EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Objectives: Humoral rejection is the B-cell-mediated production of immunoglobulin G antibody against the transplanted heart. Antibody-mediated rejection may be resistant to standard immunosuppressive therapy and is associated with high mortality and graft loss. Rituximab can be used to treat antibody-mediated rejection in heart transplant recipients. This retrospective study describes our experience with rituximab treatment in children with heart transplants.

Materials and Methods: We present 7 pediatric patients with antibody-mediated rejection who were treated with plasma exchange and rituximab therapy. Rituximab was given at a dose of 375 mg/m2 by slow infusion in the intensive care unit after 5 days of plasmapheresis, in addition to a conventional regimen consisting of steroids, mycophenolate mofetil, and tacrolimus. The peripheral blood count and sodium, potassium, serum urea nitrogen, creatinine, aspartate aminotransferase, and alanine aminotransferase levels were measured in all patients before and after treatment.

Results: Seven patients were treated with plasma exchange and rituximab. We repeated this therapy in 5 patients because of refractoriness or recurrent rejection. After diagnoses of antibody-mediated rejection, 4 patients died within 6 months (mortality rate of 57.1%). We did not observe any adverse effects or complications related to rituximab.

Conclusions: Rituximab can be used in humoral rejection after pediatric heart transplant. However, the success of the treatment is controversial, and further study is needed to find an effective treatment for antibody-mediated rejection and steroid-resistant cellular rejection in children.

Key words : Children, Humoral rejection, Treatment

Heart transplant is the optimal therapy for inoperable congenital heart disease with severe symptoms and advanced cardiac failure. Survival rates of heart transplant recipients have improved in the past 20 years with advances in immunosuppressive treatment.¹

Antibody-mediated rejection (AMR) occurs in approximately 10% to 20% of cardiac recipients and has emerged as a major complication of heart transplant, associated with high mortality.² The mechanism of graft injury is probably the activation of the complement cascade, as well as interactions of antibodies with macrophages and other leukocytes. Humoral rejection is a B-lymphocyte-mediated process that is treated with interventions to eliminate these cells or the antibodies.³

At present, the management of AMR is not standardized. The essence of treatment is desensitization to or a reduction in circulating HLA antibodies. Current options for treating AMR include steroid pulse therapy, plasma exchange, intravenous immunoglobulin, and intensifying immunosuppression.⁴ In most cases, several of these approaches are combined. Because of the poor prognosis, newer treatments that target B cells are being explored, including the proteasome inhibitor bortezomib (plasma cell depletion), humanized antibodies that antagonize complement proteins, and rituximab, a monoclonal antibody directed against the B-cell antigen CD20.⁴ A recent consensus statement on the treatment of AMR issued by the International Society for Heart and Lung Transplantation recommends the use of high-dose corticosteroids, plasmapheresis, and intravenous immunoglobulin, with rituximab, bortezomib, and anticomplement antibodies as other options.⁵

Rituximab works by depleting B cells in peripheral blood within 72 hours of administration, and the response can be sustained for as long 12 months.⁵ Therefore, rituximab has the potential to attenuate early allograft injury and may also reduce the risk of chronic or recurrent AMR.

In this study, we describe our experience with rituximab treatment in pediatric heart transplant recipients.

Materials and Methods

The study group consisted of 7 patients (3 boys, 4 girls) with humoral rejection at a median age of 7 years (range, 7-17.5 y). Heart transplant had been performed at a median age of 3 years (range, 1.5-17 y). Mean follow-up was 26.7 months (range, 4-72 mo). Rituximab was given at a dose of 375 mg/m2 by slow infusion in the intensive care unit after 5 days of plasmapheresis, in addition to a conventional regimen consisting of steroids, mycophenolate mofetil, and tacrolimus. Patients 6 and 7 also received intravenous immunoglobulin before the rituximab infusion, whereas patients 2 and 7 were given sirolimus instead of tacrolimus because of epilepsy (patient 2) and Epstein-Barr virus infection (patient 7). Antithymocyte induction therapy was not given at the time of transplant or after diagnosis of rejection. We administered the rituximab and addition conventional regimen to the 7 patients for a total of 12 times (Table 1). Peripheral blood count and sodium, potassium, serum urea nitrogen, creatinine, aspartate aminotransferase, and alanine aminotransferase levels were measured in all patients before and after treatment.

Patients 1 and 2 were admitted with acute decompensated heart failure with New York Heart Association class III to IV symptoms accompanied by orthopnea and fatigue. Echocardiography revealed severe systolic and diastolic dysfunction. We identified humoral rejection based on an endomyocardial biopsy. We performed 2 courses of plasmapheresis and rituximab therapy because of the clinical and echocardiographic findings. Because recovery was not evident, we also administered methylprednisolone. Panel reactive antibody results were negative in both patients. Both patients died within 6 months of rejection. The pulmonary artery systolic and pulmonary capillary wedge pressures were elevated in patient 1.

Patient 3 was admitted with acute decompensated heart failure with New York Heart Association class III to IV symptoms, accompanied by edema, orthopnea, and fatigue. Echocardiography revealed severe systolic and diastolic dysfunction. We identified cellular rejection with low-grade humoral rejection by endomyocardial biopsy. We administered pulse methylprednisolone. Because recovery did not occur, we also provided plasmapheresis and rituximab therapy. After treatment, we observed clinical improvement within several days and echocardiographic improvement in systolic function in approximately 2 months. After several months, we observed echocardiographic deterioration, repeated the same protocol, and observed the same results. The patient died suddenly at home 2 months after the last rejection. Echocardiography revealed normal systolic and impaired diastolic function 1 week before death. Panel reactive antibody results were negative. The pulmonary artery systolic and pulmonary capillary wedge pressures were also elevated in this patient.

Patients 4 and 6 were admitted with acute decompensated heart failure with New York Heart Association class III to IV symptoms accompanied by orthopnea and fatigue, with severe systolic and diastolic dysfunction on echocardiography. Humoral rejection was identified by endomyocardial biopsy. Panel reactive antibody results were positive in both patients. We provided plasmapheresis and gave rituximab therapy and observed immediate clinical improvement and echocardiographic improvement in systolic function at 2 months. After 3 to 4 months, we observed deterioration in systolic function, with patients symptomatic, and repeated the protocol and observed the same results. Patient 6 has been in good clinical condition for 2 months and patient 4 for 8 months. Their diastolic functions were still poor according to echocardiographic examinations before the initial rejection. Selective coronary angiography was normal in patient 4. Pulmonary capillary wedge pressure was elevated in both patients, and pulmonary artery systolic pressure was elevated in patient 4.

Patient 5 presented with symptoms of right-sided heart failure (ascites, edema, and fatigue). We observed severe tricuspid regurgitation on echocardiography and suspected humoral rejection based on an endomyocardial biopsy and diffuse right coronary artery stenosis based on coronary angiography. Panel reactive antibody results were positive. We initially administered pulse methylprednisolone therapy, followed by plasmapheresis and rituximab treatment. We did not observe any clinical or echocardiographic recovery and performed tricuspid plasty surgery, which was followed by tricuspid valve replacement and finally retransplant. The patient died after retransplant. The pathologic examination of the extracted heart was consistent with humoral rejection and chronic vascular rejection (cardiac allograft vasculopathy).

Patient 7, who underwent transplant 5 years earlier, was asymptomatic and had a normal echocardiographic examination in terms of diastolic and systolic function. We performed a routine endomyocardial biopsy, which revealed humoral rejection. Plasmapheresis was performed, and intravenous immunoglobulin and rituximab treatment were administered. Panel reactive antibody results were positive, and the patient’s pulmonary capillary wedge pressure was elevated. A follow-up biopsy after treatment was normal. Coronary angiography showed a diffuse irregularity in the left anterior descending artery.

We did not observe any hemodynamic or biochemical adverse effects related to rituximab therapy. An antihistaminic drug infusion was necessary because of itching or an urticarial rash during plasmapheresis.

Heart transplant is the optimal therapy for advanced cardiac failure. The development of more effective immunosuppressive agents has reduced the incidence of acute cellular rejection and improved survival after transplant.¹ However, a major complication is AMR, which occurs in 10% to 20% of cardiac allograft recipients and is associated with high mortality.² Graft injuries probably occur as a result of activation of the complement cascade, as well as interactions of antibodies with macrophages and other leukocytes. Although there is presently no standard of care for AMR, the treatment goal is to desensitize or reduce circulating HLA antibodies, with current treatment options being steroid pulse therapy, plasma exchange, intravenous immunoglobulin, and intensifying immunosuppression.⁴

The mechanism of action of rituximab in the treatment of humoral rejection in heart transplant patients is not clear. It is possible that B-cell populations that do not express CD20 are important during the early pathogenesis of AMR. Rituximab may eliminate memory B cells, which are the source of antibody-producing plasma cells.^6-8 Rituximab minimizes recurrent graft injury once the offending antibody has been removed by plasmapheresis. Future investigations are necessary to identify the mechanisms and durability of rituximab therapy for patients with AMR.

The AMR-related mortality rate remains very high. In our study, the overall mortality rate was 57.1% and 66.66% in hemodynamically compromised patients. In Wu and associates,⁹ the rate of survival 5 years after heart transplant was 86%. Ravichandran and associates¹⁰ calculated the probability of surviving for 1 week or more to be 100% in the rituximab group versus 80% in the group without rituximab ; at 3 years, the rates were 75% vs. 29% for patients with and without rituximab.

We observed that patients who benefited from rituximab clinically also had a significantly improved ejection fraction (patients 3, 4, and 6). We did not observe any recovery in diastolic function in any patient. Ravichandran and associates¹⁰ also found no difference in the pretreatment or follow-up ejection fraction results between patients with and without rituximab treatment. More detailed studies are needed to examine the effects of rituximab on clinical and echocardiographic parameters.

Regarding its safety, rituximab has been investigated extensively in the treatment of numerous malignant and immunologic conditions. Most of its adverse effects are mild to moderate infusion-related reactions. Leukopenia is relatively common after administration, mainly because of suppression of B-lymphocyte differentiation. Hemodynamic effects, particularly transient hypotension, have been reported. Anaphylaxis is possible but extremely rare. Initially, small test doses of rituximab were required to evaluate the risk of anaphylaxis; however, these have been relegated due to their poor positive predictive value. Because rituximab depletes CD20-positive B-cell progenitors, it may be associated with an increased risk of recurrent infections, particularly viral infections such as Cytomegalovirus. Results regarding infection risk in transplant recipients are poor. In a series of 8 cardiac transplant recipients, 3 developed infections.¹¹ We did not observe any adverse effects related to rituximab infusion or any increase in infection among the study survivors.

In general, AMR is associated with an increased development of cardiac allograft vasculopathy.^7,8,12 Although immunofluorescence for C4d staining was once considered the standard for diagnosing AMR, the presence of clinically significant AMR in the absence of C4d staining is now widely accepted.^13-17 All of our patients were positive for C4d immunofluorescence in the diagnosis of humoral rejection, and AMR was associated with cardiac allograft vasculopathy in 2 patients (patients 5 and 7). In patient 5, AMR was accompanied by hemodynamic compromise; however, the patient’s endomyocardial biopsy did not show significant pathologic changes. After retransplant, humoral rejection and cardiac allograft vasculopathy were verified by the pathology of the explanted heart. Wu and associates⁹ found that patients with AMR developed significantly less freedom from cardiac allograft vasculopathy (defined as ≥ 30% stenosis in any vessel) at 5 years than patients in the control group. By 5 years after transplant, only 52% of patients in the AMR group compared with 79% of patients in the control group did not show cardiac allograft vasculopathy. Hammond and associates¹⁷ found a significant difference in the time to cardiac allograft vasculopathy development among patients who had cellular rejection alone, mixed cellular rejection and AMR, and AMR alone.

Perhaps not all asymptomatic patients with C4d deposition have clinical sequelae.¹⁶ In our study, patient 7 was asymptomatic, and systolic function was normal by echocardiography, although endomyocardial biopsy revealed humoral rejection. In this patient, coronary angiography showed diffuse irregularity in the left anterior descending artery. In series report from Wu and associates,⁹ the ejection fraction was less than 40% in all asymptomatic but biopsy-positive patients.

In summary, our findings indicate that the AMR-related mortality rate is still very high despite new treatment modalities. The adjunctive use of rituximab with steroids, plasmapheresis, and intravenous immunoglobulin may improve outcomes in heart transplant recipients who present with AMR. Rituximab is safe for the treatment of AMR, even in small children. A multicenter, prospective, randomized trial is necessary to confirm the benefits of rituximab and guide the optimal treatment of cardiac allograft AMR.

Study limitations
The limitations of this study are its retrospective format and small sample size. A study with a larger number of AMR patients is needed to demonstrate the effectiveness of the treatment modality and survival.

Volume: 16 Issue: 2 April 2018

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