Immunohistochemical analyses of colon cancer in I1307K APC mutation carriers compared with noncarriers

The I1307K APC germline mutation is associated with an increased risk to colorectal cancer (CRC). Whether and to what extent the somatic features and the molecular pathways of cancer development in mutation carriers differ from colorectal cancer in noncarriers remains unknown. To gain insight into this issue, 52 Israeli patients with CRC, 24 of whom were I1307K APC mutation carriers, were analyzed. The expression pattern of genes known to be involved in the pathogenesis of sporadic CRC was assessed immunohistochemically: E-cadherin, ß-catenin, deleted in colon cancer (DCC), and p53. In addition, tumors were genotyped for somatic activating mutations in Ki-rasoncogene. Mutation carriers and noncarriers were comparable in age at diagnosis (64.3 ± 10.1 years for carriers and 60.8 ± 14.1 years for noncarriers), tumor location in the colon, and disease stages. Tumors of I1307K mutation carriers displayed positive p53 immunostaining and loss of ß-catenin, E-cadherin, and DCC expression more often compared with noncarriers, although none of these differences reached statistical significance. Mutation frequencies in the Ki-ras gene were similar in both groups. In conclusion, the molecular pathways in CRC in I1307K APC mutation carriers are seemingly similar to those of sporadic cases, but a larger study is clearly needed.     

The AAPC case,with an early onset of colorectal cancer

Introduction Attenuated adenomatous polyposis coli (AAPC) is a variant of the familial adenomatous polyposis (FAP) characterized by the occurrence of sparse polyps in the colon, stomach, and duodenum with a late onset of colorectal cancer. The AAPC syndrome is associated with mutations at the 5′ region of the APC gene. Until recently, the fragment encompassing codons 157 and 170 was considered as boundary for the described cases of AAPC and FAP syndromes. Materials and methods This study describes a case of the AAPC syndrome caused by a CCTT deletion at codon 173, with polyps diagnosed at the age of 17. The father and grandfather of the proband died of colorectal cancer (CRC), which developed from untreated polyps, at the age 35 and 40, respectively. Results and discussions In the case of the proband’s father, the untreated polyps led to death after 12 years. The proband revealed a low number of polyps and an extra colon feature characteristic of AAPC, but the polyps onset and the death of CRC of two family members, who refused colectomy, was very early and characteristic for FAP. An atypical course of AAPC must be taken into consideration both in genetic counseling and in qualifying the patients with AAPC for the surgical treatment.     

APC promoter methylation and protein expression in hepatocellular carcinoma

Purpose We investigated the impact of promoter methylation on APC protein expression in patients with hepatocellular carcinoma (HCC). Materials and methods 50 patients [HCC (n=19), liver metastasis (n=19), cholangiocellular cancer (n=7), and benign liver tumors (n=5)] were studied for methylation using Methylight analysis. APC mutation was investigated by protein truncation test and direct sequencing of genomic DNA. The protein expression was evaluated by immunohistochemistry and Western blot analysis. Results The APC promoter was hypermethylated in 81.8% of non-cancerous liver tissue samples. All HCC samples and ten patients with liver metastasis (52.6%) exhibited APC promoter methylation. The degree of methylation was significantly higher in samples from HCC compared to the non-cancerous liver tissue samples (63.1% vs. 24.98%; p=0.001). The level of APC protein expression was significantly reduced in HCC samples compared to that of the corresponding non-tumor liver tissue (p<0.05). Conclusions Promoter methylation of the APC gene seems to be of significance in hepatocarcinogenesis and results in reduced protein expression in HCC. Interestingly, APC promoter methylation is also present in the vast majority of non-cancerous liver tissue whose (patho)physiological function remains unresolved.     

Polymorphisms in methylenetetrahydrofolate reductase gene (MTHFR) and the age of onset of sporadic colorectal adenocarcinoma

Background and aims Evidence is accumulating for a role of folate in the aetiology of colorectal cancer (CRC). The methylenetetrahydrofolate reductase (MTHFR) gene, involved in folate metabolism, is polymorphic in humans. Since it is unknown whether the MTHFR C677T and A1298C polymorphisms alter the risk for CRC, this was the aim of our study. Materials and methods Genomic DNA from 102 sporadic colorectal adenocarcinoma (SCA) patients and 300 controls was analyzed by polymerase chain reaction followed by restriction digestion for the polymorphisms analyses. Results/findings The frequencies of MTHFR C677T and A1298C genotypes were similar in patients and controls. Similar overall risks for disease were seen in individuals with the distinct MTHFR genotypes. However, an excess of the MTHFR 677TT and 677CT genotypes was seen in patients under 50 years, compared with patients at an older age (19.2 vs 13.1% and 61.6 vs 39.5%, respectively; P = 0.04). The differences were more prominent when the frequency of the 677TT plus 677CT genotype was seen in both group of patients (80.8 vs 52.6%, respectively; P = 0.01), and in younger patients compared to controls (80.8 vs 52.3%, P < 0.01). Individuals with the combined genotype had 3.82-fold (95% confidence interval, 1.41–10.42) increased risk of developing SCA under 50 years, compared with those harboring the wild-type genotype. Interpretation/conclusion These results suggest a role for the MTHFR 677TT plus 677CT genotype in increasing SCA diagnosed at a low age in southeastern Brazil, but additional studies with larger sample sizes should be carried out to clarify this issue.     

Cholecystectomy and the Risk of Colorectal Adenomas

Cholecystectomy has been identified as a risk factor for colorectal cancer, yet little attention has been given to the relationship between cholecystectomy and colorectal adenomas. Utilizing data collected in two large cross-sectional studies of colorectal adenoma risk factors, we examined the association between cholecystectomy and colorectal adenomas. In the adjusted logistic regression model, both men and women showed no effect of cholecystectomy on risk of colorectal adenomas (men: OR 0.67 [95% CI 0.30–1.47]; women: OR 1.46 [95% CI 0.92–2.29]). No effect was seen when examining the time since cholecystectomy for men. There was a slight association found for women who had a cholecystectomy less than 10 years prior (OR 2.02 [95% CI 1.06–3.87]) but no association was seen in women with cholecystectomy at least 10 years prior (OR 1.14 [95% CI 0.62–2.09]). Thus, we conclude that, although cholecystectomy is a risk factor for colorectal cancer, cholecystectomy is not a risk factor for colorectal adenomas.     

Low Allele Frequency of MLH1 D132H in American Colorectal and Endometrial Cancer Patients

PURPOSE Hereditary nonpolyposis colon cancer is caused by mutations in DNA mismatch repair genes, predominantly MLH1 and MSH2. Classic MLH1 mutations cause an approximately 20-fold increase in colorectal cancer susceptibility. Recently, we identified a hypomorphic allele, MLH1 D132H , which impairs, but does not completely eliminate the function of MLH1 in tumor suppression. MLH1 D132H confers an approximately fivefold increase in colorectal cancer susceptibility and was first described in a cohort of Israeli colorectal cancer patients, with an estimated allele frequency of 1.3 percent. Because MLH1 D132H has only recently been described, the ethnic distribution of this risk allele is not well understood. This study was undertaken to determine both the frequencies of this risk allele in ethnic groups outside of Israel and whether families harboring this mutation have susceptibility to extracolonic cancers in the hereditary nonpolyposis colon cancer spectrum.METHODS We genotyped two independent cohorts: 629 population-based colorectal cancer patients ascertained from clinics in Orange, Imperial, and San Diego Counties, and 515 endometrial cancer patients ascertained from gynecologic oncology clinics in the Midwestern United States.RESULTS MLH1 D132H was not detected in either study cohort, which together totaled more than 1,100 American colorectal cancer and endometrial cancer patients.CONCLUSIONS The MLH1 D132H risk variant has significantly lower allele frequency in American compared with Israeli cancer patients and, alone, is unlikely to explain significant amounts of American sporadic colorectal cancer or uterine cancer susceptibility. Genetic testing for the MLH1 D132H allele exclusively is therefore unlikely to be cost effective for genetic risk assessment in American population-based and clinic-based colorectal cancer and endometrial cancer patients.Supported by grants from the American Cancer Society, Atlanta, Georgia (RSG-02-153-01), and NCI, Bethesda, Maryland (CA81488, CA098626, CA71754, and CA115231).     

Mutations of the APC,beta-catenin,and axin 1 genes and cytoplasmic accumulation of beta-catenin in oral squamous cell carcinoma

Purpose: The Wnt pathway is involved in carcinogenesis and three regulatory genes of the Wnt pathway, APC, beta-catenin and Axin are mutated in some primary human cancers. Mutations in these genes can impair the down regulation of beta-catenin, which results in the stabilization of beta-catenin, accumulation of free beta-catenin and subsequent activation of the Wnt pathway. To clarify the genetic alterations of components of the Wnt pathway in oral squamous cell carcinoma (SCC), we examined mutations in the APC, beta-catenin and Axin genes and subcellular localization of beta-catenin. Methods: 20 oral SCC tissues and four cell lines derived from oral SCC were used. Mutational analysis was performed by a single-strand conformation polymorphism (SSCP) method and direct sequecing analysis. The samples were also examined by immunohistochemical staining and immunoblot analysis. Results: In 3 of 4 cell lines, mutations were observed in the APC and Axin1 genes without amino acid substitutions. In a clinical sample, a mutation in the Axin1 gene was detected; a T insertion at codon 250 resulted in the formation of a stop codon at codon 259. In addition, cytoplasmic accumulation of beta-catenin was observed in 3 (75%) of 4 cell lines and 18 (90%) of 20 cancer tissue samples. Conclusion: The Axin1 gene may be one of the mutational target in oral SCC. In addition, the cytoplasmic accumulation of beta-catenin is a common characteristic of oral SCC, but is not closely associated with mutational alterations in the APC, beta-catenin and Axin1 genes.     

Colorectal cancer screening: Results of a 5-year program in asymptomatic subjects at increased risk

BACKGROUND AND AIMS: The province of Ferrara has one of the highest incidences of colorectal cancer (CRC) in Italy. In January 2000, we set up a colonoscopy screening program focussing on first-degree relatives of CRC patients. We now report the results 5 years after the beginning of the project. SCREENEES AND METHODS: In October 1999, we started a campaign stressing the usefulness of colonoscopy for the first-degree relatives of CRC patients. Subjects included in the screening program were aged between 45 and 75 years with at least one first-degree relative affected by CRC. They were invited to an interview where a physician suggested colonoscopy as a screening option. RESULTS: In 5 years, 776 subjects were interviewed and 733 (94.4%) agreed to an endoscopic examination (M/F:375/401; mean age 55 years): 562 colonoscopies were performed. Adenomas and cancers were found in 122 (21.7%) and 12 (2.1%) subjects, respectively. Histological examination in 181 persons with lesions (32.8%) showed (most serious lesion quoted) 47 hyperplastic polyps (26% of all lesions), 2 serrated adenomas (1.1%), 68 tubular adenomas (48%), 24 tubulovillous adenomas (13.3%), 9 adenomas with high grade dysplasia (5%) and 12 adenocarcinomas (6.6%). The majority of the cancers were at an early stage (8 Dukes A and 3 Dukes B). Sedation was used in only 42 colonoscopies (7.5%). CONCLUSIONS: A colonoscopy-based screening in this selected high-risk population is feasible. Even without sedation subjects readily agreed to the endoscopic procedure. We identified a significant number of advanced neoplasms and cancers at an early stage suggesting that this could be a useful tool in early identification of CRC.     

Elevated HbA1c Is an Independent Predictor of Aggressive Clinical Behavior in Patients with Colorectal Cancer: A Case-Control Study

AIM: The aim of this study was to seek an association between the control of type 2 diabetes mellitus (T2DM), as determined by hemoglobin A1c (HbA1c) levels, and the outcome of colorectal cancer (CRC). METHODS: We performed a retrospective review of patients with T2DM who had CRC diagnosed between 1997 and 2001. We defined well-controlled T2DM as HbA1c < 7.5% and poorly controlled T2DM as HbA1c > or = 7.5%. A group of age- and gender-matched patients who had CRC without T2DM were used as controls. Forty clinical factors were reviewed, and those associated with poor clinical outcome in each group were examined by univariate analysis (UA) and by the maximum likelihood analysis of logistic regression to determine the independent predictors of cancer outcome. RESULTS: We identified 155 patients with T2DM and CRC, and 114 control patients who had CRC without T2DM. We found no significant differences in any clinical factor by UA between the patients with well-controlled T2DM and the patients who had CRC without T2DM. Compared to both of those patients groups, in contrast, the patients with poorly controlled T2DM had more right-sided CRCs (P = 0.04, OR = 2, 95% CI = 1-4.1), more advanced CRCs (P = 0.02, OR = 2.1, 95% CI = 1-4.4), a younger age of presentation (P = 0.05), greater use of exogenous insulin (P = 0.002), and a poorer 5-year survival (P = 0.001) by UA. Logistic regression showed that poorly controlled T2DM independently predicted the early onset of CRC, a more advanced stage at the time of presentation, poorer 5-year survival, and an increased incidence of right-sided CRCs. CONCLUSIONS: In patients with T2DM who have CRC, poor glycemic control is associated with a clinically aggressive course for the cancer.     

Mutations of the APC Gene in Human Sporadic Colorectal Cancers

Background: Mutations of the APC gene are reported to occur frequently in sporadic colorectal adenomas and adenocarcinomas. We studied APC gene mutations in cases of human sporadic colorectal cancer in order to evaluate their correlation with pathologic characteristics and clinical prognosis. Methods: Most of the mutations of the APC gene (95%) are nonsense or frame shift mutations, encoding for truncated APC proteins. For this reason, mutation detection of the APC gene was performed using the in vitro synthesized protein (IVSP) assay, analysing the region between nucleotide 2058 and nucleotide 5079 of the gene, containing the mutation cluster region. Results: Out of 58 cases of colorectal cancer, 29 presented a mutated form of APC (mutation frequency 50%). We did not find a statistically significant correlation between APC gene mutation and age, sex, localization of the primary tumour, grading, Crohn-like lymphoid reaction or presence of residual adenoma. Tumours with low invasivity (Dukes' stages A and B) were less frequently mutated (12/27, 44.5%) than tumours of Dukes' stage C (15 out of 21, 71.4%), which developed macroscopically secondary metastasis with variable latency after surgery. Highly invasive tumours with synchronous metastases (Dukes' stage D) had, instead, a low frequency of APC mutations (20%, 2/10) ( P = 0.02, compared with Dukes' stages A, B and C). Conclusions: These data suggest that more aggressive Dukes' stage D tumours develop metastasis by means of an unknown mechanism, independent of APC mutation.     

粪便中p53与APC突变检测在大肠癌诊断中的意义

目的探讨在大肠癌患者粪便中检测p53、APC基因突变的可行性及其应用前景和意义。方法从36例大肠癌患者、10例大肠腺瘤患者以及30例正常对照者的粪便中分别提取DNA,应用PCR-SSCP法检测粪便中p53、APC基因突变情况。结果36例大肠癌患者粪便中p53及/或APC基因突变检出率为77.78%(19/36),二者突变率分别为52.78%(19/36)和36.11%(13/36);10例大肠腺瘤中p53基因突变检出率为0%,APC为20%;30例正常对照粪便中p53、APC基因突变检出率均为0%。p53的突变随大肠癌分化程度的降低而增高(P<0.05);APC基因突变与大肠癌组织学类型无关(P>0.05)。结论联合检测粪便中p53与APC突变在大肠癌诊断和筛查中有潜在的应用价值。     

Prospective Study Comparing Multislice CT Colonography With Colonoscopy in the Detection of Colorectal Cancer and Polyps

PURPOSE: Multislice CT colonography is an alternative to colonoscopy. The purpose of this study was to compare multislice CT colonography with colonoscopy in the detection of colorectal polyps and cancers. METHODS: Between June 2000 and December 2001, 45 males and 35 females (median age, 68 (29–83) years) with symptoms of colorectal disease were studied prospectively. All patients underwent multislice CT colonography and colonoscopy, and the findings were compared. RESULTS: Colonoscopy was incomplete in 18 (22 percent) patients because of obstructing lesions or technical difficulty, and multislice CT colonography was unsuccessful in 4 (5 percent) because of fecal residue. Colonoscopy was normal in 26 patients and detected 29 colorectal cancers and 33 polyps in 35 patients, diverticulosis in 16 patients, and colitis in 3 patients. Multislice CT colonography identified 28 of 29 colorectal cancers with one false negative and one false positive (sensitivity, 97 percent; specificity, 98 percent; positive predictive value, 96 percent; negative predictive value, 98 percent). Multislice CT colonography identified all 12 polyps measuring 10 mm in diameter (sensitivity, 100 percent), 5 of 6 measuring 6 to 9 mm in diameter (sensitivity, 83 percent), 8 of 15 polyps 5 mm (sensitivity, 53 percent), and false-positive for 8 polyps. The overall sensitivity was 74 percent and specificity 96 percent. The positive predictive value for polyps was 88 percent, and the negative predictive value was 90 percent. Multislice CT colonography also detected 5 of 16 patients with diverticulosis (sensitivity, 31 percent; specificity, 98 percent) and colitis in 2 of 3 patients (sensitivity, 67 percent; specificity, 100 percent). In ten (13 percent) patients, extracolonic findings on multislice CT colonography altered management and included five patients with colorectal liver metastases. In 15 (19 percent) patients, there were incidental findings that did not demand further investigation. CONCLUSIONS: The results from this study indicate that the efficacy of multislice CT colonography in the detection of colorectal cancers and polyps 6 mm is similar to colonoscopy. Multislice CT colonography allows clinical staging of colorectal cancers, outlines the whole length of the colon in obstructing carcinoma when colonoscopy fails, and can identify extracolonic causes of abdominal symptoms.     

Stem Cell Factor/c-<Emphasis Type="Italic">kit</Emphasis> Receptor Signaling Enhances the Proliferation and Invasion of Colorectal Cancer Cells Through the PI3K/Akt Pathway

In this study, we examined the role of c-kit receptor (KIT) signal transduction on the proliferation and invasion of colorectal cancer cells. We found that c-kit was expressed in 2 colorectal cancer cell lines as determined by RT-PCR, Western blot, and flow cytometry. In KIT-positive lines, KIT was activated by stem cell factor (SCF). SCF enhanced cellular proliferation of positive lines as demonstrated by the WST-1 proliferation assay. Furthermore, SCF enhanced the invasive ability of KIT-positive cell lines. SCF stimulation upregulated p44/42 mitogen-activated protein kinase (MAPK) and Akt as shown by Western blot. We examined the roles played by p44/42 MAPK and phosphatidylinositol 3-kinase (PI3K)/Akt pathways in proliferation and invasion. PI3K/Akt activity strongly correlated with proliferation and invasion and p44/42 MAPK was correlated with only invasion. In conclusion, the SCF-enhanced proliferation and invasion of KIT-positive colorectal cancer cells is achieved mainly through the PI3K/Akt pathway.     

Aberrant Methylation of HLTF, SOCS-1, and CDH13 Genes Is Shown in Colorectal Cancers Without Lymph Node Metastasis

PURPOSE and METHODS In this study, we examined the combined methylation status of HLTF, SOCS-1, and CDH13 in 61 resected primary colorectal cancers using methylation-specific polymerase chain reaction and correlated the number of methylated genes with the clinicopathologic features of affected patients.RESULTS We found a significant difference in lymph node metastasis (P = 0.020) when we compared the number of methylated genes in colorectal cancers with lymph node metastasis to those without it.CONCLUSIONS Colorectal cancers without lymph node metastasis frequently exhibited the aberrant methylation of HLTF , SOCS-1 , and CDH13 genes.     

Colorectal Adenomatous Polyposis Associated with <Emphasis Type="Italic">MYH</Emphasis> Mutations: Genotype and Phenotype Characteristics

Purpose Recent literature reports that several digestive diseases are associated with mutations in the base excision repair gene MYH. This study was designed to establish the prevalence of germ-line MYH mutations in a series of 56 consecutive patients with no detectable APC mutation and describe the phenotype of those with MYH mutations. Methods MYH mutations were screened by DNA sequencing after polymerase chain reaction amplification of each exon. Clinical, endoscopic, and surgical data were collected for the tested patients. Results MYH mutations were identified only in the group of patients with attenuated adenomatous polyposis with ten or more adenomatous polyps. The prevalence of MYH mutations was 34.4 percent (11 cases) in this subgroup of 30 patients. There were two homozygotes and eight compound heterozygotes. Only one patient had a monoallelic mutation. At least one of two mutational hot spots was identified in ten patients. Three patients presented with a family history of adenomatous polyposis in siblings, without vertical transmission. The median number of colorectal adenomatous polyps was 53 without preferential localization. Colorectal cancer was associated with polyposis in seven patients. Gastric and duodenal adenomas were diagnosed in one case. Ten of 11 patients underwent colectomy. Conclusions MYH mutations have been observed in one-third of patients with attenuated polyposis. The phenotype of the disease is similar to attenuated familial adenomatous polyposis. Upper gastrointestinal endoscopy also should be recommended. However, its transmission shows evidence of a recessive pattern. Presented at Digestive Disease Week, Los Angeles, California, May 20 to 25, 2006, at the Journées Francophones de Pathologie Digestive, Paris, France, March 19 to 22, 2006, and at the United European Gastroenterology Week, Berlin, Germany, October 21 to 25, 2006. Reprints are not available.     

Ex Vivo Sentinel Lymph Node Mapping in Colorectal Cancer

INTRODUCTION Sentinel lymph node mapping has been used in clinical work in malignant melanoma and breast cancer and shown an advantage over routine regional lymphadenectomy. The technique has been applied to colorectal cancer, but concerns over accuracy and high false-negative rates have restricted its use in the routine clinical setting. Most published series have used the in vivo technique and only three studies have been published in which the ex vivo technique was used. The aim of this study was to report the results of a larger study of ex vivo sentinel node mapping.METHODS All patients with colorectal cancer were considered for the trial, except patients who received preoperative radiotherapy for rectal cancer. All specimens were examined in the operating room within 30 minutes of resection. After opening the bowel, 0.5 ml of patent blue dye was injected submucosally at four sites immediately adjacent to the tumor (2 ml). The pathologic examination of the sentinel nodes and of an equal number of nonsentinel nodes consisted of standard hematoxylin and eosin sectioning, followed by multiple sectioning for further hematoxylin and eosin staining and immunohistochemistry if initial samples did not show tumor metastases.RESULTS A total of 58 tumors in 57 patients were studied. One or more sentinel nodes were found in relation to 56 tumors, with one of the two failures being attributed to gross mesenteric metastases obstructing lymphatic flow. A mean of 2.93 (0–8) sentinel nodes were found per patient. There was concordance between the sentinel nodes and nonsentinel nodes in 43 patients (76.8 percent). There were nine false-negative sentinel nodes (16 percent). Two patients were upstaged by detailed pathologic examination of the sentinel nodes (micrometastases), and in a further two patients the sentinel node was the only positive node on simple hematoxylin and eosin sectioning.CONCLUSIONS The technique of ex vivo sentinel node mapping is feasible and accurate in defining sentinel nodes in colorectal cancer. There is, however, a significant false-negative rate making the sentinel nodes not representative of the lymph node basin. This precludes the use of this technique in routine clinical practice. There may be a role in a research setting to help define the prognostic significance of micrometastases.Supported by grants from AP-HP, Paris, France, Contrat de Recherche Clinique 01018.Read at the meeting of the American Society of Colon and Rectal Surgeons, Dallas, Texas, May 8 to 13, 2004.Reprints are not available.     

Hypermethylation of <Emphasis Type="Italic">SFRP2</Emphasis> as a Potential Marker for Stool-Based Detection of Colorectal Cancer and Precancerous Lesions

DNA methylation is a key mechanism of colorectal carcinogenesis. Analysis of aberrantly methylation in stool DNA might provide a novel strategy for noninvasive detection of colorectal cancer (CRC). To explore the feasibility of this approach, we have assessed the methylation status of secreted frizzled-related protein gene 2 (SFRP2) in stool samples from patients with CRC with respect to a series of healthy individuals and patients with benign colorectal diseases, using methylation-specific polymerase chain reaction. Methylated SFRP2 occurs in 94.2%, 52.4%, 37.5%, and 16.7% of patients with CRC, adenomas, hyperplstic polyps, and ulcerative colitis, respectively. Of the 24 normal individuals, only 1 revealed methylated DNA. The pilot study revealed that aberrant methylated SFRP2 could be detected frequently in stools from patients with CRC and precancerous lesions. Methylation testing of fecal DNA may be a simple, promising, and noninvasive screening tool for colorectal neoplasia.