The need for a liquid biopsy in non‐small cell lung cancer (NSCLC) patients is rapidly increasing. We studied the relation between overall survival (OS) and the presence of four cancer biomarkers from a single blood draw in advanced NSCLC patients: EpCAM^highcirculating tumor cells (CTC), EpCAM^lowCTC, tumor‐derived extracellular vesicles (tdEV) and cell‐free circulating tumor DNA (ctDNA). EpCAM^highCTC were detected with CellSearch, tdEV in the CellSearch images and EpCAM^lowCTC with filtration after CellSearch. ctDNA was isolated from plasma and mutations present in the primary tumor were tracked with deep sequencing methods. In 97 patients, 21% had ≥2 EpCAM^highCTC, 15% had ≥2 EpCAM^lowCTC, 27% had ≥18 tdEV and 19% had ctDNA with ≥10% mutant allele frequency. Either one of these four biomarkers could be detected in 45% of the patients and all biomarkers were present in 2%. In 11 out of 16 patients (69%) mutations were detected in the ctDNA. Two or more unfavorable biomarkers were associated with poor OS. The presence of EpCAM^highCTC and elevated levels of tdEV and ctDNA was associated with a poor OS; however, the presence of EpCAM^lowCTC was not. This single tube approach enables simultaneous analysis of multiple biomarkers to explore their potential as a liquid biopsy.

We investigated whether Circulating Tumor Cells (CTCs) isolated from epithelial tumors could survive and grow in xenotransplants. To this purpose, EpCAM-positive CTCs were enriched by CellSearch platform the only FDA-cleared automated platform that quantifies tumor burden in peripheral blood and provides clinical evidence of predictive and prognostic value. The CTCs were isolated from metastatic prostate (n=6) and breast (n=2) cancer patients. The xenograft assay was developed in 8-week-old NOD/SCID mice that were subcutaneously injected with increasing amounts of CTCs (ranging from 50 to 3000). Human CTCs were found in 8 out of 8 murine peripheral blood (muPB) and in 6 out of 8 murine bone marrow (muBM) samples, after a median follow-up of 10.3 months. Six out of 8 spleens were positive for human cytokeratin. Our assay showed higher successful rate than those previously reported in breast cancer and hepatocellular carcinoma. The role of EpCAM during carcinogenesis is controversial. The identification of human CTCs in muPB, muBM and spleen demonstrates that the EpCAM-positive fraction of CTCs retains the migratory capacity. This is the first experimental evidence that as few as 50 EpCAM-positive prostate cancer CTCs putatively contain metastasis-initiating-cells (MIC).

Background: Circulating tumor cells (CTCs) correlate with adverse prognosis in patients with breast, colorectal, lung, and prostate cancer. Little data are available for renal cell carcinoma (RCC). Materials and Methods: We designed a multicenter prospective observational study to assess the correlation between CTC counts and progression-free survival (PFS) in patients with metastatic RCC treated with an antiangiogenic tyrosine kinase inhibitor as a first-line regimen; overall survival (OS) and response were secondary objectives. CTC counts were enumerated by the CellSearch system at four time points: day 0 of treatment, day 28, day 56 and then at progression, or at 12 months in the absence of progression. Results: One hundred ninety-five eligible patients with a median age of 69 years were treated with sunitinib (77.5%) or pazopanib (21%). At baseline, 46.7% of patients had one or more CTCs per milliliter (range, 1 to 263). Thirty patients had at least three CTCs, with a median PFS of 5.8 versus 15 months in the remaining patients (p = .002; hazard ratio [HR], 1.99), independently of the International Metastatic RCC Database Consortium score at multivariate analysis (HR, 1.91; 95% confidence interval [CI], 1.16–3.14). Patients with at least three CTCs had a shorter estimated OS of 13.8 months versus 52.8 months in those with fewer than three CTCs (p = .003; HR, 1.99; multivariate analysis HR, 1.67; 95% CI, 0.95–2.93). Baseline CTC counts did not correlate with response; neither did having CTC sequencing counts greater than or equal to one, two, three, four, or five. Conclusion: We provide prospective evidence that the presence of three or more CTCs at baseline is associated with a significantly shorter PFS and OS in patients with metastatic RCC. Implications for Practice: This prospective study evaluated whether the presence of circulating tumor cells (CTCs) in the peripheral blood correlates with activity of first-line tyrosine kinase inhibitors in metastatic renal cell carcinoma (RCC). This study demonstrated that almost half of patients with metastatic RCC have at least one CTC in their blood and that those patients with at least three CTCs are at increased risk of early progressive disease and early death due to RCC. Studies incorporating CTC counts in the prognostic algorithms of metastatic RCC are warranted.

Receptor-activator of nuclear-factor –κB-ligand (RANKL) and its receptor RANK have been recently identified as key players in breast cancer bone metastases. Since Circulating Tumor Cells (CTCs) are considered a crucial step of metastatic process, we explored RANK expression on CTCs in metastatic breast cancer (MBC), and the predictive value of RANK-positive CTCs in monitoring patients during treatment with denosumab (anti-RANKL antibody). To this purpose, we developed a novel CTC assay to quantify RANK-positive CTCs in forty-two bone MBC patients, candidates to denosumab treatment. Companion algorithms ΔAUC and Slope were developed, and correlated with time to first skeletal-related-events (SRE), time to bone metastasis progression and time to visceral metastasis progression. Twenty-seven patients had at least one CTC at baseline and, among these, nineteen (70%) had one or more RANK–positive CTCs. Notably, the baseline total CTCs, but not the RANK-positive, were associated with Time-to-first-SRE, Time-to-Bone-Metastasis-Progression and Time-to-Visceral-Metastasis-Progression. Conversely, during treatment monitoring, positive ΔAUC value, expression of RANK-positive CTCs persistence, correlated with longer Time-to-first-SRE (p = 0.0002) and Time-to-Bone-Metastasis-Progression (p = 0.0012). Furthermore, the early increase at second day, in RANK-positive CTCs (Positive-Slope) was associated with delay in time-to-first-SRE (p = 0.0038) and Time-to-Bone-Metastasis-Progression (p = 0.0024). We demonstrate, for the first time, the expression of RANK on CTCs in MBC patients and that the persistence of RANK expression determines denosumab effectiveness.