Multiplex Immunohistochemical Staining Research Articles

Abstract PO2-08-06: Metformin may improve the outcome of patients with breast cancer and type 2 diabetes mellitus through the effect of tumor immune microenvironment

Abstract Metformin, an anti-diabetic drug, is known to have anti-tumor effects. We examined the outcome of 177 patients with type 2 diabetes mellitus (T2DM) who received surgery for breast cancer. Among them, 49 patients were treated with drugs including metformin. In those patients, recurrence in distant organs was less frequent and postoperative disease-free survival (DFS) tended to be better than those without metformin intake. In patients who received preoperative systemic therapy (PST), rate of pathological complete response (pCR) was significantly more frequent in patients with metformin treatment (p < 0.05). Multiplex immunohistochemical staining of resected tumors revealed that the density of tumor associated macrophages (TAM), especially of CD68(+)CD163(+) M2-type TAM, was significantly lower in tumors with metformin treatment. In contrast, the rate of CD8(+) phenotype in CD3(+) tumor infiltrating lymphocytes (TILs) was significantly higher in metformin group. The results suggest that metformin can change the immune microenvironment from pro-tumorigenic to anti-tumorigenic status, which leads to the favorable outcome of the patients with breast cancer and T2DM. Citation Format: Satomi Shiba, Michiko Harao, Kasumi Ogihara, Takayo Fukuda, Masako Sakuragi, Kitayama Joji, Naohiro Sata. Metformin may improve the outcome of patients with breast cancer and type 2 diabetes mellitus through the effect of tumor immune microenvironment [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-08-06.

Abstract LT09: Integrative Multi-Omics Enhancer Activity Profiling Identifies Therapeutic Vulnerabilities in Cholangiocarcinoma of Different Etiologies

Abstract Objectives: Cholangiocarcinoma (CCA) is a heterogeneous malignancy with high mortality and dismal prognosis, and an urgent clinical need for new therapies. Knowledge of the CCA epigenome is largely limited to aberrant DNA methylation. Dysregulation of enhancer activities has been identified to affect carcinogenesis and leveraged for new therapies but is uninvestigated in CCA. Our aim is to identify potential therapeutic targets in different subtypes of CCA through enhancer profiling. Design: Integrative multi-omics enhancer activity profiling of diverse CCA was performed. A panel of diverse CCA cell lines, and patient-derived and cell line-derived xenografts were used to study identified enriched pathways and vulnerabilities. NanoString, multiplex immunohistochemistry staining, and single-cell spatial transcriptomics were used to explore immunogenicity of diverse CCA. Results: We identified 3 distinct groups, associated with different etiologies and unique pathways. Drug inhibitors of identified pathways reduced tumor growth in in-vitro and in-vivo models. The first group (ESTRO), with mostly fluke-associated CCAs, displayed activation in estrogen signaling and were sensitive to MTOR inhibitors. Another group (OXPHO), with mostly BAP1 and IDH-mutant CCAs, displayed activated oxidative phosphorylation pathways, and were sensitive oxidative phosphorylation inhibitors. Immune-related pathways were activated in the final group (IMMUN), made up of an immunogenic CCA subtype and CCA with aristolochic acid (AA) mutational signatures. Intra-tumor differences in AA mutation load were correlated to intra-tumor variation of different immune cell populations. Conclusion: Our study elucidates the mechanisms underlying enhancer dysregulation and deepens understanding of different tumorigenesis processes in distinct CCA subtypes, with potential significant therapeutics and clinical benefits. Citation Format: Jing Han Hong, Chern Han Yong, Hong Lee Heng, Jason Yongsheng Chan, Mai Chan Lau, Jianfeng Chen, Jing Yi Lee, Abner Herbert Lim, Zhimei Li, Peiyong Guan, Pek Lim Chu, Sheng Rong Ng, Xiaosai Yao, Peili Wang, Rong Xiao, Xian Zeng, Yichen Sun, Wei Liu, Joanna Koh, Felicia Yu Ting Wee, Jeffrey Chun Tatt Lim, Cedric Chuan Young Ng, Xiu Yi Kwek, Poramate Klanrit, Yaojun Zhang, Jiaming Lai, David Wai Meng Tai, Chawalit Pairojkul, Simona Dima, Irinel Popescu, Sen-Yung Hsieh, Ming-Chin Yu, Joe Yeong, Sarinya Kongpetch, Apinya Jusakul, Watcharin Loilome, Patrick Tan, Jing Tan, Bin Tean Teh. Integrative Multi-Omics Enhancer Activity Profiling Identifies Therapeutic Vulnerabilities in Cholangiocarcinoma of Different Etiologies [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr LT09.

Abstract 865: Single-cell sequencing identification of malignant cell subpopulations linked to recurrence in hepatocellular carcinoma

Abstract Introduction: Hepatocellular Carcinoma (HCC) is a highly malignant and challenging-to-treat tumor, with early recurrence affecting up to 35-50% of patients within two years after surgery, significantly impacting prognosis. Identifying high-risk patients for recurrence is critical for guiding postoperative adjuvant therapy. The rapidly advancing field of single-cell RNA sequencing technology offers an unprecedented opportunity to discover clinically relevant cell subpopulations. Here, we harnessed single-cell techniques to unveil a cell subpopulation closely linked with HCC recurrence, highlighting its clinical significance. Methods and Results: We conducted single-cell RNA sequencing on four HCC samples and harmonized data from three public datasets (GSE112271, GSE125449, CNP0000650) to elucidate the heterogeneity of malignant cells in HCC. Our investigation revealed a distinctive HCC subpopulation enriched in patients with early recurrence (11.4% vs. 3.8%, P=0.02). This subpopulation exhibited up-regulated STMN1 and EZH2, signifying high proliferative potential and a propensity for epithelial-mesenchymal transition (EMT). Applying the CIBERSORTx algorithm to the TCGA-LIHC dataset (N=323 patients), we inferred the proportion of this recurrence subpopulation among all malignant cells. Notably, patients with a higher proportion of this subpopulation (N=160) demonstrated significantly shorter recurrence-free survival (31.2 vs. 42.9 months, P=0.002), reduced median survival (45.9 vs 55.7 months, P=0.03) and a higher rate of microvascular invasion (35% vs. 23%, P=0.01). To validate this recurrence-related subpopulation in clinical samples, we conducted multiplex immunohistochemistry staining on paraffin-embedded (FFPE) HCC samples (including CD45, CD31, ARG1, STMN1, EZH2) and used machine learning for its identification. Our findings revealed that patients with a higher proportion of this recurrence subpopulation (N=40) experienced significantly shorter median survival (38.5 vs. 51.0 months, P<0.005) and progression-free survival (25.1 vs. 33.4 months, P=0.01) compared to a lower proportion (N=40). Multivariate Cox regression analysis confirmed that the proportion of this recurrence subpopulation is an independent predictor of worse prognosis (P=0.003). Conclusion: Through the power of single-cell RNA sequencing technology, we have unearthed and characterized a malignant cell subpopulation intricately connected with HCC recurrence. Importantly, this subpopulation can be identified in FFPE tissue samples and correlates robustly with patient prognosis. Our study underscores the transformative possibility of single-cell techniques in developing cell subpopulation-based biomarkers, emphasizing their clinical relevance and potential utility in guiding postoperative adjuvant therapy for HCC patients. Citation Format: Yucheng Dong, Xin Guo, Xuezhu Wang, Fan Yang, Yongchang Zheng, Yang Chen. Single-cell sequencing identification of malignant cell subpopulations linked to recurrence in hepatocellular carcinoma [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 865.

Immune checkpoint ligands expressed on mature high endothelial venules predict poor prognosis of NSCLC: have a relationship with CD8+ T lymphocytes infiltration.

An insufficient number of intratumoral CD8+ T lymphocytes is a major barrier to antitumor immunity and immunotherapy. High endothelial venules (HEVs) are the major sites through which lymphocytes enter tumors; however, the molecular mechanism through which HEVs mediate CD8+ T lymphocyte infiltration remains poorly understood. Forty-two patients with stage IIIA lung adenocarcinoma, who underwent surgery, were recruited. Multiplex immunohistochemical staining was conducted on tumor tissues to detect the immune checkpoint ligands (ICLs) expressed in the HEVs, blood vessels, and lymphatics. A new ICL score model was constructed to evaluate ligand expression. The relationship between ICL score, tumor-infiltrating CD8+ T cell frequency, and survival of patients was investigated. Mature HEVs, but not blood vessels or lymphatics, mediated CD8+ T cell infiltration. However, the ICLs expressed on mature HEVs could negatively regulate CD8+ T cell entry into tertiary lymphoid structures (TLSs). In addition, according to the results obtained using our ICLtotal score model, the expression of ICLs on HEVs was observed to be a predictor of both CD8+ T cell infiltration and survival, in which a high ICLtotal score > 1 represent a weak CD8+ T cell infiltration and a high ICLtotal score > 2 predicts poor survival. Using the ICL score model, we discovered that ICLs expressed on HEVs are indicative of CD8+ T cell subset infiltration in TLSs, as well as of patient survival with lung cancer.

Multiplex Cyclic Fluorescent Immunohistochemistry.

The tumor microenvironment involves interactions between host cells, tumor cells, immune cells, stromal cells, and vasculature. Characterizing and spatially organizing immune cell subsets and target proteins are crucial for prognostic and therapeutic purposes. This has led to the development of multiplexed immunohistochemistry staining methods. Multiplex fluorescence immunohistochemistry allows the simultaneous detection of multiple markers, facilitating a comprehensive understanding of cell function and intercellular interactions. In this paper, we describe a workflow for the multiplex cyclic fluorescent immunohistochemistry assay and its application in the quantification analysis of lymphocyte subsets. The multiplex cyclic fluorescent immunohistochemistry staining follows similar steps and reagents as standard immunohistochemistry, involving antigen retrieval, cyclic antibody incubation, and staining on a formalin-fixed paraffin-embedded (FFPE) tissue slide. During the antigen-antibody reaction, a mixture of antibodies from different species is prepared. Conditions, such as antigen retrieval time and antibody concentration, are optimized and validated to increase the signal-to-noise ratio. This technique is reproducible and serves as a valuable tool for immunotherapy research and clinical applications.

The Prognostic Value of CD39 as a Marker of Tumor-Specific T Cells in Triple-Negative Breast Cancer in Asian Women

Triple-negative breast cancer (TNBC) has a poor prognosis with limited therapeutic options available for affected patients. Efforts are ongoing to identify surrogate markers for tumor-specific CD8+ T cells that can predict the response to immune checkpoint inhibitor (ICI) therapies, such as programmed cell death protein 1 or programmed cell death ligand-1 blockade. We have previously identified tumor-specific CD39+CD8+ T cells in non–small cell lung cancer that might help predict patient responses to programmed cell death protein 1 or programmed cell death ligand-1 blockade. Based on this finding, we conducted a comparative interrogation of TNBC in an Asian cohort to evaluate the potential of CD39 as a surrogate marker of tumor-specific CD8+ T cells. Using ICI-treated TNBC mouse models (n = 24), flow cytometric analyses of peripheral blood mononuclear cells and tumor-infiltrating lymphocytes revealed that >99% of tumor-specific CD8+ T cells also expressed CD39. To investigate the relationship between CD39+CD8+ T-cell density and CD39 expression with disease prognosis, we performed multiplex immunohistochemistry staining on treatment-naive human TNBC tissues (n = 315). We saw that the proportion of CD39+CD8+ T cells in human TNBC tumors correlated with improved overall survival, as did the densities of other CD39+ immune cell infiltrates, such as CD39+CD68+ macrophages. Finally, increased CD39 expression on CD8+ T cells was also found to predict the response to ICI therapy (pembrolizumab) in a separate cohort of 11 TNBC patients. These findings support the potential of CD39+CD8+ T-cell density as a prognostic factor in Asian TNBC patients.

Integrative multiomics enhancer activity profiling identifies therapeutic vulnerabilities in cholangiocarcinoma of different etiologies

ObjectivesCholangiocarcinoma (CCA) is a heterogeneous malignancy with high mortality and dismal prognosis, and an urgent clinical need for new therapies. Knowledge of the CCA epigenome is largely limited to aberrant...

Identifying a panel of nine genes as novel specific model in endometriosis noninvasive diagnosis

To study biomarkers to develop a novel diagnosis model for endometriosis and validate it using clinical samples. We used publicly available data sets and weighted gene coexpression network analysis to identify differentially expressed genes. Ten machine learning algorithms were used to develop an integrative model for predicting endometriosis. The accuracy and robustness of the model were validated using data sets and clinical samples. Department of Obstetrics and Gynecology, Tangdu Hospital, Air Force Medical University, Xi'an, Shaanxi, China. The study included clinical patients between the ages of 20 and 40 years who required laparoscopic surgery and who had not undergone hormone therapy within the previous 3 months. All the healthy individuals had given birth to a child at least once in their lives. Patients with inflammatory conditions, malignant diseases, immune diseases, myoma, or adenomyosis were excluded. Paraffin blocks of the samples were collected (case, n = 5; control, n = 5). Blood samples of 58 individuals were collected (case, n = 28; control, n = 30). None. The areas under the receiver operator characteristic curve of our diagnostic model were measured for data sets and clinical samples. Multiplex immunohistochemical staining and real-time quantitative polymerase chain reaction assays were used for the validation of the model from tissue slides and peripheral blood samples. A nine-gene panel endometriosis messenger RNA score (EMScore), was constructed to distinguish the patients with endometriosis from healthy individuals using algorithms. The EMScore accurately predicted endometriosis, and the areas under the receiver operator characteristic curve of our diagnostic model were 0.920, and 0.942 for tissue and blood samples, respectively. Moreover, the EMScore outperformed other acknowledged signatures for predicting endometriosis across seven clinical cohorts. Overall, the EMScore constitutes a sensitive and specific noninvasive diagnostic method for endometriosis. We developed the EMScore, a novel model that can aid in the diagnosis of endometriosis using peripheral blood samples. This study will contribute to the development of improved clinical noninvasive and sensitive diagnostic tools for endometriosis. These nine genes might be potential target molecules for treating endometriosis.

Multiplex Immunohistochemistry Staining for Paraffin-embedded Lung Cancer Tissue

Multiplex Immunohistochemistry Staining for Paraffin-embedded Lung Cancer Tissue

Cytosolic DNA accumulation promotes breast cancer immunogenicity via a STING-independent pathway

BackgroundImmune checkpoint blockade (ICB) has revolutionized cancer treatment. However, ICB alone has demonstrated only benefit in a small subset of patients with breast cancer. Recent studies have shown that agents...

Clinical Usefulness of Immune Profiling for Differential Diagnosis between Crohn's Disease, Intestinal Tuberculosis, and Behcet's Disease.

It is important to make a differential diagnosis between inflammatory diseases of the bowel with similar clinical and endoscopic features. The profiling of immune cells could be helpful for accurately diagnosing inflammatory bowel diseases. We compared immune marker expression between Crohn's disease (CD), intestinal Behcet's disease (BD), and intestinal tuberculosis (TB) and evaluated the usefulness of immune profiling in differentiating between these diseases. Biopsy specimens were acquired around ulcerations on the terminal ileum or cecum from five patients with each disease. Panel 1 included multiplex immunohistochemistry staining for CD8, CD4, Foxp3, CD20, programmed death-1, and granzyme B. CD56, CD68, CD163, CD11c, and HLA-DR were analyzed in panel 2. The differences in cytotoxic T cells (CD8+CD4-Fopx3-CD20-), helper T cells (CD8-CD4+Fopx3-CD20-), and regulatory T cells (CD8-CD4+Fopx3+CD20-) were also not significant. However, M1 macrophage (CD68+CD163-HLA-DR-) cell densities were significantly higher in intestinal BD than in other diseases. The expression level of dendritic cells (CD56-CD68-CD163-CD11c+HLA-DR+) was highest in intestinal TB and lowest in intestinal BD. The expression of immune cells, including M1 macrophages and dendritic cells, was different between CD, intestinal BD, and intestinal TB. Immune profiling can be helpful for establishing differential diagnoses of inflammatory bowel diseases.

Real-time, multiplexed rendering of lymphoma diagnostic panels from unstained tissue sections using virtual staining.

115 Background: Lymphoproliferative disorders are complex, heterogeneous diseases with an ever expanding array of subtypes and variants requiring stepwise assay approach to arrive at a precise diagnosis. While extensive characterization of lesional cells and the microenvironment (TME) via immunophenotyping and genotyping remains the gold standard, a full workup may be unachievable due to limited input tissue, turnaround time pressure, or unavailability of particular markers in-house. Even when available, the interpretation of large numbers of markers by pathologists is limited by the ability to mentally spatially align markers by recall-superimposition of single-plex markers, limiting the discovery of patterns relevant for novel treatments strategies. Here we present a deep-learning based virtual staining method which transforms a single unstained tissue section into a panel of perfectly aligned virtual immunohistochemical (IHC) stained images. Methods: 4um unstained sections were cut from 30 formalin-fixed paraffin-embedded blocks. Using a standard commercial slide scanner (Axio Scan.Z1, Zeiss), autofluorescence images were captured from unstained sections and paired with brightfield images captured from the same sections after chemical staining with H&E or various IHC stains of interest. The virtual staining was performed by a deep neural network trained as a conditional GAN using accurately co-registered patches of paired images . The training dataset comprised seven types of lymphoma plus benign lymphoid tissues. Results: We successfully created models for virtual CD3, CD8, CD20 and CD30 IHC stains as well as H&E. Analysis by board certified pathologists confirmed consistent and accurate results between virtual and IHC stained slides in a range of lymphomas and reactive lymphoid tissue. We quantitatively evaluated the co-expression of CD3/CD8 and the spatial distribution of all four markers in eight holdout slides. We demonstrated that our virtual multiplex IHC staining achieves comparable performance for WSI scoring and cellular localization with actual staining across lymphoma types. Conclusions: We present the feasibility of spatially aligned, rapid virtual H&E and IHC staining of four different markers from single, label-free autofluorescence images using deep-learning techniques. To the best of our knowledge, this is the first report of a panel of virtual IHC stains, perfectly aligned and with overlaid H&E structural context, for analysis of neoplastic and reactive lymphoid tissues. Our technique provides an opportunity for a rapid, comprehensive histopathology and immunophenotyping workflow. Multiplex virtual staining greatly expands diagnostic and discovery opportunities: high accuracy TME profiling, seamless downstream computational image analysis, and screening of large biomarker pools in retrospective analysis of clinical trials.

CLDN18.2 and 4-1BB bispecific antibody givastomig exerts antitumor activity through CLDN18.2-expressing tumor-directed T-cell activation

BackgroundClaudin18.2 (CLDN18.2) is a tight junction protein that has been identified as a clinically proven target in gastric cancer. Stimulation of 4-1BB with agonistic antibodies is also a promising strategy...

Identification of the Prognostic Biomarkers CBX6 and CBX7 in Bladder Cancer

Chromobox (CBX) proteins are essential components of polycomb group proteins and perform essential functions in bladder cancer (BLCA). However, research on CBX proteins is still limited, and the function of CBXs in BLCA has not been well illustrated. We analyzed the expression of CBX family members in BLCA patients from The Cancer Genome Atlas database. By Cox regression analysis and survival analysis, CBX6 and CBX7 were identified as potential prognostic factors. Subsequently, we identified genes associated with CBX6/7 and performed enrichment analysis, and they were enriched in urothelial carcinoma and transitional carcinoma. Mutation rates of TP53 and TTN correlate with expression of CBX6/7. In addition, differential analysis indicated that the roles played by CBX6 and CBX7 may be related to immune checkpoints. The CIBERSORT algorithm was used to screen out immune cells that play a role in the prognosis of bladder cancer patients. Multiplex immunohistochemistry staining confirmed a negative correlation between CBX6 and M1 macrophages, as well as a consistent alteration in CBX6 and regulatory T cells (Tregs), a positive correlation between CBX7 and resting mast cells, and a negative correlation between CBX7 and M0 macrophages. CBX6 and CBX7 expression levels may assist in predicting the prognosis of BLCA patients. CBX6 may contribute to a poor prognosis in patients by inhibiting M1 polarization and promoting Treg recruitment in the tumor microenvironment, while CBX7 may contribute to a better prognosis in patients by increasing resting mast cell numbers and decreasing macrophage M0 content.

Lymphatic differentiation and microvascular proliferation in benign vascular lesions of skin and soft tissue: Diagnostic features following the International Society for The Study of Vascular Anomalies Classification—A retrospective study

Discrepancies have been noted between the clinical and histologic diagnosis of vascular malformations. To evaluate the effectiveness of the International Society for Study of Vascular Anomalies (ISSVA) classification in diagnosing benign vascular anomalies based on clinical and (immuno) histologic parameters, focusing on lymphatic differentiation and vascular proliferation. A retrospective study of 121 consecutive patients with benign skin and soft-tissue vascular anomalies located in the head and neck region (pyogenic granulomas and angioma senilis were excluded) by applying multiplex immunohistochemistry staining for lymph vessels (D2-40), endothelial blood vessels, and proliferating cells (Ki67). Clinical and histologic diagnosis was revised after the ISSVA classification. Initially, 64 lesions were diagnosed as tumors and 57 as malformations. Revision diagnosis following the ISSVA classification revealed 27 tumors, 90 malformations (22.2% lymphatic), and 4 non-ISSVA. Immunostaining showed lymphatic differentiation in 24 (19.8%) of 121 cases, of which 20 were malformations. Proliferative activity (Ki67+) was found in 41 (33.8%) of 121 cases, of which 8 were arteriovenous malformations. Quality and size of materials (biopsies vs resections) and clinical information. The diagnostic accuracy of combined histologic and clinical approaches for identifying vascular anomalies following the ISSVA classification can be substantially enhanced by incorporating additional immunostaining techniques to evaluate lymphatic differentiation and proliferative activity, particularly in identifying the occurrence of vascular malformations.

Abstract 2102: Metastatic-initiating signature-enriched subpopulation identified from single-cell transcriptomic sequencing of multi-timepoint esophageal squamous cell carcinoma pulmonary metastatic mice model

Abstract Esophageal squamous cell carcinoma (ESCC) metastatic-initiating cells are heterogenous subpopulations that are hypothesized to promote distal metastasis. Pulmonary metastatic human-origin ESCC mice model was established in our laboratory, chronological flow cytometry collection of tumor cells isolated from metastatic mice lungs at different timepoints inoculated via intravenous injection was followed by 10X genomics multiplex single-cell transcriptomic sequencing (scRNA-seq). Bioinformatics analyses denoted a 7-signature panel previously introduced with preliminary data, follow-up validations of signature-enriched subpopulation in silico, in vitro, and in vivo were performed in this study. In silico bulk transcriptomic sequencing (RNA-seq) of single signature-enriched KYSE30 subpopulation followed by gene ontology (GO) enrichment analyses indicated significantly (FDR<0.05) enhanced biological processes involved in cell motility, cell migration and cell adhesion, compared to signature-low subpopulation. In vitro western blotting showed elevated mesenchymal markers expression, while diminished epithelial markers expression. In vitro real-time qPCR revealed single signature-enriched subpopulation has co-enrichment of other signature marker RNAs compared to control group. Further in vitro functional assays demonstrated signature-enriched subpopulation possesses strengthened spheroid formation ability (p<0.05) and soft agar colony formation ability (p<0.05) as compared to signature-low subpopulation. In addition to previous preliminary data on early survival ability in vivo, signature-enriched subpopulation was significantly more favorable in sustained cell survival and proliferation, and formation of larger size of metastatic lesions in mice lungs at 2 months post-intravenous injection as illustrated by flow cytometry (p<0.05) and immunohistochemistry (IHC) staining analyses (p<0.05) respectively as compared to signature-low subpopulation. To validate the protein expression of signature markers, 6-colour multiplex IHC staining was performed on KYSE30 cell line and metastatic mice lungs collected at different timepoints. Multiplex IHC staining exhibited that these signature markers were expressed in subpopulation of cells as well as expressed on inoculated tumor cells in metastatic mice lungs with timeline pattern. In summary, signature-enriched subpopulation manifested the metastatic-initiating properties in ESCC via various in silico, in vitro, and in vivo experimental analyses. Further multiplex staining in clinical tissue microarray would be performed and analyzed to examine feasibility of adopting these signature markers for facilitating clinical diagnosis and molecular targeting of metastatic ESCC. Citation Format: Ching Ngar Wong, Yu Zhang, Beibei Ru, Kwong Yiu Lam, Hongyu Zhou, Xinyuan Guan. Metastatic-initiating signature-enriched subpopulation identified from single-cell transcriptomic sequencing of multi-timepoint esophageal squamous cell carcinoma pulmonary metastatic mice model [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 2102.

MUC1 promotes lung metastases of liver cancer by impairing anti-tumor immunity

PurposeMUC1 is a membrane bound protein that can regulate tumor progression but its role in tumor metastasis and the metastatic microenvironment remains unclear.MethodsWe performed differential gene analysis for primary liver cancer (n = 31) and lung metastases (n = 31) using the Gene Expression Omnibus (GEO) dataset (GSE141016) and obtained RNA sequencing data from 374 liver cancer and 50 normal tissues from The Cancer Genome Atlas (TCGA). We analyzed the prognostic value of MUC1 and the relationship between MUC1 and the TME using online databases and a clinical cohort. Immunohistochemistry detected MUC1 in normal liver, liver cancer, and lung metastases. Multiplex immunohistochemistry staining detected immune cells in the metastatic microenvironment.ResultsHigh MUC1 expression levels in hepatocellular carcinoma are associated with worse clinical prognosis and higher rates of lung metastasis. In addition, we observed a correlation between MUC1 and multiple immune cells in the metastatic microenvironment. In paired primary liver cancer and lung metastatic tumor tissues from the same patient, we observed higher MUC1 protein levels in lung metastases than in primary liver cancer. Furthermore, MUC1 was negatively correlated with CD8+T and Treg cells in the metastatic tumor microenvironment and positively correlated with DC. In addition, we found that MUC1 was associated with CD8+T cell activation and function using flow cytometry in another cohort of patients with liver cancer.ConclusionThese data confirm the potential of MUC1 as a prognostic marker and therapeutic target.

Localization and density of tertiary lymphoid structures associate with molecular subtype and clinical outcome in colorectal cancer liver metastases

BackgroundTertiary lymphoid structures (TLSs) have been proposed to assess the prognosis of patients with cancer. Here, we investigated the prognostic value and relevant mechanisms of TLSs in colorectal cancer liver...

Abstract A080: The immunological signatures of African American prostate cancer as biological determinants of disease disparity

Abstract Background: Prostate cancer (PCa) is currently the second leading cause of cancer-related deaths among men in the United States. African American men suffer the highest incidence of disease morbidity and mortality compared to other racial groups. Although socio-economic, cultural, epidemiological, and genomic factors have been described to contribute to cancer health disparities in African American men, there is a knowledge gap regarding how the tumor immune landscape shapes aggressive disease patterns in this patient population, and whether their tumor microenvironment (TME) is amenable to immune modulation. This study aims to identify distinct and targetable immunosuppressive phenotypes and immunological signatures in prostate tumors of AA patients. Methodology: Peripheral blood from consented African American (AA) and Caucasian American (CA) patients undergoing prostatectomy at Moffitt Cancer Center were analyzed by Luminex multiplex cytokine/chemokine assay and comprehensive immune profiling via multi-parameter flow cytometry. Also, multiparameter immune profiling of freshly resected PCa specimens obtained from these same patients after prostatectomy was performed. In addition, multiplex immunohistochemical staining was performed on prostate tumor tissue microarrays to compare the proportions and phenotypes of T cells in the prostate TME of AA and CA patients. Lastly, the immunomodulatory potential of AZD5153, a bivalent Bromodomain 4 (BRD4) inhibitor was investigated on orthotopic prostate tumor-bearing humanized mouse models under racially defined settings, followed by comprehensive flow cytometry immunophenotypic profiling of tumor-infiltrating immune cells in the resected mice prostate tumors at clinical endpoints. Results: Prostate tumors of AA patients harbored higher proportions of CD4+ regulatory T cells (Tregs) and lower proportions of CD8+ T cells relative to CA patients. Furthermore, the immunophenotypic analysis of T cells showed higher expression of immune checkpoint molecules in AA compared to CA patients. Serum analysis revealed cytokine and chemokine expression signatures that are skewed towards a more immunosuppressive profile in AA compared to CA patients. Treatment with AZD5153 led to an increase in the survival time and a more striking reduction in tumor volume of AA-matched prostate tumor-bearing humanized mice relative to CA-matched prostate tumor-bearing humanized mice. A decrease in the proportion of tumor-infiltrating Tregs and a reduction in the expression of immune checkpoint molecules were also observed in prostate tumors from AA-matched humanized mice relative to CA-matched humanized mice. Conclusion: Our findings support the contributory role of racially distinct immunophenotypes and immune signatures in PCa disparity, and highlight the potential immunotherapeutic benefits of BRD4 inhibitors, such as AZD5153 in AA PCa patients. Citation Format: Saheed O. Oseni, Arup Bag, Oluwaseyi Oluwatola, Natalie DeMars, Dennis Adeegbe. The immunological signatures of African American prostate cancer as biological determinants of disease disparity [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr A080.

IL2RA+VSIG4+ tumor-associated macrophage is a key subpopulation of the immunosuppressive microenvironment in anaplastic thyroid cancer

Extensive infiltration of tumor-associated macrophages was correlated poor prognosis in anaplastic thyroid cancer (ATC). However, the heterogeneity and characteristics of the ATC-associated macrophages (ATAMs) in ATC remain far from clear. We combined single-cell RNA-sequencing analysis and gene expression microarray datasets to assess the molecular signature of ATAMs. Compared with normal thyroid-associated macrophages (NTAMs), 778 differentially expressed genes (DEGs) significantly changed in ATAMs compared with NTAMs. These DEGs were correlated with oxidative phosphorylation (M2 phenotype) and phagocytosis (M1 phenotype). Moreover, ATAMs highly expressed pro-tumor genes associated with angiogenesis, fibrosis, metalloprotease activity, and metastasis. Notably, we identified one ATC-specific subset, IL2RA+ VSIG4+ ATAMs, co-expressed M1 and M2 markers. The infiltration of IL2RA+ VSIG4+ ATAMs showed strong correlation with BRAF and RAS signaling, and its high infiltration was associated with favorable prognosis in thyroid-cancer patients. IL2RA+ VSIG4+ ATAMs were associated with increased tumor-infiltrating lymphocytes (B cells, CD8+ T cells, Tregs). IL2RA+ VSIG4+ ATAMs interacted with CD8+ T cells and Tregs through immune checkpoints (such as LGALS9_HAVCR2), cytokines (such as CXCL10_CXCR3), and receptors (such as CSF1R_CSF1), thereby forming an immunosuppressive microenvironment. Multiplex immunohistochemistry staining and coculture experiment confirmed that ATC cancer cells were able to induce the polarization of IL2RA+ VSIG4+ ATAMs. Besides, we identified several novel ATC-specific immune checkpoint genes including the immunosuppressive molecule VSIG4, LAIR1, and LILRB2. Expression of VSIG4 was also significantly correlated with tumor-infiltrating lymphocytes (B cells, CD8+ T cells, Tregs). In conclusion, our study revealed an ATC-specific ATAM subset with bifunctional phenotype, which provided a comprehensive insight to delineate the molecular characteristics of ATC-associated macrophages.