Spatial Distribution and Predictive Significance of Dendritic Cells and Macrophages in Esophageal Cancer Treated With Combined Chemoradiotherapy and PD-1 Blockade

BackgroundThe first clinical study (NCT03671265) of first-line chemoradiotherapy combined with PD-1 blockade showed promising treatment outcomes in locally advanced esophageal squamous cell carcinoma (ESCC). However, partial patients did not respond to the combination treatment. The roles of dendritic cells (DCs) and macrophages in this combination treatment remain poorly understood.MethodsWe performed multiplexed immunofluorescence method to identify CD11c+ DCs, CD68+ macrophages, and their PD-L1- or PD-L1+ subpopulations in paired tumor biopsies (n = 36) collected at baseline and during the combination treatment (after radiation, 40 Gy) from the phase Ib trial (NCT03671265). We applied whole exome sequencing in the baseline tumor biopsies (n = 14) to estimate tumor mutation burden (TMB). We dynamically investigated the spatial distribution of DCs and macrophages under chemoradiotherapy combined with PD-1 blockade, and evaluated the association between their spatial distribution and combination outcome, and TMB.ResultsThe results showed that high percentages of PD-L1- DCs and macrophages in the baseline tumor compartment, but not in the stromal compartment, predicted improved OS and PFS. Chemoradiotherapy combined with PD-1 blockade promoted DCs and macrophages to migrate closer to tumor cells. During combination treatment, PD-L1- tumor cells were nearest to PD-L1- DCs and macrophages, while PD-L1+ tumor cells were next to PD-L1+ DCs and macrophages. High TMB was closely associated with a shorter distance from tumor cells to DCs and macrophages. Shorter distance between PD-L1+ tumor cells and PD-L1+ DCs or PD-L1- macrophages during the combination was correlated with better OS. Shorter distance between PD-L1- tumor cells and PD-L1- macrophages during combination was associated with both longer OS and PFS.ConclusionsPD-L1- or PD-L1+ DCs and macrophages exhibit distinct spatial distribution in ESCC. The close distance between tumor cells and these antigen-presenting cells (APCs) is critical to the clinical outcome in chemoradiotherapy combined with PD-1 blockade in ESCC patients. Our results highlight the predictive potential of spatial patterns of APCs in chemoradiotherapy combined with immunotherapy and reveal the underlying mechanism of APCs participating in chemoradiotherapy-induced antitumor immune response in ESCC.

Temporal single-cell tracing reveals clonal revival and expansion of precursor exhausted T cells during anti-PD-1 therapy in lung cancer

Anti-PD-1 treatment has shown unprecedented clinical success in the treatment of non-small-cell lung cancer (NSCLC), but the underlying mechanisms remain incompletely understood. Here, we performed temporal single-cell RNA and paired T-cell receptor sequencing on 47 tumor biopsies from 36 patients with NSCLC following PD-1-based therapies. We observed increased levels of precursor exhausted T (Texp) cells in responsive tumors after treatment, characterized by low expression of coinhibitory molecules and high expression of GZMK. By contrast, nonresponsive tumors failed to accumulate Texp cells. Our data suggested that Texp cells were unlikely to be derived from the reinvigoration of terminally exhausted cells; instead, they were accumulated by (1) local expansion and (2) replenishment by peripheral T cells with both new and pre-existing clonotypes, a phenomenon we named clonal revival. Our study provides insights into mechanisms underlying PD-1-based therapies, implicating clonal revival and expansion of Texp cells as steps to improve NSCLC treatment.

Pharmacodynamics and molecular correlates of response to glofitamab in relapsed/refractory non-Hodgkin lymphoma

Glofitamab, a novel CD20xCD3, T-cell-engaging bispecific antibody, demonstrated single-agent activity in study NP30179, a first-in-human, phase 1 trial in relapsed/refractory B-cell non-Hodgkin lymphoma. Preclinical studies showed that glofitamab leads to T-cell activation, proliferation and tumor cell killing upon binding to CD20 on malignant cells. Here, we provide evidence of glofitamab's clinical activity, including pharmacodynamics, mode of action and factors associated with clinical response, by evaluating biomarkers in patient samples from the dose-escalation part of this trial. Patients enrolled in NP30179 received single-dose obinutuzumab pretreatment (1000 mg; Gpt) 7 days prior to intravenous glofitamab (5 µg-25 mg). Glofitamab treatment lasted ≤12 cycles once every 2 or 3 weeks. Blood samples were collected at predefined time points per clinical protocol; T-cell populations were evaluated centrally by flow cytometry, and cytokine profiles were analyzed. Immunohistochemical and genomic biomarker analyses were performed on tumor biopsies. Pharmacodynamic modulation was observed with glofitamab treatment including dose-dependent induction of cytokines, and T-cell margination, proliferation and activation in peripheral blood. Gene expression analysis of pre-treatment tumor biopsies indicated that tumor cell intrinsic factors like TP53 signaling are associated with resistance to glofitamab, but may also be interlinked with a diminished effector T-cell profile in resistant tumors and thus represent a poor prognostic factor per se. This integrative biomarker data analysis provides clinical evidence of glofitamab's mode of action, supports optimal biological dose selection, and will further guide clinical development. This trial was registered at www.clinicaltrials.gov as #NCT03075696.

Immunohistochemistry study of tumor vascular normalization and anti-angiogenic effects of sunitinib versus bevacizumab prior to dose-dense doxorubicin/cyclophosphamide chemotherapy in HER2-negative breast cancer

Purpose Tumor angiogenesis controlled predominantly by vascular endothelial growth factor and its receptor (VEGF-VEGFR) interaction plays a key role in the growth and propagation of cancer cells. However, the newly formed network of blood vessels is disorganized and leaky. Pre-treatment with anti-angiogenic agents can “normalize” the tumor vasculature allowing effective intra-tumoral delivery of standard chemotherapy. Immunohistochemistry (IHC) analysis was applied to investigate and compare the vascular normalization and anti-angiogenic effects of two commonly used anti-angiogenic agents, Sunitinib and Bevacizumab, administered prior to chemotherapy in HER2-negative breast cancer patients. Methods This prospective clinical trial enrolled 38 patients into a sunitinib cohort and 24 into a bevacizumab cohort. All received 4 cycles of doxorubicin/cyclophosphamide chemotherapy and pre-treatment with either sunitinib or bevacizumab. Tumor biopsies were obtained at baseline, after cycle 1 (C1) and cycle 4 (C4) of chemotherapy. IHC was performed to assess the tumor vascular normalization index (VNI), lymphatic vessel density (LVD), Ki67 proliferation index and expression of tumor VEGFR2. Results In comparison to Bevacizumab, Sunitinib led to a significant increase in VNI post-C1 and C4 (p < 0.001 and 0.001) along with decrease in LVD post-C1 (p = 0.017). Both drugs when combined with chemotherapy resulted in significant decline in tumor proliferation after C1 and C4 (baseline vs post-C4 Ki67 index p = 0.006 for Sunitinib vs p = 0.021 for Bevacizumab). Bevacizumab resulted in a significant decrease in VEGFR2 expression post-C1 (p = 0.004). Conclusion Sunitinib, in comparison to Bevacizumab showed a greater effect on tumor vessel modulation and lymphangiogenesis suggesting that its administration prior to chemotherapy might result in improved drug delivery. Trial registry ClinicalTrials.gov: NCT02790580 (first posted June 6, 2016).

High Circulating Sonic Hedgehog Protein Is Associated With Poor Outcome in EGFR-Mutated Advanced NSCLC Treated With Tyrosine Kinase Inhibitors

IntroductionGrowing preclinical evidence has suggested that the Sonic hedgehog (Shh) pathway is involved in resistance to tyrosine kinase inhibitor (TKI) therapy for EGFR-mutated (EGFRm) non-small cell lung cancer (NSCLC). However, little is known concerning the prognostic value of this pathway in this context.Materials and MethodsWe investigated the relationship between plasma levels of Shh and EGFRm NSCLC patients’ outcome with EGFR TKIs. We included 74 consecutive patients from two institutions with EGFRm advanced NSCLC treated by EGFR TKI as first-line therapy. Plasma samples were collected longitudinally for each patient and were analyzed for the expression of Shh using an ELISA assay. The activation of the Shh–Gli1 pathway was assessed through immunohistochemistry (IHC) of Gli1 and RT-qPCR analysis of the transcripts of Gli1 target genes in 14 available tumor biopsies collected at diagnosis (baseline).ResultsAmong the 74 patients, only 61 had baseline (diagnosis) plasma samples, while only 49 patients had plasma samples at the first evaluation. Shh protein was detectable in all samples at diagnosis (n = 61, mean = 1,041.2 ± 252.5 pg/ml). Among the 14 available tumor biopsies, nuclear expression of Gli1 was observed in 57.1% (8/14) of patients’ biopsies. Shh was significantly (p &lt; 0.05) enriched in youth (age &lt; 68), male, nonsmokers, patients with a PS &gt; 1, and patients presenting more than 2 metastatic sites and L858R mutation. Higher levels of Shh correlated with poor objective response to TKI, shorter progression-free survival (PFS), and T790M-independent mechanism of resistance. In addition, the rise of plasma Shh levels along the treatment was associated with the emergence of drug resistance in patients presenting an initial good therapy response.ConclusionThese data support that higher levels of plasma Shh at diagnosis and increased levels of Shh along the course of the disease are related to the emergence of TKI resistance and poor outcome for EGFR-TKI therapy, suggesting that Shh levels could stand both as a prognostic and as a resistance biomarker for the management of EGFR-mutated NSCLC patients treated with EGFR-TKI.

Identification of Tumor Specific Neoantigens and Their Implications in Dendritic Cell Immunotherapy Using Liquid Biopsy Results: Findings from an Observational Cohort Study

Liquid biopsies can be a rapid, cost-effective and noninvasive alternative to tumor biopsies for detecting genetic mutations in somatic tumors. Genetic profiling of liquid biopsies can also be used to identify novel antigens for targeted therapy, provide updated information on disease prognosis and evaluate treatment efficacy. In this study, we aimed to examine mutations that could be identified in liquid biopsy and their potential implications for personalized dendritic cell immunotherapy using these antigens. We analyzed the genomic profiles of 99 blood samples from 85 patients with 22 different types of cancer using two commercially available liquid biopsy tests before the patients underwent standard cancer treatment and dendritic cell immunotherapy. Nonsynonymous mutations were detected in more than 90% of the samples, with an average frequency of 3.6 mutations per sample. The tumor mutations were specific to each patient, as approximately 94.7% of the mutations were so unique that there was almost no duplication among the patients. Clonal evolution was observed in two patients just before or after chemotherapy, radiotherapy and immunotherapy. These findings indicate that liquid biopsy can be a potential surrogate for tumor-specific antigen-based immunotherapy and the importance of tailoring immunotherapy in accordance with the liquid biopsy result in each treatment stage.

Genotype Distribution and Prevalence of Human Papillomavirus in Head and Neck Cancer Samples from Istanbul, Turkey

Human papillomavirus (HPV)-associated tumors account for a significant proportion of head and neck squamous cell carcinomas (HNSCC) in developed countries. In recent years, there has been a rise of HPV infections associated with HNSCC, especially HPV16, which is the most commonly detected type in oral and oropharyngeal cancers. To investigate the frequency of HPV-driven HNSCC among patients living in Turkey, HPV DNA positivity and p16INK4A expression were assessed in primary tumor biopsies (n = 106). Eighteen out of one hundred and six (19%) HNSCC tumors showed p16INK4A overexpression, and 26/106 cases (24.5%) were positive for HPV DNA. Sixteen out of twenty-six samples were positive for both HPV DNA and p16INK4A staining. HPV16 could be isolated from 22/26 samples (84.6%) and was found to be the most frequently detected HPV type. This study represents the largest cohort of Turkish patients with HNSCC characterized according to HPV status and p16INK4A expression. Our data suggest that HPV16 infection, along with smoking, contribute to the development of HNSCC.

Intra-Tumoral Pharmacokinetics of Pazopanib in Combination with Radiotherapy in Patients with Non-Metastatic Soft-Tissue Sarcoma

There is a lack of understanding whether plasma levels of anticancer drugs (such as pazopanib) correlate with intra-tumoral levels and whether the plasma compartment is the best surrogate for pharmacokinetic and pharmacodynamic evaluation. Therefore, we aimed to quantify pazopanib concentrations in tumor tissue, to assess the correlation between tumor concentrations and plasma concentrations and between tumor concentrations and efficacy. In this clinical trial, non-metastatic STS patients were treated with neo-adjuvant concurrent radiotherapy and pazopanib. Plasma samples and tumor biopsies were collected, and pazopanib concentrations were measured using liquid chromatography-tandem mass spectrometry. Twenty-four evaluable patients were included. The median pazopanib tumor concentration was 19.2 µg/g (range 0.149–200 µg/g). A modest correlation was found between tumor concentrations and plasma levels of pazopanib (ρ = 0.41, p = 0.049). No correlation was found between tumor concentrations and percentage of viable tumor cells (p > 0.05); however, a trend towards less viable tumor cells in patients with high pazopanib concentrations in tumor tissue was observed in a categorical analysis. Possible explanations for the lack of correlation might be heterogeneity of the tumors and timing of the biopsy procedure.

Identification of Tumor Specific Neoantigens and Their Implications in Dendritic Cell Immunotherapy Using Liquid Biopsy Results: Findings from an Observational Cohort Study

Liquid biopsies can be a rapid, cost-effective and noninvasive alternative to tumor biopsies for detecting genetic mutations in somatic tumors. Genetic profiling of liquid biopsies can also be used to identify novel antigens for targeted therapy, provide updated information on disease prognosis and evaluate treatment efficacy. In this study, we aimed to examine mutations that could be identified in liquid biopsy and their potential implications for personalized dendritic cell immunotherapy using neoantigens. We analyzed the genomic profiles of 99 blood samples from 85 patients with 22 different types of cancer using two commercially available liquid biopsy tests before the patients underwent standard cancer treatment and dendritic cell immunotherapy. Nonsynonymous mutations were detected in more than 90% of the samples, with an average frequency of 3.6 mutations per sample. The tumor mutations were specific to each patient, as approximately 94.7% of the mutations were so unique that there was almost no duplication among the patients. Clonal evolution was observed in two patients just before or after chemotherapy, radiotherapy and immunotherapy. These findings indicate that liquid biopsy can be a potential surrogate for neoantigen-based immunotherapy and the importance of tailoring neoantigen-based immunotherapy in accordance with the liquid biopsy result in each treatment stage.

Identification of Tumor Specific Neoantigens and Their Implications in Dendritic Cell Immunotherapy Using Liquid Biopsy Results: Findings from an Observational Cohort Study

Liquid biopsies can be a rapid, cost-effective and noninvasive alternative to tumor biopsies for detecting genetic mutations in somatic tumors. Genetic profiling of liquid biopsies can also be used to identify novel antigens for targeted therapy, provide updated information on disease prognosis and evaluate treatment efficacy. In this study, we aimed to examine mutations that could be identified in liquid biopsy and their potential implications for personalized dendritic cell immunotherapy using neoantigens. We analyzed the genomic profiles of 99 blood samples from 85 patients with 22 different types of cancer using two commercially available liquid biopsy tests before the patients underwent standard cancer treatment and dendritic cell immunotherapy. Nonsynonymous mutations were detected in more than 90% of the samples, with an average frequency of 3.6 mutations per sample. The tumor mutations were specific to each patient, as approximately 94.7% of the mutations were so unique that there was almost no duplication among the patients. Clonal evolution was observed in two patients just before or after chemotherapy, radiotherapy and immunotherapy. These findings indicate that liquid biopsy can be a potential surrogate for neoantigen-based immunotherapy and the importance of tailoring neoantigen-based immunotherapy in accordance with the liquid biopsy result in each treatment stage.