Metastasis and circulating tumor cells

Cancer is a prominent cause of death worldwide. In most cases, it is not the primary tumor which causes death, but the metastases. Metastatic tumors are spread over the entire human body and are more difficult to remove or treat than the primary tumor. In a patient with metastatic disease, circulating tumor cells (CTCs) can be found in venous blood. These circulating tumor cells are part of the metastatic cascade. Clinical studies have shown that these cells can be used to predict treatment response and their presence is strongly associated with poor survival prospects. Enumeration and characterization of CTCs is important as this can help clinicians make more informed decisions when choosing or evaluating treatment. CTC counts are being included in an increasing number of studies and thus are becoming a bigger part of disease diagnosis and therapy management. We present an overview of the most prominent CTC enumeration and characterization methods and discuss the assumptions made \ud about the CTC phenotype. Extensive CTC characterization of for example the DNA, RNA and antigen expression may lead to more understanding of the metastatic process

Light-scattering polarization measurements as a new parameter in flow cytometry

Polarization measurement of orthogonal light scattering is introduced as a new optical parameter in flow cytometry. \ud In the experimental setup, the electrical field of the incident laser beam is polarized in the direction of the sample flow. The intensity of the orthogonal light scattering polarized along the direction of the incoming laser beam is called depolarized orthogonal light scattering. Theoretical analysis shows that for small values of the detection aperture, the measured depolarization is caused by anisotropic cell structures and multiple scattering processes inside the cell. \ud Measurements of the orthogonal depolarized light scattering in combination with the normal orthogonal light scattering of human leucocytes revealed two populations of granulocytes. By means of cell sorting it was shown that the granulocytes with a relatively high depolarization are eosinophilic granulocytes. Similar experiments with human lymphocytes revealed a minor subpopulation of yet-unidentified lymphocytes with a relative large orthogonal light-scattering depolarization. The results were obtained with an argonion laser tuned at different wavelengths as well as with a 630-nm helium neon laser. These results show that measurement of depolarized orthogonal light scattering is a useful new parameter for flow-cytometric cell differentiation

Four-Parameter white blood cell differential counting based on light scattering measurements

Measurement of the depolarized orthogonal light scattering in flow cytometry enables one to discriminate human eosinephilic granulocytes from neutrophilic granulocytes. We use this method to perform a four-parameter differential white blood cell analysis. \ud A simple flow cytometer was built equipped with a 5-mW helium neon laser that measures simultaneously four light scattering parameters. Lymphocytes, monocytes, and granulocytes were identified by simultaneously measuring the light scattering intensity at angles between 1.0° and 2.6° and angles between 3.0° and 11.0°. Eosinophilic granulocytes were distinguished from neutrophilic granulocytes by simultaneous measurement of the orthogonal and depolarized orthogonal light scattering. \ud Comparison of a white blood cell differentiation of 45 donors obtained by the Technicon H-6000 and our instrument revealed good correlations. The correlation coefficients (r2) found were: 0.99 for lymphocytes, 0.76 for monocytes, 0.99 for neutrophilic granulocytes, and 0.98 for eosinophilic granulocytes. The results demonstrate that reliable white blood cell differentiation of the four most clinically relevant leukocytes can be obtained by measurement of light scattering properties of unstained leukocytes

Discrimination of human cytotoxic lymphocytes from regulatory and B-lymphocytes by orthogonal light scattering

Light scattering properties of human lymphocyte subpopulations selected by immunofluorescence were studied with a flow cytometer. Regulatory and B-lymphocytes showed a low orthogonal light scatter signal, whereas cytotoxic lymphocytes identified with leu-7, leu-11 and leu-15 revealed a large orthogonal light scatter signal. Two populations in light scatter histograms could be observed with monoclonal antibodies directed against determinants present on both regulatory and cytotoxic lymphocytes. By analysis of the lymphocytes of 16 individuals we found a linear relation between the number of cells with a large orthogonal light scattering and the number of cytotoxic lymphocytes identified with leu-7, leu-11 and leu-15. These observations demonstrate physical differences between cytotoxic lymphocytes and regulatory and B lymphocytes. Moreover, the results suggest a method to estimate the amount of cytotoxic lymphocytes without using monoclonal antibodies

Filter characteristics influencing circulating tumor cell enrichment from whole blood.

A variety of filters assays have been described to enrich circulating tumor cells (CTC) based on differences in physical characteristics of blood cells and CTC. In this study we evaluate different filter types to derive the properties of the ideal filter for CTC enrichment. Between 0.1 and 10 mL of whole blood spiked with cells from tumor cell lines were passed through silicon nitride microsieves, polymer track-etched filters and metal TEM grids with various pore sizes. The recovery and size of 9 different culture cell lines was determined and compared to the size of EpCAM+CK+CD45−DNA+ CTC from patients with metastatic breast, colorectal and prostate cancer. The 8 µm track-etched filter and the 5 µm microsieve had the best performance on MDA-231, PC3-9 and SKBR-3 cells, enriching >80% of cells from whole blood. TEM grids had poor recovery of ~25%. Median diameter of cell lines ranged from 10.9–19.0 µm, compared to 13.1, 10.7, and 11.0 µm for breast, prostate and colorectal CTC, respectively. The 11.4 µm COLO-320 cell line had the lowest recovery of 17%. The ideal filter for CTC enrichment is constructed of a stiff, flat material, is inert to blood cells, has at least 100,000 regularly spaced 5 µm pores for 1 ml of blood with a ≤10% porosity. While cell size is an important factor in determining recovery, other factors must be involved as well. To evaluate a filtration procedure, cell lines with a median size of 11–13 µm should be used to challenge the syste

Filtration Parameters Influencing Circulating Tumor Cell Enrichment from Whole Blood

Filtration can achieve circulating tumor cell (CTC) enrichment from blood. Key parameters such as flow-rate, applied pressure, and fixation, vary largely between assays and their influence is not well understood. Here, we used a filtration system, to monitor these parameters and determine their relationships. Whole blood, or its components, with and without spiked tumor cells were filtered through track-etched filters. We characterize cells passing through filter pores by their apparent viscosity; the viscosity of a fluid that would pass with the same flow. We measured a ratio of 5·104:102:1 for the apparent viscosities of 15 µm diameter MDA-231 cells, 10 µm white cells and 90 fl red cells passing through a 5 µm pore. Fixation increases the pressure needed to pass cells through 8 µm pores 25-fold and halves the recovery of spiked tumor cells. Filtration should be performed on unfixed samples at a pressure of ~10 mbar for a 1 cm2 track-etched filter with 5 µm pores. At this pressure MDA-231 cells move through the filter in 1 hour. If fixation is needed for sample preservation, a gentle fixative should be selected. The difference in apparent viscosity between CTC and blood cells is key in optimizing recovery of CTC

The cost of expensive breast cancer drugs

Background Increasing healthcare costs are a major challenge in medical oncology, since the total costs of oncology can account for up to 30% of the total hospital expenditures. As many novel (expensive) cancer treatments are being developed, it is important to be transparent about drug prices from an early research stage on. To assess the potential financial impact of pipeline drugs, their expected future prices can be deducted from prices of currently used drugs. As an overview of the standard prices of expensive breast cancer treatments in European countries is lacking, this review aimed to synthesize all evidence on costs of approved, expensive breast cancer drugs in the Netherlands. Methods A literature review was performed to create an overview of all approved, expensive drugs in the Netherlands. Standard drug costs were retrieved via the Dutch administrative health authority (ZINL). Drugs were considered expensive if the standard price of the drug was more than €10 per unit or if the cost of a treatment with that particular drug exceeded €1000 on average per patient. Results In the Netherlands 25 breast cancer drugs are approved with a standard price of more than €10 per unit. After excluding drugs with expected treatment costs less than €1000, 19 drugs were included in the analysis. The standard drug price is €7,943 on average (range €63 - €45,452), and the average number of cycles per patient is 10.5 (range 4 - 25.3 cycles). This results in average treatment costs per patient of expensive drugs of €17,968 (range €1,103 - €87,123). Four drugs that initially ranked low based on standard drug unit prices (rank 10-19), rank substantially higher (rank 1-10) when ranking total treatment costs. Conclusions Ranking standard drug prices per unit may not be very informative. It would be valuable to rank drug treatment costs, based on treatment length and dosage estimates. However, in the Netherlands the expected treatment length for a particular drug is not standardly reported in official approval reports. Furthermore, actual prices of expensive drugs may differ from standard drug prices, by which treatment costs might be deviant. Extending standardization of reporting and calculation of drug treatment costs would be valuable and particularly relevant when extending this type of cost calculations to other countries