Cancer Biomarkers

For full-scale analysis of Human Papillomavirus (HPV) status in humans, two minor groove binder (MGB)-based one-step multiplex real-time PCR systems were developed: one to screen 16 high-risk HPV (HR-HPV) types, and one to screen a broader spectrum of HPV types (common HPV or C-HPV).

Sensitivity and specificity were evaluated using diluted reference plasmids and 20 control human DNA samples. For clinical evaluation, 510 cervical scrape samples were evaluated.

The sensitivity assays revealed that the C-HPV detection system could detect 10 ∼ 100 plasmid copies/reaction, while the HR-HPV detection system detected 10 ∼ 500 copies. The specificity test revealed that the systems did not yield positive signals from the 20 human genomic DNA samples. Performance was tested on 510 usable clinical samples. The HR-HPV results were compared to those from the Hybrid Capture 2 (HC2) test, which assesses 13 HR-HPV types; the concordance level between the two methods was 90.8% with a kappa value of 0.813.

These results showed that our novel MGB-based one-step multiplex real-time PCR method may be used for the diagnosis and mass screening of HPV in clinical and large-scale epidemiological studies.

The aim of this study was to examine the pattern of SNPs in ECRG1 and FGFR4 gene of oral squamous cell carcinoma, and to evaluate the association between SNPs and prognostic parameters.

Total 24 cases of oral squamous cell carcinoma patients were enrolled in this study. We analyzed the pattern of SNPs in ECRG1 and FGFR4 gene using PCR and direct sequence. Also we evaluated the association between SNPs pattern and clinicopathologic parameters of oral squamous cell carcinoma.

The allele type Arg/Arg in ECRG1 gene was found in 13 (54.2%) patients, Arg/Gln in 11 (45.8%) patients and Gln/Gln in no patient. No clinical or pathological factor was associated with the SNP pattern in ECRG1. The allele types of FGFR4 amino acid 388 in 24 OSCC patients were Arg/Arg (8.3%), Arg/Gly (54.2%) and Gly/Gly (37.5%). No clinical or pathological factor was significantly associated with the SNP pattern except the nodal stage. The patients carrying FGFR4 allele Arg/Arg or Arg/Gly at amino acid 388 were associated with advanced N stage (pathologic N2+N3), compared to Gly/Gly allele carrying group (p=0.009).

This study is the first to describe a SNP pattern of both FGFR4 and ECRG1 gene with OSCC in Asian patients. In this study, FGFR4 Arg allele carrier was associated with higher N stage compared with Gly allele. If the important biological role of FGFR4 and ECRG1 in OSCC can be confirmed in further studies, this might be a rational to consider the evaluation of these genes as a therapeutic target in OSCC.

Fluorescence-activated cell sorting was commonly used for identification of cancer stem cells (CSCs), which relied on specific cell surface markers. And this approach makes it possible for us to study characteristics of CSCs in vitro. However, the pattern of membrane protein including surface makers might be vitally influenced during the dissociation of the adherent cells, thus it might heavily impact the quantity and quality of CSCs identified by flow cytometry.

To address this question, in present study, three commonly used digestive reagents and two different temperatures were performed in MCF-7 cells to assay CD44⁺CD24^- CSCs subpopulation. The potential of sorted CD44⁺CD24^- cells from different digestion to form mammosphere in culture was also compared.

The results showed that trypsin, a commonly used reagent in CSCs studies, most aggressively reduced antigenicity for surface markers and make part of CD44⁺CD24^- CSCs subpopulation cleaved into CD44^-CD24^- non-stem cancer cells (NSCCs). And it also increased the mammosphere formation efficiency of CD44^-CD24^- subpopulation. This cleavage effect is especially serious when cells are digested at 37°C. While accutase, a purified collagenase/neutral protease cocktail, provides the best balance of dissociation efficiency and antigen retention.

Taken together; these results indicate that enzymatic digestion process plays an important role in identification of CSCs with surface marker via flow cytometer, suggesting that researchers need to reconsider this process seriously.

The metronomic use of chemotherapeutic drugs is presumed to have anti-angiogenic effect. In the current study, we aimed to test the effects of lower doses of cytotoxic agents on VEGF secretion from tumor cell lines.

We tested the cytotoxic effects of widely used chemotherapeutic drugs including 5-florouracil, irinotecan, oxaliplatin, paclitaxel and docetaxel in tumor cell lines, MCF-7 (human breast cancer cell line) HT-29 (human colon cancer cell line) and a primary gastric cancer cell line and calculated the IC50 values. We’ve also assayed the effects of the lower doses of chemotherapeutic drugs on the levels of VEGF secreted by tumor cells in vitro.

The human primary gastric cancer cells were more resistant to 5-FU and oxaliplatin than the HT29 and MCF-7 cell lines (p< 0.001). No significant differences were noticed in terms of the IC50 values of the irinotecan, docetaxel and paclitaxel among the studied tumor cell lines (p > 0.05). The test drugs yielded significant decreases in VEGF levels at the doses of −2 log of IC50 values in MCF-7 and primary gastric cancer cell lines. While 5-florouracil did not inhibit the VEGF secretion of HT-29 cell line, irinotecan, oxaliplatin, docetaxel and paclitaxel significantly decreased the levels of secreted VEGF.

Our results suggest that lower doses of chemotherapeutic drugs decrease VEGF secretion from tumor cells without causing substantial cell killing. The data suggest the occurrence of a kind of selective drug-tumor cell type relationship.