Tumor Compartments Research Articles

Abstract 4293: Strategies and resources for applying a quantitative multiplex IHC imaging workflow to characterize immune contexture in solid tumors

Abstract Conventional immunohistochemistry (IHC) is a standardized diagnostic technique used in tissue pathology. However, the capacity to label only one marker per tissue section is a critical limitation. Multiplex immunohistochemistry (mIHC) is a powerful imaging technique used in basic, translational research and clinical settings to simultaneously detect the expression of multiple epitopes in a single formalin-fixed paraffin-embedded (FFPE) tissue section, allowing for characterization and quantification of cells while maintaining their spatial location. This technique allows for a comprehensive view of the immune milieu, expression patterns, and interactions between cell types that can help elucidate the complexity of the tumor-immune microenvironment (TiME). Multiplex imaging has emerged as a powerful tool to better understand tumor progression, response or resistance to therapy, and potentially identify predictive biomarkers. However, this technique requires careful tissue considerations and generates enormous amounts of hierarchical data, including single cell marker expressions, locations, and shape features, resulting in complex and challenging analyses. We previously published a validated platform using nine matched primary and recurrent head and neck squamous cell carcinoma (HNSCC) sections stained sequentially with a panel of 29 antibodies identifying malignant tumor cells, and 17 distinct leukocyte lineages and their functional states1. Here, we have optimized and consolidated important considerations, including tissue quality and QC steps necessary for generating high quality data from multiplex imaging studies. We demonstrate strategies for a streamline analytic pipeline using our quantitative workflow developed to characterize immune cells and infiltration patterns within spatial compartments of solid tumors in FFPE tissues, and herein, provide strategies and resources to practically adapt and utilize our analytic mIHC pipeline. 1. Banik, G., et al., Methods Enzymol., 2020 Citation Format: Shamilene Sivagnanam, Courtney M. Betts, Nell Kirchberger, Konjit Betre, Lisa M. Coussens. Strategies and resources for applying a quantitative multiplex IHC imaging workflow to characterize immune contexture in solid tumors. [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 4293.

Abstract 3588: Multiplex proteomics profiling with the imaging mass cytometry (IMC) identifies immune cell phenotypes associated with high NOS2/COX2 expression in ER- negative and TNBC patients

Abstract Elevated expression of Inducible Nitric oxide Synthase-2 (NOS2) occurs during acute and chronic inflammation and wound healing processes however, it is also a predictor of bad prognosis in aggressive ER-negative tumors. Overexpression of NOS2 correlates with other prognostic biomarkers making it a unique multifunctional oncoprotein. Cyclooxygenase-2 (COX2) is another enzyme associated with cancer inflammation and is overexpressed in >50% of breast cancers. In our previous studies, we have shown that NOS2/COX2 co-expression is a strong predictor of poor clinical outcome and promotes CD8+ T cell exclusion from tumor epithelia in Triple-negative breast cancers (TNBC). This observation was further explored using multiplexing proteomics technologies. Imaging Mass Cytometry (IMC) has revolutionized tumor microenvironment research by allowing the interrogation of over 35 protein markers simultaneously for the elucidation of the spatial distribution of immune and tumor cells at sub-cellular resolution. Herein, we used multiplex capability IMC to explore the interactions of immune and tumor cell counterparts in ER-negative and TNBC tumors. We employed Formalin-fixed paraffin-embedded (FFPE) tissue from 26 ER-negative and TNBC samples from a clinical cohort of African American breast cancer patients in the Baltimore MD area. We developed a panel of 15 protein metal-labeled antibody markers including antibodies targeting NOS2, COX2, T cells (CD45, CD3, CD4, CD8,) endothelium (CD31), monocytes (CD14), macrophages (CD68), basal markers (α-SMA), cancer stem cells (CD44v6), cancer cells (Pan-CK, B7H4, Cadherin), and serine proteases (granzyme B) secreted by T cells or Natural Killer (NK) cells. Single section slides of 4 µm size were stained with the cocktail of metal-labeled antibodies. Multiple regions of interest (ROIs) representing the tumor and immune cells compartments and tumor-facing stromal cell areas were ablated with the Hyperion Imaging system. The data analysis was performed by the Bodenmiller lab pipeline and VisioPharm software packages. Our preliminary data suggest that patient tumors with high or low NOS2/COX2 expression showed variable levels of CD68 positive macrophage population and T cells. We are currently analyzing the remaining data to understand the cell phenotypes associated with the immunosuppressive and immune-rich clinical samples and performing neighborhood analysis of tumor cells to immune cells and vessels. Citation Format: Milind Pore, Lisa Ridnour, Paul Mallory, Ashley Cardamone, Stephen Lockett, William Bocik, David Wink. Multiplex proteomics profiling with the imaging mass cytometry (IMC) identifies immune cell phenotypes associated with high NOS2/COX2 expression in ER- negative and TNBC patients. [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 3588.

Abstract 5106: The addition of radiation therapy to simultaneous targeting of PD-1 and IL2Rβγ enhances antigen-specific CD8 T cell anti-tumor immunity in pancreatic cancer models

Abstract Although IL-2 has been shown to critically mediate NK and T cell effector functions, when used in clinical trials this cytokine has failed due to toxicity and severe side effects of the systemic treatment. Our lab has investigated the use of a next-generation IL-2 compound, murine PD1-IL2v, which consists of an anti-PD-1 antibody fused to a IL2-variant with abolished binding to IL2Rα. Previous work shows this molecule results in tumor-antigen specific T cell expansion and differentiation towards immune effectors. Here, we report for the first time that radiation therapy amplifies the anti-tumor effect observed with PD1-IL2v treatment of murine orthotopic models of local tumor growth and metastasis. This anti-tumor effect is correlated with increases in proliferative and polyfunctional CD8 T cells across tumor, lymph node, and blood compartments. Furthermore, using an OVA-antigen specific orthotopic model, we found that radiation in combination with PD1-IL2v treatment enhances infiltration and activation of antigen-specific CD8 T cells, which have been shown to be critical for immunotherapy clinical success. A portion of tumor-bearing mice treated with radiation and PD1-IL2v fully eradicated their primary tumors and tumor rechallenges, which we show correlates with increased markers of memory in CD8 T cells of these mice. To demonstrate the clinical relevance of these preclinical findings, we performed RNA sequencing on paired pancreatic adenocarcinoma (PDAC) patient tumor tissue samples prior to and following radiation. We observed enhanced IL2Rβ and IL2Rγ expression as well as IL2R signaling in PDAC-patient responders to radiation therapy compared to non-responders. In conclusion, the antigen-specific, lasting memory response to preclinical PDAC models demonstrated here provides rationale for the treatment of PDAC with a radiation and PD1-IL2v combination. Citation Format: Maureen Hoen, Miles Piper, Laurel B. Darragh, Diemmy Nugyen, Jacob Gadwa, Maria Amann, Greta Durini, Michael Knitz, Benjamin Van Court, Tiffany Pham, Ross Kedl, Pablo Umana, Laura Codarri-Deak, Christian Klein, Angelo D’Alessandro, Sana D. Karam. The addition of radiation therapy to simultaneous targeting of PD-1 and IL2Rβγ enhances antigen-specific CD8 T cell anti-tumor immunity in pancreatic cancer models. [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 5106.

Abstract 612: Control of the Basal Molecular Subtype of Murine Bladder Cancer by Adaptive Immunity

Abstract Introduction: Developing optimized bladder cancer mouse models that can be used in the evaluation of immune based therapeutics remains a high priority in ongoing research.Experimental Procedures: In previous work by our collaborators, murine cell lines were generated that recapitulate the basal (BBN964, BBN966, and BBN975) molecular subtype of human bladder cancer (Saito et al. Cancer Res. 2018;78(14):3954-3968). Here we compared their tumorigenicity and growth rates in immunocompetent C57BL/6 mice and in immunodeficient Rag1-KO mice lacking B and T cells. Changes in the tumor and stromal compartments were correlated with the differences in growth rates observed. Observations and New Data: Tumors derived from all three BBN basal cell lines grew robustly in Rag1-KO mice (10/10 BBN964 tumors, 19/19 BBN966 tumors and 19/19 BBN975 tumors >100 mm3 by 10 weeks post-injection). Conversely, very few tumors grew in immunocompetent (C57BL/6, e.g. B6) mice (0/9 BBN964 at 8 weeks, 0/17 BBN966 at 16 weeks, 4/15 BBN975 at 9 weeks). BBN966 tumors conditioned to grow in Rag1-KO mice were also highly tumorigenic when they were subsequently transferred into immunocompetent mice (Rag1-KO ◊ B6, 16/20 mice with tumors >100 mm3 at 4 weeks), and they displayed increased tumorigenicity when they were reintroduced into immunodeficient mice (Rag1-KO ◊ Rag1-KO, 20/20 mice with tumors >100 mm3 at 3 weeks). BBN966 tumors harvested from B6 mice and passaged in vitro for 5 weeks maintained tumorigenicity when reimplanted into immunocompetent mice, and BBN966 tumors grown in female B6 mice grew robustly when transferred into male B6 mice. Bulk RNA sequencing of tumors comparing the BBN966 Rag1-KO (1st passage) to BBN966 Rag1-KO (2nd passage) demonstrated decreased expression of genes associated with tumor infiltrating macrophages in the 2nd passage tumors. Conclusions: Our results demonstrate that basal BBN syngeneic bladder tumors display markedly different growth rates in wild-type and T- and B-cell-deficient mice. The results suggest that adaptive immunity controls basal bladder cancer tumorigenicity, which is consistent with the immune infiltrated landscape observed in basal human bladder cancers. Further results described in this poster will include deeper molecular characterizations of the immune microenvironments of these tumors. Citation Format: Burles Avner Johnson, Mingxiao Feng, Woonyoung Choi, Gabriela Colocho, Alyssa Arbuiso, Samuel Jin, Adam K. Aragaki, Charles Ruland, Stanley Rapiey, Noah M. Hahn, David J. McConkey. Control of the Basal Molecular Subtype of Murine Bladder Cancer by Adaptive Immunity [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 612.

Abstract 5173: BET inhibition reprograms pancreatic cancer associated fibroblasts to promote anti-tumor immunity

Abstract BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is the most malignant neoplasm of the pancreas, and is refractory to most therapeutic strategies, including immunotherapy. We have previously demonstrated that loss of CDKN2A in PDAC tumor cells promotes an immunologically-cold tumor microenvironment, which can be counteracted through the use of bromodomain and extraterminal domain inhibitors (BETi). The immunosuppressive PDAC tumor microenvironment is also characterized by a dense desmoplastic stroma, rich in cancer associated fibroblasts (CAFs). These CAFs are a highly plastic population of cells, comprised of myofibroblastic, inflammatory, and antigen-presenting CAFs (myCAFs, iCAFs, and apCAFs, respectively). Previously research has suggested BETi can alter the function of CAFs, suggesting this therapeutic strategy may be effective at targeting both the tumor and stromal compartments of PDAC. Here we hypothesize that BETi enhances anti-tumor immunity in CDKN2A-deficient PDAC by modulating the phenotype and function of PDAC CAFs. METHODS: Isogenic wild-type and CDKN2A-deficient KPCluc pancreatic cancer cell lines were used in conjunction with KPCluc-derived murine CAF cells. CAF spheroids were used to evaluate the effects of BETi on CAF phenotype (as measured by flow cytometry) and function (as measured by NanoString analysis, checkpoint ligand expression by flow cytometry, and T cell migration assays). Additionally, the impact of BETi-induced modulation of CAF phenotype and function on wild-type and CDKN2A-deficient tumor cell spheroids was measured by flow cytometry. RESULTS: Treatment of pancreatic CAFs with BETi induced expression of genes associated with DNA damage responses and type I IFN expression. Additionally, while CAF supernatants suppressed T cell migration in vitro, treatment with BETi restored T cell migration across a transwell. This was due in part to DNA damage/STING/NF-κB signaling induced in PDAC CAF following BETi. Supernatants from BETi-treated PDAC CAFs were found to modulate PD-L1 expression on PDAC tumor cells. Direct treatment of CAFs with BETi induced a dramatic dose- and time-dependent polarization of CAF spheroids towards a myCAF phenotype. However, while BETi induced DNA damage/STING/NF-κB signaling in CAF cells, this signaling did not contribute towards this myCAF polarization, as treatment of CAF spheroids with BETi and STING or NF-κB inhibitors did not impact myCAF polarization. Additional mechanisms of polarization are currently being evaluated and will be updated at presentation. CONCLUSIONS: BET inhibition modulates CAF cell phenotype and function to promote anti-tumor immune responses. Taken together with the impact of BET inhibition on CDKN2A-deficient PDAC tumor cells, these data suggest BETi is a rational treatment approach to promote anti-tumor immunity in this genetically defined immunotherapy resistant patient population. Citation Format: Trisha Minocha, Alison Thomas, Brian M. Olson. BET inhibition reprograms pancreatic cancer associated fibroblasts to promote anti-tumor immunity. [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 5173.

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>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.

Compartmentalized spatial profiling of the tumor microenvironment in head and neck squamous cell carcinoma identifies immune checkpoint molecules and tumor necrosis factor receptor superfamily members as biomarkers of response to immunotherapy.

Mucosal head and neck squamous cell carcinoma (HNSCC) are the seventh most common cancer, with approximately 50% of patients living beyond 5 years. Immune checkpoint inhibitors (ICIs) have shown promising results in patients with recurrent or metastatic (R/M) disease, however, only a subset of patients benefit from immunotherapy. Studies have implicated the tumor microenvironment (TME) of HNSCC as a major factor in therapy response, highlighting the need to better understand the TME, particularly by spatially resolved means to determine cellular and molecular components. Here, we employed targeted spatial profiling of proteins on a cohort of pre-treatment tissues from patients with R/M disease to identify novel biomarkers of response within the tumor and stromal margins. By grouping patient outcome categories into response or non-response, based on Response Evaluation Criteria in Solid Tumors (RECIST) we show that immune checkpoint molecules, including PD-L1, B7-H3, and VISTA, were differentially expressed. Patient responders possessed significantly higher tumor expression of PD-L1 and B7-H3, but lower expression of VISTA. Analysis of response subgroups indicated that tumor necrosis factor receptor (TNFR) superfamily members including OX40L, CD27, 4-1BB, CD40, and CD95/Fas, were associated with immunotherapy outcome. CD40 expression was higher in patient-responders than non responders, while CD95/Fas expression was lower in patients with partial response (PR) relative to those with stable disease (SD) and progressive disease (PD). Furthermore, we found that high 4-1BB expression in the tumor compartment, but not in the stroma, was associated with better overall survival (OS) (HR= 0.28, p-adjusted= 0.040). Moreover, high CD40 expression in tumor regions (HR= 0.27, p-adjusted= 0.035), and high CD27 expression in the stroma (HR= 0.2, p-adjusted=0.032) were associated with better survival outcomes. Taken together, this study supports the role of immune checkpoint molecules and implicates the TNFR superfamily as key players in immunotherapy response in our cohort of HNSCC. Validation of these findings in a prospective study is required to determine the robustness of these tissue signatures.

886 Stereotactic Radiosurgery Reprograms Macrophage, Microglia, and T Cell Populations in the Glioblastoma Immune Microenvironment

INTRODUCTION: Daily ionizing radiation, the first-line treatment for glioblastoma, prevents durable immune infiltration of the tumor microenvironment. Hypofractionated radiotherapy is used to treat glioblastomas at recurrence, but the impact of hypofractionated radiotherapy on the glioblastoma immune microenvironment is incompletely understood. METHODS: Syngeneic GL261 (3x105 cells/mouse) or SB28 glioblastoma cells (3 x 104 cells/mouse) were implanted into the frontal lobe of immunocompetent C57BL/6J mice. Bioluminescence was used to assess glioblastoma growth. After tumor engraftment, glioblastomas were treated with conformal SRS (18Gy/1Fx) or sham. Glioblastomas were collected for histologic, single-cell, or molecular analyses 5 days after treatment or at the time of euthanasia after monitoring for survival. Glioblastoma immune microenvironment responses were assessed using (1) H&E, (2) single cell mass cytometry (CyTOF) or IHC to define or validate immune cell changes, respectively, or (3) multiplexed cytokine assays to elucidate molecular mechanisms reprograming the glioblastoma immune microenvironment in response to SRS. RESULTS: SRS attenuated glioblastoma growth and prolonged survival compared to sham treatment in both immunocompetent intracranial mouse models (GL261: 14 days versus 27 days, p < 0.001, SB28: 19 days versus 22 days, p = 0.001). CyTOF showed SRS decreased immunosuppressive macrophage infiltration and increased microglia or CD8+ T cell infiltration of the glioblastoma immune microenvironment in GL261 tumors , however, there were very few changes in the lymphoid compartment of SB28 tumors . Histologic analyses validated T cell and microglia infiltration after SRS . Glioblastoma cytokine analysis revealed inhibition of pro-tumor/anti-inflammatory cytokines (IL6, LIF) after SRS in SB28 tumors . CONCLUSIONS: Single-fraction SRS reprograms glioblastoma macrophage, microglia, and CD8+ T cell populations in preclinical models, suggesting SRS or inhibition of pro-tumor/anti-inflammatory mechanisms underlying the immunosuppressive glioblastoma microenvironment represent immunogenic therapies that may offer a benefit to patients with glioblastoma.

Periostin in ovarian carcinoma: from heterogeneity to prognostic value.

Periostin (POSTN), an extracellular matrix protein, is involved in tumor-associated extracellular matrix (ECM) remodeling. However, its potential value as a prognostic and/or predictive factor has not yet been confirmed. The present study aims to assess POSTN expression separately in tumor cells and stroma of different ovarian carcinoma (OC) histological types, and its relationship with clinicopathological features. 102 cases of different histological OC subtypes were immunohistochemically investigated, for POSTN expression assessment in both epithelial tumor cells and tumor stroma. Statistical analysis was performed to correlate POSTN profile with clinicopathological characteristics, therapeutic response, and survival. POSTN expression in epithelial tumor cells was significantly correlated with POSTN expression in tumor stroma. The expression of POSTN in tumor cells was associated with histological type, tumor type (type I and II), tumor recurrence, progression-free survival (PFS), and overall survival (OS), whereas stromal POSTN expression was significantly correlated with age, histological type, tumor type, grade, and stage, residual disease, tumor recurrence, response to chemotherapy, and OS. Survival analysis revealed significant differences of PFS and OS in patients with high POSTN expression in tumor cells and negative stromal POSTN expression compared to patients with low POSTN expression in tumor cells and positive stromal POSTN expression (PFS: hazard ratio (HR) = 2.11, 95% confidence interval (CI): 1.33-3.37, P = 0.002; OS: HR = 1.78, 95% CI: 1.09-2.89, P = 0.019). The comparative assessment of POSTN immunoexpression in two tumor compartments: in tumor cells and stroma, by use of different scoring systems revealed that higher stromal POSTN levels are evidently correlated with unfavorable clinical features and poorer prognosis, while POSTN expression in tumor cells seems to be associated with a better patient outcome.

A transcriptomic pan-cancer signature for survival prognostication and prediction of immunotherapy response based on endothelial senescence

BackgroundThe microvascular endothelium inherently controls nutrient delivery, oxygen supply, and immune surveillance of malignant tumors, thus representing both biological prerequisite and therapeutic vulnerability in cancer. Recently, cellular senescence emerged as a fundamental characteristic of solid malignancies. In particular, tumor endothelial cells have been reported to acquire a senescence-associated secretory phenotype, which is characterized by a pro-inflammatory transcriptional program, eventually promoting tumor growth and formation of distant metastases. We therefore hypothesize that senescence of tumor endothelial cells (TEC) represents a promising target for survival prognostication and prediction of immunotherapy efficacy in precision oncology.MethodsPublished single-cell RNA sequencing datasets of different cancer entities were analyzed for cell-specific senescence, before generating a pan-cancer endothelial senescence-related transcriptomic signature termed EC.SENESCENCE.SIG. Utilizing this signature, machine learning algorithms were employed to construct survival prognostication and immunotherapy response prediction models. Machine learning-based feature selection algorithms were applied to select key genes as prognostic biomarkers.ResultsOur analyses in published transcriptomic datasets indicate that in a variety of cancers, endothelial cells exhibit the highest cellular senescence as compared to tumor cells or other cells in the vascular compartment of malignant tumors. Based on these findings, we developed a TEC-associated, senescence-related transcriptomic signature (EC.SENESCENCE.SIG) that positively correlates with pro-tumorigenic signaling, tumor-promoting dysbalance of immune cell responses, and impaired patient survival across multiple cancer entities. Combining clinical patient data with a risk score computed from EC.SENESCENCE.SIG, a nomogram model was constructed that enhanced the accuracy of clinical survival prognostication. Towards clinical application, we identified three genes as pan-cancer biomarkers for survival probability estimation. As therapeutic perspective, a machine learning model constructed on EC.SENESCENCE.SIG provided superior pan-cancer prediction for immunotherapy response than previously published transcriptomic models.ConclusionsWe here established a pan-cancer transcriptomic signature for survival prognostication and prediction of immunotherapy response based on endothelial senescence.

Proteomic analysis of small intestinal neuroendocrine tumors and mesenteric fibrosis.

Mesenteric metastases in small intestinal neuroendocrine tumors (SI-NETs) are associated with mesenteric fibrosis (MF) in a proportion of patients. MF can induce severe abdominal complications, and an effective preventive treatment is lacking. To elucidate possible novel therapeutic targets, we performed a proteomics-based analysis of MF. The tumor cell and stromal compartment of primary tumors and paired mesenteric metastases of SI-NET patients with MF (n = 6) and without MF (n = 6) was analyzed by liquid chromatography-mass spectrometry-based proteomics. Analysis of differential protein abundance was performed. Collagen alpha-1(XII) (COL12A1) and complement component C9 (C9) expression was evaluated by immunohistochemistry (IHC) in mesenteric metastases. A total of 2988 proteins were identified. Unsupervised hierarchical clustering showed close clustering of paired primary and mesenteric tumor cell samples. Comparing MF to non-MF samples, we detected differentially protein abundance solely in the mesenteric metastasis stroma group. There was no differential abundance of proteins in tumor cell samples or primary tumor stroma samples. Analysis of the differentially abundant proteins (n = 36) revealed higher abundance in MF samples of C9, various collagens and proteoglycans associated with profibrotic extracellular matrix dysregulation and signaling pathways. Proteins involved in fatty acid oxidation showed a lower abundance. COL12A1 and C9 were confirmed by IHC to have significantly higher expression in MF mesenteric metastases compared to non-MF. In conclusion, proteome profiles of SI-NETs with and without MF differ primarily in the stromal compartment of mesenteric metastases. Analysis of differentially abundant proteins revealed possible new signaling pathways involved in MF development. In conclusion, proteome profiles of SI-NETs with and without MF differ primarily in the stromal compartment of mesenteric metastases. Analysis of differentially abundant proteins revealed possible new signaling pathways involved in MF development.

Frequency and Prognostic Relevance of Volumetric MRI Changes in Contrast- and Non-Contrast-Enhancing Tumor Compartments between Surgery and Radiotherapy of IDHwt Glioblastoma

In newly diagnosed IDH-wildtype glioblastoma, the frequency and prognostic relevance of tumor regrowth between resection and the initiation of adjuvant radiochemotherapy are unclear. In this retrospective single-center study we included 64 consecutive cases, for whom magnetic resonance imaging (MRI) was available for both the volumetric assessment of the extent of resection immediately after surgery as well as the volumetric target delineation before the initiation of adjuvant radiochemotherapy (time interval: 15.5 ± 1.9 days). Overall, a median new contrast-enhancement volume was seen in 21/64 individuals (33%, 1.5 ± 1.5 cm3), and new non-contrast lesion volume in 18/64 patients (28%, 5.0 ± 2.3 cm3). A multidisciplinary in-depth review revealed that new contrast-enhancement was either due to (I) the progression of contrast-enhancing tumor remnants in 6/21 patients or (II) distant contrast-enhancing foci or breakdown of the blood-brain barrier in previously non-contrast-enhancing tumor remnants in 5/21 patients, whereas it was unspecific or due to ischemia in 10/21 patients. For non-contrast-enhancing lesions, three of eighteen had progression of non-contrast-enhancing tumor remnants and fifteen of eighteen had unspecific changes or changes due to ischemia. There was no significant association between findings consistent with tumor regrowth and a less favorable outcome (overall survival: 14 vs. 19 months; p = 0.423). These findings support the rationale that analysis of the postsurgical remaining tumor-volume for prognostic stratification should be carried out on immediate postoperative MRI (<72 h), as unspecific changes are common. However, tumor regrowth including distant foci may occur in a subset of IDH-wildtype glioblastoma patients diagnosed per WHO 2021 classification. Thus, MRI imaging prior to radiotherapy should be obtained to adjust radiotherapy planning accordingly.

Differential Spatial Gene and Protein Expression Associated with Recurrence Following Chemoradiation for Localized Anal Squamous Cell Cancer.

The identification of transcriptomic and protein biomarkers prognosticating recurrence risk after chemoradiation of localized squamous cell carcinoma of the anus (SCCA) has been limited by a lack of available fresh tissue at initial presentation. We analyzed archival FFPE SCCA specimens from pretreatment biopsies prior to chemoradiation for protein and RNA biomarkers from patients with localized SCCA who recurred (N = 23) and who did not recur (N = 25). Tumor cells and the tumor microenvironment (TME) were analyzed separately to identify biomarkers with significantly different expression between the recurrent and non-recurrent groups. Recurrent patients had higher mean protein expression of FoxP3, MAPK-activation markers (BRAF, p38-MAPK) and PI3K/Akt activation (phospho-Akt) within the tumor regions. The TME was characterized by the higher protein expression of immune checkpoint biomarkers such as PD-1, OX40L and LAG3. For patients with recurrent SCCA, the higher mean protein expression of fibronectin was observed in the tumor and TME compartments. No significant differences in RNA expression were observed. The higher baseline expression of immune checkpoint biomarkers, together with markers of MAPK and PI3K/Akt signaling, are associated with recurrence following chemoradiation for patients with localized SCCA. These data provide a rationale towards the application of immune-based therapeutic strategies to improve curative-intent outcomes beyond conventional therapies for patients with SCCA.

Personalized 90Y-resin microspheres dose determination: a retrospective study on the impact of dosimetry software on the treatment of patients with selective internal radiotherapy.

The calculation of resin yttrium-90 (90Y-) microspheres activity for selective internal radiotherapy (SIRT) needs to be investigated. Analyses using Simplicit90Y (Boston Scientific, Natick, Massachusetts, USA) dosimetry software were performed to determine the concordance between the absorbed doses to the tumor (DT1 and DT2) and healthy liver (DN1 and DN2) during the pre-treatment and the post-treatment phases. An optimized calculation of the activity of 90Y-microspheres performed using dosimetry software was applied retrospectively to assess the impact of this calculation method on the treatment. DT1 ranged from 38.8 to 372 Gy, with a mean value of 128.9 ± 73.6 Gy and median of 121.2 Gy [interquartile range (IQR): 81.7-158.8 Gy]. The median DN1 and DN2 was 10.5 Gy (IQR: 5.8-17.6). A significant correlation was between DT1 and DT2 (r = 0.88, P < 0.001) and DN1 and DN2 (r = 0.96, P < 0.001). The optimized activities were calculated; the target dose to the tumor compartment was 120 Gy. No activity reduction was applied in accordance with the tolerance of the healthy liver. Optimization of the microspheres dosages would have resulted in a significant increase in activity for nine treatments (0.21-2.54 GBq) and a reduction for seven others (0.25-0.76 GBq). The development of customized dosimetry software adapted to clinical practice makes it possible to use dosimetry to optimize the dosage for each patient.

Abstract PD4-01: PD4-01 Spatial transcriptomics reveals a substantial heterogeneity in TNBC tumor and stroma compartments with potential clinical implications

Abstract Background Triple negative breast cancer (TNBC) is a heterogeneous disease characterized by at least five molecular subtypes, namely basal like (BL), immunomodulatory (IM), luminal androgen receptor (LAR), mesenchymal (M) and mesenchymal stem like (MSL), associated with distinct gene expression, genomic and tumor microenvironment (TME) profiles. Recent technological advances allow to investigate intratumor geographic heterogeneity ignored by bulk tumor analyses. Here, we deployed spatial transcriptomics (ST) to interrogate tumor and stroma compartments heterogeneity and assess its association with clinical outcome. Methods Spatial transcriptomics (Visium® Spatial Gene Expression, 10X Genomics) was performed on a retrospective series of 94 case-control TNBC samples matched for known clinic-pathological parameters with available long term outcome. Detailed morphological annotations spanning 11 histomorphological categories were performed by a breast dedicated pathologist assisted by the automated QuPath digital pathology software. Bioinformatic analyses were performed using in house pipelines. Results We investigated the distribution of each morphological category across the five TNBC molecular subtypes. We found that LAR, M and MSL, even though they had less tumor cells, had more patches of very small size (p&amp;lt; 0.0001). On the other hand, BL and IM had few large and dense patches. Stroma had an opposite distribution compared to tumor, with LAR and MSL being enriched with stroma (p&amp;lt; 00001). Tumor infiltrating lymphocytes were specific of the IM (p&amp;lt; 0.0001) and to a lesser extent the BL subtype. Normal structures, like fat tissue (p&amp;lt; 0.008), lactiferous ducts (p&amp;lt; 0.03) and vessels (p&amp;lt; 0.02), were more present in MSL and LAR. The differences between the molecular subtypes are mirrored at the level of their cell composition and tumor organization, suggesting the possibility to assess TNBC molecular subtypes from imaging data alone. At the gene expression level, spatial deconvolution analyses revealed the co-existence of tumor and stroma compartments from different TNBC subtypes within a tumor sample of a given subtype as defined by bulk tumor analysis. Interestingly, these different tumor-stroma combinations were associated with prognosis. For example, M tumors associated with MSL stroma seem to have a better prognosis than M tumors with an M stroma (p=0.001). Furthermore, spatial resolution of the gene expression identified 418 individual clusters (median 4 clusters per sample) associated with specific molecular and cellular features highlighting a substantial intra-patient heterogeneity. These clusters were further grouped into 11 ecotypes associated with distinct hallmarks and pathways, including EMT, angiogenesis, DNA repair and immune profiles revealing an important inter-patient heterogeneity beyond TNBC classification. Interestingly, 2 ecotypes were identified within the IM subtype associated with distinct clinical outcome, with ecotype 6 characterized by high EMT, mesenchymal stroma and worse prognosis (p = 0,021). Conclusion To our knowledge, this is the largest study demonstrating the substantial intra- and inter-patient heterogeneity characterizing TNBC at an unprecedented level, with differences both in tumor and stroma composition as well as spatial organization and clinical outcome. Our results hightlight the need to consider TNBC heterogeneity for patient care and future clinical development including immunotherapy. Citation Format: Xiaoxiao Wang, David Venet, Frédéric Lifrange, Denis Larsimont, Mattia Rediti, Linnea Stenbeck, David Gacquer, Floriane Dupont, Ghizlane Rouas, Matteo Serra, Joakim Lundeberg, Françoise Rothé, Christos Sotiriou. PD4-01 Spatial transcriptomics reveals a substantial heterogeneity in TNBC tumor and stroma compartments with potential clinical implications [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 PD4-01.

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.

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 P2-23-05: Trastuzumab-Induced Tumor Microenvironment Changes in Early HER2+ Breast Cancer

Abstract Background Trastuzumab (H) is a critical component of therapy (tx) for HER2+ breast cancers (BCs) and can induce anti-tumor immune responses that as part of its mechanism of action. Pre-clinical studies have suggested that antibody-dependent cell-mediated cytotoxicity (ADCC) through NK cells is a major driver of the immune response. However, there is also evidence that macrophages and dendritic cells (DCs) can play an important role in H immune response through antibody-dependent cellular phagocytosis (ADCP). Here, we present pre- and post-tx gene expression and immunohistochemistry (IHC) data to characterize the tumor microenvironment (TME) changes in response to a single dose of H in patients (pts) with early HER2+ BC. Methods Pts with Stage I-III HER2+ BC with minimum 1 cm tumors were eligible. Pts received 8 mg/kg IV H followed by tissue collection. There was a minimum of 14 days between (btw) H and post-tx tissue collection. Pre- and post-tx tissues were assessed for tumor-infiltrating lymphocytes (TILs), immune-related gene expression, and AE1/3, CD31, CD4, CD8, CD11b, CD68, and CD56 expression through IHC. Results Thirteen pts were enrolled and evaluable. 5/13 were estrogen-receptor positive (ER+). TILs were assessed independently by two pathologists and the results averaged. Tx with H resulted in an increase in TILs in the post-tx tissue (p &amp;lt; 0.01). Pre-tx TILs ranged from 2-80% (median 6%) and post-tx TILs ranged from 2-80% (median 20%). 6/13 pts had immune infiltration (Inf), defined as a TIL increase of greater than 1 decile btw pre- and post-tx samples. 2/6 pts with Inf were ER+. Pts with Inf had a significantly higher DC immune signature (ISS) compared to pts without Inf (p &amp;lt; 0.01, univariate analysis). No other ISS reached significance. 6 pts had paired pre- and post-tx samples evaluated by IHC. 4/6 pts had Inf per prior definition. Pts with Inf had an increase in CD11b+ (includes macrophages and some DCs) and CD56+ cells (includes NK cells) after H tx within the tumor compartment (p &amp;lt; 0.05). Pts with Inf had an increase in CD11b+, CD56+, and CD44+ cells in the stromal compartment (p &amp;lt; 0.05). Conclusions Innate immune cells such as macrophages and DCs are key players in H-induced immune response in the early HER2+ BC TME. This data is consistent with prior pre-clinical findings. Citation Format: Laura C. Kennedy, Rebeca Alvarez, Suzanne Dintzis, Vijayakrishna K. Gadi. Trastuzumab-Induced Tumor Microenvironment Changes in Early HER2+ 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 P2-23-05.

Abstract P2-21-07: Exploring spatial correlations in Breast invasive Lobular Carcinoma subtypes using a novel CAF multiplex immunofluorescence panel

Abstract Background: Recent advances in immunotherapy led to development of new technologies to identify topographical distributions and correlations in the tumor microenvironment, enabling a better understanding of the tumor immune landscape. Based on the 2019 WHO classification Invasive lobular carcinoma (ILC) has various histological presentations. The pleomorphic variant has been reported to be biologically different to the classical counterpart. Cancer associated fibroblasts (CAFs) secrete cytokines and growth factors aiding in tumor growth. CAFs have been shown to express alpha-smooth muscle actin (α-SMA), Thy1, fibroblast activation protein (FAP), S-100. IHC based studies have shown differences in CAFs subpopulation between ILC and IDC. Here, we explored different CAFs subpopulations between Pleomorphic and Classical ILC using multiplex immunofluorescence (mIF). Study design: Multiplex immunofluorescence (mIF) was performed on formalin-fixed paraffin-embedded (FFPE) tissue sections [(n=6, classic ILC; n=6 pleomorphic ILC)] stained with Opal 7-color Kit using an automated system. Sections were stained consecutively with Pancytokeratin, CD45, A-SMA, FAP, S100, Thy-1. Slides were scanned using the Vectra 3.0 spectral imaging system (PerkinElmer). Five tumor ROIs were examined with Phenochart (Akoya/PerkinElmer) viewer and subsequent images were analyzed to explore spatial distances of CK+ cells to the CAFs. Data was generated using InForm and Phenoptr softwares (Akoya Labs) as a median value. Statistical analysis data correlation was performed using SPSS version 24.0 (IBM Corp) Results: Twelve invasive lobular carcinomas cases were evaluated. All cases with classical features were nuclear grade 2 and all pleomorphic cases (n=6) were nuclear grade 3. Six were Luminal B, five were Luminal A and one case was triple negative invasive lobular carcinoma. We studied nineteen different phenotypes of CAFs in the tumor, stroma, and overall tissue compartments. We found statistically significant differences (p-value&amp;lt; 0.05) between the distances of CAFs from the tumor cells in classic and pleomorphic ILC in three phenotypes. The A-SMA+, A-SMA+/S100+ phenotypes were closer to the tumor cells in classic subtype and the S100 only phenotype was closer to the tumor cells in pleomorphic carcinomas. In addition, there were two other phenotypes namely A-SMA+/Thy-1+(closer to tumor cells in classic ILC) and FAP+/S-100+ (closer to tumor cells in pleomorphic ILC), which showed a trend of significance (Table 1) Discussion: TME studies in invasive breast lobular carcinoma have been primarily based on chromogenic IHC. Multiplex immunofluorescence quantifies cell phenotype’s densities and positions in different tissue compartments (tumor vs stroma vs total tissue). Knowing the proximity of an individual TME cells to the tumor cells, aids in pinpointing possible therapeutic targets. Fibroblast dysregulation in cancers, enhances their pro-tumorigenic and anti-tumorigenic potential. Due to their heterogeneity, they can express a wide array of markers. Identifying the full possibility of CAF subsets is one of the keys to discovering actionable targets. Our results showed Alpha- SMA positive, Alpha-SMA/S-100 positive, Alpha SMA/Thy-1 positive CAFs in closer proximity to the classic ILC tumor cells as compared to pleomorphic ILC. Additionally, S-100 positive and FAP/S-100 positive CAFs showed a closer proximity to tumor cells in the pleomorphic subtype. Based on these results we hypothesize that pleomorphic ILCs are closely associated with pro-tumorigenic CAFs as compared to classic ILCs. Larger datasets are needed to confirm this. In conclusion we utilized an objective approach to quantify, phenotype and spatially correlate each cell in the tumor microenvironment. This has helped identifying CAFs subsets that differs in the spatial correlation to tumor cells in ILC subtypes, which can be potential actionable targets. Table 1 Phenotypes of Cancer associated fibroblasts and their statistical correlation between classic vs pleomorphic invasive Lobular carcinomas. Citation Format: Harsh Batra, Renganayaki K. Pandurengan, Heladio P. Ibarguen, Salome A McAllen, Qingqing Ding, Aysegul Sahin, Ignacio Wistuba, Edwin Roger Parra, Maria Gabriela Raso. Exploring spatial correlations in Breast invasive Lobular Carcinoma subtypes using a novel CAF multiplex immunofluorescence panel [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 P2-21-07.

Abstract P5-02-11: Immune cell profile of tumors from patients with metastatic (met) HER2+ breast cancer (BC) with &amp;lt; 30 months overall survival (OS)

Abstract Background: The “Thousand Patient HER-2 database” project at Saint Vincent’s University Hospital (SVUH) Dublin has been used to identify HER2+ BC patients with durable complete response (never relapsed) to trastuzumab-based therapy. ~10% of met HER2+ BC patients achieve a durable complete response to trastuzumab, meaning the majority of patients progress on treatment. Higher stromal tumour immune infiltrate has been associated with longer OS in met HER2+ BC. There is limited tumor immune profile and PD-1 expression data available for patients with met HER2+ BC with short OS. Using the SVUH database, we have identified a preliminary cohort of 21 met HER2+ BC patients that received trastuzumab and had an OS &amp;lt; 30 months. This study examines the levels of pan T cell marker CD3, cytotoxic T cell marker CD8, Natural Killer (NK) cell marker CD56 and immune checkpoint PD-1 by immunohistochemistry (IHC) in this preliminary cohort. Methods: Formalin-fixed, paraffin-embedded (FFPE) biopsy specimens (n=21 primary, n=7 matched metastatic biopsies) and associated clinico-pathological data were curated. Tumor biopsies were processed for IHC staining of CD8 (Agilent IR62361-2), CD3 (Agilent IR50361-2), CD56 (Agilent IR62861-2) and PD-1 (Roche 07099029001). PD-1 staining was available for 20/21 samples. Staining was performed using the DAKO Link 48 Autostainer as per the manufacturer’s instructions using positive (tonsil tissue) and negative controls (isotype controls). Slides were processed using the Aperio AT2 Digital Slide Scanner (Leica Biosystems), reviewed using Aperio ImageScope 12.4 software (Leica Biosystems) and analyzed in QuPath (University of Edinburgh). Images were annotated to outline tumor areas and an algorithm was trained to identify cells and classify them as either tumor or stromal. Data was expressed as number of positively stained cells/mm2 breast tumor or stromal tissue. Survival studies utilized the Kaplan Meier method. The paired Student’s T test was utilized for primary vs metastatic site comparisons. Results: Designating samples with &amp;gt; 1 stained cell/mm2 breast tumor as positive (pos) and zero stained cells as negative (neg), 19/21 (90.5%) primary samples were pos for CD3, 15/21 (71.4%) for CD56, 14/21 (66.6%) for CD8, and 10/20 (50%) for PD-1. Within the stromal compartment, 20/21 (95.2%) primary samples were pos for CD8, 18/21 (85.7%) for CD56, 16/21 (76.2%) for CD3 and 8/20 (40%) for PD-1. PD-1 expression in the primary tumor (median OS PD-1pos 7.85 mo vs PD-1neg 5.39 mo, hazard ratio (HR) 0.642 (95% CI 0.256-1.613), p=0.346) or the stroma (median OS PD-1pos 8.84 mo vs PD-1neg 5.39 mo, HR 0.495 (95% CI 0.197-1.244), p=0.135) was not significantly associated with OS. When comparing matched primary and metastatic samples (n=7), increased stromal levels of CD3 (4/7), CD8 (4/7), CD56 (5/7) and PD-1 (4/7) were observed. Increased levels of CD3 and CD8 were observed for 2/7 samples, and increased levels of CD56 and PD-1 for 4/7 samples. With the exception of tumor CD8 levels which decreased, mean values for tumor and stromal CD3, CD56, PD-1 and stromal CD8 levels were higher in metastatic sites but all differences were not found to be significant (p&amp;gt;0.05). Conclusions: Our results suggest that met HER2+ BC patients with &amp;lt; 30 months OS have significant T cell and NK cell presence in the tumor and stromal compartments in both primary and metastatic sites. Further expansion of this limited dataset is planned to gain greater insight in to the immune cell profiles and PD-1 status of met HER2+ BC patients with short OS. Citation Format: Denis M. Collins, Janet McCormack, Laura P. Ivers, Jose Javier Berenguer Pina, Jo Ballot, Cecily Quinn, Darko Skrobo, Alex J. Eustace, Naomi Walsh, Aurelie Fabre, John Crown. Immune cell profile of tumors from patients with metastatic (met) HER2+ breast cancer (BC) with &amp;lt; 30 months overall survival (OS). [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 P5-02-11.