DNA alterations in human aberrant crypt foci and colon cancers by random primed polymerase chain reaction

Colon cancers are the result of the accumulation of multiple genetic alterations. To evaluate the role genomic instability plays during tumor development, we compared DNA fingerprints of 44 aberrant crypt foci (ACF; the earliest identified neoplastic lesion in the colon), 23 cancers, and normal crypts generated by random primers with PCR. The PCR products, separated by PAGE and viewed after silver staining, demonstrate altered fingerprints for 23.3% of the ACF and 95.7% of the cancers. In this first study of human ACF with this approach, the finding of altered DNA fingerprints in these microscopic lesions suggests that genomic instability can occur very early in human colon tumorigenesis.     

人直肠异常隐窝病灶的2年随访研究

  目的  异常隐窝病灶（aberrant crypt foci,ACF）目前被广泛认为是光镜下可视最早期的大肠癌癌前病变。本研究在2年时间通过结肠镜下动态观察ACF的变化来初步探讨ACF与结直肠癌的关系。  方法  选取2013年1月至2014年12月在山西医科大学第二医院接受结肠镜检查并排除常见致癌及抑癌因素的100例患者,常规在退镜时进行直肠靛胭脂染色,观察并记录初次结肠镜检查、1年及2年复查肠镜时直肠ACF的数目及单个ACF的自然发展变化过程。  结果  初次检查及1年和2年后的ACF阳性率比较,差异无统计学意义。ACF随时间的变化大部分继续存在或自行消失,少数发展为息肉、腺瘤,ACF < 6枚的病例,以及腺管开口为椭圆形的ACF自行消失率更大,而腺管开口为非椭圆形者更易发展为息肉或腺瘤。  结论  ACF变化呈异质性,其中部分可能为大肠癌癌前病变。      

变性隐窝病灶研究进展

变性隐窝病灶(ACF)是近年来提出的一种新型的结直肠癌癌前病变.其病理学上存在非增生性、单纯增生性以及各种不同程度异常增生的多种组织病理学类型改变.其生物化学及分子生物学上亦存在与结直肠肿瘤发生密切相关的一系列指标改变.对ACF的深入研究将有助于揭示结直肠肿瘤早期病变机制,寻找有效措施在肿瘤发生早期对病变发展进行限制、阻断和消除.     

Beta-catenin expression is altered in human colonic aberrant crypt foci.

The aberrant expression of beta-catenin in colon tumors and the discovery of beta-catenin mutations in small adenomas suggest that alterations of beta-catenin are early events in human colorectal carcinogenesis. Here, we describe the expression of beta-catenin in human aberrant crypt foci (ACF), the earliest identified neoplastic lesions in the colon. Paraffin-embedded sections of 94 ACF, 12 adenomas, and 10 carcinomas were evaluated for beta-catenin expression by immunohistochemistry. Normal colonic epithelial cells adjacent to these lesions showed strong membranous expression of beta-catenin and lacked cytoplasmic and nuclear expression. Cytoplasmic expression of beta-catenin was seen in 25 of 46 ACF with dysplasia and in 2 of 48 ACF with atypia. In ACF with dysplasia, reduced membranous expression of beta-catenin was associated with increased nuclear (P = 0.0013) and cytoplasmic (P = 0.0247) expression. The membranous (P = 0.0003) expression of beta-catenin was reduced, and the cytoplasmic (P = 0.0016) and nuclear (P = 0.0266) expressions increased in ACF according to their degree of dysplasia. Likewise, membranous (P = 0.0007) expression of beta-catenin was reduced, and the cytoplasmic (P = 0.0050) and nuclear (P = 0.0001) expressions increased from ACF to adenoma to carcinoma. These data suggest that ACF and their aberrant expression of beta-catenin play a role in colon tumorigenesis.     

Classification of aberrant crypt foci and microadenomas in human colon.

Aberrant crypt foci (ACF) can be observed and quantified on the mucosal surface of formalin-fixed human colon resections after staining with methylene blue. To determine whether these ACF could be identified in fresh tissue, 10 colon resections were collected after surgery for colorectal cancer. Unfixed and fixed flat normal colonic mucosa from each colon were scored for ACF under a dissecting microscope after methylene blue staining. The number of ACF per cm2 and the average number of crypts per foci correlated highly in unfixed and fixed mucosa (r = 0.93 and 0.78, respectively). A significantly higher frequency of lesions was found in left-sided compared to right-sided colon resections. To determine whether the topographic features of the ACF gave an indication of the histological appearance, 68 specimens containing ACF or normal mucosa were examined histologically. The presence of slit-like lumen in the crypts of ACF on the mucosal surface correlated with the presence of dysplasia at histology, thus identifying microadenomas. These two observations suggest that the topographic classification of ACF in vivo could be used to distinguish microadenomas, a putative precursor lesion of colon cancer.     

异常隐窝灶治疗的研究进展

异常隐窝灶(Aberrant crypt foci,ACF)在表型和分子学变化上与结直肠癌(Colorectal cancer,CRC)高度相似,与CRC发生风险呈正相关,被视为CRC的癌前病变。很多化合物对ACF具有治疗效果,抑制其发生和恶性变,可以视为对CRC的预防作用。本文将对近年来各种化合物通过不同途径对ACF的治疗做一综述。     

Instability of X chromosome methylation in aberrant crypt foci of the human colon

Aberrant crypt foci (ACF) in the colon have long been thought to be precancerous lesions and therefore monoclonal, but this is unresolved. Eleven ACF were isolated from five female patients. From these ACF, 178 individual aberrant crypts (ACs) were obtained and assessed for clonality using a method based on X chromosome inactivation of the polymorphic X-linked human androgen receptor (HUMARA) gene. Ten ACF were found to be mixtures of monoclonal and polyclonal types. The HUMARA analysis indicated that almost all ACF were polyclonal lesions. Simultaneously, we investigated K-ras mutations in each AC. We found that seven of the ACF harbored the K-ras mutation; strikingly, this was concordant for all of the ACs from a single ACF. These results, by contrast to the results of HUMARA analysis indicate that ACF lesions are monoclonal. This discrepancy suggests that ACF are apparently polyclonal because of de novo methylation on the active X chromosome. To confirm this possibility, we investigated the methylation status of the X chromosome in male ACF using a competitive PCR assay. One hundred nineteen individual ACs were isolated from eight ACF derived from four male patients. A total of 47 of 119 (39%) of male ACs showed de novo methylation of the HUMARA gene. We found that six of the eight male ACF harbored the K-ras mutation, and this was concordant for all of the ACs from a single ACF. We conclude that X chromosome methylation is unstable in ACF and that this might be an early event in colon carcinogenesis.     

A xanthine oxidase inhibitor 1'-acetoxychavicol acetate inhibits azoxymethane-induced colonic aberrant crypt foci in rats

The modifying effect of dietary administration of a xanthine oxidase inhibitor 1'-acetoxychavicol acetate (ACA) present in an edible plant Languas galanga in Thailand on the development of azoxymethane (AOM)- induced colonic aberrant crypt foci (ACF) was investigated in rats. Male F344 rats were given s.c. injections of AOM (15 mg/kg body wt) once a week for 3 weeks to induce colonic ACF. They were fed the diets containing 100 or 200 ppm ACA for 5 weeks, starting 1 week before the first dosing of AOM. At the termination of the study (week 5), AOM induced 118 +/- 28 ACF/colon. Dietary administration of ACA caused significant reduction in the frequency of ACF (41% inhibition by 100 ppm ACA feeding and 37% inhibition by 200 ppm ACA feeding, P<0.01). Such inhibition might be associated with suppression of the proliferation biomarkers' expression such as ornithine decarboxylase activity in the colonic mucosa, number of silver-stained nucleolar organizer regions' protein in the colonic mucosal cell nuclei and blood polyamine content. These results indicate that ACA could inhibit the development of AOM-induced ACF through its suppression of cell proliferation in the colonic mucosa and ACA might be a possible chemopreventive agent against colon tumourigenesis.      

Number of aberrant crypt foci associated with adiposity and IGF1 bioavailability

Background  Dysregulation of the insulin-like growth factor (IGF) system, a common consequence of adiposity-induced insulin resistance, may be a key underlying mechanism linking excess body weight with colon cancer. Evidence has been derived from studies of cancer and polyps. Supporting data about aberrant crypt foci (ACF), putative pre-polyp changes, have been generated only from animal studies to date. Methods  We randomly selected 26 patients with sex-specific elevated waist-hip-ratio (WHR) and 26 with normal values from a series of 150 patients seeking routine colonoscopy at the University of Connecticut Health Center. Cross-sectional analyses were performed of ACF number (<5, ≥5) in relation to total IGF1, IGF-binding protein-3 (IGFBP3), insulin, body mass index (BMI), WHR and waist circumference (WC). Visualized ACF in the 20 cm of the distal colon were counted using advanced endoscopic imaging. Results  Patients with ≥5 ACF had higher BMI, WHR, and WC compared with patients with >5 ACF (p = 0.04, p = 0.03, and p = 0.01, respectively). IGFBP3 was reduced (p = 0.02) and IGF1:IGFBP3 molar ratio was greater (p = 0.03) in patients with ≥5 ACF. We did not observe significant associations between ACF number and insulin or total IGF1. Conclusions  Our study provides the first report in humans of a possible association of ACF prevalence and IGF1 bioavailability as characterized by IGF1:IGFBP3 molar ratio and IGFBP3 level. More research is needed to determine whether this relationship is varied by ACF features (e.g., size, dysplasia, molecular changes), synchronous cancer and polyps, and is modified by colon cancer risk factors.     

Aberrant methylation of the PTCH1 gene promoter region in aberrant crypt foci

Patched homolog 1 (PTCH1) is a known tumor suppressor that regulates the Hedgehog (Hh) pathway and has been implicated in tumorigenesis. The role of PTCH1 in colon carcinogenesis, however, is controversial. The aim of the present study was to investigate epigenetic modifications of PTCH1 in aberrant crypt foci (ACF), the earliest precursor lesion of colorectal cancer (CRC). Using laser‐capture microdissection (LCM), a pure population of ACF epithelial cells was isolated and studied. The inherent protein expression levels of SHH, PTCH1, SMO and GLI1 were assessed by immunohistochemistry for 405 ACF, including 54 dysplastic ACF (d‐ACF) and 351 non‐dysplastic ACF (n‐ACF). The mRNA levels and methylation status of PTCH1 were also determined in 54 d‐ACF and 96 n‐ACF. Our data showed that the expression of SHH, SMO and GLI1 was significantly up‐regulated in d‐ACF, compared to n‐ACF. Also, the mRNA and protein levels of PTCH1 were lower in d‐ACF than n‐ACF. Using MSP or MS‐HRM, PTCH1 methylation was present in 64.8% (35/54) or 63.3% (34/54), respectively, of d‐ACF and 19.8% (19/96) or 22.9% (11/48), respectively, of n‐ACF. PTCH1 methylation was more frequent in d‐ACF than n‐ACF (p < 0.001) and was associated with PTCH1 mRNA levels (r = 0.358, p < 0.01). There was a statistically significant correlation between PTCH1 methylation status and the prevalence of colorectal neoplasms. In conclusion, this study suggests that aberrant methylation of the PTCH1 promoter may be an early, initiating event of colon carcinogenesis.     

Scanning electron microscopy of aberrant crypt foci in rat colon

The surface of the colon mucosa of 1,2-dimethylhydrazinetreatedF344 rats was examined with the scanning electron microscope.A detailed examination of the mucosal topography revealed fociwith one to several aberrant crypts. These were seen as structureselevated from the background mucosa. The shape of the luminalopenings of the aberrant crypts varied from elongated or tortuousto circular. However, we found no ultrastructural variationsbetween the different aberrant crypt foci (ACF) or between theACF and the background mucosa. There was no direct relationshipbetween the size of ACF and the number of aberrant crypts perfocus, which may be explained by the mechanism of crypt fission;in two aberrant crypts we discovered the formation of a transverseepithelial septum, dividing the large crypt into two smallercrypts. The gross morphology of the ACF observed by scanningelectron microscopy and light microscopy was in principle thesame.     

Spontaneous development of aberrant crypt foci in F344 rats

Aberrant crypt foci (ACF) have been proposed as intermediate biomarkers for colon carcinogenesis on the basis of many rodent studies. Although molecular analyses have indicated that these lesions in experimental animals are related to early events in colon carcinogenesis, their preneoplastic nature has yet to be fully elucidated. In the present study, one hundred and thirty 19-week-old male Fischer 344 rats were examined. The biological characteristics of spontaneous ACF were analyzed histopathologically, immunohistochemically and with molecular biological techniques, and compared with colon tumors found in control groups used for carcinogenicity tests. The incidences of spontaneous ACF consisting of 1, 2, 3 and 4 or more crypts were respectively 27.7%, 32.5%, 16.8% and 22.8%. Most ACF were distributed in the lower middle and upper distal colon, and proximal colon ACF was rare. Likewise, ACF frequently (42.5%) developed in untreated animals, whereas the incidence of spontaneous colorectal tumors was extremely low (0.68%) in control male rats. In addition, spontaneous ACF did not show apparent proliferative activity or c-K-ras point mutations. Our results thus suggest that spontaneous ACF rarely progress to colon tumors although long-term sequential observation might be necessary to conclude the significance of ACF.     

Polyethylene glycol reduces inflammation and aberrant crypt foci in carcinogen-initiated rats

Polyethylene glycol 8000 inhibits the formation of tumors and of aberrant crypt foci (ACF) in carcinogen-initiated rats. We asked: is the inhibition associated with a reduction of colonic inflammation and an increase in colonic cell permeability? Twenty-eight, male F 344 rats were divided into two groups, 10 control animals and 18 animals initiated with azoxymethane. Nine of the rats in the carcinogen-initiated group were given a diet with 5% PEG 8000 in an AIN-93 based, high fat diet. The other nine, and the control group received the diet without the addition of PEG. Nine weeks later, the rats receiving the diet containing PEG had a 43% reduction in ACF (P<0.001) compared with the carcinogen-initiated rats on the control diet, a result confirming earlier observations that PEG inhibits colon carcinogenesis. The animals receiving the diet containing PEG also had a 10-fold reduction in fecal granulocyte marker protein (GMP) (P<0.001) compared with both the carcinogen-treated and the control animals. PEG reduced inflammation below the levels of carcinogen-treated and of untreated animals. Fecal water from the rats receiving PEG did not reduce transepithelial resistance of, or manitol flux through, human Caco-cells grown as monolayers in vitro. PEG may reduce colon carcinogenesis through a mechanism involving colonic inflammation.     

Genetic and epigenetic changes in aberrant crypt foci and serrated polyps

Aberrant crypt foci (ACF) in colorectal mucosa are the earliest known morphological precursors to colorectal cancer and can be subclassified as dysplastic, heteroplastic (non-dysplastic), and mixed types. Serrated adenoma (SA) is a polyp with serrated architecture and dysplasia, and can be subclassified as traditional SA or sessile SA. Sessile SA is thought to be preneoplastic and differs from most lesions in the traditional SA category because of their flat morphology and general lack of cytological dysplasia. Serrated polyps include hyperplastic polyps (HP), SA, and admixed hyperplastic-adenomatous polyps and are considered a morphological continuum encompassing heteroplastic ACF, HP, admixed hyperplastic-adenomatous polyps, and SA. Recent studies have uncovered other developmental pathways including a heteroplastic ACF-HP/SA-carcinoma sequence and a heteroplastic ACF–adenoma–carcinoma sequence. Heteroplastic ACF histopathologically resemble HP and SA. Sporadic HP are usually present in the left colon, are small, and are considered benign. However, adenocarcinoma arising in the setting of colorectal HP or SA, especially in patients with hyperplastic polyposis, has been described. The relationship between heteroplastic ACF, HP, and colorectal cancer is less certain than that of dysplastic ACF. Here, we discuss the current understanding of genetic and epigenetic alterations in the development of colorectal cancer. Our goal is to provide a conceptual framework for understanding the heteroplastic ACF–HP/SA–carcinoma sequence. ( Cancer Sci 2008; 99: 1071–1076)     

Deoxycholic acid promotes the growth of colonic aberrant crypt foci

AKR/J mice are resistant to the tumorigenic properties of the colon carcinogen, azoxymethane (AOM). Following AOM exposure, limited numbers of preneoplastic lesions, referred to as aberrant crypt foci (ACF), are formed in the colon, and their progression to tumors rarely occurs. To determine whether genetic resistance can be overcome by exposure to a dietary tumor promoter, AOM-exposed AKR/J mice were fed a diet containing 0.25% deoxycholic acid (DCA). DCA exposure was begun 1 wk prior to or 1 wk after tumor initiation with AOM. Mice placed on the DCA diet prior to AOM treatment developed a significantly higher multiplicity of ACF compared to AOM-exposed mice fed a control diet (15.50 +/- 0.96 vs. 6.17 +/- 0.48, respectively; P < 0.05). When DCA exposure was begun after AOM treatment (post-initiation), ACF formation was further enhanced (34.00 +/- 1.22). Interestingly, increased numbers of ACF were associated with the presence of nuclear beta-catenin, assessed by immunohistochemistry. While approximately 33% of ACF from mice exposed to DCA prior to AOM treatment contained positive nuclear beta-catenin staining, approximately 77% of ACF from mice fed DCA after AOM were positive. Accumulation of nuclear beta-catenin was not associated with a loss of E-cadherin from the plasma membrane, although loss of APC staining was a consistent feature of most AOM-induced ACF, regardless of DCA exposure. These results demonstrate that exposure to DCA, an important digestive component, is sufficient to sensitize the resistant AKR/J colon to formation of high-grade dysplasia, and that nuclear translocation of beta-catenin may play an important role in this process.     

Natural history of human aberrant crypt foci and correlation with risk factors for colorectal cancer

Although aberrant crypt foci (ACF) are estimated to have potential usefulness as a biomarker for colorectal carcinoma (CRC), this remains uncertain because the natural history of ACF has not been well-clarified. To determine the usefulness of ACF as a surrogate marker for CRC, it is necessary to understand the natural history of ACF. A total of 82 subjects who underwent total colonoscopy and whose ACF number was examined at least 2 times at Yokohama City University Hospital were enrolled. We retrospectively evaluated the changes in the ACF number at four different surveillance periods (6 months, 1 year, 2 years, 3 years) and in groups with and without colorectal neoplasms. Furthermore, we classified the subjects into an increased ACF group and a no change/decreased ACF group, and investigated the relationship between the changes in the ACF number and known risk factors for CRC. No significant differences were observed in the ACF number between the first and second observations in any surveillance period groups, and in the groups classified according to the presence or absence of colorectal neoplasms. There were no significant differences between the increased and no change/decreased ACF group in terms of gender, smoking habit, current alcohol consumption, age, BMI, HbA1c or serum triglyceride level (TG), whereas a significant difference between the groups was observed in the serum total cholesterol level (TC) (p=0.012). ACF are a reliable surrogate marker that are not affected by any risk factors for adenomas or CRC, except TC, and may therefore be considered as a useful marker in chemopreventive trials.     

Abnormal expression of gastric mucin in human and rat aberrant crypt foci during colon carcinogenesis.

Colorectal cancer is a major cause of death in Europe and the USA, and much effort is therefore devoted to improve its early detection. In this article, we report the abnormal expression of gastric mucin in aberrant crypt foci (ACF) that appear in the colon mucosae removed from colorectal cancer patients and rats treated with methyl-N'-nitro-N-nitroso-guanidine (MNNG). We performed the immunoperoxidase test using monoclonal antibodies raised against gastric M1 mucin encoded by the MUC5AC gene and against rat gastric mucins (MAb 660), respectively. In both human and rat colon, these anti-gastric mucin MAbs stained specifically goblet cells within ACF. In humans, the M1/MUC5AC mucin was expressed in the upper part of the glands in hyperplastic ACF and in the typical ACF. In addition, the anti-gastric mucin MAbs stained some rare, scattered, histologically normal glands in the human and rat colon mucosae. These glands may be regarded as precursors of ACF. The abnormal expression of the MUC5AC gene constitutes a novel change in addition to genetic modifications already observed in ACF, and supports our previous findings demonstrating the potential of this gastric mucin as an early marker of human and rat colon carcinogenesis.     

Microsatellite instability in aberrant crypt foci from patients without concurrent colon cancer

Aberrant crypt foci (ACF) are microscopic surface abnormalities that are putative precursors to colorectal cancer (CRC). ACF exhibit similar histological and molecular abnormalities to adenomas and CRC and potentially represent useful biomarkers of cancer risk. Microsatellite instability (MSI) is one molecular abnormality identified in concurrent ACF from CRC patients that may indicate a risk for progression. To determine if MSI can be detected in ACF from cancer-free subjects, we examined 45 ACF from 20 subjects undergoing colonoscopies. The group included 12 patients at elevated risk for CRC based on family history of CRC or personal history of CRC or advanced adenoma and 8 patients with no known risk factors. ACF were identified using close-focus magnifying chromendoscopy and collected by biopsy in situ. Genomic DNA was prepared from ACF and adjacent normal colonic epithelium isolated by laser capture microdissection and analyzed for MSI. MSI was identified in at least one marker from 9 of 30 (30%) lesions from patients at elevated risk for CRC and in 2 of 15 (13%) lesions from average risk patients. Using methylation-specific PCR analysis, we also examined the ACF for promoter hypermethylation of the DNA repair genes hMLH1 and MGMT and found moderate changes (8/39 and 3/32, respectively). Although we found only a limited relationship between hMLH1 hypermethylation and MSI, all the lesions with MGMT hypermethylation displayed an MSI-low phenotype. These lesions may be precursors to MSI-low CRC, providing a potential early biomarker to assess the effects of cancer prevention strategies.