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Abstract

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Lymphocytes play an active role in natural immunity against hepatitis C virus (HCV). We hypothesized that a lower absolute lymphocyte count (ALC) may alter HCV outcome after liver transplantation (LT). The aim of this study was to investigate the impact of peritransplant ALC on HCV recurrence following LT. A total of 289 LT patients between 2005 and 2011 were evaluated. Peritransplant ALC (pre-LT, 2-week, and 1-month post-LT) and immunosuppression were analyzed along with recipient and donor factors in order to determine risk factors for HCV recurrence based on METAVIR fibrosis score. When stratifying patients according to pre- and post-LT ALC (<500/μL versus 500-1,000/μL versus >1,000/μL), lymphopenia was significantly associated with higher rates of HCV recurrence with fibrosis (F2-4). Multivariate Cox regression analysis showed posttransplant ALC at 1 month remained an independent predictive factor for recurrence (P = 0.02, hazard ratio [HR] = 2.47 for <500/μL). When peritransplant ALC was persistently low (<500/μL pre-LT, 2-week, and 1-month post-LT), patients were at significant risk of developing early advanced fibrosis secondary to HCV recurrence (F3-4 within 2 years) (P = 0.02, HR = 3.16). Furthermore, severe pretransplant lymphopenia (<500/μL) was an independent prognostic factor for overall survival (P = 0.01, HR = 3.01). The use of rabbit anti-thymocyte globulin induction (RATG) had a remarkable protective effect on HCV recurrence (P = 0.02, HR = 0.6) despite its potential to induce lymphopenia. Subgroup analysis indicated that negative effects of posttransplant lymphopenia at 1 month (<1,000/μL) were significant regardless of RATG use and the protective effects of RATG were independent of posttransplant lymphopenia. Conclusion: Peritransplant ALC is a novel and useful surrogate marker for prediction of HCV recurrence and patient survival. Immunosuppression protocols and peritransplant management should be scrutinized depending on peritransplant ALC. (Hepatology 2014;58:35–45)

Abbreviations
AA

African American

ACR

acute cellular rejection

ALC

absolute lymphocyte count

DCD

donation after cardiac death

HCV

hepatitis C virus

IVC

inferior vena cava

LDLT

living donor liver transplantation

LKT

liver and kidney transplantation

LT

liver transplantation

RATG

rabbit anti-thymocyte globulin

Hepatitis C virus (HCV) infection-associated cirrhosis is the most common indication for liver transplantation (LT) in the United States. Despite enormous efforts to prevent reinfection and recurrence of hepatitis, there are no established strategies that consistently control posttransplant recurrence of HCV.[1-3] Donor age, recipient gender, and HCV viral levels have all been identified as possible risk factors.[4, 5] In addition, numerous studies show an association between treatment of rejection and HCV recurrence.[6-8] Although these reports suggest potential immunosuppression strategies for patients with HCV, further investigations are necessary to reach a consensus.

The role of innate and cellular immunity in HCV recurrence after LT has not been well established. Lymphocytes, especially T cells, play an important role in the eradication of HCV in infected hepatocytes.[9-11] Patients with chronic hepatitis and liver cirrhosis are likely to suffer from an immunocompromised state from general debilitation, poor nutrition, and pancytopenia with lymphopenia. After transplant, patients are exposed to surgical stress, perioperative immunosuppression, and malnourishment. These circumstances presumably compromise the immune response to HCV, leading to the more aggressive recurrence pattern seen after LT than with primary HCV infection. A patient's perioperative immune status may be important for improving HCV outcome following LT; however, few reports have addressed these aspects.

In clinical settings, the absolute lymphocyte count (ALC), which includes the total number of T cells, B cells, and natural killer cells, is considered to reflect nutritional status and may be a surrogate marker for human immunity.[12] We hypothesized that peritransplant (both pre- and posttransplant) lymphopenia may be associated with poor outcomes following LT. The aim of this study was to investigate the clinical impact of peritransplant ALC on HCV outcome in LT patients.

Patients and Methods

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information
Patient Selection

From 2005 to 2011, 711 patients underwent LT at Henry Ford Hospital, of which 289 were infected with HCV. Prospectively collected data for these 289 consecutive patients were retrospectively reviewed. This study was approved by the Institutional Review Board at the Henry Ford Hospital (No. 8048). Of the 289 patients, 246 (85.1%) underwent an initial LT with a deceased donor, 10 patients (3.5%) received a liver graft from a living donor, eight patients (2.8%) underwent a combined liver and kidney transplantation, and 25 patients (8.7%) underwent a second LT with a deceased donor. Seventy-one patients also had hepatocellular carcinoma (24.6%).

Posttransplant Management

Standard maintenance immunosuppression regimen consisted of tacrolimus, mycophenolate mofetil, and steroids. The target trough levels of tacrolimus were 8-12 ng/mL for the first 3 months, 6-10 ng/mL during months 3-12, and 5-8 ng/mL after 12 months. Corticosteroids were tapered off by 3 months. Mycophenolate mofetil was started at 500 mg twice a day and withdrawn by 1 year. In the absence of cellular rejection, the maintenance immunosuppression protocol was applied uniformly to all patients. From January 2005 to June 2011, rabbit anti-thymocyte globulin (RATG) was used as our standard induction immunosuppression (0.5-1.0 mg/kg, 3 doses on postoperative days 1, 3, and 5). Some patients were given basiliximab or were not given induction agents, depending on surgeon preference and clinical indication. Complete blood count with differential, including ALC, was routinely checked on the day of LT, then daily during the initial hospitalization and during outpatient follow-up. HCV RNA levels were analyzed at baseline (before transplantation) and at 2 and 6 months after transplantation. Protocol liver biopsy was performed at 6 months post-LT on all patients as well as for clinical findings including elevated liver function levels. Recurrent HCV and acute cellular rejection (ACR) were diagnosed based on liver biopsy results. Banff criteria were applied to the diagnosis of ACR and diagnosis of HCV recurrence.[13] Severity of HCV histological recurrence was categorized according to the METAVIR score.

Analysis of Risk Factors for Post-LT HCV Recurrence and Overall Survival

Recipient, donor, and perioperative characteristics were evaluated for their impact on HCV recurrence and overall survival. Perioperative ALC were recorded on the day of LT and 2 weeks and 1 month after LT. Pre- and posttransplant ALC were categorized into three groups: less than 500/μL, 500-1,000/μL, and more than 1,000/μL. The cutoff level between high and low viral load was set at 800,000 IU/mL. The endpoint was HCV recurrence with fibrosis stage 2-4 (F2-4). Early advanced fibrosis was defined as stage 3-4 fibrosis (F3-4) within 2 years, and risk factors were investigated. Patients who received treatment for HCV recurrence and achieved sustained virological response (SVR) were right-censored at the time of clearance of HCV in the risk factor analysis for HCV recurrence, so that bias regarding posttransplant HCV therapy was minimized. Overall patient survival was also analyzed for each factor in order to elucidate the impact of ALC on outcome.

Statistical Analysis

Data were summarized using mean with standard deviation or median with range for continuous variables and percentage for discrete variables. Student t tests were used for two group comparisons. Analysis of risk factors for recurrence of HCV was performed using Cox proportional-hazards regression model. The recurrence rates and overall survival rates were estimated using the Kaplan-Meier method and differences in the curves were analyzed using a log-rank test. Logistic regression model was used for the analysis of the association with posttransplant lymphopenia. The cutoff levels were decided based on clinical relevance. For the multivariate analysis, we first performed univariate analysis for each potential independent variable. Any independent variables with a P-value less than 0.1 of significant level were used to build the multivariate analysis. In addition, clinically relevant factors likely associated with recurrence of HCV or with patient survival were also included in the multivariate analysis. SPSS v. 19.0 (Chicago, IL) was used for statistical analysis and the level of significance was set at 0.05.

Results

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information
HCV Histological Recurrence and Overall Survival in the Entire Cohort

A total of 289 patients were evaluated with a median follow-up of 2.8 years (range = 1 month to 7.7 years). Of these, 49.5% (143 patients) developed F2-4 recurrence (median time = 10.8 months: range = 1.1 to 86.9 months). Clinically significant fibrosis recurrence (F2-4) occurred in 13.1%, 29.9%, and 45.8% within 6 months, 1 year, and 2 years, respectively. Forty-five patients (15.6%) developed advanced fibrosis (F3 or F4) within 2 years. Fifty-eight patients received antiviral therapy for HCV and, of these, 25 achieved a SVR. Overall 1-, 2-, and 3-year patient survival rates were 90.5%, 85.0%, and 80.2%, respectively.

Risk Factors for Fibrosis Recurrence (F2-4)

Fibrosis stage 2-4 was set as an endpoint for the risk factor analysis. Pre- and posttransplant ALC were analyzed (Table 1). Patients were classified according to pre- and posttransplant ALC (<500/μL versus 500-1,000/μL versus >1,000/μL). One-year HCV recurrence rates were at 54%, 31%, and 20% in patients with pretransplant ALCs of <500/μL, 500-1,000/μL, and >1,000/μL, respectively (P = 0.007) (Fig. 1A). Posttransplant ALCs at 1 month also showed remarkable associations with F2-4 recurrence (39%, 28%, and 18%, respectively) (P = 0.004) (Fig. 1B). On univariate Cox regression analysis, pretransplant ALC and posttransplant ALC at 1 month were significantly associated with occurrence of F2-4 recurrence (P = 0.008 and 0.004, respectively). HCV genotype 1 was also associated with a higher incidence of F2-4 recurrence (P = 0.02). An episode of ACR (moderate or severe) prior to HCV recurrence significantly increased the risk of recurrence (Table 1), when treated with RATG (P = 0.007) or steroids (P = 0.008).

image

Figure 1. Histological HCV recurrence (F2-4) according to peritransplant ALC. (A) The risk of F2-4 recurrence according to pretransplant ALC (<500/μL versus 500-1,000/μL versus >1,000/μL, P = 0.007). (B) The risk of F2-4 recurrence according to posttransplant ALC at 1 month (<500/μL versus 500-1,000/μL versus >1,000/μL, P = 0.004).

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Table 1. Risk Factor Analysis for Fibrosis Stage 2-4 HCV Recurrence Following Liver Transplantation
VariableObservation (%)Univariate AnalysisMultivariate Analysis
P ValueHR (95% CI)P ValueHR (95% CI)
  1. a

    Moderate or severe rejection.

Recipient age (per year)2890.21.02 (0.99-1.05)  
Recipient, female79 (27.3)0.520.88 (0.60-1.30)  
Recipient, African American69 (23.8)0.371.19 (0.81-1.76)  
MELD (per score 10 up)2890.441.07 (0.90-1.27)  
Type of transplantation
Primary LT (Ref)246 (85.1)    
LDLT10 (3.5)0.420.62 (0.20-1.96)  
Simultaneous LKT8 (2.8)0.840.91 (0.37-2.24)  
Re-LT25 (8.7)0.200.61 (0.33-1.27)  
Pretransplant ALC
>1,000/μL (Ref)125 (43.5)    
500-1,000/μL122 (42.5)0.061.41 (0.98-2.03)0.671.10 (0.71-1.69)
< 500/μL40 (14.0)0.0022.15 (1.31-3.51)0.151.60 (0.85-3.00)
Posttransplant ALC at 2 weeks
> 1,000/μL (Ref)60 (20.9)    
500-1,000/μL112 (39.0)0.311.27 (0.80-2.04)0.820.93 (0.48-1.79)
< 500/μL115 (40.1)0.031.65 (1.04-2.61)0.800.90 (0.41-1.98)
Posttransplant ALC at 1 month
> 1,000/μL (Ref)78 (27.2)    
500-1,000/μL92 (32.2)0.0561.55 (0.99-2.42)0.031.97 (1.07-3.62)
< 500/μL116 (40.6)0.0012.06 (1.34-3.16)0.022.47 (1.16-5.28)
Induction immunosuppression
No induction (Ref)99 (34.3)    
Basiliximab34 (11.8)0.321.34 (0.75-2.41)0.991.00 (0.52-1.93)
RATG156 (53.9)0.130.76 (0.54-1.08)0.020.60 (0.40-0.92)
ACRa prior to recurrence
No episode of ACR (Ref)248 (85.8)    
Treatment with RATG18 (6.2)0.0072.06 (1.22-3.48)0.151.60 (0.84-3.06)
Treatment with steroids6 (2.1)0.0083.36 (1.36-8.28)0.112.09 (0.84-5.69)
Treatment without RATG or steroids17 (5.9)0.71.14 (0.58-2.25)0.320.50 (0.68-3.33)
Donor age (per year)2890.091.01 (1.00-1.02)0.071.01 (0.99-1.02)
Donor, African American53 (18.3)0.490.86 (0.55-1.34)  
Combination of recipient-donor race
Caucasian recipient of AA donor (Ref)26 (9.0)    
AA recipient of AA donor21 (7.3)0.660.84 (0.37-1.87)  
AA recipient of Caucasian donor40 (13.8)0.771.11 (0.55-2.23)  
Caucasian recipient of Caucasian donor126 (43,6)0.90.96 (0.53-1.76)  
DCD donor13 (4.5)0.940.97 (0.45-2.08)  
HCV genotype 1194 (82.6)0.021.83 (1.08-3.10)0.061.69 (0.97-2.93)
Pretransplant HCV RNA
< 800,000 IU/mL123 (79.3)    
> 800,000 IU/mL32 (20.7)0.231.38 (0.82-2.31)  
Ischemia time
Cold (per hour)2890.981.00 (0.92-1.09)  
Warm (per min)2890.741.00 (0.99-1.02)  

Based on these results, pre- and posttransplant ALC, HCV genotype 1, RATG induction, episode of ACR, and donor age were included in a multivariate Cox regression analysis (Table 1). Posttransplant ALC at 1 month was considered an independent predictive factor for F2-4 HCV recurrence (P = 0.03, hazard ratio [HR] = 1.97 for 500-1,000/μL; P = 0.02, HR = 2.47 for <500/μL), whereas pretransplant ALC did not remain an independent risk factor. RATG induction therapy showed remarkable protective effects against HCV recurrence in comparison with no-induction (P = 0.02, HR = 0.60).

Early Advanced Fibrosis (F3-4 Within 2 Years)

Risk factors for early advanced fibrosis (F3-4 within 2 years), which is associated with dismal prognosis after LT, were also investigated (Table 2). Three-year patient survival was 61.8% and 83.4%, respectively, in patients with and without early advanced fibrosis (P = 0.001). ALC in patients with early advanced fibrosis was significantly lower than in those without (Fig. 2); before LT (867/μL versus 1,090/μL: P = 0.03), at 2 weeks after LT (626/μL versus 730/μL: P = 0.15) and 1 month after LT (712/μL versus 905/μL: P = 0.03). Comparing ALC at 2 weeks and 1 month after LT, the count was significantly improved in patients without early advanced recurrence (730/μL to 905/μL, P < 0.001), whereas no significant recovery was observed in the early advanced recurrence group (626/μL to 712/μL: P = 0.06). Univariate analysis demonstrated significant differences in the incidence of early advanced fibrosis between patients with pretransplant ALC <500/μL and those >1,000/μL (P = 0.004), and between patients with posttransplant ALC at 1 month <500/μL and those >1,000/μL (P = 0.03). Persistent lymphopenia was independently associated with development of early advanced fibrosis (P = 0.02, HR = 3.16). Steroids treatment for ACR (P = 0.01, HR = 4.87) and donor age (P = 0.01, HR = 1.03 per year) were also independent risk factors (Table 2).

image

Figure 2. Trend of peritransplant ALC in patients with or without early advanced fibrosis of HCV recurrence (F3-4 within 2 years). ALC pretransplant and 1 month posttransplant were significantly lower in the patients with early advanced fibrosis. Recovery of ALC from 2 weeks to 1 month after LT was remarkable in those without early advanced fibrosis, but not in those with early advanced fibrosis.

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Table 2. Risk Factor Associated With Early Advanced Hepatic Fibrosis (F3-4 Within 2 Years)
VariableObservation (%)Univariate AnalysisMultivariate Analysis
P ValueHR (95% CI)P ValueHR (95% CI)
  1. a

    ALC < 500/μL before and at 2 weeks and 1 month after LT.

  2. b

    Moderate or severe rejection.

Persistent Lymphopenia (< 500/μLa)23 (8.1)0.231.76 (0.70-4.48)0.023.16 (1.17-8.55)
Pretransplant ALC   N/A 
> 1,000/μL (Ref)125 (43.5)    
500-1,000/μL122 (42.5)0.131.70 (0.85-3.39)  
< 500/μL40 (14.0)0.0043.33 (1.46-7.60)  
Posttransplant ALC at 2 weeks   N/A 
> 1,000/μL (Ref)60 (20.9)    
500-1,000/μL112 (39.0)0.501.35 (0.56-3.26)  
< 500/μL115 (40.1)0.201.78 (0.74-4.10)  
Posttransplant ALC at 1 month   N/A 
> 1,000/μL (Ref)78 (27.2)    
500-1,000/μL92 (32.2)0.421.44 (0.60-3.48)  
< 500/μL116 (40.6)0.032.47 (1.09-5.39)  
Induction immunosuppression
No induction (Ref)99 (34.3)    
Basiliximab34 (11.8)0.801.14 (0.42-3.09)0.740.81 (0.24-2.75)
RATG156 (53.9)0.430.78 (0.42-1.45)0.740.89 (0.43-1.81)
ACR prior to recurrenceb
No episode of ACR (Ref)248 (85.8)    
Treatment with RATG18 (6.2)0.122.20 (0.83-5.43)0.671.30 (0.39-4.35)
Treatment with steroids6 (2.1)0.014.60 (1.41-15.01)0.014.87 (1.43-16.57)
Treatment without RATG or steroids17 (5.9)0.331.68 (0.60-4.74)0.212.13 (0.66-6.83)
Donor age (per year)2890.031.02 (1.00-1.04)0.0091.03 (1.01-1.05)
HCV Genotype 1194 (82.6)0.12.71 (0.83-8.84)0.12.77 (0.82-9.39)
Survival

Overall patient survival was significantly lower in patients with pretransplant ALC less than 500/μL. The 1- and 3-year survival was 74% and 60% for the ALC <500/μL group, 93% and 85% for the 500-1,000/μL group, and 94% and 82% for the >1,000/μL group (P = 0.001 for ALC > versus <500/μL) (Fig. 3A). Patients with a posttransplant ALC at 1 month less than 500/μL showed relatively lower survival rate (P = 0.08), but the difference was not significant (Fig. 3B). Multivariate analysis demonstrated that pretransplant ALC less than 500/μL (compared with pretransplant ALC >1,000/μL) remained a risk factor for diminished survival (P = 0.01, HR = 3.01), along with increased cold ischemia time (P = 0.03, HR = 1.19 per hour) and older donor age (P < 0.001, HR = 1.04 per year) (Table 3). We evaluated causes of death according to pretransplant ALC. Recurrent HCV accounted for 33% of all deaths but the deaths were evenly distributed by pretransplant ALC group. Recurrent HCV accounted for 4 of 16 deaths (25%) in the low ALC group (<500/μL), 6 of 21 deaths (29%) in the moderate ALC (500-1,000/μL), and 11 of 27 (40%) in the high ALC group (>1,000/μL) (P = 0.95). Other leading causes of death, including recurrent hepatocellular carcinoma (14%) and sepsis (11%), were also spread evenly across ALC groups.

image

Figure 3. Overall patient survival according to peritransplant ALC. (A) Pretransplant lymphopenia had a significant impact on patient survival (P = 0.001 for ALC > versus <500/μL) (1- and 3-year survival rate: ALC <500/μL, 74% and 60%; 500-1,000/μL, 93% and 85%; >1,000/μL, 94% and 82%, respectively). (B) Patient survival in the group of posttransplant ALC less than 500/μL was relatively lower compared with the group of ALC more than 500/μL (P = 0.08) (1- and 3-year survival rate: ALC <500/μL, 85% and 76%; 500-1,000/μL, 97% and 85%; >1,000/μL, 94% and 85%, respectively).

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Table 3. Risk Factor Analysis for Patient Survival
VariableObservation (%)Univariate AnalysisMultivariate Analysis
P ValueHR (95% CI)P ValueHR (95% CI)
  1. a

    Moderate or severe rejection.

Recipient age (per year)2890.721.01 (0.97-1.05)  
Recipient, female79 (27.3)0.191.40 (0.84-2.34)  
Recipient, African American69 (23.8)0.670.88 (0.49-1.58)  
MELD (per score 10 up)2890.590.93 (0.72-1.21)  
Type of transplantation
Primary LT (Ref)246 (85.1)    
LDLT10 (3.5)0.760.74 (0.10-5.34)  
Simultaneous LKT8 (2.8)0.911.07 (0.33-3.44)  
Re-LT25 (8.7)0.531.29 (0.59-2.83)  
Pretransplant ALC
>1,000/μL (Ref)125 (43.5)    
500-1,000/μL122 (42.5)0.380.78 (0.44-1.37)0.470.79 (0.42-1.49)
<500/μL40 (14.0)0.0082.31 (1.25-4.27)0.013.01 (1.55-5.86)
Posttransplant ALC at 2 weeks
>1,000/μL (Ref)60 (20.9)    
500-1,000/μL112 (39.0)0.601.19 (0.62-2.28)  
<500/μL115 (40.1)0.651.17 (0.61-2.25)  
Posttransplant ALC at 1 month
>1,000/μL (Ref)78 (27.2)    
500-1,000/μL92 (32.2)0.611.19 (0.61-2.32)  
<500/μL116 (40.6)0.111.67 (0.90-3.09)  
Induction immunosuppression
No induction (Ref)99 (34.3)    
Basiliximab34 (11.8)0.550.69 (0.21-2.31)  
RATG156 (53.9)0.991.00 (0.60-1.64)  
ACR prior to recurrencea40 (13.8)0.150.54 (0.23-1.26)  
Donor age (per year)289<0.0011.03 (1.02-1.05)<0.0011.04 (1.02-1.05)
Donor, African American53 (18.3)0.080.51 (0.24-1.07)0.270.64 (0.29-1.41)
DCD donor13 (4.5)0.510.62 (0.15-2.53)  
HCV Genotype 1194 (82.6)0.112.11 (0.84-5.33)  
Pretransplant HCV RNA
<800,000 IU/mL123 (79.3)    
>800,000 IU/mL32 (20.7)0.141.87 (0.82-4.24)  
Ischemia time
Cold (per hour)2890.071.13 (0.99-1.28)0.031.19 (1.01-1.39)
Warm (per min)2890.520.99 (0.97-1.01)  
Risk Factors Associated With Posttransplant Lymphopenia

Recipient and donor characteristics, immunological, virological, and surgical factors were analyzed in order to determine their impacts on posttransplant ALC (Supporting Table 1). A high Model for Endstage Liver Disease (MELD) score (P < 0.001, odds ratio [OR]= 2.0 per score 10 up), RATG induction (P < 0.001, OR = 3.27), pretransplant ALC <1,000/μL (P < 0.001, OR = 4.60), requirement of intraoperative red blood cell transfusion (P = 0.004, OR = 2.36), and requirement of peritransplant dialysis (P = 0.03, OR = 4.04) were considered risk factors for posttransplant ALC <1,000/μL at 1 month.

Impact of Lymphopenia on Response to Antiviral Therapy

There were 58 patients who received antiviral therapy and 25 achieved SVR. Mean ALC prior to therapy in the patients with SVR was 1,387/μL compared to 749/μL in those without SVR (P < 0.001). HCV genotype 1 was found to be a negative predictive factor for SVR (P = 0.033). When the cutoff level of ALC was 1,000/μL, 6 of 30 patients (20%) in the low ALC group achieved SVR, compared to 19 of 26 patients (73%) in the high ALC group (P < 0.001). Lymphopenia before initiating antiviral therapy adversely affected achievement of SVR.

Relationship Between Lymphopenia and RATG Induction Immunosuppression and Association With HCV Histological Recurrence

Lymphopenia was associated with higher recurrence of HCV. Paradoxically, RATG induction showed a protective effect against HCV recurrence (Table 1) despite its ability to induce lymphopenia (Supporting Table 1). Therefore, subanalysis was performed in order to elucidate the independent effects of lymphopenia and RATG on HCV recurrence. Patients were classified into four groups: Group 1: no-RATG induction with posttransplant lymphopenia (<1,000/μL at 1 month); Group 2: RATG induction with posttransplant lymphopenia; Group 3: no-RATG induction without posttransplant lymphopenia; Group 4: RATG induction without posttransplant lymphopenia. Figure 4 shows the cumulative recurrence in each group. Comparisons between Groups 1 and 3 and between Groups 2 and 4 show a significant adverse effect of lymphopenia regardless of RATG use (P = 0.007 and 0.02, respectively). The impact of RATG is seen by comparing the recurrence rates between Groups 1 and 2 and between Groups 3 and 4. In Groups 1 and 2, with posttransplant lymphopenia, RATG induction was associated with a lower recurrence rate (P = 0.01), whereas the positive effect is less obvious in the comparison between groups 3 and 4 without posttransplant lymphopenia (P = 0.12). Overall, patients who received RATG induction and did not have lymphopenia 1 month after LT had the lowest recurrence rates of 8% at 1 year and 12% at 2 years. Patients who did not receive RATG induction but who had lymphopenia had the highest recurrence rates (40% at 1 year and 66% at 2 years) (P < 0.001).

image

Figure 4. Histological HCV recurrence (F2-4) according to posttransplant ALC and induction immunosuppression therapy. Patients were classified as follows: Group 1: no-RATG induction with posttransplant lymphopenia (<1,000/μL); Group 2: RATG induction with posttransplant lymphopenia; Group 3: no-RATG induction without posttransplant lymphopenia; Group 4: RATG induction without posttransplant lymphopenia. The negative impact of lymphopenia on recurrence was significant regardless of RATG use (Groups 1 versus 3, P = 0.007; Groups 2 versus 4, P = 0.02).

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Influence of Lymphopenia and RATG on Posttransplant Viral Load

Associations among HCV RNA levels, lymphopenia, and RATG induction were analyzed. Prior to transplant, patients with low and high pretransplant ALC (< and >1,000/μL), had comparable HCV RNA levels with values of 1,263,105 and 2,159,398 IU/mL (P = 0.31), respectively. After transplant, when comparing viral loads between the low and high ALC groups (< and >1,000/μL at 1 month), the HCV RNA levels were significantly higher in the low ALC group than in the high ALC group 2 months after transplant (18,526,415 IU/mL versus 3,932,466 IU/L: P < 0.001) and 6 months after LT (13,029,584 IU/mL versus 6,705,260 IU/mL: P = 0.01) (Fig. 5A). When classifying patients based on induction therapy, levels were comparable between the RATG group and the no-RATG group at 2 months (14,038,942 IU/mL versus 18,827,988 IU/mL: P = 0.38) and 6 months (13,028,029 IU/mL versus 9,315,735 IU/mL: P = 0.2) (Fig. 5B). Next, the viral levels were compared among Groups 1, 2, 3, and 4 (Fig. 5C). At 2 months, the negative impact of lymphopenia on viral replication was statistically significant regardless of RATG induction use (24,038,638 IU/mL in Group 1 versus 2,544,709 IU/mL in Group 3, P = 0.001; 15,394,471 IU/mL in Group 2 versus 5,518,475 IU/mL in Group 4, P = 0.02). However, RATG induction did not significantly influence viral level (24,038,638 IU/mL in Group 1 versus 15,394,471 IU/mL in Group 2, P = 0.2; 2,544,709 IU/mL in Group 3 versus 5,518,475 IU/mL in Group 4, P = 0.17). At 6 months the difference between Groups 1 and 3 was still statistically significant (12,022,070 IU/mL versus 5,461,259 IU/mL: P = 0.045), whereas there was no statistical difference among other groups (Groups 2 versus 4, P = 0.5; Groups 1 versus 2, P = 0.67; Groups 3 versus 4, P = 0.37).

image

Figure 5. Comparison of pre- and posttransplant HCV levels according to ALC and induction immunosuppression. (A) Viral levels according to posttransplant ALC. Viral levels at 2 months and 6 months were significantly higher in the low ALC group (P < 0.001 and = 0.01, respectively). (B) Viral levels according to induction immunosuppression. The posttransplant viral levels were comparable between the RATG and no-RATG induction groups. (C) In both no-RATG induction and RATG induction groups, viral level at 2 months were significantly higher in patients with posttransplant lymphopenia (P = 0.001 for Group 1 versus Group 3; P = 0.02 for Group 2 versus Group 4).

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Discussion

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

The natural history of HCV infection is markedly accelerated after LT. Overall, 20%-40% of patients will progress to cirrhosis within 5 years compared with less than 5% of nontransplantation patients.[3] One explanation for this is that both innate and adaptive immunity against HCV may be compromised after LT by immunosuppressive medications, malnutrition, and surgical stress, leading to enhanced HCV hepatitis and graft loss.[2] This study focused on ALC as a useful surrogate marker for patient immune and nutritional status and investigated the impact of peritransplant lymphopenia on HCV recurrence after LT. We hypothesized that, given the role of lymphocytes (e.g., T cells and natural killer cells) in immunity against HCV, patients with low peritransplant ALC may be more susceptible to viral replication and hepatitis.[9, 10, 14] The results of this study indicate that posttransplant ALC markedly affected histological HCV recurrence after LT. Moreover, the association between viral load and low posttransplant ALC strengthened our hypothesis.[15] To our knowledge, this is the first report that describes posttransplant ALC as a predictive factor for HCV recurrence after LT. These findings should contribute to the identification of groups at risk for early HCV recurrence and poor outcome in posttransplant management.

Many studies suggest that compromised HCV-specific immunity is correlated with accelerated fibrosis after LT.[10, 11, 16-18] One study utilized the Cylex ImmuKnow assay to assess cellular immunity by measuring adenosine triphosphate production from CD4+ T cells in response to mitogens.[12] In HCV-infected LT patients, there was a clear correlation between CD4+ T cell ATP levels at 4 and 12 months after LT and fibrosis progression. Bharat et al.[16] looked specifically at HCV-specific T cells in the peripheral blood of LT patients and found a clear correlation between a lack of HCV specific, Th1-type, CD4+ T cells, and progression to allograft cirrhosis. Rasmussen et al.[18] made use of the novel singular value decomposition-initialized multidimensional scaling (SVD-MDS) analysis to evaluate the transcriptome of liver biopsy specimens from HCV-infected LT patients. They found genes associated with immune and inflammatory responses were notably down-regulated in patients with accelerated fibrosis. This effect occurred early after LT and before fibrosis occurred.[18] The ALC may provide a readily available and inexpensive marker for poor immunity and risk of HCV recurrence after LT.

It is of note that pretransplant lymphopenia was an independent risk factor for survival, but excess mortality in this group was not related to HCV recurrence. In evaluating causes of death, no one etiology, including HCV recurrence, HCC, and sepsis, accounted for the increased mortality in patients with pretransplant lymphopenia. Rather, we feel that pretransplant lymphopenia, especially an ALC <500/μL, is a marker for numerous factors, including malnutrition, debilitation, and immune compromise, which, in toto, portend a poor prognosis after LT.

Many factors may contribute to perioperative lymphopenia in addition to immunosuppression, including malnutrition, sepsis, and surgical stress.[19-21] We identified factors associated with posttransplant lymphopenia which include an elevated MELD score, peritransplant dialysis, and surgical factors including intraoperative red blood cell transfusion and reoperation. Our results suggest that peritransplant lymphopenia due to suboptimal patient condition might result in greater susceptibility to HCV recurrence than lymphopenia induced by immunosuppression. It should be noted that severe lymphopenia prior to transplant, at a time when patients had not received immunosuppressive medications, was an independent risk factor for patient survival. Maintaining peritransplant ALC by optimizing patient status, both before and immediately after LT, may be important in reducing HCV recurrence and improving patient survival.

The positive effects of RATG, despite its ability to induce lymphopenia, are of interest. The use of RATG induction with a resulting delay in calcineurin inhibitor introduction may help preserve renal function after LT, and may reduce requirement for peritransplant dialysis, which was a risk factor for posttransplant lymphopenia. Episodes of ACR after LT are a risk factor for HCV recurrence. As a potent immunosuppressive agent, RATG may decrease the incidence of rejection in the immediate posttransplant period. This benefit may be especially pronounced in patients with renal insufficiency in whom there may be a temptation to underdose calcineurin inhibitors. In an analysis of the UNOS database, in which induction was shown to improve survival in HCV-infected patients, the benefit was primarily seen in HCV patients with renal insufficiency.[22] Finally, it has been reported that RATG can alleviate damage from ischemia-reperfusion injury,[23, 24] which may also be a potential risk factor for HCV recurrence.[25, 26] Thus, the role of RATG in optimizing liver graft condition, especially in the first month, may override its effect on lymphocyte counts.[1]

The cell-specific impact of RATG may also mitigate its lymphopenic effect. Pearl et al.[27] reported that RATG reduced CD8+, CD4+ naive cells and natural killer cells, but CD4+ effector memory T cells, which are reputed to play a major role in immunity against viral infection, were relatively spared. LT patients with HCV have had prolonged exposure to viral antigen and memory immunity should be well established. When posttransplant lymphopenia is due to RATG, memory immunity may be relatively resistant compared to when lymphopenia is due to factors such as sepsis, malnutrition, and surgical complications. It should also be noted that RATG induced lymphopenia is most pronounced early and the number of circulating T cells gradually increase, reaching pretreatment levels after 1-6 weeks.[28] HCV RNA levels dramatically decrease immediately after transplant.[29] Patients may be able to tolerate lymphopenia when HCV RNA levels are low but not when they increase. This is consistent with our finding that posttransplant lymphopenia at 1 month was a risk factor for fibrosis stage 2-4 but lymphopenia at 2 weeks was not.

Based on these findings, posttransplant management can be altered and individualized. One of the most promising approaches would be nutritional intervention.[30] Nutritional status and management may alter mechanistic as well as absolute number of lymphocytes and affect host immunity.[12, 17, 31, 32] Because lymphopenia negatively affected outcomes, perioperative nutritional managements should be keenly monitored and intervened. ALC would be a good surrogate marker in such situations. Another approach would be to alter the immunosuppression protocol.[33-35] RATG use might be warranted with careful monitoring of ALC. The emphasis on avoiding ACR may be further increased in HCV patients. While it is difficult to suggest the most reasonable and appropriate immunosuppression protocol for HCV patients based on the results of this study, peritransplant ALC may allow us to individualize the immunosuppression protocol. The association between ALC and the success of antiviral therapy is also an important consideration. Maintaining ALC may be important not only during peritransplant period, but also in long-term follow-up.

The limitations of this study should also be mentioned. This is a single-center, retrospective study without randomization for induction therapies. In terms of ALC, lymphocyte subsets were not measured and alteration of the mechanistic number of lymphocytes was not available. It is possible that specific lymphocytes are associated with immunity against HCV and related to recurrence. This will be an interesting next step to further investigate the impact of lymphocytes on HCV recurrence following LT. At present, our simple risk assessment model consisting of peritransplant ALC, which is universally available in all facilities, can be a valuable tool to identify and stratify patients who can benefit from close monitoring and aggressive therapy after LT for HCV.

In conclusion, peritransplant ALC can be a useful surrogate marker for prediction of HCV recurrence and survival. Maintaining peritransplant ALC could lead to improved HCV outcome following LT. Since ALC is easily measured without additional effort or cost, this laboratory test can be useful not only for transplant physicians and surgeons but also primary care doctors in daily clinical settings. Immunosuppression regimens should be scrutinized in the setting of LT for patients with HCV. Our results suggest that RATG induction may improve the prognosis for HCV recurrence after LT. Further investigations into optimization of immunosuppression protocols are necessary.

References

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Patients and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
hep26536-sup-0001-suppinfo.doc93KSupporting Table 1. Risk factors for Post-transplant Lymphopenia at one month (<1,000/μL)

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