Diagnostic accuracy of folate receptor-positive circulating tumor cells in differentiating between benign and malignant pulmonary nodules

Introduction

Pulmonary nodules are small, round, or irregular lesions in the lungs measuring 3 cm or less in diameter (1). They are often indicative of early-stage lung cancer and are being detected with increasing frequency due to advancements in computed tomography (CT) imaging technology (2). Lung cancer remains the most prevalent and lethal form of cancer globally (3). Due to the lack of obvious early symptoms, most patients are not diagnosed until the disease has progressed to later stages (4), having missed the optimal window for surgical intervention. Therefore, early detection and treatment of lung cancer are critical areas of research.

Although tissue biopsy stands as the gold standard for lung cancer diagnosis (5), its invasive nature, limited accessibility, and lack of repeatability present significant challenges. Blood biomarkers such as neuron-specific enolase (NSE), carcinoembryonic antigen (CEA), and squamous cell carcinoma antigen (SCCA) are used for early lung cancer detection (6). However, these markers have limited sensitivity and may only be elevated in the later stages of the disease. There is an urgent need for more sensitive, specific, and accessible blood biomarkers for clinical applications.

Circulating tumor cells (CTCs) have recently gained attention as promising biomarkers for early cancer detection. These cells can be identified in the blood of patients with early-stage tumors and are easily collected through a simple blood draw, facilitating noninvasive and repeatable sampling for tumor diagnosis (7,8). The folate receptor (FR) is a membrane glycoprotein that is overexpressed on the surface of 72–83% of lung cancer cells (9,10). It is expressed by only a small number of activated macrophages in the bloodstream (11), making it a potential biomarker for identifying CTCs in the peripheral blood of patients with lung cancer.

Previous studies have explored the diagnostic potential of folate receptor-positive circulating tumor cells (FR⁺CTCs) in lung cancer (12-14). However, relying solely on FR⁺CTCs as a diagnostic marker for lung cancer in patients with lung nodules has limitations. Previous studies have demonstrated that the accuracy of differentiating lung cancer from benign occupational lung disease can be improved by constructing diagnostic models that include FR-CTC and other biomarkers, but the relevant diagnostic models only incorporate blood biomarkers such as NSE and CEA and have small sample sizes (12,15-18). Studies have indicated that cancer is closely related to inflammation (19,20). In the present study, we included indicators of inflammation in the analyses and enlarged the sample sizes with the aim of obtain a more comprehensive assessment. This study thus aimed to retrospectively assess the feasibility and challenges of using FR⁺CTC in diagnosing lung cancer in patients with lung nodules. Our objective was to develop a composite index that incorporates FR⁺CTCs, offering a novel diagnostic tool to aid in the clinical diagnosis and treatment of early-stage lung cancer. We present this article in accordance with the STARD reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2493/rc).

Methods

Study design and patient enrollment

A total of 3,848 patients who underwent FR⁺CTC detection at The First Affiliated Hospital of Guangzhou Medical University from January 1, 2019 to December 31, 2023 were included through our medical record system.

The inclusion criteria were as follows: (I) Asian ethnicity; (II) age ≥18 years old; (III) definitive pathological assessment of diseased tissue obtained by tissue biopsy or surgical excision; and (IV) maximum diameter of nodule ≤3 cm (as confirmed by X-ray, CT, or magnetic resonance imaging). Meanwhile, the exclusion criteria were as follows: (I) missing key information; (II) lung metastasis from other primary cancers, central lung cancer, and hilar or mediastinal lymphadenopathy; and (III) conditions other than lung cancer and benign lung diseases. The pathological classification was based on the National Comprehensive Cancer Network (NCCN) guidelines (21), and lung cancer staging was established according to the eighth edition the tumor, node, metastasis (TNM) staging system created by the International Association for the Study of Lung Cancer (IASLC) (22). This study was conducted in accordance with the Declaration of Helsinki (as revised in 2013) and was approved by the Ethics Committee of The First Affiliated Hospital of Guangzhou Medical University (No. ES-2024-K108). The requirement for individual consent was waived due to the retrospective nature of the analysis.

Data collection

The following information was collected retrospectively from hospital medical records: gender; age; serum indicators including levels of FR⁺CTCs, white blood cells (WBCs), NSE, CEA, carbohydrate antigen 125 (CA125), CA153, cytokeratin 19 fragment antigen 21-1 (CYFRA21-1), SCCA, procalcitonin (PCT), interleukin-6 (IL-6), IL-8, IL-10, tumor necrosis factor (TNF), and lactate dehydrogenase (LDH); CT findings; and pathological results.

Analysis of tumor biomarkers

CA125, CEA, CYFRA21-1, CA153 and NSE and SCCA were analysed by electrochemiluminescence (Roche Diagnostics, Shanghai, China).

FR⁺CTC analysis

Before any treatment, a volume of 3 milliliters of peripheral blood was drawn from each participant with a 6-milliliter tube that contained ethylenediaminetetraacetic acid (EDTA) for the purpose of FR⁺CTC detection. The FR⁺CTC levels were determined within a 24-hour window following the blood draw through use of a CytoploRare Kit (GenoSsaber Biotech, Shanghai, China). The procedure adhered to the guidelines provided by the kit’s manufacturer.

In brief, the process began with a negative enrichment technique to remove red blood cell and WBC from the samples. Subsequently, the samples underwent ligand-targeted polymerase chain reaction (PCR) to count the FR⁺CTCs. This involved the use of a unique detection probe, which was a folate receptor alpha-targeting folate ligand linked to a specific oligonucleotide to tag the FR⁺CTCs, as described in a literature (11). Finally, a standard PCR was conducted to measure the signal from the oligonucleotide that was attached to the FR⁺CTCs.

Statistical analysis

Numerical variables are presented as numbers and percentages, the mean ± standard deviation, or as the median (with the 25^th and 75^th percentiles). The independent samples t-test or analysis of variance was used to evaluate the statistical significance of numerical variables that conformed to a normal distribution. The Mann-Whitney test or Kruskal-Wallis nonparametric test was used to analyze variables with a nonnormal distribution. The χ² test was used for categorical variables, and the Spearman correlation was used to analyze the correlation between blood biomarkers. The diagnostic value of blood biomarkers was evaluated with the receiver operating characteristic (ROC) curve and area under the curve (AUC). The Hanley-McNeil method was used to evaluate the statistical significance between different curves, and the Youden index was used to determine the optimal cutoff. Random forest model was used to screen the indicators, the variables with weights greater than 0.10 in the random forest model were selected, the variables were arranged and combined to form different combinations of variables, the ROC curve analysis of the combined multiple indicators was carried out by SPSS, the optimal cut-off point was determined by using Jordon’s index, and the sensitivity and specificity corresponding to the optimal cut-off point were the sensitivity and specificity of the combination of variables. Sensitivity = number of true positives/(number of true positives + number of false negatives) and specificity = number of true negatives/(number of true negatives + number of false positives). Statistical analysis was performed using SPSS 25.0 (IBM Corp., Armonk, NY, USA), GraphPad Prism 9.5.0 (GraphPad Software, Inc., San Diego, CA, USA). Finally, bioinformatics tools (https://hiplot.cn, https://hiplot.com.cn, and https://xiantao.love) were used for data visualization. A P value <0.05 was considered statistically significant.

Results

Comparison of basic characteristics between the lung cancer group and benign pulmonary nodule group

Patient general information

A total of 1,135 patients with lung nodules were included, with the enrollment procedure detailed in Figure 1. In this study, patients were categorized into a lung cancer group (n=612) and benign pulmonary nodule group (n=523). The median age was 54 years in the lung cancer group, with 264 (43.1%) being males; the benign pulmonary nodule group had a median age of 53 years old, with 245 (46.8%) being males. There was no notable difference in gender or age between the two groups (P>0.05) (Table 1).

Figure 1 Flowchart of the patient enrollment process. FR⁺CTC, folate receptor-positive circulating tumor cell; COPD, chronic obstructive pulmonary disease; CT, computed tomography.

Patients’ blood indicators

The level of FR⁺CTC was found to be significantly higher in patients with lung cancer compared to those with benign lung nodules (P<0.001). The levels of WBC and cytokeratin 19 fragment antigen 21-1 (CYFRA21-1) were also significantly elevated in the lung cancer group as compared to the benign lung nodule group (P<0.001 and P=0.01, respectively). Other inflammatory markers and tumor biomarkers did not show significant differences between the two groups (Table 1). Correlation analysis showed that the FR⁺CTC level was not correlated with other tumor markers or inflammatory indicators in the benign lung nodule group (P>0.05) (Figure 2A) but was positively correlated with WBC and LDH levels in the lung cancer group (P<0.001 and P=0.01, respectively) (Figure 2B-2D).

Figure 2 Distribution of FR⁺CTCs among enrolled patients. (A) A chord plot illustrating the correlation between FR⁺CTCs and biomarkers in patients with benign pulmonary nodules. The width of the connection arcs represents the strength of the relationship between biomarkers. (B) A chord plot illustrating the correlation between FR⁺CTCs and biomarkers in patients with lung cancer. Scatter plots demonstrate the correlation between FR⁺CTC and (C) WBC and (D) LDH in patients with lung cancer. SCCA, squamous cell carcinoma antigen; PCT, procalcitonin; IL-6, interleukin-6; IL-8, interleukin-8; IL-10, interleukin-10; TNF, tumor necrosis factor; LDH, lactate dehydrogenase; FR⁺CTC, folate receptor-positive circulating tumor cell; WBC, white blood cell; NSE, neuron-specific enolase; CEA, carcinoembryonic antigen; CA125, carbohydrate antigen 125; CA153, carbohydrate antigen 153; CYFRA21-1, cytokeratin 19 fragment antigen 21-1.

The level of FR⁺CTCs in different pathological types and clinical stages of lung cancer

Lung squamous cell carcinoma (LUSC) [FR⁺CTC: 11.9 (9.4, 15.7) FU/3 mL] showed a higher FR⁺CTC level compared to lung adenocarcinoma (LUAD) [FR⁺CTC: 9.5 (6.8, 12.6) FU/3 mL] (P=0.02). However, there was no significant difference in FR⁺CTC levels between LUAD and small cell lung cancer (SCLC) [FR⁺CTC: 10.3 (10.2, 10.6) FU/3 mL] (P=0.61) nor between LUSC and SCLC (P=0.48) (Figure 3).

Figure 3 Distribution of FR⁺CTCs in different pathological types of lung cancer, including LUAD (n=584), LUSC (n=17), and SCLC (n=5). Cases of non-small cell carcinoma with no specific pathological classification (n=1), cases with no precise pathological classification (n=2), cases of ASC (n=1) and LCLC (n=2) were not included in further analysis due to the limited number of cases. NS, no significance, indicates P>0.05; *, P<0.05. LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; SCLC, small-cell lung cancer; FR⁺CTC, folate receptor-positive circulating tumor cell; ASC, adenosquamous carcinoma; LCLC, large-cell lung cancer.

Given that the majority of patients in the lung cancer group had adenocarcinoma (n=584), we conducted an in-depth analysis of the cohort. It was revealed that a large number of patients were diagnosed at stage I (n=458), with a median age of 53 years, and no gender discrepancy was noted among patients with different stages of LUAD. Moreover, there was no statistical variance in FR⁺CTC levels between the various stages (P=0.29) (Table 2).

Comparison of FR⁺CTCs in patients with single or multiple nodules

In this study, chest spiral CT was used to screen for lung nodules, with patients being placed into either a single-nodule group or a multiple-nodule group based on the findings. There were higher FR⁺CTC levels in the single-nodule group compared to the multiple-nodule group in both the benign lung nodule group [single nodule group 7.0 (5.3, 9.9) FU/3 mL; multiple-nodule group 6.5 (4.7, 7.7) FU/3 mL; P=0.002] and the lung cancer group [single-nodule group 9.9 (7.0, 12.8) FU/3 mL; multiple-nodule group 8.9 (6.6, 12.1) FU/3 mL; P=0.040] (Figure 4).

Figure 4 Comparison of FR⁺CTC levels between the single-nodule group and the multiple-nodule group. The distribution of FR⁺CTC levels in the benign pulmonary nodule group (single: n=365; multiple: n=158) and the lung cancer group (single: n=365; multiple: n=247). *, P<0.05; **, P<0.01. FR⁺CTC, folate receptor-positive circulating tumor cell.

Diagnostic efficacy analysis

The ROC analysis (Table 3) revealed that the cutoff value of FR⁺CTC level for diagnosing lung cancer was determined to be 8.7 FU/3 mL (Figure 5A). At this threshold, the sensitivity of diagnosis was 61.9% with a specificity of 75.0% [AUC =0.684; 95% confidence interval (CI): 0.653–0.715]. As the cutoff value increased, the specificity of diagnosis also increased. Specifically, at 1.5 times the cutoff (13.1 FU/3 mL) and 2 times the cutoff (17.4 FU/3 mL), the specificity of diagnosis rose to 90.8% and 95.6%, respectively (Figure 5B-5D; Table 3).

Figure 5 Optimal cutoff values for diagnosing lung cancer from FR⁺CTCs in pulmonary nodules. (A) Line chart of Youden index of FR⁺CTC levels. The ordinate of the point with the highest value on the curve is 0.369 (i.e., the maximum value of the Youden index is 0.369). The corresponding abscissa is 8.7 (i.e., the FR⁺CTC level is 8.7 FU/3 mL). In brief, the cutoff value of FR⁺CTC is 8.7 FU/3 mL. The cutoff values were used to analyze the percentage of patients in the pulmonary nodule and lung cancer groups (B-D). The Y-axis on the percentage stacked bar chart represents the percentage of patients in each group out of the total number of patients, and patients were divided into two groups based on the different cutoff values. (B) Correspondence with 1 time the cutoff value, (C) 1.5 times the cutoff value, and (D) 2 times the cutoff value. FR⁺CTC, folate receptor-positive circulating tumor cell.

Among the clinical indicators commonly in used, FR⁺CTC level demonstrates the highest AUC value. However, the efficacy of a single blood biomarker in diagnosing lung cancer in patients with lung nodules is generally limited, often yielding an AUC value below 0.700. To bolster the precision of lung cancer diagnosis, we employed a random forest model to identify the top three blood biomarkers with the most substantial influence: WBC, PCT, and LDH. We then crafted a composite indicator by amalgamating FR⁺CTC level with the levels of WBC, PCT, and LDH. The results showed that this composite indicator significantly improved the ability to diagnose lung cancer, yielding an AUC value of 0.976. Furthermore, the sensitivity (100%) of the composite indicator was notably enhanced compared to that of any single blood biomarker used alone (all P values <0.05) (Table 4 and Figure 6).

Figure 6 ROC curve of the different blood biomarkers in diagnosing lung cancer. FR⁺CTC, folate receptor-positive circulating tumor cell; WBC, white blood cell; NSE, neuron-specific enolase; CEA, carcinoembryonic antigen; CA125, carbohydrate antigen 125; CA153, carbohydrate antigen 153; CYFRA21-1, cytokeratin 19 fragment antigen 21-1; SCCA, squamous cell carcinoma antigen; PCT, procalcitonin; IL-6, interleukin-6; IL-8, interleukin-8; IL-10, interleukin-10; TNF, tumor necrosis factor; LDH, lactate dehydrogenase; ROC, receiver operating characteristic.

Discussion

With the progress in CT imaging technology, the identification of lung nodules as an indicator of early-stage lung cancer has increased. Quickly establishing whether patients with lung nodules have early-stage lung cancer is critical to effective treatment. The use of blood-based biomarkers as an auxiliary diagnostic method is gaining traction. Previous studies have demonstrated that FR⁺CTCs are more effective than are traditional blood biomarkers, such as CEA and NSE, in diagnosing and predicting the prognosis of patients with lung cancer (12-14). However, the related studies often contrast patients with lung cancer with either healthy individuals or those with indeterminate pathology, which represents an inherent limitation.

In this study, we used FR⁺CTCs to assess individuals with imaging manifestations suggestive of lung nodules and with subsequent confirmation of benign or malignant pathology. Our results revealed a notably elevated level of FR⁺CTCs among those with lung cancer as opposed to the benign lung nodules, supporting the potential of FR⁺CTCs to be distinctive biomarkers for lung cancer diagnosis. The levels of WBC and CYFRA21-1 mirrored observations of FR⁺CTCs. An elevated WBC level is associated with an elevated risk of lung cancer (23), while an elevated CYFRA21-1 level is associated with non-small cell lung cancer (NSCLC) (24).

The debate persists regarding the expression patterns of FR⁺CTC across various lung cancer histology subtypes. Our findings indicated that FR⁺CTC levels were markedly higher in squamous cell carcinoma than in adenocarcinoma, aligning with Xu et al.’s earlier work (25). In contrast, a study has noted a modest, but nonsignificant, increase in FR⁺CTC levels in LUAD (26). Some researchers suggest that the histological characteristics of adenocarcinoma and EGFR mutations may contribute to the higher expression of FR in individuals with LUAD (9,27,28), yet others contend that it is equally high in both LUAD and LUSC (29). The precise biological underpinnings of these disparities have yet to be elucidated. Despite our study’s relatively small sample size, it provides pivotal clues for ongoing investigations into the differential expression of FR⁺CTC among lung cancer subtypes.

Peripheral adenocarcinoma is the most prevalent subtype of lung cancer encountered in clinical settings (30). Our study exclusively included patients with peripheral lung cancer, with adenocarcinoma constituting 95.4% of the diagnoses. A subgroup analysis of LUAD also revealed that FR⁺CTC levels tended to increase as LUAD differentiation decreased, although no significant difference was observed. This could be attributed to the fact that most patients included in the study were in the early stages (93.7% had stage 0–I disease).

Chang et al. (31) discovered an intriguing inverse correlation between lung cancer risk and the count of lung nodules. Along a similar vein, McWilliams et al. crafted a predictive model that revealed a surprising link: fewer lung nodules may actually signal a higher susceptibility to lung cancer (32). In our present study, we observed that patients with solitary lung nodules in the lung cancer group had a significantly higher level of FR⁺CTCs as compared to those with multiple lung nodules. This finding points to a higher likelihood of single lung nodules evolving into lung cancer as opposed to their multiple-nodule counterparts. Intriguingly, even within the benign nodule group, those with a single nodule showed elevated FR⁺CTC levels. Yet, more scrutiny is needed to ascertain whether elevated FR⁺CTC levels are an indicator of impending cancer development.

Additionally, it was found that FR⁺CTC level had the highest AUC when juxtaposed with traditional blood biomarkers such as NSE, CEA, CA125, CYFRA21-1, and SCCA (33). This suggests that FR⁺CTC level can effectively distinguish patients with NSCLC from the controls, even at the disease’s early stages. The AUC of FR⁺CTC level in lung cancer screening was superior to that of other blood biomarkers, indicating good diagnostic efficacy. However, relying solely on FR⁺CTC for specificity and sensitivity may not be sufficient for accurate clinical diagnosis of early lung cancer. Clinical insights have shown that the FR⁺CTC levels in patients with confirmed lung cancer tend to increase significantly (25). By setting a cutoff value and tracking pathological results, it was found that when the cutoff value of the FR⁺CTC level was doubled, the specificity of diagnosis increased from 75.0% to 95.6%. This suggests that if a patient with lung nodules has an abnormally high FR⁺CTC level, there is a high likelihood of cancer. Nonetheless, it is crucial to recognize that with an increase in specificity, the sensitivity of diagnosis using only FR⁺CTC decreases.

A study has illuminated that combining the detection of FR⁺CTC level and tumor markers can amplify lung cancer diagnosis accuracy (34). In our study, we used the random forest model to identify the top three blood biomarkers—WBC, PCT, and LDH—for an integrated index analysis in lung cancer diagnosis. The combined detection of FR⁺CTC level with WBC, PCT, and LDH significantly bolstered the AUC and sensitivity, effectively countering the limitation of relying on elevated FR⁺CTC level alone. This methodology offers new insights into the clinical diagnosis of early lung cancer. It has been shown that WBC level plays a pivotal role in tumor progression and metastasis across both the blood and the tumor microenvironment (35,36), LDH level is increased in lung cancer cells (37), and PCT level has the capacity to differentiate inflammatory nodules from lung cancer (38). This may explain the positive correlation between the FR⁺CTC, WBC, and LDH levels in the lung cancer group that was indicated by the Spearman analysis in our study.

A limitation to this study was the use of a retrospective, single-center design, which might have led to deviations in the data. Future validation of these combined indicators will necessitate a broader collection of patient data.

Conclusions

FR⁺CTC level was found to be a robust blood biomarker for aiding in the early detection of lung cancer among those with lung nodules. The strategic integration of FR⁺CTC level with WBC, PCT, and LDH levels significantly amplified the accuracy of lung cancer diagnosis within this population.

Acknowledgments

Funding: This work was supported by The Guangzhou Municipal Bureau of Science and Technology Basic Research Program Municipal School (Institute) Joint Funding Project (Guangdong Zhong Nanshan Medical Foundation) (No. 202201020445), and Undergraduate Innovation Ability Improvement Program of Guangzhou Medical University “Guangzhou Medical Dafa (2022) 66” (No. 02-408-2304-01024XM).

Footnote

Reporting Checklist: The authors have completed the STARD reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2493/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2493/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2493/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2024-2493/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. This study was conducted in accordance with the Declaration of Helsinki (as revised in 2013) and was approved by the Ethics Committee of The First Affiliated Hospital of Guangzhou Medical University (No. ES-2024-K108). The requirement for individual consent was waived due to the retrospective nature of the analysis.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: J. Gray)