| A considerable body of evidence indicates that tumor cells are shed from a primary tumor mass at the earliest stages of malignant progression. Some of these cells will travel via the peripheral blood to sites anatomically distant from the primary tumor and form metastases. As individual disseminated tumor cells present in low numbers, they can be occult to standard methods of investigation. However, these circulating tumor cells (CTCs) are understood to be a source of eventual lethal metastases, the major cause of treatment failure in cancer patients.;Many studies have concluded that the presence of CTCs provides important prognostic information predictive of disease-free and overall survival in various malignancies. By contrast, other reports have found no statistically significant relationship between CTC detection and prognosis. This discrepancy is most likely because even in metastatic patients, the frequency of CTCs is extremely low (estimated to be in the range of 1-10 CTCs/ml of PB). We hypothesize that the issue of varying conclusions regarding the prognostic significance of CTCs may be attributed to the technical difficulties associated with the reliable detection of such rare events.;The main goal of our work was to develop and improve approaches for CTC detection in the PB of cancer patients and to use this technology in patient studies to investigate the prevalence and characteristics of CTCs. The aims were to build methods that provide improved sensitivity and specificity of CTC detection while meeting the requirements of a practical clinical CTC assay. Both cell-based and molecular methods of CTC detection were investigated for areas of improvement. In addition, steps of pre-analytical and analytical procedures were dissected in order to identify potential sources of inaccuracy and irreprocubility. The developed instrumentations and preparation methods were used to investigate the prevalence of various rare cells, such as CTCs and Cytomegalovirus (CMV)-infected leukocytes. Furthermore, together with four different European laboratories a pilot study was conducted to assess the variation and inconsistencies of CTC detection and to formulate the basis of a standardized, quantitative CTC detection protocol.;Results from our investigations provided novel insights into the molecular and cell-based detection and quantitation of rare cells. Through the application of the procedures and models described in this thesis a potentially more accurate assessment of level signals may be obtained. This would facilitate the earlier diagnosis and more accurate therapy monitoring of infectious and cancerous diseases. |
