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.

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.

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.

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.

DPYD, TYMS, TYMP, TK1, and TK2 genetic expressions as response markers in locally advanced rectal cancer patients treated with fluoropyrimidine-based chemoradiotherapy.

This study is to investigate multiple chemotherapeutic agent- and radiation-related genetic biomarkers in locally advanced rectal cancer (LARC) patients following fluoropyrimidine-based concurrent chemoradiotherapy (CCRT) for response prediction. We initially selected 6 fluoropyrimidine metabolism-related genes (DPYD, ORPT, TYMS, TYMP, TK1, and TK2) and 3 radiotherapy response-related genes (GLUT1, HIF-1α, and HIF-2α) as targets for gene expression identification in 60 LARC cancer specimens. Subsequently, a high-sensitivity weighted enzymatic chip array was designed and constructed to predict responses following CCRT. After CCRT, 39 of 60 (65%) LARC patients were classified as responders (pathological tumor regression grade 2 ~ 4). Using a panel of multiple genetic biomarkers (chip), including DPYD, TYMS, TYMP, TK1, and TK2, at a cutoff value for 3 positive genes, a sensitivity of 89.7% and a specificity of 81% were obtained (AUC: 0.915; 95% CI: 0.840-0.991). Negative chip results were significantly correlated to poor CCRT responses (TRG 0-1) (P = 0.014, hazard ratio: 22.704, 95% CI: 3.055-235.448 in multivariate analysis). Disease-free survival analysis showed significantly better survival rate in patients with positive chip results (P = 0.0001). We suggest that a chip including DPYD, TYMS, TYMP, TK1, and TK2 genes is a potential tool to predict response in LARC following fluoropyrimidine-based CCRT.

Overexpression of S100B, TM4SF4, and OLFM4 genes is correlated with liver metastasis in Taiwanese colorectal cancer patients.

Distant metastasis of colorectal cancer (CRC) occurs mainly in the liver and is the major cause of death. This study explored the overexpression of liver metastasis-associated mRNAs in human CRC by using a well-established, weighted enzymatic chip array platform. Analysis of 10 CRC tissue specimens compared with their normal adjacent tissues revealed that ATP2A2, ELAVL4, hTERT, KCTD2, MUC1, OLFM4, S100B, and TM4SF4 genes were upregulated (gene expression ratio of cancer tissue to paired normal tissue was >2) by microarray and bioinformatics analysis. A gene chip including eight candidate genes was constructed to investigate the circulating tumor cells in blood specimens of 103 preoperative CRC patients and further validated by reverse transcriptase-polymerase chain reaction. Liver metastasis was significantly correlated with overexpression of S100B (p=0.001, OR=9.217), TM4SF3 (p=0.011, OR=4.385), and OLFM4 (p=0.015, OR=3.438). These results suggest that S100B, TM4SF3, and OLFM4 overexpression may affect metastatic behavior of tumor cells in Taiwanese CRC patients.

EVI2B, ATP2A2, S100B, TM4SF3, and OLFM4 as potential prognostic markers for postoperative Taiwanese colorectal cancer patients.

Undetected micrometastasis may play a key role in the early relapse of colorectal cancer (CRC) patients. The aim of this study was to detect circulating tumor cells (CTCs) for predicting early relapse of CRC patients by a weighted enzymatic chip array (WEnCA) and analyze 15 candidate genes associated with CRC carcinogenesis. The genes of 105 postoperative CRC patients were analyzed by membrane array and direct sequencing. We constructed a WEnCA platform including five prognosis-related genes and analyzed the detection rate of WEnCA for CTCs in 30 clinically confirmed CRC relapse patients. Postoperative relapse was significantly correlated with gene overexpression, including EVI2B (p=0.001, OR=4.622), ATP2A2 (p=0.006, OR=4.688), S100B (p=0.001, OR=11.521), TM4SF3 (p=0.001, OR=6.756), and OLFM4 (p=0.008, OR=3.545). Using WEnCA (weighting score of each gene: 5 to EVI2B, 5 to ATP2A2, 12 to S100B, 7 to TM4SF3, and 4 to OLFM4), we could detect CTCs presenting these genotypes in relapsed CRC patients. The sensitivity, specificity, and accuracy were 94.7%, 93.5%, and 97%, respectively. The results of the present study suggest that EVI2B, ATP2A2, S100B, TM4SF3, and OLFM4 could be potential prognostic markers for CRC patients.

CDC25A, VAV1, TP73, BRCA1 and ZAP70 gene overexpression correlates with radiation response in colorectal cancer.

Radiotherapy is increasingly used in adjuvant approaches for colorectal cancer (CRC) to reduce local recurrence and improve survival. However, the principal limitation is the large variability in response among different individuals due to tumor heterogeneity. In the present study, we compared gene expression profiles between radiosensitive and radioresistant colorectal cancer cell lines to identify radiation-related molecules that can be used to evaluate the effects of radiation. The CRC cell line SW620 was irradiated with a high-energy photo beam. Following radiation treatment, RNA was extracted from non-irradiated and irradiated cells, respectively, and gene expression analysis was performed by oligonucleotide microarray and the DAVID bioinformatics method. To further confirm the results, an additional 4 CRC cell lines, COLO205, T84, HCT116, SW480 and SW403 were purchased from ATCC. The radiosensitivities of each were determined by the survival fraction at 2 Gray (SF2) of the surviving cells using the ATPLite assay, and the gene expression profiles after irradiation among the radiosensitive and radioresistant cell lines were analyzed by membrane arrays. The relationships between gene expression and patient clinicopathological features were also analyzed using membrane arrays and RT-PCR. The results from oligonucleotide microarray analysis show that 1601 genes were up-regulated (gene expression ratio of post- to pre-radiation treatment>2). By bioinformatic database analysis, 30 up-regulated genes were identified as involved in DNA damage response pathways, immune response pathways and the complement and coagulation cascades pathway. Fifteen genes showed differential gene expression profiles between radiosensitive (HCT116 and SW620) and radioresistant CRC cell lines (SW403 and SW480). In 110 CRC tissues, we detected five genes CDC25A, VAV1, TP73, BRCA1 and ZAP70 from 15 overexpressed genes that significantly related to prognostic factors (tumor size, advanced stage, invasive depth, lymph node metastasis and differentiation). These findings suggest that CDC25A, VAV1, TP73, BRCA1 and ZAP70 may be novel markers for predicting the effectiveness of radiotherapy in 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.

Simultaneous detection of multiple single-nucleotide polymorphisms by a simple membrane chip.

Technologies that screen multiple single-nucleotide polymorphisms (SNPs) could be very valuable in predicting patients' susceptibilities to diseases or responses to therapeutic interventions. In this study, we developed a chip that can accurately detect four SNPs at same time. This chip is cost-effective and user-friendly because it uses a detection protocol analogous to dot blotting and does not require sophisticated instruments. To establish this chip, we designed and blotted onto a nylon membrane SNP-specific oligonucleotide probes for human angiotensinogen, cholesteryl ester transfer protein, and apolipoprotein E. This chip detected the corresponding SNPs harbored within the angiotensinogen, cholesteryl ester transfer protein, and apolipoprotein E sequences from 20 donors. Importantly, the SNPs detected by our chip matched exactly with the direct sequencing results, thereby highlighting the accuracy of this chip. In conclusion, our chip is a robust tool for multiple SNP screening and holds the potential to future refinement in detecting diseases-associating genes in patients.

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.

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.

Endocytosis of PEGylated agents enhances cancer imaging and anticancer efficacy.

PEGylated nanoparticles and macromolecules are increasingly used in cancer imaging and anticancer treatment. The role of receptor-mediated endocytosis in the efficacy of these agents, however, has not been clearly defined. Here, we developed a matched pair of endocytic and nonendocytic receptors to directly and unambiguously assess this issue. The ligand-binding domains of the low-density lipoprotein receptor (LDLR) or a truncated LDLR lacking the NPXY endocytosis motif (DeltaLDLR) were replaced with an anti-polyethylene glycol antibody (alphaPEG) to form endocytic alphaPEG-LDLR and nonendocytic alphaPEG-DeltaLDLR receptors. The receptors were stably expressed at similar levels on the surface of HCC36 cells. HCC36/alphaPEG-LDLR cells, but not HCC36/alphaPEG-DeltaLDLR cells, rapidly endocytosed PEG-quantum dots and PEG-liposomal doxorubicin (Lipo-Dox) in vitro and in vivo. Lipo-Dox was significantly more cytotoxic to HCC36/alphaPEG-LDLR cells than to HCC36/alphaPEG-DeltaLDLR cells. HCC36/alphaPEG-LDLR tumors also accumulated significantly more PEGylated near-IR probes (PEG-NIR797) and PEG-liposomal-(111)In than HCC36/alphaPEG-DeltaLDLR tumors in vivo. Furthermore, Lipo-Dox more significantly suppressed the growth of established HCC36/alphaPEG-LDLR tumors as compared with HCC36/alphaPEG-DeltaLDLR tumors. Our data show that endocytosis of PEGylated probes and drugs enhances both cancer imaging and anticancer efficacy, indicating that endocytic receptors are superior targets for the design of cancer imaging probes and immunoliposomal drugs.

Overexpression of EGFR pathway-related genes in the circulation is highly correlated with EGFR mutations and overexpression in paired cancer tissue from patients with non-small cell lung cancer.

Epidermal growth factor receptor (EGFR)-directed tyrosine kinase inhibitors (TKIs) have been established as a treatment option in patients with advanced non-small cell lung cancer (NSCLC). Clinically, PCR and RFLP are commonly used to evaluate the efficacy of TKIs, and these methods require cancer tissues to proceed. In the event a peripheral blood test is able to replace current evaluation methods, a greater clinical application advantage may be achieved. Therefore, in this study, we selected 30 EGFR pathway-related genes and constructed activated EGFR chips to identify overexpression of EGFR pathway-related genes from the peripheral blood of 72 NSCLC patients and 100 normal subjects. According to ROC curve analysis, the best chip interpretation cutoff value was 11 genes. Correlation analysis showed high significance among EGFR mutations, overexpression and the overexpression of EGFR pathway-related genes (p<0.0001). The potential application of this new technique may provide an accurate, instantaneous and convenient drug evaluation tool.

Common polymorphisms of the peroxisome proliferator-activated receptor-gamma (Pro12Ala) and peroxisome proliferator-activated receptor-gamma coactivator-1 (Gly482Ser) and the response to pioglitazone in Chinese patients with type 2 diabetes mellitus.

We investigated the effects of the common polymorphisms in the peroxisome proliferator-activated receptor-gamma (PPAR-gamma; Pro12Ala) and in PPAR-gamma coactivator-1(PGC-1; Gly482Ser) genes on the response to pioglitazone in Chinese with type 2 diabetes mellitus. A total of 250 patients with type 2 diabetes mellitus were treated with pioglitazone (30 mg/d) for 24 weeks without a change in previous medications. All patients were genotyped for the PPAR-gamma Pro12Ala and PGC-1 Gly482Ser polymorphisms. The Ala12Ala and Pro12Ala genotypes (26.0% vs 13.5%, P = .025) and Ala allele (15.6% vs 7.3%, P = .008) were significantly more frequent in pioglitazone responders than in nonresponders. The distribution of PGC-1 genotypes and alleles was not significantly different between responders and nonresponders. The decrease in fasting glucose (50.4 +/- 52.2 vs 43.3 +/- 51.7 mg/dL, P < .001) and hemoglobin A(1c) (0.57% +/- 1.44% vs 0.35% +/- 1.10%, P = .004) levels was significantly greater in subjects with the Ala12 carriers (Pro12Ala and Ala12Ala) than in those without the allele (Pro12Pro). Baseline fasting glucose and triglyceride levels were related to the response of pioglitazone. Only the PPAR-gamma Pro12Ala polymorphism was found to be associated with the response of pioglitazone by multiple logistic regression analysis. The PPAR-gamma Pro12Ala gene polymorphism is associated with the response to pioglitazone in Chinese patients with type 2 diabetes mellitus. These findings may be helpful for targeted treatment of diabetes by identifying patients who are likely to respond to pioglitazone.

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.

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.

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.

Association of the functional A118G polymorphism of OPRM1 in diabetic patients with foot ulcer pain.

BACKGROUND: Diabetic foot ulcer (DFU) patients may experience moderate or severe pain. A single-nucleotide polymorphism, at nucleotide 118 for opioid receptor mu 1 (OPRM1), has been reported to alter the opioid effects to relieve acute or chronic pain. The purpose of this study was to elucidate the correlation between nucleotide 118 variants and foot ulcer pain in DFU patients. METHODS: Sixty-five DFU patients with Grade 2-5 Wagner-Meggitt classification were enrolled. The occurrence of pain in activities was categorized into five grades. Patients were allocated either into the painless DFU group, with a visual analog scale (VAS) pain score or=4 and Grades 3-5 of occurrence of pain in daily activities. DNA was extracted from blood samples of analyzed patients. Using the polymerase chain reaction-single-strand conformation polymorphism analysis and DNA sequencing of nucleotide 118, we identified the genotype distribution and allelic frequencies in DFU patients. The sequences of the forward and the reverse primer are designed as follows: 5'-TAATACGACTCACTATAGGG-3' and 5'-ACGCACACGATGGAGTAGAG-3', respectively. RESULTS: Fifteen patients were classified into the painful DFU group and 50 patients were classified into the painless DFU group. The amplified DNA fragments showed 26 homozygous (AA), 34 heterozygous (AG), and 5 mutant homozygous (GG) genotypes, with overall A and G allelic frequencies of 66.2% and 33.8%, respectively. The painful DFU group included 10 AA subjects, 4 AG subjects, and 1 GG subject, while the painless DFU group had 16 AA, 30 AG, and 4 GG subjects (P=.038). CONCLUSION: The A118G polymorphism of mu-opioid receptor may be closely associated with DFU pain in 34 out of 50 patients in the painless group and in 5 out of 15 patients in the painful group. This indicates that the nucleotide 118 variant patients may suffer less DFU pain.

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.