Tertiary Lymphoid Structures Research Articles

Distinct genomic features between osteosarcomas firstly metastasing to bone and to lung

BackgroundOsteosarcoma initially metastasing to bone only shows distinct biological features compared to osteosarcoma that firstly metastasizes to the lung, which suggests us underlying different genomic pathogenetic mechanism. MethodsWe analyzed whole-exome sequencing (WES) data for 38 osteosarcoma with paired samples in different relapse patterns. We also sought to redefine disease subclassifications for osteosarcoma based on genetic alterations and correlate these genetic profiles with clinical treatment courses to elucidate potential evolving cladograms. ResultsWe investigated WES of 12/38 patients with high-grade osteosarcoma (31.6%) with initial bone metastasis (group A) and 26/38 (68.4%) with initial pulmonary metastasis (group B), of whom 15/38 (39.5%) had paired samples of primary lesions and metastatic lesions. We found that osteosarcoma in group A mainly carries single-nucleotide variations displaying higher tumor mutation burden and neoantigen load and more tertiary lymphoid structures, while those in group B mainly exhibits structural variants. High conservation of reported genetic sequencing over time in their evolving cladograms. ConclusionsOsteosarcoma with mainly single-nucleotide variations other than structural variants might exhibit biological behavior predisposing toward bone metastases as well as better immunogenicity in tumor microenvironment.

096 The lymphoid follicles in primary cutaneous plasmacytosis may function as a tertiary lymphoid structure, playing a critical role in the differentiation of B cells and class-switch recombination

096 The lymphoid follicles in primary cutaneous plasmacytosis may function as a tertiary lymphoid structure, playing a critical role in the differentiation of B cells and class-switch recombination

106 The lymphoid follicle in pseudolymphoma, primary cutaneous follicle center lymphoma and primary cutaneous marginal zone B-cell lymphoma may activate the differentiation of B-cells and class-switching reaction as tertiary lymphoid structures

106 The lymphoid follicle in pseudolymphoma, primary cutaneous follicle center lymphoma and primary cutaneous marginal zone B-cell lymphoma may activate the differentiation of B-cells and class-switching reaction as tertiary lymphoid structures

278 Tumor immune microenvironment of cutaneous angiosarcoma with cancer-testis antigens and tertiary lymphoid structures

278 Tumor immune microenvironment of cutaneous angiosarcoma with cancer-testis antigens and tertiary lymphoid structures

037 The intra-niche activation of clonal CXCL13+CD4+ T cells in tertiary lymphoid structures associated with non-healing blisters of pemphigus

037 The intra-niche activation of clonal CXCL13+CD4+ T cells in tertiary lymphoid structures associated with non-healing blisters of pemphigus

272 Tertiary lymphoid structures in primary cutaneous squamous cell carcinomas are associated with subsequent metastasis

272 Tertiary lymphoid structures in primary cutaneous squamous cell carcinomas are associated with subsequent metastasis

1250 Induction of tertiary lymphoid structures in a mouse melanoma model under a combination of CXCL13 and anti-PD-1 antibody

1250 Induction of tertiary lymphoid structures in a mouse melanoma model under a combination of CXCL13 and anti-PD-1 antibody

Nanovaccines Fostering Tertiary Lymphoid Structure to Attack Mimicry Nasopharyngeal Carcinoma.

Tertiary lymphoid structures (TLSs) are formed in inflamed tissues, and recent studies demonstrated that the appearance of TLSs in tumor sites is associated with a good prognosis for tumor patients. However, the process of natural TLSs' formation was slow and uncontrollable. Herein, we developed a nanovaccine consisting of Epstein-Barr virus nuclear antigen 1 (EBNA1) and a bi-adjuvant of Mn2+ and cytosine-phosphate-guanine (CpG) formulated with tannic acid that significantly inhibited the development of mimicry nasopharyngeal carcinoma by fostering TLS formation. The nanovaccine activated LT-α and LT-β pathways, subsequently enhancing the expression of downstream chemokines, CCL19/CCL21, CXCL10 and CXCL13, in the tumor microenvironment. In turn, normalized blood and lymph vessels were detected in the tumor tissues of the nanovaccine group, correlated with increased infiltration of lymphocytes. Especially, the proportion of the B220+ CD8+ T, which was produced via trogocytosis between T and B cells during activation of T cells, was increased in tumors of the nanovaccine group. Furthermore, the intratumoral effector memory T cells (Tem), CD45+, CD3+, CD8+, CD44+, and CD62L-, did not decrease after blocking the egress of T cells from tumor-draining lymph nodes by FTY-720. These results demonstrated that the nanovaccine can foster TLS formation, which thus enhances local immune responses significantly, delays tumor outgrowth, and prolongs the median survival time of murine models of mimicry nasopharyngeal carcinoma, demonstrating a promising strategy for nanovaccine development.

Antibodies against endogenous retroviruses promote lung cancer immunotherapy

B cells are frequently found in the margins of solid tumours as organized follicles in ectopic lymphoid organs called tertiary lymphoid structures (TLS)1,2. Although TLS have been found to correlate with improved patient survival and response to immune checkpoint blockade (ICB), the underlying mechanisms of this association remain elusive1,2. Here we investigate lung-resident B cell responses in patients from the TRACERx 421 (Tracking Non-Small-Cell Lung Cancer Evolution Through Therapy) and other lung cancer cohorts, and in a recently established immunogenic mouse model for lung adenocarcinoma3. We find that both human and mouse lung adenocarcinomas elicit local germinal centre responses and tumour-binding antibodies, and further identify endogenous retrovirus (ERV) envelope glycoproteins as a dominant anti-tumour antibody target. ERV-targeting B cell responses are amplified by ICB in both humans and mice, and by targeted inhibition of KRAS(G12C) in the mouse model. ERV-reactive antibodies exert anti-tumour activity that extends survival in the mouse model, and ERV expression predicts the outcome of ICB in human lung adenocarcinoma. Finally, we find that effective immunotherapy in the mouse model requires CXCL13-dependent TLS formation. Conversely, therapeutic CXCL13 treatment potentiates anti-tumour immunity and synergizes with ICB. Our findings provide a possible mechanistic basis for the association of TLS with immunotherapy response.

The roles of tertiary lymphoid structures in chronic diseases.

Tertiary lymphoid structures (TLSs) are ectopic lymphoid tissues that drive antigen-specific immune responses at sites of chronic inflammation. Unlike secondary lymphoid organs such as lymph nodes, TLSs lack capsules and have their own unique characteristics and functions. The presumed influence of TLSs on the disease course has led to widespread interest in obtaining a better understanding of their biology and function. Studies using single-cell analyses have suggested heterogeneity in TLS composition and phenotype, and consequently, functional correlates with disease progression are sometimes conflicting. The presence of TLSs correlates with a favourable disease course in cancer and infection. Conversely, in autoimmune diseases and chronic age-related inflammatory diseases including chronic kidney disease, the presence of TLSs is associated with a more severe disease course. However, the detailed mechanisms that underlie these clinical associations are not fully understood. To what extent the mechanisms of TLS development and maturation are shared across organs and diseases is also still obscure. Improved understanding of TLS development and function at the cellular and molecular levels may enable the exploitation of these structures to improve therapies for chronic diseases, including chronic kidney disease.

Evaluation of tumor-infiltrating T & B lymphocytes and their association and distribution in oral squamous cell carcinoma tumor microenvironment: An in vitro immunohistochemical study

The immune interaction between host immunity and the tumor microenvironment is complex, and a thorough understanding of tumor-infiltrating lymphocyte selection in oral cancer, including T and B cells, is urgently required. Within the tumor microenvironment, tumor cells escape immune surveillance and grow uncontrollably. The study examined the relationship and distribution of tumor-infiltrating T and B lymphocytes. Retrospective data of paraffin-embedded tissue samples of 47 primary oral squamous cell carcinoma (OSCC) cases were retrieved. Hematoxylin and eosin evaluation, along with all clinicopathologic data, were collected. Immunohistochemical CD3 and CD20 markers were used and evaluated for association and distribution in given OSCC cases. The intermediate type of inflammatory infiltrate was seen primarily in Well DIfferentiated Squamous cell Carcinoma grade and positive and negative lymph nodes. Compared with T-cell density, B-cell density showed an aggregate pattern rather than a scattered pattern, indicating a statistically significant association between T-cell and B-cell infiltrate. B-cell infiltrates were also found to have a statistically significant relationship with tertiary lymphoid structure. A strong, positive association and correlation exists between B- and T-lymphocyte infiltration in both the stroma and the invasive front. When compared with T-cell density, B-cell density is more predominantly in aggregates.

Abstract 2211: The prognostic role of tertiary lymphoid structure (TLS) in locally advanced esophageal squamous cell carcinoma (ESCC)

Abstract Introduction: The presence of TLS in tumors is associated with improved efficacy of immunotherapy in various types of cancer. The significance of TLS in patients with ESCC has been sporadically reported. The study aimed to investigate the prognostic significance of TLS in patients with locally advanced ESCC receiving neoadjuvant chemoradiotherapy (CRT). Patients and Methods: We included two cohorts of locally advanced ESCC patients from two referral centers of Taiwan. Cohort A consisted of 99 patients treated with paclitaxel/cisplatin-based CRT, 69 of them being neoadjuvant CRT and 30 of them being definitive CRT, between 2002 and 2015; cohort B consisted of 67 patients treated with paclitaxel/carboplatin-based neoadjuvant CRT between 2012 and 2018. Primary tumor tissues were retrospectively collected for immunohistochemical stains by CD20 (clone L26; Zytomed, Berlin, Germany) and CD23 (clone DAK-CD23; agilent Dako, California, U.S.A.). TLS was defined as aggregation of CD20 B cells, and mature TLS was defined as TLS with CD23-positively stained germinal center. Patients’ characteristics were compared between those with or without TLS or mature TLS. The impact of TLS or mature TLS on patients’ overall survival (OS) was analyzed by Kaplan-Meier method and log rank test. Results: In cohort A (median age of 56 years, M:F= 88:11), no TLS, immature TLS, and mature TLS were seen in 52, 35, and 12 patients, respectively. In cohort B (median age of 55 years, M:F= 64:3), no TLS, immature TLS, and mature TLS were seen in 27, 25, and 15, respectively. In cohort A, the presence of TLS is more commonly seen in ESCC with the primary site at upper (58%) and lower segments (63%) than middle segment of esophagus (35%) (p=0.039), and the mature TLS is more commonly seen at lower segment (25%) than other segments (8%) (p=0.033). The pathological complete response (pCR) was not significant different between patients with vs without TLS in cohort A (39% vs 43 %, p= 0.75), but trended to be lower in patients with TLS than without in cohort B (28% vs 52 %, p= 0.061). Besides, pCR rate trended to be lower in patients with mature TLS than without in both cohorts (13% vs 45 %, p= 0.080, for cohort A; 28% vs 43 %, p= 0.23, for cohort B). As patients with or without TLS did not differ in their OS in both cohorts, patients with mature TLS trended to have worse overall survival in both cohort A (HR: 1.4 [95% CI 0.33-3.05], p=0.29) and cohort B (HR: 1.6 [95% CI 0.81-2.98], p=0.15). Conclusion: In two independent cohorts of locally advanced ESCC patients receiving paclitaxel/platinum-based CRT, we did not find the correlation of the presence of TLS or mature TLS in pre-treatment primary ESCC tumor tissue with the favorable treatment outcomes. (The study was supported by the grant NTUH 110-S5021, the grant MOST 108-2314-B-002-076-MY3, and grant MOHW 111-TDU-B-221-114006) Citation Format: Ta-Chen Huang, Cher-Wei Liang, Yu-I Li, Jhe-Cyuan Guo, Chia-Chi Lin, Jang-Ming Lee, Yin-Kai Chao, Chih-Hung Hsu. The prognostic role of tertiary lymphoid structure (TLS) in locally advanced esophageal squamous cell carcinoma (ESCC) [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 2211.

Abstract 5444: Integrated analysis of gene expression signatures and AI-based detection of tertiary lymphoid structures and stromal tumor-infiltrating lymphocytes in breast cancer H&E samples

Abstract Background: Transcriptomic and morphological features of the tumor microenvironment (TME) are associated with favorable prognosis and can serve as surrogate markers for immune checkpoint inhibitor response in certain breast cancer subtypes. Tertiary lymphoid structures (TLS) and stromal tumor-infiltrating lymphocytes (sTILs) are integral components of the TME. New techniques for TLS characterization could improve reproducibility and automate the current workflow. Here, we correlated AI-based imaging platform predictions of TLS and sTILs on breast cancer H&E whole slide images (WSIs) with expression data. Design: WSIs from 390 TCGA breast cancer samples were used to detect normalized tumoral TLS and tumor stroma infiltration areas by a convolutional neural network (CNN). High and low groups were defined by IQR1 and IQR3. Gene counts were compared using differential expression analysis followed by gene set enrichment analysis (GSEA) with hallmark gene sets. Kassandra's cell type percentage predictions, functional gene signature (Fges) single-sample GSEA, and PROGENy pathway activity median-scaled scores were compared with the Mann-Whitney U test. The Fges consisted of 4 TLS and 29 TME signatures and were clustered into 4 groups: fibrotic, myeloid, T cell, and TLS. Group medians were analyzed. Somatic deep amplifications, deletions, and missense mutations of cancer pathways were compared with Fisher’s exact test. The FDR method was used to adjust p-values. Results: FDCSP, CXCL13, CD79A, CCL19, and IGLL5 were upregulated in the TLS-high group (logFC > 1.5, p < 0.003), and CXCL13, CXCL9, CCL19, CD3E, and CD79A were upregulated in the sTIL-high group (logFC > 2, p < 4e-9). GSEA identified interferon γ and α response and allograft rejection signatures in TLS- and sTIL-high groups (p = 0.006, NES > |2.5|). sTIL-high samples expressed high levels of E2F targets, while the sTIL-low group was enriched with myogenesis and epithelial-mesenchymal transition gene sets. The TLS-high group was enriched in the TLS Fges cluster (p = 1e-5) and contained high B, CD4+ T, and CD8+ T cell content (p < 2e-4). sTIL samples expressed high levels of B, CD4+ T, CD8+ T, and myeloid cells (p < 0.001) and TLS and myeloid clusters (p < 1e-5). TGFβ and VEGF pathways were more active in sTIL-low samples (p < 0.001), while JAK-STAT was most active in the sTIL-high group (p = 0.0006). In the basal subtype (p < 0.001), the TLS-high group was associated with NOTCH2 amplification; the sTIL-low group had increased NRG1 deletions; and the sTIL-high group contained BRCA2 mutations. Conclusions: RNA-based methods used were concordant with AI-based methods of TLS and sTIL detection. Our CNN-based method of identifying immune structures in the TME with morphological features on H&E WSIs may automate traditional pathology workflows with additional validation. Citation Format: Nadezhda Lukashevich, Vladimir Kushnarev, Daniil Dymov, Anastasia Zotova, Anna Belozerova, Ivan Valiev, Lev Popyvanov, Anna M. Love, Nathan Fowler, Alexander Bagaev, Ekaterina Postovalova. Integrated analysis of gene expression signatures and AI-based detection of tertiary lymphoid structures and stromal tumor-infiltrating lymphocytes in breast cancer H&E samples. [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 5444.

Abstract SY16-02: Generation of B cell immunity in tertiary lymphoid structures supports response to immunotherapy

Abstract SY16-02: Generation of B cell immunity in tertiary lymphoid structures supports response to immunotherapy

Abstract 6767: Cancer-associated fibroblasts promote T-cell exclusion and resistance to immunotherapy in non-small cell lung cancer with tertiary lymphoid structures

Abstract Background: We and others have reported the major role of mature tertiary lymphoid structures (TLS) in the efficacy of PD-L1 blockade in NSCLC. The objective response rate (ORR) to inhibition of PD1/PDL1 used as single agents observed in TLS-positive NSCLC is similar to ORRs observed in all-comers NSCLC treated with a combination of immune checkpoint inhibitor (ICI) and chemotherapy. However, a substantial proportion of patients with TLS-positive NSCLC did not display clinical benefit. The aim of our study was to identify the determinants of response to ICI in TLS-positive NSCLC. Methods: We spatially profiled the expression of >18,000 protein-coding genes across six patients with mature TLS-positive NSCLC (three with objective response and three with progressive disease as the best response) using the GeoMx whole-transcriptome atlas (WTA) assay. Using two consecutive slides stained with CD3/CD20 and CD45/PanCK markers, a first set of regions of interest (ROIs) selected the TLS (CD3+/CD20+) as an entire area of illumination (AOIs). A second set of ROIs was selected outside the TLS region and further segmented into ‘tumor’ (PanCK+/CD45-) versus ‘stroma’ (PanCK-/CD45+) AOIs. To further validate our findings, we performed multiplex immunofluorescence (mIF) on 70 mature TLS-positive NSCLC tumors. Results: Comparative transcriptomic analysis of TLS between non-responders and responders revealed upregulation in responders of CCL21 which plays a crucial role in T cell recruitment, and of IGHM indicating the presence of unswitched B cells. Outside of TLS, in the stromal compartment, the most significantly differentially expressed genes between responders and non-responders were those belonging to a subset of cancer associated fibroblasts (CAFs) expressing both FAP and αSMA markers, and recently described as linked to more inflammatory activated immune cells and involved in T cell exclusion in NSCLC. We then used FFPE tissue mIF to histologically profile CAF subset composition and spatial distribution of T cells in a cohort of 70 mature TLS-positive NSCLC. In tumor lesions enriched in FAP+αSMA+ CAFs, the tumor-to-stroma ratio of infiltrating CD8+ cells was significantly lower, consistent with a decreased infiltrating CD8+ T cell density in the tumor. Strikingly, ORR and survival of patients with mature TLS NSCCLC enriched in FAP+αSMA+ CAFs were significantly lower than those of patients with low abundance of CAFs. Conclusions: We report here the first study investigating the mechanisms of resistance to immunotherapy in TLS-positive tumors. Our analysis of human tumor samples suggests a crucial role of FAP+αSMA+ CAFs in excluding T cells and in driving resistance to immune checkpoint inhibition in mature TLS-positive NSCLC. The development of novel CAF-targeting strategies may be particularly relevant in this subset of tumors. Citation Format: Florent Peyraud, Jean-Philippe Guegan, Christophe Rey, Oren Ngouala, Ophélie Odin, Emma Clot, Lucile Vanhersecke, Maxime Brunet, Thomas Grellety, Marie Del Castillo, Sylvestre Le Moulec, François Le Loarer, Alban Bessede, Antoine Italiano. Cancer-associated fibroblasts promote T-cell exclusion and resistance to immunotherapy in non-small cell lung cancer with tertiary lymphoid structures [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 6767.

Abstract 4622: Tumor microenvironment differences between lung cancer subtypes revealed by spatial transcriptomics

Abstract Lung cancer is a leading cause of cancer deaths worldwide and has complex underlying genetic drivers, subtypes and immune cell types. Molecular analysis of bulk tumors has repeatedly identified key somatic driver genes and subtypes in lung cancer. However, these key molecular strata of bulk lung tumors still contain significant heterogeneity which if characterized in finer detail may reveal new tumor microenvironment factors and lead to improved patient prognostication and therapy options. Here, we sought to compare the tumor microenvironments of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) through spatial transcriptomics. Using four frozen lung tumors and three with replicated sections (n = 7), we sequenced spatial transcriptomes using the 10X Visium platform and illumina sequencing to measure up to 5,000 latticed-spots throughout 6.5 mm2 of the tumor surface area. Data analysis revealed a large number of latticed-spots per tumor (median 3,486) with a large number of genes detected per median spot (tumor median 4,427). Through unsupervised clustering of spots in each tumor, we found between 8 and 10 clusters per tumor with distinct pathway activities, including multiple immune-enriched clusters per tumor (range: 3-5). Immune-enriched clusters in one LUAD tumor displayed a spatial shape consistent with tertiary lymphoid structures (TLS). Concordantly, this cluster overexpressed both B cell and T cell pathways and as well as a TLS signature from liver cancer. Interestingly by bulk tumor RNA analysis, this TLS+ tumor was classified to be in the terminal respiratory unit expression subtype, which is an immune-mild bulk subtype. This supports that the TLS signal can be a unique property of spatial expression in lung cancer that may be unobservable by bulk tumor RNA sequencing. Then to compare global spatial heterogeneity among tumors, we calculated an index of expression spatial continuity and found LUSC tumors to have more contiguous expression patterns than LUAD tumors (mean 0.60 vs 0.54). We also quantified expression diversity across all tumor latticed-spots and found that LUSC tumors had greater values compared to LUAD tumors (mean 0.37 vs 0.27). Together, our results suggest that LUSC has a more contiguous and heterogeneous tumor expression microenvironment than LUAD. TLS are predictive of immune checkpoint inhibitor response in many other tumor types, and our results suggest that spatial transcriptomics may also identify this responsiveness in lung cancer. Future, larger cohorts of lung tumors are needed to determine recurrent spatial properties associated with patient outcome and treatment response. The views expressed in this abstract are solely of the authors and do not reflect the official policy of the Departments of Army/Navy/Air Force, Department of Defense, USUHS, HJF, or U.S. Government. Citation Format: Matthew D. Wilkerson, Savannah Kounelis-Wuillaume, Camille Alba, Teri J. Franks, Martin L. Doughty, Robert L. Kortum, Robert F. Browning, Clifton L. Dalgard, Craig D. Shriver. Tumor microenvironment differences between lung cancer subtypes revealed by spatial transcriptomics. [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 4622.

Abstract 4341: Indoleamine 2,3-dioxygenase 1 is highly expressed in tertiary lymphoid structures and associated with improved outcome in patients with advanced non-small cell lung cancer treated with anti-PD1/PDL1 inhibitors

Abstract Background: Immune checkpoint inhibitors (ICI) has revolutionized the management of patients with non-small cell lung cancer (NSCLC). However, most patients with advanced NSCLC patients displayed primary resistance to ICI. Indoleamine 2,3-dioxygenase 1 (IDO1) may play a role in primary resistance by catabolizing tryptophan to several immunosuppressive metabolites such as kynurenine. The aim of our study was to investigate the role of IDO1 expression in the outcome of NSCLC patients treated with immunotherapy. Methods: We analyzed the largest NSCLC gene expression (GE) dataset worldwide including 891 pre-treatment tumors from POPLAR and OAK studies, two RCT assessing the efficacy of atezolizumab vs docetaxel in NSCLC. We then confirm our findings with multiplex immunofluorescence (mIF) and digital pathology analysis to investigate the correlation between expression of IDO1 by tumor cells and spatial distribution of CD8+ T cells. Results: The PFS of IDO1high patients was significantly higher than IDO1low (3.81 vs 1.89months, p=7.13e-09). Strikingly, high IDO1 expression was also associated with better ORR (19.9% vs 5.2%, p=9.93e-05) and improved OS (15.9 vs 9.65 months, p=6.35e-06). When adjusting for covariates (histological subtype, TLS signature, PD-L1 expression), IDO1 remained independently associated with PFS (HR 1.54, p=4.1e-4) and OS (HR 1.43, p=8.2e-3). None of these correlations were observed in the docetaxel arm, suggesting a predictive value of IDO1 GE specifically for response to atezolizumab. Using a deconvolution approach, the tumor microenvironment composition differed significantly between IDO1high and IDO1low tumors. Indeed, IDO1high tumors were characterized by the highest expression of genes specific to immune populations such as interferon-gamma-producing T helper 1 (Th1) cells, CD8+ T cells, natural killer cells, cytotoxic lymphocytes, and B cells. To further validate our findings, we performed mIF on 53 NSCLC tumors. Overall, elevated IDO1 expression by tumor cells was correlated with increased CD8+/PD1+ T cells infiltration (p=0.001). Spatial cell distribution analysis also revealed that the relative distance between tumor cells and CD8+ T cells was significantly lower in IDO1high compared to IDO1low patients. Interestingly, 52% of tumors were characterized by the presence of TLS and IDO1 was observed in most TLS analyzed. Conclusions: IDO1 expression by tumor cells in NSCLC is associated with improved outcome in patients with NSCLC, independently of PDL1 expression score. IDO1 overexpression in NSCLC is associated with increased immune activity and may represent a negative feedback mechanism to CD8+ T cell infiltration. Citation Format: Jean-Philippe Guegan, Florent Peyraud, Christophe Rey, Oren Ngouala, Ophélie Odin, Imane Nafia, Isabelle Soubeyran, Sylvestre Le Moulec, Alban Bessede, Antoine Italiano. Indoleamine 2,3-dioxygenase 1 is highly expressed in tertiary lymphoid structures and associated with improved outcome in patients with advanced non-small cell lung cancer treated with anti-PD1/PDL1 inhibitors. [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 4341.

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 3273: Guadecitabine plus ipilimumab in unresectable melanoma: Five-year follow-up and correlation with integrated, multi-omic analysis in the NIBIT-M4 trial

Abstract DNA hypomethylating agents (DHA) combined with immune-checkpoint inhibitors (ICI) is a promising strategy to improve the effect of immunotherapy. Providing initial support to this notion, the phase Ib NIBIT-M4 study, a dose-escalation trial of the DHA guadecitabine plus ipilimumab, has shown this regimen to be safe, tolerable and with promising immunomodulatory and anti-tumor activity in advanced melanoma. Here we report the 5-year clinical outcome of NIBIT-M4 patients and its correlation with an integrated multi-omic analysis. The NIBIT-M4 enrolled unresectable Stage III or IV cutaneous melanoma patients. Median Overall Survival (OS), Progression Free Survival (PFS), 5-year OS and PFS rate, and median Duration of Response (DoR) were assessed. Tumor biopsies were performed at baseline and at week 4 and 12 on-treatment. RNA-seq, whole exome sequencing (WES), RRBS methylation profiling, and tumor immune contexture data generated by immunohistochemistry (IHC), were integratively analyzed and correlated with clinical outcome. Patients were classified as responder (R) or non-responder (NR) based on Disease Control. Nineteen patients were enrolled in the NIBIT-M4 study between October 2015, and August 2018. As of July 1, 2022, median OS was 25.6 months (mo) (95% CI, 0.0-52.9), median PFS was 5.2 mo (95% CI, 4.0-6.4); 5-year OS rate was 28.9% and 5-year PFS rate was 5.3 %; median DoR was 20.6 mo (95% CI, 12.4-28.8). Tumor biopsies for correlative analyses were available from 14 patients. Compared to NR, R patients showed higher mutation frequency in NRAS, while NR patients harbored more frequent somatic alterations in genes involved in Epithelial to Mesenchymal Transition (EMT) and chromatin organization pathways. By tumor transcriptome analysis, NR patients showed enrichment for proliferation/differentiation/EMT pathways associated with suppression of INF-ɣ/HLA-II/immune-related transcriptional modules. R patients displayed a dynamic pattern of activated immune signatures related to CD8+ Tex cells, B cells, tertiary lymphoid structures, TFH cells and IFN-ɣ, reaching highest expression levels in week 12 biopsies. Deconvolution of transcriptomic data showed higher CD8+ and NK cell content and higher correlation of TCRB clonality with CD8+ content in R patients. By integration of a genetic immunoediting index (GIE) with an adaptive immunity signature (ICR) lesions were stratified into four distinct subsets. Interestingly, the ICR/GIE classification discriminated 5-year OS and PFS while the classification based on response groups did not. Moreover, patients with a “high ICR/non-GIE” (i.e., without immunoediting) showed lower expression of antigen presentation and processing-related genes associated with defective HLA-class I expression in the lesions, in spite of high CD8+ content. Citation Format: Teresa Noviello, Anna Maria Di Giacomo, Francesca Pia Caruso, Alessia Covre, Giovanni Scala, Luigi Ferraro, Sandra Coral, Wolf H. Fridman, Catherine Sautes-Fridman, Roberta Mortarini, Elena Simonetti, Maria Fortunata Lofiego, Davide Bedognetti, Andrea Anichini, Michele Ceccarelli, Michele Maio. Guadecitabine plus ipilimumab in unresectable melanoma: Five-year follow-up and correlation with integrated, multi-omic analysis in the NIBIT-M4 trial [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 3273.

Abstract 6624: 3D assessment of the lung cancer microenvironment using multi-resolution open-top light-sheet microscopy

Abstract Background: Non-small cell lung cancer (NSCLC) tissue is a valuable resource for diagnosis, treatment planning, and drug development. Current 2D histopathological techniques introduce under-sampling error (i.e., a single 5 um section represents 0.5% of a 1 mm thick biopsy), interobserver variability, and fail to capture the biology contained within the entire tissue sample. We have developed a suite of technologies to stain, chemically clarify, image, visualize, and analyze entire intact NSCLC tissue samples. Methods: Human NSCLC tissue, stored frozen in OCT, was fixed in 4% paraformaldehyde, stained with nuclear (TOPRO-3) and general protein (Eosin) fluorescent dyes, and optically cleared using a modified iDISCO protocol with ethyl cinnamate as the refractive index matching solution. Whole, intact tissue samples, roughly 1-5mm3 in volume, were imaged at 2 microns/pixel resolution with an open-top light-sheet microscope (3Di, Alpenglow Biosciences). Smaller regions of interest (ROIs) with key pathologic features were reimaged at higher resolution, 0.17 microns/pixel, to reveal subnuclear features and for cell typing. Visualization was performed using Aivia software. Results: NSCLC tissue samples were successfully imaged in 3D. Low resolution images (2 microns/pixel) were obtained within 4-31 minutes, depending on the tissue volume. The 3D distribution of cancer cells, immune cells, vessels, and fibrosis varied substantially throughout the volume of the tissue. Recognizable histologic features, including nests of tumor cells surrounded by vasculature and immune cells, were readily visualized. Squamous and adenocarcinoma with its subtypes (solid, acinar, lepidic, and micropapillary) morphologies were recognizable in 2D optical sections of the 3D datasets. Imaging quality degraded in tissue deeper than 1 mm due to light scattering. Conclusion: We assessed intact NSCLC tissue samples measuring up to 5 mm3 using our custom light-sheet microscope and tissue clearing techniques. This novel method enables us to visualize key features of NSCLC such as the tumor interfaces, tertiary lymphoid structures, vessels and fibrosis in the entire tissue sample, preventing under sampling error, and potentially enabling new biologic insights. Next steps include segmentation and quantification of key tissue structures such as tumor volume, immune cell distribution, and fibrosis/immune cell exclusion. This proof-of-concept study provides motivation for further investigation into the significance of 3D tissue features in NSCLC tissue samples. Citation Format: Alexandra Alvarsson, Carl Storey, Brandy Olin Pope, Caleb Stoltzfus, Robert Vierkant, Jessica Tufariello, Aaron Bungum, Julia Naso, Cheuk Ki Chan, Eric Edell, Christopher Hartley, Janani Reisenauer, Nicholas Reder. 3D assessment of the lung cancer microenvironment using multi-resolution open-top light-sheet microscopy [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 6624.