Personalized oncology through integrative high-throughput sequencing: a pilot study

Abstract

Individual cancers harbor a set of genetic aberrations that can be informative for identifying rational therapies currently available or in clinical trials. We implemented a pilot study to explore the practical challenges of applying high-throughput sequencing in clinical oncology. We enrolled patients with advanced or refractory cancer who were eligible for clinical trials. For each patient, we performed whole-genome sequencing of the tumor, targeted whole-exome sequencing of tumor and normal DNA, and transcriptome sequencing (RNA-Seq) of the tumor to identify potentially informative mutations in a clinically relevant time frame of 3 to 4 weeks. With this approach, we detected several classes of cancer mutations including structural rearrangements, copy number alterations, point mutations, and gene expression alterations. A multidisciplinary Sequencing Tumor Board (STB) deliberated on the clinical interpretation of the sequencing results obtained. We tested our sequencing strategy on human prostate cancer xenografts. Next, we enrolled two patients into the clinical protocol and were able to review the results at our STB within 24 days of biopsy. The first patient had metastatic colorectal cancer in which we identified somatic point mutations in NRAS, TP53, AURKA, FAS, and MYH11, plus amplification and overexpression of cyclin-dependent kinase 8 (CDK8). The second patient had malignant melanoma, in which we identified a somatic point mutation in HRAS and a structural rearrangement affecting CDKN2C. The STB identified the CDK8 amplification and Ras mutation as providing a rationale for clinical trials with CDK inhibitors or MEK (mitogen-activated or extracellular signal-regulated protein kinase kinase) and PI3K (phosphatidylinositol 3-kinase) inhibitors, respectively. Integrative high-throughput sequencing of patients with advanced cancer generates a comprehensive, individual mutational landscape to facilitate biomarker-driven clinical trials in oncology.

Figure 1. Exploratory integrative sequencing of tumors for personalized oncology

A, The MI-ONCOSEQ study recruits cancer patients and provides up-front genetic counseling. Patients are tracked through a biospecimen and clinical database. B, A multi-disciplinary Sequencing Tumor Board was instituted including expertise in clinical oncology, genomics, bioinformatics, pathology, bioethics, and genetics. C, Clinically relevant timeframe from tumor biopsy to available results. D, Integration of whole genome sequencing (blue), whole exome capture sequencing for 1-2% of the genome (red), and transcriptome or messenger RNA sequencing (green). Each sequencing strategy can be integrated (bottom) for analysis of tumor aberrations including structural rearrangements, copy number alteration, point mutations, and gene expression.

Figure 2. Integrative sequencing of a patient with metastatic colorectal cancer enrolled on the MI-ONCOSEQ protocol

Patient 3 is a 46-year-old man with metastatic colorectal cancer and the first patient enrolled. A, CT scan abdomen demonstrates liver metastases and biopsy site. B, Representative histology from liver biopsy demonstrates poorly differentiated adenocarcinoma and estimated tumor content 60-70%. C, Summary of genetic aberrations identified includes an activating point mutation of NRAS, an inactivating point mutation of TP53, and amplification of CDK8. Wildtype genes included KRAS and BRAF. D, Integrated copy number analysis based on exome and whole genome data. E, Amplification in region of chromosome 13q including CDK8 is displayed as estimated copy number based on integrative analysis of whole genome (green) and exome (orange) data. F, CDK8 is highly expressed based on RNA-seq compared with benign or other cancer samples. G, Schema shows integrative analysis used to identify activating NRAS mutation with number of variant reads on right. H, Schema of probable inactivating rearrangement involving PPP2R3B based on integrative analysis of RNA-seq and whole genome data.

Figure 3. Aberrations reported in Patient 4 (Melanoma)

Patient 4 is a 48-year-old woman with metastatic melanoma. A, Multiple skin metastases and sites of biopsy. B, Representative histology from skin biopsy demonstrates dermal proliferation of ovoid to spindle cells with frequent prominent nucleoli. C, Summary of mutations reveals an activating HRAS mutation and an ETS transcription factor (ELK1) mutation. Wildtype genes included BRAF, CKIT, MEK, and NRAS. D, Copy number landscape across chromosomes derived from whole genome and exome sequencing. E, Circos plot derived from whole genome sequencing depicts structural variations including deletions (green), interchromosomal (orange) and intrachromosomal (blue) rearrangements. F, RNA-seq data support a possible rearrangement involving CDKN2C, WIPI1, and FSHR, and is predicted to inactivate CDKN2C. G, Integrative analysis identifies the activating HRAS mutation.