The influence of wire localisation for non-palpable breast lesions on visualisation of the sentinel node.

PURPOSE: In our clinic, patients with occult breast lesions are treated with a sentinel node biopsy combined with wire-guided tumour excision. The aim of this retrospective study was to determine the influence of the sequence of wire localisation and sentinel node procedure on visualisation of the sentinel node. METHODS: A total of 136 patients had a wire-guided tumour excision combined with a sentinel node procedure. Sixty-six patients had guide wire localisation prior to the sentinel node procedure. Seventy patients had sentinel node visualisation before insertion of the guide wire. RESULTS: The sentinel node was visualised in 41 (62%) of the patients who first underwent guide wire localisation. In the group of patients who underwent visualisation of the sentinel node before placement of the guide wire, the sentinel node was visualised in 62 (89%). This is a significant difference in visualisation (p<0.001). CONCLUSION: This study shows that guide wire localisation prior to the sentinel node procedure negatively influences visualisation of the sentinel node.

[Survival of patients with aggressive non-hodgkin's lymphoma: no difference between first line treatment in a prospective randomised phase III clinical trial and first line treatment according to routine clinical practice]. / Overleving van patiënten met agressief non-hodgkinlymfoom: geen verschil tussen eerstelijnsbehandeling in klinisch prospectief gerandomiseerd fase-III-onderzoek en eerstelijnsbehandeling buiten studieverband.

OBJECTIVE: To compare survival of patients with disseminated aggressive non-Hodgkin's lymphoma (NHL) who were treated either as part of a clinical trial or in routine clinical practice. DESIGN: Retrospective. METHOD: The survival was studied of patients with disseminated NHL of an intermediate or high degree of malignancy who were treated in the Meander Medical Centre, Amersfoort, the Netherlands, in the years 1994-2001 with chemotherapy consisting of cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP). This took place either in routine clinical practice (RCP) or as part of a clinical trial where patients < or = 60 years of age received intensified CHOP and patients > 60 years received CHOP with growth factors. Treatment data, the response to therapy, survival and prognostic factors according to the International Prognostic Index for aggressive NHL were collected by a review of the patient records. RESULTS: Fifty-nine patients were eligible for this analysis: 32 men and 27 women with a median age of 63 years (range 30-83). Of these, 35 were treated within a clinical trial and 24 were treated in RCP. The patient characteristics in the two groups were comparable. There was no difference in median survival between the trial and RCP groups, this being 27 months for all patients, 34 months for the younger patients, 20 months for the elderly patients, and 42 months for patients who achieved complete remission following chemotherapy. CONCLUSION: No difference in overall survival was found between patients with disseminated aggressive NHL who underwent treatment according to either RCP or as part of a clinical trial. It demonstrates that both patients in clinical trials and patients treated according to RCP received equally effective therapy. Recent developments in NHL treatments are promising, and therefore participation in clinical trials should be encouraged.

Molecular quantification of viral load in plasma allows for fast and accurate prediction of response to therapy of Epstein-Barr virus-associated lymphoproliferative disease after allogeneic stem cell transplantation.

Epstein-Barr virus lymphoproliferative disease (EBV-LPD) following allogeneic stem cell transplantation (allo-SCT) has a poor prognosis. We used a sensitive real-time polymerase chain reaction (PCR) assay for quantitative detection of EBV-DNA in plasma and serially measured EBV-DNA levels to assess the response to treatment in allo-SCT recipients with EBV-LPD. Fourteen allo-SCT recipients with EBV-LPD who received a T cell-depleted (TCD) sibling (n = 5) or matched unrelated donor (n = 9) graft were monitored from the time of EBV-LPD diagnosis, during therapy and assessment of clinical response. Seven patients had complete responses of EBV-LPD to therapy, of whom 21% (3 out of 14) survived beyond 6 months from EBV-LPD diagnosis. Clinically responding patients showed a rapid decline of EBV-DNA plasma levels within 72 h from the start of therapy. In contrast, all clinical non-responders showed an increase of EBV-DNA levels. Absolute EBV-DNA levels at the time of EBV-LPD diagnosis did not predict for response, but the pattern of EBV-DNA levels within 72 h from the start of therapy (> 50% decrease versus increase) strongly predicted for clinical response (P = 0.001). Quantitative monitoring of EBV-DNA levels from the start of and during therapy for EBV-LPD rapidly and accurately predicts for response to therapy as early as within 72 h. It may thus provide a powerful tool to adjust and select treatment in individuals with EBV-LPD following allo-SCT.

Increased proteasome degradation of cyclin-dependent kinase inhibitor p27 is associated with a decreased overall survival in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an aggressive neoplasm characterized by the deregulated expression of cyclin D1 by t(11;14). The molecular mechanisms responsible for MCL's clinical behavior remain unclear. The authors have investigated the expression of p53, E2F-1, and the CDK inhibitors p27 and p21 in 110 MCLs, relating their expression to proliferative activity (Ki-67). For comparison, they have similarly analyzed low-grade (12 MALT, 16 CLL/SLL) and high-grade (19 DLCL) lymphomas. p53 was detected more frequently in large-cell MCL (l-MCL; 5 of 7) than in classical MCL (s-MCL; 13 of 103) and DLCL (8 of 19). In MCL and DLCL, the percentage of E2F-1+ nuclei was high, correlating with high Ki-67 expression. Most MCLs (91 of 112) and DLCLs (12 of 19) showed a loss of p27; MALT and CLL/SLL, however, were p27 positive. Reverse transcription-polymerase chain reaction and in vitro protein degradation assays demonstrated that MCLs have normal p27 mRNA expression but increased p27 protein degradation activity via the proteasome pathway. Correlation of MCL p53 and p27 expression with clinical data showed an association between reduced overall survival rates and the overexpression of p53 (P =.001), the loss of p27 (P =. 002), or both. Loss of p27 identified patients with a worse clinical outcome among p53 negative cases (P =.002). These findings demonstrated that MCL has a distinct cell cycle protein expression similar to that of high-grade lymphoma. The loss of p27 and the overexpression of p53 in MCL are prognostic markers that identify patients at high risk. The demonstration that low levels of p27 in MCL result from enhanced proteasome-mediated degradation should encourage additional clinical trials. (Blood. 2000;95:619-626) (Blood. 2000;95:619-626)

Hematopoietic growth factor receptors: structure variations and alternatives of receptor complex formation in normal hematopoiesis and in hematopoietic disorders.

Receptors of most hematopoietic growth factors are structurally related and grouped in the hematopoietin or cytokine receptor superfamily. In this paper, we will first review the general principles of hematopoietin receptor complex formation and cytoplasmic signaling. Subsequently, the significance of defective hematopoietic growth factor receptors for the development of hematological diseases will be discussed.

Interleukin-3 (IL-3) receptors on rhesus monkey bone marrow cells: species specificity of human IL-3, binding characteristics, and lack of competition with GM-CSF.

The relative affinity of recombinant human interleukin-3 (IL-3) binding to normal rhesus monkey bone marrow cells was found to be 25- to 50-fold less than that of homologous IL-3, which explained the species specificity of human IL-3 observed when tested in Macaca species. In contrast, only a small difference was found between human and rhesus monkey IL-3 in relative binding affinity for receptors on human acute myelogenous leukemia (AML) cells, which confirmed that the species specificity of IL-3 is largely unidirectional. The biological significance of the findings was demonstrated by direct in vivo comparison of the effects of high-dose recombinant rhesus monkey and human IL-3. The binding characteristics of IL-3 receptors on rhesus monkey bone marrow and peripheral blood cells were further characterized by specific binding of radiolabeled rhesus monkey IL-3. Scatchard analysis of two bone marrow samples demonstrated high-affinity IL-3 receptors (25 and 80 sites/cell, respectively; equilibrium dissociation constants [Kd] of 8 and 3 pM/L) as well as low-affinity receptors (1070 and 1290 sites/cell; equilibrium dissociation constants of 2600 and 1200 pM/L). In addition, IL-3 receptor expression was also detected on purified CD34-positive bone marrow cells. Competition by human granulocyte-macrophage colony-stimulating factor (GM-CSF) of IL-3 binding to high- or low-affinity receptors on rhesus monkey peripheral blood and bone marrow cells could not be demonstrated, which may indicate that the growth factor-specific alpha-subunits of the GM-CSF and IL-3 receptors are expressed predominantly on different cell types.

Granulocyte-macrophage colony-stimulating factor receptors alter their binding characteristics during myeloid maturation through up-regulation of the affinity converting beta subunit (KH97).

Acute myeloid leukemia blasts express dual affinity (high and low) granulocyte-macrophage colony-stimulating factor (GM-CSF) binding, and the high affinity GM-CSF binding is counteracted by excess interleukin-3 (IL-3). Neutrophils express a single class of GM-CSF-R with intermediate affinity that lack IL-3 cross-reactivity. Here we demonstrate the differentiation associated changes of GM-CSF binding characteristics in three models representative of different stages of myeloid maturation. We find that high affinity GM-CSF binding is converted into intermediate affinity binding, which still cross-reacts with IL-3, beyond the stage of promyelocytes. During terminal maturation towards neutrophils, IL-3 cross-reactivity is gradually lost. We sought to determine the mechanism underlying the affinity conversion of the GM-CSF-R. Northern and reverse transcriptase-polymerase chain reaction analysis of GM-CSF-R alpha and -beta c (KH97) transcripts did not provide indications for the involvement of GM-CSF-R splice variants in the formation of the intermediate affinity GM-CSFR complex. In COS-cell transfectants with increasing amounts of beta c in the presence of a fixed number of GM-CSF-R alpha chains, the high affinity GM-CSF binding converted into intermediate affinity GM-CSF binding. These results are discussed in view of the concept that increasing expression of beta c subunits may cause alternative oligomerization of the GM-CSF-R alpha and -beta c subunits resulting in the formation of intermediate rather than high affinity GM-CSFR alpha.beta c complexes.

Effects of mast cell growth factor on acute myeloid leukemia cells in vitro: effects of combinations with other cytokines.

We have investigated the stimulative effects of mast cell growth factor (MGF) in primary acute myeloid leukemia (AML) in vitro. MGF stimulated DNA synthesis of purified leukemic blasts in eight out of 10 cases and colony formation in four cases in serum-free (SF) culture. MGF synergized with interleukin-3 (IL-3; four out of 10 cases), granulocyte-macrophage colony-stimulating factor (GM-CSF; three out of 10 cases), granulocyte colony-stimulating factor (G-CSF; six out of 10 cases), macrophage colony-stimulating factor (M-CSF; one out of 10 cases) and erythropoietin (EPO; one out of 10 cases) when added to culture in combination. Synergistic effects of MGF in combination with other CSFs were also seen in the colony assay. Antibodies against GM-CSF, M-CSF, G-CSF, and IL-6 did not inhibit the MGF response, suggesting that the stimulative effect of MGF was not mediated through autocrine release of those cytokines. Cell recovery data in liquid cultures that contained MGF, IL-3, or MGF + IL-3, indicated that both MGF and IL-3 augmented the maintenance of clonogenic cells as compared to nonsupplemented cultures, but the effect of the combination of IL-3 + MGF did not show synergy. In contrast, activation of DNA synthesis by MGF was abrogated in the presence of tumor necrosis factor (TNF; four out of 10 cases) and interleukin-4 (IL-4; two out of 10 cases). Fluorescence-activated cell sorting (FACS) analysis with anti c-kit antibodies revealed MGF receptor expression in eight out of nine cases, often in a subpopulation of the cells. Scatchard analysis of MGF receptors in two cases indicated the presence of 1460 and 41,500 (mean) binding sites, respectively, of high affinity (Kd 40-160 pmol/l). The MGF dose-response curve in the presence of IL-3 or GM-CSF resulted in a higher plateau of DNA synthesis, however no shift in the dose response was apparent. The respective reciprocal dose response relations to GM-CSF, IL-3, or G-CSF were similarly elevated when MGF was added. MGF did not alter IL-3 and GM-CSF receptor expression, nor did IL-3, GM-CSF, G-CSF, TNF, or IL-4 influence MGF binding to AML cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Tumor necrosis factor downregulates granulocyte-colony-stimulating factor receptor expression on human acute myeloid leukemia cells and granulocytes.

Tumor necrosis factor (TNF) inhibits granulocyte-colony-stimulating factor (G-CSF)-induced human acute myeloid leukemia (AML) growth in vitro. Incubation of blasts from three patients with AML in serum-free medium with TNF (10(3) U/ml), and subsequent binding studies using 125I-G-CSF reveal that TNF downregulates the numbers of G-CSF receptors by approximately 70%. G-CSF receptor numbers on purified blood granulocytes are also downmodulated by TNF. Downregulation of G-CSF receptor expression becomes evident within 10 min after incubation of the cells with TNF at 37 degrees C and is not associated with an apparent change of the dissociation constant (Kd). The TNF effect does not occur at 0 degrees C and cannot be induced by IL-2, IL-6, or GM-CSF. TNF probably exerts its effect through activation of protein kinase C (PKC) as the TNF effect on G-CSF receptor levels can be mimicked by 12-O-tetradecanoylphorbol-13- acetate. The PKC inhibitor Staurosporine (Sigma Chemical Co., St. Louis, MO) as well as protease inhibitors can completely prevent G-CSF receptor downmodulation. Thus, it appears TNF may act as a regulator of G-CSF receptor expression in myeloid cells and shut off G-CSF dependent hematopoiesis. The regulatory ability of TNF may explain the antagonism between TNF and G-CSF stimulation.

Tumor necrosis factor regulates the expression of granulocyte-macrophage colony-stimulating factor and interleukin-3 receptors on human acute myeloid leukemia cells.

Tumor necrosis factor (TNF) acts as a potent enhancer of granulocyte-macrophage colony-stimulating factor (GM-CSF)- and interleukin-3 (IL-3)-induced human acute myeloid leukemia (AML) growth in vitro. We have analyzed the effects of TNF alpha on the expression of GM-CSF and IL-3 receptors on AML cells. Incubation of blasts from seven patients with AML in serum-free medium with TNF (10(3) U/mL) and subsequent binding studies using 125I-GM-CSF and 125I-IL-3 show that TNF increases the specific binding of GM-CSF (30% to 280%) and IL-3 (40% to 600%) in all cases. From Scatchard plot analysis it appears that TNF upregulates (1) low-affinity GM-CSF binding sites, (2) common high-affinity IL-3/GM-CSF binding sites, and (3) unique (non-GM-CSF binding) IL-3 binding sites. The effect of TNF is dose dependent and is half maximal at a concentration of 100 U/mL, and becomes evident at 18 hours of incubation with TNF at 37 degrees C, but not at 0 degree C. The GM-CSF dose-response curve of AML-colony-forming units plateaus at a higher level in the presence of TNF, which indicates that additional numbers of cells become responsive to GM-CSF. Incubation of AML blasts with the phorbol ester 12-0-tetradecanoylphorbol-13-acetate or formyl-Met-Leu-Phe (protein kinase C activators) does not influence GM-CSF receptor expression, suggesting that receptor upregulation by TNF is not mediated through activation of protein kinase C. On the other hand, the protein synthesis inhibitor cycloheximide abrogates receptor upregulation induced by TNF. In contrast to these findings in AML, TNF does not upregulate GM-CSF receptor numbers on blood granulocytes or monocytes. We conclude that TNF exerts positive effects on growth factor receptor expression of hematopoietic cells.

Common binding structure for granulocyte macrophage colony-stimulating factor and interleukin-3 on human acute myeloid leukemia cells and monocytes.

Granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) control the proliferation of human acute myeloid leukemia (AML) cells in vitro. Previously, we have shown that receptors for GM-CSF and IL-3 are often coexpressed on AML cells. Here we present experiments with purified AML blasts, normal monocytes, and granulocytes that were conducted to analyze the properties of GM-CSF and IL-3 binding proteins in more detail. On AML cells from eight cases we demonstrate two types of GM-CSF receptors: one with low affinity (dissociation constant [kd] 5.1 to 24.8 nmol/L) and one with a high affinity (kd 31 to 104 pmol/L). These AML cells also expressed high affinity receptors for IL-3 (kd 24 to 104 pmol/L). Cross-competition experiments showed that an excess concentration of nonlabeled IL-3 completely prevented the high affinity binding of radiolabled GM-CSF. This competition occurred at 37 degrees C as well as 4 degrees C. Low affinity GM-CSF binding was not affected by IL-3. Binding of radiolabeled IL-3 could be prevented by nonlabeled GM-CSF. In certain cases, this competition was complete, whereas in others only partial (49% to 77%) reduction of the radiolabeled IL-3 binding was seen. On the basis of these ligand binding features, we propose the existence of three receptor types on AML cells: (1) low affinity GM-CSF receptors that do not bind IL-3, (2) dual high affinity GM-CSF/IL-3 receptors, and (3) high affinity IL-3 receptors that do not bind GM-CSF. We could also demonstrate these receptor types on normal monocytes. In addition, a fourth type of receptor was apparent on normal granulocytes (4), incapable of binding IL-3 and with an intermediate affinity for GM-CSF (approximately 400 pmol/L). Chemical crosslinking showed that GM-CSF and IL-3 both bind to proteins with molecular weight values of 130, 105, and 75, which provides additional evidence for the existence of a common GM-CSF/IL-3 receptor complex.

Granulocyte colony-stimulating factor receptors in human acute myelocytic leukemia.

The binding of granulocyte colony-stimulating factor (G-CSF) to normal and human acute myeloid leukemia (AML) cells was investigated with radiolabeled recombinant human G-CSF (rhG-CSF). In all 14 cases of primary AML specific receptors for G-CSF were demonstrated on purified blast cells. The average numbers of G-CSF receptors ranged from very low to 428 receptors per cell (mean). Normal granulocytes showed G-CSF binding sites on their surface at higher densities (703 to 1,296 sites per cell). G-CSF receptors appeared to be of a single affinity type with a dissociation constant (kd) ranging between 214 and 378 pmol/L for AML blasts and 405 to 648 pmol/L for granulocytes. In 12 of 14 cases, including those with relatively low specific binding, G-CSF was a potent inducer of DNA synthesis of blasts in vitro; therefore, apparently relatively few receptors are required to permit activation of AML cell growth. However, in two cases cell cycling was not activated in response to G-CSF despite G-CSF receptor availability. The results show that G-CSF receptors of high affinity are frequently expressed on the blasts of human AML, but their presence may not be a strict indicator of the proliferative responsiveness of the cells to G-CSF.

Interleukin-3 and granulocyte-monocyte colony-stimulating factor receptors on human acute myelocytic leukemia cells and relationship to the proliferative response.

Interleukin-3 (IL-3) and granulocyte-monocyte-colony-stimulating factor (GM-CSF) stimulate proliferation of human acute myeloid leukemia (AML) in vitro, although patterns of response among clinical cases are diverse. Whether regulatory abnormalities related to growth factor responses in human AML may establish the outgrowth of the neoplasm is unclear. We determined receptor numbers and affinity for IL-3 and GM-CSF on human AML cells using human recombinant IL-3 (rIL-3) and GM-CSF (rGM-CSF). In 13 of 15 cases of primary AML high-affinity (kd 26 to 414 pmol/L) receptors for IL-3 were demonstrable on the cells. The average numbers of IL-3 receptors ranged from 21 to 145 receptors per cell. Normal WBCs showed IL-3 receptors on their surface at similar densities. IL-3 receptor positivity often correlated with GM-CSF receptor positivity of AML; GM-CSF receptors were demonstrated on the cells of 11 of 15 cases, although average numbers of GM-CSF receptors were ten times greater. The in vitro response of the cells to exogenous IL-3 or GM-CSF was examined by measuring thymidine uptake. Because IL-3 and GM-CSF were potent inducers of DNA synthesis in vitro, apparently relatively few receptors are required to permit activation of growth. These experiments did not provide evidence for overexpression or increased receptor sensitivity as an explanation for AML growth. In a minority of cases, however, the cells were unable to respond to IL-3 (four of 15 cases) or GM-CSF (four of 15 cases) despite normal receptor availability on the cell surface.