Clonality of primary pulmonary lymphoproliferative disorders; using in situ hybridization and polymerase chain reaction for immunoglobulin

Primary pulmonary lymphoproliferative disorders (PLDs) are histologically divided into a neoplastic state of high and low grade malignant lymphoma (ML), and a reactive state of follicular bronchitis/bronchiolitis (FB) and lymphoid interstitial pneumonia (LIP). We reviewed 19 cases with PLDs, including 4 cases each of high and low grade B cell ML, 6 FB cases, and 5 cases of LIP. To clarify the clonality of the proliferating cells, we performed an immunohistochemical examination (IHC), in situ hybridization (ISH) for the immunoglobulin light chain and a polymerase chain reaction (PCR) analysis of the immunoglobulin heavy chain gene using DNA obtained from paraffin sections. In addition, a Southern blot analysis was also performed in 6 cases using fresh materials. In IHC, all ML were positive for L26 (CD20), while the monoclonality of the kappa light chain was observed in only one high grade case. However, using ISH we could detect the clonality in three of four high grade ML cases and in one of four low grade ML cases. In FB and LIP, no clonality of immunoglobulin by ISH was observed. In a PCR analysis for the immunoglobulin heavy chain gene, we could detect one or two prominent bands in all 8 cases of high and low grade ML. On the other hand, in all cases of FB and LIP, we could only detect either an oligoclonal or polyclonal population. In summary, the presence of monoclonality of ISH and/or PCR for the immunoglobulin heavy chain gene were limited in the neoplastic state, but not in the reactive state.     

Clonality of Primary Pulmonary Lymphoproliferative Disorders; Using in Situ Hybridization and Polymerase Chain Reaction for Immunoglobulin

Primary pulmonary lymphoproliferative disorders (PLDs) are histologically divided into a neoplastic state of high and low grade malignant lymphoma (ML), and a reactive state of fol-licular bronchitis/ bronchiolitis (FB) and lymphoid interstitial pneumonia (LIP). We reviewed 19 cases with PLDs, including 4 cases each of high and low grade B cell ML, 6 FB cases, and 5 cases of LIP. To clarify the clonality of the proliferating cells, we performed an immunohis-tochemical examination (IHC), in situ hybridization (ISH) for the immunoglobulin light chain and a polymerase chain reaction (PCR) analysis of the immunoglobulin heavy chain gene using DNA obtained from paraffin sections. In addition, a Southern blot analysis was also performed in 6 cases using fresh materials. In IHC, all ML were positive for L26 (CD20). while the monoclonality of the kappa light chain was observed in only one high grade case. However, using ISH we could detect the clonality in three of four high grade ML cases and in one of four low grade ML cases. In FE3 and LIP, no clonality of immunoglobulin by ISH was observed. In a PCR analysis for the immunoglobulin heavy chain gene, we could detect one or two prominent bands in all 8 cases of high and low grade ML. On the other hand, in all cases of FB and LIP, we could only detect either an oligoclonal or polyclonal population. In summary, the presence of monoclonality of ISH and/or PCR for the immunoglobulin heavy chain gene were limited in the neoplastic state, but not in the reactive state.     

原位杂交技术在淋巴瘤研究中的应用

原位杂交是在组织切片或细胞涂片上检测核酸分子的一种方法，它是病理学研究的重要手段。本文介绍原杂交的特点及其在淋巴瘤研究中的应用，包括病毒感染、染色体异常、淋巴瘤相关基因及转录体的检测。     

Detection of Epstein-Barr virus in gastric carcinoma cells and surrounding lymphocytes

Background. Epstein-Barr virus (EBV), the etiological agent of infectious mononucleosis, has an important role in the oncogenesis of EBV-related malignant diseases. The association of EBV with gastric carcinoma cells has become well known recently, but there are only a few reports concerning its association with surrounding epithelia and infiltrating lymphocytes. In this study, we investigated the association of EBV with gastric carcinoma and surrounding cells. Methods. One hundred and two cases of gastric carcinoma were studied. The specimens were studied for the presence of the EBV genome by polymerase chain reaction (PCR), and then by in situ hybridization (ISH) technique to determine the localization of EBV. Results. Of 97 informative cases, EBV was detected in 21 cases (21.6%) by the PCR method. ISH studies showed that EBV RNA was expressed in 5 of the 97 cases (5.2%) and was localized to the nuclei of carcinoma cells. All these 5 lesions were found in male patients. In these 5 cases, 3 were diffuse type and 2 were intestinal type, and all cases arose in the proximal region of the stomach. EBV RNA was not detected in non-neoplastic epithelia, but it was detected in 24 of the 97 cases (24.7%) in small lymphocytes. Conclusion. EBV was detected in 5.2% of gastric carcinomas and in 24.7% of infiltrating lymphocytes by the ISH method. The high positive rate (21.6%) by the PCR method corresponds to the presence of the EBV genome in surrounding lymphocytes.     

Rapid Diagnosis of Gastric Malignant Lymphoma from Biopsy Specimens: Detection of Immunoglobulin Heavy Chain Rearrangement by Polymerase Chain Reaction

The endoscopic appearances of the gastrointestinal lymphomas differ widely, and it is often difficult to make the distinction between a benign lymphoproliferative disorder and a malignant lymphoma even with a histologic evaluation. Since almost all primary malignant lymphomas of the gastrointestinal tract are of B-cell origin, the confirmation of monoclonality in immunoglobulin (Ig) is helpful for differential diagnosis. Rearrangements of the Ig heavy chain gene were examined by polymerase chain reaction (PCR) analysis in frozen biopsy specimens of human stomach. The sensitivity of the analysis was sufficient to detect even a 5% clonal B-cell proliferation and results could be obtained within 17 h. In a clinical investigation, seven of eight cases (88%) of primary gastric malignant lymphoma showed a single band in polyacrylamide gel electrophoresis (PAGE) after PCR, suggesting a monoclonal proliferation of B-cell lineage. By contrast, all seven cases of reactive lymphoreticnlar hyperplasias showed a broad smear pattern in PAGE, which is thought to reflect polyclonal proliferation. None of the lymphocytes infiltrating around gastritis (7 cases), gastric ulcers (12 cases) and gastric carcinomas (15 cases) showed a monoclonal proliferation pattern. These findings suggest that detection of monoclonality in Ig heavy gene rearrangement by PCR is useful for the differential diagnosis of B-cell lymphoproliferative diseases in the gastrointestinal tract.     

乳腺癌前哨淋巴结CK19 mRNA和蛋白的表达

目的 探讨CK19微转移分子在乳腺癌前哨淋巴结(SLN)的表达情况及意义.方法 采用免疫组化S-P法及原位杂交技术检测66例SLN中CK19微转移分子的表达.同时与常规病检法比较其检测敏感性.并比较转移组、微转移组、无转移组患者的临床资料.结果 66例SLN在常规病理检查阴性38例淋巴结中6例表达CK19蛋白15.8%(6/38),8例21.1%(8/38)表达CK19mRNA.原位杂交法与常规病检法转移的检出率相比较,差异有统计学意义(P＜0.05).CK19蛋白和mRNA表达之间差异无统计学意义(P＞0.05).同时乳腺癌转移组与微转移组患者在肿物大小与淋巴管浸润上有相似性,而同无转移组差异有统计学意义(P＜0.05).结论 免疫组化法和原位杂交法较常规病理检查更为敏感.通过免疫组化法和原位杂交法的联合使用,可明显提高乳腺癌SLN微转移的检出率,同时也证明原位杂交法是可靠的,比免疫组化更为敏感的技术.SLN微转移有可能作为肿瘤预后的指标.     

Epstein-Barr virus is seldom found in mammary epithelium of breast cancer tissue using in situ molecular methods

Epstein–Barr virus (EBV) has been proposed as a possible etiological agent of breast cancer based on 21 reports of EBV in malignant breast tissues. Most of these studies used standard and nested solution polymerase chain reaction (PCR) techniques, both disadvantaged by susceptibility to contamination from laboratory EBV, and the inability to localize the signal to a specific cell type. To avoid these issues, we used in situ molecular methods of viral detection to reassess the frequency of EBV in malignant breast tissue. We used a commercial in situ hybridization (ISH) system with an EBER genome target, and a non-commercial in situ PCR (IS-PCR) method using primers specific for the BamH1 region. The assays were performed on malignant breast tissue sections from 70 breast cancer patients at the M. D. Anderson Cancer Center, Houston, TX. EBV was found in mammary epithelial cells, the cell type from which most breast cancers arise, in 2/70 (2.9%) of specimens using IS-PCR and in none of the specimens using ISH. Based on these findings that EBV was present in human mammary epithelial cells so infrequently, it is unlikely to play a causative role in most types of breast cancer.     

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.     

171例淋巴瘤病变组织中EB病毒表达情况的分析

目的:探讨不同类型淋巴瘤与EB病毒(Epstein-Barr virus,EBV)感染的关系。方法:收集淋巴瘤组织171例,包括弥漫大B细胞淋巴瘤(DLBC)106例;结外NK/T细胞淋巴瘤,鼻型22例;霍奇金淋巴瘤(HL)19例;血管免疫母细胞性T细胞淋巴瘤(AITL)13例;黏膜相关淋巴组织B细胞淋巴瘤(MALT)11例。应用EBV Lmp-1单抗免疫组化(IHC)和生物素标记的EBER1寡核苷酸探针原位杂交(ISH)分析EBV感染与淋巴瘤的关系。结果:淋巴瘤组织中EBV Lmp-1蛋白与EBER1 mRNA总阳性率分别为11.1%(19/171)、25.7%(44/171)。其中AITL为30.8%(4/13)、61.5%(8/13);HL为47.4%(9/19)、57.9%(11/19);结外NK/T细胞淋巴瘤为22.7%(5/22)、81.8%(18/22);DLBC为0.94%(1/106)、5.7%(6/106);MALT为0(0/11)、9.1%(1/11)。结果显示EBV在DLBC及MALT中的表达率低于AITL、HL及结外NK/T细胞淋巴瘤,差异有统计学意义(P0.05);且原位杂交检测EBER1 mRNA比免疫组化检测Lmp-1蛋白更为敏感(P0.01)。结论:EBV感染与淋巴瘤有密切关系,不同类型淋巴瘤与EBV感染的关系有差异。     

The prognostic significance of overexpression of the decoy receptor for Fas ligand (DcR3) in patients with gastric carcinomas

Abstract. Background: The FasL-Fas system has an important role in mediating immune-cytotoxic killing of cells such as virus-infected or tumor cells. It was recently reported that there is a soluble decoy receptor (DcR3), which binds to FasL and inhibits FasL-induced apoptosis, and certain tumors may escape FasL-dependent immune-cytotoxic attack by expressing a decoy receptor that blocks FasL. We evaluated whether DcR3 has clinical relevance in actual human gastric cancers. Methods: . The expression of DcR3 was investigated by Northern blot analysis in a series of 84 primary gastric carcinomas and compared with clinicopathological features and prognosis. The DcR3 expression level was analyzed and quantified densitometrically. The location of DcR3 mRNA in gastric carcinoma tissue was detected by in situ hybridization. Results: The frequency of DcR3 overexpression was 26% (22 of 84 surgical specimens). The DcR3 expression level was significantly associated with lymph node metastasis and pathological stage, but did not correlate with tumor size, metastatic status, or histological type. In situ hybridization demonstrated that DcR3 mRNA was expressed in tumor cells. When the patients were followed up for 63 months, DcR3 overexpression was found to be associated with a significantly shortened duration of overall survival compared with findings in patients having normal DcR3 expression. Conclusion: The DcR3 decoy receptor for FasL may be involved in the progression of gastric cancer. Further evaluation of these possible roles of DcR3 and the regulation of DcR3 expression in malignant cells will be critically important for the development of new strategies for controlling the growth of malignant cells that escape host immune surveillance. Received: July 19, 2001 / Accepted: December 21, 2001     

Long-term persistence of monoclonal B cells after cure of Helicobacter pylori infection and complete histologic remission in gastric mucosa-associated lymphoid tissue B-cell lymphoma.

PURPOSE: Cure of Helicobacter pylori infection is associated with remission induction in the majority of patients with low-grade gastric mucosa associated lymphoid tissue (MALT) lymphoma in localized stages; however, limited data exist as to whether these patients may be cured of their lymphoma. The present study was performed to investigate whether the polymerase chain reaction (PCR) for the rearranged immunoglobulin heavy chain region may be used to define "molecular" remission. PATIENTS AND METHODS: Ninety-seven patients who suffered from low-grade gastric MALT lymphoma stage I(E) were observed with central pathology and molecular biology after cure of H pylori infection. PCR was performed with the use of consensus primers for the framework regions 1, 2, and 3 and monoclonality was corroborated by sequence analysis. In selected cases, microdissection was performed to study the origin of the monoclonal B cells. RESULTS: Of the 97 patients, 77 obtained complete endoscopic and histologic remission (CR). Twenty of 44 patients with PCR monoclonality at diagnosis and with sufficient molecular follow-up displayed monoclonal bands for a median time of 20.5 months after CR (range, 0 to 50.4 months). These B cells were related to the original lymphoma clone by sequence analysis. Microdissection analysis identified basal lymphoid aggregates as the source of these monoclonal B cells. Local relapse occurred in and was observed by PCR in four patients. All four patients displayed monoclonal PCR before relapse, and three of these four showed ongoing PCR monoclonality throughout their course, indicating the persistence of malignant cells. CONCLUSION: Half of all patients with gastric MALT lymphoma show long-term PCR monoclonality up to several years after cure of H pylori infection and CR. Patients with monoclonal PCR should be observed closely, whereas long-term PCR negativity may indicate cure of the disease.     

胃原发性恶性淋巴瘤31例临床分析

目的 探讨胃原发性恶性淋巴瘤临床及内镜表现。方法 回顾性总结本院近十年来31例原发性胃恶性淋巴瘤的临床资料。结果 31例病人临床表现无特异性，上腹痛最常见，其次为上消化道出血等。胃镜表现：病变发生2个以上部位多见，其次为胃体、胃窦等，幽门螺杆菌（Hp）阳性检出率90．3％。结论 胃原发性恶性淋巴瘤临床表现无特异性；其镜下形态多种多样，其中累及2个以上部位者多见，病变范围较大。胃镜活检加免疫组化病理检查是诊断本病的重要手段，Hp感染与胃原发性恶性淋巴瘤的发生具有相关性。     

鼻NK/T细胞淋巴瘤的免疫表型、EBV感染及TCRγ基因重排的检测

 目的 检测鼻NK/T细胞淋巴瘤（NK/TCL）的免疫表型、EBV感染及TCRγ基因重排,为诊断和鉴别诊断提供依据。方法 收集诊断鼻NK/TCL48例患者石蜡包埋标本,用免疫组化SP法标记LCA、CD79α、CD20、CD56、CD3、CD45RO及EBV抗体研究其免疫表型;EBER探针原位杂交方法检测EBV编码的小分子RNA（EBER）;聚合酶链式反应扩增方法检测TCRγ基因重排。结果 48例鼻NK/TCL均表达LCA,CD3、CD45RO、CD56和EBV阳性率分别为44%、52%、73%和19%,CD79α和CD20均阴性;EBER阳性率为81%;TCRγ基因重排阳性率为19%。结论 鼻NK/TCL免疫表型不一致,并非所有病例CD56阳性,石蜡切片中CD3阳性定位于细胞质;EBER在肿瘤细胞中高表达,提示它们可能为NK细胞来源;部分TCRγ基因重排阳性病例应为鼻NK样T细胞淋巴瘤。     

Clinical relevance of telomerase activity in primary gastric lymphoma

Background. To distinguish between low-grade lymphoma and reactive lymphoid infiltrate can be demanding for pathologists. Demonstration of B-cell monoclonality by polymerase chain reaction (PCR) analysis of immunoglobulin heavy chain is useful as referential data. Telomerase activity, which is frequently detected in malignant tumors while being undetectable in normal tissues, may also have a supportive role in the diagnostic procedure, but has not been investigated in specimens of primary gastric lymphoma. Methods. Telomerase activity was qualitatively and quantitatively evaluated, using fluorescence-based telomeric amplification assay protocol (TRAP) analysis, in 16 malignant lymphoma and related specimens, including 7 specimens of low-grade mucosa-associated lymphoid tissue (MALT) lymphoma, among which specimens before and after Helicobacter pylori eradication were evaluated from five patients. Results. Telomerase activity was detected more frequently (6 of 7 specimens) at higher levels in high-grade lymphoma compared with low-grade MALT lymphoma. It was detected in 3 of 7 specimens of low-grade MALT lymphoma, but was undetectable either at the stage of Helicobacter pylori -induced gastritis or after H. pylori eradication therapy. Conclusion. Telomerase activity evaluated by fluorescence-based TRAP analysis was detectable late in the process of evolution of MALT lymphoma and was undetectable early in the process of regression. It was frequently and overtly elevated in high-grade lymphoma. Telomerase activity may have a role in confirming the histologic diagnosis of primary gastric lymphoma. Received: March 16, 2000 / Accepted: May 16, 2000     

Aspiration biopsy-nucleic acid diagnosis of thyroid malignant lymphoma by vectorette PCR: experience of eight cases

The usefulness of a new method to detect B-cell monoclonality named VR, which is a combination of the digestion with restriction enzymes and vectorette PCR, in aspiration biopsy-nucleic acid diagnosis (ABND) of thyroid malignant lymphoma was investigated. ABND was performed in 12 patients in whom malignant lymphoma was suspected. Among the patients who underwent open surgery, monoclonality was detected in the aspirates in four (50%) of eight patients with malignant lymphoma, but was not detected in one patient with Hashimoto disease. ABND using VR is a useful adjunct of aspiration biopsy cytology in the diagnosis of thyroid malignant lymphoma.