Luca Roncucci

Identification and quantification of aberrant crypt foci and microadenomas in the human colon

The objective of the present study was to determine whether aberrant crypt foci (ACF) similar to those observed in the colons of experimental animals exposed to colon carcinogens could be identified and quantified in the human colon. Twenty-seven colon resections from patients affected by familial adenomatous polyposis (FAP, five cases), colorectal cancer (CRC, 12 cases), and benign diseases of the large bowel (BD, 10 cases) were collected from a pathology repository or immediately after operation. Ten or more 1-cm2 formalin-fixed, methylene-blue--stained samples of colonic mucosa from each colon were scored under light microscopy for ACF. The number of ACF per cm2 and the number of crypts per ACF for each colon were calculated. The average number of ACF per cm2 in the FAP group (20 +/- 19, mean +/- SD) was significantly higher (P less than 0.01) than those of the CRC (0.37 +/- 0.41) and BD (0.18 +/- 0.35) groups. At least one ACF was found in every colon resection from CRC patients and in six out of 10 colon resections from the BD group. The average number of crypts per ACF ranged from five to 35 with absolute values from 1 to over 100. Fifty-five histologic specimens, 43 with ACF of various size and 12 without, were prepared by sectioning the colon parallel to the mucosal surface. There was a close correlation between the number of crypts per ACF in each specimen as scored by methylene-blue and histologic examination. Twenty-six aberrant crypt foci displayed dysplasia as evident by histologic analysis. In these instances we feel the term microadenoma is appropriate and, using this unique approach of examining the human colon, they can be easily identified and quantified. These lesions may well be precursors for adenomatous polyps and colorectal cancer.

Microsatellite Instability and Colorectal Cancer Prognosis

Purpose: Many studies have evaluated the role of high levels of microsatellite instability (MSI) as a prognostic marker and predictor of the response to chemotherapy in colorectal cancer (CRC); however, the results are not conclusive. The aim of this study was to analyze the prognostic significance of high levels of MSI (MSI-H) in CRC patients in relation to fluorouracil-based chemotherapy. Experimental Design: In three different institutions, 1,263 patients with CRC were tested for the presence of MSI, and CRC-specific survival was then analyzed in relation to MSI status, chemotherapy, and other clinical and pathologic variables. Results: Two hundred and fifty-six tumors were MSI-H (20.3%): these were more frequently at a less advanced stage, right-sided, poorly differentiated, with mucinous phenotype, and expansive growth pattern than microsatellite stable carcinomas. Univariate and multivariate analyses of 5-year–specific survival revealed stage, tumor location, grade of differentiation, MSI, gender, and age as significant prognostic factors. The prognostic advantage of MSI tumors was particularly evident in stages II and III in which chemotherapy did not significantly affect the survival of MSI-H patients. Finally, we analyzed survival in MSI-H patients in relation to the presence of mismatch repair gene mutations. MSI-H patients with hereditary non–polyposis colorectal cancer showed a better prognosis as compared with sporadic MSI-H; however, in multivariate analysis, this difference disappeared. Conclusions: The type of genomic instability could influence the prognosis of CRC, in particular in stages II and III. Fluorouracil-based chemotherapy does not seem to improve survival among MSI-H patients. The survival benefit for patients with hereditary non–polyposis colorectal cancer is mainly determined by younger age and less advanced stage as compared with sporadic MSI-H counterpart.

Antioxidant vitamins or lactulose for the prevention of the recurrence of colorectal adenomas

Colonic adenomas represent the natural precursor lesions of most colorectal cancers. The treatment of choice is endoscopic polypectomy. However, after endoscopic removal, polyps recur in a large fraction of cases. Thus, we evaluated the effect of antioxidant vitamins or lactulose on the recurrence rate of adenomatous polyps. After polypectomy, 255 individuals were randomized into three groups. Group 1 was given vitamin A (30,000 IU/ day), vitamin C (1 g/day), and vitamin E (70 mg/day); Group 2 was given lactulose (20 g/day); Group 3 received no treatment. Forty-six subjects had to be excluded because the histologic diagnosis was not consistent with adenoma. The remaining 209 individuals were included in the analysis according to the “intention to treat” criterion, though 34 did not adhere to the scheduled treatment or were lost during the follow-up. Subjects were followed at regular intervals for an average of 18 months. Polyps recurring before one year from index colonoscopy were considered missed by the endoscopist. In the 209 evaluable subjects, the percentages of recurrence of adenomas were 5.7 percent, 14.7 percent, and 35.9 percent in the vitamins, lactulose, and untreated groups, respectively. The fraction of subjects remaining free of adenomas, estimated by Kaplan-Meier survival curves, was significantly different among the three groups (log-rank chisquared =17.138;P< 0.001). Using Coxs regression analysis, treatment was the only variable that significantly contributed to the model (regression coefficient=0.905;P<0.001). In conclusion, either antioxidant vitamins or, to a lesser extent, lactulose lower the recurrence rate of adenomas of the large bowel and can be proposed as chemopreventive agents, at least in high-risk individuals.

Identification of Muir-Torre Syndrome among Patients with Sebaceous Tumors and Keratoacanthomas Role of Clinical Features, Microsatellite Instability, and Immunohistochemistry

The Muir–Torre syndrome (MTS) is an autosomal‐dominant genodermatosis characterized by the presence of sebaceous gland tumors, with or without keratoacanthomas, associated with visceral malignancies. A subset of patients with MTS is considered a variant of the hereditary nonpolyposis colorectal carcinoma, which is caused by mutations in mismatch‐repair genes. The objective of the current study was to evaluate whether a combined clinical, immunohistochemical, and biomolecular approach could be useful for the identification of Muir–Torre syndrome among patients with a diagnosis of sebaceous tumors and keratoacanthomas.

Molecular Screening for Hereditary Nonpolyposis Colorectal Cancer: A Prospective, Population-Based Study

PURPOSE Germline mutations in mismatch repair genes predispose to hereditary nonpolyposis colorectal cancer (HNPCC). To address effective screening programs, the true incidence of the disease must be known. Previous clinical investigations reported estimates ranging between 0.5% and 13% of all the colorectal cancer (CRC) cases, whereas biomolecular studies in Finland found an incidence of 2% to 2.7% of mutation carriers for the disease. The aim of the present report is to establish the frequency of the disease in a high-incidence area for colon cancer. PATIENTS AND METHODS Through the data of the local CRC registry, we prospectively collected all cases of CRC from January 1, 1996, through December 31, 1997 (N = 391). Three hundred thirty-six CRC cases (85.9% of the incident cases) were screened for microsatellite instability (MSI) with six to 12 mono- and dinucleotide markers. MSI cases were subjected to MSH2 and MLH1 germline mutation analysis and immunohistochemistry; the methylation of the promoter region was studied for MLH1. RESULTS Twenty-eight cases (8.3% of the total) showed MSI. MSI cases differed significantly from microsatellite-stable (MSS) cases for their proximal location (P <.01), high mucinous component (P <.01), and poor differentiation (P =.002). Of MSI cases studied (n = 12), only one with a family history compatible with HNPCC had a germline mutation (in MSH2). Five other patients with a family history of HNPCC (two with MSI and three with MSS tumors) did not show germline mutations. CONCLUSION We conclude that the incidence of molecularly confirmed HNPCC (one [0.3%] of 336) in a high-incidence area for CRC is lower than in previous biomolecular and clinical estimates.

Aberrant crypt foci in colorectal carcinogenesis. Cell and crypt dynamics

Aberrant crypt foci (ACF) have been identified on the colonic mucosal surface of rodents treated with colon carcinogens and of humans after methylene‐blue staining and observation under a light microscope. Several lines of evidence strongly suggest that ACF with certain morphological, histological, cell kinetics, and genetic features are precursor lesions of colon cancer both in rodents and in humans. Thus, ACF represent the earliest step in colorectal carcinogenesis. This paper has the main purpose of reviewing the evidence supporting this view, with particular emphasis on cell and crypt dynamics in ACF. ACF have been used as intermediate biomarkers of cancer development in animal studies aimed at the identification of colon carcinogens and chemopreventive agents. Recently, evidence has also shown that ACF can be effectively employed in chemopreventive studies also in humans.

Identification of hereditary nonpolyposis colorectal cancer in the general population : the 6-year experience of a population-based registry

Background. Hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome) is an autosomal dominant disease characterized by early‐onset intestinal neoplasms, localization of tumors in the proximal colon, and frequent association with cancers at other sites, especially the endometrium, skin, and stomach. The identification of HNPCC is often difficult, owing to the lack of biomarkers and the extreme frequency of sporadic colorectal cancer in the Western World.

Microsatellite instability in multiple colorectal tumors.

Tumor multiplicity is a hallmark of hereditary cancers: in the colon‐rectum multiple tumors represent 5–10% of all colorectal cancer cases. A portion of these cases belongs to hereditary non‐polyposis colorectal cancer (HNPCC), a genetic cancer syndrome due to mismatch repair (MMR) gene mutations, phenotypically expressed as microsatellite instability (MSI); the majority of multiple tumors, however, is apparently without any family history. We analyzed 78 (38 synchronous and 40 metachronous) neoplasms from 37 patients with multiple tumors of the large bowel, both HNPCC and sporadic, with the aim of identifying a common genetic basis in multiple tumors. DNA was extracted from normal and cancerous formalin‐fixed tissue and was analyzed for MSI using 6 markers. Tumors showing MSI in at least 2 of 6 microsatellite loci were defined as MSI(+). The overall number of MSI(+) tumors was 22 (28.2% of the total). A significant difference in the rate of MSI(+) between HNPCC and sporadic tumors was observed (85% vs. 17%). In the same patients, the MSI phenotype of synchronous tumors (both HNPCC and sporadic) tended to be more concordant than that of the metachronous ones. The higher frequency of MSI in HNPCC than in sporadic tumors, even when multiple, suggests that the involvement of MMR genes in the pathogenesis of the sporadic cases may be uncommon, thus confirming that screening for MSI in multiple colorectal tumors could be a useful tool in the identification of HNPCC in the general population. Int. J. Cancer 81:1–5, 1999.

Characterization of MSH2 and MLH1 mutations in Italian families with hereditary nonpolyposis colorectal cancer

Mismatch repair genes MSH2 and MLH1 are considered to be the two major genes that are responsible for hereditary nonpolyposis colorectal cancer (HNPCC). Germline heterozygous inactivating mutations of MSH2 and MLH1 have been identified previously in a substantial fraction of individuals who are predisposed genetically to colorectal carcinoma (CRC) and other tumors of the HNPCC spectrum. With the aim of determining the relevance of these two genes in the Italian population, we submitted to mutational analysis a set of 17 HNPCC families, all of which fulfilled the “Amsterdam criteria.” A combination of different techniques, including reverse transcription‐polymerase chain reaction (RT‐PCR) of long fragments and single‐strand conformation polymorphism (SSCP) on cDNA and genomic DNA, allowed the identification of ten molecular variants, seven of which are predicted to inactivate mismatch repair function. The mutated predisposing gene was MSH2 in two families and MLH1 in five other families. All of the mutations were characterized by DNA sequencing and appeared to involve different molecular mechanisms, such as short in‐frame and out‐of‐frame deletions, splicing errors, and nonsense mutations. This study also demonstrates that, in the Italian population, a considerable fraction of HNPCC families (at least 41%) is linked to MSH2 and MLH1 mutations. Genes Chromosom. Cancer 18:8–18, 1997.

Histology of aberrant crypt foci in the human colon

Aberrant crypt foci (ACF) have been identified in the methylene‐blue stained mucosa of the human colon. Some lines of evidence suggest that ACF may be precursors of colon cancer. The objective of the present study was to establish morphological criteria able to define and classify ACF in histological sections. Twenty‐four colectomy specimens were collected after operation for colorectal cancer and fixed in 10% formalin. Strips of grossly normal mucosa were stained in a 0.2% solution of methylene blue in saline for 5–10 min. The strips were measured, put on a glass slide and observed under a light microscope at ×25. One hundred and fourteen ACF identified by topology were sectioned parallel to the muscularis mucosae. Eighty‐four ACF were evident at histological examination and could be classified into three main groups: group A (61 ACF, 72.6%) including foci whose epithelial cells had regular nuclei, with only mild or focal crowding but no stratification, no mucin depletion and no dysplasia; group B (16 ACF, 19.1%), in which features of hyperplasia were evident; and group C (seven ACF, 8.3%) including foci with enlarged, crowded and stratified nuclei, mucin depletion, frequent mitoses, and evident dysplasia, diffuse or focal (mild in five cases, moderate in two) representing microadenomas. Finally, hyperplastic foci were significantly larger than foci of group A and C. Group B ACF were also more frequent in the rectum than in the colon. In conclusion, selected histological features allow the definition of groups of ACF, which may represent sequential steps in the development of human colorectal tumours.