| Lung cancer is one of the most prevalent cancer and the leading cause of malignant tumor related death in China and worldwide. The overall5-year survival rate remains at approximately15%, and the majority of patients still present with advanced disease. Low-dose computed tomography is an effective mean for early diagnosis and screening, which could reduce the mortality of lung cancer by20%. However, a high rate of false positives to small nodules and central tumors still represents a distinct clinical entity. Therefore, we need to find a novel biomarker for early screening and diagnosis in lungcancer.Circulating tumor cells (CTCs) have recently emerged as important potential biomarkers of diagnosis, evaluation of treatment effect, and prognosis in several epithelial cancers including lung cancer. Previous study confirmed that CTCs could be detected in very early stage during tumor development, even at a premalignant stage in a pancreatic cancer model, which implied that CTCs could be a potential biomarker for early diagnosis of lung cancer by a sensitive technique.The CytoploRare circulating lung cancer cell kit was used in our study which was provided by GenoSaber Biotech Co. Ltd.(Shanghai, China). In our study, we used this kit to evaluate its clinical value in healthy people and patients with lung cancer or benign lung diseases, to detect the dynamic change of CTCs level in patients with lung cancer during the perioperative period, as well as its clinical value in the other malignant tumors, such as esophageal cancer, breast cancer, colorectal cancer, gastric cancer, and liver cancer. The fundamental principles of this kit was comprises two components:one is for CTCs enrichment that is based on negative enrichment by immunomagnetic beads, and the other is for CTCs detection and quantification that is based on ligand-targeted polymerase chain reaction (PCR).3mL of whole blood samples was lysed of erythrocytes with red cell lysis buffer and then depleted of leukocytes with magnetic beads. Following that, enriched CTCs were incubated with labeling buffer that contained conjugates of a tumor-specific ligand folate receptor (FR) and a synthesized oligonucleotide. After washing off free conjugates, the bound conjugates were stripped from CTCs and then analyzed by PCR.Three hundred and five patients with lung cancer were enrolled into the study (including296patients with non-small cell lung cancer and12with small cell lung cancer). In addition, peripheral blood samples were obtained from107healthy donors who exhibited no evidence of any clinically detectable disease after health checkups and102patients who were diagnosed with benign lung disease based on imaging or pathologic examinations. Our results showed that the median CTC units were5.71[3.7-8.68],6.47[4.78-7.83], and10.51[7.5-15.55] in healthy donors, benign diseases, and patients with lung cancer, respectively. Compared with patients with lung cancer, the CTCs levels were significantly lower in healthy donors and benign diseases (p<0.001; Mann-Whitney U test). However, there was no significant difference between the two control groups (p=0.132). The median CTC unit was9.93[7.03-14.41] in patients with stage I lung cancer, which was higher than control groups (p<0.001).To investigate the significance of CTCs levels in patients with lung cancer, we found that the CTCs levels of patients with bigger tumor (maximum tumor diameter>3.4cm) or advanced stage diseases (stage â…¢/â…£) or non-adenocarcinoma (squamous cell carcinoma or small cell lung cancer) were higher than those with smaller tumor (maximum tumor diameter<3.4cm) or early-stage diseases (stage â… /â…¡) or adenocarcinoma (all at p<0.05). ROC curves were plotted to determine the diagnostic efficiency of CTCs levels for lung cancer, the area under the curve (AUC) is0.781(95%CI:0.743-0.816). The Youden index was used to identify the cutoff point, which meant to choose the "optimal" threshold value when the threshold value for sensitivity+specificity-1was maximized, and the cutoff value is8.5CTC units, with the sensitivity of68.8%, the specificity of79.4%. With the cutoff point of8.5CTC units, the sensitivity was65.2%in the diagnosis of patients with stage I lung cancer. The FR-based CTCs detection method displays the highest AUC and Youden index compared with other five biomarkers (NSE, CEA, CA125,cyfra21-1,SCC Ag).Postoperative blood samples was obtained in forty-four patients with lung cancer and CTCs level>8.5units. Our results showed that CTCs levels in postoperative blood samples were6.65[4.07-9.84], which was lower than those in preoperative blood samples of13.41[11.11-17.35](p<0.001).Finally, we further evaluated the efficacy and feasibility of FR-based CTCs detection method in patients with other malignant tumors. Our results showed that the median CTC units were8.5[4.76-9.87],7.37[5.35-9.43],8.08[5.4-10.33],6.51[5.17-10.02], and6.84[4.93-9.12] in patients with esophageal cancer, breast cancer, colorectal cancer, gastric cancer, and liver cancer, respectively. Mann-Whitney U test showed that the CTCs levels of patients with breast cancer (p=0.013), colorectal cancer (p=0.007), gastric cancer (p=0.049) were higher that healthy donors. However, there were no significant differences between patients with esophageal cancer (p=0.063) or liver cancer (p=0.163) and healthy donors.In conclusion, our results suggested that FR-positive CTCs were feasible diagnostic biomarkers in lung cancer, even in early-stage tumors. The CTCs levels of patients with lung cancer would decline after the tumor load was removed. The FR-based CTCs detection method may be also useful in patients with breast cancer, colorectal cancer and gastric cancer, and further investigation is required. |
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