EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Objectives: Although liver transplant is highly effective for early-stage hepatocellular carcinoma, guidance on tailored posttransplant management to optimize outcomes is lacking. We examined the incidence and pretransplant radiological scans and indicators of tumor marker associated with posttransplant hepatocellular carcinoma recurrence.
Materials and Methods: We reviewed outcomes of 34 hepatocellular carcinoma candidates aged ≥18 years who underwent living-donor liver transplant between January 2016 and January 2023. The primary outcome was biopsy-proven posttransplant hepatocellular carcinoma recurrence at any site. We used Kaplan-Meier analysis to calculate cumulative incidence and Cox regression to identify predictors of posttransplant hepatocellular carcinoma recurrence.
Results: Among 34 transplant candidates, median age was 44 years, 84% had hepatitis C, median laboratory Model for End-Stage Liver Disease score was 18, and median pretransplant α-fetoprotein level was 235 ng/dL. From imaging scans pretransplant, 74% of candidates met Milan criteria. Median wait time to transplant was 67 days, and 23% received pretransplant locoregional therapy. Seven (20.5%) had hepatocellular carcinoma recurrence after median of 1.4 years, with cumulative incidences of 4 (11.7%) and 3 (8.8%) at 1 and 2 years posttransplant. Pretransplant number of lesions (P = .015), largest lesion diameter (P = .008), and higher amount of tumor markers (P = .002) were significant predictors of hepatocellular carcinoma recurrence after adjusting for pretransplant locoregional therapy and wait time. Posttransplant hepatocellular carcinoma recurrence (P < .001) and higher amount of tumor markers (P = .029) were associated with lower posttransplant survival.
Conclusions: Risk of hepatocellular carcinoma recurrence was significantly associated with the number and size of lesions at the time of living-donor liver transplant and amount of tumor markers. Risk stratification using a predictive model for posttransplant hepatocellular carcinoma recurrence based on pretransplant imaging and tumor markers may help guide candidate selection and tailoring of hepatocellular carcinoma surveillance strategy after living-donor liver transplant.

Key words : Hepatocellular carcinoma, Milan criteria, Tumor markers

Hepatocellular carcinoma (HCC) is a common cause of morbidity in patients with cirrhosis and the third leading cause of cancer mortality worldwide.1 Hepatocellular carcinoma is the most common primary liver cancer with 782 000 new cases and 745 000 deaths annually worldwide.² The best treatments for HCC include liver resection and liver transplant (LT). The rates of living-donor liver transplant (LDLT) for candidates with HCC have significantly increased in the Model for End-Stage Liver Disease (MELD) era because of the extra priority given to these candidates. Although LT for HCC is a highly effective cure for early-stage disease, guidance regarding tailored posttransplant management of this unique population to optimize outcomes is lacking. However, most patients present at advanced stage and are not candidates for these potentially curative therapies. Liver transplant is the treatment of choice for patients with unresectable early HCC, with 5-year survival rate of 70%.^3,4

Posttransplant HCC recurrence, however, is still a cause of morbidity and mortality among these patients. Even after the adoption of restrictive selection criteria, HCC recurrence rates posttransplant, in most recent studies, have been reported to range from 8% to 20%.^5-8 Recurrence of HCC posttransplant occurs because of progression of occult metastases months or years after transplant or secondary to the release of tumor cells at the time of surgery.⁵ The presence of vascular invasion and satellite nodules in the explant and the size and number of tumors are recognized risk factors related to HCC recurrence posttransplant.^6-8 Most of these recurrences occur within 2 years after transplant. Although there are no clear guidelines on how to treat these recurrences, surgical resection is the preferred treatment option. Unfortunately, little evidence is available of survival benefits of treatment for recurrent HCC posttransplant. In addition, few studies have examined risk factors in recurrent HCC after transplant or prognostic factors for survival after recurrence. Moreover, although tumor recurrence tends to happen within the first 2 years posttransplant, late recurrence can occur and the pathobiology underlying these cases is not well understood.⁹ In transplant recipients with HCC recurrence, survival is reduced. Some factors related to worse prognosis were early recurrence (<2 years after LT) and presence of bone metastases.⁸ Early tumor recurrence (TR) may lead to an increase in survival, as it allows surgical or locoregional treatment with curative intent.^5-8 However, the optimal management of these patients has not been well established.

Materials and Methods

In this retrospective analysis, we analyzed the outcomes of HCC candidates aged ≥18 years who underwent LDLT between January 2016 and January 2023 (n = 34). All donor-recipient pairs in the study group were exclusively related. Hepatocellular carcinoma was diagnosed histologically by percutaneous biopsy or at surgery. Diagnosis of HCC was made with only imaging if a contrast-enhanced study (dynamic computed tomography or magnetic resonance imaging) showed typical arterial enhancement with “washout” in the venous phase as described by guidelines from the American Association for the Study of Liver Disease.¹⁰ All patients received transplant livers from living donors. For immunosuppression after LT, all patients received basiliximab for induction, steroids, tacrolimus, and mycophenolate mofetil. All patients discontinued steroids after 6 months, and maintenance immunosuppression was continued with tacrolimus or tacrolimus and mycophenolate mofetil.

We collected information on demographics, medical history, laboratory results, tumor characteristics, treatment, and survival via clinical medical records. Demographic data included age and sex. Data collected on medical history included diabetes mellitus, hyperlipidemia, smoking, and risk factors for HCC, including viral hepatitis, alcohol abuse, and other chronic liver diseases. Information was based on available medical records. Laboratory data collected included serum bilirubin, albumin, prothrombin time, creatinine, alanine aminotransferase, aspartate aminotransferase, platelet count, and α-fetoprotein (AFP). We obtained laboratory data from within 2 weeks of initial visits or at the time of visits. Serum bilirubin, prothrombin time with international normalized ratio, and creatinine were used to calculate the MELD score. As risk factors for TR, we reviewed factors related to tumor staging (number and size of the nodules, vascular invasion, AFP levels), factors related to the patient (obesity, viral etiology, hepatitis C virus treatment), and factors related to treatment (time to transplant, donor age, immunosuppression, and ischemia time).

Dynamic imaging with computed tomography or magnetic resonance imaging was performed to determine whether Milan criteria were met (single tumor ≤5 cm or up to 3 tumors ≤3 cm each, no vascular invasion, no extrahepatic spread). Patients who met Milan criteria initially or who could be down-staged with locoregional therapy to meet Milan criteria were considered for LT. For data analysis, we used Microsoft Excel and the Statistical Package for the Social Sciences software to identify potential predictors for recurrent HCC. We performed Fisher test and chi-square analysis, and P < .05 was considered significant. The primary outcome was biopsy-proven posttransplant HCC recurrence at any site. Kaplan-Meier analysis was used to calculate the cumulative incidence, and Cox regression was used to identify the predictors of posttransplant HCC recurrence.

Results and Discussion
Although LT for HCC has been limited by the shortage of donor livers, LT has shown superior disease-free survival, with improved 5-year survival rate of 70% compared with 10% in untreated HCC.¹¹ However, despite optimal therapy with transplant, up to 20% of patients may experience recurrent HCC. Generally, HCC relapse occurs within the first 2 years after LT and may be either intrahepatic or extrahepatic, especially to bone, lung, and lymph nodes.^5-8

Factors related to the tumor
Staging, number, and size of nodules
In clinical practice, with compliance to Milan criteria, patients with well-differentiated tumors without vascular invasion have shown similar 5-year survival rate to survival rate among transplant recipients for nontumor causes.12,13 The increased risk of TR with higher numbers of nodules is not linear, because, with 3 or more nodules, the increase in risk tends to be attenuated.14 In the 34 LDLT patients in our study, only 7 (20%) exceeded the Milan criteria (2 had >3 nodes, 4 had nodule size >3 cm, and in 1 had nodule size of 5 cm). Cancer recurrence developed in only 3 patients (43%).

Vascular invasion
Macrovascular tumoral invasion can be identified by imaging studies and is considered a contraindication to LT. In turn, microvascular invasion can only be detected by explant analysis, which is unavailable preoperatively. However, microvascular invasion tends to be associated with tumor staging, with 16.6% of tumors shown in patients within Milan criteria and 50.2% shown in those beyond the up-to 7 criteria group (sum of the diameter of the largest node with the number of nodules smaller than 7). Microvascular invasion is a determining factor in the risk of TR and survival, doubling the risk of death.14,15 In our study, final identification of microvascular invasion was based on histomorphological data obtained after hepatectomy, in which we found 4 cases (12%), with 2 (50%) developing recurrence.

α-Fetoprotein
Levels of AFP are high in approximately 60% of HCC cases.¹⁶ Levels of ATP, although not important for diagnosis, play a role in the prognosis of HCC. A retrospective analysis, based on the United Network for Sharing Organs (UNOS) data, reported an inverse relationship between AFP level (from 16 ng/mL) and survival post-LT.^17,18 In patients with tumors within the Milan criteria, a monthly increase in AFP greater than 7.5 ng/mL, despite locoregional therapy (LRT), was associated with the presence of microvascular invasion.¹⁹ Reduction of AFP levels after locoregional therapy is associated with a good prognosis.^17,18,20 Even patients with initial levels of AFP above 1000 ng/mL attained good survival, as long as the AFP levels were reduced to less than 400 ng/mL after locoregional therapy.21 Among our patients, 14 (41%) had AFP levels <100 ng/ml, with 1 case (7%) of relapse, 7 (20%) had levels of 100 to 200 ng/mL, with 1 case (14%) of relapse, 8 (23.5%) had levels of 200 to 400 ng/mL, with 2 cases (12.5%) of relapse, and 5 had levels >500 ng/mL, with 3 cases (60%) of relapse. These results clearly demonstrate the absolute correlation of the increase in AFP with the probability of recurrence.

Factors related to the patient
Obesity
One study reported that 25% of patients with HCC who underwent LT were obese and had twice the risk of death, a higher frequency of microvascular invasion, and tendency for a higher of TR, suggesting that the increased expression of vascular endothelial growth factor induced by the adipose tissue may stimulate tumor angiogenesis.²² Another study confirmed the increased risk of TR among overweight patients, suggesting that obesity induces a pro-oncogenic state, via reduction of adiponectin and increase of leptin, which would stimulate HCC proliferation, migration, and invasion.²³ We suggest that these results are controversial because the correlation of weight distribution with cancer recurrence was not confirmed among our patients. Among our patients, 24 (70%) had BMI <30, with recurrence developing in 5 patients (71.5%), whereas 10 patients (30%) had BMI >30, with recurrence developing in only 2 patients (28.5%).

Viral etiology
A study from Taiwan described a strong association between failure of prophylactic therapy against reactivation of hepatitis B in the posttransplant period and the risk of TR, both of which are related to the presence of a specific mutation of the virus, which seems to induce a procarcinogenic state.²⁴ Another study found a 2.45-fold higher risk of TR in patients with hepatitis B and viral load above 5 log and reported an association between reactivation of hepatitis B in the post-LT period and risk of TR.^25-27 There are controversial reports on the influence of hepatitis C on the risk of TR after LT due to HCC. In our study patients, only 9 (26.5%) had HCC developed on the background of hepatitis B, with only 1 (14%) developing recurrence. Only 4 patients had antiviral therapy; thus, we do not consider it valid to appeal to viral (hepatitis B) etiology in cancer recurrence after LT.

Hepatitis C virus treatment
There are few reports on the effects of hepatitis C virus treatment in the post-LT period on risk of TR. In a small case series, treatment with interferon-based schemes was suggested to be associated with lower risk of TR.²⁸ In the CUPILT cohort, 314 patients transplanted for HCC were treated with direct-acting antiviral (DAA) agents about 67 months posttransplant, with 96.8% attaining sustained virological response and with only 2.2% having TR,29 suggesting the use of antiviral treatment in patients with HCC during the wait time for transplant. In an Italian cohort, 94% attained sustained virological response after treatment during the wait time, with TR being observed in only 8.5% of patients after 20 months of follow-up.^30,31 Another study compared patients treated or not with DAAs while waiting for transplant, with no difference in dropout risk, characteristics of the explant, or TR.³² In our study group, with regard to cancer development and posttransplant recurrence of hepatitis C virus, 22 of 34 patients (65%) diagnosed with HCC had hepatitis C virus infection, with liver damage at stage 3 and stage 4 of fibrosis. All of these patients had undergone hepatitis C virus treatment in the last 2 years against the background of which the disease developed. Viral activity was low before transplant in all patients, but the main relapse (71.5%) came from those with low viral activity. Results suggested an increased risk of TR with antiviral therapy, both during primary cancer development and during recurrence.

Factors related to treatment
Time to transplant
Studies based on the UNOS database reported an association between a short time to transplant and increased risk of TR after LT, with decreased survival, suggesting that rigorous image monitoring during the wait time could select tumors with more favorable biological behavior.^33,34 A multicenter study found a dropout rate of 3.2% and 12.4% when time between HCC diagnosis and LT was greater than 6 and 18 months, respectively, despite the completion of locoregional therapy. The risk of 5-year TR was greater in patients who had LT 6 months before or 18 months after diagnosis of HCC.³⁵ We divided our study patients with HCC into 2 subgroups, with 20 (59%) who underwent transplant <6 months after diagnosis and 14 (41%) who underwent transplant 6 to 12 months after diagnosis. Recurrence was shown in 4 patients (57%) in the first group and 3 patients (43%) in the second group. Naturally, the patients included in both groups were evaluated in the same way by the Milan criteria, and the cases when we exceeded were proportional to it in both groups. Thus, if patients are correctly selected by Milan criteria and the tumor does not come out of the liver due to the loss of time or the eligibility criteria, we did not find a difference in increased time.

Donor age
Patients who developed TR after LT had higher median donor age, which remained significant after multivariate analysis; thus, older livers may have less tolerance to the preservation injury and have increased susceptibility to cold ischemia.³⁶ In a survey of the UNOS database, a 70% higher risk of TR was found in patients who received grafts from donors older than 60 years, regardless of the etiology of liver disease.³⁷ Because our study patients all had LDLT, the maximum age of the donor was 55 years according to our criterion, with average age of 36 years. When we divided donors into those aged 18 to 36 years (22 donors; 65%) and those aged 36 to 55 years (12 donors; 35%), we observed recurrence in 4 patients (57%) in the young donor group and recurrence in 3 patients (43%) in the older donor group (not statistically significant).

Ischemia time
Warm and cold ischemia times are related to intensity of ischemia-reperfusion injury, which stimulates immune and inflammatory phenomena. Nagai and colleagues38 observed a gradual increase in the risk of TR with increased ischemia time, with a significant difference after 10 hours of cold ischemia and 50 minutes of warm ischemia. A German group found an association between warm ischemia time >50 minutes and risk of TR.³⁹ Ischemia-reperfusion injury can accelerate growth and implantation of HCC micrometastases present at the time of LT. Because all of our patients had LDLT, cold ischemia phase was not long and the period of implantation was prolonged compared with deceased donor procedures. When we analyzed 19 patients (56%) with cold ischemia time of <1 hour and reperfusion start time of ≤1 hour versus 15 patients (44%) with cold ischemia of >1 hour and reperfusion start time of ≤2 hours, cancer recurrence was shown in 3 patients (43%) in the short cold ischemia group and in 4 patients (57%) in the longer cold ischemia group, although results were not significantly different.

Immunosuppression
In the transplant scenario for treatment of a neoplasia, a balance must be sought between immunological risks (graft rejection) and oncological risks (TR). An association has been shown between serum level of tacrolimus in the first month after LT and risk of TR, with patients with a level above 10 ng/mL having a 2.8-fold higher risk of TR.⁴⁰ Because mechanistic target of rapamycin inhibitors (sirolimus and everolimus) inhibit cell proliferation and angiogenesis, these drugs could reduce the risk of TR after LT. A meta-analysis of 5 cohort studies found 70% lower risk of TR in patients who used sirolimus and a calcineurin inhibitor.⁴¹ Another meta-analysis of 42 studies showed a lower frequency of TR among patients treated with mechanistic target of rapamycin inhibitors, although this difference was only significant among patients with tumors within the Milan criteria.⁴² At our center, for immunosuppression, we aim to have first week tacrolimus blood level of 10 to 12 ng/mL, with level of 8 to 10 ng/mL at up to month 1 and 5 to 6 ng/mL after month 1. We also use everolimus only when tacrolimus is contraindicated for any reason. Thus, we did not analyze this among our patients.

In our study, the median age of the 34 candidates was 44 years, 84% had hepatitis C, median MELD score was 18, and median pre-LT AFP was 235 ng/dL. Based on pre-LDLT imaging, 74% candidates met the Milan criteria. The median wait time to transplant was 67 days, and 23% received pre-LDLT locoregional therapy. Seven patients (20.5%) developed HCC recurrence after median of 1.4 years, with cumulative incidence of 4 and 3 (11.7% and 8.8%) at 1 and 2 years post-LDLT. The pre-LDLT number of lesions (P = .015), largest lesion diameter (P = .008), vascular invasion (P = .0025), and higher amount of tumor marker (P = .002) were significant predictors of HCC recurrence after adjusting for pre-LT locoregional therapy and wait time. Posttransplant HCC recurrence (P < .001) and higher amount of tumor marker (P = .029) were associated with lower post-LT survival. The risk of TR has been shown to be proportional to the diameter of the larger nodule, with no association with the number of nodules, probably because multiple nodules, however small, do not present a higher frequency of vascular invasion.⁴³ These findings were confirmed in a retrospective cohort study that showed an increase of 36% in the risk of TR for each extra centimeter in the diameter of the larger nodule, with no association with the number of nodules.⁴⁴

Conclusions
The risk of HCC recurrence was significantly associated with the number of lesions and size of the largest lesion at the time of LDLT, as well as number of tumor markers. Risk stratification using a predictive model for posttransplant HCC recurrence based on imaging and tumor markers before transplant may help guide candidate selection and tailoring of HCC surveillance strategy after LDLT.

Volume: 22 Issue: 1 January 2024 - Supplement - 1

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