Quantitative expression of thrombopoietin receptor on leukaemia cells from patients with acute myeloid leukaemia and acute lymphoblastic leukaemia

Using a non-isotopic ligand binding assay, we quantitatively examined the amount of human thrombopoietin (TPO) receptor (TPO-R) on leukaemia cells from 128 patients with acute myeloid leukaemia (AML) or acute lymphoblastic leukaemia (ALL). The TPO-R was expressed in 53 (47%) of 114 AML cases, and an in vitro treatment with TPO led to proliferation (stimulation index >1.5) of leukaemia cells in 13 (20%) of 66 AML cases examined. The TPO levels had no relation to the FAB classification except for FAB-M7 AML. All five FAB-M7 cases expressed TPO-R, and one of three FAB-M7 examined showed in vitro proliferative response to TPO. Although there was no significant correlation ( r  = 0.3125) between the amount of TPO-R and the proliferative response, all of the AML cases which showed the in vitro response had TPO-R expression. There was no relationship between TPO-R amount and CD phenotypes, or the amount of granulocyte-colony stimulating factor (G-CSF) receptor. TPO-R was also expressed in two (14%) of 14 cases of ALL, and only these two cases had in vitro proliferative response to TPO. One had only lymphoid antigens, and the other had both lymphoid and myeloid antigens. Our results suggest that some leukaemia cells express functionally active TPO-R.     

Blood dendritic cells in patients with acute lymphoblastic leukaemia

Myeloid and plasmacytoid dendritic cells (MDCs, PDCs) play a key role in the initiation of immune responses. In this study, we show a severe reduction of MDCs and PDCs in patients with B lineage acute lymphoblastic leukaemia (B-ALL; P = 0.01 vs. controls). DCs from patients with T lineage ALL (T-ALL) were quantitatively and functionally comparable to healthy donors, as demonstrated by secretion of interleukin (IL)-12p70 and interferon-alpha. In vitro, the circulating CD34(+) fraction of B-ALL cases did not generate either CD1a(+) MDCs or PDCs, suggesting that DC development is probably affected in B-ALL, but not in T-ALL.     

Inducibility of lymphokine activated killer (LAK) cells in patients with acute myelogenous leukaemia in complete remission and its clinical relevance

Peripheral blood mononuclear cells (PBMC) from 42 patients with acute myelogenous leukaemia (AML) in complete remission (CR) and from normal donors were activated into LAK cells in the presence of 1000 U/ml of recombinant interleukin-2 (rIL-2). Cytotoxicity of LAK cells was assayed against K562, Daudi, and Raji cell lines, and autologous and/or allogeneic thawed leukaemic blasts. Fresh unactivated PBMC from normal donors and AML patients served as controls. Mean +/- standard deviation (SD) percentage lysis of the different targets by patient LAK cells were: K562 61 +/- 20%, Daudi 62 +/- 23%, Raji 48 +/- 24%, autologous blast cells 12 +/- 16% and allogeneic blast cells 13 +/- 10%. Lysis of the different targets by LAK cells from normal donors was similar to that achieved with LAK cells from AML patients. Overall there was a good correlation between the lysis of the different targets. There was no significant difference between the percentage lysis of autologous and allogeneic thawed blast cells, although LAK cells from seven out of the 18 patients tested were unable to lyse autologous leukaemic cells. Activity of patient LAK cells did not correlate with the initial characteristics of the patient nor with the time spent in CR before harvesting PBMC for activation. At the time of analysis, 32 patients were in continuing CR and 10 had relapsed. Multivariant analysis for prognostic factors showed that patients whose LAK cells had more lytic activity on K562 (P = 0.005) and fresh blast cell (P = 0.02) targets had significantly less risk of relapse than patients with little inducible LAK cell activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Down-regulation of liver regeneration by LAK cells--a study for effect of neuraminidase treated LAK cells on liver regeneration]

Both effect of LAK cells and neuraminidase treated LAK (N-LAK) cells on liver regeneration were investigated after 70% partial hepatectomy in mice. Intravenous transfusion of LAK cells suppressed the liver regeneration depending on cell numbers injected. N-LAK cells accumulated into regenerating liver 1.7 times in cell number compared with LAK cells. Injection of 5 x 10(7) LAK cells and N-LAK cells into hepatectomized mice suppressed the liver regeneration by 16.5% and 53.8% respectively. These results indicated that suppression of the liver regeneration by LAK cells was dependent upon the number of injected LAK cells and the degree of accumulation of LAK cells into the liver, namely, LAK cells down-regulate the regeneration of liver cells in the micro-milieu.     

免疫因子对慢性免疫性血小板减少性紫癜骨髓巨核祖细胞体外生长的影响

目的 :探讨慢性免疫性血小板减少性紫癜 (CITP)骨髓巨核祖细胞生长成熟与免疫因素的关系。方法 :通过巨核祖细胞体外培养 ,观察CITP骨髓巨核祖细胞生长和成熟情况 ,并观察抗CD80抗体及干扰素α 2b(IFN α 2b)对CITP和对照组巨核祖细胞生长和成熟的影响 ,并与对照组比较。结果 :①CITP组的各种集落数(CFU MK、BFU MK、mCFU MK)均低于对照组 ,两组间集落数的均值比较差异有统计学意义 (P <0 .0 5 )。CITP组的直径和面积均低于对照组 ,两组间直径和面积均值比较差异有统计学意义 (P <0 .0 5 )。②加入抗CD80抗体的CITP的巨核祖细胞集落生成数明显增多 (P <0 .0 5 )。③IFN α 2b对两组的总集落均有抑制 ,CITP比对照组的巨核祖细胞集落生成受抑制程度低 (P <0 .0 5 )。结论 :CITP巨核祖细胞的集落生成和成熟度较低。抗CD80抗体和IFN α 2b对CITP巨核祖细胞的集落形成均有影响     

Neoplastic circulating endothelial-like cells in patients with acute myeloid leukaemia

Accumulating evidence suggests that angiogenesis may play a key role in the pathogenesis of leukaemic disorders. Several studies have shown that bone marrow-derived endothelial cells (EC) may contribute to tumour angiogenesis and that in the peripheral blood of cancer patients there is an increased amount of circulating ECs (CECs) that may participate to new vessel formation. In this report, we showed that, in seven acute myeloid leukaemia (AML) patients with known cytogenetic abnormalities, CEC levels were significantly increased in comparison with controls and that a significant proportion of these CECs carried the same chromosomal aberration as blast cells (20-78%, mean value 42.1% of CECs). Most of CECs (mean value 74.4%) displayed immunophenotypic features of endothelial progenitor cells as they expressed CD133, a marker gradually lost during EC differentiation and absent on mature EC. These findings suggest a possible direct contribution of AML-related CECs to tumour vasculogenesis and possibly to the spreading and progression of the disease.     

In-vitro differentiation of cells of patients with acute undifferentiated leukaemia

The diagnosis of acute undifferentiated leukaemia (AUL) is made when the cells of patients with acute leukaemias cannot be classified as myeloid or lymphoid by means of morphological, cytochemical and immunological criteria. The mononuclear cells of eight different AUL patients were cultured in suspension for 3 d with or without TPA. After culture, especially in the presence of TPA, the cells of all patients expressed at least one myeloid membrane antigen. It was shown that this antigen expression was dependent on de novo protein synthesis and not influenced by inhibition of proliferation.     

Survival in 222 adult patients with acute leukaemia treated with intermittent combination chemotherapy programme

222 patients aged 15-59 years with acute leukaemia were treated with intermittent combination chemotherapy consisting of six 5-day courses of cytotoxic drugs as induction treatment followed by 3 years of maintenance therapy in patients who obtained complete remission (CR). CR was achieved in 50.3% of 161 patients (early deaths included) with acute myelogenous leukaemia (AML). The observed cumulative 5-yr survival rate (observation time 1-12.25 yr) calculated by the life table method was 12% in AML. Among patients who obtained CR, those aged 40-59 yr appeared to fare better than younger patients (5-yr survival: 24%). The M-3 subtype was an adverse prognostic factor. In acute lymphoblastic leukaemia (ALL) the CR rate was 83.6% and the observed cumulative 5-yr survival rate was 14%. Women fared better than men.     

Deoxycytidine kinase and cN-II nucleotidase expression in blast cells predict survival in acute myeloid leukaemia patients treated with cytarabine

Summary. The cytotoxic activity of cytarabine (ara-C) in leukaemic blasts depends on activating enzymes such as deoxycytidine kinase (dCK) and inactivating enzymes such as the 5'-nucleotidases. We have analysed dCK and 'high-Km' 5'-nucleotidase (cN-II) mRNA expression by the quantitative real-time polymerase chain reaction at diagnosis in leukaemic blasts from 115 acute myeloid leukaemia (AML) patients treated with ara-C. The prognostic value of these parameters as well as that of the cN-II/dCK ratio was determined. In univariate analyses: (1) low levels of dCK, high levels of cN-II and a high cN-II/dCK ratio predicted shorter disease-free survival (DFS); (2) low levels of dCK and cN-II/dCK ratio also predicted shorter overall survival (OS). In a multivariate analysis taking into account other clinical and laboratory variables: (1) high cN-II expression, a high cN-II/dCK ratio, age ≥ 60 years and an unfavourable karyotype were independent prognostic factors for DFS; and (2) a high cN-II/dCK ratio, age ≥ 60 years and an unfavourable karyotype predicted shorter OS. Age, karyotype and cN-II/dCK ratio were used to define a prognostic score that permitted the identification of high- and low-risk groups. Our results suggest that dCK and cN-II mRNA expression in leukaemic blasts at diagnosis is correlated with clinical outcome and may play a functional role in the resistance to ara-C in patients with AML.     

回生口服液对人IL-2水平及LAK细胞活性的影响

目的探讨回生口服液对人体ＩＬ２水平及ＬＡＫ细胞活性的影响．方法取健康献血者外周静脉血，分离出单个核细胞（ＰＢＭＣ），加入植物血凝素（ＰＨＡ），于９６孔微量培养板中，分别加入不同浓度的回生口服液，孵育２４ｈ，以ＣＴＬＬ２细胞作为检测细胞，用ＭＴＴ法测定ＩＬ２水平，同时设空白对照及阴性对照组；分离出的ＰＢＭＣ加入γＩＬ２，于２５ｍｌ培养瓶中共同孵育９６ｈ，制备ＬＡＫ细胞，然后将其与经不同浓度回生口服液处理后的ＳＭＭＣ７７２１，Ｈｅｌａ细胞株于９６孔微量培养板中共同孵育２０ｈ，用ＬＤＨ释放法测ＬＡＫ细胞活性，同时设空白对照．全部数据采用ｔ检验方法进行处理．结果回生口服液在浓度为１０ｍｇ／ｍｌ以上时能促进ＰＢＭＣ在ＰＨＡ作用下分泌ＩＬ２（Ｐ＜００１），在１ｍｇ／ｍｌ时即能拮抗１％（Ｐ＜００１）、５％（Ｐ＜００５）人血清ＩＬ２抑制物的作用，且都与药物浓度呈正相关，在高浓度（１００ｍｇ／ｍｌ）时还能增强ＬＡＫ细胞的活性（Ｐ＜００１）．结论回生口服液可提高人ＩＬ２水平，并增强ＬＡＫ细胞活性，以实现抗癌作用．     

Zygomycoses in patients with acute leukaemia

Zygomycoses are rare invasive mould infections which mainly occur in immunocompromised patients, especially during prolonged neutropenia. The high mortality rate is due to a high failure rate of both intravital diagnosis and treatment. Exact diagnosis requires microscopic examination and proof by culture. The treatment consists of amphotericin B and surgical debridement. We report four recent cases of zygomycosis among 89 patients with intensively treated acute leukaemia at our institution. Three cases were breakthrough infections since the patients were under voriconazole treatment prior to diagnosis of zygomycosis. Only one patient had premortal diagnosis (paranasal sinus infection) and showed clinical response with amphotericin B and surgical debridement. A review of the literature of these emerging fungal infections is given and is focused on patients with acute leukaemia. In addition, the importance of autopsy as a tool for quality control and epidemiological studies is pointed out.     

Evaluation of natural killer and lymphokine-activated killer (LAK) cell activity in vivo in patients treated with high-dose interleukin-2 and adoptive transfer of autologous LAK cells

Summary This study investigated the development of peripheral blood natural killer (NK) and lymphokineactivated killer (LAK) cell activity in vivo in cancer patients treated with high doses of recombinant interleukin-2 and autologous LAK cell infusion. It was found that interleukin-2 administration by bolus injection resulted in an early but transient rise of NK and LAK cell activity in vivo during the first 5–9 days of treatment (postpriming period), whereas continuous infusion of interleukin-2 caused an increase in both cytotoxic activities in the second phase of the treatment, concomitant with infusions of autologous LAK cells. Elevated NK but not LAK cell activity in vivo in the continuous-infusion patients persisted up to 180 days after completion of therapy. In both bolus and continuous interleukin-2 protocols, augmented NK cell activity in vivo appeared to be correlated with a beneficial response to therapy.Supported by the US-Poland Cancer Program (NCI) and by NCI Contract NO1-CM-73705     

Glycophorin A mutations and risk of secondary leukaemia in patients treated for childhood acute lymphoblastic leukaemia

Survivors of childhood acute lymphoblastic leukaemia (ALL) have a higher than expected risk of developing secondary acute myeloid leukaemia (AML). The glycophorin A (GPA) mutation assay measures the frequency of variant NO and NN erythrocytes in MN heterozygotes. A raised variant frequency (Vf) has been shown in patients treated with chemotherapy known to be at risk of secondary leukaemia. ALL patients were investigated for increased Vf using the GPA assay. Vf's at diagnosis were not significantly different from controls (NO Vf P  = 0.193; NN Vf P  = 0.790). During treatment Vf's increased significantly (NO Vf P  = 0.001; NN Vf P  = 0.001). NO Vf returned to control values ( P  = 0.169) within 5 years from diagnosis but NN Vf remained significantly raised ( P  = 0.014). Three study patients developed secondary AML. At diagnosis of AML all three had significantly increased Vf. The first had a significantly raised Vf at routine follow-up 19 years following diagnosis of ALL then developed AML 3.5 years later. The second had a significantly raised NN Vf at diagnosis of ALL indicating possibly prior exposure to a mutagen or defective DNA repair involving erythroid stem cells. We conclude that a raised Vf detected by the GPA assay can act as a marker for the development of secondary induced leukaemia and can be used to screen individuals at a known high risk of this complication.     

Increased regulatory T cells in acute lymphoblastic leukaemia patients

Introduction: Regulation in adaptive immune response balances a fine line that prevents instigation of self-damage or fall into unresponsiveness permitting abnormal cell growth. Mechanisms that keep this balance in check include regulatory T cells (Tregs). Tregs consist of a small but heterogeneous population, which may be identified by the phenotype, CD3+CD4+CD25+CD127?. The role of Tregs in pathogenesis of cancers is thus far supported by evidence of increased Tregs in various cancers and may contribute to poorer prognosis. Tregs may also be important in acute leukaemias.

Objective: A review of the literature on Tregs in acute leukaemias was conducted and Tregs were determined in B-cell acute lymphoblastic leukaemias (ALLs).

Results: Studies on Tregs in B-cell ALL are few and controversial. We observed a significantly increased percentage of Tregs (mean±SD, 9.72?±?3.79% vs. 7.05?±?1.74%; P?=?0.047) in the bone marrow/peripheral blood of ALL (n?=?17) compared to peripheral blood of normal controls (n?=?35). A positive trend between Tregs and age (R?=?0.474, P?=?0.055, n?=?17) implicates this factor of poor prognosis in B-cell ALL.

Discussion: Tregs in cancer are particularly significant in immunotherapy. The manipulation of the immune system to treat cancer has for a long time ignored regulatory mechanisms inducible or in place. In lymphoma studies, tumour-specific mechanisms that are unlike conventional methods in the induction of Tregs have been hypothesized. In addition, tumour-infiltrating Tregs may present different profiles from peripheral blood pictures. Tregs will continue to be dissected to reveal its mysteries and their impact on clinical significance.     

Human CD80/IL2 lentivirus-transduced acute myeloid leukaemia (AML) cells promote natural killer (NK) cell activation and cytolytic activity: implications for a phase I clinical study

Immunotherapeutic strategies may promote T and/or natural killer (NK) cell cytotoxicity. NK cells have the potential to exert a powerful anti-leukaemia effect, as demonstrated by studies of allogeneic transplantation. We have previously shown that CD80/interleukin 2 (IL2) lentivirus (LV)-transduced AML cells stimulate in-vitro T cell activation. The present study demonstrated that allogeneic and autologous culture of peripheral blood mononuclear cells with CD80/IL2-expressing AML cells also promoted NK cell cytotoxicity. Expression of the activation receptors NKp30, NKp44, CD244, CD25, CD69 and HLA-DR significantly increased following allogeneic culture and a consistent increased expression of NKp30, NKp44, NKp46, NKG2D, NKG2C and CD69, and up-regulation of the cytolytic marker CD107a was detected following autologous culture with LV-CD80/IL2 AML cells. Furthermore, increased NK cell lysis of K562 and primary AML blasts was detected. The lytic activity increased by twofold against K562 (from 46·6% to 90·4%) and allogeneic AML cells (from 11·8% to 20·1%) following in-vitro stimulation by CD80/IL2-expressing AML cells. More importantly for potential therapeutic applications, lysis of primary AML cells by autologous NK cells increased by more than 40-fold (from 0·4% to 22·5%). These studies demonstrated that vaccination of patients with CD80/IL2-transduced AML cells could provide a powerful strategy for T/NK cell-mediated stimulation of anti-leukaemic immunological responses.     

Depressed fibrinolysis in patients with acute leukaemia

The fibrinolytic system was studied in 46 patients with acute leukaemia at diagnosis. Untreated patients (with the sole exception of the M3 subgroup) showed an inhibition of fibrinolytic activity, measured by the euglobulin lysis time and area. This inhibition was accompanied by reduced t-PA antigen and t-PA inhibitor activity. No correlation was found between the above-mentioned fibrinolytic parameters and the biochemical haematological values considered, nor with clinical and/or laboratory features of DIC, fever, liver failure. The decrease in immunological plasminogen and functional alpha 2-antiplasmin, showed a significant correlation with the presence of clinical and/or laboratory signs of DIC, as diagnosed on the basis of concomitant increase in fibrin monomers, plasmatic fibrinopeptide A and serum FDP.     

Hepatosplenic candidiasis in patients with acute leukaemia

A retrospective study of 23 patients with acute leukaemia and hepatosplenic candidiasis (HSC) was conducted to evaluate clinical treatment characteristics in terms of amount and duration of antifungal agents and to assess treatment outcome. Patients were admitted to two major tertiary care centres between 1990 and 1998. The diagnosis of HSC was based on clinical, blood cultures, histologic and imaging studies. Patients were treated with amphotericin B without interruption of the planned chemotherapy regimens. Serial magnetic resonance imaging (MRI) studies were the main tool for following patients' response and activity of the fungal lesions in conjunction with clinical and laboratory parameters. Treatment with amphotericin B was continued until resolution of all clinical symptoms and signs attributable to HSC, obtaining negative blood cultures and the appearance of at least healed lesions on MRI. Amphotericin B was discontinued in four patients because of severe nephrotoxicity (two patients), or continuous fever and persistent fungal lesions on MRI (two patients). Amphotericin B lipid complex (ABELCET) was successfully used as salvage therapy for these refractory patients. Four patients died with evidence of HSC despite treatment and supportive measures. The response rate for treatment of HSC was 82%. The mean total dose of amphotericin B including empirical treatment was 4 g and the median duration of treatment for responding patients was 112 d. The median number of days of anti- fungal treatment before the disappearance of fever was 19 d. Our results confirmed the need for protracted courses of antifungal agents for the successful eradication of HSC. Chemotherapy for the underlying disorder should not be interrupted or delayed in order to treat HSC.