Co-expression of cancer-testis antigens of MAGE-A6 and MAGE-A11 is associated with tumor aggressiveness in patients with bladder cancer

Melanoma antigen gene (MAGE)-A6 and MAGE-A11 are two of the most cancer-testis antigens overexpressed in various types of cancers. However, the clinical and prognosis value of MAGE-A6 and MAGE-A11 co-expression in the pathophysiology of the bladder is unknown. Three studies were selected from GEO databases in order to introduce the common genes that are involved in bladder cancer. Then immunohistochemical analysis for staining pattern and clinicopathological significance of suggested markers, MAGE-A6 and MAGE-A11, were performed in 199 and 213 paraffin-embedded bladder cancer with long adjacent normal tissues, respectively. A significant and positive correlation was found between both nuclear and cytoplasmic expressions of MAGE-A6 as well as expression of cytoplasmic MAGE-A11 with histological grade, PT stage, lamina propria invasion, and LP/ muscularis (L/M) involvement (all of the p-values in terms of H-score were < 0.0001). Additionally, significant differences were found between both nuclear and cytoplasmic MAGE-A6/MAGE-A11 phenotypes with tumor size (P = 0.007, P = 0.043, respectively), different histological grades, PT stage, LP involvement, and L/M involvement (all of the p-values for both phenotypes were < 0.0001). The current study added the value of these novel markers to the bladder cancer clinical settlement that might be considered as an admirable target for immunotherapy.

Growth differentiation factor 1-induced tumour plasticity provides a therapeutic window for immunotherapy in hepatocellular carcinoma

Tumour lineage plasticity is an emerging hallmark of aggressive tumours. Tumour cells usually hijack developmental signalling pathways to gain cellular plasticity and evade therapeutic targeting. In the present study, the secreted protein growth and differentiation factor 1 (GDF1) is found to be closely associated with poor tumour differentiation. Overexpression of GDF1 suppresses cell proliferation but strongly enhances tumour dissemination and metastasis. Ectopic expression of GDF1 can induce the dedifferentiation of hepatocellular carcinoma (HCC) cells into their ancestral lineages and reactivate a broad panel of cancer testis antigens (CTAs), which further stimulate the immunogenicity of HCC cells to immune-based therapies. Mechanistic studies reveal that GDF1 functions through the Activin receptor-like kinase 7 (ALK7)-Mothers against decapentaplegic homolog 2/3 (SMAD2/3) signalling cascade and suppresses the epigenetic regulator Lysine specific demethylase 1 (LSD1) to boost CTA expression. GDF1-induced tumour lineage plasticity might be an Achilles heel for HCC immunotherapy. Inhibition of LSD1 based on GDF1 biomarker prescreening might widen the therapeutic window for immune checkpoint inhibitors in the clinic.

Role and Clinical Utility of Cancer/Testis Antigens in Head and Neck Squamous Cell Carcinoma

Cancer/testis (CT) antigens exhibit selective expression predominantly in immunoprivileged tissues in non-pathological contexts but are aberrantly expressed in diverse cancers. Due to their expression pattern, they have historically been attractive targets for immunotherapies. A growing number of studies implicate CT antigens in almost all hallmarks of cancer, suggesting that they may act as cancer drivers. CT antigens are expressed in head and neck squamous cell carcinomas. However, their role in the pathogenesis of these cancers remains poorly studied. Given that CT antigens hold intriguing potential as therapeutic targets and as biomarkers for prognosis and that they can provide novel insights into oncogenic mechanisms, their further study in the context of head and squamous cell carcinoma is warranted.

Role and Clinical Utility of Cancer/Testis Antigens in Head and Neck Squamous Cell Carcinoma

Cancer/testis (CT) antigens exhibit selective expression predominantly in immunoprivileged tissues in non-pathological contexts but are aberrantly expressed in diverse cancers. Because of their expression pattern, they have historically been attractive targets for immunotherapies. The investigation of mechanistic roles of CT antigens in promoting oncogenesis has historically been a prominent research question, and a growing number of studies implicate CT antigens in promoting almost all the hallmarks of cancer. This suggests that CT antigens may act as cancer drivers. CT antigens are expressed in head and neck squamous cell carcinomas, although their role in the pathogenesis, prognostication, and treatment for this family of cancers remains poorly studied. Given that CT antigens hold intriguing potential as therapeutic targets and as biomarkers for prognosis and therapeutic response and that they can provide novel insights into oncogenic mechanisms, their further study in the context of head and squamous cell carcinoma is warranted.

P06.02 Cancer-specific differences of tertiary lymphoid structures and cellular responses against frequently expressed cancer testis antigens

BackgroundTertiary lymphocyte structures (TLS) can be detected in the tumor microenvironment across a wide range of cancer types and are associated with increased patient survival and susceptibility to immune checkpoint inhibition. However, evidence for the functional significance of TLS on humoral and cellular immunity is scarce. In this study, we combine assessment of abundance and spatial distribution of TLS with expression levels of 10 tumor associated antigens (TAAs) and functional analyses of T cell responses to these antigens.Materials and Methods52 treatment naïve cancer patients across 5 tumor types (NSCLC, CRC, RCC, HCC and BCA) were included. Presence and localization of TLS was assessed in immunohistochemical stainings (CD20) of whole section slides from FFPE embedded tumor samples. B cell clusters were quantified in the whole tumor region and in two different tumor margins (300 µm, 2000 µm). A panel of 30 cancer testis antigens was selected via GEPIA software (TCGA Database) and their expression in our cohort was determined using NanoString based RNA expression analysis of tumor samples and patient-matched healthy tissue. The 10 peptide pools with the largest cross-cancer overlap were selected based on our NanoString results. 2-color Fluorospot assays (IFN-γ and IL-2) were applied to assess the frequency of tumor-specific T-cell responses in patient PBMCs (triplicates for each TAA).ResultsCD20 immunohistochemistry and enumeration of intra- and peritumoral TLS revealed different distribution patterns of TLS/mm2 with the largest proportion in the 300 µm margin (p < 0.01) in most of the cancer types. This effect was particularly observed in patients with non-small cell lung cancer (NSCLC). The 10 tumor antigens CEP55, CT83, GAGE1, IGF2BP3, MAGEA1, MAGEA3/6, PBK, PRAME, Survivin and TTK were selected as they showed the highest overlap across different cancer types and the most pronounced differential expression between tumor and matched normal tissue. While 31/52 (59.6%) patients showed an IFN-γ, only 11/52 (21.2%) patients exhibited an IL-2 response against at least one of the tested CTAs. Survivin was the CTA presenting the highest frequency of responses (18/52 IFN-γ and 5/52 IL-2 responses). PBMCs of patients with NSCLC showed the highest frequency of T-cell responses (83.3% with at least one IFN-γ response) and patients with HCC and BCA the lowest proportion (40.0%).ConclusionsThe observed findings underline the importance of TLS as a novel biomarker and a possible association to cellular responses may even enhance their prognostic value. Our planned analyses of combined humoral immune responses will further elucidate the role of TLS in anti-tumor immune response of the analyzed cancer types. A combined targeting of a predefined or personalized set of included TAAs appears promising across the different cancer types.Disclosure InformationE. Preugszat: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Koeln Fortune Program, Faculty of Medicine, University of Cologne. M. Thelen: None. M. Garcia-Marquez: None. J. Lehmann: None. D. Keller: None. M. von Bergwelt-Baildon: Other; Modest; Honoraria for advisory boards, for invited talks from BMS and financial support for research projects from Astellas, Roche and MSD. H.A. Schlößer: Other; Significant; Financial support for research projects from Astra Zeneca.

P06.05 Endogenous T-cell responses to ten major cancer testis antigens are frequent in esophago-gastric adenocarcinoma and antigen-specific T cells can be expanded using CD40-activated B cells

BackgroundTumor-associated antigens (TAAs) and especially cancer testis antigens (CTAs) are classical tumor-specific targets for immunotherapies. As TAAs are shared between patients, strategies aiming to exploit these targets are scalable and potentially applicable across different types of cancer. Loss of target antigens and other mechanisms of immune escape have limited the success of CTA-directed immunotherapy. CAR T cells and other highly effective cellular therapies have renewed the interest in TAAs. Especially combined targeting of multiple antigens appears highly promising as recently shown in lymphoma. In our study, we aimed to characterize CTA-expression patterns and their impact on endogenous T-cell responses, T-cell abundance and antigen-presentation in esophago-gastric adenocarcinoma (EGA).Materials and Methods41 treatment-naïve EGA patients were included in our study. RNA of tumor and patient-matched healthy tissue was isolated and used for NanoString based RNA expression analysis of 26 CTAs and 25 genes associated with antigen-presentation. Based on CTA expression, 10 peptide pools were selected and co-cultured with peripheral blood mononuclear cells (PBMCs, n=21) to determine cellular anti-tumor immune responses in a FluoroSpot assay. T-cell abundance was assessed using immunohistochemistry (CD3, CD8) and digital image analyses of tumor area and invasive margin. Autologous CD40 activated B cells were used to expand antigen-specific T cells using peptide pools of CTAs.ResultsNanoString analysis revealed pronounced differences regarding CTA expression, with CEP55 and MAGEA3/6 showing strong expression, while NY-ESO-1 or MAGEA1 were only weakly expressed. 68.3% (28/41) of the patients showed expression of ≥ 5/10 analyzed TAAs simultaneously. In line with the frequent expression, 75.0% of the patients showed a cellular response against at least one of the TAAs. T-cell responses were most frequently detected to Survivin and NY-ESO-1 (65.0% and 52.6% of patients, respectively), while only 20.0% responded to CEP55 or TTK peptide pools. Overall, 6/20 patients showed cellular responses against ≥5 TAAs simultaneously. We found a strong correlation of T-cell abundance and antigen-presentation. In addition, patients with a high Immune-Score showed increased TAA expression. Finally, we demonstrate feasibility of TAA-specific T-cell expansion using CD40 activated B cells as potential strategy to induce or enhance TAA immune responses in EGA.ConclusionsOur study highlights the importance of TAAs in EGA. The identified antigens are highly relevant for immunomonitoring of clinical trials and as targets for immunotherapy. Personalized immunotherapeutic strategies targeting EGA-specific or even patient specific TAAs appear highly promising in this challenging disease.Disclosure InformationM. Thelen: None. M. Garcia-Marquez: None. J. Lehmann: None. D. Keller: None. E. Preugszat: None. M. von Bergwelt-Baildon: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Modest; Astellas, Roche, MSD. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; BMS. F. Consultant/Advisory Board; Modest; BMS. H.A. Schlößer: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Astra Zeneca.

PRAMEF2-mediated dynamic regulation of YAP signaling promotes tumorigenesis

PRAMEF2 is a member of the PRAME multigene family of cancer testis antigens, which serve as prognostic markers for several cancers. However, molecular mechanisms underlying its role in tumorigenesis remain poorly understood. Here, we report that PRAMEF2 is repressed under conditions of altered metabolic homeostasis in a FOXP3-dependent manner. We further demonstrate that PRAMEF2 is a BC-box containing substrate recognition subunit of Cullin 2–based E3 ubiquitin ligase complex. PRAMEF2 mediates polyubiquitylation of LATS1 kinase of the Hippo/YAP pathway, leading to its proteasomal degradation. The site for ubiquitylation was mapped to the conserved Lys860 residue in LATS1. Furthermore, LATS1 degradation promotes enhanced nuclear accumulation of the transcriptional coactivator YAP, resulting in increased expression of proliferative and metastatic genes. Thus, PRAMEF2 promotes malignant phenotype in a YAP-dependent manner. Additionally, elevated PRAMEF2 levels correlate with increased nuclear accumulation of YAP in advanced grades of breast carcinoma. These findings highlight the pivotal role of PRAMEF2 in tumorigenesis and provide mechanistic insight into YAP regulation.