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Background: Low muscle mass quantity and quality (myosteatosis) can be evaluated by computed tomography (CT) by measuring skeletal muscle area and muscular attenuation, respectively, at the third lumbar vertebra. We aimed to define cut-off points of skeletal muscle area and muscular attenuation to predict mortality in non-dialysis chronic kidney disease (CKD) patients., Methods: We conducted a retrospective study including non-dialysis CKD patients over two years, who underwent an opportunistic computed tomography within a two year period, and for whom creatinine was measured within 90 days of CT. Skeletal muscle area was normalized for stature to calculate the skeletal muscle index. Area under the receiver operating characteristic (AuROC) curve and Youden's index were used, to identify the cut-point, separately according to sex., Results: One hundred sixty-seven patients (50.9% male, mean age of 68.3 ± 16.4 years) were included, most with CKD stages 3 and 4. During a median follow-up of 4.9 (4.2) years, 39 (23.4%) patients died. Muscular attenuation showed a better ability to predict mortality (AuROC curve 0.739 [95% CI 0.623-0.855] in women and 0.744 in men [95% CI 0.618-0.869]) than skeletal muscle index (AuROC curve 0.491 [95% CI 0.332-0.651] in women and 0.711 [95% CI 0.571-0.850] in men). For muscular attenuation, the best cut-off values to predict mortality were 27.56 Hounsfield units in women and 24.58 Hounsfield units in men. For skeletal muscle index, the best cut-off values were 38.47 cm 2 /m 2 in women and 47.81 cm 2 /m 2 in men. In univariable Cox-regression both low muscle mass and myosteatosis were associated with increased mortality. In multivariable Cox-regression models only myosteatosis maintained a significant association with mortality (Hazard Ratio 2.651 (95% CI 1.232-5.703, p = 0.013))., Conclusions: We found sex-specific cut-off values for muscle parameters using CT analysis in non-dialysis CKD patients that were associated with mortality. In this population, myosteatosis may be more closely associated with mortality than muscle quantity., (© 2024. The Author(s) under exclusive licence to Italian Society of Nephrology.)

Colorectal cancer (CRC) responses to KRAS-targeted inhibition have been limited due to low response rates, the mechanisms of which remain unknown. Herein, we explored the cancer-associated fibroblasts (CAFs) secretome as a mediator of resistance to KRAS silencing. CRC cell lines HCT15, HCT116, and SW480 were cultured either in recommended media or in conditioned media from a normal colon fibroblast cell line (CCD-18Co) activated with rhTGF-β1 to induce a CAF-like phenotype. The expression of membrane stem cell markers was analyzed by flow cytometry. Stem cell potential was evaluated by a sphere formation assay. RNAseq was performed in KRAS-silenced HCT116 colonospheres treated with either control media or conditioned media from CAFs. Our results demonstrated that KRAS-silencing up-regulated CD24 and down-regulated CD49f and CD104 in the three cell lines, leading to a reduction in sphere-forming efficiency. However, CAF-secreted factors restored stem cell marker expression and increased stemness. RNA sequencing showed that CAF-secreted factors up-regulated genes associated with pro-tumorigenic pathways in KRAS-silenced cells, including KRAS, TGFβ, NOTCH, WNT, MYC, cell cycle progression and exit from quiescence, epithelial-mesenchymal transition, and immune regulation. Overall, our results suggest that resistance to KRAS-targeted inhibition might derive not only from cell-intrinsic causes but also from external elements, such as fibroblast-secreted factors.

Nanostructured lipid carriers (NLCs) have the potential to increase the bioavailability and reduce the side effects of docetaxel (DTX). However, only a small fraction of nanoparticles given intravenously can reach a solid tumor. In situ-forming gels combined with nanoparticles facilitate local administration and promote drug retention at the tumor site. Injectable hydrogels based on poloxamer 407 are excellent candidates for this hybrid nanoparticle-hydrogel system because of their thermoresponsive behavior and biocompatibility. Therefore, this work aimed to develop injectable poloxamer hydrogels containing NLCs for intratumoral delivery of DTX. To ensure sterility, the obtained hydrogels were autoclaved (121 °C for 15 min) after preparation. Then, the incorporation of NLCs into the poloxamer hydrogels and the impact of steam sterilization on the nanocomposite hydrogels were evaluated concerning sol-gel transition, injectability, and physicochemical stability. All formulations were extruded through the tested syringe-needle systems with acceptable force (2.2-13.4 N) and work (49.5-317.7 N·mm) of injection. Following steam sterilization, injection became easier in most cases, and the physicochemical properties of all hydrogels remained practically unchanged according to the spectroscopical and thermal analysis. The rheological evaluation revealed that the nanocomposite hydrogels were liquid at 25 °C and underwent rapid gelation at 37 °C. However, their sterilized counterparts gelled at 1-2 °C above body temperature, suggesting that the autoclaving conditions employed had rendered these nanocomposite hydrogels unsuitable for local drug delivery.

The specific interaction between cell surface receptors and corresponding antibodies has driven opportunities for developing targeted cancer therapies using nanoparticle systems. It is challenging to design and develop such targeted nanomedicines using antibody ligands, as the final nanoconjugate's specificity hinges on the cohesive functioning of its components. The multicomponent nature of antibody-conjugated nanoparticles also complicates the characterization process. Regardless of the type of nanoparticle, it is essential to perform physicochemical characterization to establish a solid foundation of knowledge and develop suitable preclinical studies. A meaningful physicochemical evaluation of antibody-conjugated nanoparticles should include determining the quantity and orientation of the antibodies, confirming the antibodies' integrity following attachment, and assessing the immunoreactivity of the obtained nanoconjugates. In this review, the authors describe the various techniques (electrophoresis, spectroscopy, colorimetric assays, immunoassays, etc.) used to analyze the physicochemical properties of nanoparticles functionalized with antibodies and discuss the main results.

The widespread push to invest in local cancer therapies comes from the need to overcome the limitations of systemic treatment options. In contrast to intravenous administration, local treatments using intratumoral or peritumoral injections are independent of tumor vasculature and allow high concentrations of therapeutic agents to reach the tumor site with minimal systemic toxicity. Injectable biodegradable hydrogels offer a clear advantage over other delivery systems because the former requires no surgical procedures and promotes drug retention at the tumor site. More precisely, in situ gelling systems based on poloxamers have garnered considerable attention due to their thermoresponsive behavior, biocompatibility, ease of preparation, and possible incorporation of different anticancer agents. Therefore, this review focuses on the use of injectable thermoresponsive hydrogels based on poloxamers and their physicochemical and biological characterization. It also includes a summary of these hydrogel applications in local cancer therapies using chemotherapy, phototherapy, immunotherapy, and gene therapy.

Objective: We investigated the association between body composition upon diagnosis and complicated phenotypes and time until surgery in patients with Crohn's disease (CD)., Methods: We conducted a retrospective cohort study including patients with CD who had a computed tomography enterography or a magnetic resonance enterography performed ≤6 mo of diagnosis. Skeletal muscle and visceral and subcutaneous adipose tissue cross-sectional areas were determined with computed tomography or magnetic resonance images at the third lumbar vertebral level, processed with the sliceOmatic (TomoVison, Magog, QC, Canada) and ABACS plugin., Results: We included 63 patients: 33 (52%) men, median age 35 y. Disease location (L) and behavior (B) according to the Montreal classification were L1 (ileal disease) = 28 (44%), L2 (colonic disease) = 13 (21%), L3(ileocolonic disease) = 18 (28%), L1 + L4 (ileal and isolated upper disease) = 1 (2%), L3 + L4 (ileocolonic and isolated upper disease) = 3 (5%), B1 (non-stricturing) = 39 (62%), B2 (stricturing) = 11 (17%), and B3 (penetrating)= 13 (21%); 20 (32%) patients had perianal disease. Visceral obesity was present in 12 (19%) patients and was associated with higher age of CD onset (median 60 versus 34 y; P = 0.002) and complicated disease behavior (B2 and B3) (66.7% versus 31.7%; P = 0.021). After adjusting for age and perianal disease, total adipose tissue was associated with a 4% increase in the odds of complicated behavior per 10 cm 2 of total adipose tissue (odds ratio [OR] = 1.004; 95% confidence interval [CI], 1.00-1.008; P = 0.043). Median follow-up time was 3.35 y, during which 15 (24%) of patients underwent abdominal surgery. Visceral obesity was associated with 5.10-times higher risk of abdominal surgery (95% CI, 1.52-17.09; P = 0.008); after adjusting for disease behavior, visceral obesity maintained a near-significant association with a 2.90-times higher risk of surgery (95% CI, 0.83-10.08; P = 0.09)., Conclusion: Total fat was associated with complicated disease phenotype and visceral obesity, with higher risk of abdominal surgery and shorter time until surgery., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Nanotechnology takes the lead in providing new therapeutic options for cancer patients. In the last decades, lipid-based nanoparticles-solid lipid nanoparticles (SLNs), nanostructured lipid carriers (NLCs), liposomes, and lipid-polymer hybrid nanoparticles-have received particular interest in anticancer drug delivery to solid tumors. To improve selectivity for target cells and, thus, therapeutic efficacy, lipid nanoparticles have been functionalized with antibodies that bind to receptors overexpressed in angiogenic endothelial cells or cancer cells. Most papers dealing with the preclinical results of antibody-conjugated nanoparticles claim low systemic toxicity and effective tumor inhibition, which have not been successfully translated into clinical use yet. This review aims to summarize the current "state-of-the-art" in anticancer drug delivery using antibody-functionalized lipid-based nanoparticles. It includes an update on promising candidates that entered clinical trials and some explanations for low translation success.

We investigated the impactof microsatellite instability (MSI) and Epstein-Barr virus (EBV) status in gastric cancer (GC), regarding response to perioperative chemotherapy (POPChT), overall survival (OS), and progression-free survival (PFS). We included 137 cases of operated GC, 51 of which were submitted to POPChT. MSI status was determined by multiplex PCR and EBV status by EBV-encoded RNA in situ hybridization. Thirty-seven (27%) cases presented as MSI-high, and seven (5.1%) were EBV+. Concerning tumor regression after POPChT, no differences were observed between the molecular subtypes, but females were more likely to respond ( p = 0.062). No significant differences were found in OS or PFS between different subtypes. In multivariate analysis, age (HR 1.02, IC 95% 1.002-1.056, p = 0.033) and positive lymph nodes (HR 1.82, IC 95% 1.034-3.211, p = 0.038) were the only prognostic factors for OS. However, females with MSI-high tumors treated with POPChT demonstrated a significantly increased OS compared to females with MSS tumors ( p = 0.031). In conclusion, we found a high proportion of MSI-high cases. MSI and EBV status did not influence OS or PFS either in patients submitted to POPChT or surgery alone. However, superior survival of females with MSI-high tumors suggests that sex disparities and molecular classification may influence treatment options in GC.

Genetic alterations influence the malignant potential of cancer cells, and so does the tumor microenvironment. Herein, we combined the study of KRAS oncogenic effects in colorectal cancer cells with the influence of fibroblast-derived factors. Results revealed that mutant KRAS regulates cell fate through both autonomous and nonautonomous signaling mechanisms. Specifically, processes such as proliferation and cell-cell aggregation were autonomously controlled by mutant KRAS independently of the stimulation with fibroblasts conditioned media. However, cancer cell invasion revealed to be a KRAS-dependent nonautonomous effect, resulting from the cooperation between fibroblast-derived HGF and mutant KRAS regulation of C-MET expression. C-MET downregulation upon KRAS silencing rendered cells less responsive to HGF and thus less invasive. Yet, in one cell line, KRAS inhibition triggered invasion upon stimulation with fibroblasts conditioned media. Inhibition of PIK3CA oncogene did not promote invasion, thus showing a KRAS-specific effect. Moreover, the invasive capacity also depended on the HGF-C-MET axis. Overall, our study awards oncogenic KRAS an important role in modulating the response to fibroblast-secreted factors either by promoting or impairing invasion, and depicts the HGF-C-MET axis as a putative therapeutic target to impair the invasive properties of mutant KRAS cancer cells., (© 2022 UICC.)

KRAS mutations have been shown to extend their oncogenic effects beyond the cancer cell, influencing the tumor microenvironment. Herein, we studied the impact of mutant KRAS on the modulation of the pro-tumorigenic properties of cancer-associated fibroblasts (CAFs), including α-SMA expression, TGFβ1 and HGF production, extracellular matrix components and metalloproteinases expression as well as collagen contraction and migration capacities. To do so, CCD-18Co normal-like colon fibroblasts were challenged with conditioned media from control and KRAS silenced colorectal cancer (CRC) cells. Our results showed that the mutant KRAS CRC cell-secreted factors were capable of turning normal-like fibroblasts into CAF-like by modulating the α-SMA expression, TGFβ1 and HGF production and migration capacity. Oncogenic KRAS played a secondary role as its silencing did not completely impair the capacity of CRC cells to modulate most of the fibroblast properties analyzed. In summary, our work suggests that mutant KRAS does not play a major role in controlling the CRC cell-secreted factors that modulate the behavior of fibroblasts. The fact that CRC cells retain the capacity to modulate the pro-tumorigenic features of fibroblasts independently of KRAS silencing is likely to negatively impact their response to KRAS inhibitors, thus standing as a putative mechanism of resistance to KRAS inhibition with potential therapeutical relevance.

Understanding how mutant KRAS signaling is modulated by exogenous stimuli is of utmost importance to elucidate resistance mechanisms underlying pathway inhibition failure, and to uncover novel therapeutic targets for mutant KRAS patients. Hence, aiming at perceiving KRAS-autonomous versus -non autonomous mechanisms, we studied the response of two mutant KRAS colorectal cancer cell lines (HCT116 and LS174T) upon KRAS silencing and treatment with rhTGFβ1-activated fibroblasts secretome. A proteomic analysis revealed that rhTGFβ1-activated fibroblast-secreted factors triggered cell line-specific proteome alterations and that mutant KRAS governs 43% and 38% of these alterations in HCT116 and LS174T cells, respectively. These KRAS-dependent proteins were localized and displayed molecular functions that were common to both cell lines (e.g., extracellular exosome, RNA binding functions). Moreover, 67% and 78% of the KRAS-associated proteome of HCT116 and LS174T cells, respectively, was controlled in a KRAS-non-autonomous manner, being dependent on fibroblast-secreted factors. In HCT116 cells, KRAS-non-autonomously controlled proteins were mainly involved in proteoglycans in cancer, p53, and Rap1 signaling pathways; whereas in LS174T cells, they were associated with substrate adhesion-dependent cell-spreading and involved in metabolic processes. This work highlights the context-dependency of KRAS-associated signaling and reinforces the importance of integrating the tumor microenvironment in the study of KRAS-associated effects.

More than a physical structure providing support to tissues, the extracellular matrix (ECM) is a complex and dynamic network of macromolecules that modulates the behavior of both cancer cells and associated stromal cells of the tumor microenvironment (TME). Over the last few years, several efforts have been made to develop new models that accurately mimic the interconnections within the TME and specifically the biomechanical and biomolecular complexity of the tumor ECM. Particularly in colorectal cancer, the ECM is highly remodeled and disorganized and constitutes a key component that affects cancer hallmarks, such as cell differentiation, proliferation, angiogenesis, invasion and metastasis. Therefore, several scaffolds produced from natural and/or synthetic polymers and ceramics have been used in 3D biomimetic strategies for colorectal cancer research. Nevertheless, decellularized ECM from colorectal tumors is a unique model that offers the maintenance of native ECM architecture and molecular composition. This review will focus on innovative and advanced 3D-based models of decellularized ECM as high-throughput strategies in colorectal cancer research that potentially fill some of the gaps between in vitro 2D and in vivo models. Our aim is to highlight the need for strategies that accurately mimic the TME for precision medicine and for studying the pathophysiology of the disease.

The ability of some hydrogels to exhibit a phase transition or change their structure in response to stimuli has been extensively explored for drug depot formation and controlled drug release. Taking advantage of the unique features of the tumor microenvironment (TME) or externally applied triggers, several injectable stimuli-responsive hydrogels have been described as promising candidates for intratumoral drug delivery. In this review, we provide a brief overview of the TME and highlight the advantages of intratumoral administration, followed by a summary of the reported strategies to endow hydrogels with responsiveness to physical (temperature and light), chemical (pH and redox potential), or biological (enzyme) stimuli., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

We aimed to evaluate the changes in eating behaviours of the adult population across 16 European countries due to the COVID-19 confinement and to evaluate whether these changes were somehow related to the severity of the containment measures applied in each country. An anonymous online self-reported questionnaire on socio-demographic characteristics, validated 14-items Mediterranean diet (MedDiet) Adherence Screener (MEDAS) as a reference of a healthy diet, eating and lifestyle behaviours prior to and during the COVID-19 confinement was used to collect data. The study included an adult population residing in 16 European countries at the time of the survey. Aggregated Stringency Index (SI) score, based on data from the Oxford COVID-19 Government Response Tracker, was calculated for each country at the time the questionnaire was distributed (range: 0-100). A total of 36,185 participants completed the questionnaire (77.6% female, 75.2% with high educational level and 42.7% aged between 21 and 35 years). In comparison to pre-confinement, a significantly higher adherence to the MedDiet during the confinement was observed across all countries (overall MEDAS score prior to- and during confinement: 5.23 ± 2.06 vs. 6.15 ± 2.06; p < 0.001), with the largest increase seen in Greece and North Macedonia. The highest adherence to MedDiet during confinement was found in Spain and Portugal (7.18 ± 1.84 and 7.34 ± 1.95, respectively). Stricter contingency restrictions seemed to lead to a significantly higher increase in the adherence to the MedDiet. The findings from this cross-sectional study could be used to inform current diet-related public health guidelines to ensure optimal nutrition is followed among the population, which in turn would help to alleviate the current public health crisis., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 Elsevier Ltd. All rights reserved.)

Chemokines are essential mediators of immune cell trafficking. In a tumor microenvironment context, chemotactic cytokines are known to regulate the migration, positioning and interaction of different cell subsets with both anti- and pro-tumor functions. Additionally, chemokines have critical roles regarding non-immune cells, highlighting their importance in tumor growth and progression. CCL18 is a primate-specific chemokine produced by macrophages and dendritic cells. This chemokine presents both constitutive and inducible expression. It is mainly associated with a tolerogenic response and involved in maintaining homeostasis of the immune system under physiological conditions. Recently, CCL18 has been noticed as an important component of the complex chemokine system involved in the biology of tumors. This chemokine induces T regulatory cell differentiation and recruitment to the tumor milieu, with subsequent induction of a pro-tumor (M2-like) macrophage phenotype. CCL18 is also directly involved in cancer cell-invasion, migration, epithelial-to-mesenchymal transition and angiogenesis stimulation, pinpointing an important role in the promotion of cancer progression. Interestingly, this chemokine is highly expressed in tumor tissues, particularly at the invasive front of more advanced stages (e.g. colorectal cancer), and high levels are detected in the serum of patients, correlating with poor prognosis. Despite the promising role of CCL18 as a biomarker and/or therapeutic target to hamper disease progression, its pleiotropic functions in a context of cancer are still poorly explored. The scarce knowledge concerning the receptors for this chemokine, together with the insufficient insight on the downstream signaling pathways, have impaired the selection of this molecule as an immediate target for translational research. In this Review, we will discuss recent findings concerning the role of CCL18 in cancer, integrate recently disclosed molecular mechanisms and compile data from current clinical studies., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The cell membrane glycoprotein CD26 with peptidase activity (DPP4) and/or its soluble CD26/DPP4 counterpart expression and/or activity are altered in several cancers. Its role in metastasis development was recently highlighted by the discovery of CD26 + cancer stem cell subsets and the fact that clinical DPP4 inhibitors showed antimetastatic effects in animal models. Also, diabetic patients treated with the DPP4 inhibitor sitagliptin showed greater overall survival after colorectal or lung cancer surgery than patients under other diabetic therapies. However, the mechanism of action of these inhibitors in this context is unclear. We studied the role of CD26 and its DPP4 enzymatic activity in malignant cell features such as cell-to-cell homotypic aggregation, cancer cell motility, and invasion in a panel of human colorectal cancer (CRC) cell lines, avoiding models that include the physiological role of DPP4 in chemotaxis. Present results indicate that CD26 participates in the induction of cell invasion, motility, and aggregation of CD26-positive CRC cell lines. Moreover, only invasion and motility assays, which are collagen matrix-dependent, showed a decrease upon treatment with the DPP4 inhibitor sitagliptin. Sitagliptin showed opposite effects to those of transforming growth factor-β1 on epithelial-to-mesenchymal transition and cell cycle, but this result does not explain its CD26/DPP4-dependent effect. These results contribute to the elucidation of the molecular mechanisms behind sitagliptin inhibition of metastatic traits. At the same time, this role of sitagliptin may help to define areas of medicine where DPP4 inhibitors might be introduced. However, they also suggest that additional tools against CD26 as a target might be used or developed for metastasis prevention in addition to gliptins., (© 2021 International Union of Biochemistry and Molecular Biology.)

Introduction: Pancreatic surgery still carries a high morbidity and mortality even in specialized centers. The aim of this study was to evaluate the influence of patients' body composition on postoperative complications and survival after pancreatic surgery., Methods: This was a retrospective study on patients undergoing pancreatic surgery between March 2012 and December 2017. Demographics, clinical data, and postoperative complications classified according to Clavien-Dindo were recorded. Body composition was assessed using routine diagnostic or staging computed tomography (CT). Multiple Cox proportional hazards models were adjusted., Results: Ninety patients were included, 55% were male, and the mean age was 68 ± 10.9 years. Of these 90, 92% had a total pancreatectomy or pancreaticoduodenectomy, 7% a distal pancreatectomy, and 1% a pancreaticoduodenectomy with multi-visceral resection; 84% had malignant disease. The incidence of major complications was 27.8% and the 90-day mortality was 8.8%. The ratio of visceral fat area/skeletal muscle area (VFA:SMA) was associated with an increased risk of complications (OR 2.24, 95% CI 1.14-4.87, p = 0.03) and 90-day survival (HR 2.13, 95% CI 1.13-4.01, p = 0.019). On simple analysis, shorter overall survival (OS) was observed in patients aged ≥70 years ( p = 0.0009), with postoperative complications ≥IIIb ( p = 0.01), an increased VFA:SMA ( p = 0.007), and decreased muscle radiation attenuation ( p = 1.6 × 10 -5 ). In an OS model adjusted for age, disease malignancy, postoperative complications, and body composition parameters, muscle radiation attenuation remained significantly associated with survival (HR 0.94, 95% CI 0.90-0.98, p = 0.0016). A model which included only body composition variables had a discrimination ability ( C -statistic 0.76) superior to a model which comprised conventional clinical variables ( C -statistic 0.68)., Conclusion: Body composition is a major determinant of postoperative complications and survival in pancreatic surgery patients., Competing Interests: There were no conflicts of interest., (Copyright © 2020 by Sociedade Portuguesa de Gastrenterologia. Published by S. Karger AG, Basel.)

Background: Patients with locally advanced rectal adenocarcinoma (LARC) are treated with neoadjuvant chemoradiotherapy (CRT). However, biomarkers for patient selection are lacking, and the association between miRNA expression and treatment response and oncological outcomes is unclear. Objectives: To investigate miRNAs as predictors of response to neoadjuvant CRT and its association with oncological outcomes. Methods: This retrospective study analyzed miRNA expression (miR-16, miR-21, miR-135b, miR-145, and miR-335) in pre- and post-chemoradiation rectal adenocarcinoma tissue and non-neoplastic mucosa in 91 patients treated with neoadjuvant CRT (50.4 Gy) and proctectomy. Two groups were defined: a pathological complete responders group (tumor regression grade-TRG 0) and a pathological incomplete responders group (TRG 1, 2, and 3). Results: miR-21 and miR-135b were upregulated in tumor tissue of incomplete responders comparing with non-neoplastic tissue ( p = 0.008 and p < 0.0001, respectively). Multivariate analysis showed significant association between miR-21 in pre-CRT tumor tissue and response, with a 3.67 odds ratio (OR) of incomplete response in patients with higher miR-21 levels ( p = 0.04). Although with no significance, patients treated with 5-fluorouracil (5-FU) presented reduced odds of incomplete response compared with those treated with capecitabine (OR = 0.19; 95% confidence interval (CI) 0.03-1.12, p = 0.05). Moreover, significant differences were seen in overall survival (OS) in relation to clinical TNM stage ( p = 0.0004), cT ( p = 0.0001), presence of distant disease ( p = 0.002), mesorectal tumor deposits ( p = 0.003), and tumor regression grade ( p = 0.04). Conclusion: miR-21 may predict response to CRT in rectal cancer (RC)., (Copyright © 2020 Ourô, Mourato, Velho, Cardador, Ferreira, Albergaria, Castro, Maio and Rodrigues.)

Standard cancer therapies sometimes fail to deliver chemotherapeutic drugs to tumor cells in a safe and effective manner. Nanotechnology takes the lead in providing new therapeutic options for cancer due to major potential for selective targeting and controlled drug release. Antibodies and antibody fragments are attracting much attention as a source of targeting ligands to bind specific receptors that are overexpressed on cancer cells. Therefore, researchers are devoting time and effort to develop targeting strategies based on nanoparticles functionalized with antibodies, which hold great promise to enhance therapeutic efficacy and circumvent severe side effects. Several methods have been described to immobilize antibodies on the surface of nanoparticles. However, selecting the most appropriate for each application is challenging but also imperative to preserve antigen binding ability and yield stable antibody-conjugated nanoparticles. From this perspective, we aim to provide considerable knowledge on the most widely used methods of functionalization that can be helpful for decision-making and design of conjugation protocols as well. This review summarizes adsorption, covalent conjugation (carbodiimide, maleimide and "click" chemistries) and biotin-avidin interaction, while discussing the advantages, limitations and relevant therapeutic approaches currently under investigation., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objectives: The use of exclusive enteral nutrition (EEN) in patients with Crohn's disease (CD) before surgical resection can reduce disease activity and improve nutritional status. The mechanism of EEN action is unclear, but it might involve modulation of the intestinal microbiota. The aim of this study was to evaluate the effects (namely changes in gut microbiota) of preoperative EEN in adults with complicated CD referred to surgery., Methods: This was a prospective study of adult patients with CD referred to surgery. Patients with body mass index <18.5 kg/m 2 , weight loss >10 %, serum albumin <3 g/dL, or a combination of some or all three, received EEN for ≥2 wk. The effects of EEN on clinical (Harvey-Bradshaw Index [HBI]) and laboratory markers (C-reactive protein [CRP], serum albumin, and fecal calprotectin) and fecal microbiota were analyzed after EEN (before surgery) and 6 mo later. We used 16 S rRNA gene sequencing to determine changes in the fecal microbiota., Results: Fifteen patients were included, of whom 60% were men with a mean age of 45.4 ± 19.1 y. Of those, 10 received EEN. The median duration of preoperative EEN was 41.5 d (15-70 d). During EEN, there was a significant reduction in mean HBI (8.7 ± 1.9 versus 4.1 ± 2.4; P = 0.001) and CRP (11.7 ± 10.3 versus 0.8 ± 0.8 mg/dL; P = 0.008) and an increase in serum albumin (3.1 ± 0.6 versus 4 ± 0.6 g/dL; P = 0.022). Two patients did not require surgery after EEN. The overall microbial composition changed after EEN (Permutational analysis of variance test, P = 0.046) and there was a significant reduction in α diversity (8 ± 2.3 versus 5.2 ± 1.5; P = 0.023). EEN significantly changed the relative abundance of 11 taxonomic operational units (OTUs). At the family level, we found this was mainly due to the decrease in the Enterobacteriaceae family (7 OTUs). Six months after surgery, α diversity was not different from that before or after EEN; at this time point 6 OTUs were significantly different, mainly due to the decrease of Clostridiales order (3 OTUs). The incidence of postoperative complications and hospital length of stay were similar in EEN and immediate surgery groups, as well as clinical and endoscopic recurrence rates 6 mo after surgery., Conclusions: Preoperative EEN improved disease activity and nutritional status in patients with CD referred to surgery. Despite being malnourished, patients given EEN did not have increased postoperative complications compared with well-nourished patients. During EEN, overall microbiota composition changed and α diversity decreased. EEN did not influence postoperative recurrence and gut microbiota 6 mo after surgery., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Cancers are complex tissues composed by genetically altered cancer cells and stromal elements such as inflammatory/immune cells, fibroblasts, endothelial cells and pericytes, neuronal cells, and a non-cellular component, the extracellular matrix. The complex network of interactions and crosstalk established between cancer cells and the supportig cellular and non-cellular components of the microenvironment are of extreme importance for tumor initiation and progression, strongly impacting the course and the outcome of the disease. Therefore, a better understanding of the tumorigenic processes implies the combined study of the cancer cell and the biologic, chemical and mechanic constituents of the tumor microenvironment, as their concerted action plays a major role in the carcinogenic pathway and is a key determinant of the efficacy of anti-cancer treatments. The use of animal models (e.g. Mouse, Zebrafish and Drosophila) to study cancer has greatly impacted our understanding of the processes governing initiation, progression and metastasis and allowed the discovery and pre-clinical validation of novel cancer treatments as it allows to recreate tumor development in a more pathophysiologic environment.

Current evidence strongly suggests that cancer cells depend on the microenvironment in order to thrive. In fact, signals from the surrounding tumor microenvironment are crucial for cancer cells´ aggressiveness, altering their expression profile and favoring their metastatic potential. As such, targeting the tumor microenvironment to impair cancer progression became an attractive therapeutic option. Interestingly, it has been shown that oncogenic KRAS signaling promotes a pro-tumorigenic microenvironment, and the associated crosstalk alters the expression profile of cancer cells. These findings award KRAS a key role in controlling the interactions between cancer cells and the microenvironment, granting cancer a poor prognosis. Given the lack of effective approaches to target KRAS itself or its downstream effectors in the clinic, exploring such interactions may open new perspectives on possible therapeutic strategies to hinder mutant KRAS tumors. This review highlights those communications and their implications for the development of effective therapies or to provide insights regarding response to existing regimens.

Background: E-cadherin has been awarded a key role in the aetiology of both sporadic and hereditary forms of gastric cancer. In this study, we aimed to identify molecular interactors that influence the expression and function of E-cadherin associated to cancer., Methods: A data mining approach was used to predict stomach-specific candidate genes, uncovering S100P as a key candidate. The role of S100P was evaluated through in vitro functional assays and its expression was studied in a gastric cancer tissue microarray (TMA)., Results: S100P was found to contribute to a cancer pathway dependent on the context of E-cadherin function. In particular, we demonstrated that S100P acts as an E-cadherin positive regulator in a wild-type E-cadherin context, and its inhibition results in decreased E-cadherin expression and function. In contrast, S100P is likely to be a pro-survival factor in gastric cancer cells with loss of functional E-cadherin, contributing to an oncogenic molecular program. Moreover, expression analysis in a gastric cancer TMA revealed that S100P expression impacts negatively among patients bearing Ecad - tumours, despite not being significantly associated with overall survival on its own., Conclusions: We propose that S100P has a dual role in gastric cancer, acting as an oncogenic factor in the context of E-cadherin loss and as a tumour suppressor in a functional E-cadherin setting. The discovery of antagonist effects of S100P in different E-cadherin contexts will aid in the stratification of gastric cancer patients who may benefit from S100P-targeted therapies.

Background and Aim: Preoperative biliary drainage (PBD) in patients with pancreatic cancer remains debatable. The aim of this study was to analyse the indications for PBD in patients performing pancreaticoduodenectomy (PD) and to evaluate the impact of this procedure on postoperative outcome., Methods: Observational retrospective cohort study of patients undergoing PD for pancreatic cancer. Clinical data and postoperative outcome, namely complications and 90-day mortality, were prospectively collected and compared between patients performing PBD or direct surgery (DS)., Results: Eighty-two patients were included: 40 underwent PBD and 42 performed DS. Major complications (27.5% vs 33.3%, P=0.156) and 90-day mortality (10% vs 16.7%, P=0.376) were similar between the two groups. There was a trend for higher mean total bilirubin in patients with PBD (P=0.073). The indication for PBD was suspicion of cholangitis/choledocholithiasis or need to perform neoadjuvant chemotherapy in 24 (60%) patients. In the remaining, elevated bilirubin was probably the only reason to perform PBD. Length of hospital stay was longer in PBD group (P=0.003). On multiple logistic regression, 90-day mortality was not related with preoperative bilirubin levels, biliary drainage or its indication, but solely with age (OR 1.15, 95%CI 1.05-1.31, P=0.008)., Conclusions: PBD is often performed in patients undergoing PD without a formal indication, mainly due to high bilirubin levels. No increased morbidity/mortality was observed but length of hospital stay was prolonged in patients performing PBD., Competing Interests: The authors declare that they have no conflict of interest, (© Acta Gastro-Enterologica Belgica.)

Objectives: The purpose of this exploratory study was to identify the main dietary patterns of a Portuguese population of patients with gastrointestinal cancer and to analyze their association with sarcopenia., Methods: This was a prospective study with a consecutive sample of 100 patients with gastrointestinal cancer enrolled at diagnosis. Dietary intake was assessed with a semiquantitative Food Frequency Questionnaire, and dietary patterns were obtained with principal component analysis. Nutritional assessment was done using the Patient-Generated Subjective Global Assessment, and body composition was evaluated with anthropometric measures and computed tomography image processing obtained at the third lumbar vertebrae. Sex and body mass index specific cutoffs were used to define sarcopenia., Results: Four major patterns were identified: high-fat dairy products, fried snacks, and processed meat diet; legumes, vegetables, and fruit diet; fat and fish diet; and alcohol, cereal, and animal protein diet. On simple logistic regression, the occurrence of sarcopenia in participants in the second tertile (odds ratio [OR] 0.30; 95% confidence interval [CI] 0.10-0.83; P = 0.02) and third tertile (OR 0.24; 95% CI 0.08-0.69; P = 0.01) of adherence to the high-fat and fish diet was reduced compared with the first tertile. On multiple logistic regression, the second tertile (OR 0.38, 95% CI 0.11-1.19; P = 0.10) of the fat and fish dietary pattern maintained a trend toward a reduction of the odds of sarcopenia compared with the first tertile, independently of calorie intake, age, disease location, and stage., Conclusions: The fat and fish dietary pattern was associated with lower odds of sarcopenia in this population of patients with gastrointestinal cancer., (Copyright © 2019 Elsevier Inc. All rights reserved.)

KRAS is one of the most frequently mutated oncogenes in cancer, being a potent initiator of tumorigenesis, a strong inductor of malignancy, and a predictive biomarker of response to therapy. Despite the large investment to understand the effects of KRAS activation in cancer cells, pharmacologic targeting of KRAS or its downstream effectors has not yet been successful at the clinical level. Recent studies are now describing new mechanisms of KRAS-induced tumorigenesis by analyzing its effects on the components of the tumor microenvironment. These studies revealed that the activation of KRAS on cancer cells extends to the surrounding microenvironment, affecting the properties and functions of its constituents. Herein, we discuss the most emergent perspectives on the relationship between KRAS-mutant cancer cells and their microenvironment components. Cancer Res; 78(1); 7-14. ©2017 AACR ., (©2017 American Association for Cancer Research.)

Background and Aims: The prognostic value of body composition analysis in patients with Crohn's disease (CD) is poorly explored. The aims of the present study were to assess fat and skeletal muscle compartments including muscle radiation attenuation (MA) in patients with CD, and to analyze its predictive value to identify complicated phenotypes., Methods: Seventy one patients with CD who have had an abdominal CT within one month of clinical, laboratory, and endoscopic evaluation were included. Skeletal muscle area (SMA) and index (SMI), visceral fat area (VFA) and index (VFI), subcutaneous fat area (SFA), and mean MA were measured using appropriate software. Sarcopenia, as defined by Martin's criteria was assessed. Montreal classification was used to characterize disease phenotype., Results: Mean MA was lower in patients >40 years (p = 0.001), L2 (p = 0.09) and stricturing/penetrating disease (p = 0.03) whereas SMA and SMI were significantly lower in patients with positive C-reactive protein and previous hospital admissions (p < 0.01). On multivariate analysis, higher MA was protective against the complicated disease phenotype (stricturing/penetrating disease and/or previous surgeries) (OR 0.81; p = 0.002) whereas a high visceral fat index increased such risk (OR 26.1; p = 0.02). A ROC curve showed a 82.4% sensibility, 90.3% specificity, 17.6% positive predictive value, 9.7% negative predictive value and an area under the curve (AUC) of 0.91 for body composition analysis to predict complicated disease., Conclusions: A lower muscle attenuation and a high visceral fat index seem to be associated with more severe phenotypes in patients with CD., (Copyright © 2017 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved.)

Present-day and projected future changes in mean radiant temperature, T mrt in one northern, one mid-, and one southern European city (represented by Gothenburg, Frankfurt, and Porto), are presented, and the concept of hot spots is adopted. Air temperature, T a , increased in all cities by 2100, but changes in solar radiation due to changes in cloudiness counterbalanced or exacerbated the effects on T mrt . The number of days with high T mrt in Gothenburg was relatively unchanged at the end of the century (+1 day), whereas it more than doubled in Frankfurt and tripled in Porto. The use of street trees to reduce daytime radiant heat load was analyzed using hot spots to identify where trees could be most beneficial. Hot spots, although varying in intensity and frequency, were generally confined to near sunlit southeast-southwest facing walls, in northeast corner of courtyards, and in open spaces in all three cities. By adding trees in these spaces, the radiant heat load can be reduced, especially in spaces with no or few trees. A set of design principles for reducing the radiant heat load is outlined based on these findings and existing literature.

Purpose: Neoadjuvant chemotherapy has been shown to improve survival in locally advanced gastric cancer, but it is associated with significant toxicity. Sarcopenia and sarcopenic obesity have been studied in several types of cancers and have been reported to be associated with higher chemotherapy toxicity and morbi-mortality. The aim of this study was to assess the prevalence of sarcopenia/sarcopenic obesity in patients with gastric cancer, as well as its association with chemotherapy toxicity and long-term outcomes., Materials and Methods: A retrospective analysis was performed using an academic cancer center patient cohort diagnosed with locally advanced gastric cancer between January 2012 and December 2014 and treated with neoadjuvant chemotherapy. We analyzed body composition (skeletal muscle and visceral fat index) in axial computed tomography images., Results: A total of 48 patients met the inclusion criteria. The mean age was 68±10 years, and 33 patients (69%) were men. Dose-limiting toxicity was observed in 22 patients (46%), and treatment was terminated early owing to toxicity in 17 patients (35%). Median follow-up was 17 months. Sarcopenia and sarcopenic obesity were found at diagnosis in 23% and 10% of patients, respectively. We observed an association between termination of chemotherapy and both sarcopenia (P=0.069) and sarcopenic obesity (P=0.004). On multivariate analysis, the odds of treatment termination were higher in patients with sarcopenia (odds ratio=4.23; P=0.050). Patients with sarcopenic obesity showed lower overall survival (median survival of 6 months [95% confidence interval {CI}=3.9-8.5] vs. 25 months [95% CI=20.2-38.2]; log-rank test P=0.000)., Conclusions: Sarcopenia and sarcopenic obesity were associated with early termination of neoadjuvant chemotherapy in patients with gastric cancer; additionally, sarcopenic obesity was associated with poor survival., Competing Interests: Conflict of Interest: No potential conflict of interest relevant to this article was reported.

Colorectal cancer (CRC) is a leading cause of cancer mortality worldwide. It is often associated with activating mutations in KRAS leading to deregulation of major signaling pathways as the RAS-RAF-MAPK and PI3K-Akt. However, the therapeutic options for CRC patients harboring somatic KRAS mutations are still very limited. It is therefore urgent to unravel novel therapeutic approaches for those patients. In this study, we have awarded PI3K p110α a key role in CRC cells harboring KRAS/PIK3CA mutations or KRAS mutations alone. Specific silencing of PI3K p110α by small interfering RNA (siRNA) reduced viability and induced apoptosis or cell cycle arrest. In agreement with these cellular effects, PI3K p110α silencing led to alterations in the expression levels of proteins implicated in apoptosis and cell cycle, namely XIAP and pBad in KRAS/PIK3CA mutant cells and cyclin D1 in KRAS mutant cells. To further validate our data, a specific PI3K p110α inhibitor, BYL719, was evaluated. BYL719 mimicked the in vitro siRNA effects on cellular viability and on the alterations of apoptotic- and cell cycle-related proteins in CRC mutant cells. Overall, this study demonstrates that specific inhibition of PI3K p110α could provide an alternative therapeutic approach for CRC patients, particularly those harboring KRAS mutations.

Both cancer and tumour-associated host cells are exposed to ionizing radiation when a tumour is subjected to radiotherapy. Macrophages frequently constitute the most abundant tumour-associated immune population, playing a role in tumour progression and response to therapy. The present work aimed to evaluate the importance of macrophage-cancer cell communication in the cellular response to radiation. To address this question, we established monocultures and indirect co-cultures of human monocyte-derived macrophages with RKO or SW1463 colorectal cancer cells, which exhibit higher and lower radiation sensitivity, respectively. Mono- and co-cultures were then irradiated with 5 cumulative doses, in a similar fractionated scheme to that used during cancer patients' treatment (2 Gy/fraction/day). Our results demonstrated that macrophages sensitize RKO to radiation-induced apoptosis, while protecting SW1463 cells. Additionally, the co-culture with macrophages increased the mRNA expression of metabolism- and survival-related genes more in SW1463 than in RKO. The presence of macrophages also upregulated glucose transporter 1 expression in irradiated SW1463, but not in RKO cells. In addition, the influence of cancer cells on the expression of pro- and anti-inflammatory macrophage markers, upon radiation exposure, was also evaluated. In the presence of RKO or SW1463, irradiated macrophages exhibit higher levels of pro-inflammatory TNF, IL6, CCL2 and CCR7, and of anti-inflammatory CCL18. However, RKO cells induce an increase of macrophage pro-inflammatory IL1B, while SW1463 cells promote higher pro-inflammatory CXCL8 and CD80, and also anti-inflammatory VCAN and IL10 levels. Thus, our data demonstrated that macrophages and cancer cells mutually influence their response to radiation. Notably, conditioned medium from irradiated co-cultures increased non-irradiated RKO cell migration and invasion and did not impact on angiogenesis in a chicken embryo chorioallantoic membrane assay. Overall, the establishment of primary human macrophage-cancer cell co-cultures revealed an intricate cell communication in response to ionizing radiation, which should be considered when developing therapies adjuvant to radiotherapy.

Loss of DNA mismatch repair (MMR) function, due to somatic or germline epi/genetic alterations of MMR genes leads to the accumulation of numerous mutations across the genome, creating a molecular phenotype known as microsatellite instability (MSI). In gastric cancer (GC), MSI occurs in about 15% to 30% of the cases. This review summarizes the current knowledge on the molecular mechanisms underlying the acquisition of MSI in GC as well as on the clinic, pathologic and molecular consequences of the MSI phenotype. Additionally, current therapeutic strategies for GC and their applicability in the MSI subset are also discussed.

Background: MLK3 gene mutations were described to occur in about 20% of microsatellite unstable gastrointestinal cancers and to harbor oncogenic activity. In particular, mutation P252H, located in the kinase domain, was found to have a strong transforming potential, and to promote the growth of highly invasive tumors when subcutaneously injected in nude mice. Nevertheless, the molecular mechanism underlying the oncogenic activity of P252H mutant remained elusive., Methods: In this work, we performed Illumina Whole Genome arrays on three biological replicas of human HEK293 cells stably transfected with the wild-type MLK3, the P252H mutation and with the empty vector (Mock) in order to identify the putative signaling pathways associated with P252H mutation., Results: Our microarray results showed that mutant MLK3 deregulates several important colorectal cancer- associated signaling pathways such as WNT, MAPK, NOTCH, TGF-beta and p53, helping to narrow down the number of potential MLK3 targets responsible for its oncogenic effects. A more detailed analysis of the alterations affecting the WNT signaling pathway revealed a down-regulation of molecules involved in the canonical pathway, such as DVL2, LEF1, CCND1 and c-Myc, and an up-regulation of DKK, a well-known negative regulator of canonical WNT signaling, in MLK3 mutant cells. Additionally, FZD6 and FZD10 genes, known to act as negative regulators of the canonical WNT signaling cascade and as positive regulators of the planar cell polarity (PCP) pathway, a non-canonic WNT pathway, were found to be up-regulated in P252H cells., Conclusion: The results provide an overall view of the expression profile associated with mutant MLK3, and they support the functional role of mutant MLK3 by showing a deregulation of several signaling pathways known to play important roles in the development and progression of colorectal cancer. The results also suggest that mutant MLK3 may be a novel modulator of WNT signaling, and pinpoint the activation of PCP pathway as a possible mechanism underlying the invasive potential of MLK3 mutant cells.

Aim: To identify clinical and/or genetic predictors of response to several therapies in Crohn's disease (CD) patients., Methods: We included 242 patients with CD (133 females) aged (mean ± standard deviation) 39 ± 12 years and a disease duration of 12 ± 8 years. The single-nucleotide polymorphisms (SNPs) studied were ABCB1 C3435T and G2677T/A, IL23R G1142A, C2370A, and G9T, CASP9 C93T, Fas G670A and LgC844T, and ATG16L1 A898G. Genotyping was performed with real-time PCR with Taqman probes., Results: Older patients responded better to 5-aminosalicylic acid (5-ASA) and to azathioprine (OR 1.07, p = 0.003 and OR 1.03, p = 0.01, respectively) while younger ones responded better to biologicals (OR 0.95, p = 0.06). Previous surgery negatively influenced response to 5-ASA compounds (OR 0.25, p = 0.05), but favoured response to azathioprine (OR 2.1, p = 0.04). In respect to genetic predictors, we observed that heterozygotes for ATGL16L1 SNP had a significantly higher chance of responding to corticosteroids (OR 2.51, p = 0.04), while homozygotes for Casp9 C93T SNP had a lower chance of responding both to corticosteroids and to azathioprine (OR 0.23, p = 0.03 and OR 0.08, p = 0.02,). TT carriers of ABCB1 C3435T SNP had a higher chance of responding to azathioprine (OR 2.38, p = 0.01), while carriers of ABCB1 G2677T/A SNP, as well as responding better to azathioprine (OR 1.89, p = 0.07), had a lower chance of responding to biologicals (OR 0.31, p = 0.07), which became significant after adjusting for gender (OR 0.75, p = 0.005)., Conclusions: In the present study, we were able to identify a number of clinical and genetic predictors of response to several therapies which may become of potential utility in clinical practice. These are preliminary results that need to be replicated in future pharmacogenomic studies.

Objective: We aimed to identify the clinical and genetic [IL23 receptor (IL23R) single nucleotide polymorphisms (SNPs)] predictors of response to therapy in patients with ulcerative colitis., Patients and Methods: A total of 174 patients with ulcerative colitis, 99 women and 75 men, were included. The mean age of the patients was 47±15 years and the mean disease duration was 11±9 years. The number of patients classified as responders (R) or nonresponders (NR) to several therapies was as follows: 110 R and 53 NR to mesalazine (5-ASA), 28 R and 20 NR to azathioprine (AZT), 18 R and 7 NR to infliximab. Clinical and demographic variables were recorded. A total of four SNPs were studied: IL23R G1142A, C2370A, G43045A, and G9T. Genotyping was performed by real-time PCR using Taqman probes., Results: Older patients were more prone to respond to 5-ASA (P=0.004), whereas those with pancolitis were less likely to respond to such therapies (P=0.002). Patients with extraintestinal manifestations (EIMs) were less likely to respond to 5-ASA (P=0.001), AZT (P=0.03), and corticosteroids (P=0.06). Carriers of the mutant allele for IL23R SNPs had a significantly higher probability of developing EIMs (P<0.05), a higher probability of being refractory to 5-ASA (P<0.03), but a higher likelihood of responding to AZT (P=0.05). A significant synergism was observed between IL23R C2370A and EIMs with respect to nonresponse to 5-ASA (P=0.03)., Conclusion: Besides extent of disease and age at disease onset, the presence of EIMs may be a marker of refractoriness to 5-ASA, corticosteroids, and AZT. IL23R SNPs are associated both with EIMs and with nonresponse to 5-ASA and corticosteroids.

N-RAS is one member of a family of oncoproteins that are commonly mutated in cancer. Activating mutations in NRAS occur in a subset of colorectal cancers, but little is known about how the mutant protein contributes to the onset and progression of the disease. Using genetically engineered mice, we find that mutant N-RAS strongly promotes tumorigenesis in the context of inflammation. The protumorigenic nature of mutant N-RAS is related to its antiapoptotic function, which is mediated by activation of a noncanonical mitogen-activated protein kinase pathway that signals through STAT3. As a result, inhibition of MAP-ERK kinase selectively induces apoptosis in autochthonous colonic tumors expressing mutant N-RAS. The translational significance of this finding is highlighted by our observation that NRAS mutation correlates with a less favorable clinical outcome for patients with colorectal cancer. These data show for the first time the important role that N-RAS plays in colorectal cancer.

Background/objectives: To assess the distribution of interleukin (IL)-1β, IL-6, tumour necrosis factor (TNF)-α and C-reactive protein (CRP) according to the different definitions of metabolically healthy obesity (MHO)., Subjects/methods: A total of 881 obese (body mass index (BMI) > or =30 kg/m2) subjects derived from the population-based CoLaus Study participated in this study. MHO was defined using six sets of criteria including different combinations of waist, blood pressure, total high-density lipoprotein cholesterol or low-density lipoprotein -cholesterol, triglycerides, fasting glucose, homeostasis model, high-sensitivity CRP, and personal history of cardiovascular, respiratory or metabolic diseases. IL-1β, IL-6 and TNF-α were assessed by multiplexed flow cytometric assay. CRP was assessed by immunoassay., Results: On bivariate analysis some, but not all, definitions of MHO led to significantly lower levels of IL-6, TNF-α and CRP compared with non-MH obese subjects. Most of these differences became nonsignificant after multivariate analysis. An posteriori analysis showed a statistical power between 9 and 79%, depending on the inflammatory biomarker and MHO definition considered. Further increasing sample size to overweight+obese individuals (BMI > or =25 kg/m2, n=2917) showed metabolically healthy status to be significantly associated with lower levels of CRP, while no association was found for IL-1β. Significantly lower IL-6 and TNF-α levels were also found with some but not all MHO definitions, the differences in IL-6 becoming nonsignificant after adjusting for abdominal obesity or percent body fat., Conclusions: MHO individuals present with decreased levels of CRP and, depending on MHO definition, also with decreased levels in IL-6 and TNF-α. Conversely, no association with IL-1β levels was found.

Background: COPD morbidity is a good example of how the urban form may interfere with a disease's severity. Then, it may play an important role as a stimulus to increase the acceptability of several policy actions that aim to upgrade urban resilience. Despite the multiple dimensions of wellbeing, health is surely a key variable attracting everyone's attention, which is thus more likely to be able to persuade people that actions that may at first seem undesirable are fundamental in improving urban sustainability and well-being., Methods: After creating a short list of socio-economic and environmental factors relating to the onset and aggravation of COPD, daily admissions distributions were compared using both a non-weighted and a weighted multi-criteria hierarchical analysis procedure., Results: Porto's COPD Social and Environmental Inequalities Index (SEII), calculated with a hierarchical analysis procedure, accurately illustrates a great relationship between COPD admissions and adverse urban form variables., Conclusion: COPD may be an important communication tool to stimulate the acceptability of some otherwise unpopular planning measures to improve urban resilience (sustainability and well-being)., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Much of our current state of knowledge pertaining to the mechanisms controlling intestinal epithelial homeostasis derives from epidemiological, molecular genetic, cell biological, and biochemical studies of signaling pathways that are dysregulated during the process of colorectal tumorigenesis. Activating mutations in members of the RAS oncoprotein family play an important role in the progression of colorectal cancer (CRC) and, by extension, intestinal epithelial homeostasis. Mutations in K-RAS account for 90% of the RAS mutations found in CRC. As such, the study of RAS protein function in the intestinal epithelium is largely encompassed by the study of K-RAS function in CRC. In this review, we summarize the data available from genetically defined in vitro and in vivo models of CRC that aim to characterize the oncogenic properties of mutationally activated K-RAS. These studies paint a complex picture of a multi-functional oncoprotein that engages an array of downstream signaling pathways to influence cellular behaviors that are both pro- and anti-tumorigenic. While the complexity of K-RAS biology has thus far prevented a comprehensive understanding of its oncogenic properties, the work to date lays a foundation for the development of new therapeutic strategies to treat K-RAS mutant CRC., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: High dose chemotherapy with autologous stem cell transplantation (HDCT-ASCT) has been administered to patients with high-risk germ cell tumours (GCT). The role of this treatment for GCT still remains unclear, including the identification of subgroups more likely to benefit from such strategy., Methods: A retrospective review was conducted of all male patients with gonadal and extra gonadal GCT treated with HDCT-ASCT between 1996 and 2008 at the Instituto Português de Oncologia de Lisboa Francisco Gentil (IPOLFG)., Results: Twenty patients were treated with HDCT-ASCT, 17 with primary testicular tumours, two mediastinal and one retroperitoneal GCT. According to the International Germ Cell Cancer Consensus Group (IGCCCG) classification, at diagnosis three patients had good risk, four intermediate, eight poor but for the patients left the risk group could not be ascertained. In eight patients HDCT-ASCT was used upfront, after induction with first-line conventional chemotherapy, and in the remaining 12 for relapsed GCT. One patient had platinum-resistant and another platinum-refractory disease. Only two patients had Beyer score > 2. All but one patient were treated with ICE (Ifosfamide, Carboplatin, Etoposide). Six patients underwent a second HDCT-ASCT course. The 5-year overall survival and progression free survival were respectively 53% and 44%; both patients with mediastinal GCT are long term survivors., Conclusion: Randomized studies to date have failed to indicate a survival advantage for HDCT-ASCT in GCT. This series is small and heterogeneous which prevents us from drawing conclusions regarding the benefit of this treatment for GCT. However, we could confirm the lack of benefit of HDCT-ASCT for platinum-resistant GCT and to question the absolute contraindication to this therapeutic modality in mediastinal GCT. HDCT-ASCT should therefore be performed exclusively in experienced centers and, preferably, in the setting of clinical trials.

Aim: Mitogen-activated protein kinase (MAPK) cascade and phosphatidylinositol 3-kinase (PI3K) survival pathways are frequently activated in the progression of gastrointestinal malignancies. In this study, we aimed to determine the frequency of gene mutations in members of these pathways--Epithelial Growth Factor Receptor (EGFR), KRAS, BRAF, PIK3CA and MLK3 in a series of 63 gastric carcinomas with high levels of microsatellite instability (MSI)., Methods: Gene mutation analysis was performed by PCR amplification followed by direct sequencing. In selected tumour cases, EGFR expression was evaluated by immunohistochemistry. Association studies between molecular data and clinicopathologic characteristics were performed., Results: Mutations in EGFR (3'-untranslated region [UTR] polyA repeat), KRAS, PIK3CA and MLK3 genes occurred in 30 (47.6%), 11 (17.5%), 9 (14.3%) and 2 (3.2%) of the MSI gastric cancer (GC) cases, respectively. No BRAF or EGFR hotspot mutations were identified. Overall, mutations in at least one of these genes were found in 55.6% (35/63) of gastric carcinomas. From those mutant cases 40.0% (14/35) of them had concomitant gene mutations, always involving EGFR polyA deletions. Interestingly, we observed significant associations between oncogenic mutations and female gender (p = 0.046) old age of diagnosis (p = 0.001) and intestinal subtype (p = 0.043)., Conclusion: Our results show that MSI gastric carcinoma frequently shows activation of EGFR-MAPK and PI3K pathways. Within all alterations found, deletions of the A13 repeats of EGFR were common, suggesting this molecular event as an important biomarker for stratification of GC patients for treatment with EGFR inhibitors., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Objective: To estimate the prevalence of metabolically healthy obesity (MHO) according to different definitions., Methods: Population-based sample of 2803 women and 2557 men participated in the study. Metabolic abnormalities were defined using six sets of criteria, which included different combinations of the following: waist; blood pressure; total, high-density lipoprotein or low-density lipoprotein-cholesterol; triglycerides; fasting glucose; homeostasis model assessment; high-sensitivity C-reactive protein; personal history of cardiovascular, respiratory or metabolic diseases. For each set, prevalence of MHO was assessed for body mass index (BMI); waist or percent body fat., Results: Among obese (BMI 30 kg/m(2)) participants, prevalence of MHO ranged between 3.3 and 32.1% in men and between 11.4 and 43.3% in women according to the criteria used. Using abdominal obesity, prevalence of MHO ranged between 5.7 and 36.7% (men) and 12.2 and 57.5% (women). Using percent body fat led to a prevalence of MHO ranging between 6.4 and 43.1% (men) and 12.0 and 55.5% (women). MHO participants had a lower odd of presenting a family history of type 2 diabetes. After multivariate adjustment, the odds of presenting with MHO decreased with increasing age, whereas no relationship was found with gender, alcohol consumption or tobacco smoking using most sets of criteria. Physical activity was positively related, whereas increased waist was negatively related with BMI-defined MHO., Conclusion: MHO prevalence varies considerably according to the criteria used, underscoring the need for a standard definition of this metabolic entity. Physical activity increases the likelihood of presenting with MHO, and MHO is associated with a lower prevalence of family history of type 2 diabetes.

Mixed lineage kinase 3 (MLK3) is a serine/threonine kinase, regulating MAPkinase signalling, in which cancer-associated mutations have never been reported. In this study, 174 primary gastrointestinal cancers (48 hereditary and 126 sporadic forms) and 7 colorectal cancer cell lines were screened for MLK3 mutations. MLK3 mutations were significantly associated with MSI phenotype in primary tumours (P = 0.0005), occurring in 21% of the MSI carcinomas. Most MLK3 somatic mutations identified were of the missense type (62.5%) and more than 80% of them affected evolutionarily conserved residues. A predictive 3D model points to the functional relevance of MLK3 missense mutations, which cluster in the kinase domain. Further, the model shows that most of the altered residues in the kinase domain probably affect MLK3 scaffold properties, instead of its kinase activity. MLK3 missense mutations showed transforming capacity in vitro and cells expressing the mutant gene were able to develop locally invasive tumours, when subcutaneously injected in nude mice. Interestingly, in primary tumours, MLK3 mutations occurred in KRAS and/or BRAF wild-type carcinomas, although not being mutually exclusive genetic events. In conclusion, we have demonstrated for the first time the presence of MLK3 mutations in cancer and its association to mismatch repair deficiency. Further, we demonstrated that MLK3 missense mutations found in MSI gastrointestinal carcinomas are functionally relevant.

Microsatellite instability (MSI) occurs in about 15% of gastrointestinal cancers and it is associated with specific clinic, pathologic, and molecular features of the tumors. MSI-high (MSI-H) carcinomas also follow specific tumor development pathways. This review is focused on the molecular profile of alterations in members of the KRAS signaling pathway (EGFR, KRAS, BRAF, PIK3CA, RASSF1A, and MLK3 genes) in MSI gastrointestinal carcinomas. Alterations in these genes characterize more than half of gastrointestinal cancers and frequently occur simultaneously in the same tumor, pinpointing the KRAS signaling pathway as one of the most frequently altered pathways in this subset of cancers. Nowadays, many and novel inhibitors targeting molecules of this signaling pathway are being described; therefore, it is worthwhile to test their efficacy in MSI gastrointestinal cancers in order to develop new and more directed targeted therapies for patients affected by this disease., (Copyright © 2010 Elsevier Inc. All rights reserved.)

About 90% of gastric cancer (GC) cases appear in a sporadic setting. Nonetheless, in high incidence areas high familial aggregation rates have been recently described. Microsatellite instability (MSI) is thought to be an important molecular phenotype both in sporadic GC and in tumors of the HNPCC spectrum. The aim of this study was to assess the frequency of MSI in GC with familial aggregation. Five quasimonomorphic mononucleotide repeats (BAT-26, BAT-25, NR-24, NR-21 and NR-27) were analyzed in 250 GC patients. Seventy-five patients (30%) had at least one-first-degree family member affected by GC and 63 patients (25.2%) showed MSI. The frequency of MSI was significantly higher in patients with a positive family history of GC (38.7%) compared to patients with other tumor types within the family (15.7%) or with a negative oncological familial history (21.9%, P = 0.004). Within cases with a positive familial oncological history, the MSI frequency in families with GC only was similar to the one observed in families with GC and colon cancer (P = 0.96). Nonetheless, in families with GC and lung cancer, the frequency of MSI was significantly lower (5.6%, P = 0.007). MSI occurs in GCs with familial aggregation. Similar MSI rates have been observed in GC patients with other family members affected by GC or colon cancer. The same does not occur in families with other members affected by lung cancer. Our data seem to suggest that familial aggregation for either GC alone or gastric and colon cancer share common etiological factors in contrast to families with gastric and lung cancers.

Background: BRAF, KRAS and PIK3CA mutations are frequently found in sporadic colorectal cancer (CRC). In contrast to KRAS and PIK3CA mutations, BRAF mutations are associated with tumours harbouring CpG Island methylation phenotype (CIMP), MLH1 methylation and microsatellite instability (MSI). We aimed at determine the frequency of KRAS, BRAF and PIK3CA mutations in the process of colorectal tumourigenesis using a series of colorectal polyps and carcinomas. In the series of polyps CIMP, MLH1 methylation and MSI were also studied., Methods: Mutation analyses were performed by PCR/sequencing. Bisulfite treated DNA was used to study CIMP and MLH1 methylation. MSI was detected by pentaplex PCR and Genescan analysis of quasimonomorphic mononucleotide repeats. Chi Square test and Fisher's Exact test were used to perform association studies., Results: KRAS, PIK3CA or BRAF occur in 71% of polyps and were mutually exclusive. KRAS mutations occur in 35% of polyps. PIK3CA was found in one of the polyps. V600E BRAF mutations occur in 29% of cases, all of them classified as serrated adenoma. CIMP phenotype occurred in 25% of the polyps and all were mutated for BRAF. MLH1 methylation was not detected and all the polyps were microsatellite stable. The comparison between the frequency of oncogenic mutations in polyps and CRC (MSI and MSS) lead us to demonstrate that KRAS and PIK3CA are likely to precede both types of CRC. BRAF mutations are likely to precede MSI carcinomas since the frequency found in serrated polyps is similar to what is found in MSI CRC (P = 0.9112), but statistically different from what is found in microsatellite stable (MSS) tumours (P = 0.0191)., Conclusion: Our results show that BRAF, KRAS and PIK3CA mutations occur prior to malignant transformation demonstrating that these oncogenic alterations are primary genetic events in colorectal carcinogenesis. Further, we show that BRAF mutations occur in association with CIMP phenotype in colorectal serrated polyps and verified that colorectal serrated polyps and MSI CRC show a similar frequency of BRAF mutations. These results support that BRAF mutations harbour a mild oncogenic effect in comparison to KRAS and suggest that BRAF mutant colorectal cells need to accumulate extra epigenetic alterations in order to acquire full transformation and evolve to MSI CRC.