Reference miRNAs for colorectal cancer: analysis and verification of current data.

MicroRNAs (miRNAs) hold great promise in cancer research. The use of appropriate reference miRNAs for normalization of qPCR data is crucial for accurate expression analysis. We present here analysis and verification of current data, proposing a workflow strategy for identification of reference miRNAs in colorectal cancer (CRC). We performed a systematic review of studies aimed to identify stable reference miRNAs in CRC through high-throughput screening. Among the candidate miRNAs selected from the literature we excluded those predicted to target oncogenes or tumor suppressor gene. We then assessed the expression levels of the remaining candidates in exosomes, plasma and tissue samples from CRC patients and healthy controls. The expression stability was evaluated by box-plot, ∆Cq analysis, NormFinder and BestKeeper statistical algorithms. The effects of normalisers on the relative quantification of the oncogenic miR-1290 was also assessed. Our results consistently showed that different combinations of miR-520d, miR-1228 and miR-345 provided the most stably expressed reference miRNAs in the three biological matrices. We identified suitable reference miRNAs for future miRNA expression studies in exosomes plasma and tissues CRC samples. We also provided a novel conceptual framework that overcome the need of performing ex novo identification of suitable reference genes in single experimental systems.

Epigenetic alteration: new insights moving from tissue to plasma - the example of PCDH10 promoter methylation in colorectal cancer.

BACKGROUND: Tumour-released DNA in blood represents a promising biomarker for cancer detection. Although epigenetic alterations such as aberrant promoter methylation represent an appealing perspective, the discordance existing between frequencies of alterations found in DNA extracted from tumour tissue and cell-free DNA (cfDNA) has challenged their practical clinical application. With the aim to explain this bias of agreement, we investigated whether protocadherin 10 (PCDH10) promoter methylation in tissue was associated with methylation pattern in matched cfDNA isolated from plasma of patients with colorectal cancer (CRC), and whether the strength of concordance may depend on levels of cfDNA, integrity index, as well as on different clinical-pathological features. METHODS: A quantitative methylation-specific PCR was used to analyse a selected CpG site in the PCDH10 promoter of 67 tumour tissues, paired normal mucosae, and matched plasma samples. The cfDNA integrity index and cfDNA concentration were assessed using a real-time PCR assay. RESULTS: The PCDH10 promoter methylation was detected in 63 out of 67 (94.0%) surgically resected colorectal tumours and in 42 out of 67 (62.7%) plasma samples. The median methylation rate in tumour tissues and plasma samples was 43.5% (6.3-97.8%) and 5.9% (0-80.9%), respectively. There was a significant correlation between PCDH10 methylation in cfDNA and tumour tissue in patients with early CRC (P<0.0001). The ratio between plasma and tissue methylation rate increases with increasing cfDNA integrity index in early-stage cancers (P=0.0299) and with absolute cfDNA concentration in advanced cancers (P=0.0234). CONCLUSION: Our findings provide new insight into biological aspects modulating the concordance between tissues and plasma methylation profiles.

The trochanteric fat pad.

Technological developments based on the use of autologous white adipose tissue (WAT) attracted attention to minor fat depots as possible sources of adipose tissue. In plastic surgery, the trochanteric fatty pad is one of the most used WAT depots for its location and organoleptic characteristics that make it particularly suitable for reconstructive procedures. Despite its wide use in clinic, the structure of this depot has never been studied in detail and it is not known if structural differences exist among trochanteric fat and other subcutaneous WAT depots. The present study was performed on trochanteric fat pad with the aim to clarify the morphology of its adipocytes, stroma and microcirculation, with particular reference to the stem niches. Histological and ultrastructural studies showed that the main peculiar feature of the trochanteric fat concerns its stromal component, which appears less dense than in the other subcutaneous WATs studied. The intra-parenchymal collagen stroma is poor and the extracellular compartment shows large spaces, filled with electron-light material, in which isolated collagen bundles are present. The adipocytes are wrapped in weak and easily detachable collagen baskets. These connective sheaths are very thin compared to the sheaths in other subcutaneous WAT depots. The capillaries are covered by large, long and thin elements surrounded by an external lamina; these perivascular cells are poor in organelles and mainly contain poly-ribosomes. In conclusion, when compared to other WAT deposits, the trochanteric fatty pad shows structural peculiarities in its stroma and microcirculation suggesting a high regenerative potential. Resistance, dissociability, microvascular weft and high regenerative potential make the trochanteric fatty pad a privileged source for harvesting in autologous WAT-based regenerative procedures.

A new model of rectal cancer with regional lymph node metastasis allowing in vivo evaluation by imaging biomarkers.

OBJECT: The work is aimed to develop a murine model of rectal cancer, which could be used to monitor lymph node metastasis development by magnetic resonance imaging (MRI) and optical imaging (OI) techniques. SUBJECTS AND METHODS: Ht-29 cancer cells were directly injected into the submucosal layer of the rectum of athymic nude mice using trans-anal rectal cancer cell injection (TARCI). Thirty-six mice were inoculated with 10×10(5) cells and five mice were treated with sterile phosphate buffer solution. One to 4 weeks after cell injection, tumor growth was evaluated in vivo using T2-weighted MRI at 4.7T. A further group of animal (n=6) treated with ht-29_luc cells, with the same protocol, was monitored by optical imaging. In both groups, the presence of the primary tumor and of lymph nodes metastasis was confirmed by histology. RESULTS: In all animals, primary tumors were detectable by MRI, 1 week from TARCI. After 4 weeks primary tumors showed a mean longitudinal diameter of about 2cm. All animals developed regional lymph node metastases. Others organs (e.g. lung or liver) were not affected. In fat-suppressed, T2-weighted MRI, lymph nodes appeared as small areas characterized by hyper-intense signal compared to muscle. OI permitted evaluation of the primary tumor growth in perineal region. CONCLUSIONS: TARCI of ht-29 cells into the rectum of nude mice is a feasible way to obtain a easily reproducible model of regional lymph node metastases could be monitored by magnetic resonance and optical imaging techniques.

[Endoscopic diverticulostomy for Zenker's diverticulum. Experience on 123 cases]. / Diverticulostomia endoscopica per diverticolo di Zenker. Esperienza su 123 casi.

AIM: To evaluate the short-term clinical outcome of the patients with Zenker 's diverticulum undergone to endoscopic esophago-diverticulostomy according to Collard. PATIENTS AND METHODS: A retrospective analysis evaluated 123 patients. The most common symptom was dysphagia, severe in 50 patients (40.6%) and moderate in 73 (59.4%), with a mean dysphagia score of 2.3 (range 0-4). Regurgitation was present in 70 cases (56.9%), with a mean score of 0.8 (range 0-2). The mean diameter of diverticula was 4.1 cm (range 2.5-10). The procedure was undertaken in 87 male and 36 female with a mean follow-up of 69.1 months (range 1-168). RESULTS: Good results were obtained in 82 patients (66.7%), and the improvement of symptoms in 24 (19.5%), data confirmed by pre versus postoperative dysphagia mean score (2.3 vs 0.4) and by regurgitation score (0.8 vs 0.2). The failures have been seen in 17 patients (13.8%). The mean time of the intervention was 18 minutes (range 15-40), while the mean length of postoperative hospital stay was 2.5 days (range 1-5), with a oral intake in first postoperative day (mean). Major complications were documented in 2 patients (1.6%), minor ones in 10 patients (6.1%), without mortality. CONCLUSION: Nowadays esophago-diverticulostomy according to Collard, based on our results and on the literature data, represent the most effective and safe technique for the patients with Zenker's diverticulum, with low rate of morbidity and mortality compared to the others surgical on endoscopic procedures.

Early laparoendoscopic rendezvous for acute biliary pancreatitis: preliminary results.

INTRODUCTION: Early restored patency of the papilla has been hypothesized to reduce complications and mortality of acute biliary pancreatitis. The aim of this study was to evaluate the role of urgent laparoscopic cholecystectomy with intraoperative cholangiography and rendezvous when necessary in acute biliary pancreatitis natural history. PATIENTS AND METHODS: Patients observed in the early stage of an acute biliary pancreatitis were included in the study. Operative risk assessment based on American Society of Anesthesiologists (ASA) score allowed the performance of urgent laparoscopic cholecystectomy within 72 h from onset of symptoms in 55 patients and a delayed intervention during the same admission in 21 patients. Intraoperative cholangiography was performed in all cases, and clearance of common bile duct was performed by flushing when possible, or rendezvous when necessary. Evolution of pancreatitis was evaluated with clinical and radiological monitoring. RESULTS: Urgent laparoscopic cholecystectomy was performed in all cases without conversion. At intraoperative cholangiography common bile duct was free in 25 patients, a papillary spasm was observed in 9, and common bile duct stones in 21 patients. Patency of the papilla was restored by flushing in 13 patients, while a rendezvous was necessary in 17 patients. The rate of organ failure and pancreatic necrosis was 1.8%, overall mortality was 1.8%, and overall morbidity 21.8%. No infectious complications of peripancreatic collections were observed. CONCLUSION: Urgent laparoscopic cholecystectomy with selective intraoperative rendezvous may be considered as a treatment option in the early stage of acute biliary pancreatitis.

Treatment of abdominal aortic aneurysms associated with pancreatic tumors: personal experience and review of the literature (1967-2006).

The aim of the present study was to discuss the approach to a rare, but challenging, clinical situation: the coexistence of an abdominal aortic aneurysm (AAA) and a pancreatic tumor. The authors present their experience and a review of the literature of the last 40 years. From January 1988 to December 2006 the authors faced 3 cases of associated AAA and pancreatic neoplasia. Through a Medline search the authors found 15 cases of this comorbidity reported in the literature from 1967 to 2006, obtaining a total number of 18 cases. The treatment of the two diseases was in a single stage in 4 cases (22%) and in two stages in 5 cases (28%), while only one pathology was treated in 7 cases (39%) and no treatment at all was attempted in 2 cases (11%). Mortality was 0%, while morbidity was 22%, i.e. in 4 cases out of 18, although no aortic prosthesis infection was recorded. From literature analysis and their experience the authors concluded that the surgical strategy in cases of AAA and a pancreatic tumor is to be chosen depending on the pancreatic tumor prognosis, the AAA dimensions and the schedule of chemotherapy. According to the authors, AAA surgical repair is recommended in case of pancreatic cystic adenoma and neuroendocrine neoplasia, in view of their good prognosis, while endovascular repair (EVAR), when feasible, is better in patients with pancreatic adenocarcinoma.

Treatment of abdominal aortic aneurysm associated with colorectal cancer: presentation of 14 cases and literature review.

PURPOSE: The coexistence of abdominal aortic aneurysm (AAA) and cancer is observed with increasing frequency, raising several questions about therapeutic and surgical strategies for management of both diseases. In this study, we present our experience on 14 patients affected by both colorectal cancer (CRC) and AAA, and we have also reviewed the literature from 1988 to 2005 for clinical experiences on this matter. MATERIALS AND METHODS: From January 1988 to May 2006, 1,012 AAA and 1,480 CRC were observed and treated in our department; in 14 patients (1.3% of AAA and 0.9% of CRC), both diseases were coexistent. We also performed a literature review from 1987 to 2005, and we found 254 cases of AAA associated with CRC. RESULTS: Priority was given for treatment of vascular disease. The diseases were treated in one stage in nine cases and in two stage in four patients; in the remaining case, only the CRC was treated due to patient's poor cardiac conditions. Postoperative (30-day) complications were seen in 1 of 14 patients (7.1%), whereas there were no postoperative deaths or prosthetic infections. In the literature review, treatment in one stage was performed in 102 cases and in two stage in 118 cases; in the remaining 25 cases, only one disease was treated (in 24 cases, for different reasons, only CRC was treated, whereas in the last case, only the AAA was treated, and the patient died in the postoperative period). Postoperative (30-day) morbidity and mortality in one-stage treatment were 8 and 4.5%, respectively, and 21.3 and 6% in two-stage treatments, respectively. In patients treated for only one disease, 30-day morbidity and mortality were 4 and 24%, respectively. Only one case of prosthetic infection was reported after a two-stage treatment. CONCLUSIONS: From the analysis of the literature and our experience, it is evident that, when AAA and CRC are coexistent with preoperative diagnosis of both diseases, single-stage intervention, when feasible for patient in general and local conditions, has to be preferred due to the lower morbidity. Single-stage treatment avoids a second surgical and anesthesiologic trauma and eliminates the risks joined with the non-treated lesion, increasing, however, the magnitude of the operation. Endovascular therapy, for its less invasiveness, appears to be an adequate solution for one-stage treatment of the two diseases but its role is still subject of ongoing discussions.

Laparoscopic hepatic resection.

BACKGROUND: Although laparoscopy in general surgery is increasingly being performed, only recently has liver surgery been performed with laparoscopy. We critically review our experience with laparoscopic liver resections. METHODS: From January 2000 to April 2004, we performed laparoscopic hepatic resection in 16 patients with 18 hepatic lesions. Nine lesions were benign in seven patients (five hydatid cysts, three hemangiomas, and one simple cyst), five were malignant in five patients (five hepatocarcinoma), and four patients had an uncertain preoperative diagnosis (one suspected hemangioma and three suspected adenomas). The mean lesion size was 5.2 cm (range, 1-12). Twelve lesions were located in the left lobe, three were in segment VI, one was in segment V, one was in segment IV, and one was in the subcapsular part of segment VIII. RESULTS: The conversion rate was 6.2%; intraoperative bleeding requiring blood transfusions occurred in two patients. Mean operative time was 120 min. Mean hospital stay was 4 days (range, 2-7). There were no major postoperative complications and no mortality. CONCLUSIONS: Hepatic resection with laparoscopy is feasible in malignant and benign hepatic lesions located in the left lobe and anterior inferior right lobe segments (IV, V, and VI). Results are similar to those of the open surgical technique in carefully selected cases, although studies with large numbers of patients are necessary to drawn definite conclusions.