This is the first publication reporting on the detection rate and the prognostic significance of CTC according to primary tumor sidedness in metastatic colorectal cancer patients. Although the term “colorectal cancer” is currently referred to a single tumor type, increasing evidence is emerging concerning the prognostic impact of primary tumor sidedness. A recent meta-analysis of 66 clinical studies compared the overall survival of RCC versus LCC in over 1.4 million patients and demonstrated a 20% reduced risk of death for patients whose tumors arise from the left side [11
]. To date, the molecular background of proximal and distal colorectal cancers has been only preliminary unraveled and translational efforts to gain knowledge about the biological underpinnings of colorectal cancer sidedness are critically important. It has been shown that baseline detection of CTC in metastatic colorectal cancer is an independent prognostic factor for progression-free survival (PFS) and overall survival (OS) with CellSearch®
]. We confirmed here that the presence of CTC is a significant prognostic factor in metastatic colorectal cancer patients regardless of tumor sidedness. The whole population was then sub-grouped according to primary tumor sidedness. In particular, left-sided colon cancers and rectal cancers were considered separately in this retrospective evaluation, in line with recently released data supporting the colorectal cancer “three entities” hypothesis [13
]. In our series of patients, CTC were not uniformly distributed in the three subgroups, showing the highest prognostic impact in patients with left-sided colon cancer, while being the lowest in number in this subgroup of patients. We hypothesized that this unanticipated result could reflect a difference at the molecular level between CTC shed from left-sided as compared to right-sided primary tumors. Indeed, we found that CTC exhibit phenotypic heterogeneity, with a predominant mesenchymal phenotype found in CTCs from distal compared to proximal primary tumors. It is well recognized that the EMT program enables epithelial cancer cells to acquire properties that are critical to invasion and metastatic dissemination, such as increased motility, invasiveness, and the ability to degrade components of the extracellular matrix. The EMT program that allows cancer cells to disseminate from a primary tumor also promotes their self-renewal capability, usually depicted as the defining trait of cancer stem cells. Such EMT process seems almost invariably triggered by heterotypic signals, including TGF-β, Wnt, and interleukins, which cancer cells receive from the tumor reactive stroma, which thus plays a substantial role in dictating cancer progression [16
]. Tumor cells can reach the circulation by either active invasion, which requires the acquisition of certain mesenchymal traits, or by passive shedding, due to cancer cell pushing by tumor expansive growth and facilitated by the abundance of highly abnormal blood vessels. Tumors are likely able to use both active and passive methods to enter the vasculature, depending on the site of tumor initiation, the aggressiveness of tumor cells, and the tumor micro-environmental conditions. It has been reported that most of the cancer cells that entered into the vasculature by passive shedding are non-viable cells, thus incapable of completing the efficient colonization of distant sites [17
]. Distinct metastatic patterns in colorectal cancer patients based on primary tumor location have been recently demonstrated, with higher rates of liver and lung metastases in left-sided colon cancers and rectal cancers, respectively, as compared to right-sided tumors, which appear to be associated with higher rates of peritoneal metastases. We could hypothesize that proximal and distal colorectal cancers may also differ in the early steps of the metastatic cascade and that alternative modalities of tumor cells intravasation might be adopted, depending on primary tumor location. As far as we know, substantial differences exist in the tumor microenvironment of distal and proximal colorectal cancer, with the former being inflamed and with marked stromal infiltration and the latter characterized by lower inflammatory status and higher expression of pro-angiogenic factors [18
]. With this in mind, we could envisage that cancer cells from primary distal cancers receive abundant signals from the surrounding reactive stroma, which are able to activate their latent EMT programs and equip them with the ability to actively intravasate into the circulation. Conversely, proximal cancers, which are frequently larger in size and plenty of disorganized and leaky blood vessels, might release into the circulation a higher number of cancer cells, which are not necessarily viable and able to sustain the following steps of the metastatic cascade. As expected, we found that the vast majority of CTC in RCC patients exhibited a clear apoptotic pattern, thus, possibly providing the rationale for the limited prognostic impact of these cells in patients with primary proximal cancers. Although we did not use any apoptosis-specific marker to identify apoptotic CTC, several reports have described apoptotic CTC as a specific CTC subtype well identifiable at CellSearch®
]; they are characterized by altered morphological parameters such as speckled pattern of keratin staining and/or fragmented or disintegrated nuclei.
Our data suggest that the poor prognosis of right-sided colorectal cancer might not be determined by the hematogenous dissemination of tumor cells, which appears to predominantly be a passive shedding of non-viable cells in the blood vessels. We also demonstrate that a subgroup of poor-prognosis left-sided colon cancer exists, which is reliably identified by the presence of CTC in the blood vessels. Particularly, CTC found in left-sided colon cancer patients exhibit a phenotype with different levels of mesenchymal differentiation. This might imply a substantial difference in the biology of proximal and distal cancers, mainly related to the tumor microenvironment and strongly associated with different patterns of tumor cells dissemination from primary tumors. Although EMT is certainly triggered by stromal signals, EMT-specific traits in CTC, which are someway "stromal independent", might indicate that cancer cells in the blood are able to transcriptionally control their nature by cell-autonomous mechanisms, as recently advocated by the new CRIS-B subtype of colorectal cancer [20
]. The main limitation of the study is the small population of samples available for molecular analysis, and our results need to be confirmed in a larger cohort. Investigating the intracellular properties of CTC, in their fluid microenvironment, might contribute to define how and to what extent cancer cell-specific traits contribute to the creation of accurate molecular subtypes of CRC and to the definition of reliable prognostic indicators.