Identification of dysplasia in human colonic aberrant crypt foci.

American Journal of Pathology, Vol. 150, No. 5, May 1997 Copyright © American Society for Investigative Pathology

Identification of Dysplasia in Human Colonic Aberrant Crypt Foci

I-Mei Siu,* Thomas G. Pretlow,* Saeid B. Amini,t and Theresa P. Pretlow* From the Institute of Pathology* and Department of Epidemiology and Biostatistics,t Case Western Reserve University Medical Center, Cleveland, Ohio

Aberrant crypt foci (ACF) are grossly invisible putative premalignant lesions in the colon. As dysplasia is considered an important precursor of colon carcinoma, we wanted to determine the presence and severity of dysplasia in human ACF. Fifty ACF from 28 patients were embedded in paraffin, cut serialy, and stained with hematoxylin and eosin. Multiple slides from eacb ACF were evaluatedfor dysplasia according to a defined set of criteria. Of 50ACF, 3 (6%) contained focal areas with severe dysplasia, ie, carcinoma in situ, 4 (8%) containedfocal areas with moderate dysplasia, and 20 (40%) contained focal areas with mild dysplasia. Twenty-three ACF (46%) contained no detectable dysplasia. In 15 of 27ACF with dysplasia, less than 50% (eg, 4 of 28, 10 of54, and 10 of3O sections) of the sections cut and evaluatedfrom each ACF demonstrated dysplasia. The presence of dysplasia in a large proportion ofACF supports the hypothesis that they may be precarcinomatous. (Am J Pathol 1997,

150:1805-1813) Aberrant crypt foci (ACF) are putative precursors of colon cancer. First identified in unembedded murine colon after treatment with colon carcinogen,1 ACF were postulated to be preneoplastic lesions. These microscopically elevated lesions are grossly invisible and defined as being composed of crypts that 1) have altered luminal openings, 2) exhibit thickened epithelia, and 3) are larger than adjacent normal crypts.2 ACF are specifically induced by colon carcinogens3 in a dose-dependent fashion4; at higher doses of carcinogen, more ACF are formed. Rat ACF and colon neoplasms share some similar genetic alterations such as mutations in K-ras,5 changes in

enzymatic activity,6'7 and increases in proliferation.8 ACF, identified in human patients with colon cancer,9-11 exhibit mutations in K-ras,12-15 enzymatic changes,10 increases in proliferative activity,16 and genomic instability1718 similar to those observed previously in human colon tumors. Mutations in the adenomatous polyposis coli (APC) gene, associated with inherited familial adenomatous polyposis coli and postulated to be the earliest genetic alteration in colon carcinogenesis,19 have been found in some human ACF. 13,14 A group of colonic pathologists20 have expressed the view that "precarcinomatous change (dysplasia) in the epithelium of the large bowel [is] a histologic marker for increased cancer risk." Dysplasia in ACF has been investigated,1113152122 but some studies have used only unstated criteria for dysplasia, whereas others have examined ACF only in cross sections. Analysis of cross sections of ACF is often less useful than analysis of longitudinal sections that permit the evaluation of the entire lengths of the crypts, as some ACF in longitudinal sections exhibit dysplasia only at the lower portions of the crypts.23 In testing the hypothesis that ACF are important precarcinomatous lesions, it is desirable to determine the frequency of dysplasia in ACF. We examined the histological features of 50 human, serially sectioned ACF to determine the presence and degree of dysplasia in these putative preneoplastic lesions.

Materials and Methods Dysplasia Assessment Scale Based upon the attempts of others2024 to quantify dysplasia, the following scale was created. NondysSupported by National Institutes of Health grants CA48032, CA66725, DK51347, CA43703, and CA54031. Accepted for publication January 7, 1997. Address reprint requests to Dr. Theresa P. Pretlow, Institute of Pathology B-30, Case Western Reserve University Medical Center, 2085 Adelbert Road, Cleveland, OH 44106.

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plastic, or atypical, ACF may exhibit enlarged nuclei and some loss of mucin. Mildly dysplastic ACF exhibit enlarged nuclei, increased loss of mucin as compared with ACF that are not dysplastic, and some nuclear stratification. Moderately dysplastic ACF exhibit pleomorphic and more stratified nuclei with a more advanced degree of mucin loss as compared with mildly dysplastic ACF. Severely dysplastic ACF, or carcinoma in situ, exhibit highly pleomorphic nuclei that are greatly stratified, a higher degree of loss of mucin, and occasionally increased mitotic activity. For this study, two slides, 20 sections apart (approximately 100 ,um), had to meet the criteria for dysplasia for the ACF to be classified as dysplastic.

Specimens Human resected colons were placed in saline at 40C in the operating room of University Hospitals of Cleveland and transported to the pathologist by the Western Division of the Cooperative Human Tissues Network of the National Cancer Institute located at Case Western Reserve University. Grossly normal segments, 2 x 30 cm, of colonic mucosa were stripped from the submucosa by the pathologist, snap-frozen over liquid nitrogen, and stored at -1950C. Specimens, 5 cm or greater from the tumor and averaging 25 cm2 per patient, were thawed in ice-cold 1% paraformaldehyde in 0.1 mol/L sodium phosphate buffer (pH 7.4), fixed flat in formalin for 1 hour at room temperature, and stained for 3 minutes in 0.2% methylene blue.23 ACF (Figure 1) were identified in the fixed specimens with a standard binocular light microscope (American Optical) with a magnification of x40 as described previously.10 They were marked with permanent yellow ink (The Davidson Marking System, Bradley Products, Bloomington, MN) with a microprobe while viewed directly with the microscope,6'10 dissected out, and embedded in paraffin. The ACF were composed of 55 + 56 (mean + SD) crypts (range, 7 to 225 crypts) and occupied a mean area of 1.8 + 1.7 mm2 (range, 0.2 to 8.3 mm2). To avoid weighting this study with too many ACF from single patients, a maximum of three ACF per patient was taken. The 50 ACF evaluated for the presence of dysplasia were obtained from 28 patients with a mean age of 70 ± 15 years (range, 34 to 98 years). Of the 28 patients, 26 had sporadic colorectal cancer: 11 in the right colon, 2 in the transverse colon, and 13 in the left colon. Two patients did not have cancer; one had focal chronic ulcers with local reactive inflammation, and one had diverticulosis.

Figure 1. ACF in the methylene-blue-stained unembedded human colotn. A: An ACF uwith mild dysplasia in histological sections. The luminal openings of the crypts (arrows) are noticeably different from those of surrounding normal crypts. Magnification, x27. B: An ACF wvith moderate dysplasia in histological sections. The crvpts of the ACF (black arrows) are enlarged anzd elevated conmpared uvith the adjacent normal cijpts (white arrow). Magnification, X27 C: An ACF with severe dvsplasia in histological sectionts is well circumscribed and elevated (arrows). Magnification, X 27.

Dysplasia In Human Aberrant Crypt Foci

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Histological Preparation Serial 5-,um sections from each ACF were cut and mounted, two sections per slide. The number of sections per ACF ranged from 80 sections (from the smallest ACF) to 640 sections (from the largest ACF). At intervals of 20 sections, 2 sections were stained with hematoxylin and eosin (H&E). These slides were then assessed histopathologically twice by three observers, the second or subsequent times without knowing the results of the first or previous examinations. Criteria used for grading dysplasia are given above. If the two results did not agree precisely, the case was analyzed a third time. Of the 15 ACF that required a third evaluation, all were classified the first time as either mildly dysplastic or without dysplasia (atypical). The final classification for these 15 ACF was 7 with atypia and 8 with mild dysplasia. Similar difficulty in distinguishing between atypia and mild dysplasia was reported by Riddell et al.20 Representative slides were immunostained for the presence of carcinoembryonic antigen (CEA) as performed previously23 with a mouse anti-CEA monoclonal antibody (MAb; Zymed Immunologicals, San Francisco, CA). Overexpression of CEA has been demonstrated in a high percentage of ACF23 and was used to identify the ACF in histological sections. The lack of CEA expression eliminated 7 of 57 ACF from inclusion in this study. To determine whether severity of dysplasia correlated with increased proliferation and/or a shift in the proliferative zone, representative lesions with all grades of dysplasia were immunostained as reported previously25 for the presence of Ki-67, an antigen associated with proliferative activity.26 Briefly, slides from these cases were deparaffinized for 7 minutes in xylene twice and then rehydrated through absolute ethanol, 95% ethanol, and distilled water. Slides were then microwaved for 3 minutes in 50 mmol/L sodium phosphate/citric acid buffer, pH 3.Q,27 six times. After cooling for 1 to 2 minutes in distilled water, the slides were incubated 20 minutes at 37°C with the blocking solution (10% horse serum in 10 mmol/L phosphate-buffered saline (PBS), pH 7.4). All dilutions were performed with blocking solution unless mentioned otherwise. The slides were incubated for 2 hours at 370C with either MIB-1 (Biogenex, San Ramon, CA), a mouse MAb that recognizes Ki-67, diluted 1:10, or with mouse anti-human prostate-specific antigen MAb (Dako Antibodies, Carpinteria, CA) as an inappropriate antibody of the same Ig class as MIB-1, diluted 1:100 to match the concentration of MIB-1. The slides were washed for 5 minutes in PBS three times and incubated with bioti-

nylated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA), diluted 1:400, for 30 minutes at 22°C. They were then washed in PBS, incubated in 30% methanol with 0.3% H20228 for 10 minutes to block endogenous peroxidase, and rinsed briefly in distilled water. The slides were incubated in streptavidin-biotinylated horseradish peroxidase complex (Amersham, Arlington Heights, IL), diluted 1:100, for 30 minutes at 22°C and washed three times in PBS. The immune complexes were visualized with the diaminobenzidine substrate after heavy metal enhancement as used previously in our laboratory.23

Statistical Analysis Statistical analyses were performed with Statview Version 4.01 software (Abacus Concepts, Berkeley, CA) and with StatXact Version 2.2 (Cytel Software Corp., Cambridge, MA). The data are presented as proportions or as means ± SD. As most of the data do not follow a normal distribution, the Mann-Whitney U test statistics were used to compare the medians of two continuous variables. Nonparametric Spearman rank correlation was used to correlate ACF characteristics with other continuous variables. The X2 test and Fisher's exact test were used to compare proportions among groups. Armitage trend analysis29 was used to correlate location of the ACF in the colon with severity of dysplasia.

Results ACF were found at a higher frequency in the left colon than in the right colon (Table 1; P < 0.0001), which is in agreement with previous studies on the distribution of ACF in the human colon.Y1015,22 ACF tended to increase in size with advancing age of the patients (r = 0.28, P = 0.049, Spearman correlation). ACF in the right colon (2.8 ± 2.0 mm2) were larger (P = 0.0074) than ACF in the left colon (1.5 ± 1.4 mm2), and ACF with dysplasia (2.2 ± 1.6 mm2) were larger (P = 0.0036) than ACF without dysplasia (1.4 ± 1.7 mm2). The four smallest ACF ( 0.0001, Mann-Whitney)

had only atypia or mild dysplasia. In any category based on the number of crypts per ACF, a wide range of pathologies was observed. For example, four ACF had 16 crypts per ACF; one of those ACF had severe dysplasia, one ACF had moderate dysplasia, and two ACF had only atypia. The surface topology observed in the methylene blue, whole-mount specimens before embedding (Figure 1) did not permit us to distinguish between ACF with and without dysplasia as determined in histological sections. The ACF in Figure 1 A (mildly dysplastic) is composed of crypts that have a vast array of unusual shapes and sizes; in contrast, the ACF shown in Figure 1B (moderately dysplastic) has crypts that are much more uniform in shape and size. The ACF in Figure 1C is severely dysplastic, ie, shows carcinoma in situ. It is plainly demarcated and elevated above the normal mucosa; and most of the crypts, enlarged with slitshaped lumina, are relatively uniform in size and appearance. For 42 (84%) of the 50 ACF studied, the average crypt surface area (the ratio of the size of the ACF in square millimeters to number of crypts in the ACF) was 0.02 to 0.04 mm2. Of the remaining eight ACF, one had smaller crypts and seven had larger crypts. It is interesting to note that three of the four ACF with the largest surface areas per crypt (0.08 mm2 or greater) were severely dysplastic and the fourth was moderately dysplastic. Representative examples of ACF that are mildly (Figure 2, A to C), moderately (Figure 2, D and E), or severely (Figure 3) dysplastic are illustrated. Artifacts were frequently present in histological sections of moderately and severely dysplastic ACF; examples of such artifacts can be seen in Figure 3 (asterisks). Some degree of dysplasia was seen in 27 (54%) of 50 ACF; 20 exhibited mild dysplasia, 4 exhibited moderate dysplasia, and 3 (from two different patients) exhibited severe dysplasia, ie, carcinoma in situ (Figure 3). Serial sections of ACF did not reveal uniform pathology throughout individual lesions; ie, in 15 (56%) of 27 ACF with dysplasia, less

than 50% (eg, 4 of 28, 10 of 54, and 10 of 30 sections) of the sections analyzed demonstrated dysplasia. Four ACF showed dysplasia in less than 20% of the sections analyzed; at the opposite extreme, three ACF showed dysplasia in 90% or more of the sections. Dysplasia needed to be observed in at least two slides, 20 sections apart, for an ACF to be designated dysplastic. If all serial sections of ACF had been examined, it is likely that the presence of dysplasia would have been found to be even greater than that reported in this study. Of the 15 ACF from the right colon, 5 (33.3%) exhibited severe or moderate dysplasia, 6 (40%) showed mild dysplasia, and 4 (26.7%) showed atypia (Figure 4). Of the 31 ACF from the left colon (composed of the descending colon, the sigmoid colon, and the rectum), none exhibited severe dysplasia, only 2 (6.4%) showed moderate dysplasia, 13 (42%) exhibited mild dysplasia, and 16 (51.6%) exhibited atypia (Figure 4). There was a trend toward a greater proportion of the ACF in the right colon to exhibit a higher grade of dysplasia than ACF in the left colon (P = 0.004, Armitage trend test). Of the 15 ACF from Black patients, 73% were dysplastic compared with 44% of 34 ACF from White patients (P = 0. 06, x2 test). This increased dysplasia in ACF from Blacks was not a reflection of increased samples from the right colon as 14 of the 15 ACF from Black patients were from the left colon. The two ACF from the two patients without colon cancer were indistinguishable from ACF from cancer patients; ie, one was moderately dysplastic and one had only atypia. ACF that were graded as not dysplastic or mildly dysplastic did not exhibit an extension of the proliferation zone when stained for the presence of Ki-67 with MIB-1 MAb. One mildly dysplastic ACF demonstrated a marked increase of proliferation and extension of the proliferative zone to the lumen of the colon; this unusual staining pattern may be related to the presence of a large number of inflammatory cells throughout this specimen. A marked extension of the proliferation zone


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Figure 2. Histological sections of dysplastic ACF(H&f). The inik uised to markACFin unembedded colon is indicated bvarrowheads. A to C: Ani ACF uith mild dysplasia shown in Figure IA. The nuclei of the gland indicated by arrows are enlarged and sho'wsome stratification, and there is some loss of mlcin. D and E: An ACF uith mode-rate dyvsplasia. The nutclei (arrows) are niot on/y enlarged btut a/so pleomnorphic, anid some have migrated toward the lumen of'the crypt or become stratyied. Thsere is an increased los:s of'mucin as well. Magnification, x 34 (A), x 65 (B), X 165 (C and D), an d X260 (E).

toward the lumen of the colon was seen in three of four ACF with moderate dysplasia and all three ACF with severe dysplasia, ie, carcinoma in situ (Figure 5).

Discussion ACF are postulated to be premalignant lesions. Dysplasia is defined by a consensus of authorities as a "precarcinomatous change" and as "an unequivocal

neoplastic alteration of the colonic epithelium."20 Occult carcinomas have been found in dysplasia-associated lesions or masses, which demonstrates the strength of dysplasia as a marker for malignancy.30 Three ACF were found to contain carcinoma in situ, or noninvasive cancer. Most dysplastic ACF are only mildly dysplastic, whereas the ACF with more advanced dysplasia are far fewer in number. The range of severity of dysplasia found in ACF suggests that a

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Figure 3. Histological sections of ACF with severe dysplasia or carcinoma in situ (H&E). Glatnds with severe dysplasia are marked by arrows. The nuclei are highly pleomorphic and stratified, and there is a marked loss of mucin. Typical artifactsfound in severely dysplastic ACF are marked with asterisks. The ink used to mark ACF in unlembedded coloni is indicated by arrowheads. A and B: An AC,Fshoun in Figuire IC C and D: Another ACF twith severe dysplasia. Magnificatiotn, X 65 (A and C), X 165 (B), and x 260 (D).

certain percentage of ACF may progress to cancer. In the rat model, ACF have been found to contain invasive carcinoma. 7 The increased frequency of ACF in the left colon (Table 1) agrees with previous reports. 10,15'22 ACF in the ascending or right colon exhibit a trend toward more dysplasia than did ACF found in the left colon (Figure 4); this suggests that ACF in the ascending colon may progress differently than ACF in the left colon. It has been hypothesized that the etiology of colon cancer may be different for different locations in the bowel.31 Additional studies with a much larger number of ACF are needed to confirm the regionally based differences in the biology of ACF. There are conflicting views concerning the presence of dysplasia and/or hyperplasia in ACF. The

finding of only hyperplasia in ACF15 would suggest that ACF are not premalignant, but the finding of dysplasia in ACF11 13,22,23 supports the theory that ACF are precursors of colon cancer. Early studies found dysplasia in 26 of 35 (74%)11 and in 12 of 46 (26%)22 human ACF, all of which had been cut in cross section. In the first study,1 1 more severely dysplastic ACF were from familial adenomatous polyposis coli patients, whereas less severely dysplastic ACF were from patients with sporadic colorectal cancer. In the second study,22 all ACF were from patients with sporadic colorectal cancer, but only the superficial 20 ,tm of the ACF was assessed for presence of dysplasia; the severity was not specified. A strong correlation was found between the presence of dysplasia in ACF and a slit-shaped lumen.22 One


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Figure 5. Proliferative activity in ACF. A section is shown from an ACF u'ith severe dysplasia immunostained with MIB-1 MAbfor the expression of Ki-67. Magnification, X 65.

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Loc.ation in Colon Figure 4. Dysplasia in ACF bhy locationi. ACF were groutped hy both location ( right, transverse (t), left includes descending ( d) plus sigmoid (s) plus rectum ( r)) in the colon and severity ofdysplasia to determine uhether the presence and degree of dysplasia were related to location. ACFfrom the right coloni tend to be more dysplastic than ACFfrom the left colon (P = 0.004, Arm itage trend test).

group15 found hyperplasia but no dysplasia in ACF from patients with sporadic colorectal cancer. Another study13 of patients with sporadic colorectal cancer found 19 (95%) of 20 human ACF with hyperplasia and only 1 (5%) ACF with changes characteristic of dysplasia. In that study,13 only two 12-p.m sections from each lesion in an unspecified orientation were analyzed for dysplasia; however, we have found that sections from different parts of the same lesion often exhibit different degrees of dysplasia. It has been shown that when some ACF with serrated openings are viewed longitudinally, dysplasia

can be seen in the lower portions of some crypts that have a serrated, hyperplastic appearance near the surfaces of the crypts.23 The presence of dysplasia probably would be missed in crypts viewed in cross section near the luminal surfaces of the crypts. These serrated adenomatous crypts in ACF differ from the serrated crypts in hyperplastic polyps (eg, those described by Lewin et al32). The changes observed in dysplastic ACF most commonly involve at least two-thirds of the thickness of the colonic mucosa. Commonly, they involve the middle third of the mucosa with at least as much alteration as seen at the bases of the crypts. None of the studies reviewed above mention the sizes of individual crypts in the ACF analyzed, which we found to be unusually large in the ACF with severe dysplasia. However, the severely dysplastic ACF were not the ACF with the largest number of crypts, nor were they the ACF with the greatest surface areas (Figure 1). This suggests that progression may not be dependent upon the size of the ACF as a whole; progression may be related to the sizes of individual crypts. The artifacts marked by asterisks in Figure 3 are most likely due to

this abnormal enlargement, which somehow renders the tissue more vulnerable to the knife of the microtome.

Otori et al33 described some ACF from patients with sporadic colorectal cancer that were hyperplastic and had an extended proliferative zone. They33 "suggest that some hyperplastic ACF may develop into adenomatous ACF by way of stage abnormality ACF" that are "characterized by their extension of the

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proliferative compartment to the surface of crypts but with no changes in the major site of proliferation." In our study, immunostaining for the presence of Ki-67, a proliferation-associated antigen, has shown that ACF with moderate or severe dysplasia have an extension of the major site of proliferation in the aberrant crypts in addition to an increase in proliferation (Figure 5). As in our previous study,23 we also found that some ACF exhibited both dysplasia and hyperplasia (data not shown), suggesting that the two are not mutually exclusive. The coexistence of dysplasia with serrated crypts in the same lesions has been described34 and emphasizes the need for careful pathological evaluation of lesions that have a hyperplastic appearance. In this study, serial sections of ACF, with crypts cut longitudinally, were analyzed for the presence and degree of dysplasia. We found that dysplasia in an ACF is usually a focal change that does not affect the entire lesion. With the use of longitudinal sections and rigorous criteria for the severity of dysplasia, this study demonstrates that significant proportions of ACF are dysplastic; a subpopulation of these ACF may progress to cancer.

Acknowledgments We thank Sunny McClellan for her assistance in preparing this manuscript.

References 1. Bird RP: Observation and quantification of aberrant crypts in the murine colon treated with a colon carcinogen. Cancer Lett 1987, 37:147-151 2. Bird RP, McLellan EA, Bruce WR: Aberrant crypts, putative precancerous lesions, in the study of the role of diet in the aetiology of colon cancer. Cancer Surv 1989, 8:189-200 3. McLellan EA, Bird RP: Specificity study to evaluate induction of aberrant crypts in murine colons. Cancer Res 1988, 48:6183-6186 4. McLellan EA, Medline A, Bird RP: Dose response and proliferative characteristics of aberrant crypt foci: putative preneoplastic lesions in rat colon. Carcinogenesis 1991, 12:2093-2098 5. Stopera SA, Murphy LC, Bird RP: Evidence for a ras gene mutation in azoxymethane-induced colonic aberrant crypts in Sprague-Dawley rats: earliest recognizable precursor lesions of experimental colon cancer.

Carcinogenesis 1992, 13:2081-2085 6. Pretlow TP, O'Riordan MA, Kolman MF, Jurcisek JA: Colonic aberrant crypts in azoxymethane-treated F344 rats have decreased hexosaminidase activity. Am J Pathol 1990, 136:13-16

7. Pretlow TP, O'Riordan MA, Pretlow TG, Stellato TA: Aberrant crypts in human colonic mucosa: putative preneoplastic lesions. J Cell Biochem Suppl 1992, 16G:55-62 8. Pretlow TP, Cheyer C, O'Riordan MA: Aberrant crypt foci and colon tumors in F344 rats have similar increases in proliferative activity. Int J Cancer 1994, 56: 599-602 9. O'Riordan MA, Barow BJ, Jurcisek JA, Stellato TA, Pretlow TP: Aberrant crypts in the colons of humans and carcinogen-treated rats are enzyme-altered. Proc Am Assoc Cancer Res 1990, 31:85 10. Pretlow TP, Barrow BJ, Ashton WS, O'Riordan MA, Pretlow TG, Jurcisek JA, Stellato TA: Aberrant crypts: putative preneoplastic foci in human colonic mucosa. Cancer Res 1991, 51:1564-1567 11. Roncucci L, Stamp D, Medline A, Cullen JB, Bruce WR: Identification and quantification of aberrant crypt foci and microadenomas in the human colon. Hum Pathol 1991, 22:287-294 12. Pretlow TP, Brasitus TA, Fulton NC, Cheyer C, Kaplan EL: K-ras mutations in putative preneoplastic lesions in human colon. J Natl Cancer Inst 1993, 85:2004-2007 13. Jen J, Powell SM, Papadopoulos N, Smith KJ, Hamilton SR, Vogelstein B, Kinzler KW: Molecular determinants of dysplasia in colorectal lesions. Cancer Res 1994, 54:5523-5526 14. Smith AJ, Stern HS, Penner M, Hay K, Mitri A, Bapat BV, Gallinger S: Somatic APC and K-ras codon 12 mutations in aberrant crypt foci from human colons. Cancer Res 1994, 54:5527-5530 15. Yamashita N, Minamoto T, Ochia A, Onda M, Esumi H: Frequent and characteristic K-ras activation and absence of p53 protein accumulation in aberrant crypt foci of colon. Gastroenterology 1995, 108:434-440 16. Roncucci L, Pedroni M, Fante R, Di Gregorio C, Ponz de Leon M: Cell kinetic evaluation of human colonic aberrant crypts. Cancer Res 1993, 53:3726-3729 17. Augenlicht LH, Richards C, Corner G, Pretlow TP: Evidence for genomic instability in human colonic aberrant crypt foci. Oncogene 1996, 12:1767-1772 18. Heinen CD, Shivapurkar N, Tang Z, Groden J, Alabaster 0: Microsatellite instability in aberrant crypt foci from human colons. Cancer Res 1996, 56:5339-5341 19. Powell SM, Zilz N, Beazer-Barclay Y, Bryan TM, Hamilton SR, Thibodeau SN, Vogelstein B, Kinzler KW: APC mutations occur early during colorectal tumorigenesis. Nature 1992, 359:235-237 20. Riddell RH, Goldman H, Ransohoff DF, Appelman HD, Fenoglio CM, Haggitt RC, Ahren C, Correa P, Hamilton SR, Morson BC, Sommers SC, Yardley JH: Dysplasia in inflammatory bowel disease: standardized classification with provisional clinical applications. Hum Pathol 1983, 14:931-966 21. McLellan EA, Medline A, Bird RP: Sequential analyses of the growth and morphological characteristics of aberrant crypt foci: putative preneoplastic lesions. Cancer Res 1991, 51 :5270-5274


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22. Roncucci L, Medline A, Bruce WR: Classification of aberrant crypt foci and microadenomas in human colon. Cancer Epidemiol Biomarkers Prev 1991, 1:57-60 23. Pretlow TP, Roukhadze E, O'Riordan MA, Chan JC, Amini SB, Stellato TA: Carcinoembryonic antigen in human colonic aberrant crypt foci. Gastroenterology 1994, 107:1719-1725 24. Konishi F, Morson B: Pathology of colorectal adenomas: a colonoscopic survey. J Clin Pathol 1982, 35:830-841 25. Konstantakos AK, Siu l-M, Pretlow TG, Stellato TA, Pretlow TP: Human aberrant crypt foci with carcinoma in situ from a patient with sporadic colon cancer. Gastroenterology 1996, 111:772-777 26. Gerdes J, Ulrich S, Lemke H, Stein H: Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation. Int J Cancer 1983, 31:13-20 27. Shi S-R, Imam SA, Young G, Cote RJ, Taylor CR: Antigen retrieval immunohistochemistry under the influence of pH using monoclonal antibodies. J Histochem Cytochem 1995, 43:193-201 28. Beckstead JH: Optimal antigen localization in human

29.

30.

31.

32.

33.

34.

tissues using aldehyde-fixed plastic-embedded sections. J Histochem Cytochem 1985, 33:954-958 Armitage P: Test for linear trend in proportions and frequencies. Biometrics 1955, 11:375-386 Blackstone MO, Riddell RH, Rogers BHG, Levin B: Dysplasia-associated lesion or mass (DALM) detected by colonoscopy in long-standing ulcerative colitis: an indication for colectomy. Gastroenterology 1981, 80: 366-374 Bufill JA: Colorectal cancer: evidence for distinct categories based on proximal or distal tumor location. Ann Intern Med 1990, 113:779-788 Lewin KJ, Riddell RH, Weinstein WM: Non-neoplastic polyps. Gastrointestinal Pathology and Its Clinical Implications. New York, Igaku-Shoin, 1992, pp 12011208 Otori K, Sugiyama K, Hasebe T, Fukushima S, Esumi H: Emergence of adenomatous aberrant crypt foci (ACF) from hyperplastic ACF with concomitant increase in cell proliferation. Cancer Res 1996, 55:4743-4746 Longacre TA, Fenoglio-Preiser CM: Mixed hyperplastic adenomatous polyps/serrated adenomas: a distinct form of colorectal neoplasia. Am J Surg Pathol 1990, 14:524-537