[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.

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.

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.

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.

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.

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 (AU)

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The relevance of circulating epithelial tumor cells (CETC) for therapy monitoring during neoadjuvant (primary systemic) chemotherapy in breast cancer.

BACKGROUND: Having demonstrated in a previous report that the response of circulating epithelial tumor cells (CETC) during the first cycles of primary (neoadjuvant) chemotherapy perfectly reflects the response of the tumor, in the present study the changes in cell numbers during subsequent cycles and their possible impact on the therapy's outcome were examined. PATIENTS AND METHODS: In 58 breast cancer patients CETC were quantified during therapy with either EC (epirubicin/ cyclophosphamid) or dose intensified E (epirubicin) followed by taxane, with or without trastuzumab, and subsequent CMF (cyclophosphamid/methorexate/ fluorouracil). RESULTS: CETC numbers declined more than 10-fold (good response) in 65% (her2/neu-negative) and 55% (her2/neu-positive) of patients during EC, and in 60% during dose intensified E, respectively, followed by an increase of CETC in all patients. CETC remained increased, decreasing only when adding CMF. A good initial response correlated with estrogen-receptor negativity, a poor response with early distant relapse (P < 0,0001, hazard ratio = 11.91). CONCLUSION: Response of CETC already during the first cycles of neoadjuvant treatment predicts the final response of the tumor. Hitherto unknown effects of the release of tumor cells during therapy further our understanding of tumor-blood interaction and may improve access of agents like antibodies to cells. The impact on the further course of disease remains to be evaluated.

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.

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.

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.

Detection and quantification of small numbers of circulating tumour cells in peripheral blood using laser scanning cytometer (LSC).

The detection of circulating tumour cells disseminated from solid tumours requires extremely sensitive methods. Molecular genetic methods, which are most sensitive, are not applicable to solid tumours because no tumour-specific genetic markers are available. Detection of disseminated tumour cells by immunocytochemistry is time-consuming, whereas fluorimetry is fast and quantitative. The laser scanning cytometer (LSC) provides an automated microscopic procedure for screening up to 5x10(4) cells in suitable time. Using this system together with an enrichment procedure which allows up to ten thousand-fold enrichment, we have quantified minimal numbers of tumour cells. In a model system, breast cancer cell line cells diluted into peripheral blood mimicked seeding of tumour cells into the periphery. After staining with fluorochrome-conjugated anti-epithelial antibody, slides were screened for positive events directly or after enrichment with antibody-coated magnetic beads. One positive cell was unequivocally detectable in 10(4) cells and 50 out of 60 tumour cells were reliably recovered from a 20 ml blood volume, equal to 1-2 cells per 10(7), after magnetic bead enrichment. This method allows quantitation of tumour cells in peripheral blood and bone marrow in reasonable time and will, for the first time, enable extensive investigation of the seeding behaviour of tumours.

Expression of bcr-abl mRNA in individual chronic myelogenous leukaemia cells as determined by in situ amplification.

We present the results of a novel method developed for evaluation of in situ amplification, a molecular genetic method at the cellular level. Reverse transcription polymerase chain reaction (RT-PCR) was used to study bcr-abl transcript levels in individual cells from patients with chronic myelogenous leukaemia (CML). After hybridizing a fluorochrome-labelled probe to the cell-bound RT-PCR product, bcr-abl mRNA-positive cells were determined using image analysis. A dilution series of bcr-abl-positive BV173 into normal cells showed a good correlation between expected and actual values. In 25 CML samples, the percentage of in situ PCR-positive cells showed an excellent correlation with cytogenetic results (r = 0.94, P < 0.0001), interphase fluorescence in situ hybridization (FISH) (r = 0.95, P = 0.001) and hypermetaphase FISH (r = 0.81, P < 0.001). The fluorescence intensity was higher in residual CML cells after interferon (IFN) treatment than in newly diagnosed patients (P = 0.004), and was highest in late-stage CML resistant to IFN therapy and lowest in CML blast crisis (P = 0.001). Mean fluorescence values correlated with bcr-abl protein levels, as determined by Western blot analysis (r = 0.62). Laser scanning cytometry allowing automated analysis of large numbers of cells confirmed the results. Thus, fluorescence in situ PCR provides a novel and quantitative approach for monitoring tumour load and bcr-abl transcript levels in CML.

Applications of consensus polymerase chain reaction with subsequent electrophoretic distinction of amplificates.

Conserved sequences within gene families permit the design of consensus primers that match several members of a given class of homologous genes. Polymerase chain reaction (PCR) products obtained with such consensus primers were characterized by restriction mapping or single-strand conformation polymorphism (SSCP) analysis, using precast polyacrylamide minigels and automated silver staining. Examples for the electrophoretic distinction of consensus amplificates are presented in the fields of guanylyl cyclase expression studies and in the determination of B-cell clonality in human blood samples. Guanylyl cyclase expression in inner ear tissues of guinea pigs was investigated by reverse transcription PCR using consensus primers with specificity for the subclass of particulate guanylyl cyclases. The resulting PCR products were assigned to three representatives of this group by restriction mapping. The consensus PCR approach enabled the detection of an unexpected receptor type, namely guanylyl cyclase C, in the inner ear. The distinction by SSCP analysis of denatured consensus amplificates was appropriate for the identification of clone-specifically rearranged immunoglobulin heavy chain genes of B-lymphocytes. Genomic DNA isolated from blood samples of leukemia patients served as the template for the consensus amplification of clone-specific VDJ rearrangements. Rapid distinction and re-identification of consensus PCR products was achieved by SSCP analysis for regular antigen receptor rearrangements and for t(14; 18) translocations. The potential of these procedures for detecting leukemia or lymphoma clones when monitoring minimal residual disease was assessed.

Complementary anchor PCR of rearranged variable T-cell receptor beta-chain cDNA regions.

Sequencible amplificates comprising the variable cDNA sequences of the rearranged T-cell receptor (TCR) beta-chain were obtained from the T-leukemia cell line Jurkat using a single-sided PCR approach based on five synthetic oligonucleotides derived from the flanking constant sequence. Double-stranded cDNA was cleaved by a restriction enzyme creating cohesive ends, to which an anchor oligonucleotide was ligated. Since this anchor was complementary to the antisense strand of the known constant region, exclusively the desired ligation product folded into a stem-loop-structure that was enzymatically extended to yield a PCR template, now flanked at both ends by primer binding sites appropriate for nested PCR.

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.

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.

Influence of OM-85 BV on different humoral and cellular immune defense mechanisms of the respiratory tract.

To clarify the mode of action of an oral bacterial extract (OM-85 BV) on local airway immunity pre- and posttherapeutic washings from bronchoalveolar lavage (BAL) fluid of 28 adult patients with nonobstructive chronic bronchitis were analysed. In comparison to healthy controls, an elevation of total cell count due to an increased number of PMN leukocytes, and an impaired activity of the alveolar macrophages measured by the chemiluminescence response to opsonized zymosan was observed in patients with chronic bronchitis. After treatment with OM-85 BV, the BAL CD4+/CD8+ lymphocyte ratio and BAL interferon-gamma levels were increased. The alveolar macrophage activity was normalized and the BAL IgA was regulated from a reduced or hyperelevated to a moderately increased level.

In situ amplification of single copy gene segments in individual cells by the polymerase chain reaction.

A method is described using the polymerase chain reaction (PCR) to amplify defined nucleic acid strands in individual cells in situ in conventional smears of bone marrow and peripheral cells. Using radioactively labeled precursors, the incorporation into newly synthesized strands by PCR can be detected by microautoradiography. The specificity of the method can be monitored by gel electrophoresis of the material shed into the reaction mixture. Thus it could be shown that even single genes in individual cells can be amplified to visibility. In a mixture of HIV infected and non infected cells both can be clearly distinguished from one another.