Objectives: Graft-versus-host disease is a serious, fatal complication following liver transplantation. The diagnosis is challenging, owing to nonspecific clinical features and invasive procedures. High-throughput proteomics could provide an effective approach to identifying potential serum biomarkers for graft-versus-host disease. Materials and Methods: We retrospectively analyzed the clinical information of 3 patients with graft-versus-host disease treated at our center from 2016 to 2018. We compared serum samples from the 3 patients with the disease, patients with excellent posttransplant outcomes, and healthy controls using mass spectrometry-based proteomics in discovery study. Probable peptides were further identified by a tandem mass spectrometry system and verified by enzyme-linked immunosorbent assay. Results: Of 343 patients, 3 patients (0.875%) had graft-versus-host disease. Two of these patients died of sepsis and multiorgan failure despite intensive therapy. We observed no correlation between severity of clinical manifestation and prognosis; however, the patients with graft-versus-host disease had early onset and infection and showed worse outcome. Serum peptidome profiling showed 65 differentially expressed peaks among the 3 groups; the 2 peptides with the most significant changes (m/z values of 1950.29 and 2088.16) were further sequenced and identified as ATP citrate lyase and fibrinogen alpha chain. Western blot and enzyme-linked immunosorbent assay showed that both peptides gradually decreased among all groups. Conclusions: Graft-versus-host disease is a complication of organ and tissue transplantation with a high mortality rate. Our identification of potential biomarkers for graft-versus-host disease associated with liver transplant may aid in diagnosis and help to reduce patient mortality in those cases.
Key words : Biomarkers, Mass spectrometry, Posttransplant mortality
Graft-versus-host disease (GVHD) is a serious, fatal complication following liver transplantation (LT); acute GVHD usually occurs within the first few weeks after LT.1,2 The exact mechanisms are still unclear, with mechanisms depending on the balance between the donor and recipient immune systems. Humoral GVHD is mediated by antibodies against the red cell antigen, resulting in self-limiting hemolytic anemia,3 whereas cellular GVHD occurs as a result of a destructive cellular immune response by immunocompetent donor T lymphocytes against the recipient tissue.4 Skin, gastrointestinal tract, and bone marrow are the targeted organs of cellular GVHD.5,6
The diagnosis of GVHD relies on clinical suspicion and is confirmed by pathology; however, diagnosis may be delayed because of infections and drug reactions with similar presentations.7 There is growing interest in the detection of chimerism with the use of molecular techniques, such as polymerase chain reaction-based techniques or fluorescence in situ hybridization.8 However, it is still controversial whether persistence of chimerism correlates with GVHD.9,10 Alexander and colleagues reported that chimerism functions as a nonspecific diagnostic marker, not only for GVHD but also for immune tolerance.10 Monitoring chimerism may just be a tool in the presence of symptoms. With nonspecific clinical manifestations, invasive biopsies, and limited applications of chimerism, the diagnosis of GVHD remains a challenge. Further investigations on early recognition and diagnosis of GVHD are critical for improvement of patient outcomes.
Ideal biomarkers should be accurate, noninvasive, rapid, and inexpensive. There are numerous strategies in the search for GVHD biomarkers, with technological advancements in chemistry, engineering, and bioinformatics. Over the past decade, proteomic technologies have been successful in elucidating pathogenesis and discovering clinical biomarkers in many diseases such as cancers.11,12 Antibody microarray profiling and mass spectrometry (MS) have been employed as classical proteomic tools for biomarker study.13,14 Serum, which may reflect the patient’s particular pathophysiological state, is readily used as a classical clinical sample. Today, tumor biomarker discovery in serum is well established and is widely used in the clinic. We hypothesized that identification of serum biomarkers in GVHD after LT could also be a promising application of proteomic research.
In this discovery study, we identified differentially expressed proteins discriminating healthy controls from GVHD patients by MS-based proteomics. The potential biomarkers were then analyzed by bioinformatics and validated by enzyme-linked immunosorbent assay (ELISA) and Western blot. We identified 2 peptides, ATP citrate lyase (ACLY) and fibrinogen alpha chain (FGA), that could function as noninvasive diagnostic tools for GVHD after LT.
Materials and Methods
Patient selection and sample preparation
All patients included in our study had long-term follow-up after standard orthotopic LT. Diagnostic criteria for GVHD in our center include acute onset (within 2 months), typical clinical manifestation (fever, rash, diarrhea, and pancytopenia), and histopathology after exclusion of differential diseases. Skin biopsy revealed skin squamous cell dyskeratosis associated with dermal chronic inflammatory cell infiltration.15
In discovery study, we screened 3 manifestations and confirmed GVHD through pathology in 3 patients (patients 1-3); our study also included 10 patients without any complications (excellent posttransplant outcomes) and 10 control patients matched by age and sex; all patients were seen between 2016 and 2018. For this study, GVHD refers specifically to acute cellular-mediated GVHD.
Serum samples for the 3 patients with GVHD were collected immediately after the diagnosis (days 15, 15, and 32); serum for patients with excellent posttransplant outcomes were collected 14 to 30 days after LT. Serum from control patients were matched based on age and sex. Serum samples were centrifuged at 3500 g for 20 minutes and stored at -80 °C until use.
Mass spectrometry and analysis systems
We used the matrix-assisted laser desorption ionization time of flight (MALDI-TOF) MS technique. Serum samples were separated with the use of magnetic bead-based weak cation exchange (Bruker). A mixture of same volume eluted peptides and matrix were placed on the MALDI AnchorChip surface (Bruker).
All targets were analyzed by Autoflex analysis software (version 3.0; Bruker), with an optimized protocol of FlexControl software (version 3.0; Bruker). Peptide patterns were identified with the use of ClinProTools software (version 2.2; Bruker).
Peptide identification and bioinformatic analysis
Peptides were measured using liquid chromatography/electrospray ionization tandem MS/MS system, consisting of an EASY-nLC 1000 (Thermo Fisher Scientific) coupled with a nano-electrospray ion source to a Q-Exactive HF Orbitrap mass spectrometer (Thermo Fisher Scientific), as described previously by Wang and colleagues.15
We used gene ontology (GO) enrichment analysis to analyze the identified proteins. We used the STRING database as the source to establish an interaction network of potential biomarkers.
Enzyme-linked immunosorbent assay and Western blot
We determined ACLY and FGA levels using a human ACLY ELISA kit and a human FGA ELISA kit (H-12925 and H-12843; both from Hengyuan Biotech). Standard curves were generated to determine the concentrations of ACLY and FGA. All serum samples mentioned above were analyzed blindly and run in triplicate.
Proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequently transferred to polyvinylidene difluoride membranes. After a blocking procedure, membranes were incubated with primary antibodies overnight and then incubated with secondary antibodies.
Statistical analysis was performed with GraphPad Prism version 6.0. Data are expressed as mean ± SD. We used t tests to compare means between 2 groups and one-way analysis of variance with subsequent Bonferroni correction to analyze multiple comparisons. Spearman correlation analysis was used to determine the correlation between expression of serum biomarkers and clinical information. P < .05 was considered statistically significant.
All procedures performed were approved by the ethics committee of the First Affiliated Hospital, Xi’an Jiaotong University (No. 20151006). Signed consent was obtained from all participants. Donations after brain death were the source for LT in the study.
Among 343 LT patients seen at our center between 2016 and 2018, there were 3 patients (0.875%) with GVHD. Two of the 3 patients died (66.67%) (Table 1).
Table 2 provides background information on all study patients. All 3 patients with GVHD were men versus 7 of 10 patients were men in the group with excellent posttransplant outcomes; patients with GVHD were also older: 52.7 ± 9.8 versus 47.4 ± 8.6 years old. Among patients with GVHD, indications for LT included cirrhosis and hepatocellular carcinoma (HCC); in the patient group with excellent posttransplant outcomes, indications were cirrhosis (8/10), HCC (1/10), and acute hepatic failure (1/10). Two patients with GVHD (66.7%) underwent previous transcatheter arterial chemoembolization (TACE) and 1 patient required retransplant due to tumor recurrence. Patients with excellent posttransplant outcomes did not have any surgical history. Furthermore, patients with GVHD seemed to have relatively higher mean immunosuppression concentration in the first week. In the control group, there were 7 men and 3 women with an average age of 47.3 ± 8.7 years. No differences were found regarding the other factors.
The clinical characteristics and treatment of patients with GVHD are summarized in Table 1. Average time from surgery until clinical symptom of GVHD was 20.7 days. All patients with GVHD had fever, skin rash, and pancytopenia, which appeared early and shortly after onset of fever; 2 patients (66.67%) had diarrhea and 2 (66.67%) developed infection (1 bacterial, 1 candidiasis); however, liver, renal, and coagulation functions were not remarkable affected (Table 3). Patients also had maculopapular rashes that covered the trunk and spread to other parts of the body (Figure 1A and 1B). There was a rapid drop in white blood cell count, reaching a minimum of 0.01 to 0.75 × 109/L, and platelet count, reaching a minimum of 5 to 26 × 109/L, with patients eventually becoming profoundly pancytopenic (Table 1). We found no correlation between grade of fever, range of rash, degree of pancytopenia, and outcome; however, patients with GVHD had early onset of symptoms (postoperative day 15) and complications of infection.
Diagnosis of GVHD was confirmed by clinical manifestation and skin biopsies.16 Skin biopsy revealed many keratinocytes, perivascular lymphocytic infiltration, and spongiosis (Figure 1C to 1E). Although there is no clear treatment protocol, our treatment strategies included high-dose methylprednisolone in 3 patients, antithymocyte globulin in 2 patients, and stopping/reducing current immunosuppressive protocol. Empirical antibiotics and antifungal agents play a vital part in bacterial and fungal infections after LT. Hematopoietic cytokine was administered to treat pancytopenia. Patients also received supportive therapy, such as isolation and nutritional support, with the goal of benefiting the entire condition. Despite intensive treatment, 2 of 3 patients (66.67%) with GVHD died due to sepsis and multiorgan failure. The remaining patient was alive after 300 days of follow-up without complications.
Comparison of serum proteomic profiles among the study groups
We tested the system stabilization first, and the results showed that the mean value of the coefficient of variance was <20%, with maximum and minimum values of 20.76% and 8.73%, respectively. We analyzed 3 representative serum samples and found close reproducibility and stability of the mass spectra for the mass range of 1 to 10 kDa (Figure 2A). Figure 2B shows the differentially expressed peaks among the control group, the patients with excellent posttransplant outcomes, and the patients with GVHD. Distribution among the 3 groups showed small overlapping areas, indicating that patients with GVHD could be distinguished from control patients (Figure 2C and 2D).
Selection of differential expressed peptides
When we compared the patient groups, the ClinProTools software identified 65 different peaks, of which 2 were significantly different among the 3 groups (fold-change >1.5; P < .001). Peak 1 (m/z: 1950.29) and peak 4 (m/z: 2088.16) were both downregulated in patients with GVHD compared with control patients (Table 4) and showed the same trend when compared with patients with excellent posttransplant outcomes. Peptide mass spectrum comparisons of the 2 peaks in all samples (Figure 3A and 3C) were in line with the results shown in Table 4. The area under the curve values of the 2 peaks were both 1 (peak 1, m/z: 1950.29; peak 4, m/z: 2088.16) (Figure 3B and 3D). Relative expressions of peak 1 and peak 4 are shown in Figure 3E.
Gene ontology and STRING interaction analysis
Figure 5 shows the results of GO analysis of identified proteins. The identified proteins were scattered among various cellular components, including cell and cell parts, organelle and organelle parts, membrane-enclosed lumen, extracellular region and parts, membrane and membrane parts, protein-containing complex, and synapse (Figure 5A). The molecular function identified protein patterns, including binding, structural molecular, and catalytic activity (Figure 5A). In addition, the proteins were involved in a wide range of biological processes, including metabolic process, cellular process, multicellular organismal process, biological regulation, regulation of biological process, positive regulation of biological process, localization, signaling, response to stimulus, negative regulation of biological process, multiorgan process, immune system process, cellular component organization or biogenesis, biological adhesion, and developmental progress (Figure 5A). Moreover, the STRING database was used to explore the network of ACLY and FGA, which showed a close protein-protein interaction network (Figure 5B).
Protein expression of identified peptides
Serum concentrations of ACLY and FGA were examined by ELISA in samples from control patients, patients with excellent posttransplant outcomes, and patients with GVHD. The concentration of ACLY in patients with GVHD (571.6 ± 183.0 pg/mL) was notably lower than in patients with excellent posttransplant outcomes (837.2 ± 124.7 pg/mL) (Figure 6A). There was a lower level of FGA in serum in patients with GVHD (428.1 ± 159.4 ng/mL) than in patients with excellent posttransplant outcomes (880.8 ± 137.8 ng/mL) and control patients (1042.2 ± 237.3 ng/mL) (Figure 6B). Furthermore, Western blot was performed to identify the same expression trend for the ACLY and FGA from 3 serum samples chosen randomly from the control, excellent posttransplant, and GVHD groups (Figure 6C). Results of ELISA and Western blot indicated that ACLY and FGA might be potential diagnostic serum biomarkers for GVHD after LT.
Clinical correlation analysis
Spearman analysis was used to compare the correlations between biomarkers (ACLY and FGA) and clinical information. Our study showed that ACLY was not correlated with any laboratory value; however, FGA was inversely correlated with lymphocyte (P = .018) and monocyte (P = .003) levels (Table 6), which likely was due to the small sample size.
Graft-versus-host disease was first discovered as a common complication after hematopoietic stem cell transplant, with an estimated incidence of 50%.17 The incidence of GVHD after LT is only 0.1% to 2%; however, the mortality rate is over 75% despite intensive treatment protocols. With the increased number of procedures and the advanced immunosuppressor options, decreased host immune defenses have increased the incidence of GVHD after LT. Treatment strategies remain controversial, and treatments are mostly based on experience. The most common causes of mortality in GVHDs are sepsis-associated complications and bleeding. Risk factors include complete HLA match, recipient age >65 years, age difference >20 years, autoimmune hepatitis, alcoholic liver disease, HCC, retransplant, and glucose intolerance.18 In our LT center, prevalence of GVHD was 0.875% and mortality rate was 66.67%. Elderly recipients (52.7 ± 9.8 years), large age differences between donors and recipients (16.0 ± 7.3 years), and state of immunosuppression (HCC, TACE history, and higher mean immunosuppression concentration) were factors for more likely development of GVHD (Table 2), which were consistent with previous reports.19,20 Compatibility testing of HLA between the donors and recipients was not routinely done, so we did not have data on HLA matching in our patients.
In our patients with GVHD, the most common symptoms were fever (100%), followed by skin rash (100%), pancytopenia (100%), and diarrhea (66.67%) (Table 1). We observed that the first symptom of fever occurred on average in 20.67 days, but patients were histopathologically diagnosed with GVHD about 1 to 2 weeks after clinical findings were noted. Late-onset GVHD is associated with a relatively mild and self-limiting process and better prognosis than early-onset GVHD.16 Because chimerism detection was not yet available in our institute, we employed symptom and histopathological evaluations of skin to diagnose GVHD. Early diagnosis was difficult; the lack of a biomarker and the delay in diagnosis and complexity in treatment of GVHD after LT could influence the prognosis of this disease.
During the past decade, many efforts have been devoted to identifying potential biomarkers to diagnose GVHD without invasive tissue biopsies. For example, several different immune cells, particularly T cells, may serve as promising cellular biomarkers that could diagnose, predict risk, and evaluate responsiveness to treatment in GVHD.21-23 Furthermore, because of the potential cytokine storm occurring early after LT, interleukin 2 (IL-2) and tumor necrosis factor α have been tested as potential GVHD biomarkers, which was helpful to understanding pathophysiology and target therapy.24 However, cytokines were not specific for GVHD, often being elevated in inflammation and other complications. In addition to cytokines, microRNA-based diagnostic panel (miR-155 and miR-146a) and lymphocyte surface molecules (CD30 and α4β7 integrin) were found to be upregulated in acute GVHD, showing association with T-cell function, and thus could be biomarkers for GVHD, from mechanism to diagnostic and prognosis.25-29
Proteomic studies have provided promising insights into biomarker discovery for GVHD, from mechanism to diagnostic and prognosis. Recent studies have revealed elafin to be a biomarker of skin GVHD30 and regenerating islet-derived 3-alpha (reg3α) as a relevant biomarker in gastrointestinal acute GVHD31; both of these molecules were secreted as a result of end-stage organ damage. These studies mainly focused on patients who underwent hematopoietic stem cell transplant; however, there is still no validated diagnostic blood test that could be used in routine clinical visits. In a study from Meng and colleagues,32 IL-2, IL-18, and interferon Γ were identified as potential biomarkers for early diagnosis and for monitoring the effects of anti-GVHD treatment; however, this was the only other study (other than ours) involved in study of GVHD biomarkers after LT.
Our previous study15 identified biomarkers after LT by a novel but robust method using untargeted MALDI-TOF proteomics with network analysis and validation by ELISA. Based on this previous method, in this study, we explored the differentially expressed proteins in a small cohort with GVHD of LT. As a result, our identification of ACLY and FGA for GVHD associated with LT might also aid in diagnosis.
The key metabolic enzyme ACLY interconnects glucose and lipid metabolism; ACLY is increased or activated in many different kinds of tumors, whereas inhibition of ACLY could arrest cancer cell proliferation.33 In addition, ACLY may play an important role in various chronic diseases, including cardiovascular diseases, inflammation, and neurodegenerative diseases.34
Fibrinogen is a plasma glycoprotein, which is involved in many physiopathological processes, such as blood coagulation, inflammation, and angiogenesis.35 Drew and colleagues found that fibrinogen may regulate tissue repair via supplying the matrix and accelerating cell proliferation and migration.36 Through evaluation in cancer and other diseases, FGA was identified as an alpha component of human fibrinogen.37,38
Our study had several limitations. First, although powerful statistical tests are useful, they are not robust enough to identify biomarkers in such a small population. Sufficient validation is still needed in an independent and diverse cohort. To overcome the challenge of a small sample size and to further confirm the results in patients with GVHD after LT, additional serum and liver tissue samples should be obtained and patients with GVHD after other solid-organ transplant procedures and bone marrow transplant should be investigated. Screening LT recipients who show some symptoms of GVHD is helpful but not for those who have GVHD-like febrile complications or allergic skin rash or cytopenia due to drug toxicity. We acknowledge that ACLY and FGA may be altered in cancers, inflammation, and other conditions, and their use should be combined with other clinical indicators in practice.
In our study, when we reviewed 65 peptide peaks distinguishing patients with GVHD from patients with excellent posttransplant outcomes and control patients, we identified 2 significantly differently peaks as potential biomarkers. These 2 downregulated peptides were identified as ACLY and FGA and validated by ELISA and Western blot. As far as we are aware, this study is the first to show the downregulation of ACLY and FGA in serum samples from patients with GVHD after LT through untargeted MALDI-TOF proteomics with network analysis, which were thereafter validated by ELISA and conventional statistical analyses, suggesting that ACLY and FGA are potential serum biomarkers for GVHD.
Volume : 19
Issue : 10
Pages : 1048 - 1057
DOI : 10.6002/ect.2021.0073
From the 1Department of Cell Biology and Genetics, School of Basic Medical Sciences, Xi’an Jiaotong University, Health Science Center; the 2Department of Hepatobiliary Surgery and the 3Department of Pathology, the First Affiliated Hospital of Xi’an Jiaotong University; the 4Institute of Genetics and Developmental Biology, Translational Medicine Institute, Xi’an Jiaotong University; the 5Key Laboratory of Environment and Genes Related to Diseases (Xi’an Jiaotong University), Ministry of Education of China; and the 6Key Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi’an Jiaotong University, Xi’an, PR China
Acknowledgements: This work was supported by The National Natural Science Foundation of China (grant number: 81702765, 51837008). The authors have no conflicts of interest to declare. Author contributions: HC, BG, XGZ, and JY designed and supervised all experiments. WL, XGZ, and WW collected the samples and clinical information. WW, JZ, WL, XX, and XW carried out all experiments. WW analyzed data and drafted the manuscript. HC and BG revised the manuscript. All authors reviewed and approved the final manuscript. *Bo Guo and Chen Huang contributed equally to this work.
Corresponding author: Bo Guo or Chen Huang, Department of Cell Biology and Genetics, School of Basic Medical Sciences, Xi’an Jiaotong University, Health Science Center, Shaanxi, Xi’an 710061, PR China
Phone: +86 029 82655190 or +86 029 82655190
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Table 1. Clinical Course and Treatment of 3 Patients With Graft-Versus-Host Disease After Liver Transplant
Table 2. Demographics and Characteristics of Patients With Graft-Versus-Host Disease and Patients With Excellent Posttransplant Outcomes
Figure 1. Appearance of Maculopapular Rashes and Histopathology of Skin Lesion Biopsy of Graft-Versus-Host Disease After Liver Transplant
Table 3. Laboratory Tests in Study Patients
Figure 2. Proteomic Profiling in the Different Patient Study Groups
Table 4. Mean Levels of Different Peptides Among the Different Patient Study Groups
Table 5. Amino Acid Sequences of Identified Proteins That Showed Abundance Among the Patient Study Groups
Figure 3. Representative Spectra of Peak 1 and Peak 4 in the Different Patient Study Groups
Figure 4. Tandem Mass Spectrometry System Spectrometry Fragment Map
Figure 5. Bioinformatics Analysis of Identified Proteins
Figure 6. Concentration of ACLY and FGA in Validation Study
Table 6. Spearman Correlation Analysis Between Identified Serum Biomarkers and Clinical Information