Ralf Häfer

Monitoring of Epstein-Barr virus load after hematopoietic stem cell transplantation for early intervention in post-transplant lymphoproliferative disease.

Epstein‐Barr virus (EBV)‐associated post‐transplant lymphoproliferative disease is a life‐threatening complication following hematopoietic stem cell transplantation. A quantitative polymerase chain reaction to evaluate EBV‐genome copy numbers based on a nested polymerase chain reaction and an end‐point dilution was used. Applying this assay EBV load was prospectively screened weekly in 123 patients after transplantation. The results demonstrate that EBV reactivations with more than 1,000 EBV‐genome copies measured in 105 peripheral blood mononuclear cells were observed in 31 patients (25.2%). Three patients developed lymphoproliferative disease with extremely high EBV‐genome copies in peripheral blood mononuclear cells (>100,000 copies/105 cells) and plasma. After combined antiviral and immune therapy two of three patients showed a dramatic decrease of EBV load and survived, while the third patient died of lymphoma. A subclinical EBV reactivation was observed in 24 cases (19.5%) with EBV‐genome copies in 105 peripheral blood mononuclear cells ranging between 2,500 and mostly 10,000. After reduction of immunosuppression the EBV levels normalized. In four patients, the high copy number of ≥80,000 copies/105 peripheral blood mononuclear cells and plasma positivity prompted us to start pre‐emptive therapy with rituximab and cidofovir for prevention of lymphoproliferative disease. After drug administration the high EBV load was reduced remarkably. Ninety‐two patients (74.8%) who had ≤1,000 copies/105 peripheral blood mononuclear cells did not develop EBV‐associated lymphoproliferative disease. In conclusion, monitoring of EBV load is a sensitive and useful parameter in the surveillance of EBV reactivation for early intervention in EBV‐associated lymphoproliferative disease as well as for follow‐up of the efficacy of therapy. J. Med. Virol. 80:441–454, 2008.

Prenatal origin of childhood acute lymphoblastic leukemia, association with birth weight and hyperdiploidy

Recent studies with very small numbers of patients showed that in some cases of childhood acute lymphoblastic leukemia (ALL), preleukemic cells are detectable on Guthrie cards that were used for newborn screening. We present here the largest series of ALL patients (n=32) in whom Guthrie cards were analyzed for the presence of preleukemic cells. Rearranged immunoglobulin heavy-chain genes were used as a marker for leukemic clones. We combined our set of patients with 17 previously published cases. Preleukemic cells were detected in 31 of all 49 patients (63%). Positive screening cards were not associated with patients age at diagnosis but were almost always found in patients with hyperdiploidy (10/11; 91%; P=0.04). High birth weight is an established risk factor for childhood ALL. Positive screening cards were strongly associated with low birth weight (P=0.01). In conclusion, the majority of childhood B-precursor ALL arise prior to birth. In the search for causes of childhood leukemia we should concentrate on prenatal factors as well as postnatal factors. Our results suggest that autologous cord bloods could be a poor choice as the source of stem cells for transplantation in leukemia, which may contain preleukemic cells. Pending the development of suitable methods, childhood leukemia is a potentially screenable disease.

Pre-emptive therapy with rituximab for prevention of Epstein-Barr virus-associated lymphoproliferative disease after hematopoietic stem cell transplantation.

Summary:Epstein–Barr virus (EBV)-associated lymphoproliferative disease (LPD) is a life-threatening complication following hematopoietic stem cell transplantation (HSCT). Therefore, early diagnosis of EBV reactivation and pre-emptive therapy may be clinically useful. We report three patients who presented with an extremely high EBV load in peripheral blood mononuclear cells and plasma without evidence of EBV disease. Following pre-emptive therapy with a single dose of rituximab, a concordant decrease of EBV-genome copies and B lymphocytes was observed. In all three patients, no EBV-associated LPD occurred. We conclude that pre-emptive therapy with rituximab appears to be effective for prevention of EBV-associated LPD after HSCT.

Messenger RNA Analysis of the Multidrug Resistance Related Protein (MRP1) and the Lung Resistance Protein (LRP) in de novo and Relapsed Childhood Acute Lymphoblastic Leukemia

In this study, 86 children (58 initial ALL and 28 children with relapsed disease) were investigated for lung resistance protein (LRP) and multidrug resistance related protein (MRP1)-mRNA expression by semiquantitative RT-PCR. The majority of investigated cases demonstrated variable LRP and MRP1 mRNA expression, when normalized for β -microglobulin expression. LRP and MRP1 mRNA expression may be coordinately regulated, as expression of both transcripts was found to be significantly correlated (p =0.0001). No differences of LRP and MRP expression were observed between initial and relapsed stage patients (LRP: p =0.89 and for MRP: p =0.09 ). The prognostic value of both resistance mechanisms was subjected to Kaplan-Meier analysis for event-free survival. For this analysis the patients were divided into groups with high or low LRP or MRP1 mRNA expression by utilizing the median value as the cut-off point. Overexpression of both resistance mechanisms had no prognostic significance in our retrospective study (log-rank test for LRP: p =0.12 and for MRP1: p =0.95 ), however, patients who showed high LRP expression exhibited a lower tendency of remaining in continuous first remission.

Successful treatment of Epstein-Barr virus-induced transverse myelitis with ganciclovir and cytomegalovirus hyperimmune globulin following unrelated bone marrow transplantation.

We report a patient who developed Epstein–Barr virus (EBV)-induced transverse myelitis 19 months after unrelated bone marrow transplantation (BMT). The disease was diagnosed by physical examination, serologic determinations, EBV-specific polymerase chain reaction in peripheral blood lymphocytes and cerebrospinal fluid, and characteristic magnetic resonance imaging scan of the spine. The patient was treated with ganciclovir and cytomegalovirus (CMV) hyperimmune globulin. He gradually improved and recovered completely within 4 weeks. This case suggests that ganciclovir and CMV hyperimmune globulin appear to be effective for the treatment of EBV-induced transverse myelitis in immunocompromised patients following BMT.

Expression of CD34 and other haematopoietic antigens on neuroblastoma cells: consequences for autologous bone marrow and peripheral blood stem cell transplantation.

Autologous peripheral blood stem cells, obtained by CD34+ stem cell selection, are being used with increasing frequency for transplantation in patients with neuroblastoma. Here, we examined the surface membrane antigens of neuroblastoma cells with a panel of hematopoietic monoclonal antibodies (mAbs), including anti-CD34 mAbs, by flow cytometric analysis. We found stronger binding of anti-CD34 mAbs to clonogenic, less differentiated, non-adherent neuroblastoma cells than to adherent neuroblastoma cells. Moreover, the majority of neuroblastoma cell lines shared hematopoietic-associated antigens with all blood cells. Because of these cross-reactions, especially found with the anti-CD34 mAbs 12.8 and ICH3, we have demonstrated that there is a potential risk of cell harvest contamination by circulating neuroblastoma cells during CD34+ stem cell selection.

Neuroblastoma cells can express the hematopoietic progenitor cell antigen CD34 as detected at surface protein and mRNA level

Recently, we have shown the expression of the hematopoietic precursor cell antigen CD34 on neuroblastoma cells. Here, we present the CD34 expression on 16 permanent neuroblastoma cell lines and primary cell lines at the mRNA level and the flow cytometric results on neuroblastoma cells grown in the same culture and split for flow cytometric analysis and total mRNA extraction. The flow cytometry was performed using a panel of anti-CD34 antibodies covering the epitope classes I to III. In eight neuroblastoma cell lines, CD34 mRNA expression could be detected and corresponded always with the protein surface expression. Alternatively, when CD34 mRNA expression was not seen, CD34 antigen expression ranged from negative to as high as 78%. Based on these results caution should be taken with transplants obtained by CD34+ stem cell selection from neuroblastoma patients.

Cyclosporin A-induced graft-versus-host disease following autologous bone marrow and stem cell transplantation in hematological malignancies of childhood

Cyclosporin A (CsA) can induce graft-versus-host disease (GVHD) following autologous bone marrow transplantation (ABMT) and autologous peripheral blood stem cell transplantation (APBSCT) in adults. We investigated whether GVHD can be induced following ABMT and APBSCT in childhood, and which cells are involved in the pathogenesis of this syndrome. We conducted a prospective study of 20 children and adolescents with hematological malignancies receiving CsA after ABMT and APBSCT. Skin biopsies were obtained on day 21 after transplantation or in the event of a rash. Immunophenotypic analysis of peripheral blood lymphocytes was performed on days 14, 21, 28 and 60 after transplantation. Clinical GVHD of the skin, confirmed by histological criteria, occurred in five patients. Five patients had no clinical GVHD but had acute GVHD alterations on routine skin biopsy. In all 10 patients with a positive skin biopsy for GVHD, CD4+ lymphocytes were the predominant cells in the epidermis. Immunophenotypic analysis of peripheral blood lymphocytes revealed a significantly increased CD4/CD8 ratio in patients with a positive skin biopsy (Pu2009<u20090.01). our findings indicate that it is possible to induce acute gvhd following abmt and apbsct in childhood. in addition, cd4+ lymphocytes play an important role in the pathogenesis of CsA-induced GVHD.

Comparison of different rabbit ATG preparation effects on early lymphocyte subset recovery after allogeneic HSCT and its association with EBV-mediated PTLD.

PurposeRabbit antithymocyte globulin (ATG) is commonly used before allogeneic hematopoietic stem cell transplantation (allo-HSCT) to prevent graft-versus-host disease. Studies comparing the effect of different ATG preparations and dosages on immune reconstitution and risk for Epstein–Barr virus (EBV)-mediated post-transplant lymphoproliferative disorder (PTLD) are rare.MethodsIn this retrospective study, we determined T and B cell subsets by flow cytometry after allo-HSCT in children, who received ATG-Genzyme (ATG-G, nxa0=xa015), ATG-Fresenius (ATG-F, nxa0=xa025) or no-ATG treatment (nxa0=xa019). Additionally, PCR-quantified EBV-genome copy counts were correlated with incidence of PTLD.ResultsWe could confirm a dose-dependent impairment of CD8+ and CD4+ T cell regeneration by ATG-G, including naïve and memory CD4+ T cells. No differences were seen between the currently applied dosages of 5–10xa0mg/kg ATG-G and 20–60xa0mg/kg ATG-F. Significantly delayed T cell subset reconstitution was determined only at high dosages of 20–60xa0mg/kg ATG-G compared to ATG-F. B cell reconstitution was comparably impaired in ATG-G- and ATG-F-treated patients. Although the incidence of EBV reactivation was similar in both ATG groups, EBV copy counts of >104 copies/105 peripheral blood mononuclear cells and the occurrence of PTLD were only found in ATG-G-treated patients.ConclusionsWe conclude that high, but importantly not currently applied low dosages of ATG-G, impair thymic T cell regeneration and memory T cell immunity to a greater extent than ATG-F in pediatric patients. In addition, our results suggest an increased risk for EBV-PTLD when treated with ATG-G. Prospective studies are warranted to compare different ATG preparations with regard to the immune reconstitution and EBV-PTLD.

Treatment of a Patient with Chronic Immune Thrombocytopenic Purpura with Rituximab and Monitoring by Flow Cytometric Analysis

Treatment modalities of patients with chronic immune thrombocytopenic purpura (ITP) include the administration of intravenous immunoglobulins (IVIG), corticosteroids, anti-D(Rh) immunoglobulin (anti-D), and splenectomy. Approximately 25–30% of patients with chronic ITP do not respond to established therapeutic regimens. We describe a 19-year-old patient with chronic ITP refractory to standard therapies treated with rituximab (anti-CD20 antibody). Initially, the therapy with rituximab appeared to be successful; however, the patient relapsed after a surveillance of 57 weeks documenting that the rituximab therapy has failed. Flow cytometric analyses during and after the administration of rituximab revealed new aspects of monitoring rituximab therapy.