Year of publication
- Reduced migration of MLH1 deficient colon cancer cells depends on SPTAN1 (2014)
- Introduction: Defects in the DNA mismatch repair (MMR) protein MLH1 are frequently observed in sporadic and hereditary colorectal cancers (CRC). Affected tumors generate much less metastatic potential than the MLH1 proficient forms. Although MLH1 has been shown to be not only involved in postreplicative MMR but also in several MMR independent processes like cytoskeletal organization, the connection between MLH1 and metastasis remains unclear. We recently identified non-erythroid spectrin αII (SPTAN1), a scaffolding protein involved in cell adhesion and motility, to interact with MLH1. In the current study, the interaction of MLH1 and SPTAN1 and its potential consequences for CRC metastasis was evaluated. Methods: Nine cancer cell lines as well as fresh and paraffin embedded colon cancer tissue from 12 patients were used in gene expression studies of SPTAN1 and MLH1. Co-expression of SPTAN1 and MLH1 was analyzed by siRNA knock down of MLH1 in HeLa, HEK293, MLH1 positive HCT116, SW480 and LoVo cells. Effects on cellular motility were determined in MLH1 deficient HCT116 and MLH1 deficient HEK293T compared to their MLH1 proficient sister cells, respectively. Results: MLH1 deficiency is clearly associated with SPTAN1 reduction. Moreover, siRNA knock down of MLH1 decreased the mRNA level of SPTAN1 in HeLa, HEK293 as well as in MLH1 positive HCT116 cells, which indicates a co-expression of SPTAN1 by MLH1. In addition, cellular motility of MLH1 deficient HCT116 and MLH1 deficient HEK293T cells was impaired compared to the MLH1 proficient sister clones. Consequently, overexpression of SPTAN1 increased migration of MLH1 deficient cells while knock down of SPTAN1 decreased cellular mobility of MLH1 proficient cells, indicating SPTAN1-dependent migration ability. Conclusions: These data suggest that SPTAN1 levels decreased in concordance with MLH1 reduction and impaired cellular mobility in MLH1 deficient colon cancer cells. Therefore, aggressiveness of MLH1-positive CRC might be related to SPTAN1.
- Promoter Methylation of MLH1, PMS2, MSH2 and p16 Is a Phenomenon of Advanced-Stage HCCs (2014)
- Epigenetic silencing of tumour suppressor genes has been observed in various cancers. Looking at hepatocellular carcinoma (HCC) specific protein silencing was previously demonstrated to be associated with the Hepatitis C virus (HCV). However, the proposed HCV dependent promoter methylation of DNA mismatch repair (MMR) genes and thereby enhanced progression of hepatocarcinogenesis has been the subject of controversial discussion. We investigated promoter methylation pattern of the MMR genes MLH1, MSH2 and PMS2 as well as the cyclin-dependent kinase inhibitor 2A gene (p16) in 61 well characterized patients with HCCs associated with HCV, Hepatitis B virus infection or alcoholic liver disease. DNA was isolated from formalin-fixed, paraffin-embedded tumour and non-tumour adjacent tissue and analysed by methylation-specific PCR. Moreover, microsatellite analysis was performed in tissues showing methylation in MMR gene promoters. Our data demonstrated that promoter methylation of MLH1, MSH2, PMS2 and p16 is present among all considered HCCs. Hereby, promoter silencing was detectable more frequently in advanced-stage HCCs than in low-stage ones. However, there was no significant correlation between aberrant DNA methylation of MMR genes or p16 and HCV infection in related HCC specimens. In summary, we show that promoter methylation of essential MMR genes and p16 is detectable in HCCs most dominantly in pT3 stage tumour cases. Since loss of MMR proteins was previously described to be not only responsible for tumour development but also for chemotherapy resistance, the knowledge of mechanisms jointly responsible for HCC progression might enable significant improvement of individual HCC therapy in the future.
- Interobserver Agreement of Thyroid Imaging Reporting and Data System (TIRADS) and Strain Elastography for the Assessment of Thyroid Nodules (2013)
- Background: Thyroid Imaging Reporting and Data System (TIRADS) was developed to improve patient management and cost-effectiveness by avoiding unnecessary fine needle aspiration biopsy (FNAB) in patients with thyroid nodules. However, its clinical use is still very limited. Strain elastography (SE) enables the determination of tissue elasticity and has shown promising results for the differentiation of thyroid nodules. Methods: The aim of the present study was to evaluate the interobserver agreement (IA) of TIRADS developed by Horvath et al. and SE. Three blinded observers independently scored stored images of TIRADS and SE in 114 thyroid nodules (114 patients). Cytology and/or histology was available for all benign (n = 99) and histology for all malignant nodules (n = 15). Results: The IA between the 3 observers was only fair for TIRADS categories 2–5 (Coheńs kappa = 0.27,p = 0.000001) and TIRADS categories 2/3 versus 4/5 (ck = 0.25,p = 0.0020). The IA was substantial for SE scores 1–4 (ck = 0.66,p<0.000001) and very good for SE scores 1/2 versus 3/4 (ck = 0.81,p<0.000001). 92–100% of patients with TIRADS-2 had benign lesions, while 28–42% with TIRADS-5 had malignant cytology/histology. The negative-predictive-value (NPV) was 92–100% for TIRADS using TIRADS-categories 4&5 and 96–98% for SE using score ES-3&4 for the diagnosis of malignancy, respectively. However, only 11–42% of nodules were in TIRADS-categories 2&3, as compared to 58–60% with ES-1&2. Conclusions: IA of TIRADS developed by Horvath et al. is only fair. TIRADS and SE have high NPV for excluding malignancy in the diagnostic work-up of thyroid nodules.
- Differential Stability of Cell-Free Circulating microRNAs: Implications for Their Utilization as Biomarkers (2013)
- Background: MicroRNAs circulating in the blood, stabilized by complexation with proteins and/or additionally by encapsulation in lipid vesicles, are currently being evaluated as biomarkers. The consequences of their differential association with lipids/vesicles for their stability and use as biomarkers are largely unexplored and are subject of the present study. Methods: The levels of a set of selected microRNAs were determined by quantitative reverse-transcription PCR after extraction from sera or vesicle- and non-vesicle fractions prepared from sera. The stability of these microRNAs after incubation with RNase A or RNase inhibitor, an inhibitor of RNase A family enzymes was studied. Results: The levels of microRNA-1 and microRNA-122, but not those of microRNA-16, microRNA-21 and microRNA-142-3p, declined significantly during a 5-h incubation of the sera. RNase inhibitor prevented the loss of microRNAs in serum as well as the degradation of microRNA-122, a microRNA not expressed in blood cells, in whole blood. Stabilization of microRNA-122 was also achieved by hemolysis. Prolonged incubation of the sera led to enrichment of vesicle-associated relative to non-vesicle-associated microRNAs. Vesicle-associated microRNAs were more resistant to RNase A treatment than the respective microRNAs not associated with vesicles. Conclusions: Serum microRNAs showed differential stability upon prolonged incubation. RNase inhibitor might be useful to robustly preserve the pattern of cell-free circulating microRNAs. In the case of microRNAs not expressed in blood cells this can also be achieved by hemolysis. Vesicle-associated microRNAs appeared to be more stable than those not associated with vesicles, which might be useful to disclose additional biomarker properties of miRNAs.
- 52-week efficacy and safety of telbivudine with conditional tenofovir intensification at week 24 in HBeAg-positive chronic Hepatitis B (2013)
- Background and Aims: The Roadmap concept is a therapeutic framework in chronic hepatitis B for the intensification of nucleoside analogue monotherapy based on early virologic response. The efficacy and safety of this approach applied to telbivudine treatment has not been investigated. Methods: A multinational, phase IV, single-arm open-label study (ClinicalTrials.gov ID NCT00651209) was undertaken in HBeAg-positive, nucleoside-naive adult patients with chronic hepatitis B. Patients received telbivudine (600 mg once-daily) for 24 weeks, after which those with undetectable serum HBV DNA (<300 copies/mL) continued to receive telbivudine alone while those with detectable DNA received telbivudine plus tenofovir (300 mg once-daily). Outcomes were assessed at Week 52. Results: 105 patients commenced telbivudine monotherapy, of whom 100 were included in the efficacy analysis. Fifty-five (55%) had undetectable HBV DNA at Week 24 and continued telbivudine monotherapy; 45 (45%) received tenofovir intensification. At Week 52, the overall proportion of undetectable HBV DNA was 93% (93/100) by last-observation-carried-forward analysis (100% monotherapy group, 84% intensification group) and no virologic breakthroughs had occurred. ALT normalization occurred in 77% (87% monotherapy, 64% intensification), HBeAg clearance in 43% (65% monotherapy, 16% intensification), and HBeAg seroconversion in 39% (62% monotherapy, 11% intensification). Six patients had HBsAg clearance. Myalgia was more common in the monotherapy group (19% versus 7%). No decrease in the mean glomerular filtration rate occurred in either treatment group at Week 52. Conclusions: Telbivudine therapy with tenofovir intensification at Week 24, where indicated by the Roadmap strategy, appears effective and well tolerated for the treatment of chronic hepatitis B. Trial Registration: ClinicalTrials.gov NCT00651209
- Serum microRNA-122 predicts survival in patients with liver cirrhosis (2012)
- BACKGROUND: Liver cirrhosis is associated with high morbidity and mortality. MicroRNAs (miRs) circulating in the blood are an emerging new class of biomarkers. In particular, the serum level of the liver-specific miR-122 might be a clinically useful new parameter in patients with acute or chronic liver disease. AIM: Here we investigated if the serum level of miR-122 might be a prognostic parameter in patients with liver cirrhosis. METHODS: 107 patients with liver cirrhosis in the test cohort and 143 patients in the validation cohort were prospectively enrolled into the present study. RNA was extracted from the sera obtained at the time of study enrollment and the level of miR-122 was assessed. Serum miR-122 levels were assessed by quantitative reverse-transcription PCR (RT-PCR) and were compared to overall survival time and to different complications of liver cirrhosis. RESULTS: Serum miR-122 levels were reduced in patients with hepatic decompensation in comparison to patients with compensated liver disease. Patients with ascites, spontaneous bacterial peritonitis and hepatorenal syndrome had significantly lower miR-122 levels than patients without these complications. Multivariate Cox regression analysis revealed that the miR-122 serum levels were associated with survival independently from the MELD score, sex and age. CONCLUSIONS: Serum miR-122 is a new independent marker for prediction of survival of patients with liver cirrhosis.
- Acoustic radiation force impulse imaging for differentiation of thyroid nodules (2012)
- Background: Acoustic Radiation Force Impulse (ARFI)-Imaging is an ultrasound-based elastography method enabling quantitative measurement of tissue stiffness. The aim of the present study was to evaluate sensitivity and specificity of ARFIimaging for differentiation of thyroid nodules and to compare it to the well evaluated qualitative real-time elastography (RTE). Methods: ARFI-imaging involves the mechanical excitation of tissue using acoustic pulses to generate localized displacements resulting in shear-wave propagation which is tracked using correlation-based methods and recorded in m/s. Inclusion criteria were: nodules $5 mm, and cytological/histological assessment. All patients received conventional ultrasound, real-time elastography (RTE) and ARFI-imaging. Results: One-hundred-fifty-eight nodules in 138 patients were available for analysis. One-hundred-thirty-seven nodules were benign on cytology/histology, and twenty-one nodules were malignant. The median velocity of ARFI-imaging in the healthy thyroid tissue, as well as in benign and malignant thyroid nodules was 1.76 m/s, 1.90 m/s, and 2.69 m/s, respectively. While no significant difference in median velocity was found between healthy thyroid tissue and benign thyroid nodules, a significant difference was found between malignant thyroid nodules on the one hand and healthy thyroid tissue (p = 0.0019) or benign thyroid nodules (p = 0.0039) on the other hand. No significant difference of diagnostic accuracy for the diagnosis of malignant thyroid nodules was found between RTE and ARFI-imaging (0.74 vs. 0.69, p = 0.54). The combination of RTE with ARFI did not improve diagnostic accuracy. Conclusions: ARFI can be used as an additional tool in the diagnostic work up of thyroid nodules with high negative predictive value and comparable results to RTE.
- Hepatitis C viral evolution in genotype 1 treatment-naïve and treatment-experienced patients receiving telaprevir-based therapy in clinical trials (2012)
- Background: In patients with genotype 1 chronic hepatitis C infection, telaprevir (TVR) in combination with peginterferon and ribavirin (PR) significantly increased sustained virologic response (SVR) rates compared with PR alone. However, genotypic changes could be observed in TVR-treated patients who did not achieve an SVR. Methods: Population sequence analysis of the NS3•4A region was performed in patients who did not achieve SVR with TVR-based treatment. Results: Resistant variants were observed after treatment with a telaprevir-based regimen in 12% of treatment-naïve patients (ADVANCE; T12PR arm), 6% of prior relapsers, 24% of prior partial responders, and 51% of prior null responder patients (REALIZE, T12PR48 arms). NS3 protease variants V36M, R155K, and V36M+R155K emerged frequently in patients with genotype 1a and V36A, T54A, and A156S/T in patients with genotype 1b. Lower-level resistance to telaprevir was conferred by V36A/M, T54A/S, R155K/T, and A156S variants; and higher-level resistance to telaprevir was conferred by A156T and V36M+R155K variants. Virologic failure during telaprevir treatment was more common in patients with genotype 1a and in prior PR nonresponder patients and was associated with higher-level telaprevir-resistant variants. Relapse was usually associated with wild-type or lower-level resistant variants. After treatment, viral populations were wild-type with a median time of 10 months for genotype 1a and 3 weeks for genotype 1b patients. Conclusions: A consistent, subtype-dependent resistance profile was observed in patients who did not achieve an SVR with telaprevir-based treatment. The primary role of TVR is to inhibit wild-type virus and variants with lower-levels of resistance to telaprevir. The complementary role of PR is to clear any remaining telaprevir-resistant variants, especially higher-level telaprevir-resistant variants. Resistant variants are detectable in most patients who fail to achieve SVR, but their levels decline over time after treatment.
- C-terminal fluorescent labeling impairs functionality of DNA mismatch repair proteins (2012)
- The human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency.
- Long-term effects of treatment and response in patients with chronic hepatitis C on quality of life : an international, multicenter, randomized, controlled study (2012)
- Background: Hepatitis C decreases health related quality of life (HRQL) which is further diminished by antiviral therapy. HRQL improves after successful treatment. This trial explores the course of and factors associated with HRQL in patients given individualized or standard treatment based on early treatment response (Ditto-study). Methods: The Short Form (SF)-36 Health Survey was administered at baseline (n = 192) and 24 weeks after the end of therapy (n = 128). Results: At baseline HRQL was influenced by age, participating center, severity of liver disease and income. Exploring the course of HRQL (scores at follow up minus baseline), only the dimension general health increased. In this dimension patients with a relapse or sustained response differed from non-responders. Men and women differed in the dimension bodily pain. Treatment schedule did not influence the course of HRQL. Conclusions: Main determinants of HRQL were severity of liver disease, age, gender, participating center and response to treatment. Our results do not exclude a more profound negative impact of individualized treatment compared to standard, possibly caused by higher doses and extended treatment duration in the individualized group. Antiviral therapy might have a more intense and more prolonged negative impact on females.