Flow cytometry significantly improves the diagnostic value of fine needle aspiration cytology of lymphoproliferative lesions of salivary glands.

OBJECTIVE: Lymphoid proliferations of the salivary glands can be either reactive or malignant. Diagnosis based solely on fine needle aspiration (FNA) cytology may be troublesome in view of the difficulty in distinguishing low-grade B-cell and mucosa-associated lymphoid tissue (MALT) lymphomas from reactive lymphoid proliferations. We report our experience with FNA cytology combined with flow cytometry (FC) immunophenotyping for the diagnosis of lymphoproliferative processes affecting the salivary glands. METHODS: Sixty-one FNA specimens, obtained from salivary glands over a 10-year period, were analysed by cytology and FC. The results were correlated with histological follow-up if available. RESULTS: A diagnosis of lymphoma was given in 37 of 61 (61%) specimens; 22 of 61 (36%) specimens were considered as benign/reactive or non-lymphomatous processes; two of 61 (3%) specimens were considered as suspicious for lymphoma on cytological analysis and negative on FC. Histological control was available in 23 malignant, four non-lymphomatous and one cytologically suspicious case. Data obtained by the combination of cytology and FC were confirmed in all but one case: the case suspicious on cytology received a histological diagnosis of carcinoma. Four of seven cases with small populations of clonal cells (less than 15%) were histologically confirmed as lymphoma, whereas two remain under surveillance and one was reactive. Correlation with histological data showed a sensitivity of 100% and a specificity of 83% for the combination of cytology and FC. CONCLUSIONS: FC is fundamental for the diagnosis of lymphoproliferative lesions of the salivary glands. It may solve cytologically suspicious cases and detect the presence of neoplastic B or T cells. This combined approach reduces the time to therapy and may prevent unnecessary surgical biopsies.

Diagnosis of deep-seated lymphomas by endoscopic ultrasound-guided fine needle aspiration combined with flow cytometry.

OBJECTIVE: Although endoscopic ultrasound combined with fine needle aspiration (EUS-FNA) is rapidly becoming the preferred diagnostic approach for the sampling and diagnosis of gastrointestinal and mediastinal malignancies, there are limited data as to its use in the diagnosis of lymphoproliferative disorders. Therefore, we carried out a retrospective evaluation of the performance of EUS-guided FNA combined with flow cytometry (FC) as a tool to improve overall sensitivity and specificity in the diagnosis of lymphoma. METHODS: Of 1560 patients having EUS-guided FNA during the period of the study, a total of 56 patients were evaluated by cytology with FC after EUS-FNA. There was adequate material to perform FC analysis for all but one case. RESULTS: EUS-FNA-FC gave a diagnosis of lymphoma in 11 cases and of reactive lymphadenopathy in 20. A specific histological type was defined by FC alone in eight cases. The remaining cases were diagnosed later by cytology and cell block sections: 13 carcinomas, nine granulomatous lymphadenopathies and one mediastinal extramedullary haematopoiesis. One case was considered only suspicious for lymphoma on cytology and FC but was not confirmed on molecular analysis and one had insufficient material for FC. CONCLUSIONS: Our results show that a combination of EUS-FNA-FC is a feasible and highly accurate method, which may be used for the diagnosis and subtyping of deep-seated lymphoma, providing a significant improvement to cytomorphology alone both for diagnosis and treatment planning, as long as immunocytochemistry is available for non-lymphoma cases.

Kinetics of human hemopoietic cells after in vivo administration of granulocyte-macrophage colony-stimulating factor.

The kinetic changes induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) on hemopoietic cells were assessed in physiological conditions by administering GM-CSF (8 micrograms/kg per d) for 3 d to nine patients with solid tumors and normal bone marrow (BM), before chemotherapy. GM-CSF increased the number of circulating granulocytes and monocytes; platelets, erythrocytes, lymphocyte number, and subsets were unmodified. GM-CSF increased the percentage of BM S phase BFU-E (from 32 +/- 7 to 79 +/- 16%), day 14 colony-forming unit granulocyte-macrophage (CFU-GM) (from 43 +/- 20 to 82 +/- 11%) and day 7 CFU-GM (from 41 +/- 14 to 56 +/- 20%). The percentage of BM myeloblasts, promyelocytes, and myelocytes in S phase increased from 26 +/- 14 to 41 +/- 6%, and that of erythroblasts increased from 25 +/- 12 to 30 +/- 12%. This suggests that GM-CSF activates both erythroid and granulomonopoietic progenitors but that, among the morphologically recognizable BM precursors, only the granulomonopoietic lineage is a direct target of the molecule. GM-CSF increased the birth rate of cycling cells from 1.3 to 3.4 cells %/h and decreased the duration of the S phase from 14.3 to 9.1 h and the cell cycle time from 86 to 26 h. After treatment discontinuation, the number of circulating granulocytes and monocytes rapidly fell. The proportion of S phase BM cells dropped to values lower than pretreatment levels, suggesting a period of relative refractoriness to cell cycle-active antineoplastic agents.

Phosphotyrosine antibodies identify the p210c-abl tyrosine kinase and proteins phosphorylated on tyrosine in human chronic myelogenous leukemia cells.

Antibodies against phosphotyrosine are a powerful tool with which to identify proteins phosphorylated on tyrosine residues, such as viral oncogene-encoded transforming proteins and their cellular protein substrates. Probed on human leukemia cell lines, phosphotyrosine antibodies recognized a 210,000-molecular-weight protein (p210) in K562 cells, a cell line derived from a Philadelphia (Ph)'-positive chronic myelogenous leukemia (CML), but recognized no protein in control Ph'-negative non-CML leukemia cells. The p210 protein was also recognized by antisera against v-abl-encoded polypeptides and displayed kinase activity, phosphorylating itself on tyrosine, in an immunocomplex kinase assay. These data are consistent with reported findings of the expression of a recombined bcr-abl gene in Ph'-positive CML cells, leading to the synthesis of an altered p210c-abl protein endowed with tyrosine kinase activity. Phosphotyrosine antibodies also detected the expression of the p210c-abl protein in fresh bone marrow cells harvested from CML patients in blast crisis. Besides the p210c-abl protein kinase, phosphotyrosine antibodies recognized other proteins with molecular weights of 110,000, 68,000, and 36,000 (p110, p68, and p36) in K562 cells. When [gamma-32P]ATP was added to nonionic detergent-extracted cells, these proteins became phosphorylated on tyrosine, as confirmed by phosphoamino acid analysis. A comparison with fibroblasts transformed by the v-abl, v-src, and v-fps oncogenes suggested the identity of the p36 protein with the common 36-kilodalton protein substrate of viral oncogene-encoded tyrosine kinases. Enhanced tyrosine phosphorylation of cellular proteins is thus a feature shared by cells transformed by v-abl and cells expressing a rearranged bcr-abl gene.

Opposite effect of tumor necrosis factor alpha on granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor-dependent growth of normal and leukemic hemopoietic progenitors.

The effect of recombinant human tumor necrosis factor alpha (TNF-alpha) on normal and chronic myeloid leukemia granulocyte-macrophage progenitors (CFU-GM) growing in semisolid agar cultures in the presence of recombinant granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor was studied. Granulocyte-macrophage colony-stimulating factor-dependent growth of normal and chronic myeloid leukemia bone marrow CFU-GM was greatly enhanced by TNF-alpha at doses of 0.1 to 100 units/ml. Growth enhancement included neutrophil, eosinophil, and monocyte-macrophage colonies and clusters at 7 and 14 days of culture. Since similar results were achieved with highly enriched progenitor cell populations, devoid of accessory cells, an indirect effect on CFU-GM growth through the release by accessory cells of other cytokines upon TNF-alpha stimulation was thus ruled out. By contrast, the same doses of TNF-alpha inhibited the growth of normal CFU-GM in granulocyte colony-stimulating factor-dependent cultures. Taken together, our findings indicate that the final effect of TNF-alpha on normal bone marrow granulocyte-macrophage progenitor growth is dependent on the specific growth factor interacting with it, and that both normal and chronic myeloid leukemia CFU-GM are equally responsive to the combined effects of TNF-alpha and a given colony-stimulating factor.

Expression of HLA class II (DR, DQ) determinants by normal and chronic myeloid leukemia granulocyte/monocyte progenitors.

It has been suggested that the expression of certain HLA class II antigens stemming from three distinct loci (DR, DP, and DQ) is important not only in the regulation of the immune response but also on the response of hemopoietic precursors to factors inhibiting myelopoiesis. Changes in the expression of DR antigens may be involved in the pathogenesis of altered cell proliferation in chronic myeloid leukemia, since they result in decreased sensitivity of the colony forming units, granulocyte-macrophage to prostaglandin E and acidic isoferritins. In studies using monoclonal antibodies against monomorphic DR or DQ determinants, in a complement-dependent cytotoxic assay, it was found that nearly all normal and chronic myeloid leukemia bone marrow colony forming units, granulocyte-macrophage express DR antigens. The dose response curve was similar for both normal and leukemic precursors. Leukemic peripheral blood precursors were more sensitive than were normal peripheral blood precursors. Normal colony forming units, granulocyte-macrophage did not express DQ antigens, whereas these were expressed in varying quantities by leukemic cells. This study shows that, in the patients we studied, leukemic cells express DR antigens in amounts comparable to normal. In addition, varying amounts of DQ antigens may be observed on leukemic but not on normal progenitors, perhaps as a consequence of an increase in the number of antigens also expressed by normal cells, though in an amount below the detection threshold of cytotoxicity techniques.

Expression of type III receptor tyrosine kinases FLT3 and KIT and responses to their ligands by acute myeloid leukemia blasts.

The stem cell tyrosine kinase 1 (STK1) protein is the human homologue of the murine FLT3 gene product, a receptor belonging to the FMS/KIT family. FLT3 and KIT with their ligands control the growth and differentiation of early human hemopoietic cells. In the present study, 16 cases of acute myeloid leukemia (AML) were examined by flow cytometry for cell surface expression of FLT3 and KIT receptors. All cases were also tested for their proliferative response to human FLT3 ligand (FL) and KIT ligand (KL) and for colony formation in the presence of single or associated cytokines. Among 16 AML cases tested, 10/16 expressed FLT3 receptor and 12/16 expressed KIT receptor, without any correlation with FAB subtype. FL and KL stimulated the proliferation of leukemic blasts in 11/16 AML cases (including five FLT3 or KIT receptor-negative cases), with an additive effect when added simultaneously. By contrast, some receptor-expressing AMLs did not display significant proliferative responses to their respective ligands. FL and KL as single factors induced or significantly increased the colony formation by clonogenic precursor cells respectively in eight and six of 13 cases tested. In some cases growth factor association significantly enhanced colony growth. Taken together these observations provide evidence that the pattern of FLT3 and KIT receptor expression is extremely variable among the AMLs and that receptor presence is not necessarily combined with proliferative and clonogenic response or vice versa.

Stem cell factor improvement of proliferation and maintenance of hemopoietic progenitors in myelodysplastic syndromes.

Human recombinant stem cell factor (rSCF) was tested for its capability of improving the defective growth of hemopoietic progenitors in 28 cases of myelodysplastic syndromes (MDS). In vitro growth and response to rSCF were quite variable. However, in most cases, rSCF stimulated CFU-GM growth induced by rG-CSF, rGM-CSF, rIL-3, 5637 conditioned medium (50-1400% enhancement). rSCF effect was slightly more evident on day 14 CFU-GM and in the presence of rIL-3. BFU-E growth induced by rEPO or rIL-3 + rEPO was enhanced by rSCF in about 50% of cases, in linear correlation with the levels of patients' hemoglobin. rSCF did not increase CFU-E growth, whereas it slightly stimulated CFU-Mk in 33% of the cases. EPO, SCF and, particularly, their combination, enhanced the recovery of normal CFU-E and BFU-E after 7 days of liquid culture. This was less evident in cultures of MDS patients. Conversely, CFU-GM generation in long term liquid cultures, although highly variable, was stimulated by rSCF and, above all, by rSCF + rG-CSF, similarly to what was observed with normal bone marrow samples. SCF seems to enhance in vitro erythropoiesis only in MDS cases presenting without severe anemia. It has little effect on megakaryocytopoiesis, while it seems to be more active on CFU-GM growth and maintenance.

In vivo effect of granulocyte-macrophage colony-stimulating factor on the kinetics of human acute myeloid leukemia cells.

Granulocyte-macrophage colony-stimulating factor, (GM-CSF) was given at 8 micrograms/kg daily by continuous i.v. infusion for 72 h to six patients with acute myeloid leukemia (AML) in expansion and one with chronic myeloid leukemia in blastic crisis to determine whether it was possible to augment the proliferative activity of the neoplastic population. The percentage of marrow blasts in S phase (labeling index, LI) was increased in five patients (1.3-, 1.5-, 1.9-, 2.3- and 3.2-fold change). The increase in LI was similar 24 and 48 h after beginning GM-CSF. The RNA Index also increased in patients who showed an increased LI, suggesting that GM-CSF had recruited quiescent neoplastic cells into the cell cycle. Forty eight hours after beginning GM-CSF, chemotherapy was started. The fate of S phase cells, labeled in vivo with bromodeoxyuridine (BrdU) immediately before cytostatic treatment, was monitored. BrdU positive cells were identified by fluorescent antibody for up to 28 days. A preferential killing of BrdU (S phase) cells was observed in 5/7 patients who obtained a complete remission, whereas this was not apparent in the two patients who achieved only a partial remission. Chemotherapy induced a rapid and profound aplasia; its duration, however, was not significantly different from that observed in historical controls. GM-CSF may have a potential role in the treatment of AML, as this study shows that it recruits leukemic cells into the cell cycle without adversely prolonging aplasia after cycle-specific therapy.

In vivo effect of human granulocyte-macrophage colony-stimulating factor (GM-CSF) on neutrophil GM-CSF receptors.

The effect of in vivo administration of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on neutrophils GM-CSF receptor, was investigated in patients with neoplastic diseases and normal hematopoiesis. Patients were divided into two groups. Group A received a single dose of rhGM-CSF (5 micrograms/kg/day) and receptor studies were performed 90 min and 48 h after treatment. Group B received three doses, administered subcutaneously every 24 h and receptor studies were performed 90 min after first injection and 24 h after the last. Before treatment neutrophils only displayed high-affinity receptors (KD 85 +/- 53 pM; number of receptors/cell 1318 +/- 567). The first injection of rhGM-CSF produced a transient leucopenia and the internalization of GM-CSF receptor on neutrophils in both groups of patients: 90 min after s.c. administration receptors could not be detected with conventional binding studies. In group A patients, 48 h after a single dose of rhGM-CSF, receptors, albeit with a decreased affinity (KD = 240 +/- 131 pM; number of receptors/cell 783 +/- 494) were again expressed. In group B patients, 24 h after the last rhGM-CSF injection, low intermediate affinity receptors not present before treatment appeared (KD 720 +/- 175 pM; number of receptor/cell 1222 +/- 179). They were associated with a low number of high affinity receptors (KD = 9 +/- 4 pM; number of receptors/cell 106 +/- 44). These observations indicate that more than one type of GM-CSF receptor may exist on neutrophils. It may be suggested that in vivo the regulation of the GM-CSF receptor is different from that in vitro and is related to the presence of the cytokine in patient blood.

In vitro reappearance of myeloid progenitors killed by mafosfamide.

Cyclophosphamide derivatives active in vitro, such as mafosfamide, are potentially capable of reducing the number of leukemic cells remaining in marrow explanted for autografting. Although this treatment kills nearly all the committed hemopoietic progenitors, it does not prevent the reinstatement of hemopoiesis after chemoradiotherapy. This points to the persistence of more immature hemopoietic progenitors not detectable with current semi-solid culture techniques and resistant to cytotoxic treatment. Treatment of normal marrow with 80-140 micrograms/ml mafosfamide is followed in medium-term cultures by a gradual and dose-dependent reduction in total cellularity, whereas granulomonocyte progenitors (CFU-GM), virtually absent at the start of the culture, progressively reappear. The quantity of progenitors present after day 14 in liquid culture is, however, less in the treated marrows than in the controls, and the reappearance of CFU-GM is inversely related to the mafosfamide dose. In addition, the recovery of the more immature (day-14) CFU-GM is greater than that of the more mature (day 7) CFU-GM.

Responsiveness of highly enriched CFU-GM subpopulations from bone marrow, peripheral blood, and cord blood to hemopoietic growth inhibitors.

Human early and late granulocyte-monocyte progenitors (granulocyte-macrophage colony-forming units, CFU-GM), depleted of accessory cells, were physically separated using an antimyeloid monoclonal antibody (DS1.1). They were separately cultured at optimal growth conditions and tested for responsiveness to prostaglandin E2 (PGE2), recombinant tumor necrosis factor alpha (TNF alpha), and transforming growth factor beta-1 (TGF beta 1). Late (DS1.1+) CFU-GM displayed the highest sensitivity to PGE2 and TNF alpha, the first significant inhibition being evident at 10(-9)M PGE2 and 1 U/ml TNF alpha. Conversely, their growth was stimulated (211%-217%) by 0.25-2.5 ng/ml TGF beta 1. Early (DS1.1-) marrow CFU-GM evidenced a lower sensitivity to PGE2 and TNF alpha. Their growth, however, was inhibited by 0.25-2.5 ng/ml TGF beta 1. Early CFU-GM constitute the totality of peripheral blood myeloid progenitors. Cord blood CFU-GM were also demonstrated here to be entirely DS1.1-. Both adult and cord blood CFU-GM displayed the highest resistance to PGE2 and TNF alpha. By contrast, they showed the maximum sensitivity to growth inhibition by TGF beta 1, active at 0.025-0.25 ng/ml. For the first time, therefore, highly purified subsets of human CFU-GM were separated that displayed a different responsiveness to well-defined growth-regulatory molecules. Our results indicate that TGF beta 1 has a dual activity; it is inhibitory on early and stimulatory on late CFU-GM, whereas PGE2 and TNF alpha preferentially inhibit late CFU-GM growth.

Interaction of transforming growth factor-beta 1 with hemopoietic growth factors in the regulation of human normal and leukemic myelopoiesis.

We studied the effect of transforming growth factor-beta 1 (TGF-beta 1) on the growth of normal and chronic myeloid leukemia (CML) granulo-monopoietic progenitors (CFU-GM) and erythroid progenitors (BFU-E) of different origins and degrees of maturation. In the presence of the supernatant of the 5637 cell line, used as a source of growth factors, TGF-beta 1 stimulates the growth of day-7 CFU-GM from Ficoll-isolated normal bone marrow cells. Maximum stimulation (172% of controls) is observed with 2.5 ng/ml TGF-beta. The results with a highly enriched progenitor cell population stimulated by recombinant granulocyte colony-stimulating factor (rG-CSF) and recombinant granulocyte-macrophage CSF (rGM-CSF) were similar, suggesting a direct effect of TGF-beta 1 on hemopoietic progenitors. In contrast to this stimulatory effect of TGF-beta 1 on normal day-7 bone marrow CFU-GM, TGF-beta 1 does not affect the growth of day-14 CFU-GM. The growth of normal bone marrow BFU-E is strongly inhibited. In the majority of cases (11/15) of CML, bone marrow day-7 CFU-GM growth is inhibited by TGF-beta 1. In few cases (4/15) leukemic progenitors respond to TGF-beta 1 as normal cells. TGF-beta 1 always inhibits the growth of day-14 bone marrow CFU-GM from CML patients.

The effects of human FLT3 ligand on in vitro human megakaryocytopoiesis.

The human homolog of the murine flt3/flk2 gene product is a tyrosine kinase receptor that plays a role in regulating the proliferation and differentiation of cells in the hematopoietic system. Using a plasma-clot clonal assay and a long-term bone marrow culture (LTBMC) system, we studied the effects of the recently cloned human flt3 ligand (FL) alone and in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), or stem cell factor (c-kit ligand [KL]) on human megakaryocytopoiesis. The effects of FL on the primitive megakaryocyte (MK) progenitor cell, the burst-forming unit-megakaryocyte (BFU-MK), and the more differentiated colony-forming unit-megakaryocyte (CFU-MK) were determined. FL alone had no megakaryocytic colony-stimulating activity (MK-CSA), but was capable of augmenting the MK-CSA of both GM-CSF and IL-3. FL synergized with IL-3 at the level of both CFU-MK and BFU-MK and with GM-CSF and KL at the level of CFU-MK. Although FL alone exhibited a limited potential in sustaining long-term megakaryocytopoiesis in vitro, it synergistically augmented the ability of IL-3 and KL, alone or in association, to promote long-term megakaryocytopoiesis. These data indicate that multiple cytokines are necessary to optimally stimulate the proliferation of both classes of MK progenitor cells and that FL plays a significant role in this process by amplifying the MK-CSA of GM-CSF, IL-3, and KL.

In-vitro effect of retinoic acid on normal and chronic myeloid leukemia granulopoiesis.

The effect of increasing concentrations of retinoic acid (RA) on the in-vitro proliferation of normal and chronic myeloid leukemia (CML) granulo-monocyte precursors (CFU-GM) was studied. 10(-7)M RA added to semisolid cultures stimulated the growth of day 14 but not of day 7 normal CFU-GM, whereas in CML the growth of both populations was either unchanged or inhibited. Five-day and 10-day preincubation of normal bone marrow cells with RA augmented the number of day 14 CFU-GM (by up to 187% with 10(-6) M RA), whereas there was a marked decrease when CML cells were used. Total cellularity was not much affected, though a slight increase in liquid normal bone marrow cultures and a slight fall in CML cultures could be detected. These data point to a difference in the response to RA of normal and CML precursors. They may offer of preclinical basis for its employment to delay the blastic progression of CML.

Effect of interferon-gamma on HLA class II antigen expression and sensitivity to prostaglandin E1 by normal and leukemic myeloid progenitors.

Chronic myelogenous leukemia (CML) granulo-monocyte committed progenitors (CFU-GM) are markedly less sensitive than normal progenitors to the inhibitory action of prostaglandin E (PGE). This phenomenon has been ascribed to their abnormal expression of HLA class II (mainly DR) determinants. Since interferon gamma (IFN-gamma) is a potent inducer of the expression of HLA class II (DR and to a lesser extent DQ) antigens, we have sought to determine the extent to which this agent can modulate both the antigenic pattern of normal and leukemic progenitors and their sensitivity to PGE 1. 72-h preincubation of normal and CML bone marrow cells with or without IFN-gamma does not significantly change DR and DQ expression by CFU-GM. Pre-incubation for 72 h with and without IFN-gamma produces the following changes in PGE 1 sensitivity: (1) normal CFU-GM lose some sensitivity to PGE 1. This is only marginally counteracted by the presence of IFN-gamma. (2) CML CFU-GM, preincubated with IFN-gamma regain a significant sensitivity to high concentrations of PGE 1. Our data confirm the expression of DR molecules on normal and leukemic progenitors. They also show that, although incubation with IFN-gamma for 72 h in a liquid culture system does not significantly affect the expression of HLA class II molecules by progenitor cells, it may increase their sensitivity to PGE, particularly in the case of CML CFU-GM. Thus expression of HLA class II antigens and sensitivity to PGE may be dissociated.

Expression of HLA class II determinants by normal and chronic myeloid leukemia progenitors.

It has been suggested that the expression of some HLA class II antigens, derived from three loci (DR, DP, DQ) is important in the regulation of both the immune response and the response of haemopoietic progenitors to regulation factors, such as acidic isoferritins (AIF), as well as in the interaction between T lymphocytes and erythroid progenitors (BFU-E). Changes in the expression of class II antigens have been reported on the surface of granulo-monocyte progenitors in chronic myeloid leukemia (CML) and correlated to the abnormal proliferation of such cells. In this study, monoclonal antibodies against DR and DQ monomorphic determinants were used to investigate the expression of these antigens on the surface of normal and CML bone marrow and peripheral blood BFU-E by means of complement mediated cytotoxicity. It was found that most normal and leukemic BFU-E express DR antigens. Antigens density tends to be greater on marrow as opposed to peripheral precursors. In addition, leukemic BFU-E are more sensitive to cytolytic treatment than their normal counterparts. Normal BFU-E do not express detectable amounts of DQ antigens, whereas these are present on a proportion of leukemic BFU-E.

MEC1 and MEC2: two new cell lines derived from B-chronic lymphocytic leukaemia in prolymphocytoid transformation.

We report the establishment and characterization of two cell lines, MEC1 and MEC2, that grew spontaneously on two subsequent occasions from the peripheral blood (PB) of a patient with B-chronic lymphocytic leukemia (B-CLL) in prolymphocytoid transformation. The patient was EBV-seropositive, his leukemic cells were EBNA negative, but the spontaneously grown cell lines are EBNA-2 positive. In liquid culture MEC1 cells grow adherent to the vessel wall and as tiny clumps; MEC2 cells do not adhere and form large clumps. The doubling time of MEC1 is 40h and of MEC2 is 31h. Both cell lines express the same light (kappa) and heavy chains (mu, delta) as the fresh parental B-CLL cells at the same high intensity, share the expression of mature B cell markers (CD19, CD20, CD21, CD22), differ in the expression of CD23 and FMC7, are CD11a+, CD18+, CD44+, CD49d+, CD54+ and express at high levels both CD80 and CD86. CD5 is negative on MEC1 cells (as on the vast majority of parental cells) and it has been lost by MEC2 cells after several months of culture. The cells have a complex karyotype. The tumour origin of MEC1 and MEC2 has been demonstrated by Southern blot analysis of the IgH loci and by Ig gene DNA sequencing. They use the VH4 Ig family and have not undergone somatic mutations (94.8% homology with germline Ig gene 4-59). Cytofluorographic analysis and RT-PCR reveal that MEC1 and MEC2 overexpress Bcl-2 together with Bax, express large amounts of Bcl-xL and trace amounts of Bcl-xS.

Effect of Hemopoietic Growth Factors on the Proliferation of Acute Myeloid and Lymphoid Leukemias.

The effect of G-CSF, GM-CSF, IL-1 and IL-3 on the proliferation of acute leukemia cells (evaluated as (3)HTdR uptake) was investigated in short-term liquid cultures and compared with that observed on normal bone marrow (BM) populations enriched for immature cells. In acute myeloid leukemias (AML), a marked leukemic proliferation was induced in 10/18 cases by IL-3, in 9/18 by GM-CSF, in 7/18 by IL-1 and in 4/18 by G-CSF. In acute lymphoid leukemias (ALL), marked stimulation was observed in 7/11 cases with IL-3 and in 5/11 with GM-CSF, whereas IL-1 and G-CSF were ineffective. Both in AML and ALL, the combination of several factors did not result in an additive synergistic effect. Purified normal BM cells responded to all four growth factors and their combinations produced an additive effect on cell proliferation which probably relates to the heterogeneity of the cell populations studied. The effect of a G-CSF, GM-CSF, IL-1 and IL-3 on the proliferation of acute leukemia cells by different growth factors suggests that caution should be exercised in their clinical use in these diseases.

Flow cytometry in the bone marrow staging of mature B-cell neoplasms.

BACKGROUND: Even though flow cytometric (FC) analysis of bone marrow aspirates is often performed in hematolymphoid disorders at diagnosis and during disease monitoring, its role has not been defined during the staging of B-non-Hodgkin's lymphoma (B-NHL) and B-cell lymphoproliferative diseases. The goal of this study was to provide an objective evaluation of how FC might help in the detection of bone marrow involvement by the different types of B-cell malignant neoplasms. METHODS: Fifty-four staging and 156 restaging bone marrow biopsies and bone marrow aspirates, obtained from 185 consecutive patients, were analyzed retrospectively. The results of the morphologic examination and FC were reviewed independently, and their ability to detect bone marrow involvement was compared. RESULTS: FC and morphology agreed in 176 cases (83.8%), i.e., both showed 77 positive cases and 99 negative ones. Discrepant results were obtained in 30 cases (14.2%) in which morphologic examination showed 25 (11.9%) positive cases, whereas FC showed no evidence of disease. FC detected involvement in five cases (2.4%) in the presence of a histologically negative bone marrow biopsy. All morphologically undetermined bone marrow cases (four) were negative by FC. CONCLUSIONS: Neither morphologic examination nor FC alone is adequate for the detection of all cases of B-lymphoid neoplasm bone marrow involvement. FC failed to detect bone marrow involvement in those B-NHL cases having focal paratrabecular infiltration, but proved to be more sensitive than histology in detecting small clonal B-cells in B-NHL, which demonstrated fewer than 5% neoplastic infiltrates. The clinical relevance of minimal disease detected by FC alone needs further evaluation because staging of lymphomas currently is based only on morphologic data.