MSIMEP: Predicting microsatellite instability from microarray DNA methylation tumor profiles.

Deficiency in DNA MMR activity results in tumors with a hypermutator phenotype, termed microsatellite instability (MSI). Beyond its utility in Lynch syndrome screening algorithms, today MSI has gained importance as predictive biomarker for various anti-PD-1 therapies across many different tumor types. Over the past years, many computational methods have emerged to infer MSI using either DNA- or RNA-based approaches. Considering this together with the fact that MSI-high tumors frequently exhibit a hypermethylated phenotype, herein we developed and validated MSIMEP, a computational tool for predicting MSI status from microarray DNA methylation tumor profiles of colorectal cancer samples. We demonstrated that MSIMEP optimized and reduced models have high performance in predicting MSI in different colorectal cancer cohorts. Moreover, we tested its consistency in other tumor types with high prevalence of MSI such as gastric and endometrial cancers. Finally, we demonstrated better performance of both MSIMEP models vis-à-vis a MLH1 promoter methylation-based one in colorectal cancer.

Laboratory Cross-Comparison and Ring Test Trial for Tumor BRCA Testing in a Multicenter Epithelial Ovarian Cancer Series: The BORNEO GEICO 60-0 Study.

Germline and tumor BRCA testing constitutes a valuable tool for clinical decision-making in the management of epithelial ovarian cancer (EOC) patients. Tissue testing is able to identify both germline (g) and somatic (s) BRCA variants, but tissue preservation methods and the widespread implementation of NGS represent pre-analytical and analytical challenges that need to be managed. This study was carried out on a multicenter prospective GEICO cohort of EOC patients with known gBRCA status in order to determine the inter-laboratory reproducibility of tissue sBRCA testing. The study consisted of two independent experimental approaches, a bilateral comparison between two reference laboratories (RLs) testing 82 formalin-paraffin-embedded (FFPE) EOC samples each, and a Ring Test Trial (RTT) with five participating clinical laboratories (CLs) evaluating the performance of tissue BRCA testing in a total of nine samples. Importantly, labs employed their own locally adopted next-generation sequencing (NGS) analytical approach. BRCA mutation frequency in the RL sub-study cohort was 23.17%: 12 (63.1%) germline and 6 (31.6%) somatic. Concordance between the two RLs with respect to BRCA status was 84.2% (gBRCA 100%). The RTT study distributed a total of nine samples (three commercial synthetic human FFPE references, three FFPE, and three OC DNA) among five CLs. The median concordance detection rate among them was 64.7% (range: 35.3-70.6%). Analytical discrepancies were mainly due to the minimum variant allele frequency thresholds, bioinformatic pipeline filters, and downstream variant interpretation, some of them with consequences of clinical relevance. Our study demonstrates a wide range of concordance in the identification and interpretation of BRCA sequencing data, highlighting the relevance of establishing standard criteria for detecting, interpreting, and reporting BRCA variants.

Anti-Inflammatory Effect of Tacrolimus/Hydroxypropyl-ß-Cyclodextrin Eye Drops in an Endotoxin-Induced Uveitis Model.

BACKGROUND: Uveitis is an infrequent disease which constitutes a major cause of ocular morbidity. Correct management is essential, being corticosteroids its cornerstone. In case of contraindication to corticosteroids or treatment failure, the use of topical tacrolimus (TAC) could be an alternative which has already demonstrated safety and effectiveness in other ocular pathologies. However, TAC eye drops are not marketed, thus their elaboration must be carried out in Hospital Pharmacy Departments (HPDs). METHODS: 32 Sprague-Dawley rats were divided into 4 groups of 8 rats each: (a) untreated healthy rats (Healthy); (b) untreated Endotoxin-Induced Uveitis model-rats (EIU); (c) EIU-rats treated with standard treatment of dexamethasone ophthalmic drops (DXM) and (d) EIU-rats treated with TAC-hydroxypropyl-ß-cyclodextrin eye drops previously developed by our group (TAC-HPßCD). The mRNA expression levels of IL-6, IL-8, MIP-1α and TNF-α, quantitative analysis of leucocytes in aqueous humor and histological evaluation were performed. RESULTS: TAC-HPßCD eye drops demonstrated to reduce ocular inflammation, expression of IL-6, TNF-α, MIP-1α and leukocyte infiltration in aqueous humor. CONCLUSIONS: TAC-HPßCD eye drops showed beneficial effect in EIU model in rats, positioning as an alternative for uveitis treatment in case of corticosteroids resistance or intolerance.

Development and Characterization of Inhaled Ethanol as a Novel Pharmacological Strategy Currently Evaluated in a Phase II Clinical Trial for Early-Stage SARS-CoV-2 Infection.

Inhaled administration of ethanol in the early stages of COVID-19 would favor its location on the initial replication sites, being able to reduce the progression of the disease and improving its prognosis. Before evaluating the efficacy and safety of this novel therapeutic strategy in humans, its characterization is required. The developed 65° ethanol formulation is stable at room temperature and protected from light for 15 days, maintaining its physicochemical and microbiological properties. Two oxygen flows have been tested for its administration (2 and 3 L/min) using an automated headspace gas chromatographic analysis technique (HS-GC-MS), with that of 2 L/min being the most appropriate one, ensuring the inhalation of an ethanol daily dose of 33.6 ± 3.6 mg/min and achieving more stable concentrations during the entire treatment (45 min). Under these conditions of administration, the formulation has proven to be safe, based on histological studies of the respiratory tracts and lungs of rats. On the other hand, these results are accompanied by the first preclinical molecular imaging study with radiolabeled ethanol administered by this route. The current ethanol formulation has received approval from the Spanish Agency of Medicines and Medical Devices for a phase II clinical trial for early-stage COVID-19 patients, which is currently in the recruitment phase (ALCOVID-19; EudraCT number: 2020-001760-29).

3D Printed Tacrolimus Rectal Formulations Ameliorate Colitis in an Experimental Animal Model of Inflammatory Bowel Disease.

The aim of this study was to fabricate novel self-supporting tacrolimus suppositories using semisolid extrusion 3-dimensional printing (3DP) and to investigate their efficacy in an experimental model of inflammatory bowel disease. Blends of Gelucire 44/14 and coconut oil were employed as lipid excipients to obtain suppository formulations with self-emulsifying properties, which were then tested in a TNBS (2,4,6-trinitrobenzenesulfonic acid) induced rat colitis model. Disease activity was monitored using PET/CT medical imaging; maximum standardized uptake values (SUVmax), a measure of tissue radiotracer accumulation rate, together with body weight changes and histological assessments, were used as inflammatory indices to monitor treatment efficacy. Following tacrolimus treatment, a significant reduction in SUVmax was observed on days 7 and 10 in the rat colon sections compared to non-treated animals. Histological analysis using Nancy index confirmed disease remission. Moreover, statistical analysis showed a positive correlation (R2 = 71.48%) between SUVmax values and weight changes over time. Overall, this study demonstrates the effectiveness of 3D printed tacrolimus suppositories to ameliorate colitis and highlights the utility of non-invasive PET/CT imaging to evaluate new therapies in the preclinical area.

Evaluation of the therapeutic activity of melatonin and resveratrol in Inflammatory Bowel Disease: A longitudinal PET/CT study in an animal model.

Inflammatory Bowel Disease (IBD) is a group of chronic disorders of the gastrointestinal tract, which two main types are Crohn's disease and ulcerative colitis. Although conventional therapeutic strategies have demonstrated to be effective in the IBD treatment, it is necessary to incorporate novel therapeutic agents that target other mechanisms involved in the pathogenesis of the disease, such as oxidative stress. For this reason, the efficacy in vivo of two antioxidant compounds, melatonin and resveratrol, has been investigated in an animal model of TNBS (2, 4, 6-trinitrobenzenesulfonic acid) induced colitis. PET/CT (Positron emission tomography/Computer Tomography) scans were performed to assess disease activity and evaluate treatment response. SUVmax (Standardized Uptake Value) values, body weight changes and histological evaluation were used as inflammatory indices to measure the efficacy of both treatments. SUVmax values increased rapidly after induction of colitis, but after the beginning of the treatment (day 3) a statistically significant decrease was observed on days 7 and 10 in treated animals compared to the non-treated group. This remission of the disease was also confirmed by histological analysis of the colon tissue using the Nancy histological index (p value < 0.05 for differences between non-treated and both groups of treated animals). Moreover, statistical analysis showed a correlation (R2 = 65.52%) between SUVmax values and weight changes throughout the treatment. Overall, this study demonstrates the potential of resveratrol, and melatonin in lower extent, as therapeutic agents in the IBD treatment.

Longitudinal PET/CT evaluation of TNBS-induced inflammatory bowel disease rat model.

Inflammatory bowel disease (IBD) is a group of chronic disorders of the gastrointestinal tract, which two main types are Crohn's disease and ulcerative colitis. It has multifactorial etiologies, being essential the use of animal models and disease activity measures to develop new therapies. With this aim, the use of animal models in combination with non-invasive molecular imaging can play an important role in the development of new treatments. In this study, IBD was induced in rats using 2,4,6-trinitrobenzenesulfonic acid (TNBS) and longitudinal [18F]FDG PET/CT scans were conducted to assess disease progression post-TNBS administration. Afterwards, [18F]FDG PET/CT scans were carried out after treatment with methylprednisolone to validate the model. In non-treated rats, SUVmax (Standardized Uptake Value) rapidly increased after IBD induction, being particularly significant (p < 0.01) on days 7-13 after induction. There were no significant differences between non-treated and treated IBD rats from days 0-3. Nevertheless, treated IBD rats showed a significant decrease in SUVmax between days 7-13 (p < 0.01). Histological examination showed descending and transverse colon as the most affected regions. There was a moderate (R2 = 0.61) and strong (R2 = 0.82) correlation of SUVmax with Nancy grade (parameter for histological assessment of disease activity) and weight changes, respectively. In this study, we have performed the first longitudinal [18F]FDG PET/CT assessment of TNBS-induced IBD in rats, demonstrating the potential role of preclinical molecular imaging for the evaluation of new therapies in combination with IBD rat models.

Prognostic value of changes in the expression of stem cell markers in the peripheral blood of patients with colon cancer.

Cancer stem cells play an important role in carcinogenesis and resistance to treatment and may lead to metastasis. The isolation of circulating stem cells involves cell sorting based on the presence of cell surface markers. Many surface markers such as CD133, c-Kit, SOX, OCT4 and TWIST have been reported. In the present study, we determined the expression of different stem cell markers and their variation in expression at different stages of the treatment process. Samples of EDTA blood were collected from metastatic colorectal cancer patients, and circulating cancer stem cells were isolated for the analysis of the expression of stem cell markers using RT-PCR. These findings were correlated with the response to therapy. All statistical analyses were performed using the GraphPad Prism 5.03 software. Significant differences were found in the expression levels of the markers CD133, SOX2, OCT4 and TWIST1. No differences were found in c-Kit expression. Correlation in the expression levels of most of the markers was observed. Expression of CD133, OCT4, SOX2 and TWIST1 had a predictive value for colon cancer behavior. Evaluation of this stem cell gene expression panel may be useful for predicting the response during the process of treatment, and the relative easy access to samples facilitates this method. Moreover the correlation between CD133 and TWIST1 expression may be associated with tumor regrowth and metastatic relapse.