Epstein-Barr virus re-activation in post-kidney transplant period: risk factors and specific immune- responses Erica Franceschini

KDIGO guidelines suggest monitoring high-risk (donor EBV seropositive/recipient seronegative) kidney renal transplants for EBV by NAT Despite the lack of definitive evidence, EBV load measurements are frequently used in Europe to guide diagnostic workup and pre- emptive reduction of immunosuppression EBV monitoring 85.9% 77.4% pre-emptive treatment for patients with significant EBV DNAemia levels Background B. Kasiske et al. KDIGO clinical practice guideline for the care of kidney transplant recipients: a summary R. San-Juan et al. Clin Microbiol Infect 2015

Objectives Evaluation of the incidence of EBV re-activation in the post-kidney transplant period in the Modena cohort Evaluation of EBV re-activation risk factors Evaluation of the probability to develop a PTLD once a patient has an EBV re-activation

Patients and Methods Retrospective observational study All consecutive patients who performed a quantitative EBV-DNA assay in the post-KT period from January 2005 to December 2014 Patients with negative EBV-DNA were compared to patients with positive EBV-DNA Patients with EBV-VL >4,000 copies/ml were compared to patients with EBV-VL <4,000 copies/ml D.Rowe et al. Epstein–Barr virus load monitoring: its role in the prevention and management of post-transplant lymphoproliferative disease

Results 523 kidney transplants, person year follow-up (PYFU) 265 kidney transplants (50.7%) performed in Modena 340 (65.0%) males Median age 46 years (IQ 35-57) 44 (8.4%) living-donor transplant 190 (36.3%) screened for EBV-DNA, PYFU 128 (67.4%) out of 190 EBV-DNA positive

Characteristics of EBV-positive and EBV-negative patients EBV-positive N= 128 EBV-negative N= 62 p-value Sex Women, n (%) Men, n (%) 45 (35.16) 83 (64.84) 16 (25.81) 46 (74.19) Age, mean (SD) (years)46 (±14.21)50 (±13.41)0.104 Acute rejection, n (%)20 (15.62)11 (17.74)0.834 KT primary indication, n (%) Glomerulonephritis Chronic pyelonephritis Obstructive nephropathy Haemolytic uraemic syndrome Trauma Unknown 53 (41.41) 40 (31.25) 13 (10.16) 6 (4.69) 0 (0) 16 (12.50) 26 (41.94) 21 (33.87) 7 (11.29) 3 (4.84) 0 (0) 5 (8.06) 0.928

EBV-positive N= 128 EBV-negative N= 62 p-value Epstein-Barr recipient serostatus, n (%) Positive Negative Missing 52 (40.62) 3 (2.34) 73 (57.03) 33 (53.22) 2 (3.22) 27 (43.54) Re-KT, n (%)6 (4.69)2 (0.50)0.003 HCV-positive, n (%)9 (7.03)5 (8.06)0.774 HIV-positive, n (%)5 (3.91)3 (4.84)0.717 Comorbidities, n (%)93 (72.66)39 (62.90)0.182 Graft failure, n (%)15 (11.72)9 (14.52)0.643 Time between KT and graft failure, median (IQR) (days) 1,834 (415-4,389) 522 (91-3,098) Patient death, n (%)8 (6.25)10 (16.12)0.036 CMV re-activation, n (%) Missing Negative Positive 38 (31.93) 73 (61.34) 8 (6.72) 15 (28.84) 34 (65.38) 3 (5.77) 0.926

EBV-positive N= 128 EBV-negative N= 62 p-value Polyoma virus BK re-activation, n (%) Missing Negative Positive 66 (55.46) 37 (31.09) 16 (13.44) 27 (51.92) 16 (30.77) 9 (17.31) Number of samples tested for EBV-VL, median (IQR) 2 (1-5)1 (1-1)<0.001 Induction therapy, n (%) Basiliximab Thymoglobulin Daclizumab Not done 60 (46.87) 9 (7.03) 1 (0.78) 58 (45.31) 33 (53.22) 4 (6.45) 1 (1.61) 24 (38.70) Creatinine value at EBV-VL sample, median (IQR) (mg/dl) 1.67 ( ) 1.61 ( ) PTLD, n (%)6 (4.68)3 (4.83)1.000

Univariate Poisson regression analysis for factors associated with EBV re-activation VariableIRR95% CIp Age, per 1 year <0.001 Men vs Women Creatinine Induction: Thymoglobulin vs Basiliximab None vs Basiliximab <0.001 Cyclosporine Micofenolic acid Sirolimus Everolimus <0.001 Tacrolimus Steroid <0.001 Azathioprine

Characteristics of patients with EBV-VL > 4,000 copies/ml 19 patients out of 128 (14.8%) EBV viral load superior to 4,000 copies/ml (IR 1.07/100 PYFU, 95% CI ) No statistically significant differences between patients with EBV-VL> 4,000 copies/ml and EBV-VL < 4,000 copies/ml The use of MPA and steroids resulted as risk factors for EBV re-activation with EBV-VL superior to 4,000 copies/ml in the univariable analysis

Post Transplant Lymphoproliferative Diseases 9 patients out of 523 (1.7%) had a PTLD IR of PTLD in KT population was 0.19/100 PYFU (0.09 – 0.37) IR of PTLD in patients tested for EBV-VL was 0.50/100 PYFU ( ) 1 EBV-related PTLD 8 non EBV-related PTLD

Characteristics of population with PTLD AgeSexEBV IgG Induction Therapy Manteinance Therapy Time KT- PTLD EBV-VL (copies/ ml) PTLD type SymptomsPTLD type Histological examination Patient 1 21MR-BASSteroids CSA 9 months 79B- DL L ymphonode enlargement earlypositive Patient 2 68MR+BASCSA Steroids EVR 8 years61B-NHL L ymphonode enlargement cytopenia latenegative Patient 3 31FNABAS Steroid TAC SIR 12 years 342B-DLSweats Fever L ymphonode enlargement latenegative

Patient 7 22MNAnoCSA13 yearsnegLGLcytopenialatenegative Patient 8 59FR+BSAEVR4 yearsnegLGLnolatenot done Patient 9 41MNAnoCSA Steroid 15 yearsnegMMSplenomegalylatenegative Patient 4 55MR+ATGTAC Steroid 3 years80LGLnolatenot done Patient 5 63FNAnoCSA Steroid MPA 11 years 697MMnolatenegative Patient 6 46MR+BSACSA8 years53NML L ymphonode enlargement latenegative AgeSexEBV IgG Induction Therapy Manteinance Therapy Time KT- PTLD EBV-VL (copies/ ml) PTLD type SymptomsPTLD type Histological examination

Univariate Poisson regression analysis for factors associated with PTLD VariableIRR95% CIp Age, per 1 year Men vs Women Creatinine Induction: ATG vs BAS None vs BAS CSA SIR EVR TAC Steroid

Conclusions Importance of initial EBV serologic screening of the candidate Despite the literature suggests the importance of quantitative EBV viral load monitoring for PTLD prevention in high-risk populations in the first year the only case of early PTLD in our study had a low VL Avoid EBV viral load assays performing in low risk patients or after the first year post-transplant in absence of a strong PTLD suspicion Importance of PTLD clinical suspicion

Thanks to… Mauro Codeluppi Patrizia Comoli Delia Davoli Margherita Digaetano Jessica Plessi Leonardo Potenza Elisabetta Rubbiani Antonella Santoro Stefano Zona Cristina Mussini