Methylation-based molecular margin analysis in hepatocellular carcinoma

The positive surgical margins are associated with postsurgical recurrence in hepatocellular carcinoma patients, and molecular margin analysis is considered more sensitive in detecting preneoplastic lesions than conventional histological margin examination. To evaluate the feasibility of methylation-based molecular margin analysis in HCC and explore its clinical application, we investigated CDKN2A methylation status in the surgical margins of 20 HCC patients using a nested BS-MSP protocol and compared the methylation patterns in resection margins with those in the corresponding tumor and adjacent nonmalignant tissues. The results showed that a considerable frequency (35%, 7 of 20) of CDKN2A methylation was present in histologically negative margins, and methylation pattern analysis might be valuable for studying the cellular origin of recurrent carcinoma. Therefore, methylation-based molecular surgical margin analysis offers a promising tool in prognosis for HCC patients who underwent hepatectomy.     

Assessment of methylation events during colorectal tumor progression by absolute quantitative analysis of methylated alleles

To date, the epigenetic events involved in the progression of colorectal cancer are not well described. To study, in detail, methylation during colorectal cancer development in high-risk adenomas, we developed an assay combining in situ (on-slide) sodium bisulfite modification (SBM) of paraffin-embedded archival tissue sections with absolute quantitative assessment of methylated alleles (AQAMA). We tested the performance of the assay to detect methylation level differences between paired pre-malignant and malignant colorectal cancer stages. AQAMA assays were used to measure methylation levels at MINT (methylated in tumor) loci MINT1, MINT2, MINT12, and MINT31. Assay performance was verified on cell line DNA and standard cDNA. On-slide SBM, allowing DNA methylation assessment of 1 to 2 mm(2) of paraffin-embedded archival tissue, was employed. Methylation levels of adenomatous and cancerous components within a single tissue section in 72 colorectal cancer patients were analyzed. AQAMA was verified as accurately assessing CpG island methylation status in cell lines. The correlation between expected and measured cDNA methylation levels was high for all four MINT AQAMA assays (R >or= 0.966, P<0.001). Methylation levels at the four loci increased in 11% and decreased in 36% of specimens comparing paired adenoma and cancer tissues (P<0.0001 by Kolmogorov-Smirnov test). Single-PCR AQAMA provided accurate methylation level measurement. Variable MINT locus methylation level changes occur during malignant progression of colorectal adenoma. Combining AQAMA with on-slide SBM provides a sensitive assay that allows detailed histology-oriented analysis of DNA methylation levels and may give new, accurate insights into understanding development of epigenetic aberrancies in colorectal cancer progression.     

The phosphoinositide hydrolase phospholipase C delta1 inhibits epithelial-mesenchymal transition and is silenced in colorectal cancer

In this study, we found that the phospholipase C delta1 (PLCD1) protein expression is reduced in colorectal tumor tissues compared with paired surgical margin tissues. PLCD1-promoted CpG methylation was detected in 29/64 (45%) primary colorectal tumors, but not in nontumor tissues. The PLCD1 RNA expression was also reduced in three out of six cell lines, due to PLCD1 methylation. The ectopic expression of PLCD1 resulted in inhibited proliferation and attenuated migration of colorectal tumor cells, yet promoted colorectal tumor cell apoptosis in vitro. We also observed that PLCD1 suppressed proliferation and promoted apoptosis in vivo. In addition, PLCD1 induced G1/S phase cell cycle arrest. Furthermore, we found that PLCD1 led to the downregulation of several factors downstream of β-catenin, including c-Myc and cyclin D1, which are generally known to be promoters of tumorigenesis. This downregulation was caused by an upregulation of E-cadherin in colorectal tumor cells. Our findings provide insights into the role of PLCD1 as a tumor suppressor gene in colorectal cancer (CRC), and demonstrate that it plays significant roles in proliferation, migration, invasion, cell cycle progression, and epithelial-mesenchymal transition. On the basis of these results, tumor-specific methylation of PLCD1 could be used as a novel biomarker for early detection and prognostic prediction in CRC.     

Molecular probes for fluorescence image-guided cancer surgery

In cancer surgery, complete surgical resection of tumor lesions is critical to optimize the outcome. However, it is sometimes difficult to distinguish the boundary between tumor and normal tissues, and residual tumor tissue can result in cancer recurrence. Intraoperative imaging with fluorescent molecular probes can assist surgeons to visualize tumor lesions and their boundaries during surgery. Here, we review molecular probes for fluorescence image-guided cancer surgery, focusing especially on recent developments in high-performance tumor-imaging probes and the strategies used for their design.     

Epigenetic silencing of the WNT antagonist Dickkopf 3 disrupts normal Wnt/β‐catenin signalling and apoptosis regulation in breast cancer cells

Dickkopf‐related protein 3 (DKK3) is an antagonist of Wnt ligand activity. Reduced DKK3 expression has been reported in various types of cancers, but its functions and related molecular mechanisms in breast tumorigenesis remain unclear. We examined the expression and promoter methylation of DKK3 in 10 breast cancer cell lines, 96 primary breast tumours, 43 paired surgical margin tissues and 16 normal breast tissues. DKK3 was frequently silenced in breast cell lines (5/10) by promoter methylation, compared with human normal mammary epithelial cells and tissues. DKK3 methylation was detected in 78% of breast tumour samples, whereas only rarely methylated in normal breast and surgical margin tissues, suggesting tumour‐specific methylation of DKK3 in breast cancer. Ectopic expression of DKK3 suppressed cell colony formation through inducing G0/G1 cell cycle arrest and apoptosis of breast tumour cells. DKK3 also induced changes of cell morphology, and inhibited breast tumour cell migration through reversing epithelial‐mesenchymal transition (EMT) and down‐regulating stem cell markers. DKK3 inhibited canonical Wnt/β‐catenin signalling through mediating β‐catenin translocation from nucleus to cytoplasm and membrane, along with reduced active‐β‐catenin, further activating non‐canonical JNK signalling. Thus, our findings demonstrate that DKK3 could function as a tumour suppressor through inducing apoptosis and regulating Wnt signalling during breast tumorigenesis.     

Bisulfite-modified target DNA array for aberrant methylation analysis

Aberrant DNA methylation of CpG islands is among the earliest and most frequent alterations in cancer. It is of great importance to develop simple and high-throughput methods of methylation analysis for earlier cancer diagnosis or the detection of recurrence. In this study, bisulfite-modified target DNA arrays were prepared on positively charged nylon membrane with two different procedures: fixing PCR products and fixing genomic DNA. First, a bisulfite PCR product array was prepared through fixing PCR products amplified in bisulfite sequencing primers from the bisulfite-modified genomic DNA of different clinical samples on membrane. Furthermore, bisulfite-modified genomic DNA of the different samples was directly fixed on membrane to fabricate bisulfite genomic DNA arrays. The two kinds of arrays were hybridized by probes labeled with digoxigenin, and the hybridization signals were obtained through chemiluminescent detection. The methylation statuses of the IGFBP7 gene for breast tumor and normal tissue samples and for normal human blood cell samples were detected successfully by the two procedures. It was shown that the methods are reliable and sensitive and that they have high potential in screening molecular methylation markers from a large number of clinical samples.     

DNA Methylation Combinations in Adjacent Normal Colon Tissue Predict Cancer Recurrence: Evidence from a Clinical Cohort Study

Accumulating evidence has suggested the requirement for further stratification of patients in the same tumor stage according to molecular factors. We evaluate the combination of cancer stage and DNA methylation status as an indicator of the risk of recurrence and mortality among patients with colorectal cancer (CRC). A cohort study of 215 patients with CRC (mean age 64.32 years; 50.5% of men) from Tri-Service General Hospital in Taiwan examined the association between cancer stage and risk of CRC recurrence and mortality. A Cox proportional hazard model was used to analyze patient methylation status and clinical information at study entry, and their associations with CRC recurrence and mortality during follow-up. The advanced stage patients with p16, hMLH1, and MGMT methylation were associated with higher risk of CRC recurrence compared with the local stage patients with unmethylation status in tumor tissues, with adjusted hazard ratios (HRs) (95% confidence interval [CI]) of 9.64 (2.92–31.81), 8.29 (3.40–20.22), and 11.83 (3.49–40.12), respectively. When analyzing normal tissues, we observed similar risk of CRC recurrence with adjusted HRs (95% CI) of 10.85 (4.06–28.96), 9.04 (3.79–21.54), and 12.61 (4.90–32.44), respectively. For combined analyses, the risk of recurrence in the patients in advanced stage with DNA methylation in both normal and tumor tissues, compared with local stage with unmethylation, was increased with adjusted HR (95% CI) of 9.37 (3.36–26.09). In the advanced stage patients, methylation status and tissue subtype were associated with increased risk of 5-year cumulative CRC recurrence (p < 0.001). This study demonstrates that clustering DNA methylation status according to cancer stage and tissue subtype is critical for the assessment of risk of recurrence in CRC patients and also indicated an underlying mechanism.     

Identification of proteins with altered expression in colorectal cancer by means of 2D-proteomics

Modern proteomic techniques make it possible to identify numerous changes in protein expression in tumors as compared to normal tissues. Although proteomics is currently widely used, identification of proteins differentially expressed in particular types of cancer remains a challenging task. The goal of our study was to detect novel protein markers of colorectal cancer using comparative proteomics of protein extracts obtained from primary tumors and adjacent normal tissues. Coloreetal cancer is nearly asymptomatic at the early stages, which calls for development of fast and sensitive methods for molecular diagnostics. Proteomes of 11 paired specimens of primary colorectal tumors and adjacent histologically normal tissues were studied using comparative 2D PAGE. Altogether, 16 proteins with altered expression levels were detected, including 13 proteins with increased levels and three proteins with decreased levels in tumor tissues. These proteins were identified using MALDI-TOF mass spectrometry. The proteins GPD1, RRBP1 (increased levels), HNRNPH1, and SERPINB6 (decreased levels) have been associated with colorectal cancer for the first time.     

Telomerase activity levels in the surgical margin and tumour distant tissue of the squamous cell carcinoma of the head-and-neck.

The survival of patients with a head-and-neck squamous cell carcinoma is determined by loco-regional recurrence and second primary carcinomas. As a complement to histopathology, molecular changes of tumour marginal and tumour distant tissue may confirm curative surgical tumour extirpation. We tested telomerase activity with PCR-ELISA kits.20 tumour margin biopsies were chosen by the surgeon from 20 patients. In addition, 3 tissue samples were taken from each of 20 additional patients, one from the carcinoma centre, the tumour margin and one distant from the tumour. 50% of the carcinoma centres were telomerase-positive. Thirteen of the 40 tumour margin samples showed increased telomerase levels, and in 3 of these residual carcinoma was histopathologically detected. Six of the 20 tumour distant tissues revealed increased telomerase levels. Telomerase positivity in carcinoma-free tumour margins correlated with a good prognosis. Confirmation of the results in a larger patient group is needed.     

Augmented CD133 expression in distal margin correlates with poor prognosis in colorectal cancer

Pathological assessment of excised tumour and surgical margins in colorectal cancer (CRC) play crucial role in prognosis after surgery. Molecular assessment of margins could be more sensitive and informative than conventional histopathological analysis. Considering this view, we evaluated the distal surgical margins for expression of cancer stem cell (CSC) markers. Cellular and molecular assessment of normal, tumour and distal margin tissues were performed by flow cytometry, real‐time q‐PCR and immuno‐histochemical analysis for CRC patients after tumour excision. CRC patients were evaluated for expression of CSC markers in their normal, tumour and distal tissues. Flow cytometry assay revealed CD133 and CD44 enriched cells in distal margin and tumour compared to normal colorectal tissues, which was further confirmed by immunohistochemistry. Most importantly, immunohistochemistry also revealed the enrichment of CSC markers expression in pathologically negative distal margins. Patients with distal margin enriched for CD133 expression showed an increased recurrence rate and decreased disease‐free survival. This study proposes that although distal margin seems to be tumour free in conventional histopathological analysis, it could harbour cells enriched for CSC markers. Further CD133 could be a promising molecule to be used in molecular pathology for disease prognosis after surgery in CRC patients.     

MAT2A与NDRG2基因在结直肠癌中表达情况及临床意义的研究

目的 研究MAT2A（甲硫氨酸腺苷转移酶2A）和NDRG2（N-Myc下游调节基因-2）基因在结直肠癌组织中的表达及两者的相关性,为结直肠癌的研究及治疗提供重要参考。方法 选取68例术中切除结直肠癌组织及相应的癌旁组织标本,应用RT-PCR（逆转录‒聚合酶链式反应）和Western blotting（蛋白质印迹法）检测结直肠癌中MAT2A和NDRG2表达水平。结果 结直肠癌中MAT2A和NDRG2 mRNA表达情况：MAT2A阳性表达率（66.2%,45/68）高于癌旁组织（7.4%,5/68）;NDRG2阳性表达率（36.8%,25/68）低于癌旁组织（91.2%,62/68）;两者的表达与患者年龄、性别、肿瘤生长位置等因素均无关,与癌肿临床分期、分化水平及淋巴结转移密切相关。癌肿组织中MAT2A蛋白表达量相较于癌旁组织明显增多（t=39.1152,P=0.0000）,而癌肿组织中NDRG2蛋白表达量较癌旁组织明显降低（t=46.5103,P=0.0000）;且在结直肠癌组织中两基因的表达可能有相关性（χ2=7.41,P=0.009）。结论 在结直肠癌组织中MAT2A表达增高,NDRG2表达低下,两者与肿瘤的发生发展具有一定相关性,推测可能与DNA甲基化异常有关,但具体机制仍待进一步研究。     

Estrogen and progesterone receptors in colorectal cancer and surrounding mucosa

In this prospective study we have quantified by means of ELISA-methods the cytosolic content of estrogen (ER) and progesterone receptors (PgR) in tumoral tissue and paired normal mucosa from 163 patients with resectable colorectal cancer. Survival analysis was performed in a subgroup of 120 patients and the mean follow-up period was 24.9 months. The cutoff for ER and PgR levels was set at 1 fmol/mg protein. On the basis of this cutoff 20.9% of the cancers were ER positive and 25.8% were PgR positive; normal adjacent tissue presented ER in 18.4% and PgR in 24.5%. Our results did not show any significant correlation between ER and PgR levels in neoplastic tissues. Howewer, a correlation was found in normal mucosa samples (p=0.02). Statistical analysis showed that there was no correlation between tumor ER and PgR content and patient age or sex, tumor location, Dukes' stage, histological differentiation, DNA ploidy status and S-phase fraction. Furthermore, the results did not show any statistical differences in relapse-free and overall survival curves calculated for patients classified according to the hormone receptor content of their tumors. ER and PgR were detected at low levels in normal and neoplastic colorectal tissues without any significant relationship to either clinicopathological tumor characteristics or patient outcome. Their possible role in colorectal cancer remains to be elucidated.     

Identification of differentially expressed proteins in colorectal cancer by proteomics: down-regulation of secretagogin

To identify proteins with colorectal cancer-specific regulation, comparative 2-DE of individual-matched normal and neoplastic colorectal tissue specimens was performed. We found 15 protein spots with concordantly increased and 20 protein spots with concordantly decreased intensity in tumor tissue (expression regulation more than fivefold). Nine of these proteins were identified by MS/MS. Interestingly, one of the proteins, which exhibited a marked down-regulation in colorectal cancer tissues, was the recently identified endocrine cell-expressed protein secretagogin. The reduction of the secretagogin content in colorectal cancer tissues was confirmed by comparative immunoblotting (n = 17) and RT-PCR (n = 22) as well as by immunohistochemistry (n = 45) of individual-matched neoplastic and normal colorectal tissue specimens. Immunohistochemistry revealed absence of secretagogin-expressing cells in most of the colorectal cancer tissue specimens. However, some colorectal cancers were characterized by secretagogin-expressing cells. In normal mucosa, positively stained cells exhibited a neuroendocrine cell-characteristic morphology and mucosal location. In colorectal cancer tissues, secretagogin-expressing cells were characterized by a malignant morphology. Our findings might represent the basis for the clinical application of secretagogin as a biomarker for a distinct subgroup of colorectal cancers.     

骨桥蛋白在胰腺癌中的表达及其临床意义

目的:研究骨桥蛋白OPN在胰腺癌中的表达水平及其与临床病理特征的关系。方法:采用RT-PCR和免疫组织化学方法分别检测50例胰腺癌手术切除标本和20例癌旁正常胰腺组织中OPNmRNA及其蛋白水平的表达。结果:胰腺癌组织中OPNmRNA高表达率为90%(45/50)明显高于其在癌旁正常胰腺组织中的表达15%(3/20),差异有统计学意义(P<0.01);OPN蛋白的阳性率为86.0%(43/50),明显高于癌旁正常胰腺组织中的表达30%(6/20),差异有统计学意义(P<0.01);OPN在胰腺癌组织中表达与其淋巴结转移明显相关(P<0.05)。结论:OPN在胰腺癌中的过表达对胰腺癌的生长、浸润及转移起重要作用。     

The recently suggested intestinal cancer stem cell marker DCLK1 is an epigenetic biomarker for colorectal cancer

Recently, Dclk1 expression was identified to be an intestinal cancer stem cell specific biomarker in mouse models, implicating a potential role for targeting the DCLK1-postive cancer cells as a treatment for colorectal cancer. Using quantitative methylation specific PCR (qMSP) we here demonstrated that the DCLK1 promoter is hypermethylated in the vast majority of colorectal cancers (134/164; 82%), with no methylation in the normal mucosa samples (0/106). We further showed by Affymetrix exon arrays that DCLK1 is significantly downregulated in human colorectal cancer (n = 125) compared with normal colonic mucosa (n = 15), which was further confirmed by real-time RT-PCR of a subgroup of the samples. Additionally, a significant negative correlation was observed between methylation and DCLK1 expression in 74 cancer cell lines derived from 15 different tissues, and gene expression increased significantly after epigenetic drug treatment of initially methylated cancer cell lines. These findings underscore the potential of DCLK1 as a colorectal cancer biomarker for early detection, but may also have clinical implications regarding the previously proposed therapy toward DCLK1-positive cancer cells. This therapy would at best affect the cancer stem cell population, but will, based on the present results, not be efficient to treat the bulk of the tumor.     

p16基因甲基化状态与散发性大肠癌的相关性研究

为探讨p1 6基因甲基化状态与散发性大肠癌发生发展的关系 ,用甲基化特异性的聚合酶链反应 (methylati omspecificPCR ,MSP)结合测序检测散发性大肠癌及相应癌旁组织p1 6基因甲基化状态。研究发现p1 6基因在散发性大肠癌中甲基化率为 2 8 9% (1 3 4 5 ) ,有 8例癌及癌旁组织都发生了甲基化 ;有淋巴结及远处转移的甲基化率为5 0 % (8 1 6 ) ,高于无转移的甲基化率 2 0 8(5 2 4 ) (P <0 0 5 )。p1 6基因高甲基化是散发性大肠癌中常见的分子改变之一 ,大肠癌中p1 6基因高甲基化可能发生在癌变早期并与大肠癌的恶性进展有相关性     

人类抑癌基因beclin 1在胃癌和直结肠癌中表达下调的研究

人类抑癌基因beclin 1通过自噬作用调节细胞生长,但在胃癌和直结肠癌中其表达水平和调控机制仍不清楚．通过检测胃癌和直结肠肿瘤组织中beclin 1基因的表达水平,及DNA异常甲基化和杂合子缺失对其表达的影响,发现与癌旁组织相比,35％的胃癌标本和30％的直结肠癌标本中beclin 1基因表达显著下调．同时发现,beclin 1基因5’端存在一高密度CpG岛,在胃癌和直结肠癌中beclin 1的启动子区域和第二个内含子区域存在甲基化,而杂合子缺失仅在胃癌中发生．这些发现表明beclin 1基因的异常甲基化和杂合子缺失对其在胃癌和直结肠癌中的表达起调控作用．     

Abberant methylation of p16, HIC1, N33 and GSTP1 genes in tumor epitelium and tumor-associated stromal cells of prostate cancer

The methylation status of four genes significant in prostate carcinogenesis p16, HIC1, N33 and GSTP1, were evaluated using quantitative methylationsensitive polymerase chain reaction. Tumor epithelia, tumor-associated stroma, normal epithelia, foci of PIN and benign prostate hyperplasia, and stroma adjacent to tumor tissues were isolated from whole-mount prostatectomy specimens of patients with localized prostate cancer by using laser capture microdissection. We found high levels of gene methylation in the tumor epithelium and tumor-associated stromal cells and some methylation in both hyperplastic epithelium and stromal cells in normal-appearing tissues located adjacent to tumors. Promoter methylation in the non-neoplastic cells of the prostate tumor microenvironment may play an important role in cancer development and progression. We examined the promoter methylation status of pl6, HIC1, N33 and GSTP1 in prostate biopsy fragments and prostate tissues after radical prostatectomy from patients with adenocarcinoma without laser capture microdissection. Methylation frequencies of all genes in tumor samples were considerably lower than frequencies in microdissected tumour samples (HIC1, 71 versus 89%; p16, 22 versus 78%; GSTP1, 32 versus 100%; N33, 20 versus 33%). The laser capture microdissection is required procedure in methylation studies taking into account multifocality and heterogenity of prostate cancer tissue.     

TaqMan real-time PCR assay for specific detection of Opisthorchis viverrini DNA in Thai patients with hepatocellular carcinoma and cholangiocarcinoma

The aim of this study was to develop TaqMan real-time PCR assay that detected Opisthorchis viverrini DNA from 18 normal and 18 tumor tissue specimens from Thai patients with hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), who underwent liver resection from October 2005 to May 2006. Control liver specimens were seven non-primary liver cancers. A conserved probe representing 100% sequence homology was used as a reference for O. viverrini-specific probe. Five of six tumors (83%) and all six normal tissues from CCA group; and seven of twelve tumors (58%) and ten of twelve normal tissues (83%) from HCC group were found to have O. viverrini DNA. The O. viverrini DNA detection among HCC and CCA patients were not associated (p = 0.193; 90%CI). This RT-PCR will be a useful tool for investigating the relationship between cancer type and presence of the parasite and also for conducting epidemiological surveys.