Kinetics of bone marrow cell production in human acute and chronic myeloid leukemias

Utilizing a technique of quantitative ¹⁴C-autoradiography, relative rates of cell production were determined in the various morphological cell compartments of erythro-and granulo-cytopoiesis in human myeloid leukemias. AML, chronic phase of CML as well as blastic crisis of CML were studied. From the ratios of relative cell production rates in the individual compartments of the white and red cell lineages and by comparison of the two lineages, implications concerning a reduction or increase of cell production are derived. A deficit of erythroblast production increasing with progression towards the more mature stages was attributed to ineffective erythropoiesis. The loss of erythroblasts in the proliferative pool ranged from 45% in CML to 79% in AML.The ratios of granulocytic precursor production in the various CML compartments (chronic phase) were normal. In AML and blastic crisis of CML, on the other hand, an excess of blast cell production was observed. In view of the complete analysis of all proliferative cells in these bone marrows, it appears that there is a capacity of blast cells to expand in number by self-amplification. This self-amplification is not necessarily restricted to one morphological cell type. It takes place among myeloblasts, but promyelocytes may also exhibit this ‘leukemic’ type of cell growth. The data further suggest that the stem cell influx into the red and presumably also granulocyte series is roughly normal in AML and blastic crisis of CML, whereas it is increased in CML.     

Expression of human myeloid cell nuclear differentiation antigen (MNDA) in acute leukemias

Human myeloid cell nuclear differentiating antigen (MNDA) is a Mr 55,000 non-histone basic nuclear protein expressed in myeloid leukemia cell lines that are at late stages of differentiation (HL-60 and U937) and in normal granulocytes and monocytes, but is not present in lymphoid cells or in other human cells and tissues tested. Affinity purified monospecific polyclonal antibodies and rat monoclonal antibodies have been developed for the immunocytochemical detection of MNDA. Using these antibodies, we surveyed 21 cases of acute leukemia classified by French-American-British (FAB) Group criteria, two cases of biphenotypic acute leukemia and one case of blast crisis of chronic granulocytic leukemia for the presence of MNDA. The most intense staining reactions were present in the nuclei of two cases of acute promyelocytic (FAB M3) leukemia. MNDA was not detected in three of five cases of acute myeloblastic leukemia without maturation (FAB M1). The remaining two cases of the M1 category showed weak to moderate staining. No staining reaction was seen in acute lymphocytic leukemia (ALL), biphenotypic leukemia or the lymphoid blast crisis of chronic granulocytic leukemia. Variable staining reactions were demonstrated in the remaining cases. These data suggest that the presence of MNDA is correlated with myeloid and monocytic differentiation in acute leukemia, being strongly expressed in M3 type, often not detected in M1 leukemia and absent in ALL.     

Expression of p120 nucleolar proliferating antigen in human gliomas and growth suppression of glioma cells by p120 ribozyme vector

p120 is a nucleolar proliferating antigen which is expressed in tumor cells but not normal resting cells. The expression and localization of p120 in human gliomas were studied by Northern blot analysis, Western blot analysis and immunohistochemistry. All five of the glioma cell lines and all of the glioma specimens we investigated expressed p120 at both the mRNA and protein levels. p120 expression was not detected in adjacent brain tissues. A ribozyme vector was constructed to cleave the first GUC sequence in the coding region of p120 mRNA. This p120 ribozyme vector was transfected into the glioma cell line SF188, which expresses p120. The reduced p120 expression of the transfectant at both the mRNA and protein levels was confirmed. An MTT assay indicated that the transfected cells grew more slowly than control cells. These results indicate that i) p120 has an important role in the proliferation of gliomas, and ii) the ribozyme against p120 mRNA can suppress glioma cell growth.     

Expression of VEGF-C and its receptor VEGFR-3 in the bone marrow of patients with acute myeloid leukaemia

Vascular endothelial growth factor-C (VEGF-C) has been shown to promote survival and resistance to chemotherapy of AML-cells in vitro. We investigated the expression of VEGF-C/VEGFR-3 in the bone marrow and pretherapeutic plasma levels of VEGF-C in patients with newly diagnosed AML. Expression of VEGF-C/VEGFR-3 was significantly higher in AML patients than in controls, while circulating levels did not differ. However, VEGF-C/VEGFR-3 expression was not able to predict clinical outcome. In conclusion, AML is associated with an increased expression of VEGF-C/VEGFR-3. Although expression levels display no prognostic significance in our study, strategies targeting the VEGF-C/VEGFR-3-pathway might be a promising treatment approach.     

流感疫苗促进髓系白血病骨髓细胞来源树突状细胞的功能

目的: 研究流感疫苗对髓系白血病骨髓源性树突状细胞（dendritic cells,DCs）功能的影响及其机制。方法：分离髓系白血病患者\[急性髓细胞白血病(acute myeloid leukemia, AML)19例, 慢性髓细胞白血病（chronic myeloid leukemia,CML) 8例\]骨髓单个核细胞（mononuclear cell,MNC）,用GMCSF和IL4诱导7 d,获得未成熟白血病DCs,然后加入全病毒灭活流感疫苗（whole inactivated influenza vaccine, WIV）、裂解病毒流感疫苗（split influenza vaccine,SIV）或TNFα继续培养24 h。R显带法分析DCs染色体核型,流式细胞仪检测DCs表型,ELISA法测定DCs培养上清IL12的水平,CCK8法检测DCs诱导的CTL对自体白血病细胞的细胞毒作用。结果：19例AML患者中的15例及8例CML患者的MNC全部成功诱导出DCs。与TNFα刺激的白血病DCs相比,流感疫苗刺激的白血病DCs表面分子（CD80、CD83、CD86、HLADR）表达明显上调（P<005）,培养上清中IL12的分泌水平明显增加（P<0.05）,其诱导的CTL可显著杀伤自体白血病细胞（P<0.05）;WIV刺激的DCs在表型、IL12分泌水平及细胞毒作用方面均较SIV刺激的DCs显著增高（P<0.05）。结论： 流感疫苗促进髓系白血病源DCs表型成熟及IL12的分泌,增强其诱导的CTL对自体白血病细胞的杀伤作用。     

Methotrexate kinetics in myeloid bone marrow cells and peripheral neutrophils

The pharmacokinetics of methotrexate (MTX) in the proliferating and the maturing myeloid compartments of the bone marrow and in the neutrophils of the peripheral blood was investigated in four patients with malignant non-Hodgkin's lymphoma after treatment with 24-h MTX infusions (500-790 mg/m2) followed by leukovorin rescue. The myeloid bone marrow cells were separated into two fractions, using a two-step discontinuous Percoll gradient with densities of 1.076 and 1.095 g/ml. The upper fraction consisted predominantly of immature bone marrow cells plus lymphocytes, and the lower fraction contained the mature myeloid bone marrow cells. Cells in the proliferating (immature) myeloid compartment took up and retained MTX to a much greater extent than the mature myeloid cells and the neutrophils. Two days after the MTX infusion no MTX was detected in the neutrophils in spite of a general rise in the total neutrophil count. MTX reappeared in the neutrophils on day 8 in concentrations not related to the concentrations in the proliferating myeloid cells during the MTX infusion. The time of reappearance of MTX in the neutrophils was in accordance with the time it takes for cells in the proliferating pool of the bone marrow to mature and be released into the circulation. No neutropenia was seen after the MTX infusions.     

Comparison of autoradiography and DNA histograms in assessing S-phase cells from patients with myeloid leukemias

The studies reported here were designed to determine whether DNA histogram analysis provided the same estimate for the percentage of cells in S-phase as does 3H-TdR labeling index (LI). For DNA histogram analysis high resolution flow cytometry was employed together with 3 different computer based methods for histogram analysis. When tissue culture cells were employed there was a good correlation (r = 0.89) between the estimates for the percentage of cells in S-phase by the LI and DNA histogram techniques. In contrast, when 1138 bone marrow or peripheral blood specimens obtained from leukemic patients were studied the correlation was poor. For example when 243 pretherapy bone marrow aspirate cells were studied by both methods the correlation was only 0.46. A direct comparison of the clinical relevance of the two methods for measuring S-phase cells when high dose cytosine arabinoside therapy was used to treat patients demonstrated that only LI measurements provided clinically useful information. In conclusion, DNA histogram analysis provides an inaccurate estimate of the percentage of cells in S-phase.     

伴有髓系分化抗原表达的神经母细胞瘤骨髓转移1例并文献复习

目的：报道伴有髓系抗原表达的神经母细胞瘤骨髓转移患儿1例,并进行文献复习。方法：取患者骨髓液进行细胞形态学分析及流式细胞术检测细胞分化抗原。结果：该患者骨髓形态学考虑为神经母细胞瘤髓内转移,经流式细胞术分析异常细胞同时表达髓系分化抗原CD13、CD15和CD11b,结合影像学检查确诊为神经母细胞瘤（Ⅳ期）。结论：复习相关文献并结合本例报道发现神经母细胞瘤细胞和造血细胞拥有一些相同的抗原决定簇,可伴有髓系及淋巴系分化抗原的表达,但其在神经母细胞瘤的分化程度、临床分期、疗效及预后判断上的作用尚需进一步研究。     

Prognostic significance of the glucocorticoid receptor level in the bone marrow blast cells of children with leukemias

Glucocorticoid receptor (GR) levels of bone marrow blast cells were measured in 29 pediatric patients suffering different forms of acute leukemia. A decrease in GR level was registered immediately after prednisolone treatment. Differences in cell GR levels in patients with lymphoblastic and myeloblastic leukemia were not significant. The level of GR of bone marrow blast cells was found to be of prognostic significance in acute lymphoblastic leukemia: 4 out of 5 patients with GR levels under 3,000 sights per cell relapsed within 5-7 months of follow-up whereas all 7 cases with higher levels continue in remission. No correlation between GR level and prognosis was established in 5 patients with various forms of acute myeloblastic leukemia.     

伴有髓系分化抗原表达的神经母细胞瘤骨髓转移1例并文献复习

目的:报道伴有髓系抗原表达的神经母细胞瘤骨髓转移患儿1例,并进行文献复习.方法:取患者骨髓液进行细胞形态学分析及流式细胞术检测细胞分化抗原.结果:该患者骨髓形态学考虑为神经母细胞瘤髓内转移,经流式细胞术分析异常细胞同时表达髓系分化抗原CD13、CD15和CD11b,结合影像学检查确诊为神经母细胞瘤(Ⅳ期).结论:复习相关文献并结合本例报道发现神经母细胞瘤细胞和造血细胞拥有一些相同的抗原决定簇,可伴有髓系及淋巴系分化抗原的表达,但其在神经母细胞瘤的分化程度、临床分期、疗效及预后判断上的作用尚需进一步研究.     

慢性粒细胞白血病80例骨髓活检分析

目的:对慢性粒细胞白血病骨髓活检进行分析,以便全面了解慢性粒细胞白血病的骨髓情况,有助于诊断及治疗。方法:用塑料包埋法制片,然后进行HGF,Gomori染色,观察各项指标。结果:80例病人中78例(97％)骨髓增长明显活跃;19例(61％)伴有不同程度的纤维化;19例(23％)可见3～10个原幼细胞簇。结论:骨髓活检可以全面了解慢性粒细胞白血病的骨髓增生情况、骨髓纤维化程度及原幼细胞簇数量,对判断分期及预后,指导治疗有较重要的意义。     

Survivin在多发性骨髓瘤患者骨髓细胞中的表达及其临床意义

背景与目的:Survivin是凋亡抑制蛋白家族成员之一,在多种恶性肿瘤中高表达,但在正常成人分化组织无表达。其表达与多种肿瘤的预后及肿瘤对放、化疗的耐受有关。本研究旨在通过检测Survivin在正常人、初治及复发/难治多发性骨髓瘤(multiplemyeloma,MM)患者骨髓细胞及骨髓瘤细胞系KM3中的表达,以揭示Survivin在骨髓瘤中的表达与耐药及疗效的关系。方法:RT-PCR和Westernblot法检测29例MM患者(其中初治17例,复发/难治12例)、9例正常人骨髓标本及KM3细胞系中SurvivinmRNA和蛋白质水平表达。结果:骨髓瘤细胞系KM3高表达Survivin。SurvivinmRNA在正常人、初治和难治/复发MM患者骨髓细胞中阳性率分别为22.2%、70.5%和83.3%,表达水平(Survivin/β-actin比值)分别为0.06±0.04、0.31±0.14和0.69±0.24,骨髓瘤患者表达率和表达水平显著高于正常人(P<0.01),而难治/复发MM组表达水平高于初治组(P<0.05)。所有的正常人标本中均未检测到Survivin蛋白,在初治和难治/复发MM组Survivin蛋白阳性率分别为41.1%和58.3%(P>0.05),表达水平(Survivin/α-tubulin半定量比值)分别为0.11±0.07和0.21±0.12,难治/复发MM组明显为高(P<0.05)。17例初治患者中,7例Survivin阳性者4例部分缓解(partialresponse,PR),1例微小反应(minorresponse,MR),2例无变化(nochange,NC),有效率[完全缓解(completeresponse,CR) PR MR]71.4%;10例Survivin阴性者中2例CR,4例PR,2例MR,2例NC,有效率80%,略高于阳性组。但统计学分析未发现两组有效率存在显著性差异。结论:Survivin在骨髓瘤患者中的高表达可能是部分患者对化疗不敏感的原因之一,由于Survivin仅特异性表达于骨髓瘤细胞,Survivin可作为逆转骨髓瘤细胞耐药治疗中的潜在靶点。     

STI571对慢性髓细胞白血病树突状细胞发育的影响

目的研究STI571对慢性髓细胞白血病（CML）树突状细胞（DC）发育的影响。方法分离CML患者及正常人的骨髓单个核细胞,将实验分为CML实验组、CML对照组及正常对照组。CML对照组及正常对照组：在培养体系中加入重组人粒单核细胞集落刺激因子（rhGM—CSF）和重组人白细胞介素4（rhIL-4）培养;CML实验组：在对照组的基础上,再加入药物STI571培养。于实验第8天,各组加入重组人肿瘤坏死因子α（rhTNF-α）进一步刺激成熟。Wright染色观察细胞形态,流式细胞仪检测细胞表型,荧光原位杂交（FISH）进行细胞遗传学分析,混合淋巴细胞反应（MLR）检测抗原递呈功能;ELISA法检测培养上清中血管内皮生长因子（VEGF）的浓度。结果各组均呈现典型的树突状细胞形态;CML实验组CDS0、CD86、CD83和HLA—DR的表达均显著高于CML对照组（P〈0．05）,经FISH证实,CMLDC来源于白血病细胞;各组DC均具有刺激同种异体T淋巴细胞增殖的能力,CML实验组刺激淋巴细胞增殖的能力显著高于CML对照组（P〈0．05）,而与正常对照组相似（P〉0．05）;CML实验组VEGF的浓度较CML对照组显著降低（P〈0．05）。结论STI571可促进CML骨髓来源DC的活化,可能与其抑制了CML细胞VEGF的过度分泌,从而解除了VEGF对CMLDC的分化抑制有关。     

In vitro growth response to G-CSF and GM-CSF by bone marrow cells of patients with acute myeloid leukemia.

The in vitro growth response of bone marrow and blood cells to granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) was studied in 18 acute myeloid leukemia (AML) patients using semisolid and suspension cultures. In 80% of the cases growth of leukemic progenitor cells was stimulated by GM-CSF and/or G-CSF, as judged by colony or cluster formation. In acute promyelocytic leukemia [t(15;17)], G-CSF stimulated and maintained the leukemic progenitors only transiently but fully stimulated the residual normal granulocyte/macrophage colony-forming units (CFU-GM). In some cases of M2 and M4 leukemia, G-CSF enhanced markedly the production of mature but cytochemically abnormal neutrophils. In some cases of M1 leukemia, neither CSF stimulated leukemic progenitors but instead stimulated only residual normal granulopoiesis. Spontaneous colony formation was observed in 20% of cases and was correlated with high-grade leukemic growth in vivo and a poor response to chemotherapy. The differing effects of the CSFs upon leukemic cells and residual normal granulopoiesis may have some implications for the clinical use of GM-CSF and G-CSF to overcome infectious complications.     

Expression of nucleolar protein p120 predicts poor prognosis in patients with stage I lung adenocarcinoma

Background:P120 is a proliferation-associated nucleolar proteinfound in most human malignant tumors, but not in resting normal cells. In ourprevious studies, the expression of p120 was statistically correlated with theproliferation capacity in human lung cancer cells and could be a prognosticmarker for resected lung adenocarcinoma. Patients and methods:The expression levels of p120 in tumors wereassessed by immunohistochemistry in 59 patients with stage I lungadenocarcinoma who underwent radical resection. Using clinical follow-up data,the prognostic significance of p120 calculated by labeling indices wasevaluated using Coxs proportional hazard model. Results:A mean ± SD of the labeling index of p120 was35.3 ± 14.4%. No significant correlation was found betweenthe expression levels of p120 and clinicopathological factors. Using acutoff value of 35% in the labeling index of p120, patients with highexpression of p120 experienced early recurrence and shorter survival comparedwith those having low expression of p120 (P= 0.04). Multivariateanalysis revealed that p120 served as an independent and strongest prognosticfactor for resected lung adenocarcinoma (P= 0.033). Conclusion:This article provides the first evidence that theexpression levels of p120 in tumor tissues can be used as an independent andpowerful prognostic marker for resected stage I lung adenocarcinoma.     

Ph-Chromosome-positive chronic myeloid leukemia with associated bone marrow mastocytosis

The concurrent development of chronic myeloid (CML) or myelomonocytic (CMML) leukemia in patients with systemic mastocytosis (SM) is a well recognized phenomenon. Although the leukemia often resembles CML in morphological and in clinical terms, a Ph-Chromosome-positive variant has not been reported in SM so far. We here describe a 43-year-old female patient with typical Ph-Chromosome-positive CML in whom a co-existing bone marrow mastocytosis, a special subvariant of SM, was diagnosed. RT-PCR analysis revealed the typical p210 kDa form of BCR/ABL in leukemic cells. The diagnosis SM was based on the typical focal aggregates of spindle-shaped mast cells (MC) in the bone marrow, expression of CD25 in MC, and the c-kit mutation D816V, which was detectable in microdissected bone marrow MC, but not in microdissected leukemic cells, suggesting the presence of two different (sub)clones of neoplastic cells. Therapy with the BCR/ABL-targeting drug Imatinib (STI571) resulted in complete cytogenetic remission of CML. Together, our case provides further evidence for the biologic diversity of leukemias that may occur in patients with SM. The exact knowledge of the pathology and target-profile of the associated leukemias in SM have important therapeutic implications.