Circulating Tumor Cells Predict Prognosis Following First-Generation EGFR-TKI Treatment in EGFR- and TP53-Mutant Non-Small Cell Lung Cancer

Purpose: First-generation EGFR-TKIs have become the rst-line standard treatment for advanced non-small cell lung cancer (NSCLC) with EGFR mutations. This study isolates and quanties circulating tumor cells (CTCs) and evaluates patient prognosis before and after rst-line treatment with EGFR-TKIs in advanced NSCLC with EGFR and TP53 mutation. Methods: Patients with advanced NSCLC with EGFR and TP53 mutation were treated with a rst-generation EGFR-TKI using a standard daily dose. Continuous blood samples were collected at baseline (CTCs-d0) and 28 days (CTCs-d28), and the isolation by size of tumor cells (ISET) method was used to detect CTCs. Results: The CTC results were divided into favorable (< 5 CTCs) and unfavorable ( ≥ 5 CTCs) groups. The median progression-free survival (PFS) of patients in the favorable group was signicantly longer at baseline compared to those in the unfavorable group (15 vs 7.5 months; p = 0.0055). After 28 days of treatment with rst-generation EGFR-TKI, the PFS of patients in the favorable group was 12.5 months, which was signicantly longer than the median PFS of 7 months in the unfavorable group (p = 0.0003). After treatment, the PFS of patients with reduced CTCs was signicantly better than those with no signicant change in CTCs (9 months vs 6 months, p = 0.014). In univariate and multivariate analysis, patients with CTCs-d0 ≥ 5 and CTCs-d28 ≥ 5 had signicantly lower PFS when compared to those with CTCs-d0 < 5 and CTCs-d28 < 5, respectively. Conclusion: This study conrmed for the rst time that CTC count is closely correlated with prognosis in EGFR- and TP53-mutant advanced NSCLC following rst-line treatment with rst-generation EGFR-TKIs.


Introduction
Lung cancer has the highest incidence and mortality of any malignancy (1). Approximately 85% of lung cancer patients have non-small cell lung cancer (NSCLC), for which traditional chemotherapy has limited e cacy and the 5-year survival rate is less than 15% (2). Molecular therapies targeting different driver genes, especially epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI), have become standard treatment for advanced NSCLC (3). Almost all patients become resistant to targeted therapy within 1 year(4). Compared with single mutation tumors, NSCLC patients with multiple somatic mutations in the same tumor have a worse prognosis (5). TP53 is the most commonly mutated gene in NSCLC (6,7).
Current approaches to evaluate the e cacy of targeted therapy for NSCLC mainly include imaging examinations and serum tumor markers, and all approaches have limitations. Studies have shown that circulating tumor cells (CTCs) are related to e cacy of EGFR-TKI in advanced NSCLC (11,12). However, the e cacy of rst-generation EGFR-TKIs (ge tinib, erlotinib, and icotinib) in advanced NSCLC with comutation of EGFR/TP53 remains unclear.
This study analyzed CTCs counts of 31 cases of advanced NSCLC with EGFR/TP53 co-mutation in which patients received rst-line EGFR-TKI treatment. and explored the evaluation of CTCs in the rst-line EGFR- CTC analysis A 6 ml blood collection tube was used to collect a blood sample from the median cubital vein. Immediately following blood collection, the tube was gently inverted and mixed eight times to fully mix the blood with anticoagulant. The specimen was stored and further processed within 24h. Based on differences in size and deformability of abnormal cells and blood cells, abnormal cells were enriched by membrane ltration. The blood sample was pre-processed and transferred to a cell lter. The blood in the cell lter was passed through the lter membrane through the abnormal cell separation staining instrument, where abnormal cells gathered on the surface of the lter membrane to enrich CTCs. The Romanowsky staining technique was used to stain the enriched CTCs with an abnormal cell separation staining instrument. The stained lter membrane was removed, xed on a glass slide, and incubated at 50°C for 30 minutes. After the lter membrane was dry, an appropriate amount of neutral resin mounting tablets were added and the lter was covered with glass. The whole lter was observed under a microscope and counted.
First-generation EGFR-TKIs were administered for at least 1 month; the longest treatment duration was 31 months; the average treatment duration was 11 months. Dosing was as follows: ge tinib 250 mg per os quaque die (po qd); erlotinib (150 mg po qd); and icotinib [125mg po ter in die (tid)]. CTC number was measured before treatment and again 28 days after treatment with ge tinib, erlotinib, or icotinib. According to the Response Evaluation Criteria in Solid Tumors (RECIST) 1.0, complete response (CR) and partial response (PR) were considered to be effective in imaging examinations; CR, PR and stable disease (SD) were considered to be controlled. PFS was calculated from the rst day of treatment to the date of PD.  In univariate analysis, CTCs-d0 and CTCs-d28 were signi cantly correlated with PFS (Table 2). In the multivariate analysis, CTCs-d28 ≥ 5 was the most important prognostic factor among all poor prognostic indicators, which indicates that the favorable group had a lower risk of disease progression (HR: 0.285, 95% CI: 0.121-0.672, p = 0.004, Table 2).  (15). First-generation EGFR-TKIs are the rst-line treatment for advanced NSCLC patients with EGFR mutations. However, drug resistance limits the use of rst-generation EGFR-TKIs. Studies have shown that CTC count is negatively correlated with the e cacy of EGFR-TKI treatment. With the launch of the next-generation sequencing platform, studies have shown that TP53 mutations in EGFR-mutated NSCLC range from 30% to 60% prevalence (9). When treated with a rst-generation EGFR-TKI, patients with EGFR/TP53 double mutations, especially those with missense mutations, exhibit a lower effective rate and PFS compared to advanced NSCLC patients without TP53 mutations (8,16). Predicting e cacy of rst-generation EGFR-TKIs is crucial for the treatment and prognosis of NSCLC patients with EGFR/TP53 co-mutations.

Statistical analysis
In recent years, CTC count and peripheral blood gene expression data have been used to guide the clinical treatment of NSCLC. Punnoose et al. (17) reported that in EGFR-TKI-treated NSCLC patients, the genome expression of CTCs was highly consistent with that of the primary tumor tissue. Maheswaran et al. (18,19) reported that EGFR, EGFR T790M, MET, and other gene mutations in CTCs can be used to evaluate the treatment effect and prognosis of NSCLC patients.
Common CTC detection methods include the isolation by size of tumor cells (ISET) method, the Cell SearchTM system, reverse transcription polymerase chain reaction, CTC-chip, and more. These technologies can signi cantly improve the sensitivity and speci city of CTC detection. ISET is a highspeed cell analysis and sorting technique. Because it is simple, reliable and fast, ISET has been selected as the main method for detecting CTCs (20).
In this study, ISET was used to count CTCs in the peripheral blood of patients with advanced NSCLC. For the rst time, this study demonstrated that in NSCLC patients with EGFR/TP53 mutations, low CTC count was correlated with better PFS after rst-line treatment with rst-generation EGFR-TKIs. The improved PFS was most obvious in patients with reduced CTC count after treatment.
The median PFS of the low CTC group was signi cantly higher than that of the high CTC group both before and after treatment with a rst-generation EGFR-TKI (P<0.01). These data indicate that CTC count is closely related to prognosis of advanced NSCLC in patients with EGFR/TP53 mutations following treatment with a rst-generation EGFR-TKI. This study is not without shortcomings; the small sample size must be expanded to further con rm these ndings. The study was approved by institutional ethics committee of Jiangsu Province Hospital.

Consent for publication
Not applicable.

Availability of supporting data
All data generated or analyzed during this study are available from the corresponding author on reasonable request.

Competing interests
The authors have no relevant competing interests.