Transcripts Per Million Research Articles

Vulnerability to immune therapy in BRAF- and MYB-altered pediatric gliomas.

2066 Background: The frequency and expression of predictive immune biomarkers in pediatric gliomas is relatively unknown. Here, we sought to define predictive signatures of immunotherapy responders by examining immune checkpoint expression, immune cell population signatures, and gene amplifications in a variety of pediatric gliomas. Methods: We profiled a cohort of pediatric, adolescent, and young adult glioma samples submitted to Caris Life Sciences (Phoenix, AZ) for analysis (N = 207). The cohort was further stratified by driver mutations: IDH mutant (N = 103), H3-3A mutant (N = 36), MYB-altered (N = 4), BRAF-altered (N = 39), and IDH wild type [IDH WT] (N = 25). De-identified next generation DNA sequencing (592-gene or whole exome) and RNA (whole transcriptome) sequencing were used to determine tumor-infiltrating immune cell signatures and immune checkpoint protein expression. Transcriptomic signatures predictive of response to immunotherapy (T cell inflamed score) and replication stress response defect (RSRD) score were calculated on transcripts per million (TPM) values. Immune cell fractions were estimated using RNA deconvolution (quanTIseq). Results: BRAF-altered tumors showed the highest IFN gamma signature (a clinically relevant inflammatory biomarker), a high pro-inflammatory M1 macrophage signature, and very high CD4+/CD8+ T cell signatures compared to other tumor groups. IDH mutant, but not WT gliomas, were relatively deficient in T cell infiltration but revealed an increase in dendritic cell signatures. IDH WT gliomas showed more T cell enrichment but higher immune suppressive checkpoint expression. H3-3A mutant tumors showed relatively low immune cell infiltration and immune checkpoint expression. Pro-inflammatory M1 macrophages were lower in the high-grade glioma microenvironment compared to low-grade. Immune suppressive M2 macrophages were elevated in the IDH WT microenvironment relative to other gliomas. There was no apparent difference in the frequency of PD-1+ T cells and tumor-expressed PD-L1 among the various molecularly defined pediatric glioma groups. Conclusions: In general, BRAF- and MYB-altered gliomas displayed high immune activation relative to other tumors. IDH WT tumors have an immune suppressive microenvironment with relatively high immune checkpoint expression. Based on predictive markers, BRAF- and MYB-driven gliomas show signatures suggesting the possibility of a greater response to immunotherapies than IDH WT/mutant gliomas or H3-3A gliomas.

Correlation of comprehensive molecular mapping of pancreatic ductal adenocarcinoma with XPO1 mRNA expression levels to potential clinical targets.

4144 Background: Encouraging pre-clinical efficacy targeting Exportin-1 (XPO1) - a master regulator of tumor suppressor protein export - has been reported in pancreatic ductal adenocarcinoma (PDAC) and clinical trials are currently ongoing. However, limited data is available regarding expression and function of XPO1 in PDAC. Thus, we investigated XPO1 mRNA expression and its clinical and immune correlates in PDAC. Methods: 5,488 PDACs were tested at Caris Life Sciences (Phoenix, AZ) with NGS of DNA (WES) and RNA (WTS) . TMB-H was defined as ≥10 mutations/MB. The cohort was stratified in quartiles according to XPO1 mRNA expression status, and XPO1-high ( XPO1H) and XPO1-low ( XPO1L) expression were defined as ≥top and <bottom quartile of transcripts per million (TPM). Immune cell fraction was calculated by QuantiSeq method. Gene expression profiles were analyzed for transcriptomic signatures predictive of response to immune checkpoint inhibitors (T cell-inflamed score) and MAPK pathway activation (MPA). The Mann-Whitney U and X2 tests were applied as appropriate, with p-values adjusted for multiple comparisons. Real-world overall survival (OS) data was obtained from insurance claims. TGCA data were used to validate molecular, biological and immunological findings. Single-cell RNA analyses on a publicly available dataset were performed to elaborate cellular localization of XPO1. Results: Increased XPO1 mRNA expression was observed in metastatic lesions compared to primary tumors (p<0.001). Copy number amplification and mutation prevalence was comparable between XPO1H and XPO1L samples (p>0.05), along with similar rates of MSI-H/dMMR (1.3 vs 0.9%) and TMB-H (2.0 vs 1.3%; p>0.05). XPO1H PDACs were associated with a significant higher prevalence of T cell-inflamed tumors compared to XPO1L cases (58 vs 5%; p<0.05), consistent with higher immune cell infiltration in the XPO1H quartile as compared to XPO1L (p<0.05), except for immunosuppressive regulatory T cells and monocytes (p>0.05). XPO1H was associated with a higher MPA score as compared to XPO1L (2.4 vs -2.1 Arbitrary Units; p<0.05). XPO1H was associated with shorter OS (HR 1.09 [1.1-1.4], p=0.048). Validation studies using TCGA datasets corroborate our findings and confirmed increased immune cell infiltration as a function of XPO1. Moreover, single cell data revealed predominant and high expression of XPO1 in the tumor cell compartment. Conclusions: This is the first study comprehensively mapping XPO1 mRNA expression and immune-correlates in PDAC. We found that high XPO1 is linked to increased immune cell infiltration with a T cell-inflamed pattern. Our data provided a potential rationale to combine immune checkpoint therapy (+/- XPO1 inhibitors) in XPO1H PDACs.

Impact of CLEC3B mRNA expression on genetic landscape and immune tumor-microenvironment in NSCLC.

e20517 Background: Downregulation of C-type lectin domain family 3 member B (CLEC3B) is observed in non-small cell lung cancer (NSCLC), but its role remains largely unclear. Thus, we conducted this large scale genomic and transcriptomic analysis to gain novel insights into molecular and immunological functions of CLEC3B mRNA expression in NSCLC. Methods: Five publicly available WTS/WES NSCLC datasets (TCGA LUAD/LUSC, GSE41271, GSE66863, GSE81089, BATTLE) and the lung cancer single-cell atlas (Salcher, Cancer Cell 2022) were re-analyzed to decipher various molecular and biological aspects of CLEC3B in NSCLC. Findings were validated in a cohort of 19,982 NSCLC samples, which were centrally profiled (Caris Life Sciences, Phoenix, AZ) using WES/WTS. The cohort was stratified in quartiles according to CLEC3B mRNA expression status, with CLEC3Bhigh ( CLEC3BH) and CLEC3Blow ( CLEC3BL) expression defined as top and bottom quartile of transcripts per million (TPM) for further comparison. Immune cell fractions were calculated using QuantiSeq. Real-world overall survival (OS) was calculated using data from insurance claims. Results: CLEC3B mRNA expression was diminished in NSCLC compared to matched normal tissue (p < 0.001). Adenomatous histology was associated with higher CLEC3B expression compared to squamous/large-cell carcinomas (p < 0.005). In CLEC3B H tumors we observed lower rates of somatic mutations in TP53 (44.1 vs 81.7%), TTN (54.1 vs 79%), CDKN2A (5.2 vs 15%), but higher rates in KRAS (24 vs 7.8%) and STK11 (15.8 vs 2.69%) (all, q < 0.01), which we corroborated in our validation cohort (TP53 (52.4 vs 74.7%), CDKN2A (8.3 vs 13%), RB1 (9.2 vs 13.5%), KMT2D (3.8 vs 7.3%), KRAS (31.8 vs 25.6%), EGFR (17.2 vs 8.1%) and STK11 (15.9 vs 11.3%, all q < 0.05)). Further, CLEC3BH tumors were characterized by an immunosuppressive phenotype reflected by lower TMB and lower rates of MSI-H/dMMR, lower PD-L1 IHC expression and abundance of M2 macrophages and regulatory T-cells. Analysis of bulk and single cell transcriptomic datasets revealed that CLEC3BH was linked to endothelium-specific signaling and higher expression levels in endothelial cells. Finally, increased CLEC3B expression was associated with improved OS in the exploratory and validation cohorts. Conclusions: We herein described the molecular landscape of NSCLCs according to CLEC3B mRNA expression. We speculate that CLEC3B is mainly expressed by endothelial cells, and observed that CLEC3BH was associated with a distinct genetic profile and an immunosuppressive tumor-microenvironment. Thus, CLEC3B expression warrants further investigation as a stratification marker for NSCLC patients undergoing immune checkpoint therapy.

Fat graft survival requires metabolic reprogramming toward the glycolytic pathway

Fat grafts are widely used as natural fillers in reconstructive and cosmetic surgery. However, the mechanisms underlying fat graft survival are poorly understood. Here, we performed an unbiased transcriptomic analysis in a mouse fat graft model to determine the molecular mechanism underlying free fat graft survival. We conducted RNA-sequencing (RNA-seq) analysis in a mousefree subcutaneous fat graft model on days 3 and 7 following grafting (n = 5). High-throughput sequencing was performed on paired-end reads using NovaSeq6000. The calculated transcripts per million (TPM) values were processed for principal component analysis (PCA), unsupervised hierarchically clustered heatmap generation, and gene set enrichment analysis. PCA and heatmap data revealed global differences in the transcriptomes of the fat graft model and the non-grafted control. The top meaningful upregulated gene sets in the fat graft model were related to the epithelial-mesenchymal transition, hypoxia on day 3, and angiogenesis on day 7. Mechanistically, the glycolytic pathway was upregulated in the fat graft model at days 3 (FDR q = 0.012) and 7 (FDR q = 0.084). In subsequent experiments, pharmacological inhibition of the glycolytic pathway in mouse fat grafts with 2-deoxy-D-glucose (2-DG) significantly suppressed fat graft retention rates, both grossly and microscopically (n = 5). Free adipose tissue grafts undergo metabolic reprogramming toward the glycolytic pathway. Future studies should examine whether targeting this pathway can enhance the graft survival rate.

Abstract 5895: Analysis of microbiome in gastric MALT lymphoma

Abstract Gastric MALT (mucosa-associated lymphoid tissue) lymphoma is found at a rate of 50% or more among the types of MALT lymphoma. Majority of patients with gastric MALT lymphoma are infected with Helicobacter pylori and also have clinical improvement with eradication therapy. However, it is unclear how H. pylori cause gastric MALT lymphoma. To explore it, we prospectively collected 13 patients who are newly diagnosed with gastric MALT lymphoma with H. pylori. We gathered samples from normal regions and lesions during endoscopy for diagnosis. Samples of lesion were harvested again after eradication of H. pylori. we sequenced 10 of WES (whole exome sequencing) and 13 of WTS (whole transcriptome sequencing) with these samples. With sequencing data, we aligned the sequenced reads to human reference genome (hg19) using BWA and called the variants with Mutect2. We aligned the reads and calculated TPM (Transcripts Per Million) from WTS with STAR and RSEM. We performed DEG (Differentially Expressed Genes) and GSEA (Gene Set Enrichment Analysis) for three comparison groups which are divided into normal, lesion, and after eradication. Gene fusions were detected using STAR-Fusion and Arriba with WTS. To detect potential microbiome from the samples, we used Kraken2, a taxonomic classification tool. We found that MUC6 was mutated from all samples and some samples have mutated MUC3A, KMT2C, ZNF595, CDC27, etc., and two have BIRC3-MALT1 fusion. From DEG and GSEA analysis, G protein-coupled receptor activity was activated in the group of lesion samples and suppressed in normal samples and eradication samples. Lymphocyte activation and immune response were also activated in the cancerous lesion. Kraken2 classified the reads which were not aligned to the human reference genome as reads from microbiota including H. pylori. The reads of H. pylori were more abundant in the lesion group than the other groups. According to the result of differentially expressed species analysis between the groups of lesion and eradication, the following species were also more abundant in the lesion group; Veillonella dispar, Helicobacter hepaticus, Helicobacter winghamensis, etc. We analyzed genetic alterations and differentially expressed genes and also classified non-human reads as microbiome with sequencing data from gastric MALT lymphoma. Our results suggest how H.pylori infection is related to carcinogenesis of gastric MALT lymphoma. Citation Format: SeungJoo Yoo, Dong-Yeop Shin, Ja Min Byun, Junshik Hong, Hongseok Yun, Youngil Koh, Sung-Soo Yoon. Analysis of microbiome in gastric MALT lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5895.

Effects of organic and inorganic nitrogen sources on the transcriptome of gellan gum biosynthesis by Sphingomonas paucimobilis.

Investigate the effects of different nitrogen sources on the metabolic characteristics of Sphingomonas paucimobilis during gellan gum (GG) production was helpful for developing optimized conditions that are widely applicable to all GG production processes. We compared the effects of organic nitrogen (ON) and inorganic nitrogen (IN) sources during GG production using transcriptome sequencing. Our results showed that compared with the IN source, the ON source effectively improved the cell number and GG production of S. paucimobilis during fermentation. There were significant differences in gene transcription levels between the ON and IN groups at different fermentation times. The transcriptional levels of multiple genes in the pathways from α-D-glucose-1P to glyceraldehyde-3P were reduced in the ON group, whereas those of multiple genes in the pathways from glyceraldehyde-3P to acetyl-CoA were significantly enhanced in the ON group after 12 h of fermentation. The transcription levels of multiple genes participating in the citrate cycle and upstream of fatty acid metabolism pathways were significantly enhanced in the ON group after 12 h of fermentation. Except for the transcripts per million (TPMs) of pgm and rfbA genes in ON, which were significantly higher than those in IN at 12 h after fermentation, the TPMs of the majority of genes in ON were significantly lower than those in IN. The transcription levels of genes participating in the transformation of N-acetyl-D-glucosamine (GlcNAc) to UDP-N-acetyl-alpha-D-glucosamine (UDP-GlcNAc) were enhanced in the ON group during the fermentation process.

272 Differential expression of two Plasmodium falciparum variant surface antigen families in Malian children with cerebral malaria compared to mild malaria

OBJECTIVES/GOALS: Recent in vitro evidence suggests that diverse parasite protein families called RIFINs and STEVORs are displayed on the surface of infected red blood cells and may have a role in severe malaria, but they remain sparsely studied in natural infections. We measured the RNA expression of these antigens in Malian children with severe or mild malaria illness. METHODS/STUDY POPULATION: We collected blood samples from Malian children aged six months to five years, including 14 with cerebral malaria, 10 with severe malarial anemia, and demographic-matched controls with mild, uncomplicated malaria. We extracted total RNA from each patient and used a custom capture array to selectively enrich Plasmodium falciparum parasite RNA. We then performed Illumina next-generation RNA sequencing and reconstructed parasite transcriptomes using reference-free de novo assembly. We identified RNA encoding RIFINs and STEVORs using an in-house classifier, then measured the diversity and abundance of gene expression for each infection. Expression diversity was defined as the number of unique variants transcribed. Expression abundance was calculated as transcripts per million (TPM). RESULTS/ANTICIPATED RESULTS: Cerebral malaria cases, but not severe malarial anemia cases, had higher diversity and abundance of RIFIN expression compared to mild infections. Type A RIFINs predominated over Type B RIFINs, and the same two RIFINs were predominantly expressed in all disease phenotypes. We anticipate that predominantly expressed RIFINs share high sequence homology with variants previously shown to bind blood antigens or immune inhibitory receptors. STEVOR expression was also higher in cerebral malaria compared to mild malaria, but STEVOR transcripts were sparse overall. DISCUSSION/SIGNIFICANCE: Elevated RIFIN expression in cerebral malaria over mild malaria supports a role for these antigens in pathogenesis. Severe malarial anemia may progress through a different pathogenic mechanism. Predominantly expressed RIFIN variants may be promising targets for vaccines and therapeutics to protect children against cerebral malaria.

Predicting the severity of COVID-19 patients using the CD24-CSF1R index in whole blood samples

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, has become one of the most serious public health crises worldwide. Most infected people are asymptomatic but are still able to spread the virus. People with mild or moderate illnesses are likely to recover without hospitalization, while critically ill patients face a higher risk of organ injury or even death. In this study, we aimed to identify a novel biomarker that can predict the severity of COVID-19 patients. Clinical information and RNA-seq data of leukocytes from whole blood samples with and without a COVID-19 diagnosis (n = 100 and 26, respectively) were retrieved from the National Center for Biotechnology Information Gene Expression Omnibus database. Raw data were processed using the Transcripts Per Million (TPM) method and then transformed using log2 (TPM+1) for normalization. The CD24-CSF1R index was established. Violin plots, Kaplan-Meier curves, ROC curves, and multivariate Cox proportional hazards regression analyses were performed to evaluate the prognostic value of the established index. The CD24-CSF1R index was significantly associated with ICU admission (n = 50 ICU, 50 non-ICU) and ventilatory status (n = 42 ventilation, 58 non-ventilation) with p = 4.186e-11 and p = 1.278e-07, respectively. The ROC curve produced a relatively accurate prediction of ICU admission with an AUC of 0.8524. Additionally, patients with a high index had significantly fewer mechanical ventilation-free days than patients with a low index (p = 6.07e−07). Furthermore, the established index showed a strong prognostic ability for the risk of using a ventilator in the multivariate Cox regression model (p < 0.001). The CD24-CSF1R index was significantly associated with COVID-19 severity. The established index could have potential implications for prognosis, disease severity stratification, and clinical management.

CO2 improves the anaerobic biodegradation intensity and selectivity of heterocyclic hydrocarbons in heavy oil

Heterocyclic hydrocarbons pollution generated by oil spills and oilfield wastewater discharges threatens the ecological environment and human health. Here we described a strategy that combines the greenhouse gas CO2 reduction with microbial remediation. In the presence of nitrate, CO2 can improve the biodegradation efficiency of the resins and asphaltenes in heavy oil, particularly the biodegradation selectivity of the polar heterocyclic compounds by the newly isolated Klebsiella michiganensis. This strain encoded 80 genes for the xenobiotic biodegradation and metabolism, and can efficiently utilize CO2 when degrading heavy oil. The total abundance of resins and asphaltenes decreased significantly with CO2, from 40.816% to 26.909%, to 28.873% with O2, and to 36.985% with N2. The transcripts per million (TPM) value of accA gene was 57.81 under CO2 condition, while respectively 8.86 and 21.23 under O2 and N2 conditions. Under CO2 condition, the total relative percentage of N1-type heterocyclic compounds was selectively decreased from 32.25% to 22.78%, resulting in the heavy oil viscosity decreased by 46.29%. These results demonstrated a novel anaerobic degradation mechanism that CO2 can promote the anaerobic biodegradation of heterocyclic hydrocarbons in heavy oil, which provides a promising biotreatment technology for the oil-contaminated water.

Characterization of FOLH1 expression in renal cell carcinoma (RCC).

713 Background: The FOLH1 gene encodes prostate-specific membrane antigen (PSMA), a transmembrane glycoprotein that is highly expressed in prostate cancer cells and on endothelial cells in the neovasculature of solid tumors, including RCC. PSMA has been used as a target for diagnostic imaging and therapeutic radioligand therapy. We utilized a database of molecularly profiled RCC tumors to evaluate associations with FOLH1 expression. Methods: NextGen sequencing of DNA (592-gene/whole exome) and RNA (whole transcriptome) was performed for RCC patient specimens (n=1765) through Caris Life Sciences (Phoenix, AZ). FOLH1-High/Low expression were defined as ≥75th/&lt;25th-percentile of RNA transcripts per million (TPM). Angiogenic, T-effector, and Myeloid expression signatures were calculated using previously defined gene sets (McDermott, 2018). Immune cell infiltration in tumor microenvironments (TMEs) was estimated using MCP-Counter (Becht, 2016). Tumor cell PD-L1+ expression (≥2+, ≥%5; SP142) was assessed by IHC. Kaplan-Meier estimates were calculated from time of tissue collection or therapy start. Results: FOLH1 expression was similar between sexes (71% male/29% female, 11.2 vs. 11.3 median TPM, p=0.54) and was not correlated with patient age at time of profiling (median 63 years, range 1-90+, spearman = 0.02, P=0.42). FOLH1 expression was significantly higher in clear cell RCC (ccRCC; 71.1% prevalence) compared to non-ccRCC tumors (19.0 vs 3.3 TPM, P&lt;0.001). FOLH1 expression varied by specimen site (45% kidney/55% metastatic, 13.6 vs. 9.9 TPM, P&lt;0.001), with notably lower expression in lymph nodes (5.3 TPM, P&lt;0.001, 8.2% prevalence). FOLH1 expression was strongly correlated with angiogenic gene expression compared to T-effector and myeloid signatures (spearman = 0.76 vs 0.33 and 0.20, respectively, each P&lt;0.001), with similar correlation strength observed for endothelial cell abundance in TMEs (spearman = 0.76 vs. 0.04-0.50 for immune cell types, P&lt;0.001). PD-L1+ IHC frequency was numerically lower yet not significantly different in FOLH1-High compared to -Low tumor among ccRCC (10 vs. 17%, P=0.07), but was similar among non-ccRCC (31 vs 32%, P=0.95). For patients stratified by median FOLH1 expression, no difference in overall survival from time of tissue sampling was observed for ccRCC (HR 1.2, P=0.57) or non-ccRCC cohorts (HR 0.77, P=0.59), while FOLH1-High was associated with numerically longer cabozantinib time-on-treatment (223 vs. 61 days, HR 0.60, P=0.08). Conclusions: We observed differential patterns of FOLH1 expression by histology and tumor site. FOLH1 expression was strongly correlated with angiogenic gene expression and distinct differences in TME composition, including endothelial cell abundance . FOLH1 gene expression was positively correlated with increased duration of anti-angiogenic treatment. Additional studies are needed to test the efficacy of PSMA-based diagnostics/therapeutics in RCC.

Characterization and impact of Wnt5A signaling on outcomes of urothelial carcinoma.

560 Background: Active Wnt signaling via WNT5A through ROR1 and its partner, ROR2, or WNT5A/frizzled 2 (FZD2) is linked to processes driving tumorigenesis, disease progression and therapy resistance. The role of this pathway in the pathogenesis of urothelial carcinoma (UC) is not fully elucidated. In adult tissue, ROR1 is largely absent, which makes it ideal for targeted therapies, with several ROR1 targeting agents in early clinical development. We utilized a large dataset of molecularly characterized UC tumors to investigate the significance of Wnt5a/ ROR1, ROR2 or FZD2 transcriptional expression. Methods: NextGen Sequencing of DNA (592 genes or WES)/RNA (WTS) was performed for 4743 UC tumors submitted to Caris Life Sciences (Phoenix, AZ). PD-L1 expression (SP142; Positive (+): ³2+, ³%5) was tested by IHC. Gene expression profiles were analyzed for a transcriptional signature predictive of response to immunotherapy (T cell-inflamed; Bao, 2020). WNT5a, ROR1, ROR2, and FZD2-high and -low expression were defined as ³ top and &lt; bottom quartile of transcripts per million (TPM), respectively. Mann-Whitney U and X2/Fisher-Exact tests were applied where appropriate, with P-values adjusted for multiple comparisons ( q &lt; .05). Real-world overall survival (OS) information was obtained from insurance claims data and Kaplan Meier estimates were calculated for molecularly defined cohorts. Results: We observed similar expression of these WNT5A signaling pathway genes between upper (N=795) and lower tract UC (N=3,204): WNT5A (22.7 v. 22.2 median TMP (mTPM), q = .18), FZD2 (3.4 v 3.5, q = .93), ROR1 (2.0 v. 1.7, q = .05), and ROR2 (2.1 v 2.5, q &lt; .01). WNT family gene expression varied significantly between primary (N=2,756) and metastatic sites (N=1,361): WTN5A (25.2 v 16.8 mTPM), FZD2 (3.2 v 4.05), ROR1 (1.7 v 2.1), and ROR2 (2.4 v 2.6) for primary vs. metastatic sites respectively ( q &lt; .05 for all). Comparison of high- and low-expression subgroups revealed variation in the prevalence of TP53, FGFR3 and RB1 mutations, as well as PDL1+ staining and T cell-inflamed scores (Table). High gene expression for ROR2 (HR 0.66, 95% CI 0.56-0.78, p &lt; .001) and FZD2 (HR 0.75, 95% CI 0.63-0.89, p &lt; .001) was associated with worse OS compared to low gene expression. No significant difference in OS was observed for WNT5A (HR 0.97, 95% CI 0.82-1.15, p &lt; .76) and ROR1 (HR 0.86, 95% CI 0.72-1.01, p &lt; .068). Conclusions: Distinct genomic and immune landscapes for the four investigated WNT pathway components were observed and should be leveraged to identify combination therapies that complement the current pipeline of WNT pathway-targeting drugs. [Table: see text]

The genomic landscape of urothelial carcinoma with high and low ERBB2/HER2 expression.

555 Background: Human epidermal growth factor receptor 2 (HER2) expression has been associated with poor prognosis in urothelial carcinoma (UC). Recent data suggests that HER2-targeted antibody-drug conjugate (ADC) treatment is efficacious. Here, we explore the role of ERBB2/ HER2 expression in UC by analyzing genomic and clinical outcomes in a large database of real-world patient samples. Methods: NextGen Sequencing (NGS) of DNA (592 genes or whole exome sequencing; WES)/RNA (whole transcriptome sequencing; WTS) was performed for 4,743 UC tumors submitted to Caris Life Sciences (Phoenix, AZ). PD-L1 (SP142; Positive (+): ≥ 2+, ≥ %5) and HER2 (4B5; +: ≥3+ and &gt;10%) expression was tested by IHC. High tumor mutational burden (TMB-high) was defined as ≥10 mutations/MB. ERBB2-high and -low expression were defined as ≥ top and &lt; bottom quartile of ERBB2 transcripts per million (TPM), respectively. Mann-Whitney U and X2/Fisher-Exact tests were applied where appropriate, with P-values adjusted for multiple comparisons ( p &lt; .05). Real-world overall survival (OS) information was obtained from insurance claims data and Kaplan-Meier estimates were calculated for molecularly defined patients. Results: 2.5% (120/4743) of tumors had HER2 IHC and WTS data available, 97% (61/63) of HER2+ tumors were ERBB2-high, and 98% (51/52) were ERBB2-amplified by DNA NGS (copy number ≥4; 77% with copy number ≥6). Tumors from lower tract UC had higher ERBB2 expression compared to upper tract UC (50 v 40 median TPM (mTPM), p &lt; .001). ERBB2 expression was similar between primary and metastatic tumors (47 v 47 mTPM, p = .95) but significantly higher in lung metastases (59 v 47 mTPM, p &lt; .001). Mutation rates of ERRB2 (12%), pTERT (76%) and ELF3 (14 %) were higher in ERBB2-high compared to ERBB2-low tumors (5%, 60%, 5%, all p &lt; .001). The opposite pattern was observed for CDKN2A (4% ERBB2-high, 9% ERBB2-low), KMT2D (20%, 35%) and PIK3CA (13%, 23%) (all p &lt; .001). There was a higher incidence of PD-L1+ staining in ERBB2-low (40.3%) compared to ERBB2-high tumors (18%, p &lt; .001). 45.4% of ERBB2-high tumors were TMB-high compared to 35% of ERBB2-low ( p &lt; .001). ERBB2-high tumors had higher expression of ADC target genes NECTIN4 (12 v 8 mTPM) and TACSTD2 (366 v 74 mTPM) compared to ERBB2-low (each, p &lt; .001). ERBB2-high tumors were associated with better overall survival from time of tissue sampling than ERBB2-low (HR 1.34, 95% CI 1.14-1.60, p &lt; .001) and from start of anti-PD-L1 therapy (HR 2.26, 95% CI 1.02-5.02, p = .04) but no difference in OS from start of anti-PD1 therapy (HR 1.39, 95% CI 0.75-2.58, p = .30). Conclusions: High concordance between HER2 IHC and ERBB2 expression/amplification was observed. Differences in the genomic landscape and ADC target expression of ERBB2-high vs -low UC may provide rationale for combination treatment strategies with HER2-blocking antibodies. The association between high ERBB2 expression and survival advantage warrants further investigation.

Molecular correlates of DSCR1 expression in colorectal cancer (CRC).

185 Background: Down syndrome (DS), a genetic disorder caused by trisomy of chr 21, is associated with a considerably lower risk for solid tumors and other angiogenesis related diseases. DSCR1 belongs to a family of evolutionary conserved protein-coding genes located on chr 21 and is highly upregulated in DS patients. Its product, calcipressin-1, has been shown to reduce cancer risk by suppressing angiogenesis. We previously reported that a germline polymorphism in DSCR1 was associated with time to recurrence in resected CRC and anti-VEGF treatment outcomes in metastatic CRC. Here, we analyzed the molecular landscape of CRC according to DSCR1 expression levels. Methods: 20,237 samples from CRC tested at Caris Life Sciences (Phoenix, AZ) with WTS (Illumina NovaSeq) and NextGen DNA sequencing (NextSeq, 592 Genes and NovaSEQ, WES) were analyzed. Top quartile transcripts per million (TPM) for DSCR1 expression were considered high (Q4) while bottom quartile low (Q1). Cell infiltration (CI) in the tumor microenvironment (TME) was estimated by RNA deconvolution analysis using QuantiSEQ. Interferon-gamma and T-cell inflamed signatures were also calculated from RNA data. X2 and Fisher-Exact tests were used and statistical significance was determined as P-value adjusted for multiple comparisons ( q &lt; 0.05). Results: DSCR1 expression was higher in primary tumors than metastases (10.5 vs 8.1 median TPM, q &lt; 0.05). No significant difference was observed in right- versus left-sided tumors, however rectal tumors showed the highest DSCR1 expression ( P &lt; 0.05). Overall, high DSCR1 TPM were associated with TMB-high (11.3% vs 9.3%), dMMR/MSI-H (7.9% vs 5.9%), and PD-L1 (4.7% vs 3.1%) ( q &lt; 0.01); the association with TMB-H was not significant in pMMR/MSS. DSCR1 high was associated with lower mutation rates of APC, KRAS, TP53 and amplification of FLT1/ FLT3, while higher mutation rates of KMT2C/D, BRAF, PTEN, RNF43, and RSPO3 fusions ( q &lt; 0.0001). Gene set enrichment analysis showed that high DSCR1 expressing tumors were enriched in alterations of several pathways including hypoxia, apoptosis, DNA repair, KRAS signaling, inflammatory response, and oxidative stress-related pathways (all P &lt; 0.05, FDR &lt; 0.25). B cells, macrophages, neutrophils, NK cells, Tregs, cancer associated fibroblasts and endothelial cells were more abundant in the TME of tumors with high DSCR1 while myeloid dendritic cells were lower, regardless of MMR status (all q &lt; 0.001). DSCR1 expression was associated with a higher T-cell inflamed signature and IFN score ( q &lt; 0.05). Conclusions: This is the first and most extensive profiling study to investigate DSCR1 expression in CRC. Our data show a strong association between tumor DSCR1 gene expression and distinct molecular features and TME cell infiltration. These findings suggest that DSCR1 holds potential as a novel therapeutic target for CRC and may be an important player in TME modulation.

SETD2 gene expression and the molecular landscape of colorectal cancer (CRC).

184 Background: SETD2, a key methyltransferase modulating histone 3 gene transcription, has been shown to act as a tumor suppressor gene by reducing oxidative stress, colonic inflammation and tumorigenesis in animal models. SETD2 gene expression has been reported to be significantly downregulated in CRC and linked with poorer patient survival. Additionally, SETD2 plays an important role in DNA repair and loss of function mutations have been associated with increased tumor mutational burden (TMB), mismatch repair (MMR) deficiency, and benefit from immunotherapy. Hence, we aimed to characterize the molecular features associated with SETD2 gene expression in CRC. Methods: 15,425 CRC tumors tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing on DNA (592 genes or whole exome sequencing), RNA (whole transcriptome sequencing), and IHC were analyzed. SETD2-high and -low expression were defined as ≥ top and &lt; bottom quartile of SETD2 transcripts per million (TPM), respectively. Cell infiltration in the tumor microenvironment (TME) was estimated by QuantiSEQ. Mann-Whitney U and X2/Fisher-Exact tests were applied where appropriate, with P-values adjusted for multiple comparisons ( q &lt; .05). Gene expression profiles were analyzed for transcriptional signatures predictive of response to immunotherapy (T cell-inflamed) and MAPK pathway activation (MPAS). Real-world overall survival information was obtained from insurance claims data and Kaplan-Meier estimates were calculated for molecularly defined patients. Results: SETD2 expression was significantly increased in left-sided/rectal compared to right-sided tumors (median TPM [mTPM] 55.8 vs 51.1, q &lt; .001). SETD2 mutations were associated with reduced SETD2 expression in pMMR/MSS CRC (mTPM 37.9 vs 54.3 in WT, q &lt; .001), although not statistically significant in dMMR/MSI-H CRC (mTPM 43.6 vs 51.7 in WT, P = .17). Compared to SETD2-high tumors, SETD2-low was associated with increased rates of TMB-high (10.4% vs 8.2%, P = .009), dMMR/MSI-H (7.3% vs 5.6%, P = .02), and PD-L1 IHC levels (4.4% vs 2.5%, q &lt; .002). Only minor differences in gene mutation and copy number rates were observed between SETD2-high and -low tumors , whereas SETD2-high TME was associated with increased immune cell infiltration, including neutrophils, B cells, NK cells, M2 macrophages, and dendritic cells ( q &lt; .05). SETD2-high was also associated with increased T cell-inflamed and MPAS signatures (152% and 159% median increase, respectively, q &lt; .001), regardless of tumor MMR status. Among dMMR/MSI-H CRC, SETD2-high tumors receiving immune checkpoint inhibitors had longer time-on-treatment (HR: 0.39, 95% CI: 0.2-0.76, P = .005) and a trend towards longer OS (HR: 0.29, 95% CI: 0.07-1.2, P = .069). Conclusions: Our data show distinct immune biomarkers and TME cell infiltration associated with SETD2 gene expression in CRC. These findings support a key role for SETD2 in modulating anti-tumor immunity and TME.

P247 Oncostatin M and its receptor are markers of endoscopic and histological disease activity in Inflammatory Bowel Disease

Abstract Background Oncostatin M (OSM) and its receptor (OSMR) have been associated with active disease in patients with inflammatory bowel disease (IBD). This study compared the expression of OSM and OSMR in intestinal tissue according to clinical, endoscopic, and histological disease activity. Methods A multi-centre IBD database containing RNA-sequencing datasets, was used to analyse OSM and OSMR expression in ileal and/or colonic tissue, expressed as transcripts per million (TPM). Clinical scoring systems included the Harvey Bradshaw Index for Crohn’s disease (CD) and partial Mayo score for ulcerative colitis (UC). Endoscopic scoring systems included the simple endoscopic score for CD (SES-CD) and UC endoscopic index of severity (UCEIS). Histological scoring included the modified Naini and Cortina, and Riley scores. Graphs were presented in log transformed TPM+1 values. Results 155 patients (108 CD, 47 UC) and 38 controls were included. For IBD patients, 49% were men, median age was 35 years and intestinal tissue was collected at endoscopy (73%) and surgery (27%). Compared to controls, patients with CD and UC had significantly elevated median expression of OSM (0.08, 0.29 and 0.34 TPM respectively) and OSMR (1.21, 1.69 and 3.53 TPM respectively). OSMR expression was higher in UC patients than CD (p&amp;lt;0.001). There was no difference in OSM or OSMR between Harvey Bradshaw Index categories (0-4 vs 5-7 vs 8-16) or partial Mayo scores (0 vs 1-3 vs 4-7). Endoscopically, OSM was higher for SES-CD scores of 16-51 (1.11 TPM), compared to SES-CD scores of 0-3 (0.14 TPM, p=0.003) and controls (0.08 TPM, p&amp;lt;0.0001). Median OSMR increased non-significantly with increasing SES-CD scores (score 0-3: 1.59 TPM; score 4-15: 1.90 TPM; score 16-51: 2.04 TPM). There was no difference in OSM and OSMR expression between UCEIS scores, but the most severe disease (UCEIS score 5-8) had higher expression than controls for both OSM (3.92 vs 0.08 TPM, p&amp;lt;0.001) and OSMR (6.10 vs 1.21 TPM, p&amp;lt;0.0001). Increasing expression of OSM and OSMR was found with increasing histological scores, see Figure. Using the modified Naini and Cortina score for CD, OSM and OSMR were most expressed in the highest scores compared to the lowest scores (OSM: 1.48 vs 0.09 TPM, p&amp;lt;0.001; OSMR: 5.50 vs 1.27 TPM, p=0.003). For the modified Riley score for UC, both OSM and OSMR were also able to differentiate between the highest and lowest score groups (OSM: 3.14 vs 0.12 TPM, p=0.0001; OSMR: 6.61 vs 1.58 TPM, p&amp;lt;0.0001). Conclusion This study showed that OSM and OSMR expression in intestinal tissue is associated with inflammation both histologically and endoscopically. Further investigations are needed to confirm our observations and explore the OSM pathway’s relevance in intestinal inflammation.

P109 Using baseline Peripheral blood mononuclear cell (PBMC) transcriptomics of Crohn Disease patients receiving Ustekinumab treatment as a tool to assess potential response at 1 year

Abstract Background Ustekinumab, a monoclonal antibody to the p40 subunit of interleukin-12 and interleukin-23, is used to treat Crohn Disease (CD) and clinical remission after one year was observed in about 50% of patients. We aimed to identify predictors for clinical response using PBMC transcriptomics obtained from Crohn Disease patients just prior to Ustekinumab treatment initiation. Methods RNA extraction from leukocytes was performed using Qiagen AllPrep RNA/DNA Mini Kit, followed by PolyA-RNA selection, fragmentation, cDNA synthesis, adaptor ligation, TruSeq RNA sample library preparation (Illumina, San Diego, CA), and paired-end 75bp sequencing. mRNA genes with transcripts per million (TPM) values above 1 in at least 20% of the samples were used. Differential gene expression was performed using DESeq2. Results We processed blood samples from 36 CD patients (13 males, [36%], median age 35 [IQR 29-41] years). Samples were obtained at baseline just before starting Ustekinumab treatment. 22/36 (61%) were define as responders and 14/36 (39%) as non-responders after 1 year based on Physician Global Assessment (PGA). Small bowel disease was significantly more common among responders 82% versus 36%, p=0.004. Principal component analysis (PCA) using the 12773 genes that passed expression filtering showed partial separation (Fig. 1). Differential gene expression between responders (n=22) and non-responders (n=14), did not show gene expression signature that passed FDR correction, however the analyses identified 68 genes including CXCL1/2/3 that were induced in non-responders vs. responders with p&amp;lt;0.05 and fold change above 1.5. Functional annotation enrichments of these 68 genes using ToppGene indicated enrichment (Fig. 2) for cytokine activity (FDR=1.98E-05), CXCR chemokine receptor binding (FDR=2.11E-05), Interleukin-10 signaling (FDR=5.03E-07), Genes encoding secreted soluble factors (FDR=1.73E-05), Myeloid Cells, granulocytes CD11b+ (FDR=6.64E-12), and Myeloid Cells Dendritic cells (FDR=1.80E-08). Conclusion No strong differences were found in the blood transcriptomics between responders and non-responders. However, among the non-responders we noted an increased inflammatory response enriched for pathways linked with cytokine activity and chemokine receptor binding and innate granulocytes and Myeloid signature. A larger cohort is required to validate and farther explore these findings. Fig. 1: Principal component analysis (PCA) using the 12773 genes that passed expression filtering showed partial separation.  Fig. 2: Functional annotation enrichments of the 68 genes induced in PBMC. 

Deciphering key genomic regions controlling flag leaf size in wheat via integration of meta-QTL and in silico transcriptome assessment

BackgroundGrain yield is a complex and polygenic trait influenced by the photosynthetic source-sink relationship in wheat. The top three leaves, especially the flag leaf, are considered the major sources of photo-assimilates accumulated in the grain. Determination of significant genomic regions and candidate genes affecting flag leaf size can be used in breeding for grain yield improvement.ResultsWith the final purpose of understanding key genomic regions for flag leaf size, a meta-analysis of 521 initial quantitative trait loci (QTLs) from 31 independent QTL mapping studies over the past decades was performed, where 333 loci eventually were refined into 64 meta-QTLs (MQTLs). The average confidence interval (CI) of these MQTLs was 5.28 times less than that of the initial QTLs. Thirty-three MQTLs overlapped the marker trait associations (MTAs) previously reported in genome-wide association studies (GWAS) for flag leaf traits in wheat. A total of 2262 candidate genes for flag leaf size, which were involved in the peroxisome, basal transcription factor, and tyrosine metabolism pathways were identified in MQTL regions by the in silico transcriptome assessment. Of these, the expression analysis of the available genes revealed that 134 genes with > 2 transcripts per million (TPM) were highly and specifically expressed in the leaf. These candidate genes could be critical to affect flag leaf size in wheat.ConclusionsThe findings will make further insight into the genetic determinants of flag leaf size and provide some reliable MQTLs and putative candidate genes for the genetic improvement of flag leaf size in wheat.

Transcriptomic analysis reveals biosynthesis genes and transcription factors related to leaf anthocyanin biosynthesis in Aglaonema commutatum

BackgroundAglaonema commutatum ‘Red Valentine’, as a foliage ornamental plant, is widely used for interior and exterior decoration because of its easy cultivation and management. However, reduced proportion of red foliage during large-scale production of A. commutatum seedlings is a frequent occurrence, which has considerable implications on the plant’s ornamental and market value. However, the molecular mechanisms underlying this phenomenon remain unclear.ResultsTo explore the molecular basis of the variation in leaf color of A. commutatum Red Valentine, we performed transcriptome sequencing with the Illumina platform using two different varieties of A. commutatum, namely Red Valentine and a green mutant, at three different stages of leaf development. We annotated 63,621 unigenes and 14,186 differentially expressed genes by pairwise comparison. Furthermore, we identified 26 anthocyanin biosynthesis structural genes. The transcript per million (TPM) values were significantly higher for Red Valentine than for the green mutant in all three developmental stages, consistent with the high anthocyanin content of Red Valentine leaves. We detected positive transcription factors that may be involved in the regulation of anthocyanin biosynthesis using BLAST and through correlation analysis. Downregulation of these transcription factors may downregulate the expression of anthocyanin genes. We obtained full-length cDNA of the anthocyanin biosynthesis and regulatory genes and constructed phylogenetic trees to ensure accuracy of the analysis.ConclusionsOur study provides insights into the molecular mechanisms underlying leaf variation in A. commutatum Red Valentine and may be used to facilitate the breeding of ornamental cultivars with high anthocyanin levels.

Abstract A010: Deciphering molecular and cellular differences between black and white colorectal cancers through in-depth transcriptomic analyses

Abstract Background Compared to all other racial groups, black Americans are most likely to be diagnosed with colorectal cancer (CRC) and are most likely to die from the disease. These racial disparities have been largely attributed to social and economic conditions that impact the prevalence of co-morbidities and access to quality health care. However, the molecular and cellular differences in CRC tumors between black and white subjects remain largely uncharacterized. Methods We analyzed paired tumor and adjacent normal RNA sequencing reads collected from 15 black and 18 white subjects at Downstate Medical Center and Stony Brook University Hospital (DSSBU). 273 additional RNA sequencing and clinicopathologic data from The Cancer Genome Atlas (TCGA) were used as a validation cohort. In the TCGA dataset, 54 black subjects were propensity score matched to 108 white subjects by age, sex, tumor location and tumor stage. Differential expression analysis was performed with EdgeR and Wilcoxon rank-sum test, which was recently recommended for human population samples. Gene Ontology (GO) enrichment analysis of DEGs (absolute log 2-fold change &amp;gt; 1, adjust p value &amp;lt; 0.05) was performed using enrichR. Gene set enrichment analysis (GSEA) of hallmark gene sets was applied to the gene list ranked by fold changes. The TPM (transcript per million) data was used to estimate relative cell abundance via xCell. Antitumor immune response likelihood scores were generated via the Estimate Systems Immune Response (EaSIeR) R package. Results EdgeR analysis identified 15 and 80 DEGs in the DSSBU and TCGA datasets, respectively. Two DEGs, CXCL10 and RPPH1 have decreased expression while EEF1A1P13 has higher expression in black samples from both datasets. CXCL10 remained a DEG under the Wilcoxon test in the TCGA dataset and in propensity score matched samples. GO enrichment analysis identified “Chemokine-mediated signaling pathway” as a significantly different biological process between tumor groups with a FDR of 0.043 and 0.0004 in TCGA and DSSBU respectively. GSEA computed a negative enrichment score for IL6 JAK STAT3 signaling, Interferon alpha response, Interferon gamma response, Allograft rejection and Myc targets V1 in the black group from both datasets. xCell analysis of the DSSBU data revealed significant enrichment for endothelial cells, adipocytes and hematopoietic stem cells in black tumor samples, while white samples were significantly enriched for basophils, T helper 2 cells and common lymphoid progenitor cells. No significant differences in cell type enrichment were identified in the TCGA dataset. Predicted immunotherapy response scores were not significantly different between races in both cohorts. Conclusion Our results show differences in genes and pathways associated with inflammation and immune cell recruitment which may impact anti-tumor activity. Our future work will include experimental validation of interesting genes accounting for the differences. Citation Format: Dimitri F. Joseph, Ellen Li, Bin Chen, Joseph F. LaComb, Alexandra Guillaume. Deciphering molecular and cellular differences between black and white colorectal cancers through in-depth transcriptomic analyses [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr A010.

Chloroplast gene expression level is negatively correlated with evolutionary rates and selective pressure while positively with codon usage bias in Ophioglossum vulgatum L

BackgroundCharacterization of the key factors determining gene expression level has been of significant interest. Previous studies on the relationship among evolutionary rates, codon usage bias, and expression level mostly focused on either nuclear genes or unicellular/multicellular organisms but few in chloroplast (cp) genes. Ophioglossum vulgatum is a unique fern and has important scientific and medicinal values. In this study, we sequenced its cp genome and transcriptome to estimate the evolutionary rates (dN and dS), selective pressure (dN/dS), gene expression level, codon usage bias, and their correlations.ResultsThe correlation coefficients between dN, dS, and dN/dS, and Transcripts Per Million (TPM) average values were -0.278 (P = 0.027 < 0.05), -0.331 (P = 0.008 < 0.05), and -0.311 (P = 0.013 < 0.05), respectively. The codon adaptation index (CAI) and tRNA adaptation index (tAI) were significantly positively correlated with TPM average values (P < 0.05).ConclusionsOur results indicated that when the gene expression level was higher, the evolutionary rates and selective pressure were lower, but the codon usage bias was stronger. We provided evidence from cp gene data which supported the E-R (E stands for gene expression level and R stands for evolutionary rate) anti-correlation.