**2. Selection Criteria for LT Based on HCC Morphological Characteristics**

A common theme in the morphological criteria adopted to select patients with HCC was to exclude those patients presenting HCC with macrovascular invasion and/or extrahepatic spread. The main reason why morphological pre-LT criteria improved the accuracy in the selection of patients with HCC for LT derives from the demonstration that both the number and the size of HCC nodules can be considered surrogate markers of histologic microvascular invasion (MVI) and/or poor tumor differentiation [28,29], which are the main determinants of HCC recurrence and death after LT [30]. In addition to MC, two pretransplant expansive morphological criteria were adopted and validated in cohorts of patients of various geographical origins. The Shanghai Fudan criteria were originally developed in patients with chronic HBV infection. Patients transplanted with a single HCC nodule ≤ 9 cm in diameter or with up to three lesions (the largest ≤ 5 cm) but with a maximum tumor diameter of ≤9 cm experienced 1- and 3-year post-LT survival and HCC recurrence-free survival comparable with those transplanted within the MC [20]. These criteria were subsequently validated in more than 1000 patients enrolled in liver transplant centers in Shanghai [31]. In the United States, a further criterion aimed at expanding the original MC was developed at the University of California, San Francisco (UCSF) [14]. The UCSF criteria were developed after the observation that patients transplanted with a single nodule of HCC ≤ 6.5 cm or with up to 2–3 lesions ≤ 4.5 cm each, maintaining the total tumor diameter ≤ 8 cm, experienced a 5-year post-LT recurrence-free survival of 80.7%, which was not significantly different than that obtained in patients transplanted within the MC.

Both new criteria increased, albeit slightly, the size and/or the number of HCC nodules to select patients for LT compared to what was originally established by the MC. The certainty of always being able to carry out an accurate and reproducible measurement of the size and number of nodules to capture millimeter differences with respect to the MC probably represents the greatest limitation of the new criteria based exclusively on the morphological characteristics of HCC. There is great heterogeneity and different accuracies of liver imaging techniques applied to detect liver nodules and to properly characterize them, such as contrast-enhanced computed tomography (CT) or magnetic resonance imaging (MRI) [19]. Several reports have indicated that as many as 20–25% of patients undergoing LT for HCC have been inaccurately staged when only imaging techniques were used [32,33]. To overcome this important drawback, the American College of Radiology created the Liver Imaging Reporting and Data System (LI-RADS), aimed at standardizing the process of acquisition, interpretation, reporting, and data collection for liver imaging [34]. LI-RADS stratified the characteristics of liver lesions into five categories starting from LR-1 (definitely benign) to LR-5 (definitely HCC). This system is adopted in many countries to allow a standardized differential diagnosis of liver nodules in patients at high risk of HCC. Despite this improvement in the radiological categorization of liver nodules, a recent meta-analysis showed that LI-RADS was only 67% sensitive and 92% specific in diagnosing HCC [35].

#### **3. Selection Criteria for LT Based on HCC Histological Characteristics**

In patients transplanted beyond MC, a greater incidence of MVI, which is associated with higher post-LT tumor recurrence and death, has been demonstrated [36]. When the explanted livers of patients within and outside the MC were compared, MVI was present

in 11% of the former group and in 42% of the latter group [37]. A potential tool to detect the presence of MVI of HCC before LT is to perform liver biopsy of the nodules. Liver biopsy of confirmed HCC in the explanted liver was performed before LT in a series of 155 patients [33]. MVI was significantly more frequent in patients with larger nodules or with multinodular HCC; furthermore, 68% of patients who experienced HCC recurrence after LT were positive for MVI. In addition to the detection of MVI, another potential tool employing liver biopsy of HCC is to evaluate tumor histological grading. It has been demonstrated that well-differentiated HCCs may be successfully transplanted, assuring a recipient 5-year post-LT survival of 75% despite approximately 30% of these patients being beyond MC at the explant [38]. Although the clinical usefulness of pre-LT histologic assessment of tumor grading and MVI may be important, routine biopsy of HCC is not often feasible, mainly due to the presence of multiple nodules or the risk of promoting seeding of cancer cells [39].

The current challenging question is whether the presence of MVI may be predicted by means of noninvasive methods. To answer this question, several authors propose the use of imaging techniques such 18F-FDG PET/CT or gadoxetic acid enhanced dynamic MRI as noninvasive methods to predict the presence of MVI [40,41]. The rationale for using 18F-FDG PET/CT in detecting MVI derives from the observation that the HCC growth rate and the activity of glycolytic enzymes are related [42]. This explains why poorly differentiated HCC shows low glucose-6 phosphatase activity and high 18F-FDG uptake [43]. It has been proposed that good cutoff values for the SUVmax of HCC (SUVmax T) and SUVmax of the normal liver (SUVmax L) in predicting the presence of MVI may be 3.80 and 1.49, respectively [44]. The SUVmax T/SUVmean L ratio ≥ 1.2 was demonstrated to be significantly associated with the presence of MVI [45]. Compared with 18F-FDG PET/CT alone, the addition of gadoxetic acid enhanced dynamic MRI is a promising technology that may further improve the sensitivity and specificity of MVI detection. This was confirmed by applying the combination of MRI and PET/CT, as the sensitivity and specificity in predicting the presence of MVI were 78.6% and 80%, respectively [40]. These observations have been confirmed in clinical studies conducted in Asia in living donor liver transplantation. In Japan, 182 living donor liver transplanted patients with HCC were studied by means of 18F-FDG PET/CT and the serum levels of alpha-fetoprotein (AFP) before LT. In recipients transplanted beyond MC who presented negative 18F-FDG PET/CT and AFP serum levels < 115 ng/mL, the 5-year HCC recurrence rate was comparable to those transplanted within MC [46]. Very similar results were obtained in a Korean study also conducted in patients transplanted outside the Milan and UCSF criteria using living donors. The authors demonstrated that these two groups of patients, if they presented a negative 18F-FDG PET/CT, experienced 5-year post-LT HCC-free survival rates of 73.3% and 72.8%, respectively [47]. Although these results are encouraging, it is necessary to confirm them in populations of different ethnic groups, where the causes of LT are often different from those present in Asia, as well as in patients transplanted using deceased donors. At the present time, the combined use of 18F-FDG PET/CT and gadoxetic acid enhanced MRI could be applied in addition to the morphological and biological characteristics of HCC to stratify the risk of MVI.
