Liang In Lin

The survival and colonization of tumor cells at new locations involve a variety of complex genetic, epigenetic, and microenvironmental factors. TRIM24 was originally named transcription intermediary factor 1-alpha (TIF1α), which was associated with cellular proliferation and was an oncogene in tumor development. Here we provide the first evidence of the expression profile and clinicopathological significance of TRIM24 in patients with hepatocellular carcinoma (HCC). Immunohistochemistry was employed to determine the expression level of TRIM24 in HCC tissues and noncancerous liver tissues. Elevated TRIM24 level was found in 61.4% HCC samples (51/83) correlating with AFP (P = 0.036), poor differentiation (P = 0.004), intrahepatic metastasis (P = 0.004), recurrence (P = 0.000006), and shorter tumor-free survival time (P = 0.002). Small interfering RNA induced down-regulation of TRIM24 promoted apoptosis in HCC cell line HepG2. Moreover, western blotting analysis revealed that knockdown of TRIM24 increased the protein levels of p53, Bax, and Caspase-8, and decreased Bcl-2, Survivin, Cyclin D1, and CDK4. Depletion of TRIM24 decreased Snail, Slug, β-catenin, and Vimentin, and increased E-cadherin expression, which suggested the involvement of TRIM24 in EMT. These results indicated that TRIM24 plays an important role in the pathogenesis of human HCC.

Molecular biomarkers to determine the effectiveness of targeted therapies in cancer treatment have been widely adopted in colorectal cancer (CRC), but those to predict chemotherapy sensitivity remain poorly defined. We tested our hypothesis that KRAS mutation may be a predictor of oxaliplatin sensitivity in CRC. KRAS was knocked-down in KRAS-mutant CRC cells (DLD-1(G13D) and SW480(G12V)) by small interfering RNAs (siRNA) and overexpressed in KRAS-wild-type CRC cells (COLO320DM) by KRAS-mutant vectors to generate paired CRC cells. These paired CRC cells were tested by oxaliplatin, irinotecan and 5FU to determine the change in drug sensitivity by MTT assay and flow cytometry. Reasons for sensitivity alteration were further determined by western blot and real-time quantitative reverse transcriptase polymerase chain reaction (qRT -PCR). In KRAS-wild-type CRC cells (COLO320DM), KRAS overexpression by mutant vectors caused excision repair cross-complementation group 1 (ERCC1) downregulation in protein and mRNA levels, and enhanced oxaliplatin sensitivity. In contrast, in KRAS-mutant CRC cells (DLD-1(G13D) and SW480(G12V)), KRAS knocked-down by KRAS-siRNA led to ERCC1 upregulation and increased oxaliplatin resistance. The sensitivity of irinotecan and 5FU had not changed in the paired CRC cells. To validate ERCC1 as a predictor of sensitivity for oxaliplatin, ERCC1 was knocked-down by siRNA in KRAS-wild-type CRC cells, which restored oxaliplatin sensitivity. In contrast, ERCC1 was overexpressed by ERCC1-expressing vectors in KRAS-mutant CRC cells, and caused oxaliplatin resistance. Overall, our findings suggest that KRAS mutation is a predictor of oxaliplatin sensitivity in colon cancer cells by the mechanism of ERCC1 downregulation.

Annexin A10 (ANXA10) is a member of the ANX family that is normally expressed in gastric mucosa. ANXA10 was recently observed to be upregulated in sessile serrated adenoma, a precursor to microsatellite-unstable colorectal cancer. We investigated the use of ANXA10 in diagnosing colorectal carcinoma. In an immunohistochemical analysis, the intensity and quantity of ANXA10, MUC5AC, MUC6 and CDX2 in 123 colorectal carcinomas were graded. We determined the molecular status of BRAF and KRAS mutations, as well as the microsatellite instability status and the CpG island methylator phenotype in all colorectal carcinomas, and subcategorized into four molecular subgroups according to the molecular derangements. Nuclear ANXA10 staining was present in 36 colorectal carcinomas, exhibiting a strong significant association with the BRAF mutation status (P<0.0001) and positive CpG island methylator phenotype (P<0.0001), and a borderline significant association with high levels of microsatellite instability (P=0.072). The ANXA10-positive colorectal carcinomas were frequently positive for MUC5AC and MUC6, and were associated with absent or reduced CDX2 expression (all P<0.0001). According to a classification and regression tree analysis, ANXA10 is a superior marker for the molecular subtyping of colorectal carcinomas and represents a specific marker for colorectal cancers of the serrated pathway. Our results indicated that ANXA10 expression is implicated in gastric programming in serrated-pathway-associated colorectal carcinoma. ANXA10-positive colorectal carcinoma is highly associated with the molecular features of the serrated neoplasia pathway.

Aims To understand the role of and differences in molecular alterations between endometrial and ovarian endometrioid adenocarcinomas. Methods and results We investigated the microsatellite status of 26 ovarian endometrioid adenocarcinomas ( OVEM s), 42 endometrial endometrioid adenocarcinomas ( EMCA s), and 19 concurrent (endometrial and ovarian) endometrioid adenocarcinomas. We evaluated the expression of the mismatch repair proteins, PTEN and ARID 1A, and mutations of PTEN , KRAS , CTNNB 1 , and PIK 3 CA . High levels of microsatellite instability ( MSI ‐H) were present in one of 26 OVEM s, 12 of 42 EMCA s, and four of 19 concurrent endometrioid adenocarcinomas. Only four of 19 concurrent endometrioid adenocarcinomas showed identical molecular alterations in their endometrial and ovarian components. Loss of ARID 1A or loss of PTEN expression, and MSI ‐H, were more common in EMCA s than OVEM s ( P = 0.044, P = 0.004, and P = 0.012, respectively). MSI ‐H in endometrial endometrioid adenocarcinomas was also related to loss of ARID 1A expression ( P &lt; 0.001). In the cohort of MSI ‐H endometrioid adenocarcinomas involving the endometrium ( n = 16), MSH 6‐deficient cases showed higher frequencies of CTNNB 1 and PIK 3 CA mutations ( P = 0.008 and P = 0.036, respectively), but lower frequencies of KRAS mutation ( P = 0.011), than PMS 2‐deficient cases. Conclusions The different frequencies of molecular genetic alterations between endometrial endometrioid adenocarcinomas and ovarian endometrioid adenocarcinomas imply that distinct processes may be involved in their tumorigenesis or tumour progression.

Background: Cigarette smoking is a major risk factor in the development and further progression of periodontal diseases. Heme oxygenase‐1 (HO‐1) is known as a stress‐inducible protein and functions as an antioxidant enzyme. There is limited information on the expression of HO‐1 in smoking‐associated periodontal disease. Objectives: The aim of the present study was to investigate the effects of nicotine on the expression of HO‐1 protein in cultured human gingival fibroblasts in vitro and further to compare HO‐1 expression in gingival tissues obtained from cigarette smokers and non‐smokers in vivo . Methods: Western blot assay was used to investigate the effects on human gingival fibroblasts exposed to nicotine. In addition, antioxidants catalase, superoxide dismutase (SOD), and N ‐acetyl‐ l ‐cysteine (NAC) were added to test how they modulated the effects on nicotine‐induced HO‐1 expression. Gingival biopsies taken from the flap surgery of 20 male patients with periodontal disease (10 cigarette smokers and 10 non‐smokers) were examined by immunohistochemistry. Results: The exposure of quiescent human gingival fibroblasts to 10 m m nicotine resulted in the induction of HO‐1 protein expression in a time‐dependent manner ( p &lt; 0.05). The addition of glutathione (GSH) precursor NAC inhibited the nicotine‐induced HO‐1 protein expression ( p &lt; 0.05). However, SOD and catalase did not decrease the nicotine‐induced HO‐1 protein expression ( p &gt; 0.05). The results from immunohistochemistry demonstrated that HO‐1 expression was significantly higher in cigarette smokers ( p &lt; 0.05). HO‐1 was noted in the basal layers of epithelium, inflammatory cells, and fibroblasts in specimens from cigarette smoking. Conclusions: Taken together, these results suggest that HO‐1 expression is significantly up‐regulated in gingival tissues from cigarette smokers, and nicotine may, among other constituents, be responsible for the enhanced HO‐1 expression in vivo . The regulation of HO‐1 expression induced by nicotine is critically dependent on the intracellular GSH concentration.

ABSTRACT Stefins have been reported to be associated with the progression and metastasis of various malignant tumors. However, the expressions of stefins in hepatocellular carcinoma (HCC) have not been well‐defined. In this study, the protein levels of stefin A and stefin B were assessed by immunohistochemical staining, and the mRNA levels were quantified by real‐time polymerase chain reaction in 85 primary HCC tissues, 85 surrounding non‐cancerous tissues, and 9 normal hepatic tissues. The immunohistochemical staining of cathepsin B and cathepsin D, and the ratio of cathepsins to stefins were assessed. The mRNA expressions of stefin A and stefin B in HCC tissues were significantly higher than surrounding noncancerous tissues and normal hepatic tissues, respectively. A significant positive relationship of stefin A and stefin B was found with node metastasis, tumor size, and Edmondson grade for HCC. Univariate and multivariate analyses revealed that Edmondson grade and stefin B expression were independent factors associated with the risk of lymph node metastasis in HCC. The ratios of cathepsin B to stefin A, cathepsin D to stefin A, cathepsin B to stefin B and cathepsin D to stefin B of the HCC group were significantly higher than that of the surrounding noncancerous group. A significant positive correlation between the ratio of cathepsins to stefins (cathepsin B/stefin A, cathepsin B/stefin B and cathepsin D/stefin B) and node metastasis was demonstrated. We concluded that high expressions of stefin A and stefin B may be an important factor contributing to the development and metastasis of HCC. Anat Rec, 299:428–438, 2016. © 2016 Wiley Periodicals, Inc.

The transcription factor Twist protein has been found to be correlated with metastasis in various carcinomas, including hepatocellular, breast and prostate carcinomas. However, the role of Twist in head and neck squamous cell carcinomas (HNSCC) remains unknown. Head and neck cancer tissue microarrays (TMAs) of tumors from 50 patients with HNSCC were examined. Immunohistochemical (IHC) stain analysis showed that, out of the 50 patients, twenty (40%) showed Twist-positive staining in the tumor cells, and Twist expression was positively associated with differentiation status (p=0.027), lymph node metastasis (p=0.032) and disease progression (p=0.029). Further analysis revealed that the expression of Twist was positively correlated with CXCR4 (Spearman, r=0.408, p=0.003) and CCR7 (r=0.417, p=0.003). FindPatterns analysis suggested that the transcription factor Twist, as a basic helix-loop-helix (bHLH) protein, might regulate CXCR4 and CCR7 expression in squamous cell carcinomas, which in turn might be associated with lymph node metastasis.

Resveratrol (3,5,4′-trihydroxy-trans-stilbene), a phytoalexin found in grapes and other plants, plays a protective role in human atherosclerosis and carcinogenesis. We examined the effects of resveratrol on the anaplastic large-cell lymphoma (ALCL) cell line SR-786. Resveratrol inhibited growth and induced cellular differentiation, as demonstrated by morphological changes and elevated expression of T cell differentiation markers CD2, CD3, and CD8. Resveratrol also triggered cellular apoptosis, as demonstrated by morphological observations, DNA fragmentation, and cell cycle analyses. Further, the surface expression of the death receptor Fas/CD95 was increased by resveratrol treatment. Our data suggest that resveratrol may have potential therapeutic value for ALCL.

To identify better regimens in currently available chemotherapy would be beneficial to KRAS mutant metastatic colorectal cancer (mCRC) patients because they have fewer treatment options than KRAS wild-type mCRC patients. Clinicopathologic features and overall survival (OS) of KRAS mutant and wild-type mCRC patients who had used oxaliplatin-based, irinotecan-based, bevacizumab-based, as well as cetuximab-based regimens were compared to those who had never-used oxaliplatin-based, irinotecan-based, bevacizumab-based, as well as cetuximab-based regimens respectively. Between 2007 and 2012, a total of 394 mCRC patients, in whom 169 KRAS mutant and 225 KRAS wild-type, were enrolled. In KRAS mutant patients who had used oxaliplatin-based regimens (N = 131), the OS was significantly longer than that in KRAS mutant patients who had never-used oxaliplatin-based regimens (N = 38). The OS was 28.8 months [95% confidence interval (CI): 23.2–34.4] in KRAS mutant patients who had used oxaliplatin-based regimens versus 17.8 months [95% CI: 6.5–29.1] in KRAS mutant patients who had never-used oxaliplatin-based regimens (P = 0.026). Notably, OS in KRAS wild-type mCRC patients who had used oxaliplatin-based regimens (N = 185) was not significantly longer than that in KRAS wild-type mCRC patients who had never-used oxaliplatin-based regimens (N = 40) (P = 0.25). Furthermore, the OS in KRAS mutant patients who had used either irinotecan-based, bevacizumab-based or cetuximab-based regimens was not significantly different than that in KRAS mutant patients who had never-used either irinotecan-based, bevacizumab-based or cetuximab-based regimens respectively. In multivariate analyses, patients who had used oxaliplatin-based regimens remains an independent prognostic factor for longer OS in KRAS mutant mCRC patients. In conclusion, oxaliplatin-based regimens are more beneficial in KRAS mutant than in KRAS wild-type mCRC patients.

This study investigated the clinical implications of SETDB 1 (also known as KMT 1E) in human colon adenocarcinoma. Expression levels of SETDB 1 proteins were analyzed by immunohistochemistry staining, and tissue microarrays were used to examine expression profiles in human patients. Our results revealed that SETDB 1 protein expression was significantly higher in tumor tissue than in normal tissue for the breast, colon, liver, and lung (p &lt; 0.05). Moreover, an analysis with SurvExpress software suggested that elevated expression of SETDB 1 mRNA was significantly associated with the overall survival of colon adenocarcinoma patients (p &lt; 0.05); and additional analysis involving 90 paired samples of colon adenocarcinoma tissue and normal tissue revealed that SETDB 1 protein expression was 82% higher in cancerous cells (p &lt; 0.001). High SETDB 1 expression was also found to be significantly correlated with histological grade (p = 0.005), TNM stage (p = 0.003), T‐class/primary tumor (p = 0.001), and N‐class/regional lymph nodes (p = 0.017); and Kaplan–Meier survival curves indicated that SETDB 1 protein expression was significantly associated with poor survival. Finally, univariate analysis demonstrated that SETDB 1 protein expression was related to TNM stage (p = 0.004) and SETDB 1 score (p = 0.001), whereas multivariate analysis showed that the influence of SETDB 1 on overall colon adenocarcinoma survival was independent from other risk factors. Taken together, our results suggest that the SETDB 1 protein could serve as a clinical prognostic indicator for colon adenocarcinoma.

The purpose of this study was to examine JMJD2B expression in human hepatocellular carcinoma (HCC) and elucidate relationships between various expression patterns and clinicopathological parameters of HCC patients. Immunohistochemical techniques were performed to detect JMJD2B expression in a tissue microarray from patients with breast, cerebrum, colon, esophagus, kidney, liver, lung, prostate, stomach, and uterus cancers. We performed immunohistochemical staining of a multiple tissue array to examine the expression profile of JMJD2B. Our results demonstrate that JMJD2B protein levels were upregulated in malignant human tumors, including breast, colon, liver, and lung. Immunohistochemistry staining examination of liver tumor tissue microarray revealed that the expression of JMJD2B is significant according to the histological grade and TNM stage of liver tumor. Moreover, JMJD2B was also correlated with Ki-67 expression in HCC samples. These results reveal that JMJD2B is dramatically upregulated in HCC, making it a potential diagnostic marker for the further development of HCC treatment therapies.

Epigenetic inactivation of tumor-suppressor genes, often in association with aberrant DNA methylation of CpG islands in the promoter region of these genes, is a key factor in tumorigenesis. CCAAT/enhancer binding protein alpha (CEBPA) methylation is a favorable prognostic biomarker for acute myeloid leukemia; however, rather than the complete methylation observed in inherited disorders, CEBPA methylation is heterogeneous. In this study, we established an algorithm called the “methylation index,” deduced from high-resolution melting profiles, which includes Tm shifting (ΔTm) and Tm width ratio (fold of width), to evaluate the heterogeneous methylation status. The methylation index was highly correlated with the exact methylation levels detected by using the MassARRAY method (R2 = 0.80; P < 0.001). Within-run reproducibility for the methylation index was 0.9% as the coefficient of variation, and between-run reproducibility was 2.6%. It was determined that with a cutoff methylation index of 1.412, the best measures of sensitivity and specificity could be obtained (97.14% and 95.89%, respectively) to discern low or high CEBPA methylation status. This novel algorithm for calculation of the methylation index from high-resolution melting profiles for CEBPA methylation is compatible with measurement of the methylation level as assayed using MassARRAY and could be a simple and efficient screening method for determination of CEBPA methylation status in acute myeloid leukemia. Epigenetic inactivation of tumor-suppressor genes, often in association with aberrant DNA methylation of CpG islands in the promoter region of these genes, is a key factor in tumorigenesis. CCAAT/enhancer binding protein alpha (CEBPA) methylation is a favorable prognostic biomarker for acute myeloid leukemia; however, rather than the complete methylation observed in inherited disorders, CEBPA methylation is heterogeneous. In this study, we established an algorithm called the “methylation index,” deduced from high-resolution melting profiles, which includes Tm shifting (ΔTm) and Tm width ratio (fold of width), to evaluate the heterogeneous methylation status. The methylation index was highly correlated with the exact methylation levels detected by using the MassARRAY method (R2 = 0.80; P < 0.001). Within-run reproducibility for the methylation index was 0.9% as the coefficient of variation, and between-run reproducibility was 2.6%. It was determined that with a cutoff methylation index of 1.412, the best measures of sensitivity and specificity could be obtained (97.14% and 95.89%, respectively) to discern low or high CEBPA methylation status. This novel algorithm for calculation of the methylation index from high-resolution melting profiles for CEBPA methylation is compatible with measurement of the methylation level as assayed using MassARRAY and could be a simple and efficient screening method for determination of CEBPA methylation status in acute myeloid leukemia. CpG islands, composed of multiple repeats of CpG dinucleotides and regulated by DNA methylation, are predominately located in the promoter and first exon of a given gene. Epigenetic inactivation of tumor-suppressor genes, often in association with aberrant DNA methylation of CpG islands in the promoter region, is a key factor in tumorigenesis.1Herman J.G. Baylin S.B. Gene silencing in cancer in association with promoter hypermethylation.N Engl J Med. 2003; 349: 2042-2054Crossref PubMed Scopus (2782) Google Scholar Acute myeloid leukemia (AML) is a hematologic malignant disease caused by uncontrolled proliferation and blocked differentiation of myeloid progenitors.2Tenen D.G. Disruption of differentiation in human cancer: AML shows the way.Nature Rev. 2003; 3: 89-101Google Scholar Hypermethylation of the CpG island associated with CCAAT/enhancer binding protein alpha (CEBPA), a gene that induces myeloid differentiation, results in silencing of CEBPA expression and has frequently been observed in a substantial percentage of patients with AML,3Hackanson B. Bennett K.L. Brena R.M. Jiang J. Claus R. Chen S.S. Blagitko-Dorfs N. Maharry K. Whitman S.P. Schmittgen T.D. Lubbert M. Marcucci G. Bloomfield C.D. Plass C. Epigenetic modification of CCAAT/enhancer binding protein alpha expression in acute myeloid leukemia.Cancer Res. 2008; 68: 3142-3151Crossref PubMed Scopus (132) Google Scholar lung cancer, and head and neck squamous cell carcinoma. CEBPA methylation is a favorable prognostic biomarker for AML.4Lin T.C. Hou H.A. Chou W.C. Ou D.L. Yu S.L. Tien H.F. Lin L.I. CEBPA methylation as a prognostic biomarker in patients with de novo acute myeloid leukemia.Leukemia. 2011; 25: 32-40Crossref PubMed Scopus (38) Google Scholar Unlike the case for inherited disorders, in which abnormal gene imprinting manifests in complete methylation of a specific DNA sequence region, most cancer cells exhibit heterogeneous methylation of CpG dinucleotides located in the promoter region of tumor-suppressor genes, which may skew the measure of CpG island methylation.5Mikeska T. Candiloro I.L. Dobrovic A. The implications of heterogeneous DNA methylation for the accurate quantification of methylation.Epigenomics. 2010; 2: 561-573Crossref PubMed Scopus (112) Google Scholar Currently, several methods are used to detect abnormal DNA methylation and include methylation-specific PCR,6Herman J.G. Graff J.R. Myohanen S. Nelkin B.D. Baylin S.B. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.Proc Natl Acad Sci USA. 1996; 93: 9821-9826Crossref PubMed Scopus (5229) Google Scholar combined bisulfite restriction analysis,7Sadri R. Hornsby P.J. Rapid analysis of DNA methylation using new restriction enzyme sites created by bisulfite modification.Nucleic Acids Res. 1996; 24: 5058-5059Crossref PubMed Scopus (72) Google Scholar bisulfite sequencing,8Frommer M. McDonald L.E. Millar D.S. Collis C.M. Watt F. Grigg G.W. Molloy P.L. Paul C.L. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.Proc Natl Acad Sci USA. 1992; 89: 1827-1831Crossref PubMed Scopus (2522) Google Scholar matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry with base-specific cleavage (MassARRAY),9Ehrich M. Nelson M.R. Stanssens P. Zabeau M. Liloglou T. Xinarianos G. Cantor C.R. Field J.K. van den Boom D. Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry.Proc Natl Acad Sci USA. 2005; 102: 15785-15790Crossref PubMed Scopus (692) Google Scholar pyrosequencing,10Colella S. Shen L. Baggerly K.A. Issa J.P. Krahe R. Sensitive and quantitative universal pyrosequencing methylation analysis of CpG sites.BioTech. 2003; 35: 146-150PubMed Google Scholar MethyLight assay,11Eads C.A. Danenberg K.D. Kawakami K. Saltz L.B. Blake C. Shibata D. Danenberg P.V. Laird P.W. MethyLight: a high-throughput assay to measure DNA methylation.Nucleic Acids Res. 2000; 28: e32Crossref PubMed Scopus (1210) Google Scholar and methylation-sensitive high-resolution melting (MS-HRM).12Wojdacz T.K. Dobrovic A. Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation.Nucleic Acids Res. 2007; 35: e41Crossref PubMed Scopus (426) Google Scholar, 13Wojdacz T.K. Dobrovic A. Hansen L.L. Methylation-sensitive high-resolution melting.Nat Protoc. 2008; 3: 1903-1908Crossref PubMed Scopus (221) Google Scholar Of these, bisulfite sequencing, as a criterion standard method, requires cloning for quantification and is expensive, labor-intensive, and time-consuming. Furthermore, this method does not offer sufficient sensitivity to recognize the heterogeneous methylation pattern of malignant cells. Whereas methylation-specific PCR and combined bisulfite restriction analysis are highly sensitive and cost-effective, these methods can be applied to only a few CpG sites in a single reaction at a time.14Laird P.W. The power and the promise of DNA methylation markers.Nature Rev. 2003; 3: 253-266Google Scholar MassARRAY and pyrosequencing can enable determination of methylation levels of several continuous CpG sites; however, their use is restricted to specific instrumentation. High-resolution melting (HRM) is a simple closed-tube PCR assay that precisely monitors changes in fluorescence on the basis of various sizes and nucleotide components of the amplicons during melting of the DNA duplex. This procedure can provide highly sensitive, high-throughput, cost-effective genotyping and detection of genetic mutations15Audrezet M.P. Dabricot A. Le Marechal C. Ferec C. Validation of high-resolution DNA melting analysis for mutation scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.J Mol Diagn. 2008; 10: 424-434Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar and single-nucleotide polymorphisms and DNA methylation,16Wittwer C.T. Reed G.H. Gundry C.N. Vandersteen J.G. Pryor R.J. High-resolution genotyping by amplicon melting analysis using LCGreen.Clin Chem. 2003; 49: 853-860Crossref PubMed Scopus (1019) Google Scholar, 17Liew M. Pryor R. Palais R. Meadows C. Erali M. Lyon E. Wittwer C. Genotyping of single-nucleotide polymorphisms by high-resolution melting of small amplicons.Clin Chem. 2004; 50: 1156-1164Crossref PubMed Scopus (513) Google Scholar, 18Balic M. Pichler M. Strutz J. Heitzer E. Ausch C. Samonigg H. Cote R.J. Dandachi N. High quality assessment of DNA methylation in archival tissues from colorectal cancer patients using quantitative high-resolution melting analysis.J Mol Diagn. 2009; 11: 102-108Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar in particular in imprinting disorders.19White H.E. Hall V.J. Cross N.C. Methylation-sensitive high-resolution melting-curve analysis of the SNRPN gene as a diagnostic screen for Prader-Willi and Angelman syndromes.Clin Chem. 2007; 53: 1960-1962Crossref PubMed Scopus (85) Google Scholar, 20Wojdacz T.K. Dobrovic A. Algar E.M. Rapid detection of methylation change at H19 in human imprinting disorders using methylation-sensitive high-resolution melting.Human Mutat. 2008; 29: 1255-1260Crossref PubMed Scopus (46) Google Scholar Some HRM assays based on methylation-dependent or methylation-independent PCR have been used to evaluate aberrant DNA hypermethylation in malignant tumors12Wojdacz T.K. Dobrovic A. Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation.Nucleic Acids Res. 2007; 35: e41Crossref PubMed Scopus (426) Google Scholar, 21Kristensen L.S. Mikeska T. Krypuy M. Dobrovic A. Sensitive melting analysis after real-time methylation specific PCR (SMART-MSP): high-throughput and probe-free quantitative DNA methylation detection.Nucleic Acids Res. 2008; 36: e42Crossref PubMed Scopus (147) Google Scholar; however, these methods require generation of a standard curve of various methylation levels and are unsuitable in cases with heterogeneous methylation patterns frequently observed in malignant lesions. In the present study, we established an algorithm deduced from HRM profiles to evaluate the status of heterogeneous methylation. By using this novel protocol, we studied CEBPA methylation status and compared this method with the well-known MassARRAY method. From 2002 to 2006, bone marrow samples with at least 90% leukemia cells were collected from 181 patients with AML at diagnosis and from 33 patients with AML at various stages after treatment in the Department of Internal Medicine, National Taiwan University Hospital. Patients with acute promyelocytic leukemia (AML type M3) received all-trans retinoic acid with or without concomitant induction chemotherapy, and patients with non–AML type M3 subtypes received conventional induction chemotherapy. On the basis of tri-lineage cell regeneration with < 5% blasts in the bone marrow, normalization of the peripheral blood cell count, and absence of leukemic infiltration in the tissue, complete remission was considered to have been achieved. Hematologic relapse was defined by recurrence of blasts in peripheral blood or by infiltration of bone marrow by more than 5% blasts. The study was approved by our institutional review board. HL-60 and K562 cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum. Bisulfite treatment of genomic DNA was performed using the Epitect Bisulfite Kit (Zymo Research Corp., Irvine, CA) according to the manufacturer's instructions. The bisulfite-converted DNA was used for subsequent MassARRAY analysis and HRM assay. Quantitative MassARRAY analysis of CEBPA methylation was performed according to the manufacturer's instructions. The bisulfite-treated DNA was prepared for PCR amplification using primer pairs according to the Standard EpiPanel (CEBPA_04; Sequenom, Inc., San Diego, CA). The PCR mixture contained 10 ng bisulfite-treated DNA, 200 μmol/L deoxyribonucleotide triphosphate, 0.2 U HotStar Taq DNA polymerase (Qiagen, Inc., Valencia, CA), 0.2 μmol/L forward (5′-AGGAAGAGAGGTTTAGATGGAGTTTTGGGAGTTTT-3′) and reverse (5′-CAGTAATACGACTCACTATAGGGAGAAGGCTCCTTACCAAACCTAAAACCACT-3′) primers, and 1X reaction buffer with the enzyme in a total volume of 5 μL. The PCR reaction mixture was preheated for 15 minutes at 94°C, followed by 45 cycles at 94°C for 20 seconds, 62°C for 30 seconds, and 72°C for 1 minute, followed by one final extension cycle at 72°C for 1 minute. The redundant deoxyribonucleotide triphosphates were digested by adding 1.7 μL H2O and 0.3 U shrimp alkaline phosphatase (Sequenom, Inc.). The reaction was performed at 37°C for 20 minutes, and shrimp alkaline phosphatase was inactivated by heating at 85°C for 5 minutes. Subsequently, the in vitro transcription mixture and RNase A digestion were performed at 37°C for 3 hours by using a MassCLEAVE kit (Sequenom, Inc.) including 0.22 μL T-cleavage mix, 3.14 mmol/L dithiothreitol, 20 U T7 RNA and DNA polymerase, 0.09 mg/mL RNase A, 1X T7 polymerase buffer, and 2 μL product of the PCR/shrimp alkaline phosphatase reaction as the template in a total volume of 7 μL. The reaction mixture was further diluted by adding H2O to a final volume of 27 μL, purified with CLEAN resin, and robotically dispensed onto silicon chips preloaded with matrix (SpectroCHIP; Sequenom, Inc.). The matrix-associated laser desorption/ionization–time-of-flight mass spectrometry spectra were collected and analyzed using EpiTyper analyzer software (Sequenom, Inc.). For quantification analysis, methylation levels were calculated as the mean methylation values of three CpG units (units 2, 3, and 4) covering seven CpG sites (see Supplemental Figure S1 at http://jmd.amjpathol.org). For qualification analysis, two-way hierarchic clustering based on the methylation values of units 2, 3, and 4 was presented in a double dendrogram by using Gene Expression Statistical System software (NCSS, Kaysville, UT). PCR amplification and HRM were performed using the LightCycler 480 instrument (Roche Applied Science, Indianapolis, IN). The primer sequences for this PCR amplification are the same as those used for MassARRAY. PCR was performed in 20 μL reaction mixture containing 1X LightCycler 480 High-Resolution Melting Master mix (Roche Applied Science), 200 nmol/L of each primer, 2.5 mmol/L MgCl2, and 10 ng bisulfite-treated DNA. The PCR condition was as follows: preheating at 95°C for 10 minutes, followed by 50 cycles at 95°C for 10 seconds, 62°C for 15 seconds, and 72°C for 10 seconds. The subsequent melting protocol was as follows: 95°C for 1 minute, 40°C for 1 minute, and temperature ramping from 65°C to 95°C accompanied by continuous data acquisition 25 times at 1°C. All reactions were performed in triplicate, and bisulfite-treated marrow mononuclear cellular DNA from healthy individuals was used as control. The HRM data were analyzed using the Tm Calling software module in LightCycler 480 software (Roche Applied Science), which could automatically indicate the values of melting temperature (Tm) and width. Tm is defined as the point at which half of the amplicons are double-stranded and single-stranded, and width is the width at one-half height of the melting peak (Figure 1A), representing the distribution of dissociation temperature. The significance of association between the Tm value or methylation index from the HRM assay and the methylation level from the MassARRAY assay was analyzed using simple linear regression analysis. To differentiate the high methylation group from the low methylation group, the receiver operating characteristic (ROC) curve and the area under the ROC curve were calculated for the best sensitivity and specificity by using commercially available software (MedCalc Software bvba, Mariakerke, Belgium). In a previous study,4Lin T.C. Hou H.A. Chou W.C. Ou D.L. Yu S.L. Tien H.F. Lin L.I. CEBPA methylation as a prognostic biomarker in patients with de novo acute myeloid leukemia.Leukemia. 2011; 25: 32-40Crossref PubMed Scopus (38) Google Scholar we demonstrated that K562 cells were fully methylated in the distal promoter region of CEBPA; however, HL-60 cells exhibited no methylation. DNA samples with various methylation levels (0%, 1%, 5%, 10%, 50%, and 100%) were prepared from serial dilution of K562 DNA with HL-60 genomic DNA. After bisulfite treatment, the HRM assay demonstrated both the methylation peak and the unmethylation peak. The melting profile indicated that K562 with CEBPA methylation demonstrated a higher Tm, whereas HL-60 without CEBPA methylation exhibited a lower Tm (Figure 1B). It was further validated that the ratio of the methylation peak area over the unmethylation peak area was positively correlated with the dilution percentage (R2 = 0.9722; Figure 1C); however, patients with AML with heterogeneous methylation exhibited only one broad methylation peak (Figure 1D), the methylation level of which could not be calculated by the areas of methylated and unmethylated peaks stated above. After bisulfite treatment, unmethylated cytosine is converted to uracil and is subsequently amplified as thymine via PCR reaction, whereas methylated cytosine remains unchanged and is amplified as cytosine. Therefore, the amplicon derived from the methylated DNA has high G-C (guanine-cytosine) content, suggestive of a higher Tm, whereas the unmethylated fragment with low G-C content exhibits a lower Tm. To determine whether the Tm value of the amplified fragments could be used to indicate heterogeneous methylation patterns, the methylation status of CEBPA promoter in 181 patients with AML was evaluated by using MassARRAY and Tm assay from HRM profiles. In these 181 studied patients with AML, the exact methylation levels detected by using the MassARRAY method ranged from 0.0978 to 0.9711 (median, 0.2397). After two-way hierarchic clustering using a complete linkage algorithm, these patients could be classified into a high CEBPA methylation group (n = 35) and a low CEBPA methylation group (n = 146), which corresponded to a cutoff methylation level of 0.486 (Figure 2A). Afterward, the Tm values determined automatically from the HRM profile ranged from 77.76°C to 81.74°C (median, 78.18°C). The ROC curve was determined to differentiate the high methylation group from the low methylation group. A Tm cutoff value of 78.67oC, corresponding to an area under the ROC curve value of 0.887 (95% confidence-interval, 0.831 to 0.929; P < 0.001) accompanied with the best sensitivity and specificity of 71.4% and 99.3%, respectively (see Supplemental Figure S2A at http://jmd.amjpathol.org). Quantification analysis revealed that the Tm values demonstrated moderate correlation with the exact methylation levels (R2 = 0.68; P < 0.001; see Supplemental Figure S2B at http://jmd.amjpathol.org). In addition to the Tm shifting for heterogeneous methylation, the wide distribution of dissociation temperature might be the reason why only the Tm value could not achieve good sensitivity in assessing methylation status (Figure 1D). We further assessed the correlation between the exact methylation levels from the MassARRAY method and the HRM assays, including the Tm values and the distribution of dissociation temperature, both of which are automatically called by the LightCycler 480 software as Tm and width, respectively. The pooled bisulfite-converted DNA from four healthy volunteer donors was used as reference control in the HRM assay. We deduced an algorithm, named “methylation index,” that collectively considered both Tm shifting and Tm width ratio for assessment of the heterogeneous CEBPA methylation pattern in AML. The algorithm is (Widthpatient/Widthreference) + (Tmpatient – Tmreference), where Widthreference and Tmreference are, respectively, the mean values of width and Tm from triplicate reference controls in each run. The threshold of methylation index was determined by using ROC analysis according to the dichotomization using the MassARRAY method. With the cutoff methylation index of 1.412, corresponding to an area under the ROC curve of 0.983 (95% confidence interval, 0.952 to 0.996; P < 0.001), the best sensitivity and specificity were 97.14% and 95.89%, respectively (Figure 2B), which demonstrates that the methylation index had better sensitivity than the Tm value alone (97.14% versus 71.4%). In addition, simple regression analysis suggested that the deduced methylation index was highly correlated with the exact methylation levels (R2 = 0.80; P < 0.001; Figure 2C), demonstrating the potential of HRM profiles for quantitative evaluation in the case of heterogeneous methylation. The within-run reproducibility for Tm and width was determined in both the reference control and a sample with high CEBPA methylation by making nine replicate measurements. The coefficient of variation for Tm and width of the reference control was 0.04% and 4.0%, respectively, and for the sample with high CEBPA methylation was 0.04% and 2.56%, respectively. The between-run reproducibility was also determined for the same samples by performing five separate reactions, and with coefficients of variation for Tm and width of 0.1% and 5.0%, respectively, for the reference control, and 0.2% and 5.1%, respectively, for the sample with high CEBPA methylation. Furthermore, within-run and between-run reproducibility was reduced to 0.9% and 2.6%, respectively, when calculating the methylation index in the sample with high CEBPA methylation. This novel algorithm for calculating the methylation index based on HRM profiles for CEBPA methylation is compatible with the methylation level from the MassARRAY method and could be a simple and efficient screening technique for determination of CEBPA methylation status in AML. We subsequently determined CEBPA methylation status in patients with AML at various disease stages by using this novel HRM assay. Of the 181 patients, 77 samples from 33 patients were serially studied for CEBPA methylation (Figure 3). In patients with CEBPA hypermethylation, the CEBPA methylation index decreased significantly at complete remission [n = 15; range, 0.726 to 2.000 (median, 0.953)] compared with that at initial diagnosis [range, 1.460 to 4.403 (median, 2.674)]. The methylation index increased again at relapse in the six patients who demonstrated a high methylation index at diagnosis. In two of these six patients, the CEBPA methylation index decreased again at second complete remission. These results suggest that the HRM assay could be used to monitor CEBPA methylation status; however, CEBPA methylation level as a biomarker for disease status must be further evaluated in large studies. We previously reported that CEBPA methylation was present in 14.5% of patients with AML and acted as a prognosis factor for better outcome.4Lin T.C. Hou H.A. Chou W.C. Ou D.L. Yu S.L. Tien H.F. Lin L.I. CEBPA methylation as a prognostic biomarker in patients with de novo acute myeloid leukemia.Leukemia. 2011; 25: 32-40Crossref PubMed Scopus (38) Google Scholar In the present study, we established a novel algorithm to quantitatively evaluate CEBPA methylation. This algorithm was deduced from the information of Tm shifting (Tmpatient – Tmreference) and the Tm width ratio (fold of width, Widthpatient/Widthreference). The distribution of dissociation temperature is similar to a Gaussian distribution (Figure 1A). For a Gaussian curve, the width is equal to 2.35σ, where σ is the standard deviation in this population. Consequently, the Widthsample/Widthreference in our deduced algorithm for evaluating methylation status could reflect the distribution of dissociation temperature in a patient sample compared with a reference control. The high-sensitivity HRM instruments and high-resolution fluorescence dyes intercalating double-stranded DNA with saturated concentrations could give Tm and width higher resolution to evaluate fine methylation changes. Most patients demonstrated a single unique melting peak accompanied by different Tm and peak width. Only four patients exhibited relatively homogeneous methylation and represented a biphasic melting pattern showing two distinct peaks with low and high Tm, equivalent to the unmethylated and methylated amplicons, respectively. For these conditions, the lower Tm and the sum of both widths were used to calculate the methylation index. However, more patients will be necessary to confirm this alternative calculation. Several points should be noted when using the HRM assay to evaluate methylation status. First, the primer design should meet the principle of methylation-independent PCR, excluding CpG sites and including a few cytosines from non-CpG sites. Although we could not confirm the bisulfite conversion efficiency by using the HRM assay, only bisulfite-treated DNA converted completely could be amplified by the designed primers covering the non-CpG cytosines. Thus, amplification of incomplete bisulfite conversion would be excluded and did not affect the methylation index calculation. Second, single-nucleotide polymorphisms in the analyzed region should be avoided because of their interference with the melting profiles. Third, one melting domain of the amplicons would be suggested because more than one melting domain would result in complication of the melting profile at analysis. Poland's algorithm (http://www.biophys.uni-duesseldorf.de/local/POLAND/; accessed March 25, 2011) could be used to predict melting domains for amplicons and to determine the suitability for designed primers. In the present study, pooled bone marrow genomic DNA from four healthy volunteers was used as the reference control. For future clinical usage, an in vitro diagnostic kit including reference controls (prepared from pooled control DNA) and low and high methylation controls from patient DNA with a known range of methylation index could be expected. The methylation levels and the methylation indexes were highly correlated with each other but were not perfectly matched. One reason is that the methylation index determines the mean methylation information for all of the 12 CpG sites in this region, whereas the information of methylation level includes only 7 of these sites because of the limitation of cleaved fragment mass (see Supplemental Figure S1 at http://jmd.amjpathol.org). In addition, the different detection limit and detection sensitivity might account for the discrepancy between the methylation index using the HRM assay and the methylation level using MassARRAY. HRM is an easy, fast, and inexpensive method for methylation detection in the clinical setting. MS-HRM,12Wojdacz T.K. Dobrovic A. Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation.Nucleic Acids Res. 2007; 35: e41Crossref PubMed Scopus (426) Google Scholar and digital MS-HRM22Candiloro I.L. Mikeska T. Hokland P. Dobrovic A. Rapid analysis of heterogeneously methylated DNA using digital methylation-sensitive high resolution melting: application to the CDKN2B (p15) gene.Epigenet Chromatin. 2008; 1: 7Crossref PubMed Google Scholar are the HRM assays developed for evaluation of heterogeneous DNA methylation in cancer. However, MS-HRM requires construction of a standard curve in each assay to estimate the approximate methylation status, and digital MS-HRM demands multiple PCR reactions for each sample that could obtain a methylation value for each allele for total methylation evaluation. In summary, the HRM assay coupled with methylation index calculation is a highly sensitive, specific, and reproducible method and could be used in the future as a modern method for evaluating methylation status, in particular in samples with heterogeneous methylation. We thank the medical technologists in the Division of Molecular Diagnostics at National Taiwan University Hospital (NTUH) for consultation on the high-resolution melting application, and the faculty and staff of the Division of Hematology at NTUH for referring their patients with AML for participation in this study. Download .pdf (.03 MB) Help with pdf files Supplemental Figure S1CEBPA distal promoter analyzed using the MassARRAY and HRM methods. Vertical bars indicate individual CpG sites; arrows represent CpG sites analyzed using MassARRAY and HRM. Seven CpG sites in units 2, 3, and 4 were examined using MassARRAY, and 12 CpG sites in units 1, 2, 3, 4 and 5 were examined using the HRM method. Nucleotides are numbered according to GenBank Accession No. AC008738. Download .pdf (.05 MB) Help with pdf files Supplemental Figure S2Tm cutoff value, ROC curve, sensitivity, and selectivity. A: ROC curve of the Tm value in patients with high and low methylation. Open circle indicates that the cutoff Tm value was 78.67, with the best sensitivity and specificity, 71.4% and 99.3%, respectively. The positive and negative case number was determined using MassARRAY, and Tm is shown in the lower table. B: Simple linear regression analysis between the Tm from HRM and the methylation level from MassARRAY in 181 patients with AML. Dotted lines, cutoff (C-O) methylation level from MassARRAY and the cutoff Tm.

Occurrence of early-onset colorectal cancer (EOCRC) under the age of 30 is very rare and the molecular characteristics are poorly understood. A low BRAF mutation rate has been noted in several studies of EOCRC from Western countries.To determine the clinicopathological and molecular features of EOCRCs in Taiwan.KRAS/BRAF gene mutation, mismatch repair protein immunohistochemistry, microsatellite instability and CpG island methylation phenotype analyses were examined to determine the molecular characteristics of EOCRC.Sixty-six patients with EOCRC at our hospital between 2000 and 2012 were studied. BRAF mutation was detected in 11 of the 59 tumours analysed (19%) and the rate was significantly higher than the overall BRAF mutation rate of colorectal cancer in patients older than 30 years (p<0.001). Clinically, 9 of 11 patients with BRAF-mutated tumours presented with advanced-stage diseases and they presented significantly more frequently with stage IV disease than those with BRAF wild-type tumours (p=0.042). Histologically, BRAF mutation was associated with a poorly differentiated histology, a serrated precursor polyp and focal signet ring cell differentiation (p=0.042, 0.008 and 0.008, respectively). None of the BRAF-mutated tumours was mismatch repair protein-deficient and/or microsatellite instability-high. Overall survival of patients with BRAF-mutated tumours was significantly worse than that of patients with BRAF wild-type tumours, despite adjustment for the disease stages and tumour differentiation.BRAF mutation was frequent in EOCRCs in Taiwan and was associated with distinct clinicopathological and molecular features.

Lung cancer is the most common cancer-related death worldwide. In 2022, the number of daily deaths of lung cancer was estimated to reach around 350 in the United States. Lung adenocarcinoma is the main subtype of lung cancer and patients with malignant pleural effusion (MPE) suffer from poor prognosis. Microbiota and its metabolites are associated with cancer progression. However, the effect of pleural microbiota on pleural metabolic profile of MPE in lung adenocarcinoma patients remains largely unknown.Pleural effusion samples collected from lung adenocarcinoma patients with MPE (n = 14) and tuberculosis pleurisy patients with benign pleural effusion (BPE group, n = 10) were subjected to microbiome (16S rRNA gene sequencing) and metabolome (liquid chromatography tandem mass spectrometry [LC-MS/MS]) analyses. The datasets were analyzed individually and integrated for combined analysis using various bioinformatic approaches.The metabolic profile of MPE in lung adenocarcinoma patients were clearly distinguished from BPE with 121 differential metabolites across six significantly enriched pathways identified. Glycerophospholipids, fatty and carboxylic acids, and derivatives were the most common differential metabolites. Sequencing of microbial data revealed nine significantly enriched genera (i.e., Staphylococcus, Streptococcus, Lactobacillus) and 26 enriched ASVs (i.e., species Lactobacillus_delbrueckii) in MPE. Integrated analysis correlated MPE-associated microbes with metabolites, such as phosphatidylcholine and metabolites involved in the citrate cycle pathway.Our results provide substantial evidence of a novel interplay between the pleural microbiota and metabolome, which was drastically perturbed in MPE in lung adenocarcinoma patients. Microbe-associated metabolites can be used for further therapeutic explorations.

The identification of better regimens in currently available chemotherapeutic agents is crucial for treating patients with KRAS mutant metastatic colorectal cancer (mCRC). Records of mCRC patients who received first-line oxaliplatin- based or irinotecan-based regimens were reviewed retrospectively. Clinicopathologic features and treatment outcome of patients with first-line progression-free survival (PFS) and overall survival (OS) in association with KRAS mutation status were analyzed using the Cox proportional hazard model. Between 2007 and 2010, a total of 118 mCRC patients were enrolled. Among them, 67 were males and 51 were females. In patients who received first-line oxaliplatin-based regimens, the PFS was significantly longer in KRAS mutant patients (N = 32) than that in KRAS wild-type patients (N = 51). The median PFS was 8.5 months in KRAS mutant versus 5.8 months in KRAS wild-type patients (P = .008). In contrast, in patients who received first-line irinotecan-based regimens, the PFS was shorter in KRAS mutant patients (N = 15) than that in KRAS wild-type patients (N = 20). Median PFS was 3.9 months in KRAS mutant versus 6.0 months in KRAS wild-type patients (P = .23). Median OS between KRAS mutant and wild-type patients was not significantly different in both oxaliplatin-based and irinotecan-based regimens. In multivariate analyses, KRAS mutation remains an independent predictive factor for longer PFS in first-line oxaliplatin-based regimens. In conclusion, oxaliplatin-based chemotherapy in KRAS mutant mCRC might result in longer PFS than in KRAS wild-type mCRC.

OBJECTIVE To detect the expression of Nusap1 of surgical margins in hepatocellular carcinoma (HCC) and investigate its association with early tumor recurrence. METHODS The expression of Nusap1 in the surgical margins of HCC, which were histopathologically negative for tumor cells, was examined using immunohistochemistry in 61 HCC cases. RESULTS Fifteen of 21 (71.4%) cases with immunohistochemical positivity for Nusap1 expression in the surgical margins had early recurrence of HCC, a rate significantly higher than that in patients with negative Nusap1 expression (12/40, 30%) (P<0.05). CONCLUSIONS Nusap1 expression in the surgical margins of HCC is closely correlated to early postoperative recurrence and can serve as an indicator for predicting early recurrence of HCC.

To explore the expressions of indoleamine 2, 3-dioxygenase (IDO) in hepatocellular carcinoma and analyze its relationship with clinicopathological parameters.Quantitative real-time polymerase chain reaction (PCR), fluorogenic quantitative PCR, immunohistochemical and immunofluorescence were used to detect the expression of indoleamine 2, 3-dioxygenase in hepatocellular carcinoma.The IDO mRNA expression in cancerous tissues increased markedly than that in the corresponding non-cancerous tissues (2(-ΔΔCT) = 1.71, P = 0.001) . The immunohistochemical and immunofluorescence results showed that IDO protein was expressed in cytoplasm of hepatocellular carcinoma and tumor-surrounding tissues. But there was no expression in normal liver tissues from benign hepatic lesions and corresponding non-cancerous tissues. An over-expression of IDO protein was detected in 43 patients (48.3%) , a low expression in 25 (28.1%) and no expression in 21 (23.6%). Relationship between IDO expression and clinicopathological parameters: an over-expression of IDO in HCC was associated with recurrence, survival time, metastasis and TNM stage (P < 0.05), but not associated with patient's cirrhosis, AFP level, histological differentiation type, Barcelona clinic liver cancer stage, gender, age, HbsAg positivity, number of tumors and tumor size (P > 0.05).An over-expression of IDO in HCC patients may affect patient prognosis.

This study was purpose to investigate the B7-H3 expression in multiple myeloma cell lines and CD138 cells of patients with multiple myeloma, and explore its clinical significance. Three myeloma cell lines (RPMI8226, U266 and H929) were used. Forty-five patients with multiple myeloma were enrolled in the study. The expression of B7-H3 was detected by flow cytometry and RT-PCR. The relationship between B7-H3 and clinical prognostic factor was analyzed. The results showed that (1)In myeloma cell lines, high expression of B7-H3 was seen in RPMI8226 (92.30 ± 1.1)% and U266 (79.03 ± 1.2)% but not in H929 cell line (4.26 ± 0.2)%. (2) Exogenous IL-6 had no effect on upregulation of B7-H3 in myeloma cell lines. (3) In multiple myeloma patients, the proportions of B7-H3 positive cells in newly diagnosed, remission and relapsed patients were (48.58 ± 33.593)%, (22.16 ± 18.853)%, and (57.65 ± 28.296)%, respectively. The difference between the newly diagnosed and remission patients, and remission and relapsed patients was significant (P = 0.023, P = 0.004). (4)High B7-H3 expression was correlated with high numbers of bone destruction and high levels of serum calcium (P = 0.027, P = 0.046, respectively). It is concluded that the relation of B7-H3 molecule expression with prognosis of multiple myeloma may be negative, but with degree of bone destruction is positive, thus the high expression of B7-H3 may correlated with disease progression and bone destruction of patients with multiple myeloma.

Anti-epidermal growth factor receptor (EGFR) monoclonal antibodies benefit patients with wild-type KRAS exon 2 metastatic colorectal cancer (mCRC). However, their effect in KRAS-mutant mCRC remains unclear.This was a retrospective study enrolling 163 patients with unresectable KRAS-mutant mCRC diagnosed at the National Taiwan University Hospital between 2007 and 2011.The median overall survival (mOS) was 29.5 months in patients who had never used cetuximab and 19.0 months in those who had (p=0.040). The mOS was 32.0 months in patients with mutant KRAS codon 12 who had never used cetuximab and 17.5 months in those who had (p=0.017). In patients with mutant KRAS codon 13, the mOS was not significantly different. Univariate and multivariate Cox proportional hazards analysis revealed that absence of cetuximab treatment was an independent prognostic factor for longer mOS in patients with unresectable KRAS-mutant mCRC.Cetuximab usage might be detrimental to patients with mCRC with mutant KRAS codon 12.

To study the expression of special AT-rich sequence binding protein 1 (SATB1) in hepatocellular carcinoma (HCC) cell lines with different invasive capacities.SATB1 expression was detected using real-time fluorescence quantitative PCR, RT-PCR, Western blotting and immunofluorescence in immortalized liver cell line HL-7702, noninvasive HCC cell lines HepG2 and SMMC-7721, MHCC97L cells with low invasiveness, and highly invasive cell lines MHCC97H and HCCLM3.In comparison with HL-7702 cells, all the 5 HCC cell lines showed overexpression of SATB1 mRNA, which was the highest in the highly invasive HCCLM3 and MHCC97H cells, followed by MHCC97L cell line, and then by SMMC-7721 and HepG2 cell lines (P<0.001). The relative expression quantity of SATB1 protein in HepG2, SMMC-7721, MHCC97L, MHCC97H, and HCCLM3 cell lines was 0.271±0.002, 0.351±0.023, 0.621±0.026, 0.878±0.026, and 1.236±0.006, respectively. SATB1 expression level in HCCLM3 cell line was 4.6-fold higher than that in HepG2 cell line (P<0.001). SATB1 was found to localize in the cytoplasm and cell nuclei of the 5 HCC cell lines, and the highly invasive HCCLM3 and MHCC97H cell lines showed a strong positive staining for SATB1 in immunofluorescence assay.SATB1 expression levels differ distinctly between the HCC cell lines with different invasive capacities and are possibly associated with the metastatic potentials of the cells.

To investigate the expression of miR-126/miR-126* in hepatocellular carcinoma (HCC) tissues and its clinical significance.The expressions of miR-126/miR-126* in the tumor tissues and non-cancer tissues from 74 HCC cases were detected using real-time quantitative PCR. The correlation between miR-126/miR-126* expression and the clinicopathological features of the patients was analyzed.Compared with the non-cancer tissues, HCC tissues showed significantly lowered expression levels of miR-126/miR-126*. A positive correlation was found between the expressions of miR-126 and miR-126* in the tumor tissues. Lower expressions of miR-126/miR-126* were significantly correlated with tumor recurrence and poor survival of the HCC patients.The down-regulation of miR-126/miR-126* may play an important role in HCC metastasis and contributes to poor survival of HCC patients.

Tartrate-resistant acid phosphatase 5a (TRACP5a) is mainly secreted by activated macrophages in chronic inflammation. Serum TRACP5a is associated with symptom distress in lung cancer patients during chemotherapy. Therefore, this study aimed to investigate whether chemotherapy drugs modulate TRACP5a as an inducible marker for symptom distress in lung cancer patients during chemotherapy. In clinical analysis, lung cancer participants completely received the six-cycle chemotherapy process (n = 42). Clinical determinations for TRACP5a, C-reactive protein (CRP), interleukin-6 (IL-6), white blood cells, monocytes, and hemoglobin were analyzed at six time points: BL, C1d8, C2d1, C4d1, C4d8, and Ed28. Meanwhile, five questionnaires for fatigue, sleep disturbance, pain, depression, and confusion were finished before drug treatment. For monocyte-to-macrophage differentiation, THP-1 cells were treated with phorbol 12-myristate 13-acetate (PMA). TRACP5a secretion in THP-1 cells was determined at the following days up to 6 days after 1-day incubation of chemotherapy drugs by dot blotting. Clinical analysis revealed that TRACP5a significantly increased at C1d8 and C4d8, but dropped at C2d1 and Ed28. CRP and IL-6 displayed a broad-range variation, resulting in no significant difference among the assessment time points. In contrast, monocytes decreased at C1d8 and C4d8, but rose again at C2d1 and Ed28. In symptom distress, the changes only in fatigue and sleep disturbance were positively associated with the trend in TRACP5a. In PMA-treated THP-1 cells, TRACP5a significantly increased after stimulation with gemcitabine and paclitaxel. Taken together, induction of TRACP5a by chemotherapy drugs might be generated from monocyte-differentiated macrophages, further causing clinical symptom distress in lung cancer patients.

Objective To investigate the role of miRNA expression in the acute rejection of liver transplantation in rats.Methods The rat orthotopic liver transplantation (ROLT) was performed using the two-cuff technique described by Kamada with modification.Rats were randomly divided into two groups,a rejection group (n=12) and a control group (n=12).MicroRNA microarray technology was used to screen the microRNA expression profile in peripheral blood on the 7th postoperative day,and the bioinformatics microRNA screening method detected that miR-206 had the biggest difference in expression level.Peripheral blood samples were gathered before,3 days,5 days,and 7 days after ROLT to analyze the expression level of miR 206 quantitatively with qRT PCR technology.Dynamic contrast analysis was made between the quantitative results of miR-206 and the results of pathological examination and liver function tests.Results There was no significant difference in miR-206 expression level between the rejection group and control group before the operation (t=0.105,P=0.921).MiR-206 expression was significantly higher in the rejection group than in the control group 3 days after ROLT (t=8.875,P=0.001).The expression level of miR 206 in the rejection group also increased gradually over time (F=32.154,P＜0.01).However,pathological changes and liver function index did not show obvious differences until the 5th and 7th day postoperatively.The expression of miR-206 in peripheral blood and graft was strongly and positively correlated with the rejection active index (RAI) of acute liver rejection pathology (0.812 and 0.881 respectively,P＜0.01).In the rejection group,the miR-206 level in peripheral blood and graft were positively associated with each other.Conclusions Detection of miR-206 in peripheral blood was significantly better than pathological examination and liver function test in the diagnosis of acute rejection of rat liver transplantation.Moreover,miR-206 was an early sensitive and specific marker for making this diagnosis. Key words: MicroRNA;  Liver transplantation;  Rat;  Acute rejection

To explore progesterone-induced blocking factor (PIBF) expression in the placenta and blood of patients with severe preeclampsia and its relationship with immune tolerance imbalance.Forty-seven patients admitted between January and December, 2012 were enrolled in this study, including 25 patients with early-onset severe preeclampsia (EOPE) and 22 with late-onset severe preeclampsia (LOPE), with 25 women with normal pregnancy serving as control group. The antenatal blood and postpartum placenta were collected for immunohistochemical staining to detect PIBF expression in the placenta and for testing serum PIBF level using ELISA. Flow cytometry was used to detect the percentage of circulating Th1 and Th2 cells and the Th1/Th2 ratio was calculated.PIBF was expressed in decidual cells, syncytiotrophoblasts and partial cytotrophablasts. The serum PIBF levels were 213.58 ± 44.93 ng/ml in EOPE group, 243.00∓61.19 ng/ml in LOPE group and 273.91 ± 48.57 ng/ml in control group. There were significant differences in serum PIBF, blood Th1/Th2 and placenta PIBF-IOD among the 3 groups (P<0.05). EOPE group had significantly lower serum PIBF, lower llacental PIBF quantity (PIBF-IOD) and higher blood Th1/Th2 than the control group (P<0.05). Serum PIBF in women with severe preeclampsia was positively correlated with placenta PIBF-IOD and negatively with blood Th1/Th2 ratio (P<0.05), but a negative correlation between serum PIBF and 24-hour urinary protein was found only in EOPE group (P<0.05).The immune tolerance imbalance mediated by PIBF may participate in the pathogenesis of severe preeclampsia. PIBF, the immune suppressor secreted by lymphocytes of pregnancy women, is also a protective factor against severe preeclampsia, which is expected to be a new target in therapy.

Objective To explore the expression of NVM-1(novel metastasis-promoting gene 1) in hepatocellular carcinoma (HCC) tissue and examine its clinical significance. Methods The expression of NVM-1 in 92 cases of HCC tumor tissues and non-tumor tissues was detected by immunohistochemical staining with SP (streptavidin-perosidase). And its clinical significance was evaluated. Results The staining of NVM-1 was predominantly located in the nucleus of HCC tumor tissue. The positive rate of NVM-1 was 52.2% in all detected cancers and it was higher than that in control non-tumor tissues(28.3%) and benign hepatic lesions(0). A significant difference of positive rates existed between tumor and non-tumor tissues (P<0.05). The expression of NVM-1 in HCC was not correlated with hepatic B virus (HBV), size, amount or cirrhosis, etc. However, it was correlated with TNM(P=0.016), BCLC stage (P=0.015), poor differentiation (P=0.000), intrahepatic metastasis (P=0.002), portal vein tumor thrombus (P=0.023) and recurrence (P=0.001). By Kaplan-Meier analysis, the median tumor-free survival time was 5 months in positive expression group and it was significantly shorter than 13 months in negative expression group (P=0.001). Conclusion There is probably a close relation between recurrence and metastasis and the expression of NVM-1 in HCC. Key words: Carcinoma, hepatocellular; Novel metastasis-promoting gene 1 protein, human; Recurrence; Neoplasm metastasis

BACKGROUND Anti-epidermal growth factor receptor (EGFR) monoclonal antibodies benefit patients with wild-type KRAS exon 2 metastatic colorectal cancer (mCRC). However, their effect in KRAS-mutant mCRC remains unclear. PATIENTS AND METHODS This was a retrospective study enrolling 163 patients with unresectable KRAS-mutant mCRC diagnosed at the National Taiwan University Hospital between 2007 and 2011. RESULTS The median overall survival (mOS) was 29.5 months in patients who had never used cetuximab and 19.0 months in those who had (p=0.040). The mOS was 32.0 months in patients with mutant KRAS codon 12 who had never used cetuximab and 17.5 months in those who had (p=0.017). In patients with mutant KRAS codon 13, the mOS was not significantly different. Univariate and multivariate Cox proportional hazards analysis revealed that absence of cetuximab treatment was an independent prognostic factor for longer mOS in patients with unresectable KRAS-mutant mCRC. CONCLUSION Cetuximab usage might be detrimental to patients with mCRC with mutant KRAS codon 12.

This paper presents an easily produced, cost-efficient chip-level device which has the ability to perform real-time PCR with single copy sensitivity. In order to integrate the high sensitivity and accuracy of real-time PCR and advantages of a miniaturized device, a simple chip-level device is built. We used this device to perform real-time PCR with single copy sensitivity. The minimum off-chip work will decrease the handling and human error during the whole process which will help to increase the successful rate and sensitivity of the chip.

Objective To investigate the fibrosis-associated genes expressed in the tissue of extensive fibrotic kidney of patients with severe hydronephrosis.Methods Twelve extensive fibmtic kidneys in patients with severe hydronephrosis(hydrnephrosis group)were compared with 6 normal renal tissues (control group)by using real-time polymerase chain reaction(PCR)assay.All samples were collected from our hospital from Oct.2005 to Aug.2007.The results of real time PCR assay were processed with ΔΔCt method.When 2~(ΔΔc1) wag≤0.5 or≥2.the difference Was considered significant.Then the expression of left-right determination factor A(LEFTY-A)in both groups were detected by immunohistochemistry.Results A total of 49 differential genes expressed in the fibrotic renal tissues were screened out.of which,25 were up-regulated and 24 were down-regulated.The differential genes included members and their receptors of transforming growth factor(TGF)-β super family or bone morphogenetic protein(BMP) family,target molecular of TGF-β or BMP signal pathway and several relative regulators.And the gene of LEFTY-1.one of the most markedly-changed genes,in hydrnephrosis group Was 13.55-fold down-regulated as compared with control group.Immunohistochemistry analysis reveled that LEGTU-A protein in the sample of hydronephrosis group was significantly decreased as compared with that in control group(P＜0.05).Conclusion The LEFTY mRNA and protein expression Was decreased in human severely hydronephrotic renal tissue.indicating that LEFTY may play an important role in the inhibition of renal fibrosis resulting from long-term hydronephrosis. Key words: Hydronephrosis; Reflal fibrosis; Gene chip; Left-right determination factor

Abstract The transcription factor CCAAT/enhancer binding protein alpha (C/EBPa) encoded by the CEBPA gene, is crucial for the differentiation of immature granulocytes. Diminished or abnormal C/EBPa activity resulting from CEBPA gene mutations is widely known to contribute to the transformation of myeloid progenitors via reduction of their differentiation potential. The CEBPA mutations have been detected in approximately 7% of total acute myeloid leukemia (AML) and in 15% of those with intermediate-risk cytogenetics or those with normal karyotype. However, the age distribution of the patients with the CEBPA mutations and the immunophenotype of their leukemic cells are not known. Sequential studies of the CEBPA gene in AML patients are also limited. In this study, 104 patients with de novo acute myeloid leukemia (AML) were evaluated for the CEBPA mutation by direct sequencing. Excluding the silent mutations, 16 (15%) of the total 104 AML patients, 15 (25%) of the 61 patients with intermediate-risk cytogenetics and 11 (35%) of the 31 patients with normal karyotype showed CEBPA mutations, frequencies higher than those reported in the West. Further cloning and subsequent nucleotide sequence analysis revealed that 14 patients had heterozygous biallelic mutations: 11 had mutations involving both the N-terminal transactivation domain (TAD) and the C-terminal basic leucine zipper domain (bZIP) and three, in either the TAD region or the bZIP region. The remaining two patients had only one allele mutation in the TAD1 region. Most mutations in TAD region were repeat-number changes of simple sequence repeats and those in bZIP region were internal tandem duplications. Sequence analysis revealed that in the region spanning the bZIP mutations, there was hot spot for concensus topoisomerase II sites, which has also been shown in other AML-related mutations FLT3-ITD and MLL duplication. All but one patient with CEBPA mutations had M1 or M2 subtype of AML. The patients with CEBPA mutations had significantly higher incidences of CD7 (73%), CD15 (100%), CD34 (93%) and HLA-DR (93%) expression than others and the majority of them showed a distinct immunophenotype of the leukemic cells: HLA-DR+ CD7+ CD13+ CD14− CD15+ CD33+ CD34+. The incidence of the CEBPA mutation in children with AML was similar to that in adults. The CEBPA mutation was serially analyzed in 27 patients; the mutations disappeared at CR, but reappeared at relapse. No one developed novel mutation during the follow-up period. In conclusion, the CEBPA mutation may play an important role in the development, but not progression, of AML. The patients with the CEBPA mutations showed a distinct immunophenotype of the leukemic cells. Potential topoisomerase II cleavage sites locating in the bZIP region were first reported and we propose that this is relevant to the process of illegitimate recombination generating the internal tandem duplication pattern of bZIP mutations.