Abstract
BackgroundQuantification of circulating tumor cells (CTC) is valuable for evaluation of non-small cell lung cancer (NSCLC). The sensitivity of current methods constrains their use to detect rare CTCs in early stage. Here we evaluate a novel method, ligand-targeted polymerase chain reaction (LT-PCR), that can detect rare CTCs in NSCLC patients.MethodsCTCs were enriched by immunomagnetic depletion of leukocytes and then labeled by a conjugate of a tumor-specific ligand and an oligonucleotide. After washing off free conjugates, the bound conjugates were stripped from CTCs and then analyzed by qPCR. To evaluate the clinical utility, blood samples were obtained from 72 NSCLC patients (33 initially diagnosed and 39 on chemotherapy), 20 benign patients, and 24 healthy donors.ResultsExperiments with healthy blood spiked with tumor cells indicated the LT-PCR allows specific detection of CTC. The clinical study showed that the initially diagnosed patients have an average of 20.8 CTC units with metastatic diseases, 11.8 CTC units with localized diseases, and 6.0 CTC units with benign diseases. With the threshold of 8.5 CTC units, the assay can detect 80% of stage I/II, 67% of stage III, and 93% of stage IV cancer. With the benign patients and healthy donors as control group, the method can detect cancer with a sensitivity of 81.8% and a specificity of 93.2%.ConclusionThe LT-PCR would allow quantification of CTC in NSCLC patients at a more sensitive level, providing a potential tool for stratifying malignant lung diseases, especially at early stage.
Highlights
Circulating tumor cells (CTC), known as ‘‘liquid biopsy’’, is a readily accessible marker for monitoring cancer progression, response to therapy and recurrence of malignant diseases
PCR calibration and circulating tumor cells (CTC) unit As an external calibration curve, six standards containing a serial of concentrations of the conjugated oligonucleotides were used to calculate the quantity of folate receptors on CTCs (Figure S1)
The intra-assay coefficient of variability (CV) is from 2.6% to 3.8%, and interassay CV is from 3.3% to 5.3% (Table S2 in File S1)
Summary
Circulating tumor cells (CTC), known as ‘‘liquid biopsy’’, is a readily accessible marker for monitoring cancer progression, response to therapy and recurrence of malignant diseases. Clinical significance of CTC in NSCLC has been widely reported in recent studies. High numbers of detected CTC were reported to associate with poor prognosis in metastatic lung cancer [1,2,3,4,5]. Detection of certain mRNA or multi-gene in CTC can be used for prognosis of the outcome of debulking surgery and radiotherapy in NSCLC patients [6,7,8,9,10]. Quantification of circulating tumor cells (CTC) is valuable for evaluation of non-small cell lung cancer (NSCLC). We evaluate a novel method, ligand-targeted polymerase chain reaction (LT-PCR), that can detect rare CTCs in NSCLC patients
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