Targeted Gene Expression Profiling Research Articles

A targeted gene expression biomarker predicts clinic low-risk meningioma recurrence.

Despite reassuring clinical and histological features, low grade meningiomas can recur after surgery. Targeted gene expression profiling improves risk stratification of meningiomas, but the utility of this approach for clinical low-risk meningiomas is incompletely understood. This was a multicenter retrospective cohort study of meningiomas from patients who were treated at 4 institutions from 1992 to 2023. Adult patients with newly diagnosed or recurrent World Health Organization (WHO) grade 1 meningiomas that were treated with gross total resection (GTR) or subtotal resection (STR), or newly diagnosed WHO grade 2 meningiomas that were treated with GTR, were included. A 34-gene expression biomarker and gene expression risk score (continuous from 0 to 1) was evaluated in all samples. The study cohort was comprised of 723 patients, none of which were used for discovery or training of the gene expression biomarker and 265 of which were previously unreported. There were 626 WHO grade 1 meningiomas, 490 with GTR and 126 with STR, and 97 WHO grade 2 meningiomas with GTR. Targeted gene expression profiling classified 51.3% of clinical low-risk meningiomas as molecular intermediate-risk and 9.5% as molecular high-risk. Combining the gene expression biomarker with extent of resection revealed 19.8% of clinical low-risk meningiomas had unfavorable local freedom from recurrence (LFFR) and overall survival (OS), including 7.1% of newly diagnosed WHO grade 1 meningiomas with GTR. The risk score was prognostic for LFFR (HR per 0.1 increase in risk score 1.89, 95% CI 1.58-2.25) across all WHO grades, extents of resection, and newly diagnosed or recurrent presentations. Targeted gene expression profiling can identify clinical low-risk meningiomas that are likely to recur after surgery.

OKN-007 is an Effective Anticancer Therapeutic Agent Targeting Inflammatory and Immune Metabolism Pathways in Endometrial Cancer.

Advanced-stage endometrial cancer patients typically receive a combination of platinum and paclitaxel chemotherapy. However, limited treatment options are available for those with recurrent disease, and there is a need to identify alternative treatment options for the advanced setting. Our goal was to evaluate the pre-clinical efficacy and mechanism of action of Oklahoma Nitrone 007 (OKN-007) alone and in combination with carboplatin and paclitaxel in endometrial cancer. The effect of OKN-007 on the metabolic viability of endometrial cancer cells in both two- and three-dimensional (2D and 3D) cultures, as well as on clonogenic growth, in vitro was assessed. We also evaluated OKN-007 in vivo using an intraperitoneal xenograft model and targeted gene expression profiling to determine the molecular mechanism and gene expression programs altered by OKN-007. Our results showed that endometrial cancer cells were generally sensitive to OKN-007 in both 2D and 3D cultures. OKN-007 displayed a reduction in 3D spheroid and clonogenic growth. Subsequent targeted gene expression profiling revealed that OKN-007 significantly downregulated the immunosuppressive and immunometabolic enzyme indolamine 2,3-dioxygenase 1 (IDO1) (-11.27-fold change) and modulated upstream inflammatory pathways that regulate IDO1 expression (interferon- (IFN-), Jak-STAT, TGF-β, and NF-kB), downstream IDO1 effector pathways (mTOR and aryl hydrocarbon receptor (AhR)) and altered T-cell co-signaling pathways. OKN-007 treatment reduced IDO1, SULF2, and TGF-β protein expression in vivo, and inhibited TGF-β, NF-kB, and AhR- receptor-mediated nuclear signaling in vitro. These findings indicate that OKN-007 surmounts pro-inflammatory, immunosuppressive, and pro-tumorigenic pathways and is a promising approach for the effective treat endometrial cancer. Significance Statement Women with advanced and recurrent endometrial cancer have limited therapeutic options. OKN-007, which has minimal toxicity and is currently being evaluated in early-phase clinical trials for the treatment of cancer, is a potential new strategy for the treatment of endometrial cancer.

Digital assessment of tertiary lymphoid structures and therapeutic responses in gastric cancer: A multi-centric retrospective study.

Tertiary lymphoid structures (TLSs) are associated with favorable prognosis and enhanced response to anti-cancer therapy. A digital assessment of TLSs could provide an objective alternative that mitigates variability inherent in manual evaluation. This study aimed to develop and validate a digital gene panel based on biological prior knowledge for assessment of TLSs, and further investigate its associations with survival and multiple anti-cancer therapies. The present study involved 1,704 patients with gastric cancer from seven cancer centers. TLSs were identified morphologically through hematoxylin-and-eosin staining. We further developed a digital score based on targeted gene expression profiling to assess TLSs status, recorded as gene signature of tertiary lymphoid structures (gsTLS). For enhanced interpretability, we employed the SHapley Additive exPlanation (SHAP) analysis to elucidate its contribution to the prediction. We next evaluated the signature's associations with prognosis, and investigated its predictive accuracy for multiple anti-cancer therapies, including adjuvant chemotherapy and immunotherapy. The gsTLS panel with nine gene features achieved high accuracies in predicting TLSs status in the training, internal and external validation cohorts (area under the curve, range: 0.729-0.791). In multivariable analysis, gsTLS remained an independent predictor of disease-free and overall survival (hazard ratio, range: 0.346-0.743, all P < 0.05) after adjusting for other clinicopathological variables. SHAP analysis highlighted gsTLS as the strongest predictor of TLSs status compared with clinical features. Importantly, patients with high gsTLS (but not those with low gsTLS) exhibited substantial benefits from adjuvant chemotherapy (P < 0.05). Furthermore, we found that the objective response rate to anti-programmed cell death protein 1 (anti-PD-1) immunotherapy was significantly higher in the high-gsTLS group (40.7%) versus the low-gsTLS group (5.6%, P = 0.036), and the diagnosis was independent from Epstein-Barr virus (EBV), tumor mutation burden (TMB), and programmed cell death-ligand 1 (PD-L1) expression. The gsTLS digital panel enables accurate assessment of TLSs status, and provides information regarding prognosis and responses to multiple therapies for gastric cancer.

Immunogenic cell death mediated TLR3/4-activated MSCs in U87 GBM cell line

Background and aimsGlioblastoma (GBM) is an aggressive primary brain cancer with no promising curative therapies. It has been indicated that MSCs can interact with the tumour microenvironment (TME) through the secretion of soluble mediators regulating intercellular signalling within the TME. TLRs are a multigene family of pattern recognition receptors with evolutionarily conserved regions and are widely expressed in immune and other body cells. MSCs by TLRs can recognize conserved molecular components (DAPMPs and PAPMPs) and activate signalling pathways, which regulate immune and inflammatory responses. MSCs may exert immunomodulatory functions through interaction with their expressed toll-like receptors (TLRs) and exert a protective effect against tumour antigens. As an emerging approach, we aimed to monitor the U87 cell line growth, migration and death markers following specific TLR3/4-primed-MSCs-CMs treatment. Methods and resultsWe investigated the phenotypic and functional outcomes of primed-CMs and glioma cell line co-culture following short-term, low-dose TLR3/4 priming. The gene expression profile of target genes, including apoptotic markers and related genes, was analyzed by qRT-PCR. MicroRNA-Seq examined the miRNA expression patterns, and flow cytometry evaluated the cell viability and cycle stages. The results showed significant changes in apoptosis and likely necroptosis-related markers following TLR3/4-primed-MSCs-CMs exposure in the glioma cell line. Notably, we observed a considerable induction of selective pro-apoptotic markers and both the early and late stages of apoptosis in treated U87 cell lines. Additionally, the migration rate of glioma cells significantly decreased following MSCs-CM treatment. ConclusionOur findings confirmed that the exposure of TLR3/4-activated-MSCs-CMs with glioma tumour cells possibly changes the immunogenicity of the tumour microenvironment and induces immunogenic programmed cell death. Our results can support the idea that TLR3/4-primed-MSCs can lead to innate immune-mediated cell death and modify tumour cell biology in invasive and metastatic cancers.

400 Brain AVMs-Related MicroRNAs: Machine Learning Algorithm for Expression Profiles of Target Genes

INTRODUCTION: microRNAs (miRNAs) are a class of non-coding RNAs playing a myriad of important roles in regulating gene expression. Of note, recent work demonstrated a critical role of miRNAs in the genesis and progression of brain arteriovenous malformations (bAVMs). METHODS: A PRISMA-based literature review was performed using PubMed/Medline database with the following search terms: "brain arteriovenous malformations", "cerebral arteriovenous malformations", "microRNA", and "miRNA". All preclinical and clinical studies written in English, regardless of date, were selected. For our bioinformatic analyses, miRWalk and miRTarBase machine learning algorithms were employed; the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was quired for associated pathways/functions. RESULTS: 4 studies were ultimately included in the final analyses. Sequencing data consistently revealed the decreased expression of miR-18a in bAVM-endothelial cells, resulting in increased levels of vascular endodermal growth factor (VEGF), Id-1, matrix metalloproteinase, and growth signals. Our analyses also suggest that the downregulation of miR-137 and miR-195* within vascular smooth muscle cells (VSMCs) may foster the activation of inflammation, aberrant angiogenesis, and phenotypic switching. In the peripheral blood, the overexpression of miR-7-5p, miR-629-5p, miR-199a-5p, miR-200b-3p, and let-7b-5p may contribute to endothelial proliferation and nidus development. The machine learning algorithms employed confirmed associations between miRNA-related target networks, vascular rearrangement, and bAVM progression. CONCLUSIONS: miRNAs expression appears to be critical in managing bAVMs' post-transcriptional signals. Targets of microRNAs regulate canonical vascular proliferation and reshaping. Although additional scientific evidence is needed, the identification of bAVM miRNA signatures may facilitate the development of novel prognostic/diagnostic tools and molecular therapies for bAVMs.

Selection of reliable reference genes for gene expression studies involving peripheral blood mononuclear cells in small ruminants

Quantitative PCR (qPCR) is a highly sensitive, cost effective, and routinely used molecular assay for analyzing the gene expression patterns of specific target genes across tissues, pathological conditions, treatment regimes, and physiological states. However, normalization of expression profiles of target genes using stable reference genes (RGs) is a critical step to ensure the accuracy of relative quantification using qPCR. In this study, we evaluated the stability of fourteen potential existing reference genes (ACTB, BACH1, B2M, GAPDH, HMBS, PGK1, PPIA, PPIB, RPLP0, RPL19, RPS9, RPS15, RPS28, and UXT) in the peripheral blood mononuclear cells (PBMCs) of healthy sheep and goats to determine the most stable RGs. These candidate genes belong to different functional classes and were chosen based on published literature on commonly used RGs in different livestock species. Four different analytical approaches (geNorm, NormFinder, BestKeeper, and ΔCt analysis) as well as RefFinder, an online tool which integrates the geometric means of these four prominent stability algorithms were utilized to determine a comprehensive ranking of the investigated genes. Our data indicates that PPIB, BACH1, ACTB, and PPIA are the most suitable RGs, while RPLP0, GAPDH and RPS15 are the most variable and unsuitable genes for normalization of qPCR data in the PBMCs of sheep and goats. The results of this study provide useful resource for researchers engaged in unravelling the transcriptional landscape of PBMCs of small ruminants for various scientific investigations.

Identification and validation of stable reference genes for expression profiling of target genes in diverse ovine tissues

Quantitative PCR (qPCR) is a widely-used technique for quantifying the expression of target genes across various tissues, as well as under different pathological and physiological conditions. One of the challenges associated with this method is the need to identify optimal reference genes (RGs) that maintain consistent expression levels under diverse experimental settings, thereby ensuring accurate biological interpretation. In this study, we conducted a thorough analysis of 18 candidate RGs (ACTB, BACH1, B2M, GAPDH, HMBS, HPRT1, PGK1, PPIA, PPIB, RPLP0, RPL19, RPS9, RPS15, RPS28, SDHA, TBP, UXT, and YWHAZ) across 10 ovine tissues (muscle, skin, kidney, liver, intestine, rumen, lung, testis, heart, and spleen) obtained from five individual sheep. We aimed to identify genes with stable expression across these tissues. A literature-based survey helped us shortlist candidate genes representing various functional classes from multiple livestock species. We employed four algorithms: geNorm, NormFinder, BestKeeper, and Delta Ct (ΔCt), to rank these genes based on their stability. A consistent trend in the rankings was observed across these different algorithms. RefFinder was then used for a comprehensive ranking, integrating the outputs from the various methods. ACTB, PPIB, BACH1, and B2M emerged as the most stable RGs, while RPS9, RPS15, and PGK1 displayed variable expression. We validated our findings through qPCR analysis of four target genes (ACTN2, CRYAB, DLK1, and TRIM54) in the skin samples from two different sheep breeds. Based on these results, we recommend ACTB, PPIB, BACH1, and B2M as reliable internal control genes for qPCR experiments involving diverse ovine tissues.

Whole blood RNA signatures in tuberculosis patients receiving H56:IC31 vaccine as adjunctive therapy.

Therapeutic vaccination in tuberculosis (TB) represents a Host Directed Therapy strategy which enhances immune responses in order to improve clinical outcomes and shorten TB treatment. Previously, we have shown that the subunit H56:IC31 vaccine induced both humoral and cellular immune responses when administered to TB patients adjunctive to standard TB treatment (TBCOX2 study, NCT02503839). Here we present the longitudinal whole blood gene expression patterns in H56:IC31 vaccinated TB patients compared to controls receiving standard TB treatment only. The H56:IC31 group (N=11) and Control group (N=7) underwent first-line TB treatment for 182 days. The H56:IC31 group received 5 micrograms of the H56:IC31 vaccine (Statens Serum Institut; SSI, Valneva Austria GmbH) intramuscularly at day 84 and day 140. Total RNA was extracted from whole blood samples collected in PAXgene tubes on days 0, 84, 98, 140, 154, 182 and 238. The expression level of 183 immune-related genes was measured by high-throughput microfluidic qPCR (Biomark HD system, Standard BioTools). The targeted gene expression profiling unveiled the upregulation of modules such as interferon (IFN) signalling genes, pattern recognition receptors and small nucleotide guanosine triphosphate (GTP)-ases in the vaccinated group compared to controls two weeks after administration of the first H56:IC31 vaccine. Additionally, the longitudinal analysis of the Adolescent Cohort Study-Correlation of Risk (ACS-COR) signature showed a progressive downregulation in both study arms towards the end of TB treatment, in congruence with reported treatment responses and clinical improvements. Still, two months after the end of TB treatment, vaccinated patients, and especially those developing both cellular and humoral vaccine responses, showed a lower expression of the ACS-COR genes compared to controls. Our data report gene expression patterns following H56:IC31 vaccination which might be interpreted as a lower risk of relapse in therapeutically vaccinated patients. Further studies are needed to conclude if these gene expression patterns could be used as prognostic biosignatures for therapeutic TB vaccine responses.

Immune-Related Gene Expression Signatures Identify a Patient's Subset Achieving Durable Complete Response to Brentuximab Vedotin in Relapsed/Refractory Hodgkin Lymphoma

Background: In classical Hodgkin lymphoma (cHL), 20%-30% of patients experience relapse or progressive disease after standard chemotherapy, requiring salvage treatment strategies including targeted immunotherapy with brentuximab vedotin (BV). Molecular mechanisms underlying treatment resistance to BV in cHL are still poorly understood. The unique characteristics of cHL microenvironment, which represents &amp;gt;90% of the cellular infiltrate, make targeted gene expression profiling (T-GEP) a suitable technique to investigate immune-related determinants of BV sensitivity. Methods: 31 consecutive patients (pts) affected by relapsed-refractory (R/R) cHL treated at 2 italian Institutions from 2013 to 2018 with available formalin-fixed paraffin embedded (FFPE) tissue collected right before the start of BV therapy, were included in this retrospective study. FFPE tissue was profiled by T-GEP on the Nanostring platform, using the PanCancer Immune Profiling panel, encompassing 730 genes regulating the most relevant immunologic pathways. Disease response after BV treatment was evaluated by FDG-PET after 4, 8, 12 and 16 cycles. Results: Median age of the whole cohort was 38 years (y) (range 18-76). 18 pts (58%) were male. Median number of BV cycles was 8 (range 2-16). After a median follow-up of 3.5 y from the start of Brentuximab treatment, the overall survival (OS) and progression-free survival (PFS) rates were 87% and 32% respectively. Pts were classified as BV-responders in case of complete metabolic response (CMR) lasting more than 6 months (m); pts achieving less than CMR or short lasting (&amp;lt; 6 m) CMR were classified as non-responders (NR). By applying these criteria, 12 pts (39%) were classified as responders and 19 as non-responders. Differential expression (DE) analyses were performed and differentially expressed (DE) genes were selected by applying two statistical filters: (log2FC &amp;gt; 1 and p-value &amp;lt; 0.05). 61 genes were differentially expressed between responders and non-responders. The 61-gene signature included genes involved in JAK-STAT signaling, IL-17 signaling and cytokine-cytokine receptor interaction. Unsupervised clustering analyses identified two different pts clusters with clearly different BV-sensitivity profiles: cluster1 (22 pts) and cluster2 (9 pts). Among the 22 pts of cluster 1, only 4 pts were responders while 18 were non-responders. As opposite, 8 of 9 patients of Cluster2 were responders, while only 1 was non-responder (p=0.0005). In line with this, 4-y PFS rate of the 22 pts included in cluster1 was 19%, while the 4-y PFS rate of the 9 pts included in cluster2 was 66% (p=0.007). Median PFS of Cluster1 was only 5.7 months. The composition of the two clusters was not significantly different in terms of number of prior therapies and disease stage at the time of BV treatment. After achieving a CMR 6 of 9 pts were consolidated with autologous stem cell transplantation (ASCT) in cluster2. In cluster1, 1 patient was consolidated with ASCT after CMR, and 2 pts received allogeneic stem cell transplantation (allo-SCT) after BV-failure. Conclusions: With the limitation of the small sample size, these data suggest that the composition of cHL microenvironment could influence the activity of single agent BV. Pre-treatment microenvironment profiling with T-GEP could be a useful tool for selecting patients who may derive maximal benefit from BV in cHL.

Clinical and Immunological Factors Associated with Poor Graft Function after Allogeneic Hematopoietic Stem Cell Transplantation in Myelofibrosis Patients

Allogeneic hematopoietic stem cell transplantation (HSCT) is widely recognized as the sole curative treatment for myelofibrosis (MF). However, patients with MF often experience graft function impairment despite successful engraftment and absence of relapse signs. Although it has been reported that poor graft function can have a negative impact on the outcomes of patients with MF after HSCT, the specific factors that can influence poor graft function are not yet clearly understood. In this background, we analyzed various clinical and immunological factors that may contribute to poor graft function after transplantation. The study included 93 patients who underwent HSCT for the treatment of MF at Seoul St. Mary's Hospital between the years 2000 and 2022. By cumulative incidence analysis of poor graft function (death, relapse, graft failure, and loss of chimerism as competing risks), we evaluated which pre-HSCT factors has significant impact on poor graft function after HSCT. The definition of poor graft function and graft failure in this study followed the criteria set by the European Society for Blood and Marrow Transplantation (EBMT). We measured the longitudinal diameter of spleen up to 2 months prior to HSCT, and classified the patients into pre-HSCT splenomegaly (≥ 14cm) or not. Next, we investigated the association of immune-related gene expression signature in bone marrow immune-microenvironment with the occurrence of poor graft function, by performing targeted gene expression profiling and gene set enrichment analysis using a custom NanoString panel composed of 579 immune-related genes in pre- and post-HSCT BM samples from patients with poor graft function and good graft function (total 24 samples). A median age of 93 patients was 57 years, and 55.9% were male. Secondary MF was observed in 30.1% of the cases. Fifty-three (57.0%) received graft from a matched sibling donor graft and 40 (43.0%) from a matched unrelated donor. Sixty-three (67.7%) patients were classified to the pre-HSCT splenomegaly group. In terms of ABO match, 45 (48.4%) patients were matched, 10 (10.8%) had bi-directional mismatch, 17 (18.3%) had major mismatch, and 19 (20.4%) had minor mismatch. The median infused CD34 (+) cell dose was 6.4 x 10^6/kg (Interquartile range 5.3 x 10^6/kg-8.3 x 10^6/kg). In our patients, cumulative incidence of poor graft function after 5 years from HSCT was 32.0% (95% CI: 23.4-42.7). Infused CD34 (+) cell dose, the presence of splenomegaly before HSCT were revealed to have an impact on the occurrence of poor graft function after HSCT. The other significant factor associated with poor graft function was ABO mismatch, in particular, with a substantial difference between matched patients and patients with bi-directional ABO mismatch. (Figure A). In NanoString panel analysis, although the expression of immune-related genes mostly upregulated after HSCT, genes related to processes such as complement system, inflammasomes, phagocytosis and degradation, and MHC class II antigen presentation pathways were significantly downregulated in PGF. Whereas, oxidative stress, inflammasomes, and autophagy pathways were downregulated in GGF (Figure B). In this study, we could observe that a significant proportion of MF patients undergoing HSCT experienced poor graft function. Factors that significantly associated with the occurrence of poor graft function included the CD34 (+) cell dose at the time of HSCT, the presence of splenomegaly before HSCT, and ABO mismatch. Interestingly, distinct gene expression signatures present in IME between pre- and post-SCT in both PGF and GGF. Further research is warranted to investigate the impact of poor graft function caused by these factors on outcomes such as transfusion requirements and quality of life.

Targeted gene expression profiling predicts meningioma outcomes and radiotherapy responses.

Surgery is the mainstay of treatment for meningioma, the most common primary intracranial tumor, but improvements in meningioma risk stratification are needed and indications for postoperative radiotherapy are controversial. Here we develop a targeted gene expression biomarker that predicts meningioma outcomes and radiotherapy responses. Using a discovery cohort of 173 meningiomas, we developed a 34-gene expression risk score and performed clinical and analytical validation of this biomarker on independent meningiomas from 12 institutions across 3 continents (N = 1,856), including 103 meningiomas from a prospective clinical trial. The gene expression biomarker improved discrimination of outcomes compared with all other systems tested (N = 9) in the clinical validation cohort for local recurrence (5-year area under the curve (AUC) 0.81) and overall survival (5-year AUC 0.80). The increase in AUC compared with the standard of care, World Health Organization 2021 grade, was 0.11 for local recurrence (95% confidence interval 0.07 to 0.17, P < 0.001). The gene expression biomarker identified meningiomas benefiting from postoperative radiotherapy (hazard ratio 0.54, 95% confidence interval 0.37 to 0.78, P = 0.0001) and suggested postoperative management could be refined for 29.8% of patients. In sum, our results identify a targeted gene expression biomarker that improves discrimination of meningioma outcomes, including prediction of postoperative radiotherapy responses.

Formononetin attenuates high glucose-induced neurotoxicity by negatively regulating oxidative stress and mitochondrial dysfunction in Schwann cells via activation of SIRT3

High glucose induces Schwann cells death and neurotoxicity. Formononetin was originally found in Astragalus membranaceus and showed anti-tumor and anti-neuroinflammation properties. The aim of this study is to explore the molecular mechanism underlying the neuroprotective effects of formononetin and identify its direct protein target. The effects of formononetin on oxidative stress and mitochondrial dysfunction in Schwann cells induced by high glucose were investigated. High glucose treatment significantly induced oxidative stress, mitochondrial dysfunction and apoptosis in Schwann cells, while these effects were partially or completely prevented by co-treatment with formononetin. Mechanistically, we found that SIRT3/PGC-1α/SOD2 pathway was activated by formononetin under high glucose conditions as evidenced by western blotting. Knockdown of SIRT3 by siRNA delivery reversed the protective effects of formononetin on high glucose-induced Schwann cells injury and changes in expression profile of SIRT3 downstream target genes. Molecular docking, thermal shift assay and surface plasmon resonance assay revealed a direct binding between formononetin and SIRT3. Taken together, we identified a novel SIRT3 activator formononetin and revealed its beneficial effects on high glucose-induced neurotoxicity, suggesting that targeting SIRT3 in Schwann cells may be a new approach for treatment of peripheral nerve regeneration related diseases such as diabetic peripheral neuropathy.

Targeting doublecortin-like kinase 1 reveals a novel strategy to circumvent chemoresistance and metastasis in ovarian cancer

Ovarian cancer (OvCa) has a dismal prognosis because of its late-stage diagnosis and the emergence of chemoresistance. Doublecortin-like kinase 1 (DCLK1) is a serine/threonine kinase known to regulate cancer cell “stemness”, epithelial-mesenchymal transition (EMT), and drug resistance. Here we show that DCLK1 is a druggable target that promotes chemoresistance and tumor progression of high-grade serous OvCa (HGSOC). Importantly, high DCLK1 expression significantly correlates with poor overall and progression-free survival in OvCa patients treated with platinum chemotherapy. DCLK1 expression was elevated in a subset of HGSOC cell lines in adherent (2D) and spheroid (3D) cultures, and the expression was further increased in cisplatin-resistant (CPR) spheroids relative to their sensitive controls. Using cisplatin-sensitive and resistant isogenic cell lines, pharmacologic inhibition (DCLK1-IN-1), and genetic manipulation, we demonstrate that DCLK1 inhibition was effective at re-sensitizing cells to cisplatin, reducing cell proliferation, migration, and invasion. Using kinase domain mutants, we demonstrate that DCLK1 kinase activity is critical for mediating CPR. The combination of cisplatin and DCLK1-IN-1 showed a synergistic cytotoxic effect against OvCa cells in 3D conditions. Targeted gene expression profiling revealed that DCLK1 inhibition in CPR OvCa spheroids significantly reduced TGFβ signaling, and EMT. We show in vivo efficacy of combined DCLK1 inhibition and cisplatin in significantly reducing tumor metastases. Our study shows that DCLK1 is a relevant target in OvCa and combined targeting of DCLK1 in combination with existing chemotherapy could be a novel therapeutic approach to overcome resistance and prevent OvCa recurrence.

Combined Transcriptomic and Metabolomic Approach Revealed a Relationship between Light Control, Photoprotective Pigments, and Lipid Biosynthesis in Olives.

Olive possesses excellent nutritional and economic values for its main healthy products. Among them, a high content of antioxidant compounds, balanced during the ripening process, are produced under genetic and environmental control, resulting in high variability among cultivars. The genes involved in these complex pathways are mainly known, but despite many studies which indicated the key role of light quality and quantity for the synthesis of many metabolites in plants, limited information on these topics is available in olive. We carried out a targeted gene expression profiling in three olive cultivars, Cellina di Nardò, Ruveia, and Salella, which were selected for their contrasting oleic acid and phenolic content. The -omics combined approach revealed a direct correlation between a higher expression of the main flavonoid genes and the high content of these metabolites in 'Cellina di Nardò'. Furthermore, it confirmed the key role of FAD2-2 in the linoleic acid biosynthesis. More interestingly, in all the comparisons, a co-regulation of genes involved in photoperception and circadian clock machinery suggests a key role of light in orchestrating the regulation of these pathways in olive. Therefore, the identified genes in our analyses might represent a useful tool to support olive breeding, although further investigations are needed.

Analyzing Flow Cytometry or Targeted Gene Expression Data Influences Clinical Discoveries-Profiling Blood Samples of Pancreatic Ductal Adenocarcinoma Patients.

Monitoring the therapeutic response of pancreatic ductal adenocarcinoma (PDAC) patients is crucial to determine treatment strategies. Several studies have examined the effectiveness of FOLFIRINOX as a first-line treatment in patients with locally advanced pancreatic cancer, but little attention has been paid to the immunologic alterations in peripheral blood caused by this chemotherapy regimen. Furthermore, the influence of the measurement type (e.g., flow cytometry and targeted gene expression) on the clinical discoveries is unknown. Therefore, we aimed to scrutinize the influence of using flow cytometry or targeted immune gene expression to study the immunological changes in blood samples of PDAC patients who were treated with a single-cycle FOLFIRINOX combined with lipegfilgrastim (FFX-Lipeg). Whole-blood samples from 44 PDAC patients were collected at two time points: before the first FOLFIRINOX cycle and 14 days after the first cycle. EDTA blood tubes were used for multiplex flow cytometry analyses to quantify 18 immune cell populations and for complete blood count tests as the standard clinical routine. The flow cytometry data were analyzed with FlowJo software. In addition, Tempus blood tubes were used to isolate RNA and measure 1230 immune-related genes using NanoString Technology®. Data quality control, normalization, and analysis were performed using nSolver™ software and the Advanced Analysis module. FFX-Lipeg treatment increased the number of neutrophils and monocytes, as shown by flow cytometry and complete blood count in concordance with elevated gene expression measured via targeted gene expression profiling analysis. Interestingly, flow cytometry analysis showed an increase in the number of B and T cells after treatment, while targeted gene expression analysis showed a decrease in B and T cell-specific gene expression. Targeted gene expression complements flow cytometry analysis to provide a comprehensive understanding of the effects of FFX-Lipeg. Flow cytometry and targeted gene expression showed increases in neutrophils and monocytes after FFX-Lipeg. The number of lymphocytes is increased after treatment; nevertheless, their cell-specific gene expression levels are downregulated. This highlights that different techniques influence clinical discoveries. Therefore, it is important to carefully select the measurement technique used to study the effect of a treatment.

Targeted gene expression profiling for accurate endometrial receptivity testing

Expressional profiling of the endometrium enables the personalised timing of the window of implantation (WOI). This study presents and evaluates a novel analytical pipeline based on a TAC-seq (Targeted Allele Counting by sequencing) method for endometrial dating. The expressional profiles were clustered, and differential expression analysis was performed on the model development group, using 63 endometrial biopsies spanning over proliferative (PE, n = 18), early-secretory (ESE, n = 18), mid-secretory (MSE, n = 17) and late-secretory (LSE, n = 10) endometrial phases of the natural cycle. A quantitative predictor model was trained on the development group and validated on sequenced samples from healthy women, consisting of 52 paired samples taken from ESE and MSE phases and five LSE phase samples from 31 individuals. Finally, the developed test was applied to 44 MSE phase samples from a study group of patients diagnosed with recurrent implantation failure (RIF). In validation samples (n = 57), we detected displaced WOI in 1.8% of the samples from fertile women. In the RIF study group, we detected a significantly higher proportion of the samples with shifted WOI than in the validation set of samples from fertile women, 15.9% and 1.8% (p = 0.012), respectively. The developed model was evaluated with an average cross-validation accuracy of 98.8% and an accuracy of 98.2% in the validation group. The developed beREADY screening model enables sensitive and dynamic detection of selected transcriptome biomarkers, providing a quantitative and accurate prediction of endometrial receptivity status.

Expression profile of plasma microRNAs and target genes in patients with complicated pregnancy

Aim: to assess features of placental transcriptome in patients with complicated pregnancy course and outcome.Materials and Methods. A prospective observational comparative study in parallel groups was carried out by examining 44 patients divided into three groups: group 1 included 13 pregnant women with moderate and severe preeclampsia (PE), group 2 consisted of 12 patients with fetal growth retardation (FGR), control group included 19 clinically healthy women with uncomplicated pregnancy and delivery. All women enrolled to the study underwent a comprehensive examination, including history collection, general and obstetric-gynecological examination, laboratory and instrumental studies. Special research methods were used during the study, which included placental tissue sampling followed by analyzed expression profile of 12 microRNAs using real-time polymerase chain reaction (PCR).Results. The PCR data indicated about lack of expression for analyzed miR-210-3p, miR-320-3p, miR-1304-5p and miR-375-5p in placental tissue samples in FGR patients. No significant differences in placental miRNA levels were observed in FGR vs. PE and control group. Analysis of placenta-specific microRNAs in women with PE vs. control group showed a significantly down modulated expression level for miR-517a-3p (p = 0.025), miR-517c-3p (p = 0.036), miR-574-5p (p = 0.015) along with upregulated expression of miR-20a-5p (p = 0.046).Conclusion. The data obtained evidence that pregnancy-related complications are characterized by specific molecular changes at placental transcriptome level.

In situ immune impact of nivolumab + ipilimumab combination before standard chemoradiation therapy (RTCT) for FIGO IB3-IVA in patients (pts) with cervical squamous carcinoma: COLIBRI trial, a GINECO study.

5501 Background: We report the results of a window-of-opportunity phase 2 trial using neoadjuvant nivolumab + ipilimumab followed by standard chemoradiation therapy (RTCT) in cervical squamous cell carcinoma pts using the CD8+/FOXP3+ ratio as a surrogate marker of immune checkpoint blockade (ICB) in situ effect. Association with better clinical outcome after neoadjuvant chemotherapy and higher CD8+/FOXP3+ ratio was described in cervical cancer pts. However, immune modulation after ICB and correlation with treatment response needs to be characterized. Methods: The primary objective was to explore the changes in the ratio of CD8+/FOXP3+ in tumor biopsies performed before and after ICB (prior to standard RTCT) by multiplex-immunofluorescence (mIF) analyzing densities of CD8 effector T cells (CD8+: CD3+CD8+FOXP3-) and CD4 regulatory T cells (FOXP3+: CD3+CD8-FOXP3+). To normalize the distribution of the values, mIF data were log-transformed (log10). Targeted gene expression profiling of 2,549 genes using HTG technology allowed us to evaluate the 27-gene based ‘HOT’ score reported to be associated with immunologically active tumors (Foy J-P. et al., 2022, Eur J Can). Results: Pts received 1 cycle of nivolumab 3 mg/kg (D1D15) + ipilimumab 1 mg/kg (D1) before starting RTCT + brachytherapy. After RTCT, pts could continue nivolumab in maintenance 480 mg total dose every 28 days for 6 months. Forty pts were treated (including 50% FIGO III-IV). There were no new or unexpected toxicity. Grade≥3 AEs related to ICB occurred in 3 pts. mIF data of the 28 evaluable pts revealed an increase of total CD8+ cells (Wilcoxon, p=0.009), proliferating CD8+ cells (p=0.002) and CD8+/FOXP3+ ratio (p=0.03) between baseline and before RTCT. In the 37 pts evaluable by HTG, a significant increase of expression of the CD8A gene (paired t-test, p=2.2e-05) and the ‘HOT’ score (paired t-test, p=3.1e-06) was observed after ICB. The Objective Response Rate on primary tumor before, after, at the end of treatment (EOT) is reported in table. Correlation between CD8+/FOXP3+ ratio, the ‘HOT’ score and response to treatment will be presented. Conclusions: These data indicate that neoadjuvant nivolumab + ipilimumab is safe and orchestrates de novo immune responses in cervical squamous cell carcinoma. The 82.5% CR rate on primary tumor 6 months post RTCT suggests favorable clinical outcomes. Clinical trial information: NCT04256213 . [Table: see text]

Temporal transcriptional control of neural induction in human induced pluripotent stem cells.

Neural induction of human induced pluripotent stem cells represents a critical switch in cell state during which pluripotency is lost and commitment to a neural lineage is initiated. Although many of the key transcription factors involved in neural induction are known, we know little of the temporal and causal relationships that are required for this state transition. Here, we have carried out a longitudinal analysis of the transcriptome of human iPSCs undergoing neural induction. Using the temporal relationships between the changing profile of key transcription factors and subsequent changes in their target gene expression profiles, we have identified distinct functional modules operative throughout neural induction. In addition to modules that govern loss of pluripotency and gain of neural ectoderm identity, we discover other modules governing cell cycle and metabolism. Strikingly, some of these functional modules are retained throughout neural induction, even though the gene membership of the module changes. Systems analysis identifies other modules associated with cell fate commitment, genome integrity, stress response and lineage specification. We then focussed on OTX2, one of the most precociously activated transcription factors during neural induction. Our temporal analysis of OTX2 target gene expression identified several OTX2 regulated gene modules representing protein remodelling, RNA splicing and RNA processing. Further CRISPRi inhibition of OTX2 prior to neural induction promotes an accelerated loss of pluripotency and a precocious and aberrant neural induction disrupting some of the previously identified modules. We infer that OTX2 has a diverse role during neural induction and regulates many of the biological processes that are required for loss of pluripotency and gain of neural identity. This dynamical analysis of transcriptional changes provides a unique perspective of the widespread remodelling of the cell machinery that occurs during neural induction of human iPSCs.

Expression profile of plasma microRNAs and target genes in patients with complicated pregnancy

Aim: comparative analysis of the expression profile of plasma microRNAs and target genes in patients with complicated pregnancy.Materials and Methods. A prospective observational comparative study in parallel groups was carried out. The study included 73 women divided into three groups: the main group – 42 patients with preeclampsia (PE), the comparison group – 12 pregnant women with fetal growth retardation (FGR), the control group – 19 clinically healthy women with uncomplicated pregnancy. An examination was performed, which included the analysis of clinical characteristics and the study of microRNA expression in blood plasma using the real-time polymerase chain reaction.Results. MicroRNA hsa-miR-210-5p and hsa-miR-1972 were not identified in any plasma sample. Analyzing plasma microRNAs in group of women with PE showed significant changes in the expression levels of hsa-miR-517a-3p (p = 0.025), hsa-miR-517c-3p (p = 0.036), hsa-miR-574-5p (p = 0.015), hsa-miR-517a-3p (p &lt; 0.001) and an increase in miR-20a-5p (p = 0.046) compared to control group. No significant differences were found in the miRNA expression profile in group of women with FGR compared to control group. Assessing an influence of the studied microRNAs on regulatory signaling pathways allowed to establish that hsa-miR-miR-146a-5p, -181a-5p, -210-3p, -517a-3p, -517c-3p, -574-3p, -574-5p, -1304-5p are potential regulators of the reaction cascades involved in the PE pathogenesis.Conclusion. The changes revealed in the circulating blood plasma miRNA level indicate the presence of specific transcriptomic alterations during complicated course of pregnancy.