Enhancing detection of circulating tumor cells with activating KRAS oncogene in patients with colorectal cancer by weighted chemiluminescent membrane array method.

BACKGROUND: In 2008, the National Comprehensive Cancer Network suggested conducting a KRAS mutations test in metastatic colorectal cancer (mCRC) patients prior to administering therapy that uses anti-epidermal growth factor receptor (EGFR) monoclonal antibody. However, tests of KRAS mutations have been limited when traditional molecular techniques, such as polymerase chain reaction (PCR) combined direct sequencing, are used to obtain and analyze patients' cancer tissues. If the primary tumor or metastatic tissues of patients with mCRC is unavailable, then such analysis will not be feasible. Our laboratory has successfully established a colorimetric membrane array analysis platform that could detect activating KRAS mutations from the peripheral blood of patients with various malignancies. METHODS: The current research aims to improve the above-mentioned technique not only by using chemiluminescence detection to replace color development, but also to add scores weighted according to the relevance of each gene to activating KRAS mutations. RESULTS: Our results show that the described weighted chemiluminescent membrane array (WCHMA) can detect circulating tumor cells (CTCs) harboring activating KRAS mutations in the peripheral blood in CRC. The sensitivity, specificity, and accuracy were 90.2, 94.9, and 93.5%, respectively, and the detection limitation was three colon tumor cells per millimeter of blood. The current study would significantly improve the detection sensitivity and accuracy over that of our previously designed membrane array method. CONCLUSIONS: These findings also highlight the need to prompt further prospective studies on more cases of CRC to further establish the clinical relevance of activating KRAS mutation detection from peripheral blood in anti- EGFR-based chemotherapy that uses activating KRAS detection chips and the WCHMA analysis method.

Differential gene expression profile of MAGE family in taiwanese patients with colorectal cancer.

BACKGROUND: The melanoma-associated antigen (MAGE) gene family consists of different expression patterns in various tumor types. They are considered tumor-specific antigens and are ideal targets for cancer immunotherapy. The purpose of this study is to identify the expression profiles of the MAGE family genes in Taiwanese colorectal cancer patients. METHODS: In this study, a well-constructed chip array platform was used to analyze the expression of the MAGE family genes of 100 colorectal cancer tissues. Statistical analysis of the experimental results and patients' clinical manifestations were also conducted. RESULTS: The results showed MAGE-A2 (87%), -A7 (83%), -A8 (75%), -A12 (71%), -B2 (75%), -B3 (79%), -D2 (75%), -F1 (79%), and -H1 (70%) were significantly overexpressed genes in colorectal cancer tissues. MAGE-A2 was the most highly overexpressed gene among the MAGE family. MAGE-B3 gene expression is statistically correlated with tumor size, lymph node, and UICC stage. In addition, the overexpression of MAGE-D2 and -H1 genes are statistically correlated to the tumor size and depth, respectively (P < 0.05). CONCLUSIONS: This is the first comprehensive report to clarify the differential expression profile of whole MAGE family in CRCs, and it might provide some crucial information about the carcinogenesis and progression in Taiwanese patients with CRC.

MMP13 is potentially a new tumor marker for breast cancer diagnosis.

Within the past decade, the incidence of breast cancer in Taiwan has been rising year after year. Breast cancer is the first most prevalent cancer and the fourth leading cause of cancer-related deaths among women in Taiwan. The early stage of breast cancer not only have a wider range of therapeutic options, but also obtain a higher success rate of therapy than those with advanced breast cancer. A test for tumor markers is the most convenient method to screen for breast cancer. However, the tumor markers currently available for breast cancer detection include carcinoembryonic antigen (CEA), carbohydrate antigen 15.3 (CA15.3), and carbohydrate antigen 27.29 (CA27.29) exhibited certain limitations. Poor sensitivity and specificity greatly limits the diagnostic accuracy of these markers. This study aims to identify potential tumor markers for breast cancer. At first, we analyzed genes expression in infiltrating lobular carcinoma, metaplastic carcinoma, and infiltrating ductal carcinoma of paired specimens (tumor and normal tissue) from breast cancer patients using microarray technology. We selected 371 overexpressed genes in all of the three cell type. In advanced breast cancer tissue, we detected four genes MMP13, CAMP, COL10A1 and FLJ25416 from 25 overexpressed genes which encoded secretion protein more specifically for breast cancer than other genes. After validation with 15 pairs of breast cancer tissue and paired to normal adjacent tissues by membrane array and quantitative RT-PCR, we found MMP13 was 100% overexpressed and confirmed to be a secreted protein by Western blot analysis of the cell culture medium. The expression level of MMP13 was also measured by immunohistochemical staining. We suggest that MMP13 is a highly overexpressed secretion protein in breast cancer tissue. It has potential to be a new tumor marker for breast cancer diagnosis.

Significance of the glycolytic pathway and glycolysis related-genes in tumorigenesis of human colorectal cancers.

We investigated gene expressions involved in the glycolytic pathways in colorectal cancer. The study was designed to use gene ontology and its relevant bioinformatics tools to analyze the microarray data obtained from CRC tissues and their corresponding normal tissues, in order to explore the correlation between the glycolytic metabolic pathway and possible pathogenesis of this disease. The overexpression of glycolysis-related genes was observed in over 76% of CRC tissues. In addition, we stimulated the SW480 and SW620 CRC cell lines with 15 mM D-(+)-glucose and 10 mM 2-deoxy-D-glucose respectively. The results indicate that the proliferation response of both the SW480 and SW620 cell lines increased remarkably with a time-dependent effect by D-(+)-glucose administration. In contrast, the proliferation response of both the SW480 and SW620 cell lines was significantly inhibited by 2-DG administration. Likewise, further analyses of the expression of related genes triggered by the D-(+)-glucose in vivo show that the activation process of these eight genes - GLUT1, HK1, GPI, GAPD, PGK1, PGK2, ENO2, PKM2 - prominently increased with a time-dependent effect. In conclusion, this study demonstrates that the glycolytic pathway and glycolysis-related genes may play an important role in the tumorigenesis of CRC, but their molecular mechanisms need further investigation to verify this.

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.

A fast and convenient new technique to detect the therapeutic target, K-ras mutant, from peripheral blood in non-small cell lung cancer patients.

Activating mutation of the K-ras gene was one of the earliest discoveries of genetic alterations in lung cancer. Moreover, K-ras somatic mutations might be suggested for predicting resistance to molecular antibodies targeting the epidermal growth factor receptor (EGFR). However, activated K-ras mutant detection methods are limited to traditional techniques. The techniques are complicated and are used only in tissue samples, which are limited for clinical applications. In a previous study, we established a low-cost, convenient, and easy technique for detecting activated K-ras in a small number of circulating tumor cells by the colorimetric membrane array method (CLMA). However, the sensitivity still needs further improvement. The aim of this study is to develop a new platform with chemiluminescence as reporter and weighted values of target genes on the chip in order to achieve a more sensitive, easier to read, and more accurate platform-weighted chemiluminecent membrane array (WCHMA). In advance, we collected 209 peripheral blood samples of non-small cell lung cancer (NSCLC) from patients to evaluate clinical K-ras activation detection using Activating KRAS Detection Chip both conducted by CLMA and WCHMA. Results show 71 specimens with K-ras mutation, of which 59 were identified as positive through CLMA and 66 were positive through WCHMA. After statistical analysis, the sensitivity of CLMA was found to be 83% and the specificity was 96%. On the other hand, the sensitivity of WCHMA increased to 93% and the specificity remained at 94%. Results of the detection limitation of peripheral blood on two platforms are: 3cancer cells/cm(3) blood using WCHMA, which is better than 5cancer cells/cm(3) blood using CLMA. Further analysis on the correlation between the test results and clinical pathological features shows that the mean score obtained using WCHMA is significantly correlated to TNM stage, tumor size, and metastasis.

MMP13 is a potential prognostic marker for colorectal cancer.

Matrix metalloproteinase 13 (MMP13), a member of the matrix metalloproteinase family, is considered to play a role in the tumor cell proliferation and invasion. The purpose of this study was to verify the expression of MMP13 in colorectal cancer (CRC) in vitro and in vivo, and subsequently analyze whether the MMP13 expression levels correlate with the clinicopathological features and prognosis of CRC patients. We assessed MMP13 mRNA expression profile in human colorectal adenocarcinoma cell lines by quantitative RT-PCR, and further verified if it was a secreted protein or not by Western blot analysis of cell culture medium. By immunohistochemical staining the immunoreactivity of MMP13 showed that MMP13 was localized in the cytoplasm of CRC cells. MMP13 mRNA expression of 80 cancerous tissues collected from UICC stage I to III CRC patients were examined by membrane array. The correlations between MMP13 mRNA expression and patients' clinicopathological features were analyzed. MMP13 was confirmed to be a secreted protein by Western blot analysis. The larger tumor size (P<0.0001), advanced clinical stage (P=0.002), tumor invasive depth (P=0.039), lymph node metastasis (P=0.001) and post-operative relapse (P<0.0001) were significantly correlated with the MMP13 mRNA overexpression. Patients with MMP13 mRNA overexpression have a higher risk of postoperative relapse (P<0.0001; OR=7.989; 95% CI, 2.607-24.481). The results of the present study highly suggest that MMP13 is a secreted protein with a significant correlation to development of postoperative relapse; hence it could be a potential prognostic marker for CRC patients.