Targeted Gene Expression Profiling Research Articles

Selection of stable reference genes for quantitative real-time PCR in the Varroa mite, Varroa destructor.

To investigate the acaricide toxicity and resistance mechanisms in the Varroa mite, it is essential to understand the genetic responses of Varroa mites to acaricides, which are usually evaluated by transcriptional profiling based on quantitative real-time polymerase chain reaction(qPCR). In this study, to select reference genes showing consistent expression patterns regardless of the acaricide treatment or the type of tissue, Varroa mites treated with each of the three representative acaricides (coumaphos, fluvalinate, and amitraz) were processed for transcriptomic analysis, from which eight genes (NADH dehydrogenase[NADHD], glyceraldehyde-3-phosphate dehydrogenase[GAPDH], eukaryotic translation elongation factor 1 α 1[eEF1A1], eukaryotic translation elongation factor 2[eEF2], ribosomal protein L5[RpL5], Actin, tubulin α-1D chain[α-tubulin], and Rab1) were selected as candidates. The transcription profiles of these genes, depending on the treatment of the three acaricides or across different tissues (cuticle, legs, gut/fat bodies, and synganglion), were analyzed using qPCR with four validation programs, BestKeeper, geNorm, NormFinder, and RefFinder. Following acaricide treatment, eEF1A1 and NADHD showed the least variation in their expression levels, whereas the expression levels of α-tubulin and RpL5 were the most stable across different tissue groups. Rab1/GAPDH and Actin/eEF2 showed the least stable expression patterns following acaricide treatments and across different tissues, respectively, requiring precautions for use. When vitellogenin gene expression was analyzed by different reference genes, its expression profiles varied significantly depending on the reference genes, highlighting the importance of proper reference gene use. Thus, it is recommended using eEF1A1 and NADHD as reference genes for the comparison of the effects of acaricide on the whole body, whereas α-tubulin and RpL5 are recommended for investigating the tissue-specific expression profiles of target genes.

Identification and validation of suitable housekeeping genes for gene expression studies in BCR-ABL1 positive B-lineage acute lymphoblastic leukemia.

The stability of the housekeeping gene (HKG) expression is an absolute prerequisite for accurate normalization of target gene expression in a quantitative real-time polymerase chain reaction (RQ-PCR). In RQ-PCR, the widely used normalization approach involves the standardization of target genes to the most stable HKG control genes. According to the recent literature, in different experimental conditions the HKGs exhibit either up or down-regulation and thus affecting the gene expression profiles of target genes which leads to erroneous results. This implies that it is very important to select the appropriate HKG and verify the expression stability of the HKG before quantification of the target gene. The present study aims to analyze six different HKGs for their expression profiles and stability in BCR-ABL1 negative cases and validate them in BCR-ABL1 positive cases, detected by multiplex reverse transcribed polymerase chain reaction (RT-PCR). Six commonly used reference genes (GAPDH, ABL1, RNA18S, ACTB, GUSB, and EEF2) were selected in this study. RQ-PCR was performed on 24 BCR-ABL1 negative cases and the outcomes were validated on 24 BCR-ABL1 positive cases. RefFinder™, a web-based composite software was used to check the stability of HKG genes by different algorithms and comprehensive ranking of each HKG gene in BCR-ABL1 negative cases and finally validated in BCR-ABL1 positive cases. It was found that RNA18S, ABL1 and GUSB are good stable HKG genes, which showed minimum variability in gene expression compared to GAPDH, EEF2, and ACTB, the most commonly used HKG.

Targeted Gene Expression Profiling of Human Myeloid Cells From Blood and Lung Compartments of Patients With Tuberculosis and Other Lung Diseases.

Myeloid-derived suppressor cells (MDSC) have been identified in the peripheral blood and granulomas of patients with active TB disease, but their phenotype-, function-, and immunosuppressive mechanism- spectrum remains unclear. Importantly, the frequency and signaling pathways of MDSC at the site of disease is unknown with no indication how this compares to MDSC identified in peripheral blood or to those of related myeloid counterparts such as alveolar macrophages and monocytes. Most phenotypic and functional markers have been described in oncological studies but have not yet been validated in TB. Using a panel of 43 genes selected from pathways previously shown to contribute to tumor-derived MDSC, we set out to evaluate if the expression of these additional functional markers and properties may also be relevant to TB-derived MDSC. Differential expression was investigated between MDSC, alveolar macrophages and monocytes enriched from bronchoalveolar lavage fluid and peripheral blood of patients with active TB, patients with other lung diseases (OLD). Results demonstrated that anatomical compartments may drive compartment-specific immunological responses and subsequent MDSC immunosuppressive functions, demonstrated by the observation that MDSC and/or monocytes from PB alone can discriminate, via hierarchical clustering, between patients with active TB disease and OLD. Our data show that the gene expression patterns of MDSC in peripheral blood and bronchoalveolar lavage fluid do not cluster according to disease states (TB vs OLD). This suggests that MDSC from TB patients may display similar gene expression profiles to those found for MDSC in cancer, but this needs to be validated in a larger cohort. These are important observations for TB research and may provide direction for future studies aimed at repurposing and validating cancer immunotherapies for use in TB.

Clinical and Molecular Profiling to Develop a Potential Prediction Model for the Response to Alemtuzumab Therapy for Acute Kidney Transplant Rejection.

Alemtuzumab, a monoclonal antibody that depletes CD52‐bearing immune cells, is an effective drug for the treatment of severe or glucocorticoid‐resistant acute kidney transplant rejection (AR). Patient‐specific predictions on treatment response are, however, urgently needed, given the severe side effects of alemtuzumab. This study developed a multidimensional prediction model with the aim of generating clinically useful prognostic scores for the response to alemtuzumab. Clinical and histological characteristics were collected retrospectively from patients who were treated with alemtuzumab for AR. In addition, targeted gene expression profiling of AR biopsy tissues was performed. Least absolute shrinkage and selection operator (LASSO) logistic regression modeling was used to construct the ALEMtuzumab for Acute Rejection (ALEMAR) prognostic score. Response to alemtuzumab was defined as patient and allograft survival and at least once an estimated glomerular filtration rate (eGFR) > 30 mL/min/1.73 m2 during the first 6 months after treatment. One hundred fifteen patients were included, of which 84 (73%) had a response to alemtuzumab. The ALEMAR‐score accurately predicted the chance of response. Gene expression analysis identified 13 differentially expressed genes between responders and nonresponders. The combination of the ALEMAR‐score and selected genes resulted in improved predictions of treatment response. The present preliminary prediction model is potentially helpful for the development of stratified alemtuzumab treatment for acute kidney transplant rejection but requires validation.

Identifying Molecular Changes in Early Cervical Cancer Samples of Patients That Developed Metastasis

Cervical cancer is one of the most common cancers in women worldwide. Patients diagnosed with early-stage cervical cancer have a good prognosis, however, 10-20% suffer from local or distant recurrent disease after primary treatment. Treatment options for recurrent cervical cancer are limited. Therefore, it is crucial to identify factors that can predict patients with an increased risk of recurrence to optimize treatment to prevent the recurrence of cervical cancer. We aimed to identify biomarkers in early-stage primary cervical cancer which recurred after surgery. Formalin-Fixed, Paraffin-Embedded surgical specimens of 34 patients with early-stage cervical cancer (FIGO 2009 stage 1B1) and 7 healthy controls were analyzed. Targeted gene expression profiling using the PanCancer IO 360 panel of NanoString Technology was performed. The findings were confirmed by performing immunohistochemistry stainings. Various genes, namely GLS, CD36, WNT5a, HRAS, DDB2, PIK3R2, and CDH2 were found to be differentially highly expressed in primary cervical cancer samples of patients who developed distant recurrence. In addition, The relative infiltration score of CD8+ T cells, CD80+CD86+ macrophages, CD163+MRC1+ macrophages, and FOXP3+IL2RA+ regulatory T cells were significantly higher in this group of samples. In contrast, no significant differences in gene expression and relative immune infiltration were found in samples of patients who developed local recurrence. The infiltration of CD8 and FOXP3 cells were validated by immunohistochemistry using all samples included in the study. We identified molecular alterations in primary cervical cancer samples from patients who developed recurrent disease. These findings can be utilized towards developing a molecular signature for the early detection of patients with a high risk to develop metastasis.

Treating Pancreatic Ductal Adenocarcinoma Patients with Rintatolimod: Hitting Two Targets with One Arrow?

Introduction: Rintatolimod (Ampligen©), a mismatched double-stranded RNA molecule, is a selective toll-like receptor 3 (TLR3) agonist that boosts antitumor immunity. Recently, the U.S. Food and Drug Administration granted Orphan Drug Designation status to Rintatolimod for the treatment of patients with pancreatic ductal adenocarcinoma (PDAC). This study aimed to investigate the direct effect of Rintatolimod on PDAC cells. Methods: Three PDAC cell lines (CFPAC-1, MIAPaCa-2, and PANC-1) were treated with various concentrations of Rintatolimod and their corresponding vehicle control. The proliferation and migration effects were examined using in-vitro assays and the molecular effect was examined by targeted gene expression profiling. Furthermore, human PDAC samples were used to validate the expression of TLR3 by immunohistochemistry. Results: Rintatolimod decreased the proliferation and migration ability of CFPAC-1 cells. In addition, it decreased the proliferation of MIAPaCa-2 cells and the migration of PANC-1 cells. However, it did not have a dual effect in MIAPaCa-2 and PANC-1 cells. Interestingly, TLR3 was highly expressed in CFPAC-1 cells, low expressed in MIAPaCa-2 and not expressed in PANC-1. Gene expression analysis revealed the upregulation of interferon-related genes, chemokines, interleukins and cell cycle regulatory genes. The heterogeneity of TLR3 expression was confirmed in human PDAC samples. Conclusion: intatolimod decreases the proliferation without increasing the migration ability of PDAC cells that express TLR3. The direct effect of Rintatolimod on tumor cells combined with its boosting effect on the immune system highlights the potential therapeutic effect in PDAC patients.

Transcriptomic signatures induced by the Ebola virus vaccine rVSVΔG-ZEBOV-GP in adult cohorts in Europe, Africa, and North America: a molecular biomarker study

SummaryBackgroundA recombinant vesicular stomatitis virus vector expressing the Zaire Ebola virus glycoprotein (rVSVΔG-ZEBOV-GP) vaccine has been reported as safe, immunogenic, and highly protective in a ring vaccination trial. We aimed to identify transcriptomic immune response biomarker signatures induced by vaccination and associated signatures with its immunogenicity and reactogenicity to better understand the potential mechanisms of action of the vaccine.Methods354 healthy adult volunteers were vaccinated in randomised, double-blind, placebo-controlled trials in Europe (Geneva, Switzerland [November, 2014, to January, 2015]) and North America (USA [Dec 5, 2014, to June 23, 2015]), and dose-escalation trials in Africa (Lambaréné, Gabon [November, 2014, to January, 2015], and Kilifi, Kenya [December, 2014, to January, 2015]) using different doses of the recombinant vesicular stomatitis virus vector expressing the Zaire Ebola virus glycoprotein (rVSVΔG-ZEBOV-GP; 3 × 105 to 1 × 108 plaque-forming units [pfu]). Longitudinal transcriptomic responses (days 0, 1, 2, 3, 7, 14, and 28) were measured in whole blood using a targeted gene expression profiling platform (dual-colour reverse-transcriptase multiplex ligation-dependent probe amplification) focusing on 144 immune-related genes. The effect of time and dose on transcriptomic response was also assessed. Logistic regression with lasso regularisation was applied to identify host signatures with optimal discriminatory capability of vaccination at day 1 or day 7 versus baseline, whereas random-effects models and recursive feature elimination combined with regularised logistic regression were used to associate signatures with immunogenicity and reactogenicity.FindingsOur results indicated that perturbation of gene expression peaked on day 1 and returned to baseline levels between day 7 and day 28. The magnitude of the response was dose-dependent, with vaccinees receiving a high dose (≥9 × 106 pfu) of rVSVΔG-ZEBOV-GP exhibiting the largest amplitude. The most differentially expressed genes that were significantly upregulated following vaccination consisted of type I and II interferon-related genes and myeloid cell-associated markers, whereas T cell, natural killer cell, and cytotoxicity-associated genes were downregulated. A gene signature associated with immunogenicity (common to all four cohorts) was identified correlating gene expression profiles with ZEBOV-GP antibody titres and a gene signatures associated with reactogenicity (Geneva cohort) was identified correlating gene expression profiles with an adverse event (ie, arthritis).InterpretationCollectively, our results identify and cross-validate immune-related transcriptomic signatures induced by rVSVΔG-ZEBOV-GP vaccination in four cohorts of adult participants from different genetic and geographical backgrounds. These signatures will aid in the rational development, testing, and evaluation of novel vaccines and will allow evaluation of the effect of host factors such as age, co-infection, and comorbidity on responses to vaccines.FundingInnovative Medicines Initiative 2 Joint Undertaking.

Follicular Lymphoma Microenvironment Signatures Define Patients Subsets Obtaining Long Term Clinical Benefit after Single-Agent First-Line Anti-CD20 Immunotherapy

The role of first-line single agent Rituximab immunotherapy in follicular lymphoma (FL) is still a matter of debate. Although a subset of patients (pts) may obtain long term benefit with upfront immunotherapy, first-line therapy with standard chemoimmunotherapy offers better results in terms of progression free survival (PFS) compared to single agent Rituximab. On the other hand, there is no clear demonstration of sizeable long term benefit with first-line Rituximab compared to an initial wait and see approach, as most FL pts will ultimately be exposed to chemotherapy at some point in their disease course.Here we show that the efficacy of front line anti CD20 immunotherapy in FL could be dependent on microenvironmental factors, and that specific immune signatures could define FL pts subsets obtaining maximal benefit from upfront anti CD20 immunotherapy.First, we retrospectively analyzed the outcome of our single center cohort of 81 FL pts treated with first-line single agent Rituximab therapy with (n=53) or without (n=28) maintenance. The vast majority of pts considered in this analysis were treated in the context of several clinical trials exploring the efficacy of upfront single agent originator (SAKK 35/98, 35/03, 35/10: n= 68) or biosimilar (JASMINE and REFLECTIONS B328/06 n= 9) Rituximab, which were ran at our center from 2000 to 2018. Four patients received off-label single agent Rituximab. Median age was 55 years (y), 52% (42) of patients were female, 72% (58) stage III-IV, 76% (62) were Follicular international prognostic index (FLIPI) score 0-2, 26% (21) were bulky, 52% (42) were high tumor burden according to the GELF criteria. After a median follow-up of 9.5 y, the overall survival (OS) and PFS rates were 82% and 35% respectively. With a long follow-up, these data are in line with previous findings indicating that a sizeable fraction of FL pts derive long term benefit from upfront single agent Rituximab maintaining a continuous complete remission. Paraffin embedded tissue from initial diagnosis was available in 39 pts. In order to dissect determinants of Rituximab immunotherapy efficacy in this homogeneous chemo-naive population, we analyzed FL diagnostic biopsies (n=39 FL + 5 healthy controls) with targeted gene expression profiling (T-GEP) on the NanoString platform, using the PanCancer Immune Profiling panel, which includes 730 genes belonging to the most relevant immunologic checkpoints and pathways.A 20-gene signature including genes involved in chemokine-cytokine signaling, T-regulatory cells, natural-killer cell activity and interleukin-17 signaling was significantly associated with the achievement of a complete response after induction +/- maintenance treatment. A simple 6-gene immune signature (hereafter ImSig) was found to be significantly associated with PFS, with IL22RA2, CCL22, TNFRSF4, IL17RB, CCL19 overexpression and CD209 downregulation being associated with worse outcome. By applying the maxstat package 10-y PFS was 65% for ImSig low pts vs 6% for ImSig high pts (p<0.0001). In multivariate analysis only the 6-gene ImSig and Rituximab maintenance retained independent prognostic value (p<0.001 and 0.002 respectively). As opposite, GELF criteria (present/absent = 27/12) and FLIPI score (0-2/3-5 = 23/16) were not associated with PFS. The 6-gene immune signature was validated in silico in 2 independent publicly available cohorts of FL pts treated with upfront chemoimmunotherapy: a cohort of 137 pts (Silva et al 2019, Affimetryx platform) and a cohort of 50 pts (Bararia et al. 2020), the latter analyzed on the NanoString Platform with the same T-GEP panel (PanCancer Immune Profiling) used in our discovery cohort. The 6-gene signature was confirmed to be a powerful outcome predictor in these 2 chemoimmunotherapy-treated cohorts, and notably the 10-y PFS rates of ImSig low vs high patients mirrored the results observed with single agent immunotherapy in the discovery cohort.The results here reported indicate that pts with a favorable immune signature could derive maximal benefit from a first-line chemo-free treatment approach with single agent Rituximab, achieving and maintaining complete remission in the long term, irrespective of the tumor burden and other clinical variables. Thus, profiling of FL microenvironment with T-GEP could provide a useful tool for selecting patients who may be suitable for a chemo-free upfront treatment with anti CD20 immunotherapy. DisclosuresDerenzini: TG-THERAPEUTICS: Research Funding; ASTRA-ZENECA: Consultancy, Other: ADVISORY-BOARD; ADC-THERAPEUTICS: Research Funding; TAKEDA: Research Funding; BEIGENE: Other: ADVISORY BOARD. Pileri: CELGENE: Other: ADVISORY BOARD; NANOSTRING: Other: ADVISORY BOARD; ROCHE: Other: ADVISORY-BOARD. Tarella: ADC-THERAPEUTICS: Other: ADVISORY BOARD; Abbvie: Other: ADVISORY BOARD. OffLabel Disclosure:First line single agent Rituximab in Follicular Lymphoma

Integration of Targeted Gene Expression Profiling and FDG-PET Radiomics Uncovers Radiometabolic Signatures Associated with Outcome in Diffuse Large B-Cell Lymphoma

Metabolic rewiring is a hallmark of cancer and a predominant feature of aggressive lymphoproliferative disorders such as diffuse large B-cell lymphomas (DLBCL), which need a reshaped metabolism in order to meet the increased demands related to rapid cell proliferation.Emerging evidence indicates that chemoresistance is closely related to altered metabolism in cancer. However, the relationship between metabolic rewiring and chemoresistance in lymphoma is yet to be elucidated. Radiomic analysis applied to functional imaging with fluoroedoxyglucose positron emission tomography (FDG-PET) provides a unique opportunity to explore DLBCL metabolism. In this study we hypothesized that distinct gene expression (GEP) signatures might be correlated with specific FDG-PET radiomics signatures, which in turn could be associated with resistance to standard chemoimmunotherapy and DLBCL outcome.First, we retrospectively analyzed a discovery cohort of 48 consecutive DLBCL patients (pts) treated at our center with standard first line R-CHOP/R-CHOP-like chemoimmunotherapy from 2010 to 2018, with available formalin-fixed paraffin embedded (FFPE) tissue from the initial diagnostic biopsy and FDG-PET radiomics data extracted from the same target lesion. Median follow-up was 55 months (range 18-110). We profiled this cohort with targeted-GEP (T-GEP) (NanoString platform), using a custom panel to define the cell of origin (COO) and MYC/BCL-2 levels, and a dedicated panel comprising 180 genes encompassing the most relevant cancer metabolism pathways. By applying the maxstat package we found that a 6-gene metabolic signature was strongly associated with outcome and outperformed the COO, the MYC/BCL-2 status and the International Prognostic Index (IPI) score for progression free survival (PFS) and overall survival (OS) in multivariate analysis. The 6-gene metabolic signature included genes regulating oxidative metabolism and fatty acid oxidation (SCL25A1, PDK4, PDPR) which were upregulated, and was inversely associated with genes involved in glycolytic pathways (MAP2K1, HIF1A, GBE1) which were downregulated. Notably 5-year PFS and OS were 100% and 95% in metabolic signature (met-Sig) low pts vs 24% and 45% in met-Sig high pts respectively (p<0.0001 for PFS and OS). There was no significant association between the COO, MYC/BCL-2 levels, standardized uptake value (SUV), and the 6-gene signature. The prognostic value of the 6-gene signature for OS was validated in 2 large publicly available cohorts of 469 (Sha et al. J Clin Oncol 2019) and 233 (Lenz et al. N Eng J Med 2005) pts. Next, we integrated PET radiomics and T-GEP data. Radiomics analysis (LifeX package) was performed by applying regions of interest semi-automatically, using a 25% SUV max threshold for segmentation. Fifty-five radiomic features (RFs) were extracted and 10 RFs significantly correlated either positively or negatively with the T-GEP metabolic signature (Spearman). After stability evaluation, applying a stepwise feature selection procedure, 4 RFs (Histo Curtosis, Histo Energy, Shape Sphericity, NGLDM Contrast) were used to generate a radiomic signature (hereafter called radiometabolic signature) characterized by the most significant correlation with both the metabolic T-GEP signature (r=0.43, p=0.0027) and PFS (p=0.004). These results (obtained analyzing the lesion of the initial diagnostic biopsy), were confirmed using different target lesions (i.e. the most FDG-avid and the largest lesion), and were validated in a second independent cohort of 64 patients (validation cohort) treated at our center in the same period of time (with no FFPE tissue available). A multivariate analysis performed in the whole cohort of 112 pts (discovery + validation) indicated that the radiometabolic signature retained independent prognostic value in relation to the IPI score and metabolic tumor volume. The robustness of the radiometabolic signature was further confirmed by using a second segmentation method (fixed 2.5 SUV max threshold).These data indicate that oxidative metabolic rewiring could be a powerful adverse prognostic predictor, suggesting the possibility of targeting oxidative metabolism to overcome chemorefractoriness in DLBCL. This study provides the proof of principle for the use of FDG-PET radiomics as a tool for non-invasive assessment of cancer metabolism, and for predicting metabolic vulnerabilities in DLBCL. [Display omitted] DisclosuresTarella: ADC-THERAPEUTICS: Other: ADVISORY BOARD; Abbvie: Other: ADVISORY BOARD. Pileri: CELGENE: Other: ADVISORY BOARD; ROCHE: Other: ADVISORY-BOARD; NANOSTRING: Other: ADVISORY BOARD. Derenzini: TAKEDA: Research Funding; BEIGENE: Other: ADVISORY BOARD; ASTRA-ZENECA: Consultancy, Other: ADVISORY-BOARD; TG-THERAPEUTICS: Research Funding; ADC-THERAPEUTICS: Research Funding.

Multi-Run Concrete Autoencoder to Identify Prognostic lncRNAs for 12 Cancers.

Background: Long non-coding RNA plays a vital role in changing the expression profiles of various target genes that lead to cancer development. Thus, identifying prognostic lncRNAs related to different cancers might help in developing cancer therapy. Method: To discover the critical lncRNAs that can identify the origin of different cancers, we propose the use of the state-of-the-art deep learning algorithm concrete autoencoder (CAE) in an unsupervised setting, which efficiently identifies a subset of the most informative features. However, CAE does not identify reproducible features in different runs due to its stochastic nature. We thus propose a multi-run CAE (mrCAE) to identify a stable set of features to address this issue. The assumption is that a feature appearing in multiple runs carries more meaningful information about the data under consideration. The genome-wide lncRNA expression profiles of 12 different types of cancers, with a total of 4768 samples available in The Cancer Genome Atlas (TCGA), were analyzed to discover the key lncRNAs. The lncRNAs identified by multiple runs of CAE were added to a final list of key lncRNAs that are capable of identifying 12 different cancers. Results: Our results showed that mrCAE performs better in feature selection than single-run CAE, standard autoencoder (AE), and other state-of-the-art feature selection techniques. This study revealed a set of top-ranking 128 lncRNAs that could identify the origin of 12 different cancers with an accuracy of 95%. Survival analysis showed that 76 of 128 lncRNAs have the prognostic capability to differentiate high- and low-risk groups of patients with different cancers. Conclusion: The proposed mrCAE, which selects actual features, outperformed the AE even though it selects the latent or pseudo-features. By selecting actual features instead of pseudo-features, mrCAE can be valuable for precision medicine. The identified prognostic lncRNAs can be further studied to develop therapies for different cancers.

A Targeted Gene Expression Risk Score Predicts Meningioma Outcomes and Responses to Radiotherapy

Indications for postoperative radiotherapy after meningioma resection are controversial. DNA methylation profiling identifies meningiomas at risk for recurrence, but logistic and technical barriers have encumbered clinical translation of this approach. The aim of this study was to optimize and validate a clinically-tractable targeted gene expression biomarker to predict meningioma outcomes and responses to postoperative radiotherapy.Targeted gene expression and Illumina 850k DNA methylation profiling were performed on a discovery cohort of 173 meningiomas (median follow-up 7.8 years) and an external validation cohort of 331 meningiomas (median follow-up 5.8 years) from patients treated with surgery (n = 504) and postoperative radiotherapy (n = 73) at independent, international institutions (70% WHO grade 1, 24% WHO grade 2, 6% WHO grade 3). RNA sequencing was performed on the discovery cohort, and 125 genes were selected for targeted gene expression profiling based on associations with meningioma recurrence. Regularized Cox regression was used to develop continuous gene expression risk score for local freedom from recurrence (LFFR). The model (34 meningioma genes and 7 housekeeping genes) and risk quartiles (low, low-intermediate, high-intermediate, and high) were locked and validated on the external cohort. Multivariate regressions (MVR) incorporating WHO grade, DNA methylation grouping, extent of resection, primary versus recurrent presentation, and postoperative radiotherapy were used to assess the risk score across clinical contexts.The gene expression risk score (concordance-index 0.78 ± 0.03) outperformed DNA methylation grouping (0.71 ± 0.03) and WHO grade (0.65 ± 0.03) in stratifying meningioma LFFR in the validation cohort (n = 331). The risk score classified 43% of WHO grade 1 meningiomas as high-intermediate (n = 82) or high risk (n = 34), with 5-year LFFR and overall survival (OS) of 83% and 89% for high-intermediate risk WHO grade 1 meningiomas, and 41% and 73% for high risk WHO grade 1 meningiomas, respectively. Low risk WHO grade 1 meningiomas (n = 50) had 5-year LFFR and OS of 92% and 88%, respectively. High and high-intermediate risk scores were independently prognostic for LFFR (HR 2.9, 95% CI 1.2-4.4) and OS (HR 2.7, 95% CI 1.3-5.6) on MVR. An interaction term between postoperative radiotherapy and risk score was predictive for LFFR (P = 0.046) and OS (P = 0.001) on MVR in the validation cohort, suggesting meningiomas with higher risk scores derived greater benefit from postoperative radiotherapy.Here we use targeted gene expression profiling to develop a risk score predicting meningioma outcomes and responses to postoperative radiotherapy. This cost-effective assay outperforms DNA methylation grouping and WHO grade in discriminating meningioma outcomes, and may be useful for guiding clinical trial design.

A Comprehensive Analysis of SE-lncRNA/mRNA Differential Expression Profiles During Chondrogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells.

Objective: Articular cartilage injury is common and difficult to treat clinically because of the characteristics of the cartilage. Bone marrow-derived mesenchymal stem cell (BMSC)-mediated cartilage regeneration is a promising therapy for treating articular cartilage injury. BMSC differentiation is controlled by numerous molecules and signaling pathways in the microenvironment at both the transcriptional and post-transcriptional levels. However, the possible function of super enhancer long non-coding RNAs (SE-lncRNAs) in the chondrogenic differentiation of BMSCs is still unclear. Our intention was to explore the expression profile of SE-lncRNAs and potential target genes regulated by SE-lncRNAs during chondrogenic differentiation in BMSCs.Materials and Methods: In this study, we conducted a human Super-Enhancer LncRNA Microarray to investigate the differential expression profile of SE-lncRNAs and mRNAs during chondrogenic differentiation of BMSCs. Subsequent bioinformatic analysis was performed to clarify the important signaling pathways, SE-lncRNAs, and mRNAs associated with SE-lncRNAs regulating the chondrogenic differentiation of BMSCs.Results: A total of 77 SE-lncRNAs were identified, of which 47 were upregulated and 30 were downregulated during chondrogenic differentiation. A total of 308 mRNAs were identified, of which 245 were upregulated and 63 were downregulated. Some pathways, such as focal adhesion, extracellular matrix (ECM)–receptor interaction, transforming growth factor-β (TGF-β) signaling pathway, and PI3K–Akt signaling pathway, were identified as the key pathways that may be implicated in the chondrogenic differentiation of BMSCs. Moreover, five potentially core regulatory mRNAs (PMEPA1, ENC1, TES, CDK6, and ADIRF) and 37 SE-lncRNAs in chondrogenic differentiation were identified by bioinformatic analysis.Conclusion: We assessed the differential expression levels of SE-lncRNAs and mRNAs, along with the chondrogenic differentiation of BMSCs. By analyzing the interactions and co-expression, we identified the core SE-lncRNAs and mRNAs acting as regulators of the chondrogenic differentiation potential of BMSCs. Our study also provided novel insights into the mechanism of BMSC chondrogenic and cartilage regeneration.

Targeted gene expression profiling of inverted papilloma and squamous cell carcinoma

Inverted papilloma (IP) is a sinonasal tumor with a well-known potential for malignant transformation. The purpose of this study was to identify the genes and pathways associated with IP, with progression to carcinoma-in-situ and invasive carcinoma. To determine genes and molecular pathways that may indicate progression and correlate with histologic changes, we analyzed six IP without dysplasia, five IP with carcinoma-in-situ, and 13 squamous cell carcinoma ex-IP by targeted sequencing. The HTG EdgeSeq Oncology Biomarker Panel coupled with next-generation sequencing was used to evaluate 2560 transcripts associated with solid tumors. Progressive upregulation of 11 genes were observed (CALD1, COL1A1, COL3A1, COL4A2, COL5A2, FN1, ITGA5, LGALS1, MMP11, SERPINH1, SPARC) in the order of invasive carcinoma>carcinoma-in-situ>IP without dysplasia. When compared with IP without dysplasia, more genes are differentially expressed in invasive carcinoma than carcinoma-in-situ samples (341 downregulated/333 upregulated vs. 195 downregulated/156 upregulated). Gene set enrichment analysis determined three gene sets in common between the cohorts (epithelial mesenchymal transition, extracellular matrix organization, and coagulation). Progressive upregulation of genes specific to IP malignant degeneration has significant clinical implications. This panel of 11 genes will improve concordance of histologic classification, which can directly impact treatment and patient outcomes. Additionally, future studies on larger tumor sets may observe upregulation in the gene panel that preceded histologic changes, which may be useful for further risk stratification.

RhoC Modulates Cell Junctions and Type I Interferon Response in Aggressive Breast Cancers.

Metastases are the leading cause of death in cancer patients. RhoC, a member of the Rho GTPase family, has been shown to facilitate metastasis of aggressive breast cancer cells by influencing motility, invasion, and chemokine secretion, but as yet there is no integrated model of the precise mechanism of how RhoC promotes metastasis. A common phenotypic characteristic of metastatic cells influenced by these mechanisms is dysregulation of cell-cell junctions. Thus, we set out to study how RhoA- and RhoC-GTPase influence the cell-cell junctions in aggressive breast cancers. We demonstrate that CRISPR-Cas9 knockout of RhoC in SUM 149 and MDA 231 breast cancer cells results in increased normalization of junctional integrity denoted by junction protein expression/colocalization. In functional assessments of junction stability, RhoC knockout cells have increased barrier integrity and increased cell-cell adhesion compared to wild-type cells. Whole exome RNA sequencing and targeted gene expression profiling demonstrate decreased expression of Type I interferon-stimulated genes in RhoC knockout cells compared to wild-type, and subsequent treatment with interferon-alpha resulted in significant increases in adhesion and decreases in invasiveness of wild-type cells and a dampened response to interferon-alpha stimulation with respect to adhesion and invasiveness in RhoC knockout cells. We delineate a key role of RhoC-GTPase in modulation of junctions and response to interferon, which supports inhibition of RhoC as a potential anti-invasion therapeutic strategy.

Dual targeting of the DNA damage response pathway and BCL-2 in diffuse large B-cell lymphoma

Standard chemotherapies for diffuse large B-cell lymphoma (DLBCL), based on the induction of exogenous DNA damage and oxidative stress, are often less effective in the presence of increased MYC and BCL-2 levels, especially in the case of double hit (DH) lymphomas harboring rearrangements of the MYC and BCL-2 oncogenes, which enrich for a patient’s population characterized by refractoriness to anthracycline-based chemotherapy. Here we hypothesized that adaptive mechanisms to MYC-induced replicative and oxidative stress, consisting in DNA damage response (DDR) activation and BCL-2 overexpression, could represent the biologic basis of the poor prognosis and chemoresistance observed in MYC/BCL-2-positive lymphoma. We first integrated targeted gene expression profiling (T-GEP), fluorescence in situ hybridization (FISH) analysis, and characterization of replicative and oxidative stress biomarkers in two independent DLBCL cohorts. The presence of oxidative DNA damage biomarkers identified a poor prognosis double expresser (DE)-DLBCL subset, characterized by relatively higher BCL-2 gene expression levels and enrichment for DH lymphomas. Based on these findings, we tested therapeutic strategies based on combined DDR and BCL-2 inhibition, confirming efficacy and synergistic interactions in in vitro and in vivo DH-DLBCL models. These data provide the rationale for precision-therapy strategies based on combined DDR and BCL-2 inhibition in DH or DE-DLBCL.

Stability evaluation of reference genes for real-time quantitative PCR normalization in Spodoptera frugiperda (Lepidoptera: Noctuidae)

Real-time quantitative PCR (qPCR) is a reliable and widely used technique for analyzing the expression profiles of target genes in different species, and reference genes with stable expressions have been introduced for the normalization of the data. Therefore, stability evaluation should be considered as the initial step for qPCR experiments. The fall armyworm Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) is a polyphagous pest that consumes many plant species and seriously threatens corn production around the world. However, no studies thus far have examined the stability of reference genes in this pest. In this study, the expression profiles of the eight candidate reference genes of Actin, elongation factor 1 alpha (EF1α), elongation factor 2 (EF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L3 (RPL3), ribosomal protein L13 (RPL13), alpha-tubulin (α-TUB), and beta-1-tubulin (β-1-TUB) were obtained from S. frugiperda in different samples and the stability was evaluated by ΔCt, BestKeeper, geNorm, NormFinder, and RefFinder methods. The results of pairwise variation (V) calculated by GeNorm indicated two reference genes could be selected for normalization. Therefore, the combinations of the most stable reference genes for different experimental conditions of S. frugiperda were shown as follows: EF2 and RPL13 for developmental stages, RPL3 and β-1-TUB for larval tissue samples, EF2 and EF1α for the larval samples treated with different temperatures, RPL3 and EF1α for the larval samples under starvation stress, and RPL13 and EF1α for all the samples. Our results lay the foundation for the normalization of qPCR analyses in S. frugiperda and could help guarantee the accuracy of subsequent research.

Rintatolimod Induces Antiviral Activities in Human Pancreatic Cancer Cells: Opening for an Anti-COVID-19 Opportunity in Cancer Patients?

Simple SummarySpecific treatment for COVID-19 infections in cancer patients is lacking while the demand for treatment is increasing. Therefore, we explored the effect of Rintatolimod, a Toll-like receptor 3 (TLR3) agonist, on human epithelial cancerous cells. Our results demonstrated that Rintatolimod stimulated an anti-viral effect by producing RNase L that blocks virus replication. Moreover, Rintatolimod activated the innate and the adaptive immune systems by activating a cascade of actions in human cancerous cells. We believe that Rintatolimod should be considered in the treatment regimens of cancer patients who suffer from SARS-CoV-2 infection.Severe acute respiratory virus-2 (SARS-CoV-2) has spread globally leading to a devastating loss of life. Large registry studies have begun to shed light on the epidemiological and clinical vulnerabilities of cancer patients who succumb to or endure poor outcomes of SARS-CoV-2. Specific treatment for COVID-19 infections in cancer patients is lacking while the demand for treatment is increasing. Therefore, we explored the effect of Rintatolimod (Ampligen®) (AIM ImmunoTech, Ocala, FL, USA), a Toll-like receptor 3 (TLR3) agonist, to treat uninfected human pancreatic cancer cells (HPACs). The direct effect of Rintatolimod was measured by targeted gene expression profiling and by proteomics measurements. Our results show that Rintatolimod induces an antiviral effect in HPACs by inducing RNase-L-dependent and independent pathways of the innate immune system. Treatment with Rintatolimod activated the interferon signaling pathway, leading to the overexpression of several cytokines and chemokines in epithelial cells. Furthermore, Rintatolimod treatment increased the expression of angiogenesis-related genes without promoting fibrosis, which is the main cause of death in patients with COVID-19. We conclude that Rintatolimod could be considered an early additional treatment option for cancer patients who are infected with SARS-CoV-2 to prevent the complicated severity of the disease.

A gene expression signature associated with B cells predicts benefit from immune checkpoint blockade in lung adenocarcinoma

ABSTRACT Immune checkpoint blockade (ICB) expands the therapeutic options for metastatic lung cancer nowadays representing a standard frontline strategy as monotherapy or combination therapy, as well as an option in oncogene-addicted NSCLC after exhaustion of targeted therapies. Predictive markers are urgently needed, since only a minority of patients benefits from ICB, while serious adverse effects of immunotoxicity may occur. The study cohort included 43 ICB-treated metastatic lung adenocarcinoma showing long-term response (n = 16), rapid progression (n = 21) or intermediate patterns of response (n = 6). Lung biopsies acquired before initiation of ICB were analyzed by targeted mRNA expression profiling of 770 genes. Level and proportions of 14 immune cell types were estimated using characteristic gene expression signatures. Abundance of B cells (HR = 0.66, p = .00074), CD45+ cells (HR = 0.61, p = .01) and total TILs (HR = 0.62, p = .025) was associated with prolonged progression-free survival after ICB treatment. In a ROC analysis, B cells (AUC = 0.77, p = .0055) and CD45+ cells (AUC = 0.73, p = .019) predicted benefit of ICB, which was not the case for PD-L1 mRNA (AUC = 0.54, p = .72) and PD-L1 protein expression (AUC = 0.68, p = .082). Clustering of 79 candidate predictive markers identified among 770 investigated genes revealed two distinct predictive clusters which included cytotoxic cell or macrophage markers, respectively. In summary, targeted gene expression profiling was feasible using routine diagnostics biopsies. This study proposes B cells and total TILs as complementary predictors of ICB benefit in NSCLC. While further preferably prospective validation is required, gene expression profiling could be integrated in the routine diagnostic work-up complementing existing NGS protocols.

Molecular investigation of Tuscan sweet cherries sampled over three years: gene expression analysis coupled to metabolomics and proteomics

Sweet cherry (Prunus avium L.) is a stone fruit widely consumed and appreciated for its organoleptic properties, as well as its nutraceutical potential. We here investigated the characteristics of six non-commercial Tuscan varieties of sweet cherry maintained at the Regional Germplasm Bank of the CNR-IBE in Follonica (Italy) and sampled ca. 60 days post-anthesis over three consecutive years (2016-2017-2018). We adopted an approach merging genotyping and targeted gene expression profiling with metabolomics. To complement the data, a study of the soluble proteomes was also performed on two varieties showing the highest content of flavonoids. Metabolomics identified the presence of flavanols and proanthocyanidins in highest abundance in the varieties Morellona and Crognola, while gene expression revealed that some differences were present in genes involved in the phenylpropanoid pathway during the 3 years and among the varieties. Finally, proteomics on Morellona and Crognola showed variations in proteins involved in stress response, primary metabolism and cell wall expansion. To the best of our knowledge, this is the first multi-pronged study focused on Tuscan sweet cherry varieties providing insights into the differential abundance of genes, proteins and metabolites.

Loss of SMAD4 Is Sufficient to Promote Tumorigenesis in a Model of Dysplastic Barrett's Esophagus.

Background & AimsEsophageal adenocarcinoma (EAC) develops from its precursor Barrett’s esophagus through intermediate stages of low- and high-grade dysplasia. However, knowledge of genetic drivers and molecular mechanisms implicated in disease progression is limited. Herein, we investigated the effect of Mothers against decapentaplegic homolog 4 (SMAD4) loss on transforming growth factor β (TGF-β) signaling functionality and in vivo tumorigenicity in high-grade dysplastic Barrett’s cells.MethodsAn in vivo xenograft model was used to test tumorigenicity of SMAD4 knockdown or knockout in CP-B high-grade dysplastic Barrett’s cells. RT2 polymerase chain reaction arrays were used to analyze TGF-β signaling functionality, and low-coverage whole-genome sequencing was performed to detect copy number alterations upon SMAD4 loss.ResultsWe found that SMAD4 knockout significantly alters the TGF-β pathway target gene expression profile. SMAD4 knockout positively regulates potential oncogenes such as CRYAB, ACTA2, and CDC6, whereas the CDKN2A/B tumor-suppressor locus was regulated negatively. We verified that SMAD4 in combination with CDC6-CDKN2A/B or CRYAB genetic alterations in patient tumors have significant predictive value for poor prognosis. Importantly, we investigated the effect of SMAD4 inactivation in Barrett’s tumorigenesis. We found that genetic knockdown or knockout of SMAD4 was sufficient to promote tumorigenesis in dysplastic Barrett’s esophagus cells in vivo. Progression to invasive EAC was accompanied by distinctive and consistent copy number alterations in SMAD4 knockdown or knockout xenografts.ConclusionsAltogether, up-regulation of oncogenes, down-regulation of tumor-suppressor genes, and chromosomal instability within the tumors after SMAD4 loss implicates SMAD4 as a protector of genome integrity in EAC development and progression. Foremost, SMAD4 loss promotes tumorigenesis from dysplastic Barrett’s toward EAC.