Preservation of cfRNA in cytological supernatants for cfDNA & cfRNA double detection in non-small cell lung cancer patients

Abstract

Backgroud: Supernatants from various cytological samples, including body cavity effusion, sputum, bronchoalveolar lavage fluid (BALF), and needle aspiration, have been validated for detecting genetic alterations using cell-free DNA (cfDNA) in patients with non-small cell lung cancer (NSCLC). However, the sensitivity of fusion variations detection remains challenging. The protection of cell-free RNA (cfRNA) is critical for resolving the issue.

Methods: A protective solution (PS) was applied for preserving cfRNA in cytological supernatant (CS), and the quality of protected cfRNA was assessed by cycle threshold (CT) values from reverse transcription quantitative polymerase chain reaction (RT-qPCR). Furthermore, we collected an additional set of malignant cytological and matched tumor samples from 84 NSCLC patients, cfDNA & cfRNA extraction and double detection for driver gene mutations was validated using the multi-gene mutations detection by RT-qPCR.

Results: Under the optimal protection system, 91.0% (101/111) of cfRNA were protected effectively. Among the 84 NSCLC patient samples, seven cytological samples failed the tests. In comparison with tumor samples, the overall sensitivity and specificity of detecting driver genes of supernatant cfDNA and cfRNA were 93.8% (74/77) and 100% (77/77), respectively. Notably, when focusing exclusively on patients with fusion gene changes, both sensitivity and specificity reached 100% (11/11) for EML4-ALK, ROS1, RET fusions, and MET ex14 skipping.

Conclusion: These findings suggest that cfDNA & cfRNA extraction and double detection strategy recommended in this study improve the accuracy of driver genes mutations test, especially for RNA-based assay.

Keywords: NSCLC; cell‐free RNA; driver gene; fusion; liquid biopsy.

FIGURE 1

Flowcharts of study design. (A) cfRNA protection system of testing and validation set; (B) processing of preparation for CS samples; (C) detection of 11 driver genes mutations in CS and matched tumor samples. CS, cytological supernatant; PS, protective solution.

FIGURE 2

Exploration and validation the optimal proportions of PS:CS for cfRNA protection. CS samples were protected with different proportions of PS:CS before cfRNA extraction. The efficacy of cfRNA PS was evaluated through RT‐qPCR CT values across different PS:CS proportions. Paired Wilcoxon signed‐rank test was used for evaluation. (A) CT values for cfRNA in PS:CS proportions of 0:1, 1:1, 1:2, and 1:3 in 19 CS samples; (B) 92 CS samples for validation with the proportion of PS:CS = 1:1 and 0:1.

FIGURE 3

Hotspot mutations detection profile in cytological supernatant compared to tumor samples. Hotspot mutations detection profile in cytological supernatant compared to tumor samples. (Top) Plot shows the mutation profiles, the colors indicate the mutation types. (Bottom) Plot illustrates the sample type, patients stage and treatment status. Multi_Hit indicates multi types of variation.