title: Massively-parallel sequencing assists the diagnosis and guided treatment of cancers of unknown primary.
abstract: The clinical management of patients with cancer of unknown primary (CUP) is hampered by the absence of a definitive site of origin. We explored the utility of massively-parallel (next-generation) sequencing for the diagnosis of a primary site of origin and for the identification of novel treatment options. DNA enrichment by hybridization capture of 701 genes of clinical and/or biological importance, followed by massively-parallel sequencing, was performed on 16 CUP patients who had defied attempts to identify a likely site of origin. We obtained high quality data from both fresh-frozen and formalin-fixed, paraffin-embedded samples, demonstrating accessibility to routine diagnostic material. DNA copy-number obtained by massively-parallel sequencing was comparable to that obtained using oligonucleotide microarrays or quantitatively hybridized fluorescently tagged oligonucleotides. Sequencing to an average depth of 458-fold enabled detection of somatically acquired single nucleotide mutations, insertions, deletions and copy-number changes, and measurement of allelic frequency. Common cancer-causing mutations were found in all cancers. Mutation profiling revealed therapeutic gene targets and pathways in 12/16 cases, providing novel treatment options. The presence of driver mutations that are enriched in certain known tumour types, together with mutational signatures indicative of exposure to sunlight or smoking, added to clinical, pathological, and molecular indicators of likely tissue of origin. Massively-parallel DNA sequencing can therefore provide comprehensive mutation, DNA copy-number, and mutational signature data that are of significant clinical value for a majority of CUP patients, providing both cumulative evidence for the diagnosis of primary site and options for future treatment.
authors.affiliation: The Peter MacCallum Cancer Centre, East Melbourne, VIC, Australia; The Department of Pathology, University of Melbourne, Parkville, VIC, Australia.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Tothill
authors.forename: Jason
authors.initials: J
authors.lastname: Li
authors.forename: Linda
authors.initials: L
authors.lastname: Mileshkin
authors.forename: Ken
authors.initials: K
authors.lastname: Doig
authors.forename: Terence
authors.initials: T
authors.lastname: Siganakis
authors.forename: Prue
authors.initials: P
authors.lastname: Cowin
authors.forename: Andrew
authors.initials: A
authors.lastname: Fellowes
authors.forename: Timothy
authors.initials: T
authors.lastname: Semple
authors.forename: Stephen
authors.initials: S
authors.lastname: Fox
authors.forename: Keith
authors.initials: K
authors.lastname: Byron
authors.forename: Adam
authors.initials: A
authors.lastname: Kowalczyk
authors.forename: David
authors.initials: D
authors.lastname: Thomas
authors.forename: Penelope
authors.initials: P
authors.lastname: Schofield
authors.forename: David D
authors.initials: DD
authors.lastname: Bowtell
mesh_terms: D000328:Adult; D000368:Aged; D056915:DNA Copy Number Variations / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods; D004273:DNA, Neoplasm / Q000235:genetics; D019317:Evidence-Based Medicine / Q000379:methods; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods; D052138:Genes, Neoplasm / Q000235:genetics; D006801:Humans; D008297:Male; D008875:Middle Aged; D058990:Molecular Targeted Therapy / Q000379:methods*; D009154:Mutation; D009382:Neoplasms, Unknown Primary / Q000175:diagnosis / Q000235:genetics* / Q000628:therapy; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods
chemical_list: D004273:DNA, Neoplasm
keywords: cancer diagnostic; cancer of unknown primary; massively-parallel sequencing; mutation profiling; next-generation sequencing; targeted therapy