Human CD4+ iNKT cell adoptive immunotherapy induces anti-tumour responses against CD1d-negative EBV-driven B lymphoma.

Invariant natural killer T (iNKT) cells are a conserved population of innate T lymphocytes that are uniquely suitable as off-the-shelf cellular immunotherapies due to their lack of alloreactivity. Two major subpopulations of human iNKT cells have been delineated, a CD4- subset that has a TH1/cytolytic profile, and a CD4+ subset that appears polyfunctional and can produce both regulatory and immunostimulatory cytokines. Whether these two subsets differ in anti-tumour effects is not known. Using live cell imaging, we found that CD4- iNKT cells limited growth of CD1d+ Epstein-Barr virus (EBV)-infected B-lymphoblastoid spheroids in vitro, whereas CD4+ iNKT cells showed little or no direct anti-tumour activity. However, the effects of the two subsets were reversed when we tested them as adoptive immunotherapies in vivo using a xenograft model of EBV-driven human B cell lymphoma. We found that EBV-infected B cells down-regulated CD1d in vivo, and administering CD4- iNKT cells had no discernable impact on tumour mass. In contrast, xenotransplanted mice bearing lymphomas showed rapid reduction in tumour mass after administering CD4+ iNKT cells. Immunotherapeutic CD4+ iNKT cells trafficked to both spleen and tumour and were associated with subsequently enhanced responses of xenotransplanted human T cells against EBV. CD4+ iNKT cells also had adjuvant-like effects on monocyte-derived DCs and promoted antigen-dependent responses of human T cells in vitro. These results show that allogeneic CD4+ iNKT cellular immunotherapy leads to marked anti-tumour activity through indirect pathways that do not require tumour cell CD1d expression and that are associated with enhanced activity of antigen-specific T cells.

Combining Dual Checkpoint Immunotherapy with Ablative Radiation to All Sites of Oligometastatic Non-Small Cell Lung Cancer: Toxicity and Efficacy Results of a Phase 1b Trial.

PURPOSE: Ablative local treatment of all radiographically detected metastatic sites in patients with oligometastatic non-small cell lung cancer (NSCLC) increases progression-free survival (PFS) and overall survival (OS). Prior studies demonstrated the safety of combining stereotactic body radiation therapy (SBRT) with single-agent immunotherapy. We investigated the safety of combining SBRT to all metastatic tumor sites with dual checkpoint, anticytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4), and anti-programmed cell death ligand 1 (anti-PD-L1) immunotherapy for patients with oligometastatic NSCLC. METHODS AND MATERIALS: We conducted a phase 1b clinical trial in patients with oligometastatic NSCLC with up to 6 sites of extracranial metastatic disease. All sites of disease were treated with SBRT to a dose of 30 to 50 Gy in 5 fractions. Dual checkpoint immunotherapy was started 7 days after completion of radiation using anti-CTLA-4 (tremelimumab) and anti-PD-L1 (durvalumab) immunotherapy for a total of 4 cycles followed by durvalumab alone until progression or toxicity. RESULTS: Of the 17 patients enrolled in this study, 15 patients received at least 1 dose of combination immunotherapy per protocol. The study was closed early (17 of planned 21 patients) due to slow accrual during the COVID-19 pandemic. Grade 3+ treatment-related adverse events were observed in 6 patients (40%), of which only one was possibly related to the addition of SBRT to immunotherapy. Median PFS was 42 months and median OS has not yet been reached. CONCLUSIONS: Delivering ablative SBRT to all sites of metastatic disease in combination with dual checkpoint immunotherapy did not result in excessive rates of toxicity compared with historical studies of dual checkpoint immunotherapy alone. Although the study was not powered for treatment efficacy results, durable PFS and OS results suggest potential therapeutic benefit compared with immunotherapy or radiation alone in this patient population.

A clinical-grade liquid biomarker detects neuroendocrine differentiation in prostate cancer.

BackgroundNeuroendocrine prostate cancer (NEPC) is an aggressive subtype, the presence of which changes the prognosis and management of metastatic prostate cancer.MethodsWe performed analytical validation of a Circulating Tumor Cell (CTC) multiplex RNA qPCR assay to identify the limit of quantification (LOQ) in cell lines, synthetic cDNA, and patient samples. We next profiled 116 longitudinal samples from a prospectively collected institutional cohort of 17 patients with metastatic prostate cancer (7 NEPC, 10 adenocarcinoma) as well as 265 samples from 139 patients enrolled in 3 adenocarcinoma phase II trials of androgen receptor signaling inhibitors (ARSIs). We assessed a NEPC liquid biomarker via the presence of neuroendocrine markers and the absence of androgen receptor (AR) target genes.ResultsUsing the analytical validation LOQ, liquid biomarker NEPC detection in the longitudinal cohort had a per-sample sensitivity of 51.35% and a specificity of 91.14%. However, when we incorporated the serial information from multiple liquid biopsies per patient, a unique aspect of this study, the per-patient predictions were 100% accurate, with a receiver-operating-curve (ROC) AUC of 1. In the adenocarcinoma ARSI trials, the presence of neuroendocrine markers, even while AR target gene expression was retained, was a strong negative prognostic factor.ConclusionOur analytically validated CTC biomarker can detect NEPC with high diagnostic accuracy when leveraging serial samples that are only feasible using liquid biopsies. Patients with expression of NE genes while retaining AR-target gene expression may indicate the transition to neuroendocrine differentiation, with clinical characteristics consistent with this phenotype.FundingNIH (DP2 OD030734, 1UH2CA260389, R01CA247479, and P30 CA014520), Department of Defense (PC190039 and PC200334), and Prostate Cancer Foundation (Movember Foundation - PCF Challenge Award).

Longitudinal Molecular Profiling of Circulating Tumor Cells in Metastatic Renal Cell Carcinoma.

PURPOSE: Liquid biopsies in metastatic renal cell carcinoma (mRCC) provide a unique approach to understand the molecular basis of treatment response and resistance. This is particularly important in the context of immunotherapies, which target key immune-tumor interactions. Unlike metastatic tissue biopsies, serial real-time profiling of mRCC is feasible with our noninvasive circulating tumor cell (CTC) approach. METHODS: We collected 457 longitudinal liquid biopsies from 104 patients with mRCC enrolled in one of two studies, either a prospective cohort or a phase II multicenter adaptive immunotherapy trial. Using a novel CTC capture and automated microscopy platform, we profiled CTC enumeration and expression of HLA I and programmed cell death-ligand 1 (PD-L1). Given their diametric immunological roles, we focused on the HLA I to PD-L1 ratio (HP ratio). RESULTS: Patients with radiographic responses showed significantly lower CTC abundances throughout treatment. Furthermore, patients whose CTC enumeration trajectory was in the highest quartile (> 0.12 CTCs/mL annually) had shorter overall survival (median 17.0 months v 21.1 months, P < .001). Throughout treatment, the HP ratio decreased in patients receiving immunotherapy but not in patients receiving tyrosine kinase inhibitors. Patients with an HP ratio trajectory in the highest quartile (≥ 0.0012 annually) displayed significantly shorter overall survival (median 18.4 months v 21.2 months, P = .003). CONCLUSION: In the first large longitudinal CTC study in mRCC to date to our knowledge, we identified the prognostic importance of CTC enumeration and the change over time of both CTC enumeration and the HP ratio. These insights into changes in both tumor burden and the molecular profile of tumor cells in response to different treatments provide potential biomarkers to predict and monitor response to immunotherapy in mRCC.

Analytical validation and initial clinical testing of quantitative microscopic evaluation for PD-L1 and HLA I expression on circulating tumor cells from patients with non-small cell lung cancer.

INTRODUCTION: PD-L1 expression in non-small cell lung cancer (NSCLC) predicts response to immune checkpoint blockade, however is an imperfect biomarker given tumor heterogeneity, and the antigen presentation pathway requiring other components including HLA I expression. HLA I downregulation may contribute to resistance, warranting its evaluation in attempts to guide patient selection. In addition, earlier detection of acquired resistance could prompt earlier change in treatment and prolong patient survival. Analysis of circulating tumor cells (CTCs) captures heterogeneity across multiple sites of metastases, enables detection of changes in tumor burden that precede radiographic response, and can be obtained in serial fashion. METHODS: To quantify the expression of both PD-L1 and HLA I on CTCs, we developed exclusion-based sample preparation technology, achieving high-yield with gentle magnetic movement of antibody-labeled cells through virtual barriers of surface tension. To achieve clinical-grade quantification of rare cells, we employ high quality fluorescence microscopy image acquisition and automated image analysis together termed quantitative microscopy. RESULTS: In preparation for clinical laboratory implementation, we demonstrate high precision and accuracy of these methodologies using a diverse set of control materials. Preliminary testing of CTCs isolated from patients with NSCLC demonstrate heterogeneity in PD-L1 and HLA I expression and promising clinical value in predicting PFS in response to PD-L1 targeted therapies. CONCLUSIONS: By confirming high performance, we ensure compatibility for clinical laboratory implementation and future application to better predict and detect resistance to PD-L1 targeted therapy in patients with NSCLC.

BAF155 methylation drives metastasis by hijacking super-enhancers and subverting anti-tumor immunity.

Subunits of the chromatin remodeler SWI/SNF are the most frequently disrupted genes in cancer. However, how post-translational modifications (PTM) of SWI/SNF subunits elicit epigenetic dysfunction remains unknown. Arginine-methylation of BAF155 by coactivator-associated arginine methyltransferase 1 (CARM1) promotes triple-negative breast cancer (TNBC) metastasis. Herein, we discovered the dual roles of methylated-BAF155 (me-BAF155) in promoting tumor metastasis: activation of super-enhancer-addicted oncogenes by recruiting BRD4, and repression of interferon α/γ pathway genes to suppress host immune response. Pharmacological inhibition of CARM1 and BAF155 methylation not only abrogated the expression of an array of oncogenes, but also boosted host immune responses by enhancing the activity and tumor infiltration of cytotoxic T cells. Moreover, strong me-BAF155 staining was detected in circulating tumor cells from metastatic cancer patients. Despite low cytotoxicity, CARM1 inhibitors strongly inhibited TNBC cell migration in vitro, and growth and metastasis in vivo. These findings illustrate a unique mechanism of arginine methylation of a SWI/SNF subunit that drives epigenetic dysregulation, and establishes me-BAF155 as a therapeutic target to enhance immunotherapy efficacy.

Development and initial clinical testing of a multiplexed circulating tumor cell assay in patients with clear cell renal cell carcinoma.

Although therapeutic options for patients with advanced renal cell carcinoma (RCC) have increased in the past decade, no biomarkers are yet available for patient stratification or evaluation of therapy resistance. Given the dynamic and heterogeneous nature of clear cell RCC (ccRCC), tumor biopsies provide limited clinical utility, but liquid biopsies could overcome these limitations. Prior liquid biopsy approaches have lacked clinically relevant detection rates for patients with ccRCC. This study employed ccRCC-specific markers, CAIX and CAXII, to identify circulating tumor cells (CTC) from patients with metastatic ccRCC. Distinct subtypes of ccRCC CTCs were evaluated for PD-L1 and HLA-I expression and correlated with patient response to therapy. CTC enumeration and expression of PD-L1 and HLA-I correlated with disease progression and treatment response, respectively. Longitudinal evaluation of a subset of patients demonstrated potential for CTC enumeration to serve as a pharmacodynamic biomarker. Further evaluation of phenotypic heterogeneity among CTCs is needed to better understand the clinical utility of this new biomarker.

Versatile exclusion-based sample preparation platform for integrated rare cell isolation and analyte extraction.

Rare cell populations provide a patient-centric tool to monitor disease treatment, response, and resistance. However, understanding rare cells is a complex problem, which requires cell isolation/purification and downstream molecular interrogation - processes challenged by non-target populations, which vary patient-to-patient and change with disease. As such, cell isolation platforms must be amenable to a range of sample types while maintaining high efficiency and purity. The multiplexed technology for automated extraction (mTAE) is a versatile magnetic bead-based isolation platform that facilitates positive, negative, and combinatorial selection with integrated protein staining and nucleic acid isolation. mTAE is validated by isolating circulating tumor cells (CTCs) - a model rare cell population - from breast and prostate cancer patient samples. Negative selection yielded high efficiency capture of CTCs while positive selection yielded higher purity with an average of only 95 contaminant cells captured per milliliter of processed whole blood. With combinatorial selection, an overall increase in capture efficiency was observed, highlighting the potential significance of integrating multiple capture approaches on a single platform. Following capture (and staining), on platform nucleic acid extraction enabled the detection of androgen receptor-related transcripts from CTCs isolated from prostate cancer patients. The flexibility (e.g. negative, positive, combinatorial selection) and capabilities (e.g. isolation, protein staining, and nucleic acid extraction) of mTAE enable users to freely interrogate specific cell populations, a capability required to understand the potential of emerging rare cell populations and readily adapt to the heterogeneity presented across clinical samples.

Integration of Magnetic Bead-Based Cell Selection into Complex Isolations.

Magnetic bead-based analyte capture has emerged as a ubiquitous method in cell isolation, enabling the highly specific capture of target populations through simple magnetic manipulation. To date, no "one-size fits all" magnetic bead has been widely adopted leading to an overwhelming number of commercial beads. Ultimately, the ideal bead is one that not only facilitates cell isolation but also proves compatible with the widest range of downstream applications and analytic endpoints. Despite the diverse offering of sizes, coatings, and conjugation chemistries, few studies exist to benchmark the performance characteristics of different commercially available beads; importantly, these bead characteristics ultimately determine the ability of a bead to integrate into the user's assay. In this report, we evaluate bead-based cell isolation considerations, approaches, and results across a subset of commercially available magnetic beads (Dynabeads FlowComps, Dynabeads CELLection, GE Healthcare Sera-Mag SpeedBeads streptavidin-blocked magnetic particles, Dynabeads M-270s, Dynabeads M-280s) to compare and contrast both capture-specific traits (i.e., purity, capture efficacy, and contaminant isolations) and endpoint compatibility (i.e., protein localization, fluorescence imaging, and nucleic acid extraction). We identify specific advantages and contexts of use in which distinct bead products may facilitate experimental goals and integrate into downstream applications.

High Specificity in Circulating Tumor Cell Identification Is Required for Accurate Evaluation of Programmed Death-Ligand 1.

BACKGROUND: Expression of programmed-death ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC) is typically evaluated through invasive biopsies; however, recent advances in the identification of circulating tumor cells (CTCs) may be a less invasive method to assay tumor cells for these purposes. These liquid biopsies rely on accurate identification of CTCs from the diverse populations in the blood, where some tumor cells share characteristics with normal blood cells. While many blood cells can be excluded by their high expression of CD45, neutrophils and other immature myeloid subsets have low to absent expression of CD45 and also express PD-L1. Furthermore, cytokeratin is typically used to identify CTCs, but neutrophils may stain non-specifically for intracellular antibodies, including cytokeratin, thus preventing accurate evaluation of PD-L1 expression on tumor cells. This holds even greater significance when evaluating PD-L1 in epithelial cell adhesion molecule (EpCAM) positive and EpCAM negative CTCs (as in epithelial-mesenchymal transition (EMT)). METHODS: To evaluate the impact of CTC misidentification on PD-L1 evaluation, we utilized CD11b to identify myeloid cells. CTCs were isolated from patients with metastatic NSCLC using EpCAM, MUC1 or Vimentin capture antibodies and exclusion-based sample preparation (ESP) technology. RESULTS: Large populations of CD11b+CD45lo cells were identified in buffy coats and stained non-specifically for intracellular antibodies including cytokeratin. The amount of CD11b+ cells misidentified as CTCs varied among patients; accounting for 33-100% of traditionally identified CTCs. Cells captured with vimentin had a higher frequency of CD11b+ cells at 41%, compared to 20% and 18% with MUC1 or EpCAM, respectively. Cells misidentified as CTCs ultimately skewed PD-L1 expression to varying degrees across patient samples. CONCLUSIONS: Interfering myeloid populations can be differentiated from true CTCs with additional staining criteria, thus improving the specificity of CTC identification and the accuracy of biomarker evaluation.