IMPACT OF NON-ONCOLOGICAL AND ONCOLOGICAL FACTORS ON TUMOR RECURRENCE IN HCC LIVER TRANSPLANTED PATIENTS-SINGLE LIVER TRANSPLANT CENTRE EXPERIENCE

Corresponding author: Gabriela Smira, MD E-mail: smira.gabriela@gmail.com ABSTRACT Background. The aim of this study was to identify pre-transplant risk factors that lead to tumor recurrence and to evaluate mortality in liver transplanted patients from the single Liver Transplant Center in Romania. Methods. 81 patients who underwent liver transplantation between 2011 and 2015 at the Fundeni Clinical Institute were assessed based on age, sex, type of operation, histopathological staging, pre-transplant bridging therapies, etiology of cirrhosis, San Francisco and Up to seven scores, AFP and CA19-9. Results. Between 2011 and 2015, 81 out of 454 liver transplants were performed for hepatocarcinoma (17.84%). The most frequent etiology was the HVB and HVD co-infection 39.7%. The 1 and 3 years-survival rates were 81.15%, respectively 72.5%. The 1 year and 3 years tumor recurrence rate were 14.49%, respectively 15. Conclusions. We identified as risk factors for 1 year-tumor recurrence the following: pre-transplant Milan, San Francisco and Up to Seven scores, pre-transplant AFP > 200 ng/ml and Edmonson/Steiner staging. Age over 65 at transplant was not correlated to cancer recurrence, 1 year and 3 years mortality rates or postoperative complications.


INTRODUCTION
HCC constitutes the fifth most frequent form of cancer worldwide and it holds the second place in malignancy-related mortality. HCC occurs two to six times more frequently in men than in women (1).The key risk for HCC is liver cirrhosis, approximately 80% of which are related to hepatitis B and C.In Eastern Europe, Romania is on the first place in hepatocarcinoma related mortalityand second regarding liver cancer incidence (2).
Chronic hepatitis B is the major risk factor for developing HCC in Africa and Asia, while in the US, Europe and Japan chronic hepatitis C, alcohol and non-alcoholic steatohepatitis (NASH) are leading causes of HCC (6,7). Eighty percent of liver cancers are found in cirrhotic livers, which themselves carry a high risk for HCC. Chronic carriers of hepatitis B virus (HBV) have a 100-fold increased risk as compared to a non-infected healthy reference population.There is growing evidence that the hepatitis B virus (HBV) contributes to hepatocarcinogenesis via direct malignant transformation and other indirect effects. Furthermore, persistent HBV infection can increase genetic instability by causing hepatocyte destruction and regeneration.
Moreover, HBV load is involved in post-LT HCC recurrence and increases the risk of post-LT recurrence through an inflammatory effect after HBV or hepatitis C virus (HCV) allograft re-infection (5,8). Li et al. retrospectively analyzed 340 HBV-positive patients who underwent orthotropic LT (OLT) and found that HBV relapse was an independent predictor of HCC recurrence (P = 0.03), and that high pre-transplant levels of HBV DNA were associated with HCC recurrence. Wu et al. also performed a retrospective study of 78 patients with HBV-related HCC who underwent LT, and found that 13 patients (16.6%) experienced HCC recurrence and 18 patients (23.1%) experienced HBV relapse. Therefore, the authors concluded that HBV relapse was closely related to HCC recurrence (P = 0.004) and led to a shorter OS after LT. Thus, HBV relapse and HCC recurrence may have a reciprocal causative relationship in the post-transplantation setting .
Approximately 130-170 million people globally are infected with the hepatitis C virus (HCV), 20 to 30% of whom will develop liver cirrhosis which carries a 3-5% annual risk of ultimately progressing to liver cancer. Unlike HBV, there is no close relationship between HCV and RNA and the risk of developing HCC. As a general rule, patients will not develop liver cancer in chronic HCV before their disease has progressed to advanced fibrosis and cirrhosis. The risk of HCV-induced HCC appears to be related to the degree of inflammation and necrosis, while HBV-related HCC does not correlate well with inflammation and seems rather to involve activation of specific oncogenes by the virus.
The need for LT exceeds the number of deceased donors, which increases waiting times and contributes to a high drop-out rate among patients who experience tumor progression while awaiting surgery (9,11).Thus, living donor LT (LDLT) provides patients with HCC better access totimely treatment. However, several recent studies have demonstrated that LDLT is associated with an increased incidence of post-LT HCC recurrence, compared to deceased donor LT (DDLT) (11)(12)(13).
There are three suggested mechanisms by which LDLT might increase the risk of HCC recurrence. The first mechanism is the release of growth factors that mediate the regeneration of the hemiliver and increase the vascular inflow during the rapid regeneration of the partial grafts from living donors, which might contribute to tumor progression and recurrence. Furthermore, small-sized grafts are more likely to cause acute phase graft injury, which results in cell adhesion, angiogenesis, and migration; all of these factors may promote tumor recurrence (14)(15)(16)17).
The second mechanism is the "fast-tracking effect", whereby patients who undergo LDLT have a shorter waiting time, which might preclude the detection of an aggressive tumor before surgery and increase the risk of recurrence (18,19).
The third mechanism is the LDLT technique itself might directly contribute to a higher recurrence rate, due to the sparing of the inferior vena cava (which is necessary for complete tumor removal) and more extensive liver manipulation during the LDLT. All of these factors might contribute to the high recurrence rates after LDLT compared to after DDLT (12,20).
Despite these potential mechanisms by which LDLT might increase HCC recurrence, other studies have reported that LDLT recipients have a similar recurrence rate and comparable recurrence-free survival compared to patients who underwent DDLT. In these studies, the authors attributed the inferior outcomes after LDLT for HCC to the tumor's characteristics and biology. Although there is no clear evidence regarding whether LDLT is associated with a higher recurrence rate, the conflicting data suggest that different indication criteria may be appropriate for LDLT and DDLT (21)(22)(23).
In general, there is an arbitrary age limit for LT, due to the increased incidence of age-related comorbidities among elderly patients with HCC (24,25).Several studies have reported that elderly patients who underwent LT exhibited a lower survival rate and higher rates of HCC malignancy, which may be associated with their increased risk of adverse outcomes due to chronic comorbidities, immunosuppression, and immunosenescence. (25,26). Age-related immunological changes and immunosenescence can increase the susceptibility of elderly patients to infection, autoimmune disease, and cancer; and long-term immunosuppressive therapy after LT might increase these patients' risks of morbidity and mortality compared to their younger counterparts. However, other studies have reported that LT is not contraindicated for elderly patients, and Ballarin et al. reported similar shortand middle-term survival outcomes and morbidities (e.g., HCC recurrence) among young and elderly patients.
Moreover, Kim et al. demonstrated that OS was prolonged among younger patients who underwent OLT for HCC, although there were no significant differences in HCC-specific survival among the various age groups (27).Therefore, these findings suggest that carefully selected elderly patients with HCC could experience a benefit from OLT that is equal to the benefit that is experienced by younger patients.
Alpha-fetoprotein (AFP) is a serum tumor marker for hepatocellular carcinoma (HCC) and can also be used to monitor HCC recurrence after liver transplantation (LT). Although AFP can be slightly elevated in several other conditions, such as chronic hepatitis and liver cirrhosis , high preoperative levels of AFP are associated with poorer outcomes following liver transplantation for HCC (5,6,7). Recently, the significance of AFP decline after curative resection for HCC has been reported as a predictor of tumor recurrence (8,9). Nobuoka et al. (9) emphasized that a positive level of AFP within 4 months after resection was a strong predictor of post-operative tumor recurrence. Little is known, however, about the relationship between peri-operative change in AFP level and tumor recurrence or the clinical usefulness of peri-operative change as an early marker of HCC recurrence.
Based on the studies in resection, we hypothesized that a rapid decline in AFP may be associated with better recurrence-free survival (RFS) in patients who have undergone LT.
This study, therefore, evaluated the clinical significance of change in peri-transplant serum AFP level as a predictor of tumor recurrence in HCC patients who underwent liver transplantation.
The aim of this study was to identify pre-transplant risk factors that lead to tumor recurrenceand to evaluate mortality in liver transplanted patients from the single Liver Transplant Center in Romania.

MATERIALS AND METHODS
81 patients who underwent liver transplantation between 2011 and 2015 at Fundeni Clinical Institute were assessed based on age, sex, type of operation performed, histopathological staging, pretransplant bridging therapies, etiology of cirrhosis, San Francisco and Up to seven scores, AFP and CA19 9 values before and at 1, 3, 6 and 12 months post-transplant. The prevalence of cirrhosis among patients with hepatocellular carcinoma has been 95% and the HCC incidence rate among patients with cirrhosis has been shown to be 2-4 % per year.

RESULTS
Between 2011 and 2015, 81 out of 454 liver transplants were performed for hepatocarcinoma (17.84%), 26 of them received living related liver transplantation. The most frequent etiology was the HVB and HVD co-infection 39.7 %, followed in close range by the HVC infection 25.4% and alcoholism 22.2% (Fig. 1). The median follow-up period was 36 months. The 1 year and 3 years-survival rates were 81.15%, respectively 72.5% (Fig. 2). No death after the first year post liver transplant was associated to tumor recurrence. The 1 year and 3 years tumor recurrence rate were 14.49%, respectively 15.94%. Milan, San Francisco and Up to Seven scores, pre-transplant AFP > 200 ng/ml and Edmonson/Steiner staging were found to be statistically significant associated to tumor recurrence (p=0.01; p=0.00032; p=0.01) (Fig. 3).

DISCUSSION
Liver transplantation has been shown to be the best therapeutic option in selected patients with HCC. Although transplants are usually performed in patients who meet the Milan criteria, a substantial subset of patients who do not meet these criteria have the potential for good outcomes after liver transplantation, leading to the proposal of several new criteria (5,10). These latter criteria emphasize the importance of the inclusion of new markers of biological behavior, along with morphological tumor size and number, in formulating new sets of criteria that can better predict the risk of recurrence. AFP is one of strong biological markers and used for early diagnosis of HCC and for recurrence after LT (28,29). Numerous study groups have empha-sized that high preoperative levels of AFP are associated with poorer outcomes from liver transplantation for HCC.
Pre-LT high AFP is an important risk factor for HCC recurrence (30).
Patients undergoing liver transplantation for hepatocellular carcinoma within the Milan criteria (single tumour ≤5 cm in size or ≤3 tumours each ≤3 cm in size, and no macrovascular invasion) have an excellent outcome. Age over 65 at transplant was not correlated to cancer recurrence.
In conclusion, pre-transplant AFP is significantly prognostic of tumor recurrence after liver transplantation for HCC in patients with elevated pre-LT AFP level. (28)(29)(30).

CONCLUSIONS
Our results about survival and cancer-free survival at one year post-transplant correspond to specialty literature (81.15% vs. 82%) / (84.06% vs. 80%). We identified as risk factors for 1 year-tumor recurrence the following: pre-transplant Milan, San Francisco and Up to Seven scores, pre-transplant AFP> 200 ng/ml and Edmonson/Steiner staging. Age over 65 at transplant was not correlated to cancer recurrence.
1 year and 3 years mortality rates or postoperative complications and that supports the current European trend to discard this variable during the liver graft allocation process.