Analysis Of Individual Genes Research Articles

Abstract 2145: Genomic and transcriptomic analyses related to the clinical efficacy of first-line nivolumab in advanced hepatocellular carcinoma from the phase 3 CheckMate 459 trial

Abstract In the phase 3 CheckMate 459 study (NCT02576509), first-line nivolumab (NIVO) treatment demonstrated higher overall response rate (ORR) and a favorable safety profile, but no significant improvement in overall survival (OS) compared with sorafenib (SORA) in patients with advanced hepatocellular carcinoma (HCC). Here, we report exploratory biomarker analyses from the trial with a potential to identify patients who may benefit from NIVO. Archival or fresh tumor samples were collected before treatment and subjected to whole exome sequencing (WES) (400/730: 55%) for genetic alterations including high microsatellite instability (MSI-H) and WNT/beta-catenin pathway components, as well as whole transcriptome RNA sequencing (469/730: 64%) for analysis of individual genes, inflammation signatures and Gene Set Enrichment Analysis (GSEA). Associations between biomarkers and best overall response (BOR), progression free survival (PFS) and OS were evaluated at a minimum follow-up of 33·6 months. RNA sequencing analysis demonstrated that high Gajewski inflammation gene signature score of ≥0.47 (30% of tumors) was associated with an improvement in BOR (p=0.0043), PFS (p=0.00065) and OS (p=0.0073) within the NIVO arm only. Upon comparison of NIVO versus SORA, only patients whose tumors were inflammation high demonstrated NIVO benefit for BOR (p=0.017), PFS (p=0.0063) and OS (p=0.037). Responses to NIVO were associated with higher expression of CD8A, CD8B, PDCD1 (PD-1) and other inflammation-associated genes within the tumor (false discovery rate, FDR<0.01). GSEA of 50 hallmark gene sets for ORR showed that responders to NIVO and SORA were enriched in similar gene sets such as inflammatory response, interferon alpha/gamma response, E2F targets and G2M checkpoint (FDR<0.01). In contrast, GSEA for OS showed inflammatory response and IL6-JAK-STAT3 signaling gene sets were associated with benefit from NIVO, but shorter OS for SORA (FDR<0.01). WES analysis demonstrated that the prevalence of MSI-H was low (12/400: 3%) and insufficient to confirm relationship to clinical outcomes (no radiographic responses observed in 9 NIVO- and 3 SORA-treated patients). Alterations within beta-catenin gene were not associated with survival outcomes for NIVO. Overall, patients with advanced HCC whose tumors had high Gajewski inflammation gene signature demonstrated improved outcomes with NIVO versus SORA. NIVO responders had higher expression of individual inflammation-associated genes. Expression of inflammatory response and IL6-JAK-STAT3 signaling gene sets showed association with longer OS for NIVO, but not SORA. These exploratory analyses support further studies of tumor inflammation-related biomarkers to identify patients most likely to benefit from PD-1 inhibition in advanced HCC. Citation Format: Jaclyn Neely, Jin Yao, Masatoshi Kudo, Richard S. Finn, Bruno Sangro, Ignacio Melero, Anthony El-Khoueiry, Marina Tschaika, Damir Begic, Ashwin Sama, Parul Doshi, Thomas Yau, Robin Kate Kelley. Genomic and transcriptomic analyses related to the clinical efficacy of first-line nivolumab in advanced hepatocellular carcinoma from the phase 3 CheckMate 459 trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2145.

The Use of Next-generation Sequencing in the Diagnosis of Rare Inherited Anaemias: A Joint BSH/EHA Good Practice Paper.

The Use of Next-generation Sequencing in the Diagnosis of Rare Inherited Anaemias: A Joint BSH/EHA Good Practice Paper.

23-OR: Loss of the Transcription Factor Tcf21 in Adult Podocytes Leads to Susceptibility in Diabetic Kidney Disease

Background: Diabetic kidney disease (DKD) is the most common cause of dialysis induction in the United States. Tcf21 is a basic helix-loop-helix transcription factor that is essential for the development of the lungs, heart, and kidneys. Previously, we reported that Tcf21 regulates podocyte development. However, the precise role of Tcf21 in mature individuals and DKD remains unclear. Method: We generated mice that lacked Tcf21 only in podocytes after birth (iKO) using a doxycycline induction system. We also constructed a diabetes mouse model using streptozotocin injections. In addition, we used cultured human podocytes to investigate the effects of Tcf21 overexpression and the factors that regulate Tcf21 expression. Results: When the Tcf21 gene was deleted specifically in podocytes in 3-week-old mice, urinary protein was not observed. In contrast, when diabetes was induced in these mice, massive urinary protein and strong glomerulosclerosis was present in 60% of iKO mice. Ultrastructural analysis revealed glomerular basement membrane thickening and podocyte foot process effacement, suggesting that iKO mice are susceptible to DKD. In addition, RNA-seq analysis of Tcf21-overexpressing podocytes showed accumulation of genes involved in the interferon pathway, extracellular matrix production, senescence, and autophagy in gene ontology analysis. Analysis of individual genes showed upregulation of interferon-related genes such as IFI6 and IFI27, increased expression of PAI-1, which is one of the senescence-associated secreted phenotypes, and decreased expression of HMGA2, whose SNIP is known to correlate with susceptibility to DKD. In addition, Tcf21 expression was suppressed by 58% and 27% after TGF-β and high glucose plus insulin stimulation, respectively. Conclusion: Our results suggest that Tcf21 protects against DKD in mature individuals. Elucidation of the mechanism by which Tcf21 prevents DKD may lead to the identification of novel therapeutic targets. Disclosure N.Teramoto: None. Y.Maezawa: None. T.Minamizuka: None. M.Koshizaka: None. K.Yokote: None.

Functional Analysis of Mmd2 and Related PAQR Genes During Sex Determination in Mice

Introduction: Sex determination in eutherian mammals is controlled by the Y-linked gene Sry, which drives the formation of testes in male embryos. Despite extensive study, the genetic steps linking Sry action and male sex determination remain largely unknown. Here, we focused on Mmd2, a gene that encodes a member of the progestin and adipoQ receptor (PAQR) family. Mmd2 is expressed during the sex-determining period in XY but not XX gonads, suggesting a specific role in testis development. Methods: We used CRISPR to generate mouse strains deficient in Mmd2 and its 2 closely related PAQR family members, Mmd and Paqr8, which are also expressed during testis development. Following characterization of Mmd2 expression in the developing testis, we studied sex determination in embryos from single knockout as well as Mmd2;Mmd and Mmd2;Paqr8 double knockout lines using quantitative RT-PCR and immunofluorescence. Results: Analysis of knockout mice deficient in Sox9 and Nr5a1 revealed that Mmd2 operates downstream of these known sex-determining genes. However, fetal testis development progressed normally in Mmd2-null embryos. To determine if other genes might have compensated for the loss of Mmd2, we analyzed Paqr8 and Mmd-null embryos and confirmed that in both knockout lines, sex determination occurred normally. Finally, we generated Mmd2;Mmd and Mmd2;Paqr8 double-null embryos and again observed normal testis development. Discussion: These results may reflect functional redundancy among PAQR factors, or their dispensability in gonadal development. Our findings highlight the difficulties involved in identifying genes with a functional role in sex determination and gonadal development through expression screening and loss-of-function analyses of individual candidate genes and may help to explain the paucity of genes in which variations have been found to cause human disorders/differences of sex development.

Abstract P5-13-18: Upregulation of immune response biomarkers by ribociclib plus endocrine therapy (ET) in paired tumor samples from phase I studies

Abstract Background: In addition to inhibiting cell cycle progression, CDK4/6 inhibitors have been found to modulate anti-cancer immune response (Goel S, et al. Nature. 2017;548:471-475). Data from preclinical studies suggested multiple mechanisms—involving changes in both cancer cells and immune cells—by which CDK4/6 inhibition can enhance anti-cancer immunity. These mechanisms include increased antigen presentation, interferon response triggering, and regulatory T-cell suppression. Clinical correlates to preclinical observations have so far been obtained using paired biopsies from neoadjuvant breast cancer trials (Johnston S, et al. J Clin Oncol. 2019;37:178-189; Hurvitz S, et al. Clin Cancer Res. 2020; ;26:566-80). Here, we describe a gene expression analysis of paired pre- and on-treatment biopsies from CLEE011A2115C and CLEE011X2107, two phase I clinical trials evaluating ribociclib in combination with ET in the metastatic breast cancer (MBC) setting. Methods: The nCounter PanCancer IO 360 Panel (NanoString) was used to quantify expression of 770 genes in 7 pairs of tumor samples (baseline vs. cycle 1 day 15) from two phase I trials in the MBC setting (5 patients from CLEE011A2115C and 2 from CLEE011X2107). All patients were treated with ribociclib and an ET. Pairwise differential gene expression analysis of individual genes and previously published immune-related gene signatures was conducted (Bedognetti D, et al. Curr Opin Oncol. 2015;27:433-444). Results: Many genes that were markedly suppressed with treatment (eg, MKI67, MYC, CDK2, CCNB1, TYMS, and DNMT1) were related to proliferation, DNA replication, and G1/S transition of the cell cycle, as expected. Interestingly, expression of numerous genes involved in immune response was increased. These were mostly interferon-regulated genes; particularly, a gene signature indicative of a T cell-inflamed tumor microenvironment (Ayers M, et al. J Clin Invest. 2017;127:2930-2940) was upregulated. Immune-related genes tended to preferentially increase with treatment in patients who later experienced a clinical response. Since bulk RNA from tumor biopsies was analyzed, we could not distinguish whether the observed expression changes reflected increased infiltration by immune cells or an endogenous interferon response in cancer cells. The latter could be triggered by DNMT1 suppression (as previously described), which then promotes T cell-mediated immunity by increasing antigen presentation and chemokine production in cancer cells. Conclusions: This analysis was conducted with a small sample size and is considered hypothesis generating; further investigation is needed. Cell cycle and proliferation markers showed robust suppression by ribociclib-based treatment regimens, as expected. Conversely, a notable increase in immune-related genes was detected that tended to occur preferentially in patients who later experienced more favorable clinical outcomes. Both changes occurred early (cycle 1 day 15). To our knowledge, this is the first clinical biomarker report of immune activation mediated by CDK4/6 inhibition in MBC. Citation Format: Dejan Juric, Chong Ma, Ralph Tiedt, Yoon-Sim Yap, Joanne Chiu, Pamela Munster, Roohi Ismail-Khan, Laura Garcia-Estevez, Ingrid A. Mayer, Carlos Becerra, Nadia Solovieff, Agnes Lteif, Faye Su, Yen-Shen Lu. Upregulation of immune response biomarkers by ribociclib plus endocrine therapy (ET) in paired tumor samples from phase I studies [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-18.

Protease-activated receptor-2 dependent and independent responses of bone cells to prostate cancer cell secretory products.

Metastatic prostate cancer lesions in the skeleton are frequently characterized by excessive formation of bone. Prostate cancer cells secrete factors, including serine proteases, that are capable of influencing the behavior of surrounding cells. Some of these proteases activate protease-activated receptor-2 (PAR2 ), which is expressed by osteoblasts (bone-forming cells) and precursors of osteoclasts (bone-resorbing cells). The aim of the current study was to investigate a possible role for PAR2 in regulating the behavior of bone cells exposed to metastatic prostate cancer cells. The effect of medium conditioned by the PC3, DU145, and MDA-PCa-2b prostate cancer cell lines was investigated in assays of bone cell function using cells isolated from wildtype and PAR2 -null mice. Osteoclast differentiation was assessed by counting tartrate-resistant acid phosphatase-positive multinucleate cells in bone marrow cultured in osteoclastogenic medium. Osteoblasts were isolated from calvariae of neonatal mice, and BrdU incorporation was used to assess their proliferation. Assays of alkaline phosphatase activity and quantitative PCR analysis of osteoblastic gene expression were used to assess osteoblast differentiation. Responses of osteoblasts to medium conditioned by MDA-PCa-2b cells were analyzed by RNAseq. Conditioned medium (CM) from all three cell lines inhibited osteoclast differentiation independently of PAR2 . Media from PC3 and DU145 cells had no effect on assays of osteoblast function. Medium conditioned by MDA-PCa-2b cells stimulated BrdU incorporation in both wildtype and PAR2 -null osteoblasts but increased alkaline phosphatase activity and Runx2 and Col1a1 expression in wildtype but not PAR2 -null cells. Functional enrichment analysis of RNAseq data identified enrichment of multiple gene ontology terms associated with lysosomal function in both wildtype and PAR2 -null cells in response to MDA-PCa-2b-CM. Analysis of individual genes identified osteogenesis-associated genes that were either upregulated by MDA-PCa-2b-CM selectively in wildtype cells or downregulated selectively in PAR2 -null cells. Factors secreted by prostate cancer cells influence bone cell behavior through both PAR2 -dependent and -independent mechanisms. Both PAR2 -independent suppression of osteoclast differentiation and PAR2 -dependent stimulation of osteogenesis are likely to determine the nature of prostate cancer metastases in bone.

Abstract PD6-05: Characterization of the immune landscape of primary vs metastatic breast cancer in a real-world data cohort

Abstract Breast cancer is considered to be a relatively immunologically “silent” tumor type. However, there is substantial heterogeneity in breast cancer, including differences in the immune microenvironment of tumors. For example, differences in the immune profile of primary and metastatic breast tumors have been observed. A deeper investigation of the immune profile of primary and metastatic breast tumors would enhance our understanding of late-stage breast cancer and provide insights that can guide the development of immunotherapies for primary or metastatic disease. In the current study, we explored differences in the immune microenvironment of primary and metastatic breast tumors in a clinico-genomic, real-world cohort (Tempus) of 420 subjects with breast cancer. Use of a real-world cohort for this study provided a diverse set of samples across subtypes, stages, and sites, and in particular a relatively large number of biopsies from metastatic sites as compared to other large breast cancer cohorts. Analyses were conducted using gene expression data generated by bulk RNA-sequencing of biopsies from lesions at primary (stage I-IV, N = 110) and metastatic (N = 310) sites, along with subject medical histories compiled from a variety of sources, including clinical records, pathology images, and lab reports. Differences in the immune microenvironment of assayed tumors were characterized by evaluating gene expression signatures representing immune-related processes as well as individual immune cell types, with emphasis on signatures known to be associated with response to checkpoint blockade, as well as differential expression analysis of individual genes. We observed higher expression of inflammation signatures in early stage and locally advanced primary tumors as compared to metastatic tumors (excluding lymph node biopsies), as well as enrichment of a number of immune cell types, including CD8+ T cells, regulatory T cells, B cells, and dendritic cells. We further examined individual metastatic sites (including lymph nodes, liver, brain, bone, and lung, among others), and noted variability in the immune contexture of metastatic tumors across sites. For example, we observed a trend to reduced inflammation and infiltration of certain cell types in brain and liver metastases as compared to primary tumors. Differential gene expression analysis of immune checkpoint molecules and other key immune-related genes was also conducted, revealing, for example, higher expression of IL-8 at certain metastatic sites; elevated IL-8 expression has been linked to reduced response to PD-1/PD-L1 agents and an immunosuppressive microenvironment. Finally, transcriptome-wide differential gene expression and gene set enrichment analyses were conducted using several gene-set and pathway databases (Gene Ontology, KEGG, Reactome, and Molecular Signatures Database hallmark collection). The results from these hypothesis-free analyses confirmed observations from the gene signature analyses described above; for example, inflammation-related pathways like the Hallmark “Interferon Gamma Response” and Gene Ontology “Regulation of Inflammatory Response” were enriched in primary tumors. Overall, the results of this study provide insights into the immune microenvironment of breast tumors and may be further used to inform the development of immune-oncology therapies in the space. Citation Format: Aparna Chhibber, Kim Van Naarden Braun, Celine Han, Han Chang, Mustimbo Roberts, Bin Li. Characterization of the immune landscape of primary vs metastatic breast cancer in a real-world data cohort [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD6-05.

Gene Expression Analysis in Stem Cell-derived Cortical Neuronal Cultures Using Multi-well SYBR Green Quantitative PCR Arrays.

To optimize differentiation protocols for stem cell-based in vitro modeling applications, it is essential to assess the change in gene expression during the differentiation process. This allows controlling its differentiation efficiency into the target cell types. While RNA transcriptomics provides detail at a larger scale, timing and cost are prohibitive to include such analyses in the optimization process. In contrast, expression analysis of individual genes is cumbersome and lengthy. Here, we developed a versatile and cost-efficient SYBR Green array of 27 markers along with two housekeeping genes to quickly screen for differentiation efficiency of human induced pluripotent stem cells (iPSCs) into excitatory cortical neurons. We first identified relevant pluripotency, neuroprogenitor, and neuronal markers for the array by literature search, and designed primers with a product size of 80-120 bp length, an annealing temperature of 60°C, and minimal predicted secondary structures. We spotted combined forward and reverse primers on 96-well plates and dried them out overnight. These plates can be prepared in advance in batches and stored at room temperature until use. Next, we added the SYBR Green master mix and complementary DNA (cDNA) to the plate in triplicates, ran quantitative PCR (qPCR) on a Quantstudio 6 Flex, and analyzed results with QuantStudio software. We compared the expression of genes for pluripotency, neuroprogenitor cells, cortical neurons, and synaptic markers in a 96-well format at four different time points during the cortical differentiation. We found a sharp reduction of pluripotency genes within the first three days of pre-differentiation and a steady increase of neuronal markers and synaptic markers over time. In summary, we built a gene expression array that is customizable, fast, medium-throughput, and cost-efficient, ideally suited for optimization of differentiation protocols for stem cell-based in vitro modeling.

Gene Expression Analysis in Stem Cell-derived Cortical Neuronal Cultures Using Multi-well SYBR Green Quantitative PCR Arrays

To optimize differentiation protocols for stem cell-based in vitro modeling applications, it is essential to assess the change in gene expression during the differentiation process. This allows controlling its differentiation efficiency into the target cell types. While RNA transcriptomics provides detail at a larger scale, timing and cost are prohibitive to include such analyses in the optimization process. In contrast, expression analysis of individual genes is cumbersome and lengthy.Here, we developed a versatile and cost-efficient SYBR Green array of 27 markers along with two housekeeping genes to quickly screen for differentiation efficiency of human induced pluripotent stem cells (iPSCs) into excitatory cortical neurons. We first identified relevant pluripotency, neuroprogenitor, and neuronal markers for the array by literature search, and designed primers with a product size of 80-120 bp length, an annealing temperature of 60°C, and minimal predicted secondary structures. We spotted combined forward and reverse primers on 96-well plates and dried them out overnight. These plates can be prepared in advance in batches and stored at room temperature until use. Next, we added the SYBR Green master mix and complementary DNA (cDNA) to the plate in triplicates, ran quantitative PCR (qPCR) on a Quantstudio 6 Flex, and analyzed results with QuantStudio software.We compared the expression of genes for pluripotency, neuroprogenitor cells, cortical neurons, and synaptic markers in a 96-well format at four different time points during the cortical differentiation. We found a sharp reduction of pluripotency genes within the first three days of pre-differentiation and a steady increase of neuronal markers and synaptic markers over time. In summary, we built a gene expression array that is customizable, fast, medium-throughput, and cost-efficient, ideally suited for optimization of differentiation protocols for stem cell-based in vitro modeling., [摘要] 为了优化基于干细胞的体外建模应用的分化方案，必须评估分化过程中基因表达的变化。这允许控制其分化为目标细胞类型的效率。虽然 RNA 转录组学提供了更大规模的细节，但在优化过程中包含此类分析的时间和成本令人望而却步。相比之下，单个基因的表达分析既繁琐又冗长。 在这里，我们开发了一个多功能且具有成本效益的 SYBR Green 阵列，该阵列包含 27 个标记以及两个管家基因，以快速筛选人类诱导多能干细胞 (iPSC) 分化为兴奋性皮层神经元的效率。我们首先通过文献检索确定了阵列的相关多能性、神经祖细胞和神经元标记，并设计了产物大小为 80-120 bp 长度、退火温度为 60°C 和最小预测二级结构的引物。我们在 96 孔板上发现了组合的正向和反向引物，并将它们干燥过夜。这些板可以提前分批制备，并在室温下储存直至使用。接下来，我们将 SYBR Green 预混液和互补 DNA (cDNA) 一式三份添加到板中，在Quantstudio 6 Flex 上运行定量 PCR (qPCR)，并使用QuantStudio 软件分析结果。 我们比较了皮层分化过程中四个不同时间点在 96 孔格式中多能性、神经祖细胞、皮层神经元和突触标记的基因表达。我们发现在预分化的前三天内多能性基因急剧减少，并且随着时间的推移神经元标志物和突触标志物稳定增加。总之，我们构建了一个可定制、快速、中等通量且具有成本效益的基因表达阵列，非常适合优化基于干细胞的体外建模的分化方案。[背景] 实时定量 PCR (qPCR) 技术检测靶核酸序列的扩增，被认为是灵敏的、可重复的和特异的(Arya et al ., 2005) 。 在这里，我们使用多孔 qPCR 分析通过 SYBR Green 技术扩增多个基因（Arikawa et al ., 2011） 。 SYBR Green 是一种 DNA 结合染料，可与双链 DNA (dsDNA) 非特异性结合(Boone et al ., 2015) 。 基于 RT 2 Profiler TM阵列(Arikawa et al ., 2011) ，我们设计了一个多孔 SYBR Green qPCR panel，通过强制表达 Neurogenin 2 来分析参与皮层神经元与人类 iPSCs 分化的基因的表达。 (Ngn2)，它是一种神经元转录因子，支持人类胚胎干细胞或 iPSCs 的神经元分化为皮质样神经元(Zhang et al ., 2013) 。在这里，我们使用了人类 iPSC 细胞系，该细胞系将多西环素诱导型小鼠 Ngn2 转基因工程化到安全港基因座中(Wang et al ., 2017) 。我们收集了不同时间点的细胞（iPSC、第0 天前神经元、第15天和第30 天皮质神经元），并在单个 96 孔板上分析了标记多能干细胞、中间神经祖细胞和成熟皮质神经元的基因。扩增子/引物设计范围为 80-120 bp，Tm 介于 63°C 和 66°C 之间。多孔 SYBR Green qPCR 检测的内部制备允许根据需要定制引物以适应特定实验和检测修改。这种多孔 SYBR Green qPCR 检测可用于对任何一组感兴趣的基因进行定量分析。 用于通路分析的不同 RT2 分析器阵列可商购获得，但是，它们都没有经过优化以跟踪源自 iPSC 的皮质神经元的成熟。我们表明，多孔 SYBR Green qPCR 很容易适应实验室的定制。它是一个经济的平台，非常适合优化体外干细胞建模的分化方案。

Identification of Diagnostic Markers Correlated With HIV+ Immune Non-response Based on Bioinformatics Analysis.

Background: HIV-infected immunological non-responders (INRs) are characterized by their inability to reconstitute CD4+ T cell pools after antiretroviral therapy. The risk of non-AIDS-related diseases in INRs is increased, and the outcome and prognosis of INRs are inferior to that of immunological responders (IRs). However, few markers can be used to define INRs precisely. In this study, we aim to identify further potential diagnostic markers associated with INRs through bioinformatic analyses of public datasets. Methods: This study retrieved the microarray data sets of GSE106792 and GSE77939 from the Gene Expression Omnibus (GEO) database. After merging two microarray data and adjusting the batch effect, differentially expressed genes (DEGs) were identified. Gene Ontology (GO) resource and Kyoto Encyclopedia of Genes and Genomes (KEGG) resource were conducted to analyze the biological process and functional enrichment. We performed receiver operating characteristic (ROC) curves to filtrate potential diagnostic markers for INRs. Gene Set Enrichment Analysis (GSEA) was conducted to perform the pathway enrichment analysis of individual genes. Single sample GSEA (ssGSEA) was performed to assess scores of immune cells within INRs and IRs. The correlations between the diagnostic markers and differential immune cells were examined by conducting Spearman’s rank correlation analysis. Subsequently, miRNA-mRNA-TF interaction networks in accordance with the potential diagnostic markers were built with Cytoscape. We finally verified the mRNA expression of the diagnostic markers in clinical samples of INRs and IRs by performing RT-qPCR. Results: We identified 52 DEGs in the samples of peripheral blood mononuclear cells (PBMC) between INRs and IRs. A few inflammatory and immune-related pathways, including chronic inflammatory response, T cell receptor signaling pathway, were enriched. FAM120AOS, LTA, FAM179B, JUN, PTMA, and SH3YL1 were considered as potential diagnostic markers. ssGSEA results showed that the IRs had significantly higher enrichment scores of seven immune cells compared with IRs. The miRNA-mRNA-TF network was constructed with 97 miRNAs, 6 diagnostic markers, and 26 TFs, which implied a possible regulatory relationship. Conclusion: The six potential crucial genes, FAM120AOS, LTA, FAM179B, JUN, PTMA, and SH3YL1, may be associated with clinical diagnosis in INRs. Our study provided new insights into diagnostic and therapeutic targets.

Transcriptomic analysis of human skin wound healing and rejuvenation following ablative fractional laser treatment.

Ablative fractional laser treatment is considered the gold standard for skin rejuvenation. In order to understand how fractional laser works to rejuvenate skin, we performed microarray profiling on skin biopsies to identify temporal and dose-response changes in gene expression following fractional laser treatment. The backs of 14 women were treated with ablative fractional laser (Fraxel®) and 4 mm punch biopsies were collected from an untreated site and at the treated sites 1, 3, 7, 14, 21 and 28 days after the single treatment. In addition, in order to understand the effect that multiple fractional laser treatments have on skin rejuvenation, several sites were treated sequentially with either 1, 2, 3, or 4 treatments (with 28 days between treatments) followed by the collection of 4 mm punch biopsies. RNA was extracted from the biopsies, analyzed using Affymetrix U219 chips and gene expression was compared between untreated and treated sites. We observed dramatic changes in gene expression as early as 1 day after fractional laser treatment with changes remaining elevated even after 1 month. Analysis of individual genes demonstrated significant and time related changes in inflammatory, epidermal, and dermal genes, with dermal genes linked to extracellular matrix formation changing at later time points following fractional laser treatment. When comparing the age-related changes in skin gene expression to those induced by fractional laser, it was observed that fractional laser treatment reverses many of the changes in the aging gene expression. Finally, multiple fractional laser treatments, which cover different regions of a treatment area, resulted in a sustained or increased dermal remodeling response, with many genes either differentially regulated or continuously upregulated, supporting previous observations that maximal skin rejuvenation requires multiple fractional laser treatments. In conclusion, fractional laser treatment of human skin activates a number of biological processes involved in wound healing and tissue regeneration.

T2-DAG: a powerful test for differentially expressed gene pathways via graph-informed structural equation modeling.

A major task in genetic studies is to identify genes related to human diseases and traits to understand functional characteristics of genetic mutations and enhance patient diagnosis. Compared with marginal analyses of individual genes, identification of gene pathways, i.e. a set of genes with known interactions that collectively contribute to specific biological functions, can provide more biologically meaningful results. Such gene pathway analysis can be formulated into a high-dimensional two-sample testing problem. Given the typically limited sample size of gene expression datasets, most existing two-sample tests tend to have compromised powers because they ignore or only inefficiently incorporate the auxiliary pathway information on gene interactions. We propose T2-DAG, a Hotelling's T2-type test for detecting differentially expressed gene pathways, which efficiently leverages the auxiliary pathway information on gene interactions from existing pathway databases through a linear structural equation model. We further establish its asymptotic distribution under pertinent assumptions. Simulation studies under various scenarios show that T2-DAG outperforms several representative existing methods with well-controlled type-I error rates and substantially improved powers, even with incomplete or inaccurate pathway information or unadjusted confounding effects. We also illustrate the performance of T2-DAG in an application to detect differentially expressed KEGG pathways between different stages of lung cancer. The R (R Development Core Team, 2021) package T2DAG which implements the proposed T2-DAG test is available on Github at https://github.com/Jin93/T2DAG. Supplementary data are available at Bioinformatics online.

Chronic liquid fructose supplementation does not cause liver tumorigenesis but elicits clear sex differences in the metabolic response in Sprague-Dawley rats.

BackgroundNon-alcoholic fatty liver disease (NAFLD) has increased over the last decades and may evolve into hepatocellular carcinoma (HCC). As HCC is challenging to treat, knowledge on the modifiable risk factors for NAFLD/HCC (e.g. hyper caloric diets rich in fructose) is essential.Objective and designWe used a model of diethyl nitrosamine-induced hepatocarcinogenesis to investigate the liver cancer-promoting effects of a diet supplemented with 10% liquid fructose, administered to male and female rats for 11 months. A subset of the fructose-supplemented rats received resveratrol (RVT) in the last 4 months of treatment.Results and discussionRat livers showed no de visu or histological evidence of liver tumorigenesis. However, we observed metabolic abnormalities that could be related to cancer development mainly in the female fructose-supplemented rats, such as increases in weight, adiposity and hepatic triglyceride levels, as well as hyperglycaemia, hyperuricemia, hyperleptinemia and a reduced insulin sensitivity index, which were partially reversed by RVT. Therefore, we performed a targeted analysis of 84 cancer-related genes in the female liver samples, which revealed expression changes associated with cancer-related pathways. Analysis of individual genes indicated that some changes increased the risk of hepatocarcinogenesis (Sfrp2, Ccl5, Socs3, and Gstp1), while others exerted a protective/preventive effect (Bcl2 and Cdh1).ConclusionOur data clearly demonstrate that chronic fructose supplementation, as the sole dietary intervention, does not cause HCC development in rats.

Evolutionary study of maize dwarf mosaic virus using nearly complete genome sequences acquired by next-generation sequencing

Next-generation sequencing is a robust approach to sequence plant virus genomes in a very short amount of time compared to traditional sequencing methods. Maize dwarf mosaic virus (MDMV) is one of the most important plant viruses worldwide and a significant threat to maize production. In this study, we sequenced 19 MDMV isolates (10 from Johnsongrass and 9 from maize) collected in Oklahoma and Missouri during 2017–2019 using Illumina sequencing and determined the genetic diversity. Sequence reads were assembled and 19 nearly complete genome sequences of MDMV isolates were obtained. Phylogenetic analysis based on complete genomes nucleotide and amino acid sequences revealed two main clusters and a close evolutionary relationship among 19 MDMV isolates. Statistical analysis of individual genes for site-specific selection revealed that all genes are under negative selection. The fixation index (FST) analysis of the MDMV isolates revealed no gene flow between the two main phylogenetic clusters, which emphasizes the divergence of MDMV isolates from the USA. Among the USA MDMV isolates, the mean genetic distance (d) and nucleotide diversity ((π) were highest in the P1 gene coding region. This is the first detailed study on the evolutionary relationship of MDMV isolates based on the nearly complete genome analysis from maize and Johnsongrass.

Salivary DNA Methylation as an Epigenetic Biomarker for Head and Neck Cancer. Part I: A Diagnostic Accuracy Meta-Analysis.

DNA hypermethylation is an important epigenetic mechanism for gene expression inactivation in head and neck cancer (HNC). Saliva has emerged as a novel liquid biopsy representing a potential source of biomarkers. We performed a comprehensive meta-analysis to evaluate the overall diagnostic accuracy of salivary DNA methylation for detecting HNC. PubMed EMBASE, Web of Science, LILACS, and the Cochrane Library were searched. Study quality was assessed by the Quality Assessment for Studies of Diagnostic Accuracy-2, and sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (dOR), and their corresponding 95% confidence intervals (CIs) were calculated using a bivariate random-effect meta-analysis model. Meta-regression and subgroup analyses were performed to assess heterogeneity. Eighty-four study units from 18 articles with 8368 subjects were included. The pooled sensitivity and specificity of salivary DNA methylation were 0.39 and 0.87, respectively, while PLR and NLR were 3.68 and 0.63, respectively. The overall area under the curve (AUC) was 0.81 and the dOR was 8.34. The combination of methylated genes showed higher diagnostic accuracy (AUC, 0.92 and dOR, 36.97) than individual gene analysis (AUC, 0.77 and dOR, 6.02). These findings provide evidence regarding the potential clinical application of salivary DNA methylation for HNC diagnosis.

Phylogeny of the supertribe Nebriitae (Coleoptera, Carabidae) based on analyses of DNA sequence data.

The phylogeny of the carabid beetle supertribe Nebriitae is inferred from analyses of DNA sequence data from eight gene fragments including one nuclear ribosomal gene (28S), four nuclear-protein coding genes (CAD, topoisomerase 1, PEPCK, and wingless), and three mitochondrial gene fragments (16S + tRNA-Leu + ND1, COI (“barcode” region) and COI (“Pat/Jer” region)). Our taxon sample included 264 exemplars representing 241 species and subspecies (25% of the known nebriite fauna), 39 of 41 currently accepted genera and subgenera (all except Notiokasis and Archileistobrius), and eight outgroup taxa. Separate maximum likelihood (ML) analyses of individual genes, combined ML analyses of nuclear, nuclear protein-coding, and mitochondrial genes, and combined ML and Bayesian analyses of the eight-gene-fragment matrix resulted in a well-resolved phylogeny of the supertribe, with most nodes in the tree strongly supported. Within Nebriitae, 167 internal nodes of the tree (out of the maximum possible 255) are supported by maximum-likelihood bootstrap values of 90% or more. The tribes Notiophilini, Opisthiini, Pelophilini, and Nebriini are well supported as monophyletic but relationships among these are not well resolved. Nippononebria is a distinct genus more closely related to Leistus than Nebria. Archastes, Oreonebria, Spelaeonebria, and Eurynebria, previously treated as distinct genera by some authors, are all nested within a monophyletic genus Nebria. Within Nebria, four major clades are recognized: (1) the Oreonebria Series, including eight subgenera arrayed in two subgeneric complexes (the Eonebria and Oreonebria Complexes); (2) the Nebriola Series, including only subgenus Nebriola; (3) the Nebria Series, including ten subgenera arrayed in two subgeneric complexes, the Boreonebria and Nebria Complexes, with the latter further subdivided into three subgeneric subcomplexes (the Nebria, Epinebriola, and Eunebria Subcomplexes)); and (4) the Catonebria Series, including seven subgenera arrayed in two subgeneric complexes (the Reductonebria and Catonebria Complexes). A strong concordance of biogeography with the inferred phylogeny is noted and some evident vicariance patterns are highlighted. A revised classification, mainly within the Nebriini, is proposed to reflect the inferred phylogeny. Three genus-group taxa (Nippononebria, Vancouveria and Archastes) are given revised status and seven are recognized as new synonymies (Nebriorites Jeannel, 1941 and Marggia Huber, 2014 = Oreonebria Daniel, 1903; Pseudonebriola Ledoux & Roux, 1989 = Boreonebria Jeannel, 1937; Patrobonebria Bänninger, 1923, Paranebria Jeannel, 1937 and Barbonebriola Huber & Schmidt, 2017 = Epinebriola Daniel & Daniel, 1904; and Asionebria Shilenkov, 1982 = Psilonebria Andrewes, 1923). Six new subgenera are proposed and described for newly recognized clades: Parepinebriola Kavanaugh subgen. nov. (type species: Nebria delicata Huber & Schmidt, 2017), Insulanebria Kavanaugh subgen. nov. (type species: Nebria carbonaria Eschscholtz, 1829), Erwinebria Kavanaugh subgen. nov. (type species Nebria sahlbergii Fischer von Waldheim, 1828), Nivalonebria Kavanaugh subgen. nov. (type species: Nebria paradisi Darlington, 1931), Neaptenonebria Kavanaugh subgen. nov. (type species: Nebria ovipennis LeConte, 1878), and Palaptenonebria Kavanaugh subgen. nov. (type species: Nebria mellyi Gebler, 1847). Future efforts to better understand relationships within the supertribe should aim to expand the taxon sampling of DNA sequence data, particularly within subgenera Leistus and Evanoleistus of genus Leistus and the Nebria Complex of genus Nebria.

Mitochondrial genome announcements need to consider existing short sequences from closely related species to prevent taxonomic errors

The reconstruction of complete mitochondrial genomes (mitogenomes) has considerable potential to clarify species relationships in cases where morphological analysis and DNA sequencing of individual genes are inconclusive. However, the trend to use only mitogenomes for the phylogenies presented in mitogenome announcements carries the inherent risk that the study species’ taxonomy is incorrect because no mitogenomes have yet been reconstructed for its sister species. Here, I illustrate this problem using the mitogenomes of two seahorses, Hippocampus capensis and H. queenslandicus. Both specimens used for mitogenome reconstruction originated from traditional Chinese medicine markets rather than native habitats. Although mitogenome phylogenies placed these specimens correctly among the seahorses from which mitogenomes were available at the time, incorporating single-marker sequence from closely related species into the phylogenies revealed that both mitogenomes are problematic. The mitogenome of the endemic South African H. capensis did not cluster among single-marker DNA sequences of seahorses from the species’ native habitat, but among sequences submitted under the names H. casscsio, H. fuscus and H. kuda that originated from all over the Indo-Pacific, including China. Phylogenetic placement of the mitogenome of H. queenslandicus within a cluster of seahorses that also included H. spinosissimus confirms an earlier finding that H. queenslandicus is a synonym of H. spinosissimus, a widespread Indo-Pacific species that also occurs in China. It is recommended that mitogenome announcements incorporate available single-marker sequences of closely related species, not only mitogenomes. The reconstruction of mitogenomes can exacerbate taxonomic confusion if existing information is ignored.

Abstract PO-059: Investigating gene expression profiles associated with clinical radiation resistance in KEAP1/NFE2L2 wildtype lung cancer

Abstract Background: We previously reported that approximately half of local recurrences (LR) after radiotherapy for localized NSCLC harbor mutations in KEAP1 or NFE2L2. Here we sought to explore factors associated with LR after radiotherapy in KEAP1/NFE2L2 wildtype NSCLC. Methods: We identified consecutive patients with stage IA1-IIIC NSCLC treated at our institution with chemoradiotherapy (CRT) or stereotactic ablative radiotherapy (SABR) from 2009-2018 and who had genotyping performed on tumor tissue with full coverage of common recurrent lung cancer driver genes including TP53, KRAS, KEAP1, and NFE2L2. We defined LR as tumor regrowth within the prescription dose radiotherapy volume. Our primary objective was to identify factors associated with LR in KEAP1/NFE2L2WT tumors. We performed RNA-seq on a subset of cases with available tissue using the SMARTer Stranded total RNA-seq Kit v2 (Takara Bio USA, Inc.). Statistical analysis was performed using R (version 3.6) with differential gene expression performed using ‘DESeq2’. All P-values were two-sided and considered significant at P<0.05 with adjustment for multiple hypothesis testing when appropriate. Results: We identified 139 consecutive patients with localized NSCLC who received tumor genotyping and were treated with CRT for stage IIB-IIIC NSCLC (n=58) or SABR for stage I-IIB NSCLC (n=81). 26 (18.7%) of these patients harbored KEAP1/NFE2L2WT tumors. Clinical factors such as tumor volume (P=0.18), histology (P=0.87), and radiation dose (P=0.3) were not associated with LR in this subset. Similarly, somatic mutation analysis did not reveal association of any recurrent driver mutations with LR in these KEAP1/NFE2L2WT tumors, including in TP53 (n=19; P=0.73) or KRAS (n=9; P=0.98). Tissue was available for RNA-seq analysis of 38 KEAP1/NFE2L2WT tumors, of which the majority were adenocarcinomas (n=25, 65.8%) and approximately half each received CRT (n=20, 52.6%) and SABR (n=18, 47.4%). Gene set enrichment analysis revealed a trend for association of LR with expression of hypoxia genes (P=0.07, Q=0.28). Similarly, a previously reported 10-gene radiation sensitivity index (RSI) was not associated with LR (P=0.34). Individual gene analysis identified KRT14 as being significantly less expressed in cases with LR (adjusted P=2.2e-9). In a validation cohort of 24 stage I-IIA KEAP1/NFE2L2WT patients from the TCGA who were treated with radiotherapy, those who died had lower expression of KRT14 than those who did not (P=0.0003). Conclusions: In summary, we identify low expression of KRT14, a squamous cell carcinoma differentiation gene, as a potential biomarker for increased risk of LR after definitive radiotherapy of KEAP1/NFE2L2WT NSCLC. Validation in larger cohorts and biological characterization will be required to determine if this biomarker could be useful for guiding precision radiotherapy approaches. Citation Format: Michael S. Binkley, Young-Jun Jeon, Monica Nesselbush, Everett J. Moding, Barzin Nabet, Diego Almanza, Christopher Yoo, David M. Kurtz, Susie Grant Owen, Leah M. Backhus, Mark F. Berry, Joseph B. Shrager, Kavitha J. Ramchandran, Sukhmani K. Padda, Millie Das, Joel W. Neal, Heather A. Wakelee, Ash A. Alizadeh, Billy W. Loo, Maximilian Diehn. Investigating gene expression profiles associated with clinical radiation resistance in KEAP1/NFE2L2 wildtype lung cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-059.

SLC39A8/Zinc Suppresses the Progression of Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most frequent and lethal subtype, which has high risk of metastasis or recurrence, accounting for 75–83% of renal cell carcinoma (RCC). Zrt‐ and Irt‐like proteins (ZIP) family members (SLC39A1-14) function to pass zinc into the cytoplasm for many critical biological processes when cellular zinc is depleted. However, the functional analysis of individual ZIP family genes in ccRCC is not clarified. This study aimed to investigate whether ZIP family genes are related to the clinicopathological features and survival of ccRCC patients, and to identify the function of key gene of ZIP family in ccRCC in vitro. Through bioinformatics analysis of tumor databases, SLC39A8 was identified as a key gene of ZIP family in ccRCC, which could be used as an effective indicator for diagnosing ccRCC and judging its prognosis. With the progression of tumor, the expression of SLC39A8 decreased progressively. The prognosis of patients with low expression of SLC39A8 is significantly worse. Furthermore, we found that overexpression of SLC39A8 or treatment with low concentration of zinc chloride could effectively inhibit the proliferation, migration and invasion of ccRCC cells. Moreover, the inhibition effect of SLC39A8 overexpression could be enhanced by low concentration zinc supplement. Therefore, this study provides a novel understanding for the role of SLC39A8/zinc in the regulation of ccRCC progression. These findings provide a new direction and target for progressive ccRCC drug development and combination therapy strategies.

Ex vivo visualization of RNA polymerase III-specific gene activity with electron microscopy

The direct study of transcription or DNA–protein-binding events, requires imaging of individual genes at molecular resolution. Electron microscopy (EM) can show local detail of the genome. However, direct visualization and analysis of specific individual genes is currently not feasible as they cannot be unambiguously localized in the crowded, landmark-free environment of the nucleus. Here, we present a method for the genomic insertion of gene clusters that can be localized and imaged together with their associated protein complexes in the EM. The method uses CRISPR/Cas9 technology to incorporate several genes of interest near the 35S rRNA gene, which is a frequently occurring, easy-to-identify genomic locus within the nucleolus that can be used as a landmark in micrographs. As a proof of principle, we demonstrate the incorporation of the locus-native gene RDN5 and the locus-foreign gene HSX1. This led to a greater than 7-fold enrichment of RNA polymerase III (Pol III) complexes associated with the genes within the field of view, allowing for a significant increase in the analysis yield. This method thereby allows for the insertion and direct visualization of gene clusters for a range of analyses, such as changes in gene activity upon alteration of cellular or external factors.