Publications by authors named "Carlos de Andrea"

47 Publications

Circulating Levels of the Interferon-γ-Regulated Chemokines CXCL10/CXCL11, IL-6 and HGF Predict Outcome in Metastatic Renal Cell Carcinoma Patients Treated with Antiangiogenic Therapy.

Cancers (Basel) 2021 Jun 7;13(11). Epub 2021 Jun 7.

IDISNA and Program in Solid Tumors, Center for Applied Medical Research (CIMA), University of Navarra, 31008 Pamplona, Spain.

Sunitinib and pazopanib are standard first-line treatments for patients with metastatic renal cell carcinoma (mRCC). Nonetheless, as the number of treatment options increases, there is a need to identify biomarkers that can predict drug efficacy and toxicity. In this prospective study we evaluated a set of biomarkers that had been previously identified within a secretory signature in mRCC patients. This set includes tumor expression of c-Met and serum levels of HGF, IL-6, IL-8, CXCL9, CXCL10 and CXCL11. Our cohort included 60 patients with mRCC from 10 different Spanish hospitals who received sunitinib ( = 51), pazopanib ( = 4) or both ( = 5). Levels of biomarkers were studied in relation to response rate, progression-free survival (PFS) and overall survival (OS). High tumor expression of c-Met and high basal serum levels of HGF, IL-6, CXCL11 and CXCL10 were significantly associated with reduced PFS and/or OS. In multivariable Cox regression analysis, CXCL11 was identified as an independent biomarker predictive of shorter PFS and OS, and HGF was an independent predictor of reduced PFS. Correlation analyses using our cohort of patients and patients from TCGA showed that HGF levels were significantly correlated with those of IL-6, CXCL11 and CXCL10. Bioinformatic protein-protein network analysis revealed a significant interaction between these proteins, all this suggesting a coordinated expression and secretion. We also developed a prognostic index that considers this group of biomarkers, where high values in mRCC patients can predict higher risk of relapse (HR 5.28 [2.32-12.0], < 0.0001). In conclusion, high plasma HGF, CXCL11, CXCL10 and IL-6 levels are associated with worse outcome in mRCC patients treated with sunitinib or pazopanib. Our findings also suggest that these factors may constitute a secretory cluster that acts coordinately to promote tumor growth and resistance to antiangiogenic therapy.
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http://dx.doi.org/10.3390/cancers13112849DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8201218PMC
June 2021

Heterogenous presence of Neutrophil Extracellular Traps in human solid tumours is partially dependent on Interleukin-8.

J Pathol 2021 Jun 29. Epub 2021 Jun 29.

Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Spain.

Neutrophil extracellular traps (NETs) are webs of extracellular nuclear DNA extruded by dying neutrophils infiltrating tissue. NETs constitute a defence mechanism to entrap and kill fungi and bacteria. Tumours induce the formation of NETs to the advantage of the malignancy via a variety of mechanisms shown in mouse models. Here, we investigated the presence of NETs in a variety of human solid tumours and their association with IL-8 (CXCL8) protein expression and CD8 T-cell density in the tumour microenvironment. Multiplex immunofluorescence panels were developed to identify NETs in human cancer tissues by co-staining with the granulocyte marker CD15, the neutrophil marker myeloperoxidase, and citrullinated histone H3 (H3Cit), as well as IL-8 protein and CD8 T cells. Three ELISA methods to detect and quantify circulating NETs in serum were optimized and utilized. Whole tumour sections and tissue microarrays from patients with non-small cell lung cancer (NSCLC) (n = 14), bladder cancer (n = 14), melanoma (n = 11), breast cancer (n = 31), colorectal cancer (n = 20), and mesothelioma (n = 61) were studied. Also, serum samples collected retrospectively from patients with metastatic melanoma (n = 12) and NSCLC (n = 34) were ELISA-assayed to quantify circulating NETs and IL-8. NETs were detected in six different human cancer types with wide individual variation in terms of tissue density and distribution. At least in NSCLC, bladder cancer, and metastatic melanoma, NET density positively correlated with IL-8 protein expression and inversely correlated with CD8 T-cell densities. In a series of serum samples from melanoma and NSCLC patients, a positive correlation between circulating NETs and IL-8 was found. In conclusion, NETs are detectable in formalin-fixed human biopsy samples from solid tumours and in the circulation of cancer patients with a considerable degree of individual variation. NETs show a positive association with IL-8, and a trend towards a negative association with CD8 tumour-infiltrating lymphocytes. This article is protected by copyright. All rights reserved.
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http://dx.doi.org/10.1002/path.5753DOI Listing
June 2021

Characterizing the Invasive Tumor Front of Aggressive Uterine Adenocarcinoma and Leiomyosarcoma.

Front Cell Dev Biol 2021 3;9:670185. Epub 2021 Jun 3.

Cancer CIBER (CIBERONC), Madrid, Spain.

The invasive tumor front (the tumor-host interface) is vitally important in malignant cell progression and metastasis. Tumor cell interactions with resident and infiltrating host cells and with the surrounding extracellular matrix and secreted factors ultimately determine the fate of the tumor. Herein we focus on the invasive tumor front, making an in-depth characterization of reticular fiber scaffolding, infiltrating immune cells, gene expression, and epigenetic profiles of classified aggressive primary uterine adenocarcinomas (24 patients) and leiomyosarcomas (11 patients). Sections of formalin-fixed samples before and after microdissection were scanned and studied. Reticular fiber architecture and immune cell infiltration were analyzed by automatized algorithms in colocalized regions of interest. Despite morphometric resemblance between reticular fibers and high presence of macrophages, we found some variance in other immune cell populations and distinctive gene expression and cell adhesion-related methylation signatures. Although no evident overall differences in immune response were detected at the gene expression and methylation level, impaired antimicrobial humoral response might be involved in uterine leiomyosarcoma spread. Similarities found at the invasive tumor front of uterine adenocarcinomas and leiomyosarcomas could facilitate the use of common biomarkers and therapies. Furthermore, molecular and architectural characterization of the invasive front of uterine malignancies may provide additional prognostic information beyond established prognostic factors.
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http://dx.doi.org/10.3389/fcell.2021.670185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8209546PMC
June 2021

PD-L1 in Cytological Samples: A Review and a Practical Approach.

Front Med (Lausanne) 2021 7;8:668612. Epub 2021 May 7.

Department of Pathology, Clinica University of Navarra, Pamplona, Spain.

With a growing number of predictive biomarkers needed to manage patients with non-small cell lung cancer (NSCLC), there has been a paradigm shift in care and handling of diagnostic samples. Among the various testing methods, immunohistochemistry (IHC) is the most cost- effective and widely available. Furthermore, over the past decade immunotherapy has emerged as one of the most promising cancer treatments. In this scenario IHC is the most used testing method available for PDL-1/PD1 immunotherapy. Several monoclonal antibodies targeting programmed death 1 (PD-1)/programmed death ligand-1 (PD-L1) pathways have been integrated into standard-of-care treatments of a wide range of cancer types, once provided evidence of PD-L1 expression in tumor cells by immunohistochemistry (IHC). Since currently available PD-L1 assays have been developed on formalin-fixed paraffin embedded (FFPE) histological specimens, a growing body of research is being dedicated to confirm the feasibility of applying PDL-1 assays also to cytological samples. Albeit promising results have been reported, several important issues still need to be addressed. Among these are the type of cytological samples, pre-analytical issues, cyto-histological correlation, and inter-observer agreement. This review briefly summarizes the knowledge of the role of cytopathology in the analysis of PD-L1 by immunocytochemistry (ICC) and future directions of cytopathology in the immunotherapy setting.
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http://dx.doi.org/10.3389/fmed.2021.668612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8139418PMC
May 2021

Whole exome sequencing characterization of individuals presenting extreme phenotypes of high and low risk of developing tobacco-induced lung adenocarcinoma.

Transl Lung Cancer Res 2021 Mar;10(3):1327-1337

Health Research Institute of Navarra (IdisNA), Pamplona, Spain.

Background: Tobacco is the main risk factor for developing lung cancer. Yet, some heavy smokers do not develop lung cancer at advanced ages while others develop it at young ages. Here, we assess for the first time the genetic background of these clinically relevant extreme phenotypes using whole exome sequencing (WES).

Methods: We performed WES of germline DNA from heavy smokers who either developed lung adenocarcinoma at an early age (extreme cases, n=50) or did not present lung adenocarcinoma or other tumors at an advanced age (extreme controls, n=50). We selected non-synonymous variants located in exonic regions and consensus splice sites of the genes that showed significantly different allelic frequencies between both cohorts. We validated our results in all the additional extreme cases (i.e., heavy smokers who developed lung adenocarcinoma at an early age) available from The Cancer Genome Atlas (TCGA).

Results: The mean age for the extreme cases and controls was respectively 49.7 and 77.5 years. Mean tobacco consumption was 43.6 and 56.8 pack-years. We identified 619 significantly different variants between both cohorts, and we validated 108 of these in extreme cases selected from TCGA. Nine validated variants, located in relevant cancer related genes, such as , or , among others, achieved statistical significance in the False Discovery Rate test. The most significant validated variant (P=4.48×10) was located in the tumor-suppressor gene .

Conclusions: We describe genetic variants associated with extreme phenotypes of high and low risk for the development of tobacco-induced lung adenocarcinoma. Our results and our strategy may help to identify high-risk subjects and to develop new therapeutic approaches.
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http://dx.doi.org/10.21037/tlcr-20-1197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8044482PMC
March 2021

Utilisation of cytological samples for multiplex immunofluorescence assay.

Cytopathology 2021 Apr 18. Epub 2021 Apr 18.

Department of Pathology, Clínica Universidad de Navarra, Pamplona, Spain.

Objective: Understanding the immune environment of non-small cell lung cancer (NSCLC) is important for designing effective anticancer immunotherapies. We describe the use of multiplex immunofluorescence (mIF) assays to enable characterisation of the tumour-infiltrating immune cells and their interactions, both across and within immune subtypes.

Methods: Six cytological samples of NSCLC taken by transoesophageal ultrasound-guided fine needle aspiration were tested with an mIF assay designed to detect the expression of key immune cell markers such as CD3, CD8, CD20, CD11b, and CD68. Pan-cytokeratin was used to detect the NSCLC cells. Fluorescence images were acquired on a Vectra-Polaris Automated Quantitative Pathology Imaging System (Akoya Biosciences).

Results: MIF assay was able to reliably detect and quantify the myeloid cell markers CD11b, CD68, CD3+ and CD8+ T cells, and CD20+ B lymphocytes on cytological samples of NSCLC. Whole-tissue analysis and its correlation with the corresponding H&E stains allowed a better understanding of the tissue morphology and the relationship between tumour and stroma compartments. Additionally, a uniform, specific, and correct staining pattern was seen for every immune marker.

Conclusion: The implementation of mIF assay on cytological samples taken with minimally invasive methods seems feasible and can be used to explore the immune environment of NSCLC.
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http://dx.doi.org/10.1111/cyt.12979DOI Listing
April 2021

TargetPlex FFPE-Direct DNA Library Preparation Kit for SiRe NGS panel: an international performance evaluation study.

J Clin Pathol 2021 Mar 25. Epub 2021 Mar 25.

Department of Pathology, Katholieke Universiteit Leuven UZ Leuven, Leuven, Belgium.

Aim: Next generation sequencing (NGS) represents a key diagnostic tool to identify clinically relevant gene alterations for treatment-decision making in cancer care. However, the complex manual workflow required for NGS has limited its implementation in routine clinical practice. In this worldwide study, we validated the clinical performance of the TargetPlex FFPE-Direct DNA Library Preparation Kit for NGS analysis. Impressively, this new assay obviates the need for separate, labour intensive and time-consuming pre-analytical steps of DNA extraction, purification and isolation from formalin-fixed paraffin embedded (FFPE) specimens in the NGS workflow.

Methods: The TargetPlex FFPE-Direct DNA Library Preparation Kit, which enables NGS analysis directly from FFPE, was specifically developed for this study by TargetPlex Genomics Pleasanton, California. Eleven institutions agreed to take part in the study coordinated by the Molecular Cytopathology Meeting Group (University of Naples Federico II, Naples, Italy). All participating institutions received a specific Library Preparation Kit to test eight FFPE samples previously assessed with standard protocols. The analytical parameters and mutations detected in each sample were then compared with those previously obtained with standard protocols.

Results: Overall, 92.8% of the samples were successfully analysed with the TargetPlex FFPE-Direct DNA Library Preparation Kit on Thermo Fisher Scientific and Illumina platforms. Altogether, in comparison with the standard workflow, the TargetPlex FFPE-Direct DNA Library Preparation Kit was able to detect 90.5% of the variants.

Conclusion: The TargetPlex FFPE-Direct DNA Library Preparation Kit combined with the SiRe panel constitutes a convenient, practical and robust cost-saving solution for FFPE NGS analysis in routine practice.
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http://dx.doi.org/10.1136/jclinpath-2021-207450DOI Listing
March 2021

Proteomic Analysis of Low-Grade, Early-Stage Endometrial Carcinoma Reveals New Dysregulated Pathways Associated with Cell Death and Cell Signaling.

Cancers (Basel) 2021 Feb 14;13(4). Epub 2021 Feb 14.

Department of Pathology, Hospital Universitario La Paz, IdiPAZ, 28046 Madrid, Spain.

Low-grade, early-stage endometrial carcinoma (EC) is the most frequent malignant tumor of the uterine corpus. However, the molecular alterations that underlie these tumors are far from being fully understood. The purpose of this study is to describe dysregulated molecular pathways from EC patients. Sixteen samples of tumor tissue and paired healthy controls were collected and both were subjected to mass spectrometry (MS)/MS proteomic analysis. Gene ontology and pathway analysis was performed to discover dysregulated pathways and/or proteins using different databases and bioinformatic tools. Dysregulated pathways were cross-validated in an independent external cohort. Cell signaling, immune response, and cell death-associated pathways were robustly identified. The SLIT/ROBO signaling pathway demonstrated dysregulation at the proteomic and transcriptomic level. Necroptosis and ferroptosis were cell death-associated processes aberrantly regulated, in addition to apoptosis. Immune response-associated pathways showed a dominance of innate immune responses. Tumor immune infiltrates measured by immunofluorescence demonstrated diverse lymphoid and myeloid populations. Our results suggest a role of SLIT/ROBO, necroptosis, and ferroptosis, as well as a prominent role of innate immune response in low-grade, early-stage EC. These results could guide future research in this group of tumors.
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http://dx.doi.org/10.3390/cancers13040794DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7917913PMC
February 2021

SRC family kinase (SFK) inhibitor dasatinib improves the antitumor activity of anti-PD-1 in NSCLC models by inhibiting Treg cell conversion and proliferation.

J Immunother Cancer 2021 Mar;9(3)

IDISNA and Program in Solid Tumors, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain

Introduction: The use of immune-checkpoint inhibitors has drastically improved the management of patients with non-small cell lung cancer (NSCLC), but innate and acquired resistances are hurdles needed to be solved. Immunomodulatory drugs that can reinvigorate the immune cytotoxic activity, in combination with antiprogrammed cell death 1 (PD-1) antibody, are a great promise to overcome resistance. We evaluated the impact of the SRC family kinases (SFKs) on NSCLC prognosis, and the immunomodulatory effect of the SFK inhibitor dasatinib, in combination with anti-PD-1, in clinically relevant mouse models of NSCLC.

Methods: A cohort of patients from University Clinic of Navarra (n=116) was used to study immune infiltrates by multiplex immunofluorescence (mIF) and YES1 protein expression in tumor samples. Publicly available resources (TCGA, Km Plotter, and CIBERSORT) were used to study patient's survival based on expression of SFKs and tumor infiltrates. Syngeneic NSCLC mouse models 393P and UNSCC680AJ were used for in vivo drug testing.

Results: Among the SFK members, YES1 expression showed the highest association with poor prognosis. Patients with high YES1 tumor levels also showed high infiltration of CD4+/FOXP3+ cells (regulatory T cells (Tregs)), suggesting an immunosuppressive phenotype. After testing for YES1 expression in a panel of murine cell lines, 393P and UNSCC680AJ were selected for in vivo studies. In the 393P model, dasatinib+anti-PD-1 treatment resulted in synergistic activity, with 87% tumor regressions and development of immunological memory that impeded tumor growth when mice were rechallenged. In vivo depletion experiments further showed that CD8+ and CD4+ cells are necessary for the therapeutic effect of the combination. The antitumor activity was accompanied by a very significant decrease in the number of Tregs, which was validated by mIF in tumor sections. In the UNSCC680AJ model, the antitumor effects of dasatinib+anti-PD-1 were milder but similar to the 393P model. In in vitro assays, we demonstrated that dasatinib blocks proliferation and transforming growth factor beta-driven conversion of effector CD4+ cells into Tregs through targeting of phospholymphocyte-specific protein tyrosine kinase and downstream effectors pSTAT5 and pSMAD3.

Conclusions: YES1 protein expression is associated with increased numbers of Tregs in patients with NSCLC. Dasatinib synergizes with anti-PD-1 to impair tumor growth in NSCLC experimental models. This study provides the preclinical rationale for the combined use of dasatinib and PD-1/programmed death-ligand 1 blockade to improve outcomes of patients with NSCLC.
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http://dx.doi.org/10.1136/jitc-2020-001496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7931761PMC
March 2021

The mutational load and a T-cell inflamed tumour phenotype identify ovarian cancer patients rendering tumour-reactive T cells from PD-1 tumour-infiltrating lymphocytes.

Br J Cancer 2021 Mar 5;124(6):1138-1149. Epub 2021 Jan 5.

Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona, Spain.

Background: Adoptive immunotherapy with tumour-infiltrating lymphocytes (TIL) may benefit from the use of selective markers, such as PD-1, for tumour-specific T-cell enrichment, and the identification of predictive factors that help identify those patients capable of rendering tumour-reactive TILs. We have investigated this in ovarian cancer (OC) patients as candidates for TIL therapy implementation.

Methods: PD-1 and PD-1 CD8 TILs were isolated from ovarian tumours and expanded cells were tested against autologous tumour cells. Baseline tumour samples were examined using flow cytometry, multiplexed immunofluorescence and Nanostring technology, for gene expression analyses, as well as a next-generation sequencing gene panel, for tumour mutational burden (TMB) calculation.

Results: Tumour-reactive TILs were detected in half of patients and were exclusively present in cells derived from the PD-1 fraction. Importantly, a high TIL density in the fresh tumour, the presence of CD137 cells within the PD-1CD8 TIL subset and their location in the tumour epithelium, together with a baseline T-cell-inflamed genetic signature and/or a high TMB, are features that identify patients rendering tumour-reactive TIL products.

Conclusion: We have demonstrated that PD-1 identifies ovarian tumour-specific CD8 TILs and has uncovered predictive factors that identify OC patients who are likely to render tumour-specific cells from PD-1 TILs.
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http://dx.doi.org/10.1038/s41416-020-01218-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7961070PMC
March 2021

Delta-24-RGD, an Oncolytic Adenovirus, Increases Survival and Promotes Proinflammatory Immune Landscape Remodeling in Models of AT/RT and CNS-PNET.

Clin Cancer Res 2021 Mar 29;27(6):1807-1820. Epub 2020 Dec 29.

Health Research Institute of Navarra (IdiSNA), Pamplona, Navarra, Spain.

Purpose: Atypical teratoid/rhabdoid tumors (AT/RT) and central nervous system primitive neuroectodermal tumors (CNS-PNET) are pediatric brain tumors with poor survival and life-long negative side effects. Here, the aim was to characterize the efficacy and safety of the oncolytic adenovirus, Delta-24-RGD, which selectively replicates in and kills tumor cells.

Experimental Design: Delta-24-RGD determinants for infection and replication were evaluated in patient expression datasets. Viral replication and cytotoxicity were assessed in a battery of CNS-PNET and AT/RT cell lines. , efficacy was determined in different orthotopic mouse models, including early and established tumor models, a disseminated AT/RT lesion model, and immunocompetent humanized mouse models (hCD34-NSG-SGM3).

Results: Delta-24-RGD infected and replicated efficiently in all the cell lines tested. In addition, the virus induced dose-dependent cytotoxicity [IC value below 1 plaque-forming unit (PFU)/cell] and the release of immunogenic markers. , a single intratumoral Delta-24-RGD injection (10 or 10 PFU) significantly increased survival and led to long-term survival in AT/RT and PNET models. Delta-24-RGD hindered the dissemination of AT/RTs and increased survival, leading to 70% of long-term survivors. Of relevance, viral administration to established tumor masses (30 days after engraftment) showed therapeutic benefit. In humanized immunocompetent models, Delta-24-RGD significantly extended the survival of mice bearing AT/RTs or PNETs (ranging from 11 to 27 days) and did not display any toxicity associated with inflammation. Immunophenotyping of Delta-24-RGD-treated tumors revealed increased CD8 T-cell infiltration.

Conclusions: Delta-24-RGD is a feasible therapeutic option for AT/RTs and CNS-PNETs. This work constitutes the basis for potential translation to the clinical setting.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-3313DOI Listing
March 2021

IL8, Neutrophils, and NETs in a Collusion against Cancer Immunity and Immunotherapy.

Clin Cancer Res 2021 May 29;27(9):2383-2393. Epub 2020 Dec 29.

Program of Immunology and Immunotherapy, Cima Universidad de Navarra, Pamplona, Navarra, Spain.

One of the most important mechanisms by which cancer fosters its own development is the generation of an immune microenvironment that inhibits or impairs antitumor immune responses. A cancer permissive immune microenvironment is present in a large proportion of the patients with cancer who do not respond to immunotherapy approaches intended to trigger preexisting antitumor immune responses, for instance, immune checkpoint blockade. High circulating levels of IL8 in patients with cancer quite accurately predict those who will not benefit from checkpoint-based immunotherapy. IL8 has been reported to favor cancer progression and metastases via different mechanisms, including proangiogenesis and the maintenance of cancer stem cells, but its ability to attract and functionally modulate neutrophils and macrophages is arguably one of the most important factors. IL8 does not only recruit neutrophils to tumor lesions, but also triggers the extrusion of neutrophil extracellular traps (NET). The relevance and mechanisms underlying the contribution of both neutrophils and NETs to cancer development and progression are starting to be uncovered and include both direct effects on cancer cells and changes in the tumor microenvironment, such as facilitating metastasis, awakening micrometastases from dormancy, and facilitating escape from cytotoxic immune cells. Blockade of IL8 or its receptors (CXCR1 and CXCR2) is being pursued in drug development, and clinical trials alone or in combination with anti-PD-L1 checkpoint inhibitors are already ongoing.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-1319DOI Listing
May 2021

Id1 and PD-1 Combined Blockade Impairs Tumor Growth and Survival of -mutant Lung Cancer by Stimulating PD-L1 Expression and Tumor Infiltrating CD8 T Cells.

Cancers (Basel) 2020 Oct 28;12(11). Epub 2020 Oct 28.

Department of Oncology, Clínica Universidad de Navarra, 31008 Pamplona, Spain.

The use of PD-1/PD-L1 checkpoint inhibitors in advanced NSCLC is associated with longer survival. However, many patients do not benefit from PD-1/PD-L1 blockade, largely because of immunosuppression. New immunotherapy-based combinations are under investigation in an attempt to improve outcomes. (inhibitor of differentiation 1) is involved in immunosuppression. In this study, we explored the potential synergistic effect of the combination of inhibition and pharmacological PD-L1 blockade in three different syngeneic murine -mutant lung adenocarcinoma models. TCGA analysis demonstrated a negative and statistically significant correlation between PD-L1 and expression levels. This observation was confirmed in vitro in human and murine -driven lung cancer cell lines. In vivo experiments in -mutant syngeneic and metastatic murine lung adenocarcinoma models showed that the combined blockade targeting and PD-1 was more effective than each treatment alone in terms of tumor growth impairment and overall survival improvement. Mechanistically, multiplex quantification of CD3/CD4/CD8 T cells and flow cytometry analysis showed that combined therapy favors tumor infiltration by CD8 T cells, whilst in vivo CD8 T cell depletion led to tumor growth restoration. Co-culture assays using CD8 cells and tumor cells showed that T cells present a higher antitumor effect when tumor cells lack expression. These findings highlight that blockade may contribute to a significant immune enhancement of antitumor efficacy of PD-1 inhibitors by increasing PD-L1 expression and harnessing tumor infiltration of CD8 T lymphocytes.
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http://dx.doi.org/10.3390/cancers12113169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7693788PMC
October 2020

Endoscopical and pathological dissociation in severe colitis induced by immune-checkpoint inhibitors.

Oncoimmunology 2020 05 13;9(1):1760676. Epub 2020 May 13.

Department of Oncology, Clinica Universidad de Navarra (CUN), Pamplona, Spain.

Checkpoint inhibitors have improved the survival of patients with advanced tumors and show a manageable toxicity profile. However, auto-immune colitis remains a relevant side effect, and combinations of anti-PD1/PDL1 and anti-CTLA-4 increase its incidence and severity. Here, we report the case of a 50-year-old patient diagnosed with stage IV cervical cancer that relapsed following radical surgery, external radiation/brachytherapy and standard chemotherapy. She was subsequently treated with Nivolumab and Ipilimumab combination and developed grade 2 colitis presenting a dissociation between endoscopic and pathological findings. At cycle 10 the patient reported grade 3 diarrhea and abdominal discomfort, without blood or mucus in the stools. Immunotherapy was withheld and a colonoscopy was performed, showing normal mucosa in the entire colon. Puzzlingly, histologic evaluation of randomly sampled mucosal biopsy of the distal colon showed an intense intraepithelial lymphocyte infiltration with crypt loss and some regenerating crypts with a few apoptotic bodies set in a chronically inflamed lamina propria, consistent with the microscopic diagnosis of colitis. Treatment with methylprednisolone 2 mg/kg was initiated which led to a decrease in the number of stools to grade 1. Additional investigations to exclude other causes of diarrhea rendered negative results. The patient experienced a major partial response and, following the resolution of diarrhea, she was re-challenged again with immunotherapy, with the reappearance of grade 2 diarrhea, leading to permanent immunotherapy interruption. We conclude and propose that performing random colonic biopsies should be considered in cases of immune checkpoint-associated unexplained diarrhea, even when colonoscopy shows macroscopically normal colonic mucosa inflammatory lesions.
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http://dx.doi.org/10.1080/2162402X.2020.1760676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7466860PMC
May 2020

Histopathological findings in fatal COVID-19 severe acute respiratory syndrome: preliminary experience from a series of 10 Spanish patients.

Thorax 2020 12 24;75(12):1116-1118. Epub 2020 Aug 24.

Pathology, University of Navarra, Pamplona, Navarra, Spain.

In December 2019, an outbreak of severe acute respiratory syndrome associated to SARS-CoV2 was reported in Wuhan, China. To date, little is known on histopathological findings in patients infected with the new SARS-CoV2. Lung histopathology shows features of acute and organising diffuse alveolar damage. Subtle cellular inflammatory infiltrate has been found in line with the cytokine storm theory. Medium-size vessel thrombi were frequent, but capillary thrombi were not present. Despite the elevation of biochemical markers of cardiac injury, little histopathological damage could be confirmed. Viral RNA from paraffin sections was detected at least in one organ in 90% patients.
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http://dx.doi.org/10.1136/thoraxjnl-2020-215577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7447113PMC
December 2020

A comprehensive diagnosis of a desmoplastic small round cell tumor of unusual location based on fine-needle aspiration cytology: Report of a case arising in the parotid gland and review of the literature.

Diagn Cytopathol 2020 Sep 13;48(9):827-832. Epub 2020 Jul 13.

Medical Oncology, University Hospital of San Sebastian, San Sebastian, Spain.

Desmoplastic small round cell tumor (DSRCT) is rare and a highly aggressive neoplasm that typically involves the soft tissues of the abdomen or pelvis in children or young adults, showing a male predilection. Although it can occurs over a wide age range, the peak incidence is in the third decade of life. DSRCT usually shows widespread abdominal serosal involvement, and overall patient survival is poor. On the other hand, extra-abdominal DSRCT is very rare. DSRCT in major salivary glands has been reported, but it is extremely rare. In the majority of reported series diagnosis is made by the histological analysis of FFPE tissues together with immunohistochemistry (IHC) and molecular analysis, particularly the demonstration of chromosomal translocation involving EWSR1. Very few cases have been diagnosed so far by Fine Needle Aspiration (FNA) cytology. Moreover ancillary studies have been performed in all reported cases in FFPE samples. There is still controversy and lack of consensus regarding the suitability of cytological samples especially smears for immunocytochemical (ICC) and fluorescence in situ hybridization (FISH), what makes its standardization difficult. We report a case of a primary DSRCT of parotid gland in a 17-year-old male diagnosed by FNA cytology. The cytomorphological diagnosis was coupled with ICC and FISH analysis performed on stained smears. We emphasize the feasibility and reliability of cytological smears for the application of immunocytochemical and molecular techniques.
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http://dx.doi.org/10.1002/dc.24542DOI Listing
September 2020

Diverse immune environments in human lung tuberculosis granulomas assessed by quantitative multiplexed immunofluorescence.

Mod Pathol 2020 12 26;33(12):2507-2519. Epub 2020 Jun 26.

Department of Pathology, Clínica Universidad de Navarra, University of Navarra, Pamplona, Spain.

The precise nature of the local immune responses in lung tuberculosis (TB) granulomas requires a comprehensive understanding of their environmental complexities. At its most basic level, a granuloma is a compact, organized immune aggregate of macrophages surrounded by myeloid, B and T cells. We established two complementary multiplex immunolabeling panels to simultaneously evaluate the myeloid and lymphocytic contexture of 14 human lung TB granulomas in formalin-fixed paraffin-embedded tissue samples. We observed diverse CD3+ and CD8+ T-cell and CD20+ B lymphocyte compositions of the granuloma immune environment and a relatively homogeneous distribution of all myeloid cells. We also found significant associations between CD8+ T-cell densities and the myeloid marker CD11b and phagocytic cell marker CD68. In addition, significantly more CD68+ macrophages and CD8+ T cells were found in Mycobacterium tuberculosis-infected granulomas, as detected by Ziehl-Neelsen staining. FOXP3 expression was predominately found in a small subset of CD4+ T cells in different granulomas. As the success or failure of each granuloma is determined by the immune response within that granuloma at a local and not a systemic level, we attempted to identify the presence of reactive T cells based on expression of the T-cell activation marker CD137 (4-1BB) and programmed cell death-1 (PD-1). Only a small fraction of the CD4+ and CD8+ T cells expressed PD-1. CD137 expression was found only in a very small fraction of the CD4+ T cells in two granulomas. Our results also showed that multinucleated giant cells showed strong PD-L1 but not CTLA-4 membrane staining. This study offers new insights into the heterogeneity of immune cell infiltration in lung TB granulomas, suggesting that each TB granuloma represents a unique immune environment that might be independently influenced by the local adaptive immune response, bacterial state, and overall host disease status.
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http://dx.doi.org/10.1038/s41379-020-0600-6DOI Listing
December 2020

Human CD8 T cells are susceptible to TNF-mediated activation-induced cell death.

Theranostics 2020 15;10(10):4481-4489. Epub 2020 Mar 15.

Program of Immunology and Immunotherapy, Cima Universidad de Navarra, Pamplona, Spain.

Activation-induced cell death (AICD) is a complex immunoregulatory mechanism that causes the demise of a fraction of T-lymphocytes upon antigen-driven activation. In the present study we investigated the direct role of TNF in AICD of CD8 T lymphocytes. : Human peripheral mononuclear cells were isolated from healthy donors and fresh tumor-infiltrating lymphocytes were obtained from cancer patients undergoing surgery. T cells were activated with anti-CD3/CD28 mAbs or with a pool of virus peptides, in combination with clinical-grade TNF blocking agents. : A portion of CD8 T cells undergoes apoptosis upon CD3/CD28 activation in a manner that is partially prevented by the clinically used anti-TNF agents infliximab and etanercept. TNF-mediated AICD was also observed upon activation of virus-specific CD8 T cells and tumor-infiltrating CD8 T lymphocytes. The mechanism of TNF-driven T cell death involves TNFR2 and production of mitochondrial oxygen free radicals which damage DNA. : The use of TNF blocking agents reduces oxidative stress, hyperpolarization of mitochondria, and the generation of DNA damage in CD8 T celss undergoing activation. The fact that TNF mediates AICD in human tumor-reactive CD8 T cells suggests that the use of TNF-blocking agents can be exploited in immunotherapy strategies.
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http://dx.doi.org/10.7150/thno.41646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7150490PMC
April 2021

Prognostic value of macrophage polarization markers in epithelial neoplasms and melanoma. A systematic review and meta-analysis.

Mod Pathol 2020 08 14;33(8):1458-1465. Epub 2020 Apr 14.

Navarra Institute for Health Research (IDISNA), Pamplona, Spain.

Macrophage polarization is relevant for tumor biology. M2 polarized macrophages favor tumor growth and survival, while M1 macrophages support tumor destruction and antigen presentation. Markers identifying M1/M2 polarization are a subject of debate. We conducted a systematic review and meta-analysis to investigate the association of proposed macrophage markers with prognosis across epithelial tumors and melanoma. The Medline search engine was used and 195 articles were recovered for full review. Only articles which measured markers using immunohistochemistry or immunofluorescence and had overall survival (OS) as the primary endpoint were included. One hundred and thirteen articles were finally accepted for analysis. CD68 was associated with worse survival across tumors (hazard ratio (HR) = 1.24, 95% CI = 1.11-1.37). Tumor anatomical location influenced this association. Colorectal tumors showed an inverse association between CD68 and OS in contrast to the rest of cancer types (HR = 0.56 vs. 1.34). The approach taken to measure CD68 had an impact on prognosis; when macrophages were measured at the tumor invasion front prognosis was more favorable than when they were measured intratumorally (HR = 0.94 vs. 1.4). CD163, CD204, and CD206 showed a robust association with worse OS (HR = 1.63, 1.95, 1.65, respectively). Tumors arising in the lung and the liver showed a weaker association between CD163 and OS as compared with other locations (β = -0.5401 for the lung and -0.5940 for the liver compared with other anatomical locations). The counting strategy also had an impact on CD163 association with OS, with hot-spot counting having higher HRs compared with averaging macrophage counts across spots or absolute cell counting (β = -0.4678). In conclusion, proposed M2 markers are associated with worse survival across epithelial tumors and melanoma. The anatomical origin of tumors influences this association. The compartment where the macrophages were scored and counting strategy influenced the association with OS of CD68 and CD163, respectively.
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http://dx.doi.org/10.1038/s41379-020-0534-zDOI Listing
August 2020

CXCR1 and CXCR2 Chemokine Receptor Agonists Produced by Tumors Induce Neutrophil Extracellular Traps that Interfere with Immune Cytotoxicity.

Immunity 2020 05 13;52(5):856-871.e8. Epub 2020 Apr 13.

Program for Immunology and Immunotherapy Department, CIMA, Universidad de Navarra, 31008 Pamplona, Spain; Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), Madrid, Spain; Navarra Institute for Health Research (IDISNA), 31008 Pamplona, Spain. Electronic address:

Neutrophils are expanded and abundant in cancer-bearing hosts. Under the influence of CXCR1 and CXCR2 chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8 T cells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells.
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http://dx.doi.org/10.1016/j.immuni.2020.03.001DOI Listing
May 2020

Topological Tumor Graphs: A Graph-Based Spatial Model to Infer Stromal Recruitment for Immunosuppression in Melanoma Histology.

Cancer Res 2020 03 24;80(5):1199-1209. Epub 2019 Dec 24.

Centre for Evolution and Cancer, The Institute of Cancer Research, London, United Kingdom.

Despite the advent of immunotherapy, metastatic melanoma represents an aggressive tumor type with a poor survival outcome. The successful application of immunotherapy requires in-depth understanding of the biological basis and immunosuppressive mechanisms within the tumor microenvironment. In this study, we conducted spatially explicit analyses of the stromal-immune interface across 400 melanoma hematoxylin and eosin (H&E) specimens from The Cancer Genome Atlas. A computational pathology pipeline (CRImage) was used to classify cells in the H&E specimen into stromal, immune, or cancer cells. The estimated proportions of these cell types were validated by independent measures of tumor purity, pathologists' estimate of lymphocyte density, imputed immune cell subtypes, and pathway analyses. Spatial interactions between these cell types were computed using a graph-based algorithm (topological tumor graphs, TTG). This approach identified two stromal features, namely stromal clustering and stromal barrier, which represented the melanoma stromal microenvironment. Tumors with increased stromal clustering and barrier were associated with reduced intratumoral lymphocyte distribution and poor overall survival independent of existing prognostic factors. To explore the genomic basis of these TTG-derived stromal phenotypes, we used a deep learning approach integrating genomic (copy number) and transcriptomic data, thereby inferring a compressed representation of copy number-driven alterations in gene expression. This integrative analysis revealed that tumors with high stromal clustering and barrier had reduced expression of pathways involved in naïve CD4 signaling, MAPK, and PI3K signaling. Taken together, our findings support the immunosuppressive role of stromal cells and T-cell exclusion within the vicinity of melanoma cells. SIGNIFICANCE: Computational histology-based stromal phenotypes within the tumor microenvironment are significantly associated with prognosis and immune exclusion in melanoma.
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http://dx.doi.org/10.1158/0008-5472.CAN-19-2268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7985597PMC
March 2020

TMPRSS4: A Novel Tumor Prognostic Indicator for the Stratification of Stage IA Tumors and a Liquid Biopsy Biomarker for NSCLC Patients.

J Clin Med 2019 Dec 3;8(12). Epub 2019 Dec 3.

IDISNA and Program in Solid Tumors, Center for Applied Medical Research (CIMA), University of Navarra, 31008 Pamplona, Spain.

Relapse rates in surgically resected non-small-cell lung cancer (NSCLC) patients are between 30% and 45% within five years of diagnosis, which shows the clinical need to identify those patients at high risk of recurrence. The eighth TNM staging system recently refined the classification of NSCLC patients and their associated prognosis, but molecular biomarkers could improve the heterogeneous outcomes found within each stage. Here, using two independent cohorts (MDA and CIMA-CUN) and the eighth TNM classification, we show that TMPRSS4 protein expression is an independent prognostic factor in NSCLC, particularly for patients at stage I: relapse-free survival (RFS) HR, 2.42 (95% CI, 1.47-3.99), < 0.001; overall survival (OS) HR, 1.99 (95% CI, 1.25-3.16), = 0.004). In stage IA, high levels of this protein remained associated with worse prognosis ( = 0.002 for RFS and = 0.001 for OS). As expression is epigenetically regulated, methylation status could be used in circulating tumor DNA from liquid biopsies to monitor patients. We developed a digital droplet PCR (ddPCR) method to quantify absolute copy numbers of methylated and unmethylated CpGs within the and (as control) promoters in plasma and bronchoalveolar lavage (BAL) samples. In case-control studies, we demonstrated that hypomethylation can be used as a diagnostic tool in early stages, with an AUROC of 0.72 ( = 0.008; 91% specificity and 52% sensitivity) for BAL and 0.73 ( = 0.015; 65% specificity and 90% sensitivity) for plasma, in early stages. In conclusion, TMPRSS4 protein expression can be used to stratify patients at high risk of relapse/death in very early stages NSCLC patients. Moreover, analysis of methylation status by ddPCR in blood and BAL is feasible and could serve as a non-invasive biomarker to monitor surgically resected patients.
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http://dx.doi.org/10.3390/jcm8122134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6947244PMC
December 2019

Identification of a novel synthetic lethal vulnerability in non-small cell lung cancer by co-targeting TMPRSS4 and DDR1.

Sci Rep 2019 10 28;9(1):15400. Epub 2019 Oct 28.

IDISNA and Program in Solid Tumors, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona, Spain.

Finding novel targets in non-small cell lung cancer (NSCLC) is highly needed and identification of synthetic lethality between two genes is a new approach to target NSCLC. We previously found that TMPRSS4 promotes NSCLC growth and constitutes a prognostic biomarker. Here, through large-scale analyses across 5 public databases we identified consistent co-expression between TMPRSS4 and DDR1. Similar to TMPRSS4, DDR1 promoter was hypomethylated in NSCLC in 3 independent cohorts and hypomethylation was an independent prognostic factor of disease-free survival. Treatment with 5-azacitidine increased DDR1 levels in cell lines, suggesting an epigenetic regulation. Cells lacking TMPRSS4 were highly sensitive to the cytotoxic effect of the DDR1 inhibitor dasatinib. TMPRSS4/DDR1 double knock-down (KD) cells, but not single KD cells suffered a G0/G1 cell cycle arrest with loss of E2F1 and cyclins A and B, increased p21 levels and a larger number of cells in apoptosis. Moreover, double KD cells were highly sensitized to cisplatin, which caused massive apoptosis (~40%). In vivo studies demonstrated tumor regression in double KD-injected mice. In conclusion, we have identified a novel vulnerability in NSCLC resulting from a synthetic lethal interaction between DDR1 and TMPRSS4.
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http://dx.doi.org/10.1038/s41598-019-51066-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6817908PMC
October 2019

Amylin as a potential link between type 2 diabetes and alzheimer disease.

Ann Neurol 2019 10 19;86(4):539-551. Epub 2019 Aug 19.

Neurology Department, Clínica Universidad de Navarra, Pamplona, Spain.

Objective: Alzheimer disease (AD) is the leading cause of dementia, and although its etiology remains unclear, it seems that type 2 diabetes mellitus (T2DM) and other prediabetic states of insulin resistance could contribute to the appearance of sporadic AD. As such, we have assessed whether tau and β-amyloid (Aβ) deposits might be present in pancreatic tissue of subjects with AD, and whether amylin, an amyloidogenic protein deposited in the pancreas of T2DM patients, might accumulate in the brain of AD patients.

Methods: We studied pancreatic and brain tissue from 48 individuals with no neuropathological alterations and from 87 subjects diagnosed with AD. We examined Aβ and tau accumulation in the pancreas as well as that of amylin in the brain. Moreover, we performed proximity ligation assays to ascertain whether tau and/or Aβ interact with amylin in either the pancreas or brain of these subjects.

Results: Cytoplasmic tau and Aβ protein deposits were detected in pancreatic β cells of subjects with AD as well as in subjects with a normal neuropathological examination but with a history of T2DM and in a small cohort of control subjects without T2DM. Furthermore, we found amylin deposits in the brain of these subjects, providing histological evidence that amylin can interact with Aβ and tau in both the pancreas and hippocampus.

Interpretation: The presence of both tau and Aβ inclusions in pancreatic β cells, and of amylin deposits in the brain, provides new evidence of a potential overlap in the mechanisms underlying the pathogenesis of T2DM and AD. ANN NEUROL 2019;86:539-551.
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http://dx.doi.org/10.1002/ana.25570DOI Listing
October 2019

Programmed death-ligand 1 expression on direct Pap-stained cytology smears from non-small cell lung cancer: Comparison with cell blocks and surgical resection specimens.

Cancer Cytopathol 2019 07 27;127(7):470-480. Epub 2019 Jun 27.

Department of Pathology, University of Navarra Clinic, University of Navarra, Pamplona, Spain.

Background: Programmed death-ligand 1 (PD-L1) expression, as assessed by immunohistochemistry (IHC), is used to select patients with non-small cell lung cancer (NSCLC) for anti-programmed cell death protein 1 (PD-1)/PD-L1 therapy. The current study evaluated the feasibility and efficacy of PD-L1 immunostaining and quantitation on direct Papanicolaou-stained cytological smears compared with formalin-fixed paraffin-embedded samples (cytological cell blocks and surgical resection specimens) in NSCLC cases using 2 commercially available assays: the PD-L1 IHC 22C3 pharmDx assay (Agilent Technologies/Dako, Carpinteria, CA, USA) and the Ventana SP263 Assay (Ventana Medical Systems Inc, Tucson, Arizona).

Methods: PD-L1 immunostaining using either both or one of the assays was tested in 117 sets of paired samples obtained from 62 NSCLC cases. The tumor proportion score was reported in every case following the recommendations of the International Association for the Study of Lung Cancer (IASLC).

Results: In 57 sets of samples, both PD-L1 assays were used. Due to the availability of samples, only 1 assay was performed in 3 sets of samples and in 2 cases, only cytology smears were used and tested for both assays. A total of 113 sets of paired samples finally were evaluated; 4 cases could not be studied due to intense nonspecific background staining. A significant concordance between the 2 assays on cytological smears was found. Concordance between paired cytological smears and formalin-fixed paraffin-embedded samples was observed in 97.3% of the cases.

Conclusions: The quantification of PD-L1 expression on direct Papanicolaou-stained cytology smears is feasible and reliable for both PD-L1 assays.
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http://dx.doi.org/10.1002/cncy.22155DOI Listing
July 2019

Prophylactic TNF blockade uncouples efficacy and toxicity in dual CTLA-4 and PD-1 immunotherapy.

Nature 2019 05 1;569(7756):428-432. Epub 2019 May 1.

Program of Immunology and Immunotherapy, Cima Universidad de Navarra, Pamplona, Spain.

Combined PD-1 and CTLA-4-targeted immunotherapy with nivolumab and ipilimumab is effective against melanoma, renal cell carcinoma and non-small-cell lung cancer. However, this comes at the cost of frequent, serious immune-related adverse events, necessitating a reduction in the recommended dose of ipilimumab that is given to patients. In mice, co-treatment with surrogate anti-PD-1 and anti-CTLA-4 monoclonal antibodies is effective in transplantable cancer models, but also exacerbates autoimmune colitis. Here we show that treating mice with clinically available TNF inhibitors concomitantly with combined CTLA-4 and PD-1 immunotherapy ameliorates colitis and, in addition, improves anti-tumour efficacy. Notably, TNF is upregulated in the intestine of patients suffering from colitis after dual ipilimumab and nivolumab treatment. We created a model in which Rag2Il2rg mice were adoptively transferred with human peripheral blood mononuclear cells, causing graft-versus-host disease that was further exacerbated by ipilimumab and nivolumab treatment. When human colon cancer cells were xenografted into these mice, prophylactic blockade of human TNF improved colitis and hepatitis in xenografted mice, and moreover, immunotherapeutic control of xenografted tumours was retained. Our results provide clinically feasible strategies to dissociate efficacy and toxicity in the use of combined immune checkpoint blockade for cancer immunotherapy.
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http://dx.doi.org/10.1038/s41586-019-1162-yDOI Listing
May 2019

Consistency and reproducibility of next-generation sequencing in cytopathology: A second worldwide ring trial study on improved cytological molecular reference specimens.

Cancer Cytopathol 2019 05 25;127(5):285-296. Epub 2019 Apr 25.

Department of Pathology, University Clinic of Navarra, Pamplona, Spain.

Background: Artificial genomic reference standards in a cytocentrifuge/cytospin format with well-annotated genomic data are useful for validating next-generation sequencing (NGS) on routine cytopreparations. Here, reference standards were optimized to be stained by different laboratories before DNA extraction and to contain a lower number of cells (2 × 10 ). This was done to better reflect the clinical challenge of working with insufficient cytological material.

Methods: A total of 17 worldwide laboratories analyzed customized reference standard slides (slides A-D). Each laboratory applied its standard workflow. The sample slides were engineered to harbor epidermal growth factor receptor (EGFR) c.2235_2249del15 p.E746_A750delELREA, EGFR c.2369C>T p.T790M, Kirsten rat sarcoma viral oncogene homolog (KRAS) c.38G>A p.G13D, and B-Raf proto-oncogene, serine/threonine kinase (BRAF) c.1798_1799GT>AA p.V600K mutations at various allele frequencies (AFs).

Results: EGFR and KRAS mutation detection showed excellent interlaboratory reproducibility, especially on slides A and B (10% and 5% AFs). On slide C (1% AF), either the EGFR mutation or the KRAS mutation was undetected by 10 of the 17 laboratories (58.82%). A reassessment of the raw data in a second-look analysis highlighted the mutations (n = 10) that had been missed in the first-look analysis. BRAF c.1798_1799GT>AA p.V600K showed a lower concordance rate for mutation detection and AF quantification.

Conclusions: The data show that the detection of low-abundance mutations is still clinically challenging and may require a visual inspection of sequencing reads to detect. Genomic reference standards in a cytocentrifuge/cytospin format are a valid tool for regular quality assessment of laboratories performing molecular studies on cytology with low-AF mutations.
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http://dx.doi.org/10.1002/cncy.22134DOI Listing
May 2019

Targeting of TMPRSS4 sensitizes lung cancer cells to chemotherapy by impairing the proliferation machinery.

Cancer Lett 2019 07 21;453:21-33. Epub 2019 Mar 21.

IDISNA and Program in Solid Tumors, Center for Applied Medical Research (CIMA), University of Navarra, 31008, Pamplona, Navarra, Spain; Department of Pathology, Anatomy and Physiology, School of Medicine, University of Navarra, 31008, Pamplona, Navarra, Spain; CIBERONC, ISC-III, Spain. Electronic address:

High mortality rates caused by NSCLC show the need for the identification of novel therapeutic targets. In this study we have investigated the biological effects and molecular mechanisms elicited by TMPRSS4 in NSCLC. Overexpression of TMPRSS4 in LKR13 cells increased malignancy, subcutaneous tumor growth and multiorganic metastasis. In conditional knock-down (KD) experiments, abrogation of TMPRSS4 in H358 and H2170 cells altered proliferation, clonogenicity, tumor engraftment and tumor growth. Reduction in S and G2/M phases of the cell cycle, decreased BrdU incorporation and increased apoptosis was also found. Transcriptomic analysis in KD cells revealed downregulation of genes involved in DNA replication, such as MCM6, TYMS and CDKN1A (p21). In patients, expression of a signature of MCM6/TYMS/TMPRSS4 genes was highly associated with poor prognosis. Downregulation of TMPRSS4 significantly increased sensitivity to chemotherapy agents. In experiments using cisplatin, apoptosis and expression of the DNA-damage marker γ-H2A was higher in cells lacking TMPRSS4. Moreover, in vivo assays demonstrated that tumors with no TMPRSS4 were significantly more sensitive to cisplatin than controls. These results show that TMPRSS4 can be considered as a novel target in NSCLC, whose inhibition increases chemosensitivity.
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http://dx.doi.org/10.1016/j.canlet.2019.03.013DOI Listing
July 2019

Neoadjuvant nivolumab modifies the tumor immune microenvironment in resectable glioblastoma.

Nat Med 2019 03 11;25(3):470-476. Epub 2019 Feb 11.

Centro de Investigación Biomedica en Red de Oncología (CIBERONC), Madrid, Spain.

Glioblastoma is the most common primary central nervous system malignancy and has a poor prognosis. Standard first-line treatment, which includes surgery followed by adjuvant radio-chemotherapy, produces only modest benefits to survival. Here, to explore the feasibility, safety and immunobiological effects of PD-1 blockade in patients undergoing surgery for glioblastoma, we conducted a single-arm phase II clinical trial (NCT02550249) in which we tested a presurgical dose of nivolumab followed by postsurgical nivolumab until disease progression or unacceptable toxicity in 30 patients (27 salvage surgeries for recurrent cases and 3 cases of primary surgery for newly diagnosed patients). Availability of tumor tissue pre- and post-nivolumab dosing and from additional patients who did not receive nivolumab allowed the evaluation of changes in the tumor immune microenvironment using multiple molecular and cellular analyses. Neoadjuvant nivolumab resulted in enhanced expression of chemokine transcripts, higher immune cell infiltration and augmented TCR clonal diversity among tumor-infiltrating T lymphocytes, supporting a local immunomodulatory effect of treatment. Although no obvious clinical benefit was substantiated following salvage surgery, two of the three patients treated with nivolumab before and after primary surgery remain alive 33 and 28 months later.
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http://dx.doi.org/10.1038/s41591-018-0339-5DOI Listing
March 2019

Immunodivergence in Metastatic Colorectal Cancer.

Cancer Cell 2018 12;34(6):876-878

Clinica Universidad de Navarra, Pamplona, Spain; CIBERONC, Madrid, Spain; Center for applied Medical Research (CIMA), Pamplona, Spain; IDISNA, Pamplona, Spain. Electronic address:

Van den Eynde et al. publish in this issue of Cancer Cell that metastatic colorectal cancer shows marked heterogeneity in T cell infiltration among different lesions and patients. Measurements of T cell infiltration in metastases by immunoscore offer some prognostic information and support immune editing by coevolving adaptive immune responses.
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http://dx.doi.org/10.1016/j.ccell.2018.11.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7385539PMC
December 2018
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