Characterization of ecto-nucleotidases in human oviducts with an improved approach simultaneously identifying protein expression and in situ enzyme activity.

Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those taking place in oviducts, including contraction, beating of cilia, and maintenance of fluid composition that, in turn, influences sperm capacitation and hyperactivation, as well as oocyte and embryo nourishing. Ecto-nucleotidases are the enzymes that regulate extracellular ATP and adenosine levels, thus playing a role in reproduction. We have optimized a convenient method for characterizing ecto-nucleotidases that simultaneously localizes the protein and its associated enzyme activity in the same tissue slice and characterizes ecto-nucleotidases in human oviducts. The technique combines immunofluorescence and in situ histochemistry, allowing precise identification of ecto-nucleotidases at a subcellular level. In oviducts, remarkably, ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) and NTPDase3, with the ability to hydrolyze ATP to AMP, are expressed in ciliated epithelial cells but with different subcellular localization. Ecto-5'nucleotidase/CD73 is also expressed apically in ciliated cells. CD73, together with alkaline phosphatase, also expressed apically in oviductal epithelium, complete the hydrolysis sequence by dephosphorylating AMP to adenosine. The concerted action of these enzymes would contribute to the local increase of adenosine concentration necessary for sperm capacitation. The use of this method would be an asset for testing new potential therapeutic drugs with inhibitory potential, which is of great interest presently in the field of oncology and in other clinical disciplines.

Preliminary evaluation of the safety and efficacy of glucose solution infusion through the hepatic artery on irreversible electroporation focusing.

Due to electrical features of the tissue, such as impedance, which have a significant impact on irreversible electroporation (IRE) function, the administration of glucose solution 5% (GS5%) through the hepatic artery would focus IRE on scattered liver tumors. By creating a differential impedance between healthy and tumor tissue. This study aimed to determine the effects of the GS5% protocol on healthy liver tissue and its safety. 21 male Athymic nude rats Hsd: RH-Foxn1mu were used in the study. Animals were split into two groups. In group 1, a continuous infusion through the gastroduodenal artery of GS5% was performed to measure the impedance with a dose of 0.008 mL/g for 16 min. In group 2, the animals were divided into two subgroups for infusions of GS5%. Group 2.1, at 0.008 mL/g for 16 min. Group 2.2 at 0.03 mL/g for 4 min. Blood samples were collected after anesthesia has been induced. The second sample, after catheterization of the artery, and the third after the GS5% infusion. All the animals were sacrificed to collect histological samples. The survival rate during the experiment was 100%. A considerable impact on the impedance of the tissue was noticed, on average up to 4.31 times more than the baseline, and no side effects were observed after GS5% infusion. In conclusion, impedance alteration by Glucose solution infusion may focus IRE on tumor tissue and decrease IRE's effects on healthy tissue.

KRAS Assessment Following ESMO Recommendations for Colorectal Liver Metastases. Is It Always Worth It?

BACKGROUND: Genetic evaluation is essential in assessing colorectal cancer (CRC) and colorectal liver metastasis (CRLM). The aim of this study was to determine the pragmatic value of KRAS on oncological outcomes after CRLM according to the ESMO recommendations and to query whether it is necessary to request KRAS testing in each situation. METHODS: A retrospective cohort of 126 patients who underwent surgery for hepatic resection for CRLM between 2009 and 2020 were reviewed. The patients were divided into three categories: wild-type KRAS, mutated KRAS and impractical KRAS according to their oncological variables. The impractical (not tested) KRAS group included patients with metachronous tumours and negative lymph nodes harvested. Disease-free survival (DFS), overall survival (OS) and hepatic recurrence-free survival (HRFS) were calculated by the Kaplan-Meier method, and a multivariable analysis was conducted using the Cox proportional hazards regression model. RESULTS: Of the 108 patients identified, 35 cases had KRAS wild-type, 50 cases had a KRAS mutation and the remaining 23 were classified as impractical KRAS. Significantly longer medians for OS, HRFS and DFS were found in the impractical KRAS group. In the multivariable analyses, the KRAS mutational gene was the only variable that was maintained through OS, HRFS and DFS. For HRFS (HR: 13.63; 95% confidence interval (CI): 1.35-100.62; p = 0.010 for KRAS), for DFS (HR: 10.06; 95% CI: 2.40-42.17; p = 0.002 for KRAS) and for OS (HR: 4.55%; 95% CI: 1.37-15.10; p = 0.013). CONCLUSION: Our study considers the possibility of unnecessary KRAS testing in patients with metachronous tumours and negative lymph nodes harvested. Combining the genetic mutational profile (i.e., KRAS in specific cases) with tumour characteristics helps patient selection and achieves the best prognosis after CRLM resection.

The impact of additional margin coagulation with radiofrequency in liver resections with subcentimetric margin: can we improve the oncological results? A propensity score matching study.

BACKGROUND: Whereas the usefulness of radiofrequency (RF) energy as haemostatic method in liver surgery has become well established in the last decades, its intentional application on resection margins with the aim of reducing local recurrence is still debatable. Our goal was to compare the impact of an additional application of RF energy on the top of the resection surface, namely additional margin coagulation (AMC), on local recurrence (LR) when subjected to a subcentimeter margin. METHODS: We retrospectively analyzed 185 patients out of a whole cohort of 283 patients who underwent radical hepatic resection with subcentimetric margin. After propensity score adjustment, patients were classified into two balanced groups according to whether RF was applied or not. RESULTS: No significant differences were observed within groups in baseline characteristics after PSM adjustment. The LR rate was significantly higher in the Control than AMC Group: 12 patients (14.5%) vs. 4 patients (4.8%) (p = 0.039). The estimated 1, 3, and 5-year LR-free survival rates of patients in the Control and AMC Group were: 93.5%, 86.0%, 81.0% and 98.8%, 97.2%, 91.9%, respectively (p = 0.049). Univariate Cox analyses indicated that the use of the RF applicator was significantly associated with lower LR (HR = 0.29, 95% confidence interval 0.093-0.906, p = 0.033). The Control Group showed smaller coagulation widths than the AMC group (p < 0.001). CONCLUSIONS: An additional application of RF on the top of the resection surface is associated with less local hepatic recurrence than the use of conventional techniques.

Microwave versus radiofrequency ablation for the treatment of liver malignancies: a randomized controlled phase 2 trial.

Microwave (MWA) and radiofrequency ablation (RFA) are main ablative techniques for hepatocellular carcinoma (HCC) and colorectal liver metastasis (MT). This randomized phase 2 clinical trial compares the effectiveness of MWA and RFA as well as morphology of corresponding ablation zones. HCC and MT patients with 1.5-4 cm tumors, suitable for ablation, were randomized into MWA or RFA Groups. The primary endpoint was short-to-long diameter ratio of ablation zone (SLR). Primary technical success (TS) and a cumulative local tumor progression (LTP) after a median 2-year follow-up were compared. Between June 2015 and April 2020, 82 patients were randomly assigned (41 patients per group). For the per-protocol analysis, five patients were excluded. MWA created larger ablation zones than RFA (p = 0.036) although without differences in SLR (0.5 for both groups, p = 0.229). The TS was achieved in 98% (46/47) and 90% (45/50) (p = 0.108), and LTP was observed in 21% (10/47) vs. 12% (6/50) (OR 1.9 [95% CI 0.66-5.3], p = 0.238) of tumors in MWA vs. RFA Group, respectively. Major complications were found in 5 cases (11%) vs. 2 cases (4%), without statistical significance. MWA and RFA show similar SLR, effectiveness and safety in liver tumors between 1.5 and 4 cm.

Pancreatic duct ligation reduces premalignant pancreatic lesions in a Kras model of pancreatic adenocarcinoma in mice.

Pancreatic duct ligation (PDL) in the murine model has been described as an exocrine pancreatic atrophy-inducing procedure. However, its influence has scarcely been described on premalignant lesions. This study describes the histological changes of premalignant lesions and the gene expression in a well-defined model of pancreatic ductal adenocarcinoma by PDL. Selective ligation of the splenic lobe of the pancreas was performed in Ptf1a-Cre(+/ki); K-ras LSLG12Vgeo(+/ki) mice (PDL-Kras mice). Three experimental groups were evaluated: PDL group, controls and shams. The presence and number of premalignant lesions (PanIN 1-3 and Atypical Flat Lesions-AFL) in proximal (PP) and distal (DP) pancreas were studied for each group over time. Microarray analysis was performed to find differentially expressed genes (DEG) between PP and PD. Clinical human specimens after pancreaticoduodenectomy with ductal occlusion were also evaluated. PDL-Kras mice showed an intense pattern of atrophy in DP which was shrunk to a minimal portion of tissue. Mice in control and sham groups had a 7 and 10-time increase respectively of risk of high-grade PanIN 2 and 3 and AFL in their DP than PDL-Kras mice. Furthermore, PDL-Kras mice had significantly less PanIN 1 and 2 and AFL lesions in DP compared to PP. We identified 38 DEGs comparing PP and PD. Among them, several mapped to protein secretion and digestion while others such as Nupr1 have been previously associated with PanIN and PDAC. PDL in Ptf1a-Cre(+/ki); K-ras LSLG12Vgeo(+/ki) mice induces a decrease in the presence of premalignant lesions in the ligated DP. This could be a potential line of research of interest in some cancerous risk patients.