Biomedical subjects

Shiu-Ru Lin

Publications and source records attributed to Shiu-Ru Lin.

At least 19 recordsLinked to original sources

PubMed2007

Differential expression profile of MAGE family in non-small-cell lung cancer.

The expression of the melanoma-associated antigen (MAGE) genes consists of variables in all tumor types, such as lung cancer, which are relevant to be silent in all normal tissues except germ cells. They are considered as tumor-specific antigens, and are ideal targets for cancer immunotherapy. A complete MAGE genes differential expression profile analysis of lung cancer can provide this study not only various target genes for immunotherapy, but also valuable markers for further diagnosis and prognosis. This research has constructed a membrane array, which was consisted 32 MAGE genes, to detect whether the differential expression profile occurred in 52 pairs of non-small-cell lung cancer (NSCLC) samples. Nearly 32 MAGE genes have been differential expressed in NSCLC except MAGE-B1 and -E2. MAGE-B, -C, -D, and subgroup -B6, -D4 have showed prominences in lung adenocarcinoma. High-frequent expression of MAGE-D, and subgroup -A2, -D2 has also been discovered in non-metastasis group (p<0.05). However, there is no significant difference of MAGE genes differential expression shown among different primary tumor (T), nodal involvement (N) and overall stages. Several MAGE subgroup genes, such as MAGE-A5, -A7, -A8, -A9, -A11, -B3, -B4, -B10, -D2, -D3, -F1, -G1, -H1, and -L2, have been first discovered to show differential expression in NSCLC. Although the small size of the sample may limit the diagnostic and prognostic value of MAGE genes, the function of the membrane array can provide this study a high-throughput method to detect the whole MAGE genes differential expression profile.

Antigens, Neoplasm↗

PubMed2007

Combination of multiple mRNA markers (PTTG1, Survivin, UbcH10 and TK1) in the diagnosis of Taiwanese patients with breast cancer by membrane array.

OBJECTIVE: Early detection is a prerequisite to the effective reduction of morbidity and mortality from breast cancer. The present study intended to employ a high-throughput membrane array to detect a panel of mRNA markers expressed by circulating tumor cells (CTCs) in the peripheral blood of female patients with breast cancer. METHODS: Peripheral blood was sampled from 92 breast cancer patients and 100 normal persons. CTCs were detected by using a membrane array technique. The markers used included the pituitary tumor transforming gene 1, survivin, UbcH10 and thymidine kinase 1. RESULTS: The results showed that the membrane array could positively detect 5 cancer cells per 1 ml of peripheral blood in breast cancer cell dilution experiments. For the panel of 4 mRNA markers, sensitivity and specificity were elevated up to 86 and 88%, respectively. Furthermore, it was found that the patients' clinicopathological characteristics tumor size (p = 0.006), histologic grade (p = 0.012), lymph node metastasis (p = 0.001) and TNM stage (p = 0.006) significantly correlated with the positive detection rate of the multimarker panel. CONCLUSIONS: These findings demonstrated that our multimarker membrane array method could detect CTCs in the circulation of breast cancer patients with considerably high sensitivity and specificity.

Biomarkers, Tumor↗

PubMed2006

Development of a membrane array-based multimarker assay for detection of circulating cancer cells in patients with non-small cell lung cancer.

Combination of multiple mRNA markers has been largely investigated for detection of circulating cancer cells. However, current PCR-based methods are relatively expensive and time consuming. The aim of this study was to develop a membrane array-based multimarker assay for detection of circulating cancer cells in nonsmall cell lung cancer (NSCLC) patients. At first, we selected 22 candidate genes by means of suppression subtractive hybridization and Northern blot analysis. The diagnostic value of each candidate gene was then preliminarily evaluated in 50 pairs of blood samples by membrane array method. Accordingly, 17 genes with area under the ROC curve (AUC) > or = 0.8 were selected as target genes to reconstruct the diagnostic membrane array, which was then used to test peripheral blood samples from 100 NSCLC patients and 147 control subjects. ROC curve analysis demonstrated that the optimal threshold number of overexpressed markers on membrane array for discrimination between NSCLC patients and control subjects was 12. As a result, the diagnostic membrane array could detect circulating cancer cells in 90 (90%) of 100 NSCLC patients and in 14 (9.5%) of 147 control subjects (including 6 of 100 normal persons, 3 of 20 breast cancer patients, 3 of 15 colorectal cancer patients and 2 of 12 gastric cancer patients). Moreover, the detection rate was significantly correlated with NSCLC patients' metastatic status and overall stage (p = 0.028 and 0.014, respectively). These results suggested that our blood-based membrane array assay for molecular detection of circulating lung cancer cells has great potential for clinical applications.

Adult↗

PubMed2006

Development of a high-throughput membrane-array method for molecular diagnosis of circulating tumor cells in patients with gastric cancers.

Recently several noninvasive methods have been employed to detect circulating tumor cells (CTCs) in cancer patients. In this study, we have developed a highly sensitive, high-throughput colorimetric membrane-array method that was designed to detect a panel of mRNA markers including human telomerase reverse transcriptase (hTERT), cytrokeratin-19 (CK-19), carcinoembryonic antigen (CEA) and mucin 1 (MUC1) mRNA for the presence of CTCs in the peripheral blood of patients with gastric cancer (GC). Digoxigenin-labeled cDNA targets synthesized following total RNA isolation from peripheral blood samples of 64 GC patients and 80 healthy individuals were subjected to membrane-array hybridization. The results showed that membrane array could positively detect 5 cancer cells/ml of peripheral blood in GC cell-dilution experiments. The sensitivity, specificity and diagnostic accuracy for hTERT, CK-19, CEA and MUC1 mRNA ranged from 78.1% to 82.8%, 76.3% to 85% and 81.3% to 83.3%, respectively. Both CEA and MUC1 mRNA expression was correlated significantly with all malignant biological properties of GC, such as macroscopic type, depth of tumor invasion, lymph-node metastasis, TNM stage and coexisting distant metastasis (all p < 0.05). Using these 4 markers in combination, the sensitivity, specificity and diagnostic accuracy of membrane array were raised to 89.1%, 91.3% and 90.3%, respectively. The expression of all 4 mRNA markers was an independent predictor for postoperative recurrence/metastasis. GC patients with the expression of all the 4 mRNA markers showed a poorer survival rate than those without the expression of any 1 mRNA marker (p = 0.0223). These findings demonstrated that our membrane-array method could detect CTCs in the circulation of GC patients with considerably high sensitivity and specificity. The identification of CTCs in the peripheral blood may be useful in the auxiliary cancer diagnostics or postoperative surveillance of GC patients for recurrence/metastasis.

Adult↗

PubMed2006

Combined detection of CEA, CK-19 and c-met mRNAs in peripheral blood: a highly sensitive panel for potential molecular diagnosis of non-small cell lung cancer.

OBJECTIVE: Detection of tumor-related mRNA in blood has become a potential cancer diagnostic approach. However, the sensitivity of single-marker assays is not high enough for clinical applications. The present study was aimed to evaluate the efficacy of a multimarker panel for molecular diagnosis of non-small cell lung cancer (NSCLC). METHODS: Carcinoembryonic antigen (CEA), cytokeratin 19 (CK-19), c-met and heterogeneous nuclear ribonucleoprotein (hnRNP) B1 mRNAs were quantified by quantitative real-time reverse transcriptase polymerase chain reaction in 34 tumor tissues and 69 peripheral blood samples of NSCLC patients. RESULTS: All four markers displayed high overexpression rates (range 82.3-97.1%) in NSCLC tumors. When used as single markers in blood for NSCLC diagnosis, CEA, CK-19, c-met and hnRNP B1 could only reach sensitivities of 52.2, 50.7, 42 and 17.4%, respectively. However, the sensitivity was enhanced up to 85.5% when CEA, CK-19 and c-met were combined in a 3-marker panel. Moreover, the expression of c-met and hnRNP B1 in blood was significantly correlated with patients' pathological stages. CONCLUSIONS: The combined detection of CEA, CK-19 and c-met mRNAs in blood provided a valuable tool for molecular diagnosis of NSCLC. In addition, our results also suggested that hnRNP B1 was not a valuable diagnostic marker but a potential prognostic marker for NSCLC.

Actins↗

PubMed2006

Fatty acid metabolism pathway play an important role in carcinogenesis of human colorectal cancers by Microarray-Bioinformatics analysis.

The present study systematically explored metabolic pathways and altered expressions of genes speculatively participating in colorectal carcinogenesis by using a Microarray-Bioinformatic analysis methods. The results revealed that 157 genes were up-regulated and 281 genes were down-regulated in colorectal cancer (CRC). Gene Ontology (GO) and relevant bioinformatics tools indicated that the functional category to which 438 genes (12%; 438/3800) of the most frequent alteration belonged was metabolism. The analysis of 10 colorectal cancer tissue specimens demonstrated that genes involved in fatty acid metabolic pathways had high rates of overexpression. In addition, we stimulated CRL-1790 cell line with linoleic acid (a polyunsaturated fatty acid) for 12, 24, 48 and 72 h. Cell proliferation was elevated by 5, 25, 28 and 31% (P<0.05), respectively. Further analyses revealed that the genes increasingly expressed in the cell line included enoyl-Coenzyme A, hydratase/3-hydroxyacyl Coenzyme A dehydrogenase (EHHADH), enoyl Coenzyme A hydratase, short chain, 1, mitochondrial (ECHS1); glutaryl-Coenzyme A dehydrogenase (GCDH), acyl-Coenzyme A oxidase 2, branched chain (ACOX2); acyl-Coenzyme A dehydrogenase, C-2 to C-3 short chain precursor (ACADS); carnitine palmitoyltransferase 1B (CPT1B), acyl-CoA synthetase long-chain family member 5 (ACSL5), and cytochrome P450, family 4, subfamily A, and polypeptide 11 (CYP4A11) genes. This indicated that the stimulating effect of linoleic acid on cell proliferation was due to interference with the metabolic pathway of fatty acid metabolism. In conclusion, genes with altered expression levels in CRC were mainly associated with fatty acid metabolic pathways speculated to have an important role linked to carcinogenesis.

Aged↗

PubMed2006

Clinical significance of MUC1 and c-Met RT-PCR detection of circulating tumor cells in patients with gastric carcinoma.

BACKGROUND: Recently, several reverse transcription-polymerase chain reaction (RT-PCR) techniques have been proven to be useful in the detection of circulating tumor cells (CTCs) in cancer patients. We attempted to detect CTCs in patients with gastric cancer (GC) using a RT-PCR assay for c-Met and MUC1 and to evaluate their clinical value. METHODS: Using a RT-PCR assay, c-Met and MUC1 mRNAs were amplified in 52 GC patients and 36 healthy individuals. Analyses were carried out for their correlation with the patients' clinicopathologic features, the occurrence of new post-operative metastasis, as well as the overall survival rates. RESULTS: In the RT-PCR analysis of peripheral blood, 61.5% (32/52) and 71.2% (37/52) of GC patients were positive for c-Met and MUC1 mRNA, respectively. The sensitivity and specificity of either mRNA detected in peripheral blood is 82.7% and 86.1%, respectively, with an accuracy of 84.1%. The detection of c-Met or MUC1 mRNA was significantly correlated with the depth of tumor invasion, lymph node metastases, TNM stage, vessel invasion, perineural involvement, and post-operative metastasis (all P<0.05). Kaplan-Meier analysis demonstrated that the overall survival rate of patients with positive c-Met or MUC1 mRNA expression in the peripheral blood was significantly shorter than in patients negative for c-Met or MUC1 mRNA expression (both P<0.05). CONCLUSIONS: Our findings suggest that using RT-PCR for the detection of c-Met or MUC1 mRNA may be a promising tool for the early detection of micro-metastatic CTCs in GC patients. Combination of these 2 tumor-specific mRNA markers would increase the detection rate and may be clinically helpful in predicting the outcome in GC patients.

Adult↗

PubMed2006

Molecular detection of circulating tumor cells in the peripheral blood of patients with colorectal cancer using RT-PCR: significance of the prediction of postoperative metastasis.

BACKGROUND: Approximately 20%-45% of colorectal cancer (CRC) patients ultimately develop local recurrence or metastasis following curative surgical resection. The latter is caused by tumor cells shed from the primary carcinoma prior to or during operation, currently undetected by standard clinical staging. Fortunately, the presence of tumor cells in peripheral blood can be detected by molecular methods and is being regarded increasingly as a clinically relevant prognostic factor. MATERIALS AND METHODS: To detect the presence of circulating tumor cells and evaluate their relationship to postoperative metastatic relapse, we simultaneously examined human telomerase reverse transcriptase (hTERT), cytokeratin-19 (CK-19), cytokeratin-20 (CK-20), and carcinoembryonic antigen (CEA) mRNA (messenger RNA) in the peripheral blood of 72 CRC patients and 30 healthy individuals. Using a reverse-transcriptase polymerase chain reaction (RT-PCR), these tumor-related mRNAs were amplified; in addition, analyses were carried out for their correlation with patients' clinicopathologic features, as well as the occurrence of postoperative metastasis. RESULTS: In RT-PCR analysis of the peripheral blood, 69.4% (50 out of 72), 66.7% (48 out of 72), 52.8% (38 out of 72), and 72.2% (52 out of 72) of CRC patients were positive for hTERT, CK-19, CK-20, and CEA mRNA respectively. All 30 healthy individuals were negative for hTERT and CEA mRNA expression, while 2 were positive for either CK-19 mRNA or CK-20 mRNA expression. The detection of CEA mRNA was significantly correlated with depth of tumor invasion (P=0.012), vessel invasion (P=0.035), TNM stage (P<0.0001), and postoperative metastasis (P<0.0001), while positive hTERT mRNA was correlated with TNM stage (P=0.037) and CK-19 was correlated with depth of tumor invasion (P=0.039) and postoperative metastasis (P=0.017). In addition, multivariate logistic regression showed that only CEA mRNA was an independent and significant predictor of postoperative metastasis (P=0.006). Our findings suggest that CEA mRNA may be a more reliable marker than hTERT, CK-19, and CK-20 for the detection of circulating cancer cells in the peripheral blood of CRC patients. CONCLUSIONS: Using RT-PCR for the detection of CEA mRNA is feasible and may be a promising tool for early detection of micrometastatic circulating tumor cells in CRC patients. CRC patients expressing positive CEA mRNA in peripheral blood have a significantly higher risk of postoperative metastasis. Nevertheless, confirmation of CEA mRNA as a prognostic predictive factor requires the continuation of patient follow-up.

Adult↗

PubMed2006

Mitogen-activated protein kinase pathway was significantly activated in human bronchial epithelial cells by nicotine.

Nicotine is potentially associated with the onset of chronic obstructive pulmonary disease (COPD) and lung cancer. To gain insights into the molecular mechanism underlying such nicotine-induced conditions, microarray- bioinformatics analysis was carried out in the present study to explore the gene expression profiles in human bronchial epithelial cells (HBECs) treated with 5 microM nicotine for 4, 8, and 10 h. Of 1,800 assessed genes overall, 260 (14.4%) were upregulated and 17 (0.9%) down regulated significantly. Gene ontology analysis demonstrated that most of the differentially expressed genes belonged to the category of molecular function, especially to the subcategories of enzyme activity. The integration of obtained information with bioinformatics tools in DAVID and KEGG databases indicated that the greatest number of overexpressed genes was involved in mitogen-activated protein kinase (MAPK) pathway. Membrane array analysis subsequently suggested that both extracellular signal-regulated kinase (ERK) 1/2 and c-Jun-NH(2)-terminal kinase (JNK) signalings but not p38 MAPK signaling were activated in response to nicotine. Pretreatment of HBECs with specific inhibitors against ERK 1/2 and JNK but not p38 could significantly inhibit nicotine-induced interleukin- 8 production. These results suggest that MAPK pathway may mediate the effect of nicotine through ERK 1/2 and JNK but not p38 in HBECs treated with nicotine.

Base Sequence↗

PubMed2006

Sarco/endoplasmic reticulum calcium-ATPase 2 expression as a tumor marker in colorectal cancer.

Maintaining a high calcium concentration in the endoplasmic reticulum through the action of sarco/endoplasmic reticulum calcium-ATPases (SERCAs) is crucial in many cell functions involved in intracellular signal transduction, control of proliferation, programmed cell death, or the synthesis of mature proteins. Recent studies have found that many SERCAs have altered expression patterns in various malignancies. The purpose of the current study was to quantify the expression of SERCA2 in colorectal cancer (CRC) tissues and the corresponding noncancerous tissues, and to statistically analyze whether the SERCA2 expression levels correlate with the clinico-pathologic features and prognosis of CRC patients. Paired colorectal tissue samples from cancerous and the corresponding noncancerous tissues were obtained from 50 patients who underwent surgical resection. Semiquantitative measurements of SERCA2 messenger RNA (mRNA) expression were done using the multiplex reverse transcriptase-polymerase chain reaction. CRC tissues were analyzed through immunohistochemistry for the SERCA2 protein. SERCA2 mRNA overexpression in cancerous tissues compared with normal counterparts was observed in 45 of 50 (90%) patients. The mean expression level of SERCA2 mRNA in cancerous tissues was significantly higher than that in noncancerous tissues (P = 0.01). Increased SERCA2 protein expression was significantly correlated with serosal invasion (P = 0.012), lymph node metastasis (P = 0.009), and advanced tumor stage (P = 0.004). Furthermore, patients with high SERCA2 expression had a significantly poorer overall survival rate than patients with low SERCA2 (P = 0.032). Multivariate analyses indicated that tumor stage (P = 0.015) and SERCA2 expression were independently correlated with overall survival (P = 0.018). The result of this study indicated that SERCA2 may be a molecular determinant in the development and progression of CRC. The molecular mechanisms underlying the SERCA-dependent calcium accumulation and CRC tumorigenesis are worthy of further investigations.

Adenocarcinoma↗

PubMed2006

Molecular detection of circulating cancer cells in the peripheral blood of patients with colorectal cancer by using membrane array with a multiple mRNA marker panel.

The objective of this study was mainly to evaluate the simultaneous detection of expression levels of a multiple mRNA marker panel in the peripheral blood of colorectal cancer (CRC) patients for use in complementary CRC diagnosis. Twenty-seven tumor tissue specimens and 80 peripheral blood specimens were collected from CRC patients. Firstly, the levels of multiple molecular markers in the tumor tissue and blood specimens were evaluated by using real-time quantitative PCR (RT-QPCR) and membrane array. The result of linear regression showed a high degree of correlation (r=0.954, P<0.0001) between the data of these two methods. CK-19 was the marker with the highest detection rate (87.5%) in the peripheral blood, followed by CEA (82.6%), REG4 (80.8%), and then uPA (80.0%) and TLAM1 (80.0%). The levels of the six markers in the peripheral blood were extensively explored. In the 80 patients, the frequency of CK-19, CK-20, CEA, REG4, uPA, and TIAM1 mRNA overexpression was 82.5% (66/80), 78.8% (63/80), 82.5% (66/80), 80.0% (64/80), 78.8% (63/80), and 80.0% (64/80), respectively. Then, a panel combining these 6 mRNA markers was evaluated for its utility in the clinical diagnosis of CRC. The sensitivity, specificity, and accuracy of membrane array-based diagnostic method were 88.8%, 87.8%, and 88.2%, respectively; much higher than those of examinations with single markers. Finally, lymph node metastasis (P=0.024) and TNM stage (P=0.009) were found to be significantly correlated with overexpression of the multiple mRNA marker panel. The detection rates of stage-I and -II CRC by using the multi-marker membrane array were 54.5% (6/11) and 92.0% (23/25), respectively. In conclusion, the results of the present study have shown that this innovative membrane array technique with a multiple mRNA marker panel can significantly improve the diagnosis rate of early colorectal cancer.

Adult↗

PubMed2006

Development and evaluation of a colorimetric membrane-array method for the detection of circulating tumor cells in the peripheral blood of Taiwanese patients with colorectal cancer.

Early detection is the hallmark of successful cancer treatment. Evidence is accumulating that primary cancers begin shedding neoplastic cells in the circulation at an early stage. To date, a high-sensitivity and high-throughput method for the detection of circulating tumor cells (CTCs) is deficient. In this study, we have developed a high-sensitivity colorimetric membrane-array method to detect CTCs in the peripheral blood of colorectal cancer (CRC) patients as a potential diagnostic tool. Previously, we identified a set of 18 oligonucleotide clones, significantly overexpressed in CRC, which were synthesized and applied to a nylon membrane. Digoxigenin (DIG)-labeled cDNA were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) from the peripheral blood of 88 Taiwanese CRC patients and 50 healthy subjects, and were then hybridized to the membrane-array. Hybridization signals were detected by color development. Meanwhile, blood samples were analyzed by real-time quantitative PCR (Q-PCR). Subsequently, both methods were compared regarding their correlation, sensitivity and specificity in the detection of CTCs by statistics. The results of membrane-arrays were demonstrated to be closely related to that of Q-PCR (P<0.001). The sensitivity and specificity of membrane-arrays for the detection of CTCs were 94.3% (95% CI, 86.4-102.2%) and 94% (95% CI, 85.9-102.1%), respectively. Moreover, the accuracy of membrane-arrays is higher than that of any one gene by Q-PCR. The detection rate of membrane-arrays was significantly associated with the depth of tumor invasion (P=0.002), the presence of lymph node metastasis (P=0.016), and TNM stage (P=0.005). The preliminary results indicated that the accuracy of membrane-arrays was sufficient to distinguish Taiwanese CRC patients from normal individuals with the advantages of time-saving, cost-effectiveness and high-throughput. Thus, the constructed colorimetric membrane-array could be a promising approach for the future detection of CTCs.

Adult↗

PubMed2006

Great potential of a panel of multiple hMTH1, SPD, ITGA11 and COL11A1 markers for diagnosis of patients with non-small cell lung cancer.

Research on molecular mechanisms underlying the carcinogenesis of non-small cell lung cancer (NSCLC) may provide gene targets in critical pathways valuable for improving the efficacy of therapy and survival of patients with NSCLC. However, the molecular markers highly sensitive for the prognosis and treatment evaluation of NSCLC are not yet available. To explore candidates, we conducted an oligonucleotide microarray study with three pairs of NSCLC and normal lung tissue, and determined 8 differentially expressed genes including the Human MutT homologue (hMTH1), Surfactant protein D (SPD), Human hyaluronan binding protein 2 (HABP2), Crystalline-mu (CRYM), Ceruloplasmin (CP), Integrin alpha-11 subunit (ITGA11), Collagen type XI alpha I (COL11A1), and Lung-specific X protein (Lun X). Four lung cancer-related markers MUC-1, hTERT, hnRNP B1, and CK-19 were also incorporated for further analysis. The expression profiles of the twelve genes in seventy pairs of NSCLC tumor and normal lung tissue were then detected quantitatively by using membrane array and quantitative real-time PCR (qRT-PCR). The data of the membrane array and qRT-PCR were compared for consistency and the potential of these mRNA markers in clinical application. The results showed that membrane array and qRT-PCR obtained consistent data for the tested genes in both sensitivity and specificity (correlation coefficient 0.921, p<0.0001). For patients' clinicopathological characteristics, the overexpression of hMTH1, SPD, HABP 2, ITGA11, COL11A1, and CK-19 was significantly correlated with the pathological stage (p<0.05). In addition, the overexpression of hMTH1, SPD, ITGA11, and COL11A1 was correlated with lymph node metastasis and poor prognosis. This is the first report relating SPD to a prognosis marker for NSCLC. Moreover, the combined detection of these four mRNA markers by membrane array had a sensitivity of 89% and a specificity of 84% for NSCLC, significantly higher than these markers had achieved separately. In conclusion, we identified mRNA markers for NSCLC prognosis and therapy evaluation from differentially expressed genes determined by using micro-array. Further studies are needed to collect the data of the mRNA markers used in clinical practice.

Adult↗

PubMed2006

Molecular mechanisms underlying the tumorigenesis of colorectal adenomas: correlation to activated K-ras oncogene.

Mutations of K-ras gene have been demonstrated in 40-50% of colorectal cancer and large adenoma (>1 cm). This study was intended to clarify the correlation between the existence of K-ras oncogene and the pathological features of colorectal adenomas using our recently developed membrane arrays. Moreover, the downstream genes regulated by K-ras oncogene were explored to serve as potential biomarkers in the early diagnosis and risk assessment of patients with colorectal adenoma. Specimens were collected from 70 patients with colorectal adenoma. The alterations of K-ras oncogene were analyzed by direct sequencing and our constructed membrane arrays, respectively. The results of direct sequencing showed that 21 of 70 samples (30%) had K-ras gene mutations. The most frequently mutated sites included codons 12, 13, 15 and 18. Furthermore, activated K-ras oncogene was identified in 18 of 70 (25.7%) adenoma by membrane arrays. Statistical analyses showed that the membrane array had the accuracy of 90.0%, sensitivity of 88.9%, and specificity of 90.4%. The frequency of the mutational sites of K-ras gene was located as follows: codon 12, 100% (4/4); codon 13, 100% (4/4); codon 15, 75% (6/8); and codon 18, 100% (2/2). The analysis of the correlation between the experimental data and pathological characteristics of adenoma showed that activated K-ras oncogenes were significantly associated with the size, number and histology of adenomas (all P<0.001). Finally, we found the downstream genes activated by K-ras oncogene, including B-cell CLL/lymphoma 2 (BCL2), Homo sapiens H2A histone family, member Z (H2AFZ), Homo sapiens RAP1B, member of RAS oncogene family (RAP1B), Homo sapiens T-box 19 (TBX19), Homo sapiens E2F transcription factor 4, p107/p130-binding (E2F4) and matrix metallopeptidase 1 (MMP1), of which were overexpressed in most of all examined adenomas. These genes were then suggested to have functions involved in cell growth. The preliminary results indicated that the accuracy of membrane arrays was comparable to conventional DNA sequencing in the detection of activated K-ras oncogenes. Therefore, we propose that activated K-ras oncogene in colorectal adenomas may play an important role in the subsequent colorectal carcinogenesis through a group of K-ras-related molecular targets.

Adenoma↗

PubMed2005

Simultaneous detection of multiple mRNA markers CK19, CEA, c-Met, Her2/neu and hMAM with membrane array, an innovative technique with a great potential for breast cancer diagnosis.

The objective of this study was mainly to develop and evaluate a membrane array-based method simultaneously detecting the expression levels of a multiple mRNA marker panel in the peripheral blood for used in complementary breast cancer diagnosis. The mRNA markers employed included cytokeratin 19 (CK-19), carcinoembryonic antigen (CEA), c-Met, Her2/neu, and mammaglobin (hMAM). The specimens of peripheral blood were collected from 80 healthy women and 102 female patients with breast cancer. The expression levels of molecular markers were evaluated by real-time Q-PCR and membrane array. Data obtained from real-time Q-PCR and membrane array were subjected to linear regression analysis, revealing that there was a high degree of correlation between the results of these two methods (r=0.979, P<0.0001). The result of membrane array assay with a combined panel of five mRNA markers was demonstrated to achieve sensitivity of 80.6%, and specificity of 83.8% for breast cancer detection, much higher than those of analysis of single marker. In addition, we demonstrated that the membrane array method could detect circulating cancer cells at a density as low as five cancer cells per 1 ml of blood. The analysis of correlation between the outcome of membrane array and clinicopathological characteristics indicated that overexpression of the multiple marker panel was significantly correlated with tumor size (P=0.030) and TNM stage (0.009). In conclusion, the detection of circulating cancer cells by means of membrane array simultaneously monitoring five mRNA markers could significantly enhance the sensitivity and specificity for cancer cell detection.

Biomarkers, Tumor↗

PubMed2005

High frequency of somatic missense mutation of BRCA2 in female breast cancer from Taiwan.

Somatic mutation of BRCA2 has been thought to be rare in breast cancers, though common allelic deletions in the BRCA2 locus (13q12-q13) imply an important role of somatic mutation in these tumors. Reasons of the reported rare incidence could be related to very few studies focusing on the mutational analysis of BRCA2 in sporadic tumors. The mutational status of the BRCA2 gene in exon11, the largest exon harboring the RAD51 interacting BRC domains which are critical for BRCA2 function, was screened by polymerase chain reaction/single strand conformation polymorphism (PCR-SSCP) analysis followed by direct sequencing. Tumor and paired normal tissue sample from 175 patients unselected for family history or age were taken after mastectomy for breast cancer and evaluated. There were 20 mutations of BRCA2 gene in exon 11 in 15 cases (15/175, 8.6%). Most mutations we identified were point mutation (19/20, 95%), except for one nucleotide insertion. Furthermore, among these mutations, missense mutations comprised 80% (16/20) of the BRCA2 mutations. All mutations we found were novel mutation after searched in the BIC database. There were three recurrent mutations at codon 1904; and two recurrent mutations at 1907, 1936, 1937 and 1968, respectively. The mutations were associated with ductal carcinoma in situ (P=0.038) and borderline with low histological grade (P=0.072). Besides, there were three cases possessing multiple mutations in the region we studied and one of them demonstrated aggressive lymph node metastasis. These findings implicated that somatic mutations of BRCA2 genes may play a significant role in the pathogenesis of breast carcinoma in Taiwan. In addition, those events were associated with some early clinicopathological features.

Adult↗

PubMed2005

APC, K-ras, and p53 gene mutations in colorectal cancer patients: correlation to clinicopathologic features and postoperative surveillance.

Current researches have proposed a genetic model for colorectal cancer (CRC), in which the sequential accumulation of mutations in specific cancer-related genes, including adenomatous polyposis coli (APC), K-ras, and p53, drives the transition from normal epithelium through increasing adenomatous dysplasia to colorectal cancer. To identify patients with an increased risk of tumor recurrence or metastasis and evaluate the prognostic values of APC, K-ras, and p53 gene mutations, we investigated the frequency of these three mutated genes in tumors and sera of CRC patients. APC, K-ras, and p53 gene mutations in primary tumor tissues and their paired preoperative serum samples of 118 CRC patients were detected by using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, followed by direct DNA sequencing of the PCR-amplified genomic DNA. Subsequently, serum molecular markers were analyzed for their correlation with patients' clinicopathologic features and presence of postoperative recurrence/metastasis. We did not observe any significant difference in the association of APC or K-ras or p53 gene mutations in primary tumors with patients' demographic data (all were P > 0.05). In contrast, both serum APC and p53 molecular markers were closely correlated with lymph node metastasis and TNM stage (both P < 0.05). Moreover, the serum overall molecular markers (at least one of the three markers) were prominently associated with depth of tumor invasion (P = 0.033), lymph node metastasis (P < 0.001), and TNM stage (P < 0.001). In addition, a significantly higher postoperative metastasis/recurrence rate in patients positive for overall molecular markers compared to those negative for these molecular markers were also demonstrated (P < 0.001). APC and K-ras molecular markers were more frequently observed in patients with locoregional metastasis (both P < 0.05), while p53 molecular marker was usually detected in the cases of peritoneal metastasis (P = 0.004). Our findings suggest that serum molecular markers are potentially useful in the determination of colorectal cancer patients harboring gene mutations at high risk of metastasis. Serial analysis is warranted in order to assess their long-term prognostic significance and the therapeutic implications.

Biomarkers, Tumor↗

PubMed2005

Analysis of progressively overexpressed genes in tumorigenesis of colorectal cancers using cDNA microarray.

The identification of differentially expressed genes has important implications in understanding the biology of colorectal tumorigenesis and progression, as well as developing new diagnostic and therapeutic strategies. In this study, cDNA microarray technology was used to identify colorectal tumor-related functional genes, which are overexpressed continuously from colorectal adenoma to adenocarcinoma. A set of 23 genes with progressive overexpression in the development of colorectal cancer (CRC) was identified by cDNA microarray, then analyzed by sequencing and Northern blot analysis. Validation of our array results was simultaneously performed by exploring the SAGEmap database. Furthermore, the gradually over-expressed genes from adenoma to adenocarcinoma were validated by Northern blot analysis with additional samples from three patients with synchronous colorectal adenocarcinoma and adenoma and four patients with CRC. Of these 23 genes, one was a function-unknown gene, designated as Homo sapiens chromosome 21q22.1 anonymous mRNA sequence (Genbank accession no. AF003738), and 22 were function-known genes. Searching through the Gene Ontology Browser at the Cancer Genome Analysis Project website revealed that the biological roles of these 22 function-known genes are involved in cell motility, cell adhesion, chemokine activity, signal transduction, cytoskeleton organization, proteolysis, apoptosis, and cell proliferation. The genes identified in the present study offer valuable information on colorectal carcinogenesis and metastasis, and represent a potential source of novel targets for new strategies for CRC diagnosis and therapy.

Blotting, Northern↗