Fingerprinting Acute Digestive Diseases by Untargeted NMR Based Metabolomics.

Precision medicine may significantly contribute to rapid disease diagnosis and targeted therapy, but relies on the availability of detailed, subject specific, clinical information. Proton nuclear magnetic resonance (¹H⁻NMR) spectroscopy of body fluids can extract individual metabolic fingerprints. Herein, we studied 64 patients admitted to the Florence main hospital emergency room with severe abdominal pain. A blood sample was drawn from each patient at admission, and the corresponding sera underwent ¹H⁻NMR metabolomics fingerprinting. Unsupervised Principal Component Analysis (PCA) analysis showed a significant discrimination between a group of patients with symptoms of upper abdominal pain and a second group consisting of patients with diffuse abdominal/intestinal pain. Prompted by this observation, supervised statistical analysis (Orthogonal Partial Least Squares⁻Discriminant Analysis (OPLS-DA)) showed a very good discrimination (>90%) between the two groups of symptoms. This is a surprising finding, given that neither of the two symptoms points directly to a specific disease among those studied here. Actually herein, upper abdominal pain may result from either symptomatic gallstones, cholecystitis, or pancreatitis, while diffuse abdominal/intestinal pain may result from either intestinal ischemia, strangulated obstruction, or mechanical obstruction. Although limited by the small number of samples from each of these six conditions, discrimination of these diseases was attempted. In the first symptom group, >70% discrimination accuracy was obtained among symptomatic gallstones, pancreatitis, and cholecystitis, while for the second symptom group >85% classification accuracy was obtained for intestinal ischemia, strangulated obstruction, and mechanical obstruction. No single metabolite stands up as a possible biomarker for any of these diseases, while the contribution of the whole ¹H⁻NMR serum fingerprint seems to be a promising candidate, to be confirmed on larger cohorts, as a first-line discriminator for these diseases.

Totally robotic intracorporeal side-to-side isoperistaltic strictureplasty for Crohn's disease.

The development of bowel-sparing techniques (strictureplasties) for extended stricturing Crohn's disease (CD) and the increased use of minimally invasive surgery (wound sparing) represent the two most important improvements in inflammatory bowel disease surgery from the origin. Nevertheless, the minimally invasive approach for extended stricturing forms is usually avoided primarily because of difficulties in performing complex intracorporeal sutures. We describe a totally intracorporeal robotic ileocecal resection with a yet described modified side-to-side isoperistaltic strictureplasty for an extended ileocecal CD. The strictureplasty was 6 cm long including the stricture in its middle part. Adopting this approach, the preserved small bowel was about 10 cm longer. Operative time was about 4 h, with a blood loss of about 50 ml. The patients' post-operative course was uneventful, enteral nutrition started at post-operative day 2 and gradual oral food intake from day 3. She was discharged on post-operative day 6. Histology confirmed a stricturing CD, and the patient is recurrence free at 34 months' follow-up. Our report suggests that robotic-assisted intracorporeal strictureplasty is feasible and that robotics could represent an interesting instrument for allowing the intersection between minimally invasive and bowel-sparing surgery for CD.

Results of Surgical Salvage Treatment for Anal Canal Cancer: A Retrospective Analysis with Overview of the Literature.

BACKGROUND AND AIM: Chemoradiotherapy (CRT) is the gold standard treatment for anal cancer, which permits the maintenance of the anal function. However, about 30-40% of patients develop local disease progression, for which surgery represents a good salvage therapy. The aim of this study is to evaluate survival and morbidity rate in patients who undergo salvage surgery in our single institution, with an overview of the literature. METHODS: A retrospective study was carried out on patients who underwent surgical treatment of anal canal cancer after failure of CRT. We evaluated overall survival at 1, 3, and 5 years and postoperative morbidity rate. RESULTS: Twenty patients who underwent radical surgery with abdominoperineal resection were included in the study. The survival rates at 1, 3, and 5 years were 75, 60, and 37.4%; with a disease-free survival of 67, 53, and 35%, respectively. There was no postoperative mortality. The morbidity rate was 35%. CONCLUSION: Surgery represents the recommended therapy for persistent or recurrent anal canal cancer after CRT, with a good survival rate and an acceptable morbidity.

Rationalisation of the surgical technique for minimally invasive laparoscopic ileal pouch-anal anastomosis after previous total colectomy for ulcerative colitis.

INTRODUCTION: No previous study clearly focuses on laparoscopic technique to perform the second stage surgery (proctectomy with ileal pouch-anal anastomosis [IPAA]) after total colectomy for acute/severe ulcerative colitis (UC). We describe the procedural steps for a simple and rational minimally invasive second stage surgery, reporting intra- and short-term post-operative results. PATIENTS AND METHODS: During the period December 2014-December 2015, 10 consecutive patients (8 males and 2 females) with mean age of 48 years underwent laparoscopic proctectomy and IPAA adopting our novel approach. They were operated 3 months after the previous total colectomy which has been performed, respectively, for acute (three patients) or severe (seven patients) UC. Intraoperative data and post-operative complications, divided as minor and major, were recorded and analysed. A body image questionnaire was administered to all patients to evaluate the cosmetic results of the procedure. RESULTS: Overall mean surgical time was 235 ± 49 min. During the post-operative course, three patients required morphine for >48 h, no patient needed blood transfusion and bowel movements recovery happened as mean during the 2nd day. No early major complications happened. Two patients (20%) developed peri-ileostomic wound infection at the right flank. Only one patient (10%) suffered from ileal-anal anastomotic dehiscence, conservatively treated till resolution. The average length of hospital stay was 8 ± 2 days. The body image questionnaire showed in all patients an extreme satisfaction about the results obtained (mean value = 59/64 points). CONCLUSIONS: Through three standardised surgical steps easily reproducible, we describe an almost scar-less procedure able to optimise the intraoperative time with good post-operative results in terms of complications and cosmesis.

Intra-tumoral IFN-γ-producing Th22 cells correlate with TNM staging and the worst outcomes in pancreatic cancer.

PDAC (pancreatic ductal adenocarcinoma) is the fifth leading cause of cancer-related death. The causes of this cancer remain unknown, but increasing evidence indicates a key role of the host immune response and cytokines in human carcinogenesis. Intra-tumoral IL (interleukin)-22 levels have been shown to be elevated in PDAC patients. However, little is known regarding the expression and clinical relevance of Th22 cells in human PDAC and, furthermore, which TILs (tumour-infiltrating lymphocytes) are the main producers of IL-22 is unknown. In the present study, we characterized the functional proprieties of the different subsets of IL-22-producing TILs and analysed their relationship with the TNM staging system and patient survival. We have demonstrated for the first time that, in PDAC patients, the T-cells co-producing IFN-γ (interferon γ) and exerting perforin-mediated cytotoxicity are the major intra-tumoral source of IL-22. In addition, isolated Th22 cells were able to induce apoptosis, which was antagonized by IL-22. Finally, we observed that the IL-22-producing T-cells were significantly increased in tumour tissue and that this increase was positively correlated with TNM staging of PDAC and poorer patient survival. These novel findings support the dual role of the anti-tumour immune system and that IL-22-producing cells may participate in PDAC pathogenesis. Therefore monitoring Th22 levels could be a good diagnostic parameter, and blocking IL-22 signalling may represent a viable method for anti-PDAC therapies.

Safer intestinal invagination for a solid pancreatico-jejunal anastomosis in presence of a soft texture pancreatic remnant and non-dilated duct.

Pancreatico-jejunal anastomosis after pancreatoduodenectomy still represents the Achilles' heel of the procedure: the failure of this anastomosis is relatively common and it is the main cause of post-operative morbidity and mortality. Studies have described different reconstruction strategies for the control of the development of post-operative pancreatic fistula, but the strategy to obtain a safer pancreatico-jejunal anastomosis is still far from satisfaction. We report a novel variation of the invagination technique based on preliminary clinical experience in 8 patients who underwent pancreatico-jejunal anastomosis after pancreatoduodenectomy in our hepatobiliopancreatic center from 2008 to 2014. The variation could obtain a safer intestinal invagination for a solid pancreatico-jejunal anastomosis even in the presence of soft pancreatic remnant.

Reconstructive strategy after pancreaticoduodenectomy in partially gastrectomized patients.

CONTEXT: Pancreaticoduodenectomy in partially-gastrectomized patients presents some peculiarities of the reconstructive phase. Above all, in B II and Roux-en-Y partial gastrectomies, a gastric re-resection with a redo gastrojejunal anastomosis should be avoided because it is often needlessly time-consuming and risky. In our series of 7 consecutive patients, either one of two reconstruction methods was used, depending upon the length of the pre-existing afferent loop. CASE REPORTS: In order to better illustrate this strategy, two cases of carcinoma of the duodenal papilla are reported. Both of the patients had previously undergone partial gastrectomy with B II reconstruction for peptic ulcers. Both were admitted to our hospital with a past history of jaundice. However, whereas in Case #2 a sufficiently long pre-existent afferent loop could be utilized for the pancreatic and bile duct anastomoses, in Case #1 a shorter afferent loop was removed and the efferent loop was utilized for the anastomoses. The postoperative course was uneventful in both patients. CONCLUSIONS: This reconstructive strategy for pancreaticoduodenectomy in gastrectomized patients, which uses either of the methods described above, has produced good results in our series of 7 patients and appears to be rational and straightforward.

Cyclooxygenase-2 and inflammation mediators have a crucial role in reflux-related esophageal histological changes and Barrett's esophagus.

BACKGROUND: Gastroesophageal reflux (GER) causes injury of the esophageal squamous epithelium, a condition called reflux esophagitis. The sequence reflux-esophagitis-intestinal metaplasia-dysplasia-invasive cancer is widely accepted as the main adenocarcinogenetic pathway in the esophagus; however, the mechanisms of this progression need to be better defined. AIMS: We evaluated COX-2 expression and activity in biopsies from patients affected with GER, and these parameters have been correlated with the stage of the disease, ceramide expression, apoptotic process, and angiogenesis. The effects of celecoxib on bile acid- and EGF-induced mucosal proliferation, apoptosis and angiogenesis have been also investigated. METHODS: Four groups of patients were distinguished: non esophagitis, non erosive esophagitis, erosive esophagitis, and Barrett's esophagus. COX-2 expression, basal PGE2 levels, proliferative activity, VEGF expression and apoptosis were evaluated in esophageal biopsies. RESULTS: COX-2 expression, basal PGE2 levels, proliferative activity, VEGF expression and apoptosis progressively increase from non esophagitis patients to patients with non erosive and erosive esophagitis, to those with BE. Incubation of the cells with DCA/EGF increases PGE2 production, proliferative activity and VEGF production, effects prevented by celecoxib pretreatment. Ceramide expression increased from non esophagitis patients to patients with non erosive and erosive esophagitis, and decreased in BE; caspase-3 activity progressively decreased from non esophagitis to BE patients, suggesting an impairment of the apoptotic process with disease progression. CONCLUSION: These results stand for a close relationship between progression of initial steps of gastroesophageal reflux disease (GERD) and COX-2, proliferative activity and EGF/VEGF expression and could have implications in GERD treatment in order to prevent its neoplastic evolution.

Ex vivo analysis of pancreatic cancer-infiltrating T lymphocytes reveals that ENO-specific Tregs accumulate in tumor tissue and inhibit Th1/Th17 effector cell functions.

Pancreatic cancer (PC) is an aggressive disease with dismal prognosis. Surgical resection is the recommended treatment for long-term survival, but patients with resectable PC are in the minority (with a 5-year survival rate of 20 %). Therefore, development of novel therapeutic strategies, such as anti-PC immunotherapy, is crucial. α-Enolase (ENO1) is an enzyme expressed on the surface of pancreatic cancer cells and is able to promote cell migration and cancer metastasis. The capacity of ENO1 to induce an immune response in PC patients renders it a true tumor-associated antigen. In this study, we characterized the effector functions of ENO1-specific T cells isolated from PC patients, and we specifically evaluated the successful role of intra-tumoral T helper 17 (Th17) cells and the inhibitory role of regulatory T (Tregs) cells in respectively promoting or reducing the cancer-specific immune response. In this ex vivo study, we have demonstrated, for the first time, that ENO1-specific Th17 cells have a specific anti-cancer effector function in PC patients, and that there are decreased levels of these cells in cancer compared to healthy mucosa. Conversely, there are elevated levels of ENO1-specific Tregs in PC patients which lead to inhibition of the antigen-specific effector T cells, thus highlighting a possible role in promoting PC progression. These results may be relevant for the design of novel immunotherapeutic strategies in pancreatic cancer.

The Cholegas Study: safety of prophylactic cholecystectomy during gastrectomy for cancer: preliminary results of a multicentric randomized clinical trial.

BACKGROUND: Cholelithiasis is more frequent in patients after gastrectomy, due to dissection of vagal branches and gastrointestinal reconstruction. METHODS: A randomized controlled trial was conducted from November 2008 to March 2012. Patients were randomized into two groups: prophylactic cholecystectomy (PC) and standard gastric surgery only (SS) for curable cancers. We planned three end points: evaluation of the number of patients who developed symptoms and needed further surgery for cholelithiasis after standard gastric cancer surgery, evaluation of the incidence of cholelithiasis overall after standard gastric cancer surgery and perioperative complications or costs of prophylactic cholecystectomy. The present study answers to the last end point only. RESULTS: After 40 months from the beginning of study, 172 patients were eligible from 9 Centers. Ten patients refused consent and 32 were excluded due to flawing of inclusion criteria (not confirmed adenocarcinomas and no R0 surgery). Therefore, final analysis included 130 patients: 65 in PC group and 65 in SS. Among PC group, 12 patients had surgical complications during the perioperative period; only 1 biliary leakage, conservatively treated, might have been caused by prophylactic cholecystectomy. 6 patients had surgical complications in SS group. One postoperative death occurred in PC group due to pulmonary embolism. Differences were not statistically significant. Similarly, no differences were significant in duration of surgery, blood loss, hospital stay. CONCLUSIONS: Concomitant cholecystectomy during standard surgery for gastric malignancies seemed to add no extra perioperative morbidity, mortality and costs to the sample included in the study.

Robotic Versus Open Liver Resection in Hepatocarcinoma: Surgical and Oncological Outcomes.

BACKGROUND: Minimally invasive approaches are spreading in every field of surgery, including liver surgery. However, studies comparing robotic hepatectomy with the conventional open approach regarding oncologic outcomes for hepatocellular carcinoma are limited. MATERIALS AND METHODS: We retrospectively reviewed demographics characteristics, pathologic features, surgical, and oncological outcomes of patients who underwent robotic and conventional open liver resection for hepatocellular carcinoma. RESULTS: No significant differences in demographics features, tumor size, tumor location, and type of liver resection were found. The morbidity rate was similar, 23% for the open group versus 17% of the robotic group (P=0.605). Perioperative data analysis showed a greater estimated blood loss in patients who underwent open resection, if compared with robotic group (P=0.003). R0 resection and disease-free resection margins showed no statistically significant differences. The 3-year disease-free survival of the robotic group was comparable with that of the open group (54% vs. 37%; P=0.592), as was the 3-year overall survival (87% vs. 78%; P=0.203). CONCLUSIONS: The surgical and the oncological outcomes seem to be comparable between minimally invasive and open hepatectomy. Robotic liver resections are effective, and do not compromise the oncological outcome, representing a reasonable alternative to the open approach.

Significant and Conflicting Correlation of IL-9 With Prevotella and Bacteroides in Human Colorectal Cancer.

Background and aim: Gut microbiota (GM) can support colorectal cancer (CRC) progression by modulating immune responses through the production of both immunostimulatory and/or immunosuppressive cytokines. The role of IL-9 is paradigmatic because it can either promote tumor progression in hematological malignancies or inhibit tumorigenesis in solid cancers. Therefore, we investigate the microbiota-immunity axis in healthy and tumor mucosa, focusing on the correlation between cytokine profile and GM signature. Methods: In this observational study, we collected tumor (CRC) and healthy (CRC-S) mucosa samples from 45 CRC patients, who were undergoing surgery in 2018 at the Careggi University Hospital (Florence, Italy). First, we characterized the tissue infiltrating lymphocyte subset profile and the GM composition. Subsequently, we evaluated the CRC and CRC-S molecular inflammatory response and correlated this profile with GM composition, using Dirichlet multinomial regression. Results: CRC samples displayed higher percentages of Th17, Th2, and Tregs. Moreover, CRC tissues showed significantly higher levels of MIP-1α, IL-1α, IL-1ß, IL-2, IP-10, IL-6, IL-8, IL-17A, IFN-γ, TNF-α, MCP-1, P-selectin, and IL-9. Compared to CRC-S, CRC samples also showed significantly higher levels of the following genera: Fusobacteria, Proteobacteria, Fusobacterium, Ruminococcus2, and Ruminococcus. Finally, the abundance of Prevotella spp. in CRC samples negatively correlated with IL-17A and positively with IL-9. On the contrary, Bacteroides spp. presence negatively correlated with IL-9. Conclusions: Our data consolidate antitumor immunity impairment and the presence of a distinct microbiota profile in the tumor microenvironment compared with the healthy mucosa counterpart. Relating the CRC cytokine profile with GM composition, we confirm the presence of bidirectional crosstalk between the immune response and the host's commensal microorganisms. Indeed, we document, for the first time, that Prevotella spp. and Bacteroides spp. are, respectively, positively and negatively correlated with IL-9, whose role in CRC development is still under debate.

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 NO 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).

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.

Poor pathogenetic role of luminal obstruction in the development of appendicitis: A case report.

RATIONALE: In developed countries, the incidence of acute appendicitis is about 95 cases out of 100,000 per year, being one of the most common urgencies in general surgery worldwide. However, its pathogenesis is still poorly understood. Direct luminal obstruction (by a fecalith, lymphoid hyperplasia, or impacted stool) is reported to be the primary and principal cause of acute appendicitis. PATIENT CONCERNS: During October 2016 a 58-year-old woman was operated because of a clinical recurrence of Crohn's disease. At surgery, performed through single incision laparoscopy, we observed an exceptional finding. DIAGNOSES: Despite a previous ileo-cecal resection, the appendix was still present and vascularized by small vessels within the mesoappendix connected to the neo-terminal ileum mesentery; it was about 5 cm long and macroscopically not inflamed even if its base was clearly no longer connected with the cecum. OUTCOMES: The patient underwent ileo-colic resection with en-bloc removal of the appendix. With a narrow metallic stylet probe we carefully tried to enter the appendix lumen through the opposite side from its fundus but we were not able to enter it before cutting the wall with scissors. Pathological examination confirmed the Crohn's disease recurrence affecting the small bowel and the appendix lumen obstructed in the presence of a fecalith but without any sign of inflammation. LESSONS: This finding seems to highlight the poor pathogenetic role of luminal obstruction in the development of acute appendicitis.

Comparing laparoscopic surgery with open surgery for long-term outcomes in patients with stage I to III colon cancer.

BACKGROUND: Although the short-term advantages of laparoscopy for colon cancer (CC) over open surgery have been clearly demonstrated, there is little evidence available concerning the long-term outcomes. This study aimed to compare the long-term results of laparoscopic surgery versus open surgery in a cohort of CC patients from a single center. METHODS: A series of 443 patients consecutively operated on for stage I to III CC between January 2006 and December 2013 were followed up. Patients were divided into two groups according to the surgical technique and were compared for disease-free survival (DFS) and overall survival (OS) before and after 1:1 propensity score matching. RESULTS: Due to exclusions and drop-outs, the statistical analysis of the study is based on 398 patients. Open surgery was performed in 133 patients, and laparoscopic surgery was performed in 265. After propensity score matching, two comparable groups of 89 patients each were obtained. The 5-year DFS was 64.3% and 78.2% for patients in the open and laparoscopic resection groups, respectively [hazard ratio (HR) 0.63, 95% confidence interval (CI) 0.33-1.19; P = 0.148]. A 5-year OS of 72.1% and 86.8% was observed in the open and laparoscopic resection groups, respectively (HR 0.43, 95%CI 0.20-0.94; P = 0.026). The multivariate survival analysis demonstrated better results of laparoscopy compared with open surgery for both DFS (HR 0.43, 95%CI 0.23-0.78; P = 0.004) and OS (HR 0.28, 95%CI 0.14-0.59; P < 0.001). CONCLUSIONS: Despite the limitations of a retrospective analysis, our study confirms better results for laparoscopic surgery in terms of DFS and OS compared with open surgery in CC treatment.

Analysis of Prognostic Factors for Resected Synchronous and Metachronous Liver Metastases from Colorectal Cancer.

BACKGROUND: Surgical treatment is the cornerstone in the management of colorectal cancer (CRC) liver metastases. The aim of this study is to identify clinicopathological factors affecting disease-free (DFS) and overall survival (OS) in patients undergoing potentially curative liver resection for CRC metastasis. METHODS: All consecutive patients undergoing liver resection for first recurrence of CRC from February 2006 to February 2018 were included. Prognostic impact of factors related to the patient, primary and metastatic tumors, was retrospectively tested through univariate and multivariate analyses. RESULTS: Seventy patients were included in the study. Median postoperative follow-up was 37 months (range 1-119). Median DFS and OS were 15.2 and 62.7 months, and 5-year DFS and OS rates were 16% and 53%. In univariate analysis, timing of metastasis presentation/treatment (combined colorectal and liver resection, "bowel first" approach or metachronous presentation) (p < 0.0001), ASA score (p = 0.003), chemotherapy after liver surgery (p = 0.028), T stage (p = 0.021), number of resected liver lesions (p < 0.0001), and liver margin status (p = 0.032) was significantly associated with DFS while peritoneal resection at colorectal surgery (p = 0.026), ASA score (p = 0.036), extension of liver resection (p = 0.024), chemotherapy after liver surgery (p = 0.047), and positive nodes (p = 0.018) with OS. In multivariate analysis, timing of metastasis presentation/treatment, ASA score, and chemotherapy (before and after liver surgery) resulted significantly associated with DFS and timing of metastasis presentation/treatment, positive nodes, peritoneal resection at colorectal surgery, and surgical approach (open or minimally invasive) of colorectal resection with OS. CONCLUSIONS: Surgery may provide good DFS and OS rates for CRC liver metastasis. Patient selection for surgery and correct timing of intervention within a multidisciplinary approach may be improved by taking into account negative prognostic factors which stress the importance of systemic therapy.

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.