Mutational analysis reveals the origin and therapy-driven evolution of recurrent glioma

Abstract

Tumor recurrence is a leading cause of cancer mortality. Therapies for recurrent disease may fail, at least in part, because the genomic alterations driving the growth of recurrences are distinct from those in the initial tumor. To explore this hypothesis, we sequenced the exomes of 23 initial low-grade gliomas and recurrent tumors resected from the same patients. In 43% of cases, at least half of the mutations in the initial tumor were undetected at recurrence, including driver mutations in TP53, ATRX, SMARCA4, and BRAF; this suggests that recurrent tumors are often seeded by cells derived from the initial tumor at a very early stage of their evolution. Notably, tumors from 6 of 10 patients treated with the chemotherapeutic drug temozolomide (TMZ) followed an alternative evolutionary path to high-grade glioma. At recurrence, these tumors were hypermutated and harbored driver mutations in the RB (retinoblastoma) and Akt-mTOR (mammalian target of rapamycin) pathways that bore the signature of TMZ-induced mutagenesis.

Fig. 1. Genetic landscapes of low-grade gliomas and their patient-matched recurrences

(A) Total number of mutations private to or shared between the initial and first recurrent glioma of 23 patients. (B to D) Shared and private somatic mutations in paired initial and recurrent tumors (x and y axes respectively) as a function of the estimated fraction of tumor cells carrying the mutant allele. Mutations present in all the cells in both tumors are represented by a single point whose radius is scaled by the log count of such mutations. Shared and private CNAs are indicated (red and blue are gains and losses respectively, white is copy-neutral). In panel C, clonal TP53 and ATRX mutations in the initial tumor were not identified in the recurrent tumor, but different clonal mutations in these two genes were acquired. (D) Inset shows the DNA sequence encompassing BRAF V600E in the normal tissue and in 15 geographically distinct samples of the initial and recurrent tumors.

Fig. 2. The temporal and spatial patterns of clonal evolution in the tumors of two glioma patients

(A and B). A timeline of treatment histories for patient 17 (A) and patient 04 (B) (top, intervals labeled in months). Vertical bars correspond to the time of tumor resection and are labeled with the tumor diagnosis and grade. Representative MRIs are also shown. A phylogenetic tree (bottom) depicts the patterns of clonal evolution of these tumors inferred from the pattern and frequency of somatic mutations, highlighting genes frequently mutated in cancer.

Fig. 3. Recurrent tumors from patients treated with TMZ harbor genetic alterations in the RB and AKT-mTOR signaling pathways

(A) The number of TMZ-associated mutations and other mutations identified in the six patients with hypermutated recurrent tumors. (B) Somatic mutations and CNAs acquired upon recurrence in key genes of pathways associated with GBM. (C) Expression level of RB1 at each exon and exon-exon junction in the initial and recurrent tumor of patient 01 showing aberrant splicing of the RB1 transcript in the recurrent tumor harboring the RB1 c.2520+1G>A splice-site mutation. The RB1 exon and exon junctions with significant differential usage (red) and the location of the splice-site mutation are shown. (D) Gene set enrichment analysis shows significant enrichment of genes down-regulated by RB1 and up-regulated by E2F in the recurrent tumors of patients 01 (blue) and 10 (green), coincident with the acquisition of TMZ-associated mutations in the RB pathway. (E) Hematoxylin and eosin (H&E)-stained tumor sample from the first recurrent tumor of patient 01. A dotted line separates the two morphologically distinct regions. IHC for phospho-RPS6, phospho-4E-BP1 and Ki-67 show differential activation of mTORC1 targets and proliferation rates in the two adjacent regions. Bars represent 100 microns.