Circulating tumor cells (CTCs) were first documented by Thomas Ashworth in 1869 (Ashworth, 1869), when he first documented the presence of cells with the morphology of the tumor of origin in the blood of a patient with severe cancer. The observation of nonhematopoietic blood CTCs with characteristics comparable to those of tumors was strengthened in 1958–59 (Roberts et al., 1958), when Roberts et al, using a protocol based on “Proto”-Ficoll combined with morphologic analysis by Papanicolau staining, was able to compare hematologic samples enriched with tumor cells with tumor smear to demonstrate the existence of CTCs in the blood of cancer patients. The concept of “classic” CTCs was better defined forty decades later on the basis of a series of mAbs recognizing tissue-specific molecules that differ in carcinomas (of ectodermal origin) versus leukocytes (of mesodermal origin) (Racila et al., 1998). Based on this principle, protocols for identifying and counting CTCs were developed. In 2004, the FDA approved the protocol for counting CTCs with the CellSearch™ platform (Allard et al., 2004), which works as follows: an initial immunomagnetic enrichment step for EpCAM-expressing cells, followed by detection of the nucleus and expression of cytoskeletal cytokeratin protein (CK+), combined with the absence of the common leukocyte antigen CD45 (CD45-). This approach represents a milestone in the study of CTCs, as it represents a standardized method for the identification of CTCs. Translational research performed in the past 20 years suggests that CTCs represent attractive tumor markers (Alix-Panabières and Pantel, 2021, Krebs et al., 2014). Translational studies on “classical CTCs” confirmed the association between CTC-count and survival in metastatic breast (Hayes et al., 2006), colorectal (Cohen et al., 2008) and prostate cancer patients (De Bono et al., 2008). In spite of this, CTC-guided chemotherapy administration failed in clinical trials SWOG S0500 (Smerage et al., 2014), Circe01 (Cabel et al., 2021) and STIC (Bidard et al., 2021). Specifically, SWOG S0500 trial observed that the evaluation of “classical” CTC counts in patients with metastatic breast cancer starting first-line chemotherapy did not improve overall survival (Smerage et al., 2014). Circe01 study reported a lack of benefit in chemotherapy switch for advanced breast cancer patients based on CTC count (Cabel et al., 2021). STIC trial demonstrated the potential clinical utility of “classical” CTC count as a biomarker in therapy choice for hormone receptor positive, HER2 negative metastatic breast cancer patients (Bidard et al., 2021). The failing of “conventional-CTC” count as an early measure of therapy response doesn’t necessarily entail that CTCs are ineffective biomarkers, rather leads to hypothesize that the classical definition of CTCs needs to be revised, based on the observation of the existence of “atypical” CTCs. Indeed, the concept of “atypical CTCs” was put forth for the first time in 1959 (Sandberg et al., 1959). The authors identified cells with immature appearance, large nuclei and nucleoli and resemblance to cells observed in tumor smears (Sandberg et al., 1959). The Authors suggested that, although they were difficult to be classified, the cells classified as “atypical” were referable as tumor-derived cells that were not present in the blood of healthy people.

The aim of this chapter is to propose biologic rationales for the existence of unconventional CTCs.