Abstract Comprehensive molecular profiling by next-generation sequencing has revolutionized tumor classification and biomarker evaluation. However, routine implementation is challenged by the scant nature of diagnostic material obtained through minimally invasive procedures. Here, we describe our long-term experience in profiling cytology samples with an in-depth assessment of the performance, quality metrics, biomarker identification capabilities, and potential pitfalls. We highlight the impact of several optimization strategies to maximize performance with 4,871 prospectively sequenced clinical cytology samples tested by MSK-IMPACT TM . Special emphasis is given to the use of residual supernatant cell-free DNA (ScfDNA) as a valuable source of tumor DNA. Overall, cytology samples are similar in performance to surgical samples in identifying clinically relevant genomic alterations, achieving success rates up to 93% with full optimization. While cell block (CB) samples have excellent performance overall, low-level cross-contamination is identified in a small proportion of cases (4.7%), a common pitfall intrinsic to the processing of paraffin blocks, suggesting that more stringent precautions and processing modifications should be considered in quality control initiatives. By contrast ScfDNA samples have negligible contamination. Finally, ScfDNA testing exclusively used as a rescue strategy, delivered successful results in 71% of cases where tumor tissue from CB was depleted.

Maximizing the clinical utility and performance of cytology samples for comprehensive genetic profiling