石蜡包埋组织DNA含量的流式细胞分析对恶性淋巴瘤的诊断价值

 目的 探讨流式细胞术（FCM）对恶性淋巴瘤的DNA含量分析在肿瘤诊断、临床分期、分类中的意义。方法 采用流式细胞仪检测48例恶性淋巴瘤（ML）和30例淋巴结反应性增生（RLN）,石蜡包埋组织,并分析它们的DNA倍体及细胞周期变化。结果 48例恶性淋巴瘤石蜡包埋组织样本中17例为异二倍体,31例为二倍体,30例RLN为二倍体。恶性淋巴瘤的S期细胞比率（SPF）、增生指数（PI）均高于淋巴结反应性增生（P＜0.05）。结论 异倍体的出现对恶性淋巴瘤的诊断具有较高特异性。SPF,PI对恶性淋巴瘤和淋巴结反应性增生的鉴别具有重要意义。     

Cytogenetic abnormalities in reactive lymphoid hyperplasia: byproducts of the germinal centre reaction or indicators of lymphoma?

Non‐random karyotypic abnormalities associated with non‐Hodgkin lymphomas (NHLs) have been described in cases of reactive lymphoid hyperplasia (RLH). However, the frequency and types of cytogenetic aberrations detected and their clinical relevance are unknown. To address these questions, we undertook a retrospective analysis of a large series of RLH diagnosed at our institute over 8 years. Cytogenetic abnormalities were identified in 20 of 116 (17%) cases with informative karyotypes, comprising 14 (70%) structural and 11 (55%) numerical changes. Clonal (n = 14, 70%) and non‐clonal (n = 6, 30%) abnormalities were observed. Aberrations of chromosome 14 were the most frequent (n = 8, 42%, 7 represented IgH translocations), followed by chromosome 3 (n = 4, 3 represented BCL6 translocations), and chromosome 12 (n = 4). Abnormal karyotypes were most often associated with florid follicular hyperplasia. Isolated lymphoid organ (lymph node, tonsil or spleen) enlargement (12/20, 60%) was more common, no specific etiology was identified in 10/20 (50%) cases and only 1 of 18 patients with clinical follow‐up (range 2–107 months, median 60 months) developed lymphoma. In our experience, cytogenetic abnormalities involving loci associated with B‐cell NHL are not infrequently detected in RLH. Their occurrence portends low risk for lymphomagenesis, however longer follow‐up is prudent to further evaluate the natural history of such cases. Copyright © 2010 John Wiley & Sons, Ltd.     

1号染色体克隆性异常在恶性血液病中的发生及其临床意义

 【摘要】　目的　分析1号染色体克隆性异常在恶性血液病中的发生频率、类型,并了解其临床和生物学意义。方法　对256例恶性血液病患者细胞遗传学R显带核型分析结果进行回顾性分析,将累及1号染色体的异常核型及临床资料进行总结,并结合文献复习。结果　256例中涉及1号染色体异常的恶性血液病25例（9.8 %）,分别见于急性淋巴细胞白血病（ALL）-L2、急性非淋巴细胞白血病（ANLL）-M2、骨髓增生异常综全征（MDS）、多发性骨髓瘤（MM）、淋巴瘤骨髓浸润、慢性粒细胞加速急变期、浆细胞白血病、慢性粒单核细胞白血病。1号染色体与其他染色体之间易位11例,其中t（1;19）3例,t（1;14） 2例,1号染色体部分增加或缺失7例,增加1条完整或部分缺失的1号染色体7例,1q等臂染色体3例。t（1;19）见于B淋巴细胞增殖性疾病,t（1;14）见于急性T淋巴细胞白血病。随访涉及1号染色体异常的20例患者,临床疗效差,生存期短。结论　发生1号染色体克隆性异常的恶性血液病主要见于急性白血病、MDS、MM,非特异性的异常多见,特异性的异常主要是与其他染色体之间的易位和1q三体。具有重现性的异常有t（1;19）、t（1;14）,均与白血病免疫表型相关;而1q三体和1q21扩增分别对MDS和MM的治疗、预后有指导意义。     

Cell surface markers on lymphoid cells from Warthin's tumors.

T- and B-cell markers were studied on suspensions of lymphoid cells obtained from two Warthin's tumors. Each tumor was composed predominantly of T- lymphocytes and a smaller percentage of B-lymphocytes with a polyclonal distribution of their surface immunoglobulin. These results do not differ from those obtained in normal or reactive lymph nodes. Together with the finding of normal lymph node structures in these tumors, our findings support the concept that the lymphoid component of Warthin's tumor represents pre-existing lymph node tissue.     

细针穿刺细胞学联合流式细胞术在B细胞非霍奇金淋巴瘤诊断及分类中的应用

目的 探讨细针穿刺（FNA）细胞学（FNAC）联合流式细胞术（FCM）在B细胞非霍奇金淋巴瘤（B-NHL）诊断及分类中的价值.方法 对17例经组织病理诊断为B-NHL的患者行FNA,获取穿刺细胞后迅速涂片,经瑞特-吉姆萨染色及显微镜观察细胞形态;同时,制备细胞悬液,标记相应抗体后在流式细胞仪上检测,结果 与组织病理诊断比较.结果 17例B-NHL患者中,FNAC联合FCM可诊断15例,l例因取材不佳无法诊断,另1例考虑为反应性淋巴组织增生,与病理诊断符合率为93.8％;进一步分类诊断符合率为93.3％.结论 在临床高度怀疑为淋巴瘤但无法行病理活组织检查的特殊情况下,FNAC联合FCM可为B-NHL的诊断及分类提供重要的信息.     

Tissue-specific markers in flow cytometry of urological cancers. III. Comparing chromosomal and flow cytometric DNA analysis of bladder tumors

Thirty-seven transitional-cell carcinomas (TCC) of the urinary bladder were analyzed by DNA flow cytometry (FCM). After labelling of the cell suspensions with antibodies to cytokeratin, the cytokeratin-positive cells and the non-epithelial cytokeratin-negative cells could be analyzed separately. After estimation of S- and G2M phase, 3/17 cases (18%) with a normal DNA index showed elevated proliferative levels, among cytokeratin-labelled suspensions only. Of these 17 cases, 14 showed chromosomal abnormalities. The remaining 20 cases were abnormal, irrespective of the technique used. Although immuno-labeling of tumor cells for cytokeratin in FCM increases the sensitivity of this method in detecting aneuploid tumors or tumors with high proliferation fractions, the discriminating power of chromosomal analysis of TCC is greater than FCM.     

DNA diagnosis of human cancers: lymphoid malignancies and leukemia

Usefulness of DNA analysis in diagnosis of hematopoietic malignancy was discussed. Examination on the presence of rearrangement in immunoglobulin (Ig) and T cell receptor (TCR) was the first DNA analysis used for clinical diagnosis of lymphoid malignancy to determine the cell-lineage and clonality of proliferating lymphoid cells. One point mutation in ras oncogene has also been used to detect residual leukemic cells as well as diagnosis of the early relapse of leukemia, although not all leukemic cells have this mutation. Presence of BCR-abl fused gene is a genetic marker for Ph1 chromosome. Analysis of BCR-abl gene has made it possible to diagnose the Ph1 ALL and masked Ph1 CML. Development of PCR technique markedly increased the possibility for the use of DNA analysis in clinical medicine. In addition to Ph1 chromosome, various chromosomal abnormalities resulted in a reciprocal translocation between Ig or TCR gene and other genes in various lymphoid malignancies, such as Burkitt lymphoma and follicular lymphoma. These translocations can be analyzed by Southern hybridization and used for clinical diagnosis.     

Flow cytometric analysis of DNA in bone and soft-tissue tumors using nuclear suspensions

Ninety-four bone and soft tissue tumors were analyzed for their DNA content using flow cytometry (FCM). A simple, rapid method for preparing isolated nuclear suspensions was used. Tissues, minced in a hypotonic solution containing detergent and propidium iodide as a fluorescent probe for DNA, provided in most instances high nuclear yields from only 0.02 to 0.03 g of solid tumor. Whereas all nonneoplastic samples had a diploid DNA content, various degrees of abnormal DNA distributions were detected in 90% of the neoplastic samples and were present in benign as well as malignant tumors. Our findings demonstrate that FCM DNA analysis is practical in most musculoskeletal tumors and support the observations of others that abnormal DNA content may serve as a general neoplastic marker in these tumors.     

Decoupling of normal CD40/interleukin-4 immunoglobulin heavy chain switch signal leads to genomic instability in SGH-MM5 and RPMI 8226 multiple myeloma cell lines.

The processes mediating genomic instability and clonal evolution are obscure in multiple myeloma (MM). Acquisition of new chromosomal translocations into the switch region of the immunoglobulin heavy chain (IgH) gene (chromosome 14q32) in MM, often heralds transformation to more aggressive disease. Since the combined effects of CD40 plus interleukin-4 (IL-4) mediate IgH isotype class switch recombination (CSR), and this process involves DNA double strand break repair (DSBR), we hypothesized that CD40 and/or IL-4 activation of MM cells could induce abnormal DNA DSBR and lead to genomic instability and clonal evolution. In this study, we show that MM cell lines that are optimally triggered via CD40 and/or IL-4 demonstrate abnormal decoupling of IL-4 signal transduction from CD40. Specifically, CD40 alone was sufficient to trigger maximal growth of tumor cells. We further demonstrate that CD40 triggering induced both DNA DSBs as well as newly acquired karyotypic abnormalities in MM cell lines. Importantly, these observations were accompanied by induction of activation induced cytidine deaminase expression, but not gross apoptosis. These data support the role of abnormal CD40 signal transduction in mediating genomic instability, suggesting a role for the CD40 pathway and intermediates in myelomagenesis and clonal evolution in vivo.     

Chromosomal abnormalities in patients with Hodgkin's disease: evidence for frequent involvement of the 14q chromosomal region but infrequent bcl-2 gene rearrangement in Reed-Sternberg cells.

BACKGROUND: Rearrangements of the bcl-2 gene (also known as BCL2) have been detected in up to 40% of cases of Hodgkin's disease, and it has been speculated that such rearrangements may have a role in the pathogenesis of Hodgkin's disease. PURPOSE: The purposes of this study were (a) to assess the frequency of clonal chromosomal abnormalities in Hodgkin's disease, (b) to identify recurrent changes, (c) to determine whether the bcl-2 gene rearrangement was present in Reed-Sternberg cells (the neoplastic cells of Hodgkin's disease) and their variants, and (d) to analyze whether the presence of t(14;18) translocations in Reed-Sternberg cells explains the observed bcl-2 gene rearrangements in Hodgkin's disease. METHODS: A cytogenetic study was performed on biopsy specimens from 28 consecutive untreated patients with Hodgkin's disease. The same patients were analyzed for bcl-2 gene rearrangement by a polymerase chain reaction (PCR) technique. To ascertain whether the abnormal karyotypes were present in and restricted to Reed-Sternberg cells, we also performed in situ hybridization with chromosome-specific probes. RESULTS: Abnormal metaphases were identified in 23 of the 28 patients. In 11 patients, the chromosome 14q region was abnormal; in six of these patients, there was involvement of the 14q32 region that comprises the gene encoding for heavy-chain immunoglobulin. Only one patient had a t(14;18) translocation, whereas almost 40% of these 28 patients showed bcl-2 gene rearrangements by a PCR method. The in situ hybridization method showed that the abnormal karyotype was present in and restricted to Reed-Sternberg cells. CONCLUSIONS: We conclude that the majority of cases of Hodgkin's disease contain a clonal population with an abnormal karyotype, comprising the Reed-Sternberg cells. The q32 region of chromosome 14 is frequently involved, but a t(14;18) translocation is extremely infrequent. The occurrence of a bcl-2 gene rearrangement in Hodgkin's disease most likely results from the presence of sporadic, small bystander B lymphocytes that carry the translocation and that also can be frequently detected in reactive lymphoid tissue such as tonsils. Also, a range of different chromosomal translocations may provide growth or survival advantages to Reed-Sternberg cells.     

Examination of clonal variants from human malignant melanoma studied by chromosome banding analysis.

Multiple metastatic melanoma lesions from three patients were cytogenetically characterized in order to assess the degree of intra-patient karyotypic heterogeneity. A total of 20 specimens were analysed: 12 samples from patient No. 1, five samples from patient No. 2, and three samples from patient No. 3. Sufficient mitoses were obtained to perform detailed analysis in 19/20 specimens following short-term culture. The modal chromosome number of all three cases was near-diploid, with all samples demonstrating multiple structural abnormalities. Abnormalities shared by all three patients were alterations of chromosomes 1 and 8. Other structural abnormalities common to two of the three patients involved chromosomes 6, 7, 9 and 10. Minor intra-tumour karyotypic variation was detected in all three cases. However, the majority of clonal alterations were retained in all metastatic lesions, clearly indicating the karyotypically stable and clonal nature of this neoplasm.     

Feasibility and reliability of flow-cytometry (fcm) DNA analysis of fresh and fixed urine samples

The qualitative results of FCM DNA analysis on fresh and fixed urine specimens (28 and 97, respectively) from 68 normal subjects and 10 patients with a past history of bladder cancer were compared. FCM DNA evaluability was not significantly different in fresh and fixed samples (63% vs 73%, respectively) whereas mean CV was significantly higher (7.3% vs 5.7%, respectively; p=0.04). A double FCM analysis on fresh and fixed urine was also performed in 16 cases. In this subgroup, the percentage of evaluable histograms from fixed urine specimens was slightly higher than that from fresh specimens. Aneuploid cases were found only in the fixed urine samples but the CVs from fresh and fixed cell suspensions did not differ. The absence of inflammatory cells with cytological analysis of the same samples was associated with low percentages of FCM evaluability and higher CVs. The use of fixed samples improves the quality of FCM DNA analysis permitting its use for screening programs.     

用IgH、TCR基因重排技术检测疑难淋巴组织增生性病变

目的探讨IgH、TCR基因重排技术检测疑难淋巴组织增生性病变的意义。方法采用IgHTCR-β、TCR-γ基因重排标志检测,36例经常规HE、免疫组化不能诊断的疑难淋巴组织增生性病变,并进行克隆性分析。结果29例淋巴组织增生性病变呈克隆阳性,其中IgH、TCR双重排阳性者为1例,IgH单一阳性8例,TCR单阳性为8例。克隆阳性病例与免疫组化结果一致的为24/29例,7例阴性者,4例为反应性增生,2例经随诊为猫抓病淋巴结炎,1例为不典型增生。结论IgH、TCR基因重排技术对于疑难淋巴组织增生性病变的诊断和鉴别诊断有较大的意义。     

Primary B-cell malignant lymphoma of the maxilla with a sarcomatous pattern and multilobated nuclei

Three cases of primary malignant lymphoma of the maxilla are reported. The primary intraosseous origin of these tumors was demonstrated by x-ray examination and surgical exploration. The initial interpretation as odontogenic infection led to a delay in starting therapy of 9 months in one case. Biopsies of two cases were initially interpreted as sarcoma because of a dense reactive fibrosis between the tumor cells. Subsequently, hemimaxillectomy was performed in one case. Histologically and ultrastructurally the tumor cells showed marked nuclear abnormalities with cleavage, folding, and lobulation. Immunohistochemical studies of two cases showed a monoclonal immunoglobulin expression, IgG-K; T-lymphocyte-associated antigens were not detected on the tumor cells. The findings indicate the existence of a primary B-cell malignant lymphoma of bone with multilobated nuclei. The lymphoid nature may be masked by a dense proliferation of connective tissue. The relation of these tumors to the classifications for malignant lymphoma of lymph node is discussed.     

Immunoglobulin gene rearrangement investigations in the diagnosis of lymphoid malignancies from formaldehyde-fixed biopsies

Determination of the biologic potential of lymphoid proliferations in biopsies can be difficult by standard histological or even immunohistochemical examination. Polymerase chain reaction (PCR) has been used with increasing frequency to detect clonal rearrangements of the immunoglobulin heavy chain (IgH) in formaldehyde fixed, paraffin wax embedded tissues. Sensitivity ranges between 50 and 80%, and therefore at least 20% of neoplasms remain undetected by these approaches. Few investigators have attempted to detect immunoglobulin light chain (IgL) gene rearrangements by PCR using paraffin wax embedded samples. We studied 29 cases of B-cell neoplasms, along with 21 cases with equivocal histology and 4 reactive biopsies, using degenerate oligoprimers to amplify Ig κand Ig λlight chain genes, along with IgH (Fr 1, 2 and 3) gene rearrangement analysis. The combination of these methods detected clonality in 93% of cases (27/29) with histological diagnosis of B-NHL. Fr2 and Fr3 primers detected clonality in 79% (23/29) of cases. IgL chain rearrangements detected 4 cases (14%), negative for IgH rearrangements, improving sensitivity from 79 to 93%. Clonality was detected in 52% (11/21) of histologically equivocal lymphoid proliferations, including one case detected by IgL rearrangements which was negative for IgH rearrangements. Archival material from 4 cases with reactive histology produced polyclonal results. These results confirm that PCR based immunoglobulin gene rearrangement is a sensitive and specific method for demonstrating B-cell clonality in paraffin-wax embedded sections. The addition of IgL analysis to the IgH assay allows the detection of greater than 90% of B-cell lymphoproliferative disorders from routine histological specimens with poor preservation of genomic DNA.     

Aneuploidy as a marker of minimal residual disease in leukemia

Aneuploidy as an indication of abnormal cellular DNA content has recently been confirmed to be a reliable marker of malignant cells in human solid tumors and hematologic malignancies. Flow cytometry (FCM), measuring cellular DNA content in thousands of cells within seconds, is able to safely detect the "rare event cell," the rare aneuploid cell in a diploid cell population. This very fast and sensitive technique was combined with a newly developed cell separation technique. Cell separation prior to FCM enabled us to detect malignant cells at concentrations of 0.05% in blood, bone marrow, and lymph node cell suspensions of patients with leukemia. An illustration of this method is presented in conjunction with first clinical applications demonstrating that patients with minimal residual disease in clinically complete remission had significantly shorter survival times than patients in whom no minimal residual disease was detected with this new method.     

Flow analysis of hairy cell leukemia

Cellular DNA content, Coulter volume and light scatter were measured in cell suspensions from spleens or peripheral blood in 6 patients with hairy cell leukemia. In 2 of 6 cases, the DNA distribution obtained by flow microfluorometry demonstrated an abnormal cellular DNA content of the G₀?G₁ cells. This abnormality was confirmed by mixing the patients' cells with normal blood mononuclear cells. This is the first report of cellular DNA abnormalities in hairy cell leukemia demonstrated by flow microfluorometry. A relatively low number of cells were observed in the S phase of the cell cycle in all cases. On Coulter analysis, “hairy” cells displayed a modal volume 2 to 3 times larger than that of non-malignant lymphoid populations. Differences between “hairy” cell populations and normal lymphoid cells were also noted using light scatter measurements of ethanol-fixed cells, but they were less marked than those observed by Coulter analysis.     

BIOMED-2体系检测免疫球蛋白重链基因重排在黏膜相关淋巴组织结外边缘区B细胞淋巴瘤诊断中的应用

 【摘要】　目的　探讨BIOMED-2标准化体系检测免疫球蛋白重链（IGH）基因重排对黏膜相关淋巴组织结外边缘区B细胞淋巴瘤（MALT淋巴瘤）诊断的应用价值。方法　45例标本包括不同部位原发的MALT淋巴瘤36例、淋巴结外淋巴组织增生性病变3例、幽门螺杆菌（HP）感染相关性重度胃炎6例。用DNA提取试剂盒从石蜡包埋组织中提取所选病例的基因组DNA,通过BIOMED-2体系质控混合引物检测其质量,应用IGH VH-JH 3组引物组合进行IGH基因克隆性重排检测;比较分析该体系在MALT淋巴瘤诊断中的敏感性和特异性。结果　45例中31例（MALT淋巴瘤22例,淋巴组织增生性病变3例、重度胃炎6例）提取的基因组DNA可扩增出300 bp片段,其余14例DNA均严重降解。22例MALT淋巴瘤中,16例检测出IGH基因重排,灵敏度为72.7 %;6例严重胃炎病例未检测出IGH克隆性重排,其特异度是100 %;3例淋巴组织增生性病变中1例检测出IGH克隆性重排,2例未检测出重排。结论　在MALT淋巴瘤的诊断和淋巴组织增生性疾病的鉴别诊断中,BIOMED-2标准化体系检测IGH基因克隆性重排是一种快速可靠的方法,具有重要的临床应用价值。     

Ig-Gene and T-Cell Receptor Gene Rearrangements in a Secondary, Mono-Histiocytic Malignancy

In 1984, a 21-year-old male was diagnosed with an actue lymphoblastic leukemia of pre-B cell type. Treatment with chemotherapy, including alkylating agents and prophylactic radiotherapy to the central nervous system, induced a complete remission. In June 1990, a biopsy from a supraclavicular node revealed a malignancy of mono-histiocytic type with erythrophagocytosis. Soon thereafter bone marrow involvement was found. No remission was achieved and the patient died in December 1990. DNA from bone marrow and lymph node obtained 1990 showed clonal rearrangements of both the immunoglobulin heavy-chain gene and the T-cell receptor gamma chain gene. This unusual case illustrates a typical secondary malignancy proven to be separate from the primary neoplasm judged by morphological appearance, immunophenotype and cytogenetic constitution. Coexistent clonal rearrangements of immunoglobulin and T-cell receptor genes have been reported in acute non-lymphoblastic leukemias and notably in cases expressing TdT, interpreted as a predominant lymphoid commitment of the tumor cells. In the present case, however, the malignant cells had a differentiated phenotype and showed erythrophagocytosis, indicating a more mature mono-histiocytic cell type. However, also CD3 expression was found by immunohistochemistry of frozen sections which might indicate a biphenotypic malignancy.     

Hairy cell leukemia has a B-cell genotype

The phenotype and, by inference, the cell of origin of some lymphocytic neoplasms has been defined by surface marker studies; however, the precise cellular origin of other neoplasms of the lymphoid system is still unknown. For example, with reference to hairy cell leukemia (HCL), cell marker data has been used in support of a monocytic, a T cell, or a B cell origin. If hairy cell leukemia is a B cell-derived neoplasm, the controversy may be resolved by genotyping the cells, using the rearrangement of immunoglobulin genes as a marker of the B cell nature of the process. Rearrangement of these genes is detected using the Southern blot technique and cloned probes specific for the JH segment of the immunoglobulin genes. In this study, the arrangement of the immunoglobulin genes was analysed in normal tissue, in two accepted B cell lymphomas and in nine cases of hairy cell leukemia. DNA from peripheral blood leukocytes (two patients) and from the spleen (seven patients) revealed a discrete new JH restriction fragment length in the leukocytes of hairy cell leukemia cases. The presence of rearranged restriction fragments is interpreted as evidence of the existence of clonal B cell populations. Three of six samples had rearranged kappa light chain fragments. We conclude that most cases of hairy cell leukemia have a B cell genotype. The use of genotyping has wider application in the analysis of hematological malignancies.