Performance of the Idylla microsatellite instability test in endometrial cancer.

CONTEXT: DNA mismatch repair (MMR) deficiency (dMMR) testing is now recommended in endometrial cancer. Defect identification in the molecules participating in this pathway, or the presence of microsatellite instability, are commonly employed for this purpose. Novel methods are continuously evolving to report dMMR/microsatellite instability and to easily perform routine diagnoses. OBJECTIVE: The main aim of this study was to compare the concordance of the Idylla microsatellite instability test for the identification of dMMR endometrial cancer samples defined by immunohistochemistry and MMR genomic status. DESIGN: We applied the Idylla MSI test to 126 early-stage endometrial cancer cases with MMR testing by immunohistochemistry and genomic characterization (methylation in MLH1 and sequence alterations in MLH1, PMS2, MSH2 and MSH6). Individual markers and overall specific performance indicators were explored. RESULTS: The Idylla platform achieved a higher global concordance rate with MMR genomic status than with immunohistochemistry (75 % and 66 %, respectively). Sensitivity and specificity are also higher (75 % vs 66 % and 96 % vs 90 %, respectively). Clustering analysis split the patients into 2 well-differentiated clusters, the pMMR and the dMMR group, represented by MLH1/PMS2 loss and the MLH1 methylated promoter. Overall, immunohistochemistry and MMR genomic status identified more dMMR cases than did the Idylla test, although correlations were improved with a modified Idylla test cut-off. CONCLUSIONS: Performance of the Idylla test was better correlated with MMR genomic status than MMR immunohistochemistry status, which improved with a modified test cut-off. Further studies are needed to confirm the cut-off accuracy.

Comparison of Methods for Testing Mismatch Repair Status in Endometrial Cancer.

Approximately 20-30% of endometrial carcinomas (EC) are characterized by mismatch repair (MMR) deficiency (dMMR) or microsatellite instability (MSI), and their testing has become part of the routine diagnosis. The aim of this study was to establish and compare the MMR status using various approaches. Immunohistochemistry (IHC), PCR-based MSI, and the detection of defects in the four key MMR genes (MLH1, PMS2, MSH2, and MSH6) via methylation-specific multiplex ligation-dependent probe amplification (MLPA) and targeted next-generation sequencing (NGS) were performed. MSH3 expression was also evaluated. A set of 126 early-stage EC samples were analyzed, 53.2% of which were dMMR and 46.8% of which were proficient MMR (pMMR) as determined using IHC, whereas 69.3% were classified as microsatellite stable, while 8.8% and 21.9% were classified MSI-low (MSI-L) and MSI-high (MSI-H), respectively. In total, 44.3% of the samples showed genetic or epigenetic alterations in one or more genes; MLH1 promoter methylation was the most common event. Although acceptable concordance was observed, there were overall discrepancies between the three testing approaches, mainly associated with the dMMR group. IHC had a better correlation with MMR genomic status than the MSI status determined using PCR. Further studies are needed to establish solid conclusions regarding the best MMR assessment technique for EC.

Characterisation of new in vitro models and identification of potentially active drugs in angiosarcoma.

Angiosarcoma is a rare soft tissue sarcoma originating from endothelial cells. Given that current treatments for advanced disease have shown limited efficacy, alternative therapies need to be identified. In rare diseases, patient-derived cell models are crucial for screening anti-tumour activity. In this study, cell line models were characterised in 2D and 3D cultures. The cell lines' growth, migration and invasion capabilities were explored, confirming them as useful tools for preclinical angiosarcoma studies. By screening a drug library, we identified potentially effective compounds: 8-amino adenosine impacted cell growth and inhibited migration and invasion at considerably low concentrations as a single agent. No synergistic effect was detected when combining with paclitaxel, gemcitabine or doxorubicin. These results suggest that this compound could be a potentially useful drug in the treatment of AGS.

Efficiency of breeding olives for resistance to Verticillium wilt.

Olive trees are the most cultivated evergreen trees in the Mediterranean Basin, where they have deep historical and socioeconomic roots. The fungus Verticillium dahliae develops inside the vascular bundles of the host, and there are no effective applicable treatments, making it difficult to control the disease. In this sense, the use of integrated disease management, specifically the use of resistant cultivars, is the most effective means to alleviate the serious damage that these diseases are causing and reduce the expansion of this pathogen. In 2008, the University of Cordoba started a project under the UCO Olive Breeding Program whose main objective has been to develop new olive cultivars with high resistance to Verticillium wilt. Since 2008, more than 18,000 genotypes from 154 progenies have been evaluated. Only 19.9% have shown some resistance to the disease in controlled conditions and only 28 have been preselected due to their resistance in field condition and remarkable agronomic characteristics. The results of this study represent an important advancement in the generation of resistant olive genotypes that will become commercial cultivars currently demanded by the olive growing sector. Our breeding program has proven successful, allowing the selection of several new genotypes with high resistance to the disease and agronomical performance. It also highlights the need for long-term field evaluations for the evaluation of resistance and characterization of olive genotypes.

High Input of Nitrogen Fertilization and Short Irrigation Frequencies Forcefully Promote the Development of Verticillium Wilt of Olive.

It is known that high N doses, N/K imbalances, and frequent irrigation favor Verticillium wilt. The influence of fertilization and its interaction with the frequency of irrigation on the development of Verticillium wilt of olive (VWO) has been evaluated. A split-split-plot design in microplots with two naturally infested soils of different texture was established for studying three fertilization treatments (NO3Ca, NPK and without fertilization), plus two irrigation frequencies (daily and deficit). The treatments were applied by means of fertigation, evaluating the susceptible cultivar Picual. Final disease incidence in plants subjected to NO3Ca daily treatment was 100% regardless of soil texture. However, final mortality in these plants was 37% and 85.2% in clay and sandy loam soils, respectively. In addition, the area under the disease progress curve values were significantly higher (49.1%) in plants subjected to NO3Ca fertilization compared to those not fertilized or fertilized with N-P-K when plants were grown in clay soil. This value in the sandy loam soil was significantly higher in the NO3Ca daily irrigation treatment (94.3%), followed by the N-P-K-daily treatment (61.1%) which also was significantly higher than the unfertilized daily, N-deficit and NPK-deficit treatments (37.8, 42.6 and 44.9%, respectively). The plants submitted to unfertilized-deficit treatment reached the lowest value (9.6%). In this work it can be concluded that the application of fertilizer or the application of fertilizer with daily irrigation in naturally infested soils increases the development of VWO in Picual.

Effect of Previous Crops and Soil Physicochemical Properties on the Population of Verticillium dahliae in the Iberian Peninsula.

The soil infestation of Verticillium dahliae has significant Verticillium wilt of olive (VWO) with epidemiological consequences which could limit the expansion of the crop. In this context, there is a misunderstood history of the crops and soil property interactions associated with inoculum density (ID) increases in the soil. In this study, the effect of the combination of both factors was assessed on the ID of V. dahliae in the olive-growing areas of the Iberian Peninsula. Afterwards, the relationship of the ID to the mentioned factors was explored. The detection percentage and ID were higher in Spain than Portugal, even though the fields with a very favourable VWO history had a higher ID than that of the fields with a barely favourable history, regardless of the origin. The soil physicochemical parameters were able to detect the degree to which the ID was increased by the previous cropping history. By using a decision tree classifier, the percentage of clay was the best indicator for the V. dahliae ID regardless of the history of the crops. However, active limestone and the cation exchange capacity were only suitable ID indicators when <2 or 4 host crops of the pathogen were established in the field for five years, respectively. The V. dahliae ID was accurately predicted in this study for the orchard choices in the establishment of the olive.

The Role of Volatile Organic Compounds and Rhizosphere Competence in Mode of Action of the Non-pathogenic Fusarium oxysporum FO12 Toward Verticillium Wilt.

Verticillium wilts caused by Verticillium spp. are among the most challenging plant diseases to control and affect numerous hosts worldwide. Due to the lack of effective, conventional control methods, integrated control strategies provide a promising approach to manage these diseases. The non-pathogenic Fusarium oxysporum strain FO12 was reported in previous studies to be an effective biocontrol agent against Verticillium dahliae, however, its mode of action remains to be elucidated. In this study, complementary in vitro and in vivo experiments were conducted in order to explore the implications of inhibitory substances and rhizosphere competence in antagonistic effects of FO12 against V. dahliae and V. longisporum. Volatile organic compounds and soluble substances produced by FO12, which caused significant inhibition of mycelial growth and microsclerotia viability in the two tested Verticillium species, were identified by means of gas and liquid chromatography-mass spectrometry. We showed that the antagonistic effect of F. oxysporum FO12 is partially due to the production of bioactive compounds such as 3-methyl-1-butanol and 2-methyl-1-butanol, among others. Several metabolic pathways of FO12 were altered upon contact with V. dahliae ELV22 volatiles. The reduced production of alpha, alpha-trehalose, a metabolite used in starch and sucrose metabolism, suggests that the biocontrol agent activates its stress response in the presence of the phytopathogen. Microscopic analysis using sGFP-tagged FO12 on oil seed rape as a model plant suggests that the biocontrol strain is an efficient root colonizer, which could compete with V. dahliae in the same ecological niche. The findings obtained in this study provide new insights into the mode of action of this potential biocontrol agent, which are relevant for controlling Verticillium wilt through an ecologically friendly approach.