Density Of Immune Cells Research Articles

Impact of vitamin d on the colon tumor immune microenvironment: Results of a randomized clinical trial of preoperative vitamin D supplementation in patients with stage I-III colon cancer.

3607 Background: Although the anti-tumor properties of vitamin D in colorectal cancer (CRC) have been broadly reported, its potential role as a tumor microenvironment (TME) immunomodulator has not been well established. Vitamin D receptor (VDR) and CYP27B1 (1-α-hydroxylase; required to convert vitamin D to its active form) are critical factors in the vitamin D signaling pathway and harbor prognostic significance for CRC. Pre-clinical studies suggest that higher stromal VDR expression enhances chemotherapy efficacy. To characterize the effects of vitamin D on the TME in humans, we conducted a randomized placebo-controlled trial of preoperative high-dose vitamin D supplementation in stage I-III colon cancer patients. Methods: Forty-two patients were randomized to receive vitamin D3 50,000 IU/day x 7 days followed by 10,000 IU/day versus placebo prior to resection. Paired pre-treatment biopsy specimens and post-treatment surgical resection specimens from 19 patients who completed the assigned treatment and had sufficient tissue for analysis were collected for spatially-resolved immune cell profiling and evaluation of VDR and CYP27B1 expression using custom multiplex immunofluorescence. Blood samples were collected pre- and post-treatment to assess plasma 25-hydroxyvitamin D [25(OH)D] levels. Linear mixed effects models were used to compare tissue parameters; plasma 25(OH)D level changes were assessed using the Wilcoxon rank-sum test. Results: Patients randomized to high-dose vitamin D had increased post-treatment plasma 25(OH)D (median 18.0 to 63.6 (ng/ml)) whereas placebo-randomized patients did not (median 19.1 to 18.6 (ng/ml)) ( p <0.001). Analysis of both biopsy and resection tissue revealed highly heterogeneous VDR and CYP27B1 expression. VDR expression was highest in tumor cells, followed by immune and then stromal cells. Although resection tissue from vitamin D-treated patients showed lower VDR expression than specimens from placebo-treated patients in all cells ( p= 0.03), VDR was preferentially expressed by tumor cells at the tumor invasive front compared to tumor center in the vitamin D arm, while the opposite was observed in the placebo arm. Vitamin D supplementation did not alter CYP27B1 expression. While treatment did not change total immune cell density, resection tissue from vitamin D-treated patients had higher overall CD3+CD8+ T cell density ( p= 0.04), particularly CD3+CD8+ memory T cell density ( p= 0.02). Additionally, vitamin D supplementation led to greater co-localization between CD3+CD8+ T cells and tumor cells ( p< 0.001) compared to placebo. Conclusions: In patients with stage I-III CRC, vitamin D supplementation led to changes in vitamin D pathway signaling and evidence of an anti-tumor effect in the TME. Further investigation into the underlying mechanism of action is warranted. Clinical trial information: NCT02172651 .

Spatial Immunoprofiling of Adenoid Cystic Carcinoma Reveals B7-H4 Is a Therapeutic Target for Aggressive Tumors.

Adenoid cystic carcinoma (ACC) is a heterogeneous malignancy, and no effective systemic therapy exists for metastatic disease. We previously described two prognostic ACC molecular subtypes with distinct therapeutic vulnerabilities, ACC-I and ACC-II. In this study, we explored the ACC tumor microenvironment (TME) using RNA-sequencing and spatial biology to identify potential therapeutic targets. Tumor samples from 62 ACC patients with available RNA-sequencing data that had been collected as part of previous studies were stained with a panel of 28 validated metal-tagged antibodies. Imaging mass cytometry (IMC) was performed using the Fluidigm Helios CyTOF instrument and analyzed with Visiopharm software. The B7-H4 antibody-drug conjugate AZD8205 was tested in ACC patient-derived xenografts (PDX). RNA deconvolution revealed that most ACCs are immunologically "cold," with approximately 30% being "hot." ACC-I tumors with a poor prognosis harbored a higher density of immune cells; however, spatial analysis by IMC revealed that ACC-I immune cells were significantly restricted to the stroma, characterizing an immune-excluded TME. ACC-I tumors overexpressed the immune checkpoint B7-H4, and the degree of immune exclusion was directly correlated with B7-H4 expression levels, an independent predictor of poor survival. Two ACC-I/B7-H4-high PDXs obtained 90% complete responses to a single dose of AZD8205, but none were observed with isotype-conjugated payload or in an ACC-II/B7-H4 low PDX. Spatial analysis revealed that ACC subtypes have distinct TMEs, with enrichment of ACC-I immune cells that are restricted to the stroma. B7-H4 is highly expressed in poor-prognosis ACC-I subtype and is a potential therapeutic target.

Impact of microvessel patterns and immune status in NSCLC: a non-angiogenic vasculature is an independent negative prognostic factor in lung adenocarcinoma.

Non-small cell lung carcinomas (NSCLC) exhibit different microvessel patterns (MVPs). Basal (BA), diffuse (DA) and papillary (PA) patterns show signs of angiogenesis (new blood vessels), while an alveolar pattern indicates that tumors are co-opting existing normal vessels (non-angiogenic alveolar, NAA). NAA tumor growth is known to exist in NSCLC, but little is known about its prognostic impact in different histological subgroups, and about associations between MVPs and immune cell infiltration. Detailed patterns of angiogenic and non-angiogenic tumor growth were evaluated by CD34 immunohistochemistry in whole tissue slides from 553 surgically treated patients with NSCLC stage I-IIIB disease. Associations with clinicopathological variables and markers related to tumor immunology-, angiogenesis- and hypoxia/metabolism were explored, and disease-specific survival (DSS) was analyzed according to histological subtypes. The predominant MVP was angiogenic in 82% of tumors: BA 40%, DA 34%, PA 8%, while a NAA pattern dominated in 18%. A contribution of the NAA pattern >5% (NAA+), i.e., either dominant or minority, was observed in 40.1% of tumors and was associated with poor disease-specific survival (DSS) (p=0.015). When stratified by histology, a significantly decreased DSS for NAA+ was found for adenocarcinomas (LUAD) only (p< 0.003). In multivariate analyses, LUAD NAA+ pattern was a significant independent prognostic factor; HR 2.37 (CI 95%, 1.50-3.73, p< 0.001). The immune cell density (CD3, CD4, CD8, CD45RO, CD204, PD1) added prognostic value in squamous cell carcinoma (LUSC) and LUAD with 0-5% NAA (NAA-), but not in LUAD NAA+. In correlation analyses, there were several significant associations between markers related to tumor metabolism (MCT1, MCT4, GLUT1) and different MVPs. The NAA+ pattern is an independent poor prognostic factor in LUAD. In NAA+ tumors, several immunological markers add prognostic impact in LUSC but not in LUAD.

Frequent Overexpression of HER3 in Brain Metastases from Breast and Lung Cancer.

HER3 belongs to a family of receptor tyrosine kinases with oncogenic properties and is targeted by a variety of novel anticancer agents. There is a huge unmet medical need for systemic treatment options in patients with brain metastases (BM). Therefore, we aimed to investigate HER3 expression in BM of breast (BCa) and non-small cell lung cancer (NSCLC) as the basis for future clinical trial design. We analyzed 180 BM samples of breast cancer or NSCLC and 47 corresponding NSCLC extracranial tissue. IHC was performed to evaluate protein expression of HER3, and immune cells based on CD3, CD8, and CD68. To identify dysregulated pathways based on differential DNA methylation patterns, we used Infinium MethylationEPIC microarrays. A total of 99/132 (75.0%) of BCa-BM and 35/48 (72.9%) of NSCLC-BM presented with HER3 expression. Among breast cancer, HER2-positive and HER2-low BM showed significantly higher rates of HER3 coexpression than HER2-negative BM (87.1%/85.7% vs. 61.0%, P = 0.004). Among NSCLC, HER3 was more abundantly expressed in BM than in matched extracranial samples (72.9% vs. 41.3%, P = 0.003). No correlation of HER3 expression and intratumoral immune cell density was observed. HER3 expression did not correlate with overall survival from BM diagnosis. Methylation signatures differed according to HER3 status in BCa-BM samples. Pathway analysis revealed subtype-specific differences, such as TrkB and Wnt signaling pathways dysregulated in HER2-positive and triple-negative breast cancer BM, respectively. HER3 is highly abundant in BM of breast cancer and NSCLC. Given the promising results of antibody-drug conjugates in extracranial disease, BM-specific trials that target HER3 are warranted. See related commentary by Kabraji and Lin, p. 2961.

Prognostic value of various immune cells and Immunoscore in triple-negative breast cancer.

This study aimed to evaluate the expression status and prognostic role of various immunoregulatory cells and test in triple-negative breast cancer (TNBC). The expression of five markers (CD3/CD4/CD8/CD19/CD163) of tumor immune cells was evaluated retrospectively in tumor sections from 68 consecutive cases of TNBC by immunohistochemistry. Computational image analysis was used to quantify the density and distribution of each immune marker within the tumor region, tumor invasive margin, and expression hotspots. Immunoscores were calculated using an automated approach. Other clinical characteristics were also analyzed. For all patients, Kaplan-Meier survival analysis showed that high CD3+ signals in the tumor region (disease-free survival (DFS), P=0.0014; overall survival (OS), P=0.0031) and total region (DFS, P=0.0014; OS, P=0.0031) were significantly associated with better survival. High CD4+ levels in the tumor region and total regions were significantly associated with better survival (P<0.05). For Hotspot analysis, CD3+ was associated with significantly better survival for all Top1, Top2, and Top3 densities (DFS and OS, P<0.05). High CD4+ levels were significantly associated with better prognosis for Top1 and Top3 densities (DFS and OS, P<0.05). For stage IIB and IIIC patients, CD3+ in the tumor region and all Top hotspots was found to be significantly correlated with survival (DFS and OS, P<0.05). CD4+ cells were significantly associated with survival in the tumor region, total region, and Top3 density (DFS, P=0.0213; OS, P=0.0728). CD8+ cells were significantly associated with survival in the invasive margin, Top2 density, and Top3 density. Spatial parameter analysis showed that high colocalization of tumor cells and immune cells (CD3+, CD4+, or CD8+) was significantly associated with patient survival. Computational image analysis is a reliable tool for evaluating the density and distribution of immune regulatory cells and for calculating the Immunoscore in TNBC. The Immunoscore retains its prognostic significance in TNBC later than IIB stage breast cancer. Future studies are required to confirm its potential to predict tumor responses to chemotherapy and immune therapy.

Abstract 5786: Spatial relationships in the tumor microenvironment predict response to immune checkpoint inhibitors in urothelial and head and neck cancer

Abstract Background: Immune checkpoint inhibitors (ICI) can achieve remarkable clinical responses in urothelial cancer (UC). However, it remains unclear which aspects of the tumor microenvironment (TME) determine a patient’s response. The TME is usually characterized by immune cell density, which ignores cells’ spatial relationships relative to each other. Methods: Using multiplex immunofluorescence data (PanCK, CD20, CD68, CD3, CD8, and FoxP3 antibody panel) of 24 pre-ICI UC transurethral resections retrieved from the NABUCCO trial (NCT03387761), we spatially profiled cancer cells, macrophages, B-cells, and distinct T-cell populations. We first quantified the TME immune cell densities at the tumor and stroma tissue compartments. We then quantified the TME spatial relationships with a novel approach based on fitting a Weibull function to the first nearest neighbor (1-NN) distance distribution, allowing us to uniquely summarize spatial relationships with two parameters (Weibull approach). We compared this approach to conventional techniques (G-functions) that rely upon a predefined distance threshold. We performed a simulation study to identify sources of variation in the spatial relationship parameters. Lastly, we associated the TME parameters with ICI (ipilimumab + nivolumab) response. Results: Unlike the Weibull approach, the G-function quantifications manifested a variable effect size and statistical power in association studies because of its distance threshold dependence. We found that variation in density affected the spatial relationship metrics of rare cell types (i.e., B-cells) but not of abundant cell types (i.e., cancer cells). The spatial relationship metrics from the Weibull approach outperformed immune cell density in ICI response prediction. Specifically, immune cell density did not discriminate between ICI response groups (FDR&amp;gt;10%). In contrast, the spatial relationship between either CD8+ T-cells or macrophages to their closest cancer cell did associate with response to ICI (FDR=1%). Furthermore, non-responding tumors were characterized by CD8+ T-cells close to B-cells (FDR=9%). Importantly, we validated the association between proximity and response from CD8+ T-cells to cancer cells (FDR=1%) and from macrophages to cancer cells (FDR=1%) using data from 25 pre-ICI head and neck squamous cell carcinoma tumors from the IMCISION trial (NCT03003637, arm B, ipilimumab + nivolumab). Conclusion: We created a framework to quantify, study, interpret and analyze spatial relationships in the TME and illustrated its superior clinical relevance compared to density metrics for predicting ICI treatment response. Our findings emphasize the importance of spatial relationships in the TME for response and suggest that proximity between either macrophages or CD8+ T-cells to cancer cells are candidate biomarkers for ICI response. Citation Format: Alberto Gil-Jimenez, Nick van Dijk, Yoni Lubeck, Maurits L. van Montfoort, Dennis Peters, Erik Hooijberg, Annegien Broeks, Joris L. Vos, Charlotte L. Zuur, Bas van Rhijn, Daniel J. Vis, Michiel S. van der Heijden, Lodewyk F. Wessels. Spatial relationships in the tumor microenvironment predict response to immune checkpoint inhibitors in urothelial and head and neck cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5786.

Abstract 5172: Tumor-immune microenvironmental profiling during chemo- and targeted therapy for head and neck squamous cell carcinoma

Abstract Understanding longitudinal changes of tumor-immune microenvironment during chemo/targeted therapies contributes to the development of optimized combinations of immunotherapy with chemotherapy and targeted therapy for patients with head and neck squamous cell carcinoma (HNSCC). We previously reported a chromogenic sequential immunohistochemical (IHC) platform enabling quantitative and spatial assessment of 29+ biomarkers in a single tissue section (Tsujikawa T et al. Cell Reports 2017, Banik G et al. Methods Enzymology 2020). Using this platform, densities, phenotypes, and distributions of tumor and immune cells were evaluated, comparing baseline and post-treatment specimens from the same individual treated by paclitaxel, carboplatin, and cetuximab (PCE) for advanced HNSCC (N = 30). Immune cell density analyses based on CD8+ T cells, helper T cells, regulatory T cells, B cells, natural killer cells, macrophages, dendritic cells, mast cells, granulocytes revealed the presence of differential immune cell compositions, where immune profiles were divided into hypo-, lymphoid-, and myeloid-inflamed groups according to the same criteria as in our previous report (Tsujikawa et al. Cell Reports 2017). Lymphoid and hypo-inflamed groups exhibited significant tumor volume reduction, increased CD45+ immune cell densities and elevated combined positive scores of PD-L1 at the post-treatment status, suggesting the potential involvement of immunogenic mechanisms related to therapeutic response. On the other hand, the myeloid group exhibited no significant tumor volume reduction, together with higher expression of HIF1α and ZEB2 on tumor cells which are potentially associated with hypoxia and epithelial-mesenchymal transition. In conclusion, longitudinal tissue-based monitoring revealed the presence of differential tumor-immune complexity profiles related to therapeutic efficacy and resistance. Hypo-inflamed profiles might require upfront chemo/targeted therapy before immunotherapy, and myeloid-inflamed profiles might require myeloid cell-targeted therapies, mandating the establishment of rapid clinical assessment of tumor-immune microenvironment. Citation Format: Alisa Kimura, Takahiro Tsujikawa, Junichi Mitsuda, Aya Miyagawa-Hayashino, Hiroki Morimoto, Sumiyo Saburi, Kanako Yoshimura, Gaku Ohmura, Sigeyuki Mukudai, Hikaru Nagao, Yoichiro Sugiyama, Hiroshi Ogi, Saya Shibata, Eiichi Konishi, Kyoko Itoh, Shigeru Hirano. Tumor-immune microenvironmental profiling during chemo- and targeted therapy for head and neck squamous cell carcinoma. [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 5172.

Abstract 5902: The immune features of Helicobactor pylori-positive gastric cancer provides insights into immunotherapy

Abstract Introduction: Helicobactoer pylori (H. pylori) is one of the most important risk factors of gastric cancer (GC). However, the effects of Helicobacter pylori on the immune microenvironment and its potential roles in tumor immunotherapy responses is still lacking. Methods: We included 61 patients, 46 of whom received anti-PD-1/PD-L1 based immunotherapy. Patients’ samples at baseline were collected and examined with multiplex immunothistochemistry. Four detecting panels including 16 kinds of antibodies were used to label CD8+ T cells, CD4+ T cells, B cells, macrophages, neutrophils, and their functional status. Immunotherapy-related overall survival (irOS) is the interval from the initiation of immunotherapy to death or the last following up. mmunotherapy-related progression-free survival (irPFS) is the interval from the initiation of immunotherapy to disease progression or the last following up. The Student’s t test is used to compared the average density of immune cells. The Chi-squared test was used to compare the objective response rate (ORR) between two groups. Results: H. pylori-positive rate was 29/61. In 46 patients received immunotherapy, 23 patients were infected with H. pylori. There was a increasing trend for the density of CD8+ T cells, B cells, and neutrophils in H. pylori-positive patients compared with negative group. We observed that the densities of total CD4+ T cells, CD4+PD-L1+, and CD4+CTLA-4+ T cells, were higher in H. pylori-positive group compared with negative group (522.58 vs. 268.77/mm^2, P=0.020, 118.94 vs. 265.98/mm^2, P=0.0091; 61.30 vs. 18.56/mm^2, P=0.035). In addition, STING positive macrophages (CD68+STING+ cells) were also enriched in H. pylori-positive group than negative group (138.45 vs. 44.15/mm^2, P=0.0012). Next, we found H. pylori-positive patients harbored higher ORR than negative group (47.62% vs. 17.39%, P=0.032). However, there were no significant difference of irPFS and irOS between positive and negative group (median irPFS: 5.97 vs. 6.83 months, hazard ratio: 1.02, P=0.96; median irOS: 12.83 vs. 10.97 months, hazard ratio: 0.855, P=0.59). Conclusions: We observed the enrichment of total CD4+ T cells, CD4+PD-L1+ T cells, CD4+CTLA-4+ T cells, and CD68+STING+ cells in H. pylori-positive patients, indicating the combination of anti-CTLA-4 or STING may enhance the efficacy of immunotherapy in H. pylori-positive GC. Citation Format: Keren Jia, Yang Chen, Xiaoyi Chong, Yilin Li, Jiajia Yuan, Yanyan Li, Xiaotian Zhang, Jian Li, Lin Shen. The immune features of Helicobactor pylori-positive gastric cancer provides insights into immunotherapy. [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 5902.

Abstract 4471: Reprogramming “cold” NF1 malignancies into “hot” tumors for immunotherapy

Abstract Introduction: Neurofibromatosis Type 1 (NF1) results from mutations in the NF1 gene that inactivate the tumor suppressor, neurofibromin. This leads to hyperactive RAS signaling that predisposes NF1 patients to tumor development. Over time, some of these tumors can develop into malignant peripheral nerve sheath tumors (MPNST) which are currently incurable.Immune checkpoint blocking (ICB) programs a patient’s immune system to enhance tumor destruction. However, MPNSTs are cold tumors characterized by low immune cell presence in the tumor microenvironment. Interestingly, MPNSTs show checkpoint protein PD-L1 expression suggesting ICB as potential therapy for MPNST, given the ability to increase immune cell density in the tumor microenvironment. Activation of the intracellular receptor stimulator of interferon genes (STING) enhances antitumor immunity through the induction of pro-inflammatory cytokines and chemokines, including type I interferons. Cyclic GMP-AMP synthase (cGAS) is an enzyme that once bound to cytosolic DNA, synthesizes cyclic GMP-AMP (cGAMP), which activates STING to induce inflammatory cytokines and other immune mediators. Preclinical studies using mouse tumor models have assessed the efficacy of STING agonists that trigger the cGAS-STING-IFN axis, ultimately leading to augmented innate immunity and a T cell-rich tumor environment. Additionally, combining STING agonists with ICB has a synergistic effect in treating tumors. Therefore, we hypothesize that treatment with STING agonists would turn “cold” MPNSTs into “hot” tumors making them susceptible to targeting with ICB. Methods: We treated MPNST cell lines with synthetic STING agonist ADU-S100 for varying durations of 8, 18, 24, and 48 hours. At the end of the treatment, cells were harvested for qRT-PCR and immunoblot analysis. Results: We measured STING-IFN pathway activation through immunoblotting and observed that phosphorylation of STING effector proteins - a readout of STING activation – was increased followingby 8 hours after treatment. qRT-PCR analysis showed that STING target cytokine/chemokine gene expression was also upregulated by 8 hours after ADU-S100 treatment. Conclusions: Theise data demonstrates that ADU-S100 was able to activate the STING pathway in MPNST cell lines leading to proinflammatory cytokine/chemokine production. We will next test STING agonist in vivo use to determine whether ADU-S100 treatment inin mouse MPNST modelss of MPNST to is able to reprogram the tumor microenvironment into an immune inflamed one amenable to immunotherapy. Citation Format: Laasya Madana, Lu Q. Le, Nipunika Somatilaka, Renee McKay. Reprogramming “cold” NF1 malignancies into “hot” tumors for immunotherapy. [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 4471.

Abstract 5706: ImmunoPROFILE: A prospective implementation of clinically validated, quantitative immune cell profiling test identifies tumor-infiltrating CD8+ and PD-1+ cell densities as prognostic biomarkers across a 2,023 patient pan-cancer cohort treated with different therapies

Abstract Tumor-infiltrating lymphocyte (TIL) density has been identified as a prognostic and predictive biomarker in select tumors treated with defined therapies. These observations suggest that TILs may be general markers of patient outcomes, but evidence in support of this hypothesis has been limited by small cohorts. We validated ImmunoPROFILE, a multiplexed immunofluorescence (MIF)-based assay coupled with machine-learning-based image analysis, to identify and quantify tumor cells (cytokeratin, PAX5, PAX8, SOX10), T cells (CD8), T-regulatory cells (FOXP3), exhausted cells (PD-1) and immunosuppressive tumor and immune cells (PD-L1). We applied the MIF panel to specimens from patients collected prospectively over three years and analyzed 2,023 cases across 27 tumor types. The association between biomarkers and overall survival (OS) was investigated using Cox models controlling for patient risk factors such as cancer type, metastatic vs. primary disease, age, and gender. Multivariable biomarker selection was based on likelihood ratios. The assay was highly robust (success rate 97%), reproducible (inter-scanning and intra-staining density controls within 1 SD, inter-staining PD-L1 scores ≤11% CV), and operator-independent (R2 &amp;gt;0.7 to &amp;gt;0.9 for each biomarker and 95% concordance in PD-L1 score-based interpretation between technicians). From whole slide images, a total of 11,932 individual regions of interest were analyzed across the cohort, resulting in &amp;gt;50 million spatially-resolved single cells which were summarized into cell population densities and PD-L1 scores. High densities of CD8+ (&amp;gt;64/mm2, p&amp;lt;0.0001), PD-1+ (&amp;gt;50/mm2, p&amp;lt;0.0001), and FOXP3+ (&amp;gt;30/mm2, p&amp;lt;0.0001) T cells were associated with longer overall survival (OS) irrespective of therapy and across all cancer types. PD-L1 metrics were not associated with OS (p=0.43). Compared to patients with low densities of CD8+ and PD-1+ cells, high densities of at least one of these cell types had better OS (Both high, HR: 0.49, 95% CI: 0.41 - 0.59; CD8+ high, HR: 0.63, (0.48 - 0.82); PD-1+ high, HR: 0.71, (0.54 - 0.93)). The results were consistent in the subset of patients (N=1572) who did not receive immunotherapy (IO). In patients who received IO therapy (N=451), only PD-1+ T-cell density associated with OS (HR: 0.48, (0.36 - 0.65)). To our knowledge, this is the first enterprise-level immune biomarker assay using multiplexed staining, digital imaging, and machine learning to be applied in a prospective manner to clinical specimens at scale. We found that select immune cell densities are prognostic across cancer types and therapies and demonstrated that quantification of multiple cell populations yields better prognostic power than single marker analyses. Citation Format: James Lindsay, Bijaya Sharma, Kristen D. Felt, Anita Giobbie-Hurder, Ian Dryg, Jason L. Weirather, Jennifer Altreuter, Tali Mazor, Priti Kumari, Joao V. Alessi, Ajit J. Nirmal, Michael P. Manos, Ananth R. Kumar, William Lotter, Ethan Cerami, Burce E. Johnson, Neil I. Lindeman, Lynette M. Sholl, Jonathan A. Nowak, Scott J. Rodig. ImmunoPROFILE: A prospective implementation of clinically validated, quantitative immune cell profiling test identifies tumor-infiltrating CD8+ and PD-1+ cell densities as prognostic biomarkers across a 2,023 patient pan-cancer cohort treated with different therapies. [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 5706.

Abstract 1186: Contraception and ovarian cancer risk by tumor immune profiles

Abstract Introduction: An estimated 38 million US women currently use contraception, a critical component of women’s health. Yet, the impact of changing contraceptive types and patterns of use on ovarian cancer incidence is unclear and the biologic pathway through which contraception influences ovarian cancer risk is not well understood. Using multiplex immunofluorescence (mIF) technology, we evaluated how contraception influences the tumor immune microenvironment (e.g., T cells) to better understand the association of intrauterine device (IUD) and oral contraception (OC) use on ovarian cancer risk. Methods: We measured tumor immune profiles via mIF on tissue microarrays including tumor cores from 214 women with ovarian cancer (85% high grade serous) and 414 population-based controls frequency matched on age who participated in the New England Case Control Study. T cell types were defined by expression of CD3+ (total T cells), CD3+CD4+ (helper), CD3+CD8+ (cytotoxic), CD3+CD4+FoxP3+ (regulatory), CD3+CD4+CD69+ or CD3+CD8+CD69+ (recently activated), or CD3+Tim3+ or CD3+Lag3+(exhausted). The average immune cell density across 3 cores per tumor was used to categorize tumors as high or low abundance based on the median across all tumors. Contraception use was self-reported and included having ever used an IUD and/or OCs and duration of use. Polytomous logistic regression models were used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for ovarian cancer defined by tumor immune cell abundance adjusted for age at diagnosis or interview, parity, and family history of breast or ovarian cancer in first degree relative. Results: Overall, ever use of IUD or OC was not differentially associated with ovarian cancer risk by T cell tumor immune profiles. However, for OC use of 5 years or longer, we observed a significant reduction in risk for low-abundance total T cell (ORlow=0.44, 95% CI 0.21-0.94), cytotoxic T cell (ORlow=0.44, 95% CI:0.21-0.94), regulatory T cell (ORlow=0.31, 95% CI: 0.14-0.68), and recently activated T cell (CD3+CD4+CD69+ ORlow=0.34, 95% CI: 0.17-0.68) tumors with significant trends with longer duration. There was no significant decrease in risk of tumors with high abundance of these T cell types, although statistically significant heterogeneity was only observed in the association for duration of OC use and risk by low and high abundance of recently activated helper T cells (CD3+CD4+CD69+, p-het=0.006). Conclusion: These results suggest that ever use of IUD and OCs are not associated with risk of ovarian tumors by immune phenotype, though risk by T cell abundance may vary with longer duration. Analyses are ongoing and will be expanded to include types and patterns of contraception, other immune markers, and additional studies. Citation Format: Jennifer Mongiovi, Ana Babic, Naoko Sasamoto, Mary Townsend, Allison Vitonis, Jonathan Hecht, Jose Conejo-Garcia, Brooke Fridley, Shelley Tworoger, Kathryn Terry. Contraception and ovarian cancer risk by tumor immune profiles [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 1186.

Abstract 2254: Immunotopography characterization after neoadjuvant therapy in the pancreatic cancer microenvironment

Abstract Pancreatic cancer is one of the most common cancers worldwide and its incidence is increasing globally. Treatment consists of surgical resection followed by adjuvant or neoadjuvant therapy. Neoadjuvant therapy (neoTx) has dramatically improved the prognosis of patients with locally advanced and borderline resectable pancreatic cancer. NeoTX has been shown to reverse the immunosuppression exerted by malignant cells and selectively deplete regulatory T cells and myeloid-derived suppressor cells in the peritumoral niche. In addition, neoTx mediates a significant decrease in the severity of neural invasion and stroma activation. However, the effect of neoTx on the topographical interactions between the different populations of tumor-infiltrating immune cells, the degree of intratumoral immune infiltration and distance to tumor cells as well as their spatial interactions with other key features of the tumor microenvironment (TME) remained unknown. In this study, we employed a multiplex immunofluorescence approach on the COMET™ platform from Lunaphore. COMET™ performs full-automated sequential immunofluorescence (seqIF™) assays, which consist of cycles of staining, imaging, and elution. We developed a customized 12-plex panel on COMET™ to characterize the TME of paraffin-embedded sections of pancreatic cancer samples. COMET™ outputs are OME.TIFF files that were analyzed and quantified with QuPath and CellProfiler for cell segmentation and cell phenotyping respectively. The optimized panel was used to analyze the immune architecture of neoadjuvant-treated pancreatic cancer patients compared to primary resected ones who underwent surgical resection with curative intention. We detected the following immune phenotypes: cytotoxic T cells (CD8+, CD3+), helper T cells (CD4+, CD3+), B cells (CD20+, CD3-), regulatory T cells (FoxP3+, CD4+), macrophages (CD68+), neutrophils (CD163+, CD11b+), and myeloid-derived suppressor cells (CD163, CD11b+, CD66b+). We also quantified the presence of both immune checkpoints (PD1+) cells and their apoptotic rate (Caspase-3), and how those correlate with the immunotopography after neoadjuvant therapy. Our optimized 12-plex panel was successfully transferred to an initial cohort of 10 patients. Our preliminary analysis shows interesting results on the effect of neoTx treatment on the immune microenvironment, such as decreased immune cell density, induction of apoptosis in lymphoid cells, and increased PD1 expression in T-killer cells compared to samples from primary resected patients. Our approach demonstrates the importance of a deep topographic characterization to understand the TME composition of pancreatic cancers, with or without neoTx treatment. Our preliminary findings highlight new differential cell identities in neoTx-treated patients, confirming their suitability for application in future clinical immunotherapy trials. Citation Format: Carmen Mota Reyes, Maximilian Kiessler, Pino Bordignon, Samuel Aubert, Pamela Pulimeno, Helmut Friess, Rouzanna Istvanffy, Ihsan Ekin Demir. Immunotopography characterization after neoadjuvant therapy in the pancreatic cancer microenvironment [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 2254.

Abstract 6776: Spatial transcriptomics of response to tepotinib treatment in a patient with NSCLC

Abstract Background: While cancer therapies are becoming increasingly more targeted, our understanding of the genomic changes occurring during these treatments is lacking. Moreover, the tumoral heterogeneity and its spatial distribution remain important obstacles in predicting tumor evolution and treatment responses. In recent years, genomic spatial profiling techniques have shed light on this complexity by detecting gene expression changes in heterogenous tumor regions and their microenvironment over the course of treatment. Methods: Tepotinib is a highly selective and potent MET class Ib inhibitor, approved for the treatment of patients with NSCLC with MET exon 14 skipping alterations. To detect genomic changes during neoadjuvant treatment with tepotinib, a GeoMx® digital spatial profiler (DSP; Nanostring Technologies) analysis was conducted in paired baseline and on-treatment patient samples. GeoMx® DSP was performed by selecting regions of interest with nuclear, TTF-1, CD3 and CD33 morphology markers followed by next-generation sequencing (NGS) using the Cancer Transcriptome Atlas RNA panel (&amp;gt;1,800 cancer-related genes). Results: Following a poor response to chemotherapy, neoadjuvant tepotinib treatment was initiated in a 54-year-old female, never-smoker with NSCLC harboring a MET exon 14 alteration. Owing to a good response to tepotinib, surgical resection of the tumor was decided by the tumor board given the downgrading (from IIIB to IA1) and resulting in a major pathological response. This resection allowed spatial transcriptomics to be compared to a baseline biopsy from the same patient. MET signaling inhibition by tepotinib treatment increased immune cell infiltration in the tumor microenvironment compared to the baseline sample. This enhanced tumoral immune cell density was shown by the fluorescent morphology markers and confirmed through spatial deconvolution of the NGS results. It was further characterized by the significantly altered gene expression levels between the two samples. Gene ontology (GO) assessment of the differentially expressed genes revealed GO terms associated with tumorigenesis in the basal sample, such as cell mobility and motility, vascular epithelial growth factor production and cell communication. In turn, the tepotinib-treated tissue mainly exhibited tumor-clearing characteristics, including antigen processing and presentation through major histocompatibility complex classes I &amp; II, T cell-mediated toxicity, and granulocyte and leukocyte activation. Conclusion: This is the first spatial genomic characterization of the effect of tepotinib on the tumor and its microenvironment in a patient with NSCLC over time. Results from this case report highlight the impact of MET inhibition by tepotinib on the tumor immune microenvironment, likely enabling the recruitment of antigen-presenting cells for tumor clearance, while reducing the expression of tumor-promoting genes. Citation Format: Manon A. Simard, Felix Geist, Carlos Cabrera, Santiago Viteri, Joachim Albers, Michael Zuehlsdorf, Niki Karachaliou. Spatial transcriptomics of response to tepotinib treatment in a patient with NSCLC. [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 6776.

Immune regulation mechanism of Saposhnikoviae Radix polysaccharide based on zebrafish model

The immunomodulatory effect of Saposhnikoviae Radix polysaccharide(SRP) was evaluated based on the zebrafish mo-del, and its mechanism was explored by transcriptome sequencing and real-time fluorescence-based quantitative PCR(RT-qPCR). The immune-compromised model was induced by navelbine in the immunofluorescence-labeled transgenic zebrafish Tg(lyz: DsRed), and the effect of SRP on the density and distribution of macrophages in zebrafish was evaluated. The effect of SRP on the numbers of macrophages and neutrophils in wild-type AB zebrafish was detected by neutral red and Sudan black B staining. The content of NO in zebrafish was detected by DAF-FM DA fluorescence probe. The content of IL-1β and IL-6 in zebrafish was detected by ELISA. The differentially expressed genes(DEGs) of zebrafish in the blank control group, the model group, and the SRP treatment group were analyzed by transcriptome sequencing. The immune regulation mechanism was analyzed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment, and the expression levels of key genes were verified by RT-qPCR. The results showed that SRP could significantly increase the density of immune cells in zebrafish, increase the number of macrophages and neutrophils, and reduce the content of NO, IL-1β, and IL-6 in immune-compromised zebrafish. The results of transcriptome sequencing analysis showed that SRP could affect the expression level of immune-related genes on Toll-like receptor pathway and herpes simplex infection pathway to affect the release of downstream cytokines and interferon, thereby completing the activation process of T cells and playing a role in regulating the immune activity of the body.

Tumoral Densities of T-Cells and Mast Cells Are Associated With Recurrence in Early-Stage Lung Adenocarcinoma

IntroductionLung cancer is the deadliest cancer in the United States and worldwide, and lung adenocarcinoma (LUAD) is the most prevalent histologic subtype in the United States. LUAD exhibits a wide range of aggressiveness and risk of recurrence, but the biological underpinnings of this behavior are poorly understood. Past studies have focused on the biological characteristics of the tumor itself, but the ability of the immune response to contain tumor growth represents an alternative or complementary hypothesis. Emerging technologies enable us to investigate the spatial distribution of specific cell types within the tumor nest and characterize this immune response. This study aimed to investigate the association between immune cell density within the primary tumor and recurrence-free survival (RFS) in stage I and II LUAD. MethodsThis study is a prospective collection with retrospective evaluation. A total of 100 patients with surgically resected LUAD and at least 5-year follow-ups, including 69 stage I and 31 stages II tumors, were enrolled. Multiplexed immunohistochemistry panels for immune markers were used for measurement. ResultsCox regression models adjusted for sex and EGFR mutation status revealed that the risk of recurrence was reduced by 50% for the unit of one interquartile range (IQR) change in the tumoral T-cell (adjusted hazard ratio per IQR increase = 0.50, 95% confidence interval: 0.27–0.93) and decreased by 64% in mast cell density (adjusted hazard ratio per IQR increase = 0.36, confidence interval: 0.15–0.84). The analyses were reported without the type I error correction for the multiple types of immune cell testing. ConclusionsAnalysis of the density of immune cells within the tumor and surrounding stroma reveals an association between the density of T-cells and RFS and between mast cells and RFS in early-stage LUAD. This preliminary result is a limited study with a small sample size and a lack of an independent validation set.

Tumor cell density dependent IL-8 secretion induces the fluctuation of tregs/CD8 + T cells infiltration in hepatocellular carcinoma: one prompt for the existence of density checkpoint

BackgroundTumor cell density is a basic pathological feature of solid tumors. Chemotherapy, radiotherapy, and targeted therapy reduce tumor cell density, whereas unrestricted tumor cell proliferation promotes this feature. The impact of tumor cells on the microenvironment following changes in tumor cell density is still unclear. In this study, we focused on the response of key immune cell subsets to tumor cell density in hepatocellular carcinoma (HCC).MethodsWe determined the density of tumor and immune cells in the same area by section staining. We then identified potential mediators using polymerase chain reaction (PCR), enzyme-linked immunofluorescence assay (ELISA), 3D and co-culture, flow cytometry, and lentivirus intervention. The mechanism of lactate promotion was verified using lactate tests, bioinformatics, western blotting, and the above methods. The IL-8/DAPK1/lactate/regulatory T cell (Treg) axis was verified using a mouse liver cancer model. Tumor mutation burden was calculated using maftools in R.ResultsWe found that the Treg/CD8 + T cell ratio is not consistent with tumor cell density in HCC, and a decreased Treg/CD8 + T cell ratio in the range of 5000–6000 cells/mm2 may elicit the possibility for immunotherapy in an immunosuppressive microenvironment. We showed that IL-8 mediates this immune fluctuation and promotes the infiltration of Tregs through the DAPK1/pyruvate kinase activity/lactate axis in HCC. Based on tumor ploidy and mutation burden data, we discussed the potential significance of immune fluctuation in the homeostasis of HCC mutation burden and proposed a “density checkpoint” and “entropy model” to describe this phenomenon.ConclusionsIn summary, we report the mode of infiltration of Tregs/CD8 + T cells in response to tumor cell density and provide a new theoretical basis for IL-8 as a therapeutic target and the selection of an immunotherapy window in HCC.

Abstract P1-04-02: Gut microbiome diversity correlates with tumor PD-L1 status in metastatic triple negative breast cancer (mTNBC): correlative analysis of gut microbiome and tumoral biomarkers

Abstract Background: Greater gut microbiome diversity is observed in patients with a clinical response to immune checkpoint inhibitor (ICI) therapy in solid tumors and in preclinical models targeting the gut microbiome is a promising strategy to improve ICI efficacy. The investigation of the immunomodulatory effects of the gut microbiome and tumor biology is in its infancy and whether interindividual microbiome variations correlate with tumor biology in breast cancer is unknown. Here we report a correlative analysis of gut microbiome diversity with tumor biomarkers: PD-L1, tumor immune cell density, tumor infiltrating lymphocytes (TILs), MutSig3, and interferon (IFN) gene signatures in a cohort of mTNBC patients treated with olaparib and durvalumab of the Adaptive Multi-Drug Treatment of Evolving Cancers (AMTEC) trial (NCT03801369). Methods: AMTEC participants undergo a baseline biopsy (bx) and fecal sample collection, then start olaparib monotherapy on a 28-day cycle. Following the first cycle, an on-treatment (on-tx) bx and fecal sample are collected, then as part of enrollment to Arm 1, durvalumab is added to the treatment regimen. Ten patients with stool collections and tissue biopsies were used in this analysis, eight had paired pre- and on-tx collections. At interim-analysis, participants were categorized as responders (PR/SD) or non-responders (PD). Gut microbiome composition in fecal samples collected pre- and on-olaparib monotherapy was assessed by 16S rRNA sequencing and alpha diversity was quantified by observed diversity, Fisher, Inverse Simpson, Pielou, and Shannon indices. Response, PDL1 expression (Positive CPS &amp;gt;1), multiplex immunohistochemistry (mIHC) immune cell density and immune cell signature, TILs, RNA signatures including interferon (INF) family signatures, and mutational significance (MutSig3) were correlated to alpha diversity by T-test. Results: Average baseline alpha diversity of the fecal microbiome did not vary based on response (6 non-responses and 4 responses). However, in the on-tx samples, Fisher (CB: mean = 21.81, std. dev. = 3.6; PD: mean = 17.04, std. dev. = 2.3; p=0.048) and Observed alpha diversity (CB: mean = 260, std. dev. = 38.8; PD: mean = 209, std. dev. = 25.8; p = 0.049) were higher in responders. Average baseline alpha diversity of the gut microbiome as measured across all examined indices was significantly lower in participants with PD-L1 expressing tumors (p &amp;lt; 0.05 for each, T-test). Among on-tx samples, there was a trend toward lower alpha diversity in PD-L1 expressing tumors, but PD-L1 expression did not correlate with clinical response. There was a notable trend toward greater alpha diversity in on-tx bx and high TILs. Alpha diversity did not differ by immune cell densities by mIHC (CD8+ T cell, B cell, and regulatory T cells), hypo-inflamed/pro-inflammatory signature, IFN gene signature, nor MutSig3 score. Conclusion: Our preliminary data reveal that higher alpha diversity following olaparib monotherapy correlated with response to therapy with combination olaparib and durvalumab and correlated with tumor PD-L1 status. Whether gut microbiome features represent a meaningful biomarker for ICI treatment of mTNBC warrants further study. Enrollment to stage 2 of AMTEC Arm 1 is ongoing. Citation Format: Brie Chun, Shaun Goodyear, Travis Rice-Stitt, Lisa Karstens, Erin Dahl, Allen Li, Evthokia Hobbs, Mitri Zahi. Gut microbiome diversity correlates with tumor PD-L1 status in metastatic triple negative breast cancer (mTNBC): correlative analysis of gut microbiome and tumoral biomarkers [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-04-02.

Abstract OT1-20-02: Intralesional injection of anti-PD-1 (pembrolizumab) and OX40L/IL-23/IL-36 mRNAs (mRNA-2752) results in regression of DCIS characterized by lymphocytic infiltrates

Abstract Background: High-grade DCIS with immune infiltrates may represent lesions that are able to be kept in check by the immune system, but still are at risk for progression to invasive disease. We and others have demonstrated that the presence of T cells, and in particular the spatial proximity of CD8+/PD-1+ T cells and PD-L1+ cells predicts response to chemotherapy and PD-1 inhibitors in the setting of invasive triple negative breast cancer. DCIS with high-risk features (e.g. large, palpable, high grade, HR-, or HER2+) often have T cell infiltrates. We hypothesized that it might be possible to potentiate the immune response in high-risk DCIS with immune checkpoint blockade. Since mortality for DCIS is extremely low, we proposed an intralesional pembrolizumab (pembro) treatment approach to avoid systemic adverse effects. In a phase 1 dose escalation study of single agent pembro we found 2 doses of 8 mg, administered intralesionally 2-3 wks apart, was tolerable, induced immunological changes within the DCIS lesions, but had little clinical benefit. Herein, we expanded the number of injections to 4 and also tested a combination with mRNA-2752 (Moderna), an mRNA-based therapeutic encoding a T cell co-stimulator OX40L, and pro-inflammatory cytokines IL-23 and IL-36 gamma, to determine if we could find a dose that was both tolerable and elicited anti-tumor responses. Methods Women eligible for this study had DCIS with at least 2 of the following high-risk features: age&amp;lt; 45; high grade, extensive comedonecrosis; palpable mass; hormone receptor negative [HR-]; HER2+; size &amp;gt;5 cm, or microinvasion. A dose expansion cohort was performed using 4 doses of 8 mg pembro, 2-3 wks apart (n=5). We then combined pembro with mRNA-2752 (n=8 cases), initially at 8 and 4 mg, respectively. Dose reductions were implemented based on AEs. Patients received an MRI before and after 2 injections, spaced 2-3 wks apart. A total of 4 injections were allowed. Core biopsy or surgery was conducted after the last MRI. Results We observed an increase in T cell density in the dose expansion cohort (pembro only), except when there was a paucity of T cells in the pre-treatment biopsy. However, only 1 patient in this cohort demonstrated a reduction in lesion size (clinically and on MRI). As of 22SEP22, 8 patients have received the combination of pembro and mRNA-2752; 7 are evaluable. Two patients with minimal T cell infiltrates at baseline (both HR+) failed to respond, based on imaging or pathology. 4/5 patients with moderate to high T cell infiltrates at baseline had partial (2) or complete responses (2) based on imaging (surgery pending), one of whom had absence of DCIS on post-therapy core biopsy and a clear MRI 4 months later. Correlative studies (immune cell densities, spatial proximities) will be presented at the meeting along with complete data (including post-treatment pathology) on the first 8 patients. Side effects were independent of response and included Gr1/2 fever, myalgias, and fatigue starting within 12 hours of injection (3-4 days), enlargement of regional nodes by day 2, and Gr 1/2 erythema and induration of the breast starting at 4 days and lasting 4-20 days. Earlier onset and persistence of symptoms with subsequent exposure required dose reductions for the majority of patients to maintain tolerability (avoid fevers over 39.4°C, intense erythema and swelling of the breast lasting &amp;gt; 4 days). No AEs were higher than grade 2. Conclusion: The combination of intralesional pembro and mRNA-2752 demonstrated modulation of the tumor immune microenvironment and robust antitumor activity in high-risk DCIS (typically HR- or HER2+) with existing T cell infiltrates. This is an ongoing study; the optimal Phase 2 dose for the combination will be based on the totality of evolving safety and translational data. Citation Format: Laura J. Esserman, Alexa Glencer, Kirithiga Ramalingam, Christopher J. Schwartz, Alexander Borowsky, Gillian L. Hirst, Rachel Woody, Nicole Schindler, Nola M. Hylton, Michael Campbell. Intralesional injection of anti-PD-1 (pembrolizumab) and OX40L/IL-23/IL-36 mRNAs (mRNA-2752) results in regression of DCIS characterized by lymphocytic infiltrates [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr OT1-20-02.

Abstract PD10-08: PD10-08 Immune cell infiltration associated with poor anti-proliferative response to aromatase inhibitors in postmenopausal women with primary ER-positive HER2-negative breast cancer

Abstract Background: Aromatase inhibitors (AIs) are one of the main treatment strategies for the clinical management of estrogen receptor-positive (ER+) breast cancer (BC). Despite prolonged time to recurrence and initial clinical responses, &amp;gt;20% of patients eventually relapse, and previous studies have shown an association of poor anti-proliferative response to AIs and worse outcome. High immune activity in ER+ tumors may be associated with worse outcome, in contrast to ER-negative BC where immune infiltration is a feature associated with better outcome. Our work focused on understanding the correlations between immune cell infiltration and response to AI. Methods: All patients with ER+ HER2- tumors within the bottom 15% of Ki67 anti-proliferative responders to AIs (poor responders [PRs]; n=177) were selected from the PeriOperative Endocrine Therapy for Individualizing Care (POETIC) trial and matched on baseline Ki67 levels to good responders (GRs) within the 50% showing the best response (n=190). Response to AI was measured by the Ki67 percentage change after 2 weeks of treatment. PRs were further divided into groups expressing high ESR1 (PRs ESR1HIGH; n=119) and low ESR1 (PRs ESR1LOW; n=58) levels to represent PR subgroups that showed partial or no response to AIs. The percentage of stromal tumor-infiltrating lymphocytes (TILs) was assessed. Multiple immunofluorescence was performed for ER, CD3, CD20, CD68, FOXP3, and CD3/FOXP3 in 15 baseline samples from each of the GR, PR ESR1HIGH, and PR ESR1LOW populations and immune cell density in stromal or tumor compartments was estimated. Spearman correlations of TILs with Consensus tumor microenvironment (TME) deconvolution and Molecular Signatures Database hallmark gene sets were conducted. The relationship between the immune markers’ density and genes, hallmark gene sets and Consensus TME was assessed. Results: The percentage of TILs was significantly higher in the PR ESR1HIGH and PR ESR1LOW compared to the GRs (adjusted p&amp;lt; 0.05). As expected, TILs were highly correlated with T cells (particularly T-regulatory cells) and immune hallmark gene sets. There was a tendency for higher density of each of the immune markers in PRs compared to GRs, with significant differences being observed in stromal B-cell marker CD20 density (p&amp;lt; 0.05). Analysis showed a significant correlation between TILs and stromal FOXP3 marker density (FDR&amp;lt; 0.05), and stromal biomarker density was highly correlated to the gene expression of the encoding genes of the same tumors (CD3/CD3D, FOXP3/FOXP3, and CD20/MS4A1) (FDRs&amp;lt; 0.05). There was also a strong and significant correlation between the stromal expression of CD20, CD3, FOXP3, and CD3/FOXP3 with the immune hallmark gene sets (FDRs&amp;lt; 0.05). Finally, the immune phenotyping showed the expected correlations with TME deconvolution, with particularly strong correlations of CD20 and CD3 with B- and T-cell gene signatures, respectively (FDRs&amp;lt; 0.05). Conclusions: Different immune features indicated a broad involvement of several immune cell types in PRs to AIs, suggesting that the immune system might be associated with resistance of ER+ breast tumors to AI treatment. Spatial gene expression profiling is ongoing to characterize these tumors further and investigate potential mechanisms of AI resistance. Citation Format: Anastasia Alataki, Gene Schuster, Lila Zabaglo, Perry Maxwell, Elena López-Knowles, Elizabeth Folkerd, David Evans, Kally Sidhu, Holly Tovey, Nicholas Turner, Stephen Johnston, Maggie Chon U Cheang, John Robertson, Manuel Salto-Tellez, Ian Smith, Judith Bliss, Mitch Dowsett. PD10-08 Immune cell infiltration associated with poor anti-proliferative response to aromatase inhibitors in postmenopausal women with primary ER-positive HER2-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD10-08.

Abstract P2-03-08: Deconstructing the molecular characteristics of ER+ HER2+ early breast cancer in the POETIC trial using multiplex immunofluorescence and gene expression profiles

Abstract Background: POETIC was a phase III clinical trial, with patients randomised 2:1 to 2-week perioperative aromatase inhibitor (POAI) vs control for postmenopausal women with oestrogen receptor positive (ER+) early breast cancer (BC) (Smith et al., Lancet Oncology 2020). Our previous study on POETIC trial patients with ER+ human epidermal growth factor receptor 2 positive (HER2+) BC suggested both HER2 enriched subtype (HER2-E) and immune enrichment pre-POAI (baseline, B) are main drivers of poor early response to POAI (Bergamino et al., 2022). However, some patients with HER2-E or immune enriched BC at B still showed good response to POAI. In this study, we aim to further investigate a sub-cohort of ER+ HER2+ BC from the POETIC trial, including a subset of aforementioned HER2-E tumours, to further explore the multi-modal molecular characteristics of the tumours resistant to POAI. Methods: Proliferation rate was assessed as percentage of cancer cells stained by Ki67. Patient POAI response was determined by Ki67 reduction at 2 weeks of treatment. A sub-cohort of 37 patients were selected based on response and classified as poor responders (PR, reduction &amp;lt; 30%, n=18), good responders (GR, reduction &amp;gt; 90%, n=11) and good responders with HER2-E BC at B (GR, reduction &amp;gt; 65%, n=8). Paired B and post-POAI (surgery, S) samples were taken from each patient of the sub-cohort. Multiplex immunofluorescence (mIF) was performed on these samples, measuring the immune cell densities in stroma and tumour compartments using five biomarkers: CD3 (all T cells), CD20 (B cells), CD68 (Macrophages), FOXP3 (regulatory T cells), and CD3 FOXP3 co-expression. The samples were also profiled using Breast Cancer 360TM (NanoString, BC360), covering the expressions of 758 genes and 46 biological signatures. Wilcoxon test, hierarchical clustering and spearman correlation test were performed to compare the tumour characteristics of GR and PR. Results: In this study, two B and four S samples were not achievable for mIF experiments due to low tumour content. At B (n = 35), among the five mIF biomarker measurements in stroma and tumour, only the stromal CD3 density was significantly different between GR (median = 0.0013) and PR (median = 0.0003, p = 0.041). In GR, HER2-E BC at B were separated into immune-high and immune-low groups with mIF biomarkers at B; the immune-high group was more likely to change into luminal subtypes post-POAI, while the immune-low group remained HER2-E. After POAI, the density changes in five mIF biomarkers in stroma and CD68 in tumour were all significantly higher in PR than GR (Table 1, n of paired samples = 62). The BC360 signatures of BC p53 (p &amp;lt; 0.001), BC proliferation (p &amp;lt; 0.001), LumB (p &amp;lt; 0.001) and HER2-E correlation coefficients (p &amp;lt; 0.001) were significantly downregulated in GR after POAI, while LumA correlation coefficients (p &amp;lt; 0.001) were notably increased. Conclusions: Our results suggest that for this sub-cohort, increased stromal immune response is associated with poor response to 2-week POAI in ER+ HER2+ early BC. HER2-E GR display visible immune heterogeneity at B. Lower-risk BC characteristics were exhibited in GR after the 2-week treatment. Further integrating mIF imaging data and additional digital spatial profiling are ongoing to reveal additional characteristics of ER+ HER2+ BC and tumour microenvironment predicting POAI resistance. Table 1: List of medians of log2 fold changes in mIF biomarker densities between GR and PR among the 62 paired samples, and Wilcoxon test p-values. Citation Format: Xixuan Zhu, Hui Xiao, Elena López-Knowles, Milana A. Bergamino Sirvén, Anastasia Alataki, Perry Maxwell, Holly Tovey, Lucy Kilburn, Chris Holcombe, Anthony Skene, Ian Smith, John Robertson, Katherine A Hoadley, Roberto Salgado, Judith Bliss, Nicholas Turner, Manuel Salto-Tellez, Gene Schuster, Mitch Dowsett, Maggie Chon U Cheang. Deconstructing the molecular characteristics of ER+ HER2+ early breast cancer in the POETIC trial using multiplex immunofluorescence and gene expression profiles [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-03-08.