Patient Selection For Immunotherapy Research Articles

Quantitative indicators of TREC and KREC excision circles in malignancies: a prospective cohort study

Background. In oncology, of particular interest is the study of the T-cell receptor excision circles (TREC) and the κ-deletion B-cell receptor excision circles (KREC), which are extrachromosomal DNA structures. In many malignancies, the effectiveness of immune checkpoint inhibitors depends on the mutational load of the tumor, which correlates with the formation of specific antitumor immunity. Quantitative indicators of recombination excision circles reflect the occurrence of a different repertoire of T-cell receptors, an integral component in the formation of specific immunity. Understanding the change in quantitative values of TREC and KREC in cancer patients can improve the selection of patients for immunotherapy. Aim. To determine quantitative indicators of TREC and KREC for immunological evaluation of patients with malignancies. Materials and methods. The study included 55 healthy individuals and 180 patients with malignancies. Among healthy individuals, 49.1% (27/55) were males and 50.9% (28/55) females. Among patients with malignancies, 20.5% (37/180) were males and 79.5% (143/180) females. The median age in healthy individuals was 36 years [Q1–Q3: 26–58]. The median age in the group of patients with malignancies was 57 years [Q1–Q3: 47.5–67]. Results. In the general population of healthy individuals, the median TREC level was 60.1 [Q1-Q3: 31.3-188.9] and the median KREC level was 256 [Q1-Q3: 149.8-353]. In the general population of patients with malignancies, the median TREC rate was 4.6 [Q1-Q3: 0.9-17.3] and the median KREC was 111.9 [Q1-Q3: 29.3-339.28]. According to the results of the study, we noted statistically significant differences in TREC and KREC indices between all patients with malignancies and healthy individuals (p0.001, p=0.001). Analysis of TREC and KREC indices in patients with malignancies of various localizations (breast cancer, ovarian cancer, lung cancer, colorectal cancer, skin melanoma, lymphomas) in comparison with healthy individuals statistically significant differences in TREC level were noted (p=0.001, p0.001). When analyzing the KREC level in the studied groups, statistically significant differences in patients with ovarian malignancies (p0.001), lymphoma (p0.001), colorectal cancer (p=0.001) and melanoma (p=0.039) in comparison with healthy individuals were obtained. When comparing groups pairwise, it was found that TREC level in patients with malignancies in the age group of 25–44 years was significantly higher than in the age group of 45–60 years (p=0.03); TREC level in the age group of 25–44 years was significantly higher than in the age group of persons over 60 years (p0.001); TREC level in the age group of 45–60 years was significantly higher than in the age group over 60 years (p0.001). Statistically significant differences of KREC level in the studied patients with malignancies depending on the age group were not established (p=0.16), there were no age differences of groups by KREC level. Conclusion. The results demonstrate a significant decrease in TREC and KREC levels in patients with malignancies compared to healthy individuals. The study of TREC and KREC excision circles in peripheral blood is one of the promising approaches for the immunological evaluation of cancer patients.

Tumor purity as confounder for the determination of tumor mutational burden (TMB).

e14656 Background: TMB determination is a prerequisite to stratify advanced cancer patients for pembrolizumab based on the histology-agnostic FDA approval. The accuracy of TMB measurement is challenged by low tumor purity resulting in a lower number of detected somatic variants for a fixed threshold of variant allele frequency (VAF) and a lower measured TMB level as a consequence. Methods: We derived a mathematical formula describing the influence of tumor purity on the VAF of somatic variants, in which the DNA copy number of the corresponding region of the tumor genome is a parameter. We simulated the effect of tumor purity in a cohort of 4,900 tumors from the TCGA project with tumor purity of at least 80%. TMB was calculated as the number of missense mutations with VAF greater or equal than 5%. Tumors were classified as TMB-high when harboring at least 199 missense mutations. This cutpoint corresponds to 10 mut/Mb as derived in the TMB bridging study. We compared tumors with a simulated tumor purity of 70%, 60%, ..., and 10% with tumors with a tumor purity of 80%, which served as reference. Results: The median of TMB in the pan-cancer cohort was reduced by 2%, 10%, and 32% for tumor purity of 60%, 40%, and 20% compared to the reference of 80%. For the 10% tumors with the worst estimation, TMB was reduced even by 12%, 35%, and 77% or more, respectively. In the pan-cancer cohort, the sensitivity to detect high TMB was 98%, 90%, and 62% for tumor purity of 60%, 40%, and 20%, respectively. For head and neck cancer, lung adenocarcinoma, lung squamous cell carcinoma, and cutaneous melanoma, the sensitivity to detect high TMB was 80%, 89%, 92%, and 91%, respectively, for tumors of tumor purity 40%. Conclusions: High tumor purity is critical for accurate determination of the TMB and to reach a high sensitivity concerning the selection of patients for immunotherapy. As shown by the simulations, the sensitivity to detect high TMB is close to 100% for tumor purity of at least 60%, while a substantial proportion of patients that could receive immunotherapy is missed for tumors with purity of 40% or less. Potential ways out of the TMB underestimation include increasing the sequencing coverage and lowering the VAF threshold for mutation calling or using clonal TMB with an adapted cutpoint instead of total TMB as biomarker for immunotherapy guidance.

Options for enhancing specific antitumor immunity in patients with melanoma.

e21547 Background: The special interest in oncology is the research of the immune status of patients. Quantification of T-cell receptor excision rings (TREC) and B-cell receptor κ deletion elements (KREC) allows to evaluate a humoral and a cellular immunity. In melanoma, the effectiveness of the use of drugs to control immune points depends on the formation of specific antitumor immunity. Quantitative indicators of excision recombination rings reflect the formation of a different repertoire of T-cell receptors, which are an integral component in the formation of specific immunity. The understanding of changes in TREC levels in patients with melanoma during therapy with various drugs can improve the selection of patients for immunotherapy. The purpose of this study is to assess the dynamics of changes in quantitative indicators of the T-cell receptor during adjuvant therapy for melanoma. Methods: The study included 82 patients with melanoma. Among patients with melanoma, the proportion of male patients was 36.5% (30/82), female - 63.5% (52/82). The median age in the group of patients with cancer was 63 years [Q1-Q3: 52.8-75]. Results: From the results obtained, it follows that in cancer patients, a decrease in the number of TREC in the blood indicates the presence of immunodeficiency. The data obtained indicate significant changes in TREC in melanoma. Targeted therapy (dabrafenib + trametinib) and interferon therapy are accompanied by a significantly significant increase in the level of TREC, which indicates an increase in the formation of specific antitumor immunity. Conclusions: The results obtained demonstrate a significant decrease in TREC content in patients with melanoma compared to healthy individuals. Determination of TREC levels during adjuvant therapy demonstrated the effectiveness of dabrafenib in combination with trametinib and interferon α-2β, which is reflected in a significant increase in the T-cell receptor repertoire. The presented data indicate the possibility of using the method for determining the TREC level to predict the risk of cancer progression and the effectiveness of prescribed therapy.

Apparent diffusion coefficient and tissue stiffness are associated with different tumor microenvironment features of hepatocellular carcinoma.

To investigate associations between tissue diffusion, stiffness, and different tumor microenvironment features in resected hepatocellular carcinoma (HCC). Seventy-two patients were prospectively included for preoperative magnetic resonance (MR) diffusion-weighted imaging and MR elastography examination. The mean apparent diffusion coefficient (ADC) and stiffness value were measured on the central three slices of the tumor and peri-tumor area. Cell density, tumor-stroma ratio (TSR), lymphocyte-rich HCC (LR-HCC), and CD8 + T cell infiltration were estimated in resected tumors. The interobserver agreement of MRI measurements and subjective pathological evaluation was assessed. Variables influencing ADC and stiffness were screened with univariate analyses, and then identified with multivariable linear regression. The potential relationship between explored imaging biomarkers and histopathological features was assessed with linear regression after adjustment for other influencing factors. Seventy-two patients (male/female: 59/13, mean age: 56 ± 10.2 years) were included for analysis. Inter-reader agreement was good or excellent regarding MRI measurements and histopathological evaluation. No correlation between tumor ADC and tumor stiffness was found. Multivariable linear regression confirmed that cell density was the only factor associated with tumor ADC (Estimate = -0.03, p = 0.006), and tumor-stroma ratio was the only factor associated with tumor stiffness (Estimate = -0.18, p = 0.03). After adjustment for fibrosis stage (Estimate = 0.43, p < 0.001) and age (Estimate = 0.04, p < 0.001) in the multivariate linear regression, intra-tumoral CD8 + T cell infiltration remained a significant factor associated with peri-tumor stiffness (Estimate = 0.63, p = 0.02). Tumor ADC surpasses tumor stiffness as a biomarker of cellularity. Tumor stiffness is associated with tumor-stroma ratio and peri-tumor stiffness might be an imaging biomarker of intra-tumoral immune microenvironment. Tissue stiffness could potentially serve as an imaging biomarker of the intra-tumoral immune microenvironment of hepatocellular carcinoma and aid in patient selection for immunotherapy. Apparent diffusion coefficient reflects cellularity of hepatocellular carcinoma. Tumor stiffness reflects tumor-stroma ratio of hepatocellular carcinoma and is associated with tumor-infiltrating lymphocytes. Tumor and peri-tumor stiffness might serve as imaging biomarkers of intra-tumoral immune microenvironment.

Inflammatory markers predict survival in patients with postoperative urothelial carcinoma receiving tislelizumab (PD-1 inhibitor) adjuvant therapy

Background In the management of urothelial carcinoma, patient selection for immunotherapy, particularly with immune checkpoint inhibitors such as PD-1 (programmed cell death protein 1), is important for treatment efficacy. Inflammatory markers are useful for predicting treatment outcomes and immune-related adverse events (irAEs). This study aims to retrospectively explore the associations between inflammatory markers and outcomes in patients with postoperative urothelial carcinoma undergoing tislelizumab (PD-1 inhibitor) adjuvant therapy.Methods A retrospective analysis was conducted on 133 patients with postoperative urothelial carcinoma who received tislelizumab adjuvant therapy at the Affiliated Hospital of Xuzhou Medical University from April 2020 to August 2023. The prognostic effects of the neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and monocyte-to-lymphocyte ratio (MLR) on disease-free survival (DFS) and overall survival (OS) were assessed using Cox regression models. The correlation between inflammatory markers and the onset of irAEs was analyzed using logistic regression models.ResultsNLR < 5 and MLR < 0.31 were significantly associated with better outcomes compared to NLR >5 and MLR >0.31, respectively. Multivariate analysis revealed that an NLR < 5 was independently associated with better DFS and OS. However, there was no significant effect on the DFS and OS between PLR < 135 and PLR >135. Patients who experienced irAEs had longer DFS and OS. Multivariate analysis demonstrated that irAEs were an independent prognostic risk factor for DFS and OS. There was no significant difference in the occurrence of irAEs among different NLR, PLR, and MLR groups.ConclusionIn patients with postoperative urothelial carcinoma receiving tislelizumab adjuvant therapy, the assessment of NLR and MLR before treatment may serve as valuable predictive markers of clinical outcome.

Intra-tumoral T cells in pediatric brain tumors display clonal expansion and effector properties.

Brain tumors in children are a devastating disease in a high proportion of patients. Owing to inconsistent results in clinical trials in unstratified patients, the role of immunotherapy remains unclear. We performed an in-depth survey of the single-cell transcriptomes and clonal relationship of intra-tumoral T cells from children with brain tumors. Our results demonstrate that a large fraction of T cells in the tumor tissue are clonally expanded with the potential to recognize tumor antigens. Such clonally expanded T cells display enrichment of transcripts linked to effector function, tissue residency, immune checkpoints and signatures of neoantigen-specific T cells and immunotherapy response. We identify neoantigens in pediatric brain tumors and show that neoantigen-specific T cell gene signatures are linked to better survival outcomes. Notably, among the patients in our cohort, we observe substantial heterogeneity in the degree of clonal expansion and magnitude of T cell response. Our findings suggest that characterization of intra-tumoral T cell responses may enable selection of patients for immunotherapy, an approach that requires prospective validation in clinical trials.

Gustave Roussy immune score is a prognostic marker in patients with small cell lung cancer undergoing immunotherapy: a real-world retrospective study.

The utilization of the Gustave Roussy Immune Score (GRIm-Score) in patient selection for immunotherapy was initially reported. The objective of this retrospective study is to assess the potential of the GRIm-Score, a novel prognostic score based on nutritional and inflammatory markers, as a prognostic predictor in patients with small cell lung cancer (SCLC) undergoing immunotherapy. This retrospective study conducted at a single center included 159 patients with SCLC who received immunotherapy. The objective of the study was to investigate potential differences in overall survival (OS) and progression-free survival (PFS) among patients stratified by their GRIm-Score, utilizing the Kaplan-Meier survival analysis and the log-rank test. The final independent prognostic factors were identified through both propensity score matching (PSM) analysis and multivariable Cox proportional hazards regression analysis. Our analysis of the 159 patients revealed that there was a significant decrease in both OS and PFS with each increase in the GRIm-Score group, displaying a stepwise pattern. Moreover, even after conducting PSM analysis, the significant associations between the modified three-category risk scale-based GRIm-Score and survival outcomes remained significant. Both the total cohort and PSM cohort were subjected to multivariable analysis, which demonstrated that the three-category risk assessment-based GRIm-Score was a valuable predictor of both OS and PFS. In addition, the GRIm-Score may serve as a valuable and non-invasive prognostic predictor for SCLC patients undergoing PD1/PD-L1 immunotherapy.

Abstract 5445: Segmentation-free analysis of multiplexed images with unbiased spatial analytics and explainable AI for predicting disease outcomes

Abstract Background: The spatial intratumor heterogeneity (ITH) is widely acknowledged as driving therapeutic response and providing fuel for drug resistance. Currently, patient selection for immunotherapy is driven mostly by PD-1/PD-L1 based IHC tests and mutational analysis. These oversimplified approaches fail to predict the risk of recurrence, therapeutic response and drug resistance with high accuracy. We hypothesize that functional responses of heterogeneous non-random spatial arrangements of tumor, stromal and immune cells in the tumor microenvironment are determined by distinct combinations of their internal states and spatial interactions within neighborhoods. The interactions generate distinct cell-cell communication networks forming functional spatial configurations of cells, termed microdomains, as organizational units of spatial ITH. Deriving the spatial networks within each microdomain with unbiased spatial analytics and the underlying network biology through explainable AI, is key to understanding tumor initiation, tumor progression, and response to therapy. Problem: There has been an explosion of spatial imaging technologies using immunofluorescence and/or mass spectrometry for intact tissues measuring protein expressions, DNA and RNA probes. To extract high-value knowledge from these multiplexed datasets, a key first step is to segment cells accurately. Despite decades of research, this step remains elusive due to imaging artifacts arising from issues with tile stitching, incorrect registration, signal oversaturation, non-uniform illumination, poor or high-background nuclei signal, defocus, varying cell and nuclear shapes, fluorescence emission efficiency variation, overlapping and/or superimposed nuclei and low resolution (e.g., mass cytometry). Those artifacts may lead to incorrect cell phenotypes, incomplete cell phenotype atlases, and missing rare cell, fusion cell or transition cell types. Solution: We present TumorMapr™, a segmentation-free, unbiased spatial analytics and explainable AI platform that extracts information and creates knowledge from patient disease pathology samples imaged with any technology including fluorescence, mass spectrometry etc. Results: We apply TumorMapr on hyperplexed immunofluorescence dataset of colorectal cancer and hyperplexed imaging mass cytometry dataset of triple negative breast cancer to discover functional cell prototypes involving spatial collections of neighboring pixels highly predictive of disease progression and response to therapy, tumor promoting and tumor restraining microdomains and microdomain-specific network biology predictive of disease outcomes. We further compare these results with segmentation-based approaches to showcase the discovery of novel cell prototypes. Citation Format: Filippo Pullara, Brian Falkenstein, Bruce Campbell, Samantha Panakkal, Akif Burak Tosun, Jeffrey Fine, S. Chakra Chennubhotla. Segmentation-free analysis of multiplexed images with unbiased spatial analytics and explainable AI for predicting disease outcomes. [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 5445.

Baseline immune signature score of Tregs × HLA-DR+CD4+ T cells × PD1+CD8+ T cells predicts outcome to immunotherapy in cancer patients.

The use of immunotherapy (IT) is rapidly increasing across different tumor entities. PD-L1 expression is primarily used for therapy evaluation. The disadvantages of PD-L1 status are spatial and temporal heterogeneity as well as tumor type-dependent variation of predictive value. To optimize patient selection for IT, new prediction markers for therapy success are needed. Based on the systemic efficacy of IT, we dissected the immune signature of peripheral blood as an easily accessible predictive biomarker for therapeutic success. We conducted a retrospective clinical study of 62 cancer patients treated with IT. We assessed peripheral immune cell counts before the start of IT via flow cytometry. The predictive value for therapy response of developed immune signature scores was tested by ROC curve analyses and scores were correlated with time to progression (TTP). High score values of "Tregs ÷ (CD4+/CD8+ ratio)" (Score A) and high score values of "Tregs × HLA-DR+CD4+ T cells × PD1+CD8+ T cells" (Score B) significantly correlated with response at first staging (p = 0.001; p < 0.001). At the optimal cutoff point, Score A correctly predicted 79.1% and Score B correctly predicted 89.3% of the staging results (sensitivity: 86.2%, 90.0%; specificity: 64.3%, 87.5%). A high Score A and Score B statistically correlated with prolonged median TTP (6.13 vs. 2.17 months, p = 0.025; 6.43 vs. 1.83 months, p = 0.016). Cox regression analyses for TTP showed a risk reduction of 55.7% (HR = 0.44, p = 0.029) for Score A and an adjusted risk reduction of 73.2% (HR = 0.27, p = 0.016) for Score B. The two identified immune signature scores showed high predictive value for therapy response as well as for prolonged TTP in a pan-cancer patient population. Our scores are easy to determine by using peripheral blood and flow cytometry, apply to different cancer entities, and allow an outcome prediction before the start of IT.

Integrative Genomic Tests in Clinical Oncology.

Many clinical decisions in oncology practice rely on the presence or absence of an alteration in a single genetic locus, be it a pathogenic variant in a hereditary cancer gene or activating mutation in a drug target. In addition, there are integrative tests that produce continuous variables and evaluate complex characteristics of the entire tumor genome. Microsatellite instability (MSI) analysis identifies tumors with the accumulation of mutations in short repetitive nucleotide sequences. This procedure is utilized in Lynch syndrome diagnostic pipelines and for the selection of patients for immunotherapy. MSI analysis is well-established for colorectal malignancies, but its applications in other cancer types lack standardization and require additional research. Homologous repair deficiency (HRD) indicates tumor sensitivity to PARP inhibitors and some cytotoxic drugs. HRD-related "genomic scars" are manifested by a characteristic pattern of allelic imbalances, accumulation of deletions with flanking homology, and specific mutation signatures. The detection of the genetic consequences of HRD is particularly sophisticated and expensive, as it involves either whole genome sequencing (WGS) or the utilization of large next-generation sequencing (NGS) panels. Tumor mutation burden (TMB) can be determined by whole exome sequencing (WES) or middle-throughput NGS multigene testing. Although TMB is regarded as an agnostic indicator of tumor sensitivity to immunotherapy, the clinical utility of this test is proven only for a few cancer types.

KRAS mutation predict response and outcome in advanced non-small cell lung carcinoma without driver alterations receiving PD-1 blockade immunotherapy combined with platinum-based chemotherapy: a retrospective cohort study from China.

The selection of patients for immunotherapy remains challenging given the lack of highly specific and highly sensitive biomarkers. Kirsten rat sarcoma (KRAS) mutation is the most frequent molecular alteration found in advanced non-small cell lung carcinoma (NSCLC). We explored whether KRAS mutation status predicted the effects of first-line immune checkpoint inhibitor (ICI) treatment and platinum-based chemotherapy in Chinese patients with advanced NSCLC. Clinical data were extracted from medical records of patients with advanced NSCLC at the First Affiliated Hospital of Guangzhou Medical University in China between January 2019 and March 2020. Overall survival (OS) and progression-free survival (PFS) rates were compared via log-rank tests, and independent prognostic factors were identified via Cox regression. Patients with advanced NSCLC without driver alterations who were treated with ICI and platinum-based chemotherapy (N=80) were identified, including 28.7% with KRAS mutations and 71.3% with non-KRAS mutations. Tumor programmed death-ligand 1 (PD-L1) expression was analyzed using a 1% cutoff, and 32.5% of patients were negative and 67.5% were positive. The median tumor mutational burden (TMB) was 7.29 mutations per megabase (muts/Mb) (range, 0.08-44.8 muts/Mb), with 32.5% of cases <5 muts/Mb and 67.5% ≥5 muts/Mb. The median PFS and OS for the entire cohort were 9.8 (95% CI: 9.1-10.5) and 17.6 (95% CI: 14.4-20.8) months, respectively. The 6-month PFS rate was 67.5% and the 1-year OS rate was 72.5%. Thirty-five patients survived until the last follow-up. TheOS and PFS of patients with KRAS mutations were significantly higher than those in the non-KRAS mutant group (P<0.05). The Cox multivariate analyses showed that brain metastasis [hazard ratio (HR) =0.232, 95% CI: 0.102-0.530; P=0.001], TMB (HR =5.675, 95% CI: 1.948-16.535; P=0.001), KRAS mutation (HR =2.552, 95% CI: 1.141-5.708; P=0.023) were independent predictors of OS in patients treated with ICIs and platinum-based chemotherapy. Liver metastasis (HR =0.344, 95% CI: 0.191-0.619; P<0.001) and KRAS/tumor protein p53 (TP53) co-mutation (HR =0.220, 95% CI: 0.067-0.725; P=0.013) were the prognostic factor for PFS of qualified patients. This work provides evidence that KRAS mutation in advanced NSCLC may be served as a potential predictive biomarker for immunotherapeutic efficacy.

Comparison of three validated PD-L1 immunohistochemical assays in urothelial carcinoma of the bladder: interchangeability and issues related to patient selection.

Different programmed cell death-ligand 1 (PD-L1) assays and scoring algorithms are being used in the evaluation of PD-L1 expression for the selection of patients for immunotherapy in specific settings of advanced urothelial carcinoma (UC). In this paper, we sought to investigate three approved assays (Ventana SP142 and SP263, and Dako 22C3) in UC with emphasis on implications for patient selection for atezolizumab/pembrolizumab as the first line of treatment. Tumors from 124 patients with invasive UC of the bladder were analyzed using tissue microarrays (TMA). Serial sections were stained with SP263 and SP142 on Ventana Benchmark Ultra and with 22C3 on Dako Autostainer Link 48. Stains were evaluated independently by two observers and scored using the combined positive score (CPS) and tumor infiltrating immune cells (IC) algorithms. Differences in proportions (DP), overall percent agreement (OPA), positive percent agreement (PPA), negative percent agreement (NPA), and Cohen κ were calculated for all comparable cases. Good overall concordance in analytic performance was observed for 22C3 and SP263 with both scoring algorithms; specifically, the highest OPA was observed between 22C3 and SP263 (89.6%) when using CPS. On the other hand, SP142 consistently showed lower positivity rates with high differences in proportions (DP) compared with 22C3 and SP263 with both CPS and IC, and with a low PPA, especially when using the CPS algorithm. In conclusion, 22C3 and SP263 assays show comparable analytical performance while SP142 shows divergent staining results, with important implications for the selection of patients for both pembrolizumab and atezolizumab.

A Quantitative Digital Analysis of Tissue Immune Components Reveals an Immunosuppressive and Anergic Immune Response with Relevant Prognostic Significance in Glioblastoma.

Objectives: Immunostimulatory therapies using immune checkpoint blockers show clinical activity in a subset of glioblastoma (GBM) patients. Several inhibitory mechanisms play a relevant role in the immune response to GBM. With the objective of analyzing the tumor immune microenvironment and its clinical significance, we quantified several relevant immune biomarkers. Design: We studied 76 primary (non-recurrent) GBMs with sufficient clinical follow-up, including a subgroup of patients treated with a dendritic cell vaccine. The IDH-mutation, EGFR-amplification, and MGMT methylation statuses were determined. Several relevant immune biomarkers, including CD163, CD8, PD1, and PDL1, were quantified in representative selected areas by digital image analysis and semiquantitative evaluation. The percentage of each immune expression was calculated with respect to the total number of tumor cells. Results: All GBMs were wild-type IDH, with a subgroup of classical GBMs according to the EGFR amplification (44%). Morphologically, CD163 immunostained microglia and intratumor clusters of macrophages were observed. A significant direct correlation was found between the expression of CD8 and the mechanisms of lymphocyte immunosuppression, in such a way that higher values of CD8 were directly associated with higher values of CD163 (p < 0.001), PDL1 (0.026), and PD1 (0.007). In a multivariate analysis, high expressions of CD8+ (HR = 2.05, 95%CI (1.02–4.13), p = 0.034) and CD163+ cells (HR 2.50, 95%CI (1.29–4.85), p = 0.007), were associated with shorter survival durations. The expression of immune biomarkers was higher in the non-classical (non-EGFR amplified tumors) GBMs. Other relevant prognostic factors were age, receipt of the dendritic cell vaccine, and MGMT methylation status. Conclusions: In accordance with the inverse correlation between CD8 and survival and the direct correlation between effector cells and CD163 macrophages and immune-checkpoint expression, we postulate that CD8 infiltration could be placed in a state of anergy or lymphocytic inefficient activity. Furthermore, the significant inverse correlation between CD163 tissue concentration and survival explains the relevance of this type of immune cell when creating a strong immunosuppressive environment. This information may potentially be used to support the selection of patients for immunotherapy.

Abstract 6142: Gain-of-function p53R172H mutation drives accumulation of neutrophils in the pancreatic tumor microenvironment promoting resistance to immunotherapy

Abstract The immune microenvironment plays a critical role in cancer development, progression and resistance to therapy. Specific tumor genotypes can profoundly influence the composition of the immune microenvironment. After oncogenic KRAS, TP53 is the most frequently mutated gene in pancreatic ductal adenocarcinoma (PDAC). KRASG12D mutations in tumor cells are known to modify the immune landscape of PDAC, however the immune effects of neomorphic TP53 mutations have not been defined. Focusing on the most common neomorphic TP53 mutation in human PDAC, we sought to evaluate the non-cell-autonomous role of gain-of-function mutant Trp53R172H in modulating the immune microenvironment of pancreatic tumors by using KrasG12D-mutated mouse pancreatic ductal epithelial cells (PDEC) genetically engineered to express the Trp53R172H gain-of-function. Orthotopically implanted tumors derived from CRISPR-generated KrasG12D/+;Trp53R172H/+ PDEC had a distinct immune profile in comparison to KrasG12D/+;Trp53+/+ tumors, characterized by an influx of intratumoral CD11b+Ly6G+ neutrophils and concomitant decreases in CD3+ T cells, CD8+ T cells, and CD4+ T helper 1 (Th1) cells. Analysis of publicly available human PDAC cohorts revealed enrichment of genes in neutrophil-related pathways in TP53-mutated tumors. Knockdown of CXCL2, a neutrophil chemoattractant, in the tumor epithelial compartment of CRISPR KrasG12D/+;Trp53R172H/+ PDEC tumors reversed the neutrophil phenotype. Depleting neutrophils in mice bearing CRISPR KrasG12D/+;Trp53R172H/+ PDEC tumors augmented sensitivity to combined CD40 immunotherapy and chemotherapy. Collectively, these data link gain-of-function mutant Trp53R172H to the presence of intratumoral neutrophils in pancreatic cancer and suggests that tumor genotypes could inform patient selection for immunotherapy. Citation Format: Despina Siolas, Emily Vucic, Emma Kurz, Cristina Hajdu, Dafna Bar-Sagi. Gain-of-function p53R172H mutation drives accumulation of neutrophils in the pancreatic tumor microenvironment promoting resistance to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6142.

Prevalence of Epstein-Barr Virus Infection and Mismatch Repair Protein Deficiency and the Correlation of Immune Markers in Tibetan Patients with Gastric Cancer.

Background Gastric cancer (GC) is a major cause of cancer-related death in China. Immunotherapies based on PD-1/PD-L1 inhibitors have improved the survival of some patients with GC. Epstein–Barr virus (EBV) infection, mismatch repair (MMR) deficiency, and tumor immune microenvironment (TIME) markers (such as CD3, CD8, and PD-L1) may help to identify specific patients who will respond to PD-1/PD-L1 inhibitors. Considering racial heterogeneity, the pattern of TIME markers in Tibetan patients with GC is still unclear. We aimed to identify the prevalence of EBV infection and the MMR status and their association with immune markers in Tibetan GC to aid in patient selection for immunotherapy. Materials and Methods From 2001 to 2015, we retrospectively collected 120 tissue samples from consecutive Tibetan GC patients and constructed tissue microarrays. EBV infection was assessed by Epstein–Barr-encoded RNA (EBER) in situ hybridization, and MMR protein levels were measured. Immune markers (including CD3 and CD8) in intraepithelial, stromal, and total areas were detected by immunohistochemistry (IHC). PD-L1 expression was assessed by the combined positive score (CPS). We also analyzed the relationships of EBV infection and MMR status with immune markers. Results Of the 120 samples, 11 (9.17%) were EBV positive (+), and 6 (5%) were MMR deficient (dMMR). PD-L1 CPS ≥1% was found in 32.5% (39/120) of Tibetan GC patients. EBV infection was associated with higher numbers of CD3+ T cells (P < 0.05) and CD8+ T cells (P < 0.05) and higher PD-L1 expression (P < 0.05). For the limited number of dMMR patients, no significant relationship was observed between dMMR and TIME markers (P > 0.05). Conclusions In Tibetan GC patients, the rates of EBV infection, dMMR, and positive PD-L1 expression were 9.17%, 5%, and 32.5%, respectively. EBV infection was associated with the numbers of CD3+ T cells and CD8+ T cells and PD-L1 expression within the tumor. These markers may guide the selection of Tibetan GC patients for immunotherapy.

Comparison of 3 PCR-based assays for microsatellite instability detection in formalin-fixed paraffin-embedded tissues of patients with colorectal cancer.

e15042 Background: Routine assessment of microsatellite instability (MSI) status in colorectal cancer (CRC) patients can help with initial screening for Lynch syndrome, prediction of tumor prognosis and selection of patients for immunotherapy. Immunohistochemistry (IHC) and PCR-based approaches represent the current clinical laboratory standard for the evaluation of MSI status. Both Bethesda and Promega marker panels have been used widely in the world and the Bethesda panel and the six mononucleotides panel (NR21/BAT25/NR24/NR27/MONO27/BAT26) have been approved by the National Medical Products Administration (NMPA) in China. The Idylla MSI Test, a fully automated system based on real-time PCR, which incorporates 7 novel MSI loci (ACVR2A/BTBD7/DIDO1/MRE11/RYR3/SEC31A/SULF2) can perform MSI analysis within 150min using 1-3 tissue slides. The aim of this study was to evaluate the performance of the three 3 PCR-based assays for MSI detection including Idylla MSI Test, Bethesda and that six mononucleotides panel. Methods: One hundred and fifty-three formalin-fixed paraffin-embedded (FFPE) tumor tissues from patients with CRC were collected and their MSI status were analyzed using 3 PCR-based assays, respectively, i.e. the Idylla MSI, “2B3D” NCI panel and the six mononucleotides panel. All samples were previously evaluated by IHC method. Results: In the all 153 patients, IHC identified 27 cases to be MMR-deficient (dMMR), and 126 to be MMR-proficient (pMMR). Using IHC as a reference, the Idylla MSI had a higher consistency that showed overall agreement, sensitivity and specificity as 93.46%, 92.89% and 93.65%, respectively, while the corresponding values of the six mononucleotides panel were 84.31%, 92.59% and 82.54% respectively, and the “2B3D” NCI panel showed a sensitivity of 92.16%, a specificity of 88.89% and an overall concordance of 92.86%. The PCR-based approaches identified 11 more MSI-H cases than IHC while 22 dMMR cases identified by IHC were detected by all the 3 assays, 2 dMMR by 2 assays, 1 dMMR by one assay only and 1 dMMR by neither assays above. Moreover, the Idylla MSI showed advantages in ease of use, turnaround time (TAT) and small dose of tissue input. Conclusions: Our findings support that the Idylla MSI assay provides a rapid, reliable, and ease-to-use solution to MSI detection in Chinese CRC patients with high sensitivity and specificity. The inconsistent cases between IHC and PCR-based approaches need a further analysis.

A Glycolysis-Related Gene Signature Correlates With the Characteristics of the Tumor Immune Microenvironment and Predicts Prognosis in Patients With Hepatocellular Carcinoma.

Aim: To develop a glycolysis-related gene signature that correlated with the characteristics of the tumor immune microenvironment and had good predictive power for overall survival (OS) in hepatocellular carcinoma (HCC). Methods: Gene expression profiles, RNA sequencing data, clinical characteristics and survival information for 407 patients with HCC and 58 healthy controls were downloaded from the TCGA database. GSEA 4.1.0 software was used to evaluate the glycolysis-related pathways enriched in HCC compared to normal liver tissue. Univariate Cox, Least Absolute Shrinkage, Selection Operator, and two-step multivariate Cox analyses were used to construct a glycolysis-related gene signature for prognostic prediction. The glycolysis-related gene signature was combined with clinical characteristics to generate a nomogram. Tumor-infiltrating immune cell profiles and PD-L1 protein expression in HCC tissues were investigated. Results: The gene expression profiles of HCC tissues were enriched in glycolysis-related pathways. A glycolysis-related gene signature was used to categorize patients as high-risk or low-risk, where high-risk patients had significantly worse OS. Receiver operating characteristic curves confirmed the predictive capability of the glycolysis-related gene signature for OS (AUC >0.80). There was a significant difference in M0 macrophage (p = 0.017), dendritic cell (p = 0.043), B cell (p = 0.0018), CD4 T cell (p = 0.003), Treg (p = 0.01) and mast cell (p = 0.02) content and PD-L1 protein expression (p = 0.019) between HCC tissues in patients in the high-risk and low-risk groups. Conclusion: We established a glycolysis-related gene signature for OS in HCC that was predictive in training and test TCGA cohorts and correlated with the characteristics of the HCC tumor immune microenvironment. The glycolysis-related gene signature may guide clinical decision-making concerning patient selection for immunotherapy in HCC.

Prognostic Value of PD-L1 Immunohistochemical Marker in Gastric Carcinoma and Its Correlation with HER2 Status.

Objective:Programmed death-ligand 1 (PD-L1) and human epidermal growth factor receptor 2 (HER2) are currently considered as prognostic markers and therapeutic targets in many human cancers. This study aims to evaluate immunohistochemical (IHC) expression of PD-L1 in gastric cancer (GC) and explore its prognostic role in terms of association with HER2 expression, different clinico-pathological variables, in particular density and cluster designation (CD)8 positivity in tumor infiltrating lymphocytes (TILs) and with patients’ disease-free and overall survival (DFS, OS). Methods:This retrospective cohort study included 111 diagnosed primary GC patients who underwent surgical resection at the Gastrointestinal Surgery Center (GISC), Faculty of Medicine, Mansoura University, Egypt. After demographic, clinicopathological and survival data collection, histopathological evaluation was done for GC typing, staging and assessment of the histopathological prognostic parameters. IHC was performed for PD-L1, HER2 and CD8. PDL-1 was scored using the Combined Positive Score (CPS). Results: PD-L1 was expressed in 43.2% of GCs at a CPS cut-off value ≥ 1. PDL-1 positivity was significantly associated with high TILs and CD8+ TILs (p=0.008, 0.016 respectively), indicating its contribution to tumor microenvironment along with the TILs. Multivariate analysis spotted PD-L1 positivity as an independent prognostic predictor for shorter OS in GC (p=0.013), with a tendency toward shorter DFS. Only 9.9% GCs were HER2 positive (score +3) with no significant association with PD-L1. Conclusion:PDL-1 is a promising prognostic and therapeutic target in GC that may direct the selection of patients for immunotherapy and checkpoint-blockade (pembrolizumab) therapy.

Inclusion of Immunotherapy in the Management of Primary and Metastatic Malignant Tumours of the Kidney Including Adult Renal Cell Carcinoma: A Review and Update

Inclusion of Immunotherapy in the Management of Primary and Metastatic Malignant Tumours of the Kidney Including Adult Renal Cell Carcinoma: A Review and Update

Common DNA methylation changes in biliary tract cancers identify subtypes with different immune characteristics and clinical outcomes

BackgroundDNA methylation-associated studies on biliary tract cancer (BTC), including cholangiocarcinoma (CCA) and gallbladder cancer (GBC), may improve the BTC classification scheme. We proposed to identify the shared methylation changes of BTCs and investigate their associations with genomic aberrations, immune characteristics, and survival outcomes.MethodsMulti-dimensional data concerning mutation, DNA methylation, immune-related features, and clinical data of 57 CCAs and 48 GBCs from Eastern Hepatobiliary Surgery Hospital (EHSH) and 36 CCAs in the TCGA-CHOL cohort were analyzed.ResultsIn our cohort including 24 intrahepatic CCAs (iCCAs), 20 perihilar CCAs (pCCAs), 13 distal CCAs (dCCAs), and 48 GBCs, 3369 common differentially methylated regions (DMRs) were identified by comparing tumor and non-tumor samples. A lower level of methylation changes of these common DMRs was associated with fewer copy number variations, fewer mutational burden, and remarkably longer overall survival (OS, hazard ratio [HR] = 0.07, 95% confidence interval [CI] 0.01–0.65, P = 0.017). Additionally, a 12-marker model was developed and validated for prognostication after curative surgery (HR = 0.21, 95% CI 0.10–0.43, P < 0.001), which exhibited undifferentiated prognostic effects in subgroups defined by anatomic location (iCCAs, d/pCCAs, GBCs), TNM stage, and tumor purity. Its prognostic utility remained significant in multivariable analysis (HR = 0.26, 95% CI 0.11–0.59, P = 0.001). Moreover, the BTCs with minimal methylation changes exhibited higher immune-related signatures, infiltration of CD8+ lymphocytes, and programmed death-ligand 1 (PD-L1) expression, indicating an inflamed tumor immune microenvironment (TIME) with PD-L1 expression elicited by immune attack, potentially suggesting better immunotherapy efficacy.ConclusionsIn BTCs, DNA methylation is a powerful tool for molecular classification, serving as a robust indicator of genomic aberrations, survival outcomes, and tumor immune microenvironment. Our integrative analysis provides insights into the prognostication after curative surgery and patient selection for immunotherapy.