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Functional diversity and cooperativity between subclonal populations of pediatric glioblastoma and diffuse intrinsic pontine glioma cells

DOI:10.1038/s41591-018-0086-7
Authors:
Maria Vinci at Ospedale Pediatrico Bambino Gesù
Maria Vinci
Anna Burford at Institute of Cancer Research
Anna Burford
Valeria Molinari at Institute of Cancer Research
Valeria Molinari
Ketty Kessler at Institute of Cancer Research
Ketty Kessler
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Abstract and Figures

The failure to develop effective therapies for pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG) is in part due to their intrinsic heterogeneity. We aimed to quantitatively assess the extent to which this was present in these tumors through subclonal genomic analyses and to determine whether distinct tumor subpopulations may interact to promote tumorigenesis by generating subclonal patient-derived models in vitro and in vivo. Analysis of 142 sequenced tumors revealed multiple tumor subclones, spatially and temporally coexisting in a stable manner as observed by multiple sampling strategies. We isolated genotypically and phenotypically distinct subpopulations that we propose cooperate to enhance tumorigenicity and resistance to therapy. Inactivating mutations in the H4K20 histone methyltransferase KMT5B (SUV420H1), present in <1% of cells, abrogate DNA repair and confer increased invasion and migration on neighboring cells, in vitro and in vivo, through chemokine signaling and modulation of integrins. These data indicate that even rare tumor subpopulations may exert profound effects on tumorigenesis as a whole and may represent a new avenue for therapeutic development. Unraveling the mechanisms of subclonal diversity and communication in pGBM and DIPG will be an important step toward overcoming barriers to effective treatments.
| DIPGs infiltrate the brain through branching evolution and genotypic convergence. a, Thirteen different tumor-harboring regions of HSJDDIPG-010 were sampled post mortem, from within and outside the pons. Scale bars, 100 µ m. b, Exome sequencing was carried out for all regions. CCFs plotted as a heat map for all variants found in at least one specimen, with anatomical location highlighted and color-coded. c, Phylogenetic trees were reconstructed using neighbor-joining algorithms based on the nested subpopulation phylogenies calculated as part of EXPANDS, with evident laterally directed evolution and early escape from the pons of tumor cells found in distinct anatomical sites. GL, germline. d-f, Eight different tumor-harboring regions of HSJD-DIPG-014 subjected to the same analysis. Scale bars, 100 µ m. g-i, Eight different tumor-harboring regions of HSJD-DIPG-015 subjected to the same analysis. Scale bars, 100 µ m.
| DIPGs infiltrate the brain through branching evolution and genotypic convergence. a, Thirteen different tumor-harboring regions of HSJDDIPG-010 were sampled post mortem, from within and outside the pons. Scale bars, 100 µ m. b, Exome sequencing was carried out for all regions. CCFs plotted as a heat map for all variants found in at least one specimen, with anatomical location highlighted and color-coded. c, Phylogenetic trees were reconstructed using neighbor-joining algorithms based on the nested subpopulation phylogenies calculated as part of EXPANDS, with evident laterally directed evolution and early escape from the pons of tumor cells found in distinct anatomical sites. GL, germline. d-f, Eight different tumor-harboring regions of HSJD-DIPG-014 subjected to the same analysis. Scale bars, 100 µ m. g-i, Eight different tumor-harboring regions of HSJD-DIPG-015 subjected to the same analysis. Scale bars, 100 µ m.
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| Distinct infiltrative phenotypes of genotypically divergent DIPG subclones in vivo. a, Heterogeneous HSJD-DIPG-007 bulk cells and NS-F10 and NS-F8 subclones were implanted directly into the pons of NOD-SCID mice and tumors allowed to form over 8 months. At weeks 23-24, bulk cells and NS-F10 formed diffusely infiltrating tumors throughout the brain, as seen by H&E staining as well as immunohistochemistry with anti-human nuclear antigen (HNA) or astrocyte marker GFAP, whereas NS-F8 had formed considerably less infiltrative lesions even at 30 weeks. Representative images from a total of n = 4 mice per group. Main scale bars, 1,000 µ m; inset scale bars, 50 µ m. b, Tumor-bearing animals implanted with NS-F8 subclones had significantly longer survival than heterogeneous HSJD-DIPG-007 bulk cells and NS-F10 (P = 0.0236, log-rank test, n = 4 mice per group). *P < 0.05. c, Digital droplet PCR. Plot of assay for KMT5B wild-type (x axes) and R187* mutation (y axes) for normal human astrocytes and tumors from mice implanted with heterogeneous bulk cells, and subclones NS-F10 and NS-F8. Mutant reads are present in 51.33% droplets from NS-F10 and 0.23% droplets from the original bulk preparation. Taken from n = 3 independent experiments. d, Heterogeneous SU-DIPG-VI bulk cells and A-D10 and A-E6 subclones were implanted directly into the pons of nude mice and tumors allowed to form over 8 months. At week 10, bulk cells and A-D10 formed highly cellular, infiltrating tumors, as seen by H&E staining or immunohistochemistry with anti-HNA, whereas A-E6 had formed considerably less cellular lesions even at 14 weeks. Representative images from a total of n = 8 mice per group. Main scale bars, 1,000 µ m; inset scale bars, 50 µ m. e, Tumorbearing animals implanted with A-E6 subclones had significantly longer survival than heterogeneous SU-DIPG-VI bulk cells and A-D10 (P = 0.037, logrank test, n = 8 mice per group).
| Distinct infiltrative phenotypes of genotypically divergent DIPG subclones in vivo. a, Heterogeneous HSJD-DIPG-007 bulk cells and NS-F10 and NS-F8 subclones were implanted directly into the pons of NOD-SCID mice and tumors allowed to form over 8 months. At weeks 23-24, bulk cells and NS-F10 formed diffusely infiltrating tumors throughout the brain, as seen by H&E staining as well as immunohistochemistry with anti-human nuclear antigen (HNA) or astrocyte marker GFAP, whereas NS-F8 had formed considerably less infiltrative lesions even at 30 weeks. Representative images from a total of n = 4 mice per group. Main scale bars, 1,000 µ m; inset scale bars, 50 µ m. b, Tumor-bearing animals implanted with NS-F8 subclones had significantly longer survival than heterogeneous HSJD-DIPG-007 bulk cells and NS-F10 (P = 0.0236, log-rank test, n = 4 mice per group). *P < 0.05. c, Digital droplet PCR. Plot of assay for KMT5B wild-type (x axes) and R187* mutation (y axes) for normal human astrocytes and tumors from mice implanted with heterogeneous bulk cells, and subclones NS-F10 and NS-F8. Mutant reads are present in 51.33% droplets from NS-F10 and 0.23% droplets from the original bulk preparation. Taken from n = 3 independent experiments. d, Heterogeneous SU-DIPG-VI bulk cells and A-D10 and A-E6 subclones were implanted directly into the pons of nude mice and tumors allowed to form over 8 months. At week 10, bulk cells and A-D10 formed highly cellular, infiltrating tumors, as seen by H&E staining or immunohistochemistry with anti-HNA, whereas A-E6 had formed considerably less cellular lesions even at 14 weeks. Representative images from a total of n = 8 mice per group. Main scale bars, 1,000 µ m; inset scale bars, 50 µ m. e, Tumorbearing animals implanted with A-E6 subclones had significantly longer survival than heterogeneous SU-DIPG-VI bulk cells and A-D10 (P = 0.037, logrank test, n = 8 mice per group).
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| DIPG subclones cooperate to enhance tumorigenic phenotypes. Individual subclones of SU-DIPG-VI (a-d) and HSJD-DIPG-007 (e-h) were differentially labeled and cultured either as pure populations or mixed in equal ratios. a, Growth of cocultured (yellow) and monocultured E6 (green) and D10 (red) cells plated as single neurospheres after 96 h, measured as diameter of the sphere, with representative images provided from the Celigo S cytometer under phase contrast (phase) and fluorescence (fluor). Data derived and representative images taken from n = 3 independent experiments. Scale bars, 500 µ m. b, Invasion of cocultured (yellow) and monocultured E6 (green) and D10 (red) cells into Matrigel over 168 h, with area assessed by ImageJ software from representative images provided from the Celigo S cytometer under phase contrast and fluorescence. Cocultures and D10 have significantly enhanced invasive capabilities compared to E6. Data derived and representative images taken from n = 3 independent experiments. Scale bars, 500 µ m. c, Migration of mono-and cocultured E6 (green) and D10 (red) cells on Matrigel, assessed by the number of differentially labeled distant cells at 24 h, with representative images provided from the IncuCyte Zoom live-cell analysis system under phase contrast and fluorescence. Cells from individual subclones have enhanced migratory properties when cultured together compared to alone. Data derived and representative images taken from n = 3 independent experiments. Scale bars, 500 µ m. d, Confocal microscopy analysis of invasion of mono-and cocultured E6 (green) and D10 (red) cells into Matrigel after 4 d, with nuclei stained with DAPI. Poorly motile E6 cells are found to invade further and in greater numbers alongside D10 cells than when cultured alone. Representative images taken from n = 3 independent experiments. Scale bars, 200 µ m. e, Growth of cocultured (yellow) and monocultured NS-F8 (green) and NS-F10 (red) cells plated as single neurospheres after 96 h, measured as diameter of the sphere, with representative images provided from the Celigo S cytometer under phase contrast and fluorescence. Data derived and representative images taken from n = 3 independent experiments. Scale bars, 500 µ m. f, Invasion of cocultured (yellow) and monocultured NS-F8 (green) and NS-F10 (red) cells into Matrigel over 72 h, with area assessed by ImageJ software from representative images provided from the Celigo S cytometer under phase contrast and fluorescence. Cocultures and NS-F10 have significantly enhanced invasive capabilities compared to NS-F8. Data derived and representative images taken from n = 3 independent experiments. Scale bars, 500 µ m. g, Migration of mono-and cocultured NS-F8 (green) and NS-F10 (red) on fibronectin, assessed by the number of differentially labeled distant cells at 48 h, with representative images provided from the IncuCyte Zoom live-cell analysis system under phase contrast and fluorescence. Cells from NS-F8 have enhanced migratory properties when cultured with NS-F10 compared to alone. Data derived and representative images taken from n = 3 independent experiments. Scale bars, 500 µ m. h, Confocal microscopy analysis of migration of mono-and cocultured NS-F8 (green) and NS-F10 (red) cells on fibronectin after 3 d, with nuclei stained with DAPI. Poorly motile NS-F8 cells are found to migrate further and in greater numbers alongside NS-F10 cells than when cultured alone. Representative images taken from n = 3 independent experiments. Scale bars, 200 µ m. All comparisons carried out by ANOVA, **P < 0.01. ***P < 0.001. All graphs show mean ± s.d.
| DIPG subclones cooperate to enhance tumorigenic phenotypes. Individual subclones of SU-DIPG-VI (a-d) and HSJD-DIPG-007 (e-h) were differentially labeled and cultured either as pure populations or mixed in equal ratios. a, Growth of cocultured (yellow) and monocultured E6 (green) and D10 (red) cells plated as single neurospheres after 96 h, measured as diameter of the sphere, with representative images provided from the Celigo S cytometer under phase contrast (phase) and fluorescence (fluor). Data derived and representative images taken from n = 3 independent experiments. Scale bars, 500 µ m. b, Invasion of cocultured (yellow) and monocultured E6 (green) and D10 (red) cells into Matrigel over 168 h, with area assessed by ImageJ software from representative images provided from the Celigo S cytometer under phase contrast and fluorescence. Cocultures and D10 have significantly enhanced invasive capabilities compared to E6. Data derived and representative images taken from n = 3 independent experiments. Scale bars, 500 µ m. c, Migration of mono-and cocultured E6 (green) and D10 (red) cells on Matrigel, assessed by the number of differentially labeled distant cells at 24 h, with representative images provided from the IncuCyte Zoom live-cell analysis system under phase contrast and fluorescence. Cells from individual subclones have enhanced migratory properties when cultured together compared to alone. Data derived and representative images taken from n = 3 independent experiments. Scale bars, 500 µ m. d, Confocal microscopy analysis of invasion of mono-and cocultured E6 (green) and D10 (red) cells into Matrigel after 4 d, with nuclei stained with DAPI. Poorly motile E6 cells are found to invade further and in greater numbers alongside D10 cells than when cultured alone. Representative images taken from n = 3 independent experiments. Scale bars, 200 µ m. e, Growth of cocultured (yellow) and monocultured NS-F8 (green) and NS-F10 (red) cells plated as single neurospheres after 96 h, measured as diameter of the sphere, with representative images provided from the Celigo S cytometer under phase contrast and fluorescence. Data derived and representative images taken from n = 3 independent experiments. Scale bars, 500 µ m. f, Invasion of cocultured (yellow) and monocultured NS-F8 (green) and NS-F10 (red) cells into Matrigel over 72 h, with area assessed by ImageJ software from representative images provided from the Celigo S cytometer under phase contrast and fluorescence. Cocultures and NS-F10 have significantly enhanced invasive capabilities compared to NS-F8. Data derived and representative images taken from n = 3 independent experiments. Scale bars, 500 µ m. g, Migration of mono-and cocultured NS-F8 (green) and NS-F10 (red) on fibronectin, assessed by the number of differentially labeled distant cells at 48 h, with representative images provided from the IncuCyte Zoom live-cell analysis system under phase contrast and fluorescence. Cells from NS-F8 have enhanced migratory properties when cultured with NS-F10 compared to alone. Data derived and representative images taken from n = 3 independent experiments. Scale bars, 500 µ m. h, Confocal microscopy analysis of migration of mono-and cocultured NS-F8 (green) and NS-F10 (red) cells on fibronectin after 3 d, with nuclei stained with DAPI. Poorly motile NS-F8 cells are found to migrate further and in greater numbers alongside NS-F10 cells than when cultured alone. Representative images taken from n = 3 independent experiments. Scale bars, 200 µ m. All comparisons carried out by ANOVA, **P < 0.01. ***P < 0.001. All graphs show mean ± s.d.
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Pediatric GBM and DIPG harbor a complex subclonal architecture a, Representative images (from n = 142) of six cases of pGBM and DIPG from different anatomical locations and with different histone H3 mutation status. For each case, a CIRCOS plot shows chromosomal ideograms on the outermost ring, with banding in black and gray and centromere in red, and highlights somatic SNVs and insertions or deletions on the next ring, DNA copy number changes (dark red, amplification; light red, gain; dark blue, homozygous deletion; light blue, single-copy loss) and loss of heterozygosity (yellow) on the inner rings, and intra- or interchromosomal translocations inside the circle (orange). The CCF for each somatic coding mutation is plotted as a histogram with a kernel density overplotted. In all cases, in addition to a peak of mutations present in 100% of cells (clonal), there is a complex pattern of subclonal mutations (<95% CCF) forming peaks at low frequencies within a given tumor. b, Violin plot of CCFs for a given series of gene mutations across all 142 independent cases of pGBM and DIPG (H3.3 G34R or G34V, n = 10; H3.3 K27M, n = 61; H3.1 K27M, n = 23; ATRX, n = 22; NF1, n = 4; ACVR1, n = 27; TP53, n = ; ATM, n = 5; PIK3R1, n = 8; PPM1D, n = 11; PDGFRA, n = 7; BRAF, n = 5; PIK3CA, n = 15). The shaded area represents a CCF of 95–100% to indicate a clonal mutation. Purported drivers such as histone H3 mutations, ATRX and NF1 are almost wholly found to be clonal (though there are single outliers in some instances). Other genes such as PIK3CA, BRAF and PDGFRA are frequently found to be mutated in smaller subclonal compartments of the tumors. Kernel densities of CCFs are plotted for all samples harboring a given mutation (number of independent cases listed on figure). c, The number of subclones present in 142 pGBM and DIPG is calculated from somatic mutation data using the EXPANDS package²⁷ and ordered first by the number of subclones (colored using a rainbow palette) and then by the proportion of the tumor defined by the main clone in each tumor. A single case was clonal, with more than 85% of cases harboring 3–10 subclones. d, Dot plot of the number of somatic coding SNVs (y axis) against the number of subclones (x axis), demonstrating a significant positive relationship (Pearson r² = 0.2188, P = 4.36 × 10⁻⁹, n = 142 independent samples). Horizontal bar, median. Individual tumors are colored by their histone H3 mutation status, with outliers often seen to harbor H3.3 G34R (blue). e, Clinical and molecular correlates of subclonal numbers. Box plots highlight lack of difference in the number of subclones on the basis of anatomical location, but an increased number in H3.3 G34R tumors (P = 0.044, t-test) and a reduced number in infants (<3 years, P = 0.0108, t-test) across all n = 142 independent samples. The thick line within the box is the median, the lower and upper limits of the boxes represent the first and third quartiles, whiskers 1.5 times the interquartile range, and individual points outliers. Hemi, hemispheric. f, Prognostic implications. Kaplan–Meier curves demonstrate that H3.3 G34R tumors have a longer overall survival than other pGBM and DIPG (P = 3.94 × 10⁻⁶, log-rank test); however, despite the association of this subgroup with an increased number of tumor subclones, an elevated subclonal diversity shows a trend toward shorter survival across all pGBM and DIPG (P = 0.068, log-rank test). Comparisons included all n = 142 independent samples. *P < 0.05. **P < 0.01.
Pediatric GBM and DIPG harbor a complex subclonal architecture a, Representative images (from n = 142) of six cases of pGBM and DIPG from different anatomical locations and with different histone H3 mutation status. For each case, a CIRCOS plot shows chromosomal ideograms on the outermost ring, with banding in black and gray and centromere in red, and highlights somatic SNVs and insertions or deletions on the next ring, DNA copy number changes (dark red, amplification; light red, gain; dark blue, homozygous deletion; light blue, single-copy loss) and loss of heterozygosity (yellow) on the inner rings, and intra- or interchromosomal translocations inside the circle (orange). The CCF for each somatic coding mutation is plotted as a histogram with a kernel density overplotted. In all cases, in addition to a peak of mutations present in 100% of cells (clonal), there is a complex pattern of subclonal mutations (<95% CCF) forming peaks at low frequencies within a given tumor. b, Violin plot of CCFs for a given series of gene mutations across all 142 independent cases of pGBM and DIPG (H3.3 G34R or G34V, n = 10; H3.3 K27M, n = 61; H3.1 K27M, n = 23; ATRX, n = 22; NF1, n = 4; ACVR1, n = 27; TP53, n = ; ATM, n = 5; PIK3R1, n = 8; PPM1D, n = 11; PDGFRA, n = 7; BRAF, n = 5; PIK3CA, n = 15). The shaded area represents a CCF of 95–100% to indicate a clonal mutation. Purported drivers such as histone H3 mutations, ATRX and NF1 are almost wholly found to be clonal (though there are single outliers in some instances). Other genes such as PIK3CA, BRAF and PDGFRA are frequently found to be mutated in smaller subclonal compartments of the tumors. Kernel densities of CCFs are plotted for all samples harboring a given mutation (number of independent cases listed on figure). c, The number of subclones present in 142 pGBM and DIPG is calculated from somatic mutation data using the EXPANDS package²⁷ and ordered first by the number of subclones (colored using a rainbow palette) and then by the proportion of the tumor defined by the main clone in each tumor. A single case was clonal, with more than 85% of cases harboring 3–10 subclones. d, Dot plot of the number of somatic coding SNVs (y axis) against the number of subclones (x axis), demonstrating a significant positive relationship (Pearson r² = 0.2188, P = 4.36 × 10⁻⁹, n = 142 independent samples). Horizontal bar, median. Individual tumors are colored by their histone H3 mutation status, with outliers often seen to harbor H3.3 G34R (blue). e, Clinical and molecular correlates of subclonal numbers. Box plots highlight lack of difference in the number of subclones on the basis of anatomical location, but an increased number in H3.3 G34R tumors (P = 0.044, t-test) and a reduced number in infants (<3 years, P = 0.0108, t-test) across all n = 142 independent samples. The thick line within the box is the median, the lower and upper limits of the boxes represent the first and third quartiles, whiskers 1.5 times the interquartile range, and individual points outliers. Hemi, hemispheric. f, Prognostic implications. Kaplan–Meier curves demonstrate that H3.3 G34R tumors have a longer overall survival than other pGBM and DIPG (P = 3.94 × 10⁻⁶, log-rank test); however, despite the association of this subgroup with an increased number of tumor subclones, an elevated subclonal diversity shows a trend toward shorter survival across all pGBM and DIPG (P = 0.068, log-rank test). Comparisons included all n = 142 independent samples. *P < 0.05. **P < 0.01.
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Isolation of genotypically and phenotypically diverse single stem-like cell-derived subclones of pediatric GBM and DIPG a, Isolation of subclonal populations: disaggregation of heterogeneous mixtures of patient-derived tumor cells, flow sorting into single cells in 96-well plates, and colony formation as either 2D cultures, adherent on laminin, or 3D neurospheres, all under stem cell conditions. Individual subclonal colonies are subjected to high-throughput phenotypic analysis and targeted resequencing, and further cultured for detailed in vitro and in vivo mechanistic comparison with heterogeneous bulk populations. b, Percentage of single cells that formed colonies under 2D laminin and 3D neurosphere stem cell conditions are given for six pGBM and DIPG primary patient-derived cell cultures, labeled by anatomical location, histone H3 mutation subgroup (dark green, HIST1H3B; light green, H3F3A) and name of the cell line. Mid, midline. c, 3D neurosphere culture from single-cell-derived colonies from SU-DIPG-VI assessed by Celigo S imaging cytometer. d, Growth of single-cell-derived colonies over time, assessed as diameter of neurosphere, labeled and color-coded. e, Targeted sequencing contingency plot of somatic mutations common to all subclones (blue), shared among certain subclones (yellow) and private to individuals (red). f, 2D laminin culture from single-cell-derived colonies from SU-DIPG-VI assessed by Celigo S imaging cytometer. g, Growth of single-cell-derived colonies over time, assessed as diameter of neurosphere, with subclones taken for later analysis highlighted: A-D10 (fast, purple), A-B8 (intermediate, pink) and A-E6 (slow, violet). h, Targeted sequencing contingency plot of somatic mutations common to all subclones (blue), shared among certain subclones (yellow) and private to individuals (red). Gene names are colored to highlight private mutations in selected subclones or common to A-D10 and A-B8 (brown). i, Time course for growth of selected subclones replated and grown over 160 h, highlighting statistically significant differences among subclones and heterogeneous bulk cell populations of SU-DIPG-VI (blue). Representative images at 72 h are provided from the Celigo S cytometer, with tumor cells marked in green. Data derived and representative images taken from n = 3 independent experiments. Scale bars, 500 µm. j, Time course of invasion of cells into a Matrigel matrix over 72 h, either as percentage of the total area in the field of view covered by invading cells, or as a percentage of time zero. Representative images given at 72 h, with extent of tumor cell invasion marked in green. Data derived and representative images taken from n = 3 independent experiments. Scale bars, 500 µm. k, Time course of tumor cell migration onto Matrigel over 72 h, either as percentage of the total area of the well covered by migrating cells or as a percentage of that at time zero. Representative images given at 72 h, with extent of tumor cell migration marked in green. Data derived and representative images taken from n = 3 independent experiments. Scale bars, 500 µm. ANOVA. *P < 0.05. **P < 0.01. ***P < 0.001. All graphs show mean ± s.d.
+1
Isolation of genotypically and phenotypically diverse single stem-like cell-derived subclones of pediatric GBM and DIPG a, Isolation of subclonal populations: disaggregation of heterogeneous mixtures of patient-derived tumor cells, flow sorting into single cells in 96-well plates, and colony formation as either 2D cultures, adherent on laminin, or 3D neurospheres, all under stem cell conditions. Individual subclonal colonies are subjected to high-throughput phenotypic analysis and targeted resequencing, and further cultured for detailed in vitro and in vivo mechanistic comparison with heterogeneous bulk populations. b, Percentage of single cells that formed colonies under 2D laminin and 3D neurosphere stem cell conditions are given for six pGBM and DIPG primary patient-derived cell cultures, labeled by anatomical location, histone H3 mutation subgroup (dark green, HIST1H3B; light green, H3F3A) and name of the cell line. Mid, midline. c, 3D neurosphere culture from single-cell-derived colonies from SU-DIPG-VI assessed by Celigo S imaging cytometer. d, Growth of single-cell-derived colonies over time, assessed as diameter of neurosphere, labeled and color-coded. e, Targeted sequencing contingency plot of somatic mutations common to all subclones (blue), shared among certain subclones (yellow) and private to individuals (red). f, 2D laminin culture from single-cell-derived colonies from SU-DIPG-VI assessed by Celigo S imaging cytometer. g, Growth of single-cell-derived colonies over time, assessed as diameter of neurosphere, with subclones taken for later analysis highlighted: A-D10 (fast, purple), A-B8 (intermediate, pink) and A-E6 (slow, violet). h, Targeted sequencing contingency plot of somatic mutations common to all subclones (blue), shared among certain subclones (yellow) and private to individuals (red). Gene names are colored to highlight private mutations in selected subclones or common to A-D10 and A-B8 (brown). i, Time course for growth of selected subclones replated and grown over 160 h, highlighting statistically significant differences among subclones and heterogeneous bulk cell populations of SU-DIPG-VI (blue). Representative images at 72 h are provided from the Celigo S cytometer, with tumor cells marked in green. Data derived and representative images taken from n = 3 independent experiments. Scale bars, 500 µm. j, Time course of invasion of cells into a Matrigel matrix over 72 h, either as percentage of the total area in the field of view covered by invading cells, or as a percentage of time zero. Representative images given at 72 h, with extent of tumor cell invasion marked in green. Data derived and representative images taken from n = 3 independent experiments. Scale bars, 500 µm. k, Time course of tumor cell migration onto Matrigel over 72 h, either as percentage of the total area of the well covered by migrating cells or as a percentage of that at time zero. Representative images given at 72 h, with extent of tumor cell migration marked in green. Data derived and representative images taken from n = 3 independent experiments. Scale bars, 500 µm. ANOVA. *P < 0.05. **P < 0.01. ***P < 0.001. All graphs show mean ± s.d.
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Articles
https://doi.org/10.1038/s41591-018-0086-7
1Centre for Evolution and Cancer, The Institute of Cancer Research, London, UK. 2Division of Molecular Pathology, The Institute of Cancer Research,
London, UK. 3Division of Cancer Therapeutics, The Institute of Cancer Research, London, UK. 4Department of Cellular Pathology, University Hospital of
Wales, Cardiff, UK. 5Stanford University School of Medicine, Stanford, CA, USA. 6CRUK Gene Function Laboratory and Breast Cancer Now Toby Robins
Research Centre, The Institute of Cancer Research, London, UK. 7UCL Cancer Institute, University College London, London, UK. 8Paediatric Oncology
Drug Development Team, Children and Young People’s Unit, Royal Marsden Hospital, Sutton, UK. 9UQ Child Health Research Centre, The University
of Queensland, Brisbane, Queensland, Australia. 10Oncology Services Group, Children’s Health Queensland Hospital and Health Service, Brisbane,
Queensland, Australia. 11The University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Queensland, Australia. 12Department
of Anatomical and Cellular Pathology, The Chinese University of Hong Kong, Hong Kong, China. 13Department of Cytogenetics and Reproductive Biology,
Farhat HACHED Hospital, Sousse, Tunisia. 14Faculty of Medicine, Sousse, Tunisia. 15Centre Hospitalier Régional et Universitaire Hautepierre, Strasbourg,
France. 16Department of Radiotherapy, Royal Marsden Hospital, Sutton, UK. 17Department of Cellular Pathology, St George’s Hospital NHS Trust, London,
UK. 18Department of Neurosurgery, St George’s Hospital NHS Trust, London, UK. 19Department of Neuropathology, Kings College Hospital, London, UK.
20Department of Neurosurgery, Kings College Hospital, London, UK. 21Hospital Sant Joan de Deu, Barcelona, Spain. 22Department of Neurology, Stanford
University School of Medicine, Stanford, CA, USA. 23Present address: Bambino Gesù Children’s Hospital–IRCCS, Rome, Italy. 24Present address: Department
of Pediatric Hematology Oncology, Columbia University Medical Center, New York, NY, USA. *e-mail: chris.jones@icr.ac.uk
pGBM and DIPG are a highly heterogeneous group of high-
grade glial tumors with no effective treatments1. Integrated
molecular profiling2–7 has revealed unique, specific and highly
recurrent mutations in genes encoding histone H3 variants that
mark robust subgroups of pGBM and DIPG with distinct age of
onset, anatomical distribution, clinical outcome, and histopatho-
logical and radiological features8,9. A paradigm shift away from
extrapolating from inappropriate adult GBM data and toward
a more pediatric-biology-specific approach to developing new
therapies has been a positive consequence of the discovery of these
mechanisms of tumorigenesis10–12.
Despite these advances in our understanding of the unique bio-
logical drivers of these diseases13, a major challenge to improving
outcomes for children with these tumors is likely to overlap with
morphologically similar tumors in adults: their extensive intratu-
moral heterogeneity14. This has been demonstrated spatially by the
application of genomic analyses of topographically distinct areas
of the tumor at resection15, through longitudinal studies of tumor
Functional diversity and cooperativity between
subclonal populations of pediatric glioblastoma
and diffuse intrinsic pontine glioma cells
Mara Vinci1,2,3,23, Anna Burford1,2,3, Valeria Molinari1,2,3, Ketty Kessler1,2,3, Sergey Popov1,2,3,4,
Matthew Clarke1,2,3, Kathryn R. Taylor1,2,3,5, Helen N. Pemberton6, Christopher J. Lord6,
Alice Gutteridge7, Tim Forshew7, Diana Carvalho 1,2,3, Lynley V. Marshall8, Elizabeth Y. Qin5,
Wendy J. Ingram 9,10, Andrew S. Moore 9,10,11, Ho-Keung Ng12, Saoussen Trabelsi13,
Dorra H’mida-Ben Brahim13,14, Natacha Entz-Werle15, Stergios Zacharoulis8,24, Sucheta Vaidya8,
Henry C. Mandeville16, Leslie R. Bridges17, Andrew J. Martin18, Safa Al-Sarraj19,
Christopher Chandler20, Mariona Sunol21, Jaume Mora21, Carmen de Torres21, Ofelia Cruz 21,
Angel M. Carcaboso 21, Michelle Monje 22, Alan Mackay1,2,3 and Chris Jones1,2,3*
The failure to develop effective therapies for pediatric glioblastoma (pGBM) and diffuse intrinsic pontine glioma (DIPG) is
in part due to their intrinsic heterogeneity. We aimed to quantitatively assess the extent to which this was present in these
tumors through subclonal genomic analyses and to determine whether distinct tumor subpopulations may interact to promote
tumorigenesis by generating subclonal patient-derived models in vitro and in vivo. Analysis of 142 sequenced tumors revealed
multiple tumor subclones, spatially and temporally coexisting in a stable manner as observed by multiple sampling strategies.
We isolated genotypically and phenotypically distinct subpopulations that we propose cooperate to enhance tumorigenic-
ity and resistance to therapy. Inactivating mutations in the H4K20 histone methyltransferase KMT5B (SUV420H1), present
in < 1% of cells, abrogate DNA repair and confer increased invasion and migration on neighboring cells, in vitro and in vivo,
through chemokine signaling and modulation of integrins. These data indicate that even rare tumor subpopulations may exert
profound effects on tumorigenesis as a whole and may represent a new avenue for therapeutic development. Unraveling the
mechanisms of subclonal diversity and communication in pGBM and DIPG will be an important step toward overcoming barri-
ers to effective treatments.
NATURE MEDICINE | VOL 24 | AUGUST 2018 | 1204–1215 | www.nature.com/naturemedicine
1204
Content courtesy of Springer Nature, terms of use apply. Rights reserved

Supplementary resources (9)

Supplementary Video S2
Data
August 2018
Maria Vinci · Anna Burford · Valeria Molinari · Ketty Kessler · Chris Jones
Supplementary Video S1
Data
August 2018
Maria Vinci · Anna Burford · Valeria Molinari · Ketty Kessler · Chris Jones
Supplementary Table S6
Data
August 2018
Maria Vinci · Anna Burford · Valeria Molinari · Ketty Kessler · Chris Jones
Supplementary Table S5
Data
August 2018
Maria Vinci · Anna Burford · Valeria Molinari · Ketty Kessler · Chris Jones
Supplementary Table S3
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August 2018
Maria Vinci · Anna Burford · Valeria Molinari · Ketty Kessler · Chris Jones
Supplementary Material
Data
August 2018
Maria Vinci · Anna Burford · Valeria Molinari · Ketty Kessler · Chris Jones
Supplementary Table S4
Data
August 2018
Maria Vinci · Anna Burford · Valeria Molinari · Ketty Kessler · Chris Jones
Supplementary Table S2
Data
August 2018
Maria Vinci · Anna Burford · Valeria Molinari · Ketty Kessler · Chris Jones
Supplementary Table S1
Data
August 2018
Maria Vinci · Anna Burford · Valeria Molinari · Ketty Kessler · Chris Jones
... A significant degree of genetic and phenotypic, interand intra-tumour heterogeneity, at both spatial and temporal levels, has been described for PDHGG, representing one of the most challenging aspects in the effort of developing effective therapeutic strategies for these diseases [5][6][7][8][9][10]. The sub-clonal architecture of PDHGG has been demonstrated using whole genome and exome sequencing analysis of longitudinally and multi-region resected samples [7,10]. ...
... A significant degree of genetic and phenotypic, interand intra-tumour heterogeneity, at both spatial and temporal levels, has been described for PDHGG, representing one of the most challenging aspects in the effort of developing effective therapeutic strategies for these diseases [5][6][7][8][9][10]. The sub-clonal architecture of PDHGG has been demonstrated using whole genome and exome sequencing analysis of longitudinally and multi-region resected samples [7,10]. Moreover, using primary patient-derived cell lines and multifluorescent optical barcoded-derived clones, we have shown that PDHGGs are composed of heterogeneous cell subpopulations that behave like functional networks conferring a more aggressive phenotype compared to the individual derived sub-clones [10,11]. ...
... The sub-clonal architecture of PDHGG has been demonstrated using whole genome and exome sequencing analysis of longitudinally and multi-region resected samples [7,10]. Moreover, using primary patient-derived cell lines and multifluorescent optical barcoded-derived clones, we have shown that PDHGGs are composed of heterogeneous cell subpopulations that behave like functional networks conferring a more aggressive phenotype compared to the individual derived sub-clones [10,11]. ...
Inhibition of exosome biogenesis affects cell motility in heterogeneous sub-populations of paediatric-type diffuse high-grade gliomas
Article
Full-text available
  • Nov 2023
Background Paediatric-type diffuse High-Grade Gliomas (PDHGG) are highly heterogeneous tumours which include distinct cell sub-populations co-existing within the same tumour mass. We have previously shown that primary patient-derived and optical barcoded single-cell-derived clones function as interconnected networks. Here, we investigated the role of exosomes as a route for inter-clonal communication mediating PDHGG migration and invasion. Results A comprehensive characterisation of seven optical barcoded single-cell-derived clones obtained from two patient-derived cell lines was performed. These analyses highlighted extensive intra-tumour heterogeneity in terms of genetic and transcriptional profiles between clones as well as marked phenotypic differences including distinctive motility patterns. Live single-cell tracking analysis of 3D migration and invasion assays showed that the single-cell-derived clones display a higher speed and longer travelled distance when in co-culture compared to mono-culture conditions. To determine the role of exosomes in PDHGG inter-clonal cross-talks, we isolated exosomes released by different clones and characterised them in terms of marker expression, size and concentration. We demonstrated that exosomes are actively internalized by the cells and that the inhibition of their biogenesis, using the phospholipase inhibitor GW4689, significantly reduced the cell motility in mono-culture and more prominently when the cells from the clones were in co-culture. Analysis of the exosomal miRNAs, performed with a miRNome PCR panel, identified clone-specific miRNAs and a set of miRNA target genes involved in the regulation of cell motility/invasion/migration. These genes were found differentially expressed in co-culture versus mono-culture conditions and their expression levels were significantly modulated upon inhibition of exosome biogenesis. Conclusions In conclusion, our study highlights for the first time a key role for exosomes in the inter-clonal communication in PDHGG and suggests that interfering with the exosome biogenesis pathway may be a valuable strategy to inhibit cell motility and dissemination for these specific diseases.
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... SU-DIPG-13P* (hereafter called DIPG 13P*) [26,27] were kindly provided by Dr. Koschmann (Pediatrics, Michigan Medicine, University of Michigan), HSJD-DIPG 007 (hereafter called DIPG 007; RRID:CVCL_VU70) [28] by Dr. Venneti (Neuropathology, Michigan Medicine, University of Michigan) and SU-DIPG-XIII (hereafter called DIPG-XIII; RRID:CVCL_IT41) [29] by Drs. Maachani and Souweidane (Pediatric Neurological Surgery, Weill Cornell Medicine) and cultured as described in [16]. ...
... Nevertheless, we were able to show efficient brain penetrance, drug delivery to the brain tumor, and kinase target inhibition, indicating a promising compound for the treatment of DIPG. Furthermore, our data indicate the potential of MTX-241F not only for radiosensitization, but also for re-sensitizing DIPG to radiotherapy, since the patient derived cells we tested stemmed from radioresistant, recurrent tumors [28,29]. ...
Targeting DNA Repair and Survival Signaling in Diffuse Intrinsic Pontine Gliomas to Prevent Tumor Recurrence
Article
Full-text available
  • Sep 2023
Therapeutic resistance remains a major obstacle to successful clinical management of diffuse intrinsic pontine glioma (DIPG), a high-grade pediatric tumor of the brain stem. In nearly all patients, available therapies fail to prevent progression. Innovative combinatorial therapies that penetrate the blood–brain barrier and lead to long-term control of tumor growth are desperately needed. We identified mechanisms of resistance to radiotherapy, the standard of care for DIPG. On the basis of these findings, we rationally designed a brain-penetrant small molecule, MTX-241F, that is a highly selective inhibitor of EGFR and PI3 kinase family members, including the DNA repair protein DNA-PK. Preliminary studies demonstrated that micromolar levels of this inhibitor can be achieved in murine brain tissue and that MTX-241F exhibits promising single-agent efficacy and radiosensitizing activity in patient-derived DIPG neurospheres. Its physiochemical properties include high exposure in the brain, indicating excellent brain penetrance. Because radiotherapy results in double-strand breaks that are repaired by homologous recombination (HR) and non-homologous DNA end joining (NHEJ), we have tested the combination of MTX-241F with an inhibitor of Ataxia Telangiectasia Mutated to achieve blockade of HR and NHEJ, respectively, with or without radiotherapy. When HR blockers were combined with MTX-241F and radiotherapy, synthetic lethality was observed, providing impetus to explore this combination in clinically relevant models of DIPG. Our data provide proof-of-concept evidence to support advanced development of MTX-241F for the treatment of DIPG. Future studies will be designed to inform rapid clinical translation to ultimately impact patients diagnosed with this devastating disease.
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... Some tumors, such as ependymoma, can be characterized by intratumoral heterogeneity as various genetic alterations and histological patterns can be comprised within the same tumor. 105,106 Anatomical information about surrounding brain structures, tumor location, and global morphology extracted from radiology imaging in combination with microscopic information about cellular subpopulations from histopathology imaging could lead to sophisticated 3D models of the non-uniform evolution of a given tumor over time. In this way, radio-pathomics may better capture the spatial heterogeneity of pediatric brain tumors and be used to construct individualized predictive modeling of tumor growth and infiltration. ...
Radio-pathomic approaches in pediatric neuro-oncology: Opportunities and challenges
Article
Full-text available
  • Sep 2023
With medical software platforms moving to cloud environments with scalable storage and computing, the translation of predictive artificial intelligence (AI) models to aid in clinical decision-making and facilitate personalized medicine for cancer patients is becoming a reality. Medical imaging, namely radiologic and histologic images, has immense analytical potential in neuro-oncology, and models utilizing integrated radiomic and pathomic data may yield a synergistic effect and provide a new modality for precision medicine. At the same time, the ability to harness multi-modal data is met with challenges in aggregating data across medical departments and institutions, as well as significant complexity in modeling the phenotypic and genotypic heterogeneity of pediatric brain tumors. In this paper, we review recent pathomic and integrated pathomic, radiomic, and genomic studies with clinical applications. We discuss current challenges limiting translational research on pediatric brain tumors and outline technical and analytical solutions. Overall, we propose that to empower the potential residing in radio-pathomics, systemic changes in cross-discipline data management and end-to-end software platforms to handle multi-modal data sets are needed, in addition to embracing modern AI-powered approaches. These changes can improve the performance of predictive models, and ultimately the ability to advance brain cancer treatments and patient outcomes through the development of such models.
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... Response to chemoradiation is poor and palliative radiotherapy remains the only standard-of-care treatment with proven benefit 6 , resulting in a median overall survival (OS) between 10 and 15 months after initial diagnosis 7 . Immune checkpoint inhibitors (ICIs), such as PD-1 blockade are successfully used in combinatorial immunotherapeutic approaches in high-grade gliomas 8 ; however, in DMG intratumoral heterogeneity 9 , low PD-L1 expression 10 , low mutational burden 11 and the nature of chemotherapy-induced mutations 12,13 may explain why no survival benefit has been observed using ICI monotherapy 14 so far, though several clinical trials investigating the efficacy and safety of PD-1 blockade are ongoing (NCT02359565, NCT02793466, NCT03130959 and NCT01952769). ...
A H3K27M-targeted vaccine in adults with diffuse midline glioma
Article
Full-text available
Substitution of lysine 27 to methionine in histone H3 (H3K27M) defines an aggressive subtype of diffuse glioma. Previous studies have shown that a H3K27M-specific long peptide vaccine (H3K27M-vac) induces mutation-specific immune responses that control H3K27M⁺ tumors in major histocompatibility complex-humanized mice. Here we describe a first-in-human treatment with H3K27M-vac of eight adult patients with progressive H3K27M⁺ diffuse midline glioma on a compassionate use basis. Five patients received H3K27M-vac combined with anti-PD-1 treatment based on physician’s discretion. Repeat vaccinations with H3K27M-vac were safe and induced CD4⁺ T cell-dominated, mutation-specific immune responses in five of eight patients across multiple human leukocyte antigen types. Median progression-free survival after vaccination was 6.2 months and median overall survival was 12.8 months. One patient with a strong mutation-specific T cell response after H3K27M-vac showed pseudoprogression followed by sustained complete remission for >31 months. Our data demonstrate safety and immunogenicity of H3K27M-vac in patients with progressive H3K27M⁺ diffuse midline glioma.
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... 16 More recently, intratumor genetic heterogeneity was proposed to underlie some form of cooperativity between co-existing tumor clones, in which minor tumor populations instruct other tumor cells to infiltrate. 17 Yet, it is unclear whether these alterations, retrieved in a very limited number of patients, illustrate general druggable mechanisms driving DMG invasiveness. ...
Diffuse Midline Glioma Invasion and Metastasis Rely on Cell-autonomous Signaling
Article
Full-text available
  • Sep 2023
Background Diffuse midline gliomas (DMG) are pediatric tumors with negligible two-year survival after diagnosis characterized by their ability to infiltrate the central nervous system. In the hope of controlling the local growth and slowing the disease all patients receive radiotherapy. However, distant progression occurs frequently in DMG patients. Current clues as to what causes tumor infiltration circle mainly around the tumor microenvironment, but there are currently no known determinants to predict the degree of invasiveness. Methods In this study we use patient-derived glioma stem cells (GSCs) to create patient-specific 3D avatars to model interindividual invasion and elucidate the cellular supporting mechanisms. Results We show that GSC models in 3D mirror the invasive behavior of the parental tumors, thus proving the ability of DMG to infiltrate as an autonomous characteristic of tumor cells. Furthermore, we distinguished two modes of migration, mesenchymal and amoeboid-like, and associated the amoeboid-like modality with GSCs derived from the most invasive tumors. Using transcriptomics of both organoids and primary tumors, we further characterized the invasive amoeboid-like tumors as oligodendrocyte progenitor-like, with highly contractile cytoskeleton and reduced adhesion ability driven by crucial over-expression of BMP7. Finally, we deciphered MEK, ERK and Rho/ROCK kinases activated downstream of the BMP7 stimulation as actionable targets controlling tumor cell motility. Conclusions Our findings identify two new therapeutic avenues. First, patient-derived GSCs represent a predictive tool for patient stratification in order to adapt irradiation strategies. Second, autocrine and short-range BMP7-related signaling becomes a druggable target to prevent DMG spread and metastasis.
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... and a member of the histone-lysine methyltransferases (KMTs) family, which are essential in many tightly regulated cellular processes. Dysregulation of KMTs has been associated with many cancers [47,48]. Using the eQTLGen consortium [49], the authors showed this SNP to be associated with expression of 12 of the 47 genes mapped in 500KB of the SNP on Chr 11q13.2; ...
Current Landscape of Genome-Wide Association Studies in Acute Myeloid Leukemia: A Review
Article
Full-text available
  • Jul 2023
Acute myeloid leukemia (AML) is a clonal hematopoietic disease that arises from chromosomal and genetic aberrations in myeloid precursor cells. AML is one of the most common types of acute leukemia in adults; however, it is relatively rare overall, comprising about 1% of all cancers. In the last decade or so, numerous genome-wide association studies (GWAS) have been conducted to screen between hundreds of thousands and millions of variants across many human genomes to discover genetic polymorphisms associated with a particular disease or phenotype. In oncology, GWAS has been performed in almost every commonly occurring cancer. Despite the increasing number of studies published regarding other malignancies, there is a paucity of GWAS studies for AML. In this review article, we will summarize the current status of GWAS in AML.
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... In the last 10 years, research on liquid biopsy, especially non-invasive detection and monitoring of cancer using biomarkers, has risen dramatically. This is due to the development of highly sensitive analytical techniques for measuring and identifying these biomolecules in biofluids [3]. For the diagnosis of solid cancers, collecting biofluid non-invasively is beneficial for most patients. ...
The Role of Liquid Biopsy in the Diagnosis and Prognosis of WHO Grade 4 Astrocytoma
Article
Full-text available
  • Jul 2023
Liquid biopsy, as a non-invasive diagnostic tool, has recently gained significant attention in the field of oncology. It involves the analysis of various biomarkers present in bodily fluids, such as blood or cerebrospinal fluid, to provide information about the underlying cancer. In the case of WHO grade 4 astrocytomas, liquid biopsy has the potential to significantly impact the diagnosis and prognosis of this aggressive malignant brain tumor. By detecting specific genetic mutations, such as IDH1 or EGFR, and monitoring levels of circulating tumor DNA, liquid biopsy can aid in the early detection and monitoring of disease progression. This innovative approach is gradually being acknowledged as a less invasive and cost-effective procedure for cancer diagnosis and management to improve patient outcomes and quality of life. Various kinds of biomarkers circulating in cerebrospinal fluid (CSF), such as circulating tumor cells (CTC) and different types of nucleic acids like cell-free DNA (cfDNA), cell-free RNA (ctRNA), and microRNAs (miRNA), have been identified. These biomarkers, which require dependable detection methods, are comparatively simple to obtain and allow for repeated measurements, making them significantly superior for disease monitoring. This review aims to compare the latest liquid biopsy analysis tools for both CSF and plasma in the central nervous system.
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... pHGG are not only genetically and molecularly distinct from adult pHGG, but also exhibit a high degree of intra-and intertumoral heterogeneity. Sequencing studies analyzing either multiple brain regions or comparing diagnostic and recurrent tumors taken from individual pHGG patients reveal spatially and temporally heterogeneous DNA mutations even among samples acquired from the same patient affecting genes, such as ATM, PPM1D, BCOR, ATRX, MYC, and KMT5B (Hoffman et al., 2016;Nikbakht et al., 2016;Salloum et al., 2017;Vinci et al., 2018). pHGG also exhibit heterogeneity in the presence of copy number variations in PDGFRA and other genes regulating oncogenic signaling and the cell cycle (Bax et al., 2010;Paugh et al., 2011;Koschmann et al., 2016), and cell to cell variation in epigenetic regulation reflected by differences in histone post-translational modifications and DNA methylation in pHGG (Sturm et al., 2012;Mackay et al., 2017;Castel et al., 2018;Huang et al., 2018). ...
Applying single cell multi-omic analyses to understand treatment resistance in pediatric high grade glioma
Article
Full-text available
  • May 2023
Despite improvements in cancer patient outcomes seen in the past decade, tumor resistance to therapy remains a major impediment to achieving durable clinical responses. Intratumoral heterogeneity related to genetic, epigenetic, transcriptomic, proteomic, and metabolic differences between individual cancer cells has emerged as a driver of therapeutic resistance. This cell to cell heterogeneity can be assessed using single cell profiling technologies that enable the identification of tumor cell clones that exhibit similar defining features like specific mutations or patterns of DNA methylation. Single cell profiling of tumors before and after treatment can generate new insights into the cancer cell characteristics that confer therapeutic resistance by identifying intrinsically resistant sub-populations that survive treatment and by describing new cellular features that emerge post-treatment due to tumor cell evolution. Integrative, single cell analytical approaches have already proven advantageous in studies characterizing treatment-resistant clones in cancers where pre- and post-treatment patient samples are readily available, such as leukemia. In contrast, little is known about other cancer subtypes like pediatric high grade glioma, a class of heterogeneous, malignant brain tumors in children that rapidly develop resistance to multiple therapeutic modalities, including chemotherapy, immunotherapy, and radiation. Leveraging single cell multi-omic technologies to analyze naïve and therapy-resistant glioma may lead to the discovery of novel strategies to overcome treatment resistance in brain tumors with dismal clinical outcomes. In this review, we explore the potential for single cell multi-omic analyses to reveal mechanisms of glioma resistance to therapy and discuss opportunities to apply these approaches to improve long-term therapeutic response in pediatric high grade glioma and other brain tumors with limited treatment options.
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... Through epigenetic silencing of oncogenes such as IL13RA2, overexpression of KMT5B reduced glioblastoma cell proliferation, cell viability, clonogenic potential in vitro, and tumor growth in vivo (López et al., 2021;Hulen et al., 2022). Two inactivating mutations of KMT5B (R187* and R699*) were identified in glioblastoma and diffuse intrinsic pontine glioma (DIPG) samples, which abrogated DNA repair and increased invasion and migration in neighboring cells (Vinci et al., 2018). Notably, 5 of 6 patients with altered KMT5B in our study had the same mutation of Overall survival of pediatric-type glioma based on the 2021 WHO classification of CNS tumors. ...
Spotlights on adult patients with pediatric-type diffuse gliomas in accordance with the 2021 WHO classification of CNS tumors
Article
Full-text available
  • May 2023
Introduction The fifth edition of the World Health Organization (WHO) classification of central nervous system (CNS) tumors released in 2021 formally defines pediatric-type diffuse gliomas. However, there is still little understanding of pediatric-type diffuse gliomas, and even less attention has been paid to adult patients. Therefore, this study describes the clinical radiological, survival, and molecular features of adult patients with pediatric-type glioma. Methods Adult patients who underwent surgery from January 2011 to January 2022, classified as pediatric-type glioma, were included in this study. Clinical, radiological, histopathological, molecular pathological, and survival data were collected for analysis. Results Among 596 adult patients, 20 patients with pediatric-type glioma were screened, including 6 with diffuse astrocytoma, MYB - or MYBL1 -altered, 2 with diffuse midline glioma, H3 K27-altered, and 12 with diffuse pediatric-type high-grade glioma, H3-wildtype and IDH-wildtype. Pediatric high-grade glioma (pHGG) frequently showed tumor enhancement, peritumoral edema, and intratumoral necrosis. Adult patients with pHGG showed a longer life expectancy than adult patients with glioblastoma. Common molecular alterations included chromosome alterations and CDKN2A/B , PIK3CA , and PTEN , while altered KMT5B and MET were found to affect the overall survival. Conclusion Our study demonstrated adult patients with pediatric-type glioma. Notably, our research aims to expand the current understanding of adult patients with pediatric-type diffuse gliomas. Furthermore, personalized therapies consisting of targeted molecular inhibitors for MET and VEGFA may exhibit beneficial effects in the corresponding population.
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Clinical and translational relevance of intratumor heterogeneity
Article
  • May 2023
Intratumor heterogeneity (ITH) is a driver of tumor evolution and a main cause of therapeutic resistance. Despite its importance, measures of ITH are still not incorporated into clinical practice. Consequently, standard treatment is frequently ineffective for patients with heterogeneous tumors as changes to treatment regimens are made only after recurrence and disease progression. More effective combination therapies require a mechanistic understanding of ITH and ways to assess it in clinical samples. The growth of technologies enabling the spatially intact analysis of tumors at the single-cell level and the development of sophisticated preclinical models give us hope that ITH will not simply be used as a predictor of a poor outcome but will guide treatment decisions from diagnosis through treatment.
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Characterizing temporal genomic heterogeneity in pediatric high-grade gliomas
Article
Full-text available
  • Oct 2017
Pediatric high-grade gliomas (pHGGs) are aggressive neoplasms representing approximately 20% of brain tumors in children. Current therapies offer limited disease control, and patients have a poor prognosis. Empiric use of targeted therapy, especially at progression, is increasingly practiced despite a paucity of data regarding temporal and therapy-driven genomic evolution in pHGGs. To study the genetic landscape of pHGGs at recurrence, we performed whole exome and methylation analyses on matched primary and recurrent pHGGs from 16 patients. Tumor mutational profiles identified three distinct subgroups. Group 1 (n = 7) harbored known hotspot mutations in Histone 3 (H3) (K27M or G34V) or IDH1 (H3/IDH1 mutants) and co-occurring TP53 or ACVR1 mutations in tumor pairs across the disease course. Group 2 (n = 7), H3/IDH1 wildtype tumor pairs, harbored novel mutations in chromatin modifiers (ZMYND11, EP300 n = 2), all associated with TP53 alterations, or had BRAF V600E mutations (n = 2) conserved across tumor pairs. Group 3 included 2 tumors with NF1 germline mutations. Pairs from primary and relapsed pHGG samples clustered within the same DNA methylation subgroup. ATRX mutations were clonal and retained in H3G34V and H3/IDH1 wildtype tumors, while different genetic alterations in this gene were observed at diagnosis and recurrence in IDH1 mutant tumors. Mutations in putative drug targets (EGFR, ERBB2, PDGFRA, PI3K) were not always shared between primary and recurrence samples, indicating evolution during progression. Our findings indicate that specific key driver mutations in pHGGs are conserved at recurrence and are prime targets for therapeutic development and clinical trials (e.g. H3 post-translational modifications, IDH1, BRAF V600E). Other actionable mutations are acquired or lost, indicating that re-biopsy at recurrence will provide better guidance for effective targeted therapy of pHGGs.
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Integrated Molecular Meta-Analysis of 1,000 Pediatric High-Grade and Diffuse Intrinsic Pontine Glioma
Article
Full-text available
  • Sep 2017
We collated data from 157 unpublished cases of pediatric high-grade glioma and diffuse intrinsic pontine glioma and 20 publicly available datasets in an integrated analysis of >1,000 cases. We identified co-segregating mutations in histone-mutant subgroups including loss of FBXW7 in H3.3G34R/V, TOP3A rearrangements in H3.3K27M, and BCOR mutations in H3.1K27M. Histone wild-type subgroups are refined by the presence of key oncogenic events or methylation profiles more closely resembling lower-grade tumors. Genomic aberrations increase with age, highlighting the infant population as biologically and clinically distinct. Uncommon pathway dysregulation is seen in small subsets of tumors, further defining the molecular diversity of the disease, opening up avenues for biological study and providing a basis for functionally defined future treatment stratification.
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Spatial heterogeneity in medulloblastoma
Article
Full-text available
Spatial heterogeneity of transcriptional and genetic markers between physically isolated biopsies of a single tumor poses major barriers to the identification of biomarkers and the development of targeted therapies that will be effective against the entire tumor. We analyzed the spatial heterogeneity of multiregional biopsies from 35 patients, using a combination of transcriptomic and genomic profiles. Medulloblastomas (MBs), but not high-grade gliomas (HGGs), demonstrated spatially homogeneous transcriptomes, which allowed for accurate subgrouping of tumors from a single biopsy. Conversely, somatic mutations that affect genes suitable for targeted therapeutics demonstrated high levels of spatial heterogeneity in MB, malignant glioma, and renal cell carcinoma (RCC). Actionable targets found in a single MB biopsy were seldom clonal across the entire tumor, which brings the efficacy of monotherapies against a single target into question. Clinical trials of targeted therapies for MB should first ensure the spatially ubiquitous nature of the target mutation.
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Spatial and temporal homogeneity of driver mutations in diffuse intrinsic pontine glioma
Article
Full-text available
  • Apr 2016
Diffuse Intrinsic Pontine Gliomas (DIPGs) are deadly paediatric brain tumours where needle biopsies help guide diagnosis and targeted therapies. To address spatial heterogeneity, here we analyse 134 specimens from various neuroanatomical structures of whole autopsy brains from nine DIPG patients. Evolutionary reconstruction indicates histone 3 (H3) K27M - including H3.2K27M - mutations potentially arise first and are invariably associated with specific, high-fidelity obligate partners throughout the tumour and its spread, from diagnosis to end-stage disease, suggesting mutual need for tumorigenesis. These H3K27M ubiquitously-associated mutations involve alterations in TP53 cell-cycle (TP53/PPM1D) or specific growth factor pathways (ACVR1/PIK3R1). Later oncogenic alterations arise in sub-clones and often affect the PI3K pathway. Our findings are consistent with early tumour spread outside the brainstem including the cerebrum. The spatial and temporal homogeneity of main driver mutations in DIPG implies they will be captured by limited biopsies and emphasizes the need to develop therapies specifically targeting obligate oncohistone partnerships.
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Immune Checkpoint Inhibition for Hypermutant Glioblastoma Multiforme Resulting From Germline Biallelic Mismatch Repair Deficiency
Article
Full-text available
  • Mar 2016
Purpose: Recurrent glioblastoma multiforme (GBM) is incurable with current therapies. Biallelic mismatch repair deficiency (bMMRD) is a highly penetrant childhood cancer syndrome often resulting in GBM characterized by a high mutational burden. Evidence suggests that high mutation and neoantigen loads are associated with response to immune checkpoint inhibition. Patients and methods: We performed exome sequencing and neoantigen prediction on 37 bMMRD cancers and compared them with childhood and adult brain neoplasms. Neoantigen prediction bMMRD GBM was compared with responsive adult cancers from multiple tissues. Two siblings with recurrent multifocal bMMRD GBM were treated with the immune checkpoint inhibitor nivolumab. Results: All malignant tumors (n = 32) were hypermutant. Although bMMRD brain tumors had the highest mutational load because of secondary polymerase mutations (mean, 17,740 ± standard deviation, 7,703), all other high-grade tumors were hypermutant (mean, 1,589 ± standard deviation, 1,043), similar to other cancers that responded favorably to immune checkpoint inhibitors. bMMRD GBM had a significantly higher mutational load than sporadic pediatric and adult gliomas and all other brain tumors (P < .001). bMMRD GBM harbored mean neoantigen loads seven to 16 times higher than those in immunoresponsive melanomas, lung cancers, or microsatellite-unstable GI cancers (P < .001). On the basis of these preclinical data, we treated two bMMRD siblings with recurrent multifocal GBM with the anti-programmed death-1 inhibitor nivolumab, which resulted in clinically significant responses and a profound radiologic response. Conclusion: This report of initial and durable responses of recurrent GBM to immune checkpoint inhibition may have implications for GBM in general and other hypermutant cancers arising from primary (genetic predisposition) or secondary MMRD.
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Spatial genomic heterogeneity in diffuse intrinsic pontine and midline high-grade glioma: Implications for diagnostic biopsy and targeted therapeutics
Article
Full-text available
  • Jan 2016
Introduction: Diffuse intrinsic pontine glioma (DIPG) and midline high-grade glioma (mHGG) are lethal childhood brain tumors. Spatial genomic heterogeneity has been well-described in adult HGG but has not been comprehensively characterized in pediatric HGG. We performed whole exome sequencing on 38-matched primary, contiguous, and metastatic tumor sites from eight children with DIPG (n = 7) or mHGG (n = 1) collected using a unique MRI-guided autopsy protocol. Validation was performed using Sanger sequencing, Droplet Digital polymerase-chain reaction, immunohistochemistry, and fluorescent in-situ hybridization. Results: Median age at diagnosis was 6.1 years (range: 2.9–23.3 years). Median overall survival was 13.2 months (range: 11.2–32.2 months). Contiguous tumor infiltration and distant metastases were observed in seven and six patients, respectively, including leptomeningeal dissemination in three DIPGs. Histopathological heterogeneity was evident in seven patients, including intra-pontine heterogeneity in two DIPGs, ranging from World Health Organization grade II to IV astrocytoma. We found conservation of heterozygous K27M mutations in H3F3A (n = 4) or HIST1H3B (n = 3) across all primary, contiguous, and metastatic tumor sites in all DIPGs. ACVR1 (n = 2), PIK3CA (n = 2), FGFR1 (n = 2), and MET (n = 1) were also intra-tumorally conserved. ACVR1 was co-mutated with HIST1H3B (n = 2). In contrast, PDGFRA amplification and mutation were spatially heterogeneous, as were mutations in BCOR (n = 1), ATRX (n = 2), and MYC (n = 1). TP53 aberrations (n = 3 patients) varied by type and location between primary and metastatic tumors sites but were intra-tumorally conserved. Conclusion: Spatial conservation of prognostically-relevant and therapeutically-targetable somatic mutations in DIPG and mHGG contrasts the significant heterogeneity of driver mutations seen in adult HGG and supports uniform implementation.
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Recurrent MET fusion genes represent a drug target in pediatric glioblastoma
Article
  • Oct 2016
Pediatric glioblastoma is one of the most common and most deadly brain tumors in childhood. Using an integrative genetic analysis of 53 pediatric glioblastomas and five in vitro model systems, we identified previously unidentified gene fusions involving the MET oncogene in ∼10% of cases. These MET fusions activated mitogen-activated protein kinase (MAPK) signaling and, in cooperation with lesions compromising cell cycle regulation, induced aggressive glial tumors in vivo. MET inhibitors suppressed MET tumor growth in xenograft models. Finally, we treated a pediatric patient bearing a MET-fusion-expressing glioblastoma with the targeted inhibitor crizotinib. This therapy led to substantial tumor shrinkage and associated relief of symptoms, but new treatment-resistant lesions appeared, indicating that combination therapies are likely necessary to achieve a durable clinical response. © 2016 Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.
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Abstract LB-B06: Functionally defined therapeutic targets in diffuse intrinsic pontine glioma
Article
  • Dec 2015
Diffuse intrinsic pontine glioma (DIPG) is a fatal childhood cancer that has a limited response to treatment (median survival after diagnosis is only 9 months). Historically, DIPG research has been limited by a dearth of tumor tissue available for study and a lack of experimental model systems. Recently, both cell cultures derived from patients with DIPG and orthotopic xenograft models have been established. We performed a chemical screen in 16 patient-derived DIPG cultures using 83 drugs, selected by pediatric neurooncologists as either promising small-molecule compounds or traditional chemotherapeutic agents. Of these 83 drugs, 14 showed activities against three or more DIPG cultures. Notable ‘misses’ that highlight the resistance of this tumor to traditional chemotherapies included temozolomide, carboplatin, and vincristine, while DIPG cell cultures were reported to have substantial sensitivity to histone deacetylase inhibitors, consistent with the highly recurrent H3K27M driving mutations in ∼80% of DIPG. Panobinostat was one of the most effective drugs screened, with 12/16 DIPG cultures showing sensitivity to this drug. Compared to vorinostat, another HDAC inhibitor, panobinostat exhibited substantially greater potency against DIPG cells. Treatment of DIPG cultures with panobinostat yielded a time and dose-dependent decrease in viability (RNA-mediated knockdown of HDAC1 and HDAC2 confirmed the mechanism of action). Treatment of DIPG cultures with panobinostat also increased H3 acetylation and H3K27 trimethylation, and led to a partial rescue of the H3K27M induced global hypotrimethylation phenotype. Increased K27 trimethylation was an unexpected effect of the drug, but it is consistent with recent findings that acetylated H3K27 can ‘detoxify’ K27M-induced inhibition of PCR2. RNA-seq performed on panobinostat- or vehicle-treated DIPG cells revealed sweeping changes in gene expression, including normalization of the K27M gene expression signature and decrease of the oncogenic MYC target gene-expression signature. In addition, the multi-histone deacetylase inhibitor panobinostat demonstrated efficacy in DIPG orthotopic xenograft models administered using convection enhanced delivery. Combination testing of panobinostat and the histone demethylase inhibitor GSK-J4, recently shown to decrease viability of mutant DIPG cells, revealed that the two had synergistic effects. Together, these data suggest a promising FDA-approved therapeutic strategy for DIPG that could be rapidly translated for use in the clinic, further indicating that epigenetic modifying therapies are emerging as the most promising class of agents for the treatment of DIPG. Citation Format: Catherine S. Grasso. Functionally defined therapeutic targets in diffuse intrinsic pontine glioma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-B06.
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