caspase-3、bcl-2蛋白在非霍奇金淋巴瘤组织的表达及其与细胞凋亡和增殖的关系

目的探讨caspase-3、bcl-2蛋白在非霍奇金淋巴瘤(NHL)发生、发展中的可能作用及相互关系.方法应用TdT介导的dUTP缺口末端标记(TUNEL)技术和免疫组织化学链霉素抗生物素-过氧化酶(SP)法,检测5例反应性增生淋巴组织和119例NHL组织中的细胞凋亡和增殖细胞核抗原(PCNA)、caspase-3、bcl-2蛋白的表达水平.结果 caspase-3和bcl-2在119例NHL中表达的阳性率分别为86.6%(103例)和53.8%(64例).二者在良、恶性淋巴组织中的表达方式相反在反应性增生淋巴滤泡中心caspase-3高表达而bcl-2阴性表达,滤泡套区caspase-3阴性表达而bcl-2高表达;在肿瘤性滤泡中心bcl-2常高表达而caspase-3常表达减弱或不表达;在NHL中二者的表达与肿瘤恶性度呈相反关系,高度恶性组中caspase-3的表达(44.4%)高于低度恶性组(23.7%,P<0.01),而B细胞性淋巴瘤中bcl-2的表达(42.6%)低于低度恶性组(75.5%,P<0.01);caspase-3的表达与凋亡指数呈正相关(r=0.512, P <0.01)),bcl-2的表达与凋亡指数呈负相关(r=-0.436, P<0.01).此外,NHL的凋亡指数与增殖指数呈显著正相关(r=0.710, P<0.01).结论 caspase-3可能参与了NHL的凋亡调节机制.caspase-3和bcl-2蛋白在良、恶性淋巴组织中常呈现相反的表达方式,提示二者在淋巴细胞增殖动力学调节中可能存在着密切联系.     

The pattern of Ki-67 and bcl-2 expression in lymphoid malignancies

The International Working Formulation divides non-Hodgkin's lymphoma (NHL) into three grades: low, intermediate and high. This grading system implies rate of tumour growth and hence prognosis. Ki-67 antigen is a proliferation-related nuclear antigen and bcl-2 oncogene product is known to inhibit apoptosis. This study aimed to determine the pattern of expression of Ki-67 antigen and bcl-2 oncoprotein in various grades of NHL. Paraffin-embedded tissues from 42 cases of NHL (7 low, 15 intermediate, 20 high grade) were retrieved from the files of the Department of Pathology, University of Malaya. Ki-67 antigen and bcl-2 oncoprotein were detected using immunohistochemistry. The percentage of positively stained neoplastic cells was determined by semi-quantitative estimation and given scores ranging from 0 to 6. Partition chi square test demonstrated the association of Ki-67 antigen expression and histological grade (p = 0.007). There was no significant difference in Ki-67 antigen expression between intermediate and high grade malignant lymphomas (p = 0.28), whereas significant difference was demonstrated between low and intermediate/high grade tumours (p = 0.003). Bcl-2 oncoprotein expression in the neoplastic cells varied widely within the three histological grades. Statistical analysis showed no association between the expression of bcl-2 oncoprotein and histological grade (p = 0.25). Ki-67 immunostaining is therefore a useful adjunct to histological grading of NHL.     

胶质瘤bcd—2基因表达水平与其细胞增殖和凋亡关系的研究

目的 探讨胶质瘤细胞ｂｃｌ－２基因表达水平与肿瘤恶性程度、细胞增殖活性及凋亡程度的关系。方法 以６９例不同级别的人胶质瘤组织为研究对象,用原位杂交及免疫组化染色ＡＢＣ法分别检测ｂｃｌ－２ｍＲＮＡ、ｂｃｌ－２蛋白和增殖细胞核抗原（细胞增殖活性标记物）的表达,并用３’末标记法做原位细胞凋亡检测。结果 ６４例（９２．８％）表达ｂｃｌ－２ｍＲＮＡ,６０例（８７．０％）表达ｂｃｌ－２蛋白,两者的表达水平呈正     

非霍奇金淋巴瘤中survivin、bcl-2、bax、p53表达及其与细胞增殖和凋亡的关系

目的研究非霍奇金淋巴瘤(non-Hndgkin’s lymphoma,NHL)中survivin、bcl-2、bax和p53指标表达的情况,比较它们在B-NHL和T-NHL中的差异,探讨它们在临床病理实践中的应用价值。方法应用免疫组化、原位凋亡检测及组织芯片技术对190例NHL进行检测。结果①survivin、bcl-2、bax、p53在75例B-NHL中表达的总阳性率分别为70·67%、57·33%、46·67%和36·00%,在115例T-NHL中表达的总阳性率分别为81·74%、44·35%、46·09%和40·00%,其中bcl-2表达在B-NHL和T-NHL之间差异有显著性(P=0·045);②随着B-NHL生物侵袭性的增强,survivin表达率和表达强度升高(P=0·001),bcl-2表达率和表达强度下降(P=0·018);③在低中度侵袭组中,随着NHL生物侵袭性的增强,突变型p53表达率升高;在高侵袭组中p53的表达率反而降低;④随着NHL生物侵袭性的增强,Ki-67表达升高(P=0·014和P=0·002)。结论①survivin是判断NHL恶性及侵袭性有意义的指标,联合检测survivin和bcl-2蛋白的表达情况对判断B-NHL的侵袭性可能有互补作用;②p53表达可用于评价低中度侵袭性NHL的生物学行为;③Ki-67蛋白表达强度是评估NHL肿瘤细胞增殖的良好指标,它对NHL侵袭性的分级具有较为重要的参考价值。     

Pleomorphic lobular carcinoma of the breast: its cell kinetics, expression of oncogenes and tumour suppressor genes compared with invasive ductal carcinomas and classical infiltrating lobular carcinomas

AIMS: The study addresses whether pleomorphic lobular breast carcinomas represent a distinct entity with respect to proliferation and apoptosis as well the expression of the p53, bcl-2 and Her2 protein. METHODS AND RESULTS: The study included 30 cases of pleomorphic lobular carcinoma (PLC; G2 n=15, G3 n=15). Poorly differentiated invasive ductal carcinomas (IDC; n=15) and well-differentiated infiltrating lobular carcinomas (ILC; n=15) were used as controls. Lymph node metastases were present equally in all groups. MIB-1 labelling was counted as: PLC (G2) 8.36%; PLC (G3) 11.3%; IDC 44.26%; ILC 2.19% (P=0.0001, P=0.004, P=0.001). Apoptotic index was: PLC (G2) 0.82%; PLC (G3) 1.2%; IDC 2.09%; ILC 0.6% (P=0.009, P=0.001). Over-expression of Her2 protein was detected in 53% of PLC (G3) tumours and was present only in scattered cases in the other groups. PLCs and ILCs were strongly positive for bcl-2 and for hormone receptors, while p53+ cells were rare. IDCs exhibited a heterogeneous staining pattern for bcl-2 and for hormone receptors, while p53+ cells occurred considerably more frequently. Stage could not be linked directly to proliferation or apoptosis. CONCLUSION: Our data suggest that more frequent over-expression of Her2 among PLCs (G3) as well as the generally low apoptosis can contribute to their aggressive behaviour.     

Erythropoietic activities in acute leukemia and in malignant lymphoma with or without bone marrow involvement

Erythropoietic activities and immature reticulocyte production were investigated in a total of 157 patients (81 men, 76 women, median age = 42 yr, range = 23 to 65 yr) with acute lymphoid leukemia (ALL, n = 31), acute myeloid leukemia (AML, n = 39), or non-Hodgkin's lymphoma (NHL, n = 87), based on assays of the hemogram, red cell indices, reticulocyte subpopulations, and intramedullary erythroid precursors. There were no significant differences in red blood cell (RBC) counts, blood hemoglobin levels, or erythroid precursors between ALL and AML patients. Reticulocytes in AML patients averaged 1.7 +/- 0.8%, which was higher than in patients with ALL (0.8 +/- 0.3%, p <0.01); the proportion of high-fluorescence reticulocytes (HFR) averaged 4-fold higher in AML versus ALL (p <0.01) and the reticulocyte maturity index (RMI) was higher in AML (20.8 +/- 8.3%) versus ALL (12.4 +/- 6.5%, p <0.01). The RMI was higher in NHL patients with bone marrow (BM) involvement (15.6 +/- 9.4%), compared to those without BM involvement (4.3 +/- 2.1%, p <0.01). The proportion of HFR averaged 11-fold higher in NHL with BM involvement versus NHL without BM involvement. In summary, erythropoietic activity is significantly more active in patients with AML compared to ALL and in patients with NHL with BM involvement, compared to NHL without BM involvement.     

bcl-2 expression by multicolor flow cytometric analysis assists in the diagnosis of follicular lymphoma in lymph node and bone marrow

The expression of bcl-2, CD10, and CD20 was examined by multicolor flow cytometry in 78 samples including lymph node or other tissue biopsy specimens containing follicular lymphoma (FL; n = 17), reactive hyperplasia (RH; n = 28), or other malignant lymphomas (n = 20), as well as bone marrow aspirates (n = 13). The presence of CD10+ cells with high bcl-2 expression predicted the presence of FL rather than RH with a positive predictive value of 100% and negative predictive value of 96%. CD10+ cells with high bcl-2 expression also were found in a subset of diffuse large B-cell lymphomas and were otherwise rare in other types of malignant lymphoma. In contrast with immunohistochemical studies, a reduced but apparently measurable level of bcl-2 was present in benign follicular center cells. Hematogones showed lower bcl-2 levels than did FL cells in the bone marrow, and neutrophils were bcl-2-. Measurement of bcl-2 expression levels by multiparameter flow cytometry offers a rapid, quantitative assessment that may assist in the diagnosis of FL in lymph nodes or bone marrow, even when other CD10+ cells or admixed normal B cells are present.     

The immunolocalization of bcl-2 at the maternal-fetal interface in healthy and failing pregnancies

Programmed cell death by apoptosis occurs in both fetal and maternal tissues during early pregnancy. To investigate a role for apoptosis at the maternal-fetal interface, we have immunolocalized the bcl-2 protein in formalin-fixed decidual and placental tissue collected from women undergoing surgical termination of pregnancy (n = 22), from women undergoing a sporadic miscarriage (n = 16) and from women with a history of recurrent pregnancy loss (more than three consecutive pregnancy losses; n = 22) undergoing a further miscarriage. In all three groups, bcl-2+ cells were found in aggregates and dispersed in the stroma, and immunoreactivity was observed in glandular epithelium. Double immunostaining revealed that a majority of stromal bcl-2+ cells were CD56+ large granular lymphocytes. A computerized image analysis revealed no significant differences in percentage area of bcl-2 or CD56+ immunostaining. Significantly more biopsies from the surgical termination group (4/10) had > 20% positive immunostaining for CD56 compared with 0% in the other two groups combined (0/20; P < 0.05). Bcl- 2 immunoreactivity was observed in the villi syncytiotrophoblast, and staining intensity was consistently greater in the surgical termination group. The possible roles of bcl-2 at the maternal-fetal interface are discussed.      

Plasma soluble interleukin-2 receptor (sIL-2R) levels in patients with acute leukemia

The plasma soluble interleukin-2 receptor (sIL-2R) level was higher in 137 patients with acute leukemia (1,489 +/- 1,798 U/ml, including 98 cases of acute myeloid leukemia (AML), 1,063 +/- 1,414 U/ml, and 39 cases of acute lymphoblastic leukemia (ALL), 2,561 +/- 2,194 U/ml), compared to 49 normal control subjects, 421 +/- 151 U/ml). The ALL patients showed elevated plasma sIL-2R levels more frequently than the AML patients (92.3% vs 44.9%). No patient with either hypoplastic AML or AML with multilineage dysplasia and only 1 of 13 patients with acute promyelocytic leukemia (APL) had an elevated plasma sIL-2R level. All the My+ ALL patients (15 cases) showed elevated plasma sIL-2R levels. Plasma sIL-2R levels were significantly lower after chemotherapy in the ALL patients, but were not significantly lower in the AML patients. IL-2R was expressed on the leukemic cells in 36 (53.7%) of 67 AML and in 9 (21.4%) of 42 ALL cases. None of the AML M3, M4, M5, M6, or M7 subgroups showed IL-2R expression. The My+ ALL patients (42.9%, 6/14) showed IL-2R expression more frequently than the other ALL subgroups (10.7%, 3/28) (p = 0.025). The plasma sIL-2R level was correlated with the proportion of leukemic cells expressing IL-2R in acute leukemia. However, there were many cases, particularly ALL cases, who had elevated plasma sIL-2R levels without IL-2R expression on their leukemic cells. These results suggest that the plasma sIL-2R level is a valuable marker for monitoring ALL after chemotherapy, particularly in My+ ALL cases, and that the T cell immune reaction to leukemia appears to be much higher in ALL patients than in AML patients.     

Characterization of immune complexes in acute lymphoblastic leukaemia.

Sera from 120 children and young adults with acute leukaemia (59), various other tumours (53) and histiocytosis X (eight) were studied for the presence and characteristics of circulating immune complexes (CIC). Serial and parallel testing was performed using: C1q binding (solid phase), Raji cell radioimmunoassay and anti-C3 (solid phase). CIC were detected in 36 of 56 (64%) patients with acute lymphoblastic leukaemia (ALL) and in 62% of other tumour subjects. In the ALL sera, the mean positive C1q binding was 5.4 s.d., Raji cell 4.2 s.d. and anti-C3 4.4 s.d. In 12 ALL sera CIC were characterized for molecular size by sucrose gradient centrifugation. Most samples showed high molecular weight (19S) complexes but intermediate (11-14S) and smaller (8-9S) complexes were also detected. There was no apparent relationship between the presence, amount or physical size of the detectable CICs and clinical course of the patients studied; 12 patients with ALL in long term remission showed presence of CIC at some time during their course. Immune complexes precipitated from leukaemic sera were also examined for the presence of common ALL antigen (cALL) and Ia(DR) antigens utilizing rabbit antisera and mouse monoclonal antibodies. Experiments with isolated immune complexes from ALL sera provided no positive evidence for the presence of cALL antigen or Ia antigen within immune complex materials from ALL patients.     

Developmental regulation of bcl-2 expression in the thymus.

An important factor in shaping the T-cell receptor (TcR) repertoire during thymocyte development is the susceptibility of double-positive (CD4+ CD8+) thymocytes to induction of apoptosis (negative selection) when the TcR is engaged by 'self'-antigens. Recent evidence has suggested that this susceptibility to apoptosis may be influenced by the expression of bcl-2, a proto-oncogene known to increase the resistance to apoptosis in various cell systems. Using a semi-quantitative polymerase chain reaction (PCR) technique in conjunction with staged embryonic material and purified thymocyte subpopulations we have investigated patterns of bcl-2 expression during normal T-cell development. Our results show that while bcl-2 alpha gene expression is readily detectable in immature CD3-CD4-CD8- thymocytes and in mature single-positive TcRhi cells, it is drastically reduced in TcR negative double-positive (CD3- CD4+ CD8+) cortical thymocytes of intermediate maturity. Careful mapping of bcl-2 alpha re-expression in relation to the onset of TcR expression within the population of embryonic thymocytes indicates that bcl-2 alpha is up-regulated as soon as TcR molecules are expressed on the surface of CD4+ CD8+ thymocytes. Therefore, thymocytes susceptible to apoptosis on TcR ligation express bcl-2 alpha mRNA suggesting that changing levels of bcl-2 expression are unlikely to be the only determinant regulating susceptibility to apoptosis in the thymus. The possible implications of these changes in bcl-2 expression regarding other facets of thymocyte development will be discussed.     

bcl-2 immunoreactivity, human papillomavirus DNA, and cervical intraepithelial neoplasia.

The aim of this study was to identify the role of bcl-2 protein expression in precancerous lesions of the cervix in patients from Johannesburg, South Africa and to correlate this expression with human papillomavirus (HPV) status. Archival cervical biopsy specimens (n = 107) of normal squamous epithelia (n = 18), pure HPV squamous epithelial lesions (n = 15), cervical intraepithelial neoplasia (CIN) I lesions (n = 17), CIN II lesions (n = 26), and CIN III lesions (n = 31) underwent bcl-2 immunohistochemical analysis with use of the streptavidin-biotin complex/horseradish peroxidase system and nonisotopic in situ hybridization for the detection of HPV DNA. Although 45 (61%) of the 74 CIN lesions demonstrated bcl-2 protein expression in the epithelia, most seemed to be in a patchy basal cell distribution, with a 1+ to 2+ intensity. Furthermore, comparison of bcl-2 immunoreaction between the low and high grades of the CIN lesions did not reveal significant differences. In addition, there was no apparent link between the presence of HPV DNA and bcl-2 expression in the CIN lesions. In contrast to previous studies that showed an increase in bcl-2 immunostaining intensity with increasing severity of CIN, only 4 (5.4%) of our 74 CIN specimens satisfied this pattern. Hence, we suggest that bcl-2 protein expression might not play a significant role in the majority of CIN lesions in this population group and that it might not correlate with HPV status.     

Immunohistochemical expression of p53, bcl-2, and p21waf1 in 48 Argentinean children with non-Hodgkin lymphoma.

BACKGROUND: Lymphoma accounts for 10% of all childhood cancers in developed countries. In Argentina, the incidence is 13.6%; of these, 53% are non-Hodgkin lymphomas (NHL) and 47% are Hodgkin lymphomas. Overexpression of p53, p21, and bcl-2 has been studied mainly in adults with NHL and correlated with clinical features and survival. The authors' aim was to analyze overexpression of these cellular oncogenes in children with NHL. METHODS: Formalin-fixed paraffin-embedded lymph node biopsies from 48 children with NHL were studied by immunohistochemistry with monoclonal antibodies for p53, bcl-2, and p21. RESULTS: Overexpression of p53 was detected in 81% of cases; bcl-2 positivity was 46% and p21 positivity was 42%. The p53/p21 expression patterns (types IV and V patterns), which correspond to the overexpression of nonfunctional p53, accounted for 46% of NHL. None of the studied oncogene patterns correlated with poor clinical patient outcome or histologic subtype. CONCLUSIONS: Of 81% of cases with p53 overexpression, 46% exhibited a nonfunctional p53 pattern, and this may contribute to dysregulation of proliferation, bcl-2 expression in up to 46% of cases may reflect a failure of bcl-2 downregulation in pre-B and immature B cells.     

Prenatal origin of TEL-AML1-positive acute lymphoblastic leukemia in children born in California

Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer. The peak incidence of ALL between ages 2 and 5 is accounted for by one subtype, referred to as common acute lymphoblastic leukemia (cALL). About 25% of cALL patients have the TEL-AML1 gene fusion derived from the t(12;21) chromosomal translocation. Recent evidence from retrospective analysis of neonatal blood spots (Guthrie cards) in Europe has demonstrated that this chromosome translocation may arise prenatally. The aim of our study was to determine whether TEL-AML1 fusions arise prenatally in a U.S. population of cALL patients. TEL-AML1-positive cALL cases (n = 14) were identified by fluorescence in situ hybridization, and the genomic breakpoints were identified by a streamlined long-distance PCR approach and sequenced. Clonotypic primers were designed for each patient breakpoint, and a nested PCR assay was used to determine the presence of the TEL-AML1 fusion sequence in neonatal Guthrie cards. Seven of 14 cases demonstrated clonotypic sequences on the archival Guthrie cards. The oldest patient that was positive was 6.7 years old at the time of diagnosis of leukemia. These results confirm previously published findings of a prenatal origin of TEL-AML1 in Europe by demonstrating its occurrence in a California-born population. Secondary changes were also similar to those described previously, with deletion of the second TEL allele being the most common. Other secondary changes included duplication of the fusion gene, trisomy 21, and monosomy X.     

Apoptosis and bcl-2 expression in normal human endometrium, endometriosis and adenomyosis

Apoptosis has been implicated in the pathogenesis of several diseases and is partly regulated by bcl-2, which blocks the apoptotic pathway and promotes cell survival. Apoptosis and bcl-2 expression were examined in paired eutopic and ectopic endometrium from women with endometriosis (n = 30 samples) or adenomyosis (n = 15 samples) and compared with control endometrium (n = 30 samples). Apoptotic cells were detected using the dUTP nick-end labelling (TUNEL) assay for DNA fragmentation; bcl-2 expression was demonstrated with a streptavidin- biotin peroxidase immunohistochemical technique. Apoptotic cells were rare in eutopic, ectopic and control endometrium; there were no significant differences between subject groups nor between eutopic and ectopic endometrium. Stromal bcl-2 expression increased in the late secretory phase in control and eutopic endometrium in endometriosis; double labelling studies revealed that most stromal bcl-2+ cells were leukocytes. Stromal bcl-2 expression in endometriotic foci was significantly increased compared with the paired eutopic endometrium, did not vary with menstrual cycle and included a significant population of non-leukocytic bcl-2+ stromal cells. In contrast, stromal bcl-2 expression in adenomyosis remained at low levels and did not show significant cyclical variation. Glandular epithelial bcl-2 expression also varied with menstrual cycle phase and peaked in the proliferative phase; in contrast, surface epithelial bcl-2 expression increased in the late secretory phase. Elevated stromal bcl-2 expression in ovarian endometriotic lesions could have implications for the growth and survival of ectopic endometrial tissue at these sites.      

Significance of microRNA-16 and bcl-2 expression in T lymphoblastic lymphoma/leukemia and its relation with prognosis北大核心CSCD

Objective: To study the expression of miR-16 and bcl-2 in T lymphoblastic lymphoma/leukemia (T-LBiyALL) and its relationship to prognosis. Methods: 70 cases of T-LBL/ALL with follow-up data were studied by using immunohistochemical EnVision method for CDla,CD3, cCD3, CD7, CD10, CD20, CD23, CD34, CD43, CD45RO, CD99, TDT, MPO, bcl-2 and Ki-67. The expression levels of miR-16 were examined by TaqMan real-time polymerase chain reaction (RT-PCR). Thirty cases of reactive lymph node were selected as control. Results: Among the 70 cases of T-LBL/ALL, the percentages of tumor cells expression of TDT, CD99, CD3, CD7.CD10, CD34, CDla,cCD3, bcl-2, CD45RO and CD43 were 94. 3% (66/70), 94. 3% (66/70), 68. 6% (48/70), 92. 9% (65/70), 32. 9% (23/70), 24. 3% (17/70), 40. 0% (28/70), 51. 4% (36/70), 34. 3 % (24/70), 37. 1 % (26/70), and 48. 6% (34/70). Separately, while tumor cells expression of MPO, CD20 and CD23 was all negative. A figure of Ki-67 expression > 80% was found in 24 cases and ≤ 80% in 46 cases. The expression of miR-16 was up-regulated in T-LBL/ALL, and it was 5. 07 times of the reactive lymph node(P = 0. 001). The high expression group of miR-16 was significantly correlated with longer over survival (P = 0. 041). The prognosis of negative bcl-2 group was better than bcl-2 positive one(P =0. 904). The relationship of miR-16 and bcl-2 was significant P =0. 042, x2=4. 147). Survival multivariate COX proportional hazard regression analysis revealed that the low expression of miR-16 might be a independent poor prognosis factor (P =0.049). Conclusions: While the high expression group of miR-16 has longer OS than that in low expression group. The prognosis of bcl-2 negative was better than bcl-2 positive. miR-16 may be a independent prognosis factor. The relationship of miR-16 and bcl-2 might suggested that gene regulation may be influenced by them.     

Receptors for the third component of complement: their association with maturation stage in non-Hodgkin lymphomas (NHL) and their possible implication with the development of follicular structures

In the majority of 188 non-Hodgkin lymphomas (NHL) investigated in this study, we found a simultaneous expression of the receptors for c3b and c3d (CR1 & CR2; cccorr = 0.69, P less than 0.0005). An analysis of the different histological entities of the Kiel classification revealed that this coexpression was most pronounced for germinal centre-derived (cccorr = 0.63, P = 0.0004) and immunocytic NHL (cccorr = 0.86, P = 0.0024), whereas in chronic lymphocytic leukaemias there tended to be a more heterogeneous pattern of complement receptor (CR) expression (cccorr = 0.29, P greater than 0.10). In contrast to these NHL of mid B cell stage, most of the NHL of early (i.e. acute lymphocytic leukaemia, lymphoblastic NHL) and late B cell stage (i.e. hairy cell leukaemia, immunoblastic NHL, multiple myeloma) did not express either of these receptors. CR positive NHL often showed a follicular arrangement of the neoplastic cells and had higher numbers of T helper/inducer (T4) lymphocytes (PCR1 less than 0.00005, PCR2 less than 0.05). Some cases of mid B cell NHL and all cases of hairy cell leukaemia reacted with antibodies against CR3 (i.e. the ic3b receptor).     

The immunological classification of leukaemia based on a rapid microcytotoxicity test.

We describe a rapid, accurate, and reproducible cytotoxic antibody test for the immunological classification of leukaemia. Well characterized heteroantisera and monoclonal antibodies were distributed in a microcytotoxicity tray referred to as a leukaemia screening tray (LST). Leukaemia cells were tested for the presence of Ia-like, ''blast'', thymocyte, common acute lymphoblastic leukaemia (cALL), and acute myeloblastic leukaemia (AML) antigens. Utilizing this technique, T ALL could be distinguished from non-T ALL, ALL and AML could be differentiated, the myeloid blast crisis of chronic myelogenous leukaemia (CML) could be distinguished from the lymphoid blast crisis, and the T lymphocyte and B lymphocyte lymphoid leukaemias were readily identified. It has been shown that subclassification of leukaemia according to surface markers, as described here, offers considerable improvement in the diagnosis and treatment of leukaemia over the classical morphological methodologies. Because the test can be completed in 2 hr and unlimited amounts of monoclonal antibodies are available, the LST or similar tests should become universally available in the future to supplement morphological data and replace other lengthy enzyme and rosetting tests.     

Assessment of proliferative activity in leukaemic bone marrow using the monoclonal antibody Ki-67.

AIMS--To investigate proliferative activity in leukaemic and lymphomatous bone marrow infiltrates and to assess the feasibility of transport of specimens among institutions. METHODS--Proliferative activity in bone marrow trephine cryosections from 99 patients with non-Hodgkin''s lymphoma (NHL), 23 patients with acute myeloid leukaemia (AML), 11 with acute lymphoblastic leukaemia (ALL), and two with acute undifferentiated leukaemia (AUL) was investigated. Infiltration was seen in 52 out of 99 cases of NHL on bone marrow cryosections. A score was devised to assess pathological infiltrates in bone marrow trephine cryosections using the monoclonal antibody Ki-67. This method of scoring gave a measure of non-erythroid proliferative activity. RESULTS--Mean Ki-67 positivity in bone marrow infiltrates in 31 low grade B cell lymphomas (Kiel classification) was 0.3% before and 4.7% after treatment, 16.4% in seven high grade B cell lymphomas, and 17.8% in 12 peripheral T cell lymphomas. In 48 cases of NHL, bone marrow cryosections had not been infiltrated, and in all but one case the percentage of Ki-67 positive cells in normal marrow was less than 3%; the remaining case showed coexistent myelodysplasia and 8% bone marrow Ki-67 positivity. In eight cases of common ALL at diagnosis, the mean Ki-67 positivity in marrow cryosections was 24.9%, significantly higher than the 2.4% Ki-67 positivity seen in AML (p < 0.05). One of the two cases of common ALL with less than 1% Ki-67 positivity was refractory to treatment. CONCLUSIONS--Proliferative activity of erythroid elements in the bone marrow varies greatly. Immunostaining of bone marrow cryosections using Ki-67 permits accurate assessment of non-erythroid proliferative activity in lymphomas and leukaemia. High grade B cell lymphomas and peripheral T cell lymphomas invading the marrow have very similar mean proliferative activities. Such levels of proliferation are of the same order as those seen in common ALL, but much higher than those seen in AML.