Expression Levels In Tumor Cells Research Articles

Abstract 1328: AccuBaseTM (ceBE), a second-generation high-fidelity base editor, can proficiently modify genes in a human primary NK cells with minimizing off-target effect

Abstract Natural Killer (NK) cells hold great promise for cancer therapy owing to their innate ability to detect cells expressing signs of stress or infection. However, NK cell-based therapeutics face significant challenges: limited expansion and genetic modification resistance. In response to these challenges, we present our pioneering work in the development of Base Editing-Directed NK (BED-NK) cell products, referred to as NK510, which is an off-the-shelf allogeneic cell therapy product containing peripheral blood mononuclear cell (PBMC)-derived TIGIT knock-out NK cells. Through a meticulously planned optimization process, we have achieved over 80% editing efficiency in NK510s using our high-fidelity Base Editor, AccuBase™. By knocking out TIGIT (an inhibitory receptor in NK cells), we have prompted tumor cell-derived CD155 (an inhibitory ligands in tumor cells) to engage with CD226 (an activating receptor in NK cells), triggering activating signals in NK510s. Our results showed that CD155 positive tumor cells were more susceptible to NK510s than CD155 negative tumor cells. And target killing percentage is positively related to CD155 expression level in tumor cells. This transformation effectively converted an inhibitory ligand into an activating one, resulting in tumor regression in multiple mouse models, including non-small cell lung cancer (NSCLC) and sarcoma. We developed a GMP-compliant NKBoost+ expansion system to generate a millionfold expansion of NK510 within a 21-day expansion protocol. To investigate the safety of NK510 product, we conducted repeated dosing toxicity, tumorigenicity, and oncogenicity study in a GLP facility, and the results validated the safety of NK510. Additionally, we developed an analytical protocol based on Droplet Digital PCR (ddPCR) technology to investigate the pharmacokinetics (PK) and biodistribution of NK510s in NSCLC tumor-bearing mice. We observed an enrichment of NK510-derived genomic DNA (gDNA) in lung tissues of tumor-bearing mice at day 1. The concentration of NK510-derived gDNA was up to 2000 copies/ug DNA. The concentration of NK510-derived gDNA sharply decreased from day 2 to day 6, and then decreased to below the lower limit of quantification (LLOQ). Distinct from the PK data of NK510s, the concentration of NK510-derived gDNA started low at day 1 and reached peak concentration at day 2 (in blood samples) and at day 4 (in liver tissues), respectively. These results demonstrated the efficacy and safety of BED-NK product and underscore the feasibility of combining base editing with ex vivo expansion of NK cells, significantly enhancing their therapeutic potential in clinical settings. Citation Format: Han Ying, Chao Yao, Cheng Fang, Guanglei Li, Lixin Wang, Yufu Zhou, Chenyuan Gong, Juewan Li, Yiming Wang, Wenwen Fan, Fenghua Lv, Xiaohu Shao, Haiyang Zhao, Liling Wang, Jun Yan, Tianhong Xu, Shiguo Zhu. AccuBaseTM (ceBE), a second-generation high-fidelity base editor, can proficiently modify genes in a human primary NK cells with minimizing off-target effect [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1328.

Association of Androgen Receptor and PD-L1 Expression in Upper Urinary Tract Urothelial Carcinoma.

The response to immune checkpoint inhibitors (ICIs) or enfortumab vedotin is limited in patients with upper urinary tract urothelial carcinoma (UTUC), and the development of new targeted therapy for UTUC is eagerly needed. Several biomarkers, including programmed cell death-ligand 1 (PD-L1), have already been reported as predictors of response to ICIs therapy for UTUC. Recently, several studies have shown that steroid hormone receptors, including the androgen receptor (AR), are associated with progression of urothelial carcinoma. We prepared tissue microarrays (TMA) from paraffin blocks of UTUC specimens in 99 non-metastatic UTUC patients who underwent radical nephroureterectomy. With these TMA sections, we performed immunohistochemical staining for PD-L1 and AR and examined PD-L1 and AR expression levels in tumor cells. In addition, we analyzed the correlation between these markers and clinical prognosis in UTUC cases. PD-L1 was positive in 24 (24%) of the 99 samples, whereas AR was positive in 20 (20%) patients. AR-negative samples had significantly higher PD-L1 expression level than that the AR-positive samples (mean value 4.70% versus 2.55%, p=0.0324). Among AR-positive cases, patients with absence of PD-L1 expression had significantly lower cancer-specific survival (CSS) than that in PD-L1 expression-positive cases (p=0.049), although PD-L1 expression had no significant impact on CSS in AR-negative cases (p=0.920). Our findings suggest that AR is the promising target for UTUC treatment, especially in PD-L1-negative cases.

An innovative gene expression modulating strategy by converting nucleic acids into HNC therapeutics using carrier-free nanoparticles.

Cell fate and microenvironmental changes resulting from aberrant expression of specific proteins in tumors are one of the major causes of inadequate anti-tumor immune response and poor prognosis in head and neck cancer (HNC). Eukaryotic initiation factor 3C (eIF3c) has emerged as a promising therapeutic target for HNC due to its ability to regulate protein expression levels in tumor cells, but its drug development is difficult to achieve by targeting traditional protein-protein interactions. siRNA has emerged as a highly promising modality for drug development targeting eIF3c, while its application is hindered by challenges pertaining to inadequate stability and insufficient concentration specifically within tumor sites. We employed a method to convert flexible siRNAs into stable and biologically active infinite Auric-sulfhydryl coordination supramolecular siRNAs (IacsRNAs). Through coordinated self-assembly, we successfully transformed eIF3C siRNAs into the carrier-free HNC nanotherapeutic agent Iacs-eif3c-RNA. The efficacy of this agent was evaluated in vivo using HNC xenograft models, demonstrating promising antitumor effects. Iacs-eif3c-RNA demonstrated the ability to overcome the pharmacological obstacle associated with targeting eIF3C, resulting in a significant reduction in eIF3C expression within tumor tissues, as well as effective tumor cell proliferating suppression and apoptosis promotion. In comparison to monotherapy utilizing the chemotherapeutic agent cisplatin, Iacs-eif3c-RNA exhibited superior anti-tumor efficacy and favorable biosafety. The utilization of Iacs-eif3c-RNA as a carrier-free nanotherapeutic agent presents a promising and innovative approach for addressing HNC treating challenges. Moreover, this strategy demonstrates potential for the translation of therapeutic siRNAs into clinical drugs, extending its applicability to the treatment of other cancers and various diseases.

Predictive indicators of immune therapy efficacy in hepatocellular carcinoma based on neutrophil-to-lymphocyte ratio

Hepatocellular carcinoma (HCC) exhibits high incidence and mortality rates in China. Most cases are often diagnosed at late stages and require multi-strategy therapies. In recent years, immune checkpoint inhibitors (ICIs), particularly programmed cell death protein 1 (PD-1) antibodies, have demonstrated effectiveness in comprehensive HCC treatment. However, the efficacy and prognosis vary greatly among patients. Screening suitable patients and predicting outcomes are crucial for improving the efficacy of ICIs. Although PD-L1 expression levels in tumor cells have been used as predictors of PD-1/PD-L1 antibody therapy, they may not consistently correlate with clinical response in some studies; thus, exploring new biomarkers is necessary. The neutrophil-to-lymphocyte ratio (NLR) emerged as a new predictor of ICI immunotherapy efficacy, and its application in HCC is worth exploring. This study utilizes the Cancer Genome Atlas Liver Hepatocellular Carcinoma Collection (TCGA-LIHC) project in the Genomic Data Commons (GDC) database for methylation and transcriptome data analysis. The correlation between NLR and ICI immunotherapy efficacy for HCC was evaluated, identifying differentially expressed genes. Analysis revealed 74 up-regulated and 445 down-regulated genes in the high-NLR group compared to the low-NLR group. NLR-related differential methylation analysis identified 68 hypermethylated and 65 hypomethylated probes in the NLR high group. Furthermore, a machine learning model using 27 intersecting genes predicted PD-1 antibody therapy efficacy, achieving an AUC value of 0.813. In summary, we established a predictive model for HCC immunotherapy based on 27 genes related to differential expressions and NLR-associated methylation, showing significant potential for clinical research potential in this field.

Abstract A173: ASP2074, a novel tetraspanin 8 x CD3 bispecific antibody, demonstrates selectivity and antitumor activity in preclinical cancer models

Abstract ASP2074 is a novel bispecific antibody that targets the tumor-associated antigen tetraspanin 8 (TSPAN8) and CD3, which is expressed on the plasma membrane of all T cells. By engaging with both TSPAN8 and CD3, ASP2074 enables the retargeting of cytotoxic T cells specifically against TSPAN8-expressing tumor cells in a human leukocyte antigen-independent manner. TSPAN8 is a member of the tetraspanin superfamily of proteins that plays a crucial role in intracellular signal transduction and regulates various cellular functions, including leukocyte trafficking, angiogenesis, and wound repair. TSPAN8 is highly expressed in various types of cancer, such as colorectal cancer, gastric cancer, pancreatic cancer, esophageal adenocarcinoma, and gastroesophageal junction (GEJ) adenocarcinoma. Its overexpression has been associated with tumor cell growth, angiogenesis, invasion, metastasis, and poor prognosis, making it an attractive target for antibody-based cancer immunotherapy. ASP2074 is currently undergoing phase 1 clinical trials for the treatment of patients with colorectal cancer, esophageal adenocarcinoma, pancreatic cancer, gastric cancer, and GEJ adenocarcinoma. In this study, we examined the selectivity and antitumor activity of ASP2074 in vitro and in vivo. Methods: Retrogenix Cell Microarray Technology was used to evaluate the selective binding of ASP2074 to TSPAN8 and CD3 in 6018 full-length human plasma membrane proteins, secreted and cell surface-tethered human secreted proteins, and 397 human heterodimers containing homologs and isoforms of tetraspanin. Cytotoxicity and T-cell activation of ASP2074 were examined using TSPAN8-expressing tumor cells in combination with human peripheral blood mononuclear cells (PBMCs) as effector cells in a redirected T-cell cytotoxicity assay. Furthermore, the antitumor effect of ASP2074 was examined in a human PBMC-engrafted NOG mouse model bearing human TSPAN8-expressing colorectal cancer cells. Results: Our results showed that ASP2074 selectively bound to TSPAN8 and CD3 without significant binding to other human plasma membrane proteins, secreted proteins, and heterodimers. ASP2074 exhibited cytotoxicity against TSPAN8-expressing cancer cells and increased interferon-gamma production and expression of T-cell activation markers. The cytotoxic activities were dependent on the TSPAN8 expression levels in tumor cells. In the NOG mouse model, ASP2074 exhibited an antitumor effect on human TSPAN8-expressing colorectal cancer cells in vivo. Conclusion: Our findings suggest that ASP2074 has potent antitumor activity through cytotoxicity against TSPAN8-expressing tumor cells by T-cell activation via selective binding to TSPAN8 and CD3. These preclinical results support the continued development of ASP2074 as a promising immunotherapy for the treatment of TSPAN8-expressing cancers. Citation Format: Yoshiyuki Tenda, Yuichi Hanada, Hiroaki Tanaka, Masashi Shimazaki, Kenna Shirasuna. ASP2074, a novel tetraspanin 8 x CD3 bispecific antibody, demonstrates selectivity and antitumor activity in preclinical cancer models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A173.

Low-density lipoprotein receptor promotes crosstalk between cell stemness and tumor immune microenvironment in breast cancer: a large data-based multi-omics study

BackgroundTumor cells with stemness in breast cancer might facilitate the immune microenvironment’s suppression process and led to anti-tumor immune effects. The primary objective of this study was to identify potential targets to disrupt the communication between cancer cell stemness and the immune microenvironment.MethodsIn this study, we initially isolated tumor cells with varying degrees of stemness using a spheroid formation assay. Subsequently, we employed RNA-seq and proteomic analyses to identify genes associated with stemness through gene trend analysis. These stemness-related genes were then subjected to pan-cancer analysis to elucidate their functional roles in a broader spectrum of cancer types. RNA-seq data of 3132 patients with breast cancer with clinical data were obtained from public databases. Using the identified stemness genes, we constructed two distinct stemness subtypes, denoted as C1 and C2. We subsequently conducted a comprehensive analysis of the differences between these subtypes using pathway enrichment methodology and immune infiltration algorithms. Furthermore, we identified key immune-related stemness genes by employing lasso regression analysis and a Cox survival regression model. We conducted in vitro experiments to ascertain the regulatory impact of the key gene on cell stemness. Additionally, we utilized immune infiltration analysis and pan-cancer analysis to delineate the functions attributed to this key gene. Lastly, single-cell RNA sequencing (scRNA-seq) was employed to conduct a more comprehensive examination of the key gene’s role within the microenvironment.ResultsIn our study, we initially identified a set of 65 stemness-related genes in breast cancer cells displaying varying stemness capabilities. Subsequently, through survival analysis, we pinpointed 41 of these stemness genes that held prognostic significance. We observed that the C2 subtype exhibited a higher stemness capacity compared to the C1 subtype and displayed a more aggressive malignancy profile. Further analysis using Lasso-Cox algorithm identified LDLR as a pivotal immune-related stemness gene. It became evident that LDLR played a crucial role in shaping the immune microenvironment. In vitro experiments demonstrated that LDLR regulated the cell stemness of breast cancer. Immune infiltration analysis and pan-cancer analysis determined that LDLR inhibited the proliferation of immune cells and might promote tumor cell progression. Lastly, in our scRNA-seq analysis, we discovered that LDLR exhibited associations with stemness marker genes within breast cancer tissues. Moreover, LDLR demonstrated higher expression levels in tumor cells compared to immune cells, further emphasizing its relevance in the context of breast cancer.ConclusionLDLR is an important immune stemness gene that regulates cell stemness and enhances the crosstalk between breast cancer cancer cell stemness and tumor immune microenvironment.

Correlation of Beta2-Glycoprotein I With Tumor Prognosis in Breast Cancer Patients.

Beta2-glycoprotein I (β2-GPI) is a plasma glycoprotein, which has been implicated in a variety of physiological functions. However, the connection between β2-GPI and breast cancer is mostly unknown. Breast cancer is a malignant tumor that severely impairs women's health worldwide. The aim of the study was to investigate the role of β2-GPI in tumor cells of breast cancer patients and its correlation with tumor prognosis. A total of 125 female patients diagnosed with breast cancer were enrolled in the study. The expression of β2-GPI in resected breast tissues was determined by immunohistochemistry (IHC) and correlated with clinicopathological variables by the Chi-squared test. The prognostic value of β2-GPI for overall survival (OS) and disease-free survival (DFS) was determined by Kaplan-Meier estimates and the significance of differences was evaluated by the log-rank test. β2-GPI staining was predominantly observed in tumor cells of breast cancer patients and significantly correlated with tumor stage and lymph node metastasis of breast cancer. High β2-GPI expression was significantly correlated with better OS and DFS. Moreover, DFS was found to be significantly better in patients with higher β2-GPI expression, especially those in the early tumor stage groups. High β2-GPI expression levels in tumor cells of breast cancer patients were independent factors predicting a better OS and DFS. β2-GPI activation in high-risk patients may be a potential strategy for reducing breast cancer progression.

Tumor Area Positivity (TAP) score of programmed death-ligand 1 (PD-L1): a novel visual estimation method for combined tumor cell and immune cell scoring

BackgroundDetermination of programmed death-ligand 1 (PD-L1) protein expression level in tumor cells and tumor-associated immune cells is critical for identifying patients eligible for immunotherapy. PD-L1 manual scoring algorithms can generally be divided into two categories: cell counting and visual estimation. Cell counting can be time-consuming and is not in sync with pathology practice, which classically uses a Gestalt approach based on pattern recognition and visual estimation. In this study, we introduce the Tumor Area Positivity (TAP) score, which is a novel, straightforward method for scoring tumor cells and immune cells together using visual estimation.MethodsTo demonstrate the reproducibility of TAP scoring among pathologists, between- and within-reader precision studies were performed both within (internal) and outside of (external) our organization. We also compared the TAP score to the Combined Positive Score (CPS), which is based on cell counting, for concordance and time efficacy.ResultsThe average positive agreement, average negative agreement, and overall percent agreement between and within readers were all above 85% for both internal and combined external reader precision studies. TAP score had high concordance rate at 5% cutoff compared with CPS at cutoff 1: positive percent agreement, negative percent agreement, and overall percent agreement were all above 85%.ConclusionsOur study showed the TAP scoring method to be straightforward, significantly less time-consuming, and highly reproducible with a high concordance rate between TAP score and CPS.

Clinical Importance of the lncRNA NEAT1 in Cancer Patients Treated with Immune Checkpoint Inhibitors.

mAbs targeting the PD-1/PD-L1 immune checkpoint are powerful tools to improve the survival of patients with cancer. Understanding the molecular basis of clinical response to these treatments is critical to identify patients who can benefit from this immunotherapy. In this study, we investigated long noncoding RNA (lncRNA) expression in patients with cancer treated with anti-PD-1/PD-L1 immunotherapy. lncRNA expression profile was analyzed in one cohort of patients with melanoma and two independent cohorts of patients with glioblastoma (GBM) undergoing anti-PD-1/PD-L1 immunotherapy. Single-cell RNA-sequencing analyses were performed to evaluate lncRNA expression in tumor cells and tumor-infiltrating immune cells. We identified the lncRNA NEAT1 as commonly upregulated between patients with melanoma with complete therapeutic response and patients with GBM with longer survival following anti-PD-1/PD-L1 treatment. Gene set enrichment analyses revealed that NEAT1 expression was strongly associated with the IFNγ pathways, along with downregulation of cell-cycle-related genes. Single-cell RNA-sequencing analyses revealed NEAT1 expression across multiple cell types within the GBM microenvironment, including tumor cells, macrophages, and T cells. High NEAT1 expression levels in tumor cells correlated with increased infiltrating macrophages and microglia. In these tumor-infiltrating myeloid cells, we found that NEAT1 expression was linked to enrichment in TNFα/NFκB signaling pathway genes. Silencing NEAT1 suppressed M1 macrophage polarization and reduced the expression of TNFα and other inflammatory cytokines. These findings suggest an association between NEAT1 expression and patient response to anti-PD-1/PD-L1 therapy in melanoma and GBM and have important implications for the role of lncRNAs in the tumor microenvironment.

PD-L1 translocation to the plasma membrane enables tumor immune evasion through MIB2 ubiquitination.

Programmed death-ligand 1 (PD-L1), a critical immune checkpoint ligand, is a transmembrane protein synthesized in the endoplasmic reticulum of tumor cells and transported to the plasma membrane to interact with programmed death 1 (PD-1) expressed on T cell surface. This interaction delivers coinhibitory signals to T cells, thereby suppressing their function and allowing evasion of antitumor immunity. Most companion or complementary diagnostic devices for assessing PD-L1 expression levels in tumor cells used in the clinic or in clinical trials require membranous staining. However, the mechanism driving PD-L1 translocation to the plasma membrane after de novo synthesis is poorly understood. Herein, we showed that mind bomb homolog 2 (MIB2) is required for PD-L1 transportation from the trans-Golgi network (TGN) to the plasma membrane of cancer cells. MIB2 deficiency led to fewer PD-L1 proteins on the tumor cell surface and promoted antitumor immunity in mice. Mechanistically, MIB2 catalyzed nonproteolytic K63-linked ubiquitination of PD-L1, facilitating PD-L1 trafficking through Ras-associated binding 8-mediated (RAB8-mediated) exocytosis from the TGN to the plasma membrane, where it bound PD-1 extrinsically to prevent tumor cell killing by T cells. Our findings demonstrate that nonproteolytic ubiquitination of PD-L1 by MIB2 is required for its transportation to the plasma membrane and tumor cell immune evasion.

Proteomic and single-cell landscape reveals novel pathogenic mechanisms of HBV-infected intrahepatic cholangiocarcinoma

Despite the epidemiological association between intrahepatic cholangiocarcinoma (ICC) and hepatitis B virus (HBV) infection, little is known about the relevant oncogenic effects. A cohort of 32 HBV-infected ICC and 89 non-HBV-ICC patients were characterized using whole-exome sequencing, proteomic analysis, and single-cell RNA sequencing. Proteomic analysis revealed decreased cell-cell junction levels in HBV-ICC patients. The cell-cell junction level had an inverse relationship with the epithelial-mesenchymal transition (EMT) program in ICC patients. Analysis of the immune landscape found that more CD8 Tcells and Th2 cells were present in HBV-ICC patients. Single-cell analysis indicated that transforming growth factor beta signaling-related EMT program changes increased in tumor cells of HBV-ICC patients. Moreover, ICAM1+ tumor-associated macrophages are correlated with a poor prognosis and contributed to the EMT in HBV-ICC patients. Our findings provide new insights into the behavior of HBV-infected ICC driven by various pathogenic mechanisms involving decreased cell junction levels and increased progression of the EMT program.

Novel pyroptosis-associated genes signature for predicting the prognosis of sarcoma and validation.

Sarcoma is a rare mesenchymal malignant tumor. Recently, pyroptosis has been reported to be a mode of programmed cell death. Nonetheless, levels of pyroptosis-associated genes in sarcoma and its relevance to prognostic outcomes are yet to be elucidated. Sarcoma cases were classified into two subtypes with regards to differentially expressed genes. We established a profile composed of seven genes and classified the sarcoma patients into low- and high-risk groups through least absolute shrinkage and selection operator Cox regression. Survival rate of low-risk sarcoma patients was markedly higher, relative to high-risk group (P<0.001). In combination with clinical features, the risk score was established to be an independent predictive factor for OS of sarcoma patients. Chemotherapeutic drug sensitivity response analysis found 65 drugs with higher drug sensitivity in low-risk, than in high-risk group and 14 drugs with higher drug sensitivity in the high-risk patient group, compared with low-risk patient group. In addition, functional enrichment, pathway and gene mutation of the two modules were analyzed. Finally, we used qRT-PCR to detect the expression of seven pyroptosis-related genes in tumor cells, and human skeletal muscle cells, compared with human skeletal muscle cells, PODXL2, LRRC17, GABRA3, SCUBE3 and RFLNB genes show high expression levels in tumor cells, while IGHG2 and hepatic leukemia factor show low expression levels in tumor cells. Our research suggest that pyroptosis is closely associated with sarcoma, and these findings confirm that pyroptosis-associated seven genes have a critical role in sarcoma and are potential prognostic factors for sarcoma.

Pseudohypoxia in paraganglioma and pheochromocytoma is associated with an immunosuppressive phenotype.

Metastatic pheochromocytoma and paraganglioma (PPGL) have poor prognosis and limited therapeutic options. The recent advent of immunotherapies showing remarkable clinical efficacies against various cancer types offers the possibility of novel opportunities also for metastatic PPGL. Most PPGLs are pathogenically linked to inactivating mutations in genes encoding different succinate dehydrogenase (SDH) subunits. This causes activation of the hypoxia-inducible factor 2 (HIF2)-mediated transcriptional program in the absence of decreased intratumoral oxygen levels, a phenomenon known as pseudohypoxia. Genuine hypoxia in a tumor creates an immunosuppressive tumor microenvironment. However, the impact of pseudohypoxia in the immune landscape of tumors remains largely unexplored. In this study, tumoral expression of programmed death-ligand 1 (PD-L1) and HIF2α and tumor infiltration of CD8 T lymphocytes (CTLs) were examined in PPGL specimens from 102 patients. We assessed associations between PD-L1, CTL infiltration, HIF2α expression, and the mutational status of SDH genes. Our results show that high PD-L1 expression levels in tumor cells and CTL tumor infiltration were more frequent in metastatic than nonmetastatic PPGL. However, this phenotype was negatively associated with SDH mutations and high HIF2α protein expression. These data were validated by analysis of mRNA levels of genes expressing PD-L1, CD8, and HIF2α in PPGL included in The Cancer Genome Atlas database. Further, PD-L1 and CD8 expression was lower in norepinephrine than epinephrine-secreting PPGL. This in silico analysis also revealed the low PD-L1 or CD8 expression levels in tumors with inactivating mutations in VHL or activating mutations in the HIF2α-coding gene, EPAS1, which, together with SDH-mutated tumors, comprise the pseudohypoxic molecular subtype of PPGL. These findings suggest that pseudohypoxic tumor cells induce extrinsic signaling toward the immune cells promoting the development of an immunosuppressive environment. It also provides compelling support to explore the differential response of metastatic PPGL to immune checkpoint inhibitors. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

ABCL-371 STR Loci Allele Imbalance at 9p24.1 in Primary Mediastinal B-Cell Lymphoma

Molecular factors that determine the prognosis of immunotherapy response in primary mediastinal B-cell lymphoma (PMBCL) are not yet described. PD-L1/L2 expression levels in tumor samples have demonstrated clinical utility for many solid tumors. PD-L1/L2 expression levels could be affected by chromosomal lesions on 9p24.1 (losses or gains). Short tandem repeat (STR) analysis of appropriate markers is the simplest way to detect these lesions. To analyze possible associations between STR loci allele imbalance (AI) on 9p24.1 and PD-L1 expression levels in tumor cells from PMBCL patients. Two STR Loci (gt) n and (ttat)m on 9p24.1 in tumor cells were analyzed for a cohort of 36 PMBCL patients admitted to the National Medical Research Center for Hematology (Moscow, Russia) from 2013 to 2022. For six patients, STR data were compared with PD-L1 expression assessed by immunohistochemistry. DNA was isolated from tumor biopsy samples taken at diagnosis. Control DNA samples were taken from the blood of patients in complete remission or from the buccal epithelium. STR analysis for AI was performed by PCR with original primers. The fragment analysis was performed on an ABI3130 Genetic Analyzer. Inclusion criteria: de novo diagnosed PMBCL patients. pretreatment. The heterozygosity level for the (gt)n locus was 31 of 36 patients (86%), and AI was found in 16 of 31 (51.6%). The heterozygosity level for the (ttat)m locus was 20 of 36 patients (56%), and AI was found in 9 of 20 (45%). One patient was homozygous for both loci. No patients were found with AI at one locus and a normal heterozygous variant at the second locus. PD-L1 expression in tumor cells was evaluated for six patients. In two patients with aberrant STR loci, PD-L1 expression was found in 75%-100% of tumor cells. None of the four patients with normal 9p24.1 STR markers had PD-L1-positive cells. The high incidence of AI in the 9p24.1 locus indicates its possible involvement in the pathogenesis of PMBCL. The association between AI and PD-L1 expression in tumor cells requires confirmation in an extended cohort of patients. This study was supported by Rakfond grant 2/2020.

An individual nomogram can reliably predict tumor spread through air spaces in non-small-cell lung cancer

BackgroundTumor spread through air spaces (STAS) has been shown to adversely affect the prognosis of lung cancer. The correlation between clinicopathological and genetic features and STAS remains unclear.MethodWe retrospectively reviewed 3075 NSCLC patients between2017-2019. We evaluated the relationship between STAS and patients’ clinicopathological and molecular features. The chi-square test was performed to compare categorical variables. Univariate analysis and multivariate logistic regression analysis were performed to investigate the association of clinical factors with STAS. A nomogram was formulated to predict the presence of STAS.ResultsSTAS was identified in 617 of 3075 patients (20.07%). STAS was significantly related to sex (p < 0.001), smoking (p < 0.001), CEA (p < 0.001), differentiation (p < 0.001), histopathological type (p < 0.001), lymphatic vessel invasion (p < 0.001), pleural invasion (p < 0.001), T stage (p < 0.001), N stage (p < 0.001), M stage (p < 0.001), and TNM stage (p < 0.001). STAS was frequently found in tumors with wild-type EGFR (p < 0.001), KRAS mutations (p < 0.001), ALK rearrangements (p < 0.001) or ROS1 rearrangements (p < 0.001). For programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1), STAS was associated with PD-L1 expression level in tumor cells (p < 0.001) or stromal cells (p < 0.001), while PD-1 only in stromal cells (p < 0.001). Multivariable analyses demonstrated significant correlations between STAS and CEA level (p < 0.001), pathological grade (p < 0.001), lymphatic vessel invasion (p < 0.001), pleural invasion (p = 0.001), and TNM stage (p = 0.002). A nomogram was formulated based on the results of the multivariable analysis.ConclusionsTumor STAS was associated with several invasive clinicopathological features. A nomogram was established to predict the presence of STAS in patients with NSCLC.

Comparison of Efficacy and Specificity in Cancer‐Targeting Prodrug Conjugates

Current chemotherapy methods are non‐specific, killing cancerous and healthy cells alike. This drug‐related cytotoxicity results in many side effects such as hair loss or lower blood cell count. Prodrug conjugates have been developed with the goal of preferentially targeting diseased cells to minimize interactions with healthy cells, thereby mitigating deleterious off‐target effects. One powerful prodrug strategy exploits higher vitamin receptor expression levels in tumor cells. These abundant receptors drive their rapid, nutrient‐demanding growth rates. As such, a vitamin structural motif may be included in the prodrug conjugate design to target the ever‐growing tumor: a “targeting group” that acts as an appealing disguise for the drug. Biotin, folate, cyclic and acyclic RGD peptides have been found to be effective in targeting tumors over non‐cancerous cells. To systematically compare the selectivity and potency of these targeting groups, we intend to construct fluorogenic prodrug conjugates based on a previously established scaffold. The conjugates include one of the four respective targeting groups, the anticancer drug (camptothecin), a naphthalimide fluorophore component to track drug delivery, and an activatable disulfide linker that cleaves upon entering the reducing cellular environment, activating both the drug and the fluorophore. After these conjugates have been synthesized, comparisons of efficacy and specificity among targeting groups will be made to gain a better understanding of targeted cancer prodrug conjugates. Currently, these conjugates are still in the synthesis stages.

Dissection of PD-L1 promoter reveals differential transcriptional regulation of PD-L1 in VHL mutant clear cell renal cell carcinoma

AbstractProgrammed death-ligand 1 (PD-L1) is constitutively expressed by hypoxia-inducible factor 2α (HIF2α). It can be induced by interferon gamma (IFNγ) signaling in clear cell renal cell carcinoma (ccRCC). Clinical trials of metastatic ccRCCs have suggested that a canonical IFNγ signature is a better biomarker for therapeutic response to immune checkpoint inhibitors (ICIs) than PD-L1 expression levels in tumor cells. To understand the therapeutic response to ICIs according to PD-L1 expression levels, we analyzed transcriptional regulation of the PD-L1 promoter by HIF2α and IFNγ-inducible interferon regulatory factor-1 (IRF-1) in ccRCC cells. Here, we present two ccRCC cell models showing differential PD-L1 expression levels in response to IFNγ and hypoxia. Analysis of The Cancer Genome Atlas RNA-sequencing data revealed that PD-L1 expression correlated with JAK2 and STAT1 expression of the canonical IFNγ signature in ccRCC tissues. Upon IFNγ stimulation, PD-L1 was induced by sequential activation of JAK2/STAT1/IRF-1 signaling in both WT- and Mut- VHL ccRCC cells. IFNγ activated the IRF-1α site of the PD-L1 promoter. The IFNγ-mediated increase of PD-L1 expression in Mut-VHL cells was 4.8-fold greater than that in WT-VHL cells. Under normoxia condition, PD-L1 expression in Mut-VHL cells was significantly higher than that in WT-VHL cells due to high basal HIF2α expression. Under hypoxia condition, PD-L1 expression in WT-VHL cells was induced up to 1.8-fold through activation of hypoxia-response elements 2 and 3. In contrast, although PD-L1 in Mut-VHL cells was already highly expressed in the basal state through activation of hypoxia-response elements 2, 3, and 4, it was no longer induced by hypoxia. In conclusion, Mut-VHL ccRCC cells displayed higher PD-L1 expression due to high basal HIF2α expression and a stronger response to IFNγ stimulation than WT-VHL cells. The fact that HIF2α antagonists can potentially reduce PD-L1 expression levels should be considered in ICI combination therapy.Through analysis of the PD-L1 promoter, the authors describe two clear cell renal cell carcinoma (ccRCC) models that exhibit differential PD-L1 expression levels upon IFNγ and hypoxia stimulation. Mutant-VHL ccRCC cells display higher PD-L1 expression than wild-type-VHL ccRCC cells due to high basal HIF2α expression and a stronger response to IFNγ stimulation. Thus, mutant-VHL ccRCC cells are expected to respond well to immune checkpoint inhibitors.

Reduced Expression of the Tissue-Specific Oct-IL Isoform Exerts an Antitumor Effect on Namalwa Burkitt's Lymphoma Cells

Increased expression levels of the Oct-1 transcription factor is considered to be one of the key markers of poor cancer prognosis. In addition to the ubiquitous Oct-1A isoform, which is found in all cells, there also exists a tissue-specific Oct-1L isoform, which is expressed in hematopoietic cells. Oct-1L increases cell resistance to different stresses and also regulates the expression of genes controlling differentiation of hematopoietic and immune system cells. The tissue-specific Oct-1L isoform levels are significantly increased in the B-cell lymphoblastoma Namalwa and Raji lines and the T-cell lymphoblastoma Jurkat line compared to normal B and T cells. Apparently, aberrant Oct-1L overexpression not only enhances stress resistance but also leads to the disruption of developmental pathways in the cells promoting their malignant transformation. We report here that targeted suppression of the tissue-specific Oct-1L isoform expression reduces the proliferation rate of Namalwa B-lymphoblastic Burkitt's lymphoma cells, significantly increases cell death rate under hypoxic conditions, and makes cells more sensitive to chemotherapeutic agents such as docetaxel and doxorubicin. These results indicate that targeted therapy aimed at the suppression of the Oct-1 isoforms with increased expression levels in tumor cells rather than the total Oct-1, thus avoiding the traumatic effects of total Oct-1 knockdown, may be promising. Selective suppression of Oct-1 isoforms is a promising strategy in the treatment of lymphoid tumors and may contribute to mitigating the disease course and increasing survival rates in cancer patients.

Differential tumor inhibitory effects induced by HER3 extracellular subdomain-specific mouse monoclonal antibodies.

The therapeutic potential of targeting the human epidermal growth factor receptor-3 (ErbB3/HER3) has long been ignored due to impaired tyrosine kinase function and low expression level in tumor cells compared with EGFR and HER2. Although recent investigations have explored the potential benefit of HER3 targeting and several anti-HER3 agents have been developed, there is still a critical need to design and produce more efficient therapeutics. This study was designed to develop tumor inhibitory monoclonal antibodies (MAbs) against different extracellular subdomains of HER3. Distinct extracellular subdomains of HER3 (DI+II and DIII+IV) were utilized to produce MAbs by hybridoma technology. Biochemical and functional characteristics of these MAbs were then investigated by various methodologies, including immunoblotting, flow cytometry, cell proliferation, cell signaling, and enzyme-linked immunosorbent assays. Four anti-DI+II and six anti-DIII+IV MAbs were obtained, selected based on their ability to bind recombinant full HER3 extracellular domain (ECD). Our data showed that only one anti-DI+II and four anti-DIII+IV MAbs recognized the native form of HER3 by immunoblotting. Four MAbs recognized the membranous HER3 by flow cytometry leading to induction of different levels of receptor internalization and subsequent degradation. Results of cell proliferation assays using these MAbs indicated that they differentially inhibited proliferation of HER3-expressing cancer cells and showed considerable synergistic effects in combination with trastuzumab. Selected MAb with the highest inhibitory effect significantly inhibited the phosphorylation of AKT and ERK1/2 molecules. Some of the anti-HER3 MAbs produced in this study displayed tumor inhibitory function and may be considered promising candidates for future HER3-targeted cancer therapy.

Design and Rationale for a Phase III, Double-Blind, Placebo-Controlled Study of Neoadjuvant Durvalumab + Chemotherapy Followed by Adjuvant Durvalumab for the Treatment of Patients With Resectable Stages II and III non-small-cell Lung Cancer: The AEGEAN Trial

For patients with resectable, early-stage non-small-cell lung cancer (NSCLC), surgery is the primary treatment; however, 5-year survival rates remain poor. Postoperative adjuvant platinum-doublet chemotherapy is associated with a statistically significant but modest improvement in survival of ∼5% at 5 years and is widely accepted as standard of care in patients with resectable, Stage II-III NSCLC. Neoadjuvant chemotherapy has been associated with similar improvements in overall survival to adjuvant therapy in this setting. Durvalumab, a high-affinity PD-L1 inhibitor, has become the standard of care for patients with unresectable, Stage III NSCLC following chemoradiotherapy based on improved progression-free and overall survival in the phase III PACIFIC trial. AEGEAN is a phase III, double-blind, placebo-controlled, international study that will assess pathological and clinical outcomes of durvalumab plus chemotherapy prior to surgery, followed by durvalumab monotherapy after surgery in adults with resectable, Stage II-III NSCLC. Approximately 800 patients will be randomized (1:1) to receive durvalumab or placebo every 3 weeks (q3w) alongside platinum-based chemotherapy (≤4 cycles) prior to surgery, followed by durvalumab or placebo monotherapy q4w, for an additional 12 cycles post surgery, stratified by disease stage (IASLC 8th Edition, Stage II vs. Stage III) and PD-L1 tumor cell expression levels (<1% vs. ≥1%). Primary endpoints include pathological complete response and event-free survival for patients with wild-type EGFR and ALK. Key secondary efficacy endpoints include major pathologic response, disease-free survival and overall survival.