Glioblastoma (GBM) is a prototypical heterogeneous brain tumor refractory to conventional therapy. A small residual population of cells escapes surgery and chemoradiation, resulting in a typically fatal tumor recurrence ∼7 mo after diagnosis. Understanding the molecular architecture of this residual population is critical for the development of successful therapies. We used whole-genome sequencing and whole-exome sequencing of multiple sectors from primary and paired recurrent GBM tumors to reconstruct the genomic profile of residual, therapy resistant tumor initiating cells. We found that genetic alteration of the p53 pathway is a primary molecular event predictive of a high number of subclonal mutations in glioblastoma. The genomic road leading to recurrence is highly idiosyncratic but can be broadly classified into linear recurrences that share extensive genetic similarity with the primary tumor and can be directly traced to one of its specific sectors, and divergent recurrences that share few genetic alterations with the primary tumor and originate from cells that branched off early during tumorigenesis. Our study provides mechanistic insights into how genetic alterations in primary tumors impact the ensuing evolution of tumor cells and the emergence of subclonal heterogeneity.

Cutis laxa is a rare group of inherited and acquired disorders characterized by loose and redundant skin with reduced elasticity. Mutations in the elastin coding gene have been shown to cause autosomal dominant cutis laxa in three families. A homozygous mutation in the fibulin-5 coding gene was discovered in a Turkish pedigree showing recessive inheritance, and a different mutation in this gene was found in the heterozygous state in a sporadic case of the disease. Here, we report the third case of a mutation in the fibulin-5 coding gene in a recessive Iranian cutis laxa pedigree. The mutation is the same as previously reported in the Turkish pedigree, further confirming that it is causative of disease. A haplotype consisting of seven intragenic sequence variations common to both pedigrees is described for the mutation-carrying fibulin-5 allele.

Although mutations in TACSTD2 among Iranian patients with GDLD were heterogeneous, E227K was found to be a common mutation. It is suggested that E227K may be a founder mutation in this population. Based on positions of known mutations in TACSTD2, significance of the thyroglobulin domain of the TACSTD2 protein in the pathogenesis of GDLD is suggested.

To evaluate evolutionary patterns in progression and therapy-resistance of GBM, we analyzed the genomic profiles of 252 GBM samples from The Cancer Genome Atlas (TCGA)1, including 48 multi-sector and recurrent tumor biopsies taken from 17 pairs of pre- and post-treatment GBMs, to understand 1) the intratumoral heterogeneity of GBM and 2) how GBM responds to therapeutic intervention. We integrated variant allele fraction, DNA copy number and genotype information to determine clonality of all mutations and found that 69.5% of mutations (median across samples 70.1% ± 19.6%) were classified as clonal and 30.5% as subclonal. To verify our classification approach, we classified mutations detected in two non-overlapping biopsies from 11 tumors into clonal and subclonal categories. Of mutations detected in both tumor sectors 86.2% were classified as clonal and 45.2% of sample-private mutations were categorized as clonal, which was a strongly significant difference (P = 1.8×10-87). Separating patients into discrete age groups by an interval of 10 years, we found a significant linear correlation between clonal mutations and age (P < 0.001). This observation supports the notion that clonal mutations predominantly accumulated over the life span of the cell population that gave rise to the cell of origin before neoplastic onset2. No correlation with age was found for subclonal mutations. In contrast, the frequency of subclonal mutations was strongly correlated with the presence of alterations related to the p53 pathway (P < 0.001), particularly TP53 point mutation or amplification of MDM2. This observation raises the possibility that p53 pathway alterations stimulate subclonal evolution, possibly by providing greater tolerance to DNA damage and/or suppression of apoptosis3. To evaluate the effects of clonal heterogeneity on disease recurrence, we analyzed matched pairs of primary and recurrent GBM, including five pairs with mutated TP53 and nine pairs with wildtype TP53. Recurrent TP53 mutant GBM showed a further and significant increase in the subclonal mutation frequency. In contrast, TP53 wildtype tumors showed an increase in the frequency of clonal mutations compared to their matched primary tumor. These data suggest that TP53 mutant GBM became increasingly clonally complex at time of recurrence, whereas TP53 wildtype GBM showed a reduced level of intratumoral heterogeneity. We observed an ultramutator phenotype in three recurrent tumors, associated with temozolomide treatment. Our results suggest that mutations in the p53 pathway affect the response to therapy by supporting greater intratumoral heterogeneity. Paradoxically we observe a trend towards improved event free survival in samples with high subclonal mutation frequency. Further research is needed to assess whether the level of intratumoral heterogeneity is a reflection of the molecular portrait of GBM progression. Citation Format: Hoon Kim, Siyuan Zheng, Seyed S. Amini, Selene M. Virk, Tom Mikkelsen, Daniel J. Brat, Jonna Grimsby, Carrie Sougnez, Andrew E. Sloan, Mark L. Cohen, Erwin G. Van Meir, Lisa Scarpace, The Cancer Genome Research Network, Peter W. Laird, John N. Weinstein, Eric Lander, Stacey Gabriel, Gad Getz, Matthew Meyerson, Lynda Chin, Jill S. Barnholtz-Sloan, Roel G.w. Verhaak. Alteration of the p53 pathway is associated with subclonal tumor progression in glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 984. doi:10.1158/1538-7445.AM2014-984

To investigate therapy induced evolutionary patterns in glioblastoma (GBM), we completed whole exome sequencing of all samples, as well as transcriptome sequencing, DNA copy number, DNA methylation, and mRNA from at least one primary sample, and one (post-therapy) recurrent sample. Sample collection was enabled through The Cancer Genome Atlas Research Network (TCGA). Multiple spatially distinct samples were available for five primary and six recurrent cases, allowing the integration of intratumoral heterogeneity with the evolutionary processes that shape the tumor recurrence after therapeutic intervention. Sample unique mutations were common, but most of the somatic variants in driver genes (like TP53 and EGFR) were preserved across the tumor and after therapy. We found that the therapeutic bottleneck resulted in proportionally higher numbers of (mostly clonal) mutations, reduced clonal complexity and changes in the mutation spectrum. GBM recurrence could be grouped into two categories, characterized by drastically different clonal evolution patterns. Our findings suggest that multi-sector sequencing sharpens the analysis but is not essential for precise delineation of the tumor progression process. Projecting our results on genomic data from 253 primary GBMs, we observed that the number of clonal, but not subclonal, mutations increased with age at diagnosis, and the two categories differed in their mutation spectrum. This study represents an important advance toward comprehensive characterization of the genomic alterations of GBM before and after cytotoxic treatment and surgery. Our analysis of spatially and temporally distinct samples provides new insights into the relevance of intratumoral heterogeneity on disease progression in GBM. Acknowledgements: This work is supported by Award Numbers 5 P50 CA127001 and 5 P50 CA083639-12 from the National Cancer Institute (NCI) to RGWV. H.K. is supported in part by the Odyssey Program and Theodore N. Law Endowment for Scientific Achievement at The University of Texas MD Anderson Cancer Center. The results published here are in whole or part based upon data generated by The Cancer Genome Atlas project established by the NCI and NHGRI. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B130. Citation Format: Hoon Kim, Siyuan Zheng, Seyed S. Amini, Selene Virk, Tom Mikkelsen, Daniel J. Brat, Jonna Grimsby, Carrie Sougnez, Andrew E. Sloan, Mark L. Cohen, Erwin G. Van Meir, Lisa Scarpace, Peter W. Laird, John N. Weinstein, Eric S. Lander, Stacey Gabriel, Gaddy Getz, Matthew Meyerson, Lynda Chin, Jill S. Barnholtz-Sloan, Roeland GW Verhaak. The intratumoral heterogeneity of glioblastoma suggests a pivotal role for clonal evolution. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B130.

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