Successful In Vitro Priming of EBV-Specific CD8+ T Cells Endowed with Strong Cytotoxic Function from T Cells of EBV-Seronegative Children

Epstein‐Barr virus (EBV)‐seronegative transplant recipients are at high risk of developing EBV‐associated post‐transplant lymphoproliferative disorder (PTLD), and would maximally benefit from an EBV‐directed T‐cell therapy for prevention or treatment of PTLD. So far, efforts to activate CD8+ EBV‐specific cytotoxic T lymphocytes (CTL) endowed with high specific cytotoxicity from EBV‐seronegative children have failed. We compared the CD8+ CTL priming efficiency of three different modified activation protocols, based on lymphoblastoid cell lines (LCL) stimulation potentially enhanced by either LCL presentation through dendritic cells, or selection of IFN‐γ+ cultured cells, or culture in the presence of rhIL‐12 and rhIL‐7, according to the standard protocol for reactivation of EBV‐specific CTL. We found that only specific LCL stimulation in the presence of rhIL‐12 and rhIL‐7 was able to reproducibly expand EBV‐specific CD8+ CTL endowed with strong cytotoxic activity from truly EBV‐seronegative children. The lines thus activated, which included specificities toward EBV latent and lytic proteins, showed high percentage CD8+ T cells, with <10% naïve CD8+/CCR7+/CD45RA+ cells. Overall, the total number of CD8+ central memory cells, and of CCR7 T‐cell effectors was comparable to that observed in healthy EBV‐seropositive controls. In conclusion, it is feasible to activate EBV‐specific CD8+ CTL with suitable characteristics for in vivo employment from EBV‐seronegative children.     

Preemptive Therapy of EBV-Related Lymphoproliferative Disease after Pediatric Haploidentical Stem Cell Transplantation

The treatment of Epstein-Barr virus (EBV)-related post-transplant lymphoproliferative disease (PTLD) after hematopoietic stem cell transplantation (HSCT) is still unsatisfactory. We conducted a prospective trial to evaluate the impact of routine EBV surveillance and preemptive treatment with the anti-CD20 monoclonal antibody rituximab on the development of PTLD in pediatric recipients of extensively T-cell depleted HSCT from an HLA-haploidentical relative. Twenty-seven patients were included in the surveillance program, 12 developed EBV DNA positivity, with 8 of 12 presenting with sustained viral DNA levels requiring treatment with rituximab. Treatment was well tolerated, and induced clearance of EBV DNA in all patients. However, 4/8 patients showed a new increase in EBV load, coincident with the emergence of CD20(-)/CD19(+) B cells in peripheral blood, accompanied by overt PTLD in 3 patients. The latter cleared PTLD after receiving donor EBV-specific cytotoxic T-lymphocytes (CTLs), and persist in remission at a median 30-month follow-up. EBV-specific T-cell frequency, undetectable at time of EBV DNA positivity, was restored by T-cell therapy to levels comparable with controls. We conclude that preemptive therapy with rituximab is safe, but only partly effective in haplo-HSCT recipients. Patients who progress to PTLD under rituximab treatment can be rescued permanently by infusion of EBV-specific CTLs.     

Post-transplant Lymphoproliferative Syndrome in the Pediatric Liver Transplant Population

Post-transplant lymphoproliferative disease remains a complication with a high morbidity and mortality. The present study examined 291 pediatric liver transplants performed in 263 children from October 1984 to December 1999. Post-transplant lymphoproliferative disease has an overall incidence of 12%. Tacrolimus and cyclosporine had a similar incidence of post-transplant lymphoproliferative disease. Fifty-six per cent of patients who developed post-transplant lymphoproliferative disease were Epstein-Barr virus negative at the time of transplantation. Mean time of conversion to Epstein-Barr virus positivity was 1.1 years after liver transplantation. Ten per cent of those who developed post-transplant lymphoproliferative disease never had Epstein-Barr virus detected. Mean time from Epstein-Barr virus positivity to detection of post-transplant lymphoproliferative disease was 2.68 years, and 3.13 years from liver transplantation (OLTx) to post-transplant lymphoproliferative disease. There was a 35% incidence of mortality. Deaths occurred a mean of 0.76 years after diagnosis of post-transplant lymphoproliferative disease. Most cases of post-transplant lymphoproliferative disease had extranodal location. There was one recurrence in 10% of patients, and two in 3%. All recurrent cases were seen in recipients who became Epstein-Barr virus positive after transplantation. There has been a decrease in the incidence of post-transplant lymphoproliferative disease from 15% to 9% to 4%. Post-transplant lymphoproliferative disease should be diagnosed promptly and treated aggressively. The best treatment, however, seems to be prevention, starting in the immediate postoperative period. Survivors should be monitored for both recurrence of post-transplant lymphoproliferative disease and acute cellular rejection.     

Quantitative EBV Viral Loads and Immunosuppression Alterations can Decrease PTLD Incidence in Pediatric Liver Transplant Recipients

Epstein-Barr virus (EBV) is a common viral infection in pediatric liver transplant patients and can lead to development of post-transplant lymphoproliferative disorder (PTLD). Differing studies have used immunosuppression reduction, antiviral medications or i.v. CMV-immunogloublin for EBV prevention and treatment. The purpose of this study was to determine whether implementation of a protocol for frequent EBV monitoring and EBV viral load-driven immunosuppression reduction could decrease the incidence of PTLD in our patient population. All data were prospectively collected between 2001 and 2004 at a single institution. Seventy-three patients were entered into the study. Patients were divided into a historical control group (pre-2001, 30 patients) and a treatment group (post-2001, 43 patients). Approximately 1271 blood samples of 73 patients were collected between 2001 and 2004. Eleven out of 43 patients received immunosuppression tapering due to high EBV viral loads (>4000 copies/microg DNA). One patient developed allograft rejection after immunosuppression modulation. Prior to 2001, the incidence of PTLD at our institution was 16%. After instituting a protocol for EBV monitoring, the incidence of PTLD decreased to 2% (p-value<0.05). These findings illustrate that frequent EBV viral load monitoring and preemptive immunosuppression modulation have an integral role in preventing PTLD in the pediatric liver transplant population.     

Management of Epstein–Barr Virus-induced Post-transplant Lymphoproliferative Disease in Recipients of Solid Organ Transplantation

The optimal management of Epstein-Barr virus (EBV)-induced post-transplant lymphoproliferative diseases (PTLD) remains controversial. While withdrawal or reduction of immunosuppression is widely accepted as the strategy for the treatment of EBV/PTLD, the role of additional therapeutic interventions remains less clear. Newer strategies, including anti-B-cell monoclonal antibodies and adoptive immunotherapy offer the promise of impaired efficacy and outcome against EBV disease, but lack data demonstrating how and when to use these approaches. The current review provides an overview of potential strategies and presents guidelines for the management of EBV/PTLD in solid-organ transplant recipients.     

Successful Renal Retransplantation after Post-Transplant Lymphoproliferative Disease

Post-transplant lymphoproliferative disease (PTLD) is a rare but severe complication of renal transplantation. Reduction of immunosuppression is essential for controlling PTLD but may induce graft loss. Retransplantation after PTLD is considered dangerous, because immunosuppressive treatment resumption may trigger hematological relapse. We retrospectively report six patients (five adults, one child) who underwent a second renal transplantation after successfully treated PTLD. Epstein-Barr virus (EBV) serology was positive before the first transplantation in all patients except the child. Post-transplant lymphoproliferative disease was detected 6.6 months (range 4.5-9.4) after transplantation. Lymphoproliferation was always monomorphic, B-cell, and EBV-related. Post-transplant lymphoproliferative disease was confined to the renal allograft (n = 4), multilocular (n = 1) or cerebral (n = 1). Immunosuppression tapering (6/6) and transplantectomy (5/6) led to hematological remission in all patients. Retransplantation was performed 77 months (range 50-128) after PTLD diagnosis. Immunosuppression included steroids (n = 6), ATG (n = 2), anti-CD25 (n = 2), cyclosporine (n = 4), tacrolimus (n = 2), mycophenolate mofetil (n = 4) and azathioprine (n = 1). After a median follow up of 30 months (range 24-47) patient survival was 100%, with no recurrence of PTLD. In conclusion, renal retransplantation can be considered in patients with monomorphic PTLD history, without major risk of hematological recurrence.     

Use of EBV PCR for the Diagnosis and Monitoring of Post-Transplant Lymphoproliferative Disorder in Adult Solid Organ Transplant Patients

Epstein-Barr virus (EBV) is known to be involved in the majority of patients who develop post-transplant lymphoproliferative disorder after solid organ transplant. We conducted a retrospective study to determine the utility of qualitative and quantitative Epstein-Barr virus polymerase chain reaction (PCR) for the diagnosis and monitoring of post-transplant lymphoproliferative disorder in adult solid organ transplant patients. Peripheral blood leukocytes obtained from 35 adult solid organ transplant patients consecutively referred for evaluation of possible post-transplant lymphoproliferative disorder, were tested by EBV PCR at the time of initial evaluation and at time points thereafter. Eighteen of 35 (51%) patients were ultimately diagnosed with post-transplant lymphoproliferative disorder by tissue biopsy. Fifteen of 18 (83%) patients were found to have EBER-1 positive tumors by in situ hybridization. EBV PCR was positive in 7 of 15 patients, suggesting a sensitivity of 39%. Seventeen patients without post-transplant lymphoproliferative disorder and three with EBER-1 negative post-transplant lymphoproliferative disorder all had negative EBV PCR tests, suggesting a specificity of 100%. We observed that declines in EBV DNA load were associated with response to therapeutic interventions, such as reduction in immunosuppression, rituximab therapy and chemotherapy. We conclude that peripheral blood EBV PCR may have a role in the diagnosis and monitoring of post-transplant lymphoproliferative disorder in adult solid organ transplant patients.     

Polyomavirus interstitial nephritis and concurrent post‐transplant lymphoma in a renal allograft: coincidence or more?

We describe a case of an Epstein-Barr virus (EBV)-negative post-transplant large B-cell non-Hodgkin lymphoma located in the renal allograft, spleen, liver and left inguinal lymph node of a renal recipient and accompanied by a simultaneous polyomavirus-associated nephropathy. To our knowledge, this is the first report of a simultaneous polyomavirus infection and post-transplant lymphoproliferative disorder.     

Use of cytokine polymorphisms and Epstein-Barr virus viral load to predict development of post-transplant lymphoproliferative disorder in paediatric liver transplant recipients

OBJECTIVE: Currently there are no tests to accurately identify paediatric liver transplant patients at risk for post-transplant lymphoproliferative disorder (PTLD). Herein we describe the use of cytokine polymorphisms and real-time quantitative polymerase chain reaction (qPCR) Epstein-Barr virus (EBV) viral load to identify patients at risk for PTLD development. METHODS: Between 2001 and 2004, approximately 1047 patient samples were collected for qPCR for EBV in 59 patients. EBV viral load was reported in three groups: low EBV (<4,000 copies/microg DNA), high EBV/no PTLD (>4000 copies/microg DNA) and biopsy-proven PTLD. All 59 patients also had cytokine polymorphism genotyping performed for six cytokine polymorphisms (transforming growth factor (TGF)-beta, tumor necrosis factor (TNF)-alpha, interleukins (IL)-6, IL-10, IL-2, and interferon (IFN)-gamma) from DNA isolated from peripheral blood mononuclear cells. Positive predictive value (PPV) and negative predictive value (NPV) were calculated using qPCR and cytokine polymorphism results. Data are reported as a mean +/- standard error of the mean. RESULTS: There were 35 males and 24 females with a mean follow-up of 34.9 months. EBV viral load had a PPV and NPV of 29 and 95%, respectively. The low IFN-gamma (A/A) polymorphism was found to be present in 4/6 PTLD patients (67%) and only 17/53 (33%) non-PTLD patients. When the low A/A IFN-gamma polymorphism was combined with EBV viral load for prediction of PTLD, PPV and NPV were 57 and 93%, respectively. DISCUSSION: Use of cytokine genotyping in conjunction with qPCR for EBV viral load can significantly improve the predictive value of diagnostic tests for identification of patients at high risk for PTLD.     

Presentation and early detection of post-transplant lymphoproliferative disorder after solid organ transplantation

Post-transplant lymphoproliferative disorder (PTLD) is a serious and still frequently observed complication of solid organ transplantation. Despite the recent introduction of anti B-cell monoclonal antibody therapy (rituximab) for treatment of PTLD, mortality rates remain high. Because PTLD often presents in a nonspecific way in clinically unsuspected patients, it is a major challenge to diagnose PTLD at an early stage. Epstein-Barr virus (EBV)-DNA load monitoring is a promising tool for the identification of patients at risk for PTLD development. However, there are some limitations of this method, and not all patients at risk for PTLD can be identified by EBV-DNA measurements alone. Therefore, it is of major importance to recognize early clinical signs and symptoms of PTLD. In this review, risk factors for PTLD development, disease presentation, and methods for early detection will be discussed. Special attention is given to allograft and digestive tract localization and the relation with time of onset of PTLD. The value and pitfalls of EBV-DNA load monitoring are discussed. In addition, because fluorodeoxyglucose (FDG)-positron emission tomography (PET) has shown to be a powerful tool for staging and response evaluation of malignant lymphoma, the role of FDG-PET for early diagnosis and staging of PTLD is addressed.     

Review of Epstein-Barr virus and post-transplant lymphoproliferative disorder post-solid organ transplantation

Post-transplant lymphoproliferative disorder (PTLD) following solid organ transplantation is an important form of post-transplant malignancy. PTLD is typically associated with Epstein-Barr virus (EBV) and occurs in the setting of profound immunosuppression resulting in a deficiency of EBV-specific cytotoxic T lymphocytes (CTL). Predisposing factors include EBV mismatch between donor and recipient, use of immunosuppression especially T-cell depletive therapies and genetic predisposition of recipients. The standard approach has been to reduce immunosuppression but is often insufficient to induce tumour regression. Further understanding of the immunobiology of PTLD has resulted in improved monitoring techniques (including EBV viral load determined by polymerase chain reaction) and newer treatment options. Recent work has highlighted a potential role for dendritic cells in both the pathogenesis and treatment of PTLD. Current treatment modalities include adoptive immunotherapy using ex vivo generated autologous EBV-specific CTL or allogeneic CTL, cytokine therapies, antiviral agents, and more recently, rituximab and dendritic-cell based therapies. This review focuses on the developments and progress in the pathogenesis, diagnosis and treatment of PTLD.     

Successful Treatment of T-cell Post-Transplant Lymphoproliferative Disorder with the Retinoid Analog Bexarotene

T-cell post-transplant lymphoproliferative disorder (PTLD) is a rare life threatening complication of organ transplantation. It is usually resistant to treatment with reduction in immunosuppression or chemotherapy and carries a poor prognosis. We report on a combined kidney and pancreas transplant patient with Epstein–Barr virus (EBV) positive T-cell PTLD that had recurred after chemotherapy and reduction in immunosuppression. The patient was successfully treated with bexarotene, a novel synthetic retinoid analog, achieving a complete clinical response. Bexarotene may be a promising treatment for T-cell PTLD.     

Post-transplant lymphoproliferative disorders in kidney transplantation: histological and molecular genetic assessment

Abstract:  We performed histopathological and immunohistochemical studies, in situ hybridization (ISH) for Epstein–Barr virus (EBV) and molecular genetic analysis using the PCR method for the immunoglobulin heavy chain gene in six patients with post-transplant lymphoproliferative disorders (PTLD) to clarify these problems. In two patients, PTLD developed in the graft, and in the remaining four it developed in systemic lymphoid tissues or organs. In terms of morphological classification, lesions in the graft of two patients were polymorphic with features of rejection in the background. In the other four patients, three of them presented monomorphic lesions and the remaining one presented features of reactive lymphoid hyperplasia. All the patients were positive for EBV as determined by ISH. The molecular genetic study could be performed for four patients, the cells in lesions of two patients were found to be monoclonal and those of the remaining two were polyclonal in IgH as determined by PCR. We conclude that PTLD are of two types, namely, intragraft PTLD and extra-graft PTLD. In the cases of intragraft PTLD their diagnosis is relatively difficult, because lesions are mixed with features of rejection. Molecular genetic analysis and detection of EBV by ISH are useful for diagnosis. In contrast, the diagnosis of extra-graft PTLD is easier than that of intragraft PTLD, by basing of the latest classification of malignant lymphoma. Grading of severity of PTLD should be carried out according to the newest system and protocols for treatment based on the grade of the lesion should be established as soon as possible.     

EBV-associated recurrent Hodgkin''s disease after renal transplantation

Hodgkin's disease is recognized as part of the spectrum of post-transplantation lymphoproliferative disorders (PTLD), although it is still an uncommon de novo malignancy in this population. Epstein-Barr virus (EBV) has been linked to both post-transplant non-Hodgkin's lymphomas and Hodgkin's disease. We report a case of recurrent Hodgkin's disease in a patient who received a renal transplant in childhood and later developed EBV-associated Hodgkin's disease with remission after chemotherapy until subsequent relapse 9 years later that was successfully treated. To our knowledge, this is the first report of recurrent Hodgkin's disease in a transplant recipient. We briefly discuss the pathogenesis of and risk factors for EBV-related PTLD, utility of EBV load surveillance, and the options for treatment of PTLD including immunosuppression reduction, antiviral therapy, anti-CD20 monoclonal antibodies, cytotoxic T cells, and the possible roles of interferon-alpha and rapamycin.     

Post-Transplant Lymphoproliferative Disorders Following Liver Transplantation: Incidence, Risk Factors and Survival

This study investigates retrospectively the incidence, risk factors and mortality of post-transplant lymphoproliferative disorders (PTLD) in adult orthotopic liver transplant (OLT) recipients. Among 1206 OLT recipients at a single institution, 37 developed a PTLD. The incidence of PTLD was highest during the first 18 months and relatively constant thereafter with cumulative incidence of 1.1% at 18 months and 4.7% at 15 years. The risk of PTLD was approximately 10% to 15% of the risk of death without PTLD. During the first 4 years following OLT, PTLD were predominantly related to EBV, while afterward most PTLD were EBV negative. Significant risk factors for PTLD in OLT recipients were transplantation for acute fulminant hepatitis during the first 18 months following OLT (HR = 2.6, p = 0.007), and rejection therapy with high-dose steroids (HR = 4.5, p = 0.049) and OKT3 (HR = 3.9, p = 0.016) during the previous year. Therapy with high-dose steroids or OKT3 (HR = 3.6, p = 0.0071) were also significant risk factors for PTLD-associated mortality. OLT recipients remain at risk for PTLD years after transplantation. The strong association of PTLD with rejection therapy and the worse post-PTLD prognosis among recipients of rejection therapy indicate the need to balance the risk of immunosuppression against the risk of PTLD following rejection treatment.     

An autopsy case of late-onset Epstein–Barr virus associated post-transplant lymphoproliferative disorders 11 yr after kidney transplantation

Abstract:  A 42-yr-old Japanese female presented with right cervical lymphadenopathy. She had been diagnosed with IgA glomerulonephritis at the age of 19 and had a one-yr history of hemodialysis after giving birth to her first child. At the age of 31, she had a living-related renal transplantation from her mother. On admission, she was diagnosed with diffuse large B-cell lymphoma (DLBL) by lymph node biopsy and was treated with Rituxymab plus CHOP (Rituximab/Cyclophosphamide/Doxorubicin/Vincristine/Prednisone). However, the therapy was not successful, and the patient had a rapidly deteriorating clinical course with elevated Epstein–Barr virus (EBV) loads in her peripheral blood. She died four months after the onset of lymphoma, because of disseminated intravascular coagulation and multiple organ failure. In kidney transplantation, the incidence of post-transplant lymphoproliferative disorders (PTLD) is between 0.4% and 2% and mostly occurs within a year after transplantation, in strong association with EBV infection. Late-onset PTLD, however, is more like standard lymphomas and is rarely induced by EBV. The present case was quite atypical of late-onset PTLD because the patient's high viral load was first detected 11 yr after transplantation and presented as a monomorphic B-cell lymphoma.     

Associations of serum EBV DNA and gammopathy with post-transplant lymphoproliferative disease

Abstract: Background: Post‐transplant lymphoproliferative disease (PTLD) is a life‐threatening complication of immunosuppression following transplantation. Epstein–Barr virus (EBV) and gammopathy in serum are associated with PTLD, but these two parameters have not been evaluated in parallel for their association with PTLD. Methods: We evaluated the incidence of EBV load positivity, gammopathy, and protein expression in sera from all PTLD patients diagnosed at our hospital during the past seven yr. Results were compared with those of a control group including matched transplanted patients who did not develop PTLD. Results: Seven of 10 PTLD patients presented EBV⁺ PTLD, for which five patients had detectable serum EBV DNA levels compared with none of 38 controls (RR between two groups =121, p < 0.0001). Five out of 10 patients had gammopathy at PTLD diagnosis compared with 5/38 controls (RR between two groups = 6.6, p = 0.022). Additionally, protein serum analysis by high‐resolution two‐dimensional gel electrophoresis and image examination failed to evidence specific abnormality in patients with PTLD compared with controls. Conclusions: Our results confirm an association between EBV in sera and gammopathy with PTLD, and highlight the high specificity of the former analysis. Whether a combination of both analyses will improve the clinical detection of PTLD remains to be evaluated in a larger prospective cohort study.     

Post-Transplant Lymphoproliferative Disorders Occurring After Renal Transplantation in Adults: Report of 230 Cases From the French Registry

Post-transplant lymphoproliferative disorders (PTLD) are a rare but serious complication after organ transplantation. A French Registry of PTLD was set up in a nationwide population of kidney transplant recipients. We prospectively enrolled all adult kidney recipients developing PTLD between January 1, 1998, and December 31, 2003. We analyzed the incidence, risk and prognostic factors of PTLD by Kaplan-Meier and Cox analyses. Totally 230 cases of PTLD were referred to the French Registry. Cumulative incidence was 1.18% after 5 years. Older age (per year, AHR = 2.19, CI = 1.22-3.94) and recipient Epstein-Barr virus seronegativity (AHR = 3.01, CI = 1.57-5.08) were associated with an increased risk of PTLD. Patients with PTLD had a reduced survival rate (61% at 5 years). Graft PTLD had the best prognosis with an 81% survival rate after 5 years. Infection with hepatitis C or B virus (HCV or HBV), late-onset PTLD, multiple sites involvement and high Ann Arbor staging were risk factors for patient death. Use of azathioprine was associated with a poorer survival rate. PTLD incidence and risk factors in French recipients are in line with the international or American PTLD series. We highlighted the role of HBV or HCV in patient mortality and described the relevant prognosis factors for patients with post-transplant lymphoproliferations.     

Human herpesvirus 8 and Epstein-Barr virus-related monotypic large B-cell lymphoproliferative disorder coexisting with mixed variant of Castleman's disease in a lymph node of a renal transplant recipient

Human herpesvirus 8 (HHV8) has been related to some malignant lymphoproliferations, including post-transplant lymphoproliferative disorders (PTLD). We describe a case of a HHV8 and Epstein-Barr virus (EBV) positive large B-cell lymphoproliferation coexisting with Castleman's disease in the same lymph node of a long-term renal transplant recipient. Biopsy revealed mixed type of Castleman's disease and anaplastic cells showing IgA restriction, although molecular analysis failed to detect monoclonality. Only large cells were co-infected by both EBV and HHV8. After reduction of immunosuppression, the lesion partially regressed. After 1 yr, local evolution required surgery followed by irradiation. The present case represents a unique form of localized monotypic but polyclonal large cell PTLD associated with Castleman's disease. It can be added to PTLD with HHV8 and EBV co-infection.     

Analysis of Factors that Influence Survival with Post-Transplant Lymphoproliferative Disorder in Renal Transplant Recipients: The Israel Penn International Transplant Tumor Registry Experience

Significant mortality is associated with post-transplant lymphoproliferative disorder (PTLD) in kidney transplant recipients (KTX). Univariate/multivariate risk factor survival analysis of US PTLD KTX reported to Israel Penn International Transplant Tumor Registry from November 1968 to January 2000 was performed. PTLD presented 18 (median) (range 1–310) months in 402 KTX. Death rates were greater for those diagnosed within 6 months (64%) versus beyond 6 months (54%, p = 0.04). No differences in death risk for gender, race, immunosuppression, EBV, B or T cell positivity were identified. Death risk increased for multiple versus single sites (73% vs. 53%, hazards ratio (HR) 1.4). A 1-year increase in age increased HR for death by 2%. Surgery was associated with increased survival (55% vs. 0% without surgery) (p < 0.0001). Patients with allograft involvement, treated with transplant nephrectomy alone (n = 20), had 80% survival versus 53% without allograft removal (n = 15) (p < 0.001). Overall survival was 69% for allograft involvement alone versus 36% for other organ involvement plus allograft (n = 19 alive) (p < 0.0001). Death risk was greater for multiple site PTLD and increasing age, and risks were additive. Univariate analysis identified increased death risk for those not receiving surgery, particularly allograft involvement alone.