This study showed that in ddPCR of liquid biopsy samples, BWF samples are more reliable than blood samples for reflecting the tumor mEGFR status. Additionally, BWF can be used to analyze both L858R and E19del and is more sensitive than plasma in detecting every stage of lung cancer.
Although tissue biopsy is the gold standard method for molecular genotyping in lung cancer, liquid biopsy may play an important role as a complementary method for detecting targeted genes and predicting the clinical course or outcome[13, 14] and to detect lung cancer in an early stage. Liquid biopsy is relatively non-invasive, safe, and simple procedure. Therefore, it can be performed in patients with poor medical condition or when the location or size of lung cancer is difficult to biopsy.
Many studies have shown the feasibility of liquid biopsy in lung cancer, but some limitations remain.[16, 17] Blood samples are mainly used for liquid biopsy, including ctDNA, circulating tumor cells, platelets, exosomes, and microRNAs, which provide specific genetic information of the tumor. Although the proportion of ctDNA can vary depending on the tumor burden, stage, vascularization, and biologic feature, this value is generally only 0.1–1.0%. Furthermore, ctDNA has a relatively short half-life, from approximately 16 to 150 min. Thus, if sample processing is delayed, ctDNA would not reflect the precise experimental results. To overcome this limitation, we froze the BWF and blood samples within 3 and 8 h, respectively, and we used Streck tube®, which maintained the stability of cfDNA for up to 14 days and CTC for up to 7 days. Recently, Krug el al. demonstrated that compared to ctDNA, combined isolation of exosomal RNA (exoRNA) and cfDNA detected EGFR mutation and T790M with improved sensitivity (96% in activating EGFR mutation and 90% for T790M). They showed the largest improvement in sensitivity (26% to 74%), particularly in patients with intrathoracic metastatic diseases by using combined exoRNA and ctDNA. Therefore, applying exoRNA and ctDNA in BWF specimens may greatly improve the detection of EGFR mutations, even in the early stage of lung cancer, but further studies are needed to evaluate this.
BAL plays a supporting role in the diagnosis of lung cancer. Since the 1980s, various studies have shown the usefulness of BAL in diagnosing lung malignancy. BAL showed a diagnostic yield of 33–90% in diffuse malignant pulmonary infiltrates, although the value differed according to the cancer type. Particularly, in NSCLC such as squamous cell carcinoma and adenocarcinoma, the diagnostic yield was 50% and 77%, respectively. Park et al. suggested that BAL fluid was effective for detecting the EGFR mutation status. Although their study involved a small number of subjects (n = 20), a high concordance rate (91.7%) was observed between the BALF and tissue for detecting EGFR mutations by using PNA-mediated PCR clamping and PANAMutyper with fluorescence melting curve analysis. However, their study included only three patients with early stage lung cancer, and they did not report the difference in the detection rate between the BALF and plasma. Our study included 38 patients with an early stage of lung cancer, and the sensitivity was 65% with BWF, while it was only 15.0% with plasma. There was a greater improvement in the sensitivity and diagnostic yield with BWF compared to that with blood in an early stage of lung cancer. Therefore, we suggest that, rather than plasma, BWF can be used to detect mutational variations, regardless of the lung cancer stage.
Our study showed BWF could be substituted for biopsy to confirm EGFR mutation and may shorten the time from diagnosis to treatment by avoiding the delay for biopsy and confirmation of biopsy results. BWF specimens are simple to collect, and obtaining BWF is safer than lung biopsy because only simple bronchoscopy needs to be performed, for which hospitalization is not required.
Additionally, we investigated the relationship between tumor size and mEGFR detection rate. There may be a significant agreement of the mEGFR status between liquid biopsy and tissue biopsy for larger-sized tumors, but no significant association was noted between tumor size and concordance of mEGFR for liquid biopsy and tissue biopsy (data not shown). This suggests that the diagnostic yield of liquid biopsy with BWF depends mainly on staging rather than on tumor size. However, as our study sample size was small, further studies using large sample sizes are needed.