Bladder cancer circulating tumor cell concentration fluctuates in the absence of systemic therapy or surgery

INTRODUCTION AND OBJECTIVES: 00 Liquid biopsies 00 such as circulating tumor cells (CTCs) aim to monitor response to therapy or provide a noninvasive measure of disease progression.In bladder cancer, it is not known to what extent CTC counts fluctuate over time during the same treatment phase (in the absence of systemic therapy or surgery).Understanding the baseline variation in CTC counts is a crucial step towards defining what constitutes a meaningful change in CTC detection.Here, we collected peripheral blood (PB) samples from metastatic bladder cancer patients with replicates from the same draw and also at multiple time points over different days.METHODS: 3 replicate PB samples (A, B, C) were collected on day 1 from 5 metastatic bladder cancer patients.On days 2 and 3, PB was available from 80% (n¼4/5) patients.The median CTC count of the 3 replicates from day 1 was used.Each PB was processed with the AccuCyteâ system to isolate and transfer the buffy coat onto 8 slides.Slides were stained with DAPI (nuclear), anti-pan-cytokeratin and anti-EpCAM (epithelial), and anti-CD45/CD66b/CD34/CD14/CD11b (white blood cell) antibodies.Slides were scanned and analyzed using the CyteFinderâ system, an assay validated to have high recovery (>80%) and sensitivity for detecting individual CTCs (defined as DAPI positive, epithelial-marker positive, and white blood cell marker negative).RESULTS: For replicate samples, the range of CTC counts/7.5mL PB for Patient 1 was 70 (A¼43, B¼102, C¼32), Patient 2 was 5 (A¼6, B¼2, C¼1), Patient 3 was 1 (A¼1, B¼0, C¼0), Patient 4 was 2 (A¼2, B¼4, C¼2), and Patient 5 was 1 (A¼1, B¼1, C¼0).The median of the ranges (70, 5, 1, 2, 1) was 2 CTCs/7.5 mL PB.For PB collected on different days, the range of CTC counts for Patient 1 was 192 (43, 170, 235), Patient 2 was 11 (2, 13, 5), Patient 3 was 3 (0, 3, 3), and Patient 4 was 4 (2, 5, 6).The median of the ranges (192, 11, 3, 4) was 7 CTCs/7.5 mL PB.CONCLUSIONS: While the median fluctuations of 2 and 7 CTCs/7.5mLPB were low, in certain cases we did detect considerable variation in CTC count from sample to sample and from day to day.Ongoing work is necessary to study the natural fluctuation in CTC counts and the heterogeneity of blood CTC concentration from moment to moment.Given the many challenges of CTC detection, ctDNA may emerge as the preferred liquid biopsy in bladder cancer.