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.

Detection and quantitation of circulating tumor cells from solid epithelial tumors could become a valuable tool for therapy monitoring if the procedure can be standardized. In the present work we assessed the influence of pre-analytical handling, storage and white blood cell isolation on analysis of a population of spiked tumor cell-line cells and intrinsically present epithelial cells in the peripheral blood of breast and lung cancer patients and the sensitivity of their detection. Sucrose density separation did not enrich epithelial cells, and even depleted them, leading to a gross underestimation of their numbers (3/13 positive, between 2.9 and 50 cells/mL) in comparison to red blood cell lysis (13/13 positive, between 77,200 and 800 cells/mL). Short-term storage of whole blood samples for up to 7 days had little influence on the number of epithelial cells recovered. The effectiveness of magnetic bead enrichment was dependent on the number of relevant cells and the volume used for enrichment. Red blood cell lysis and fluorochrome-labeled antibody staining in a no-wash procedure with subsequent laser scanning cytometry allowed the detection of circulating epithelial cells in 92% of breast and lung cancer patients. Two examples of how this method can be applied for the longitudinal analysis in individual patients are shown, with an increase in numbers preceding relapse and a decrease paralleling tumor reduction. The proposed simple and easy method allows close monitoring, which may help in real-time analysis of the response of solid tumors, especially their systemic component, to therapy and hopefully will contribute to more individually tailored therapy.

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.

BackgroundThe current cancer research strongly focuses on immune therapies, where the PD-1, with its ligands plays an important role. It is known that PD-L1 is frequently up-regulated in a number of different cancers and the relevance of this pathway has been extensively studied and therapeutic approaches targeting PD-1 and PD-L1 have been developed. We used a non-invasive, real-time biopsy for determining PD-L1 and PD-L2 expression in CETCs of solid cancer patients.MethodsCETCs were determined from blood of 128 patients suffering from breast (72), prostate (27), colorectal (18) and lung (11) cancer. The number of vital CETCs and the expression of PD-L1 and PD-L2 were investigated using the maintrac® method.ResultsPD-L1 expressing CETCs were detected in 94.5% of breast, 100% of prostate, 95.4% of colorectal and 82% of lung cancer patients whereas only 75% of breast cancer patients had PD-L2 positive CETCs. In the PD-L1 and PD-L2 expressing patients the cell fraction of PD-L1 positive CETCs is significantly higher than the fraction of PD-L2 positive CETCs (54.6% vs. 28.7%; p<0.001). Breast cancer patients with metastatic disease had significantly more PD-L1 positive CETCs as compared to patients without metastasis (median 75% vs. 61.1%; p<0.05).ConclusionPD-L1 seems to be a major factor in immune evasion and is highly expressed on CETCs regardless of the type of cancer. Monitoring the frequency of PD-L1 positive CETCs could reflect individual patient's response for an anti-PD-1/PD-L1 therapy and may be a promising target of anticancer treatment.

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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.

Introduction In 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.

BackgroundThe 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.MethodsIn 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).ResultsBefore 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 during the complete course of therapy had a significantly lower OS than patients with values