Patcharee Jearanaikoon

The use of viral load as a surrogate marker in predicting disease progression for patients with early invasive cervical cancer with integrated human papillomavirus type 16.

OBJECTIVE The purpose of this study was to assess the effectiveness of the use of human papillomavirus type 16 (HPV16) physical status and viral load in combination to predict clinical outcome during cervical development. STUDY DESIGN A follow-up study was monitored in association with HPV integration and viral load in 121 cervical samples with the use of multiplex quantitative polymerase chain reaction. RESULTS A significant increase of viral load was found earlier from preinvasive to invasive groups compared with normal groups, except with clinical staging and clinical outcome. High occurrence of integrated HPV16 was observed in preinvasive (27/44 samples) and invasive cervical carcinoma (40/68 samples). Cervical progression was observed significantly in most preinvasive (18/27 samples) and invasive cases (25/40 samples) that were infected with integrated HPV. Integrated HPV16 with significant viral load can be used as a predictive marker for tumor progression in the early stage of invasive cervical carcinoma. CONCLUSION Integrated HPV16 in combination with viral load is a predictive indicator for tumor progression in early invasive stage but not in preinvasive and advanced invasive stage.

Characterization of 5-Fluorouracil-Resistant Cholangiocarcinoma Cell Lines

Background: Although 5-fluorouracil (5-FU) is the drug of choice for the palliative treatment of cholangiocarcinoma (CCA), resistance to the drug is a therapeutic obstacle. The aim of this study was to explore the mechanisms underlying 5-FU resistance of CCA using cell lines derived from CCA associated with liver fluke infection. Methods: A stepwise exposure was used for inducing 5-FU-resistant CCA cell lines, and the expression of nine genes associated with 5-FU resistance was analyzed using real-time (RT)-PCR. Results: Altered expression of several genes involved in 5-FU resistance in CCA cell lines was observed. The expression levels of almost all target genes investigated including TP, DPD, ENT1, UNG1, TOP2A, BIRC5, TP73 and DeltaNp73 appeared to be significantly altered in these resistant strains. The expression of the TS gene tended to be increased but the fold change was not significantly different from their parental cell lines. UNG1 (a DNA repairing enzyme) and BIRC5 (an apoptotic inhibitor) expressions were increased whereas TP73 (a proapoptotic factor) expression levels decreased concomitantly. Conclusion: Our study showed that increases in UNG1 and BIRC5 expression and concomitant decreases in TP73 expression may be associated with development of acquired 5-FU resistance in CCA lines and their phenotypes.

The development of DNA-based quartz crystal microbalance integrated with isothermal DNA amplification system for human papillomavirus type 58 detection.

To address the effect of dramatic change in temperature and viscosity during PCR process on quartz crystal microbalance (QCM) sensor and to increase the sensitivity, isothermal amplification was employed in the system. We combined loop-mediated isothermal amplification (LAMP) technique with QCM, called as LAMP-QCM, for detection of high-risk human papillomavirus viral DNA type 58 (HPV-58) which is commonly found in Asian women. The liquid-phase LAMP-QCM prototype comprised the frequency counter, a temperature control device and housing of the quartz crystal with polished gold electrodes on both sides. QCM detection signal was monitored in real-time based on an avidin-biotin binding between avidin coated QCM surface and specific biotinylated LAMP products. Analytical performance was evaluated for precision, sensitivity and specificity. A plasmid clone containing the HPV-58 sequence was diluted from 10(6) to 1 copy and used for detection limit. Cut-off value was estimated at 28.8 Hz from negative viral template. The system could detect 100 copies with Δf at 34.0±3.6 Hz compared to 1000 copies detected by conventional LAMP. No cross-reaction was observed with other HPV types. The HPV-58 detection was compared among LAMP-QCM, conventional LAMP and nested PCR in 50 cervical cancer tissues. The positive rate of LAMP-QCM was higher than that of conventional LAMP with 100% sensitivity and 90.5% specificity. The integrated LAMP-QCM system has improved the detection limit up to ten times compared to conventional LAMP with less-time consuming.

Trefoil factors: Tumor progression markers and mitogens via EGFR/MAPK activation in cholangiocarcinoma

AIM To investigate trefoil factor (TFF) gene copy number, mRNA and protein expression as potential biomarkers in cholangiocarcinoma (CCA). METHODS TFF mRNA levels, gene copy number and protein expression were determined respectively by quantitative reverse transcription polymerase chain reaction (PCR), quantitative PCR and immunohistochemistry in bile duct epithelium biopsies collected from individuals with CCA, precancerous bile duct dysplasia and from disease-free controls. The functional impact of recombinant human (rh)TFF2 peptide treatment on proliferation and epidermal growth factor receptor (EGFR)/mitogen-activated protein kinase (MAPK) signaling was assessed in the CCA cell line, KMBC, by viable cell counting and immunoblotting, respectively. RESULTS TFF1, TFF2 and TFF3 mRNA expression was significantly increased in CCA tissue compared to disease-free controls, and was unrelated to gene copy number. TFF1 immunoreactivity was strongly increased in both dysplasia and CCA, whereas TFF2 immunoreactivity was increased only in CCA compared to disease-free controls. By contrast, TFF3 immunoreactivity was moderately decreased in dysplasia and further decreased in CCA. Kaplan-Meier analysis found no association of TFF mRNA, protein and copy number with age, gender, histological subtype, and patient survival time. Treatment of KMBC cells with rhTFF2 stimulated proliferation, triggered phosphorylation of EGFR and downstream extracellular signal related kinase (ERK), whereas co-incubation with the EGFR tyrosine kinase inhibitor, PD153035, blocked rhTFF2-dependent proliferation and EGFR/ERK responses. CONCLUSION TFF mRNA/protein expression is indicative of CCA tumor progression, but not predictive for histological sub-type or survival time. TFF2 is mitogenic in CCA via EGFR/MAPK activation.

Chitinase 3 like 1 is associated with tumor angiogenesis in cervical cancer.

Elevated serum levels of a secreted glycoprotein chitinase 3 like 1 (CHI3L1) are associated with poor prognosis and short survival time of patients with cervical cancer (CxCa). Our previous microarray data showed the increased expression of CHI3L1 in invasive CxCa compared to normal tissue, implicating a potential role of CHI3L1 in CxCa. To establish the pathological role of CHI3L1 in the development of CxCa, this study focused on its expression in CxCa and angiogenic impacts in tumor vessel formation. CHI3L1 activated angiogenesis by promoting endothelial cell migration and tube formation in vitro but failed to protect CxCa cell lines, CaSki and HeLa against apoptosis induced by γ-irradiation. In addition, the capability of CHI3L1 to induce proliferation and migration of CaSki and HeLa cells was cell type specific. In an analysis of 103 specimens from CxCa patients, increased expression levels of CHI3L1 mRNA and protein in invasive CxCa were 4-fold (P<0.05) and 2-fold (P<0.01), respectively, stronger than those in normal subjects. The immunostaining of CHI3L1 was positively correlated with VEGF expression (P=0.0019) and microvessel density (P=0.0110). Moreover, CHI3L1 expression was also positively associated with cancer metastasis (P=0.011). The data suggest the crucial role of CHI3L1 by promoting angiogenesis, which may contribute to the development and progression of CxCa. The findings help establish CHI3L1 as a prognostic biomarker and therapeutic target for CxCa patients.

Ratio disruption of the ∆133p53 and TAp53 isoform equilibrium correlates with poor clinical outcome in intrahepatic cholangiocarcinoma

All p53 family members are expressed in several isoforms through alternative promoters and alternative splicing. However, the significance of these isoforms is not yet well understood in cholangiocarcinoma (CCA). In this study, we investigated the expression of p53, p63, p73 and their isoforms at the mRNA and protein levels in CCA. The overexpression of ∆133p53 was observed in the CCA cell lines and clinical specimens. Moreover, the high expression of ∆133p53/TAp53 correlated with short overall survival (p<0.001). Defective p53, including mutant and ∆Np53, was associated with poor prognosis (p<0.024). Multivariate analysis demonstrated that ∆133p53/TAp53 and mutant p53 protein may be used as independent prognostic factors for CCA. To our knowledge, this is the first report of the use of ∆133p53/TAp53 as a potential biomarker in CCA.

Preferentially different mechanisms of inactivation of 9p21 gene cluster in liver fluke―related cholangiocarcinoma

Cholangiocarcinoma in northeast Thailand is associated with liver fluke infection. Mechanisms of inactivation of the p15(INK4b), p16(INK4a), and p14(ARF) have been reported in many human cancers but have not hitherto been studied in liver fluke-related cholangiocarcinoma, particularly genetic and epigenetic effects on protein expression. We investigated loss of heterozygosity and microsatellite instability and performed fine mapping of the chromosomal region 9p21-pter in 94 microdissected cholangiocarcinoma samples using polymerase chain reaction based-microsatellite markers. Methylation and protein expression of p14(ARF), p15(INK4b), and p16(INK4a) was determined using methylation-specific polymerase chain reaction and immunohistochemistry, respectively. Genetic and epigenetic alterations, including loss of protein expression, were correlated with clinicopathological data. Fine mapping at 9p21-pter showed a distinctive region between D9S286 and D9S1752 of common loss. Methylation frequency was 40.2% for p14(ARF), 48.9% for p15(INK4b), and 28.3% for p16(INK4a). Loss of protein expression of p14(ARF), p15(INK4b), and p16(INK4a) was 30.9%, 58%, and 81.5%, respectively. Both p14(ARF) methylation and allelic loss at 9p21 were associated with loss of p14(ARF) expression. Poor prognosis was associated with loss of p16(INK4a) expression. In conclusion, mechanisms of inactivation of p14(ARF), p15(INK4b), and p16(INK4a) in liver fluke-related cholangiocarcinoma are preferentially different, by which epigenetic event being the main mechanism of p14(ARF), whereas p16(INK4a) and p15(INK4b) inactivation occurs through genetic and both genetic and epigenetic events, respectively.

Microsatellite alterations in liver fluke related cholangiocarcinoma are associated with poor prognosis.

We have characterized the role of genetic alterations in the development of liver fluke related cholangiocarcinoma. We analyzed the loss of heterozygosity (LOH) and microsatellite instability (MSI) of hMSH2, hMLH1, and p53 genes in 55 patients with intrahepatic cholangiocarcinoma by using polymerase chain reaction based microsatellite markers D2S119, D3S1611, and TP53, respectively and determined the association between microsatellite alterations and patient survival. A total of 27 (49.1%) out of 55 cases exhibited microsatellite alterations in one locus or more. Of 55 samples, 11 (20%) demonstrated MSI at D2S119 and four (7%) showed MSI at D3S1611. LOH was shown in seven out of 36 (19%) informative cases for D3S1611 and 16 out of 50 (32%) for TP53. Microsatellite alterations at loci studied were significantly associated with poor survival (P=0.0098). This study suggests that genetic alterations of DNA mismatch repair genes and tumor suppressor gene p53 may be involved in cholangiocarcinogenesis and these alterations may be of value as prognostic indicators for liver fluke related cholangiocarcinoma.

Rapid detection of the most common high-risk human papillomaviruses by loop-mediated isothermal amplification

Persistent infection with high-risk human papillomavirus (HPV) is a major risk factor for development of cervical cancer. At present, polymerase chain reaction (PCR)-based methods, the most widely molecular tools used for HPV detection, are time-consuming and require expensive instruments. In this study, loop-mediated isothermal amplification (LAMP) was established for detection of HPV types 16, 18, 45 and 58 which are frequently found in Thailand. The optimal condition for detection of these high risk HPVs was 63°C for 60min. Since a white magnesium pyrophosphate precipitate is a characteristic by product of the LAMP reaction which can be visualized directly by the naked eye, the entire assay time of LAMP is 1h compared to 6-8h of for a nested PCR detection. The detection limit of LAMP assay was shown to be equivalent to nested PCR that could amplify 10(2) copies of HPV-18 and 10(3) copies of HPV 16, 45 and 58, as determined by either turbidity detection or agarose gel electrophoresis. No cross-reaction was observed, indicating that LAMP assay has high type-specificity. The assay showed successful detection of HPV in 56 clinical specimens. Using nested PCR as the gold standard, the sensitivity, specificity, negative predictive values and positive predictive values of LAMP assay were 100%. In conclusion, LAMP assay is a high efficiency, low cost diagnostic tool, useful for rapid, accurate, direct detection of HPV for clinical diagnosis.

Associations of MICB with cervical cancer in north‐eastern Thais: identification of major histocompatibility complex class I chain‐related gene B motifs influencing natural killer cell activation

The expression of MICB, a member of the major histocompatibility complex class I chain‐related gene B family, is induced in response to cellular stress. It is one of the ligands to the NKG2D receptor. MICB is polymorphic, but the distribution of MICB polymorphism in north‐eastern Thais and their potential associations with cancer have not yet been elucidated. In this study, polymerase chain reaction–sequence‐specific primers were developed to identify 15 MICB alleles and one group of alleles. We performed MICB typing in 100 healthy north‐eastern Thai females (NETF) and 99 cervical cancer patients to evaluate the association of MICB polymorphisms and the risk of developing cervical cancer. Eight and nine alleles were detected in the NETF and cervical cancer respectively. MICB*00502 was associated negatively with a corrected P‐value of 0·0009, suggesting the existence of a protective allele in cervical cancer. Amino acid substitutions carried by this allele were investigated for their potential involvement in natural killer (NK) cell activation. Although lysine at amino acid position 80 (Lys80) and aspartic acid at position 136 (Asp136) were associated negatively with cervical cancer, only MICB carrying Asp136 could induce NK cell killing more efficiently than MICB‐Lys80 when the NK cells were blocked by anti‐NKG2D. This result suggested that aspartic acid at position 136 may affect NKG2D binding, leading to different degrees of immune cell activation.