Kuo-Chien Tsao

Cell-free DNA: measurement in various carcinomas and establishment of normal reference range.

BACKGROUND Cell-free DNA is detectable in circulating blood. Numerous reports in the literature have pointed out that cell-free DNA in plasma or serum has the clinical potential to be a more specific tumor marker for the diagnosis and prognosis, as well as the early detection, of cancer. METHODS In order to adapt cell-free DNA to a routine clinical laboratory test, we used commercial kits such as the QIAamp blood kit for DNA extraction and the PicoGreen DNA kit for DNA quantification. This was done so our results and the normal reference value established would allow to be compared by other laboratories. We have established the normal reference level of cell-free DNA for females and males from age 20-70 years. We also detected elevated cell-free DNA in all cancers that were tested in this study, including carcinomas, leukemia and lymphoma. RESULTS Our study indicates that the elevation of serum cell-free DNA was usually detected in specimens containing elevated tumor markers and is most likely associated with tumor metastases. The electrophoretic pattern of cell-free DNA showed that cell-free DNA from cancer patient is fragmented, containing smaller DNA (100 bp) not found in normal cell-free DNA. CONCLUSIONS Measuring cell-free DNA may complement currently used tumor markers for the management of cancer patients.

Urinary 8-hydroxydeoxyguanosine and its analogs as DNA marker of oxidative stress: development of an ELISA and measurement in both bladder and prostate cancers

BACKGROUND 8-hydroxydeoxyguanosine (8-OHdG) is the most frequently detected and studied DNA lesion. Upon DNA repair, 8-OHdG is excreted in the urine. Urinary 8-OHdG is now considered as a biomarker of generalized, cellular oxidative stress and is linked to degenerative diseases including cancer. METHODS We developed a competitive enzyme-linked immunosorbent assay (ELISA) for urinary 8-OHdG by coating BSA conjugated 8-hydroxyguanine (8-OHG) on a microplate. Urine specimens containing 8-OHdG and monoclonal anti-8-OHdG antibody were incubated together in the microwell. Final quantification of bound anti-8-OHdG antibody was estimated by the addition of HRP-conjugated sheep-anti-mouse antibody. RESULTS The concentration range of the calibration curve was 0-60 ng/ml. The sensitivity of the assay was 0.5 ng/ml. The within-day precision and day-to-day precision were <10%. The ELISA correlated well with a commercial kit (r=0.9). Our assay measured not only 8-OHdG but also 8-OHG and 8-hyroxyguanine in urine. Increased urinary concentration of 8-OHdG and its analogs were detected in both patients with bladder cancer (70.5+/-38.2 ng/mg creatinine) and prostate cancer (58.8+/-43.4 ng/mg creatinine) as compared to the healthy control (36.1+/-24.5 ng/mg creatinine). CONCLUSION Our preliminary data suggest that the competitive ELISA for 8-OHdG and its analogs appears to be a simple method for quantifying the extent of oxidative stress and may have potential for identifying cancer risk.

Elevated cell-free serum DNA detected in patients with myocardial infarction

BACKGROUND Cell-free DNA is detectable in the circulation. Increased cell-free DNA has been detected in cancer patients and individuals with trauma. We want to know whether patients with myocardial infarction (MI) also have increased cell-free DNA in their blood. METHODS We used a QIAamp blood kit for DNA extraction from serum and the PicoGreen DNA kit for quantification. DNA patterns of serum DNA were established by gel electrophoresis on 2.5% metarphor gel. RESULTS The average serum DNA in MI patients (N=55) was 511+/-398 ng/ml, more than 10-fold higher than normal (36.3+/-23.8 ng/ml, n=274). Patients with increased CK-MB (>4%) were associated with highly increased concentrations of cell-free DNA (93.4%). There was no correlation between the concentration of cell-free DNA and the concentrations of CK-MB, troponin I and C-reactive protein. In serial specimens, we found that the cell-free DNA rose early, but peaked behind CK-MB. A slightly diffused DNA ladder could be found with pooled cell-free DNA from MI patients by electrophoresis with the smallest DNA band at only a few hundred base pairs. CONCLUSIONS Cell-free DNA in MI patients is increased in patients diagnosed with MI, and may complement troponin and CK-MB in a multiple marker test format.

Development of ELISA on microplate for serum C-reactive protein and establishment of age-dependent normal reference range

BACKGROUND C-reactive protein (CRP), a marker of systemic inflammation, has been proposed to predict outcome in patients with unstable angina; and elevated levels of CRP were found to be associated with an increased risk of coronary events. METHODS Two enzyme-linked immunosorbent assays (ELISA) of different sensitivities were developed on microplate for CRP. Both ELISA established used Dako polyclonal anti-CRP antibody for coating and Dako horse radish peroxidase (HRP)-conjugated polyclonal anti-CRP antibody for detection. RESULTS The sensitivity of the high and regular sensitivity ELISA was 0.16 and 0.6 mg/l, respectively. Our assays demonstrated an excellent correlation with commercial CRP assays performed on a Behring Nephelometer Analyzer II (BNII) at both regular and ultrasensitive levels, with both correlation coefficients above 0.98 and slopes of approximately 1. Using our microplate assays, we established normal reference value for serum CRP. Based on ANOVA statistical test, we found that the mean +/- S.D. was 1.3 +/- 1.27 mg/l (n=202) for normal individuals of 50-80 years and 0.43 +/- 0.42 mg/l (n=148) for the group of 20-50 years. CONCLUSIONS The normal serum CRP mean concentrations for two age groups were distinctively different (p value<0.001). Our study suggests two different normal cutoffs of serum CRP to be employed for individuals in different age groups.

Human papillomavirus-16 infection in advanced oral cavity cancer patients is related to an increased risk of distant metastases and poor survival.

Background Human papillomavirus (HPV) is an oncogenic virus causing oropharyngeal cancers and resulting in a favorable outcome after the treatment. The role of HPV in oral cavity squamous cell carcinoma (OSCC) remains ambiguous. Objective This study aimed to examine the effect of HPV infection on disease control among patients with OSCC following radical surgery with radiation-based adjuvant therapy. Patients and Method We prospectively followed 173 patients with advanced OSCC (96% were stage III/IV) who had undergone radical surgery and adjuvant therapy between 2004 and 2006. They were followed between surgery and death or up to 60 months. Surgical specimens were examined using a PCR-based HPV blot test. The primary endpoints were the risk of relapse and the time to relapse; the secondary endpoints were disease-free survival, disease-specific survival, and overall survival. Results The prevalence of HPV-positive OSCC was 22%; HPV-16 (9%) and HPV-18 (7%) were the genotypes most commonly encountered. Solitary HPV-16 infection was a poor predictor of 5-year distant metastases (hazard ratio, 3.4; 95% confidence interval, 1.4–8.0; P = 0.005), disease-free survival (P = 0.037), disease-specific survival (P = 0.006), and overall survival (P = 0.010), whereas HPV-18 infection had no impact on 5-year outcomes. The rate of 5-year distant metastases was significantly higher in the HPV-16 or level IV/V metastasis group compared with both the extracapsular spread or tumor depth ≥11-mm group and patients without risk factors (P<0.001). Conclusions HPV infections in advanced OSCC patients are not uncommon and clinically relevant. Compared with HPV-16-negative advanced OSCC patients, those with a single HPV-16 infection are at higher risk of distant metastases and poor survival despite undergoing radiation-based adjuvant therapy and require a more aggressive adjuvant treatment and a more thorough follow-up.

Serum total homocysteine increases with the rapid proliferation rate of tumor cells and decline upon cell death: a potential new tumor marker

BACKGROUND We were interested to know why cancer patients are frequently associated with elevated circulating total homocysteine (tHcy) even though they are not treated with anti-folate drugs. METHODS We employed tissue cultures to compare both the homocysteine (Hcy)-released and production of tumor markers between tumor and normal cell lines. RESULTS We detected much higher concentrations of homocysteine (Hcy) released by the tumor cells. However, much less difference was found between normal and tumor cell lines when Hcy concentration was expressed per the same number of cells. During the cell culture, the increase of Hcy and the increase of tumor marker concentration paralleled each other for the first 7 days. After the seventh day of the culture when cells started dying, tumor markers continued to rise, whereas levels of Hcy and cell numbers leveled off. We found that the serum concentration of Hcy fluctuated in circulation coinciding with that of tumor marker in individual cancer patients unless taking anti-neoplastic drug. CONCLUSIONS The elevation of tHcy concentration may be caused by the rapid tumor cell proliferation and reflect only the number of live cells. Serum Hcy may be a potentially useful tumor marker to monitor tumor activity.

Comparison of Clinical Features Between Coxsackievirus A2 and Enterovirus 71 During the Enterovirus Outbreak in Taiwan, 2008: A Children's Hospital Experience

BACKGROUND/PURPOSE Coxsackievirus A2 (Cox A2) was the predominant serotype in the enterovirus outbreak in Taiwan, 2008. However, detailed clinical features of Cox A2 infection have not been reported. In this study, we compared Cox A2 with enterovirus 71 (EV71) in terms of clinical manifestation and epidemiology during the 2008 enterovirus outbreak in Taiwan. METHODS A total of 280 hospitalized patients (97 with culture-proven EV71 infection and 183 with culture-proven Cox A2 infection) in 2008 at the Chang Gung Childrens Medical Center were enrolled in this study. Epidemiologic data, clinical manifestations, and outcomes for these patients were collected and compared. RESULTS Both Cox A2 and EV71 serotypes peaked in June and declined soon afterwards. Seventy-one percent of the patients were younger than 3 years of age. Both groups had the same male-to-female ratio of 1.6:1. Patients with EV71 infection had a significantly longer hospitalization period (4.1 vs. 3.0 days, p< 0.001). Fever, fever for more than 3 days with a temperature above 39 degrees C, lethargy, poor activity, poor appetite and a myoclonic jerk were significantly associated with EV71 infection. Fever, or fever with a temperature above 39 degrees C, febrile seizure, elevated white cell counts, and elevated serum C-reactive protein concentrations were significantly associated with Cox A2 infection. Most patients with EV71 infection presented with hand-foot-mouth disease (78.3%), while most Cox A2-infected patients presented with herpangina (83.6%). Central nervous system complications were found in 18.6% of EV71-infected children, but only in 1.1% of Cox A2-infected children. All the patients with Cox A2 infection showed total recovery. One patient with EV71 infection died from encephalitis with cardiopulmonary failure, and 6.2% of EV71-infected children had neurologic sequelae. CONCLUSION Both Cox A2 and EV71 serotypes accounted for the enterovirus outbreak in Taiwanese children in 2008. Compared with those infected by EV71, the children with Cox A2 infection mostly presented with herpangina, had fewer central nervous system complications, and had better overall outcome.

Complementary serum test of antibodies to Epstein-Barr virus nuclear antigen-1 and early antigen: a possible alternative for primary screening of nasopharyngeal carcinoma.

This hospital-based cohort study evaluated the efficacy of three Epstein-Barr virus (EBV) - associated assays for nasopharyngeal carcinoma (NPC) primary screening and monitoring treatment outcome. Five hundred and seventeen consecutive subjects, including 156 NPC patients, 264 healthy volunteers and 97 patients with head and neck squamous cell carcinoma (HNSCC) were enrolled. The sensitivity and specificity of EBV IgAs to viral capsid antigen (VCA), complementary EBV IgAs to early antigen and nuclear antigen-1 (EA+EBNA-1), and EBV DNA load were examined by immunofluorescent assays, enzyme-linked immunosorbent assays, and quantitative real-time PCR, respectively. After constructing the receiver operating characteristics to demonstrate screening efficacy, EBV EA+EBNA-1 IgA (AUC: 0.952; 95% CI, 0.930-0.974) was proved superior to EBV VCA IgA (AUC: 0.888; 95% CI, 0.854-0.922) or EBV DNA load (AUC: 0.893; 95% CI, 0.854-0.932) in differentiating NPC patients from controls. Comparison of screening efficacy between NPC patients and HNSCC patients revealed EBV EA+EBNA-1 IgA (AUC: 0.964; 95% CI, 0.943-0.985) still outperformed EBV VCA IgA (AUC: 0.884; 95% CI, 0.845-0.923). In subjects with higher serum titer or level equal to or above 1:80 and 6 EU/ml for EBV VCA IgA and EA+EBNA-1 IgA, the specificity reached as high as 99.2% and 95.1%, respectively, in the control groups. However, correlation of these three assays with clinicopathological manifestations of NPC, revealed only EBV DNA load significantly associated with N stage and overall stage in NPC patients. Additionally, EBV DNA load could be used to further raise the specificity of EBV EA+EBNA-1 IgA assays and was also the only assay to be consistently predictive of tumor relapse in post-treatment patients according to serial test results by time frame. Consequently, an EBV EA+EBNA-1 IgA-based protocol is recommended for mass screening, but EBV DNA load should be used solely for post-treatment monitoring for NPC in endemic areas.

Paper-based enzyme-free immunoassay for rapid detection and subtyping of influenza A H1N1 and H3N2 viruses.

Development of rapid screening in the ambulatory environment is the most pressing needs for the control of spread of infectious disease. Despite there are many methods to detect the immunoassay results, quantitative measurement in rapid disease screening is still a great challenge for point-of-care applications. In this work, based on the internal structural protein, i.e., nucleoprotein (NP), and outer surface glycoproteins, i.e., H1 and H3, of the influenza viruses, specific and sensitive immunoassay on paper-based platform was evaluated and confirmed. Detection and subtyping of influenza A H1N1 and H3N2 viruses found in people were demonstrated by colorimetric paper-based sandwich immunoassay. Concentration-dependent response to influenza viruses was shown and the detection limits could achieve 2.7×10(3) pfu/assay for H1 detection and 2.7×10(4) pfu/assay for H3 detection, which are within the clinical relevant level. Moreover, detection of influenza virus from infected cell lysate and clinical samples was demonstrated to further confirm the reliability of the paper-based immunoassay. The use of paper for the development of diagnostic devices has the advantages of lightweight, ease-of-use, and low cost and paper-based immunoassay is appropriate to apply for rapid screening in point-of-care applications.

Epidemiologic features and virus isolation of enteroviruses in Northern Taiwan during 2000–2008

The susceptibility of five cell lines, RD, MRC-5, MK-2, Hep-2 and A549, for various serotypes of human enteroviruses was evaluated. RD cells were susceptible to most serotypes of enteroviruses, especially for human enterovirus A. Consequently, a high prevalence of human enterovirus A in Taiwan may reflect the relative importance of RD cells in the clinical virology laboratory. However, susceptibilities of RD cells to coxsackievirus A9 and A24, and echovirus 9 and 11 were exceptionally low. Alternatively, MRC-5 or MK-2 cells can improve the isolation of these four enteroviruses.