组织细胞性坏死性淋巴结炎病理组织学分析

目的探讨组织细胞性坏死性淋巴结炎(HNL)的临床病理特征。方法复习36例HNL淋巴结活检标本的HE切片,并用免疫组化SP法检测病灶内细胞的免疫表型。结果HNL组织学上副皮质区,尤其是滤泡间区有散在大小不等或融合病灶,由多种形态的组织细胞、转化的淋巴细胞和凋亡碎屑构成,缺乏中性粒细胞、浆细胞和/或嗜酸性粒细胞反应。部分淋巴细胞表达CD45RO,组织细胞表达CD68,残留的生发中心细胞表达CD20。结论HNL形态学上显示由多种组织细胞、转化T淋巴细胞及凋亡碎片组成的病灶,不见中性粒细胞浸润。应与恶性淋巴瘤、淋巴结的非肿瘤性病变鉴别。     

CD138/Syndecan-1在多发性骨髓瘤免疫表型中的意义

为了建立多发性骨髓瘤（MM）免疫分型的测定方法,分析各种抗原的表达特点,并分选高纯度原代骨髓瘤细胞,应用CD45/侧向角（SSC）设门的三色免疫荧光流式细胞术（FCM）测定18例MM患者、20例急性白血病（AL）患者和7例正常人骨髓免疫分型特点;应用抗CD138和免疫磁珠从MM患者的骨髓中分选骨髓瘤细胞.结果表明：骨髓瘤细胞CD45阴性或弱阳性,SSC介于有核红细胞和中性粒细胞之间,CD138和CD38均阳性,T、B细胞及髓系抗原多为阴性,CD56阳性率为83.3%,HLA-DR阳性率为44.4%.与MM患者相比,AL患者CD138均阴性,而CD38为100%阳性,CD56阳性率为25%.正常人CD138和CD56均为阴性.磁珠分选获得的原代骨髓瘤细胞纯度在73%-95%之间,平均为86%.结论：CD45/SSC设门的三色免疫荧光FCM是测定MM免疫分型的稳定可靠方法,CD138是骨髓瘤细胞表面较为特异的抗原标志,应用抗CD138和免疫磁珠可以获得高度富集的原代骨髓瘤细胞.     

多发性骨髓瘤流式细胞术免疫表型及微量残留病研究

目的 探讨MM细胞免疫表型特征及MRD检测的临床意义.方法 应用多参数流式细胞术检测16名健康对照者和172例MM患者的免疫表型.32例MM患者缓解后作MRD分析,同时进行跟踪随访,分析MRD阳性和阴性对MM患者的复发率和无病生存时间是否存在影响.结果 流式细胞术检测结果显示,16名健康对照者正常浆细胞CD38、CD 138、CD19、CD45均阳性,172例MM患者骨髓瘤细胞主要免疫表型为CD+38 172例(100.0%)、CD+138172例(100.0%)、CD-19 167例(97.1%)、CD+56 152例(88.4%)、CD-45 166例(96.5%),骨髓瘤细胞具有显著的特征性表型,即CD+38、CD+138、CD+56、CD-19、CD-45.32例MM患者缓解后进行MRD检测,其中14例MRD阴性,18例MRD阳性.随访2～16个月后,MRD阴性组4例复发(28.6%),MRD阳性组13例复发(72.2%),两组复发率比较,差异有统计学意义(χ2=6.03,P＜0.05).MRD阴性组累积无病生存时间中位数16.23(10.37～21.62)个月,明显长于MRD阳性组的10.07(3.79～16.20)个月,两组比较差异有统计学意义(χ2=7.53,P＜0.05).结论 MM患者骨髓瘤细胞主要免疫表型特征是CD+38、CD+138、CD1-19、CD-45、CD+56.多发性骨髓瘤MRD检测可以作为临床预后的参考指标.

Abstract：

Objective To investigate the characteristics of immunophenotyping and clinical significance of MRD analysis in MM patients. Methods Multi-parameter flow cytometry was applied to analyze the immunophenotyping of malignant plasma cells from 172 MM patients, and normal plasma cells from 16 healthy individuals. MRD was analyzed in 32 MM patients with remission. Meanwhile, the effects of MRD status on the disease relapse and patient disease free survival ( DFS ) time was evaluated by following up patients. Results The immunophenotyping of normal plasma cells were CD38, CD138, CD19 and CD45 positive, while the predominant phenotype of MM cells were CD+38( 100.0% ), CD+138( 100.0% ), CD-19 ( 167/172, 97. 1% ), CD+56( 152/172, 88.4% ) and CD-45( 166/172, 96. 5% ). The characteristic markers for MM cells were CD+38, CD-138, CD-19, CD-45 and CD+56. MRD analysis revealed that, among 32 MM patients with remission, 14 patients were MRD negative and 18 patients were MRD positive. During follow-up of 2 to 16 months, the relapse rate in MRD negative patients was significantly lower ( 4/14, 28.6% ) than that of MRD positive patients ( 13/18, 72. 2% ;χ2 =6. 03, P ＜0. 05 ). Furthermore, the DFS time was significantly longer in MRD negative patients[ 16. 23( 10. 37-21.62 )months ] than that of the MRD positive patients [ 10. 07( 3. 79-16. 20 )months,χ2 =7. 53,P ＜0. 05 ]. Conclusions CD+38, CD+138, CD-19, CD-45 and CD+56 are the characteristic markers of MM cells compared to those of the normal plasma cells. MRD analysis is a valuable prognostic factor for MM patients.     

Identification of malignant plasma cell precursors in the bone marrow of multiple myeloma.

Precursors of plasma cells were studied in the bone marrow of 28 patients with multiple myeloma, plasma cell leukemia, and benign monoclonal gammopathy. Pre-B and B cell populations were analyzed with anti-B monoclonal antibodies corresponding to the clusters standardized at the Leucocyte Typing Workshops in Paris and Boston (CD9, CD10, CD19-22, CD24). In advanced forms of plasma cell malignancies, such as cases of multiple myeloma in stages II and III and of plasma cell leukemia, some cells of lymphoid morphology expressed common acute lymphoblastic leukemia antigen (CALLA, CD10) and HLA-DR, but contained no detectable terminal deoxynucleotidyl transferase enzyme. These CALLA+ cells were absent in benign monoclonal gammopathies. In multiple myeloma, the CALLA+ cells were negative for surface and cytoplasmic immunoglobulins (Ig), and, unlike CALLA+, terminal deoxynucleotidyl transferase (TdT+) pre-B cells in the normal bone marrow also failed to react with antibodies to B cell-associated antigens such as CD9, CD19, CD22, and CD24. The CALLA+, Ig- cells could be regarded as preplasmacytic since, after having been separated and stimulated with the phorbol ester 12-0-tetradecanoyl-phorbol-13 acetate in vitro, they transformed into plasma cells and synthesized the same heavy and light chains as myeloma cells.     

应用流式细胞术评估多发性骨髓瘤预后的研究进展

流式细胞术在多发性骨髓瘤方面的临床应用很为广泛,例如可以应用于与反应性浆细胞增多的区别诊断、疾病进展的监测、疾病预后的评估、微小残留病的监测等等.以流式细胞术为基础的免疫表型克隆分析,促进了更严格的诊断和随访技术的发展.本综述主要是介绍最新的骨髓瘤的恶性浆细胞的免疫表型、最广泛的抗原（如CD19,CD33,CD28,CD45,CD56及CD117）在正常的和恶性的浆细胞的表达的差别.本综述还着重介绍了恶性浆细胞表型和染色体异常之间的相关性,明确多发性骨髓瘤的预后因素.     

组织细胞性坏死性淋巴结炎的临床病理分析

目的 探讨组织细胞性坏死性淋巴结炎(HNL)的临床病理特征.方法 复习10例HNL淋巴结活检标本的HE切片,并用免疫组化SP法检测病灶内细胞的免疫表型.结果 HNL组织学上副皮质区,尤其是滤泡间区有散在大小不等或融合病灶,由多种形态的组织细胞、转化的淋巴细胞和凋亡碎屑构成,缺乏中性粒细胞、浆细胞和(或)嗜酸性粒细胞反应.部分淋巴细胞表达CD45RO,组织细胞表达CD68,残留的生发中心细胞表达CD20.结论 淋巴结活检显示有多种组织细胞、转化T细胞以及凋亡碎片组成的病灶,不合并有粒细胞浸润,有利于HNL的诊断.

Abstract：

Objective To describe the clinicopathological features of histiocytic necrotizing lymphadenitis ( HNL). Methods Routine lymph node biopsies of 10 HNL cases were reviewed and their immunophenotyping were performed using S-P immunohistochemical staining. Results Histologically, HNL had discrete or integrated nodules variable in size in the paracortex,expecially in the interfollicular area,which were full of proliferating pleomorphic histocytes,transformed lymphocytes,and karyorrhectic debris without infiltration of the neutrophils, plasma cells and/or eosinophils. Immunohistochemistry revealed CD45RO + for transformed lymphocytes,CD68 +for histiocytes.and CD20 +for lymphocytes in the residual germinal centers. Conclusion The presence of pleomorphic histiocytes, transformed T-cells, and karyor-rhectic debris in the biopsy of lymph nodes, together with the absence of neutrophils support the diagnosis of HNL.     

The Normal Counterpart of IgD Myeloma Cells in Germinal Center Displays Extensively Mutated IgVH Gene, Cμ–Cδ Switch, and λ Light Chain Expression

Human myeloma are incurable hematologic cancers of immunoglobulin-secreting plasma cells in bone marrow. Although malignant plasma cells can be almost eradicated from the patient's bone marrow by chemotherapy, drug-resistant myeloma precursor cells persist in an apparently cryptic compartment. Controversy exists as to whether myeloma precursor cells are hematopoietic stem cells, pre–B cells, germinal center (GC) B cells, circulating memory cells, or plasma blasts. This situation reflects what has been a general problem in cancer research for years: how to compare a tumor with its normal counterpart. Although several studies have demonstrated somatically mutated immunoglobulin variable region genes in multiple myeloma, it is unclear if myeloma cells are derived from GCs or post-GC memory B cells. Immunoglobulin (Ig)D-secreting myeloma have two unique immunoglobulin features, including a biased λ light chain expression and a Cμ–Cδ isotype switch. Using surface markers, we have previously isolated a population of surface IgM⁻IgD⁺CD38⁺ GC B cells that carry the most impressive somatic mutation in their IgV genes. Here we show that this population of GC B cells displays the two molecular features of IgD-secreting myeloma cells: a biased λ light chain expression and a Cμ–Cδ isotype switch. The demonstration of these peculiar GC B cells to differentiate into IgD-secreting plasma cells but not memory B cells both in vivo and in vitro suggests that IgD-secreting plasma and myeloma cells are derived from GCs.     

多发性骨髓瘤免疫表型流式细胞术研究新进展

多发性骨髓瘤(multiple myeloma,MM)是一种常见的血液系统恶性肿瘤,以浆细胞恶性增殖、溶骨破坏为特征,表现为M蛋白、骨骼破坏、贫血、肾功能受损和免疫功能异常.通过流式细胞术(flow cytometry,FCM)检测其免疫表型特点,已经作为血液系统疾病的常用检测手段,在辅助诊断、评估预后以及监测微小残留病灶方面的价值,已经被越来越多的学者认同.本文就将免疫表型分析在MM中的应用进展方面作一个综述,包括流式细胞术在MM中的应用和MM中流式免疫表型的特点.     

Vigorous premalignancy-specific effector T cell response in the bone marrow of patients with monoclonal gammopathy

Most approaches targeting the immune system against tumors have focused on patients with established tumors. However, whether the immune system can recognize preneoplastic stages of human cancer is not known. Here we show that patients with preneoplastic gammopathy mount a vigorous T cell response to autologous premalignant cells. This preneoplasia-specific CD4+ and CD8+ T cell response is detected in freshly isolated T cells from the BM. T cells from myeloma marrow lack this tumor-specific rapid effector function. These data provide direct evidence for tumor specific immune recognition in human preneoplasia and suggest a possible role for the immune system in influencing the early growth of transformed cells, long before the development of clinical cancer.     

2-甲氧基雌二醇对原代骨髓瘤细胞分化的作用

本研究观察2-甲氧基雌二醇对多发性骨髓瘤患者原代细胞诱导的分化作用。采用CD138+磁珠分离纯化7例骨髓瘤患者原代骨髓瘤细胞,通过形态观察、细胞表面标志分析和细胞分泌轻链蛋白的情况,观察2-甲氧基雌二醇对多发性骨髓瘤原代骨髓瘤细胞分化的影响。结果表明:患者原代骨髓瘤细胞经0.5μmol/L2-甲氧基雌二醇分别作用36及72小时后,细胞形态向成熟阶段发展,表现为细胞胞核缩小,胞浆丰富,核浆比例下降,核仁减少或消失,核染色质变粗、变密。CD49e阳性细胞率由(11.02±1.75)%(DMSO对照组)增加到(23.80±1.62)%(36小时组)及(35.68±1.85)%(72小时组),差异具有统计学意义(P<0.05);细胞分泌轻链蛋白由(225.7±30.4)ng/ml(DMSO对照组)升高到(296.7±18.8)ng/ml(36小时组)及(378.9±15.8)ng/ml(72小时组),差异具有统计学意义(P<0.05)。结论:较低浓度2-甲氧基雌二醇可诱导骨髓瘤患者原代骨髓瘤细胞向成熟阶段分化。     

Diagnosis of plasma cell myeloma.

Diagnosis of myeloma depends on identification of malignant plasma cells and the product of these cells, a monoclonal immunoprotein. Of the clinical manifestations of plasma cell myeloma, skeletal pain and anemia are two of the more common. Unexplained anemia and osteoporosis noted in the elderly should suggest the possibility of myeloma; this combination of symptoms certainly warrants obtaining a protein electrophoresis. Hypercalcemia and renal insufficiency are frequent sequelae of myeloma.     

A tumor-associated antigen specific for human kappa myeloma cells

A monoclonal antibody (K-1-21) raised against a kappa Bence Jones protein exhibits unique binding properties to malignant plasma cells. K-1-21 is an IgG1 kappa antibody that reacts with human kappa light chains in free form, but shows no reactivity with heavy chain-associated kappa light chains. By immunofluorescence, K-1-21 binds to the surface of LICR LON/HMy2 (HMy2) kappa myeloma cells and to plasma cells from a majority (8/11) of patients with various types of kappa myeloma; it did not bind to the surface of normal cells, nor to malignant cells of non-kappa myeloma origin. Flow cytometry analysis of K-1-21 binding to HMy2 cells indicated that the surface reactivity of K-1-21 could be completely inhibited by preincubation of the antibody with purified kappa light chains, whereas no inhibition occurred after preincubation with lambda chains or intact human IgG. Thus, the epitope recognized by K-1-21 on the cell surface may be similar, if not identical, to the determinant recognized on soluble free kappa light chains, and constitutes a tumor-associated antigen with selectivity for kappa myeloma cells. K-1-21 may therefore have clinical potential in patients with kappa myeloma.     

Phagocytic myeloma cells in asymptomatic multiple myeloma

A newly observed case of asymptomatic multiple myeloma in which phagocytic myeloma cells were observed is described. Bone marrow aspirates contained 16% myeloma cells, 2% of which engulfed red blood cells, lymphocytes, and platelets. The possibility is discussed that phagocytizing ability may be one of the markers for malignant plasma cells. Nothing is so far known of the phagocytosis by plasma cells in benign monoclonal gammopathy which is strictly defined.     

多发性骨髓瘤细胞系CZ-1的建立及其生物学特性

目的 建立多发性骨髓瘤（MM）细胞系并鉴定其生物学特性。方法 分离1例晚期λ轻链型MM患者外周血和骨髓的单个核细胞，用液体培养法进行培养，用瑞特-姬姆萨染色和细胞化学染色确定细胞形态；用免疫固定电泳技术检测细胞是否分泌单克隆λ轻链，用荧光定量PCR法检测细胞是否被EB病毒感染；用流式细胞仪分析细胞的免疫表型；用染色体G分带法研究细胞的遗传学特征。结果 所得细胞系在饲养细胞或条件培养液存在时，可以持久增殖，该细胞分泌λ轻链，EB病毒阴性；细胞在瑞特-姬姆萨染色下呈典型的原，幼浆细胞形态，细胞化学染色，酸性磷酸酶染色（ ），髓过氧化物酶（-）；细胞免疫表型为CD10^ ,CD28^ ,CD38^ ,CD138^ ,CD56^ ,CD49d^ ,CD54^ ,CD44^ ,CD58^ ，而不表达CD19，CD40，CD95，CD95L，CD34，CD2，CD5，骨髓来源和外周血来源的细胞系免疫表型无明显不同，核型分析结果均为i(lq ),8q 13q ,i(17q),i(18q), M。结论 用1例MM患者的外周血和骨髓细胞同时建立了一个生物学特性相同的分泌λ轻链的MM细胞系CZ-1。     

Notch信号通路和多发性骨髓瘤

Notch信号通路是介导细胞和细胞之间直接接触的主要信号通路之一,调控了多细胞机体的细胞凋亡、增殖和分化,是造血微环境调节造血细胞增殖与分化的重要信号通路,与多种血液系统恶性肿瘤的发病有关。多发性骨髓瘤（multiple myeloma,MM）是以浆细胞克隆性增殖为特征的B系肿瘤。由于骨髓瘤细胞的增殖比例低及多药耐药的形成,因此其对常规剂量的化疗药耐药,使得MM的临床治疗仍十分困难。近年来的研究发现,多发性骨髓瘤病人肿瘤细胞和骨髓瘤细胞株都大量表达Notch的配体Jagged-2,而正常的浆细胞或是其它恶性疾病来源的病人肿瘤细胞低量表达Jagged-2。Jagged-2可诱导基质细胞分泌白介素6（IL-6）、血管内皮生长因子（vascular endothelial growth factor,VEGF）、胰岛素样生长因子－1（Insulin—like growth factor 1,IGF－1）。Notch信号通路激活后可通过与NF－κB,C—myc的相互作用,调节细胞的增殖,促进骨髓瘤细胞的生长,从而抑制骨髓瘤细胞的凋亡,它与骨髓瘤的发生和耐药有关。抑制Notch信号通路能诱导骨髓瘤细胞的凋亡,增强化疗药的细胞毒作用。研究表明,单独使用Notch信号通路的特异性化学抑制剂γ－分泌酶抑制剂（γ－secrctase inhibitors,GSI）可通过特异性阻滞Notch信号通路从而诱导骨髓瘤细胞的凋亡,并且与传统化疗药物联合应用可以增强骨髓瘤细胞对化疗药的敏感性。本文就Notch信号通路的组成、Notch信号通路的作用机制及Notch信号通路与多发性骨髓瘤的关系进行综述．     

IMMUNOCHEMICAL STUDIES OF TWENTY MOUSE MYELOMA PROTEINS: EVIDENCE FOR TWO GROUPS OF PROTEINS SIMILAR TO GAMMA AND BETA-2A GLOBULINS IN MAN

The serum myeloma proteins associated with 20 mouse plasma cell tumors in C₃H or BALB/c mice that had proved transplantable were characterized by electrophoretic and immunochemical techniques. Although the myeloma proteins ranged in electrophoretic mobility from gamma to alpha globulins, they could be divided into two groups, the gamma type and the beta type myeloma globulins, on the basis of characteristic immunochemical properties. Gamma type myeloma proteins (5563, MPC-11) showed a close immunochemical relationship to normal mouse gamma globulins. Eighteen beta type mouse myeloma proteins migrated as beta or alpha globulins on zone electrophoresis. These proteins shared common antigenic features which permitted their recognition, separate from gamma myeloma proteins. The beta type myeloma proteins were shown to be related to a beta globulin component present in normal serum. Strain differences were observed for the normal beta globulin component believed to be formed in plasma cells. The proteins formed in mouse plasma cells were found to be antigenically complex. Shared antigenic determinants as well as distinctive antigenic determinants were detected when representative myeloma proteins were purified and compared by the Ouchterlony double diffusion technique. The myeloma proteins associated with each of the transplantable plasma cell tumors in mice are regarded as distinctive and characteristic products of plasma cell metabolism. The variety of myeloma globulins was similar for plasma cell tumors arising in C₃H as well as in BALB/c mice, indicating that differences in mouse strains would not account for the differences among the myeloma globulins. These differences, however, may be due to differences among the normal plasma cells from which the malignant cells are derived. If this is so, the variety of myeloma globulins reflect the variety of plasma cells present normally.     

应用骨髓细胞免疫表型分析诊断和监测多发性骨髓瘤

用流式细胞仪分析浆细胞轻链限制性和细胞表面的一些免疫标记(如CD138、CD38、CD56、CD117)能够清楚地识别多发性骨髓瘤,监测治疗后的微小残留病.这些方法很可能成为多发性骨髓瘤重要的临床诊疗标志.

Abstract：

Immunophenotyping with flow cytometry can clearly identify multiple myeloma cells and monitor minimal residual disease by using the characteristic of light chain restriction in plasma cells and some cell surface markers( eg CD138, CD38, CD56, CD117 ). This technology may provide important evidence in the diagnosis of multiple myeloma.     

Monoclonal gammopathies--from MGUS to myeloma

Monoclonal gammopathies are characterized by the overproduction of a monoclonal immunoglobulin (M-Protein), which may be detected in serum or urine by protein electrophoresis and immunofixation. The presence of an M-Protein results from the proliferation of a single abnormal clone of differentiated B lymphocytes or plasma cells, and is associated with a variety of clinical conditions, ranging from asymptomatic to malignant disease. Recent years have witnessed considerable advances in the treatment of plasma cell myeloma, the most common malignant disorder of the monoclonal gammopathies. As compared with conventional-dose treatments, high-dose chemotherapy with autologous stem-cell transplantation increases response rates and overall survival of patients with myeloma who are younger than 65 years of age. Progress in supportive therapies and the development of promising new drugs such as proteasome inhibitors and thalidomide analogues may provide further benefits for myeloma patients in the future.     

Marginal zone B cell lymphomas with extensive plasmacytic differentiation are neoplasms of precursor plasma cells

Occasional marginal zone lymphomas have extensive plasmacytic differentiation (P-MZL). We present an argument based on our data and previous published observations that P-MZL represent neoplasms of precursor plasma cells. Five P-MZL were analyzed by flow cytometry and the phenotype was compared with conventional MZL, plasma cell myeloma (PCM), reactive plasmacytoses (RP), and normal marrow plasma cells. The clonal cells in four of five P-MZL were CD19+, CD20(dim/?), CD38(bright), CD45++, CD56?, CD138?, and surface light chain(dim/?). One case was CD56+, CD138+, and CD19?. A separate clonal mature B cell population [CD20+, CD79b+] was not detected in any of these tumors. Hierarchical clustering demonstrated similarity between the neoplastic cells in four of the five P-MZL with plasma cells in RP comprised of precursor plasma cells. Analysis of normal bone marrow plasma cells revealed a CD45++CD38(bright)CD19+CD138?/+ precursor plasma cell population similar to the plasma cells in RP and P-MZL and a very small population of mature CD45+/?CD38(bright)CD138+CD19? plasma cellsresembling the neoplastic cells in PCM. The findings suggest that P-MZL represents a malignancy of a plasma cell stage of differentiation distinct from the plasma cell stage corresponding to PCM. We propose the term precursor plasma cell neoplasm for these tumors.