B细胞淋巴瘤患者骨髓IgH基因克隆性重排检测的临床意义

目的 探讨半巢式聚合酶链反应(PCR)检测B细胞淋巴瘤患者骨髓中IgH基因克隆性重排的可行性,并初步评价其临床价值.方法 选用FR2、FR3A引物,采用半巢式PCR方法检测105例B细胞淋巴瘤患者骨髓中IgH基因的单克隆性重排,与骨髓穿刺细胞形态学检测结果进行比较,并评价PCR检测结果与临床病理特征的关系.结果 105例B细胞淋巴瘤患者中,IgH基因克隆性重排PCR检测48例(45.7%)阳性,而骨髓细胞形态学只检测出22例(21.0%),两者差异有统计学意义(P<0.05),符合率为71.4%(75/105).弥漫大B细胞性淋巴瘤(DLBCL)、滤泡性淋巴瘤(FL)及小淋巴细胞性淋巴瘤(SLL)初治患者PCR检测阳性率分别为30.8%、25.0%和100.0%.PCR检测结果与Ann Arbor分期有关,早期B细胞淋巴瘤患者lgH基因克隆性重排PCR检出阳性率低于晚期患者(P=0.02).PCR检测阳性和阴性患者的近期疗效差异无统计学意义(P>0.05),但CR率(23.3%和46.3%)差异有统计学意义(P=0.019).结论 IgH基因克隆性重排PCR检测可能是判断B细胞淋巴瘤患者骨髓异常的有效方法,较骨髓细胞形态学敏感;Ann Arbor分期晚的患者PCR检测阳性率高于分期早的患者;PCR检测阳性者治疗后获得CR的机会低于阴性者.     

Detection of immunoglobulin heavy chain genes rearrangements in B-cell leukemias, lymphomas, multiple myelomas, monoclonal and polyclonal gammopathies

Polymerase chain reaction (PCR) based assays were found to be a realistic alternative to Southern blot hybridization for the assessment of clonal immunoglobulin heavy chain gene rearrangements. However, a comparison of the different PCR based studies reveals considerable variation in experimental design and marked differences in the reported results. This study compared different single- and double-step PCR assays relying on various FR3, FR2, FR1 and JH based primers for the detection of B cell clonality in acute lymphoblastic leukemias (ALL), non-Hodgkin's-lymphoma (NHL), multiple myeloma (MM), monoclonal gammopathies of unknown significance (MGUS) and three polyclonal gammopathies (PG). The highest monoclonality rate was observed using seminested CDR-III region amplification. This method achieved a monoclonal product in 6 of 13 pro-B ALL 21 of 29 c-ALL, 7 of 8 pre-B-ALL, 18 of 21 B-ALL, 14 of 17 B-NHL (intermediate or high grade) with bone marrow involvement, 0 of 9 B-NHL without bone marrow involvement, 9 of 9 low grade B-NHL (immunocytoma and including chronic lymphocytic leucemia), 13 of 19 MM, 2 of 9 MGUS, and 0 of 3 PG. Additional monoclonality was detected with nested CDR I PCR in 1 pro-B-ALL, 1 c-ALL, and 2 MM. CDR III IgH PCR has been confirmed as an efficient method for determining clonality in B-cell neoplasias. Some additional monoclonal products can be seen with CDR I-based PCR. Detection of monoclonality depends on the maturation grade of the neoplastic B-cell population.     

An improved method for detection of B-lymphoid clonality by polymerase chain reaction

Several groups have recently described methods for the detection of clonal immunoglobulin heavy chain (IgH) gene rearrangements in B-cell malignancies by polymerase chain reaction (PCR) gene amplification using variable region-(VH) and joining (JH) region-specific primers. The simplest methods utilize a single VH primer specific for sequences present in most VH regions corresponding to the third framework region (FR3). An alternative approach is to use a panel of VH family-specific primers specific for the first framework regions (FR1). In the course of nucleotide sequence analysis of IgH gene rearrangements amplified using a VH FR1 primer panel, these authors previously observed 3' VH region deletion and/or base mis-matches sufficient to prevent efficient priming from the VH FR3 primer target sequence in a significant minority of cases of B-lineage malignancy. An improved PCR method has therefore been developed by using a panel of seven VH FR1 family-specific primers incorporated in a single reaction. By using this method clonal IgH gene rearrangement is detected in 15 of 16 cases of B-lineage malignancy. Significantly, this series included four cases of B-lymphoma in which previous attempts to detect PCR clonal IgH gene rearrangements using a VH FR3 primer were unsuccessful. In two of these cases, nucleotide sequence analysis of the amplified DNA showed that failure to prime with the VH FR3 primer was likely to be attributable to insufficient homology with the target sequence. The use of the approach described in this paper should significantly improve the reliability of detection of B-lymphoid clonality by PCR.     

Heterogeneous in situ immunophenotyping of follicular dendritic reticulum cells in malignant lymphomas of B-cell origin

The phenotype of follicular dendritic reticulum cells (DRC) was analyzed with monoclonal antibodies (DRC-1, OKB7, BA-2, Leu-M3, and antidesmoplakin 1 and 2) in 28 frozen biopsy specimens of both morphologically and phenotypically analyzed B-cell lymphomas and 21 normal or reactive controls. The former included 15 follicular center cell lymphomas (FCCL), four intermediately differentiated lymphocytic lymphomas (ILL), four mantle zone lymphomas (MZL), and five well-differentiated lymphocytic lymphomas (WDLL). In controls, DRC-1+ and OKB7+ DRC were localized in both follicular centers (FC) and mantle zones (MZ), but BA-2+ and Leu-M3+ DRC were confined to FC only. FCCL were usually accompanied by DRC-1+, OKB7+, and BA-2+ DRC, and either lost or maintained positively with Leu-M3 from case to case. By contrast, MZL consistently lacked BA-2+ and Leu-M3+ DRC, and was associated with DRC-1+ and OKB7+ DRC only. Desmoplakin-positive DRC occurred in variable proportions in both FCCL and MZL. As opposed to FCCL and MZL, all WDLL and all but one of the ILL (associated only with DRC-1+, OKB7+, and desmoplakin+ DRC) did not show any DRC as identifiable with the antibody panel used. Remarkably, the difference in the distribution of BA-2+ and Leu-M3+ DRC in the normal FC and MZ appears to be maintained in their neoplastic counterparts (FCCL and MZL) also. Such a difference represents an example of the possible interactions between lymphoma cells of different phenotype and their microenvironment, as portrayed by phenotypically heterogeneous DRC.     

Significantly improved PCR-based clonality testing in B-cell malignancies by use of multiple immunoglobulin gene targets. Report of the BIOMED-2 Concerted Action BHM4-CT98-3936.

Polymerase chain reaction (PCR) assessment of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements is an important diagnostic tool in mature B-cell neoplasms. However, lack of standardized PCR protocols resulting in a high level of false negativity has hampered comparability of data in previous clonality studies. In order to address these problems, 22 European laboratories investigated the Ig/TCR rearrangement patterns as well as t(14;18) and t(11;14) translocations of 369 B-cell malignancies belonging to five WHO-defined entities using the standardized BIOMED-2 multiplex PCR tubes accompanied by international pathology panel review. B-cell clonality was detected by combined use of the IGH and IGK multiplex PCR assays in all 260 definitive cases of B-cell chronic lymphocytic leukemia (n=56), mantle cell lymphoma (n=54), marginal zone lymphoma (n=41) and follicular lymphoma (n=109). Two of 109 cases of diffuse large B-cell lymphoma showed no detectable clonal marker. The use of these techniques to assign cell lineage should be treated with caution as additional clonal TCR gene rearrangements were frequently detected in all disease categories. Our study indicates that the BIOMED-2 multiplex PCR assays provide a powerful strategy for clonality assessment in B-cell malignancies resulting in high Ig clonality detection rates particularly when IGH and IGK strategies are combined.     

Detection of clonality in lymphoproliferations using PCR of the antigen receptor genes: does size matter?

A biopsy of a nasal mass that had morphologic and immunostaining features consistent with a B-cell lymphoma was studied for clonality using PCR of the IgH gene. An unexpectedly low molecular weight DNA fragment of approximately 140bp (acceptable size limit: 250-295bp) was obtained using FR2 and JH primers. The sequence of this DNA was consistent with a clonal IgH rearrangement followed by a deletion that removed most of the downstream portion of the V segment. Thus, the biopsy contained a monoclonal population of B-lymphocytes, consistent with a diagnosis of lymphoma. This work illustrates that bands outside of the size range expected from PCR of the antigen receptor genes may still be consistent with a monoclonal result.     

IgH和IgL引物联合检测对提高石蜡组织淋巴瘤基因重排检出率的价值

背景与目的：通过聚合酶链反应（polymerase chain reaction,PCR）扩增免疫球蛋白重链（immunoglobulin heavy chain,IgH）基因对其克隆性的检测,可以辅助诊断淋巴瘤。缺点是假阴性率较高,在石蜡包埋组织中尤为明显。本研究拟采用手工显微切割、免疫球蛋白重链和轻链（immunoglohulin light chain,IgL）联合测定等方式,探讨该方法在石蜡包埋组织中非霍奇金淋巴瘤（non-Hodgkin’s lymphoma,NHL）诊断中的价值。方法：选用1对IgH引物、1对T细胞受体γ（Tcell receptor γ,TCRγ）、TCRγ引物、2对新设计的轻链引物,通过PCR、琼脂糖和聚丙烯酰胺凝胶电泳（PAGE）及银染技术,检测经形态学及免疫组织化学确诊的58例石蜡包埋组织标本,包括39例B细胞淋巴瘤、16例T细胞淋巴瘤和3例淋巴结反应性增生组织。以DG75和Jurkat淋巴瘤细胞系DNA作为对照。结果：IgH引物P1在39例B细胞淋巴瘤检出阳性率79．5％（31／39）。假阳性率6．25％（1／16）,IgL引物在B细胞淋巴瘤检出阳性率71．8％（28／39）。假阳性率12．5％（2／16）,经统计学分析二者检出率无显著性差异（P〉0．05）。二者联合检测,B细胞淋巴瘤阳性检出率可以达到92．3％,假阳性率并无明显提高（12．5％）。以上重排引物在反应性增生淋巴结组织中均未检出。结论：IgH与IgL引物联合检测可明显提高石蜡包埋组织中B细胞淋巴瘤的检出率,并为B-NHL的诊断及鉴别诊断提供了有效的辅助手段。     

The Polymerase Chain Reaction in Diagnosis of Small B-Cell Non-Hodgkin Lymphomas

Background: Small B-cell non-Hodgkins lymphoma (NHL) is difficult to be distinguished from non-neoplastic reactive processes using conventional haematoxylin-eosin (HE) staining due to different interpretations among pathologists with diagnosis based on morphologic features. Ancillary examinations such as immunohistochemical (IHC) staining are essential. However, negative or doubtful results are still sometimes obtained due to unsatisfactory tissue processing or IHC technique. The polymerase chain reaction (PCR) as a molecular diagnostic technique is very sensitive and specific. Clonality detection of heavy chain immunoglobulin (IgH) gene rearrangement has been widely used to establish diagnosis of B-cell NHL. Aims: To elaborate interobserver variation in small B-cell NHL diagnosis based on morphologic features only and to confirm sensitivity and specificity of the PCR technique as an ancillary method. Materials and Methods: A toptal of 28 samples of small B cell NHL and suspicious lymphoma were interpreted by 3 pathologists in Sardjito General Hospital based on their morphology only. The reliability of assessment and the coefficient of interobserver agreement were calculated by Fleiss kappa statistics. Interpretation results were confirmed with IHC staining (CD20, CD3, Bcl2). PCR was performed to analyze the clonality of IgH gene rearrangement. Results: Interobserver agreement in morphologic evalution of small B cell NHL and chronic lymphadenitis revealed kappa coefficient 0.69 included in the substantial agreement category. The cases were divided into 3 groups based on morphology and IHC results; lymphoma, reactive process and undetermined group. PCR analysis showed 90% sensitivity and 60% specificity. Conclusions: The present study revealed a substantial agreement among pathologists in small B-cell NHL diagnosis. For difficult cases, PCR is useful as complementary method to morphologic and IHC examinations to establish definitive diagnosis.     

Detecting clonal rearrangement in non-Hodgkin's lymphomas in Taiwan by polymerase chain reaction

The detection of monoclonal expansions of the immunoglobulin heavy chain (IgH) or the T-cell receptor-gamma (TCRgamma) chain genes is an important supplement for the diagnosis of the non-Hodgkin's lymphomas (NHLs). Detection of monoclonality by polymerase chain reaction (PCR) method has offered an efficient approach for rapid diagnosis and monitoring of the therapeutic effects. Here we conducted a retrospective PCR clonality study on 49 cases of NHLs including 23 B-cell lymphomas (BCLs), 20 peripheral T-cell lymphomas (PTCLs), 6 natural killer (NK)/T-cell lymphomas and 3 reactive lymphoid tissues from southern Taiwan. Genomic DNAs from paraffin sections were extracted and analyzed by the IgH- and TCR-specific PCR reactions. The results showed that 20 of 23 (87.5%) BCLs exhibited IgH gene rearrangements and were all germline for TCRgamma. 15 of 20 (75.0%) PTCLs exhibited TCRgamma gene rearrangements while 1 case (5%) was positive for IgH gene rearrangement. The 6 NK/T-cell lymphomas and 3 reactive lymphoid tissues were all germline for either IgH or TCRgamma genes. Our results were similar to other Western reports in terms of sensitivity and cell-lineage specificity. This is the first large series of PCR clonality study of IgH and TCRgamma gene rearrangements on NHLs from Taiwan. We have confirmed that this rapid method is a sensitive diagnostic tool for NHLs.     

Expression of inhibitor of apoptosis proteins in B-cell non-Hodgkin and Hodgkin lymphomas

BACKGROUND: Inhibitor of apoptosis proteins (IAPs) inhibit apoptosis by binding specific caspases, and possibly by other mechanisms. Eight IAPs have been identified in humans, of which cIAP1, cIAP2, and XIAP are well known. IAPs are being investigated as potential treatment targets in cancer patients. METHODS: cIAP1, cIAP2, and XIAP were assessed in lymphoma cell lines, 240 B-cell non-Hodgkin lymphoma (NHL) tumors, and 40 Hodgkin lymphoma (HL) tumors. RESULTS: All IAPs were expressed in most NHL and all HL cell lines. In NHL tumors, cIAP1 was expressed in 174 (73%), cIAP2 in 115 (48%), and XIAP in 37 (15%). cIAP1 was positive in all precursor B-cell lymphoblastic lymphoma/leukemia (LBL) and nodal marginal zone B-cell lymphoma (MZL), over 90% of follicular lymphoma and diffuse large B-cell lymphoma (DLBCL), and approximately 50% to 60% of myeloma, Burkitt lymphoma (BL), lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia (LPL/WM), small lymphocytic lymphoma/ chronic lymphocytic leukemia (SLL/CLL), extranodal marginal zone B-cell lymphoma of mucosa associated lymphoid tissue (MALT-lymphoma), splenic MZL, and mantle cell lymphoma. cIAP2 was positive in all MALT-lymphoma, over 90% of precursor B-cell LBL (94%), most BL (75%), LPL/WM (71%), and SLL/CLL (67%), and approximately 40% to 60% of follicular lymphoma, myeloma, and DLBCL. XIAP was positive most cases of precursor B-cell LBL (57%) and approximately 30% to 40% of nodal MZL, BL, and DLBCL. In HL tumors, cIAP1 was positive in 30 (75%), cIAP2 in 27 (68%), and XIAP in 23 (58%), and did not correlate with histologic type. CONCLUSIONS: Differential expression of IAPs in B-cell lymphomas suggests differences in pathogenesis that may have implications for novel treatment strategies targeting IAPs.     

The use of archival bone marrow specimens in detecting B-cell non-Hodgkin's lymphomas using polymerase chain reaction methods

The detection of B-cell non-Hodgkin's lymphoma (B-NHL) involving the bone marrow (BM) can be enhanced by assessing immunoglobulin heavy chain (IgH/JH) gene rearrangement using PCR. While the fresh BM aspirate has been the most commonly used specimen, the utility of archival BM tissues has not been extensively evaluated. We studied the BM from 13 patients with nodal B-NHL (7 low-grade and 6 intermediate grade), which were categorized into three groups based on the histologic finding of lymphoma (H) and the presence of a monoclonal IgH/JH band by PCR using fresh BM aspirates (M): (1) H(+)/M(+), 4 cases; (2) H(+)/M(-), 4 cases; and (3) H(equivocal)/M(-), 5 cases. Archival tissues available for study included paraffin-embedded trephine biopsy (TB)/aspirate clots (AC) and air-dried aspirate smears (AS). All TB (13/13) and a subset of AC (5/13) were B5-fixed, and all these tissues failed to yield analyzable DNA. In contrast, sufficient DNA was consistently obtained in AC that were formalin-fixed (8/13). Of these 8 cases, 2/3 of group 1, 3/3 of group 2, and 0/2 of group 3 had a monoclonal IgH band. Using DNA extracted from microdissected lymphoid aggregates morphologically evident in the AC sections, additional positive cases were identified: 1/3 of group 1 and 2/2 of group 3. In those 5 cases that did not have formalin-fixed TB/AC, sufficient DNA was extracted from AS in all cases; one additional positive case was identified in group 1. Overall, 4/4 (100%) of group 1, 3/4 (75%) of group 2, and 2/5 (40%) of group 3 showed molecular evidence of lymphoma. To conclude, archival BM specimens are a useful source of DNA for molecular detection of B-NHL involvement, and formalin appears to be a better fixative than B5. The use of these samples may improve the overall detection sensitivity.     

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.     

Clonal analysis of B-cell leukemias and lymphomas using the polymerase chain reaction for the third complementarity determining region of the IgH gene: a study of 75 cases from Nagasaki Japan.

Using semi-nested polymerase chain reaction (PCR), we examined 75 Japanese cases of hematologic malignancies with B-cell antigens including 25 common acute lymphoblastic leukemia (ALL), 13 chronic lymphocytic leukemia (CLL), 28 B-cell malignant lymphoma (B-ML), 2 hairy cell leukemia (HCL), 7 acute myelogenous leukemia with B-cell antigens (AML-B), and 23 controls. When amplified products were analysed by a standard polyacrylamide gel electrophoresis, the sensitivity for detection of clonal IgH rearrangements in each group of ALL, CLL, B-ML, HCL, and AML-B was 88%, 92.3%, 71.4%, 100%, and 57.1%, respectively, with an overall sensitivity of 80.0%. There were no false positive results in any of the control samples. Single strand conformation polymorphism (SSCP) analysis of the amplified products gave rise to a much greater sensitivity, up to 84% overall. The false negative samples were mainly encountered in B-ML with SmIgG and non-Ig, suggesting miss-annealing between the primers used and the template DNA because of somatic hypermutation of IgH genes in such clones. This indicates that PCR analysis is very useful in detecting the clonal IgH rearrangements in B-cell malignancies, especially in ALL and CLL, but not in B-ML corresponding to neoplasms originating from pre-germinal center naive B-cells.     

Histomorphology and cytomorphology of cutaneous B-cell lymphomas

This article summarizes the conceptual and morphologic background for the diagnosis of cutaneous B-cell lymphomas (CBCL). The architectural pattern of the infiltrate and cytomorphology can indicate the B-cell nature of a skin lymphoma. Virtually all types of B-cell lymphomas, including pre-B-cell lymphoma; Burkitt-like lymphoma; malignant lymphoma, small lymphocytic; the many forms of germinal center cell-derived lymphomas; immunocytoma; plasmacytoma; and immunoblastic lymphoma (B type)-may involve the skin and can be classified according to modern lymphoma classifications.     

Powerful strategy for polymerase chain reaction-based clonality assessment in T-cell malignancies Report of the BIOMED-2 Concerted Action BHM4 CT98-3936.

Polymerase chain reaction (PCR) assessment of clonal T-cell receptor (TCR) and immunoglobulin (Ig) gene rearrangements is an important diagnostic tool in mature T-cell neoplasms. However, lack of standardized primers and PCR protocols has hampered comparability of data in previous clonality studies. To obtain reference values for Ig/TCR rearrangement patterns, 19 European laboratories investigated 188 T-cell malignancies belonging to five World Health Organization-defined entities. The TCR/Ig spectrum of each sample was analyzed in duplicate in two different laboratories using the standardized BIOMED-2 PCR multiplex tubes accompanied by international pathology panel review. TCR clonality was detected in 99% (143/145) of all definite cases of T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, peripheral T-cell lymphoma (unspecified) and angioimmunoblastic T-cell lymphoma (AILT), whereas nine of 43 anaplastic large cell lymphomas did not show clonal TCR rearrangements. Combined use of TCRB and TCRG genes revealed two or more clonal signals in 95% of all TCR clonal cases. Ig clonality was mostly restricted to AILT. Our study indicates that the BIOMED-2 multiplex PCR tubes provide a powerful strategy for clonality assessment in T-cell malignancies assisting the firm diagnosis of T-cell neoplasms. The detected TCR gene rearrangements can also be used as PCR targets for monitoring of minimal residual disease.     

Comparative analysis of minimal residual disease detection using four-color flow cytometry, consensus IgH-PCR, and quantitative IgH PCR in CLL after allogeneic and autologous stem cell transplantation.

The clinically most suitable method for minimal residual disease (MRD) detection in chronic lymphocytic leukemia is still controversial. We prospectively compared MRD assessment in 158 blood samples of 74 patients with CLL after stem cell transplantation (SCT) using four-color flow cytometry (MRD flow) in parallel with consensus IgH-PCR and ASO IgH real-time PCR (ASO IgH RQ-PCR). In 25 out of 106 samples (23.6%) with a polyclonal consensus IgH-PCR pattern, MRD flow still detected CLL cells, proving higher sensitivity of flow cytometry over PCR-genescanning with consensus IgH-primers. Of 92 samples, 14 (15.2%) analyzed in parallel by MRD flow and by ASO IgH RQ-PCR were negative by our flow cytometric assay but positive by PCR, thus demonstrating superior sensitivity of RQ-PCR with ASO primers. Quantitative MRD levels measured by both methods correlated well (r=0.93). MRD detection by flow and ASO IgH RQ-PCR were equally suitable to monitor MRD kinetics after allogeneic SCT, but the PCR method detected impending relapses after autologous SCT earlier. An analysis of factors that influence sensitivity and specificity of flow cytometry for MRD detection allowed to devise further improvements of this technique.     

Application of laser capture microdissection to cytologic specimens for the detection of immunoglobulin heavy chain gene rearrangement in patients with malignant lymphoma

BACKGROUND: The demonstration of the monoclonality of immunoglobulin heavy chain (IgH) gene rearrangement is an indispensable method for the diagnosis of B-cell lymphoma as well as histocytochemical analysis. For the detection of IgH gene rearrangement, the extraction of DNA from a homogenous cell population is necessary. Recently, the laser capture microdissection (LCM) technique was shown to isolate specific cells from histopathologic specimens for molecular analysis. However, to the authors' knowledge the applicability of LCM to cytologic specimens has not yet been well established. METHODS: Using LCM, a homogenous population of B-cell lymphoma cells as both histologic sections and cytologic specimens was captured, and genomic DNA was extracted from the captured cells. IgH gene rearrangement was analyzed by the polymerase chain reaction (PCR)-based single-strand conformational polymorphism (SSCP) method. RESULTS: Genomic DNAs were extracted successfully from ethanol-fixed cytologic specimens, but cells were not captured from air-dried specimens. Using PCR-SSCP analysis, the monoclonality of the IgH gene rearrangement was detected in five cases of tissue sections among nine analyzed cases of malignant lymphoma diagnosed immunohistochemically. However, analysis of the cytologic specimens with LCM demonstrated the monoclonality of the IgH gene rearrangement in seven cases of lymphoma. CONCLUSIONS: The results of the current study suggest that the novel application of LCM to cytologic specimens occasionally exhibits high sensitivity for the detection of IgH gene rearrangement monoclonality compared with the use of histologic sections.     

PCR和FISH技术在子宫颈淋巴瘤与淋巴瘤样病变诊断和鉴别诊断中的作用

 目的　探讨PCR和FISH检测技术在原发性子宫颈淋巴瘤与淋巴瘤样病变的诊断与鉴别诊断中的作用。方法　收集3例原发性子宫颈弥漫性大B细胞淋巴瘤（DLBCL）与2例子宫颈淋巴瘤样病变,进行PCR-IgH重排及FISH检测。结果　PCR检测显示3例DLBCL和1例淋巴瘤样病变中出现单克隆性IgH基因重排。间期FISH检测显示3例DLBCL均发生了IgH和bcl-6基因断裂,而2例淋巴瘤样病变均未检测到特定的染色体断裂。结论　PCR-IgH重排并非仅见于子宫颈细胞淋巴瘤。间期FISH检测IgH和bcl-6基因的断裂对于子宫颈B细胞淋巴瘤和淋巴瘤样病变的诊断和鉴别诊断有帮助。     

Quantitative assessment of contaminating tumor cells in autologous peripheral blood stem cells of B-cell non-Hodgkin lymphomas using immunoglobulin heavy chain gene allele-specific oligonucleotide real-time quantitative-polymerase chain reaction

A real-time quantitative-polymerase chain reaction (RQ-PCR) targeting the immunoglobulin heavy chain (IgH) gene has been used for the quantification of minimal residual disease (MRD) in B-cell hematological malignancies. In non-Hodgkin lymphoma (NHL), experimental costs are increased, as a large number of primer-probe sets are required because of diversity, due to somatic and ongoing mutations of the IgH gene. We developed an allele-specific oligonucleotide (ASO) combined with a germline consensus probe-based RQ-PCR assay and examined MRD in peripheral blood stem cells (PBSC). The IgH consensus probes were adapted in seven (50%) of 14 amplifiable cases. Patients with heavily contaminating tumor cells in PBSC relapsed after PBSC transplantation. Our strategy will contribute to the development of a cost-efficient, precisely quantitative and systemic detection assay for MRD in NHL.