Liquid biopsy, consisting in the non-invasive analysis of circulating tumor-derived material (the Tumor Circulome), represents an innovative tool in precision oncology to overcome current limitations associated with tissue biopsies. Within the tumor circulome, ctDNA and CTCs are the only components whose clinical application is FDA-cleared. Extracellular vesicles, ctRNA and tumor-educated platelets are relatively novel tumor circulome constituents with promising potential at each stage of cancer management. Here, we discuss the clinical applications of each element of the tumor circulome and the prevailing factors that currently limit implementation in clinical practice. We also detail the most recent technological developments in the field, which demonstrate potential in improving the clinical value of liquid biopsies. Commented [GDR3R2]: Thanks for doing that Commented [MK(2]: Please include all weblinks in a separate section before "References", entitled "Resources" Commented [MK(1]: Please include all weblinks in a separate section before "References", entitled "Resources" Commented [MK(4]: Since this is how it is depicted in the Figure. Commented [MK(5]: I suggest that this be converted into "Key Figure". The Figure represents the contents of the review well. Commented [MK(6]: Are the authors talking about advances in sample isolation? I'd encourage you to give examples to align the readers to what advancements the authors are referring to. Commented [GDR7R6]: Done with some of the innovations discussed in the following chapters.

Cancer management paradigms are shifting towards a personalized approach thanks to the advent of the -omics technologies. Liquid biopsies, consisting in the sampling of blood and other bodily fluids, are emerging as a valid alternative to circulating tumor biomarkers and tumor tissue biopsies for cancer diagnosis, routine monitoring and prognostication. The content of a liquid biopsy is referred to as the "tumor circulome". Among its components, circulating tumor DNA (ctDNA), including both cell-free and exosome-associated DNA, is the most widely characterized element. ctDNA analysis has a tremendous capability in the diagnostic arena. Its potential has been demonstrated at each level of disease staging and management and supported by a recent FDA approval for companion diagnostic, and the investments being made by pharmaceutical companies in this sector are numerous. The approaches available for ctDNA analysis allow both quantitative and qualitative studies and range from PCR and dPCR-mediated single/multiple gene mutational assessment to whole genome next generation sequencing and methylation mapping. Although the principal object of a liquid biopsy is blood, other body fluids such as urine and saliva show potential as complementary DNA sources for tumor analysis. In this review we provide a synopsis on the state of play of current ctDNA application. We discuss the clinical significance of ctDNA analysis and review the state of the art of technologies being currently developed to this aim. We also discuss the current issues limiting ctDNA application and highlight the promising approaches being developed to overcome these.

Multiple myeloma is an incurable cancer of bone marrow plasma cells, with a 5-year survival rate of 43%. Its incidence has increased by 126% since 1990. Treatment typically involves high-dose combination chemotherapy, but therapeutic response and patient survival are unpredictable and highly variable-attributed largely to the development of multidrug resistance (MDR). MDR is the simultaneous cross-resistance to a range of unrelated chemotherapeutic agents and is associated with poor prognosis and survival. Currently, no clinical procedures allow for a direct, continuous monitoring of MDR. We identified circulating large extracellular vesicles (specifically microparticles (MPs)) that can be used to monitor disease burden, disease progression and development of MDR in myeloma. These MPs differ phenotypically in the expression of four protein biomarkers: a plasma-cell marker (CD138), the MDR protein, P-glycoprotein (P-gp), the stem-cell marker (CD34); and phosphatidylserine (PS), an MP marker and mediator of cancer spread. Elevated levels of P-gp + and PS + MPs correlate with disease progression and treatment unresponsiveness. Furthermore, P-gp, PS and CD34 are predominantly expressed in CD138 − MPs in advanced disease. In particular, a dual-positive (CD138 − P-gp + CD34 + ) population is elevated in aggressive/unresponsive disease. Our test provides a personalised liquid biopsy with potential to address the unmet clinical need of monitoring MDR and treatment failure in myeloma.

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The molecular complexes formed by specific members of the family of CARMA proteins, the CARD domain-containing adapter molecule BCL10 and MALT1 (CBM complex) represent a central hub in regulating activation of the pleiotropic transcription factor NF-κB. Recently, missense mutations in CARMA2sh have been shown to cause psoriasis in a dominant manner and with high penetrancy. Here, we demonstrate that in human keratinocytes CARMA2sh plays an essential role in the signal transduction pathway that connects pathogen-associated molecular patterns recognition to NF-κB activation. We also find that the serine/threonine kinase ULK2 binds to and phosphorylates CARMA2sh, thereby inhibiting its capacity to activate NF-κB by promoting lysosomal degradation of BCL10, which is essential for CARMA2sh-mediated NF-κB signaling. Remarkably, CARMA2sh mutants associated with psoriasis escape ULK2 inhibition. Finally, we show that a peptide blocking CARD-mediated BCL10 interactions reduces the capacity of psoriasis-linked CARMA2sh mutants to activate NF-κB. Our work elucidates a fundamental signaling mechanism operating in human keratinocytes and opens to novel potential tools for the therapeutical treatment of human skin disorders.