The hTERT protein as a marker for malignancy in meningiomas

The meningioma evolution remains problematic as 6 to 19% relapse after total resection. We have no criterion or marker to predict with certainty the tumour behaviour, and the WHO grading system is to a certain degree controversial. Telomerase expression seems to play an active role in conferring to the tumour cell indefinite life span. Telomerase activity has been documented via TRAP protocol and telomerase messenger expression (hTERT mRNA). In meningiomas the protein hTERT itself has not been studied directly. Thirty tumour samples of meningiomas operated in our Neurosurgical Department are reviewed with a mean follow-up of 4 years. Specifically hTERT protein, resection type, proliferation markers (Ki-67), and recurrences are evaluated. MRI is used for recurrence controls. Seven samples appeared to be hTERT-positive and all seven showed recurrence. Four patients had undergone a subtotal resection (STR). Among them two were hTERT-positive; only these showed recurrence and malignancy. Of the five macroscopically total resections (MTR), two were initially histologically benign and progressed to malignancy. A strong correlation was found between hTERT and recurrences (coefficient=0.989; p=0,01) with the Spearman's rho test, and weaker one between the Ki-67 and hTER (coefficient=0.672; p<0.0001). The hTERT staining revealed the presence of the hTERT protein not only in their nucleoli but sometime outside as nuclear speckles. The presence of nucleolar or subnuclear hTERT is directly correlated to recurrence and progression towards malignancy. Relocalisation of this protein was confirmed. A distinction is proposed between regrowth, based on normal proliferation (Ki-67) which can accompany subtotal resection and recurrence. Recurrence appears to be pathologic proliferation linked to hTERT presence. The hTERT presence predicts a sombre clinical outcome at mid-term for the individual patient.     

MUC1在泌尿系肿瘤的相关研究进展

MUC1是一种高相对分子质量糖蛋白,属于黏蛋白家族成员,正常情况下存在于许多人上皮细胞膜的腺腔面,具有多种功能,其核心肽由胞外段、跨膜段和胞内段组成。短的跨膜段和胞内段在不同种属间的结构是高度保守的,长的胞外段含有20~125个连续重复序列,连续重复序列由20个氨基酸组成,富含丝氨酸、苏氨酸和脯氨酸残基。在多种肿瘤组织中,MUC1异常表达和异常糖基化。虽然,在泌尿系肿瘤,如膀胱癌、肾癌、前列腺癌,其确切功能尚不清楚,但MUC1可作为肿瘤标志物和靶点应用于放免诊断和治疗,MUC1同这些肿瘤的浸润、转移和预后也密切相关。其在泌尿系肿瘤中特征性变化具有一定的临床应用价值。     

Immunogenicity of the hydrophilic region of the MUC1 mucin protein core

This report is an analysis of data relating to the epitopes of 28 murine monoclonal antibodies reactive with the protein core of human carcinoma-associated MUC1 mucins. All anti-MUC1 antibodies define epitopes of linear sequences of 3, 4 or 5 amino acids within the hydrophilic domain, APDTRPAP, which is expressed multiple times in a highly conserved 20 amino acid repeat sequence of the MUC1 core. The R residue is present in the epitopes defined by all of the 28 anti-MUC1 monoclonal antibodies. Epitopes of antibodies originally prepared against immunogens containing human milk fat globule membranes include the motif DTR in over 90% of the examples studied.     

黏蛋白7作为潜在膀胱癌标志物的研究进展

通过对黏蛋白7（MUC7）特异性表达的检测对膀胱癌进行无创诊断已成为近年来的研究热点。研究表明,MUC7在膀胱癌恶性转化、移行上皮细胞癌（TCC）诊断、膀胱癌分期分级监控以及复发监控等方面均具有潜在的标志物作用,对于膀胱肿瘤的临床诊断具有应用价值。     

Variant serum beta-hexosaminidase as a biochemical marker of malignancy

Previous studies have demonstrated a variant form of beta-hexosaminidase in metastatic tumor tissue of human liver and in sera from cancer patients with liver metastases. The current investigation examined sera for the presence of the variant form of beta-hexosaminidase from a large and heterogeneous group of cancer patients (with different primary sites and differing degrees of metastatic involvement), from normal controls and pathological controls with nonmalignant diseases. Comparison of the total serum beta-hexosaminidase activity levels and the percentage of the total activity comprised of beta-hexosaminidase B (Hex B) revealed no significant differences (P greater than 0.01) between the three groups. Analytical isoelectric focusing indicated that the variant beta-hexosaminidase was present in 80% of 108 cancer patients, 37% of 27 pathological controls and 11% of 18 normal controls. Examination of subgroups of the cancer patients based on the extent of metastasis revealed that there was a significant increase in total serum beta-hexosaminidase activity and the presence of variant beta-hexosaminidase in the sera as the disease progressed. These results suggest that serum beta-hexosaminidase may be a useful marker for monitoring the progression of malignant disease.     

Monoclonal antibodies reacting with the MUC2 mucin core protein.

This study sought to produce monoclonal antibodies (MAbs) which reacted with the MUC2 core protein. Two MAbs [3A2 (IgG1) and 4F1 (IgM)] were produced by immunising female BALB/c mice with gel-formed mucin from the LS174T colon cancer cell line followed by a KLH conjugate of a 29 amino acid synthetic peptide whose sequence was derived from the variable number of tandem repeats (VNTR) region of a MUC2 cDNA clone. The MAbs reacted with synthetic MUC2 VNTR peptides but not synthetic MUC1 or MUC3 VNTR peptides, and showed specific reactivity in Western blotting with a high molecular weight protein produced by the LS174T colon carcinoma cell line. The use of shorter peptides indicated that the minimum peptide epitopes for these MAbs were different. Mab 3A2 reacted with amino acids 5-19 of the MUC2 VNTR by inhibition ELISA but not by direct ELISA, while 4F1 reacted with this peptide in both assays. Furthermore, 4F1 reacted in direct ELISA when a larger (29 amino acid) MUC2-derived peptide was coated onto the assay plate by incubating in carbonate buffer or by drying the peptide onto the assay plate, while 3A2 only reacted when this peptide was coated in carbonate buffer. The different specificity of the MAbs was also illustrated by the reactivity of 4F1 but not 3A2 with partially deglycosylated cystic fibrosis mucin. Immunohistochemical analysis with these MAbs revealed a strong reactivity with lung, gastric and colon tumours relative to normal tissue, with some breast and ovarian tumours also reacting. Both MAbs stained some normal goblet cells in the perinuclear region but not the mucin droplet or secreted mucin, indicating a reaction with immature (poorly glycosylated) mucin in the endoplasmic reticulum and/or golgi, but not with mature (fully glycosylated) mucin. In contrast, tumours showed strong diffuse cytoplasmic staining. 4F1 also showed weak apical cytoplasmic staining in some goblet cells and stained some tumours which showed no reactivity with 3A2. These antibodies should prove useful in the study of MUC2 structure and function, and in the diagnosis of some tumours.     

Plasminogen activator inhibitor-1 as a potential marker for the malignancy of colorectal cancer

To test the hypothesis that plasminogen activator inhibitor-1 (PAI-1) may serve as a candidate marker for the malignancy of colorectal cancer (CRC), we performed a quantitative RT-PCR for PAI-1 gene and evaluated the possible relationship between PAI-1 gene expression levels and clinicopathological findings in CRC. A significant increase in PAI-1 expression scores was observed in lymph node metastasis-positive CRCs (2.19 +/- 0.43) compared to negative ones (0.35 +/- 0.42) (P = 0.0037) as well as in distant metastasis-positive CRCs (3.50 +/- 1.18) compared to negative ones (0.99 +/- 0.30). The PAI-1 expression score markedly increased with the tumour stage (P = 0.0063; ANOVA test). Moreover, multivariate analysis revealed the PAI-1 expression score to be a strong and independent prognostic factor for CRC (P = 0.0432). These results suggested that PAI-1 might serve as a new parameter for the prediction of prognoses in CRC.     

RCAS1 as a tumour progression marker: an independent negative prognostic factor in gallbladder cancer.

Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) induces apoptosis in immune cells bearing the RCAS1 receptor. We sought to determine RCAS1 involvement in the origin and progression of gallbladder cancer, and also implications of RCAS1 for patient survival. RCAS1 expression was examined immunohistochemically in 110 surgically resected gallbladder specimens. The gallbladders represented 20 cases of cholecystitis with no associated pancreaticobiliary maljunction; 23 cases of cholecystitis with pancreaticobiliary maljunction; 14 cases of adenomyomatosis; 7 adenomas; and 46 cancers. High expression of RCAS1 (immunoreactivity in over 25% of cells) was observed in 32 of the 46 cancers (70%), but not in other diseases, including pre-cancerous conditions. RCAS1 immunoreactivity was associated with depth of tumour invasion (P = 0.0180), lymph node metastasis (P = 0.0033), lymphatic involvement (P = 0.0104), venous involvement (P = 0.0224), perineural involvement (P = 0.0351) and stage by the tumour, nodes and metastases (TNM) classification (P = 0.0026). Thus, RCAS1 expression may be a relatively late event in gallbladder carcinogenesis, possibly promoting tumour progression. Cox regression multivariate analysis demonstrated RCAS1 positivity to be an independent negative predictor for survival (P = 0.0337; risk ratio, 12.690; 95% confidence interval, 1.216-132.423). High expression of RCAS1 significantly correlated with tumour progression and predicted poor outcome in gallbladder cancer.     

MUC1和MUC16在胆囊癌中的表达及临床意义

目的:研究MUC1和MUC16在正常胆囊黏膜、胆囊腺瘤及胆囊癌中的表达情况,以及胆囊癌中MUC1和MUC16的表达与胆囊癌Nevin分期及组织分化程度的关系。方法 :收集因胆囊息肉行胆囊切除术病例60例、胆囊腺瘤病例60例及胆囊癌病例60例,进行MUC1和MUC16的免疫组织化学染色,结合病理图像,分析其在正常胆囊黏膜组织、胆囊腺瘤及胆囊癌中的表达情况。结果:MUC1在正常胆囊黏膜组、胆囊腺瘤组和胆囊癌组中,阳性表达率分别为6.67%、38.33%、81.67%。MUC16在正常胆囊黏膜组和胆囊腺瘤组中,无阳性表达。在胆囊癌组中,阳性表达率为36.67%。MUC1和MUC16在Nevin分期 I期组、II期组、III期及III期以上组中,阳性表达率分别为62.50%、80.00%、95.83%和18.75%、30.00%、54.17%。MUC1和MUC16在高分化组、中分化组、低分化组中阳性表达率分别为60.00%、78.95%、96.15%和13.33%、26.32%、57.69%。结论:MUC1和MUC16的阳性表达在胆囊癌的发生发展中起着重要作用,MUC1和MUC16的阳性表达促进胆囊癌的发生发展。MUC1和MUC16作为一种胆囊癌的肿瘤标志物,并且预示胆囊癌的恶性程度及浸润程度。     

MUC1 and MUC2 expression in human gallbladder carcinoma: a clinicopathological study and relationship with prognosis

Mucins are a group of high molecular weight glycoproteins consisting of a mucin core protein and O-linked carbohydrates. To date, nine apomucins (MUC1-8, and MUC5B) have been identified. Recent studies have demonstrated that MUC1 is expressed in tumors of various human organs, and may function as an anti-adhesion molecule that inhibits cell-to-cell adhesion, inducing tumor metastasis. MUC2 is a major secreted mucin of colon and is known to be expressed in cells showing intestinal metaplasia in the stomach and other organs. MUC2 expression in the mucosal epithelia is an apparently abnormal phenomenon related to the neoplastic process. In this study, we examined MUC1 and MUC2 expression in human gallbladder adenocarcinoma and its clinicopathological significance and relationship with the prognosis of the patients. MUC1 immunoreactivity was detected not only in the cancer cells but also in the cancer stroma. Cytoplasmic MUC1 expression was significantly relation to lymphatic invasion and lymph node metastasis (p < 0.001), and was associated with a poor outcome. In contrast, MUC2 was rarely expressed in gallbladder carcinomas, and its immunoreactivity was detected only in the cancer goblet cells. Overexpression of MUC2 was not significantly related to lymphatic invasion or lymph node metastasis, or prognosis of patients. These observations suggested that MUC1 expression plays a more important role than MUC2 expression in cancer cell growth and metastasis of human gallbladder adenocarcinomas.     

Genome-wide profiling of papillary thyroid cancer identifies MUC1 as an independent prognostic marker

Clinicopathological variables used at present for prognostication and treatment selection for papillary thyroid carcinomas (PTCs) do not uniformly predict tumor behavior, necessitating identification of novel prognostic markers. Complicating the assessment is the long natural history of PTC and our rudimentary knowledge of its genetic composition. In this study we took advantage of differences in clinical behavior of two distinct variants of PTC, the aggressive tall-cell variant (TCV) and indolent conventional PTC (cPTC), to identify molecular prognosticators of outcome using complementary genome wide analyses. Comparative genome hybridization (CGH) and cDNA microarray (17,840 genes) analyses were used to detect changes in DNA copy number and gene expression in pathological cPTC and TCV. The findings from CGH and cDNA microarray analyses were correlated and validated by real-time PCR and immunohistochemical analyses on a series of 100 cases of cPTC and TCV. Genes identified by this approach were evaluated as prognostic markers in cPTC by immunohistochemistry on tissue arrays. CGH identified significant differences in the presence (76 versus 27%; P = 0.001) and type of DNA copy number aberrations in TCV compared with cPTC. Recurrent gains of 1p34-36, 1q21, 6p21-22, 9q34, 11q13, 17q25, 19, and 22 and losses of 2q21-31, 4, 5p14-q21, 6q11-22, 8q11-22, 9q11-32, and 13q21-31 were unique to TCV. Hierarchical clustering of gene expression profiles revealed significant overlap between TCV and cPTC, but further analysis identified 82 dysregulated genes differentially expressed among the PTC variants. Of these, MUC1 was of particular interest because amplification of 1q by CGH correlated with MUC1 amplification by real-time PCR analysis and protein overexpression by immunohistochemistry in TCV (P = 0.005). Multivariate analysis revealed a significant association between MUC1 overexpression and treatment outcome, independent of histopathological categorization (P = 0.03). Analysis of a validation series containing a matched group of aggressive and indolent cPTCs confirmed the association between MUC1 overexpression and survival (relative risk, 2.3; 95% confidence interval, 1.1-5.5; P = 0.03). Our data suggest that MUC1 dysregulation is associated with aggressive behavior of PTC and may serve as a prognostic marker and potential therapeutic target in this disease.     

DNA aptamers that bind to MUC1 tumour marker: design and characterization of MUC1-binding single-stranded DNA aptamers.

Agents able to bind tightly and selectively to disease markers can greatly benefit disease diagnosis and therapy. Aptamers are functional molecules, usually DNA or RNA oligonucleotides, with the appropriate sequence and structure to form a complex with a target molecule. MUC1 is a well-known tumour marker present in a variety of malignant tumours and it has been a target of interest for many years. In this work we report the selection of DNA aptamers that bind with high affinity and selectivity to the MUC1 peptides. Combinatorial chemistry techniques based on the SELEX methodology were used for the identification of the specific aptamers. These were selected from an initial library containing a 25-base-long variable region, resulting in 4(25) random sequences of single-stranded DNA molecules, for their ability to bind to synthetic forms of MUC1. Ten rounds of in vitro selection were performed enriching for MUC1 binding. By round ten more than 90% of the pool of sequences consisted of MUC1-binding molecules. Selected aptamer families were cloned, sequenced and found to be unique, sharing no sequence consensus. The binding properties of these aptamers were quantitated by enzyme-linked immunosorbent assay and surface plasmon resonance, whereas their specificity for MUC1-expressing cancer cells has been validated using fluorescent microscopy. Aptamers offer significant advantages over existing antibody-based recognition procedures in that they offer higher binding affinity (higher retention/reduced dissociation) and specificity to the target (ability to determine variations on the protein target down to single amino acid changes), higher selectivity against mutated protein epitopes and potentially reduced immunogenicity and increased tumour penetration associated with their size.     

Correlation between expression of MUC1 core protein and outcome after surgery in mass-forming intrahepatic cholangiocarcinoma

BACKGROUND: It has been reported that MUC1 is an important prognostic factor in several cancers. This study investigated the importance of MUC1 as a prognostic factor in mass-forming intrahepatic cholangiocarcinoma (m-ICC). METHODS: In 50 patients with m-ICC who had undergone hepatectomy, expression of MUC1 was investigated. Expression of MUC1 was examined by immunohistochemical staining with monoclonal antibody HMPV, which recognizes the MUC1 core peptide. The immunohistochemical staining patterns of MUC1 were classified into three types: ductal type (the luminal surface membrane of neoplastic cells was stained), cytoplasmic type (the cytoplasm of neoplastic cells was stained dominantly), and negative type. RESULTS: Expression of MUC1 was detected immunohistochemically in 38 (76%) of 50 cases of m-ICC (ductal type, 18; cytoplasmic type, 20; and negative type, 12). Seventy-five percent of patients with lymph node metastasis had the cytoplasmic type MUC1 expression. Lymph node dissection was performed in only 20 patients, but significant correlation was demonstrated between MUC1 expression and lymph node metastasis (P = 0.0227). The location of MUC1 expression correlated with surgical outcome in m-ICC. Patients with the cytoplasmic type expression showed significantly lower survival rates. Univariate analysis revealed that MUC1 expression was a statistically significant risk factor affecting outcome in m-ICC (P = 0.0028). Furthermore, expression of MUC1 was found to be a statistically significant independent risk factor in multivariate analysis (P = 0.0063). CONCLUSIONS: The results suggest that evaluation of MUC1 expression may be very useful in predicting the surgical outcome in m-ICC.     

MicroRNA profiling of adrenocortical tumors reveals miR-483 as a marker of malignancy

BACKGROUND:

The authors are interested in identifying molecular markers that can aid in the diagnosis of adrenocortical carcinoma (ACC). The aim of this study was to identify microRNAs (miRNAs or miRs) that are differentially expressed in malignant adrenocortical tumors as compared with benign tumors and assess their potential as diagnostic predictors.

METHODS:

Differentially expressed miRNAs were identified using microarray profiling of adrenocortical tumors and validated by quantitative real‐time RT‐PCR.

RESULTS:

Microarray profiling in benign and primary malignant adrenocortical tumors revealed several significant differences between these histological groups. By using directed quantitative RT‐PCR analysis on a subset of these differentially expressed miRNAs, the authors determined that miRs ‐100, ‐125b, and ‐195 were significantly down‐regulated, whereas miR‐483‐5p was significantly up‐regulated in malignant as compared with benign tumors. Furthermore, the current study shows that miR‐483‐5p expression can accurately categorize tumors as benign or malignant.

CONCLUSIONS:

The authors identified 4 miRNAs that are dysregulated in adrenocortical carcinoma. The high expression of one of these, miR‐483‐5p, appears to be a defining characteristic of adrenocortical malignancies, and can thus be used to accurately distinguish between benign and malignant adrenocortical tumors. Cancer 2011. © 2010 American Cancer Society.     

Human DF3/MUC1 carcinoma-associated protein functions as an oncogene

The human DF3/MUC1 mucin-like glycoprotein is aberrantly overexpressed by most carcinomas of the breast and other epithelia. The contribution of MUC1 overexpression to the malignant phenotype is, however, not known. In the present studies, we have stably expressed MUC1 in rat 3Y1 fibroblasts. MUC1-positive cells were selected from independent transfections. The results demonstrate that, as found in human carcinomas, MUC1 is expressed on the cell surface and as a complex with beta-catenin in the nucleus of the transfectants. Colony formation in soft agar demonstrates that cells expressing MUC1, but not the empty vector, exhibit anchorage-independent growth. The results also show that MUC1 expression confers tumor formation in nude mice. These findings provide the first evidence that MUC1 induces cellular transformation.