Intratumoral T-cell Research Articles

Impact of Chemoembolic Regimen on Immune Cell Recruitment and Immune Checkpoint Marker Expression following Transcatheter Arterial Chemoembolization in a VX2 Rabbit Liver Tumor Model

PurposeTo characterize the effects of commonly used transcatheter arterial chemoembolization (TACE) regimens on the immune response and immune checkpoint marker expression using a VX2 rabbit liver tumor model. Materials and MethodsTwenty-four VX2 liver tumor-bearing New Zealand white rabbits were assigned to 7 groups (n = 3 per group) undergoing locoregional therapy as follows: (a) bicarbonate infusion without embolization, (b) conventional TACE (cTACE) using a water-in-oil emulsion containing doxorubicin mixed 1:2 with Lipiodol, drug-eluting embolic-TACE with either (c) idarubicin-eluting Oncozene microspheres (40 μm) or (d) doxorubicin-eluting Lumi beads (40–90 μm). For each therapy arm (b–d), a tandem set of 3 animals with additional bicarbonate infusion before TACE was added, to evaluate the effect of pH modification on the immune response. Three untreated rabbits served as controls. Tissue was harvested 24 hours after treatment, followed by digital immunohistochemistry quantification (counts/μm2 ± SEM) of tumor-infiltrating cluster of differentiation 3+ T-lymphocytes, human leukocyte antigen DR type antigen-presenting cells (APCs), cytotoxic T-lymphocyte–associated protein-4 (CTLA-4), and programmed cell death protein-1 (PD-1)/PD-1 ligand (PD-L1) pathway axis expression. ResultsLumi-bead TACE induced significantly more intratumoral T-cell and APC infiltration than cTACE and Oncozene-microsphere TACE. Additionally, tumors treated with Lumi-bead TACE expressed significantly higher intratumoral immune checkpoint markers compared with cTACE and Oncozene-microsphere TACE. Neoadjuvant bicarbonate demonstrated the most pronounced effect on cTACE and resulted in a significant increase in intratumoral cluster of differentiation 3+ T-cell infiltration compared with cTACE alone. ConclusionsThis preclinical study revealed significant differences in evoked tumor immunogenicity depending on the choice of chemoembolic regimen for TACE.

Intratumoral Electroporation of Plasmid Encoded IL12 and Membrane-Anchored Anti-CD3 Increases Systemic Tumor Immunity.

This DNA-encodable polyclonal T-cell stimulator (membrane-anchored anti-CD3 plasmid) may represent a key addition to intratumoral IL12 therapies in the clinic.

Preclinical PET Imaging of Granzyme B Shows Promotion of Immunological Response Following Combination Paclitaxel and Immune Checkpoint Inhibition in Triple Negative Breast Cancer.

Advancements in monitoring and predicting of patient-specific response of triple negative breast cancer (TNBC) to immunotherapy (IMT) with and without chemotherapy are needed. Using granzyme B-specific positron emission tomography (GZP-PET) imaging, we aimed to monitor changes in effector cell activation in response to IMT with chemotherapy in TNBC. TNBC mouse models received the paclitaxel (PTX) ± immune checkpoint inhibitors anti-programmed death 1 (anti-PD1) and anti-cytotoxic T-lymphocyte 4 (anti-CTLA4). GZP-PET imaging was performed on treatment days 0, 3, and 6. Mean standard uptake value (SUVmean), effector cell fractions, and SUV histograms were compared. Mice were sacrificed at early imaging timepoints for cytokine and histological analyses. GZP-PET imaging data revealed differences prior to tumor volume changes. By day six, responders had SUVmean ≥ 2.2-fold higher (p < 0.0037) and effector cell fractions ≥ 1.9-fold higher (p = 0.03) compared to non-responders. IMT/PTX resulted in a significantly different SUV distribution compared to control, indicating broader distribution of activated intratumoral T-cells. IMT/PTX resulted in significantly more necrotic tumor tissue and increased levels of IL-2, 4, and 12 compared to control. Results implicate immunogenic cell death through upregulation of key Th1/Th2 cytokines by IMT/PTX. Noninvasive PET imaging can provide data on the TNBC tumor microenvironment, specifically intratumoral effector cell activation, predicting response to IMT plus chemotherapy.

Abdominopelvic FLASH Irradiation Improves PD-1 Immune Checkpoint Inhibition in Preclinical Models of Ovarian Cancer

Treatment of advanced ovarian cancer using PD-1/PD-L1 immune checkpoint blockade shows promise; however, current clinical trials are limited by modest response rates. Radiotherapy has been shown to synergize with PD-1/PD-L1 blockade in some cancers but has not been utilized in advanced ovarian cancer due to toxicity associated with conventional abdominopelvic irradiation. Ultrahigh-dose rate (FLASH) irradiation has emerged as a strategy to reduce radiation-induced toxicity, however, the immunomodulatory properties of FLASH irradiation remain unknown. Here, we demonstrate that single high-dose abdominopelvic FLASH irradiation promoted intestinal regeneration and maintained tumor control in a preclinical mouse model of ovarian cancer. Reduced tumor burden in conventional and FLASH-treated mice was associated with an early decrease in intratumoral regulatory T cells and a late increase in cytolytic CD8+ T cells. Compared with conventional irradiation, FLASH irradiation increased intratumoral T-cell infiltration at early timepoints. Moreover, FLASH irradiation maintained the ability to increase intratumoral CD8+ T-cell infiltration and enhance the efficacy of αPD-1 therapy in preclinical models of ovarian cancer. These data highlight the potential for FLASH irradiation to improve the therapeutic efficacy of checkpoint inhibition in the treatment of ovarian cancer.

Time-spatial analysis of T cell receptor repertoire in esophageal squamous cell carcinoma patients treated with combined radiotherapy and PD-1 blockade

ABSTRACT T cell receptor (TCR) repertoire as a biomarker for predicting immunotherapy efficiency has been widely studied. However, its dynamics during radiotherapy combined with PD-1 blockade is little known. Using paired tumor and blood samples from the phase Ib clinical study (NCT03222440), we investigate the time-spatial TCR repertoire in esophageal squamous cell carcinoma (ESCC) patients treated with first-line definitive radiotherapy concurrently with anti-PD-1 antibody camrelizumab, and also evaluate the association between TCR repertoire and clinical outcomes. TCR sequencing was performed on tumor biopsies (n = 34, 15 pairs) and peripheral CD8+ T cells (n = 36, 18 pairs) collected at baseline and during treatment (after 40 Gy radiation and 2 rounds of camrelizumab). Whole exome sequencing was applied to estimate genomic mutations and tumor mutation burden. We show that the intratumoral TCR repertoire at baseline was correlated with tumor microenvironment and presented heterogeneity inter-individually. T-cell clones inflowed mutually between tumors and peripheral blood under combination treatment, resulting in an elevation of intratumoral TCR diversity. The peripheral CD8+ TCR diversity at baseline, increased tumor-peripheral Morisita-Horn overlap during treatment, and expansion of persistent intratumoral T-cell clones during treatment predicted improved survival. While it is unclear whether radiation contributed to the TCR changes versus PD-1 therapy alone, our results firstly reveal radiotherapy combined with PD-1 blockade greatly promoted time-spatial alteration of TCR repertoire between tumor and peripheral blood, which demonstrate the peripheral CD8+ TCR diversity at baseline and dynamic alteration of intratumoral TCRs acted as potential effective biomarkers of radiotherapy combined with immunotherapy in ESCC.

NKG7 Is Required for Optimal Antitumor T-cell Immunity.

Tumor antigen-specific CD8+ T cells play a critical role in antitumor immunity. Clinical trials reinvigorating the immune system via immune checkpoint blockade (ICB) have shown remarkable clinical promise. Numerous studies have identified an association between NKG7 expression and patient outcome across different malignancies. However, aside from these correlative observations, very little is known about NKG7 and its role in antitumor immunity. Herein, we utilized single-cell RNA sequencing (scRNA-seq) datasets, NKG7-deficient mice, NKG7-reporter mice, and mouse tumor models to investigate the role of NKG7 in neoantigen-mediated tumor rejection and ICB immunotherapy. scRNA-seq of tumors from patients with metastatic melanoma or head and neck squamous cell carcinoma revealed that NKG7 expression is highly associated with cytotoxicity and specifically expressed by CD8+ T cells and natural killer (NK) cells. Furthermore, we identified a key role for NKG7 in controlling intratumor T-cell accumulation and activation. NKG7 was upregulated on intratumor antigen-specific CD8+ T cells and NK cells and required for the accumulation of T cells in the tumor microenvironment. Accordingly, neoantigen-expressing mouse tumors grew faster in Nkg7-deficient mice. Strikingly, efficacy of single or combination ICB was significantly reduced in Nkg7-deficient mice.See related article by Wen et al., p. 162.

T-Cell Receptor Repertoire Analysis Reveals Positive Correlation between Intra-Tumoral T-Cell Receptor Diversity and Overall Survival in Patients with Pancreatic Ductal Adenocarcinoma

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy that is refractory to immune therapy. Nevertheless, it is established that intra-tumoral T-cells correlate with improved overall survival (OS). This highlights the importance of the T-cell receptor (TCR) repertoire. We hypothesized that a more clonal TCR repertoire would correlate with increased survival.

IMMU-45. COMBINATION OF EGFRVIII CAR T-CELL THERAPY AND PARACRINE GAM MODULATION FOR THE TREATMENT OF GBM

Abstract The GBM immune tumor microenvironment mainly consists of protumoral glioma-associated microglia and macrophages (GAMs). We have previously shown that blockade of CD47, a ‘don't eat me’-signal overexpressed by GBM cells, rescued GAMs' phagocytic function in mice. However, monotherapy with CD47 blockade has been ineffective in treating human solid tumors to date. Thus, we propose a combinatorial approach of local CAR T cell therapy with paracrine GAM modulation for a synergistic elimination of GBM. We generated humanized EGFRvIII CAR T-cells by lentiviral transduction of healthy donor human T-cells and engineered them to constitutively release a soluble SIRPγ-related protein (SGRP) with high affinity towards CD47. Tumor viability and CAR T-cell proliferation were assessed by timelapse imaging analysis in co-cultures with endogenous EGFRvIII-expressing BS153 cells. Tumor-induced CAR T-cell activation and degranulation were confirmed by flow cytometry. CAR T-cell secretomes were analyzed by liquid chromatography-mass spectrometry. Immunocompromised mice were orthotopically implanted with EGFRvIII+ BS153 cells and treated intratumorally with a single CAR T-cell injection. EGFRvIII and EGFRvIII-SGRP CAR T-cells killed tumor cells in a dose-dependent manner (72h-timepoint; complete cytotoxicity at effector-target ratio 1:1) compared to CD19 controls. CAR T-cells proliferated and specifically co-expressed CD25 and CD107a in the presence of tumor antigen (24h-timepoint; EGFRvIII: 59.3±3.00%, EGFRvIII-SGRP: 52.6±1.42%, CD19: 0.1±0.07%). Differential expression analysis of CAR T-cell secretomes identified SGRP from EGFRvIII-SGRP CAR T-cell supernatants (-Log10qValue/Log2fold-change= 3.84/6.15). Consistent with studies of systemic EGFRvIII CAR T-cell therapy, our data suggest that intratumoral EGFRvIII CAR T-cells were insufficient to eliminate BS153 tumors with homogeneous EGFRvIII expression in mice (Overall survival; EGFRvIII-treated: 20%, CD19-treated: 0%, n= 5 per group). Our current work focuses on the functional characterization of SGRP binding, SGRP-mediated phagocytosis, and on the development of a translational preclinical model of heterogeneous EGFRvIII expression to investigate an additive effect of CAR T-cell therapy and GAM modulation.

HHV-6 in the Lymphoma Microenvironment: Both Chicken and Egg?

HHV-6 in the Lymphoma Microenvironment: Both Chicken and Egg?

Molecular Evolution of Classical Hodgkin Lymphoma Revealed Though Whole Genome Sequencing of Hodgkin and Reed-Sternberg Cells

Introduction: Classical Hodgkin lymphoma (cHL) is characterized by a small fraction of Hodgkin and Reed-Sternberg (HRS) tumor cells (~1%) which are surrounded by an extensive immune infiltrate. The rare nature of HRS cells limits the ability to study the genomics of cHL using standard platforms. To circumvent this, our group has optimized fluorescence-activated cell sorting to isolate HRS cells and intratumor B- and T- cells and to perform whole exome sequencing (WES; Reichel, Blood 2015). To date, however, there have been no reports on whole genome sequencing (WGS) of cHL.Methods: We performed flow-sorting of HRS cells and WGS to define the genomic landscape of cHL including: i) mutational processes involved in pathogenesis, ii) large and focal copy number variants, iii) structural variants including complex events, iv) the sequence and evolution of molecular events in cHL. We interrogated WGS from 25 cases of cHL: 10 pediatric patients (age<18), 9 adolescents and young adults (AYA, age 18-40), and 6 older adults (age>40). Intra-tumoral T-cells were used as germline control. An additional 36 cHL cases were evaluated by WES.Results: The average depth of coverage among the 25 WGS cases was 27.5x. After having identified and removed amplification-based palindromic sequencing artifacts, we observed a median of 5006 single base substitutions (SBS; range 1763-18436). Pediatric and AYA patients had a higher SBS burden compared to older adults (median 5279 vs. 2945, p=0.009). Five main SBS signatures were identified: SBS1 and SBS5 (aging), SBS2 and SBS13 (APOBEC), and SBS25 (chemotherapy, in a relapsed case). A dNdScv driver discovery analysis performed on the combined WES and WGS cases identified 24 driver genes including BCL7A and CISH which had not been previously reported as drivers in cHL.An investigation of copy number alterations (CNAs) confirmed high ploidy in cHL (median 2.95, range 1.66-5.33). Whole genome duplication was identified in 64% cases. We also observed clear evidence of complex events such as chromothripsis (n=4), double minutes (dm, n=2), breakage-fusion-bridge (bfb; n=4). Some of these events were responsible for the acquisition of distinct drivers. For example, we observed one dm and one bfb responsible for CD274 and REL gains, respectively (>10 copies).Leveraging the high prevalence of large chromosomal gains, we performed an investigation of the relative timing of acquisition of driver mutations. Clonal mutations within chromosomal gains can be defined as duplicated (VAF~66%; acquired before the gain) or non-duplicated (VAF~33%; acquired before or after the gain). Sixty-one percent (152/249) of driver genes were duplicated suggesting that they were acquired prior to large chromosomal gains. Next, we used the corrected ratio between duplicated and non-duplicated mutations within large chromosomal gains to estimate the molecular time of each duplicated segment (Rustad, Nat Comm 2020). In 11/22 genomes the final CNA profile was acquired through at least two temporally distinct events. To convert these relative estimations into absolute timing (i.e., the age at which events occurred), we leveraged the clock-like mutation signatures (SBS1, SBS5). We first confirmed that the SBS1 and SBS5 mutation rate were constant over time (R 2=0.84; p<0.0001 in Peds/AYA; R 2 =0.82; p=0.002 in older adults). We observed a higher mutation rate in Pediatric/AYA cases compared to older adults (p=0.01), which is consistent with the higher mutational burden observed in this age group. By estimating the SBS1- and SBS5-based molecular time for large chromosomal gains and converting relative estimates to absolute time, we are able to estimate the age in years at the time of the first multi-chromosomal gain event. We observed that the first multi-chromosomal gain in cHL is often acquired several years before the diagnosis/sample collection: median latency of 19.5 (range 12-27) and 5.6 (range 1.8-16) years in older adults and pediatric/AYA patients respectively.Conclusion: Here we report the first WGS in cHL. We identify novel drivers and genomic mechanisms involved in cHL pathogenesis. We found that mutations in driver genes are often acquired earlier then chromosomal gains, potentially preceding the cHL diagnosis by several years. In addition, we observed key differences in biology of cHL across age groups including accelerated mutagenesis and increased mutational burden among younger patients. DisclosuresMaura: OncLive: Honoraria; Medscape: Consultancy, Honoraria. Oberley: Caris LIfe Science: Current Employment. Lim: EUSA Pharma: Honoraria. Landgren: Janssen: Other: IDMC; Celgene: Research Funding; Janssen: Honoraria; Amgen: Honoraria; Janssen: Research Funding; Amgen: Research Funding; Takeda: Other: IDMC; GSK: Honoraria. Moskowitz: Merck & Co., Inc.: Research Funding. Roshal: Celgene: Other: Provision of services; Auron Therapeutics: Other: Ownership / Equity interests; Provision of services; Physicians' Education Resource: Other: Provision of services. Elemento: Owkin: Consultancy, Other: Current equity holder; AstraZeneca: Research Funding; Champions Oncology: Consultancy; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; One Three Biotech: Consultancy, Other: Current equity holder; Eli Lilly: Research Funding; Johnson and Johnson: Research Funding; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Janssen: Research Funding. Roth: Janssen: Consultancy; Merck: Consultancy.

1029TiP Phase II trial of pembrolizumab (pembro) and brentuximab vedotin (BV) in patients with metastatic solid malignancies after progression on prior programmed cell death protein (PD)-1 inhibitors (SGN35-033)

Intratumoral regulatory T-cells (Tregs) contribute to resistance to checkpoint inhibitors through various mechanisms (Saleh 2019). Intratumoral Tregs have a unique phenotype expressing higher levels of CD30 than other T-cells and non-tumor infiltrating Tregs in several malignancies including non-small cell lung cancer (NSCLC; De Simone 2016, Vasanthakumar 2017). BV (ADCETRIS®), a CD30-directed antibody-drug conjugate, has been shown to reduce Tregs in clinical trials of classical Hodgkin’s lymphoma and cutaneous T-cell lymphoma, to be uniquely toxic to CD30-expressing Tregs, and to allow for expansion of co-cultured CD8 T-cells in vitro (Romano 2019, Heister 2018 [AACR]).

Associations of immune cell homing gene signatures and infiltrates of lymphocyte subsets in human melanomas: discordance with CD163+ myeloid cell infiltrates

BackgroundImmune cells in the tumor microenvironment have prognostic value. In preclinical models, recruitment and infiltration of these cells depends on immune cell homing (ICH) genes such as chemokines, cell adhesion molecules, and integrins. We hypothesized ICH ligands CXCL9-11 and CCL2-5 would be associated with intratumoral T-cells, while CXCL13 would be more associated with B-cell infiltrates.MethodsSamples of human melanoma were submitted for gene expression analysis and immune cells identified by immunohistochemistry. Associations between the two were evaluated with unsupervised hierarchical clustering using correlation matrices from Spearman rank tests. Univariate analysis performed Mann–Whitney tests.ResultsFor 119 melanoma specimens, analysis of 78 ICH genes revealed association among genes with nonspecific increase of multiple immune cell subsets: CD45+, CD8+ and CD4+ T-cells, CD20+ B-cells, CD138+ plasma cells, and CD56+ NK-cells. ICH genes most associated with these infiltrates included ITGB2, ITGAL, CCL19, CXCL13, plus receptor/ligand pairs CXCL9 and CXCL10 with CXCR3; CCL4 and CCL5 with CCR5. This top ICH gene expression signature was also associated with genes representing immune-activation and effector function. In contrast, CD163+ M2-macrophages was weakly associated with a different ICH gene signature.ConclusionThese data do not support our hypothesis that each immune cell subset is uniquely associated with specific ICH genes. Instead, a larger set of ICH genes identifies melanomas with concordant infiltration of B-cell and T-cell lineages, while CD163+ M2-macrophage infiltration suggesting alternate mechanisms for their recruitment. Future studies should explore the extent ICH gene signature contributes to tertiary lymphoid structures or cross-talk between homing pathways.

An Engineered IL15 Cytokine Mutein Fused to an Anti-PD1 Improves Intratumoral T-cell Function and Antitumor Immunity.

The use of cytokines for immunotherapy shows clinical efficacy but is frequently accompanied by severe adverse events caused by excessive and systemic immune activation. Here, we set out to address these challenges by engineering a fusion protein of a single, potency-reduced, IL15 mutein and a PD1-specific antibody (anti-PD1-IL15m). This immunocytokine was designed to deliver PD1-mediated, avidity-driven IL2/15 receptor stimulation to PD1+ tumor-infiltrating lymphocytes (TIL) while minimally affecting circulating peripheral natural killer (NK) cells and T cells. Treatment of tumor-bearing mice with a mouse cross-reactive fusion, anti-mPD1-IL15m, demonstrated potent antitumor efficacy without exacerbating body weight loss in B16 and MC38 syngeneic tumor models. Moreover, anti-mPD1-IL15m was more efficacious than an IL15 superagonist, an anti-mPD-1, or the combination thereof in the B16 melanoma model. Mechanistically, anti-PD1-IL15m preferentially targeted CD8+ TILs and single-cell RNA-sequencing analyses revealed that anti-mPD1-IL15m treatment induced the expansion of an exhausted CD8+ TIL cluster with high proliferative capacity and effector-like signatures. Antitumor efficacy of anti-mPD1-IL15m was dependent on CD8+ T cells, as depletion of CD8+ cells resulted in the loss of antitumor activity, whereas depletion of NK cells had little impact on efficacy. The impact of anti-hPD1-IL15m on primary human TILs from patients with cancer was also evaluated. Anti-hPD1-IL15m robustly enhanced the proliferation, activation, and cytotoxicity of CD8+ and CD4+ TILs from human primary cancers in vitro, whereas tumor-derived regulatory T cells were largely unaffected. Taken together, our findings showed that anti-PD1-IL15m exhibits a high translational promise with improved efficacy and safety of IL15 for cancer immunotherapy via targeting PD1+ TILs.See related Spotlight by Felices and Miller, p. 1110.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Non-terminally exhausted tumor-resident memory HBV-specific T cell responses correlate with relapse-free survival in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) often develops following chronic hepatitis B virus (HBV) infection and responds poorly to immune checkpoint blockade. Here, we examined the antigen specificities of HCC-infiltrating Tcells and their relevance to tumor control. Using highly multiplexed peptide-MHC tetramer staining of unexpanded cells from blood, liver, and tumor tissues from 46 HCC patients, we detected 91 different antigen-specific CD8+ Tcell populations targeting HBV, neoantigen, tumor-associated, and disease-unrelated antigens. Parallel high-dimensional analysis delineated five distinct antigen-specific tissue-resident memory T (Trm) cell populations. Intratumoral and intrahepatic HBV-specific Tcells were enriched for two Trm cell subsets that were PD-1loTOXlo, despite being clonally expanded. High frequencies of intratumoral terminally exhausted Tcells were uncommon. Patients with tumor-infiltrating HBV-specific CD8+ Trm cells exhibited longer-term relapse-free survival. Thus, non-terminally exhausted HBV-specific CD8+ Trm cells show hallmarks of active involvement and effective antitumor response, implying that these cells could be harnessed for therapeutic purposes.

Abstract NG12: From cholesterol regulation to tumor suppression: Pcsk9 as a novel target for cancer immunotherapy

Abstract NG12: From cholesterol regulation to tumor suppression: Pcsk9 as a novel target for cancer immunotherapy

Abstract SY17-01: Analysis of immune contexture to refine prognosis in patients with colon cancer: Implications for immunotherapy

Abstract Current classification of colorectal cancer (CRC) is based on TNM staging which does not account for considerable within stage variability in clinical outcome. Currently, there is no immune-based classification of colon cancer although data suggest that intratumoral T-cell densities can provide prognostic information that is more robust than that has been provided by genomic or transcriptomic profiling. A subset of CRCs show microsatellite instability (MSI) that is a predictive biomarker for immune checkpoint inhibitors whose indication in CRC is limited to metastatic tumors. In a prior study, we compared the density and localization of CD3+ and cytotoxic CD8+ T-cell densities in non-metastatic colon cancers with deficient (dMMR) vs proficient (pMMR) DNA mismatch repair (MMR). CD3+ at the tumor invasive margin (IM) was most strongly associated with patient overall survival, and dichotomization of CD3+ prognostically stratified both dMMR and pMMR tumors. The ability to stratify dMMR tumors was a novel observation and revealed a poorer prognosis dMMR group. By multivariable (MV) analysis, CD3+ density and N (nodal) stage contributed the most to patient survival. In separate studies, expression of FOXP3+ Tregs in CRC has paradoxically been associated with more favorable survival. Interestingly, we found that colon cancers with low FOXP3+ and low CD8+ are a very poor prognostic subset. Immunoscore (IS; HalioDx) is a validated assay that examines CD3+ and CD8+ using digital pathology with conversion of densities into percentile scores using a software algorithm. In patients with stage III colon cancers from a completed adjuvant trial, recurrence rates were inversely related to IS score (0-4), and the dichotomized IS was associated with significantly better survival across all disease subgroups (Forest plot). Stage III colon cancers are categorized into low risk (T1-3N1) and high risk (T4 and/or N2) groups for guiding the duration of adjuvant chemotherapy (3 vs 6 months). IS was shown to prognostically stratify patients within both risk groups and was only a significant variable in low risk tumors by MV analysis. These data suggest the potential of IS to further risk stratify patients and to potentially inform treatment decisions. In patients with metastatic CRC, analysis of IS was shown to predict tumor metastatic burden which had prognostic value. Data also suggest that potential for IS to provide predictive information for chemotherapy (fluoropyrimidine + oxaliplatin) response in CRC, yet further studies are needed. A potential mechanism is immunogenic cell death induced by some anti-cancer drugs, including oxaliplatin. Regarding immune checkpoint inhibitors (ICPs), only 40-50% of patients with metastatic CRC respond to such therapy, and small studies in suggest that TIL densities may be a factor in observed variability in response to ICP inhibitors. To further explore this issue, we are examining tumor immunophenotypes in relationship to response and outcome in CRC patients treated with anti-PD-1 antibodies. We aim to develop a MV model, and to validate it in an independent dataset. Important and relevant issues include the fact that only a fraction of mutations result in neoantigens recognized by T-cells, the potential for poor presentation of neoantigens by MHC1, as well as activating or suppressive cytokines. Citation Format: Frank A. Sinicrope. Analysis of immune contexture to refine prognosis in patients with colon cancer: Implications for immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr SY17-01.

Pivotal factors associated with the immunosuppressive tumor microenvironment and melanoma metastasis.

BackgroundConsidering melanoma is the deadliest malignancy among dermatoma and presently lacks effective therapies, there is an urgent need to investigate the potential mechanisms underlying melanoma metastasis and determine prospective therapeutic targets for precise treatment of melanoma.MethodHub genes in melanoma metastasis were identified by analyzing RNA‐seq data (mRNA, miRNA, and lncRNA) obtained from TCGA database. Then the identified hub genes were validated in human tissues with qRT‐PCR, followed by survival analysis. Competing endogenous RNAs of the hub genes were defined to clarify potential molecular mechanism of melanoma progression. Then central gene‐related signaling pathways were analyzed, followed by immune cell abundance analysis in tumor microenvironment with CYTERSORTx.ResultA tetrad of IL2RA, IL2RG, IFNG, and IL7R genes were determined as hub genes and verified by qRT‐PCR, which were significantly associated with unfavorable prognosis in melanoma. LINC02446, LINC01857, and LINC02384 may act as competing endogenous lncRNAs of IL2RA and IL7R through absorbing their shared miR.891a.5p and miR.203b.3p. JAK—STAT signaling pathway identified as the most relevant pathway in melanoma metastasis, as well as a wealthy of genes including TNFRSF 13B, TNFRSF17, TNFRSF9, TNFRSF8, TNFRSF13C, TNFRSF11B, LAG3, NRP1, ENTPD1, NT5E, CCL21, and CCR7, may induce tumor autoimmune suppression through enhancing regulatory T‐cell abundance and performance in the tumor microenvironment. And regulatory T‐cell proportion was indeed critically elevated in metastatic melanoma relative to primary melanoma, as well as in highly expressed IL2RA, IL2RG, IL7R, and IFNG group than their respective counterparts.ConclusionElevated IL2RA, IL2RG, IL7R, and IFNG expression may play a central role in promoting melanoma metastasis through up regulation of intratumoral regulatory T—cell proportion mainly by activation of JAK—STAT signaling pathway. LINC02446, LINC01857, and LINC02384 may stimulate melanoma progression by reducing tumor‐protecting miR.891a.5p and miR.203b.3p. A number of identified molecules including TNFRSF13B, LAG3, NRP1, ENTPD1, NT5E, CCL21, and CCR7 can serve as future therapeutic targets in melanoma treatment.

Infiltration by CXCL10 Secreting Macrophages Is Associated With Antitumor Immunity and Response to Therapy in Ovarian Cancer Subtypes.

Ovarian carcinomas (OCs) are poorly immunogenic and immune checkpoint inhibitors (ICIs) have offered a modest benefit. In this study, high CD3+ T-cells and CD163+ tumor-associated macrophages (TAMs) densities identify a subgroup of immune infiltrated high-grade serous carcinomas (HGSCs) with better outcomes and superior response to platinum-based therapies. On the contrary, in most clear cell carcinomas (CCCs) showing poor prognosis and refractory to platinum, a high TAM density is associated with low T cell frequency. Immune infiltrated HGSC are characterized by the 30-genes signature (OC-IS30) covering immune activation and IFNγ polarization and predicting good prognosis (n = 312, TCGA). Immune infiltrated HGSC contain CXCL10 producing M1-type TAM (IRF1+pSTAT1Y701+) in close proximity to T-cells. A fraction of these M1-type TAM also co-expresses TREM2. M1-polarized TAM were barely detectable in T-cell poor CCC, but identifiable across various immunogenic human cancers. Single cell RNA sequencing data confirm the existence of a tumor-infiltrating CXCL10+IRF1+STAT1+ M1-type TAM overexpressing antigen processing and presentation gene programs. Overall, this study highlights the clinical relevance of the CXCL10+IRF1+STAT1+ macrophage subset as biomarker for intratumoral T-cell activation and therefore offers a new tool to select patients more likely to respond to T-cell or macrophage-targeted immunotherapies.

IL-6 signaling in macrophages is required for immunotherapy-driven regression of tumors

BackgroundHigh serum interleukin (IL-6) levels may cause resistance to immunotherapy by modulation of myeloid cells in the tumor microenvironment. IL-6 signaling blockade is tested in cancer, but as this inflammatory...