Changes In Tumor Microenvironment Research Articles

Abstract C029: Window-of-opportunity trial of metastatic pancreatic cancer reveals potential mechanisms of response to targeting RAS/MEK signaling

Abstract KRAS is mutated in ∼85% of pancreatic ductal adenocarcinoma (PDAC) patient's tumors, suggesting that it is a key node of PDAC targeted therapy. RAF/MEK/ERK signaling is downstream of KRAS signaling. Preclinical studies targeting MEK showed promises but clinical trials failed to show the benefit compared to Standard-of-Care treatment chemotherapy. To elucidate the mechanism of action and resistance to several targeted therapies including MEK inhibition in human PDAC, we conducted a Window-Of-Opportunity trial for Metastatic pancreatic cancer (WOOM trial). In this trial, metastatic PDAC patients received 10 days of Cobimetinib, an FDA approved MEK inhibitor, therapy and specimens of pre- and post- treatment were obtained by biopsy to evaluate the response to targeted treatment using deep multi-omic analytics including DNA-seq, RNA-seq, multiplex IHC, cyclic IF and Digital Spatial Profiling (DSP). Four of 13 patient’s tumors were defined as Responsive tumors by using CA19- 9 and KI67 as a readout. Genomic sequence showed that KRAS amplifications were frequently seen in non-responsive tumors and KrasG12R mutations were seen with higher frequency in Responsive tumors. RNA-seq analysis showed that these Responsive tumors had lower KRAS activity and were more likely to be classical-like subtype compared to non-responsive tumors. DSP analysis confirmed the downregulation of ERK phosphorylation with paradoxical phospho- MEK activation after treatment in all examined cases which is consistent with effective target inhibition. Phospho-MYC protein expression and MYC transcriptomic activity were significantly decreased in responsive tumors, which suggest MYC downregulation could contribute to an active response to MEK inhibition. Single cell RNA-seq and spacial analysis showed tumor microenvironment changes after Cobimetinib treatment including the increase of CD8 T cell infiltration in responsive tumors. Furthermore, PDX from patient’s biopsy recapitulated patient's response to Cobimetinib and faithfully metastasized to the liver. Interestingly, Cobimetinib resistant PDX was also resistant to KRAS inhibitor. This model could be a great tool for testing new therapeutic strategies targeting the KRAS/MEK pathway. Here, we summarize the multi- omics data and discuss potential mechanisms of response and resistance to targeting KRAS signaling and related microenvironmental changes. Citation Format: Motoyuki Tsuda, Colin Daniel, Xiaoyan Wang, Carl Peltz, Hayley Zimmny, Alexander Smith, John Muschler, Xi Li, Tugba Yildiran Ozmen, Furkan Ozmen, Dove Keith, Christopher Corless, Koei Chin, Jonathan Brody, Charles Lopez, Gordon Mills, Rosalie Sears. Window-of-opportunity trial of metastatic pancreatic cancer reveals potential mechanisms of response to targeting RAS/MEK signaling [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C029.

Single-cell RNA sequencing reveals the change in cytotoxic NK/T cells, epithelial cells and myeloid cells of the tumor microenvironment of high-grade serous ovarian carcinoma

BackgroundThe heterogeneity of high-grade serous ovarian carcinoma (HGSOC) has hindered the clinical treatment, and our current study aims to characterize the change in tumor microenvironment (TME) with the progression of HGSOC via single cell RNA sequencing (scRNA-seq).MethodsThe single-cell landscape in HGSOC was downloaded from the dataset GSE184880, which included 7 HGSOC and 5 normal samples and then applied for the filtering and annotation of cell clusters. The differentially expressed marker genes in these clusters were analyzed via “FindAllMarker” function in Seurat package and the functional enrichment analyses were implemented using clusterProflier package. Finally, the CellChat package was applied for the cell–cell communication analysis. Cellular experimental were determined Real-time Reverse Transcription Polymerase Chain Reaction (RT-qPCR).Results45,448 single cells were categorized into 10 cell clusters. The proportion of NK/T cells (49.5%), epithelial cells (15.3%) and myeloid cells (14%) was higher in the HGSOC samples. The heterogeneity and different enriched pathways of epithelial cells have been revealed with the progression of HGSOC from early to late stage, concurrent with the reduced activity of cytotoxic NK/T cells and the decreased capabilities of recruiting immune cells and presenting antigens in macrophages. Besides, the cell–cell communication analysis has revealed a strong communication of CXCL and CCL signal between M1 macrophages and cytotoxic NK/T cells in early stage of HGSOC. Moreover, RT-qPCR indicated that CCL4/5 and CCR1/5 levels were upregulated in tumor cell SK-OV-3.ConclusionThe investigation using scRNA-seq has depicted the change in cytotoxic NK/T cells, epithelial cells and myeloid cells of the TME of HGSOC, which may provide another insight into the specific mechanisms underlying the progression of HGSOC.

Deciphering single-cell transcriptomic landscape of tumors in colorectal cancer treated with photothermal semiconducting polymers to design the combination therapy with checkpoint blockades for inhibition of tumor progression and metastasis

Immunotherapy is often less effective for tumors with “immune-excluded,” or “immune-desert” microenvironment. To circumvent this hurdle, new therapeutic strategies need to be designed to reprogram the tumor microenvironment (TME). It is well known that photothermal therapy (PTT) can prime the TME and provoke the systemic anti-tumor immunity. But few studies perform extensive exploration in depth to decipher the changes in TME after PTT with cutting-edge technology besides conventional flow cytometry analysis. In this study, we designed a nanosystem consisting of organic semiconductor polymer liposome nanoparticles (Y8 NPs) as a multimodal imaging and photothermal agent to image and treat tumors. Upon laser irradiation, Y8 NPs in tumors could rapidly increase the temperature, induce massive cancer cell apoptosis and also promote the immunogenic cell death (ICD), ultimately remodulating the TME. We employed single cell RNA sequencing (scRNA-seq) technology to comprehensively explore transcriptomic profiling of individual cells constituting TME, demonstrating heterogenous alterations in the subtypes of immune cells, even tumor cells. Considering the findings by scRNA-seq and TME remodulation, the combinational strategy with checkpoint blockade PD-1 antibody was designed to treat the tumors, demonstrating that the combination not only impedes primary tumor progression but also produce profound abscopal anti-tumor effect. In conclusion, we provide a simple strategy for the remodulation of TME with a photothermal nanosystem to achieve activation of anti-tumor immunity, and suppress primary and metastatic tumor progression.

Hypoxia-Driven Changes in Tumor Microenvironment: Insights into Exosome-Mediated Cell Interactions.

Hypoxia, as a prominent feature of the tumor microenvironment, has a profound impact on the multicomponent changes within this environment. Under hypoxic conditions, the malignant phenotype of tumor cells, the variety of cell types within the tumor microenvironment, as well as intercellular communication and material exchange, undergo complex alterations. These changes provide significant prospects for exploring the mechanisms of tumor development under different microenvironmental conditions and for devising therapeutic strategies. Exosomes secreted by tumor cells and stromal cells are integral components of the tumor microenvironment, serving as crucial mediators of intercellular communication and material exchange, and have consequently garnered increasing attention from researchers. This review focuses on the mechanisms by which hypoxic conditions promote the release of exosomes by tumor cells and alter their encapsulated contents. It also examines the effects of exosomes derived from tumor cells, immune cells, and other cell types under hypoxic conditions on the tumor microenvironment. Additionally, we summarize current research progress on the potential clinical applications of exosomes under hypoxic conditions and propose future research directions in this field.

Characterizing the Linkage of Systemic Hypoxia and Angiogenesis in High-Grade Glioma to Define the Changes in Tumor Microenvironment for Predicting Prognosis.

High-grade gliomas (HGG) comprising WHO grades 3 and 4 have a poor overall survival (OS) that has not improved in the past decade. Herein, markers representing four components of the tumor microenvironment (TME) were identified to define their linked expression in TME and predict the prognosis in HGG, namely, interleukin6 (IL6, inflammation), inducible nitric oxide synthase(iNOS), heat shock protein-70 (HSP70, hypoxia), vascular endothelial growth receptor (VEGF), and endothelin1 (ET1) (angiogenesis) and matrix metalloprotease-14 (MMP14) and intercellular adhesion molecule1 (ICAM1, extracellular matrix). To establish a non-invasive panel of biomarkers for precise prognostication in HGG. Eighty-six therapy-naive HGG patients with 45 controls were analyzed for the defined panel. Systemic expression of extracellular/secretory biomarkers was screened dot-immune assay (DIA), quantified by ELISA, and validated by immunocytochemistry (ICC). Expression of iNOS, HSP70, IL-6, VEGF, ET1, MMP14, and ICAM1 was found to be positively associated with grade. Quantification of circulating levels of the markers by ELISA and ICC presented a similar result. The biomarkers were observed to negatively correlate with OS (p < 0.0001). Cox-regression analysis yielded all biomarkers as good prognostic indicators and independent of confounders. On applying combination statistics, the biomarker panel achieved higher sensitivity than single markers to define survival. The intra-association of all seven biomarkers was significant, hinting of a cross-talk between the TME components and a hypoxia driven systemic inflammation upregulating the expression of other components. This is a first ever experimental study of a marker panel that can distinguish between histopathological grades and also delineate differential survival using liquid biopsy, suggesting that markers of hypoxia can be a cornerstone for personalized therapy. The panel of biomarkers of iNOS, HSP70, IL-6, VEGF, ET1, MMP14, and ICAM1 holds promise for prognostication in HGG.

BAG6 restricts pancreatic cancer progression by suppressing the release of IL33-presenting extracellular vesicles and the activation of mast cells

Recent studies reveal a critical role of tumor cell-released extracellular vesicles (EVs) in pancreatic cancer (PC) progression. However, driver genes that direct EV function, the EV-recipient cells, and their cellular response to EV uptake remain to be identified. Therefore, we studied the role of Bcl-2-associated-anthanogene 6 (BAG6), a regulator of EV biogenesis for cancer progression. We used a Cre recombinase/LoxP-based reporter system in combination with single-cell RNA sequencing to monitor in vivo EV uptake and tumor microenvironment (TME) changes in mouse models for pancreatic ductal adenocarcinoma (PDAC) in a Bag6 pro- or deficient background. In vivo data were validated using mouse and human organoids and patient samples. Our data demonstrated that Bag6-deficient subcutaneous and orthotopic PDAC tumors accelerated tumor growth dependent on EV release. Mechanistically, this was attributed to mast cell (MC) activation via EV-associated IL33. Activated MCs promoted tumor cell proliferation and altered the composition of the TME affecting fibroblast polarization and immune cell infiltration. Tumor cell proliferation and fibroblast polarization were mediated via the MC secretome containing high levels of PDGF and CD73. Patients with high BAG6 gene expression and high protein plasma level have a longer overall survival indicating clinical relevance. The current study revealed a so far unknown tumor-suppressing activity of BAG6 in PDAC. Bag6-deficiency allowed the release of EV-associated IL33 which modulate the TME via MC activation promoting aggressive tumor growth. MC depletion using imatinib diminished tumor growth providing a scientific rationale to consider imatinib for patients stratified with low BAG6 expression and high MC infiltration.EVs derived from BAG6-deficient pancreatic cancer cells induce MC activation via IL33/Il1rl1. The secretome of activated MCs induces tumor proliferation and changes in the TME, particularly shifting fibroblasts into an inflammatory cancer-associated fibroblast (iCAF) phenotype. Blocking EVs or depleting MCs restricts tumor growth.

Change in tumor microenvironment and PD-L1 expression with rucaparib treatment in triple-negative breast cancer.

e12569 Background: Triple-Negative Breast Cancers (TNBCs) represent a diverse subset of tumors characterized by varied responses to therapies including immunotherapy (IO). While IO is approved in early-stage and advanced TNBC, responses are modest. Modulation of the tumor microenvironment to improve responses to IO is an active area of research. In-vitro and in-vivo studies have shown Poly-ADP Ribose Polymerase inhibitors (PARPi) to increase PD-L1 expression in sporadic, BRCA1 and BRCA2 mutated TNBC cell lines improving response to IO. In addition, TNBCs have diverse mechanisms leading to Homologous Recombination Deficiency and the role of PARPi in sporadic TNBCs is still unclear. Methods: To further investigate the role of PARPi in tumor immuno-modulation we conducted a single-arm, open-label, window of opportunity (WOO) trial in patients with early-stage TNBC (NCT03911453). This study was approved by our institutional review board. Patients with histologically documented stage I-III TNBC were eligible for definitive surgery were enrolled. Patients were treated with 600mg BID rucaparib for 21 days. Pre- and post-treatment tumor biopsies were collected. Post-treatment, patients went to surgery or proceeded with neoadjuvant chemotherapy (NAC) followed by definitive surgery. Our primary endpoint was to assess change in PD-L1 expression. In addition, change in tumor microenvironment was by evaluating change in percentage of B cells, T cells and macrophages was calculated. Results: Twenty patients were enrolled. Nineteen patients completed 21 days and 1 patient went on extended duration PARPi during the COVID19 pandemic. Almost 50% of patients went to surgery while the rest went onto NAC after PARPi therapy. Pre-treatment tumor samples were obtained from the diagnostic core biopsies while post-treatment, fresh frozen and Formalin Fixed Paraffin Embedded were obtained either at time of surgery or a research biopsy prior to NAC initiation. Primary biomarkers data was successfully done in 14 paired samples. Treatment with rucaparib has increased expression of PD-L1 from median of 15% in pre-treatment tumor samples to a median of 26% in post-treatment tumor samples. Table below summarizes our results. Conclusions: Our study revealed that treatment with PARPi, rucaparib, changed the tumor microenvironment as noted by changes in expression of PD-L1, and immune milieu in and around the tumor. This could lead to change in responsiveness to therapies including CKI. This strategy should be further investigated to evaluate improving responses to immunotherapies. Clinical trial information: NCT03911453 . [Table: see text]

The efficacy of recombinant human adenovirus type 5 (H101) intra-tumor therapy in persistent/recurrent/metastatic gynecological cancer and the exploration of changes in tumor microenvironment.

The efficacy of recombinant human adenovirus type 5 (H101) intra-tumor therapy in persistent/recurrent/metastatic gynecological cancer and the exploration of changes in tumor microenvironment.

Feasibility and safety of neoadjuvant nivolumab and chemotherapy for resectable diffuse pleural mesotheliomas: Results of a prospective pilot study.

8003 Background: Diffuse pleural mesotheliomas (DPM) are an aggressive malignancy where even patients with potentially resectable disease have poor outcomes. With an evolving understanding of the role of pleurectomy/decortication (P/D) as the standard of care for resectable disease, the need to improve upfront therapies is more urgent. Here we report the results of our single institution prospective pilot study of nivolumab with platinum (cisplatin/carboplatin) and pemetrexed prior to P/D for patients with resectable DPM (NCT04162015). Methods: Patients deemed operable and resectable by a multidisciplinary team received 2 cycles of neoadjuvant nivolumab, pemetrexed, and platinum followed by P/D. Subsequent therapy was at the physician's discretion. Using a minimax Simon two-stage design, the primary objective was defined as attempted P/D within 30 days of the planned surgery date. Secondary objectives included evaluating safety, overall survival (OS) from diagnosis, and progression free survival (PFS). Tumor tissue and plasma were collected for exploratory analyses such as immune cell profiling and pathologic response. Using single cell RNA sequencing (scRNAseq), we explored DPM transcriptional heterogeneity and treatment-associated tumor microenvironment (TME) changes. Results: 22 patients received neoadjuvant chemo-immunotherapy and 19 underwent attempted P/D (2 progressed and 1 declined surgery). 68% were men (13/19), 89% were epithelioid (17/19; remaining 2 were biphasic), and the median age at diagnosis was 72 (range: 33-80). No patient experienced pre-operative toxicity precluding P/D. 95% (18/19) of patients underwent P/D within 30 days of the planned date; 1 patient had biopsy-proven tumor flare after cycle 2 and had surgery at day +37. As of January 2024, median OS for the 19 patients that went for attempted P/D was 41.0 mos (median follow up: 25.3mos). OS for those who underwent R1 resections (n = 12) was not reached and for R2 (n = 7) was 31.4 mos (p = 0.67; HR 1.43, 95% CI 0.28-7.30). 26% of specimens (5/19) had ≤ 10% viable tumor cells on the resection; there was no clear correlation with outcomes. scRNAseq was performed on 15 resection specimens with available material. Comparing patients with PFS greater than or less than 12mos (PFS12), we found significantly increased abundance of CD8 cytotoxic T cells, and ISG+ (interferon stimulated) T cells. Conclusions: Neoadjuvant platinum, pemetrexed, and nivolumab was reliably able to be given to patients with DPM without impacting their ability to undergo P/D. The encouraging OS of this cohort to date, and tolerance of the regimen, highlights the importance of patient selection and preoperative treatments when considering P/D. Ongoing exploratory analyses will profile the cellular and transcriptional landscape of DPM and identify correlates of response to neoadjuvant chemo-immunotherapy. Clinical trial information: NCT04162015 .

FAT1 as a tumor mutation burden specific gene affects the immunotherapy effect in head and neck squamous cell cancer

BackgroundResponse to immunotherapy is the main challenge of head and neck squamous cancer (HNSCC) treatment. Previous studies have indicated that tumor mutational burden (TMB) is associated with prognosis, but it is not always a precise index. Hence, investigating specific genetic mutations and tumor microenvironment (TME) changes in TMB-high patients is essential for precision therapy of HNSCC. MethodsA total of 33 HNSCC patients were enrolled in this study. We calculated the TMB score based on next-generation sequencing (NGS) sequencing and grouped these patients based on TMB score. Then, we examined the immune microenvironment of HNSCC using assessments of the bulk transcriptome and the single-cell RNA sequence (scRNA-seq) focusing on the molecular nature of TMB and mutations in HNSCC from our cohort. The association of the mutation pattern and TMB was analyzed in The Cancer Genome Atlas (TCGA) and validated by our cohort. Results33 HNSCC patients were divided into three groups (TMB-low, -medium, and -high) based on TMB score. In the result of 520-gene panel sequencing data, we found that FAT1 and LRP1B mutations were highly prevalent in TMB-high patients. FAT1 mutations are associated with resistance to immunotherapy in HNSCC patients. This involves many metabolism-related pathways like RERE, AIRE, HOMER1, etc. In the scRNA-seq data, regulatory T cells (Tregs), monocytes, and DCs were found mainly enriched in TMB-high samples. ConclusionOur analysis unraveled the FAT1 gene as an assistant predictor when we use TMB as a biomarker of drug resistance in HNSCC. Tregs, monocytes, and dendritic cells (DCs) were found mainly enriched in TMB-high samples.

Elucidating immunological characteristics of the adenoma-carcinoma sequence in colorectal cancer patients in South Korea using a bioinformatics approach

Colorectal cancer (CRC) is one of the top five most common and life-threatening malignancies worldwide. Most CRC develops from advanced colorectal adenoma (ACA), a precancerous stage, through the adenoma-carcinoma sequence. However, its underlying mechanisms, including how the tumor microenvironment changes, remain elusive. Therefore, we conducted an integrative analysis comparing RNA-seq data collected from 40 ACA patients who visited Dongguk University Ilsan Hospital with normal adjacent colons and tumor samples from 18 CRC patients collected from a public database. Differential expression analysis identified 21 and 79 sequentially up- or down-regulated genes across the continuum, respectively. The functional centrality of the continuum genes was assessed through network analysis, identifying 11 up- and 13 down-regulated hub-genes. Subsequently, we validated the prognostic effects of hub-genes using the Kaplan–Meier survival analysis. To estimate the immunological transition of the adenoma-carcinoma sequence, single-cell deconvolution and immune repertoire analyses were conducted. Significant composition changes for innate immunity cells and decreased plasma B-cells with immunoglobulin diversity were observed, along with distinctive immunoglobulin recombination patterns. Taken together, we believe our findings suggest underlying transcriptional and immunological changes during the adenoma-carcinoma sequence, contributing to the further development of pre-diagnostic markers for CRC.

Abstract PO3-07-01: Multiscale Computational Analysis of Patient-Matched Multi-Modal Imaging of Breast Tissue Microenvironment

Abstract Introduction: Mammographic breast density is a strong quantitative cancer risk factor, but its link to underlying tissue and cellular level changes are weakly understood. Understanding the complex restructuring changes in the breast tumor microenvironment and its impact on breast density is integral to improving risk assessment and treatment strategies. A novel approach was developed to stratify mammographic dense breast tissue into areas of active dense tissue and passive dense tissue. Active dense tissue is structurally reorganizing and links to cancer dynamics, whereas passive dense mammographic tissue remains organized.1,2 A complementary wavelet-based analysis technique to measure the multiscale anisotropy of collagen fibers from Second-Harmonic Generation (SHG) imaging demonstrated clinical cancer diagnostic potential in mouse carcinoma and human pancreatic cancer.3,4 This tool is now leveraged for a multi-modal study in which patient-matched H&amp;E tissue biopsies are imaged using both bright-field microscopy and SHG imaging to compare with the patient’s corresponding mammograms. Stratifying the mammographic density into active and passive dense areas, a correlation between biopsy tissue anisotropy and mammographic density subtypes can be calculated to help understand how tumor microenvironment changes affect a patient’s mammographic breast tissue composition. Methods: SHG and brightfield-imaged biopsy slides from 10 patients (five malignant, five benign) collected from Maine Medical Center (Maine, US) were analyzed using the 2D Wavelet Transform Modulus Maxima (WTMM) Anisotropy Method generating a large-scale and a small-scale anisotropy factor as well as a scale-combined anisotropy calculated from their large- and small-scale anisotropy separation. A multi-modal anisotropy score was calculated by adding the imaged area’s H&amp;E combined anisotropy score with its respective SHG combined anisotropy score. This analysis was done on eight randomly selected ductal areas for each imaging modality on a slide and correlated with the measured areas of density subtypes from the whole mammographic view. Linear regression was used for determining correlation between H&amp;E and SHG anisotropy factors as well as with active and passive mammographic density subtype measurements. Results: The malignant patients’ small-scale H&amp;E anisotropy factor positively correlated with their small scale SHG anisotropy factor (R2=0.571). No correlation was found for the benign patients (R2=0.011). The strongest correlations were found between benign patients’ mammographic passive (positive correlation) and active (negative correlation) dense tissues vs the H&amp;E anisotropy scale-combined factor (R2=0.858 and R2=0.961, respectively). The largest malignant patient correlation was a negative relationship between SHG anisotropy scale-combined factor and overall mammographic density (inclusive of both active and passive dense tissue) (R2=0.891). The multi-modal anisotropy score correlated with overall mammographic density for both benign (positive correlation, R2=0.698) and malignant patients (negative correlation, R2=0.773). Combining the multi-modal anisotropy factor from SHG and H&amp;E with the active dense tissue measurement using K-Means clustering, 5/5 malignant and 4/5 benign patients were correctly classified. Conclusion: Density in mammograms correlates with underlying quantitative changes in a patient’s tissue and collagen anisotropy. Combining both H&amp;E and SHG analysis on tissue biopsy with active and passive density metrics from computational mammography leads to the best predictive potential than any one method on their own.

Abstract PO1-01-09: Neoadjuvant endocrine therapy and avelumab with or without palbociclib in stage II/III endocrine receptor-positive breast cancer: the ImmunoADAPT trial

Abstract Background: Pre-clinical and clinical data suggest that CDK4/6 inhibitors can promote an immunogenic tumor microenvironment (TME) and when combined with programmed cell death ligand 1 (PD-L1) inhibitors synergistic anti-cancer effects are observed. To understand the differential effects of CDK4/6 inhibition in the context of immunotherapy we designed a study evaluating neoadjuvant endocrine therapy (ET) with a PD-L1 inhibitor (avelumab) with or without a CDK4/6 inhibitor (palbociclib, palbo) in patients (pts) with stage II/III endocrine receptor (ER)-positive breast cancer. Methods: In this randomized phase 2 pilot study, pts with stage II/III ER+, HER2-negative breast cancer were randomized 2:1 to ET (aromatase inhibitor, AI, for post-menopausal pts; tamoxifen+/-ovarian function suppression for pre-menopausal pts) with avelumab, with or without palbo (palbo vs. control arms). Avelumab was added after a 1-month lead-in of ET +/- palbo, and all drugs were continued for additional 3-month-long cycles prior to surgery. We obtained tumor tissue and breast MRIs on C1D1, C2D1, and at the end of treatment. Imaging assessment was done according to RECIST 1.1 criteria at each time point. The primary endpoint was clinical complete response (cCR) by MRI in the palbo arm (H0: cCR=10% vs. 40% at a two-sided alpha level of 0.1). Secondary endpoints included pathologic CR (pCR), overall response rates (ORR), percent sum diameter changes, and adverse effects (AE). We report descriptive statistics on patient characteristics, clinical response rates, and changes in the sum of the longest diameters of the target lesions (SLD). Translational correlatives including imaging mass cytometry, spatial transcriptomic assessments, and other pathologic biomarkers will be reported at the meeting. Results: From 2018-2023, 33 pts were enrolled, and 30 were eligible for the primary analysis, including 20 pts on the palbo arm (1 pt with bilateral breast cancer, response to each breast cancer evaluated separately) and 10 to the control arm (1 pt with missing data). Clinical and pathologic characteristics were well balanced between arms, however, there was a higher proportion of patients with node-positive disease on the palbo arm (80% vs 60%). The study did not meet its primary endpoint as only 1 pt achieved a cCR and pCR in the palbo arm (cCR/pCR rate 4.8%) vs. no pts in the control arm. ORR was numerically higher in the palbo arm, 42.9% vs. 11.1% (p=0.204). After 1-month of ET+palbo, the mean SLD decreased by 11.6% compared to 9.4% in the ET-only control group (p=0.704). After avelumab was added to both arms, the mean SLD decreased by a further 15.2% in the palbo arm in contrast to a 6.9% increase in the control arm (p=0.018). Subgroup analysis in the palbo arm did not identify clinical or pathologic variables significantly associated with responses. No new safety or toxicity signals were observed. Notably, however expected, grade 3 or higher immune-related adverse events were observed including autoimmune diabetes (n=1), hepatitis (n=1), and colitis (n=1). Conclusions: As shown in previous studies, the addition of palbo to ET did not improve responses in the neoadjuvant setting; however, increased responses to palbo+ET were seen after PD-L1 inhibition, whereas not to ET, suggesting synergy between ET/palbo and avelumab. Translational correlative analysis will aim to decipher TME changes that augment PD-L1 inhibitor responses. Future studies in high-risk pts, where the toxicity of immunotherapy is acceptable, are warranted. Citation Format: Phaedon Zavras, Ruizhe Chen, Hanfei Qi, Mary Kate Jones, Ann Folmer, Hayden Chae, Allen Khodab, Ashley Cimino-Mathews, Lisa Jacobs, Lisa Mullen, Christie Hiton, Ahmed Elkhanany, Katia Khoury, Massimo Cristofanilli, Elizabeth Jaffee, Vered Stearns, Cesar Santa-Maria. Neoadjuvant endocrine therapy and avelumab with or without palbociclib in stage II/III endocrine receptor-positive breast cancer: the ImmunoADAPT trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-01-09.

Abstract PS07-08: Bazedoxifene plus conjugated estrogen reduces mammary proliferation markers and improves adipocyte size, gut microbiome, and metabolic health: Findings from a preclinical model of obesity and breast cancer risk

Abstract Background: Many women at high risk for breast cancer will not take tamoxifen or aromatase inhibitors for cancer prevention due to concern of side effects including hot flashes. Further, tamoxifen has detrimental metabolic effects in some overweight/obese women. Duavee®, a tissue-selective complex of bazedoxifene + conjugated estrogen, is FDA-approved for relief of hot-flashes and prevention of osteoporosis. Preclinical studies suggest favorable metabolic effects and potential for breast cancer risk reduction. In a single arm clinical trial, BZA+CE reduced Ki-67 and mammographic density (PMID: 31420361), and this combination is currently being assessed further in a Phase IIB trial of postmenopausal high risk women with vasomotor symptoms. Here, we examined the modifying effects of obesity on response to BZA+CE in a rodent model of obesity and breast cancer risk. Methods: Rats received carcinogen at 7-weeks of age to induce mammary tumors and were fed a high-fat diet (46% kcal fat) to promote obesity. Lean and obese rat were selected based on adiposity at 16 weeks. Separate cohorts of lean and obese ovary-intact or ovariectomized (OVX) rats were randomized to a daily dose of BZA+CE or placebo control for 8 weeks. We assessed tumor development throughout the study and end of study RNA from mammary glands was analysed by gene expression microarray (Affymetrix). We also analyzed body weight/composition, markers of metabolic health (circulating glucose, insulin, adiponectin), as well as changes in the gut microbiome (metagenomic sequencing on DNA isolated from baseline and end-of-study fecal samples) in response to BZA+CE. Results: BZA+CE improved metabolic health in both ovary intact and OVX rats, including reduced body weight and % body fat, particularly in the visceral regions. These effects were greater in obese rats compared to lean. BZA+CE reduced the number of large adipocytes and increased small (insulin sensitive) adipocytes in the mammary adipose, indicating benefical changes in local tumor microenvironment. We found no evidence that Duavee® promoted tumor development or growth in ovary-intact or OVX’d rats. Gene set enrichment analysis (GSEA) of microarray data showed enrichment of cell proliferation pathways in MG from obese rats and these same pathways were downregulated with BZA+CE, consistent with anti-cancer effects. Analysis of the microbiome found that BZA+CE increased proportional abudance of Odoribacter laneus in the gut, regardless of lean/obese or intact/OVX status. Previous studies have demonstrated that this bacterim improves glucose control and reduces inflammatory cytokines when administered to obese mice, suggesting a possible mechanistic link in this study. Conclusions: Unlike traditional SERMs that can have negative metabolic effects, BZA+CE improved whole body and mammary gland metabolic health and reduced expression of cell proliferation pathways, particularly in obese rats. Prelminary data suggest that changes in the gut microbiome could contribute, at least in part, to these effects. Together this supports BZA+CE (Duavee) as an agent with potential beneficial effects on breast cancer risk reduction and improvements in metabolic health in women with obesity. Further analyses will guide assessment of outcomes in an ongoing parallel clinical study in postmenopausal women at high risk for breast cancer. Citation Format: Katherine L. Cook, Ramsey Jenshcke, Karen Corleto, Carol Fabian, Stephen Hursting, Bruce Kimler, Danilo Landrock, Erin Giles. Bazedoxifene plus conjugated estrogen reduces mammary proliferation markers and improves adipocyte size, gut microbiome, and metabolic health: Findings from a preclinical model of obesity and breast cancer risk [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS07-08.

Impacts of tumor microenvironment during neoadjuvant chemotherapy in patients with esophageal squamous cell carcinoma.

With the advent of immune checkpoint inhibitors (ICIs), a better understanding of tumor microenvironment (TME) is becoming crucial in managing esophageal squamous cell carcinoma (ESCC) patients. We investigated the survival impact of TME status and changes in patients with ESCC who underwent neoadjuvant chemotherapy (NAC) followed by surgery (n = 264). We examined immunohistochemical status (CD4+, CD8+, CD20+, Foxp3+, HLA class-1+, CD204+, and programmed death ligand-1 [PD-L1+]) on 264 pre-NAC and 204 paired post-NAC specimens. Patients were classified by their pre- and post-NAC immune cell status and their changes following NAC. Our findings showed that pre-NAC TME status was not significantly associated with survival outcomes. In contrast, post-NAC TME status, such as low level of T cells, CD4+ T cells, and high PD-L1 combined positive score (CPS), were significantly associated with poor overall survival (OS). Notably, TME changes through NAC exerted significant survival impacts; patients with consistently low levels of T cells, low levels of CD4+ T cells, or high levels of PD-L1 (CPS) had very poor OS (3-year OS: 35.5%, 40.2%, and 33.3%, respectively). Tumor microenvironment changes of consistently low T cells, low CD4+ T cells, and high PD-L1 were independent predictors of poor OS in multivariate Cox hazards analyses, while factors indicating post-NAC status (T cells, CD4+, and PD-L1 [CPS]) alone were not. Therefore, we suggest that the consistently low T/high PD-L1 group could benefit from additional therapies, such as ICIs, and the importance of stratification by the TME, which has recently been recognized.

Latest Developments in Magnetic Resonance Imaging for Evaluating the Molecular Microenvironment of Gliomas.

The 2021 World Health Organization (WHO) Classification of Tumors of the Central Nervous System has brought a transformative shift in the categorization of adult gliomas. Departing from traditional histological subtypes, the new classification system is guided by molecular genotypes, particularly the Isocitrate Dehydrogenase (IDH) mutation. This alteration reflects a pivotal change in understanding tumor behavior, emphasizing the importance of molecular profiles over morphological characteristics. Gliomas are now categorized into IDH-mutant and IDH wildtype, with significant prognostic implications. For IDH-mutant gliomas, the concurrent presence of Alpha-Thalassemia/mental retardation syndrome X-linked (ATRX) gene expression and co-deletion of 1p19q genes further refine classification. In the absence of 1p19q co-deletion, further categorization depends on the phenotypic expression of CDKN2A/B. Notably, IDH wildtype gliomas exhibit a poorer prognosis, particularly when associated with TERT promoter mutations, EGFR amplification, and +7/-10 co-deletion. Although not part of the new guidelines, the methylation status of the MGMT gene is crucial for guiding alkylating agent treatment. The integration of structural and functional Magnetic Resonance Imaging (MRI) techniques may play a vital role in evaluating these genetic phenotypes, offering insights into tumor microenvironment changes. This multimodal approach may enhance diagnostic precision, aid in treatment planning, and facilitate effective prognosis evaluation of glioma patients.

Abstract P28: Comprehensive Molecular Phenotyping of ARID1A-deficient Gastric Cancer Reveals Pervasive Epigenomic Reprogramming and Therapeutic Opportunities

Abstract Objective Gastric cancer (GC) is a leading cause of cancer mortality, with ARID1A being the second most frequently mutated driver gene in GC. We sought to decipher ARID1A-specific GC regulatory networks and examine therapeutic vulnerabilities arising from ARID1A loss. Design Genomic profiling of GC patients including a Singapore cohort (&amp;gt;200 patients) was performed to derive mutational signatures of ARID1A inactivation across molecular subtypes. Single-cell transcriptomic profiles of ARID1A-mutated GCs were analyzed to examine tumor microenvironmental changes arising from ARID1A loss. Genome-wide ARID1A binding and chromatin profiles (H3K27ac, H3K4me3, H3K4me1, ATAC-seq) of gastric cell lines were generated to identify gastric-specific epigenetic landscapes regulated by ARID1A. Distinct cancer hallmarks of ARID1A-mutated GCs were converged at the genomic, single-cell, and epigenomic level, and targeted by pharmacological inhibition. Results We observed prevalent ARID1A inactivation across GC molecular subtypes, with distinct mutational signatures and linked to a NFKB-driven pro-inflammatory tumour microenvironment. ARID1A-depletion caused loss of H3K27ac activation signals at ARID1A-occupied distal enhancers, but unexpectedly gain of H3K27ac at ARID1A-occupied promoters in genes such as NFKB1 and NFKB2. Promoter activation in ARID1A-mutated GCs was associated with enhanced gene expression, increased BRD4 binding, and reduced HDAC1 and CTCF occupancy. Combined targeting of promoter activation and tumour inflammation via bromodomain and NFKB inhibitors confirmed therapeutic synergy specific to ARID1A-genomic status. Conclusion Our results suggest a therapeutic strategy for ARID1A-mutated GCs targeting both tumour-intrinsic (BRD4-assocatiated promoter activation) and extrinsic (NFKB immunomodulation) cancer phenotypes. Citation Format: Chang Xu, Kie Kyon Huang, Jia Hao Law, Joy Shijia Chua, Taotao Sheng, Natasha M. Flores, Melissa Pool Pizzi, Atsushi Okabe, Angie Lay Keng Tan, Feng Zhu, Vikrant Kumar, Xiaoyin Lu, Ana Morales Benitez, Benedict Shi Xiang Lian, Haoran Ma, Shamaine Wei Ting Ho, Kalpana Ramnarayanan, Chukwuemeka George Anene-Nzelu, Milad Razavi-Mohseni, Siti Aishah Binte Abdul Ghani, Su Ting Tay, Xuewen Ong, Ming Hui Lee, Yu Amanda Guo, Hassan Ashktorab, Duane Smoot, Shang Li, Anders Jacobsen Skanderup, Michael A. Beer, Roger Sik Yin Foo, Joel Shi Hao Wong, Kaushal Sanghvi, Wei Peng Yong, Raghav Sundar, Atsushi Kaneda, Shyam Prabhakar, Pawel Karol Mazur, Jaffer A. Ajani, Khay Guan Yeoh, Jimmy Bok-Yan So, Patrick Tan, Singapore Gastric Cancer Consortium. Comprehensive Molecular Phenotyping of ARID1A-deficient Gastric Cancer Reveals Pervasive Epigenomic Reprogramming and Therapeutic Opportunities [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P28.

Abstract 2726: A novel trispecific antibody HC010 targeting PD-1/CTLA-4/VEGF for the potential treatment in anti-PD-1 antibody resistant NSCLC patients

Abstract Background and Aim: Immune checkpoint inhibitors (anti-PD-1/CTLA-4 antibody) have been approved and widely used for the first line treatment of non-small cell lung cancer (NSCLC). However, most patients are experiencing resistant to PD-1 blockade. In recent clinical trials, anti-VEGF antibody has shown to improve the clinical outcomes of anti-PD-1 antibodies and overcome resistance to checkpoint blockade. We developed a PD-1/CTLA-4/VEGF trispecific antibody (HC010) by site-specifically fusing anti-PD-1 scFv and anti-CTLA-4 nanobody to the defined site of anti-VEGF antibody. The aim of this study was to evaluate the potential use of HC010 for treatment of anti-PD-1 antibody resistant NSCLC patients. Method: Humanized mice were established in NCG mice by reconstituting human PBMC before inoculating the PDX tissue, which was derived from a NSCLC patient treated with two cycles of an anti-PD-1 antibody sintilimab. When tumors were approx. 170mm3, mice were dosed with HC010 twice weekly for 3 weeks. Pembrolizumab, sintilimab and a Standard of Care (SoC) agent docetaxel were included as reference. For IHC analysis, tumor samples were harvested 24h post final dose and fixed with 10% NFB overnight and subsequently stained with CD31 and CD8 to explore the change in tumor microenvironment after HC010 treatment. For preclinical toxicity assessment, HC010 was intravenously administered once weekly for four weeks in naïve cynomolgus monkeys and immunogenicity/local tolerance/clinical pathology were evaluated during the dosing and recovery phase. Results: HC010 showed a significant tumor inhibition (TGI=81%) when compared with the vehicle control. Pembrolizumab and sintilimab alone exhibited limited anti-tumor effect. Docetaxel only showed a partial response during the dosing phase. By analyzing tumor samples, there was a significant increase in CD8+ positive T cells in HC010 treated tumors in contrast to the vehicle group and pembrolizumab/sintilimab treated groups, suggesting that HC010 can significantly improve CD8+ T cell infiltration. Expression of CD31 as an angiogenic and vasculogenic marker, was significantly reduced in HC010 treated tumors. In the toxicity study, HC010 at selected doses showed no obvious toxicity in cynomolgus monkeys. Conclusion: Our trispecific antibody HC010 targeting PD-1/CTLA-4/VEGF showed a superior anti-tumor effect in a sintilimab resistant PDX animal model when compared with pembrolizumab and sintilimab as well as SoC. By analyzing tumor microenvironment, HC010 overcame acquired resistance by modulating tumor microenvironment such as increase in intra-tumoral cytotoxic T cell infiltration and normalization of blood vessels. Together with preclinical efficacy and safety data, HC010 showed potential benefits in treatment of anti-PD-1 antibody resistant NSCLC patients. Citation Format: Diandong Jiang, Yue Xi, Chen Xu, Xingding Wang, Qiaoshan Yin, Zhaohui Li, Teddy Yang. A novel trispecific antibody HC010 targeting PD-1/CTLA-4/VEGF for the potential treatment in anti-PD-1 antibody resistant NSCLC patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2726.

Abstract 1621: Integrated analysis of genetic, transcriptional and TME evolution of ccRCC: TRACERx Renal

Abstract Background: Genetic evolution of clear cell renal cell carcinoma (ccRCC) follows distinct trajectories, with varying levels of intratumor heterogeneity (ITH) and chromosomal complexity (WGII). While these patterns associate with clinical outcomes, it remains unknown whether they fully reconcile tumor behavior and how genetic and transcriptional features co-evolve in relation to the tumor microenvironment (TME). Methods: To analyze the patterns of transcriptional and TME heterogeneity, we performed bulk whole-transcriptome sequencing on 244 samples, including 22 metastatic and 12 tumor-adjacent normal samples, from 79 ccRCC patients recruited to the TRACERx Renal study. We integrated transcriptional data with previously published genetic, phylogenetic, spatial and clinical information. Results: Transcriptional distances between paired samples from the same primary tumor mirrored but were not fully determined by genetic distance (p-value &amp;lt; 0.001); and increased from primary-primary to primary-metastasis and primary-normal pairs. Within primary-metastasis pairs, metastasis-seeding primary tumor regions were transcriptionally closest to their matched metastasis (p-value &amp;lt; 0.001), suggesting that an important fraction of metastatic transcriptional traits were acquired in the primary tumor. Regarding the tumor clonal structure, transcriptional evolution followed a conserved path through increasing cell proliferation and oxidative phosphorylation and downregulating DNA repair from earlier to later clones. Further, within tumors with increasing WGII we observed upregulation and downregulation of repressors and downstream effectors, respectively, of the canonical cGAS-STING pathway. Combining the presence of this transcriptional pattern with WGII predicted shorter PFS in TRACERx Renal (p-value &amp;lt; 0.001) and in TCGA-KIRC (p-value &amp;lt; 0.001). Clonal evolution was also linked to changes in TME, with each of the previously defined genetic evolutionary trajectories associated to a specific TME (p-value &amp;lt; 0.001). For example, ccRCCs on a PBRM1-SETD2 trajectory demonstrated increased infiltration of cytotoxic immune cells. TME ITH was pervasive and associated with shorter PFS (p-value = 0.03). A recurrent trend from earlier to later clones was progressive T cell depletion (p-value &amp;lt; 0.001). The evolution of the TCR repertoire mirrored the tumor clonal structure (p-value = 0.002), suggesting the thus far elusive antigenic source in ccRCC is heritable. Accordingly, the TCR repertoire in metastasis-seeding primary tumor regions resembled the closest the TCR repertoire of matched metastasis (p-value = 0.06). Conclusion: Integrated analysis of genetic and transcriptional data in TRACERx Renal showed i) transcriptional and TME ITH not fully recapitulated by genetic ITH, ii) conserved paths of transcriptional and TME evolution and iii) a heritable nature of part of the ccRCC antigen source. Citation Format: Ángel Fernández Sanromán, Lewis Au, Benjy Jek Yang Tan, Charlotte Spencer, Anne-Laure Catin, Irene Lobon, Husayn Pallikonda, Kevin Litchfield, Fiona Byrne, James Larkin, Annika Fendler, Samra Turajlic. Integrated analysis of genetic, transcriptional and TME evolution of ccRCC: TRACERx Renal [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1621.

Abstract 5490: Spatial profiling of inflamed tumor microenvironment of clear cell renal cell carcinoma

Abstract Clear cell renal cell carcinoma (ccRCC) presents a distinctive tumor microenvironment (TME) characterized by extensive vascularization and immune infiltration. This unique TME makes ccRCC an attractive target for anti-angiogenesis and immune checkpoint blockade (ICB). To better understand the complexity of ccRCC and the associated cell types and states involved in disease progression, we conducted high-throughput single-cell immunofluorescence imaging on tissue sections from 437 localized tumors that were surgically resected. These samples included regions from the tumor center, tumor edge, and adjacent benign areas (TMA cores, n=1859). Our primary focus was on tumors with elevated immune cell enrichment (CD45high), as these tumors are known to be aggressive and may respond well to immunotherapy. Our study reveals the intricate interactions between tumor, vascular, stromal, and immune cells within ccRCC. We find that CD45high tumors, upon losing epithelial marker expression, acquire tumor cell PD-L1 and mesenchymal markers such as Fibroblast activation protein (FAP). This shift is indicative of the epithelial-mesenchymal transition (EMT) process. These CD45high and EMThigh tumors are enriched with changes in TME, including elevated FAP+ cancer-associated fibroblasts (CAFs) and subsets of immune suppressive T cells and macrophages. We report that ccRCC patients with high tumor PD-L1 expression or abundant FAP+ CAFs are associated with earlier metastasis and, in sunitinib-treated patients, shorter progression-free survival. This study illuminates the intricate connection between immune cell infiltration, stromal biology, and EMT in ccRCC, offering a deeper understanding of the disease's complex biology and potentially guiding future therapeutic approaches and strategies. Citation Format: Teijo Pellinen, Lassi Luomala, Kalle Mattila, Jenni Säilä, Annabrita Hemmes, Katja Valimaki, Oscar Bruck, Lassi Paavolainen, Harry Nisen, Petrus Järvinen, Olli Kallioniemi, Paula Vainio, Panu Jaakkola, Tuomas Mirtti. Spatial profiling of inflamed tumor microenvironment of clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5490.