ABO-Incompatible Adult Living Donor Liver Transplantation Under the Desensitization Protocol With Rituximab

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1 American Journal of Transplantation 2016; 16: Wiley Periodicals Inc. C Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons doi: /ajt ABO-Incompatible Adult Living Donor Liver Transplantation Under the Desensitization Protocol With Rituximab G.-W. Song 1, S.-G. Lee 1, *, S. Hwang 1, K.-H. Kim 1, C.-S. Ahn 1, D.-B. Moon 1, T.-Y. Ha 1, D.-H. Jung 1, G.-C. Park 1, W.-J. Kim 1, M.-H. Sin 1, Y.-I. Yoon 1, W.-H. Kang 1, S.-H. Kim 1 and E.-Y. Tak 2 1 Division of Liver Transplantation and Hepato-Biliary Surgery, Department of Surgery, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea 2 Asan Center for Life Science, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea Corresponding author: Sung-Gyu Lee, sglee2@amc.seoul.kr ABO incompatibility is no longer considered a contraindication for adult living donor liver transplantation (ALDLT) due to various strategies to overcome the ABO blood group barrier. We report the largest singlecenter experience of ABO-incompatible (ABOi) ALDLT in 235 adult patients. The desensitization protocol included a single dose of rituximab and total plasma exchange. In addition, local graft infusion therapy, cyclophosphamide, or splenectomy was used for a certain time period, but these treatments were eventually discontinued due to adverse events. There were three cases (1.3%) of in-hospital mortality. The cumulative 3-year graft and patient survival rates were 89.2% and 92.3%, respectively, and were comparable to those of the ABO-compatible group (n ¼ 1301). Despite promising survival outcomes, 17 patients (7.2%) experienced antibody-mediated rejection that manifested as diffuse intrahepatic biliary stricture; six cases required retransplantation, and three patients died. ABOi ALDLT is a feasible method for expanding a living liver donor pool, but the efficacy of the desensitization protocol in targeting B cell immunity should be optimized. Abbreviations: ABOc, ABO compatible; ABOi, ABO incompatible; ACLF, acute-on-chronic liver failure; ACR, acute cellular rejection; ALC, alcoholic liver cirrhosis; ALDLT, adult living donor liver transplantation; AMR, antibody-mediated rejection; BMI, body mass index; BPACR, biopsy-proven acute cellular rejection; BS, biliary stricture; BTZ, bortezomib; CD20þ, CD20-positive B lymphocyte; COD, cause of death; COGF, cause of graft failure; CMV, cytomegalovirus; CR, chronic rejection; Cx, complication; D, donor; DDLT, deceased donor liver transplantation; DIHBS, diffuse intrahepatic biliary stricture; DSZ, desensitization; F, female; FHF, fulminant hepatic failure; GSR, graft survival rate; GRWR, graft-torecipient weight ratio; HA, hepatic artery; HAS, hepatic artery stenosis; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HVS, hepatic vein stenosis; IA, isoagglutinin; ICU, intensive care unit; LDLT, living donor liver transplantation; LFT, liver function test; LGIT, local graft infusion therapy; LL, left lobe; LT, liver transplantation; M, male; MELD, Model for End-Stage Liver Disease; MMF, mycophenolate mofetil; MRL, modified right lobe; PCLD, polycystic liver disease; Pn, pneumonia; PSR, patient survival rate; PV, portal vein; PVS, portal vein stenosis; R, recipient; RBC, red blood cell; RIT, rituximab; RL, right lobe; SBC, secondary biliary cirrhosis; SFSG, small-forsize graft; SPL, splenectomy; T/F, transfusion; TPE, total plasma exchange; yr, year Received 16 March 2015, revised 29 May 2015 and accepted for publication 24 June 2015 Introduction Despite great potential for donor-pool expansion, the application of ABO-incompatible (ABOi) adult living donor liver transplantation (ALDLT) has been limited because of concerns about the poor survival outcomes of recipients. When Starzl et al (1) breached the ABO blood group barrier in liver transplantation (LT), the liver was regarded as an immunologically privileged organ; however, the actual outcomes of ABOi LT were disappointing, with a high incidence of early graft loss due to antibody-mediated rejection (AMR) manifesting as hemorrhagic necrosis with diffuse intravascular coagulation within the graft, so-called single organ diffuse intravascular coagulation (2,3). To avoid AMR, unselective overimmunosuppression such as wholebody irradiation, scheduled steroid pulse therapy, or extremely high trough levels of calcineurin inhibitor have been used, but these approaches failed to show any positive impact on the prevention of AMR or improvement in survival outcome. Most deaths in the earlier experiences of ABOi LT were associated with AMR or infections caused by inefficacious overimmunosuppression. Consequently, ABOi LT became unpopular and was reserved for urgent LT, 157

2 Song et al particularly for pediatric patients (4). Since the introduction of desensitization (DSZ) with the anti-cd20 monoclonal antibody rituximab (RIT), the risk of hepatic necrosis caused by full-blown AMR has almost disappeared, and overimmunosuppression aimed at avoiding AMR is no longer required (5,6). Nonetheless, most reports on the clinical outcomes of ABOi ALDLT in the literature still include unfavorable outcome data from before the RIT era, leading to overestimation of the morbidity rate and underestimation of the survival rate of current ABOi ALDLT (7). As such, misconceptions about unacceptable clinical outcomes of ABOi ALDLT persist and limit the widespread application of ABOi ALDLT. In this study, we reported the largest singlecenter experience showing recent achievements of ABOi ALDLT under RIT DSZ. Materials and Methods Study population This retrospective study included adult patients (aged 18 years) who underwent ABOi living donor liver transplantation (LDLT) at a single center between November 2008 and December Adult patients who underwent ABO-compatible (ABOc) LDLT during the same period were included in a control group for comparison of outcomes. DSZ protocol and immunosuppression The DSZ protocol for ABO incompatibility consisted of a single dose of RIT ( mg/m 2 per body surface area) and total plasma exchange (TPE). RIT was infused intravenously 2 3 weeks prior to LDLT. The first pretransplant TPE was usually performed 1 week after the administration of RIT. The frequency and timing depended on the recipient s isoagglutinin (IA) titer before DSZ. The target IA titer before LT was 1:8. For the first 20 cases, we used local graft infusion therapy (LGIT) in which methylprednisolone and prostaglandin E1 were administered through a catheter installed through the hepatic artery (HA) or portal vein (PV) branch. We continued LGIT for 3 weeks (8). Additional components of DSZ such as cyclophosphamide (CSP; 2 mg/kg per day) or splenectomy (SPL) were used for a period of time but eventually were eliminated because of adverse events. Posttransplant immunosuppression comprised tacrolimus, corticosteroid and mycophenolate mofetil (MMF) ( g/day). Corticosteroid was usually tapered off over 3 months after LT. At the beginning of our program, we adopted the DSZ protocol used by a Japanese group (9); however, we modified the protocol after trial and error, as presented in Figure 1. Monitoring for AMR The CD20-positive B lymphocyte (CD20þ) count was measured prior to DSZ and LDLT, twice a week during the first week after LDLT, once a week while the patient remained in the hospital, and at every visit to the outpatient clinic after discharge. The IA titer was measured prior to DSZ and just before subsequent TPE. During the first week after LT, the IA titer was checked daily. Thereafter, it was measured twice a week until discharge and at every visit to the outpatient clinic after discharge. IA titers were measured by Figure 1: Evolution of the desensitization and immunosuppression protocol for ABO-incompatible adult living donor liver transplantation at Asan Medical Center. HA, hepatic artery; IA, isoagglutinin; LDLT, living donor liver transplantation; MMF, mycomophetilphenolate; PV, portal vein; TPE, total plasma exchange. 158 American Journal of Transplantation 2016; 16:

3 ABOi ALDLT standard blood-banking procedures. Anti-A and anti-b IA titers were determined by incubating a standard type A and B red blood cell suspension in saline with two-fold serial dilutions of patient serum at room temperature followed by centrifugation and agglutination scoring. Diagnosis and management of AMR AMR in ABOi groups can have two clinical manifestations: hepatic necrosis occurring within 1 week after LDLT or diffuse intrahepatic biliary stricture (DIHBS) usually occurring 1 or 2 months later (10,11). In this study, we did not perform protocol biopsy. If there was any clinical suspicion of AMR (IA titer 1:64, aminotransferase or total bilirubin levels 2 3 times baseline or higher, no technical reason for an abnormal liver function test [LFT]), we increased the dosage of tacrolimus and steroids and performed TPE and liver biopsy. We repeated TPE until the IA titer decreased to 1:32 and stabilized. If patients did not respond to the initial treatment or if liver biopsy revealed histologic evidence of AMR, we administered high-dose intravenous immunoglobulin ( mg/kg per day) and high-dose steroid bolus (5 10 mg/kg per day) and performed TPE daily. For accurate histologic diagnosis of AMR, a liver specimen should contain at least five portal areas. AMR was suspected when periportal edema and necrosis or hemorrhagic edema was associated with an increase in IA titers on hematoxylin and eosin staining (12). In such cases, we performed special immunohistochemical staining of formalin-fixed paraffin-embedded tissue by using a polyclonal antibody against C4d complement. The intensity of the C4d staining was quantitatively assessed by the percentage of portal tracts containing distinctly stained stroma and/or endothelium. Biopsies containing 50% stromal-positive portal tracts were considered positive (13). DIHBS was considered if there was evidence of multiple strictures and diffusely scattered dilatation of intrahepatic bile ducts on computed tomography without HA thrombosis. If the stricture with dilatation of the biliary tree on endoscopic retrograde or percutaneous transhepatic cholangiography was diffuse and multifocal and located in the center proximal to the hepatic hilum, the definite diagnosis of DIHBS was made (10,14,15). considered for ABOi ALDLT and underwent DSZ; however, two patients (0.8%) were excluded due to TPE-related anaphylactic shock. Of these, one patient required cardiopulmonary resuscitation due to circulatory collapse. In two patients, hepatic encephalopathy and hepatorenal syndrome occurred in each due to disease progression during DSZ. Of these, one patient underwent deceased donor LT (DDLT) and one died. In one patient (0.4%) with extremely high IA (1:2048), the IA titer did not decrease to below 1:256, and the patient was excluded. After the exclusion of five patients, the mean age of the 235 recipients of ABOi ALDLT was years (range: years), and 173 (73.6%) were men. The mean Model for End-Stage Liver Disease (MELD) score was (range: 5 34). No patient with acute hepatic failure, pre-lt stay at an intensive care unit, pre-lt use of vasopressor or ventilator care underwent ABOi ALDLT. The most common origin of disease was hepatitis B virus associated liver cirrhosis (n ¼ 170, 72.3%), and 117 patients (49.8%) had hepatocellular carcinoma, which was the most common reason for LT. The most commonly used graft type in the ABOi group was modified right lobe (n ¼ 206, 87.7%). Dual-graft LDLT was performed in 23 patients (9.8%), of whom 17 underwent ABOi/ABOc graft combinations and 6 underwent ABOi/ABOi combinations. In one case of dual-graft LDLT, the ABOi split graft came from a deceased 15-yearold male donor. Consequently, a total of 258 donors including one deceased donor were involved. The mean age of the 258 donors was years (range: years), and 179 (69.4%) were men. Donor selection and ethical considerations The donor selection criteria and evaluation process have been described elsewhere (16). We evaluated an ABOi donor candidate only if an ABOc candidate was unavailable. Each transplantation procedure was evaluated and approved by the local authorities and the Korean Network for Organ Sharing affiliated with the Ministry of Health and Welfare of the Republic of Korea, and this study was reviewed and approved by the institutional review board of our institute. The requirement for informed consent was waived because of the retrospective nature of the study. Statistical analysis Data are expressed as mean plus or minus standard deviation. Statistical analysis was performed using SPSS 14.0 for Windows (IBM Corp, Armonk, NY). Patient and graft survival rates were calculated using the Kaplan Meier method, and the data were compared using the log-rank test. All other parameters were compared using the Student t test or Fisher exact test. For multivariate analysis, Cox regression was used. Results Demographic data of the ABOi group From November 2008 to December 2013, a total of 1536 ALDLTs were performed at our institute, and 235 recipients (15.3%) received ABOi grafts. Initially, 240 patients were Comparison of demographic and other clinical parameters between the ABOi and ABOc groups Comparison of demographic and clinical parameters between the ABOi and ABOc groups is shown in Table 1. The mean pretransplant MELD score was significantly lower (12.7 vs. 16.9, p < 0.001), and the mean pretransplant length of hospital stay was longer (14.2 vs. 9.7 days, p < 0.001) in the ABOi group than in the ABOc group. The prevalence of pretransplant bacteremia and pneumonia and the necessity for renal replacement therapy were significantly lower in the ABOi group. No significant differences in donor profiles and operative and graft factors were noted between the ABOi and ABOc groups. Changes in the CD20þ count and IA titer in the ABOi group A mean dosage of mg/m 2 (range: mg/m 2 ) of RIT was administered at an average of days (range: 8 43 days) prior to LDLT. The mean pre-dsz level of CD20þ was 13.1% 7.2% (range: %) and decreased to 0.2% 0.7% (range: %) at the time of LDLT. The CD20þ level increased to a highest mean value of 2.1% 3.0% (range: %) at a mean of American Journal of Transplantation 2016; 16:

4 Song et al Table 1: Comparison of recipient and donor clinical characteristics between ABO-incompatible and -compatible adult living donor liver transplantation recipients Variable ABO-incompatible group (n ¼ 235) ABO-compatible group (n ¼ 1301) p value Recipient age (range) (25 68) (19 70) Recipient sex (male/female) 173 (73.6%)/ (75.5%)/ ABO blood type A 59 (25.1%) A 537 (41.3%) <0.001 B 47 (20.0%) B 342 (26.3%) O 129 (54.9%) O 262 (20.1%) AB 0 (0.0%) AB 160 (12.3%) Original disease HBV 170 (72.3%) HBV 879 (67.6%) HCV 17 (7.2%) HCV 87 (6.7%) Nonviral disease 48 (20.4%) Nonviral disease 335 (25.7%) Combined HCC (yes/no) 116 (49.4%)/ (51.1%)/ MELD score (range) (5 34) (5 40) <0.001 Pre-LT hospital stay (days) (5 63) (0 111) <0.001 Pre-LT combined factor (yes/no) Bacteremia 1 (0.7%) 17 (1.3%) Pneumonia 1 (0.7%) 59 (4.5%) Renal replacement therapy 4 (2.8%) 78 (6.0%) Ventilator care 0 (0.0%) 71 (5.5%) ICU stay 0 (0.0%) 85 (6.5%) <0.001 Use of vasopressor 0 (0.0%) 26 (2.0%) FHF (yes/no) 0 (0.0%) 72 (5.5%) ACLF (yes/no) 1 (0.04%) 68 (5.2%) Emergency LT (yes/no) 0 (0.0%) 85 (6.5%) Liver retransplantation 3 (1.3%) 11 (0.8%) Donor age (years) ( ) (16 57) Donor sex (male/female) 179 (69.4)/ (69.2%)/ Donor BMI (range) ( ) ( ) Intraoperative RBC T/F (unit) (0 170) (0 133) Cold ischemic time (min) (23 270) (23 271) Warm ischemic time (min) (18 126) (17 164) Graft type (RL/LL/dual) 266 (87.7%)/6/ (90.6%)/23/ GRWR (%) ( ) ( ) Graft steatosis (%) ( ) ( ) ACLF, acute-on-chronic liver failure; BMI, body mass index; FHF, fulminant hepatic failure; GRWR, graft-to-recipient body weight ratio; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; ICU, intensive care unit; LL, left lobe; LT, liver transplantation; MELD, Model for End-Stage Liver Disease; RBC, red blood cell; RL, right lobe; T/F, transfusion. 1 Deceased donor. 2 Left lateral section graft in dual-graft living donor LT days (range: 1 30 days) after LDLT. The trends in CD20þ changes before and after DSZ and LDLT are depicted in Figure 2. Patients were categorized into three groups according to pre-ldlt IA titer: low (1:64), intermediate (1:128 1:512) and high (1:1024). The pre-dsz IA titer was low in 89 patients (37.8%), intermediate in 140 (59.6%) and high in 6 (2.6%) (Figure 3A). A summary of the clinical data of the six patients with high pre-dsz IA titer is shown in Table 2. After a mean sessions (range: 1 15 sessions) of pre- LDLT TPE, all patients achieved a target pre-ldlt IA level (1:8). After LDLT, 103 patients (43.8%) maintained an IA titer of <1:16. Fifty patients (23.5%) showed a rebound rise in IA titer to >1:8 (Figure 3B) and underwent a mean sessions (range: 0 20 sessions) of TPE at a mean days (range: 1 19 days) after LDLT. Three patients (1.3%) showed an elevation in IA titer of >1:512. A summary of the clinical data of the three patients with a posttransplant IA titer higher than 1:512 is shown in Table 2. Analysis for survival outcomes The in-hospital mortality rate in the ABOi group was 1.3% (3 of 235), which was significantly lower than that of the ABOc group (4.7%, p ¼ 0.007). The retransplantation rates of the ABOi and ABOc groups were 1.6% and 3.4%, respectively, with no statistically significant difference between the groups. Detailed clinical information regarding in-hospital mortality and retransplantation in the ABOi group is presented in Tables 3 and 4, respectively. The 1- and 3- year graft survival rates of the ABOi group were 93.3% and 89.2%, respectively, whereas the 1- and 3-year patient survival rates were 96.5% and 92.3%, respectively. No 160 American Journal of Transplantation 2016; 16:

5 ABOi ALDLT the ABOc group (12.0%, p < 0.001); however, the incidence of pure anastomotic stricture was similar between the two groups (12.4% vs. 12.0%, p ¼ 0.988). There was no difference in the overall incidence of infectious complications or surgical complications between the two groups. Comparison of the incidence of morbidity between the two groups is presented in Table 5. Figure 2: CD20-positive B cell levels before and after desensitization and living donor liver transplantation. significant differences in the graft and patient survival rates were noted between the two groups. To adjust for significant differences in recipients pretransplant conditions between the ABOc and ABOi groups, we conducted propensity score matched analysis. There were no significant differences in the patient and graft survival rates between the two groups in the propensity score matched cohort (Figure 4). Morbidity The most common source of morbidity in the ABOi group was biliary stricture (19.6%) including DIHBS, which was significantly higher than the incidence of biliary stricture in In ABOi recipients, LGIT-related complications occurred in 6 of 20 patients (30.0%). Of the nine patients with an HA catheter, two received injuries to the HA on catheter insertion. Both cases required immediate surgical revision of the HA anastomosis. Of the 11 patients with a PV catheter, four developed intra-abdominal bleeding due to catheter dislocation, and two of those patients underwent surgical exploration. In this study, 107 patients underwent simultaneous SPL. Of those, 15 patients (14.0%) experienced SPL-associated surgical complications including bleeding (n ¼ 7), pancreatic fistula (n ¼ 4) and PV thrombosis (n ¼ 3). Analysis of risk factors for AMR A total of 50 patients showed a rebound rise of IA titer 1:64. Of those, 27 patients (54%) showed elevated serum transaminase or total bilirubin 2 3 times baseline or more. We performed liver biopsy in the 27 patients with clinical suspicion of AMR. Of those, only one patient had marked periportal edema and necrosis and diffuse positivity of C4d immunohistochemical staining. All 27 patients responded to TPE with stabilization of the IA titer 1:32 and LFT, but DIHBS eventually occurred in 7 patients. An additional 10 patients developed DIHBS without antecedent clinical suspicion of AMR. Consequently, AMR manifesting as DIHBS was diagnosed in 17 patients (7.2%) in the ABOi Figure 3: Distribution of patients according to their isoagglutinin titer (A) before desensitization and (B) after living donor liver transplantation. American Journal of Transplantation 2016; 16:

6 Song et al Table 2: Summary of clinical information for patients with high predesensitization and post liver transplantation isoagglutinin titers Patient final Graft survival period (mo) Post-LT TPE (frequency) LGIT SPL ACR AMR BS Pre-LT TPE (frequency) Post-LT peak IA titer Initial IA titer ABO mismatch (D!R) MELD Patient number Sex Age High predesensitization isoagglutinin titers 1 F 51 A!O No No No No No 40.8 Alive 2 M 53 B!O No No No No No 37.4 Alive 3 M 48 A!O No No No No No 26.8 Alive 4 M 54 A!O No No No No No 26.1 Alive 5 M 61 B!O No Yes No No No 12.6 Alive 6 M 48 A!O No No No No No 36.1 Alive Post-LT isoagglutinin titers 1 M 53 B!A No No Yes Yes Yes 46.6 Alive 2 M 50 A!O No No No No No 40.7 Alive 3 M 64 A!B No Yes No Yes Yes 18.9 Alive ACR, acute cellular rejection; AMR, antibody-mediated rejection; BS, biliary stricture; D, donor; F, female; IA, isoagglutinin; LGIT, local graft infusion therapy; LT, liver transplantation; M, male; MELD, Model for End-Stage Liver Disease; R, recipient; SPL, splenectomy; TPE, total plasma exchange. group. The clinical information of those patients is detailed in Table 6. Of the 17 patients with DIHBS, two died without retransplantation and one died after re-lt (DDLT), and at the time of writing, six were alive after re-lt (one LDLT and five DDLT), one remained active on a waiting list for DDLT, and seven were alive with a well-functioning graft. The mean follow-up period of the seven patients with well-functioning grafts despite DIHBS was months (range: months); however, the 1-year graft survival rate of patients with AMR was significantly lower than that of patients without AMR (55.8% vs. 98.8%, p < 0.001). In a risk factor analysis for AMR, the type of DSZ and immunosuppression protocol, the use of LGIT, and the performance of SPL did not affect the incidence of AMR (Figure 5A C). When we analyzed the incidence of infectious complications according to the evolving steps of the DSZ and IS protocol, as described in Figure 1, the incidence was highest in phase I (Figure 5D). Modification of the DSZ protocol including CSP, LGIT or SPL in addition to RIT increased the incidence of infectious complications (Figure 5C E); however, the difference was not statistically significant. The interval of LDLT from the administration of RIT had no impact on the incidence of AMR (Table 7). In addition, initial and post-lt peak levels of IA titer were not associated with the incidence of AMR in univariate analysis (Table 7). Discussion Although this study did not include an exact quantitative assessment of the impact of including ABOi donors on living liver donor pool expansion, we found a 15% increase in the number of LDLTs over 5 years in our experience. In addition, the survival outcomes of this 5-year single-center experience, which included the largest number of patients to date (n ¼ 235), were excellent, with 3-year graft and patient survival rates of 89.2% and 92.3%, respectively. Such findings provide evidence of the feasibility and effectiveness of ABOi ALDLT. These results, however, should be interpreted with caution. The mean MELD score of the ABOi group in this study was 12.7, which is significantly lower than the mean MELD in the ABOc group. The survival outcomes following LDLT largely depend on the patient s pretransplant condition, and that is also true in ABOi ALDLT. Several previous studies reported that pretransplant intensive care unit stay and high MELD score were the most significant risk factors for patient survival (17 19). The most common cause of death was not AMR but rather infection, which may be attributed to overimmunosuppression; therefore, we cannot exclude the possibility that ABO incompatibility negatively affects survival outcomes in patients with poor pretransplant conditions. Consequently, we performed propensity score matched analysis to adjust for factors affecting the survival outcome. We observed no difference in survival outcomes in the propensity score matched cohort. 162 American Journal of Transplantation 2016; 16:

7 ABOi ALDLT Table 3: Case summary of in-hospital mortalities among 235 ABO-incompatible adult living donor liver transplantation recipients Sex Age ABO mismatch (D!R) Disease MELD Graft type GRWR (%) LGIT SPL Initial IA Peak IA AMR BS Survival period (mo) COD M 56 A!O HBV 19 Dual (LL þ LL) 1.02 Yes No 16 1 No No 0.90 Pn M 56 AB!B HBV 24 MRL 1.36 No Yes 64 1 No No 1.90 Pn F 59 B!O SBC 20 MRL 2.92 No No No No 0.80 Sepsis AMR, antibody-mediated rejection; BS, biliary stricture; D, donor; COD, cause of death; F, female; GRWR, graft-to-recipient body weight ratio; HBV, hepatitis B virus; IA, isoagglutinin; LGIT, local graft infusion therapy; LL, left lobe; M, male; MELD, Model for End-Stage Liver Disease; MRL, modified right lobe; Pn, pneumonia; R, recipient; SBC, secondary biliary cirrhosis; SPL, splenectomy. Table 4: Case summary of retransplantation among 235 ABO-incompatible adult living donor liver transplantation recipients Sex Age ABO mismatch (D! R) Disease MELD GRWR (%) Graft type LGIT SPL Initial IA Peak IA AMR COGF Graft survival period (mo) Re-LT Type Patient survival period (mo) Final M 50 B!O PCLD MRL No No No SFSG 1.10 DDLT 35.0 Alive M 56 A!O HBV MRL No No Yes DIHBS 3.40 DDLT 30.5 Alive M 57 A!O ALC MRL No No Yes DIHBS 5.10 DDLT 29.1 Alive M 44 B!O HBV MRL No No Yes DIHBS 6.27 LDLT 21.4 Alive F 57 B!O HBV MRL No Yes Yes DIHBS DDLT 13.5 Dead M 60 B!O HBV MRL No Yes Yes DIHBS 5.33 DDLT 15.1 Alive M 49 A!O ALC MRL No No Yes DIHBS 2.13 DDLT 4.9 Alive ALC, alcoholic liver cirrhosis; AMR, antibody-mediated rejection; COGF, cause of graft failure; D, donor; DDLT, deceased donor liver transplantation; DIHBS, diffuse intrahepatic biliary stricture; F, female; GRWR, graft-to-recipient body weight ratio; HBV, hepatitis B virus; IA, isoagglutinin; LDLT, living donor liver transplantation; LGIT, local graft infusion therapy; LT, liver transplantation; M, male; MELD, Model for End-Stage Liver Disease; MRL, modified right lobe; PCLD, polycystic liver disease; R, recipient; SFSG, small-for-size graft; SPL, splenectomy. American Journal of Transplantation 2016; 16:

8 Song et al Figure 4: Comparison of the (A) 1-, 3- and 5-year patient survival rates and (B) graft survival rate between ABO-compatible (n ¼ 470) and ABO-incompatible (n ¼ 235) adult living donor liver transplantation recipients in a propensity-matched cohort. ABOc, ABO compatible; ABOi, ABO incompatible; GSR, graft survival rate; PSR, patient survival rate; yr, year. Table 5: Comparison of post liver transplantation morbidities between ABO-incompatible and -compatible adult living donor liver transplantation recipients Variable ABO-incompatible group (n ¼ 235) ABO-compatible group (n ¼ 1301) p value BPACR 19 (8.1) 122 (9.4) CR 2 (0.9) 10 (0.9) AMR 17 (7.2) 0 (0.0) <0.001 Biliary Cx 46 (19.6) 156 (12.0) <0.001 Infectious Cx 24 (10.2) 139 (10.6) Bacterial Pn 9 (3.8) 61 (4.7) Fungal Pn 4 (2.8) 30 (2.3) PCP Pn 3 (1.3) 9 (0.7) Pulmonary 2 (0.9) 4 (0.3) tuberculosis CMV 3 (1.3) 13 (1.0) infection Other 3 (1.3) 22 (1.7) infections Surgical Cx 20 (8.5) 88 (6.8) Bleeding 11 (4.9) 59 (4.5) HVS 6 (2.8) 21 (1.6) PVS 1 (0.7) 4 (0.3) HAS 2 (1.4) 4 (0.3) Data are shown as n (%). AMR, antibody-mediated rejection; BPACR, biopsy-proven acute cellular rejection; CMV, cytomegalovirus; CR, chronic rejection; Cx, complication; HAS, hepatic artery stenosis; HVS, hepatic vein stenosis; Pn, pneumonia; PVS, portal vein stenosis. Accordingly, we currently perform ABOi ALDLT, even for patients with high MELD scores and regardless of the patient s pretransplant condition. Further study is required with an intention-to-treat design to compare the clinical outcomes of DDLT and ABOi LDLT in seriously ill patients, with consideration of the local situation of deceased donor organ availability and waiting times. For DSZ, we adopted the protocol from Japanese experiences. The initial version included LGIT and CSP. In 20 patients with LGIT, two HA injuries and four intraabdominal bleedings occurred. Although all patients were rescued by timely surgical intervention, HA injury or massive intra-abdominal bleeding is a potentially lifethreatening complication in LDLT; therefore, we subsequently abandoned LGIT. At that time, the successful experience without LGIT in ABOi ALDLT reported by Ikegami et al (20) influenced the change in our DSZ protocol. With CSP, 12 patients experienced severe leukopenia and 10 patients experienced increased serum creatinine (>2.0 mg/dl). Although we are not sure that CSP was the definite cause of the severe leukopenia and increased serum creatinine, all patients improved after discontinuation of CSP. Consequently, we stopped using CSP. During our experience with the phase I and II protocols, we experienced a 6.2% incidence (8 of 127 patients) of AMR; therefore, we added SPL, expecting a significant decrease in AMR incidence. The spleen is one of the major organs that harbor antibody-secreting B cells. CD20-negative B lymphocytes (eg, plasma cells or memory B cells) can 164 American Journal of Transplantation 2016; 16:

9 ABOi ALDLT Table 6: Summary of the clinical information for 17 patients with antibody-mediated rejection among 235 ABO-incompatible adult living donor liver transplantation recipients Post-LT peak IA Re-LT Final Initial IA Post-LT peak B cell (%) Initial B cell (%) Interval LT AMR (month) Post-LT TPE Pre-LT TPE Interval RIT LT (days) Donor ABO LGIT SPL Recipient ABO Sex Age M 54 A B Yes No No Dead M 58 O A No No No Dead M 61 O A No No No Alive M 53 A B No No No Alive M 56 O A No No Yes Alive M 57 O A No No Yes Alive F 57 O A No No No Alive M 44 O B No No Yes Alive F 57 A B No Yes No Dead F 57 O B No Yes Yes Dead M 48 O A No Yes No Alive F 58 O B No Yes No Alive M 57 O AB No Yes No Alive M 64 B A No Yes No Alive M 60 O B No Yes Yes Alive F 41 O B No Yes No Alive M 49 O A No No Yes Alive AMR, antibody-mediated rejection; F, female; LGIT, local graft infusion therapy; LT, liver transplantation; M, male; RIT, rituximab; TPE, total plasma exchange. survive treatment with high-dose RIT, mostly in the spleen. Ramos et al (21) conducted an in vivo study of the effect of a DSZ protocol on human splenic B cell subsets and found that plasma cells (CD138þ) and memory cells (CD27þ) survived within the spleen. SPL has been used as a rescue therapy in ABOi kidney transplantation recipients with intractable AMR, and a successful outcome was achieved in some patients (22). In our experience, however, SPL did not decrease the incidence of AMR. With a phase III protocol, the incidence of AMR was slightly higher, although the increase was not statistically significant. In addition, we experienced SPL-associated complications in a substantial number of patients; therefore, the benefit and risk of SPL in ABOi LT should be balanced. RIT works by depleting CD20þ from the circulation and lymphoid tissues, thus RIT acts as a form of chemical SPL. Raut et al (23) found no statistically significant difference in the titers of anti-abo IgM and IgG antibodies between an SPL group and a non-spl group. Toki et al (24) studied the impact of low-dose RIT on the splenic B cell population in recipients of ABOi kidney transplantation and reported the near-complete depletion of CD20þ from the spleen. Consequently, we have recently abandoned SPL. Outcome data of ABOi LT can be divided into before and after RIT. A Japanese nationwide survey revealed that the 3-year survival rate increased from 30% to 80% after the introduction of RIT (17). A recent Japanese multicenter study revealed that the only risk factor for AMR was a DSZ protocol without RIT (5). In that study, the incidence of AMR decreased from 23.5% to 6.2% after the introduction of RIT. Timing, number of doses and total dose did not affect patient survival or the incidence of AMR. Meanwhile, regular dosage and multiple doses of RIT increased the incidence of fungal and cytomegalovirus infection. In the present study, most patients maintained very low CD20þ levels until 3 months after LDLT with a single infusion of RIT (Figure 2). We found similar results in terms of the incidence of infection according to DSZ type (Figure 5). At this point, we need to improve our understanding of the pharmacokinetic properties of RIT in transplant recipients. RIT can suppress cells in all stages of B lymphocyte differentiation except stem cells and long-lived plasma cells. Several previous studies revealed that the effect of RIT on B cells in peripheral blood was very rapid, eliminating cells within hours, and persisted for several months in transplant recipients (25). Furthermore, most data indicate that a single dose of RIT, as opposed to repeated doses, is sufficient for sustained suppression of B cells in peripheral blood (5,9,25,26). Consequently, repeated dosing of RIT may be unnecessary and may increase the risk of serious infection due to prolonged hypogammaglobulinemia. Pre-LT DSZ was usually required for >2 weeks in this study population; therefore, acute liver failure patients requiring an urgent LT were considered ineligible for ABOi LDLT in this study. The shortening of the DSZ period, which is American Journal of Transplantation 2016; 16:

10 Song et al Figure 5: Comparison of the incidence of antibody-mediated rejection according to (A) the phase of the desensitization and immunosuppression protocol, (B) the use of local graft infusion therapy, (C) the performance of splenectomy, (D) the phase of the desensitization and immunosuppression protocol, and (E) the modification in the rituximab prophylaxis protocol. Local graft infusion therapy, splenectomy, cyclophosphamide, or repeated dosing of rituximab. AMR, antibody-mediated rejection; LGIT, local graft infusion therapy; RIT, rituximab; SPL, splenectomy. determined mainly by the interval from RIT infusion to first TPE, is the key point for extending the indication of ABOi LDLT to acute liver failure. Egawa et al (9) suggested that the timing of RIT administration was closely related to the rebound rise of IA titer. They emphasized early RIT prophylaxis, with the assumption that TPE immediately after RIT infusion would diminish the effect; however, only sparse data are available to evaluate the impact of TPE on the pharmacokinetic properties of RIT. Nevertheless, TPE clearly has the potential to eliminate RIT from the plasma because TPE involves nonselective filtration, and all antibodies are small enough to pass through the pores of the membrane (27); however, the minimal time interval to minimize RIT waste by TPE is still unclear. Several studies have shown a remarkable increase in RIT clearance in thrombotic thrombocytopenic purpura or kidney transplant recipients by TPE performed within 1 3 days of RIT infusion (28,29); however, these studies did not analyze the impact of TPE on the therapeutic efficacy of RIT. Darabi et al (30) suggested that regularly scheduled TPE did not diminish the immunosuppressive effects of RIT due to the rapid effects of RIT on circulating CD20þ in a study with two thrombocytopenic purpura patients who were given RIT hours before TPE. In renal or liver transplant recipients, peripheral CD20þ also rapidly disappeared within hours after RIT infusion (25,31). Furthermore, a recent Japanese multicenter study showed that the timing of RIT use was not related to the incidence of AMR (5); therefore, we can cautiously consider an earlier start of the first TPE after infusion of RIT in acute liver failure patients requiring urgent LT. In addition, successful emergent LTs by the modified DSZ protocol, including immunoglobulin, routine daily post-lt TPE and ABO antigen-specific immunoadsorption with immunoadsorbent columns, have been reported, but the efficacy and feasibility of this approach should be validated in a larger cohort (32 34). Despite remarkable improvements in survival outcomes in ABOi LT, serious concerns persist about AMR. The diagnosis of rejection in LT is usually confirmed by histologic findings; however, confirmation of the diagnosis of AMR is often difficult because there is no consensus for the histologic diagnostic criteria of AMR in ABOi LT. Staining with C4d, an immunohistochemical stain for a fragment of activated complement particle, may be a special finding of AMR in ABOi LT (12,13); however, C4d staining alone cannot be considered specific for the diagnosis of AMR because focal C4d deposits in the arteriolar wall can be found in approximately 30% of liver biopsies from normal livers. Diffuse endothelial or sinusoidal C4d deposition is a finding of AMR, but it can be found in other inflammatory processes such as chronic or acute 166 American Journal of Transplantation 2016; 16:

11 ABOi ALDLT Table 7: Univariate analysis to identify risk factors for antibody-mediated rejection in 232 ABO-incompatible adult living donor liver transplantation recipients excluding three cases of in-hospital mortality Variable 1-Yr AMR-Free Survival Rate (%) Incidence of AMR (%) p value Recipient age <55 years (n ¼ 138) years (n ¼ 94) Recipient sex Male (n ¼ 171) Female (n ¼ 61) Original disease Viral (n ¼ 186) Nonviral (n ¼ 46) MELD score <20 (n ¼ 194) (n ¼ 38) Recipient ABO blood type A/B (n ¼ 105) O(n¼127) ABO blood barrier Anti-A (n ¼ 112) Anti-B (n ¼ 107) Anti-A and -B (n ¼ 13) Use of LGIT Yes (n ¼ 19) No (n ¼ 213) Splenectomy Yes (n ¼ 98) No (n ¼ 134) DSZ protocol type Phase I (n ¼ 19) Phase II (n ¼ 115) Phase III (n ¼ 98) Antecedent BPACR Yes (n ¼ 14) No (n ¼ 218) Interval from RIT to LT <2 weeks (n ¼ 39) weeks (n ¼ 193) IA titer Initial >1:1024 (n ¼ 6) :128 1:512 (n ¼ 139) :64 (n ¼ 87) Post-LT >1:64 (n ¼ 101) :16 1:64 (n ¼ 109) :8 (n ¼ 22) AMR, antibody-mediated rejection; BPACR, biopsy-proven acute cellular rejection; DSZ, desensitization; IA, isoagglutinin; MELD, Model for End-Stage Liver Disease; LGIT, local graft infusion therapy; LT, liver transplantation; RIT, rituximab. cellular rejection, biliary obstruction and viral hepatitis (35 37). The diagnosis of AMR should be based on the combination of multiple parameters including clinical (eg, LFT and radiologic finding), serological (IA titer) and histological findings. In general, AMR in ABOi LT can manifest in two clinical forms: (1) hepatic necrosis occurring within 1 2 weeks and leading to massive graft necrosis within 1 month or (2) DIHBS occurring several months after LT, with development of diffuse and multiple strictures of the intrahepatic bile duct (10). Since the introduction of RIT, the incidence of hepatic necrosis has become negligible. In this study, no patient experienced AMR manifesting as hepatic necrosis. In contrast to hepatic necrosis, DIHBS is not always fatal, but it frequently results in grave outcomes due to refractory cholangitis leading to sepsis and graft failure. In most cases, DIHBS cannot be resolved by conventional biliary interventions. The only proven effective treatment is retransplantation. Furthermore, the American Journal of Transplantation 2016; 16:

12 Song et al occurrence of DIHBS significantly affects patient quality of life because of reoccurrences of cholangitis and the need for biliary intervention and readmissions. Even though the role of the ABO antibody titer is still not well defined, the general belief is that a higher IA titer may result in a higher risk of AMR (4 6,17,26).The role of IA titers in the AMR of ABOi LT has been controversial. Mor et al (38) showed a possible link between a high IA titer and AMR, in particular, in A-to-O transplants, whereas Skogsberg et al (39) showed no correlation between a high IA titer and AMR. This may be due to heterogeneity in the direction of the ABO mismatch as well as ABO antigen levels, as may be the case in A-to-O transplants when high IA titers and high levels of antigen expression contribute to a higher risk of AMR. In our present study, the initial IA titer in patients with blood type O was significantly higher than that of patients with other blood types; however, we did not identify any significant correlation among the incidence of AMR, blood type and IA titer. Nonetheless, a high pretransplant IA titer can be lowered by repetitive TPE, and the posttransplant rebound rise in IA can be suppressed efficiently by RIT prophylaxis and posttransplant TPE. Consequently, the impact of IA titer on AMR may be masked, and no relationship between IA titer and patient survival or between recipient blood type or mismatch pattern and AMR or patient survival has been found. A recent large multicenter Japanese study by Egawa et al (5) and a single-center study by Song et al (6) failed to prove the impact of IA on AMR. Nevertheless, there is no doubt about the principal role of antidonor ABO IA in the pathophysiology of AMR. The question remains as to why antidonor ABO IA is detected after LT despite clearance or significant reduction in peripheral B lymphocytes by RIT. When we consider B lymphocyte immunology and the pharmacological effect of RIT, plasma cells that do not express CD20 may be the main cause of the persistence or rebound rise of IA after ABOi LT. The current DSZ protocol including RIT is very effective for the suppression of peripheral B lymphocytes in most patients but has not been shown to be effective in targeting the IA production of plasma cells. The occurrence of AMR in a substantial proportion of ABOi ALDLT recipients may be associated with the production of antidonor ABO IA by plasma cells, which are either newly generated from memory or na ıve B cells or from those that existed prior to transplant. Consequently, plasma cell depleting agents, such as bortezomib (BTZ), which is a proteasome inhibitor approved by the US Food and Drug Administration for the treatment of plasma cell dyscrasia, may be used in ABOi LT recipients with caution. BTZ selectively induces apoptosis among plasma cells, decreasing IA production (40). Several case reports and series have been published regarding the use of BTZ in the treatment or prevention of AMR associated with anti-hla antibody (41,42). As such, BTZ combined with RIT in the DSZ protocol for ABO incompatibility may be effective based on this theoretical benefit. Clinical experience with this drug in the context of AMR in ABOi LT is scarce, and well-controlled prospective studies are needed to prove the safety and efficacy of the DSZ protocol with BTZ. In conclusion, the use of ABOi living liver donors is a very effective and safe method for expanding the donor pool in LDLT. DIHBS, an attenuated form of AMR, remains an unresolved problem despite the fairly effective DSZ protocol. Further studies are required to improve the efficacy of the DSZ protocol and to identify definite risk factors and preventive measures for DIHBS. Disclosure The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation. References 1. Gordon RD, Iwatsuki S, Esquivel CO, Tzakis A, Todo S, Starzl TE. Liver transplantation across ABO blood groups. Surgery 1986; 100: Gugenheim J, Samuel D, Reynes M, Bismuth H. Liver transplantation across ABO blood group barriers. Lancet 1990; 336: Demetris AJ, Jaffe R, Tzakis A, et al. Antibody-mediated rejection of human orthotopic liver allografts. A study of liver transplantation across ABO blood group barriers. Am J Pathol 1988; 132: Raut V, Uemoto S. Management of ABO-incompatible living-donor liver transplantation: Past and present trends. 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Transplant Proc 2012; 44: Supporting Information Additional Supporting Information may be found in the online version of this article. Table S1: Baseline characteristics of the propensity score matched patients. Table S2: Comparison of patient and graft survival rates in the propensity-matched cohort. Figure S1: Annual number and ratio of ABO-incompatible adult living donor liver transplantations at Asan American Journal of Transplantation 2016; 16: