Comparison of EGFR signaling pathway somatic DNA mutations derived from peripheral blood and corresponding tumor tissue of patients with advanced non-small-cell lung cancer using liquidchip technology.

Somatic DNA mutations affecting the epidermal growth factor receptor (EGFR) signaling pathway are known to predict responsiveness to EGFR-tyrosine kinase inhibitor drugs in patients with advanced non-small-cell lung cancers. We evaluated a sensitive liquidchip platform for detecting EGFR, KRAS (alias Ki-ras), proto-oncogene B-Raf, and phosphatidylinositol 3-kinase CA mutations in plasma samples, which were highly correlated with matched tumor tissues from 86 patients with advanced non-small-cell lung cancers. Either EGFR exon 19 or 21 mutations were detected in 36 patients: 23 of whom had identical mutations in both their blood and tissue samples; whereas mutations in the remaining 13 were found only in their tumor samples. These EGFR mutations occurred at a significantly higher frequency in females, never-smokers, and in patients with adenocarcinomas (P ≤ 0.001). The EGFR exon 20 T790M mutation was detected in only one of the paired samples [100% (95% CI, 96% to 100%) agreement]. For KRAS, proto-oncogene B-Raf, and phosphatidylinositol 3-kinase CA mutations, the overall agreements were 97% (95% CI, 90% to 99%), 98% (95% CI, 92% to 99%), and 97% (95% CI, 90% to 99%), respectively, and these were not associated with age, sex, smoking history, or histopathologic type. In conclusion, mutations detected in plasma correlated strongly with mutation profiles in each respective tumor sample, suggesting that this liquidchip platform may offer a rapid and noninvasive method for predicting tumor responsiveness to EGFR-tyrosine kinase inhibitor drugs in patients with advanced non-small-cell lung cancers.

Detection of EGFR somatic mutations in non-small cell lung cancer (NSCLC) using a novel mutant-enriched liquidchip (MEL) technology.

We have developed and standardized a novel technology, mutant-enriched liquidchip (MEL), for clinical detection of EGFR mutations. The MEL integrates a mutant-enriched PCR procedure with liquidchip technology for detections of EGFR exon 19 deletions and L858R mutation on both formalin-fixed and paraffin-embedded (FFPE) slides and plasma samples from patients with non-small cell lung cancer (NSCLC). The detection sensitivity was 0.1% of mutant DNA in the presence of its wild-type DNA. The cross-reaction rate was lower than 5%. To evaluate the MEL platform, the EGFR mutation status of 59 patients with advanced NSCLC treated with EGFRTKIs (Tyrosine Kinase Inhibitors) were tested on their FFPE samples. EGFR exon 19 deletions and L858R were detected in 21 patients (21/59) and 76.2% (16/21) of them had partial response to the EGFR-TKIs, while by sequencing method, only 4 (4/59) mutations were detected. Plasma samples from 627 patients with various stages of NSCLC were examined with the MEL and 22% of EGFR exon 19 deletions and L858R were detected. Furthermore, in patients with advanced disease there are more mutations detected in plasma samples than in patients with less advanced disease. In conclusion, the MEL is a sensitive, stable, and robust technology for detecting EGFR DNA mutations from both FFPE and plasma samples from patients with NSCLC and is now routinely used for clinical diagnosis.

Somatic mutation analysis of EGFR, KRAS, BRAF and PIK3CA in 861 patients with non-small cell lung cancer.

The prevalence of EGFR, KRAS, BRAF and PIK3CA somatic mutations in 861 randomly selected Chinese patients with non-small cell lung cancer (NSCLC) was assayed by the SurPlex®-xTAG70plex platform and analyzed. The results showed that the occurrence rates were 41.0, 8.0, 0.7 and 3.7%, respectively. The mutation rates significantly correlated with gender, histology and smoking history. The EGFR exon 19, 20 and 21 mutations were higher in females compared to males (p< 0.001, exon 19 and 21; p=0.018, exon 20), higher in adenocarcinomas compared to other forms of lung cancers (p< 0.001, exon 19 and 21; p=0.035, exon 20), and higher in non-smokers compared to smokers (p< 0.001, exon 19 and 21; p=0.029, exon 20). Conversely, the KRAS mutations were higher in males compared to females (p=0.004), higher in adenocarcinomas compared to other forms of lung cancers (p< 0.001), and higher in smokers compared to non-smokers (p< 0.001). The PIK3CA mutation rate was lower in adenocarcinomas compared to other forms of lung cancers (p=0.003).

A novel liquidchip platform for simultaneous detection of 70 alleles of DNA somatic mutations on EGFR, KRAS, BRAF and PIK3CA from formalin-fixed and paraffin-embedded slides containing tumor tissue.

BACKGROUND: DNA somatic mutations of EGFR, KRAS, BRAF and PIK3CA in the epidermal growth factor receptor (EGFR) signaling pathway play critical roles in the response or resistance of tumors to targeted therapy with tyrosine kinase inhibitors (EGFR-TKIs). To provide a high-throughput (HTP) clinical testing service for detecting these mutations, we developed a novel platform, SurPlex®-xTAG70plex-EGFR liquidchip. METHODS: This platform was developed based on a universal 100-tag system. The procedures for multiplex PCR, allele specific primer extension (ASPE) and hybridization were optimized and standardized. RESULTS: A total of 70 alleles of somatic mutations of EGFR, KRAS, BRAF and PIK3CA can be detected simultaneously in one reaction from one formalin-fixed and paraffin-embedded (FFPE) slide within one day. Cross-reaction was < 8% between individual amplimers and 70 different ASPE primers. The sensitivity for detecting mutants in the wild-type DNA was 1%-5%. Seventy-three FFPE samples with somatic mutations were used to validate the 70plex. Seventy-one showed a complete match, while two were not detected. CONCLUSIONS: A simple, accurate, sensitive HTP technology was developed and standardized for detecting simultaneously 70 different alleles of EGFR, KRAS, BRAF and PIK3CA gene mutations from FFPE tumor slides.

A novel mutant-enriched liquidchip technology for the qualitative detection of somatic mutations in KRAS gene from both serum and tissue samples.

BACKGROUND: Somatic mutations in the KRAS gene have been reported to confer drug resistance to epidermal growth factor receptor tyrosine kinase inhibitors and some monoclonal antibodies. However, current DNA mutation detection technologies are primarily DNA sequencing-based and not high throughput, nor sensitive enough to meet clinical needs. METHODS: A mutant-enriched PCR method was designed by introducing a unique restriction enzyme site to the PCR product. This allowed the wild-type KRAS sequence to be selectively removed by restriction enzyme digestion before application to the Luminex liquidchip system. RESULTS: A total of 100 copies of mutant KRAS DNA fragment mixed with 1x10(5) copies of the wild-type KRAS DNA could be detected to achieve a sensitivity of 0.1%. This technology is currently used for clinical testing of KRAS somatic mutations for the purpose of pharmacogenomic evaluation. Serum samples from 109 patients with non-small cell lung cancer were tested and 34 mutations were detected (34/109). The formalin-fixed and paraffin-embedded samples from 60 patients with colorectal cancer were tested and 19 mutations were detected (19/60). CONCLUSIONS: A novel, qualitative, sensitive, reliable and high throughput liquidchip technology has been developed for detecting KRAS mutations using clinical serum and formalin-fixed and paraffin-embedded samples.

EpCAM expression in retinoblastoma: a novel molecular target for therapy.

PURPOSE: This study was conducted to investigate the potential of targeting epithelial cell adhesion molecules (EpCAMs) in the treatment of retinoblastoma. It was first determined whether EpCAM is expressed in retinoblastoma and then whether EpCAM reactivity correlates with tumor aggressiveness. METHODS: EpCAM reactivity was evaluated by immunohistochemistry in 43 retinoblastoma specimens from 43 patients, by using the monoclonal antibody GA733.2. The tumors were divided into two groups. There were 20 tumors with no invasion of the choroid and optic nerve (group A) and 23 tumors with invasion of the choroid, optic nerve, and orbit (group B). EpCAM reactivity was correlated with invasion and differentiation of the tumors. RESULTS: Among the 43 tumors, EpCAM reactivity was observed in 100% (43/43) tumors. EpCAM reactivity was significantly higher in the invasive than the noninvasive tumors (P < 0.05) and in poorly differentiated than in well-differentiated tumors (P < 0.005). Non-neoplastic retina also expressed EpCAM. CONCLUSIONS: The results confirm that EpCAM is vastly expressed in retinoblastoma and point to its use as a target for therapy in the future.

Redirected T-cell cytotoxicity to epithelial cell adhesion molecule-overexpressing adenocarcinomas by a novel recombinant antibody, E3Bi, in vitro and in an animal model.

BACKGROUND: To redirect cytotoxic T cells to target a broad range of adenocarcinomas, the authors constructed a novel, recombinant, bispecific antibody, E3Bi, directed at the tumor-associated antigen, epithelial cell adhesion molecule (EpCAM), and the CD3 receptor on T cells. METHODS: T cells were prepared from healthy blood donors. The cytotoxicity of activated T cells (ATC) redirected to tumor cells by E3Bi was measured with in vitro (51)Cr release assays. In vivo studies were performed in a severe combined immunodeficient (SCID)/Beige mouse xenograft model. Tumor-bearing mice were treated with low doses (1 mg/kg) or high doses (10 mg/kg) of E3Bi along with ATC (2 x 10(9) cells/kg), and treatment efficacy was evaluated both by ex vivo tumor cell survival assay after in vivo treatments and by in vivo tumor growth delay studies. RESULTS: In vitro, targeting the EpCAM-overexpressing human tumor cell lines with E3Bi increased specific cytotoxicity of ATC by > 70% at an effector-to-target ratio of 2.5 (P < 0.001); this cytotoxicity was abolished competitively in the presence of an anti-EpCAM monoclonal antibody. In contrast, E3Bi did not enhance ATC cytotoxicity toward the low EpCAM-expressing tumor cell line. In ex vivo tumor cytotoxicity assays, a significant reduction in tumor cell survival (40% with low-dose E3Bi; 90% with high-dose E3Bi) was observed in E3Bi/ATC-treated mice compared with control mice that were treated with ATC only. In addition, SCID/Beige mice xenografted with LS174T tumors demonstrated a significant tumor growth delay (P = 0.0139) after receiving E3Bi/ATC/interleukin 2 (IL-2) compared with mice that received ATC/IL-2 alone. CONCLUSIONS: E3Bi specifically and very efficiently redirected T cells to destroy EpCAM-overexpressing tumors both in vitro and in an animal model. These results suggest a therapeutic utility for E3Bi in the treatment of adenocarcinomas.

Comparison of the TCR zeta-chain with the FcR gamma-chain in chimeric TCR constructs for T cell activation and apoptosis.

A promising strategy for cancer treatment is adoptive gene therapy/immunotherapy by genetically modifying T cells with a chimeric T cell receptor (cTCR). When transduced T cells (T-bodies) specifically bind to tumor antigens through cTCR, they will become cytotoxic T lymphocytes (CTL) and lyse the tumor cells in a non-major histocompatibility complex (MHC)-restricted manner. Both the FcR gamma-chain and the TCR zeta-chain have been used to construct such cTCR, and both have shown specific cytolytic functions against tumor cells. However, most researchers believe that the zeta-chain generates stronger cytolytic activities against tumor than the gamma-chain and therefore would be a better candidate for cTCR construction. On the other hand, because of the lack of costimulation signaling in such constructs, the T-body might cause activation-induced T cell death (AICD) when bound to tumor antigens. Therefore, one can argue that the gamma-chain might generate less AICD than the zeta-chain because the gamma-chain has only one immunoreceptor tyrosine-based activation motif (ITAM), and the cytolytic activities can be therefore recycled. Two cTCR, GAHgamma and GAHzeta, were constructed and evaluated for cytokine production, specific cytolytic function and AICD in T-bodies after exposure to tumor cells. Using EGP-2-positive LS174T colorectal carcinoma cells as targets, there was no substantial difference observed between a gamma-chain or zeta-chain as the T-body signaling moiety in terms of specific cytolytic functions and induced cytokine production. This paper also demonstrates that, in the absence of a costimulation system, tumor antigen may not trigger apoptosis of T cells transduced with a cTCR carrying either an FcR gamma-chain or a TCR zeta-chain. These observations challenge current ideas about the role of ITAM in T cell activation.