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The diagnostic criteria for CLL rely on morphology and immunophenotype. Current approaches have limitations affecting reproducibility and there is no consensus on the role of new markers. The aim o ...

18F-fluorodeoxyglucose PET/CT (PET/CT) is the current state-of-the-art in the staging of diffuse large B-cell lymphoma (DLBCL) and has a high sensitivity for extranodal involvement. Therefore, reassessment of extranodal involvement and the current prognostic indices in the PET/CT era is warranted. We screened patients with newly diagnosed DLBCL seen at the academic centers of Aalborg, Copenhagen, and British Columbia for eligibility. Patients that had been staged with PET/CT and treated with R-CHOP(-like) 1(st) line treatment were retrospectively included. In total 443 patients met the inclusion criteria. With a median follow-up of 2.4 years, the 3-year overall (OS) and progression-free survival (PFS) were 73% and 69%, respectively. The Ann Arbor classification had no prognostic impact in itself with the exception of stage IV disease (HR 2.14 for PFS, P 2 extranodal sites, including HR 7.81 (P 3 sites. Bone/bone marrow involvement was the most commonly involved extranodal site identified by PET/CT (29%) and was associated with an inferior PFS and OS. The IPI, R-IPI, and NCCN-IPI were predictive of PFS and OS, and the two latter could identify a very good prognostic subgroup with 3-year PFS and OS of 100%. PET/CT-ascertained extranodal involvement in DLBCL is common and involvement of >2 extranodal sites is associated with a dismal outcome. The IPI, R-IPI, and NCCN-IPI predict outcome with high accuracy.

Patients with multiple myeloma (MM) suffer from a general impaired immunity comprising deficiencies in humoral responses, T-cell responses as well as dendritic cell (DC) function. Thus, to achieve control of tumour growth through immune therapy constitutes a challenge. Careful evaluation of the immune status in patients with MM seems crucial prior to active immune therapy. We evaluated the proportion of both, DC, Treg cells and myeloid-derived suppressor cells (MDSC) in peripheral blood from patients with MM at diagnosis and in remission as well as patients with monoclonal gammopathy of undetermined significance (MGUS). We found that the proportion of both myeloid (m) DC and plasmacytoid (p) DC in patients at diagnosis was lowered compared to control donors, while only the proportion of pDC in patients in remission and with MGUS was significantly lower than in controls. The proportion of CD4+FOXP3+ Treg cells was increased in patients at diagnosis and not in patients in remission or with MGUS. Also, Treg cells from patients with MM were functionally intact as they were able to inhibit proliferation of both CD4 and CD8 T cells. Finally, we observed an increase in the proportion of CD14+HLA-DR⁻/low MDSC in patients with MM at diagnosis, illustrating that this cell fraction is also distorted in patients with MM. Taken together, our results illustrate that, both mDC, pDC, Treg cells and MDSC are affected in patients with MM underlining the fact that the immune system is dysregulated as a consequence of the disease.

Alloreactive cytotoxic lymphocytes present in peripheral blood of two normal humans have been studied by rosette fractionation experiments. It is shown that the effector cells, have receptors for SRBC, low avidity FcR, are nylon non-adherent and without CR. These results reveal a phenotype of membrane markers which is very much like the phenotypes of the major part of K cells, NK cells and the effector cells in mitogen induced cell-mediated cytotoxicity, indicating a common ancestor in the immune system. From a functional point of view all these cytotoxic effector cells may be T cells, some specific others non specific, in parallel to T helper and suppressor lymphocytes.

In a study of lymphocyte subpopulations in peripheral blood during infectious mononucleosis the lymphocytosis was found to be of T-cell origin (i.e. E-RFC), while the number of non T lymphocytes (i.e. EA-, EAC-RFC and Smlg + ve cells) was normal in 7 out of 8 patients. Ten patients were tested for the presence of HLA-DR determinants on their B and T cells and not only B lymphocytes but also a great part (31–75 96) of T cells were lysed by the anti HLA-DR testsera, indicating that HLA-DR determinants are expressed on a population of T cells in IM patients. After recovery all patients were retested and showed a normal pattern of HLA-DR typing. This indicates an increase of HLA-DR antigens on T cells or a vigorous proliferation of a small DR-positive T-cell subpopulation during the acute stage of IM.

In a randomized study of 18 adult patients with high-risk or advanced acute myeloid leukemia (AML) we investigated the effect of supplementing conventional induction chemotherapy with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF). For comparison, a historical control group of 90 patients treated for de novo AML with conventional chemotherapy during the previous period, 1984–1990, was also analyzed. Before induction chemotherapy, 10 patients were randomized to receiving rhGM-CSF, starting on day 1 to 3 before chemotherapy and continued for a maximum of 21 days after the start of induction treatment. Fatal complications and treatment outcome did not differ between the study groups and historical controls. Nor were there any differences between the groups in terms of hematological toxicity, e.g. time to three-lineage regeneration and need for supportive therapy. However, sequential weekly bone marrow examinations revealed a prolonged reduction of the relative number of myeloid (CD33-positive) marrow cells in the rhGM-CSF treated group. Although the small number of patients studied may not permit a definite conclusion, this randomized study did not demonstrate major beneficial effects of combining rhGM-CSF with standard induction chemotherapy in high-risk patients with AML.

3 children who fulfilled the criteria for the diagnosis of paroxysmal cold haemoglobinuria of the classical Donath-Landsteiner type are described. The benign clinical course of this illness is confirmed. A serological study of two of the Donath-Landsteiner antibodies revealed the anti-P specificity in one, but not in another serum.

The in vitro immune functions of peripheral blood lymphocytes have been studied in 70 splenectomized patients. 45 patients were splenectomized due to traumatic rupture of the spleen; in 22 of these patients residual splenic tissue was detected, employing a selective spleen scintigraphy. 14 patients were splenectomized due to hereditary spherocytosis and 11 patients due to immune thrombocytopenia or autoimmune haemolytic anaemia; they were all without ectopic splenic tissue. The study revealed that splenectomized patients have (i) an elevated number of blood lymphocytes, (ii) an elevated relative number of EA-RFC, but normal %s of E, EAC-RFC and SmIg positive cells, (iii) normal T-cell mitogenic responses induced by PHA, but enhanced responses induced by ConA and PWM, (iv) normal cell-mediated enhancement of the PWM-induced proliferative B-cell response, (v) no cell-mediated inhibition of the T-cell dependent and PWM-induced proliferative B-cell response and (vi) an impaired number of PFC after stimulation with PWM. The findings were unrelated to the cause of the splenectomies or to the presence of residual splenic tissue. It is possible that the impaired B-cell response as shown by the reduced number of PFC after stimulation with PWM may be of significance for the in vivo resistance to infections in splenectomized patients.

A method for preparation of purified human PWM stimulated B lymphoblasts and their usefulness as targets in CML is described. The purified B lymphoblasts were found to carry surface membrane immunoglobulin (SmIg) and HLA-DR antigens and were non E-rosette forming. In contrast, PHA-stimulated lymphoblasts were found to be E-rosetting, SmIg negative and without serologically detectable HLA-DR antigens, and thus were characterized as T lymphoblasts. Using B and T lymphoblasts in parallel as targets in CML, it was possible to demonstrate target determinants exclusively expressed by the B lymphoblasts.

Aiming at the production of anti HLA--DR test sera, eight healthy human volunteers were immunized by repeated intradermal injections of lymphocytes which were selected to be incompatible for one HLA--DR antigen, and matched as well as possible for HLA--A,--B,--C antigens. One out of 3 recipients immunized exclusively against HLA--DR produced lymphocytotoxic HLA-DR antibodies. The remaining 5 recipients were immunized against 1 or more HLA--A,--B,--C antigens in addition to one HLA--DR antigen. After 3 immunizations, 3 of these reacted with strong HLA--A or --B antibody production; however, only one showed a parallel anti HLA-DR antibody response detectable by complement dependent lymphocytotoxicity. Testing of the recipient sera in the antibody dependent cell-mediated cytotoxicity (ADCC) assay revealed that 6 of the 8 recipients did react early to the immunizations with HLA specific antibody production. However, in spite of repeated booster injections it was not possible to obtain more than the above-mentioned 2 sera with HLA--DR antibodies strong enough to react in the lymphocytotoxicity microtechnique.

The present study analyses the influence of high-dose chemotherapy (HD) and autologous stem cell transplantation on natural and vaccine induced specific immunity in multiple myeloma patients. Peripheral blood was collected from six multiple myeloma (MM) patients at serial time points in connection with treatment and during a follow-up period of 3 months. T-cell response to cytomegalovirus (CMV), varicella zoster virus (VZV) and tetanus toxoid (TT) was determined by flow cytometry analysis for CD69, TNFalpha, IFNgamma, IL-4 expression and cell proliferation. At diagnosis and prior to induction chemotherapy TNFalpha expressing T cells in 5/6 patients were found specific for CMV, 3/6 for VZV and 4/6 for TT. Serial analyses during treatment conclude impaired immune response, however, 3 months post-transplantation all but one patient had regained cytokine expressing CD8(+) T cells specific for CMV, VZV and TT. The highest percentages of cytokine responding T cells were observed after stimulation with CMV antigen. A striking observation was the low cytokine reactivity (close to zero) measured in G-CSF mobilized blood at the time of leukapheresis. In spite of a general reduction of the CD4/CD8 ratio following transplantation, recovery of antigen specific CD4(+) T cells reactivity generally occurred prior to CD8(+) recovery and often to a higher level. In conclusion, the study demonstrates that natural as well as vaccine induced specific immunity present prior to HD was regained after stem cell transplantation, hence identifying a possible window for future vaccination trials.

The frequently detected t(4;14)(p16.3;q32) translocation in multiple myeloma (MM) results in a dysregulation of two potential oncogenes: multiple myeloma SET domain (MMSET) and fibroblast growth factor receptor 3 (FGFR3). As the expression of FGFR3 is undetectable in 30% of the t(4;14)+ MM patients, MMSET has been suggested to play an important role in the malignant transformation associated with the t(4;14) translocation. Screening with a real-time polymerase chain reaction (PCR) found complex expression patterns of the MMSET transcripts in fluorescence-activated cell sorted (FACS)-purified plasma cells (PCs) from 15 t(4;14)+ MM patients. In addition, potential target genes of MMSET type I and II were identified, using microarray analyses of MMSET transfected cell lines. Subsequently, the expression of potential target genes was verified by real-time PCR in FACS-purified PCs from 15 t(4;14)+ and 22 t(4;14)- MM patients. We suggest that the inhibitor of differentiation 1 (ID-1) is a target gene of MMSET, based on its upregulation in MMSET transfected cell lines and a significant association between the t(4;14) translocation and ID-1 expression in MM patients (P = 0.002). As high levels of ID-1 are associated with cancer, our findings indicate that MMSET promotes oncogenic transformation in t(4;14)+ MM patients by transcriptional activation of ID-1 expression.

Dendritic cells (DC) are promising candidates for cancer immunotherapy. However, it is not known whether in vitro-generated monocyte-derived DC from cancer patients are altered compared with DC from healthy donors. In a clinical phase I/II study, monocyte-derived DC were generated in vitro utilizing granulocyte macrophage colony-stimulating factor and rh-interleukin-4 (IL-4) and used for cancer immunotherapy. In this study, we tested the effect of various maturation cocktails and performed a comparative evaluation of the DC phenotype and functional characteristics. Polyriboinosinic polyribocytidylic acid (Poly I:C) + tumour necrosis factor-alpha (TNF-alpha) induced significant IL-12 p70 secretion, which was increased after addition of a decoy IL-10 receptor. The lymph node homing chemokine receptor CCR-7 expression was induced by TNF-alpha + IL-1beta + IL-6 + prostaglandin E2 but was not induced by Poly I:C + TNF-alpha. In general, DC from patients had an intermediate maturity phenotype with a significantly higher expression of CD40 and CD54 compared with healthy donors. In vitro analyses showed an unimpaired capacity of the patient-derived DC for antigen-specific (cytomegalovirus, tetanus and keyhole limpet haemocyanin) T-cell stimulation, whereas the allostimulatory capacity of patient-derived DC was significantly decreased. These data suggest that patient-derived DC are more differentiated but are less sensitive to maturation-inducing agents than DC obtained from healthy individuals.

SummaryPrevious findings of megakaryocytic hypog ranulation and dysmegakaryocytopoieticfeatures in acute myeloid leukaemia (AML) strongly indicate defects inplatelet production. The bleeding tendency of these patients may result fromdysregulated platelet production, resulting in thrombocytopenia as well asqualitative platelet defects. The present study examined platelet function atdiagnosis in 50 AML patients by whole blood flow cytometry. Following invitro platelet agonist stimulation, platelet activation markers were analysedand compared with 20 healthy individuals. To detect recent in vivo plateletactivation, plasma soluble P-selectin (sP-selectin) was measured. Flowcytometric analysis of platelet activation markers demonstrated reducedCD62P [35AE6 vs. 118AE5 · 10 3 molecules of equivalent soluble fluorochrome(MESF); P

This study investigated the expression pattern in primary plasma cells (PCs) of putative oncogenes suggested to be involved in multiple myeloma (MM) development. cDNA archives were generated by global reverse transcription polymerase chain reaction from CD38++/CD19-/CD56-/++ aberrant PCs of a prospective cohort of 96 subjects, including healthy individuals, patients with monoclonal gammopathies of undetermined significance (MGUS), MM and MM with extramedullary manifestations (ExMM). The cDNA archives were analysed quantitatively for expression of the cyclin D1, fibroblast growth factor receptor 3 (FGFR3), C-MYC, C-MAF and cyclin D3 oncogenes. In addition, all patients were screened for IGH-MMSET hybrid transcripts. None of the analysed oncogenes was randomly distributed. C-MYC and cyclin D3 expression increased at the extramedullary transformation stage. Furthermore, C-MYC and cyclin D3 expression in CD56+ MM was similar to MGUS, whereas CD56- MM was similar to ExMM. FGFR3/IGH-MMSET was only observed among CD56+ MM patients, whereas an increased frequency of C-MAF dysregulation was seen among CD56- MM. High cyclin D1 expression levels were identified at similar frequencies at all stages, whereas the frequency of patients with low cyclin D1 levels increased during MM development. These data support the stepwise transformation model accumulating genetic alterations and proliferative capacity during MM initiation and development resulting in different clinical entities.

Multiple myeloma (MM) is considered to be an essentially incurable haematological malignant disease, probably because of the existence of resistant clonal precursor cell with self-renewal capacity. Recent data have indicated that the myeloma cell hierarchy includes circulating clonal memory B cells, which differ considerably from the classical end-stage plasma cells, infiltrating the bone marrow. The pathophysiological significance of these cells is unknown, but hypothetically they may serve as ‘sleeping’ myeloma stem cells responsible for and ‘feeding’ post-treatment relapse and progression. The present study evaluates the toxicity and feasibility of fludarabine, added to the VAD-induction regimen in MM, and investigates the effect on the myeloma cell hierarchy. Nineteen patients were randomised to receive either four cycles of VAD (n = 9) or two cycles of VAD, followed by two cycles of VAD combined with 5 days fludarabine 25 mg/m2/day i.v. (n = 10). Toxicity evaluation showed more profound neutropenia in the fludarabine-treated patients and two infectious episodes in each study arm: three were fever of unknown origin while one, in the fludarabine-arm, was a local skin infection at the insertion site of the central venous line. Nine of the fludarabine-treated patients responded to treatment (two complete remission, seven partial remission), compared with five responders (all PR) in the control-arm. The effects on the blood circulating myeloma compartments identified an increased reduction of CD19+ B cells and myeloma plasma cells in the fludarabine-arm. In conclusion, adding fludarabine to VAD induction in multiple myeloma is feasible and may be clinically effective by reducing the myeloma clone.

Background Cryopreservation is an acknowledged procedure to store vital cells for future biomarker analyses. Few studies, however, have analyzed the impact of the cryopreservation on phenotyping. Methods We have performed a controlled comparison of cryopreserved and fresh cellular aliquots prepared from individual healthy donors. We studied circulating B-cell subset membrane markers and global gene expression, respectively by multiparametric flow cytometry and microarray data. Extensive statistical analysis of the generated data tested the concept that “overall, there are no phenotypic differences between cryopreserved and fresh B-cell subsets.” Subsequently, we performed an uncontrolled comparison of tonsil tissue samples. Results By multiparametric flow analysis, we documented no significant changes following cryopreservation of subset frequencies or membrane intensity for the differentiation markers CD19, CD20, CD22, CD27, CD38, CD45, and CD200. By gene expression profiling following cryopreservation, across all samples, only 16 out of 18708 genes were significantly up or down regulated, including FOSB, KLF4, RBP7, ANXA1 or CLC, DEFA3, respectively. Implementation of cryopreserved tissue in our research program allowed us to present a performance analysis, by comparing cryopreserved and fresh tonsil tissue. As expected, phenotypic differences were identified, but to an extent that did not affect the performance of the cryopreserved tissue to generate specific B-cell subset associated gene signatures and assign subset phenotypes to independent tissue samples. Conclusions We have confirmed our working concept and illustrated the usefulness of vital cryopreserved cell suspensions for phenotypic studies of the normal B-cell hierarchy; however, storage procedures need to be delineated by tissue-specific comparative analysis. © 2014 International Clinical Cytometry Society

Background. Cells belonging to the malignant clone are found in the peripheral blood in myeloma patients. In order to minimise the content of tumour cells in the stem cell product it is crucial to perform stem cell harvest at a time when tumour cells in the peripheral blood are at a minimum. Objective: The aim of the study was to compare the mobilisation kinetics of normal CD34 + cells and myeloma plasma cells during mobilisation with either G-CSF alone or high-dose cyclophosphamide (HDCy) plus G-CSF. Design and methods: Morning blood samples were drawn each day during mobilisation from start of G-CSF or HDCy and to the end of leukapheresis, and were analysed by flow cytometry for content of CD34 + cells and myeloma plasma cells (CD38 + +CD45 - ). Tumour cells were also estimated by a patient-specific real-time polymerase chain reaction (PCR) method based on the 5' nuclease TaqMan technology. Results: Flow cytometry data from 16 patients showed concomitant mobilisation of CD34 + cells and myeloma plasma cells. Seven patients were mobilised twice; first with G-CSF alone and then with HDCy plus G-CSF. There was no difference between the two mobilisation regimens regarding tumour cell mobilisation kinetics. Real-time PCR was performed in one patient and confirmed the mobilisation of tumour cells at the time when CD34 + blood cells were at a maximum. Conclusions: Tumour cells are mobilised to the peripheral blood at the same time as CD34 + cells in multiple myeloma patients after priming with both G-CSF alone and HDCy in combination with G-CSF.

Objective Cyclin D1 dysregulation has been found with varying frequencies in multiple myeloma (MM) and has been suggested to be associated with a poor prognosis. The aim of this study was to investigate the frequency of cyclin D1 dysregulation in patients being treated for MM and to test whether cyclin D1 dysregulation is a prognostic factor for MM patients. Methods To achieve the above aims we designed a highly sensitive and reproducible real-time reverse-transcription polymerase chain reaction (RT-PCR) assay for quantitation of cyclin D1 mRNA. Using this assay, 110 diagnostic bone marrow (BM) samples from patients with MM were screened for cyclin D1 dysfunction. Results The real-time assay was able to detect the presence of 0.01% cyclin D1 positive cells allowing a safe detection in MM BM samples. In 42% (46/110) of MM BM samples a greater-than-or-equals 3-fold increase in cyclin D1 mRNA was observed compared to the cyclin D1 level in normal BM. In the remaining group of MM patients the cyclin D1 mRNA levels were comparable to normal donors. Follow-up of 76 MM patients showed no significant (P = 0.35) difference in survival between cyclin D1 positive and negative MM patients. In addition, cyclin D1 dysregulation did not correlate with known prognostic factors. Conclusion The developed real-time RT-PCR assay for detection of cyclin D1 mRNA levels offers a fast and safe screening for cyclin D1 dysfunction. When a large cohort of MM patients was screened, the cyclin D1 gene was found to be frequently dysregulated, but there was no significant correlation to survival or known prognostic parameters.

A sufficient number of CD34+ cells in the peripheral blood stem cell product is important to achieve a rapid and sustained engraftment. The purpose of the present work was to study CD34+ cell kinetics during leukapheresis. Blood samples before and after leukapheresis were analysed for CD34+ cells in 205 procedures. The number of CD34+ cells after plus the number of CD34+ cells harvested was 1.5-fold greater than the number available at the beginning of the procedure, indicating recruitment of CD34+ cells during leukapheresis. In a subgroup of 66 procedures, granulocytes and platelets were measured. In contrast to CD34+ cells, these cell fractions were not recruited to the blood stream during leukapheresis. An additional nine patients were studied with serial blood measurements during leukapheresis, showing an initial decline that was followed by an increase in CD34+ cells during leukapheresis. In conclusion, CD34+ cells are recruited to the blood during the leukapheresis procedure in contrast to granulocytes and platelets.

The peripheral blood (PB) mononuclear cells in patients with multiple myeloma (MM) have been reported to include CD34-expressing cells that are clonally related to the myeloma cells. To determine whether there were elevated levels of CD34 mRNA or whether CD34+ cells in the PB include myeloma-related cells, we developed a quantitative real-time and a competitive CD34 RT-PCR assay working on single flow-sorted cells. Myeloma-specific cells were detected with allele-specific oligonucleotides (ASO) IgH PCR. PBSC products and mononuclear cell fractions in blood from normal donors, untreated and treated myeloma patients were analysed. When measured by flow cytometry, the numbers of CD34+/CD19+ cells were consistently < 0.1% of the mononuclear cells. In addition, no significant difference was found in the levels of CD34 mRNA between normal subjects and untreated MM patients (P = 0.935). In the treated group of MM patients the CD34 mRNA levels were significantly reduced (P = 0.052) because of the stem cell toxicity of melphalan. Further, no cells clonally related to the MM clone were found within the CD34 compartment, defined by a sort-gate that included all cells expressing CD34 mRNA, as no cells outside the used CD34 sort-gate had a detectable level of CD34 mRNA. We conclude that in myeloma patients, the myeloma clone is not found within the CD34 compartment.

The need for a rapid and reliable marker for the engraftment potential of hematopoietic stem and progenitor cell (HPC) transplants has led to the development of flow cytometric assays to quantitate such cells on the basis of their expression of CD34. The variability associated with enumeration of low-frequency cells (i.e., as low as 0.1% or 5 cells/microl) is exceedingly large, but recent developments have improved the accuracy and precision of the assay. Here, we review and compare the major techniques. Based on the current state of the art, we recommend 1) bright fluorochrome conjugates of class II or III monoclonal antibodies (mAbs) that detect all glycoforms of CD34, 2) use of a vital nucleic acid dye to exclude platelets, unlysed red cells, and debris or use of 7-amino actinomycin D to exclude dead cells during data acquisition, 3) counterstaining with CD45 mAb to be included in the definition of HPC, 4) during list mode data analysis, Boolean gating to resolve the CD34+ HPCs from irrelevant cell populations on the basis of the low levels of CD45 expression and low sideward light-scatter signals of HPCs, 5) inclusion of CD34dim and CD34bright populations in the CD34+ cell count, 6) omission of the negative control staining, and 7) for apheresis products, enumeration of at least 100 CD34+ cells to ensure a 10% precision. Unresolved technical questions are 1) the replacement of conventional dual-platform by single-platform assay formats, i.e., derivation of absolute CD34+ cell counts from a single flow cytometric assessment instead of from combined flow cytometer (percent CD34+) and hematology analyzer (absolute leukocyte count) data, 2) the cross-calibration of the available single-platform assays, and 3) the optimal method for sample preparation. An important clinical question to be addressed is the definition of the precise phenotypes and required numbers of HPCs responsible for short- and long-term recovery to optimize HPC transplant strategies.

To day it is possible to predict the probability of fast engraftment based on a very simple flow cytometry standard analysis of CD34+ cells as documented by the 28 laboratories within the NSCL-G. However, the risk for delayed platelet engraftment still needs to be predicted in clinical practice for patients receiving less than 10 x 10(6) CD34+ cells/kg. Here we present data from our center supporting that identification by double staining of uncommitted (CD34+/CD38-) and lineage specific (CD34+/CD61+) progenitors may allow us to predict patients at high risk for prolonged platelet recovery. Following high dose therapy more than 30% of patients with haematological malignancies do suffer from disease recurrence within the first 3-6 months following high dose therapy. Today there are strong indications that such patients may have been transplanted with an autograft contaminated with a high number of potentially malignant B cells. Here we present a novel methodology for quantitation of blood circulating tumor cells by combining flow cytometry, cell sorting, limiting dilution and single cell RT-PCR. Such methodology has documented mobilization of clonal B cells following priming of the peripheral blood stem cell harvest and it can be used to identify minor populations and predict the efficacy of patient specific purging strategy. Consequently, quality assessment of autografts may include techniques which can predict fast three lineage engraftment as well as the risk for prolonged platelet recovery and can identify the group of patients/autografts with a strong contamination of potential tumor cells with a risk of early relapse. The future supportive cell therapy may depend upon improvements of such technologies and strategies including the selective administration of lineage specific growth factors e.g., trombopoietin as well as patient specific controlled purging strategies.

The determination of CD34-expressing cells by multiparameter flow cytometry is now widely used to estimate the reconstitution potential of cells harvested by cytapheresis for peripheral blood stem cell and progenitor cell transplantation. There is a correlation between the number of CD34-expressing cells collected and committed progenitor cells (CFU-GM and BFU-E) capable of forming colonies in vitro, but there is considerable variation in the proportion of CD34-expressing cells capable of clonogenic growth. The data in this study of 782 cytapheresis samples indicates that there is a negative correlation between the clonogenicity of the CD34-expressing cells and the absolute number or the proportion of CD34-expressing cells within the harvest. In 116 samples the proportion of CD34-expressing cells co-expressing the CD45-RA-antigen (a subset of CD34-expressing cells which includes virtually all clonogenic cells in terms of CFU-GM) was determined, but this did not help to identify the clonogenicity of a given sample. These findings may have clinical relevance, particularly when mobilization is judged to be relatively poor or when a good harvest is to be divided for multiple high-dose procedures.

The t(4:14) translocation affects two potential oncogenes, FGFR3 and MMSET, in multiple myeloma (MM). We investigated the frequency of FGFR3 dysregulation and its prognostic value in MM. FGFR3 mRNA levels were determined in 110 diagnostic bone marrow (BM) samples from MM patients. In addition, selected BM samples were screened for elevated MMSET mRNA levels. 14.5% (16/110) of MM BM samples showed dysregulated FGFR3 expression. Follow-up of 76 MM patients showed no significant difference between FGFR3 dysfunction and survival (P = 0.3) or correlation with known prognostic factors. Further, no linear relation was observed between FGFR3 and MMSET levels.

Recent studies concerning the numbers of circulating clonal B cells in patients with multiple myeloma (MM) have reported conflicting data regarding the exact levels of clonal B cells and the existence of clonal cells in the CD34 compartment. In this report we show that high numbers of clonal cells with a phenotype of late-stage B cells or pre-plasma cells were present in the peripheral blood (PB) of a patient with MM. During treatment the initial high level of PB clonal cells was markedly reduced and remained low (

A 34-year-old male with hypereosinophilic syndrome (HES) and cardiac complications was treated with recombinant human alpha-interferon (rhIFN) and recombinant human granulocyte colony-stimulating factor (rhG-CSF) in the attempt to suppress the eosinophilic cell clones and stimulate the neutrophil myelopoiesis in the bone marrow, respectively. After 1 month of pretreatment with rhIFN, rhG-CSF was administered daily for 22 days. Within a few days the combined treatment with rhIFN and rhG-CSF was followed by a marked increase in absolute neutrophil count but complicated by abdominal pain and an increase in plasma creatinine and blood urea nitrogen. The renal failure persisted when rhIFN therapy was stopped but resolved after discontinuing rhG-CSF. The pathogenesis of this reversible renal involvement needs further investigation. In that hematological improvement in vivo as well as in vitro followed the administration of rhIFN and rhG-CSF, a role for combined therapy with these cytokines may be advocated. However, caution with regard to kidney function should be taken with this combination therapy.

Interleukin 2 (IL-2) was shown to induce a small but significant increase in the level of cGMP after 20 min stimulation and a subsequent fall after 1 h in activated T lymphocytes. No change in the level of cAMP was observed. Addition of the cyclic nucleotide analogues dbcAMP or dbcGMP did not stimulate DNA synthesis. On the contrary, IL-2-induced [3H]thymidine incorporation was inhibited by these drugs. Further, the phosphodiesterase inhibitor theophylline inhibited proliferation of activated T lymphocytes. Our results indicate that neither cAMP nor cGMP act as 'second messengers' for IL-2 but support the theory that cAMP is a negative regulator of cell proliferation.

The B blast specific target determinant phenotype defined by cytotoxic lymphocytes and the HLA-DR phenotype was tested for correlation in two panel studies. First, CTL reagents with B blast specificity were selected from CTLs educated between HLA-A, B, (C) identical unrelated donors giving no cytotoxicity with specific T blasts, resulting in free selection of panel B blasts used as targets. Second, CTLs educated between HLA non-identical unrelated donors giving cytotoxicity against specific T as well a B blast targets could be seen with negative reactions against T blasts but positive reactions against B blasts of selected panel members giving, in this set up, a free selection of donors cocultured for education of CTLs. These panel studies showed that HLA-DR associated B blast specific target determinants can be identified by CTLs, indicating that the HLA system codes for these determinants either through gene products of a locus closely linked to or identical with the HLA-DR locus.

The enhanced stimulation of human B lymphocytes by pokeweed mitogen in the presence of irradiated T helper lymphocytes has been studied, revealing that the proliferative responses measured by incorporation of thymidine in cultures of B lymphocytes and irradiated T lymphocytes in a 1:2 or 1:4 ratio is mostly a function of the B cells. Only a minimal number, if any, of the T cells contaminating the B cell suspensions is stimulated to proliferation. This is in contrast to stimulation of the B-cell suspensions without addition of irradiated T cells, where both B cells and T cells proliferate. The irradiated T helper cells have no FcR for antigen-bound IgG, and as well allogeneic as autologous T cells exhibit helper capacity of equal strength. The test system described makes it possible selectively to test one B-cell function and the corresponding T helper capacity.

In a study of the role of Ca++ in the stimulation of activated T lymphocytes with interleukin 2 (IL-2) it was found that IL-2-induced proliferation can occur independently of extracellular calcium. Further, there was no correlation between triggering of DNA synthesis and an increase in free cytoplasmic calcium. However, IL-2 induced an increased uptake of 45Ca++ from the extracellular medium. Since there is no increase in free cytoplasmic calcium, it must be assumed that this is caused by an increase in membrane-associated calcium. Further, the calcium channel-blocking agent, verapamil, and TMB-8, a putative inhibitor of mobilization of calcium from intracellular pools, both exerted a dose-dependent inhibition of IL-2-induced DNA synthesis in activated T lymphocytes. We conclude that calcium is not a second messenger in activated T lymphocytes stimulated by IL-2, but our results indicate that calcium may play a role at membrane level.