SEARCH

SEARCH BY CITATION

Keywords:

  • Epstein–Barr virus;
  • organ transplantation;
  • polymerase chain reaction;
  • transplant lymphoproliferative disorder

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Post-transplant lymphoproliferative disorder (PTLD) is a potentially devastating complication of the immunosuppression used in organ and bone-marrow transplant patients. Between 1 and 15% of transplant patients develop PTLD, with a mortality rate of between 40 and 70% (1–5). When detected in an early state, reduction in immunosuppression is an effective therapy, with response rates as high as 89% in low-risk patients (6). However, in patients with poor prognostic factors, such as significant organ dysfunction, multiorgan tumor involvement and high lactate dehydrogenase (LDH), the chance for response to reduction in immunosuppression is low (6). Chemotherapy is the standard salvage therapy after failure of reduction in immunosuppression, but carries with it significant mortality and morbidity rates in the organ transplant population (7,8). Early identification of patients with PTLD is important in the treatment of this disease, as it allows for early intervention with reduction in immunosuppression, and/or the use of anti-B-cell antibodies, antiviral drugs, or chemotherapy when the patient is best able to tolerate treatment and has the highest chance for response.

Unlike most non-Hodgkin's lymphomas, PTLD is characterized by the presence of transformed lymphocytes of B-cell origin which are frequently infected by the Epstein-Barr virus (EBV) (9,10). Early detection of EBV DNA in peripheral blood leukocytes or plasma using quantitative polymerase chain reaction (PCR) may provide an indirect method of identifying patients at risk for PTLD and to monitor their response to therapy. PCR assays for various EBV targets such as EBNA-1, EBER-1, LMP have been used to identify patients at risk for PTLD, especially in the pediatric and allogeneic bone marrow transplant populations (11–16). These two patient populations are particularly at high risk for PTLD, given the lack of EBV immunity in most pediatric patients and the high level of immunosuppression and the altered immune system seen in T-cell depleted or mismatched allogeneic bone marrow transplants. The utility of this technique in the larger adult solid organ transplant population is less well studied.

We present our experience with qualitative and quantitative EBV PCR in 35 adult solid organ transplant patients evaluated for possible PTLD.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Patients

The series is a retrospective analysis of 35 adult patients at our institution who had previously undergone solid organ transplantation and who were consecutively referred for evaluation for possible PTLD, between February 1999 and March 2001. Data were collected from a review of the patients' medical records. Whenever possible, patients were interviewed and examined at the initial diagnostic evaluation and during follow-up.

Evaluation for PTLD

All patients underwent evaluation of their symptoms and laboratory findings, which prompted the initial PTLD evaluation. Routine studies consisted of computed tomographic (CT) scans of the chest, abdomen and pelvis, EBV PCR, complete blood count and serum chemistries. Tissue biopsy by needle aspirate or excisional biopsy of enlarged lymph nodes or visceral masses was done whenever possible. Bone marrow biopsies, CT scan of the head, and bronchoscopy or gastrointestinal endoscopy were performed when medically indicated.

The diagnosis of PTLD required positive tissue biopsy. Morphologic criteria were used to classify the PTLD subtype according to the criteria of Frizzera and Knowles (17,18). Immunohistochemistry by immunoglobulin staining and flow cytometry were performed whenever feasible. In situ hybridization for EBV EBER-1 transcripts was performed on all tissue samples to evaluate for the presence of EBV in the lymphocytes. PTLD was excluded in patients with no evidence of mass lesions or adenopathy, who were found to have other diagnoses that explained their initial symptoms and findings.

EBV PCR assay

Sample preparation and DNA purification: A total of 5 mL of whole blood was collected in a sterile tube containing ethylenediaminetetraacetic acid (EDTA) as the anticoagulant. The blood was immediately stored at 4 °C and processed within 8 h of collection. Peripheral blood leukocytes were pelleted by centrifugation for 2 min at 1000 × g following a one-step lysis of erythrocytes for 5 min at room temperature in a solution composed of 0.8% (w/v) ammonium chloride, 10 mm potassium bicarbonate, and 0.1 mm EDTA. A total of 4.0 mL of blood was lysed in 60 mL of erythrocyte lysis solution for each specimen. The leukocytes were then washed once in 60 mL of phosphate-buffered saline (PBS), centrifuged again for 2 min at 1000 × g, and resuspended in 2.0 mL of PBS. The cells were counted in a hemacytometer and the concentration was adjusted in PBS to 2.0 × 106 cells/mL. Aliquots of 250 µL each of the cell suspensions were stored at − 70 °C.

DNA was extracted from 200 µL each of patient leukocyte suspensions and positive and negative controls using the QIAamp DNA Blood Mini Kit (QIAGEN Inc., Valencia, CA, USA) as recommended by the manufacturer. Prior to extraction of the DNA, known amounts of either an internal amplification standard (IAS; qualitative assay) or internal calibration standard (ICS; quantitative assay) were added to each control and patient sample. The positive control consisted of linearized plasmid containing partial EBER 1 EBV sequences and carrier DNA, while the negative control was composed of only carrier DNA. Solutions of purified DNA were eluted in 200 µL volumes of sterile, nuclease-free water. The extracted DNA was used immediately for amplification or was stored at − 70 °C until used.

EBV qualitative and quantitative PCR: Commercial qualitative and quantitative PCR-based assays (Qualitative EBV Viral Detect™ and Quantitative EBV Viral Quant™, BioSource International, Inc., Camarillo, CA) were performed according to the manufacturer's instructions for the detection and quantification of EBV-specific amplicons from blood (Figure 1). Briefly, a single primer pair targeting a conserved sequence of EBER 1 was used to efficiently amplify both the EBV and IAS (qualitative assay) or ICS (quantitative assay) DNA targets; one of the primers was biotinylated. The IAS or ICS are synthetic oligonucleotides that have been constructed to contain the same primer binding sites as those for the EBV target and unique capture binding sites that allow the resulting IAS or ICS amplicons to be distinguished from the EBV amplicon. A total of 5 µL of DNA from each patient sample and controls was analyzed by PCR in a total reaction mixture of 100 µL of master mix containing 50 mm KCl, 10 mm Tris-HCl, pH 8.3, 1.5 mm MgCl2, 200 nm dATP, dCTP, dGTP, and dTTP, 2.5 University of AmpliTaq Gold, and 500 nm of each primer. After 1 cycle at 95 °C for 2.5 min, amplification involved a three-step procedure consisting of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min. This was repeated for 40 cycles for the qualitative assay and 34 cycles for the quantitative assay. The final step involved a single cycle at 72 °C for 15 min, followed by cooling at 4 °C. If the PCR products were not used immediately, they were stored at − 70 °C.

image

Figure 1. Schema for qualitative and quantitative EBV PCR assay.

Download figure to PowerPoint

Following PCR amplification, the biotinylated amplicons were hybridized to EBV, IAS, or ICS sequence-specific capture oligonucleotide probes prebound to microtiter wells, and detected by addition of a streptavidin-horse radish peroxidase conjugate followed by a 3,3′,5,5′-tetramethylbenzidine substrate. The reaction was stopped with the addition of acid and the intensity of color developed was read using a spectrophotometer set at 450 nm. To acquire quantitative results over a broad range for the quantitative assay, four serial 5-fold dilutions (1 : 20, 1 : 100, 1 : 500, 1 : 2500) of each sample and control amplicon were made in the EBV and ICS oligonucleotide capture wells prior to detection of the amplified products. For both qualitative and quantitative assays, the optical density (OD) in each well was proportional to the amount of EBV or IAS amplicon present in the original reaction. EBV target OD values < 0.30 and IAS target values > 0.30 indicated successful extraction and amplification and that EBV DNA was not detected. EBV target OD values > 0.30 with IAS target values > 0.30 indicated the presence of EBV DNA in the sample tested. EBV copy number was calculated in the quantitative assay as:

where the Total EBV and Total ICS OD values represent the EBV and ICS microtiter well with the lowest corrected OD450 that was > 0.30 but < 1.50 OD units multiplied by its dilution factor after subtraction of the OD values of an EBV or ICS blank well. Final results of the quantitative assay were expressed in DNA copies/2 × 106 PBL.

All patient specimens were first screened by qualitative EBV PCR and all positive samples were then tested by quantitative EBV PCR. Ten-fold dilutions of purified EBV DNA (Advanced Biotechnologies, Columbia, MD) at 0–108 copies/2 × 106 PBL (peripheral blood lymphocytes) were used to measure the detection limits of both the qualitative and quantitative assays. The lower and upper limits of detection for the qualitative and quantitative EBV PCR assays were determined to be 100 copies/2 × 106 PBL (peripheral blood lymphocytes) to 1 × 106 copies/2 × 106 PBL and 1000 copies/2 × 106 PBL to 1 × 106 copies/2 × 106 PBL, respectively. The primer and probe sequences for the qualitative and quantitative EBV PCR assays are proprietary and the manufacturer has asked that these sequences not be disclosed.

Treatment and monitoring of PTLD

Patients with the diagnosis of PTLD underwent treatment with reduction in immunosuppression, chemotherapy or rituximab (anti-CD 20 monoclonal antibody). Reduction in immunosuppression was specifically tailored for each patient, based on their immunosuppression regimen at diagnosis and organ transplant type. In general, reduction in immunosuppression consisted of deletion of any azathioprine or mycophenolate mofetil and reduction in cyclosporine, tacrolimus and prednisone. Chemotherapy consisted of standard CHOP (cyclophosphamide 750 mg/m2 day 1, vincristine 1.4 mg/m2 day 1, doxorubicin 50 mg/m2 on day 1, and prednisone 100 mg qd for 5 days) chemotherapy given every 3 weeks as is standard for NHL (19). Rituximab was given as 4 weekly intravenous infusions of 375 mg/m2 (20). Restaging CT scans and EBV PCR were performed at various intervals after initiation of treatment.

Complete response was defined as the disappearance of evidence of any active tumor, including all abnormal masses and adenopathy associated with the PTLD and the absence of any new lesions. Partial response was defined as a greater than 50% reduction in the bi-dimensional measurement of all detectable lesions and the absence of any new lesions. Stable disease was defined as less than a 25% change in the size of indicator lesions in the absence of new lesions. Progressive disease was defined as the enlargement of indicator lesions by over 25% or the occurrence of new lesions. Response to treatment, as measured by changes in CT scans, was correlated to EBV PCR.

Data analysis

EBV test results were examined with respect to patient status, survival (dead or alive), response to PTLD treatment, and disease characteristics: multivisceral involvement, organ failure, initial stage of disease, presence of baseline symptoms, LDH levels and age at diagnosis. Fisher's exact test was used for 2-way tables, and the Chi-square approximation of the Kruskal–Wallis test was used for continuous variables. Kappa statistics were calculated to measure agreement between EBV PCR and PTLD evaluations, and confirmed with StatXact-4 statistical software. All other analyses were performed using SAS/Stat Software version 6.12 on the UNIX platform. All statistical tests were performed at the 0.05 significance level.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Patient demographics

Between 1966 and March 2001, 4356 organ transplants [kidney (2769), liver (639), heart (387), lung (263), pancreas/kidney (142), pancreas (13) and allogeneic bone marrow (143)] were performed at the University of Pennsylvania Medical Center. Between February 1999 and March 2001, 35 consecutive adult solid organ transplant patients were evaluated for suspected PTLD and tested with peripheral blood EBV PCR (Table 1). Any abnormal adenopathy or mass lesions were biopsied as part of the PTLD evaluation. Reasons for the initial evaluations included mass/adenopathy 22 (63%), fever 8 (23%), abnormal liver function tests 2 (6%), pancytopenia 1(3%), positive monospot 1 (3%), and abnormal allograft biopsy 1 (3%). Seventeen evaluated patients were found to have diagnoses other than PTLD including bacterial infection 5 (14%), hepatitis 3 (9%), undefined viral syndrome 3 (9%), other malignancy 2 (6%), cytomegalovirus 2 (6%), allograft rejection 1 (3%), and fungal infection 1 (3%). Eighteen (51%) patients were ultimately diagnosed as having PTLD by tissue biopsy. At presentation, the majority of PTLD patients were characterized as having advanced stage (61%), extranodal disease (78%), high LDH (61%), B-cell histology (100%), and EBV positive tumor (83%) (Table 2).

Table 1. : Patient characteristics at initial evaluation
CharacteristicAll patients (n = 35)PTLD patients (n = 18)
Age (years)
 Median5252
 Range26–6726–65
Gender
 Male28 (80%)14 (78%)
 Female 7 (20%) 4 (22%)
Race
 Caucasian31 (88%)15 (83%)
 African-American 3 (9%) 3 (17%)
 Asian 1 (3%) 0 (0%)
Organ transplant type and rationale
 Lung13 (37%) 7 (39%)
  COPD 6 2
  Cystic fibrosis 3 2
  Pulmonary hypertension 1 1
  Idiopathic bronchiectasis 1 1
  Lymphangioleiomyomatosis 1 1
  Idiopathic pulmonary fibrosis 1 0
 Heart 6 (17%) 2 (11%)
  Coronary artery disease 5 2
  Cardiomyopathy 1 0
 Liver10 (29%) 4 (22%)
  Hepatitis 7 3
  Idiopathic cirrhosis 3 1
 Kidney 5 (14%) 1 (22%)
  Glomerulonephritis 1 1
  Glomerulosclerosis 1 1
  Systemic lupus erythematosus 11
  Sarcoid 1 1
  Polycystic kidney disease 1 0
 Kidney/pancreas 1 (3%) 1 (6%)
  Diabetes mellitus 1 1
Table 2. : PTLD presentation
Characteristics of PTLD presentationPTLD patients
(n = 18)
Time from transplantation to PTLD (months)
 Median43
 Range 2–176
Histology
 Polymorphic B cell hyperplasia 4
 Polymorphic B cell lymphoma 3
 Monomorphic B cell lymphoma (NHL)11
Disease stage
 I 3 (17%)
 II 4 (22%)
 III 1 (6%)
 IV10 (55%)
Multiorgan involvement
 Yes 2 (11%)
 No16 (89%)
Disease sites
 Extranodal14 (78%)
 Allograft 5 (28%)
 Lymph node10 (56%)
 Lung 6 (33%)
 GI tract 4 (22%)
 Skin 3 (17%)
 Oral pharynx 2 (11%)
 Pancreas 2 (11%)
 Spleen 1 (6%)
 Liver 1 (6%)
 Adrenal gland 1 (6%)
 Kidney 0 (0%)
 CNS 0 (0%)
 Bone marrow (n = 3) 0 (0%)
Number of B symptoms: (fever, night sweats & weight loss)
 011 (61%)
 1 + 7 (39%)
Elevated LDH
 Yes11 (61%)
 No 6 (33%)
 Unknown 1 (6%)
Organ failure
 Yes 1 (6%)
 No17 (94%)
EBV status of PTLD
 Positive15 (83%)
 Negative 3 (17%)

EBV PCR results as part of PTLD evaluation

Seventeen of 35 patients in this series were found not to have PTLD and the qualitative EBV PCR at initial evaluation was negative (less than 100 copies/2 × 106 PBL) in all cases, suggesting a specificity of 100%. Eighteen patients were diagnosed with PTLD by histologic evaluation of tissue biopsy. In seven PTLD patients (Figure 2), the EBV was positive with viral loads greater than 1000 copies/2 × 106 PBL, suggesting a sensitivity of 39% (Table 3). The value of Cohen's Kappa correlation coefficient was 0.38 (95% CI 0.14, 0.62), which indicates a fair amount of agreement. The exact 2-sided p-value was 0.0078. In no case did a patient have a positive EBV PCR (load greater than 1000 copies/2 × 106 PBL) at initial evaluation and not have PTLD (Table 4A). In our experience, EBV loads of greater than 1000 copies/2 × 106 PBL are suggestive of PTLD, while a negative EBV PCR does not rule out its presence.

image

Figure 2. EBV peripheral blood loads for 7 PTLD patients with positive EBV PCR. Each point represents a positive EBV load value.

Download figure to PowerPoint

Table 3. : Characteristics of patients with PTLD
Patient no.Age/ sexAllograft typeMonths post transplantPTLD typeEBV tumor statusInitial EBV PCRHighest EBV PCRTreatmentsOutcomeFollow-up (wks)
  1. The clinical features of 35 patients with PTLD evaluations are summarized above. Patients are arranged by outcome. Months Post transplant is the time from organ transplantation to diagnosis of PTLD or relapse. EBV PCR is listed as DNA Copies/mL whole blood. Treatments are arranged in the order that they were given in order to obtain the outcome noted. Patient no. 2 was treated with RI and CHOP concurrently.

  2. Follow-up is the time from diagnosis of PTLD to most recent clinic visit. Abbreviations: CR, complete response; PR, partial response; PD, progressive disease; RI, reduction in immunosuppression; CHOP, Cyclophosphamide/Vincristine/Doxorubicin/Prednisone.

  3. Chemotherapy; NHL, non-Hodgkin's lymphoma; PMBCH, polymorphous B cell hyperplasia; PMBCL, polymorphous B cell lymphoma.

 159/MLiver  7NHLPositive  < 100    3950RICR 86
 266/MHeart 56NHLPositive11 576   11 576RI + CHOPCR 19
 352/MKidney176NHLPositive  7226    8119RICR 48
 440/MKidney 47PMBCHPositive29 259   29 259RICR 68
 555/FLung 47NHLPositive11 791   11 791RICR 58
 652/FLung 32PMBCHPositive51 1181132 653RI-RituximabCR132
 750/FKidney  7NHLPositive  < 100    < 100RICR 24
 841/MLung 72NHLPositive  < 100    < 100RI-CHOPCR 25
 927/MKidney114NHLNegative  < 100    < 100RICR 56
1048/MKidney170PMBCLNegative  < 100    < 100RICR 64
1164/FLung 30PMBCHPositive  7882    7882RIPR  5
1254/MLung 95PMBCLPositive  9005   15 827RI-RituximabPR 37
1326/MLung  2PMBCHPositive  < 100    < 100RIPR 67
1460/MLiver 40NHLPositive  < 100    < 100RI-RituximabPR 56
1536/MLung 10PMBCLPositive  < 100    < 100RIPR 40
1662/MHeart 49NHLNegative  < 100    < 100RI-RituximabPD 13
1764/MLiver 24NHLPositive  < 100    < 100RIPD 12
1849/MLiver  3NHLPositive  < 100    < 100RI-RituximabPD 15
Table 4A. : Correlation between EBV PCR results and diagnosis of PTLD
  Diagnosis of any PTLD YesNo 
EBV PCR+ 7 0 7
Results111728
181735

Correlation between EBER-1 in situ hybridization and EBV PCR assay

Fifteen (83%) of 18 patients with PTLD had EBER-1 positive tumors by in situ hybridization (Table 4B). All three (17%) EBER-1 negative PTLD patients had EBV loads of less than 100 copies/2 × 106 PBL. Seven of 15 patients with EBER-1 positive tumors had positive initial EBV loads greater than 1000 copies/2 × 106 PBL (range 7226–29 259), giving a sensitivity of 47%. Cohen's Kappa correlation coefficient was 0.50 (95% CI 0.24, 0.76), demonstrating moderate amount of agreement. The exact 2-sided p-value was 0.0010. Five patients with EBER-1 positive tumors but negative initial EBV loads underwent a second confirmatory EBV PCR a median of 14 days after the first test. All 5 second EBV PCR tests remained negative despite all patients still having persistent disease at the time of repeat study. A negative EBV PCR does not rule out the possibility of having PTLD, and the tumor may be EBV negative or positive. A positive EBV PCR is suggestive of the presence of EBER-1 positive PTLD.

Table 4B. : Correlation between EBER-1 in situ hybridization and EBV PCR assay
  Diagnosis of EBER-1 + PTLD YesNo 
EBV PCR+ 7 0 7
Results 82028
152035

Reduction in immunosuppression and EBV PCR

All seven patients with positive EBV PCR initially underwent a reduction in immunosuppression (Table 3). Two patients (nos 6 and 12) developed progressive disease within 4 weeks after reduction in immunosuppression. In these cases (nos 6 and 12), EBV loads increased accordingly during that time period from 51 118 and 9005 copies/2 × 106 PBL to 1132 653 and 15 827 copies/2 × 106 PBL, respectively. In four patients (nos 3, 4, 5, and 11) who responded to reduction in immunosuppression, the EBV loads fell from 7226, 29 259, 11 791, and 7882 copies/2 × 106 PBL to zero by 45, 29, 27, and 27 days from the initial reduction in immunosuppressive medications, respectively (Figure 3). EBV load decreases as patients clinically respond to reduction in immunosuppression and increases in patients with progressive PTLD.

image

Figure 3. Results in 4 patients treated with reduction in immunosuppression. Week 0 represents EBV PCR done within 3 weeks of initiation of reduction in immunosuppression. Radiographic and physical exam assessments of disease status are indicated: Complete Response (CR) and Partial Response (PR).

Download figure to PowerPoint

Chemotherapy and EBV PCR

One patient (no. 2) with a positive EBV load was treated with CHOP chemotherapy concurrently with reduction in immunosuppression (Table 3). His EBV load went from 11 791 copies/2 × 106 PBL to zero copies/2 × 106 PBL 9 days after initiation of chemotherapy. The patient's adenopathy also decreased and he was found to have achieved a complete response after 4 cycles of chemotherapy. He has received a total of 6 cycles of CHOP with RI, and remained in a complete response 25 weeks after initial diagnosis of PTLD.

Rituximab therapy and EBV PCR

Five patients with CD20 positive PTLD were treated with rituximab after failing primary therapy with reduction in immunosuppression (Table 3). Two patients (nos 14 and 18) had EBER-1 positive tumors and negative EBV PCR. One patient (no. 16) had EBER-1 negative tumor and negative qualitative EBV PCR. Two patients (nos 6 (Figure 4), and 12) had EBER-1 positive tumors and positive initial qualitative EBV PCR with initial values of 51 118 and 9005 copies/2 × 106 PBL. After failing reduction in immunosuppression, the two EBV PCR positive patients developed increased EBV load, with values increasing to 1132 653 and 15 827 copies/2 × 106 PBL, respectively. With initiation of rituximab, both had a significant decrease in EBV copy number in their peripheral blood, as indicated by quantitative EBV PCR. By day 22 and 76, respectively, EBV load was zero in both patients. This pattern corresponded with the patients' clinical status, and both showed significant shrinkage in their tumors though neither had achieved a complete response at that point. Peripheral blood EBV loads as assessed by EBV PCR appeared to correspond with clinical response to rituximab and may suggest a therapeutic response prior to radiographic remission.

image

Figure 4. EBV PCR results in patient no. 6. PTLD treatments including reduction in immunosuppression and rituximab therapy are noted. Radiographic response at time points are indicated: Complete Response (CR), Partial Response (PR) and Progressive Disease (PD).

Download figure to PowerPoint

EBV PCR and PTLD prognosis

Ultimate clinical outcomes were analyzed with respect to EBV loads in 15 patients with EBV positive tumors. For seven patients with EBV load greater than 1000 copies/2 × 106 PBL, there were 5 complete responses and 2 partial responses (Table 3). In patients with EBV load less than 100 copies/2 × 106 PBL, there were 3 complete responses, 3 partial responses and 2 progressive diseases. There was no statistically significant difference between these two groups (p = 0. 36) though the total sample size of 15 was small. Additional analysis correlating EBV load to other prognostic variables such as age and LDH was not significant. In both EBV load positive and negative groups, patients were able to achieve complete responses to reduction in immunosuppression, chemotherapy or rituximab.

EBV PCR and increases in immunosuppression

Patient no. 4 developed an episode of renal allograft rejection while on reduced immunosuppression. He was treated with pulse steroids and an increase in his cyclosporine (Figure 5). His PTLD was in complete remission at the time of this episode. Sixteen days after treatment for rejection his EBV load was found to be 6857 copies/2 × 106 PBL. Upon follow-up on day 30 the EBV load was less than 1000 copies/2 × 106 PBL and has remained negative since that time. During this period of time there was no radiologic evidence of recurrent PTLD. EBV loads are sensitive to significant increases in immunosuppression associated with the treatment of allograft rejection, but do not necessarily indicate PTLD recurrence.

image

Figure 5. EBV PCR results in patient no. 4. PTLD treatments including reduction in immunosuppression are noted. Radiographic and physical exam assessments of disease status are indicated: Complete Response (CR) and Partial Response (PR).

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Advances in immunosuppression and surgical techniques have made organ transplantation a safer and more routine procedure than in the past. However, as a result of more aggressive immunosuppression and the larger number of transplants being performed, PTLD has become an increasingly common occurrence. Unlike most cancers, PTLD occurs almost exclusively in a relatively small, well-defined population of pharmacologically immunosuppressed organ transplant patients. In addition, the peak time for PTLD occurrence is within the first year after organ transplantation (5,6). In a closely monitored, small patient population, this provides an ideal situation for performing cancer screening with a noninvasive test such as EBV PCR. Unlike most cancers, PTLD is in most cases virally driven, with EBV being present within tumor cells of almost all patients with PTLD. Many of these patients would be expected to shed live virus or harbor infected lymphocytes in their blood, providing a unique tumor marker that might be followed noninvasively. There have been a number of reports using EBV PCR to study patients with PTLD or at risk for PTLD, mostly in the setting of pediatric solid organ transplant patients or adult bone marrow transplant patients, both high-risk populations for developing this disease (16,21,22). Our series aimed to evaluate this new technology in the larger population of adult solid-organ transplant patients.

PTLD is a spectrum of lymphoproliferative disorders that range from hyperplastic growths to non-Hodgkin's lymphoma. It often presents with symptoms such as fatigue, malaise, fever, weight loss, masses, lymphadenopathy, and organ dysfunction that may easily mimic more common etiologies such as infection or allograft rejection. Evaluation for these other etiologies often consumes considerable health-care resources and delays the correct diagnosis of PTLD, resulting in more extensive PTLD by the time of diagnosis. Currently, the definitive method of PTLD diagnosis is through tissue biopsy. Unfortunately, the enlarged lymph nodes and mass lesions are often not easily accessible without invasive operative procedures. EBV PCR has been shown in other series to be useful in identifying patients who are at high risk for developing PTLD. In series of pediatric liver and bone-marrow transplant patients, EBV PCR may identify patients at risk for PTLD, so that early interventions such as reduction in immunosuppression or EBV cytotoxic T cells may be employed (16,22–24). Other small series have shown that EBV load increases in adult solid organ transplant patients with PTLD (11,25).

In our study, we concur that EBV PCR may be an effective and noninvasive way of rapidly and effectively identifying patients with EBER-1 positive PTLD. All seven patients who presented with a positive EBV load of > 1000 copies/2 × 106 PBL were ultimately found to have PTLD. Unfortunately, this assay did not diagnose patients with EBV negative PTLD and was negative in approximately half the patients with EBER-1 positive PTLD in our series. The low sensitivity of the assay makes it unsuitable as a screening test, but its high specificity makes it potentially useful as a definitive diagnostic test for PTLD. It is unclear why our assay did not identify all patients with known EBV positive PTLD. In our series, our EBV PCR assay did not allow for accurate quantitation of EBV loads below 1000 copies/2 × 106 PBL. While it is not clear what the optimal EBV load cut-off should be, other authors have suggested EBV load cut-offs for PTLD similar to ours (24). By lowering the EBV load standard for determining a positive test we might have identified more EBV PCR positive patients, but at the expense of false positive results for patients without PTLD.

Our finding that patients with EBV-positive PTLD may have either positive or negative EBV PCR likely reflects the heterogeneous nature of this disease. The possible reasons for this finding are many. It is ultimately related to variability in the level of infected lymphocytes in the peripheral blood of patients with PTLD. Some tumors may be more prone to releasing infected lymphocytes than others. Alternatively, EBV may present as a lytic infection with active viral replication and shedding vs. a quiescent latent infection with little viral replication or shedding. Finally the host immune response may be able to control and limit peripheral blood EBV infection in some cases but not others. PTLD represents a wide spectrum of lymphoproliferative disorders that reflect the different manners in which EBV may infect B cells in combination with random mutations to the B cell's DNA, all in the setting of a given patient's immune system and immunosuppression. With this varied presentation and immune response, it would not be surprising that some tumors are more likely to release virus or circulating infected B cells than others, or that some patients' immune systems may be able to clear circulating virus or infected cells.

EBER-1 was the target for both our peripheral blood PCR and our in situ hybridization of the tumor. The function of this RNA is unknown and it is expressed in both lytic and latent infection, and hence does not differentiate between the two. We examined patient outcome with EBV viral load in the hope of correlating between EBV load and outcome. There seemed to be more patients with progressive disease and fewer complete responses in patients with a negative EBV PCR (Table 3). Statistical analysis in this very small sample was unrevealing, however. How these two types of EBV infections and their relative importance affect the prognosis of PTLD is unclear, and further work in this area is warranted. Despite these issues, our series still suggests that the presence of a positive EBV PCR in the setting of adult solid organ transplant patient, especially those with mass or adenopathy, is indicative of PTLD. In this setting, EBV PCR would be a reasonable test to employ as part of the diagnostic evaluation for PTLD.

Tumor markers are frequently used in oncology to follow the clinical course of patients as they undergo treatment and afterwards to monitor for relapse. In essence, tumor markers represent materials shed into the blood by active tumor and provide an indirect measure of tumor bulk. PTLD is unique in that the released marker is EBV itself. In our series there seemed to be a direct correlation between the patient's clinical course and the EBV load. Patients with progressive disease showed increased EBV loads and those with disease responding to therapy developed undetectable EBV loads. In addition, the EBV load often decreased prior to complete radiographic resolution of the tumor, suggesting the possible role of EBV load as a predictive and correlative test in the clinical setting. This finding is in contrast to a report that EBV load in patients treated with rituximab (anti-CD20 monoclonal antibody) did not correlate well with response (26). A possible explanation may be that therapy with rituximab may cause depletion of peripherally circulating B cells, which harbor EBV, thus causing a therapy-induced EBV false negative test result. This may occur in some patients, but we did not see this in our series. Otherwise, our results confirm the findings of other small series that EBV load correlates with disease response. Further study in this area should be done to fully evaluate the utility of EBV PCR in the monitoring of PTLD.

Our study has a number of limitations that must be recognized. First, the number of patients studied with the diagnosis of PTLD was small. This limited our ability to detect statistically significant associations. Secondly, the patients in this series, while consecutive, were studied retrospectively in an uncontrolled manner. The specimens were initially obtained for routine clinical purposes, and thus the timing and number of the specimens in relationship to PTLD diagnosis were irregular, making direct comparisons from patient to patient difficult. Despite these limitations, our series suggests a role for EBV PCR in the diagnosis and monitoring of PTLD in adult solid organ transplant patients.

We find that EBV PCR may be a useful tool in the diagnosis of EBV positive PTLD and in monitoring the therapeutic response during reduction in immunosuppression, rituximab therapy or chemotherapy. Alternative methods of EBV evaluation or other serologic tests should be developed to evaluate patients with EBV negative PTLD and those EBV positive patients who do not shed large amounts of EBV into the peripheral blood. We recommend that EBV PCR be performed on all adult solid organ transplant patients as part of their evaluation for PTLD. In those found to have EBV positive PTLD with positive EBV PCR, serial EBV PCR tests should be done to monitor their clinical course. Larger multicenter studies to evaluate the true potential of EBV PCR for the diagnosis and monitoring of PTLD are warranted.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

This publication was supported in part by a Young Investigator Award from the American Society of Clinical Oncology (DT). We like to thank Toby Laiken for her assistance in this study.

Presented in part at the Annual Meeting of the American Society of Clinical Oncology, 2001 in San Francisco, California.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • 1
    Benkerrou M, Durandy A, Fischer A. Therapy for transplant-related lymphoproliferative diseases. Hematol Oncol Clin North Am 1993; 7: 467475.
  • 2
    Bhatia S, Ramsay NK, Steinbuch M et al. Malignant neoplasms following bone marrow transplantation. Blood 1996; 87: 36333639.
  • 3
    Zutter MM, Martin PJ, Sale GE et al. Epstein-Barr virus lymphoproliferation after bone marrow transplantation. Blood 1988; 72: 520529.
  • 4
    Shapiro RS, McClain K, Frizzera G et al. Epstein-Barr virus associated B cell lymphoproliferative disorders following bone marrow transplantation. Blood 1988; 71: 12341243.
  • 5
    Armitage JM, Kormos RL, Stuart RS et al. Posttransplant lymphoproliferative disease in thoracic organ transplant patients. ten years of cyclosporine-based immunosuppression. J Heart Lung Transplant 1991; 10: 877886. Discussion 886–877.
  • 6
    Tsai DE, Hardy CL, Tomaszewski JE et al. Reduction in immunosuppression as initial therapy for posttransplant lymphoproliferative disorder. analysis of prognostic variables and long- term follow-up of 42 adult patients. Transplantation 2001; 71: 10761088.
  • 7
    Swinnen LJ, Mullen GM, Carr TJ, Costanzo MR, Fisher RI. Aggressive treatment for postcardiac transplant lymphoproliferation. Blood 1995; 86: 33333340.
  • 8
    Mamzer-Bruneel MF, Lome C, Morelon E et al. Durable remission after aggressive chemotherapy for very late post-kidney transplant lymphoproliferation. A report of 16 cases observed in a single center. J Clin Oncol 2000; 18: 36223632.
  • 9
    Starzl TE, Nalesnik MA, Porter KA et al. Reversibility of lymphomas and lymphoproliferative lesions developing under cyclosporin-steroid therapy. Lancet 1984; i: 583587.
  • 10
    Nalesnik MA, Makowka L, Starzl TE. The diagnosis and treatment of posttransplant lymphoproliferative disorders. Curr Probl Surg 1988; 25: 367472.
  • 11
    Riddler SA, Breinig MC, McKnight JL. Increased levels of circulating Epstein-Barr virus (EBV)-infected lymphocytes and decreased EBV nuclear antigen antibody responses are associated with the development of posttransplant lymphoproliferative disease in solid-organ transplant recipients. Blood 1994; 84: 972984.
  • 12
    Savoie A, Perpete C, Carpentier L, Joncas J, Alfieri C. Direct correlation between the load of Epstein-Barr virus-infected lymphocytes in the peripheral blood of pediatric transplant patients and risk of lymphoproliferative disease. Blood 1994; 83: 27152722.
  • 13
    Rooney CM, Smith CA, Ng CY et al. Use of gene-modified virus-specific T lymphocytes to control Epstein- Barr-virus-related lymphoproliferation. Lancet 1995; 345: 913.
  • 14
    Kenagy DN, Schlesinger Y, Weck K, Ritter JH, Gaudreault-Keener MM, Storch GA. Epstein-Barr virus DNA in peripheral blood leukocytes of patients with posttransplant lymphoproliferative disease. Transplantation 1995; 60: 547554.
  • 15
    Lucas KG, Burton RL, Zimmerman SE et al. Semiquantitative Epstein-Barr virus (EBV) polymerase chain reaction for the determination of patients at risk for EBV-induced lymphoproliferative disease after stem cell transplantation. Blood 1998; 91: 36543661.
  • 16
    McDiarmid SV, Jordan S, Kim GS et al. Prevention and preemptive therapy of posttransplant lymphoproliferative disease in pediatric liver recipients. Transplantation 1998; 66: 16041611.
  • 17
    Knowles DM, Cesarman E, Chadburn A et al. Correlative morphologic and molecular genetic analysis demonstrates three distinct categories of posttransplantation lymphoproliferative disorders. Blood 1995; 85: 552565.
  • 18
    Frizzera G, Hanto DW, Gajl-Peczalska KJ et al. Polymorphic diffuse B-cell hyperplasias and lymphomas in renal transplant recipients. Cancer Res 1981; 41: 42624279.
  • 19
    McKelvey EM, Gottlieb JA, Wilson HE et al. Hydroxyldaunomycin (adriamycin) combination chemotherapy in malignant lymphoma. Cancer 1976; 38: 14841493.
  • 20
    McLaughlin P, Grillo-Lopez AJ, Link BK. Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: half of patients respond to a four-dose treatment program. J Clin Oncol 1998; 16: 28252833.
  • 21
    Rooney CM, Loftin SK, Holladay MS, Brenner MK, Krance RA, Heslop HE. Early identification of Epstein-Barr virus-associated post- transplantation lymphoproliferative disease. Br J Haematol 1995; 89: 98103.
  • 22
    Green M, Bueno J, Rowe D et al. Predictive negative value of persistent low Epstein-Barr virus viral load after intestinal transplantation in children. Transplantation 2000; 70: 593596.
  • 23
    Rooney CM, Smith CA, Ng CY et al. Infusion of cytotoxic T cells for the prevention and treatment of Epstein-Barr virus-induced lymphoma in allogeneic transplant recipients. Blood 1998; 92: 15491555.
  • 24
    Rowe DT, Qu J, Reynes N et al. Use of quantitative competitive PCR to measure Epstein-Barr virus genome load in the peripheral blood of pediatric transplant patients with lymphoproliferative disorders. J Clin Microbiol 1997; 35: 16121615.
  • 25
    Bladanti F, Grossi P, Furione M et al. High Levels of Epstein-Barr virus DNA in blood of solid-organ transplant recipients and their value in predicting posttransplant lymphoproliferative disorders. J Clin Microbiol 2000; 38: 613619.
  • 26
    Yang J, Tao Q, Flinn IW et al. Characterization of Epstein-Barr virus-infected B cells in patients with posttransplantation lymphoproliferative disease: disappearance after rituximab therapy does not predict clinical response. Blood 2000; 96: 40554063.