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.

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.

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.

Evaluation of perforated and nonperforated appendicitis with CT.

Fifty-three patients with 38 cases of perforated appendicitis and 15 cases of appendicitis without perforation were evaluated based on the computed tomography (CT) appearances of appendiceal diameter, phlegmon, abscess, extraluminal air, appendiceal wall enhancement, lateroconal fascial thickening, appendicolith, bowel wall thickening, ascites, ileal wall enhancement, peritoneal enhancement, periappendiceal fluid, omental haziness, retrocecal appendix, intraluminal air, and the combination of intraluminal air and appendicolith. The result of appendiceal diameter was compared using two-sample Student's t test, and the other CT findings were analyzed by Fisher's Exact Test. Our results showed that appendix was larger in caliber in perforated appendix (P< .05). Direct CT signs (i.e., phlegmon, abscess, and extraluminal air) were more specific for perforated appendicitis (P< .05). Indirect signs (bowel wall thickening, ascites, ileal wall enhancement, intraluminal air, and combined intraluminal air and appendicolith) were also found in higher incidence in appendiceal perforation (P< .05). Appendiceal enlargement and ileal wall enhancement were the two predominant findings in one case of perforation. We concluded that direct and indirect CT appearances can differentiate appendicitis with and without perforation. Indirect signs may be helpful in difficult case.

CT evaluation of gastrointestinal tract perforation.

The purpose of this study is to review the computed tomography (CT) appearance of gastrointestinal tract (GI) perforation. Forty-two patients with 10 cases of proximal GI perforation and 32 cases of distal GI perforation were evaluated based on the CT findings of extraluminal air (which was subdivided into the CT-falciform ligament sign crossing the midline and scattered pockets of air), bowel wall thickening (>8 mm in gastroduodenal wall, >3 mm in the small bowel wall, >6 mm in the caliber of the appendix and >5 mm in the colonic wall), associated abscess formation, ascites and adjacent fat stranding. The results were compared using Fisher's Exact Test. Detection of extraluminal air in the upright plain films and CT was analyzed by Z test. Our results showed that CT-falciform ligament sign was more frequent in the proximal GI perforation, while pockets of extraluminal air (excluding the cases accompanying CT-falciform ligament sign), bowel wall thickening and fat stranding were found in higher incidence in distal GI perforation (P<.05). CT detected extraluminal air in more cases than the upright plain films did (69% vs. 19%; Z=4.62>Z(0.01)=2.326). We concluded that CT is a good imaging tool to differentiate the various GI perforations.