Augusto Giannini

Interobserver agreement in the histologic diagnosis of colorectal polyps: the experience of the multicenter adenoma colorectal study (SMAC)

Current clinical practice guidelines for patients with colorectal polyps are mainly based on the histologic characteristics of their lesions. However, interobserver variability in the assessment of specific polyp characteristics was evaluated in very few studies. The purpose of this study was to evaluate the interobserver agreement of four pathologists in the diagnosis of histologic type of colorectal polyps and in the degree of dysplasia and of infiltrating carcinoma in adenomas. A stratified random sample of 100 polyps was obtained from the 4,889 polyps resected within the Multicentre Adenoma Colorectal Study (SMAC), and the slides were blindly reviewed by the four pathologists. Agreement was analyzed using kappa statistics. A median kappa of 0.89 (range 0.79-1.0) was estimated for the interobserver agreement for the diagnosis of hyperplastic polyp vs. adenoma. The agreement in the diagnosis of tubular, tubulovillous, and villous type, was given by median kappa values of 0.50, 0.15, and 0.36, respectively. The median kappa for the diagnosis of infiltrating carcinoma was 0.78 (range 0.73-0.84). Agreement on diagnosis of adenoma histologic subtypes, degrees of dysplasia, or infiltrating carcinoma in adenoma was moderate. A simpler classifications might help to better identify patients at different risk of colorectal cancer.

Mutations of the Apc gene in experimental colorectal carcinogenesis induced by azoxymethane in F344 rats

We investigated in the rat the role of the Apc gene, which is mutated in familial adenomatous polyposis and sporadic colon cancer in the process leading from normal colonic mucosa to aberrant crypt foci (ACF) and finally to adenomas and adenocarcinomas. We analysed mutations in exon 15 of the rat Apc gene using in vitro synthesized protein assay in 66 ACF and in 28 colon tumours induced by azoxymethane. No Apc mutations were found in ACF, whereas five mutations were found in the tumours. The data suggest that mutations of the Apc gene are associated with the transition from ACF to adenoma and adenocarcinoma and not from normal mucosa to ACF.

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.

Correlations between histopathological and biological findings in nasopharyngeal carcinoma and its prognostic significance.

Forty‐five consecutive cases of nasopharyngeal carcinoma were morphologically and immunocyto‐chemically studied using monoclonal (anti‐B and anti‐T cell) and polyclonal (anti‐SlOO protein and antilysozyme) antibodies with the peroxidase‐anti‐peroxidase method to identify infiltrating lymphocytes (T and B cell) and histiocytes (monocytic/mac‐rophagic and dendritic cells) in nasopharyngeal carcinoma.

Mucin-depleted foci have β-catenin gene mutations, altered expression of its protein, and are dose- and time-dependent in the colon of 1,2-dimethylhydrazine-treated rats

Mucin‐depleted foci (MDF) are purported preneoplastic lesions that can be easily visualized in the unsectioned colon of carcinogen‐treated rats stained with high‐iron diamine alcian blue (HID‐AB). In F344 rats treated twice with 150 mg/kg of 1,2‐dimethylhydrazine (DMH) and sacrificed after 5, 9, 13 and 28 weeks, MDF increased over time from 5 to 13 weeks, whereas they decreased at 28 weeks, when tumors appear. MDF multiplicity (crypts/MDF) linearly increased with time. Increasing doses of DMH (100, 150 and 200 mg/kg × 2 times) caused a dose‐related increase in MDF. Mutations in Ctnnb1 gene codifying for β‐catenin were identified with PCR amplification and direct sequencing in 6/15 tumors (40%), 7/28 MDF (25%) and 2/27 (7%) aberrant crypt foci (ACF) identified in HID‐AB‐stained colon. All mutations in tumors and MDF caused amino acid substitution, while one mutation in ACF was silent. β‐catenin detected at membrane level by immunohistochemistry was markedly reduced in MDF and tumors and, to a lesser extent, in ACF identified with HID‐AB. By contrast, nuclear localization of β‐catenin was significantly increased in MDF and tumors, while no variation was observed in ACF. β‐catenin cytoplasmic expression was also significantly increased in MDF and tumors but to a lesser extent in ACF. In conclusion, MDF are induced dose‐dependently by DMH, increase in size with time, have mutations in the β‐catenin gene and marked alterations in β‐catenin cellular localization. Since all these phenomena are considered specific steps for colon tumorigenesis, these results further support the hypothesis that MDF are cancer precursors and can be proposed as endpoints in short‐term carcinogenesis experiments.

Prognostic Significance of Accessory Cells and Lymphocytes in Nasopharyngeal Carcinoma

Forty-five consecutive biopsy specimens of nasopharyngeal carcinoma (NPC) and 10 biopsies of healthy nasopharyngeal mucosa obtained from non-cancer patients were investigated by immunohistochemical methods. Monoclonal (B2, T1) and policlonal antibodies (against S-100 protein and lysozyme) with reference to infiltrating lymphocytes and accessory cells (monocytic/macrophagic and dendritic cells) were used. Variable population densities of dendritic cells (S100+) were demonstrated in 22 out of the 45 cases (49%) of NPC; the distribution of these cells was typically within the cancer nests. Monocytic/macrophagic cells (Lys+) were found along the tumor margins and interspersed among the tumor cells in 14 out of 45 (31%) cases. No significant statistical correlation between density of accessory cells and histological type of NPC (classified according to Micheau criteria) was found. Cases with a moderate to marked density of dendritic and monocytic/macrophagic cells survived longer than those with a slight one (mean survival of 63%, 67% and 29%, 27% respectively). In NPC tissues T-lymphocyte infiltration was prevalent. In contrast, B cells were numerous and T cells rare in normal control tissues. The intensity of T-cell infiltration was significantly high in cases with a marked density of S-100+ cells, according to the ability of these cells to present antigens to sensitized T-cells. This study suggests a prognostic significance for reactive cells infiltrating NPC, which means longer survival for cases associated with marked infiltration density of accessory cells.

Distal hyperplastic polyps do not predict proximal adenomas: results from a multicentric study of colorectal adenomas☆☆☆★★★♢

BACKGROUND The association between distal hyperplastic polyps and proximal adenomas is still a matter of debate. We investigated this association while taking into account patient characteristics. METHODS After exclusion of patients with inflammatory bowel diseases, familial adenomatous polyposis, or any cancer, 3088 eligible consecutive subjects aged 18 to 69 years underwent total colonoscopy in four gastroenterology units. The odds ratios (OR) of having proximal adenomas according to patient characteristics (age, sex, medical center, year of endoscopy, reasons for referral, and distal findings) were estimated in univariate and multivariate analyses. RESULTS Patients with distal polyps of any type showed an adjusted OR of 2.5 (95% CI [1.9, 3.1] p < .001) of having proximal adenomas as compared with those without distal polyps. When distal adenomas and distal hyperplastic polyps were included in the multivariate model as independent factors, the presence of adenomas significantly increased the risk of proximal adenomas (OR = 2.8: 95% CI [2.2, 3.6] p < .001), whereas the presence of hyperplastic polyps did not (OR = 1.1: 95% CI [0.8, 1.5] p = .64). No association with number, size, or location of distal hyperplastic polyps was seen. CONCLUSIONS Our data show that the presence of hyperplastic polyps should not be the sole indication for total colonoscopy because they are not associated with proximal adenomas when adjusting for patient characteristics and presence of distal adenomas.

Identification of Mucin Depleted Foci in the Human Colon

Aberrant crypt foci (ACF) originally described in rodents treated with colon-specific carcinogens have been identified also in humans at high risk of colon cancer (CRC) and are extensively used as cancer biomarkers. However, studies documenting the heterogeneity of ACF have questioned their precancerous nature. Recently, we described dysplastic foci depleted of mucins (MDF) in the colon of rats treated with colon-specific carcinogens. Like colon tumors, MDFs show activation of Wnt signaling driven by mutations in the β-catenin gene and Apc, a key gene in colorectal carcinogenesis. Because MDFs have been identified thus far only in rodents, we wanted to search for similar lesions in humans. Familial adenomatous polyposis (FAP) subjects, carrying germ-line mutations in the APC gene, are at high risk of CRC. Therefore, we first searched for MDF-like lesions in unsectioned colon samples from FAP patients and then in patients with sporadic CRC. MDFs were present in the colon of FAP patients (average of 0.0577 lesions/cm2) and at a much lower density in CRC patients (average of 0.0006 lesions/cm2). ACFs were also observed in all patients. Histologic preparations of all the MDFs identified in FAP and CRC consisted of microadenomas at variable grades of dysplasia. The occurrence of MDF-like lesions in high-risk patients provides evidence that these lesions have a counterpart in human pathology and, as observed in rodents, may represent the very early stages of CRC.

Gene expression profile and genomic alterations in colonic tumours induced by 1,2-dimethylhydrazine (DMH) in rats

BackgroundAzoxymethane (AOM) or 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in rats shares many phenotypical similarities with human sporadic colon cancer and is a reliable model for identifying chemopreventive agents. Genetic mutations relevant to human colon cancer have been described in this model, but comprehensive gene expression and genomic analysis have not been reported so far. Therefore, we applied genome-wide technologies to study variations in gene expression and genomic alterations in DMH-induced colon cancer in F344 rats.MethodsFor gene expression analysis, 9 tumours (TUM) and their paired normal mucosa (NM) were hybridized on 4 × 44K Whole rat arrays (Agilent) and selected genes were validated by semi-quantitative RT-PCR. Functional analysis on microarray data was performed by GenMAPP/MappFinder analysis. Array-comparative genomic hybridization (a-CGH) was performed on 10 paired TUM-NM samples hybridized on Rat genome arrays 2 × 105K (Agilent) and the results were analyzed by CGH Analytics (Agilent).ResultsMicroarray gene expression analysis showed that Defcr4, Igfbp5, Mmp7, Nos2, S100A8 and S100A9 were among the most up-regulated genes in tumours (Fold Change (FC) compared with NM: 183, 48, 39, 38, 36 and 32, respectively), while Slc26a3, Mptx, Retlna and Muc2 were strongly down-regulated (FC: -500; -376, -167, -79, respectively). Functional analysis showed that pathways controlling cell cycle, protein synthesis, matrix metalloproteinases, TNFα/NFkB, and inflammatory responses were up-regulated in tumours, while Krebs cycle, the electron transport chain, and fatty acid beta oxidation were down-regulated. a-CGH analysis showed that four TUM out of ten had one or two chromosomal aberrations. Importantly, one sample showed a deletion on chromosome 18 including Apc.ConclusionThe results showed complex gene expression alterations in adenocarcinomas encompassing many altered pathways. While a-CGH analysis showed a low degree of genomic imbalance, it is interesting to note that one of the alterations concerned Apc, a key gene in colorectal carcinogenesis. The fact that many of the molecular alterations described in this study are documented in human colon tumours confirms the relevance of DMH-induced cancers as a powerful tool for the study of colon carcinogenesis and chemoprevention.

K-ras mutations and mucin profile in preneoplastic lesions and colon tumors induced in rats by 1,2-dimethylhydrazine

K‐ras and mucin profile variations, associated with intestinal carcinogenesis, were studied in the preneoplastic lesions, mucin‐depleted foci (MDF) and aberrant crypt foci (ACF), and in colonic tumors induced in rats by 1,2‐dimethylhydrazine (DMH). The frequency of lesions with K‐ras mutations was 23% (3/13), 5.5% (1/18) and 100% (14/14) in MDF, tumors and ACF, respectively. Two of three MDF mutated in K‐ras also carried a missense mutation in Apc. We also tested the expression of MUC2, a mucin abundantly expressed in normal colon and M1/MUCA5C, up‐regulated in colon carcinogenesis, using immunohistochemistry. MDF and tumors showed a dramatic reduction in the expression of MUC2, whereas ACF showed only a slight reduction. The expression of M1/MUC5AC was almost absent in normal mucosa, but was increased in all the lesions (MDF, tumors and ACF). The expression of the intestinal trefoil factor (ITF), a marker of goblet cell lineage, was reduced in MDF and tumors compared to normal mucosa but not in ACF. In conclusion, although K‐ras mutations are present in all ACF, they are less frequent in MDF and tumors; M1/MUC5AC is a marker associated with all preneoplastic events while the reduction of MUC2 and ITF expression is selectively associated with more advanced lesions such as MDF and tumors.