Treatment-associated TP53 DNA-binding domain missense mutations in the pathogenesis of secondary gliosarcoma

Abstract

Background: Gliosarcoma is a rare variant of glioblastoma (GBM) that exhibits frequent mutations in TP53 and can develop in a secondary fashion after chemoradiation of a primary GBM. Whether temozolomide (TMZ)-induced mutagenesis of the TP53 DNA-binding domain (DBD) can drive the pathogenesis of gliosarcoma is unclear.

Methods: We identified a case of a primary GBM that rapidly progressed into secondary gliosarcoma shortly after chemoradiation was initiated. Bulk tumor was collected and gliomasphere cultures derived from both the pre- and post-treatment tumors. We performed targeted DNA sequencing and transcriptome analyses of the specimens to understand their phylogenetic relationship and identify differentially expressed gene pathways. Gliomaspheres from the primary GBM were treated with TMZ and then analyzed to compare patterns of mutagenesis in vivo and ex vivo.

Results: The pre- and post-treatment tumors shared EGFR, CDKN2A, and PTEN mutations, but only the secondary gliosarcoma exhibited TP53 DBD missense mutations. Two mutations, R110C, and R175H, were identified, each in distinct clones. Both were base transitions characteristic of TMZ mutagenesis. Gene expression analysis identified increased JAK-STAT signaling in the gliosarcoma, together with reduced expression of microRNAs known to regulate epithelial-mesenchymal transition. Ex vivo treatment of the GBM spheres with TMZ generated numerous variants in cancer driver genes, including TP53 and CDH1, which were mutated in the post-treatment tumor.

Conclusions: TMZ-induced TP53 gain-of-function mutations can have a driving role in secondary gliosarcoma pathogenesis. Analysis of variants identified in ex vivo TMZ-treated gliomaspheres may have utility in predicting GBM evolutionary trajectories in vivo during standard chemoradiation.

Keywords: TP53 mutation; glioblastoma; gliosarcoma; mutagenesis; temozolomide.

Figure 1. Radiographic and immunohistochemical characterization of GBM1 and SGS1

(A) Preoperative and serial postoperative axial T1 contrast-enhanced brain MRIs. (B) Stains of GBM1 demonstrating astrocytic morphology of tumor cells on H&E, p53 positive staining of 5% of tumor cells, amplification of EGFR on in situ hybridization (ISH), and positive staining for the astrocytic marker GFAP. (C) Stains of SGS1 demonstrating spindle cell morphology on H&E, p53 positive staining of 30% of tumor cells, lack of EGFR amplification on ISH, and positive reticulin staining of sarcoma-like tumor regions. Scale bars 100μm.

Figure 2. Targeted DNA sequencing reveals relationship between GBM1 and SGS1

(A) SNVs common and private to each of the bulk specimens and derived cultures obtained from GBM1 and SGS1 were used to construct a phylogenetic tree. GBM1 specimens are denoted in red and SGS1 specimens are denoted in blue. Dashed boxes indicate cultured specimens from bulk tissues, which are denoted by solid boxes. (B) Sanger chromatograms of five single cell-derived colonies from SGS1 sector B sequenced at TP53 exons 4 (left) and 5 (right). Colonies 9, 15 and 21 exhibit the R175H (CGC>CAC) but not the R110C (CCG>CTG) mutation.

Figure 3. Growth rate and chemoresistance of GBM1 and SGS1 gliomaspheres

(A) Gliomasphere cultures were serially passaged and cumulative PDL was plotted against time. (B) Phase contrast micrographs at 4x (top) and 10x (bottom) magnification. (C) Neurofilament expression by immunofluorescence (20x). Normalized cell viability (D) and interpolated TMZ IC50 values (E) from three independent experiments comparing GBM1 and SGS1 gliomaspheres. Expression of γH2AX and β-actin control in gliomaspheres by immunoblot at the indicated number of hours after exposure to TMZ 10 μg/ml for 24 hours (F) or 2 Gy ionizing radiation (G). (H) Immunoblot for MGMT expression in gliomaspheres. A=cultured normal human astrocytes, G1=GBM1, S1=SGS1, S2=SGS2.

Figure 4. Comparative transcriptome analysis

(A) GBM molecular subtype gene set enrichment scores on bulk tumor and gliomasphere samples. B=bulk, S=gliomasphere. (B) Volcano plot indicating significance and fold-change of expression level of 84 transcription factors in two gliosarcomas compared to GBM1. Y-axis is -log (p-value). (C) IC50 values of temozolomide and ruxolitinib for the gliomasphere lines indicated (p-value<0.0001 for interaction of drug and gliomasphere line by 2-way ANOVA). Each bar represents a biological replicate. Error bars are standard error.