Bortezomib retreatment in relapsed multiple myeloma - results from a retrospective multicentre survey in Germany and Switzerland.

OBJECTIVES: This multicenter, retrospective survey evaluated the efficacy and safety of bortezomib retreatment in patients with relapsed multiple myeloma who had responded to initial bortezomib treatment. METHODS: Clinical records of 94 patients receiving bortezomib retreatment in Germany and Switzerland were reviewed. RESULTS: Sixty patients were included according to prespecified criteria. Patients had received a mean 3.7 ± 2.3 therapies prior to initial bortezomib. Overall response rate to bortezomib retreatment was 63.3%; 8 (13.3%), 3 (5.0%) and 27 (45.0%) patients achieved complete response (CR), near-CR and partial response, respectively. Response to retreatment was associated with response to initial treatment (75.0% of patients with CR to initial treatment responded to retreatment) and treatment-free interval (TFI) after initial treatment (76.9 vs. 38.1% overall response rate for patients with TFI >6 vs. ≤ 6 months). After retreatment, median time to progression was 9.3 months. Median TFI was 5.7 months; 31.7, 25.0 and 15.0% of patients experienced a TFI longer than 6, 9 and 12 months, respectively. Reported adverse drug reactions were consistent with the known safety profile of bortezomib and most resolved completely. CONCLUSIONS: These results demonstrate that relapsed multiple myeloma patients who respond to initial bortezomib treatment have a sustained susceptibility to bortezomib and do not experience uncommon toxicity to retreatment.

A CD19-specific single-chain immunotoxin mediates potent apoptosis of B-lineage leukemic cells.

CD19 is a B-lineage-specific transmembrane signaling protein participating in the control of proliferation and differentiation. It is present at high surface density on chronic B-lymphocytic leukemia (B-CLL) cells and cells of other B-cell malignancies, and is a prime target for therapy with antibody-derived agents. Many attempts have been made to target malignant cells via CD19, but to date none of these agents have received drug approval. Here we report the design of a monovalent immunotoxin consisting of a CD19-specific single-chain Fv antibody fragment fused to a derivative of Pseudomonas Exotoxin A. This fusion protein induced efficient antigen-restricted apoptosis of several human leukemia- and lymphoma-derived cell lines including Nalm-6, which it eliminated at an effective concentration (EC(50)) of 2.5 nM. The agent displayed synergistic toxicity when used in combination with valproic acid and cyclosporin A in cell-culture assays. It induced apoptosis of primary malignant cells in 12/12 samples from B-CLL patients, including patients responding poorly to fludarabine, and of cells from one pediatric acute lymphoblastic leukemia patient. In NOD/SCID mice transplanted with Nalm-6 cells, the toxin prevented engraftment and significantly prolonged survival of treated mice. Owing to its efficient antigen-restricted antileukemic activity, the agent deserves further development towards clinical testing.

Evidence for cell adhesion-mediated drug resistance of multiple myeloma cells in vivo.

BACKGROUND/AIMS: Multiple myeloma is an incurable disease and patients eventually die of disease progression due to drug resistance. VLA-4 (very late antigen 4), VCAM (vascular adhesion molecule), LFA-1 (leukocyte function-associated antigen 1), and ICAM-1 (intercellular adhesion molecule 1)-mediated adhesion of myeloma cells to bone marrow stromal cells induces primary multidrug resistance in vitro. Based on these preclinical data we hypothesized that myeloma cells with strong adhesion - due to strong expression of adhesion molecules on the cell surface - are selected by chemotherapy in patients. To prove this hypothesis we determined the expression levels of adhesion molecules in 31 multiple myeloma patients by flow cytometry. METHODS: A 3-color stain with CD38, CD138 and antibodies against VLA-4, ICAM-1, LFA-1, and VCAM was performed. The patients were either at diagnosis (chemo-naive; n=17) or at relapse (pre-treated; n=15). Furthermore, the response to the next chemotherapy of chemo-naive patients was correlated with the expression levels of adhesion molecules. RESULTS: ICAM-1, VLA-4, and VCAM expression was higher in pre-treated patients than in chemo-naive patients and the expression levels increased with the number of chemotherapy regimens. Primarily multidrug-resistant patients had significantly higher expression levels of VLA-4 and ICAM-1 than responders. CONCLUSION: This study suggests that multiple myeloma cells expressing high levels of VLA-4 and ICAM-1 are drug resistant and that such a subpopulation of cells is selected by chemotherapy.

Activation of Src kinases p53/56lyn and p59hck by p210bcr/abl in myeloid cells.

Chronic myeloid leukemia is characterized by the Philadelphia (Ph1) translocation t(9;22) that generates a hybrid gene, bcr/abl, translated to a Mr210,000 tyrosine kinase (p210bcr/abl) with transforming activity for hematopoietic cells. Hematopoietic cell transformation by p2l0bcr/abl seems to involve activation of the Ras signaling pathway by at least two different signaling intermediates, growth factor receptor-bound protein 2 and Src homology and collagen protein, but additional signaling proteins are likely to be required as well. In an effort to identify additional phosphoproteins activated by p210bcr/abl, we studied the murine, interleukin 3-dependent, myeloid cell line, 32D, and a bcr/abl-transfected, factor-independent subline, 32Dp210. The analysis of whole-cell lysates of 32D and 32Dp210 cells showed that several proteins with a molecular weight of Mr50,000-60,000 were phosphorylated on tyrosine residues in 32Dp210 cells. Because Src family kinases have an apparent molecular weight of Mr50,000-60,000, we asked whether they could become activated by p2l0bcr/abl. Two Src family kinases, p53/56lyn and p59hck, showed a severalfold higher phosphokinase activity in 32Dp210 cells than in 32D cells. Coimmunoprecipitation experiments with anti-Lyn, anti-Hck, and anti-Abl antibodies demonstrated an intracellular association of p210bcr/abl with p53/56lyn and p59hck. Moreover, the phosphokinase activity of p53/56lyn was higher in bcr/abl-positive myeloid cell lines (K562, BV173, and LAMA84) than in the bcr/abl-negative myeloid cell line JOSK-M. In conclusion, the results show that p210bcr/abl induces the activation of at least two Src family kinases, P53/56lyn and p59hck, in myeloid cells. These findings extend the range of potential targets of p210bcr/abl that might mediate its transforming effects.

CD30 ligand signal transduction involves activation of a tyrosine kinase and of mitogen-activated protein kinase in a Hodgkin's lymphoma cell line.

CD30 is a transmembrane receptor of the nerve growth factor/tumor necrosis factor receptor superfamily. Its expression associated with Hodgkin's lymphoma and a subset of non-Hodgkin's lymphoma. Recently, its ligand (CD30L) has been cloned. CD30L enhances the proliferation of peripheral T cells and the Hodgkin's cell line HDLM-2 but seems to exert antiproliferative effects on large cell anaplastic lymphoma cell lines. Since tyrosine kinases are critical regulators of cell growth, we investigated whether CD30L induced changes in cellular tyrosine phosphorylation in CD30-positive lymphoma cell lines. Stimulation with CD30L or with an agonistic mAb against CD30, M44, induced a rapid, transient, and concentration-dependent tyrosine phosphorylation of a cytosolic protein of M(r) 42,000 (p42) in the Hodgkin's lymphomas cell line HDLM-2 but not in other CD30-positive lymphomas. In HDLM-2 cells, the phrobol ester phorbol 12-myristate 13-acetate also stimulated tyrosine phosphorylation of p42, and this effect was enhanced by M44. In marked contrast, agents stimulating the protein kinase A pathway, like forskolin or dibutyryl cAMP, did not affect tyrosine phosphorylation of P42. By immunoprecipitation with mAbs against mitogen-activated protein kinase (MAPK; p42ERKII), a M(r) 42,000 protein was identified which comigrated with p42 on SDS gels and which was phosphorylated on tyrosine residues in response to stimulation of CD30. Immune complex kinase assays showed that M44 mAb induced the activation of MAPK (p42ERKII) and the phosphorylation of a MAPK substrate, myelin basic protein. Taken together, the results suggest that CD30L induces the tyrosine phosphorylation and activation of the MAPK p42ERKII isoform in HDLM-2 cells. These findings may have implications for the understanding of the pathogenesis of Hodgkin's disease.

Different susceptibility of protein synthesis to inhibitors of elongation in cell-free systems from plasma cell tumours and reticulocytes.

Plasma cells and reticulocytes are mammalian cell systems which have specialized in the synthesis of a single protein during their differentiation from one common stem cell. To study whether there is a difference in cell susceptibility at the level of elongation, dose vs. inhibition curves of sparsomycin, cycloheximide and emetine in cell-free systems with S-30 fractions from plasma cell tumours (MOPC 63, MOPC 41, RPC 20, MOPC 104 E), reticulocytes and liver were compared. The experiments revealed: (1) all the selected systems are equally sensitive to sparsomycin; (2) the susceptibility of the reticulocyte systems to cycloheximide and emetine is higher than that of the plasma cell tumours. In the dose range of 1 - 10(-7) --5 - 10(-5) M cycloheximide and 1 - 10(-6)--1 - 10(-4) M emetine the reticulocyte system is preferentially inhibited; (3) the sensitivities of all plasma cell tumours are equal; (4) the liver system is more sensitive to emetine than to cycloheximide; (5) the site of the different susceptibility to these antibiotics could be located on the ribosomes; (6) however, when the extracts of the plasma cell tumours were prepared in the presence of hemin, their susceptibility rises and is like that of reticulocytes. These results show that hemin promotes in a cell-specific manner the sensitivity to some inhibitors of protein synthesis.

Prognostic factors in chronic lymphocytic leukemia.

The clinical course of chronic lymphocytic leukemia (CLL) shows a marked heterogeneity, with a median survival ranging from 2 to 20 years at different disease stages. This unpredictable course has inspired clinical hematologists and oncologists to search for parameters which predict survival and disease progression. This effort resulted in different staging systems, two of which, the Rai and Binet staging systems, have become most popular because of their simplicity. They identify three major groups of patients with different survival. Since patients at early stages have a relatively good prognosis, only advanced stages used to be treated by chemotherapy with alkylating agents. With the advent of potentially curative, but more aggressive treatment options for CLL, additional prognostic criteria are required to predict the outcome more precisely, in particular in young patients with early disease. It is the intent of this review to summarize the current knowledge on both established and new prognostic factors in CLL, some of which might help to define risk-adapted treatment protocols in the near future.

Phosphodiesterase type 4 inhibitor suppresses expression of anti-apoptotic members of the Bcl-2 family in B-CLL cells and induces caspase-dependent apoptosis.

B cell chronic lymphocytic leukemia (B-CLL) is an incurable clonal disease which shows initial responsiveness to a number of chemotherapeutic drugs. However, in most patients the disease becomes resistant to treatment. Rolipram, a specific inhibitor of phosphodiesterase (PDE) type 4, the PDE predominantly expressed in B-CLL cells, has been shown to induce cAMP-dependent apoptosis in these cells. In the present study, we demonstrate that the extent of rolipram-induced apoptosis is similar to fludarabine-induced apoptosis in vitro. The combination of rolipram and fludarabine results in an enhancement in the number of apoptotic cells compared to apoptosis induced by either agent alone. Second, rolipram suppresses the expression of anti-apoptotic members of the Bcl-2 family and induces the pro-apoptotic protein Bax, thereby shifting the balance between pro- and anti-apoptotic members of the Bcl-2 family towards a pro-apoptotic direction. Finally rolipram-induced apoptosis is caspase-dependent. PDE 4 inhibitors are currently under investigation for chronic obstructive pulmonary disease and asthma in phase III clinical trials showing promising results with tolerable side-effects. In conclusion, by inducing apoptosis, by enhancing apoptosis induced by fludarabine, by suppressing Bcl-2, Bcl-X and by inducing Bax expression, PDE 4 inhibitors may add a new therapeutic option for patients with B-CLL.

T cell receptor alpha expression in B-type chronic lymphocytic leukemia.

Normal B lymphocytes are characterized by rearrangement and expression of immunoglobulin genes, but not of T cell receptor genes. These properties might assist in lineage assignment, but there are examples of fresh leukemic cells and of cell lines where exceptions to this rule have been noted. We have studied cell samples of patients with B-CLL for expression of TCR alpha and beta chain genes. Using in situ hybridization with fluorescein-labeled probes, TCR alpha mRNA was found to be expressed in 14 of 18 samples and TCR beta mRNA in 7 of 16 samples. Specificity of hybridization was demonstrated by near complete blockade of TCR alpha hybridization with unlabeled TCR alpha, but not with unlabeled TCR beta probe. Furthermore, in Northern blot analysis a truncated 1,4 kb message for TCR alpha was readily detectable. No significant cell surface staining with the anti-TCR alpha/beta monoclonal antibody WT31 was observed. A contribution of T cells within the leukemic sample could be excluded since only samples with leukemic cell counts of greater than 50,000 cells/mm3 and only samples with 5% or less CD2+ T lymphocytes were studied. Our data show that a large proportion of B-CLL samples may express a truncated version of the TCR alpha message, indicating that this gene can be activated in leukemic B cells frozen at a late stage of differentiation.

Consolidation with alemtuzumab in patients with chronic lymphocytic leukemia (CLL) in first remission--experience on safety and efficacy within a randomized multicenter phase III trial of the German CLL Study Group (GCLLSG).

Patients with CLL responding to initial chemotherapy with fludarabine alone (F) or in combination with cyclophosphamide (FC) were randomized for treatment with alemtuzumab (30 mg i.v. TIW, 12 weeks) or observation. Of 21 evaluable patients, 11 were randomized to alemtuzumab before the study was stopped due to severe infections in seven of 11 patients. These infections (one life-threatening pulmonary aspergillosis IV; four CMV reactivations III requiring i.v. ganciclovir; one pulmonary tuberculosis III; one herpes zoster III) were successfully treated and not associated with cumulative dose of alemtuzumab. In the observation arm, one herpes zoster infection II and one sinusitis I were documented. At 6 months after randomization, two patients in the alemtuzumab arm converted to CR, while three patients in the observation arm progressed. After alemtuzumab treatment, five of six patients achieved a molecular remission in peripheral blood while all patients in the observation arm remained MRD-positive (P=0.048). At 21.4 months median follow-up, patients receiving alemtuzumab showed a significant longer progression-free survival (no progression vs mean 24.7 months; P=0.036). In conclusion, a consolidation therapy with alemtuzumab is able to achieve molecular remissions and longer survival in CLL, but a safe treatment regimen needs to be determined.

Urinary excretion of proteolyzed alpha1-antitrypsin: specificity, quantitation, and relation to therapy response in patients with acute myeloid leukemia.

During remission induction chemotherapy, a 41-kDa cleavage product of alpha1-antitrypsin (alpha1-AT41) can be found in the urine of patients with acute myeloid leukemia. By using immunoblotting with antibodies against this protein, 27 patients with acute myeloid leukemia were screened for the excretion of this fragment and the amount of alpha1-AT41 compared with treatment response assessed by therapy-induced cytoreduction in the bone marrow and time to reach remission. Patients with acute lymphoblastic leukemia, malignant lymphomas, and solid tumors receiving chemotherapy, patients with nonmalignant diseases like sepsis and kidney dysfunction, and healthy subjects were probed to evaluate the specificity of this phenomenon. In 74% of the acute myeloid leukemia patients, the truncated inhibitor was detected. Mean concentration of peak excretion was found to be 6.7 microgram/mg creatinine (range, 1.1-41 microgram/mg). Among the patients treated with induction chemotherapy, those who responded completely (<5% residual marrow blast cells) exhibited significantly higher alpha1-AT41 concentrations than the nonresponders (P < 0.03). Patients who showed a partial response (6-25% residual blasts) excreted intermediate values of the protein. The probability of median time to reach remission was 40 days in patients excreting the truncated inhibitor in measurable amounts compared to 100 days in patients negative for alpha1-AT41 (P < 0.02). The 41-kDa fragment was also found in one of 10 patients with acute lymphoblastic leukemia and in 3 of 18 lymphoma patients but not in those with solid tumors, infections, or kidney disease or in healthy individuals.

Modulation of malignant B cell activation and apoptosis by bcl-2 antisense ODN and immunostimulatory CpG ODN.

Inhibition of bcl-2 expression by antisense oligodeoxynucleotides (ODN) might render bcl-2 overexpressing malignant B cells more susceptible to chemotherapy. ODN containing unmethylated CG dinucleotides (CpG) are known to activate B cells. We studied the effects of two bcl-2 antisense ODN, with (G3139) or without CG dinucleotides (NOV 2009) within the sequence, and the effects of a nonantisense, CpG-containing ODN (ODN 2006) on activation and apoptosis of malignant B cell lines and primary B-CLL cells. Without cationic lipids, no antisense-mediated inhibition of bcl-2 synthesis was achieved with G3139 and NOV 2009. Instead, G3139, but not NOV 2009, induced similar changes as ODN 2006 in proliferation, expression of costimulatory and antigen-presenting molecules, as well as in bcl-2 and bcl-xL levels of primary B-CLL cells. G3139 and ODN 2006 inhibited in vitro, spontaneous apoptosis in B-CLL cells of patients with high serum thymidine kinase activity (s-TK, marker for proliferative activity of malignant B cells), whereas in patients with low s-TK activity, apoptosis was induced. In conclusion, our results suggest that modulation of malignant B cell apoptosis by G3139 depends on its immunostimulatory properties rather than on antisense-mediated reduction of bcl-2 expression. Immunostimulatory CpG ODN may have a therapeutic potential in patients with B-CLL, especially those with low s-TK activity.

Signal transduction of interleukin-6 involves tyrosine phosphorylation of multiple cytosolic proteins and activation of Src-family kinases Fyn, Hck, and Lyn in multiple myeloma cell lines.

Binding of interleukin-6 to its receptor (IL-6R) induces the association of the IL-6R alpha chain (IL-6Ralpha) with a 130-kDa transmembrane glycoprotein, gp130. This event activates tyrosine kinases of the Janus kinase (JAK) family and transduces signals to the cytosol or nucleus. To further characterize the biochemical mechanisms by which IL-6 promotes cell proliferation, we investigated the effects of IL-6 on the growth and transmembrane signaling of several lymphoid cell lines. In the IL-6-dependent cell line B-9, IL-6 induced a rapid, transient, and concentration-dependent tyrosine phosphorylation of several cytosolic proteins as detected by antiphosphotyrosine immunoblots. The molecular weight of major bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 44, 65, 70, 80, 137, 148, 184, and 190 kDa, respectively. Similar effects of IL-6 on tyrosine phosphorylation were observed in the human multiple myeloma cell line LP-1. Because JAKs were unlikely to mediate all the biological effects of IL-6, we investigated whether members of the Src family of tyrosine kinases were also activated in B-9 or LP-1 cells. IL-6 induced the activation and tyrosine phosphorylation of p59Fyn, p56/59Hck, and p56Lyn. Coprecipitation experiments with anti-Hck, anti-Lyn, anti-Fyn, and anti-gp130 antibodies revealed a physical association with gp130 of p56/59Hck and p56Lyn, but not p59Fyn, in LP-1 cells. Together, these results show for the first time that several Src kinases may become activated by IL-6 (p59Fyn, p56/59Hck, and p56Lyn) and associate with gp130 (p56/59Hck and p56Lyn).

PCR identification of HIV-1 DNA sequences in brain tissue of patients with AIDS encephalopathy.

We analyzed brain tissue from 39 patients for the presence of proviral HIV-1 sequences, using the polymerase chain reaction (PCR) for the amplification of segments of the viral LTR and gag genes. A novel primer extension procedure allowed the detection of a single HIV-1 copy in 1 micrograms DNA. We detected proviral HIV-1 DNA in 16 of 25 brain samples from AIDS patients. Semiquantitative evaluation of the amplified DNAs indicated considerable variation in viral load. Highest levels of proviral DNA were present in brain samples from six patients with clinical evidence of HIV-associated cognitive/motor complex and the histopathologic correlate of HIV leukoencephalopathy or HIV encephalitis. An additional 11 brain samples contained smaller amounts of proviral DNA. In these patients, clinical data were inconclusive regarding the diagnosis of HIV-1 encephalopathy and histopathologically there was no evidence of HIV-1-induced tissue lesions. In nine of 25 seropositive patients with AIDS (36%), brain samples scored negative or did not contain an unequivocal signal indicating the presence of proviral DNA. HIV-1 sequences were not detected in any of 14 control brain samples from HIV-1 seronegative patients. Our data indicate that HIV-1 is present in the central nervous system of the majority (two thirds) of AIDS patients and that the highest levels of proviral DNA in brain tissue are associated with HIV encephalopathy.

Reduced responsiveness of adenylate cyclase to forskolin in human lymphoma cells.

The beta 2-adrenergic transmembrane signal transduction was investigated in malignant B-cells from 15 patients with low grade non-Hodgkin's lymphoma as compared with normal lymphocytes of seven healthy adults. The number of beta 2-adrenoceptors and the response of adenylate cyclase (AC) to isoproterenol were slightly decreased in lymphoma cells. The responsiveness of AC to forskolin was 8-fold lower in lymphoma cells, whereas the response to cholera toxin showed no difference. These findings demonstrate an impairment of the beta 2-adrenergic signal transduction in low grade lymphoma cells that particularly affects the function of AC. The comparison with forskolin resistant mutants of an adrenocortical tumor cell line, Y1 (Schimmer et al., J Biol Chem 262: 15521-15526, 1987), suggests that the availability of functional active alpha subunits of stimulatory G proteins (Gs) might be reduced in human B-cell lymphoma, although other mechanisms known to inhibit the AC activity might be involved.

Multicentre study on intensified remission induction therapy for acute myeloid leukemia.

In a cooperative study at 13 centres in the Federal Republic of Germany, 213 adult patients with AML were treated for remission induction by a 9-day regimen consisting of cytosine arabinoside, daunorubicin and thioguanine (TAD) according to previously described sequencing. Complete remission was achieved in 70% of all patients. Complete remission rate was 57% in the 49 patients 60 years of age and older and 74% in the 164 patients under 60 years. Sixty-eight per cent of all complete remissions and 75% of those in the higher age group were induced by one induction course. Median survival was 10 months for all patients treated and 16 months for responders. Median remission duration was 13 months with 72 patients still in continuous remission for 1-31 months. Remission duration was not significantly different for patients treated either by monthly maintenance therapy or induction type consolidation without further therapy. However, patients completing two consolidation courses had a significantly longer remission duration of 22 months. Compared to similar multicentre studies on AML therapy the intensified induction regimen applied in this study shows an improvement even in older patients.

Assessment and characterization of the cytolytic T lymphocyte response against Epstein-Barr virus in patients with non-Hodgkin's lymphoma after autologous peripheral blood stem cell transplantation.

The cytolytic T lymphocyte (CTL) response has often been used to assess the reconstitution of T cell function after allogeneic or autologous bone marrow transplantation (BMT). Less is known, however, about the reconstitution of the CTL response after peripheral blood stem cell transplantation (PBSCT). Therefore, we investigated the CTL response against Epstein-Barr virus (EBV) of patients undergoing autologous PBSCT. CTLs of six patients with relapsed non-Hodgkin's lymphoma and multiple myeloma were established before and at different times after PBSCT by in vitro stimulation of peripheral blood lymphocytes with autologous EBV-transformed lymphoblastoid cell lines (LCLs). The efficiency of T cell priming by LCLs was assessed at the time of initiation of CTL lines; the proliferative response was strongly reduced during the first 4 months and increased 5 months or more following PBSCT. Cytolytic activity was measured after three or four restimulations of CTLs. All patients investigated had a detectable EBV-specific CTL response which was poor during the first weeks after transplantation, accompanied by a strong non-MHC-restricted cytotoxic activity and a high proportion of CD56-positive T cells. Five or more months after PBSCT, a specific CTL response against EBV was seen which was similar to the situation prior to PBSCT, while the unspecific cytotoxic response decreased. Blocking experiments with monoclonal anti-CD3, anti-CD8 or anti-MHC I antibodies resulted in substantial inhibition of autologous LCL lysis, whereas anti-CD4 or anti-MHC II antibodies had no effect. Finally, autologous PHA blasts of a patient with the HLA haplotype A1/9+, B5/8+, Cw4/7+, were loaded with various EBNA-derived nonapeptides known to be presented by HLA B8 or A11, and exposed to autologous, EBV-directed CTLs. Specific lysis by CTLs only occurred with HLA B8-, but not with HLA A11-restricted nonapeptides. This demonstrated the existence of an MHC I-restricted anti-EBV CTL response after PBSCT. Taken together, the results show that the anlaysis of the EBV-directed CTL activity may serve as a surrogate marker to assess the reconstitution of the cellular immune response in patients undergoing autologous PBSCT.

Target value tailored (TVT) apheresis approach for blood progenitor cell collection after high-dose chemotherapy and rh-G-CSF.

Twenty-eight patients with different hematological diseases (17 non-Hodgkin's lymphoma, one Hodgkin's disease and 10 multiple myeloma) underwent peripheral blood progenitor cell (PBPC) collection after cyclophosphamide 7 g/m2 and rh-G-CSF. Fifty-eight leukaphereses were carried out with a fully automated PBPC collection procedure. Progenitor cell release was monitored by standardized determination of CD34+ cells in the peripheral blood. After a profound aplasia, a continuous increase in CD34+ cells in the peripheral blood was seen for at least 3-4 days. In 82% of our patients more than 2.5 x 10(6) CD34/kg could be collected using a standard apheresis of 10 l. There was a high correlation between the CD34+ cells in the peripheral blood and CD34+ cells/kg harvested. (r2 = 0.91). A relatively constant ratio (median 14.3, range 3.2-22.6) was found between CD34+ cells/kg and CFU-GM/kg. Based on the CD34 values of the pre-apheresis blood and the body weight of an individual patient and using the mathematical model of regression analysis (y = mx + b) for the correlation between the CD34+ cells/microliter in the pre-apheresis blood and the CD34+ cells/kg, it was possible to create a formula allowing for target value tailored apheresis. Using this formula, the blood volume which needs to be processed in order to harvest a desired number of CD34+ cells/kg can be calculated. This strategy can be applied to reduce the time for and the number of aphereses. Nineteen leukaphereses were carried out applying the formula. In 18 of 19 leukaphereses the expected CD34+/kg values were correctly achieved or exceeded. The formula was most reliable when the CD34 value was higher than 15/microliter and when the WBC count was below 20 x 10(9)/l in the pre-apheresis blood. For mobilizations using hematopoietic growth factors alone our formula is not applicable, because in most cases the pre-apheresis white blood cell count is higher than 20 x 10(9)/l and the collection efficacy of lymphomonocytoid cells decreases with a high pre-apheresis white blood cell count. The formula also works with other mobilization regimens that induce a pronounced aplasia.

Cancer in pregnancy: maternal-fetal conflict.

The occurrence of malignancies during pregnancy has increased over the last decades. They complicate approximately 1 per 1000 pregnancies. The most common malignancies associated with pregnancy include malignant melanoma, malignant lymphomas and leukemia, and cancer of the cervix, breast, ovary, colon and thyroid. Since it is impossible for prospective randomized clinical trials to be conducted in this field, relevant data have been generated from case reports and matched historical cohort studies in order to evaluate the treatment outcomes and the issues complicating the management of malignancy in the pregnant patient. There is almost always a conflict between optimal maternal therapy and fetal well-being. The maternal interest is for an immediate treatment of the recently diagnosed tumor. However, the optimal therapy, be it chemotherapy, radiotherapy or surgery, may impose great risks on the fetus. Consequently, either maternal or fetal health, or both, will be compromised. Therefore, both the pregnant patient and her physician are often in a dilemma as to the optimal course. On the basis of the medical facts, we discuss the issues raising potential ethical conflicts and present a practical ethical approach which may help to increase clarity in maternal-fetal conflicts. We review the available data informing the incidence and impact of the most common malignancies during pregnancy and their treatment on both the pregnant woman and her fetus. The optimal therapy for the tragic diagnosis of cancer in pregnancy requires a collaborative and interdisciplinary approach between gynecologists, oncologists, obstetricians, surgeons, neonatologists, psychologists, nursing staff and other disciplines. The purpose of this article is not to answer specific questions or to construct management schemes for specific tumors but to provide a framework for approaching some of these complex issues.

Chromosome analyses in chronic lymphocytic leukemia and related B-cell neoplasms.

Chromosome analyses were performed by routine G-banding in 29 patients with B-cell chronic lymphocytic leukemia (B-CLL), six with immunocytoma (IC), three with centroblastic-centrocytic (cb-cc) lymphoma, and one with hairy cell leukemia (HCL). Ages of the patients were between 46 and 81 years (mean, 63 years). 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was used as a mitogen to stimulate leukemic B-cells in 72-hour cultures. Twenty-one patients had one or more chromosomal abnormalities; and in 13 patients, they were clonal; 18 patients had a normal karyotype. Seven patients had trisomy 12 (three B-CLL, two IC, two cb-cc lymphoma); two (B-CLL) had it as the sole abnormality. One patient with B-CLL had trisomy 18 as the sole abnormality, and one with IC had trisomy 18 in combination with trisomy 19. One patient with B-CLL had t(1;6)(p36;p21) as a clonal structural abnormality. A t(11;14)(q13;q32) was consistently observed in one patient with cb-cc lymphoma together with inv(1) (p22p36), der(4)t(4;?)(p16;?), del(6)(q13) and other variable changes. One patient with morphologically atypical B-CLL had t(1;11)(p36;q13) together with der(X)t(X;?)(q26;?), der(3)t(3;?)(q29;?), der(8)t(4;8)(q12;q24.1) and additional variable changes. Both patients with these complex karyotypes were in an advanced stage of disease (Binet stage C) and died within 3-6 months after chromosome analysis.