Cluster Analysis Research Articles

Abstract 1253: Single-cell analysis reveals intricate subclonal heterogeneity and evolution in response to standard of care treatments in glioblastoma

Abstract Glioblastoma multiforme (GBM) is the most common and lethal brain cancer. Standard of care treatments (SOC) are limited to surgery, radiation and chemotherapies, providing an average overall survival of only ∼16 months. Tumor heterogeneity and cellular plasticity are major contributions to treatment resistance in this disease. We constructed an integrated patient-level and cohort-level framework and analyzed single-nucleotide RNA sequencing data in matched primary and recurrence specimens to understand tumor heterogeneity, evolution, and drug resistance in SOC treated GBM patients (N=23). Patient-level tumor analysis revealed distinct tumor cell transcriptional subclones representing widespread intra-patient heterogeneity. Tumor evolution analysis from primary to recurrence revealed diverse treatment responses representing sensitive, intrinsic resistant, and acquired resistant subclones. Further characterization of tumor subclones and clustering analyses indicated that tumor heterogeneity under SOC treatments was linked to diverse biological mechanisms including neural development and oncogenic pathways, contributing to varying levels of subclonal resistance. Our analysis also highlighted intrinsic resistance was prominent as primary tumors often harbored substantial proportion of resistant tumor cells. This insight on resistant status at cell level then allowed us to compare resistant to sensitive cells and identify both established and potentially novel mechanisms of SOC resistance that were not always obvious in the comparison between primary and recurrence samples. These included activation of PI3K/AKT, MAPK, TGF-β, EMT, and WNT signaling pathways. Taken together, our novel analytical strategy revealed complex tumor heterogeneity and evolution landscape in SOC treated GBM, offering insights on potential mechanisms driving these phenomena and providing a resource for further exploration and development of therapeutic strategies. Citation Format: Yueshan Zhao, Xiaoyu Lu, Verah Nyarige, Junfei Zhao, Gonzalo Lopez, Ann Forslund, Maria Ortiz-Estevez, Jorge Benitez-Hernandez, Wei Zhang, Celia Fontanillo, Kai Wang, Ha Dang. Single-cell analysis reveals intricate subclonal heterogeneity and evolution in response to standard of care treatments in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1253.

Abstract 3353: Predicting the prognosis of ductal carcinoma in situ through chemical analysis of breast microcalcifications and soft tissue using infrared and Raman spectroscopy

Abstract Introduction: Ductal carcinoma in situ (DCIS) is a potential precursor to invasive breast cancer (IBC). The trajectory of an individual’s DCIS, if it will progress to IBC or remain as DCIS is difficult to predict. Currently >80% of DCIS is detected through mammographic screening of breast calcifications. Despite the close association of calcifications with DCIS, their role in the development of DCIS and/or its progression to IBC remains largely unexplored. In this study, we present a spectroscopy based analytical approach to probe chemical compositional changes of both DCIS associated breast calcifications and surrounding soft tissue aiming to identify a cohort at increased risk for invasive progression. Experimental Procedures: Tissue samples from 316 DCIS patients without invasive cancer were obtained from multiple centres as part of the PRECISION consortium (The Netherlands, UK and USA). Three consecutive tissue sections were obtained each of the tissue biopsies. Mid-infrared (mid-IR) and Raman hyperspectral imaging was performed independently on two sections that were left unstained. The third H&E-stained section was used to annotate calcifications and histopathological features. All DCIS samples had known outcome (i) ‘pure DCIS as controls’ (DCIS without progression to invasion) (n=193), (ii) ‘DCIS with progression to invasion as cases’ (DCIS from patients who subsequently developed invasive disease after initial treatment) (n=123). Spectral features of DCIS calcifications and surrounding soft tissue were used as inputs for analysis. Data was divided into a discovery and a validation set. Cluster analysis followed by Principal component analysis fed linear discriminant analysis was carried out on the discovery set to develop DCIS prediction models. Results: For the Raman data, a mean area under the receiver operating characteristic curve (AUROC) value of 0.85 was obtained using calcification spectral features, and 0.75 using soft tissue spectral features in distinguishing controls from cases (N=118 vs 52). Similar analysis on the IR data showed a mean AUROC value of 0.68 for calcification, 0.78 for epithelial and 0.80 for stromal components (N=97 vs 61). Preliminary analysis shows changes in phosphate to carbonate ratio and variations in magnesium whitlockite content in calcifications, and protein secondary structural changes in soft tissue, between the two groups. The models will be tested independently on the validation set and the outcomes will be presented at the AACR conference. Conclusion: Spectroscopic chemical analysis of breast calcifications and soft tissue show promise in predicting the likely progression of DCIS to IBC. Pending further independent validation, these techniques appear to be novel image-based risk assessment tools that can potentially be utilised to inform DCIS prognosis and treatment options. Citation Format: Jayakrupakar Nallala, Doriana Calabrese, Sarah Gosling, Esther Lips, Ihssane Bouybayoune, Rachel Factor, Sarah Pinder, Lorraine King, Jeffrey Marks, Thomas Lynch, Donna Pinto, Alastair Thompson, Elinor Sawyer, Jelle Wesseling, Shelley Hwang, Keith Rogers, Nick Stone, Grand Challenge PRECISION consortium. Predicting the prognosis of ductal carcinoma in situ through chemical analysis of breast microcalcifications and soft tissue using infrared and Raman spectroscopy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3353.

Abstract 5303: Temporal dynamics of transcriptional and spatial landscape in tumor microenvironment of esophageal squamous cell carcinoma during chemotherapy treatment

Abstract Although esophageal squamous cell carcinoma (ESCC) is a highly aggressive malignancy, there is little knowledge about its tumor heterogeneity affecting treatment response. To decipher transcriptomic heterogeneity of tumor microenvironment (TME) from a therapeutic perspective, we conducted CITE-seq and immunostaining by Hyperion in ESCC with or without chemotherapeutic stimuli. Tumor biopsies of ESCC were sequentially collected by endoscopy from 3 patients during chemotherapy treatments. CITE-seq samples were processed with Chromium Next GEM Single Cell 5′ Reagent Kit v2 (10 x Genomics) and TotalSeq Universal Cocktails (BioLegend) as per the manufacturer′s recommendations, respectively. Cell-to-cell interaction analyses in CITE-seq and neighborhood analyses in Hyperion were performed. Clinical responses of 3 patients for ESCC tumor biopsies were PD, SD, and PR, respectively. Cell annotation analyses in TME components revealed that populations of myeloid cells exhibited an overall decrease during the treatment across three patients, while the decrease in myeloid cell populations was more pronounced in the PR patient. The cluster analyses in epithelial cells exhibited 6 unique subclusters, which were transcriptionally characterized in proliferation, EMT, inflammation, and metabolic pathways. In spatial analyses, the epithelial cells in the PD patient adhered tightly to each other, while the epithelial cells in the SD or PR patient clustered relatively mixed with immune and stromal cells. In SD or PR patients, furthermore, varieties of T cell populations were more accumulated in response to the chemotherapy treatments compared to the PD patient. Neighborhood analysis and cell-cell interaction analysis together identified cell type pairs that showed consistent changes in physical and molecular perspectives. In PD patient, dendritic cell (DC)-macrophage pair was enriched in the proximity of each other, and also increased molecular interactions after chemotherapy. In contrast, activated T-Treg pair was depleted and decreased molecular interactions. Interestingly not just activated T-Treg interaction decreased, but proliferating T cells were also depleted from the tumor. We conducted integrative analyses with cutting-edge technologies to determine temporal dynamics of TME landscape in ESCC during chemotherapy treatment. Our observations may indicate that chemotherapy increased DC-macrophage interaction to suppress antigen presentation by DC, which suppressed T cell activation and proliferation. The mechanism might contribute to the chemotherapy resistance. These findings will shed light on the molecular insights to deeply understand complexity of the TME networks for chemotherapy resistance in ECSS. Citation Format: Hiroki Yamashita, Chiaki Mashima, Maiko Narahara, Ryo Kamata, Kenji Watanabe, Ryuichi Nakamura, Sumie Muramatsu, Ayumi Tezuka, Eri Otsuka, Chisa Wada, Yoko Ishimoto, Daisuke Kotani, Takashi Kojima, Naoya Wada, Hiroyuki Sumi, Tomonori Yano, Gensuke Takayama, Akihiro Ohashi. Temporal dynamics of transcriptional and spatial landscape in tumor microenvironment of esophageal squamous cell carcinoma during chemotherapy treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5303.

Abstract 7475: A comprehensive single-cell atlas of gastric cancer reveals malignant cell state dynamics and plasticity in tumor progression and metastasis

Abstract Background: Gastric cancer exhibits diverse malignant cell states that contribute to intratumor heterogeneity (ITH), a fundamental factor that drive tumor progression, metastasis, and treatment resistance. While previous single-cell RNA sequencing (scRNA-seq) studies have provided novel insights into ITH in gastric cancer, they are often constrained by small sample sizes and limited transcriptional coverage. Methods: In this study, we curated, annotated, and integrated scRNA-seq datasets from 21 studies, comprising 626 samples from 316 individuals, including healthy donors and patients with precancerous lesions and gastric cancer. Within the epithelial compartment, malignant and non-malignant cells were distinguished using three complementary computational methods. To refine malignant cell subclustering, we developed a novel approach that incorporates patient-specific copy number variations, minimizing the impact of inter-patient heterogeneity on clustering analysis. Results: Following rigorous quality control and data integration, unsupervised clustering analysis classified 2, 088, 266 high-quality cells into six major cell types or compartments: B cells, T cells, innate lymphoid cells, myeloid cells, stromal cells, and epithelial cells. Within the epithelial cell compartment, we identified 12 distinct malignant cell states characterized by unique expression profiles and phenotypic characteristics, such as cell proliferation capacity and lineage-specific plasticity. These cancer cell states exhibited substantial variation in the activity of hallmark cancer signaling pathways, with their presence and composition closely linked to tumor aggressiveness and clinical outcomes. Proliferative cancer cells and translationally active cancer cells predominated in intermediate tumor stages, dormant cells were prevalent in advanced stages, and epithelial-mesenchymal transition (EMT) states were enriched in metastatic tumors. Additionally, we identified a malignant cell population with high expression of intestinal lineage markers in patients with signet ring cell carcinoma, and these findings were validated using spatial transcriptomic. Conclusions: This study presents a comprehensive single-cell transcriptomic atlas of gastric cancer epithelial cells, providing valuable insights into transcriptional ITH. Our findings define distinct malignant cell states, elucidate their dynamics, and highlight their clinical significance, establishing a framework for understanding how these states contribute to disease progression and metastasis. Citation Format: Enyu Dai, Yanshuo Chu, Yang Liu, Ruiping Wang, Yibo Dai, Jaffer A. Ajani, Linghua Wang. A comprehensive single-cell atlas of gastric cancer reveals malignant cell state dynamics and plasticity in tumor progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7475.

Islamic Financing Mechanisms for Renewable Energy Projects: Mapping Research Trends and Future Directions

This paper discusses the emergent trends in Islamic financing mechanisms for renewable energy projects, highlights their role in promoting economic growth, and discusses sustainability. By applying bibliometric analysis, cluster analysis (VOSviewer), and co-citation analysis, this study explores literature between 2008 and 2024 on the impact of Islamic finance on renewable energy projects. Data is collected from two major databases; Scopus and Web of Science Core Collection; key trends; influential publications; and future research directions. Findings show that Islamic financing mechanisms; including green sukuk and impact investing; are essential in supporting renewable energy investments. The study identifies key contributors such as Saudi Arabia; Malaysia; UAE; and Malaysia; with research focusing on the relationship between Islamic finance; ESG performance; renewable energy; and climate change mitigation. Future research may explore the integration of digital technologies; green innovation; and technological advancements in Islamic financing for renewable energy projects. The limitations include the reliance on Scopus and Web of Science databases, with further suggestions for incorporating additional databases and expanding keyword searches. This paper contributes to a better understanding of the intersection between Islamic finance and renewable energy, hence providing a good foundation for further research and practical applications in the field.

Abstract 1940: EpCAM and L1CAM expression signatures in circulating tumor cells of advanced colorectal cancer

Abstract Background: Colorectal cancer (CRC) is the third most common deadliest cancer among both men and women in the United States. Hence, to improve CRC clinical management, there is an unmet need for affordable and personalized approaches, such as non-invasive blood tests that analyze circulating tumor cells (CTCs). However, there is limited data about the expression of markers associated with epithelial-mesenchymal transition (EMT) of CTCs in advanced CRC. In this study, we sought to evaluate EMT markers signatures in CTCs of advanced cases of CRC. Method: Peripheral blood samples (10-20 mL) were collected from five patients with metastatic CRC. CTCs were isolated using the Single-cell CellSelect Slides™ enrichment or in-house protocol, followed by FACS (Fluorescence-Activated Cell Sorting). The following markers were analyzed: Ghost Dye™, CD45, CD133, LGR5, L1CAM, CD326 (EpCAM), CD325 (CDH2), CD274 (PD-L1), CD87 (VIM), and Cytokeratin-7/-8. FACS data analysis was done in FlowJo and plugins needed. Results: The average age of diagnosis was 51.6 years. At the time of blood collection, all CRC patients had stage IV disease and were progressing. Self-organizing map clustering and dimensionality reduction algorithm analysis of 10, 197 sorted CD45 negative living cells showed that LGR5 was the least expressed marker. The highest expression of CD133 was associated with highest expression of either EpCAM or L1CAM, which were both inversely correlated with LGR5. Co-expression of EpCAM and L1CAM were negatively associated with CD133, but positively associated with LGR5. EpCAM and L1CAM expression had significant association with CDH2, VIM, Cytokeratin-7/-8 and PD-L1. Conclusion: In advanced CRC, the mutually exclusive expression of EpCAM and L1CAM in the presence of CD133, and absence of LGR5 could be linked to increased plasticity leading to EMT, and increased risk of metastasis and recurrence. In addition, it is important to consider other markers alongside EpCAM for the identification of CTCs in advanced CRC. Citation Format: Gabriela Felix, Jun Gong, Francesca Aguirre, Lisa Zhou, Rocio Ramirez, Megan P. Hitchins. EpCAM and L1CAM expression signatures in circulating tumor cells of advanced colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1940.

Abstract 774: Deciphering the biology of colorectal metastasis to the peritoneal surface

Abstract Background. Colorectal cancer (CRC) is the third most frequently diagnosed cancer with an annual incidence of 1.4 million new cases and 700, 000 deaths worldwide. The high death toll of CRC patients is due to metastatic disease with liver being the preferred site of metastasis (LM), followed by lung and peritoneal metastasis (PM). Roughly 25% of CRC patients present with, or will eventually develop PM, and while survival for LM and lung metastasis has significantly improved over time, survival following PM remains poor. Mucinous and non-mucinous adenocarcinoma have different propensity to form PM or LM, and surprisingly little is known about the biology of CRC PM. Aims and Objectives. To perform deep mapping using Nanostring’s GeoMx Cancer Transcriptome Atlas assay to measure gene expression in resected normal colon (healthy) primary tumors (primary), LM, and PM samples collected from the KUMC Biospecimen Repository. Morphological staining using Pan-cytokeratin, CD45, & ELA2 was performed to identify epithelial tumor cells, immune cells, and neutrophils, respectively. Data processing and resulting DCC files were imported to the GeoMx DSP software for statistical analyses of the transcriptomic data using a linear mixed model. Results. We performed spatial transcriptomic analysis on paired tissue samples (surgical tissue from primary and metastatic sites) from 4 patients with stage IV CRC. Our results unveiled diverse gene expression patterns across different PM and LM tumor sub-regions. Cluster analysis revealed significant differences in gene expression between specimen types. Specifically, in mucinous adenocarcinoma samples, PKM, CEACAM6 and LYZ were upregulated in primary tumors while ADH1A/B/C, Muc4 and PCK1 were upregulated in healthy tissues. Interestingly, we also observed significant upregulation of CEACAM6 and SERPINA1 in PM vs primary tumor cells. In non-mucinous adenocarcinoma, CEACAM6 and H3C2/10 were upregulated in primary vs metastatic tissues. When comparing either primary or metastatic vs healthy stromal areas, COL1A1 and COL6A3 were significantly upregulated in primary and or metastatic regions hinting at extracellular matrix remodeling during PM. In LM, CEACAM6, SLC1A5 and CD74 were significantly upregulated when compared to PM, indicating an inflammatory role in LM. As neutrophil extracellular traps (NETs) promote metastasis, we discovered significant infiltration of ELA2+ neutrophils in the primary mucinous adenocarcinoma while ELA2+ cells were predominantly localized at the leading edge of the non-mucinous adenocarcinoma. These spatio-temporal relationships were further validated in the metastatic sites of the two CRC subtypes. Conclusions. Mucinous vs non-mucinous adenocarcinomas differ in terms of their proclivity to metastasize to the peritoneal surfaces and that the differences could be attributed to the intra-tumoral vs leading edge distribution of ELA2+ neutrophils. Citation Format: Shahid Umar, Badal C. Roy, Amrita Mitra, Jill Haley, Harsh B. Pathak, Andrew K. Godwin, Luke V. Selby, Mazin Al-Kasspooles. Deciphering the biology of colorectal metastasis to the peritoneal surface [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 774.

Abstract 1156: Real-world clinical multi-omics analyses reveal bifurcation of ER-independent and ER-dependent drug resistance to CDK4/6 inhibitors

Abstract To better understand the molecular mechanisms of drug resistance to CDK4/6 inhibitors (CDK4/6i) and inform precision medicine strategies, we retrospectively analyzed a multi-omics dataset of 400 HR+/HER2- mBC patients who received CDK4/6i plus endocrine therapies in routine clinical practice, including 200 pre-treatment (Pre), 227 post-progression (Post) samples. Integrative clustering analysis identified three subgroups of tumors harboring different drug resistance mechanisms: ER driven, ER co-driven and ER independent. The ER independent subgroup increased the most in prevalence from 4% pre-treatment to 23% post-progression and is characterized by down-regulation of estrogen signaling and enrichment of drug resistance markers including TP53 and RB1 mutations, Basal-like and Her2-like subtypes and CCNE1 over-expression. We deconvoluted bulk tumor expression profiles to derive cancer-specific expression (CSE) profiles for investigating tumor intrinsic mechanisms that give rise to ER independent drug resistance. We then performed trajectory inference analyses often used in single-cell transcriptomics on CSEs and identified a latent pseudotime variable strongly correlated with ER independence and disease progression. Elastic principal graph (EPG) analysis further revealed bifurcated evolutionary trajectories for drug resistance that correspond to ER-dependent vs. ER-independent mechanisms. Using elastic net models trained on in vitro CRISPR knockout screen data, we predicted a shift in therapeutic dependency on ESR1 and CDK4 among ER-dependent tumors to CDK2 among ER-independent tumors. These predictions were experimentally validated using genetically modified isogenic models. Citation Format: Zhengyan Kan, Ji Wen, Vinicius Bonato, Jennifer Webster, Wenjing Yang, Vladimir Ivanov, Kimberly Hyunjung Kim, Whijae Roh, Chaoting Liu, Xinmeng Jasmine Mu, Jennifer Lapira-Miller, Jon Oyer, Todd VanArsdale, Paul A. Rejto, Jadwiga Bienkowska. Real-world clinical multi-omics analyses reveal bifurcation of ER-independent and ER-dependent drug resistance to CDK4/6 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1156.

Abstract 1823: Spatial transcriptome profiling shows a decrease in heterogeneity of GBM xenografts that regrow after irradiation and suggests novel therapeutic targets

Abstract Radiation remains a primary treatment for glioblastoma, yet tumors frequently recur within two years. As a model for investigating the biology of recurrent GBMs, we used orthotopic xenografts initiated of GBM-stem like cells (GSCs) implanted into the right striatum of nude mice. Thirty-five days post-implant, mice bearing brain tumor xenografts were locally irradiated (10Gy) and brains collected 14 and 21 days later. In untreated mice, tumors were highly diffuse with tumor cells invading from the injection site in the right hemisphere (RH) to the anterior part of the brain, invading the corpus callosum, cortex, and olfactory bulb (OB). A different growth pattern was detected in tumors that regrew after irradiation. At 14 days post-10Gy, tumors were located primarily in the OB with an increased cell density and a clearly demarcated border. 21 days post-10Gy tumor cell density increased with the demarcated border extending further into the hemisphere, i.e., farther away from the OB. This growth pattern suggests that tumor regrowth after irradiation is initiated in the OB, consistent with previous results showing that GBM cells in the OB are radioresistant compared to those in the RH. To compare transcriptomes of untreated and recurrent tumors we used digital spatial profiling (DSP). Tumor cells were identified by Sox2 staining, and regions of interest (ROIs) were selected in various locations within the OB and the RH of untreated tumors, and throughout recurrent tumors (D21 post-10Gy). Cluster analysis showed that ROIs across the OB and RH of untreated tumors were more similar to each other as compared to ROIs from the tumors that regrew after irradiation. Variance in the expression of individual genes across the ROIs was reduced in the recurrent tumors compared to the untreated tumors. These results suggest a reduction in phenotypic heterogeneity among the cells of the regrown tumor. Comparative gene expression analysis identified 463 differentially expressed genes with 319 downregulated and 144 upregulated in the recurrent versus untreated tumors. GSEA revealed a significant enrichment of pathways related to cell cycle regulation in the regrown tumors as compared to non-irradiated tumors. Further examination of the cell cycle control related pathways indicated an enrichment in proteasome genes. Specifically, 22 genes encoding proteasome proteins were significantly upregulated in the recurrent tumors compared to the untreated group, suggesting that the proteasome may provide a therapeutic target unique to the recurrent GBM. Altogether, these results suggest that this xenograft model of GBM regrowth after irradiation, characterized by an altered growth pattern and a distinct molecular profile may be useful for investigations into the biology and treatment of recurrent GBM. Citation Format: Charlotte Marie Degorre. Spatial transcriptome profiling shows a decrease in heterogeneity of GBM xenografts that regrow after irradiation and suggests novel therapeutic targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1823.

Abstract 2348: Unveiling dichotomies between liver cancer and adjacent normal tissues in Wilson’s disease model

Abstract Objective: The Long-Evans Cinnamon (LEC) rat is a model for Wilson’s disease (WD) and is associated with chronic inflammation and hepatocellular carcinoma. This study performed a gene expression microarray using total RNA extracted from LEC liver tissue at 84 weeks late chronic hepatitis, tumor-adjacent normal (84 weeks N), and 84 weeks tumor (84 weeks T). Understanding the difference between a tumor and adjacent normal within the same animal can be very intriguing as both are in the same environment, and one becomes and sustains a tumor status, but the other still maintains its normalcy. Experiment: Total RNA was extracted from liver tissue of three LEC rats at 84 weeks N and 84 weeks T and hybridized to the Affymetrix GeneChip Rat Genome 230 2.0 Array (Affymetrix Inc, Santa Clara). GeneGO Pathways software was used to analyze the differentially expressed genes using a cutoff of P-value ≤0.05 and FDR <0.25% and a fold change +/- 2. A volcano plot was generated to visualize significant gene change expression between 84 weeks N vs 84 weeks T. Agglomerative Hierarchical and K-means clustering were used to group similarly expressed genes into clusters and build a hierarchy of clusters. Each cluster obtained from K-means clustering analysis within the up or down-regulated groups were checked for pathway analysis to understand the interplay between biological pathways by QIAGEN Ingenuity® Pathway Analysis (QIAGEN IPA). In addition − log(p) > 1.3, a z-score cut-off͕ ≥ 2 for activation and a z-score cutoff ≤ -2 for inhibition was used to call a pathway activation, and inhibition is significant. Result: The seven most significant pathways that were activated are the S100 Family Signaling Pathway, Extracellular Matrix Organization, Response to Elevated platelet cytosolic Ca2+, HMGB1 Signaling, Sertoli Cell-Sertoli Cell Junction Signaling, Cachexia Sunaina Pathway, and Nicotine Degradation II. Except for Nicotine Degradation II pathway, all other pathways are shown activated in non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC) patients previously but shown in the WD model for the first itme. The Nicotine Degradation II pathway is shown to be activated in the NAFLD patient before. Conclusion: To our knowledge, this is the first report where gene expression analysis was done between tumor-adjacent normal and tumor within the same animal in the WD model. Our results identified multiple pathways in the model that could lead us to new diagnostics and therapeutic targets when validated. Citation Format: Ritam Adhikari, Chiranjeev Dash, Rabindra Roy. Unveiling dichotomies between liver cancer and adjacent normal tissues in Wilson’s disease model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2348.

Abstract 5686: Revealing the unique malignant mechanism of gastric-type adenocarcinoma of the cervix

Abstract Objective: Gastric-type adenocarcinoma of the cervix (GAS) is an HPV-independent malignancy that accounts for 20-25 % of cervical adenocarcinomas. Compared to usual-type endocervical adenocarcinoma (UEA), GAS is resistant to chemotherapy and radiotherapy, and has a poor prognosis. However, its molecular biological mechanisms remain unclear. This study aimed to identify specific RNA profiles associated with GAS pathophysiology to discover diagnostic and therapeutic targets. Methods: We examined 15 tumor samples from patients who underwent primary hysterectomy (7 GAS, 8 UEA). From FFPE specimens, only the tumor region was obtained, and miRNA-seq and mRNA-seq were performed. RNA was also extracted from the GAS-derived KGAS cell line, UEA-derived HCA-1 cell line, and normal cervical epithelial HCK1T cell line for miRNA-seq. Extracellular vesicles (EVs) from each cell line were collected, and miRNA-seq was performed by using EV-RNA. Detail functional analyses were performed using cell lines. Results: miRNA sequencing of FFPE samples, cell lines, and EVs derived from GAS and UEA identified 48 miRNAs highly expressed in GAS tissue, 37 in GAS cell lines, and 41 in GAS-EVs. Further target exploration revealed one miRNA specifically highly expressed in GAS. Additionally, mRNA sequencing analysis of tumor tissue samples with 10 cases (5 GAS, 5 UEA) identified a total of 14, 541 genes, and clustering analysis showed that GAS and UEA had clearly distinct expression profiles. Differential expression analysis identified 449 upregulated genes in GAS and 577 downregulated genes in UEA, meeting the criteria of FC ≥ 1 and p-value < 0.05. Among these, the top 30 genes with the highest FC values and basemean ≥ 50 were selected. IPA and GSEA analyses significantly detected cancer-related pathways, including RTK-RAS-PI3K, PDGF signaling, and cell cycle-related pathways. The top 10 significant genes were validated by qRT-PCR, and protein expression was confirmed by immunohistochemistry. Functional validation in cell lines showed that GAS exhibited significantly higher cisplatin resistance (p < 0.05) than other cell lines, and suppression of newly identified specific target gene in GAS cells resulted in increasing sensitivity of cisplatin. Conclusion: This study revealed detailed miRNA and mRNA profiles of GAS and UEA, identifying GAS-specific miRNA and gene expression. Identified genes were confirmed as not only useful as disease markers but also functional targets. These findings contribute to understanding the unique malignant mechanisms of GAS and highlight promising targets for future therapeutic strategies. Citation Format: Hiroaki Yamada, Akira Yokoi, Kazuhiro Suzuki, Kosuke Yoshida, Eri Inami, Masami Kitagawa, Naotake Tsuda, Hiroaki Kajiyama. Revealing the unique malignant mechanism of gastric-type adenocarcinoma of the cervix [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5686.

Abstract 5795: Dynamic proteomic biomarkers of survival outcomes with anti-PD-1 immunotherapy in head and neck squamous cell carcinoma

Abstract Background: Despite advances in anti-programmed death (PD)-1 based immunotherapy in recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC), challenges remain in identifying who will benefit from these therapies. Current tissue-based biomarkers have many limitations. Due to its accessibility and ease of longitudinal monitoring, there is growing interest in blood-based dynamic biomarkers for immunotherapy outcomes in R/M HNSCC. Methods: In a prospective multicenter study of patients with R/M HNSCC (n=38) receiving anti-PD-1 based therapy, peripheral blood was collected pre-treatment and post-treatment (cycle 2 day 1). A total of 83 serum-derived cytokine biomarkers were evaluated with the Olink Target 96 Immuno-Oncology panel. Associations of pre-treatment, post-treatment, and the change in cytokine levels (post minus pre) were assessed with overall survival (OS) and progression-free survival (PFS). Unsupervised clustering analysis identified subgroups based on cytokine profiles. Further analyses were conducted using univariable and multivariable Cox proportional hazards and Cox Elastic Net machine learning models. Results: Among the 83 cytokines, 49 (59%) exhibited a statistically significant within-patient increase from pre- to post-treatment. Further, several cytokines were significantly associated with OS and PFS and 10 associated with OS were shared in both pre- and post-treatment analyses. Generally, increased cytokine levels were associated with poorer outcomes. In analyses of cytokine level change (post minus pre), seven cytokines were significantly associated (FDR0.05) with OS (i.e., VEGFA, HGF, PGF, CCL19, CCL23, MCP3, TNFRSF12A) and three with PFS (i.e., NOS3, MMP12, and CCL23) after adjustment for potential confounders. An unsupervised clustering analysis of patients based on the change in the levels of all 83 cytokines uncovered two subgroups that differed significantly with respect to OS and PFS even after adjustment of potential confounders. A prognostic cytokine score was derived using machine learning analysis using elastic net regularization. Not only were cytokine scores significantly associated with platelet count and, in some cases, neutrophil count, they also provided additional prognostic value beyond established prognostic factors. Conclusion: Dynamics of serum cytokines related to blood platelets during anti-PD-1-based therapy may serve as prognostic biomarkers in patients with R/M HNSCC. Citation Format: Md Saiful Islam Sai, Kartik Sehgal, Ze Zhang, Rondi Butler, John Wiencke, Annette Molinaro, Juliana Miller, Hannah Stolrow, Keisuke Shirai, Lucas Salas, Devin Kosetler, Robert Haddad, Brock C. Christensen, Karl Kelsey. Dynamic proteomic biomarkers of survival outcomes with anti-PD-1 immunotherapy in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5795.

Abstract 1982: Transcriptomic subtype identification of circulating tumor cells by label-free capture and single-cell RNA sequencing

Abstract Introduction: Single-cell RNA sequencing (scRNA-Seq) of rare cells, such as circulating tumor cells (CTCs), is critical for identifying various cell subtypes and understanding intercellular heterogeneity at the genetic level. Despite recent advances in scRNA-Seq, transcriptomic profiling of single CTCs has been technically demanding due to its limited cell utilization and background noise from unwanted cells during sequencing. In addition, conventional CTC isolation methods often miss important rare cell subtypes due to their low abundance and reliance on specific surface markers for detection. This limitation hinders comprehensive transcriptomic analysis, especially for CTCs undergoing the critical epithelial-mesenchymal transition (EMT), which may not express typical epithelial markers. To address these challenges, we present an optimized workflow that integrates high-purity label-free CTC capture with in-droplet barcoding-based scRNA-seq to achieve higher cell utilization and lower background sequencing noise. Methods: To model heterogeneous CTC subpopulations at different EMT status, samples were prepared by spiking in two types of cancer cells, MCF7 (epithelial CTCs) and HeLa (mesenchymal CTCs), into a total of 7.5 ml of whole blood from a healthy donor at a concentration of 100 cells/ml. CTCs were captured using the Genesis Cell Isolation System and the enriched CTCs were further purified to reduce leukocyte contamination or red blood cell (RBC) contamination using CD45 pre-conjugated magnetic beads or RBC lysis reagent, respectively. The processed CTC sample was then used to create scRNA-Seq libraries following the Bio-Rad ddSEQ Single-Cell 3’ RNA-Seq Kit protocol, which includes single-cell in-droplet barcoding, reverse transcription, and cDNA synthesis. Results: This Genesis System allowed for capturing both epithelial and mesenchymal CTCs to be sequenced. After sequencing and Omnition Software Analysis, individual cells were visualized using a Uniform Manifold Approximation and Projection (UMAP) plot and clustering analysis showed a distinct separation between epithelial and mesenchymal CTCs while each different cell population revealed expression of representative genes for each cell type. Discussion and Conclusions: By combining rare single cell capture with scRNA-seq library prep and analysis, our workflow provides an easy, highly sensitive and cost-effective solution for CTC single-cell transcriptomic studies, compatible with commercially available instruments and kits. This scRNA-seq workflow can be integrated into a broad spectrum of applications in liquid biopsy across many different cancer types and empowering researchers to gain deeper insights into tumor heterogeneity and gene expression patterns with greater efficiency and within the same budget.​ Citation Format: Yoon-Tae Kang, Patricia Schnepp, Rashika Shrestha, Rain Yu Hong, Errile Pusod, Will Chow, Floyd Watkins, Kyle Thurston, Dominique Winston, Marta Gonzalez-Plasky, Angelica Olcott, David Coe, Michelle Racey, Elizabeth Dreskin. Transcriptomic subtype identification of circulating tumor cells by label-free capture and single-cell RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1982.

Abstract 2051: Baseline and adaptive activated kinome profiling identifies PTK6 as a mediator of resistance to vemurafenib and dabrafenib but not encorafenib in BRAFV600-mutant metastatic melanoma

Abstract To investigate the landscape of the activated kinome in MM, we applied a proteomic approach that interrogates the activation status of more than half of the entire kinome (∼300), termed multiplexed kinase inhibitor bead affinity chromatography coupled with mass spectrometry (MIB/MS) on 45 MM patient (pt) tumors (BRAFV600, n=25; NRASQ61, n=5, NF1 loss-of-function, n=4; Triple wild-type n=11) in conjunction with whole exome sequencing and RNA sequencing (RNAseq). We performed exploratory analyses (Wilcoxon rank sum test for each kinase) on the untreated tumors to identify differentially activated kinases in BRAFV600-mutant MM vs. all others. While no activated kinases were differentially expressed in BRAFV600-mutant MM after adjustment for multiple testing, AKT1, PDGFRA, and MAP3K1 kinases exhibited higher MIB binding (i.e., more activated) in BRAFV600E-mutant pts (unadjusted p-value < 0.05). Spearman’s rank correlation tests between the activated kinase and its gene expression by RNAseq and adjusted for multiple testing did not identify a significant correlation for any kinase, suggesting a complex mechanism of kinase activation other than sole gene expression. K-means clustering analysis to classify melanomas into 4 subtypes using a 6-gene discriminant expression signature showed that BRAFV600-mutant tumors were more frequently classified as melanocytic (9/24, MLANA/MITF/SOX10high, AXLlow). In contrast, triple wild-type tumors were more frequently classified as transitory (5/10, MITF/SOX10/ETV4high, AXLlow). We then performed similar exploratory analyses on the baseline (untreated) tumors from the subset of the 20 BRAFV600E-mutant pts who received D+T as part of a prospective clinical trial (NCT01726738; median follow-up 30.5 months, range 2.0-116.1 months) to identify differentially activated kinases that would predict shorter (<12 months) vs. longer progression-free survival. Again, while no kinases survived multiple comparison testing, the bromodomain protein TRIM28, known to regulate melanoma plasticity, exhibited a more consistent loss of MIB binding in BRAFV600E-mutant pts who progressed shorter than in pts who progressed longer than 12 months on D+T (unadjusted p-value < 0.05). 7 baseline-progression tumor pairs were evaluated for MIB/MS, and 6 were for RNAseq. Although diverse resistance mechanisms were identified, as previously described, we also identified melanoma subtype switch in 4/6 pt tumors and a consistent loss of MIB binding of the non-receptor tyrosine kinase PTK6 in response to D+T, an off-target of D and vemurafenib but not encorafenib. Our in vitro findings revealed that off-target PTK6 inhibition by D could activate beta-catenin signaling and contribute to the development of resistance. Citation Format: Steve P. Angus, Naim U. Rashid, Andrew J. Walther, David L. Corcoran, Steven D. Rhodes, Frances A. Collichio, C. Paige Jones, Mikaela J. Bauer, Joel S. Parker, Noah Sciaky, 1Alisha R. Coffey, Samantha M. Bevill, Brian T. Golitz, v Timothy J. Stulhmiller, Nancy E. Thomas, David W. Ollila, Norman E. Sharpless, Carrie B. Lee, Gary L. Johnson, Stergios J. Moschos. Baseline and adaptive activated kinome profiling identifies PTK6 as a mediator of resistance to vemurafenib and dabrafenib but not encorafenib in BRAFV600-mutant metastatic melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2051.

Abstract 2089: Spatial profiling of exhausted T cells using high-plex imaging mass cytometry

Abstract The spatial organization and cellular composition of the tumor microenvironment (TME) has the potential to inform clinical and translational researchers about mechanisms of disease progression and therapeutic success. Of particular interest are immune cells, especially T cells, which can become exhausted due to chronic stimulation. This poses a significant challenge in cancer therapy as exhausted T cells have reduced effector functions and sustained expression of inhibitory receptors such as PD-1 and CTLA-4, resulting in failure to effectively eliminate tumor cells. Imaging Mass Cytometry™ (IMC™) technology is a high-plex imaging technique that enables deep characterization of the heterogeneity and complexity of the TME. The Hyperion™ XTi Imaging System utilizes IMC technology to provide signal intensities over a wide dynamic range and entails one-step detection of 40-plus markers without issues of tissue autofluorescence, making it ideally suited for spatial biology applications. Whole slide imaging modes and an automated slide loader function enable a streamlined, versatile scalable workflow for high-throughput analysis. We used IMC technology to characterize immune cell populations and the spatial distribution of T cell exhaustion markers across various cancer types. We applied a 40-plus-marker IMC panel by integrating the Human Immuno-Oncology IMC Panel with the Human T Cell Exhaustion IMC Panel to study the TME of multiple human tissues. IMC analysis revealed striking heterogeneity with distinct tumor and immune-rich niches in the TME. In colon adenocarcinoma, we detected functional effector T cells and multiple tertiary lymphoid structures (TLSs). In contrast, we observed fewer TLSs and more T cells expressing PD-1 and TIM-3 at the tumor periphery in the urothelial carcinoma tissue, suggesting that they are in advanced exhaustion stages. We also observed expression of PD-L1, IDO and VISTA in the tumor cells. t-SNE and PhenoGraph clustering analysis of Cell Mode data segregated and spatially resolved the T cells into effector and exhausted T cell subpopulations, which were mapped back to the segmented cell masks. Unsupervised pixel clustering and analyses of Tissue Mode data further delineated distinct tumor areas based on the presence of infiltrating immune cells, tumor replicative activity and spatial proximity to stromal components. This work characterizing T cell exhaustion markers in multiple cancers showcases the capabilities of IMC technology and establishes it as a reliable high-plex, high-throughput spatial biology imaging platform. IMC technology is ideally suited for developing future translational and clinical applications and has the potential to help guide personalized therapeutic strategies for cancer treatment. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Thomas D. Pfister, Jyh Yun Chwee, Qanber Raza, Nikesh Parsotam, David Howell, Liang Lim, Christina Loh. Spatial profiling of exhausted T cells using high-plex imaging mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2089.

Multivariate time-series clustering analysis of the Global Dietary Database to uncover patterns in dietary trends (1990-2018).

Understanding country-level nutrition intake is crucial to global nutritional policies that aim to reduce disparities and relevant disease burdens. Still, there are limited numbers of studies using clustering techniques to analyse the recent Global Dietary Database. This study aims to extend an existing multivariate time-series clustering algorithm to allow for greater customisability and to provide the first cluster analysis of the Global Dietary Database to explore temporal trends in country-level nutrition profiles (1990-2018). Trends in sugar-sweetened beverage intake and nutritional deficiency were explored using the newly developed program 'MTSclust'. Time-series clustering algorithms are different from simple clustering approaches in their ability to appreciate temporal elements. Nutritional and demographical data from 176 countries were analysed from the Global Dietary Database. Population representative samples of the 176 in the Global Dietary Database. In a 3-class test specific to the domain, the MTSclust program achieved a mean accuracy of 71.5% (Adjusted Rand Index [ARI]=0.381) while the mean accuracy of a popular algorithm, DTWclust, was 58% (ARI=0.224). The clustering of nutritional deficiency and sugar-sweetened beverage intake identified several common trends among countries and found that these did not change by demographics. Multivariate time-series clustering demonstrated a global convergence towards a Western diet. While global nutrition trends are associated with geography, demographic variables such as sex and age, are less influential to the trends of certain nutrition intake. The literature could be further supplemented by applying outcome-guided methods to explore how these trends link to disease burdens.

Abstract 1896: Spatial proteomics and tumor-immune phenotyping identifies a diverse immune landscape in syngeneic murine tumor models

Abstract Syngeneic mouse models have a functionally intact immune system, and therefore, serve as a crucial tool for evaluation of the immune response to tumors and pre-clinical evaluation of immunotherapeutic strategies. However, the immune response varies between different syngeneic models. Identification and selection of appropriate models for pre-clinical evaluation is challenging. To understand the dynamic interplay between the tumor and immune system, we implemented multiplexed spatial proteomics across various syngeneic tumor models. Spatial mapping of multiple tumor and immune markers in untreated tumor provides an overview of the tumor immune architecture. This information can be applied to identify optimal models for preclinical immunotherapy approaches, to understand the response to therapies at the preclinical stage, and to identify potential biomarkers for clinical evaluation. The Cell DIVE Multiplexed Imaging Solution, in combination with IHC-validated antibody conjugates from Cell Signaling Technology (CST), enables profiling of immune cell populations in the context of human and mouse tumor tissue. Here, we demonstrate multiplexed Cell DIVE imaging of syngeneic mouse tumor models using a novel CST® panel of mouse-reactive antibodies. We apply segmentation and clustering analysis to identify spatially co-localized populations of cells and discuss the complexities of immune cells and their interactions. Cell type specific markers combined with multiplexed tissue imaging and spatial mapping is an approach that enables a better understanding of the anti-tumor immune response, which can be applied to address challenges associated with developing immune-based therapies. The study highlights the importance of simultaneously observing multiple immune cell types and tumor characteristics in spatial context. Citation Format: Arindam Bose, Emily Quann Alonzo, Sophie Struble, Jeremy Fisher, Richard A. Heil-Chapdelaine, Natasha F. Diaz Granados, Vasundhara Agrawal. Spatial proteomics and tumor-immune phenotyping identifies a diverse immune landscape in syngeneic murine tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1896.

Abstract 750: Exploring PD1/PDL1 interactions and macrophage-tumor barriers in head and neck cancer: A spatial approach to immunotherapy response

Abstract With nearly a million new cases expected, the global death rate from mucosal head and neck cancer is projected to reach around 50% in 2024. In the U.S., patients with localized tumors have a five-year survival rate of 50-60%. While immune checkpoint inhibitors (ICIs) have shown promise in extending survival, significant challenges remain, particularly the limited effectiveness of PD-1/PD-L1 blockade therapies. Since many studies have shown that PD-L1 protein expression alone may not be a reliable predictor of response to ICI therapy, understanding the spatial context of PD-1/PD-L1 interactions might be crucial for identifying immune evasion mechanisms and predicting responses to ICIs in head and neck cancer. To address this, we aimed to take a more comprehensive approach by mapping PD-1/PD-L1 interactions across n=35 mucosal head and neck squamous cell carcinoma (HNSCC) tissue samples collected before ICI treatment, using a combination of high-plex spatial proteomics and the in situ Navinci Proximity Ligation Assay (isPLA). Formalin-fixed, paraffin-embedded (FFPE) tissue samples were stained using multiplex immunofluorescence and the Navinci Diagnostics isPLA assay to detect PD-1/PD-L1 interactions. After incubation with primary anti-PD1 and anti-PD-L1 antibodies, secondary probes tagged with oligonucleotides were applied, enabling PLA to highlight the interactions through a ligation step and an amplification process to enhance the visibility of the protein interactions. Finally, a detection solution was applied to visualize the amplification product at the sites of PD1/PD-L1 interaction.We identified distinct spatial patterns of PD-1/PD-L1 interactions at the tumor-stroma interface, particularly in progressive disease (PD) patients, who exhibited dense layers of isPLA+ macrophages, CD3e T cells, and tumor cells, suggesting an immunosuppressive barrier in this region. In contrast, complete response (CR) patients showed rare or absent barriers, with enriched immune cell aggregates, including B cells and T cells, near the tumor boundary. These findings were corroborated through neighborhood clustering analysis, revealing significant associations between isPLA+ regions and poor prognosis.The spatially resolved nature of our analysis underscores the critical role of PD-1/PD-L1 interactions in shaping the tumor microenvironment (TME) and highlights their potential as predictive biomarkers for ICI therapy efficacy. Unlike methods that detect mere colocalization, isPLA technology enables us to detect true PD-1/PD-L1 interactions, which are more likely to affect immune suppression directly. Our study also emphasizes the need for further exploration of macrophage and T cell subtypes, as well as PD-1/PD-L1 interactions across tumor-stroma boundaries in various cancers to improve immunotherapy strategies. Citation Format: Vahid Yaghoubi Naei, Rafael Tubelleza, James Monkman, Habib Sadeghirad, Meg L. Donovan, Tony Blick, Agata Wicher, Sara Bodbin, Subham Basu, Robert Stad, Catherine Barnett, Ken O’Byrne, Rahul Ladwa, Caroline Cooper, Majid Ebrahimi Warkiani, Brett GM Hughes, Arutha Kulasinghe. Exploring PD1/PDL1 interactions and macrophage-tumor barriers in head and neck cancer: A spatial approach to immunotherapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 750.

Study on the status and countermeasures of disease self-management ability in chronic kidney disease patients in cold regions

Abstract Objective Effective Chronic Kidney Disease (CKD) management is particularly important in cold regions of China, where climate and lifestyle factors play significant roles. However, there is a lack of relevant studies in this area. Therefore, the purpose of this study was: (1) to assess the status of self-management capacity in individuals with CKD in cold regions of China and analyze the factors influencing it; (2) to identify strategies to improve CKD management in primary care settings in these regions; and (3) to understand patients' attitudes toward eHealth services. Methods This was a regional, cross-sectional observational study. A questionnaire measuring CKD patients' self-management abilities was derived from the Perceived Kidney Disease Self-Management Scale, the Kidney Disease Behavioral Inventory (KDBI), and the Health Literacy Questionnaire. Data were collected from hospitalized CKD patients in Heilongjiang Province and analyzed using One-Way Analysis of Variance (ANOVA), Hierarchical Regression Analysis, and K-prototype cluster analysis. Results A total of 957 participants were tested. Of these, 70.64% had less than a bachelor's degree, and 56.27% had been diagnosed with hypertensive or diabetic nephropathy. The KDBI scale showed a lower overall score compared to the PKDSMS. Factors such as CKD stage 4 (F = 2.367, P = 0.042), last year's medical expenses (F = 3.974, P = 0.004), and poor self-rated health (F = 33.352, P < 0.01) were found to influence scores on both scales. The health literacy questionnaire revealed significant differences (P < 0.01) in health knowledge, except by sex. Additionally, healthcare expenditures and poor self-rated health were negatively associated with self-management capacity. Conclusions This study provides valuable insights into the self-management challenges faced by CKD patients in cold regions of China. Despite some difficulties in improving self-management, patients showed positive attitudes toward enhancing CKD management services in primary care and developing digital management tools. These findings offer useful references and recommendations for future clinical practice and research in this field.

Abstract 7458: Germline-p53-mutant adrenocortical carcinoma is a possible candidate for the novel use of arsenic trioxide as a first targeted anticancer therapy for Li-Fraumeni syndrome

Abstract Background: Li-Fraumeni Syndrome (LFS) is a cancer predisposition disorder associated with germline mutations in the tumor suppressor gene TP53, which encodes the protein p53. Despite a near-guarantee for individuals with LFS to develop cancer during their lifetime, there are still no specific antitumor therapies for LFS. As a possible approach to address this unmet clinical need, FDA-approved anticancer agent arsenic trioxide (ATO) has recently been shown to rescue a wide spectrum of p53 mutants back to wild-type structure and function. However, ATO-rescuable p53 mutants have not been examined in a germline context to date, preventing an understanding in the field of ATO’s capacity to serve as a first targeted therapy for LFS. Methods: To address this gap, we integrated published research data on 103 ATO-rescuable p53 mutants with public clinical and mutational data on 842 individuals with germline-p53-mutant cancers in the TP53 Database and 12, 793 individuals with somatic-p53-mutant cancers in the cBioPortal database. Ensuing in silico analyses included p53 mutant and cancer prevalence calculations, statistical tests of proportions and associations, unsupervised cluster analysis, and Cox proportional hazards modeling. Results: ATO-rescuable p53 mutants overall were significantly enriched in germline-p53-mutant cancers compared to somatic-p53-mutant cancers. In both cohorts, ATO-rescuable p53 mutants overall were significantly associated with damaged function, dominant-negative effects, and loss of function, but were not significantly linked with sex or earlier ages of diagnosis. At the mutational level, ATO-rescuable p53M133T, p53P152L, p53R158H, and (also hotspot mutant) p53G245S were significantly enriched in germline-p53-mutant cancers compared to somatic-p53-mutant cancers. Notably, ATO-rescuable p53R175H and p53R282W, both also hotspot mutants, were equally highly prevalent in both cohorts. Within germline-p53-mutant cancers, ATO-rescuable p53 mutants were significantly enriched in adrenocortical carcinoma (ACC) compared to the full germline cohort and to other cancers frequently diagnosed in LFS. Unsupervised cluster analysis identified a cluster of three p53 mutants, two of which were ATO-rescuable (p53P152L and p53R158H), specifically associated with germline-p53-mutant ACC. Cox proportional hazards modeling subsequently revealed this cluster was significantly associated with a higher risk for individuals to develop ACC independent of other p53 mutants, sex, type of p53 mutant, and p53 domain of residues affected. Conclusions: This work provides the first insights in the field on potential germline p53 mutant and cancer candidates for the novel use of ATO as a first targeted antitumor therapy for individuals with LFS. Citation Format: Neha Wali, Richard P. Owen, Curtis C. Harris, Xin Lu. Germline-p53-mutant adrenocortical carcinoma is a possible candidate for the novel use of arsenic trioxide as a first targeted anticancer therapy for Li-Fraumeni syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7458.