Whole-genome and multisector exome sequencing of primary and post-treatment glioblastoma reveals patterns of tumor evolution

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

Probing the <it>Xenopus laevis</it> inner ear transcriptome for biological function

<p><b>Abstract</b></p> <p><b>Background</b></p> <p>The senses of hearing and balance depend upon mechanoreception, a process that originates in the inner ear and shares features across species. Amphibians have been widely used for physiological studies of mechanotransduction by sensory hair cells. In contrast, much less is known of the genetic basis of auditory and vestibular function in this class of animals. Among amphibians, the genus <it>Xenopus</it> is a well-characterized genetic and developmental model that offers unique opportunities for inner ear research because of the amphibian capacity for tissue and organ regeneration. For these reasons, we implemented a functional genomics approach as a means to undertake a large-scale analysis of the <it>Xenopus laevis</it> inner ear transcriptome through microarray analysis.</p> <p><b>Results</b></p> <p>Microarray analysis uncovered genes within the <it>X. laevis</it> inner ear transcriptome associated with inner ear function and impairment in other organisms, thereby supporting the inclusion of <it>Xenopus</it> in cross-species genetic studies of the inner ear. The use of gene categories (inner ear tissue; deafness; ion channels; ion transporters; transcription factors) facilitated the assignment of functional significance to probe set identifiers. We enhanced the biological relevance of our microarray data by using a variety of curation approaches to increase the annotation of the <it>Affymetrix</it> GeneChip® <it>Xenopus laevis</it> Genome array. In addition, annotation analysis revealed the prevalence of inner ear transcripts represented by probe set identifiers that lack functional characterization.</p> <p><b>Conclusions</b></p> <p>We identified an abundance of targets for genetic analysis of auditory and vestibular function. The orthologues to human genes with known inner ear function and the highly expressed transcripts that lack annotation are particularly interesting candidates for future analyses. We used informatics approaches to impart biologically relevant information to the <it>Xenopus</it> inner ear transcriptome, thereby addressing the impediment imposed by insufficient gene annotation. These findings heighten the relevance of <it>Xenopus</it> as a model organism for genetic investigations of inner ear organogenesis, morphogenesis, and regeneration.</p

Identification of variants in primary and recurrent glioblastoma using a cancer-specific gene panel and whole exome sequencing.

Glioblastoma (GBM) is an aggressive, malignant brain tumor typically resulting in death of the patient within one year following diagnosis; and those who survive beyond this point usually present with tumor recurrence within two years (5-year survival is 5%). The genetic heterogeneity of GBM has made the molecular characterization of these tumors an area of great interest and has led to identification of molecular subtypes in GBM. The availability of sequencing platforms that are both fast and economical can further the adoption of tumor sequencing in the clinical environment, potentially leading to identification of clinically actionable genetic targets. In this pilot study, comprised of triplet samples of normal blood, primary tumor, and recurrent tumor samples from three patients; we compared the ability of Illumina whole exome sequencing (ExomeSeq) and the Ion AmpliSeq Comprehensive Cancer Panel (CCP) to identify somatic variants in patient-paired primary and recurrent tumor samples. Thirteen genes were found to harbor variants, the majority of which were exclusive to the ExomeSeq data. Surprisingly, only two variants were identified by both platforms and they were located within the PTCH1 and NF1 genes. Although preliminary in nature, this work highlights major differences in variant identification in data generated from the two platforms. Additional studies with larger samples sizes are needed to further explore the differences between these technologies and to enhance our understanding of the clinical utility of panel based platforms in genomic profiling of brain tumors

Patient demographics and clinical information.

<p>All patients received total resection, followed by temozolomide and external beam radiation of 6,000 Gray in 30 fractions.</p><p>^aW/NH, white not Hispanic or Latino; W/H, white Hispanic or Latino.</p><p>^bSample purity determined by histological review.</p><p>Patient demographics and clinical information.</p

Read alignment view of <i>PTEN</i> (A) and <i>TP53</i> (B) genes zoomed out to exon level.

<p>The AmpliSeq data did not produce any reads covering the variant base in <i>PTEN</i>; however, a single read not shown in the graphic did cover the variant base in <i>TP53</i> in the recurrent tumor. Data from primary tumors are shown. (A) View displayed spans chr10: 89,653,700–89,653,900 of <i>PTEN</i>; variant is T to G mutation located at base 89,653,783. (B) View of <i>TP53</i> displays region of chr17: 7,578,400–7,578,600; variant is G to A mutation located at base 7,578,475. The location of the variant base is indicated by the vertical line crossing the reads.</p

Overview of patient samples and sequencing methods.

<p>Sequencing for the AmpliSeq CCP samples was done using the Ion 318 Chip and whole exome sequencing was preformed using the Genome Analyzer II or HiSeq 2000, both from Illumina.</p

Summary of mutated AmpliSeq CCP genes.

<p>Genes containing variants and the associated sequencing platforms are shown. Most variants were associated with ExomeSeq data and there were a total of 409 genes on the AmpliSeq CCP.</p

Variants identified in Ion AmpliSeq Comprehensive Cancer Panel Genes.

<p>Summary of all variants identified in this study, the majority of which were found in primary tumors. R, recurrent; Chr, chromosome; NS, nonsynonymous; P, primary; SNV, single nucleotide variant.</p><p>Variants identified in Ion AmpliSeq Comprehensive Cancer Panel Genes.</p