Multi-omics analysis of adamantinomatous craniopharyngiomas reveals distinct molecular subgroups with prognostic and treatment response significance

Abstract

Background: Adamantinomatous craniopharyngioma (ACP) is the commonest pediatric sellar tumor. No effective drug is available and interpatient heterogeneity is prominent. This study aimed to identify distinct molecular subgroups of ACP based on the multi-omics profiles, imaging findings, and histological features, in order to predict the response to anti-inflammatory treatment and immunotherapies.

Methods: Totally 142 Chinese cases diagnosed with craniopharyngiomas were profiled, including 119 ACPs and 23 papillary craniopharyngiomas. Whole-exome sequencing (151 tumors, including recurrent ones), RNA sequencing (84 tumors), and DNA methylome profiling (95 tumors) were performed. Consensus clustering and non-negative matrix factorization were used for subgrouping, and Cox regression were utilized for prognostic evaluation, respectively.

Results: Three distinct molecular subgroups were identified: WNT, ImA, and ImB. The WNT subgroup showed higher Wnt/β-catenin pathway activity, with a greater number of epithelial cells and more predominantly solid tumors. The ImA and ImB subgroups had activated inflammatory and interferon response pathways, with enhanced immune cell infiltration and more predominantly cystic tumors. Mitogen-activated protein kinases (MEK/MAPK) signaling was activated only in ImA samples, while IL-6 and epithelial-mesenchymal transition biomarkers were highly expressed in the ImB group, mostly consisting of children. The degree of astrogliosis was significantly elevated in the ImA group, with severe finger-like protrusions at the invasive front of the tumor. The molecular subgrouping was an independent prognostic factor, with the WNT group having longer event-free survival than ImB (Cox, P = 0.04). ImA/ImB cases were more likely to respond to immune checkpoint blockade (ICB) therapy than the WNT group ( P <0.01). In the preliminary screening of subtyping markers, CD38 was significantly downregulated in WNT compared with ImA and ImB ( P = 0.01).

Conclusions: ACP comprises three molecular subtypes with distinct imaging and histological features. The prognosis of the WNT type is better than that of the ImB group, which is more likely to benefit from the ICB treatment.

Figure 1

ACPs and PCPs are driven by CTNNB1 and BRAF somatic mutations, respectively. (A) Mutation status and VAFs of CTNNB1 and BRAF in all WES-profiled tumors (122 ACPs and 26 PCPs, from 119 ACP patients and 22 PCP patients, respectively) and copy number variation results. The "mutation status" indicates the final result through all means and the "GATK pipeline" indicates the original Mutect2 result. The samples enclosed by the red square were "wildtype" in the GATK pipeline but found to harbor CTNNB1 mutations by RNAseq; the samples inside the black square were "wildtype" in both the initial WES and RNAseq data and subject to a second round of WES with newly cut FFPE specimens. Somatic copy number variants include both the results of WES and DNAm profiles (EPIC). (B) Distribution of mutant amino acids in CTNNB1 variants. The color indicates the property of the side chain residue. (C) VAFs of CTNNB1 point mutations in RNAseq correlated with WES data (Spearman R = 0.40, P <0.01). ACPs: Adamantinomatous craniopharyngiomas; FFPE: Formalin-fixed paraffin-embedded; PCP: Papillary craniopharyngioma; RNAseq: RNA-sequencing; VAF: Variant allele frequencies; WES: Whole exome sequencing; DNAm: DNA methylome.

Figure 2

RNAseq and DNAm profiles revealed that ACPs comprise three distinct molecular subgroups. (A) Dimensionality reduction plots with t-SNE of RNAseq profiles; (B) The consenus matrix heatmap in the M3C analysis of RNAseq; (C) Dimensionality reduction plot with t-SNE of DNAm profles. (D) VAFs of CTNNB1 mutations are significantly elevated in the WNT subgroup compared with the immune subgroups (ImA and ImB), which also consist of wildtype tumors (Kruskal–Wallis χ² = 36.8, P <0.01, n = 24, 15 and 32 for the WNT, ImA, and ImB subgroups, respectively). (E) xCell immune scores estimated from RNAseq are strongly correlated with the proportions of immune cells inferred from DNAm analysis (Spearman R = 0.57, P <0.01). (F) Proteomic profiles showed that CD38 is expressed in both the ImA and ImB subgroups, but not in the WNT subgroup (Kruskal–Wallis χ² = 9.19, P = 0.01); CD14 expression was elevated in the ImB subgroup (Kruskal–Wallis χ² = 7.81, P = 0.02, n = 6, 6, and 4 for the WNT, ImA, and ImB subgroups, respectively). (G) Heatmap of signature genes in various subgroups, xCell scores for epithelial, immune, and stromal cells, and single-sample GSEA scores for representative tumor hallmark pathways. Samples in each subgroup are sorted in a decreasing order of VAF of CTNNB1 mutations. The colors in A, C, D, E, F follow the same convention and they are indicated in the legend of G. ACPs: Adamantinomatous craniopharyngiomas; CD14: cluster of differentiation 14; CD38: cluster of differentiation 38; t-SNE: t-distributed stochastic neighbor embedding; VAFs: Variant allele frequencies; DNAm: DNA methylome.

Figure 3

ACP subgroups show distinct histological and imaging characteristics. (A) Typical IHC staining of β-catenin in the three ACP subgroups (arrow heads, whorl-like cell cluster; scale bar = 50 μm). (B) Typical H&E images demonstrate immune infiltration and gliosis characteristics in the ACP subgroups (left for immune infiltration, scale bar = 20 μm; right for gliosis, scale bar = 50 μm). The degree of immune infiltration was significantly more severe in the ImA and ImB subgroups than in the WNT subgroup (Kruskal–Wallis χ² = 7.90, P = 0.02, Supplementary Table 11, http://links.lww.com/CM9/B635). Tumors with a significant amount of multinucleated giant cells (indicated by arrows in ImB) were mainly detected in the ImB subgroup. Gliosis (indicated by arrows in the WNT and ImA subgroups) was more pronounced in the ImA subgroup. (C) Typical IHC staining images for CD3 and CD38 detection, respectively (scale bar = 50 μm). Additional IHC images for other markers are depicted in Supplementary Figure 14, http://links.lww.com/CM9/B629. (D) Both the proportions of cells with β-catenin nuclear accumulation (ImB vs. WNT, Welch t = 2.38, P = 0.02) and numbers of whorl-like clusters (ImB vs. WNT, Welch t = 3.68, P <0.01), indicated by arrows in (A) were significantly lower in ImB (n = 23, 15, and 32 for the WNT, ImA, and ImB subgroups, respectively). ImA samples had higher infiltration rates of CD3+ cells (Kruskal–Wallis χ² = 2.66, P = 0.26), and ImA and ImB had more CD38+ cells infiltrated (Kruskal–Wallis χ² = 6.21, P = 0.04, n = 5 for each subgroup). (E) Enrichment with xCell showed that macrophages were enriched in ImA and ImB samples (Kruskal–Wallis χ² = 47.06, P <0.01), while NKT cells were enriched in ImA samples only (Kruskal–Wallis χ² = 44.92, P <0.01, n = 24, 15, 32, and 9 for WNT, ImA, ImB, and PCP, respectively). (F) Typical CT and MRI images for predominantly solid and predominantly cystic ACPs, respectively. (G) The WNT subgroup consisted of more predominantly solid tumors, while the other two subgroups consisted of more predominantly cystic tumors (Fisher's exact test, P <0.01). (H) Receiver operating characteristic curve analysis to classify 35 ACPs based on the proportion of non-tumor area in H&E images (area under the receiver operating curve, AUC = 0.866). ACP: Adamantinomatous craniopharyngioma; CD3: cluster of differentiation 3; CD38: cluster of differentiation 38; CT: computed tomography; H&E: Hematoxylin and eosin; IHC: Immunohistochemical; Immune inf.: immune infiltration; NKT: Natural killer T cell; PCP: Papillary craniopharyngioma.

Figure 4

Integrated correlation analysis of DNAm, RNAseq, and proteomics profiles. (A) Distribution of Spearman correlation coefficients between mRNA expression and beta values for the corresponding promoter CpG sites and between mRNA and protein expression levels. Genes with a coefficient below -0.50 in the former or above 0.55 in the latter were considered strongly correlated genes. (B) These two sets shared 19 common genes. (C) Most of the common genes showed distinct expression or methylation pattern among ACP subgroups; the mRNA expression levels of nine genes and the beta values of the corresponding probes are plotted in the heatmap. (D) Detailed RNA-seq transcription and DNA methylation data are shown for SMAD3, an important regulator of β-catenin signaling. (E) Plot of the normalized protein level of SMAD3 versus its transcription level measured by RNAseq. Red lines are the best linear fits, and the gray shade shows 95% confidence interval. ACP: Adamantinomatous craniopharyngioma; DNAm: DNA methylome; RNAseq: RNA-sequencing; SMAD3: SMAD family member 3.

Figure 5

Clinical relevance of ACP subgrouping. (A,B) Comparison of EFS curves among three ACP subgroups and the degree of resection. The WNT subgroup had a better prognosis compared with the ImB subgroup (log-rank P = 0.10). Patients with complete resection had a significantly longer EFS (log-rank P <0.01). Additional survival curves are shown in Supplementary Figure 15, http://links.lww.com/CM9/B629 (C) Multivariate Cox regression found that ImB had a significantly higher recurrence hazard ratio than WNT as an independent prognostic factor other than the degree of resection. (D) Frequencies of predicted responders and non-responders to ICB therapy in ACP subgroups and PCP. (E) Enrichment analysis by ssGSEA showed that WNT samples had significantly lower scores for INFα response than other subgroups (Kruskal–Wallis χ² = 35.78, P <0.01, n = 24, 15, 32, and 9 for WNT, ImA, ImB, and PCP, respectively). (F) SOX10 expression was higher in the ImA group compared with the other subgroups (Kruskal–Wallis χ² = 39.73, P <0.01), same sample sizes as in (E). ACP: Adamantinomatous craniopharyngioma; CR: Complete resection; EFS: Event-free survival; ICB: Immune checkpoint blockade; IR: Incomplete resection; PCP: Papillary craniopharyngioma; IFNα: interferon alpha; SOX10: sex determining region Y-box 10.