Background:
Diffuse midline glioma (DMG) H3K27 alteration is the most difficult grade 4 glioma, and this tumor type is now divided into four subtypes. However, h3The 3-K27M subgroup remains clinically and molecularly heterogeneous. Studies have shown that rare H3Patients with 3K27M and alterations in BRAF or FGFR1 tend to have a better prognosis. Single cases or small cohort samples with changes in the H3-K27M and MAPK pathways may have longer survival compared with patients with DMG H3K27 alterations, BRAF, and FGFR1 wild-type. To understand how these alterations improve tumor outcomes, the investigators analyzed cohorts of cases with larger DMG H3K27 alterations (imaging, histology, genomics, transcriptomics, and DNA methylation analysis), including 29 tumors containing BRAF V600E or FGFR1mut.
Findings:
1.The genomic landscape of DMG H3K27 changes with BRAF FGFR1
Clinical and molecular characteristics of a cohort of patients with 1DMG H3-K27M combined with BRAF and FGFR1 mutations. Overview of clinical and molecular annotations in 60 children with BRAF or FGFR1 mutations and **DMG H3-K27 patients. Cases are represented in columns, and gene status is expressed in rows. Age is measured in years. With the exception of immunohistological data, including histological analysis, EZHip, and ATRX expression, all molecular information is derived from DNA or RNA sequencing analysis. Patients who are still alive at the last follow-up are indicated by a square of oblique cuts.
The investigators analyzed a cohort of H3K27-altered gliomas containing BRAF (n=22), FGFR (n=37), or mutations containing both (n=1). Of these, 43 were children (< 18 years old) and 16 were **. At the same time, control group cases of Brafwt FGFRWT DMG H3-K27 patients from the corresponding cohort were analyzed. All DMG H3-K27M with BRAF alterations carry the somatic V600E mutation but without fusion (Figure 1). In terms of FGFR, all cases showed FGFR1 hotspot substitution. N546K D (73%, 27 37) and K656E M (23%, 11 37) preferentially occur in CNS tumors, but there are neither FGFR1 fusion duplications nor FGFR23 alterations (Figure 1).
Brafmut or FGFR1mut is mainly associated with H33-K27M mutation is associated with H3The 1-K27M mutation is not related. BRAF and FGFR1 mutations are mutually exclusive because only one tumor has both outcomes (case 23;).Figure 1). In 90% (9 10) of FGFR1mut cases, FGFR1 mutations were cloned with H3-3A mutations with similar variant allele frequencies, suggesting a role in these early steps of tumorigenesis.
Fewer cases occur in patients with DMG H3-K27M BRAFV600E, with only 33% (2, 6) of tumors having BRAF mutation cloning to H3-K27M mutation. In addition, H3-K27 FGFR1mut tumors often present other mutations in the MAPK pathway**, NF1 (13, 31;32%) or PTPN11 (3-22;13.6%) is the most common mutated gene (Figure 1). Among BRAFMUT and FGFR1mut tumors, there are 5% (1, 20) and 9TP53 mutations were found in 3% (3 32) (Figure 1).
In addition, ATRX is at 59Mutations or loss of IHC expression occurred in 3% (16, 27) of FGFR1mut DMGs. In 19% of FGFR1mut tumors of H3-K27M (4 21), mutations are present in members of the PI3K AKT MTOR signaling pathway (Figure 1).
brafmutfgfr1mutH3K27-altered tumors are histologically heterogeneous, often with mixed diffuse-localized manifestations and calcifications
Figure 2 has histopathological features of DMG H3-K27 with BRAF or FGFR1 mutations. Case 14aGlial neuronal proliferation with ganglion cells, eosinophil granules, and microcalcifications. Case 11bProliferation of glial neurons with a large number of ganglion cells. Case 13·cGlial neuronal proliferation with massive ganglion cell and lymphocyte swelling. Case 31·dIt is mainly localized. Case 32·eIt is mainly localized hyperplasia with a fusion component around the tumorfgSolubilize chromogranin immunoreactive staining of neuronal cells. hExpression of BRAF V600E in all tumor cells, including ganglion cells. iExpression of H3K27M in all tumor cells including ganglion cells. Case 7·jGlial hyperplasia with hypocalcification-like and microcalcification. Case 11kThe overall absence of h3k27me3. lDeletion of ATRX in tumor cells. mGFAP vortex (20). nGFAP vortex (400),oGFAP antibody staining.
Next, the investigators analyzed the histopathological characteristics of the cases in their cohort. The 29 tumors were initially diagnosed with DMG H3K27 (n=13), ganglioglioma grade 1 (n=5), anaplastic ganglioglioma grade 3 (n=4), or other gliomas (GBM n=3;).Pilocytic astrocytoma n=1;LGG n=1 or oligoastrocytoma grade 3, n=2). All Brafmut FGFR1mut DMGs showed loss of H3K27 trimethylation as expected and positive H3K27M staining, with the exception of cases of Ezhip overexpression.
Three main histological patterns were observed:413% GG sample (12-29), 344% DMG-like (10-29), 243% HGG (Piloid-HGG) with hairy astrocyte components (7, 29). Brafmut and FGFR1mut tumors exhibited typical DMG histological features in only 25% (4 16) and 46% (6 13) of cases, respectively. In BRAFMUT or FGFR1mut cases, 50% (8, 16) and 30% (4, 13) are characterized by mixed neuronal and glial tumor cells (Figure 2B-I). 25% (4 16) of BraFMUT and 23% (3 13) of FGFR1mut DMG exhibited a piloid-hgg morphology with hairy cell morphology (Figure 2J,K).
The remaining FGFR1mut DMG cases (3-13) contain a mix of DMG-like and GG-like or piloid-hgg, GG-like and piloid-hgg (Figure 2L). Although rare in classical DMG, microcalcifications were observed in 56% (36) and 38% (5 13) DMGs (Figure 2J). GFAP vortex forms a small nodule that represents a rare but exceptional case (n=4) (Figure 2M-O).
Figure 3 DMG H3-K27M BRAF V600E orfgfr1mutImaging specificity. Depending on BRAF or FGFR1 mutation status, AT2-FLAIR MR image sequence, or CT. b Compare tumor imaging manifestations (confluent, localized, or mixed) of DMG according to their genotype. Compare the presence of large calcifications on DMG CT scans based on the presence of MAPK alterations.
On imaging, DMG H3-K27 BRAFMUT or FGFR1mut were mostly diffuse, but the mixed nodular type was significantly enriched compared with the control DMG H3-K27 (Fig. 3A-B). Few tumors are completely localized (Fig. 3a, b), and imaging analysis at the time of progression reveals half of the diffuse evolutionary patterns (2, 4).
They showed more calcifications than DMG H3-K27 (Figure 3A,C). Overall, in histological and imaging analysis, DMG H3K27M BRAFMUT or FGFR1mut were 8 11 (72.)7%) and 6 9 (667%).
3.DNA methylation analysis distinguishes a subset of DMG H3K27 altered by MAPK activation mutations
Figure 4 DMG H33-k27m brafmut/fgfr1mutDNA methylation profiling. Classification of tumors of the central nervous system based on DNA methylation analysis.
Analyzing the DNA methylation profile of the entire cohort, 54% (7 13) of BRAFMUT and 67% (10 15) of FGFRMUT tumors were classified as DMG K27, and the rest corresponded to other categories or unclassified (score < 0.).9）。
4.BRAF and FGFR1 mutation status are prognostic factors in children and **DMG H3K27 altered form
Figure 5 DMG H3-K27M BRAFmut/fgfr1mutand clinical characteristics of patients with transcriptomic tumor features. aCompare OS according to histone H3, BRAF, FGFR1, and TP53 mutation status. bAge distribution at diagnosis based on BRAF and FGFR1 mutation status. cTumor locations are compared based on mutation status of BRAF and FGFR1. dGSA plot showing DMG H3Co-transcriptome characterization of 3-K27M.
The TP53 mutation and the BRAFV600E FGFR1 mutation are mostly mutually exclusive in DMG. In order to avoid TP53 mutations on H3 without MAPK activation changes3-K27M DMG results were confounded, and patients with histone H3 and TP53 genotypes were stratified in the Kaplan-Meier overall survival (OS) assay. with DMG H33-K27M TP53WT was significantly better OS for children with BRAF-activating (median OS 37 months) or FGFR1 mutation (median OS 36 months) and patients with DMG H3-K27 compared with other DMG subtypes (median OS 12 months) (Figure 5A).
Table 1 Multivariate Cox proportional hazards regression model for OS in patients with H3-K27M DMG
Multivariate analysis showed that histone H3 and TP53 status were significantly correlated with OS (Table 1). Age was significantly associated with prognosis, but its overall effect was limited compared to other variables. Braf V600E and FGFR1mut are strong and independent prognostic markers in H3K27-altered DMG (Table 1).
5.Clinical differences in children and ** patients with DMG H3K27M BRAF FGFR1 mutations
In DMGs with MAPK activating mutations, the ratio of males and females is equal. At the time of diagnosis, h3There was a significant age difference between 3-K27M DMG, BRAFMUT, and FGFR1mut DMG (Figure 5B). In contrast, patients with FGFR1mut had a significantly higher age at diagnosis than those with FGFR1WT, with a median age of 148 years and 98 years (Figure 5b). with DMG H3DMGs with BRAF and FGFR1 mutations are significantly more common in the thalamus compared to 3-K27M (Figure 5C).
6.During tumorigenesis, h3The 3K27M mutation precedes BRAF V600E
Next, we wanted to know if the sequences of the mutations in these tumors were the same, and more importantly whether they corresponded to (i) DMG (H3-K27M as the "first hit") or (ii) atypical aggressive low-grade gliomas (BRAFV600E as the "first hit"). The investigators used a digital droplet PCR on a BRAFV600E H33-K27m tumor (case 2) and one H3Genomic DNA cloned from 3-K27M BRAFWT was analyzed for BRAF copy number variation (CNV). The BraFWT clone has two BRAF alleles and is therefore composed of H3 only3-K27M was caused by re-expansion of ancestral clones rather than by genetic loss of the Braf v600e allele, suggesting that in 1 case of DMG H33-K27M BRAF V600E, H33-K27m is the first mutation.
7.DMG H3 with MAPK alterations3K27M shows transcriptomic characterization of senescence and is upregulatedcdkn1a（p21）
h3.3K27M BrafwtDMG with H33-K27M BRAFV600E DMG ratio, 676 significantly up-regulated genes and 633 down-regulated genes, H33-K27M FGFR1WTDMG with H33-K27M FGFR1mut DMG ratio, 228 up-regulated genes and 274 down-regulated genes. The 94 up-regulated genes and 111 down-regulated genes were common to the two comparisons. Enrichment of MAPK signal and PI3K AKT MTOR signal tags in Brafmut and FGFR1mut DMG (Figure 5D) as well as angiogenesis and hypoxia tag enrichment. In addition, with only h3The CDKN1A gene, which encodes the aging marker P21, was overexpressed in Brafmut and FGFR1mut gliomas compared to patients with the 3-K27M mutation.
Discuss
The data from this study support another novel subtype of DMG with unique histological, radiological, clinical, genomic, transcriptomic, and epigenetic signatures, which we tentatively refer to as DMG, with co-alterations of H3 K27 and BRAF FGFR1 (DMG K27-BRAF FGFR1), which may represent 20% of the DMG H3 K27 variant. DMG K27-BRAF FGFR1 may have distinct cells** capable of exhibiting mixed differentiation of glia and neurons, most pronounced in the BRAF subclass. Genotype-morphotype correlation supports the distinction between classical DMG, H3K27 alterations, and glial neuronal tumor MAPK-alterations. Several other clinical and biological features support the new classification from classical DMG, H3K27-altered tumors. First of all, the OS is significantly different from the classic DMG K27. In addition, multivariate analyses have shown for the first time that the presence of these mutations is an independent prognostic factor for OS improvement in DMG K27. A cohort of BRAF FGFR1-mutated DMG K27 received a prolonged period of considerable heterogeneity** with no specific statistical conclusions. Therefore, it remains to be determined whether these patients will respond to targeting BAFV600E or FGFR1.
Another meaningful difference is that from H3DMG K27-BRAF FGFR1 is more common in the thalamus than in the brainstem compared to DMG of the 3-K27M subtype. The age at the time of diagnosis also varies depending on the presence of MAPK alterations. The researchers also observed a feature of aging. Notably, DMG K27-BRAF FGFR1 shares other features in common with LGGs, such as preferential association of calcifications and mutations. h3.3K27M is the first mutational event in tumorigenesis, and BRAF and FGFR1 mutations will be secondary driver events for tumorigenesis and may be H33-K27M ancestral clones provide proliferative advantages. Expanding the analysis of mutational sequence acquisition in DMG K27-MAPK tumorigenesis is essential for the design of future ** interventions.
References: Auffret L, Ajlil Y, Tauziède-Espariat A, et al a new subtype of diffuse midline glioma, h3 k27 and braf/fgfr1 co-altered: a clinico-radiological and histomolecular characterisation. acta neuropathol. 2023;147(1):2. published 2023 dec 8. doi:10.1007/s00401-023-02651-4
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Written by: Zhao Chuan.
Reviewer: Zhang Junping, Zhao Chuan.
Typesetting: Zhang Yaqi.