Localization of immunoglobulin light chain mRNA expression in Hodgkin's disease by in situ hybridization

In situ hybridization techniques were used to detect immunoglobulin light chain messenger RNA (mRNA) in 28 formalin-fixed, paraffin-embedded samples of Hodgkin's disease. Cocktails of biotinylated oligonucleotide probes specific for the constant regions of kappa and lambda light chain mRNA were used. None of the Reed-Sternberg cells or their variants in any of the cases studied showed positive staining with either probe, in contrast to normal plasma cells which showed strong staining in the same sections. It was concluded, therefore, that the cytoplasmic immunoglobulin frequently detected within these cells by immunocytochemistry is present not as a result of synthesis, but as a result of some other mechanism.     

Combination of Hodgkin's disease and diffuse large cell lymphoma: an in situ hybridization study for immunoglobulin light chain messenger RNA

It is not clear whether the rare combination of Hodgkin's disease with non-Hodgkin lymphomas are true composite lymphomas or differentiation stages of one tumour cell clone. We used in situ hybridization and immunohistochemistry for the demonstration of immunoglobulin light chains in order to investigate the relationship between the two lymphoma components. In three cases of nodular lymphocyte predominance Hodgkin's disease combined with diffuse large B-cell lymphoma the Hodgkin cells, as well as the tumour cells in the diffuse large B-cell lymphoma, showed the same messenger RNA for one light chain. Thus, using in situ hybridization in nodular lymphocyte predominance Hodgkin's disease combined with diffuse large B-cell lymphoma in a small number of cases a possible genetic relationship between the two components could be shown. In nodular sclerosis combined with diffuse large B-cell lymphoma, in situ hybridization did not support a common clonal origin of both tumour parts. However, a unique clonal derivation cannot be excluded by the techniques applied.     

Serum free light chain analysis in multiple myeloma and plasma cell dyscrasias

After the development of a reliable method to detect free light chains in serum, several investigations have been conducted to explore their importance in plasma cell dyscrasias (PCD). Detection of monoclonal proteins is very important in the diagnosis and management of PCD, which include a broad spectrum of diseases such as multiple myeloma and also benign, premalignant disorders like monoclonal gammopathy of undetermined significance. The aim of this article is to summarize the recent studies and to highlight the importance of free light chain analysis in the diagnosis of PCD, its prognostic value and role in the management of this group of diseases.     

In situ hybridization of immunoglobulin light chain mRNA in paraffin sections using biotinylated or hapten-labelled oligonucleotide probes

An in situ hybridization technique has been developed for the detection of immunoglobulin light chain mRNA in routine pathology specimens. The method detects kappa or lambda constant region sequences using a cocktail of synthetic oligonucleotide probes labelled with biotin or fluorescein 5-isothiocyanate (FITC) reporter molecules. The probes were labelled at flanking sites chemically by primary amine directed acylation and by 'homopolymer tailing' with terminal deoxynucleotidyl transferase using non-radioactive nucleotide analogues. The mRNA was unmasked in the formalin-fixed tissue sections by digestion with varying concentrations of proteinase K, and the hybrids were demonstrated using alkaline phosphatase with either a streptavidin/biotin based four-stage system or an anti-FITC antibody based detection system. Alkaline phosphatase was visualized using a Fast Red naphthol-capture method and the sections were counterstained with haematoxylin. The results confirm that the method is specific for kappa or lambda mRNA and show that specific mRNAs can be detected in routine formalin-fixed sections using non-radioactive techniques with retention of good morphology. The method reliably detects light chain mRNA in cells expressing secretory immunoglobulin. The protocol can also be applied to tissue rich in endogenous biotin by using hapten-labelled probes.     

Serum free light chain analysis in multiple myeloma and plasma cell dyscrasias

After the development of a reliable method to detect free light chains in serum, several investigations have been conducted to explore their importance in plasma cell dyscrasias (PCD). Detection of monoclonal proteins is very important in the diagnosis and management of PCD, which include a broad spectrum of diseases such as multiple myeloma and also benign, premalignant disorders like monoclonal gammopathy of undetermined significance. The aim of this article is to summarize the recent studies and to highlight the importance of free light chain analysis in the diagnosis of PCD, its prognostic value and role in the management of this group of diseases.     

原位杂交对胃粘膜相关组织淋巴瘤的免疫球蛋白轻链的检测

目的 评价mRNA原位杂交方法检测免疫球蛋白轻链限制性在胃粘膜相关组织淋巴瘤诊断中的应用价值。方法 用非同位素标记寡核苷酸mRNA探针原位杂交方法检测27例原发怀胃粘膜相关淋巴瘤和5例慢性胃炎,对其中的肿瘤细胞、淋巴细胞及浆细胞的免疫球蛋白κ和λ轻链的比例进行分析,以确定肿瘤细胞的轻链限制性及浆细胞的单克隆性。结果 27例原发性胃粘膜相关淋巴瘤中有10例出现轻链限制性（37％）,其中5例（5／9）为低度恶性病例,5例（5／18,28％）为高度恶性病例,单克隆性的浆细胞只在低度恶性病例中检出。慢性胃炎标本及肿瘤旁组织中未检测出轻链限制性。结论 mRNA原位杂交方法检测轻链限制性是诊断胃淋巴瘤的一个有用工具,低度恶性淋巴瘤中单克隆性浆细胞的存在,可作为诊断早期胃粘膜相关淋巴瘤的依据之一。     

用细胞原位杂交方法检测NPY mRNA在PC12细胞中的表达

目的：建立ＮＰＹ ｍＲＮＡ物细胞原位杂交检测方法,并以ＮＧＦ为诱导因子研究Ｄｅｘ对大鼠嗜铬细胞瘤ＰＣ１２细胞中ＮＰＹｍＲＮＡ表达的影响。方法：采用细胞原位杂交方法。方法：ＮＧＦ具有诱导ＮＰＹ基因表达的生物学效应且呈剂量效应关系;Ｄｅｘ对ＮＰｘＲＮＡ表达具有双相调节效应,即早期（〈８ｈ）可促进ＮＧＦ的诱导作用,而晚期（〉１６ｈ）则具有抑制作用（Ｐ〈０．０１）,但单独Ｄｅｘ对ＮＰＹｍＲＮＡ表达无显著影     

Translocation (2;14) associated with complex rearrangements of the Ig heavy chain in non-Hodgkin lymphoma.

Cytogenetic analysis of a patient with non-Hodgkin lymphoma revealed the following karyotype: 49,XXX,t(2;14)(q21;q32),+4,+8,del(13)(q14q21). Southern blot analysis with an Ig region probe showed non-productive rearrangements indicative of a translocation involving the Ig locus. However, molecular cloning of the abnormal rearrangements did not show novel sequences derived from chromosome 2 but showed that the BCL-6 gene was juxtaposed to the IgH enhancer. Three further clones with abnormal rearrangements involving the Ig locus, particularly Iggamma3, were isolated. This suggests that the mature lymphoid cells, in this patient, were capable of undergoing indiscriminate switch cleavage and religation.     

Detection of immunoglobulin light chain mRNA by in situ hybridisation using biotinylated tyramine signal amplification.

A highly sensitive method for the light microscopic in situ hybridisation of immunoglobulin light chain mRNA in formalin fixed, paraffin wax embedded sections is reported. This method is based on signal amplification using horseradish peroxidase catalysed deposition of biotinylated tyramine at the sites of hybridisation. kappa and lambda light chain immunoglobulin mRNA in situ hybridisation was performed with fluorescein isothiocyanate conjugated oligonucleotide probe cocktails. The hybridisation signal was detected using a biotinylated tyramine signal amplification procedure with streptavidinbiotin-horseradish peroxidase complex as the final layer. Peroxidase was demonstrated using 3,3'-diaminobenzidine. The biotinylated tyramine signal amplification method resulted in the sensitive detection of immunonoglobulin light chain mRNA, with the whole procedure being completed in one day. Moreover, the use of peroxidase as the final reporter molecule also allowed haemamatoxylin to be used as counterstain, thereby permitting the evaluation of cellular morphology.     

Detection of immunoglobulin light chain mRNA by in situ hybridisation using biotinylated tyramine signal amplification.

A highly sensitive method for the light microscopic in situ hybridisation of immunoglobulin light chain mRNA in formalin fixed, paraffin wax embedded sections is reported. This method is based on signal amplification using horseradish peroxidase catalysed deposition of biotinylated tyramine at the sites of hybridisation. kappa and lambda light chain immunoglobulin mRNA in situ hybridisation was performed with fluorescein isothiocyanate conjugated oligonucleotide probe cocktails. The hybridisation signal was detected using a biotinylated tyramine signal amplification procedure with streptavidinbiotin-horseradish peroxidase complex as the final layer. Peroxidase was demonstrated using 3,3'-diaminobenzidine. The biotinylated tyramine signal amplification method resulted in the sensitive detection of immunonoglobulin light chain mRNA, with the whole procedure being completed in one day. Moreover, the use of peroxidase as the final reporter molecule also allowed haemamatoxylin to be used as counterstain, thereby permitting the evaluation of cellular morphology.     

Nodular lymphocyte-predominant Hodgkin's disease: study of immunoglobulin light chain protein and mRNA expression.

The techniques of immunohistochemistry and in situ hybridization were applied to 10 formalin- or B5-fixed, paraffin-embedded cases of nodular lymphocyte-predominant Hodgkin's disease to determine whether lymphohistiocytic (L&H) cells contain any detectable amount of immunoglobulin light chain protein or messenger RNA. None of the cases studied demonstrated any detectable amount of either kappa or lambda light chain mRNA within L&H cells or Reed-Sternberg cells despite positive labeling of plasma cells, immunoblasts, and germinal center cells. Polyclonal kappa light chain antibody studies showed positive staining of L&H cells in seven cases, including three costaining with polyclonal lambda light chain antibody. Monoclonal kappa and lambda light chain antibody studies, however, showed no staining of L&H cells despite positive staining of immunoblasts and plasma cells. It is suggested that L&H cells do not synthesize appreciable amounts of light chain immunoglobulin protein and are not closely related to reactive immunoblasts or germinal center cells.     

Determination of light chain restriction in fine-needle aspiration-type preparations of B-cell lymphomas by mRNA in situ hybridization.

The purpose of this study was to investigate whether B-cell clonality could be demonstrated in fine-needle aspiration (FNA)-type preparations by using automated and manual in situ hybridization (ISH) systems. FNA-like preparations were made from 10 cases of B-cell lymphoma and 5 cases of reactive lymphoid hyperplasia. Kappa/lambda expression was determined using an automated mRNA ISH assay or a manual ISH system. Other variables tested included type and length of fixation, protease digestion, and time in chromogen. Clonality data were corroborated by either flow cytometry or tissue-based analysis. Optimal conditions required formalin fixation, strong protease digestion, and prolonged hybridization and chromogen times; under these conditions, monoclonality was demonstrated by in situ in 8 of 10 cases. Each of the five cases of reactive lymphoid hyperplasia showed polyclonal light chain expression by automated mRNA ISH. In situ kappa/lambda mRNA analysis of FNA-type specimens allows direct determination of monoclonality in cytologic preparations.     

Determination of HER2 gene amplification by chromogenic in situ hybridization (CISH) in archival breast carcinoma.

PURPOSE: To compare the efficacy of chromogenic in situ hybridization (CISH(TM)) with fluorescence in situ (FISH) hybridization and immunohistochemistry (IHC) in determination of the HER2 status in human breast cancer. MATERIALS AND METHODS: HER2 gene amplification was determined on formalin-fixed paraffin-embedded (FFPE) sections of 62 invasive breast cancers by FISH and followed by CISH using a digoxigenin (DIG)-labeled HER2 DNA probe generated by Subtraction Probe Technology (SPT(TM)), and a biotin-labeled chromosome 17 centromeric (chr.17cen) probe. The sections were heat treated and enzyme digested. After in situ hybridization, the HER2 probe was detected with fluorescein (FITC)-anti-DIG for FISH, followed by peroxidase-anti-FITC and diaminobenzidine (DAB) for CISH. The chr.17cen probe was detected with peroxidase-streptavidin and DAB. For CISH application, HER2 gene copies or chromosome 17 centromeres and morphology of cells were easily visualized simultaneously with a 40x objective under bright-field microscope in hematoxylin-counterstained sections. IHC study of HER2 overexpression was performed on adjacent sections using a panel of three HER2 antibodies (TAB 250, CB11, A0485), and staining was scored according to the criteria specified in the HercepTest. RESULTS: HER2 gene amplification detected by CISH was visualized typically as large DAB-stained clusters or by many dots in the nucleus. FISH and CISH identified HER2 gene amplification in 19% of the tumors. Chromosome 17 polysomy was detected in 31% of the tumors. HER2 overexpression was demonstrated in 19% (TAB 250), 23% (CB11), and 36% (A0485) of the tumors. Complete concordance between the results of CISH with FISH, TAB 250, CB11, and A0485 was seen in 100%, 97%, 94%, and 84% of the cases, respectively. CONCLUSION: By permitting observation of morphology using a bright-field microscope, CISH is an accurate, practical, and economical approach to screen HER2 status in breast cancers. It is a useful methodology for confirming ambiguous IHC results.     

Chromogenic in situ hybridization (CISH) to detect HER2 gene amplification in breast and gastric cancer: Comparison with immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH)

The chromogenic in situ hybridization (CISH) assay, designed to detect the amplification of the HER2 gene in formalin‐fixed, paraffin‐embedded (FFPE) breast cancer (BC) and gastric cancer (GC) tissue specimens, was evaluated in 125 FFPE BC cases and 198 FFPE GC cases for which the HER2 status had been predetermined using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). In the 125 BC cases and the 198 gastric cases, we found a very good concordance (98.4% and 99.0%, respectively) between CISH and FISH. In particular, we evaluated the polysomy cases, as these cases often have ambiguous treatment options in clinical practice. The polysomy of chromosome 17 was defined as the presence of three or more CEP17 signals in at least 10% of the tumor cells. In the 50 BC cases and 54 GC cases displaying chromosome 17 polysomy, the concordance between FISH and CISH was 98.0% and 98.1%, respectively. These results indicate that CISH could provide an accurate and practical alternative to FISH for the clinical diagnosis of HER2 gene amplification in FFPE BC and FFPE GC samples.     

聚合酶链反应技术检测非霍奇金淋巴瘤抗原受体基因重排的局限性

病理形态学和免疫组织化学有效标记,结合分子病理学的检测是当前临床病理诊断的发展趋势。近年来这种三结合的诊断模式已在淋巴瘤的病理诊断中得到应用。各类淋巴瘤的病理形态学特征是病理诊断的基石,免疫组织化学有助于正确的诊断及分类,仅靠这二者,恶性淋巴瘤的误诊率仍较高,采用分子病理学技术检测病理标本中淋巴瘤特征性的分子标记具有重要的参考价值。其中运用聚合酶链反应（PCR）技术检测IgH／TCR基因的重排是目前常用的淋巴瘤的分子病理学的方法之一。     

Identification of MYCN gene amplification in neuroblastoma using chromogenic in situ hybridization (CISH): an alternative and practical method.

Chromogenic in situ hybridization (CISH) is a recently developed technique, which utilizes the general principles of in situ hybridization and a detection system similar to immunohistochemistry. To assess the utility of CISH for analysis of MYCN gene amplification, we compared this assay with established diagnostic assays such as Southern blot analysis (SB) and fluorescent in situ hybridization (FISH). CISH was performed on 67 cases of neuroblastoma using tissue microarray (65 cases) and whole tissue sections (2 cases). Unequivocal, high-level amplification (> or =10 gene copies per tumor nucleus) was identified in 19 of 67 (28.4%) tumors. Two (3%) tumors showed low-level amplification (6-9 gene copies per tumor nucleus). No amplification was seen in 46 of 67 (68.6%) tumors. SB data were available in 44 tumors. Forty-one of the 44 tumors (93%) showed concordant results between CISH and SB. Three tumors showed MYCN amplification by CISH but no amplification by SB, most likely due to dilution effect of nonneoplastic tissue in the test samples. Two of these three tumors also showed MYCN amplification by FISH, and the third tumor was not analyzed by FISH. FISH data were available in total of 30 tumors. All 30 tumors showed concordant results between CISH and FISH for classifying a tumor as MYCN amplified or not amplified. We conclude that CISH is an accurate method for determining MYCN gene amplification, with added advantages that make it a more practically useful method.