Inflamed Group Research Articles

Tissue-matched analysis of MRI evaluating the tumor infiltrating lymphocytes in hepatocellular carcinoma.

Tumor-infiltrating lymphocytes (TILs) play critical roles in the tumor microenvironment and immunotherapy response. This study aims to explore the feasibility of multi-parametric magnetic resonance imaging (MRI) in evaluating TILs and to develop an evaluation model that considers spatial heterogeneity. Multi-parametric MRI was performed on hepatocellular carcinoma (HCC) mice (N = 28). Three-dimensional (3D) printing was employed for tissue sampling, to match the multi-parametric MRI data with tumor tissues, followed by flow cytometry analysis and next-generation RNA-sequencing. Pearson's correlation, multivariate logistic regression, and receiver operating characteristic (ROC) curve analyses were utilized to model TIL-related MRI parameters. MRI quantitative parameters, including T1 relaxation times and perfusion, were correlated with the infiltration of leukocytes, T-cells, CD4+ T-cells, CD8+ T-cells, PD1 + CD8+ T-cells, B-cells, macrophages, and regulatory T-cells (correlation coefficients ranged from -0.656 to 0.482, p <.05) in tumor tissues. TILs were clustered into inflamed and non-inflamed subclasses, with the proportion of T-cells, CD8+ T-cells, and PD1 + CD8+ T-cells significantly higher in the inflamed group compared to the non-inflamed group (43.37% vs. 25.45%, 50.83% vs. 34.90%, 40.45% vs. 29.47%, respectively; p <.001). The TIL evaluation model, based on the Z-score combining Kep and T1post, was able to distinguish between these subgroups, yielding an area under the curve of 0.816 (95% confidence interval 0.721-0.910) and a cut-off value of -0.03 (sensitivity 68.4%, specificity 91.3%). Additionally, the Z-score was related to the gene expression of T-cell activation, chemokine production, and cell adhesion. The tissue-matched analysis of multi-parametric MRI offers a feasible method of regional evaluation and can distinguish between TIL subclasses.

Evaluating serum proteinogram methodologies for the diagnosis of inflammation in fish: Acute and chronic patterns in gilthead seabream (Sparus aurata) injected with λ-carrageenan.

Proteinogram is a semiquantitative method specially used in clinic to separate the serum proteins from patients for use in the diagnosis of diseases. However, this methodology has only been applied very recently with this approach in farmed fish. Thus, the aim of this study was to explore the changes in the serum proteinogram of gilthead seabream (Sparus aurata), after triggering an acute or chronic sterile inflammation. For this, two experiments were carried out: i) Acute inflammation experiment: seabream specimens were injected intramuscularly with 50 μL of λ-carrageenan (0.5 mg fish-1) or buffer (control) and blood samples were collected at 3, 6 and 24 h post-injection; ii) Chronic inflammation experiment: specimens were injected at 0, 7 and 14 days with 500, 250 and 250 μL of λ-carrageenan, respectively (20 mg fish-1) or buffer, and blood samples were collected at 15 days post-injection. In both cases, serum was obtained and processed by electropherograms and HPLC-mass spectrometry. Results of electropherograms of control fish revealed four major proteins of 19.5, 76.3, 104.4, and 156.7 kDa in the serum. These four proteins were correlated with apolipoprotein A-II (II (the counterpart of mammalian albumin, Apo fraction), serotransferrin (β fraction), inter-α-trypsin inhibitor heavy chain H3-like (α1 fraction) and α-2-macroglobulin-like (α2 fraction) according to the results obtained with HPLC-mass spectrometry. In a statistical view (p<0.05), no variations were detected in the four major serum protein bands between the control and the acutely inflamed groups. However, in chronically inflamed fish, the Apo fraction decreased statistically compared to the control group. In contrast, the α1 and α2 fractions were statistically increased in the serum of fish sampled 15 days after λ-carrageenan injection, compared to those found in the control fish. α1 and α2 protein fractions are recognized indicators of inflammation in mammals. Consequently, our study presents a novel method for assessing both acute and chronic λ-carrageenan-induced sterile inflammation in gilthead seabream, which could be applicable to other marine species for diagnostic purposes.

The anti-angiogenic, anti-inflammatory and anticoagulant potential of a polysaccharide extracted from the brown alga Cystoseira humilis

The present study aimed to evaluate the properties of a sulfated polysaccharide (PSCH) extracted from the brown alga Cystoseira humilis. We analyzed the chemical composition, anti-angiogenesis activities, anticoagulant, and anti-inflammatory effects of the PSCH, using various techniques encompassing UV–visible spectroscopy, fourier transform infrared (FT-IR) spectroscopy, X-ray diffraction (X-RD), scanning electron microscopy (SEM), steric exclusion chromatography (SEC) and high-performance liquid chromatography coupled to refractive index detector (HPLC-RID). The study found that PSCH was a sulfated heteropolysaccharide consisting of glucuronic acid, arabinose, galacturonic acid, xylose and fructose, and has a semi-crystalline nature with a molecular weight of about 379.44 kDa. The PSCH contains 41.77 % carbohydrates, 12.33 % proteins, and 4.59 % sulfates. The PSCH fraction has anti-angiogenic potential highlighted through assays on the chorioallantoic membrane, where vascular branching was inhibited by about 41.65 % and 61.38 % at concentrations of 50 and 100 μg/egg, respectively, compared to the untreated group. PSCH displayed an anti-edematous effect in vivo by reducing malondialdehyde and advanced oxidation protein products in paw tissue after five hours of carrageenan injection. When rats were co-treated with PSCH, there was evidence of a significant increase in antioxidant enzyme activity (superoxide dismutase, glutathione peroxidase, and glutathione) in edematous paw tissues compared to untreated rats. Furthermore, PSCH significantly influenced hematological parameters and the rate of anti-inflammatory serum protein fractions compared to the inflamed group. Interestingly, PSCH may exhibit anticoagulant properties by extending activated partial thrombosis time, thrombin time and prothrombin time. The present study has shown that Cystoseira humilis polysaccharides have antioxidant, anti-angiogenic, anti-inflammatory, and anti-coagulant properties, making them suitable for therapeutic purposes.

Artificial Intelligence-Powered Spatial Analysis of Tumor-Infiltrating Lymphocytes as a Potential Biomarker for Immune Checkpoint Inhibitors in Patients with Biliary Tract Cancer.

Recently, anti-programmed cell death-1/anti-programmed cell death ligand-1 (anti-PD1/L1) immunotherapy has been demonstrated for its efficacy when combined with cytotoxic chemotherapy in randomized phase 3 trials for advanced biliary tract cancer (BTC). However, no biomarker predictive of benefit has been established for anti-PD1/L1 in BTC. Here, we evaluated tumor-infiltrating lymphocytes (TIL) using artificial intelligence-powered immune phenotype (AI-IP) analysis in advanced BTC treated with anti-PD1. Pretreatment hematoxylin and eosin (H&E)-stained whole-slide images from 339 patients with advanced BTC who received anti-PD1 as second-line treatment or beyond, were employed for AI-IP analysis and correlative analysis between AI-IP and efficacy outcomes with anti-PD1. Next, data and images of the BTC cohort from The Cancer Genome Atlas (TCGA) were additionally analyzed to evaluate the transcriptomic and mutational characteristics of various AI-IP in BTC. Overall, AI-IP were classified as inflamed [high intratumoral TIL (iTIL)] in 40 patients (11.8%), immune-excluded (low iTIL and high stromal TIL) in 167 patients (49.3%), and immune-desert (low TIL overall) in 132 patients (38.9%). The inflamed IP group showed a substantially higher overall response rate compared with the noninflamed IP groups (27.5% vs. 7.7%, P < 0.001). Median overall survival and progression-free survival were significantly longer in the inflamed IP group than in the noninflamed IP group (OS, 12.6 vs. 5.1 months; P = 0.002; PFS, 4.5 vs. 1.9 months; P < 0.001). In the TCGA cohort analysis, the inflamed IP showed increased cytolytic activity scores and IFNγ signature compared with the noninflamed IP. AI-IP based on spatial TIL analysis was effective in predicting the efficacy outcomes in patients with BTC treated with anti-PD1 therapy. Further validation is necessary in the context of anti-PD1/L1 plus gemcitabine-cisplatin.

Comparison of Biological Augmentation in Rotator Cuff Repair Using Inflamed Versus Noninflamed Bursal Tissue in Rats

PurposeTo examine how augmentation of a rotator cuff repair with inflamed versus non-inflamed bursal tissue affects tendon-to-bone healing in a rat model of rotator cuff repair. Methods136 Sprague-Dawley rats were randomly assigned to an inflamed or non-inflamed bursal tissue application group. After detachment the supraspinatus tendon was re-attached with bursal tissue sewn onto the tendon-to-bone interface. The specimens were analysed biomechanically 6 and at 7 weeks and immunohistologically at 1 and at 7 weeks after surgery. ResultsImmunohistological results showed no significant difference in the percentage of collagen type II in the tendon-to-bone interface at 1 (p = 0.97) and 7 weeks (p = 0.42) when utilising autologous non-inflamed bursal tissue in comparison to inflamed bursal tissue specimens. The inflamed bursa group also showed no significant difference in collagen I to III quotient (p= 0.14) after surgery in comparison to post-surgery non-inflamed bursa groups. Biomechanical assessment showed that tendon stiffness (p = 0.87 resp. p = 0.1) and the tendon viscoelasticity (p = 0.12 resp. p = 0.07) was the same after 6 and 7 weeks comparing inflamed bursa to the non-inflamed bursa group. There was no significant difference (p = 0.8 resp. p = 0.97) in load to failure between in both inflamed and non-inflamed bursa groups after 6 and 7 weeks. ConclusionAutologous inflamed bursal tissue derived from the Achilles bursa and implanted to the tendon-to-bone interface after rotator cuff repair facilitates the same histological and biomechanical healing response as using a non-inflamed bursa interposition in rats. Clinical relevanceDuring augmentation of a rotator cuff repair, it is irrelevant whether the bursa tissue is inflamed or not.

Dietary Tryptophan Plays a Role as an Anti-Inflammatory Agent in European Seabass (Dicentrarchus labrax) Juveniles during Chronic Inflammation.

Where teleost fish are concerned, studies in tryptophan immunomodulation generally point to immunosuppressive properties, thus presenting a potential anti-inflammatory dietary strategy. The goal of the present work was to evaluate the effects of tryptophan dietary supplementation on immune and neuroendocrine responses of the European seabass, Dicentrarchus labrax, undergoing chronic inflammation. Juvenile European seabass were intraperitoneally injected with either Freund's Incomplete Adjuvant (FIA, inflamed group) or a saline solution (control group). Within each group, fish were fed a control (CTRL) and a CTRL-based diet supplemented with tryptophan (0.3% DM basis; TRP) for 4 weeks. Different tissues were sampled every week for the assessment of immune-related parameters. When TRP was provided to FIA-injected fish, mcsfr gene expression increased from 1 to 2 weeks and remained high until the end of the experiment. The same fish showed a concurrent increase in peripheral monocyte counts. Moreover, il34 expression at 1 week post-FIA injection was higher in TRP-fed than in CTRL-fed fish. After one week, molecular patterns of anti-inflammatory processes seemed to be favoured by TRP (mcsfr, gr1, il34 and tgfβ). Altogether, the results show that the feeding period seems to be critical where tryptophan supplementation is concerned since at later inflammatory stages-and longer feeding periods-fish fed TRP displayed a molecular profile similar to that of the CTRL group. In contrast, shorter administration periods might accelerate immune regulatory pathways.

Recombinant probiotic Lactococcus lactis delivering P62 mitigates moderate colitis in mice.

p62 is a human multifunctional adaptor protein involved in key cellular processes such as tissue homeostasis, inflammation, and cancer. It acts as a negative regulator of inflammasome complexes. It may thus be considered a good candidate for therapeutic use in inflammatory bowel diseases (IBD), such as colitis. Probiotics, including recombinant probiotic strains producing or delivering therapeutic biomolecules to the host mucosal surfaces, could help prevent and mitigate chronic intestinal inflammation. The objective of the present study was to combine the intrinsic immunomodulatory properties of the probiotic Lactococcus lactis NCDO2118 with its ability to deliver health-promoting molecules to enhance its protective and preventive effects in the context of ulcerative colitis (UC). This study was realized in vivo in which mice were supplemented with the recombinant strain. The intestinal barrier function was analyzed by monitoring permeability, secretory IgA total levels, mucin expression, and tight junction genes. Its integrity was evaluated by histological analyses. Regarding inflammation, colonic cytokine levels, myeloperoxidase (MPO), and expression of key genes were monitored. The intestinal microbiota composition was investigated using 16S rRNA Gene Sequencing. No protective effect of L. lactis NCDO2118 pExu:p62 was observed regarding mice clinical parameters compared to the L. lactis NCDO2118 pExu: empty. However, the recombinant strain, expressing p62, increased the goblet cell counts, upregulated Muc2 gene expression in the colon, and downregulated pro-inflammatory cytokines Tnf and Ifng when compared to L. lactis NCDO2118 pExu: empty and inflamed groups. This recombinant strain also decreased colonic MPO activity. No difference in the intestinal microbiota was observed between all treatments. Altogether, our results show that recombinant L. lactis NCDO2118 delivering p62 protein protected the intestinal mucosa and mitigated inflammatory damages caused by dextran sodium sulfate (DSS). We thus suggest that p62 may constitute part of a therapeutic approach targeting inflammation.

MONITORING IN VITRO AND EX VIVO INFLAMMATION USING SINGLE-WALLED CARBON NANOTUBES (SWCNTS) SENSOR TECHNOLOGY

Osteoarthritis (OA) is a degenerative joint disease affecting millions worldwide. Early detection of OA and monitoring its progression is essential for effective treatment and for preventing irreversible damage. Although sensors have emerged as a promising tool for monitoring analytes in patients, their application for monitoring the state of pathology is currently restricted to specific fields (such as diabetes). In this study, we present the development of an optical sensor system for real-time monitoring of inflammation based on the measurement of nitric oxide (NO), a molecule highly produced in tissues during inflammation.Single-walled carbon nanotubes (SWCNT) were functionalized with a single-stranded DNA (ssDNA) wrapping designed using an artificial intelligence approach and tested using S-nitroso-N-acetyl penicillamine (SNAP) as a standard released-NO marker. An optical SWIR reader with LED excitation at 650 nm, 730 nm and detecting emission above 1000 nm was developed to read the fluorescence signal from the SWCNTs. Finally, the SWCNT was embedded in GelMa to prove the feasibility of monitoring the release of NO in bovine chondrocyte and osteochondral inflamed cultures (1–10 ng/ml IL1β) monitored over 48 hours. The stability of the inflammation model and NO release was indirectly validated using the Griess and DAF-FM methods. A microfabricated sensor tag was developed to explore the possibility of using ssDNA-SWCNT in an ex vivo anatomic set-up for surgical feasibility, the limit of detection, and the stability under dynamic flexion.The SWCNT sensor was sensitive to NO in both in silico and in vitro conditions during the inflammatory response from chondrocyte and osteochondral plug cultures. The fluorescence signal decreased in the inflamed group compared to control, indicating increased NO concentration. The micro-tag was suitable and stable in joints showing a readable signal at a depth of up to 6 mm under the skin.The ssDNA-SWCNT technology showed the possibility of monitoring inflammation continuously in an in vitro set-up and good stability inside the joint. However, further studies in vivo are needed to prove the possibility of monitoring disease progression and treatment efficacy in vivo.Acknowledgments: The project was co-financed by Innosuisse (grant nr. 56034.1 IP-LS)

The influence of inflammation on the characteristics of adipose-derived mesenchymal stem cells (ADMSCs) and tissue repair capability in a hepatic injury mouse model

BackgroundMesenchymal stem cells (MSCs) are adult stem cells with self-renewal and multi-directional differentiation potential and possess the functions of immunomodulation, regulation of cell growth, and repair of damage. Over recent years, MSCs have been found to regulate the secretion of inflammatory factors and to exert regulatory effects on various lymphocytes in inflammatory states, and on the subsequent repair of tissue damage caused by inflammation. In the present study, we analyzed the effects of tissue inflammation on the characteristics of MSCs.MethodsHuman fat derived from the infrapatellar fat pad (IPFP) of knees with differing degrees of inflammation was extracted from specimens derived from total knee arthroplasties. HE and immunohistochemical staining was performed to directly observe the evidence and degree of inflammation in human infrapatellar fat pad tissue in order to classify MSCs cells, by their origin, into highly inflamed and lowly inflamed groups, and to study the effect of tissue inflammation on cell acquisition rates via cellular counting data. Flow cytometry assays were performed to investigate the effect of tissue inflammation on MSC surface marker expression. Trilineage differentiation, including osteogenesis, adipogenesis, and chondrogenesis, was performed to assess the effect of tissue inflammation on the ability of MSCs to undergo directed differentiation. The effect of tissue inflammation on the ability of MSCs to proliferate was investigated via clone formation studies. RNA-sequencing was performed to evaluate the transcriptomes of MSCs derived from different areas of inflammation. The effect of tissue inflammation on tissue repair capacity and safety of MSCs was investigated via a murine model of acute liver injury.ResultsThe results of cell count data indicate that a high degree of tissue inflammation significantly decreases the acquisition rate of MSCs, and the proportion of CD34+ and CD146+ cells. The results of our trilineage differentiation assay show that a higher degree of inflammation decreases osteogenic differentiation and enhances adipogenic and chondrogenic differentiation of MSCs. However, these differences were not statistically significant. Clone formation assays indicate that the degree of tissue inflammation at the MSC source does not significantly affect the proliferative capacity of MSCs. The transcriptomes of MSCs remain relatively stable in fat pad tissues derived from both highly and lowly inflamed samples. The results of acute liver injury investigations in mice indicate that MSCs of high and low inflammatory tissue origin have no significant difference in their tissue repair capability.ConclusionsHigh tissue inflammation at the source of MSCs reduces the acquisition rate of MSCs and the percentage of CD34+ and CD146+ cells acquisition. However, source tissue inflammation may not significantly affect trilineage differentiation potential and proliferative capacity of MSCs. Also, MSCs obtained from differing source degrees of inflammation retain stable and similar transcriptomic profile and are both safe and efficacious for tissue repair/regeneration without detectable differences.Graphical

Mesenchymal Stem Cell Conditioned Medium Modulates Inflammation in Tenocytes: Complete Conditioned Medium Has Superior Therapeutic Efficacy than Its Extracellular Vesicle Fraction.

Tendinopathy, a prevalent overuse injury, lacks effective treatment options, leading to a significant impact on quality of life and socioeconomic burden. Mesenchymal stem/stromal cells (MSCs) and their secretome, including conditioned medium (CM) and extracellular vesicles (EVs), have shown promise in tissue regeneration and immunomodulation. However, it remains unclear which components of the secretome contribute to their therapeutic effects. This study aimed to compare the efficacy of CM, EVs, and the soluble protein fraction (PF) in treating inflamed tenocytes. CM exhibited the highest protein and particle concentrations, followed by PF and EVs. Inflammation significantly altered gene expression in tenocytes, with CM showing the most distinct separation from the inflamed control group. Treatment with CM resulted in the most significant differential gene expression, with both upregulated and downregulated genes related to inflammation and tissue regeneration. EV treatment also demonstrated a therapeutic effect, albeit to a lesser extent. These findings suggest that CM holds superior therapeutic efficacy compared with its EV fraction alone, emphasizing the importance of the complete secretome in tendon injury treatment.

More Than Pigments: The Potential of Astaxanthin and Bacterioruberin-Based Nanomedicines.

Carotenoids are natural products regulated by the food sector, currently used as feed dyes and as antioxidants in dietary supplements and composing functional foods for human consumption. Of the nearly one thousand carotenoids described to date, only retinoids, derived from beta carotene, have the status of a drug and are regulated by the pharmaceutical sector. In this review, we address a novel field: the transformation of xanthophylls, particularly the highly marketed astaxanthin and the practically unknown bacterioruberin, in therapeutic agents by altering their pharmacokinetics, biodistribution, and pharmacodynamics through their formulation as nanomedicines. The antioxidant activity of xanthophylls is mediated by routes different from those of the classical oral anti-inflammatory drugs such as corticosteroids and non-steroidal anti-inflammatory drugs (NSAIDs): remarkably, xanthophylls lack therapeutic activity but also lack toxicity. Formulated as nanomedicines, xanthophylls gain therapeutic activity by mechanisms other than increased bioavailability. Loaded into ad hoc tailored nanoparticles to protect their structure throughout storage and during gastrointestinal transit or skin penetration, xanthophylls can be targeted and delivered to selected inflamed cell groups, achieving a massive intracellular concentration after endocytosis of small doses of formulation. Most first reports showing the activities of oral and topical anti-inflammatory xanthophyll-based nanomedicines against chronic diseases such as inflammatory bowel disease, psoriasis, atopic dermatitis, and dry eye disease emerged between 2020 and 2023. Here we discuss in detail their preclinical performance, mostly targeted vesicular and polymeric nanoparticles, on cellular models and in vivo. The results, although preliminary, are auspicious enough to speculate upon their potential use for oral or topical administration in the treatment of chronic inflammatory diseases.

An inflamed subtype of difficult-to-treat depression

BackgroundChronic low-grade inflammation may play a role in the pathophysiology of depression, at least in a subset of patients. High-sensitivity C-reactive protein (hs-CRP) has been used to define an inflamed subgroup of depression with specific clinical characteristics and symptoms. In this study we investigated biochemical and clinical characteristics in patients with difficult-to-treat depression with and without chronic low-grade inflammation. MethodWe assayed plasma levels of interferon-gamma, tumor necrosis factor-alpha, Interleukin (IL)-10, IL-6, IL-8, and vitamin D in a clinically well-characterized sample of patients with difficult-to-treat depression (n = 263) and healthy controls (n = 46). Serum hs-CRP levels were available in the patient group and were used to define “inflamed depression” (hs-CRP > 3 mg/L). Based on previous studies correlating specific depressive symptoms to inflammatory markers, we calculated a composite score of inflammatory depressive symptoms (Infl-Dep score). A principal component analysis (PCA) was performed to identify patterns of variance in cytokines and vitamin D among patients. ResultsMean levels of IL-6 and IL-8 were significantly higher in depressed patients compared to controls, also after adjusting for sex, smoking, BMI, and age. None of the other inflammatory markers differed significantly between depressed patients and controls. Two components were extracted using PCA; one showed general cytokine elevations and one represented a pattern where IL-6 and IL-8 were inversely related to vitamin D (IL6-IL8-VitD component). The inflamed subgroup (hs-CRP > 3, n = 51) exhibited significantly higher BMI, higher Infl-Dep scores and higher IL6-IL8-VitD component scores than uninflamed patients (hs-CRP ≤ 3, n = 212). There were no significant differences in overall depression severity or suicidality between the inflamed and uninflamed groups. ConclusionOur results support the hypothesis of an inflamed subgroup of depression as a meaningful construct. This subgroup may have certain biological and clinical characteristics and more studies are needed to determine potential clinical implications.

Abstract 5446: Immune phenotypes classified by deep learning-based H&amp;E tissue analyzer demonstrate distinct immune landscape and transcriptomic features in ovarian cancer

Abstract Introduction: Deep learning-based H&amp;E slide analyzer, Lunit-SCOPE IO can classify the tumor microenvironment as three immune phenotypes (IPs): inflamed, immune-excluded and immune-desert. In our previous study, the IPs demonstrated distinct survival outcome and immunologic landscape in endometrial cancer. To further explore the application of IPs in other type of cancer, we applied Lunit-SCOPE in ovarian cancer (OV) and compared tumor microenvironments based on the IPs. Methods: H&amp;E slide images, RNA-sequencing data, and clinical data were obtained from TCGA OV cohort. Lunit-SCOPE IO is a H&amp;E slide analyzer using deep learning algorithm and was trained with 1,824 H&amp;E slides of advanced NSCLC. We used the analyzer to classify H&amp;E slide images from TCGA OV cohort to three IPs. Cibersort was used to assess immune cell infiltration across the IPs. The cytolytic activity score was obtained from geometric mean of GZMA and PRF1 RNA expression. We further assessed transcriptomic characteristics across IPs via differentially expressed genes analysis and gene set enrichment analysis (GSEA). Additionally, we compared CD274 gene expression across IPs. Finally, survival analysis was performed to evaluate survival outcome based on IPs. We used Mann-Whitney method to compare non-parametric variables between the IPs. Results: The proportion of inflamed, immune-excluded and immune-desert group in TCGA OV were 7 (10.2%), 13 (18.8%), and 49 (71%), respectively. Inflamed group showed significantly higher proportion of M1 macrophage compared with immune-desert group (6.6% vs. 2%, P=.012). We observed higher cytolytic activity score in inflamed group compared to immune-desert group (7.21 vs. 5.27, P=.016). In GSEA, inflamed group is significantly enriched in IL6 JAK STAT3 signaling, interferon gamma response, KRAS signaling up, and inflammatory response pathway (NES 2.19; adjusted P=.021, NES 1.70; adjusted P=.021, NES 1.62; adjusted P=.037, and NES 1.52; adjusted P=.037, respectively). Immune-desert is related to upregulated MYC target V2 pathway (NES -1.95; adjusted P=.037). Inflamed group did not show significant difference in CD274 expression, compared with others (Inflamed vs. immune-excluded, 4.97 vs. 4.19, P=.115 and Inflamed vs. immune-desert, 4.97 vs. 3.95, P=.058). Inflamed group did not show any significant difference in overall survival, compared with non-inflamed group (Inflamed vs. non-inflamed, HR 0.98; 95% CI 0.45 - 2.14; P=.958). Conclusion: Deep learning-based H&amp;E slide analyzer can be used to classify tumor H&amp;E slide images with OV into three IPs. In this study, the classification demonstrated distinct immunologic traits and transcriptomic characteristics in tumor microenvironment. The enrichment of interferon gamma response pathway in inflamed OV group may indicate opportunities for targeted immunotherapy. Citation Format: Horyun Choi, Youjin Oh, Chan-Young Ock, Kyunghyun Paeng, Young Kwang Chae. Immune phenotypes classified by deep learning-based H&amp;E tissue analyzer demonstrate distinct immune landscape and transcriptomic features in ovarian cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5446.

Abstract 4333: Spatial analysis of tumor-infiltrating lymphocytes in tumor microenvironment as biomarker for immune checkpoint inhibition in biliary tract cancer

Abstract Background: Recently, anti-PD-L1 in combination with cytotoxic chemotherapy has shown significant survival benefit in a randomized phase 3 trial for unresectable or metastatic biliary tract cancer (BTC). However, no biomarker including PD-L1 expression has been established to predict clinical outcomes, and there is an unmet need for a novel predictive biomarker for anti-PD-1 or PD-L1 therapy. Here, we assessed TILs using artificial intelligence (AI)-powered spatial analysis in advanced BTC treated with anti-PD-1 beyond 1st line treatment. Methods: An AI-powered whole-slide image (WSI) analyzer (Lunit SCOPE IO, Lunit, Seoul, Korea) was used to segment tumor epithelium and stroma, and identification of intratumor TIL (iTIL) and stromal TIL (sTIL). H&amp;E stained WSI from pre-treatment samples was acquired from Asan Medical Center (n =166), and a total of 154 samples (92.8%) after quality control were used for the final analysis. Immune phenotypes (IP) were defined as follow: inflamed as high iTIL and sTIL; immune-excluded as low iTIL and high sTIL; immune-desert as low TIL overall. Among them, 20 patients were available for multi-color flow cytometry analysis (FACS) using peripheral blood mononuclear cells, collected at baseline, C1D8, and C2D1. Results: All patients (n=154) were treated with anti-PD-1 (pembrolizumab or nivolumab) monotherapy, and 72 of 154 patients (46.8%) were treated as 2nd line. Gemcitabine plus cisplatin (GemCis) was used prior to anti-PD-1 as first-line therapy in all patients. Overall, 15 (9.7%) patients showed inflamed IP. With median follow-up duration of 15.4 months, the inflamed IP group showed better overall survival (17.2 vs. 6.6 months, P=0.03), and progression-free survival (PFS; 4.5 vs. 2.6 months, P=0.09) along with higher PFS rate at 12 months (33.3% vs. 11.5%, P=0.035), and overall response rate (26. 7% vs. 8.6%, P=0.053) than other phenotype groups. There was no significant difference in median PFS with GemCis among IP groups (P=0.74). In the FACS available subgroup, inflamed IP showed higher baseline central memory T (Tcm)+effector memory T (Tem)/Tnaive ratio than other IPs. With the administration of anti-PD-1, Tcm+Tem/Tnaive ratio was increased, while the proportion of PD1+CD8+T, CD39+CD8+T, CD103+CD8+T and Treg were decreased in the inflamed IP group than other phenotype groups. Conclusions: Immune phenotypes classified by AI-powered spatial TIL analysis was effective to predict the clinical outcomes of patients with advanced BTC treated with anti-PD-1 therapy. Citation Format: Yeong Hak Bang, Kyunghye Bang, Jin Ho Shin, Hyunseok Yoon, Kyu-Pyo Kim, Inkeun Park, Jae Ho Jeong, Heung-Moon Chang, Baek-Yeol Ryoo, Chiyoon Oum, Seulki Kim, Yoojoo Lim, Gahee Park, Chan-Young Ock, Changhoon Yoo. Spatial analysis of tumor-infiltrating lymphocytes in tumor microenvironment as biomarker for immune checkpoint inhibition in biliary tract cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4333.

Construction and analysis of a lncRNA–miRNA–mRNA competing endogenous RNA network from inflamed and normal synovial tissues after anterior cruciate ligament and/or meniscus injuries

Background: Despite ample evidence demonstrating that anterior cruciate ligament (ACL) and meniscus tears are associated with posttraumatic osteoarthritis (PTOA) development, the contributing factors remain unknown. Synovial inflammation has recently been recognized as a pivotal factor in the pathogenesis of OA. However, there is a lack of data on synovial profiles after ACL or meniscus injuries, which may contribute to PTOA. Methods: Twelve patients with ACL tears and/or meniscus injuries were recruited. During surgery, synovial tissues were obtained from the injured knees. The inflammation status of the synovium was characterized according to macroscopic criteria and histological synovitis grades. Then the synovial tissues were classified as control group or inflamed group. High-throughput RNA sequencing of the synovial samples (3 vs. 3) was conducted to identify differentially expressed (DE) RNAs. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and protein-protein interaction (PPI) analyses were performed to investigate DE mRNAs. Next, competing endogenous RNA (ceRNA) networks were constructed based on bioinformatics analyses. Associations of the identified DE genes (DEGs) with infiltrating immune cells were explored using Pearson correlation analysis. Results: The results showed that 2793 mRNAs, 3392 lncRNAs and 211 miRNAs were significantly DE between two groups. The top 3 significantly upregulated GO terms and KEGG pathways were immune response, adaptive immune response and immune system process, systemic lupus erythematosus, haematopoietic cell lineage and cytokine-cytokine receptor interaction, respectively. In PPI networks, the top 10 hub genes were IL6, CCR7, C3, CCR5, CXCR3, CXCL8, IL2, CCR3, CCR2 and CXCL1. Seven mRNAs (EPHA5, GSN, ORC1, TLN2, SOX6, NKD2 and ADAMTS19), 4 lncRNAs (MIR4435-2HG, TNXA, CEROX1 and TMEM92-AS1) and 3 miRNAs (miR-486-5p, miR-199a-3p and miR-21-3p) were validated by quantitative real-time polymerase chain reaction and sub-networks were constructed. In correlation analysis, MMP9 correlated positively with M0 macrophages and plasma cells, NKD2 positively with CD8 T cells, and CCR7 and IL2RB positively with naive B cells. Conclusion: Our study provides foundational synovial inflammation profiles following knee trauma. The ceRNA and PPI networks provide new insight into the biological processes and underlying mechanisms of PTOA. The differential infiltration profiles of immune cells in synovium may contribute to PTOA development. This study also highlights immune-related DEGs as potential PTOA treatment biomarkers.

IMMU-04. Transcriptional analysis reveals distinct microenvironmental subgroups across pediatric nervous system tumors

INTRODUCTION: Recent clinical trials of immune checkpoint inhibitors indicated 5-11% response rate in pediatric patients depending on cancer type and expression of target proteins. Currently, a systematic analysis characterizing the immune microenvironment of childhood tumors is lacking. The main objective of this study is to uncover the features of immune microenvironment in pediatric nervous system tumors (pedNST). METHODS: We compiled transcriptomes of 925 tumors from three initiatives, Therapeutically Applicable Research To Generate Effective Treatments (TARGET, n = 149), International Cancer Genome Consortium (ICGC, n = 195) and Children Brain Tumor Tissue Network (CBTN, n = 581). We analyzed the performance of immune deconvolution tools and used publicly available datasets to define immune genesets. We conducted a consensus analysis to assign genes to cell-types and identify immunological groups. RESULTS: We found wide variability in immune infiltration across and within cancer types ranging from cold tumors such as medulloblastoma (2.7% infiltrate) to infiltrated entities such as neurofibroma (22.6%). Consensus clustering revealed four distinct immune clusters. The pediatric inflamed group (10%) included MYCN non-amplified neuroblastoma and ATRT. The myeloid-predominant group (30%) showed decreased infiltration of lymphoid cells but enrichment of myeloid cell genesets. The pediatric-cold group (42%) harbored no enrichment of immune genesets and included 72% of ependymomas and 65% of medulloblastomas. The immune excluded group (18%) showed depletion of immune cell-types and included sonic-hedgehog medulloblastoma. 71% of pedNST belonged to the lymphocyte depleted or immunologically quiet clusters, indicating the cold immune microenvironment in pedNST compared to adult cancers. CONCLUSION: We report characteristics of the immune microenvironment in pedNST. We found an overall cold microenvironment with low lymphocyte infiltration in this population compared to common adult cancers. We identified ~10% of tumors harboring a relatively inflamed microenvironment. Our data uncover characteristics of immune infiltration in pediatric tumors with potential implications to guide therapy.

Comprehensive genomic and immune profiling defines immunotherapy treatment in patients with NSCLC with low PD-L1 IHC.

2623 Background: Immune checkpoint inhibitors (ICIs) have emerged as effective treatments in non-small cell lung cancer (NSCLC). While the clinical utility of single agent ICI or in combination with chemotherapy has been well established, there remains an unmet need for the development of biomarkers that can better predict response. To address this need, we developed and applied a combination genomic and immune biomarker strategy to ICI-treated NSCLC patients which identified distinct patient subgroups with differential benefit among single agent or combination ICI treatment strategies. Methods: A discovery cohort (DC) of 5450 tumors across 37 histologies were evaluated by comprehensive genomic and immune profiling of the tumor immune microenvironment. Individual and combination biomarker assessment included PD-L1 IHC, TMB, tumor inflammation (TIGS), cell proliferation (CP) and cancer testis antigen burden (CTAB). From this cohort, combinations of molecular and immune biomarkers were identified and applied to a retrospective cohort (RC) of 225 metastatic NSCLC patients treated with pembrolizumab + chemo or pembrolizumab alone to correlate with response. Comparison of objective response rates (ORR) was performed using Chi-square test. Kaplan-Meir analysis was performed to test for differences in overall survival (OS) and 1-year OS. Results: Unsupervised analysis of the DC revealed four distinct biomarker combination groups that describe underlying tumor immunobiology: tumor dominant (CTAB, TMB, CP High), proliferative (CP High), inflamed (TIGS High), and checkpoint (PDL1, TIGS and TMB High). Application of these biomarker groups to the RC demonstrated significant differences in response to ICI regimens between groups (p = 0.04). Patients in the proliferative group (35.1%, 79/225; median PD-L1 = 20% TPS) treated with single agent pembrolizumab showed a significantly higher ORR (59%; 16/27) compared to pembrolizumab + chemo (27%; 14/52; p = 0.005), significantly improved 1-yr OS (p = 0.03), and trend towards better OS (p = 0.14). Importantly, patients in the inflamed group (16%, 36/225; median PD-L1 = 1% TPS), suggested that pembrolizumab + chemo (ORR 26.1%; 6/23) was not associated with ORR compared to pembrolizumab (ORR 31%; 4/13, p = 0.76), or OS (p = 0.37) and 1-yr OS (p = 0.57). Conclusions: Comprehensive genomic and immune profiling may identify PD-L1 low NSCLC patients who benefit from single agent pembrolizumab. PD-L1 low NSCLC patients with a proliferative phenotype may benefit from single agent pembrolizumab, whereas PD-L1 low cases with an inflamed phenotype may benefit from both single agent and combination pembrolizumab. Although further clinical validation of these predictive biomarker combinations is required, this data-driven approach demonstrates the potential to provide treatment decision support when selecting an ICI therapeutic strategy in lung cancer.

Anatomical Variations of Vermiform Appendix on Plain MDCT and Its Association with Acute Appendicitis in Adult Urban Population of Karachi, A Tertiary Care Hospital Experience

Objective: To evaluate the anatomical positions of vermiform appendix and its association with acute appendicitis on Multidetector computed tomography (MDCT). Study design and setting: A cross sectional study based on hospital database was carried out in Department of Anatomy in collaboration with Department of Radiology, Liaquat National Hospital. Methodology: A total of three hundred and six 306 adult urban patients CT axial images were evaluated retrospectively over period of 6 months from March 2021 to August 2021 who had abdominal MDCT scan for acute abdomen after getting approval from ethical committee to find out anatomical variations of position of vermiform appendix in a tertiary care hospital among adult urban Karachi population and its association with appendicitis. Data was analyzed using SPSS version 25. All categorical variables were summarized as frequencies and percentages. Result: In present study 159(52%) were males and 147(48%) were females with mean age of 32.97 years. The subcecal anatomical position of vermiform appendix on MDCT was most common n=85(27.8%), followed by postileal n=78(25.5%), pelvic n=61(19.9%), postcecal n= 36(11.8%), preileal n=29(9.5%), ectopic n=17(5.6%). The association between appendicitis with anatomical position of vermiform appendix and age was analyzed by chi- square and was statistically significant (p =0.05). There was no significant association between position of vermiform appendix on MDCT and gender. Conclusion: The subcecal position of vermiform appendix is most common on MDCT and the postileal position was more frequent in the inflamed group of vermiform appendix

Clinical phenotypes of depressed patients with evidence of inflammation and somatic symptoms

Whether depressed patients with evidence of inflammation are more appropriate candidates for immunotherapies is being tested in several clinical trials, which are selecting patients based on elevated C-reactive protein (CRP) and inflammation-related symptoms. However, studies of the clinical and phenotypic profile of depressed patients with elevated CRP are relatively scarce. We have investigated detailed clinical characteristics of 84 depressed patients, grouped as those with (CRP≥3 mg/L) and without (CRP<3 mg/L) inflammation. All patients met the International Classification of Diseases 10th Revision criteria for current depressive episode and had somatic symptoms of depression. We report that depressed patients with inflammation are more likely to be older (P=0.04), have higher body mass index (P<0.01), and be on non-selective serotonin reuptake inhibitor antidepressants (P=0.04). After adjusting for potential confounders, the inflammation group had higher depression severity (adjusted mean difference, 8.82; 95% CI, 3.91–13.72), somatic symptoms (adjusted mean difference, 3.25; 95% CI, 1.58–4.92), state anxiety (adjusted mean difference, 9.25; 95% CI, 3.82–14.67), perceived stress (adjusted mean difference, 4.58; 95% CI, 1.98–7.18), and fatigue (adjusted mean difference, 9.71; 95% CI, 3.09–6.33), but not anhedonia. The inflamed group also had poorer quality of life (adjusted mean difference, −0.18; 95% CI, −0.32–0.05). At individual depressive symptom level, the inflammation group had increased guilty feelings (adjusted odds ratio [OR], 7.28; 95% CI, 2.09–31.17), pessimism (adjusted OR, 5.38; 95% CI, 1.53–22.73), concentration difficulties (adjusted OR, 4.56; 95% CI, 1.32–19.02), and indecisiveness (adjusted OR, 4.21; 95% CI, 1.15–18.54). Our findings highlight the clinical features associated with inflammation in depressed patients with somatic symptoms, including poor quality of life, supporting the need for intervention targeting this group. These results could also aid patient and outcome selection in future clinical trials testing immunotherapies in depression. Replication of these findings in larger samples is required.

Identification of Diagnostic Biomarkers by Bioinformatics Analysis in the Inflamed and Non-inflamed Intestinal Mucosa of Patients With Crohn’s Disease

Background: Crohn’s Disease (CD) is a type of inflammatory bowel disease that, despite its unknown etiology, is generally associated with genetics, immune system, and environmental factors. In this study, we uncover transcriptional signatures in patients with CD and subsequently explain the putative molecular pathways in the inflamed and non-inflamed intestinal mucosa. Materials and Methods: We obtain GSE83448 gene expression profiles from the Omnibus gene expression database. Also, for the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of Differentially Expressed Gene (DEG) pathways, we used DAVID software. DEGs were detected in the inflamed and non-inflamed intestinal mucosa of CD patients compared to the control group using the GEO2R instrument. Significant modules and hub genes were identified after producing protein-protein interaction (PPIs) networks of DEGs using Cytoscape software. Results: The 10 specific hub genes of CD, including Matrix Metallopeptidase 2 (MMP2), Cadherin 1 (CDH1), Periostin (POSTN), Collagen type I alpha 2 chain (COL1A2), C-X-C motif chemokine ligand 8 (CXCL8), Collagen type III alpha 1 chain (COL3A1), JUN, Serine Protease Inhibitor clade E member 1 (SERPINE1), Integrin alpha M (ITGAM), and Connective Tissue Growth Factor (CTGF), were used as biomarkers to discriminate between inflamed and non-inflamed intestinal mucosa groups in patients. Conclusion: These findings could lead to new molecular targets and diagnostic biomarkers for both inflamed and non-inflamed intestinal mucosa in CD patients.