Jiasen Xu

Classification of Circulating Tumor Cells by Epithelial-Mesenchymal Transition Markers

In cancer, epithelial-mesenchymal transition (EMT) is associated with metastasis. Characterizing EMT phenotypes in circulating tumor cells (CTCs) has been challenging because epithelial marker-based methods have typically been used for the isolation and detection of CTCs from blood samples. The aim of this study was to use the optimized CanPatrol CTC enrichment technique to classify CTCs using EMT markers in different types of cancers. The first step of this technique was to isolate CTCs via a filter-based method; then, an RNA in situ hybridization (RNA-ISH) method based on the branched DNA signal amplification technology was used to classify the CTCs according to EMT markers. Our results indicated that the efficiency of tumor cell recovery with this technique was at least 80%. When compared with the non-optimized method, the new method was more sensitive and more CTCs were detected in the 5-ml blood samples. To further validate the new method, 164 blood samples from patients with liver, nasopharyngeal, breast, colon, gastric cancer, or non-small-cell lung cancer (NSCLC) were collected for CTC isolation and characterization. CTCs were detected in 107(65%) of 164 blood samples, and three CTC subpopulations were identified using EMT markers, including epithelial CTCs, biophenotypic epithelial/mesenchymal CTCs, and mesenchymal CTCs. Compared with the earlier stages of cancer, mesenchymal CTCs were more commonly found in patients in the metastatic stages of the disease in different types of cancers. Circulating tumor microemboli (CTM) with a mesenchymal phenotype were also detected in the metastatic stages of cancer. Classifying CTCs by EMT markers helps to identify the more aggressive CTC subpopulation and provides useful evidence for determining an appropriate clinical approach. This method is suitable for a broad range of carcinomas.

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

Abstract 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 1×105 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. Clin Chem Lab Med 2010;48:1103–6.

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

Abstract 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.

Enrichment and enumeration of circulating tumor cells by efficient depletion of leukocyte fractions

Abstract Background: Enumeration and characterization of circulating tumor cells (CTCs) can provide information on patient prognosis and treatment efficacy. However, CTCs are rare, making their isolation a major technological challenge. We developed a technique for enrichment, and subsequent characterization of CTCs based on efficient depletion of human leukocytes. Methods: The technique (CanPatrolTM CTC enrichment) we developed is based on red blood cell lysis to remove erythrocytes, followed by depletion of CD45+ leukocytes using a magnetic bead separation method, and subsequent isolation of CTCs by virtue of their larger size, compared with leukocytes. We also demonstrated that fluorescence in situ hybridization (FISH) and genetic abnormalities analysis could be performed on the isolated CTCs. Results: The spiking experiments showed that the average efficacy of leukocytes depletion was 99.98% and the average tumor cells recovery was not lower than 80%. FISH could be used to perform ALK gene rearrangement analysis on the collected NCI-H2228 cells, and EGFR Exon 19 deletion was detected by PCR-based analysis in isolated HCC827 cells. The in vivo feasibility of this technique had been demonstrated in patients with non-small cell lung cancer, breast, colon, and esophageal cancers. CTCs were detected in 13 of 59 blood samples. Tumor microemboli was also detected in three breast cancer samples. Conclusions: The technique we developed allowed isolation and characterization of circulating epithelial tumor cells that do not express classical epithelial antigens. This potentially leads to a more accurate enumeration of the number of CTCs and is suitable for application to a broad range of 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.

Abstract 385: Predictive factors of c-kit-positive cancer stem cells, epithelial-mesenchymal transition and circulating tumor cells in distant metastasis formation in non-small cell lung cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Cancer stem cells (CSC) are a rare subpopulation of undifferentiated cells that are responsible for tumor initiation and tumor regeneration after chemotherapy. Although a universal marker for CSCs has not been identified, previous studies showed that c-kit (CD117) functioned as a stem cell factor (SCF) receptor, and SCF-ckit signaling axis was essential for self-renewal and proliferation of lung CSCs. During tumorigenesis, subsets of tumor cells disseminate from primary tumors by undergoing phenotypic changes that allow the cells to penetrate blood vessels. These changes are accompanied by a process described as epithelial-mesenchymal transition (EMT). EMT endows epithelial cells with enhanced invasive potential by the loss of their epithelial characteristics and the acquisition of a mesenchymal phenotype. The aim of this study is to correlate c-kit-positive CSCs and EMT-positive, subpopulations of circulating tumor cells (CTCs) with clinicopathological parameters to verify whether these biomarkers contribute to cancer distant metastasis in non-small cell lung cancer (NSCLC). A total of 31 patients with NSCLC were enrolled into this study and evaluated for CTC levels at baseline. Peripheral blood samples (5 ml, anticoagulated with EDTA) were collected, transferred to the CanPatrol CTC tube, and then processed within 8 hours. The CanPatrol CTC enrichment technique was used to isolate and characterize CTCs. The first step of this technique was to isolate CTCs via a filter-based method; then, an quantifiable, quadruple-colorimetric RNA in situ hybridization (ISH) method was used to examine CTCs for expression of four epithelial (E) biomarkers (cytokeratins (CKs)8,18 and 19; and epithelial cell adhesion molecule (EpCAM)), two mesenchymal (M) biomarkers (vimentin and twist), c-kit and CD45. Using this technique, CTCs were detected in 25(80.6%) of 31 blood samples, and five categories of CTCs were defined using EMT biomarkers ranging from exclusively epithelial (E) to intermediate (E > M, E = M, M > E) and exclusively mesenchymal (M). Of the CTC-positive samples, 13(52%) samples contained c-kit-positive CTCs, and the percentage of cells with c-kit expression was 27.8%, with 2.1%, 4.6%, 2.1%, 18.3% and 0.8% in exclusively E, E > M, E = M, M > E and exclusively M CTCs, respectively. The expression of c-kit showed a high statistically significant correlation with distant metastasis (P = 0.0001). Compared with the non metastatic or regional metastatic carcinoma, mesenchymal CTCs (including M > E and exclusively M) were predominant in patients with distant metastatic carcinoma (P = 0.033). However, the sample size of this study was small, further validation needed to be done with a larger sample size. In summary, mesenchymal CTCs or/and c-kit may be potential predictive factors for invasion and metastatic spread of NSCLC. Citation Format: Shiyang Wu, Jianyu Sun, Weihua Qiu, Suyan Liu, Yujie Ma, Jiaming Che, Beili Gao, Jiasen Xu, David K. Ann. Predictive factors of c-kit-positive cancer stem cells, epithelial-mesenchymal transition and circulating tumor cells in distant metastasis formation in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 385. doi:10.1158/1538-7445.AM2015-385

A multiplexer liquidchip technology for detecting single nucleotide polymorphisms from metabolism of anti-thrombotic drugs in dried blood spots on filter paper.

A single nucleotide polymorphism (SNP) is the most frequent type of variation in the genome. There are around 10 million SNPs that have been identified in the human genome [1]. Because SNPs are highly conserved throughout evolution and within a population, the map of SNPs serves as an excellent genotypic marker for research. The elucidation of SNP information will contribute to an individual’s susceptibility to disease and responsiveness to drug toxicity and medical intervention [2,3]. Nowadays, a variety of techniques have been used to perform SNP genotyping, but these techniques required whole blood as the sample. Dried blood spot (DBS) specimens require less material and are substantially more stable (several months at room temperature) than whole blood [4]. Thus, the simplicity of sample preparation, long time storage and convenient transport make DBS to be a cost-effective and suitable alternative tool for collecting blood sample. Plavix (clopidogrel) and warfarin are wildly used antithrombotic drugs in the treatment and prevention of thrombotic diseases, including myocardial infarction, ischemic stroke, and venous thrombosis [5]. However, because of genetic variation, the two drugs have wide inter-individual variation in dose resulting in the risk of serious bleeding complications [6,7].Many studies have shown that genetic status can greatly influence an individual patient’s warfarin or Plavix dosing [8–15]. So the Food and Drug Administration changed the warfarin label to encourage lower initial doses in patients who have the VKORC1–1639G.A, CYP2C9*2, or CYP2C9*3 allele in August 2007 [16] and added pharmacogenetic information to the Plavix product label because of genetic differences in CYP2C19 in March 2010. In this study, we developed a method to detect genotyping of DBS specimens about Plavix and warfarin using SurPlexTM-xTAGmethod. All oligonucleotides were synthesized by Invitrogen Corporation (Carlsbad, USA). PCR primers used for the genotyping assay (Table 1) were unmodified. Allele-specific primer extension (ASPE) primers consist of two sequences: a tag sequence (according to Luminex tag sequence) and the allele-specific sequence (Table 1). Universal anti-tags (probes) were amino (NH2)-modified for coupling to carboxylated microspheres. Tag sequences used for validation of the universal array were labeled with biotin. To evaluate the specificity of this method, the plasmids (pGEM-T-Easy; Promega, Madison, USA) containing wild-type (WT) alleles of CYP2C9, CYP2C19, VKORC1, and ABCB1 were constructed. The mutant-type plasmids were derived from WT plasmids using the TaKaRa Mutant Best kit (TaKaRa, Dalian, China). Approximately 30 ml of whole blood was spotted on 3-mm filter paper (Whatman, Wisconsin, USA). After drying, a 6-mm disk was cut out from the sampling paper. Additionally, the 6-mm disk was placed into a 1.5-ml Eppendorf cup (Hamburg, Germany), with 100 ml sterile water, and then placed in a boiling water bath for 15 min to make the cells lyse. After gentle vortexes, the mixture was centrifuged at 10,000 g for 5 min, then 10 ml supernatants were pipetted and used as DNA source for amplification. Multiplex PCR about warfarin was performed in a 50 ml volume containing: 1 Ex Taq polymerase buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, 0.2 mM each of the primers, 1.0 U Ex Taq HS DNA polymerase (TaKaRa) and 10 ml supernatant of boiling DBS as template DNA. Thermo cycling was performed using 30 cycles of 958C for 30 s, 568C for 30 s, and 728C for 30 s. The reaction was concluded with a final extension step of 728C for 10 min and the product was kept at 48C until use. A similar approach was used to enrich DNA fragments of Plavix-related genes using 3 mM MgCl2. Exonuclease I and shrimp alkaline phosphatase (EXO-SAP) reaction was performed in a 25-ml volume containing: 1 SAP buffer, 1 U shrimp alkaline phosphatase (inactivating any remaining nucleotides) (TaKaRa), 10 U exonuclease I (degrading any remaining PCR primers) (New England Biolabs, Ipswich, USA) and 7.5 ml PCR product. Samples Acta Biochim Biophys Sin 2014, xx: 1–4 |a The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmu028.

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

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 patient9s 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.