Cancer Testis Research Articles

Correlation of cancer testis antigen and interleukin expression with survival in small bowel neuroendocrine tumors.

e16247 Background: Patients with small bowel neuroendocrine tumors (SBNETs) frequently present with metastatic disease, and unfortunately, the range and efficacy of available therapies is limited. Immunotherapeutic checkpoint inhibitors have demonstrated benefit in other malignancies and may also play a role in SBNETs, although relatively little is known about the immune infiltrate in these tumors. Toward a goal of developing novel immunomodulatory strategies, we sought to evaluate the tumor immune microenvironment of SBNETs utilizing Nanostring transcriptional profiling. Methods: Patients with SBNETs who underwent surgical resection at MD Anderson Cancer Center from 2003 to 2016 were retrospectively analyzed. Clinicopathologic data was collected, and patients were stratified by survival. Overall survival (OS) from date of resection was assessed using the Kaplan-Meier method, and p-values were calculated using the log-rank test. Multivariate (MV) analysis was performed using the Cox proportional hazards model. Transcription expression from bulk RNA was analyzed using the Nanostring PanCancer Immune Profiling Panel. Results: Resected SBNETs from 45 patients were selected for transcriptional analysis. Unsupervised clustering of RNA expression data revealed separation into high and low expression groups in two distinct transcription panels: cancer testis antigens (CTA) and interleukins (IL). All patients in the IL-high group also were in the CTA-high group. CTA-high patients (n=13) had significantly improved overall survival compared to CTA-low patients (n=32; median OS not reached vs 1975 days, p=0.0032). MV analysis controlling for age, gender, metastatic disease and history of carcinoid syndrome confirmed CTA-high status as an independent predictor of improved OS (hazard ratio 6.6, 95% confidence interval 1.7-26.4, p=0.008). CTA-high patients had significantly elevated expression of CTAs (such as PRAME and GAGE1) and cytokines (such as IL2 and CXCR1). High normalized expression levels of these single genes were independent predictors of improved OS in MV analysis (PRAME p=0.008, GAGE1 p=0.006, IL2 p=0.008, CXCR1 p<0.0001). Conclusions: High expression of CTA and IL signatures in resected SBNETs identified patients with improved survival agnostic of stage. While CTA expression across multiple tumor subtypes have been implicated in their immunogenicity and potential for therapeutic targeting, this is the first work to identify a clinically relevant signal in SBNET. The concurrent increase of key cytokines (which are known to mediate anti-tumor activity) among CTA-high patients suggests an immune-mediated component to their improved survival. Further work is underway to elucidate the epigenetic mechanisms of CTA expression and silencing, with the goal of validating key CTAs such as PRAME and GAGE1 as predictive and therapeutic targets for immunologic intervention.

Single-Cell Evolutionary Analysis Reveals Drivers of Plasticity and Mediators of Chemoresistance in Small Cell Lung Cancer.

Understanding the evolutionary dynamics of SCLC can shed light on key mechanisms such as cellular plasticity, heterogeneity, and chemoresistance.

In vivo CRISPR screens reveal Serpinb9 and Adam2 as regulators of immune therapy response in lung cancer

How the genetic landscape governs a tumor’s response to immunotherapy remains poorly understood. To assess the immune-modulatory capabilities of 573 genes associated with altered cytotoxicity in human cancers, here we perform CRISPR/Cas9 screens directly in mouse lung cancer models. We recover the known immune evasion factors Stat1 and Serpinb9 and identify the cancer testis antigen Adam2 as an immune modulator, whose expression is induced by KrasG12D and further elevated by immunotherapy. Using loss- and gain-of-function experiments, we show that ADAM2 functions as an oncogene by restraining interferon and TNF cytokine signaling causing reduced presentation of tumor-associated antigens. ADAM2 also restricts expression of the immune checkpoint inhibitors PDL1, LAG3, TIGIT and TIM3 in the tumor microenvironment, which might explain why ex vivo expanded and adoptively transferred cytotoxic T-cells show enhanced cytotoxic efficacy in ADAM2 overexpressing tumors. Together, direct in vivo CRISPR/Cas9 screens can uncover genetic alterations that control responses to immunotherapies.

Chemical features of melanoma tumor resident TRG CDR3s associated with better survival probabilities.

We assessed the T-cell receptor gamma (TRG) recombination reads from the cancer genome atlas melanoma tumor exome files and the TRG recombination reads from an independent, melanoma exome file dataset, from the Moffitt Cancer Center. TRG complementarity determining region-3 (CDR3) amino acid (AA) sequences were assessed for chemical complementarity to cancer testis antigens, with such complementarity for FAM133A and CRISP2 associated with better survival probabilities for both datasets. These results, along with related TRG CDR3 AA chemical feature assessments provided in this report, have indicated opportunities for melanoma patient stratifications based on the recovery of TRG recombination reads from both tumor and blood samples, and the results may point towards novel, effective melanoma antigens.

Higher Expression Levels of SSX1 and SSX2 in Patients with Colon Cancer: Regulated In Vitro by the Inhibition of Methylation and Histone Deacetylation.

Background and Objectives: Colon cancer (CC) has a high mortality rate and is often diagnosed at an advanced stage in Saudi Arabia. Thus, the identification and characterization of potential new cancer-specific biomarkers are imperative for improving the diagnosis of CC by detecting it at an early stage. Cancer-testis (CT) genes have been identified as potential biomarkers for the early diagnosis of various cancers. Among the CT genes are those belonging to the SSX family. In order to assess the usefulness of SSX family genes as cancer biomarkers for the detection of early-stage CC, the goal of this research was to validate the expressions of these genes in patients with CC and in matched patients with normal colons (NCs). Materials and Methods: RT-PCR assays were used to analyze the SSX1, SSX2, and SSX3 family gene expression levels in 30 neighboring NC and CC tissue samples from male Saudi patients. Epigenetic alterations were also tested in vitro using qRT-PCR analysis to determine whether reduced DNA methyltransferase or histone deacetylation could stimulate SSX gene expression via 5-aza-2'-deoxycytidine and trichostatin treatments, respectively. Results: The RT-PCR results showed SSX1 and SSX2 gene expression in 10% and 20% of the CC tissue specimens, respectively, but not in any of the NC tissue specimens. However, no SSX3 expression was detected in any of the examined CC or NC tissue samples. In addition, the qRT-PCR results showed significantly higher SSX1 and SSX2 expression levels in the CC tissue samples than in the NC tissue samples. The 5-aza-2'-deoxycytidine and trichostatin treatments significantly induced the mRNA expression levels of the SSX1, SSX2, and SSX3 genes in the CC cells in vitro. Conclusions: These findings suggest that SSX1 and SSX2 are potentially suitable candidate biomarkers for CC. Their expressions can be regulated via hypomethylating and histone deacetylase treatments, subsequently providing a potential therapeutic target for CC.

Immunohistochemical expression of PRAME is a marker of poor prognosis in uveal melanoma: A clinico-pathologic and immunohistochemical study on a series of 85 cases

PReferentially expressed Antigen in Melanoma (PRAME) is a cancer testis antigen, first isolated in tumor-reactive T-cell clones from a metastatic melanoma patient. It has been widely studied in skin pathology as an immunohistochemical marker capable of distinguishing between benign nevi and malignant melanomas. PRAME has been found to be also expressed in non-melanocytic tumors, including lung, breast, kidney and ovarian cancer. However, less is known about the diagnostic and/or prognostic role of this protein in uveal melanoma (UM); few studies have reported that PRAME expression seems to give to UM patients an additional metastatic risk beyond the other already-known prognostic parameters. In the present retrospective study, we aimed to correlate PRAME immunoreactivity to other clinico-pathologic features and follow-up data on a large series of 85 cases (45 non-metastasizing and 40 metastasizing tumors) of primary UM. A statistically significant correlation was found between PRAME expression and higher metastatic risk and lower metastasis-free survival. We propose to include PRAME in the immunohistochemical panel of UM as an easily usable marker capable of predicting higher metastatic risk and stratifying patients’ outcome.

Sperm protein antigen 17 and Sperm flagellar 1 cancer testis antigens are expressed in a rare case of ciliated foregut cyst of the common hepatic duct

IntroductionCiliated foregut cysts (CFCs) are frequently described in liver, pancreas and gallbladder and generally considered benign although one case of squamous cell metaplasia and five cases of squamous cell carcinoma arising from a ciliated hepatic foregut cyst have been reported. Here we explore two cancer-testis antigens (CTAs), Sperm protein antigen 17 (SPA17) and Sperm flagellar 1 (SPEF1) expression in a rare case of CFC of the common hepatic duct Materials and Methods3 µm-thick CFC sections were immunohistochemically treated with antibodies raised against human SPA17 or SPEF1. In silico Protein-Protein Interaction (PPI) network and differential protein expression were also investigated ResultsImmunohistochemistry revealed SPA17 and SPEF1 in the cytoplasm of ciliated epithelium. SPA17, but not SPEF1, was also detected in cilia. The PPI networks demonstrated that other CTAs are significantly predicted functional partners with SPA17 and SPEF1. The differential protein expression demonstrated that SPA17 was higher in breast cancer, cholangiocarcinoma, liver hepatocellular carcinoma, uterine corpus endometrial carcinoma, gastric adenocarcinoma, cervical squamous cell carcinoma, bladder urothelial carcinoma. SPEF1 expression was higher in breast cancer, cholangiocarcinoma, uterine corpus endometrial carcinoma and kidney renal papillary cell carcinoma ConclusionsOur study suggests that further characterization of SPA17 and SPEF1 in patients with CFCs might provide significant insights to understand the mechanisms underlying their potential to malignant transformation.

Establishment, characterization, and drug screening of low-passage patient individual non-small cell lung cancer in vitro models including the rare pleomorphic subentity.

For pre-clinical drug development and precision oncology research, robust cancer cell models are essential. Patient-derived models in low passages retain more genetic and phenotypic characteristics of their original tumors than conventional cancer cell lines. Subentity, individual genetics, and heterogeneity greatly influence drug sensitivity and clinical outcome. Here, we report on the establishment and characterization of three patient-derived cell lines (PDCs) of different subentities of non-small cell lung cancer (NSCLC): adeno-, squamous cell, and pleomorphic carcinoma. The in-depth characterization of our PDCs included phenotype, proliferation, surface protein expression, invasion, and migration behavior as well as whole-exome and RNA sequencing. Additionally, in vitro drug sensitivity towards standard-of-care chemotherapeutic regimens was evaluated. The pathological and molecular properties of the patients' tumors were preserved in the PDC models HROLu22, HROLu55, and HROBML01. All cell lines expressed HLA I, while none were positive for HLA II. The epithelial cell marker CD326 and the lung tumor markers CCDC59, LYPD3, and DSG3 were also detected. The most frequently mutated genes included TP53, MXRA5, MUC16, and MUC19. Among the most overexpressed genes in tumor cells compared to normal tissue were the transcription factors HOXB9, SIM2, ZIC5, SP8, TFAP2A, FOXE1, HOXB13, and SALL4; the cancer testis antigen CT83; and the cytokine IL23A. The most downregulated genes on the RNA level encode the long non-coding RNA LANCL1-AS1, LINC00670, BANCR, and LOC100652999; the regulator of angiogenesis ANGPT4; the signaling molecules PLA2G1B and RS1; and the immune modulator SFTPD. Furthermore, neither pre-existing therapy resistances nor drug antagonistic effects could be observed. In summary, we successfully established three novel NSCLC PDC models from an adeno-, a squamous cell, and a pleomorphic carcinoma. Of note, NSCLC cell models of the pleomorphic subentity are very rare. The detailed characterization including molecular, morphological, and drug-sensitivity profiling makes these models valuable pre-clinical tools for drug development applications and research on precision cancer therapy. The pleomorphic model additionally enables research on a functional and cell-based level of this rare NCSLC subentity.

LENS: Landscape of Effective Neoantigens Software.

Elimination of cancer cells by T cells is a critical mechanism of anti-tumor immunity and cancer immunotherapy response. T cells recognize cancer cells by engagement of T cell receptors with peptide epitopes presented by major histocompatibility complex molecules on the cancer cell surface. Peptide epitopes can be derived from antigen proteins coded for by multiple genomic sources. Bioinformatics tools used to identify tumor-specific epitopes via analysis of DNA and RNA-sequencing data have largely focused on epitopes derived from somatic variants, though a smaller number have evaluated potential antigens from other genomic sources. We report here an open-source workflow utilizing the Nextflow DSL2 workflow manager, Landscape of Effective Neoantigens Software (LENS), which predicts tumor-specific and tumor-associated antigens from single nucleotide variants, insertions and deletions, fusion events, splice variants, cancer-testis antigens, overexpressed self-antigens, viruses, and endogenous retroviruses. The primary advantage of LENS is that it expands the breadth of genomic sources of discoverable tumor antigens using genomics data. Other advantages include modularity, extensibility, ease of use, and harmonization of relative expression level and immunogenicity prediction across multiple genomic sources. We present an analysis of 115 acute myeloid leukemia samples to demonstrate the utility of LENS. We expect LENS will be a valuable platform and resource for T cell epitope discovery bioinformatics, especially in cancers with few somatic variants where tumor-specific epitopes from alternative genomic sources are an elevated priority. More information about LENS, including code, workflow documentation, and instructions, can be found at (https://gitlab.com/landscape-of-effective-neoantigens-software).

The cancer testis antigen TDRD1 regulates prostate cancer proliferation by associating with the snRNP biogenesis machinery.

Prostate cancer is the most commonly diagnosed noncutaneous cancer in American men. TDRD1, a germ cell-specific gene, is erroneously expressed in more than half of prostate tumors, but its role in prostate cancer development remains elusive. In this study, we identified a PRMT5-TDRD1 signaling axis that regulates the proliferation of prostate cancer cells. PRMT5 is a protein arginine methyltransferase essential for small nuclear ribonucleoprotein (snRNP) biogenesis. Methylation of Sm proteins by PRMT5 is a critical initiation step for assembling snRNPs in the cytoplasm, and the final snRNP assembly takes place in Cajal bodies in the nucleus. By mass spectrum analysis, we found that TDRD1 interacts with multiple subunits of the snRNP biogenesis machinery. In the cytoplasm, TDRD1 interacts with methylated Sm proteins in a PRMT5-dependent manner. In the nucleus, TDRD1 interacts with Coilin, the scaffold protein of Cajal bodies. Ablation of TDRD1 in prostate cancer cells disrupted the integrity of Cajal bodies, affected the snRNP biogenesis, and reduced cell proliferation. Taken together, this study represents the first characterization of TDRD1 functions in prostate cancer development and suggests TDRD1 as a potential therapeutic target for prostate cancer treatment.

Electrostatic Complementarities of Glioblastoma-Resident T-Cell Receptors and Cancer Testis Antigens Linked to Poor Outcomes and High Levels of Sphingosine Kinase-2 Expression.

Glioblastoma (GBM) is the most aggressive primary brain tumor in adults. Despite a growing understanding of glioblastoma pathology, the prognosis remains poor. In this study, we used a previously extensively benchmarked algorithm to retrieve immune receptor (IR) recombination reads from GBM exome files available from the cancer genome atlas. The T-cell receptor complementarity determining region-3 (CDR3) amino acid sequences that represent the IR recombination reads were assessed and used for the generation of chemical complementarity scores (CSs) that represent potential binding interactions with cancer testis antigens (CTAs), which is an approach particularly suited to a big data setting. The electrostatic CSs representing the TRA and TRB CDR3s and the CTAs, SPAG9, GAGE12E, and GAGE12F, indicated that an increased electrostatic CS was associated with worse disease-free survival (DFS). We also assessed the RNA expression of immune marker genes, which indicated that a high-level expression of SPHK2 and CIITA genes also correlated with high CSs and worse DFS. Furthermore, apoptosis-related gene expression was revealed to be lower when the TCR CDR3-CTA electrostatic CSs were high. Adaptive IR recombination reads from exome files have the potential to aid in GBM prognoses and may provide opportunities to detect unproductive immune responses.

Abstract 2311: Extensive molecular heterogeneity in circulating tumor cells: A key player in oral squamous cell carcinoma prognosis

Abstract Background: Oral Squamous cell carcinoma (OSCC) often results in unfavorable outcomes due to locoregional relapse. While circulating tumor cells (CTCs) may be attributed to these locoregional metastases, detecting CTCs and their molecular profiling remains challenging. Here we report a novel strategy for isolating CTCs in OSCC. Secondly, we characterized the CTCs to prognosticate the disease outcome using their transcriptomic signature. Methods: Keeping in view the inherent disadvantages of currently available CTC-isolation techniques, like the use of single marker or size-based isolation, we developed a novel FACS–based strategy, combining pre-enrichment by immunomagnetic depletion of CD45+ cells, followed by positive selection of CTCs using multiple FITC-conjugated epithelial markers (EpCAM, EGFR, CK) and EMT marker (Vimentin), with a novel gating strategy for isolating pure CTCs. Isolated CTCs were reconfirmed using various methods. CTCs and Primary tumors were isolated in 63 OSCC patients, and their transcriptomic signature was correlated with disease outcome. Results: Seventy-one percent (45/63) of patients were positive for CTCs, and associated with poor OS and DFS, while there is no difference between the proportion of CTC-positivity between early (23/33;69%) and late (22/30;73%) TNM stage. CTC-specific markers (EpCAM, EGFR, CK, and Vim) are expressed in various combinations, indicating extensive phenotypic heterogeneity. Importantly, EpCAM-positive CTCs were detected only in 46% (29/63) patients, and patients with EpCAM-negative CTCs had worse OS (p=0.044*), indicating the importance of a multi-marker approach. On unsupervised hierarchical clustering of RNAseq data of isolated CTCs and primary tumors, we found that CTCs and primary tumors clustered separately, indicating that the CTCs are transcriptionally different from the primary tumors. Moreover, CTCs were clustered into two distinct sub-clusters with significantly different OS (p=0.0004***) and DFS (p=0.0097***), suggesting that the inter-patient transcriptomic heterogeneity of CTCs is an important prognostic factor. We found coagulation, IL-6 JAK STAT3 signaling, allograft rejection, complement, EMT, inflammatory response, heme metabolism, UV response, and ECM receptor interaction pathways significantly enriched in CTCs compared to the primary tumor. TNF-alpha signaling via NF-kB and KRAS signaling pathways were also positively enriched in CTCs with poor prognostic traits. Interestingly, for the first time, we found various Cancer/Testis (CT) antigens upregulated in CTCs, while not detected in primary tumors. Conclusion: Here we report a novel, technologically less difficult, cost-effective CTC isolation technique suitable for developing countries. We explored the prognostic implications of the extensive heterogeneity in CTCs isolated from OSCC patients for the first time. Citation Format: Anshika Chauhan, Geeta S. Boora, Sahana Ghosh, Monil Parsana, Subrata K. Patra, Roshan K. Verma, Jaimanti Bakshi, Debajyoti Chatterjee, Radhika Srinivasan, Sushmita Ghoshal, Arindam Maitra, Arnab Pal. Extensive molecular heterogeneity in circulating tumor cells: A key player in oral squamous cell carcinoma prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2311.

Abstract 345: Cutaneous T-cell lymphoma: ectopic expression of gametocyte specific factor 1 (GTSF1) stimulates the Th phenotype

Abstract Diagnosis of Cutaneous T-Cell Lymphomas (CTCL) can take up to 6 years, increasing the risk of disease progression in these patients. Transcriptomic analyses have helped identify several diagnosis biomarkers, such as TOX, KIRD3DL2, GTSF1 and others. Although not officially a Cancer-Testis (CT) antigen, ectopic expression of Gametocyte Specific Factor 1 (GTSF1), presents the same advantages than CT antigens. Its germline-cell restricted expression potentially makes this gene easily identifiable by the immune system and a therapeutic target with less side effects. The role of GTSF1 has been elucidated in mice, where it controls retrotransposon expression during germ-cell development. Here, we investigate the molecular function of GTSF1 in CTCL. Using shRNA-mediated GTSF1 knockdown in CTCL cell lines, our current work demonstrates an unexpected role of GTSF1 regulating the Th phenotype of CTCL cells. With multiple approaches (qRT-PCR, Western blotting and a dual-luciferase based retrotransposition assay) we concluded that GTSF1 knockdown does not lead to retrotransposon reactivation. Transcriptome analysis with RNA-Seq, followed by differential gene expression analysis with DESeq2 and pathway enrichment analysis with GO, KEGG, Reactome and WikiPathways, suggested an involvement of GTSF1 in the Th phenotype of CTCL cells. Differential gene expression analysis revealed 12,496 differentially expressed genes (p adjusted < 0.05). Pathway enrichment analysis demonstrated an enrichment of several pathways including response to stimulus, immune response, response to cytokine and inflammatory response. More specifically, GTSF1 knockdown leads to an upregulation of the key cytokines associated with a Th1 phenotype, IFNG and TNF. Disease progression of CTCL is associated with a decrease in Th1 cytokine production and an enhanced Th2 cytokine production by malignant T cells. These findings shed light on the molecular function of GTSF1 as a regulator of the Th phenotype in CTCL and paves the way for the development of a valuable treatment target for CTCL patients. Citation Format: Amelia Martínez Villarreal, Ivan Litvinov. Cutaneous T-cell lymphoma: ectopic expression of gametocyte specific factor 1 (GTSF1) stimulates the Th phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 345.

Tumor immune microenvironment of cutaneous angiosarcoma with cancer testis antigens and the formation of tertiary lymphoid structures.

Cutaneous angiosarcoma (CAS) is a highly malignant tumor with few effective treatments. Although the indication for immune checkpoint inhibitors such as anti-PD-1 antibodies is expected to expand, there are many unknowns regarding the tumor immune microenvironment in CAS, which is generally considered an immunologically "cold" tumor. Our previous study demonstrated that tertiary lymphoid structures (TLSs) were associated with a favorable prognosis in CAS. However, we still don't know what the difference is between cases of TLS-rich and TLS-poor. Furthermore, the number of TLSs can vary significantly between lesions in the same case, for example, between primary and recurrence. To analyze the changes in the tumor immune microenvironment in CAS in more detail, we performed comprehensive RNA sequencing using a Next-generation sequencer (NGS). Sixty-two samples from 31 cases of CAS treated at Nagoya City University were collected. NGS and gene set enrichment analysis (GSEA) were performed on 15 samples among them. Immunohistochemistry and prognostic analysis by Kaplan-Meier method were performed on all 62 samples. NGS results showed that NY-ESO-1 (CTAG1B) was significantly upregulated in the TLS-positive cases. Immune checkpoint molecules including programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) were upregulated in TLS-negative or TLS-low cases and seemed to associate with the suppression of TLS formation. In a comparison of primary and recurrent lesions, other cancer-testis antigens (CTAs) including XAGE-1B were significantly upregulated in recurrent lesions. The number of infiltrating CD8-positive cells and TLSs showed no significant trend between primary and recurrent lesions. However, the PD-L1 expression of tumor cells was significantly lower in recurrent than in primary lesions. Chemokines correlated with NY-ESO-1 expression were CCL21 and CXCL8, and only CCL21 correlated with the number of TLS. There was no chemokine associated with XAGE-1. NY-ESO-1 and XAGE-1 are detectable by immunohistochemistry. Although each cannot be a prognostic marker by itself, they can be a helpful marker in combination with the number of TLSs. CTAs play an essential role in forming the tumor immune microenvironment in CAS. These findings are evidence that CAS is an immunologically "hot" tumor and provides us with potential therapeutic targets and encourages the expansion of immunotherapy indications.

Abstract 5948: MAGE-A3 expression with MMR-MSH6 screening suggests MAGE-A3 as a potential target for microsatellite-stable colorectal cancer

Abstract Colorectal Cancer (CRC) is the second leading cause of death worldwide. Recent developments in cancer immunotherapy have obtained encouraging clinical responses, including PD-1/PD-L1 blockade therapy, although response rates vary depending on tumor type. For example, despite the lack of response by microsatellite-stable CRC, a subset of CRC with deficient mismatch repair (dMMR) and high microsatellite instability (MSI-H) responds especially well to the PD-1/PD-L1 blockade therapy. For other types of CRC which do not respond to PD-L1 therapy, new targets are being investigated. One family of targets is the cancer testis antigen MAGE-A whose expression is restricted to germline cells in normal tissue but is overexpressed in many cancer cells. In this study, immunohistochemical analysis of MAGE-A3 expression in colon cancer along with IHC screening of mismatch protein MSH6 was performed. Many of the cases in this study observed a co-expression in the tissue. In the tumors, MAGE-A3 was observed to have weak cytoplasmic expression while MSH6 had strong nuclear expression in the epithelial cells. The immune cells adjacent to the epithelial cells were also positive for MAGE-A3. This result suggests that MAGE-A3 may be a promising target for microsatellite-stable colorectal cancer immunotherapy. Citation Format: Jina Yom, Rachel Gonzalez, Eden Zewdu, Aubrey Su, Patrick Yin, Bailey Gilmore, Dehe Kong, Andy Han, Zhaoying Guo, Tianli Qu, Zoe Zhao, Eric Christenson, Yan Ma, Hailey Guo, Xiaomin Hu, Qi Ren, Zhaohui Wu, Xuan Liu, Wei Fu. MAGE-A3 expression with MMR-MSH6 screening suggests MAGE-A3 as a potential target for microsatellite-stable colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5948.

Abstract 2269: Bexmarilimab induces B-cell activation and autoantibody production

Abstract Introduction: Bexmarilimab, a Clever-1 targeting humanized antibody, is a macrophage checkpoint inhibitor promoting antigen presentation and pro-inflammatory cytokine secretion. Data from the first-in-human clinical Phase I/II study (MATINS; NCT03733990) demonstrate that single agent bexmarilimab is able to ignite an interferon response with survival benefit in 30% of patients with advanced gastric cancer, cutaneous melanoma and cholangiocarcinoma (ASCO 2022). Since Clever-1 knockout mice have been reported to have enhanced T-cell antibody production and bexmarilimab increases peripheral B-cell populations, we investigated B-cell phenotype, clonality and autoantibody formation in bexmarilimab treatmented MATINS patients. Methods: We first performed comprehensive phenotyping of peripheral B-cells from a colorectal carcinoma patient showing partial response (per RECIST v1.1). Autoantibody reactivities were measured in 80 serum samples of bexmarilimab treated patients (37 pre-treatment and 43 post-treatment samples from cycles 3 and 4) and compared to serums from 53 healthy individuals using Oncimmune's SeroTag multiplex technology with an immune-oncology (IO) specific protein array comprised of 1162 antigens. Results: Single-cell sequencing together with BCR sequencing of the responder’s B-cells during cycle 4 revealed an induced and activated B-cell population consisting of four transcriptionally similar clusters expressing IGHM, IGHD, CD23 but not CD43 and one cluster expressing CD79B, PLD4 and MZB1, which was distant from the naïve CCR7 expressing B-cells and IGHA1 and IGHG1 expressing plasma blasts. The expansion of B-cells was not due to clonal expansion as no overlapping BCR clones were identified between B-cells at pre-dose and cycle 4. Autoantibody profiling revealed great inter-individual heterogeneity in the number and targets of induced antibodies among patients treated with bexmarilimab reflecting individual patterns in self- and tumor antigens. However, shared autoantibody changes against targets such as cancer testis antigens (GAGE2), typical autoimmune disease antigens (SNRPC, TPO, TOP1) and antigens related to the induction of innate immunity and interferon responses, e.g. TRIM21 were observed. Importantly, we identified a set of pre-treatment autoantibodies that were associated with longer time to disease progression and were predictive of clinical response (disease control rate [DCR], consisting of CR, PR, SD) and progression-free survival (PFS) on bexmarilimab therapy. Conclusions: Our data implies that bexmarilimab can induce activation and secondary Ig rearrangements in mature B-cells, which has been reported to occur in germinal centres during T-cell dependent antibody responses to increase B-cell diversity and affinity of antigen receptors. B cell diversity and activation was reflected in patient autoantibody production and may enhance cancer immune recognition. Citation Format: Elisa M. Vuorinen, Mari L. Björkman, Reetta Virtakoivu, Juho Jalkanen, Sofia Aakko, Akira Takeda, Petra Budde, Hans-Dieter Zucht, Manuel Brautigam, Behnaz Ahangarianabhari, Petri Bono, Maija Hollmén. Bexmarilimab induces B-cell activation and autoantibody production [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2269.

Abstract 6071: Whole-exome RNA sequencing of metastatic synovial sarcomas reveals heterogeneous transcriptomic profile and targetable co-alterations: Cohort study from the French Sarcoma Group

Abstract Introduction: Synovial Sarcoma (SS) is a rare and aggressive disease that predominantly occurs in young adult. SS are characterized by a pathognomonic t(X:18) translocation leading SS18: SSX1/2/4 fusion. Small studies (n<100) described molecular background beyond this fusion in patients and the transcriptomic profile of SS remains poorly known. The aim of our study is to provide a large cohort analyzing transcriptomic and co-alterations to better understand and treat SS. Methods: We present a clinico-biological cohort study including all adult patients with histologically confirmed diagnosis of afvanced SS from January 2000, registered in the French Sarcoma database (NETSARC+) and with available formalin-fixed paraffin embedded (FFPE) archival tumor samples and clinical data. FFPE tumor samples were analyzed by dedicated whole-exome RNA-sequencing (WERS) to assess transcriptomic, small nucleotide variation (SNV) and fusions. Results: 122 patients (133 samples) met the study criteria, including 11 patients with paired primary-metastatic tumor samples. Clinical characteristics (age, sex, tumor grade, primary and metastasis site distribution) were consistent with expected SS population. The SS18:SSX1/2/4 fusion was found in all patient (90% by WERS and if negative, by FISH). Unsupervised analyses of transcriptomic data by principal component analyses, hierarchical sample clustering or UMAP revealed heterogeneity in gene expression. Clinical factors (age, sex, grade, tumor type, complete response to treatment, survival) did not correlate with transcriptomic profiles. Immune cell analyses confirmed a low infiltration of immune cells, notably poor in CD8+ T cells. Cancer testis antigens such as NYESO-1, MAGE-A4 were both expressed with a heterogeneous co-expression of other CTAs across samples. In the 11 primary-metastatic paired- samples, no gene pathway was found differentially expressed between primary and metastatic samples cohort with the exception of lung tissue specific genes. Comparing chemotherapy-naïve versus pre-treated samples did not identify differential expression of specific genes across samples. Single Nucleotide Variant analyses was reliable on 84% FFPE samples and revealed an overall low Tumor Mutational Burden with some samples harboring canonical oncogene pathogenic mutations of BTK, RAS, NF1, RB1 as well as in DNA repair pathway(15% spanning over ATM ATR,CHK2, BRCA1/2 PALB2 CHK2, FANCM, RAD51), PI3K pathways (PI3KCA E545K) and other poorly described in SS. Conclusion: Investigating the largest cohort of metastatic SS by whole-exome RNA-sequencing, revealed Synovial Sarcoma, usually classified as “simple genomic sarcoma” is a translocation-related sarcoma harboring heterogeneous transcriptomic and numerous SNV co-alterations including targetable mutations. Citation Format: Helene Vanacker, Mehdi Brahmi, Yannick Le Meitour, Julien Bollard, Valery Attignon, Alexandra Meurgey, Myriam Jean-Denis, Laurie Tonon, Shibani Pokras, Erika Klohe, Michael Nathenson, Kristin Blouch, Ioanna Eleftheriadou, Jean-Yves Blay, Franck Tirode, Armelle Dufresne. Whole-exome RNA sequencing of metastatic synovial sarcomas reveals heterogeneous transcriptomic profile and targetable co-alterations: Cohort study from the French Sarcoma Group. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6071.

Abstract 892: Afamitresgene autoleucel (afami-cel; formerly ADP-A2M4) demonstrates durable clinical responses by inducing broad immune engagement with anti-tumor activity

Abstract Afami-cel is a mixed CD4+ CD8+ autologous T-cell therapy engineered to target the cancer testis antigen melanoma-associated antigen A4 in HLA-A*02-positive patients with advanced/metastatic synovial sarcoma or myxoid/round cell liposarcoma (MRCLS). Pooled data from the Phase 1 (NCT03132922) and Phase 2 (SPEARHEAD-1, NCT04044768) trials of afami-cel showed an acceptable benefit to risk profile with an overall response rate of 36.2% and a median duration of response of 52.0 weeks.1 To support the continued investigation of potential mechanisms of durable anti-tumor activity, we previously showed that afami-cel induces broad and enduring peripheral cytokine responses2 and that afami-cel tumoral infiltration is associated with increased presence of activated and proliferative cytotoxic T-cells in the tumor microenvironment.3 Here, we report the results of translational analyses exploring the cooperation between afami-cel-induced innate and adaptive immune responses pooled from the Phase 1 and 2 trials. Methods included measurement of 92 biomarkers related to apoptosis, chemotaxis, metabolism, tumor immunity promotion/suppression, and vascular/tissue remodeling in pre- and post-infusion serum samples from 38 patients. We also conducted multiplex immunofluorescence and gene set variation analysis of Reactome immune system pathway categories and microenvironment cell populations in RNA sequencing data from pre- and post-infusion biopsies from ≥15 patients. Serum analyses showed that patients with a clinical benefit as defined by RECIST v1.1 had significantly greater post-infusion levels of chemotactic markers (Kruskal-Wallis; p<0.05 for partial response [PR] compared to progressive disease, p<0.01 for PR compared to stable disease), indicating higher signaling related to immune-cell recruitment towards lesions. Tumor analyses showed increased expression of genes associated with innate and adaptive immunity, and cytokine signaling, in post-infusion biopsies, including T-cell receptor signaling-related expression, which was consistent with relatively greater spatial protein detection of pro-immune infiltrate. This profile was associated with longer progression-free survival. In conclusion, our data suggest that afami-cel induces peripheral and tumoral innate and adaptive immune responses, a hallmark of durable anti-tumor activity. Updated patient sample data will be presented. 1. D'Angelo SP, et al. J Clin Oncol. 2022;40:16_suppl:11562. 2. D’Angelo SP, et al. Poster 146 presented at: CTOS 2021; Virtual. 3. Van Tine, BA et al. Paper 61 presented at: CTOS 2022; Vancouver, BC, Canada. Citation Format: Cheryl McAlpine, Martin Isabelle, Robyn Broad, Revashnee Naidoo, Ashley Liddle, Elizabeth Duperret, Paul Noto, Ruoxi Wang, Dzmitry Batrakou, Sumit Middha, Chris Evans. Afamitresgene autoleucel (afami-cel; formerly ADP-A2M4) demonstrates durable clinical responses by inducing broad immune engagement with anti-tumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 892.

Standardized Computer-Assisted Analysis of PRAME Immunoreactivity in Dysplastic Nevi and Superficial Spreading Melanomas.

PRAME (PReferentially expressed Antigen in MElanoma) is a cancer testis antigen that is frequently expressed in melanoma compared to benign melanocytic proliferations and nevi. However, the interpretation of the intensity and distribution of PRAME immunostaining is not standardized a lot, which makes interpretation difficult. PRAME-stained histological slides of superficial spreading melanomas (SSM) and dysplastic nevi (DN) were digitized and analyzed using the digital pathology and image platform QuPath. t-tests and ROC AUCs were performed with SPSS. A p-value of <0.05 was used for statistical significance, and a ROC AUC score of >0.8 was considered a good result. A cut-off score was defined in an evaluation cohort and subsequently analyzed in an independent validation cohort. In total, 81 PRAME-stained specimens were included. The evaluation cohort included 32 (50%) SSM and 32 (50%) DN, and the mean of PRAME-positive cells/mm2 for the entire lesion was 455.3 (SD 428.2) in SSM and 60.5 (SD 130.1; p < 0.001) in DN. The ROC AUC of PRAME-positive cells of the entire lesion was 0.866, and in the epidermis it was 0.901. The defined cut-off score to distinguish between DN and SSM was 97.67 cells/mm2. In the validation cohort, 16 out of 17 cases (94.1%) were correctly classified by the cut-off score. The computer-aided assessment of PRAME immunostaining is a useful tool in dermatopathology to distinguish between DN and SSM. Lesions with a moderate expression and indifferent morphologic features will remain a challenge for dermatopathologists.

A Therapeutic Vaccine Targeting Rat BORIS (CTCFL) for the Treatment of Rat Breast Cancer Tumors.

Cancer testis antigens are ideal for tumor immunotherapy due to their testis-restricted expression. We previously showed that an immunotherapeutic vaccine targeting the germ cell-specific transcription factor BORIS (CTCFL) was highly effective in treating aggressive breast cancer in the 4T1 mouse model. Here, we further tested the therapeutic efficacy of BORIS in a rat 13762 breast cancer model. We generated a recombinant VEE-VRP (Venezuelan Equine Encephalitis-derived replicon particle) vector-expressing modified rat BORIS lacking a DNA-binding domain (VRP-mBORIS). Rats were inoculated with the 13762 cells, immunized with VRP-mBORIS 48 h later, and then, subsequently, boosted at 10-day intervals. The Kaplan-Meier method was used for survival analysis. Cured rats were re-challenged with the same 13762 cells. We demonstrated that BORIS was expressed in a small population of the 13762 cells, called cancer stem cells. Treatment of rats with VRP-BORIS suppressed tumor growth leading to its complete disappearance in up to 50% of the rats and significantly improved their survival. This improvement was associated with the induction of BORIS-specific cellular immune responses measured by T-helper cell proliferation and INFγ secretion. The re-challenging of cured rats with the same 13762 cells indicated that the immune response prevented tumor growth. Thus, a therapeutic vaccine against rat BORIS showed high efficacy in treating the rat 13762 carcinoma. These data suggest that targeting BORIS can lead to the elimination of mammary tumors and cure animals even though BORIS expression is detected only in cancer stem cells.