Quantification of Rare Circulating Tumor Cells in Non-Small Cell Lung Cancer by Ligand-Targeted PCR

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.

Survivin mRNA-circulating tumor cells are associated with prostate cancer metastasis

“…All patient samples were tested in duplicates with six standards and three quality controls. The observed CTC counts were computed as previously described [17].…”

Detection of circulating tumor cells in non-small cell lung cancer

“…The LT-PCR-FOLR1 method has been investigated for detection of CTCs in NSCLC in several trials (22). The sensitivity of this method was applied to detect rare CTCs in early stage NSCLC patients.…”

A preliminary study on the relationship between circulating tumor cells count and clinical features in patients with non-small cell lung cancer

“…The latter utilized differential expression of folate receptor on cell surface of cell types present in the sample: unlike blood cells, lung cancer cells tend to abundantly express folate receptor on their surface. This allowed previously-established average concentration of folate receptor per tumor cell (16) to be used to design a calibrator curve and to infer from the fluorescence detected in the quantitative PCR for folate receptor CTCs (expressed as CTC unit) how many were present in each sample. Remarkably, the number of CTC units detected corresponded tightly to the stage of NSCLC.…”

“…While Tanaka's study discriminated between stage I and stage IV disease using CellSearch (9), Lou showed that CTC number correlated with stage I/II versus stage III and stage IV disease (16). With the technique used in their study, Wan and colleagues achieved even further distinction between stages by CTC number, being able to clearly determine CTC unit cut-offs delineating even stage I from stage II, in addition to being able to distinguish stage III and IV (15).…”

“…Initially, CTCs were associated with the process of metastasis and indeed their numbers detectable in blood are the highest in the advanced disease (3,4). However, the notion that CTCs enter blood stream early in the process of carcinogenesis is supported by the detection of CTCs at earlier stages in cancer development suggesting that CTC presence in blood is an early event (9,15,16). This opens an exciting area of potential future utilization for early diagnosis of lung cancer.…”