APC and K-ras gene mutation in aberrant crypt foci of human colon

AIM To study the genetic alteration in ACF andto define the possibility that ACF may be a veryearly morphological lesion with molecularchanges,and to explore the relationshipbetween ACF and colorectal adenoma evencarcinoma.METHODS DNA from 35 CRC,15 adenomas,34ACF and 10 normal mucus was isolated by meansof microdissection.Direct gene sequencing of K-ras gene including codon 12,13 and 61 as well asthe mutation cluster region(MCR)of APC genewas performed.RESULTS K-ras gene mutation frequency inACF,adenoma and carcinoma was 17.6%(6/34),13.3%(2/15),and 14.3%(5/35)respectively,showing no difference(P>0.05)in K-ras gene mutation among three pathologicprocedures.The K-ras gene mutation inadenoma,carcinoma and 4 ACF restricted incodon 12(GGT→GAT),but the other 2 mutationsfrom ACF located in codon 13(GGC→GAC).K-ras gene mutation was found more frequently inolder patients and patients with polypoidcancer.No mutation in codon 61 was found in thethree tissue types.Mutation rate of APC gene inadenoma and carcinoma was 22.9%(8/35)and26.7%(4/15),which was higher than ACF(2.9%)(P<0.05).APC gene mutation incarcinoma was not correlated with age ofpatients,location,size and differentiation oftumor.CONCLUSION ACF might be a very early morphological lesion in the tumorogenesis ofcolorectal tumor.The morphological feature andgene mutation status was different in ACF andadenoma.ACF is possibly putative“microadenoma”that might be the precursor ofadenoma.In addition,the development of asubgroup of colorectal carcinomas mightundergo a way of“normal epithelium→ACF→carcinomas”.     

直肠异常隐窝病灶对结肠肿瘤的预警作用

目的 评判内镜下直肠异常隐窝病灶(ACF)与结肠病变及高癌变潜能肿瘤(AN)的关系.方法 接受全结肠镜检查的正常、息肉、腺瘤及癌的患者212例,在退镜时用0.4%靛胭脂对直肠进行染色,根据直肠ACF的数目对患者分级,无ACF者为0级,ACF数目为1～4,5～9,≥10者分别为Ⅰ、Ⅱ、Ⅲ级,统计分析ACF级别与结肠病变(息肉、腺瘤和癌),高癌变潜能肿瘤(≥1 cm,绒毛状、管状绒毛状,高度异型增生或浸润癌)的关系.结果 212例患者中,72例直肠ACF为0级,48例为Ⅰ级,41例为Ⅱ级,51例为Ⅲ级.直肠有ACF(包括Ⅰ、Ⅱ、Ⅲ级)的患者发生结肠病变及结肠高癌变潜能肿瘤的概率较无ACF者明显升高,其OR值(95%CI)分别为22.352(6.716～74.395),7.982(1.838～34.672).结论 直肠ACF对结肠病变及高癌变潜能肿瘤有预示作用.     

Identification and quantification of aberrant crypt foci in the colon of Min mice--a murine model of familial adenomatous polyposis

Min mice are heterozygous for a nonsense mutation in the murine adenomatous polyposis coli (APC) gene and spontaneously develop multiple intestinal neoplasms similar to the familial adenomatous polyposis (FAP) syndrome in humans. Aberrant crypt foci (ACF) are assumed to be preneoplastic lesions in both murine and human colon carcinogenesis and have been observed in FAP patients. Light microscopic examination of the colonic mucosa of 42 Min mice did not show even a single 'classical' ACF on the basis of previously defined criteria, specifying that they are elevated above the surrounding mucosa. However, in Min mice we discovered aberrant crypt foci of a different type, which we denoted ACF(Min). In contrast to the classical type, ACF(Min) were not elevated above the surrounding mucosa, their detection was totally dependent on methylene blue staining and transillumination, and they could not be identified with scanning electron microscopy. Histopathologic examination of ACF(Min) showed dysplastic crypts, similar to those found in larger lesions--that is, microadenomas in the Min mouse. The number of ACF(Min) increased up to the age of 6 weeks and then seemed to remain at a constant level of approximately four per colon. In conclusion, by transillumination of whole-mount preparations stained with methylene blue, we have identified and quantified small microscopic lesions that may be precursors of colonic adenomas in Min mice.     

Natural history of aberrant crypt foci

BACKGROUND: The aberrant crypt focus (ACF) appears to be an important early step in colorectal carcinogenesis. Our objectives were to determine the natural history of ACF in a surgical model. METHODS: The natural history of ACF was followed by marking the lesions in vivo with tattoos. Rats were given four weekly injections of azoxymethane (AOM; 20 mg/kg). One hundred days after the first injection of AOM, rats were anesthetized, and individual aberrant crypt focus was identified by staining with methylene blue. A 3× 3 mm area, identifying one large (4–8 crypts) ACF was marked with a tattoo dye in each colon. Control animals received saline or AOM injections and were tattooed in areas without ACF. At 200 days, colons were examined for the presence of macroscopic lesions. RESULTS: A total of 54 tumors were found in the study group of 38 animals, and 21 of these were in the transverse and proximal descending colon. The marked areas (all in transverse and proximal descending colon) yielded 6 tumors and 2 ACF, but in 30 instances no abnormality was noted. Probability of observing a tumor in the 3×3 mm area of the colon that was identified as containing ACFs was 17 times greater than expected from the observed tumor rate in approximately the same zone (16 vs. 1.7 percent; 95 confidence interval, 10 to 22 and 0.5 to 1.3 percent). Twenty control animals receiving saline had no tumors of epithelial origin. Nine control animals that were carcinogen-treated and tattooed in areas without ACF had no tumors in the marked areas. CONCLUSION: Results thus show regression of many ACF identified early in the carcinogenesis process. Results also support the hypothesis that some ACF are precursor lesions for adenomas and cancers.Supported by S. Lederman Fellowship Foundation, American Physician Fellowship, and, in part, by National Cancer Institute of Canada.     

Azoxymethane-induced rat aberrant crypt foci： Relevance in studying chemoprevention of colon cancer

The pathogenesis of colon cancer involves sequential and multistep progression of epithelial cells initiated to a cancerous state with defined precancerous intermediaries. Aberrant crypt foci （ACF） represent the earliest identifiable intermediate precancerous lesions during colon carcinogenesis in both laboratory animals and humans. ACF are easily induced by colon-specific carcinogens in rodents and can be used to learn more about the process of colon carcinogenesis. For over two decades, since its first discovery, azoxymethane （AOM）-induced rodent ACF have served as surrogate biomarkers in the screening of various anticarcinogens and carcinogens. Several dietary constituents and phytochemicals have been tested for their colon cancer chemopreventive efficacy using the ACF system. There has been substantial effort in defining and refining ACF in terms of understanding their molecular make-up, and extensive research in this field is currently in progress. In chemoprevention studies, AOM-induced rat ACF have been very successful as biomarkers, and have provided several standardized analyses of data. There have been several studies that have reported that ACF data do not correlate to actual colon tumor outcome, however, and hence there has been an ambiguity about their role as biomarkers. The scope of this mini-review is to provide valuable insights and limitations of AOM-induced rat ACF as biomarkers in colon cancer chemoprevention studies. The role of the dynamics and biological heterogeneity of ACF is critical in understanding them as biomarkers in chemoprevention studies.     

塞来昔布对二甲基肼诱导的大鼠畸变隐窝灶的预防作用

目的　明确塞来昔布 (选择性COX 2抑制剂 )对二甲基肼 (DMH)诱导的大鼠畸变隐窝灶(ACF)的化学预防作用 ,并与舒林酸 (一种非甾体类抗炎药 )进行比较。方法　 32只 8周龄雌性SD大鼠 ,随机分为 4组 ,每组 8只。第 1组单次腹腔注射DMH(每千克体重 12 0mg) ;第 2组单次腹腔注射生理盐水 (1ml/只 ) ;第 3组先舒林酸 (每千克饲料 32 0mg)喂养 7d ,然后注射DMH ;第 4组先塞来昔布(每千克饲料 15 0 0mg)喂养 7d ,然后注射DMH。注射DMH 5周后处死大鼠 ,美蓝染色后计数每只大鼠大肠中ACF和畸变隐窝 (AC)个数。结果　第 1组平均每只大鼠结肠中ACF和AC分别为 (182 .4 0± 93.4 3)和 (2 6 2 .80± 197.80 )个。第 2组没有发现ACF。第 3组ACF和AC分别为 (91.2 5± 4 8.98)和(139.6 0± 6 8.5 2 )个 ,与第 1组相比差异有显著性 (P <0 .0 5 )。第 4组ACF和AC分别为 (6 5 .83± 38.5 4 )和 (10 6 .0 0± 6 1.0 3)个 ,与第 1组相比差异有显著性 (P <0 .0 1)。第 3组与第 4组相比 ,ACF及AC个数差异无显著性。结论　选择性COX 2抑制剂塞来昔布对实验诱导的大鼠ACF具有预防作用 ,其作用与舒林酸相当。     

异常隐窝病灶与大肠肿瘤关系及发生途径的初步研究

目的 实体镜下连续观察Wistar鼠大肠癌模型中异常隐窝病灶(ACF)的发生情况,探讨ACF与大肠肿瘤的相关性及其发生途径.方法 60只Wismr鼠给予二甲肼皮下注射,每周1次,连续18周,分组处死.将美蓝染色后的大肠组织在实体镜下观察.结果 发现2种不同镜下表现的ACF,即cACF和dACF.dACF与大肠肿瘤形成早期有相似的镜下形态及病理特点,而cACF则无类似特征.cACF与dACF在β-catenin中的表达异常率分别为4.8%和100.0%(P=0.000),在MMP-7中的阳性表达率分别为7.9%和81.8%(P=0.000),dACF与肿瘤在β-catenin的表达异常率以及在MMP-7的阳性表达率上的差异均无统计学意义(P>0.05).结论 cACF与肿瘤发生无明显相关,dACF与肿瘤发生关系密切,其发生遵循Wnt途径.     

256例大肠异常隐窝灶的内镜与病理特征分析

目的了解大肠异常隐窝灶的内镜和病理特点,探讨ACF与结直肠肿瘤的关系。方法随机选择2011年8月-2012年3月就诊于江西省萍乡市人民医院准备接受结肠镜检查的患者370例,在距肛门25～30 cm以下给予0.4%靛胭脂染色后观察、记录发现远端大肠ACF病例数及ACF数目,并且行病理检查。结果 370例患者共发现256例ACF病例(69.19%)。正常黏膜组、增生性息肉组、腺瘤组、结肠癌组ACF患病率分别为58.21%、78.18%、82.05%、88.89%。256例ACF患者Ⅰ级106例(41.41%)、Ⅱ级82例(32.03%)、Ⅲ级68例(26.56%)。病理有异型增生病例数39例(10.54%),均为轻度异型增生;病理无异型增生217例(58.65%)。有ACF组增生性息肉的发生率为16.8%,腺瘤的发生率25.0%,结直肠癌的发生率12.5%;而无ACF组分别为10.53%、12.58%、3.51%。结论异常隐窝灶是常见结肠镜下病变,异型增生ACF病变并不少见。ACF可能是结肠腺瘤和大肠癌的独立预测因素。     

Reduced susceptibility to azoxymethane-induced aberrant crypt foci formation and colon cancer in growth hormone deficient rats

ObjectivesTo evaluate the role of GH in colon carcinogenesis, we examined the formation of aberrant crypt foci (ACFs) and tumor development in wild type (WT) and GH-deficient, spontaneous dwarf rats (SDRs) exposed to the carcinogen azoxymethane (AOM).DesignACF were quantified by stereomicroscopy and tumor number and weights were recorded for each animal. Cell proliferation was measured by vincristine metaphase arrest, flow cytometry, and bromodeoxyuridine (BrdU) incorporation. Apoptosis was measured by TUNEL staining and cleaved caspase-3 immunohistochemistry. IGF-I was measured by radioimmunoassay (RIA). Hexokinase activity was measured by spectrophotometric assay. PARP cleavage, and IGF-IR, and p27^kip/cip expression were measured by Western blotting.ResultsACFs detected by stereomicroscopy were markedly reduced (～85%) in SDRs vs. WT rats at 10, 25, and 28 weeks after AOM. Tumor incidence, number, and weight also were reduced in SDR vs. WT animals. AOM treatment increased cell proliferation in the distal colon (where tumors occur) of WT rats but not SDRs, and these changes corresponded to increased ACF and tumor formation. Apoptosis rates were similar in AOM-treated WT and SDRs. Alterations in serum IGF-I levels may contribute to differences in the proliferative response to AOM and decreased ACF formation in SDR vs. WT rats.ConclusionsWe conclude that early neoplastic lesions (ACFs) were reduced in GH-deficient animals. This effect corresponds with differences in AOM-induced proliferation, but not apoptosis. These data indicate that GH is required for the full effect of AOM on colon ACF and tumor development, and that the SDR rat is a promising model for studies regarding the role ofGH/IGF system in the initiation and promotion of colon cancer.     

Role of aberrant crypt foci detected using high-magnification-chromoscopic colonoscopy in human colorectal carcinogenesis

Abstract Liaison between gastrointestinal endoscopists and histopathologists is essential to provide the highest standards of diagnostic accuracy and patient management. The histopathologist needs to be aware of the endoscopic findings when interpreting endoscopic biopsies. High-magnification-chromoscopic-colonoscopy (HMCC) is a new technology that provides the endoscopists with much greater resolution and functional staining of the gastrointestinal tract. Using HMCC, the endoscopist is now able to identify subtle changes in the colorectal luminal openings or crypts. Changes in crypt appearances now allow detection of aberrant crypt foci (ACF) in the colon, which might themselves be precancerous lesions but additionally might serve as a valid biomarker of subsequent adenoma and colorectal cancer formation. This article describes the role of the aberrant crypt focus in colorectal carcinogenesis and discusses the clinical impact of HMCC techniques as applicable to ACF.     

ACF在结肠癌早期病变中的变化及其与PPAR-γ和β-catenin的关系

目的: 观察结肠癌前病变序列的增殖凋亡演变过程及其与PPAR-γ、β-catenin异常表达的关系.方法: 选用SPF级SD大鼠,经改良的DMH结肠肿瘤诱导方法诱导结肠ACF和腺瘤,然后对"正常-ACF-腺瘤"序列各阶段的组织进行免疫组织化学分析其PPAR-γ和β-catenin的表达,同时选择Ki-67表示增殖水平,Bcl-2表示凋亡水平.结果: 在DMH诱导的"正常-ACF-腺瘤"序列中,Ki-67在"正常-ACF"转变阶段即出现异常高表达,呈整个隐窝广泛分布,显著高于正常隐窝,而接近腺瘤组织. Bcl-2则在"ACF-腺瘤"转变阶段才出现异常高表达,呈广泛的胞质阳性颗粒. PPAR-γ在"正常-ACF"转化阶段发生广泛的高表达,呈广泛的核阳性.β-catenin则是在"ACF-腺瘤"转化阶段出现异常表达,呈广泛的核阳性.结论: 在"正常-ACF"转化阶段主要是增殖水平的升高,而且可能与PPAR-γ的异常表达有关. 在"ACF-腺瘤"转化阶段主要是凋亡抑制增加,β-catenin与Bcl-2同期出现异常表达对腺瘤的形成可能有一定的协同作用.     

Suppression of azoxymethane-induced aberrant crypt foci in rat colon by nimesulide,a selective inhibitor of cyclooxygenase 2

Non-steroidal anti-inflammatory drugs, such as piroxicam and sulindac, are known to inhibit development of aberrant crypt foci (ACF) and cancer in the colon. However, these agents cause gastrointestinal side-effects. Nimesulide is a selective inhibitor of cyclooxygenase 2 and has been shown to have a more potent anti-inflammatory action than piroxicam, but be less ulcerogenic and, therefore, a potentially more useful chemopreventive agent. To assess this possibility the inhibitory effects of nimesulide on the formation of ACF induced by azoxymethane in rat colon were investigated, and compared with those of piroxicam and sulindac. Male F344 rats were treated s.c. with 15 mg/kg body weight azoxymethane once a week for 2 weeks and given 50, 100 or 200 ppm nimesulide, 200 ppm piroxicam, or 200 ppm sulindac in their diet from the day before the first carcinogen treatment until the end of the experiment at week 4. At this time, nimesulide at doses of 50, 100 and 200 ppm had reduced the numbers of azoxymethane-induced ACF to 75%, 71% and 65% respectively compared to the control. The number of azoxymethane-induced ACF per colon in the group given 200 ppm nimesulide was almost the same as in those given 200 ppm piroxicam, and lower than that in the group given 200 ppm sulindac. These results suggest that nimesulide, a selective cyclooxygenase 2 inhibitor, warrants attention as a candidate for chemopreventive agent with low toxicity, active against colon carcinogenesis.Abbreviations ACF aberrant crypt foci - COX cyclooxygenase - NSAID non-steroidal anti-inflammatory drugs Work dedicated to Haruo Sugano on the occasion of his 70th birthday. The material of this paper was essentially presented at the 60th Anniversary Symposium of the Cancer Institute and the Cancer Institute Hospital, Tokyo, held in September 1994.The work was supported by Grants-in-Aid for Cancer Research from the Ministry of Health and Welfare, Japan, and the Ministry of Health and Welfare for the Second-Term Comprehensive 10-Year Strategy for Cancer Control, Japan, and a grant from the Bristol-Myers Squibb Foundation     

Chemopreventive effect of aspirin on growth of aberrant crypt foci in rats

Nonsteroidal anti-inflammatory drugs display a chemopreventive effect on polyps and cancer of the large bowel. This study evaluated the inhibitory effect of aspirin on the distribution and growth of aberrant crypt foci (ACF), the earliest putative preneoplastic and early neoplastic lesions in a rat model. For initiation of ACF, Sprague Dawley rats were injected with azoxymethane (30 mg/kg), a well-established rat carcinogen. After the second injection the rats were allocated to three groups, which received 0.2% or 0.6% aspirin or the solvent only (control group). After 6 weeks the animals were killed, and their colons removed, fixed in formalin, and screened for distribution and size of ACF, separately for middle and distal parts of the large intestine. The rats injected with azoxymethane showed a 100% incidence of ACF. Administration of 0.2% and 0.6% aspirin resulted in 55% and 54% reduction, respectively, in overall frequency of ACF. Aspirin significantly reduced the frequency of medium-sized (four to six crypts per focus) and large (three to six crypts per focus) but not the small (one to three crypts per focus) ACF. In the control group the ACF of the same multiplicity were larger than those in the aspirin-treated rats. No statistically significant difference in ACF-inhibiting effect was noted between 0.6% and 0.2% aspirin solution. Aspirin given at a concentration of either 0.2% of 0.6% thus has a chemopreventive effect on ACF, acting on postinitiation stage of azoxymethane-induced colonic carcinogenesis model in rats. Accepted: 20 July 1998     

Folate deficiency diminishes the occurrence of aberrant crypt foci in the rat colon but does not alter global DNA methylation status

BACKGROUND AND AIMS: It has been suggested that a diminished folate status may enhance colorectal carcinogenesis by causing DNA hypomethylation. The aims of the present study were to assess the impact of different levels of folate depletion on azoxymethane (AOM)-induced aberrant crypt foci (ACF) formation and DNA hypomethylation in the colon of male Sprague-Dawley rats. METHODS: Rats, aged 4 weeks, were divided into four groups and were fed semipurified diets either containing adequate folate (control), devoid of folate (FD) or FD + 1% succinylsulfathiazole before AOM treatment (FD1) or during the last 4 weeks of the study (FD2). At 8 weeks of age, all animals received subcutaneous injections of AOM once weekly for 3 weeks at a dose rate of 15 mg/kg bodyweight. Animals were necropsied 6 weeks after the last AOM injection and the ACF were visualized under light microscopy in formalin-fixed, methylene blue-stained colons. RESULTS: Blood folate concentrations were significantly depleted (P < 0.001) in the treatment groups consuming folate deplete diets, with the FD2 treatment group having significantly lower folate levels compared with all other groups. Higher plasma homocysteine concentrations (P < 0.001) were observed in the groups that exhibited diminished blood folate levels. There were no significant differences in global DNA methylation in the liver or colonic mucosa between the four groups, despite some groups exhibiting marked folate depletion. Animals with the most severe folate deficiency (FD2) had a lower final bodyweight and had significantly fewer ACF in their colon (P < 0.05) compared with control animals. Total (mean +/- SEM) ACF counts were as follows: control 286+/-24; FD 290+/-25; FD1 218+/-32; and FD2 205+/-27. CONCLUSIONS: In this model, folate deficiency diminished the occurrence of ACF but did not alter global DNA methylation status in the colon.     

Suppressive effect of aspirin on aberrant crypt foci in patients with colorectal cancer

BACKGROUND: and aims: Aspirin and other non-steroidal anti-inflammatory drugs have been shown to reduce the risk of colorectal cancer (CRC). Animal models have shown that aspirin is also effective in reducing the density of aberrant crypt foci (ACF). The aim of the study was to evaluate the effect of chronic administration of aspirin on the distribution pattern and histological characteristics of ACF in patients with CRC. METHODS: Our study compared the distribution patterns and histomorphological characteristics of ACF between a group of CRC patients treated with low dose aspirin (n=59) and a control group without aspirin (n=135). ACF were visualised on methylene blue stained macroscopically normal mucosa, microdissected, and serially cut. RESULTS: ACF were found in 75.8% of mucosal samples from the control group and in 36% of mucosal samples from the aspirin treated group, indicating a 47% decline in prevalence of ACF in colonic samples of patients treated with aspirin. A significant reduction from 92.5% to 40% (p<0.0001) was found in distal large bowel samples containing one or more ACF. Similarly, the aspirin treated group showed a reduction in ACF density of 64% and 82%, respectively, in both proximal and distal parts of the colon, indicating a significant reduction in ACF/cm(2) in distal colon samples (p<0.01). The aspirin treated group displayed a 52% reduction in dysplastic ACF although this difference was not statistically significant. CONCLUSIONS: Our study has provided evidence of the effective chemopreventive action of low dose aspirin on ACF in humans.