Potential Predictive Marker Research Articles

Correlation of PKM2 Expression With HER2/neu and Additional Breast Cancer Biomarkers and its Prognostic Significance.

Pyruvate kinase M2 (PKM2) has a central role in both tumor development and metastasis, and it has increasingly become a valuable subject for many cancer studies due to its important prognostic value in various tumor types. In this study, we aimed to elucidate the impact of PKM2 expression level on breast cancer prognosis and survival rates and its association with various clinicopathologic characteristics and tumor markers in breast cancer patients. This retrospective study included sample tissues from patients with breast cancer who did not receive chemotherapy or radiotherapy before surgery. Expression levels of PKM2, estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2 (HER2), and Ki-67 were analyzed using tissue microarray and immunohistochemistry. A total of 164 patients were included with an age range from 28 to 82 years. High PKM2 was observed in 48.8% of cases (80/164). A significant association was found between PKM2 expression and breast cancer molecular subtype and HER2 status ( P <0.001). In HER2-negative tumors, there was a significant association between PKM2 expression and tumor grade, TNM stage, pN stage, lymphovascular invasion, and estrogen receptor/progesterone receptor status. Survival analysis revealed that high PKM2 expression levels were associated with decreased overall survival rate in HER2-positive cases with high Ki-67 index. Moreover, in the HER2-positive group, low PKM2 expression level impacted the survival outcome of metastasis ( P =0.002). PKM2 is a valuable prognostic and a potential diagnostic and predictive marker in breast cancer. Moreover, the combination of PKM2 with Ki-67 provides excellent prognostic accuracy in HER2-positive tumors.

Discovery of novel metabolic signatures for early identification of women at risk of developing gestational hypertension

Gestational hypertension (GH) is defined as the presence of systolic blood pressure (BP) ≥ 140mm Hg and/or diastolic BP ≥ 90mm Hg, measured at least 4h apart after 20 weeks of gestation. Early identification of women at high-risk of developing GH could contribute significantly towards improved maternal and fetal outcomes. To determine early metabolic biomarkers in women with GH as compared with normotensive women. Serum samples were collected from subjects during three stages of their pregnancy: 8-12 weeks, 18-20 weeks and after 28 weeks (< 36 weeks) of gestation and studied using nuclear magnetic resonance (NMR) metabolomics approach. Multivariate and univariate analyses were performed to determine the significantly altered metabolites in GH women. A total of 10 metabolites, including isoleucine, glutamine, lysine, proline, histidine, phenylalanine, alanine, carnitine, N-acetyl glycoprotein and lactic acid were observed to be significantly downregulated during all pregnancy stages in women with GH as compared with controls. Furthermore, expression of 5 metabolites in the first trimester i.e., phenylalanine [area under the curve (AUC) = 0.745], histidine [AUC = 0.729], proline [AUC = 0.722], lactic acid [AUC = 0.722], and carnitine [AUC = 0.714] exhibited highest potential in discriminating GH from normotensive women. The present study is the first of its kind to identify significantly altered metabolites that have the potential to discriminate between women at risk of developing GH and normotensive women across three trimesters of pregnancy. This opens up the possibility of exploring these metabolites as potential early predictive markers of GH.

Effects of prenatal exposure to (es)citalopram and maternal depression during pregnancy on DNA methylation and child neurodevelopment

Studies assessing associations between prenatal exposure to antidepressants, maternal depression, and offspring DNA methylation (DNAm) have been inconsistent. Here, we investigated whether prenatal exposure to citalopram or escitalopram ((es)citalopram) and maternal depression is associated with differences in DNAm. Then, we examined if there is an interaction effect of (es)citalopram exposure and DNAm on offspring neurodevelopmental outcomes. Finally, we investigated whether DNAm at birth correlates with neurodevelopmental trajectories in childhood. We analyzed DNAm in cord blood from the Norwegian Mother, Father and Child Cohort Study (MoBa) biobank. MoBa contains questionnaire data on maternal (es)citalopram use and depression during pregnancy and information about child neurodevelopmental outcomes assessed by internationally recognized psychometric tests. In addition, we retrieved ADHD diagnoses from the Norwegian Patient Registry and information on pregnancies from the Medical Birth Registry of Norway. In total, 958 newborn cord blood samples were divided into three groups: (1) prenatal (es)citalopram exposed (n = 306), (2) prenatal maternal depression exposed (n = 308), and (3) propensity score-selected controls (n = 344). Among children exposed to (es)citalopram, there were more ADHD diagnoses and symptoms and delayed communication and psychomotor development. We did not identify differential DNAm associated with (es)citalopram or depression, nor any interaction effects on neurodevelopmental outcomes throughout childhood. Trajectory modeling identified subgroups of children following similar developmental patterns. Some of these subgroups were enriched for children exposed to maternal depression, and some subgroups were associated with differences in DNAm at birth. Interestingly, several of the differentially methylated genes are involved in neuronal processes and development. These results suggest DNAm as a potential predictive molecular marker of later abnormal neurodevelopmental outcomes, but we cannot conclude whether DNAm links prenatal (es)citalopram exposure or maternal depression with child neurodevelopmental outcomes.

Distribution characteristics and clinical significance of infiltrating Tcells in the tumor microenvironment of pancreatic cancer.

Tumor-infiltrating lymphocytes (TILs) are important components of the tumor microenvironment (TME). However, the distribution characteristics of TILs and their significance in pancreatic cancer (PC) remain largely unexplored. The levels of TILs, including the total number of T cells, cluster of differentiation (CD)4+ T cells, CD8+ cytotoxic T lymphocytes (CTLs), regulatory T-cells (Tregs), programmed cell death protein 1+ T cells and programmed cell death ligand 1 (PD-L1)+ T cells, in the TME of patients with PC were detected using multiple fluorescence immunohistochemistry. The associations between the number of TILs and the clinicopathological characteristics were investigated using χ2 tests. In addition, Kaplan-Meier survival and Cox regression analyses were used to assess the prognostic value of these TIL types. Compared with paracancerous tissues, in PC tissues, the proportions of total T cells, CD4+ T cells and CD8+ CTLs were markedly decreased, while those of Tregs and PD-L1+ T cells were significantly increased. The levels of CD4+ T cell and CD8+ CTL infiltrates were inversely associated with tumor differentiation. Higher infiltrates of Tregs and PD-L1+ T cells were closely associated with advanced N and TNM stages. It is important to note that the infiltrates of total T cells, CD4+ T cells, Tregs and PD-L1+ T cells in the TME were independent risk factors for the prognosis of PC. PC was characterized by an immunosuppressive TME with a decrease in the number of CD4+ T cells and CD8+ CTLs, and an increase in the number of Tregs and PD-L1+ T cells. Overall, the number of total T cells, CD4+ T cells, Tregs and PD-L1+ T cells in the TME was a potential predictive marker for the prognosis of PC.

MiRNA implication in the pathogenesis and the outcome of Tunisian endemic pemphigus foliaceus.

Pemphigus foliaceus (PF) is a bullous autoimmune skin disease diagnosed through sera and skin analyses. PF severity is associated with maintained anti-Dsg1 sera levels and its prognosis is unpredictable. MicroRNA (miRNA), dynamic regulators of immune function, have been identified as potential biomarkers for some autoimmune diseases. This study aimed to assess the miRNA expression of miR-17-5p, miR-21-5p, miR-146a-5p, miR-155-5p and miR-338-3p using quantitative real-time PCR in peripheral blood mononuclear cells (PBMC) and lesional skin samples from untreated and treated PF patients (both remittent and chronic) over 3 months. Overall, miRNA expression was significantly higher in PBMC than in biopsy samples. Blood miR-21 expression was increased in untreated patients compared to controls and had a diagnostic value with an AUC of 0.78. After 6 weeks, it decreased significantly, similar to anti-Dsg1 antibodies and the PDAI score. In addition, a positive correlation was observed between cutaneous miR-21 expression and the disease activity score. Conversely, cutaneous expressions of miR-17, miR-146a and miR-155 were significantly higher in treated chronic patients compared to remittent ones. The cutaneous level of miR-155 positively correlated with pemphigus activity, making it a potential predictive marker for patients' clinical stratification with an AUC of 0.86.These findings suggest that blood miR-21 and cutaneous miR-155 can be used as supplemental markers for PF diagnosis and activity, respectively in addition to classical parameters.

Dissecting the single-cell transcriptome network of macrophage and identifies a signature to predict prognosis in lung adenocarcinoma.

The tumor immune microenvironment (TME) plays a vital role in tumorigenesis, progression, and treatment. Macrophages, as an important component of the tumor microenvironment, play an essential role in antitumor immunity and TME remodeling. In this study, we aimed to explore the different functions of different origins macrophages in TME and their value as potential predictive markers of prognosis and treatment. We performed single-cell analysis using 21 lung adenocarcinoma (LUAD), 12 normal, and four peripheral blood samples from our data and public databases. A prognostic prediction model was then constructed using 502 TCGA patients and explored the potential factors affecting prognosis. The model was validated using data from 4 different GEO datasets with 544 patients after integration. According to the source of macrophages, we classified macrophages into alveolar macrophages (AMs) and interstitial macrophages (IMs). AMs mainly infiltrated in normal lung tissue and expressed proliferative, antigen-presenting, scavenger receptors genes, while IMs occupied the majority in TME and expressed anti-inflammatory, lipid metabolism-related genes. Trajectory analysis revealed that AMs rely on self-renew, whereas IMs originated from monocytes in the blood. Cell-to-cell communication showed that AMs interacted mainly with T cells through the MHC I/II signaling pathway, while IMs mostly interacted with tumor-associated fibrocytes and tumor cells. We then constructed a risk model based on macrophage infiltration and showed an excellent predictive power. We further revealed the possible reasons for its potential prognosis prediction by differential genes, immune cell infiltration, and mutational differences. In conclusion, we investigated the composition, expression differences, and phenotypic changes of macrophages from different origins in lung adenocarcinoma. In addition, we developed a prognostic prediction model based on different macrophage subtype infiltration, which can be used as a valid prognostic biomarker. New insights were provided into the role of macrophages in the prognosis and potential treatment of LUAD patients.

DEC1 is a potential marker of early metastasis in Oral squamous cell carcinoma

ObjectiveThis study aimed to investigate the role of differentiated embryonic-chondrocyte expressed gene 1 (DEC1) in early oral squamous cell carcinoma (OSCC) metastasis. Methods and materialsThis study collected normal oral mucosas (NOM) and OSCC tissues from Xiangya Hospital for immunohistochemistry to detect the expressions of DEC1 and epithelial-mesenchymal transition (EMT) -related molecules. Correlation analysis between the expressions of the cytoplasmic DEC1 and EMT-related molecules was performed. Kaplan-Meier analysis was conducted to estimate Recurrence-free survival (RFS). After knocking down DEC1, cell migration and the expressions of EMT-related molecules were evaluated in HN6 cells by cell scratch assay, qRT-PCR, and western blot. ResultsImmunohistochemistry showed that the subcellular location of DEC1 expression was different between OSCC and NOM tissues. The cytoplasmic expression of DEC1 in OSCC tissues was significantly higher than in NOM tissues, and its expression was highest in early OSCC patients with metastasis. In addition, the cytoplasmic DEC1 was negatively correlated with the E-cadherin and β-catenin, but positively correlated with the N-cadherin in OSCC and NOM tissues. In vitro assays showed that DEC1 knockdown inhibited cell migration and EMT in HN6 cells. ConclusionDEC1 could serve as a potential predictive marker for early OSCC metastasis.

TGF-β as Predictive Marker and Pharmacological Target in Lung Cancer Approach

Lung cancer (LC) represents the leading cause of cancer incidence and mortality worldwide. LC onset is strongly related to genetic mutations and environmental interactions, such as tobacco smoking, or pathological conditions, such as chronic inflammation. Despite advancement in knowledge of the molecular mechanisms involved in LC, this tumor is still characterized by an unfavorable prognosis, and the current therapeutic options are unsatisfactory. TGF-β is a cytokine that regulates different biological processes, particularly at the pulmonary level, and its alteration has been demonstrated to be associated with LC progression. Moreover, TGF-β is involved in promoting invasiveness and metastasis, via epithelial to mesenchymal transition (EMT) induction, where TGF-β is the major driver. Thus, a TGF-β-EMT signature may be considered a potential predictive marker in LC prognosis, and TGF-β-EMT inhibition has been demonstrated to prevent metastasis in various animal models. Concerning a LC therapeutic approach, some TGF-β and TGF-β-EMT inhibitors could be used in combination with chemo- and immunotherapy without major side effects, thereby improving cancer therapy. Overall, targeting TGF-β may be a valid possibility to fight LC, both in improving LC prognosis and cancer therapy, via a novel approach that could open up new effective strategies against this aggressive cancer.

Clustering analysis and prognostic model based on PI3K/AKT-related genes in pancreatic cancer.

Pancreatic cancer is one of most aggressive malignancies with a dismal prognosis. Activation of PI3K/AKT signaling is instrumental in pancreatic cancer tumorigenesis. The aims of this study were to identify the molecular clustering, prognostic value, relationship with tumor immunity and targeting of PI3K/AKT-related genes (PARGs) in pancreatic cancer using bioinformatics. The GSEA website was searched for PARGs, and pancreatic cancer-related mRNA data and clinical profiles were obtained through TCGA downloads. Prognosis-related genes were identified by univariate Cox regression analysis, and samples were further clustered by unsupervised methods to identify significant differences in survival, clinical information and immune infiltration between categories. Next, a prognostic model was constructed using Lasso regression analysis. The model was well validated by univariate and multivariate Cox regression analyses, Kaplan-Meier survival analysis and ROC curves, and correlations between risk scores and patient pathological characteristics were identified. Finally, GSEA, drug prediction and immune checkpoint protein analyses were performed. Pancreatic cancers were divided into Cluster 1 (C1) and Cluster 2 (C1) according to PARG mRNA expression. C1 exhibited longer overall survival (OS) and higher immune scores and CTLA4 expression, whereas C2 exhibited more abundant PD-L1. A 6-PARG-based prognostic model was constructed to divide pancreatic cancer patients into a high-risk score (HRS) group and a low-risk score (LRS) group, where the HRS group exhibited worse OS. The risk score was defined as an independent predictor of OS. The HRS group was significantly associated with pancreatic cancer metastasis, aggregation and immune score. Furthermore, the HRS group exhibited immunosuppression and was sensitive to radiotherapy and guitarbine chemotherapy. Multidrug sensitivity prediction analysis indicated that the HRS group may be sensitive to PI3K/AKT signaling inhibitors (PIK-93, GSK2126458, CAL-101 and rapamycin) and ATP concentration regulators (Thapsigargin). In addition, we confirmed the oncogenic effect of protein phosphatase 2 regulatory subunit B'' subunit alpha (PPP2R3A) in pancreatic cancer in vitro and in vivo. PARGs predict prognosis, tumor immune profile, radiotherapy and chemotherapy drug sensitivity and are potential predictive markers for pancreatic cancer treatment that can help clinicians make decisions and personalize treatment.

Translational relevance of SOS1 targeting for KRAS-mutant colorectal cancer.

It has been challenging to target mutant KRAS (mKRAS) in colorectal cancer (CRC) and other malignancies. Recent efforts have focused on developing inhibitors blocking molecules essential for KRAS activity. In this regard, SOS1 inhibition has arisen as an attractive approach for mKRAS CRC given its essential role as a guanine nucleotide exchange factor for this GTPase. Here, we demonstrated the translational value of SOS1 blockade in mKRAS CRC. We used CRC patient-derived organoids (PDOs) as preclinical models to evaluate their sensitivity to SOS1 inhibitor BI3406. A combination of in silico analyses and wet lab techniques was utilized to define potential predictive markers for SOS1 sensitivity and potential mechanisms of resistance in CRC. RNA-seq analysis of CRC PDOs revealed two groups of CRC PDOs with differential sensitivities to SOS1 inhibitor BI3406. The resistant group was enriched in gene sets involving cholesterol homeostasis, epithelial-mesenchymal transition, and TNF-α/NFκB signaling. Expression analysis identified a significant correlation between SOS1 and SOS2 mRNA levels (Spearman's ρ 0.56, p < 0.001). SOS1/2 protein expression was universally present with heterogeneous patterns in CRC cells but only minimal to none in surrounding nonmalignant cells. Only SOS1 protein expression was associated with worse survival in patients with RAS/RAF mutant CRC (p = 0.04). We also found that SOS1/SOS2 protein expression ratio >1 by immunohistochemistry (p = 0.03) instead of KRAS mutation (p = 1) was a better predictive marker to BI3406 sensitivity of CRC PDOs, concordant with the significant positive correlation between SOS1/SOS2 protein expression ratio and SOS1 dependency. Finally, we showed that GTP-bound RAS level underwent rebound even in BI3406-sensitive PDOs with no change of KRAS downstream effector genes, thus suggesting upregulation of guanine nucleotide exchange factor as potential cellular adaptation mechanisms to SOS1 inhibition. Taken together, our results show that high SOS1/SOS2 protein expression ratio predicts sensitivity to SOS1 inhibition and support further clinical development of SOS1-targeting agents in CRC.

Abstract 954: PIK3CA mutation induces immunogenicity and increases the immune checkpoint inhibitor response in urothelial carcinoma

Abstract Background: Immune checkpoint inhibitor (ICI) has been widely used for advanced urothelial carcinoma (mUC) globally. However, little is known about the potential predictive factors of the ICI response. The study aims to identify a potential biomarker to predict ICI response in UC. Material and methods: We performed targeted next generation sequencing (NGS) of pre-treatment tumor Formalin-fixed paraffin-embedded (FFPE) samples by using ACTOnco® comprehensive genomic panel (ACT Genomics, Co. Ltd., Taiwan) to target all coding exons of 440 cancer-associated genes. We retrospective analyzed a total of 70 patients with mUC underwent at least one cycle of ICI with or without chemotherapy at Kaohsiung Chang Gung Memorial Hospital between April 2016 and November 2019. Patients were defined as responders (CR/PR) and non-responders (SD/PD) according to the RECIST evaluation. TMB was estimated by calculating all coding exons variants, including somatic non-synonymous, frameshift and splice site variants. Results: A total of 55 UC tumor samples passed QC process were analyzed. The median age was 67, and 74% of all patients were in ICI monotherapy cohort. The most common genetic alterations were TP53 (66%), KMT2D (42%) and MUC16 (32%). In ICI monotherapy group, responders had a higher TMB than non-responders (median 19.5 vs 6.5 mt/Mb; p = 0.004). Patients with high TMB on ICI monotherapy had a significantly longer PFS (14.6 vs 2.9 months; p = 0.003) and OS (NR vs 11.0 months; p = 0.01) than TMB low patients. We found that PIK3CA (32% vs 3%), UBR5 (23% vs 0%), PRKDC (32% vs 10%) were the top 3 enriched mutations between responders and non-responders. All PIK3CA mutation were hot-spot mutation, and tumor harbored PIK3CA mutation had a median high TMB than PIK3CA wild type in UC, NSCLC, breast and colorectal cancer. By analyzing tumor and immune microenvironment by TISIDB platform, UC with PIK3CA mutation was highly associated with increasing MHC molecules expression (HLA-A, HLA-B, HLA-C, B2M) in UC and gastric cancer. In vitro study, we confirmed anti-PDL1 with PBMC had a synergistic cytotoxicity in PIK3CA mutated cell lines (BFTC-909, AGS and DLD-1), but not in PIK3CA wild type cell lines (T24, RT4, UMUC-14, N87 and SW837). Knocking-down PIK3CA expression diminished the cytotoxic activity of anti-PDL1 with PBMC in BFTC-909 and AGS cells. PIK3CA down-regulation inhibits antigen presentation machinery process. We confirmed that knockout PIK3CA gene function by CRISPR/Cas9 decreased MHC1 and B2M molecules expression, and showing modest resistance to ICI than mice with preserved PIK3CA function. Conclusion: UC with PIK3CA missense mutation exhibits a higher immunogenicity through enhancing MHC class I and B2M expression. PIK3CA missense mutation is a potential predictive marker for ICI response in UC. Citation Format: Harvey Yu-Li Su, Ling-Yi Xiao, Shih-Yu Huang, Li-Chung Chang, Yi-Hua Chen, Hao-Lun Luo, Chia-Ling Wu. PIK3CA mutation induces immunogenicity and increases the immune checkpoint inhibitor response in urothelial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 954.

Abstract 2127: TERT C228T and KDM6A alterations are potential predictive biomarkers in non-muscle-invasive bladder cancer treated with intravesical Bacillus Calmette-Guérin instillation

Abstract Introduction: Bacillus Calmette-Guérin (BCG) is the standard of care for high-risk non-muscle-invasive bladder cancer (NMIBC) following transurethral resection. However, patients often have heterogeneous responses. Even among those who initially respond well to BCG, 10-20% relapse. Identification of reliable biomarkers predicting the efficacy of BCG remains an unmet need. En bloc resection is a novel technique representing a substantial advancement in the surgical management of NMIBC. We sought to investigate genomic and tumor microenvironmental (TME) profiles in NMIBC and explore potential predictive markers for BCG treatment following en-block resection. Methods: A total of 40 patients with high-risk NMIBC (cTis-T1N0M0) were retrospectively enrolled who underwent en bloc resection followed by BCG instillation. Surgical samples were subjected to NGS sequencing using a 520-gene panel (Burning Rock Biotech, Guangzhou) and multiplex immunofluorescence (mIF) assay. Results: The cohort had a median age of 63 years, and 80% were male. After a median follow-up of 21.8 months, 19/40 patients relapsed with a one-year relapse-free survival (RFS) rate of 57.5%. All tumors were microsatellite stable and showed a median TMB of 7.98muts/Mb. Genomic profiling revealed a high prevalence of alterations in TERT (55%), KDM6A (32.5%), KMT2D (32.5%), FGFR3(30%), PIK3CA (30%), TP53(27.5%), KMT2C (25%), and ARID1A (20%). TME analysis showed higher proportions of M1 macrophages and CD56 dim NK cells in the tumoral compartment and more intense infiltration of CD8+ T cells, exhausted CD8+T, CD56 bright NK cells, and M2 macrophages in the stromal compartment. Multivariate analysis identified TERT C228T mutation (HR=3.28 [95%CI:1.225-8.79], p=0.0181) and alteration in KDM6A (HR=2.94 [95%CI:1.040-8.29], p=0.042) as two independent factors associated with inferior RFS. Patients with concomitant TERT C228T and KDM6A alteration had the shortest RFS (median RFS:5.83months) compared with those who were free of (median RFS: NR) or harbored either one of the two alterations (median RFS:9.13months) (p=0.0022). We also found that tumoral infiltration of CD8+T cells was positively associated with RFS (HR=0.29 [95%CI:0.097-0.885], p=0.0208). Conclusion: The study comprehensively depicted the genomic and TME profiles in NMIBC and identified potential predictive biomarkers for BCG treatment. Our findings may facilitate the stratification of patients and better guide the clinical decision-making on the management of NMIBC. Citation Format: Qi-Dong Xia, Yao-Bing Chen, Jian-Xuan Sun, Chen-Qian Liu, Jin-Zhou Xu, Zhi-Peng Yao, Ye An, Meng-Yao Xu, Si-Han Zhang, Xing-Yu Zhong, Na Zeng, Si-Yang Ma, Hao-Dong He, Heng-Long Hu, Jia Hu, Yi Lu, Lin Shao, Si-Qi Li, Zheng Liu, Shao-Gang Wang. TERT C228T and KDM6A alterations are potential predictive biomarkers in non-muscle-invasive bladder cancer treated with intravesical Bacillus Calmette-Guérin instillation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2127.

Abstract 1392: Integrative biomarker analysis of regorafenib plus nivolumab (RegoNivo) in unresectable hepatocellular carcinoma (uHCC): A multicenter phase 2 RENOBATE trial

Abstract Regorafenib plus nivolumab (RegoNivo) combination has shown promising anti-cancer activity in multiple cancer types. REBNOBATE trial is a single-arm multicenter phase 2 trial of first-line RegoNivo in patients (pts) with uHCC (NCT04310709). Here we report the clinical outcomes of the RENOBATE study and integrative biomarker analysis using circulating tumor DNA (ctDNA) and single cell RNA sequencing (scRNA-seq) analysis. Method: Adult pts with ECOG PS 0 or 1, BCLC stage B or C, and no prior systemic therapy were eligible for the study. Pts received nivolumab 480 mg iv, every 4 wks, and regorafenib 80 mg daily po, 3 wks on/1 wk off, every 4 wks. Tumor response was assessed according to RECIST v1.1 every 8 wks. ctDNA analysis was performed using the Guardant 360 CDx (n=42). scRNA-seq was performed using PBMCs at baseline (e.g., C1D1) and on treatment (eg., C1D15) from early progression (EP) group (14 pts with PD or tumor burden increases towards PD on the 1st evaluation) and long-term response (LR) group (15 pts with PR or SD &amp;gt; 6 months). Results: 42 pts were enrolled. Response rates were 31.0% and median progression-free survival (was 7.4 mo (95% CI, 4.2-13.0) and 1-year OS rate was 80.5% (95% CI, 63.0-90.3%). Aberrations of Wnt/β-Catenin and PI3K/mTOR pathways identified in ctDNA were not associated with efficacy outcomes. scRNA-seq revealed that genes upregulated in immune cells on C1D15 (vs. C1D1) were associated with immune activation and regulation of angiogenesis. Unsupervised clustering identified 13 immune subsets; among them, classical monocyte showed the most prominent changes in their proportion and gene expression profiles. After RegoNivo treatment, CD8 clusters had enhanced expressions of genes related to cytotoxicity, tissue homing (i.e., CXCR6, CX3CR1, and CXCR3) and proliferation (MKI67), and monocyte clusters showed an enrichment of the gene signatures representing polarization towards M1-related features. Importantly, the enrichment of cytotoxic features of the T cell clusters and M1-polarizing features of monocyte clusters were more pronounced in LR group than in EP group. From the analysis of differentially expressed genes monocyte clusters between the EP and LR groups, a surface molecule ‘X’ was identified as a potential biomarker involved in resistance to RegoNivo. RegoNivo showed promising efficacy outcomes in pts with uHCC. Through scRNA-seq of serial PBMCs, we identified enhanced cellular immune responses induced by RegoNivo and a potential predictive marker for immunotherapy in pts with uHCC. Our study highlighted that regorafenib might synergize with anti-PD-1 in pts with uHCC. Association between differential features of T cell and monocyte clusters induced by this combination and distinct clinical outcomes highlights the importance of immune-modulation in HCC pts suggesting a potential to further harness these signals Citation Format: Hyung-Don Kim, Seyoung Jung Jung, Baek-Yeol Ryoo, Min-Hee Ryu, Beodeul Kang, Hong Jae Chon, Jung Yong Hong, Ho Yeong Lim, Jeong Seok Lee, June-Young Koh, Changhoon Yoo. Integrative biomarker analysis of regorafenib plus nivolumab (RegoNivo) in unresectable hepatocellular carcinoma (uHCC): A multicenter phase 2 RENOBATE trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1392.

Abstract 1848: A novel murine tumor model for assessing the efficacy of neoadjuvant dosing regimens for immunotherapies

Abstract Immunotherapy extends survival for patients with several cancer types, especially in combination with surgery. Consistently, recent evidence demonstrated improved efficacy with neoadjuvant/adjuvant pembrolizumab in combination with debulking surgery in patients with melanoma (Patel et al, ESMO 2022). However, evaluating these regimens preclinically is challenging due to the rarity of appropriate models. To address this question, we have established a novel murine tumor model involving partial tumor-debulking surgery which enables monitoring of the temporal response to immunotherapy in neoadjuvant, neoadjuvant/adjuvant and adjuvant settings. We utilized the MC38 tumor model which represents a ‘hot’ tumor microenvironment (TME), analogous to the TME in melanoma patients. MC38 tumor-bearing C57Bl/6 mice were subcutaneously administered murine nemvaleukin alfa (mNemva), an engineered IL-2 immunotherapy that selectively activates and expands cytotoxic NK cells and antigen-experienced CD8+ T cells. Partial tumor-debulking surgery was performed under general anesthesia 3 days after the first dose of mNemva along with neoadjuvant and neoadjuvant/adjuvant regimens, or 1 day before the first dose of mNemva in the adjuvant regimen. Whereas a single dose of mNemva was administered to mice in the neoadjuvant setting, four and five doses were given in the adjuvant and neoadjuvant/adjuvant settings, respectively. Two types of control groups were also evaluated: vehicle in combination with partial tumor-debulking surgery, and five doses of mNemva without the debulking procedure.The neoadjuvant/adjuvant treatment regimen was superior to neoadjuvant or adjuvant in all aspects with 28% of animals exhibiting complete responses, tumor growth inhibition (TGI) of 82% and prolonged median survival from 17 to 27 days. While the neoadjuvant regimen yielded 10% complete responders, TGI was only 7% and median survival was not extended. No complete responders were observed with the adjuvant regimen. Importantly, tumors in both control groups grew to the predefined endpoint irrespective of treatment and surgical procedure. Following tumor re-challenge, 75% of prior complete responders were protected, suggesting that mNemva induced immunological memory in most mice. Moreover, long-term immunity was 100% effective if the re-challenge experiment was performed out to 5 months after the last dose of mNemva had been administered.Therefore, the neoadjuvant/adjuvant dosing regimen exhibits a superior response in this preclinical model, when combined with partial tumor-debulking surgery, similar to the recent findings in patients with melanoma that were treated with pembrolizumab. In addition, this strategy enables sampling of tumor tissue for analyses of the immune response and the identification of potential predictive markers of response. Citation Format: Sara Prijic, Jason Wood, Arthur Liberzon, Harikiran Nistala, Raymond J. Winquist, Jared E. Lopes. A novel murine tumor model for assessing the efficacy of neoadjuvant dosing regimens for immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1848.

Abstract 3238: Integrative biomarker analysis of regorafenib plus nivolumab (RegoNivo) in unresectable hepatocellular carcinoma (uHCC): A multicenter phase 2 RENOBATE trial

Abstract RegoNivo combination has shown promising anti-cancer activity in multiple cancer types. REBNOBATE is a single-arm multicenter phase 2 trial of first-line RegoNivo in patients (pts) with uHCC (NCT04310709). In this study, we present the final clinical outcomes of RENOBATE and integrative biomarker analysis. Pts with &amp;gt;19 years, ECOG PS 0 or 1, BCLC stage B or C, and no prior systemic therapy were eligible. Pts received Nivo 480 mg iv, every 4 wks, and Rego 80 mg daily po, 3 wks on/1 wk off, every 4 wks. Tumor response was assessed according to RECIST v1.1 every 8 wks. ctDNA analysis was performed using the Guardant 360 CDx platform (n=42). scRNA-seq was performed using peripheral blood mononuclear cells (PBMCs) at baseline (e.g., C1D1, n=28) and C1D15 (n=19) from early progression (EP) group (pts with PD or tumor burden increases towards PD on the first evaluation) and long-term response (LR) group (pts with PR or SD &amp;gt; 6 months). A total of 42 pts was enrolled. Response rates were 31.0% and median progression-free survival (PFS) was 7.4 mo (95% CI, 4.2-13.0) and 1-year OS rate was 80.5% (95% CI, 63.0-90.3%). Aberrations of Wnt/β-Catenin and PI3K/mTOR pathways identified in ctDNA were not associated with efficacy outcomes. scRNA-seq revealed that genes upregulated in immune cells on C1D15 (vs. baseline) were associated with immune activation and regulation of angiogenesis. Unsupervised clustering identified 13 immune subets; among them, classical monocyte showed most prominent changes in their proportion and gene expression profiles. After RegoNivo treatment, CD8 T cell clusters had enhanced expressions of genes related to cytoxicity, tissue homing (i.e., CXCR6, CX3CR1, and CXCR3) and proliferation (MKI67), and monophaghocyte clusters showed an enrichment of the gene signatures representing polarization towards M1-related features. Importantly, enrichment of cytotoxic features of the T cell clusters and M1-polarizing features of monophagocyte clusters were more pronounced in LR group than in EP group. From the analysis of differentially expressed genes monophagocyte clusters between the EP and LR groups, a surface molecule ‘X’ was identified as a potential biomarker involved in resistance to RegoNivo. RegoNivo showed promising efficacy outcomes in pts with uHCC. Through scRNA-seq of serial PBMCs, we identified enhanced cellular immune responses induced by RegoNivo and found a potential predictive marker for immunotherapy in pts with uHCC. Our study highlighted that Rego, a multikinase inhibitor, might synergize with anti-PD-1 in pts with uHCC. Association between differential features of T cell and monophagocyte clusters induced by this combination and distinct clinical outcomes highlights the importance of immune-modulation in HCC pts and suggests a potential to further harness these signals. Citation Format: Hyung-Don Kim, Seyoung Jung, Baek-Yeol Ryoo, Min-Hee Ryu, Beodeul Kang, Hong Jae Chon, Jung Yong Hong, Ho Yeong Lim, Jeong Seok Lee, June-Young Koh, Changhoon Yoo. Integrative biomarker analysis of regorafenib plus nivolumab (RegoNivo) in unresectable hepatocellular carcinoma (uHCC): A multicenter phase 2 RENOBATE trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3238.

Abstract 4405: Tumor mutational burden, as a potential predictive marker for the efficacy of immunotherapy in advanced gastric cancer

Abstract Background: The optimal value of tumor mutational burden (TMB) for predicting treatment response of immunotherapy in advanced gastric cancer (AGC) is still unclear. We aimed to establish the optimal cut-off value of TMB, and evaluate the efficacy of immunotherapy in AGC according to TMB and other markers. Method: From October 1, 2020, to July 27, 2021, at Samsung medical center in Korea, patients with AGC, who received pembrolizumab or nivolumab were included. TMB was measured by next-generation sequencing (NGS)-based assays, and PD-L1 is tested using immune-histochemical assay 22C3 pharmDx. Based on receiver operating characteristic analysis, the cut-point value of TMB was determined as the point where the Youden’s index is maximum. Result: A total of 53 patients were analyzed. The cut-off value of TMB for predicting the overall response of immune checkpoint inhibitors was defined as 13.31 mutations/megabase (mt/mb), with 56% of sensitivity and 95% of specificity. Under this definition of TMB, 7 patients were TMB-high group (≥13.31 mt/mb), while 46 patients were TMB-low group (&amp;lt;13.31 mt/mb). The overall response rate (ORR) had a statistically significant difference between TMB-low (8.7%, n=4/46) and TMB-high patients (71.4%, n=5/7; p=0.001). The progression-free survival (PFS) and overall survival (OS) for 53 patients were 1.93 months (95% CI, 1.600-2.268) and 4.26 months (95% CI, 2.992-5.532). The OS was longer in the TMB-high group with the median of 20.8 months (95% CI, 2.292-39.281) compared to the TMB-low group with the median of 3.31 months (95% CI, 1.604-5.019; p=0.049). The ORR of the patients whose PD-L1 CPS was 1 or above was not different from the patients whose PD-L1 CPS was lower than 1. Conclusion: In this study, the optimal value of TMB for predicting the objective response rate of immunotherapy in AGC was determined as 13.31 mt/mb. TMB-high (≥13.31 mt/mb) was associated with a better objective response rate, and TMB-high patients exhibited better overall survival. TMB has the potential for predicting therapeutic response to immunotherapy in advanced gastric cancer. Citation Format: Jaeyeon Jang, Youngkyung Jeon, Sun Young Jeong, Ye Ji Jung, Daeho Choi, Joohyun Hong, Jeeyun Lee, Won Ki Kang, Seung Tae Kim. Tumor mutational burden, as a potential predictive marker for the efficacy of immunotherapy in advanced gastric cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4405.

Generation of induced pluripotent stem cells from two ADHD patients and two healthy controls

Attention-deficit hyperactivity disorder is a neurodevelopmental disorder which prevalence has been increasing in the past decades, affecting more than 5% of children, adolescents worldwide. Regarding etiology, polygenic, environmental factors contribute to the occurrence of ADHD even though molecular mechanisms are not known. Understanding the pathophysiology in patient-specific cells is crucial for the discovery of potential predictive markers, the establishment of new therapeutic targets. In this study, we generated further lines from ADHD patients, healthy controls using Sendai virus transduction, which may help on the study of ADHD at the molecular, cellular levels.

Rasd2 Mediates Acute Fasting-Induced Antidepressant-Like Effects via Dopamine D2 Receptor Activation in Ovariectomized Mice.

Previous studies have shown that estrogen and acute fasting for 9 hours have antidepressant-like effects by reducing immobility time in the forced swimming test. Estrogen and acute fasting share a common regulatory gene, Rasd2. RASD2 regulates dopamine D2 receptor (DRD2) transmission, but the role of Rasd2 in the DRD2-mediated antidepressant-like effect of acute fasting has not been examined. In this study, open field test, forced swimming test, tail suspension test and sucrose preference test were used for behavioral assessments. RNA-seq, western blot, enzyme-linked immunosorbent assay, and co-immunoprecipitation were used to explore the role of Rasd2 in a depression model induced by ovariectomy and the antidepressant-like effects of 9-hour fasting. The RNA seq results showed that acute fasting induced a significant change in Rasd2 gene expression. Depression-like behaviors induced by ovariectomy were associated with decreased RASD2 and DRD2 protein levels in the hippocampus, and Rasd2 overexpression in the hippocampus alleviated depression-like behaviors and increased DRD2 expression. Nine-hour fasting had antidepressant-like effects in ovariectomized mice by upregulating the protein levels of RASD2, DRD2, CREB-BDNF, Akt, and estrogen receptor beta, and these effects can be blocked by DRD2 antagonists. Our results suggest that Rasd2 and DRD2 play pivotal roles in depression-like behavior induced by ovariectomy. Rasd2 regulates DRD2-mediated antidepressant-like effects of acute fasting in ovariectomized mice. Rasd2 can therefore be postulated to be a potential therapeutic target for depression and perhaps also a potential predictive marker for depression.

Comparison of the Aroma-Active Compounds and Sensory Characteristics of Different Grades of Light-Flavor Baijiu.

This study comprehensively characterized and compared the aroma differences between four different grades of Fenjiu (FJ, the most representative light-flavor Baijiu). Aroma-active compounds were analyzed by liquid-liquid extraction (LLE) coupled with gas chromatography-olfactometry-mass spectrometry (GC-O-MS). A total of 88 aroma-active compounds were identified, and 70 of them were quantified. The results showed that a majority of aroma compounds in high-grade FJ had higher aroma intensities and concentrations. Among these compounds, there were 28 compounds with odor activity values (OAVs) greater than one in all four wines, which indicated that they might contribute to the characteristic aroma of FJ. Temporal dominance of sensation (TDS) and quantitative descriptive analysis (QDA) were used to characterize the sensory differences. The results suggested that high-grade FJ had a rich, pleasant and lasting retronasal aroma perception and exhibited pleasant orthonasal aroma of floral, fruity, sweet and grassy. Partial least squares regression (PLSR) analysis effectively distinguished four kinds of FJ and revealed associations between the orthonasal aroma attributes and the aroma compounds with OAVs >1. There were 15 compounds with variable importance in projection (VIP) values >1, and they were considered potential aroma markers for quality prediction.

Low expression of SEMA4D as a potential predictive molecular marker of poor survival in patients with melanoma combined with liver cancer.

This study explored the correlation between semaphorin 4D (SEMA4D) and the prognosis and survival time of patients with melanoma combined with liver cancer. A total of 272 patients were recruited, and clinical and follow-up data were recorded. The expression levels of SEMA4D and SEMA3B were determined. Pearson's χ2 test and Spearman's rank correlation coefficient were used to analyze the relationship between prognosis and the assessed parameters of melanoma patients. Univariate and multivariate Logistic regression and Cox proportional risk regression analyses were used for further analysis. Additionally, receiver operating characteristic curve and survival curves of subjects were plotted. The Pearson's χ2 test showed that the prognosis of melanoma patients was significantly correlated with age, tumor grade, and decreased SEMA4D expression. Additionally, Spearman's correlation coefficient analysis showed that age, tumor grade, and SEMA4D expression were significantly correlated with prognosis. Univariate logistic regression analysis showed that age and tumor grade, and SEMA4D expression, were significantly correlated with prognosis. Older patients, a higher tumor grade, and lower SEMA4D expression were associated with a poorer prognosis. Multivariate logistic regression analysis showed that older patients had a poorer prognosis, and patients with lower SEMA4D expression levels had a significantly worse prognosis than patients with higher SEMA4D expression levels. Kaplan-Meier analysis showed that the survival time of older patients was lower than that of the younger patients. The survival times of patients with lower SEMA4D expression levels were significantly lower than that of patients with higher SEMA4D expression levels. Multivariate Cox regression analysis showed that the survival time of older patients was lower than that of younger patients. The survival time of melanoma patients with low SEMA4D expression was significantly lower than that of patients with higher SEMA4D expression. SEMA4D was significantly associated with melanoma, and lower SEMA4D expression was associated with a poorer survival prognosis in melanoma patients.