Katharina Pachmann

Monitoring the Response of Circulating Epithelial Tumor Cells to Adjuvant Chemotherapy in Breast Cancer Allows Detection of Patients at Risk of Early Relapse

PURPOSE To demonstrate that it is possible to monitor the response to adjuvant therapy by repeated analysis of circulating epithelial tumor cells (CETCs) and to detect patients early who are at risk of relapse. PATIENTS AND METHODS In 91 nonmetastatic primary breast cancer patients, CETCs were quantified using laser scanning cytometry of anti-epithelial cell adhesion molecule-stained epithelial cells from whole unseparated blood before and during adjuvant chemotherapy. RESULTS Numbers of CETCs were analyzed before therapy, before each new cycle, and at the end of chemotherapy. The following three typical patterns of response were observed: (1) decrease in cell numbers (> 10-fold); (2) marginal changes in cell numbers (< 10-fold); and (3) an (sometimes saw-toothed) increase or an initial decrease with subsequent reincrease (> 10-fold) in numbers of CETCs. Twenty relapses (22%) were observed within the accrual time of 40 months, including one of 28 patients from response group 1, five of 30 patients from response group 2, and 14 of 33 patients from response group 3. The difference in relapse-free survival was highly significant for CETC (hazard ratio = 4.407; 95% CI, 1.739 to 9.418; P < .001) between patients with decreasing cell numbers and those with marginal changes and between patients with marginal changes and those with an increase of more than 10-fold (linear Cox regression model). CONCLUSION These results show that peripherally circulating tumor cells are influenced by systemic chemotherapy and that an increase (even after initial response to therapy) of 10-fold or more at the end of therapy is a strong predictor of relapse and a surrogate marker for the aggressiveness of the tumor cells.

Increase in number of circulating disseminated epithelial cells after surgery for non-small cell lung cancer monitored by MAINTRAC ® is a predictor for relapse: A preliminary report

BackgroundLung cancer still remains one of the most commonly occurring solid tumors and even in stage Ia, surgery fails in 30% of patients who develop distant metastases. It is hypothesized that these must have developed from occult circulating tumor cells present at the time of surgery, or before. The aim of the present study was to detect such cells in the peripheral blood and to monitor these cells following surgery.Methods30 patients treated for lung cancer with surgery were monitored for circulating epithelial cells (CEC) by taking peripheral blood samples before, 2 weeks and 5 months after surgery and/or radiotherapy (RT) chemotherapy (CT) or combined RT/CT using magnetic bead enrichment and laser scanning cytometry (MAINTRAC®) for quantification of these cells.ResultsIn 86% of the patients CEC were detected before surgery and in 100% at 2 weeks and 5 months after surgery. In the control group, which consisted of 100 normal donors without cancer, 97 % were negative for CEC. A significantly higher number of CEC was found preoperatively in patients with squamous cell carcinoma than in those with adenocarcinoma. In correlation to the extent of parenchymal manipulation 2 weeks after surgery, an increase in numbers of CEC was observed with limited resections (18/21) whereas pneumonectomy led to a decrease (5/8) of CEC, 2 weeks after surgery. The third analysis done 5 months after surgery identified 3 groups of patients. In the group of 5 patients who received neo- or adjuvant chemo/radiotherapy there was evidence that monitoring of CEC can evaluate the effects of therapy. Another group of 7 patients who underwent surgery only showed a decrease of CEC and no signs of relapse. A third group of 11 patients who had surgery only, showed an increase of CEC (4 with an initial decrease after surgery and 7 with continuous increase). In the group with a continuous increase during the following 24 months, 2 early relapses in patients with stage Ia adenocarcinoma were observed. The increase of CEC preceded clinical detection by six months.ConclusionWe consider, therefore, that patients with adenocarcinoma and a continuous increase of CEC after complete resection for lung cancer are at an increased risk of early relapse.

Quantification of the response of circulating epithelial cells to neodadjuvant treatment for breast cancer: a new tool for therapy monitoring

IntroductionIn adjuvant treatment for breast cancer there is no tool available with which to measure the efficacy of the therapy. In contrast, in neoadjuvant therapy reduction in tumour size is used as an indicator of the sensitivity of tumour cells to the agents applied. If circulating epithelial (tumour) cells can be shown to react to therapy in the same way as the primary tumour, then this response may be exploited to monitor the effect of therapy in the adjuvant setting.MethodWe used MAINTRAC® analysis to monitor the reduction in circulating epithelial cells during the first three to four cycles of neoadjuvant therapy in 30 breast cancer patients.ResultsMAINTRAC® analysis revealed a patient-specific response. Comparison of this response with the decline in size of the primary tumour showed that the reduction in number of circulating epithelial cells accurately predicted final tumour reduction at surgery if the entire neoadjuvant regimen consisted of chemotherapy. However, the response of the circulating tumour cells was unable to predict the response to additional antibody therapy.ConclusionThe response of circulating epithelial cells faithfully reflects the response of the whole tumour to adjuvant therapy, indicating that these cells may be considered part of the tumour and can be used for therapy monitoring.

A study of Docetaxel-induced effects in MCF-7 cells by means of Raman microspectroscopy

AbstractChemotherapies feature a low success rate of about 25%, and therefore, the choice of the most effective cytostatic drug for the individual patient and monitoring the efficiency of an ongoing chemotherapy are important steps towards personalized therapy. Thereby, an objective method able to differentiate between treated and untreated cancer cells would be essential. In this study, we provide molecular insights into Docetaxel-induced effects in MCF-7 cells, as a model system for adenocarcinoma, by means of Raman microspectroscopy combined with powerful chemometric methods. The analysis of the Raman data is divided into two steps. In the first part, the morphology of cell organelles, e.g. the cell nucleus has been visualized by analysing the Raman spectra with k-means cluster analysis and artificial neural networks and compared to the histopathologic gold standard method hematoxylin and eosin staining. This comparison showed that Raman microscopy is capable of displaying the cell morphology; however, this is in contrast to hematoxylin and eosin staining label free and can therefore be applied potentially in vivo. Because Docetaxel is a drug acting within the cell nucleus, Raman spectra originating from the cell nucleus region were further investigated in a next step. Thereby we were able to differentiate treated from untreated MCF-7 cells and to quantify the cell–drug response by utilizing linear discriminant analysis models. FigureRaman microspectroscopy in combination with powerful chemometric methods (e.g. artificial neural networks) indicates morphological (nucleus fragmentation) and spectral changes in Docetaxel treated breast cancer cells (MCF-7) in comparison to untreated cell samples

Longtime Recirculating Tumor Cells in Breast Cancer Patients

only RBC lysis and no other enrichment apart from a single step of centrifugation, we detect considerable numbers of circulating epithelial cells in 92% of patients with breast or lung cancer and none in 97% of normal controls. Among 16 patients with complete continuous remission, 13 had detectable numbers of circulating epithelial cells which were significantly lower (P = 0.005) than in patients with metastatic disease. Meng et al. speculate that, due to the rapid turnover of circulating tumor cells with a half-life of 1 to 2.5 hours after surgery of the primary tumor, there must be remote tumors, feeding these cells into the circulation. In breast cancer patients, we observe an up to 1,000-fold increase in epithelial cells during the first 3 days after surgery (Fig. 1; 2 out of the now >50 cases). A considerable part of these cells then disappears after another 2 to 4 days, consistent with a rapid turnover of these initially appearing cells. Subsequently, however, the number of epithelial cells remains stable over long periods of time (>1 year, as shown below). The same is true for remnant circulating epithelial cells after completion of adjuvant therapy and after completion of neoadjuvant therapy and surgery (Fig. 2). Others have shown (3) that dormant tumor cells can be long-lived. This agrees with our observations that the number of circulating cells remains at identically high levels for long times without any indication of relapse in the absence of additional interventions. We therefore favor the hypothesis that part of the tumor cells detected in the blood belong to a population of resting, longtime recirculating dormant breast cancer (stem) cells.

Prognostic Role of Circulating Tumor Cells during Induction Chemotherapy Followed by Curative Surgery Combined with Postoperative Radiotherapy in Patients with Locally Advanced Oral and Oropharyngeal Squamous Cell Cancer

Background The prognostic role of circulating tumor cells (CTCs) after induction chemotherapy using docetaxel, cisplatin and fluorouracil (TPF) prior to surgery and adjuvant (chemo)radiation in locally advanced oral squamous cell cancer (OSCC) was evaluated. Methods In this prospective study, peripheral blood samples from 40 patients of the phase II study TISOC-1 (NCT01108042) with OSCC before, during, and after treatment were taken. CTCs were quantified using laser scanning cytometry of anti– epithelial cell adhesion molecule–stained epithelial cells. Their detection was correlated with clinical risk factors, recurrence-free (RFS) and overall survival (OS). Results Before starting the treatment CTCs were detected in 32 of 40 patients (80%). The median number at baseline was 3295 CTCs/ml. The median maximal number of CTCs during treatment was 5005 CTCs/ml. There was a significant increase of CTCs before postoperative radiotherapy compared to baseline before 1st cycle of IC (p = 0.011), 2nd cycle of IC (p = 0.001), 3rd cycle of IC (p = 0.004), and before surgery (p = 0.002), but not compared to end of therapy (p = 0.118). CTCs at baseline >median was also associated to risk of recurrence (p = 0.014). Maximal CTCs during therapy >median was more frequently observed in tumors of the oral cavity (p=0.022) and related to higher risk of death during follow-up (p = 0.028). Patients with CTCs at baseline >median value had significant lower RFS than patients with CTCs at baseline <median value (p = 0.025). Patients with maximal CTCs values >median during the complete course of therapy had a significantly lower OS than patients with values <median (p = 0.049). Finally, the multivariate analysis revealed that OS was significantly lower in patients with maximal CTCs during treatment higher than the median value (HR=6.151; CI: 1.244-30.420). Conclusions Baseline CTCs and maximal CTCs during therapy both seem to be good prognostic markers for OSCC when treated by TPF induction chemotherapy, surgery, and postoperative (chemo)radiation.

Improvement of the separation of tumour cells from peripheral blood cells using magnetic nanoparticles

Circulating tumour cells are a key challenge in tumour therapy. Numerous approaches are on the way to achieving the elimination of these potential sources of metastasis formation. Antibody-directed magnetic cell sorting is supposed to enrich tumour cells with high selectivity, but low efficiency. The short term application of carboxymethyl dextran (CMD) coated magnetit/maghemit nanoparticles allows the discrimination of tumour cells from leukocytes. In the present work we show that the interaction of CMD nanoparticles is cell-type specific and time dependent. The breast cancer cell line MCF-7 and the CML cell line K-562 are characterized by a rapid and high interaction rate, whereas leukocytes exhibit a decelerated behaviour. The addition of carboxymethyl dextran or glucose stimulated the magnetic labelling of leukocytes. The variation of the degree of substitution of dextran with carboxymethyl groups did not affect the labelling profile of leukocytes and MCF-7 cells. In order to verify the in vitro results, whole blood samples from 13 cancer patients were analysed ex vivo. Incubation of the purified leukocyte fraction with CMD nanoparticles in the presence of low amounts of plasma reduced the overall cell content in the positive fraction. In contrast, the absolute number of residual tumour cells in the positive fraction was 90% of the initial amount.

Insulin-like growth factor receptor I (IGF-IR) and vascular endothelial growth factor receptor 2 (VEGFR-2) are expressed on the circulating epithelial tumor cells of breast cancer patients.

Background Circulating epithelial tumor cell (CETC) analysis is a promising diagnostic field for estimating the risk for metastatic relapse and progression in patients with malignant disease. CETCs characterization can be used as a liquid biopsy for prognostic and predictive purposes in breast and other cancers. IGF-IR and VEGFR-2 play an important role in tumor growth and the progression of cancer disease. The purpose of the current study was therefore to investigate their expression on CETCs. Methods CETCs were determined from the blood of 50 patients suffering from breast cancer. The number of vital CETCs and the expression of IGF-IR and VEGFR-2 were investigated using the maintrac® method. Results IGF-IR and VEGFR-2 expression on the surface of CETCs were detected in 84% of patients. A statistically high correlation was found between IGF-IR and VEGFR-2 (r = 0.745 and p<0.001) on the CETCs. The co-expression of both receptors was confirmed in some experiments and ranged between 70% and 100%. Statistically significant correlations were observed between the number of CETCs and IGF-IR (r = 0.315 and p<0.05) and VEGFR-2 (r = 0.310 and p<0.05) expression. The presence of CETCs and the level of IGF-IR and VEGFR-2 expression were not associated with tumor stage, hormone receptor status or nodal/distant metastasis. Summary In this study, a parallel and co-expression of IGF-IR and VEGFR-2 was examined on the surface of CETCs in breast cancer patients for the first time. Characterization of CETCs may be a promising approach for the rational design of targeted anticancer therapies.

Monitoring circulating epithelial tumor cells (CETCs) during primary systemic chemotherapy including trastuzumab for early prediction of outcome in patients with Her2/neu-positive tumors

In Her2/neu growth factor overexpressing poor prognosis breast cancers trastuzumab has shown to improve disease-free survival as well as overall survival in metastatic and primary diseases. On the basis of these positive results, trastuzumab has also been included into studies of neo-adjuvant therapy of Her2/neu-positive T2 tumors [1].

Short‐Term Application of Magnetic Core‐Shell Nanoparticles—Effect on Immune Cells

The effect of magnetic nanoparticles on the survival of leukocytes in general and especially the lymphocytes as important parts of the immune system during incubation and separation was analyzed. Primary leukocytes were inoculated with magnetic core/carboxymethyl‐dextran (CMD) nanoparticles for 4 to 30 minutes. Labelled cells were separated by MACS, counted and analyzed by FACS. T‐Lymphocytes were identified by CD3 and B‐Lymphocytes by CD19. After magnetic separation granulocytes represented the majority of cells in both, the positive and negative fraction. Most of the lymphocytes were detected in the negative fraction. The number of T‐Lymphocytes in the positive fraction increased 2.5‐fold from 4 to 16 minutes, whereas the amount of B‐Lymphocytes remains constant. T cells could be expanded after short‐term incubation with nanoparticles indicating full biological activity. Our enrichment procedure of tumor cells from peripheral blood preserves the integrity and biological activity of leukocytes in the neg...