DNA sequencing and gene-expression profiling assists in making a tissue of origin diagnosis in cancer of unknown primary

Cancer of unknown primary (CUP) constitutes a group of metastatic cancers in which standardized clinical investigations fail to identify a tissue of origin (TOO). Gene-expression profiling (GEP) has been used to resolve TOO, and DNA sequencing to identify potential targeted treatments; however, these methods have not been widely applied together in CUP patients. To assess the diagnostic utility of DNA and RNA tests for TOO classification, we applied GEP classification and/or gene-panel DNA sequencing in 215 CUP patients. Based on a retrospective review of pathology reports and clinical data, 77% of the cohort were deemed True-CUPs (T-CUP). Among the remaining cases, a latent primary diagnosis (10%) (LP-CUP) or TOO was highly suspected based on combined clinicopathological data (13%) (histology-resolved CUP, HR-CUP). High-medium confidence GEP classifications were made for 80% of LP/HR-CUPs, and these classifications were consistent with a pathologist-assigned diagnosis in 94% of cases, while only 56% of T-CUPs had high-medium confidence predictions. The frequency of somatic mutations in cancer genes was similar to 2,785 CUPs from AACR GENIE Project. DNA features, GEP classification, and oncovirus detection assisted making a TOO diagnosis in 37% of T-CUPs. Gene mutations and mutational signatures of diagnostic utility were found in 31% T-CUPs. GEP classification was useful in 13% of cases and viral detection in 4%. Among resolved T-CUPs, lung and biliary were the most frequently identified cancer types, while kidney cancer represented another minor subset. Multivariate survival analysis showed that unresolved T-CUPs had poorer overall survival than LP/HR-CUPs (Hazard ratio=1.9, 95% CI 1.1 − 3.2, p=0.016), while the risk of death was similar in genomically-resolved T-CUPs and LP/HR-CUPs. In conclusion, combining DNA and RNA profiling with clinicopathological data supported a putative TOO diagnosis in over a third of T-CUPs. DNA sequencing benefited T-CUP tumors with atypical transcriptional patterns that hindered reliable GEP classification. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement The study was supported by funding from Cancer Australia (APP1048193, APP1082604) and the Victorian Cancer Agency (TRP13062). RWT was supported by funding from the Victorian Cancer Agency (TP828750). The Westmead, Blacktown and Nepean study sites were supported by the Cancer Institute NSW 11/TRC/1-06, 15/TRC/1-01 and 15/RIG/1-16. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All participating patients provided informed consent to partake in the study. Peter MacCallum Cancer Centre (PMCC) human research ethics committee gave ethical approval for this work (HREC protocol: 13/62). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.