Detection of EpCAM-Negative and Cytokeratin-Negative Circulating Tumor Cells in Peripheral Blood

Enrichment of rare circulating tumor cells (CTCs) in blood is typically achieved using antibodies to epithelial cell adhesion molecule (EpCAM), with detection using cytokeratin (CK) antibodies. However, EpCAM and CK are not expressed in some tumors and can be downregulated during epithelial-to-mesenchymal transition. A micro-fluidic system, not limited to EpCAM or CK, was developed to use multiple antibodies for capture followed by detection using CEE-Enhanced (CE), a novel in situ staining method that fluorescently labels the capture antibodies bound to CTCs. Higher recovery of CTCs was demonstrated using antibody mixtures compared to anti-EpCAM. In addition, CK-positive breast cancer cells were found in 15 of 24 samples (63%; range 1–60 CTCs), while all samples contained additional CE-positive cells (range 1–41; median = 11; P = .02). Thus, antibody mixtures against a range of cell surface antigens enables capture of more CTCs than anti-EpCAM alone and CE staining enables the detection of CK-negative CTCs.

[1]  G. Sauter,et al.  High Ep-CAM Expression is Associated with Poor Prognosis in Node-positive Breast Cancer , 2004, Breast Cancer Research and Treatment.

[2]  Anne Vincent-Salomon,et al.  Enrichment methods to detect bone marrow micrometastases in breast carcinoma patients: clinical relevance , 2004, Breast Cancer Research.

[3]  M. Keeney,et al.  Circulating Tumor Cell Analysis: Technical and Statistical Considerations for Application to the Clinic , 2009, Journal of oncology.

[4]  Oscar Lin,et al.  Fluorescence In situ Hybridization Analysis of Circulating Tumor Cells in Metastatic Prostate Cancer , 2009, Clinical Cancer Research.

[5]  Annibale Biggeri,et al.  Correlation of HER2 status between primary tumors and corresponding circulating tumor cells in advanced breast cancer patients , 2009, Breast Cancer Research and Treatment.

[6]  K. Tai,et al.  DNA methylation and histone modification regulate silencing of epithelial cell adhesion molecule for tumor invasion and progression , 2007, Oncogene.

[7]  M. Slijper,et al.  Full-length cytokeratin-19 is released by human tumor cells: a potential role in metastatic progression of breast cancer , 2009, Breast Cancer Research.

[8]  S. Digumarthy,et al.  Isolation of rare circulating tumour cells in cancer patients by microchip technology , 2007, Nature.

[9]  M. Kowalewska,et al.  Implications of cancer-associated systemic inflammation for biomarker studies. , 2010, Biochimica et biophysica acta.

[10]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[11]  Brigitte Rack,et al.  Detection of Circulating Tumor Cells in Peripheral Blood of Patients with Metastatic Breast Cancer: A Validation Study of the CellSearch System , 2007, Clinical Cancer Research.

[12]  T. Kroll,et al.  Assessing the efficacy of targeted therapy using circulating epithelial tumor cells (CETC): the example of SERM therapy monitoring as a unique tool to individualize therapy , 2010, Journal of Cancer Research and Clinical Oncology.

[13]  K. Pienta,et al.  Circulating Tumor Cells Predict Survival Benefit from Treatment in Metastatic Castration-Resistant Prostate Cancer , 2008, Clinical Cancer Research.

[14]  T. Molina,et al.  Preoperative Circulating Tumor Cell Detection Using the Isolation by Size of Epithelial Tumor Cell Method for Patients with Lung Cancer Is a New Prognostic Biomarker , 2010, Clinical Cancer Research.

[15]  D. Dearnaley,et al.  Characterization of ERG, AR and PTEN gene status in circulating tumor cells from patients with castration-resistant prostate cancer. , 2009, Cancer research.

[16]  I. Runnebaum,et al.  Monitoring circulating epithelial tumour cells (CETC) to gauge therapy: in patients with disease progression after trastuzumab persisting CETC can be eliminated by combined lapatinib treatment , 2009, Journal of Cancer Research and Clinical Oncology.

[17]  Miss A.O. Penney.  (b) , 1974, The New Yale Book of Quotations.

[18]  Alessandro Lugli,et al.  Frequent EpCam protein expression in human carcinomas. , 2004, Human pathology.

[19]  Peter Kuhn,et al.  Cytomorphology of Circulating Colorectal Tumor Cells:A Small Case Series , 2010, Journal of oncology.

[20]  H. Scher,et al.  Detection of androgen receptor mutations in circulating tumor cells: highlights of the long road to clinical qualification. , 2010, Clinical chemistry.

[21]  Hartwig Huland,et al.  Circulating prostate tumor cells detected by reverse transcription-PCR in men with localized or castration-refractory prostate cancer: concordance with CellSearch assay and association with bone metastases and with survival. , 2009, Clinical chemistry.

[22]  T. Fehm,et al.  Determination of HER2 status using both serum HER2 levels and circulating tumor cells in patients with recurrent breast cancer whose primary tumor was HER2 negative or of unknown HER2 status , 2007, Breast Cancer Research.

[23]  K. Isselbacher,et al.  Isolation of circulating tumor cells using a microvortex-generating herringbone-chip , 2010, Proceedings of the National Academy of Sciences.

[24]  E. D. de Vries,et al.  Detection of micrometastatic breast cancer by means of real time quantitative RT‐PCR and immunostaining in perioperative blood samples and sentinel nodes , 2003, International journal of cancer.

[25]  P. Paterlini-Bréchot,et al.  Circulating tumor cells (CTC) detection: clinical impact and future directions. , 2007, Cancer letters.

[26]  Tanja Fehm,et al.  Circulating Tumor Cells in Patients with Breast Cancer Dormancy , 2004, Clinical Cancer Research.

[27]  M. Rots,et al.  Persistent downregulation of the pancarcinoma‐associated epithelial cell adhesion molecule via active intranuclear methylation , 2008, International journal of cancer.

[28]  H. Scher,et al.  Circulating Tumor Cell Analysis in Patients with Progressive Castration-Resistant Prostate Cancer , 2007, Clinical Cancer Research.

[29]  J. Bono,et al.  All circulating EpCAM+CK+CD45- objects predict overall survival in castration-resistant prostate cancer. , 2010, Annals of oncology : official journal of the European Society for Medical Oncology.

[30]  Jonathan W. Uhr,et al.  Tumor Cells Circulate in the Peripheral Blood of All Major Carcinomas but not in Healthy Subjects or Patients With Nonmalignant Diseases , 2004, Clinical Cancer Research.

[31]  N. Lane,et al.  Methods for isolating circulating epithelial cells and criteria for their classification as carcinoma cells. , 2005, Cytotherapy.

[32]  Klaus Pantel,et al.  Down-Regulated Expression of Cytokeratin 18 Promotes Progression of Human Breast Cancer , 2004, Clinical Cancer Research.

[33]  G. Sauter,et al.  Changes in Cytoskeletal Protein Composition Indicative of an Epithelial-Mesenchymal Transition in Human Micrometastatic and Primary Breast Carcinoma Cells , 2005, Clinical Cancer Research.

[34]  T. Hirano,et al.  Analysis of polypeptide expression in benign and malignant human breast lesions: down-regulation of cytokeratins. , 1996, British Journal of Cancer.

[35]  D. Wallwiener,et al.  Rare expression of epithelial cell adhesion molecule on residual micrometastatic breast cancer cells after adjuvant chemotherapy. , 2003, Clinical cancer research : an official journal of the American Association for Cancer Research.

[36]  Xiaoyu Jia,et al.  Circulating tumour cells as prognostic markers in progressive, castration-resistant prostate cancer: a reanalysis of IMMC38 trial data. , 2009, The Lancet. Oncology.

[37]  T. Fehm,et al.  Detection and characterization of circulating tumor cells in blood of primary breast cancer patients by RT-PCR and comparison to status of bone marrow disseminated cells , 2009, Breast Cancer Research.

[38]  R. Moll,et al.  The human keratins: biology and pathology , 2008, Histochemistry and Cell Biology.

[39]  K. Pachmann,et al.  Seeding of epithelial cells into circulation during surgery for breast cancer: the fate of malignant and benign mobilized cells , 2006, World journal of surgical oncology.