Background. In different solid cancers, CTC have proved to be surrogates of tumor aggressiveness and predictors of treatment response. More exactly, CTC have shown to correlate with overall survival (OS) and response rate (RR). The evidence in the context of HCC are less strong even if some studies have produced encouraging results. End-points of the Study. Endpoints of the study are defining the possible role of CTC in the prediction of OS and RR in patients with intermediate and advanced HCC candidate to undergo TACE, sorafenib and TARE. Population and Methods. Patients of both sexes aged over 18 years with intermediate and advanced HCC candidate to TACE or sorafenib or TARE will be included in the study. Patients who undergo orthotopic liver transplantation will be excluded. A baseline and subsequent measurements of CTC concentration in peripheral venous blood before and after the treatments for HCC will be performed according to a regular schedule. ScreenCell Cyto kit will be used for the detection of CTC in peripheral blood and an immunofluorescence analysis will follow. Parameters of Efficacy. OS is the major parameter of efficacy in the present study while secondary parameter of efficacy is RR judged according to RECIST and mRECIST criteria. RR will be evaluated with contrast TC or RMN. The main object of the study is the potential role of CTC in the prediction of OS and RR.
Even if HCC is still today a malignancy with poor prognosis especially in the advanced stage, sorafenib represents a treatment option with proved efficacy in prolonging survival respect to placebo. However, despite its high cost (in Italy €5878,77/month) the RR is quite low and variable between patients, for reasons that are not entirely clear1. Moreover, the unpredictable occurrence of adverse events often represent an important drawback of the treatment. All considered, a surrogate of response, a tool to predict the outcome is required with urgency. A variety of studies have shown that the inactivation of Ras/Raf/extracellular signal-regulated kinase (ERK) pathway and the activation of the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway play a critical role in the resistance to sorafenib2,3. Thus, these pathways may provide biomarkers to assess sorafenib resistance before and throughout the course of treatment. Currently, tumor tissues obtained by an excisional or needle biopsy are used for detection of drug targets. However, invasive sampling is potentially harmful and expensive, and cannot be performed repeatedly. So, the collection of CTCs provides a viable alternative. The survival of CTCs in blood is hypothesized to be short, resulting in real-time information of the state of the disease. Because the cells can be individually captured and analyzed, heterogeneity can be determined as well as the combinations of aberrations present in each cell. Also when therapy resistance occurs and a change in therapy is needed, the CTCs will most probably reflect the resistant population of cells. This is the first study (included those registered on clinicaltrial.gov) to propose, for the isolation of CTCs in HCC, the association between ScreenCell (in alternative to CellSearch) and antibodies direct against targets specific of HCC cells (in alternative to EpCAM) like Glipican-3, Hep-par1, Akt and p-ERK. The potential for this choice stands on the observation that CellSearch captures a clinically relevant group of CTCs only in EpCAM(+) malignancies while several studies indicate that there are tumor cells (like those of HCC) expressing lower EpCAM that are missed by CellSearch. Even if different studies ongoing hypnotize to use waste blood from the CellSearch system, depleted from EpCAM(+) cells, to capture manually and automatically the EpCAM(-) CTCs, significant results still lack4,5. After depletion of white blood cells by anti-CD45, the ScreenCell cyto kit filters the cells on a dimensional basis thus permitting to subsequently run an immunofluorescence analysis through the incubation with antibodies against cell markers that have been clearly recognized to be specifically associated to HCC cells in immunohisrochemical studies on stained liver tissue (Glipican-3, HepPar1). The adjunctive analysis of Akt and p-ERK phenotype of CTC will provide further information useful to explore the potential role of these features in predicting response to sorafenib treatment and innate or achieved resistance to this drug, as suggested elsewhere6 but never proven in an occidental population of patients with HCC. In conclusion, we found that the study proposed has the potential to succeed in isolate and detect HCC CTCs with an innovative combination of tools that promises maximal efficacy in respect to a relatively simple and direct method. The rationale of the study relies on the strong believe that a solid and effective detection of CTCs represents the essential condition to further explore the correlation between their concentration in the bloodstream and OS and RR, both in patients treated with sorafenib, and in those candidate to loco-regional treatment as TACE and TARE.
1. Llovet JM, Ricci S, Mazzaferro V et al. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med. 2008; 359:378¿390.
2. De Luca A, Maiello MR, D¿ Alessio A et al. The RAS/RAF/MEK/ERK and the PI3K/ AKT signalling pathways: role in cancer pathogenesis and implications for therapeutic approaches. Expert Opin Ther Targets. 2012; 2:S17¿27.
3. Newell P, Toffanin S, Villanueva A, et al. Ras pathway activation in hepatocellular carcinoma and anti-tumoral effect of combined sorafenib and rapamycin in vivo. J Hepatol. 2009; 51:725¿733.
4. Swennenhuis JF, van Dalum G, Zeune LL et al. Improving the CellSearch® system. Expert Rev Mol Diagn. 2016 Dec;16(12):1291-1305.
5. de Wit S, van Dalum G, Lenferink ATM, et al. The detection of EpCAM(+) and EpCAM(-) circulating tumor cells. Sci Rep. 2015;5:12270.
6. Jun Li, Lehua Shi, Xiaofeng Zhang et al. ERK/pAkt phenotyping in circulating tumor cells as a biomarker for sorafenib efficacy in patients with advanced hepatocellular carcinoma. Oncotarget 2015, Vol. 7, No. 3