Expression Of Cancer-testis Antigens Research Articles

Expression of Cancer Testis Antigens in Tumor-Adjacent Normal Liver Is Associated with Post-Resection Recurrence of Hepatocellular Carcinoma.

Simple SummaryHigh recurrence rates after resection of liver cancer (hepatocellular carcinoma) with curative intent impair clinical outcomes of patients diagnosed with liver cancer. Cancer/testis antigens (CTAs) are expressed in cancer and can serve as therapeutic targets. We identified 12 CTAs expressed in 80% of liver cancer patients, and each one individually in at least 10%. Furthermore, we found that patients with expression of CTAs in macroscopically tumor-free liver tissue, experience more tumor recurrence and poor survival after surgical tumor removal. The increased risk of tumor recurrence in patients with CTA expression in tumor-free liver suggests that these patients already have micro-metastasis at the time of operation. These CTA-expressing (pre-)malignant cells may thus be a source of liver cancer recurrence, reflecting the relevance of targeting these to prevent liver cancer recurrence.High recurrence rates after resection of hepatocellular carcinoma (HCC) with curative intent impair clinical outcomes of HCC. Cancer/testis antigens (CTAs) are suitable targets for cancer immunotherapy if selectively expressed in tumor cells. The aims were to identify CTAs that are frequently and selectively expressed in HCC-tumors, and to investigate whether CTAs could serve as biomarkers for occult metastasis. Tumor and paired tumor-free liver (TFL) tissues of HCC-patients and healthy tissues were assessed for mRNA expression of 49 CTAs by RT-qPCR and protein expression of five CTAs by immunohistochemistry. Twelve CTA-mRNAs were expressed in ≥10% of HCC-tumors and not in healthy tissues except testis. In tumors, mRNA and protein of ≥ 1 CTA was expressed in 78% and 71% of HCC-patients, respectively. In TFL, CTA mRNA and protein was found in 45% and 30% of HCC-patients, respectively. Interestingly, CTA-expression in TFL was an independent negative prognostic factor for post-resection HCC-recurrence and survival. We established a panel of 12 testis-restricted CTAs expressed in tumors of most HCC-patients. The increased risk of HCC-recurrence in patients with CTA expression in TFL, suggests that CTA-expressing (pre-)malignant cells may be a source of HCC-recurrence, reflecting the relevance of targeting these to prevent HCC-recurrence.

Single cell multiomic analysis and immune cell type profiling of multiple myeloma with t(4;14).

e20013 Background: Multiple Myeloma (MM) is an incurable plasma cell (PC) malignancy and high risk (HR) MM remains an unmet clinical need. Translocation 4;14 occurs in 15% of MM and is associated with an adverse prognosis. A deeper understanding of the biology and immune micro-environment of t(4;14) MM is necessary for the development of effective targeted therapies. Single Cell multi-omics provides a new tool for phenotypic characterization of MM. Here we used Proteona’s ESCAPE™ single cell multi-omics platform to study a cohort of patients with t(4;14) MM. Methods: Diagnostic bone marrow (BM) samples from 14 patients with t(4;14) MM were analysed using the ESCAPE platform from Proteona which simultaneously measures gene and cell surface protein expression in single cells. Cryopreserved BM samples were stained with 65 DNA barcoded antibodies and subsequently sorted on CD138 expression. The CD138 positive and negative fractions were recombined at a known ratio for analysis using the 10x Genomics 3’ RNAseq kit. Resulting data were analyzed with Seurat and MapCell. Results: The patients had a median age of 63 years. All received novel agent based induction. Median progression free and overall survival (PFS and OS) were 22 and 34 months respectively. MMSET was overexpressed in all PCs while FGFR3 expression could be categorized into zero cells expressing FGFR3, low expression (< 10% of cells expressing FGFR3) or high expression (> 80% of cells expressing FGFR3). We also found heterogeneity in the expression of cancer testis antigens (CTA) such as FA133A and CTAG2 between PC clusters across samples. Variation in the immune microenvironment of the BM was seen across all patient samples with no correlation between cell types and PFS or OS. However, an analysis of BM samples at diagnosis and relapse in one patient showed a shift in the ratio of T cells to CD14 monocytes with a ratio of 5.7 at diagnosis compared to 0.6 at relapse. Further analysis of PCs in this patient found 8 PC populations, each containing variable numbers of cells from both the diagnostic and relapse samples. This suggests that all populations present at relapse were also present at diagnosis, although at variable proportions. Increased expression of RCAN3 (associated with cereblon depletion) was detected at relapse. Conclusions: We present the first application of single cell multi-omics immune profiling in high risk MM. The heterogeneity in expression of CTA has implications for the application of immunotherapies, while the upregulation of RCAN3 may explain failure of immunomodulatory therapy. Our small sample size may explain the lack of correlation between gene or protein expression with clinical outcomes. We propose that t(4;14) MM is a genomically and immunologically heterogeneous disease. Single cell analysis of larger cohorts is required to build on our findings.

Expression of cancer testis antigens in thymic epithelial tumors

Cancer testis antigens (CTAs) are detected in cancer cells but not in healthy normal tissues, with the exception of gametogenic tissues. However, to our knowledge, expression of the antigens in thymic epithelial tumors has not been examined yet. We examined the immunohistochemical expression of five CTAs (MAGE-A, NY-ESO-1, MAGE-C1, SAGE and GAGE7) in 192 cases of thymic epithelial tumor. The CTAs were variably expressed in the thymic epithelial tumors. Type B component of type AB thymomas, type B1/B2/B3 thymomas, and thymic carcinomas showed a generally positive correlation between the malignancy grades and positive expression rates in four CTAs other than MAGE-C1. In thymic squamous cell carcinomas (SqCCs), four antigens except for MAGE-C1 showed high expression rates ranging from 23.1% to 43.6%. In the prognostic analysis, a positive expression of SAGE (P = 0.0485) and GAGE7 (P = 0.0289) were associated with a shorter overall survival in type B2/B3 thymomas, respectively. In thymic SqCC, a positive MAGE-A expression was significantly associated with an increased level of programmed death ligand in tumor-infiltrating lymphocytes (P = 0.0181). We showed (i) a frequent CTA expression, (ii) a general correlation of CTA expression with tumor malignancy grades and (iii) a prognostic impact in some of the CTAs.

Cancer/testis antigens: from serology to mRNA cancer vaccine.

Cancer/testis antigens (CTAs) are a group of tumor antigens expressed in numerous cancer tissues, as well as in the testis and placental tissues. There are over 200 CTAs supported by serology and expression data. The expression patterns of CTAs reflect the similarities between the processes of gametogenesis and tumorigenesis. It is notable that CTAs are highly expressed in three types of cancers (lung cancer, bladder cancer, and skin cancer), all of which have a metal etiology. Here, we review the expression, regulation, and function of CTAs and their translational prospects as cancer biomarkers and treatment targets. Many CTAs are highly immunogenic, tissue-specific, and frequently expressed in cancer tissues but not under physiological conditions, rendering them promising candidates for cancer detection. Some CTAs are associated with clinical outcomes, so they may serve as prognostic biomarkers. A small number of CTAs are membrane-bound, making them ideal targets for chimeric antigen receptor (CAR) T cells. Mounting evidence suggests that CTAs induce humoral or cellular immune responses, providing cancer immunotherapeutic opportunities for T-cell receptors (TCRs), CAR T cell, antibody-based therapy and peptide- or mRNA-based vaccines. Indeed, CTAs are the dominating non-mutated targets in mRNA cancer vaccine development. Clinical trials on CTA TCR and vaccines have shown effectiveness, safety, and tolerance, but these successes are limited to a small number of patients. In-depth studies on CTA expression and function are needed to improve CTA-based immunotherapy.

Immunohistochemical Detection of Cancer-Testis Antigen PRAME

Cancer-testis (CT) antigens were identified by their ability to elicit T- or B-cell immune responses in the autologous host. They are typically expressed in a wide variety of neoplasms and in normal adult tissues are restricted to testicular germ cells. PReferentially expressed Antigen of Melanoma (PRAME) is a member of the family of nonclassical CT antigens being expressed in a few other normal tissues besides testis. Interestingly, knowledge about the protein expression of many CT antigens is still incomplete due to the limited availability of reagents for their immunohistochemical detection. Here, we tested several commercially available serological reagents and identified a monoclonal antibody suitable for the immunohistochemical detection of PRAME in formalin-fixed paraffin-embedded specimens. We also tested a wide array of normal and neoplastic tissues. PRAME protein expression in normal tissues is congruent with original molecular data being present in the testis, and at low levels in the endometrium, adrenal cortex, and adult as well as fetal ovary. In tumors, there is diffuse PRAME immunoreactivity in most metastatic melanomas, myxoid liposarcomas, and synovial sarcomas. Other neoplasms such as seminomas and carcinomas of various origins including endometrial, serous ovarian, mammary ductal, lung, and renal showed an intermediate proportion of cases and variable extent of tumor cells positive for PRAME protein expression. As seen with other CT antigens, hepatocellular and colorectal carcinoma, Leydig cell tumors, mesothelioma, and leiomyosarcoma are poor expressers of PRAME.

P35.02 Lung Adenocarcinoma with Oncogenic SMARCA4 Mutation: Possible Target of Cancer Immunotherapy

SWI/SNF complex, responsible for the transcriptional regulation through chromatin remodeling, is known to be altered in several malignancies. SMARCA4 gene, encoding a subunit of SWI/SNF complex, has been mutated in a subset of malignancies and its alteration may confer sensitivities to the several anti-neoplastic agents in preclinical model. However, the clinical and genomic characteristics of SMARCA4 gene-mutant lung adenocarcinoma have not been fully characterized. We investigated the proportions of SMARCA4 gene mutation both in lung adenocarcinoma cases in Asan Medical Center clinical next-generation sequencing cohort (AMC-LUAD) (n=249) and TCGA-LUAD (N=510) cohorts. The cases in the cohorts were divided into SMARCA4-likely oncogenic (SMARCA4-LO), SMARCA4-unknown and SMARCA4-wild type (SMARCA4-WT) groups according to the presence of SMARCA4 mutation and its oncogenic potential, and their clinicopathologic and mutational characteristics were compared. Differential gene expression (DEG) analysis between the three groups was performed using RNA-seq expression profile in TCGA-LUAD dataset. SMARCA4 gene mutation was identified in 25 (10%) and 43 (8.4%) cases and, of these, 15 (6.0%) and 24 (4.7%) cases were classified as SMARCA4-LO group in in AMC-LUAD and TCGA-LUAD cohorts, respectively. There were significant male and smoker predominance of SMARCA4-mutated groups in AMC-LUAD cohort (92.0% each), but these tendencies were non-significant in TCGA-LUAD cohort (60.5% and 76.7%, respectively). Large proportion of SMARCA4-LO group exhibited solid-predominant histologic features both in AMC-LUAD (13 of 15 cases; 87%) and TCGA-LUAD cohorts (19 of 24 cases; 79%). No concurrent driver gene alteration was not found in SMARCA4-LO group of AMC-LUAD cohort; however, oncogenic KRAS (3 cases), BRAF (1 case) and MAP2K1 (1 case) mutations were accompanied in a subset of SMARCA4-LO group of TCGA-LUAD cohort. The tumor mutation burden (TMB) of SMARCA4-LO group was significantly increased compared to that of the SMARCA4-WT patients both in AMC-LUAD and TCGA-LUAD cohorts (P<0.0001 each), and the case with high TMB (>10 mutations/Mb) was also frequent in SMARCA4-LO group of both cohorts (AMC-LUAD, P=0.0003; TCGA-LUAD, P=0.001). DEG analysis between SMARCA4-LO and SMARCA4-WT groups in TCGA-LUAD revealed significant upregulation of cancer-testis antigen genes and downregulation of lineage-specific genes in SMARCA4-LO group. Oncogenic SMARCA4 mutation may be identified in about 5% of lung adenocarcinoma and strongly associated with solid-predominant histologic pattern. The case with oncogenic SMARCA4 mutation may show high TMB and cancer-testis antigen expression, which serve as a target of immune checkpoint inhibitor therapy and cancer vaccine treatment.

Expression of Cancer-testis Antigens in Adenoid Cystic Carcinoma of the Salivary Glands Correlates with Clinical Outcomes

Expression of Cancer-testis Antigens in Adenoid Cystic Carcinoma of the Salivary Glands Correlates with Clinical Outcomes

Unraveling the Regulation of Cancer/Testis Antigens in Tumorigenesis Through an Analysis of Normal Germ Cell Development in Rodents.

Cancer/testis (CT) antigens are proteins aberrantly overexpressed in various tumorigenic cells, but they can also be normally expressed in the mammalian germline. Most CT antigens are highly immunogenic and known to be involved in cancer cell proliferation and tumor metastasis. A recent genome-wide analysis systematically identified CT antigen expression in 19 cancer types, significantly expanding the repertoire of CT antigens by 5-fold,from over 200 to approximately 1000. However, their function and regulation in tumorigenesis remain poorly understood. The shared functional characteristics between germ cells and cancer cells, if methodically defined, offer a unique gateway to understanding the regulation of CT antigens in cancers by studying gametogenesis. Nonetheless, such studies also provide insightful information on the role of CT antigens in spermatogenesis. Herein, we analyzed publicly available next generation sequencing datasets generated from normal adult testes in rodents, primordial germ cells and cancer samples across a series of published studies and databases. Based on these analyses, we report that a subset of CT antigens belonged to the core fitness gene family. Furthermore, super-enhancers both in normal testes and various cancers controlled specific CT antigens. We found that DNA methylation of CT antigens, such as TEX101 and TAF7L, was inversely correlated with their expression in both normal primordial germ cells and various cancers, which was mediated at least partly by DNA methyltransferase1 (DNMT1). By analyzing data from a testis knockout model, we showed that TAF7L could further influence the expression of additional CT antigens, which also held true in tumors. These findings not only confirmed the previous notion that CT antigens regulate cancer dynamics, but also showed that understanding the regulation of CT antigens during gametogenesis can offer new insights for cancer research.

Cancer-testis antigen MAGE-C1/ CT7 in multiple myeloma: literature review

Group of tumor-associated antigens, which is normally expressed in placental cells and testicular germ cells, is called cancer-testis antigens. To date, more than 40 gene families have been identified that encode cancer-testis antigens, and their expression has been studied in many types of malignant diseases. It is assumed that the expression of cancer-testis antigens can contribute to the development of the tumor transformation process, including hematological diseases. Of particular interest in the pathogenesis of multiple myeloma is the MAGE-C1/CT7 antigen, the expression of which is most often detected in this case. According to data published by various authors, the expression of MAGEC1 in multiple myeloma can be considered as an additional marker of a poor disease prognosis, represent the effectiveness of chemotherapy approaches, and, possibly, be an earlier predictor of relapse or progression.

Circulating tumor cell characterization of lung cancer brain metastases in the cerebrospinal fluid through single-cell transcriptome analysis.

BackgroundBrain metastases explain the majority of mortality associated with lung cancer, which is the leading cause of cancer death. Cytology analysis of the cerebrospinal fluid (CSF) remains the diagnostic gold standard, however, the circulating tumor cells (CTCs) in CSF (CSF‐CTCs) are not well defined at the molecular and transcriptome levels.MethodsWe established an effective CSF‐CTCs collection procedure and isolated individual CSF cells from five lung adenocarcinoma leptomeningeal metastases (LUAD‐LM) patients and three controls. Three thousand seven hundred ninety‐two single‐cell transcriptomes were sequenced, and single‐cell RNA sequencing (scRNA‐seq) gene expression analysis was used to perform a comprehensive characterization of CSF cells.ResultsThrough clustering and expression analysis, we defined CSF‐CTCs at the transcriptome level based on epithelial markers, proliferation markers, and genes with lung origin. The metastatic‐CTC signature genes are enriched for metabolic pathway and cell adhesion molecule categories, which are crucial for the survival and metastases of tumor cells. We discovered substantial heterogeneity in patient CSF‐CTCs. We quantified the degree of heterogeneity and found significantly greater among‐patient heterogeneity compared to among‐cell heterogeneity within a patient. This observation could be explained by spatial heterogeneity of metastatic sites, cell‐cycle gene, and cancer‐testis antigen (CTA) expression profiles as well as the proportion of CTCs displaying mesenchymal and cancer stem cell properties. In addition, our CSF‐CTCs transcriptome profiling allowed us to determine the biomarkers during the progression of an LM patient with cancer of unknown primary site (CUP).ConclusionsOur results will provide candidate genes for an RNA‐based digital detection of CSF‐CTCs from LUAD‐LM and CUP‐LM cases, and shed light on the therapy and mechanism of LUAD‐LM.

RNA expression profiling reveals PRAME, a potential immunotherapy target, is frequently expressed in solitary fibrous tumors.

Solitary fibrous tumors are a type of translocation-associated sarcoma with up to 30% rates of metastasis and poor response to conventional chemotherapy. Other translocation-associated sarcomas have been shown to display elevated expression of various cancer-testis antigens which may render them susceptible to immunotherapy strategies such as cancer vaccines and adoptive T-cell therapy. After an RNA sequencing assay brought the cancer-testis antigen Preferentially Expressed Antigen In Melanoma (PRAME) to our attention as possibly being upregulated in aggressive TERT promoter-mutated solitary fibrous tumors, we used tissue microarrays to asses PRAME expression in a large series of previously characterized solitary fibrous tumors, with correlation to various clinicopathologic features, as well as with tumor-infiltrating macrophages and the associated signal regulatory protein α (SIRPα)-CD47 regulatory checkpoint. We found that PRAME was expressed in 165/180 solitary fibrous tumors, with high expression seen in 58%, irrespective of TERT promoter status. Elevated PRAME expression was more frequent in primary intrathoracic solitary fibrous tumors and correlated with older age at primary diagnosis. Elevated PRAME was also associated with features suggestive of immune evasion, including lower numbers of antigen-presenting CD163+ and CD68+ macrophages, and expression of the "don't eat me" receptor CD47 on tumor cells. Taken together, these features suggest that strategies targeting PRAME with or without concomitant SIRPα-CD47 axis inhibition may represent a potential future therapeutic option in aggressive solitary fibrous tumor.

Comprehensive analysis of the immunological landscape of pituitary adenomas: implications of immunotherapy for pituitary adenomas.

Immunotherapies for solid tumor are gaining traction in the clinic, however, the immunological landscape of pituitary adenomas (PAs) is not well defined. In the present study, we used the RNA-seq data of PAs to investigate the impact of immunological landscape on clinical features of pituitary adenomas and aim to evaluate the potential immunotherapy for PAs. We analyzed tumor-infiltrating immune cells in 115 PA samples using RNA-seq. Main immune cell types (B cells, CD8+ T cells, CD4+ T cells, macrophages and NK cells) were detected from the expression of genes. The association between immune cells abundance and immune checkpoint, as well as inflammatory factors were analyzed. 10 additional patients were enrolled for validation. In RNA sequencing data, landscape of PAs were identified. Our computationally inferred immune infiltrates significantly associate with patient clinical features. Growth hormone-secreting adenomas (GHomas) were found with higher B cells and CD8+ T cells infiltration. Moreover, GHomas showed relative different genetic background, significant invasive behavior and independently correlated with reduced progress-free time. Tumor progression was related to increased expression of PD-1/PD-L1 and was associated with higher immune infiltration. Analysis of cancer-testis antigen expression and CD8+ T-cell abundance suggested CTAG2 and TSPYL6 were potential immunotherapeutic targets in GHomas and non-functioning adenomas, respectively. Tumor-infiltrating immune cells confer important clinical and biological implications. Our results of immune-infiltrate levels in PAs may inform effective cancer vaccine and checkpoint blockade therapies and make it possible to take immunotherapy into invasive PAs.

Abstract 913: Defining melanoma patients unresponsive to single agent anti-PD-1 therapy but responsive to combination anti-PD-1 + anti-CTLA-4 therapy

Abstract Background: A proportion of patients that fail single agent anti-PD-1 therapy will subsequently receive and respond to combination anti-PD-1 and anti-CTLA-4 immunotherapy. The clinical and biological characteristics of these patients remain poorly defined and as such there are presently no reliable validated biomarkers to distinguish patients requiring the addition of anti-CTLA-4 in the context of anti-PD-1 therapy. Clinical cohort and methods: We performed RNA sequencing, immunofluorescence-IHC, and multiparameter image-mass cytometry (IMC) on formalin-fixed paraffin embedded (FFPE) tumors at baseline and post anti-PD-1 therapy from n=15 stage IV melanoma patients who failed anti-PD-1 therapy and went on to receive combination immunotherapy (7 non-responders; 8 responders). Results: Tumor pathological parameters including TIL and neutrophil densities, fibrosis, necrosis and mitotic rates identified were not different between responders and non-responders. While T-reg, cytotoxic CD8 and CD39+ resident CD8 T cells densities were not significantly different, the proportion of TCF7+ CD8 T cells was significantly higher at baseline (p=0.01) and in all tumor samples (p=0.007) in responders compared to non-responders. RNA differential gene expression analysis revealed higher expression of cancer testis antigens (CTAs) in responding patient tumors. Hierarchical clustering of differentially expressed genes separated responding patients from non-responding patients. Conclusions: The proportion of TCF7+ CD8 T cells and the presence of melanoma cancer testis antigens may be a predictive biomarker of response to combination immunotherapy in patients failing anti-PD-1 monotherapy but requires further validation. Citation Format: Jarem J. Edwards, Inês Pires da Silva1, Angela Ferguson, Peter Johansson, Jordan Conway, Grace Attrill, Robyn P.M Saw, John F. Thompson, Alexander M. Menzies, Umaimainthan Palendira, Georgina V. Long, Richard A. Scolyer, James S. Wilmott. Defining melanoma patients unresponsive to single agent anti-PD-1 therapy but responsive to combination anti-PD-1 + anti-CTLA-4 therapy [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 913.

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.

A survey of cancer testis antigen (CTA) expression across T-cell lymphoma subtypes.

e15246 Background: T-cell lymphomas (TCL) are a rare and heterogeneous group of non-Hodgkin lymphomas (NHL) that originate from T- and NK-cells and portends a poor prognosis. One promising therapeutic strategy involves generation of tumor associated antigen directed T-cells (TAA-T) against specific CTA. RNAseq have established that combinations of histone deacetylase inhibitors and hypomethylating agents uniquely induce genes characterized as cancer testes antigens (CTA), which may increase immunogenicity. There is a lack of data on the baseline expression of CTA across the TCL, limiting application of TAA-T. We sought to establish the baseline expression of CTA in PTCL using immunohistochemistry to better understand treatment effects. Methods: A tissue microarray was created using formalin-fixed paraffin embedded tissue from 40 patients with diverse T-cell neoplasms. Expression of WT1, SCP1, survivin, PLAC1, SSX2, and PRAME was evaluated by immunohistochemistry. The percentage of positive tumor cells (divided into quartiles), staining intensity (weak, moderate, strong), and subcellular localization (nuclear, cytoplasmic) was assessed. Results: Forty patients with T-cell lymphomas were included in the analysis. The median age of diagnosis and median number of treatments was 60.2 years and 2 (0-9), respectively. Thirty-nine (98%) patients expressed at least one CTA. A high frequency of survivin, which is associated with cell proliferation and is expressed on normal lymphoid tissue, was expressed in 98% of TCL, 5% expressed WT1, and 12.5% expressed SCP1, and expression of other CTA was not observed. Of five patients with anaplastic large cell lymphoma, 3 samples were positive for SCP1 and one was also positive for WT1. One of 9 angioimmunoblastic T-cell lymphoma samples was positive for SCP1. Of 4 T-lymphoblastic lymphoma patients, 1 was positive for WT1, and of 3 transformed mycosis fungoides specimens, 1 demonstrated SCP1 expression. In this sample set, we did not find a correlation with expression and response to chemotherapy. There were no patients with CTA expression who were treated with epigenetic treatments. Conclusions: We described low expression of CTA on T-cell lymphomas, which is consistent with the literature on expression by RT-PCR. By establishing this baseline expression of CTA, we have the foundation on which to measure changes in CTA expression pre- and post-treatment by immunohistochemistry.

YAP1 positive small-cell lung cancer subtype is associated with the T-cell inflamed gene expression profile and confers good prognosis and long term survival.

9019 Background: The dominant expression of transcription factors ASCL1, NeuroD1, YAP1 or POU2F3 characteristically defines four small cell lung cancer (SCLC) subtypes (SCLC-A, SCLC-N, SCLC-Y and SCLC-P). The clinical validation and biological relevance of these emerging SCLC subtypes is currently lacking. Methods: Using the Illumina TruSeq RNA Exome Kit, we generated RNA-Seq data from 61 cases of SCLC and pulmonary carcinoid to interrogate gene expression differences in SCLC subtypes as well as in survival outliers (top and bottom decile) matched for clinically relevant prognostic factors and treatment. We also assessed YAP1 protein expression in a blinded fashion by immunohistochemistry in 130 SCLC cases. Results: We successfully classified 68% of SCLC into one of the four SCLC subtypes whereas 81.5% of carcinoids did not fit into any of these categories. GSEA for differentially expressed genes between outlier subgroups showed significant upregulation of interferon gamma and interferon alpha response genes in late survivors. Moreover, a previously validated 18-gene T-cell inflamed gene expression profile was upregulated in late survivors and in the SCLC-Y subtype. Furthermore, the SCLC-Y subtype and late survivors showed higher expression of HLA gene family and reduced expression of cancer testis antigens. The median (95%CI) OS was 14 (4.3, 28.8), 16.7 (0.9, NA), 8.1 (2, 9.7) and 20.1 (0.6, 39.5) months respectively, for SCLC-A, N, P and Y subtypes. YAP-1 protein expression was positive in 17 of 130 (13%) SCLC cases. The majority of cases with positive YAP1 expression by immunohistochemistry, 12 of 17 cases (70.6%), were limited stage SCLC at the time of original diagnosis. Conclusions: SCLC subtypes have clinical implication as predictive and prognostic biomarker. SCLC-Y subtype is enriched for T-cell inflamed phenotype and long term survival, and may predict for clinical benefit of immunotherapy.

The Integration of PD1 blockade with epigenetic therapy is highly active and safe in heavily treated patients with T-cell lymphoma (PTCL) and cutaneous T-cell lymphoma (CTCL).

8049 Background: Our group has demonstrated that combinations of epigenetic modifiers produce potent synergy in pre-clinical models of PTCL and induce the expression of cancer testis antigen, suggesting a role in the addition of the immune-checkpoint inhibitor, pembrolizumab. Methods: This is a phase 1b study of pembrolizumab combined with pralatrexate alone (Arm A), with pralatrexate + decitabine (Arm B), or decitabine alone (Arm C) in patients with relapsed and refractory PTCL and CTCL. A standard 3+3 dose-escalation is applied in the triplet Arm (Arm B) while in the doublet Arms (A and C) de-escalation is applied in case of toxicity. Pharmacokinetic and pharmacodynamic studies are ongoing. Results: We treated a total of 12 patients with 4 patients in each Arm. All patients that received at least one dose of drug were evaluable for toxicity. There was a dose limiting toxicity (DLT) in each arm including prolonged grade 3 thrombocytopenia (Arm A), febrile neutropenia (Arm B), grade 3 hyponatremia, and rash (Arm C). There were no treatment-related deaths. Six patients out of 12 were evaluable for response at the time of this analysis. One patient achieved a complete remission, 2 had partial remission, 1 had stable disease, and 2 experienced progression of disease. Interestingly, all of the responses were seen in the triple combination of pralatrexate, decitabine, and pembrolizumab. Table summarizes the patient characteristics, toxicities, and response rates. Conclusions: These preliminary clinical data suggest that the integration of pembrolizumab on an epigenetic backbone is safe and demonstrates encouraging responses in patient with PTCL and CTCL. Clinical trial information: 03240211 . [Table: see text]

Modulation of CT-genes transcriptional activity in HT-29 cells by irradiation with a linear accelerator.

e15218 Background: Cancer-testis antigens (CTAs) are used as targets in immunotherapeutic approaches based on gene-cell vaccines (GCV). In some cases, it is possible to use experimental combinatorial therapy regimens that combine radiation and GCV. Despite numerous studies of the expression of CTA in tumors of various locations, including colon tumors, the transcriptional activity of CT-genes in tumor tissue of the colon after radiation therapy remains poorly studied. The aim of the study was to analyze changes in the expression of CT-genes in HT-29 cells after irradiation with a linear accelerator. Methods: The study used human cell culture HT-29. For the model experiment, 7 Gy fractionation option was used (irradiation was carried out 5 times every 24 hours). Irradiation was performed on a Novalis TX linear accelerator (Varian, USA). The ratio of living and dead cells was calculated in a Goryaev chamber using a 0.4% trypan blue dye solution. After microscopy on the 5th day of irradiation, the cell line was removed from the vial substrate by Trypsin/Versen solution. RNA was isolated by the Chomczynski&amp;Sacchi method. For cDNA synthesis a set of "REVERTA-L" reagents used. Expression of 16 CT genes (MAGE-A1, -A2, -A3, -A4, MAGE-B1, -B2, GAGE-1, -3, -4, MAGEC1, BAGE, XAGE3, NYESO1, SSX2, SCP1, PRAME1) was determined using the Real-Time qPCR method (reference genes — GAPDH and B2M). Statistical analysis was performed using univariate analysis of variance (ANOVA) with repeated measurements. Results: After 5 days of exposure at a dose of 7 Gy, only 20% of the initial number of HT-29 cells remained viable. It was found that irradiation was statistically significantly (p &lt; 0.05) influenced the increase in the expression of genes MAGEA1 by 1.8 times, MAGEB1 by 6.8 times, BAGE by 1.6 times, CTAGE1 by 3.3 times and SSX2 by 5.8 times relative to intact cells. Conclusions: Thus, tumor cells exposed to radiation at a dose of 7 Gy show an increase in the expression of 5 CT-genes, which, in turn, can increase their immunogenic potential.

Tumor antigen expression and survival of patients with previously treated advanced non-small cell lung cancer (NSCLC) receiving viagenpumatucel-L (HS-110) plus nivolumab.

9546 Background: Viagenpumatucel-L (HS-110) is an allogeneic cellular vaccine derived from a human lung adenocarcinoma cell line transfected with gp96-Ig fusion protein. Gp96-Ig functions as an antigen chaperone for dendritic cell activation and direct CD8+T cell expansion via cross presentation. DURGA is a multi-cohort study evaluating HS-110 plus anti-PD-1 mAbs in patients (pts) with advanced NSCLC. We report on Cohort A, which enrolled previously-treated pts who had not received an anti-PD(L)1 prior to study entry. Methods: Primary objectives were safety and objective response rate (ORR). Overall Survival (OS) was a secondary endpoint. Pts received 1 X 107 HS-110 cells intradermally every week for 18 wks and nivolumab until tumor progression. To determine cancer testis antigen (CTA) overexpression from baseline pt tumor samples, hybrid-capture RNA-seq libraries were prepared from macrodissected formalin fixed paraffin embedded tumor tissue and sequenced on an Illumina NovaSeq 6000. Gene-level transcripts were quantified using the Salmon software package. Results: 47 pts were enrolled into Cohort A. ORR and clinical benefit rate (CR + PR + SD) were 21% and 43%, respectively, with a 17.2 month median duration of response. Median OS was 28.7 months (mos), with a median follow up of 15.7 mos. One and 2-year survival were 57% and 36%, respectively. A prespecified exploratory analysis of CTA expression level in baseline pt tumor tissue was performed. 50% of pts shared at least 8 of the 39 total antigens overexpressed by HS110. Although there was no difference in ORR between these groups, mOS was higher in pts with tumors that shared ≥ 8 antigens with HS-110 (not reached (NR) [95%CI: 10.3 mos, NR] vs 6.7 mos [95%CI: 1.4 mos, NR]), p = 0.028. Pts whose tumors expressed the ZNF492 antigen also had improved OS (NR [95%CI: 11.6 mos, NR] vs 7.2 mos [95%CI: 1.6 mos, NR]), p = 0.03. All pts experienced at least one adverse event (AE), and the most common AEs were fatigue (28%), arthralgia (19%) and cough (17%). There were 2 grade 5 AEs not related to treatment. Conclusions: The combination of HS-110 and nivolumab appears safe and well tolerated. OS was improved in pts whose tumors express ≥ 8 shared antigens with HS110, as well as in those who specifically expressed ZNF492. Further exploration of antigen expression as a predictor for treatment outcome with HS110 plus nivolumab is ongoing. Clinical trial information: NCT02439450 .

Copy number variation and expression of CT-genes in patients with colorectal cancer.

e16104 Background: Cancer-testis antigens (CTAs) can be used for immunotherapeutic approaches and early detection of malignant tumors. Despite numerous studies of CTA expression in various tumors, including colon tumors, the mechanisms of transcriptional activity regulation in colorectal cancer (CRC) remain poorly studied. One of the possible mechanisms of regulation of gene expression is a change in their copy number (CNV). The aim of the study was to analyze the changes in the copy number and expression of CT-genes in patients with CRC. Methods: Tumor and normal colon tissues of 81 patients were used in the study. DNA was isolated by phenol-chloroform extraction. RNA was isolated by the Chomczynski&amp;Sacchi method (2006). The REVERTA-L reagent kit was used for the cDNA synthesis. Determination of expression and copy number of 16 CT genes ( MAGE-A1, -A2, -A3, -A4, MAGE-B1, -B2, GAGE-1, -3, -4, MAGEC1, BAGE, XAGE3, NYESO1, SSX2, SCP1, PRAME1) was performed using the Real-Time qPCR method (reference genes - GAPDH and GUSB). Differences were evaluated using the Mann-Whitney test, correlation analysis - using Spearman's rank correlation coefficient (r). Results: An analysis of CT-gene expression and CNV in tumor and normal colon tissues (n = 81) revealed a statistically significant (p &lt; 0.05) difference between these parameters in tumor tissue relative to normal one: for MAGEB1 an increase of 2.0 and 2.7 times, GAGE3 an increase of 2.0 and 2.5 times, GAGE4 decreased by 5.0 and 6.8 times, BAGE decreased by 2.4 and 1.7 times, SSX2 increased by 1.8 and 2.1 times, SCP1 increased copy number by 4.4 times and PRAME1 increased expression and copy number by 2.9 and 2.3 times, respectively. When comparing CNV and expression of 16 CT genes, a positive correlation was observed (r = 0.875). Conclusions: Thus, the aberrant expression of MAGEB1, GAGE3, GAGE4, BAGE, SSX2 and PRAME1 found in the tumor tissue of CRC patients depends on the copy number of these CT-genes.