Abstract
Post-transplant lymphoproliferative disorder (PTLD) is a life-threatening complication after solid organ transplantation. Reduction of immunosuppression (RI) is accepted as a first step treatment with a long-term complete response rate observed in 23-50% of patients. Chemotherapy for diseases refractory to RI is based on small cohorts treated with different regimens. This paper reports on 10 consecutive cases of PTLD after liver transplantation. The median time from transplantation to PTLD diagnosis was 5 years. PTLD was frequently extranodal involving the transplanted liver. Seven monomorphic PTLD, 2 polymorphic and one Hodgkin disease were observed. Epstein Barr virus was present in tumour tissue only in one case. Initial therapy included RI in all patients. Chemotherapy was used in eight patients. No treatment-related mortality was observed and no patient developed graft rejection during chemotherapy. At a median follow-up period of 25 months, 6 of the 10 patients were alive and without evidence of disease.
Post-transplant lymphoproliferative disorder (PTLD) is a clearly recognized and potentially life-threatening complication after solid organ transplantation or bone marrow transplantation (1). PTLD comprises a spectrum of diseases ranging from infectious mononucleosis and lymphoid hyperplasia to highly aggressive lymphoma. The disease is clinically important in view of the constantly rising number of organ transplant recipients and the development of more potent and specific immunosuppressive drugs (2-4). PTLD is a common malignancy after transplantation, with an incidence ranging from 2% to 10% and an overall mortality rate often exceeding 50% (5-7) .
Registry-based reports do not usually provide details of treatment and outcome: single institution studies are largely reports of limited scope and only a few studies include significant numbers of patients with PTLD. Most cases are associated with Epstein Barr virus (EBV), leading to uncontrolled B-cell proliferation in patients with a decreased function of EBV-specific T-cells due to the use of immunosuppressive drugs. PTLD cases that developed late after transplantation (8-10) were often EBV negative.
PTLD differs from lymphoma in the general population in histopathological findings: PTLD is characterized by increased extranodal involvement, a more aggressive clinical course and poorer response to conventional treatment (11-17).
Treatment of PTLD consists of reduction of immunosuppression (RI) as a first step with positive response rates ranging from 23 to 50%. In the past, chemotherapy was reserved for patients for whom other treatments failed because of the increased toxicities of cytotoxic agents and the high susceptibility to life-threatening infections. In fact, most authors consider new anti-CD20 monoclonal antibody essential for treatment as single agent or in association with chemotherapy but there is no definitive agreement about schedules, duration of treatment and setting of patients.
The present study focuses on the clinical presentation and outcome of PTLD after liver transplantation observed in a single centre, the usefulness of a specific algorithm for treatment, and the efficacy and tolerability of a weekly chemotherapeutic regimen.
Patients and Methods
Study population. This study was carried out in the Azienda Ospedaliera-Padua University and included 10 patients who developed PTLD after liver transplantation from January 1990 to January 2009 and were treated at the Medical Oncology Division (Istituto Oncologico Veneto, I.R.C.C.S. since 1/03/2006). All patients, ≥18 year old age, underwent a baseline evaluation at diagnosis with a complete blood cell count, a screening battery of chemistry tests, a computed tomography (CT) of the chest and abdomen, and bone marrow biopsy. The stage was reported according to the Ann Arbor staging system. International Prognostic Index (IPI) was also evaluated (18, 19). In situ hybridisation was performed for the detection of EBV in tumour tissues. The immunosuppressive drugs were stopped or reduced whenever possible (with introduction of steroids), with their dosage reduced by at least 50% and/or the number of drugs reduced to no more than 2 according to the local protocol. Patients were eligible for chemotherapy if they had EBV-negative PTLD in advanced stage or EBV-positive/not available PTLD in progressive (PD) or stable (SD) disease after reduction or withdrawal of immuno-suppression (Figure 1). Patients had a performance status of 0-2 according to the Eastern Cooperative Oncology Group criteria (20). Patients were not eligible to take part in this study if they had central nervous system (CNS) involvement or any serious concomitant disease. All gave informed consent to change immunosuppressive therapy and then, in cases of lack of response (within 2-4 weeks), to start chemotherapy.
Response criteria and follow-up. Tumour response was classified as complete response, unconfirmed complete response, partial response, SD or PD according to the revised International Workshop criteria (21). Complete response (CR) was defined as the disappearance of all measurable lesions and radiological or biological abnormalities observed at diagnosis and the absence of new lesions. Unconfirmed CR was defined as a CR with the persistence of some radiological abnormalities, which had to have regressed in size by at least 75% [this definition was omitted after introduction of positron-emission tomography (PET-CT)]. Partial response (PR) was defined as the regression of all measurable lesions by more than 50%, the disappearance of any measurable lesions and the absence of new lesions. SD was defined as the regression of any measurable lesions by 50% or less or no change for the non measurable lesions, but without growth of existing lesions or the appearance of new lesions. PD was defined as the appearance of a new lesion, any growth of the initial lesion by more than 25%, or growth of any measurable lesion that had regressed during treatment by more than 50% from its smallest dimension. Response to treatment was assessed 6 weeks after the start and at the end of chemotherapy. After 2006, staging and response evaluation was performed by PET-CT scan.
Histology and immunohistochemestry. The diagnosis of PTLD was always established by histological examination of biopsies or surgical specimens of tumour.
All specimens were fixed in 10% neutral formalin, embedded in paraffin and stained with standard hematoxylin and eosin. Histological diagnosis was performed based on criteria established by the World Health Organisation (22).
Sections were stained for immunohistochemistry using the biotin-avidin technique in which diaminobenzidine was used as a chromogen (23). Commercially obtained primary antibodies were used directed against the following antigens: CD45 (Dakocytomation, Dako, Denmark), CD20 (Dakocytomation), CD3 (Novocastra, Leica Microsystems Srl, Milano, Italy), CD30 (Dakocytomation) and CD15 (Dakocytomation). Antigen retrieval was achieved through heat pre-treatment for CD30 and CD15.
Normal rabbit and/or mouse serum was used as negative control. Primary antibody was omitted in additional negative controls.
EBV in situ hybridisation analysis. EBV in situ hybridisation was performed on paraffin sections with fluorescein isothiocyanate (FITC)-conjugated EBER1 and EBER-2 probes. Known cases of EBV-positive and negative lymphomas were used as positive and -negative controls. The stained sections were examined under light microscopy, and unequivocal brown-black staining of the tumour cell nuclei was interpreted as positive.
Assessment of clonality. Clonality was determined by the presence of kappa or lambda light chain restriction in tissue samples.
DNA was extracted from formalin-fixed, paraffin-embedded tissues and analysed for immunoglobulin heavy chain (IgH) and T-cell receptor γ and δ chain (TCR γ and δ) gene rearrangements using PCR-based methods. For the analysis of the IgH gene, 3 sets of fluorescent-labelled consensus variable region (V) primers, framework regions (FR) I, II, and III, and a mixture of joining (J) region primers were used. For the analysis of the TCR γ gene, a mixture of fluorescent-labeled consensus V primers and J primers were used following PCR, capillary electrophoresis and GeneScan (Applied Biosystems, Foster City, CA, USA) analysis. A segment of the β-globin gene was amplified as an internal control.
Treatment. Patients were managed according to the local protocol (Figure 1). At the time of the initial suspected diagnosis of PTLD, patients were treated promptly with reduction/withdrawal of immunosuppression. Chemotherapy was started after 2-4 weeks in cases of EBV negative PTLD in advanced stage or EBV positive PTLD in progressive or stable disease after RI. Patients with non Hodgkin's lymphoma (NHL) received VACOP-B, a third chemotherapeutic generation regimen including doxorubicin 50 mg/m2 at weeks 1, 3, 5, 7, 9, and 11; cyclophosphamide 350 mg/m2 at weeks 1,5, and 9; etoposide 50 mg/m2 i.v. (d1) at weeks 3, 7 and 11; etoposide 100 mg/m2 (d2 and d3) per os at weeks 3, 7 and 11; vincristine 1.4 mg/m2 (maximum dose, 2 mg) and bleomycin 10 units/m2 at weeks 2, 4, 6, 8, 10, and 12. Prednisone 40 mg/m2 was administered orally every other day in weeks 1 through 10 and tapered during weeks 11 and 12 (24). At the end of chemotherapy, patients received rituximab 375 mg/m2 weekly for four weeks.
Patients with Hodgkin lymphoma were treated according to the Stanford V chemotherapy regimen followed by radiotherapy (25).
Chemotherapy was given weekly for 12 weeks as according to the following schedule: vinblastine 6 mg/m2 and doxorubicin 25 mg/m2 at weeks 1, 3, 5, 7, 9 and 11; vincristine 1.4 mg/m2 (maximum dose, 2 mg) and bleomycin 5 units/m2 at weeks 2, 4, 6, 8, 10 and 12; mustard 6 mg/m2 at weeks 1, 5 and 9; and etoposide 60 mg/m2 two times daily weeks 3, 7 and 11. Prednisone 40 mg/m2 was administered orally every other day weeks 1 through 10 and tapered during weeks 11 and 12. In both regimens, doses of doxorubicin, cyclophosphamide, vinblastine, mustard, and etoposide were reduced to 65% if the absolute neutrophil count (ANC) was less than 1,000/μl, and treatment was delayed if the ANC was less than 500/μl. Granulocyte colony-stimulating factor (GCSF) was allowed after the first delay and was started 48 h after myelosuppressive chemotherapy in order to prevent additional reductions or delays of chemotherapy.
Prophylactic medication included trimethoprim-sulfamethoxazole, ranitidine, fluconazole and acyclovir or valaciclovir throughout the treatment period.
Statistical analysis. The date of PTLD diagnosis was designed as the date of biopsy. Overall survival was defined as the time from diagnosis of PTLD to death by any cause or to the date they were last confirmed to be alive. Survival curves were plotted using the Kaplan-Meier method.
Results
Patients' characteristics. A total of 826 orthotopic liver transplantations were performed in 766 patients at the Hospital of Padua University between January 1990 and January 2009. Of these, ten patients who developed PTLD (1.3%) were identified. The median age at the time of transplantation was 53 years (range 26-62 years).
The indication for liver transplantation was hepatic C virus (HCV)-related cirrhosis in 6 patients, alcoholic-related cirrhosis in 2, HBV-related cirrhosis in 1 patient and sclerosing cholangitis in the final patient.
Current immunosuppressive treatment in patients who developed PTLD consisted of cyclosporine in 4, tacrolimus in 5 and mycofenolate mofetil and tacrolimus in 1. The target level for either tacrolimus or cyclosporine treatment was modulated on an individual basis according to rejection history, age, renal and hepatic function.
The median time from transplantation to diagnosis of PTLD was 5 (range: 1-8) years, with two early PTLD (Table I).
One Hodgkin lymphoma, 7 monomorphic PTLD and 2 polymorphic PTLD cases were observed.
Eight PTLD cases were of B-cell origin and 1 presented as T-cell lymphoma. EBV was present only in 1/6 sampled tissues. Serology/EBV DNA was negative in 6 patients and positive in 1 with absence of EBV in tissue (Table II). Molecular analysis revealed that the samples were monoclonal in 5 patients. Symptoms at presentation were non-specific with considerable variation; B symptoms (weight loss >10%, night sweating or fever) occurred in three patients (patients 2, 4 and 6). Symptoms of local involvement such as bleeding and diarrhoea were present in patients with gastrointestinal involvement (patients 8 and 10) and shortness of breath in the patient with lung lesion (patient 3).
The tumour had nodal involvement in 4 patients. Patient 5 also presented right leg paraesthesia due to sciatic nerve compression. Six patients presented with early-stage PTLD (stage I-II) and 4 with advanced-stage PTLD (stage III-IV). None of the patients had CNS involvement at diagnosis. Four patients had an intermediate-high, high IPI. One patient (patient 9) developed fulminant PTLD with disseminated systemic disease and died suddenly a few days after diagnosis. Patient 8 underwent a colonoscopy procedure, suggesting colon adenocarcinoma, and received chemotherapy according to the FOLFOX schedule (26) but died 15 days after chemotherapy due to hepatorenal syndrome (Table III). Patients were initially treated with RI: this resulted in the discontinuance of all immunosuppressive drugs to restore T lymphocyte function and the commencement of prednisone in order to prevent organ rejection and to take advantage of the anti-lymphoma activity of steroids.
Patient 7 only underwent hepatectomy and RI. Patient 8 received one dose of rituximab and died suddenly from disease progression. Eight patients were treated weekly with chemotherapy: 6 after failure of RI and 2 were initially treated with surgery and RI (patients 3 and 10). Chemotherapy started according to VACOP-B schedule after 120 and 60 days respectively.
The median time from diagnosis of PTLD and the start of chemotherapy was 24 days.
No cases of rejection were observed during chemotherapy administration. At the end of treatment, patients 3, 4, 5 and 7 maintained only prednisone, while patients 2 and 6 started tacrolimus again even though with lower serum levels.
Response rate. After chemotherapy, 6 patients showed CR, 1 patient PR and 1 PD (Table IV).
Patient 1 had an early relapse and died from disease progression. Patient 2 relapsed 18 months after the end of chemotherapy and radiotherapy. At relapse, disease involved only left laterocervical lymph nodes and the patient received radiation treatment resulting in a complete remission. Patient 3 relapsed in the left lung 4 months after a right lung bi-lobectomy. This patient started chemotherapy according to the VECOP-B schedule (epirubicin instead of doxorubicin because of low ejection fraction) obtaining a complete remission (27). Patients 4, 5 and 6 were alive at the time of writing, without evidence of disease. For patient 7, immunosuppressive treatment was reduced after hepatectomy for a monoclonal, monomorphic, late PTLD. This was the only case with in situ hybridisation positive for EBV. Patients 8, 9 and 10 died of disease progression.
Toxicity during chemotherapy. No cases of treatment-related mortality were observed. There was only a single case of G4 mucositis (patient 1) and a single case of cutaneous G4 toxicity (patient 4). No patient developed allograft rejection while on chemotherapy and all maintained a level of lowered immunosuppression for almost three months after the end of therapy. Patient 8 started chemotherapy with the FOLFOX (26) schedule because colonoscopy suggested colon adenocarcinoma. Revision of histological samples did not confirm diagnosis of adenocarcinoma but a monomorphic PTLD.
Survival rate. At a median follow-up of 25 months (range 1-76 months), 6 out of the 10 patients who achieved CR were alive and without evidence of disease. The median overall survival was 14 months, with a 3-year projected survival rate of 57% (Figure 2). The remaining 4 out of the 10 died patients died: 2 of relapse and disease progression, and 1 with fulminant disease. One patient died suddenly 15 days after administration of chemotherapy according to the FOLFOX schedule. All deaths were observed during the first year after diagnosis. Figure 3 shows similar survival between patients with PTLD after liver transplantation and patients with PTLD after heart or kidney transplantation treated in our centre (28).
Discussion
PTLD is a serious complication after organ transplantation, associated with a high mortality rate, with an incidence ranging from 1% to 10% (5-7). The risk of developing PTLD is greater within the first year of transplantation and declines thereafter (29).
Risk factors include EBV, cytomegalovirus, the degree of immunosuppression and the type of organ transplanted. In this study 6 out of the 10 patients received transplantation for HCV-related cirrhosis: previous experience indicates HCV infection as a possible risk factor for the development of PTLD (30, 31).
Clinical presentation is highly variable: most patients present B symptoms and extra nodal involvement including the allograft (32, 33).
Prognosis remains poor with first year mortality rates of 50% and 40% in heart and renal transplantation recipients respectively (29). Recent data indicate CNS or bone marrow involvement as risk factors for poor survival (34).
There are no established approaches to the treatment of PTLD: RI can be justified as first step in most cases, with a long-term CR observed in 23-50% of patients (35). Chemotherapy is considered as a last chance therapy for patients with diseases refractory to RI but results are discordant because studies are based on small cohorts treated with different regimens. However complete remission rates range from 50% to >60% (11-17) .
Treatment-related death is significant, occurring in about 25% of patients. The factors influencing tumour-related mortality include sepsis, cardiac complication, graft dysfunction and opportunistic infections (36, 37). Rituximab shows promising efficacy either as single agent, especially in early onset PTLD, or in combination with chemotherapy (38-44).
In the present study 10 consecutive cases of PTLD developed in 19 years of liver transplantation activity in Padua Hospital are reported. Monomorphic PTLD, polymorphic PTLD and Hodgkin lymphoma cases were included. EBV was present only in 1 out of 6 tissues sampled (16%) while, in our previous experience among heart and kidney transplant recipients with PTLD (28), EBV was positive in 12 out of 20 patients (60%). This difference is probably related to less intensive immunosuppressive therapy in liver transplant recipients.
Six out of 10 patients had an HCV-related liver cirrhosis: viral infection probably plays a role in lymphomagenesis as reported by literature (30-31).
Eight patients received weekly chemotherapy (VACOP-B for non Hodgkin Lymphomas and Stanford V plus radiotherapy for Hodgkin's disease): 6 at diagnosis and the other 2 were treated initially with surgery and RI. At relapse, the last 2 patients received chemotherapy: 1 achieved complete durable remission and the other partial remission.
No treatment-related mortality was observed, probably due to prophylactic uses of trimethoprim-sulfamethoxazole, fluconazole, acyclovir and growth factors. No rejection occurred during chemotherapy.
Six patients achieved a CR, 1 patient a PR and one PD (ORR 87.5%). At a median follow-up of 25 (range 1-76) months, 6 out of the 10 patients were alive and without evidence of disease.
There is a clear lack of consensus in the management of patients with PTLD. Nevertheless, RI is considered the initial treatment, especially in patients with early PTLD (45) where regression of the disease following RI is observed frequently. In fact, restoring T-cell function may lead to regression of disease. However, how much reduction, for how long, and how to predict response to such reduction are unknown. EBV-negative PTLD arises later in the post transplant course, RI is less effective and chemotherapy with or without rituximab remains the primary treatment (46-48). In this study, patients awaiting histological demonstration of EBV in tumour tissue received RI treatment: they started chemotherapy after diagnosis of EBV negative PTLD and/or because of failure of RI. The only patient with EBV-positive PTLD sample tissue underwent central hepatectomy, and after surgery he remained only on steroid treatment and is currently in good health, without evidence of disease.
Table V summarises the findings of several studies of PTLD after liver transplantation reported in literature (17, 49-53). All studies were retrospective. Most patients received anthracycline-based chemotherapy and only two cases of treatment-related mortality were reported both due to neutropenic sepsis. CR rate ranged from 50 to 80%.
In the study by Norin et al., 6 out of 12 patients were alive and without evidence of disease at a median follow-up of 4 years. More recently Patel et al. (17) obtained a disease-free survival of four years in 4 out of 5 patients treated with CHOP schedule.
These studies and our data confirm that patients with PTLD can be treated with chemotherapy with an overall response rate exceeding 70%, with long-term disease-free survival.
This is the first study of weekly chemotherapy in the treatment of PTLD after liver transplantation. The patient were assigned to chemotherapy treatment on the basis of a decisional algorithm (Figure 1). The authors' experience demonstrates that most cases of PTLD after solid organ transplantation require chemotherapy. A weekly regimen is clinically effective and safe, with no graft loss from rejection and no case of treatment-related mortality. It also allows the reduction or discontinuance of all immunosuppressive drugs for the entire duration of treatment.
These data also confirm the Authors' previous experience in management of PTLD after heart and kidney transplantation (28). No second malignancies were observed but the short median follow-up must be considered.
From 2006, four doses of weekly rituximab were added to the end of chemotherapy. Rituximab was administered after the end of chemotherapy, 3 months after reduction/withdrawal of immunosuppression, thus taking advantage of a long period free from immunosuppressive drug to reduce the risk of infectious diseases and to obtain a response consolidation.
We considered the algorithm proposed as a valid aid to orient the therapeutic strategy. The weekly chemotherapy regimen is also safe and effective in the treatment of PTLD. Moreover, rituximab represents a favourable option in the treatment of PTLD, but there are only a few published studies with small populations. Therefore schedule, timing and duration of rituximab administration is not well established.
- Received December 21, 2009.
- Revision received March 29, 2010.
- Accepted March 29, 2010.
- Copyright© 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved