Immunohistochemical Analysis Research Articles

The Immunohistochemical and Bioinformatics Analysis of the Placental Expressions of Vascular Cell Adhesion Protein 1 (VCAM-1) and High Mobility Group Box 1 (HMGB1) Proteins in Gestational Diabetic Mothers.

We aimed to examine both the expression levels of high mobility group box 1 (HMGB1) and vascular cell adhesion molecule-1 (VCAM-1) proteins in the placentas of pregnant women with gestational diabetes mellitus (GDM) and control groups by immunohistochemical (IHC) method. An experimental case-control study was conducted, including 40 pregnant women complicated with GDM and 40 healthy pregnant women. Placental tissues obtained following cesarean delivery were subjected to routine tissue monitoring. The placental sections were stained with VCAM-1 and HMGB1 immunostains and subjected to IHC examination under a light microscope. H-score (HS) was used to evaluate the results of IHC staining by semi-quantitative analysis. Pathway analysis in Cytoscape software identified GDM-associated proteins within HMGB1 and VCAM-1 interaction networks, followed by GO analysis to explore associated biological processes. Placental HGMB1 expression was significantly increased in the GDM group compared to the control group (p<0.001). However, placental VCAM-1 expression was found to be statistically similar in GDM and control groups (p=0.584). The shared 19 proteins were identified between HMGB1 and GDM, and 13 between VCAM-1 and GDM, with notable GO biological process terms such as immune system activation for HMGB1 and interleukin-6 regulation for VCAM-1 associated with GDM. We consider that GDM-related inflammation and oxidative stress may contribute to tissue damage and inflammation by increasing placental HMGB1 expression. The blockade of HMGB1 and its receptors might represent a promising therapeutic approach to control inflammation in GDM. Understanding the distinct roles of HMGB1 and VCAM-1 may provide valuable insights for the development of targeted therapies aimed at mitigating the inflammatory processes associated with GDM and improving maternal and fetal outcomes.

Prediction model of male reproductive function damage caused by CHOP chemotherapy regimen for non-Hodgkin's lymphoma.

The CHOP combined chemotherapy regimen (cyclophosphamide, doxorubicin, vincristine, and prednisone) is commonly used to treat non-Hodgkin Lymphoma (NHL). While these drugs are effective for cancer treatment, they may have side effects on the reproductive system that are poorly studied. This study used a mouse model to investigate the mechanisms of reproductive function impairment induced by the CHOP regimen and developed a predictive model for assessing reproductive damage with a non-invasive procedure. From 2022 to 2023, we statistically analyzed the changes of reproductive function of NHL patients before and after receiving CHOP regimen in the First Affiliated Hospital of Xiamen University. The NHL mouse model was established and divided into CHOP treatment group and control group. The weight of testis and epididymis, sperm quality and motility were compared between the two groups. Histopathological examination of testicular tissue was performed to determine pathological changes. ELISA was used to measure the expression of cytokines and cytokine pathways in serum, protein expression was analyzed by immunohistochemistry, and protein and mRNA levels of cytokines and pathways were evaluated by Western blotting and qPCR. Using stepwise regression method to select important factors, a prediction model of reproductive system damage was constructed. Fifty-two NHL patients included in the questionnaire showed significant reproductive system damage after CHOP regimen treatment. The weight of testis and epididymis, as well as the number and vitality of sperm in the mouse model treatment group were significantly lower than those in the control group. Serum LH, FSH, estradiol and progesterone levels decreased significantly, while inhibin B levels increased significantly. There was no significant change in testosterone or prolactin levels. Inflammatory markers such as CSF-1, IL-1, IL-6, TGF-β1 and GDNF increased significantly, while the level of SOD1 decreased significantly. Immunohistochemical staining analysis showed that CAMP, Caspase3, CSF-1, GDNF, IL-1, IL-6, PRKACB, TGF-β1 and TXNDC5 were all expressed in spermatocytes, and the expression of therapeutic histones was significantly higher than that of the control group. Western blot analysis further detected the protein expression, and QPCR detected the mRNA content. The results showed that the expression of histone and mRNA in the treatment group was significantly higher than that in the control group. Stepwise regression method determined that estradiol (E2) was the most important variable in the prediction model, and the AUC for predicting reproductive damage was 1. The CHOP regimen induces male reproductive toxicity, potentially mediated through alterations in hormone levels and increased expression of inflammatory cytokines and oxidative stress. Using E2 as the sole predictor in the model accurately predicts the extent of reproductive damage, offering a non-invasive method for detecting reproductive system damage.

Abstract 4136598: Osteochondrogenic transdifferentiation of vascular smooth muscle cells and microenvironmental dynamics in medial arterial calcification at single-cell resolution

Background: Medial arterial calcification (MAC) significantly contributes to cardiovascular mortality, yet effective treatments remain limited due to unclear molecular mechanisms. This study aimed to develop a mouse model of MAC using O-ring-induced transverse aortic constriction (OTAC) and to elucidate key genes, pathways, and cellular interactions involved in MAC formation through lineage tracing and single-cell RNA sequencing (scRNA-seq). Methods: An OTAC mouse model was established to replicate MAC. Adult C57BL/6J male mice underwent OTAC, and subsequent analyses were conducted at various time points. Histological and immunohistochemical assessments were performed to monitor changes in vascular smooth muscle cells (VSMCs). Lineage tracing was utilized to verify the origin of osteochondrogenic VSMCs. Furthermore, scRNA-seq was employed to capture dynamic changes in VSMCs and other cell types contributing to MAC development. Results: The OTAC model successfully induced osteochondrogenic transdifferentiation of VSMCs, leading to MAC. Immunohistochemical analysis demonstrated time-dependent decreases in α-SMA expression and increases in SOX9 and RUNX2 expression. Lineage tracing using Myh11CreERT2;ROSA26-EGFP mice confirmed that osteochondrogenic VSMCs originated from contractile VSMCs. scRNA-seq identified osteochondrogenic VSMCs and revealed dynamic changes in surrounding cells, including macrophages and fibroblasts, which were implicated in calcification-related and inflammatory processes. Conclusion: The OTAC method effectively mimicked human MAC pathology, offering comprehensive insights into the microenvironmental dynamics of MAC progression at the single-cell level. This model serves as a robust platform for future research on therapeutic interventions.

Abstract 4136692: Spatial Transcriptomics Identifies Novel Clinical Markers to Classify the Etiology of Rare and Aggressive Cardiac Sarcomas

Background: Cardiac sarcomas, such as leiomyosarcoma (LMS), synovial sarcoma (SS), and undifferentiated pleomorphic sarcoma (UPS), lack sufficient diagnostic markers for early diagnosis and tailored therapeutic interventions. Understanding their molecular signatures with spatial-temporal resolution compared to normal heart tissue is crucial. Hypothesis: Spatial transcriptomics will reveal a comprehensive set of novel clinical markers, including changes in cellular composition, stemness, and metabolic profiles unique for each cardiac sarcoma subtypes. Aims: Identify novel molecular markers for the accurate diagnosis of cardiac sarcoma subtypes. Methods: Using 10X Visium spatial transcriptomics, we analyzed three cardiac sarcoma subtypes (LMS, SS, UPS) and compared them with normal heart tissue. Differential expression analysis identified novel target genes suitable for clinical disease stratification. Subsequently, machine learning models reveal an unbiased transcriptomic profile to distinguish normal from sarcoma tissues. Finally, immunohistochemistry analysis validated the differential marker gene expression. Results: We identify several novel clinical markers, such as IGKC, PCOLCE, NET1, TLE2, MDFI, BGN, TNNC1, and CNN3, that classify normal heart tissue from sarcoma subtypes. Machine learning models and immunostaining analysis confirm the transcriptomic signature in each sarcoma subtype. Pathway enrichment analysis show LMS enriched in antigen processing and B cell-mediated immunity, SS in protein folding and response to incorrect proteins, and UPS in axon development and Wnt signaling regulation. Trajectory Reconstruction Analysis indicate higher stemness scores in all sarcomas, with SS highest, followed by UPS and LMS. Additional cell cycling analysis show LMS cells in G2M phase, SS in G1, and UPS in S phase. SS express a high number of cancer stem markers (EZH2, BMI1, KDM5B, LGR5, HIF1A), while UPS express KDM5B and CTNNB1, and LMS show fewer stem-related marker genes (EZH2, BMI1, KDM5B, CTNNB1). Both SS and LMS express CD44. Glucose and oxidative pathways are also differentially regulated in each sarcoma subtype. Conclusion: Spatial transcriptomics uncovers distinct clinical markers, stemness characteristics, and metabolic activity profiles in cardiac sarcomas, offering insights for fast, accurate and improved diagnostics and therapeutics.

NIMG-10. THE ROLE OF FIBROBLAST ACTIVATION PROTEIN IN GLIOBLASTOMA AND GLIOSARCOMA – A COMPARISON OF TISSUE, 68GA-FAPI-46 POSITRON EMISSION TOMOGRAPHY AND SURVIVAL DATA

Abstract BACKGROUND Despite their unique histological features,gliosarcomas belong to the group of glioblastomas and are treated according to the same standards. Fibroblast-activation-protein(FAP) is a component of a tumor-specific subpopulation of fibroblasts that play a critical role in tumor growth and invasion.However,the prognostic impact of FAP in glioblastoma and gliosarcoma is unclear. METHODS In a retrospective analysis,patients diagnosed with glioblastoma without sarcomatous differentiation and gliosarcoma were enrolled.Histological examination was performed using immunohistochemical FAP-staining and quantitative analysis with a standardized FAP-score.In a subset of patients,FAPI46-PET has been performed.The SUV-values are planned to be correlated with FAP-expression in the tumor tissue.Data obtained from immunohistochemical analysis and SUV-values are planned to be brought into perspective with clinically relevant prognostic factors,including survival estimates. RESULTS We enrolled 61 patients,including 13 diagnosed with gliosarcoma.Our analysis revealed that gliosarcomas exhibit significantly higher FAP-expression (median FAP-score: 3) compared to glioblastomas without sarcomatous differentiation (median FAP-score: 0.5, p&amp;lt;0.0001),where FAP was predominantly observed in the perivascular space.In gliosarcomas,FAP was also expressed by neoplastic cells.Moreover,a significant relationship was established between the immunohistochemical FAP-scores and the SUVmean and SUVpeak values on PET,suggesting that the PET tracer uptake accurately reflects the tumor’s FAP-expression.However,the differential expression of FAP suggests its potential as both a diagnostic marker and a therapeutic target.This hypothesis is further supported by the application of 68Ga-FAPI-46-PET in a 66-year-old female patient with recurrent gliosarcoma,alongside immunohistochemical analysis of tumor tissue.Both methods identified elevated FAP-expression and uptake,underscoring the feasibility of FAP as a target for nuclear medicine therapies in brain tumors. CONCLUSIONS Our study establishes a significant correlation between SUVmean/SUVpeak in 68Ga-FAPI-46 PET scans and FAP-expression in glioblastoma.Gliosarcomas express significantly more FAP than other glioblastomas. These insights suggest FAP’s potential as a theranostic target.We plan to investigate the effectiveness of FAP-specific tracers in treating recurrent gliosarcoma,aiming to open new therapeutic avenues.

Assessment of the Concentration of Transforming Growth Factor Beta 1–3 in Degenerated Intervertebral Discs of the Lumbosacral Region of the Spine

The purpose of this study was to evaluate the feasibility of using the expression profile of transforming growth factor beta (TGF-β-1-3) to assess the progression of L/S spine degenerative disease. The study group consisted of 113 lumbosacral (L/S) intervertebral disc (IVD) degenerative disease patients from whom IVDs were collected during a microdiscectomy, whereas the control group consisted of 81 participants from whom IVDs were collected during a forensic autopsy or organ harvesting. Hematoxylin and eosin staining was performed to exclude degenerative changes in the IVDs collected from the control group. The molecular analysis consisted of reverse-transcription real-time quantitative polymerase chain reaction (RT-qPCR), an enzyme-linked immunosorbent assay (ELISA), Western blotting, and an immunohistochemical analysis (IHC). In degenerated IVDs, we noted an overexpression of all TGF-β-1-3 mRNA isoforms with the largest changes observed for TGF-β3 isoforms (fold change (FC) = 19.52 ± 2.87) and the smallest for TGF-β2 (FC = 2.26 ± 0.16). Changes in the transcriptional activity of TGF-β-1-3 were statistically significant (p &lt; 0.05). Significantly higher concentrations of TGF-β1 (2797 ± 132 pg/mL vs. 276 ± 19 pg/mL; p &lt; 0.05), TGF-β2 (1918 ± 176 pg/mL vs. 159 ± 17 pg/mL; p &lt; 0.05), and TGF-β3 (2573 ± 102 pg/mL vs. 152 ± 11 pg/mL) were observed in degenerative IVDs compared with the control samples. Determining the concentration profiles of TGF-β1-3 appears to be a promising monitoring tool for the progression of degenerative disease as well as for evaluating its treatment or developing new treatment strategies with molecular targets.

IMMU-44. EXTRINSIC ELECTRICAL MODULATION OF THE GLIOBLASTOMA TUMOR MICROENVIRONMENT

Abstract BACKGROUND Approximately 30 to 50% of patients with glioblastoma (GBM) present with glioma-related epilepsy (GRE). Despite morbidity associated with seizures, GRE constitutes an independent positive predictive predictor for survival in GBM. Our prior results showed that repeated seizure induction through extrinsic electrical modulation (EEM, a technique in which an electrical stimulus sufficient to produce a therapeutic seizure is applied) slows intracranial tumor growth and prolongs overall survival in immunocompetent CT-2A glioma-bearing mice. However, the overall survival advantage following EEM treatment was diminished in immunocompromised mice bearing human patient derived xenografts. We hypothesized that EEM induces immune-related tumor microenvironment (TME) changes that prolong survival in mice. METHODS C57BL/6J mice were orthotopically implanted with CT-2A xenografts and subjected to EEM of 2.0 millicoulombs or sham treatment. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed at 3 and 24 hours to determine mRNA level of inflammatory cytokines. Immunohistochemistry (IHC) and bulk RNA sequencing analysis were utilized to assess CD3+ T-cell infiltration and identify immune-related secreted genes, respectively. Flow cytometry at 3 and 96 hours was utilized to characterize tumor-infiltrating immune cell populations. Samples were subjected to Percoll gradient enrichment of immune cell populations and stained for extracellular (CD45, CD4, CD8, CD11b, F4/80, CD80, Ly6G, Ly6C, and CX3CR1) and intracellular (TNF-a, Il-1b, CCL2, and Arg1) antigens. RESULTS RT-qPCR showed an increase in pro-inflammatory cytokines TNF-α (P = 0.0087) and IL-1β (P = 0.000349). IHC analysis demonstrated an increase in intratumoral CD3+ T-cells in EEM-treated samples (P = 0.0171). From 1855 upregulated genes, 3 immune-related secreted genes (C1QTNF4, FAM19A4, and GREM2) were identified. Flow cytometric analysis showed a significant increase in CD11b+, CX3RC1+ microglia (P = 0.0306) and CD45+, CD11b+ tumor-associated myeloid cells (TAMCs) (P = 0.0192) at 3 hours post-EEM. CONCLUSIONS EEM treatment promotes an inflammatory TME with increased intratumoral microglia, TAMCs and T-cell infiltration. These inflammatory changes may prolong survival of CT-2A glioma-bearing mice.

Intraductal Papillary Mucinous Neoplasm: New Insights Into Its Origin and Nomenclatures.

Mucinous cells can be detected sporadically or may constitute the primary tumor component in salivary gland tumors, as observed in the intraductal papillary mucinous neoplasm (IPMN). This low-grade tumor is composed of mucinous columnar cells organized into papillary cystic structures. The present study aimed to compare the mucous cells in IPMN with mucous cells present in mucoepidermoid carcinoma (MEC) and papillary cystadenoma (PC). Immunohistochemistry analysis was carried out to compare the mucous cells in IPMN with the sporadic mucous cells in MEC (n = 4) and PC (n = 3). The results indicated that IPMN cells were positive for CK7, CK18, DOG1, and NKX3.1 and negative for CK14, SMA, and p63. The mucous cells in both MEC and PC were positive for CK7 and negative for CK18, SMA, DOG1, and NKX3.1. The positive expression of CK14 and p63 revealed the presence of basal cells both in PC, cystic areas of MEC, and normal mucous salivary glands. The immunohistochemical profile of IPMN closely resembles that of the mucous cells of the minor salivary glands yet differs from the mucous cells observed in MEC and PCX. This suggests that IPMN is probably derived from the transformation of secretory cells of the minor mucous salivary gland and has no rimming/basal cells. For this reason, we propose that this tumor is designated as mucous acinic cell carcinoma.

Lack of Evidence for Transmission of Atypical H-Type Bovine Spongiform Encephalopathy Prions (H-BSE Prions) by Intracranial and Oral Challenges to Nonhuman Primates.

Bovine spongiform encephalopathy (BSE) is a prion disease in cattle caused by classical-type (C-), L-type (L-), or H-type (H-) BSE prions. While C-BSE prions are zoonotic agents responsible for variant Creutzfeldt-Jakob disease, L- and H-BSE prions are believed not to be connected to human prion diseases. However, L-BSE prions have been shown to transmit to cynomolgus monkeys (Macaca fascicularis), suggesting they may have zoonotic potential. In the present study, we examined whether H-BSE prions are transmissible to cynomolgus monkeys. The monkeys were injected intracranially (n = 2) or given orally (n = 2) with brain homogenates from a cow infected with H-BSE prions. After asymptomatic observation periods of 4-6 years, the monkeys were euthanized for autopsy. Histological examination of the brain did not reveal any pathological changes. Immunohistochemical and Western blot analyses did not detect disease-associated forms of prion protein (PrPSc) in the brain, peripheral neurons, or lymphatic tissues. The unsuccessful transmission indicates an effective barrier against the transmission of cattle H-BSE prions to cynomolgus monkeys. Based on the results obtained in this nonhuman primate model, we estimated that the potential transmission of H-BSE prions to humans is substantially lower than C- and L-BSE prions.

INNV-06. ADULT GLIOBLASTOMA WITH MISMATCH REPAIR DEFICIENCY FORMS A DISTINCT HIGH-RISK MOLECULAR SUBGROUP FOR WHICH TREATMENT WITH IPILIMUMAB-NIVOLUMAB SHOWS SURVIVAL ADVANTAGE

Abstract Glioblastoma is the most frequent and malignant primary brain tumor. Although the survival is generally dismal for glioblastoma patients, risk stratification and the identification of high-risk subgroups is important for prompt and aggressive management. The G1-G7 molecular subgroup classification based on the MAPK pathway activation has offered for the first time a non-redundant, all-inclusive classification of adult glioblastoma. Five patients from the large, 220-patient prospective cohort showed germline mutations in mismatch repair (MMR) genes and significantly worse median survival (3.25 months post-surgery) than those from other subgroups or from the rest of the cohort adjusted for age. These tumors were assigned to a new subgroup, G3/MMR, a spin-off from the major G3/NF1 subgroup, as they generally show genomic alterations leading to RAS activation. An integrated clinical, histological, immunohistochemical and molecular analysis of the G3/MMR tumors showed distinct characteristics as compared to other glioblastomas, including those with iatrogenic high tumor mutation burden (TMB), warranting a separate subgroup. Among these, prior history of cancer, midline location or multifocality, presence of multinucleated giant cells, positive p53 and MMR immunohistochemistry, and specific molecular characteristics, including high TMB, alert to this high-risk G3/MMR subgroup. High TMB constitutes the criterium for treatment with immune checkpoint inhibitors in solid cancers. The FDA-approved first-line therapy for advanced non-small cell lung cancer and high-TMB colorectal cancer is with the dual ipilimumab-nivolumab regimen. One of the five G3/MMR subgroup patients received the dual ipilimumab-nivolumab regimen and survived much longer than the rest of the G3/MMR patients, setting a proof-of-principle example for the treatment of these very aggressive G3/MMR glioblastomas.

BIOM-56. HEAT SHOCK PROTEINS PREDICT CLINICAL RESPONSE AND POTENTIATE LOCAL IMMUNOGENICITY AFTER LITT FOR GLIOBLASTOMA

Abstract Glioblastoma (GBM) remains one of the deadliest cancers despite advances in therapies. Laser interstitial thermal therapy (LITT) is a minimally invasive technique used to ablate intra-axial brain tumors. Non-ablative LITT-induced hyperthermia (33-43˚C) increases intra-tumoral mutational burden and neoantigen production, promoting neoantigen presentation via heat shock proteins (HSPs). HSPs are protein-folding chaperones that present novel intracellular antigens for immune recognition. We investigated HSPs as predictors of local immune response and clinical outcomes of GBM patients undergoing LITT. We conducted immune deconvolution of bulk RNA-sequencing data from The Cancer Genome Atlas to determine the correlation between 97 unique HSPs and tumor infiltration of CD8+, CD4+, B, macrophage, neutrophil, and dendritic cells. We evaluated the association between HSP expression and overall survival (OS). Pre-LITT biopsies from 9 patients (responders and non-responders) were analyzed via immunohistochemistry (IHC) to identify spatial HSP expression. Next-generation sequencing was utilized to validate findings and identify other predictive biomarkers. We identified two HSPs for further investigation: SACS and HSPA5. SACS was associated with improved survival based on TCGA data (14.9 vs. 11 months, HR:1.3, p&amp;lt;0.0001). HSPA5 was associated with worse outcomes (12.1 vs. 15.0 months, HR:0.73, p&amp;lt;0.0001). Immune deconvolution showed HSPA5 positively correlated with dendritic cell infiltration (r=0.50, p=6.1E-27) and SACS with CD8+ infiltration (r=0.31, p=6.8E-11). IHC of 9 patients demonstrated high SACS expression was associated with improved OS (p&amp;lt;0.05) after LITT. Multiplex IHC illustrated that immune cell infiltration was associated with therapeutic response post-LITT. Our ongoing immunohistochemical and genomic analyses aim to clarify the role of HSPs in enhancing immunogenicity in GBM. Further investigation into SACS and immune cell infiltration may reveal how hyperthermia via LITT improves response through neoantigen presentation.

Regulatory T cells require peripheral CCL2-CCR2 signaling to facilitate the resolution of medication overuse headache-related behavioral sensitization.

Medication overuse headache (MOH) is the most common secondary headache disorder, resulting from chronic and excessive use of medication to treat headaches, for example, sumatriptan. In a recent study, we have shown that the peripheral C-C motif ligand 2 (CCL2), C-C motif chemokine receptor 2 (CCR2) and calcitonin-gene-related peptide (CGRP) signaling pathways interact with each other and play critical roles in the development of chronic migraine-related behavioral and cellular sensitization. In the present study, we investigated whether CCL2-CCR2 and CGRP signaling pathways play a role in the development of sumatriptan overuse-induced sensitization, and whether they are involved in its resolution by the low-dose interleukin-2 (LD-IL-2) treatment. Mice received daily sumatriptan administration for 12 days. MOH-related behavioral sensitization was assessed by measuring changes of periorbital mechanical thresholds for 3 weeks. CCL2-CCR2 and CGRP signaling pathways were inhibited by targeted gene deletion or with an anti-CCL2 antibody. Ca2+-imaging was used to examine whether repetitive sumatriptan treatment enhances CGRP and pituitary adenylate cyclase-activating polypeptide (PACAP) signaling in trigeminal ganglion (TG) neurons. LD-IL-2 treatment was initiated after the establishment of sumatriptan-induced sensitization. Immunohistochemistry and flow cytometry analyses were used to examine whether CCL2-CCR2 signaling controls regulatory T (Treg) cell proliferation and/or trafficking. CCL2, CCR2 and CGRPα global KO mice exhibited robust sumatriptan-induced behavioral sensitization comparable to wild-type controls. Antibody neutralization of peripheral CCL2 did not affect sumatriptan-induced behaviors either. Repeated sumatriptan administration did not enhance the strength of CGRP or PACAP signaling in TG neurons. Nevertheless, LD-IL-2 treatment, which facilitated the resolution of sumatriptan-induced sensitization in wild-type and CGRPα KO mice, was completely ineffective in mice with compromised CCL2-CCR2 signaling. In CCL2 KO mice, we observed normal LD-IL-2-induced Treg expansion in peripheral blood, but the increase of Treg cells in dura and TG tissues was significantly reduced in LD-IL-2-treated CCL2 KO mice relative to wild-type controls. These results indicate that the endogenous CCL2-CCR2 and CGRP signaling pathways are not involved in sumatriptan-induced behavioral sensitization, suggesting that distinct molecular mechanisms underlie chronic migraine and MOH. On the other hand, peripheral CCL2-CCR2 signaling is required for LD-IL-2 to reverse chronic headache-related sensitization.

Orbital and eyelid inflammatory myofibroblastic tumors: a clinicopathological analysis of 13 cases

Objective: To investigate the clinicopathological features of orbital and eyelid inflammatory myofibroblastic tumors (IMTs). Methods: A retrospective analysis was conducted among 13 patients with IMTs treated at Tianjin Eye Hospital between January 2000 and October 2023. The clinicopathological data, immunohistochemical staining characteristics, molecular phenotypes, and follow-up outcomes were collected and analyzed. Results: The study cohort comprised 7 males and 6 females. The median age was 44 (22, 68) years. The tumor was observed in the left eye in 6 cases and the right eye in 7 cases. The age at onset ranged from 5 to 76 years, with a disease course of 1 month to 2 years before tumor excision. The tumor was located within the orbit in 10 cases (including one case of a systemic multifocal lesion) and in the eyelid in 3 cases. Pathological characteristics of the tumors were mainly proliferation of spindle or ovoid fibroblasts and myofibroblasts, with varying degrees of mucoid degeneration or collagenization of the mesenchyme, and varying amounts of lymphocytes and plasma cells infiltrated in the background. Immunohistochemical analysis revealed that Vimentin and α-SMA were positive in all cases (13/13, 13/13), ALK1 was positive in 5 cases (5/11), CK was positive in 1 case (1/1), and Desmin was positive in 1 case (1/6). ALK fluorescence in situ hybridization was positive in 2 out of 3 cases, and EBER in situ hybridization was negative in 6 cases. Nine patients were available for the follow-up of 7 to 139 months. Seven patients remained tumor-free, 2 experienced relapses, and no deaths or metastases were reported. Conclusions: Orbital and eyelid IMTs predominantly occurred in adults, with localized lesions in most cases and favorable prognoses. The disease was histopathologically characterized by proliferation of fibroblasts and myofibroblasts. The accurate diagnosis depended on a comprehensive assessment of the morphological features of the tumor histopathology as well as immunohistochemical markers such as ALK and α-SMA, and molecular detection.

TMOD-35. EGFRVIII OVEREXPRESSION AND LOSS OF MOUSE SPECIFIC CDKN2A IN GLIAL CELLS LEADS TO SPONTANEOUS GLIOMAGENESIS IN A NOVEL MOUSE MODEL

Abstract A new mouse model for the classical subtype of human glioblastoma (GBM) has been bred using Cre-mediated EGFRvIII overexpression and homozygous p19-ARF deletion, the mouse homolog of human p14ARF/CDKN2A, in GFAP expressing cells. Transgenic mice develop intraparenchymal and/or leptomeningeal brain lesions with some spinal cord invasion as early as 1 month old and 95% of mice die by 6 months due to hydrocephalus and/or paralysis. Mice with high grade tumors have worse survival and similar features to human classical GBM such as necrosis, high levels of mitosis and infiltration of tumor cells into normal brain. Immunohistochemical analysis confirms EGFRvIII overexpression and p19-ARF loss in Cre-expressing cells, along with patchy positive GFAP and high proliferation by Ki67. Bulk RNA sequencing reveals GEC3 tumors cluster closely with human classical GBM and have significant upregulation of downstream markers in the JAK/STAT and P13K/AKT pathways. Adherent and neurosphere primary culture of dissociated tumors indicate that tumor cells maintain EGFRvIII expression in culture and generate xenograft tumors by 3 weeks after intracranial injections into NODSCID mice. Xenograft tumors are reminiscent of the primary tumor, with similar histopathological features and immunohistochemical staining. Our spontaneous glioma model is a powerful tool for future studies focused on the role of the immune microenvironment on glioma recurrence and resistance.

NCMP-06. GENOTYPE AND SEX IMPACT MEMORY FUNCTION AFTER BRAIN IRRADIATION

Abstract PURPOSE/OBJECTIVE Cognitive changes are a serious complication that can occur after brain radiotherapy. There is a need to better understand which individuals are more susceptible to develop strategies to preserve their function. The apolipoprotein E type 4 allele (APOEe4) APOE4 is the most prevalent genetic risk factor for Alzheimer Disease. We hypothesized that APOE genotype influences memory function after a subtherapeutic dose of whole brain irradiation in male and female mice. MATERIALS/METHODS Age-matched adult female and male mice engineered with either human APOE3 or APOE4 gene knocked-in were randomized to no treatment or whole brain irradiation (0 Gy or 5 Gy x 2), then evaluated with longitudinal neurobehavioral tests of learning, memory, pain threshold and anxiety. We performed immunohistochemical analyses of mouse brain sections at 1 month and at 4 months post-radiation to quantify extracellular amyloid beta plaques and other neuropathologic hallmarks associated with neurodegeneration. RESULTS Neurobehavioral testing identified significant sex and genotype-dependent changes in memory and anxiety measures developing longitudinally after brain irradiation. At baseline (unirradiated), APOE4 mice (male and female) performed worse on multiple memory measures. However after irradiation, sex and genotype each independently impacted multiple neurobehavioral function tests and also interacted with each other to significantly influence the timing and magnitude of changes in memory function. ApoE4 significantly impacted the toxicity of radiation in a sex-dependent manner. Neuropathologic analyses including amyloid beta plaque deposition and ApoE levels are ongoing. CONCLUSIONS After brain irradiation of relatively moderate dose, neurobehavioral performance including memory are impacted in a genotype and sex-dependent manner and dynamically over time. In addition to suggesting important sex-dependent differences in memory vulnerability, these data point to new opportunities for personalizing care for patients receiving brain irradiation.

Head-to-head study of [18F]FAPI-04 PET/CT and [18F]FDG PET/CT for non-invasive assessment of liver cancer and its immunohistochemical markers.

To compare the performance of [18F]FDG and [18F]FAPI-04 in PET/CT evaluation for liver cancer lesions, with a further exploration of the associations between PET semiquantitative data and immunohistochemical markers to liver cancer. Patients with suspected malignant liver lesions (MLL) underwent [18F]FDG and [18F]FAPI-04 PET/CT scanning. Liver lesions were visually classified as positive or negative based on their uptake level exceeding that of adjacent normal liver tissue. SUVmax and tumor-to-background ratio (TBR) were recorded for semi-quantitative analysis. Sensitivity, specificity and accuracy of each tracer were determined using pathological findings as the gold standard. Furthermore, immunohistochemical analysis provided the molecular characteristics of all MLLs. Comprehensive analysis explored correlations between these molecular markers and PET semiquantitative parameters (SUVmax andTBR) to identify potential associations. The study enrolled 44 patients, with 39 confirmed cases of MLL, comprising 28 hepatocellular carcinomas (HCC) and 11 intrahepatic cholangiocarcinomas (ICC). For MLL detection, [18F]FAPI-04 and [18F]FDG exhibited sensitivities of 84.6% (33/39) and 76.9% (30/39), specificitiesy of 60% (3/5) and 100%(5/5), and accuracy of 81.8% (36/44) and 79.5%(35/44). Across all liver lesions, [18F]FAPI-04 significantly surpassed [18F]FDG in SUVmax(10.54 ± 6.72 VS. 7.68 ± 6.79) and TBR(4.35 ± 3.78 Vs. 3.17 ± 3.05). Notably, [18F]FAPI-04 displayed markebly elevated SUVmax in benign liver lesions (BLLs) (P = 0.032), HCCs (P = 0.005), and ICCs (P = 0.011). Lesions with hepatocyte negativity (P = 0.023), CD34 negativity(P = 0.044), and high Ki67 expression (> 30%) (P = 0.001) had higher SUVmax on [18F]FAPI-04. Additionally, ARG-1-negative lesions demonstrated higher TBR on [18F]FAPI-04 than ARG-1-positive lesions(P = 0.018). No significant SUVmax/TBR differences were observed with [18F]FDG based on these markers. A linear relationship was identified between Ki67 scores and SUVmax of [18F]FAPI-04 (R = 0.603, P < 0.001). [18F]FAPI-04 exhibits superior performance over [18F]FDG in PET/CT evaluation of liver cancer, characterized by increased sensitivity and SUVmax/TBR. Significant correlations with molecular markers, including Ki67, suggest [18F]FAPI-04's potential for characterizing liver cancer subtypes and assessing tumor proliferation. However, further research is required to validate these findings and their clinical significance. NCT05485792, Registered 01 August 2022.

EXTH-24. THERAPEUTIC TARGETING OF MISLOCALIZED NUCLEAR ENVELOPE PROTEINS INMYC-DRIVEN GROUP 3 MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is the most common, malignant pediatric brain tumour comprised of four distinct molecular subgroups (WNT, SHH, Group 3, and Group 4). A subset of Group 3 MB tumors harbors focal amplifications of the MYC oncogene (MYC-G3MB) and are particularly prone to tumour recurrence due to their highly proliferative nature linked to its embryonic stem cell-of-origin and programming. We discovered that LBR, an inner nuclear transmembrane protein, while present in all cells at the nuclear envelope (NE), is aberrantly presented to the cell surface in MYC-G3MBs. Transcriptomic profiling of publicly available datasets identified significant NE protein enrichment in MYC-G3MB compared to normal tissue and high expression during fetal development that dampens postnatally and in adult samples. Immunohistochemistry analysis of LBR on patient tumors was able to significantly prognosticate overall and progression free survival. Flow cytometry analysis identified LBR cell surface expression in MYC-G3MBs with low to no expression in human neural stem cells and other MB subgroups. High resolution microscopy of endogenously tagged LBR revealed cell surface presentation to be linked to ER-like vesicles that directly trafficked to the cell surface in MYC-G3MBs. Transcriptomic analysis of LBR cell surface positive and negative cells identified LBR to be linked to mitotic processes and mislocalization was significantly enriched following exposure to chemotherapy and radiation in vitro and in vivo. Therefore, we demonstrate that LBR is an ideal therapeutic target for patients facing treatment-refractory MB and have generated single domain antibodies (sdAb) to develop LBR-CAR T cells. Validation of LBR sdAbs demonstrate on-target binding and comparable affinity and avidity to commercial LBR antibodies. There is an undeniable paucity of targets for MB patients stemming from the lack of neoantigens and genomic aberrations in pediatric tumors. The preclinical development and validation of novel immunotherapeutic targets such as LBR provides alternative therapies for patients otherwise facing palliation.

Repeated inhalation of GM-CSF by nonhuman primates induces bronchus-associated lymphoid tissue along the lower respiratory tract

BackgroundRepeated inhalation of granulocyte-macrophage colony-stimulating factor (GM-CSF) was recently approved in Japan as a treatment for autoimmune pulmonary alveolar proteinosis. However, the detailed physiological and pathological effects of repeated inhalation in the long term, especially at increasing doses, remain unclear.MethodsIn this chronic safety study, we administered 24 cynomolgus monkeys (Macaca fascicularis) aged 2–3 years with aerosolized sargramostim (a yeast-derived recombinant human GM-CSF [rhGM-CSF]) biweekly for 26 weeks across four dosing groups (0, 5, 100, and 500 µg/kg/day). We measured the serum GM-CSF antibody (GM-Ab) concentration by an ELISA and assessed the neutralizing capacity of GM-Ab using the GM-CSF-dependent cell line TF-1. We subjected lung tissue samples taken from all monkeys at 27 weeks to histopathological assessment using a sargramostim-specific monoclonal antibody to detect localization of residual sargramostim.ResultsAll the animals maintained good body condition and showed steady weight gain throughout the study. The pathological analyses of the lung revealed the formation of induced bronchus-associated lymphoid tissue (iBALT) in the lower respiratory tract, even at the clinical dose of 5 µg/kg/day. There was a relationship between the number or size of BALT and sargramostim dose or the serum GM-Ab levels. Immunohistochemical analyses revealed GM-Ab–producing cells in the follicular region of iBALT, with residual sargramostim in the follicles. Leucocyte counts were inversely correlated with GM-Ab levels in the high-dose groups. Additionally, serum GM-Ab from the treated animals significantly suppressed the alveolar macrophage proliferation activity of both Cynomolgus recombinant and rhGM-CSF in vitro.ConclusionLong-term repeated inhalation of sargramostim led to iBALT formation in the lower respiratory tract, even at the clinical dose of 5 µg/kg/day, with the extent of iBALT formation increasing in a dose-dependent manner. Inhaled sargramostim was localized to the follicular region of iBALT nodules, which may induce the production of GM-Ab.

Efficient Production of Chondrocyte Particles from Human iPSC-Derived Chondroprogenitors Using a Plate-Based Cell Self-Aggregation Technique

The limited capacity of articular cartilage for self-repair is a critical challenge in orthopedic medicine. Here, we aimed to develop a simplified method of generating chondrocyte particles from human-induced pluripotent stem cell-derived expandable limb-bud mesenchymal cells (ExpLBM) using a cell self-aggregation technique (CAT). ExpLBM cells were induced to form chondrocyte particles through a stepwise differentiation protocol performed on a CAT plate (prevelex-CAT®), which enables efficient and consistent production of an arbitrary number of uniformly sized particles. Histological and immunohistochemical analyses confirmed that the generated chondrocyte particles expressed key cartilage markers, such as type II collagen and aggrecan, but not hypertrophic markers, such as type X collagen. Additionally, when these particles were transplanted into osteochondral defects in rats with X-linked severe combined immunodeficiency, they demonstrated successful engraftment and extracellular matrix production, as evidenced by Safranin O and Toluidine Blue staining. These data suggest that the plate-based CAT system offers a robust and scalable approach to produce a large number of chondrocyte particles in a simplified and efficient manner, with potential application to cartilage regeneration. Future studies will focus on refining the system and exploring its clinical applications to the treatment of cartilage defects.

Primary hepatic alpha-fetoprotein-producing neuroendocrine neoplasm harboring FGFR2 and TP53 mutations: a case report and literature review.

This report presents a rare case of a 45-year-old man diagnosed with a primary hepatic alpha-fetoprotein-producing neuroendocrine neoplasm, a condition rarely reported in the literature. The patient presented with initial symptoms of back and epigastric pain, after which multiple liver lesions were discovered on contrast-enhanced computed tomography, suggesting intrahepatic cholangiocarcinoma. Histopathological and immunohistochemical analyses confirmed the diagnosis of alpha-fetoprotein-producing neuroendocrine neoplasm that was further supported by genetic testing, which revealed FGFR2 and TP53 mutations commonly encountered in intrahepatic cholangiocarcinoma. Despite receiving various chemotherapeutic regimens, the patient exhibited a progressive disease. This case underscores the importance of accurate differential diagnosis from hepatocellular carcinoma and intrahepatic cholangiocarcinoma due to differences in treatment approaches and prognoses and highlights the necessity for increased awareness of AFP-producing primary hepatic neuroendocrine neoplasms among clinicians and pathologists. It emphasizes the significance of comprehensive histopathological evaluation, immunohistochemical profiling, and genetic analysis for precise diagnosis and tailored therapeutic strategies. Further research is warranted to elucidate the molecular mechanisms underlying this rare liver tumor subtype and develop targeted treatments.