Lothar Bergmann

Initial results in the assessment of multiple myeloma using 18F-FDG PET.

Abstract. This prospective study was undertaken to investigate the appearance of multiple myeloma on fluorine-18 fluorodeoxyglucose positron emission tomography (FDG-PET). Furthermore, the accuracy of FDG-PET in detecting myeloma lesions and its influence on patient management were evaluated. Forty-three patients with known multiple myeloma (n=28) or solitary plasmacytoma (n=15) underwent FDG-PET. The results of routinely performed radiographs and of scans obtained using all available imaging modalities (MRI, CT), as well as the clinical course, were used for verification of detected lesions. Focally increased tracer uptake was observed in 38 of 41 known osteolytic bone lesions (sensitivity 92.7%) in 23 patients. In addition, 71 further bone lesions which were negative on radiographs were detected in 14 patients. Twenty-six (36.6%) of these lesions could be confirmed in ten patients. As a result of FDG-PET imaging, clinical management was influenced in five (14.0%) patients. The positive predictive value for active disease was 100% in patients with focal or mixed focal/diffuse skeletal FDG uptake and 75% in patients with diffuse bone marrow uptake. Depending on the interpretation of the PET scans in patients with diffuse bone marrow uptake, the sensitivity ranged from 83.8% to 91.9% and the specificity from 83.3% to 100%. FDG-PET thus proved highly accurate in detecting multiple myeloma, and revealed a greater extent of disease than routine radiographs in 14 of 23 (60.9%) patients who had osteolytic bone lesions. FDG-PET might contribute to the initial staging of solitary plasmacytoma.

High expression of bcl-2 mRNA as a determinant of poor prognosis in acute myeloid leukemia

BACKGROUND The bcl-2 oncoprotein is suggested to be directly involved in the emergence of drug resistance by disrupting or delaying the apoptotic program and promoting tumor survival. PATIENTS AND METHODS In order to define the clinical relevance of the bcl-2 mRNA expression in acute myeloid leukemia (AML) and its correlation to therapy outcome and prognosis, we analyzed 219 AML bone marrow (BM) samples, including 119 patients with de novo AML at presentation, 37 with AML following myelodysplastic syndrome (MDS), as well as 42 BM samples of AML in relapse and 21 in complete remission (CR) using RT-PCR. For performing quantitative measurements of bcl-2 mRNA, we developed a quantitative RT-PCR. RESULTS Bcl-2 mRNA was detectable in 133 of 156 (84%) patients at diagnosis and 40 of 42 (95%) at relapse. AML patients with high bcl-2 mRNA expression achieved lower CR rates than those with no or low expression. Concerning the long-term outcome, the overall (OS) and disease-free survival (DFS) was significantly worse in AML patients with high expression levels of bcl-2 mRNA. The three-year OS for all newly diagnosed AML patients was 49% and 10% (P = 0.028), respectively, and 71% and 15% (P = 0.0004) for patients < 60 years. Comparable significant differences were observed for the DFS. In AML following MDS and patients > 60 years, the bcl-2 expression was not associated with remission rate or survival. CONCLUSIONS The expression of bcl-2 mRNA may serve as a prognostic factor predicting remission outcome and long-term prognosis in AML.

Receptor for hyaluronan acid–mediated motility (RHAMM) is a new immunogenic leukemia-associated antigen in acute and chronic myeloid leukemia

OBJECTIVE Identification of leukemia-associated antigens (LAA) eliciting an immune response in patients is a prerequisite for specific immunotherapy of leukemias. To identify new LAA, we used the method of serologic screening of cDNA expression libraries (SEREX). MATERIALS AND METHODS A SEREX library of the cell line K562 was subjected to allogeneic screening with sera from patients with acute myeloid leukemia (AML) or chronic myeloid leukemia (CML) vs sera from healthy volunteers. RESULTS The receptor for hyaluronan acid-mediated motility (RHAMM) involved in cell growth and metastasis was identified as a new LAA. Serologic responses to RHAMM were observed in patients with AML (42%), CML (31%), melanoma (83%), renal cell carcinoma (40%), breast cancer (67%), and ovarian carcinoma (50%), but not in HV or patients with autoimmune diseases. RHAMM mRNA was detectable in peripheral blood mononuclear cells (PBMN) of 60% of newly diagnosed AML patients. Western blotting stained positive for RHAMM protein in 70% of AML patients. mRNA expression of RHAMM also was found in patients with CML (40%), renal cell carcinoma (73%), breast carcinoma (60%), and ovarian carcinoma (50%). In melanoma, RHAMM mRNA expression was detected in metastases (80%) but not in primary tumors. RHAMM is differentially expressed: significant mRNA expression was not found in normal tissues, except from testis, placenta, and thymus, or in PBMN- and CD34-separated cell samples of healthy volunteers. CONCLUSIONS RHAMM is an immunogenic antigen in leukemias and solid tumors and might be a potential target structure for cellular immunotherapies and antibody therapies.

Positron emission tomography (PET) for staging of solitary plasmacytoma.

PURPOSE This study was undertaken to investigate the clinical value in staging patients with plasmacytoma (PC) using positron emission tomography (PET). METHODS Fifteen patients with known PC underwent PET using F-18 fluorodeoxyglucose (FDG-PET). FDG-PET was done for staging in 11 patients and for restaging in 4 patients. Eleven patients had PC of bone, and 4 patients had extramedullary PC. The results of all available imaging modalities, such as x-ray, magnetic resonance imaging (MRI), computed tomography (CT), bone scans, and of the clinical course, were used for verification of detected lesions. RESULTS Intensively increased tracer uptake was observed in 9 of 11 patients with bone lesions. One osteolytic lesion showed only slightly increased FDG-uptake and another PC of a rib (30 mm diameter) presented without FDG-uptake. The four extramedullary PC showed an intensively increased tracer uptake. In addition, 20 (10 confirmed) further plasmacytoma lesions, which were negative on the standard staging methods, were detected in 4 patients (27%). Since the diagnosis was changed from PC to multiple myeloma, a potentially curative local therapy was changed to a palliative systemic therapy. CONCLUSION Although FDG-PET was false negative in 1 patient (7%) and indeterminate in another patient, additional lesions were detected in 5 (33%) of the patients, resulting in a change in the therapy regimen in 4 (27%) of the 15 patients.

Simultaneous expression of different immunogenic antigens in acute myeloid leukemia

Identification of immunogenic leukemia-associated antigens as target structures is mandatory for specific immunotherapy of leukemia. Here, we define acute myeloid leukemia (AML) antigens eliciting a humoral immune response in the autologous host. We applied the method of serologic screening of cDNA expression libraries with autologous serum (SEREX). To date, this technique has been used to characterize antigen structures in solid tumors. The mRNA expression pattern of these newly in AML isolated antigens and previously described leukemia antigens (PRAME, MAGE-1, and Wt-1) was evaluated by reverse transcriptase polymerase chain reaction. For Wt-1, Western blotting also was performed. Screening of a cDNA expression library prepared from a patient with AML FAB M2 using autologous and allogeneic sera, followed by sequencing of positive clones, yielded three autoantigens (Prp1p/Zer1p, L19H1, and one without homology to previously described genes) and two antigens reactive with allogeneic sera (MAZ, PINCH). PRAME mRNA was expressed in 47% of 34 AML patients, but not in 13 CD34(+) cell samples or in peripheral blood mononuclear cells of 13 healthy volunteers. mRNA expression of MAZ was detected in 44% of AML patients, but only in 8% of healthy donors. Humoral responses to MAZ were detected in 35%. More than 80% of the screened AML patients showed simultaneous expression of two or more of these antigens.Differential expression in AML patients vs healthy volunteers suggests that the immunogenic antigens PRAME and MAZ are potential candidates for immunotherapy in AML.

Curcumin Inhibits Constitutive STAT3 Phosphorylation in Human Pancreatic Cancer Cell lines and Downregulation of Survivin/BIRC5 Gene Expression

ABSTRACT The purpose of this study was to determine the effect of curcumin on Survivin/BIRC5 and on the role of signal transducer and activator of transcription 3 (STAT3) activation in Survivin/ BIRC5. We incubated two pancreatic cancer cell lines with different amounts of curcumin. This resulted in a downregulation of proliferation in all cell lines tested. The expression of Survivin/BIRC5 on mRNA and protein level was significantly downregulated and the phosphorylation of STAT3 was blocked. Treatment of pancreatic cancer cells with curcumin resulted in an induction of apoptosis. The results indicate that curcumin inhibits several key factors in cancer cellular pathways and may be of interest in pancreatic cancer.

Positron Emission Tomography with 18-Fluorodeoxyglucose in the Staging and Follow-up of Lymphoma in the Chest

The purpose of this retrospective study was to evaluate the accuracy of positron emission tomography (PET) using 18-F-fluorodeoxyglucose (FDG) in predicting lymphomatous involvement in the hilar and mediastinal regions in the staging and follow-up of patients with malignant lymphoma. One hundred forty-seven thoracic PET studies in 89 consecutive lymphoma patients were reviewed. Static FDG-PET imaging was performed following application of 270 MBq FDG (mean). Results of FDG-PET were compared with the findings of computed tomography (CT) in all patients and clinical follow-up examination. Eighty-nine of 147 (60%) PET studies showed no FDG uptake in the hilar or mediastinal regions, while 58 (40%) studies did detect FDG uptake in these regions. In 52 of 58 abnormal studies (90%), lymphomatous involvement of the hilar and/or mediastinal regions seen by CT was present. In the remaining six abnormal PET studies (10%), FDG uptake was considered as false-positive because of missing lesions on corresponding CT scans. In four patients false-positive FDG uptake was observed before treatment, in two patients after completion of therapy. In these two patients FDG uptake after therapy was caused by thymus hyperplasia. The remaining four cases before treatment remained unresolved. Sensitivity of FDG-PET was 96%, specificity 94%, positive predictive value 90%, and negative predictive value 98%, respectively. The present study suggests that FDG-PET has potential value in predicting lymphomatous involvement in the hilar and mediastinal regions. FDG-PET may obviate invasive diagnostic procedures in patients with lymphoma.

Mechanisms of Bcr-Abl-mediated NF-κB/Rel activation

Bcr-Abl constitutes a deregulated tyrosine kinase involved in the pathogenesis of chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemia (ALL). Although activation of the transcription factor NF-kappaB/Rel has been demonstrated, mechanisms of NF-kappaB/Rel activation by Bcr-Abl remain obscure. In this paper we demonstrate activation of NF-kappaB/Rel by Bcr-Abl and for the first time by v-Abl. Furthermore, we investigated mechanisms of NF-kappaB/Rel induction by Bcr-Abl and v-Abl. Both Bcr-Abl and v-Abl induced NF-kappaB/Rel DNA binding in Ba/F3 cells. DNA binding was a result of nuclear translocation of p65/RelA, whereas p65/RelA expression was unaffected. Nuclear translocation of p65/RelA is at least partially due to increased IkappaBalpha degradation, which is independent of IkappaB kinase (IKK) activity. IKK activity is not deregulated by Bcr-Abl and v-Abl. NF-kappaB/Rel transactivation was dependent on abl kinase activity but independent of Grb2 and Grb10 binding tobcr sequences. In addition, NF-kappaB/Rel activation was dependent on Ras activity. Primary CML blasts showed constitutive p65/RelA NF-kappaB/Rel DNA binding activity. Thus NF-kappaB/Rel represents a potential target for molecular therapies in CML.

Real-time RT-PCR for the detection and quantification of AML1/MTG8 fusion transcripts in t(8;21)-positive AML patients.

AML1/MTG8 was quantified relative to the expression of the GAPDH housekeeping gene by real‐time RT‐PCR in 22 patients with t(8;21)‐positive acute myeloblastic leukaemia (AML) at initial diagnosis and in seven of these patients also during/after chemotherapy and allogeneic bone marrow transplantation. Real‐time PCR was able to specifically detect and quantify AML1/MTG8 over a 5 log range. The detection limit for t(8;21)‐positive cells was a dilution of 1:105. The AML1/MTG8 expression varied considerably among the 22 AML patients at intial diagnosis with a ratio AML1/MTG8:GAPDH of 0.5135±0.536 (range 0.1–2.14, median 0.318). In six patients with t(8;21)‐positive AML a marked decline of AML1/MTG8 could be induced by chemotherapy. These patients are in ongoing complete haematological remission (CR) with a constant low‐level AML1/MTG8 expression. In another patient a rapid rise of AML1/MTG8 transcripts could be detected in CR after allogeneic bone marrow transplantation and the patient relapsed 10 weeks later. In conclusion, real‐time RT‐PCR is a suitable approach for the quantification of AML1/MTG8 transcripts in the monitoring of AML patients with t(8;21) during/after chemotherapy and can provide data of prognostic relevance.

CD30-mediated cell cycle arrest associated with induced expression of p21(CIP1/WAF1) in the anaplastic large cell lymphoma cell line Karpas 299.

One of the major characteristics of anaplastic large cell lymphomas (ALCL) is the expression of the Ki-1/CD30 antigen. While the receptor mediates NF-κB-activation in Hodgkins lymphomas, some data suggest the CD30-mediated apoptosis of other CD30-expressing cells. We were able to demonstrate that activation of CD30 leads to different effects regarding cell proliferation of the ALCL-derived cell lines Karpas 299 and JB6. Western and Northern blotting analysis revealed that CD30-induced growth inhibition of Karpas 299 cells correlated with a strong upregulation of the cell cycle inhibitor p21CIP1/WAF1. We found a non activating point mutation at codon 273 in exon 8 of the p53 gene in Karpas 299 cells which indicates an p53-independent mechanism for induced p21 expression. Abundant p21 protein expression resulted in hypophosphorylation of the retinoblastoma protein (Rb) and inhibition of the proliferating cell nuclear antigen (PCNA). CD30-stimulated cells showed no indications of apoptotic cell death, like genomic DNA fragmentation or cleavage of the caspase-3 target protein poly (ADP-ribose) polymerase (PARP). Our results indicate that CD30 is able to mediate an p21-associated cell cycle arrest in ALCL with possible implications for prognosis and clinical treatment.