Finding The Seeds Of Recurrence: The Role Of The Liquid Biopsy To Detect Circulating Tumor Cells As Markers Of Advanced Disease And Prognosis In HCC

Liquid biopsy may allow a better stratification of patients according to their estimated prognosis. As well in other tumors (e.g. breast cancer), molecular characterization is permitting not only to better understand the pathological pathways which lead tumorigenesis and progression, but also to drive specific treatments tailored on the specificity of the patient disease. Liquid biopsy is very easy and cheap to perform, since it consist in a simple venous blood sample which will be investigated by cells sorter to identify specific populations of cells.

In hepatocellular carcinoma is common to assist on an elevated recurrence rate: patients who have been treated with a curative intent, in 70% of cases relapse. In these cases, patients often had an underlying inflammatory liver disease, as cirrhosis, which may drive the relapse as an oncogenetic factor. Cirrhosis is a well-know risk factor for developing hepatocellular carcinoma in general. Because of this, in clinical practice is almost difficult to distinguish if the new nodule we have diagnosed should be considered as a real recurrence of the first treated tumor, or a de-novo occurrence linked to the unchanged underlying tumorigenesis boost, which may be effectively cured only after liver transplantation. Several retrospective studies already demonstrated the advantage, in terms of disease-free-survival and overall-survival, of a de-novo occurrence in respect to a real metastatic presentation. However, no one has reached any specific clinical or morphological markers to distinguish between these two conditions. Characterization of the nature of the recurrent nodule has been obtained only by genetic and molecular evaluation of the tumors heterogeneity. Nevertheless, this characterization nowadays is possible only with research intent, and on histological specimen, which could be provided only after an eventual decision of redo-surgery to treat the considered recurrence. Indication on how to treat HCC recurrence after surgery still lacks. Our hypothesis is founded on the "seed and oil" theory, which is basically founded on the evidence of the presence of circulating tumor cells (CTCs) in the blood stream. These cells may be spread from the original tumors as an hallmark of advance cancer, which has already developed the invasiveness characteristic. It is our opinion that tumors in an early stage do not release, or not in the same rate or quality, CTCs in the bloodstream as advanced tumor does. We considered an advanced tumor the one which may has already microvascular invasion and/or satellitosis, which may be the histological signs of an advanced and already systemic stage. Patients with CTCs identified at the liquid biopsy may be associated with a worsen prognosis, accounting for an increased rate of early recurrence and overall recurrence, even in case of transplantation. In addition, the "seed and oil" theory, associated with the "rehoming theory" may justified, from a bio-oncological point of view, the cases of recurrence in the transplant setting, when the whole organ, and the relative oncogenetic risk factors, is treated by substitution. Finally, from a pathophysiological point of view, we want to demonstrate that recurrent intra-hepatic nodules (IM) are due to circulating tumour cells'seeding, permitting a better understanding of carcinogenesis in those tumours.

• Primary objective: to evaluate the association between variation in CTCs - determined in blood samples - and overall survival and disease-free-survival in HCC patients before and after surgery (either resection and transplant).
• Secondary objective: to evaluate if the positive identification of CTCs in blood samples is associated with the intra-hepatic metastatic pattern of recurrence in either resected patients and transplanted one.
• Tertiary objective: to evaluate and to monitor the role of different clinical, biochemical, radiological and histopathological variables in determining the recurrence after surgery and their eventual relationship with the presence and rate of CTCs Patients enrollment will be assessed by the surgical divisions of this study, usually during the outpatient clinic visit before the surgical operation. In that moment patients will be candidate for this study, and, if they accept to participate, researchers will acquire the written consent. Enrolled patients will be codified by an alpha-numeric number to preserve the identity. Researchers who will be employed in the laboratory where the CTCs will be analyzed will not be able to trace the owner of the sample, and they will not have the access to the clinical data (blind analysis).

Before surgery for each patient liquid biopsy from peripheral blood will be collected (in the same time of routine blood analysis). Samples will be used to analyze CTCs rate and type by FACSARIA III (BD bioscience). During surgery, a fresh slice of the tumor and near health liver will be collected by an expert pathologist, who will use section(s) for the usual histological evaluation as provided by the best clinical practice and international guidelines. The remaining sections will be maintained on ice during shipment to the laboratory unit. Researchers will perform alive hepatic cells isolation and storage under sterile conditions. Then, hepatic cells will be analyzed with FACSARIA III and used for further in-vitro experiments.

Liquid biopsy will be performed again at 30th post-operative day during the first outpatient visit, and then every six months, in accordance and in parallel with the local follow-up protocols. Liquid biopsy performed during follow-up will be managed as the pre-operative one, during the normal tests provided for clinical purpose. Follow-up will be closed at 30 months.