Gene-expression-based T-Cell-to-Stroma Enrichment (TSE) score predicts response to immune checkpoint inhibitors in urothelial cancer.

Immune checkpoint inhibitors (ICI) improve overall survival in patients with metastatic urothelial cancer (mUC), but therapeutic success at the individual patient level varies significantly. Here we identify predictive markers of response, based on whole-genome DNA (n = 70) and RNA-sequencing (n = 41) of fresh metastatic biopsy samples, collected prior to treatment with pembrolizumab. We find that PD-L1 combined positivity score does not, whereas tumor mutational burden and APOBEC mutagenesis modestly predict response. In contrast, T cell-to-stroma enrichment (TSE) score, computed from gene expression signature data to capture the relative abundance of T cells and stromal cells, predicts response to immunotherapy with high accuracy. Patients with a positive and negative TSE score show progression free survival rates at 6 months of 67 and 0%, respectively. The abundance of T cells and stromal cells, as reflected by the TSE score is confirmed by immunofluorescence in tumor tissue, and its good performance in two independent ICI-treated cohorts of patients with mUC (IMvigor210) and muscle-invasive UC (ABACUS) validate the predictive power of the TSE score. In conclusion, the TSE score represents a clinically applicable metric that potentially supports the prospective selection of patients with mUC for ICI treatment.

Retroviral Transduction of Human Primary T Cells Followed by Real-Time T-Cell-Mediated Cancer Cell Cytolysis Analysis.

Retroviral transduction is a highly useful tool to genetically engineer hard-to-transfect human primary cells. Here, we transduce human primary T cells with a tumor-specific T cell receptor. This creates a useful tool to analyze T cell-cancer cell interactions, such as cytolysis analysis using xCELLigence technology.

Analyzing Flow Cytometry or Targeted Gene Expression Data Influences Clinical Discoveries-Profiling Blood Samples of Pancreatic Ductal Adenocarcinoma Patients.

INTRODUCTION: Monitoring the therapeutic response of pancreatic ductal adenocarcinoma (PDAC) patients is crucial to determine treatment strategies. Several studies have examined the effectiveness of FOLFIRINOX as a first-line treatment in patients with locally advanced pancreatic cancer, but little attention has been paid to the immunologic alterations in peripheral blood caused by this chemotherapy regimen. Furthermore, the influence of the measurement type (e.g., flow cytometry and targeted gene expression) on the clinical discoveries is unknown. Therefore, we aimed to scrutinize the influence of using flow cytometry or targeted immune gene expression to study the immunological changes in blood samples of PDAC patients who were treated with a single-cycle FOLFIRINOX combined with lipegfilgrastim (FFX-Lipeg). MATERIAL AND METHODS: Whole-blood samples from 44 PDAC patients were collected at two time points: before the first FOLFIRINOX cycle and 14 days after the first cycle. EDTA blood tubes were used for multiplex flow cytometry analyses to quantify 18 immune cell populations and for complete blood count tests as the standard clinical routine. The flow cytometry data were analyzed with FlowJo software. In addition, Tempus blood tubes were used to isolate RNA and measure 1230 immune-related genes using NanoString Technology®. Data quality control, normalization, and analysis were performed using nSolver™ software and the Advanced Analysis module. RESULTS: FFX-Lipeg treatment increased the number of neutrophils and monocytes, as shown by flow cytometry and complete blood count in concordance with elevated gene expression measured via targeted gene expression profiling analysis. Interestingly, flow cytometry analysis showed an increase in the number of B and T cells after treatment, while targeted gene expression analysis showed a decrease in B and T cell-specific gene expression. CONCLUSIONS: Targeted gene expression complements flow cytometry analysis to provide a comprehensive understanding of the effects of FFX-Lipeg. Flow cytometry and targeted gene expression showed increases in neutrophils and monocytes after FFX-Lipeg. The number of lymphocytes is increased after treatment; nevertheless, their cell-specific gene expression levels are downregulated. This highlights that different techniques influence clinical discoveries. Therefore, it is important to carefully select the measurement technique used to study the effect of a treatment.

HYpofractionated, dose-redistributed RAdiotherapy with protons and photons to combat radiation-induced immunosuppression in head and neck squamous cell carcinoma: study protocol of the phase I HYDRA trial.

BACKGROUND: Radiotherapy (RT) is the standard of care for most advanced head and neck squamous cell carcinoma (HNSCC) and results in an unfavorable 5-year overall survival of 40%. Despite strong biological rationale, combining RT with immune checkpoint inhibitors does not result in a survival benefit. Our hypothesis is that the combination of these individually effective treatments fails because of radiation-induced immunosuppression and lymphodepletion. By integrating modern radiobiology and innovative radiotherapy concepts, the patient's immune system could be maximally retained by (1) increasing the dose per fraction so that the total dose and number of fractions can be reduced (HYpofractionation), (2) redistributing the radiation dose towards a higher peak dose within the tumor center and a lowered elective lymphatic field dose (Dose-redistribution), and (3) using RAdiotherapy with protons instead of photons (HYDRA). METHODS: The primary aim of this multicenter study is to determine the safety of HYDRA proton- and photon radiotherapy by conducting two parallel phase I trials. Both HYDRA arms are randomized with the standard of care for longitudinal immune profiling. There will be a specific focus on actionable immune targets and their temporal patterns that can be tested in future hypofractionated immunoradiotherapy trials. The HYDRA dose prescriptions (in 20 fractions) are 40 Gy elective dose and 55 Gy simultaneous integrated boost on the clinical target volume with a 59 Gy focal boost on the tumor center. A total of 100 patients (25 per treatment group) will be recruited, and the final analysis will be performed one year after the last patient has been included. DISCUSSION: In the context of HNSCC, hypofractionation has historically only been reserved for small tumors out of fear for late normal tissue toxicity. To date, hypofractionated radiotherapy may also be safe for larger tumors, as both the radiation dose and volume can be reduced by the combination of advanced imaging for better target definition, novel accelerated repopulation models and high-precision radiation treatment planning and dose delivery. HYDRA's expected immune-sparing effect may lead to improved outcomes by allowing for future effective combination treatment with immunotherapy. TRIAL REGISTRATION: The trial is registered at ClinicalTrials.gov; NCT05364411 (registered on May 6th, 2022).

Transcriptomics of Epstein-Barr virus aids to the classification of T-cell evasion in nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) contributes to oncogenesis and immune evasion in nasopharyngeal carcinoma (NPC). At present, an aggregated, higher-level view on the impact of EBV genes toward the immune microenvironment of NPC is lacking. To this end, we have interrogated tumor-derived RNA sequences of 106 treatment-naive NPC patients for 98 EBV transcripts, and captured the presence of 10 different immune cell populations as well as 23 different modes of T-cell evasion. We discovered 3 clusters of EBV genes that each associate with distinct immunophenotypes of NPC. Cluster 1 associated with gene sets related to immune cell recruitment, such as those encoding for chemoattractants and their receptors. Cluster 2 associated with antigen processing and presentation, such as interferon-related genes, whereas cluster 3 associated with presence of M1-like macrophages, absence of CD4+ T cells, and oncogenic pathways, such as the nuclear factor kappa light-chain enhancer of activated B-cell pathway. We discuss these 3 EBV clusters regarding their potential for stratification for T-cell immunity in NPC together with the next steps needed to validate such therapeutic value.

Frequency of Peripheral CD8+ T Cells Expressing Chemo-Attractant Receptors CCR1, 4 and 5 Increases in NPC Patients with EBV Clearance upon Radiotherapy.

Radiotherapy (RT) is the standard-of-care for Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC), where the post-RT clearance of plasma EBV DNA is prognostic. Currently, it is not known whether the post-RT clearance of plasma EBV DNA is related to the presence of circulating T-cell subsets. Blood samples from NPC patients were used to assess the frequency of T-cell subsets relating to differentiation, co-signaling and chemotaxis. Patients with undetectable versus detectable plasma EBV DNA levels post-RT were categorized as clearers vs. non-clearers. Clearers had a lower frequency of PD1+CD8+ T cells as well as CXCR3+CD8+ T cells during RT compared to non-clearers. Clearers exclusively showed a temporal increase in chemo-attractant receptors CCR1, 4 and/or 5, expressing CD8+ T cells upon RT. The increase in CCR-expressing CD8+ T cells was accompanied by a drop in naïve CD8+ T cells and an increase in OX40+CD8+ T cells. Upon stratifying these patients based on clinical outcome, the dynamics of CCR-expressing CD8+ T cells were in concordance with the non-recurrence of NPC. In a second cohort, non-recurrence associated with higher quantities of circulating CCL14 and CCL15. Collectively, our findings relate plasma EBV DNA clearance post-RT to T-cell chemotaxis, which requires validation in larger cohorts.

Triple-Negative Breast Cancer and Predictive Markers of Response to Neoadjuvant Chemotherapy: A Systematic Review.

Around 40-50% of all triple-negative breast cancer (TNBC) patients achieve a pathological complete response (pCR) after treatment with neoadjuvant chemotherapy (NAC). The identification of biomarkers predicting the response to NAC could be helpful for personalized treatment. This systematic review provides an overview of putative biomarkers at baseline that are predictive for a pCR following NAC. Embase, Medline and Web of Science were searched for articles published between January 2010 and August 2022. The articles had to meet the following criteria: patients with primary invasive TNBC without distant metastases and patients must have received NAC. In total, 2045 articles were screened by two reviewers resulting in the inclusion of 92 articles. Overall, the most frequently reported biomarkers associated with a pCR were a high expression of Ki-67, an expression of PD-L1 and the abundance of tumor-infiltrating lymphocytes, particularly CD8+ T cells, and corresponding immune gene signatures. In addition, our review reveals proteomic, genomic and transcriptomic markers that relate to cancer cells, the tumor microenvironment and the peripheral blood, which also affect chemo-sensitivity. We conclude that a prediction model based on a combination of tumor and immune markers is likely to better stratify TNBC patients with respect to NAC response.

Suitability of tumor-associated antibodies as predictive biomarker for response to immune checkpoint inhibitors in patients with melanoma: a short report.

In 2019, Fässler et al showed in this journal that the presence of tumor-associated antibodies correlated with response to immune checkpoint inhibitor treatment in patients with metastatic melanoma. The results of this study suggested that tumor-associated antibodies directed against melanocyte-differentiation antigens and the cancer-germline antigen NY-ESO-1 should be further investigated as candidate biomarkers for response to immune checkpoint inhibitors. The aim of the current study was to validate and extend these previous findings. Therefore, we examined the correlation between serum levels of tumor-associated antibodies and tumor response after treatment with immune checkpoint inhibitors in patients with metastatic melanoma.All patients included in this prospective study were diagnosed with advanced stage melanoma and treated with nivolumab or pembrolizumab monotherapy. Blood samples were collected before and during treatment. Serum levels of tumor-associated antibodies against the melanocyte differentiation antigen Melan-A and the cancer germline antigens NY-ESO-1, MAGE-C2, MAGE-A6 and ROPN1B were measured at baseline and during treatment. Differences between responders and non-responders were assessed using the Mann-Whitney U-test, and differences between different overall survival categories with the Kruskal-Wallis test. P values ≤0.05 were considered significant.Serum samples of 58 patients with advanced melanoma with long-term follow-up (>3 years) were collected. In contrast to the findings of Fässler et al, for all antibodies tested, we found no significant differences between serum levels of responders and non-responders before or during treatment with immune checkpoint inhibitors. In addition, no significant differences were found in serum levels of tumor-associated antibodies for different overall survival groups.Although our study included a larger and more mature cohort of patients with longer follow-up, we could not externally validate the findings of Fässler et al In addition, we were not able to identify other cancer germline antigens as predictive biomarkers of response to immune checkpoint inhibitors in patients advanced melanoma. Based on the results of the present study, clinical applicability of tumor-associated antibodies directed against tumor antigens as predictive biomarkers for immune checkpoint inhibitors in patients with advanced melanoma is not feasible.

Alternative normalization and analysis pipeline to address systematic bias in NanoString GeoMx Digital Spatial Profiling data.

Spatial transcriptomics is a novel technique that provides RNA-expression data with tissue-contextual annotations. Quality assessments of such techniques using end-user generated data are often lacking. Here, we evaluated data from the NanoString GeoMx Digital Spatial Profiling (DSP) platform and standard processing pipelines. We queried 72 ROIs from 12 glioma samples, performed replicate experiments of eight samples for validation, and evaluated five external datasets. The data consistently showed vastly different signal intensities between samples and experimental conditions that resulted in biased analysis. We evaluated the performance of alternative normalization strategies and show that quantile normalization can adequately address the technical issues related to the differences in data distributions. Compared to bulk RNA sequencing, NanoString DSP data show a limited dynamic range which underestimates differences between conditions. Weighted gene co-expression network analysis allowed extraction of gene signatures associated with tissue phenotypes from ROI annotations. Nanostring GeoMx DSP data therefore require alternative normalization methods and analysis pipelines.

A blood-based immune marker for resistance to pembrolizumab in patients with metastatic urothelial cancer.

PD1 inhibition is effective in patients with metastatic urothelial cancer (mUC), yet a large fraction of patients does not respond. In this study, we aimed to identify a blood-based immune marker associated with non-response to facilitate patient selection for anti-PD1. To this end, we quantified 18 immune cell populations using multiplex flow cytometry in blood samples from 71 patients with mUC (as part of a biomarker discovery trial; NCT03263039, registration date 28-08-2017). Patients were classified as responder (ongoing complete or partial response, or stable disease; n = 25) or non-responder (progressive disease; n = 46) according to RECIST v1.1 at 6 months of treatment with pembrolizumab. We observed no differences in numbers of lymphocytes, T-cells, granulocytes, monocytes or their subsets between responders and non-responders at baseline. In contrast, analysis of ratios of immune cell populations revealed that a high mature neutrophil-to-T-cell ratio (MNTR) exclusively identified non-responders. In addition, the survival of patients with high versus low MNTR was poor: median overall survival (OS) 2.2 vs 8.9 months (hazard ratio (HR) 6.6; p < 0.00001), and median progression-free survival (PFS) 1.5 vs 5.2 months (HR 5.6; p < 0.0001). The associations with therapy response, OS, and PFS for the MNTR were stronger than for the classical neutrophil-to-lymphocyte ratio (HR for OS 3.5, and PFS 3) and the PD-L1 combined positivity score (HR for OS 1.9, and PFS 2.1). In conclusion, the MNTR distinctly and uniquely identified non-responders to treatment and may represent a novel pre-treatment blood-based immune metric to select patients with mUC for treatment with pembrolizumab.

Detection of Low-Frequency Epitope-Specific T Cells in Blood of Healthy Individuals according to an Optimized In Vitro Amplification System.

Detection and amplification of epitope-specific T cells hold great promise for diagnosis and therapy of cancer patients. Currently, measurement and retrieval of epitope-specific T cells is hampered by limited availability of patients' biomaterials and lack of sensitive and easy-to-implement T cell priming and expansion. We have developed an in vitro T cell amplification system starting from healthy donor blood and tested different subsets and ratios of autologous T cells and APCs as well as the resting period between amplification cycles. We demonstrated in 10 different donors significantly enhanced frequency of T cells specific for MelanA/HLA-A2, which relied on coculturing of naive T cells and CD11c+ dendritic cells in a 1:1 ratio followed by three weekly amplification cycles using the effluent of the naive T cell sort as APCs, a 24-h rest period prior to every reamplification cycle, and IFN-γ production as a readout for epitope-specific T cells. Using this system, MelanA/HLA-A2-specific T cells were enriched by 200-fold, measuring up to 20-60% of all T cells. We extended this system to enrich NY-ESO-1/HLA-A2- and BMLF-1/HLA-A2-specific T cells, examples of a cancer germline Ag and an oncoviral Ag differing in their ability to bind to HLA-A2 and the presence of specific T cells in the naive and, in case of BMLF-1, also the Ag-experienced repertoire. Collectively, we have developed a sensitive and easy-to-implement in vitro T cell amplification method to enrich epitope-specific T cells that is expected to facilitate research and clinical utility regarding T cell diagnosis and treatments.

Intratumoral Niches of B Cells and Follicular Helper T Cells, and the Absence of Regulatory T Cells, Associate with Longer Survival in Early-Stage Oral Tongue Cancer Patients.

In early oral squamous cell carcinoma (OSCC), the occurrence of clusters between CD20 B cells and CD4 T cells in the invasive margin (IM) can be captured by using the CD20 cluster score, and is positively associated with patient survival. However, the exact contribution of different CD4 T cell subsets, as well as B cell subsets toward patient prognosis is largely unknown. To this end, we studied regulatory T cells ((Treg cells) FOXP3 and CD4), T helper-type 1 cells ((Th1 cells) Tbet and CD4), follicular helper T cells ((Tfh cells) Bcl6 and CD4), B cells (CD20), germinal center B cells ((GC B cells) BCL6 and CD20), and follicular dendritic cells ((fDCs) CD21) for their density, location, and interspacing using multiplex in situ immunofluorescence of 75 treatment-naïve, primary OSCC patients. We observed that Treg, Th1-, Tfh-, and GC B cells, but not fDCs, were abundantly present in the stroma as compared with the tumor, and in the IM as compared with in the center of the tumor. Patients with high CD20 cluster scores had a high density of all three CD4 T cell subsets and GC B cells in the stromal IM as compared with patients with low CD20 cluster scores. Notably, enriched abundance of Tfh cells (HR 0.20, p = 0.04), and diminished abundance of Treg cells (HR 0.10, p = 0.03), together with an overall short distance between Tfh and B cells (HR:0.08, p < 0.01), but not between Treg and B cells (HR 0.43, p = 0.28), were significantly associated with overall survival of patients with OSCC. Our study identified the prognostic value of clusters between CD20 B cells and Tfh cells in the stromal IM of OSCC patients, and enabled an improved understanding of the clinical value of a high CD20 cluster score, which requires validation in larger clinical cohorts.

Transient cell-in-cell formation underlies tumor relapse and resistance to immunotherapy.

Despite the remarkable successes of cancer immunotherapies, the majority of patients will experience only partial response followed by relapse of resistant tumors. While treatment resistance has frequently been attributed to clonal selection and immunoediting, comparisons of paired primary and relapsed tumors in melanoma and breast cancers indicate that they share the majority of clones. Here, we demonstrate in both mouse models and clinical human samples that tumor cells evade immunotherapy by generating unique transient cell-in-cell structures, which are resistant to killing by T cells and chemotherapies. While the outer cells in this cell-in-cell formation are often killed by reactive T cells, the inner cells remain intact and disseminate into single tumor cells once T cells are no longer present. This formation is mediated predominantly by IFNγ-activated T cells, which subsequently induce phosphorylation of the transcription factors signal transducer and activator of transcription 3 (STAT3) and early growth response-1 (EGR-1) in tumor cells. Indeed, inhibiting these factors prior to immunotherapy significantly improves its therapeutic efficacy. Overall, this work highlights a currently insurmountable limitation of immunotherapy and reveals a previously unknown resistance mechanism which enables tumor cells to survive immune-mediated killing without altering their immunogenicity.

Cancer immunotherapies use the body's own immune system to fight off cancer. But, despite some remarkable success stories, many patients only see a temporary improvement before the immunotherapy stops being effective and the tumours regrow. It is unclear why this occurs, but it may have to do with how the immune system attacks cancer cells. Immunotherapies aim to activate a special group of cells known as killer T-cells, which are responsible for the immune response to tumours. These cells can identify cancer cells and inject toxic granules through their membranes, killing them. However, killer T-cells are not always effective. This is because cancer cells are naturally good at avoiding detection, and during treatment, their genes can mutate, giving them new ways to evade the immune system. Interestingly, when scientists analysed the genes of tumour cells before and after immunotherapy, they found that many of the genes that code for proteins recognized by T-cells do not change significantly. This suggests that tumours' resistance to immune attack may be physical, rather than genetic. To investigate this hypothesis, Gutwillig et al. developed several mouse tumour models that stop responding to immunotherapy after initial treatment. Examining cells from these tumours revealed that when the immune system attacks, they reorganise by getting inside one another. This allows some cancer cells to hide under many layers of cell membrane. At this point killer T-cells can identify and inject the outer cell with toxic granules, but it cannot reach the cells inside. This ability of cancer cells to hide within one another relies on them recognising when the immune system is attacking. This happens because the cancer cells can detect certain signals released by the killer T-cells, allowing them to hide. Gutwillig et al. identified some of these signals, and showed that blocking them stopped cancer cells from hiding inside each other, making immunotherapy more effective. This new explanation for how cancer cells escape the immune system could guide future research and lead to new cancer treatments, or approaches to boost existing treatments. Understanding the process in more detail could allow scientists to prevent it from happening, by revealing which signals to block, and when, for best results.

Serum cytokine levels are associated with tumor progression during FOLFIRINOX chemotherapy and overall survival in pancreatic cancer patients.

Background: Biomarkers predicting treatment response may be used to stratify patients with pancreatic ductal adenocarcinoma (PDAC) for available therapies. The aim of this study was to evaluate the association of circulating cytokines with FOLFIRINOX response and with overall survival (OS). Methods: Serum samples were collected before start and after the first cycle of FOLFIRINOX from patients with PDAC (n=83) of all disease stages. Overall, 34 circulating cytokines were analyzed with a multiplex immunoassay. In addition, changes in peripheral blood immune cell counts were determined by flow cytometry to correlate with differences in cytokine levels. Chemotherapy response was determined by CT scans with the RECIST 1.1 criteria, as disease control (n=64) or progressive disease (n=19) within eight cycles of FOLFIRINOX. Results: Patients with high serum IL-1RA concentrations after one cycle of chemotherapy were less likely to have tumor progression during FOLFIRINOX (OR 0.25, P=0.040). Increase of circulating IL-1RA concentrations correlated with increase of total, classical (CD14+CD16-), and non-classical monocytes (CD14-CD16+), and dendritic cells. In multivariable cox regression, including the variables chemotherapy response outcome and baseline CA19-9 level, serum concentrations of IL-7 (HR 2.14, P=0.010), IL-18 (HR 2.00, P=0.020), and MIP-1ß (HR 0.51, P=0.025) after one cycle of FOLFIRINOX showed correlations with OS. Conclusions: Circulating IL-1RA, IL-7, IL-18, and MIP-1ß concentrations are biomarkers associated with FOLFIRINOX response in PDAC patients, suggesting an important role for specific immune cells in chemotherapy response and PDAC progression. Cytokine-based treatment might improve patient outcome and should be evaluated in future studies.

Associations between patient and disease characteristics and severe adverse events during immune checkpoint inhibitor treatment: An observational study.

AIM: With increasing use of immune checkpoint inhibitors (ICIs) more patients will develop severe and potentially life-threatening immune-related adverse events (irAEs). So far, predictive models for the occurrence of grade ≥3 irAEs are lacking. Therefore, we analysed associations between patient and disease characteristics, and the occurrence of grade ≥3 irAEs. METHODS: Patients with cancer who were treated with anti-PD-1 (+/-anti-CTLA-4) between July 2015 and February 2020, and who were prospectively included in the MULTOMAB-trial, were eligible for this cohort study. Time to and occurrence of grade ≥3 irAEs according to CTCAE v5.0 were retrospectively registered. The associations between patient and disease characteristics and irAE occurrence were analysed using the competing risk cox-regression model of Fine and Gray. Analyses were performed separately in patients treated with monotherapy (anti-PD-1) and combination therapy (anti-PD-1 + anti-CTLA-4). Subgroup analyses were performed in tumour types with the highest number of patients; melanoma and NSCLC. RESULTS: Out of 641 patients, 106 patients (17%) experienced grade ≥3 irAEs. None of the analysed factors were associated with grade ≥3 irAE occurrence in the monotherapy (n = 550) or the combination therapy (n = 91) groups, nor in the subgroup analyses. Of interest, none of the patients with NSCLC with a WHO performance status of 0 (n = 34) experienced grade ≥3 irAEs. Most common NSCLC histology types were adenocarcinoma (n = 99/55%) and squamous cell carcinoma (n = 39/22%). CONCLUDING STATEMENT: This study shows that patient and disease characteristics are not able to predict the occurrence of serious AEs in patients treated with ICIs.

Gene Engineering T Cells with T-Cell Receptor for Adoptive Therapy.

Prior to clinical testing of adoptive T-cell therapy with T-cell receptor (TCR)-engineered T cells, TCRs need to be retrieved, annotated, gene-transferred, and extensively tested in vitro to accurately assess specificity and sensitivity of target recognition. Here, we present a fundamental series of protocols that cover critical preclinical parameters, thereby enabling the selection of candidate TCRs for clinical testing.

Cancer germline antigens and tumor-agnostic CD8+ T cell evasion.

Cancer germline antigens (CGAs) are expressed in immune-privileged germline tissues, while epigenetically silenced in somatic tissues. CGAs become re-expressed in tumors and can promote oncogenesis. Tumors prominently exploit mechanisms similar to those in germline tissues to shield from immunosurveillance. We hypothesize that CGAs contribute towards tumor escape from immune effector CD8+ T cells. For illustrative purposes, we assessed the co-presence or -absence of CGAs with these cells in multiple tumor types. Considering a broad array of CD8+ T cell evasive mechanisms, we exemplify the co-occurrence of gene transcripts of eight CGAs with those of adhesion molecules, endothelial cells, and/or the Wnt pathway. We present a novel concept of CGAs and their association with CD8+ T cell evasion, which may be relevant for future immunotherapeutic interventions.

Rintatolimod (Ampligen®) Enhances Numbers of Peripheral B Cells and Is Associated with Longer Survival in Patients with Locally Advanced and Metastasized Pancreatic Cancer Pre-Treated with FOLFIRINOX: A Single-Center Named Patient Program.

Background: Treatment with the TLR-3 agonist rintatolimod may improve pancreatic cancer patients' survival via immunomodulation, but the effect is unproven. Methods: In this single-center named patient program, patients with locally advanced pancreatic cancer (LAPC) or metastatic disease were treated with rintatolimod (six weeks total, twice per week, with a maximum of 400 mg per infusion). The primary endpoints were the systemic immune-inflammation index (SIII), the neutrophil to lymphocyte ratio (NLR), and the absolute counts of 18 different populations of circulating immune cells as measured by flow cytometry. Secondary endpoints were progression-free survival (PFS) and overall survival (OS). Subgroup analyses were performed in long-term survivors (>1-year overall survival after starting rintatolimod) and compared to short-term survivors (≤1 year). Results: Between January 2017 and February 2019, twenty-seven patients with stable LAPC or metastatic disease were pre-treated with FOLFIRINOX and treated with rintatolimod. Rintatolimod treatment was well-tolerated. The SIII and NLR values were significantly lower in the 11 long-term survivors, versus 16 short-term survivors. The numbers of B-cells were significantly increased in long-term survivors. Numbers of T cells and myeloid cells were not significantly increased after treatment with rintatolimod. Median PFS was 13 months with rintatolimod, versus 8.6 months in a subset of matched controls (n = 27, hazard ratio = 0.52, 95% CI = 0.28−0.90, p = 0.007). The median OS was 19 months with rintatolimod, versus 12.5 months in the matched control (hazard ratio = 0.51, 95% CI = 0.28−0.90, p = 0.016). Conclusions: Treatment with rintatolimod showed a favorable effect on the numbers of peripheral B cells in patients with pancreatic cancer and improved survival in pancreatic cancer, but additional evidence is required.

Anti-PD-1 Efficacy in Patients with Metastatic Urothelial Cancer Associates with Intratumoral Juxtaposition of T Helper-Type 1 and CD8+ T cells.

PURPOSE: PD-1 inhibition results in durable antitumor responses in a proportion of patients with metastatic urothelial cancer (mUC). The majority of patients, however, do not experience clinical benefit. In this study, we aimed to identify early changes in T-cell subsets that underlie anti-PD-1 efficacy in patients with mUC. EXPERIMENTAL DESIGN: Paired samples were collected from peripheral blood, plasma, and metastatic lesions of 56 patients with mUC at baseline and weeks 6 and 12 after initiating pembrolizumab treatment (200 mg intravenously, every 3 weeks). Samples were analyzed using multiplex flow cytometry, ELISA, and in situ stainings, including cellular network analysis. Treatment response was evaluated as best overall response according to RECIST v1.1, and patients were classified as responder (complete or partial response) or nonresponder (progressive disease). RESULTS: In responders, baseline fractions of CD4+ T cells expressing cosignaling receptors were higher compared with nonresponders. The fraction of circulating PD-1+ CD4+ T cells decreased at weeks 6 and 12, whereas the fraction of 4-1BB+ CD28+ CD4+ T cells increased at week 12. In metastatic lesions of responders, the baseline density of T helper-type 1 (Th1) cells, defined as T-bet+ CD4+ T cells, was higher as compared to non-responders. Upon treatment, Th1 cells became localized in close proximity to CD8+ T cells, CD11b+ myeloid cells, and tumor cells. CONCLUSIONS: A decrease in the fraction of circulating PD-1+ CD4+ T cells, and juxtaposition of Th1, CD8+, and myeloid cells was associated with response to anti-PD-1 treatment in patients with mUC.

Treatment of early-stage breast cancer with percutaneous thermal ablation, an open-label randomised phase 2 screening trial: rationale and design of the THERMAC trial.

INTRODUCTION: Breast cancer is the most frequently diagnosed malignancy worldwide but almost half of the patients have an excellent prognosis with a 5-year survival rate of 98%-99%. These patients could potentially be treated with thermal ablation to avoid surgical excision, reduce treatment-related morbidity and increase patients' quality of life without jeopardising treatment effectiveness. Previous studies showed highest complete ablation rates for radiofrequency, microwave and cryoablation. However, due to heterogeneity among studies, it is unknown which of these three techniques should be selected for a phase 3 comparative study. METHODS AND ANALYSIS: The aim of this phase 2 screening trial is to determine the efficacy rate of radiofrequency, microwave and cryoablation with the intention to select one treatment for further testing in a phase 3 trial. Additionally, exploratory data are obtained for the phase 3 trial. The design is a multicentre open-label randomised phase 2 screening trial. Patients with unifocal, invasive breast cancer with a maximum diameter of 2 cm without lymph node or distant metastases are included. Triple negative, Bloom-Richardson grade 3 tumours and patients with an indication for neoadjuvant chemotherapy will be excluded. Included patients will be allocated to receive one of the three thermal ablation techniques. Three months later surgical excision will be performed to determine the efficacy of thermal ablation. Treatment efficacy in terms of complete ablation rate will be assessed with CK 8/18 and H&E staining. Secondary outcomes include feasibility of the techniques in an outpatient setting, accuracy of MRI for complete ablation, patient satisfaction, adverse events, side effects, cosmetic outcome, system usability and immune response. ETHICS AND DISSEMINATION: This study protocol was approved by Medical Research Ethics Committee of the Erasmus Medical Center, Rotterdam, the Netherlands. Study results will be submitted for publication in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NL9205 (www.trialregister.nl); Pre-results.