Establishment And Improvement Of The Methodology Of Circulating Tumor Cells Detection And Its Clinical Application For Solid Tumors

Circulating tumor cells (CTC) are the cells in peripheral blood that detached from the primary or metastasis cancer lesions. And as a real-time "liquid biopsy", CTC have long been considered a reflection of tumor aggressiveness. The efficacy of CTC detection among patients with solid malignancy, such as lung cancer, breast cancer, prostate cancer, colorectal cancer, has been investigated which shows CTC is an independent predictor of poor prognosis and co μ Id serve as an early marker to assess the therapeutic response in overt cancers. Several studies have shown that CTC may gain additional phenotypic and genotypic characteristics over time and develop independently from the primary tumor. Thus, direct molecular genetic analysis of CTC coμld provide important additional information before patients are stratified to expensive therapies that might have considerable side effects. In short, CTC detection in patients is of great significance.However, owing to the rarity of CTC in peripheral blood, detecting these cells requires methods combined with high sensitivity and specificity, which sets tremendous challenges for the implementation of these assays into clinical routine. Generally, CTCs detection methods are composed of two consecutive steps:isolation process and identification process. Among these, Epithelial cell surface adhesion molecμle (EpCAM) dependent method to enrichment and detection CTC has been the most common, such as CellSeach system, which has been approved by USA food and drug administration (FDA). But, as research continues, people found that expression of EpCAM on CTCs varies greatly, and epithelial CTCs co μ ld undergo epithelial-mesenchymal transition (EMT) during metastasis, causing epithelial specific antigen expression weakening or loss. CTC heterogeneity has limited the clinical application of traditional methods relying on EpCAM to detect CTC, which co μ ld fail to detect EpCAM negative cells. Therefore, we aim to establish an EpCAM independent method for CTC isolation and identification, and to further study site mutagenesis of K-RAS on those single enriched and identified tumor cells.In the first part of this study, we combined immunocytochemistry (ICC) staining of CD45, DAPI and fluorescence in situ hybridization with the centromere of chromosome8(CEP8) probe method (CD45-FISH) to improve the identification for CTCs with weak or negative CK. We found that, for detecting CTC negatively enriched with EpCAM independent magnetic beads coated with anti-CD45antibodies from lung cancers, CD45-FISH had higher sensitivity and specificity compared to ICC-CK,83.3%and43.3%,98.6%and89.5%, respectively. For CTC detection in ovarian cancers, CD45-FISH showed76.2%sensitivity. Four of six ovarian cancers showed dramatic ally decrease in both CTCs and serum CA125on the7th day after surgery. The FISH hybridization signals were different between different CTC, which provide evidence for cell heterogeneity. In brief, we have established an EpCAM-independent CD45-FISH method to identify CTC for lung cancer and ovarian cancer patients, and compared to traditional ICC-CK, CD45-FISH method had improved sensitivity and specificity. This combined detection strategy may be usefμl in detecting or monitoring CTCs after ovarian cancer surgery.In the second part of this study, we further optimized CD45-FISH method by simμltaneously immunohistochemical staining of CK and established CK/CD45-FISH method which allowed us not only to distinguish between CK positive CTCs and CTCs with reduced or absent CK expression, but also between diploid and hyperdiploid CTCs. To analysis CTC in single cell level, MiaPaCa-2cells were spiked into normal peripheral bold and then enriched and identified, which were finally collected by laser capture micro-dissection (LCM) followed by whole genome amplification, enzymatic digestion and degeneration high performance liquid chromatography (DHPLC) to further study their site mutagenesis of K-RAS. Our resμlts showed that LCM co μ1d accurately separate and purify single tumor cells. Enzymatic digestion and DHPLC can stablely detect K-ras mutant from10MiaPaCa-2cells, while fluorescent PCR kit can detect from even one tumor cell. For CTC enriched from pancreatic cancers and identified by combining CK, CD45, DAPI and CEP8method (CK/CD45/CEP8), We found that remaining cells coμld be classified into5patterns:CK+CD45-DAPI+CEP8=2, CK+CD45-DAPI+CEP8>2, CK-CD45-DAPI+CE P8>2, CK-CD45-DAPI+CEP8=2, and CK+/-CD45+DAPI+CEP8=2or>2. Patterns of CK+CD45-DAPI+CEP8=2, CK+CD45-DAPI+CEP8>2and CK-CD45-DAPI+CEP8>2were considered as CTCs, and CK-CD45-CEP8=2and CK+/-CD45+DAPI+CEP8=2or>2were considered as indeterminate cells. Among22pancreatic cancers, patterns of CK+CD45-DAPI+CEP8=2and CK+CD45-DAPI+CEP8>2were identified in2cases, and the number of CTCs was6,12cells/3.75mL and2,44cells/3.75mL, respectively. CK-CD45-CEP8>2pattern was identified in16cases with the range of1-14cells/3.75mL and the median of3cells/3.75mL. CK-CD45-CEP8=2patterns was identified in21cases with the range of1-25cells/3.75mL and the median of5cells/3.75mL. CK+/-CD45+CEP8=2/>2patterns was identified in4cases with the range of1-4cells/3.75mL and the median of2cells/3.75mL. Above all, CK/CD45-FISH method can efficiently identify various tumors CTC, and detection efficiency is not affected by either degradation of cytokeratin in tumor cells. However, because tumor cell lines were very different from clinical samples in some respects, there are still many aspects needed to be modified for CTC separation and purification by LCM for clinical application.