Inhibition of the AURKA/YAP1 axis is a promising therapeutic option for overcoming cetuximab resistance in colorectal cancer stem cells.

BACKGROUND: Primary resistance to anti-EGFR therapies affects 40% of metastatic colorectal cancer patients harbouring wild-type RAS/RAF. YAP1 activation is associated with this resistance, prompting an investigation into AURKA's role in mediating YAP1 phosphorylation at Ser397, as observed in breast cancer. METHODS: We used transcriptomic analysis along with in vitro and in vivo models of RAS/RAF wild-type CRC to study YAP1 Ser397 phosphorylation as a potential biomarker for cetuximab resistance. We assessed cetuximab efficacy using CCK8 proliferation assays and cell cycle analysis. Additionally, we examined the effects of AURKA inhibition with alisertib and created a dominant-negative YAP1 Ser397 mutant to assess its impact on cancer stem cell features. RESULTS: The RAS/RAF wild-type CRC models exhibiting primary resistance to cetuximab prominently displayed elevated YAP1 phosphorylation at Ser397 primarily mediated by AURKA. AURKA-induced YAP1 phosphorylation was identified as a key trigger for cancer stem cell reprogramming. Consequently, we found that AURKA inhibition had the capacity to effectively restore cetuximab sensitivity and concurrently suppress the cancer stem cell phenotype. CONCLUSIONS: AURKA inhibition holds promise as a therapeutic approach to overcome cetuximab resistance in RAS/RAF wild-type colorectal cancer, offering a potential means to counter the development of cancer stem cell phenotypes associated with cetuximab resistance.

Foreign body in a Zenker's diverticulum.

Zenker's diverticulum (ZD) is an uncommon disorder that can cause dysphagia with risk of aspiration. While surgical treatment has been the mainstay for many years, endoscopic diverticulotomy has emerged as a first-line option with favorable outcomes. We present the case of a 93-year-old woman with no significant past medical history who was diagnosed with a 6 cm ZD. Due to dysphagia, she experienced significant weight loss and was at risk of malnutrition. She developed aspiration pneumonia and required admission to our center. Given her condition and inability to swallow, a nasogastric tube was placed under radiological guidance for nutritional support pending definitive treatment. On radiographic localization of the ZD, a radiopaque metallic density image was observed that had not been identified in previous imaging. Suspecting a possible retained foreign body in the large diverticulum, a gastroscopy was performed. During the procedure, the ZD was accessed and a 10 mm metallic object was identified. The object was extracted using a Roth net, confirming the suspicion of a foreign body lodged in the ZD. The metallic piece was later identified as a patient's dental prosthesis. After resolution of the aspiration pneumonia, endoscopic-assisted diverticulotomy was performed. The procedure was carried out under deep sedation with cricopharyngeal myotomy without immediate complications. After 48 hours of hospitalization, the patient was discharged without requiring a nasogastric tube for feeding.

Neuropsychological profile of executive functions in autism spectrum disorder and schizophrenia spectrum disorders: a comparative group study in adults.

As assessed by numerous neuropsychological tasks, individuals with autism spectrum disorder (ASD) and schizophrenia spectrum disorders (SSDs) have similar impairments related to executive functions (EFs). The neuropsychological profile of these two conditions was examined using the three-component EFs' framework of Miyake and Friedman (Cogn Psychol 41(1):49-100, 2000). This approach assesses Inhibition (suppression of unwanted and irrelevant information/responses), Updating (use and control of contents of working memory), and Shifting (disengagement between activities or mental tasks) using nine different tasks. In line with previous research, we expected greater performance deficits in ASD in all three components compared to SSD, as well as faster responses for the SSD group. A self-paced task format allowed us to examine whether unlimited time given for a task would lead to better performance. The sample was constituted by the control group (N = 25), ASD group (N = 24), and SSD group (N = 12). Groups did not differ on Inhibition performance. In Updating, individuals with SSD performed poorer than the other groups. As for Shifting, both groups demonstrated poorer performance compared to controls, with the SSD group presenting the greatest difficulties. In terms of reaction time (RT), SSD participants' RT were the slowest on Inhibition and Shifting tasks. There was a positive correlation between performance and time spent on Inhibition and Shifting only for the SSD group, which demonstrates that their performance improves when there are no time constraints. Our work provides a better understanding of spared and impaired EFs, which could be useful for designing strategies aimed at improving specific EFs in each group.

CD44 Modulates Cell Migration and Invasion in Ewing Sarcoma Cells.

The chimeric EWSR1::FLI1 transcription factor is the main oncogenic event in Ewing sarcoma. Recently, it has been proposed that EWSR1::FLI1 levels can fluctuate in Ewing sarcoma cells, giving rise to two cell populations. EWSR1::FLI1low cells present a migratory and invasive phenotype, while EWSR1::FLI1high cells are more proliferative. In this work, we described how the CD44 standard isoform (CD44s), a transmembrane protein involved in cell adhesion and migration, is overexpressed in the EWSR1::FLI1low phenotype. The functional characterization of CD44s (proliferation, clonogenicity, migration, and invasion ability) was performed in three doxycycline-inducible Ewing sarcoma cell models (A673, MHH-ES1, and CADO-ES1). As a result, CD44s expression reduced cell proliferation in all the cell lines tested without affecting clonogenicity. Additionally, CD44s increased cell migration in A673 and MHH-ES1, without effects in CADO-ES1. As hyaluronan is the main ligand of CD44s, its effect on migration ability was also assessed, showing that high molecular weight hyaluronic acid (HMW-HA) blocked cell migration while low molecular weight hyaluronic acid (LMW-HA) increased it. Invasion ability was correlated with CD44 expression in A673 and MHH-ES1 cell lines. CD44s, upregulated upon EWSR1::FLI1 knockdown, regulates cell migration and invasion in Ewing sarcoma cells.

Targeting Galectin-1 by Aflibercept Strongly Enhances Its Antitumor Effect in Neuroendocrine Carcinomas.

BACKGROUND: Galectin-1 (Gal-1) plays major roles in cancer by modulating different processes leading to tumor development and progression. In the last years, it has been suggested as a promising target for anticancer therapy. Recently, aflibercept has shown high affinity for Gal-1. Here, we investigated how aflibercept could exert its antitumor activity via Gal-1-driven pathways in neuroendocrine carcinomas (NECs). METHODS AND RESULTS: NEC tumor xenografts were used to assess the effect of aflibercept on Gal-1 functions. Aflibercept induced a significant reduction of Gal-1 at epithelial, stromal, and extracellular localizations in lung NEC, whereas this was not observed in colon NECs, which displayed low expression of Gal-1. Additionally, aflibercept significantly reduced p-VEGFR2 protein, extracellular matrix remodeling, epithelial-mesenchymal transition, and activation of cancer-associated fibroblast hampering cell invasion in lung NEC but not in colon NEC. Gal-1 screening in human NECs confirmed that pulmonary and pancreatic tumors displayed higher levels of Gal-1 than colon NECs, becoming good candidates to benefit from aflibercept treatment. CONCLUSIONS: The lack of validated predictive markers of aflibercept is a weakness for guaranteeing the best treatment management with this drug. This work provides new mechanistic insight of aflibercept depending on Gal-1. Thus, in tumors overexpressing Gal-1, aflibercept has not only an antiangiogenic effect but also prevents Gal-1-mediated tumor-stroma cross talk. The stronger aflibercept effect in tumors with high levels of Gal-1 points out this protein as a molecular marker to predict the efficacy of this agent not only for NECs but also for other tumors with high levels of this protein.

Strong Antitumor Activity of Bevacizumab and Aflibercept in Neuroendocrine Carcinomas: In-Depth Preclinical Study.

BACKGROUND: Neuroendocrine carcinoma (NEC) is a rare and very aggressive tumor. It has been greatly understudied, and very little is known about optimal treatment strategy for patients with this disease. The purpose of this study was to evaluate in vivo whether anti-vascular endothelial growth factor (VEGF) drugs could be a therapeutic alternative for these tumors with a poor prognosis. METHODS: We have developed 2 xenograft models using either human cell line derived from lung (H460) or from colon (COLO320) NEC to assess the effect of 2 antiangiogenic drugs, aflibercept and bevacizumab, on tumor growth and their pathological characteristics. Additionally, tumors were subjected to immunohistochemistry staining and proteins were measured with Western blot and ELISA. RESULTS: Both aflibercept and bevacizumab showed significant antitumor activity (p < 0.001). In the H460 model, aflibercept resulted in 94% tumor growth inhibition (TGI) and bevacizumab treatment resulted in 72.2% TGI. Similarly, in the COLO320 model, aflibercept and bevacizumab resulted in 89.3 and 84% TGI, respectively. Moreover, antitumor activity occurs early after treatment initiation. Using Tumor Control Index score, which address the kinetics of tumor growth in a way comparable to the methods used in human clinical studies, we confirmed that both drugs inhibit significantly tumor growth. When tumor stabilization was evaluated, aflibercept shows higher ability to stabilize NEC tumors than bevacizumab. CONCLUSION: Results derived from this study strongly support anti-VEGF therapies, especially aflibercept, as a novel therapeutic option in NECs. Further studies are necessary, but our observations encourage the evaluation of antiangiogenics in clinical trials combined with standard chemotherapy.

Does nebulized epinephrine improve the efficacy of hypertonic saline solution in the treatment of hospitalized moderate acute bronchiolitis? A double blind, randomized clinical trial.

BACKGROUND: Nebulized 3% hypertonic saline solution (HSS 3%) has proven to reduce hospital stay in infants with acute bronchiolitis, as compared with nebulized physiological saline solutions. There are no studies assessing the effectiveness of nebulized epinephrine in patients treated with HSS 3%. The aim of this study was to compare the length of stay (LOS) in hospitalized patients treated with HSS 3% with placebo vs. HSS 3% with epinephrine. Secondarily we aimed to assess the effectiveness and safety of both treatments. METHODS: We performed a prospective, randomized, double-blind, parallel-group study, including infants hospitalized for moderate acute bronchiolitis. Both groups received standard life support and were randomly treated with nebulized HSS 3% (7 mL) with either placebo 3 mL or epinephrine 3 mL. Nebulizations were initially administered every four hours and this interval was modified according to the patient's response. RESULTS: Sixty-four infants were included, 32 patients in each group. No statistically significant differences were found between the two groups (P=0.948) in length of stay, disease severity, SatO2, respiratory rate or heart rate. On the third day of hospitalization, severity and respiratory rate in the HSS 3%+E presented a non statistically significant trend to an earlier improvement, (P=0.063 and P=0.096 repectively). No adverse events occurred. Four patients (two from each group) required transfer to the pediatric intensive care unit. CONCLUSIONS: With a third of the final estimated sample, we find a trend to an earlier clinical recovery in the epinephrine group, even though no statistical significant differences in LOS were found. The study needs to be continued until the total sample is recruited.

Serine versus threonine glycosylation with α-O-GalNAc: unexpected selectivity in their molecular recognition with lectins.

The molecular recognition of several glycopeptides bearing Tn antigen (α-O-GalNAc-Ser or α-O-GalNAc-Thr) in their structure by three lectins with affinity for this determinant has been analysed. The work yields remarkable results in terms of epitope recognition, showing that the underlying amino acid of Tn (serine or threonine) plays a key role in the molecular recognition. In fact, while Soybean agglutinin and Vicia villosa agglutinin lectins prefer Tn-threonine, Helix pomatia agglutinin shows a higher affinity for the glycopeptides carrying Tn-serine. The different conformational behaviour of the two Tn biological entities, the residues of the studied glycopeptides in the close proximity to the Tn antigen and the topology of the binding site of the lectins are at the origin of these differences.

Profiles of Dementia Caregivers according to Psychosocial and Resource Variables. Importance of Kinship.

The present study aims to analyze the existence of different profiles in family caregivers of people with dementia according to psychosocial and resource variables. In addition, it aims to study whether there is a greater representation of each kinship group in each of the profiles and if there are differences in emotional distress among such profiles considering the kinship with the care-recipient. Participants were 288 family dementia caregivers, divided into four kinship groups (wives, husbands, sons and daughters). Psychosocial (familism, dysfunctional thoughts and experiential avoidance), resource (leisure activities and social support) and outcomes (depressive, anxious and guilt symptomatology) variables were collected. A hierarchical cluster analysis using Ward's method, an exploratory factor analysis of two fixed factors and contingency tables were performed. Five clusters were obtained: Low psychosocial vulnerability-High resources, Low psychosocial vulnerability-Low resources, Mixed, High psychosocial vulnerability-High resources, and High psychosocial vulnerability-Low resources. Results suggested that clusters associated with lower distress were the Low psychosocial vulnerability-High resources and the High psychosocial vulnerability-High resources. Clusters associated with higher distress were the Low psychosocial vulnerability-Low resources and Mixed. High levels of dysfunctional thoughts, familism and experiential avoidance do not always have a maladaptive function. This could depend on sociocultural and resource variables such as the kinship with the caregiver or perceived social support. The identification of profiles of family caregivers potentially needing protection and vulnerable to psychological distress could help to increase the effectiveness of interventions aimed at this population.

Azithromycin reduces inflammation in a rat model of acute conjunctivitis.

PURPOSE: Macrolide antibiotics are known to have various anti-inflammatory effects in addition to their antimicrobial activity, but the mechanisms are still unclear. The effect of azithromycin on inflammatory molecules in the lipopolysaccharide-induced rat conjunctivitis model was investigated. METHODS: Twenty-four Wistar rats were divided into two groups receiving topical ocular azithromycin (15 mg/g) or vehicle. In total, six doses (25 µl) were administered as one dose twice a day for three days before subconjunctival lipopolysaccharide injection (3 mg/ml). Before the rats were euthanized, mucus secretion, conjunctival and palpebral edema and redness were evaluated. Real-time polymerase chain reaction was used to determine gene expression for interleukin-6, cyclooxygenase-2, tumor necrosis factor-α, matrix metalloproteinase (MMP)-2, and MMP-9. Interleukin-6 was determined with enzyme-linked immunosorbent assay, nuclear factor-kappa B with western blot, and MMP-2 activity with gelatin zymogram. Four eyes per group were processed for histology and subsequent periodic acid-Schiff staining and CD68 for immunofluorescence. The Student t test or the Wilcoxon test for independent samples was applied (SPSS v.15.0). RESULTS: Azithromycin-treated animals showed a significant reduction in all clinical signs (p<0.05) compared to controls. Interleukin-6 (p<0.05), nuclear factor-kappa B protein expression (p<0.01), and MMP-2 activity (p<0.05) in conjunctival homogenates were significantly reduced compared with the control animals. MMP-2 gene expression showed a tendency to decrease in the azithromycin group (p=0.063). Mucus secretion by goblet cells and the macrophage count in conjunctival tissue were also decreased in the azithromycin group (p<0.05). CONCLUSIONS: These results suggest that azithromycin administration ameliorates induced inflammation effects in a rat model of acute conjunctivitis.

Differences in daily life executive functioning between people with autism and people with schizophrenia.

This is a comparative analysis of everyday executive functioning between individuals with Autism Spectrum Disorder (ASD), Schizophrenia Spectrum Disorders (SSD) and controls using Dysexecutive Questionnaire-Spanish (DEX-Sp), to identify patterns of difficulties. Also we assessed the relationship between EF and adaptive behavior as measured by the Vineland Adaptive Behavioral Scale-II. Common areas of everyday executive functions were established as problematic in individuals with ASD and SSD related to Disinhibition and Apathy, while Disorganization and Impulsivity was gravely affected in ASD group only. The degree of Dysexecutive Syndrome was predictive of adaptive behavior in ASD group only. These suggest that DEX-Sp could be a useful tool in differentiating areas of strength and weaknesses in clinical groups such as ASD and SDD.

Executive functions in daily living skills: A study in adults with autism spectrum disorder.

Introduction: Adults with autism and adults with schizophrenia show difficulties in adaptive skills, especially those related to daily functioning. Some studies suggest that adaptive skills are associated with deficits in executive functions (EF), while others indicate that intelligence quotient (IQ) might also play a role. Literature suggests that autistic symptoms further affect adaptive skills. The interest of the current study, therefore, was to explore to what extent IQ, EFs as well as core autistic symptoms predict adaptive skills. Methods: To do this, 25 controls, 24 adults with autism, and 12 with schizophrenia were assessed on IQ (Wechsler Adult Intelligence Scale), and executive functioning. The EF was measured with neuropsychological tasks (inhibition, updating, and task switching) and with the Dysexecutive-Spanish Questionnaire (DEX-Sp) which assessed everyday life EF problems. Core ASD symptoms were measured using the Autism Diagnostic Observation Schedule, the Autism Spectrum Quotient-Short version (AQ-S), and the Repetitive Behavior Questionnaire - 3 (RBQ-3). Results: The results indicated EF difficulties in both, autism and schizophrenia. The IQ explained a high percentage of the variance found in adaptive skills, but only in the autism group. We can conclude, therefore, that high IQ is associated with low adaptive skills levels and EFs affect adaptive functioning in people with autism; however, this does not explain the difficulties in adaptive functioning in the schizophrenia group. Core features of autism assessed with self-report questionnaires (but not the ADOS-2) predicted low scores on the adaptive skills, only in the autism group. Discussion: Both EF measures predicted adaptive skills scores in autism, but not in schizophrenia. Our results suggest that different factors affect the adaptive functioning in each disorder. For instance, the EFs should be a central focus for improvement, especially for individuals with autism.

Adenine base editing efficiently restores the function of Fanconi anemia hematopoietic stem and progenitor cells.

Fanconi Anemia (FA) is a debilitating genetic disorder with a wide range of severe symptoms including bone marrow failure and predisposition to cancer. CRISPR-Cas genome editing manipulates genotypes by harnessing DNA repair and has been proposed as a potential cure for FA. But FA is caused by deficiencies in DNA repair itself, preventing the use of editing strategies such as homology directed repair. Recently developed base editing (BE) systems do not rely on double stranded DNA breaks and might be used to target mutations in FA genes, but this remains to be tested. Here we develop a proof of concept therapeutic base editing strategy to address two of the most prevalent FANCA mutations in patient hematopoietic stem and progenitor cells. We find that optimizing adenine base editor construct, vector type, guide RNA format, and delivery conditions leads to very effective genetic modification in multiple FA patient backgrounds. Optimized base editing restored FANCA expression, molecular function of the FA pathway, and phenotypic resistance to crosslinking agents. ABE8e mediated editing in primary hematopoietic stem and progenitor cells from FA patients was both genotypically effective and restored FA pathway function, indicating the potential of base editing strategies for future clinical application in FA.

The Transcription Factor FEZF1, a Direct Target of EWSR1-FLI1 in Ewing Sarcoma Cells, Regulates the Expression of Neural-Specific Genes.

Ewing sarcoma is a rare pediatric tumor characterized by chromosomal translocations that give rise to aberrant chimeric transcription factors (e.g., EWSR1-FLI1). EWSR1-FLI1 promotes a specific cellular transcriptional program. Therefore, the study of EWSR1-FLI1 target genes is important to identify critical pathways involved in Ewing sarcoma tumorigenesis. In this work, we focused on the transcription factors regulated by EWSR1-FLI1 in Ewing sarcoma. Transcriptomic analysis of the Ewing sarcoma cell line A673 indicated that one of the genes more strongly upregulated by EWSR1-FLI1 was FEZF1 (FEZ family zinc finger protein 1), a transcriptional repressor involved in neural cell identity. The functional characterization of FEZF1 was performed in three Ewing sarcoma cell lines (A673, SK-N-MC, SK-ES-1) through an shRNA-directed silencing approach. FEZF1 knockdown inhibited clonogenicity and cell proliferation. Finally, the analysis of the FEZF1-dependent expression profile in A673 cells showed several neural genes regulated by FEZF1 and concomitantly regulated by EWSR1-FLI1. In summary, FEZF1 is transcriptionally regulated by EWSR1-FLI1 in Ewing sarcoma cells and is involved in the regulation of neural-specific genes, which could explain the neural-like phenotype observed in several Ewing sarcoma tumors and cell lines.

Overcoming PLK1 inhibitor resistance by targeting mevalonate pathway to impair AXL-TWIST axis in colorectal cancer.

New therapeutic targets are revolutionizing colorectal cancer clinical management, opening new horizons in metastatic patients' outcome. Polo Like Kinase1 (PLK1) inhibitors have high potential as antitumoral agents, however, the emergence of drug resistance is a major challenge for their use in clinical practice. Overcoming this challenge represents a hot topic in current drug discovery research. BI2536-resistant colorectal cancer cell lines HT29R, RKOR, SW837R and HCT116R, were generated in vitro and validated by IG50 assays and xenografts models by the T/C ratio. Exons 1 and 2 of PLK1 gene were sequenced by Sanger method. AXL pathway, Epithelial-to-Mesenchymal transition (EMT) and Multidrug Resistance (MDR1) were studied by qPCR and western blot in resistant cells. Simvastatin as a re-sensitizer drug was tested in vitro and the drug combination strategies were validated in vitro and in vivo. PLK1 gene mutation R136G was found for RKOR. AXL pathway trough TWIST1 transcription factor was identified as one of the mechanisms involved in HT29R, SW837R and HCT116R lines, inducing EMT and upregulation of MDR1. Simvastatin was able to impair the mechanisms activated by adaptive resistance and its combination with BI2536 re-sensitized resistant cells in vitro and in vivo. Targeting the mevalonate pathway contributes to re-sensitizing BI2536-resistant cells in vitro and in vivo, raising as a new strategy for the clinical management of PLK1 inhibitors.

Therapeutic Potential of EWSR1-FLI1 Inactivation by CRISPR/Cas9 in Ewing Sarcoma.

Ewing sarcoma is an aggressive bone cancer affecting children and young adults. The main molecular hallmark of Ewing sarcoma are chromosomal translocations that produce chimeric oncogenic transcription factors, the most frequent of which is the aberrant transcription factor EWSR1-FLI1. Because this is the principal oncogenic driver of Ewing sarcoma, its inactivation should be the best therapeutic strategy to block tumor growth. In this study, we genetically inactivated EWSR1-FLI1 using CRISPR-Cas9 technology in order to cause permanent gene inactivation. We found that gene editing at the exon 9 of FLI1 was able to block cell proliferation drastically and induce senescence massively in the well-studied Ewing sarcoma cell line A673. In comparison with an extensively used cellular model of EWSR1-FLI1 knockdown (A673/TR/shEF), genetic inactivation was more effective, particularly in its capability to block cell proliferation. In summary, genetic inactivation of EWSR1-FLI1 in A673 Ewing sarcoma cells blocks cell proliferation and induces a senescence phenotype that could be exploited therapeutically. Although efficient and specific in vivo CRISPR-Cas9 editing still presents many challenges today, our data suggest that complete inactivation of EWSR1-FLI1 at the cell level should be considered a therapeutic approach to develop in the future.

A Higher Proportion of Eicosapentaenoic Acid (EPA) When Combined with Docosahexaenoic Acid (DHA) in Omega-3 Dietary Supplements Provides Higher Antioxidant Effects in Human Retinal Cells.

Retinal pigment epithelium (RPE) is a key regulator of retinal function and is directly related to the transport, delivery, and metabolism of long-chain n-3 polyunsaturated fatty acids (n3-PUFA), in the retina. Due to their functions and location, RPE cells are constantly exposed to oxidative stress. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have shown to have antioxidant effects by different mechanisms. For this reason, we designed an in vitro study to compare 10 formulations of DHA and EPA supplements from different origins and combined in different proportions, evaluating their effect on cell viability, cell proliferation, reactive oxygen species production, and cell migration using ARPE-19 cells. Furthermore, we assessed their ability to rescue RPE cells from the oxidative conditions seen in diabetic retinopathy. Our results showed that the different formulations of n3-PUFAs have a beneficial effect on cell viability and proliferation and are able to restore oxidative induced RPE damage. We observed that the n3-PUFA provided different results alone or combined in the same supplement. When combined, the best results were obtained in formulations that included a higher proportion of EPA than DHA. Moreover, n3-PUFA in the form of ethyl-esters had a worse performance when compared with triglycerides or phospholipid based formulations.

Prognostic significance of neutrophil-to lymphocyte ratio and platelet-to lymphocyte ratio in older patients with metastatic colorectal cancer.

Aging is associated with a higher risk of cancer, >70% of cancer-related deaths occur in aged patients; however, this population is underrepresented in clinical trials, therefore, clinical information regarding this age group is rather limited. OBJECTIVES: Neutrophil-to lymphocyte ratio (NLR) and platelet-to lymphocyte ratio (PLR) have been described as biomarkers in cancer, thus, we have assessed their impact in an aged cohort of patients with metastatic colorectal cancer (mCRC). PATIENTS AND METHODS: 110 patients with a mean age of 72.2 years at diagnosis were retrospectively reviewed; NLR and PLR were calculated and dichotomized using a cutoff point estimated by a ROC curve. Survival curves and Cox regression analysis were performed to assess the prognostic potential of ratios in terms of progression free survival (PFS) and overall survival (OS). RESULTS: High NLR was associated to worse outcome in terms of PFS (ten vs sixteen months; Log rank <0.001) (HR 2.00 95%CI 1. 29-3.11; p = .002) and OS (20 vs 26 months; Log rank 0.002) (HR 2.28 95%CI 1.40-3.71; p = .001). Similarly it occurs with high PLR and PFS (nine vs fifteen months; Log rank 0.04) (HR 1.55 95%CI 1.01-2.40; p = .04) and OS (nineteen vs 25 months; Log rank <0.001) (HR 2.35 95%CI 1.45-3.80; p < .001). CONCLUSION: This study confirms the role of NLR and PLR as accessible and noninvasive biomarkers that could be use as a routine tool in the clinical practice in geriatric patients with mCRC.

Preclinical Evaluation of a Cell-Based Gene Therapy Using the Sleeping Beauty Transposon System in Choroidal Neovascularization.

Age-related macular degeneration (AMD) is a progressive retinal disorder characterized by imbalanced pro- and antiangiogenic signals. The aim of this study was to evaluate the effect of ex vivo cell-based gene therapy with stable expression of human pigment epithelium-derived factor (PEDF) release using the non-viral Sleeping Beauty (SB100X) transposon system delivered by miniplasmids free of antibiotic resistance markers (pFAR4). Retinal pigment epithelial (RPE) cells and iris pigment epithelial (IPE) cells were co-transfected with pFAR4-inverted terminal repeats (ITRs) CMV-PEDF-BGH and pFAR4-CMV-SB100X-SV40 plasmids. Laser-induced choroidal neovascularization (CNV) was performed in rats, and transfected primary cells (transfected RPE [tRPE] and transfected IPE [tIPE] cells) were injected into the subretinal space. The leakage and CNV areas, vascular endothelial growth factor (VEGF), PEDF protein expression, metalloproteinases 2 and 9 (MMP-2/9), and microglial/macrophage markers were measured. Injection with tRPE/IPE cells significantly reduced the leakage area at 7 and 14 days and the CNV area at 7 days. There was a significant increase in PEDF and the PEDF/VEGF ratio with tRPE cells and a reduction in the MMP-2 activity. Our data demonstrated that ex vivo non-viral gene therapy reduces CNV and could be an effective and safe therapeutic option for angiogenic retinal diseases.