Gene Expression Profiles Of Tumors Research Articles

Abstract 3290: Evaluation of immune phenotypes and gene expression profiles in tumors from patients treated with bintrafusp alfa

Abstract Background: Bintrafusp alfa (M7824) is a first-in-class bifunctional fusion protein composed of the extracellular domain of the TGF-βRII receptor (a TGF-β “trap”) fused to a human IgG1 mAb blocking PD-L1. In expansion cohorts of global, phase 1, open-label trials (NCT02517398 and NCT02699515), extensive analysis was conducted to identify potential tumor biomarkers correlated with bintrafusp alfa efficacy. Methods: Immunohistochemistry was performed on archival or fresh formalin-fixed and paraffin-embedded tumor samples using anti-PD-L1 antibody clone 73-10. Based on H&E and PD-L1 staining with negative controls, tumors were scored as having an immune desert (<1% of the tumor microenvironment area populated by immune cells [ICs]), immune excluded (≥1% of the tumor stroma area populated by ICs that do not infiltrate the tumor), or inflamed (abundance of tumor-infiltrating ICs) phenotype by a pathologist blinded to clinical responses. Whole transcriptome messenger RNA sequencing was performed on tumor samples to determine expression levels of various genes/genes signatures associated with immune and TGF-β pathways. Results: The frequency of immune phenotypes varied by tumor type. Triple negative breast cancer (TNBC) and biliary tract cancer (BTC) had no/few inflamed tumors (0/30 and 2/28, respectively) while gastric cancer (GC) and esophageal squamous and adenocarcinoma cancers (ESCC and ESAD) had few immune desert tumors (GC, 1/30; ESCC, 1/30; ESAD, 1/27). Response to bintrafusp alfa varied by immune phenotype. The majority of responders to bintrafusp alfa in GC and squamous cell carcinoma of the head and neck (SCCHN) had inflamed phenotypes (4/5 and 3/4, respectively) while non-small cell lung cancer (NSCLC) responders had similar numbers of inflamed (7/17) and immune excluded (9/17) phenotypes. Most responders to bintrafusp alfa in ESCC, ESAD, TNBC, and BTC had immune excluded tumors (3/3, 5/6, 2/3, 5/6, respectively). Responses rarely occurred in immune desert tumors (n=1 each in NSCLC, BTC, and TNBC). Evaluation of genes/gene signatures found inflamed tumors had a significantly higher level of immune genes/gene signatures compared to immune excluded or immune desert tumors. In TNBC and ESCC there were significantly higher levels of some genes related to TGF-β biology in immune excluded tumors compared to the other two phenotypes. This potential link to TGF-β biology was less clear in other tumor types. Immune excluded and inflamed tumors could not be distinguished by molecular methods across all indications. Conclusion: The data suggest that both pathology evaluation of immune phenotypes and molecular methods are useful to understand response to bintrafusp alfa. Moreover, several complementary biomarkers are likely needed to more precisely predict response to bintrafusp alfa, a drug designed to have a bifunctional mechanism of action, across cancer indications. Citation Format: George Locke, Christian Ihling, Christoph Helwig, Laureen S. Ojalvo, Olaf Christensen, Isabelle Dussault. Evaluation of immune phenotypes and gene expression profiles in tumors from patients treated with bintrafusp alfa [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3290.

Abstract 2202: The combination of a mouse ortholog of ALKS 4230, a selective agonist of the intermediate affinity IL-2 receptor, and the angiogenesis inhibitor lucitanib enhances antitumor activity

Abstract ALKS 4230, an engineered cytokine designed for selective binding to the intermediate affinity interleukin-2 receptor (IL-2R), is being evaluated as a monotherapy and in combination with pembrolizumab in patients with advanced solid tumors. The selectivity of ALKS 4230 is achieved through stable fusion of circularly permuted IL-2 to IL-2Rα, with the goal of driving antitumor responses via selective activation of CD8+ T cells and NK cells, while avoiding activation of regulatory T cells (Tregs), which express the high affinity IL-2R. High levels of vascular endothelial growth factor (VEGF) are associated with an immunosuppressive tumor microenvironment and diminished response to high-dose IL-2. Lucitanib is an anti-angiogenic, multi-tyrosine kinase inhibitor targeting VEGFR1-3, PDGFRα/β, and FGFR1-3. We evaluated the antitumor efficacy and mechanism of the combination of RDB 1462, the mouse ortholog of ALKS 4230, and lucitanib in the MC38 mouse syngeneic tumor model. C57BL/6 mice were subcutaneously implanted with MC38 cells and treated with RDB 1462 (1.5 mg/kg and 3 mg/kg once every 3 days for 3 weeks), lucitanib (10 mg/kg daily for 28 days), or the combination of both molecules. After 11 days of treatment, lucitanib monotherapy resulted in 100% tumor growth inhibition (TGI), while 1.5 mg/kg and 3 mg/kg RDB 1462 monotherapy resulted in 25% and 51% TGI, respectively. Lucitanib monotherapy extended survival, with a mean survival time (MST) of 39 days compared to 15 days for vehicle, with no complete tumor regressions. MSTs were 18-22 days for RDB 1462 monotherapy groups, 60 days for the lower dose combination arm, and >60 days for the higher dose combination. Durable complete tumor regressions were observed in 1/20 mice treated with either dose of RDB 1462 monotherapy and 5/10 mice treated with the lower dose of RDB 1462 in combination with lucitanib. 10/10 mice treated with the higher dose of RDB 1462 in combination with lucitanib exhibited complete responses on Day 60. Flow cytometric analyses revealed that RDB 1462 treatment induced significant peripheral CD8+ T cell expansion compared to vehicle (p<0.05) without an increase in Foxp3+ Tregs. This effect was enhanced by the combination of RDB 1462 with lucitanib. The combination led to a significant (p<0.05) increase in intratumoral CD8+ T cells and dendritic cells (CD11c+F4/80−), together with a reduction in tumor-associated macrophages (CD11b+F4/80+). The combination treatment elicited a distinct gene expression profile in tumors by RNA-Seq, merging the immunostimulatory effects of RDB 1462 with the anti-angiogenic effects of lucitanib. The combination of an intermediate affinity IL-2R-selective cytokine and an angiogenesis inhibitor resulted in durable dose-dependent antitumor efficacy in the MC38 mouse tumor model. Citation Format: Jared E. Lopes, Rachel L. Dusek, Liliane Robillard, Minh Nguyen, Bruce Roth, Bryan Vought, Arthur Liberzon, Raymond J. Winquist, Heather C. Losey. The combination of a mouse ortholog of ALKS 4230, a selective agonist of the intermediate affinity IL-2 receptor, and the angiogenesis inhibitor lucitanib enhances antitumor activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2202.

Abstract 6613: Identification of targetable HLA*A2:01 restricted peptides in osteosarcoma

Abstract Background: Osteosarcoma is the most common primary tumor of bone with 800-900 new cases diagnosed annually in the United States. The development of effective chemotherapeutic agents in the early 1970's led to a dramatic increase in osteosarcoma survival from less than 10% to over 60%. Unfortunately, little progress has been made in the past several decades despite intensified treatment and the application of targeted therapies with the 5-yr overall survival stalled at ~70%. Several phase I studies have reported occasional clinical responses to immune checkpoint inhibitors in osteosarcoma patients, although the role of immunotherapy in osteosarcoma treatment is largely unexplored. The goal of this work was to perform a deep immuno-genomic and proteomic profile of osteosarcoma tumors as the basis for developing effective immune-based therapy for osteosarcoma. Methods: Osteosarcoma tumors and cell lines were subjected to whole transcriptomes analysis. Intratumoral T cell receptor sequences were identified using MiXCR and VDJtools from the RNA-seq data. Immune signatures were scored in each tumor sample using single-sample GSEA. MHC class I bound peptides were isolated by immunoprecipitation of MHC class I complex, acid elution of bound peptides, and identification using LC-MS/MS. Healthy donor T cells were transduced with a lentiviral construct targeting PRAME and co-cultured with U2OS cells to evaluate specific PRAME peptide targeting by T cells. Results: Gene expression profiling of extracranial pediatric solid tumors identified high T cell infiltration in osteosarcoma patient tumors. High CD8+ T cell infiltration were associated with favorable outcome among osteosarcoma patients, suggesting the presence of an underlying endogenous T cell response against osteosarcoma tumors. We identified a set of tumor associated antigens, such as cancer germline antigens (CGA), which are highly expressed in osteosarcomas and display low/absent expression in normal tissues. PRAME, a CGA expressed in multiple solid tumors and hematological malignancies, was identified as one of the most frequently overexpressed CGAs in osteosarcoma. Immunoprecipitation of MHC Class I complexes followed by LC-MS/MS from osteosarcoma cells identified peptides derived from PRAME and other tumor associated antigens that are presented by HLA*A2:01 in osteosarcoma. In a proof-of-concept experiment, we demonstrate effective T cell cytotoxicity against osteosarcoma using a TCR which specifically recognizes an PRAME HLA*A2:01 peptide identified by mass spectrometry. Conclusions: Osteosarcoma tumors have prognostic high CD8+ T cell infiltration, expression of cancer testis antigens, and presentation of tumor associated peptides on MHC class I complex. These data warrant a broad evaluation of the efficacy of immunotherapy in osteosarcoma. Citation Format: David Milewski, Sivasish Sindiri, Jun S. Wei, Andrew Brohl, Young Song, Xinyu Wen, Andrew Qi, Udayan Guha, Javed Khan. Identification of targetable HLA*A2:01 restricted peptides in osteosarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6613.

Abstract 5903: Clinical immune and genomic profiling of STK11 mutant non-squamous non-small cell lung cancer

Abstract Background: The use of immune PD-1/PD-L1 checkpoint inhibitors (ICI) has dramatically altered the treatment of advanced Non-Small Cell Lung Cancer (NSCLC). However, a large proportion of patients with NSCLC do not derive clinical benefit from ICI treatment. Recent studies have identified STK11 mutations leading to loss of LKB1 in NSCLC as drivers of primary resistance to ICI treatment. Using a clinical cohort of patients whose tumors underwent comprehensive genomic and immune transcriptomic analysis, we characterized the immune gene expression profile of STK11 mutant non-squamous NSCLC tumors and identified TRIM29 as a potential therapeutic target in STK11 mutant non-squamous NSCLC. Methods: We performed comprehensive analysis of the genomic and immunological landscape of 199 formalin-fixed, paraffin-embedded tumor samples (obtained prior to ICI treatment) from advanced stage non-squamous NSCLC patients treated at Roswell Park Comprehensive Cancer Center using a CLIA-certified laboratory test that included targeted NGS genomic sequencing, gene fusion analysis, and RNA-seq of 394 immune transcripts. Differential gene expression analysis was performed using R/Bioconductor package limma with Benjamini-Hochberg adjusted p-values reported. This study was approved by Roswell Park internal review board review (protocol BDR 091817) according to institutional policy for nonhuman subjects research. Results: Among the cohort of 199 cases, 32 contained truncating STK11 mutations. STK11 mutant cases showed an immunosuppressive environment with decreased PD-L1 expression (p=0.01) and decreased T-cell inflammation gene expression signature (p=0.0029) consistent with prior published cohorts. Analysis of differentially expressed immune genes signatures showed significant decreases in antigen presentation (p=0.015) and processing (p=0.044), NK cell activation (p=0.039), chemokine and cytokine signaling (p=0.044), immune checkpoint pathways (p=0.015), innate immune response (p=0.04), and interferon signaling (p=0.039). The top upregulated gene in STK11 mutant cases was TRIM29 (p=0.03). Other overexpressed targets included ID3 (p=0.009), KRT7 (p=0.02), and NOTCH3 (p=0.05). Interrogation of TCGA LUAD dataset also demonstrated elevated TRIM29 expression (p=0.006) in STK11-mutant compared to STK11-wild-type samples and we also confirmed elevated TRIM29 RNA and protein expression in a panel of STK11 mutant NSCLC human cell lines. Immunohistochemical staining of a cohort of 68 KRAS mutant NSLCLC patient samples also showed higher levels of TRIM29 protein staining in STK11 mutant samples. Knockdown of TRIM29 using shRNA inhibited growth of STK11 mutant tumor xenografts. TRIM29 is a E3 ubiquitin ligase and we evaluated known targets of TRIM29 and found decreased TAB2 expression in STK11 mutant NSCLC. Notably, TAB2 is also regulated by SIK1/3, which have been recently identified as signaling partners with STK11. TRIM29 has been implicated as a mediator of processes also involved in STK11 tumorigenesis such as cellular metabolism, DNA damage repair, innate immunity, and squamous differentiation. Conclusion: Using a large clinical cohort of non-squamous NSCLC patients we characterized the immunosuppressive environment of STK11 mutant tumors and identified the E3 ubiquitin ligase TRIM29 as a potential therapeutic target. Citation Format: Edwin Yau, Sarabjot Pabla, Sean Glenn, Antonios Papanicolau-Sengos, Mary Nesline, Grace Dy, Carl Morrison. Clinical immune and genomic profiling of STK11 mutant non-squamous non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5903.

Abstract 317: Organoid and TIL co-culture system models the human tumor immune microenvironment and drug sensitivity

Abstract BACKGROUNDS: Host tumor immune microenvironment has been shown to play a critical role on the outcome of the breast cancer patient. Among the immune cells, tumor infiltrating lymphocytes (TILs) were shown to be a predictive biomarker for the efficacy of chemotherapy by clinical trials. Triple negative breast cancer (TNBC) is known to have high infiltration of TILs compared with other subtypes. TILs were reported to be associated with the response to doxorubicin and cyclophosphamide, and with better survival in breast cancer patients. Recently, there are accumulating evidences that organoids generated from patient tumors are useful as models for drug sensitivity pre-clinical trials. Here, we hypothesized that co-culture of TILs with organoids will mimic human tumor immune microenvironment and enhance the drug sensitivity. MATERIAL AND METHODS: GSE22226 that contains both clinical data and gene expression profile of untreated tumors was used as the breast cancer patient cohort that underwent neoadjuvant chemotherapy (NAC). Relative fractions of 22 types of infiltrating immune cells were estimated from tumor gene expression with the CIBERSORT method. Organoids of the same tumors grown as mouse xenografts were generated using standard methods. Organoids (1000/well of 96-w plate) were treated with doxorubicin (0.01-100 µM) for 2 days before addition of TILs of same patient (1-16 x103 per well). Cytotoxicity was assessed after another two days with the CellTiter-Glo 3D cell viability assay. RESULTS: We first assessed the clinical relevance of TILs in response outcome of NAC by utilizing the previously reported cohort, in which Adriamycin, Cyclophosphamide and Taxane were used. We found that the number of TILs were significantly higher in the patients that achieved pathological complete response (pCR) when compared with the ones that did not. We established 18 PDX models all of which are from TNBC and we successfully expanded TILS 7 of these patients. We derived 8 organoids from the patients. We performed the experiment of adding TILs to the organoids. TILs effectively killed organoids when TILs were cultured at 1:8 or 1:16 ratio. Furthermore, the killing of organoids by doxorubicin was enhanced by the addition of TILs. CONCLUSION: Amount of TILs associated with better outcome in NAC cohort. In pre-clinical setting, addition of TIL intensified the sensitivity of organoids to doxorubicin. Citation Format: Yoshihisa Tokumaru, Masanori Oshi, Eriko Katsuta, Nobuhisa Matsuhashi, Manabu Futamura, Kazuhiro Yoshida, Kazuaki Takabe. Organoid and TIL co-culture system models the human tumor immune microenvironment and drug sensitivity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 317.

Abstract 1678: Establishment and characterization by expression microarrays of a patient-derived xenograft biobank for human pancreatic adenocarcinoma

Abstract Pancreatic cancer survival rate is poor, with a 5-year survival rate of 7% in the Republic of Ireland. Over the last 40 years, pancreatic cancer is one of the only solid tumor types to have minimal improvement in patient outcome. In Ireland, pancreatic cancer surgical resection is limited to two hospitals: Cork University Hospital and St Vincent's University Hospital (SVUH) in Dublin. In collaboration with SVUH, we have established Ireland's only pancreatic cancer patient-derived xenograft (PDX) biobank program. Tumor material from candidate patients following surgical resection was cold transferred and implanted subcutaneously into CB17/Icr-Prkdcscid mice. At time of implant and continuation of generations, snap frozen material was collected. Using miRNA and mRNA microarray technology, matched adjacent normal tissue, original tumor material and first generation (F1) PDX tumor material was interrogated. Differential expression analysis was carried out on all samples with comparisons of normal, tumor and F1 generation. Across all comparisons there were approximately 4000 genes and miRNAs found to be differential expressed. In focusing on genes that were upregulated in tumor samples compared to normal and further upregulated in the PDX F1 samples compared to the tumor samples, we identified 88 genes of interest. Biological processes such as cell division, mitotic cell cycle processes and cell cycle processes characterized a significant number of these genes. Two of the key genes of interest in this analysis were TSPAN1 and TPX2. TSPAN1 was upregulated 9.5-fold in tumor-normal comparison and upregulated a further 2.3-fold in the PDX F1 compared to the patient tumor. TPX2 was upregulated 4.4-fold in the tumor-normal comparison and a further 3.9-fold increase in the PDX tumor compared to patient tumor. TSPAN1 has previously been shown to increase the metastatic and invasive potential of pancreatic ductal adenocarcinoma cell lines. TPX2, a microtubule-associated protein, has been shown to reduce tumor growth in vivo when silenced. The pancreatic PDX biobank represents a versatile, expandable patient cohort for preclinical investigation. Analysis of gene expression profiles of normal vs tumor and tumor Vs PDX showed genes with associated tumor proliferation and aggressiveness. Citation Format: Sandra Roche, Fiona O'Neill, Niall Swan, Ninfa L. Straubinger, Neil T. Conlon, Jean Murphy, Kevin Conlon, Ray McDermott, Justine Meiller, Justin Geoghegan, Michael Moriarty, Robert M. Straubinger, Martin Clynes. Establishment and characterization by expression microarrays of a patient-derived xenograft biobank for human pancreatic adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1678.

A Pilot Study of the Predictive Potential of Chemosensitivity and Gene Expression Assays Using Circulating Tumour Cells from Patients with Recurrent Ovarian Cancer.

Circulating tumour cells (CTCs) from liquid biopsies are under current investigation in several cancers, including epithelial ovarian cancer (EOC) but face significant drawbacks in terms of non-standardised methodology, low viable cell numbers and accuracy of CTC identification. In this pilot study, we report that chemosensitivity assays using liquid biopsy-derived metastatic EOC CTCs, from 10 patients, nine with stage IIIC and one with stage IV disease, in progression after systemic chemotherapy, submitted for hypoxic isolated abdominal perfusion (HAP), are both feasible and useful in predicting response to therapy. Viable metastatic EOC CTCs (>5 cells/mL for all 10 blood samples), enriched by transient culture and identified by reverse transcription polymerase chain reaction (RT-PCR) and indirect immunofluorescence (IF), were subjected to flow cytometry-based Annexin V-PE assays for chemosensitivity to several chemotherapeutic agents and by RT-PCR for tumour gene expression profiling. Using a cut-off value of >80% cell death, CTC chemosensitivity tests were predictive of patient RECIST 1.1 responses to HAP therapy associated with 100% sensitivity, 50% specificity, 33% positive predictive, 100% negative predictive and 60% accuracy values. We propose that the methodology employed in this study is feasible and has the potential to predict response to therapy, setting the stage for a larger study.

The Contribution of Race to Breast Tumor Microenvironment Composition and Disease Progression.

Breast cancer is the second most commonly diagnosed cancer in American women following skin cancer. Despite overall decrease in breast cancer mortality due to advances in treatment and earlier screening, black patients continue to have 40% higher risk of breast cancer related death compared to white patients. This disparity in outcome persists even when controlled for access to care and stage at presentation and has been attributed to differences in tumor subtypes or gene expression profiles. There is emerging evidence that the tumor microenvironment (TME) may contribute to the racial disparities in outcome as well. Here, we provide a comprehensive review of current literature available regarding race-dependent differences in the TME. Notably, black patients tend to have a higher density of pro-tumorigenic immune cells (e.g., M2 macrophages, regulatory T cells) and microvasculature. Although immune cells are classically thought to be anti-tumorigenic, increase in M2 macrophages and angiogenesis may lead to a paradoxical increase in metastasis by forming doorways of tumor cell intravasation called tumor microenvironment of metastasis (TMEM). Furthermore, black patients also have higher serum levels of inflammatory cytokines, which provide a positive feedback loop in creating a pro-metastatic TME. Lastly, we propose that the higher density of immune cells and angiogenesis observed in the TME of black patients may be a result of evolutionary selection for a more robust immune response in patients of African geographic ancestry. Better understanding of race-dependent differences in the TME will aid in overcoming the racial disparity in breast cancer mortality.

Differences in Gene Expression Profile of Primary Tumors in Metastatic and Non-Metastatic Papillary Thyroid Carcinoma-Do They Exist?

Molecular mechanisms of distant metastases (M1) in papillary thyroid cancer (PTC) are poorly understood. We attempted to analyze the gene expression profile in PTC primary tumors to seek the genes associated with M1 status and characterize their molecular function. One hundred and twenty-three patients, including 36 M1 cases, were subjected to transcriptome oligonucleotide microarray analyses: (set A—U133, set B—HG 1.0 ST) at transcript and gene group level (limma, gene set enrichment analysis (GSEA)). An additional independent set of 63 PTCs, including 9 M1 cases, was used to validate results by qPCR. The analysis on dataset A detected eleven transcripts showing significant differences in expression between metastatic and non-metastatic PTC. These genes were validated on microarray dataset B. The differential expression was positively confirmed for only two genes: IGFBP3, (most significant) and ECM1. However, when analyzed on an independent dataset by qPCR, the IGFBP3 gene showed no differences in expression. Gene group analysis showed differences mainly among immune-related transcripts, indicating the potential influence of tumor immune infiltration or signal within the primary tumor. The differences in gene expression profile between metastatic and non-metastatic PTC, if they exist, are subtle and potentially detectable only in large datasets.

Identification of specific microRNA-messenger RNA regulation pairs in four subtypes of breast cancer.

Four subtypes of breast cancer, luminal A, luminal B, basal-like, human epidermal growth factor receptor-enriched, have been identified based on gene expression profiles of human tumours. The goal of this study is to find whether the same groups' genes would exhibit different networks among the four subtypes. Differential expressed genes between each of the four subtypes and the normal samples were identified. The overlaps between the four groups of differentially expressed genes were used to construct regulations networks for each of the four subtypes. Univariate and multivariate Cox regressions were employed to test the genes in the four regulation networks. This study demonstrated that the common genes in four subtypes showed different regulation. Also, the hsa-miR-182 and decorin pair performs different functions among the four subtypes of breast cancer. The result indicated that heterogeneity of breast cancer is not only reflected in the different expression patterns among different genes, but also in the different regulatory networks of the same group of genes.

WISP1 Predicts Clinical Prognosis and Is Associated With Tumor Purity, Immunocyte Infiltration, and Macrophage M2 Polarization in Pan-Cancer.

Cancer is becoming the leading cause of death and a major public health problem. Although many advanced treatment strategies are currently in use, the general prognosis of cancer patients remains dismal due to the high frequency of recurrence, metastasis. The identification of effective biomarkers is important for predicting survival of cancer patients and improving treatment efficacy. In this study, we comprehensively analyzed WNT1-inducible-signaling pathway protein 1 (WISP1) expression and explored its correlation with prognosis in pan-cancer using tumor IMmune Estimation Resource (TIMER) and Gene Expression Profiling Interactive Analysis 2 (GEPIA2). We also examined correlations between WISP1 and immunocyte infiltration using TIMER. We identified genes co-expressed with WISP1 using the LinkedOmics database and analyzed associated gene ontology using Metascape. Finally, we constructed protein-protein interaction networks and examined correlations between genes co-expressed with WISP1 and immunocyte infiltration in pan-cancer. WISP1 level differed between human pan-cancer tissues and normal tissues, indicating its potential as a prognostic biomarker. WISP1 expression was correlated with tumor purity and immunocyte infiltration, especially monocyte-macrophage trafficking and M2 polarization. Genes co-expressed with WISP1 were mainly associated with extracellular matrix organization, with collagen members COL6A3, COL5A1, and COL8A1 being key genes correlated with macrophage infiltration and M2 polarization in pan-cancer. Conversely, in certain types of cancer with better prognoses, WISP1 was associated with low M2 macrophage infiltration. These results suggest that WISP1 affect clinical prognosis through associations with tumor purity, immune cell infiltration, and macrophage M2 polarization in pan-cancer, with collagen member proteins may serving as effector molecules of WISP1.

Ancestry-specific gene expression profiles in TNBC tumors.

1547 Background: Due to persistent disparities in breast cancer mortality, there has been a renewed focus on investigating tumor biology. Deeper exploration has exposed distinctions in tumor biology based upon self-reported race and ancestry. The disparities associated with Triple Negative Breast Cancer (TNBC) across the modern African Diaspora suggests that there is a genetic ancestry connection between its aggressive tumor biology and clinical outcomes. Understanding this connection could hold the key to improving clinical outcomes in this group. Methods: We investigated 75 TNBC primary tumors using Self-Reported Race (SRR) groups: African American (AA, n = 42) and European American (EA, n = 33). Using best practices established by TCGA, we analyzed bulk RNA sequencing to measure changes in genome-wide expression levels. We next quantified global ancestry in a novel manner using RNAseq variants using 1000 Genomes as the reference data. We then identified African and European ancestry-associated genes using a logistic regression (adjusted FDR p < 0.05) between quantified ancestry and gene expression levels. Results: We identified > 150 genes associated with quantified African ancestry. We also found using quantified ancestry was a more robust method to screen for differentially expressed genes than SRR. Using an updated TNBC subtyping method, we noted higher incidences of Basal-like 2 tumors in AAs. Pathway analyses indicated several canonical cancer pathways; including, TP53, NFKB1 and AKT, have altered functionality in patients of African descent. For example, TP53-associated genes were activated in TNBC tumors of AA versus EA. This upregulation, rather than loss of function, is suggestive of polymorphic and/or ancestry-specific expression regulation, likely driven by population-private genetic variants. Lastly, we used TCGA data to validate a subset of African ancestry-specific genes that were upregulated in AA patients in our cohort. Specifically, PIM3, ZBTB22 and PPP2R4 each retained significant upregulation, in our cohort, but also TNBC tumors from TCGA (p = 0.0018, 0.023 and 0.022, respectively). Conclusions: Our study has uncovered ancestry-specific gene expression profiles in TNBC tumors. The distinct distribution of TNBC subtypes and altered functional oncologic pathways are evidence that biological underpinnings in TNBC can be driven by shared genetic ancestry. These findings emphasize the need to investigate patient populations of various ancestral origins in order to fully appreciate the molecular diversity in tumor biology for precision of disease management.

Panel-derived TMB and immune-related GEP are associated with the response to immune checkpoint inhibitors (ICIs) in urothelial cancers.

e17047 Background: Tumor mutational burden (TMB) and gene expression profile (GEP) have emerged as potential biomarkers for the prediction of response to the immune checkpoint inhibitor (ICI) treatment. An interferon-g gene signature was shown recently to predict ICI response in a pan-cancer setting. For TMB, not only did TMB-high patients experience better clinical outcome with ICI, but panel-derived TMB was also shown to be comparable to the gold standard, TMB derived from whole exome sequencing. Here, we evaluate possible correlations between panel-derived TMB, as well as immune-related GEP, with ICI response in urothelial cancers. Methods: FFPE tumor tissues from 30 patients with urothelial cancers who had previously received ICI as monotherapies or combination with chemotherapies at Kaohsiung Chang Gung Memorial Hospital were retrospectively tested for targeted next-generation sequencing (ACTOnco) and quantitative PCR-based GEP (ACTTME), for the identification of nonsynonymous variants across 440 cancer-associated genes and expression levels of > 90 immune-related genes, respectively. TMB was calculated by using the sequenced regions of ACTOnco to estimate the number of somatic nonsynonymous mutations per megabase of all protein-coding genes. For ACTTME, Cq values were normalized to internal control before group analysis. RECIST criteria were used to categorize tumor response to the treatment. Results: Patients were defined as responders (CR/PR) and non-responders (SD/PD) according to the RECIST criteria. In the ICI monotherapy cohort, the responders (n = 8) had significantly higher TMB than non-responders (n = 15) (Median 16.2 muts/Mb vs. 6.5 muts/Mb, p= 0.0035). In contrast, for the cohort receiving ICI combination therapy, several genes implicated in hypoxia (HIF1A), suppressive cell types (Treg & MDSC) and immune checkpoint (PD-L2) were significantly elevated ( p< 0.05) in the non-responder group by 2 to 8 folds. Conclusions: Despite the fact that the cohort size is small, this study showed panel-derived TMB, as well as immune-related GEP, can potentially serve as predictive biomarkers to identify urothelial cancer patients for immune checkpoint inhibitor therapy.

Prognostic relevance of CD4+ T-cells in the microenvironment of newly diagnosed follicular lymphoma (FL) patients is independent of the tumor gene expression profile.

8052 Background: A significant proportion of patients with FL experience an early relapse and a subsequent poor outcome. While several prognostic indices have been developed, none were designed to predict early failure. Recently, we established that lack of intrafollicular CD4+ T-cell expression predicted risk of early failure, and integrating this microenvironment biomarker with the Follicular Lymphoma International Prognostic Index, termed BioFLIPI, further improved identification of FL patients at risk of early failure ( Blood 2019;134(suppl1):121). However, the microenvironment may be influenced by the genetic composition of tumor. We investigated whether the CD4 biomarker and BioFLIPI were impacted by genetic features of the tumor as assessed by a 23-gene expression prognostic score ( Lancet Oncol 2018;19:549-61). Methods: Of the 186 cases with FL grade 1-3A treated with immunochemotherapy (IC) in our prior study, 152 had digital expression quantification of 23 selected genes (23-GEP score), which used RNA from formalin-fixed, paraffin-embedded samples. Event-free survival (EFS) was defined as time from diagnosis to progression, relapse, retreatment, or death. Early failure was defined as failing to achieve EFS at 24 months. Risk of early failure was estimated using odds ratios (ORs) and 95% confidence intervals from logistic regression models. We also used Cox regression to assess associations with continuous EFS and overall survival (OS). Results: 28% of patients failed to achieve EFS24. Lack of CD4+ intrafollicular expression (38% of patients, OR = 2.33, p = 0.024) and high risk 23-GEP score (26% of patients, OR = 3.52, p = 0.001) each predicted early failure, and in a multivariable model that included FLIPI, both CD4+ (OR = 2.26, p = 0.046) and 23-GEP score (OR = 2.26, p = 0.0.057) remained predictors. Similarly, BioFLIPI modeled as a continuous score (1-4, OR per one point increase = 2.31, p < 0.001) predicted early failure, and the association remained (OR = 2.14, p < 0.001) when the high risk 23-GEP score (OR = 2.79, p = 0.013) was included in the model. When stratified on 23-GEP score, BioFLIPI was a stronger predictor of early failure in low risk (74%, OR = 2.51, p = 0.002) relative to high risk (26%, OR = 1.55, p = 0.27) patients. Similar patterns were observed for EFS and OS. Conclusions: CD4+ T-cell infiltrate and tumor gene expression appear to be independently predictive of early failure in newly diagnosed FL patients treated with IC. Future studies should integrate and validate these measures.

Apolipoprotein A-I anti-tumor activity targets cancer cell metabolism.

Previously, we reported apolipoprotein A-I (apoA-I), the major protein component of high-density lipoprotein (HDL), has potent anti-melanoma activity. We used DNA microarray and bioinformatics to interrogate gene expression profiles of tumors from apoA-I expressing (A-I Tg+/–) versus apoA-I-null (A-I KO) animals to gain insights into mechanisms of apoA-I tumor protection. Differential expression analyses of 11 distinct tumors per group with > 1.2-fold cut-off and a false discovery rate adjusted p < 0.05, identified 176 significant transcripts (71 upregulated and 105 downregulated in A-I Tg+/– versus A-I KO group). Bioinformatic analyses identified the mevalonate and de novo serine/glycine synthesis pathways as potential targets for apoA-I anti-tumor activity. Relative to A-I KO, day 7 B16F10L melanoma tumor homografts from A-I Tg+/– exhibited reduced expression of mevalonate-5-pyrophosphate decarboxylase (Mvd), a key enzyme targeted in cancer therapy, along with a number of key genes in the sterol synthesis arm of the mevalonate pathway. Phosphoglycerate dehydrogenase (Phgdh), the first enzyme branching off glycolysis into the de novo serine synthesis pathway, was the most repressed transcript in tumors from A-I Tg+/–. We validated our mouse tumor studies by comparing the significant transcripts with adverse tumor markers previously identified in human melanoma and found 45% concordance. Our findings suggest apoA-I targets the mevalonate and serine synthesis pathways in melanoma cells in vivo, thus providing anti-tumor metabolic effects by inhibiting the flux of biomolecular building blocks for macromolecule synthesis that drive rapid tumor growth.

Prognostic role of PD-L1 and immune-related gene expression profiles in giant cell tumors of bone.

Giant cell tumor of bone (GCTB) is a locally aggressive and rarely metastatic tumor, with a relatively unpredictable clinical course. A retrospective series of 46 GCTB and a control group of 24 aneurysmal bone cysts (ABC) were selected with the aim of investigating the PD-L1 expression levels and immune-related gene expression profile, in correlation with clinicopathological features. PD-L1 and Ki67 were immunohistochemically tested in each case. Furthermore, comprehensive molecular analyses were carried out using NanoString technology and nCounter PanCancer Immune Profiling Panel, and the gene expression results were correlated with clinicopathological characteristics. PD-L1 expression was observed in 13/46 (28.3%) GCTB (and in 1/24, 4.2%, control ABC, only) and associated with a shorter disease free interval according to univariate analysis. Moreover, in PD-L1-positive lesions, three genes (CD27, CD6 and IL10) were significantly upregulated (p < 0.01), while two were downregulated (LCK and TLR8, showing borderline significance, p = 0.06). Interestingly, these genes can be related to maturation and immune tolerance of bone tissue microenvironment, suggesting a more immature/anergic phenotype of giant cell tumors. Our findings suggest that PD-L1 immunoreactivity may help to select GCTB patients with a higher risk of recurrence who could potentially benefit from immune checkpoint blockade.

The role of SRGN in the survival and immune infiltrates of skin cutaneous melanoma (SKCM) and SKCM-metastasis patients

BackgroundSkin cutaneous melanoma (SKCM) is one of most aggressive type of cancers worldwide. Serglycin (SRGN) is an intracellular proteoglycan that playing an important role in various tumors. However, its effect on immune infiltrates and whether it associates with survival of SKCM and SKCM-metastasis patients has not been explored.MethodsWe evaluated SRGN expression via the databases of Oncomine, Tumor Immune Estimation Resource (TIMER) and Gene Expression Profiling Interactive Analysis (GEPIA). The influence of SRGN expression on survival of SKCM and SKCM-metastasis patients was analyzed using TIMER database. Furthermore, the correlations between SRGN expression and immune infiltrates or gene marker sets of immune infiltrates were also analyzed via TIMER database.ResultsWe found that the expression of SRGN in SKCM and SKCM-metastasis tissues was significantly increased compared to the normal skin tissues (P < 0.001). Interestingly, it was showed that lower level of SRGN expression and lower immune infiltrates of B cell, CD8+ T cell, Neutrophil, and Dendritic cell were correlated with poor survival rate of SKCM and SKCM-metastasis patients (P < 0.001) but not SKCM primary patients. We also demonstrated that SRGN expression was positively associated with the immune infiltrates and diverse immune marker sets in SKCM and SKCM-metastasis.ConclusionsOur findings indicated that SRGN was associated with the survival of SKCM and SKCM-metastasis patients. SRGN may be a new immune therapy target for treating SKCM and SKCM-metastasis.

Regression of murine breast tumors after hetIL-15 monotherapy correlates with a novel population of intratumoral dendritic cells, in addition to increased infiltration of T, NK and cDC1 cells

Abstract Background We studied mechanisms of therapeutic efficacy of the heterodimeric IL-15 (hetIL-15) of human, macaque or mouse in several mouse tumor models, including the murine EO771 orthotopic breast cancer model in syngeneic C57BL/6 mice. Methods The effects of hetIL-15 on immune cells were compared in tumors, lymph nodes and spleen by flow cytometry, immunohistochemistry (IHC), gene expression profiling (Nanostring) and metabolic profiling of tumor infiltrating T cells. Results hetIL-15 administration resulted in tumor regression in 40% of the treated animals and resistance to tumor rechallenge, inducing long term immunological memory. Activated NK and CD8+ T cells were increased in hetIL-15 treated tumors. hetIL-15 administration resulted in increased intratumoral infiltration of conventional Dendritic cells (cDC1) and also induced a new population of intratumoral DC, (F4/80high DCs), which were correlated negatively with the tumor size. Phenotypic profiling of F4/80high DCs identified expression of several cDC1 specific markers, including CD103, IRF8 and XCR1. tSNE analysis clustered F4/80high DCs close to cDC1s and macrophages. Conclusion Complete eradication of EO771 tumors by hetIL-15 treatment correlates with different tumor infiltrating immune cell types. We propose that the anti-cancer activity of hetIL-15 in primary tumors is orchestrated by the interplay of CD8+T cells, cDC1 and a novel subset of antigen presenting cells with a distinct phenotypic profile. Effective hetIL-15 treatment leads to development of long term anti-tumor memory. hetIL-15 supported a favorable metabolic profile of intratumoral effector lymphocytes, important for their function.

Differences in gene-expression profiles in breast cancer between African and European-ancestry women.

African American (AA) women have an excess breast cancer mortality than European American (EA) women. To investigate the contribution of tumor biology to this survival health disparity, we compared gene expression profiles in breast tumors using RNA sequencing data derived from 260 AA and 155 EA women who were prospectively enrolled in the Southern Community Cohort Study (SCCS) and developed breast cancer during follow-up. We identified 59 genes (54 protein-coding genes and 5 long intergenic non-coding RNAs) that were expressed differently between EA and AA at a stringent false discovery rate (FDR) < 0.01. A gene signature was derived with these 59 genes and externally validated using the publicly available Cancer Genome Atlas (TCGA) data from180 AA and 838 EA breast cancer patients. Applying C-statistics, we found that this 59-gene signature has a high discriminative ability in distinguishing AA and EA breast cancer patients in the TCGA dataset (C-index = 0.81). These findings may provide new insight into tumor biological differences and the causes of the survival disparity between AA and EA breast cancer patients.

Comparative evaluation of network features for the prediction of breast cancer metastasis

BackgroundDiscovering a highly accurate and robust gene signature for the prediction of breast cancer metastasis from gene expression profiling of primary tumors is one of the most challenging tasks to reduce the number of deaths in women. Due to the limited success of gene-based features in achieving satisfactory prediction accuracy, many methodologies have been proposed in recent years to develop network-based features by integrating network information with gene expression. However, evaluation results are inconsistent to confirm the effectiveness of network-based features, because of many confounding factors involved in classification model learning process, such as data normalization, dimension reduction, and feature selection. An unbiased comparative evaluation is essential for uncovering the strength of network-based features.MethodsIn this study, we compared several types of network-based features obtained using different mathematical operators (Mean, Maximum, Minimum, Median, Variance) on geneset (i.e., a gene and its’ neighbors in the network) in protein-protein interaction network and gene co-expression network for their ability in predicting breast cancer metastasis using gene expression data from more than 10 patient cohorts.ResultsWhile network-based features are usually statistically more significant than gene-based feature, a consistent improvement of prediction performance using network-based features requires a substantial number of patients in the dataset. In contrary to many previous reports, no evidence was found to support the robustness of network-based features and we argue some of the robustness may be due to the inherent bias associated with node degree in the network. In addition, different types of network features seem to cover different pathways and are complementary to each other. Consequently, an ensemble classifier combining different network features was proposed and was found to significantly outperform classifiers based on gene-based feature or any single type of network-based features.ConclusionsNetwork-based features and their combination show promise for improving the prediction of breast cancer metastasis but may require a large amount of training data. Robustness claim of network-based features needs to be re-examined with network node degree and other confounding factors in consideration.