Elisa Izquierdo

Diffuse midline gliomas are a group of tumours for which no effective therapies exist. These tumours have substantial intratumoural heterogeneity and harbour beneficial subclonal interactions. Therapeutic failure due to resistance is a major concern in these tumours. We demonstrate how distinct subpopulations derived from the same tumour can interact with one another to alleviate the impacts of therapeutic intervention. This is achieved through the establishment of a mathematical extension to the Lotka-Volterra competition model, where interactions perturbing the Allee effect were demonstrated to exist. These interactions had substantial downstream consequences as they were determined to be perturbed by therapeutic interventions and strikingly, when simulating a two-drug paradigm, the interactions were demonstrated to substantially later the outcomes of treatment scheduling.

Pediatric cancer care in Spain lacked a national personalized medicine program. The SEHOP-PENCIL initiative, established by the SEHOP, was designed to address this gap. A national survey identified the genomic sequencing needs of hospitals treating pediatric patients with cancer. Results showed improved access to targeted next-generation sequencing panels but revealed persistent variability in implementation, limited availability of advanced sequencing (whole-exome sequencing, whole-genome sequencing, RNA sequencing, and DNA methylation profiling), and gaps in cancer predisposition clinics and molecular tumor boards (MTBs). SEHOP-PENCIL is organized as a network of 10 specialized genomic centers serving 45 hospitals through a centralized inclusion system. The program standardizes patient eligibility, genomic workflows, and result interpretation, supported by a national MTB. This framework enables informed clinical decision making, ensures access to molecular diagnosis and innovative therapies, and supports systematic data collection to advance pediatric cancer research and personalized care. SEHOP-PENCIL represents a pioneering national model for integrating precision oncology into pediatric cancer care in Spain. By fostering collaboration, standardizing genomic practices, and promoting equitable access, it aims to reduce disparities and improve outcomes, offering a scalable example for other decentralized health care systems.

Diffuse intrinsic pontine glioma (DIPG) is the pediatric tumor with the worst prognosis. BIOMEDE was a randomized phase 2 trial comparing the efficacy in terms of overall survival (OS) (primary endpoint) of epidermal growth factor receptor (EGFR) inhibitor erlotinib, mTOR inhibitor everolimus and multitargeted tyrosine kinase inhibitor dasatinib in combination with radiotherapy in patients with a biopsy-proven DIPG. Tumors were assessed centrally for immunohistochemical biomarkers (EGFR overexpression or PTEN loss) together with whole-exome and RNA sequencing. A cohort of 66 children with the same inclusion criteria and treated previously with temozolomide-based regimen was used to compare outcome. Treatment allocation was performed by randomization in 233 patients, designed so that a drug could not be allocated if the corresponding biomarker was absent: 36 received erlotinib, 102 received dasatinib and 95 received everolimus. The trial was ended for futility of the primary endpoint following the recommendations of the independent data monitoring committee: OS from biopsy was not different from the control cohort (median OS = 10.8 months (95% confidence interval (CI): 9.5−13.0)) in any of the three arms (median OS = 9.7 months (95% CI: 7.8−14.6) for erlotinib; 9.9 months (95% CI: 8.8−11.2) for dasatinib; and 11.9 months (95% CI: 10.7−14.2) for everolimus). Everolimus showed significantly less ocular, renal, skin and gastrointestinal side effects and treatment discontinuation for toxicity (secondary endpoint). TP53 mutations, frequently linked to multiple structural chromosomal aberrations, were the strongest predictor for poor survival in multivariate analysis (hazard ratio = 2.8 (95% CI: 1.9−4.2), P < 0.0001). Both mutations in and activation of the mTOR pathway were associated with a better response to everolimus. Four long-term survivors treated with an mTOR inhibitor were alive free of treatment over 6 years from diagnosis. With comprehensive tumor profiling, BIOMEDE validated prognostic biomarkers as well as informative theranostic biomarkers for future trials. ClinicalTrials.gov: NCT02233049.

In vitro sensitivity to trametinib in patient-derived DIPG models. A, Oncoprint representation of an integrated annotation of single-nucleotide variants, DNA copy number changes, and structural variants for patient-derived models and tumor biopsy specimens. Samples are arranged in columns with genes labeled along rows. Clinicopathologic and molecular annotations are provided as bars according to the included key. B, The t-statistic–based stochastic neighbor embedding (t-SNE) projection of a combined methylation data set comprising the in vitro models (circled) plus a reference set of glioma subtypes (n = 1,766). The first two projections are plotted on the x- and y-axes, with samples represented by dots colored by subtype as labeled on the figure. C, Drug sensitivities in the mini-screens carried out on cells grown under 2-D and 3-D conditions, visualized by heat map of normalized AUC values. Clinicopathologic and molecular annotations are provided as bars according to the included key. D, Dose–response curves for the MEK inhibitor trametinib tested against patient-derived models in vitro grown in 2-D. E, Dose–response curves for the MEK inhibitor trametinib tested against patient-derived models in vitro grown in 3-D. Cells harboring MAPK pathway alterations are highlighted in blue. Concentration of compound is plotted on a log scale (x-axis) against cell viability (y-axis). Means plus standard errors are plotted from at least n = 3 experiments.

PIK3R1 and NF1 mutations drive the sensitivity of DIPG cells to trametinib. A, Cartoon representing the protein domains of PIK3R1 showing the mutant residue for the observed hotspot N564D mutation observed in ICR-B181. B, Cartoon representing the protein domains of NF1 showing the mutant residue for the observed I1824S missense mutation observed in ICR-B184. Generated in ProteinPaint (pecan.stjude.cloud/proteinpaint). C, Dose–response validation curves for trametinib tested against ICR-B181 cells in vitro grown in 3-D (PIK3R1N564D, blue) and 2-D (PIK3R1 wild-type, gray). D, Dose–response curves for trametinib tested against ICR-B184 cells in vitro grown in 3-D (NF1I1824S, blue) and 2-D (NF1 wild-type, gray). Concentration of compound is plotted on a log scale (x-axis) against cell viability (y-axis). Mean plus standard error are plotted from at least n = 3 experiments. ****, P < 0.0001, AUC t test. E, Bar plot of quantitative capillary phospho-protein assessment of phospho-ERK1/2T202/Y204, plotted as a ratio to total ERK1/2, and normalized to the 2-D (MAPK wild-type) model in each case. F, VAF (y-axis) of PIK3R1N564D in ICR-B181 cells grown in 3-D (blue) and 2-D (gray) over time, as measured by ddPCR. Passage number of cells assessed is given on the x-axis. G, Survival curves for ICR-B181-CDX models, separated by mice implanted with cells grown as either 2-D (gray) or 3-D (blue). H, Anti–human nuclear antigen (HNA), staining for ICR-B181-CDX derived from cells grown in 3-D, with extensive tumor cell infiltration. Sagittal sections, counterstained with hematoxylin. I, Sagittal T2-weighted image (day 246 postimplantation) for ICR-B181-CDX derived from cells grown in 3-D, showing hyperintense tumor throughout the cerebellum and upper pons (indicated by arrow). J, Hematoxylin and eosin–stained section of ICR-B181–3-D CDX, showing histology consistent with high-grade glioma. Scale bar, 200 μm.

MEK1/2 mutations drive resistance to trametinib in BRAFG469V-driven DIPG cells. A, Protein structure representation of BRAF showing the mutant residue (shaded orange) for the observed G469 missense mutation observed in ICR-B169. Generated in COSMIC-3D (cancer.sanger.ac.uk/cosmic3d). B, Timeline of clinical experience for the child with BRAFG469V-mutant DIPG treated with trametinib at progression. Initial therapy with everolimus and radiotherapy is shaded in orange, later treatment with trametinib in blue. Sagittal T1-weighted postgadolinium MRI images are provided at diagnosis, the initial progression, and the later progression immediately prior to death from disease. Periods of steroid treatment are noted by purple lines. The tumor is highlighted with arrows. C, Survival curves for mice bearing ICR-B169 cell-derived orthotopic xenografts, treated with trametinib (blue), compared with vehicle-treated controls (gray). Treatment window is shaded in gray. D, Experimental design for the generation of cells resistant to trametinib by the continuous exposure model. Parental ICR-B169 cells are treated with either an exponentially increasing dose of inhibitor over time (approach 1) or a constant IC80 dose (approach 2). E, Dose–response curves for trametinib tested against ICR-B169 parental cells (gray) and resistant clones T1 (MEK2I115N, pink), T3 (MEK1I141S, purple), and T6 (MEK1K57N, red) after 7 to 9 months of exposure to inhibitor. F, Dose–response curves for trametinib tested against ICR-B169 parental cells (gray) and resistant clones (dashed lines) after 2-month withdrawal of inhibitor. Concentration of compound is plotted on a log scale (x-axis) against cell viability (y-axis). Means plus standard errors are plotted from at least n = 3 experiments. **, P < 0.001; ***, P < 0.001, AUC t test. G, Emergence of MEK1/2 mutations in clones T1 (pink), T3 (purple), and T6 (red) over time, as assessed by ddPCR. The x-axes represent passage number; left y-axes are specific mutation VAFs; right y-axes plot the concentration of trametinib that cells were exposed to (gray dashed line). H, Emergence of resistance in clones over time (T1, pink; T3, purple; T6, red), plotted as days exposed to inhibitor on the x-axis, with passage numbers labeled. The y-axis is a GI50 value for trametinib in cells harvested at the given passage. I, Pathway activation in resistant clones (T1, pink; T3, purple; T6, red) assessed by a capillary electrophoresis assay and plotted as a ratio of respective phosphorylated/total protein compared to ICR-B169 parental cells (gray).

Reciprocity of drug sensitivities and gene expression signatures between trametinib and dasatinib in DIPG cells. A, Dose–response curves for ulixertinib tested against ICR-B169 parental cells (gray) and resistant clones T1 (MEK2I115N, pink), T3 (MEK1I141S, purple), and T6 (MEK1K57N, red). B, Dose-response curves for dasatinib tested against ICR-B169 parental cells (gray) and resistant clones. Concentration of compound is plotted on a log scale (x-axis) against cell viability (y-axis). Means plus standard errors are plotted from at least n = 3 experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001, AUC t test. C, Box plot of dasatinib GI50 values (log scale, y-axis) for primary patient-derived cultures separated by trametinib sensitivity status. D, Box plot of trametinib GI50 values (log scale, y-axis) for primary patient-derived cultures separated by dasatinib sensitivity status. **, P < 0.001, t test. E, GSEA enrichment plots for the signature VERHAAK_GLIOBLASTOMA_PRONEURAL in primary patient-derived cultures separated by trametinib (top) or dasatinib (bottom) sensitivity status. The curves show the enrichment score on the y-axis and the rank list metric on the x-axis. Alongside are heat map representations of expression of significantly differentially expressed genes in the signature in all trametinib- or dasatinib-resistant versus sensitive cell cultures. F, GSEA enrichment plots for the signature HALLMARK_EPITHELIAL_ MESENCHYMAL_TRANSITION in primary patient-derived cultures separated by trametinib (top) or dasatinib (bottom) sensitivity status. The curves show the enrichment score on the y-axis and the rank list metric on the x-axis. Alongside are heat map representations of expression of significantly differentially expressed genes in the signature in all trametinib- or dasatinib-resistant versus sensitive cell cultures. G, GSEA enrichment plots for the signature HUANG_DASATINIB_RESISTANCE_UP in primary patient-derived cultures separated by trametinib (top) or dasatinib (bottom) sensitivity status. The curves show the enrichment score on the y-axis and the rank list metric on the x-axis. Alongside are heat map representations of expression of significantly differentially expressed genes in the signature in all trametinib- or dasatinib-resistant versus sensitive cell cultures.

Efficacy of combined dasatinib and trametinib on ex vivo brain slice preparations. A, Coronal slices of normal mouse brain, counterstained with Hoechst 33342 (aqua), are implanted in the pontine region with ICR-B169 parental cells, stained with human nuclear antigen (orange), and treated for 4 days with 1 μmol/L dasatinib, 0.1234 μmol/L trametinib, or both compared with vehicle control. Scale bars, 2 mm. B, Bar plot of quantification of tumor cell infiltration across the brain parenchymal tissue as measured by the calculated area invaded compared with vehicle control. Plotted is the mean of at least six independent slices; error bars represent the SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant, FDR-corrected t test.

Synergy of combined dasatinib and trametinib in BRAFG469V-driven DIPG cells. A, Cell viability matrices for ICR-B169 parental (gray) and trametinib-resistant clones T1 (MEK2I115N, pink), (T3 MEK1I141S, purple), and T6 (MEK1K57N, red), treated with distinct combinations of dasatinib (y-axes) and trametinib (x-axes) ranging from 0 to 10 μmol/L. A heat map is overlaid to the proportions of viable cells remaining, colored according to the key provided from 1.0 (black, all cells) to 0 (red, no viable cells). B, Excess above BLISS matrices for ICR-B169 parental and trametinib-resistant clones treated with distinct combinations of dasatinib (y-axes) and trametinib (x-axes) ranging from 0 to 10 μmol/L. A heat map is overlaid to the excess score, colored according to the key provided from 0.4 (red, enhanced effects) to 0 (green, no difference). C, BLISS synergy maps for ICR-B169 parental and trametinib-resistant clones treated with distinct combinations of dasatinib (y-axes) and trametinib (x-axes) ranging from 0 to 10 μmol/L. The heat map represents the δ score colored according to the key provided from 30 (red, high degree of synergy) to −30 (green, antagonism).

Integrated gene and protein expression profiling of trametinib-resistant DIPG cells. A, Coordinately downregulated genes (left), proteins (middle), and phospho-sites (right) in all three trametinib-resistant subclones of ICR-B169 BRAFG469V cells as compared with parental. B, Coordinately upregulated genes (left), proteins (middle), and phospho-sites (right) in all three trametinib-resistant subclones, as compared with ICR-B169 parental (gray). T1, MEK2I115N, pink; T3, MEK1I141S, purple; T6, MEK1K57N, red. C, GSEA enrichment plots for the signature VERHAAK_GLIOBLASTOMA_PRONEURAL in T6 MEK1K57N cells compared with ICR-B169 parental. The curves show the enrichment score on the y-axis and the rank list metric on the x-axis. Alongside is a heat map representation of expression of significantly differentially expressed genes in the signature in all three trametinib-resistant clones compared with parental. D, GSEA enrichment plots for the signature HALLMARK_EPITHELIAL_MESENCHYMAL_ TRANSITION in T6 MEK1K57N cells compared with ICR-B169 parental. The curves show the enrichment score on the y-axis and the rank list metric on the x-axis. Alongside is a heat map representation of expression of significantly differentially expressed genes in the signature in all three trametinib-resistant clones compared with parental. E, GSEA enrichment plots for the signature HUANG_DASATINIB_RESISTANCE_UP in T6 MEK1K57N cells compared with ICR-B169 parental. The curves show the enrichment score on the y-axis and the rank list metric on the x-axis. Alongside is a heat map representation of expression of significantly differentially expressed genes in the signature in all three trametinib-resistant clones compared with parental. T1, MEK2I115N, pink; T3, MEK1I141S, purple; T6, MEK1K57N, red.