Molecular characteristics and outcomes of a real-world cohort of carcinoma of unknown primary determined by tissue-of-origin assays.

3076 Background: Carcinoma of unknown primary (CUP) increasingly intersects with precision oncology as molecular profiling becomes integrated into diagnostic algorithms. However, published CUP cohorts and guideline-based series remain anchored to clinical and pathologic criteria and do not characterize patients whose tissue-of-origin (TOO) predictions remain low probability despite comprehensive molecular analysis. This population—where molecular data fail to resolve lineage—is poorly described. We evaluated clinical, molecular, and outcome features of CUP defined as < 90% probability for all predicted lineages. Methods: A retrospective chart review of TOO tests ordered at a single academic center between 2021-2025 was conducted. Demographics, pathology, molecular features including Tumor Mutational Burden (TMB), mutational profiles, predicted lineages, and treatments were abstracted from the TOO report and electronic health record. Survival status was assessed as of 12/31/2025. Results: Among 132 patients evaluated for CUP, 50 met the study definition (TOO probability <90% for a single lineage). Median age was 66 years, 56% were female and 76% were Black. Predicted lineages spanned gastrointestinal, pancreaticobiliary, lung, breast, melanoma, and genitourinary tissues, reflecting heterogeneity and lack of dominant lineage signal. Many tumors demonstrated overlapping immunophenotypes consistent with molecular ambiguity. High TMB (≥10 mut/Mb) was reported in 16% with PD-L1 positive in 4%, limiting eligibility for tissue-agnostic immunotherapy. Most patients received empiric systemic therapy rather than site-directed or molecularly targeted regimens. Targetable alterations reported included BRAF, KRAS, HER2, ATM, CHEK2, and PIK3CA. TOO primary site predictions were in concordance with final pathology in only 22 cases (44%). At last follow-up, 29 patients (57%) were deceased. Conclusions: This study characterizes a precision-oncology–relevant subset of CUP in which molecular classifiers fail to assign high-confidence lineage, a population not represented in prior CUP publications. Tumors in this cohort showed broad predicted lineages across multiple organ systems with low rates of high TMB, reflecting substantial molecular heterogeneity. Many displayed nondistinct histology with overlapping immunophenotypes, consistent with diagnostic ambiguity. Concordance between predicted TOO and final pathology was limited, underscoring the challenges of relying solely on molecular classifiers. Actionable targets and markers for immunotherapy were infrequent, and most patients received empiric chemotherapy. Outcomes were poor, underscoring the unmet need for improved integrative molecular approaches and novel precision-oncology strategies in CUP subsets that remain unresolved despite comprehensive molecular assessment.