K- ras mutations and the RAS pathway

The mutations best-characterized in non-polypoid lesions are those in K- ras . Several studies, including the following, have suggested that K- ras mutations are rarer in nonpolypoid lesions than polypoid ones. For example, Yamagata and colleagues found that K- ras mutations could be found in only 23% of flat adenomas, versus 67% of polypoid adenomas. Kaneko and colleagues similarly studied a series of 42 carcinomas. They found that p53 overexpression did not differ between the two morphologies but that the non-polypoid cancers lacked K- ras mutations, whereas they were present in 44% of the polypoid cancers. Olschwang and colleagues analyzed a series of 44 flat colorectal neoplasms for microsatellite instability and mutations in APC , K- ras , and TGF-RII and found that only K- ras mutations had a lower frequency than in polypoid neoplasms. Umetani and colleagues found K- ras mutations in none of their superficial depressed adenomas but 31% of polypoid adenomas.

Laterally spreading tumors (LSTs) also have been analyzed, although the results are less clear. For K- ras mutations, Hiraoka and colleagues and Mukawa and colleagues found a lower prevalence in flat nongranular lesions (16% and 26% respectively) versus granular lesions that are more protruded (78% and 77% respectively). In contrast, Takahashi and colleagues found that although K- ras mutation was present in 35% of flat-type LSTs, it was only present in 13% of protruded-type adenomas.

In addition to concluding that K- ras mutations correlate with polypoid growth, Yashiro and colleagues also found an association of LOH at chromosome 3p with cancers of the de novo type, a region known to contain multiple tumor suppressors or related genes, including MLH1 , β-catenin , TGFBR2 , and RASSF1A . RASSF1A is a member of a relatively recently discovered family of proteins with tumor suppressor functions, for which epigenetic inactivation by promoter hypermethylation has been described in a wide variety of cancers. Although almost universal in some cancers such as breast or small cell lung cancer, RASSF1A methylation is less frequent in CRC. Van Engeland and colleagues found that 45 of 222 (20%) sporadic CRCs had RASSF1A methylation, and of six normal epithelial samples from cancer patients, only one had RASSF1A methylation. Oliveira and colleagues found a higher frequency (22 of 51 cases, or 43%), studying polypoid tumors with microsatellite instability. Sakamoto and colleagues investigated the frequency in 48 flat tumors comprising 39 early carcinomas and nine high-grade dysplasias; 39 of 48 (81.3%) had RASSF1A methylation, but only 7 of 48 (14.6%) had K- ras mutations. In the 39 cases of tumors with RASSF1A methylation, 19 (49%) also showed RASSF1A methylation in morphologically normal mucosa. It is unclear whether this represents an abnormal background giving rise to tumors, versus a field effect. More data are needed to clarify the roles of these family members, as a separate study by Noda and colleagues showed a low (16.4%) incidence of RASSF1A methylation in all tumors, with no difference between flat and polypoid lesions. Analysis of RASSF2 methylation has been found in 43% of tumors in another series, again with no difference between flat and protruded neoplasms. Still, alternate pathways to perturbing RAS signaling may be present in flat colonic neoplasms and their background mucosa.

Role of APC and LOH at chromosome 17p

There are differing data regarding APC, as Umetani and colleagues found that in addition to a lower K- ras frequency, APC mutation was also less frequent in flat versus polypoid adenomas (13% vs 43%, encompassing both depressed and elevated flat adenomas vs polypoid ones) although the frequency was similar in carcinomas. On the other hand, Kaneko and colleagues found that although the rate of APC mutation in polypoid versus non-polypoid carcinomas was similar, the types of mutations differed. Non-polypoid carcinomas completely lacked frameshift mutations that were found in 66% of polypoid carcinomas, thus leading the authors to propose that different types of APC mutations could influence tumor morphology and development.

In a similar vein, several groups have analyzed loss of heterozygosity at multiple loci, most notably chromosome 17p. Several studies have concluded that LOH at chromosome 17p or p53 overexpression occurs with similar frequency in both flat and polypoid neoplasms. LOH at 17p, however, has been found to be the most frequent (92%) of multiple LOH found in a series of flat tumors, and Mueller and colleagues found LOH at 17p in 73% of de novo cancers versus 37% of ex-adenoma cancers.

Role of APC and LOH at chromosome 17p

There are differing data regarding APC, as Umetani and colleagues found that in addition to a lower K- ras frequency, APC mutation was also less frequent in flat versus polypoid adenomas (13% vs 43%, encompassing both depressed and elevated flat adenomas vs polypoid ones) although the frequency was similar in carcinomas. On the other hand, Kaneko and colleagues found that although the rate of APC mutation in polypoid versus non-polypoid carcinomas was similar, the types of mutations differed. Non-polypoid carcinomas completely lacked frameshift mutations that were found in 66% of polypoid carcinomas, thus leading the authors to propose that different types of APC mutations could influence tumor morphology and development.

In a similar vein, several groups have analyzed loss of heterozygosity at multiple loci, most notably chromosome 17p. Several studies have concluded that LOH at chromosome 17p or p53 overexpression occurs with similar frequency in both flat and polypoid neoplasms. LOH at 17p, however, has been found to be the most frequent (92%) of multiple LOH found in a series of flat tumors, and Mueller and colleagues found LOH at 17p in 73% of de novo cancers versus 37% of ex-adenoma cancers.

Other molecular correlates of tumor behavior

With respect to other markers, Wlodarczyk and colleagues also found more de novo cancers with decreased E-cadherin expression and extensive stromelysin-3 expression. It has recently been proposed that CD10, β-catenin, and mucin expression may correlate with flat tumor morphology and prognosis also. CD10 is a marker that in CRC correlates with a higher incidence of venous invasion or liver metastasis, although a causal relationship has not been established. In contrast, the absence of MUC5AC may be important. MUC5AC is not normally expressed in the colon, but frequently is expressed de novo in adenomas and colorectal cancers. It has been suggested that MUC5AC negativity correlates with higher metastatic potential and poorer prognosis, and it may be differentially expressed in MSI-H (77%) versus MSS cancers (28%). Finally, the β-catenin signaling pathway has key roles in development and cancer, where the protein is often stabilized due to APC or β-catenin mutation and aberrant signaling ensues. Koga and colleagues performed immunohistochemistry to assess CD10, nuclear β-catenin, MUC2, and MUC5AC in 111 flat colorectal neoplasms and 96 polypoid ones. CD10 was found in 50% of flat low-grade neoplasia (LGN) but 0% of polypoid LGN, as well as 59% of flat high-grade neoplasia versus 33% of polypoid neoplasia. Invasive lesions had, respectively, 51% and 39% CD10 positivity. Nuclear β-catenin was found in 86% of non-polypoid LGN versus 58% of polypoid LGN, but similar percentages of HGN and invasive neoplasia. For non-polypoid versus polypoid lesions, MUC5AC was present in 25% versus 50% of LGNs; 0% versus 28% of HGN; and 3% versus 32% of invasive neoplasia. Markers of more aggressive phenotype therefore may be present at an earlier stage in flat neoplasms compared with polypoid ones.