Search

PURPOSETo better understand immigration disparities among a Spanish Early-Onset Colorectal Cancer (SECOC) subset, according to the country of origin.PATIENTS AND METHODSWe selected 250 consecutive participants from the SECOC consortium. Data on baseline patient and tumor characteristics, family history of colorectal cancer (CRC), and follow-up were collected. The presence of mismatch repair deficiency was also assessed. Special data regarding country of origin, time of stay in Spain in case of other different country, and a 10-year cutoff that specifies the obtaining of Spanish nationality defined the variables of interest for comparison.RESULTSSeventy-five percent of patients with early-onset CRC (EOCRC) (188) were born in Spain, whereas the other 25% were born outside of Spain. The mean time of living in Spain until the EOCRC diagnosis was 16.5 years. Comparatively, most of the analyzed features showed equivalent proportions between cohorts. Only Spanish patients appeared to have more familial cancer component in first degree in general (32.3%; P = .01), compared with non-Spaniards, which showed a predominant sporadic component (56.4%; P < .001). Among immigrants, those patients living in Spain before CRC diagnosis ≤10 years were younger at diagnosis (39.1 v 42.5), more frequently male (77.8 v 47.7), were in more advanced stages (88.8% diagnosed at stage III and IV [P = .01]), and had a worse prognosis regarding recurrence rates (29.4% v 6.3%).CONCLUSIONAlthough there were few differences between Spanish and non-Spanish EOCRC, the most remarkable difference was that linked with the situation of those immigrants who have recently arrived in Spain, in relation to their lower health coverage, which could be associated with the delay in the diagnosis and their subsequent worse prognosis.

Abstract Differential presence of exons (DPE) by next generation sequencing (NGS) is a method of interpretation of whole exome sequencing. This method has been proposed to design a predictive and diagnostic algorithm with clinical value in plasma from patients bearing colorectal cancer (CRC). The aim of the present study was to determine a common exonic signature to discriminate between different clinical pictures, such as non-metastatic, metastatic and non-disease (healthy), using a sustainable and novel technology in liquid biopsy. Through DPE analysis, we determined the differences in DNA exon levels circulating in plasma between patients bearing CRC vs. healthy, patients bearing CRC metastasis vs. non-metastatic and patients bearing CRC metastasis vs. healthy comparisons. We identified a set of 510 exons (469 up and 41 down) whose differential presence in plasma allowed us to group and classify between the three cohorts. Random forest classification (machine learning) was performed and an estimated out-of-bag (OOB) error rate of 35.9% was obtained and the predictive model had an accuracy of 75% with a confidence interval (CI) of 56.6–88.5. In conclusion, the DPE analysis allowed us to discriminate between different patho-physiological status such as metastatic, non-metastatic and healthy donors. In addition, this analysis allowed us to obtain very significant values with respect to previous published results, since we increased the number of samples in our study. These results suggest that circulating DNA in patient’s plasma may be actively released by cells and may be involved in intercellular communication and, therefore, may play a pivotal role in malignant transformation (genometastasis).

ObjectiveThe specific effect of Adipose-Derived Mesenchymal Stem Cells (Ad-MSC) on acute joint inflammation, where the response mostly depends on innate immunity activation, remains elusive. The pathogenesis of gouty arthritis, characterized by the deposition of monosodium urate (MSU) crystals in the joints, associated to acute flares, has been associated to NLRP3 inflammasome activation and subsequent amplification of the inflammatory response. Our aim was to study the effect of human Ad-MSC administration in the clinical inflammatory response of rabbits after MSU injection, and the molecular mechanisms involved.MethodsAd-MSC were administered by intraarterial route shortly after intraarticular MSU crystal injections. Joint and systemic inflammation was sequentially studied, and the mechanisms involved in NLRP3 inflammasome activation, and the synthesis of inflammatory mediators were assessed in the synovial membranes 72h after insult. Ad-MSC and THP-1-derived macrophages stimulated with MSU were co-cultured in transwell system.ResultsA single systemic dose of Ad-MSC accelerated the resolution of local and systemic inflammatory response. In the synovial membrane, Ad-MSC promoted alternatively M2 macrophage presence, inhibiting NLRP3 inflammasome and inducing the production of anti-inflammatory cytokines, such as IL-10 or TGF-β, and decreasing nuclear factor-κB activity. Ad-MSC induced a net anti-inflammatory balance in MSU-stimulated THP-1 cells, with a higher increase in IL-10 and IDO expression than that observed for IL-1β and TNF.ConclusionOur in vivo and in vitro results showed that a single systemic dose of Ad-MSC decrease the intensity and duration of the inflammatory response by an early local COX-2 upregulation and PGE2 release. Ad-MSCs suppressed NF-kB activity, NLRP3 inflammasome, and promoted the presence of M2 alternative macrophages in the synovium. Therefore, this therapeutic approach could be considered as a pharmacological alternative in patients with comorbidities that preclude conventional treatment.

Ovarian cancer is the seventh most common cancer worldwide in women and the most lethal gynecologic malignancy due to the lack of accurate screening tools for early detection and late symptom onset. The absence of early-onset symptoms often delays diagnosis until the disease has progressed to advanced stages, frequently when there is peritoneal involvement. Although ovarian cancer is a heterogeneous malignancy with different histopathologic types, treatment for advanced tumors is usually based on chemotherapy and cytoreduction surgery. CAR T cells have shown promise for the treatment of hematological malignancies, though their role in treating solid tumors remains unclear. Outcomes are less favorable owing to the low capacity of CAR T cells to migrate to the tumor site, the influence of the protective tumor microenvironment, and the heterogeneity of surface antigens on tumor cells. Despite these results, CAR T cells have been proposed as a treatment approach for peritoneal carcinomatosis from colorectal and gastric origin. Local intraperitoneal administration of CAR T cells has been found to be superior to systemic administration, as this route is associated with increased tumor reduction, a more durable effect, protection against local relapse and distant metastases, and fewer systemic adverse effects. In this article we review the application of CAR T cells for the treatment of ovarian cancer and peritoneal carcinomatosis from ovarian cancer.

Abstract Colorectal cancer cells can transfer the oncogene KRAS to distant cells, predisposing them to malignant transformation (Genometastasis Theory). This process could contribute to liver metastasis; besides, hepatic progenitor cells (HPCs) have been found to be involved in liver malignant neoplasms. The objective of this study is to determine if mouse HPCs—Oval cells (OCs)—are susceptible to incorporate Kras GAT (G12D) mutation from mouse colorectal cancer cell line CT26.WT and if OCs with the incorporated mutation behave like malignant cells. To achieve this, three lines of OCs in different conditions were exposed to CT26.WT cells through transwell co-culture for a week. The presence of Kras G12D and capacity to form tumors were analyzed in treated samples by droplet digital PCR and colony-forming assays, respectively. The results showed that the Kras G12D mutation was detected in hepatic culture conditions of undifferentiated OCs and these cells were capable of forming tumors in vitro. Therefore, OCs are susceptible to malignant transformation by horizontal transfer of DNA with Kras G12D mutation in an undifferentiated condition associated with the liver microenvironment. This study contributes to a new step in the understanding of the colorectal metastatic process.

Objetivo: Enfermedades como la obesidad y el cáncer pueden alterar la cicatrización de las heridas. Las células madre derivadas del tejido adiposo (ASC) abren un nuevo campo de investigación ya que muchos estudios han demostrado su utilidad y alto grado de seguridad para la reparación de heridas debido a sus propiedades inmunomoduladoras y de regeneración tisular. El propósito de este estudio es determinar el impacto de la obesidad y el cáncer en el potencial terapéutico de las ASCs. Material y métodos: Aislamos y caracterizamos el fenotipo, la capacidad de diferenciación, el secretoma y la capacidad de migración in vitro de las ASC. Asimismo, analizamos la capacidad de migración in vitro asociada al plasma de los diferentes pacientes. Resultados: Observamos que las ASC aisladas de pacientes obesos y con cáncer tienen el mismo fenotipo, proliferación celular y capacidades de migración que las ASCaisladas de donantes sanos. Sin embargo, no tienen el mismo potencial de diferenciación y exhiben perfiles distintos de citoquinas secretadas tanto proinflamatorias como reguladoras. Conclusiones: Consideramos que las ASC de pacientes con obesidad o cáncer están levemente alteradas. Esta puede ser la causa de una peor cicatrización de las heridas en este tipo de pacientes.

Abstract Background Mesenchymal stromal cells (MSCs) constitute one of the cell types most frequently used in cell therapy. Although several studies have shown the efficacy of these cells to modulate inflammation in different animal models, the results obtained in human clinical trials have been more modest. Here, we aimed at improving the therapeutic properties of MSCs by inducing a transient expression of two molecules that could enhance two different properties of these cells. With the purpose of improving MSC migration towards inflamed sites, we induced a transient expression of the C-X-C chemokine receptor type 4 (CXCR4). Additionally, to augment the anti-inflammatory properties of MSCs, a transient expression of the anti-inflammatory cytokine, interleukin 10 (IL10), was also induced. Methods Human adipose tissue-derived MSCs were transfected with messenger RNAs carrying the codon-optimized versions of CXCR4 and/or IL10. mRNA-transfected MSCs were then studied, first to evaluate whether the characteristic phenotype of MSCs was modified. Additionally, in vitro and also in vivo studies in an LPS-induced inflamed pad model were conducted to evaluate the impact associated to the transient expression of CXCR4 and/or IL10 in MSCs. Results Transfection of MSCs with CXCR4 and/or IL10 mRNAs induced a transient expression of these molecules without modifying the characteristic phenotype of MSCs. In vitro studies then revealed that the ectopic expression of CXCR4 significantly enhanced the migration of MSCs towards SDF-1, while an increased immunosuppression was associated with the ectopic expression of IL10. Finally, in vivo experiments showed that the co-expression of CXCR4 and IL10 increased the homing of MSCs into inflamed pads and induced an enhanced anti-inflammatory effect, compared to wild-type MSCs. Conclusions Our results demonstrate that the transient co-expression of CXCR4 and IL10 enhances the therapeutic potential of MSCs in a local inflammation mouse model, suggesting that these mRNA-modified cells may constitute a new step in the development of more efficient cell therapies for the treatment of inflammatory diseases.

Abstract Rectal cancer (RC) appears to behave differently compared with colon cancer. We aimed to analyze existence of different subtypes of RC depending on distinct features (age of onset and the presence of synchronous primary malignant neoplasms). We compared the clinicopathological, familial and molecular features of three different populations diagnosed with RC (early-onset RC [EORC], late-onset RC, and synchronous RC [SRC]). Eighty-five RCs were identified and were evaluated according to their microsatellite instability, CpG Island Methylator Phenotype (CIMP) and chromosomal instability, as assessed by Next Generation Sequencing and microarray-based comparative genomic hybridization approaches. The results were subjected to cluster analysis. SRCs displayed the most specific characteristics including a trend for the development of multiple malignant neoplasms, a greater proportion of CIMP-High tumors (75%) and more frequent genomic alterations. These findings were confirmed by a clustering analysis that stratified RCs according to their genomic alterations. We also found that EORCs exhibited their own features including an important familial cancer component and a remarkable rate of mutations in TP53 (53%). Together, heterogeneity in RC characteristics by age of disease-onset and SRC warrants further study to optimize tailored prevention, detection and intervention strategies—particularly among young adults.

Latest advances in the field of cancer immunotherapy have developed the (Chimeric Antigen Receptor) CAR-T cell therapy. This therapy was first used in hematological malignancies which obtained promising results; therefore, the use of CAR-T cells has become a popular approach for treating non-solid tumors. CAR-T cells consist of T-lymphocytes that are engineered to express an artificial receptor against any surface antigen of our choice giving us the capacity of offering precise and personalized treatment. This leaded to the development of CAR-T cells for treating solid tumors with the hope of obtaining the same result; however, their use in solid tumor and their efficacy have not achieved the expected results. The reason of these results is because solid tumors have some peculiarities that are not present in hematological malignancies. In this review we explain how CAR-T cells are made, their mechanism of action, adverse effect and how solid tumors can evade their action, and also we summarize their use in colorectal cancer and peritoneal carcinomatosis.

Adipose-derived stem cells comprise several clinically beneficial qualities that have been explored in basic research and have motivated several clinical studies with promising results. After being approved in the European Union, UK, Switzerland, Israel, and Japan, allogeneic adipose-derived stem cells (darvadstrocel) have been recently granted a regenerative medicine advanced therapy (RMAT) designation by US FDA for complex perianal fistulas in adults with Crohn’s disease. This huge scientific step is likely to impact the future spread of the indications of allogeneic adipose-derived stem cell applications. The current knowledge on adipose stem cell harvest describes quantitative and qualitative differences that could be influenced by different donor conditions and donor sites. In this comprehensive review, we summarize the current knowledge on the topic and propose donor profiles that could provide the optimal initial quality of this living drug, as a starting point for further applications and studies in different pathological conditions.

(1) Background: Abdominal adhesions are a common disease appearing after any type of abdominal surgery and may prolong surgical time and cause intestinal obstruction, infertility, or chronic pain. We propose the use of intraperitoneal collagenase to perform chemical adhesiolysis based on the pathophysiology and histology of adhesions. (2) Methods: We generated an adhesion model with intraperitoneal polypropylene meshes. Four months later, we evaluated the efficacy of the treatment in blinded form, i.e., 0.05% collagenase vs. placebo at 37 °C for 20 min. Protocol 1: Ten rats with ten mesh fragments, in which an attempt was made to remove the maximum number of meshes in a 5-min period. Protocol 2: Six rats with four mesh fragments in the sides of the abdominal cavity in which adhesiolysis was performed using a device that measures burst pressure. (3) Results: Protocol 1: 42% efficacy in the collagenase group versus 8% in the control group (p < 0.013). Protocol 2: 188.25 mmHg (SD 69.65) in the collagenase group vs. 325.76 mmHg (SD 50.25) in the control group (p < 0.001). (4) Conclusions: Collagenase allows for the safe and effective chemical adhesiolysis in this experimental model of adhesions.

Mesenchymal stromal cells (MSCs) currently constitute the most frequently used cell type in advanced therapies with different purposes, most of which are related with inflammatory processes. Although the therapeutic efficacy of these cells has been clearly demonstrated in different disease animal models and in numerous human phase I/II clinical trials, only very few phase III trials using MSCs have demonstrated the expected potential therapeutic benefit. On the other hand, diverse controversial issues on the biology and clinical applications of MSCs, including their specific phenotype, the requirement of an inflammatory environment to induce immunosuppression, the relevance of the cell dose and their administration schedule, the cell delivery route (intravascular/systemic vs. local cell delivery), and the selected cell product (i.e., use of autologous vs. allogeneic MSCs, freshly cultured vs. frozen and thawed MSCs, MSCs vs. MSC-derived extracellular vesicles, etc.) persist. In the current review article, we have addressed these issues with special emphasis in the new approaches to improve the properties and functional capabilities of MSCs after distinct cell bioengineering strategies.

Background: The Septin 9 test analyzes the methylation status of the SEPT9 gene, which appears to be hypermethylated in patients with colorectal cancer (CRC). This has been validated as a colorectal cancer screening test. Due to the high sensitivity and specificity found, the justification was to use it as a biomarker tool for monitoring minimal residual disease after radical surgery and recurrence. Methods: A prospective study was carried out at the Fundación Jiménez Díaz University Hospital extracting peripheral blood from 28 patients and 4 healthy donors. Free circulating DNA was obtained and subsequently a PCR reaction to quantify the number of methylated genes. Samples were obtained preoperatively and postoperatively at five to seven days, one and three months after surgery. Results: A total of 32 preoperative samples were analyzed. The sensitivity of the test to detect CRC was 55.6% and specificity was 100%. There were 22 postsurgical samples obtained at 5–7 days after surgery, the sensitivity to detect tumor recurrences was 100% and specificity was 75%. There were 21 samples analyzed 1 month after surgery exhibiting a sensitivity and specificity of 100% and 94.7%, respectively. At 3 months, 31 postsurgical samples were analyzed and the sensitivity and specificity were 66.7% and 80%. Conclusions: Detection of methylation of Septin 9 gene in circulating plasma DNA, obtained from a peripheral blood sample, may be a useful, non-invasive and effective method for detecting minimal residual disease and could therefore predict CRC tumor recurrences. The optimal time in our series to obtain the best prediction results based on Septin 9 methylation levels was one month after surgery. Despite these considerable findings, a study with more patients is necessary to obtain more robust conclusions.

Background: Positive cytology has been identified as an independent negative prognostic factor in patients with peritoneal metastases (PM) of colorectal origin. Liquid biopsy in plasma may detect increasing levels of circulating tumor DNA (ctDNA) and could help predict systemic relapse in patients with colorectal cancer, but little is known about the role of liquid biopsy in peritoneal fluid. The aim of this study was to evaluate the prognostic value of peritoneal fluid and plasma liquid biopsy in patients undergoing complete cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CC-HIPEC). Methods: A longitudinal prospective study was designed in patients with KRAS- mutated colorectal or appendiceal primary tumor, including PM of colorectal origin, pseudomyxoma peritonei and patients at high risk of developing PM (selected for second-look surgery). Eleven patients were recruited according to inclusion and exclusion criteria. ctDNA from plasma and peritoneal fluid before and after HIPEC was studied by droplet digital PCR looking for KRAS mutation. A close follow-up was scheduled (mean of 28.5 months) to monitor for systemic and peritoneal recurrences. Results: All patients with positive plasma postHIPEC had systemic relapse and four patients died as a result, while those with negative plasma postHIPEC did not relapse. Patients with negative peritoneal ctDNA after CC-HIPEC did not present peritoneal relapse. Of six patients with positive peritoneal ctDNA postHIPEC, two presented peritoneal recurrence and four systemic relapses. Conclusions: Treatment with CC-HIPEC does not always neutralize ctDNA in peritoneal fluid, and its persistence after treatment may predict adverse outcome. Despite being a proof of concept, an adequate correlation between liquid biopsy in plasma and peritoneal fluid with both systemic and peritoneal relapse has been observed.

Mesenchymal stromal cells (MSCs) constitute the cell type more frequently used in many regenerative medicine approaches due to their exclusive immunomodulatory properties, and they have been reported to mediate profound immunomodulatory effects in vivo. Nevertheless, MSCs do not express essential adhesion molecules actively involved in cell migration, a phenotypic feature that hampers their ability to home inflamed tissues following intravenous administration. In this study, we investigated whether modification by fucosylation of murine AdMSCs (mAdMSCs) creates Hematopoietic Cell E-/L-selectin Ligand, the E-selectin-binding CD44 glycoform. This cell surface glycan modification of CD44 has previously shown in preclinical studies to favor trafficking of mAdMSCs to inflamed or injured peripheral tissues. We analyzed the impact that exofucosylation could have in other innate phenotypic and functional properties of MSCs. Compared to unmodified counterparts, fucosylated mAdMSCs demonstrated higher in vitro migration, an altered secretome pattern, including increased expression and secretion of anti-inflammatory molecules, and a higher capacity to inhibit mitogen-stimulated splenocyte proliferation under standard culture conditions. Together, these findings indicate that exofucosylation could represent a suitable cell engineering strategy, not only to facilitate the in vivo MSC colonization of damaged tissues after systemic administration, but also to convert MSCs in a more potent immunomodulatory/anti-inflammatory cell therapy-based product for the treatment of a variety of autoimmune, inflammatory, and degenerative diseases.

Differential presence of exons (DPE) is a method of interpretation of exome sequencing, which has been proposed to design a predictive algorithm with clinical value in patients with colorectal cancer (CRC). The goal of the present study was to examine the reproducibility in a rat model of metastatic colon cancer. DHD/K12-TRb cells were injected in syngenic immunocompetent BD-IX rats. Cells were from two stocks with low and normal metastatic potential, and injected into two separate groups of rats. Five to ten weeks after injection, blood samples were taken prior euthanasia and whole exome sequencing performed. Through DPE analysis, we identified a set of exons whose differential presence in plasma allowed us to compare both groups of tumor-bearing animals. A verification test was performed to confirm that the algorithm was able to classify extracted samples into their corresponding groups of origin. The highest mean probability was 0.8954. In conclusion, the DPE analysis in tumor-bearing animals was able to discriminate between different disease status, which fully supports previous results in CRC patients.

Background: Identification of effective treatments in severe cases of COVID-19 requiring mechanical ventilation represents an unmet medical need. Our aim was to determine whether the administration of adipose-tissue derived mesenchymal stromal cells (AT-MSC) is safe and potentially useful in these patients. Methods: Thirteen COVID-19 adult patients under invasive mechanical ventilation who had received previous antiviral and/or anti-inflammatory treatments (including steroids, lopinavir/ritonavir, hydroxychloroquine and/or tocilizumab, among others) were treated with allogeneic AT-MSC. Ten patients received two doses, with the second dose administered a median of 3 days (interquartile range-IQR- 1 day) after the first one. Two patients received a single dose and another patient received 3 doses. Median number of cells per dose was 0.98 × 106 (IQR 0.50 × 106) AT-MSC/kg of recipient's body weight. Potential adverse effects related to cell infusion and clinical outcome were assessed. Additional parameters analyzed included changes in imaging, analytical and inflammatory parameters. Findings: First dose of AT-MSC was administered at a median of 7 days (IQR 12 days) after mechanical ventilation. No adverse events were related to cell therapy. With a median follow-up of 16 days (IQR 9 days) after the first dose, clinical improvement was observed in nine patients (70%). Seven patients were extubated and discharged from ICU while four patients remained intubated (two with an improvement in their ventilatory and radiological parameters and two in stable condition). Two patients died (one due to massive gastrointestinal bleeding unrelated to MSC therapy). Treatment with AT-MSC was followed by a decrease in inflammatory parameters (reduction in C-reactive protein, IL-6, ferritin, LDH and d-dimer) as well as an increase in lymphocytes, particularly in those patients with clinical improvement. Interpretation: Treatment with intravenous administration of AT-MSC in 13 severe COVID-19 pneumonia under mechanical ventilation in a small case series did not induce significant adverse events and was followed by clinical and biological improvement in most subjects. Funding: None.

Background: Acellular pseudomyxoma peritonei ( a PMP) is a rare peritoneal malignancy characterized by the accumulation of large amounts of mucin (lacking tumor cells) in the peritoneum. Many cases account for several kilograms of mucin to be screened by the pathologist. This is a comprehensive study of three patients with a PMP, whose tumors showed KRAS mutation, allowing for the tracking of this marker by liquid biopsy. Methods: Pre and post-surgery plasma, and mucin removed during cytoreductive surgery were collected from the patients. KRAS mutations were analyzed using droplet digital polymerase chain reaction (ddPCR). Mucin was injected in mice. KRAS and cytokine levels were measured in plasma of the mice using ddPCR and a magnetic bead-based assay. Mucin microbiome was analyzed by 16S rRNA sequencing. Results: KRAS mutations were detected in mucin cell-free DNA (cfDNA) from the three patients but not in the pre or post-surgery plasma. Electron microscopy detected microparticles (diameter

Background: To examine the usefulness of green fluorescent protein (GFP) mice for studying the interactions between normal cells and tumor cells in a host, we used a melanoma model in such “green” mice [C57BL/6-Tg(CAG-EGFP)1Osb mice]. Mice were given a subcutaneous injection of B16-F10 cells, and the resultant primary tumors were removed. Then cells from individual tumors were cultured. Results: The proportion of EFGP+ cells was determined by fluorescence-activated cell sorting (FACS) and was 6.8% ± 3.2% (mean ± s.d.) on day 1 of culture, 0.6% ± 0.3% on day 2, and 0.02% ± 0.01% at day 7. In all cases, isolated cells grew at a constant rate, but fluorescence decreased over time and became undetectable on day 14. Cells were tested using PCR for the presence of an EGFP-specific sequence, and results were negative in all cases, thus indicating that the cells did not harbor the host's reporter gene. Cells were also tested for the presence of EGFP mRNA, which was consistently detected for 22 days after the start of culture. The tumorogenicity of the cultured cells was confirmed in GFP mice injected with cells from a selection of cultures. Conclusions: In a melanoma model in GFP mice, the detection of “green” cells in tumors was not equivalent to the detection of host-derived cells. Such “masking” was caused by a transient, but lasting, transfer of EGFP mRNA from the host's normal cells to tumor cells. Thus, an analysis of tumors postmortem by techniques that yield only a single snapshot can lead to incorrect interpretations and erroneous conclusions.How to cite: Garcia-Olmo D, Picazo MG, García-Olmo D. Melanoma transplants in “green” mice: fluorescent cells in tumors are not equivalent to host-derived cells. Electron J Biotechnol 2018;34. https://doi.org/10.1016/j.ejbt.2018.04.007. Keywords: Cancer models, Development of tumors, EGFP, GFP mice, Green-fluorescent cells, Melanoma, Mouse, Oncogenic transformation

Abstract Next‐generation sequencing (NGS) has been proposed as a suitable tool for liquid biopsy in colorectal cancer (CRC), although most studies to date have focused almost exclusively on sequencing of panels of potential clinically actionable genes. We evaluated the clinical value of whole‐exome sequencing (WES) of cell‐free DNA (cfDNA) circulating in plasma, with the goal of identifying differential clinical profiles in patients with CRC. To this end, we applied an original concept, “differential presence of exons” (DPE). We determined differences in levels of 379 exons in plasma cfDNA and used DPE analysis to cluster and classify patients with disseminated and localized disease. The resultant bioinformatics analysis pipeline allowed us to design a predictive DPE algorithm in a small subset of patients that could not be initially classified based on the selection criteria. This DPE suggests that these nucleic acids could be actively released by both tumor and nontumor cells as a means of intercellular communication and might thus play a role in the process of malignant transformation. DPE is a new technique for the study of plasma cfDNA by WES that might have predictive and prognostic value in patients with CRC.

Summary: Most colorectal cancer (CRC)-related deaths are due to liver metastases. PKCζ is a tumor suppressor in CRC with reduced expression in metastasis. Given the importance of microRNAs (miRNAs) in regulating cellular plasticity, we performed an unbiased screening and identified the miR-200 family as the most relevant miRNAs downregulated by PKCζ deficiency. The regulation of the intracellular levels of miR-200 by PKCζ is post-transcriptional and involves their secretion in extracellular vesicles. Here, we identified ADAR2 as a direct substrate of PKCζ in CRC cells. Phosphorylation of ADAR2 regulates its editing activity, which is required to maintain miR-200 steady-state levels, suggesting that the PKCζ/ADAR2 axis regulates miR-200 secretion through RNA editing. Loss of this axis results in epithelial-to-mesenchymal transition (EMT) and increased liver metastases, which can be inhibited in vivo by blocking miR-200 release. Therefore, the PKCζ/ADAR2 axis is a critical regulator of CRC metastases through modulation of miR-200 levels. : Shelton et al. demonstrate that the loss of the tumor suppressor PKCζ in colorectal cancer cells results in the downregulation of miR-200, leading to increased epithelial-to-mesenchymal transition, cell invasion, and liver metastasis. This is mediated by an increase in miR-200 secretion through the inactivation of the RNA editing enzyme ADAR2, identifying a key vulnerability in liver metastasis. Keywords: PRKCZ, atypical PKC, ADAR2, colorectal cancer, metastasis, EMT, mir-200, extracellular vesicles, tumor suppressors, RNA editing

Myocardial infarction has been carefully studied in numerous experimental models. Most of these models are based on electrophysiological and functional data, and pay less attention to histological discoveries. During the last decade, treatment using advanced therapies, mainly cell therapy, has prevailed from among all the options to be studied for treating myocardial infarction. In our study we wanted to show the fundamental histological parameters to be evaluated during the development of an infarction on an experimental model as well as treatment with mesenchymal stem cells derived from adipose tissue applied intra-lesionally. The fundamental parameters to study in infarcted tissue at the histological level are the cells involved in the inflammatory process (lymphocytes, macrophages and M2, neutrophils, mast cells and plasma cells), neovascularization processes (capillaries and arterioles) and cardiac cells (cardiomyocytes and Purkinje fibers). In our study, we used intramyocardial injection of mesenchymal stem cells into the myocardial infarction area 1 hour after arterial occlusion and allowed 1 month of evolution before analyzing the modifications on the normal tissue inflammatory infiltrate. Acute inflammation was shortened, leading to chronic inflammation with abundant plasma cells and mast cells and complete disappearance of neutrophils. Another benefit was an increase in the number of vessels formed. Cardiomyocytes and Purkinje fibers were better conserved, both from a structural and metabolic point of view, possibly leading to reduced morbidity in the long term. With this study we present the main histological aspects to be evaluated in future assays, complementing or explaining the electrophysiological and functional findings.

Cell therapy is a progressively growing field that is rapidly moving from preclinical model development to clinical application. Outcomes obtained from clinical trials reveal the therapeutic potential of stem cell-based therapy to deal with unmet medical treatment needs for several disorders with no therapeutic options. Among adult stem cells, mesenchymal stem cells (MSCs) are the leading cell type used in advanced therapies for the treatment of autoimmune, inflammatory and vascular diseases. To date, the safety and feasibility of autologous MSC-based therapy has been established; however, their indiscriminate use has resulted in mixed outcomes in preclinical and clinical studies. While MSCs derived from diverse tissues share common properties depending on the type of clinical application, they markedly differ within clinical trials in terms of efficacy, resulting in many unanswered questions regarding the application of MSCs. Additionally, our experience in clinical trials related to critical limb ischemia pathology (CLI) shows that the therapeutic efficacy of these cells in different animal models has only been partially reproduced in humans through clinical trials. Therefore, it is crucial to develop new research to identify pitfalls, to optimize procedures and to clarify the repair mechanisms used by these cells, as well as to be able to offer a next generation of stem cell that can be routinely used in a cost-effective and safe manner in stem cell-based therapies targeting CLI.

Adipose mesenchymal stem cells (ASC) are considered minimally immunogenic. This is due to the low expression of human leukocyte antigens I (HLA-I), lack of HLA-II expression and low expression of co-stimulatory molecules such as CD40 and CD80. The low rate of observed immunological rejection as well as the immunomodulatory qualities, position ASC as a promising cell-based therapy for the treatment of a variety of inflammatory indications. Yet, few studies have addressed relevant aspects of immunogenicity such as ASC donor-to-patient HLA histocompatibility or assessment of immune response triggered by ASC administration, particularly in the cases of presensitization. The present study aims to assess allo-immune responses in a cohort of Crohn's disease patients administered with allogeneic ASC (darvadstrocel formerly Cx601) for the treatment of complex perianal fistulas. We identified donor-specific antibodies (DSA) generation in a proportion of patients and observed that patients showing preexisting immunity were prone to generating DSA after allogeneic therapy. Noteworthy, naïve patients generating DSA at week 12 (W12) showed a significant reduction in DSA titer at week 52 (W52), whereas DSA titer was reduced in pre-sensitized patients only with no specificities against the donor administered. Remarkably, we did not observe any correlation of DSA generation with ASC therapeutic efficacy. In vitro complement-dependent cytotoxicity (CDC) studies have revealed limited cytotoxic levels based upon HLA-I expression and binding capacity even in pro-inflammatory conditions. We sought to identify CDC coping mechanisms contributing to the limited cytotoxic killing observed in ASC in vitro. We found that ASC express membrane-bound complement regulatory proteins (mCRPs) CD55, CD46, and CD59 at basal levels, with CD46 more actively expressed in pro-inflammatory conditions. We demonstrated that CD46 is a main driver of CDC signaling; its depletion significantly enhances sensitivity of ASC to CDC. In summary, despite relatively high clearance, DSA generation may represent a major challenge for allogeneic cell therapy management. Sensitization may be a significant concern when evaluating re-treatment or multi-donor trials. It is still unknown whether DSA generation could potentially be the consequence of donor-to-patient interaction and, therefore, subsequently link to efficacy or biological activity. Lastly, we propose that CDC modulators such as CD46 could be used to ultimately link CDC specificity with allogeneic cell therapy efficacy.

Comparative studies of colorectal cancer (CRC) according to the age of onset have found differences between early-onset CRC (EOCRC) and late-onset CRC (LOCRC). Using this as a starting point, we wished to determine whether intermediate-onset CRC (IOCRC) might also be considered as an independent group within CRC. We performed a retrospective comparative study of the clinicopathological and familial features, as well as of the symptoms and their duration, of a total of 272 subjects diagnosed with CRC classified into three groups according to the age-of-onset (98 EOCRC, 83 IOCRC and 91 LOCRC). The results show that from a clinicopathological point of view, IOCRC shared certain features with EOCRC (gender, prognosis), and with LOCRC (multiple primary CRCs), whereas it also had characteristics that were specific for IOCRC (mean number of associated polyps). A gradual progression was observed from EOCRC to LOCRC from a greater family aggregation to sporadic cases, in parallel with a change of Lynch Syndrome cases to the sporadic microsatellite instability pathway, with the IOCRC being a boundary group that is more related to EOCRC. With respect to symptoms, duration and correlation with stages, IOCRC appeared more similar to EOCRC. Clinically, IOCRC behaves as a transitional group between EOCRC and LOCRC, with features in common with both groups, but also with IOCRC-specific features. Excluding cases with familial cancer history, the awareness for EOCRC diagnosis should be extended to IOCRC.

Background Diabetes is one of the major risk factors for peripheral arterial disease. In patients in whom surgery cannot be performed, cell therapy may be an alternative treatment. Since time is crucial for these patients, we propose the use of allogenic mesenchymal cells. Methods We obtained mesenchymal cells derived from the fat tissue of a healthy Sprague-Dawley rat. Previous diabetic induction with streptozotocin in 40 male Sprague-Dawley rats, ligation plus left iliac and femoral artery sections were performed as a previously described model of ischemia. After 10 days of follow-up, macroscopic and histo-pathological analysis was performed to evaluate angiogenic and inflammatory parameters in the repair of the injured limb. All samples were evaluated by the same blind researcher. Statistical analysis was performed using the SPSS v.11.5 program (P

Objective: to analyze short-term outcomes and complications for our first fifty patients with fecal incontinence undergoing sacral root stimulation. Patients: fifty patients with fecal incontinence receiving sacral neuromodulation in 4 hospitals are reviewed. Discussed variables include: age, sex, incontinence duration, incontinence cause, prior surgery for incontinence, Wexner scale score, anorectal manometry parameters, and endoanal ultrasonographic findings. Following the procedure Wexner scale score, anorectal manometry parameters, and associated complications are reviewed. Results: mean age of patients is 59.9 years, with females predominating. Most common causes of incontinence include obstetric procedures, idiopathic origin, and prior anal surgery. Mean follow-up is 17.02 months. Follow-up revealed a statistically significant reduction in Wexner scale score and increase in voluntary anal pressure. Technique-derived minor complications included: 2 surgical wound infections that led to stimulator withdrawal; 2 patients with pain who were managed conservatively; 1 case of externalization in a gluteal stimulator; and 1 broken tetrapolar electrode. Conclusions: sacral nerve stimulation is a simple technique that improves Wexner scores in a statistically significant manner with a low complications rate.Objetivo: analizar los resultados y complicaciones a corto plazo de nuestros primeros cincuenta pacientes con incontinencia fecal tratados mediante estimulación de raíces sacras. Pacientes: se revisan cincuenta pacientes con incontinencia fecal tratados mediante neuromodulación de raíces sacras en 4 centros hospitalarios. Las variables analizadas son: edad, sexo, tiempo de evolución de la incontinencia, causa de la incontinencia, cirugías previas para tratar la incontinencia, puntuación en la escala de Wexner, parámetros de la manometría anorrectal y los hallazgos en la ecografía endoanal. Tras la intervención se revisa la puntuación en la escala de Wexner, los parámetros en la manometría anorrectal y las complicaciones asociadas a esta técnica. Resultados: la edad media de los pacientes es de 59,9 años con predominio del sexo femenino. Las causas más frecuentes de incontinencia son obstétrica, idiopática y cirugía anal previa. El tiempo medio de seguimiento es de 17,02 meses. Tras el tratamiento se objetivó disminución en la puntuación en la escala de Wexner y aumento de la presión anal de contracción voluntaria de forma estadísticamente significativa. Hubo complicaciones menores derivadas de la técnica: 2 infecciones de herida quirúrgica que obligaron a retirar el estimulador, 2 casos de dolor que se manejaron conservadoramente, 1 superficialización del estimulador colocado en glúteo y una rotura del electrodo tetrapolar. Conclusiones: la neuromodulación de raíces sacras es una técnica sencilla que consigue una mejoría en la escala de Wexner estadísticamente significativa con una incidencia de complicaciones baja.