EBV Copy Number Research Articles

PCR Screening for Epstein-Barr Virus-Related Lymphoproliferative Disease (EBV-LPD) among Allogeneic Stem Cell Transplant (AlloSCT) Recipients: The Importance of T Cell Depletion.

PCR monitoring for EBV reactivation is commonly used to confirm the diagnosis of EBV-LPD in alloSCT recipients. Previous studies using multiple PCR approaches have suggested a correlation between high EBV copy number and impending EBV-LPD, potentially allowing for the administration of preemptive rituximab and/or donor leukocytes prior to disease onset. However, subpopulations of alloSCT recipients with EBV reactivation at higher risk for EBV-LPD remain incompletely defined. We sought to determine the demographic factors among alloSCT recipients that affect the predictive value of our EBV PCR assay. Quantitative real-time PCR was performed on serum samples obtained from alloSCT recipients using primers that amplify a portion of the BNRF1-p143 locus. Clinical patient information was retrieved from institutional research databases. Patients who did not undergo appropriate EBV PCR screening (n=13), had a history of EBV-LPD prior to alloSCT (n=1) or received rituximab for prophylaxis against EBV-LPD (n=2) were excluded. 187 alloSCT recipients underwent EBV PCR monitoring between 7/03–7/05, of whom 58 (31.0%) were <18 years old, 117 (62.6%) were female and 111 (59.4%) received allografts depleted of T cells ex vivo (TCD). 56/187 (29.9%) had EBV reactivation (i.e., PCR >500 copies/mL) at some point after alloSCT. The prevalence of EBV reactivation was similar in recipients of TCD and conventional allografts (28.8% vs. 31.5%). Among patients with EBV reactivation, the risk for EBV-LPD did not differ based on age, sex, underlying disease, donor relation or HLA disparity. However, of the 56 patients with EBV reactivation, 5/32 (15.6%) recipients of TCD allografts and 0/24 (0.0%) recipients of conventional allografts developed EBV-LPD (p<0.03). The highest PCR values within one month prior to presentation of EBV-LPD varied ~1,000-fold between the 5 patients (799, 1007, 2380, 12K and 862K copies/mL). Of the alloSCT recipients without EBV-LPD, 8 had PCR values >500 for >6 months and 3 had ≥1 PCR value >100K. In our study population, the utility of the EBV PCR was maximized in recipients of TCD allografts with PCR values >500 (Table). In conclusion, EBV reactivation is common among recipients of both conventional and T cell depleted allografts, but preemptive therapy guided by EBV PCR should be primarily directed toward the latter. The effects of in vivo TCD with agents such as alemtuzumab on EBV reactivation and EBV-LPD are under investigation.Utility of the EBV PCR for predicting EBV-LPD and for guiding preemptive therapy in all patients and in only those who received T cell depleted allograftsPatient groupPCR cutoff (copies/mL)SensitivitySpecificityPositive predictive valueNegative predictive valueNumber needed to treatAll patients>500100%72%9%100%11.2>1,00080%77%9%99%12.5>5,00040%85%7%98%20>10,00040%88%9%98%14.6>100,00020%98%25%98%4.4T cell depleted>500100%75%16%100%6.4>1,00080%77%14%99%7.6>5,00040%85%11%97%12.7>10,00040%89%14%97%8.9>100,00020%98%33%96%3.4The highest PCR value within 1 year prior to LPD presentation was used for patients with LPD.

EBV DNAemia after Allogeneic Stem Cell Transplantation (SCT): A Prospective Study in 131 High Risk Patients.

We have studied prospectively the incidence of EBV DNAemia and its early treatment in 131 adult allogeneic high risk SCT recipients transplanted during the years 2000 to 2003. Based on our retrospective experience, high risk patients for EBV infection and post-transplant lymphoroliferative disorder (PTLD) were matched unrelated donor (MUD) recipients (of whom a large majority had received anti-thymocyte globulin (ATG) in the conditioning), and patients receiving high dose corticosteroids (HDMP) +/− ATG for the treatment of acute GVHD (aGVHD). 130 patients had a malignant hematological disease, and 1 had aplastic anemia. 23 donors were siblings and 108 unrelated. All donors were A, B, DR matched. 56 grafts were bone marrow and 75 blood stem cells. The grafts were unmanipulated. 101 patients received a standard myeloablative and 30 patients a reduced toxicity conditioning. 101 MUD recipients were given ATG (ThymoglobulineR) as part of conditioning. GVHD prophylaxis consisted of cyclosporine and methotrexate, and 23 patients with a sibling donor received MP in addition. After reduced toxicity conditioning 15 patients received cyclosporine and mycophenolate mophetil. In MUD transplantation EBV was studied weekly for 4 months after SCT and then monthly up to one year after SCT. In sibling SCT EBV was studied from the HDMP treatment of aGVHD on. EBV DNAemia was diagnosed if two consecutive plasma samples had >1000 EBV genome copies/ml detected. The median follow-up of the patients was 12 months, range 2–12. None of the 23 sibling SCT recipients had EBV DNAemia detected during the follow-up despite that 26% of them received also ATG for the treatment of GVHD. Of the 108 MUD recipients 28/108 (26%) had EBV DNAemia detected and 80/108 (74%) remained EBV negative. The activation of EBV was significantly (p<0.001) predicted by the use of high dose (12 mg/kg) ATG during the conditioning treatment but not by the age of the patients, haematological diagnoses, conditioning, the source of the graft, or of the incidence of aGVHD. The median (range) day of the detection of EBV DNAemia was 53 (10–145). The median EBV copy number/ml at detection was 3300 (1200–553700). 20/28 (71%) of the patients had symptoms associated with the DNAemia, 12 fever, 7 enlarged tender lymphnodes, 3 fever and lymphnodes, and 1 had atypical blood lymphocytes. The median day of the first symptoms was 53 (8–102). 21 patients had early EBV treatment started on day 59 (12–139). The decision to initiate treatment was based on the copy number at the onset and its development, and the clinical signs and symptoms. The EBV copy number at treatment was 20150 (1700–2847000). The treatment consisted of Rituximab (R, 375 mg/m2) and decrease of immunosuppression (IS) in 9 patients, R alone in 5, decrease of IS in 7, irradiation of the lymphoma in 4, and donor lymphocyte infusion in 4 patients. The median number of R infusions given per patient was 1.5 (1–4). 12 months after SCT 18/28 (64%) of the patients were alive. Two patients who received EBV-treatment died of PTLD. Other causes of death were 1 rejection, 3 aGVHD, 4 relapses, and 1 epileptic seizure. In conclusion, of the present patients who had received unmanipulated grafts none of the sibling recipients developed EBV DNAemia even after intensive treatment of aGVHD. 26% of the present MUD recipients developed EBV DNAemia but due to the frequent monitoring and early intervention PTLD could in most cases be prevented or cured.

Lytic viral replication as a contributor to the detection of Epstein-Barr virus in breast cancer.

Epstein-Barr virus (EBV) has an accepted association with the epithelial malignancy nasopharyngeal carcinoma and has also been reported in other more controversial carcinoma settings. Evaluation of EBV association with epithelial carcinomas such as breast cancer would benefit from a better understanding of the outcome of EBV infection of these cells. Cell-free preparations of a green fluorescent protein-expressing virus, BX1, were used to infect breast cancer cell lines, which were then examined for EBV gene expression and viral genome copy number. Reverse transcription-PCR analyses revealed that the cells supported a mixture of latency II and lytic EBV gene expression. Lytic Zta and BMRF1 protein expression was detected by immunohistochemistry, and DNA PCR analyses estimated an EBV copy number of 300 to 600 genomes per infected cell. Evidence for lytic EBV expression was also found in breast tissue, where reverse transcription-PCR analyses detected lytic Zta transcripts in 7 of 10 breast carcinoma tissues and 4 of 10 normal tissues from the same patients. Scattered cells immunoreactive for Zta protein were also detectable in breast carcinoma. Quantitative real-time PCR analysis of EBV-positive breast carcinoma tissues suggested that less than 0.1% of the cells contained viral genomes. We suggest that sporadic lytic EBV infection may contribute to PCR-based detection of EBV in traditionally nonvirally associated epithelial malignancies.