Ada Piepoli

Aberrant DNA methylation in non-neoplastic gastric mucosa of H. Pylori infected patients and effect of eradication.

BACKGROUND:Gene promoter methylation is an epigenetic event leading to gene silencing. This mechanism is particularly relevant in cancer since it can interfere with the activity of specific “suppressor” genes.AIM:To evaluate promoter methylation of CDH1, p16, APC, MLH1, and COX2 in patients with H. pylori (Hp) infection before and after eradication.METHODS:Fifty-seven dyspeptic outpatients who had never performed previous endoscopy or Hp testing and treatment underwent clinical interview, endoscopy with three paired gastric biopsy specimens from the antrum, angulus, and corpus, and 13C-urea breath test (UBT). Biopsies were scored for the presence of Hp and intestinal metaplasia (IM). DNA methylation of five tumor-related genes (CDH1, p16, MLH1, APC, and COX2) was evaluated by methylation-specific PCR in each biopsy. Infected patients were given a standard eradicating treatment and, after 1 yr, underwent endoscopy with biopsies and UBT.RESULTS:Hp infection was found in 45 patients. IM was detected in 17 out of 45 (38%) infected patients. Mean number of methylated genes was 0, 1.1 ± 0.9, and 1.6 ± 0.9 among the 12 Hp−/IM−, the 28 Hp+/IM−, and the 17 Hp+/IM+ patients, respectively (P < 0.0001). Specifically, promoter hypermethylation of CDH1, p16, APC, MLH1, and COX2 was found in 68%, 25%, 7%, 0%, and 14% of Hp+/IM− patients and in 71%, 29%, 35%, 12%, and 12% of Hp+/IM+ patients. No significant difference was found among the three groups of patients as far as age, smoking, alcohol, meat and vegetable consumption, and family history of gastric cancer were considered. Twenty-three out of 45 (51%) infected patients underwent the 1-yr follow-up endoscopy: 17 out of 23 (74%) were successfully eradicated. After Hp eradication, CDH1, p16, and APC methylation significantly decreased while COX2 methylation completely disappeared. Conversely, MLH1 methylation did not change significantly in patients with IM.CONCLUSION:Hp infection is associated with promoter methylation of genes which are relevant in the initiation and progression of gastric carcinogenesis. While CDH1 methylation seems to be an early event in Hp gastritis, MLH1 methylation occurs late along with IM. Hp eradication is able to significantly reduce gene methylation thus delaying or reversing Hp-induced gastric carcinogenesis.

Mirna Expression Profiles Identify Drivers in Colorectal and Pancreatic Cancers

Background and Aim Altered expression of microRNAs (miRNAs) hallmarks many cancer types. The study of the associations of miRNA expression profile and cancer phenotype could help identify the links between deregulation of miRNA expression and oncogenic pathways. Methods Expression profiling of 866 human miRNAs in 19 colorectal and 17 pancreatic cancers and in matched adjacent normal tissues was investigated. Classical paired t-test and random forest analyses were applied to identify miRNAs associated with tissue-specific tumors. Network analysis based on a computational approach to mine associations between cancer types and miRNAs was performed. Results The merge between the two statistical methods used to intersect the miRNAs differentially expressed in colon and pancreatic cancers allowed the identification of cancer-specific miRNA alterations. By miRNA-network analysis, tissue-specific patterns of miRNA deregulation were traced: the driving miRNAs were miR-195, miR-1280, miR-140-3p and miR-1246 in colorectal tumors, and miR-103, miR-23a and miR-15b in pancreatic cancers. Conclusion MiRNA expression profiles may identify cancer-specific signatures and potentially useful biomarkers for the diagnosis of tissue specific cancers. miRNA-network analysis help identify altered miRNA regulatory networks that could play a role in tumor pathogenesis.

Familial expression of anti-Saccharomyces cerevisiae Mannan antibodies in Crohn's disease and ulcerative colitis: a GISC study.

OBJECTIVES:Recent studies in familial Crohns disease (CD) have suggested that anti-Saccharomyces cerevisiae mannan antibodies (ASCAs) may represent a new specific marker of genetic susceptibility. In this study we aimed to assess the importance of ASCAs by comparing their presence in a large number of patients with sporadic and familial occurrence of CD or ulcerative colitis (UC) and their unaffected relatives.METHODS:Serum samples from 96 patients with sporadic CD, 97 patients with sporadic UC, and 50 unrelated healthy controls were tested for ASCAs by a standard ELISA method. Moreover, 73 families with two or more members affected by CD and/or UC were recruited. From these families 58 CD patients, 84 UC patients, and 216 unaffected first degree relatives were investigated.RESULTS:ASCAs were detected in 34 of 96 patients with sporadic CD (35%, p < 0.01 vs controls), 11 of 97 patients with sporadic UC (12%), and two of 50 controls (4%). ASCAs were significantly (p < 0.04) more frequent in patients with familial CD (55%) and familial UC (25%) than in sporadic cases. Moreover, ASCAs were found in 25% of unaffected relatives, and this rate did not significantly differ in CD, UC, and mixed families (28%, 26%, and 22%, respectively).CONCLUSIONS:In this study we confirm that ASCAs occur particularly frequently in CD patients, especially with the presence of a positive family history. However, they are also significantly increased in UC patients with a family history and in a considerable number of unaffected relatives of inflammatory bowel disease families, irrespective of the characteristics of their families (UC, CD, mixed, ASCA positive, and ASCA negative). The presence of ASCAs in unaffected relatives might point toward a genetic predisposition to either CD or UC.

On the statistical assessment of classifiers using DNA microarray data.

BackgroundIn this paper we present a method for the statistical assessment of cancer predictors which make use of gene expression profiles. The methodology is applied to a new data set of microarray gene expression data collected in Casa Sollievo della Sofferenza Hospital, Foggia – Italy. The data set is made up of normal (22) and tumor (25) specimens extracted from 25 patients affected by colon cancer. We propose to give answers to some questions which are relevant for the automatic diagnosis of cancer such as: Is the size of the available data set sufficient to build accurate classifiers? What is the statistical significance of the associated error rates? In what ways can accuracy be considered dependant on the adopted classification scheme? How many genes are correlated with the pathology and how many are sufficient for an accurate colon cancer classification? The method we propose answers these questions whilst avoiding the potential pitfalls hidden in the analysis and interpretation of microarray data.ResultsWe estimate the generalization error, evaluated through the Leave-K-Out Cross Validation error, for three different classification schemes by varying the number of training examples and the number of the genes used. The statistical significance of the error rate is measured by using a permutation test. We provide a statistical analysis in terms of the frequencies of the genes involved in the classification. Using the whole set of genes, we found that the Weighted Voting Algorithm (WVA) classifier learns the distinction between normal and tumor specimens with 25 training examples, providing e = 21% (p = 0.045) as an error rate. This remains constant even when the number of examples increases. Moreover, Regularized Least Squares (RLS) and Support Vector Machines (SVM) classifiers can learn with only 15 training examples, with an error rate of e = 19% (p = 0.035) and e = 18% (p = 0.037) respectively. Moreover, the error rate decreases as the training set size increases, reaching its best performances with 35 training examples. In this case, RLS and SVM have error rates of e = 14% (p = 0.027) and e = 11% (p = 0.019). Concerning the number of genes, we found about 6000 genes (p < 0.05) correlated with the pathology, resulting from the signal-to-noise statistic. Moreover the performances of RLS and SVM classifiers do not change when 74% of genes is used. They progressively reduce up to e = 16% (p < 0.05) when only 2 genes are employed. The biological relevance of a set of genes determined by our statistical analysis and the major roles they play in colorectal tumorigenesis is discussed.ConclusionsThe method proposed provides statistically significant answers to precise questions relevant for the diagnosis and prognosis of cancer. We found that, with as few as 15 examples, it is possible to train statistically significant classifiers for colon cancer diagnosis. As for the definition of the number of genes sufficient for a reliable classification of colon cancer, our results suggest that it depends on the accuracy required.

Clock Gene Expression Levels and Relationship With Clinical and Pathological Features in Colorectal Cancer Patients

The clock gene machinery controls cellular metabolism, proliferation, and key functions, such as DNA damage recognition and repair. Dysfunction of the circadian clock is involved in tumorigenesis, and altered expression of some clock genes has been found in cancer patients. The aim of this study was to evaluate the expression levels of core clock genes in colorectal cancer (CRC). Quantitative real-time polymerase chain reaction (qPCR) was used to examine ARNTL1, CLOCK, PER1, PER2, PER3, CRY1, CRY2, Timeless (TIM), TIPIN, and CSNK1Ε expression levels in the tumor tissue and matched apparently healthy mucosa of CRC patients. In the tumor tissue of CRC patients, compared to their matched healthy mucosa, expression levels of ARNTL1 (p = .002), PER1 (p = .002), PER2 (p = .011), PER3 (p = .003), and CRY2 (p = .012) were lower, whereas the expression level of TIM (p = .044) was higher. No significant difference was observed in the expression levels of CLOCK (p = .778), CRY1 (p = .600), CSNK1Ε (p = .903), and TIPIN (p = .136). As to the clinical and pathological features, a significant association was found between low CRY1 expression levels in tumor mucosa and age (p = .026), and female sex (p = .005), whereas high CRY1 expression levels in tumor mucosa were associated with cancer location in the distal colon (p = .015). Moreover, high TIM mRNA levels in the tumor mucosa were prevalent whenever proximal lymph nodes were involved (p = .013) and associated with TNM stages III–IV (p = .005) and microsatellite instability (p = .015). Significantly poorer survival rates were evidenced for CRC patients with lower expression in the tumor tissue of PER1 (p = .010), PER3 (p = .010), and CSNKIE (p = .024). In conclusion, abnormal expression levels of core clock genes in CRC tissue may be related to the process of tumorigenesis and exert an influence on host/tumor interactions. (Author correspondence: g.mazzoccoli@tin.it)

Mutation analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the cationic trypsinogen (PRSS1) gene, and the serine protease inhibitor, Kazal type 1 (SPINK1) gene in patients with alcoholic chronic pancreatitis.

Susceptibility to alcoholic chronic pancreatitis (ACP) could be genetically determined. Mutations in cationic trypsinogen (PRSS1), cystic fibrosis transmembrane conductance regulator (CFTR), and serine protease inhibitor, Kazal type 1 (SPINK1) genes have been variably associated with both the hereditary and the idiopathic form of chronic pancreatitis (CP). Our aim was to analyze the three genes in ACP patients. Mutational screening was performed in 45 unrelated ACP patients and 34 patients with alcoholic liver disease (ALD). No mutation of PRSS1 was found in ACP and ALD patients. Three mutations of CFTR were detected in four ACP patients with a prevalence (8.9%) not significantly different from that observed (3.0%) in ALD patients and from that expected (3.2%) in our geographical area. Neither compound heterozygotes for CFTR nor trans-heterozygotes for CFTR/SPINK1 were found. One ACP patient (2.2%) was found to carry the most common mutation (N34S) of SPINK1 compared to none of the ALD patients (P=NS). In five other patients (two with ACP and three with ALD) other rare variants, including P55S, were found. In contrast with the hereditary and the idiopathic forms of CP, in which mutations of PRSS1, CFTR, and SPINK1 genes may occur, ACP is still a ‘gene(s)-orphan’ disease. The supposed genetic susceptibility to ACP relies on other yet unknown gene(s) which could affect the alcohol metabolism or modulate the pancreatic inflammatory response to alcohol abuse.

Cancer risk associated with STK11/LKB1 germline mutations in Peutz-Jeghers syndrome patients: Results of an Italian multicenter study

BACKGROUND Germline mutations in the STK11/LKB1 gene cause Peutz-Jeghers syndrome, an autosomal-dominantly inherited condition characterized by mucocutaneous pigmentation, hamartomatous gastrointestinal polyposis, and an increased risk for various malignancies. We here report the results of the first Italian collaborative study on Peutz-Jeghers syndrome. AIMS To assess cancer risks in a large homogenous cohort of patients with Peutz-Jeghers syndrome, carrying, in large majority, an identified STK11/LKB1 mutation. METHODS One-hundred and nineteen patients with Peutz-Jeghers syndrome, ascertained in sixteen different Italian centres, were enrolled in a retrospective cohort study. Relative and cumulative cancer risks and genotype-phenotype correlations were evaluated. RESULTS 36 malignant tumours were found in 31/119 (29 STK11/LKB1 mutation carriers) patients. The mean age at first cancer diagnosis was 41 years. The relative overall cancer risk was 15.1 with a significantly higher risk (p < 0.001) in females (22.0) than in males (8.6). Highly increased relative risks were present for gastrointestinal (126.2) and gynaecological cancers (27.7), in particular for pancreatic (139.7) and cervical cancer (55.6). The Kaplan-Meier estimates for overall cumulative cancer risks were 20%, 43%, 71%, and 89%, at age 40, 50, 60 and 65 years, respectively. CONCLUSION Peutz-Jeghers syndrome entails markedly elevated cancer risks, mainly for pancreatic and cervical cancers. This study provides a helpful reference for improving current surveillance protocols.

Germline novel MSH2 deletions and a founder MSH2 deletion associated with anticipation effects in HNPCC

Hereditary non‐polyposis colorectal cancer (HNPCC) is caused by inactivating mutations of DNA mismatch repair genes. Large genomic rearrangements in these genes have been increasingly recognized as important causes of HNPCC. Using multiplex ligation‐dependent probe amplification, we identified three MSH2 deletions in Italian patients with HNPCC (proband A: exons 1–3, proband M: exon 8, and proband C: exons 1–6). Deletion breakpoint sequencing allowed us to develop rapid polymerase chain reaction‐based mutation screening, which confirmed the presence of the deletions in affected and asymptomatic individuals of families A, C, and M. While the exon 8 and exon 1–3 deletions appear to be novel, the MSH2 1–6 deletion found in family C is identical to the one recently documented in two branches of another unrelated Italian family (family V+Va). Haplotype analysis showed that the kindreds C and V+Va (both from northeastern Italy, both displaying clinical features of the Muir–Torre syndrome) shared a seven‐locus haplotype, indicating that the MSH2 1–6 deletion is probably a founder mutation. Families A, C, M, and V+Va all showed progressively earlier cancer‐onset ages in successive generations. Analysis of 23 affected parent–child pairs in the four kindreds showed median anticipation of 12 years in offsprings’ onset of cancer (p = 0.0001). No birth cohort effect was found. This is the first significant evidence of anticipation effects in HNPCC families carrying MSH2 deletions.

Changes in miR-143 and miR-21 expression and clinicopathological correlations in pancreatic cancers.

Objectives Despite advances in clinical management of pancreatic cancer (PC), there is still room for improvement in early detection, diagnosis, and treatment strategies. The role of microRNAs (miRNAs) in tumor biology might pinpoint an alteration in expression of miRNAs as new diagnostic/prognostic biomarkers. Methods Expression levels of miR-143 and miR-21 and correlations with clinicopathological features were analyzed in 26 matched pairs of tumor and adjacent noncancerous tissue samples collected from patients with PCs, including 18 pancreatic ductal adenocarcinomas (PDACs) and 8 adenocarcinomas of Vater’s papilla (PVACs). Results Compared to normal tissues, miR-143 was up-regulated in both PDAC and PVAC tumor samples (P = 0.0028 and P = 0.039, respectively). Conversely, alterations in miR-21 expression were significantly different in PDAC versus PVAC samples (P = 0.0049). Tumor levels of miR-21 were associated with preoperative serum levels of CA 19-9 (r = 0.63, P = 0.0022), whereas miR-143 expression was negatively correlated to lymph node spreading (r = −0.64; P = 0.0004). Correlation between miR-143 and miR-21 expression levels in patients with PDAC was observed (r = 0.53, P = 0.023). Conclusions Deregulation of miR-143 and miR-21 may reflect histological features and biological behavior of different PCs. Association data with clinical parameters might indicate a prognostic significance for miR-143 and miR-21 in PCs.

HLA-DRB1 Alleles May Influence Disease Phenotype in Patients With Inflammatory Bowel Disease: A Critical Reappraisal With Review of the Literature

PURPOSEThe HLA region has been implicated in determining the disease susceptibility or the clinical phenotype of inflammatory bowel disease. The aim of this study was to assess the relation between HLA-DRB1 alleles with the clinical features of Crohn’s disease and ulcerative colitis and the presence of anti-neutrophil cytoplasmic and anti-Saccharomyces cerevisiae antibodies.METHODSBlood samples were obtained from 102 Crohn’s disease patients, 114 ulcerative colitis patients, and 264 unrelated healthy controls. Anti-neutrophil cytoplasmics were detected by a standard immunofluorescence method, and anti-Saccharomyces cerevisiaes were examined by an enzyme-linked immunosorbent assay immunoglobulin G/immunoglobulin A commercial assay. HLA-DRB1 typing of 26 alleles was performed by polymerase chain reaction sequence-specific primes. Patients were phenotyped according to gender, disease location, extent, and behavior, surgical resection, need of steroid, and anti-neutrophil cytoplasmic/anti-Saccharomyces cerevisiae status.RESULTSAs a whole, after applying Bonferroni’s correction for multiple comparisons, no significant association of HLA-DRB1 alleles with Crohn’s disease or ulcerative colitis was found. After stratifying HLA-DRB1 alleles by clinical phenotypes of patients with ulcerative colitis, an excess of DRB1*1309*1320*1325*1329 allele (DR13) was found in conjunction with pancolitis (P < 0.0001), surgical resection (P < 0.0003), and extraintestinal manifestations (P < 0.0001). In Crohn’s disease patients, an excess of DRB1*0304*0305*0307*0309 allele (DR3) was found in those with colonic disease (P < 0.0001) and patients with extraintestinal manifestations (P = 0.0003). This statistical association, however, emerged in only 3 of 114 patients with ulcerative colitis and in 3 of 102 patients with Crohn’s disease. We found no association with the presence of anti-Saccharomyces cerevisiae or anti-neutrophil cytoplasmic.CONCLUSIONSSome clinical features of Crohn’s disease and ulcerative colitis may be influenced by specific HLA-DR alleles; in particular, in ulcerative colitis some alleles appear to segregate with more aggressive disease, whereas in Crohn’s disease different alleles cosegregate in patients with colonic disease and extraintestinal manifestations.