Liquid Chip Technology Research Articles

PIK3CA mutations are a predictor of docetaxel plus epirubicin neoadjuvant chemotherapy clinical efficacy in breast cancer.

This study proposed to investigate the relationship of PIK3CA somatic mutations, the most common activating mutations in human breast cancer (BC), and the efficacy of neoadjuvant chemotherapy (NCT).Using a novel liquid chip technology,PIK3CA DNA somatic mutations and HER2, PTEN, EGFR mRNA expression profiles were analyzed in formalin fixed paraffin embedded samples of 93 BC patients treated with epirubicin plus docetaxel NCT.PIK3CA mutations were found in 30 patients (32.3%), in which the point mutations of E542K, E545K, H1047L and H1047R were 4.3, 9.7, 4.3 and 14.0%respectively. The PIK3CA mutations were significantly associated with patients' clinical response; 27 of 30 PIK3CA mutated patients had either a partial or complete response (p=0.002). Multivariate analysis further confirmed that, after adjustments for age, disease stages and NCT cycles, PIK3CA was associated with clinical response (Odds Ration 0.126, 95% CI [0.029,0.691]). However, there was no significant difference between PIK3CA mutations in pathological complete response (pCR,7 /92) and non-pCR group. Furthermore, no EGFR, KRAS and BRAF mutations were detected in any of the 93 samples. Our data for the first time suggested that PIK3CA mutation status may be a predictor for better understanding clinical response to the combination of epirubicin and docetaxel NCT in patients with BC.

Identifying EGFR mutations from SCLC patient plasma by mutant-enriched liquidchip technology.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) such as erlotinib and gefitinib are targeted drugs for the kinase domain of EGFR. They are widely used for the treatment of non-small cell lung cancer (NSCLC). The EGFR exon 19 deletion mutation and the L858R mutation in exon 21 comprise approximately 90% of the somatic mutations in NSCLC patients that respond to EGFR TKI. Several recent studies have also reported that small cell lung cancer (SCLC) patients with EGFR mutations responded to gefitinib. Further study, however, has been limited due to the difficulty obtaining tumor specimens from SCLC patients. The aim of this study was to explore the EGFR mutation status in SCLC patients by plasma analysis. Plasma samples from SCLC patients were collected for mutant-enriched liquidchip (MEL) analysis to identify the EGFR mutations in exon 19 and 21. The exon 19 deletion mutation was detected in one out of 35 patients (a female non-smoker). No exon 21 mutations were found. A prevalence of EGFR mutations in SCLC is rare, and occurs most frequently in females and nonsmokers.

Bone marrow angiogenesis in patients presenting with differential Chinese medicine syndrome: Correlation with the clinico-pathological features of aplastic anemia

To explore differences in bone marrow angiogenesis seen in aplastic anemia (AA) patients presenting with differential Chinese medicine (CM) syndrome, and to correlate these differences with clinical pathology. Thirty-five patients were enrolled, including 18 with "yang deficiency syndrome" and 17 with "yin deficiency syndrome." Bone marrow biopsies and serum were collected. Microvessel density (MVD) and positive expression of vascular endothelial-derived growth factor (VEGF) were detected by immunohistochemisty. Hypoxia inducible factor -1α (HIF-1α), and VEGF expression were assayed by enzyme-linked immunoabsorbent assay (ELISA), serum lactate dehydrogenase (LDH) was tested by enzyme method and liquid chip technology was used to detected the expression of interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Counts for leukocytes, absolute neutrophils and platelets in "yin deficiency syndrome" were lower than those found in "yang deficiency syndrome" (P<0.05). MVD and VEGF expression, and the positive rate of CD34 and VEGF in bone marrow were lower in AA, especially in "yin deficiency syndrome" (P<0.01 or P<0.05). "Yin deficiency syndrome" displayed decreased VEGF and LDH expression, and enhanced expression of HIF-1α as compared to "yang deficiency syndrome" (P<0.05). Levels of IL-4 and IL-6 were higher in AA (P<0.01), but IL-10 was decreased (P<0.05). High TNF-α expression was seen in "yang deficiency syndrome" and IFN-γ expression was decreased in "yin deficiency syndrome" as compared with normals (P <0.01 and P<0.05, respectively). AA patients have lower MVD than normals, especially in "yin deficiency syndrome." MVD might differentially correlate to disease severity, and could be dependent on bone marrow or serum VEGF expression and LDH. Additionally, IL-2, IL-10, IL-4 and IFN-γ were negatively associated while IL-6 and TNF-α were positively associated with MVD.

Frequency, characterization, and prognostic analysis of PIK3CA gene mutations in Chinese esophageal squamous cell carcinoma

PIK3CA gene mutations are found in numerous cancers but correlate differently with prognosis. Although the frequency of PIK3CA gene mutation in esophageal squamous cell carcinoma (ESCC) has been previously studied, a prognostic analysis has not been reported. Ninety-six surgically resected ESCC tissues were collected from Chinese patients and DNA was extracted. Gene mutations in PIK3CA (exons 9 and 20), EGFR (exons 18, 19, 20 and 21), KRAS (exons 2 and 3), and BRAF (exons 11 and 15) were screened using mutant-enriched liquid chip technology. PIK3CA gene mutations were identified in 12 of 96 ESCC cases (12.5%). No mutations were identified in EGFR, KRAS or BRAF genes in this study. Correlations between clinicopathological features and PIK3CA mutation status were analyzed and finally, patient survival information was used to determine the prognostic significance of PIK3CA mutation. Interestingly, the frequency of PIK3CA mutation was higher in female ESCC patients (31.3%, 5/16) than in males (8.8%, 7/80), and higher in patients with non-lymph node metastasis (19.6%, 10/51, P = .013) than in patients with lymph node metastasis (4.4%, 2/45, P = .025). Furthermore, patients with PIK3CA-mutated tumors showed a trend towards favorable overall survival (P = .085) but not disease-free survival (P = .238), suggesting that PIK3CA gene status may be a favorable predictive marker in ESCC patients.

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.

The preliminary establishment and application of a new method for evaluating K-ras mutations based fluid chip

Objective A new method for detecting K-ras mutations based liquid chip was used to evaluate K-ras mutations associated with non-small cell lung cancer (NSCLC) patients,to direct the personalized treatment and prognosis evaluation. Methods Take the diagnosis technology research methods,the sensitivity and repeatability of the liquid chip K-ras gene mutation detection method were assessed.A total of 100 NSCLC patients from Nov 2011 to Feb 2012 in Shanghai Chest hospital were included in this study,the fresh tumor tissues were collected for DNA extraction.The 2nd exon 12 and 13 codons,containing 8 K-ras mutations occuring in high frequency were amplified by polymerase chain reaction (PCR),followed by ligation of the PCR products to a series of special probes using ligase detection reaction (LDR),then the PCR-LDR products were analyzed by liquid chip platform.Direct sequencing was applied to compare with the detection results. Results The sensitivity of liquid chip technology detection was 10%-20%,higher than the traditional sequencing method by 1%.Average CV value was 4%-15% and showed good repeatability.5 K-ras mutations in 100 patients (5%) were detected using multiplex PCR-LDR combined fluid chip methods,including 3 Gly12Val and 2 Gly12 Asp mutations in exon 2.The 5 K-ras mutations were verified accurately by direct sequencing. Conclusions The novel detection method of K-ras mutations based PCR-LDR and fluid chip shows high throughput,high sensitivity,good repeatability and the results are reliable and accurate.This method can be used to accurately identified K-ras mutations for NSCLC patients prior to their targeted therapy with TKIs.(Chin J Lab Med,2013,36:704-707) Key words: Microfluidic analytical techniques; Polymerase chain reaction; Ligase chain reaction; Genes; ras; Genotype; Carcinoma; non-small-cell lung

Exploratory study on the correlation between 14 lung cancer-related gene expression and specific clinical characteristics of NSCLC patients

Personalized medicine has become essential in the treatment of lung cancer. However, the lung cancer-related gene expression profiles in non-small cell lung cancer (NSCLC) patients have not been elucidated. In this study, the correlation between gene expression profiles and clinicopathological characteristics was investigated in NSCLC patients. A total of 95 patients were enrolled in this study. The mRNA expression levels of 14 genes were assessed by multiplex branched DNA liquidchip (MBL) technology and data on 9 clinicopathological characteristics of patients were collected simultaneously. The correlation between gene expression and clinicopathological characteristics was investigated. Out of the 9 clinicopathological parameters, 6 were associated with several of the 14 genes analyzed. Patient gender was associated with TYMS and TOP2A. Clinical stage was associated with VEGFR2, KIT and HER2. There was weak correlation between primary tumor size of ≤3 cm and the expression level of KIT. The mRNA expression levels of VEGFR2 and HER2 correlated with distant metastasis. BRCA1, TYMS, TOP2A and HER2 were associated with histological type. Smoking correlated with higher expression levels of BRCA1, TYMS and TOP2A and lower expression levels of PDGFRβ. The results were suggestive of correlation between the clinicopathological parameters of the NSCLC patients and the mRNA expression levels of certain lung cancer-related genes, including BRCA1, TYMS, TOP2A, PDGFRβ, VEGFR2, KIT and HER2.

Effect of TUBB3, TS and ERCC1 mRNA expression on chemoresponse and clinical outcome of advanced gastric cancer by multiplex branched-DNA liquid chip technology

To analyze the impact of β-tubulin-III (TUBB3), thymidylate synthase (TS) and excision repair cross complementation group 1 (ERCC1) mRNA expression on chemoresponse and clinical outcome of patients with advanced gastric cancer treated with TXT/CDDP/FU (DCF) regimen chemotherapy. The study population consisted of 48 patients with advanced gastric cancer. All patients were treated with DCF regimen palliative chemotherapy. The mRNA expressions of TUBB3, TS and ERCC1 of primary tumors were examined by multiplex branched-DNA liquid chip technology. The patients with low TUBB3 mRNA expression had higher response rate to chemotherapy than patients with high TUBB3 expression (P=0.011). There were no significant differences between response rate and TS or ERCC1 expression pattern. Median overall survival (OS) and median time to progression (TTP) were significantly longer in patients with low TUBB3 mRNA expression (P=0.002, P<0.001). TS or ERCC1 expression was not correlated with TTP and OS. In the combined analysis including TUBB3, TS and ERCC1, the patients with 0 or 1 high expression gene had better response rate, TTP and OS than the remaining patients (all P<0.001). Multivariate analysis revealed that ECOG (Eastern Cooperative Oncology Group)≥2 (HR=2.42, P=0.009) and TUBB3 (HR=2.34, P=0.036) mRNA expression significantly impacted on OS. High TUBB3 mRNA expression is correlated with resistance to DCF regimen chemotherapy. TUBB3 might be a predictive and prognostic factor in patients with advanced gastric cancer treated with TXT-based chemotherapy. The combined evaluation of TUBB3, TS and ERCC1 expression can promote the individual treatment in advanced gastric cancer.

Establishment of a novel mutant-enriched liquid chip technology for detecting EGFR mutations in plasma of patients with non-small cell lung cancer

Objective To establish a sensitive, specific, simple and high-throughput method for detection of the epidermal growth factor receptor (EGFR) mutation in the plasma samples of patients with non-small cell lung cancer (NSCLS) by the use of mutant-enriched liquid chip (MEL) assay. Methods The specific probes for the EGFR exon19 E746-750 deletion, exon 21 L858R mutation and wild-type sequence were designed and coupled to the microspheres coding with different fluorescent dye.The probe coupling efficiency was verified by crossing hybridization test with biotin-labeled reverse sequence.A blood-based MEL approach which integrates a sensitive mutant-enriched PCR and quantitative high throughput liquid chip assay for assessment of EGFR mutations was developed.The sensitivity and specificity of MEL was further evaluated using the mixture with different copy numbers of mutant and wild-type plasmids as template.The mutations of exon 19 and 21 of EGFR gene in plasma samples from 201 patients with stage ⅢB or Ⅳ NSCLC who enrolled in the First Affiliated Hospital of Guangzhou Medical College from September 2008 to April 2010 were analyzed by both the MEL and the mutant-enriched PCR assay.The result comparison was made between direct sequencing and MEL in 50 cases whose EGFR gene type had been tested by MEL and mutant-enriched PCR.The correlation of EGFR gene mutation and the response to the Geftinib treatment was analyzed in 16 patients with lung adenocarcinoma as well. Results The probes were successfully coupled to the microspheres encoding with different fluorescent dye, and could be specifically recognized by the corresponding target sequence.The MEL was capable of detecting as few as 10 copies of EGFR mutants (sensitivity was 0.1%).Among the enrolled 201 cases of advanced NSCLC, the detection rate of the EGFR exon19 E746-750 del and exon 21 L858R was 55.7% (112/201) by MEL assay.Compared with mutant-enriched PCR［58.2%（117/201）］, the coincidence rate was 97.5%(196/201).There was no statistically significant difference between the results of mutant-enriched PCR and MEL (χ2=3.20，P>0.05).The mutations detection rate was 22.0% (11/50) by directing sequencing, which was significantly lower than by MEL［50.0%(25/50), χ2=12.07，P<0.05］.Among the 16 patients treated with Gefitinib，9 cases who had EGFR mutation showed a higher response rate（P=0.041）and prolonged progression-free survival（χ2=6.76，P=0.009）after the treatment compared to those 7 who without EGFR mutation. Conclusions A new method of MEL with accuracy, specificity, fast and high-throughput is established for the detection of EGFR 19 E746-750 deletion and exon 21 L858R mutations in plasma from advanced NSCLC patients.It has the ability to provide the most direct and valuable guidance for clinicians to make decision on EGFR tyrosine kinase inhibitors therapy in the advanced NSCLC patients.(Chin J Lab Med, 2012,35:986-992) Key words: Carcinoma; non-small-cell lung; Receptor; epidermal growth factor; Mutation; Microchip analytical procedures

PIK3CA as a predictor of the clinical efficacy for epirubicin plus docetaxel neoadjuvant chemotherapy in breast cancer.

113 Background: PIK3CAgene mutations are the most common activating mutations in human breast cancer (BC). Its association with the resistance to herceptin in HER2-positive BC was reported, but whether it’s related to the efficacy of neoadjuvant chemotherapy is unknown. Methods: We reviewed records of 108 patients with BC treated with epirubicin plus docetaxel neoadjuvant chemotherapy between June 2005 and April 2011. 92 patients’ clinical and pathologic objective response data were collected. FFPE tumor biopsies obtained at the time of diagnoses (and before treatment) were collected. EGFR, KRAS, BRAF, PIK3CA mutations and HER2, PTEN, EGFR mRNA expression were analyzed by liquidchip technology. Chi-square test and Fisher’s exact test were used in statistical analysis. A p value less than 0.05 was considered statistically significant. Results: No mutation was detected in EGFR, KRAS and BRAF. The mutation rate of PIK3CA was 35.1% (38/108), and mutation rates of E542K, E545K, H1047L and H1047R were 3.7, 10.2, 3.7 and 17.6% respectively. More PIK3CA mutations were detected in HER2 high expression tumors (p=0.018). No correlation was found between PIK3CA mutations and PTEN, EGFR expression. Clinical response (CR+PR, 63/92) was associated with PIK3CA mutations (p=0.002). However, PIK3CA mutations rate between pathologic complete release (pCR, 7/92) and non-pCR group was not significantly different (p=0.422). Conclusions: PIK3CA mutation may predict better clinical response in BC patients treated with epirubicin plus docetaxel neoadjuvant chemotherapy. More studies are needed to validate these results and the correlation between PIK3CA mutations and pCR.

1530P - Epidermal Growth Factor Receptor Mutation in Small Cell Lung Cancer Patients Detected by Mutant-Enriched Liquidchip Technology from Plasma

ABSTRACT Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) has been widely used in non-small cell lung cancer (NSCLC), and the incidence of EGFR mutation in NSCLC is higher in China than in the United States and European countries. Mutations of EGFR exons 19 and 21 in NSCLC is related to response of tumors to EGFR TKIs, suggesting their usefulness as biomarkers. Some case studies reported a gefitinib-responsive small cell lung cancer (SCLC) with EGFR mutation. However, there are few large studies which reported the mutation status of SCLC patients. It is difficult to obtain tumor tissues to detect EGFR mutations in SCLC patients especially from surgery. The aim of this study was to determine the EGFR mutation status in SCLC patients in China, and evaluate the feasibility of EGFR mutation detection from plasma by mutant-enriched liquidchip (MEL) technology. From September 2011 to March 2012, plasma from 35 cases of SCLC were collected at the Zhejiang Cancer Hospital, Hangzhou, China. There were 7 female, 28 male; age from 46 to 74 years old and median age of 60 years old. The stage (Veterans Administration Lung Study Group, VALSG): limited disease (LD) 8 cases, extensive disease (ED) 27 cases. Smoking history: non-smoker 10 cases, light smoker 0 cases, moderate smoker 3 cases and heavy smoker 22 cases. MEL technology was used to detect EGFR exon 19 and exon 21 mutations from plasma of 35 SCLC patients. One of 35 cases was found with mutation in exon 19 of the EGFR gene. The patient with EGFR exon 19 mutation was a female and non-smoker. EGFR mutation is rare in SCLC patients, and may more easily occur in females and non-smokers. It is feasible to detect EGFR mutation for SCLC from plasma by MEL technology. Disclosure All authors have declared no conflicts of interest.

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.

Detection of EGFR and KRAS somatic mutations in tumor tissue and peripheral blood by a liquidchip technology for patients with advanced non-small cell lung cancer.

e18142 Background: Responsiveness of non-small cell lung cancer (NSCLC) to EGFR tyrosine kinase inhibitors has been confirmed to relate to mutations in the epidermal growth factor receptor (EGFR) signaling pathway genes. This study’s aim is to detect mutations in two important components of the EGFR signaling pathway, EGFR and KRAS, using a novel liquidchip technology, and to evaluate the correspondence between tumor and plasma DNA in the same patient. Methods: 58 advanced primary NSCLC ( IIIB or ‡W) tumor tissues and matched peripheral blood samples (MPBS) were obtained. The procedures for multiplex PCR, allele specific primer extension (ASPE) and hybridization were performed after DNA extraction. A novel liquidchip platform for simultaneous detection multiple alleles of DNA somatic mutations was applied to detect the EGFR and KRAS status in matched samples. Results: EGFR exon 19 and exon 21 mutation rate was detected to be 29.3% (17/58) and 17.2% (10/58) respectively in tumor tissues, while 20.7% (12/58) and 12.1% (7/58) in MPBS. Among those 27 patients who harbor either EGFR exon 19 or exon 21 mutations in tissues, 19 (70.4%) of them were detected to have the same mutations in their peripheral blood samples. EGFR exon 20 T790M mutations were found in one of the paired samples. All the patients who were detected to have wild-type EGFR in tissues were detected to have wild-type in MPBS as well. In 50 of 58 (86.2%) of the paired samples, the EGFR mutation status in the tumors was consistent with those in the MPBS. The KRAS exon 2 mutation was also found in both blood and tumor samples in one patient; however only in the blood of another patient, not in his tumor. Conclusions: The liquidchip technology may serve as an effective method for detecting multiple gene mutations. EGFR and KRAS gene mutations in blood DNA were highly consistent with the corresponding tissue samples, and therefore, peripheral blood could be used as an alternative sample to predict patients’ response to the anti-EGFR targeted therapy. Further studies to evaluate the clinical application are suggested.

Epidermal growth factor receptor mutation in small cell lung cancer patients detected by mutant-enriched liquidchip technology from plasma in China.

e17512 Background: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) has been widely used in non-small cell lung cancer (NSCLC), and the incidence of EGFR mutation in NSCLC is higher in China than in the United States and European countries. EGFR exons 19 and 21 mutation in NSCLC is related to response of tumors to EGFR TKIs, suggesting its usefulness as a biomarker. Some case studies reported that gefitinib-responsive small cell lung cancer (SCLC) with EGFR mutation. However, there are few large studies which reported the mutation status of SCLC patients. It is difficult to obtain tumor tissues to detect EGFR mutation in SCLC patients especially from surgery. This aim of the study is to know the EGFR mutation status in SCLC patients in China, and evaluate the feasibility of EGFR mutation detection from plasma by mutant-enriched liquidchip (MEL) technology. Methods: From September 2011 to January 2012, 27 cases of SCLC plasmas were collected at our Hospital, China. There are 6 female, 21 male. Age from 46 to 74 years old and median age is 60 years old. The stage (Veterans Administration Lung Study Group, VALSG): limited disease (LD) 6 cases, extensive disease (ED) 21 cases. Smoking history: non-smoker 8 cases, light smoker 0 cases, moderate smoker 3 cases and heavy smoker 16 cases. MEL technology was used to detect EGFR exon 19 and exon 21 mutations from plasma of 27 SCLC patients. Results: One of 27 cases was found with mutation in exon 19 of the EGFR gene. The patient with EGFR exon 19 mutation is a female and non-smoker. Conclusions: EGFR mutation is rare in SCLC patients, and may be more easily occurred in female and non-smokers. It is feasible to detect EGFR mutation for SCLC from plasma by MEL technology.

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

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. 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. 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). 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.

RNA expression profiling of ERCC1, BRCA1, TUBB3, and STMN1 in non-small cell lung cancer by a multiplex branched DNA liquidchip technology (MBL) for predicting the efficacy of chemotherapy.

e21020 Background: It is very important to measure these RNA levels before chemotherapy. However, tumor specimens are usually preserved in formalin-fixed and paraffin-embedded (FFPE) slides and the...

Abstract B180: A novel mutant-enriched liquidchip technology (MEL) for detecting circulating EGFR somatic mutations in non small cell lung cancer

Abstract Epidermal growth factor receptor (EGFR) is a key target for developing targeted therapeutics to treat cancer. The US FDA has approved some EGFR targeted drugs that include monoclonal antibody drugs Erbitux, Panitumumab, as well as the EGFR tyrosine kinase inhibitors Iressa and Tarceva. However, clinical data indicated that only a small percentage of patients were responsive to these targeted drugs. Further studies have revealed that up to 20% of non small cell lung cancer (NSCLC) tumors that harbor the EGFR Exon 19 or 21 somatic mutations are more likely responsive, while patients without these mutations are not responsive. Therefore, physicians have a responsibility to test whether or not the patient's EGFR is mutated before prescribing these targeted drugs. In general, a very small amount of free DNA fragments can be found in the blood, especially in patients with malignant tumors. However, it is technically very difficult to detect a very small amount of DNA somatic mutations that are usually masked under a huge amount of the wild-type (WT) sequence. To cater to the clinical need, we have successfully developed a novel technology that creatively combines a mutant-enriched method to a liquidchip platform. The principle of MEL is to create a unique restriction enzyme site at the WT gene sequence in order to remove the WT gene by the restriction enzyme digestion before the mutated gene sequence could be selectively amplified by the PCR. The mutated PCR product will then be hybridized to a specific probe on polystyrene microspheres and analyzed by the Luminex. This technology is extremely sensitive and therefore allows us to use the serum or plasma sample to detect somatic gene mutations. It is also high-throughput; which allows us to assay up to 96 samples at once and read up to 100 different mutations from one sample. MEL technology has been successfully used in the clinic for detecting other somatic mutations from serum and plasma samples as well, such as KRAS, BRAF, PIK3CA, EGFR exon 20, etc. The detection sensitivity is one mutant DNA in the presence of 1×103 wild-type copies. As low as 10 copies of mutant DNA in a sample could be detected. To evaluate the MEL, the EGFR mutation status of NSCLC was tested on tissue samples using both sequencing and MEL methods. The positive samples of EGFR mutation by sequencing method were in agreement with the results by the MEL. However, 31.8% (7/22) of the negative samples by sequencing were positive by MEL. To date, our results have shown that 32.1% (146/455) serum samples of NSCLC patients showed EGFR mutant positive, from which 9.2% (42/455) were exon 19 deletion mutations; 19.8% (90/455), exon 21 single nucleotide polymorphism mutation; and 3.1% (14/455), both. Our study also showed that EGFR mutations were detected more frequently in patients with partial response following the targeted therapy than those with stable disease or progressive disease (p=0.027). In conclusion, MEL is a valuable method for detecting circulating EGFR mutations in serum or plasma of NSCLC patients for pharmacogenomic diagnostics. This method could be used on tissue samples as well. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B180.