Abstract
Background
The aim of the study was to evaluate the prognostic value of postoperative folate receptor‐positive circulating tumor cell (FR + CTC) detection in patients with stage I–III invasive adenocarcinoma (IAC) treated with surgery.
Methods
Patients with lung adenocarcinoma (LUAD) who underwent surgical resection in Peking University Cancer Hospital and received postoperative FR + CTC analysis from July 2016 to January 2021 were retrospectively collected. Comparisons between or among groups were made using the Kruskal‐Wallis or Mann–Whitney U tests. Survival curves were estimated using the Kaplan–Meier method and compared using the log‐rank test. Cox proportional hazard regression analyses were performed to explore the factors predicting recurrence and survival.
Results
There were significant differences between the high and low groups in terms of age (p = 0.002), postoperative CA199 (p = 0.038), and postoperative SCC (p = 0.024). There were no significant differences in the other indicators (all p>0.05). N stage 1, N stage 2, and neoadjuvant therapy (NAT) were independent risk factors for disease recurrence and death; pleural invasion (PI), and nerve invasion were independent risk factors for death. The Kaplan–Meier curve showed a notable trend for a worse disease‐free survival (DFS) or overall survival (OS) for patients with high levels of FR + CTCs in our study, but none of these were statistically significant.
Conclusion
The detection of FR + CTCs postoperatively was an independent predictor of recurrence in patients treated for stage I–III IAC. Standardized detection methods and optimal time points for assessment should be established in future studies.
Keywords: folate receptor‐positive circulating tumor cells (FR + CTC), invasive adenocarcinoma (IAC), postoperative, recurrence, survival
This single‐center, retrospective, observational study was designed to assess the long‐term prognostic value of FR + CTC levels in IAC patients. A total of 188 patients were included in the final analysis. For DFS, patients with high levels of FR + CTCs had a 1.96‐fold higher risk of disease recurrence than those with low levels (hazard ratio [HR] 1.96, 95% CI: 1.05–3.69, p = 0.036). For OS, pleural invasion (PI) and nerve invasion were independent risk factors for OS. N stage 1, N stage 2, and neoadjuvant therapy (NAT) were independent risk factors for DFS and OS, simultaneity.
INTRODUCTION
Lung cancer is the main cause of cancer‐related death and the second most important tumor in terms of incidence. It is estimated that there were 2.2 million new cases and 1.8 million deaths worldwide in 2020. 1 Non‐small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers, and adenocarcinoma is the most common histological type of NSCLC. 2 Invasive adenocarcinoma of the lung (IAC) is a malignantly transformed epithelial tumor that displays adenoidal differentiation, secretes mucus, and expresses alveolar cell markers. On a pathological level, this tumor type infiltrates the surrounding tissue, contains myofibroblasts, and exhibits growth patterns such as papillae, micropapillae, and solid, all of which exceed a size of 5 mm. 3 Surgical resection is the main treatment for resectable tumors in early stages of NSCLC. Adjuvant therapy, which includes radiation, chemotherapy, and targeted therapy, is added whenever the tumor is considered resectable. 4 Little is currently known about the optimal surgical approach for early‐stage IAC, nor is it clear whether the clinical efficacy of segmentectomy is equivalent to lobectomy. 5
Prognosis mainly depends on the tumor stage at diagnosis. Despite optimal treatment, 5 years survival rates are lower than expected in early‐stage NSCLC when compared with other cancer types, probably because of the risk of relapse after surgery, which is nearly 25% for local progression and an additional 13% for relapse in stage I and II disease. 6 Pathological features have been identified that predict relative aggressive behavior and early recurrence in resected NSCLC. 7 These attributes described on surgical pathology, such as tumor diameter, differentiation, and lymphovascular invasion (LVI), 8 visceral pleural invasion (VPI), lymph node involvement, and advanced pathological stage, 9 are widely accepted as prognostic features; however, due to the fact that these features are static and represent the disease status at the time of surgical resection, they tend to lose their predictive power over time. Few biomarkers exist for biology and its application in NSCLC that predict the disease status over the adjuvant treatment and follow‐up.
Liquid biopsy is most likely to fulfill the role as a dynamic biomarker, and has been one of the most promising areas in oncology in the past decade years, providing a well‐performed noninvasive tool for diagnosis and monitoring cancer status. Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) are the main biomarkers analyzed by liquid biopsy. Others such as RNA, tumor‐educated platelets, and extracellular vesicles also hold potential. 10 ctDNA consists of small fragments of free nucleic acid harboring specific tumor mutations that can be detected by polymerase chain reaction (PCR), or next‐generation sequencing (NGS). 11 CTCs are intact and viable cells that can be distinguished from normal immune cells in blood based on size, deformability, density, and electrical charge, or by using antibodies to epithelial and/or mesenchymal proteins, by negative selection through leukocyte depletion using anti‐CD45 antibodies, 12 or maybe labeled with a conjugate of a tumor‐specific ligand folic acid and a synthesized oligonucleotide, as described in a previous study. 13
Folate receptor‐positive CTCs (FR + CTCs) are circulating rare cells with high expression of FRs on the cell surface and can be reliably quantified using commercially available kits. 14 The prognostic value of FR + CTCs has been studied in different cancer types, particularly in lung cancer, in which it has proved to be associated with a higher risk of relapse and worse prognosis. 14 , 15 , 16 However, these studies focused on the prognostic value of the level of FR + CTC before treatment. In recent years, studies have successively reported the prediction of postoperative CTC on the prognosis of cancer patients, especially recurrence and survival. 17 , 18 , 19 Surgery is the inevitable choice for stage I–III NSCLC patients, and postoperative CTCs appear to be more likely to predict prognosis than preoperative CTCs. However, few studies have been performed to determine whether postoperative FR + CTCs levels are correlated with prognosis in patients with stage I–III NSCLC. In this context, the aim of the present study was to evaluate the prognostic value of postoperative FR + CTC detection in patients with stage I–III IAC treated with surgery. To our knowledge, this is the first study to only use follow‐up FR + CTCs after surgery to predict recurrence and death in IAC patients.
METHODS
Study subjects
This was a single‐center, retrospective, observational study designed to assess the long‐term prognostic value of FR + CTC level in IAC patients. A total of 223 patients with lung adenocarcinoma (LUAD) who underwent surgical resection in Peking University Cancer Hospital and received FR + CTC analysis postoperatively were enrolled from July 2016 to January 2021. The inclusion criteria were as follows: (1) patients were between 18 and 80 years old, (2) the final pathological diagnosis confirmed LUAD, (3) the Eastern Cooperative Oncology Group (ECOG) score of the patient was 0–1 and (4) FR + CTC analysis after surgery was performed. The exclusion criteria were as follows: (1) the patient had other malignant tumors in the past and (2) the period between surgery and CTC detection was more than 2 months. The study was approved by the ethics committee of Peking University Cancer Hospital and Institute (no. 2018KT30).
Data collection
Age, gender, ECOG score, smoking history, tumor size, subtype, LVI, pleural invasion (PI), spread through air spaces (STAS), nerve invasion, therapeutic regimen, genetic mutations, and the level of tumor biomarkers and FR + CTC level were obtained from the hospital information system (HIS). Clinical disease staging was based on the eighth edition of the tumor node metastasis (TNM) classification for lung cancer. 20
FR + CTC analysis
FR + CTCs were analyzed and quantified by the CytoploRare kit (Genosaber Biotech). In brief, first, 3 mL of peripheral blood was withdrawn into an ethylenediaminetetraacetic acid (EDTA)‐containing anticoagulant tube from each subject. Second, CTCs were enriched by lysis of erythrocytes and then immunomagnetic depletion of leukocytes from the whole blood. Third, FR + CTCs were quantified by ligand‐targeted polymerase chain reaction (LT‐PCR), as previously described. 13 , 14 A self‐referenced CTC unit (denoted “FU”) derived from standard curve was used to indicate the abundance of FR + CTCs in 3 mL peripheral blood. For examples, 8.7 FU indicates 8.7 FU in 3 mL of whole blood. A series of standards containing oligonucleotides (10–14 to 10−9 M, corresponding to 2–2 × 105 CTC units/3 mL blood) were used for FR + CTC quantification.
Tumor biomarker detection
Carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA125), carbohydrate antigen 199 (CA199), carbohydrate antigen 153, neuron‐specific enolase (NSE), cytokeratin 19 fragment (CYFRA21‐1), and squamous cell carcinoma (SCC) antigen were measured by chemiluminescent immunoassay. Positive criterion: CEA ≥5.00 ng/mL, CA125 ≥ 35.00 U/mL, CA199 ≥ 27.00 U/mL, NSE ≥17.5 μg/L, CYFRA21‐1 ≥ 3.3 ng/mL and SCC ≥2 ng/L.
Follow‐up
Patients were regularly followed up for 7 years after surgery, and they were provided with standard care during the follow‐up period. Follow‐up was conducted with each patient until relapse, death or December 2023. Disease‐free survival (DFS) was defined as the period between the time of surgery and cancer recurrence or metastasis or death. Overall survival (OS) was defined as the period between the time of surgery and death from any cause or until December 2023.
Statistical analysis
Categorical data are presented as counts and percentages and were compared using Fisher's exact test. FR + CTC levels are presented as medians with interquartile ranges (IQR) and were compared using the Kruskal–Wallis or Mann–Whitney U tests. The optimal cutoff values of FR + CTC level to stratify the study population into different prognostic groups were identified by the receiver operator characteristic (ROC) curve. Survival curves were estimated using the Kaplan–Meier method and compared using the log‐rank test. Cox proportional hazard regression analysis was performed to explore the factors predicting the recurrence and survival. Potentially significant covariates (p < 0.05) in the univariate analysis were selected for subsequent multivariate analysis, and the final multivariate model was developed by stepwise regression to obtain the best result with the smallest Akaike information criterion (AIC). 21 All statistical analyses were performed using R version 4.2.1 (R Foundation for Statistical Computing, Vienna, Austria, https://cran.r-project.org). A p‐value less than 0.05 was considered statistically significant.
RESULTS
Patient characteristics
A total of 188 patients were included in the final analysis of which 35 were excluded for the following reasons: two patients had nonprimary LUAD; another two patients had a period between surgery and CTC detection of more than 2 months; one had distant metastasis; 32 had adenocarcinoma in situ (AIS) or minimally invasive adenocarcinoma (MIA) (Figure 1). In the cohort, 105 women (55.9%) and 83 men (44.1%) were included, with a median age of 57.0 years. A total of 60 patients were current or former smokers. Regarding tumor histology, acinar or lepidic accounted for 42% and 33%, which were the first and second most prevalent subtypes, respectively. For the TNM stage distribution, 77.1% of patients were stage I, 6.9% were stage II, and 16.0% were stage III. In our study, the majority of patients underwent anatomic resection (94.7%), while only 10 patients did not undergo the procedure. The full results regarding clinical characteristics, tumor biomarkers, and gene detection are shown in Table 1.
FIGURE 1.
Patient section flow chart. AIS, adenocarcinoma in situ; CTC, circulating tumor cell; LUAD, lung adenocarcinoma; MIA, minimally invasive adenocarcinoma.
TABLE 1.
Baseline information of 188 patients with invasive lung adenocarcinoma.
| Characteristics | Level | Overall (n = 188) |
|---|---|---|
| Age (median [IQR], years) | 57.00 [52.75, 63.00] | |
| Sex (n, %) | Female | 105 (55.9) |
| Male | 83 (44.1) | |
| Smoking history (n, %) | No | 128 (68.1) |
| Yes | 60 (31.9) | |
| ECOG PS (n, %) | 0 | 52 (27.7) |
| 1 | 136 (72.3) | |
| Tumor size (median [IQR], cm) | 2.00 [1.50, 2.80] | |
| Predominant histological subtype of IAC (n, %) | Papillary | 15 (8.0) |
| Solid | 15 (8.0) | |
| Micropapillary | 15 (8.0) | |
| Mucinous adenocarcinoma | 2 (1.1) | |
| Acinar | 79 (42.0) | |
| Lepidic | 62 (33.0) | |
| STAS (n, %) | No | 177 (94.1) |
| Yes | 11 (5.9) | |
| PI (n, %) | No | 143 (76.1) |
| Yes | 45 (23.9) | |
| LVI (n, %) | No | 168 (89.4) |
| Yes | 20 (10.6) | |
| Nerve invasion (n, %) | No | 184 (97.9) |
| Yes | 4 (2.1) | |
| T stage (n, %) | 1 | 126 (67.0) |
| 2 | 54 (28.7) | |
| 3 | 6 (3.2) | |
| 4 | 2 (1.1) | |
| N stage (n, %) | 0 | 148 (78.7) |
| 1 | 14 (7.4) | |
| 2 | 26 (13.8) | |
| M stage (n, %) | 0 | 188 (100.0) |
| TNM stage (n, %) | 1 | 145 (77.1) |
| 2 | 13 (6.9) | |
| 3 | 30 (16.0) | |
| EGFR (n, %) | Wild‐type | 72 (38.3) |
| Mutation | 116 (61.7) | |
| KRAS (n, %) | Wild‐type | 173 (92.0) |
| Mutation | 15 (8.0) | |
| ROS1 (n, %) | Wild‐type | 178 (94.7) |
| Mutation | 10 (5.3) | |
| NAT (n, %) | No | 174 (92.6) |
| Yes | 14 (7.4) | |
| Surgical procedure | ||
| Anatomical resection | 178 (94.7) | |
| Right upper lobectomy | 72 (40.5) | |
| Right middle lobectomy | 11 (6.2) | |
| Right lower lobectomy | 31 (17.4) | |
| Left upper lobectomy | 34 (19.1) | |
| Left lower lobectomy | 26 (14.6) | |
| Segmentectomy | 4 (2.2) | |
| No anatomical resection | Wedge resection | 10 (5.3) |
| Postoperative adjuvant treatment (n, %) | No | 140 (74.5) |
| Yes | 48 (25.5) | |
| Postoperative CTC (median [IQR], FU/3 mL) | 8.52 [6.63, 11.95] | |
| Postoperative CEA (median [IQR], ng/mL) | 1.63 [0.97, 2.16] | |
| Postoperative CA199 (median [IQR], U/mL) | 9.09 [5.73, 16.35] | |
| Postoperative CA125 (median [IQR], U/mL) | 10.48 [6.89, 13.21] | |
| Postoperative NSE (median [IQR], U/mL) | 10.55 [9.23, 12.43] | |
| Postoperative CYFRA21‐1 (median [IQR], ng/mL) | 1.88 [1.42, 2.57] | |
| Postoperative SCC (median [IQR], μg/L) | 0.60 [0.50, 0.80] | |
Abbreviations: CA125, carbohydrate antigen 125; CA199, carbohydrate antigen 199, CEA, carcinoembryonic antigen; CTC, circulating tumor cell; CYFRA21‐1, cytokeratin 19 fragment; ECOG PS, Eastern Cooperative Oncology Group performance status; IIAC, invasive adenocarcinoma; QR, interquartile range; LVI, lymphovascular invasion; NAT, neoadjuvant therapy; NSE, neuron‐specific enloase; PI, pleural invasion; SCC, squamous cell carcinoma; STAS, spread through air spaces; TNM, tumor node metastasis.
Correlation between postoperative FR + CTC levels and clinical characteristics
FR + CTC levels were detected during the period after surgery in 188 patients. According to the median of the FR + CTC level, the patients were divided into low (≤8.52 FU/3 mL) and high groups (>8.52 FU/3 mL). There were significant correlations between the two groups with respect to age (p = 0.002), postoperative CA199 (p = 0.038), and postoperative SCC (p = 0.024). There were no significant differences in sex, histological subtype, STAS, PI, LVI, nerve invasion, or TNM stage. Additionally, there were no significant differences in gene mutation status of EGFR, KRAS, or ROS1, in neoadjuvant therapy (NAT) or postoperative adjuvant treatment, and the other biomarker levels postoperatively (Table 2).
TABLE 2.
The correlation of postoperative FR + CTC and patient characteristics.
| Characteristics | Group | FR + CTC (FU/3 mL) | p‐value | |
|---|---|---|---|---|
| ≤8.52 (n = 94) | >8.52 (n = 94) | |||
| Age (median [IQR]) | 56.00 [52.00, 61.00] | 60.00 [54.00, 65.75] | 0.002 | |
| Sex (%) | Female | 49 (52.1) | 56 (59.6) | 0.378 |
| Male | 45 (47.9) | 38 (40.4) | ||
| Smoking history (%) | No | 64 (68.1) | 64 (68.1) | 1.000 |
| Yes | 30 (31.9) | 30 (31.9) | ||
| ECOG PS (%) | 0 | 27 (28.7) | 25 (26.6) | 0.870 |
| 1 | 67 (71.3) | 69 (73.4) | ||
| Tumor size (median [IQR]) | 1.90 [1.50, 2.50] | 2.10 [1.50, 3.00] | 0.127 | |
| Predominant histological subtype of IAC (n, %) | Papillary | 7 (7.4) | 8 (8.5) | 0.897 |
| Solid | 6 (6.4) | 9 (9.6) | ||
| Micropapillary | 6 (6.4) | 9 (9.6) | ||
| Mucinous adenocarcinoma | 1 (1.1) | 1 (1.1) | ||
| Acinar | 41 (43.6) | 38 (40.4) | ||
| Lepidic | 33 (35.1) | 29 (30.9) | ||
| STAS (n, %) | No | 90 (95.7) | 87 (92.6) | 0.534 |
| Yes | 4 (4.3) | 7 (7.4) | ||
| PI (n, %) | No | 70 (74.5) | 73 (77.7) | 0.732 |
| Yes | 24 (25.5) | 21 (22.3) | ||
| LVI (n, %) | No | 80 (85.1) | 88 (93.6) | 0.098 |
| Yes | 14 (14.9) | 6 (6.4) | ||
| Nerve invasion (n, %) | No | 91 (96.8) | 93 (98.9) | 0.613 |
| Yes | 3 (3.2) | 1 (1.1) | ||
| T stage (%) | 1 | 66 (70.2) | 60 (63.8) | 0.461 |
| 2 | 25 (26.6) | 29 (30.9) | ||
| 3 | 3 (3.2) | 3 (3.2) | ||
| 4 | 0 (0.0) | 2 (2.1) | ||
| N stage (%) | 0 | 72 (76.6) | 76 (80.9) | 0.696 |
| 1 | 7 (7.4) | 7 (7.4) | ||
| 2 | 15 (16.0) | 11 (11.7) | ||
| TNM stage (%) | 1 | 71 (75.5) | 74 (78.7) | 0.686 |
| 2 | 8 (8.5) | 5 (5.3) | ||
| 3 | 15 (16.0) | 15 (16.0) | ||
| EGFR (n, %) | Wild‐type | 38 (40.4) | 34 (36.2) | 0.653 |
| Mutation | 56 (59.6) | 60 (63.8) | ||
| KRAS (n, %) | Wild‐type | 89 (94.7) | 84 (89.4) | 0.282 |
| Mutation | 5 (5.3) | 10 (10.6) | ||
| ROS1 (n, %) | Wild‐type | 91 (96.8) | 87 (92.6) | 0.330 |
| Mutation | 3 (3.2) | 7 (7.4) | ||
| NAT (n, %) | No | 85 (90.4) | 89 (94.7) | 0.405 |
| Yes | 9 (9.6) | 5 (5.3) | ||
| Postoperative adjuvant treatment (n, %) | No | 67 (71.3) | 73 (77.7) | 0.403 |
| Yes | 27 (28.7) | 21 (22.3) | ||
| Postoperative CEA (median [IQR], ng/mL) | 1.63 [1.10, 2.16] | 1.59 [0.94, 2.16] | 0.389 | |
| Postoperative CA199 (median [IQR], ku/L) | 7.95 [5.18, 14.99] | 10.43 [6.92, 16.54] | 0.038 | |
| Postoperative CA125 (median [IQR], U/mL) | 10.01 [6.82, 12.43] | 10.57 [6.91, 13.89] | 0.270 | |
| Postoperative NSE (median [IQR], U/mL) | 10.37 [9.23, 12.35] | 10.70 [9.06, 12.43] | 0.737 | |
| Postoperative CYFRA21‐1 (median [IQR], ng/mL) | 1.71 [1.39, 2.50] | 2.01 [1.46, 2.84] | 0.162 | |
| Postoperative SCC (median [IQR], μg/L) | 0.70 [0.50, 0.80] | 0.60 [0.40, 0.70] | 0.024 | |
Abbreviations: CA125, carbohydrate antigen 125; C199, carbohydrate antigen 199, CEA, carcinoembryonic antigen; CTC, circulating tumor cell; CYFRA21‐1, cytokeratin 19 fragment; ECOG PS, Eastern Cooperative Oncology Group performance status; IIAC, invasive adenocarcinoma; QR, interquartile range; LVI, lymphovascular invasion; NAT, neoadjuvant therapy; NSE, neuron‐specific enloase; PI, pleural invasion; SCC, squamous cell carcinoma; STAS, spread through air spaces; TNM, tumor node metastasis.
Prognostic significance of FR + CTC levels
By drawing a ROC curve, the cutoff value of FR + CTC to distinguish DFS was 9.865 FU/3 mL (Se = 0.500, Sp = 0.669, AUC = 0.537 [0.433–0.641]). According to the cutoff, the patients were divided into the high group (FR + CTC>9.865 FU/3 mL) and low group (FR + CTC≤9.865 FU/3 mL). Kaplan–Meier curves comparing the time to recurrence or death distributions of the FR + CTC high and low groups are shown in Figure 2. We found no differences between the patients who had high postsurgical FR + CTCs and patients who had low postsurgical FR + CTCs in terms of 5‐year DFS (69.8% vs. 83.0%, p = 0.072), or 5‐year OS (86.8% vs. 93.4%, p = 0.17).
FIGURE 2.
Kaplan–Meier Curves (a) The time to recurrence for patients with high or low postoperative folate receptor‐positive circulating tumor cell (FR + CTC) levels. (b) The time to death for patients with high or low postoperative FR + CTC levels.
Cox regression analysis
For DFS, patients with high FR + CTC levels had a 1.96‐fold higher risk of disease recurrence than those with low levels (hazard ratio [HR] 1.96, 95% CI: 1.05–3.69, p = 0.036). Meanwhile, tumor size, IVI, nerve invasion, T3 versus T1 stage, N stage, TNM stage, NAT, postoperative adjuvant treatment, and postoperative CEA were factors which affected recurrence in the univariate analysis (all p < 0.05). N stage 1 (HR 9.09, 95% CI: 3.82–21.63, p < 0.001), N stage 2 (HR 6.88, 95% CI: 3.25–14.56, p < 0.001), and NAT (HR 3.53, 95% CI: 1.62–7.69, p = 0.001) were the independent risk factors for disease recurrence in the multivariate analysis. The univariate and multivariate results of the COX prediction model are summarized in Table 3.
TABLE 3.
Univariate and multivariate analyses for DFS.
| Characteristics | Univariate analyses | Multivariate analyses | ||||
|---|---|---|---|---|---|---|
| HR | 95% CI | p‐value | HR | 95% CI | p‐value | |
| Sex | ||||||
| Female | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| Male | 1.30 | 0.70–2.41 | 0.410 | ‐ | ‐ | ‐ |
| Age | 1.00 | 0.97–1.04 | 0.929 | ‐ | ‐ | ‐ |
| Smoking history | ||||||
| No | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| Yes | 1.65 | 0.88–3.09 | 0.118 | ‐ | ‐ | ‐ |
| ECOG PS | ||||||
| 0 | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| 1 | 1.61 | 0.74–3.51 | 0.226 | ‐ | ‐ | ‐ |
| Tumor size | 1.41 | 1.20–1.67 | <0.001 | ‐ | ‐ | ‐ |
| Predominant histological subtype of IAC | ||||||
| Acinar, lepidic or papillary | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| Solid or micropapillary | 1.96 | 0.95–4.03 | 0.067 | ‐ | ‐ | ‐ |
| STAS | ||||||
| No | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| Yes | 1.37 | 0.42–4.47 | 0.598 | ‐ | ‐ | ‐ |
| PI | ||||||
| No | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| Yes | 1.61 | 0.83–3.14 | 0.160 | ‐ | ‐ | ‐ |
| IVI | ||||||
| No | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| Yes | 3.32 | 1.62–6.82 | 0.001 | ‐ | ‐ | ‐ |
| Nerve invasion | ||||||
| No | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| Yes | 4.48 | 1.38–14.59 | 0.013 | ‐ | ‐ | ‐ |
| T stage | ||||||
| 1 | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| 2 | 1.78 | 0.91–3.48 | 0.091 | ‐ | ‐ | ‐ |
| 3 | 4.18 | 1.24–14.11 | 0.021 | ‐ | ‐ | ‐ |
| 4 | 4.76 | 0.64–35.64 | 0.128 | ‐ | ‐ | ‐ |
| N stage | ||||||
| 0 | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| 1 | 10.98 | 4.78–25.23 | <0.001 | 9.09 | 3.82–21.63 | <0.001 |
| 2 | 7.69 | 3.74–15.82 | <0.001 | 6.88 | 3.25–14.56 | <0.001 |
| TNM | ||||||
| I | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| II | 9.65 | 4.04–23.10 | <0.001 | ‐ | ‐ | ‐ |
| III | 8.17 | 4.00–16.67 | <0.001 | ‐ | ‐ | ‐ |
| EGFR | ||||||
| Wild‐type | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| Mutation | 0.71 | 0.38–1.33 | 0.288 | ‐ | ‐ | ‐ |
| KRAS | ||||||
| Wild‐type | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| Mutation | 1.88 | 0.73–4.81 | 0.189 | ‐ | ‐ | ‐ |
| ROS1 | ||||||
| Wild‐type | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| Mutation | 0.97 | 0.23–4.06 | 0.967 | ‐ | ‐ | ‐ |
| NAT | ||||||
| No | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| Yes | 6.18 | 2.99–12.78 | <0.001 | 3.53 | 1.62–7.69 | 0.001 |
| Postoperative adjuvant treatment | ||||||
| No | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| Yes | 2.31 | 1.22–4.37 | 0.010 | ‐ | ‐ | ‐ |
| Postoperative FR + CTC | ||||||
| <=9.865 | ||||||
| >9.865 | 1.76 | 0.94–3.26 | 0.075 | 1.96 | 1.05–3.69 | 0.036 |
| Postoperative CEA | 1.05 | 1.01–1.08 | 0.006 | ‐ | ‐ | ‐ |
| Postoperative CA199 | 1.01 | 0.99–1.04 | 0.225 | ‐ | ‐ | ‐ |
| Postoperative CA125 | 1.01 | 0.97–1.05 | 0.601 | ‐ | ‐ | ‐ |
| Postoperative NSE | 1.02 | 0.93–1.13 | 0.610 | ‐ | ‐ | ‐ |
| Postoperative CYFRA21‐1 | 0.94 | 0.67–1.31 | 0.714 | ‐ | ‐ | ‐ |
| Postoperative SCC | 0.42 | 0.12–1.48 | 0.177 | ‐ | ‐ | ‐ |
Abbreviations: CA125, carbohydrate antigen 125; CA199, carbohydrate antigen 199, CEA, carcinoembryonic antigen; CI, confidence interval; CTC, circulating tumor cell; CYFRA21‐1, cytokeratin 19 fragment; DFS, disease‐free survival; ECOG PS, Eastern Cooperative Oncology Group performance status; EGFR, epidermal growth factor receptor; FR + CT, folate receptor‐positive circulating tumor cell; HR, hazard ratio; IAC, invasive adenocarcinoma; IQR, interquartile range; LVI, lymphovascular invasion; NAT, neoadjuvant therapy; NSE, neuron‐specific enloase; PI, pleural invasion; SCC, squamous cell carcinoma; STAS, spread through air spaces; TNM, tumor node metastasis.
For OS, patients with high FR + CTC levels had a 1.92‐fold higher risk of death than those with low levels in the univariate analysis; however, there was no significant difference (p = 0.181). Meanwhile, tumor size, solid or micropapillary histological subtype, PI, IVI, nerve invasion, T3 versus T1 stage, T4 versus T1 stage, N stage, TNM stage, NAT, postoperative adjuvant treatment, and postoperative CEA were factors which affected recurrence (all p<0.05). In the multivariate analysis, PI (HR 3.47, 95% CI: 1.21–9.97, p = 0.021), nerve invasion (HR 4.29, 95% CI: 1.01–18.27, p = 0.049), N stage 1 (HR 23.90, 95% CI: 6.42–88.99, p < 0.001), N stage 2 (HR 6.03, 95% CI: 1.70–21.37, p = 0.005), and NAT (HR 8.67, 95% CI: 2.82–26.73, p < 0.001) were independent risk factors for death. The univariate and multivariate results of the COX prediction model are summarized in Table 4.
TABLE 4.
Univariate and multivariate analyses for OS.
| Characteristics | Univariate analyses | Multivariate analyses | ||||
|---|---|---|---|---|---|---|
| HR | 95% CI | p‐value | HR | 95% CI | p‐value | |
| Sex | ||||||
| Female | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| Male | 1.15 | 0.45–2.99 | 0.767 | ‐ | ‐ | ‐ |
| Age | 1.01 | 0.96–1.07 | 0.646 | ‐ | ‐ | ‐ |
| Smoking history | ||||||
| No | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| Yes | 1.95 | 0.75–5.06 | 0.169 | ‐ | ‐ | ‐ |
| ECOG PS | ||||||
| 0 | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| 1 | 1.88 | 0.54–6.56 | 0.322 | ‐ | ‐ | ‐ |
| Tumor size | 1.71 | 1.39–2.11 | <0.001 | ‐ | ‐ | ‐ |
| Predominant histological subtype of IAC | ||||||
| Acinar, lepidic or papillary | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| Solid or micropapillary | 3.92 | 1.49–10.31 | 0.006 | ‐ | ‐ | ‐ |
| STAS | ||||||
| No | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| Yes | 2.14 | 0.49–9.38 | 0.312 | ‐ | ‐ | ‐ |
| PI | ||||||
| No | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| Yes | 3.84 | 1.48–9.96 | 0.006 | 3.47 | 1.21–9.97 | 0.021 |
| IVI | ||||||
| No | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| Yes | 4.83 | 1.78–13.07 | 0.002 | ‐ | ‐ | ‐ |
| Nerve invasion | ||||||
| No | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| Yes | 11.08 | 3.17–38.68 | 0.000 | 4.29 | 1.01–18.27 | 0.049 |
| T stage | ||||||
| 1 | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| 2 | 2.83 | 0.95–8.43 | 0.061 | ‐ | ‐ | ‐ |
| 3 | 15.92 | 3.93–64.46 | 0.000 | ‐ | ‐ | ‐ |
| 4 | 18.22 | 2.17–153.03 | 0.008 | ‐ | ‐ | ‐ |
| N stage | ||||||
| 0 | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| 1 | 21.77 | 6.13–77.30 | <0.001 | 23.90 | 6.42–88.99 | <0.001 |
| 2 | 11.25 | 3.29–38.49 | <0.001 | 6.03 | 1.70–21.37 | 0.005 |
| TNM | ||||||
| I | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| II | 22.76 | 5.43–95.33 | <0.001 | ‐ | ‐ | ‐ |
| III | 17.29 | 4.67–63.98 | <0.001 | ‐ | ‐ | ‐ |
| EGFR | ||||||
| Wild‐type | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| Mutation | 0.41 | 0.16–1.09 | 0.074 | ‐ | ‐ | ‐ |
| KRAS | ||||||
| Wild‐type | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| Mutation | 1.58 | 0.36–6.93 | 0.543 | ‐ | ‐ | ‐ |
| ROS1 | ||||||
| Wild‐type | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| Mutation | 0.00 | 0.00 −s Inf | 0.997 | ‐ | ‐ | ‐ |
| NAT | ||||||
| No | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| Yes | 12.05 | 4.55–31.9 | <0.001 | 8.67 | 2.82–26.73 | <0.001 |
| Postoperative adjuvant treatment | ||||||
| No | ‐ | ‐ | ‐ | ‐ | ‐ | ‐ |
| Yes | 1.27 | 0.45–3.59 | 0.659 | ‐ | ‐ | ‐ |
| Postoperative FR + CTC | ||||||
| <=9.865 | ||||||
| >9.865 | 1.92 | 0.74–4.97 | 0.181 | ‐ | ‐ | ‐ |
| Postoperative CEA | 1.07 | 1.03–1.11 | 0.000 | ‐ | ‐ | ‐ |
| Postoperative CA199 | 1.02 | 0.99–1.05 | 0.140 | ‐ | ‐ | ‐ |
| Postoperative CA125 | 1.04 | 0.99–1.08 | 0.117 | ‐ | ‐ | ‐ |
| Postoperative NSE | 0.96 | 0.81–1.13 | 0.604 | ‐ | ‐ | ‐ |
| Postoperative CYFRA21‐1 | 1.00 | 0.60–1.66 | 0.995 | ‐ | ‐ | ‐ |
| Postoperative SCC | 0.70 | 0.12–4.10 | 0.690 | ‐ | ‐ | ‐ |
Abbreviations: Abbreviations: CA125, carbohydrate antigen 125; CA199, carbohydrate antigen 199, CEA, carcinoembryonic antigen; CI, confidence interval; CTC, circulating tumor cell; CYFRA21‐1, cytokeratin 19 fragment; ECOG PS, Eastern Cooperative Oncology Group performance status; EGFR, epidermal growth factor receptor; FR + CT, folate receptor‐positive circulating tumor cell; HR, hazard ratio; IAC, invasive adenocarcinoma; IQR, interquartile range; LVI, lymphovascular invasion; NAT, neoadjuvant therapy; NSE, neuron‐specific enloase; OS, overall survival; PI, pleural invasion; SCC, squamous cell carcinoma; STAS, spread through air spaces; TNM, tumor node metastasis.
DISCUSSION
In this study, we identified a cohort of patients who underwent surgery and had FR + CTCs detected during postoperative follow‐up. Some reports have described CTC detection in NSCLC patients after surgical manipulation, although the clinical relevance of these postoperatively detected CTCs remains controversial.
Associations of postoperative CTCs with recurrence and OS have been reported in breast, colorectal and pancreatic cancer.s 18 , 22 , 23 Distant metastasis is common in patients with CTC clusters detected immediately after surgery, which suggests that CTC clusters may predict the development of distant metastasis in patients with NSCLC. 17 We found that postoperative FR + CTCs were an independent risk factor for disease recurrence in patients with stage I–III IAC. However, there were no differences in 5‐year DFS between patients with high and low levels of postsurgical FR + CTCs. Therefore, monitoring persistent postsurgical CTCs can provide a window into residual disease and timely detection of disease recurrence. Additionally, in our study, N stage and NAT were independent risk factors for disease recurrence. PI, nerve invasion, N stage, and NAT were the independent risk factors for death. PI, nerve invasion, N stage were common factors for poor prognosis, which have been proved in previous studies. NAT was a risk factor for disease recurrence and death, and it was a controversial item. It is generally believed that neoadjuvant therapy is considered to be beneficial to the prognosis of patients; at least, it is not a risk factor for prognosis. 18 Some potential reasons for NAT being a risk factor for tumor recurrence and death include: (1) IAC is more aggressive subtype than the other subtypes, and the biology of the tumor may determine its response to treatment. Tumors with more aggressive biology may not respond well to neoadjuvant therapy, leading to a higher risk of recurrence or death. There may be residual cancer cells that are not eliminated, even after surgery and adjuvant therapy. These cells can lead to recurrence if they are not fully eliminated. (2) Neoadjuvant therapy can cause immune‐related side effects that can weaken the patient's immune system. A weakened immune system may not be able to fight off cancer cells effectively, leading to a higher risk of recurrence or death. It is important to note that these factors are not exclusive, but the risk of recurrence or death is influenced by multiple factors working together. Therefore, patients should closely follow their doctors' recommendations and continue regular check‐ups to monitor for any signs of recurrence. Moreover, the Kaplan–Meier curve showed a notable trend for a worse DFS or OS for a high level of FR + CTCs patients in our study, but the differences were not statistically significant. Further studies may be able to address these questions in the future.
In our study, FR + CTCs were detected in all patients within 2 months postoperatively. The median of FR + CTCs level (8.52 FU/3 mL) was also lower than the diagnostic positive reference value (8.70 FU/3 mL) in lung cancer. Wei et al. also recently reported that the postoperative FR + CTC increased after surgery in nearly half of the patients, regardless of the procedure and sequences of vessel ligation during surgery in NSCLC patients. 15 Sawabata et al. suggested that surgical manipulation can lead to seeding of CTCs, which become detectable after surgery. 17 These results are inconsistent with each other, possibly because the time of blood sampling after surgery was different, postoperative blood samples were harvested immediately after the chest was closed in Wei et al. study, while the blood samples were harvested within 2 months in our study. Furthermore, in the 2 months after surgery, some patients received different adjuvant therapies according to their disease condition, which will also affect the FR + CTC results.
We demonstrated that postoperative FR + CTCs were associated with age, postoperative CA199, and postoperative SCC. FR + CTC was not correlated with major pathological features, including tumor size, positive lymph nodes, histological grade, or perineural invasion. Similar results have been reported in previous studies. 18 , 24 When compared to tumor‐centered pathological features, FR + CTCs postoperatively are more likely to be seeded from micrometastases, which can occur at any time, even during the early stages before the formation of primary tumors. As a result, FR + CTCs at postoperative may not reflect the characteristics of the primary tumor at the time of resection. Instead, FR + CTCs may provide a more direct assessment of the driving source of recurrence.
Limitations
The present study had several limitations. First, the FR + CTCs detection time points were not uniform for each patient. The blood sample for FR + CTCs test was always done according to the routine of clinical treatment or follow‐up. The available CTC results within a window of one day to 2 months after surgery were used for each patient, and this may be the source of some heterogeneity. Second, since the data were collected retrospectively, we only had postoperative FR + CTC results, which resulted in the inability to analyze the impact of pre‐ and postoperative changes on prognosis. However, these may be the most common scenario clinicians would meet. Third, the small sample size from a single center and the retrospective design of the study were potential sources of bias. Lastly, some patients' follow‐up time were not long enough for recurrence to be adequately observed. Future studies should be designed prospectively, particularly in follow‐up schedules, adjuvant treatment plans, or timely interventions.
The prognostic value of postoperative CTCs in lung cancer patients is an area of active research. The present study revealed that the detection of FR + CTCs postoperatively is an independent predictor of recurrence in patients treated for stage I–III IAC at our center. Further studies are needed to establish standardized detection methods and optimal time points for assessment. The integration of postoperative FR + CTC monitoring into clinical practice may improve patient outcomes by more effective earlier interventions and more personalized therapeutic strategies.
AUTHOR CONTRIBUTIONS
Acquisition of data: All authors. Zeming Ma and Zhiwei Zhou: Data collection and manuscript writing. Zhiwei Zhou and Shijie Wang: Data analysis and manuscript writing. Zeming Ma and Shijie Wang: Data analysis and manuscript editing. Hong Ji and Dachuan Zhao: Manuscript editing. Liang Wang and Jinfeng Chen: Manuscript editing and project development. Liang Wang and Jinfeng Chen: Study concept and project development.
FUNDING INFORMATION
The study was funded by National Natural Science Foundation of China (81773144), Capital Health Research and Development of Special Funds (2018–2‐2155).
CONFLICT OF INTEREST STATEMENT
The authors have no relevant financial or nonfinancial interests to disclose.
Ma Z, Zhou Z, Wang S, Ji H, Zhao D, Wang L, et al. Clinical significance of postoperative folate receptor‐positive circulating tumor cells (FR + CTCs) for long‐term prognosis in patients with invasive adenocarcinoma (IAC) of the lung. Thorac Cancer. 2024;15(13):1060–1071. 10.1111/1759-7714.15288
Zeming Ma, Zhiwei Zhou and Shijie Wang contributed equally to the study.
Contributor Information
Liang Wang, Email: 0065610672@bjmu.edu.cn.
Jinfeng Chen, Email: chenjinfengdoctor@bjmu.edu.cn.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.