Leading Edge Analysis Research Articles

Front Line of Analytical Techniques Supporting Polymer Sciences: Special Issue on Leading-edge Analysis of Spectroscopy

Front Line of Analytical Techniques Supporting Polymer Sciences: Special Issue on Leading-edge Analysis of Spectroscopy

Thermo-Catalytic Decomposition Comparisons: Carbon Catalyst Structure, Hydrocarbon Feed and Regeneration

Thermo-catalytic decomposition (TCD) activity and stability depend upon the initial carbon catalyst structure. However, further transitions in the carbon structure depend on the carbon material (structure and composition) originating from the TCD process. In this article, reaction data are presented that illustrates the time-dependent TCD activity as TCD-formed carbon contributes and then dominates conversion. A variety of initial carbon catalysts are compared, including sugar char, a conductive carbon black (AkzoNobel Ketjenblack), a rubber-grade carbon black (Cabot R250), and its graphitized analogue as formed and partially oxidized. Regeneration of carbon catalysts by partial oxidation is evaluated using nascent carbon black as a model, coupled with subsequent comparative TCD performance relative to the nascent, non-oxidized carbon black. Activation energies for TCD with nascent and oxidized carbons are evaluated by a leading-edge analysis method applied to TCD. Given the correlation between nanostructure and active sites, two additional carbons, engine soots, are evaluated for regeneration and dependence upon nanostructure. Active sites are quantified by oxygen chemisorption, followed by X-ray photoelectron spectroscopy (XPS). The structure of carbon catalysts is assessed pre- and post-TCD by high-resolution transmission electron microscopy (HRTEM). Last, energy dispersive X-ray analysis mapping (EDS) is carried out for its potential to visualize oxygen chemisorption.

Altered Peripheral Blood Gene Expression in Childhood Cancer Survivors With Anthracycline-Induced Cardiomyopathy - A COG-ALTE03N1 Report.

Background Anthracycline-induced cardiomyopathy is a leading cause of premature death in childhood cancer survivors, presenting a need to understand the underlying pathogenesis. We sought to examine differential blood-based mRNA expression profiles in anthracycline-exposed childhood cancer survivors with and without cardiomyopathy. Methods and Results We designed a matched case-control study (Children's Oncology Group-ALTE03N1) with mRNA sequencing on total RNA from peripheral blood in 40 anthracycline-exposed survivors with cardiomyopathy (cases) and 64 matched survivors without (controls). DESeq2 identified differentially expressed genes. Ingenuity Pathway Analyses (IPA) and Gene Set Enrichment Analyses determined the potential roles of altered genes in biological pathways. Functional validation was performed by gene knockout in human-induced pluripotent stem cell-derived cardiomyocytes using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) technology. Median age at primary cancer diagnosis for cases and controls was 8.2 and 9.7 years, respectively. Thirty-six differentially expressed genes with fold change ≥±2 were identified; 35 were upregulated. IPA identified "hepatic fibrosis" and "iron homeostasis" pathways to be significantly modulated by differentially expressed genes, including toxicology functions of myocardial infarction, cardiac damage, and cardiac dilation. Leading edge analysis from Gene Set Enrichment Analyses identified lactate dehydrogenase A (LDHA) and cluster of differentiation 36 (CD36) genes to be significantly upregulated in cases. Interleukin 1 receptor type 1, 2 (IL1R1, IL1R2), and matrix metalloproteinase 8, 9 (MMP8, MMP9) appeared in multiple canonical pathways. LDHA-knockout human-induced pluripotent stem cell-derived cardiomyocytes showed increased sensitivity to doxorubicin. Conclusions We identified differential mRNA expression profiles in peripheral blood of anthracycline-exposed childhood cancer survivors with and without cardiomyopathy. Upregulation of LDHA and CD36 genes suggests metabolic perturbations in a failing heart. Dysregulation of proinflammatory cytokine receptors IL1R1 and IL1R2 and matrix metalloproteinases, MMP8 and MMP9 indicates structural remodeling that accompanies the clinical manifestation of symptomatic cardiotoxicity.

Identification of Potential Drug Targets in Erythrocyte Invasion Pathway of Plasmodium falciparum.

The erythrocyte invasion phase plays a critical role in multiplication, sexual determination, and drug resistance in Plasmodium falciparum. In order to identify the critical genes and pathways in the erythrocyte invasion phase, the gene set (GSE129949) and the RNA-Seq count data for the W2mef strain were used for further analysis. An integrative bioinformatics study was performed to scrutinize genes as potential drug targets. 487 differentially expressed genes (DEGs) with an adjusted P value < 0.001 enriched 47 Gene Ontology (GO) terms that were over-represented based on hyper-geometric analysis P value < 0.01. Protein-Protein interaction network analysis was done using DEGs with higher confidence interactions (PPI score threshold = 0.7). MCODE and cytoHubba apps were utilized to define the hub proteins and rank them based on multiple topological analyses and MCODE scores. Furthermore, Gene Set Enrichment Analysis (GSEA) was carried out by using 322 gene sets from the MPMP database. The genes involved in multiple significant gene sets were determined by leading-edge analysis. Our study identified six genes encoding proteins that could be potential drug targets involved in the erythrocyte invasion phase related to merozoites motility, cell-cycle regulation, G-dependent protein kinase phosphorylation in schizonts, control of microtubule assembly, and sexual commitment. The druggability of those proteins was calculated based on the DCI (Drug Confidence Index) and predicted binding pockets' values. The protein that showed the best binding pocket value was subjected to deep learning-based virtual screening. The study identified the best small molecule inhibitors in terms of drug-binding score against the proteins for inhibitor identification.

Microarray profiling of hypothalamic gene expression changes in Huntington's disease mouse models.

Structural changes and neuropathology in the hypothalamus have been suggested to contribute to the non-motor manifestations of Huntington's disease (HD), a neurodegenerative disorder caused by an expanded cytosine-adenine-guanine (CAG) repeat in the huntingtin (HTT) gene. In this study, we investigated whether hypothalamic HTT expression causes transcriptional changes. Hypothalamic RNA was isolated from two different HD mouse models and their littermate controls; BACHD mice with ubiquitous expression of full-length mutant HTT (mHTT) and wild-type mice with targeted hypothalamic overexpression of either wild-type HTT (wtHTT) or mHTT fragments. The mHTT and wtHTT groups showed the highest number of differentially expressed genes compared to the BACHD mouse model. Gene Set Enrichment Analysis (GSEA) with leading-edge analysis showed that suppressed sterol- and cholesterol metabolism were shared between hypothalamic wtHTT and mHTT overexpression. Most distinctive for mHTT overexpression was the suppression of neuroendocrine networks, in which qRT-PCR validation confirmed significant downregulation of neuropeptides with roles in feeding behavior; hypocretin neuropeptide precursor (Hcrt), tachykinin receptor 3 (Tacr3), cocaine and amphetamine-regulated transcript (Cart) and catecholamine-related biological processes; dopa decarboxylase (Ddc), histidine decarboxylase (Hdc), tyrosine hydroxylase (Th), and vasoactive intestinal peptide (Vip). In BACHD mice, few hypothalamic genes were differentially expressed compared to age-matched WT controls. However, GSEA indicated an enrichment of inflammatory- and gonadotropin-related processes at 10 months. In conclusion, we show that both wtHTT and mHTT overexpression change hypothalamic transcriptome profile, specifically mHTT, altering neuroendocrine circuits. In contrast, the ubiquitous expression of full-length mHTT in the BACHD hypothalamus moderately affects the transcriptomic profile.

Abstract 1321: Effects of sulindac on inflammatory and cellular responses in mammary tumors from obese mice

Abstract Background: Breast cancer is the second leading cause of cancer-related death in US women. Obesity (BMI &amp;gt; 30 kg/m2) promotes breast cancer incidence, progression, and mortality in women of all ages. The prevalence of obesity in the US has increased dramatically over recent decades and continues to rise. Although obesity is associated with increased adipose tissue inflammation and metabolic dysfunction the exact mechanisms regulating obesity-exacerbated tumor growth are relatively unknown. Nonsteroidal anti-inflammatory drugs (NSAIDs), like sulindac, inhibit cyclooxygenase-1 and cyclooxygenase-2 and modulate the secretion of pro-inflammatory cytokines. The purpose of this study was to investigate the mechanisms underlying the anticancer effects of sulindac in our E0771 mouse model of obesity and mammary tumor growth. Methods: C57BL/6J mice were fed either a high-fat diet (n=16) or a low-fat control diet (n=12) for 15 weeks. Half of the control (n=6) and DIO (n=8) mice were then treated with 140 ppm sulindac in their respective diets for 4 weeks prior to orthotopic injection of 20,000 E0771 cells. Tumors were harvested and weighed, and serum was collected 4 weeks following injection. Bulk transcriptional profile analysis was performed on E0771 tumors from untreated control and DIO mice relative to sulindac-treated control and DIO mice using Affymetrix microarrays followed by gene set enrichment analysis (GSEA) to identify significantly enriched gene sets. Serum cytokine levels were measured in all mice using the Bio-Plex Pro mouse chemokine panel 33-plex; multiple linear regression was conducted to determine cytokines associated with mammary tumor size, independent of diet or treatment group assignment. Results: DIO mice had increased tumor weight relative to control mice, while sulindac decreased tumor weight in DIO but not control mice. GSEA analysis revealed only one gene set upregulated in the DIO tumors relative to control or DIO sulindac tumors. Immune-related gene sets were commonly enriched in tumors from control and DIO sulindac relative to DIO mice, suggesting that sulindac restored immune signaling in tumors from DIO mice. Leading edge analysis of the significantly enriched gene sets showed that: 1) the reversal of DIO-mediated immunosuppression by sulindac occurred via principally overlapping genes; and 2) cytokine and immune genes predominate the transcriptional effects of sulindac treatment in DIO mice. Sulindac also modified circulating serum cytokine levels, but multiple linear regression showed that cytokine levels were not independently predictive of tumor weight in our study. Conclusions: The study suggests that sulindac can reverse transcriptional changes in mammary tumors from obese mice and restore immunosurveillance. Sulindac, or potentially other NSAIDs, may protect against obesity–driven mammary tumor growth via remodeling of antitumor immune function. Citation Format: Morgan E. Cody, Shannon B. McDonell, Qing Shi, Elaine M. Glenny, Michael F. Coleman, Stephen D. Hursting. Effects of sulindac on inflammatory and cellular responses in mammary tumors from obese mice [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 1321.

Abstract LB107: Novel transcriptional signatures associated with antitumor activity in vidutolimod (vidu)-treated patients (pts) with anti-PD-1-refractory melanoma and non-small cell lung cancer (NSCLC)

Abstract Background: Vidu is a first-in-class CpG-A TLR9 agonist in a virus-like particle that activates plasmacytoid dendritic cells (pDC), thus bridging innate and adaptive immunity. Intratumoral (IT) vidu alone or in combination with intravenous (IV) anti-PD-(L)1 has shown evidence of antitumor activity in pts with anti-PD-(L)1-refractory melanoma or NSCLC. As biomarkers associated with anti-PD-(L)1 response were not predictive of vidu activity, we sought to identify novel transcriptional signatures. Methods: RNA-Seq was performed on baseline biopsies from pts with anti-PD-(L)1-refractory melanoma (NCT02680184; RECIST v1.1 responders [R; n=20]; nonresponders [NR; n=78; 30 pts with stable disease + 48 pts with progressive disease [PD]]) or NSCLC (NCT03438318; N=11) treated with vidu ± anti-PD-(L)1. IFNg18 signature was used to characterize melanomas as non-T cell-inflamed (non-Tinfl), T cell-inflamed (Tinfl), or intermediate. Gene Set Enrichment Analysis (GSEA) using MSigDB and other signatures (&amp;gt;19,000) was performed on non-Tinfl melanoma in R vs NR (false discovery rate [FDR] &amp;lt;0.25). Deconvolution of immune cells was performed using TIMER2.0. Prediction models were generated using QLattice. Signatures were characterized using both publicly available bulk and single-cell (sc) RNA-Seq datasets of pDC subsets or PD-1/CTLA-4 blockade response datasets. Results: GSEA on non-Tinfl melanoma biopsies revealed 2 gene signatures (COPII vesicle and Golgi targeting) most strongly associated with R (FDR &amp;lt;0.16). Leading edge analysis of these signatures identified 35 common core genes (CC) that strongly differentiated RECIST 1.1 R vs PD. As independent validation, CC enrichment was also significantly associated with R in intermediate melanoma (p=0.009) and with tumor shrinkage in NSCLC (p=0.027). CC was significantly associated with R to vidu single-agent or combination treatment, but not with clinical baseline prognostic factors or IFNg18. In Tinfl melanoma, CC was not associated with R, but myeloid signatures were significantly associated with NR. A model based on CC and transcription factor ELF2 predicted R in melanoma (AUC 0.93 [95% CI 0.82-1.00]). In public datasets, CC was not associated with R to PD-1/CTLA-4 blockade, but CC was highly expressed in the type I interferon-secreting subset of pDCs and some myeloid cells. CC expression was prevalent in most tumor types and was independent of IFNg18 in TCGA. Conclusion: In pts with anti-PD-1-refractory melanoma or NSCLC, transcriptional signatures of COPII vesicle and Golgi targeting (functionally related to TLR9 activation) were associated with antitumor activity of IT vidu ± IV anti-PD-(L)1. Ongoing bulk and sc RNA-Seq analyses may clarify the underlying biology and the signature’s potential role as a predictive biomarker for clinical response to vidu. Citation Format: Hong Liu, Luping Zhao, Ping Zheng, Riyue Bao, Jason J. Luke, Marcelo V. Negrao, Shakoora A. Sabree, George J. Weiner, Sujatha Kumar, Dmitri Bobilev, James E. Wooldridge, Arthur M. Krieg. Novel transcriptional signatures associated with antitumor activity in vidutolimod (vidu)-treated patients (pts) with anti-PD-1-refractory melanoma and non-small cell lung cancer (NSCLC) [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 LB107.

Abstract 14: Resolution of obesity-driven proinflammatory adipose signaling by weight loss or an NSAID is associated with reduced tumor burden in a mouse model of colon cancer

Abstract Obesity increases the incidence and progression of colon cancer. The chronic low-grade inflammation that often accompanies obesity is purported to promote these adverse outcomes. In obesity, remodeling of visceral white adipose tissue contributes to an inflammatory state via increased immune cell recruitment and production of proinflammatory cytokines. The aim of this study in a mouse model of obesity and colon carcinogenesis was to compare the effects of weight loss versus treatment with a nonsteroidal anti-inflammatory drug (sulindac) on tumor development and adipose and tumoral transcriptional changes. Azoxymethane (10 mg/kg) was intraperitoneally injected weekly into FVB/N male mice for 5 weeks to induce colon tumors. Mice were then randomized to an ad libitum 10 kcal% fat diet (control, n=20) or 60 kcal% fat diet to promote diet-induced obesity (DIO, n=55). After 15 weeks, DIO mice were randomized again to receive for an additional 7 weeks either: a) DIO diet (n=18); b) DIO diet supplemented with 140ppm sulindac (DIO+Su, n=19), or c) 10 kcal% fat control diet to generate formerly obese (FOb-LFD) mice (n=18). Mesenteric fat and colon tumors were collected for an Affymetrix Clariom D mouse gene expression microarray (n=5-6/group). FOb-LFD mice, but not DIO+Su mice, lost significant body weight and body fat. Relative to DIO mice, tumor burden and multiplicity were lower in control, DIO+Su, and FOb-LFD mice. Gene set enrichment analysis (GSEA) using the Hallmark gene sets revealed strikingly concordant transcriptional changes in the control, DIO+Su, and FOb-LFD mesenteric fat depots compared with DIO mice. Of the 8 immune related gene sets, 7 were uniformly suppressed (Normalized enrichment score (NES) &amp;gt;1.66, FDR q&amp;lt;0.05) across all groups relative to DIO. Moreover, compared with DIO, metabolism gene sets (e.g., FATTY_ACID_ METABOLISM, GLYCOLYSIS) were significantly enriched (NES &amp;gt;1.45, FDR q&amp;lt;0.05), and none were suppressed in control, DIO+Su, and FOb+LFD tumors. Tumor transcriptional changes were less coherent with EPITHELIAL_MESENCHYMAL_TRANSITION and COAGULATION being the only gene sets commonly suppressed across all groups relative to DIO. A leading-edge analysis revealed that Lum, Pthlh, Sfrp4, Mmp8, and Serpine1 were commonly suppressed amongst tumors from groups other than DIO mice. Both weight loss and sulindac impeded the effects of chronic obesity on colon tumorigenesis. GSEA analyses of immune related gene sets indicated that weight loss and sulindac effectively suppressed inflammation in the adipose tissue but not in the tumor. The conserved immunological and metabolic transcriptional programs in the adipose tissue relative to the tumor suggests the possibility that paracrine signaling from the adipose to the tumor may underpin the observed beneficial effects of these interventions on obesity-driven colon tumor growth. Citation Format: Elaine M. Glenny, Laura W. Bowers, Michael F. Coleman, Jatin Roper, Stephen D. Hursting. Resolution of obesity-driven proinflammatory adipose signaling by weight loss or an NSAID is associated with reduced tumor burden in a mouse model of colon cancer [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 14.

Abstract 2388: Role of the primary cilium in the crosstalk between obesity and cancer

Abstract Background: Defects in primary cilium can lead to a group of disorders termed ciliopathies, with links to both obesity and cancer. Obesity causes inflammation and downregulates anti-tumor immunity generating a protumor microenvironment and increases the risk of breast cancer (BC) development and metastasis. Also, obesity affects cilium length, which could disrupt the essential role of the cilium in detecting extracellular cues, coordinating cell signaling responses, and regulating cell fate and mitochondrial function. However, there is a gap in understanding the effects of obesity on ciliogenesis in cancer cells and in regard to the impact of cilium length and localization on mitochondrial function of tumor cells. We therefore sought to elucidate the impact of obesity on the presence, localization, and function of primary cilium, using murine models of triple-negative breast cancer (TNBC). Methods: We generated mammary tumors by orthotopic transplantation of murine metM-Wntlung or E0771 TNBC cells, and evaluated the impact of obesity on ciliary specific gene sets in tumors using GSEA analysis. We also tested the abundance of cilium in tumor sections using immunofluorescence. Additionally, we developed metM-Wntlung and E0771 expressing the ciliary transmembrane protein Smo fluorescently tagged to pHluorin. Using these cells, we performed in vitro studies to investigate the length and cellular localization of the cilium in cancer cells in 3 compartments; intracellular (in), intermediate, or extracellular (out). Results: Our GSEA analysis results indicated significant upregulation of 18/19 ciliary gene sets in metM-Wntlung mammary tumors from obese mice and 19/19 ciliary gene sets in E0771 mammary tumors from obese mice compared to the corresponding tumors developed in non-obese mice. Ongoing leading-edge analysis will determine the genes driving the enrichment of ciliary-related gene sets in tumors of obese mice. Immunofluorescence analysis showed a significant number of ciliated cells in tumor sections of obese mice and in cultured cells expressing pHluorin-Smo. Ongoing studies will reveal the specific cellular localization of the cilium on cancer cells and the effects on mitochondrial function. Our research is establishing a proof-of-concept that mammary cancer cells express cilium and will elucidate key mechanistic details related to the role of the primary cilium in the crosstalk between obesity and cancer metabolism. This research is supported by R35CA197627 (SDH), AICR Marylin Gentry Fellowship (XBM). Citation Format: Ximena Bustamante-Marin, Jenna L. Merlino, Emma J. Grindstaff, Michael F. Coleman, Erika Rezeli, Kristina K. Camp, Laura Smith, Stephen D. Hursting. Role of the primary cilium in the crosstalk between obesity and cancer [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 2388.

Transcriptomic Profiling of MSI-H/dMMR Gastrointestinal Tumors to Identify Determinants of Responsiveness to Anti-PD-1 Therapy.

Transcriptomic profiling was performed for microsatellite instability-high (MSI-H)/mismatch repair-deficient (dMMR) gastrointestinal tumors to determine the predictors of response to PD-1 blockade. Thirty-six patients with MSI-H/dMMR gastrointestinal tumors, including gastric cancer, colorectal cancer, cholangiocarcinoma, small intestine cancer, and pancreatic cancer, being treated with PD-1 blockade were analyzed. We conducted the transcriptomic analysis of gastrointestinal tumors using RNA sequencing data, including the consensus molecular subtypes (CMS) of colorectal cancer. Gene set enrichment analysis (GSEA) demonstrated that non-responders had upregulations of epithelial-mesenchymal transition, angiogenesis, hypoxia, mTORC1, TNF-α, KRAS, Wnt/β-catenin, TGF-β, and various metabolism-related signaling pathways. Meanwhile, the IFNγ pathway was enriched in responders. On the basis of the leading-edge analysis of GSEA, VEGF-A was significantly correlated with enriched pathways in non-responders. Patients with high VEGF-A expression, compared with those with low expression, had significantly shorter progression-free survival [PFS; median 4.8 months vs. not reached (NR), P = 0.032] and overall survival (median 11.1 months vs. NR, P = 0.045). Among 13 patients with colorectal cancer evaluable for CMS classification, the objective response rate was 100%, 0%, 0%, and 16.7% in CMS1, CMS2, CMS3, and CMS4, respectively. Patients with CMS1 had significantly longer PFS (NR vs. 4.8 months, P = 0.017) than those with CMS2, CMS3, or CMS4. Several transcriptomic features, including CMS classification and related genes, were associated with response to PD-1 blockade in MSI-H/dMMR gastrointestinal tumors. These findings can help develop predictive biomarkers or combination immunotherapies.

The extended concurrent genes signature for disease-free survival in breast cancer

Previously we had identified concurrent genes, which highlighted the interplay between copy number variation (CNV) and differential gene expression (GE) for Han Chinese breast cancers. The merit of the approach is to discovery biomarkers not identifiable by conventional GE only data, for which phenotype-correlation or gene variability is the criteria of gene selection. Thirty-one comparative genomic hybridization (CGH) and 83GE microarrays were performed, with 29 breast cancers assayed from both platforms. Potential targets were revealed by Genomic Identification of Significant Targets in Cancer (GISTIC) from CGH arrays. Concurrent genes and genes with significant GISTIC scores were used to derive the extended concurrent genes signature, which was consensus from leading edge analysis across all studies and a supervised partial least square (PLS) regression predictive model of disease-free survival was constructed. There were 1584 concurrent genes from 29 samples with both CGH and GE microarrays. Enriched concurrent genes sets for disease-free survival were identified independently from 83GE arrays and another one with Han Chinese origin as well as three studies of Western origin. For five studies with disease-free survival follow up, prognostic discrepancy was observed between predicted high-risk and low-risk group patients. We concluded that through parallel analyses of CGH and GE microarrays, the proposed extended concurrent gene expression signature can identify biomarkers with prognostic values.

Gene-Interaction-Sensitive enrichment analysis in congenital heart disease

BackgroundGene set enrichment analysis (GSEA) uses gene-level univariate associations to identify gene set-phenotype associations for hypothesis generation and interpretation. We propose that GSEA can be adapted to incorporate SNP and gene-level interactions. To this end, gene scores are derived by Relief-based feature importance algorithms that efficiently detect both univariate and interaction effects (MultiSURF) or exclusively interaction effects (MultiSURF*). We compare these interaction-sensitive GSEA approaches to traditional χ2 rankings in simulated genome-wide array data, and in a target and replication cohort of congenital heart disease patients with conotruncal defects (CTDs).ResultsIn the simulation study and for both CTD datasets, both Relief-based approaches to GSEA captured more relevant and significant gene ontology terms compared to the univariate GSEA. Key terms and themes of interest include cell adhesion, migration, and signaling. A leading edge analysis highlighted semaphorins and their receptors, the Slit-Robo pathway, and other genes with roles in the secondary heart field and outflow tract development.ConclusionsOur results indicate that interaction-sensitive approaches to enrichment analysis can improve upon traditional univariate GSEA. This approach replicated univariate findings and identified additional and more robust support for the role of the secondary heart field and cardiac neural crest cell migration in the development of CTDs.

Abstract 2579: Links between obesity and secreted frizzled-related protein 4 (Sfrp4) in a murine model of colon cancer

Abstract Obesity is a well-established risk factor for colon cancer and the effect of obesity-mediated inflammation is particularly pronounced in colon carcinogenesis. An incomplete understanding of the biology underlying this strong obesity-colon cancer association has hampered efforts to identify effective colon cancer prevention strategies. The current study aims to identify novel colon tumor intrinsic targets that mediate critical inflammatory signals underlying the obesity-colon cancer connection. To induce colon tumors, FVB/N male mice were treated weekly with azoxymethane (10 mg/kg intraperitoneally) for 5 weeks. Mice were then randomized to either continue receiving 10 kcal% fat diet (control) or were switched to a 60 kcal% fat diet to promote diet-induced obesity (DIO) over a 15-week period. DIO mice were then randomized to either receive 0ppm (DIO) or 140ppm sulindac (DIO+Su), a non-steroidal anti-inflammatory drug, for 7 additional weeks. At the end of the 27-week study, colonic tumors were collected for histology and global transcriptomic analysis using Affymetrix Clariom D mouse gene expression microarrays. DIO increased tumor multiplicity and burden in AOM-treated mice. Moreover, long-term administration of dietary sulindac was sufficient to reduce inflammatory signals, tumor multiplicity, and tumor burden in DIO-Su versus DIO mice. Principal component analysis and hierarchical clustering based on the 1,000 most differentially expressed genes revealed distinct gene expression profiles in tumors from control, DIO, and DIO+Su mice. To determine the biological processes most up- or down-regulated in tumors from DIO mice, gene set enrichment analysis was run using the hallmark gene sets. Epithelial-mesenchymal-transition (EMT) was the most upregulated gene set in tumors from DIO mice compared with tumors from control mice (normalized enrichment score 2.00; adjusted p=0.0002) and compared with tumors from DIO+Su mice (normalized enrichment score 1.78; adjusted p=0.009), suggesting that obesity drives EMT via an inflammatory mechanism. Based on a leading-edge analysis, secreted frizzled-related protein 4 (Sfrp4) expression was significantly enriched in the EMT gene set. Sfrp4 is of biological interest given its biphasic modulation of Wnt signaling and association with poor prognosis in aggressive prostate cancer. We confirmed via immunofluorescence that Sfrp4 is produced by AOM-treated colonic intestinal epithelial and submucosal cell populations and observed by qPCR that it is expressed across multiple human colon cancer cell lines. Hence, Sfrp4 may be a novel target to mitigate obesity-induced exacerbation of colon tumorigenesis. Future work will determine the role of Sfrp4 in colon cancer cell-adipose interactions and whether ablation of Sfrp4 is sufficient to impede colon tumor growth. This research was supported by R35CA197627 to S. Hursting. Citation Format: Elaine M. Glenny, Laura W. Bowers, Diana Li, Jatin Roper, Stephen D. Hursting. Links between obesity and secreted frizzled-related protein 4 (Sfrp4) in a murine model of colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2579.

Abstract NG15: Progesterone-mediated immune evasion in breast cancer

Abstract NG15: Progesterone-mediated immune evasion in breast cancer

Abstract P4-07-12: Residual risk stratification of Taiwanese breast cancer following curative therapies with digital RNA counting and actionable mutations sequencing

Abstract Introduction: Breast cancer is the most common female malignancy in Taiwan and treatment outcomes have improved enormously with early detection and advancements in adjuvant therapy. Unfortunately, a subset of early stage breast cancer still suffers from recurrence, metastasis, or even cancer-specific death despite treatment improvement, and conventional pathological factors fail to provide sufficient explanatory power accounting for the observed prognostic discrepancy. The aim of the project is to take advantage of digital RNA counting for gene expression and targeted sequencing of actionable mutations to develop a predictive model for operable breast cancers initially treated with curative intention, and the risk of recurrence, distant metastasis, as well as breast cancer-specific mortality will be speculated. Methods: Two approaches are applied for candidate genes yield; we have identified concurrent genes from the coherent patterns between genomic and transcriptional profiles, and the synthesized signatures were prognostic. In addition, concordant genes from leading edge analysis of published gene expression signatures were retrieved, and the consensus of leading edge subsets could stratify breast cancer patients into groups with distinct survival patterns. With bioinformatics tools, the harvested genes will be used to build a risk stratification model, and the residual risk after surgery and adjuvant therapy will be evaluated from archived formalin-fixed paraffin embedded (FFPE) cancer tissues, augmenting the clinical significance of the proposed signature. And finally, actionable mutations will be identified for relapsing patients through targeted sequencing. Results: There were 1584 extended concurrent genes with the threshold MAD (medium absolute deviation) of 0.9, constituting 15.89% of common genes across copy number variation and gene expression arrays. We also constructed breast cancer risk prediction using concordance of genes from leading edge analysis of various prognostic signatures; enrolled signatures were split into either the up-regulated or down-regulated gene sets within each study, and leading edge analysis of filtered up-/down-regulated gene sets from all training data sets were performed, followed by the PLS (partial least square) risk predictive model composed of consensus of leading edge subsets among all assayed subjects. The 145-gene BCeC Sig platform designated for the current study has been established, and was prognostic in 96 Taiwanese breast cancers (log-rank test: P&amp;lt;0.01). Among 37 breast cancers categorized into the high-risk group, there were 383 SNPs, 23 deletes, and 11 insertions. Variations per breast cancer were between 91 and 162 (average: 120). When collapsed into genes, the most altered genes were: BRCA1/2 (30 variants), FANCA(27), NOTCH(21), ALK, AR (20), ATM, BSG(14), FGFR, MET(12), JAK2, PDFGR, RET(11), CSF1R, DAB2I, and TP53(10). Discussion and conclusions: Archived pathological specimens provide an invaluable source to validate the residual risk prediction model for breast cancer patients managed with contemporary multi-modality treatment including surgery, chemo-, endocrine, and targeted therapy. Degraded nucleic acid from FFPE samples could be tackled by the latest NanoString digital RNA counting. This breast cancer residual risk model, composed of concurrent genes and consensual leading edge subset of published gene expression signatures, is believed to provide clinical applicability and substantial benefits for Taiwanese breast cancer patients in terms of personalized medicine Citation Format: Chi-Cheng Huang, Ching-Shui Huang. Residual risk stratification of Taiwanese breast cancer following curative therapies with digital RNA counting and actionable mutations sequencing [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-07-12.

Transcriptome meta-analysis reveals differences of immune profile between eutopic endometrium from stage I-II and III-IV endometriosis independently of hormonal milieu

Eutopic endometrium appears to be crucial for endometriosis development. Despite of the evident importance, data regarding the cellular microenvironment remain unclear. Our objective was to explore the tissue microenvironment heterogeneity, transcripts, and pathways that are enriched in all phases of the menstrual cycle by analysing publicly deposited data derived from whole transcriptome microarrays of eutopic endometria of women with and without endometriosis. A meta-analysis of the transcriptome microarrays was performed using raw data available from a public database. Eligibility criteria included eutopic endometrium samples from women with endometriosis and healthy controls without any pathological condition reported the presence of an adequately reported normal menstrual phase, and samples containing both glandular and stromal components. Raw data were processed using a robust multiarray average method to provide background correction, normalisation, and summarisation. The batch effect was estimated by principal variant component analysis and removed using an empirical Bayes method. Cellular tissue heterogeneity was inferred using the xCell package. Differentially expressed genes were identified based on a 5% adjusted p value and a 2.0-fold change. Pathways were identified by functional enrichment based on the Molecular Signatures Database, a p value of < 5%, and an FDR q value of ≤ 25%. Genes that were more frequently found in pathways were identified using leading edge analysis. In a manner independent of cycle phase, the subpopulations of activated dendritic cells, CD4 T effector memory phenotype cells, eosinophils, macrophages M1, and natural killer T cells (NKT) were all higher in stage I-II endometriosis compared to those in healthy controls. The subpopulations of M2 macrophages and natural killer T cells were elevated in eutopic endometriums from women with stage III-IV endometriosis, and smooth muscle cells were always more prevalent in healthy eutopic endometriums. Among the differently expressed genes, FOS, FOSB, JUNB, and EGR1 were the most frequently mapped within the interaction networks, and this was independent of stage and cycle phase. The enriched pathways were directly related to immune surveillance, stem cell self-renewal, and epithelial mesenchymal transition. PI3K AKT mTOR, TGF signalling, and interferon alpha/gamma responses were enriched exclusively in stage III-IV endometriosis. The cellular microenvironments and immune cell profiles were different between eutopic endometriums from women with stage I-II and stage III-IV endometriosis, and these differences were independent of the hormonal milieu. Specifically, a pro-inflammatory profile was predominant in stage I-II endometriosis, and M1-M2 polarization into eutopic endometrium may be crucial for the progression of the disease. The higher prevalence of NKT cells in eutopic endometriums from women with endometriosis that was independent of cycle phase or staging suggested a sustained stress and/or damage to these eutopic endometriums. Based on this, the results of this meta-analysis are important for identifying challenges and opportunities for future research.

Gene transcription profiling of astheno- and normo-zoospermic sperm subpopulations.

Spermatozoa contain a repertoire of RNAs considered to be potential functional fertility biomarkers. In this study, the gene expression of human sperm subpopulations with high (F1) and low (F2) motility from healthy normozoospermic (N) and asthenozoospermic (A) individuals was evaluated using RNA microarray followed by functional genomic analysis of differentially expressed genes. Results from A–F1 versus N–F1, A–F2 versus N–F2, N–F1 versus N–F2, and A–F1 versus A–F2 comparisons showed a considerably larger set of downregulated genes in tests versus controls. Gene ontology (GO) analysis of A–F1 versus N–F1 identified 507 overrepresented biological processes (BPs), several of which are associated with sperm physiology. In addition, gene set enrichment analysis of the same contrast showed 110 BPs, 36 cellular components, and 31 molecular functions, several of which are involved in sperm motility. A leading-edge analysis of selected GO terms resulted in several downregulated genes encoding to dyneins and kinesins, both related to sperm physiology. Furthermore, the predicted activation state of asthenozoospermia was increased, while fertility, cell movement of sperm, and gametogenesis were decreased. Interestingly, several downregulated genes characteristic of the canonical pathway protein ubiquitination were involved in asthenozoospermia activation. Conversely, GO analysis of A–F2 versus N–F2 did not identify overrepresented BPs, although the gene set enrichment analysis detected six enriched BPs, one cellular component, and two molecular functions. Overall, the results show differences in gene transcription between sperm subpopulations from asthenozoospermic and normozoospermic semen samples and allowed the identification of gene sets relevant to sperm physiology and reproduction.

Abstract 5133: Identification of distinct gene expression profiles in African Americans and Caucasian men with prostate cancer

Abstract Introduction African Americans (AA) are disproportionately affected by prostate cancer (PCa) compared to European Americans (EA). Genetic and epigenetic differences have been implicated in this racial disparity. Present PCa diagnostic and treatment regimens have focused mainly on gene fusion product involving the androgen driven transmembrane protease serine 2 (TMPRSS2) and the transcription factor, ETS related gene (ERG). However, TMPRSS2:ERG (T2E) fusion product is common mainly to EA (50-80% of patients) and has lower prevalence amongst other ethnicities including AAs (10-30%), Japanese and Chinese patients. In addition, T2E negative PCa subtype has been associated with increased risk of developing castration resistant PCa (CRPC). This study aims at classifying PCa by molecular and pathologic subtypes that drive disease aggressiveness in patients of AA and EA ancestry. Methodology Prostate cancer tissue slides obtained from EA and AA patients were stained for ERG expression. Laser-capture micro-dissected RNAs from formalin-fixed paraffin-embedded tissues were used for RNA-seq. DESeq2 was used as a statistical procedure to call differentially expressed genes (DEGs) in different samples. Data set from Powell was also analyzed for DEGs between ERG positive and ERG negative samples. Leading-Edge Analysis (GSEA v2.2.4) was performed to determine the representation of DEGs across enriched gene sets. Results 32.4% of EA were ERG positive compared to 29% of AA that stained positive. More EA samples (26.4%) had high intensity staining (2≥ on a 1 - 3 scale) compared to AA samples (25.8%). Over 11% of EA samples had high (4) distribution of ERG positive staining compared 6.5% in AA samples. Analysis of the Powell dataset (GSE41696) revealed 301 DEGs and gene set enrichment analysis identified immune system process, regulation of cellular component and homeostatic process as most biological processes affected by DEGs. Other processes significantly affected included: cell motility, cell activation and regulation of locomotion. At least 15 genes including: CCL5, TLR4, PIK3CB, CD86, KIT, CD40, ITGB3, PDGFRA, LEF1, HIF1A, CLU, PLA2G6, GLI3, PIK3CA and TNNB1, were present in at least 10 enriched gene sets. The DEGs across enriched gene sets showed gene signatures involved in Wnt signaling, PI3K, chemokines and receptor tyrosine kinases. Conclusion Specific sets of genes are differentially enriched between EGR-/AR- and ERG+/AR+. These findings suggest that PCas have a distinct gene expression profile in the absence of the T2E fusion, which may act as a driver of disease aggressiveness and racial health disparities in PCa. Identifying these differences in gene-signature expression and signaling transduction pathways will allow stratification of men across races and identify, early on, those individuals who are likely to develop the most aggressive disease. Citation Format: Joakin Mori, Raven Williams, Hui-Xian Lin, Alahni Becks, Jason White, Balasubramanyam Karanam, Clayton Yates, Honghe Wang. Identification of distinct gene expression profiles in African Americans and Caucasian men with prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5133.

Prognostic modeling of the immune-centric transcriptome reveals interleukin signaling candidates contributing to differential patient outcomes.

Immunotherapy is a promising advancement in the treatment of non-small-cell lung carcinoma (NSCLC), although much of how lung tumors interact with the immune system in the natural course of disease remains unknown. We investigated the impact of the expression of immune-centric genes and pathways in tumors on patient survival to reveal novel candidates for immunotherapeutic research. Tumor transcriptomes and detailed clinical characteristics were obtained from patients with NSCLC who were participants of either the Inflammation, Health and Lung Epidemiology (INHALE) (discovery, N = 280) or The Cancer Genome Atlas (TCGA) Lung (replication, N = 1026) studies. Expressions of 2253 genes derived from 48 major immune pathways were assessed for association with patient prognosis using a multivariable Cox model and pathway effects were assessed with an in-house implementation of the Gene Set Enrichment Analysis (GSEA) algorithm. Prognosis-guided gene and pathway analysis of immune-centric expression in tumors revealed significant survival enrichments across both cohorts. The 'Interleukin Signaling' pathway, containing 430 genes, was found to be statistically and significantly enriched with prognostic signal in both the INHALE (P = 0.008) and TCGA (P = 0.039) datasets. Subsequent leading-edge analysis identified a subset of genes (N = 23) shared between both cohorts, driving the pathway enrichment. Cumulative expression of this leading-edge gene signature was a strong predictor of patient survival [discovery: hazard ratio (HR) = 1.59, P = 3.0 × 10-8; replication: HR = 1.29, P = 7.4 × 10-7]. These data demonstrate the impact of immune-centric expression on patient outcomes in NSCLC. Furthermore, prognostic gene effects were localized to discrete immune pathways, of which Interleukin Signaling had the greatest impact on overall survival and the subset of genes driving these effects have promise for future therapeutic intervention.

Role of PLK1 signaling pathway genes in gastrointestinal stromal tumors.

In previous studies by the authors, aurora kinase A (AURKA) was demonstrated as an independent poor prognostic marker for the recurrence of localized gastrointestinal stromal tumors (GISTs) and for the progression of advanced GISTs. In the present study, the prognostic effect of genes involved in cell cycle regulation in GISTs was further examined. Leading edge analysis in gene set enrichment analysis was used to identify the most common genes in the top 10 enriched gene sets of high-risk patients with GISTs in a Japanese study. The obtained gene list was uploaded to the Pathway Interaction Database to search for critical pathways. Selected genes within the pathway were subsequently verified through immunohistochemistry (IHC) in another cohort of patients. A total of 5 genes in ‘PLK1 signaling events,’ namely AURKA, polo-like kinase 1 (PLK1), cell division cycle 25C (CDC25C), budding uninhibited by benzimidazoles (BUB1), and targeting protein for Xklp2 (TPX2), were identified for subsequent study. Among the Japanese cohort, all 5 genes, except BUB1, were significant prognostic factors for poor recurrence-free survival (RFS). Among 141 patients enrolled for the IHC study, all 5 genes exhibited variable expression patterns. In the association study, only AURKA exhibited significant overexpression in non-gastric tumors. Although all 5 genes were considered as risk factors for poor RFS based on a univariate analysis, only the mitotic count and expression levels of CDC25C, BUB1, and TPX2 retained prognostic effects in the multivariate analysis. The PLK1 signaling pathway is crucial in the disease progression of GISTs. Genes within this pathway may serve as predictive markers for adjuvant therapy.