Whole-Exome Sequencing of Metastatic Cancer and Biomarkers of Treatment Response

Abstract

Importance: Understanding molecular mechanisms of response and resistance to anticancer therapies requires prospective patient follow-up and clinical and functional validation of both common and low-frequency mutations. We describe a whole-exome sequencing (WES) precision medicine trial focused on patients with advanced cancer.

Objective: To understand how WES data affect therapeutic decision making in patients with advanced cancer and to identify novel biomarkers of response.

Design, setting, and patients: Patients with metastatic and treatment-resistant cancer were prospectively enrolled at a single academic center for paired metastatic tumor and normal tissue WES during a 19-month period (February 2013 through September 2014). A comprehensive computational pipeline was used to detect point mutations, indels, and copy number alterations. Mutations were categorized as category 1, 2, or 3 on the basis of actionability; clinical reports were generated and discussed in precision tumor board. Patients were observed for 7 to 25 months for correlation of molecular information with clinical response.

Main outcomes and measures: Feasibility, use of WES for decision making, and identification of novel biomarkers.

Results: A total of 154 tumor-normal pairs from 97 patients with a range of metastatic cancers were sequenced, with a mean coverage of 95X and 16 somatic alterations detected per patient. In total, 16 mutations were category 1 (targeted therapy available), 98 were category 2 (biologically relevant), and 1474 were category 3 (unknown significance). Overall, WES provided informative results in 91 cases (94%), including alterations for which there is an approved drug, there are therapies in clinical or preclinical development, or they are considered drivers and potentially actionable (category 1-2); however, treatment was guided in only 5 patients (5%) on the basis of these recommendations because of access to clinical trials and/or off-label use of drugs. Among unexpected findings, a patient with prostate cancer with exceptional response to treatment was identified who harbored a somatic hemizygous deletion of the DNA repair gene FANCA and putative partial loss of function of the second allele through germline missense variant. Follow-up experiments established that loss of FANCA function was associated with platinum hypersensitivity both in vitro and in patient-derived xenografts, thus providing biologic rationale and functional evidence for his extreme clinical response.

Conclusions and relevance: The majority of advanced, treatment-resistant tumors across tumor types harbor biologically informative alterations. The establishment of a clinical trial for WES of metastatic tumors with prospective follow-up of patients can help identify candidate predictive biomarkers of response.

Conflict of interest statement

Conflict of Interest Disclosures: None reported.

Figures

Figure 1. Overall Schematic of the Precision Medicine Trial

The process starts with clinical examination and consent, followed by metastatic tumor biopsy for whole-exome sequencing and biobanking. Results are discussed in tumor board, returned to the patient and referring physicians to guide treatment, and also used to fuel translational research and the development of new diagnostics and therapeutics.

Figure 2. Clinical Demographic Characteristics Including Sequencing From a Wide Range of Metastatic Biopsy Sites and a Predominantly Solid Tumor Population

Numbers indicate number of patients with primary tumor types (left) and number of biopsies performed at specific locations (indicated on person). Smaller font indicates primary tumor site.

Figure 3. The Mutational Landscape of Precision Medicine Cases

Figure shows the number of somatic alterations detected per patient in each category by tumor type (51 patients with prostate cancer, 14 with urothelial cancer, and 32 with other cancers).

Figure 4. Whole-Exome Sequencing of 1 Patient's Primary and Metastatic Tumors

A, Both primary tumor (prostate, histologic subtype Gleason 9 adenocarcinoma with focal neuroendocrine differentiation) and metastatic tumor (brain, histologic subtype neuroendocrine prostate cancer) of patient PM12 were sequenced by means of whole-exome sequencing (WES). A substantial number of somatic mutations and copy number alterations were observed by both WES and whole-genome sequencing. Images are original magnification ×200. B, The whole-genome sequencing Circos plot of PM12's metastatic tumor illustrated a highly altered genome with complex structural variations and rearrangements. Chromosome number is indicated outside the circle. One important finding from WES was hemizygous deletion of the DNA repair gene FANCA in both primary tumor and metastasis, which was confirmed by fluorescence in situ hybridization (tumor panel insets; green probe = control centromeric; red probe = FANCA). C and D, Loss of heterozygosity in FANCA. In the tumor cells, in the heterozygous germline single-nucleotide polymorphism on exon 33 of FANCA (rs17233497), only the missense variant is expressed. C, Coverage of the region for germline (DNA) and 2 tumor samples (RNA). D, Magnification of the reads in 1 RNA-sequencing sample. Reads are reported as on the forward strand; hence, the A variant is indicated, corresponding to a T in the mRNA molecule because FANCA is transcribed from the reverse strand.

Figure 5. Cisplatin Sensitivity In Vitro and In Vivo

A, Cisplatin sensitivity in 22Rv1 cells following genome editing of FANCA (KO1) or a control sequence by CRISPR. Inset, Western blot of FANCA and GAPDH expression in indicated cell lines. IC50 indicates half maximal inhibitory concentration. B, Mean tumor size of the LTL545 xenograft before, during, and after treatment with vehicle (blue lines) or cisplatin (4 mg/kg, day 1 and day 8, intraperitoneal injection). C and D, FANCD2 foci formation following genome editing of FANCA (KO2) or a control sequence by CRISPR.