Ulrich Jäger

CD20 antibody (C2B8)-induced apoptosis of lymphoma cells promotes phagocytosis by dendritic cells and cross-priming of CD8+ cytotoxic T cells.

C2B8 (Rituximab, MabThera) is a chimeric mouse/human monoclonal antibody (mAb) directed against the human B cell-restricted cell surface antigen CD20 which is used as an alternative medication in the treatment of B cell non-Hodgkin lymphomas (NHL). Treatment of CD20+ B cells with C2B8 triggers different cell damaging effects including complement-dependent lysis of tumor cells, antibody-dependent cellular cytotoxicity and induction of apoptosis. Dendritic cells (DC) have recently been shown to ingest cell debris and to present associated antigens even on MHC class I molecules, a mechanism called cross-presentation. In this study, we investigated whether C2B8 treatment of lymphoma promotes the induction of CD8+ T cell responses against lymphoma cell-associated antigens via cross-presentation. We used Daudi lymphoma cells as a model system in our studies and could demonstrate, that C2B8-treated Daudi cells undergo apoptosis, are phagocytosed by DC and induce in DC typical features of maturation; among them, the induction of CD83 expression as well as the up-regulation of prominent accessory molecules (CD40, CD86) and MHC molecules. Importantly, upon co-culture of such lymphoma cell-pulsed DC with autologous T cells, we could induce efficient cytotoxic T cell (CTL) responses against Daudi cell-associated antigens. These findings suggest that antibody treatment of tumor cells can, in addition to its direct cell damaging effects, under certain conditions, contribute to an induction of potentially protective cytotoxic T cell responses.

18F-Fluorodeoxyglucose Positron Emission Tomography (18F-FDG-PET) for Staging and Follow-Up of Marginal Zone B-Cell Lymphoma

Objective: According to recent reports, nodal marginal zone lymphoma (MZL) appears to be a distinctive lymphoma entity rather than a more advanced stage of extranodal MZL of mucosa-associated lymphoid tissue (MALT). We have therefore retrospectively evaluated all patients diagnosed with nodal or extranodal MZL who have been referred to our unit for imaging using 18F-fluorodeoxyglucose positron emission tomography (18F-FDG-PET). Patients and Methods: A total of 21 patients with a diagnosis of MZL upon referral for imaging with 18F-FDG-PET were identified. Histological reassessment of biopsy specimens confirmed the diagnosis of extranodal MZL of MALT in 14 patients, while a diagnosis of nodal MZL was verified in 6 patients. Lymphoma cell proliferation was assessed immunohistochemically using a Ki-67 antibody. Whole-body 18F-FDG-PET scans were performed on a GE advanced PET scanner 40 min after intravenous injection of 300–380 MBq 18F-FDG. Results: None of the patients with extranodal MZL showed focal tracer uptake within verified tumor sites. In contrast, 5 of the 6 patients with nodal MZL showed significant FDG uptake within the affected lymph nodes. These results did not simply reflect the different growth fractions of the two lymphoma entities since the proliferation indices of the two groups did not differ significantly. Conclusion:18F-FDG-PET visualizes nodal MZL in a high proportion of patients whereas FDG uptake is undetectable in extranodal MZL. Although limited by the small number of patients, this study suggests that imaging with 18F-FDG-PET might play a potential role in the diagnostic workup of patients with nodal MZL involvement.

Prognostic significance of WT1 gene expression at diagnosis in adult de novo acute myeloid leukemia

We examined the presence of WT1-specific mRNA in bone marrow samples of 125 patients with de novo acute myeloid leukemia at diagnosis by two-step RT-PCR. The sensitivity of the assay was 1:100 (first step) and 1:10 000 (second step), respectively. WT1-specific mRNA was detected in 73% of patients. No correlation was found between WT1 gene expression and age, FAB type, LDH and karyotype at diagnosis. All patients were treated with standard induction chemotherapy. There was no difference in the CR rate between WT1-positive and -negative patients. Using Kaplan and Meier plot analysis we found no difference in disease-free survival (DFS) and overall survival (OS) between patients displaying the WT1 transcript and WT1-negative patients. Furthermore, no significant interactions between WT1 PCR results and age, FAB type, LDH and karyotype on DFS and OS were demonstrable using Cox regression analysis. Eight patients who were WT1 PCR positive at diagnosis and achieved complete hematological remission following chemotherapy were monitored during the course of the disease. Based on our limited data demonstrating a heterogenity of WT1 PCR results in CR we cannot draw any conclusions regarding the usefulness of WT1 PCR analysis for the early detection of relapse. We conclude that WT1 gene expression at diagnosis is not associated with specific characteristics of AML blast cells and is not a prognostic factor for CR, remission duration and overall survival in acute myeloid leukemia.

Detection of the WT1 transcript by RT-PCR in complete remission has no prognostic relevance in de novo acute myeloid leukemia

The WT1 gene is expressed in 73–100% of patients with acute myelogenous leukemia (AML) and is thought to play a role in maintaining the viability of leukemic cells. WT1 has been proposed as a marker for minimal residual disease in leukemia. We obtained serial blood or bone marrow samples from patients with de novo AML at diagnosis, during therapy, and up to 95 months after diagnosis and analyzed for WT1 gene expression by RT-PCR to determine whether gene expression was predictive of relapse. Forty-four patients had WT1-positive AML and achieved a complete remission (CR) following chemotherapy and 24 patients underwent unrelated donor (n = 4), sibling donor (n = 13) or autologous (n = 7) marrow transplantation. After achieving CR 62% of the patients became WT1-negative, while 38% remained WT1-positive. There was no difference in the disease-free survival (DFS) and survival from remission between WT1-positive and -negative patients (P > 0.1). Following BMT, 32% of the patients analyzed in CR within the first 100 days after transplantation were WT1 PCR positive. Detection of WT1 transcripts within 100 days following BMT did not affect DFS and overall survival (OS) after transplantation (P > 0.1). Ten of 11 patients who are in continuous CR following chemotherapy or BMT for more than 3 years were transiently WT1-positive during the observation period. Four of these patients displayed the WT1 transcript at the last examination. Thirteen of 39 patients were WT1 PCR negative within 4 months before clinical onset of relapse and eight patients were WT1 PCR negative at time of relapse. These data indicate that: (1) achievement of WT1 negativity is not associated with longer DFS, survival from remission, or OS after transplantation; (2) not all patients who relapse become WT1 positive again; (3) long-term remitters frequently display the WT1 transcript. Thus, we conclude that the monitoring of WT1 gene expression by qualitative RT-PCR during treatment and CR is of very limited value.

High expression of activation-induced cytidine deaminase (AID) mRNA is associated with unmutated IGVH gene status and unfavourable cytogenetic aberrations in patients with chronic lymphocytic leukaemia

Activation-induced cytidine deaminase (AID) is essential for somatic hypermutation of B-cells. We investigated the expression of AID mRNA by real-time polymerase chain reaction (PCR) in peripheral blood mononuclear cells of 80 patients with B-CLL. AID expression was detected in 45 of 80 patients (56%) at various levels, but was undetectable in 35 patients (44%). AID PCR positivity was associated with unmutated IGVH gene status (22 of 25 patients; P=0.002) and unfavourable cytogenetics (18 of 23 patients with deletion in 11q or loss of p53; P=0.040). Using a threshold level of 0.01-fold expression compared to Ramos control cells, even more significant associations were observed (P=0.001 for IGVH; P=0.002 for cytogenetics). A correlation was observed between individual AID levels and the percentage of VH homology (R=0.41; P=0.001). AID positivity predicted unmutated IGVH status with an odds ratio of 8.31 (P=0.003) and poor risk cytogenetics with an odds ratio of 3.46 (P=0.032). Significance was retained after adjustment for Binet or Rai stages. AID mRNA levels were stable over time. These data suggest a potential role of AID as a prognostic marker in B-CLL.

Cross-Priming of Cytotoxic T Cells Promoted by Apoptosis-Inducing Tumor Cell Reactive Antibodies?

Humanizing xenogenic monoclonal antibodies (MAbs) by genetic engineering has greatly improved their therapeutic utility and efficacy. The chimeric CD20 MAb C2B8 (Rituximab) is a prominent representative of this new generation of therapeutic MAbs and has been proposed as a treatment of choice for recurrent follicular non-Hodgkins lymphomas. Treatment of CD20+ B cells with MAb C2B8 triggers several cell-damaging actions including complement-mediated lysis (CDL), antibody-dependent cellular cytotoxicity (ADCC), and MAb-induced induction of apoptosis. We provide an overview of the most prominent mechanisms underlying the efficacy of antibody treatment. We introduce our concept of cross-priming of cytotoxic T-cell responses promoted by apoptosis incucing antibodies. Treatment of tumor cells with antibodies that are capable of inducing a proapoptotic signal via their cell surface target structure may not only contribute to their direct killing but also may induce cellular responses against the tumor, which may have a long-lasting protective effect. We report, using the example of C2B8 anti-CD20 treatment of lymphoma cells, that MAb C2B8-induced apoptosis of lymphoma cells not only kills these cells but also promotes uptake and cross-presentation of lymphoma cell-derived peptides by antigen-presenting dendritic cells (DC), induces maturation of DC, and allows the generation of specific CTL.

A comparative study on demographic, hematological, and cytogenetic findings and prognosis in acute myeloid leukemia with and without leukemia cutis

Abstract. We studied the incidence of leukemia cutis (LC) in 381 consecutive patients with acute myeloid leukemia (AML) in a single institution and compared the demographic, hematological, and cytogenetic findings in AML patients with and without LC. We also examined the response to intensive chemotherapy, overall survival, and duration of remission in this patient population with regard to the presence of LC. The prevalence of LC was 3.7% in clinically diagnosed patients and 2.9% in biopsy-proven cases, respectively. Patients with and without LC did not differ with regard to age, sex, white blood cell counts, hemoglobin, fibrinogen, and platelet counts at diagnosis, but lactate dehydrogenase (LDH) was significantly higher in patients with LC. Various karyotype abnormalities were found, but in patients with LC numerical abnormalities of chromosome 8 were significantly more common (P<0.0001). Patients with LC did not differ from patients without LC with regard to remission rate, but there was a trend towards shorter remission duration in patients with LC. We conclude that patients with LC have some features different from patients without this symptom. The increased frequency of numerical aberrations of chromosome 8 in patients with LC was the most interesting observation of our study. The pathophysiological significance of this finding remains to be determined.

Duration of second complete remission in patients with acute myeloid leukemia treated with chemotherapy: a retrospective single-center study

Abstract A total of 168 patients with de novo AML were retreated with chemotherapy at relapse following first CR; 66 patients (39%) achieved a second complete remission (CR). The probability of achieving a second CR was highly dependent on the duration of the first remission. Patients who received no or conventional postremission chemotherapy after second CR had a median remission duration of 7.5 months, and the probability of remaining in remission at 3 years was 24%. Patients with a first CR of more than 12 months had a median second remission duration of 18 months. The probability of a second CCR was 35% at 3 years and 24% at 5 years, whereas none of the patients with a first CR of less than 12 months was in remission at 3 years. Only a poor correlation (p=0.31) was found when the durations of the first and second CR were compared in patients with a second relapse. Patients with long-lasting remissions and long-term survivors after second CR are characterized by a first CR duration of >12 months and favorable or normal cytogenetics. The type of salvage treatment seems to be less important for achievment of long-term remission, but it is probably important to administer consolidation chemotherapy after second CR. Other so-far ill-defined factors may be responsible for the supression of the leukemic clone in patients with long-lasting remissions following chemotherapy for relapse after second CR.

Serum tryptase measurements in patients with myelodysplastic syndromes.

Abnormal differentiation and maturation of hemopoietic cells are characteristic features of myelodysplastic syndromes (MDS). Tryptases (α- and β-type) are lineage-restricted serine proteases primarily expressed in mast cells (MC). We have analyzed expression of tryptase in 89 de novo MDS patients (refractory anemia (RA), n = 30; RA with ringed sideroblasts (RARS), n = 21; RA with excess of blasts (RAEB/RAEB-t), n = 27; chronic myelomonocytic leukemia (CMML), n = 11). Serum levels of total tryptase (α – protryptase + β – tryptase) were measured by FIA. The numbers of tryptase+ cells were determined in paraffin-embedded bone marrow (bm) sections by immunohistochemistry and morphometry. In healthy individuals, serum total tryptase levels ranged between < 1 and 15 ng/ml (5.6 ± 2.8 ng/ml). Tryptase levels of > 20 ng/ml were detected in 5/22 patients with RA (22.7%), 4/17 with RARS (23.5%), 0/16 with RAEB/RAEB-t, and 3/8 with CMML (37.5%). Thus, serum tryptase concentrations were higher in RA (16.6 ± 14.3 ng/ml), RARS (12.9 ± 8.2), and CMML (16.5 ± 7.6) compared to RAEB/-t (8.7 ± 3.8). By morphometry, elevated numbers of tryptase+ bm cells were detected in all MDS groups (RA: 139 ± 131; RARS: 118 ± 98; RAEB/RAEB-t: 80 ± 79; CMML: 105 ± 114 cells/mm2) compared to controls (54 ± 51 cells/mm2). As assessed by Northern blotting and protein analysis, bm cells in MDS primarily produced α-(pro)tryptase, but little or no β-tryptase. Together, our data show that elevated levels of tryptase are detectable in a group of patients with MDS probably because of an increase in neoplastic (mast) cells producing the enzyme(s). In addition, serum tryptase levels appear to correlate with MDS variants. Follow up studies should clarify whether an elevated tryptase concentration in MDS is of prognostic significance.

FLAG (fludarabine, cytosine arabinoside, G-CSF) for refractory and relapsed acute myeloid leukemia.

Abstract Twenty-two patients with refractory or relapsed AML were treated with FLAG [25 mg/m2 fludarabine daily (days 1–5), 2 g/m2 daily Ara-C (days 1–5) and 400 μg/m2 daily G-CSF (day -1 till the absolute neutrophil count was >500/μl)]. Median age was 46 years (range 24–63). Eight patients had leukemia which was primarily refractory to conventional regimens, six were in first, seven were in second, and one was in third relapse.Overall, 11 of 22 (50%) patients achieved complete remission (CR), three had a partial response (PR), and seven did not respond (NR). One patient died of an early cerebral hemorrhage. The median remission duration from achievement of CR after FLAG was 9.9 months and median survival was 13.0 months. One patient is alive in CR at 31.9 months. Hematological toxicity of the regimen was severe. The median time to neutrophil recovery (ANC >500/μl) was 21 days (range 18–33). A median of seven red cell units (range 0–22) and of six platelet concentrate units (range 3–28) had to be given. Median duration of febrile neutropenia was 2 days (range 0–20 days) and patients were on i.v. antibiotics for a median of 16 days (range 0–51). There was no death from infection. Nonhematological toxicity was remarkably low, with almost no neurotoxicity and no major hepatotoxicity. In conclusion, FLAG seems to be an efficient and well tolerated regimen. It may be particularly useful for patients who have a sibling or unrelated donor for subsequent allogeneic bone marrow transplantation.