e15172 Background: The diagnosis and management of patients with cancer of unknown or unclear primary present significant clinical hurdles, often culminating in unfavorable outcomes. A randomized phase III trial (CUP001) has demonstrated that site-specific treatment guided by the 90-gene-expression assay (Canhelp-Origin Assay) resulted in more therapy options, and significantly improved progression-free survival compared with empirical chemotherapy in patients with cancer of unknown primary. This study evaluated the clinical utility of the 90-gene expression assay in the diagnosis and management of patients with cancer of unknown or unclear primary. Methods: Patients who underwent the 90-gene expression assay at Fudan University Shanghai Cancer Center (FUSCC) between February 2023 and December 2023 were enrolled in the study. Data collection encompassed patient demographics, clinical characteristics, and pathological features. Results: Patient enrollment began in February 2023 and closed in December 2023 after enrolling 303 patients with 51 inpatient cases and 244 pathological consultation cases (from 144 sites). Among them, 295 patients met all inclusion criteria, yielding an overall success rate of 97.4% (295/303). Tumor cells were enriched by either macrodissection (N = 257) or microdissection (N = 38), with median highest similarity scores of 57.5 and 67.1, respectively. Using a positive cutoff value of the highest similarity score ≥45.3, the positivity rates for macrodissection and microdissection were 66.5% (171/257) and 81.6% (31/38), respectively. Of the enrolled patients, 177 (60%) were male, and 118 (40%) were female, with a median age of 63 years (range: 25 to 85 years). Predictive results encompassed 21 tumor types, with the most common being gastroesophageal cancer (N = 40, 13.6%), lung cancer (N = 37, 12.5%), breast cancer (N = 36, 12.2%), head and neck squamous cell carcinoma (N = 29, 9.8%), and ovarian cancer (N = 28, 9.5%). Of 166 samples with an assigned grade, 89.8% were poorly differentiated or undifferentiated, 6.0% were moderately differentiated, and 4.2% were well differentiated. The consistency of prediction results and clinicopathological results will be further evaluated after follow-up. Conclusions: This study demonstrates that the 90-gene expression assay has diagnostic implications for patients with cancer of unknown or unclear primary. This affirms its clinical utility as a standard diagnostic approach in the diagnosis and management of these patients, facilitating the formulation of more precise treatment strategies and ultimately enhancing prognosis.