Graft-versus-host disease (GVHD) is a complex immunological complication that may occur after liver transplantation (LT), affecting a minority of recipients with an incidence rate estimated between 0.5 to 2% [1,2]. Despite its rarity, GVHD presents a formidable challenge due to its high mortality rate, which has been reported to be as high as 85% among affected patients, even with aggressive therapeutic interventions [3-6]. This stark statistic underlines the critical nature of GVHD in the context of LT and highlights the urgent need for a deeper understanding and more effective management strategies.

Historically, research efforts have concentrated on identifying risk factors associated with the onset of GVHD following LT. Significant predictors include the use of a donor liver from individuals at least 20 years younger than the recipient, the application of basiliximab induction therapy, and the presence of hepatocellular carcinoma in the recipient [2,7]. The pathogenesis of GVHD is believed to be initiated by donor lymphocytes transferred alongside the liver graft, with particular emphasis on the role of donor T-cells infected with human T-cell lymphotropic virus type I as a potent risk factor [4,8,9]. This insight into the etiology of GVHD has been instrumental in directing research towards potential preventive measures and therapeutic approaches.

Despite advancements in our understanding of GVHD, the lack of definitive treatment options remains a significant concern. Although interventions such as CD2-blockers and TNF-α antagonists have been proposed as potentially promising, their efficacy has yet to be conclusively established [1]. Consequently, the current consensus within the medical community emphasizes the importance of risk avoidance and the development of prophylactic strategies as the primary means of combating this life-threatening complication. The pressing nature of GVHD in LT necessitates ongoing research to identify effective treatments and mitigate the risks associated with this condition.

This study explored the treatment experiences of GVHD in adult LT patients within a single center, aiming to contribute valuable insights and potential strategies for managing this complex condition.

Study Materials

This observational study, conducted at Severance Hospital in Korea, examined 1,107 adult LT recipients from January 2009 to March 2023. Data on baseline characteristics, incidence of GVHD, and mortality were extracted from a prospectively maintained LT database. Information specific to GVHD, including diagnostic methods, treatments, and outcomes, was retrieved from electronic health records. Given the observation that GVHD typically manifested between 14 to 21 days post-operatively, patients who passed away within 14 days of LT were excluded (n=52), resulting in a cohort of 1,055 patients for analysis.

Diagnosis and Management of GVHD

For the diagnosis and therapeutic management of GVHD, the onset was anticipated based on symptoms such as unexplained fever, skin rash, cytopenia, and diarrhea. While skin biopsies were predominantly used for diagnosis, instances also included the identification of donor human leukocyte antigen (HLA) chimerism and colonoscopic biopsies. In suspected GVHD cases, high-dose corticosteroids (500 mg IV daily for 3–4 days) were administered as an immediate intervention before biopsy confirmation. Adjustments to immunosuppressive medications were considered on a case-by-case basis to control immune reactions, complemented by the use of broad-spectrum antibiotics to prevent infection.

Immunosuppression Protocol

The immunosuppression protocol adhered to by our institution began with the initiation of tacrolimus around the day of surgery, closely monitoring serum levels initially daily for a week, followed by bi-weekly to tri-weekly checks until discharge. Post-discharge, tacrolimus levels were assessed during each outpatient visit, gradually spacing out the intervals over time. Induction therapy involved an interleukin-2 receptor antagonist, with steroids introduced at 500 mg or 1,000 mg on the day of LT, tapering to a maintenance dose by the 14th postoperative day. Mycophenolate mofetil was added based on rejection or infection risk from the second postoperative day, with an mTOR inhibitor introduced later as an alternative based on specific institutional criteria.

Statistical Methods

The study conducted a propensity score matching (PSM) between the group experiencing GVHD and a control group, adhering to a 1:5 ratio. This matching approach employed the nearest neighbor method, with a caliper width set at 0.1, aiming for accurate pairings. The propensity scores were calculated through an exhaustive evaluation of initial characteristics of all patients involved. This meticulous matching process was deemed effective when the standardized mean differences across all primary variables remained below 0.1, indicating a balanced comparison across both cohorts [10]. To uphold the analytical integrity, any instances lacking an appropriate match were omitted from both the GVHD and control groups.

Statistical analysis included descriptive statistics for baseline characteristics and outcomes, with categorical variables expressed as frequencies and percentages, and continuous variables as medians with interquartile ranges (IQRs). Comparative analyses utilized the chi-square or Wilcoxon rank-sum test as appropriate. Survival outcomes were assessed using Kaplan–Meier curves and the log-rank test, with logistic regression models identifying risk factors for GVHD. Variables with a p-value<0.1 in univariate analyses were included in multivariate models to ascertain independent predictors. All statistical procedures were conducted using the R software version 4.2.0 for mac OS, with a significance threshold set at p<0.05.

Ethic Approval

This study was performed in accordance with the Declaration of Helsinki and Declaration of Istanbul and was approved by the Institutional Review Board at Severance Hospital, Yonsei University Health System (IRB No. 4-2023-1567). Informed consent was not required because of the study’s retrospective design.

Information of LT Patients who Experienced GVHD

Fourteen recipients, or 1.3% of the 1,055 eligible LT patients, developed GVHD. In Table 1, GVHD information is displayed. The average age of patients was approximately 54.9 years, with a majority being male (78.6%). Donors were equally divided between deceased and offspring (50.0% each), with an average donor age of 35.2 years, mostly male (64.3%). The HLA one-way mismatch was observed at 2 loci and 3 loci in one patient each, respectively. Diagnosis was predominantly via skin biopsy (85.7%), with one case each identified through colonoscopic biopsy and HLA chimerism. About half of the patients had their immunosuppressants stopped following GVHD diagnosis. The days from GVHD diagnosis to steroid pulse initiation averaged at 4.7 days, with an average total steroid dosage of 1,457.5 mg. Ultimately, 21.4% of the patients (3 out of 14) survived.

Table 1 . Information about GVHD.

Variable	GVHD (n=14)
Age (yr)	54.9±6.4
Sex, male	11 (78.6)
Donor relation
Deceased	7 (50.0)
Offspring	7 (50.0)
Donor age (yr)	35.2±13.3
Donor sex, male	9 (64.3)
HLA one-way mismatch
2 Loci	1 (7.1)
3 Loci	1 (7.1)
No	12 (85.7)
GVHD postoperative day	16 (16–19)
Diagnostic method
Colonoscopic biopsy	1 (7.1)
HLA chimerism	1 (7.1)
Skin biopsy	12 (85.7)
HLA donor chimerism
No	3 (21.4)
Not done	7 (50.0)
Yes	4 (28.6)
Immunosuppressant stop	7 (50.0)
Days from GVHD to steroid pulse	4.7±2.9
Total steroid dose (mg)	1,457.5±605.7
Survive	3 (21.4)

Values are presented as mean±standard deviation, number (%), or median (interquartile range)..

GVHD, graft-versus-host disease; HLA, human leukocyte antigen..

Baseline Characteristics

As shown in Table 2, Patients who experienced GVHD and controls displayed a similar median age distribution, with the GVHD group at 55 years (IQR 51–59) and the control group at 55 years (IQR 49–60), showing no significant difference (p=0.465). The sex composition was also comparable between the two groups, with females constituting 21.4% of the GVHD group and 28.3% of the control group (p=0.989). Body mass index readings indicated a slight variance, yet this difference did not reach statistical significance (23.2 [IQR 22.3–24.8] vs. 23.8 [IQR 21.9–26.1], p=0.326). In terms of underlying liver diseases, hepatitis C virus infection was more frequently observed in the GVHD group compared to that in the control group (14.3% vs. 6.4%), although these differences were not statistically significant (p=0.677). The incidence of hepatocellular carcinoma (HCC) was higher in the GVHD group (71.4%) compared to the control group (52.1%), yet this finding was not statistically significant (p=0.379). The pretransplant model for end- stage liver disease (MELD) score was significantly lower in the GVHD group (median 11, IQR 7–19) than in the control group (median 14, IQR 9–24 p=0.013). Donor to recipient HLA one way mismatch was significantly higher in the GVHD group than controls (14.3% vs. 0.7%, p<0.001).

Table 2 . Comparison of baseline characteristics according to the development of GVHD.

Variable	GVHD (n=14)	Control (n=1,041)	p-value
Age (yr)	55 (51–59)	55 (49–60)	0.465
Sex, female	3 (21.4)	295 (28.3)	0.989
BMI (kg/m2)	23.2 (22.3–24.8)	23.8 (21.9–26.1)	0.326
Diabetes mellitus	5 (35.7)	327 (31.4)	0.960
Underlying liver disease			0.677
Hepatitis B	9 (64.3)	564 (54.2)
Hepatitis C	2 (14.3)	67 (6.4)
Alcoholic	1 (7.1)	249 (23.9)
Autoimmune liver disease	0 (0)	46 (4.4)
Acute liver failure	1 (7.1)	25 (2.4)
Biliary atresia	0 (0)	4 (0.4)
Others	1 (7.1)	86 (8.3)
HCC	10 (71.4)	542 (52.1)	0.379
Pretransplant MELD	11 (7–19)	14 (9–24)	0.013
Donor relation			0.057
Living related	7 (50.0)	583 (56.0)
Living unrelated	0 (0)	165 (15.9)
Deceased	7 (50.0)	293 (28.1)
Donor age (yr)	35 (22–48)	36 (26–47)	0.083
Donor sex, female	5 (35.7)	405 (38.9)	0.899
Donor to recipient HLA one way mismatch	2 (14.3)	7 (0.7)	<0.001

Values are presented as median (interquartile range) or number (%)..

GVHD, graft-versus-host disease; BMI, body mass index; HCC, hepatocellular carcinoma; MELD, model for end-stage liver disease; HLA, human leukocyte antigen..

Patient Survival and the Risk Factors for GVHD

The GVHD group had a considerably poorer one-year patient survival rate (21.4%) compared to the control group (90.2%, p<0.001, Fig. 1). Following PSM which balanced two groups (Supplementary Table 1), the 1-year survival rate for GVHD was significantly lower (21.4% vs. 84.3%, p<0.001) compared to the control group. When compared to a living donor, the deceased donor had a significantly greater risk of GVHD in the multivariable logistic regression (odds ratio [OR] 4.54, 95% confidence interval [CI] 1.36–15.3, p=0.012). HLA one-way mismatch between donor and recipient was found to be an additional important risk factor for GVHD (OR 29.6, 95% CI 3.88–160, p<0.001, Table 3).

Figure 1. Comparison of 1 year patient survival by development of graft-versus-host disease (GVHD). (A) Before propensity score matching (PSM), (B) after PSM. LT, liver transplantation.

Table 3 . Risk factor analyses for GVHD.

Variable	Univariable			Multivariable
	OR (95% CI)	p-value		OR (95% CI)	p-value
Pretransplant MELD ≥18	0.98 (0.91–1.03)	0.491		0.95 (0.88–1.01)	0.138
Donor type
Living	Reference			Reference
Deceased	2.55 (0.87–7.52)	0.082		4.54 (1.36–15.3)	0.012
Donor to recipient HLA one way mismatch	24.6 (3.4–115)	<0.001		29.6 (3.88–160)	<0.001

GVHD, graft-versus-host disease; MELD, model for end-stage liver disease; OR, odds ratio; CI, confidence interval; HLA, human leukocyte antigen..

This study showed that those who developed GVHD exhibited significantly lower one-year survival rates (21.4%) compared to the non-GVHD group (90.2%), underscoring the severe impact of GVHD on patient outcomes. Post-stratification, the survival disparity persisted, with the GVHD group’s survival markedly lower. Deceased donor transplants and donor to recipient HLA one-way mismatches emerged as significant risk factors, highlighting the importance of careful donor selection and genetic compatibility in minimizing GVHD risk and improving survival.

The survival outcomes for GVHD following LT have generally been excremely unfavorable, with previously reported mortality rates in scholarly articles fluctuating between 75% and 91.6% [2]. Our analysis yielded similar mortality rate of 78.6% within 1 year. The predominant causes of death among the patients included complications such as multiple organ failure syndrome, infections leading to sepsis, and hemorrhagic episodes within the gastrointestinal tract, despite receiving extensive support through antimicrobial therapies and hematological interventions. The sole investigation specifically targeting mortality rates in GVHD post-LT, performed by Taylor and colleagues [11], encompassed a review of 51 case studies and pinpointed rash and fever as potential mortality risk indicators. The traditional division of GVHD post-LT into acute and chronic based on the 100-day timeline is being questioned due to overlapping clinical and histological features [12]. The reliance on the post-transplant duration as the sole criterion for classification is insufficient, necessitating detailed clinical and pathological assessments for accurate diagnosis. Chronic GVHD can emerge from acute GVHD, as a new episode after a symptom-free interval, or without any acute phase, with each pathway representing about a third of cases. This complexity underlines the need for a nuanced understanding and approach to GVHD diagnosis and management.

Our analysis showed deceased donor LT (DDLT) has higher risk of GVHD compared to living donor LT (LDLT). A study presented a single-center experience over 14 years with 6 cases of GVHD diagnosed among 604 liver transplant recipients, split between 283 DDLT and 321 LDLT cases. This research highlighted that GVHD, while rare, requires high suspicion in patients developing symptoms like rash, diarrhea, or severe pancytopenia post-transplantation. Treatment strategies should be discussed according to specific differentiation between DDLT and LDLT outcomes in terms of GVHD incidence rates [13].

Donor to recipient HLA one way mismatch, which represents the donor is HLA-homozygous while the recipient is HLA-heterozygous, emerged as strong risk factor in our cohort. Recent studies have highlighted a significant escalation in the risk of GVHD associated with a specific disparity in HLA profiles between donors and recipients [14-17]. This risk escalates notably with the involvement of three-loci one way mismatch in the context of LDLT. The underlying mechanism is thought to involve a decreased propensity for the recipient to reject donor-derived hematopoietic cells, thereby facilitating interactions between donor T cells and recipient antigen-presenting cells. Our previous reports also showed donor to recipient HLA one way mismatch has significantly higher incidence of GVHD than that of controls (14.3% vs. 1.5%), although long term patient survival was not statistically difference [18]. The approach to determining the suitability of living donors in the presence of HLA one-way mismatches for LT demands a meticulous and discerning assessment. Strategy ensures that decisions are made with a balance between immediate transplantation needs and the potential long-term health outcomes for both recipients and donors.

This study, based on data from a single center, acknowledges certain constraints, including the small number of GVHD instances, which limits the ability to derive broad conclusions. Additionally, the treatment approaches for GVHD were varied and lacked standardization. Despite these limitations, our findings demonstrated miserable outcomes and the risk factors of GVHD after LT. Advancing our comprehension of GVHD will pave the way towards developing efficacious prevention and treatment strategies, thereby ameliorating the prognosis for LT recipients grappling with this condition.

Supplementary data related to this article can be found online at https://doi.org/10.52604/alt.24.0003.

There was no funding related to this study.

Deok-Gie Kim is an editorial member of the journal but was not involved in the review process of this manuscript. Any other authors have no conflict of interest.

Conceptualization: DGK. Data curation: HHK, MK, JGL, DJJ. Formal analysis: HHK. Investigation: DGK, JGL, DJJ, MSK. Methodology: HHK, EKM, MSK. Project administration: DGK, DJJ. Resources: HHK, JGL. Software: HHK. Supervision: DGK, MSK. Validation: HHK. Visualization: HHK. Writing – original draft: HHK. Writing – review & editing: DGK.