsatellite areas which induce chromosomal abnormalities (insertions/deletions)².

It is important to study the histological features and growth patterns of colonic adenocarcinomas in combination with biological markers, which indicate the genetic events that determine initiation and progression of colorectal carcinogenesis. The histopathology of colorectal adenocarcinoma includes the macroscopic features, histological types, grade, stromal response, angiogenesis, inflammation, Iymphatic and blood vessel invasion, the stage of the disease according to the depth of invasion and margin clearance. The emerging information on the significance of various biological markers that they can increase the ability to predict biological behaviour and determine more effective treatment, follow up or surveillance of high risk individuals awaits evaluation.

Precursor lesions: The adenoma dysplasia - carcinoma sequence

Adenomas^7-10 are benign neoplasms of the colonic epithelium single or multiple with a sharp increase at the end of the 4th decade. There is a tendency to recur (21-41%) with frequent recurrence the first 2 years after polypectomy. The left colon is the commonest site but in older individuals there is increased frequency in the right colon and a tendency to clustering in the rectum. Colonic adenomas are either pedunculated (80%) or sessile but flat/depressed adenomas are currently described and they have distinct histological features despite their smaller size compared to the other types. Microscopically they are tubular, villous, tubulovillous or mixed hyperplastic-adenomatous polyps. The tubular adenomas are composed of branching, closely packed tubular glands and the villous component shows papillae supported by vascular stalk. The lining cells are mucous producing (goblet cells), absorptive and endocrine cells. Paneth cells can be seen and areas of squamous cell metaplasia are rare. It is important to recognise flat or depressed adenomas which are a subtype of tubular adenoma since it is a relatively new entity first described by Lento in 1985¹¹. Diagnosis of flat adenomas is made on a flat or slightly depressed lesion which has been completely removed and truly neoplastic confined to the mucosa. There is male preponderance and they are more frequently located in the ascending and transverse colon (48.8%).

It is debatable if flat carcinomas arise in flat adenomas but several investigations support this concept^13,14. The dispersed, sparse pericryptal myofribroblasts characterise flaudepressed adenomas in contrast to the retained normal pattern in polypoid adenomas¹². Most depressed adenomas remain small (< 5 mm), they probably develop into small flat carcinomas but with a tendency for rapid invasive growth. Despite their size, dysplasia is not an uncommon finding. Compared with polypoid type lesions they do not express ras p2l protein and p53 positivity occurs in the dysplastic foci with a pattern similar to polypoid adenomas.

Polypoid adenomas with low grade dysplasia and size < 1.5 cm rarely undergo invasive malignant transformation but more than 30% of villous adenomas > 5cms contain carcinoma. The malignant potential of an adenoma is related to the degree of dysplasia. Small adenomas even < 1cm if they show foci of severe dysplasia warrant total colonoscopy as the rate of malignancy raises to 27%. There is also strong evidence for recurrence of colonic adenomas if there is increased proliferation index along the crypt with upward extension toward the middle and upper portions of the normal colonic mucosa. It may be important to assess the proliferative state of normal mucose in every patient with adenoma as patients with higher S-phase index may form a high risk group for recurrent adenomas which requires surveillance^15,16.

In conclusion adenomas greater than 1.5 cm are the major premalignant lesions of the colon. If malignant change has occurred in the form of severe dysplasia, intraepithelial or intramucosal carcinoma radical surgery is not required as lymphatics are not present in the upper and middle lamina propria but they form along the base of the crypts above the muscularis mucosae. If there is submucosal invasion, poorly differentiated tumor, lymphatic or vascular invasion colectomy should be considered¹⁶. It is important that the pathologist's report should state the presence of invasion, the metastatic potential of the Iesion and the clearance of the surgical margin. Pseudoinvasion should always be distinguished from invasive carcinoma.

Dysplasia as classified by Riddel is an important risk factor for development of adenocarcinoma as it has been observed in chronic inflammatory bowel disease mainly ulcerative colitis or Crohn's, in juvenile polyposis, at ureterosigmoidoscopy sites and in patients with schistosomiasis. Dysplasia also shows marked reduction in the pericryptal fibroblasts as it is seen in flat adenomas and the disturbed epithelial-pericryptal fibroblast relationship is more obvious in severe dysplasia¹⁸.

Transitional mucosa

Elongation of the glands is observed adjacent to adenocarcinomas and large adenomas. The cellular phenotype is altered compared to the normal epithelium as the cells have an immature phenotype with sialomucins, reduction in secretory component, increase of CEA and MI antigen. Due to loss of o-acetyl groups and reduced fucosylation blood group antigens usually absent from normal goblet cell appear in transitional and neoplastic mucosa.

Transitional mucosa has not been widely accepted as a precancerous lesion but its presence suggests nearby malignancy, and it has been suggested that increase reccurrence rate and worse prognosis is expected in cases of adenocarcinoma with transitional type adjacent mucosa⁸.

Pathological features of colorectal adenocarcinoma

Early colorectal cancer^8,19-21 is not a distinct clinicopathological entity and presents as polypoid carcinoma, focal carcinoma in a large sessile adenoma or as a small ulcerating tumor. It may be intramucosal or submucosal but it does not invade the muscularis propria. If poorly differentiated tumors are excluded only 4% of early cancers metastasise.

The polypoid carcinoma resembles macroscopically adenoma but has all the histological features of malignancy, such as disturbed architecture, nuclear enlargement, atypia, mitotic activity, atypical mitosis, reduced cytoplasmic/nuclear ratio, reduction of cytoplasmic mucin, nuclear overlapping, pseudostratification. There is no absolute need for radical surgery if excision is complete and the tumor is well or moderately differentiated without lymphatic or vascular invasion but controversy remains and more radical therapeutic approach is considered by some centers specialised in the management of colorectal cancer. Focal carcinomas arising in adenomas must be clearly described as to the involvement of the head, neck, stalk or submucosa.

Macroscopic features of adenocarcinoma

Small carcinomas (1-2 cm) are usually granular, red, slightly raised lesions and sharply circumscribed. They have a softer consistency if adenomatous areas persist but they become paler and harder if the whole lesion is carcinoma. Larger carcinomas are raised ulcerated mushroom like lesions with flat center and rolled edges, exophytic papillary, nodular or annular constricting adenocarcinomas causing stenosis and bowel obstruction. Exophytic tumors are commoner in the R-colon, and ulcerating or constricting in the transverse and descending colon. Rarely, a diffuse infiltration of the colon resembling Linitis plastica occurs or flat infiltrative carcinoma plaque like with extensive intramural invasion. Infrequently tumor necrosis and perforation causes peritonitis.

Histological features

The usual type of colorectal adenocarcinoma shows classical features. If the malignant glands are well formed, irregular, branching, tubular the tumour is well differentiated. If at least 25% of the tumor is arranged in solid sheets, moderately differentiated carcinoma is diagnosed and in poorly differentiated carcinomas less than 25% of the tumor shows glandular arrangement. Paneth cells, neuroendocrine cells may be present. The histological grade is an important prognostic factor particularly in carcinomas with recognizable adenomatous component. Mucous production varies from minimal to abundant and then a diagnosis of mucinous carcinoma is made. A significant feature which must be described is the infiltrative irregular peripheral margin of the tumor or the expanding well circumscribed tumors which have better prognosis. The stroma may be abundant, myofibroblastic, with varying degrees of neoangiogenesis, chronic inflammation or elastosis.

Special histologic types of colorectal carcinomas

a. Mucinous carcinoma is characterised by mucous production in > 50% of the tumor. The colloid type produces ample extracellular mucin and the signet ring type is characterised by intracytoplasmic accumulation of mucin. Mucinous carcinomas (10-15%) may have worst prognosis, they are common in young patients. In patients with ulcerative colitis²², anorectal fistulae, malignant transformation of villous adenomas¹⁸. The non signet-ring type has similar prognosis to the non-mucinous adenocarcinoma, with absent inflammatory response but the signet ring type (1%) carries the worst prognosis (18% five year survival). The signet ring carcinoma infiltrates the wall causing rigid, thick segments mimicking Crohn's disease^9,10,23.

b. Adenosquamous carcinoma

Some adenocarcinomas are admixed with malignant squamous epithelium and it seems that the squamous epithelium predominates. As these neoplasms are rare, of uncertain histogenesis and without precise criteria for diagnosis they remain an interesting histological entity. Sarcomatous stroma has also been reported in rare cases⁸.

c. Carcinoma with choriocarcinomatous areas

Trophoblastic differentiation of tumor cells has also been reported but this is an extremely rare phenomenon. The histological features with immunohistochemical identification of p-HCG expression confirm the diagnosis²⁴.

d. Microglandular carcinoma

Microglandular pattern of colorectal adenocarcinoma has also been reported and this type needs differential diagnosis from carcinoid tumors⁹.

e. Undifferentiated carcinoma

Undifferentiated carcinoma²⁵ develops either as solid, hypercellular tumor arranged in broad trabeculae of large cells with vesicular nucleus with prominent nucleoli, sparse mitosis, expansive margin and good prognosis or as small cell carcinoma similar to the oat cell carcinoma of the lung. The solid type may show abortive glandular structures, occasional mucin production and scattered neuroendocrine cells and must be distinguished from malignant carcinoid and other neuroendocrine neopIasms.

Staging of colorectal carcinoma

Since 1932 when Dukes introduced tumour stage with reference to the depth of invasion through the bowel wall and pericolic tissues regardless of different classification systems for staging colorectal cancer, stage remains the most important prognostic factor in colorectal adenocarcinoma. At the moment the most widely used system is the modified Dukes's, the Astler-Coller modification or the TNM system. The Jass's proposed pathological system has not received wide accteptance yet, as its reproducibility and objectivity have been challanged. According to Duke's classification the predicable 5 year survival in resectable colorectal adenocarcinoma is 81%, 64 and 27% for stages A, B, and C respectively²⁷.

Prognostic factors in colorectal adenocarcinoma

As the stage of the disease remains the most important independent prognostic factor, there are additional factors that may determine biological behaviour^28,29.

The histoloqical grade is an important indicator of tumour growth and Grinnel's classification of well, moderately and poorly differentiated adenocarcinoma is widely used. As only 28% of carcinomas show the same grade throughout the tumor, for assessment of the histological grade the extent of the least differentiated areas are interpreted. Interobserver variation still remains a challenge in tumor diagnosis and prognosis.

Other histological features valuable for predicting biological behaviour is the expanding or infiltrative tumor margin which in Jass's classification plays a predominant role and the degree of lymphocytic infiltrate as peritumoral T-cells, with CD8 and CD4 phenotypes seem to be activated with cytotoxic capacity despite ineffective control of tumor expansion^30,31.

Marked chronic inflammatory response is accepted as an indicator of better prognosis, as well as the simultaneous expression of HLA class I and ll antigens on tumor cells or cell adhesion molecules^32-34. An emerging significant prognostic parameter is the angiogenesis of the tumoral stroma, particulary of neo-vessel formation along the peripheral tumor margin. There is strong evidence that angiogenesis is a critical step in colorectal carcinogenesis and its progression³⁵.

The assessment of tumor vessel formation and the angiogenic factors controlling endothelial growth may prove a useful marker of tumor behaviour^36,37. At present the pattern of tumour angiogenesis should be mentioned in the histological report until acceptable definite values implicating prognosis will influence treatment in less advanced stages of the disease.

Lymph node metastasis and the number of positive nodes are very important prognostic factors in colorectal carcinoma³⁸. The pathologists shoud identify and examine at the least 13-15 lymph nodes including the apical lymph node of the mesenteric segment as 66% 5 years survival is expected if only 1-3 lymph nodes show metastasis, reduced to 37% with > 4 positive nodes and to 2,1% if more than10 positive nodes are present. Some relevance has been reported in the histological pattern of lymph node response as sinus histocytosis indicates better prognosis.

Extramural vein invasion is also an important factor indicative of metastatic potential and assessement of vascular invasion of thick wall extramural veins is required in every report.

Margin clearance

A part of the obvious importance of the involvement of proximal and distal surgical margins, the circumferential (lateral) surgical margin shoud always be scrutinised for adenocarcinoma TF involvement of the serosal surface or < 1 mm clear margin are identified, 78% of resected carcinomas will recur in 5 years³⁹.

Biological markers of colorectal adenocarcinoma

Regardless of the used staging classification system and the assessment of all previously mentioned prognostic factors, the biological behaviour of an individual tumors cannot be accurately predicted. Advances in the know edge of the cellular phenotype during colorectal carcinogenesis have allowed the emergence of biological markers which gradually unravel features determining aggressive behaviour with enhanced metastatic potential, as the genetic patways of colorectal adenocarcinoma have already suggested. At chromosomal level DNA aneuploidy has been studied but conflicted data do not allow the use of DNA aneuploidy as an independent prognostic factor although there is strong evidence that diploid tumors at Dukes's stages B2 and C have better prognosis^40,41. Modern molecular biology techniques applied on tissue sections indicate that Ioss of heterozygocity of chromosomes 18 and 17 may prove a valuable factor in predicting aggressive biological behaviour^42,43.

The overexpression of P53 oncoprotein is a rather late phenomenon in colonic neoplasia, related to the grade of dysplasia. It may indicate worse prognosis particularly if it is due to mutations in the highly conserved region of the P53 gene and the codon 175^44-48. Deletion of the NM 23 gene may also contribute to the metastatic potential of the tumor⁵⁰, as well as CD4V6 proteins which is reported as valuable prognostic factor in Dukes B and C adenocarcinomas⁵¹. Combined mutations of K-ras and P53 with cytoplasmic overexpression of c-erbB2 protein may indicate full malignant potential^47,51.

As invasion of the basement membrane and cell-cell and cell-stroma matrix interactions determine tumor growth and differentiation, basement membrane components, cell adhesion molecules and factors determing stroma remodeling are also important in predicting behaviour.

The absence or marked reduction of Laminin and collagen lV⁵² from the epithelial basement membrane and overexpression of laminin receptors⁵³ characterise tumors with metatastic ability and poor survival and increased collagenase lV⁵⁴ has been associated with invasiveness. Several reports indicate that overexpession of urokinase plasminogen activator is an important factor^55,56 of aggressive biological behaviour but the significance of selective or complete HLA-class I antigen loss or HLA-DR expession have not been evaluated due to conflicting data in various studies.

The change in the mucin structure with appearance of neutral mucin and reduction of a-acylated constituents, altered Iectin binding patterns, expression of ABO antigens, C9-19 and CEA⁸ are well recognized features of neoplastic colorectal cell phenotype.

In conclusion colorectal adenocarcinoma shows marked heterogeneity and distinct genetic pathways with hereditary and sporadic cases. Stage, histological type, number of positive nodes, tumor margin, lymphocytic infiltrate, stomal angiogenesis and extramural thick vein ivasion are the most important predictive factors of biological behaviour.

lnvolvement of the circumferential surgical margins, if the proximal and distal margins are clear is of major importance for local recurrence. Emerging biological markers of tumor behaviour at chromosomal oncogene and tumor supressor gene levels regulating cell growth and communication, differentiation and full expression of malignant phenotype may in the near future prove more precise indicators of biological behaviour determing early diagnosis, surveillance programs cind more effective treatment.

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