Limitations to the use of ACF and their sub-types as a biomarker to identify cancer preventive agents is compromised as the disease progresses from early to late stages. Increased biological segregation from normal crypts increases the specificity to the diseased state but the ability of early lesions, such as the ACF to predict the differences between two or more experimental groups are limited by the fact that their number also declines. With time, ACF undergo the selection process; while some regress, remodel or even get eliminated; others progress forward to the next stage. For example as shown in Figure , consider a pool of AOM-injected rat colonic ACF with different sub-types at varied duration (8, 12 and 24 wk post-initiation). In 8 wk, ACF with 1, 2 or 3 crypts per foci appear, and at this time point the incidence of total ACF (irrespective of their crypt multiplicity) becomes a valid biomarker. In subsequent weeks, ACF with higher crypt multiplicities or those with advanced biological features appear, while those with lower crypt multiplicities regress. So, at this time point, incidences of higher crypt multiplicities or those displaying advanced features could be considered more specific. In more advanced stages of the carcinogenic process, the value or sensitivity of ACF as a biomarker to predict tumor outcome diminishes; however, their specificity as an indictor of the stage remains uncompromised. The use of ACF and its sub-types as biomarkers thus depend much on the stage of colon carcinogenesis. Only at early stages, total number of ACF and ACF displaying different biological features may be considered as a valid biomarker. At very early stages the relevance of utilizing a parameter involving advanced lesions such as aberrant crypts per ACF (AC/ACF) becomes invalid, as at early stages to begin with there may not be a significant pool of ACF with varied features. The description as ‘very early’, ‘early’ and ‘advanced’ stages of colon carcinogenesis have been used cautiously and have considered the AOM-induced rats as the model system (described in the former Section).
Figure 1 A pictorial representation of the dynamics of ACF during azoxymethane-induced rat colon carcinogenesis. A: Colon with normal crypts prior to AOM injection; B: Colon bearing early ACF at approximately 8-wk post AOM injections; C: Colon bearing early and (more ...)
ACF with severe dysplasia have been identified as the actual precursors of colonic adenomas and adenocarcinomas[22
]. The recent findings of the presence of β-catenin accumulated aberrant crypts (BCAC)[23
], mucin-depleted crypts (MDF)[24
], and flat-dysplastic ACF (FDACF)[25
] in carcinogen-treated rat colons are significant advances in the identification of events leading to tumor development. The morphological features of these lesions resemble those of advanced ACF, as described earlier[10,13,14
]; strongly suggesting that they plausibly are subtypes of advanced ACF. However, it is still unclear as to the exact evolutionary sequence of BCAC, MDF or FDACF, and their relevance to the human situation. These specific ACF-subtypes are purported to accurately predict tumor outcome[20-22
]. However using them as valid biomarkers to screen chemicals/agents for potential colon cancer chemoprevention requires further scrutiny. Indeed, the simplistic methylene blue staining method in assessing ACF in whole mounts of colon remains a valuable tool in screening compounds for their colon cancer chemopreventive potential.