Advantages of a New Taq DNA Polymerase in Multiplex PCR and Time-Release PCR

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-2Rα, IL-2/15Rβ, γc-chain, IL-4 and IL-4Rα; IL-10, IL-15 and IL-15Rα; and IL-2, IFNγ, IL-7, IL-7Rα and IL-9Rα). 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

Salvage therapy for relapsed posttransplant lymphoproliferative disorders (PTLD) with a second progression of PTLD after upfront chemotherapy: the role of single-agent rituximab

Currently no standard treatment exists for patients with posttransplant lymphoproliferative disorders relapsed or refractory to chemotherapy after failure of reduction in immunosuppression. We have analyzed the effects of single-agent rituximab treatment in eight patients (seven adult, one pediatric) in this setting. Three patients had been salvaged with rituximab several times. In the seven adults, rituximab salvage therapy achieved complete remission (CR) in three patients (43%) and partial remission in one (14%). In the pediatric patient, a PR was obtained that could be reinduced on relapse with repeated administrations of rituximab. Patients achieving CR either remained in CR or were successfully salvaged again with single-agent rituximab. At a median follow-up of 69 months, median progression-free survival was 9 months and no relevant therapy-associated toxicity was observed. Single-agent rituximab salvage therapy is an effective treatment option in this setting of intensively pretreated patients, with virtually no therapy-associated toxicity