Cornelia Coith

PURPOSE: The CellSearch system (Veridex, Warren, NJ) is designed to enrich and enumerate circulating tumor cells (CTCs) from peripheral blood. Here, we validated the analytic performance of this system for clinical use in patients with metastatic breast cancer. EXPERIMENTAL DESIGN: This prospective multicenter study conducted at three independent laboratories involved samples from 92 patients with metastatic breast cancer. Intra- and inter-assay variability using controls containing defined numbers of cells (average, 50 and 1,000, respectively), cell stability based on varying storage and shipment conditions, recovery precision from samples spiked with 4 to 12 tumor cells, inter-instrument variability, and positivity of samples from metastatic breast cancer patients were tested. RESULTS: Intra- and inter-assay precision for two sites were high: All eight positive controls analyzed in the same run and >95% of the run to run control values (n=299) were within the specified ranges. Recovery rate of spiked samples averaged between 80% and 82%. CTCs were detected in approximately 70% of metastatic breast cancer patients. CTC values of identical samples processed either immediately after blood drawing or after storage for 24, 48, or 72 h at room temperature or at 4 degrees C did not differ significantly. Shipment of samples had no influence on CTC values. When analyzing identical samples in different centers, inter-instrument accordance was high. CONCLUSIONS: The CellSearch system enables the reliable detection of CTCs in blood and is suitable for the routine assessment of metastatic breast cancer patients in the clinical laboratory. Blood samples should be shipped at room temperature and CTC counts are stable for at least 72 h.

Immune checkpoint inhibition (ICI) of the PD-1/PD-L1 axis shows durable responses in a subset of patients with metastatic urothelial carcinoma (UC). However, PD-L1 expression in tumor biopsies does not necessarily correlate with response to PD-1/PD-L1 inhibitors. Thus, a reliable predictive biomarker is urgently needed. Here, the expression of PD-L1 on circulating tumor cells (CTCs) in blood from patients with advanced UC was analyzed. For this purpose, an assay to test PD-L1 expression on CTCs using the CellSearch® system was established using cells of five UC cell lines spiked into blood samples from healthy donors and applied to a heterogeneous cohort of UC patients. Enumeration of CTCs was performed in blood samples from 49 patients with advanced UC. PD-L1 expression in ≥1 CTC was found in 10 of 16 CTC-positive samples (63%). Both intra- and inter-patient heterogeneity regarding PD-L1 expression of CTCs were observed. Furthermore, vimentin-expressing CTCs were detected in 4 of 15 CTC-positive samples (27%), independently of PD-L1 analysis. Both CTC detection and presence of CTCs with moderate or strong PD-L1 expression correlated with worse overall survival. Analyses during disease course of three individual patients receiving ICI suggest that apart from CTC numbers also PD-L1 expression on CTCs might potentially indicate disease progression. This is the first study demonstrating the feasibility to detect CTC-PD-L1 expression in patients with advanced UC using the CellSearch® system. This assay is readily available for clinical application and could be implemented in future clinical trials to evaluate its relevance for predicting and monitoring response to ICI.

PURPOSE: Over the past 5 years, several clinical studies on a total of approximately 2500 patients have shown that the immunocytochemical detection of occult metastatic tumor cells in bone marrow (BM) at primary surgery provides important prognostic information in breast cancer (e.g., Ref 13 ). Here, we evaluated whether these cells can survive first-line chemotherapy and express epithelial cell adhesion molecule (Ep-CAM), recently suggested as promising target for immunotherapeutic interventions in breast cancer. EXPERIMENTAL DESIGN: A total of 62 patients with node-negative and -positive breast cancer but without distant metastases (Tumor-Node-Metastasis stage M(0)) was treated with two or more courses of various forms of adjuvant chemotherapy (e.g., cyclophosphamide-methotrexate-5-fluorouracil, anthracyclines). After chemotherapy, BM was aspirated from the upper iliac crest and analyzed for the presence of tumor cells. A first cohort of 34 BM aspirates was enriched for tumor cells by Ficoll density gradient centrifugation, and 2-4 x 10(6) mononuclear cells were analyzed per patient. The tumor cells were detected by anticytokeratin monoclonal antibody (Mab) A45-B/B3 and double labeled with Mab 3B10 against an Ep-CAM-epitope. The subsequent 27 BM aspirates were specifically enriched for Ep-CAM(+) cells using magnetic beads coupled to Mab 3B10, and tumor cells were identified by Fab fragments of Mab A45-B/B3 directly conjugated with alkaline phosphatase. RESULTS: After chemotherapy, 10 of 35 (28.6%) Ficoll-enriched BM samples contained cytokeratin-positive tumor cells. In total, 26 cytokeratin-positive cells were detected, but none of these cells coexpressed Ep-CAM. Even within the second cohort of 27 Ep-CAM-enriched BM samples, only 2 specimens (7.4%) harbored cytokeratin-positive cells costaining with the Ep-CAM antibody. CONCLUSION: Our results indicate that disseminated breast cancer cells in BM can survive first-line adjuvant chemotherapy. Ep-CAM expression is, however, restricted to a subset of these cells, which may limit the broad applicability of Ep-CAM as target for second-line adjuvant therapy in breast cancer.

Modern technologies enable detection and characterization of circulating tumor cells (CTC) in peripheral blood samples. Thus, CTC have attracted interest as markers for therapeutic response in breast cancer. First studies have incorporated CTC analyses to guide therapeutic interventions and stratification of breast cancer patients. Aim of this study was to analyze characteristic features of CTC as biomarker for predicting resistance to HER2-targeted therapies. Therefore, CTC from metastatic breast cancer patients with HER2-negative primary tumors screened for the prospective randomized phase III trial DETECT III were explored for their HER2 status and the presence of PIK3CA mutations. Detection and characterization of HER2 expression of CTC were conducted with the CellSearch(®) system. Fifteen of 179 CTC-positive patients (8.4%) contained ≥1 CTC with strong HER2 expression. Genomic DNA from individual CTC isolated by micromanipulation was propagated by whole genome amplification and analyzed for PIK3CA mutations in exons 9 and 20 by Sanger sequencing. One or more CTC/7.5 mL were detected in 179/290 patients (61.7%). In 109 patients (34.8%), ≥5 CTC/7.5 mL were found. We detected at least one CTC with the mutation p.E542K, p.E545K, p.H1047R, p.H1047L or p.M1043V in 12/33 patients (36.4%). Thirty six of 114 CTC (31.6%) harbored one of these mutations. CTC in individual patients exhibited heterogeneity concerning PIK3CA mutations and HER2 expression. In conclusion, clinically relevant genomic aberrations such as mutations in the hotspot regions of exon 9 and 20 of the PIK3CA gene can be detected in single CTC and might provide insights into mechanisms of resistance to HER2-targeted therapies.