Background: No targeted agents except for drugs against NTRK fusion, dMMR/MSI-H or TMB-H are recommended for the treatment of cancer of unknown primary (CUP), despite of the occurrence of multiple actionable mutations identified by NGS. We aimed to explore the characteristics of circulating tumor DNA (ctDNA) and its value in guiding targeted treatment in combination with predicted cancer types by 90-gene reverse-transcription polymerase chain reaction assay for tissue origin. Methods: 172 treatment-naïve CUP patients with ctDNA testing were retrospectively included between April 2017 and Oct 2020. Of them, 98 patients had primary site predicted. Genetic alterations were reclassified based on their predicted primary site and the oncoKB scale. 153 patients treated with the first-line therapy and available survival data were used to explore prognostic value of detected genetic alterations in ctDNA. Results: We identified 82.6% (142/172) of patients had alterations detected, with the most commonly seen mutations of TP53 (51%), ARID1A (11%), KRAS (10%), RB1 (9%), APC (8%), PI3KCA (8%) and NFE2L2 (7%). The most commonly observed actionable mutations were PIK3CA (n=14, 9.8%), ERBB2 (n=7, 4.9%), BRAF (n=6, 4.2%), MET (n=6, 4.2%) and EGFR (n=5, 3.5%). After introducing predicted primary site in the 98 patients, 6.1% (n=6) of patients upgraded to a Level 1 alteration, 1.0% (n=1) to a Level 2 alteration (Table). In these 7 patients, only one patient with predicted lung cancer and EGFR 19 del received gefitinib with partial response for 5+ months. In multivariate analysis, NFE2L2 (hazard ratio [HR] = 2.96, 95%CI=1.32-6.61, P = .008) and CDKN2A mutation (HR = 2.50, 95%CI=1.26-4.96, P = .009) were independently associated with shorter PFS. Furthermore, NFE2L2 (HR = 4.96, 95%CI=1.98-12.43, P ＜ .001) and CDKN2A mutation (HR = 4.84, 95%CI=1.63-14.40, P = .005) were correlated with worse OS. Conclusions: This is the first attempt to integrate ctDNA sequencing and gene expression profiling for tissue of origin in OncoKB classification schema, resulting in 7.1% (7/98) of the genetic alterations reclassified to level 1 or 2, which might identify patients benefiting from corresponding targeted treatment. NFE2L2 and CDKN2A mutations in ctDNA were associated poor prognosis.

Change in therapeutic level of evidence of the actionable mutations with or without gene expression-based primary site prediction in patients with cancer of unknown primary.