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Introduction Diagnostic yield of pleural fluid (PF) cytology is limited, with an overall sensitivity of 58.2% 1; often necessitating invasive procedures to obtain histology. Profiling circulating tumour DNA (ctDNA) from PF represents a promising strategy for diagnosing malignant pleural effusion (MPE). This proof-of-concept study evaluated the potential of profiling PF ctDNA to improve diagnostic accuracy using a rapid and cost-effective shallow whole-genome sequencing (sWGS) approach.Methods At time of writing, matched PF and blood samples were collected from five patients with confirmed (n=2) or suspected (n=3) thoracic malignancies. PF samples were also submitted for standard cytological evaluation. Cell-free DNA (cfDNA) was extracted from PF and plasma and subjected to sWGS (300pg) using previously described methods. 2 3 Results were analysed using a tailored ichorCNA pipeline to detect genome-wide copy number (CN) aberrations indicative of ctDNA. Patient recruitment is currently ongoing.Results All 5 patients had a malignant diagnosis, PF cytology confirmed MPE in 2 (40%) patients. Two patients required a biopsy for diagnosis (1 ultrasound guided, 1 thoracoscopy) and 1 patient underwent EBUS (see table 1). sWGS analysis identified ctDNA (CN deletions) in PF in all patients, whereas plasma only showed deletions in 3 patients (60%). Notably, deletions in PF ctDNA involved chromosomal regions harbouring key lung cancer-associated genes, including RB1, SMARCA4 and CDKN2A.Abstract S34 Table 1Underlying diagnosis, PF cytology and method of final diagnosis Diagnostic or Confirmed Cytology Underlying diagnosis Method diagnosis made Confirmed NSCLC NSCLC - adenocarcinoma Pleural fluid cytology Confirmed Negative NSCLC - NOS Pleural biopsy Diagnostic Negative Sarcamatoid carcinoma Thoracoscopy Diagnostic Negative Diffuse B cell lymphoma EBUS Diagnostic Metastatic malignant epithelioid tumour Unknown origin - CK7 positive sarcoma/carcinoma - SMARCA4 Pleural fluid cytology Discussion We identified presence of ctDNA in PF samples through CN aberrations, potentially offering evidence of malignancy using a rapid, cost-effective methodology. Future work to increase sample size and profile actionable genomic alterations that may inform therapeutic strategies is warranted.References Kassirian S, Hinton SN, Cuninghame S, et al. Diagnostic sensitivity of pleural fluid cytology in malignant pleural effusions: systematic review and meta-analysis. Thorax. 2023;78(1):32–40. doi:10.1136/thoraxjnl-2021–217959Page K, Martinson LJ, Fernandez-Garcia D, et al. Circulating tumor DNA profiling from breast cancer screening through to metastatic disease. JCO Precis Oncol. 2021;5.Allsopp RC, Page K, Ambasager B, et al. Rapid shallow whole genome sequencing for limited cell-free DNA. Clin Chem. 2023;69(5):510–8.