Detection of activated KRAS from cancer patient peripheral blood using a weighted enzymatic chip array.

BACKGROUND: The KRAS oncogene was one of the earliest discoveries of genetic alterations in colorectal and lung cancers. Moreover, KRAS somatic mutations might be used for predicting the efficiency of anti-EGFR therapeutic drugs. The purpose of this research was to improve Activating KRAS Detection Chip by using a weighted enzymatic chip array (WEnCA) platform to detect activated KRAS mutations status in the peripheral blood of non-small-cell lung cancer (NSCLC) and colorectal cancer (CRC) patients in Taiwan. METHODS: Our laboratory developed an Activating KRAS Detection Chip and a WEnCA technique that can detect activated KRAS mutation status by screening circulating cancer cells in the surrounding bloodstream. We collected 390 peripheral blood samples of NSCLC patients (n = 210) and CRC patients (n = 180) to evaluate clinical KRAS activation using this gene array diagnosis apparatus, an Activating KRAS Detection Chip and a WEnCA technique. Subsequently, we prospectively enrolled 88 stage III CRC patients who received adjuvant FOLFOX-4 chemotherapy with or without cetuximab. We compared the chip results of preoperative blood specimens and their relationship with disease control status in these patients. RESULTS: After statistical analysis, the sensitivity of WEnCA was found to be 93%, and the specificity was found to be 94%. Relapse status and chip results among the stage III CRC patients receiving FOLFOX-4 plus cetuximab (n = 59) and those receiving FOLFOX-4 alone (n = 29) were compared. Among the 51 stage III CRC patients with chip negative results who were treated with FOLFOX-4 plus cetuximab chemotherapy, the relapse rate was 33.3%; otherwise, the relapse rate was 48.5% among the 23 out of 88 patients with chip negative results who received FOLFOX-4 alone. Negative chip results were significantly associated to better treatment outcomes in the FOLFOX-4 plus cetuximab group (P = 0.047). CONCLUSIONS: The results demonstrated that the WEnCA technique is a sensitive and convenient technique that produces easy-to-interpret results for detecting activated KRAS from the peripheral blood of cancer patients. We suggest that the WEnCA technique is also a potential tool for predicting responses in CRC patients following FOLFOX-4 plus cetuximab chemotherapy.

Decreasing relapse in colorectal cancer patients treated with cetuximab by using the activating KRAS detection chip.

The KRAS oncogene was among the first genetic alterations in colorectal cancer (CRC) to be discovered. Moreover, KRAS somatic mutations might be used for predicting the efficiency of anti-epidermal growth factor receptor therapeutic drugs. Because the KRAS mutations are similar in the primary CRC and/or the CRC metastasis, KRAS mutation testing can be performed on both specimen types. The purpose of this study was to investigate the clinical advantage of using a KRAS pathway-associated molecule analysis chip to analyze CRC patients treated with cetuximab. Our laboratory developed a KRAS pathway-associated molecule analysis chip and a weighted enzymatic chip array (WEnCA) technique, activating KRAS detection chip, which can detect KRAS mutation status by screening circulating cancer cells in the bloodstream. We prospectively enrolled 210 stage II-III CRC patients who received adjuvant oxaliplatin plus infusional 5-fluorouracil/leucovorin (FOLFOX)-4 chemotherapy with or without cetuximab. We compared the chip results of preoperative blood specimens with disease control status in these patients. Among the 168 CRC patients with negative chip results, 119 were treated with FOLFOX-4 plus cetuximab chemotherapy, and their relapse rate was 35.3 % (42/119). In contrast, the relapse rate was 71.4 % among the patients with negative chip results who received FOLFOX-4 treatment alone (35/49). Negative chip results were significantly correlated with better treatment outcomes in the FOLFOX-4 plus cetuximab group (P < 0.001). We suggest that the activating KRAS detection chip is a potential tool for predicting clinical outcomes in CRC patients following FOLFOX-4 treatment with or without cetuximab therapy.

Polymorphisms in XPD and ERCC1 Associated with Colorectal Cancer Outcome.

Using the comprehensive approach to selecting polymorphisms to date, we sought to examine whether recurrence in colorectal cancer was associated with inherited variation in three genes involved in DNA repair and cell proliferation. Three polymorphisms, which are excision repair cross-complementation 1 (ERCC1), xeroderma pigmentosum group D (XPD) and epidermal growth factor receptor (EGFR), were assessed in 257 postoperative stage II/III CRC patients with 5-fluorouracial chemotherapy in Taiwan. In addition, the correlations between genetic polymorphisms and patients' clinicopathological features were investigated. Genotypes of XPD codon751 A/A and ERCC1 codon118 T/T were associated with regional recurrence in a statistically significant way (p = 0.018). Patients who carried XPD AA and ERCC1 TT genotypes demonstrated a significantly greater regional recurrence risk (OR = 5.625, 95% CI, 1.557-20.32). Inherited variation in XPD and ERCC1 was associated with outcome in patients with colorectal cancer in Taiwan. As the significant association of single-nucleotide polymorphisms has not been studied previously in colorectal cancer, these findings suggest novel sites of variation, in part explaining the range of treatment responses seen in this disease.

Significant overexpression of DVL1 in Taiwanese colorectal cancer patients with liver metastasis.

Undetected micrometastasis plays a key role in the metastasis of cancer in colorectal cancer (CRC) patients. The aim of this study is to identify a biomarker of CRC patients with liver metastasis through the detection of circulating tumor cells (CTCs). Microarray and bioinformatics analysis of 10 CRC cancer tissue specimens compared with normal adjacent tissues revealed that 31 genes were up-regulated (gene expression ratio of cancer tissue to paired normal tissue > 2) in the cancer patients. We used a weighted enzymatic chip array (WEnCA) including 31 prognosis-related genes to investigate CTCs in 214 postoperative stage I-III CRC patients and to analyze the correlation between gene expression and clinico-pathological parameters. We employed the immunohistochemistry (IHC) method with polyclonal mouse antibody against DVL1 to detect DVL1 expression in 60 CRC patients. CRC liver metastasis occurred in 19.16% (41/214) of the patients. Using univariate analysis and multivariate proportional hazards regression analysis, we found that DVL1 mRNA overexpression had a significant, independent predictive value for liver metastasis in CRC patients (OR: 5.764; 95% CI: 2.588-12.837; p < 0.0001 on univariate analysis; OR: 3.768; 95% CI: 1.469-9.665; p = 0.006 on multivariate analysis). IHC staining of the immunoreactivity of DVL1 showed that DVL1 was localized in the cytoplasm of CRC cells. High expression of DVL1 was observed in 55% (33/60) of CRC tumor specimens and was associated significantly with tumor depth, perineural invasion and liver metastasis status (all p < 0.05). Our experimental results demonstrated that DVL1 is significantly overexpressed in CRC patients with liver metastasis, leading us to conclude that DVL1 could be a potential prognostic and predictive marker for CRC patients.

Multiple genetic polymorphisms in the prediction of clinical outcome of metastatic colorectal cancer patients treated with first-line FOLFOX-4 chemotherapy.

OBJECTIVES: The objective of the study is to investigate whether multiple chemotherapeutic agent-related genetic polymorphisms are associated with the clinical outcomes of Taiwanese metastatic colorectal cancers (mCRC) patients treated with the first-line FOLFOX-4 chemotherapy. METHODS: Consecutive mCRC patients were prospectively enrolled into this study. Peripheral blood samples were used for genotyping of polymorphisms in MTHFR, DPD, GSTP1, MDR1, TYMS, ERCC1, XRCC1, and ERCC2 genes by polymerase chain reaction-restriction fragment length polymorphism technique and DNA sequencing. The primary end point of the study was to investigate the association of each genetic polymorphism with progression-free survival and overall survival (OS). RESULTS: Favorable genotypes from polymorphisms in ERCC1 codon 118C/C [hazard ratio (HR)=0.061, 95% confidence interval (CI): 0.014-0.274, P<0.001] and XRCC1 codon 399G/G (HR=0.306, 95% CI: 0.103-0.905, P=0.032) that are associated with progression-free survival were identified. Furthermore, ERCC1 codon 118C/C (HR=0.065, 95% CI: 0.011-0.377, P=0.002) and XRCC1 codon 399G/G (HR=0.152, 95% CI: 0.041-0.568, P=0.005) were significantly associated with favorable OS. Combining ERCC1 and XRCC1 genetic polymorphisms, patients with both favorable genotypes of ERCC1 codon 118C/C and XRCC1 codon 399G/G were associated with the better OS than those with one or without any favorable genotypes (P<0.001). CONCLUSION: The genetic polymorphisms of ERCC1 and XRCC1 may be useful in predicting clinical outcome in Taiwanese mCRC patients treated with FOLFOX-4. However, further prospective studies will be needed for the potential clinical implication.

Activating KRAS mutations and overexpression of epidermal growth factor receptor as independent predictors in metastatic colorectal cancer patients treated with cetuximab.

OBJECTIVE: Cetuximab, a monoclonal antibody targeting epidermal growth factor receptor (EGFR), has been proven to be efficient in metastatic colorectal cancer (mCRC); however, the therapeutic response is variable and markers predictive of response are urgently required. This study was conducted to determinate the predictive values of KRAS mutation status and EGFR expression in mCRC patients treated with cetuximab plus chemotherapy. SUMMARY BACKGROUND DATA: Clinical benefit with EGFR-targeting antibodies seems to be restricted to a particular subgroup of mCRC patients. Therefore, the identification of reliable predictive factors for mCRC patients is imperative before the introduction of targeted chemotherapy. METHODS: Ninety-five mCRC patients receiving cetuximab plus the FOLFIRI or FOLFOX-4 chemotherapy were enrolled into the present study. KRAS mutation status/EGFR expression levels were analyzed using direct sequencing, immunohistochemistry (IHC), and reverse transcription-polymerase chain reaction (RT-PCR) assay, respectively. The association between clinical response, progression-free survival (PFS) and overall survival (OS) as well as KRAS mutation status/EGFR expression levels were evaluated. RESULTS: Of 95 mCRC patients, KRAS mutations were identified in 41 cases, and EGFR overexpression (protein or mRNA levels) were observed in 78 patients. Among 41 tumors with KRAS mutation, 33 were found to be activating mutants at codons 12, 13, 15 or 18, while 8 were nonactivating mutants at codons 20, 30, or 31. Fifty-five patients responded to cetuximab plus chemotherapy, 49 were EGFR overexpression and 46 were wild-type KRAS tumor status. Patients with tumors that express high EGFR levels or harbor wild-type KRAS are more likely to have a better PFS and OS when treated with cetuximab plus chemotherapy (all P < 0.05). Furthermore, patients with nonactivating KRAS mutants in tumors had a significantly better PFS and OS than patients with activating KRAS mutants (both P < 0.05). However, for patients with wild-type KRAS tumor status, EGFR expression remains a relevant predictor of clinical response. CONCLUSIONS: The study suggests that activating KRAS mutants is a particularly important independent predictive marker in mCRC patients treated with cetuximab plus chemotherapy, of which combing activating KRAS mutants and EGFR could help to identify the subgroup of patients who are most likely to respond to cetuximab plus chemotherapy.

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.

GLUT1 gene is a potential hypoxic marker in colorectal cancer patients.

BACKGROUND: Tumor hypoxia is an important factor related to tumor resistance to radiotherapy and chemotherapy. This study investigated molecules synthesized in colorectal cancer cells during hypoxia to explore the possibility of developing molecular probes capable of detecting cell death and/or the efficiency of radiotherapy and chemotherapy. METHODS: At first, we incubated two human colorectal adenocarcinoma cell lines SW480 (UICC stage II) and SW620 (UICC stage III) cells in hypoxic (< or =2% O2, 93% N2, and 5% CO2) and normoxic conditions (20% O2, 75% N2, and 5% CO2) for 24 h and 48 h. The relative expression ratio of GLUT1 mRNA in hypoxic conditions was analyzed by RT-PCR. Ten cancerous tissues collected from human colorectal cancer patients were examined. HIF-1alpha and HIF-2alpha levels were measured to indicate the degree of hypoxia, and gene expression under hypoxic conditions was determined. As a comparison, HIF-1alpha, HIF-2alpha, and GLUT1 levels were measured in the peripheral blood of 100 CRC patients. RESULTS: Hypoxia-induced lactate was found to be elevated 3.24- to 3.36-fold in SW480 cells, and 3.06- to 3.17-fold in SW620 cells. The increased relative expression ratio of GLUT1 mRNA, under hypoxic conditions was higher in SW620 cells (1.39- to 1.72-fold elevation) than in SW480 cells (1.24- to 1.66-fold elevation). HIF-1alpha and HIF-2alpha levels were elevated and GLUT1 genes were significantly overexpressed in CRC tissue specimens. The elevated ratio of GLUT1 was higher in stage III and IV CRC tissue specimens than in the stage I and II (2.97-4.73 versus 1.44-2.11). GLUT1 mRNA was also increased in the peripheral blood of stage II and III CRC patients as compared to stage I patients, suggesting that GLUT1 may serve as a hypoxic indicator in CRC patients. CONCLUSION: In conclusion, this study demonstrated that GLUT1 has the potential to be employed as a molecular marker to indicate the degree of hypoxia experienced by tumors circulating in the blood of cancer patients.

Gender-dependent effect of ACE I/D and AGT M235T polymorphisms on the progression of urinary albumin excretion in Taiwanese with type 2 diabetes.

BACKGROUND: We investigated the gender differences in the effect of ACE I/D and AGT M235T polymorphisms on the prognosis of diabetic nephropathy (DN). METHODS: A total of 525 type 2 diabetics were enrolled to participate in this prospective observational study. ACE and AGT gene polymorphisms were analyzed by polymerase chain reaction. The progression of DN was defined as a shift to a higher stage of DN or a doubling of the baseline serum creatinine level by the end of the study. RESULTS: The baseline biophysical parameters show no gender differences in progression and non-progression of DN. The women who were ACE D allele carriers were found to be at an increased risk of DN progression compared to those with II genotypes (p = 0.024, OR 2.176). No such difference was seen in male patients (p = 0.619, OR 0.833). After adjusting for confounding factors (age, SBP, DBP, BMI, HbA1c, total cholesterol, TG, HDL-C, LDL-C, ACEI, and ARB) in our multiple regression analysis, these women were still found to be at increased risk of progressing to more severe DN (p = 0.008, OR 3.082) but not the men (p = 0.183, OR 0.586). Neither the AGT TT genotype nor the T allele were associated with the progression of DN in either sex after adjusting for confounding factors. CONCLUSION: Our follow-up study suggests that female diabetic carriers of the ACE D allele might be at an increased risk of DN progression.

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.

Multiple Genetic Polymorphisms of GSTP1 313AG, MDR1 3435CC, and MTHFR 677CC highly correlated with early relapse of breast cancer patients in Taiwan.

BACKGROUND: Genetic polymorphisms in drug-metabolizing enzymes, transporters, receptors, and other drug targets have been linked to inter-individual differences in the efficacy and toxicity of many medications. In the present study, multiple chemotherapeutic agent-related genetic polymorphisms, including GSTP1, MDR1, MTHFR, and TS tandem repeats, were analyzed in breast cancer patients and studied in correlation with the clinical outcome of patients receiving FEC adjuvant chemotherapy. METHODS: The genotypes from 192 stage II and III breast cancer patients who underwent operations and received six cycles of postoperative adjuvant chemotherapy (FEC) were determined by means of PCR-RFLP. The association of each genetic polymorphism with clinicopathological data of patients and early relapse status were analyzed. RESULTS: The results showed that the genotype distribution of GSTP1 A313G, MTHFR C677T, and TS 3R3R in Taiwanese subjects differed significantly from the distribution in Caucasians. After analysis of the relationship between the genotypes and clinicopathological data of the patients, a significant correlation was observed between postoperative early relapse in patients with genetic polymorphisms of both MDR1 3435CC and MTHFR 677CC (crude OR: 2.609, P = .013) and patients with additional GSTP1 313AG genetic polymorphism (crude OR: 2.833, P = .017). CONCLUSIONS: The results of the present study highly suggest that GSTP1, MDR1, and MTHFR genotypes could be prognostic factors for Taiwanese patients with breast cancer.

Persistent presence of postoperative circulating tumor cells is a poor prognostic factor for patients with stage I-III colorectal cancer after curative resection.

AIM: To detect pre- and postoperative circulating tumor cells (CTCs) in stage I-III colorectal cancer (CRC) patients undergoing curative resection and so identify a subgroup of patients who are at high risk for relapse. METHODS: Four mRNA molecular markers including human telomerase reverse transcriptase, cytokeratin-19, cytokeratin-20, and carcinoembryonic antigen mRNA were used to detect CTCs in 438 CRC patients underwent curative resection. RESULTS: Out of 438 patients, 80 CRC patients were classified to preoperative (-)/postoperative (-), 221 patients were preoperative (+)/postoperative (-), while 137 patients were preoperative (+)/postoperative (+). Univariately, postoperative relapse was significantly correlated with depth of invasion (P = 0.032), lymph node metastasis (P < 0.001), vascular invasion (P = 0.001), perineural invasion (P = 0.013), and persistent presence of CTCs (P < 0.001). Using a multivariate proportional hazards regression analysis, the presence of lymph node metastasis (P = 0.012; HR, 7.652; 95% CI: 4.162-14.827), vascular invasion (P = 0.033; HR, 4.360; 95% CI: 2.793-10.847), and the persistent presence of CTCs (P < 0.001; HR, 29.486; 95% CI: 10.281-87.792) were demonstrated to be independent predictors for postoperative relapse. Combination of these three independent predictors showed that patients with any one positive predictor had a hazard ratio of sevenfold to develop postoperative relapse (P < 0.001; HR, 7.064; 95% CI: 4.354-11.464). Furthermore, the persistent presence of CTCs was strongly correlated with poorer relapse-free survival rates (all P < 0.001). CONCLUSION: The promising results of this study suggest that persistent presence of postoperative CTCs may be a crucial prognostic factor adjuvant to conventional tumor markers in CRC patients who have undergone curative resection. Identification of these high-risk patients of persistent CTCs positivity is important and thus could help to define patients for adjuvant therapy with this tumor entity.

ERCC2 2251A>C genetic polymorphism was highly correlated with early relapse in high-risk stage II and stage III colorectal cancer patients: a preliminary study.

BACKGROUND: Early relapse in colorectal cancer (CRC) patients is attributed mainly to the higher malignant entity (such as an unfavorable genotype, deeper tumor invasion, lymph node metastasis and advance cancer stage) and poor response to chemotherapy. Several investigations have demonstrated that genetic polymorphisms in drug-targeted genes, metabolizing enzymes, and DNA-repairing enzymes are all strongly correlated with inter-individual differences in the efficacy and toxicity of many treatment regimens. This preliminary study attempts to identify the correlation between genetic polymorphisms and clinicopathological features of CRC, and evaluates the relationship between genetic polymorphisms and chemotherapeutic susceptibility of Taiwanese CRC patients. To our knowledge, this study discusses, for the first time, early cancer relapse and its indication by multiple genes. METHODS: Six gene polymorphisms functional in drug-metabolism - GSTP1 Ile105Val, ABCB1 Ile1145Ile, MTHFR Ala222Val, TYMS double (2R) or triple (3R) tandem repeat - and DNA-repair genes - ERCC2 Lys751Gln and XRCC1 Arg399Gln - were assessed in 201 CRC patients using a polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) technique and DNA sequencing. Patients were diagnosed as either high-risk stage II (T2 and 3 N0 M0) or III (any T N1 and 2 M0) and were administered adjuvant chemotherapy regimens that included 5-fluorouracil (5FU) and leucovorin (LV). The correlations between genetic polymorphisms and patient clinicopathological features and relapses were investigated. RESULTS: In this study, the distributions of GSTP1 (P = 0.003), ABCB1 (P = 0.001), TYMS (P < 0.0001), ERCC2 (P < 0.0001) and XRCC1 (P = 0.006) genotypes in the Asian population, with the exception of MTHFR (P = 0.081), differed significantly from their distributions in a Caucasian population. However, the unfavorable genotype ERCC2 2251A>C (P = 0.006), tumor invasion depth (P = 0.025), lymph node metastasis (P = 0.011) and cancer stage (P = 0.008) were significantly correlated with early relapse. Patients carrying the ERCC2 2251AC or2251CC genotypes had a significantly increased risk of early relapse (OR = 3.294, 95% CI, 1.272-8.532). CONCLUSION: We suggest that ERCC2 2251A>C alleles may be genetic predictors of early CRC relapse.

Chronic kidney disease as a risk factor for coronary artery disease in Chinese with type 2 diabetes.

BACKGROUND: Chronic kidney disease (CKD) is associated with cardiovascular disease (CVD) in the general population. We investigated the effects of renal function on coronary artery disease (CAD) in Chinese with type 2 diabetes who have a high risk of developing diabetic nephropathy but who may have a low risk of developing CAD. METHODS: We recruited a total of 2,434 Chinese with type 2 diabetes (1,078 men and 1,356 women) and diagnosed CAD by history or with an abnormal electrocardiogram (coronary probable or possible by Minnesota codes). Renal function was evaluated by serum creatinine (SCr) levels, estimated glomerular filtration rate (eGFR) (calculated by the abbreviated Modification of Diet in Renal Disease Study Croup formula) and urinary albumin/creatinine ratio (ACR). RESULTS: We found that patients with CAD were older, had higher SCr levels and body mass index (BMI), and had lower serum high-density lipoprotein cholesterol (HDL-c) levels. After adjusting for age, BMI, blood pressure, glycosylated hemoglobin, cholesterol, LDL-c, HDL-c, and triglycerides, we found that SCr levels >1.5 mg/dl, eGFR <60 ml/min, and urinary ACR >30 mg/g were independent risk factors for CAD in diabetic men, and that SCr levels >1.4 mg/dl and eGFR <60 ml/min were independently associated with CAD in women. CONCLUSION: Our findings indicate that Chinese with type 2 diabetes and CKD are likely to have had CAD previously and CKD is 'CVD risk state' in diabetic Chinese.

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.

Multiple molecular markers as predictors of colorectal cancer in patients with normal perioperative serum carcinoembryonic antigen levels.

PURPOSE: In this study, a high-sensitivity colorimetric membrane array method was used to detect circulating tumor cells (CTC) in the peripheral blood of colorectal cancer (CRC) patients with normal perioperative serum carcinoembryonic antigen (CEA) levels. This membrane array method was evaluated as a potential diagnostic and postoperative surveillance tool. STUDY DESIGN: Membrane arrays consisting of a panel of mRNA markers that include human telomerase reverse transcriptase, cytokeratin-19, cytokeratin-20, and CEA mRNA were used to detect CTCs in the peripheral blood of 157 postoperative CRC patients with normal perioperative serum CEA levels and in 80 healthy individuals. Digoxigenin-labeled cDNA were amplified by reverse transcription-PCR from the peripheral blood samples, which were then hybridized to the membrane array. The sensitivity, specificity, and accuracy of membrane arrays for the detection of CTCs were then calculated. RESULTS: Using the four markers in combination, expression of any three markers or all the four markers in this panel was significantly correlated with the clinicopathologic characteristics, including depth of tumor invasion, lymph node metastasis, tumor-node-metastasis stage, and postoperative relapse (all P < 0.05). The interval between the detection of all four positive molecular markers and subsequent elevated CEA ranged from 3 to 8 months (median 6 months). The expression of all four mRNA markers was an independent predictor for postoperative relapse. CRC patients with all four mRNA markers expression showed a significantly poorer survival rate than those with less than four positive markers. CONCLUSIONS: The constructed membrane array method was helpful in the early prediction of postoperative relapse in CRC patients with normal perioperative serum CEA levels.

Exon 33 T/T genotype of the thyroglobulin gene is a susceptibility gene for Graves' disease in Taiwanese and exon 12 C/C genotype protects against it.

This study investigates whether Tg gene polymorphisms can be associated with Graves' disease (GD) in a Taiwanese population and identifies potential polygenic susceptive genes for GD. The findings of such a study may have important implications for prognostic prediction and treatment of GD. We performed case control association studies for the 3 discovered Tg single nucleotide polymorphisms (SNPs) (E10, E12, E33) in 215 GD patients and 141 controls. The three SNPs were identified within the Tg gene. These SNPs were analysed by a fluorescent-based restriction fragment length polymorphism method (RFLP) and PCR. The genotype and allele frequencies at E10SNP158, E12SNP and E33SNP in GD patients were compared with those of the controls. In addition, we analysed the interactions between these SNPs and the clinical and laboratory variables. We found a significant difference in the T/T genotype of E33SNP and G/G genotype of E12SNP compared with the control group (p<0.001). We also found the E33SNP T/T genotype to be positively associated with development of GD, whereas the E12SNP G/G genotype protected it.

Molecular diversity of class 1 integrons in human isolates of Aeromonas spp. from southern Taiwan.

This work studies antimicrobial resistance and class 1 integrons of Aeromonas spp. in human isolates from southern Taiwan. PCR amplification and DNA sequence analyses were performed to characterize the gene cassette regions of the class 1 integron in 204 isolates of Aeromonas hydrophila, 36 isolates of A. sobria, 23 isolates of A. veronii, and 4 isolates of A. caviae. By using Southern hybridization with an intI1 probe to determine the presence of class 1 integrons in the 9 isolates of Aeromonas spp. harboring plasmid DNA, only 2 isolates, one A. veronii AV69 harboring 176-kb plasmid DNA, and one A. hydrophila AH207 harboring 149-kb plasmid DNA were identified. A conjugation experiment was carried out with 2 isolates of A. veronii AV69 and A. hydrophila AH207. Only one transconjugant of Escherichia coli AH207, containing 149-kb plasmids obtained from A. hydrophila AH207, was identified. ERIC-PCR analysis was performed to analyze the genetic relatedness in all isolates of Aeromonas spp. that carry class 1 integrons. The results of cluster analysis in this experiment revealed that none of these isolates were clonal, which may indicate that they were not related to the outbreak. Among the 267 isolates tested, class 1 integrons were detected in 37 isolates (13.9%) of Aeromonas spp. from humans. No class 2 or class 3 integrons were detected in this study. Gene cassette structures were identified in 30 (81.0%) of 37 isolates of Aeromonas spp. containing class 1 integrons. The gene cassette of dfrA12-orfF-aadA2 was the most prevalent in the gene cassette array (16.0%), followed by arr3-aacA4 (13.3%) and dfr2d-catB3-aadA1 (10.0%). Four novel arrays of gene cassettes were also identified, namely, dfr2d-catB3-aadA1, aadA1-aac(6')-II, aadA4a, and aac(6')-II-blaOXA-21-catB3. This is the first report of Aeromonas spp. isolates from humans.

Cholesteryl ester transfer protein B1B1 genotype is associated with a parental history of cardiovascular diseases in Taiwanese people.

OBJECTIVE: To investigate the association between family history of cardiovascular disease (CVD) and cholesteryl ester transfer protein (CETP) TaqIB polymorphism in Taiwanese subjects. SUBJECTS AND METHODS: In this cross-sectional study, 240 subjects (115 men and 125 women) were divided into two groups based on whether or not they had a parental history of CVD. Polymerase chain reaction/restriction fragment length polymorphism was used to analyze the genotype of the subjects for the TaqIB polymorphism of CETP in intron 1. RESULTS: The frequency of the B1B1 genotype was significantly higher in Taiwanese subjects with a family history of CVD than in those without it (31.2 vs. 18.8%, odds ratio = 1.97, 95% confidence interval = 1.084-3.579, p = 0.035). Siblings with the B1B1 genotype had lower levels of serum high-density lipoprotein cholesterol (HDL-C) than siblings with either B1B2 (46.7 +/- 11.0 vs. 52.5 +/- 11.1 mg/dl, p = 0.034) or B2B2 genotypes (46.7 +/- 11.0 vs. 55.2 +/- 9.6 mg/dl, p = 0.01). CONCLUSION: CETP TaqIB polymorphism is associated with plasma HDL-C levels. The CETP B1B1 genotype may influence the susceptibility to CVD in Taiwan.