Hyun-Kyoung Kim

Transglutaminase 2 as a cisplatin resistance marker in non-small cell lung cancer

PurposeRecently, it was reported that expression of transglutaminase 2 plays an important role in doxorubicin/cisplatin resistance in breast and ovarian cancer. The aims of this study were to verify the role of transglutaminase 2 in cisplatin response in non-small cell lung cancer (NSCLC) and to study if transglutaminase 2 gene (TGM2) methylation can be a molecular marker for good response to cisplatin.MethodsTGM2 promoter methylation was analyzed by sodium bisulfite sequencing. Cisplatin sensitivity was analyzed by treatment of cisplatin in NSCLC cell lines with/without TGM2 or TGM2 siRNA transfection.ResultsIn one-third of NSCLC cell lines, TGase 2 gene (TGM2) was silenced by promoter methylation. The TGM2 promoter-methylated cell lines (HCC-95 and HCC-1588) showed relatively higher sensitivity to cisplatin than the TGM2-expressing cell lines (NCI-H1299 and HCC-1195). Down-regulation and over-expression of TGM2 in those NSCLC cells also suggested a positive correlation of cisplatin sensitivity and TGM2 inhibition. With doxorubicin, the relationship was quite similar.ConclusionsWe showed that good responders of cisplatin in NSCLC could be identified by the promoter methylation of TGM2 and that TGase 2 inhibition appears to be an effective cisplatin-sensitizing modality in NSCLC.

Transglutaminase 2 as an independent prognostic marker for survival of patients with non-adenocarcinoma subtype of non-small cell lung cancer

BackgroundExpression of transglutaminase 2 (TGase 2) is related to invasion and resistance to chemotherapeutic agents in several cancer cells. However, there has been only limited clinical validation of TGase 2 as an independent prognostic marker in cancer.MethodsThe significance of TGase 2 expression as an invasive/migratory factor was addressed by in vitro assays employing down-regulation of TGase 2. TGase 2 expression as a prognostic indicator was assessed in 429 Korean patients with early-stage non-small cell lung cancer (NSCLC) by immunohistochemical staining.ResultsTGase 2 expression increased the invasive and migratory properties of NSCLC cells in vitro, which might be related to the induction of MMP-9. In the analysis of the immunohistochemical staining, TGase 2 expression in tumors was significantly correlated with recurrence in NSCLC (p = 0.005) or in the non-adenocarcinoma subtype (p = 0.031). Additionally, a multivariate analysis also showed a significant correlation between strong TGase 2 expression and shorter disease-free survival (DFS) in NSCLC (p = 0.029 and HR = 1.554) and in the non-adenocarcinoma subtype (p = 0.030 and HR = 2.184). However, the correlation in the adenocarcinoma subtype was not significant.ConclusionsTGase 2 expression was significantly correlated with recurrence and shorter DFS in NSCLC, especially in the non-adenocarcinoma subtype including squamous cell carcinoma.

Chromatin CKAP2, a new proliferation marker, as independent prognostic indicator in breast cancer.

Background The level of proliferation activity is a strong prognostic or predictive indicator in breast cancer, but its optimal measurement is still in debate, necessitating new proliferation markers. In the present study, the prognostic significance of the CKAP2-positive cell count (CPCC), a new proliferation marker, was evaluated, and the results were compared with those for the mitotic activity index (MAI). Methods This study included 375 early-stage breast cancer samples collected from two institutions between 2000 and 2006. Immunohistochemical staining was performed using a CKAP2 monoclonal antibody. Cox proportional hazard regression models were fitted to determine the association between the CPCC and relapse-free survival (RFS) amongst three groups formed on the basis of the CPCC or MAI value: groups 2 and 3 showing the middle and highest values, respectively, and group 1 the lowest. Results After adjustment for age, T stage, N stage, HER2 status, estrogen receptor status, progesterone receptor status, institution, and year of surgical resection, the CPCC was associated with a significantly worse RFS {hazard ratio [HR]  = 4.10 (95% CI: 1.64–10.29) for group 2; HR  = 4.35 (95% CI: 2.04–10.35) for group 3}. Moreover, its prognostic significance was similar to or higher than that based on the MAI {HR  = 2.05 (95% CI: 0.94–4.65) for group 2; HR  = 2.35 (95% CI: 1.09–5.10) for group 3}. In subgroup analyses, the CPCC showed a prognostic significance in the luminal A and triple-negative subgroups, but not in the HER2-positive subgroup. Conclusions Chromatin CKAP2 is an independent prognostic marker for RFS in early-stage breast cancer, and could potentially replace the MAI in clinical evaluation of proliferation activity. Additionally, our study results suggest that the prognostic significance of proliferation activity differs among the various subgroups of breast cancer.

The association between bone mineral density and periodontitis in Korean adults (KNHANES 2008–2010)

OBJECTIVES This study investigated the association between bone mineral density (BMD) and periodontitis in a representative sample of Korean adults. MATERIAL AND METHODS Of 36 188 individuals who participated in the Korea National Health and Nutrition Examination Survey in 2008, 2009, and 2010, 9977 participants aged ≥40 years were included in this cross-sectional study. The associations of BMD of lumbar spine, total femur, and femoral neck with periodontitis were investigated using logistic regression analysis. Additionally, dose-response relationships with BMD divided into quintiles and the association between osteoporosis and periodontitis were investigated. RESULTS With the set of Community Periodontal Index (CPI) ≥ 3 as a dependent variable, logistic regression analysis revealed that a decrease of BMD was significantly associated with higher odds of periodontitis [range of adjusted odds ratios (AORs); 1.15-1.20, P < 0.001 for all BMD sites]. Similarly, these associations were also found in the CPI 4 model. With regard to dose-response relations, the lower the BMD quintile, the higher the AORs appeared with statistical significance in the CPI ≥ 3 model. (P for trend < 0.001) Participants with osteoporosis had 2.26 and 1.91 times higher odds for CPI ≥ 3 and CPI 4, respectively, than those with normal BMD, indicating a significant association between the two diseases. CONCLUSIONS Our results suggest that BMD is significantly associated with periodontitis.

CD3Z hypermethylation is associated with severe clinical manifestations in systemic lupus erythematosus and reduces CD3ζ-chain expression in T cells

Objective. The importance of hypomethylation in SLE is well recognized; however, the significance of hypermethylation has not been well characterized. We screened hypermethylated marks in SLE and investigated their possible implications. Methods. DNA methylation marks were screened in SLE whole-blood DNA by microarray, and two marks (CD3Z and VHL hypermethylations) were confirmed by a methylation single-base extension method in two independent ethnic cohorts consisting of 207 SLE patients and 151 controls. The correlation with clinical manifestations and the genetic influence on those epigenetic marks were analysed. Results. Two epigenetic marks, CD3Z and VHL hypermethylation, were significantly correlated with SLE: CD3Z hypermethylation (odds ratio = 7.76; P = 1.71 × 10−13) and VHL hypermethylation (odds ratio = 3.77; P = 3.20 × 10−8), and the increased CD3Z methylation was correlated with downregulation of the CD3&zgr;-chain in SLE T cells. In addition, less genetic influence on CD3Z methylation relative to VHL methylation was found in analyses of longitudinal and twin samples. Furthermore, a higher CD3Z methylation level was significantly correlated with a higher SLE disease activity index and more severe clinical manifestations, such as proteinuria, haemolytic anaemia and thrombocytopenia, whereas VHL hypermethylation was not. Conclusion. CD3Z hypermethylation is an SLE risk factor that can be modified by environmental factors and is associated with more severe SLE clinical manifestations, which are related to deranged T cell function by downregulating the CD3&zgr;-chain.

Immunohistochemical localization of LLC1 in human tissues and its limited expression in non-small cell lung cancer.

We have shown both LLC1 expression in the lung epithelium by in situ hybridization and its inactivation in lung cancer by epigenetic modification. However, LLC1 proteins cellular localization or its role in normal lung or cancer tissues has not yet been evaluated. In the present study, a monoclonal antibody against recombinant LLC1 was produced, and immunohistochemical staining was performed on arrays including various human tissues, normal lung and non-small cell lung cancer (NSCLC) tissues for LLC1 localization. The immunohistochemical results showed LLC1 expression in the cilia of normal-airway epithelial cells and in the cytoplasm of type II pneumocytes in bronchiectatic patients, but no expression in most of the NSCLC tissues, which is consistent with our previous report positing LLC1 as a tumor suppressor. However, LLC1 over-expression in NSCLC cell lines NCI-H1299 and NCI-H23 did not show any change in proliferation or migration, which does not indicate any LLC1 tumor-suppressor role. As for the other human tissues, LLC1 was localized in renal tubular cells, pancreatic acinar cells, and epithelial cells of the stomach, duodenum, and gallbladder. In summary, our findings suggest that LLC1 is not a tumor suppressor, and that it is localized in the cilia of the normal lung epithelium but is absent in most NSCLC cases, probably due to the loss of cilia during lung carcinogenesis.

Simple and versatile molecular method of copy-number measurement using cloned competitors.

Variations and alterations of copy numbers (CNVs and CNAs) carry disease susceptibility and drug responsiveness implications. Although there are many molecular methods to measure copy numbers, sensitivity, reproducibility, cost, and time issues remain. In the present study, we were able to solve those problems utilizing our modified real competitive PCR method with cloned competitors (mrcPCR). First, the mrcPCR for ERBB2 copy number was established, and the results were comparable to current standard methods but with a shorter assay time and a lower cost. Second, the mrcPCR assays for 24 drug-target genes were established, and the results in a panel of NCI-60 cells were comparable to those from real-time PCR and microarray. Third, the mrcPCR results for FCGR3A and the FCGR3B CNVs were comparable to those by the paralog ratio test (PRT), but without PRTs limitations. These results suggest that mrcPCR is comparable to the currently available standard or the most sensitive methods. In addition, mrcPCR would be invaluable for measurement of CNVs in genes with variants of similar structures, because combination of the other methods is not necessary, along with its other advantages such as short assay time, small sample amount requirement, and applicability to all sequences and genes.

Targeted next-generation sequencing at copy-number breakpoints for personalized analysis of rearranged ends in solid tumors.

Background The concept of the utilization of rearranged ends for development of personalized biomarkers has attracted much attention owing to its clinical applicability. Although targeted next-generation sequencing (NGS) for recurrent rearrangements has been successful in hematologic malignancies, its application to solid tumors is problematic due to the paucity of recurrent translocations. However, copy-number breakpoints (CNBs), which are abundant in solid tumors, can be utilized for identification of rearranged ends. Method As a proof of concept, we performed targeted next-generation sequencing at copy-number breakpoints (TNGS-CNB) in nine colon cancer cases including seven primary cancers and two cell lines, COLO205 and SW620. For deduction of CNBs, we developed a novel competitive single-nucleotide polymorphism (cSNP) microarray method entailing CNB-region refinement by competitor DNA. Result Using TNGS-CNB, 19 specific rearrangements out of 91 CNBs (20.9%) were identified, and two polymerase chain reaction (PCR)-amplifiable rearrangements were obtained in six cases (66.7%). And significantly, TNGS-CNB, with its high positive identification rate (82.6%) of PCR-amplifiable rearrangements at candidate sites (19/23), just from filtering of aligned sequences, requires little effort for validation. Conclusion Our results indicate that TNGS-CNB, with its utility for identification of rearrangements in solid tumors, can be successfully applied in the clinical laboratory for cancer-relapse and therapy-response monitoring.