Fig 2: Clinical validation of ETV6 in uterine serous cancer. a Kaplan–Meier plot for uterine serous patients stratified by ETV6-staining score. Patient samples (N = 31) were divided into two groups based on intensity of ETV6 and positivity in nuclei or cytoplasm (patients with cytoplasmic, negative or weak/medium [score = 1 or 2] nuclear ETV6 staining, N = 20; and patients with strong [score = 3] nuclear ETV6 staining, N = 11). A significant difference in survival was observed between the groups (P = 0.004, log-rank test). The median survival was 2330 days (95% CI: 104–4556 days) for the cytoplasmic or weak nuclear group and 214 days (95% CI: 53–891) for the strong nuclear group. Source data are provided as a Source Data file. b Representative image of immunofluorescence staining on a primary uterine serous tumor. Double staining for ETV6 (green) and cytokeratin (red), shows nuclear localization of ETV6 in tumor cells; DAPI (4',6-diamidino-2-phenylindole): blue. Source data are provided as a Source Data file

Fig 3: ETV6 expression in malignant B-cells compared to normal B-cell subsets. (A) Heat map representation of ETV6 and PIM2 transcript levels in DLBCL samples and normal tonsil B-cell subsets [29]. (B) Comparison of ETV6 transcript levels between DLBCL subgroups (ABC, GCB and UNCL) and normal B-cell subsets obtained from tonsils. For statistical analysis, an unpaired t-test was used. (C) Comparison of PIM2 transcript levels between DLBCL subgroups (ABC, GCB and UNCL) and normal B-cell subsets obtained from tonsils. CC = centrocytes; CB = centroblasts; PB: plasmablasts. For statistical analysis, a nonparametric t-test was used. (D) ETV6 immunohistochemical staining of reactive tonsil (top): original magnification (×10) and 40× (insets). PIM2 immunohistochemical staining of reactive tonsil (top): original magnification (×10) and 40× (insets). (E) Immunohistochemical staining for ETV6 in representative cases of DLBCL patients showing low (+1), medium (+2) and high (+3) expression levels. Original magnification ×20; inset ×40. (F) OncoPrint visualization in cBioPortal of genetic alterations (top) affecting ETV6, MYC, BCL2 and BCL6 genes for selected B-lymphoma cell lines (source: Cancer Cell Line Encyclopedia (Broad, 2019) and Lymphoma cell lines (MSKCC,2020)). Heat map showing gene expression levels of selected genes is also depicted (bottom). (G) Heat map representation of relative ETV6 and PIM2 transcript levels (normalized to RPL19 housekeeping gene) in available B-cell lymphoma cell lines as determined by qRT-PCR. (H) Evaluation of ETV6 and PIM2 and protein expression levels in DLBCL cell lines of different molecular subtypes (ABC and GCB-type) and Burkitt lymphoma (BL) cell lines using immunoblotting. Tubulin is shown as loading control. Relative protein expressions (normalized to loading control) are shown on top of appropriate panels. kD = kilodaltons.

Fig 4: Kaplan–Meier curves of OS in DLBCL patients treated with chemotherapy and stratified according to various putative prognostic markers. (A) Kaplan–Meier curves of OS in two groups identified using the PIM2-ETV6 prognostic score (Group 0: score = median; Group 1: score > median). (B) Kaplan–Meier curves of OS in the three groups identified using the tertiles of PIM2-ETV6 prognostic score (<25%, 25–75%, >75% of score). (C) Kaplan–Meier curves of OS in the same patients subdivided into GCB and non-GCB subgroups using the Hans immune-histochemical algorithm, which uses three markers (CD10, BCL6 and MUM1) to separate GCB DLBCL from non-GCB DLBCL. (D) Kaplan–Meier curves of OS in the same cohort of patients subdivided into COO subgroups (ABC, GCB, UNCL) using the Lymph2Cx assay. (E) Kaplan–Meier curves of OS in the same patients subdivided into 4 groups using the IPI clinical score. (F) IHC validation of ETV6 and PIM2 expression in an independent DLBCL patient cohort (n = 39). Kaplan–Meier curves of OS in this cohort, dividing the patients into two groups in relations to the ETV6/PIM2 ratio (high: ratio > 1; low < 1). (G) Bar graph showing the distribution of patients’ outcome (alive versus dead) in relation to ETV6 or PIM2 expression. High: staining intensity > 1; low: staining intensity = 1. The Chi square test was used to determine significance. (H) Kaplan–Meier curves of OS in the four groups identified using the PIM2 (low versus high) and ETV6 (low versus high) IHC expression levels (cohort n = 39).

Fig 5: Identification of proteins associated with survival through univariate and multivariate Cox regression analysis. (A) The list of proteins identified as associated with survival through univariate Cox regression analysis (n = 41) was used for Gene Set Enrichment Analysis (GSEA) using WEB-based GEne SeT AnaLysis Toolkit (WebGestalt) with Wikipedia cancer pathway as enrichment categories. The enriched categories are shown. (B) Network topology-based analysis for the same list of proteins was run using TCGA RNA Seq data for DLBCL samples as the functional database. The identified network was found to be enriched for numerous Gene Ontology (GO) categories, including immune system development (proteins included in this subnetwork are shown). (C) Kaplan–Meier curves of OS in patients (n = 47) subdivided into two groups on the basis of ETV6 protein expression levels (left panel) or PIM2 protein expression levels (right panel). High: >median value; low: =median value. (D) Unsupervised hierarchical clustering of DLBCL samples using ETV6 and PIM2 proteins. (E) Kaplan–Meier curves of OS in the two clusters (Cluster 1 and 0) identified using ETV6 and PIM2 protein levels. (F) Biological interaction network of ETV6 showing physical interactions obtained using GeneMANIA. (G) Biological interaction network of PIM2 showing physical interactions and associated functional pathways obtained using GeneMANIA. (H) Biological interaction network of ETV6 and PIM2 showing physical interactions and associated functional pathways obtained using GeneMANIA. (I) Over representation analysis (ORA) of ETV6 interacting proteins using Reactome pathways as functional database.