Paulo Fidalgo

Whole-gene APC deletions cause classical familial adenomatous polyposis, but not attenuated polyposis or “multiple” colorectal adenomas

Familial adenomatous polyposis (FAP) is a dominantly inherited colorectal tumor predisposition that results from germ-line mutations in the APC gene (chromosome 5q21). FAP shows substantial phenotypic variability: classical polyposis patients develop more than 100 colorectal adenomas, whereas those with attenuated polyposis (AAPC) have fewer than 100 adenomas. A further group of individuals, so-called “multiple” adenoma patients, have a phenotype like AAPC, with 3–99 polyps throughout the colorectum, but mostly have no demonstrable germ-line APC mutation. Routine mutation detection techniques fail to detect a pathogenic APC germ-line mutation in approximately 30% of patients with classical polyposis and 90% of those with AAPC/multiple adenomas. We have developed a real-time quantitative multiplex PCR assay to detect APC exon 14 deletions. When this technique was applied to a set of 60 classical polyposis and 143 AAPC/multiple adenoma patients with no apparent APC germ-line mutation, deletions were found exclusively in individuals with classical polyposis (7 of 60, 12%). Fine-mapping of the region suggested that the majority (6 of 7) of these deletions encompassed the entire APC locus, confirming that haploinsufficiency can result in a classical polyposis phenotype. Screening for germ-line deletions in APC mutation-negative individuals with classical polyposis seems warranted.

BAT-26 identifies sporadic colorectal cancers with mutator phenotype: a correlative study with clinico-pathological features and mutations in mismatch repair genes

Microsatellite instability (MSI) is present in most colorectal cancers (CRC) associated with hereditary nonpolyposis colorectal cancer (HNPCC). MSI testing in so‐called sporadic forms of CRC may become a useful tool in identifying new HNPCC kindred. The aim of this study was to analyse the utility of BAT‐26 as a marker to identify CRCs with MSI and to investigate whether sporadic CRCs with MSI have a phenotypic expression similar to HNPCC cases. MSI was detected using two methods, an association of 7 poly(CA) repeats and a poly(A) repeat alone, BAT‐26, in a series of 62 patients with apparently sporadic forms of CRC. Germ‐line and somatic mutations in the hMSH2, hMLH1, and hMSH6 genes were analysed in patients with MSI+ tumours. Patients with MSI+ at poly(CA) loci and at BAT‐26 were younger (p = 0·024 and p = 0·002), had tumours more frequently right sided (p = 0·017 and p = 0·0001) and more often mucinous (p = 0·037 and p = 0·005, respectively) than patients with MSI negative tumours. Mutation analysis allowed the identification of two patients carrying germ‐line mutations in the hMLH1 gene (both were BAT‐26+) and two other patients who had somatic mutation in the hMSH2 and in hMLH1 genes. In conclusion, the detection of MSI using poly(CA) repeats or BAT‐26 alone allowed the identification of a subset of patients with clinico‐pathological characteristics similar to those associated to HNPCC. BAT‐26 has the advantage of being a simple and less expensive method that might be used as a screening procedure before mutation analysis. Copyright

Short chain fatty acids are effective in short-term treatment of chronic radiation proctitis

PURPOSE: Short chain fatty acids are the main energy source of coloncytes and their use may be impaired in chronic radiation proctitis. The aim of the present study was to evaluate the therapeutic effect of short chain fatty acid enemas in patients with chronic radiation proctitis. METHODS: A prospective, randomized, double-blind trial comparing short chain fatty acid enemas with placebo was conducted in 19 patients with chronic radiation proctitis. Short chain fatty acid enemas contained 60 mM sodium acetate, 30 mM sodium propionate, and 40 mM sodium butyrate. The treatment period lasted five weeks and patients were followed up for six months. RESULTS: On admission, both groups were similar regarding all parameters evaluated. After five weeks short chain fatty acid-treated patients showed a significant decrease in the number of days with rectal bleeding from the previous week (4.4±1.8 to 1.4±2.2;P=0.001) and an improvement of endoscopic score (4.8±1.4 to 2.2±1.2;P=0.001). Hemoglobin values were also significantly higher in short chain fatty acid-treated patients (13.1±0.9 g/dlvs. 10.7±2.1 g/dl;P=0.02). Mucosal DNA and protein concentrations decreased in both groups but significantly so only in placebotreated patients (P=0.05). Changes in histologic parameters were not significant in either group. Although short chain fatty acid-treated patients did not get worse in the next six months, placebo-treated ones gradually improved, and at the end of six months, differences between the two groups were no longer observed. CONCLUSIONS: Short chain fatty acid enemas can accelerate the process of healing in chronic radiation proctitis, but treatment has to be continuous if a complete and sustained clinical, endoscopic, and histologic response is to be obtained.

Colorectal cancers show distinct mutation spectra in members of the canonical WNT signaling pathway according to their anatomical location and type of genetic instability.

It is unclear whether the mutation spectra in WNT genes vary among distinct types of colorectal tumors. We have analyzed mutations in specific WNT genes in a cohort of 52 colorectal tumors and performed a meta‐analysis of previous studies. Notably, significant differences were found among the mutation spectra. We have previously shown that in familial adenomatous polyposis, APC somatic mutations are selected to provide the “just‐right” level of WNT signaling for tumor formation. Here, we found that APC mutations encompassing at least two β‐catenin down‐regulating motifs (20 a.a. repeats) are significantly more frequent in microsatellite unstable (MSI‐H) than in microsatellite stable (MSS) tumors where truncations retaining less than two repeats are more frequent (P = 0.0009). Moreover, in cases where both APC hits are detected, selection for mutations retaining a cumulative number of two 20 a.a. repeats became apparent in MSI‐H tumors (P = 0.001). This type of mutations were also more frequent in proximal versus distal colonic tumors, regardless of MSI status (P = 0.0008). Among MSI‐H tumors, CTNNB1 mutations were significantly more frequent in HNPCC than in sporadic lesions (28% versus 6%, P < 10‐6) and were preferentially detected in the proximal colon, independently of MSI status (P = 0.017). In conclusion, the observed spectra of WNT gene mutations in colorectal tumors are likely the result from selection of specific levels of β‐catenin signaling, optimal for tumor formation in the context of specific anatomical locations and forms of genetic instability. We suggest that this may underlie the preferential location of MMR deficient tumors in the proximal colon.

Missense and nonsense mutations in codon 659 of MLH1 cause aberrant splicing of messenger RNA in HNPCC kindreds.

Germline mutations that give rise to premature termination codons in mRNAs have frequently been associated with aberrant processing of the nascent transcripts. This can take the form either of nonsense‐mediated mRNA decay or of aberrant splicing of the pre‐mRNA. In a family affected by hereditary nonpolyposis colorectal cancer, a two‐nucleotide deletion in codon 659, which introduces a frameshift and a new stop codon in exon 17 of the DNA mismatch repair gene MLH1, has been reported to lead to skipping of the exon. We now report that this phenomenon occurs also when there are missense or nonsense mutations in this codon. Our results thus suggest that in aberrant splicing the nature of the mutation may be less important than its position within the exon. These findings are of importance to mutation interpretation, as they imply that aberrant splicing could be associated even with silent mutations that do not lead to amino acid substitutions. Genes Chromosomes Cancer 26:372–375, 1999.

High Prevalence of Human Papillomavirus in Squamous Cell Carcinoma and Matched Normal Esophageal Mucosa Assessment by Polymerase Chain Reaction

Background. Studies using DNA technology have reported the presence of human papillomavirus (HPV) DNA in esophageal carcinomas, suggesting that it could play a role in the pathogenesis of this tumor. In the present study, in addition to DNA from neoplasms, normal mucosa was screened for viral DNA, assuming that this would increase HPV detection substantially.

The muir-torre syndrome: a rare variant of hereditary nonpolyposis colorectal cancer associated with hmsh2 mutation

The Muir-Torre syndrome is a rare autosomal dominant disorder characterized by the association of visceral malignancies with typical skin lesions. This syndrome is now considered a subtype of the more common hereditary nonpolyposis colorectal cancer syndrome (HNPCC). This last condition has been ascribed to mutations in four mismatch repair genes, and similar mutations, mostly located at hMSH2 gene, are now being described in some Muir-Torre patients. We describe the case of a 64-yr-old woman with no family history of colorectal cancer, who developed two visceral malignancies belonging to the usual spectrum of hereditary nonpolyposis colorectal cancer (colon and stomach), beginning at age 41. She additionally developed several skin tumors, including multiple keratoacanthomas, thus fulfilling Muir-Torre diagnostic criteria. Because of her cutaneous phenotype, she was screened for DNA mismatch repair gene mutations by in vitro synthetized protein assay (IVSP) and a truncating mutation was identified at hMSH2. We further discuss the clinical significance of the Muir-Torre phenotype, the association of this syndrome with hMSH2 mutations and the important implications of genetic diagnosis for the patient and her offspring.

Validation and simplification of Bethesda guidelines for identifying apparently sporadic forms of colorectal carcinoma with microsatellite instability.

Colorectal tumors with microsatellite instability (MSI) that do not comply with previously defined clinical criteria may be found in recently diagnosed hereditary nonpolyposis colorectal carcinoma families. Until recently, the indications for MSI testing were not clearly established. The objective of the current study was to validate the recently published Bethesda guidelines for MSI testing in a series of patients with apparently sporadic forms of colorectal carcinoma (CRC).

Flow cytometric DNA ploidy and S-phase fraction correlate with histopathologic indicators of tumor behavior in colorectal carcinoma

BACKGROUND: The clinical behavior of colorectal carcinoma is highly variable without reliable predictive biomarkers. Previous reports have shown that flow cytometric DNA analysis may provide valuable prognostic information in these tumors. PURPOSE AND METHODS: This study evaluates the DNA ploidy and the S-phase fraction (SPF) on frozen samples obtained from 61 patients with colorectal carcinoma by using flow cytometry, and it correlates the data with histopathologic features known to affect disease prognosis. Tumors were classified using the World Health Organizations histologic criteria and were staged according the American Joint Committee on Cancers classification system. Grade of the neoplasm, vascular invasion, and perineural tumor spread were evaluated in every case. RESULTS: Fifty-nine percent of tumors were aneuploid and showed statistically significant higher S-phase values than diploid tumors (22.5vs.11.2 percent;P <0.00001). Mean SPF of the whole series was 17.9 (range, 4.2–44.2) percent. A statistically significant association was found between SPF values and histologic grade (P< 0.0016), nodal status (P<0.0007), distant metastasis(P <0.0001), tumor stage (P<0.0001), venous invasion (P< 0.0002), and lymphatic permeation (P< 0.01) but not with perineural growth and infiltration of the neoplasm through the bowel wall (T). DNA ploidy correlated positively with tumor stage (P<0.03), and the association between aneuploidy and advanced stages of the disease was statistically significant. CONCLUSIONS: These findings showed that flow cytometric DNA ploidy and SPF, evaluated in fresh samples, are potentially useful parameters to estimate colorectal carcinoma biopathology. Aneuploidy and high replicative neoplastic activity correlated with histopathologic features that are commonly associated with the prognosis of colorectal carcinoma, being SPF-related to disease dissemination and, therefore, an indicator of clinical relevance.

Calcium regulation of colonic crypt cell kinetics: Evidence for a direct effect in mice

BACKGROUND & AIMS Oral calcium supplementation is believed to decrease colonic hyperproliferation through neutralization of fatty acids and bile acids. In the present study, the effect of oral calcium, given with low-fat diets, in the early stages of colorectal carcinogenesis is evaluated. METHODS In experiment A, mice received normal or low-calcium diets and were killed at 25 weeks. In experiment B, mice were fed the same diets but were submitted to six weekly injections of dimethylhydrazine and were killed at 10, 16, and 21 weeks. Cell proliferation was evaluated using bromodeoxyuridine immunohistochemistry. RESULTS In experiment A, mice fed low-calcium diets showed a significant upward shift of the proliferative compartment (P = 0.04) (phase 2 defect) in the absence of hyperproliferation. In experiment B, besides a phase 2 defect, dimethylhydrazine-induced hyperproliferation was also significantly enhanced in animals fed low-calcium diets (phase 1 defect) as shown by an increased number of labeled cells per column and total labeling index (P = 0.01). CONCLUSIONS Low-calcium diets induce an upward shift of the main proliferative compartment, which reflects an increased risk for malignant transformation. This effect was observed with a low-fat diet, suggesting a direct mechanism, rather than the usual indirect one, documented with high-fat diets.