Utilization of cytology smears improves success rates of RNA‐based next‐generation sequencing gene fusion assays for clinically relevant predictive biomarkers

Abstract

Background The use of RNA‐based next‐generation sequencing (NGS) assays to detect gene fusions for targeted therapy has rapidly become an essential component of comprehensive molecular profiling. For cytology specimens, the cell block (CB) is most commonly used for fusion testing; however, insufficient cellularity and/or suboptimal RNA quality are often limiting factors. In the current study, the authors evaluated the factors affecting RNA fusion testing in cytology and the added value of smears in cases with a suboptimal or inadequate CB. Methods A 12‐month retrospective review was performed to identify cytology cases that were evaluated by a targeted RNA‐based NGS assay. Samples were sequenced by targeted amplicon‐based NGS for 51 clinically relevant genes on a proprietary platform. Preanalytic factors and NGS quality parameters were correlated with the results of RNA fusion testing. Results The overall success rate of RNA fusion testing was 92%. Of the 146 cases successfully sequenced, 14% had a clinically relevant fusion detected. NGS testing success positively correlated with RNA yield (P = .03) but was independent of the tumor fraction, the tumor size, or the number of slides used for extraction. CB preparations were adequate for testing in 45% cases, but the inclusion of direct smears increased the adequacy rate to 92%. There was no significant difference in testing success rates between smears and CB preparations. Conclusions The success of RNA‐based NGS fusion testing depends on the quality and quantity of RNA extracted. The use of direct smears significantly improves the adequacy of cytologic samples for RNA fusion testing for predictive biomarkers.

A decade of change: Trends in the practice of cytopathology at a tertiary care cancer centre

“…At our institution, molecular testing, including high‐throughput assays such as next‐generation sequencing, have been validated on cytology smears, cytospin preparations, and cell block preparations. Prior studies from our group have demonstrated that including cytology smears in our molecular assays has significantly improved the number of specimens that are adequate for testing 39,43 . This is in keeping with the increased number of molecular test requests over the past decade as seen in our study.…”

“…Multiple targeted therapeutic agents have been approved by the FDA for various malignancies, and identification of these molecular targets from clinical samples is absolutely essential 37 . Molecular studies have traditionally been performed on histological blocks; however, several studies have demonstrated that cytological specimens provide excellent substrates for molecular analysis and can provide clinically relevant genomic information that helps guide patient management 38‐40 …”

Molecular Testing on Cytology for Gene Fusion Detection

“…RNA sequencing is not affected by intronic regions but RNA extraction is more complicated than DNA, especially purification from FFPE specimens, as it can be highly degraded with the possibility to invalidate the run (50). However, in this setting, cytological samples as direct smears instead of CB preparation improve the adequacy of cytological material for RNA fusion testing for predictive biomarkers, as reported (51).…”

What Is New in Biomarker Testing at Diagnosis of Advanced Non-Squamous Non-Small Cell Lung Carcinoma? Implications for Cytology and Liquid Biopsy

“…At the moment, most of the major studies use tissue samples for NTRK status assessment [109][110][111]. However, detection can be possible with cytological samples and even liquid biopsies [109,[112][113][114][115]. Several techniques of detection have been developed, including immunohistochemistry (IHC), molecular biology approaches (NGS and RT-PCR), and multiplex digital colorcoded barcode technology on tissue sections .…”

“…Depending on the quality and quantity of the nucleic acids extracted from the sample, false negative results can be obtained [78,81,109,119,121]. Depending on the case, these approaches could be performed with cytological samples, but only a few studies in this area have been performed to date and studies comparing cytological and tissue samples are strongly needed [109,[112][113][114][115]. The detection of NTRK fusions can also be envisaged with a liquid biopsy, but the sensitivity of such an approach is still unknown and needs to be determined.…”