Cancer-testis Antigen Genes Research Articles

Abstract 3361: Surfaceome and cancer testis antigen profiling of lung adenocarcinoma by large-scale transcriptomic analysis

Abstract Background: The emergence of improved modalities to target tumor specific and tumor associated antigens in solid tumors (e.g. antibody-drug conjugates, bi-specific antibodies, and adoptive cell therapy) underscores the need to efficiently identify and validate new therapeutic targets. Methods: We examined differential expression of surfaceome genes (Fonseca et al., 2016) and cancer testis (CT) antigen genes (Almeida et al., 2009) among major lung adenocarcinoma genomic subtypes; conducting comprehensive surfaceome/CT antigen profiling of 9002 NSCLC adenocarcinoma samples using data generated by RNA sequencing (whole transcriptome) at Caris Life Sciences (Phoenix, AZ). NSCLC tumors were stratified into 5 subgroups based on the presence of specific driver mutations: ALK fusion (N=325), EGFR mutant (N=1739), KRAS mutant (no STK11 or KEAP1 mutation; N=2561), KRAS + STK or KEAP1 mutation (N=1264), and Pan-WT (no mutation in ALK, EGFR, KRAS, STK11, or KEAP1 detected; N=3113). Fold-change in expression was calculated by dividing the median value for a specific subgroup by the median of all other groups combined. Significance was tested by Mann-Whitney U test, with p-values adjust for multiple hypothesis testing (Benjamini-Hochberg). Results: Surfaceome genes with the highest median expression overall included CD74, TMBIM6, and CD44, while the highest median expression among CT antigens was observed for SPAG9, KIAA0100, and RQCD1, yet the expression of these genes across the 5 driver mutation-based subgroups was similar (fold-changes ranged 0.93-1.06). To identify driver-associated candidate genes with relatively high expression, genes with subgroup-specific median expression >75th-percentile and with significant differences when compared to all other groups were ranked by fold-change. While several candidate surfaceome genes were associated with a specific subgroup, CLDN10 was identified among the top 3 candidates for both ALK fusion and KRAS + STK11/KEAP1 mutant subgroups: ALK fusion (GRIN2A, 3.03-fold; CLDN10, 2.53-fold; SLITRK6, 1.86-fold), EGFR mutant (SFTPC, 3.82-fold; MUC21, 1.99-fold; ATP13A4, 1.60-fold), KRAS mutant (VSIG1, 1.86-fold; AREG, 1.47-fold; C15orf48, 1.25-fold), and KRAS + STK11/KEAP1 mutant (ABCC2, 4.03-fold; MUC13, 3.21-fold; CLDN10, 3.00-fold). The top driver-associated CT antigen candidates included LEMD1 (1.42-fold) in ALK fusion, TMEM108 (1.27-fold) in EGFR mutant, XAGE1D (1.7-fold) in KRAS mutant, and CABYR (1.52-fold) in KRAS + STK11/KEAP1 mutant subgroups. Conclusion: Our analysis revealed several surfaceome and CT antigen candidate genes with relatively high expression across lung adenocarcinoma, as well as differential expression of candidate genes within specific genomic subtypes. These findings can help prioritize the validation and development of novel therapeutic targets in lung adenocarcinoma. Citation Format: Swartz Andrew, Andrew Elliott, George W. Sledge, Stephen V. Liu, Balazs Halmos, Vincent K. Lam, Taofeek K. Owonikoko. Surfaceome and cancer testis antigen profiling of lung adenocarcinoma by large-scale transcriptomic analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3361.

Abstract P25: Identification of biological components and novel clinical subsets of NPM1-mutated AML using an integrated, multi-omics approach

Abstract P25: Identification of biological components and novel clinical subsets of <i>NPM1</i>-mutated AML using an integrated, multi-omics approach

Biology of Cancer-Testis Antigens and Their Therapeutic Implications in Cancer

Tumour-specific antigens have been an area of interest in cancer therapy since their discovery in the middle of the 20th century. In the era of immune-based cancer therapeutics, redirecting our immune cells to target these tumour-specific antigens has become even more relevant. Cancer-testis antigens (CTAs) are a class of antigens with an expression specific to the testis and cancer cells. CTAs have also been demonstrated to be expressed in a wide variety of cancers. Due to their frequency and specificity of expression in a multitude of cancers, CTAs have been particularly attractive as cancer-specific therapeutic targets. There is now a rapid expansion of CTAs being identified and many studies have been conducted to correlate CTA expression with cancer and therapy-resistant phenotypes. Furthermore, there is an increasing number of clinical trials involving using some of these CTAs as molecular targets in pharmacological and immune-targeted therapeutics for various cancers. This review will summarise the current knowledge of the biology of known CTAs in tumorigenesis and the regulation of CTA genes. CTAs as molecular targets and the therapeutic implications of these CTA-targeted anticancer strategies will also be discussed.

Gene-based cancer-testis antigens as prognostic indicators in hepatocellular carcinoma

Cancer/testis antigens (CTAs) are reproductive tissue-restricted genes, frequently ectopic expressed in tumors. CTA genes associate with a poor prognosis in some solid tumors, due to their potential roles in the tumorigenesis and progression. However, whether CTAs relate with hepatocellular carcinoma (HCC) remains unclear. In this study, the prognostic signatures based on CTA genes were investigated and validated in three cohorts including Chinese HCC patients with hepatitis B virus infection (CHCC-HBV), International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA) cohorts. Univariate, LASSO, and multivariate Cox regression analyses were used to screen prognostic genes and develop the prognostic gene signature. A prognosis model was established with six CTA genes (SSX1, CTCFL, OIP5, CEP55, NOL4, and TPPP2) in CHCC-HBV cohort, and further validated in the ICGC and TCGA cohorts. The CTA signature was an essential prognostic predictor independent of other clinical pathological factors. High-risk group exhibited up-regulated cell cycle-related and tumor-related pathways and more M0 macrophage, activated mast cell, activated memory CD4+ T cell, and memory B cell infiltration. Furthermore, CTA signature correlated with the sensitivity to multiple chemotherapy drugs. Our results highlighted that the CTA gene profiling was a prognostic assessment tool for HCC patients.

Autoimmunity and re-expression of cancer/testis antigens: Numerous disorders one mechanism hypothesis

Cancer/Testis antigens (CTAs) are a group of antigens whose expression is restricted to male germ cells. However, the CTA gene promoters are methylated and silenced in the normal somatic cells. Since certain CTAs are immunogenic and can elicit cellular and humoralresponses, we propose that there is a link between the re-expression of CTAs and autoimmune disorders. The hypothesis assumes that the demethylation of particular CTA gene promoters is triggered by any of the risk factors predisposing to autoimmunity. Hence, those CTAs genes are expressed by the help of binding of cell-specific transcription factors. Examination of the promoter sequences revealed that CTA genes, particularly X-linked ones, possess binding motifs for the cell-specific transcription factors in the cells associated with the respective autoimmune disease. If proves true, this hypothesis will provide a unified mechanism for the numerous autoimmune disorders and will open the way for developing novel therapeutic approaches targeting the CTAs.

Identification of Cancer/Testis Antigens Related to Gastric Cancer Prognosis Based on Co-Expression Network and Integrated Transcriptome Analyses.

Gastric cancer is a worldwide life-threatening cancer. The underlying cause of it is still unknown. We have noticed that some cancer/testis antigens (CTAs) are up-regulated in gastric cancer. The role of these genes in gastric cancer development is not fully understood. The main aim of the current study was to comprehensively investigate CTAs' expression and function in stomach adenocarcinoma (STAD). A comprehensive list of CTA genes was compiled from different databases. Transcriptome profiles of STAD were downloaded from the cancer genome atlas (TCGA) database and analyzed. Differentially-expressed CTAs were identified. Pathway enrichment analysis, weighted gene correlation network analysis (WGCNA), and overall survival (OS) analysis were performed on differentially-expressed CTA genes. Pathway enrichment analysis indicates that CTA genes are involved in protein binding, ribonucleic acid processing, and reproductive tissues. WGCNA showed that six differentially-expressed CTA genes, namely Melanoma antigen gene (MAGE) family member A3, A6, A12 and chondrosarcoma associated gene (CSAG) 1, 2, and 3, were correlated. Up-regulation of MAGEA11, MAGEC3, Per ARNT SIM domain containing 1 (PASD1), placenta-specific protein 1 (PLAC1) and sperm protein associated with the nucleus X-linked family member (SPANXB1) were significantly associated with lower OS of patients. MAGEA11, MAGEC3, PASD1, PLAC1, and SPANXB1 can be investigated as prognostic biomarkers in basic and clinical studies. Further functional experiments are needed to understand the exact interaction mechanisms of these genes.

Deficiency of cancer/testis antigen gene CT55 causes male infertility in humans and mice.

The Cancer/Testis Antigen (CTA) genes comprise a group of genes whose expression under physiological conditions is restricted to the testis but is activated in many human cancers. Depending on the particular expression pattern, the CTA genes are speculated to play a role in spermatogenesis, but evidence is limited thus far. Here, we reported patients with a hemizygous nonsense mutation in cancer-testis antigen 55 (CT55) suffering from male infertility with extreme disruption in sperm production, morphology, and locomotion. Specifically, the insufficiency of sperm individualization, excessive residue of unnecessary cytoplasm, and defects in acrosome development were evident in the spermatozoa of the patients. Furthermore, mouse models with depletion of Ct55 showed accelerated infertility with age, mimicking the defects in sperm individualization, unnecessary cytoplasm removal, and meanwhile exhibiting the disrupted cumulus-oocyte complex penetration. Mechanistically, our functional experiments uncovered CT55 as a new autophagic manipulator to regulate spermatogenesis via selectively interacting with LAMP2 and GABARAP (which are key regulators in the autophagy process) and further fine-tuning their expression. Therefore, our findings revealed CT55 as a novel CTA gene involved in spermatogenesis due to its unprecedented autophagy activity.

PRAMEY: A Bovid-Specific Y-Chromosome Multicopy Gene Is Highly Related to Postnatal Testicular Growth in Hu Sheep.

Simple SummaryPRAMEY (preferentially expressed antigen in melanoma, Y-linked) is a cancer-testis antigen and is believed to play an important role in testicular development and gametogenesis. However, the structure, expression and function of PRAMEY in sheep remain unknown. In this paper, we found that PRAMEY was extremely expressed in the testis and significantly upregulated during testicular growth. The mRNA abundance of PRAMEY was significantly correlated with testicular size traits in Hu sheep at 6 months. These results imply that PRAMEY may play an important role in testicular growth.PRAMEY (preferentially expressed antigen in melanoma, Y-linked) belongs to the cancer-testis antigens (CTAs) gene family and is predominantly expressed in testis, playing important roles in spermatogenesis and testicular development. This study cloned the full-length cDNA sequence of ovine PRAMEY using the rapid amplification of cDNA ends (RACE) method and analyzed the expression profile and copy number variation (CNV) of PRAMEY using quantitative real-time PCR (qPCR). The results revealed that the PRAMEY cDNA was 2099 bp in length with an open reading frame (ORF) of 1536 bp encoding 511 amino acids. PRAMEY was predominantly expressed in the testis and significantly upregulated during postnatal testicular development. The median copy number (MCN) of PRAMEY was 4, varying from 2 to 25 in 710 rams across eight sheep breeds. There was no significant correlation between the CNV of PRAMEY and testicular size, while a significant positive correlation was observed between the mRNA expression and testicular size in Hu sheep. The current study suggests that the expression levels of PRAMEY were closely associated with testicular size, indicating that PRAMEY may play an important role in testicular growth.

Prognostic attributes of immune signatures in soft tissue sarcomas show differential dependencies on tumor mutational burden.

BackgroundCellular and intrinsic markers of sarcoma immunogenicity are poorly understood. To gain insight into whether tumor–immune interactions correlate with clinical aggressiveness, the authors examined the prognostic significance of immune gene signatures in combination with tumor mutational burden (TMB) and cancer–testis antigen (CTA) expression.MethodsRNA sequencing and clinical data of 259 soft tissue sarcomas from The Cancer Genome Atlas project were used to investigate associations between published immune gene signatures and patient overall survival (OS) in the contexts of TMB, as computed from whole‐exome sequencing data, and CTA gene expression. Multivariate Cox proportional hazards regression models and log‐rank tests were used to assess survival associations.ResultsImmune signature scores that reflected in part the intratumoral abundance of cytotoxic T cells showed significant positive associations with OS. However, the prognostic power of the T‐cell signatures was highly dependent on TMB‐high status, consistent with protective effects of tumor‐infiltrating T cells in tumors with elevated antigenicity. In TMB‐low tumors, a signature of infiltrating plasma B cells was significantly and positively associated with OS, independent of T‐cell signature status. Although tumor subtypes based on differential expression patterns of CTA genes showed different survival associations within leiomyosarcoma and myxofibrosarcoma histologies, neither CTA nor histologic subtype interacted with the T‐cell–survival association.ConclusionsSignatures of T‐cell and plasma B‐cell infiltrates were associated with a survival benefit in soft tissue sarcomas. TMB, but not CTA expression, influenced the prognostic power of T‐cell–associated, but not plasma B‐cell–associated, survival.Lay summary Clinical data and RNA analysis of 259 soft tissue sarcomas from The Cancer Genome Atlas project were used to investigate associations between five published gene immune cell expression signatures and survival in the context of tumor mutations.Activated T cells had a significant positive association with patient survival.Although high tumor mutation burden was associated with good survival, the prognostic power of T‐cell signatures was highly dependent on tumor mutational status, consistent with protective effects of tumor‐infiltrating T cells in tumors with high levels of antigens.In low tumor mutation‐bearing tumors, plasma B cells were positively associated with survival.

Abstract 5137: Cancer testis antigen burden: Pan-cancer distribution and survival implications

Abstract Purpose of Study: Cancer testis antigens (CTA) are highly immunogenic genes with the ability to cause cancer-specific immune responses when expressed. Their tumor cell-specific expression makes them a key target of natural T cell response, cancer vaccines, immune checkpoint blockade (ICB), and cell-based immunotherapies in a wide range of tumor types. In this study, we assess the pan-cancer distribution and ICB survival association of CTA burden (CTAB) in real-world solid tumors. Procedure: Three tumor sample cohorts were studied: 1) a pan-cancer discovery cohort to develop a low- and high-CTAB cutoff (n=5450, 39 tumor types), 2) a TCGA cohort (n=19923, 32 tumor types) used to validate the classifier based on CTAB distribution and serve as a non-ICB-treated population, and 3) an ICB-treated retrospective cohort to validate the classification on overall survival (OS) (n=242, 3 tumor types). The expression levels of 17 CTA were measured using targeted RNA-Seq of FFPE tumor samples and then ranked against a pan-cancer reference population. CTAB was calculated for each sample, cohort and tumor type as the sum of the 17 CTA gene expression ranks. The discovery cohort median CTAB of 171 was used to classify all three cohorts into high- and low-CTAB groups. OS analysis was performed on the TCGA and ICB-treated cohorts using a CoxPH regression model to determine the Hazard Ratio (HR). Results: The three cohorts demonstrated overlapping single-peak, left-skewed CTAB distribution curves centered at CTAB values between 170 (discovery cohort) and 256 (retrospective cohort). When grouping by tumor types and ordering by median CTAB, the CTAB distributions for tumor types within all three cohorts were comparable. CoxPH regression analysis revealed an association between the CTAB threshold classifier and OS in both the ICB-treated retrospective and non-ICB TCGA cohorts. However, the direction of this association differed between the two cohorts, with high-CTAB samples having better survival (HR=0.936, p=0.076) in the ICB-treated retrospective cohort and worse survival (HR: 1.007, p=0.084) in the non-ICB-treated cohort. Conclusion: Our studies show that the CTAB distribution was maintained across the discovery and TCGA cohorts and a wide range of tumor types, supporting that the CTAB classifier is valid and histology agnostic. Additionally, when evaluating the ICB and non-ICB-treated cohorts, CTAB demonstrated the ability to predict OS, pointing to the utility of ICB in supporting CTA-specific natural immune response. However, further studies are necessary to verify these mechanisms of response to ICB as well as cancer vaccines and cell-based immunotherapies. Additional validation is needed to establish the predictive utility of CTAB alone and in combination with other immune oncology biomarkers for resistance or response. Citation Format: R.J. Seager, Erik Van Roey, Shuang Gao, Blake Burgher, Paul DePietro, Mary Nesline, Roger Klein, Shengle Zhang, Jeffrey M. Conroy, Sarabjot Pabla. Cancer testis antigen burden: Pan-cancer distribution and survival implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5137.

Surfaceome profiling to reveal unique therapeutic vulnerabilities in transcriptional subtypes of small cell lung cancer (SCLC).

8515 Background: Effective treatment options for SCLC remain limited and new treatment approaches are needed to improve outcome. We sought to validate the initial observation in cell lines and limited tissue samples of SCLC of a differential expression of cancer/testis (CT) antigens and TACSD2 gene that encodes surface protein, Trop2 across various subtypes of SCLC. We also tested whether overall surfaceome profile as previously described in other tumor types will show hierarchical priority of expression between transcriptionally defined SCLC subtypes. Methods: We conducted a comprehensive surfaceome profiling of SCLC samples using data generated by RNA sequencing (whole transcriptome) at Caris Life Sciences (Phoenix, AZ). SCLC tumors were stratified into 5 subgroups (SCLC-A/N/Y/P and -mixed) based on the relative expression of the four transcription factors. Expression values were converted to z-scores (the expression value for each gene is normalized to the average expression of that specific gene such that the z-score reflects the number of standard deviations above or below the average). The highest positive z-score among the 4 transcription factors determined subgroup. If all transcription factor z-scores for a given sample were negative, the sample was assigned to ‘Mixed’ subgroup. Significance was tested by Chi-square, Fisher’s exact test, or Mann-Whitney U test. Results: We employed data generated from 674 SCLC samples; median age of 66 years and male (48.7%). The SCLC subtype distribution was 241 (35.8%), 120 (17.8%), 40 (5.9%), 143 (21.2%), 130 (19.3%) for types A, N, P, Y and mixed respectively. Supervised analysis for TACSTD2 expression showed highest levels in YAP1 subtype and was overall significantly increased in SCLC-Y (̃3-fold) and SCLC-P (̃2-fold) subtypes compared to A, N and mixed subtypes. Similarly, SCLC-Y subtype showed the highest median expression as well as the strongest correlation with most TACSTD2-interacting and regulatory genes. A top 10 list of candidate surface protein gene out of 3699 surfaceome genes was defined for each subtype based on the strength of correlation. The top candidate surface protein gene and CT antigen gene respectively by subtype were: SCN3A (r = 0.7033, p = 1.08E-101) and NOL4, (r = 0.574, p = 2.46E-60) for SCLC-A; SSTR2, (r = 0.742, p = 8.18E-119) and TMEFF1, (r = 0.3601, p = 4.53E-22) for SCLC-N; TMPRSS13 (r = 0.5699, p = 2.64E-59) and LY6K (r = 0.4778, p = 9.80E-40) for SCLC-P; and CYBRD1 (r = 0.8559, p = 1.18E-194) and CTAGE5 (r = 0.5521, p = 4.95E-55) for SCLC-Y. Conclusions: SCLC-Y subtype showed the highest expression of TACSTD2 and its interacting and regulatory genes. This subtype could serve as an enrichment factor for antibody-drug-construct targeting TROP2. Several candidate CT antigens and surfaceome genes showing strong correlation with lineage-defining transcription factors offer additional therapeutic targets in SCLC.

Significance of cancer testis-associated antigens (SPAG9 and FBXO39) in colon cancer.

Cancer testis antigens (CTA) are normally expressed in immune privileged tissues such as the testis. They are considered tumor-associated antigens because they are specifically expressed in different cancers. Their distinct nature rendered them appealing targets for cancer diagnosis, prognosis. and immunotherapy. We aimed to identify the association of two CTA genes with colon cancer (CC) in a cohort of Egyptian patients. We measured the relative gene expression levels of two CTAs: SPAG9 and FBXO39 in colonic tumor tissue and adjacent normal-appearing mucosa in 50 newly diagnosed colon cancer patients by real-time reverse transcription polymerase chain reaction. Gene expression was also studied in relation to demographic and pathological criteria. SPAG9 and FBXO39 were overexpressed in 22% and 40% of cases, respectively. Overexpression of both genes was evident in 14% of cases. We report the significant expression of FBXO39 (P < 0.01) in tumor tissue compared to normal tissue. SPAG9 was significantly increased in large sized tumors compared to smaller sized tumors. Otherwise, there was no significant association between gene expression and the evaluated clinicopathological features (P > 0.05). SPAG9 and FBXO39 are possible CC diagnostic biomarkers. Further studies are warranted to validate our findings.

IMMU-30. IDENTIFICATION OF COORDINATELY REGULATED IMMUNO-MODULATORY GENES USING SINGLE-CELL RNA SEQUENCING OF DECITABINE TREATED GLIOMA CELLS

Abstract INTRODUCTION The immunotherapeutic targeting of New York-esophageal squamous cell carcinoma (NY-ESO-1) and other cancer/testis antigens (CTA) is an appealing strategy for the treatment of malignant gliomas because CTA are not expressed in most normal adult tissues and their expression can be induced in tumors for targeting by T-cells. Basally, NY-ESO-1 is often poorly expressed in glioblastoma (GBM), presumably through promoter methylation. Our previous data has shown that the hypomethylating agent decitabine (DAC) is a strong sensitizer of GBM to NY-ESO-1 specific adoptive T-cell induced cytotoxicity. However, we hypothesized that DAC alters expression of other immuno-modulatory genes in addition to NY-ESO-1 that may also increase T-cell mediated killing in GBM. The extent of regulation of other immuno-modulatory genes to DAC demethylation has not yet been thoroughly investigated. METHODS We performed single-cell RNA sequencing on DAC and non-DAC treated glioma cells. We confirmed DAC treatment induced NY-ESO-1 expression with quantitative real-time PCR and demethylation using bisulfite sequencing. We analyzed our single-cell RNA sequencing data with Seurat and our customized R-based pipelines to identify coordinate differential expression of immuno-modulatory genes and their tumor subpopulations. RESULTS Using our single-cell data, we identified tumor subpopulations with coordinate differentially expressed immuno-modulatory genes including cancer testes antigens, antigen presentation proteins, and apoptosis regulators. Amongst these candidate genes, we validated their expression with qPCR and promoter demethylation with bisulfite sequencing and TA bisulfite cloning. CONCLUSION Exposure of glioma cells to DAC results in promoter demethylation of NY-ESO-1, with increased expression of CTA and other immunomodulatory genes. DAC treatment may therefore sensitize GBM to the immunotherapeutic targeting of these antigens and reveals a feasible strategy for clinical translation.

Evaluation of Cancer Testis Antigens (CTAs) As Immuno-Therapeutic Targets in Pediatric AML

The Cancer Testis Antigen (CTA) superfamily of genes have long emerged as potential targets for immunotherapy and response monitoring due to their relative restrictive expression in testis and variable expression in some cancers. Much of the data on CTA expression in cancer have been in solid tumors with little data on CTA expression in leukemias. Identification of leukemia associated antigens (LAA) may provide attractive targets for immunotherapy or monitoring of residual disease.In order to discover AML associated CTAs, we performed transcriptome sequencing on 446 de novo pediatric AML samples and 20 normal bone marrows (NBM) from healthy donors and interrogated the expression of 245 candidate CTAs obtained from the CT database (http://www.cta.lncc.br). Differential expression using Limma (https://bioconductor.org/packages/release/bioc/html/limma.html) identified 32 CTA genes to be significantly differentially expressed with 7 being up-regulated compared to NBM and 15 genes with expression in NBM which are silenced in AML. The upregulated genes include: CCNA1, PRAME , CT45A5, IGF2BP3, SPAG6, TEX14, and ZNF165 and are collectively expressed in 330 of 446 patients (75%) with the highest contributors being PRAME and CCNA1 (Fig. 1A, Fig. 1B) . These 2 CTAs are expressed in every fusion group and constitute 65% of the total cohort, with a notable enrichment in core binding factor (CBF) patients (80%). High CCNA1 expression are present in 2/3 of patients with Inv(16), 71% of patients with a NUP98 fusion, 88% of DEK - NUP214 carriers, and 64% of cytogenetic normal cohort. PRAME is expressed in 79% of patients with a RUNX1 - RUNX1T1 fusion, 57% of CBFA2T3 - GLIS2 positives, and 20% of Inv(16) patients. While the oncoprint (Fig. 1A) shows that most patients express 1 or 2 CTAs, with some genes showing exclusivity, patients with a KMT2A fusion tend to express multiple CTAs. As a group, 77% of patients with KMT2A fusions express at least 1 CTA.To determine the underlying mechanism of dysregulated CTA expression in AML, we inquired whether promoter methylation status might be associated with CTA expression. We correlated the expression level of the 7 upregulated CTA genes with the methylation of their corresponding promoters. Overall there was a strong signal demonstrating anti-correlation of expression of the above mentioned genes with their promoter methylation. In specific, for PRAME, differences in methylation at the region chr22:22899991-22901267 explain over 87% of the variation in expression, and this locus is normally methylated in NBM (Fig. 1C). Likewise, for CT45A5, it's normally methylated in NBM, and demethylation of this locus explains about 61% of the variance in expression (Fig. 1C).Additionally, we evaluated the clinical implications of CTA expression, and demonstrated that CCNA1 expression was significantly associated with clinical outcome. The range of expression for CCNA1 in the AML samples was 0-561.4 TPM (mean = 31.8 TPM), whereas expression for NBMs ranged from 0.3-2.5 TPM (mean = 0.9) (p=8.45E-08). After binning expression values to determine the best cutpoint for high and low expressors of CCNA1, patients classified with high expression had statistically significant worse event-free (EFS, Fig.1D) and overall survival (OS) (p=<0.0001, p=0.0393 respectively, Log-Rank test). As a sub-group, patients with KMT2A rearrangements and high CCNA1 expression also show prognostic significance with correlation of poor outcome (EFS, p=0.0009, Fig. 1E). In addition, cytogenetically normal (CN-AML) patients with FLT3 ITD had a much higher overall expression of CCNA1 than did CN-AML without FLT3 ITD (p=0.009, Mann-Whitney test).In this analysis we identify candidate CTA genes that are cumulatively expressed in the majority of AML cases and can provide novel therapeutic options by targeting with modified T-cell receptor therapies. They can also be used as biomarkers for response monitoring. Further, as aberrant expression of these genes are regulated by altered methylation, use of hypomethylating agents might enhance therapeutic efficacy by increasing target expression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

DUSP22 Rearrangements Identify a Molecularly Distinct Type of Anaplastic Large Cell Lymphoma Characterized By DNA Hypomethylation and Lack of Activated STAT3

Background: Anaplastic large cell lymphomas (ALCLs) comprise a group of T-cell non-Hodgkin lymphomas that share common pathologic features such as CD30 expression, but demonstrate marked heterogeneity in clinical presentation, genetic features, and outcomes. Current classification of ALCLs is based on site of presentation (systemic or localized) and ALK status (positive or negative). Rearrangement of the DUSP22 locus is seen in ~30% of ALK-negative ALCLs and is considered a prognostically favorable biomarker, but the molecular features of these cases are incompletely understood. Specifically, their relationship to convergent mechanisms that activate STAT3 in both ALK-positive and ALK-negative ALCL has not been studied.Methods: Gene expression profiling (GEP) data from 31 frozen ALCL samples were analyzed using hierarchical clustering and gene-set enrichment analysis (GSEA). Immunohistochemistry for phosphorylated (p) STAT3 (Tyr705) was performed on whole-tissue paraffin sections and scored in a blinded fashion based on percentage of tumor cell nuclei staining. Reduced representation bisulfite sequencing (RRBS) was performed on DNA extracted from the 31 frozen ALCLs and differentially methylated regions (DMRs) were identified among genetic subtypes using methylKit software. RRBS and RNA sequencing also was performed on 6 T-cell lymphoma cell lines treated with the demethylating agent decitabine or vehicle alone, and gene expression and methylation profiles were evaluated.Results: Unsupervised clustering of GEP data identified two main groups of ALCLs, Cluster 1 (n=10) and Cluster 2 (n=21). All DUSP22- rearranged ALCLs (n=7) were in Cluster 1 whereas all ALK-positive ALCLs (n=8) were in Cluster 2. Both clusters contained ALK-negative ALCLs lacking DUSP22 rearrangements. GSEA identified significant depletion of IL6-JAK-STAT3 signaling pathway genes in Cluster 1 (NES=-2.05; p<0.001). Immunohistochemistry in 100 ALCLs confirmed low or absent pSTAT3 in DUSP22- rearranged cases (n=23; 9.2 ± 21.5 % versus 55.7 ± 35.2 % for ALCLs without DUSP22 rearrangements; p<0.0001). However, absent pSTAT3 alone could not explain the favorable prognosis in DUSP22- rearranged ALCLs, and the 3 ALK-negative ALCLs in Cluster 1 that lacked DUSP22- rearrangements showed aggressive clinical behavior. We previously found that DUSP22- rearranged ALCLs showed upregulation of cancer-testis antigen genes. Because these genes are regulated largely by methylation, we performed RRBS and found marked hypomethylation of DUSP22- rearranged ALCLs across all categories of gene-associated methylation sites (p≤0.0001). Furthermore, this hypomethylation could be recapitulated in vitro using decitabine, which led to upregulation of a DUSP22- specific gene expression signature in T-cell lymphoma cell lines.Conclusions: DUSP22- rearranged ALCLs are a molecularly distinct type of ALCL. These cases belong to a group of ALCLs that show down-regulation of STAT3-associated genes and lack activated STAT3 protein. Furthermore, DUSP22- rearranged ALCLs show global hypomethylation that contributes to their unique GEP signature, including up-regulation of cancer-testis antigens. These data suggest DUSP22- rearranged ALCLs may be less sensitive to therapies targeting JAK-STAT3 that have been proposed in ALCL but might benefit from immunotherapy approaches, which have efficacy in other cancers demonstrating hypomethylation and overexpression of cancer-testis antigens. DisclosuresAnsell:Celldex: Research Funding; Affimed: Research Funding; Seattle Genetics: Research Funding; Bristol-Myers Squibb: Research Funding; Merck: Research Funding. Cerhan:Janssen: Other: Scientific Advisory Board (REMICADELYM4001); Janssen: Other: Multiple Myeloma Registry Steering .

Global and Targeted RNA-Sequencing Assessment of Cancer Testis Antigen Re-Expression Following Azacitidine in Myelodysplastic Syndromes

Background and Aim: Azanucleoside DNA-hypomethylating agents have remarkable clinical activity in myelodysplastic sindromes (MDS) and acute myeloid leukemia (AML), particularly at low, non-cytotoxic doses favoring hypomethylation over cytotoxicity. Cancer/testis antigens (CTAs) encoding immunogenic proteins are normally expressed in the testicles and trophoblastic cells of the ovary and in some malignant tissues: these antigens are expressed to variable degrees particularly in melanoma, multiple myeloma, lung and breast cancer, but expression is quite infrequent in myeloid malignancies. However, they have been shown to undergo derepression after the use of demethylating agents in cell line models. Due to their tumor-restricted expression and their potential immunogenicity, we aimed to comprehensively study CTAs re-expression following azacitidine (AZA) treatment in MDS patients.Methods and Patients: We enrolled 32 patients with MDS (n =22) and chronic myelomonocytic leukemia (n = 10). Deep RNA-sequencing (RNA-Seq) was performed on RNA extracted from blood at day 0 and at day +28 of the first AZA cycle, from 12 patients and compared with 9 healthy donors. RNA-Seq (mean 106 million reads per sample) was sequenced on a HiSeq4000. After TopHat alignment and Cufflinks assembly, expression profile was calculated as FPKM. Targeted RNA-Seq (tRNA-Seq) designed to capture 214 CTA genes was performed on a validation cohort of 20 patients (40 samples). Coverage analysis as well as mapping the reads and alignment was done using the Ion Torrent Browser Suite. We used LIMMA package or empirical analysis of digital gene expression data in R (edgeR) to identify differential expressed genes between day 0 and d+28 samples, and between responder and non-responder groups. A log2 fold change (FC) > 10 and a p<0.1 were considered as significant.Results: The median age of the cohort was 69 (range 48-81 years). The cohort consisted of MDS (75%) and MDS/MPN (25%) patients. MDS Patients were stratified based on IPSS as low (8%), intermediate-1 (23%), intermediate-2 (46%), and high (23%) risk groups and MDS/MPN patients based on CPSS as low (20%), intermediate-1 (20%) and intermediate-2 (60%) . A homogeneous treatment schedule with AZA (75 mg/m2/day x 7 days every 4 weeks) was received by each patient. Seventeen patients were classified as responders (achieving complete remission (CR), marrow CR, partial remission, or stable disease with haematological improvement according to the 2006 International Working Group (IWG) criteria for evaluating MDS. Five patients progressed within the 6 first cycles; otherwise response evaluation was performed after the sixth cycle.In the screening RNA-Seq experiment, MAGEB3, AKAP4, POTEB2 and TMPRSS12 were non-expressed in control or MDS pretreatment, but re-expressed after 1 cycle of Aza; ADAM29, and XAGE1E were non expressed in controls, but expressed in MDS samples: an upregulation which increased after the first cycle.Regarding the tRNA-Seq cohort, on average, we achieved 3.888.308 mapped reads per sample (p25-p75, 1.2x106 - 5.9x106) which represents a sufficient depth for digital gene expression profiling of 214 genes. CTAs re-expressed after one AZA cycle in tRNA-Seq validation cohort: CT45A5, BRDT, TFDP3, TSPY3, TSPY2 showed an FC >10 and a p<0.05 and ADAM29. SLCO6A1, PAGE5 and RQCD1 showed a p<0.05 but a FC<10. CTAs with an increased expression in MDS after Aza: FMR1NB, ADAM29, and XAGE1E were not expressed in controls, re-expressed in MDS before treatment and showed an upregulation after AZA. Responders vs non responders: strikingly CT45A4, and SPAG17 were markedly overexpressed in non-responders than in responders (FC >10, = 0.006; p=0.01), and viceversa for PRM1 and TFDP3 (FC>10; p=0.01, p 0.006).Conclusions: This is the first study to assess, in a global and sentitive design, the targetable re-expression of CTAs following therapeutical demethylation in MDS. Strikingly, in addition to finding attractive targets for cancer immunotherapy, some CTAs were exclusively and markedly re-expressed in non-responders. We also show that targeted RNA-Seq can overcome the test posed by the wide range of the RNA population by sequencing targeted genes providing a vast enrichment of weakly expressed transcripts. DisclosuresNo relevant conflicts of interest to declare.

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.

SPANX Regulation of LAMIN A/C Network: Perspectives in Cancer and Laminopathies

Cancer/testis antigens (CTA) are tumor antigens whose expression is normally restricted to the testis. More than half of CTA genes are located on the X chromosome and form a branch called X-CTA [1]. Unlike the remaining CTA genes located throughout the genome, multigene X-CTA families are present as clusters on the X chromosome.

Cancer/testis antigens expression during cultivation of melanoma and soft tissue sarcoma cells

BackgroundAutologous dendritic cells (DC) loaded with tumor-associated antigens (TAAs) are a promising approach for anticancer immunotherapy. Polyantigen lysates appear to be an excellent source of TAAs for loading onto the patient’s dendritic cells. Cancer/testis antigens (CTA) are expressed by a wide range of tumors, but are minimally expressed on normal tissues, and could serve as a universal target for immunotherapy. However, CTA expression levels can vary significantly in patients with the same tumor type. We proposed that patients who do not respond to DC-based therapy may have distinct features of the CTA expression profile on tumor cells.Patients and methodsWe compared the gene expression of the principal families CTA in 22 melanoma and 27 soft tissue and bone sarcomas cell lines (STBS), received from patients and used for DC vaccine preparation.ResultsThe majority (47 of 49, 95.9%) cell lines showed CTA gene activity. The incidence of gene expression of GAGE, NYESO1, MAGEA1, PRAME’s was significantly different (adj. p < 0.05) between melanoma and sarcoma cell lines. The expression of the SCP1 gene was detected neither in melanoma cells nor in the STBS cells. Clustering by the gene expression profile revealed four different expression patterns. We found three main patterns types: hyperexpression of multiple CTA, hyperexpression of one CTA with almost no expression of others, and no expression of CTA. All clusters types exist in melanoma and sarcoma cell lines. We observed dependence of killing efficacy from the PRAME (rho = 0.940, adj. p < 0.01) expression during real-time monitoring with the xCELLigence system of the interaction between melanoma or sarcoma cells with the T-lymphocytes activated by the lysate of selected allogenous melanoma cell lines with high expression of CTA.ConclusionOur results demonstrate that one can use lysates from allogeneic melanoma cell lines as a source of CTA for DC load during the production of anticancer vaccines for the STBS treatment. Patterns of CTA expression should be evaluated as biomarkers of response in prospective clinical trials.

Cancer testis antigens in myelodysplastic syndromes revisited: a targeted RNA-seq approach

ABSTRACT Cancer-Testis antigens (CTA) are named after the tissues where they are mainly expressed: in germinal and in cancer cells, a process that mimics many gametogenesis features. Mapping accurately the CTA gene expression signature in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) is a prerequisite for downstream immune target-discovery projects. In this study, we take advantage of the use of azacitidine to treat high-risk MDS and CMML to draw the CTAs landscape, before and after treatment, using an ad hoc targeted RNA sequencing (RNA-seq) design for this group of low transcript genes. In 19 patients, 196 CTAs were detected at baseline. Azacitidine did not change the number of CTAs expressed, but it significantly increased or decreased expression in nine and five CTAs, respectively. TFDP3 and DDX53, emerged as the main candidates for immunotherapeutic targeting, as they showed three main features: i) a significant derepression on day +28 of cycle one in those patients who achieved complete remission with hypomethylating treatment (FC = 6, p = .008; FC = 2.1, p = .008, respectively), ii) similar dynamics at the protein level to what was observed at the RNA layer, and iii) to elicit significant specific cytotoxic immune responses detected by TFDP3 and DDX53 HLA-A*0201 tetramers. Our study addresses the unmet landscape of CTAs expression in MDS and CMML and revealed a previously unrecognized TFDP3 and DDX53 reactivation, detectable in plasma and able to elicit a specific immune response after one cycle of azacitidine.