Biomarkers For Immunotherapy Research Articles

Short-term cultured tumor fragments to study immunotherapy combinations based on CD137 (4-1BB) agonism

ABSTRACT Biomarkers for cancer immunotherapy are an unmet medical need. The group of Daniela Thommen at the NKI recently reported on novel methodologies based on short-term cultures of patient-derived tumor fragments whose cytokine concentrations in the supernatants and activation markers on infiltrating T cells were associated with clinical response to PD-1 blockade. We set up a similar culture technology with tumor-derived fragments using mouse tumors transplanted into syngeneic immunocompetent mice to test an agonist anti-CD137 mAb and its combinations with anti-PD-1 and/or anti-TGF-β. Increases in IFNγ concentrations in the tissue culture supernatants were detected upon in-culture activation with the anti-CD137 and anti-PD-1 mAb combinations or concanavalin A as a positive control. No other cytokine from a wide array was informative of stimulation with these mAbs. Interestingly, increases in Ki67 and other activation markers were substantiated in lymphocytes from cell suspensions gathered at the end of 72 h cultures. In mice bearing bilateral tumors in which one was excised prior to in vivo anti-CD137 + anti-PD-1 treatment to perform the fragment culture evaluation, no association was found between IFNγ production from the fragments and the in vivo therapeutic outcome in the non-resected contralateral tumors. The experimental system permitted freezing and thawing of the fragments with similar functional outcomes. Using a series of patient-derived tumor fragments from excised solid malignancies, we showed IFNγ production in a fraction of the studied cases, that was conserved in frozen/thawed fragments. The small tumor fragment culture technique seems suitable to preclinically explore immunotherapy combinations.

WFDC2 is a potential prognostic and immunotherapy biomarker in lung adenocarcinoma.

The prognosis of lung adenocarcinoma (LUAD), which is the most common type of lung cancer, remains poor. Little is known about the function and mechanism of whey acidic protein four-disulfide core domain 2 (WFDC2) in LUAD. In this study, we used online databases to compare WFDC2 expression between LUAD and normal tissues, to analyze the relationship between WFDC2 and overall survival, and to investigate the potential roles of WFDC2. We found that WFDC2 protein and mRNA expression levels were significantly higher in LUAD tissue than in normal tissue, and high WFDC2 mRNA expression was associated with better overall survival. WFDC2 mRNA expression was correlated with the mutation status of TP53. The biological function of WFDC2 was associated with the cell cycle, and low WFDC2 mRNA expression was associated with an elevated tumor mutational burden and neoantigen levels. A negative relationship was observed between WFDC2 and immune gene expression, and high WFDC2 mRNA expression was found in patients with LUAD and low programed cell death 1 mRNA expression. We propose that WFDC2 is associated with clinical benefits of immune checkpoint inhibitors in LUAD.

Cuproptosis-Related Genes as Prognostic Biomarkers for Sepsis: Insights into Immune Function and Personalized Immunotherapy.

This study aimed to discover diagnostic and prognostic biomarkers for sepsis immunotherapy through analyzing the novel cellular death process, cuproptosis. We used transcriptome data from sepsis patients to identify key cuproptosis-related genes (CuRGs). We created a predictive model and used the CIBERSORT algorithm to observe the link between these genes and the septic immune microenvironment. We segregated sepsis patients into three subgroups, comparing immune function, immune cell infiltration, and differential analysis. Single-cell sequencing and real-time quantitative PCR were used to view the regulatory effect of CuRGs on the immune microenvironment and compare the mRNA levels of these genes in sepsis patients and healthy controls. We established a sepsis forecast model adapted to heart rate, body temperature, white blood cell count, and cuproptosis key genes. This was followed by a drug sensitivity analysis of cuproptosis key genes. Our results filtered three key genes (LIAS, PDHB, PDHA1) that impact sepsis prognosis. We noticed that the high-risk group had poorer immune cell function and lesser immune cell infiltration. We also discovered a significant connection between CuRGs and immune cell infiltration in sepsis. Through consensus clustering, sepsis patients were classified into three subgroups. The best immune functionality and prognosis was observed in subgroup B. Single-cell sequencing exposed that the key genes manage the immune microenvironment by affecting T cell activation. The qPCR results highlighted substantial mRNA level reduction of the three key genes in the SP compared to the HC. The prediction model, which combines CuRGs and traditional diagnostic indicators, performed better in accuracy than the other markers. The drug sensitivity analysis listed bisphenol A as highly sensitive to all the key genes. Our study suggests these CuRGs may offer substantial potential for sepsis prognosis prediction and personalized immunotherapy.

Integrative Pan-Cancer Analysis Reveals the Oncogenic Role of MND1 and Validation of MND1's Role in Breast Cancer.

Meiotic nuclear division 1 (MND1) is a meiosis-specific protein that promotes lung adenocarcinoma progression. However, its expression and biological function across cancers remain largely unexplored. The expression, prognostic significance, mutation status, and methylation profile of MND1 in various cancers were comprehensively analyzed using the TIMER, GTEX, Kaplan-Meier plotter, cBioPortal, and GSCA databases. Additionally, we constructed a PPI network, enrichment analysis and single-cell transcriptomic sequencing to elucidate the underlying mechanism of MND1. Furthermore, we investigated the association between MND1 expression and drug sensitivity using CellMiner. Moreover, we also explored the correlation between MND1 expression and immune infiltration. Finally, we validated the functional role of MND1 in breast cancer through IHC staining, CCK8, EdU, colony formation, and flow cytometry assays. MND1 has been reported to be highly expressed in Pan-cancer, High MND1 expression was significantly associated with poor prognosis in cancers. Additionally, MND1 mutation frequency is high in most cancers, and its expression correlates with methylation. Furthermore, MND1 expression significantly correlates with immune checkpoint blockade (ICB) markers, including PD-L1, PD-1, and CTLA-4. The PPI network reveals interactions between MND1 and PSMC3IP, BRCA1, and BRCA2. Enrichment analysis and single-cell sequencing indicate that MND1 positively correlates with cell cycle. ROC curve reveals favorable diagnostic efficacy of MND1 in breast cancer. In vitro, MND1 overexpression promotes breast cancer cell proliferation and increases the expression of key cell cycle regulators (CDK4, CDK6, and cyclin D3), accelerating the G1/S phase transition and leading to abnormal breast cancer cell proliferation. The immunohistochemical analysis revealed a robust expression of MND1 in breast cancer tissues, exhibiting a significant positive correlation with PD-L1 and FOXP3. MND1 is an oncogene and may serve as a biomarker for cancer prognosis and immunotherapy. Targeting MND1 may be a potential tumor treatment strategy.

Pre-Existing Immunity Predicts Response to First-Line Immunotherapy in Non-Small Cell Lung Cancer Patients.

T-cell-mediated anti-tumoral responses may have significant clinical relevance as a biomarker for response to immunotherapy. The value of peripheral blood pre-existing tumor antigen-specific T cells (PreI+) as a predictive immunotherapy biomarker in NSCLC patients was investigated, along with the frequency of various circulating immune cells. Fifty-two treatment-naïve, stage III/IV NSCLC patients, treated with front-line immune checkpoint inhibitors (ICI)-containing regimens were enrolled. PreI was calculated as the percentages of CD3+IFNγ+ cells after in vitro co-cultures of PBMCs with peptides against four different Tumor-Associated Antigens (TAA). Immunophenotyping of peripheral blood immune cells was performed using multicolor flow cytometry. PreI+ T cells were detected in 44% of patients. Median overall survival (OS) was significantly higher in PreI+ patients compared to PreI- patients (not reached vs. 321 days, respectively; p = 0.014). PreI+ patients had significantly higher numbers of possible exhausted CD3+CD8+PD-1+ cells and lower percentages of immunosuppressive Tregs compared to PreI- patients. Additionally, patients with PreI+ and low numbers of peripheral blood M-MDSCs had a significant survival advantage compared to the rest of the patients. Thus, combining pre-existing tumor antigen-specific immunity before initiation of ICI in NSCLC patients with selected immune-suppressive cells could identify patients who have a favorable clinical outcome when treated with ICI-containing regimens.

The prognostic significance and immune characteristics of bone morphogenetic proteins (BMPs) family: A pan-cancer multi-omics analysis.

Bone morphogenetic proteins (BMPs) are a group of cancer-related proteins vital for development and progression of certain cancer types. Nevertheless, function of BMP family in pan-cancer was not detailedly researched. Investigating expression pattern and prognostic value of the BMPs family (BMP1-8A and BMP8B) expression across multiple cancer types. Our research integrated multi-omics data for exploring potential associations between BMPs expression and prognosis, clinicopathological characteristics, copy number or somatic mutations, immune characteristics, tumor microenvironment (TME), tumor mutation burden (TMB), microsatellite instability (MSI), immune checkpoint genes and drug sensitivity in The Cancer Genome Atlas (TCGA) tumors. Furthermore, association of BMPs expression and immunotherapy effectiveness was investigated in some confirmatory cohorts (GSE111636, GSE78220, GSE67501, GSE176307, IMvigor210 and mRNA sequencing data from currently undergoing TRUCE01 clinical research included), and biological function and potential signaling pathways of BMPs in bladder cancer (BCa) was explored via Gene Set Enrichment Analysis (GSEA). Eventually, immune infiltration analysis was done via BMPs expression, copy number or somatic mutations in BCa, as well as validation of the expression levels by reverse transcription-quantitative PCR and western blot, and in vitro functional experiments of BMP8A. Discoveries displayed BMPs expression was related to prognosis, clinicopathological characteristics, mutations, TME, TMB, MSI and immune checkpoint genes of TCGA tumors. Anticancer drug sensitivity analysis displayed BMPs were associated with various drug sensitivities. What's more, it was discovered that expression level of certain BMP family members related to objective response to immunotherapy. By GSEA, we discovered multiple immune-associated functions and pathways were enriched. Immune infiltration analysis on BCa also displayed significant associations among BMPs copy number variations, mutation status and infiltration level of diverse immune cells. Furthermore, differential expression validation and in vitro phenotypic experiment indicated that BMP8A significantly promoted BCa cell proliferation, migration and invasion. Current results confirmed significance of both BMPs expression and genomic alteration in the prognosis and treatment of diverse cancer types, and suggested that BMPs may be vital for BCa and can possibly be utilized as biomarkers for immunotherapy.

The JAK-STAT signaling-related signature serves as a prognostic and predictive biomarker for renal cell carcinoma immunotherapy

Renal cell carcinoma (RCC) represents a significant portion of genitourinary cancers, marked by challenging prognosis and high metastasis rates. Immunotherapy has been applied in managing advanced renal cell carcinoma, but the therapeutic outcomes are unsatisfactory. In this study, we order to construct a Janus kinase/signal transduction and activator transcriptional (JAK/STAT)-related signature linked to kidney patient outcomes for better predicting the efficacy to immune checkpoint inhibitors (ICIs) and to provide guidance for effective combination therapy. We screened 25 differentially expressed genes (DEGs) that exhibited high expression in RCC samples and were enriched in the JAK-STAT signaling pathway. Among these genes, 11 key genes were identified and correlated with the expectation of Kidney Clear Cell Carcinoma (KIRC) patients and all these genes was significantly elevated in RCC tumor tissues and cancer cells compared to para-cancer tissues and normal renal cells. Utilizing these 11 genes, we divided RCC patients into high-risk and low-risk groups. We found a clear correlation between the clinicopathologic factors of KIRC patients and the JAK-STAT-related risk score. And the IHC results shown that the JAK3 and STAT4 expression of tumor was significantly higher than normal tissue in RCC patients, the level of JAK3 and STAT4 was positively related to the T stage of RCC patients. In addition, high-risk patients had a poorer prognosis and greater protumor immune cell infiltration, and benefitted less from immunotherapy than did low-risk patients. Furthermore, the JAK-STAT-related risk score can predict disease-free survival (DFS) in RCC patients according to the nomogram, which constructed in combination with other clinical features such as age, TNM-staging and stage. Our study demonstrated the JAK-STAT signaling pathway’s important regulatory function in RCC tumor immunity. This insight not only enhances our ability to accurately predict the survival rate of RCC patients, but also underscores a potential therapeutic alternative for RCC, involving the combined targeting of the JAK-STAT pathway and immune checkpoints.

The Immune Checkpoint BTLA in Oral Cancer: Expression Analysis and Its Correlation to Other Immune Modulators.

In oral squamous cell carcinoma (OSCC) tissues, an immunotolerant situation triggered by immune checkpoints (ICPs) can be observed. Immune checkpoint inhibitors (ICIs) against the PD1/PD-L axis are used with impressive success. However, the response rate is low and the development of acquired resistance to ICI treatment can be observed. Therefore, new treatment strategies especially involving immunological combination therapies need to be developed. The novel negative immune checkpoint BTLA has been suggested as a potential biomarker and target for antibody-based immunotherapy. Moreover, improved response rates could be displayed for tumor patients when antibodies directed against BTLA were used in combination with anti-PD1/PD-L1 therapies. The aim of the study was to check whether the immune checkpoint BTLA is overexpressed in OSCC tissues compared to healthy oral mucosa (NOM) and could be a potential diagnostic biomarker and immunological target in OSCC. In addition, correlation analyses with the expression of other checkpoints should clarify more precisely whether combination therapies are potentially useful for the treatment of OSCC. A total of 207 tissue samples divided into 2 groups were included in the study. The test group comprised 102 tissue samples of OSCC. Oral mucosal tissue from 105 healthy volunteers (NOM) served as the control group. The expression of two isoforms of BTLA (BTLA-1/2), as well as PD1, PD-L1/2 and CD96 was analyzed by RT-qPCR. Additionally, BTLA and CD96 proteins were detected by IHC. Expression levels were compared between the two groups, the relative differences were calculated, and statistical relevance was determined. Furthermore, the expression rates of the immune checkpoints were correlated to each other. BTLA expression was significantly increased in OSCC compared to NOM (pBTLA_1 = 0.003; pBTLA_2 = 0.0001, pIHC = 0.003). The expression of PD1, its ligands PD-L1 and PD-L2, as well as CD96, were also significantly increased in OSCC (p ≤ 0.001). There was a strong positive correlation between BTLA expression and that of the other checkpoints (p < 0.001; ρ ≥ 0.5). BTLA is overexpressed in OSCC and appears to be a relevant local immune checkpoint in OSCC. Thus, antibodies directed against BTLA could be potential candidates for immunotherapies, especially in combination with ICI against the PD1/PD-L axis and CD96.

Exploring the Molecular Mechanism of Comorbidity of Type 2 Diabetes Mellitus and Colorectal Cancer: Insights from Bulk Omics and Single-Cell Sequencing Validation.

The relationship between type 2 diabetes mellitus (T2DM) and colorectal cancer (CRC) has long been extensively recognized, but their crosstalk mechanisms based on gene regulation remain elusive. In our study, for the first time, bulk RNA-seq and single-cell RNA-seq data were used to explore the shared molecular mechanisms between T2DM and CRC. Moreover, Connectivity Map and molecular docking were employed to determine potential drugs targeting the candidate targets. Eight genes (EVPL, TACSTD2, SOX4, ETV4, LY6E, MLXIPL, ENTPD3, UGP2) were identified as characteristic comorbidity genes for T2DM and CRC, with EVPL and ENTPD3 further identified as core comorbidity genes. Our results demonstrated that upregulation of EVPL and downregulation of ENTPD3 were intrinsic molecular features throughout T2DM and CRC and were significantly associated with immune responses, immune processes, and abnormal immune landscapes in both diseases. Single-cell analysis highlighted a cancer-associated fibroblast (CAF) subset that specifically expressed ENTPD3 in CRC, which exhibited high heterogeneity and unique tumor-suppressive features that were completely different from classical cancer-promoting CAFs. Furthermore, ENTPD3+ CAFs could notably predict immunotherapy response in CRC, holding promise to be an immunotherapy biomarker at the single-cell level. Finally, we identified that droperidol may be a novel drug simultaneously targeting EVPL and ENTPD3. In conclusion, previous studies have often focused solely on metabolic alterations common to T2DM and CRC. Our study establishes EVPL and ENTPD3 as characteristic molecules and immune biomarkers of comorbidity in T2DM and CRC patients, and emphasizes the importance of considering immunological mechanisms in the co-development of T2DM and CRC.

CircZNF609 inhibited bladder cancer immunotherapy sensitivity via enhancing fatty acid uptake through IGF2BP2/CD36 pathway

Circular RNAs (circRNAs) are gaining attention for their involvement in immune escape and immunotherapy sensitivity regulation. CircZNF609 is a well-known oncogene in various solid tumours. Our previous research revealed its role in reducing the chemosensitivity of bladder cancer (BCa) to cisplatin. However, the underlying role of circZNF609 in BCa immune escape and immunotherapy sensitivity remains unknown. We conducted BCa cells-CD8 + T cells co-culture assays, cell line-derived xenograft and patient-derived xenograft mouse models with human immune reconstitution to further confirm the role of circZNF609 in BCa immune escape and immunotherapy sensitivity. Overexpression of circZNF609 promoted BCa immune escape in vitro and in vivo. Mechanistically, circZNF609 was bound to IGF2BP2, enhancing its interaction with the 3′-untranslated region of CD36. This increased the stability of the CD36 mRNA, leading to enhanced fatty acid uptake by BCa cells and fatty acid depletion within the tumour microenvironment. Additionally, the nuclear export of circZNF609 was regulated by DDX39B. CircZNF609 promoted immune escape and suppressed BCa immunotherapy sensitivity by regulating the newly identified circZNF609/IGF2BP2/CD36 cascade. Therefore, circZNF609 holds potential as both a biomarker and therapeutic target in BCa immunotherapy.

Integration of Clinical Trial Spatial Multiomics Analysis and Virtual Clinical Trials Enables Immunotherapy Response Prediction and Biomarker Discovery.

Due to the lack of treatment options, there remains a need to advance new therapeutics in hepatocellular carcinoma (HCC). The traditional approach moves from initial molecular discovery through animal models to human trials to advance novel systemic therapies that improve treatment outcomes for patients with cancer. Computational methods that simulate tumors mathematically to describe cellular and molecular interactions are emerging as promising tools to simulate the impact of therapy entirely in silico, potentially greatly accelerating delivery of new therapeutics to patients. To facilitate the design of dosing regimens and identification of potential biomarkers for immunotherapy, we developed a new computational model to track tumor progression at the organ scale while capturing the spatial heterogeneity of the tumor in HCC. This computational model of spatial quantitative systems pharmacology was designed to simulate the effects of combination immunotherapy. The model was initiated using literature-derived parameter values and fitted to the specifics of HCC. Model validation was done through comparison with spatial multiomics data from a neoadjuvant HCC clinical trial combining anti-PD1 immunotherapy and a multitargeted tyrosine kinase inhibitor cabozantinib. Validation using spatial proteomics data from imaging mass cytometry demonstrated that closer proximity between CD8 T cells and macrophages correlated with nonresponse. We also compared the model output with Visium spatial transcriptomics profiling of samples from posttreatment tumor resections in the clinical trial and from another independent study of anti-PD1 monotherapy. Spatial transcriptomics data confirmed simulation results, suggesting the importance of spatial patterns of tumor vasculature and TGFβ in tumor and immune cell interactions. Our findings demonstrate that incorporating mathematical modeling and computer simulations with high-throughput spatial multiomics data provides a novel approach for patient outcome prediction and biomarker discovery. Significance: Incorporating mathematical modeling and computer simulations with high-throughput spatial multiomics data provides an effective approach for patient outcome prediction and biomarker discovery.

Immunotherapy, prognostic, and tumor biomarker based on pancancer analysis, SMARCD3.

SMARCD3 has recently been shown to be an important gene affecting cancer, playing an important role in medulloblastoma and pancreatic ductal adenocarcinoma. Therefore, we conducted this research to investigate the potential involvement of SMARCD3 across cancers and to offer recommendations for future studies. Utilizing information on 33 malignancies in the UCSC Xena database, SMARCD3 expression and its prognostic value were assessed. The tumor microenvironment was evaluated with the "CIBERSORT" and "ESTIMATE" algorithms. SMARCD3 and immune-related genes were analyzed using the TISIDB website. The pathways related to the target genes were examined using GSEA. MSI (microsatellite instability), TMB (tumor mutational burden), and immunotherapy analysis were used to evaluate the impact of target genes on the response to immunotherapy. There is heterogeneity in terms of the expression and prognostic value of SMARCD3 among various cancers, but it is a risk factor for many cancers including uterine corpus endometrial cancer (UCEC), renal clear cell carcinoma (KIRC), and gastric adenocarcinoma (STAD). GSEA revealed that SMARCD3 is related to chromatin remodeling and transcriptional activation, lipid metabolism, and the activities of various immune cells. The TMB and MSI analyses suggested that SMARCD3 affects the immune response efficiency of KIRC, LUAD and STAD. Immunotherapy analysis suggested that SMARCD3 may be a potential immunotherapy target. RT-qPCR demonstrated the variation in SMARCD3 expression in KIRC, LUAD, and STAD. Our study revealed that SMARCD3 affects the prognosis and immunotherapy response of some tumors, providing a direction for further research on this gene.

Efficacy of immunotherapy in ARID1A-mutant solid tumors: a single-center retrospective study

BackgroundImmune checkpoint inhibitors (ICIs), especially those targeting programmed cell death-1 (PD-1) and programmed cell death ligand-1 (PD-L1), have introduced a new treatment landscape for many types of tumors. However, they only achieve a limited therapeutic response. Hence, identifying patients who may benefit from ICIs is currently a challenge.Methods47 tumor patients harboring ARID1A mutations were retrospectively studied. The genomic profiling data through next-generation sequencing (NGS) and relevant clinical information were collected and analyzed. Additionally, bioinformatics analysis of the expression of immune checkpoints and immune cell infiltration levels was conducted in ARID1A-mutant gastric cancer (GC).ResultsARID1A mutations frequently co-occur with mutations in DNA damage repair (DDR)-associated genes. Among the 35 ARID1A-mutant patients who received immunotherapy, 27 were evaluable., with the objective response rate (ORR) was 48.15% (13/27), and the disease control rate (DCR) was 92.59% (25/27). Moreover, survival assays revealed that ARID1A-mutant patients had longer median overall survival (mOS) after immunotherapy. In ARID1A-mutated GC patients, receiving ICIs treatment indicated longer progressive-free survival (PFS). Additionally, the incidence of microsatellite instability-high (MSI-H), high tumor mutation burden (TMB-H) and Epstein‒Barr virus (EBV) infection was elevated. Bioinformatic analysis showed significant enrichment of immune response and T cell activation pathway within differentially expressed genes in ARID1A-mutant GC group. Finally, ARID1A mutations status was considered to be highly correlated with the level of tumor infiltrating lymphocytes (TILs) and high expression of immune checkpoints.ConclusionsPatients with tumors harboring ARID1A mutations may achieve better clinical outcomes from immunotherapy, especially in GC. ARID1A mutations can lead to genomic instability and reshape the tumor immune microenvironment (TIME), which can be used as a biomarker for immunotherapy.

TERT-associated DNA polymerases genes link CAF and CD8+ T Cells to improve immunotherapy response rate across multiple cancers.

e14669 Background: The cancer cells exhibit heightened basal levels of responses to DNA breaks, accumulating DNA polymerases family (DNA-pol) as repair protein, thereby increasing DNA mutation and neoantigens. Based on this, we investigated the potential of DNA-pol as an immunotherapy biomarker and its underlying mechanism. Methods: We conducted a pan-cancer analysis of DNA-pol gene dysregulation in 6366 samples. Normal fibroblast, cancer-associated fibroblast, and CD8+ T cells were cultured from cancer patients (3 esophagus, 5 lung, 5 renal, and 3 liver cancer), then treated them with PD-L1 inhibitor or overexpressing TERT for RNA-seq and proteomics. 996 multiple cancer cases with ICIs were collected to test biomarker efficiency. A phase II clinical trial is evaluating and exploring our biomarker in mechanism prospectively. Results: We defined a distinct signature score of DNA-pol genes upregulated in cancer (DNA-pol-up) and linked to worse prognosis (HR=1.2). DNA-pol-up program dysregulation led to immune processes alteration and was related to upstream activation of TERT signaling (z-score=3.6) in ingenuity pathway analysis and linked to immunologically active tumors by recruiting CD8+ T cell and downregulating CAF. DNA-pol-up score was elevated in PD-L1-treated CAF and TERT-overexpressed CD8+ T cell, in MSI-H cancer, also related to more neoepitope peptides, TMB (Rho=0.54), cancer stemness (Rho=0.78), and tumor purity (Rho=0.29). Cancers that activate this program carried distinct genomic profiles with PIK3CA, KRAS, and TP53 mutations. This signature was an independent predictor for ICIs response (OR=2.52), even outperforming cytolytic activity, MSI, TMB (OR=0.9). In 12 clinical trial datasets, this signature could stratify the responding patients (maxAUC=0.762). In our phase II clinical trial, 34 liver cancer were enrolled with 4-year follow-up, it also has a satisfactory performance in predicting the ORR (AUC=0.699) and classifies mortality rate (HR=2.5), meanwhile verified its relationship with detailed potential molecular mechanism. Conclusions: Our findings identify a distinct transcriptional pattern of DNA-pol genes across cancers, which highlighted the role of DNA-pol family genes in predicting the immunotherapy response for the first time, and TERT could be a novel vaccine candidate for improving immunotherapy response.

Identification and validation of GIMAP family genes as immune-related prognostic biomarkers in lung adenocarcinoma

BackgroundThe GIMAP family genes play a key role in immune function. Increasing evidence suggests that GIMAP genes were implicated in the tumorigenesis of lung adenocarcinoma (LUAD). This study aimed to investigate the clinical significance of GIMAP family genes in LUAD. MethodsIn this study, we explored the expression, mutation, prognostic value of GIMAP family genes and the correlation with immune microenvironment in LUAD. We further investigated the relationship between GIMAP family genes expression and immunotherapy response in GEO LUAD and melanoma cohorts. ResultsAmong the GIMAP family genes, the expression levels of GIMAP1, GIMAP2, GIMAP4, GIMAP5, GIMAP6, GIMAP7, and GIMAP8 were significantly lower in LUAD tumor tissues than normal tissues. Most GIMAP genes were closely related to age, tumor grade and T stage, but not significantly related to sex, N stage and M stage. In the overall population, patients with high expression of GIMAP family genes had a significant longer overall survival (OS). GO and KEGG enrichment analysis showed that GIMAP family genes were highly enriched in immune-related biological process. The expression of GIMAP family genes was positively correlated with immune cell infiltration and immune checkpoint molecules. Furthermore, high expression of GIMAP family genes were correlated with therapeutic response to immunotherapy in LUAD and melanoma patients. ConclusionIn this study, we identified that GIMAP family genes were significantly associated with immune cell infiltration and immune checkpoint molecules. They potentially play a critical role in anti-tumor immunity and serve as immunotherapy biomarkers.

Impact of HER2 low status on genomic signatures in triple negative breast cancer (TNBC).

1092 Background: HER2 status has become more important with new treatment regimens subcategorizing HER2 to into low (1+or 2+ FISH-) or absent HER2 expression. Through comprehensive genomic profiling (CGP), we aim to identify distinctive molecular features associated with various immunohistochemistry (IHC) categories of HER2 low tumors. Methods: Retrospective review of pathology specimens submitted to Foundation Medicine were done and 514 TNBC (ER-/PR-/HER20,1+,2+FISH-) and 191 3+/HER2 amplified clinically advanced BC underwent hybrid capture-based CGP to evaluate genomic alterations (GA) in 324 genes, MSI status and tumor mutational burden (TMB, mutations/Mb). PD-L1 expression was determined by IHC (Dako 22C3 TPS). Chi-square test and Mann Whitney U test were used in the statistical comparisons of the 2 groups. Statistical significance was defined as p &lt; 0.05. Results: 53.5% were HER2 IHC 0+, 31.5% were HER2 IHC 1+ and 15.0% were IHC 2+/FISH negative. The HER2 3+/amplified BC were 59% ER+ and 41% ER-. Notable differences in GA frequencies included a significant increase in PIK3CA GA in the HER2 2+ vs the HER 0+ and 1+ groups (p=.0003). A lower frequency in BRCA1 GA in the HER2 2+ (5.2%) vs the HER2 0+ (9.8%) was significant when compared to the 1.0% in the HER2 3+/amplified cases (p&lt;.0001). BRCA2 GA frequencies did not show statistically significant differences among the TNBC HER2 IHC score groups. TP53 and RB1 had the highest frequency in HER2 0+ samples, with decreasing prevalence in the HER2 1+, 2+ and 3+/amplified cases (p&lt;.0001 for both). There was also a trend towards higher frequency of CDH1 GA as the HER2 IHC score increased from 0 to 2+ among TNBC. There were no significant differences in immunotherapy biomarkers (MSI, TMB and PD-L1) based on HER2 status among the TNBC groups. TMB &gt;= 10 mutations/Mb was significantly higher in the HER2 3+/amplified group (p=.004). Conclusions: We found that TNBC HER2 2+ positive FISH show key molecular differences from their HER2 low (1+) or absent (0+) counterparts. The most substantial difference was an increase in GA in PIK3CA. While this is targetable in the hormone positive setting, its potential role as a target in the hormone negative setting is uncertain. Also noted but did not reach significance was a decrease in GA within BRCA1/2 and a positive linear relationship with CDH1 and HER2 IHC expression. These GA differences may indicate potential treatment options for different HER2 subgroups and warrant further investigation. Limitations of our study include the retrospective nature and the lack of clinical data. [Table: see text]

Epigenetic silencing of LDHB to promote hepatocellular carcinoma by remodeling the tumor microenvironment.

e16220 Background: Hepatocellular carcinoma (HCC) is a deadly disease with limited therapeutic options. Low expression of the LDHB has been associated with poor prognosis of HCC patients, but the role and underlying mechanism remains unclear. Methods: The HCC dada from The Cancer Genome Atlas (TCGA) and GEO were obtained to analyze the LDHB expression and DNA methylation levels. Next, the correlation between LDHB and Hallmark gene sets was analyzed by GO and KEGG. RNA-seq, Western blotting, Immunohistochemistry, and Bisulfite DNA sequencing were performed to explore the relationship between DNA methylation and LDHB expression. CCK8 and colony formation assay were used to evaluate the effect of overexpression of LDHB on the proliferation of hepatocellular carcinoma cells in vitro. Finally, the role of LDHB in HCC tumor immunity was assessed in multiple mouse models in vivo. Results: Here, we found that down-regulation of LDHB was coupled with the promoter hypermethylation and knocking down the DNMT 3A restored LDHB expression levels in HCC cell lines. Bioinformatics analysis of the HCC cohort from The Cancer Genome Atlas revealed a significant positive correlation between LDHB expression and immune regulatory signaling pathways and immune cell infiltrations. Notably, HCC patients with high LDHB expression responded better to immune checkpoint inhibitors. Finally, we demonstrated that there was no difference in the tumor growth rate between the LDHB-expressing tumors and the control tumors in immunodeficient BALB/c nude mice. However, the LDHB-expressing tumors were significantly inhibited in immunocompetent C57BL/6 mice, suggesting that the host immune system was involved in the LDHB-medicated tumor suppression. Moreover, as compared with the LDHB+/+ mice, LDHB-/- mice showed promoted tumor growth in an NRAS/shp53 plasmid-induced primary liver tumor model. Conclusions: Overall, our findings indicate that DNMT3A-mediated epigenetic silencing of LDHB may contribute to HCC progression through remodeling the tumor immune microenvironment, and LDHB may become a potential prognostic biomarker and target for HCC immunotherapy.

Gut microbiome ecological topology as next-generation biomarkers for cancer immunotherapy

Numerous studies have evaluated the gut microbiome as a biomarker for predicting cancer immunotherapy, but the heterogeneity among different studies has hindered its applications. In this issue of Cell, Derosa etal. report a biomarker based on the ecological topology of the gut microbiota that can predict immunotherapy efficacy effectively.

Genomic instability in advanced non-small cell lung cancer (NSCLC) treated with maintenance durvalumab in the UNICANCER SAFIR02-Lung/ IFCT1301 trial.

2632 Background: Genomic instability is a hallmark of cancer that has been associated to immune evasion, cancer progression and multidrug resistance. In SAFIR02-Lung, advanced NSCLC pts were enrolled to receive a platinum-based chemotherapy. After 4 cycles, pts with no targetable alterations were oriented to the immunotherapy sub-study, and if stable or in response, were randomized between maintenance durvalumab or Standard of Care (SoC) (PMID 35802649). Durvalumab prolonged survival only in the PD-L1 ≥ 1% subgroup. In this study we investigated a genomic instability score based on somatic copy number alterations as determined by Comparative Genomic Hybridization (CGH) array as a biomarker for maintenance immunotherapy. Methods: SAFIR02-Lung (NCT02117167) is a multicentric, randomized phase 2 clinical trial. Tissue samples were obtained before induction chemotherapy. Extracted DNA from fresh frozen tumor was analyzed using high-resolution Affymetrix Cytoscan arrays, DNA from FFPE samples was analyzed using the Affymetrix Oncoscan CNV platform. The absolute copy-number (ACN) profile was generated by ASCAT v3.1 through EaCoN v0.3.6 ( https://github.com/gustaveroussy/EaCoN , PMID 36669143 ) on R v4.1.1 and then the genomic instability score was estimated as the sum of the segment length multiplied by the absolute difference between segment copy number and the ASCAT estimated sample ploidy. This sum is reported to the reference genome length. Progression Free Survival (PFS) was the primary endpoint, defined as the time from randomization until the date of objective radiological disease progression, clinical progression or death. The secondary endpoint was Overall Survival (OS). Results: CGH array data were available for 79 patients out of 183, 56 randomized to durvalumab and 23 to SoC. 47 were men, 72 had non-squamous histology and 12 had brain and 10 had liver metastasis. We divided patients in tertiles according to instability (INS) score. No correlation was seen between INS score and PD-L1 positivity (p = 0.5), or liver and brain metastasis. Pts with high INS had lower median PFS of 2.5 months (m) (95% CI 1.4 – 5.2) vs 4.3m (95% CI 2.6 – 13.7) for those with intermediate INS and 6.1m for those with low INS (95% CI 1.9 – 21.2), p 0.012. Similarly, low INS patients had longer OS of 29.6 m (95% CI 15.4 – NR) vs 17.9m for intermediate INS (95% CI 10 – NA) and 12.1m for high INS (95% CI 10.2 – 19.5), p = 0.039. No difference was seen in the control group (p=0.25 for PFS and p=0.4 for OS). A multivariable model showed that high INS was independently associated worse PFS (HR 2.60, 95% CI 1.36 - 4.98, p 0.004) and OS (HR 2.90, 95% CI 1.33 - 6.35, p 0.008). Conclusions: Genomic instability score based on CGH array-based copy number alterations profile is associated with worse PFS and OS in patients under response to chemotherapy who received durvalumab maintenance. Clinical trial information: NCT02117167 .

Benchmarking the transcriptomic predictive biomarkers for immunotherapy plus chemotherapy: Results from phase III RATIONALE-309 and ORIENT-11 randomized trials.

e14624 Background: The combination of PD-(L)1 inhibitors and chemotherapy (IO+Chemo) has become the standard first-line treatment for multiple types of cancer. In contrast to PD-(L)1 monotherapy, there is a lack of established biomarkers to distinguish the outcomes of IO+Chemo. The paucity of high-quality omics data in this setting has not allowed for deep biomarker exploration. We utilized RNA-seq data from two in-house randomized controlled trials (RCTs), RATIONALE-309 and ORIENT-11, with the goal to find transcriptomic biomarkers to identify patients who may not derive benefit from IO+Chemo. Methods: RATIONALE-309 (NCT03924986) was a phase 3 study conducted at 42 sites in Asia. Treatment-naive recurrent/metastatic nasopharyngeal cancer patients were randomly allocated to either PD-1 inhibitor tislelizumab or placebo plus gemcitabine and cisplatin. ORIENT-11 (NCT03607539) was a phase 3 study that enrolled patients from 47 centers in China who had previously untreated locally advanced/metastatic nonsquamous non-small cell lung cancer. Patients were randomized to PD-1 inhibitor sintilimab or placebo plus pemetrexed and platinum. We compared several immune-related biomarkers, including IFN-γ signature, PD-L1, cytolytic score (CYT), T-cell inflamed gene expression profile, B cell infiltration, and ESTIMATE immune score, and explored the optimal predictive threshold. Results: In the RATIONALE-309 and ORIENT-11 trials, 247 and 171 patients with RNA-seq data were analyzed, respectively. Although the addition of Chemo to IO could theoretically benefit tumors with low immune activation, we hypothesized that patients might not benefit from IO+Chemo if their activation level is extremely low. We first tested the predictiveness of several thresholds (20-50th quantiles) for each selected biomarker in the two RCTs. We found that low CYT and B cell are the two best indicators for identifying a lack of survival benefits from IO+Chemo versus Chemo, especially when using 20-30th quantile as threshold. To incorporate their distinct biological information and predictive value, we combined their genes into a single gene signature to represent immune activation. We found that patients in the lower quantile (&lt;20th) showed no benefit of IO+Chemo over Chemo in terms of overall survival (OS) (RATIONALE-309: HR=1.01, P=0.99; ORIENT-11: HR=1.54, P=0.31) and progression-free survival (PFS) (RATIONALE-309: HR=0.76, P=0.42; ORIENT-11: HR=1.30, P=0.52). Moreover, this lack of advantage was maintained in key subgroups such as PD-L1-positive patients and former/current smokers across the two RCTs. Conclusions: Based on the two large-scale RCTs, our findings have implications for the selection of biomarkers when identifying suitable candidates for IO+Chemo treatment. B cells and CYT are crucial components required for the efficacy of IO+Chemo.