Liquid biopsy analysis represents a powerful and noninvasive tool to uncover biomarkers for disseminated disease assessment and longitudinal monitoring of patients. Herein, we explored the value of circulating and disseminated tumor cells (CTC and DTC, respectively) and cell-free DNA (cfDNA) in pediatric rhabdomyosarcoma (RMS). Peripheral blood and bone marrow samples were analyzed to detect and enumerate CTC and DTC, respectively. We used the epithelial cellular adhesion molecule (EpCAM)-based CellSearch platform coupled with an automatic device to collect both EpCAM-positive and EpCAM-low/negative CTCs. The standard assay was implemented, including the mesenchymal marker desmin. For selected cases, we molecularly profiled primary tumors and liquid biopsy biomarkers using whole-exome sequencing and droplet digital PCR, respectively. RMS patients with metastatic disease had a significantly higher number of CTCs compared to those with localized disease, whereas DTCs were detected independently of disease presentation. The use of the desmin marker remarkably increased the identification of CTCs and DTCs in RMS samples. Of note, CTC clusters were detected in RMS patients with disseminated disease. Further, cfDNA and CTC molecular features closely reflected the molecular makeup of primary tumors and informed of disease course.

Metastasis is responsible for the majority of cancer-related deaths. Particularly, challenging is the management of metastatic cancer of unknown primary site (CUP), whose tissue of origin (TOO) remains undetermined even after extensive investigations and whose therapy is rather unspecific and poorly effective. Molecular approaches to identify the most probable TOO of CUPs can overcome some of these issues. In this study, we applied a predetermined set of 89 microRNAs (miRNAs) to infer the TOO of 53 metastatic cancers of unknown or uncertain origin. The miRNA expression was assessed with droplet digital PCR in 159 samples, including primary tumors from 17 tumor classes (reference set) and metastases of known and unknown origin (test set). We combined two different statistical models for class prediction to obtain the most probable TOOs: the nearest shrunken centroids approach of Prediction Analysis of Microarrays (PAMR) and the least absolute shrinkage and selection operator (LASSO) models. The molecular test was successful for all formalin-fixed paraffin-embedded samples and provided a TOO identification within 1 week from the biopsy procedure. The most frequently predicted origins were gastrointestinal, pancreas, breast, lung, and bile duct. The assay was applied also to multiple metastases from the same CUP, collected from different metastatic sites: The predictions showed a strong agreement, intrinsically validating our assay. The final CUPs' TOO prediction was compared with the clinicopathological hypothesis of primary site. Moreover, a panel of 13 miRNAs proved to have prognostic value and be associated with overall survival in CUP patients. Our study demonstrated that miRNA expression profiling in CUP samples could be employed as diagnostic and prognostic test. Our molecular analysis can be performed on request, concomitantly with standard diagnostic workup and in association with genetic profiling, to offer valuable indications about the possible primary site, thereby supporting treatment decisions.