Immune Cell Markers Research Articles

Bright-Field Multiplex Immunohistochemistry Assay for Tumor Microenvironment Evaluation in Melanoma Tissues.

Simple SummaryBright-field (BF) immunohistochemistry (IHC) remains the gold standard for histopathological evaluations. The development of new BF multiplex IHC could be very useful for the study and characterization of the tumor microenvironment (TME) in melanoma samples. We herein compared different BF IHC multiplex protocols for the study of TME in primary cutaneous melanoma tissues and offered the best optimized protocol for visualization and evaluation. These methodologies are studied to maximize the quality of staining considering the tissue characteristics under examination, maintaining a high level of standardization and reproducibility.The tumor microenvironment (TME) plays a crucial role in melanoma development, progression and response to treatment. As many of the most relevant TME cell phenotypes are defined by the simultaneous detection of more than two markers, the bright-field (BF) multiplex immunohistochemistry (IHC) technique has been introduced for the quantitative assessment and evaluation of the relative spatial distances between immune cells and melanoma cells. In the current study, we aimed to validate BF multiplex IHC techniques in the Ventana Discovery Ultra Immunostainer to be applied to the evaluation of the TME in variably pigmented melanoma tissues. The BF multiplex IHC staining was performed using different combinations of six immune-cell markers—CD3, CD4, CD8, CD20, CD68 and CD163—and the melanoma cell marker SOX10. Our results show that the BF double IHC Yellow/Purple protocol guarantees the maximum contrast in all the cell populations tested and the combination SOX10 (Green), CD8 (Yellow) and CD163 (Purple) of the BF triple IHC protocol ensures the best contrast and discrimination between the three stained cell populations. Furthermore, the labeled cells were clearly distinct and easily identifiable using the image analysis software. Our standardized BF IHC multiplex protocols can be used to better assess the immune contexts of melanoma patients with potential applications to drive therapeutic decisions within clinical trials.

Discoidin domain receptor 1 is a potential target correlated with tumor invasion and immune infiltration in gastric cancer.

Discoidin domain receptor 1 (DDR1) has been demonstrated to be able to promote tumor invasion and metastasis and being closely related to tumor immune infiltration. However, DDR1 has rarely been studied in gastric cancer. Here, we primarily evaluated DDR1 expression in gastric cancer and its cell lines using multiple databases. Subsequently, the cancer prognosis was investigated in relation to DDR1 expression. After analysis, we discovered that DDR1 was highly expressed and significantly connected with poor prognosis in gastric cancer. To comprehensively understand the molecular mechanism of DDR1, we explored genes and proteins interacting with DDR1 in gastric cancer using databases. Additionally, we found that the expression level of DDR1 was inversely correlated with immune infiltration and significantly relative to various immune cell markers. Overall, DDR1 was implicated in invasion, metastasis, and immune infiltration of gastric cancer. Inhibition of DDR1 may have the potential to alleviate the strong invasiveness and metastasis of advanced gastric cancer. Meanwhile, immune exclusion by DDR1 may also provide a new strategy for improving the efficacy of immune checkpoints inhibitors (ICIs), such as programmed cell death protein 1 (PD-1) antibody.

Exploration of the Immunotyping Landscape and ImmuneInfiltration-Related Prognostic Markers in Ovarian Cancer Patients.

BackgroundIncreasing evidence indicates that immune cell infiltration (ICI) affects the prognosis of multiple cancers. This study aims to explore the immunotypes and ICI-related biomarkers in ovarian cancer.MethodsThe ICI levels were quantified with the CIBERSORT and ESTIMATE algorithms. The unsupervised consensus clustering method determined immunotypes based on the ICI profiles. Characteristic genes were identified with the Boruta algorithm. Then, the ICI score, a novel prognostic marker, was generated with the principal component analysis of the characteristic genes. The relationships between the ICI scores and clinical features were revealed. Further, an ICI signature was integrated after the univariate Cox, lasso, and stepwise regression analyses. The accuracy and robustness of the model were tested by three independent cohorts. The roles of the model in the immunophenoscores (IPS), tumor immune dysfunction and exclusion (TIDE) scores, and immunotherapy responses were also explored. Finally, risk genes (GBP1P1, TGFBI, PLA2G2D) and immune cell marker genes (CD11B, NOS2, CD206, CD8A) were tested by qRT-PCR in clinical tissues.ResultsThree immunotypes were identified, and ICI scores were generated based on the 75 characteristic genes. CD8 TCR pathways, chemokine-related pathways, and lymphocyte activation were critical to immunophenotyping. Higher ICI scores contributed to better prognoses. An independent prognostic factor, a three-gene signature, was integrated to calculate patients’ risk scores. Higher TIDE scores, lower ICI scores, lower IPS, lower immunotherapy responses, and worse prognoses were revealed in high-risk patients. Macrophage polarization and CD8 T cell infiltration were indicated to play potentially important roles in the development of ovarian cancer in the clinical validation cohort.ConclusionsOur study characterized the immunotyping landscape and provided novel immune infiltration-related prognostic markers in ovarian cancer.

Pronociceptive autoantibodies in the spinal cord mediate nociceptive sensitization, loss of function, and spontaneous pain in the lumbar disk puncture model of chronic back pain.

Previously, we observed that B cells and autoantibodies mediated chronic nociceptive sensitization in the mouse tibia fracture model of complex regional pain syndrome and that complex regional pain syndrome patient antibodies were pronociceptive in fracture mice lacking mature B cells and antibodies (muMT). The current study used a lumbar spinal disk puncture (DP) model of low back pain in wild-type (WT) and muMT mice to evaluate pronociceptive adaptive immune responses. Spinal disks and cords were collected 3 weeks after DP for polymerase chain reaction and immunohistochemistry analyses. Wild-type DP mice developed 24 weeks of hindpaw mechanical allodynia and hyperalgesia, grip weakness, and a conditioned place preference response indicative of spontaneous pain, but pain responses were attenuated or absent in muMT DP mice. Spinal cord expression of inflammatory cytokines, immune cell markers, and complement components were increased in WT DP mice and in muMT DP mice. Dorsal horn immunostaining in WT DP mice demonstrated glial activation and increased complement 5a receptor expressionin spinal neurons. Serum collected from WT DP mice and injected into muMT DP mice caused nociceptive sensitization, as did intrathecal injection of IgM collected from WT DP mice, and IgM immune complexes were observed in lumbar spinal disks and cord of WT DP mice. Serum from WT tibia fracture mice was not pronociceptive in muMT DP mice and vice versa, evidence that each type of tissue trauma chronically generates its own unique antibodies and targeted antigens. These data further support the pronociceptive autoimmunity hypothesis for the transition from tissue injury to chronic musculoskeletal pain state.

SAT040 - Multiplex immunostaining and transcriptomic profiling identify novel immune cell markers for non-alcoholic steatohepatitis and primary sclerosing cholangitis

SAT040 - Multiplex immunostaining and transcriptomic profiling identify novel immune cell markers for non-alcoholic steatohepatitis and primary sclerosing cholangitis

Local and systemic immune profiles of human pancreatic ductal adenocarcinoma revealed by single-cell mass cytometry

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy in need of effective (immuno)therapeutic treatment strategies. For the optimal application and development of cancer immunotherapies, a comprehensive understanding of local...

Abstract 1235: Presence of TLS and combined high densities of PD-L1+ macrophages & CD8+ T cells predict long-term overall survival for patients with advanced NSCLC treated with durvalumab

Abstract Introduction: Predictive biomarkers of anti‒PD-(L)1 therapies have largely focused on the tumor - T cell axis where tumor cell PD-L1 expression has demonstrated its clinical utility in predicting overall survival (OS) in patients with advanced non-small cell lung cancer (NSCLC). Although, other immune cell subsets were shown to be associated with clinical efficacy, their relative impact and combined effect in predicting improved long-term survival warrant further investigation. Using computational image analysis of multiplex immunofluorescence (mIF) and immunohistochemistry (IHC) immune marker panels, we sought to identify single and combined biomarkers of the tumor immune contexture in association with long-term OS in advanced NSCLC patients treated with Durvalumab. Methods: Pre-treatment tumor samples from advanced NSCLC patients (n = 210) enrolled in durvalumab nonrandomized phase 1/2 trial (10 mg/kg Q2W, CP1108/NCT01693562), were stained using IHC and 6-marker mIF panels to detect markers of immune cells, cell functional state and tertiary lymphoid structure (TLS) (PD-L1, CD8, PD-1, Ki67, CD68, CD20, CD1c, NKp46, CD66b, ICOS, FOXP3). Cell marker density (cells/mm2), distribution and proximity were quantified and analyzed in association with overall survival. Results: Computational image analysis of the tumor immune contexture revealed a greater immune inflamed phenotype, both innate (macrophages, dendritic cells) and adaptive (T and B cells), in NSCLC patients with long OS >2 years compared to those with short OS < 1 year (fold change > 2, p < 0.0001). Patient subgroup with high density of individual immune subsets show a median OS (mOS) of 10-20 months (p < 0.01 high vs. low subgroups) while combined markers of innate and adaptive immune cells show an improved mOS > 2 years (p < 0.001). Specifically, among the key findings, combined biomarkers of CD68+ PD-L1+ macrophages and CD8+ T cells predicts for a significant increase in OS for patients with high vs low marker density (HR = 0.21, 95%CI 0.12 - 0.39, p <10-7; mOS 39.5 months [high], 6.5 months [low]). Whereas, in patients with high density of either single biomarkers CD68+ PD-L1+ macrophage or CD8+ T cells mOS is 20.2 or 18.4 months respectively. Moreover, high density of CD20+ B cells, reflective of presence of TLS, associates with improved OS (mOS NR [high], 11 months [low], p = 0.003). In addition, TLS enriched tumors show an increased level of macrophages expressing PD-L1 in synapsis with CD8+ T cells (activated PD1+ or proliferative Ki67+ T cells). Conclusion: These findings demonstrate the importance of both tertiary lymphoid structure and high pre-existing innate-adaptive immunity in driving long-term overall survival of durvalumab-treated patients with NSCLC and highlight the need for the development of multiparametric predictive biomarkers beyond tumor-T cell axis. Citation Format: Lina Meinecke, Jorge Blando, Thomas Herz, Michael Surace, Thomas Padel, Monica Azqueta Gavaldon, Harald Hessel, Farzad Sekhavati, Megha Saraiya, Anmarie Boutrin, Karma Da Costa, Jaime Rodriguez Canales, Ashok Gupta, Carl Barrett, Zachary Aaron Cooper, Ikbel Achour. Presence of TLS and combined high densities of PD-L1+ macrophages & CD8+ T cells predict long-term overall survival for patients with advanced NSCLC treated with durvalumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1235.

Abstract 6176: CD56 dim+ immune infiltration and reprogrammed TMEs associated with response to neoadjuvant anti-PD-1 immunotherapy plus concurrent chemoradiotherapy in locally advanced gastric or gastroesophageal junction adenocarcinoma

Abstract PURPOSE Neoadjuvant immunotherapy (IO) combined with concurrent chemoradiotherapy (cCRT) showed a good efficacy in treatment of locally advanced gastric or gastroesophageal junction adenocarcinoma (G/GEJC) patients. However, the mechanism of therapeutic efficacy on tumor cells and the tumor microenvironment are still unknown. We aimed to characterize the microenvironment of immune cell infiltration at baseline and post-treatment of the patients in SHARED trial (ChiCTR1900024428). METHODS We prospectively enrolled 18 advanced G/GEJC patients who received IO (sintilimab, anti-PD-L1) in combination with cCRT therapy and resection in Nanjing Drum Tower Hospital. The pre-treatment tumor biopsies samples and post-treatment surgical specimens of all patients were measured for dynamic tumor microenvironment (TME) using mIHC (immune cell markers: CD3, CD4, CD8, FoxP3, CD20, CD56, CD68, CD163, PD-1, PD-L1) in a CLIA-certified laboratory. Twenty-five samples were successfully examined,including 10 paired pre-treatment biopsies and post-treatment specimens, three sole pre-treatment biopsies and two sole post-treatment specimens. RESULTS Among 18 patients who completed neoadjuvant treatment and resection, eight (44.4%) achieved pathological complete response (pCR), and 10 (55.6%) achieved non-pCR (six MPR and four non-MPR). Evaluable pre-treatment samples revealed a significantly higher CD56 dim+ immune infiltration (P=0.018) in pCR tumors compared with non-pCR tumors. Three patients harbored tertiary lymphoid structures (TLSs) in pre-treatment biopsies, two out of three achieved pCR and one achieved MPR. Another one patient harbored TLSs in post-treatment specimens and achieved pCR. No difference was found in all evaluable post-treatment specimens, when compared with paired pretreatment biopsies. Strikingly, significant post-treatment increases in CD3+ immune infiltration (P=0.0366) and CD4+FoxP3+ immune infiltration (P=0.0277) were observed in pCR tumors, with no significant difference in non-pCR tumors. CONCLUSIONS The different response to IO plus cCRT therapy in locally advanced G/GEJC patients and be explained by the difference in TME including the baseline CD56 dim+ immune infiltration, TLSs status and the reprogrammed immune infiltration induced by IO PLUS cCRT therapy. Further large population based studies are warranted to validate the response mechanism. Citation Format: Jia Wei, Qin Liu, Hui Chen, Xin Zhang, Feilong Zhao, Yao Fu, Ju Yang, Yue Wang, Shiqing Chen, Xiaochen Zhao, Yuezong Bai, Wenxian Guan, Baorui Liu. CD56 dim+ immune infiltration and reprogrammed TMEs associated with response to neoadjuvant anti-PD-1 immunotherapy plus concurrent chemoradiotherapy in locally advanced gastric or gastroesophageal junction adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6176.

Abstract 989: Spatial profiling of androgen receptor splice variant 7 transcriptional activity in prostate cancer metastases

Abstract Introduction: Androgen receptor splice variant 7 (AR-V7) is expressed in metastases from patients with castration resistant prostate cancer (CRPC) and shows a high level of inter- and intra-tumoral variability. However, the downstream activity generated by AR-V7 in tissue has not been shown. Whether AR-V7 is active in tissue or whether AR-V7 is a non-functioning biomarker with full-length AR is not known. To address this question, we constructed a tissue microarray (TMA) of 56 metastases from 27 patients with CRPC to analyze spatial gene expression using the GeoMx Digital Spatial Profiler. Immunostaining was performed to define epithelial, vascular, and stromal compartments. The stained tissues were then hybridized with barcoded tagged oligonucleotides targeting 2093 unique genes, which included those representing AR, AR-V cryptic exons, AR and neuroendocrine activity, and immune cell markers. One 500 um region of interest (ROI) was assessed per tissue core (approximately 1200 cells). A sequential section from the same TMA was then stained with AR-V7 and AR-C-terminal specific antibodies. ROIs for RNA and protein were selected to be similar between slides. In addition to DSP, each metastasis was assessed by RNA-seq on the bulk tissue. Results: The most differentially expressed genes (FDR<0.05) based on association with AR-V7 staining were known downstream AR regulated genes including KLK2 and 3, FKBP5, NKX3.1, TMPRSS2, FASN, and TARP. Additionally, genes associated with proliferation and stemness, e.g., POLB, KRT1, SOX2, were significantly expressed. Since 93% of patients were on ADT at time of tissue collection and over 80% also had been treated with either abiraterone or enzalutamide, the increase in AR downstream genes would not be expected to occur from ligand activation of AR-FL. We also have previously shown that knock-down of AR-V7 in LNCaP95 cells results in loss of AR binding to AREs. In these metastases, then, activation of AR downstream genes would be a result of AR-V7 nuclear transport of AR-FL through AR-V7/AR-FL heterodimers or transcriptional activation by AR-V7 homodimers. Of further note, AR cryptic exons 1, 2, and 5 were also significantly expressed in AR-V7 positive ROIs (p< 0.0001). RNA-seq intron/exon junction reads were used to demonstrate that additional AR-Vs, most commonly AR-V9, were also expressed in tissues positive for AR-V7, suggesting that AR splicing is a common event in CRPC. Finally, expression of neuroendocrine (NE) genes INSM1 and TUBB2 were not expressed in AR-V7 positive ROIs (negatively correlated, p<0.001), indicating that AR-V7 and NE phenotypes cannot co-exist in the same cell. Conclusion: AR-V7 continues to drive prostate cancer through activation of the AR-cistrome. Its expression is heterogenous in metastases along with NE cells, suggesting that in the presence of AR-V7 and NE markers, therapy needs to be directed at the N-terminus of AR and NE components. Citation Format: Cynthia C. Sprenger, Ilsa Coleman, Michelle Kriner, Lauren Brady, Margaret Hoang, Martine Roudier, Mamatha Damodarasamy, Radhika Patel, Lawrence True, Peter Nelson, Michael Haffner, Stephen Plymate. Spatial profiling of androgen receptor splice variant 7 transcriptional activity in prostate cancer metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 989.

Abstract 3865: Spatial multiplex profiling of immune cell markers in FFPE tumor tissues using the RNAscope™ HiPlex v2 in situ hybridization assay

Abstract The tumor microenvironment (TME) is highly complex, comprised of tumor cells, immune cells, stromal cells, and extracellular matrix. Understanding spatial interactions between various cell types and their activation states in the TME is crucial for implementing successful immunotherapy strategies against various types of cancer. This study demonstrates a highly sensitive and specific multiplexed technique, the RNAscope HiPlex v2 in situ hybridization (ISH) assay for spatial and transcriptomic profiling of target genes to assess immune regulation in human lung, breast, cervical and ovarian FFPE tumor tissues. We have expanded our current RNAscope HiPlex assay capability of iteratively multiplexing up to 12 targets in fixed and fresh frozen samples to include formalin fixed paraffin embedded (FFPE) tissues. The novel FFPE reagent effectively reduces background autofluorescence, improving the signal to noise ratio. We have leveraged this technology to investigate spatial expression of 12 oncology and immuno-oncology target genes, including tumor markers, immune checkpoint markers, immunosuppression markers, immune cell markers and secreted chemokine RNA expression profile within the TME. The targets were simultaneously registered using HiPlex image registration software v2 that enables background subtraction. We visualized T cell infiltration and identified T cell subsets within tumors using CD3 and CD8 expression and activated T cells by IFNG expression. We further identified subsets of pro- and anti-inflammatory macrophages by CD68 and CD163 expression as well effector cells which secrete chemokines and cytokine. We also detected the hypoxia markers HIF1A and VEGF to elucidate the immunosuppressive state of tumor cells. Preliminary analysis and quantification of the HIF1A expression using HALO® image analysis software showed higher copy numbers in the lung tumor as compared to the other tumors, demonstrating the sensitivity of the assay through differential expression. We additionally showed the differential expression of immune checkpoint markers PDCD1, and CD274 within the TME. Using a highly sensitive multiplexed RNAscope HiPlex v2 ISH assay, we have demonstrated the capability of this technique to spatially resolve 12 targets in four different tumor types. The FFPE reagent efficiently quenched background autofluorescence in the tissues and identified immune cell signatures within the TME. Quantification of immunosuppressive markers further depicted differential expression among various tumors. This technology is highly beneficial for investigating complex and spatial tumor-stroma interactions in basic science and translational research. The assay can also provide valuable understanding of the biological crosstalk among various cell types in complex and heterogeneous tissues. Citation Format: Anushka Dikshit, Sayantani Basak, Ching-Wei Chang, Michaeline Bunting, Kim Collins. Spatial multiplex profiling of immune cell markers in FFPE tumor tissues using the RNAscope™ HiPlex v2 in situ hybridization assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3865.

Abstract 3895: Moxidectin unravels the role of Hippo-YAP pathway in maintaining immunity of pediatric glioblastoma

Abstract Pediatric glioblastoma multiforme (GBM) is considered to be the second most lethal childhood cancer type after leukemia. Recent preclinical, clinical and genomic studies have highlighted upon the role of Hippo-Yap pathway in the progression of pediatric GBM. In addition, recent studies have established the role of YAP in creating immune suppressive tumor microenvironment (TME) facilitating drug resistance, recurrence and metastasis of GBM tumors. Herein, we report that ‘moxidectin’ an anti-helminthic drug inhibits the proliferation of SF268, SF295, SF188 and CT-2A Luc GBM cells by inducing apoptosis. Immunoblotting and immunofluorescence microscopy studies show that moxidectin mediates its effects by inhibiting MEK-ERK pathway, a regulator of Hippo-YAP signaling. As a result, moxidectin suppressed the nuclear translocation and transcriptional activity of YAP/TAZ-TEAD complex. Oral administration of 3.5mg/kg moxidectin suppressed the growth of GBM tumors by 90% in intracranial tumor model. Ex-vivo analysis of excised tumors confirmed the observations made in in vitro studies. We further conducted immunophenotyping on the excised tumors from both control and moxidectin treated mice to evaluate the effect of moxidectin on immune cell markers in TME and Tumor Draining Lymph Nodes (TDLNs). Our analysis indicated that treatment with moxidectin suppressed the immune suppressive TME created by GBM tumor. Interestingly, moxidectin enhanced antigen presentation in the TDLN. Indicating that it might play a role in activating the peripheral immune response. Moxidectin is an FDA approved drug and any findings from our research will promote its further investigation as a potential therapeutic agent for GBM patients. Citation Format: Itishree Kaushik, Shreyas Gaikwad, Sanjay K. Srivastava. Moxidectin unravels the role of Hippo-YAP pathway in maintaining immunity of pediatric glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3895.

Abstract 6149: Prognostic tumor microenvironment subtypes in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer in which the tumor microenvironment (TME) is prognostic, with increased tumor-infiltrating lymphocytes predicting a good prognosis. Positive clinical trials of immune checkpoint inhibitors in TNBC further highlight the TME’s importance, but its single-cell composition and relationship to patient survival has not been well described. Therefore, we generated highly-multiplex cyclic immunofluorescence (CyCIF) data from 59 TNBC in tissue microarrays. Our CyCIF dataset served as a discovery cohort; the data included 35 protein markers and clinical outcomes for 18 patients. For a validation cohort, we analyzed 32 markers imaged with multiplex ion beam imaging (MIBI) in 38 TNBC patients (Keren et. al., Cell 2018). In the CyCIF cohort, we used markers’ single-cell mean intensity to generate a Umap manifold, which we clustered with the unsupervised Leiden algorithm. We annotated the resulting 28 cell types as epithelial, immune or stromal. In the MIBI cohort, we similarly clustered and annotated 21 cell types as epithelial, immune or stromal. Next, we clustered the patients based on the fraction of epithelial, immune or stromal cells in the tissue. In the CyCIF cohort, patients fell into three subtypes: epithelial-rich, stroma-rich and immune-rich. The patients in the epithelial-rich group had significantly shorter survival than the other two subtypes (log-rank p-value = 0.0008) and had shorter recurrence-free survival (log-rank p-value = 0.048). In the MIBI cohort, patients fell into the same three subtypes. As in the CyCIF cohort, the epithelial-rich patients had shorter survival than the other subtypes (log-rank p-value = 0.021), although there was no difference in recurrence-free survival (log-rank p-value = 0.47). Finally, we identified protein biomarkers with significantly different expression levels between the subtypes in one or both cohorts. The poor-prognosis, epithelial-rich subtype had higher CD31 expression in both cohorts, and in the CyCIF cohort, Glut1 was elevated, indicating a hypoxic, angiogenic TME. The immune infiltrate was primarily CD68+ macrophages and the stroma was activated, with higher levels of Ki67 in both cohorts. In contrast, the stroma-rich, good prognosis subtype had low immune infiltration, including fewer macrophages, low CD31 expression and a quiescent, non-proliferating stroma. Lastly, the good-prognosis, immune-rich subtype had high T and B cells levels, as well as markers of immune regulation, memory cells, and antigen presentation cells. In conclusion, we generated and analyzed a new multiplex CyCIF dataset with clinical annotation and confirmed our analytical findings in a second multiplex imaging dataset. We delineated three TME subtypes in TNBC with different outcomes, cell types and biomarker expression, potentially leading to better patient stratification and informing new drug targets. Citation Format: Jennifer R. Eng, Elmar Bucher, Zhi Hu, Melinda Sanders, Bapsi Chakravarthy, Summer Gibbs, Koei Chin, Jennifer Pietenpol, Joe W. Gray. Prognostic tumor microenvironment subtypes in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6149.

Abstract 1727: CD8+ T cells in the invasive margin combined with FOXP3+ T cells in the tumor center significantly associate with survival in resectable gastric cancer, a post-hoc analysis of the Dutch D1/D2 trial

Abstract Introduction: In gastric cancer, studies of tumor infiltrating lymphocytes as a prognostic biomarker show contradictory results. These results may be caused by use of variable immunohistochemistry (IHC) quantification techniques, different marker selections and particularly inconsistency in the evaluated tumor regions. To overcome these issues, we performed a comprehensive digital image analysis of 5 immune cell markers for their prognostic value in a cohort of gastric cancer patients who were treated with surgery only in the Dutch D1/D2 trial. Methods: All available surgical resection specimens of gastric cancer patients were included in this study (N=251). IHC for T-cell markers CD3, CD45RO, CD8, FOXP3 and Granzyme B was performed on serial slides. After manual annotation of the tumor area, an invasive margin was defined as 0.5 mm into the tumor still containing tumor cells (inner margin, IM) and 0.5 mm outside of the tumor not containing tumor cells (outer margin, OM). The density of positive immune cells (cells/mm2) was digitally quantified using QuPath for each 0.5x0.5 mm2 square across tumor center (TC), IM and OM, separately. A classification and regression tree (CART) model was employed to identify an optimal combination of prognostic markers from the continuous immune cell density variables with cancer specific survival (CSS) as outcome. Results: The CART decision tree identified CD8 OM (≥798 cells/mm2) as most dominant prognostic factor, followed by FOXP3 TC (≥20 cells/mm2) in the CD8 OM low subset. This resulted in 3 CART branches in the decision tree: CD8 OM high with best prognosis, CD8 OM low/FOXP3 TC high with intermediate prognosis, and CD8 OM low/FOXP3 TC low with worst prognosis (Log-rank P-value <0.0001). The CD8 OM high branch was enriched in EBV+ (38.2%) and MSI-high (17.6%) tumors, compared to the other two branches with poorer prognosis (4.2% and 3.4% for EBV+, 7.9% and 8.4% for MSI-high). The CART model was an independent predictor of CSS in a multivariable cox-regression (HR branch 2 vs 1: 4.87, 95% CI 1.96-12.07 and HR branch 3 vs 1: 7.97, 95% CI 3.20-19.86), which included T stage, N stage, Lauren subtype, EBV-status and MSI-status. The performance of the CART model was assessed by 5-fold cross-validation, where 4 out of 5 models reached a P-value < 0.05 (Likelihood-Ratio test). Conclusions: The OM in gastric cancer contains previously overlooked important prognostic information valuable for immune biomarker studies. The combination of CD8 OM and FOXP3 TC is identified as strongest prognostic factor in the risk stratification of resectable gastric cancer, and is independent of T stage, N stage, EBV-status, MSI-status and Lauren subtype. Moreover, high T-cell densities found in a proportion of EBV-/MSS tumors support further investigation of response to immunotherapy in these subgroups. Citation Format: Tanya T. Soeratram, Hedde D. Biesma, Jacqueline M. Egthuijsen, Elma Meershoek-Klein Kranenbarg, Henk H. Hartgrink, Cornelis J. van de Velde, Erik van Dijk, Yongsoo Kim, Bauke Ylstra, Hanneke W. van Laarhoven, Nicole C. van Grieken. CD8+ T cells in the invasive margin combined with FOXP3+ T cells in the tumor center significantly associate with survival in resectable gastric cancer, a post-hoc analysis of the Dutch D1/D2 trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1727.

Abstract 277: High agreement between immune profiling assays on the detection of infiltrating immune cells in ovarian tumor tissue

Abstract Detecting immune cells and estimating their heterogeneity in the tumor microenvironment (TME) is critical to inform assessments of cancer prognosis and response to immunotherapies. Many immune activity assays to detect immune cells are available, including multiplex immunofluorescence (mIF) of tissue slices to assess co-localization of immune cell markers and traditional immunohistochemistry (IHC) staining. More recently, statistical approaches to define cell types from RNAseq data (e.g., deconvolution, gene set enrichment) have been widely adopted due to the wide availability of transcriptomics profiles. Here, we interrogated the level of agreement between immune cells in the ovarian TME assessed by mIF (Vectra images processed via InForm/Halo), IHC, and deconvoluted whole exome RNAseq. We leveraged data from epithelial ovarian tumor cores embedded in tissue microarrays from participants in the Nurses’ Health Study I and II and the New England Case-Control Study (mIF, n=668 women; IHC, n=467; RNAseq, n=241). To explore agreement between technologies, we calculated the overall and compartment-specific percentages of cells positive for mIF markers CD3, CD4, CD8, CD19, CD20, CD138 and FOXP3, including cell specific phenotypes based on these markers. For IHC, a pathologist generated scores based on proportion of positive cells and/or aggregation into four levels (0, 1, 2, or 3) for markers CD8, CD68, and CD163. Deconvolution of transcriptomic profiles was performed with CIBERSORT (22 cell types) and xCell (64 cell types). Spearman correlation between technologies for cell specific markers (i.e., cytotoxic T cells, regulatory T cells (Tregs), B cells, macrophages, monocytes, among others) was used to assess agreement. We observed that detection of CD8+ and CD3+CD8+ cells from mIF (cytotoxic T cells) was consistent with the other assays (r >0.25), and the highest correlation was observed with IHC CD8+ cells (r=0.52). For deconvoluted transcriptomic predictions, CD8 T cells (xCell, CIBERSORT), and memory CD8 T cells (xCell) showed high agreement with mIF CD8+ and CD3+CD8+ cells (r=0.25 to 0.48). CD4+ and CD3+CD4+ cells detected with mIF also showed correlation with xCell CD4 memory T cells (r=0.25 to 0.34). As expected, only general class B cells from xCell showed agreement with mIF CD20+ cells (r=0.35). In addition, xCell Tregs correlated with mIF FOXP3+ cells (r=0.34). These results highlight the importance of considering the predictive power of immunological assays for the study of the TME. Compared to mIF, the robustness of deconvoluted RNAseq data varies with the cell type being predicted. Finally, RNAseq should be considered a complementary technology to staining of archival tissues given that the former does conserve spatial distribution of cell types. Citation Format: Oscar E. Ospina, Mary K. Townsend, Tianyi Wang, Naoko Sasamoto, Paul Stewart, Jose Conejo-Garcia, Kathryn Terry, Shelley S. Tworoger, B L. Fridley. High agreement between immune profiling assays on the detection of infiltrating immune cells in ovarian tumor tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 277.

Abstract 6117: Clinical and molecular correlates of immune marker profiling and PD-L1 expression in non-small cell lung cancer

Abstract Background: Understanding the genomic landscape and tumor immune microenvironment of non-small cell lung cancer (NSCLC) may provide critical insight into the biology of tumor immune response and novel strategies for overcoming immunotherapy resistance. The association of clinical and genomic features with tumor immune microenvironment in NSCLC patients remains unclear. Methods: In this retrospective cohort study, we profiled PD-L1 and immune cell marker expressions and performed targeted next-generation sequencing (Geneseeq Technology Inc., Nanjing) of baseline tissue samples from 100 NSCLC patients. The levels of CD8+, CD68+ and HLA-DR+ immune cells were assessed by multiplexed immunohistochemistry (mIHC) assay. Immune microenvironment markers and PD-L1 expression was analyzed for associations with clinical features and mutations at both the single gene and pathway levels. Results: Mutations in TP53, SETD2, RET and the p53 signaling pathway were significantly associated with high PD-L1 high expression (P<0.001; P=0.002; P=0.04; P=0.001, respectively). Patients with high PD-L1 expression showed a trend towards higher stromal CD8+ lymphocytes (P=0.1), Alterations in T cell and B cell pathways were also associated with increased stromal CD8+ T cells (P=0.03 and P=0.08, respectively). The infiltration of intratumoral CD68(+)HLA-DR(+) M1-like macrophages positively correlated with high PD-L1 high expression (P=0.07), Hippo pathway alterations (P=0.06). By contrast, the levels of intratumoral M1-like macrophages negatively correlated with alterations in the NRF2/KEAP1 pathway (P=0.005), which might account for the poor immunotherapy outcome in this subset of patients. Conclusions: Our study demonstrates the complex relationship between gene mutations and aberrations in signaling pathways with the immune microenvironment in NSCLC. Specific molecular features are associated with unique immune profiles and may impact the response to immune checkpoint inhibitors. Citation Format: Yunfei Shi, Youming Lei, Yinqiang Liu, Jin Duan, Wei Zhao, Fujun Zhang, Guoli Lv, Qian Wu, Mengmeng Wu, Jiani C. Yin, Yang Shao. Clinical and molecular correlates of immune marker profiling and PD-L1 expression in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6117.

Abstract 1723: Spatial analysis of tumor-infiltrated immune cells with highly specific RNAscope™ RNA-protein Co-detection assays

Abstract Interrogating complex tumor microenvironment requires a multi-omics approach that can provide high level of sensitivity and specificity. Identifying immune cell subsets within the tumor can be vital for predicting response and determining therapeutic efficacy. Detecting target immune cell markers using immunohistochemistry/Immunofluorescence (IHC/IF) and visualizing cytokine expression with in situ hybridization (ISH) can provide comprehensive information about the activation states of immune cells. Here, we demonstrate a newly developed integrated ISH and IHC/IF workflow compatible with manual and automated platforms that can substantially improve RNA-protein co-detection. We demonstrate the use of our RNA-Protein Co-detection assay in combination with the automated RNAscope Multiplex Fluorescent v2 assay, automated RNAscope Chromogenic Duplex assay and manual RNAscope Multiplex Fluorescent v2 assay to detect T cell markers, macrophage markers and checkpoint markers in the tumor microenvironment by using a microarray with different tumor samples. We identified CD4+ helper T cells and CD8+ cytotoxic T lymphocytes. Additionally, we determine the activation states of CD8+ T cells by visualizing IFNG, GZMB and IL-2 expression. We were also able to identify macrophages detected by CD68 protein expression and the M1 and M2 subsets were differentiated by using the M2-specific marker, CD163. We could also delineate tumor-stroma border in the samples by using the Pan-CK probe which distinctly marks the tumor cells and visualize the expression of immunoregulatory receptors PD-L1 and CTLA4 in the tumor cells. This assay is enabled for multiplexing by combining with our fluorescent assay platforms. Similarly, for researchers interested in detecting target expression while retaining morphological context, the codetection assay should be combined with our chromogenic assays. Overall, the new RNAscope-ISH-IHC co-detection workflow and reagents enable optimized simultaneous visualization of RNA and protein targets by enhancing the compatibility of antibodies with minimal optimization. Citation Format: Anushka Dikshit, Sayantani Basak, Emerald Doolittle, Ilya Kovalenko, Michaeline Bunting. Spatial analysis of tumor-infiltrated immune cells with highly specific RNAscope™ RNA-protein Co-detection assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1723.

Clinical and Gene Features of SARS-CoV-2-Positive Recurrence in Patients Recovered From COVID-19.

There are still frequent reports that a number of recovered coronavirus disease 2019 (COVID-19) patients following discharge have re-detectable positive (RP) results by RT-PCR. Understanding the clinical and molecular characteristics of RP patients may have implications for curbing the COVID-19 pandemic. In this study, 318 COVID-19 convalescent patients, including 59 RP patients and 259 non-RP (NRP) patients, were enrolled. Among RP patients, women accounted for a significantly high proportion (67.8%), and the titers of IgG and IgM antibodies in this group were also significantly high. Differentially expressed genes (DEGs), including 692 upregulated and 383 downregulated genes, overlapped in two public GEO datasets containing RP and NRP blood cell samples. Enrichment analysis indicated that these DEGs were related to several key signaling pathways, such as viral infection, immune activation, and inflammatory responses. Importantly, 59 indicator genes constituting the core network exhibited high diagnostic values and were correlated with markers of different immune cells. Among these, 12 drug-related genes were associated with the RP results. Our work suggests that, in addition to clinically available features, blood cell transcriptome sequencing can be performed to obtain gene signatures for diagnosis of RP patients.

Dynamic peripheral blood immune cell markers for predicting the response of patients with metastatic cancer to immune checkpoint inhibitors

PurposeImmune checkpoint inhibitors (ICIs) have shown durable responses in various malignancies. However, the response to ICI therapy is unpredictable, and investigation of predictive biomarkers needs to be improved.Experimental designIn total, 120 patients receiving ICI therapy and 40 patients receiving non-ICI therapy were enrolled. Peripheral blood immune cell markers (PBIMs), as liquid biopsy biomarkers, were analyzed by flow cytometry before ICI therapy, and before the first evaluation. In the ICI cohort, patients were randomly divided into training (n = 91) and validation (n = 29) cohorts. Machine learning algorithms were applied to construct the prognostic and predictive immune-related models.ResultsUsing the training cohort, a peripheral blood immune cell-based signature (BICS) based on four hub PBIMs was developed. In both the training and the validation cohorts, and the whole cohort, the BICS achieved a high accuracy for predicting overall survival (OS) benefit. The high-BICS group had significantly shorter progression-free survival and OS than the low-BICS group. The BICS demonstrated the predictive ability of patients to achieve durable clinical outcomes. By integrating these PBIMs, we further constructed and validated the support vector machine-recursive and feature elimination classifier model, which robustly predicts patients who will achieve optimal clinical benefit.ConclusionsDynamic PBIM-based monitoring as a noninvasive, cost-effective, highly specific and sensitive biomarker has broad potential for prognostic and predictive utility in patients receiving ICI therapy.

ATRT-13. An integrative analysis of the ATRT proteome unravels novel drug targets and refines molecular subgrouping

INTRODUCTION: Atypical teratoid/rhabdoid tumors (ATRT) represent frequent brain tumors in infants. In recent years, large-scale landscaping efforts on the epigenome and transcriptome of these tumors have unravelled a high degree of heterogeneity and three major molecular subgroups, termed ATRT-TYR, ATRT-SHH,ATRT-MYC, have been identified. The ATRT-proteome, in turn, still represents a largely unchartered territory. METHODS: We have performed a peptide-based screening approach to characterize the proteome of 40 ATRTs and six ATRT cell-lines. All of these samples had matching methylation data available and 28 also corresponding gene expression data. RESULTS: Unsupervised clustering recapitulated the previously described ATRT groups, revealing also a clear split of the SHH-subgroup in a supratentorial (SHH_1) and an infratentorial subgroup (SHH_2). Overall, we identified 7265 proteins, of which 1320 were differentially expressed between the groups, with an enrichment of spliceosome associated terms in SHH_1 and integrins/cell adhesions molecules in SHH_2. ATRT-MYC displayed an overrepresentation of immune cell markers and the TYR subgroup an enrichment of PI3K- as well as mTOR-signaling. Particularly, genes that have previously been described as signature genes for the ATRT-groups such as FABP7 in ATRT-SHH and OTX2 and MITF in ATRT-TYR were among the highly correlating genes that were both expressed in the proteome and the gene expression datasets. On top of this, our analysis revealed highly differentially expressed drug targets such as the tyrosine-kinase MARCKS (overexpressed in ATRT-TYR) not previously identified in ATRT transcriptome data, which warrant investigation by in vitro drug tests. CONCLUSION: Our data reveal the importance of previously described regulatory hubs in the ATRT subgroups, but additionally highlight novel drug targets that merit further exploration. Currently, drug treatment experiments in ATRT cell lines are ongoing to validate these proteins as drug targets, ultimately aiming to establish new therapeutic strategies in this deadly disease.

Intramuscular (IM) INO-5401 + INO-9012 with electroporation (EP) in combination with cemiplimab (REGN2810) in newly diagnosed glioblastoma.

2004 Background: Novel T cell-enabling therapies plus checkpoint inhibition may improve OS in GBM. INO-5401 (synthetic DNA plasmid encoding hTERT, WT-1, PSMA) plus INO-9012 (synthetic DNA plasmid encoding IL-12), with cemiplimab (PD-1 inhibitor), was given to patients with newly diagnosed GBM with MRD to evaluate tolerability, efficacy, and immunogenicity. Median OS and immunogenicity at 18 months (OS18) are reported. Methods: This is a phase I/II, single arm, two cohort (A: unmethylated MGMT and B: methylated MGMT) study. Primary endpoint is safety; efficacy and immunogenicity are secondary. Nine mg INO-5401 plus 1 mg INO-9012 (4 doses Q3W, then Q9W) was given IM with EP in combination with cemiplimab (350 mg IV Q3W). Hypofractionated RT (40 Gy over 3 weeks) with TMZ was given to all patients, followed by maintenance (Cohort B only), which was a novel therapeutic approach. Immunogenicity was assessed by quantifying INO-5401-specific peripheral cellular immune responses via IFN-g ELISpot and flow cytometry. Intra-tumoral gene expression was analyzed by RNA-Seq of FFPE GBM tissue. Differences in gene expression were analyzed using the Wilcoxon rank sum test. Results: Fifty-two subjects were enrolled: 32 in Cohort A; 20 in Cohort B (35% women; median age 60 years [range 19-78 years]). The adverse event profile was consistent with known single-agent (INO-5401, INO-9012, EP or cemiplimab) events; most events were ≤Grade 2 and no related events were Grade ≥4. Median OS durations in Cohorts A and B were 17.9 months (95% CI 14.5-19.8) and 32.5 months (95% CI 18.4-not reached), respectively. Flow cytometry revealed activated, antigen specific CD4+CD69+PD1+ and CD8+CD69+PD1+ T cells, the latter with lytic potential as defined by presence of perforin and granzyme A. Both subsets exhibited HR < 1.0 and p < 0.05 when accounting for a 0.1% T cell frequency change, translating to a 23% and 28% reduced risk of death, respectively. Gene expression levels in pre-treatment tissues were similar between alive and deceased groups for INO-5401 antigens and immune cell markers; however, the alive group displayed significantly reduced expression of genes associated with anti-apoptosis, pro-proliferation, and immune response suppression. Post-treatment tumor tissue displayed altered gene expression for immune-related markers versus pre-treatment tissue, including markers of T cell infiltration, activation, and lytic potential. Conclusions: INO-5401 + INO-9012 has an acceptable risk/benefit profile and elicits robust immune responses that correlate with enhanced survival when administered with cemiplimab and RT/TMZ to newly diagnosed GBM patients. Pre-treatment gene expression signatures in MGMT-unmethylated patients were statistically associated with OS18. Overall, INO-5401 elicits antigen-specific T cells that can infiltrate GBM tumors. Clinical trial information: NCT03491683.