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

Ovarian cancer; BRCA mutations; BRCA testingCáncer de ovarios; Mutaciones BRCA; Prueba BRCACàncer d'ovaris; Mutacions BRCA; Prova BRCAGermline 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.This research was funded by Astra Zéneca Farmacéutica Spain SA (Grant Number GEICO60-0)

Benefits of FAIMS to Improve the Proteome Coverage of Deteriorated and/or Cross-Linked TMT 10-Plex FFPE Tissue and Plasma-Derived Exosomes Samples

The proteome characterization of complex, deteriorated, or cross-linked protein mixtures as paired clinical FFPE or exosome samples isolated from low plasma volumes (250 µL) might be a challenge. In this work, we aimed at investigating the benefits of FAIMS technology coupled to the Orbitrap Exploris 480 mass spectrometer for the TMT quantitative proteomics analyses of these complex samples in comparison to the analysis of protein extracts from cells, frozen tissue, and exosomes isolated from large volume plasma samples (3 mL). TMT experiments were performed using a two-hour gradient LC-MS/MS with or without FAIMS and two compensation voltages (CV = -45 and CV = -60). In the TMT experiments of cells, frozen tissue, or exosomes isolated from large plasma volumes (3 mL) with FAIMS, a limited increase in the number of identified and quantified proteins accompanied by a decrease in the number of peptides identified and quantified was observed. However, we demonstrated here a noticeable improvement (>100%) in the number of peptide and protein identifications and quantifications for the plasma exosomes isolated from low plasma volumes (250 µL) and FFPE tissue samples in TMT experiments with FAIMS in comparison to the LC-MS/MS analysis without FAIMS. Our results highlight the potential of mass spectrometry analyses with FAIMS to increase the depth into the proteome of complex samples derived from deteriorated, cross-linked samples and/or those where the material was scarce, such as FFPE and plasma-derived exosomes from low plasma volumes (250 µL), which might aid in the characterization of their proteome and proteoforms and in the identification of dysregulated proteins that could be used as biomarkers.This research was funded by an AES-ISCIII grant from the Instituto de Salud Carlos III (PI20CIII/00019), co-financed by the European Development Regional Fund “A Way to Achieve Europe” (FEDER). A.M.-C. is supported by an FPU predoctoral contract with the Spanish Ministerio de Educación, Cultura y Deporte. R.R.-G. is supported by a predoctoral training contract (FI22CIII/00016) of the Instituto de Salud Carlos III.S

Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation

There is overwhelming evidence that in vitro three-dimensional tumor cell cultures more accurately reflect the complex in vivo microenvironment than simple two-dimensional cell monolayers, not least with respect to gene expression profiles, signaling pathway activity and drug sensitivity. However, most currently available threedimensional techniques are time consuming and/or lack reproducibility; thus standardized and rapid protocols are urgently needed. To address this requirement, we have developed a versatile toolkit of reproducible three-dimensional tumor spheroid models for dynamic, automated, quantitative imaging and analysis that are compatible with routine high-throughput preclinical studies. Not only do these microplate methods measure three-dimensional tumor growth, but they have also been significantly enhanced to facilitate a range of functional assays exemplifying additional key hallmarks of cancer, namely cell motility and matrix invasion. Moreover, mutual tissue invasion and angiogenesis is accommodated by coculturing tumor spheroids with murine embryoid bodies within which angiogenic differentiation occurs. Highly malignant human tumor cells were selected to exemplify therapeutic effects of three specific molecularly-targeted agents: PI-103 (phosphatidylinositol-3-kinase (PI3K)- mammalian target of rapamycin (mTOR) inhibitor), 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) (heat shock protein 90 (HSP90) inhibitor) and CCT130234 (in-house phospholipase C (PLC)g inhibitor). Fully automated analysis using a Celigo cytometer was validated for tumor spheroid growth and invasion against standard image analysis techniques, with excellent reproducibility and significantly increased throughput. In addition, we discovered key differential sensitivities to targeted agents between two-dimensional and three-dimensional cultures, and also demonstrated enhanced potency of some agents against cell migration/invasion compared with proliferation, suggesting their preferential utility in metastatic disease.: We have established and validated a suite of highly reproducible tumor microplate threedimensional functional assays to enhance the biological relevance of early preclinical cancer studies. We believe these assays will increase the translational predictive value of in vitro drug evaluation studies and reduce the need for in vivo studies by more effective triaging of compounds.This work was funded by The National Centre for the Replacement, Refinement and Reduction of Animals in Research (G1000121 ID no. 94513), Cancer Research UK (grant number C309/A8274), and by Red Tematica de Investigación Cooperativa en Cancer (RD06/0020/1022). We acknowledge NHS funding to the NIHR Biomedical Research Centre. MM is supported by a postdoctoral research contract (FIS, Program ‘Sara Borrell’, Instituto de Salud Carlos III), Ministerio de Ciencia e Innovación, Spain

Transcription factor NRF2 uses the Hippo pathway effector TAZ to induce tumorigenesis in glioblastomas

Transcription factor NRF2 orchestrates a cellular defense against oxidative stress and, so far, has been involved in tumor progression by providing a metabolic adaptation to tumorigenic demands and resistance to chemotherapeutics. In this study, we discover that NRF2 also propels tumorigenesis in gliomas and glioblastomas by inducing the expression of the transcriptional co-activator TAZ, a protein of the Hippo signaling pathway that promotes tumor growth. The expression of the genes encoding NRF2 (NFE2L2) and TAZ (WWTR1) showed a positive correlation in 721 gliomas from The Cancer Genome Atlas database. Moreover, NRF2 and TAZ protein levels also correlated in immunohistochemical tissue arrays of glioblastomas. Genetic knock-down of NRF2 decreased, while NRF2 overexpression or chemical activation with sulforaphane, increased TAZ transcript and protein levels. Mechanistically, we identified several NRF2-regulated functional enhancers in the regulatory region of WWTR1. The relevance of the new NRF2/TAZ axis in tumorigenesis was demonstrated in subcutaneous and intracranial grafts. Thus, intracranial inoculation of NRF2-depleted glioma stem cells did not develop tumors as determined by magnetic resonance imaging. Forced TAZ overexpression partly rescued both stem cell growth in neurospheres and tumorigenicity. Hence, NRF2 not only enables tumor cells to be competent to proliferate but it also propels tumorigenesis by activating the TAZ-mediated Hippo transcriptional program.This study was funded by the Spanish Ministry of Economy and Competitiveness (MINECO) under the grant SAF2016-76520-R. ME is recipient of a postdoctoral contract Juan de la Cierva; DL and NRA of a FPU contract of MINECO; MP and RFG of a FPI contracts of Autonomous University of Madrid. RG has been funded by the AECC Scientific Foundation

Antitumoral Effect of Plocabulin in High Grade Serous Ovarian Carcinoma Cell Line Models

Ovarian cancer (OC) is a life-threatening tumor and the deadliest among gynecological cancers in developed countries. First line treatment with a carboplatin/paclitaxel regime is initially effective in the majority of patients, but most advanced OC will recur and develop drug resistance. Therefore, the identification of alternative therapies is needed. In this study, we employed a panel of high-grade serous ovarian cancer (HGSOC) cell lines, in monolayer and three-dimensional cell cultures. We evaluated the effects of a novel tubulin-binding agent, plocabulin, on proliferation, cell cycle, migration and invasion. We have also tested combinations of plocabulin with several drugs currently used in OC in clinical practice. Our results show a potent antitumor activity of plocabulin, inhibiting proliferation, disrupting microtubule network, and decreasing their migration and invasion capabilities. We did not observe any synergistic combination of plocabulin with cisplatin, doxorubicin, gemcitabine or trabectedin. In conclusion, plocabulin has a potent antitumoral effect in HGSOC cell lines that warrants further clinical investigation.Peer reviewe

Surveillance of Helicobacter pylori resistance over 22 Years (2000-2021) in Northern Spain

Objectives Helicobacter pylori gastritis is considered an infectious disease, regardless of symptoms and stage of disease. Most consensus documents recommend empirical therapy based on local antimicrobial susceptibility patterns. We aimed to provide clinically useful information about primary and secondary antimicrobial resistance to antimicrobials commonly prescribed for H. pylori. Methods Overall, 31,406 gastroduodenal biopsies and 2,641 string tests from patients over 15 years of age were plated on selective media, isolating H. pylori in 36.7% of biopsies and 50.7% of string tests. Susceptibility testing could be performed in 96.6% (12,399/12,835) of H. pylori isolates. Polymerase chain reaction (PCR) was also used to detect H. pylori and its resistance to clarithromycin, providing susceptibility data for 112 patients with negative culture results. Results Resistance to amoxicillin and tetracycline was unusual (0.6% and 0.2%, respectively). Rates of primary resistance to clarithromycin and metronidazole remained steady over the 22-year study period, at around 14% for clarithromycin and 30% for metronidazole, while primary resistance to levofloxacin tripled (from 7.6% in 2000 to 21.7% in 2021, P < 0.001) and increased with patient age. Notably, 1.8% of isolates were multiresistant to clarithromycin, metronidazole, and levofloxacin. Overall, secondary resistance rates were higher (P < 0.0001) than primary resistance rates for clarithromycin (42.5% vs 14.1%), metronidazole (40.9% vs 32%), and levofloxacin (21.5% vs 17.1%). Conclusion Determination of susceptibility for H. pylori by culture and/or PCR in patients undergoing endoscopy could facilitate the implementation of tailored therapy and guide the choice of empirical therapy when susceptibility testing cannot be performed, potentially helping limit the emergence of antimicrobial resistance

The transcriptional and mutational landscapes of lipid metabolism-related genes in colon cancer

Metabolic alterations encountered in tumors are well recognized and considered as a hallmark of cancer. In addition to Warburg Effect, epidemiological and experimental studies support the crucial role of lipid metabolism in colorectal cancer (CRC). The overexpression of four lipid metabolism-related genes (ABCA1, ACSL1, AGPAT1 and SCD genes) has been proposed as prognostic marker of stage II CRC (ColoLipidGene signature). In order to explore in depth the transcriptomic and genomic scenarios of ABCA1, ACSL1, AGPAT1 and SCD genes, we performed a transcriptomic meta-analysis in more than one thousand CRC individuals. Additionally we analyzed their genomic coding sequence in 95 patients, to find variants that could orchestrate CRC prognosis. We found that genetic variant rs3071, located on SCD gene, defines a 9.77% of stage II CRC patients with high risk of death. Moreover, individuals with upregulation of ABCA1 and AGPAT1 expression have an increased risk of CRC recurrence, independently of tumor stage. ABCA1 emerges as one of the main contributors to signature's prognostic effect. Indeed, both high ABCA1 expression and presence of tumoral genetic variants located in ABCA1 coding region, seem to be associated with CRC risk of death. In addition the non-synonymous polymorphism rs2230808, located on ABCA1, is associated with gene expression. Patients carrying at least one copy of minor allele showed higher levels of ABCA1 expression than patients carrying homozygous major allele. This study broaden the prognostic value of ABCA1, ACSL1, AGPAT1 and SCD genes, independently of CRC tumor stage, leading to future precision medicine approaches and "omics"-guided therapiesMinisterio de Economía y Competitividad del Gobierno de España (MINECO, Plan Nacional I+D+i AGL2016-76736-C3), Gobierno regional de la Comunidad de Madrid (P2013/ABI-2728, ALIBIRD-CM) and EU Structural Fund

High-throughput 3-dimensional culture of epithelial ovarian cancer cells as preclinical model of disease

Background: Recent reports have identified distinct genomic patterns in ovarian carcinoma, including proliferative and mesenchymal-like groups, with worse outcome. The exact mechanisms driving the onset and progression of these tumors are still poorly understood. Additionally, researchers are concerned about the correct subtype stratification of the available cell line models, and the exploration of alternatives to monolayer culture. Identification of biomarkers to stratify cell lines, characterization of important processes as epithelial-mesenchymal transition (EMT), and the use of three-dimensional (3D) cultures as alternative models could be useful for cell line classification. Methods and Results: In this work, we present a descriptive analysis of 16 commonly used ovarian cancer cell lines. We have studied their morphology in 2- and 3D culture, and their response to cisplatin, observing in the majority of them an increased resistance in 3D. We have also performed an immunohistochemical analysis for proliferation marker Ki-67, and EMT related markers to establish phenotypes. Epithelial cells tend to show higher proliferative rates, and mesenchymal cells show an increase in EMT related markers, especially when cultured in 3D conditions. Conclusions: We have stated the complex heterogeneity of ovarian cancer models, resembling primary tumors, agreeing with the argument that the cell line model for in vitro experiments must be carefully chosen. Our results also support that tridimensional culture could be a very helpful alternative in ovarian cancer research. Regarding EMT, a very important process for the development of this disease, some related biomarkers might be further characterized for their role in this disease developmentThis work was funded by Instituto de Salud Carlos III (ISCIII) and Fondo Europeo de Desarrollo Regional (FEDER), as part of PN I+D+I 2008–2011 Program (#PI10/630) and Fundación Mutua Madrileña Funding Program. VHS was supported by a phD fellowship program (#FI11/00538, ISCIII) and CIBERONC CB16/12/0039