Production of growth factors by malignant lymphoma cell lines

Fourteen Epstein-Barr virus (EBV)-negative cell lines were raised from bone marrow (BM), peripheral blood (PB), or lymph node samples of patients with intermediate- or high-grade malignant lymphoma. The cell lines were propagated in liquid suspension culture. They contain clonogenic progenitors capable of forming lymphoma colonies in semi- solid culture medium. Cells of these lines were used to examine the growth factor requirements of their clonogenic progenitors and to assess their ability to produce their own growth factors. Two of the cell lines (OCI-Ly9 and OCI-Ly13.1) required addition of exogenous factors for colony growth. These factors were routinely provided by media conditioned by phytohemagglutinin-stimulated leukocytes (PHA- LCM). Three lines formed some and nine lines gave rise to optimal numbers of colonies without addition of growth factors. Eight of these factor-independent lines were able to function as feeder cells and promoted colony formation by both factor-dependent lines. Cell lines that displayed feeder cell function released activities into supernatants able to replace their cellular source. Some of these endogenously produced growth-promoting activities could be replaced by known hematopoietic growth factors. Both factor-dependent cell lines were cultured with recombinant IL-1 alpha, IL-2, IL-3, IL-6, and GM colony-stimulating factor (CSF) and semipurified B-cell growth factor (BCGF) interleukin-4 (IL-4). A heterogeneous response pattern was observed. Both lines formed colonies with IL-4. The colonies were comparable in frequency and size with colonies observed with (PHA-LCM). OCI-Ly9 responded to IL-6 but showed no growth with IL-2. In contrast, the TAC-positive line OCI-Ly13.1 gave rise to colonies with IL-2 while remaining unresponsive to IL-6. A moderate number of colonies was observed when cells of this line were cultured with GM-CSF. Colony formation of both lines was uninfluenced by IL1 alpha or IL-3.     

Growth of clonogenic myeloblastic leukemic cells in the presence of human recombinant erythropoietin in addition to various human recombinant hematopoietic growth factors

The effects of human recombinant erythropoietin (rEpo) in the presence of other stimulators on the growth of clonogenic leukemic blast cells from ten Japanese patients with acute myeloblastic leukemia were studied with an in vitro leukemic blast colony assay in methylcellulose culture. With the addition of rEpo alone, no leukemic blast colony formation was stimulated in any of the cases examined. However, when rEpo and phytohemagglutinin lymphocyte-conditioned medium (PHA-LCM) were added to the culture simultaneously, in contrast to results with PHA-LCM alone, the number of leukemic blast colonies formed was significantly increased in two of the ten cases (P less than .01). These two cases were classified as M1 according to the French-American-British (FAB) classification. This enhancing effect of rEpo was observed with human recombinant granulocyte/macrophage colony-stimulating factor (rGM-CSF) or human recombinant interleukin-3 (rIL-3) but was not observed with human recombinant granulocyte CSF (rGCSF).     

Stimulatory activities for human multilineage hemopoietic progenitors (CFU-GEMMT) derived from patients with hemochromatosis and from normal volunteers

A medium conditioned by leukocytes in the presence of phytohemagglutinin (PHA-LCM) promotes the growth of human multilineage hemopoietic progenitors (CFU-GEMMT) which form mixed hemopoietic colonies in culture containing granulocytes, erythroblasts, megakaryocytes, macrophages, and T-lymphocytes. PHA-LCM derived from six HLA-typed patients with idiopathic hemochromatosis and from six normal individuals were tested for growth-promoting activities for multilineage hemopoietic colony formation. Four out of six conditioned media obtained from patients with hemochromatosis supported mixed hemopoietic colony formation, as did four of six conditioned media from normal HLA-typed volunteers. Active PHA-LCM preparations from patients with hemochromatosis were similar with respect to the number and size of mixed colonies and their cellular composition when compared with conditioned media obtained from volunteers. The study indicates that no link exists between the HLA phenotype of a particular donor and the predictability of obtaining an active PHA-LCM promoting multilineage hemopoietic colony formation. PHA-LCM derived from patients with hemochromatosis had no advantage with respect to stimulatory activity for mixed colony formation when compared with conditioned media obtained from healthy volunteers.     

In vitro growth pattern of myeloma cells in liquid suspension or semi-solid culture containing interleukin-6

We examined in vitro growth pattern of myeloma cells from 21 patients with multiple myeloma either in liquid suspension or methylcellulose semi-solid culture, both in the presence of interleukin-6 (IL-6). The survival or growth of myeloma cells in liquid culture was classified into four categories. Type 1: myeloma cells survived for only 2-3 weeks in eight patients; Type 2: survival for 1-2 months in seven patients; Type 3: survival for more than 3 months without an obvious increase in cell number in three patients; and Type 4: continuous proliferation (cell line) in three patients. As the clinical stage advanced, the survival of myeloma cells became longer. All three myeloma cell lines were obtained from patients in an advanced clinical stage. Human plasma added to the liquid culture induced the survival or growth of myeloma cells better than fetal calf serum in any survival or growth type. Myeloma colonies or clusters were generated in seven of 21 patients, though myeloma colony formation was restricted to Type 4 patients. IL-6 neither prolonged the survival of myeloma cells nor promoted their growth in vitro except for Type 4 cells. Moreover, IL-6 did not increase the success rate of generating myeloma colonies in vitro. However, IL-6 did elevate the number of myeloma clusters in Types 1, 2 and 3 patients examined. These results suggest that IL-6 has a minor proliferative effect on myeloma cells in semi-solid culture but not in liquid suspension culture except for cells from patients with aggressive myeloma.     

Effect of recombinant GM-CSF and recombinant G-CSF on colony formation of blast progenitors in acute myeloblastic leukemia

The colony-promoting activities of recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) and recombinant granulocyte colony-stimulating factor (rG-CSF) on primary and secondary colony formation by blast progenitors (leukemic colony-forming units [L-CFU]) from 21 patients with acute myeloblastic leukemia (AML) were examined using blast colony assay and compared to colony promotion stimulated by phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM). Recombinant GM-CSF stimulated blast colonies in 13 out of 20 cases examined (1 case not done). The magnitude of stimulation by rGM-CSF varied significantly according to the type of AML, but in general was lower than that of PHA-LCM. Blast cells of type M1 did not form any colonies with rGM-CSF, although numerous colonies were produced with PHA-LCM. Type M4 blasts formed fairly large numbers of colonies, though slightly less than those stimulated by PHA-LCM. Blasts of type M2 and M5 formed colonies with the stimulation of rGM-CSF, but the numbers were considerably smaller than type M4 and those stimulated with PHA-LCM. Recombinant G-CSF stimulated blast colonies in only 5 out of 21 cases, 3 of them being type M2. The number of cases responding to rG-CSF was significantly smaller than that responding to rGM-CSF, and even in cases in which colonies were formed, the magnitude of stimulation was minimal. From these results it seems likely that blast cells of different types of AML require a different kind of CSF for their optimal growth; type M4 blasts responded to the stimulation of rGM-CSF well, but blasts of other types of AML responded poorly. Thus, except for type M4, CSF(s) other than rGM-CSF seems to be required for the sufficient growth of L-CFU. Recombinant G-CSF is not likely to play an essential role in the proliferation of leukemic blasts of most types. Previous exposure to rGM-CSF and rG-CSF did not alter the self-renewal capacity, cellular phenotype, and morphology of colony cells, indicating that the direction and degree of differentiation of L-CFU stimulated by rGM-CSF or rG-CSF were not different from those stimulated with PHA-LCM.     

Heterogeneity of in vitro growth pattern of megakaryocyte progenitors (CFU-M) in myeloproliferative disorders

In groups of 26 patients with myeloproliferative disorders (MPD), 8 with chronic myelogenous leukaemia (CML); 8 with polycythaemia vera (PV); 10 with essential thrombocythaemia (ET); and 6 patients with reactive thrombocytosis (RT), we studied the growth characteristics of bone marrow CFU-M in agar culture. The bone marrows from all the patients with MPD formed so called endogenous CFU-M colonies, in the absence of PHA-LCM, that increased in a dose-dependent manner with the addition of increasing concentrations of normal human AB-citrated plasma (NH-ABCP), while the bone marrows from all the patients with RT and from healthy controls formed few or no endogenous CFU-M colonies. In MPD, the endogenous CFU-M growth was enhanced by normal T cells in a dose-dependent fashion, and was decreased with the depletion of T cells from the marrow cells. These results suggest that the formation of endogenous CFU-M colonies is caused by hypersensitivity of CFU-M in MPD to NH-ABCP, which may contain a small amount of Meg-CSF, and/or by in vitro T cell stimulation. Among MPD, the endogenous CFU-M growth in ET was significantly lower than that of other MPD patients; however, the total number of ET CFU-M grown in the presence of PHA-LCM was the highest. These data show that the bone marrow CFU-M in MPD are heterogeneous with respect to in vitro growth pattern or sensitivity to exogenous Meg-CSF.     

Stimulation of growth of human malignant lymphoma and lymphoid leukemia cells in vitro

Summary We have analyzed cultures of malignant lymphoma cells and cells from patients with acute lymphoid leukemia in methylcellulose for their ability to from colonies. Clonogenic growth was examined in the presence or absence of fetal calf serum (FCS), platelet-poor plasma (PPP), medium conditioned by phytohemagglutininstimulated leukocytes (PHA-LCM), or irradiated allogeneic bone marrow stroma cells. Cells from 25 lymphoma patients — 17 with non-Hodgkin's lymphoma (NHL), eight with Hodgkin's disease (HD) — and from 19 patients with acute lymphocytic leukemia (ALL) were investigated. We show that colony growth can be obtained in a minority of cases (in 3 NHL, 5 HD, and 2 ALL) and that the use of FCS and allogeneic irradiated stroma cells may be required for optimal colony formation.This work was supported by theDeutsche Forschungsgemeinschaft (Str 250/2-2).     

Primary non-Hodgkin’s lymphoma of the breast: eight-year follow-up experience

The objective of this study is to analyze the clinical characteristics and treatment of patients with primary non-Hodgkin's lymphoma of the breast (PNHLB). Forty-five patients with PNHLB treated in our hospital during a 15-year period were retrospectively analyzed. Forty-four were females and one male, with a median age of 47 years. Forty-two patients were at stage I or II and 82.2% had diffuse large B cell lymphoma (DLBCL). Local control rate was 95.2 and 66.7% for patients with and without radiotherapy, respectively (P = 0.020). Median overall survival and progression-free survival (PFS) of all patients was 6.8 and 4.3 years, respectively. For patients with DLBCL or T cell lymphoma, median PFS was 6.5 years with chemoradiation and 3.9 years with chemotherapy or radiation only (P = 0.029). Patients who used rituximab had not reached median PFS, while those treated without rituximab had a median PFS of 5.1 years (P = 0.301). International prognostic index (IPI) score and bilateral breast involvement were two independent prognostic factors for survival. Chinese patients with PNHLB have early occurrence in lifespan. Radiation confers a better local control. Patients with intermediate or high-grade PNHLB might be treated with chemotherapy, radiotherapy, and for CD-20 positive disease, rituximab. Bilateral disease and IPI are two prognostic factors.     

Effect of human recombinant erythropoietin on human marrow megakaryocyte colony formation in vitro

The effect of human recombinant erythropoietin (EP) on the growth of human marrow megakaryocyte colony forming units (CFU-M) in vitro was investigated by the use of a plasma clot assay. EP as a single stimulating factor or as an additional factor to optimal concentration of leucocyte conditioned medium (PHA-LCM) had no effect on the number of CFU-M derived colonies. However, addition of EP (0.5-1 U/ml) to cultures with suboptimal concentrations of PHA-LCM increased megakaryocytic colony formation by 50-90% but had no effect on the number of granulocytic-monocytic colonies (CFU-GM). Exposure of marrow cells to EP for 24-48 h in liquid suspension cultures, followed by removal of the hormone and assaying the cells for CFU-M in plasma clots, resulted in a 50-100% increase of megakaryocyte colony formation in vitro. The augmenting effect of EP on CFU-M growth in vitro was abolished when EP was added to the medium after the third day of culture. The presence of factors in human serum and in PHA-LCM was an absolute requirement for the hormone to exert its potentiating effect on human CFU-M growth in vitro. Recombinant EP potentiates the growth of human marrow CFU-M and this effect seems to be exerted during the early stages of CFU-M development in vitro.     

Tumor cell lines established in vitro: an independent prognostic factor for survival in non-small-cell lung cancer.

OBJECTIVE: To determine the relation between in-vitro establishment of tumor cell lines and survival in patients with non-small-cell lung cancer. DESIGN: Cohort study. SETTING: Single-institution tertiary care center. PATIENTS: One hundred twenty-three consecutive patients with non-small-cell lung cancer from whom a viable tumor specimen could be obtained. INTERVENTION: Tumor tissue was removed at the time of entry into a therapeutic protocol. The tumor tissue was processed in the laboratory for attempted cell-line establishment. Patients classified as potentially curable (stages I, II, and IIIA) were treated with surgical resection, radiation therapy, or a combination. Patients suitable for palliative therapy only (stages IIIB and IV) were treated with radiation therapy with or without chemotherapy. Chemotherapy was based on in-vitro drug sensitivity when available. Cell-line establishment was correlated to clinical outcome. MEASUREMENTS AND MAIN RESULTS: Univariate analysis of survival was done using the log-rank test; multivariate analysis was done by Cox modeling step-up and step-down techniques. Cell lines were established from the tumor specimens of 25 patients (20%). Those patients experienced a median survival of 7 months compared with 18 months in patients from whom cell lines could not be established (P less than 0.001). In the 61 patients with potentially curable disease, 8 patients (13%) with cell lines established had a median survival of 8 months compared with 32 months for those without cell lines established (P = 0.001). In the 62 palliative group patients, the median survival of the 17 patients (27%) from whom tumor cell lines were established was 5 months compared with 7 months for those without cell lines (P = 0.15). Multivariate analysis in both groups showed cell-line establishment to be a significant indicator of prognosis (P less than 0.0001 for curable group; P less than 0.01 for palliative group). CONCLUSION: In-vitro tumor growth is related to decreased patient survival, which in turn reflects the biologic aggressiveness of cancers giving rise to these tumor cell lines.     

The proliferation in suspension of the progenitors of the blast cells in acute myeloblastic leukemia

A minority of blast cells in acute myeloblastic leukemia (AML) form colonies in culture in methylcellulose when stimulated by media conditioned by normal leukocytes in the presence of phytohemagglutinin (PHA-LCM). Blast colonies can be replated successfully, either as pooled cells or suspensions from single colonies. However, the plating efficiency declines with repeated passages, and more than four subcultures have not been achieved. In this study, blast populations were cultured in suspension, with fetal calf serum, alpha-minimal essential medium and PHA-LCM. In cells from 17 of 18 patients, exponential growth of clonogenic blast cells was maintained for six to seven days without reculturing. Colonies obtained from progenitors taken from liquid culture and replated in methylcellulose were replated to obtain the secondary plating efficiency (PE2). In 14 cases, this value was maintained or increased. In three instances, PE2 fell following culture in methylcellulose. When cells in suspension were recultured, exponential growth continued. In nine instances, exponential growth was maintained for from seven to 70 days. During this time, PE2 was maintained. Results from experiments using velocity sedimentation separation and analysis of single colonies were consistent with the view that the increase in clonogenic cells in suspension was a manifestation of their self-renewal capacity. The observations also support a model of blast progenitor growth that contains the postulate that these are capable not only of self-renewal but also of determination-like events leading to loss of proliferative capacity.     

Megakaryocytic colony formation in polycythaemia vera and secondary erythrocytosis

Megakaryocytic colony formation by progenitor cells of 18 patients with polycythaemia vera, seven with secondary erythrocytosis and four with erythrocytosis of unexplained origin was studied in vitro by the methyl cellulose culture assay. Fourteen of the 18 patients with polycythaemia vera showed spontaneous megakaryocytic colony formation, i.e. colony growth with normal human plasma as the only source of colony stimulation. None of the patients with secondary erythrocytosis or erythrocytosis of unknown origin or of the normal controls grew colonies in the presence of normal human plasma only. When the plasma of a patient with aplastic anaemia was used instead of normal human plasma and phytohaemagglutinin stimulated leucocyte conditioned medium (PHA-LCM) was added to the culture medium, two of the patients with polycythaemia vera and one with secondary erythrocytosis formed slightly increased numbers of megakaryocytic colonies, while the rest of the patients showed normal colony formation. All of the patients with polycythaemia vera but none of those with secondary erythrocytosis or erythrocytosis of unknown origin showed spontaneous erythroid colony growth. The present study shows that most patients with polycythaemia vera form spontaneous megakaryocytic colonies in vitro. This phenomenon has recently also been demonstrated in essential thrombocythaemia and it is apparently analogous to spontaneous erythroid colony growth seen in all myeloproliferative disorders.     

Spontaneous formation of megakaryocyte progenitors (CFU-MK) in primary thrombocythaemia

An improved plasma clot culture method for CFU megakaryocytes (MK) has been developed with a higher plating efficiency and easier identification and enumeration of MK colonies by an indirect immunoperoxidase staining using a monoclonal antibody specific to the platelet glycoprotein IIb/IIIa complex. This technique has been used to study megakaryocytopoiesis in 20 normal individuals, 4 recently diagnosed patients with untreated primary thrombocythaemia (PT) and 2 patients with secondary thrombocytosis (ST). An increased number of MK colonies was evident in peripheral blood (mean 115 +/- 31 CFU-MK/5 X 10(5) seeded cells or 206 +/- 91/ml) and to a lesser extent in bone marrow (mean 188 +/- 26 CFU-MK/5 X 10(5) seeded cells or 696 +/- 103/ml) of PT patients as compared to controls (mean 11 +/- 4/5 X 10(5) seeded cells or 13 +/- 3/ml of peripheral blood and 153 +/- 15/5 X 10(5) seeded cells or 319 +/- 43/ml of bone marrow). There was a very obvious difference between PT patients and the others (controls plus ST patients) because CFU-MK growth with no added stimulus (PHA-LCM or PHA-LCM and normal serum) could be seen in PT patients only.     

Interleukin-9 stimulates the proliferation of human myeloid leukemic cells

Human interleukin-9 (IL-9) stimulates the proliferation of primitive hematopoietic erythroid and pluripotent progenitor cells, as well as the growth of selected colony-stimulating factor (CSF)-dependent myeloid cell lines. To further address the role of IL-9 in the development of acute leukemia, we evaluated the proliferative response of three leukemic cell lines and 32 primary samples from acute myeloblastic leukemia (AML) patients to recombinant human (rh)-IL-9 alone and combined with rh-IL-3, granulocyte-macrophage CSF (GM-CSF), and stem cell factor ([SCF] c-kit ligand). The colony-forming ability of HL60, K562, and KG1 cells and fresh AML cell populations upon IL-9 stimulation was assessed by a clonogenic assay in methylcellulose, whereas the cell-cycle characteristics of leukemic samples were determined by the acridine-orange flow cytometric technique and the bromodeoxyuridine (BRDU) incorporation assay. In addition, the terminal deoxynucleotidyl transferase assay (TDTA) and standard analysis of DNA cleavage by gel electrophoresis were used to evaluate induction of prevention of apoptosis by IL-9. Il-9, as a single cytokine, at various concentrations stimulated the colony formation of the three myeloid cell lines under serum-containing and serum-free conditions, and this effect was completely abrogated by anti-IL-9 monoclonal antibodies (MoAbs). When tested on fresh AML samples, optimal concentrations of IL- 9 resulted in an increase of blast colony formation in all the cases studied (mean +/- SEM: 19 +/- 10 colony-forming unit-leukemic [CFU- L]/10(5) cells plated in control cultures v 107 +/- 32 in IL-9- supplemented dishes, P < .02). IL-9 stimulated 36.8% of CFU-L induced by phytohemagglutinin-lymphocyte-conditioned medium (PHA-LCM), and it was the most effective CSF for promoting leukemic cell growth among those tested in this study (i.e., SCF, IL-3, and GM-CSF). The proliferative activity of IL-9 was also observed when T-cell-depleted AML specimens were incubated with increasing concentrations of the cytokine. Addition of SCF to IL-9 had an additive or synergistic effect of the two cytokines in five of eight AML cases tested for CFU-L growth (187 +/- 79 colonies v 107 +/- 32 CFU-L, P = .05). Positive interaction was also observed when IL-9 was combined with IL-3 and GM-CSF. Studies of cell-cycle distribution of AML samples demonstrated that IL-9 alone significantly augmented the number of leukemic cells in S-phase in the majority of cases evaluated. IL-9 and SCF in combination resulted in a remarkable decrease of the G0 cell fraction (38.2% +/- 24% v 58.6% +/- 22% of control cultures, P < .05) and induced an increase of G1- and S- phase cells. Conversely, neither IL-9 alone nor the combination of IL-9 and SCF had any effect on induction or prevention of apoptosis of leukemic cells. In summary, our results indicate that IL-9 may play a role in the development of AML by stimulating leukemic cells to enter the S-phase rather than preventing cell death. Moreover, IL-9 acts synergistically with SCF for recruiting quiescent leukemic cells in cell cycle.     

Elaboration of granulocyte-macrophage colony-stimulating factor by human tumor cell lines and normal urothelium

A total of 72 cell conditioned media (CCM) were screened for their ability to stimulate colony formation by human granulopoietic progenitor cells. Granulocyte-macrophage (GM) colony-stimulating factor(s) (CSF) were found in CCM of nine tumor cell lines, two primary urinary bladder tumors, and three epithelial cell cultures of normal urinary tract. The most active medium came from urinary bladder carcinoma cell line 5637. CSF released by the 5637 cell line induced dose-dependent GM colony formation from human fetal, normal adult, and CML bone marrow (BM) and from mouse BM. Human fetal and normal adult BM formed more colonies when stimulated with 5637 CCM than with peripheral blood leukocyte (PBL) feeder layers, while CML BM produced more colonies with PBL feeder layers. CCM from 5637 was more active in stimulating GM colony formation than human placenta conditioned medium (PCM) and PHA-LCM. 5637 CCM produced in serum-free hormone-supplemented medium was nearly equipotent and can serve as suitable starting material for purification.     

Role of CD21 antigen in diffuse large B-cell lymphoma and its clinical significance

Recent advances in immunological and molecular technology have prompted proposals to change tumour classification and treatment strategies. Cell surface antigens are now easy to access, and tumour origins and clinical characteristics are now readily identifiable. However, in diffuse large B-cell lymphoma (DLBCL), one of the heterogeneous forms of haematological malignancy, the clinical significance of tumour surface antigens has not been well documented. We analysed the tumour surface antigens of 50 tumours from newly diagnosed DLBCL patients by flow cytometry in accordance with their clinical characteristics and followed the patients for a median 3.7 years. Statistical analysis showed that CD21 expression was significantly negatively associated with mortality in DLBCL (CD21 negative versus positive; relative risk = 2.36, P < 0.05). As a result of these clinical observations, we generated CD21-overexpressed (CD21(+)) lymphoma cell lines after gene transfection and analysed tumour cell growth in vivo in immunocompromised mice. Mice challenged with vector-only transfectants and parental cells as controls died within 50 d. In contrast, mice injected with CD21(+) transfectants exhibited significantly reduced tumour growth and 83% survived long term (versus control groups; P < 0.05). Interestingly, all established CD21(+) transfectants (six clones from different bulks) showed homotypic aggregation during in vitro cell culture, and anti-CD21 antibodies did not block this aggregation. Expression of CD21 is strongly associated with increased survival in DLBCL in vivo. CD21 expression may be indirectly concerned with the expression of additional cell adhesion molecules.     

Megakaryocytopoiesis in vitro of patients with essential thrombocythaemia: effect of plasma and serum on megakaryocyte colony formation

To clarify the mechanism of increased numbers of megakaryocytes in patients with essential thrombocythaemia (ET), we studied in vitro megakaryocytopoiesis in ET and other myeloproliferative disorders, using a megakaryocytic colony assay in methylcellulose containing plasma or serum and medium conditioned by phytohaemagglutinin (PHA) stimulated leucocytes (PHA-LCM). Megakaryocytic colony formation was supported well by heparinized or citrated plasma and citrated serum which was harvested after clot formation of citrated plasma. Whole serum was inhibitory for megakaryocytic colony growth. The addition of platelet releasates and partially purified platelet derived growth factor (PDGF) resulted in a decrease in the number of megakaryocytic colonies. These findings suggested that platelet-derived factor(s) in serum was inhibitory to megakaryocytic colony formation. ET plasma supported the megakaryocytic colony formation by normal or ET bone marrow cells better than normal plasma. Moreover, in ET bone marrow cells, spontaneous megakaryocytic colonies were formed in the absence of PHA-LCM. Increased megakaryocytopoiesis in ET may be ascribed to (i) increased megakaryocyte-colony stimulating activity (Meg-CSA) in plasma and (ii) increased sensitivity to Meg-CSA or autonomous proliferation of megakaryocytic progenitor cells.     

Serum-soluble fas level determines clinical symptoms and outcome of patients with aggressive non-Hodgkin's lymphoma

Soluble Fas (sFas) blocks apoptosis induced by Fas ligand in vitro. The serum concentration of sFas is elevated in lympho-proliferative diseases. We hypothesized that higher levels of sFas worsen the clinical symptoms and outcome of patients with aggressive non-Hodgkin's lymphoma (NHL). We prospectively measured the serum concentrations of sFas in 67 consecutive patients with aggressive NHL (59 with diffuse large cell lymphoma and 8 with diffuse small cleaved cell lymphoma). sFas was significantly elevated in patients with aggressive NHL compared to healthy controls (N = 36, P< 0.005), while sFas in patients with B symptoms (4.20 +/- 2.12 microg/l) was significantly higher than in those without B symptoms (2.66 +/- 1.08 microg/l, P < 0.005). No significant difference was observed between B-cell lymphoma and T-cell lymphoma or between patients with clinical stage I or II and those with clinical stage III or IV. Significant correlations were found between sFas concentration and both soluble interleukin-2 receptor (R = 0.400, P < 0.001) and C-reactive protein (R = 0.340, P < 0.01) levels in patients with aggressive NHL. No correlation was observed between sFas and either white blood cell count or lactate dehydrogenase. Generalized Wilcoxon analysis revealed that NHL patients with sFas less than 4 microg/l had better overall survival than those with sFas above 4 microg/l (P < 0.001). The serum concentration of sFas might be associated with clinical symptoms and the prognosis of patients with aggressive NHL.     

Clonal origin of human erythro-eosinophilic colonies in culture

We have observed the presence of erythropoietic bursts containing eosinophils and their precursors in methylcellulose culture of human peripheral blood and marrow nucleated cells in the presence of erythropoietin and medium conditioned by phytohemagglutinin-stimulated leukocytes (PHA-LCM). It was possible to identify these bursts (colonies) in situ in methylcellulose culture on the basis of their unique red and black colors. Transmission electron microscopy revealed that the constituent erythroid and eosinophilic cells lay intermixed with each other, and through close intercellular connections formed compact colonies and bursts consisting of several sub-colonies. Differential counts of individual erythro-eosinophil colonies (EEo colonies) revealed only a small percentage of blast cells in most of the colonies. Replating experiments of single EEo colonies yielded only eosinophilic colonies and clusters and erythroid colonies. The clonal nature of the EEo colonies was documented by analysis of Y-chromatin- positive cells in individual EEo colonies derived from cocultures of male and female peripheral blood mononuclear cells. Comparison of conditioned media indicated that PHA-LCM is the best stimulator for EEo colonies. These studies suggest that the differentiation capabilities of the progenitors for EEo colonies are restricted to erythroid and eosinophilic differentiation.     

Autologous stem cell transplant for relapsed and refractory peripheral T-cell lymphoma: variable outcome according to pathological subtype

The purpose of this study was to evaluate the outcome of high-dose chemotherapy (HDCT) followed by autologous haematopoietic stem cell transplant (ASCT) for patients with relapsed T-cell non-Hodgkin's lymphoma. We reviewed 36 patients with peripheral T-cell lymphoma (PTL) who underwent ASCT between January 1987 and June 2001. Patients had chemosensitive disease, and received high-dose melphalan and etoposide with or without total body irradiation supported by unpurged autologous stem cells. Comparisons were made with 97 diffuse large B-cell lymphoma (DLBL) patients. PTL patients had a median age of 46 years (19-62 years). Twenty-nine had relapsed and seven had primary refractory disease. DLBL patients were statistically similar in baseline characteristics. Of patients with PTL, six (17%) died of treatment-related complications and 14 (39%) were in remission with a median follow-up of 42 months (range 6-116 months). Three-year overall survival and event-free survival (EFS) were 48% and 37%, respectively, for PTL, compared with 53% and 42% for DLBL (P = 0.41 and 0.29 respectively). There was no significant prognostic variable found by univariate analysis for the PTL cohort. Major PTL subtypes were analysed for outcomes. The 20 patients with PTL, not otherwise specified (PTL-NOS), had an inferior EFS compared with DLBL patients (23%, P = 0.028). In contrast, the nine patients with anaplastic large T/null cell lymphoma had a non-significant trend for improved EFS (67%, P = 0.41). While ASCT in patients with relapsed or primary refractory PTL results in long-term remission rates comparable to DLBL patients, those with PTL-NOS do significantly worse.