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.     

An immunohistological study of reactive lymphoid tissue

The aim of this study was to document the patterns of cytoplasmic Ig heavy and light chain expression in reactive lymphoid tissue, using single and double immunoenzymatic labelling techniques. This investigation was undertaken, firstly, to provide information on whether the normal counterparts of high grade lymphoma cells (e.g. centroblasts, immunoblasts) ever express more than one light or heavy chain (as has been noted in the past for lymphomas) and also, secondly, to seek evidence of intraclonal 'switching' from cytoplasmic IgM to cytoplasmic IgG expression. Paraffin embedded sections, all showing substantial reactive changes, were analysed by means of immunoperoxidase stains for the three major immunoglobulin classes (IgG, IgM and IgA), both light chain classes and J chain. In addition, double immunoenzymatic labelling techniques were used to search for cells showing simultaneous expression of kappa and lambda light chains and cells expressing mu and gamma heavy chain. Large transformed lymphocytes showing cytoplasmic Ig-staining in the pulp and interfollicular areas often have nuclear morphology indistinguishable from germinal centre centroblasts. There was no evidence of primitive appearing IgM-positive cells and IgG-positive cells of more mature morphology. In addition, immunoenzymatic staining showed that cells simultaneously expressing both IgG and IgM are only rarely encountered. When such cells were detected, the morphology was not that of a blast cell, but rather of a plasma cell containing Russel bodies. Hence it is suggested that cytoplasmic IgM switching to IgG is rarely detected by immunohistological methods in reactive tissue. Double staining for kappa and lambda revealed that cells simultaneously expressing both light chain types were not detected even among cells showing the most primitive morphology.     

Detection of immunoglobulin light chain mRNA in nodular sclerosing Hodgkin''s disease by in situ hybridization with biotinylated oligonucleotide probes compared with immunohistochemical staining with poly- and monoclonal antibodies

In order to elucidate the origin of the Hodgkin's and Reed-Sternberg cells, the expression of immunoglobulin kappa- and lambda light chain mRNA in 23 cases of nodular sclerosing and two cases of mixed cellularity Hodgkin's disease was examined by in situ hybridization using biotinylated oligonucleotide probes and compared with immunohistochemical staining with mono- and polyclonal antibodies against immunoglobulin kappa- and lambda light chains. No hybridization signals were seen in Hodgkin's or Reed-Sternberg cells in any of the cases. Polyclonal staining with polyclonal anti-immunoglobulin light chain antibodies was seen in Hodgkin's and Reed-Sternberg cells in 12 cases of nodular sclerosis and in two cases of mixed cellularity and with monoclonal antibodies in three cases of nodular sclerosis, but in no cases of mixed cellularity. In all cases, there was polyclonal labelling of plasma cells with both the oligonucleotide probes and the antibodies. In five cases, the Hodgkin's and Reed-Sternberg cells were also stained with one of the B-cell antibodies L26, MB2 or LN1. Lack of mRNA signals in Hodgkin's and Reed-Sternberg cells might indicate that these cells in Hodgkin's disease of the nodular sclerosis subtype are either not B-cell derived or they are early B-cells (precursor B-cells) not yet able to produce immunoglobulin light chain mRNA, at least not at a level detectable by in situ hybridization. Immunohistochemical staining of Hodgkin's and Reed-Sternberg cells, however, with antibodies against immunoglobulin kappa and lambda light chains may be explained by cellular uptake of the light chains, but the difference in reactivity between poly- and monoclonal antibodies cannot be explained at present.     

The immunochemical characterization of the light chains in the mesangial IgA deposits in IgA nephropathy

The immunochemical characterization of the light chains of the mesangial immunoglobulin A (IgA) deposits were studied in 45 patients with IgA nephropathy. Kappa and lambda light chains were detected with direct immunofluorescence (IF) method, using monospecific rabbit anti-human anti-kappa and anti-lambda anti-sera. The glomeruli of 42 renal biopsies studied were strongly positive for lambda light chain, while only 25 specimens were positive for kappa light chain. Sixty-five percent of the biopsies showed a predominance of lambda light chain IF staining in the mesangial deposits. This IF pattern is unique as compared with similar studies on renal biopsies from patients with systemic lupus erythematosus, idiopathic membranous nephropathy, and normal postmortem renal tissue. The results indicate that mesangial IgA deposits in IgA nephropathy consist mainly of IgA with lambda light chains despite the fact that the normal ratio of kappa to lambda light-chain-containing immunoglobulin in human serum is two to one.     

Zonal distribution of immunoglobulin-synthesizing cells within the germinal centre: an in situ hybridization and immunohistochemical study

Immunohistologic studies have shown that synthesis of cytoplasmic immunoglobulin (cIg) is a normal function of some follicle centre cells (FCCs). The mechanisms regulating this synthesis of immunoglobulin and its function within the germinal centre are still poorly understood. In this study we applied a recently developed in situ hybridization method for the detection of kappa and lambda light chain mRNA to reactive lymph nodes and tonsils in order to investigate further the immunoglobulin-synthesizing cells of the germinal centre. FCCs containing detectable levels of light chain mRNA corresponded closely to cells containing cIg. The detection of light chain mRNA rather than its immunoglobulin product was found to be an advantage in that problems associated with the detection of extracellular immunoglobulin were eliminated. This was most apparent in germinal centres where the absence of 'network' immunoglobulin led to the observations that immunoglobulin-synthesizing FCCs are predominantly small centrocytes and that in a proportion of germinal centres they localize in that part of the light zone closest to the dark zone. This zonal distribution of immunoglobulin-synthesizing FCCs raises the possibility of further functional and micro-environmental subcompartments within the light zone.     

Comparison of monoclonal and polyclonal antibodies directed against immunoglobulin light and heavy chains in non-Hodgkin's lymphoma

A study comparing the usage of monoclonal and polyclonal antibodies specific for immunoglobulin light and heavy chains was performed on frozen-tissue sections of 30 B-cell non-Hodgkin's lymphomas. In 16 cases, monotypic staining for an immunoglobulin light chain was demonstrated with monoclonal antibodies using a three-step avidin-biotin peroxidase complex (ABC) method; 13 cases were positive for kappa. In 14 cases, no immunoglobulin light-chain production was demonstrated. Repeat staining of 11 of these 14 cases with polyclonal anti-sera by a direct immunoperoxidase method demonstrated monotypic staining for light chain in 10 cases, 9 of which were positive for lambda. In 22 of 30 non-Hodgkin's lymphomas, an immunoglobulin heavy chain was identified using monoclonal anti-sera. In eight cases, however, no heavy chain was found. Repeated staining with polyclonal sera of additional sections in three of eight cases demonstrated heavy-chain production in each case. Decreased sensitivity, especially for the detection of the lambda light chain, rendered this particular lot of monoclonal antibodies unsuitable for immunophenotyping non-Hodgkin's lymphomas. Variability of antigenic sites on the immunoglobulin molecule seems a likely explanation for these observations.     

Absence of B-cell or T-cell clonal expansion in nodular, lymphocyte predominant Hodgkin's disease

Recent studies based upon immunophenotypic data have provided strong evidence that nodular lymphocyte predominant Hodgkin's disease (NLPHD) represents an entity that is distinct from other subtypes of Hodgkin's disease (HD). In contract to other forms of HD, the predominance of B-lymphocytes in NLPHD has prompted the thesis that this lesion is actually an atypical B-cell hyperplasia or follicular center cell lymphoma. Three cases of NLPHD by restriction endonuclease analysis were studied in an attempt to identify a clonal B-cell or T-cell expansion in this disorder. DNA was extracted from these tumors and hybridized to probes for the immunoglobulin genes (C kappa, C lambda, JH) and the T-cell receptor beta chain gene. Gene rearrangements were not detectable in any of the cases. The results provide genotypic evidence that there is not a monoclonal or oligoclonal proliferation of small B-lymphocytes or T-lymphocytes in NLPHD. The possibility that the L&H Reed-Sternberg cells are monoclonal cannot be excluded because their small number is below the level of sensitivity of this technique.     

Detection of immunoglobulin light-chain mRNA in lymphoid tissues using a practical in situ hybridization method.

The identification of immunoglobulin protein in routinely fixed and paraffin-embedded sections using antibodies combined with immunoperoxidase or similar techniques of detection is often problematic. We developed an in situ hybridization methodology for the identification of light-chain mRNA that is applicable to formalin-fixed, paraffin-embedded tissues, using either radiolabeled or biotinylated oligonucleotide probes based on the kappa and lambda light-chain gene-constant regions. Reactive plasma cells can be consistently identified in reactive lymphoid tissues, and a monotypic pattern of light-chain mRNA restriction was seen in each of eight cases of multiple myeloma/plasmacytoma. Immunoblasts and germinal center cells also are labeled in reactive lymphoid tissues. Using 355-labeled probes, 29 of 93 cases (30%) of non-Hodgkin's lymphomas had detectable light-chain mRNA, while 19% of non-Hodgkin's lymphomas were positive using biotinylated probes.     

Immunohistochemical study of immunoglobulin light chain amyloidosis with antibodies to the immunoglobulin light chain variable region

To detect immunoglobulin (Ig) light chain amyloidosis (AL amyloidosis) in formalin-fixed, paraffin-embedded tissue sections by immunohistochemistry, polyclonal antibodies were generated against synthetic peptides corresponding to amino acids 1-19 of the Ig lambda light chain V lambda VI subgroup (anti-V lambda VI (1-19)) and the Ig kappa light chain Vkappa I subgroup (anti-Vkappa I (1-19)). Anti-V lambda VI (1-19) antibody reacted with amyloid deposits in 21 of 22 Alambda amyloidosis cases, and anti-Vkappa I (1-19) antibody reacted with amyloid deposits in 10 of 11 Akappa amyloidosis cases. Immunoreactivity varied in intensity by case and within specimens. Surprisingly, amyloid deposits were positive for anti-V kappa I (1-19) staining in one case of Alambda amyloidosis. Analysis of anti-V lambda VI (1-19) and anti-Vkappa I (1-19) antibody reactivity by ELISA showed some cross-reactivity with peptides other than antigen peptides. The antibodies were not reactive in all cases of AL amyloidosis examined but may be useful, together with anti-Ig constant region antibodies, for immunohistochemical diagnosis of AL amyloidosis.     

Multicentric giant lymph node hyperplasia: An immunohistochemical study

Biopsy specimens from five cases of multicentric giant lymph node hyperplasia were studied by standard histochemical techniques and by immunoperoxidase staining and double immunoenzyme labeling to determine the distribution of intracytoplasmic immunoglobulins and kappa and lambda light chains. Microscopically, the affected lymph nodes showed a nodular pattern characterized by multiple lymphoid follicles permeated by numerous small vessels. A striking proliferation of post-capillary venules with many plasma cells and immunoblasts was observed in the interfollicular areas. Immunoperoxidase staining revealed that the cells were positive for IgG, IgA, and IgM with both kappa and lambda chains in the normal ratio. The IgM-positive cells had a perifollicular distribution, whereas the IgG- and IgA-positive cells were located mainly in interfollicular areas. The presence and distribution of different classes of intracytoplasmic immunoglobulins seemed to reflect a normal, albeit tumultuous, immunologic response. Therefore, the disease can be considered a lymphoproliferative disorder due to an inappropriate immunologic reaction.     

Useful polyclonal antibodies against synthetic peptides corresponding to immunoglobulin light chain constant region for immunohistochemical detection of immunoglobulin light chain amyloidosis

For the immunohistochemical detection of immunoglobulin (Ig) light chain amyloidosis on formalin-fixed, paraffin-embedded tissue sections, we prepared polyclonal antibodies against synthetic peptides corresponding to positions 118-134 of Ig lambda light chain and positions 116-133 of Ig kappa light chain. Nineteen cases of systemic Ig lambda light chain amyloidosis (Alambda amyloidosis), 10 cases of systemic Ig kappa light chain amyloidosis (Akappa amyloidosis), one case of immunohistochemically unclassified systemic amyloidosis and five cases of localized Alambda amyloidosis were tested with these antibodies. Anti-lambda (118-134) antiserum and the affinity-purified antibody both reacted with 18 of the 19 cases of systemic Alambda amyloidosis and all cases of localized Alambda amyloidosis, although the immunoexpression was somewhat variable in intensity in different areas within the same specimen in both systemic and localized amyloidosis. The signal intensities in plasma cells and serum reacted for anti-lambda (118-134) antiserum were weaker than signals obtained with commercially available anti-Ig lambda light chain antibodies. Anti-kappa (116-133) antiserum and the affinity-purified antibody reacted with nine of the 10 cases of systemic Akappa amyloidosis. We conclude that these antibodies against synthetic peptides corresponding to the Ig light chain constant region are useful for the classification of amyloidosis on formalin-fixed, paraffin-embedded tissue sections.     

Immunoglobulin G, A, and M Light Chain Ratios in some Humoral Immunological Disorders

The total kappa/lambda immunoglobulin light chain ratio and the kappa/lambda ratios within each of the serum immunoglobulin classes G, A, and M were measured in thirteen patients with humoral immunological disorders. Of those patients, eight had common variable immunodeficiency whereas five patients had other forms of humoral immunological deficiencies. Eleven patients had abnormal antibody response in vivo. All but three of the thirteen patients had clearly abnormal light chain ratios in one or more of the immunoglobulin classes. We conclude that humoral immunological disorders, usually characterized by abnormal heavy chain production and a disturbed antibody response, may frequently have a concomitant abnormal synthesis of the light chains resulting in an abnormal kappa/lambda light chain ratio.     

Restricted kappa/lambda light chain ratio by flow cytometry in germinal center B cells in Hashimoto thyroiditis

To determine the diagnostic significance of the kappa/lambda ratio in germinal center (GC) B cells in Hashimoto thyroiditis (HT), we used 4-color flow cytometry to immunophenotype 27 samples (21 patients) of well-characterized HT B-cell clonality was analyzed further by polymerase chain reaction (PCR) of the immunoglobulin heavy chain (IgH) and bcl-2/IgH fusion genes using DNA extracted from aspirate smears and/or paraffin-embedded tissues. By flow cytometric analysis, the CD10+ GC B cells had a higher mean +/- SD kappa/lambda ratio than the CD10- B cells (5.1 +/- 3.3 vs 2.0 +/- 0.8; P < .0001, Student t test). In 18 samples (67%), CD10+ GC B cells had a kappa/lambda ratio greater than 3.07 (the upper limit of kappa/lambda ratio reported in reactive nodes; range, 3.2-14.4 in the 18 cases). Cases tested by PCR showed no evidence of a clonal proliferation. None of 21 cases developed lymphoma during clinical follow-up of up to 3 years. The kappa/lambda ratio of CD10+ GC B cells in HT can be skewed markedly beyond that reported in reactive lymph nodes. This finding frequently is present in HT. Pathologists should be familiar with this phenomenon to prevent misdiagnosis of follicular lymphoma in patients with HT.     

Receptor revision plays no major role in shaping the receptor repertoire of human memory B cells after the onset of somatic hypermutation.

In order to determine whether V gene replacement accompanies somatic hypermutation in the germinal center (GC) reaction in the human, we analyzed V(kappa)J(kappa) and V(lambda)J(lambda) joints and the kappa-deleting element in single lambda(+) naive and post GC B cells for rearrangements at the kappa and lambda loci. Among 265 lambda(+) post GC B cells, not a single unequivocal and only two potential examples of a cell that switched to lambda light chain expression after accumulation of (unfavorable) mutations in its productive V(kappa) rearrangement were observed. Taking the PCR efficiency into account, the frequency of such cells is likely below 3 %. In addition, heavy and light chain gene rearrangements were amplified and sequenced from the oligoclonal population of IgD-only peripheral blood post GC B cells which display extensive intraclonal sequence diversity. Among 61 IgD-only B cells belonging to 15 clones with intraclonal diversity, no combination of V gene rearrangements indicating receptor revision during clonal expansion was observed. Moreover, among 124 and 49 V(H) genes amplified from IgD-only and class-switched B cells, respectively, not a single example of V(H) revision through V(H) hybrid generation was detected. These results suggest that in the human GC reaction V gene replacement either does not usually accompany somatic hypermutation or is mostly counterselected.     

Systemic lymphoproliferative disorder with morphologic features of Castleman's disease. Immunoperoxidase study of cytoplasmic immunoglobulins

Lymph node biopsy specimens from nine patients with a systemic lymphoproliferative disorder morphologically resembling Castleman's disease were studied for the presence of cytoplasmic immunoglobulins. After trypsinization of tissue sections, the avidin-biotin-peroxidase method was used to stain for IgG, IgA, and IgM heavy chains and kappa and lambda light chains. All cases showed polyclonal proliferation of plasma cells. The predominant immunoglobulin class observed was IgG. The IgM-containing cells tended to be localized concentrically around germinal centers. No notable changes were seen in multiple biopsy specimens from five patients. Polyclonal staining was also observed in lymph nodes obtained at autopsy from a patient who died of a disseminated atypical immunoproliferative process. The polyclonal staining pattern in these cases confirms the reactive nature of this systemic lymphoproliferative disorder.     

Generation of heterogeneous rabbit anti-DNP antibodies by gene conversion and hypermutation of rearranged VL and VH genes during clonal expansion of B cells in splenic germinal centers

The mechanisms described here account for development of the heterogeneous high-affinity anti-DNP antibodies that rabbits can produce. Rearranged immunoglobulin light and heavy chain genes from single DNP-specific splenic germinal center B cells were amplified by PCR. We found that in clonal lineages, rearranged V[kappa] and V[H] are further diversified by gene conversion and somatic hypermutation. The positive and negative selection of amino acids in complementarity-determining regions observed allows emergence of a variety of different combining site structures. A by-product of the germinal center reaction may be cells with sequences altered by gene conversion that no longer react with the immunizing antigen but are a source of new repertoire. The splenic germinal center would thus play an additional role in adults similar to that of the appendix and other gut-associated lymphoid tissues of young rabbits.     

Primary lymphomas of the liver. Report of six cases and review of the literature

Six cases of diffuse large cell lymphoma (DLCL) of the liver were studied with immunohistochemistry for common leukocyte antigen (CLA), lysozyme, alpha-1-antitrypsin (AAT), and kappa and lambda light chains on paraffin-embedded tissues. All six cases were positive for CLA. Four of the six cases showed staining for lysozyme and AAT (three focal and one diffuse staining). In three cases, frozen tissue for monoclonal antibodies and glutaraldehyde-fixed tissue for electron microscopic examination were available. Two of these showed B-cell phenotypes with monoclonal antibody studies. Electron microscopic examination on these two B-cell lymphomas showed scant cytoplasm and a paucity of cytoplasmic organelles. The third case did not show definite B- or T-cell surface markers but did show strong Leu-M1 and OKM1 staining. Electron microscopic examination of the tumor cells showed a prominent Golgi apparatus, abundant cytoplasm with numerous cytoplasmic organelles and phagolysosomes. However, DNA hybridization studies on this tumor showed immunoglobulin heavy and kappa light chain gene rearrangements typical of a B-cell lymphoma. All six lymphomas were solitary liver masses without evidence of disease elsewhere. The mean age for the six patients was 56.2 years (four males, two females).     

The tumor cells in nodular lymphocyte-predominant Hodgkin disease are clonally related to the large cell lymphoma occurring in the same individual. Direct demonstration by single cell analysis

Large cell lymphoma (LCL) sometimes occurs concurrently or subsequently in patients with nodular lymphocyte-predominant Hodgkin disease (NLPHD). Although there is evidence of a clonal relationship between LCL and NLPHD, there has been no direct demonstration that the lymphocytic and histiocytic (L&H) cells in NLPHD are related to the tumor cells in LCL. We identified 2 cases of NLPHD with an associated LCL. Single L&H cells, the Reed-Sternberg cell variants in NLPHD, were isolated from immunostained tissue sections by micromanipulation, and the immunoglobulin heavy chain gene (IgH) complementarity determining region (CDR) III of the cells was amplified by the polymerase chain reaction (PCR). The products were compared with those obtained from microdissected LCL cells using polyacrylamide gel electrophoresis and nucleotide sequencing. The IgH CDRIII sequences from the L&H cells were related to each other, but also showed nucleotide substitutions, consistent with a germinal center origin. The sequences from the L&H cells also were related to those from the corresponding LCL cells. We have provided direct evidence through sequence analysis of the IgH CDRIII that the L&H cells are clonally related to the corresponding LCL arising in 2 cases of NLPHD.