Predictor of outcome after living donor liver transplantation for patients with hepatocellular carcinoma beyond the Japan criteria

Abstract Background The Japan criteria (JC, maximum tumor size within 5 cm, within five tumor nodules, AFP within 500 ng/mL or within Milan criteria) have been applied to cadaveric liver transplantation (LT) for hepatocellular carcinoma (HCC) and will be used for living donor LT (LDLT) in Japan. The aim of this study was to verify the JC in LDLT and to clarify the risk factor of HCC recurrence and mortality after LDLT beyond the JC. Patients and methods Adult patients who underwent LDLT for end‐stage liver disease with HCC until October 2019 were reviewed retrospectively (n = 246). Patients were divided into two groups according to whether they were within JC (n = 203) or beyond JC (n = 43). Recurrence‐free or overall survival rates after LDLT were compared. Univariate and multivariate analyses were performed to identify risk factors of HCC recurrence and HCC‐related mortality after LDLT for patients beyond the JC. Results Patients beyond the JC had significantly poorer 5‐year recurrence‐free (50.3% vs 95.9%, P < .001) or overall (61.7% vs 98.1%, P < .001) survival rates compared with patients within the JC. A multivariate analysis revealed that des‐gamma‐carboxy prothrombin (DCP) ≥ 300 mAU/mL (hazard ratio 9.36, 95% CI; 2.41‐36.4, P = .001) was an independent risk factor for HCC recurrence and HCC‐related mortality (hazard ratio 13.8, 95% CI; 1.92‐98.6, P = .01) after LDLT in patients beyond the JC. Conclusion The outcome of LDLT for patients within the JC was favorable. Patients beyond the JC with DCP ≥ 300 mAU/mL might be contraindicated for LDLT.


| INTRODUC TI ON
Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer and the third most common cause of cancer-related death. 1 Its incidence is increasing worldwide because of the dissemination of hepatitis virus infection and increase of alcoholic or non-alcoholic steatohepatitis. Liver transplantation (LT), which offers the theoretical advantage of removing both the tumor and the organ that are at risk of developing future malignancy, is an established therapy for HCC in patients with liver cirrhosis. 2,3 In Asian countries, religious, cultural, and political ideologies have created significant obstacles to the transplantation of cadaver organs. Organ shortages have forced patients with HCC to endure long waiting periods that are associated with tumor development. Thus, living donor LT (LDLT) is a choice for treating such HCC patients after treatments including radio frequency ablation, transarterial chemoembolization, and/or hepatic resection. 4 It is important to allocate the deceased donor livers to ensure a reasonable outcome for living donors who need to undergo invasive surgery. 3 The Milan criteria (MC) have significantly improved the outcome of LT for HCC and have become the gold standard to achieve a favorable outcome after LT for HCC. 5 Because Japanese national insurance has covered LDLT for HCC within the MC since 2004, the number of LDLT patients within the MC is increasing in Japan. LT for patients within the MC generally reach a 5-year overall survival rate of 70%-80% and a recurrence rate of around 10%. Many groups have proposed LT for patients with large and numerous tumors; however, these favorable outcomes have raised the question of whether the selection criteria might be expanded. 6,7 Another criticism against the MC is the lack of tumor-related biological indices to help dictate best oncological practice when transplanting HCC patients. Therefore, several centers have developed criteria that include tumor biological indices such as alpha-fetoprotein (AFP) or des-gamma-carboxy prothrombin (DCP) indices to predict outcomes. 8,9 The Japanese Liver Transplantation Society recently proposed expanded criteria based on a retrospective data analysis of a Japanese nationwide survey. 10 These criteria are termed the 5-5-500 rule and are as follows: maximum tumor size within 5 cm, within five tumor nodules, and AFP within 500 ng/mL. The Japan criteria (JC), which include the 5-5-500 rule or within Milan criteria, have been applied to selection criteria for cadaveric LT for HCC and will be used for LDLT very soon in Japan. Patients beyond the JC will not be covered by national insurance, but they will have a chance to undergo LDLT at personal cost.
Therefore, it is important to reveal the contraindication of LDLT for such patients.
The aim of this study was to verify the Japan criteria in LDLT and to clarify the risk factor of HCC recurrence and mortality after LDLT beyond the Japan criteria to reveal the contraindication of LDLT in patients beyond the Japan criteria.

| Patients
The study protocol was carried out in accordance with The Code Pre-transplant imaging was used to estimate the maximum tumor size and number of nodules. AFP, DCP, neutrophil-to-lymphocyte ratio (NLR), and lymphocyte-to-monocyte ratio (LMR) were measured just prior to LDLT. 12 The histological grades obtained from the explanted livers were used for tumor differentiation and the presence of vascular invasion.

| Donor and graft selection
Donors were selected from candidates who hoped to be living donors. 13 Donors were required to be within the third degree of consanguinity with recipients or spouses, and to be between 20 and 65 years of age. For a donor who was not within the third degree of consanguinity, individual approval was obtained from the Ethics Committee of Kyushu University Hospital. Good Samaritan donations were not used.
Eligible donors proceeded to the imaging studies, including chest and abdominal X-rays and 1-mm-slice computed tomography (CT)

K E Y W O R D S
alpha-fetoprotein, des-gamma-carboxy prothrombin, hepatocellular carcinoma, Japan criteria, living donor liver transplantation scans for graft volumetric analysis. Three-dimensional CT was introduced for the volumetric analysis and delineation of vascular anatomy. The standard liver weight (SLW) of recipients was calculated according to the formula of Urata. 14 Graft weight (GW) was predicted by CT volumetric analysis. Our usual decision about graft type for recipients was based on the preoperatively predicted GW to SLW ratio. 15 LL + C graft was used when the preoperatively predicted GW-SLW ratio was ≥35%. When GW-SLW ratio with LL + C graft was <35% and remnant donor liver volume after right lobectomy was ≥35%, right lobe graft was used. Posterior segment graft was considered when the donor's vascular anatomy was suitable to take a posterior segment. 13

| Postoperative management
The graft retrieval technique, recipient surgery, and perioperative management of the recipients, including immunosuppression regimens, were described previously. 4,11 Splenectomy was performed when patients had preoperative low predicted GW-SLW ratio (35% or less), portal hypertension indicated by a large portosystemic shunt, splenomegaly, risky esophagogastric varices, high portal pressure (>20 mm Hg) after unclamping, ABO blood type-incompatible donor, positive HCV RNA, or severe hypersplenism defined as a preoperative WBC count < 1000/mL and/or platelet count < 50 000/ mL. 16 Immunosuppression was initiated using a protocol based on either tacrolimus (Prograf; Astellas Pharma Inc.) or cyclosporine A (Neoral; Novartis Pharma KK) with steroid and/or mycophenolate mofetil (MMF; Chugai Pharmaceutical Co. Ltd.). A target trough level of tacrolimus was set at 10 ng/mL for 3 months after LDLT, followed by 5-10 ng/mL thereafter. A target trough level of cyclosporine A was set at 250 ng/mL for 3 months after LDLT, followed by 150-200 ng/mL thereafter. Methylprednisolone was initiated on the day of LDLT, tapered and converted to prednisolone 7 days after LDLT.
Prednisolone treatment was tapered and discontinued 6 months after LDLT. MMF was started at 1-2 g/d on the day after LDLT, tapered and discontinued until 6 months after LDLT. A trough level was not measured for MMF. Everolimus was covered by national insurance in 2018 and was used 1-3 months after LDLT for cases beyond the MC. 17 All patients had monthly follow-ups, and the median follow-up period was 2595 days, with 983 days and 4363 days as the 25th and 75th percentiles, respectively. Tumor markers were routinely checked every 3 months. Chest/abdominal CT scan and bone scintigraphy were routinely performed every 6 months after LDLT to rule out HCC recurrence.

| Post-LDLT HCC recurrence and recipient HCC-related mortality
Hepatocellular carcinoma recurrence after LDLT was set as the primary end point. HCC recurrence was defined when any imaging studies, such as chest or abdominal CT scan, or bone scintigraphy revealed the recurrence of HCC. Recurrence-free survival was defined as the period between LDLT and tumor recurrence. Patient HCC-related mortality after the LDLT was set as the secondary end point. Overall survival was defined as the time period between LDLT and recipient HCC-related death.

| Statistical analysis
Recurrence-free or overall survival rates were calculated by the Kaplan-Meier product-limited method. Data were expressed as means. All statistical analyses were performed using JMP 14.
a Twenty-six cases underwent LDLT for indications other than HCC and were included in this study because HCC was found upon explant pathology. more positive microvascular invasion, and bilobar distribution by pathology. Figure 2 shows the recurrence-free and overall survival rates among patients beyond the JC. The patients were divided into two groups according to DCP levels. The 1-, 5-, and 10-year recurrence-free survival rates in patients with DCP < 300 mAU/mL were 88.3%, 71.5%, and 66.7%, respectively. The rates in patients with DCP ≥ 300 mAU/mL were 34.4%, 13.8%, and 13.8%, respectively (P < .0001, Figure 2A). The 1-, 5-, and 10-year overall survival rates in patients with DCP < 300 mAU/mL were 100%, 83.2%, and 78.6%, respectively. In contrast, the rates in patients with DCP ≥ 300 mAU/mL were 73.3%, 26.7%, and 20.0%, respectively (P < .0001, Figure 2B).

| D ISCUSS I ON
Des-gamma-carboxy prothrombin was an independent risk factor for HCC recurrence and HCC-related mortality after LDLT for patients beyond the JC. This study revealed that patients beyond the JC had a 65% of chance of surviving for more than 10 years after LDLT when their pre-transplant DCP levels were <300 mAU/mL, even though Japanese insurance does not cover the medical cost for these cases. DCP level is well established as a sensitive and specific tumor marker in patients with HCC and is an independent predictive factor of microvascular invasion. 18 We previously reported that DCP level was significantly correlated with macroscopic invasion and intrahepatic metastasis in the explanted liver, 11 and was a predictor of microvascular invasion even for HCC of ≤3 cm. 19 As shown in Table 4, patients with DCP ≥ 300 mAU/mL had significantly more microvascular invasion. DCP enhanced cell proliferation by Met receptor and angiogenesis by vascular endothelial growth factor. 19 Patients beyond the JC with a DCP ≥ 300 mAU/mL had larger sized and more numerous tumors in bilateral lobes and higher AFP, which may reflect the ability of these tumors and the poorer prognosis. antitumor T-cell exhaustion. 22 We recently reported the impact of LMR in resection or LDLT for HCC patients. 12 in AFP that reflected the treatment response after locoregional therapy, or as a surrogate for the biological behavior of patients, without treatment on a waiting list. 25 They stated that many criteria use AFP at a single timepoint, even though patients usually wait a long time until LT and undergo locoregional therapy for HCC during the waiting period. Therefore, they hypothesized that the dynamic changes in AFP were a better predictor of recurrence and survival. As shown in Tables 1 and 4 Abbreviations: AFP, alpha-fetoprotein; DCP, des-gamma-carboxy prothrombin; ELD, model for end-stage liver disease; GRWR, graft recipient weight ratio; MR, lymphocyte-to-monocyte ratio; NLR, neutrophil-to-lymphocyte ratio; SLW, standard liver weight; W, graft weight.

ACK N OWLED G M ENTS
We thank Edanz Group (www.edanz editi ng.com/ac) for editing a draft of this manuscript.