[Circulating tumor cells in head and neck cancer]. / Zirkulierende Tumorzellen bei Kopf-Hals-Tumoren.

Circulating tumor cells are defined as tumor cells which are circulating in the peripheral blood of the cancer patient. While several large studies have investigated the role of circulating tumor cells in other solid tumors, the importance of these tumor cells in patients with head and neck cancer was turned into the focus not until the recent years. In other solid tumor the presence of circulating tumor cells often seems to be a negative prognostic marker and seems to be a marker for therapy response. The present article wants to give an overview about the knowledge on circulating tumor cells and their clinical relevance in head and neck cancer. The methodology to detect circulating tumor cells will be critically reflected. The future potential of the detection of circulating tumor cells in head and neck cancer patients will be discussed.

Circulating tumor cells in breast cancer: methodology and clinical repercussions.

Breast cancer is the most common type of cancer among women, and clinicians have long recognized its heterogeneity. Its detection and treatment in early stages allow for reduction of mortality. Despite the advances and new strategies for combining surgical, radiotherapy, and chemotherapy options, however, the percentage of patients developing metastases and advanced stages remains high. Even though serum tumor markers have been used for the early diagnosis of metastases, their systematic determination has not had an effect on survival. Methods that are more reliable are needed to detect metastases earlier than with the common clinical methods and thus start treatment before overt relapse. Early indicators of response or resistance to treatment are also an issue in clinical practice. Imaging techniques are time consuming, and it is difficult to detect changes that indicate response limited to therapy, and approaches to defining changes in tumor mass are time and resource consuming. In contrast, detection of circulating tumor cells (CTC) could be a useful tool in early detection of relapse and response to systemic chemotherapy. Extremely sensitive techniques are available that are easily applied to peripheral blood samples, which might provide enormous research possibilities in this area.

Differential interaction of magnetic nanoparticles with tumor cells and peripheral blood cells.

PURPOSE: The separation of tumor cells from healthy cells is a vital problem in oncology and hematology, especially from peripheral blood. Magnetic assisted cell sorting (MACS) is a possibility to fulfill these needs. METHODS: Tumor cell lines and leukocytes from peripheral blood were incubated with carboxymethyl dextran-coated magnetic nanoparticles under various conditions and separated by MACS. RESULTS: We studied the interaction of magnetic nanoparticles devoid of antibodies with healthy and tumor cells. The magnetic nanoparticles interact with tumor cells and leukocytes and are located predominantly within the cell cytoplasm. Incubation of cell culture cells with magnetic nanoparticles led to a labeling of these cells without reduced biological properties for at least 14 days. The interaction of the magnetic nanoparticles with cells depends on several factors. The ionic strength (osmolality) of the solvent plays an important role. We could show that an increase in osmolality led to a dramatic reduction of labeled leukocytes. Tumor cells, however, are mildly affected. This could be detected not only in pure cultures of tumor cells or leukocytes but also in mixed cell populations. CONCLUSION: This observation gives us the opportunity to selectively label and separate tumor cells but not leukocytes from the peripheral blood.

Treatment with granulocyte-colony stimulating factor in patients with acute myocardial infarction. Evidence for a stimulation of neovascularization and improvement of myocardial perfusion.

BACKGROUND: Stem cell therapy has been suggested to be beneficial in patients after acute myocardial infarction (AMI). Strategies of treatment are either a local application of mononuclear bone marrow cells (BMCs) into the infarct-related artery or a systemic therapy with the granulocyte-stimulating factor (G-CSF) to mobilize BMCs. Nevertheless, the mechanisms responsible for improvement of cardiac function and perfusion are speculative at present. This study has been performed to investigate the effect of G-CSF on systemic levels of vascular growth factors and chemokines responsible for neovascularization, that might help to understand the positive effects of a G-CSF therapy after AMI. METHODS AND RESULTS: Five patients in the treatment group and 5 patients in the control group were enrolled in this study. The patients in the treatment group received 10 microg/kg bodyweight/day of G-CSF subcutaneously for a mean treatment duration of 6.6 +/- 1.1 days. In both groups, levels of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and monocyte chemotactic protein-1 (MCP-1) were measured on day 2 to 3 and day 5 after AMI. The regional wall perfusion and the ejection fraction (EF) were evaluated before discharge and after 3 months with ECG-gated MIBI-SPECT and radionuclide ventriculography, respectively. Significant higher levels of VEGF (p < 0.01), bFGF (p < 0.05) and MCP-1 (p < 0.05) were found in the treatment group compared to the control group. Levels of VEGF and bFGF remained on a plateau during the G-CSF treatment and decreased significantly in the control group. The wall perfusion improved significantly within the treatment group and between the groups (p < 0.05), respectively. The EF improved significantly within the treatment group (p < 0.05), but the change of the EF between the groups was not significant. CONCLUSION: In patients with AMI, the treatment with G-CSF modulates the formation of vascular growth factors that might improve neovascularization and result in an improved myocardial perfusion and function.

Impact of early NK cell recovery on development of GvHD and CMV reactivation in dose-reduced regimen prior to allogeneic PBSCT.

Dose-reduced allogeneic peripheral blood stem cell transplantation (PBSCT) is a therapeutic approach for patients with haematological malignancies who are not eligible for conventional allogeneic PBSCT. We analysed early development of lymphocyte subpopulations and the occurrence of cytomegalovirus (CMV) reactivation and acute graft-versus-host reaction (GvHD) in patients undergoing the protocol according to Slavin vs conventionally treated patients. Lymphocyte status prior to conditioning and at day +30 after allogeneic PBSCT was determined in 24 out of 51 patients who received conventional allogeneic PBSCT (eg cyclophosphamide plus total body irradiation) and compared with 27 patients being treated according to the Slavin protocol (fludarabine, busulphan and ATG). There is a significant delay in CD4 (T helper) cell development and consecutive lower CD4/CD8 ratios and a better reconstitution of CD8 (T cytotoxic) and NK (natural killer) cells after the Slavin protocol. Patients undergoing this protocol and no, or only grade I, acute GvHD show an even better NK cell reconstitution compared to patients with grade II-IV GvHD. A low CD4/CD8 ratio represents a CMV risk factor only in conventionally treated patients with grade 0-I GvHD, while after preparative regimen according to the Slavin protocol, the NK/CD8 ratio might be a marker for the prediction of CMV reactivation in addition to CMV risk status.

T cell receptor alpha expression in B-type chronic lymphocytic leukemia.

Normal B lymphocytes are characterized by rearrangement and expression of immunoglobulin genes, but not of T cell receptor genes. These properties might assist in lineage assignment, but there are examples of fresh leukemic cells and of cell lines where exceptions to this rule have been noted. We have studied cell samples of patients with B-CLL for expression of TCR alpha and beta chain genes. Using in situ hybridization with fluorescein-labeled probes, TCR alpha mRNA was found to be expressed in 14 of 18 samples and TCR beta mRNA in 7 of 16 samples. Specificity of hybridization was demonstrated by near complete blockade of TCR alpha hybridization with unlabeled TCR alpha, but not with unlabeled TCR beta probe. Furthermore, in Northern blot analysis a truncated 1,4 kb message for TCR alpha was readily detectable. No significant cell surface staining with the anti-TCR alpha/beta monoclonal antibody WT31 was observed. A contribution of T cells within the leukemic sample could be excluded since only samples with leukemic cell counts of greater than 50,000 cells/mm3 and only samples with 5% or less CD2+ T lymphocytes were studied. Our data show that a large proportion of B-CLL samples may express a truncated version of the TCR alpha message, indicating that this gene can be activated in leukemic B cells frozen at a late stage of differentiation.

Particulate ANP-sensitive guanylyl cyclase: induction in cultured human peripheral CD3+ mononuclear blood cells.

There have been conflicting reports about the occurrence and/or activity of atrial natriuretic peptide (ANP) sensitive guanylyl cyclase in the immune system. This study reports on ANP-sensitive guanylyl cyclase mRNA expression and guanylyl cyclase activity in human peripheral blood mononuclear cells (PBMC). Reverse transcription polymerase chain reaction (RT-PCR) shows that activated human PBMC of healthy blood donors express functional active ANP-sensitive guanylyl cyclase after vitro culture, whereas freshly isolated PBMC show neither specific mRNA for particulate guanylyl cyclase nor ANP-sensitive activity of this enzyme. To define the subpopulation of PBMC expressing this enzyme, cultivated PBMC were subfractioned and analyzed by RT-PCR and in situ PCR. Only CD3+ PBMC showed mRNA for ANP-sensitive guanylyl cyclase. Induction of the guanylyl cyclase required coincubation with other cells, indicating that a factor or factors secreted from cells other than CD3+ cells induces this expression. In summary, ANP-sensitive guanylyl cyclase is an inducible enzyme in human CD3+ PBMC in contrast to other cells where it is considered to be constitutive.

Insulin-like growth factor I is an independent coregulatory modulator of natural killer (NK) cell activity.

We aimed to investigate the natural killer (NK) cell activity in hGH-deficient adults and to analyze the effect of insulin-like growth factor (IGF)-I in vivo and in vitro on NK cell activity. NK cell activity was measured in a 4-h nonisotopic assay with europium-labeled and cryopreserved K-562 cells. NK-cell numbers were measured after incubation with murine monoclonal CD3 and CD16 antibodies by flow cytometry analysis. In a cross-sectional study, the basal and interferon-beta (IFN-beta) stimulated (1000 IU/ml) NK cell activity of 15 hGH-deficient patients and 15 age- and sex-matched controls was measured. The percentages and absolute numbers of CD3-/16+ NK-cells were not significantly different in the patient vs. control group. The basal and IFN-beta stimulated NK cell activity however was significantly decreased in the patient vs. control group at all effector/target (E/T) cell ratios from 12.5-100 (e.g. 17 +/- 3 vs. 28 +/- 3% lysis without IFN-beta, P < 0.05, and 42 +/- 4 vs. 57 +/- 4% lysis with IFN-beta, P < 0.05; both at E/T 50). IGF-I levels of patients and controls showed a significant positive correlation with NK cell activity (r = 0.37; P < 0.05). In an IGF-I in vitro study (IGF-I in vitro 250-1250 microg/L), the basal and IFN-beta stimulated NK cell activity of 13 hGH-deficient patients and of 18 normal subjects was significantly enhanced by IGF-I in vitro (e.g. GH-deficient patients: 9 +/- 2 vs. 10 +/- 2% lysis without IFN-beta, P < 0.05 and 25 +/- 4 vs. 30 +/- 4% lysis with IFN-beta, P < 0.005; and normal subjects: 15 +/- 3 vs. 23 +/- 3% lysis without IFN-beta, P < 0.001 and 35 +/- 4 vs. 44 +/- 5% lysis with IFN-beta, P < 0.001; both at IGF-I 500 microg/L). In summary, in our cross-sectional study, adult GH-deficient patients showed a significantly lower basal and IFN-beta stimulated NK cell activity than matched controls, despite equal NK cell numbers. IGF-I levels of patients and controls showed a weak positive correlation with NK cell activity. In an in vitro study, IGF-I significantly enhanced basal and IFN-beta stimulated NK cell activity of hGH-deficient patients and also of normal subjects. The decreased NK cell activity in GH-deficient patients may be caused at least in part by low serum IGF-I levels. IGF-I appears to be an independent coregulatory modulator of NK cell activity.

An immunocytochemical method for the detection of fluorochrome-labelled DNA probes hybridized in situ with cellular RNA.

Various detection systems for in situ hybridization of nucleic acids are currently used. We report here an immunocytochemical detection system which is based on the detection of FITC-labelled DNA/mRNA hybrids and takes advantage of FITC molecules attached covalently to the DNA probes prior to hybridization. In situ hybridization on cytocentrifuge spots is followed by the application of an anti-fluorescein antibody thus permitting detection of mRNA/DNA-FITC hybrids. The anti-FITC antibody reaction is demonstrated by an indirect immunocytochemical peroxidase-staining method. The T lymphoblast cell line Jurkat and the cDNA for the TCR-beta chain were chosen to establish the technique.

Simultaneous quantitation of two antigens in mixture in individual cells by microimmunofluorimetry.

Double immunofluorescence has so far only been used qualitatively. In present work the possibility of quantitative double immunofluorescence was evaluated in model systems with the ultimate goal to enable simultaneous measurements of different antigens in mixture in biological objects. In one test system a conjugate of fluorescein isothiocyanate (FITC) was studied in combination with conjugates of dimethyl-amino-naphthalene-sulphonyl-chloride (DANSC) or with tetramethyl-rhodamine-isothiocyanate (TRITC) in solutions. In another test system FITC + DANSC antibody conjugates were analyzed after reaction with antigen mixtures in insolubilized form. Fluorescence intensities were measured in a microspectrofluorimeter using narrow band excitation and emission filters of appropriate types for different fluorochromes. Fading was no problem in this system. A formula was developed to calculate the antibody proportions from the fluorescence contribution of either fluorochrome in the region of optimal fluorescence of other fluorochrome. Experimental values obtained from analyses of FITC + DANSC and FITC + TRITC combinations in solutions agreed excellently with theoretically expected values. In the second model system polyacrylic beads were coated with different proportions of two antigens, one labelled with 125I and the other with 131I. After radioactivity measurements the beads were stained with appropriate antisera conjugated with FITC and DANSC, respectively. "Double fluorescence" intensities of individual beads were measured and a very strong correlation was found between the radioactivity and the fluorescence values. The results indicate that quantiation of double fluorescence is possible and meaningful for measurements of two different antigens in mixture on individual particles.

Insolubilization of protein antigens on polyacrylic plastic beads using poly-L-lysine.

A new model system for quantitation of immunofluorescence on single cells is described using poly-L-lysine (PLL) coated polyacrylic plastic beads of approximately cell sizes as carriers for protein antigen. By increasing PLL concentration on the beads increased amounts of 125I labeled antigen were attached to the particles. Excess binding sites of PLL could be completely blocked by unrelated proteins. After staining with FITC-conjugated antibodies and quantitative fluorescence measurements of individual beads using a microspectrofluorimeter, strong correlations were found between antibody and antigen concentration on the beads. Neither repeated washings with PBS nor storage of the beads for two months caused detectable shedding of antigen-antibody complexes. There was a strong linear correlation between fluorescence intensity and the volume of beads, but the correlation between surface area and fluorescence was nonlinear. The described procedure was shown to be a simple method for quantitative and stable coating of particles with proteins. It can be applied as a useful model system for quantitative immunofluorescence studies on intracellular antigens.

Use of antibody-coated polyacrylic plastic beads for semiquantitative determination of cell surface antigens.

Polyacrylic plastic beads coated with specific antibodies, which had previously been shown to bind specifically to cells carrying the related antigen, are shown to react quantitatively with the antigen on individual cells. The number of beads per cell which can be counted by simple microscopy is a measure of the amount of antigen accessible on the cell surface. The method may also be used in combination with other methods for cell surface antigens, such as immunofluorescence, for multiple antigen determination.

Discrimination in immunofluorescence between labeled and unlabeled cells by quantitative microfluorometry and computer analysis.

An automated procedure for discrimination in immunofluorescence between antibody-labeled and unlabeled cells has been developed on the basis of microfluorimetric determination of intensity distributions. After smoothing the raw data for irregularities caused by the scoring statistics optimum fit of the negative distribution to the corresponding positive one was achieved. The procedure was tested in a model system by mixing various known proportions of immunofluorescence-negative and -positive plastic beads. In addition, variable mixtures of T-negative CLL cells and normal mononuclear peripheral blood cells were labeled with FITC-conjugated anti-T-antiserum. The expected percentage of T-positive peripheral blood cells agreed satisfactorily with the data measured and computed. Finally, the measured percentage of Ig-positive mononuclear cells from normal peripheral blood was in agreement with the values obtained by other techniques.

Simultaneous immunofluorimetric quantitation of IgM and IgG in single splenic lymphoid cells after immunization.

The amount of IgM and IgG was determined in individual mouse spleen lymphocytes by double immunofluorimetry after "staining" with dimethylamino-naphthaline sulfonyl chloride-labeled anti-IgM and fluorescein isothiocyanate-labeled anti-IgG. During a single immune response against sheep erythrocytes there was an inverse relationship between the amounts of these two immunoglobulins in individual cells. There was an early steep increase of the cellular amount of IgM during the first 3 days after immunization. Subsequently IgG increased exceeding the initial IgG levels while IgM decreased. Most cells containing high amounts of IgM had a low IgG content and vice versa. The present results directly demonstrate a quantitative switch from IgM to IgG in individual cells on the single cell level after an antigenic stimulus. This indicates simultaneous active production of both immunoglobulins in these cells. For an explanation of this phenomenon differential mRNA splicing rather than DNA rearrangement as an underlying process for immunoglobulin expression may be proposed in these early B-cells.

The influence of radioiodine therapy on the number of circulating epithelial cells (CEC) in patients with differentiated thyroid carcinoma - a pilot study.

GOAL: The aim of this pilot study was to investigate the changes of circulating epithelial cells in the blood of patients with differentiated thyroid cancer after radioiodine-therapy with I-131. METHODS: The cells were detected by fluorescence-microscopy via the epithelial-cell-adhesion-molecule (EpCAM), a molecule described to be over-expressed in most carcinoma tissues and also present on circulating cells deriving from primary site. Epithelial cells were assessed before radioiodine-therapy, as well as 2 days, 14 days, and 3 months after therapy. 2 patient groups were examined: 1) patients with thyroid cancer receiving a first radioiodine-therapy after thyroidectomy (RITfirst, n=13), and 2) patients with thyroid cancer in need of repeated radioiodine-therapy due to local or metastatic recurrences (RITrep, n=15). Circulating epithelial cell changes were correlated to changes of serum-thyroglobulin and to clinical response evaluated 3 months after therapy. RESULTS: Patients with an early decrease of cells after radioiodine-therapy (RITfirst 7/13; RITrep 2/15) showed an increase of serum-thyroglobulin in most of the cases (RITfirst 5/7; RITrep 2/2). In the RITrep group, a decrease in cell counts 2 days after radioiodine-therapy indicated a clinical response in 90% of the cases. CONCLUSIONS: This study indicates that the number of circulating epithelial cells in differentiated thyroid cancer undergo changes in response to radioiodine-therapy. The destruction of cells through radioiodine-therapy may induce a short-term release of thyroglobulin in the blood. A clear relationship between the clinical outcome and the cell changes could not be found, but early cell decreases may help identifying patients more likely to respond to radioiodine-therapy.

Circulating epithelial cells in patients with thyroid carcinoma. Can they be identified in the blood?

GOAL: To investigate whether circulating epithelial cells (CEC) recognized via the epithelial cell adhesion molecule (EpCAM) can be identified in the blood of patients with thyroid carcinoma, given that CEC have already been detected in other types of carcinoma and are considered a potential marker of tumour dissemination. PATIENTS, METHODS: Blood samples of patients with active differentiated thyroid carcinoma (DTC) (n = 50) were compared to samples of patients with: a) recent surgical excision of a thyroid carcinoma (postOP-DTC) (n = 16); b) athyreotic, tumour-free status after radioiodine ablation (AT-DTC) (n= 33); and c) benign thyroid diseases (BTD) (n = 51). Samples of volunteers with normal thyroid parameters (NT) (n = 12) were also investigated. Cells from EDTA-blood were subjected to erythrocyte lysis, isolated by centrifugation, and incubated with a fluorescence-labeled antibody against EpCAM. The numbers of vital cells were counted via fluorescence microscopy. RESULTS: CEC were identified in all groups, with the postOP-DTC group showing the highest mean CEC numbers of all groups. The DTC group had significantly higher CEC numbers than the NT group, and numerically higher numbers than the other groups, although not reaching statistical significance. Within the DTC group there was a correlation between levels of serum thyroglobulin and numbers of CEC (r = 0.409, p = 0.003). CONCLUSIONS: High CEC numbers were not specific to thyroid carcinoma. The methodology used here, based on a single measurement does not allow to identify severe forms of DTC, emphasizing the need of longitudinal measurements throughout therapy. Detection and characterization of tumour thyroid cells in circulation should be based on additional consideration of tissue-specific characteristics.

Heterogeneity of circulating epithelial tumour cells from individual patients with respect to expression profiles and clonal growth (sphere formation) in breast cancer.

BACKGROUND: The detection of tumour cells circulating in the peripheral blood of patients with breast cancer is a sign that cells have been able to leave the primary tumour and survive in the circulation. However, in order to form metastases, they require additional properties such as the ability to adhere, self-renew, and grow. Here we present data that a variable fraction among the circulating tumour cells detected by the Maintrac(®) approach expresses mRNA of the stem cell gene NANOG and of the adhesion molecule vimentin and is capable of forming tumour spheres, a property ascribed to tumour-initiating cells (TICs). PATIENTS AND METHODS: Between ten and 50 circulating epithelial antigen-positive cells detected by the Maintrac approach were selected randomly from each of 20 patients with breast cancer before and after surgery and were isolated using automated capillary aspiration and deposited individually onto slides for expression profiling. In addition, the circulating tumour cells were cultured without isolation among the white blood cells from 39 patients with breast cancer in different stages of disease using culture methods favouring growth of epithelial cells. RESULTS: Although no epithelial cell adhesion molecule (EpCAM)-positive cells expressing stem cell genes or the adhesion molecule vimentin was detected before surgery, 10%-20% of the cells were found to be positive for mRNA of these genes after surgery. Tumour spheres from circulating cells of 39 patients with different stages of breast cancer were grown without previous isolation in a fraction increasing with the aggressivity of the tumour. SUMMARY: Here we show that among the peripherally circulating tumour cells, a variable fraction is able to express stem cell and adhesion properties and can be grown into tumour spheres, a property ascribed to cells capable of initiating tumours and metastases.