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A2ALL Provides valuable information on the outcomes of adult-to-adult living donor liver transplants (AALDLT) at 9 transplant centers, enrolled from 1998 through present Receives oversight and funding from the National Institutes of Health (NIH), a division of the U.S. Department of Health and Human Services Coordinated by the Arbor Research Collaborative for Health together with the University of Michigan, Ann Arbor, MI, USA For more information, visit A2ALL Background

A2ALL Outcomes of Living and Deceased Donor Liver Transplant Recipients with Hepatocellular Carcinoma: Results of the A2ALL Cohort L. M. Kulik, R. A. Fisher, D.R. Rodrigo, R. S. Brown, Jr., C. E. Freise, A. Shaked, J.E. Everhart, G. T. Everson, J. C. Hong, P. H. Hayashi, C. L. Berg, A. S. F. Lok and the A2ALL Study Group Am J Transplant Nov;12(11): Kulik, L. M. et al. Am J Transplant Nov;12(11):

A2ALL Background The use of living donor liver transplantation (LDLT) shortens waiting time in patients with hepatocellular carcinoma (HCC), but the rate of recurrent HCC had been reported to be higher after LDLT compared to deceased donor liver transplant (DDLT). Overall survival rates had been reported to be similar between LDLT and DDLT. Factors responsible for increased HCC recurrence in LDLT are speculative. The Adult-to-Adult Living Donor Liver Transplantation Cohort Study (A2ALL) reported a 3-year HCC recurrence was higher in LDLT recipients based on retrospective data. Kulik, L. M. et al. Am J Transplant Nov;12(11):

A2ALL Specific Aims To determine if there is a difference in patients with HCC receiving LDLT versus DDLT in the A2ALL Prospective Cohort Study in the MELD Era: - HCC recurrence rate - Survival rate after liver transplant - Recurrence-free survival rate To determine survival benefit of receiving LDLT compared to non-LDLT Kulik, L. M. et al. Am J Transplant Nov;12(11):

A2ALL Study Population and Data Collection 229 patients diagnosed with HCC prior to transplant across the 9 A2ALL centers were included. Data were obtained using retrospective chart review from 01/01/1998 to 02/28/2003 and a combination of retrospective and prospective collection from 02/28/2003 through 08/31/2010. Kulik, L. M. et al. Am J Transplant Nov;12(11):

A2ALL Methods Primary outcomes were HCC recurrence, overall survival, and recurrence-free survival from time of transplant. Kaplan-Meier estimates were computed and Cox regression was used to assess predictors of the three outcomes. –Predictors tested included transplant type, recipient age, race, AFP, Model for End-Stage Liver Disease (MELD) score, number of loco-regional therapy (LRT), pre/post MELD era, waiting time, etiology of cirrhosis, and explant characteristics. Sequential stratification from the time of donor evaluation was used to determine overall survival in LDLT versus non-LDLT. Kulik, L. M. et al. Am J Transplant Nov;12(11):

A2ALL Table 1. Characteristics of HCC patients at evaluation of the first living donor (1/2) Kulik, L. M. et al. Am J Transplant Nov;12(11):

A2ALL Table 1. Characteristics of HCC patients at evaluation of the first living donor (2/2) Kulik, L. M. et al. Am J Transplant Nov;12(11):

A2ALL LDLT (n=100)DDLT (n=97) Characteristics † Mean ± SD or % p-value ‡ At Transplant AFP (ng/mL) < > Log AFP 1.7 ± ± Donor Age (years) 35.0 ± ± Cold Ischemia Time (minutes) 65.6 ± ± 195 < MELD 13.3 ± ± Beyond Milan Beyond UCSF HCC ablation from donor evaluation to LT HCC ablation ever before LT No. of ablations since diagnosis 1.2 ± ± Days from donor evaluation to LT77.7 ± ± Kulik, L. M. et al. Am J Transplant Nov;12(11):

A2ALL LDLT (n=100)DDLT (n=97) Characteristics † Mean ± SD or % p-value ‡ Explant Pathology Tumor stage T0613 T1712 T231 T33524 T42120 Beyond Milan Beyond UCSF Bilobar HCC Diameter of largest nodule (cm) 4.3 ± ± No. of tumor nodules 2.4 ± ± Vascular invasion Total Length of Nodules (cm)6.4± ± Kulik, L. M. et al. Am J Transplant Nov;12(11): Table 2. Characteristics of HCC patients at transplant (2/3)

A2ALL LDLT (n=100)DDLT (n=97) Characteristics † Mean ± SD or % p-value ‡ Explant Pathology Tumor grade G129 G25453 G31618 † Number of missing values for [LDLT, DDLT] were: [14,12] for AFP, [1,24] for donor age, [23,3] for cold ischemia time, [9,3] for Milan, [16,5] for UCSF, and [0, 0-1] for other transplant variables, and [2-7,4-14] for explants variables except for tumor grade, [32,35]. ‡ p-values for comparison of LDLT and DDLT. Two-sample t-tests and chi-square tests were used for continuous variables and proportions, respectively. HCC=Hepatocellular carcinoma; LDLT=Living donor liver transplant; DDLT=Deceased donor liver transplant; LT=Liver transplant. Table 2. Characteristics of HCC patients at transplant (3/3) Kulik, L. M. et al. Am J Transplant Nov;12(11):

A2ALL LDLTDDLT Tumor Stage on Explant Pre-MELDMELDAllPre-MELDMELDAllOverall T01/30/31/60/20/100/121/18 T10/30/40/70/20/90/110/18 T24/130/174/300/60/230/294/59 T36/17 12/340/55/175/2217/56 T4a3/103/66/160/41/131/177/33 T4b2/32/24/51/1 2/26/7 Missing0/11/11/20/10/30/41/6 Total16/50 (32%) 12/50 (24%) 28/100 (28%) 1/21 (5%) 7/76 (9%) 8/97 (8%) 36/197 (18%) Table 3. HCC recurrence post-LT by type of transplant, tumor stage on explant and MELD era Kulik, L. M. et al. Am J Transplant Nov;12(11):

A2ALL HCC Recurrence onlyDeathHCC Recurrence or Death PredictorsHR95% CIp-valueHR95% CIp-valueHR95% CIp-value LDLT vs. DDLT2.35(1.04,5.35) (0.69,2.26) (0.88,2.59) Number of HCC nodules in the liver 1.42(1.17,1.71) Diameter of largest nodule (cm) 1.27(1.13,1.42)< (1.00,1.23) (1.04,1.26) Vascular invasion1.96(0.94,4.11) (0.92,3.07) (1.13,3.44)0.017 AFP at Transplant (ng/mL, log) (1.07,1.71) (1.02,1.63) Table 4. Cox regression models predicting HCC recurrence, death, and the combined outcome post-transplant Kulik, L. M. et al. Am J Transplant Nov;12(11): Panel A: for all HCC patients

A2ALL HCC Recurrence onlyDeathHCC Recurrence or Death PredictorsHR95% CIp-valueHR95% CIp-valueHR95% CIp-value LDLT vs. DDLT1.36(0.47,3.93) (0.33,2.19) (0.55,2.74) Number of HCC nodules in the liver 1.7(1.30,2.22) Diameter of largest nodule (cm) 1.28(1.07,1.53) (1.03,1.38) (1.06,1.37) Vascular invasion3.04(1.10,8.39) (1.43,8.34) (2.07,10.2) AFP at Transplant (ng/mL, log) (0.92,1.94) (0.80,1.64) Table 4. Cox regression models predicting HCC recurrence, death, and the combined outcome post-transplant Kulik, L. M. et al. Am J Transplant Nov;12(11): Panel B: for MELD era patients

A2ALL Figure 1. Cumulative probability over time of LDLT, DDLT, remaining alive on the waitlist, and death without transplant, from the first living donor evaluation (based on the cumulative incidence function) for (a) HCC patients in the Pre-MELD Era and (b) HCC patients in the MELD era. Kulik, L. M. et al. Am J Transplant Nov;12(11):

A2ALL Figure 2: Probability of freedom from HCC recurrence by time since LDLT or DDLT for (a) all HCC patients (unadjusted), (b) HCC patients in the MELD era (unadjusted), and (c) all HCC patients (adjusted based on a Cox regression model, with both LDLT and DDLT groups presented for the overall average baseline characteristic values of two liver nodules, largest nodule 3.5 cm, and without vascular invasion) Kulik, L. M. et al. Am J Transplant Nov;12(11):

A2ALL Figure 3. Unadjusted probability of patient survival by time since LDLT or DDLT for (a) all HCC patients and (b) HCC patients in the MELD era Kulik, L. M. et al. Am J Transplant Nov;12(11):

A2ALL Figure 4. Unadjusted probability of recurrence-free survival by time since LDLT or DDLT for (a) all HCC patients and (b) HCC patients in the MELD era Kulik, L. M. et al. Am J Transplant Nov;12(11):

A2ALL Figure 5. Adjusted probability of patient mortality by time from first living donor evaluation, for (a) all HCC patients and (b) HCC patients in the MELD era, both based on Cox regression models with both LDLT and DDLT groups presented for the average baseline characteristic values (55 years old, beyond Milan, AFP=10, lab MELD=12, and without ablation). Kulik, L. M. et al. Am J Transplant Nov;12(11):

A2ALL Supplementary Table 1. Characteristics of the 126 MELD era HCC patients at donor evaluation (1/2) LDLT (n=50)DDLT (n=76) No Transplant (n=20) Characteristics † Mean ± SD or % p-value ‡ Mean ± SD or % Male gender Age (years) 56.7 ± ± ± 11.9 Race White African American225 Other Etiology. % HCV AFP (ng/mL) < > Log AFP (ng/mL) 1.62 ± ± ± 1.19 MELD12.4 ± ± ± 4.8 Kulik, L. M. et al. Am J Transplant Nov;12(11):

A2ALL Supplementary Table 1. Characteristics of the 126 MELD era HCC patients at donor evaluation (2/2) Kulik, L. M. et al. Am J Transplant Nov;12(11): LDLT (n=50)DDLT (n=76)No Transplant (n=20) Characteristics † Mean ± SD or % p-value ‡ Mean ± SD or % Tumor stage T0036 T14156 T T T Beyond Milan Beyond UCSF Diameter of largest nodule (cm)4.7 ± ± ± 2.9 HCC ablation before donor evaluation † Numbers of missing values for [LDLT, DDLT, no transplant] were [0,0,0] for gender, age, and race, [5,6,1] for HCV etiology of cirrhosis, [1-3,2-3,0-2] for AFP and MELD, and [1-3,10-13,4- 5] for the rest of the variables. ‡ p-values for comparison of LDLT and DDLT for age, log AFP, MELD, and diameter of largest nodule computed from t-test; and for all other characteristics from chi-square test.

A2ALL Supplementary Table 2. Characteristics of the 126 MELD era HCC transplanted patients at transplant (1/3) Kulik, L. M. et al. Am J Transplant Nov;12(11): LDLT (n=50)DDLT (n=76) Characteristics† Mean ± SD or % p-value‡ At Transplant AFP (ng/mL) < > Log AFP, ng/mL 1.6 ± ± Donor Age, years 32.8 ± ± 17.6 < Cold Ischemia Time (minutes) 51.8 ± ± 197 < MELD 13.3 ± ± Beyond Milan Beyond UCSF HCC ablation from donor evaluation to LT Yes2253 No HCC ablation ever No. of ablations since diagnosis 1.1 ± ± Days from donor Evaluation to LT68.9 ± ±

A2ALL Supplementary Table 2. Characteristics of the 126 MELD era HCC transplanted patients at transplant (2/3) Kulik, L. M. et al. Am J Transplant Nov;12(11): LDLT (n=50)DDLT (n=76) Characteristics† Mean ± SD or % p-value‡ Explant Pathology Tumor stage T0614 T1812 T23432 T33423 T41619 Beyond Milan Beyond UCSF Bilobar HCC Diameter of largest nodule (cm) 4.5 ± ± No. of tumor nodules 2.3 ± ± Vascular invasion Total Length of Nodules (cm)6.9 ± ±

A2ALL Supplementary Table 2. Characteristics of the 126 MELD era HCC transplanted patients at transplant (3/3) Kulik, L. M. et al. Am J Transplant Nov;12(11): LDLT (n=50)DDLT (n=76) Characteristics† Mean ± SD or % p-value‡ Explant Pathology Tumor grade G12123 G26456 G31521 † Numbers of missing values for [LDLT, DDLT] were [0-3,0-3] for transplant variables except for: AFP [11,11], donor age [0,10], and cold ischemia time [7,1]; and were [17,28] for grade and [0-3, 2-12] for all other explants variables. ‡ p-values for comparison of LDLT and DDLT. Two-sample t-tests and chi- square tests were used for continuous variables and proportions, respectively.

A2ALL Supplementary table 3a. Single-variable Cox models for HCC recurrence Kulik, L. M. et al. Am J Transplant Nov;12(11): All HCC PatientsHCC Patients in the MELD Era Predictor Hazard Ratio 95% CIp-valueNGen. R 2 Hazard Ratio 95% CIp-valueNGen. R 2 LDLT vs DDLT3.76(1.71,8.26) %2.97(1.17,7.54) % Number of HCC Nodes in the liver1.35(1.15,1.59) %1.56(1.25,1.95) % Diameter of Largest Nodule (cm)1.27(1.15,1.41)< %1.26(1.11,1.43) % Vascular Invasion2.76(1.38,5.54) %4.53(1.79,11.42) % Years from Donor Evaluation to Transplant0.23(0.05,1.15) %0.33(0.04,2.50) % Grade 3 vs. Non-Grade 34.16(1.93,8.98) %3.41(1.21,9.60) % Ever Had an Ablation0.62(0.32,1.19) %0.43(0.17,1.05) % Etiology of Cirrhosis at Txp1.56(0.81,3.00) %1.45(0.55,3.84) % MELD at Transplant (max 40)0.96(0.90,1.04) %0.96(0.86,1.06) % Recipient Age at Txp (per 10 yrs)0.99(0.67,1.44) %1.18(0.67,2.08) % Donor Age at Txp (per 10 yrs)1.05(0.83,1.32) %1.14(0.84,1.53) % Cold Ischemia Time (hrs)0.87(0.77,0.98) %0.84(0.73,0.98) % Milan Stage at Explant6.36(2.47,16.41) %57.0*( )*< % UCSF Stage at Explant5.44(2.47,11.98)< %11.9(3.44,41.24)< % Log AFP at Transplant1.64(1.21,2.22) %1.61(1.03,2.53) % AFP>20 at Txp1.62(0.83,3.15) %2.42(0.98,5.97) % *Based on Firth estimate in the presence of separation

A2ALL Supplementary table 3b. Single-variable Cox models for death Kulik, L. M. et al. Am J Transplant Nov;12(11): All HCC PatientsHCC Patients in the MELD Era Predictor Hazard Ratio 95% CIp-valueNGen. R 2 Hazard Ratio 95% CIp-valueNGen. R 2 LDLT vs DDLT1.32(0.80,2.16) %1.28(0.62,2.65) % Number of HCC Nodes in the liver1.09(0.96,1.24) %1.28(1.06,1.53) % Diameter of Largest Nodule (cm)1.12(1.02,1.23) %1.14(1.01,1.27) % Vascular Invasion1.86(1.06,3.24) %3.36(1.57,7.20) % Years from Donor Evaluation to Transplant1.41(1.00,1.98) %2.45(1.32,4.55) % Grade 3 vs. Non-Grade 31.83(0.93,3.60) %1.82(0.72,4.61) % Ever Had an Ablation0.8(0.49,1.32) %0.52(0.25,1.07) % Etiology of Cirrhosis at Txp1.44(0.88,2.37) %0.64(0.25,1.61) % MELD at Transplant (max 40)1.06(1.02,1.11) %1.07(1.00,1.15) % Recipient Age at Txp (per 10 yrs)1.07(0.81,1.42) %1.13(0.73,1.76) % Donor Age at Txp (per 10 yrs)1.06(0.88,1.29) %1.11(0.87,1.42) % Cold Ischemia Time (hrs)0.96(0.89,1.04) %0.88(0.78,0.99) % Milan Stage at Explant1.44(0.87,2.39) %2.87(1.29,6.36) % UCSF Stage at Explant1.92(1.16,3.16) %3.15(1.47,6.73) % Log AFP at Transplant1.42(1.13,1.79) %1.45(0.99,2.11) % AFP>20 at Txp2.03(1.20,3.43) %2.11(1.03,4.33) %

A2ALL Supplementary Figure 1. Characteristics of HCC patients among those who had LDLT and recurred within one year post-transplant, all other LDLTs, and DDLTs. Tumor grade was available for 11 (79%), 57(66%), and 62(64%), in the 3 respective groups. Kulik, L. M. et al. Am J Transplant Nov;12(11):

A2ALL Results Kulik, L. M. et al. Am J Transplant Nov;12(11): HCC recurrence: –Among entire cohort, Unadjusted probability of HCC recurrence was higher in LDLT (38%) than in DDLT (11%) (p = ). Adjusted for tumor characteristics, HCC recurrence remained higher in LDLT (HR = 2.35, 95% CI ; p = 0.04). –In the MELD era, LDLT was not significantly associated with HCC recurrence after adjusted for tumor characteristics (HR = 1.36, 95% CI ; p = 0.57) Overall survival and recurrence-free survival were not significantly different in LDLT compared to DDLT for the entire cohort as well as the MELD era patients Sequential stratification from time of enrollment did not show a significant survival benefit associated with LDLT who received LDLT likely played a role in the higher HCC recurrence rate

A2ALL Results Kulik, L. M. et al. Am J Transplant Nov;12(11): Predictors of HCC recurrence: –Among entire cohort, LDLT, greater size of largest nodule, and larger number of nodules were associated with higher HCC recurrence. –In the MELD era, LDLT, greater size of largest nodule, larger number of nodules, and vascular invasion were associated with higher HCC recurrence. Predictors of mortality: –Among entire cohort, higher AFP at transplant was associated with higher mortality. –In the MELD era, greater size of largest nodule and vascular invasion were associated with higher mortality. Predictors of recurrence-free survival: –Among entire cohort, greater size of largest nodule, larger number of nodules, and higher AFP at transplant were associated with higher mortality. –In the MELD era, greater size of largest nodule and vascular invasion were associated with higher mortality.

A2ALL Conclusions Differences in tumor characteristics and management of HCC in patients who received LDLT likely played a role in the higher HCC recurrence rate observed in LDLT. in patients who received LDLT likely played a role in the higher HCC recurrence rate Kulik, L. M. et al. Am J Transplant Nov;12(11):

A2ALL Limitations Non randomized; differences in tumor characteristics between LDLT and DDLT Lack of uniformity in treatment for HCC prior to transplant Prioritization for MELD changed during the study period Kulik, L. M. et al. Am J Transplant Nov;12(11):