RESUMEN
Autologous therapeutic tumor-infiltrating lymphocyte (TIL) therapy is a promising strategy to enhance antitumor immunity. Optimization of ex vivo TIL expansion could expand current immunotherapy options. Previous attempts to generate TIL in renal cell carcinoma (RCC) have been technically challenging. We applied a second-generation manufacturing process, currently used to generate the melanoma TIL product lifileucel, in RCC. Resected primary and metastatic RCC samples were processed using the Gen 2 manufacturing process comprising of pre-Rapid Expansion Protocol (pre-REP) and REP steps. We assessed REP TILs for viability and performed phenotypic and functional characterization. We correlated the tumor immune microenvironment (TIME) with successful TIL expansion. Eight of 11 RCC samples underwent successful REP. Three failed cases demonstrated low CD8/FoxP3 ratio and high expression of PD-1 within FoxP3 cells. Expression of exhaustion markers differed between the TIME and expanded TILs; the latter had a TIM3-high/PD-1-low phenotype but retained functional capacity comparable to lifileucel. The Gen 2 manufacturing process used for lifileucel successfully expanded functional TILs from RCC samples, enabling further study in a clinical trial. TIME features such as low CD8/FoxP3 ratio and high PD-1 expression within FoxP3 cells warrant study as potential biomarkers of successful TIL expansion.
RESUMEN
PURPOSE: Programmed cell death protein 1 (PD-1) expression on CD8+TIM-3-LAG-3- tumor-infiltrating cells predicts positive response to PD-1 blockade in metastatic clear-cell renal cell carcinoma (mccRCC). Because inhibition of PD-1 signaling in regulatory T cells (Treg) augments their immunosuppressive function, we hypothesized that PD-1 expression on tumor-infiltrating Tregs would predict resistance to PD-1 inhibitors. EXPERIMENTAL DESIGN: PD-1+ Tregs were phenotyped using multiparametric immunofluorescence in ccRCC tissues from the CheckMate-025 trial (nivolumab: n = 91; everolimus: n = 90). Expression of CD8, PD-1, TIM-3, and LAG-3 was previously determined (Ficial and colleagues, 2021). Clinical endpoints included progression-free survival (PFS), overall survival (OS), and objective response rate (ORR). RESULTS: In the nivolumab (but not everolimus) arm, high percentage of PD-1+ Tregs was associated with shorter PFS (3.19 vs. 5.78 months; P = 0.021), shorter OS (18.1 vs. 27.7 months; P = 0.013) and marginally lower ORR (12.5% vs. 31.3%; P = 0.059). An integrated biomarker (PD-1 Treg/CD8 ratio) was developed by calculating the ratio between percentage of PD-1+Tregs (marker of resistance) and percentage of CD8+PD-1+TIM-3-LAG-3- cells (marker of response). In the nivolumab (but not everolimus) arm, patients with high PD-1 Treg/CD8 ratio experienced shorter PFS (3.48 vs. 9.23 months; P < 0.001), shorter OS (18.14 vs. 38.21 months; P < 0.001), and lower ORR (15.69% vs. 40.00%; P = 0.009). Compared with the individual biomarkers, the PD-1 Treg/CD8 ratio showed improved ability to predict outcomes to nivolumab versus everolimus. CONCLUSIONS: PD-1 expression on Tregs is associated with resistance to PD-1 blockade in mccRCC, suggesting that targeting Tregs may synergize with PD-1 inhibition. A model that integrates PD-1 expression on Tregs and CD8+TIM-3-LAG-3- cells has higher predictive value.
Asunto(s)
Carcinoma de Células Renales , Humanos , Carcinoma de Células Renales/patología , Nivolumab/uso terapéutico , Linfocitos T Reguladores/metabolismo , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Everolimus/uso terapéutico , Receptor de Muerte Celular Programada 1/metabolismoRESUMEN
PURPOSE: PD-L1 expression on tumor cells (TC) is associated with response to anti-PD-1-based therapies in some tumor types, but its significance in clear cell renal cell carcinoma (ccRCC) is uncertain. We leveraged tumor heterogeneity to identify molecular correlates of TC PD-L1 expression in ccRCC and assessed their role in predicting response to anti-PD-1 monotherapy. EXPERIMENTAL DESIGN: RNA sequencing was performed on paired TC PD-L1 positive and negative areas isolated from eight ccRCC tumors and transcriptomic features associated with PD-L1 status were identified. A cohort of 232 patients with metastatic ccRCC from the randomized CheckMate-025 (CM-025) trial was used to confirm the findings and correlate transcriptomic profiles with clinical outcomes. RESULTS: In both the paired samples and the CM-025 cohort, TC PD-L1 expression was associated with combined overexpression of immune- and cell proliferation-related pathways, upregulation of T-cell activation signatures, and increased tumor-infiltrating immune cells. In the CM-025 cohort, TC PD-L1 expression was not associated with clinical outcomes. A molecular RCC subtype characterized by combined overexpression of immune- and cell proliferation-related pathways (previously defined by unsupervised clustering of transcriptomic data) was enriched in TC PD-L1 positive tumors and displayed longer progression-free survival (HR, 0.32; 95% confidence interval, 0.13-0.83) and higher objective response rate (30% vs. 0%, P = 0.04) on nivolumab compared with everolimus. CONCLUSIONS: Both TC-extrinsic (immune-related) and TC-intrinsic (cell proliferation-related) mechanisms are likely intertwined in the regulation of TC PD-L1 expression in ccRCC. The quantitation of these transcriptional programs may better predict benefit from anti-PD-1-based therapy compared with TC PD-L1 expression alone in ccRCC.
Asunto(s)
Carcinoma de Células Renales , Neoplasias Renales , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Biomarcadores de Tumor/metabolismo , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Humanos , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Neoplasias Renales/patología , Nivolumab/uso terapéutico , TranscriptomaRESUMEN
Inactivation of the VHL tumor suppressor gene is the signature initiating event in clear cell renal cell carcinoma (ccRCC), which is the most common form of kidney cancer. The VHL tumor suppressor protein marks hypoxia-inducible factor 1 (HIF1) and HIF2 for proteasomal degradation when oxygen is present. The inappropriate accumulation of HIF2 drives tumor formation by VHL tumor suppressor protein (pVHL)defective ccRCC. Belzutifan, a first-in-class allosteric HIF2 inhibitor, has advanced to phase 3 testing for advanced ccRCC and is approved for ccRCCs arising in patients with VHL disease, which is caused by germline VHL mutations. HIF2 can suppress p53 function in some settings and preliminary data suggested that an intact p53 pathway, as measured by activation in response to DNA damage, was necessary for HIF2 dependence. Here, we correlated HIF2 dependence and p53 status across a broader collection of ccRCC cell lines. We also genetically manipulated p53 function in ccRCC lines that were or were not previously HIF2-dependent and then assessed their subsequent sensitivity to HIF2 ablation using CRISPR-Cas9 or the HIF2 inhibitor PT2399, which is closely related to belzutifan. From these studies, we conclude that p53 status does not dictate HIF2 dependence, at least in preclinical models, and thus is unlikely to be a useful biomarker for predicting which ccRCC patients will respond to HIF2 inhibitors.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Carcinoma de Células Renales , Indanos , Neoplasias Renales , Sulfonas , Proteína p53 Supresora de Tumor , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Indanos/farmacología , Indanos/uso terapéutico , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/genética , Neoplasias Renales/patología , Masculino , Sulfonas/farmacología , Sulfonas/uso terapéutico , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismoRESUMEN
Translocation renal cell carcinoma (tRCC) is a poorly characterized subtype of kidney cancer driven by MiT/TFE gene fusions. Here, we define the landmarks of tRCC through an integrative analysis of 152 patients with tRCC identified across genomic, clinical trial, and retrospective cohorts. Most tRCCs harbor few somatic alterations apart from MiT/TFE fusions and homozygous deletions at chromosome 9p21.3 (19.2% of cases). Transcriptionally, tRCCs display a heightened NRF2-driven antioxidant response that is associated with resistance to targeted therapies. Consistently, we find that outcomes for patients with tRCC treated with vascular endothelial growth factor receptor inhibitors (VEGFR-TKIs) are worse than those treated with immune checkpoint inhibitors (ICI). Using multiparametric immunofluorescence, we find that the tumors are infiltrated with CD8+ T cells, though the T cells harbor an exhaustion immunophenotype distinct from that of clear cell RCC. Our findings comprehensively define the clinical and molecular features of tRCC and may inspire new therapeutic hypotheses.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Carcinoma de Células Renales/genética , Neoplasias Renales/genética , Factor de Transcripción Asociado a Microftalmía/genética , Proteínas de Fusión Oncogénica/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Linfocitos T CD8-positivos/inmunología , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/patología , Regulación Neoplásica de la Expresión Génica , Fusión Génica/genética , Humanos , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/patología , Proteínas de Fusión Oncogénica/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Receptor 1 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidoresRESUMEN
Most methanotrophic bacteria maintain intracytoplasmic membranes which house the methane-oxidizing enzyme, particulate methane monooxygenase. Previous studies have primarily used transmission electron microscopy or cryo-electron microscopy to look at the structure of these membranes or lipid extraction methods to determine the per cent of cell dry weight composed of lipids. We show an alternative approach using lipophilic membrane probes and other fluorescent dyes to assess the extent of intracytoplasmic membrane formation in living cells. This fluorescence method is sensitive enough to show not only the characteristic shift in intracytoplasmic membrane formation that is present when methanotrophs are grown with or without copper, but also differences in intracytoplasmic membrane levels at intermediate copper concentrations. This technique can also be employed to monitor dynamic intracytoplasmic membrane changes in the same cell in real time under changing growth conditions. We anticipate that this approach will be of use to researchers wishing to visualize intracytoplasmic membranes who may not have access to electron microscopes. It will also have the capability to relate membrane changes in individual living cells to other measurements by fluorescence labelling or other single-cell analysis methods.