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Pseudomyxoma peritonei (PMP) is a rare malignant growth characterized by the production of mucin and the potential for peritoneal relapse. This study aimed to investigate the immunohistochemical and biological characteristics of mucin in patients with cellular and acellular PMP. We prospectively analyzed mucin specimens obtained from our patient cohort and described the composition and type of mucin present in each sample. A metagenomic analysis of the samples was performed to investigate the bacterial composition of the PMP microbiome. Secreted mucins 2 and 5AC and membrane-associated mucin-1 were the primary components of mucin in both cellular and acellular tumor specimens. The metagenomic study revealed a predominance of the phylum Proteobacteria and the genus Pseudomonas. Notably, Pseudomonas plecoglossicida, a species not previously reported in the human microbiome, was found to be the most abundant organism in the mucin of pseudomyxoma peritonei. Our findings suggest that the presence of MUC-2 and mucin colonization by Pseudomonas are characteristic features of both cellular and acellular disease. These results may have significant implications for the diagnosis and treatment of this rare entity.

Pseudomyxoma peritonei (PMP) is a rare malignant growth characterized by the production of mucin and the potential for peritoneal relapse. This study aimed to investigate the immunohistochemical and biological characteristics of mucin in patients with cellular and acellular PMP. We prospectively analyzed mucin specimens obtained from our patient cohort and described the composition and type of mucin present in each sample. A metagenomic analysis of the samples was performed to investigate the bacterial composition of the PMP microbiome. Secreted mucins 2 and 5AC and membrane-associated mucin-1 were the primary components of mucin in both cellular and acellular tumor specimens. The metagenomic study revealed a predominance of the phylum Proteobacteria and the genus Pseudomonas. Notably, Pseudomonas plecoglossicida, a species not previously reported in the human microbiome, was found to be the most abundant organism in the mucin of pseudomyxoma peritonei. Our findings suggest that the presence of MUC-2 and mucin colonization by Pseudomonas is a characteristic feature of both cellular and acellular disease. These results may have significant implications for the diagnosis and treatment of this rare entity.

BACKGROUND Digestive tract resections are usually followed by an anastomosis. Anastomotic leakage, normally due to failed healing, is the most feared complication in digestive surgery because it is associated with high morbidity and mortality. Despite technical and technological advances and focused research, its rates have remained almost unchanged the last decades. In the last two decades, stem cells (SCs) have been shown to enhance healing in animal and human studies; hence, SCs have emerged since 2008 as an alternative to improve anastomoses outcomes. AIM To summarise the published knowledge of SC utilisation as a preventative tool for hollow digestive viscera anastomotic or suture leaks. METHODS PubMed, Science Direct, Scopus and Cochrane searches were performed using the key words “anastomosis”, “colorectal/colonic anastomoses”, “anastomotic leak”, “stem cells”, “progenitor cells”, “cellular therapy” and “cell therapy” in order to identify relevant articles published in English and Spanish during the years of 2000 to 2021. Studies employing SCs, performing digestive anastomoses in hollow viscera or digestive perforation sutures and monitoring healing were finally included. Reference lists from the selected articles were reviewed to identify additional pertinent articles. Given the great variability in the study designs, anastomotic models, interventions (SCs, doses and vehicles) and outcome measures, performing a reliable meta-analysis was considered impossible, so we present the studies, their results and limitations. RESULTS Eighteen preclinical studies and three review papers were identified; no clinical studies have been published and there are no registered clinical trials. Experimental studies, mainly in rat and porcine models and occasionally in very adverse conditions such as ischaemia or colitis, have been demonstrated SCs as safe and have shown some encouraging morphological, functional and even clinical results. Mesenchymal SCs are mostly employed, and delivery routes are mainly local injections and cell sheets followed by biosutures (sutures coated by SCs) or purely topical. As potential weaknesses, animal models need to be improved to make them more comparable and equivalent to clinical practice, and the SC isolation processes need to be standardised. There is notable heterogeneity in the studies, making them difficult to compare. Further investigations are needed to establish the indications, the administration system, potential adjuvants, the final efficacy and to confirm safety and exclude definitively oncological concerns. CONCLUSION The future role of SC therapy to induce healing processes in digestive anastomoses/sutures still needs to be determined and seems to be currently far from clinical use.

It has been proposed that cell-free nucleic acids in the plasma participate in tumorigenesis and the development of metastases via transfection-like uptake of such nucleic acids by susceptible cells. This putative phenomenon is tentatively referred to as “genometastasis.” In the present study, we examined the effects on cultured cells of plasma from healthy individuals and from patients with colon cancer. Cultures of NIH-3T3 cells and human adipose-derived stem cells (hASC) were supplemented with samples of plasma from patients with K-ras–mutated colorectal tumors or from healthy subjects using two different protocols: direct addition of plasma to cultures in standard plates and addition in the absence of contact between plasma and cells, which were separated by a membrane with 0.4-μm pores. In plasma-treated hASCs, no K-ras–mutated sequences were detected by real-time PCR. In contrast, in most cultures of plasma-treated NIH-3T3 cells (murine cells), the transfer of human DNA occurred, as verified by the detection of human K-ras sequences, p53 sequences, and β-globin–encoding sequences. Moreover, NIH-3T3 cells that had been cultured with plasma from patients with colon cancer were oncogenically transformed, as shown by the development of carcinomas in nonobese diabetic–severe combined immunodeficient mice after the injection of such cells. Microscopic analysis of membranes that had separated plasma from cultured cells confirmed the complete absence of cells in the plasma. We only observed noncell particles, having diameters of

Supplementary Methods from Cell-Free Nucleic Acids Circulating in the Plasma of Colorectal Cancer Patients Induce the Oncogenic Transformation of Susceptible Cultured Cells

Certain diseases such as obesity and cancer can cause impaired wound healing. Adipose tissue derived stem cells (ASCs) are a novel field of research. Many studies have evidenced their high degree of safety and potential for wound repair due to their immunomodulatory and tissue-regeneration properties. The purpose of this study is to determine the impact of obesity and cancer on the therapeutic potential of ASCs.We isolated and characterized the phenotype, differentiation capacities, secretome, and in vitro migration capacities of ASCs. Furthermore, we analyze their capacity of in vitro migration associated with the plasma of the different patients.We observed that ASCs isolated from obese and cancer patients have the same phenotype, cell proliferation, and migration capacities as ASCs derived from healthy donors. However, they do not have the same differentiation potential and exhibit distinct profiles of both pro-inflammatory and regulatory secreted cytokines, which, together with the signals received from the bloodstream, could account for the impaired healing in patients with these diseases.We consider the ASCs from patients with either obesity or cancer are slightly altered, and this may be the cause of worse wound healing in these patients.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.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.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.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.

BCR-ABL1/ABL1 p210 measurement by quantitative polymerase chain reaction (qPCR) is used worldwide to monitor the molecular response in chronic myeloid leukemia (CML) patients. Droplet digital polymerase chain reaction (ddPCR) seems to show a greater sensitivity than qPCR, probably due to the high number of replicates analyzed in ddPCR for the comparison. Additionally, in a recently published comparison, ddPCR measurements were not adequately transformed into International Scale (IS). We have analyzed 50 CML patients and ten non-CML donors in parallel by qPCR and ddPCR. To the best of our knowledge, this is the first study comparing both techniques under similar conditions, with BCR-ABL1/ABL1 measurements performed via both techniques transformed into IS. Qualitative and quantitative comparisons showed excellent results. The qualitative correlation showed a Kappa index of 0.94 (95% confidence interval [CI] 0.90–0.98) (P

We evaluated the safety and feasibility of adipose‐derived mesenchymal stem cells to treat endoscopically urinary incontinence after radical prostatectomy in men or female stress urinary. We designed two prospective, nonrandomized phase I‐IIa clinical trials of urinary incontinence involving 9 men (8 treated) and 10 women to test the feasibility and safety of autologous mesenchymal stem cells for this use. Cells were obtained from liposuction containing 150 to 200 g of fat performed on every patient. After 4 to 6 weeks and under sedation, endoscopic intraurethral injection of the cells was performed. On each visit (baseline, 1, 3, 6, and 12 months), clinical parameters were measured, and blood samples, urine culture, and uroflowmetry were performed. Every patient underwent an urethrocystoscopy and urodynamic studies on the first and last visit. Data from pad test, quality‐of‐life and incontinence questionnaires, and pads used per day were collected at every visit. Statistical analysis was done by Wilcoxon signed‐rank test. No adverse effects were observed. Three men (37.5%) and five women (50%) showed an objective improvement of >50% (P, We evaluated the safety and feasibility of adipose‐derived mesenchymal stem cells (MSCs) to treat endoscopically urinary incontinence after radical prostatectomy in men or women stress urinary. Our conclusion is intraurethral application of MSCs are a safe and feasible procedure to treat urinary incontinence. A statistically significant difference was obtained for pad‐test improvement in 3/8 men and 5/10 women.

Objectives. To confirm that patients affected by colorectal cancer have the V2 region of Septin 9 (SEPT9) gene hypermethylated in the circulating free DNA from a peripheral blood sample before surgery and to determine if this hypermethylated DNA disappears from the patients after complete resection of the tumour. Methods. Plasma from 10 patients with colorectal cancer was collected preoperative and three months after surgery. The analysis of the methylation status of the promoter region of the SEPT9 gene was performed using a 7500 Fast Real-Time PCR System. Results. Hypermethylation of SEPT9 gene was detected in 8 out of 10 preoperative samples (one negative result was probed to be a Lynch syndrome) and in 4 out of 10 postoperative samples matching with the cases of recurrence or persistence of disease. This means that, in this sample, the preoperative sensitivity and specificity of the test were 88.9% and 100%, respectively, and there is 100% correlation between the positive results of the SEPT9 test and a recurrence/persistence of the disease in patients after surgical resection. Conclusions. Our study shows that circulating hypermethylated SEPT9 is a specific colorectal cancer biomarker. This hypermethylated SEPT9 DNA disappears around three months after surgery and that circulating hypermethylated SEPT9 may be the first noninvasive marker for postsurgical diagnosis; this conclusion must be confirmed with a more significant number of patients.

The use of mesenchymal stem cells has resulted in a breakthrough for the treatment of complex perianal fistulas in Crohn’s disease. This novel treatment is associated with a minimally invasive surgical technique that can be well defined. However, our previous experience has taught us that neglecting any of the critical steps in the operation can result in frequent treatment failure. We have put together a comprehensive guide, a stepwise algorithm, for our minimally invasive approach to identify common pitfalls and reduce treatment failures. Using data we have collected over the past 15 years from drug development, execution of clinical trials, and enacting an advanced educational training program, we spelt out each stage of the minimally invasive surgical intervention for stem cell delivery for perianal Crohn’s disease. In this article, we provide 21 tips for a correct approach during the five major phases of the surgical procedure. To optimize the efficacy of mesenchymal stem cells for perianal Crohn’s disease, a standardized minimally invasive technique including a reliable and reproducible series of key steps should be utilized.

Background This study aimed to measure the toxicity resulting from collagenase administration to the peritoneal cavity in a pig model as a preliminary step to break down the stroma surrounding tumors. Methods Eight pigs were treated with 2 different collagenase concentrations previously tested in rats by our group. Time and temperature were controlled using a peritoneal lavage system (PRS System, Combat Medical Ltd.) identical to that used in human surgeries through hyperthermic intraperitoneal chemotherapy (HIPEC); 2 additional pigs were treated with peritoneal lavage only. Samples of blood and peritoneal fluid were collected pre-treatment, immediately after treatment, and 24 h postoperatively. In addition, histological studies and blood collagenase levels were measured. Results No complications were observed during the surgeries. Intraoperative images evidenced the release of peritoneal tissue during collagenase treatment. After surgery, the animals showed no signs of pain. Diet and mobility were normal at 4 h postoperatively, and there were no significant differences in hematologic or biochemical parameters. Quantification of MMP1 and MMP2 in all samples as measured by absorbance showed no differences in blood collagenase levels between pre-treatment, post-treatment, and 24 h postoperatively. None of the animals treated with collagenase showed peritoneal adhesions during the second surgery. Histologically, peritoneal organs and serous structures did not show any microscopic alterations associated with collagenase treatment in any group. Conclusion Lavage of the peritoneal cavity with doses of up to 100,000 collagen digestion units/animal for 30 min is safe and removes connective tissue from the peritoneal cavity.

In the era of personalized medicine and targeted therapies for the management of patients with cancer, ultrasensitive detection methods for tumor genotyping, such as next-generation sequencing or droplet digital polymerase chain reaction (ddPCR), play a significant role. In the search for less invasive strategies for diagnosis, prognosis and disease monitoring, the number of publications regarding liquid biopsy approaches using ddPCR has increased substantially in recent years. There is a long list of malignancies in which ddPCR provides a reliable and accurate tool for detection of nucleic acid-based markers derived from cell-free DNA, cell-free RNA, circulating tumor cells, extracellular vesicles or exosomes when isolated from whole blood, plasma and serum, helping to anticipate tumor relapse or unveil intratumor heterogeneity and clonal evolution in response to treatment. This updated review describes recent developments in ddPCR platforms and provides a general overview about the major applications of liquid biopsy in blood, including its utility for molecular response and minimal residual disease monitoring in hematological malignancies or the therapeutic management of patients with colorectal or lung cancer, particularly for the selection and monitoring of treatment with tyrosine kinase inhibitors. Although plasma is the main source of genetic material for tumor genomic profiling, liquid biopsy by ddPCR is being investigated in a wide variety of biologic fluids, such as cerebrospinal fluid, urine, stool, ocular fluids, sputum, saliva, bronchoalveolar lavage, pleural effusion, mucin, peritoneal fluid, fine needle aspirate, bile or pancreatic juice. The present review focuses on these "alternative" sources of genetic material and their analysis by ddPCR in different kinds of cancers.

Aim. To report our experience in a compassionate use program for complex perianal fistula. Methods. Under controlled circumstances and approved by European and Spanish laws, a compassionate use program allows the use of stem cell therapy for patients with nonhealing diseases, mostly complex fistula-in-ano, who do not meet criteria to be included in a clinical trial. Candidates had previously undergone multiple surgical interventions that had failed. The intervention consisted of surgery (with closure of the internal opening or a surgical flap performance), followed by stem cell injection. Three types of cells were used for implant: stromal vascular fraction, autologous expanded adipose-derived, or allogenic adipose-derived stem cells. Healing was evaluated at 6th month follow-up. Outcome was classified as partial response or healing. Relapse was evaluated 1 year later. Maximum follow-up period was 48 months. Results. 45 patients (24 male) were included; the mean age was 45 years, which ranged from 24 to 69 years. Since some of them received repeated doses, 52 cases were considered (42 fistula-in-ano, 7 rectovaginal fistulas, 1 urethrorectal fistula, 1 sacral fistula, and 1 hidradenitis suppurativa). Regarding fistula-in-ano, there were 18 Crohn’s-associated and 24 cryptoglandular. 49 cases (94.2%) showed partial response starting 6.5 weeks of follow-up. 24 cases (46.2%) healed in a mean time of 5.5 months. A year later, all patients cured remained healed. No adverse effects related to stem cell therapy were reported. Conclusion. Stem cells are safe and useful for treating anal fistulae. Healing can be achieved in severe cases.

Advanced therapies hold substantial promise for the treatment of periodontal conditions. Gene therapy has the potential to transfer “therapeutic” genes, which express proteins such as bone morphogenetic proteins, osteoprotegerin, and tissue nonspecific alkaline phosphatase, which is deficient in patients with hypophosphatasia, a condition that affects mineralization of teeth and bone. Transferred genes may also express platelet-derived growth factor, which modulates the growth of periodontal tissue and the alveolar bone. As regards cell therapy, several clinical trials have shown that mesenchymal stem cells, when used with different kinds of scaffolds to enable the required three-dimensional environment, possess a bone regeneration potential that is particularly useful in such disorders as osteoporosis and osteonecrosis, or for regenerating alveolar bone (osseointegration) prior to placing a dental implant. However, much work is still required before these new therapies become true alternatives in routine clinical dental practice. Medical advances require investments, which are usually influenced by the priorities of both politicians and society at large. This will contribute to promoting innovation, efficient treatments, medium- and long-term savings, and a higher quality of life.

Objective. The aims of this study are to compare 2 origins of adipose-derived mesenchymal stem cells (MSCs) (omentum and subcutaneous) from 2 pathologies (morbid obesity and cancer) vs healthy donors. Adipose tissue has revealed to be the ideal MSC source. However, in developing adipose-derived stem cells (ASCs) for clinical use, it is important to consider the effects of different fat depots and also the effect of donor variability. Methods. We isolated and characterized the membrane markers and differentiation capacities of ASCs obtained from patients with these diseases and different origin. During the culture period, we further analysed the cells’ proliferation capacity in an in vitro assay as well as their secretome. Results. Adipose-derived stem cells isolated from obese and cancer patients have mesenchymal phenotype and similar cell proliferation as ASCs derived from healthy donors, some higher in cells derived from subcutaneous fat. However, cells from these 2 types of patients do not have the same differentiation potential, especially in cancer patients from omentum, and exhibit distinct secretion of both pro-inflammatory and regulatory cytokines, which could explain the differences in use due to origin as well as pathology associated with the donor. Conclusion. Subcutaneous and omentum ASCs are slightly different; omentum generates fewer cells but with greater anti-inflammatory capacity. Adipose-derived stem cells from patients with either obesity or cancer are slightly altered, which limits their therapeutic properties.

Background aims Successful cell cryopreservation and banking remain a major challenge for the manufacture of cell therapy products, particularly in relation to providing a hermetic, sterile cryovial that ensures optimal viability and stability post-thaw while minimizing exposure to toxic cryoprotective agents, typically dimethyl sulfoxide (Me2SO). Methods In the present study, the authors evaluated the effectiveness and functionality of Limbo technology (Cellulis S.L., Santona, Spain). This system provides a hermetic vial with two compartments (one for adding cells with the cryoprotective agent solution and the other for the diluent solution) and an automated defrosting device. Limbo technology (Cellulis S.L.) allows reduction of the final amount of Me2SO, sidestepping washing and dilution steps and favoring standardization. The study was performed in several Good Manufacturing Practice laboratories manufacturing diverse cell therapy products (human mesenchymal stromal cells, hematopoietic progenitor cells, leukapheresis products, fibroblasts and induced pluripotent stem cells). Laboratories compared Limbo technology (Cellulis S.L.) with their standard cryopreservation procedure, analyzing cell recovery, viability, phenotype and functionality. Results Limbo technology (Cellulis S.L.) maintained the viability and functionality of most of the cell products and preserved sterility while reducing the final concentration of Me2SO. Conclusions Results showed that use of Limbo technology (Cellulis S.L.) offers an overall safe alternative for cell banking and direct infusion of cryopreserved cell products into patients.

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations, 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, This work was supported by the following public grants: Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III Ministerio de Ciencia, Innovación y Universidades and Fondo Europeo de Desarrollo Regional (FEDER) ((RETICS-RD16/0011/0011, PIE15/00048, PI18-01379), and Dirección General de Investigación de la Comunidad de Madrid (AvanCell-CM; Ref S2017/BMD-3692)

Background Mesenchymal stromal cells (MSC) have been proposed as a promising complement to standard immunosuppression in solid organ transplantation because of their immunomodulatory properties. The present work addresses the role of adipose-derived MSC (Ad-MSC) in an experimental model of acute rejection in small bowel transplantation (SBT). Material/methods heterotopic allogeneic SBT was performed. A single dose of 1.5x106 Ad-MSC was intra-arterially delivered just before graft reperfusion. Animals were divided into CONTROL (CTRL), CONTROL+Ad-MSC (CTRL_MSC), tacrolimus (TAC), and TAC+Ad-MSC (TAC_MSC) groups. Each Ad-MSC groups was subdivided in autologous and allogeneic third-party groups. Results Rejection rate and severity were similar in MSC-treated and untreated animals. CTRL_MSC animals showed a decrease in macrophages, T cell (CD4, CD8 and Foxp3 subsets) and B cell counts in the graft compared with CTRL, this decrease was attenuated in TAC_MSC animals. Pro- and anti-inflammatory cytokines and some chemokines and growth factors increased in CTRL_MSC animals, especially in the allogeneic group, whereas milder changes were seen in the TAC groups. Conclusion Ad-MSC did not prevent rejection when administered just before reperfusion. However, they showed immunomodulatory effects that could be relevant for a longer-term outcome. Interference between tacrolimus and the MSC effects should be addressed in further studies.

The usefulness of local collagenase in therapeutic approaches to solid tumors has been tested recently. In this study, we evaluate the safety and efficacy of intraperitoneal collagenase associated or not to mitomycin for treatment of colorectal peritoneal metastases in an experimental rat model. Using a fixed-dose procedure, we found that a dose of collagenase of 37 IU/mL administered for 15 min with a hyperthermia pump at 37.5 °C, both in isolation or associated to sequential treatment with intraperitoneal mitomycin, led to a macroscopic decrease in tumor volume as evaluated by the modified peritoneal cancer index (mPCI). Concerning the safety of the procedure, the animals showed no physiological or behavioral disorders during 8 weeks of follow-up. Local treatment for peritoneal metastases of colorectal origin with intraperitoneal collagenase has proved safe and effective in an experimental murine model. Therefore, the stroma-first approach by enzymatic breakdown of collagen from the tumor's extracellular matrix provides a new therapeutic target for colorectal peritoneal metastases.

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.

Venous leg ulcers (VLU) represent an uphill economic, health and social burden, aggravated in the elderly. Best-practice care interventions are often insufficient and alternative therapies need to be explored. Herein, we have treated for the first time a chronic VLU in an elderly patient by combining cell therapy and tissue engineering in the context of a compassionate use. The administration of allogeneic adipose-derived mesenchymal stromal cells (MSCs) embedded in a plasma-based bioengineered dermis covering the ulcer bed and also injected into the ulcer margins led to the complete closure of a 10-year recalcitrant VLU in an 85-year-old patient. Regenerative properties of MSCs might be boosted by the use of bioengineered matrices for their delivery.

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. We would like to acknowledge the Instituto de Salud Carlos III (ISCIII) through the project “RD16/0011: Red de Terapia Celular”, from the sub-program RETICS, integrated in the “Plan Estatal de I+D+I 2013-2016” and co-financed by the European Regional Development Fund “A way to make Europe”, groups RD16/0011/0001, -/0002, -/005, -/0013, -/0015, -/0029), the Centro en Red de Medicina Regenerativa y Terapia Celular de Castilla y León, Spain and AvanCell-CM (Red de Investigación de Terapia Celular de la Comunidad de Madrid, Spain), for supporting some personnel and networking activities.

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., This studywas funded by two grants from"Instituto de Salud Carlos III", Spain (FIS; refs. PS09/01815 and PI13/01924)

Background: Acellular pseudomyxoma peritonei (aPMP) 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 aPMP, 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, The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was funded by a grant from the ‘FIS-ISCIII-FEDER’ [Fondo de Investigaciones Sanitarias-Instituto de Salud Carlos III-Fondo Europeo de Desarrollo Regional (European Regional Development Fund)], Ministry of Health, Spain (grant number PI17/01233).

Faecal continence is a complex function involving different organs and systems. Faecal incontinence is a common disorder with different pathogeneses, disabling consequences and high repercussions for quality of life. Current management modalities are not ideal, and the development of new treatments is needed. Since 2008, stem cell therapies have been validated, 36 publications have appeared (29 in preclinical models and seven in clinical settings), and six registered clinical trials are currently ongoing. Some publications have combined stem cells with bioengineering technologies. The aim of this review is to identify and summarise the existing published knowledge of stem cell utilization as a treatment for faecal incontinence. A narrative or descriptive review is presented. Preclinical studies have demonstrated that cellular therapy, mainly in the form of local injections of muscle-derived (muscle derived stem cells or myoblasts derived from them) or mesenchymal (bone-marrow- or adipose-derived) stem cells, is safe. Cellular therapy has also been shown to stimulate the repair of both acute and subacute anal sphincter injuries, and some encouraging functional results have been obtained. Stem cells combined with normal cells on bioengineered scaffolds have achieved the successful creation and implantation of intrinsically-innervated anal sphincter constructs. The clinical evidence, based on adipose-derived stem cells and myoblasts, is extremely limited yet has yielded some promising results, and appears to be safe. Further investigation in both animal models and clinical settings is necessary to drawing conclusions. Nevertheless, if the preliminary results are confirmed, stem cell therapy for faecal incontinence may well become a clinical reality in the near future.