Cell-inflamed Tumor Research Articles

Abstract 177: Targeting murine or human KLRG1 exerts potent anti-tumor efficacy involving both CD8T cells and NK cells

Abstract Clinical response to anti-PD-1 immunotherapy is correlated with patients who have a T cell-inflamed tumor microenvironment. Some patients with immune cell infiltration fail to respond, and some patients experiencing an initial clinical response develop acquired resistance. It is likely that additional immune-regulatory pathways need to be targeted to expand the efficacy of immunotherapies. To identify a broader array of therapeutic relevant immunotherapy molecules in T cell-inflamed tumors, our lab utilized gene expression profiling to characterize tumor antigen-specific dysfunctional CD8+ T cells in the tumor microenvironment. One molecule that was found to be overexpressed on these CD8+ T cells was C-type lectin inhibitory receptor, KLRG1. KLRG1 is an ITIM domain-containing receptor mostly explored on NK cells, and the known ligands are N- and E- cadherins which are broadly expressed in solid cancers. Analysis of single cell RNA-Seq data from tumor biopsies of melanoma patients classified as responders or non-responders to anti-PD-1 therapy revealed significantly higher expression of KLRG1 expression on tumor-infiltrating T cell subsets in non-responders, suggesting a possible salvage immune suppressive role. To determine the potential functional relevance of KLRG1 on tumor control, we generated KLRG1 knockout (KO) mice, then implanted tumors and assessed tumor growth rate in vivo. KLRG1 KO mice showed markedly slowed B16 melanoma tumor growth compared to WT mice, arguing it is a critical negative regulator of anti-tumor immunity. Both antigen-specific CD8+ T cells and NK cells were expanded in these tumors, and depletion of CD8+ T cells or NK cells eliminated this tumor control. Genetic deletion of N-cadherin from B16 cells also resulted in slowed tumor growth, suggesting that tumor cells are providing the functionally relevant ligand. To assess the degree of inhibitory function of human KLRG1, we generated a humanized KLRG1 knock-in mouse model. We confirmed that human KLRG1 can biochemically interact with murine cadherins. Knock-in mice with the humanized KLRG1 protein had significantly increased tumor growth compared to both WT and KLRG1 KO mice, indicating a stronger immunosuppressive effect of human KLRG1 compared to murine KLRG1. An antagonistic antibody blocking interaction of human KLRG1 with its ligands was generated, which potentiated human T cell activation in vitro. Administration of anti-human KLRG1 Ab to tumor-bearing humanized KLRG1 mice led to markedly reduced tumor growth. In summary, our data support KLRG1 as a functionally critical negative regulator of the anti-tumor immune response and that blocking KLRG1-cadherin interactions has potential as a therapeutic candidate. Citation Format: Tyler A. Jones, Stephen Sinicrope, Kei Yasuda, Jan Pinkas, Thomas F. Gajewski. Targeting murine or human KLRG1 exerts potent anti-tumor efficacy involving both CD8T cells and NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 177.

Abstract C016: Pre-existing T cell inflammation is a determinant of response to mesothelin chimeric antigen receptor T cell therapy in pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) has a 12.5% 5-year survival rate and is predicted to become the 2nd leading cause of cancer-related death by 2030. PDAC is known to be resistant to immunotherapy. Thus, while chimeric antigen receptor (CAR) T cell therapy shows the potential to mediate durable responses against some hematologic and solid tumors, in PDAC, the efficacy of CAR T therapy has remained limited. PDAC tumors show heterogenous T cell infiltration with some tumors containing a high number of T cells. As T cell infiltration associates with prolonged survival, we hypothesized that the density of pre-existing T cell infiltration would predict response to CAR T therapy. Methods: Previously established clonal mouse PDAC cell lines, which show a T cell inflamed or non-inflamed tumor microenvironment upon in vivo implantation, were used. Mouse CAR T cells specific to mesothelin, a tumor antigen expressed by PDAC, were generated using retroviral transduction. Mice were implanted subcutaneously with T cell inflamed or non-inflamed tumor cell lines. Ten days later mesothelin CAR T cells were administered intravenously. Flow cytometry and multiplex immunohistochemistry was used to define the density and spatial location of CAR T cell infiltration. The impact of CAR T cells on tumor growth and survival was also measured. Results: CAR T cells were found to traffic to T cell inflamed tumors, which led to delayed tumor outgrowth. However, such results were not seen in T cell non-inflamed tumors. Rather in mice with T cell non-inflamed tumors, CAR T cells accumulated in the liver. Further, CAR T cells effectively eliminated T cell inflamed and non-inflamed tumor cells in vitro, demonstrating that tumor intrinsic mechanisms of resistance to T cell killing were not a major determinant of treatment efficacy. Conclusions: Our findings suggest the therapeutic potential of CAR T cells in PDAC but highlight mechanisms of T cell exclusion as a therapeutic barrier wherein CAR T cells accumulate in host organs. Citation Format: Jacqueline B. Plesset, Heather Coho, Kelly Markowitz, Dhruv Patel, Max M. Wattenberg, Meredith L. Stone, Devora Delman, Gregory L. Beatty. Pre-existing T cell inflammation is a determinant of response to mesothelin chimeric antigen receptor T cell therapy in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C016.

T cell-mediated curation and restructuring of tumor tissue coordinates an effective immune response

Antigen-specific Tcells traffic to, are influenced by, and create unique cellular microenvironments. Here we characterize these microenvironments over time with multiplexed imaging in a melanoma model of adoptive Tcell therapy and human patients with melanoma treated with checkpoint inhibitor therapy. Multicellular neighborhood analysis reveals dynamic immune cell infiltration and inflamed tumor cell neighborhoods associated with CD8+ Tcells. Tcell-focused analysis indicates Tcells are found along a continuum of neighborhoods that reflect the progressive steps coordinating the anti-tumor immune response. More effective anti-tumor immune responses are characterized by inflamed tumor-T cell neighborhoods, flanked by dense immune infiltration neighborhoods. Conversely, ineffective Tcell therapies express anti-inflammatory cytokines, resulting in regulatory neighborhoods, spatially disrupting productive Tcell-immune and -tumor interactions. Our study provides in situ mechanistic insights into temporal tumor microenvironment changes, cell interactions critical for response, and spatial correlates of immunotherapy outcomes, informing cellular therapy evaluation and engineering.

Uncovering the Potential of CD40 Agonism to Enhance Immune Checkpoint Blockade.

In this CCR Translations, we discuss the therapeutic potential of CD40 agonism, which stimulates antigen-presenting cells (APC) to activate effector T and NK cells. CD40 agonism may lead to development of an interferon-activated, T cell-inflamed tumor microenvironment and has the potential to facilitate long-term response with immune checkpoint blockade. See related article by Weiss et al., p. 74.

220P HDAC1 inhibition sensitizes refractory tumor cells to anti-PD-1 therapy by upregulation chemokine expression and stimulating T cells infiltration into tumor beds

Immune checkpoint inhibitors (ICIs), exemplified by anti-PD-1 are promising treatments for many tumors. However, the majority of patients lack effective responses because of the emergence of immune-refractory tumors that disrupt the amplification of antitumor immunity. In many cases, one of the major causes of resistance to these agents is the limited tumor penetrance of effector T cells. Durable clinical responses using anti–PD-1 have been associated with T cell–inflamed tumor microenvironment (TME) favoring the infiltration of functional cytotoxic T lymphocytes (CTLs).

Stromal remodeling regulates dendritic cell abundance and activity in the tumor microenvironment.

SUMMARYStimulatory type 1 conventional dendritic cells (cDC1s) engage in productive interactions with CD8+ effectors along tumor-stroma boundaries. The paradoxical accumulation of “poised” cDC1s within stromal sheets is unlikely to simply reflect passive exclusion from tumor cores. Drawing parallels with embryonic morphogenesis, we hypothesized that invasive margin stromal remodeling generates developmentally conserved cell fate cues that regulate cDC1 behavior. We find that, in human T cell-inflamed tumors, CD8+ T cells penetrate tumor nests, whereas cDC1s are confined within adjacent stroma that recurrently displays site-specific proteolysis of the matrix proteoglycan versican (VCAN), an essential organ-sculpting modification in development. VCAN is necessary, and its proteolytic fragment (matrikine) versikine is sufficient for cDC1 accumulation. Versikine does not influence tumor-seeding pre-DC differentiation; rather, it orchestrates a distinctive cDC1 activation program conferring exquisite sensitivity to DNA sensing, supported by atypical innate lymphoid cells. Thus, peritumoral stroma mimicking embryonic provisional matrix remodeling regulates cDC1 abundance and activity to elicit T cell-inflamed tumor microenvironments.

Inhibition of stearoyl-CoA desaturase 1 (SCD1) enhances the antitumor T cell response through regulating β-catenin signaling in cancer cells and ER stress in T cells and synergizes with anti-PD-1 antibody

BackgroundUnderstanding the mechanisms of non-T cell inflamed tumor microenvironment (TME) and their modulation are important to improve cancer immunotherapies such as immune checkpoint inhibitors. The involvement of various immunometabolisms has...

Abstract 5018: A comprehensive analysis of genomic determinants of response to immune checkpoint inhibitor-based immunotherapy

Abstract A low response rate is the major challenge for existing cancer immunotherapies. A number of biomarkers have been reported to be associated with the likelihood of patient responses to immune checkpoint inhibitors (ICI), including the expression of PD-L1, tumor mutational burden (TMB), tumor infiltration lymphocytes, and T cell repertoire. However, these biomarkers showed variable performance even conflicting conclusions in different cohorts. And a large proportion of patients exhibited an immune-exclusion or immune-desert phenotype that cannot be explained by current biomarkers. In addition, establishing a cohort with adequate sample size requires multiple years of multi-center efforts. Therefore, it is still a challenge and a lack for the evaluation of efficacy of known biomarkers and the discovery of new signature(s) in a large-scale study. We hypothesized that a data-driven meta-analysis approach integrating multi-cohorts with multi-omics data will help to characterize the predictive genomic features and discover the mechanisms of antitumor immune responses. In this work, we collected 3,037 ICI therapy samples with genetic and transcriptomics data for 14 cancer types. Then a data-driven strategy was designed to explore each type of genomic feature which included gene mutations, pathway mutations, TMB, mutational signatures, gene expression, pathway expression, interaction among checkpoint blockade, and immune cell components. Then a comprehensive analysis, which contained a meta-analysis for the identification of consistent biomarkers across multiple cohorts and a data aggregation-analysis for the identification of rare biomarkers on aggregated data, was performed to explore the associations between ICI response and omics features. Furthermore, we validated these signatures as biomarkers in three independent cohorts. Finally, we identified five types of biomarkers, including 1) two biomarkers regarding source of antigen (TMB and mutations of MYO7B), 2) one feature of responding to antigen (cytotoxic T lymphocytes pathway), 3) three biomarkers indicating macrophages M1 infiltrations (macrophages M1 component, and two marker genes of macrophages M1, CXCL11 and CCL19), 4) one biomarker regarding immune interactions (expression relations among immune checkpoint genes), and 5) two biomarkers about T cell-inflamed tumor microenvironment (IFNG and ligands genes of chemokine receptor CXCR3). In addition, a predictive module was built based on these signatures and outperformed than TMB or PD-L1 expression which were approved molecular biomarkers by FDA. We found high TMB patients had more likelihood to active the cytotoxic T lymphocytes which secret IFNG to kill tumor cell and promote the recruitment of macrophage M1. And the T cells were stimulated by macrophage M1 via the binding of CXCR3 of T cell surface and receptor of CXCR3 of macrophage M1 surface. Citation Format: Jing Yang, Qi Liu, Yu Shyr. A comprehensive analysis of genomic determinants of response to immune checkpoint inhibitor-based 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 5018.

Which angiosarcoma subtypes may benefit from immunotherapy?

11572 Background: Angiosarcomas (AS) are aggressive mesenchymal tumors arising from cells with endothelial properties. They include de novo primary AS (pAS), and secondary AS (sAS) due to prior radiotherapy, UV exposure or chronic lymphedema. Treatment options are limited and their prognosis is poor. Development of new treatment strategies is difficult due to the heterogeneity and rarity of AS. We hypothesize that immunological and genomic profiles are significantly different between pAS and sAS and may result in different immune checkpoint inhibition (ICI) based treatment strategies. Methods: Tumor samples from AS patients were retrospectively collected. Patients were categorized as pAS or sAS. Lymphocytes were analyzed using multiplex immunohistochemistry on tissue microarrays. Genomic profiling was performed in a selected subgroup with “TruSight Oncology 500”, a Next Generation Sequencing panel containing 523 cancer related genes. Results: Immunological data were analyzed from 257 AS patients. The cohort comprised 80 pAS patients and 177 sAS patients. The median density of CD3+ T cells was 250 cells/mm2 in pAS vs 452 cells/mm2 in sAS (p< 0.001). Median CD4+ T helper cell density was 128 cells/mm2 in pAS vs 246 cells/mm2 in secondary AS (p< 0.001). The median density of CD8+ cytotoxic T cells was 85 cells/mm2 in pAS vs 111 cells/mm2 in sAS ( p= 0.057). Density of FoxP3+ T regulatory cells was higher in sAS (median 42 cells/mm2) compared to pAS (median 23 cells/mm2) (p< 0.001). The median count of CD20+ B cells in pAS was 24 cells/mm2 compared to 32 cells/mm2 in sAS ( p= 0.533). Genomic analysis was performed on tumor DNA from 51 patients (25 pAS and 26 sAS). Median tumor mutational burden (TMB) was 3.2 (range 0.8-11.9) mutations per megabase (mut/Mb) in pAS vs 3.9 (range 0.0-99.6) in sAS ( p= 0.485). No microsatellite instability was detected. A pathogenic mutation, gene amplification or gene loss was identified in 82% of all patients (n = 42, 70% of pAS vs 100% of sAS ( p< 0.01)). In 36 patients (71%) at least one (likely) pathogenic mutation was detected (54% pAS vs 88% sAS, ( p= 0.013)). In 20 patients (39%) mutations in the DNA damage response (DDR) pathway were detected (12% pAS vs 68% sAS ( p< 0.01)). The most frequently found mutated genes were TP53 (10%), BRAF (6%), ERCC4 (6%), PTPRD (6%), WETD2 (6%) and SETD2 (6%). Amplifications were found in 49% (n = 25) of all patients (15% pAS vs 84% sAS, ( p< 0,01)). MYC amplifications were most common and were detected in 15% of pAS and 68% of sAS. Immune profiles of the 51 genomically characterized patients are currently under further investigation. Conclusions: We showed a clear distinction in immunological and genomic profiles between pAS and sAS. The potential benefit of ICI seems to be most promising in sAS with a T cell inflamed tumor microenvironment, frequent MYC amplifications, DDR mutations, and high mutational load, while in pAS boosting strategies to enhance susceptibility to ICI might be interesting for further investigation.

TcellInflamedDetector: an R package to distinguish T cell inflamed tumor types from non-T cell inflamed tumor types.

A major issue in the use of immune checkpoint inhibitors is their lack of efficacy in many patients. Previous studies have reported that the T cell inflamed signature can help predict the response to immunotherapy. Thus, many studies have investigated mechanisms of immunotherapy resistance by defining the tumor microenvironment based on T cell inflamed and non–T cell inflamed subsets. Although methods of calculating T cell inflamed subsets have been developed, valid screening tools for distinguishing T cell inflamed from non–T cell inflamed subsets using gene expression data are still needed, since general researchers who are unfamiliar with the details of the equations can experience difficulties using extant scoring formulas to conduct analyses. Thus, we introduce TcellInflamedDetector, an R package for distinguishing T cell inflamed from non–T cell inflamed samples using cancer gene expression data via bulk RNA sequencing.

Abstract P5-13-18: Upregulation of immune response biomarkers by ribociclib plus endocrine therapy (ET) in paired tumor samples from phase I studies

Abstract Background: In addition to inhibiting cell cycle progression, CDK4/6 inhibitors have been found to modulate anti-cancer immune response (Goel S, et al. Nature. 2017;548:471-475). Data from preclinical studies suggested multiple mechanisms—involving changes in both cancer cells and immune cells—by which CDK4/6 inhibition can enhance anti-cancer immunity. These mechanisms include increased antigen presentation, interferon response triggering, and regulatory T-cell suppression. Clinical correlates to preclinical observations have so far been obtained using paired biopsies from neoadjuvant breast cancer trials (Johnston S, et al. J Clin Oncol. 2019;37:178-189; Hurvitz S, et al. Clin Cancer Res. 2020; ;26:566-80). Here, we describe a gene expression analysis of paired pre- and on-treatment biopsies from CLEE011A2115C and CLEE011X2107, two phase I clinical trials evaluating ribociclib in combination with ET in the metastatic breast cancer (MBC) setting. Methods: The nCounter PanCancer IO 360 Panel (NanoString) was used to quantify expression of 770 genes in 7 pairs of tumor samples (baseline vs. cycle 1 day 15) from two phase I trials in the MBC setting (5 patients from CLEE011A2115C and 2 from CLEE011X2107). All patients were treated with ribociclib and an ET. Pairwise differential gene expression analysis of individual genes and previously published immune-related gene signatures was conducted (Bedognetti D, et al. Curr Opin Oncol. 2015;27:433-444). Results: Many genes that were markedly suppressed with treatment (eg, MKI67, MYC, CDK2, CCNB1, TYMS, and DNMT1) were related to proliferation, DNA replication, and G1/S transition of the cell cycle, as expected. Interestingly, expression of numerous genes involved in immune response was increased. These were mostly interferon-regulated genes; particularly, a gene signature indicative of a T cell-inflamed tumor microenvironment (Ayers M, et al. J Clin Invest. 2017;127:2930-2940) was upregulated. Immune-related genes tended to preferentially increase with treatment in patients who later experienced a clinical response. Since bulk RNA from tumor biopsies was analyzed, we could not distinguish whether the observed expression changes reflected increased infiltration by immune cells or an endogenous interferon response in cancer cells. The latter could be triggered by DNMT1 suppression (as previously described), which then promotes T cell-mediated immunity by increasing antigen presentation and chemokine production in cancer cells. Conclusions: This analysis was conducted with a small sample size and is considered hypothesis generating; further investigation is needed. Cell cycle and proliferation markers showed robust suppression by ribociclib-based treatment regimens, as expected. Conversely, a notable increase in immune-related genes was detected that tended to occur preferentially in patients who later experienced more favorable clinical outcomes. Both changes occurred early (cycle 1 day 15). To our knowledge, this is the first clinical biomarker report of immune activation mediated by CDK4/6 inhibition in MBC. Citation Format: Dejan Juric, Chong Ma, Ralph Tiedt, Yoon-Sim Yap, Joanne Chiu, Pamela Munster, Roohi Ismail-Khan, Laura Garcia-Estevez, Ingrid A. Mayer, Carlos Becerra, Nadia Solovieff, Agnes Lteif, Faye Su, Yen-Shen Lu. Upregulation of immune response biomarkers by ribociclib plus endocrine therapy (ET) in paired tumor samples from phase I studies [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-18.

The Prognostic and Predictive Role of Xeroderma Pigmentosum Gene Expression in Melanoma.

BackgroundAssessment of immune-specific markers is a well-established approach for predicting the response to immune checkpoint inhibitors (ICIs). Promising candidates as ICI predictive biomarkers are the DNA damage response pathway genes. One of those pathways, which are mainly responsible for the repair of DNA damage caused by ultraviolet radiation, is the nucleotide excision repair (NER) pathway. Xeroderma pigmentosum (XP) is a hereditary disease caused by mutations of eight different genes of the NER pathway, or POLH, here together named the nine XP genes. Anecdotal evidence indicated that XP patients with melanoma or other skin tumors responded impressively well to anti-PD-1 ICIs. Hence, we analyzed the expression of the nine XP genes as prognostic and anti-PD-1 ICI predictive biomarkers in melanoma.MethodsWe assessed mRNA gene expression in the TCGA-SKCM dataset (n = 445) and two pooled clinical melanoma cohorts of anti-PD-1 ICI (n = 75). In TCGA-SKCM, we applied hierarchical clustering on XP genes to reveal clusters, further utilized as XP cluster scores. In addition, out of 18 predefined genes representative of a T cell inflamed tumor microenvironment, the TIS score was calculated. Besides these scores, the XP genes, immune-specific single genes (CD8A, CXCL9, CD274, and CXCL13) and tumor mutational burden (TMB) were cross-correlated. Survival analysis in TCGA-SKCM was conducted for the selected parameters. Lastly, the XP response prediction value was calculated for the two pooled anti-PD-1 cohorts by classification models.ResultsIn TCGA-SKCM, expression of the XP genes was divided into two clusters, inversely correlated with immune-specific markers. A higher ERCC3 expression was associated with improved survival, particularly in younger patients. The constructed models utilizing XP genes, and the XP cluster scores outperformed the immune-specific gene-based models in predicting response to anti-PD-1 ICI in the pooled clinical cohorts. However, the best prediction was achieved by combining the immune-specific gene CD274 with three XP genes from both clusters.ConclusionOur results suggest pre-therapeutic XP gene expression as a potential marker to improve the prediction of anti-PD-1 response in melanoma.

Ratio of the interferon-\u03b3 signature to the immunosuppression signature predicts anti-PD-1 therapy response in melanoma

Immune checkpoint inhibitor (ICI) treatments produce clinical benefit in many patients. However, better pretreatment predictive biomarkers for ICI are still needed to help match individual patients to the treatment most likely to be of benefit. Existing gene expression profiling (GEP)-based biomarkers for ICI are primarily focused on measuring a T cell-inflamed tumor microenvironment that contributes positively to the response to ICI. Here, we identified an immunosuppression signature (IMS) through analyzing RNA sequencing data from a combined discovery cohort (n = 120) consisting of three publicly available melanoma datasets. Using the ratio of an established IFN-γ signature and IMS led to consistently better prediction of the ICI therapy outcome compared to a collection of nine published GEP signatures from the literature on a newly generated internal validation cohort (n = 55) and three published datasets of metastatic melanoma treated with anti-PD-1 (n = 54) and anti-CTLA-4 (n = 42), as well as in patients with gastric cancer treated with anti-PD-1 (n = 45), demonstrating the potential utility of IMS as a predictive biomarker that complements existing GEP signatures for immunotherapy.

Triggering interferon signaling in T cells with avadomide sensitizes CLL to anti-PD-L1/PD-1 immunotherapy

AbstractCancer treatment has been transformed by checkpoint blockade therapies, with the highest anti-tumor activity of anti-programmed death 1 (PD-1) antibody therapy seen in Hodgkin lymphoma. Disappointingly, response rates have been low in the non-Hodgkin lymphomas, with no activity seen in relapsed/refractory chronic lymphocytic leukemia (CLL) with PD-1 blockade. Thus, identifying more powerful combination therapy is required for these patients. Here, we preclinically demonstrate enhanced anti-CLL activity following combinational therapy with anti-PD-1 or anti-PD-1 ligand (PD-L1) and avadomide, a cereblon E3 ligase modulator (CELMoD). Avadomide induced type I and II interferon (IFN) signaling in patient T cells, triggering a feedforward cascade of reinvigorated T-cell responses. Immune modeling assays demonstrated that avadomide stimulated T-cell activation, chemokine expression, motility and lytic synapses with CLL cells, as well as IFN-inducible feedback inhibition through upregulation of PD-L1. Patient-derived xenograft tumors treated with avadomide were converted to CD8+ T cell-inflamed tumor microenvironments that responded to anti-PD-L1/PD-1-based combination therapy. Notably, clinical analyses showed increased PD-L1 expression on T cells, as well as intratumoral expression of chemokine signaling genes in B-cell malignancy patients receiving avadomide-based therapy. These data illustrate the importance of overcoming a low inflammatory T-cell state to successfully sensitize CLL to checkpoint blockade-based combination therapy.

Multiplex immunofluorescence to assess the tumor microenvironment in bladder cancer

A T cell-inflamed tumor microenvironment (TME) is linked to improved prognosis and response to immunotherapy in bladder cancer. Batf3+ dendritic cells (DCs) have been observed in murine models to be critical for both priming an immune response and recruiting effector CD8+ T cells to the TME. However, the role of Batf3+ DCs in human bladder cancers, especially in relation to effector CD8+ T cells, remains unknown. We performed multiplex immunofluorescence imaging on 47 surgically resected muscle invasive bladder cancer specimens to evaluate the population of tumor infiltrating immune cells (TIICs) marked by BATF3, CD8, FoxP3, PD-1, and PD-L1. The relationship of TIICs to a previously described immune gene signature based on RNA sequencing and survival outcomes were investigated. The proportion of T cell-inflamed tumors determined by RNA sequencing was higher in groups with above median TIICs versus below except for PD-1 (70.8% vs 21.7% for CD8 [P = 0.003], 66.7% vs 26.1% for Batf3 [P = 0.010], and 58.3% vs 34.8% for PD-1 [P = 0.266], 66.7% vs 26.1% for FOXP3 [P = 0.010], and 62.5% vs 30.4% for PD-L1 [P = 0.056], respectively). Analyzing Batf3 and CD8 jointly identified the group with the highest proportion of T cell-inflamed tumors (86.7% for CD8highBatf3high vs 34.8% for others [P = 0.001]) and showed a stronger association than with CD8 alone. In a spatial analysis using K function betweenCD8+ cells and Batf3+ cells, the median area under the curves of the cases were significantly higher than that of a hypothetically calculated random distribution curve, indicating that CD8+ cells clustered in proximity to Batf3+ cells. In survival analysis, CD8highBatfhigh population showed a significantly better RFS and OS than others (HR 0.307 [P = 0.019] for RFS, HR 0.361 [P = 0.044] for OS), which was more differentiating CD8 status alone (HR 0.393 [P = 0.021] for RFS, HR 0.436 [P = 0.045] for OS). Most of the investigated immune cell types correlated with a T cell-inflamed TME by gene expression profiling. CD8highBatf3high tumors were most associated with a T cell-inflamed TME and showed better survival outcomes. Further analyses to assess associations between TME and immunotherapy response are ongoing.

Molecular correlates and therapeutic targets in T cell-inflamed versus non-T cell-inflamed tumors across cancer types

BackgroundThe T cell-inflamed tumor microenvironment, characterized by CD8 T cells and type I/II interferon transcripts, is an important cancer immunotherapy biomarker. Tumor mutational burden (TMB) may also dictate response, and some oncogenes (i.e., WNT/β-catenin) are known to mediate immunosuppression.MethodsWe performed an integrated multi-omic analysis of human cancer including 11,607 tumors across multiple databases and patients treated with anti-PD1. After adjusting for TMB, we correlated the T cell-inflamed gene expression signature with somatic mutations, transcriptional programs, and relevant proteome for different immune phenotypes, by tumor type and across cancers.ResultsStrong correlations were noted between mutations in oncogenes and tumor suppressor genes and non-T cell-inflamed tumors with examples including IDH1 and GNAQ as well as less well-known genes including KDM6A, CD11c, and genes with unknown functions. Conversely, we observe genes associating with the T cell-inflamed phenotype including VHL and PBRM1. Analyzing gene expression patterns, we identify oncogenic mediators of immune exclusion across cancer types (HIF1A and MYC) as well as novel examples in specific tumors such as sonic hedgehog signaling, hormone signaling and transcription factors. Using network analysis, somatic and transcriptomic events were integrated. In contrast to previous reports of individual tumor types such as melanoma, integrative pan-cancer analysis demonstrates that most non-T cell-inflamed tumors are influenced by multiple signaling pathways and that increasing numbers of co-activated pathways leads to more highly non-T cell-inflamed tumors. Validating these analyses, we observe highly consistent inverse relationships between pathway protein levels and the T cell-inflamed gene expression across cancers. Finally, we integrate available databases for drugs that might overcome or augment the identified mechanisms.ConclusionsThese results nominate molecular targets and drugs potentially available for further study and potential immediate translation into clinical trials for patients with cancer.

Abstract 1563: Prediction of immune-checkpoint blockade response in gastrointestinal cancer patients using a machine learning-based RNA signature

Abstract Background: Currently, the objective response rate of immune checkpoint blockade (ICB) immunotherapy in gastrointestinal (GI) cancers was still unsatisfying, underscoring the urgent need for the development of novel biomarkers. To date, candidate biomarkers in GI cancers include microsatellite instability (MSI), tumor mutational burden (TMB), program death ligand 1 (PD-L1) expression, and RNA signatures associated with T cell-inflamed tumor microenvironment. Here, aiming to precisely capture the pre-existing “responsive feature” in GI cancers, we adopted a machine learning strategy based on RNA expression profiling data. Methods: This retrospective study included 96 metastatic GI patients treated with ICBs, who were randomly assigned into the discovery (75%) and validation (25%) cohorts. All formalin-fixed paraffin-embedded (FFPE) tumor specimens were subjected to the 395-plex immune oncology (IO)-related gene target sequencing. Based on the RNA profiling data of patients with durable clinic benefit (DCB) or no durable benefit (NDB), a linear support vector machine (linearSVM) strategy was applied for predictive model construction. Results: Notably, the linearSVM strategy helps construct a 24-gene RNA signature (IO-score) for discriminating DCB and NDB, which comprises genes involving in tumor antigen, tumor suppressor/oncogene, lymphocyte markers, interferon and checkpoint signaling pathways. In specific, patients with higher IO-score demonstrated stronger DCB rates than lower IO-score (discovery cohort: 92.0% [23 of 25] vs. 4.3% [2 of 47], P < 0.001; validation cohort: 85.7% [6 of 7] vs. 17.6% [3 of 17], Fisher's exact test, P =0.004). Moreover, compared with IO-score low subgroup, IO-score high subgroup displayed improved overall survival (discovery cohort: hazard ratio (HR), 0.29; 95% CI, 0.15-0.56; P =0.003; validation cohort: HR, 0.32, 95% CI, 0.10-1.05, P =0.04) as well as progression free survival (discovery cohort: HR, 0.22; 95% CI, 0.13-0.38; P <0.001; validation cohort: HR, 0.23, 95% CI, 0.09-0.60, P =0.007). Importantly, IO-score reveals a higher predictive value in both cohorts (discovery: area under the receiver operating characteristic curve (AUC)=0.97, 95% CI, 0.93-1.0; validation: AUC=0.74, 95% CI, 0.51-0.97), when compared with TMB-High (AUC=0.69, 95% CI, 0.57-0.80), MSI-High (AUC=0.59, 95% CI, 0.45-0.73) and PD-L1 positivity (AUC=0.52, 95% CI, 0.37-0.67). Conclusion: The machine learning methodologies offer a promising approach for capturing the pre-existing “responsive feature” in both tumor and infiltrating immune cells. The novel RNA signature, IO-score, may help facilitate individualized management in immunotherapy in GI cancer patients. A larger cohort study would be useful to optimize the cutoff value of IO-score. Citation Format: Lin Shen, Henghui Zhang, Zhihao Lu, Huan Chen, Xi Jiao, Wei Zhou, Shuang Li, Ying Yang, Dandan Liang. Prediction of immune-checkpoint blockade response in gastrointestinal cancer patients using a machine learning-based RNA signature [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1563.

Abstract IA24: Molecular correlates of T cell-inflamed and non-T cell-inflamed tumors

Abstract IA24: Molecular correlates of T cell-inflamed and non-T cell-inflamed tumors

Abstract B04: Molecular correlates of the non-T cell-inflamed tumor microenvironment in head and neck squamous cell carcinoma

Abstract Background: Immunotherapy confers clinical benefit in head and neck squamous cell carcinoma (HNSCC); however, only a fraction of patients respond. The non-T cell-inflamed tumor microenvironment is associated with lack of response, and tumor-intrinsic molecular signaling pathways may be critical drivers of immune exclusion. Therefore, understanding correlations between T-cell inflammation and mutated or activated oncogenic pathways may inform combination immunotherapy approaches. Methods: RNAseq, somatic mutations, and clinical data were downloaded from The Cancer Genome Atlas (TCGA) database for 484 HNSCC patients (88 HPV-positive, 396 HPV-negative). Gene expression was used to segregate tumors into non-T cell-inflamed, T cell-inflamed, or intermediate groups and genes more frequently mutated in non-T cell-inflamed versus inflamed (or vice versa) were identified. Differentially expressed genes (DEGs) between the two groups were detected by limma voom with precision weights. Pathway activation was identified by Ingenuity Pathway Analysis® (IPA). Association between molecular markers and patients’ overall survival (OS) was tested using Cox proportional-hazards regression models. Results: 132 (27%) and 183 (38%) tumors were categorized into the non-T cell-inflamed and inflamed group, respectively, with the rest intermediate. Survival analysis revealed that among age, sex, tumor grade, and HPV status, age was the strongest prognostic factor (p=0.003), with older patients showing a decreased 5-year survival. The absence of a T cell-inflamed signature was significantly associated with poorer prognosis within patients ≤65 years of age (p=0.03, HR=1.18) and remained significant after adjusting for covariates such as HPV status. This association was not observed in older patients. Comparison of somatic mutational profiles between the non-T cell-inflamed and inflamed groups identified top mutated genes enriched (TP53, COL11A1, NSD1) and depleted (CASP8) in noninflamed relative to inflamed (FDR-corrected p<0.05). Comparing gene expression profiles of non-T cell-inflamed to inflamed patient groups identified 4092 DEGs (FDR-corrected p<0.05, fold change ≥1.5 or ≤-1.5). Utilizing these DEGs as downstream target molecules in IPA causal network analysis, activation of MYC, SOX2, NFE2L2, and CTNNB1 transcriptional programs correlated with the non-T cell-inflamed tumor microenvironment (z-score ≥2.0, p<0.05). Of note, NFE2L2 (nuclear factor erythroid-2-related factor 2; NRF2) has been described as a context-dependent regulator of innate immune responses and modulator of checkpoint inhibitor response in murine models. Conclusions: A discrete list of oncogenes, some with known targeted therapies, associates with immune exclusion, suggesting rational immunotherapy combinations to overcome non-T cell-inflamed tumors in HNSCC. Citation Format: Riyue Bao, Jason J. Luke. Molecular correlates of the non-T cell-inflamed tumor microenvironment in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B04.

Henoch-schönlein Purpura (HSP) in a patient on Abemaciclib

Henoch-Schonlein Purpura (HSP), also known as IgA vasculitis, is a small-vessel vasculitis associated with IgA-dominant immune deposits in the skin, gut and kidney [[1]Rigante D. Castellazzi L. Bosco A. Esposito S. Is there a crossroad between infections, genetics, and henoch-schönlein purpura?.Autoimmun Rev. 2013; 12: 1016-1021Crossref PubMed Scopus (69) Google Scholar]. It is usually characterized by a triad of symptoms including purpuric skin rash, abdominal pain or renal involvement and arthritis. In adults, HSP is seen in 3.4–14.3 cases per million [[1]Rigante D. Castellazzi L. Bosco A. Esposito S. Is there a crossroad between infections, genetics, and henoch-schönlein purpura?.Autoimmun Rev. 2013; 12: 1016-1021Crossref PubMed Scopus (69) Google Scholar]. Approximately two thirds of patients have a triggering event, most often an infection or an allergic reaction to drugs such as targeted therapies [[1]Rigante D. Castellazzi L. Bosco A. Esposito S. Is there a crossroad between infections, genetics, and henoch-schönlein purpura?.Autoimmun Rev. 2013; 12: 1016-1021Crossref PubMed Scopus (69) Google Scholar]. The cyclin-dependent kinases 4/6 (CDK4/6) inhibitors are a new class of drugs recently approved for the treatment of hormonal receptor positive/HER2 negative (HR+/HER2-) advanced breast cancer (BC) [[2]The role of CDK4/6 inhibitors in breast cancer. Conleth G. Murphy MB, BCh, BAO1,2.Curr Treat Options Oncol. 2019; volume 20 (Article number: 52)Google Scholar]. To date, no case report of HSP in patient on CDK4/6 inhibitors plus endocrine therapies has been described. In May 2019, an otherwise healthy 65-years-old female was diagnosed with stage IV HR+/HER2- BC with liver, lung and bone metastasis. Hypertension treated with ACE inhibitors was the only known comorbidity. From June 2019, she started first line treatment with a combination of CDK4/6 inhibitor (Abemaciclib 300 mg daily) and Letrozole. She was on treatment without relevant side effects and good clinical response. In October 2019, she presented to outpatient clinic referring one-day history of joint pain, severe asthenia, fever and spreading purpuric papules on lower limbs (Fig. 1A). Laboratory tests revealed a nephritic syndrome manifesting with a progressive renal failure (Creatinine 3,13 mg/dL), microhematuria and proteinuria. Over the next 6 hours, the purpura spread further over the sides and buttocks (Fig. 1B). Assuming a provisional diagnosis of HSP, the patient was started on intravenous dexamethasone 1 mg/kg body weight and systemic antibiotics, with clinical improvement. Blood cultures, serological and antiantibodies tests resulted negative. Renal biopsy was performed. Light microscopy revealed mesangial hypercellularity accompanied by diffuse IgA and C3 deposits in the mesangiu. The diagnosis of Abemaciclib-induced HSP was made. Finally, corticosteroid tapering was initiated and the patient was discharged after 15 days of hospitalization with complete normalization of renal function. One month later Abemaciclib was re-started at the lower dose (100 mg BID), without any other side effect. We speculate that the Abemaciclib could have triggered an abnormal IgA-mediated immune response against the small vascular walls endothelial cells [[3]Immunomodulatory roles of cell cycle regulators. Laphanuwat P and Jirawatnotai SImmunomodulatory roles of cell cycle regulators. Laphanuwat P and Jirawatnotai S (2019) Immunomodulatory Roles of Cell Cycle Regulators.Front. Cell Dev. Biol. 2019; 7: 23PubMed Google Scholar]. Recent research provides cumulative evidence for functions of cell cycle regulators in immune defense. Pre-clinical evidence reported an upregulation of select cytokines and chemokines (such as PD-L1, PD-L2), an increase of IFNγ-dependent enzymes and an upregulation of MHC class I and II in tumors treated with Abemaciclib [[4]Schaer D.A. Beckmann R.P. Dempsey J.A. Huber L. Forest A. Amaladas N. et al.The CDK4/6 inhibitor abemaciclib induces a T cell inflamed tumor microenvironment and enhances the efficacy of PD-L1 checkpoint blockade.Cell Rep. 2018; 22: 2978-2994Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar]. Although uncommonly, HSP may follow treatment with Abemaciclib, which use is further increasing in BC treatment strategy. Hence, an increased awareness among oncologists could allow for early recognition and appropriate treatment. Informed consent was obtained from the patient included in the study. I declare that we have no conflict of interest.