Immune Profile Research Articles

Exercise as an anti-inflammatory Therapy in Axial Spondyloarthritis Therapeutic Intervention (EXTASI study): a randomised control trial

Abstract Objectives Axial Spondyloarthritis (AxSpA) is a chronic inflammatory disease, yet despite known anti-inflammatory effects of exercise, the effect of exercise on inflammatory immune cell populations and associated inflammatory profiles in AxSpA is unknown. This randomised-control trial investigated the effect of 12-weeks of walking on symptom severity, cardiometabolic health, inflammatory biomarkers, and immune cell populations. Methods Twenty people (60% male) living with AxSpA who were on stable dose non-steroidal anti-inflammatory (NSAIDs) medication participated. Participants were randomly assigned to control or exercise (30 min of walking five x per week). Participants were invited back every four-weeks for assessment. Results There was a 0% drop out and no adverse events in the exercise group showing walking exercise was well tolerated. Home-based walking for 12-weeks lowered the proportion of pro-inflammatory monocytes, whereas they increased in the control group. Changes were associated with lower interleukin-6 (IL-6) and C-reactive protein (CRP) concentrations, lower spinal pain, and lower systolic blood pressure in the exercise group, whereas these markers increased in the control group. Reductions in IL-6 and pro-inflammatory monocytes with exercise were independent of lower body fat percentage. Conclusion Supplementing NSAID therapy with walking exercise can improve inflammatory immune profiles in people with AxSpA coinciding with reductions in spinal pain. Importantly, the exercise was well tolerated, suggesting walking exercise can be used as an adjuvant anti-inflammatory therapy for NSAID treatments. This should now be explored in people living with AxSpA who have had high enough disease activity to necessitate the prescription of biologic or synthetic DMARD treatments. Clinical trials registration ClinicalTrials.Gov, NCT04368494

Ciita Regulates Local and Systemic Immune Responses in a Combined rAAV-α-synuclein and Preformed Fibril-Induced Rat Model for Parkinson's Disease.

Parkinson's disease (PD) is characterized by alpha-synuclein (α-Syn) pathology, neurodegeneration and neuroinflammation. Human leukocyte antigen (HLA) variants associated with PD and α-Syn specific CD4+ T lymphocytes in PD patients highlight the importance of antigen presentation in PD etiology. The class II transactivator (CIITA) regulates major histocompatibility complex class II (MHCII) expression. Reduced Ciita levels significantly increase α-Syn pathology, nigrostriatal neurodegeneration and behavioral deficits in α-Syn-induced rat PD models. Characterize immune profiles associated with enhanced PD-like pathology observed in rats expressing lower Ciita levels (DA.VRA4) compared to the background strain (DA). To model PD, we combined rAAV-mediated α-Syn overexpression in the substantia nigra with striatal injection of α-Syn preformed fibrils. Immune profiles in brain and blood were analyzed by flow cytometry and multiplexed ELISA in naïve rats, 4- and 8 weeks post rAAV injection. Flow cytometry showed Ciita-dependent regulation of MHCII on microglia, brain macrophages and circulating myeloid cells. The MHCII-dependent microglial response was highest at 4 weeks post rAAV injection, whereas the MHCII levels in circulating myeloid cells was highest at 8 weeks. There was no major infiltration of macrophages or T lymphocytes into the CNS in response to α-Syn and only subtle Ciita- and/or α-Syn-dependent changes in the T lymphocyte compartment. Lower Ciita levels were consistently associated with higher TNF levels in serum. Ciita regulates susceptibility to PD-like pathology through minor but detectable changes in resident and peripheral immune cells and TNF levels, indicating that mild immunomodulatory therapies could have therapeutic effects in PD.

Analysis and validation of biomarkers of immune cell-related genes in postmenopausal osteoporosis: An observational study.

Postmenopausal osteoporosis (PMOP) is a common metabolic inflammatory disease. In conditions of estrogen deficiency, chronic activation of the immune system leads to a hypo-inflammatory phenotype and alterations in its cytokine and immune cell profile, although immune cells play an important role in the pathology of osteoporosis, studies on this have been rare. Therefore, it is important to investigate the role of immune cell-related genes in PMOP. PMOP-related datasets were downloaded from the Gene Expression Omnibus database. Immune cells scores between high bone mineral density (BMD) and low BMD samples were assessed based on the single sample gene set enrichment analysis method. Subsequently, weighted gene co-expression network analysis was performed to identify modules highly associated with immune cells and obtain module genes. Differential analysis between high BMD and low BMD was also performed to obtain differentially expressed genes. Module genes are intersected with differentially expressed genes to obtain candidate genes, and functional enrichment analysis was performed. Machine learning methods were used to filter out the signature genes. The receiver operating characteristic (ROC) curves of the signature genes and the nomogram were plotted to determine whether the signature genes can be used as a molecular marker. Gene set enrichment analysis was also performed to explore the potential mechanism of the signature genes. Finally, RNA expression of signature genes was validated in blood samples from PMOP patients and normal control by real-time quantitative polymerase chain reaction. Our study of PMOP patients identified differences in immune cells (activated dendritic cell, CD56 bright natural killer cell, Central memory CD4 T cell, Effector memory CD4 T cell, Mast cell, Natural killer T cell, T follicular helper cell, Type 1 T-helper cell, and Type 17 T-helper cell) between high and low BMD patients. We obtained a total of 73 candidate genes based on modular genes and differential genes, and obtained 5 signature genes by least absolute shrinkage and selection operator and random forest model screening. ROC, principal component analysis, and t-distributed stochastic neighbor embedding down scaling analysis revealed that the 5 signature genes had good discriminatory ability between high and low BMD samples. A logistic regression model was constructed based on 5 signature genes, and both ROC and column line plots indicated that the model accuracy and applicability were good. Five signature genes were found to be associated with proteasome, mitochondria, and lysosome by gene set enrichment analysis. The real-time quantitative polymerase chain reaction results showed that the expression of the signature genes was significantly different between the 2 groups. HIST1H2AG, PYGM, NCKAP1, POMP, and LYPLA1 might play key roles in PMOP and be served as the biomarkers of PMOP.

Identification of driving genes of familial adenomatous polyposis by differential gene expression analysis and weighted gene co-expression network analysis.

Despite the advancement of new screening strategies and the advances in pharmacological therapies, the cancerization rates of familial adenomatous polyposis (FAP) are stable and even increased in the last years. Therefore, it necessitates additional research to characterize and understand the underlying mechanisms of FAP. To determine the genes that drive the pathogenesis of familial adenomatous polyposis (FAP). We performed on a cohort (GSE111156) gene profile, which consist of four group of gene expressions (the gene expressions of cancer, adenoma and normal tissue of duodenal cancer from patients with FAP were defined as Case N, Case A and Case C respectively, while that of adenoma tissue from patients with FAP who did not have duodenal cancer was Ctrl A). Tracking Tumor Immunophenotype (TIP) website was applied to reveal immune infiltration profile and signature genes of FAP. We merged the genes of key module (pink and midnight module) with signature genes to obtained the biomarkers related with FAP pathogenesis. The expression of these five biomarkers in FAP intratumoral region (IT) and tumor rim (TR) was detected with Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). In total, 220, 23 and 63 DEGs were determined in Cases C, A and N, in comparison to Ctrl A. In total, 196 and 10 DEGs were determined in Cases C and A, separately, as compared to Case N. A total of four biomarkers including CCL5, CD3G, CD2 and TLR3 were finally identified associated with pink module, while only one biomarker (KLF2) associated with midnight module was identified. All biomarkers were evidently raised in FAP IT tissues utilizing qRT-PCR. We identified five potential biomarkers for pathogenesis of FAP to understand the fundamental mechanisms of FAP progression and revealed some probable targets for the diagnosis or treatment of FAP.

Immunoinformatics assisted profiling of West Nile virus proteome to determine immunodominant epitopes for the development of next-generation multi-peptide vaccine

Emerging infectious diseases represent a significant threat to global health, with West Nile virus (WNV) being a prominent example due to its potential to cause severe neurological disorders alongside mild feverish conditions. Particularly prevalent in the continental United States, WNV has emerged as a global concern, with outbreaks indicating the urgent need for effective prophylactic measures. The current problem is that the absence of a commercial vaccine against WNV highlights a critical gap in preventive strategies against WNV. This study aims to address this gap by proposing a novel, multivalent vaccine designed using immunoinformatics approaches to elicit comprehensive humoral and cellular immune responses against WNV. The objective of the study is to provide a theoretical framework for experimental scientists to formulate of vaccine against WNV and tackle the current problem by generating an immune response inside the host. The research employs reverse vaccinology and subtractive proteomics methodologies to identify NP_041724.2 polyprotein and YP_009164950.1 truncated flavivirus polyprotein NS1 as the prime antigens. The selection process for epitopes focused on B and T-cell reactivity, antigenicity, water solubility, and non-allergenic properties, prioritizing candidates with the potential for broad immunogenicity and safety. The designed vaccine construct integrates these epitopes, connected via GPGPG linkers, and supplemented with an adjuvant with the help of another linker EAAAK, to enhance immunogenicity. Preliminary computational analyses suggest that the proposed vaccine could achieve near-universal coverage, effectively targeting approximately 99.74% of the global population, with perfect coverage in specific regions such as Sweden and Finland. Molecular docking and immune simulation studies further validate the potential efficacy of the vaccine, indicating strong binding affinity with toll-like receptor 3 (TLR-3) and promising immune response profiles, including significant antibody-mediated and cellular responses. These findings present the vaccine construct as a viable candidate for further development and testing. While the theoretical and computational results are promising, advancing from in-silico predictions to a tangible vaccine requires comprehensive laboratory validation. This next step is essential to confirm the vaccine’s efficacy and safety in eliciting an immune response against WNV. Through this study, we propose a novel approach to vaccine development against WNV and contribute to the broader field of immunoinformatics, showcasing the potential to accelerate the design of effective vaccines against emerging viral threats. The journey from hypothesis to practical solution embodies the interdisciplinary collaboration essential for modern infectious disease management and prevention strategies.

Assessing the role of programmed cell death signatures and related gene TOP2A in progression and prognostic prediction of clear cell renal cell carcinoma

Kidney Clear Cell Carcinoma (KIRC), the predominant form of kidney cancer, exhibits a diverse therapeutic response to Immune Checkpoint Inhibitors (ICIs), highlighting the need for predictive models of ICI efficacy. Our study has constructed a prognostic model based on 13 types of Programmed Cell Death (PCD), which are intertwined with tumor progression and the immune microenvironment. Validated by analyses of comprehensive datasets, this model identifies seven key PCD genes that delineate two subtypes with distinct immune profiles and sensitivities to anti-PD-1 therapy. The high-PCD group demonstrates a more immune-suppressive environment, while the low-PCD group shows better responses to PD-1 treatment. In particular, TOP2A emerged as crucial, with its inhibition markedly reducing KIRC cell growth and mobility. These findings underscore the relevance of PCDs in predicting KIRC outcomes and immunotherapy response, with implications for enhancing clinical decision-making.

Breast Cancer Immune Landscape: Interplay Between Systemic and Local Immunity.

Breast cancer (BC) is one of the most common malignancies in women worldwide. Numerous studies in immuno-oncology and successful trials of immunotherapy have demonstrated the causal role of the immune system in cancer pathogenesis. The interaction between the tumor and the immune system is known to have a dual nature. Despite cytotoxic lymphocyte activity against transformed cells, a tumor can escape immune surveillance and leverage chronic inflammation to maintain its own development. Research on antitumor immunity primarily focuses on the role of the tumor microenvironment, whereas the systemic immune response beyond the tumor site is described less thoroughly. Here, a comprehensive review of the formation of the immune profile in breast cancer patients is offered. The interplay between systemic and local immune reactions as self-sustaining mechanism of tumor progression is described and the functional activity of the main cell populations related to innate and adaptive immunity is discussed. Additionally, the interaction between different functional levels of the immune system and their contribution to the development of the pro- or anti-tumor immune response in BC is highlighted. The presented data can potentially inform the development of new immunotherapy strategies in the treatment of patients with BC.

Molecular landscape and clinical implication of CCNE1-amplified esophagogastric cancer.

Cyclin E overexpression as a result of CCNE1 amplification is a critical driver of genomic instability in gastric cancer, but its clinical implication is largely unknown. Thus, we integrated genomic, transcriptomic, and immune profiling analysis of 7,083 esophagogastric tumors and investigated the impact of CCNE1 amplification on molecular features and treatment outcomes. We identified CCNE1 amplification in 6.2% of esophageal adenocarcinoma samples, 7.0% of esophagogastric junction carcinoma, 4.2% of gastric adenocarcinoma samples, and 0.8% of esophageal squamous cell carcinoma. Metastatic sites such as lymph node and liver showed an increased frequency of CCNE1 amplification relative to primary tumors. Consistent with a chromosomal instability phenotype, CCNE1 amplification was associated with decreased CDH1 mutation and increased TP53 mutation and ERBB2 amplification. We observed no differences in immune biomarkers such as PD-L1 expression and tumor mutational burden comparing CCNE1-amplified and non-amplified tumors, although CCNE1 amplification was associated with changes in immune populations such as decreased B cells and increased M1 macrophages from transcriptional analysis. Real-world survival analysis demonstrated that patients with CCNE1-amplified gastric cancer had worse survival after trastuzumab for HER2-positive tumors, but better survival after immunotherapy. These data suggest that CCNE1-amplified gastric cancer has a distinct molecular and immune profile with important therapeutic implications, and therefore further investigation of CCNE1 amplification as a predictive biomarker is warranted.

Multi-Omics Characterization of Colon Mucosa and Submucosa/Wall from Crohn’s Disease Patients

Crohn’s disease (CD) is a subtype of inflammatory bowel disease (IBD) characterized by transmural disease. The concept of transmural healing (TH) has been proposed as an indicator of deep clinical remission of CD and as a predictor of favorable treatment endpoints. Understanding the pathophysiology involved in transmural disease is critical to achieving these endpoints. However, most studies have focused on the intestinal mucosa, overlooking the contribution of the intestinal wall in Crohn’s disease. Multi-omics approaches have provided new avenues for exploring the pathogenesis of Crohn’s disease and identifying potential biomarkers. We aimed to use transcriptomic and proteomic technologies to compare immune and mesenchymal cell profiles and pathways in the mucosal and submucosa/wall compartments to better understand chronic refractory disease elements to achieve transmural healing. The results revealed similarities and differences in gene and protein expression profiles, metabolic mechanisms, and immune and non-immune pathways between these two compartments. Additionally, the identification of protein isoforms highlights the complex molecular mechanisms underlying this disease, such as decreased RTN4 isoforms (RTN4B2 and RTN4C) in the submucosa/wall, which may be related to the dysregulation of enteric neural processes. These findings have the potential to inform the development of novel therapeutic strategies to achieve TH.

Characteristics of RNA Stabilizer RNApro for Peripheral Blood Collection

Peripheral blood is the most practical tissue for human immune system gene expression profiling because it is easily accessible, whereas the site of primary infection in certain diseases may not be easily accessible. Due to the ex vivo instability of RNA transcripts, a key challenge in the gene expression analysis of blood samples is the rapid sample handling and stabilization of the mRNA by adding an RNA preservative (PAXgeneTM Blood RNA Tubes, TempusTM Blood RNA tubes, RNAlater Stabilization Reagent, RNAgard® Blood Tubes). BioMole (Turin, Italy) has developed a novel blood stabilizer, called RNApro, in which RNA is stabilized during phlebotomy and sample storage. In this study, RNApro performance intended as RNA yield, integrity, and stability was evaluated. Our results show that blood samples stored at −80 °C and re-extracted after 7 years show no differences in terms of quantity, quality, and amplificability. The samples in the RNAlater stabilization solution can be stored at room temperature for up to one week or at 4 °C for up to one month. Similar results can also be observed for PAXgene tubes, Tempus tubes, and RNAgard tubes. In agreement with these data, the RNApro stabilization solution preserves the RNA from degradation for up to 1 month at 4 °C and 1 week at room temperature. RNApro can be stored indifferently at −80, −20, 4 °C, or room temperature for up to 2 months after, and then could be stored at −80 °C for up to seven years. In summary, our study is the first to analyze the performance of an RNA stabilizer called RNApro. We can conclude that several studies have shown significant differences in gene expression analysis when the sample was preserved in different RNA stabilizers. Therefore, it is desirable to standardize the method of nucleic acid conservation when comparing data from transcriptomic analyses.

Distinct cellular immune responses in children en route to type 1 diabetes with different first-appearing autoantibodies

Previous studies have revealed heterogeneity in the progression to clinical type 1 diabetes in children who develop islet-specific antibodies either to insulin (IAA) or glutamic acid decarboxylase (GADA) as the first autoantibodies. Here, we test the hypothesis that children who later develop clinical disease have different early immune responses, depending on the type of the first autoantibody to appear (GADA-first or IAA-first). We use mass cytometry for deep immune profiling of peripheral blood mononuclear cell samples longitudinally collected from children who later progressed to clinical disease (IAA-first, GADA-first, ≥2 autoantibodies first groups) and matched for age, sex, and HLA controls who did not, as part of the Type 1 Diabetes Prediction and Prevention study. We identify differences in immune cell composition of children who later develop disease depending on the type of autoantibodies that appear first. Notably, we observe an increase in CD161 expression in natural killer cells of children with ≥2 autoantibodies and validate this in an independent cohort. The results highlight the importance of endotype-specific analyses and are likely to contribute to our understanding of pathogenic mechanisms underlying type 1 diabetes development.

KRAS Allelic Variants in Biliary Tract Cancers

Biliary tract cancers (BTCs) contain several actionable molecular alterations, including FGFR2, IDH1, ERBB2 (formerly HER2), and KRAS. KRAS allelic variants are found in 20% to 30% of BTCs, and multiple KRAS inhibitors are currently under clinical investigation. To describe the genomic landscape, co-sequence variations, immunophenotype, genomic ancestry, and survival outcomes of KRAS-mutated BTCs and to calculate the median overall survival (mOS) for the most common allelic variants. This retrospective, multicenter, pooled cohort study obtained clinical and next-generation sequencing data from multiple databases between January 1, 2017, and December 31, 2022. These databases included Princess Margaret Cancer Centre, MD Anderson Cancer Center, Foundation Medicine, American Association for Cancer Research Project GENIE, and cBioPortal for Cancer Genomics. The cohort comprised patients with BTCs who underwent genomic testing. The main outcome was mOS, defined as date of diagnosis to date of death, which was measured in months. A total of 7457 patients (n = 3773 males [50.6%]; mean [SD] age, 63 [5] years) with BTCs and genomic testing were included. Of these patients, 5813 had clinical outcome data available, in whom 1000 KRAS-mutated BTCs were identified. KRAS allelic variants were highly prevalent in perihilar cholangiocarcinoma (28.6%) and extrahepatic cholangiocarcinoma (36.1%). Thirty-six KRAS allelic variants were identified, and the prevalence rates in descending order were G12D (41%), G12V (23%), and Q61H (8%). The variant G12D had the highest mOS of 25.1 (95% CI, 22.0-33.0) months compared with 22.8 (95% CI, 19.6-31.4) months for Q61H and 17.8 (95% CI, 16.3-23.1) months for G12V variants. The majority of KRAS-mutated BTCs (98.9%) were not microsatellite instability-high and had low tumor mutational burden (ranging from a median [IQR] of 1.2 (1.2-2.5) to a mean [SD] of 3.3 [1.3]). Immune profiling through RNA sequencing of KRAS and NRAS-mutated samples showed a pattern toward a more immune-inflamed microenvironment with higher M1 macrophage activation (0.16 vs 0.12; P = .047) and interferon-γ expression compared with wild-type tumors. The G12D variant remained the most common KRAS allelic variant in all patient ancestries. Patients with admixed American ancestry had the highest proportion of G12D variant (45.0%). This cohort study found that KRAS allelic variants were relatively common and may be potentially actionable genomic alterations in patients with BTCs, especially perihilar cholangiocarcinoma and extrahepatic cholangiocarcinoma. The findings add to the growing data on genomic and immune landscapes of KRAS allelic variants in BTCs and are potentially of value to the planning of specific therapies for this heterogeneous patient group.

Mature cystic teratoma with co-existent mucinous cystadenocarcinoma: describing a diagnostic challenge—a case report

BackgroundMature cystic teratoma co-existing with a mucinous cystadenocarcinoma is a rare tumor that few cases have been reported until now. In these cases, either a benign teratoma is malignantly transformed into adenocarcinoma or a collision tumor is formed between a mature cystic teratoma and a mucinous tumor, which is either primarily originated from epithelial-stromal surface of the ovary, or secondary to a primary gastrointestinal tract tumor. The significance of individualizing the two tumors has a remarkable effect on further therapeutic management.Case presentationIn this case, a mature cystic teratoma is co-existed with a mucinous cystadenocarcinoma in the same ovary in a 33-year-old Iranian female. Computed Tomography (CT) Scan with additional contrast of the left ovarian mass suggested a teratoma, whereas examination of resected ovarian mass reported an adenocarcinoma with a cystic teratoma. A dermoid cyst with another multi-septate cystic lesion including mucoid material was revealed in the gross examination of the surgical specimen. Histopathological examination revealed a mature cystic teratoma in association with a well-differentiated mucinous cystadenocarcinoma. The latter showed a CK7−/CK20 + immune profile. Due to the lack of clinical, radiological, and biochemical discoveries attributed to a primary lower gastrointestinal tract tumor, the immune profile proposed the chance of adenocarcinomatous transformation of a benign teratoma.ConclusionsThis case shows the significance of large sampling, precise recording of the gross aspects, histopathological examination, immunohistochemical analysis, and the help of radiological and clinical results to correctly diagnose uncommon tumors.

Positive impacts of Nannochloropsis oculata supplementation on gene expression of immune and antioxidant markers and metabolic profile of Barki sheep in the transition period and lipogenic effects on progeny.

Nannochloropsis species should be given priority when it comes to microalgae that should be added to feed since they are suitable for intense culture and have a high concentration of PUFAs (especially EPA), antioxidants, and certain vitamins. This study investigated the possible immune and antioxidant impacts of Nannochloropsis supplementation on Barki ewes during transition period and their newly born lambs. Three weeks prior to the expected time of lambing, the researched ewes were divided into two equal groups of thirty ewes each. The second group, on the other hand, was fed the same base diet as the first group plus 10g of commercially available Nannochloropsis powder per kg of concentrate, given daily to each ewe's concentrate. Findings revealed that supplementation of ewes with Nannochloropsis significantly up-regulated the expression pattern of immune (NFKB, RANTES, HMGB1, TNF-α, IRF4, TLR7, CLA-DRB3.2, IL1B, IL6, CXCL8, S-LZ, and Cathelicidin), and antioxidant (SOD1, CAT, GPX1, GST, ATOX1, Nrf2 and AhpC/TSA) markers in ewes post-lambing and their newly born lambs. Additionally, mRNA levels of lipogenic (ACACA, FASN SCD, LPL, and BTN1A) markers were significantly up-regulated in lambs from supplemented ewes than control ones. There was a significant increase in the WBCs, Hb, RBc count, serum level of glucose, total protein, triacylglycerol and total cholesterol, GPx, catalase, IL1α and IL6 with significantly decreased serum level of TNF-α and MDA in supplemented ewes after lambing as compared with control ones. There was also a significant increase in WBCs, Hb, RBc count, birth weight and body temperature with significantly decreased in the serum levels of TNF-α and stillbirth of newly born lambs from supplemented ewes as compared to other lambs from control ones.

Bioinformatics analysis of PANoptosis regulators in the diagnosis and subtyping of steroid-induced osteonecrosis of the femoral head.

In this study, we aimed to investigate the involvement of PANoptosis, a form of regulated cell death, in the development of steroid-induced osteonecrosis of the femoral head (SONFH). The underlying pathogenesis of PANoptosis in SONFH remains unclear. To address this, we employed bioinformatics approaches to analyze the key genes associated with PANoptosis. Our analysis was based on the GSE123568 dataset, allowing us to investigate both the expression profiles of PANoptosis-related genes (PRGs) and the immune profiles in SONFHallowing us to investigate the expression profiles of PRGs as well as the immune profiles in SONFH. We conducted cluster classification based on PRGs and assessed immune cell infiltration. Additionally, we used the weighted gene co-expression network analysis (WGCNA) algorithm to identify cluster-specific hub genes. Furthermore, we developed an optimal machine learning model to identify the key predictive genes responsible for SONFH progression. We also constructed a nomogram model with high predictive accuracy for assessing risk factors in SONFH patients, and validated the model using external data (area under the curve; AUC = 1.000). Furthermore, we identified potential drug targets for SONFH through the Coremine medical database. Using the optimal machine learning model, we found that 2 PRGs, CASP1 and MLKL, were significantly correlated with the key predictive genes and exhibited higher expression levels in SONFH. Our analysis revealed the existence of 2 distinct PANoptosis molecular subtypes (C1 and C2) within SONFH. Importantly, we observed significant variations in the distribution of immune cells across these subtypes, with C2 displaying higher levels of immune cell infiltration. Gene set variation analysis indicated that C2 was closely associated with multiple immune responses. In conclusion, our study sheds light on the intricate relationship between PANoptosis and SONFH. We successfully developed a risk predictive model for SONFH patients and different SONFH subtypes. These findings enhance our understanding of the pathogenesis of SONFH and offer potential insights into therapeutic strategies.

Abstract PO2-25-05: Spatial multiplexing of protein biomarkers for immune cell profiling of the tumor microenvironment with ChipCytometry

Abstract Understanding the spatial distribution of key immune cell populations is critical in advancing our understanding of cancer and the development of novel therapeutics. Historically, the spatial analysis of the tumor microenvironment (TME) has been limited to relatively low-plex immunohistochemical (IHC) or immunofluorescent (IF) assays, which were inadequate for deep immune cell profiling of the TME. Here, we present the analysis of fresh-frozen human breast tissue using spatial multiplexing technology called ChipCytometry, which combines iterative immuno-fluorescent staining using open-source antibodies for quantitative measure of virtually unlimited protein biomarkers on the same sample and high dynamic range imaging to facilitate quantitative phenotyping with single-cell resolution. In addition to traditional pathology review of the high-plex dataset, subsequent image analysis can be done using open-source or commercial software for advanced spatial analyses, including cluster analysis, neighborhood analysis and advanced data visualization. The results show precise expression levels for each of the protein biomarkers in the assay for each individual cell within the sample while maintaining spatial information about each cell. Dozens of immune cell subtypes were identified and quantified based on protein expression profiles. Spatial analysis of the samples revealed quantifiable heterogeneity of immune cell infiltration within the tumor samples, demonstrating the utility of the ChipCytometry platform for in-depth immune profiling in clinical samples. The ChipCytometry platform enables simultaneous detection of multiple protein biomarkers on a single tissue section for deep immune cell profiling in the TME. Combined with the single-cell spatial information, such datasets provide an opportunity for the discovery of new complex multiplexed biomarker signatures to inform therapeutic development and personalized medicine. Citation Format: Benton Berigan. Spatial multiplexing of protein biomarkers for immune cell profiling of the tumor microenvironment with ChipCytometry [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-25-05.

Abstract PO3-19-02: White Button Mushroom and Biomarkers of Immune Cell and Inflammatory Responses in Obese Postmenopausal Women at High Risk of Breast Cancer

Abstract Background: White Button Mushroom (WBM), Agaricus bisporus, has anti-tumor and anti-inflammatory effects. Oral WBM mitigated the effects of a high fat diet on hepatic steatosis and insulin resistance in ovariectomized mice. A Phase I trial of WBM in men with prostate cancer demonstrated decreased myeloid derived suppressor cells (MDSC) in those with decreased PSA; MDSC have been linked to different cancers, including breast, and conditions of chronic inflammation. Design: This is a preliminary evidence trail in postmenopausal, obese women at high risk of breast cancer with a single intervention arm of 14g of WBM taken as 28 tablets daily for 3 months to assess effects on inflammation. Participants undergo a physical exam, measuring height, weight, and waist circumference, an optional fine needle aspiration (FNA) of breast tissue, a blood draw, a food frequency questionnaire and a 24-hour food recall at baseline and final visits. Participants are contacted monthly for tolerance and adherence. Blood samples and FNA adipose are used for metabolic measurements and processed for biomarkers of inflammation and immune response. Peripheral blood mononuclear cells (PBMC) will be used for RNA single-cell analysis to assess for immune cell profiles and expression with an interest in evaluating MDSC. Red blood cells will be assessed for fatty acid profiles. Eligibility: Postmenopausal, obese (BMI ≥30, Asian ≥25) women with high risk for breast cancer who can swallow pills are eligible. Patients are high risk if their relative risk of breast cancer is ≥2 times that of the general population; this includes women with a strong family history, a genetic mutation associated with hereditary breast cancer, a high-risk breast histology, Gail model 5-year risk >1.67, or a previous history of stage I-II breast cancer. Participants are excluded for an active malignancy within 5 years, ongoing active or preventative cancer treatment, or lab abnormalities of serum creatinine and total bilirubin >1.5 the upper limit of normal, hemoglobin of < 9.0 g/dL, platelets of < 100,000/mm3, or a total white blood cell count of < 3,500/mm3. Participants cannot be taking mushroom supplements, immunosuppressants, or non-steroidal anti-inflammatories. Specific Aims: This study aims to evaluate the effects of WBM on regulating immune response and inflammation in obese women at high risk for breast cancer. Our primary objective is the assessment of PBMC, specifically MDSC immune profiles before and after treatment. Our secondary objective is to evaluate changes in biomarkers of inflammation, immune cell profiles in breast adipose tissue, BMI, waist circumference, and metabolic biomarkers. Additionally, the study aims to assess tolerability of the WBM dose and effects on diet quality. Statistical Methods: 26 patients will provide 80% power to detect a change in %MDSC of -0.67, assuming a standard deviation of 1.15, and a 2-sided type I error of 5%. We will have similar power to evaluate changes in other immune subsets, inflammatory markers, metabolic markers, anthropometric measurements, and diet quality. FNA adipose tissue analysis will be used as a safety signal to evaluate the potential for unwanted inflammation with taking WBM. 13 patients with FNA will provide 80% power to detect a change of 85% of the standard deviation of the difference in the measure of biomarkers such as Interleukin-6 with a 2-sided type I error of 5%. As a safety signal, the signal will be reported without adjustment for multiple comparisons. Any conclusions based on such secondary endpoints will make note of the multiple comparison issue. Accrual: The accrual target is 26 evaluable patients with an estimated need to enroll 31. The study has enrolled 27 patients, 14 completed the study and 5 are currently on treatment. 8 participants were removed; 3 never started treatment, 1 was diagnosed with breast cancer, and 4 had other medical issues preventing continuation. Contact: Jessica Dzubnar, MD: jdzubnar@coh.org Citation Format: Jessica Dzubnar, Lisa Yee, Shiuan Chen. White Button Mushroom and Biomarkers of Immune Cell and Inflammatory Responses in Obese Postmenopausal Women at High Risk of Breast Cancer [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 PO3-19-02.

Abstract PS03-01: Single cell analysis of paired lymph nodes and primary tumors in breast cancer patients

Abstract A sentinel lymph node (SN) is the primary node draining the tumor and is assumed to be affected early in the metastatic process. The sentinel node holds a key position in the immune response against tumor in breast, and represents a unique connection between the tumor and the host immune response. We hypothesize that the immune profile in the primary tumor and the paired lymph node (LN) is different during tumor progression. 3.7 million single cells from paired primary tumor and lymph nodes from 33 breast cancer patients (Oslo2 cohort) was analyzed by single cell mass cytometry (CyTOF) with a 47 antibody immune panel and characterized by a semi-automatic gating approach (FlowSOM). Tumor cells were found in 11 LN. When analyzing the leucocytes from LN we identified a significant difference in immune cell type skewing towards higher abundance of memory CD4 and CD8 T cells expressing an exhausted phenotype in LN with metastasis. In addition, a higher abundance of activated Tregs and significantly lower abundance of resting Tregs was found in the metastatic LNs compared to the sentinel lymph nodes. The change in immune composition and exhaustion was correlated to the metastatic tumor burden. The skewing towards an exhausted immune profile was also found in larger primary tumors compared to smaller primary tumors. We further analyzed tumor cells from 8 patients with paired primary tumor and LN. No differences were identified in the primary tumor when stratified by LN status, but LNs with smaller metastasis expressed lower levels of epithelial markers essential in the Epithelial-to-Mesenchymal Transition such as E-cadherin, Pan Cytokeratin and EpCAM – this in contrast to the LN with manifested metastasis in the axilla which expressed higher levels of epithelial markers and lower levels of mesenchymal markers such as Vimentin and CD44. We identified a skewing in the immune profile from a naive phenotype towards a memory and exhausted phenotype in CD8 and CD4 T cell population in LN with manifested metastasis, as well as in larger primary tumors. We also identified that tumor cells in smaller metastatic tumors resembled a “mesenchymal like” phenotype compared to in the larger manifested tumors. These results suggest that the immune suppression is correlated with the tumor burden. Citation Format: Marit Fjørtoft, Inga Rye, Kanutte Huse, June Myklebust, Hege Russnes, Margit Riis, Ole Christian Lingjærde, Øystein Garred, Karin Teien Lande, Inger Riise Bergheim, Ellen Schlichting. Single cell analysis of paired lymph nodes and primary tumors in breast cancer patients [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 PS03-01.

Abstract PO5-25-03: Immune cell infiltrate profiles in primary Triple Negative Breast Cancer and co-relation with treatment response and survival outcomes

Abstract Introduction Tumor microenvironment (TME) in breast cancer is a heterogeneous landscape with multiple cell types, each modulating the tumor progression and the disease prognosis. These cell types include lymphocytes, RBCs, stromal cells, macrophages, cancer-associated fibroblasts (CAFs), and cancer stem cells. Each cell type in the TME influences tumor progression and clinical outcomes for patients in a good or bad way, depending on the interaction with tumor cells. Tumor-infiltrating lymphocytes (TILs) are shown to be prognostic for therapy response in many cancers in the western cohorts. Our recent study showed that a higher proportion of Indian TNBC patients had intra-tumoral TILs compared to Western cohorts, and these are predictive of better response to NACT and longer disease-free survival in patients, making it a distinct finding from Western reports. In the follow-up study, we are characterizing infiltrating immune cell subtypes and their comparative association with treatment response in TNBC patients. Results from preliminary analysis and comparative analysis between responders and non-responders will be presented here. We speculate that the pro- and anti-tumorigenic immune cell population and its spatial information would help us understand TME and its precise role in response to therapy. Methods A retrospective cohort of breast cancer patients from a single one-surgeon breast cancer clinic with a uniform treatment strategy was evaluated for this study. FFPE primary tissue samples with variable TILs scores (moderate to high), taken from patients who showed complete response to the NACT treatment (n=6) and non-responding group (n=8) determined by H&E stained slides from our cohort, were further stained for immune cells (Pan-B cell, Pan-T cells, Pan-macrophages) and T cells (CD4; Helper T cell marker, CD8; Cytotoxic T cell marker), FOXP; Regularly CD4+ cell marker) and PanCK (Pan-tumor marker) and counterstained with DAPI for multiplex imaging. Multispectral images for these slides were obtained using the Leica Aperio Versa system. Parameters such as individual cell types of identification and scoring, spatial proximity to tumor cells, and individual cell types will be determined by HALO software using the High plex module. Results A total of 14 TNBC samples were stained for the T-cell subtypes panel and Immune cell subtype s panel to determine the TIME. DAPI stain was used for nuclear counting based the parameters such as threshold intensity, nuclear size, roundness, and aggressiveness. The cytoplasmic size was selected as 2um beyond the outside nuclear boundary. Using Halo-Indica software, around 50000-300000 cells/tissue were evaluated across all samples from mIF whole slide scans of biopsy samples. Cell count was validated by cross-checking individual ROIs and manual cell count using Fiji. A Highplex module was used to identify individual signal positivity for each marker based on threshold intensity and cell completeness percent. Preliminary data showed a higher proportion of CD8+ cells and macrophages population are spatially closer to the tumor cells in Responders compared to non-responders. Conclusion Immune profiles are distinct among responding and non-responding patients in the Indian TNBC cohort. The association of these profiles with long-term survival in patients and various clinical and pathological parameters will help us determine the clinical relevance of these profiles and identify distinct immune subgroups as prognostic in Indian TNBCs. Citation Format: Pooja Vaid, Devaki Kelkar, LS Shashidhara, C B Koppiker, Madhura Kulkarni. Immune cell infiltrate profiles in primary Triple Negative Breast Cancer and co-relation with treatment response and survival outcomes [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 PO5-25-03.

Abstract PO3-19-03: Addressing USPSTF 2023 Identified Key Gaps in Knowledge in Breast Cancer Screening through TMIST (ECOG-ACRIN EA1151) or its Ancillary Studies

Abstract Background The United States Preventative Services Task Force in their 2023 recommendations identified areas where more research data is needed to inform future breast cancer screening recommendations. Research areas identified are: improve clinicians and patients understanding and evaluation of dense breast tissue on a screening mammogram, benefits and harms of supplemental screening using ultrasound or MRI for women with dense breasts, health outcomes such as rates of breast cancer diagnosis requiring treatment, rates of advanced breast cancers diagnosed across consecutive screening rounds, and breast cancer-associated morbidity and mortality, causes of increased risk of breast cancer mortality in black women across spectrum of stages and biomarker patterns, understand why black women are more likely to be diagnosed with breast cancers that have biomarker patterns that are indicative of poor health outcomes, assess benefits/harms differences between annual and biennial screening for breast cancer in women overall and if there are differences between black and white women, approaches to reduce the risk of overdiagnosis leading to overtreatment of breast lesions found at screening that may not cause morbidity and mortality, natural history of DCIS, and identify prognostic indicators of breast tumors that are unlikely to affect quality or length of life. Methods The ongoing TMIST study, currently with 88,801 asymptomatic women presenting for screening mammography ages 45-74 enrolled out of 128,905, could contribute to scientific evidence to support the above research areas through existing study aims and planned ancillary studies. Supplemental Screening with US and MRI: TMIST PreSCRIB will utilize Machine Learning applied to TMIST and All of Us data, including genetics, mammograms, social determinates of health and other data to recommend individualized screening strategies for women. DxMRI is a study where women will get AbMRI at time of Dx work-up. There are plans to use these examinations plus supplemental screening MRIs performed on TMIST subjects in an enriched reader study to evaluate the role of supplemental screening MRI in moderate risk women. Rates of breast cancer treatment, consecutive screening, morbidity, and mortality: TMIST’s primary outcome is the proportion of women experiencing an advanced breast cancer and needing treatment. TMIST is also collecting information on health care utilization following a cancer diagnosis, including types of treatment given, and costs data from the screening and diagnostic work-up visits, and mortality data for study participants. Increased risk of breast cancer mortality in black women: TMIST is performing PAM50 plus p53 status, immune profile, DNA repair phenotype, and 21-gene recurrence assay on all breast cancers and a subset of benign tissue. Blood and buccal smears might also help address this issue. Ongoing work, funded by the Susan B. Komen Foundation, focuses on improving Black participation in TMIST Biorepository (currently about 45% participation of the 21% of TMIST US black subjects). Surveys are planned on perceived racism and social determinates of health as part of DxMRI Study. Screening Frequency: We are developing a collaboration with the UK-based clinical trial PROSPECTS to compare rates of all cancers and advanced cancers for annual, biennial, and 3-year screening. Overdiagnosis, natural history of DCIS, prognostic indicators of breast tumors not impacting quality of life: PRoGram- will use radiomics, genomics and pathomics to develop a greater understanding of the variability of the non-advanced cancers diagnosed in the TMIST population, including DCIS. It is hoped that this model will provide greater understanding of the risk of poor outcomes for women diagnosed with lower risk cancers, including DCIS. Citation Format: Etta Pisano, Constantine Gatsonis, Mitchell Schnall, Melissa Troester, Elodia Cole, Jean Cormack, Ilana Gareen, Martin Yaffe, Laura Collins, Amarinthia Curtis, Ruth Carlos, Kathy Miller, Christopher Comstock. Addressing USPSTF 2023 Identified Key Gaps in Knowledge in Breast Cancer Screening through TMIST (ECOG-ACRIN EA1151) or its Ancillary Studies [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 PO3-19-03.