PI3Kδ Inhibition Augments the Efficacy of Rapamycin in Suppressing Proliferation of Epstein−Barr Virus (EBV)+ B Cell Lymphomas

Posttransplant lymphoproliferative disorder (PTLD) continues to be a devastating and potentially life‐threatening complication in organ transplant recipients. PTLD is associated with EBV infection and can result in malignant B cell lymphomas. Here we demonstrate that the PI3K/Akt/mTOR pathway is highly activated in EBV+ B cell lymphoma lines derived from patients with PTLD. Treatment with the mTORC1 inhibitor Rapamycin (RAPA) partially inhibited the proliferation of EBV+ B cell lines. Resistance to RAPA treatment correlated with high levels of Akt phosphorylation. An mTORC1/2 inhibitor and a PI3K/mTOR dual inhibitor suppressed Akt phosphorylation and showed a greater anti‐proliferative effect on EBV+ B lymphoma lines compared to RAPA. EBV+ B cell lymphoma lines expressed high levels of PI3Kδ. We demonstrate that PI3Kδ is responsible for Akt activation in EBV+ B cell lymphomas, and that selective inhibition of PI3Kδ by either siRNA, or a small molecule inhibitor, augmented the anti‐proliferative effect of RAPA on EBV+ B cell lymphomas. These results suggest that PI3Kδ is a novel, potential therapeutic target for the treatment of EBV‐associated PTLD and that combined blockade of PI3Kδ and mTOR provides increased efficacy in inhibiting proliferation of EBV+ B cell lymphomas.     

Monitoring Infection with Epstein–Barr Virus among Seromismatch Adult Renal Transplant Recipients

Patients who undergo Epstein–Barr virus (EBV) seromismatch (D+/R − ) transplants have a higher risk for the development of post‐transplant lymphoproliferative disorder (PTLD). Adult renal transplant recipients at a single institution were prospectively monitored for EBV during the first year post‐transplant. Over a 2‐year period, 34 patients (7.78%) were identified as being EBV D+/R − recipients. Patients who developed symptoms or had persistent viremia were pre‐emptively administered rituximab. Six recipients were discharged without monitoring on the protocol. Of those six, three (50%) developed PTLD and all three lost their grafts. Twenty (60.6%) of the 34 recipients developed viremia during the first year post‐transplant. Of the recipients who became viremic, six (30%) received rituximab. None of the six who received rituximab‐developed PTLD. We found that recipients who were not monitored on the protocol were more likely to have PTLD and graft loss compared to those who were (p = 0.008). Post‐transplant monitoring of adults who undergo EBV D+/R − kidney transplants for viremia and symptoms associated with EBV infection may prompt intervention which reduces the incidence of PTLD within the first year. Use of rituximab in preventing PTLD among patients with primary EBV infection requires further prospective study to determine its overall safety and efficacy.     

Epstein–Barr Virus‐Related Post‐Transplant Lymphoproliferative Disorders: Pathogenetic Insights for Targeted Therapy

Post‐transplant lymphoproliferative disorder (PTLD) is a spectrum of major, life‐threatening lymphoproliferative diseases occurring in the post‐transplant setting. The majority of PTLD is of B‐cell origin and is associated with several risk factors, the most significant being Epstein‐Barr virus (EBV) infection. EBV's in vitro transforming abilities, distinctive latency, clonality within the malignant cells and response to targeted therapies implicate a critical role in the biology of PTLD. This minireview focuses on EBV‐related PTLD pathogenesis, in particular the interplay between aspects of the EBV life cycle and latency with nonviral factors resulting in the wide spectrum of histology and clinical presentations encountered in PTLD. With the increased prevalence of transplantation a rise in the incidence of PTLD may be expected. Therefore the importance of laboratory and animal models in the understanding of PTLD and the development of novel therapeutic approaches is discussed.     

Circulating Antibody Free Light Chains and Risk of Posttransplant Lymphoproliferative Disorder

Posttransplant lymphoproliferative disorder (PTLD) is a major complication of solid‐organ transplantation. With human immunodeficiency virus infection (an analogous immunosuppressive state), elevated kappa and lambda immunoglobulin free light chains (FLCs) in peripheral blood are associated with increased risk of lymphoma. To assess the role of B‐cell dysfunction in PTLD, we measured circulating FLCs among Canadian transplant recipients, including 29 individuals with PTLD and 57 matched transplant recipients who were PTLD‐free. Compared with controls, PTLD cases had higher kappa FLCs (median 1.53 vs. 1.07 times upper limit of normal) and lambda FLCs (1.03 vs. 0.68). Using samples obtained on average 3.5 months before PTLD diagnosis, cases were more likely to have polyclonal FLC elevations (i.e. elevated kappa and/or lambda with normal kappa/lambda ratio: odds ratio [OR] 4.2, 95%CI 1.1–15) or monoclonal elevations (elevated kappa and/or lambda with abnormal ratio: OR 3.0, 95%CI 0.5–18). Strong FLC‐PTLD associations were also observed at diagnosis/selection. Among recipients with Epstein–Barr virus (EBV) DNA measured in blood, EBV DNAemia was associated with FLC abnormalities (ORs 6.2 and 3.2 for monoclonal and polyclonal elevations). FLC elevations are common in transplant recipients and associated with heightened PTLD risk. FLCs likely reflect B‐cell dysfunction, perhaps related to EBV‐driven lymphoproliferation.     

Subclinical Epstein–Barr Virus Viremia Among Adult Renal Transplant Recipients: Incidence and Consequences

The natural history and clinical significance of posttransplant Epstein‐Barr virus (EBV) infection remain largely unknown. The aims of this study are to describe the incidence, risk factors and consequences of EBV infection after kidney transplantation. A total of 383 consecutive patients having received a kidney transplant between January 2002 and December 2010 were included. EBV polymerase chain reaction (PCR) was performed every 2 weeks for 3 months, and every 4 weeks for the next 9 months. A total of 155 of the 383 patients (40%) had at least one positive viremia during the first year posttransplant. The median time to viremia was day 31 posttransplant (14–329). A total of 73 (47%) had EBV viremia > 10³ log and 23 (15%) had positive viremia for more than 6 months. EBV D+/R? patients (12/18 (67%) versus 143/365 (39%), p = 0.02) and those having received antithymocyte globulins (ATG) (54% vs. 35%; p<0.001) were more likely to develop EBV infection. EBV infection (hazard ratio [HR], 3.03; 95% confidence interval [CI], 1.72–8.29; p = 0.01) was associated with the occurrence of opportunistic infections. A positive EBV PCR during the first 6 months posttransplant was associated with graft loss (HR, 3.04; 95% CI, 1.36–6.79; p = 0.014). EBV reactivation is frequent after transplantation and reflects overimmunosuppression. Prospective studies should examine the association between EBV and graft loss.     

The Impact of EBV Status on Characteristics and Outcomes of Posttransplantation Lymphoproliferative Disorder

We examined the associations of Epstein–Barr virus (EBV) status with characteristics and outcomes of posttransplantation lymphoproliferative disorder (PTLD) by studying 176 adult solid organ transplant recipients diagnosed with PTLD between 1990 and 2013 (58 [33%] EBV‐negative; 118 [67%] EBV‐positive). The proportion of EBV‐negative cases increased over time from 10% (1990–1995) to 48% (2008–2013) (p < 0.001). EBV‐negative PTLD had distinct characteristics (monomorphic histology, longer latency) though high‐risk features (advanced stage, older age, high lactate dehydrogenase, central nervous system involvement) were not more common compared to EBV‐positive PTLD. In multivariable analysis, EBV negativity was not significantly associated with worse response to initial therapy (adjusted odds ratio, 0.84; p = 0.75). The likelihood of achieving a complete remission (CR) was not significantly different for EBV‐negative versus EBV‐positive PTLD including when therapy was reduction of immunosuppression alone (35% vs. 43%, respectively, p = 0.60) or rituximab (43% vs. 47%, p = 1.0). EBV negativity was also not associated with worse overall survival (adjusted hazard ratio, 0.91; p = 0.71). Our findings indicate that EBV status is not prognostic or predictive of treatment response in adults with PTLD. The high proportion of EBV‐negative disease diagnosed in recent years highlights the need for new strategies for prevention and management of EBV‐negative PTLD.     

Peripheral Blood Epstein–Barr Viral Nucleic Acid Surveillance as a Marker for Posttransplant Cancer Risk

Several viruses, such as Epstein–Barr virus, are now known to be associated with several human cancers, but not all patients with these viral infections develop cancer. In transplantation, such viruses often have a prolonged time gap from infection to cancer development, and many are preceded by a period of circulating and detectable nucleic acids in the peripheral blood compartment. The interpretation of a viral load as a measure of posttransplant risk of developing cancer depends on the virus, the cancer and associated pathogenic factors. This review describes the current state of knowledge regarding the utility and limitations of peripheral blood nucleic acid testing for Epstein–Barr virus in surveillance and risk prediction for posttransplant lymphoproliferative disorders.     

The Role of Antiviral Prophylaxis for the Prevention of Epstein–Barr Virus–Associated Posttransplant Lymphoproliferative Disease in Solid Organ Transplant Recipients: A Systematic Review

The role of antiviral prophylaxis for the prevention of posttransplant lymphoproliferative disease (PTLD) remains controversial for solid organ transplantation (SOT) recipients who are seronegative for Epstein–Barr virus (EBV) but who received organs from seropositive donors. We performed a systematic review and meta‐analysis to address this issue. Two independent assessors extracted data from studies after determining patient eligibility and completing quality assessments. Overall, 31 studies were identified and included in the quantitative synthesis. Nine studies were included in the direct comparisons (total 2366 participants), and 22 were included in the indirect analysis. There was no significant difference in the rate of EBV‐associated PTLD in SOT recipients among those who received prophylaxis (acyclovir, valacyclovir, ganciclovir, valganciclovir) compared with those who did not receive prophylaxis (nine studies; risk ratio 0.95, 95% confidence interval 0.58–1.54). No significant differences were noted across all types of organ transplants, age groups, or antiviral use as prophylaxis or preemptive therapy. There was no significant heterogeneity in the effect of antiviral prophylaxis on the incidence of PTLD. In conclusion, the use of antiviral prophylaxis in high‐risk EBV‐naive patients has no effect on the incidence of PTLD in SOT recipients.     

Epstein–Barr virus reactivation in allogeneic stem cell transplantation is highly related to cytomegalovirus reactivation

Monitoring of Epstein–Barr virus (EBV) load and pre‐emptive rituximab is an appropriate approach to prevent post‐transplant lymphoproliferative disease (PTLD) occurring after hematopoietic stem cell transplantation (HSCT). This pre‐emptive approach, based on EBV‐DNA monitoring through a quantitative polymerase chain reaction, was applied to 101 consecutive patients who underwent allo HSCT at our Institute (median age 50). A single infusion of rituximab was administered to 11 of 16 patients who were at high risk for progression to PTLD, defined as a DNA value >10 000 copies/mL. All patients cleared EBV DNAemia, without any recurrences. Main factors significantly associated with high risk for PTLD were as follows: (i) unrelated vs. sibling (26% vs. 7%; p = 0.011); (ii) T‐cell depletion (29% vs. 6%; p = 0.001); (iii) graft versus host disease (GVHD; 30% vs. 7%; p = 0.002); and (iv) cytomegalovirus (CMV) reactivation (29% vs. 4%; p = 0.001). Multivariate analysis showed that CMV reactivation was the only independent variable associated with EBV reactivation. We conclude that: (i) a single infusion of rituximab is able to prevent the risk of progression into EBV‐related PTLD; and (ii) CMV reactivation is strongly associated with EBV reactivation; therefore, an intensive EBV monitoring strategy could be advisable only in case of CMV reactivation.     

Epstein–Barr virus DNA load in tumour tissues correlates with poor differentiation status in non‐muscle invasive urothelial carcinomas

What’s known on the subject? and What does the study add? Epstein‐Barr virus (EBV) could be detected in bladder cancer. In our paper, we showed that DNA of EBV could be detected more in the high grade urothelial carcinoma. In superficial bladder cancer, although high grade tumour associated with higher EBV titers, the recurrence free survival was not compromised by high EBV DNA load.

OBJECTIVE

To detect the correlation between the clinical staging, grading and genomic Epstein–Barr virus (EBV) viral numbers in tumour tissues of urothelial carcinoma patients.

PATIENTS AND METHODS

From June 2004 to May 2008, 60 urothelial carcinoma patients (50 cases of bladder carcinoma and 10 of upper tract urothelial carcinoma (UTUC) were enrolled in the study. Eight patients who underwent transurethral resection of prostate for prostate hyperplasia and two patients receiving nephrectomy for non‐function kidney were used as normal controls. The EBV viral copy numbers in genomic DNA were evaluated using a real‐time PCR‐based study. The BamHI W region of the Namalwa cell line was constructed to the plasmid clone and was used as standard curve for absolute quantitative PCR (Q‐PCR).

RESULTS

Epstein–Barr virus DNA was detected in 56% (28/50) and 60% (6/10) of the bladder and UTUC patients, respectively. The EBV DNA could not be detected in the normal control group. By pooling the UTUC and bladder patients in stage Ta,T1, the high copy number in fixed genomic DNA amount (100 ng/20 µL) was correlated with the high grading in stage Ta,T1 urothelial carcinoma (P= 0.014). The overall grading was not statistically associated with EBV copy number (P= 0.25). Although the copy numbers between paired tumour and normal tissues were not statistically different (P= 0.169), there were more copies of EBV in the normal tissues adjacent to the tumours than in those free from urothelial carcinoma. There was no significant difference between recurrence of non‐muscle invasive bladder cancer and the presence of EBV (P > 0.05).

CONCLUSIONS

Epstein–Barr virus DNA could be detected in the genome of the urothelial carcinoma specimens. The poor differentiation status was correlated with the high load of the EBV genome in non‐muscle invasive urothelial carcinoma. However, recurrence‐free survival was not greater in EBV‐positive patients than in EBV‐negative patients.     

Epstein–Barr virus‐positive post‐transplant lymphoproliferative disorder of the central nervous system,after renal transplantation with a discrepancy in viral load between peripheral blood and cerebrospinal fluid

A 43‐year‐old female developed an Epstein–Barr virus (EBV)‐positive post‐transplant lymphoproliferative disorder (PTLD) in the central nervous system (CNS), 14 years after renal transplantation. One year prior to presentation, the patients’ treatment regimen was altered from cyclosporine, azathioprine, and prednisone to mycophenolate mofetil and prednisone. Magnetic resonance imaging of the brain revealed lesions suspect for malignant lymphoma. The EBV real‐time polymerase chain reaction (PCR) on peripheral blood was negative, but highly positive on cerebrospinal fluid. EBV‐positive PTLD was confirmed using histological analysis of cerebral biopsies. Despite tapering of immune suppressive medication and treatment with rituximab and chemotherapy, the patient deceased 50 days after presentation. This case illustrates that vigilance is required when presented with a negative EBV PCR result on peripheral blood when PTLD of the CNS is suspected. This late presentation suggests a relation to the switch in immunosuppressive regimen 1 year earlier.     

Characteristics of Epstein–Barr viraemia in adult liver transplant patients: A retrospective cohort study

Therapeutic immunosuppression following solid organ transplantation increases the risk of Epstein–Barr (EBV) viraemia, which is implicated in post‐transplant lymphoproliferative disease (PTLD). We retrospectively analysed the incidence of EBV viraemia and clinical outcomes in 98 liver transplant recipients. Patients underwent EBV DNA monitoring by whole‐blood PCR: EBV levels were correlated with clinical parameters and outcomes for a median of 249 days. 67% patients developed EBV viraemia (EBV DNA ≥100 copies/ml) and 30% had sustained viraemia. There was a trend towards higher hazard ratios for viraemia with exposure to aciclovir (HR 1.57, P = 0.12) or in recipients of a poorly HLA‐matched graft (HR 1.62, P = 0.10). These associations became significant in the subgroup with >90 days surveillance; HR 2.54 (P = 0.0015) for aciclovir and HR 1.99 (P = 0.03) for poorly matched grafts. The converse was true with ganciclovir (HR 0.56 P = 0.13). Viraemia was more prolonged in men (median duration 7 days vs 1; P = 0.01) and in those with lower UKELD scores (11 days vs 1 day; P = 0.001) but shortened with ganciclovir exposure (P = 0.06). Younger patients were more likely to have high peak viral loads (P = 0.07). No clinical signs or symptoms or adverse outcomes were associated with EBV reactivation.     

Impact of antiviral prophylaxis in adults Epstein–Barr Virus‐seronegative kidney recipients on early and late post‐transplantation lymphoproliferative disorder onset: a retrospective cohort study

Post‐transplantation lymphoproliferative disorder (PTLD ) pathogenesis is related to EBV infection. Mismatch with the donor (EBV D+/R?) is the main risk factor for both early PTLD (<1 year post‐transplantation) and late (>1 year). In these at‐risk patients, the role of antiviral prophylaxis for preventing PTLD remains controversial. We analyzed the impact of antiviral drugs given to prevent CMV disease in a monocentric retrospective cohort of 73 adult kidney or kidney–pancreas EBV ‐seronegative recipients, transplanted between 01/01/2000 and 01/01/2016. Thirty‐seven (50.7%, prophylaxis group) received (val‐)aciclovir or (val‐)ganciclovir for 3–6 months and 36 (49.3%, no‐prophylaxis group) received no‐prophylaxis. Mean follow‐up was 69 ± 7.2 months in the prophylaxis group and 91 ± 10.3 months in the no‐prophylaxis group. Monitoring of EBV PCR revealed that prophylaxis delayed primary infection at 100 days (43% vs. 84%, P = 0.02). Early PTLD incidence was not different between groups (4/37 vs. 4/36, P = 0.99). Concerning late events, EBV ‐related neoplasia incidence was significantly lower in treated patients among whom no cases were observed, while in the no‐prophylaxis group 6 cases were reported (P = 0.02). Despite a weak level of evidence our study suggests that antiviral prophylaxis could prevent late onset PTLD .     

Synchronous hepatic,mesenteric and pulmonary Epstein–Barr virus‐associated smooth muscle tumors in a renal transplant recipient

Al Hussain T, Haleem A, Alsaad KO. Synchronous hepatic, mesenteric and pulmonary Epstein–Barr virus‐associated smooth muscle tumors in a renal transplant recipient.
Clin Transplant 2010. DOI: 10.1111/j.1399‐0012.2009.01206.x
© 2010 John Wiley & Sons A/S. Abstract: Epstein–Barr virus‐associated smooth muscle tumors (EBV‐SMT) are distinct lesions that occur in immunocompromised patients. EBV‐SMT following solid organ transplantation are rare and generally have an indolent biological behavior. Post‐transplant EBV‐SMT have been reported in various anatomical locations. This report describes a synchronous and multicentric development of EBV‐SMT in liver, mesentery, and lung of a 33‐yr‐old male patient, 10 yr after a deceased allograft renal transplantation. The hepatic and mesenteric tumors were available for study. These tumors were composed of bland looking, desmin‐positive, spindle‐shaped cells which showed a strong nuclear staining for EBV with in situ hybridization technique. A literature review of post solid organ transplant EBV‐SMT in the liver and lung, particularly regarding their pathogenesis, synchronicity and biological behavior would be provided.     

Expression of Epstein–Barr virus in children with sacrococcygeal pilonidal sinus determined by immunohistochemical methods

In this study, we probed whether chronic infections of skin such as pilonidal sinus could be a potential site of Epstein–Barr virus (EBV) replication. Pilonidal sinus is associated with a high recurrence rate. Therefore, we decided to determine the role of EBV's presence to explain whether it is correlated with the recurrence of pilonidal sinuses. This study was conducted on 36 patient samples with sacrococcygeal pilonidal sinus. Samples were immunohistochemically stained for EBV, CD3 and CD20 expression. Thirty‐six adolescents with pilonidal disease were evaluated. EBV‐positive cells were located in dermis with high inflammatory activity. EBV‐positive cells stained positive for the B‐cell antigen CD20 and were detected in 10 of 36 (27%) pilonidal sinus specimens. Among those who had experienced a relapse, three were positive for EBV expression. In addition, EBV expression was detected in eight cases with severe inflammation, and in two with minimal or moderate inflammation. Our study advances the field by demonstrating that similar to gastrointestinal mucosa, skin could be a reservoir for EBV. EBV was found to be restricted to B cells in skin lesions, and it was found that skin lesions with severe inflammation showed higher frequency of EBV expression in comparison to minimal or moderately inflammed skin lesions. Additionally, recurrence was more frequently observed among EBV‐positive cases. These findings point out for a role of EBV infection in the recurrence of pilonidal sinuses.