Abstract: The CpG island methylator phenotype (CIMP) in colorectal tumours can be recognized by an increased frequency of aberrant methylation in a specific set of genomic loci. Because of the strong association of CIMP with high microsatellite instability (MSI-H), the identification of CIMP+ tumours within microsatellite stable (MSS) colorectal cancers may not be straightforward. To overcome this potential limitation, we have built an improved seven-locus set of methylation markers that includes CACNA1G, IGF2, RUNX3, HTR6, RIZ1, MINT31, and MAP1B. This new set of CIMP markers revealed a bimodal distribution of methylation frequencies in a group of 95 MSS colorectal cancers, which allowed a clearer separation between CIMP classes. Correlation of MSS CIMP+ tumours with bio-pathological traits revealed significant associations with location to the proximal colon, mucinous histology, BRAF mutation, and chromosomal stability. A potential trend towards an adverse prognosis of CIMP+ cases was associated with the high frequency of BRAF mutations present within this cohort of tumours. Microarray analysis revealed that CIMP+ tumours are characterized by a unique expression profile, a result that confirms that CIMP+ tumours represent a truly distinct molecular class within MSS colorectal cancers.

Abstract: BACKGROUND: Nidogens are highly conserved proteins of basement membranes. Two nidogen proteins, nidogen 1 and nidogen 2, are known in mammals. RESULTS: We show that CpG islands of both NID1 and NID2 genes are aberrantly methylated in human cancer samples and cancer cell lines. For both genes, methylation was correlated with loss of gene transcription in human cell lines. Furthermore, demethylation of the NID1 and NID2 promoters restored gene transcription, demonstrating that methylation was responsible for silencing nidogen genes. In primary tumors, we detected NID1 promoter methylation in 67% of colon cancer samples and in 90% of gastric cancers. NID2 promoter was methylated in 29% of colon and 95% of gastric cancers. Immuno-staining for nidogen-2 confirmed the correlation between aberrant methylation and loss of nidogen expression also in primary tumors, implying that aberrant methylation was a mechanism for inhibiting nidogens expression in human gastrointestinal tumors. CONCLUSION: These results suggest that loss of nidogens expression has a potential pathogenetic role in colon and stomach tumorigenesis. Nidogens are believed to connect laminin and collagen IV networks, hence stabilizing the basement membrane structure. Nidogens are also important for cell adhesion, as they establish contacts with various cellular integrins. Loss of nidogen expression may favor invasion and metastasis of cancer cells by loosening cell interaction with basal membrane and by weakening the strength of the basement membrane itself, first barrier from the connective vascularized matrix.

Abstract: BACKGROUND: Colorectal cancer develops through two main genetic instability pathways characterized by distinct pathologic features and clinical outcome. RESULTS: We investigated colon cancer samples (23 characterized by microsatellite stability, MSS, and 16 by high microsatellite instability, MSI-H) for genome-wide expression of microRNA (miRNA) and mRNA. Based on combined miRNA and mRNA gene expression, a molecular signature consisting of twenty seven differentially expressed genes, inclusive of 8 miRNAs, could correctly distinguish MSI-H versus MSS colon cancer samples. Among the differentially expressed miRNAs, various members of the oncogenic miR-17-92 family were significantly up-regulated in MSS cancers. The majority of protein coding genes were also up-regulated in MSS cancers. Their functional classification revealed that they were most frequently associated with cell cycle, DNA replication, recombination, repair, gastrointestinal disease and immune response. CONCLUSION: This is the first report that indicates the existence of differences in miRNA expression between MSS versus MSI-H colorectal cancers. In addition, the work suggests that the combination of mRNA/miRNA expression signatures may represent a general approach for improving bio-molecular classification of human cancer.

Abstract: PURPOSE: To evaluate the prognostic significance of DNA mismatch repair (MMR) status in a large series of stage II and III colorectal cancer patients. The relationship among MMR status, adjuvant chemotherapy, and clinical outcome was also investigated. PATIENTS AND METHODS: The study included 718 patients with colorectal adenocarcinoma (393 stage II and 325 stage III) who underwent curative surgical resection. MMR status was determined by immunohistochemical analysis of MLH1 and MSH2 expression. Microsatellite instability (MSI) was assessed in 363 patients using mononucleotide and dinucleotide markers. RESULTS: One hundred fourteen (15.9%) carcinomas showed abnormal MMR protein (MMRP) expression (96 MLH1 negative and 18 MSH2 negative) and were classified as MMRP negative, whereas 604 tumors demonstrated normal MLH1/MSH2 immunoreactivity (MMRP positive). MLH1/MSH2 expression was closely related to MSI status (P < .001) and several clinicopathologic features. Patients with MMRP-negative carcinomas demonstrated a marked reduction in the risk of cancer-related death with respect to patients with MMRP-positive tumors (hazard ratio, 0.2579; 95% CI, 0.1289 to 0.5159). A better clinical outcome for patients with MMRP-negative tumors was observed in both stage II (P = .0006) and stage III (P = .0052) disease. In stage III disease, the survival advantage conferred by MMRP-negative tumors was more evident among patients treated with surgery alone than among patients who received adjuvant chemotherapy. A nonsignificant trend for survival benefit from adjuvant chemotherapy was observed among patients with MMRP-positive carcinomas but not among those with MMRP-negative carcinomas. CONCLUSION: Immunohistochemical testing for MLH1/MSH2 expression provides useful prognostic information for the management of stage II and III colorectal cancer patients.

Abstract:

Abstract: 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.

Abstract: Gene promoter methylation causes loss of tumor suppressor genes function in human cancer. Here, we show that the CDH4 gene, a member of the cadherin family encoding for R-cadherin, contains a CpG island located at the 5' of the first exon, which functions as a promoter element and is frequently affected by methylation in human cancer. By using methylation-specific PCR and reverse transcription-PCR in human cancer cell lines, promoter methylation could be directly linked to loss of gene expression. After treatment with the demethylating agent 5-aza-2-deoxycytidine, expression could be restored. Analysis of human primary tumors revealed that the CDH4 gene is methylated in 78% (38 of 49) of colorectal and 95% (20 of 21) of gastric carcinomas. CDH4 methylation was not detected in nonneoplastic colonic (0 of 10) and stomach (0 of 10) tissues or in peripheral blood (0 of 17). CDH4 methylation was detected in histologically normal tissues located in proximity of the neoplasms, indicating that CDH4 methylation is an early event in gastrointestinal tumor progression. We also proved that CDH4 methylation can be revealed in the peripheral blood of cancer patients. Our results indicate that CDH4 may act as a tumor suppressor gene in human gastrointestinal tumors and can potentially be used as an early diagnostic marker for gastrointestinal tumorigenesis.

Abstract: PURPOSE: Adenosine is a ubiquitous nucleoside that accumulates at high levels in hypoxic regions of solid tumors, and A(3) adenosine receptors have been recently demonstrated to play a pivotal role in the adenosine-mediated inhibition of tumor cell proliferation. In the present work, we addressed the question of the putative relevance of A(3) subtypes in colorectal adenocarcinomas. EXPERIMENTAL DESIGN: Seventy-three paired samples of tumor and surrounding peritumoral normal mucosa at a distance of 2 and 10 cm from the tumor and blood samples obtained from a cohort of 30 patients with colorectal cancer were investigated to determine the presence of A(3) receptors by means of binding, immunocytochemistry, and real-time reverse transcription-polymerase chain reaction studies. RESULTS: As measured by receptor binding assays, the density of A(3) receptor was higher in colon carcinomas as compared with normal mucosa originating from the same individuals (P < 0.05). Overexpression of A(3) receptors at the protein level was confirmed by immunohistochemical studies, whereas no changes in A(3) mRNA accumulation in tumors as compared with the corresponding normal tissue were revealed. The overexpression of A(3) receptors in tumors was reflected in peripheral blood cells, where the density was approximately 3-fold higher compared with healthy subjects (P < 0.01). In a cohort of 10 patients studied longitudinally, expression of A(3) receptors in circulating blood cells returned to normal after surgical resection for colorectal cancer. CONCLUSIONS: This study provides the first evidence that A(3) receptor plays a role in colon tumorigenesis and, more importantly, can potentially be used as a diagnostic marker or a therapeutic target for colon cancer.

Abstract: PURPOSE: Gene silencing by hypermethylation plays an important role in proximal colon carcinogenesis. Conversely, DNA hypomethylation has been associated with distal colon cancer (CLC). Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of 5',10'-methylenetetrahydrofolate to 5'-methyl tetrahydrofolate, which serves as methyl donor in the remethylation of homocysteine to methionine. A common MTHFR 677 C-->T polymorphism is characterized by reduced catalytic activity, which affects methionine synthesis and DNA methylation. The aim of the study was to investigate the role of MTHFR 677 C-->T gene polymorphism in the tumorigenesis of proximal and distal CLC in a monoinstitutional group of patients in North Italy. EXPERIMENTAL DESIGN: One-hundred thirty four consecutive proximal and 142 consecutive distal CLC patients, and 279 control subjects without cancer were genotyped for MTHFR using PCR-restriction fragment-length polymorphism analysis. RESULTS: The prevalence of the 677 TT genotype was significantly (P = 0.005) lower in patients with proximal tumors (10 of 134, 7%) than in subjects with distal tumors (28 of 142, 20%). Case/control approach indicated that individuals homozygous for the 677 TT allele had a significantly reduced risk (2.8-fold) (adjusted odds ratio, 0.36; 95% confidence intervals, 0.14-0.91) of developing proximal CLC compared with those harboring the wild-type or heterozygous genotype (677 CC or 677 CT). No significant association between CLC risk and TT genotype was observed in patients with distal tumors (odds ratio, 1.01; 95% confidence interval, 0.48-2.14). CONCLUSIONS: Our findings support a role for MTHFR 677 TT genotype in reducing proximal CLC risk in North Italy.

Abstract: BACKGROUND: Gene promoter methylation is a mechanism for tumor suppressor gene silencing and inactivation. The development of highly sensitive methods for revealing aberrant cancer-associated DNA methylation allows the identification of tumor markers not only in tumor samples, but also in body fluid, an approach that can be useful in the early detection of neoplasms. METHODS: We analyzed the methylation status at 16 loci in tumor samples of the gastrointestinal tract and in early or pre-neoplastic lesions of the colon. RESULTS: Tumor samples revealed that methylation at the transmembrane protein containing epidermal growth factor and follistatin domains (TPEF) locus had the best ratio of discrimination between tumor samples versus normal tissues (83 versus 0%). Its combination with hypermethylated in cancer 1 (HIC1), death-associated protein kinase (DAPK) and O-6-methylguanine DNA methyltransferase (MGMT), allowed the detection of aberrant methylation in 98% of colorectal carcinomas and 100% of gastric carcinomas. The same alterations were also detected in colon adenomas and tissues surrounding the adenomas, indicating that hypermethylation at these loci occurred early in tumor progression. Analysis of DNA from peripheral blood revealed that TPEF methylation was detectable in colorectal tumor patients and patients with early or pre-neoplastic lesions, but not in healthy volunteers. CONCLUSIONS: Our results identify TPEF as a tumor marker that could be useful in the follow-up of gastrointestinal cancer patients or the screening of individuals at risk of developing gastrointestinal neoplasms.

Abstract: Detection of colorectal carcinomas with high-frequency microsatellite instability (MSI-H) is clinically important for several reasons. Recent studies suggested that immunohistochemical analysis of MLH1 and MSH2 expression is a rapid and accurate method for identifying large bowel tumors of the MSI-H phenotype. In this study, we evaluated by immunohistochemistry MLH1 and MSH2 protein expression in 132 MSI-H, 23 MSI-L (low-frequency MSI), and 150 microsatellite stable (MSS) colorectal adenocarcinomas. Loss of MLH1 or MSH2 expression was detected in 120 (90.9%) MSI-H carcinomas, whereas all MSI-L and MSS tumors showed normal expression of both proteins. Lack of MLH1 nuclear staining was observed much more often than absence of MSH2 nuclear staining (106 and 14 cases, respectively). Among MSI-H carcinomas, MLH1/MSH2 pattern of expression was significantly related to several clinical and pathological variables. In particular, MSI-H MLH1/MSH2-positive carcinomas were more often located in the distal colon, were more frequently classified as ordinary adenocarcinomas, and were more likely to be well or moderately differentiated, p53 positive, and <7 cm in diameter than were MLH1-negative and MSH2-negative carcinomas. In addition, MLH1-negative carcinomas were less common among patients with hereditary nonpolyposis colorectal cancer (HNPCC) or suspected HNPCC and in the group of patients aged <50 years. Patients with MLH1-negative carcinomas more frequently died of disease than did patients with MLH1/MSH2-positive and MSH2-negative MSI-H tumors, but the difference was not statistically significant. The results of the present investigation strongly indicate that immunohistochemical analysis of MLH1 and MSH2 expression is a practical and reliable method for the routine detection of the vast majority of MSI-H large bowel adenocarcinomas. Our data also point out that MSI-H MLH1/MSH2-positive colorectal carcinomas are characterized by distinctive pathological features.

Abstract: Colorectal cancers with high-frequency microsatellite instability show peculiar clinicopathological features and a favorable clinical outcome. We investigated whether the improved prognosis for these cancers is related to the content of activated cytotoxic intraepithelial T lymphocytes. Microsatellite instability and the amount of activated cytotoxic lymphocytes were analyzed according to clinicopathological features, survival, and disease recurrence in 109 right-sided colon carcinomas from 245 consecutive patients with stage II/III colon cancer that underwent radical surgery. High-frequency microsatellite instability was found in 43% of stage II/III proximal colon cancers and was associated with significantly higher numbers of activated cytotoxic lymphocytes. High-frequency microsatellite instability, as well as the content of intratumoral-activated cytotoxic T lymphocytes correlated with improved overall and disease-free survival, particularly in patients with stage III tumors. Multivariate analysis revealed that patients with both features had a risk of death and relapse markedly lower than that associated with microsatellite status or intratumoral cytotoxic lymphocytes separately. The presence of local cytotoxic immune responses is probably the major determinant of the good clinical course of patients with microsatellite unstable colon cancer. Furthermore, high-frequency microsatellite instability coupled with a high content of intratumoral cytotoxic lymphocytes may identify a subset of colon cancer patients with a favorable clinical outcome, particularly in stage III disease.

Abstract: Lymph node status has great clinical importance in the management of patients with colorectal cancer. Several pathologic factors may affect the accuracy of nodal status assessment in this tumor type. The aim of the present study was to evaluate, in a series of 166 stage II and stage III colorectal adenocarcinomas, the following pathologic parameters: number of lymph nodes recovered and examined, number of lymph nodes with metastases, and number of tumor nodules (TNs) in the perirectal or pericolic adipose tissue greater or smaller than 3 mm in diameter (TNs > 3 mm and TNs < 3 mm, respectively). The prognostic significance of these parameters, as well as of other histopathologic variables, was determined using univariate and multivariate survival analyses. Our results indicate that the examination of a small number of regional lymph nodes may result in understaging of tumors classified as pN0 (stage II). In addition, our data suggest that TNs > 3 mm and TNs < 3 mm represent distinct pathologic entities. TNs > 3 mm should be considered the prognostic equivalent of lymph node metastases as recommended by the 1997 TNM classification. In contrast, TNs < 3 mm probably originate by intravascular or perivascular tumor extension. Their presence is associated with adverse clinical outcome in stage III patients, regardless of the number of lymph node metastases.

Abstract: Colon carcinomas with microsatellite mutator phenotype exhibit specific genetic and clinico-pathological features. This report describes the analysis of 63 "microsatellite instability-high" (MSI-H) tumors for the presence of mutations in microsatellites located in the coding regions (CDRs) of 6 genes: TGFbetaRII, BAX, hMSH3, hMSH6, IGFIIR, and BLM. The following frequencies of mutations were detected: TGFbetaRII (70%), BAX (54%), hMSH3 (36.5%), IGFIIR (22%), hMSH6 (17.5%), and BLM (16%). The overall picture revealed combinations of mutations suggestive of a progressive order of accumulation, with mutations of TGFbetaRII and BAX first, followed by frameshifts in hMSH3, hMSH6, IGFIIR, and BLM. Correlations with 12 clinico-pathological parameters revealed that tumors with frameshifts in 1 or 2 CDRs were significantly better differentiated than tumors with frameshifts in more than 2 CDRs. We also found that mutations in the hMSH3 gene were significantly associated with decreased wall invasiveness and aneuploidy, and frameshifts in the BLM gene were significantly associated with the mucinous histotype. A trend toward an association between hMSH3 and IGFIIR with the medullary and conventional adenocarcinoma histotypes, respectively, was seen. Our results strengthen the concept that mutations in target genes have a role in the tumorigenic process of MSI-H tumors, and indicate that frameshifts in microsatellites located in CDRs occur in a limited number of combinations that could determine distinct clinico-pathological traits.

Abstract: BACKGROUND: Widespread microsatellite instability (MSI) occurs in nearly 15% of sporadic colorectal cancers. Large bowel carcinomas with high-frequency MSI (MSI-H) (instability at > or = 30% of microsatellite loci) are believed to display distinctive pathologic features and to behave less aggressively than microsatellite-stable (MSS) tumors and carcinomas with low-frequency MSI (MSI-L) (instability at < 30% of microsatellite loci). The aim of the current study was to accurately define the clinicopathologic and biologic features of MSI-H sporadic colorectal carcinomas. METHODS: MSI status was evaluated in 216 large bowel adenocarcinomas using polymerase chain reaction (PCR) and 6 microsatellite markers. Tumors that showed instability with at least two microsatellite markers were classified as MSI-H, whereas the other tumors were classified as MSI-L (instability at one locus) or MSS (no instability). Expression of p53, hMLH1, and hMSH2 gene products was determined by immunohistochemistry, and DNA ploidy pattern was determined by flow cytometry. The prognostic significance of MSI status was assessed by univariate and multivariate survival analyses. RESULTS: The significantly different pathologic features of MSI-H carcinomas were proximal location; large size; mucinous and medullary histotype; poor differentiation; expanding pattern of growth; more frequent Crohn-like conspicuous lymphoid reaction; and low incidence of extramural vein invasion. Most MSI-H tumors were DNA diploid (33 of 40 tumors; 82.5%) and p53 negative (34 of 44 tumors; 77.3%). Conversely, DNA aneuploidy and p53 overexpression were observed in 82.3% (130 of 158 tumors; P < 0.0001) and 54.1% (93 of 172 tumors; P = 0.0002) of MSI-L/MSS tumors, respectively. Loss of hMLH1 or hMSH2 expression was detected in a high fraction of MSI-H carcinomas (86. 0%). Patients with MSI-H tumors showed a better clinical outcome than patients with MSI-L/MSS tumors (P = 0.0017). Furthermore, in multivariate analysis that included conventional clinicopathologic parameters, MSI status, and p53 expression as covariates, MSI status was a significant independent prognostic indicator of disease specific survival. CONCLUSIONS: Assessment of MSI status is an essential step in the genetic characterization of large bowel carcinomas and identifies a subset of tumors with distinct clinical, pathologic, and biologic features.

Abstract: 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.

Abstract: Adenocarcinoma in association with chronic anal fistula is a rare disease which gives rise to difficult problems of diagnosis and treatment. A case of mucinous adenocarcinoma arising on a long standing fistula in ano is described. A patient with a long history of mucinous discharge, pain and perianal induration underwent a biopsy of the external opening of the fistula that showed mucinous infiltrating adenocarcinoma. After a colonoscopy and a preoperative abdominal CT scan, she underwent a successful abdominoperineal resection with adjuvant chemoradiation therapy. Diagnosis of this condition is often difficult; deep and multiple biopsies of the fistulous tracks or perianal mass are necessary to establish the diagnosis. An accurate staging of the neoplasm, using endorectal ultrasound, NMR or CT scans is needed to plan the appropriate treatment. Recent studies have shown that locally advanced anal adenocarcinomas could benefit from pre or postoperative chemoradiation therapy. However, an accurate and complete removal of the tumor, which usually entails abdominoperineal resection, is often necessary to achieve radicality. Despite new therapy protocols, the prognosis of mucinous adenocarcinoma is still poor, mostly due to its advanced nature at the time of diagnosis. This reinforces the importance of biopsy of all perianal abscesses and fistulas for early detection and treatment.

Abstract: PURPOSE: Recent studies suggest the existence of a distinct class of poorly differentiated large bowel adenocarcinomas, usually termed medullary-type adenocarcinomas (MTAs). The aim of the present study was to accurately define the clinical, histopathologic, biologic, and genetic features of this tumor type. MATERIALS AND METHODS: Among 1,265 surgically resected sporadic colorectal carcinomas, 45 MTAs were identified on the basis of the following criteria: predominantly solid growth pattern (at least 70% of the tumor area) and lack of marked nuclear pleomorphism. The clinicopathologic, biologic, and genetic characteristics of MTAs were compared with those of a series of 457 common glandular colorectal adenocarcinomas. RESULTS: The significantly different clinicopathologic features of MTAs were proximal location, large size, invasion into adjacent organs, expanding pattern of growth, low incidence of distant metastases, more frequent conspicuous peritumoral lymphocytic infiltration, and Crohn's-like lymphoid reaction. Furthermore, young patients with MTAs often demonstrated a family history highly suggestive of a hereditary background. Unlike glandular adenocarcinomas, the large majority of MTAs were DNA diploid by flow cytometric analysis (21 of 25, 84%) and p53 negative by immunohistochemistry (36 of 41, 87.8%). In addition, 18 of the 20 MTAs examined by DNA microsatellite analysis demonstrated widespread microsatellite instability (90% of cases). Patients with MTAs showed a better clinical outcome with respect to patients with common poorly differentiated adenocarcinomas (PDAs) (P <.0001) and well- or moderately differentiated adenocarcinomas (WMDAs) (P =.133). In particular, none of the 33 patients with completely resectable stage II and III MTAs developed tumor recurrence during the observation period. Conversely, 24.7% of patients with stage II and III WMDAs and 48.9% of patients with stage II and III PDAs, who had undergone curative surgical resection, died of recurrent disease (P =.01 and P <.0001, respectively). CONCLUSION: All these data strongly indicate that MTAs represent a distinct pathologic entity, with specific histologic appearance and peculiar clinical and genetic features. These tumors need to be classified separately from other poorly differentiated colorectal carcinomas.

Abstract: BACKGROUND: The prognostic value of flow cytometric DNA ploidy in colorectal carcinoma has not been defined clearly. Most previous studies were conducted retrospectively using archival formalin fixed, paraffin embedded tumor samples. Conversely, few data on prospective studies employing fresh or frozen tissue specimens are available. There is general agreement that fresh/frozen material is more reliable than paraffin embedded tissue for DNA ploidy analysis by flow cytometry. METHODS: In the current investigation we evaluated the prognostic significance of nuclear DNA content in a prospective series of 191 patients with curatively resected TNM Stage II (n = 107) or Stage III (n = 84) sporadic colon carcinomas. DNA ploidy status was assessed by flow cytometry utilizing multiple frozen tumor samples. Mean follow-up in surviving patients was 48.5 months (median, 46.9 months; range, 29-77 months). The Cox proportional hazards model was used to adjust for several clinical and pathologic covariates. RESULTS: Of the 191 carcinomas examined, 47 (24.6%) were classified as DNA diploid and 144 (75.4%) as DNA aneuploid. DNA ploidy pattern was significantly related to tumor site (P < 0.0001), histologic type (P = 0.0002), and grade of differentiation (P = 0.009), but not to other clinical and pathologic variables. Patients with DNA diploid tumors showed a better disease free (P = 0.013) and overall survival (P = 0.021) than patients with DNA aneuploid adenocarcinomas. In particular, patients with Stage II DNA diploid tumors (n = 30) had an excellent clinical outcome, with an overall 5-year survival rate of 97%. When patients were analyzed according to the anatomic site of the tumor, a significant relationship between DNA ploidy status and disease free and overall survival was observed in the group of patients with carcinomas of the proximal colon (n = 84) (P = 0.004 and P = 0.002, respectively), but not among patients whose tumors were sited distally to the splenic flexure (n = 107). In multivariate analysis, nuclear DNA content was demonstrated to be an independent prognostic variable for both disease free and overall survival. Furthermore, in the group of patients with tumors of the proximal colon, DNA ploidy pattern was the single most important prognostic factor. CONCLUSIONS: Our results confirm that flow cytometric DNA ploidy status is a significant and independent prognostic factor in patients with colon carcinoma. These findings may have clinical implications for the management of affected patients, especially those with Stage II disease.

Abstract: To further evaluate the role of the p53 gene in the development of colorectal carcinoma we examined by immunohistochemistry p53 protein expression in a series of 136 colorectal adenomas, 25 adenomas containing early invasive carcinoma (pT1) and 160 advanced adenocarcinomas (pT3-pT4). p53 overexpression was detected in 22% of adenomas, 64% of adenomas with invasive carcinoma and 60% of advanced adenocarcinomas. In colorectal adenomas p53 expression was related to tumour size (P = 0.0013), histologic type (P < 0.0001) and grade of dysplasia (P < 0.0001). Only 7.5% of adenomas with low grade dysplasia were found to be p53 positive, whereas 73.3% of adenomas with high grade dysplasia demonstrated p53 overexpression. Most p53 positive adenomas and adenomas with invasive carcinoma showed intratumoural heterogeneity of p53 immunoreactivity. Conversely advanced adenocarcinomas were always uniformly p53 positive or negative. Our data show that p53 protein overexpression occurs at the transition from low grade dysplasia to high grade dysplasia, indicating a likely role for the p53 gene in the conversion of benign adenoma to malignant carcinoma.

Abstract: The prognostic significance of chromosome 18q allelic loss was evaluated in a series of 118 patients with curatively resected TNM stage II or stage III colon cancer. Chromosome 18q status was determined on frozen tumour samples, using microsatellite markers and the polymerase chain reaction (PCR). Mean follow-up in surviving patients was 75.9 months. Chromosome 18q allelic loss was significantly related to tumour site, extramural venous invasion, flow cytometric nuclear DNA content and p53 protein expression. Patients whose tumour had no evidence of chromosome 18q allelic loss showed a better disease-free and overall survival than patients whose tumour demonstrated 18q allelic loss. When patients were stratified by tumour stage, a significant survival advantage for patients whose tumour had no allelic loss on chromosome 18q was observed in stage II as well as in stage III disease. In particular, patients with stage II disease whose tumour had no chromosome 18q allelic loss demonstrated an excellent clinical outcome, with a 5-year disease-free survival rate of 96%. In contrast, the 5-year disease-free survival rate of patients with stage II disease and chromosome 18q allelic loss was only 54%. In multivariate analysis, status of chromosome 18q was the only significant independent prognostic factor for both disease-free and overall survival. These results indicate that assessment of chromosome 18q status provides relevant prognostic information in colon cancer and might be employed in the selection of patients for adjuvant therapy.

Abstract: A rare case of lymphoepithelial cyst of the pancreas is reported. Microscopically the cyst content consisted of keratinous material and the walls were lined by mature squamous epithelium surrounded by dense lymphoid tissue. Immunohistochemistry showed diffuse reactivity for CD20 and CD3 in the lymphoid tissue and uniform positivity for cytokeratins in the squamous epithelium. Although the histogenesis of lymphoepithelial cysts of the pancreas is not understood, awareness of this lesion is helpful in differentiating it from other pancreatic cystic lesions.

Abstract: p53 protein expression was evaluated in a series of 204 primary colorectal adenocarcinomas by immunohistochemistry using frozen tissue sections and monoclonal antibody DO-7. Nuclear staining of more than 5% of neoplastic cells was observed in 124 (60.8%) adenocarcinomas, which were classified as p53 positive. p53 immunoreactivity was found to he unrelated to several clinical and pathologic variables, including age and sex of patient, tumor site, tumor stage, grade of differentiation, pattern of growth, degree of peritumoral lymphocytic infiltration, and venous invasion. A strong association was demonstrated between p53 immunostaining and tumor type. Only 4 of 21 mucinous carcinomas examined (19%) were p53 positive. Conversely, 120 of 183 (65.6%) nonmucinous adenocarcinomas showed positive p53 immunostaining (P <.0001). p53 expression also was related to the flow cytometric DNA ploidy pattern, aneuploid carcinomas with DI >1.20 showing higher frequency of p53 overexpression than DNA diploid, and aneuploid tumors with DI < or = 1.20 (P = .0003). No relationship was found between p53 expression and the Ki-67 proliferation index. With respect to the total study population (mean follow-up 33.4 months; range 19-47 months) the duration of overall survival was independent of p53 expression. In the group of 141 patients with stage I, stage II, and stage III disease who had undergone curative resection, positive p53 immunostaining was associated with poorer overall survival (P = .029). Subgroup analysis showed that the reduced survival conferred by p53 overexpression was confined to patients with stage III tumors (P = .027). However, in multivariate analysis, p53 expression failed to demonstrate independent prognostic significance. Our results indicate that immunohistochemical analysis of p53 expression provides valuable information for the understanding of colorectal cancer biology and clinical behavior.

Abstract: The clinical, pathological and biologic features of 79 mucinous colorectal carcinomas were compared with those of 602 non-mucinous adenocarcinomas. The two groups did not show appreciable differences in patients' age, stage distribution, extent of lymph node involvement, grade of differentiation, pattern of growth and venous invasion. Mucinous carcinomas occurred more frequently among female patients (P < 0.05) and in the proximal colon (P < 0.01). Moreover, mucinous carcinomas more often demonstrated origin within villous adenomas (P < 0.0001) and lacked pronounced peritumoural lymphocytic infiltration (P < 0.001). A strong association was found between tumour type and flow cytometric nuclear DNA content. A high proportion of mucinous carcinomas showed DNA index (DI) values < or = 1.20 (26/38, 68.4%); conversely only 103 of 322 (32%) non-mucinous carcinomas had a DI < or = 1.20 (P < 0.0001). In addition mucinous carcinomas were characterized by infrequent p53 overexpression (4/21, 19% versus 120/183, 65.6%; P < 0.001) and higher levels of proliferative activity (P < 0.0001) compared to non-mucinous adenocarcinomas. Our data support the hypothesis that mucinous carcinoma represents a distinct clinicopathologic and genetic entity.

Abstract: The prognostic value of DNA ploidy in large bowel cancer is still controversial. In the present investigation we have evaluated the nuclear DNA content in 123 colorectal adenocarcinomas by flow cytometry using multiple frozen tumour samples. Thirty-three (26.8%) carcinomas were classified as diploid and 90 as aneuploid (73.2%). Presence of DNA aneuploidy was found to be unrelated to age and sex of patients, tumour stage and grade of differentiation, as well as to several other histopathological variables. However, multiploid tumours (20/123, 16.3%) resulted to be more frequently in advanced stages of disease (stages III and IV, P < 0.025) and more often showed unfavourable histopathological features, especially an infiltrating pattern of growth (P < 0.05), compared to diploid and single aneuploid carcinomas. Nuclear DNA content was found to be closely related to tumour site. Carcinomas of the proximal colon were more frequently diploid (P < 0.005) and more often displayed a DI < or = 1.20 (P < 0.001) than tumours of the distal colon. Nuclear DNA content was also found to be related to tumour type. In fact, a high proportion (66.7%) of mucinous carcinomas showed DI values < or = 1.20; conversely only 31.4% of nonmucinous adenocarcinomas had a DI < or = 1.20 (P < 0.01). Intratumoural heterogeneity in nuclear DNA content was found in 23% of cases. These results seem to suggest that the DNA ploidy pattern probably reflects different genetic mechanisms involved in the development of carcinomas in the proximal and distal colon. Furthermore our data support the hypothesis that mucinous carcinoma represents a distinct clinicopathologic entity, possibly related to pathogenetic factors different from those acting in the majority of nonmucinous adenocarcinomas. Finally, the analysis of multiple tissue samples taken from different areas of each tumour is necessary to assess carefully the DNA ploidy pattern of large bowel carcinomas.

Abstract: A xanthomatous lesion of vermis cerebelli was removed successfully from the posterior fossa of a 19 years old girl with insulin-dependent diabetes and hypercholesterolemia, admitted for cerebellar hemorrhage. A review of literature shows that this is the first case of xanthomatous lesion presented with cerebellar hemorrhage. The pathogenesis of this lesion is reviewed as it relates to the presentation of this case.