Comparison of microbiological profile of enterotoxigenic Bacteroides fragilis (ETBF) isolates from subjects with colorectal cancer (CRC) or intestinal pre-cancerous lesions versus healthy individuals and evaluation of environmental factors involved in intestinal dysbiosis.

OBJECTIVE: The aim of this study was to analyze enterotoxigenic Bacteroides fragilis (ETBF) isolates from colorectal biopsies of subjects with a histological analysis positive for colorectal cancer (CRC), pre-cancerous lesions (pre-CRC) or with a healthy intestinal tissue and to evaluate the environmental factors that may not only concur to CRC development but may also affect gut microbiota composition. METHODS: ETBF isolates were typed using the ERIC-PCR method, while PCR assays were performed to investigate the bft alleles, the B. fragilis pathogenicity island (BFPAI) region and the cepA, cfiA and cfxA genes. Susceptibility to antibiotics was tested using the agar dilution method. Environmental factors that could play a role in promoting intestinal dysbiosis were evaluated throughout a questionnaire administered to the subjects enrolled. RESULTS: Six different ERIC-PCR types were identified. The type denominated C in this study was the most prevalent, in particular among the biopsies of subjects with pre-CRC, while an isolate belonging to a different type, denominated F, was detected in a biopsy from a subject with CRC. All the ETBF isolates from pre-CRC or CRC subjects had a B. fragilis pathogenicity island (BFPAI) region pattern I, while those from healthy individuals showed also different patterns. Furthermore, 71% of isolates from subjects with pre-CRC or CRC were resistant to two or more classes of antibiotics vs 43% of isolates from healthy individuals. The B. fragilis toxin BFT1 was the most frequently detected in this study, confirming the constant circulation of this isoform strains in Italy. Interestingly, BFT1 was found in 86% of the ETBF isolates from patients with CRC or pre-CRC, while the BFT2 was prevalent among the ETBF isolates from healthy subjects. No substantial differences based on sex, age, tobacco and alcohol consumption were observed between healthy and non-healthy individuals included in this study, while most of the subjects with CRC or pre-CRC lesions were subjected to pharmacological therapy (71%) and showed a body mass index (BMI) that falls within the overweight range (86%). CONCLUSIONS: Our data suggest that some types of ETBF seem to better adapt and colonize the human gut and that the selective pressure exerted by factors related to lifestyle, such as pharmacological therapy and weight, could facilitate their persistence in the gut and their possible involvement in CRC development.

DCLK1, a Putative Stem Cell Marker in Human Cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a very aggressive cancer showing the presence of high cancer stem cells (CSCs). Doublecortin-like kinase1 (DCLK1) has been demonstrated as a CSC marker in different gastroenterological solid tumors. Our aim was to evaluate in vitro the expression and the biological function of DCLK1 in intrahepatic CCA (iCCA) and perihilar CCA (pCCA). APPROACH AND RESULTS: Specimens surgically resected of human CCA were enzymatically digested, submitted to immunosorting for specific CSC markers (LGR5 [leucine-rich repeat-containing G protein-coupled receptor], CD [clusters of differentiation] 90, EpCAM [epithelial cell adhesion molecule], CD133, and CD13), and primary cell cultures were prepared. DCLK1 expression was analyzed in CCA cell cultures by real-time quantitative PCR, western blot, and immunofluorescence. Functional studies have been performed by evaluating the effects of selective DCLK1 inhibitor (LRRK2-IN-1) on cell proliferation (MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay, cell population doubling time), apoptosis, and colony formation capacity. DCLK1 was investigated in situ by immunohistochemistry and real-time quantitative PCR. DCLK1 serum concentration was analyzed by enzyme-linked immunosorbent assay. We describe DCLK1 in CCA with an increased gene and protein DCLK1 expression in pCCALGR5+ and in iCCACD133+ cells compared with unsorted cells. LRRK2-IN-1 showed an anti-proliferative effect in a dose-dependent manner. LRRK2-IN-1 markedly impaired cell proliferation, induced apoptosis, and decreased colony formation capacity and colony size in both iCCA and pCCA compared with the untreated cells. In situ analysis confirmed that DCLK1 is present only in tumors, and not in healthy tissue. Interestingly, DCLK1 was detected in the human serum samples of patients with iCCA (high), pCCA (high), HCC (low), and cirrhosis (low), but it was almost undetectable in healthy controls. CONCLUSIONS: DCLK1 characterizes a specific CSC subpopulation of iCCACD133+ and pCCALGR5+ , and its inhibition exerts anti-neoplastic effects in primary CCA cell cultures. Human DCLK1 serum might represent a serum biomarker for the early CCA diagnosis.

Association of Polygenic Risk Score and Bacterial Toxins at Screening Colonoscopy with Colorectal Cancer Progression: A Multicenter Case-Control Study.

Colorectal cancer (CRC) is a leading cause of cancer death worldwide, and its incidence is correlated with infections, chronic inflammation, diet, and genetic factors. An emerging aspect is that microbial dysbiosis and chronic infections triggered by certain bacteria can be risk factors for tumor progression. Recent data suggest that certain bacterial toxins implicated in DNA attack or in proliferation, replication, and death can be risk factors for insurgence and progression of CRC. In this study, we recruited more than 300 biopsy specimens from people undergoing colonoscopy, and we analyzed to determine whether a correlation exists between the presence of bacterial genes coding for toxins possibly involved in CRC onset and progression and the different stages of CRC. We also analyzed to determine whether CRC-predisposing genetic factors could contribute to bacterial toxins response. Our results showed that CIF toxin is associated with polyps or adenomas, whereas pks+ seems to be a predisposing factor for CRC. Toxins from Escherichia coli as a whole have a higher incidence rate in adenocarcinoma patients compared to controls, whereas Bacteroides fragilis toxin does not seem to be associated with pre-cancerous nor with cancerous lesions. These results have been obtained irrespectively of the presence of CRC-risk loci.

Metformin exerts anti-cancerogenic effects and reverses epithelial-to-mesenchymal transition trait in primary human intrahepatic cholangiocarcinoma cells.

Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive cancer with marked resistance to chemotherapeutics without therapies. The tumour microenvironment of iCCA is enriched of Cancer-Stem-Cells expressing Epithelial-to-Mesenchymal Transition (EMT) traits, being these features associated with aggressiveness and drug resistance. Treatment with the anti-diabetic drug Metformin, has been recently associated with reduced incidence of iCCA. We aimed to evaluate the anti-cancerogenic effects of Metformin in vitro and in vivo on primary cultures of human iCCA. Our results showed that Metformin inhibited cell proliferation and induced dose- and time-dependent apoptosis of iCCA. The migration and invasion of iCCA cells in an extracellular bio-matrix was also significantly reduced upon treatments. Metformin increased the AMPK and FOXO3 and induced phosphorylation of activating FOXO3 in iCCA cells. After 12 days of treatment, a marked decrease of mesenchymal and EMT genes and an increase of epithelial genes were observed. After 2 months of treatment, in order to simulate chronic administration, Cytokeratin-19 positive cells constituted the majority of cell cultures paralleled by decreased Vimentin protein expression. Subcutaneous injection of iCCA cells previously treated with Metformin, in Balb/c-nude mice failed to induce tumour development. In conclusion, Metformin reverts the mesenchymal and EMT traits in iCCA by activating AMPK-FOXO3 related pathways suggesting it might have therapeutic implications.

Laparoscopic Liver Surgery: What Are the Advantages in Patients with Cirrhosis and Portal Hypertension? Systematic Review and Meta-Analysis with Personal Experience.

Background: Laparoscopic surgery is a choice of treatment for liver diseases; it can decrease postoperative morbidity and length of hospital stay (LOS). Hepatocellular carcinoma (HCC) in patients with cirrhosis and portal hypertension may benefit from minimally invasive liver resections (MILRs) instead of open liver resections (OLRs). Whether minimally invasive approaches are superior to conventional ones is still a matter of debate. We thus aimed to gather the available literature on this specific topic to achieve greater clarity. Materials and Methods: PubMed, EMBASE and Web of Sciences databases were assessed for studies comparing OLRs versus MILRs for HCC in cirrhotic patients up to February 2020. Data from our surgical experience from June 2010 to February 2020 were also included. Demographic characteristics, liver function, the presence of portal hypertension, tumor number, and tumor size and location were assessed; operative time, need for Pringle maneuver, estimated blood loss (EBL), major or minor hepatectomy performance, and conversion rate were evaluated for operative findings. Postoperative outcomes and liver-related complications, surgical site infection (SSI) rate, blood transfusion (BT) rate, need for reintervention, LOS, in-hospital or 30-day mortality, and radicality of resection were also considered. Meta-analysis was performed employing Review Manager 5.3 software. Results: One thousand three hundred twenty-one patients from 13 studies and our own series were considered in the meta-analysis. At preoperative settings, the OLR and MILR groups differed significantly only by tumor size (4.4 versus 3.0, P = .006). Laparoscopic procedures resulted significantly faster (120.32-330 minutes versus 146.8-342.75 minutes, P = .002) and with lower EBL than open ones (88-483 mL versus 200-580 mL, P < .00001), thus requiring less BTs (7.9% versus 13.2%, P = .02). In terms of overall morbidity, minimally invasive surgeries resulted significantly favorable (19.32% versus 38.04%, P < .00001), as well as for ascites (2.7% versus 12.9% P < .00001), postoperative liver failure (7.51% versus 13.61% P = .009), and SSI (1.8% versus 5.42%, P = .002). Accordingly, patients who had undergone MILRs had significantly shorter postoperative hospitalization than patients who underwent conventional open surgery (2.4-36 days versus 4.2-19 days P < .00001). Both groups did not differ in terms of mortality rate and radicality of resection (OLR 93.8% versus 96.1% laparoscopic liver resection, P = .12). Conclusions: Based on the available evidence in the literature, laparoscopic resections rather than open liver ones for HCC surgery in cirrhotic patients seem to reduce postoperative overall morbidity, liver-specific complications, and LOS. The lack of randomized studies on this topic precludes the possibility of achieving defining statements.

Primary leiomyoma of the liver in an immunocompetent patient.

Primary leiomyoma of the liver (PLL) is a rare benign tumor occurring in immunosuppressed people. From 1926 less than fifty cases are reported in the scientific literature and about half are in immunocompetent patients. Etiology of this kind of lesion is not yet well known. We report a case of primary hepatic leiomyoma in a 60-year-old immunocompetent woman. The patient presented with lipothymia with unexpected vomiting. She underwent an ultrasound (US), and a computed tomography (CT) scan that revealed the presence of a single, solid lesion about 9 cm located between the S5 and S8 segment of the liver. It showed a well-defined, heterogeneous hypodensity with internal and peripheral enhancement and various central hypoattenuating areas and no wash-out in the portal and the late phases. Because of her symptoms and the risk of malignancy, the patient underwent a surgical liver resection. Histological diagnosis was primary leiomyoma of the liver. The patient had an uneventful recovery and was discharged after 7 days. At 30 months follow-up there were no symptoms and no evidence of disease. Leiomyoma of the liver is a rare benign neoplasm of which clinical symptoms are nonspecific and the exact radiological diagnosis still remains a challenge for radiologists. Etiology is still unclear and usually PLL represents an incidental diagnosis. Surgery plays a primary role not only in the treatment algorithm, but also in the diagnostic workout.

Robotic Liver Resections: Application of Difficulty Score Systems to an Initial Experience. Is a Specific Robotic Difficulty Score Necessary?

Background: Recently, the minimally invasive surgical approach has been available for performing liver resections (LRs) with laparoscopic and robotic techniques. The robotic approach for LRs seems to overcome several laparoscopic limitations, which is a valid alternative when performed in high volume and specialized centers. Laparoscopic difficulty score systems (DSSs) should serve to guide the surgeon's choice in the best surgical approach to adopt for every single patient, giving the possibility to switch to the open approach when needed. To this day, no specific robotic difficulty scores exist. The aim of our study was to verify the feasibility of applying these scores and related updates on robotic LRs performed in our Institute. Materials and Methods: Out of a total of 683 LRs performed from June 2010 to July 2019, 60 were performed through using a mini invasive approach and of these 18 were performed robotically. The Ban DSS and subsequently the modified Iwate DSS were applied to our cases. Results: Based on our findings, applying the DSS we divided our series into two groups: a low difficulty level group (1-3) made up of 5 patients, and an intermediate difficulty level group (4-6) consisting of 13 patients. Average Ban DSS and subsequently updated score system results were 4.6 ± 1.5 points (range 2-6) for both scores. Conclusions: Difficulties were encountered in applying the score when simultaneous multiple wedge resections were performed. The laparoscopic DSS is applicable to robotic LRs with some limitations due to the peculiarity of the two different minimally invasive approaches. A specific robotic difficulty rating score could be necessary to include these elements.

Anterior robotic approach in en-bloc sacrectomy: a preliminary experience.

En-bloc sacrectomy is a highly demanding surgical procedure necessary to obtain wide margin in sacral tumor. The double approach, anterior and posterior approach, is usually preferred for tumors extending proximally to S3 level where iliac internal vessels are at a higher risk for damage during posterior surgery. It can be justified also in selected cases to decrease the risk of posterior approach as in local recurrence or in patients who already underwent laparotomy. Our intent was to apply robotic-assisted techniques for performing anterior preparatory approach for sacrectomy surgery. Between December 2010 and December 2014, three cases of sacrectomies were performed in a previous robotic-assisted preparatory approach to separate the rectum from the tumor. Dissections were successfully performed in all cases close to the pelvic floor. The surgeon was able to position a Gore-Tex spacer between the anterior tumor surface and the rectum in all cases. The anterior dissections were performed with a perfect control of bleeding. No complications related to the anterior approach were reported. Robot-assisted surgery can be considered a valid and minimally invasive technique which allows a safe anterior dissection of the pelvic structures dividing tumors from surrounding tissues. It allows to place a spacer to protect organs during posterior sacral resection performed on the same day or at a later time. Further experiences are advocated to evaluate its efficiency in sacral tumors of greater size.