Leo M. Budel

Molecular quantification of viral load in plasma allows for fast and accurate prediction of response to therapy of Epstein-Barr virus-associated lymphoproliferative disease after allogeneic stem cell transplantation

Epstein‐Barr virus lymphoproliferative disease (EBV‐LPD) following allogeneic stem cell transplantation (allo‐SCT) has a poor prognosis. We used a sensitive real‐time polymerase chain reaction (PCR) assay for quantitative detection of EBV‐DNA in plasma and serially measured EBV‐DNA levels to assess the response to treatment in allo‐SCT recipients with EBV‐LPD. Fourteen allo‐SCT recipients with EBV‐LPD who received a T cell‐depleted (TCD) sibling (n = 5) or matched unrelated donor (n = 9) graft were monitored from the time of EBV‐LPD diagnosis, during therapy and assessment of clinical response. Seven patients had complete responses of EBV‐LPD to therapy, of whom 21% (3 out of 14) survived beyond 6 months from EBV‐LPD diagnosis. Clinically responding patients showed a rapid decline of EBV‐DNA plasma levels within 72 h from the start of therapy. In contrast, all clinical non‐responders showed an increase of EBV‐DNA levels. Absolute EBV‐DNA levels at the time of EBV‐LPD diagnosis did not predict for response, but the pattern of EBV‐DNA levels within 72 h from the start of therapy (> 50% decrease versus increase) strongly predicted for clinical response (P = 0·001). Quantitative monitoring of EBV‐DNA levels from the start of and during therapy for EBV‐LPD rapidly and accurately predicts for response to therapy as early as within 72 h. It may thus provide a powerful tool to adjust and select treatment in individuals with EBV‐LPD following allo‐SCT.

Tumor necrosis factor downregulates granulocyte-colony-stimulating factor receptor expression on human acute myeloid leukemia cells and granulocytes.

Tumor necrosis factor (TNF) inhibits granulocyte-colony-stimulating factor (G-CSF)-induced human acute myeloid leukemia (AML) growth in vitro. Incubation of blasts from three patients with AML in serum-free medium with TNF (10(3) U/ml), and subsequent binding studies using 125I-G-CSF reveal that TNF downregulates the numbers of G-CSF receptors by approximately 70%. G-CSF receptor numbers on purified blood granulocytes are also downmodulated by TNF. Downregulation of G-CSF receptor expression becomes evident within 10 min after incubation of the cells with TNF at 37 degrees C and is not associated with an apparent change of the dissociation constant (Kd). The TNF effect does not occur at 0 degrees C and cannot be induced by IL-2, IL-6, or GM-CSF. TNF probably exerts its effect through activation of protein kinase C (PKC) as the TNF effect on G-CSF receptor levels can be mimicked by 12-O-tetradecanoylphorbol-13- acetate. The PKC inhibitor Staurosporine (Sigma Chemical Co., St. Louis, MO) as well as protease inhibitors can completely prevent G-CSF receptor downmodulation. Thus, it appears TNF may act as a regulator of G-CSF receptor expression in myeloid cells and shut off G-CSF dependent hematopoiesis. The regulatory ability of TNF may explain the antagonism between TNF and G-CSF stimulation.

Prognostic relevance of immunohistochemical subclassification of diffuse large B-cell lymphoma in two prospective phase III clinical trials.

PURPOSE Until now molecular biologic techniques have not been easily used in daily clinical practice to stratify patients for therapeutic purposes. Therefore, we have investigated the prognostic relevance of the immunohistochemical (IHC) germinal center B-cell (GCB) versus non-GCB diffuse large B-cell lymphoma (DLBCL) subtypes. PATIENTS AND METHODS We have analyzed tumor samples from patients treated in 2 prospective multicenter phase III trials, ie HOVON 25 (patients≥65 years, n=153) and HOVON 26 (patients<65 years, n=144) using whole sections (WS) or tissue microarray (TMA). CD10, BCL6, and MUM1 were applied in a specific IHC algorithm. The effect on clinical outcome using WS or TMA and variations in cut-off levels of these markers was also investigated. RESULTS The GCB subtype was not associated with a better OS in either trial. Small differences were observed in the HOVON 25 trial between techniques, with TMA showing a better outcome for GCB than did WS. Variation of cut-off levels in the specific algorithm did not improve the prediction of clinical outcome. CONCLUSION We did not observe a consistent predictive power of the GCB and non-GCB classification by IHC in this large series of DLBCL patients treated with CHOP. This underscores the need to determine the biologic variation and the standardization of the protein expression levels and to further study the relevance of prognostic IHC classifications, preferably in phase III clinical trials.

The expression of the peripheral cannabinoid receptor on cells of the immune system and non-Hodgkin's lymphomas.

The peripheral cannabinoid receptor CB2 is expressed highly on normal human B-lymphocytes. C-terminal specific anti-CB2 antibody recognises a non-phosphorylated inactive receptor on naïve and resting B-lymphocytes. Another, N-terminal specific CB2 antibody, primarily recognises B-cells present in the germinal centres of secondary follicles in lymph nodes. We hypothesise that N-terminal specific CB2 antibody recognises activated CB2 receptors. In this study, we showed using these antibodies, that expression of CB2 is generally absent on T-lymphocytes in reactive, non-malignant human lymphoid tissues. Applying single and dual immunohistochemistry, CD23+ follicular dendritic cells and a small but significant subpopulation of CD68+ macrophages showed positive staining with the N-terminal specific CB2 antibody but not with the C-terminal specific CB2 antibody. This may indicate the presence of an active CB2 receptor on these cells with possible involvement in immunomodulation. In contrast to the low expression on normal T-cells, abundant levels of CB2 protein were present on T-non-Hodgkins lymphomas (NHL). Moreover, in many B-NHL, high CB2 protein expression was found as well. In contrast to the distinct expression patterns in normal immune tissues using the two different CB2 antibodies, NHL specimens in general stained positively with both. We conclude that CB2 receptor expression pattern may be abnormal in NHL.

The expression of the peripheral cannabinoid receptor CB2 has no effect on clinical outcome in diffuse large B-cell lymphomas.

Background:  The peripheral cannabinoid receptor (CB2) is mainly detected on B cells in the germinal centers (GCs) of the immune system, using an antibody directed against the extra cellular N‐terminal domain of the receptor. We retrospectively investigated the CB2 receptor expression in diffuse large B‐cell lymphomas (DLBCL) and its clinical relevance for treatment outcome.