The Cancer Genome Atlas Pan-Cancer Research Articles

Investigation of SPOCD1 as A Suitable Diagnostic and Prognostic Biomarker in Various Common Cancer Types: Bioinformatics and Practical Analysis.

The objective of this study was to assess whether Spen paralogue and orthologue C-terminal domain containing 1 (SPOCD1) gene expression could serve as a valuable prognostic and diagnostic biomarker in common cancers, drawing from insights in recent literature. We sought to verify this concept by utilizing data sourced from The Cancer Genome Atlas (TCGA) alongside clinical samples. In this bioinformatics and experimental study, SPOCD1 RNA-seq data from 12 common cancers were collected from TCGA Pan-Cancer Atlas using the R package "TCGA BIOLINKS" and normalized for analysis. Various analytical tools, including receiver operating characteristic (ROC) curves, Kaplan-Meier and Coxregression analyses, and pathway enrichment analysis via the molecular signatures database (MSigDB), were applied. Drug resistance/sensitivity correlations with SPOCD1 expression were explored using the Gene Expression Omnibus (GEO) database. Clinical colorectal cancer (CRC) samples, including both colon and rectal malignant samples, were also evaluated. The results showed elevated SPOCD1 expression in most cancers (9/12), with notable prognostic value in COAD, HNSC, KICH, and LIHC, and a correlation with poor prognosis in COAD for disease-free survival. ROC curve analysis suggested SPOCD1 as a diagnostic biomarker in the majority of cases (7/12), although this pattern was inconsistent in clinical CRC samples. Pathway enrichment analysis revealed a strong correlation between SPOCD1 expression and critical molecular pathways. Unlike former results, we found that SPOCD1 upregulated when interacting with PD-0325901. However, treating with Panobinostat led to downregulation. Both are as anticancer reagents. This study confirms the potential of SPOCD1 as a diagnostic and prognostic biomarker in prevalent cancers. However, extensive clinical data, particularly for CRC, are required to validate its reliability. Different COAD subtypes may exhibit varying correlations with SPOCD1 expression levels, underscoring the need for further investigation to fully understand its diagnostic and prognostic value.

Integrated analysis of single-cell and bulk RNA sequencing data reveals a myeloid cell-related regulon predicting neoadjuvant immunotherapy response across cancers

BackgroundImmunotherapy has brought about a paradigm shift in the treatment of cancer. However, the majority of patients exhibit resistance or become refractory to immunotherapy, and the underlying mechanisms remain to be explored.MethodsSing-cell RNA sequencing (scRNA‑seq) datasets derived from 1 pretreatment and 1 posttreatment achieving pathological complete response (pCR) patient with lung adenocarcinoma (LUAD) who received neoadjuvant immunotherapy were collected, and pySCENIC was used to find the gene regulatory network (GRN) between cell types and immune checkpoint inhibitor (ICI) response. A regulon predicting ICI response was identified and validated using large‑scale pan-cancer data, including a colorectal cancer scRNA‑seq dataset, a breast cancer scRNA‑seq dataset, The Cancer Genome Atlas (TCGA) pan-cancer cohort, and 5 ICI transcriptomic cohorts. Symphony reference mapping was performed to construct the myeloid cell map.ResultsThirteen major cluster cell types were identified by comparing pretreatment and posttreatment patients, and the fraction of myeloid cells was higher in the posttreatment group (19.0% vs. 11.8%). A PPARG regulon (containing 23 target genes) was associated with ICI response, and its function was validated by a colorectal cancer scRNA‑seq dataset, a breast cancer scRNA‑seq dataset, TCGA pan-cancer cohort, and 5 ICI transcriptomic cohorts. Additionally, a myeloid cell map was developed, and cluster I, II, and III myeloid cells with high expression of PPARG were identified. Moreover, we constructed a website called PPARG (https://pparg.online/PPARG/ or http://43.134.20.130:3838/PPARG/), which provides a powerful discovery tool and resource value for researchers.ConclusionsThe PPARG regulon is a predictor of ICI response. The myeloid cell map enables the identification of PPARG subclusters in public scRNA-seq datasets and provides a powerful discovery tool and resource value.

The expression of immune co-stimulators as a prognostic predictor of head and neck squamous cell carcinomas and oral squamous cell carcinomas

Background/purposeT cells require second immune checkpoint molecules for activation and immune memory after antigen presentation. We found that inducible co-stimulator (ICOS) has been a favorable prognostic factor amongst B7 immune checkpoint co-stimulators (ICSs) families in head and neck squamous cell carcinoma (HNSCC) and oral SCC (OSCC). Materials and methodsThis study analyzed the expression of non-B7 tumor necrosis factor (TNF) superfamily ICSs in the Cancer Genome Atlas (TCGA) HNSCC cohort, our OSCC cohort, and TCGA pan-cancer datasets. The correlation in expression, prognosis, and immune status was assessed. ResultsThe higher expression of CD27, CD30, CD40L, death domain 3 (DR3), and OX40, presumably on the T cell surface, defined better overall survival of HNSCC patients. Besides, CD27, CD30, CD40L, and OX40 were highly correlated with ICOS expression in tumors. CD27, CD40L, and DR3 expression are higher in HPV+ HNSCC tumors than in HPV- tumors. The combined expression level of CD27/OX40 or CD27/CD40L/OX40 enables the potent survival prediction of small, less nodal involvement, early stage, and HPV + tumor subsets. Tumors expressing high CD27, CD30, CD40L, ICOS, and OX40 exhibited enhanced immune cell infiltration. The high correlation in the expression of these ICSs was also noted in the vast majority of tumor types in TCGA datasets. ConclusionThe findings of this study not only confirm the potential of the concordant stimulation of CD27, CD30, CD40L, ICOS, and OX40 as a crucial strategy in cancer immunotherapy but also inspire further exploration into the field, highlighting the promising future of cancer treatment.

Characterization of zinc finger protein 536, a neuroendocrine regulator, using pan-cancer analysis

BackgroundPrevious studies suggested that zinc finger protein 536 (ZNF536) was abundant in the central brain and regulated neuronal differentiation. However, the role of ZNF536 in cancer has remained unclear.MethodsZNF536 mutation, copy number alteration, DNA methylation, and RNA expression were explored using public portals. Data from The Cancer Genome Atlas (TCGA) were utilized to analyze pathways and tumor microenvironment (TME), with a focus on prognosis in both TCGA and immunotherapy pan-cancer cohorts. Methylated ZNF536 from small cell lung cancer (SCLC) cell lines were utilized to train with probes for conducting enrichment analysis. Single-cell RNA profile demonstrated the sublocalization and co-expression of ZNF536, and validated its targets by qPCR.ResultsGenetic alterations in ZNF536 were found to be high-frequency and a single sample could harbor different variations. ZNF536 at chromosome 19q12 exerted a bypass effect on CCNE1, supported by CRISPR data. For lung cancer, ZNF536 mutation was associated with longer survival in primary lung adenocarcinoma (LUAD), but its prognosis was poor in metastatic LUAD and SCLC. Importantly, ZNF536 mutation and amplification had opposite prognoses in Stand Up To Cancer-Mark Foundation (SU2C-MARK) LUAD cohort. ZNF536 mutation altered the patterns of genomic alterations in tumors, and had distinct impacts on the signaling pathways and TME compared to ZNF536 amplification. Additionally, ZNF536 expression was predominantly in endocrine tumors and brain tissues. High-dimensional analysis supported this finding and further revealed regulators of ZNF536. Considering that the methylation of ZNF536 was involved in the synaptic pathway associated with neuroendocrine neoplasms, demonstrating both diagnostic and prognostic value. Moreover, we experimentally verified ZNF536 upregulated neuroendocrine markers.ConclusionsOur results showed that ZNF536 alterations in cancer, including variations in copy number, mutation, and methylation. We proved the involvement of ZNF536 in neuroendocrine regulation, and identified highly altered ZNF536 as a potential biomarker for immunotherapy.

NECTIN4 Amplification Is Frequent in Solid Tumors and Predicts Enfortumab Vedotin Response in Metastatic Urothelial Cancer.

The anti-NECTIN4 antibody-drug conjugate enfortumab vedotin (EV) is approved for patients with metastatic urothelial cancer (mUC). However, durable benefit is only achieved in a small, yet uncharacterized patient subset. NECTIN4 is located on chromosome 1q23.3, and 1q23.3 gains represent frequent copy number variations (CNVs) in urothelial cancer. Here, we aimed to evaluate NECTIN4 amplifications as a genomic biomarker to predict EV response in patients with mUC. We established a NECTIN4-specific fluorescence in situ hybridization (FISH) assay to assess the predictive value of NECTIN4 CNVs in a multicenter EV-treated mUC patient cohort (mUC-EV, n = 108). CNVs were correlated with membranous NECTIN4 protein expression, EV treatment responses, and outcomes. We also assessed the prognostic value of NECTIN4 CNVs measured in metastatic biopsies of non-EV-treated mUC (mUC-non-EV, n = 103). Furthermore, we queried The Cancer Genome Atlas (TCGA) data sets (10,712 patients across 32 cancer types) for NECTIN4 CNVs. NECTIN4 amplifications are frequent genomic events in muscle-invasive bladder cancer (TCGA bladder cancer data set: approximately 17%) and mUC (approximately 26% in our mUC cohorts). In mUC-EV, NECTIN4 amplification represents a stable genomic alteration during metastatic progression and associates with enhanced membranous NECTIN4 protein expression. Ninety-six percent (27 of 28) of patients with NECTIN4 amplifications demonstrated objective responses to EV compared with 32% (24 of 74) in the nonamplified subgroup (P < .001). In multivariable Cox analysis adjusted for age, sex, and Bellmunt risk factors, NECTIN4 amplifications led to a 92% risk reduction for death (hazard ratio, 0.08 [95% CI, 0.02 to 0.34]; P < .001). In the mUC-non-EV, NECTIN4 amplifications were not associated with outcomes. TCGA Pan-Cancer analysis demonstrated that NECTIN4 amplifications occur frequently in other cancers, for example, in 5%-10% of breast and lung cancers. NECTIN4 amplifications are genomic predictors of EV responses and long-term survival in patients with mUC.

Abstract 4768: Differential expression of triptolide gene targets in human papillomavirus positive and negative head and neck squamous cell carcinomas

Abstract Head and neck squamous cell carcinoma (HNSCC) is the fifth most common cancer in the US. HPV represents a more recent risk factor for which treatment may be individualized. HPV expresses viral E6 and E7 oncoproteins which downregulate wild type p53 and Rb respectively. Triptolide, a derivative of a traditional Chinese medicinal herb, has been shown to inhibit viral E6, thereby reactivating wild type p53. Therefore, triptolide may target HPV positive HNSCC as a putative treatment. To explore this, we compared mutation, methylation, and expression of triptolide gene targets between HPV positive and HPV negative samples from The Cancer Genome Atlas (TCGA) program. Patient data was queried from the TCGA PanCancer Atlas for HNSCC using cBioPortal. This included a total of 523 patients with 487 meeting inclusion criteria. 415 patients were HPV (-) and 72 were HPV (+).Differences in direct genomic alterations, DNA methylation, mRNA expression, and protein expression of the 34 known triptolide targets were compared between these two groups using the Benjamini-Hochberg Procedure with a significance level of q &amp;lt; 0.05. Significant differences of the studied genes (q &amp;lt; 0.05) are shown in Table 1. There were no significant differences in protein expression. Of the 34 known triptolide targets, HPV positive and negative samples show variable enrichment. Triptolide targets exhibit greater methylation and mRNA expression in HPV positive samples supporting triptolide’s efficacy in these samples. However, other triptolide targets are the differentially expressed and methylated in HPV negative samples, so the compound may have a beneficial effect in this context as well. Further studies are required to test efficacy of triptolide against HPV positive and HPV negative HNSCC in vitro and in vivo models. Table 1. Significant genomic differences between HPV (+) and HPV (-) samples shown. Enriched in HPV (+) Enriched in HPV (-) Gene Log2 ratio q-value Gene Log2 ratio q-value DNA methylation Direct genomic mutations VEGFA 0 5.85E-03 TP53 3.08 4.53E-29 BCL2 0.07 9.78E-18 DNA methylation JUN 0 4.89E-02 RELA 0.07 5.06E-04 CD80 0.06 2.56E-02 STAT3 0.24 3.19E-20 BIRC3 0.01 1.29E-04 TNF 0.07 4.31E-04 CD274 0.07 4.40E-03 XIAP 0.07 8.02E-06 CCR7 0.11 3.56E-08 CXCR4 0.04 3.58E-03 CD1A 0.10 2.36E-05 CD40 0.06 1.00E-05 C3 0.06 6.91E-03 CD14 0.04 8.12E-03 CD86 0.03 2.45E-02 mRNA expression mRNA expression RELA 0.16 1.90E-03 STAT3 0.25 4.22E-03 CDKN1A 0.28 3.00E-02 BCL2 1.95 1.68E-14 PLAU 1.37 3.24E-12 CASP3 0.45 2.55E-07 CXCL8 1.05 1.51E-04 TP53 1.45 3.16E-25 MCL1 0.23 3.59E-04 IL2 0.52 2.13E-06 TGFB1 0.77 3.27E-11 INFG 1.15 2.08E-05 DEFB4A 1.10 1.77E-02 CXCR4 1.18 8.03E-07 BIRC3 1.20 6.14E-06 IL23A 0.77 3.88E-04 CCR7 1.18 2.10E-06 CD40 0.54 2.27E-04 Citation Format: William E. Kamm, Lindsey J. Mortenson, Frank G. Ondrey. Differential expression of triptolide gene targets in human papillomavirus positive and negative head and neck squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4768.

Gene alterations in the nuclear transport receptor superfamily: A study of head and neck cancer.

In cancer cells, the nuclear transport system is often disrupted, leading to abnormal localization of nuclear proteins and altered gene expression. This disruption can arise from various mechanisms such as mutations in genes that regulate nuclear transport, altered expression of transport proteins, and changes in nuclear envelope structure. Oncogenic protein build-up in the nucleus due to the disturbance in nuclear transport can also boost tumor growth and cell proliferation. In this study, we performed bioinformatic analyses of 23 key nuclear transport receptors using genomic and transcriptomic data from pancancer and head and neck squamous cell carcinoma (HNSCC) datasets from The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia and found that the total alteration frequency of 23 nuclear transport receptors in 2691 samples of the PCAWG Consortium was 42.1% and a high levels of genetic alterations was significantly associated with poor overall survival. Amplification was the most common type of genetic alterations, and results in the overexpression of nuclear transport receptors in HNSCC compared to normal tissues. Furthermore, our study revealed that seven out of eight cell cycle genes (CDK1, CDK2, CDK4, CDK6, CCNA1, CCNB1, and CCNE2) were significantly and positively correlated with nuclear transport receptor genes in TCGA pancancer and CCLE datasets. Additionally, functional enrichment analysis showed that nuclear transport receptor genes were mainly enriched in the adhesion junction, cell cycle, ERBB, MAPK, MTOR and WNT signaling pathways.

Artificial intelligence (AI) –powered spatial analysis of macrophages in tumor microenvironment and its association with interferon-gamma (IFNG) signature and immune phenotype (IP) in pan-cancer dataset.

2621 Background: Macrophages are known to play a crucial role in cancer progression, but few studies have reported macrophage spatial distribution and its association with genomic signatures and tumor-infiltrating lymphocytes (TIL). Here, we applied an AI-powered Whole Slide Image (WSI) analyzer in The Cancer Genome Atlas (TCGA) pan-cancer dataset to show correlation between specific localization of macrophage and anti-tumor activity, which is reflected by IFNG signature and IP based on spatial TIL density. Methods: For spatial macrophage analysis in TCGA pan-cancer dataset, we used Lunit SCOPE IO, an AI-powered H&amp;E WSI analyzer, which identifies and quantifies various classes of cells such as tumor cell, macrophage, and TIL within cancer or stroma area. IPs were defined as follows: inflamed IP (IIP) as high intratumoral TIL (iTIL) and stromal TIL (sTIL); immune-excluded IP (IEP) as low iTIL and high sTIL; immune-desert IP (IDP) as low TIL overall. The immune cell profiling and quantification of the IFNG signature score were analyzed by using CIBERSORT and RSEM. Results: In TCGA dataset (N = 7,402), median macrophage density in total TME area (tMP) was 19.9 /mm2 (interquartile range [IQR] 9.0-44.9), and it was highly enriched in lung adenocarcinoma (median 65.1, IQR 34.3-110.0), lung squamous cell carcinoma (61, 32.4-101.3), and head and neck squamous cell carcinoma (39.4, 19.6-77.3). Based on IP classification, tMP was highly enriched in IIP (37.8, 18.7-73.8) compared to IEP (25.5, 13.8-48.6) and IDP (8.9, 4.4-17.1). Interestingly, spatial analysis showed that intratumoral macrophage density (iMP) in IIP was highly enriched compared to the others (mean fold change 2.57) and the difference of stromal macrophage density (sMP) among IPs was relatively smaller (mean fold change x1.85). Classified into four groups based on median values of iMP and sMP, high-iMP/high-sMP group had the highest proportion of IIP (47.3% vs 18.7%, p &lt; 0.001), whereas low-iMP/high-sMP group had the highest one of IEP (54.9% vs 32.3%, p &lt; 0.001) compared to the others, respectively. Moreover, the high-iMP/high-sMP group had the highest level of IFNG signature score among 4 groups (x2.13 times higher, p &lt; 0.001). Notably, both iMP and sMP were significantly correlated with CD68 expression (rho 0.292 and 0.287) but CSF1R expression was more strongly correlated with iMP (rho 0.259) compared to sMP (rho 0.078). Conclusions: IIP, IFNG signature, and CSF1R expression are more strongly correlated with intratumoral macrophage density versus stromal macrophage density, suggesting a relationship between macrophage location and function in the TME warranting further investigation.

Abstract 4266: Genomic landscape of prostate cancer: Bioinformatic analysis of TCGA PanCancer data by race and identification of key pathways and genes in PTEN mutation

Abstract Issue: Prostate cancer (Pca) is still among the most frequently diagnosed cancers in adults. The most often mutated gene in PCa is PTEN (Phosphatase and Tensin Homolog), making it a possible biomarker for prospective customized therapy. The root causes of health inequalities in PCa have not received much attention. Numerous online databases, like The Cancer Genome Atlas (TCGA), provide gene expression data and other data, allowing researchers to further investigate new processes or therapy options. However, there hasn't been much study on tailored therapy up to this point since there may have been difficulties with access to genomic information and bioinformatic tools. Methods: The PCa data in cBioPortal comprises 494 samples. We selected 154 samples comprising 7 Blacks and 147 Whites. We used the TCGA database to obtain the PCa gene expression and DNA methylation data for study. In order to examine the data by race/ethnicity, gene expression, DNA methylation, and genomic changes, we evaluated at the differentially expressed and methylated genes using online tools (cBioPortal). To evaluate gene methylation, expression, and alterations, log ratios of means in Blacks to means in Whites were computed across the data. Additionally, Kaplan-Meier survival analysis, protein interaction analysis, and multivariable data analysis were carried out. Results: PCa data in the TCGA database showed PTEN mutation in 20% patients. The median age of participants was 63 years (range: 42-78). For every category of data assessed, there were significant differences by race/ethnicity (q &amp;lt; 0.05). In the survival analysis, SLC2A1, NUDT12, CUTALP, and other genes were associated with survival. Conclusion: Genetic alteration, expression, and DNA methylation are each significantly different by the two race groups compared. According to our research, PTEN mutation in PCa may influence several genes and biological processes. Citation Format: Olumide Arigbede, Sarah G. Buxbaum, Sara Falzarano, Suhn K. Rhie. Genomic landscape of prostate cancer: Bioinformatic analysis of TCGA PanCancer data by race and identification of key pathways and genes in PTEN mutation. [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 4266.

IKBIP is a Predictive Biomarker Related to Immunosuppressive Microenvironment in Digestive System Malignancies.

IKBKB-interacting protein (IKBIP) has rarely been reported in tumor research. This study aimed to evaluate IKBIP role in tumor progression. mRNA (messenger ribonucleic acid) expression, clinical characteristics and predictive values of IKBIP were assessed. R package "clusterProfiler" was used to examine the potential mechanisms in which IKBIP may involve. Immune cell infiltration and its correlation with IKBIP was also analyzed. We further evaluated IKBIP influence on drug resistance. It was found that IKBIP was overexpressed and related to poorer survival in most types of tumors. IKBIP expression was strongly related to immunosuppressive cells in the TCGA (The Cancer Genome Atlas) pan-cancer samples. These immunosuppressive cells included tumor-related macrophages, tumor-related fibroblasts, and regulatory T cells. Moreover, immunosuppressive genes and immune checkpoints were positively related to IKBIP expression in several tumor types. Furthermore, patients with IKBIP overexpressed did not respond to most anti-cancer medications. It was also found that compared to control group, the number of invasive cells is four times that of IKBIP overexpression group, and the number of clone forming cells is six times that of IKBIP overexpression group. IKBIP overexpression promoted colon cancer cells invasiveness and clonogenesis by Transwell assay and colon formation assay. According to current findings, IKBIP is a probable oncogene and predictive marker for most of tumor types. High IKBIP expression is associated with tumor immunosuppression.

Contribution of the Testosterone Androgen Receptor-PARD3B Signaling Axis to Tumorigenesis and Malignance of Glioblastoma Multiforme through Stimulating Cell Proliferation and Colony Formation.

Background: Glioblastoma multiforme (GBM) is the most common and malignant brain tumor with very poor prognoses. After surgical resection of the primary tumor, rapid proliferation of residual glioblastoma cells is a critical cause explaining tumor malignance and recurrence. In this study, we evaluated de novo roles of the testosterone androgen receptor (AR)–PARD3B signaling axis in the tumorigenesis and malignance of human GBM and the possible related mechanisms. Methods: AR and PARD3B gene expressions and their correlations were mined from The Cancer Genome Atlas (TCGA) database and analyzed using the UALCAN system. Analyses using a real-time PCR, cell proliferation, and colony formation and a loss-of-function strategy by suppressing AR activity with its specific inhibitor, enzalutamide, were then carried out to determine roles of the testosterone AR–PARD3B signaling axis in tumor malignance. Results: Expressions of AR, PARD3B mRNA, and proteins in human GBM tissues were upregulated compared to normal human brain tissues. In contrast, levels of AR and PARD3B mRNA in most TCGA pan-cancer types were downregulated compared to their respective normal tissues. Interestingly, a highly positive correlation between AR and PARD3B gene expressions in human GBM was identified. The results of a bioinformatics search further showed that there were five AR-specific DNA-binding elements predicted in the 5′ promoter of the PARD3B gene. Regarding the mechanisms, exposure of human glioblastoma cells to testosterone induced AR and PARD3B gene expressions and successively stimulated cell proliferation and colony formation. Suppressing AR activity concurrently resulted in significant attenuations of testosterone-induced PARD3B gene expression, cell proliferation, and colony formation in human glioblastoma cells. Conclusions: This study showed the contribution of the testosterone AR–PARD3B signaling axis to the tumorigenesis and malignance of human GBM through stimulating cell proliferation and colony formation. Therefore, the AR-PARD3B signaling axis could be targeted for potential therapy for human GBM.

Comprehensive Analysis Reveals USP45 as a Novel Putative Oncogene in Pan-Cancer

Background: Deubiquitinating enzymes specifically removes ubiquitin molecules from ubiquitin-tagged target proteins, thereby inhibiting the degradation of target proteins and playing an important role in tumor. However, the mechanism of deubiquitinating enzyme USP45 in tumors remains unclear. Methods: Based on the RNA-seq data of tissues and cell lines in The Cancer Genome Atlas (TCGA) database, GTEx and CCLE database, the pan-cancer analysis of USP45 expression and survival outcome were performed using R software and Kaplan-Meier Plotter. The structural variants, gene mutations and gene copy number alteration of USP45 were analyzed using the TCGA Pan-Cancer Atlas Studies dataset in the cBioPortal database. The relationships between USP45 and mRNA methylation, tumor heterogeneity, tumor stemness, and tumor immunity were performed by Sangerbox platform and TIMER2.0 using Pearson correlation analysis. Through the ENCORI database and string database, we constructed the ceRNA regulatory mechanism and protein-protein interaction network for USP45. Based on the RNA-seq data in TCGA and GTEx databases, we also constructed the downstream regulatory network for USP45 using the Limma and ClusterProfiler packages of R software. At last, the protein expression levels of USP45 were detected by immunohistochemistry in tumor tissue microarrays. Results: USP45 is upregulated in most types of tumors and negatively correlated with the overall survival and recurrence-free survival of patient. Furthermore, the structural variation, gene mutations and gene copy number variation of USP45 were identified in different types of tumors. The pan-cancer analysis showed that USP45 was closely related to mRNA methylation, tumor heterogeneity and tumor stemness. In most types of tumors, the expression of USP45 was positively correlated with many immune checkpoint molecules and immune regulators such as PD-L1, while negatively correlated with the infiltration levels of NK cells, Th1 cells, macrophages, and dendritic cells in the tumor microenvironment. Finally, we constructed the ceRNA regulatory network, protein-protein interaction network and downstream regulatory network for USP45 in different types of tumors. Conclusion: Our study firstly explored the putative oncogenic role of USP45 in pan-cancer, and provided insights for further investigation of USP45.

Role of genetic ancestry in endometrial cancers: Understanding disparities in black women.

e17631 Background: Endometrial cancer is the most common gynecological malignancy in the United States. Endometrial cancer disproportionately affects black women in terms of incidence and survival despite advancement in treatment, and preventive strategies. Identifying distinct tumor markers in patients with African ancestry will help distinguish biological determinants underlying the disparities in endometrial cancer. In this study, we investigated the genetic alterations in endometrial cancer in individuals of African (AFR) ancestry using The Cancer Genome Atlas (TCGA). Methods: The genomic and clinical data of TCGA PanCancer Atlas uterine corpus endometrial carcinoma (UCEC) were explored using CBioportal (http://www.cbioportal.org/). We utilized The Cancer Genome Ancestry Atlas (TCGAA) and LAMP for estimates of genetic ancestry and quantitative ancestral composition. This dataset contains 529 patients, including 357 self-reported whites and 107 blacks. For each case the proportion of European, West African, East Asian, Native American ancestry was estimated. The dominant ancestry was defined as ≥50% of admixture from one reference population. Differences in gene mutation frequency were analyzed based on AFR ancestry proportion. The Kaplan-Meier curves were generated, and Cox regression analyses were performed. Results: Global genetic ancestry analysis identified 115 AFR ancestry cases with mean ancestry of 80.4%. The dominant AFR ancestry subject matched the self-reported race with 94% accuracy. We identified 23 subjects with ≥90% AFR ancestry, 43 subjects with 80-90% AFR ancestry, 34 subjects with 70-80% AFR ancestry, and 15 subjects with 50-70% AFR ancestry. TP53 was the most frequently mutated gene in patients with AFR ancestry(49.5%). ≥90% AFR ancestry had the highest rate of TP53 mutations (52.2%) compared with 80-90% AFR ancestry (51.2%), 70-80% AFR ancestry (43.8%), and 50-70% AFR ancestry (41.7%). PTEN was less commonly mutated in patients with higher proportion of AFR ancestry (47.8% in ≥90% AFR ancestry, 39.0% in 80-90% AFR ancestry, 59.4% in 70-80% AFR ancestry, and 66.7% in 50-70% AFR ancestry). PIK3CA mutation frequency was almost identical in all AFR ancestry groups studied. No significant differences in overall survival was observed between AFR ancestry groups. Conclusions: We aimed to investigate the etiology of endometrial cancer disparities by analyzing the effect of ancestry on genetic alterations in endometrial cancer. This study demonstrated differences in the mutation frequency among patients with AFR ancestry. We have shown that TP53 mutations were seen more frequently in patients with higher AFR ancestry, which may confer a poorer prognosis. Nonetheless, to validate the findings of this study, future studies should be conducted with larger sample sizes and more diverse ancestral groups to explore how genetic ancestry impacts tumorigenesis and cancer progression.

Reduced expression of lncRNA DLEU7-AS1 is a novel favorable prognostic factor in acute myeloid leukemia.

The objective of our study was to measure DLEU7-AS1 expression in de novo acute myeloid leukemia (AML) whilst also analyzing its clinical relevance. We used gene expression data from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Cancer Cell Line Encyclopedia (CCLE) and Genotype-Tissue Expression project (GTEx) to assess the expression profile of DLEU7-AS1 in pan-cancers, cancer cell lines and normal tissues. Reverse transcription-quantitative PCR was used to measure DLEU7-AS1 expression in bone marrow from 30 normal individuals and 110 patients with de novo AML. DLEU7-AS1 expression was found to be markedly reduced in the AML samples of the TCGA pan-cancer datasets. In our PCR validation, DLEU7-AS1 expression was significantly decreased in the AML samples compared with that in controls (P<0.001). Low DLEU7-AS1 expression (DLEU7-AS1low) correlated positively with lower blood platelet counts (P=0.029). In addition, low DLEU7-AS1 expression was more frequently observed in the intermediate (58%; 44/76) and favorable karyotypes (65%; 15/23) compared with that in the poor karyotype (10%; 1/10; P=0.005). In particular, patients with high expression levels of DLEU7-AS1 (DLEU7-AS1high) showed lower complete remission rates (P=0.002) than patients with DLEU7-AS1low. Survival analysis revealed that patients with DLEU7-AS1low had longer overall survival (OS) than patients with DLEU7-AS1high (P<0.05). Multivariate Cox analysis demonstrated that in patients with non-acute promyelocytic leukemia (non-M3) who were ≤60 years old, DLEU7-AS1 expression was an independent prognostic factor for OS. Furthermore, we found distinct correlations among the expression of DLEU7-AS1, infiltration by immune cells and immune checkpoint genes in AML.

Abstract P062: PI3Kγδ inhibitor plus radiation enhances the antitumor immune effect of PD-1 blockade in syngenic murine breast cancer and humanized patient-derived xenograft model

Abstract Purpose: Breast cancer is generally viewed as immunologically ‘cold', imposing an immune-suppressive tumor microenvironment (TME) and responding poorly to lone immune checkpoint blockade (ICB). As an adjunct to ICB, radiation therapy (RT) holds promise in terms of in situ tumor vaccination effect, although it is known to promote immune suppression, increasing regulatory T cells (Treg), myeloid-derived suppressor cells (MDSCs), and M2 tumor-associated macrophages (TAMs). It was our contention that combined use of RT and a PI3Kγδ inhibitor to combat immune suppression might enhance the efficacy of ICB. Methods: Murine breast cancer cells (4T1) were grown in both immune-competent and -deficient BALB/c mice, and tumors were irradiated by 3 fractions of 24 Gy. A PD-1 blockade and a PI3Kγδ inhibitor were then administered every other day for 2 weeks. Tumors from humanized patient-derived breast cancer xenograft (PDX) model was sequenced to identify immune-related pathways and to profile infiltrated immune cells. Transcriptomic and clinical data were acquired from The Cancer Genome Atlas (TCGA) pan-cancer cohort, and the deconvolution algorithm was used to profile immune cell repertoire. Results: In the immune-competent syngenic 4T1 murine tumor model, PD-1 blockade alone led to tumor hyperprogression, whereas a three-pronged strategy of PI3Kγδ inhibitor, RT, and PD-1 blockade significantly delayed primary tumor growth, boosted abscopal effect, and improved animal survival by comparison. The immune-deficient syngenic 4T1 murine tumor model failed to show this synergism in delaying tumor growth and the abscopal effect. According to FACS analysis, RT significantly increased not only CD8+cytotoxic T-cell fractions but also immune-suppressive Tregcells, MDSCs, and M2 TAMs. However, PI3Kγδ inhibitor significantly lowered proportions of Treg, MDSCs, and M2 TAMs, achieving dramatic gains in splenic, nodal, and tumor CD8+ T-cell populations after triple combination therapy. There were significantly decreased tumor expressions of p-AKT, PD-L1, and HIF1α by PI3Kγδ inhibition. Triple combination therapy significantly delayed primary tumor growth in humanized PDX model as well and analyses of RNA sequencing data of humanized PDX samples showed decreased immune suppressive pathways with decreased and M2 macrophage and increased CD8+ T-cell by triple combination therapy. In the TCGA pan-cancer cohort, high Treg/CD8+T-cell and M2/M1 TAM ratios and poor overall patient survival was associated with high PIK3CG (PI3Kγ) or PIK3CD (PI3Kδ) gene expression. Conclusion: These findings collectively indicate that PI3Kγ and PI3Kδ are clinically relevant targets in an immunosuppressive TME. Combining PI3Kγδ inhibitor, RT, and PD-1 blockade may thus be a viable approach, helping to overcome the therapeutic resistance of immunologically cold tumors such as breast cancer. Citation Format: In Ah Kim, Min Guk Han, Bum Sup Jang, Mi Hyun Kang. PI3Kγδ inhibitor plus radiation enhances the antitumor immune effect of PD-1 blockade in syngenic murine breast cancer and humanized patient-derived xenograft model [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P062.

Peritumoral CD90+CD73+ cells possess immunosuppressive features in human non-small cell lung cancer.

SummaryBackgroundAlthough T cell abundance in solid tumours is associated with better outcomes, it also correlates with a stroma-mediated source of immune suppression driven by TGFβ1 and poor overall survival. Whether this also is observed in non-small cell lung cancer (NSCLC) is unknown.MethodsWe utilized molecular analysis of The Cancer Genome Atlas (TCGA) NSLCC cohort to correlate immune activation (IA) gene expression and extracellular matrix/stromal (ECM/stromal) gene expression with patient survival. In an independent cohort of NSCLC samples, we used flow cytometry to identify mesenchymal subsets and ex vivo functional studies to characterize their immune regulatory function.FindingsWe observed a high enrichment in a core set of genes defining an IA gene expression signature in NSCLC across TCGA Pan-cancer cohort. High IA signature score correlates with enrichment of ECM/stromal gene signature across TCGA NSCLC datasets. Importantly, a higher ratio of ECM/stromal to IA gene signature score was associated with shorter overall survival. In tumours resected from a separate cohort of NSCLC patients, we identified CD90+CD73+ peritumoral cells that were enriched in the ECM/stromal gene signature, which was amplified by TGFβ1. IFNγ and TNFα-primed peritumoral CD90+CD73+ cells upregulate immune checkpoint molecules PD-L1 and IDO1 and secrete an array of cytokines/chemokines including TGFβ1. Finally, immune primed peritumoral CD90+CD73+ cells suppress T cell function, which was relieved following combined blockade of PD-L1 and TGFβ1 with IDO1 inhibition but not PD-L1 or anti-CD73 alone.InterpretationOur findings suggest that targeting PD-L1 together with independent biological features of the stroma may enhance host antitumor immunity in NSCLC.FundingLW and HY are supported by a 4-year China Scholarship Council award. This work was funded, in part, by a grant from the Cancer League of Bern, Switzerland to SRRH. Laser scanning microscopy imaging was funded by the R'Equip grant from the Swiss National Science Foundation Nr. 316030_145003.

Pan-cancer analysis of non-coding transcripts reveals the prognostic onco-lncRNA HOXA10-AS in gliomas.

Long non-coding RNAs (lncRNAs) are increasingly recognized as functional units in cancer and powerful biomarkers; however, most remain uncharacterized. Here, we analyze 5,592 prognostic lncRNAs in 9,446 cancers of 30 types using machine learning. We identify 166 lncRNAs whose expression correlates with survival and improves the accuracy of common clinical variables, molecular features, and cancer subtypes. Prognostic lncRNAs are often characterized by switch-like expression patterns. In low-grade gliomas, HOXA10-AS activation is a robust marker of poor prognosis that complements IDH1/2 mutations, as validated in another retrospective cohort, and correlates with developmental pathways in tumor transcriptomes. Loss- and gain-of-function studies in patient-derived glioma cells, organoids, and xenograft models identify HOXA10-AS as a potent onco-lncRNA that regulates cell proliferation, contact inhibition, invasion, Hippo signaling, and mitotic and neuro-developmental pathways. Our study underscores the pan-cancer potential of the non-coding transcriptome for identifying biomarkers and regulators of cancer progression.

Tumor-Infiltrating PD-1hiCD8+-T-Cell Signature as an Effective Biomarker for Immune Checkpoint Inhibitor Therapy Response Across Multiple Cancers.

BackgroundStratification of patients who could benefit from immune checkpoint inhibitor (ICI) therapy is of much importance. PD-1hiCD8+ T cells represent a newly identified and effective biomarker for ICI therapy response biomarker in lung cancer. Accurately quantifying these T cells using commonly available RNA sequencing (RNA-seq) data may extend their applications to more cancer types.MethodWe built a transcriptome signature of PD-1hiCD8+ T cells from bulk RNA-seq and single-cell RNA-seq (scRNA-seq) data of tumor-infiltrating immune cells. The signature was validated by flow cytometry and in independent datasets. The clinical applications of the signature were explored in non-small-cell lung cancer, melanoma, gastric cancer, urothelial cancer, and a mouse model of breast cancer samples treated with ICI, and systematically evaluated across 21 cancer types in The Cancer Genome Atlas (TCGA). Its associations with other biomarkers were also determined.ResultsSignature scores could be used to identify the PD-1hiCD8+ T subset and were correlated with the fraction of PD-1hiCD8+ T cells in tumor tissue (Pearson correlation, R=0.76, p=0.0004). Furthermore, in the scRNA-seq dataset, we confirmed the capability of PD-1hiCD8+ T cells to secrete CXCL13, as well as their interactions with other immune cells. In 581 clinical samples and 204 mouse models treated with ICIs, high signature scores were associated with increased survival, and the signature achieved area under the receiver operating characteristic curve scores of 0.755 (ranging from 0.61 to 0.91) in predicting therapy response. In TCGA pan-cancer datasets, our signature scores were consistently correlated with therapy response (R=0.78, p<0.0001) and partially explained the diverse response rates among different cancer types. Finally, our signature generally outperformed other mRNA-based predictors and showed improved predictive performance when used in combination with tumor mutational burden (TMB). The signature score is available in the R package “PD1highCD8Tscore” (https://github.com/Liulab/PD1highCD8Tscore).ConclusionThrough estimating the fraction of the PD-1hiCD8+ T cell, our signature could predict response to ICI therapy across multiple cancers and could serve as a complementary biomarker to TMB.

Single-cell analysis of a tumor-derived exosome signature correlates with prognosis and immunotherapy response

BackgroundTumor-derived exosomes (TEXs) are involved in tumor progression and the immune modulation process and mediate intercellular communication in the tumor microenvironment. Although exosomes are considered promising liquid biomarkers for disease diagnosis, it is difficult to discriminate TEXs and to develop TEX-based predictive biomarkers.MethodsIn this study, the gene expression profiles and clinical information were collected from The Cancer Genome Atlas (TCGA) database, IMvigor210 cohorts, and six independent Gene Expression Omnibus datasets. A TEXs-associated signature named TEXscore was established to predict overall survival in multiple cancer types and in patients undergoing immune checkpoint blockade therapies.ResultsBased on exosome-associated genes, we first constructed a tumor-derived exosome signature named TEXscore using a principal component analysis algorithm. In single-cell RNA-sequencing data analysis, ascending TEXscore was associated with disease progression and poor clinical outcomes. In the TCGA Pan-Cancer cohort, TEXscore was elevated in tumor samples rather than in normal tissues, thereby serving as a reliable biomarker to distinguish cancer from non-cancer sources. Moreover, high TEXscore was associated with shorter overall survival across 12 cancer types. TEXscore showed great potential in predicting immunotherapy response in melanoma, urothelial cancer, and renal cancer. The immunosuppressive microenvironment characterized by macrophages, cancer-associated fibroblasts, and myeloid-derived suppressor cells was associated with high TEXscore in the TCGA and immunotherapy cohorts. Besides, TEXscore-associated miRNAs and gene mutations were also identified. Further experimental research will facilitate the extending of TEXscore in tumor-associated exosomes.ConclusionsTEXscore capturing tumor-derived exosome features might be a robust biomarker for prognosis and treatment responses in independent cohorts.

Abstract 1554: PI3Kγδ inhibitor combined with radiation enhances the antitumor immune effect of PD-1 blockade in syngenic murine breast cancer model and humanized patient-derived xenograft model

Abstract Purpose: Breast cancer is generally viewed as immunologically ‘cold', imposing an immune-suppressive tumor microenvironment (TME) and responding poorly to lone immune checkpoint blockade (ICB). As an adjunct to ICB, radiation therapy (RT) holds promise in terms of in situ tumor vaccination effect, although it is known to promote immune suppression, increasing regulatory T cells (Treg), myeloid-derived suppressor cells (MDSCs), and M2 tumor-associated macrophages (TAMs). It was our contention that combined use of RT and a PI3Kγδ inhibitor to combat immune suppression might enhance the efficacy of ICB. Methods: Murine breast cancer cells (4T1) were grown in both immune-competent and -deficient BALB/c mice, and tumors were irradiated by 3 fractions of 24 Gy. A PD-1 blockade (10 mg/kg) and a PI3Kγδ inhibitor (IPI145; 15 mg/kg) were then administered every other day for 2 weeks. We analyzed mRNA-sequencing data from humanized patient-derived xenograft (PDX) model to identify differentially activated immune-related pathways. Transcriptomic and clinical data were acquired from The Cancer Genome Atlas (TCGA) pan-cancer cohort, and the ‘xCell' deconvolution algorithm was used to profile immune cellular distributions at certain levels of PIK3C and PIK3CD expression. Results: In the immune-competent syngenic 4T1 murine tumor model, PD-1 blockade alone led to tumor hyperprogression, whereas a three-pronged strategy of PI3Kγδ inhibitor, RT, and PD-1 blockade significantly delayed primary tumor growth, boosted abscopal effect, and improved animal survival by comparison. The immune-deficient syngeneic 4T1 murine tumor model failed to show this synergism in delaying tumor growth and the abscopal effect. RT significantly increased not only CD8+cytotoxic T-cell fractions but also immune-suppressive Tregcells, MDSCs, and M2 TAMs. However, PI3Kγδ inhibitor significantly lowered proportions of Treg, MDSCs, and M2 TAMs, achieving dramatic gains in splenic, nodal, and tumor CD8+ T-cell populations after triple combination therapy. Triple combination therapy significantly delayed tumor growth in humanized PDX model as well. Analyses of RNA sequencing data of humanized PDX samples showed triple combination increased immune response pathways and increased CD8+ T-cell and decreased Treg and M2 TAM. In the TCGA pan-cancer cohort, higher tumor purity-adjusted Treg/CD8+T-cell and M2/M1 TAM ratios and worse overall patient survival were associated with high PIK3CG (PI3Kγ) or PIK3CD (PI3Kδ) gene expression. Conclusion: These findings collectively indicate that PI3Kγ and PI3Kδ are clinically relevant targets in an immunosuppressive TME. Combining PI3Kγδ inhibitor, RT, and PD-1 blockade may thus be a viable approach, helping to overcome the therapeutic resistance of immunologically cold tumors such as breast cancer. Citation Format: Min Guk Han, Bum-Sup Jang, Mi Hyun Kang, In Ah Kim. PI3Kγδ inhibitor combined with radiation enhances the antitumor immune effect of PD-1 blockade in syngenic murine breast cancer model and humanized patient-derived xenograft model [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 1554.