Patient Selection For Immunotherapy Research Articles

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.

PD-L1 expression in muscle invasive urothelial carcinoma: Comparison of SP142 and SP263 assay.

High-grade urothelial carcinoma has a different molecular pathway than superficial low grade urothelial carcinoma, and is characterized by genomic instability. The high tumor mutation burden leads to neoantigen formation, evoking an immune response. The immune response has been keenly studied in last two decades and programmed death ligand-1 (PDL-1) has emerged as acceptable immunohistochemical marker for assessment of response to therapy, prognostication and patient selection for immunotherapy. The targeting of PD-1 and PDL-1 by checkpoint inhibitors (CPIs) is an attractive strategy to unblock the inhibitor and induce cytotoxic cell death. However, the presence of complementary and companion diagnostic testing with multiple PDL-1 assays and platforms for various CPIs make a diagnostic quagmire. Thus, it is the need of hour to harmonize these assays. In this undertaken study we evaluated the concordance in PD-L1 expression between the two PD-L1 clones: SP263 and SP142, in treatment naïve muscle invasive bladder cancer (MIBC). We evaluated Ventana PD-L1 "SP263 and SP142" qualitative immunohistochemical assay using rabbit monoclonal anti-PD-L1 clones in evaluation of PDL-1 immunoexpression on Ventana autostainer platform. The study includes 30 muscle invasive urothelial carcinomas, with 10 of 30 having nodal metastasis. SP263 assay was statistically more sensitive than SP142 for tumor cell (TC) scoring (P = 0.0009), whereas SP142 was more sensitive for immune cell (IC) scoring (P = 0.0067). There was no statistical significant discordance for TC or IC scoring between primary tumor and metastatic lymph node. PD-L1 testing status can be done on both primary tumor and metastatic site, however in metachronous metastatic setting, testing on recent metastatic site should be preferred. The harmonization of immunoexpression between 2 PD-L1 clones could not be achieved.

M6A Modification Patterns Identify a Subset of Follicular Lymphoma Harboring an Exhausted Tumor Microenvironment

Background: Epigenetic dysregulation is important for the pathogenesis of follicular lymphoma (FL). N6-methyladenosine (m6A) is the most common post-transcriptional modification of mRNA. The m6A modification affects a wide range of cellular processes, including RNA stability, export, splicing or translation, and is extensively involved in the development and progression of multiple non-hematological and hematological malignancies. However, the role of m6A modification in FL remain largely unknown.Methods: We comprehensively evaluated the m6A modification patterns of 351 FL patients from three cohorts based on 16 m6A regulators and constructed the m6A gene signature the by unsupervised clustering analysis. The m6A score was then established using principal component analysis. The association between m6A modification patterns and prognostic implications, biological processes, and microenvironment characterizations in FL were further investigated.Results: A total of 16 m6A regulators were finally identified in this study, including 8 writers, 7 readers and one eraser. Two distinct m6A modification patterns, termed as m6A cluster A and m6A cluster B respectively, were identified based on the similarity displayed by the expression levels of 16 m6A regulators and the proportion of ambiguous clustering measure. Patients with m6A cluster A had significantly poorer survival than those with m6A cluster B (p=0.0085) and showed enrichment in cell cycle, DNA replication and mismatch repair biological processes. However, patients with m6A cluster B were markedly enriched in MAPK pathway, WNT pathway, mTOR pathway, natural killer cell mediated cototoxicity, cytokine-cytokine receptor interaction and T-cell receptor pathway. Additionally, myeloid dendritic cell and CD8 + T cell were significantly enriched in m6A cluster A, nevertheless CD4 + T cell was abundant in m6A cluster B. Base on the DEGs between m6A cluster A and m6A cluster B, a m6A gene signature was further constructed, namely m6A gene signature A and m6A gene signature B. Patients with m6A gene signature B experienced worse outcome than those with m6A gene signature A. Simultaneously, a m6A scoring model was also established to quantify the m6A modification pattern of individual patients with FL. Patients with low m6A score presented a prominent inferior survival than those with high m6A score (p< 0.0001). The median survival times for patients with low and high m6A score were 8.84 (95% confidence interval [CI]: 7.251-10.429) and 15.73 years (95% CI: 11.729-19.731), respectively. High m6A score group was remarkably riched in macrophage cell and CD4 + T cell infiltration, while low m6A score group exhibited significantly increased myeloid dendritic cell and CD8 + T cell infiltration, suggesting that m6A score could distinguish populations with distinct immune cell infiltration characteristics. GSVA analysis showed that the expression of genes involved in the immune and inflammatory response was significantly upregulated in low m6A score group. But we finally found that the CD8 + T cells in low m6A score group kept in a state of exhaustion, leading to that exhausted T cells were enriched in low m6A score group. The expression of PD-1 and PD-L1 in low m6A score group was also higher than those in high m6A score group. GSEA analysis showed that low m6A score group exhibited an enhanced IFN-γ response. Finally, we verified the value of the m6A score for predicting response to anti-PD-L1 antibody in external immunotherapy cohort (IMvigor210), and found that patients with low m6A score exhibited significantly clinical benefits and a markedly prolonged survival from anti-PD-L1 therapy.Conclusions: This is the first time to comprehensively investigate the role of m6A modification in FL. The m6A score identifies a subset of follicular lymphoma harboring an exhausted tumor microenvironment and helps for the selection of patients for immunotherapy, guiding the personalized immunotherapy in FL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Radiosensitivity, Immune Activation, and Mutational Frequency of Melanoma Metastases Across Tissue Types

<h3>Purpose/Objective(s)</h3> Melanoma is a more radioresistant and immunogenic histology, associated with a higher mutational burden. However, the influence of metastasis tissue location is unknown. We hypothesize exploiting these differences may allow therapy personalization. <h3>Materials/Methods</h3> We identified metastatic melanoma samples across 6 tissues (brain, liver, lung, lymph node [LN], skin, soft tissue [ST]), which were profiled for gene expression by microarray chips. Intrinsic radiosensitivity was estimated by the radiosensitivity index (RSI), a rank-based gene expression signature, where a higher RSI (scale 0-1) indicates greater radioresistance, with a cut point of 0.375 in other histologies. Degree of inflammation in the microenvironment was calculated by an existing 12-gene chemokine signature (12CK) where a higher 12CK score indicates greater inflammation. A subset (n = 94) underwent targeted exome sequencing (1,327 cancer-associated genes) and somatic mutations (MF) were enriched after filtering known germline/silent mutations and artifacts. Differences by tissue were analyzed with the Kruskal-Wallis H test. Correlations between RSI, 12CK, and MF were tested using Spearman's rho. <h3>Results</h3> From 1995 to 2011, 337 metastatic samples were identified from unique patients. Median age at diagnosis was 61 (range 20-97). With a median follow-up of 162 months (95% CI 142.6-181.4), median survival was 59.8 months (95% CI 49.2-70.4). Median values for RSI, 12CK, and MB were 0.451 (interquartile range [IQR] 0.331-0.552), 8.700 (IQR 7.218-9.746), and 101.5 (IQR 73.75-137), respectively. Significant differences existed among tissues for both RSI (<i>P</i> = 0.003) and 12CK (<i>P</i> < 0.001), but not MF (<i>P</i> = 0.498) (Table 1). No pair-wise comparisons were significant after Bonferroni correction for RSI, but for 12CK, Brain-ST (<i>P</i> = 0.044), Brain-Lung (<i>P</i> = 0.013), Brain-LN (<i>P</i> = 0.000), and Liver-LN (<i>P</i> = 0.023) were significant. Correlation between RSI and 12CK was significant (rho = -0.661, <i>P</i> < 0.001), but correlations between MB and RSI/12CK were not significant. <h3>Conclusion</h3> In this novel composite genomic analysis of melanoma metastases, all were relatively radioresistant, but higher RSI values for liver and skin metastases may warrant radiotherapy dose escalation. Similarly, the lower inflammatory scores of liver and brain metastases may have implications for selection of patients for immunotherapy. More inflamed tumors may be more responsive to radiation. In contrast to current homogenous approaches to metastases, these results indicate personalized approaches to types/sequencing of therapy for melanoma metastases by tissue type may be warranted.

Liquid Biopsy for Biomarker Testing in Non-Small Cell Lung Cancer: A European Perspective

The development of targeted therapies has improved survival rates for patients with advanced non-small cell lung cancer (NSCLC). However, tissue biopsy is unfeasible or inadequate in many patients, limiting biomarker testing and access to targeted therapies. The increasing numbers of established and emerging biomarkers with available targeted treatments highlights the challenges associated with sequential single-gene testing and limited tissue availability. Multiplex next-generation sequencing (NGS) offers an attractive alternative and represents a logical next step, and in cases where the tumour is inaccessible, tissue biopsy yields insufficient tumour content, or when the patient’s performance status does not allow a tissue biopsy, liquid biopsy can provide valuable material for molecular diagnosis. Here, we explore the role of liquid biopsy (i.e., circulating cell-free DNA analysis) in Europe. Liquid biopsies could be used as a complementary approach to increase rates of molecular diagnosis, with the ultimate aim of improving patient access to appropriate targeted therapies. Expert opinion is also provided on potential future applications of liquid biopsy in NSCLC, including for cancer prevention, detection of early stage and minimum residual disease, monitoring of response to therapy, selection of patients for immunotherapy, and monitoring of tumour evolution to enable optimal adaptation/combination of drug therapies.

DNA Methylation of Biliary Tract Cancer Identifies Subtypes with Different Immune Characteristics and Clinical Outcomes

DNA methylation-associated studies on biliary tract cancer (BTC), including cholangiocarcinoma (CCA) and gallbladder cancer (GBC), may improve the BTC classification scheme. In our cohort including 57 CCAs and 48 GBCs, lower level of methylation changes were associated with fewer copy number variations, fewer mutations of cell cycle pathway, CCND/E1, FGF/FGFR family, and NOTCH pathway, and remarkably longer overall survival (OS, HR=0.07, 95% CI 0.01-0.65, P =0.017). Additionally, we constructed a 12-feature prognostic prediction model that effectively predicted OS (HR=0.21, 95% CI 0.10-0.43, P <0.001). Moreover, the BTCs with minimal methylation changes exhibited higher immune-related signatures, infiltration of CD8+ lymphocytes, and PD-L1 expression, indicating an inflamed tumor immune microenvironment (TIME) with PD-L1 expression elicited by immune attack, potentially suggesting better immunotherapy efficacy. In 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 prognostication after curative surgery and patient selection for immunotherapy. Funding Statement: This work was supported by the Innovation Group Project of Shanghai Municipal Health Commission (2019CXJQ03) and the Shanghai Municipal Health Commission (202040045). Declaration of Interests: Y. Xu, G. Wang, C. Li, Y. Zhang, J. Zhao, C. Wang, X. Wen, Z. Zhang, B. Li, H. Zhang, Z. Zhang, and S. Cai are employees of Burning Rock Biotech. Other authors declare no potential conflicts of interest. Ethics Approval Statement: All collection and usage of human samples and clinical data were in accordance with the principles of the Declaration of Helsinki and approved by the Ethics Committee of Eastern Hepatobiliary Surgery Hospital (EHBHKY2018-02-014). The written consents were received from all the participated patients.

Quantitative Analysis of Multiplexed Immunofluorescence in T-Cell Lymphoma

IntroductionImmunohistochemistry provides biologically and clinically useful information on protein expression and cellular localization in cancer cells, but quantification of the expression by visual scores on light microscopy is subjective and suffers from poor reproducibility. There is increasing need for multiplex immunohistochemistry to study the co-localization of antigens in cell of interest. However, interpretation and quantification of co-localizing signals using conventional IHC is challenging. Novel methods in automated microscopy using machine learning algorithms have been utilized in epithelial cancers to quantitate the signals from immunohistochemistry. However, these have not been well tested in lymphoid malignancies, where IHC plays a critical role in the diagnosis and classification. Here we describe the application of quantitative analysis of immunofluorescence in lymphomas, using an example of a T- cell lymphoma, which can be challenging in terms of diagnosis and treatment.MethodsWe developed a multiplexed panel of immunofluorescent stains including CD4, CD8 and CD20 (T and B-lymphocyte markers) along with T-follicular helper markers (TFH) such as BCL6 and PD1. These were optimized and validated using benign tonsillar tissue to demonstrate appropriate cellular localization of each of these markers. (Figure1.) We then show the applicability of such a multiplexed panel in Angioimmunoblastic T-cell Lymphoma (AITL), a peripheral T-cell lymphoma characterized by cellular heterogeneity in the tumor microenvironment which often impedes IHC interpretation and quantification (figure not shown)Results and DiscussionIn AITL, we generated a tissue map which allows visualization of the spatial relationship of the different lymphocyte subsets and the quantification of TFH markers in tumor and non-tumor cell populations. We demonstrate the feasibility of an automated and quantitative imaging of this multiplexed panel of stains in formalin-fixed paraffin embedded tissue sections, which allows the study of the distribution of tumor cells in relationship to surrounding immune cells and the quantification of the expression of different biomarkers of interest in tumor compared to non-tumor cells in the microenvironment. Quantitative multiplex immunofluorescence and/or immunohistochemistry is likely to improve diagnostic accuracy and reproducibility compared to visual scoring in conventional single marker IHC. Furthermore, the ability to quantify the expression of multiple immune markers in tumor and surrounding immune cells and correlation with treatment response will play a vital role in the selection of patients for immunotherapy (such as anti PD1 or anti CD30 antibodies) which is becoming an increasingly important modality in lymphoma treatment.Figure legend. (Figure 1)Multiplexed composite immunofluorescence of CD20, CD8, CD4 and BCL6 in benign tonsil tissue (A). Unmixed images of CD20 (B, green, cell membrane), CD8 (C, magenta, cell membrane), CD4 (D, yellow, cell membrane), BCL6 (E, red, nuclear) and CD4/BCL6 (F) demonstrating the normal distribution of B and T cells and localization of BCL6-positive cells within germinal centres. The number of CD4-positive (G, red), BCL6-positive (H, green) and CD4/BCL6 double positive cells (I, yellow) are obtained after the images are segmented and scored using the InForm 2.0 software. [Display omitted] DisclosuresChng:Janssen China R&D: Research Funding.

A Tumor Progression Related 7-Gene Signature Indicates Prognosis and Tumor Immune Characteristics of Gastric Cancer.

BackgroundGastric cancer is a common gastrointestinal malignancy. Since it is often diagnosed in the advanced stage, its mortality rate is high. Traditional therapies (such as continuous chemotherapy) are not satisfactory for advanced gastric cancer, but immunotherapy has shown great therapeutic potential. Gastric cancer has high molecular and phenotypic heterogeneity. New strategies for accurate prognostic evaluation and patient selection for immunotherapy are urgently needed.MethodsWeighted gene coexpression network analysis (WGCNA) was used to identify hub genes related to gastric cancer progression. Based on the hub genes, the samples were divided into two subtypes by consensus clustering analysis. After obtaining the differentially expressed genes between the subtypes, a gastric cancer risk model was constructed through univariate Cox regression, least absolute shrinkage and selection operator (LASSO) regression and multivariate Cox regression analysis. The differences in prognosis, clinical features, tumor microenvironment (TME) components and immune characteristics were compared between subtypes and risk groups, and the connectivity map (CMap) database was applied to identify potential treatments for high-risk patients.ResultsWGCNA and screening revealed nine hub genes closely related to gastric cancer progression. Unsupervised clustering according to hub gene expression grouped gastric cancer patients into two subtypes related to disease progression, and these patients showed significant differences in prognoses, TME immune and stromal scores, and suppressive immune checkpoint expression. Based on the different expression patterns between the subtypes, we constructed a gastric cancer risk model and divided patients into a high-risk group and a low-risk group based on the risk score. High-risk patients had a poorer prognosis, higher TME immune/stromal scores, higher inhibitory immune checkpoint expression, and more immune characteristics suitable for immunotherapy. Multivariate Cox regression analysis including the age, stage and risk score indicated that the risk score can be used as an independent prognostic factor for gastric cancer. On the basis of the risk score, we constructed a nomogram that relatively accurately predicts gastric cancer patient prognoses and screened potential drugs for high-risk patients.ConclusionsOur results suggest that the 7-gene signature related to tumor progression could predict the clinical prognosis and tumor immune characteristics of gastric cancer.

PD-L1 expression and immune cells in anaplastic carcinoma and poorly differentiated carcinoma of the human thyroid gland: A retrospective study.

Anaplastic thyroid carcinoma (ATC) and poorly differentiated thyroid carcinoma (PDTC) have limited treatment options, and immune profiling may help select patients for immunotherapy. The prevalence and relevance of programmed death-1 ligand (PD-L1) expression and the presence of immune cells in ATC and PDTC has not yet been well established. The present study investigated PD-L1 expression (clone 22C3) and cells in the tumor microenvironment (TME), including tumor-infiltrating lymphocytes (TILs), tumor-associated macrophages (TAMs) and dendritic cells, in whole tissue sections of 15 cases of ATC and 13 cases of PDTC. Immunohistochemical PD-L1 expression using a tumor proportion score (TPS) with a 1% cut-off was detected in 9/15 (60%) of ATC cases and 1/13 (7.7%) of PDTC cases (P=0.006). PD-L1 expression in TILs was limited to the ATC group (73.3 vs. 0% in ATC and PDTC, respectively). In the ATC group, the TPS for tumor positive PD-L1 expression revealed a non-significant trend towards worse survival, but no difference was observed when investigating PD-L1 expression in TILs and TAMs. In addition to increased PD-L1 expression, all ATC cases exhibited significantly increased CD3+ and CD8+ T cells, CD68+ and CD163+ macrophages, and S100+ dendritic cells compared with the PDTC cases. Loss of mutL homolog 1 and PMS1 homolog 2 expression was observed in one ATC case with the highest PD-L1 expression, as well as in the only PDTC case positive for PD-L1. Notably, the latter was the only PDTC case exhibiting positivity for p53 and a cellular microenvironment similar to ATC. The current results indicated that PD-L1 expression was frequent in ATC, but rare in PDTC. In addition to PD-L1, the present study suggested that microsatellite instability may serve a role in both the TME and the identification of immunotherapy candidates among patients with PDTC.

Abstract LT005: Tumor-intrinsic gain of function p53R172H mutation drives accumulation of neutrophils in the pancreatic tumor microenvironment that promotes resistance to immunotherapy

Abstract KRASG12D mutations in tumor cells can modify the immune microenvironment of pancreatic ductal adenocarcinoma (PDAC), though the effects of secondary genetic alterations have been unexplored. After oncogenic KRAS, TP53 is the most frequently mutated gene in PDAC, however the immune effects of neomorphic TP53 mutations have not been defined. We sought to evaluate the non-cell-autonomous role of mutant p53 in modulating the immune microenvironment of pancreatic cancer by comparing the immune profiles generated by KrasG12D-mutated mouse pancreatic ductal epithelial cells (PDEC) genetically engineered to express the Trp53R172H gain-of-function mutation to their p53 wildtype control. We found orthotopically implanted tumors derived from 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 KrasG12D;Trp53R172H PDEC tumors reversed the neutrophil phenotype. Depleting neutrophils in mice bearing KrasG12D;Trp53R172H PDEC tumors augmented sensitivity to combined CD40 immunotherapy and chemotherapy. Collectively, these data link mutant p53 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. Tumor-intrinsic gain of function p53R172H mutation drives accumulation of neutrophils in the pancreatic tumor microenvironment that promotes resistance to immunotherapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT005.

Abstract PO067: The immune cell infiltration and landscape predicts clinical outcomes in gynecologic cancers

Abstract Introduction: Immunotherapy has changed the standard of treatment for many cancers. However, only a small number of gynecologic cancer patients benefit from immunotherapy. The intratumoral immune landscapes are suggested as a predictor of the response to immunotherapies. However, there are no studies that provide a comprehensive immune characterization for gynecologic cancers. Aims: To characterize cellular compositions of the immune infiltrates and investigate if the immune landscape is a predictor for patient prognosis in gynecologic cancers. Methods: Clinical data for ovarian cancer, cervical cancer, and uterine cancer were downloaded via TCGA. RNA data are download from http://gdac.broadinstitute.org. Immune cell infiltration was analyzed using QuanTIseq and EPIC. Immune subtype clusters of patients were identified by Graph-based consensus clustering. Statistical analysis was conducted by GraphPad Prism. Results: Ovarian cancer had the highest percentage of total immune cells (approximately 21%, including CD4 T-Cells, Monocytes, CD8 T-Cells, Dendritic Cells, Macrophages, Neutrophils, NK Cells, B-Cells). Cervical cancer and uterine corpus endometrial carcinoma have lower percentages of immune cells with 17% and 16%, respectively. Furthermore, ovarian cancer had a significantly higher Monocyte and M2-liked Macrophage percentage, but lower percentage for CD8 T cells and Neutrophils compared to cervical cancer and uterine cancer. Cervical cancer had the highest percentage for M1-liked Macrophages and lowest CD4 T cells. Uterine cancer had the highest percentage of dendritic cells. In cervical cancer, higher cell infiltration of CD8 T-Cells and M2-liked Macrophages was associated with a better prognosis. In uterine cancer, patients with higher number of dendritic cells and CD8 T-Cells had significantly better clinical outcomes. However, higher CD4 T-cell infiltration was associated with poor prognosis in uterine cancer. Interestingly, patient’s survival was not affected by the infiltration of any individual immune cells which we analyzed in ovarian cancer. We identified and validated four immune subtypes associated with distinct immune cell infiltration in gynecologic cancers. Cervical and uterine cancer patients from an immune-desert subtype that had the least amount of lymphocyte infiltration and a high level of Monocyte had the worst prognosis. By contrast, Cervical and uterine cancer patients from an immune-warm subtype that had higher infiltration of CD8 T-cell, NK Cell, and dendritic cells had the best prognosis. However, the survival rate of ovarian cancer patients is similar among four different subtypes. Conclusion: Our study provides a conceptual framework to understand the tumor immune microenvironment of different gynecologic cancers. This work also suggests that the immune microenvironment should be considered for the design of combination treatment strategies and guiding the optimal selection of patients for immunotherapy. Citation Format: Wai Chung Chen, Tuo Hu, Chunbo He. The immune cell infiltration and landscape predicts clinical outcomes in gynecologic cancers [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO067.

Characterization of hyperprogressive disease in patients with advanced biliary tract cancer treated with anti-PD-1 inhibitor: A multicenter retrospective study.

339 Background: Immunotherapy, including anti-PD-1 inhibitor, represents a promising breakthrough treatment in poor prognostic cancers such as biliary tract cancer (BTC). However, subset of pts suffer from unexpected acceleration of tumor growth following the initiation of immunotherapy, termed hyperprogressive disease (HPD). We assessed HPD in BTC pts treated with anti-PD-1 inhibitor in association with clinicopathologic features. Methods: This retrospective study included pts with BTC who were treated with pembrolizumab 200mg IV as palliative second or third line treatment in 4 tertiary hospitals in South Korea. Previous proposed tumor dynamic parameters including time to treatment failure (TTF), tumor growth kinetics (TGK) and tumor growth rate (TGR) were calculated per RECIST v1.1. Neural network modeling technique was adopted to find clinicopathologic features that can predict HPD. Results: Total of 223 pts with BTC were treated with palliative second or third line pembrolizumab between December 2015 to August 2020. ORR was 11.2% (n=1 for CR, n=24 for PR, n=70 for SD, n=118 for PD, and 10 pts not evaluable). Among patients with best response as PD (n=118), 41 patients (18.4% from total pts) met the criteria for HPD definition (&gt;2-fold increase in both TGK and TGR in the experimental period compared to the reference period). HPD pts had worse prognosis compared to non-HPD pts in terms of TTF (median 1.4 vs 2.7 months, P =&lt;0.0001) and OS (median 3.57 vs 5.27 months, P =0.03). Clinicopathologic features including age, ECOG performance status, primary tumor site, pathology, metastatic organ, previous treatment history (surgery or radiotherapy), pretreatment PD-L1 score, tumor markers, and baseline laboratory results (LDH, albumin, or neutrophil-to-lymphocyte ratio) did not predict HPD (compared to non-HPD pts or PD without HPD pts). By neural network modeling and plotting with hidden vectors, we could define HPD-high-risk cluster (n=37) and HPD-lower-risk cluster (n=80). Higher CA 19-9-to-lymphocyte ratio ( P =0.0004) or higher platelet-to-PD-L1 CPS (combined positivity score) ratio ( P =0.002) predicted HPD-high-risk-cluster. HPD pts (n=41) tend to have progression due to liver (73.2% vs 45.6%, P =0.0026) or peritoneum (53.7% vs 21.4%, P=&lt;0.0001) metastases, compared to non-HPD pts (n=182). Conclusions: This study for the first time describe the clinicopathologic features of HPD among pembrolizumab treated BTC pts. Baseline pathology and laboratory tests may help optimal patient selection for immunotherapy to avoid HPD in BTC pts. Further validation of BTC-specific HPD predictive features and definition (TGK or TGR ratio cut-off) will be presented after comparison with clinicopathologic features among second line 5FU-based chemotherapy treated BTC pts.

Development and Validation of a Combined Ferroptosis and Immune Prognostic Classifier for Hepatocellular Carcinoma.

BackgroundImmunotherapy and sorafenib exert anti-tumor effects via ferroptosis, but reliable biomarkers for the individual treatment and prognosis prediction of hepatocellular carcinoma (HCC) based on the ferroptosis and immune status remain lacking.MethodsFerroptosis-related genes (FRGs) were identified by downloading data from FerrDb and by searching and reading original articles from PubMed. Immune-related genes (IRGs) were downloaded from ImmPort. Prognostic FRGs and IRGs in the GSE14520 (n = 220) and The Cancer Genome Atlas (TCGA, n = 365) datasets were identified. Least absolute shrinkage and selection operator (LASSO) Cox regression and multivariate Cox regression were used for model construction. Ferroptosis expression profiles, the infiltration of immune cells, and the somatic mutation status were analyzed and compared.ResultsTwenty-seven prognostic ferroptosis- and immune-related signatures were included to construct a comprehensive index of ferroptosis and immune status (CIFI). A subgroup of patients was identified as having a high CIFI value, which was associated with a worse prognosis. This subgroup of patients had significantly up-regulated expressions of many suppressors of ferroptosis and higher fractions of immunosuppressive cells, such as cancer-associated fibroblasts (CAFs) and myeloid-derived suppressor cells (MDSCs). Notably, somatic mutation analysis indicated that high-CIFI patients had higher levels of tumor heterogeneity and higher mutation frequencies of genes like TP53.ConclusionIn this work, a novel prognostic classifier was developed based on ferroptosis- and IRGs in HCC, and this classifier could be used for prognostic prediction and the selection of patients for immunotherapies and targeted therapies.

Abstract PR-005: Tumor intrinsic p53 mutation drives accumulation of neutrophils in the pancreatic tumor microenvironment promoting resistance to immunotherapy

Abstract Intratumoral genetic alterations can cause remodeling of local immune cell populations through distinct cellular mechanisms. After oncogenic KRAS, TP53 is the gene most frequently mutated in pancreatic ductal adenocarcinoma (PDAC). These mutations in TP53 are neomorphic and confer phenotypes distinct from those due to homozygous TP53 deletion. We sought to evaluate the non-cell-autonomous role of mutant p53 protein in the immune microenvironment by studying KrasG12D-mutated mouse pancreatic ductal epithelial cells (PDEC) engineered using CRISPR/Cas gene editing to express the Trp53R172H gain-of-function mutation. We found distinct immune profiles associated with orthotopically implanted tumors derived from KrasG12D;Trp53R172H PDEC, 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 KrasG12D;Trp53R172H PDEC tumors reversed the neutrophil phenotype. Depleting neutrophils in mice bearing KrasG12D;Trp53R172H PDEC tumors augmented sensitivity to combined CD40 immunotherapy and chemotherapy. Collectively, these data link mutant p53 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. Tumor intrinsic p53 mutation drives accumulation of neutrophils in the pancreatic tumor microenvironment promoting resistance to immunotherapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PR-005.

Mismatch Repair System Genomic Scars in Gastroesophageal Cancers: Biology and Clinical Testing

Alterations in the mismatch repair (MMR) system result in genomic instability, neoantigen production, and immune response in cancer. There is evidence that gastroesophageal tumors with MMR deficiency may be susceptible to immune-checkpoint inhibitors treatment, especially in those presenting at advanced-stage disease. Although a number of biomarkers have been developed in histology-agnostic settings to assess MMR status, there is evidence that a tumor-specific testing approach would improve the selection of patients for immunotherapy. However, no testing methods have been developed specifically for gastroesophageal cancers so far. Here, we discuss the state of the art, current advances, and future perspectives of MMR-related biomarkers’ biologic and clinical role in gastroesophageal cancers.

PREDICTIVE RESPONSE MARKERS FOR IMMUNE RESPONSE BLOCKS

Despite the unprecedented success in using immune checkpoint inhibitors in the treatment of lung cancer, melanoma, hypermutable tumors of various localization, etc., a significant proportion of patients receiving these drugs do not respond to treatment. Predictive markers routinely used in the selection of patients for immunotherapy, in particular, the level of expression of PD -L1 and the presence of microsatellite instability, have certain limitations. Over the past decade, many other biomarkers designed to predict response to immunotherapy have been proposed, namely: tymor mutation burden, composition of lymphocytic infiltrate; allelic composition of the major histocompatibility complex; relationship between the numbers of different formed elements of blood as well as between its biochemical parameters; microflora of the digestive tract, etc. These markers can directly or indirectly reflect the immunogenicity of the tumor itself, as well as the state of systemic and intratumoral immune response. The predictive power and reliability of these markers are extremely different. When preparing this review, we conducted a literature search for recent studies regarding predictors of efficacy for immune checkpoint inhibitors published in the journals included in the databases, such as Pubmed, Web of Science, and Scopus.

Digital Pathology and PD-L1 Testing in Non Small Cell Lung Cancer: A Workshop Record.

A meeting among expert pathologists was held in 2019 in Rome to verify the results of the previous harmonization efforts on the PD-L1 immunohistochemical testing by scoring a representative series of non-small cell lung cancer (NSCLC) digital slides. The current paper shows the results of this digital experimental meeting and the expertise achieved by the community of Italian pathologists. PD-L1 protein expression was determined using tumor proportion score (TPS), i.e., the percentage of viable tumor cells showing partial or complete membrane staining at any intensity. The gold standard was defined as the final PD-L1 score formulated by a panel of seven lung committed pathologists. PD-L1 status was clustered in three categories, namely negative (TPS < 1), low (TPS 1–49%), and high (TPS ≥ 50%). In 23 cases (71.9%) PD-L1 staining was performed using the companion diagnostic 22C3 pharmDx kit on Dako Autostainer, while in nine (28.1%) cases it was performed using the SP263 Ventana kit on BenchMark platform. A complete PD-L1 scoring agreement between the panel of experts and the participants was reached in 57.1% of cases, whereas a minor disagreement in 16.1% of cases was recorded. Italian pathologists performed best in strong positive cases (i.e., tumor proportion score TPS > 50%), whereas only 10.8% of disagreement with the gold standard was observed, and 55.6% regarded a single challenging case. The worst performance was achieved in the negative cases, with 32.0% disagreement. A significant difference resulted from the analysis of the data separated by the different clones used: 22.3% and 38.1% disagreement (p = 0.01) was found in the group of cases analyzed by 22C3 and SP263 antibody clones, respectively. In conclusion, this workshop record proposed the application of a digital pathology platform to share controversial cases in educational meetings as an alternative possibility for improving the interpretation and reporting of specific histological tools. Due to the crucial role of PD-L1 TPS for the selection of patients for immunotherapy, the identification of unconventional approaches as virtual slides to focus experiences and give more detailed practical verifications of the standard quality reached may be a considerable option.

PD-L1 and neutrophil to lymphocyte ratio (NLR) as predictive panel of prognosis in bladder cancer.

e17040 Background: PD-1/PD-L1 blockade significantly improved survival for bladder cancer. PD-L1 expression might not be an ideal marker for patient selection in isolation. Several studies demonstrated that alternative markers such as NLR, a biomarker of systemic inflammation response to therapy, correlated with outcomes in a variety of cancers including bladder cancer, might be a predictor for patient selection and the response to immunotherapy. However, no reporters have been made combination of NLR and PD-L1 in predicting prognosis in bladder cancer. Methods: Suitable tissue of 100 FFPE blocks of bladder cancer patients treated in Sun Yat-Sen Memorial Hospital Sun Yat-sen University were stained with PD-L1 antibody. Clinicopathological data and pretreatment complete blood count were retrospectively collected. All patients were classified into high NLR group and low NLR group at cut-off 3.0. Kaplan-Meier and Cox proportional hazard models analyses were used to assessed the predictor of combined PD-L1/NLR for disease-free survival (DFS) and overall survival (OS). Results: Patients were mostly male (79%) with high grade () lymph node positive (21%), T3-4 (33%) . The positivity of PD-L1 expression was 22% (22/100) at cut-off 1%. Univariate analysis showed that PD-L1 expression was positively associated with larger tumor size, higher grade, positive lymph node metastases. Median DFS and OS were 35 months and 37 months, respectively. Both PD-L1 expression and NLR were associated with DFS and OS. COX analysis model showed that PD-L1 and NLR were independent prognostic factors for tumor prognosis. Of interest, when patients were divided in two groups based on PD-L1/NLR: patients with PD-L1+/high NLR as group 1 and other patients as group 2, group1 had significantly shorter DFS and OS (DFS, X2 = 4.146, P = 0.042; OS, X2 = 10.274, P = 0.001). COX proportional hazards regression models indicated that PD-L1+/high NLR was correlated with a significantly shorter DFS and OS (DFS, hazard ration [ HR] = 2.572, P = 0.05; OS, HR = 3.811, P = 0.003). Conclusions: The study indicated that combined use of PD-L-1/ NLR as predictive biomarker for survival. This feasible panel may be optional maker applied to select patients for immunotherapy.

Immunofocus-PD-L1 high throughput quantitative next generation sequencing assay.

e13521 Background: Immunohistochemical (IHC) assays are presently used as the gold standard predictive tests for immunotherapy but are compromised due to a number of potential variables. Comparative studies have demonstrated differing levels of PD-L1 staining between assays which appears independent of the antibody binding epitope. Secondly, inter-reader reliability even between expert pathologists is problematic particularly for assessment of PD-L1 positive immune cell populations. Methods: To improve predictive testing for anti PD-L1/PD1 immunotherapies we have developed and validated a Next Generation Sequencing Platform, Immunofocus, able to perform high-throughput quantitative PD-L1 gene expression levels in routine diagnostic PWET biopsies. We applied Immunofocus to a cohort of 130 NSCLCs and compared PD-L1 gene expression levels with PD-L1 IHC scores generated using the VENTANA PD-L1 (SP142) Assay. The PD-L1 IHC assessment was carried out double blinded by an independent laboratory. PD-L1 IHC scores were calculated using an algorithm combining tumour proportion score (TPS) with a PD-L1 positive immune cell (IC) score and immune cell area. Results: An exceptionally high degree of correlation was observed between the NGS PD-L1 levels with the combined PD-L1 IHC scores (P &lt; 0.001). Therapeutic cut points for NGS PD-L1 levels were identified corresponding to PD-L1 IHC defined clinical cut points. Notably, ~20% of patients with negative PD-L1 IHC scores showed high NGS PD-L1 expression levels. We hypothesize that these cases represent false negatives and identify a cohort of patients who have shown significant response rates to anti-PD-L1/PD-directed immunotherapies. Conclusions: The Immunofocus NGS PD-L1 assay has potential to greatly improve patient selection for immunotherapy by removing the IHC assay variables and inter-reader variability which compromise current PD-L1 IHC tests while also providing standardized high throughput in the clinical setting. Immunofocus is able to integrate gene expression with somatic mutation analysis allowing capture of networks regulating the immune-checkpoint including for example adaptive and innate resistance pathways, JAK1/2 pathways, differential MHC expression, TEFF gene signature, neoantigen surrogates such as DDR defects and TMB. The integration of NGS PD-L1 expression with other putative biomarkers of response is presently ongoing to further improve prediction of response.

New laboratory parameters in the diagnosis of acute mesenteric ischemia.

Lack of clear clinical and laboratory picture of subjective evaluation of bowel viability, the progression of bowel necrosis in the postoperative period with acute mesenteric ischemia (АМI) contributes to the high mortality rate. Earlier experiments we proved that gut necrosis leads to changes in the subpopulation structure of blood lymphocytes. This prompted us to determine the clinical significance of the subpopulation structure of venous blood lymphocytes in patients with acute mesenteric ischemia. The paper is based on a retrospective analysis of the results of a controlled clinical and immunological examination of 18 patients aged 62 to 78 years (control group and a group of АМI). Evaluation lymphocyte subpopulation structure was performed by the standard method of direct immunofluorescence staining of whole blood. The obtained data were processed with nonparametric statistical methods. Study of lymphocyte subpopulation structure in patients with АМI patients showed a decrease in the absolute and relative number of CD8, CD4, B, NK cells on the indicators in the control group. Ischemia and necrosis of the intestinal mucosa accompanied by a massive translocation of intestinal microflora through the impaired intestinal barrier along with the migration of lymphocytes into the lesion and death, which is manifested in a decrease in the number of lymphocytes of the peripheral blood. Comprehensive assessment of venous blood lymphocyte subpopulation structure can be used as an additional diagnostic criterion necrotic step АМI, serve as criteria for selection of patients for immunotherapy.