Dynamic changes of Receptor activator of nuclear factor-κB expression in Circulating Tumor Cells during Denosumab predict treatment effectiveness in Metastatic Breast Cancer

Abstract

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 Timeto-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. Receptor activator of nuclear factor-kappaB ligand (RANKL)/RANK axis represents the main characterized therapeutic target to prevent bone metastasis in solid tumors 1. About 70% metastatic breast cancer (MBC) patients develops skeletal metastases often associated to skeletal related events (SREs) with a serious negative impact on patients' quality of life 2. Accumulating evidences suggest that RANKL/RANK signaling, not only influences bone microenvironment, mediating osteoclast activity and survival, but also promotes BC metastases 1,3. Indeed, RANK-RANKL blocking antibodies showed antitumor efficacy in bone MBC preclinical models both in preventive 4,5 and therapeutic settings 4,6,7. This antitumor effect could be either "indirect" through the inhibition of bone resorption that, in

Pediatric sarcomas display a variable EpCAM expression in a histology-dependent manner

“…Moreover, to investigate the co-expression of both epithelial and mesenchymal markers in CTCs of pediatric sarcomas, we used the anti-Desmin Ab (clone Y66; abcam, UK), in conjunction with the CTC standard assay, as previously reported for other integrated tests [ 25 , 26 ]. Results were expressed as the total number of CTCs and Desmin-positive CTCs per 7.5 ml of blood.…”

Current Status of Circulating Tumor Cells, Circulating Tumor DNA, and Exosomes in Breast Cancer Liquid Biopsies

“…Today, breast cancer patients with bone metastases are treated with denosumab, an inhibitory antibody targeting RANKL. Recently, Pantano et al showed that RANK-expressing CTCs were present in blood from breast cancer patients with skeletal metastases [ 50 ]. Moreover, the presence of RANK-positive CTCs was associated with an increased response to denosumab treatment.…”

Cellular Mechanisms of Circulating Tumor Cells During Breast Cancer Metastasis

“…The presence of CTCs increases the risk of metastasis, and higher numbers of CTCs are inversely associated with progression-free survival and overall survival in patients with breast cancer [ 12 , 14 , 15 ]. CTCs have been suggested as a prognostic tool for monitoring metastasis or the efficacy of chemotherapy [ 16 , 17 , 18 , 19 ]. Studies have shown that potential diagnostic biomarkers representing stemness [ 20 ], immunogenic CTC [ 18 ], and signaling molecules that promote breast cancer metastasis [ 19 ] are found in CTCs.…”

“…CTCs have been suggested as a prognostic tool for monitoring metastasis or the efficacy of chemotherapy [ 16 , 17 , 18 , 19 ]. Studies have shown that potential diagnostic biomarkers representing stemness [ 20 ], immunogenic CTC [ 18 ], and signaling molecules that promote breast cancer metastasis [ 19 ] are found in CTCs. The mutation and expression levels of breast cancer-associated genes such as BRCA 1/2 are also detectable by liquid biopsy [ 15 , 21 ].…”