Josef D. Schwarzmeier

High expression of lipoprotein lipase in poor risk B-cell chronic lymphocytic leukemia.

We investigated the pattern of lipoprotein lipase (LPL) expression in B-cell chronic lymphocytic leukemia (B-CLL) and assessed its prognostic relevance. Expression of LPL mRNA as well as protein was highly restricted to leukemic B cells. The intensity of intracellular immunoreactivity of LPL was higher in samples of patients with unmutated immunoglobulin heavy-chain variable region genes (IGVH) compared to those with mutated IGVH genes. LPL mRNA levels in peripheral blood mononuclear cells (PBMNC) from 104 CLL patients differed by 1.5 orders of magnitude between cases with mutated (N=51) or unmutated (N=53) IGVH (median: 1.33 vs 45.22 compared to normal PBMNC). LPL expression correlated strongly with IGVH mutational status (R=0.614; P<0.0001). High LPL expression predicted unmutated IGVH status with an odds ratio of 25.90 (P<0.0001) and discriminated between mutated and unmutated cases in 87 of 104 patients (84%). LPL expression was higher in patients with poor risk cytogenetics. High LPL expression was associated with a shorter treatment-free survival (median 40 vs 96 months, P=0.001) and a trend for a shorter median overall survival (105 months vs not reached). Our data establish LPL as a prognostic marker and suggest functional consequences of LPL overexpression in patients with B-CLL.

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.

Reconstitution of PTEN activity by CK2 inhibitors and interference with the PI3-K/Akt cascade counteract the antiapoptotic effect of human stromal cells in chronic lymphocytic leukemia

Evidence suggests that tumor microenvironment is critically involved in supporting survival of chronic lymphocytic leukemia (CLL) cells. However, the molecular mechanisms of this effect and the clinical significance are not fully understood. We applied a microenvironment model to explore the interaction between CLL cells and stromal cells and to elucidate the role of phosphatidylinositol 3 kinase (PI3-K)/Akt/phosphatase and tensin homolog detected on chromosome 10 (PTEN) cascade in this process and its in vivo relevance. Primary human stromal cells from bone marrow, lymph nodes, and spleen significantly inhibited spontaneous apoptosis of CLL cells. Pan-PI3-K inhibitors (LY294002, wortmannin, PI-103), isotype-specific inhibitors of p110α, p110β, p110γ, and small interfering RNA against PI3-K and Akt1 counteracted the antiapoptotic effect of the stromal cells. Induction of apoptosis was associated with a decrease in phosphatidylinositol-3,4,5-triphosphate, PI3-K-p85, and dephosphorylation of phosphatidylinositol-dependent kinase-1 (PDK-1), Akt1, and PTEN. Freshly isolated peripheral blood mononuclear cells from patients with CLL (n = 44) showed significantly higher levels of phosphorylated Akt1, PDK-1, PTEN, and CK2 than healthy persons (n = 8). CK2 inhibitors (4,5,6,7-tetrabromo-1H-benzotriazole, apigenin, and 5,6-dichloro-1-β-D-ribofuranosylbenzimidazol) decreased phosphorylation of PTEN and Akt, induced apoptosis in CLL cells, and enhanced the response to fludarabine. In conclusion, bone marrow microenvironment modulates the PI3-K/Akt/PTEN cascade and prevents apoptosis of CLL cells. Combined inhibition of PI3-K/Akt and recovery of PTEN activity may represent a novel therapeutic concept for CLL.

Induction of apoptosis by proteasome inhibitors in B-CLL cells is associated with downregulation of CD23 and inactivation of Notch2.

Recently, proteasome inhibitors (PI) have attracted interest as novel anticancer agents in B-cell chronic lymphocytic leukemia (B-CLL). A prominent feature of B-CLL cells is the high expression of CD23, which is closely related to cell survival and is regulated by Notch2. Since several components of the Notch signaling cascade are tightly regulated by proteasomal degradation, we studied the effect of PI on Notch2 activity and CD23 expression. Exposure of B-CLL cells to PI led to induction of apoptosis, a time- and dose-dependent downregulation of CD23 expression and a decline in DNA binding of transcriptionally active Notch2. In contrast, the transcription factor NF-AT and its putative target gene CD5, which is highly expressed in B-CLL cells, were unaffected. When the late phase of PI-induced apoptosis was arrested by inhibition of caspase 3, the reduction of Notch2 activity was still observed, indicating that reduction of active Notch2 took place already during an earlier phase of apoptosis. Enforced expression of constitutively active Notch2 decreased PI-mediated apoptosis in a human B-cell line. These data indicate that downregulation of CD23 and loss of Notch2 activity are early steps in PI-induced apoptosis of B-CLL lymphocytes and may be part of the full apoptotic response.

TGF-β1 induces bone marrow reticulin fibrosis in hairy cell leukemia

The mechanisms that lead to reticulin fibrosis of bone marrow (BM) in hairy cell leukemia (HCL) are not fully understood. We therefore investigated the involvement of TGF-β1, a potent fibrogenic cytokine, in this process. Immunoassays revealed that TGF-β1 is present at higher concentrations in BM, serum, and plasma of HCL patients in comparison with healthy donors (P < 0.001). RT-PCR and immunofluorescence studies showed that TGF-β1 is overexpressed at the mRNA and protein levels in peripheral blood, spleen, and BM mononuclear cells and that hairy cells (HCs) are the main source of TGF-β1. Active TGF-β1 correlated significantly with grades of BM fibrosis, infiltration with HCs, and serum procollagen type III aminoterminal propeptide (PIIINP). Ex vivo studies demonstrated that TGF-β1 significantly enhances the production and deposition of reticulin and collagen fibers by BM fibroblasts. In addition, BM plasma of HCL patients increased the synthesis of type I and type III procollagens, the main components of reticulin fibers, at the mRNA and protein levels. This fibrogenic activity of BM plasma was abolished by neutralizing anti–TGF-β1 antibodies. These results show, for the first time to our knowledge, that TGF-β1 is highly expressed in HCs and is directly involved in the pathogenesis of BM reticulin fibrosis in HCL.

Effect of combined spa-exercise therapy on circulating TGF-beta1 levels in patients with ankylosing spondylitis.

ZusammenfassungZIEL: Um einen objektiven Indikator für das biologische Ansprechen von Patienten mit ankylosierender Spondylitis (AS, M. Bechterew) auf die Heilstollen-Behandlung in Badgastein zu finden, wurde die Rolle des anti-inflammatorischen Zytokins TGF-β1 (Transforming Growth Factor β 1) untersucht. METHODIK: 83 Patienten mit AS wurden 3–4 Wochen lang einer Therapie bestehend aus Hyperthermie, Exposition mit niedrigen Radon-Dosen und gymnastischen Übungen unterzogen. Das Ansprechen auf die Behandlung wurde durch das Ausmaß der Schmerzreduktion ermittelt. Als Kontrollen wurden 10 AS-Patienten, die eine konventionelle Therapie ohne Heilstollen-Exposition erhielten und 10 Patienten mit Rückenschmerzen ohne entzündliches Substrat (low back pain, LBP) in die Untersuchungen eingeschlossen. Vor und nach der Therapie wurden mittels Immunassays die Serumkonzentrationen von TGF-β1 bestimmt. RESULTATE: Nach Therapie wurde im Serum der ASPatienten ein signifikanter Anstieg von TGF-β1 (total und aktiviert) festgestellt. Die mittlere Konzentration des totalen TGF-β1 stieg von 28715 pg/ml auf 43136 pg/ml (p < 0,01) und die des aktivierten TGF-β1 von 77 auf 1096 pg/ml (p < 0.001). Bei Unterteilung der AS-Patienten hinsichtlich des Ausmaßes der Schmerzreduktion in zwei Gruppen, zeigte die Gruppe 1 (Schmerzreduktion: n = 46) einen 17-fachen Anstieg der Konzentration von aktiviertem TGF-β1 (96 bis 1654 pg/ml, p < 0.0001), während die Gruppe 2 (keine Schmerzreduktion: n = 37) nur einen 7-fachen Anstieg (53 bis 402 pg/ml, p < 0.01) aufwies. Bei LBP-Patienten wurde nur ein geringfügiger Anstieg des aktivierten TGF-β1 von 31 auf 42 pg/ml (p < 0.01) registriert und bei AS-Patienten, die lediglich einer konventionellen Therapie unterzogen wurden, fand sich keine signifikante Änderung der TGF-β1 Werte. SCHLUSSFOLGERUNG: Nach Heilstollen-Therapie in Badgastein fand sich im Blut von Patienten mit ankylosierender Spondylitis ein signifikanter Anstieg des zirkulierenden TGF-β1. Das Zytokin könnte auf Grund seiner antiinflammatorischen Eigenschaften auf den Heilungsprozess einwirken und somit zu einer Besserung der Schmerzen und der Mobilität der Patienten beitragen.SummaryBACKGROUND: Ankylosing spondylitis (AS) is a chronic inflammatory disease of the axial joints with no satisfactory therapy. Reduction of joint pain has been reported after a course of therapy at a spa, Gasteiner Heilstollen, in Badgastein in Austria. The mechanism underlying this beneficial effect is not clearly understood and objective evidence for the biological response to therapy is lacking. The aim of this study was to find evidence for a biological response to speleotherapy in patients with AS and to study the involvement of the antiinflammatory cytokine TGF-β1 in this response. PATIENTS AND METHODS: 83 patients with AS were treated in Badgastein for 3–4 weeks. Therapy included active exercises, hyperthermia and exposure to low doses of radon in a former mine. Response to therapy was assessed from measurement of morning pain and immunoassay of serum levels of TGF-β1 before and after therapy. Ten AS patients who received conventional therapy and 10 patients with low back pain (LBP) served as controls. RESULTS: A significant increase in TGF-β1 (total and active) was found in AS patients after spa therapy. Mean concentration of total TGF-β1 increased from 28,715 pg/ml to 43,136 pg/ml, (P < 0.01) and active TGF-β1 increased from 77 pg/ml to 1096 pg/ml (P < 0.001). When the AS patients were divided into two groups according to pain reduction, group 1 (decrease in morning pain, responders: n = 46) exhibited a 17-fold increase of active TGF-β1 levels (96 pg/ml to 1654 pg/ml, P < 0.0001) whereas group 2 (no change or an increase in morning pain: nonresponders: n = 37), showed only 7-fold increase (53 pg/ml to 402 pg/ml, P < 0.01). There was a moderate increase in active TGF-β1 from 31 pg/ml to 42 pg/ml (P < 0.05) in patients with LBP and no significant change was observed in the patients treated with conventional therapy. CONCLUSION: These results demonstrate a significant increase in circulating TGF-β1 in patients with AS after the combined spa-exercise therapy in Badgastein. The results also provide evidence for a biological response to speleotherapy and suggest that TGF-β, through its antiinflammatory function, may play a role in this response.

In vitro cytotoxic effect of proteasome inhibitor bortezomib in combination with purine nucleoside analogues on chronic lymphocytic leukaemia cells

Abstract:  Objective: The anti‐tumour in vitro activity of proteasome inhibitor bortezomib (PS‐341, VELCADE) in combination with purine nucleoside analogues, cladribine (2‐CdA) and fludarabine (FA) was tested in lymphocytes derived from 26 patients with B‐cell chronic lymphocytic leukaemia (B‐CLL). Methods: Cell viability was assessed by propidium iodide staining, and apoptosis by annexin‐V and caspase activation flow cytometry assays. Additionally, expression of the apoptosis‐regulating proteins Bax, Bak, Bid, Bcl‐w, Bcl‐2, XIAP and Mcl‐1 was evaluated in B‐CLL lymphocytes. Results: Bortezomib alone induced significant, dose‐dependent cytotoxicity starting from the low concentration 2.5 nm, inducing apoptosis of B‐CLL cells. Combination of this agent with 2‐CdA or FA resulted in an increase of cytotoxicity when compared with that mediated by single drugs. The observed increase was especially evident when 5 nm of bortezomib were combined with suboptimal doses of 2‐CdA or FA. The combination index (CI) was 0.87 for bortezomib + 2‐CdA and 0.82 for bortezomib + FA, indicating an evident additive effect of these combinations. Moreover, B‐CLL cells were more sensitive to proteasome inhibitor used alone or combined with 2‐CdA or FA comparing to CD3+ lymphocytes. Corresponding to enhanced apoptosis, the expression levels of several apoptosis‐regulating proteins were altered. The most pronounced changes were down‐regulation of XIAP and up‐regulation of Bid proteins by the combination of bortezomib with either 2‐CdA or FA. Conclusions: This study suggest that the in vitro cytotoxic effect through proteasome inhibition by bortezomib can be increased substantially with low doses of the purine nucleoside analogues, 2‐CdA and FA, and that this effect on B‐CLL cell is selectively higher than on normal, CD3‐positive lymphocytes.

Association of CD38 Antigen Expression with Other Prognostic Parameters in Early Stages of Chronic Lymphocytic Leukemia

The expression of the surface molecule CD38 on B cell chronic lymphocytic leukemia (B-CLL) cells has recently been described as a prognostic marker for patient survival. We have analyzed CD19/CD38 expression in 81 patients with predominantly early stages of B-CLL (69 Binet A, seven Binet B, five Binet C). Sixty-two patients (77%) had less than 30% CD38+/CD19+ cells, while 19 (23%) had ≥30%. There was a significant association between Binet stages (A vs. B+C, p < 0.0001), Rai stages (0-II vs. III+IV, p < 0.001) and CD38 expression, confirming the published cut-off level of 30%. A particularly strong association between CD38 expression was found with soluble CD23 (sCD23) levels of ≥2000 U/ml (p < 0.0001) and β2-microglobulin (β2 MG) serum levels of ≥3 mg/l (p < 0.0001) indicating that CD38 is a marker of tumor mass as well as disease progression. A borderline association was found with lymphocyte doubling time (LDT) < 12 months (p = 0.05) due to low patient numbers, while there was no association with age, sex or immunoglobulin deficiency. Discordant results were obtained in a number of patients: 10 of 69 patients (14%) with Binet A had a CD38 ≥30% while three of seven patients with Binet B had a CD38 < 30%. In these two subgroups CD38 and other prognostic factors gave discrepant results. Due to the early stage and short median observation time (12 months, range 1–24 months), calculations concerning patient survival were not performed. However, our data show a strong association between CD38 and other known prognostic factors. The results also suggest that this factor is not always reliable in Binet A patients.

Distinct lymphoblastic and myeloblastic populations in TdT positive acute myeloblastic leukemia: evidence by double-fluorescence staining.

Double-immunofluorescent staining for the enzyme terminal deoxynucleotidyl transferase (TdT) as a marker of primitive lymphoblasts, and for the VIM-D5 antigen as a differentiation antigen of the myeloid system gave direct evidence for distinct lymphoblastic and myeloblastic populations (mixed leukemic cell populations) in seven patients with acute leukemia. The percentage of malignant TdT positive cells contributing to a leukemic cell bulk with unequivocal signs of myeloid origin was between 10 and 80%. A defect at the level of a common progenitor cell giving rise to both the TdT and the VIM-D5 positive blast cell population is discussed.

Prediction of the response to chemotherapy in acute leukemia by a short‐term testin vitro

To detect sensitivity or resistance of leukemic cells to chemotherapy prior to treatment, a short‐term incubation method was employed. Blast cells from the peripheral blood or bone marrow of adult patients presenting with different forms of acute leukemia were analyzed for in vitro responsiveness to anticancer drugs in terms of suppression of 3H‐uridine incorporation into cellular nucleic acids. The following agents were tested over a wide range of concentrations: Adriamycin, cytosine arabinoside, thioguanine, 6‐mercaptopurine, prednisone, and 4‐hydroperoxycyclophosphamide. Retrospectively, the in vitro data were compared to the clinical response of the patients to polychemotherapy. In the majority of the patients, in vitro cytotoxic effectiveness of Adriamycin (doxorubicin) and cytosine arabinoside reflected the in vivo situation. The levels of in vitro inhibition that could distinguish between drug‐sensitive and drug‐resistant diseases appeared to be 30% for Adriamycin and 20% for cytosine arabinoside. No correlation was found between the Adriamycin effect in vitro and the proliferative state (rate of 3H‐thymidine incorporation) of the leukemic cell population. Serial in vitro sensitivity testing during the course of disease of various patients proved the ability of the test system to detect acquired resistance to chemotherapeutic agents. Therefore, the assay might serve as a reliable tool in the selection of effective chemotherapy in individual patients suffering from acute leukemia.