This is the first report of the detection of EGFR mutations of primary lung adenocarcinoma using ddPCR from prospectively collected sputum samples and compared it with an EGFR mutation in surgically resected lung cancer. In addition, this study is the first report to analyze the clinicopathological features of patients with primary lung adenocarcinoma who are candidates for collecting sputum for the detection of EGFR mutations via ddPCR. EGFR mutations can be detected with high sensitivity by ddPCR if the sputum cytology is positive. Since a CT tumor size ≥ 29 mm is a potential predictive factor for sputum cytology positive, sputum should be collected in such cases for the EGFR mutation analysis.
Bronchoscopic, CT-guided, and surgical biopsies are currently performed in clinical practice to obtain tumor tissue for molecular analysis. However, these methodologies are invasive. An overall complication rate of 1.55% was reported for bronchoscopic biopsy, and included bleeding (0.63%) and pneumothorax (0.44%), with a mortality rate of 0.003% [8]. The reported rates of mortality and serious complications for CT-guided percutaneous needle biopsy were 0.07% and 0.75%, respectively, with complication rates of 35% for pneumothorax, 0.15% for pulmonary hemorrhage/pleural bleeding, 0.06 to 0.4% for air embolism, and 0.1 to 0.56% for needle track implantation [9, 10]. Furthermore, surgical biopsy requires general anesthesia and is more invasive. The reported mortality rate due to surgery was 0.5%, and the rate of complications that included pneumonia, air leakage, and atelectasis was 9.6% [11]. It is important to collect tumor cells safely because these complications may delay or prevent the initiation of treatment for lung cancer.
Sputum can be collected non-invasively, which is a greater advantage for cancer patients compared to other methodologies. The detection sensitivity of lung cancer in sputum cytology was reported to be 40 to 66% [13, 14]. Presently, the sensitivity was 11.0%. There are two reasons of the low sensitivity we observed. Firstly, all the patients had lung adenocarcinoma. Sputum cytology is highly effective for central type squamous cell carcinoma in patients with hemoptysis [16]. Sing et al. reported that the detection rate of sputum in adenocarcinoma in 64 patients was 25.0% [14]. Secondly, patients with early staged lung cancer were included. The sensitivity of sputum cytology for patients with advanced stages are reported to be higher than for early stage cancer [14]. The diagnostic sensitivity of bronchoscopic biopsy was reported to be 88% (78% for peripheral lung cancer) [25]. The sensitivity of percutaneous needle biopsy was 86.1% [26]. Although sputum can be collected non-invasively from lung cancer patients, the detection sensitivity of lung cancer was lower compared to other methodologies. In this study, ROC curve analysis revealed that the sensitivity increased to 69.5% in sputum cytology if the CT tumor size was ≥ 29 mm. Risse et al. similarly reported that the detection sensitivity of primary lung cancer was high when the tumor size exceeded 24 mm [16].
Tumor cells in sputum specimens were reportedly detected in < 1% of the cells contained in sputum [15], and sputum has been considered unsuitable for molecular analysis. The present study demonstrates that ddPCR can detect EGFR mutations in primary lung adenocarcinoma with high sensitivity (80.0%) and high specificity (100%) in SC (+) cases. One discordant case in SC (+) (Case 4, cytology Class IIIa) might have been a false positive because it was suspected to be squamous cell carcinoma based on the sputum cytology. In contrast, the sensitivity of EGFR mutation detection was as low as 3.1%, and it was considered irrelevant to perform EGFR mutation analysis unless the cytology was positive.
Hubers et al. reported that the sensitivity of EGFR mutation detection was 30 to 50% in 10 sputum samples using four different EGFR mutation analyses (Cycleave PCR, COLD-PCR, Pangaea Biotech SL Technology, and High Resolution Melting) [27]. Su, et al. performed amplification refractory mutation system (ARMS)-PCR for 35 sputum samples containing tumor cells collected from stage III-IV lung cancer patients and reported a 90.9% sensitivity [28]. Wu et al. reported a 63% sensitivity when an EGFR sensitizing mutation was analyzed in 50 sputum samples using next-generation sequencing [29]. Presently, ddPCR analysis of 80 sputum samples revealed an 80.0% detection sensitivity for EGFR mutations if the sputum cytology was positive. Recently, Wang et al. reported that detection sensitivity and specificity for EGFR mutations of 46.2% and 100%, respectively, as detected using SuperARMS using sputum cell-free DNA from 102 sputum samples [30]. It is necessary to prospectively examine in large scale which methodology can detect EGFR mutations more accurately in sputum.
This is the first study to analyze the correlation between pathological findings of surgically resected specimens and SC (+). SC (+) status was strongly associated with STAS. The presence of STAS was higher in patients with SC (+) than in patients with SC (-) (92.3% vs. 34.3%). STAS was a potential predictive factor for SC (+) in multivariate analysis (Table 2). STAS is a risk factor for recurrence of primary lung adenocarcinoma and squamous cell carcinoma [31, 32]. Previously, we examined the morphology and EGFR mutation status of tumor cells in airway secretions collected from segmental or lobar bronchus of surgically resected specimens and compared the results with FFPE tumor tissue. The study demonstrated that STAS may be spread to the respiratory tract as far as segmental or lobar bronchus of the tumor [33]. Because STAS was a risk factor for SC (+) in the present study, we suggest that malignant tumors can be efficiently detected from sputum if STAS is predicted in preoperative radiological findings. Toyokawa et al. reported that the presence of notch and the absence of ground glass opacity were CT findings that were related to the presence of STAS [34]. Kim et al. reported that solid component ratio ≥ 90% in CT was a potential predictive factor of STAS [35]. Performing sputum cytology for tumors that display these CT findings may increase the detection sensitivity of sputum cytology in patients with primary lung cancer.
It is unclear which sputum collection method was appropriate for the analysis of EGFR mutations. Hubers et al. used Saccomanno's fixative (2% polyethylene glycol in 50% ethanol) for 3-day pooled sputum [27, 36]. Fei et al. collected spontaneous sputum in a 1.5 mL container [28]. Wu et al. collected approximately 5 mL of spontaneous sputum in a mixed solution with an equal volume of Saccomanno's fixative and 0.005% dithiothreitol solution at a 1:1 ratio [29, 37]. We collected spontaneous sputum using the YM fixative solution for 3 days. It has been reported that the detection sensitivity of lung cancer in sputum is increased by a longer duration of sputum collection and with the induction of sputum by nebulization with hypertonic saline [36, 38]. A future study should examine whether the sensitivity of EGFR mutation detection can be improved by different sputum collection methodologies.
There were several limitations in our study. First, this study was conducted at a single institution with a small number of patients. Second, we have not investigated the efficacy of EGFR-tyrosine kinase inhibitor based on the EGFR status detected in sputum samples. Third, the methodology of detecting EGFR mutation differed between surgical resected tumor samples and sputum samples. The discordance of EGFR mutations between the two samples might have occurred if the number of tumor cells harboring EGFR mutations with mutations was too small to detect these mutations, due to heterogeneity in FFPE tumor sections by conventional PCR. Fourth, this study examined only the Ex21 and Ex19 mutations. Future studies will need to detect other EGFR mutations, such as T790M, and other driver gene mutations by ddPCR from sputum, and evaluate the usefulness of ddPCR for sputum in clinical practice.