High T-cell Infiltration Research Articles

Whole genome sequencing refines stratification and therapy of patients with clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer, but a comprehensive description of its genomic landscape is lacking. We report the whole genome sequencing of 778 ccRCC patients enrolled in the 100,000 Genomes Project, providing for a detailed description of the somatic mutational landscape of ccRCC. We identify candidate driver genes, which as well as emphasising the major role of epigenetic regulation in ccRCC highlight additional biological pathways extending opportunities for therapeutic interventions. Genomic characterisation identified patients with divergent clinical outcome; higher number of structural copy number alterations associated with poorer prognosis, whereas VHL mutations were independently associated with a better prognosis. The observations that higher T-cell infiltration is associated with better overall survival and that genetically predicted immune evasion is not common supports the rationale for immunotherapy. These findings should inform personalised surveillance and treatment strategies for ccRCC patients.

An update on central nervous system tumors in germline replication-repair deficiency syndromes.

DNA replication-repair deficiency (RRD) arises from pathogenic variants in the mismatch repair and/or polymerase-proofreading genes. Multiple germline cancer predisposition syndromes in children and young adults, including constitutional mismatch repair deficiency (CMMRD), Lynch, polymerase-proofreading deficiency, and rare digenic syndromes can lead to RRD cancers. The most frequent brain tumors in these children are high-grade gliomas. Embryonal tumors like medulloblastoma have also been described. Lower-grade tumors are reported from cancer surveillance initiatives. The latter has an extremely high rate of malignant transformation. Novel functional assays quantifying the genomic microsatellite indel load have been demonstrated to be highly sensitive and specific for the diagnosis of RRD cancers and children with germline CMMRD. Importantly, RRD brain tumors uniformly harbor high mutation and microsatellite burden. High T-cell infiltration makes these aggressive cancers amenable to immune checkpoint inhibition, irrespective of their germline genetic background. Synergistic combinations are reported to be successful in patients failing checkpoint inhibitor monotherapy. Future directions include the development of innovative approaches to improve immune surveillance for RRD brain cancers. Additionally, the use of novel tools including circulating tumor DNA and quantifying microsatellite indel load over time can be useful to monitor disease burden and treatment responses in patients.

Multi-omics integration analysis unveils heterogeneity in breast cancer at the individual level

ABSTRACT Identifying robust breast cancer subtypes will help to reveal the cancer heterogeneity. However, previous breast cancer subtypes were based on population-level quantitative gene expression, which is affected by batch effects and cannot be applied to individuals. We detected differential gene expression, genomic, and epigenomic alterations to identify driver differential expression at the individual level. The individual driver differential expression reflected the breast cancer patients’ heterogeneity and revealed four subtypes. Mesenchymal subtype as the most aggressive subtype harbored deletion and downregulated expression of genes in chromosome 11q23 region. Specifically, silencing of the SDHD gene in 11q23 promoted the invasion and migration of breast cancer cells in vitro by the epithelial–mesenchymal transition. The immunologically hot subtype displayed an immune-hot microenvironment, including high T-cell infiltration and upregulated PD-1 and CTLA4. Luminal and genomic-unstable subtypes showed opposite macrophage polarization, which may be regulated by the ligand–receptor pairs of CD99. The integration of multi-omics data at the individual level provides a powerful framework for elucidating the heterogeneity of breast cancer.

Immunologic Significance of CD80/CD86 or Major Histocompatibility Complex-II Expression in Thymic Epithelial Tumors

IntroductionUnresectable or recurrent thymic epithelial tumors (TETs) have a poor prognosis, and treatment options are limited. This study aimed to investigate the immunologic significance of CD80/CD86 or major histocompatibility complex class II (MHC-II) expression in TETs, as potential predictive biomarkers for immune checkpoint inhibitors (ICIs). MethodsWe analyzed CD80, CD86, MHC class I (MHC-I), and MHC-II expression in TETs using immunohistochemistry and investigated their association with T-cell infiltration or ICI efficacy. In addition, we generated CD80- or MHC-II–expressing mouse tumors, evaluated the effects of ICIs, and analyzed tumor-infiltrating lymphocytes. We also performed tumor-rechallenge experiments in vivo. ResultsWe found that approximately 50% and 30% of TETs had high expression of CD80/CD86 and MHC-II in tumor cells, respectively, and that this expression was related to T-cell infiltration in clinical samples. In mouse models, both CD80 and MHC-II increase the effects of ICIs. In addition, senescent T cells and long-lived memory precursor effector T cells were significantly decreased and increased, respectively, in tumor-infiltrating lymphocytes from CD80-expressing tumors, and rechallenged tumors were completely rejected after the initial eradication of CD80-expressing tumors by programmed cell death protein 1 blockade. Indeed, patients with CD80-high thymic carcinoma had longer progression-free survival with anti–programmed cell death protein 1 monoclonal antibody. ConclusionsHalf of the TETs had high expression of CD80/CD86 or MHC-II with high T-cell infiltration. These molecules could potentially increase the effects of ICIs, particularly inducing a durable response. CD80/CD86 and MHC-II can be predictive biomarkers of ICIs in TETs, promoting the development of drugs for such TETs.

Immune checkpoint inhibitor treatment of brain metastasis associated with a less invasive growth pattern, higher T-cell infiltration and raised tumor ADC on diffusion weighted MRI

BackgroundBrain metastases are the most common intracranial tumors with an increasing incidence. They are an important cause of morbidity and mortality in patients with solid organ cancer and a focus of recent clinical research and experimental interest. Immune checkpoint inhibitors are being increasingly used to treat solid organ cancers.MethodsTo determine whether immune checkpoint inhibitors were biologically effective in the brain, we compared melanoma brain metastasis samples where treatment with ipilimumab had occurred preoperatively to those who had not received any immune modulating therapy and looked for histopathological (invasion, vascularity, metastasis inducing proteins, matrix metalloproteinases, immune cell infiltration, tissue architecture) and advanced MRI differences (diffusion weighted imaging).ResultsCo-localized tissue samples from the same regions as MRI regions of interest showed significantly lower vascularity (density of CD34 + vessels) in the core and higher T-cell infiltration (CD3 + cells) in the leading edge for ipilimumab-treated brain metastasis samples than for untreated cases and this correlated with a higher tumor ADC signal at post-treatment/preoperative MRI brain.ConclusionsTreatment of a melanoma brain metastasis with ipilimumab appears to cause measurable biological changes in the tumor that can be correlated with post-treatment diffusion weighted MRI imaging, suggesting both a mechanism of action and a possible surrogate marker of efficacy.

Whole-body bioluminescence imaging of T-cell response in PDAC models.

The location of T-cells during tumor progression and treatment provides crucial information in predicting the response in vivo. Here, we investigated, using our bioluminescent, dual color, T-cell reporter mouse, termed TbiLuc, T-cell location and function during murine PDAC tumor growth and checkpoint blockade treatment with anti-PD-1 and anti-CTLA-4. Using this model, we could visualize T-cell location and function in the tumor and the surrounding tumor microenvironment longitudinally. We used murine PDAC clones that formed in vivo tumors with either high T-cell infiltration (immunologically 'hot') or low T-cell infiltration (immunologically 'cold'). Differences in total T-cell bioluminescence could be seen between the 'hot' and 'cold' tumors in the TbiLuc mice. During checkpoint blockade treatment we could see in the tumor-draining lymph nodes an increase in bioluminescence on day 7 after treatment. In the current work, we showed that the TbiLuc mice can be used to monitor T-cell location and function during tumor growth and treatment.

Activated tissue resident memory T-cells (CD8+CD103+CD39+) uniquely predict survival in left sided "immune-hot" colorectal cancers.

Characterization of the tumour immune infiltrate (notably CD8+ T-cells) has strong predictive survival value for cancer patients. Quantification of CD8 T-cells alone cannot determine antigenic experience, as not all infiltrating T-cells recognize tumour antigens. Activated tumour-specific tissue resident memory CD8 T-cells (TRM) can be defined by the co-express of CD103, CD39 and CD8. We investigated the hypothesis that the abundance and localization of TRM provides a higher-resolution route to patient stratification. A comprehensive series of 1000 colorectal cancer (CRC) were arrayed on a tissue microarray, with representative cores from three tumour locations and the adjacent normal mucosa. Using multiplex immunohistochemistry we quantified and determined the localization of TRM. Across all patients, activated TRM were an independent predictor of survival, and superior to CD8 alone. Patients with the best survival had immune-hot tumours heavily infiltrated throughout with activated TRM. Interestingly, differences between right- and left-sided tumours were apparent. In left-sided CRC, only the presence of activated TRM (and not CD8 alone) was prognostically significant. Patients with low numbers of activated TRM cells had a poor prognosis even with high CD8 T-cell infiltration. In contrast, in right-sided CRC, high CD8 T-cell infiltration with low numbers of activated TRM was a good prognosis. The presence of high intra-tumoural CD8 T-cells alone is not a predictor of survival in left-sided CRC and potentially risks under treatment of patients. Measuring both high tumour-associated TRM and total CD8 T-cells in left-sided disease has the potential to minimize current under-treatment of patients. The challenge will be to design immunotherapies, for left-sided CRC patients with high CD8 T-cells and low activate TRM,that result in effective immune responses and thereby improve patient survival.

Abstract 6418: xWU-NK-101 as salvage therapy post immune checkpoint blockade (ICB)

Abstract ICB therapy has transformed cancer treatment; durable responses in difficult-to-treat cancers have been observed. Despite this most patients (pts) don’t respond to ICB (primary resistance), and many pts who initially respond eventually relapse (acquired resistance). Primary resistance is associated with tumor cell extrinsic factors, e.g., the immunosuppressive nature of the tumor microenvironment (TME), while acquired resistance is associated with tumor intrinsic factors, e.g., downregulation of MHC1, preventing T cell recognition. Treatments to overcome ICB resistance are necessary. WU-NK-101 is a PBMC-derived, cytokine-reprogrammed, expanded, and cryopreserved off-the-shelf memory NK cell product. WU-NK-101 cells capture the memory-like NK cell biology of cytokine-induced memory-like (CIML) NK cells, exhibiting enhanced cytotoxicity, metabolic fitness/flexibility, and resistance to TME immunosuppression (Muth et al. EHA 2022; Rutella et al. ESMO 2022). Bone marrow biopsies, collected from R/R AML pts who received CIML-NK cells (NCT01898793), were interrogated using immune gene expression (GE) profiling and spatially resolved proteomics (IO360® panel, n = 740 genes, and GeoMx® DSP; NanoString Technologies). Higher T-cell infiltration was noted post CIML-NK. CIBERSORT deconvoluted GE data inferred higher abundance of macrophages, γδT cells and activated dendritic cells on day 28 post-treatment. GE signatures showed downregulation of NFIL3 and FAM30A (log2 fold-change <1.0; p<0.05) post CIML-NK. TIDE algorithm modelling indicated that lower expression of NFIL3 and FAM30A correlated with high CTL infiltration and improved outcomes in many TCGA tumors (Jiang et al. Nat. Med. 2018). Hence, CIML-NK treatment led to modifications in the TME towards a more T-cell amenable environment. Deleting MHC1 on NALM6 cells significantly improved WU-NK-101 killing compared to WT (p<0.0001), highlighting one mechanism whereby WU-NK-101 overcomes acquired ICB resistance. In transwell assays, co-incubation of tumor cells with WU-NK-101 in the lower chamber led to dose dependent killing; in the upper chamber (tumor cells alone), viability was preserved but a dose-dependent increase of MHC1/PDL1 expression was noted. Similar results were observed using cell-free conditioned media from WU-NK-101 cytotoxicity assays, suggesting that soluble factors released from WU-NK-101 augment MHC1/PDL1 expression. Treatment of tumor cells with IFNγ alone led to similar increases in MHC1/PDL1 expression. Importantly, in vivo experiments revealed that residual tumor cells post WU-NK-101 treatment exhibited higher levels of MHC1/PDL1. Overall, these data highlight a further mechanism through which WU-NK-101 overcomes acquired ICB resistance. In summary, WU-NK-101 has the potential to reverse primary and acquired mechanisms of ICB resistance. A Phase 1b clinical trial of WU-NK-101 as salvage therapy post-ICB is in development. Citation Format: Tom A. Leedom, Barbara Muz, Jaykumar Vadakekolathu, John J. Muth, Xiao-Hua Li, Gregory Watson, Kristaan Magee, Ryan P. Sullivan, Melissa Berrien-Elliott, Todd Fenigher, Sergio Rutella, Matthew L. Cooper, Ayman Kabakibi, Jan K. Davidson-Moncada. xWU-NK-101 as salvage therapy post immune checkpoint blockade (ICB) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6418.

Abstract 6619: Virtual staining enabled combined morphological and spatial transcriptomic analysis of individual malignant B cells and local tumor microenvironments

Abstract B-cell lymphomas, commonly classified by histological presentation and B-cell lineage, are in fact heterogeneous diseases with respect to gene expression and tumor microenvironment. Certain lymphomas, such as Hodgkin and T-cell rich B-cell lymphoma, may contain fewer than 1% malignant cells in a largely inflammatory background. These rare malignant cells have unique interactions with local tumor microenvironments (TME), in some cases driving distinct gene expression patterns for individual B-cells. This presents a challenging fundamental biology landscape, which can create uncertainty in directing patient care or developing new therapies. The lack of tools allowing subtyping of malignant B-cells while simultaneously preserving the context of local TMEs and providing deep spatial transcriptomic sequencing further complicates analysis. Here we have developed an integrated, single-slide workflow of virtual H&E staining using the inherent tissue autofluorescence (PictorLabs, Los Angeles, CA) of formalin-fixed, paraffin embedded lymphoma tissue sections on the GeoMx® Digital Spatial Profiler (Nanostring, Seattle, WA) combined with spatial expression analysis of 1,800 genes using the Cancer Transcriptome Atlas (CTA) panel. The H&E virtual staining allows for the assessment of B-cell lymphoma subtypes, identification and phenotyping of malignant B-cells and the immediately proximal (within 30µm) TME regions by a certified pathologist. These regions of interest (ROI) are spatially profiled for transcriptional expression determined by NGS sequencing of the RNA target specific DNA oligo tags. We benchmarked our system by analyzing four lymphoma cases: Nodular sclerosis classical Hodgkin, Nodular lymphocyte-predominant Hodgkin, T-cell/histiocyte rich large B-cell, and Diffuse large B-cell lymphomas. Using virtual H&E staining on the GeoMx Digital Spatial Profiler platform, a pathologist correctly identified the diagnosis and subtype based on the morphology. Regions of inflammatory B- and T-cells could be distinguished from lymphoma cells on virtual staining, allowing subset segregation for digital transcriptional expression profiling with the GeoMx CTA panel. Additionally, background TME more distant to B-cell lymphoma as well as regions of high T-cell infiltration were selected for profiling. Analysis of the ROIs revealed distinct transcriptional profiles of malignant B-cells (including cell-of-origin signature), the signaling contributions of local TMEs (versus non-adjacent regions) and confirmed that the expression patterns are further influenced by the presence of T-cell infiltration. Citation Format: Michael E. Kallen, Alyssa Rosenbloom, Zihang Fang, Raymond Kozikowski, Yair Rivenson, Kevin de Haan, Serge Alexanian, Mark Conner, Daniel Newhouse, Felicia New, Yan Liang, Kyla Teplitz, Joachim Schmid, Jaemyeong Jung. Virtual staining enabled combined morphological and spatial transcriptomic analysis of individual malignant B cells and local tumor microenvironments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6619.

Abstract 1320: Immunogenomic analysis identifies the complement pathway as a therapeutic target in STK11 mutant non-small cell lung cancer

Abstract Background: The use of immune PD-1/-L1 checkpoint inhibitors (ICI) has dramatically altered the treatment of advanced non-small cell lung cancer (NSCLC). However, a large proportion of patients with NSCLC do not derive clinical benefit from ICI treatment. Recent studies have identified certain genomic subsets of NSCLC as drivers of primary resistance to ICI treatment. Methods: We evaluated NSCLC patients treated at Roswell Park Comprehensive Cancer Center from 2017-2020 with successful genomic and immune profiling using a CLIA-certified laboratory developed test that included targeted next-generation genomic sequencing, PD-L1 assessment by immunohistochemistry (IHC), and targeted RNA-seq of 394 immune transcripts. Results: A total of 379 treatment-naive non-squamous NSCLC subjects were identified with 113 subjects treated with an FDA approved ICI regimen and evaluable for response. Using a gene expression signature based on differential gene expression analysis in the 113 subjects with and without disease control with ICI treatment, we performed unsupervised clustering of the larger 379 subject cohort and identified a responder (R) cluster and non-responder (NR) cluster. The R cluster was associated with high PD-L1 expression, T-cell infiltration, and KRAS mutations. The NR cluster was associated with negative PD-L1 expression, lack of T-cell infiltration, and mutations in STK11, APC/CTNNB1, and NFE2L2. Of the mutation subsets, STK11 was most associated with NR with a trend towards lower PD-L1 expression by clinical IHC, but much more significantly associated by gene expression signatures with low PD-L1 and T-cell inflammation. In our gene panel, we identified elevated TRIM29 expression in STK11 mutant NSCLC tumors and confirmed TRIM29 expression by IHC on a separate 64 subject cohort with known STK11 mutation status and archival formalin fixed paraffin embedded (FFPE) tissue. A subset of STK11 mutant NSCLC cell lines also demonstrated elevated TRIM29 expression and we generated knockouts of TRIM29 with CRISPR-Cas9 in these cell lines which resulted in decreased tumor growth in xenograft models. Using Nanostring nCounter multiplex inflammation gene expression panel on both the 64 patient FFPE cohort and TRIM29 knockout cell lines, we identified the innate immune complement pathway as a target of therapeutic interest and specifically the high expression of complement component C3 by TRIM29 high tumor cells. We then generated a syngeneic mouse model where loss of Stk11 resulted in elevated expression of Trim29 and C3 for preclinical studies to evaluate the therapeutic modulation of the complement pathway on response to ICI. Conclusion: Using a large clinical cohort of non-squamous NSCLC patients we characterized the immune environment of mutational subsets of NSCLC and identified the complement pathway as a potential therapeutic target in STK11 mutant NSCLC. Citation Format: Edwin H. Yau, Bojidar Kandar, Lei Deng, Te-An Chen, Wiam Bshara, Sean Glenn, Sarabjot Pabla, Antonios Papanicolau-Sengos, Mary Nesline, Hongbin Chen, Amy Early, Carl Morrison, Grace Dy. Immunogenomic analysis identifies the complement pathway as a therapeutic target in STK11 mutant non-small cell lung cancer [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 1320.

New Artificial Intelligence Score and Immune Infiltrates as Prognostic Factors in Colorectal Cancer With Brain Metastases.

Incidence of brain metastases has increased in patients with colorectal cancer (CRC) as their survival has improved. CD3 T-cells and, lately, DGMate (DiGital tuMor pArameTErs) score, have been identified as prognostic factors in locally advanced CRC. Until now, there is no data concerning the prognostic value of these markers in patients with CRC-derived brain metastases. All consecutive patients with CRC-derived brain metastases diagnosed between 2000 and 2017 were retrospectively included. Staining for CD3, CD8, PD-1, PD-L1 and DGMate analyses were performed using tissue micro-array from primary tumors and, if available, brain metastases. All in all, 83 patients were included with 80 primary tumor samples and 37 brain metastases samples available. CD3 and CD8 T-cell infiltration was higher in primary tumors compared to brain metastases. We observed a significant higher DGMate score in rectal tumors compared to colon tumors (p=0.03). We also noted a trend of higher CD3 T-cell infiltration in primary tumors when brain metastases were both supra and subtentorial compared to brain metastases that were only subtentorial or supratentorial (p=0.36 and p=0.03, respectively). No correlation was found between CD3 or CD8 infiltration or DGMate score in primary tumors or brain metastases and overall survival (OS) in the overall population. In patients with rectal tumors, a high DGMate score in brain metastases was associated with longer OS (13.4 ± 6.1 months versus 6.1 ± 1.4 months, p=0.02). High CD3 T-cell infiltration in brain metastases was associated with lower OS in patients with supratentorial brain metastases (9.8 ± 3.3 months versus 16.7 ± 5.9 months, p=0.03). PD-L1 overexpression was rare, both in primary tumors and brain metastases, but PD-L1 positive primary tumors were associated with worse OS (p=0.01). In contrast to breast and lung cancer derived brain metastases, CD3 and CD8 infiltration and DGMate score are not major prognostic factors in patients with CRC-derived brain metastases.

Fresh Tissue Multi-omics Profiling Reveals Immune Classification and Suggests Immunotherapy Candidates for Conventional Chondrosarcoma.

There is still no standard nonsurgical regimen for conventional chondrosarcoma (CHS). We aimed to identify whether any CHSs have a favored microenvironment for immunotherapy via multidimensional evaluation of the immunologic characteristics of this tumor. We obtained 98 newly-diagnosed CHS fresh tumors from several institutions and performed comprehensive analysis of data from CyTOF, whole-exome sequencing, and flow cytometry in 22 cases. Clinical data from immunotherapy responders and nonresponders were compared to explore possible biomarkers of immunotherapy response. Mechanism studies were conducted to interpret the biomarker phenotype. Based on the integrated data of single-cell CyTOF and flow cytometry, the CHS immune-microenvironment phenotypes were classified into three groups: subtype I, the "granulocytic-myeloid-derived suppressor cell (G-MDSC) dominant" cluster, with high number of HLA-DR- CD14- myeloid cells; subtype II, the "immune exhausted" cluster, with high exhausted T-cell and dendritic-cell infiltration; and subtype III, the "immune desert" cluster, with few immune cells. Immune cell-rich subtypes (subtype I and II) were characterized by IDH mutation, pathologic high grade, and peritumoral edema, while subtype I cases were exclusively featured by myxoid transformation. In clinical practice involving 12 individuals who received PD-1 antibody immunotherapy, all of the 3 cases with controlled diseases were retrospectively classified as subtype II. In mechanism, IDH mutation significantly elevated chemokine levels and immune-cell infiltration in immune-inactivated tumors. This study is the first to provide immune characterization of CHS, representing a major step to precise immunotherapy against this malignancy. Immunotherapy is promising for the "immune exhausted" subtype of patients with CHS.

High T-cell infiltration in tumor tissue and younger age predict the response to pembrolizumab in recurrent urothelial cancer.

Targeting the programmed cell death-1 signaling pathway has been approved for the anti-cancer therapy in several cancers including urothelial cancer. To determine predictive factors of the responsiveness to pembrolizumab in urothelial cancer patients, a retrospective study that used clinical information and paraffin-embedded samples obtained from patients diagnosed with urothelial cancer between 2015 and 2020 were performed. Seventeen patients who underwent total cystectomy or nephroureterectomy of the primary lesion and were treated with pembrolizumab for chemo-resistant disease were enrolled, and immunohistochemical analysis was performed. A key difference in the characteristics between the non-responder group and the responder group was the age of the patients (74 vs. 63years, p = 0.0194). Although there was no statistically significant difference, the histological subtype with sarcomatoid and micropapillary components was only seen in the non-responder group, and squamous differentiation and lymph node metastasis were only seen in cases with a complete response. In the results of immunohistochemistry, the density of CD8-positive T-cells and Tregs was significantly increased in the responder group than in the non-responder group. In conclusion, younger age and a high number of tumor-infiltrating lymphocytes were predictive factors of a good response to immune checkpoint inhibitors, although further studies with more enrolled patients are necessary.

Increased infiltration of CD8 T cells in recurrent glioblastoma patients is a useful biomarker for assessing the response to combined bevacizumab and lomustine therapy

PurposeTreatment options for recurrent glioblastoma (rGBM) remain scarce, which may be due to the limited understanding of its molecular characteristics. MethodsBased on gene expression profiling, the infiltration scores of 26 immune cell types were calculated using gene set variation analysis. The differences between rGBM and other cancer subtypes were estimated to characterize the specific immune characteristics of rGBM, and the prognostic value of immune cells in rGBM was estimated using univariate and multivariate Cox analysis. Subgroup analyses and Kaplan-Meier analyses were performed to identify whether CD8 T-cell infiltration could be useful in selecting treatment options for rGBM patients. ResultsWe found that rGBM patients were associated with enrichment of activated CD8 T cells, and high CD8 T-cell infiltration was associated with superior overall survival. Patients exhibiting high CD8 T-cell infiltration who received treatment with bevacizumab and lomustine combination therapy experienced a significant benefit in overall survival and progression-free survival, whereas patients with low CD8 T-cell infiltration did not experience such a benefit. CD8 T cells remained an independent prognostic factor in multivariate analyses (cohort 1: hazard ratio [HR] = 0.546, 95% confidence interval [CI]: 0.316–0.945, P = 0.031; cohort 3: HR = 0.615, 95% CI: 0.387–0.978, P = 0.040) after adjusting for clinicopathological and molecular factors. ConclusionsActivated CD8 T-cells is a promising biomarker for predicting overall survival in rGBM patients and could be used for assisting treatment selection.

Safety, pharmacokinetics, efficacy, and preliminary biomarker data of first-in-class BI 765063, a selective SIRPα inhibitor: Results of monotherapy dose escalation in phase 1 study in patients with advanced solid tumors.

2623 Background: BI 765063 is a humanized IgG4 monoclonal antibody antagonist of SIRPα (Signal Regulatory Protein α), which blocks the “don't eat me” signal of the SIRPα/CD47 axis, a critical innate immune checkpoint. SIRPα is expressed on myeloid cells. BI 765063 binds to the V1 SIRPα allele with high affinity and to the V2 SIRPα allele with low affinity. BI 765063 lacks SIRPγ binding to preserve T-cell activation. We report results of the completed BI 765063 monotherapy dose escalation in patients with advanced solid tumors. Methods: This study involves a step 1 dose escalation to determine the dose-limiting toxicities (DLT) and maximum tolerated dose (MTD), then a step 2 dose-confirmation expansion at recommended phase 2 dose. In Step 1, BI 765063 ascending doses, given IV every 3 weeks, were tested using a Bayesian Logistic Regression Model (BLRM) approach with overdose control. The endpoints were safety, pharmacokinetics, receptor occupancy (RO) in peripheral CD14+ monocytes and efficacy (RECIST 1.1). Results: Fifty patients (26 V1/V1, 24 V1/V2) received at least one dose of BI 765063. The most frequent tumors were ovarian (9), colorectal (8), lung (5), breast (4), melanoma (3), and kidney (3). No DLTs were reported up to the highest dose tested. MTD was not reached. The most frequent related adverse events were infusion related reaction (IRR) (46%), fatigue (12%), headache (10%), arthralgia and diarrhea (8% each). All related adverse events were mild to moderate, except one case of IRR Grade 3. No related anemia nor thrombocytopenia were observed. BI 765063 showed dose proportional exposure and full RO saturation in Cycle 1 after the fourth dose level. Clinical benefit was observed in 21/47 (45%) patients evaluable per RECIST 1.1. One patient with hepatocellular carcinoma (HCC) with liver and lung metastases and 7 prior lines of therapy showed a durable partial response maintained for 27 weeks treatment (ongoing). The baseline tumor biopsy of this patient showed high CD8 T-cell and macrophage infiltration. There was an increase in CD8 T-cell infiltration and activation on treatment. An increase in PD-L1 expression on tumor cells 2 weeks after first dosing was also observed. Analysis of paired tumor biopsies in other patients is ongoing. Conclusions: The first-in-class SIRPα inhibitor BI 765063 was well-tolerated, showed monotherapy activity, and sustained RO saturation. A durable partial response was observed in an advanced HCC patient. The on-treatment biopsy of the responder showed an increase in CD8 T-cell infiltration and activation. PD-L1 expression on tumor cells also increased. BI 765063 dose escalation in combination with ezabenlimab (anti-PD1 antibody) is ongoing. Clinical trial information: NCT03990233.

Characterizing the tumor microenvironment of metastatic ovarian cancer by single-cell transcriptomics.

Understanding the cellular composition of the tumor microenvironment and the interactions of the cells is essential to the development of successful immunotherapies in cancer. We perform single-cell RNA sequencing (scRNA-seq) of 9,885 cells isolated from the omentum in 6 patients with ovarian cancer and identify 9 major cell types, including cancer, stromal, and immune cells. Transcriptional analysis of immune cells stratifies our patient samples into 2 groups: (1) high Tcell infiltration (high Tinf) and (2) low Tcell infiltration (low Tinf). TOX-expressing resident memory CD8+ T (CD8+ Trm) and granulysin-expressing CD4+ Tcell clusters are enriched in the high Tinf group. Concurrently, we find unique plasmablast and plasma B cell clusters, and finally, NR1H2+IRF8+ and CD274+ macrophage clusters, suggesting an anti-tumor response in the high Tinf group. Our scRNA-seq study of metastatic tumor samples provides important insights in elucidating the immune response within ovarian tumors.

High Stroma T-Cell Infiltration is Associated with Better Survival in Stage pT1 Bladder Cancer.

Stage pT1 bladder cancer (BC) shows highly diverse outcomes. Predictive markers are required to stratify patients for personalized treatment. The present study aimed to validate immune response quantification as a prognostic marker. Patients with pT1 BC (n = 167) treated by transurethral resection of the bladder (TURB) were enrolled. Formaldehyde-fixed paraffin-embedded material was stained for CD3 and CD8. Corresponding T cells were counted in three regions with the highest immune response. Numbers of tertiary lymphoid structures (TLS) and lymphocyte aggregates (LA) were quantified. High CD3+ stroma T-cell infiltration was associated with improved survival (p = 0.045), especially in the G3 subgroup (p = 0.01). Cluster with higher immune response showed less recurrence (p = 0.034) and favorable overall survival (OS) (p = 0.019). In contrast, higher CD3+ and CD8+ tumor T-cell infiltration seemed to have a negative impact on prognosis. TLS and LA were more frequently observed in G3 tumors, indicating an increased anti-tumoral immune response. We proved the role of immune cell infiltration and showed that higher infiltration numbers of CD3+ (not CD8+) lymphocytes in the stroma are associated with favorable outcome. Immune cell quantification could be used as a marker to help stratify patients’ risk and therefore, to optimize patients’ management and follow-up examination as well as possible therapies.

Abstract A11: Generation and characterization of immune-competent syngeneic model of muscle-invasive bladder cancer (MIBC)

Abstract Immunologically, MIBC tumors can be divided into inflamed and noninflamed phenotype. Inflamed tumors show high immune cell infiltration. In contrast, noninflamed tumors have little to no immune infiltration. Clinical evidence shows that response to therapy, particularly immune therapy, is associated with the immune phenotype of the tumor. In our preliminary analysis of the TCGA tumor expression data, we observed that patients with high CD8 T-cell infiltration expression signature show an improved overall survival irrespective of treatment. To study the molecular and immunologic drivers of CD8+ T-cell tumor infiltration in MIBC, we have generated immune-competent syngeneic models of bladder cancer. The mouse models were generated by inducing invasive disease by treating C57BL/6 mice with carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN). The induced tumors were grafted into sex-matched immune-competent syngeneic hosts establishing allograft tumor lines. The models represent different clinical subtypes both molecularly and histologically. Currently we have 8 different models with squamous, sarcamatoid, or a more differentiated phenotype. RNA-seq analysis of the tumor lines was used to classify the models into basal-like and luminal-like tumors using the TCGA classifier system. Using the RNA-seq data, we applied a deconvolution algorithm to identify models that had inflamed or noninflamed immune phenotype. Flow cytometry data indicate that the inflamed tumors have a higher infiltration of the myeloid population. We treated 4/8 models with cisplatin, the current standard of care. The models show variable response to cisplatin, indicating the variability between the different models. In conclusion, we have successfully developed immune-competent syngeneic mouse models of MIBC that can be used to study the molecular and immune-driven processes of the disease. Citation Format: Shruti D. Shah, Bryan Gillard, Ellen Karasik, Michelle Wrobel, Michael T. Moser, Barbara A. Foster. Generation and characterization of immune-competent syngeneic model of muscle-invasive bladder cancer (MIBC) [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A11.

Profiling the Immune Tumor Microenvironment of Newly Diagnosed Diffuse Large B Cell Lymphoma Identifies Three Major Immune Infiltration Patterns

Background: Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous disease of malignant B cells most often classified by tumor gene expression and/or mutations. DLBCL is also characterized by a tumor microenvironmental influence that has not been well described. Immuno-oncology-targeting agents such as immune checkpoint inhibitors have limited clinical activity in DLBCL, highlighting the need for a better understanding of the DLBCL tumor microenvironment for rational drug development, combinations, and disease stratification. Here, we systematically characterize the immune composition of more than 100 DLBCL tumors using 2 imaging technologies and provide insight into the complexity of DLBCL disease biology. Methods: A total of 110 cases of newly diagnosed DLBCL were analyzed by multiplex immunohistochemistry (IHC; n=70) and multiplexed ion beam imaging (MIBI) (n=40), each with 10 common markers (CD20, CD3, CD8, Foxp3, CD56, CD163, CD11c, CD56, PD-1, PD-L1) and a few platform-specific markers. Both IHC and MIBI images were digitalized to generate marker-positive cell counts (including single, double, or triple positivity), cell density (cell count/mm2), and population fraction (% of total nucleated cells). Marker density was analyzed for the major components of tumor-infiltrating cell types and correlation between any pair of markers. In addition, RNAseq data and fluorescence in situ hybridization (FISH) data on MYC, BCL-2, and BCL-6 translocation were generated. Results: In the IHC cohort, T cells (CD3+), dendritic cells (DCs; by CD11c+), and macrophages (CD163+)were the major immune components, with median population fractions of 22%, 16%, and 2.7%, respectively. Natural killer cells (CD56+CD20−) were a minor component at a median of 0.1%. A significant negative correlation was observed between tumor cells (CD20+) and CD4+ (Spearman ρ = −0.47; P = 1.3 × 10−04), and CD8+ T cells (Spearman ρ = −0.42; P = 1.4 × 10−03) cells, and an unexpectedly negative correlation between DCs (CD11c+) and macrophages (CD163+, r = −0.63; P = 9.8 × 10−06) was found. Similar to follicular lymphoma, 2 PD-1+ T-cell populations were identified: PD-1bright and PD-1dim. The PD-1bright cells co-stained with CXCR5, indicating T follicular helper cells (Tfh). The PD-1dim were expressed on exhausted effector cells that co-stained with Tim3 or Lag3. The median population fraction of Tim3+ or Lag3+ among T cells was 25%, whereas that of PD-1dim among T cells was 0.2%, indicating that PD-1 was not a useful marker for exhausted T cells. PD-L1 was predominantly found on DCs and macrophages, and the median population fraction of PD-L1+ among tumor cells was only 6.2%. By unsupervised hierarchical clustering on marker density, 3 major immune-infiltration patterns (P1, P2, and P3) were identified. The first 2 segments (P1 and P2) were 20% and 25% of the total cases, respectively. Both were characterized by high T-cell, macrophage, and DC infiltration. P1 was enriched for PD-1+ T cells, whereas P2 was void of any PD-1+ cells. The third segment (P3) that comprised 55% of the cases was predominantly tumor cells with low T-cell, macrophage, and DC infiltration. PD-L1+ cells were primarily found in segments P1 and P2 but rare in segment P3. Additional analysis on the associations between the 3 immune-infiltration patterns and prognostic DLBCL molecular features such as cell-of-origin, double-hit gene signature, and double-hit FISH will also be presented. Conclusions: These data show the complexity of DLBCL disease biology and show classification of DLBCL at the immune-infiltration level as 3 distinct patterns. The overall low expression of PD-1+ T cells and the restricted pattern of PD-L1+ tumor cells provide a possible explanation for the lack of clinical activity of PD-1/PD-L1 blockade in DLBCL. We also observed other exhausted T cells expressing Lag3 and Tim3, suggesting alternative therapeutic opportunities in stratified populations. These data also highlight the opportunity to develop rational immuno-oncology-targeted agents based on the immune infiltration pattern of DLBCL and selection of patients who may respond more favorably to particular agents. Disclosures Huang: Celgene Corporation: Employment, Equity Ownership. Nakayama:Celgene Corporation: Employment, Equity Ownership. Stokes:Celgene Corporation: Employment, Equity Ownership. Towfic:Celgene Corporation: Employment, Equity Ownership. Lee:Celgene Corporation: Employment, Equity Ownership. Ren:Celgene Corporation: Employment, Equity Ownership. Marella:Celgene Corporation: Employment, Equity Ownership. Wang:Celgene Corporation: Employment, Equity Ownership. Hagner:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Couto:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties. Newhall:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corporation: Employment, Equity Ownership, Patents & Royalties.

Importance of Multiparametric Evaluation of Immune-Related T-Cell Markers in Renal-Cell Carcinoma.

Immunotherapeutic therapies such as immune checkpoint inhibitors have been used in patients with renal cell carcinoma (RCC). To overcome therapeutic resistance or identify predictive markers, a comprehensive understanding of the immunologic condition in the tumor microenvironment is important. We reviewed the latest scientific findings on the comprehensive immunologic condition within the tumor microenvironment in patients with RCC and its clinical significance. The immunologic condition evaluated by 3 different methods (flow cytometry, mass cytometry, and next-generation sequencing) in 4 different cohorts of patients with RCC could commonly divide the immunologic condition into 2 or 3 groups, all of which were significantly correlated with tumor aggressiveness and patient prognosis. In particular, patients with high T-cell infiltration and immunosuppressive cells including regulatory T cells had the worst prognosis in each cohort. This classification correlated with angiogenesis and metabolism and glycolysis, and it suggested that distinct biology exists in each immunologic classification. Moreover, around 20% to 30% of the RCC patients had intratumor immunologic diversity within each individual; this might help in understanding the presence of radiologic heterogeneity for immunotherapies. In conclusion, a comprehensive understanding of the immune condition is needed for the upcoming era of novel cancer immunotherapy using not only genetic but also phenotypic and functional classifications.