Post-transplant lymphoproliferative diseases (PTLD) are severe complications of immunosuppressive treatment after solid organ transplantation (SOT). Most pediatric cases are associated with Epstein-Barr virus (EBV). Despite morphologic features similar to de novo lymphomas PTLDs are sensitive to immune interventions alone. We conducted a non-randomized prospective multicenter study to test a response-adapted sequential treatment with rituximab +/- chemotherapy in pediatric patients with CD20+ PTLD (Ped-PTLD Pilot 2005). Pediatric patients (<18 years) with biopsy proven CD20+ PTLD without CND involvement were treated with 3 weekly infusions of rituximab 375 mg/m2. If at least a partial response was obtained in week 3 patients received 3 further rituximab administrations every 3 weeks. In case of stable disease or progression patients were stratified to receive a moderate chemotherapy regimen (mCOMP: day 1 vincristine, prednisone, cyclophosphamide, and day 15 methotrexate; repeat every 4 weeks for 6 cycles). Overall (OS) and event-free (EFS) survival at 2 years were the primary endpoints of the trial. Epstein-Barr virus (EBV) in tumor tissue was evaluated by EBER in situ hybridization, immunohistochemistry for LMP-1 or EBNA-2, and/or EBV-PCR. 49 patients (mean age 9.8 [0.8-17.4] years) were included, among them 18 renal, 11 liver graft recipients, and 20 patients after heart or lung transplantation. Median time from SOT to PTLD was 4.7 [0.15 – 15] years (17 early, 32 late PTLDs). Tumor cells contained EBV products in 44 of 49 cases. Histology was polymorphic in 12 patients, diffuse large B cell lymphoma (DLBCL) in 24, Burkitt lymphoma in 7, and other high grade B cell lymphomas in 6 patients upon central review. Median follow-up is 4.5 years. 32 patients (64%) received rituximab only, of which 26 (81% of responders, 53% of total study population) remain alive and in continuous complete remission with a median follow up of 4.9 years. The remaining 6 rituximab responders experienced relapse (n=4), secondary malignancy (n=1), or death unrelated to PTLD (n=1). 15 patients had stable or progressive disease after initial rituximab treatment and proceeded to chemotherapy. Of these, 10 patients (66%) converted to complete remission while 4 patients (27%) progressed and went off study to receive intensive chemotherapy. 1 patient died during mCOMP treatment, which could not be distinguished between tumor progression and infection. 2/49 patients died of PTLD before rituximab response could be evaluated. 2-year OS was 86% +/- 5% with 67% +/- 7% surviving event-free. Estimated 5-year OS and EFS were 83% +/-5% and 67% +/-7%, respectively. 6/7 patients with Burkitt histology required mCOMP chemotherapy. Patients after thoracic organ transplantation had a significantly inferior outcome compared to liver or renal graft recipients (5-years EFS 79%+/-8% vs 44% +/-11%, p=0.01). There was no significant impact of tumor EBV-status, histology, stage or early/late PTLD development. Treatment-related mortality was attributed to bowel perforation (n=1) and infection (n=2). No episode of graft rejection occurred during treatment for PTLD. In conclusion, response-adapted immuno-chemotherapy is an effective and well tolerated treatment regimen for CD20-positive PTLD after SOT in children. Chemotherapy could be spared in half of the PTLD patients, but was necessary in almost all Burkitt PTLD patients. Prognosis was significantly worse in patients after heart or lung transplantation compared to renal or liver allograft recipients. Future studies are envisioned to evaluate whether the inclusion of cellular immunotherapy may contribute to avoid chemotherapy in the majority of patients. Disclosures No relevant conflicts of interest to declare.

Extensive diagnostic and scientific investigations are often restricted by limited availability of material. Therefore, methods like multiplex PCR strategies are needed to conserve as much sample as possible. Unfortunately, the establishment of such procedures poses several difficulties. Here we describe the advantages of a new enzyme, AmpliTaq Gold DNA Polymerase, in multiplex and time-release PCR. The application of this thermostable recombinant Taq DNA polymerase allows the specific amplification of DNA/cDNA targets with very high sensitivity. With our protocol, the specific amplification of 13 different cDNAs of cytokines and cytokine receptors can be realized in three multiplex PCRs (IL-2R alpha, IL-2/15R beta, gamma c-chain, IL-4 and IL-4R alpha; IL-10, IL-15 and IL-15R alpha; and IL-2, IFN gamma, IL-7, IL-7R alpha and IL-9R alpha). The novel application of AmpliTaq Gold DNA Polymerase in a time-release PCR protocol allows specific amplification of target DNA/cDNA when only limited amounts of material are available or only low-copy-number DNA/cDNA is suspected. No IL-9 cDNA can be detected in peripheral blood mononuclear cells (PBMC) in the absence of any stimulation, thus it was difficult to amplify this target with routine PCR protocols. Here we demonstrate the reliable and reproducible amplification of IL-9 cDNA in the Hodgkin's lymphoma cell line KM-H2, in PBMC and in stimulated PBMC. Results with AmpliTaq Gold DNA Polymerase were more sensitive and specific compared with AmpliTaq DNA Polymerase, with and without manual hot-start procedure.

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