Giancarlo Freschi

Expression of Estrogen Receptor β in Colon Cancer Progression

Colon cancer is the most frequent neoplasia of the intestine. This pathology is the third highest cause of death from cancer with 430,000 deaths globally per year. Estrogen has also been implicated in the development and progression of colon cancer. Also sex-specific differences have been suggested to be involved in the process. Previous studies have shown the estrogen β receptor to be the dominant receptor type in normal colonic tissue and its down-regulation along with the progression of colorectal cancer. The presence of estrogen receptors and products of estrogen-related genes in the colon suggests that estrogens have direct effects on the colonic tissue. However, the specific effect of estrogens on a normal colon and the role in the colon carcinogenesis are far from clear. The aim of this study is to analyze by real-time polymerase chain reaction, the relative quantitative expression of the estrogen receptors β, β1, β2, and β5 in colon adenocarcinomas and to compare this expression with the respective in normal tissues. Moreover, we evaluate a possible correlation between estrogens receptor expressions and disease stages. Normal tissues show estrogen receptor β expression greater than pathologic tissues and the estrogen receptor β result as most expressed in the lower disease stages.

The esophageal microbiota in health and disease

The esophageal mucosa is among the sites colonized by human microbiota, the complex microbial ecosystem that colonizes various body surfaces and is increasingly recognized to play roles in several physiological and pathological processes. Our understanding of the composition of the esophageal microbiota in health and disease is challenged by the need for invasive sampling procedures and by the dynamic nature of the esophageal environment and remains limited in comparison with the information available for other body sites. Members of the genus Streptococcus appear to be the major components of the microbiota of the healthy esophagus, although the presence of several other taxa has also been reported. Dysbiosis, consisting of enrichment in some Gram‐negative taxa (including Veillonella, Prevotella, Haemophilus, Neisseria, Campylobacter, and Fusobacterium), has been reported in association with gastroesophageal reflux disease and is hypothesized to contribute to the evolution of this condition toward Barretts esophagus (which is the most common esophageal precancerous lesion) and, eventually, adenocarcinoma. Some Campylobacter species (mostly C. concisus) are also putatively involved in the progression of disease toward adenocarcinoma. However, variable findings have recently been reported in additional studies. Causative relationships between dysbiosis or specific bacterial species and esophageal diseases remain controversial and warrant further investigations.

Real-time PCR analysis of RNA extracted from formalin-fixed and paraffin-embeded tissues: effects of the fixation on outcome reliability.

In many pathologic circumstances, quantitative mRNA expression levels are important for evaluation of possible genome mutations. The development of real-time polymerase chain reaction (RT-PCR) technology has facilitated the realization of nucleic acid quantification. Potentially, quantitative PCR offers a number of advantages over traditional methods because it permits the use of small amounts of genetic material. In the present study, we optimize a RNA purification technique on specimens that are formalin-fixed, paraffin-embedded and we examine prolonged formalin fixation effects on quantitative RT-PCR analysis. We compared RNA levels with 70 colic mucosa samples using the cyclooxygenase 2 gene as marker. The difference in amplification successes between formalin-fixed tissues and formalin-fixed, paraffin-embedded tissues was not statistically significant. Moreover, we compared the expression of formalin-fixed samples with the expression of each fresh tissue. Wilcoxon Mann-Whitney test shows that only the difference in the expression levels of 1- or 3-hour formalin-fixed samples is not statistically significant with respect to other fixation times. We found that the mRNA can be reliably extracted from formalin fixed, paraffin-embedded tissue sections but that prolonged formalin fixation produces different results in quantitative RT-PCR. It can be related to difference in RNA sequences length and the generation of secondary structures that are more susceptible to the prolonged formalin fixation. We suppose that the paraffin do not influence the RNA extraction yield because there are no statistical significant differences between amplification success of formalin-fixed tissues and paraffin-embedded tissues. Therefore, in relative expression quantization, we confirm that it is appropriate to use specimens with same protocols and time for formalin fixation.

Barrett's esophagus: endoscopic diagnosis.

This collection of summaries on endoscopic diagnosis of Barretts esophagus (BE) includes the best endoscopic markers of the extent of BE; the interpretation of the diagnosis of ultra‐short BE; the criteria for endoscopic grading; the sensitivity and specificity of endoscopic diagnosis; capsule and magnifying endoscopy; narrow band imaging; balloon cytology; the distinction between focal and diffuse dysplasia; the techniques for endoscopic detection of dysplasia and the grading systems; and the difficulty of interpretation of inflammatory or regenerative changes.

Barrett's esophagus: proton pump inhibitors and chemoprevention II.

The following on proton pump inhibitors (PPIs) and chemoprevention in relation to Barretts esophagus includes commentaries on 48‐h pH monitoring, pH‐impedence, bile acid testing, dyspepsia, long/short segment Barretts esophagus, nonerosive reflux disease (NERD), functional heartburn, dual‐release delivery PPIs, immediate‐release PPIs, long‐term PPI use, prokinetic agents, obesity, baclofen, nocturnal acid breakthrough, nonsteroidal anti‐inflammatory drugs (NSAIDs), and new PPIs.

Barrett's esophagus: histology and immunohistology

The following on histology and immunohistology of Barretts esophagus (BE) includes commentaries on the various difficulties remaining in reaching a consensus on the definition of BE; the difficulties in the characterization of intestinal and cardiac mucosa, and in the role of submucosal glands in the development of BE; the importance of a new monoclonal antibody to recognize esophageal intestinal mucosa; the importance of pseudo goblet cells; the best techniques for the endoscopic detection of Barretts epithelium; and the biomarkers for identification of patients predisposed to the development of BE.

NF2 expression levels of gastrointestinal stromal tumors: a quantitative real-time PCR study

Gastrointestinal stromal tumors are the most common mesenchymal tumors of the gastrointestinal tract. Until today, there have been few markers specific for the tumor. This has complicated the differential diagnosis of the neoplasm from tumors of smooth muscle origin. Recently, the proto-oncogene c-kit has been shown to be a very relevant marker as it almost invariably is expressed in gastrointestinal stromal tumors. Radiation exposure, hormonal and genetic factors, particularly neurofibromatosis 2, have been implicated in their development and growth. GIST initiation, either in NF2-associated or in sporadic cases, is linked to inactivation of members of the proteins 4.1 superfamily. The majority of the mutations identified in the NF2 gene result in a truncated protein and are clinically associated with a severe phenotype. Occasionally, missense mutations associated with a mild phenotype may occur. We compared NF2 gene expression in 5 cases with gastrointestinal stromal tumors by quantitative real-time polymerase chain reaction analysis. NF2 gene mRNA expression was assessed in fresh tissue of stomach from 5 consecutive patients. We detected no alterations in NF2 gene expression in the quantitative analyses of the 5 tumors.

TNM staging and T-cell receptor gamma expression in colon adenocarcinoma. Correlation with disease progression?

Aims and Background Colorectal cancer is the second most common cause of cancer-related death in Europe and the United States. Several studies have evaluated the immune response to colorectal cancer, with contradictory results. Some studies showed that lymphocyte infiltration in colorectal cancer seemed to be an important prognostic parameter, a finding not confirmed by other studies. Several studies showed the gamma-delta T-cell receptor repertoire of intestinal adenocarcinoma. In this study, we hypothesize that the presence of T cells with the T-cell receptor gamma complex may play a particular role in carcinogenesis and tumor progression. Methods A total of 58 patients with colon adenocarcinoma was included in the analysis. We used the TNM staging system to grade colon cancer. Results Thirty samples (52.6%) revealed a polyclonal rearrangement of T-cell receptor gamma. In the N0 cases, only 5 samples revealed a T-cell receptor gamma molecular assessment; in N1/N2 cases, 25 revealed a T-cell receptor gamma molecular assessment. Conclusions The results showed statistical significance between the presence of T-cell receptor gamma and N1/N2 stage lymph nodes (P = 0.001).

Barrett's esophagus: Genetic and cell changes

The following includes commentaries on how genetic code of Barretts esophagus (BE) patients, the mechanisms for GERD‐induced esophageal expression of caudal homeobox, and the development of Barretts metaplasia are increasingly better known, including the role of stromal genes in oncogenesis. Additional lessons have been learned from in vitro models in nonneoplastic cell lines, yet there are limitations to what can be expected from BE‐derived cell lines. Other topics discussed include clonal diversity in Barretts esophagus; the application of peptide arrays to clinical samples of metaplastic mucosa; proliferation and apoptosis of Barretts cell lines; tissue biomarkers for neoplasia; and transcription factors associated with BE.

hERG1 behaves as biomarker of progression to adenocarcinoma in Barrett’s esophagus and can be exploited for a novel endoscopic surveillance

Barretts esophagus (BE) is the only well-known precursor lesion of esophageal adenocarcinoma (EA). The exact estimates of the annual progression rate from BE to EA vary from 0.07% to 3.6%. The identification of BE patients at higher risk to progress to EA is hence mandatory, although difficult to accomplish. In search of novel BE biomarkers we analyzed the efficacy of hERG1 potassium channels in predicting BE progression to EA. Once tested by immunohistochemistry (IHC) on bioptic samples, hERG1 was expressed in BE, and its expression levels increased during progression from BE to esophageal dysplasia (ED) and EA. hERG1 was also over-expressed in the metaplastic cells arising in BE lesions obtained in different BE mouse models, induced either surgically or chemically. Furthermore, transgenic mice which over express hERG1 in the whole gastrointestinal tract, developed BE lesions after an esophago-jejunal anastomosis more frequently, compared to controls. A case-control study was performed on 104 bioptic samples from newly diagnosed BE patients further followed up for at least 10 years. It emerged a statistically significant association between hERG1 expression status and risk of progression to EA. Finally, a novel fluorophore- conjugated recombinant single chain variable fragment antibody (scFv-hERG1-Alexa488) was tested on freshly collected live BE biopsies: it could recognize hERG1 positive samples, perfectly matching IHC data. Overall, hERG1 can be considered a novel BE biomarker to be exploited for a novel endoscopic surveillance protocol, either in biopsies or through endoscopy, to identify those BE patients with higher risk to progress to EA.