What Is Known?
- Progressive familial intrahepatic cholestasis type 2 is an important cause of cholestasis and end-stage liver disease in pediatric patients.
- After liver transplantation recurrence of bile salt export pump disease due to alloimmune liver disease has been described.
What Is New?
- Disease recurrence can be refractory to aggressive immunosuppressive treatment.
- Stem cell transplantation may be a therapeutic option in these cases.
See “Steps Forward in the Management” by Hartley and Baumann on page 155.
Chronic cholestasis is 1 of the main causes for end-stage liver disease leading to liver transplantation (LT) in children (1). While progressive familial intrahepatic cholestasis (PFIC) is responsible for severe cholestasis in about 10% to 15% of these patients; incidence varies between 1/50,000 and 1/100,000 births (2). Furthermore, PFIC can be divided in 2 subgroups, presenting with low γ-glutamyltransferase (γ-GT) levels (eg, PFIC 1, 2 and Tight junction protein [TJP2]-deficiency) or high γ-GT -levels (PFIC 3). PFIC 2 is a recessively inherited disorder of the bile salt export pump (BSEP) and is encoded by the ABCB11 gene. BSEP is localized exclusively on the luminal side of the hepatocyte membrane and is essential for the secretion of bile acids by hepatocytes into the canaliculi. So far, a number of premature termination, missense and frameshift mutations have been discovered (3).
Absence or lack of function of BSEP results in intrahepatic accumulation of bile acids, and finally rapid development of liver cirrhosis. If PFIC-2 is diagnosed at an early stage, some children benefit from external partial biliary diversion, which may even reverse liver fibrosis (4). But the long-term success is dependent on multiple variables (5,6) and in some patients, LT is the only option (7,8).
The occurrence of antibody formation against BSEP in the transplanted liver in patients with PFIC-2 was first reported in 2009 (1,10). Since then, more than 20 patients have been reported with recurrent low γ-GT cholestasis following LT for PFIC-2 mimicking the original disease (9–13). In most of these cases, intensifying the immunosuppression led to normalization of graft function (13). Other patients needed further treatment, including rituximab or plasmapheresis/immunoadsorption to control anti-BSEP antibody titers, cholestasis and stop disease progression (14).
Here we report on 3 pediatric patients with PFIC-2 who developed so-called antibody-induced BSEP deficiency (AIBD) after LT, resulting in graft failure and the need for retransplantation despite intensive immunosuppressive treatment and immunological attempts to clear the anti-BSEP antibodies.
Keitel et al first described this female patient (and the index case for AIBD) in 2009 (1). She was the third child of consanguineous parents from Saudi Arabia and was diagnosed with PFIC-2 in early childhood. Genetic analysis revealed 3 homozygous missense changes (V444A, Y818F, and G982R). The missense mutation G982R has been shown to lead to intracellular retention of the BSEP protein (15). Her 2 older sisters harbored the same mutations and had already undergone LT with successful outcomes. Installation of a partial biliary diversion was performed at the age of 15 month but was not effective because of a recurrent prolapse of the jejunal loop, elevated bile acids and no relief from itching. Therefore, regular anatomy was reconstructed after 14 month and she was listed for LT. At that time, liver histology showed no cirrhosis. The patient received her first LT (post mortem split, segments 2 and 3) at the age of 3 5/12 years. She presented with primary dysfunction and extensive liver necrosis due to poor arterial graft perfusion and underwent a second LT (whole organ, large for size) 17 days later. Apart from the need for an abdominal patch, the postoperative course was uneventful. Immunosuppression consisted of a standard regimen with 3 doses of basiliximab at days 0, 4, and 28, steroids (10 mg/kg intraoperatively followed by 15 mg/m2 body surface area [BSA] daily, then weekly tapering to 1 mg/m2, withdrawal after 1 year) and tacrolimus (trough levels 6–8 μg/L) (Fig. 1). Fifteen months after the re-LT, the girl presented with jaundice, pruritus, and diarrhea. The histology of the performed liver biopsy showed toxic damage and cholestasis but no signs of rejection. Laboratory results revealed elevated transaminases (ALT 503 U/L [−50 U/L]) and bilirubin (bilirubin 128 μmol/L [−17]), while the γ-GT remained normal (γ-Gt 33 U/L) and a third transplantation had to be performed 17 months after the first LT (post mortem split, segments 2 and 3). Three months after retransplantation, the child again presented with signs of cholestatic liver disease like pruritus, jaundice, and low γ-GT cholestasis. Histology then revealed signs of rejection and mycophenolat mofetil (MMF) was added, but had to be discontinued due to worsening diarrhea. One month later AIBD was suspected, and via immunofluorescence staining antibodies against BSEP could be detected in blood samples and liver tissue (1).
In the following months, immunosuppressive therapy was switched from tacrolimus to cyclosporine (CSA) and then to sirolimus while cholestasis did not improve. Several attempts to decrease the alloimmune mediated cholestatic disease were performed, plasmapheresis was conducted for 5 times (18 month after re-re-transplant), followed by anti-CD20-antibody therapy (rituximab, 375 mg/m2 BSA for 4 times on a weekly basis) with a supplementary steroid therapy (dexamethasone 1.5 mg/kg bodyweight for 2 days). After treatment with rituximab, repeated immunoglobulin doses were administered (0.7 g/kg bodyweight). Low γ-GT cholestasis and pruritus, however, did not improve. Molecular adsorbent recirculation therapy (MARS) was performed for 6 times, but did not lead to adequate relief from itching and cholestasis. The patient went back to the family's home country Saudi Arabia at the age of 6 years. At that time, she was again suffering from itching and diarrhea. Histology showed marked intralobular cholestasis and hepatocellular damage, but no fibrosis or cirrhosis. She was lost to our follow-up and died after a third transplantation.
This boy initially presented with jaundice and pruritus at the age of 10 months in Riga, Latvia. PFIC-2 was diagnosed based on a homozygous BSEP truncation mutation c.3084A>G (p.Q976X, NM_003742.2). In addition to this mutation, the patient was also homozygous for the common BSEP polymorphism c.1331T>C (V444A) (16). Due to cirrhosis with reduced synthetic function and severe pruritus, a living-donor LT from the mother was performed and a standard initial immunosuppressive therapy, consisting of basiliximab, CSA (trough levels 120–150 μg/L) and prednisolone as in patient 1 has been applied. The clinical course during the first months after LT was uneventful. Nine months after LT, the patient had a relapse of pruritus and laboratory investigation revealed cholestasis with normal γ-GT but high bilirubin (10-fold above normal) and high bile acids (50-fold above normal). The onset was associated with a gastrointestinal infection and low trough levels of CSA. Liver histology showed a combination of moderate to severe rejection (Banff score 5/9), cholestasis, and toxic damage. The medication was switched from CSA to tacrolimus and the patient received high-dose intravenous steroids for 6 days. MMF was added and secondarily switched to azathioprine as the cholestasis did not improve and anti-BSEP antibodies were detected in the patient‘s serum and liver tissue (via immunochemistry). Tacrolimus was switched back to CSA as a cholestatic effect of tacrolimus was discussed (17). Cholestasis persisted despite intensification of the immunosuppression, and the titers of anti-BSEP-antibodies remained high even with administration of high-dose immunoglobulins, plasmapheresis (performed 10 times pre re-LT in our center and 4 times perioperatively), immunoadsorption (23 times altogether) and B-cell depletion (rituximab) in a dose of 375 mg/m2 BSA (Fig. 2). Histology showed progressive fibrosis, finally leading to micronodular cirrhosis.
Retransplantation was performed at the age of 34 months due to chronic graft failure, and a third LT was necessary at the age of 36 months after arterial hypoperfusion leading to recurrent graft dysfunction. Due to the second relapse of AIBD refractory to intensified immunosuppressive medication, immunoglobulins and anti-B cell-directed therapy, a fourth LT was not considered to be a promising therapeutic option in the long-term. In extensive interdisciplinary discussions, haploidentical stem cell transplantation (SCT) was assumed to be the only reliable treatment option in order to restore stable liver function. The aim was to establish a new immune system not recognizing BSEP as neo-antigen and thereby terminate the ongoing alloimmune reaction after the pretransplant alloantibodies had disappeared. At that time histology presented with low grade and therefore potentially reversible fibrosis. The liver function was deemed suitable for the medication necessary for conditioning and to have enough capacity for regeneration after SCT. SCT was performed 4 years after the first LT at the age of 5. At that time the treatment consisted of tacrolimus, everolimus, steroids as well as ursodeoxycholic acid and 3 antihypertensive drugs. No relevant complications occurred during the 3 years follow-up. This case has been published recently by our group and has been described in detail (18). After stem cell transplantation, anti-BSEP antibodies have not been detectable and the patient shows excellent graft function on low-dose CSA monotherapy (through-levels 60–80 μg/L) without pathological findings in liver histology 27 month after re-re-LT.
This boy is the third child of non-consanguineous parents. The first child of the family died due to cholestatic liver disease at the age of 2 years. The second child remained healthy. Our patient first presented as a newborn with prolonged jaundice in Russia. At the age of 12 months he was referred to our center with end-stage liver disease. Immunohistochemistry of the liver revealed absence of BSEP and the patient was diagnosed with PFIC-2. The genetic testing confirmed the diagnosis by showing a homozygous V444A mutation and the coding exons 6–9 could not be detected by next generation- or sanger sequencing.
He was subjected to LT (living-related from his grandfather) at the age of 16 months. The initial immunosuppression consisted of basiliximab at days 0, 4, and 28, steroids (according to patient 1) and CSA (trough levels about 150 μg/L). Maintenance immunosuppression in the first year after LT consisted of low-dose steroids and CSA.
The postoperative course was uneventful. One year after the first LT, a liver biopsy was performed as protocol procedure. Histology showed minimal rejection (Banff score 3/9) and fibrosis 1/4. Laboratory findings and ultrasound examinations showed no abnormalities during a 3-year follow-up. After an intercurrent infection, the boy again developed low-γ-GT cholestasis (γ-GT levels 31–53 U/L, serum bile acids up to 370 μmol/L), pruritus, and jaundice. Liver biopsy showed progression of cellular rejection (Banff score 5/9). Anti-BSEP-antibodies were detected (titer 1:10,000) in the patient's serum and in liver tissue via immunochemistry (anti-Human-IgG-Cy3 and MRP2AK; University Hospital, Düsseldorf, Germany).
Triple therapy consisting of steroids, CSA, and MMF was administered; immunoadsorption (10 cycles, starting 43 months after first LT) and anti-CD20 therapy (rituximab, 375 mg/m2 BSA) were initiated. Nevertheless, the anti-BSEP antibody titer increased to 1:16,000 and finally liver failure developed, resulting in the need for retransplantation 56 months after the first LT (Fig. 3). During the follow-up of 3 years after retransplantation the patient showed stable liver function and no detectable anti-BSEP antibodies under a dual immunosuppressive regimen with tacrolimus (through levels 4–6 μg/L) and MMF. Recently, he developed elevated liver enzymes and anti-BSEP antibodies (titer of 1:25,000) after a viral infection, while the γ-GT -levels remained normal. There were no signs of jaundice or pruritus. We adjusted the immunosuppressive medication by adding prednisolone (10 mg/day initially) and increased trough levels of tacrolimus to 8 μg/L. This led to a decrease of liver enzymes to normal values and the possibility to reduce the steroid doses again.
PFIC-2 is an inherited cholestatic liver disorder in which several identified genetic mutations lead to dysfunction or complete absence of BSEP (16). Our 3 patients developed refractory recurrence of low-γ-GT cholestasis and graft failure due to anti-BSEP antibodies after LT. Over the last few years, several patients have been reported with proven antibodies against BSEP and recurrence of cholestatic liver disease following LT for PFIC-2 mimicking the original disease (9–13). The reason for this alloimmune process seems to be multifactorial. Genetic mutation in the BSEP gene can lead to total loss of the protein or to premature truncation (12,19). The total absence seems to increase the likelihood of an immune reaction directed against BSEP that presents a new antigen to the immune system after LT (11). Patients with splice site mutations, stop codon mutations and frameshift mutations (which lead to a complete absence of BSEP from the patient's liver) therefore seem to be at particular risk of developing anti-BSEP antibodies after LT (1,20). Immunostaining showed absence of BSEP in all of our 3 patients, supporting this theory. In accordance to the findings in PFIC patients 3% to 5% of patients with Alport syndrome develop post kidney transplant anti-GBM nephritis, and it is postulated that the susceptibility to that condition is depending on the mutation type. Further risk factors have not been identified so far supporting this hypothesis (21).
Moreover, graft rejection or damage due to poor graft perfusion could lead to release of components of BSEP after LT and therefore may initiate a strong immune response directed against the “de novo” antigen. This theory is also supported by the case of a patient described by Masahata et al in 2016 (13). Their patient developed AIBD after a period of histology proven acute cellular rejection, massive lobular necrosis, and portal vein occlusion (15).
Infections stimulating the immune system and associated low immunosuppressive trough levels may also trigger the development of anti-BSEP antibodies. In our patients 2 and 3, infections and fluctuating levels of CSA were reported before the development of AIBD. Moreover, the histology of patient 2 showed signs of rejection at the beginning of the alloimmune process.
Several case reports have been described where intensifying the immunosuppressive therapy led to stabilization of graft function or at least downregulated antibody titers and delayed retransplantation (11,14). Until now, specific risk factors predicting poor outcome in patients with AIBD after LT are not yet identified. On the one hand, there were patients who were switched to a more potent immunosuppressive regimen and recovered, while on the other hand, extensive treatment (as in our patients with plasmapheresis, rituximab and immunoglobulin administration) could not prevent progression of the disease.
The outcomes of affected patients do not seem to depend on the measurable extent of anti-BSEP antibody levels, as seen in our patients. Our 3 patients had different titers ranging from 1:80 up to 1:25,000), highest in patient 3, while patient 2 had the most complicated course of disease with rapid progression of AIBD. Further studies are needed to determine the main risk factors for the development of recurrent AIBD and whether there are options to prevent the disease.
Moreover, the question needs to be addressed, what should be done as soon as anti-BSEP antibodies are detected. If the underlying mutation determines the extent of the immunoreaction after LT, it should also influence the choice of the initial immunosuppressive regimen after LT and maybe thereby influence the prevalence of antibody development. In our center, the initial immunosuppressive regimen for small children is traditionally based on CSA. Our patient 1 had 2 sisters with the same mutations and good long-term graft function after LT. These patients received an initial combination treatment with tacrolimus, MMF and low-dose steroids in contrast to our patients. Intensifying initial immunosuppression to prevent the development of anti-BSEP antibodies in patients transplanted for PFIC-2 may be an option, but also involves the risk of severe side effects associated with the immunosuppressive drugs, for example, infections, impaired kidney function, or even post-transplant proliferative disease. It remains to be seen, whether a more intense immunosuppressive therapy should be applied routinely in patients with poor graft perfusion or with an underlying genotype leading to complete absence of BSEP. A tacrolimus-based combination therapy with MMF, however, seems to be crucial after retransplantation.
Patient 2 was finally cured by stem cell transplantation (SCT) after a very complicated course of AIBD without reasonable perspective to recover and after all other options were exploited. This is somehow a proof of concept of the underlying alloimmune process. The result is impressive with currently low-dose immunosuppression and recovery of the transplant function and histology. SCT appears to be an option for patients who are refractory to conservative treatment, but the risk of severe infections, graft-versus-host disease or organ damage during this procedure should be taken into account. Basak et al describe the feasibility of allo SCT after solid organ transplantation due to various reasons including aplastic anemia, acute myelogenous leukemia or myelodysplastic syndrome (22). It is also described as a salvage strategy in autoimmune diseases leading to re-induction of self-tolerance (23).
In conclusion, the treatment of transplanted PFIC-2 patients must be decided on a case-by-case basis. Initial immunosuppression after LT should be tacrolimus-based. Nevertheless, patients need to be monitored for AIBD and immunosuppression should be adjusted promptly to prevent transplant damage due to antibodies. More investigation has to be undertaken to identify risk factors for poor outcome.
1. Keitel V, Burdelski M, Vojnisek Z, et al. De novo bile salt transporter antibodies as a possible cause of recurrent graft failure after liver transplantation: a novel mechanism of cholestasis. Hepatology
2. Jacquemin E. Progressive familial intrahepatic cholestasis. Clin Res Hepatol Gastroenterol
2012; 36 (suppl 1):S26–S35.
3. Strautnieks SS, Byrne JA, Pawlikowska L, et al. Severe bile salt export pump deficiency: 82 different ABCB11 mutations in 109 families. Gastroenterology
4. Arnell H, Papadogiannakis N, Zemack H, et al. Follow-up in children with progressive familial intrahepatic cholestasis after partial external biliary diversion. J Pediatr Gastroenterol Nutr
5. Verkade HJ, Bezerra JA, Davenport M, et al. Biliary atresia and other cholestatic childhood diseases: advances and future challenges. J Hepatol
6. Bull LN, Pawlikowska L, Strautnieks S, et al. Outcomes of surgical management of familial intrahepatic cholestasis 1 and bile salt export protein deficiencies. Hepatol Commun
7. Mehl A, Bohorquez H, Serrano MS, et al. Liver transplantation and the management of progressive familial intrahepatic cholestasis in children. World J Transplant
8. Wanty C, Joomye R, Van Hoorebeek N, et al. Fifteen years single center experience in the management of progressive familial intrahepatic cholestasis of infancy. Acta Gastroenterol Belg
9. Kubitz R, Droge C, Kluge S, et al. High affinity anti-BSEP antibodies after liver transplantation for PFIC-2: successful treatment with immunoadsorption and B-cell depletion. Pediatr Transplant
10. Maggiore G, Gonzales E, Sciveres M, et al. Relapsing features of bile salt export pump deficiency after liver transplantation in two patients with progressive familial intrahepatic cholestasis type 2. J Hepatol
11. Siebold L, Dick AA, Thompson R, et al. Recurrent low gamma-glutamyl transpeptidase cholestasis following liver transplantation for bile salt export pump (BSEP) disease (posttransplant recurrent BSEP disease). Liver Transpl
12. Stindt J, Kluge S, Droge C, et al. Bile salt export pump-reactive antibodies form a polyclonal, multi-inhibitory response in antibody-induced bile salt export pump deficiency. Hepatology
13. Masahata K, Uehara S, Ibuka S, et al. Recurrence of progressive familial intrahepatic cholestasis type 2 phenotype after living-donor liver transplantation: a case report. Transplant Proc
14. Lin HC, Alvarez L, Laroche G, et al. Rituximab as therapy for the recurrence of bile salt export pump deficiency after liver transplantation. Liver Transpl
15. Wang L, Soroka CJ, Boyer JL. The role of bile salt export pump mutations in progressive familial intrahepatic cholestasis type II. J Clin Invest
16. Droge C, Bonus M, Baumann U, et al. Sequencing of FIC1, BSEP and MDR3 in a large cohort of patients with cholestasis revealed a high number of different genetic variants. J Hepatol
17. Ganschow R, Albani J, Grabhorn E, et al. Tacrolimus-induced cholestatic syndrome following pediatric liver transplantation and steroid-resistant graft rejection. Pediatr Transplant
18. Brinkert F, Pukite I, Krebs-Schmitt D, et al. Allogeneic hematopoietic stem cell transplantation
eliminates alloreactive inhibitory antibodies after liver transplantation for bile salt export pump deficiency. J Hepatol
19. Davis AR, Rosenthal P, Newman TB. Nontransplant surgical interventions in progressive familial intrahepatic cholestasis. J Pediatr Surg
20. Kubitz R, Droge C, Kluge S, et al. Autoimmune BSEP disease: disease recurrence after liver transplantation for progressive familial intrahepatic cholestasis. Clin Rev Allergy Immunol
21. Kashtan CE. Renal transplantation in patients with Alport syndrome. Pediatr Transplant
22. Basak GW, Wiktor-Jedrzejczak W, Labopin M, et al. Allogeneic hematopoietic stem cell transplantation
in solid organ transplant recipients: a retrospective, multicenter study of the EBMT. Am J Transplant
23. Alexander T, Arnold R, Hiepe F, et al. Resetting the immune system with immunoablation and autologous haematopoietic stem cell transplantation
in autoimmune diseases. Clin Exp Rheumatol
2016; 34 (4 suppl 98):S53–S57.