Frequent mutation of Apc gene in rat colon tumors and mucin-depleted foci, preneoplastic lesions in experimental colon carcinogenesis

Mucin-depleted foci (MDF) are microscopic dysplastic lesions induced in the colon of rodents by specific colon carcinogens. Most MDF show Wnt pathway activation, whereas only a subset shows mutations in the Ctnnb1 gene, coding for beta-catenin. Because Apc is a member of the Wnt pathway and the most frequent mutated gene in human colon cancer, we tested whether MDF harbor Apc mutations. F344 rats were treated twice with 150 mg/kg of 1,2-dimethylhydrazine. After 15 or 28 weeks, MDF, aberrant crypt foci (ACF), and tumors were collected. We screened a segment of the Apc gene comprising the region homologous to the mutation cluster region (MCR) of human APC, which frequently shows mutations in experimental colon tumors. Mutations were identified by PCR amplification and sequencing in 6:24 MDF (25%), 7:23 tumors (30%), 0:24 ACF (0%). Most of the mutations (92%) in MDF and tumors were localized in a region upstream from the MCR. All mutations were single-base substitutions and mainly formed by G:C-->A:T and C:G-->T:A transitions. The pattern of nucleotide changes was similar in MDF and tumors, and, interestingly, the same mutation in codon 1047 was found in two MDF and in three tumors. Four out of the six mutations found in MDF were nonsense mutations, and two were missense. All mutations in tumors determined a protein truncation. These results show that Apc mutations are present in MDF with a frequency similar to that of tumors, strengthening the evidence that they are precancerous lesions in colon carcinogenesis.     

Aberrant crypt foci in patients with a positive family history of sporadic colorectal cancer

Early detection is crucial in the prevention of colorectal cancer (CRC) deaths. The earliest detectable neoplastic lesion in the colon is the aberrant crypt foci (ACF). A major question is whether ACF are precursors of CRC, and thus, early biomarkers for CRC risk. If so, we hypothesized that the number of ACF would be higher in patients who had a family history of CRC compared to patients without. We counted ACF in the distal 20cm of colon/rectum during 103 colonoscopic examinations using a prototype Close Focus Colonoscope (Olympus Corp.) with methylene blue chromendoscopy. Each patient was asked whether they had a family history of CRC in a first degree relative, or a personal history of CRC or adenoma. Patients answering 'no' to these questions (n=17) had a mean number of ACF of 4.4; the mean was significantly higher in the patients with a positive family history of CRC (9.0, p<0.01; n=43) or a personal history of advanced adenoma (7.5, p<0.05; n=34).     

Survival of hereditary non-polyposis colorectal cancer patients compared with sporadic colorectal cancer patients

Background

Patients with hereditary non-poliposys colorectal cancer (HNPCC) have better prognosis than sporadic colorectal cancer (CRC). Aim of our retrospective study was to compare the overall survival between sporadic CRC and HNPCC patients.

Methods

We analyzed a cohort of 40 (25 males and 15 females) HNPCC cases with a hospital consecutive series of 573 (312 males and 261 females) sporadic CRC observed during the period 1970–1993. In 15 HNPCC patients we performed mutational analysis for microsatellite instability. Survival rates were calculated by Kaplan-Meier method and compared with log rank test.

Results

The median age at diagnosis of the primary CRC was 46.8 years in the HNPCC series versus 61 years in sporadic CRC group. In HNPCC group 85% had a right cancer location, vs. 57% in the sporadic cancer group. In the sporadic cancer group 61.6% were early-stages cancer (Dukes'' A and B) vs. 70% in the HNPCC group (p = ns). The crude 5-years cumulative survival after the primary CRC was 94.2% in HNPCC patients vs. 75.3% in sporadic cancer patients (p < 0.0001).

Conclusion

Our results show that overall survival of colorectal cancer in patients with HNPCC is better than sporadic CRC patients. The different outcome probably relates to the specific tumorigenesis involving DNA mismatch repair dysfunction.     

Identification of mucin-depleted foci in the unsectioned colon of azoxymethane-treated rats: correlation with carcinogenesis

We tested the association between aberrant crypt foci (ACF) and tumor induction by feeding azoxymethane-induced rats (15 mg/kg x 2, s.c.) with synbiotics (Raftilose Synergy 1, a derivative of inulin, 10% of the diet, along with lactobacilli and bifidobacteria). After 16 weeks of feeding, synbiotics significantly increased ACF multiplicity. On the contrary, after 32 weeks, synbiotics significantly decreased intestinal tumors. When the same unsectioned colon used for ACF determination was stained with high-iron diamine Alcian blue, foci of crypts with scarce or absent mucins were identified. We defined these lesions as mucin-depleted foci (MDF), and they were visible in all azoxymethane-treated rats and correlated with tumor induction (MDF/colon: 8.2 +/- 0.9 and 3.8 +/- 0.9 in controls and synbiotic-fed rats, respectively, P < 0.01; crypts/MDF: 12.2 +/- 2 and 6.4 +/- 1 in controls and synbiotic-fed rats, respectively, P < 0.05, means +/- SE, n = 7). There were fewer MDF/colon than ACF, and they were histologically more dysplastic than mucinous lesions identified as ACF in high-iron diamine Alcian blue-stained colon. In conclusion, MDF may be premalignant lesions that predict colon carcinogenesis.     

Aberrant crypt foci in patients with colorectal cancer.

Aberrant crypt foci (ACF) are clusters of abnormally large colonic crypts identified on the mucosal surface of the human colon. They are thought to be preneoplastic lesions. The aim of the present study was to compare density (number of ACF per square cm of mucosal surface), crypt multiplicity (number of crypts per ACF) and histology of ACF in colonic resections of colorectal cancer patients resident in two Italian provinces with a twofold difference in colorectal cancer incidence rates. Thirty-two and 26 colonic resections were collected after operation in Ragusa (Southern Italy) and Modena (Northern Italy), respectively, and fixed in 10% formalin. Mucosal layers were observed under a light microscope at 25x after staining with methylene blue. Density of ACF was significantly higher in Modena (median 0.101 ACF cm(-2)) than in Ragusa (0.049, P = 0.001), whereas there was no difference in crypt multiplicity. ACF were classified into three groups according to histological features: ACF with mild alterations (hypertrophic ACF, 73%), ACF with hyperplasia (hyperplastic ACF, 17%) and ACF with dysplasia (microadenomas, 10%). The proportions of ACF in the three groups were similar in the two provinces. Density of ACF was higher and crypt multiplicity lower proceeding from proximal to distal large bowel. Microadenomas were observed only in the colon, whereas hyperplastic ACF were more frequent in the rectum. In conclusion, density of ACF correlates with colorectal cancer rates in two Italian provinces, and shows a positive gradient from proximal to distal large bowel. Histology of ACF suggests that they may be precursors of both hyperplastic and adenomatous polyps. These data provide further evidence of the role of ACF in human colorectal carcinogenesis.     

Early genetic aberrations in patients with sporadic colorectal cancer

Chromosome instability (CIN) is widely observed in both sporadic and hereditary colorectal cancer (CRC). Defects in APC and WNT signaling are primarily associated with CIN in hereditary CRC, but the genetic causes for CIN in sporadic CRC remain elusive. Using high‐density SNP array and exome data from The Cancer Genome Atlas (TCGA), we characterized loss of heterozygosity (LOH) and copy number variation (CNV) in the peripheral blood, normal colon, and corresponding tumor tissue in 15 CRC patients with proficient mismatch repair (MMR) and 24 CRC patients with deficient MMR. We found a high frequency of 18q LOH in tumors and arm‐specific enrichment of genetic aberrations on 18q in the normal colon (primarily copy neutral LOH) and blood (primarily copy gain). These aberrations were specific to the sporadic, pMMR CRC. Though in tumor samples genetic aberrations were observed for genes commonly mutated in hereditary CRC (eg, APC, CTNNB1, SMAD4, BRAF), none of them showed LOH or CNV in the normal colon or blood. DCC located on 18q21.1 topped the list of genes with genetic aberrations in the tumor. In an independent cohort of 13 patients subjected to Whole Genome Sequencing (WGS), we found LOH and CNV on 18q in adenomatous polyp and tumor tissues. Our data suggests that patients with sporadic CRC may have genetic aberrations preferentially enriched on 18q in their blood, normal colon epithelium, and non‐malignant polyp lesions that may prove useful as a clinical marker for sporadic CRC detection and risk assessment.     

Survival of patients with hereditary colorectal cancer: Comparison of HNPCC and colorectal cancer in FAP patients with sporadic colorectal cancer

Conflicting data exist on the prognosis of hereditary colorectal cancer. HNPCC patients, in particular, are often reported to have a better survival. We examined 2,340 colorectal‐cancer patients treated in our Institution: 144 HNPCC patients (Amsterdam Criteria), 161 FAP patients and 2,035 patients with sporadic cancer. Data on hereditary‐cancer patients treated between 1980 and 1995 was collected in a registry. The 2,035 sporadic colorectal‐cancer patients (controls) included all new cases treated in the Department of Gastrointestinal‐Tract Surgery during the same period. Observed survival was estimated using the Kaplan‐Meier method. Cumulative survival probability was estimated at 5 years within each group and stratified by various clinical and pathological variables. The age distribution at diagnosis of sporadic patients was significantly higher than that of FAP and HNPCC patients (median 60 years vs. 43 and 49 years; p < 0.0001). In the HNPCC group, 40% had a right cancer location, vs. 14% in the FAP group and 13% in the sporadic‐cancer group. In the sporadic group, 51% were early‐stage cancers (Dukes A or B) vs. 48.4% and 52.1% in the FAP and HNPCC groups respectively. In the HNPCC, FAP and sporadic‐cancer groups, the 5‐year cumulative survival rate was 56.9%, 54.4% and 50.6% respectively. Survival analysis by the Cox proportional‐hazards method revealed no substantial survival advantage for HNPCC and FAP patients compared with the sporadic group, after adjustment for age, gender, stage and tumor location. The hazard ratio for HNPCC was 1.01 (95% CI 0.72–1.39) and 1.27 (95% CI 0.95–1.7) for FAP patients compared with the sporadic‐colorectal‐cancer group. Int. J. Cancer 80:183–187, 1999. © 1999 Wiley‐Liss, Inc.     

Aberrant crypt foci as precursors in colorectal cancer progression

Colorectal cancer progression originates when accumulated genetic and epigenetic alterations cause genomic instability and a malignant phenotype. Subsequent molecular pathway deregulation leads to histopathologic changes that are clinically evident as aberrant crypt foci (ACF) and visualized by high-magnification chromoscopic colonoscopy. ACF are biomarkers of increased colorectal cancer risk, particularly those with dysplastic features. Genetic profiling using genomic instability, loss of heterozygosity, and methylation analysis has revealed a minority population of ACF genotypically analogous to cancer.     

Deficient mismatch repair system in patients with sporadic advanced colorectal cancer

A deficient mismatch repair system (dMMR) is present in 10–20% of patients with sporadic colorectal cancer (CRC) and is associated with a favourable prognosis in early stage disease. Data on patients with advanced disease are scarce. Our aim was to investigate the incidence and outcome of sporadic dMMR in advanced CRC. Data were collected from a phase III study in 820 advanced CRC patients. Expression of mismatch repair proteins was examined by immunohistochemistry. In addition microsatellite instability analysis was performed and the methylation status of the MLH1 promoter was assessed. We then correlated MMR status to clinical outcome. Deficient mismatch repair was found in only 18 (3.5%) out of 515 evaluable patients, of which 13 were caused by hypermethylation of the MLH1 promoter. The median overall survival in proficient MMR (pMMR), dMMR caused by hypermethylation of the MLH1 promoter and total dMMR was 17.9 months (95% confidence interval 16.2–18.8), 7.4 months (95% CI 3.7–16.9) and 10.2 months (95% CI 5.9–19.8), respectively. The disease control rate in pMMR and dMMR patients was 83% (95% CI 79–86%) and 56% (30–80%), respectively. We conclude that dMMR is rare in patients with sporadic advanced CRC. This supports the hypothesis that dMMR tumours have a reduced metastatic potential, as is observed in dMMR patients with early stage disease. The low incidence of dMMR does not allow drawing meaningful conclusions about the outcome of treatment in these patients.     

Genome-Wide search for loss of heterozygosity in Chinese patients with sporadic colorectal cancer

In an attempt to integrally investigate the loss of tumor suppressor genes and search for putative suppressor loci associated with tumor occurrence and progression, we conducted a genome-wide loss of heterozygosity (LOH) study of 83 tumor samples obtained from Chinese patients with sporadic colorectal cancer. We employed 400 fluorescence-labeled microsatellite marker primers to amplify the corresponding loci of the genomic DNA and then electrophoresed the polymerase chain reaction products and analyzed the fluorescent signals. The LOH frequencies were high (>35%) but were not associated with the tumor stage and progression in 20 loci, including the regions where TP53, E-cadherin, deleted in colorectal carcinoma (DCC), phosphatase and tensin homolog deleted on chromosome 10 (PTEN), mothers against decapentaplegic, Drosophila, homolog of 2 (MADH2) and mothers against decapentaplegic, Drosophila, homolog of 4 (MADH4) reside. Loss of other loci, including two narrow regions on chromosome 2, was found to relate to the tumor stage, suggesting that this genomic instability may contribute to tumor progression.     

Extracolonic features of familial adenomatous polyposis in patients with sporadic colorectal cancer.

We have investigated the occurrence of attenuated extracolonic manifestations (AEMs) of familial adenomatous polyposis (FAP) in patients with non-polyposis colorectal cancer. In a prospective case-control study, we observed that significantly more colorectal cancer patients exhibited AEM than did age and sex-matched controls (19.5% vs 7.5%, P < 0.004). However patients with AEMs do not have occult FAP, as we found no heterozygous adenomatous polyposis coli (APC) gene mutations despite extensive analysis of constitutional DNA. Genome-wide DNA replication errors (RERs) occur in a proportion of colorectal cancers, particularly right-sided lesions and in almost all tumours from hereditary non-polyposis colorectal cancer (HNPCC) patients. As AEMs have been reported in familial colon cancer cases, we investigated the relationship of AEMs to tumour RER phenotype. There was indeed an excess of AEMs in patients with right-sided tumours (30.2% of 53 patients vs 14.7% of 116 patients, P < 0.03) and in those with RER tumours (3 out of 12 patients with RER tumours vs none out of 21 patients with non-RER tumours, P < 0.05). Two patients with AEM were from HNPCC families compared with none of those without AEM (P < 0.05). The association of AEMs with colorectal cancer is intriguing, and we speculate that it may be a manifestation of mutational mosaicism of the APC gene, perhaps associated with a constitutional defect in DNA mismatch pair.     

散发性结直肠癌患者5号染色体杂合缺失分析

目的：探讨散发性结直肠癌患者5号染色体上与抑癌基因相关的杂合缺失（LOH）情况,并探讨新的抑癌基因位点,方法：对83例散发性结直肠癌患者基因组DNA,以15个不同荧光标记的高度多态性微卫星引物（平均遗传距离12．67cm)扩增相应的微卫星位点,用ABI PRISM 377测序仪进行基因扫描,统计各位点杂合缺失率。结果：在15个微卫星位点中,平均杂合缺失率为25．80％,5p中最高为D5S416,占48．15％,5q中最高为D5S471,占38．71％,D5S471周围的3个位点（D5S428,D5S2027 和 D5S2115）也存在高频的杂合缺失（＞30．00％）,结论：5号染色体上存在着高频的杂合缺失,其中5q13.3-31.1区域中,有与结直肠癌发生密切相关的APC,MCC,CTNNA1及IL家族等基因,而在5p15.1上的D5S416的杂合缺失率高达48．15％,此区域至今尚未发现与结直肠癌相关的基因位点,估计可能有未知的抑癌基因存在。     

FOLFOX as adjuvant chemotherapy after curative resection of distant metastatic lesions in patients with colorectal cancer

We investigated the effectiveness of prophylactic FOLFOX after curative resection of synchronous metastases in patients with colorectal cancer (CRC). Clinicopathological information including postoperative chemotherapy, such as a therapeutic regimen, relapse-free survival (RFS), site of recurrence, etc., was retrospectively analyzed in 116 CRC patients with synchronous distant metastases, and 63 patients with metachronous metastases who had received surgery in our hospital between 2000 and 2009. Fifty-three patients (84%) out of 63 without adjuvant chemotherapy, and 38 (83%) out of 46 patients that received oral or intravenous 5-fluorouracil (5-FU) (alone or with leucovorin (LV)or isovorin) developed recurrent tumor(s) afterwards. The median RFSs were 119 and 281 days, respectively. By contrast, a single patient among 6 who underwent FOLFOX (up to 12 therapeutic courses) showed recurrence 476 days after surgery. The RFS of the FOLFOX was significantly higher than that of the 5-FU (+LV) or surgery alone (p=0. 03, p=0. 007, respectively). In conclusion, the FOLFOX regimen is more beneficial for CRC patients with synchronous metastasis as adjuvant chemotherapy than 5-FU (+LV) or other followup strategies.     

Promoter hypermethylation of tumor-related genes in sporadic colorectal cancer in young patients

Sporadic colorectal cancer (SCRC) occurring in young patients represent a subset with a higher proportion of advanced tumors and a poor prognosis, however, the genetic basis of SCRCs has not yet been sufficiently studied. We assigned 16 SCRC patients aged 40 years or less to group 1, and 30 SCRCs patients aged 65 years or more to group 2. The methylation status in the promoter of 7 tumor suppressor genes regarding these two groups was then examined. The average number of hypermethylated tumor-related genes per sample in group 1 was 1.50 +/- 0.07, which was significantly lower than that in group 2 of 2.73 +/- 1.24 (p = 0.0040). The frequencies of the promoter hypermethylation of hMLH1, p15INK4b, p16INK4a, and RASSF1A in group 1 were 12.5%, 12.5%, 12.5%, 6.3%, and 0.0%, which were substantially less frequent than those same rates observed in group 2. In contrast, the frequencies of the promoter hypermethylation of APC, MGMT, p14ARF, in group 1 were 43.8%, 37.5%, and 31.3%, which were as frequent as those seen in group 2. The promoter hypermethylation of APC, MGMT, and pl4ARF is therefore considered to be closely related to the development of SCRCs in young patients, regardless of aging.     

Single nucleotide polymorphisms in sporadic colorectal cancer

Colorectal cancer (CRC) etiology involves multiple disease pathways. An important research element for unraveling these carcinogenic pathways to CRC is to gain an understanding of genetic susceptibility to CRC at the population level; single nucleotide polymorphisms (SNPs) are central to this research. Examination of a broad spectrum of SNPs along multiple disease pathways should help our understanding of cancer biology and the carcinogenic process. However, one of the greatest research challenges today will be to implement and interpret studies that utilize SNPs to gain insight into the genetic susceptibility of CRC.     

hMLHl and hMSH2 somatic inactivation mechanisms in sporadic colorectal cancer patients

Much is known about the role of germline inactivation in mismatch repair (MMR) genes in hereditary non-polyposis colorectal cancer (HNPCC), but the impact of somatic MMR gene changes on sporadic colorectal cancer remains to be elucidated. In hereditary cases the hMLHl and hMSH2 genes were shown to have a great importance, and in order to examine the somatic inactivation mechanisms of the two MMR genes hMLHl and hMSH2 we screened 37 Hungarian sporadic colorectal cancer patients for allelic imbalance (AI), microsatellite instability (MSI), hMLHl promoter hypermethylation and somatic mutations. Thirteen of the examined tumours (35%) were characterized by low-level MSI and none of the cases belonged to the high MSI group. Nine (24%) and seven (19%) cases had AI at the hMLHl and hMSH2 genes, respectively. Seven tumours (19%) showed dense promoter hypermethylation of hMLHl, but only two patients had somatic mutations, one for each MMR gene. According to our study on this limited set of cases the most prominent mismatch repair inactivation mechanism in sporadic colorectal cancer patients is the hMLHl promoter hypermethylation which may have a role in the carcinogenesis of sporadic colorectal cancer.     

Dose response and proliferative characteristics of aberrant crypt foci: putative preneoplastic lesions in rat colon

Foci of aberrant crypts (ACF) have been identified in the unsectioned methylene blue stained rodent colons and hypothesized to represent precursor lesions of colon cancer. In the present study, induction and growth characteristics of ACF were investigated in response to a single injection of varying dosages of 1,2-dimethylhydrazine-2HCl (DMH), a colon carcinogen. Female Sprague-Dawley rats were given a single injection of DMH (5-150 mg/kg). Two and 19 weeks after the injection, animals were killed and their distal 10 cm of colons were enumerated for the number and crypt multiplicity of ACF. Number of ACF increased with increasing dosages of DMH plateauing at 100 mg/kg. However, percentage of ACF exhibiting different crypt multiplicity (1 to greater than 4) were similar among different dose groups. Aberrant crypts and normal crypts were enumerated for total number of cells and number and distribution of S-phase cells along the crypt height 19 weeks after DMH injection after autoradiography. The labeling index (LI) (percentage of S-phase cells) and LI along the crypt height were determined. Compared to the surrounding normal crypts, aberrant crypts exhibited significantly higher (P less than 0.05) number of cells (1122 +/- 81 versus 411 +/- 28) and higher (P less than 0.05) LI (21 +/- 1 versus 12 +/- 1). For the eight ACF analysed in the present study, the distribution of S-phase cells in the aberrant crypts were similar to that of normal crypts in that S-phase cells were restricted to the lower two-thirds of the crypts rather than distributed throughout the height of the crypts as reported for adenomatous epithelium.     

Highlights in the prevention of sporadic colorectal cancer

Colorectal cancer is one of the most often diagnosed malignant tumors in humans. Undoubtedly, dietary patterns and lifestyle are strongly implicated in the pathogenesis of sporadic colorectal cancer. Evidence is emerging for the participation of human gut microflora in the pathogenesis of colorectal cancer. Chemoprevention of colorectal cancer with medications is an attractive opportunity, although the results from clinical trials are inconclusive. This review discusses the association between the colorectal cancer and some of the most important dietary and lifestyle risk factors, as well as the role of gut microbiota and chemoprevention with acetylsalicylic acid in colorectal cancer.