Analysis Of Tumor Tissue Research Articles

Are Ki-67 and Procalcitonin Expression Levels Useful in Predicting the Biological Behavior of Hepatocellular Carcinoma After Liver Transplantation?

Background: Examinations of procalcitonin (PCT) and Ki-67 expression levels in hepatocellular carcinoma (HCC) patients who have undergone liver transplantation (LT) through immunohistochemical analyses of tumor tissue may reveal the biological characteristics of the tumor, thus informing the selection of HCC patients for LT. Methods: Hepatectomy specimens from 86 HCC patients who underwent LT were obtained and analyzed immunohistochemically for the expression of PCT and Ki-67. The percentage and intensity of PCT staining, as well as the percentage of Ki-67 expression, were assessed for each patient. The impacts of PCT and Ki-67 expression on disease-free survival, overall survival, and the recurrence rate were studied, as well as their correlations with other clinicopathological features. Results: The recurrent HCC group showed a higher Ki-67 level (p < 0.001), larger maximum dominant tumor diameter (p < 0.001), and higher rate of vascular invasion (p = 0.001). The pre-transplant AFP (p = 0.001), maximum dominant tumor diameter (p < 0.001), number of tumor nodules (p < 0.001), rate of vascular invasion (p = 0.001), and Ki-67 level (p = 0.044) were higher in patients beyond the Milan criteria. Similarly, the pre-transplant AFP (p < 0.001); maximum dominant tumor diameter (p < 0.001); number of tumor nodules (p < 0.001); rates of portal vein tumor thrombus (p = 0.002), poor differentiation (p = 0.021), and vascular invasion (p < 0.001); and Ki-67 level (p = 0.010) were higher in patients beyond the expanded Malatya criteria. The maximum dominant tumor diameter (p = 0.006); Ki-67 level (p = 0.003); rates of vascular invasion (p < 0.001), cases beyond the Milan criteria (p = 0.042) and the expanded Malatya criteria (p = 0.027), and portal vein tumor thrombus (p = 0.020); and presence of recurrence (p < 0.001) were higher in HCC patients with mortality. The Kaplan-Meier estimates indicated that Ki-67 levels exceeding 5% significantly affected DFS and OS. Although the Kaplan-Meier estimates indicated that a PCT staining percentage of ≥25% did not have a statistically significant effect on DFS or OS, the outcomes may be considered clinically significant. Conclusions: This study demonstrated that the Ki-67 proliferation index can be used as a predictive biomarker of the biological behavior of HCC. Furthermore, we claim that PCT expression over a particular threshold might impact recurrence and survival, and we believe that further multicenter prospective studies focused on standardized PCT antibody staining are crucial in order to determine its potential as a biomarker for HCC.

The Efficacy of Liquid Biopsy of Total cfDNA for Predicting Systemic Metastasis in Japanese Patients With Oral Squamous Cell Carcinoma.

The use of liquid biopsy of total cell-free DNA (cfDNA) to identify otherwise undetectable cancers has attracted interest; however, its efficacy remains unknown. We explored whether analysis using total cfDNA is efficacious for Japanese patients with oral squamous cell carcinoma (OSCC). We collected total cfDNA from nine patients with OSCC preoperatively, 1 month postoperatively, and every 3 months thereafter to analyze this association. We used a target DNA sequence for genetic mutation analysis of tumor tissues collected from 33 patients, including the aforementioned nine patients. Patients with good disease control showed negligible changes in preoperative and postoperative total cfDNA concentrations. A rapid increase in total cfDNA concentration was observed in patients who developed systemic metastases. Patients whose tumor tissue DNA showed genetic mutations had the same mutations in preoperative circulating tumor DNA. Our data suggested that analyzing total cfDNA is efficacious for patients with OSCC.

SPP1+ macrophages promote head and neck squamous cell carcinoma progression by secreting TNF-α and IL-1β

BackgroundHead and neck squamous cell carcinoma (HNSCC) is a very aggressive disease characterized by a heterogeneous tumor immune microenvironment (TIME). Tumor-associated macrophages (TAMs) constitute the major innate immune population in the TIME where they facilitate crucial regulatory processes that participate in malignant tumor progression. SPP1 + macrophages (SPP1 + Macs) are found in many cancers, but their effects on HNSCC remain unknown. This study aimed to identify and validate the role and function of SPP1 + Macs in the malignant progression of HNSCC.MethodsIn this study, we applied single-cell RNA sequencing (scRNA-seq) analyses of paired tumor and normal tissues from 5 HNSCC patients to identify tumor-specific SPP1 + Macs. RT-qPCR and multiplex immunohistochemical and multiplex immunofluorescence staining were used to verify the presence of SPP1 + Macs in the clinical samples. Gene set variation analysis suggested that SPP1 + Macs were actively involved in cytokine production. Thus, we constructed SPP1-OE macrophages and SPP1-KD macrophages (both differentiated from THP-1 cells), performed a Luminex liquid suspension chip detection assay to detect differential cytokines, and further assessed their biological functions and mechanisms in several HNSCC cell lines and adjacent macrophages. An in vivo experiment was used to verify the function of SPP1 + Macs in HNSCC progression.ResultsThe scRNA-seq results revealed that myeloid cells were heterogeneous and strongly correlated with tumor cells in the TIME in HNSCC and identified tumor-specific SPP1 + Macs, which were positively correlated with poor prognosis of HNSCC patients. Gene set variation analysis (GSVA) suggested that SPP1 + Macs were actively involved in cytokine production. Luminex liquid suspension chip detection assay indicated that SPP1 + Mac-derived TNF-α and IL-1β played important roles. Both in vitro and in vivo experiments and the use of VGX-1027, an inhibitor of macrophage-derived TNF-α and IL-1β, confirmed that SPP1 + Mac-derived TNF-α and IL-1β promoted HNSCC progression by supporting tumor cell proliferation and migration. Mechanistically, we found that TNF-α and IL-1β were upregulated due to NF-kappa B signaling pathway activation in SPP1 + Macs. Moreover, SPP1 + Mac-derived TNF-α and IL-1β promoted the expression of OPN in both tumor cells and other adjacent macrophages through different signaling pathways.ConclusionsSPP1 + Macs increase the secretion of TNF-α and IL-1β via the NF-kappa B pathway to promote HNSCC cell proliferation, and TNF-α and IL-1β in turn upregulate the expression of OPN in tumor cells and macrophages; thus, SPP1 + Macs may be a candidate target through which antitumor efficacy can be enhanced.

Daidzein Inhibits Non-small Cell Lung Cancer Growth by Pyroptosis.

Non-Small-Cell Lung Cancer (NSCLC) represents the leading cause of cancer deaths in the world. We previously found that daidzein, one of the key bioactivators in soy isoflavone, can inhibit NSCLC cell proliferation and migration, while the molecular mechanisms of daidzein in NSCLC remain unclear. We developed an NSCLC nude mouse model using H1299 cells and treated the mice with daidzein (30 mg/kg/day). Mass spectrometry analysis of tumor tissues from daidzein-treated mice identified 601 differentially expressed proteins (DEPs) compared to the vehicle-treated group. Gene enrichment analysis revealed that these DEPs were primarily associated with immune regulatory functions, including B cell receptor and chemokine pathways, as well as natural killer cell-mediated cytotoxicity. Notably, the NOD-like receptor signaling pathway, which is closely linked to pyroptosis, was significantly enriched. Further analysis of key pyroptosis-related molecules, such as ASC, CASP1, GSDMD, and IL-1β, revealed differential expression in NSCLC versus normal tissues. High levels of ASC and CASP1 were associated with a favorable prognosis in NSCLC, suggesting that they may be critical effectors of daidzein's action. In NSCLC-bearing mice treated with daidzein, RT-qPCR and Western blot analyses showed elevated mRNA and protein levels of ASC, CASP1, and IL-1β but not GSDMD, which was consistent with the proteomic data. In summary, this study demonstrated that daidzein inhibits NSCLC growth by inducing pyroptosis. Key pathway modulators ASC, CASP1, and IL-1β were identified as primary targets of daidzein. These findings offer insights into the molecular mechanisms underlying the anti-NSCLC effects of daidzein and could offer dietary recommendations for managing NSCLC.

Prolonged clinical response is possible with regorafenib in refractory osteosarcoma: A case report

&lt;b&gt;Background:&lt;/b&gt; Treatment options for metastatic unresectable osteosarcoma are limited, and there is no standard approach after the failure of first-line chemotherapy. Conventional chemotherapy in the second and subsequent lines typically yield a median progression-free survival (PFS) of less than 4 months, with objective response rates ranging from 3 to 29%. The activity of anti-vascular endothelial growth factor receptor multi-tyrosine kinase inhibitors (TKIs) in bone sarcomas has been demonstrated. We present a case of metastatic osteosarcoma exhibiting a durable response to regorafenib treatment.&lt;br /&gt; &lt;b&gt;Case: &lt;/b&gt;We discuss a 30-year-old male with metastatic osteosarcoma who had extensive bone and lung metastases and was ineligible for metastasectomy. The patient had received all active chemotherapy regimens in the early and metastatic stages except ifosfamide. Next generation sequencing analysis of resected tumor tissue revealed a possibly pathogenic missense mutation (P.G472E) in NOTCH1 gene. Due to the presence of heart failure with a reduced ejection fraction, chemotherapy was not considered, and regorafenib was initiated. After four cycles of regorafenib, a partial metabolic response was achieved. The patient was followed up without progression under regorafenib for 15 months, without requiring dose reduction. Unfortunately, the treatment had to be interrupted for an extended period due to challenges with recurrent tumor infection and the necessity for repeated surgeries. Following successful wound site control, we restarted full-dose regorafenib and achieved a metabolic partial response again.&lt;br /&gt; &lt;b&gt;Conclusion: &lt;/b&gt;To our knowledge, this case represents the longest achieved PFS with regorafenib in metastatic osteosarcoma. TKIs can provide a durable clinical response in metastatic osteosarcoma patients. Clinical and molecular biomarkers are required to determine which patients are most likely to benefit from these treatments.

2′-Hydroxycinnamaldehyde induces ROS-mediated apoptosis in cancer cells by targeting PRX1 and PRX2

2′-Hydroxycinnamaldehyde (HCA) is a component of the commonly used spice cinnamon, which has beneficial effects on cancer, allergies, bacterial/viral infections, and Alzheimer’s disease. Our previous study showed that HCA induced reactive oxygen species (ROS) and apoptosis in cancer cells, and pretreatment of cancer cells with antioxidants abolished HCA-mediated ROS production and apoptosis. This indicates that ROS are critical effector for HCA activity. However, the molecular target of HCA for ROS induction has not been identified. In the present study, we identified peroxiredoxin 1 (PRX1) and peroxiredoxin 2 (PRX2) as target proteins of HCA using affinity chromatography, and further confirmed these association using a cellular thermal shift assay (CETSA). In addition, we used mutagenesis to identify important cysteine residues in PRX1 for HCA binding. PRX1 has four cysteines (Cys52, Cys71, Cys83, and Cys173), and when Cys173 (but not the other cysteine sites) was mutated to serine, it was unable to bind biotin-conjugated HCA, suggesting that Cys173 is important for HCA binding. Treatment of SW620 cancer cells transfected by control vector with 20 μM HCA increased ROS levels by 5.2-fold compared to DMSO-treated cells. However, downregulation of target proteins PRX1 and PRX2 using shRNAs (short hairpin RNA) significantly reduced HCA-mediated ROS induction (1.6-fold), supporting that PRX1 and PRX2 are targets of HCA for ROS elevation. Additionally, intraperitoneal injection of 50 mg/kg HCA inhibited SW620 tumor growth, resulting in a 59.9 % reduction in tumor volume. CETSA analysis of tumor tissues showed that PRX1 and PRX2 were bound and thus inactivated by HCA in a mouse xenograft model. These findings demonstrate that PRX1 and PRX2 are molecular target proteins responsible for HCA-induced ROS elevation and cancer cell death.

Synergistic Inhibition of Nasopharyngeal Carcinoma by Biyan Qingdu Granule and Micro-Radiation: An Experimental Study

Objective: To investigate the role of Biyan Qingdu Granule combined with micro-radiation in suppressing nasopharyngeal carcinoma and to explore its mechanism of action. Methods: FAT cells of nasopharyngeal carcinoma were injected into the hind limb axilla of F344 rats to establish a rat nasopharyngeal carcinoma tumor model. X-ray radiation was applied to the oral cavity of the rats, delivering a micro-dose of radiation to the distant hind limb axilla tumor. The general condition of the rats, including oral mucosal inflammation, the size of the nasopharyngeal tumor mass, pathological changes in the tumor tissue, and the levels of TNF-α, IL-1β, IL-6, IL-18, IL-12, ICAM-1, and VCAM-1 in the tumor tissue, were assessed. Results: Oral administration of Biyan Qingdu Granule improved the rats’ overall condition and reduced oral mucosal inflammation. Pathological examination revealed tumor cell degeneration and necrosis. ELISA analysis of tumor tissue showed significant reductions in TNF-α, IL-1β, IL-6, ICAM-1, and VCAM-1 levels, along with a notable increase in IL-18 levels. Conclusion: Biyan Qingdu Granule, when combined with micro-radiation, can inhibit tumor growth and reduce tumor volume. Its mechanism of action may involve enhancing immune function and suppressing inflammatory factors.

Comprehensive Clinical Genetics, Molecular and Pathological Evaluation Efficiently Assist Diagnostics and Therapy Selection in Breast Cancer Patients with Hereditary Genetic Background.

Using multigene panel testing for the diagnostic evaluation of patients with hereditary breast and ovarian cancer (HBOC) syndrome often identifies clinically actionable variants in genes with varying levels of penetrance. High-penetrance genes (BRCA1, BRCA2, CDH1, PALB2, PTEN, STK11, TP53) inform specific clinical surveillance and therapeutic decisions, while recommendations for moderate-penetrance genes (ATM, BARD1, BRIP1, CHEK2, MLH1, MSH2, MSH6, PMS2, EPCAM, NF1, RAD51C, RAD51D) are more limited. A detailed disease history, including pedigree data, helps formulate the most appropriate and personalised management strategies. In this study, we evaluated the clinical benefits of comprehensive hereditary cancer gene panel testing and a pre-sent questionnaire in Hungarian patients with suspected HBOC syndrome. We prospectively enrolled 513 patients referred for HBOC testing. Of these, 463 met the genetic testing criteria, while 50 did not but were tested due to potential therapeutic indications. Additionally, a retrospective cohort of 47 patients who met the testing criteria but had previously only been tested for BRCA1/2 was also analysed. Among the 463 patients in the prospective cohort, 96 (20.7%) harboured pathogenic/likely pathogenic (P/LP) variants-67 in high-penetrance genes and 29 in moderate-penetrance genes. This ratio was similar in the retrospective cohort (6/47; 12.7%). In patients who did not meet the testing criteria, no mutations in high-penetrance genes were found, and only 3 of 50 (6%) harboured P/LP variants in moderate-penetrance genes. Secondary findings (P/LP variants in non-HBOC-associated genes) were identified in two patients. In the prospective cohort, P/LP variants in BRCA1 and BRCA2 were the most prevalent (56/96; 58.3%), and the extended testing doubled the P/LP detection ratio. Among moderate-penetrance genes, five cases (three in the prospective and two in the retrospective cohorts) had P/LP variants in Lynch syndrome-associated genes. Further immunohistochemistry analysis of breast tumour tissues helped clarify the causative role of these variants. Comprehensive clinical and molecular genetic evaluation is beneficial for the diagnosis and management of patients with P/LP variants in hereditary tumour-predisposing genes and can serve as a basis for effective therapy selection, such as PARP inhibitors or immunotherapy.

Abstract B033: Identification of cell-specific DNA methylation alterations in the breast tumor microenvironment

Abstract The tumor microenvironment (TME) comprises diverse cell types whose interactions are crucial for cancer progression. Traditional bulk tissue analyses mask cell-type-specific epigenetic changes that may drive disease progression. We used DNA methylation data and a reference- based cell-type deconvolution algorithm — hierarchical tumor immune microenvironment epigenetic deconvolution (HiTIMED) — to quantify cell types in the TME. Then, we employed CellDMC, a statistical interaction testing framework that can use cell type proportions in conjunction with differential methylation analysis to identify cell-specific differential methylation. Genome-scale DNA methylation profiles from breast cancer tissue (n=609) and normal breast tissue (n=230) samples were accessed in GEO and GTEx. Our novel approach leverages bulk DNA methylation data to infer cell-type-specific epigenetic landscapes, circumventing challenges and limitations associated with direct single-cell methylation profiling, such as high costs and technical variability. Reference-based computational tools allow us to dissect complex tissue compositions and reveal distinct methylation patterns and pathways that may be unique to individual cell types, providing insights that are not discernible from traditional bulk tumor analysis approaches. Compared with nontumor normal samples, we identified significant DNA methylation alterations (FDR≤0.05) across tumor, stromal, endothelial, epithelial, lymphocytes, and myeloid cells in breast tumors. Tumor cells exhibited the highest number, 8,510, differentially methylated cytosines (DMCs) with 1,966 of these unique to tumor cells. Stromal, endothelial, and epithelial cells also showed considerable methylation alterations, with a total of 530, 440, 339 DMCs, respectively, with 5 DMCs unique to epithelial cells. Despite lower immune cell proportions in our samples, we identified 104 lymphocyte specific DMCs and 185 myeloid specific DMCs, with 14 and 20 DMCs unique to these cell types respectively. The unique DMCs identified in each cell type suggest distinct epigenetic landscapes that may drive specific cellular functions and interactions within the TME. Importantly, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the annotated cell-type-specific DMCs revealed distinct pathways that were not apparent in the pathway analysis of bulk tumor versus normal tissue, underscoring the value of examining epigenetic alterations at the cell-type level. Our findings highlight the opportunity to leverage DNA-based cell typing with DNA methylation data to discern cell-specific molecular alterations. By focusing on cell-type-specific alterations, we can better understand the epigenetic mechanisms driving TME crosstalk, such as the activation of stromal cells and the modulation of immune responses by tumor cells. Our findings underscore the need for targeted therapeutic strategies that consider the unique epigenetic landscapes of individual cell types within the TME, offering new avenues for improving cancer treatment outcomes. Citation Format: Barbara Karakyriakou, Brock C. Christensen, Lucas A. Salas. Identification of cell-specific DNA methylation alterations in the breast tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B033.

Abstract C035: Spatially limited stroma-mediated resistance potentiates targeted therapy resistance through an integration of multiple juxtacrine and paracrine mechanisms

Abstract Despite the ability to induce strong clinical responses, therapies that target mutational drivers of oncogenic signaling are not curative in advanced cancers, as a subset of cancer cells is capable of surviving therapy and evolving resistance. While therapy resistance is commonly viewed as a cell-intrinsic phenomenon, multiple factors produced by tumors can also confer strong therapy resistance. In contrast to the detailed elucidation of molecular mediators of cell-intrinsic and stroma-mediated (SM) resistance, the relative importance of cell-intrinsic and SM resistance to in vivo therapeutic responses remains poorly defined. To address this gap of knowledge, we used a well-characterized experimental model of ALK+ lung cancer H3122 cell line that exemplifies a duality observed across common models of targetable cancers. Under standard stroma-free in vitro cultures, H3122 cells can survive under therapeutically relevant concentrations of ALK inhibitors (ALKi), eventually acquiring cell-intrinsic therapy resistance. At the same time, co-culture with stromal fibroblasts drastically reduces the sensitivity of H3122 cells to ALKi, indicating the relevance of SM resistance. To understand the impact of SM resistance on in vivo responses, we sought to identify the mechanism(s) responsible for the therapy-protective effects of stromal fibroblasts, subsequently interrogating the impact of the disruption of this mechanism on in vivo therapeutic responses. Our in vitro studies with a large panel of primary stromal fibroblast isolates identified the HGF-cMET axis as the major mechanism responsible for ALKi desensitization. Surprisingly, xenograft validation studies demonstrated a weak effect of HGF-cMET modulation. Histological analyses of tumor tissues revealed that this relative weakness is attributable to a strong, spatially limited HGF-independent component of SM resistance. Mechanistic follow-up demonstrated that the HGF-independent component of SM resistance integrates the effect of multiple well-known juxtacrine and paracrine-acting mechanisms. Whereas the multifactorial nature of stroma-mediated resistance prevented a clear assessment of its relative contribution to in vivo therapy responses, our spatial analyses indicate that SM resistance dominates the ability of tumors to avoid therapeutic elimination. Surprisingly, we found that SM resistance was also a substantial contributor to tumor relapse, indicating that in vivo therapy resistance can integrate both cell-intrinsic and cell-extrinsic effects. This duality of therapy resistance and the multifactorial underpinning of both cell-intrinsic and stroma-mediated resistance present a challenge to therapeutic strategies focused on identifying and targeting specific resistance mechanisms. Our studies suggest that this limitation can be overcome by shifting the therapeutic focus towards shared orthogonal therapeutic sensitivities of tumor cells within residual disease. Citation Format: Bina Desai, Tatiana Miti, Sandhya Prabhakaran, Daria Miroshnychenko, Menkara Henry, Viktoriya Marusyk, Chandler Gatenbee, Marilyn Bui, Jacob Scott, Philipp M. Altrock, Eric Haura, Alexander R.A. Anderson, David Basanta, Andriy Marusyk. Spatially limited stroma-mediated resistance potentiates targeted therapy resistance through an integration of multiple juxtacrine and paracrine mechanisms [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C035.

Gadoxetic Acid-Enhanced Magnetic Resonance Imaging Features Can Predict Immune-Excluded Phenotype of Hepatocellular Carcinoma

Introduction: Immunotherapy is the first-line treatment for intermediate-advanced stage hepatocellular carcinoma (HCC), although its outcomes vary. This study aimed to identify imaging biomarkers of immunotherapy susceptibility linked to gadoxetic acid-enhanced magnetic resonance imaging (EOB-MRI) and immune phenotypes, particularly immune-excluded phenotypes, with a tumor immune barrier. Methods: We performed immunohistochemical staining with a CD8+ antibody, and samples were classified into immune-inflamed, -intermediate, -excluded, and -ignored phenotypes. We assessed EOB-MRI findings obtained from 104 patients who underwent hepatectomy for HCC and evaluated the relationship between MRI findings and immune phenotype. Spatial transcriptome analysis of tumor tissues in each immune phenotype was performed to characterize the MRI findings. For validation, we analyzed the treatment effect on 60 nodules in another cohort of 27 patients who received combined immunotherapy using anti-programmed death-ligand 1 and anti-vascular endothelial growth factor (VEGF) antibodies. Results: HCCs with rim arterial phase hyperenhancement (APHE) (odds ratio [OR] 17.3, p = 0.009), peritumoral enhancement in the arterial phase (OR: 8.6, p < 0.004), and intermediate intensity on the hepatobiliary phase (HBP) measured with a visual 3-point scale (OR: 28.2, p = 0.002) were associated with immune-excluded phenotype, where tumors tended to be larger and of the single nodular type with extranodular growth and confluent multinodular rather than the simple nodular type. Spatial transcriptome analysis revealed a spatial relationship among cytotoxic T lymphocytes, VEGF signals, and cancer-associated fibroblasts at the tumor-invasive margins in this phenotype. From the validation study, nodules with any one of these three imaging findings had a significantly prolonged time to-nodular progression (p = 0.007, median not reached vs. 226 days). Conclusion: HCCs with rim APHE, peritumoral enhancement in arterial phase, and intermediate intensity on HBP with visual 3-point scale could be non-invasive biomarkers to predict the immune-excluded phenotype with the tumor immune barrier. These HCCs were most likely to respond to combined immunotherapy.

IMMU-03. EXPLORING CANCER-SPECIFIC T CELLS AND ANTIGENS IN GLIOBLASTOMA BASED ON SINGLE-CELL SEQUENCING OF CD8+ TILS

Abstract BACKGROUND The effectiveness of cancer immunotherapy against glioblastoma (GBM) remains limited. This study aims to identify cancer-specific antigens to develop antigen-based cancer immunotherapies for GBM. We investigated candidate tumor antigen-specific T cells in GBM by single-cell RNA sequencing (scRNA-seq) and single-cell TCR sequencing (scTCR-seq), and we explored candidate antigens through genetic analysis of tumor tissues. METHODS Flow cytometry analysis was conducted on fresh tumor digest samples to evaluate tumor-infiltrating lymphocytes (TILs) of GBM. Single-cell RNA and TCR sequencing were performed on CD8+ T cells in TILs. Both data generated by Cell Ranger software were loaded into Seurat at R studio. The dimensional reduction for clustering was performed with uniform manifold approximation and projection (UMAP). Additionally, we performed bulk RNA sequencing (RNA-seq) and whole exome sequencing (WES) on tumor tissues. RESULTS Two out of 20 patients (10%) exhibited abundant TILs in GBM. From these two patients, approximately 20,000 CD8+ T cells in total were analyzed in single-cell analysis. Clustering based on UMAP revealed a predominance of clusters expressing exhaustion markers, with high TCR clonality centered on exhausted T cell (TEX) clusters, like our previous date of lung cancer TILs recognizing tumor antigens. Moreover, within the TEX clusters, the expression of CXCL13, often reported as an antigen-specific marker in recent years, was significantly higher in our data compared to two publicly available datasets. Subsequently, based on RNA-seq and WES, 61 candidate neoantigens were estimated using machine-learning prediction algorithms from NEC Corporation. Furthermore, 5 overexpressed cancer/testis antigens, and 3 bacterial species-derived microbial peptides using Kraken2 were selected as antigen candidates. CONCLUSIONS We identified prospective tumor antigen-specific T cell subsets with high CXCL13 expression from two GBM patients. We plan to identify cancer-specific T cells and antigens from these candidates by T cell activation assay.

BIOM-09. CLINICAL EFFECT OF CD25 ON THE MTX-BASED CHEMOSENSITIVITY OF PRIMARY CENTRAL NERVUS SYSTEM LYMPHOMA

Abstract INTRODUCTION Primary central nervous system diffuse large B-cell lymphoma (PCNSL) is treated with chemotherapy, mainly with high-dose methotrexate (HD-MTX), but some cases are resistant to treatment and relapse in a short period. However, a biomarker that predicts the chemosensitivity and recurrence has not yet been identified. In this study, we used surface marker analysis of tumor tissue using flow cytometry to search for biomarkers related to chemosensitivity and early recurrence. METHODS From 2017 to 2023, 25 patients were diagnosed with PCNSL by biopsy in Kobe University Hospital, and flow cytometry of the biopsy tissues were performed. Cerebrospinal fluid (CSF) examination including sIL2R, β2MG, and IL-10 was performed in all cases before surgery, and treatment based on methotrexate was administered postoperatively. We retrospectively analyzed patient characteristics, flow cytometry data, treatment details, response rate, time to recurrence, and overall survival. RESULTS The mean age of the patients was 71 (range, 37-80) years. In univariate analysis, high expression level of CD25 (interleukin-2 receptor, IL-2R) in the tissue was associated with a lower response rate to MTX-based chemotherapy (Fisher’s exact test, p=0.0072) and had a tendency of shorter time to recurrence. However, there were no significant differences in progression-free survival and overall survival. There was no correlation between expression level of CD25 in tissue and sIL-2R in CSF. Expression level of sIL-2R in CSF was independent of CD25 expression in tissue. CONCLUSION There are some reports that CD25 can be a prognostic factor in follicular lymphoma, but no reports in PCNSL. Here, we suggested that high expression level of CD25 in PCNSL tissue was associated to chemoresistance and early recurrence. In cases of PCNSL with high CD25 expression level, additional whole-brain irradiation and transition to tirabrutinib be considered because there are possibilities of chemoresistance and early recurrence.

Imaging the main molecular biological subtypes of breast cancer: comparison of mammographic data and histological findings

Background. Breast cancer (BC) stands as one of the most prevalent malignancies affecting women, posing a significant threat to health and life. Timely diagnosis and treatment of BC play a pivotal role in enhancing patient survival rates.Aim. To explore such a method of visualization of BC as mammography and the correlation of its results with the data of histological and immunohistochemical studies, and their significance in planning organ-conserving operations.Materials and methods. The study involved 217 patients diagnosed with nodular BC (T1-2N1M0). AH patients underwent digital mammography, histological examination of biopsy and surgical specimens, and immunohistochemical analysis of tumor tissue (determination of the expression of sex hormone receptors (estrogen and progesterone), HER2/neu status and the status of the Ki-67 marker, reflecting the proliferative activity of tumor cells).Results. Comparison of mammographic and histological/immunohistochemical findings revealed significant differences in tumor visualization among major molecular subtypes of BC. A statistically significant association (p &lt;0.001) was established between carcinoma in situ and radiological features such as spiculated margins and calcifications on mammography.Conclusion. Mammography emerges as an objective and accessible visualization method for BC, enabling assessment of tumor size and peritumoral region. However, for planning breast-conserving surgery for luminal and HER2-positive BC subtypes, a multimodal diagnostic approach is recommended to assess tumor spread, incorporating ultrasound and contrast-enhanced magnetic resonance imaging.

Modification of the hi-c technology for molecular genetic analysis of formalin-fixed paraffin-embedded sections of tumor tissues

Molecular genetic analysis of tumor tissues is the most important step towards understanding the mechanisms of cancer development, and it is also necessary for the choice of targeted therapy. Hi-C (high-throughput chromatin conformation capture) technology can be used to detect various types of genomic variants, including balanced chromosomal rearrangements: inversions and translocations. In this work, we propose a modification of the Hi-C method for the analysis of chromatin contacts in formalin-fixed paraffin-embedded (FFPE) sections of tumor tissues. Our results demonstrate that this protocol allow to generate high-quality Hi-C data and detect all types of chromosomal rearrangements. We have analyzed various databases to compile a comprehensive list of translocations that hold clinical importance for targeted therapy selection. The practical value of molecular genetic testing is its ability to influence patient treatment strategies and provide prognostic insights. Detecting specific chromosomal rearrangements can guide the choice of targeted therapies, which is a critical aspect of personalized medicine in oncology.

Prognostic biomarker validation in high grade serous carcinoma with the ovarian tumor tissue analysis consortium and the multidisciplinary ovarian cancer outcomes group

Prognostic biomarker validation in high grade serous carcinoma with the ovarian tumor tissue analysis consortium and the multidisciplinary ovarian cancer outcomes group

Potential mechanisms of interstitial lung disease induced by antibody-drug conjugates based on quantitative analysis of drug distribution.

Antibody-drug conjugates (ADCs) are a rapidly advancing category of therapeutic agents with notable anti-cancer efficacy. However, the emergence of interstitial lung disease (ILD) as a severe ADC-associated adverse event highlights the need to better understand the underlying mechanisms. In this study, xenograft model mice with tumors expressing different levels of the trophoblast antigen 2 (TROP2) were generated by subcutaneously transplanting the various TROP2-expression cancer lines. The mice received different doses of TROP2-eribulin, a novel TROP2-targeting ADC, composed of an anti-TROP2 antibody and the eribulin payload, joined by a cleavable linker. The concentration and distribution of TROP2-eribulin, as well as the pharmacokinetics of eribulin release, were assessed in tumor and lung tissues. Analysis of tumor tissue showed that the concentration of released eribulin was approximately 10-fold higher in NCI-H2110 (high TROP2 expression) than in A549 (low TROP2 expression), while analysis of lung tissue showed that TROP2-eribulin was distributed in lung tissue in a dose-dependent manner of TROP2-eribulin regardless of TROP2 expression, with significantly more eribulin released in the high-dose group than in the other dose groups (P < 0.05). Immunofluorescence assay analysis showed that TROP2-eribuilin localized to alveolar macrophages. In the analysis using human- leukemia monocytic cell, the concentration of eribulin released from TROP2-eribuilin was significantly reduced by the use of an Fc receptor inhibitor (P < 0.05). These results revealed that Fcγ-receptor-mediated uptake by alveolar macrophages releases cytotoxic payload into lung tissue, helping to clarify the pathogenesis of ADC-induced ILD.

A proteogenomic surfaceome study identifies DLK1 as an immunotherapeutic target in neuroblastoma

Cancer immunotherapies produce remarkable results in B cell malignancies; however, optimal cell surface targets for many solid cancers remain elusive. Here, we present an integrative proteomic, transcriptomic, and epigenomic analysis of tumor and normal tissues to identify biologically relevant cell surface immunotherapeutic targets for neuroblastoma, an often-fatal childhood cancer. Proteogenomic analyses reveal sixty high-confidence candidate immunotherapeutic targets, and we prioritize delta-like canonical notch ligand 1 (DLK1) for further study. High expression of DLK1 directly correlates with a super-enhancer. Immunofluorescence, flow cytometry, and immunohistochemistry show robust cell surface expression of DLK1. Short hairpin RNA mediated silencing of DLK1 in neuroblastoma cells results in increased cellular differentiation. ADCT-701, a DLK1-targeting antibody-drug conjugate (ADC), shows potent and specific cytotoxicity in DLK1-expressing neuroblastoma xenograft models. Since high DLK1 expression is found in several adult and pediatric cancers, our study demonstrates the utility of a proteogenomic approach and credentials DLK1 as an immunotherapeutic target.

Results of expression microarray analysis of tumor tissue from patients with colorectal cancer

The aim of the study was to evaluate the expression profile and search for markers of progression in tumor tissue in colorectal cancer. Materials and Methods - Tumor tissue samples obtained from videocolonoscopy in patients with verified colorectal cancer served as the material for the study. A total of 37 specimens were involved in the study. Of these, there were 14 samples with a “favorable” prognosis and 23 with an “unfavorable” prognosis. The quality and quantity of ribonucleic acid in the eluted solution were evaluated using an IMPLEN nanospectrophotometer (Germany). A SurePrint G3 HumanGeneExpv3 ArrayKit microarray kit (Agilent, USA) was used to assess gene expression. Microarrays were scanned on an InnoScan 1100 AL (USA) with subsequent image processing using Mapix Software (USA). For the procedure of searching for differentially expressed genes, we used the Moderated t-statistics method, which is implemented in the limma package. Results - Based on the scatter plot, the most divergent genes in samples with and without progression were selected. The following markers were selected: Marker9 (GZMB), Marker5 (CXCL11), Marker3 (SYNE4), Marker20 (MIR4432HG), Marker19 (ZDHHC11), Marker11 (COL17A1), Marker13 (ZDHHC11B), Marker17 (AGR3). Low-expressing genes mainly affected DNA replication and cell cycle pathways, whereas high-expressing genes affected metabolic pathways. Conclusions - We found an association of long-term outcomes with the expression of the gene combination ZDHHC11, MIR4432HG, and GZMB. Further study of this gene combination and increased sampling will allow the construction of a test system for predicting the course and response to treatment of patients with colorectal cancer.

Elucidating the power of arginine restriction: taming type I interferon response in breast cancer via selective autophagy

BackgroundType I interferons (IFN-I) are potent alarm factors that initiate cancer cell elimination within tumors by the immune system. This critical immune response is often suppressed in aggressive tumors, thereby facilitating cancer immune escape and unfavorable patient outcome. The mechanisms underpinning IFN-I suppression in tumors are incompletely understood. Arginase-1 (ARG1)-expressing immune cells that infiltrate tumors can restrict arginine availability by ARG1-mediated arginine degradation. We hypothesized that arginine restriction suppresses the IFN-I response in tumors.MethodsComprehensive, unbiased open approach omics analyses, various in vitro techniques, including microscopy, qPCR, immunoblotting, knock-down experiments, and flow cytometry were employed, as well as ex vivo analysis of tumor tissue from mice. Several functional bioassays were utilized to assess metabolic functions and autophagy activity in cancer cells.ResultsArginine restriction potently induced expression of selective autophagy receptors, enhanced bulk and selective autophagy and strongly suppressed the IFN-I response in cancer cells in an autophagy-dependent manner.ConclusionOur study proposes a mechanism for how tumor-infiltrating immune cells can promote cancer immune escape by dampening the IFN-I response. We suggest ARG1 and autophagy as putative therapeutic targets to activate the IFN-I response in tumors.