|Year : 2018 | Volume
| Issue : 7 | Page : 1620-1626
Folate-receptor-positive circulating tumor cells as an efficacious biomarker for the diagnosis of small pulmonary nodules
Yang Xue, Wei Cong, Shenglong Xie, Jun Shu, Gang Feng, Hong Gao
Department of Thoracic Surgery, Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital, Chengdu, Sichuan, China
|Date of Web Publication||19-Dec-2018|
Department of Thoracic Surgery, Sichuan Academy of Medical Sciences and Sichuan Provincial People's Hospital, No. 32 West Second First Ring Road, Chengdu, Sichuan 610072
Source of Support: None, Conflict of Interest: None
Objective: The objective of this study is to investigate the clinical significance of folate-receptor-positive circulating tumor cells (FR+CTC) for the diagnosis of lung cancer, especially in early-stage patients.
Materials and Methods: A total of 72 lung cancer patients, including 31 with stage I diseases and two with stage 0 diseases, were enrolled in this study. Twenty-four patients with benign lung diseases and two healthy volunteers served as the control group. Three milliliters of peripheral blood were collected from each participant for FR+CTC analysis on enrollment. FR+CTC enumeration was performed using immunomagnetic leukocyte depletion and ligand-targeted polymerase chain reaction techniques.
Results: The study results revealed that using a cutoff value of 8.7 CTC Units/3 mL, the sensitivity, and specificity of FR+CTC for diagnosis of lung cancer were 81.94% and 73.08%, respectively. Such high sensitivity (74.19%) and specificity (73.08%) persisted even if only stage I lung cancer patients were retained in the analysis. In receiver operating characteristic analysis, the performance of FR+CTC (area under the curve = 0.8153) was superior to other clinical biomarkers such as carcinoembryonic antigen, neuron-specific enolase, and cytokeratin 19 fragments. In a subgroup analysis, patients with nodule size of >3 cm showed an improved sensitivity (88.46%); although, the specificity appeared to decrease (40%). All five patients with benign diseases in this subgroup had inflammatory diseases, indicating that large inflammatory nodules may also release FR -expressing cells into the circulatory system.
Conclusion: FR+CTC is a reliable biomarker for the early diagnosis of small-sized lung cancer. Further study with larger sample size is required to assess the diagnostic efficiency of FR+CTC in patients with large nodule sizes.
Keywords: Biomarkers, circulating tumor cells, diagnosis, folate receptor, lung cancer
|How to cite this article:|
Xue Y, Cong W, Xie S, Shu J, Feng G, Gao H. Folate-receptor-positive circulating tumor cells as an efficacious biomarker for the diagnosis of small pulmonary nodules. J Can Res Ther 2018;14:1620-6
|How to cite this URL:|
Xue Y, Cong W, Xie S, Shu J, Feng G, Gao H. Folate-receptor-positive circulating tumor cells as an efficacious biomarker for the diagnosis of small pulmonary nodules. J Can Res Ther [serial online] 2018 [cited 2021 Jan 27];14:1620-6. Available from: https://www.cancerjournal.net/text.asp?2018/14/7/1620/247733
| > Introduction|| |
Lung cancer is the most common malignant disease and the leading cause of cancer-related death in China. For stage I patients, who are eligible to undergo curative surgical resection and the 5-year survival rate is estimated to be 45%–65%. In contrast, the 5-year survival rate for patients with regional or distant metastases remains dismal (4.5%–28.9%). Unfortunately, most patients have late-stage disease at initial diagnosis, and lack of an effective screening method contributes significantly to the poor prognosis of lung cancer. Therefore, early screening of lung cancer in high-risk population is essential for improving prognosis and reducing the mortality rate of lung cancer.
Since the National Lung Screening Trial reported that low-dose computed tomography (LDCT) reduced the mortality of lung cancer by 20% as compared to chest radiography, LDCT has been the standard imaging technique for lung cancer screening. Nevertheless, the low specificity of LDCT has greatly hampered its clinical utility. Studies have shown that over 90% of the nodules identified by LDCT were indeed benign tumors as confirmed by pathological assessment which remains the gold standard of lung cancer diagnosis till date. The high false-positive rate of LDCT would lead to overdiagnosis, increase medical cost, and cause unnecessary mental anguish to patients. As such, the need for a better diagnosis of lung cancer remains substantial.
Circulating tumor cell (CTC) has recently emerged as a hot topic in clinical oncology research. CTCs are tumor cells that shed from the primary or metastatic lesions and entered the blood circulatory system. As a noninvasive “liquid biopsy” approach, CTC detection has shown promises in cancer diagnosis, prognosis, and prediction of treatment efficacy.,,,, CellSearch, the only U. S. Food and Drug Administration approved CTC detection platform, is a semiautomated device for CTC capture, and enumeration based on epithelial cell adhesion molecule (EpCAM) expression. The CellSearch platform has proven its clinical utility in predicting prognosis of breast cancer, colorectal cancer, and prostate cancer.,, However, the platform suffers from ineffective CTC capturing due to its dependence on the EpCAM marker. Most of the cancer cells undergo epithelial-mesenchymal transition (EMT) and lose the expression of EpCAM during cancer development. Thus, the representativeness of EpCAM-positive CTCs is questionable in lung cancer which possesses a high rate of EMT.
Folate receptor (FR) has the potential to be a universal “tag” of CTCs. Studies showed that FR is highly expressed in several types of cancer such as ovarian cancer and lung cancer. More importantly, normal cells present in the blood circulatory system seldom express FR, except a rare subtype of activated macrophage. Based on the high expression level and the high specificity of FR in tumor cells, an assay utilizing negative enrichment and ligand-targeted polymerase chain reaction (PCR) is established to quantify FR+CTC in whole blood samples. A previous study reported high sensitivity (72.46%–76.37%) and specificity (82.39%–88.65%) of the FR+CTC assay in the diagnosis of lung cancer. In the present study, we sought to further investigate the diagnostic efficiency of FR+CTC in lung cancer patients with different nodule sizes.
| > Materials and Methods|| |
In this study, 72 newly-diagnosed, treatment-naïve lung cancer patients were enrolled as the “Lung Cancer Group.” All patients included in this group were pathologically diagnosed with lung cancer, including 50 adenocarcinomas and 14 squamous cell carcinomas. Other pathological subtypes included four cases of non-small cell lung cancers (NSCLC) not otherwise specified, two cases of large cell carcinomas, one case of adenosquamous cell carcinoma, and one case of small cell lung cancer. The 7th edition of the American Joint Committee cancer staging manual was used for clinical staging. In addition, 24 patients with benign lung diseases and two healthy volunteers were enrolled as the “Control Group.” Benign lung diseases included hamartoma, inflammatory nodule, tuberculosis, chondroma, and sclerosing hemangioma. The two healthy controls exhibited no evidence of any clinically detectable disease after health checkups. Detailed characteristics of these individuals are summarized in [Table 1]. The present study was carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. The study protocol was approved by the Institutional Review Board and all patients provided informed consent.
Three milliliters of peripheral blood sample was collected from all the participants using 6 mL EDTA anticoagulant tubes (BD Diagnostics, Sparks, MD, USA). Samples were stored at 4°C and processed within 24 hours. An “anonymized” identifier code was assigned to each sample and information of the patients can be found on the sample collection tube. As such, FR+CTC analysis was performed in a single-blinded fashion.
Folate receptor + Circulating tumor cell enumeration
FR+CTC analysis was performed with the CytoploRare® FR-positive cell detection kit (Genosaber, Shanghai, China) according to the manufacturer's instruction as previously described. In brief, CTCs were negatively enriched from the 3 mL of blood sample by immunomagnetic depletion of leukocytes and then labeled with the proprietary probe (conjugates of a tumor-specific folic acid ligand and a synthesized oligonucleotide). After washing off unbound probes, the bound conjugates were stripped, and the oligonucleotide was analyzed using quantitative PCR with an ABI7300 instrument (ThermoFisher, Waltham, MA, USA). The quantity of FR+CTC was expressed using an empirically defined FR+CTC unit in the form of the number of FR+CTCs detected in 3 mL of blood. A serial of standards containing oligonucleotides (10−14 to 10−19 M, corresponding to 2 to 2 × 105 CTC Units/3 mL) was used for calibration of the FR+CTC units in the blood samples.
The difference in FR+CTC units or biomarker expression levels between two groups was compared using the Mann–Whitney U-test. Receiver operating characteristic (ROC) analysis was performed, and the area under the ROC curve (AUC) was calculated to assess the diagnostic efficiency of FR+CTCs and serum biomarkers. According to the instruction manual of the CytoploRare® FR-positive cell detection kit, 8.7 CTC Units/3 mL was used as the cutoff threshold to differentiate lung cancers from benign lung diseases. The Youden's index was used to identify the optimal cutoff value of serum biomarkers. A value of P < 0.05 was considered to indicate a statistically significant difference. Statistical analysis was performed using Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA).
| > Results|| |
The diagnostic efficiency of Folate receptor + Circulating tumor cell in patients with lung cancer
In this study, the median FR+CTC units in the Lung Cancer Group was 10.71 CTC Units/3 mL, which was significantly higher than that in the Control Group median = 6.315 CTC Units/3 mL, P < 0.0001, [Figure 1]a. Using 8.7 CTC Units/3 mL as the cutoff threshold, the sensitivity and specificity of FR+CTC were 81.94% and 73.08%, respectively. In ROC analysis, the AUC of FR+CTC was 0.8221 (95% confidence interval [CI]: 0.7208–0.9235), [Figure 1]b.
|Figure 1: Comparison of folate-receptor-positive circulating tumor cells count in lung cancer group and control group. (a) The box-plot of folate-receptor-positive circulating tumor cells count in lung cancer group and control Group. Dotted line indicates the cutoff threshold used to differentiate positive and negative folate-receptor-positive circulating tumor cells count (8.7 CTC units/3 mL). (b) The receiver operating characteristic curve of folate-receptor-positive circulating tumor cells in lung cancer diagnosis|
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For the 31 patients with stage I NSCLC, the median FR+CTC unit was 9.730 CTC Units/3 mL. The FR+CTC count of stage I NSCLC patients was significantly higher than that of the Control Group [P = 0.0005, [Figure 2]a. ROC analysis revealed that the sensitivity and specificity of FR+CTC in differentiating stage I NSCLC from the Control Group were 74.19% and 73.08%, respectively [Figure 2]b. The AUC was 0.7699 (95% CI: 0.6429–0.8968).
|Figure 2: Comparison of folate-receptor-positive circulating tumor cells count in patients with different clinical stages. (a) The box-plot of folate-receptor-positive circulating tumor cells count in patients with Stage I non-small cell lung cancers and Control Group. (b) The receiver operating characteristic curve of folate-receptor-positive circulating tumor cells in the diagnosis of Stage I non-small cell lung cancers. (c) The box-plot of folate-receptor-positive circulating tumor cells count in early-stage and late-stage lung cancer|
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In addition, we examined the correlation of FR+CTC count with clinical stage. Late-stage lung cancer patients (stage III/IV) showed a numerically higher FR+CTC count than early-stage patients (stage I/II). The median FR+CTC counts were 12.29 CTC Units/3 mL and 9.765 CTC units/3 mL for stage III/IV and stage I/II patients, respectively. However, the difference was not statistically significant [P = 0.0660, [Figure 2]c.
Comparison of the performance between Folate receptor + Circulating tumor cell and serum biomarkers
Fifty-six patients from the Lung Cancer Group and 16 individuals from the Control Group were tested for serum carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), and cytokeratin 19 fragments (CYFRA21-1) levels. Within this subgroup, only FR+CTC and CYFRA21-1 showed a significantly higher expression level in the Lung Cancer Group as compared to the Control Group [Table 2]. In ROC analysis, FR+CTC showed the highest AUC value among the tested biomarkers [AUC = 0.8153, 95% CI: 0.6815–0.9491, [Figure 3]. The AUC of CEA, NSE, and CYFRA21-1 ranged from 0.5206–0.7137. Consistent with the results in the entire study cohort, the sensitivity and specificity of FR+CTC in lung cancer diagnosis in this subgroup using 8.7 CTC Units/3 mL as the threshold were 85.71% and 75.00%, respectively. These results suggested that FR+CTC are a reliable biomarker which has a better performance than other routine clinical biomarkers in lung cancer diagnosis.
|Figure 3: The receiver operating characteristic curves of folate-receptor-positive circulating tumor cells and serum biomarkers. Red line indicates the receiver operating characteristic curve of folate-receptor-positive circulating tumor cells; green line indicates the receiver operating characteristic curve of carcinoembryonic antigen; blue line indicates the receiver operating characteristic curve of neuron-specific enolase; and yellow line indicates the receiver operating characteristic curve of cytokeratin 19 fragments|
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In addition, 45 patients from the Lung Cancer Group and 11 individuals from the Control Group within the above subgroup were concurrently tested for serum squamous cell carcinoma antigen (SCC), progastrin-releasing peptide, and heat-shock protein 90-α (Hsp90α) levels. No significant difference in the serum expression levels of these biomarkers was observed between the Lung Cancer Group and the Control Group [Table 2]. In ROC analysis, the AUC of these biomarkers ranged from 0.5343 to 0.6465.
The diagnostic efficiency of Folate receptor + Circulating tumor cell in patients with different nodule sizes
We next analyzed the performance of FR+CTC in patients with different nodule sizes was assessed by LDCT. A tumor diameter of 3 cm was chosen as the cutoff value to divide participants into two groups [Table 3]. In the Lung Cancer Group, a significantly higher FR+CTC count was observed in patients with nodule size of >3 cm (n = 26, median FR+CTC count = 13.02 CTC Units/3 mL) when compared to patients with nodule size of <3 cm [n = 45, median FR+CTC count = 9.750 CTC Units/3 mL, P = 0.0401, [Figure 4]. For participants with nodule size of ≤3 cm, the AUC, sensitivity, and specificity were 0.8063 (95% CI: 0.6769–0.9356), 80.00%, and 75.00%, respectively. While for participants with a nodule size of >3 cm, the AUC, sensitivity, and specificity were 0.6923 (95% CI: 0.3768–1.008), 88.46%, and 40.00%, respectively. Interestingly, in patients with nodule size of >3 cm, the specificity of FR+CTC was lower than that in patients with nodule size of <3 cm due to the fact that three of five patients with benign lung diseases in this subgroup (nodule size >3 cm) had FR+CTC count higher than the threshold of 8.7 CTC Units/3 mL. Due to the small sample size (only five patients have nodule size of >3 cm in the Control Group), the impact on the specificity of FR+CTC analysis is limited.
|Figure 4: Comparison of folate-receptor-positive circulating tumor cells count in lung cancer patients with different nodule sizes. The Box-plot of folate-receptor-positive circulating tumor cells count in lung cancer patients with nodule size of ≤3 cm and >3 cm. One patient in the lung cancer group did not have information available on nodule size|
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A further investigation on the clinicopathological characteristics of the five patients, in this subgroup, indicated that all three false-positive results were found in patients with fairly large inflammatory nodules (4.3 cm, 5.0 cm, and 6.0 cm, respectively). One patient showed a marginally positive FR+CTC level (8.99 CTC Unit/3 mL). In addition, one patient showed an unusually high level of Hsp90α (177.79 ng/mL) and FR+CTC level (27.74 CTC Unit/3 mL). Detailed clinical characteristics of these five individuals are shown in [Table 4]. In general, we observed a trend toward higher FR+CTC counts in lung cancer patients with increasing nodule sizes. Furthermore, the sensitivity of FR+CTC analysis increases with nodule size.
|Table 4: Characteristics of the benign patients with a nodule size of >3 cm|
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| > Discussion|| |
The low specificity of LDCT and clinical biomarkers in early screening of lung cancer remains an unsolved problem. In this study, we analyzed the performance of FR+CTC in the diagnosis of lung cancer. The sensitivity and specificity of FR+CTC were 81.94% and 73.08% for the entire study cohort, and 74.19% and 73.08% for the stage I-only population, respectively. The median FR+CTC count in the Lung Cancer Group and Control Group were consistent with previous studies.,, Moreover, the diagnostic efficiency of FR+CTC was superior to serum biomarkers, including conventional lung cancer biomarkers (CEA, NSE, CYFRA21-1, and SCC) and novel biomarkers (ProGRP and Hsp90α).
We also evaluated the performance of FR+CTC in differentiating malignant and benign tumors with different nodule sizes. The solitary pulmonary nodule is defined as a discrete, rounded opacity of <3 cm with a clear margin. Tumors with a diameter of >3 cm are more likely malignant, while that of ≤3 cm are more likely benign. Therefore, we divided patients into two subgroups using 3 cm as a cutoff value and analyzed the corresponding diagnostic efficiency of FR+CTC in each subgroup. Consistent with previously reported, the FR+CTC count in lung cancer patients with a nodule size of >3 cm was significantly higher than those with a nodule size of ≤3 cm. The diagnostic efficiency of FR +CTC in patients with nodule size of ≤3 cm was equivalent to that of the entire study cohort, with sensitivity and specificity of 80.00% and 75.00%, respectively. These results revealed the clinical significance of FR+CTC as a companion diagnostic assay with LDCT for lung cancer screening.
For the subgroup of patients with nodule size of >3 cm, we observed better sensitivity (88.46%) and lower specificity (40.00%). The low specificity is likely an artifact of the small sample size of the Control Group. Within this subgroup, three of five patients showed high FR+CTC count, leading to the low specificity observed. All three false-positive results were found in patients with fairly large inflammatory nodules. One of the patients also showed an unusually high level of Hsp90α (177.79 ng/mL) in addition to the high FR+CTC count (27.74 CTC Units/3 mL). These large inflammatory nodules may release FR-expressing cells into the blood circulatory system, trigger inflammatory responses, and recruit FR-expressing macrophages, resulting in false-positives. Further, Hsp90α is associated with overexpression of growth factor receptors and growth factor signaling pathways, such as the PI3K/AKT pathway which is known to be involved in cancer development. However, because the sample size is too small, the impact on the specificity of this subgroup is limited, and should be investigated in a further study with larger sample size.
| > Conclusion|| |
FR+CTC is a reliable biomarker for the differential diagnosis of small-sized SPNs. Further study with a larger sample size is required to verify the diagnostic efficacy of FR+CTC in subgroup with a nodule size of >3 cm.
The authors would like to thank Genosaber Biotech Co. Ltd., offered technical support in this study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2], [Table 3], [Table 4]