Circulating epithelial cell enumeration facilitates the identification and follow-up of a patient with early stage papillary thyroid microcarcinoma: A case report.

BACKGROUND: This study examines whether the measurement of circulating epithelial cells (CECs) facilitates the identification and follow-up of a patient with thyroid cancer. METHODS: A 29-y-old woman with no cancer history was enrolled as a healthy control in a CEC study. CECs were enriched from the peripheral blood by the negative selection system PowerMag. Various medical examinations were performed on the patient to establish the diagnosis and to follow-up her disease status during treatment. RESULTS: This patient had unexpectedly high CEC counts that were sustained for more than two weeks. Thyroid gland ultra-sonography revealed lesions in the left lobe that could not be confirmed as cancer by magnetic resonance imaging, (18)F-fludeoxyglucose-positron emission tomography-computed tomography or cytopathological analysis, but were histologically confirmed after thyroidectomy as papillary thyroid microcarcinoma. Both the CEC count and serum thyroglobulin (Tg) concentration were significantly decreased after thyroidectomy, and they and the patient's disease status were correlated during remnant ablation therapy. The CEC count returned to normal when the patient was disease-free 10 months after thyroidectomy. CONCLUSIONS: CEC testing facilitates the identification of individuals at risk for cancer. Longitudinal follow-up of the CEC count may complement serum Tg testing for monitoring the status of patients with thyroid cancer.

Combined analysis of circulating epithelial cells and serum thyroglobulin for distinguishing disease status of the patients with papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) accounts for about 80% of the cases in thyroid cancer. Routine surveillance by serum thyroglobulin (Tg) and medical imaging is the current practice to monitor disease progression of the patients. Whether enumeration of circulating epithelial cells (CECs) helps to define disease status of PTC patients was investigated. CECs were enriched from the peripheral blood of the healthy control subjects (G1, n = 17) and the patients at disease-free status (G2, n = 26) or with distant metastasis (G3, n = 22). The number of CECs expressing epithelial cell adhesion molecule (EpCAM) or thyroid-stimulating hormone receptor (TSHR) was determined by immunofluorescence microscopy analyses. The medium number of EpCAM+-CECs was 6 (interquartile range 1-11), 12 (interquartile range 7-16) and 91 (interquartile range 31-206) cells/ml of blood for G1, G2 and G3, respectively. EpCAM+-CEC counts were significantly higher in G3 than in G1 (p < 0.05) and G2 (p < 0.05). The medium number of TSHR+-CECs was 9 (interquartile range 3-13), 16 (interquartile range 10-24) and 100 (interquartile range 31-226) cells/ml of blood for G1, G2 and G3, respectively. The TSHR+-CEC counts also distinguished G3 from G1 (p < 0.05) and G2 (p < 0.05). With an appropriate cut off value of CEC count, the disease status for 97.9% (47/48) of the cases was clearly defined. Notably, the metastatic disease for all patients in G3 (22/22) was revealed by combined analysis of serum Tg and CEC. This study implicates that CEC testing can supplement the current standard methods for monitoring disease status of PTC.

Prognostic value of circulating tumor cells with podoplanin expression in patients with locally advanced or metastatic head and neck squamous cell carcinoma.

BACKGROUND: Podoplanin (PDPN) is a prognostic factor for head and neck squamous cell carcinoma (HNSCC). However, PDPN expression in circulating tumor cells (CTCs) and its prognostic value are not clear. METHODS: The PowerMag system was used to enumerate CTCs from 53 patients with HNSCC prechemotherapy and 61 healthy donors. PDPN expression was determined by immunofluorescence staining. Results were correlated with clinicopathological parameters and clinical outcome, such as patient survival by receiver operating characteristic (ROC) and univariate and multivariate analyses. RESULTS: PDPN was expressed in a subset of CTCs. Both EpCAM-positive CTC and PDPN-positive CTC counts were statistically different between the disease and nondisease groups (p < .0001) with no prognostic value. After a median follow-up of 10.5 months (range, 6.6-18.5 months), the PDPN-positive/EpCAM-positive CTC ratio >20% was a significant prognostic factor for death within 6 months (p = .011) and was correlated with poor progression-free survival (p = .016) and overall survival (p = .015). CONCLUSION: PDPN-positive/EpCAM-positive CTC ratio is a prognostic factor and defining the ratio in patients with HNSCC might be valuable to clinical management.

An efficient strategy for broad-range detection of low abundance bacteria without DNA decontamination of PCR reagents.

BACKGROUND: Bacterial DNA contamination in PCR reagents has been a long standing problem that hampers the adoption of broad-range PCR in clinical and applied microbiology, particularly in detection of low abundance bacteria. Although several DNA decontamination protocols have been reported, they all suffer from compromised PCR efficiency or detection limits. To date, no satisfactory solution has been found. METHODOLOGY/PRINCIPAL FINDINGS: We herein describe a method that solves this long standing problem by employing a broad-range primer extension-PCR (PE-PCR) strategy that obviates the need for DNA decontamination. In this method, we first devise a fusion probe having a 3'-end complementary to the template bacterial sequence and a 5'-end non-bacterial tag sequence. We then hybridize the probes to template DNA, carry out primer extension and remove the excess probes using an optimized enzyme mix of Klenow DNA polymerase and exonuclease I. This strategy allows the templates to be distinguished from the PCR reagent contaminants and selectively amplified by PCR. To prove the concept, we spiked the PCR reagents with Staphylococcus aureus genomic DNA and applied PE-PCR to amplify template bacterial DNA. The spiking DNA neither interfered with template DNA amplification nor caused false positive of the reaction. Broad-range PE-PCR amplification of the 16S rRNA gene was also validated and minute quantities of template DNA (10-100 fg) were detectable without false positives. When adapting to real-time and high-resolution melting (HRM) analytical platforms, the unique melting profiles for the PE-PCR product can be used as the molecular fingerprints to further identify individual bacterial species. CONCLUSIONS/SIGNIFICANCE: Broad-range PE-PCR is simple, efficient, and completely obviates the need to decontaminate PCR reagents. When coupling with real-time and HRM analyses, it offers a new avenue for bacterial species identification with a limited source of bacterial DNA, making it suitable for use in clinical and applied microbiology laboratories.