Resultados de investigación

Instituto de Salud Carlos III Consejo Superior de Investigaciones Científicas Consejería de Educación, Junta de Castilla y León CRIS Cancer Foundation ACMUMA UCCTA ALMOM, Peer reviewed

Instituto de Salud Carlos III Consejo Superior de Investigaciones Científicas Consejería de Educación, Junta de Castilla y León CRIS Cancer Foundation ACMUMA UCCTA ALMOM, Peer reviewed

Supplementary Table 1: Overview of the excluded patients (N=147. Supplementary Table 2: Study population demographics. Supplementary Table 3: Manual Gating strategy for the identification of lymphoid populations in blood according to the EuroFlow guidelines for analysis of blood samples stained with PIDOT. Supplementary Table 4: The clinical characteristics of the non-PID disease controls (DC) (n=116). Supplementary Table 5: Verification of the EuroFlow reference values using an independent healthy control group (N=68). Supplementary Figures Supplementary Figure 1: Box plots of serum immunoglobulin levels at time of PIDOT analysis. Supplementary Figure S2: Box plots of total memory and switched memory B-cells (% as expressed on the B-cells) measured by the PIDOT. Supplementary Figure 3: Box plots of frequency of total defective lymphoid populations (over the 22 FCM PIDOT variables). Supplementary Figure 4: Box plots of frequency of total increased cell counts (over the 22 FCM PIDOT variables). Supplementary Figure 5: Receiver Operating Characteristic (ROC) curve to assess the performance of the decision-tree algorithm in relation to the predicted probabilities for lymphoid-PID., [Introduction]: Multiparameter flow cytometry (FCM) immunophenotyping is an important tool in the diagnostic screening and classification of primary immunodeficiencies (PIDs). The EuroFlow Consortium recently developed the PID Orientation Tube (PIDOT) as a universal screening tool to identify lymphoid-PID in suspicious patients. Although PIDOT can identify different lymphoid-PIDs with high sensitivity, clinical validation in a broad spectrum of patients with suspicion of PID is missing. In this study, we investigated the diagnostic performance of PIDOT, as part of the EuroFlow diagnostic screening algorithm for lymphoid-PID, in a daily practice at a tertiary reference center for PID., [Methods]: PIDOT was tested in 887 consecutive patients suspicious of PID at the Ghent University Hospital, Belgium. Patients were classified into distinct subgroups of lymphoid-PID vs. non-PID disease controls (non-PID DCs), according to the IUIS and ESID criteria. For the clinical validation of PIDOT, comprehensive characterization of the lymphoid defects was performed, together with the identification of the most discriminative cell subsets to distinguish lymphoid-PID from non-PID DCs. Next, a decision-tree algorithm was designed to guide subsequent FCM analyses., [Results]: The mean number of lymphoid defects detected by PIDOT in blood was 2.87 times higher in lymphoid-PID patients vs. non-PID DCs (p < 0.001), resulting in an overall sensitivity and specificity of 87% and 62% to detect severe combined immunodeficiency (SCID), combined immunodeficiency with associated or syndromic features (CID), immune dysregulation disorder (ID), and common variable immunodeficiency (CVID). The most discriminative populations were total memory and switched memory B cells, total T cells, TCD4+cells, and naive TCD4+cells, together with serum immunoglobulin levels. Based on these findings, a decision-tree algorithm was designed to guide further FCM analyses, which resulted in an overall sensitivity and specificity for all lymphoid-PIDs of 86% and 82%, respectively., [Conclusion]: Altogether, our findings confirm that PIDOT is a powerful tool for the diagnostic screening of lymphoid-PID, particularly to discriminate (S)CID, ID, and CVID patients from other patients suspicious of PID. The combination of PIDOT and serum immunoglobulin levels provides an efficient guide for further immunophenotypic FCM analyses, complementary to functional and genetic assays, for accurate PID diagnostics., Peer reviewed

Table S1. Differentially expressed genes in Vav2(Onc) and Vav2(–/–);Vav3(–/–) skin stem cells. Table S2A. Differentially expressed genes in WT TSCs vs SSCs. Table S3. Differentially expressed genes in Vav2(Onc) and WT tumor stem cells. FIGURE S1. Vav function regulates SSC numbers. FIGURE S2. The Vav-dependent skin phenotype is mostly keratinocyte-autonomous. FIGURE S3. Vav proteins regulate transcriptional programs involved in stem cell homeostasis. FIGURE S4. Vav2Onc SSC transcriptome cross-comparison. FIGURE S5. Impact of Vav2Onc catalysis-dependent signaling in tumor stem cells., Peer reviewed

Supplementary Figure 1 | Identification of K-RAS most common genomic alterations in patients with NSCLC by molecular subtypes. (i) Percentage of amplifications, mutations and deletions of K-RAS gene in patients with Squamous Cell Carcinoma or Adenocarcinoma. (ii) Graphical representation of the percentage of K-RAS genomic alterations according to TCGA, Firehose Legacy and MSKCC, 2020 databases in (A) or to TCGA, PanCancer Atlas and TCGA, Firehose Legacy databases in (B). Supplementary Figure 2 | Negative correlation between the expression of upregulated genes (CLRF1, HOPX, IRS2, KIT, PDE4D, and SMOC1) and most of immune infiltrates (CD8+ T cells, Neutrophils, Macrophages and dendritic cells). A green square encircles the dots with higher expression and little infiltration level. Supplementary Figure 3 | Positive correlation between the expression of downregulated genes (CD24, CDK6, HDAC9, TIAM1, TRFC, VTRC1, and VAV3) and most of immune infiltrates (CD8+ T cells, Neutrophils, Macrophages and dendritic cells). A yellow square encircles the dots with less expression and more infiltration level. Supplementary Figure 4 | Expression of CLDN10 and TMPRSS6 in different human cancer types. Bar graph showing the expression of individual CLDN10 (in (A), TMPRSS6 in (B) or both genes combined (C) in those cancer types where expression is significantly higher in tumor samples than in normal tissue (D). No correlation between CLDN10 and TMPRSS6 gene expression with most of immune infiltrates. Supplementary Figure 5 | Higher expression of CLDN10 and TMPRSS6 in K-RAS p.G12C. (A) Table presenting 15 NSLC cell lines that present the G12C variant in K-RAS gene. Bar graph showing the expression of CLDN10 in (B), and TMPRSS6 in (C) in 92 NSLC cell lines compared to those 15 selected in A. Expression of CLDN10 in (D), and TMPRSS6 in (E) in LUAD tumor samples comparing those that harbor or not the G12C mutation. Supplementary Table 1 | Investigational and approved drugs against K-RAS identified mutations. Specific K-RAS mutation, name of the drug, status (approved or investigational), identification code (NCT) and clinical studies with links and phases are included. Intervention is included if the drug is given in combination with others. Supplementary Table 2 | Gene functions of thirteen selected deregulated genes. Supplementary Table 3 | Upregulation details of cell surface-related genes analyzed. Name of the gene, mean of expression in mutant and wildtype K-RAS tumors, fold change (FC), direction and p-value are included. Supplementary Table 4 | Kaplan-Meier survival values of cell surface-related genes. Table includes the name of the gene, the hazard ratio (HR) (in blue, significant good prognosis, and in red, bad one), p-value and fold discovery rate (FDR) for FP and in LUAD patients., Targeting K-RAS-mutant non-small cell lung cancer (NSCLC) with novel inhibitors has shown promising results with the recent approval of sotorasib in this indication. However, progression to this agent is expected, as it has previously been observed with other inhibitors. Recently, new immune therapeutics, including vectorized compounds with antibodies or modulators of the host immune response, have demonstrated clinical activity. By interrogating massive datasets, including TCGA, we identified genes that code for surface membrane proteins that are selectively expressed in K-RAS mutated NSCLC and that could be used to vectorize novel therapies. Two genes, CLDN10 and TMPRSS6, were selected for their clear differentiation. In addition, we discovered immunologic correlates of outcome that were clearly de-regulated in this particular tumor type and we matched them with immune cell populations. In conclusion, our article describes membrane proteins and immunologic correlates that could be used to better select and optimize current therapies., Peer reviewed

Supplementary Table 1 | Investigational and approved drugs against K-RAS identified mutations. Specific K-RAS mutation, name of the drug, status (approved or investigational), identification code (NCT) and clinical studies with links and phases are included. Intervention is included if the drug is given in combination with others. Supplementary Table 2 | Gene functions of thirteen selected deregulated genes. Supplementary Table 3 | Upregulation details of cell surface-related genes analyzed. Name of the gene, mean of expression in mutant and wildtype K-RAS tumors, fold change (FC), direction and p-value are included. Supplementary Table 4 | Kaplan-Meier survival values of cell surface-related genes. Table includes the name of the gene, the hazard ratio (HR) (in blue, significant good prognosis, and in red, bad one), p-value and fold discovery rate (FDR) for FP and in LUAD patients., Targeting K-RAS-mutant non-small cell lung cancer (NSCLC) with novel inhibitors has shown promising results with the recent approval of sotorasib in this indication. However, progression to this agent is expected, as it has previously been observed with other inhibitors. Recently, new immune therapeutics, including vectorized compounds with antibodies or modulators of the host immune response, have demonstrated clinical activity. By interrogating massive datasets, including TCGA, we identified genes that code for surface membrane proteins that are selectively expressed in K-RAS mutated NSCLC and that could be used to vectorize novel therapies. Two genes, CLDN10 and TMPRSS6, were selected for their clear differentiation. In addition, we discovered immunologic correlates of outcome that were clearly de-regulated in this particular tumor type and we matched them with immune cell populations. In conclusion, our article describes membrane proteins and immunologic correlates that could be used to better select and optimize current therapies., Peer reviewed

Supplementary Tables Supplementary Table 1: Clinical data of the study cohort. Supplementary Table 2: Functional enrichment by trend of soluble proteins for Diagnostic group study. Supplementary Table 3: Summary of significant soluble proteins for each study after classical statistical analysis. Supplementary Table 4: Functional enrichment of significant soluble proteins for comparison Monoclonal B-cell lymphocytosis (MBLhi) vs. Chronic Lymphocytic Leukemia (CLL) and CLL in progression (p-CLL) vs. Stable/constant CLL (c-CLL) vs. MBLhi and combinations. Supplementary Table 5: Functional enrichment of significant soluble proteins according to trend for Diagnostic group study. Supplementary Table 6: Protein correlation. Supplementary Table 7: Top 20/30 proteins after random forest analyses. Supplementary Table 8: Summary of significant soluble proteins for each study after linear model. Supplementary Table 9: Functional enrichment by trend of soluble proteins for Diagnostic group and treatment line. Supplementary Table 10: Functional enrichment of significant soluble proteins for all combination combinations between Monoclonal B-cell lymphocytosis (MBLhi), Stable/constant Chronic Lymphocytic Leukemia (c-CLL), CLL in progression previously to 1st line treatment (CLL-PFT) and CLL in progression to time from 1st line treatment (CLL-TFT). Supplementary Table 11: Functional enrichment by trend of soluble proteins for Immunoglobulin Heavy chain Variable (IGHV) gene status study. Supplementary Table 12: Functional enrichment of significant soluble proteins for Immunoglobulin Heavy chain Variable (IGHV) gene status study., Chronic lymphocytic leukemia (CLL) is a lymphoid neoplasm characterized by the accumulation of mature B cells. The diagnosis is established by the detection of monoclonal B lymphocytes in peripheral blood, even in early stages [monoclonal B-cell lymphocytosis (MBLhi)], and its clinical course is highly heterogeneous. In fact, there are well-characterized multiple prognostic factors that are also related to the observed genetic heterogenicity, such as immunoglobulin heavy chain variable region (IGHV) mutational status, del17p, and TP53 mutations, among others. Moreover, a dysregulation of the immune system (innate and adaptive immunity) has been observed in CLL patients, with strong impact on immune surveillance and consequently on the onset, evolution, and therapy response. In addition, the tumor microenvironment is highly complex and heterogeneous (i.e., matrix, fibroblast, endothelial cells, and immune cells), playing a critical role in the evolution of CLL. In this study, a quantitative profile of 103 proteins (cytokines, chemokines, growth/regulatory factors, immune checkpoints, and soluble receptors) in 67 serum samples (57 CLL and 10 MBLhi) has been systematically evaluated. Also, differential profiles of soluble immune factors that discriminate between MBLhi and CLL (sCD47, sCD27, sTIMD-4, sIL-2R, and sULBP-1), disease progression (sCD48, sCD27, sArginase-1, sLAG-3, IL-4, and sIL-2R), or among profiles correlated with other prognostic factors, such as IGHV mutational status (CXCL11/I-TAC, CXCL10/IP-10, sHEVM, and sLAG-3), were deciphered. These results pave the way to explore the role of soluble immune checkpoints as a promising source of biomarkers in CLL, to provide novel insights into the immune suppression process and/or dysfunction, mostly on T cells, in combination with cellular balance disruption and microenvironment polarization leading to tumor escape., Peer reviewed

Chronic lymphocytic leukemia (CLL) is a lymphoid neoplasm characterized by the accumulation of mature B cells. The diagnosis is established by the detection of monoclonal B lymphocytes in peripheral blood, even in early stages [monoclonal B-cell lymphocytosis (MBLhi)], and its clinical course is highly heterogeneous. In fact, there are well-characterized multiple prognostic factors that are also related to the observed genetic heterogenicity, such as immunoglobulin heavy chain variable region (IGHV) mutational status, del17p, and TP53 mutations, among others. Moreover, a dysregulation of the immune system (innate and adaptive immunity) has been observed in CLL patients, with strong impact on immune surveillance and consequently on the onset, evolution, and therapy response. In addition, the tumor microenvironment is highly complex and heterogeneous (i.e., matrix, fibroblast, endothelial cells, and immune cells), playing a critical role in the evolution of CLL. In this study, a quantitative profile of 103 proteins (cytokines, chemokines, growth/regulatory factors, immune checkpoints, and soluble receptors) in 67 serum samples (57 CLL and 10 MBLhi) has been systematically evaluated. Also, differential profiles of soluble immune factors that discriminate between MBLhi and CLL (sCD47, sCD27, sTIMD-4, sIL-2R, and sULBP-1), disease progression (sCD48, sCD27, sArginase-1, sLAG-3, IL-4, and sIL-2R), or among profiles correlated with other prognostic factors, such as IGHV mutational status (CXCL11/I-TAC, CXCL10/IP-10, sHEVM, and sLAG-3), were deciphered. These results pave the way to explore the role of soluble immune checkpoints as a promising source of biomarkers in CLL, to provide novel insights into the immune suppression process and/or dysfunction, mostly on T cells, in combination with cellular balance disruption and microenvironment polarization leading to tumor escape., Peer reviewed

Chronic lymphocytic leukemia (CLL) is a lymphoid neoplasm characterized by the accumulation of mature B cells. The diagnosis is established by the detection of monoclonal B lymphocytes in peripheral blood, even in early stages [monoclonal B-cell lymphocytosis (MBLhi)], and its clinical course is highly heterogeneous. In fact, there are well-characterized multiple prognostic factors that are also related to the observed genetic heterogenicity, such as immunoglobulin heavy chain variable region (IGHV) mutational status, del17p, and TP53 mutations, among others. Moreover, a dysregulation of the immune system (innate and adaptive immunity) has been observed in CLL patients, with strong impact on immune surveillance and consequently on the onset, evolution, and therapy response. In addition, the tumor microenvironment is highly complex and heterogeneous (i.e., matrix, fibroblast, endothelial cells, and immune cells), playing a critical role in the evolution of CLL. In this study, a quantitative profile of 103 proteins (cytokines, chemokines, growth/regulatory factors, immune checkpoints, and soluble receptors) in 67 serum samples (57 CLL and 10 MBLhi) has been systematically evaluated. Also, differential profiles of soluble immune factors that discriminate between MBLhi and CLL (sCD47, sCD27, sTIMD-4, sIL-2R, and sULBP-1), disease progression (sCD48, sCD27, sArginase-1, sLAG-3, IL-4, and sIL-2R), or among profiles correlated with other prognostic factors, such as IGHV mutational status (CXCL11/I-TAC, CXCL10/IP-10, sHEVM, and sLAG-3), were deciphered. These results pave the way to explore the role of soluble immune checkpoints as a promising source of biomarkers in CLL, to provide novel insights into the immune suppression process and/or dysfunction, mostly on T cells, in combination with cellular balance disruption and microenvironment polarization leading to tumor escape., Peer reviewed

Chronic lymphocytic leukemia (CLL) is a lymphoid neoplasm characterized by the accumulation of mature B cells. The diagnosis is established by the detection of monoclonal B lymphocytes in peripheral blood, even in early stages [monoclonal B-cell lymphocytosis (MBLhi)], and its clinical course is highly heterogeneous. In fact, there are well-characterized multiple prognostic factors that are also related to the observed genetic heterogenicity, such as immunoglobulin heavy chain variable region (IGHV) mutational status, del17p, and TP53 mutations, among others. Moreover, a dysregulation of the immune system (innate and adaptive immunity) has been observed in CLL patients, with strong impact on immune surveillance and consequently on the onset, evolution, and therapy response. In addition, the tumor microenvironment is highly complex and heterogeneous (i.e., matrix, fibroblast, endothelial cells, and immune cells), playing a critical role in the evolution of CLL. In this study, a quantitative profile of 103 proteins (cytokines, chemokines, growth/regulatory factors, immune checkpoints, and soluble receptors) in 67 serum samples (57 CLL and 10 MBLhi) has been systematically evaluated. Also, differential profiles of soluble immune factors that discriminate between MBLhi and CLL (sCD47, sCD27, sTIMD-4, sIL-2R, and sULBP-1), disease progression (sCD48, sCD27, sArginase-1, sLAG-3, IL-4, and sIL-2R), or among profiles correlated with other prognostic factors, such as IGHV mutational status (CXCL11/I-TAC, CXCL10/IP-10, sHEVM, and sLAG-3), were deciphered. These results pave the way to explore the role of soluble immune checkpoints as a promising source of biomarkers in CLL, to provide novel insights into the immune suppression process and/or dysfunction, mostly on T cells, in combination with cellular balance disruption and microenvironment polarization leading to tumor escape., Peer reviewed