Cold acclimation increases cold tolerance independently of diapause programing in the bean bug, Riptortus pedestris.

The bean bug (Riptortus pedestris) is a pest of soybeans and other legumes in Japan and other Asian countries. It enters a facultative adult diapause on exposure to short days. While photoperiodism and diapause are well understood in R. pedestris, knowledge of cold tolerance is very limited, as is information on the effect of diapause on cold tolerance. We examined the effect of photoperiod, cold acclimation, and feeding status on cold tolerance in R. pedestris. We found that cold acclimation significantly increased survival at -10°C in both long- and short-day adult R. pedestris. Since the difference in cold survival between long- and short-day cold-acclimated groups was only marginal, we conclude that entering diapause is not crucial for R. pedestris to successfully pass through cold acclimation and become cold tolerant. We observed similar effects in 5th instar nymphs, with both long- and short-day cold-acclimated groups surviving longer cold exposures compared with non-acclimated groups. Starvation, which was tested only in adult bugs, had only a negligible and negative impact on cold survival. Although cold tolerance significantly increased with cold acclimation in adult bugs, supercooling capacity unexpectedly decreased. Our results suggest that changes in supercooling capacity as well as in water content are unrelated to cold tolerance in R. pedestris. An analysis of metabolites revealed differences between the treatments, and while several metabolites markedly increased with cold acclimation, their concentrations were too low to have a significant effect on cold tolerance.

How fast is optically induced electron transfer in organic mixed valence systems?

The rate of thermally induced electron transfer in organic mixed valence compounds has thoroughly been investigated by e.g. temperature dependent ESR spectroscopy. However, almost nothing is known about the dynamics of optically induced electron transfer processes in such systems. Therefore, we investigated these processes in mixed valence compounds based on triphenylamine redox centres bridged by conjugated spacers by NIR transient absorption spectroscopy with fs-time resolution. These experiments revealed an internal conversion (IC) process to be on the order of 50-200 fs which is equivalent to the back electron transfer after optical excitation into the intervalence charge transfer band. This IC is followed by ultrafast cooling to the ground state within 1 ps. Thus, in the systems investigated optically induced electron transfer is about 3-4 orders of magnitude faster than thermally induced ET.

Establishing ecological reference conditions and tracking post-application effectiveness of lanthanum-saturated bentonite clay (Phoslock®) for reducing phosphorus in aquatic systems: an applied paleolimnological approach.

Innovative management strategies for nutrient enrichment of freshwater are important in the face of this increasing global problem, however many strategies are not assessed over long enough time periods to establish effectiveness. Paleolimnological techniques using diatoms as biological indicators were utilized to establish ecological reference conditions, environmental variation, and the effectiveness of lanthanum-saturated bentonite clay (brand name: Phoslock(®)) applied to reduce water column phosphorus (P) concentrations in four waterbodies in Ontario, Canada, and eastern Australia. In sediment cores from the two Canadian sites, there were short-lived changes to diatom assemblages, relative to inferred background conditions, and a temporary reduction in both measured and diatom-inferred total phosphorus (TP) before returning to pre-application conditions (particularly in the urban stormwater management pond which has a high flushing rate and responds rapidly to precipitation and surface run-off). The two Australian sites (a sewage treatment pond and a shallow recreational lake), recorded no reduction in diatom-inferred TP. Based on our pre-application environmental reconstruction, changes to the diatom assemblages and diatom-inferred TP appeared to be driven by larger, climatic factors. While laboratory tests involving this product showed sharp reductions in water column TP, management strategies require detailed information on pre-application environmental conditions and variations in order to accurately assess the effectiveness of new technologies for lake management.

Prevalence and risk factors for Salmonella in veal calves at Danish cattle abattoirs.

The study's objectives were to determine herd- and animal-level prevalence and herd-level risk factors for Salmonella in dairy-bred veal calves at slaughter in Denmark. In total, 1296 faecal samples were collected at five cattle abattoirs in Denmark during 2007-2008. The animals came from 71 randomly selected specialized veal-calf producers that delivered more than 100 animals to slaughter per year. Salmonella Dublin bacteria were isolated from 19 samples from 12 herds and Salmonella Typhimurium was isolated from one sample. The apparent prevalence of herds delivering Salmonella-shedding animals to slaughter was 18% (95% CI 9-27). The overall estimated true prevalence of shedding calves at slaughter was 1.3%. Veal-calf herds that purchased animals from herds not classified as low risk in the Danish Salmonella surveillance programme had significantly (P=0.03) higher risk of delivering Salmonella-shedding calves to slaughter. The results emphasize the importance of efforts in the dairy industry to ensure food safety for consumers.

Analysis of the emission profile in organic light-emitting devices.

In this paper, numerical algorithms for extraction of optoelectronic material and device parameters in organic light-emitting devices (OLEDs) are presented and tested for their practical use. Of particular interest is the extraction of the emission profile and the source spectrum. A linear and a nonlinear fitting method are presented and applied to emission spectra from OLEDs in order to determine the shape of the emission profile and source spectrum. The motivation of the work is that despite the existence of advanced numerical models for optical and electronic simulation of OLEDs, their practical use is limited if methods for the extraction of model parameters are not well established. Two fitting methods are presented and compared to each other and validated on the basis of consistency checks. Our investigations show the impact of the algorithms on the analysis of realistic OLED structures. It is shown that both fitting methods p form reasonably well, even if the emission spectra to be analyzed are noisy. In some cases the nonlinear method performs slightly better and can achieve a perfect resolution of the emission profile. However, the linear method provides the advantage that no assumption on the mathematical shape of the emission profile has to be made.

Astrocyte-derived TGF-beta 2 and NGF differentially regulate neural recognition molecule expression by cultured astrocytes.

Because of the importance of neural recognition molecules expressed by glial cells to mediate interactions with neurons, growth factors and cytokines known to be functional during morphogenesis and in diseases of the nervous system were studied for their effects on recognition molecule expression by cultured immature and mature astrocytes from several brain regions. In cultures of immature astrocytes, transforming growth factors-beta 1 (TGF-beta 1) and -beta 2 (TGF-beta 2) and nerve growth factor (NGF) increased expression of the neural adhesion molecule L1, leading to a glia-mediated L1-specific increase in neurite outgrowth of dorsal root ganglion neurons on the astrocyte substrate. L1 expression induced by TGF-beta was inhibited by addition of antibodies to NGF, suggesting that TGF-beta influences L1 expression by modulating production of NGF by astrocytes. TGF-beta 1 and -beta 2 decreased expression of N-CAM by immature astrocytes. Since N-CAM expression was not affected by NGF and antibodies to NGF did not abolish the TGF-beta-induced decrease in N-CAM expression, NGF did not appear to be the mediator for regulating expression of N-CAM. Expression of the adhesion molecule on glia (AMOG) was not affected by any factor. NGF and TGF-beta 2 in latent form, but not TGF-beta 1 were found in the culture supernatants. Addition of interferon-gamma (IFN-gamma), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), platelet-derived growth factor (PDGF), or basic fibroblast growth factor (bFGF) to the cultures did not change recognition molecule expression. REcognition molecule expression by mature astrocytes was not found to be modified by any of the factors tested. In view of the observation that levels of L1 and N-CAM expression correlated with the presence of TGF-beta 2 and NGF in the culture supernatants of immature astrocytes, an autocrine regulatory mechanism for recognition molecule expression by these cells is suggested to play a crucial role in regulation of neuron-glia interactions.

The adhesion molecule on glia (AMOG) is a homologue of the beta subunit of the Na,K-ATPase.

AMOG (adhesion molecule on glia) is a Ca2(+)-independent adhesion molecule which mediates selective neuron-astrocyte interaction in vitro (Antonicek, H., E. Persohn, and M. Schachner. 1987. J. Cell Biol. 104:1587-1595). Here we report the structure of AMOG and its association with the Na,K-ATPase. The complete cDNA sequence of mouse AMOG revealed 40% amino acid identity with the previously cloned beta subunit of rat brain Na,K-ATPase. Immunoaffinity-purified AMOG and the beta subunit of detergent-purified brain Na,K-ATPase had identical apparent molecular weights, and were immunologically cross-reactive. Immunoaffinity-purified AMOG was associated with a protein of 100,000 Mr. Monoclonal antibodies revealed that this associated protein comprised the alpha 2 (and possibly alpha 3) isoforms of the Na,K-ATPase catalytic subunit, but not alpha 1. The monoclonal AMOG antibody that blocks adhesion was shown to interact with Na,K-ATPase in intact cultured astrocytes by its ability to increase ouabain-inhibitable 86Rb+ uptake. AMOG-mediated adhesion occurred, however, both at 4 degrees C and in the presence of ouabain, an inhibitor of the Na,K-ATPase. Both AMOG and the beta subunit are predicted to be extracellularly exposed glycoproteins with single transmembrane segments, quite different in structure from the Na,K-ATPase alpha subunit or any other ion pump. We hypothesize that AMOG or variants of the beta subunit of the Na,K-ATPase, tightly associated with an alpha subunit, are recognition elements for adhesion that subsequently link cell adhesion with ion transport.

Heparan sulphate proteoglycans are essential for the myeloma cell growth activity of EGF-family ligands in multiple myeloma.

The epidermal growth factor (EGF)/EGF-receptor (ErbB1-4) family is involved in the biology of multiple myeloma (MM). In particular, ErbB-specific inhibitors induce strong apoptosis of myeloma cells (MMC) in vitro. To delineate the contribution of the 10 EGF-family ligands to the pathogenesis of MM, we have assessed their expression and biological activity. Comparing Affymetrix DNA-microarray-expression-profiles of CD138-purified plasma-cells from 65 MM-patients and 7 normal individuals to those of plasmablasts and B-cells, we found 5/10 EGF-family genes to be expressed in MMC. Neuregulin-2 and neuregulin-3 were expressed by MMC only, while neuregulin-1, amphiregulin and transforming growth factor-alpha were expressed by both MMC and normal plasma-cells. Using real-time polymerase chain reaction, we found HB-EGF, amphiregulin, neuregulin-1 and epiregulin to be expressed by cells from the bone marrow-environment. Only the EGF-members able to bind heparan-sulphate proteoglycans (HSPGs) - neuregulin-1, amphiregulin, HB-EGF - promote the growth of MMC. Those ligands strongly bind MMC through HSPGs. The binding and the MMC growth activity was abrogated by heparitinase, heparin or deletion of the HS-binding domain. The number of HS-binding EGF ligand molecules bound to MMC was higher than 10(5) molecules/cell and paralleled that of syndecan-1. Syndecan-1, the main HSPG present on MM cells, likely concentrates high levels of HS-binding-EGF-ligands at the cell membrane and facilitates ErbB-activation. Altogether, our data further identify EGF-signalling as promising target for MM-therapy.

Lack of CD56 expression on myeloma cells is not a marker for poor prognosis in patients treated by high-dose chemotherapy and is associated with translocation t(11;14).

Lack of CD56 expression was reported to be associated with a poor prognosis in multiple myeloma (MM) patients treated with conventional chemotherapy. Aim of our retrospective study was to analyse whether CD56 expression on MM cells reveals as a prognostic factor in patients treated with high-dose chemotherapy. MM cells of 99 patients prior to treatment with high-dose chemotherapy were analysed for CD56 expression by flow cytometry. Multivariable analysis of event-free survival in these patients showed no statistically significant difference between the CD56(-) (n=28) and the CD56(+) (n=71) group. The lack of CD56 expression on MM cells of these patients correlated significantly with the presence of translocation (11;14) (t(11;14)) (estimated correlation coefficient=0.655 95%, confidence interval (0.481; 0.779)). In summary, our results indicate that lack of CD56 expression on MM cells is not a prognostic marker in patients treated with high-dose chemotherapy, but is associated with t(11;14).

Single-step affinity purification, partial structure and properties of human platelet cGMP inhibited cAMP phosphodiesterase.

The human platelet cilostamide- and cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) was rapidly purified approximately 19,000-fold to apparent homogeneity using single step affinity chromatography on the isothiocyanate derivative of cilostamide coupled to aminoethyl agarose. Within 24 h, 30 micrograms of enzyme protein was obtained from 20 ml of packed platelets. Vmax for cAMP and cGMP was 6.1 and 0.9 mumol/min per mg protein, respectively. Several polypeptides (110/105, 79, 62, 55/53 kDa) were identified after SDS-PAGE, all of which were immunologically related to cGI-PDE and represented approx. 5, 20, 50 and 20% of the total protein, respectively. Limited proteolysis of the cGI-PDE with chymotrypsin produced a major fragment of approximately 47 kDa (and at least two smaller peptides) with catalytic activity and sensitivity to cGMP and OPC 3911 similar to controls. Phosphorylation of the cGI-PDE by cAMP-dependent protein kinase (A-kinase) resulted in maximal incorporation of 0.6-1.8 mol of 32P/mol 110/105 and 79 kDa polypeptides; much lower and variable amounts of phosphate were incorporated into the 62 and 55/53 kDa polypeptides. After digestion of cGI-PDE with several proteinases a number of peptides were isolated and sequenced. Most of the peptide sequences obtained could be aligned within the carboxy terminal domain of the deduced sequence of the human cardiac cGI-PDE. These and other results suggest that the subunit size of the intact platelet cGI-PDE is 110 kDa and that proteolytic fragments of 79, 62 and 55/53 kDa are produced during purification. The smaller fragments (62 and 55/53 kDa) contain the catalytic domain; the larger fragments (110 and 79 kDa) also contain the regulatory domain with phosphorylation sites for A-kinase.

Sequential high-dose therapy with peripheral-blood progenitor-cell support in low-grade non-Hodgkin's lymphoma.

PURPOSE: To evaluate the feasibility of a sequential high-dose therapy with peripheral-blood progenitor-cell (PBPC) support in patients with follicular lymphoma. PATIENTS AND METHODS: Since July 1991, we have included 30 patients (17 men and 13 women) with a median age of 41 years (range, 26 to 55) in the study. At the time of study entry, 17 patients were in first and six in second or higher remission. Another six patients had relapse of disease and one had tumor progression. PBPC were collected during filgrastim-supported leukocyte recovery following high-dose cytarabine (ara-C)/mitoxantrone (HAM). RESULTS: A median of two leukaphereses (range, one to seven) resulted in a median of 5.7 x 10(6) CD34+ cells/kg (range, 2.9 to 23.7 x 10(6). A distinct population of B-lymphoid progenitors (CD34+/CD19+) was not detectable in the autografts, and the content of CD19+ B cells was remarkably low, comprising a median of 0.07% of the mononuclear cells. Using the polymerase chain reaction (PCR) assay for the major breakpoint regions (MBR) of the bcl-2/immunoglobulin H (IgH) translocation, 22 patients had autografts positive for the t(14;18) translocation, whereas seven patients had PCR-negative transplants. The autograft of one patient could not be assessed. Following myeloablative therapy, hematologic recovery was rapid without cytokine support. The median times to reach a platelet count > or = 20 x 10(9)/L and neutrophil count > or = 0.5 x 10(9)/L were 11 and 13 days, respectively. Nonhematologic toxicity was moderate. Twenty-nine patients were alive in remission after a median follow-up duration of 6 months (range, 1 to 18). Of 22 patients autografted with t(14;18)-positive harvests, 11 had PCR-detectable cells in bone marrow and/or peripheral blood as long as 16 months posttransplantation. In contrast, six patients became PCR-negative between 3 and 16 months after reinfusion. Follow-up examinations with PCR data for the remaining five patients are not yet available. CONCLUSION: Conversion to PCR negativity in patients autografted with PCR-positive harvests suggests that the myeloablative regimen is effective and that any reinfused t(14;18)-positive cells may not be sustained. Because conventional chemotherapy provides no cure, we believe that high-dose therapy including total-body irradiation (TBI) should be explored in these particularly radiosensitive lymphomas.

High-dose chemotherapy in multiple myeloma.

The median survival of conventionally treated patients with multiple myeloma is 3 years. Modifications of conventional chemotherapy have failed to show an improved survival rate in most randomized trials. Therapy regimens with dose-escalated alkylating agents (ie melphalan) have induced higher remission rates in comparison to conventional treatment modalities. With the support of autologous or allogeneic hematopoietic progenitor cells, it has been possible to reduce the hematoxicity of these dose-escalated treatments. The transplantation of autologous peripheral blood progenitor cells results in faster hematopoietic reconstitution with decreased high-dose therapy-related morbidity compared to autologous bone marrow. The randomized French myeloma trial and the pair-mate analysis of the results of the 'total therapy' including double autografting of the Barlogie group with data from the South Western Oncology Group (SWOG) showed a significant survival advantage for patients following autologous transplantation. Although a graft-versus-myeloma effect was described, the benefit of high-dose treatment with allogeneic transplantation is less clear, mainly due to the high transplantation-related mortality rate. In this paper, results of transplantation trials are summarized. Prognostic factors and future treatment modalities for myeloma are discussed.

The remission status before and the PCR status after high-dose therapy with peripheral blood stem cell support are prognostic factors for relapse-free survival in patients with follicular non-Hodgkin's lymphoma.

It was the aim of our study to examine the clinical significance of t(14;18)-positive cells in samples from 47 patients with follicular non-Hodgkin's lymphoma (NHL) who underwent high-dose therapy with autologous peripheral blood stem cell (PBSC) transplantation. At the time of PBSC mobilization, 25 patients were in first remission, while 22 patients had a history of previous treatment failure. At the same time, 43 patients had polymerase chain reaction (PCR)-positive cells in samples from bone marrow (BM) and/or peripheral blood (PB). Independent of the remission status, high-dose cytarabine and mitoxantrone with granulocyte colony-stimulating factor (G-CSF) support were administered for PBSC mobilization. Following high-dose conditioning therapy which consisted of cyclophosphamide (200 mg/kg) and hyperfractionated total body irradiation (TBI, 14.4 Gy) or BEAM (carmustine, etoposide, cytarabine, melphalan), 34 patients received PCR-positive and 13 patients received PCR-negative autografts. After a median follow-up time of 20 months (range, 6-50) post-transplantation, 33 patients were in remission, while 14 patients had relapsed after a median time of 14.5 months (range, 10-42). Using the Andersen-Gill proportional hazards regression model for the analysis of relapse-free survival, we found that PCR-positive findings in samples from BM and/or PB at any given time-point after transplantation were associated with an increased estimated hazard ratio of 4.5 in comparison with a PCR-negative finding (P=0.013). On the other hand, patients included while they were in first remission had a smaller estimated hazard ratio of 0.3 when compared with patients with a history of previous treatment failure (P=0.048). For the latter group of patients, this translates into a significantly smaller probability of relapse-free survival in comparison to patients who were in first remission at the time of PBSC-mobilization (P=0.012). In conclusion, the remission status of the patients before autografting and the PCR status as assessed on the occasion of follow-up examinations are significant prognostic parameters for relapse-free survival in patients with follicular lymphoma undergoing high-dose therapy with PBSC autografting.

A rationale for positive selection of peripheral blood stem cells in multiple myeloma: highly purified CD34+ cell fractions of leukapheresis products do not contain malignant cells.

In multiple myeloma (MM), the presence of tumor cells in leukapheresis products (LP) has been demonstrated with highly sensitive molecular biological tools in up to 100% of cases. Therefore methods to reduce the tumor load of LP by CD34+ selection are envisaged. However, there is controversy as to whether the CD34+ cell is already involved in the malignant process. We have established a PCR assay with allele-specific oligonucleotide primers (ASO) complementary to the CDR3-hypervariable region of the immunoglobulin heavy chain gene of each patient's myeloma clone. Using this ASO-PCR, 43 LP of 10 patients with MM eligible for high-dose therapy were assessed for malignant cells. Furthermore, in an experimental setting we have examined 10 CD34+ and four CD19+ fractions obtained from PCR-positive LP by sequential preparative magnetic and fluorescence activated cell sorting (purity >96%) for the presence of the tumor-specific CDR3 region. The majority of LP harbored cells of the myeloma clone (93%), while all CD34+ fractions were PCR-negative. In all CD19+ fractions malignant cells were detected. These results confirm that CD34+ selection can be considered for LP in MM. The sensitivity of the ASO-PCR (up to 10[-5]) enables us further to monitor the efficacy of CD34+ enrichment protocols in the clinical setting.

Leukapheresis products in multiple myeloma: lower tumor load after mobilization with cyclophosphamide plus granulocyte colony-stimulating factor (G-CSF) compared with G-CSF alone.

High-dose therapy with autografting of peripheral blood stem cells (PBSCs) has become an accepted treatment modality. However, gene-marking studies in patients with acute myeloid leukemia and neuroblastoma have revealed that malignant cells reinfused along with leukapheresis products (LPs) contribute to relapse. Thus, a reduction in the number of malignant cells in autografts is desirable. We analyzed the percentage of malignant cells and the number of CD34+ PBSCs in LPs mobilized by granulocyte colony-stimulating factor (G-CSF) alone (LP-S) compared with high-dose cyclophosphamide plus G-CSF (LP-CY) in patients with multiple myeloma (MM). A quantitative polymerase chain reaction assay involving CDR3-specific primers based on the method of limiting dilutions was used to determine the tumor loads of LPs. Sixteen LPs from eight patients with MM were analyzed intraindividually in matched pairs. The percentage of malignant cells was lower in LP-CY (p = 0.017; median 0.0067 vs. 0.009%), whereas the number of CD34+ cells was higher (p = 0.012; median 0.3 vs. 0.095%). The calculated number of malignant cells per CD34+ cell was significantly lower in LP-CY as well (p = 0.017). We conclude that mobilization by cyclophosphamide plus G-CSF leads to a lower number of malignant cells per CD34+ cell in LPs compared with G-CSF alone.

DNA sequencing from single phage plaques using solid-phase magnetic capture.

Many operations encountered in molecular cloning are labor-intensive and time-consuming. One case that is often troublesome is the subcloning of cDNA clones from lambda gt11 phage into plasmid vectors. In situations where several clones have been isolated, time could be saved by a means of assessing insert size and sequence unambiguously without subcloning, particularly where degenerate PCR or low-stringency hybridization approaches are taken to identify multiple members of a gene family. We describe a simple and reliable strategy for efficient sequencing of small amounts of lambda phage DNA, lysates or individual phage plaques. The strategy combines the advantages of universal lambda phage primers, rapid air thermal cycling, streptavidin magnetic bead capture of highly purified single-stranded templates and the unparalleled clarity of T7 DNA polymerase sequence. We routinely obtain 350-500 bases of unambiguous sequence from each reaction. It takes only hours from lifting phage plaques to finishing the sequencing reactions. The method provides an alternative to thermal cycle sequencing that has comparable sensitivity and affords sequence data of much higher clarity.

First and second apheresis in patients with multiple myeloma: no differences in tumor load and hematopoietic stem cell yield.

Autologous peripheral blood stem cells (PBSC) are now widely used to support myeloablative therapy in patients with multiple myeloma (MM). The presence of malignant cells in these autografts has been demonstrated. Characteristic kinetics with differential and concomitant mobilization of CD34+ and malignant cells after high-dose (HD) chemotherapy and hematopoietic growth factor administration have been reported. We determined the amounts of tumor cells and PBSC in leukapheresis products (LP) collected on day 1 (LP1) and 2 (LP2) from 16 MM patients harvested after HD chemotherapy and G-CSF. Furthermore, LP from six patients collected on day 5 (LP5) could be examined. The content of clonotypic cells was quantitated by an allele-specific oligonucleotide (ASO)-PCR assay based on limiting dilutions. CD34+ PBSC were determined by flow cytometry. The percentages of malignant cells in the leukapheresis products were in the range of 0% to 0.713% (mean 0.047%). CD34+ cells ranged between 0.06% and 5.4% (mean 1.23%). Comparing LP1 with LP2, no differences in the quantity of tumor cells (mean 0.0538% vs 0.0448%; P = 0.96) and CD34+ cells (mean 1.49% vs 1.33%; P= 0.50) were seen. The calculated number of tumor cells per CD34+ cell did not differ significantly (mean 0.0420 vs 0.0249; P = 0.65). Analyzing LP5 revealed no changes in the number of tumor cells per CD34+ cell (0.0511 vs 0.1044; P = 0.46) indicating a relatively constant ratio of PBSC to tumor cells during the course of PBSC harvesting. These results offer the possibility of combining LP harvested over several days without increasing the tumor load per CD34+ cell.

Molecular monitoring of the tumor load predicts progressive disease in patients with multiple myeloma after high-dose therapy with autologous peripheral blood stem cell transplantation.

The clinical relevance of the assessment of minimal residual disease (MRD) in patients with multiple myeloma (MM) to predict disease recurrence has not been proven. In the present study, we retrospectively analyzed the tumor load in peripheral blood (PB) and bone marrow (BM) samples of 13 patients with MM both in remission after high-dose therapy (HDT) with autologous PBSC transplantation (PBSCT) and at the time of progressive disease (PD). For six patients, subsequent samples obtained in remission could be included in the study. Tumor cells were assessed by means of quantitative PCR with allele-specific oligonucleotides (ASO-qPCR) based on the method of limiting dilutions. PD was documented with ASO-qPCR in BM samples (median concentration of tumor cells in remission vs at PD: 0.18% vs 4.6%) representing a significant increase by a median factor of 8.7. In PB, the median tumor load was 799 cells/ml in remission and 23 400 cells/ml at PD. With a median factor of 45, the increase was much more pronounced. Comparing the results of the molecular monitoring in PB with those of the determination of the monoclonal protein, routinely applied as parameter for the course of the disease, revealed a superiority of the molecular monitoring because of the significantly higher increase in the tumor load. Analyzing the subsequent remission samples showed an increase of the malignant cells in four out of six PB samples and in all four BM samples available, indicating the potential of ASO-qPCR for an early PD recognition.

Analysis of circulating tumor cells in patients with multiple myeloma during the course of high-dose therapy with peripheral blood stem cell transplantation.

In multiple myeloma (MM) circulating CD19+ cells have been considered as myeloma precursors. As these cells are also possibly a reservoir of treatment resistant disease evaluation of the CD19+ cells during the course of high-dose therapy has to be a major concern. We determined the number of tumor cells in the CD19+ as well as CD19- fractions of PB of eight patients with disease sensitive to VA[I]D chemotherapy, of 10 patients who achieved partial or complete remission post-high-dose therapy (HDT) with peripheral blood stem cell transplantation (PBSCT) and of a further seven patients with disease progression post-transplantation. CD19+ cell fractions were obtained by preparative sequential magnetic and fluorescence activated cell sorting with a median purity of 97.1%. In addition, PB samples of seven patients post-transplantation were sorted for CD20+ cells (median purity, 98.7%). The number of tumor cells in the CD19+, the CD19- and the CD20+ fractions were determined using a quantitative CDR3 PCR assay. The number of CD19+ tumor cells in patients in remission post-HDT was similar to those of the patients post-VA[I]D (median, 1.05 vs 0.92 CD19+ tumor cells/ml PB, P = 0.72) providing evidence for the persistence of this tumor cell fraction during the course of HDT. This was in contrast to the CD19- compartment, in which the number of tumor cells was significantly reduced in those patients in remission post-transplantation (median, 53 vs 0 CD19- tumor cells/ml PB; P = 0.006). In patients with progressive disease the number of tumor cells in both cell fractions was significantly higher (CD19+: median, 1.05 vs 21 tumor cells/ml PB, P = 0.05; CD19-: 0 vs 63 tumor cells/ml PB, P = 0.008). While the absolute number of CD19+ cells was reduced in the group of patients after VA[I]D treatment, a polyclonal CD19+ reconstitution had occurred in patients responding to HDT. The tumor cell content in the CD19+ fractions could be confirmed by the results obtained analyzing the CD20+ cell fractions. In conclusion, these results indicate that disease progression after PBSCT in MM is accompanied by an expansion of tumor cells in both the CD19+ and CD19- fractions. Similar numbers of CD19+ clonotypic cells post-HDT suggest that these cells persist and thus, contribute to disease dissemination and relapse.

Evaluation of the kinetics of the bone marrow tumor load in the course of sequential high-dose therapy assessed by quantitative PCR as a predictive parameter in patients with multiple myeloma.

The aim of this investigation was to examine the possible clinical significance of the kinetics of bone marrow (BM) tumor load during the course of sequential high-dose therapy (HDT) as assessed by quantitative PCR in patients with multiple myeloma. In 20 patients with multiple myeloma (MM) treated with two consecutive cycles of HDT followed by autologous peripheral blood stem cell transplantation (PBSCT), clonotypic cells in the peripheral blood (PB) and BM were quantitated by PCR using allele-specific oligonucleotides (ASO) prior to the first, immediately prior to the second, and after the second HDT. The median proportion of clonotypic cells in the BM was 1.27% before the first HDT (range, 0.03-70%), 0.17% after the first (range, 0.001-22%), and 0.05% after the second HDT (range, 0.00009-1.44%). The median number of circulating clonotypic cells was 65/ml (range, 0.9-10842) prior to HDT, 2.7/ml (range, 0-315) after the first, and 3.5/ml PB (range, 0.7-97) after the second HDT. While the median BM tumor load decreased during the first (P = 0.03) and second (P = 0.044) HDT cycles, only the first cycle resulted in a reduction of clonotypic cells in the PB (P = 0.00078 and P= 1.0, respectively). In seven patients, the BM tumor load did not decrease below the initial level after one or two cycles of HDT. All of these patients developed progressive disease (median, 19 months post first cycle; range, 10-21). Of the remaining 13 patients, only four relapsed (18, 19, 21 and 22 months after the first cycle of HDT), while nine remain in response (median followup, 29 months; range, 18-41) (log-rank test P = 0.0009). Our results indicate that the kinetics of the BM tumor load is a predictive parameter in patients with MM and identifies those patients who could benefit from further therapy including new treatment modalities.