Molecular profiling of body fluid cfDNA: Advancing diagnostics and therapeutic decisions.

3054 Background: Effusions in cancer patients pose several critical challenges for clinicians. In known cancer patients, an effusion may signal recurrence, whereas in newly diagnosed, seemingly localized cases, it indicates a more advanced stage. In many patients the effusion may be secondary to complications of treatment or comorbidities, rather than malignant. Diagnosing malignant involvement of body fluids remains a challenge due to the limitations of conventional cytology. This study explores the potential of molecular profiling of body fluids to identify actionable molecular alterations and its role in diagnosing malignant effusions. Methods: We analyzed cfDNA from body fluids—ascitic fluid (N=26), cerebrospinal fluid (N=7), pleural fluid (N=11), and pericardial fluid (N=1), collected from 45 patients with solid tumors, including lung (N=12), breast (N=9), ovarian (N=9), pancreas (N=4), gastrointestinal cancers (N=4), cervix (N=2), and one each of CNS, endometrial cancer, HCC, liposarcoma, and melanoma. In a subset, results from fluid samples were compared with tissue and plasma samples to assess concordance across different sample types. Results: Pathogenic alterations were identified in 89% (40/45) of fluid samples. The most frequently mutated genes were TP53 (53%), EGFR (20%), KRAS (18%), PIK3CA (9%), CTNNB1 (7%), FGFR3 (7%), GNAS (7%), MYC (7%), and ESR1 (4%). Simultaneous analysis of body fluid and tissue samples (n=11) revealed that 7 patients (64%) had at least one concordant pathogenic alteration. Similarly, analysis of body fluid and plasma samples (n=16) showed that 8 patients (50%) had at least one concordant pathogenic alteration. Body fluid analysis identified acquired resistance alterations, such as EGFR T790M and ALK C1156Y , which influenced therapy decisions. Among the alterations detected exclusively in fluid samples were ERBB2 amplification and ESR1 D538G mutation in two breast cancer patients. In evaluating molecular profiling against cytology for detecting malignant effusions, 17 of 25 samples were positive by both methods, while 4 of 5 cytology-negative samples were ctDNA-positive. Notably, 3 of 4 ctDNA-negative cases were cytology-positive. These results emphasize the potential role of molecular profiling for diagnosis when cytology is inconclusive. Conclusions: This study highlights the importance of body fluid ctDNA profiling (ascites, pleural, pericardial, CSF) in identifying actionable mutations, including unique druggable alterations not found tissue or liquid biopsies. The ability to detect ctDNA in cytology-negative samples underscores the potential of body fluid ctDNA as a valuable complement to fluid cytology for diagnosing malignant involvement. ESCAT classification of pathogenic variants identified in body fluids from 45 patients. Tier Level Incidence (%) Number of unique patients IA 28.9% 13 IIA 0% 0 IIIA 35.6% 16 IIIB 4.4% 2 IVA 55.6% 25 IVB 2.2% 1 X 22.2% 10