Immunohistochemical comparison of CD5, lambda,and kappa expression in primary and recurrent buccal Mucosa-associated lymphoid tissue (MALT) lymphomas

Mucosa-associated lymphoid tissue (MALT) lymphoma is a type of extranodal marginal zone B-cell lymphoma and is a distinct subtype of non-Hodgkin's lymphoma.Primary MALT lymphomas can also occur in the oral cavity, although their appearance in this location is rare. The neoplastic cells of which MALT lymphomas are composed express B-cell antigens and show monotypic immunoglobulin expression with light-chain restriction.Although neoplastic MALT lymphoma cells do not express CD5, previous studies have shown that CD5 positive MALT lymphomas are more prone to dissemination than those that do not express CD5. Moreover, there are some reports that describe kappa- and lambda- dual light chain expression in B cell malignant neoplasms.A 66-year-old Japanese woman with swelling of the right buccal mucosa was referred to our hospital. The lesion was excised and was pathologically diagnosed as a MALT lymphoma tumor with a t(11;18)(q21;q21) chromosome translocation.Swelling of the right buccal mucosa recurred 2 years later. The recurrent tumor was then excised and pathologically diagnosed as MALT lymphoma.Immunohistochemical examination of CD5, lambda, and kappa expressions revealed that the primary tumor was positive for CD5, kappa, and lambda, but the recurrent tumor was weakly positive for CD5 and kappa.With respect to lambda positivity, the recurrent tumor showed negativity.Our study suggests that immunohistochemical expression of CD5, kappa, and lambda in oral MALT lymphoma have the risk of recurrence.We first described the recurrence of CD5 positive MALT lymphoma in the oral cavity and compared the immunohistochemical expressions of CD5, lambda, and kappa between the primary and recurrent tumors.     

Morphologic and immunocytochemical evaluation of 220 fine needle aspirates of malignant lymphoma and lymphoid hyperplasia

A total of 220 fine needle aspiration (FNA) specimens from 212 patients with clinically suspected or previously histologically confirmed lymphoma were evaluated by cytology in conjunction with immunophenotyping analysis of the aspirate; the results were compared with the histologic diagnosis made on previous or current accessions of lymph node or extranodal tissue. Smears of the aspirates were stained with the Diff-Quik and Papanicolaou stains while immunoperoxidase staining using antibodies against kappa and lambda immunoglobulin light chains and Leu-4 was routinely performed on Cytospin preparations. Where indicated, additional marker studies (including T-200, Leu-1, Leu-2a, Leu-3a + 3b, Leu-M1, B1, Leu-12, IgM, CALLA and TdT) were performed. For the non-Hodgkin's lymphomas, specimens were classified by the cytologic characteristics of the neoplastic cells according to the International Working Formulation scheme. The combination of cytologic smears and immunoperoxidase studies resulted in a diagnosis of lymphoma in 173 cases (79%). The remaining aspirates were interpreted as suspicious for lymphoma (7%), benign (10%) or inadequate for diagnosis (4%). Of the 15 suspicious aspirates, 5 proved to be Hodgkin's disease and 2 to be T-cell lymphoma by subsequent biopsy. The cause of failure in the nine inadequate aspirates were necrosis (3 cases), sclerosis (2 cases) and faulty technique (4 cases). In the cases that had concurrent tissue biopsies, no false-positive diagnoses were rendered. These results indicate that FNA used in association with immunocytochemistry is a reliable tool for establishing the diagnosis and classification of the majority of cases of lymphoma. Optimal immunoglobulin light-chain ratios for defining monoclonality in FNA specimens of B-cell lymphomas are proposed.     

Detection of intracytoplasmic immunoglobulin by flow cytometry in B-cell malignancies

The aim of this study was to compare the results of flow cytometric (FCM) determination of heavy and light chain cytoplasmic immunoglobulin (cIg) with those obtained by the peroxidase-antiperoxidase (PAP) method. Fifty-one patients, including five non-T-acute lymphoblastic leukemias, 16 B-chronic lymphocytic leukemias (CLL), 13 non-Hodgkin's lymphomas, seven hairy cell leukemias, four multiple myeloma/plasma cell leukemias, and six T-cell leukemia/lymphomas, as well as 12 normal controls, were studied. Saponin-permeabilized cell suspensions were indirectly stained with monoclonal antibodies and analyzed by flow cytometry. Acetone-fixed cytocentrifuge smears were stained for cIg by the PAP method. The results obtained indicate that: (a) detection of cIg by FCM is a feasible and useful technique to confirm the B-cell lineage of leukemias and lymphomas, particularly those characterized by low-density surface immunoglobulin, such as CLL; and (b) cIg detection by FCM and PAP staining are complementary methods to recognize with certainty the monoclonality of B-cell malignancies.     

Three spontaneous lymphomas in a colony of cotton-top tamarins (Saguinus oedipus)

Cotton-top tamarins are well known for their prevalence to idiopathic colitis and adenocarcinomas. At the same time, information on the incidence of spontaneous lymphomas in this highly endangered species is rare. Records, 212 in total, of cotton-top tamarins (Saguinus oedipus) necropsied at the German Primate Centre between 1979 and 1998 were viewed to establish the prevalence of lymphoid neoplasms. Neoplastic lymphoid cell growth was mentioned in three necropsy records. Immunohistology was performed in all three cases on the remaining formalin-fixed, paraffin-embedded tissue using antibodies against CD20, CD3, lysozyme, Ki-67, IgM, IgG, kappa, lambda and EBNA-2. Combining histological and immunohistological results, the lymphomas could be differentiated into two low-grade T-cell lymphomas and one high-grade multicentric polymorphic B-cell lymphoma. This corresponds to a 1.4% incidence of lymphomas in our cotton-top tamarin population over a period of 19 years. Although frozen material was not available and virological testing could not be carried out, clinical or histological evidence did not support an aetiological role of Herpes (H.) saimiri, H. ateles, simian T-cell leukaemia virus type 1 (STLV-1) or Epstein-Barr-related herpesvirus in any of these cases. The lymphomas were considered to be spontaneous.     

Antigen-induced murine B cell lymphomas. I. Induction and characterization of CH1 and CH2.

Two unusual murine lymphomas, designated CH1 and CH2, were produced in the newly developed double congenic strain of mice, B10 H-2a H-4b p/Wts. Both tumors lack the T cell-specific antigen (thy-1), but express cell surface immunoglobulin and the H-2K, H-2D, and Ia specificities determined by the H-2a haplotype. Further studies have demonstrated that these tumors represent "early" B cells in that they express surface IgM (mu heavy and lambda light chains), but do not bear surface delta, gamma, or alpha heavy chains. CH1 and CH2 lack surface C3 receptors and results from assays for Fc receptors have proven variable. A competition radioimmunoassay directed against the gp71 group-specific antigen of Friend leukemia virus has shown that there is a murine leukemia virus associated with these tumors, however, we have been unable to establish a causal relationship between the virus and this malignancy. A comparison of the surface characteristics of these tumors with other mammalian B cell lymphomas is presented.     

Long-term-cultured mouse B-lymphocyte line. II. Modulation of Ia-antigen expression on B-lymphocyte line

Mouse B-cell line, established by culturing anti-Thy-1 and complement-treated splenic B cells with concanavalin A-stimulated conditioned medium, expressed immunoglobulins and Ia antigens on its surface. The long-term-cultured B-cell line was split in two and maintained with or without 3300 R X-irradiated T-cell-depleted syngeneic splenic adherent cells (SAC). Interestingly, the B-cell line cultured without SAC lost its Ia antigen but not its Ig expression, whereas the cell line with SAC maintained both Ia and Ig expression. The ability to express Ia antigens was restored by culturing them only in the presence of Ia-positive feeder cells. Neither recombinant interferon-gamma or lectin-stimulated conditioned medium nor cell-free culture supernatant SAC had the ability to restore Ia antigen expression on the B-cell line. Incubation of Ia-negative B-cell line with phorbol esters restored the Ia expression. It is suggested that the expression of Ia antigen on B lymphocytes was controlled differently from that on macrophage lineage. The B-cell line expressing Ia antigens acts as stimulator cells for alloantigen-activated T lymphocytes and as antigen-presenting cells on the KLH-specific Ia-restricted proliferative T-cell clone in the presence of a specific antigen.     

Histopathology of the spleen in non-Hodgkin's lymphoma

The human spleen has several different lymphoid compartments, which may be preferentially involved by non-Hodgkin's lymphomas. Low grade B-cell lymphomas tend to involve the compartments of the spleen where their normal physiological counterparts are found. High grade B-cell lymphomas grow more destructively without respecting lymphoid compartments. T-cell lymphomas involve mainly the T-cell areas, but may also involve follicles and the red pulp. Since non-Hodgkin's lymphomas can be regarded as clonal proliferations of lymphocytes frozen at a stage in differentiation, one possible conclusion is that low grade lymphomas are governed by normal homing mechanisms. High grade lymphomas may have lost their homing properties.     

Differentiating large cell lymphoma from indolent small B-cell lymphoma in fine needle aspirates using p53, PCNA and transformed lymphocyte count

OBJECTIVE: To determine the usefulness of proliferating cell nuclear antigen (PCNA), p53 protein expression and transformed lymphocyte count (TLC) as adjunctive tests to differentiate indolent small B-cell lymphoma from large cell lymphoma in fine needle aspiration biopsies. STUDY DESIGN: Aspirates of lymphoproliferative disorders from April 1993 to January 1997 were reviewed. The percentage of TLCs was determined on the Papanicolaou smear. The percentage and intensity of p53 and PCNA immunocytochemical staining was evaluated on cell block sections. These results were compared and correlated with the final diagnoses based on available morphology, flow cytometry and clinical history. RESULTS: There were 40 cases of non-Hodgkin's lymphoma and 12 reactive lymph nodes. Adequate cell blocks were available on 16 large cell lymphomas, 7 grade 1-2 follicular center cell lymphomas, 6 mucosal associated lymphoid tissue lymphomas, 2 small lymphocytic lymphomas and 2 mantle cell lymphomas. Average TLC and p53 nuclear staining was highest in large cell lymphomas (57% TLC and 24% p53), followed by grades 1 and 2 follicular lymphomas (14% TLC and 15% p53) and lowest in other indolent lymphomas (< 10% TLC and < 1% p53). Average PCNA staining was highest in large cell lymphomas (46%) and lowest in small lymphocytic lymphomas (7%); however, TLC was the best parameter for differentiating large cell lymphoma from indolent small B-cell lymphoma. CONCLUSION: TLC differentiated large cell lymphoma from indolent small B-cell lymphoma better than either p53 or PCNA alone or in combination. Significant overlap between categories limits usefulness of these immunocytochemical stains for differentiating these entities.     

Expression of v-rel induces mature B-cell lines that reflect the diversity of avian immunoglobulin heavy- and light-chain rearrangements.

The infection of newly hatched chickens with reticuloendotheliosis virus strain T (REV-T) and a nonimmunosuppressive helper virus, chicken syncytial virus, induces rapidly metastatic B-cell lymphomas. In vivo analysis of these tumors with monoclonal antibodies detected the expression of the B-cell surface markers immunoglobulin M (IgM), CIa, Bu2, and CLA-1, but not IgG, Bu1, or a T-cell surface marker, CT-1. Cell lines derived from tumors exhibited the same pattern of staining, suggesting that expression of cell surface markers does not change during in vitro cell line development. All cell lines examined synthesized IgM in varying amounts. Northern (RNA blot) analysis confirmed abundant expression of v-rel mRNA, and Southern analysis revealed rearrangement of both heavy- and light-chain immunoglobulin loci. Analysis of the light-chain locus demonstrated that 20 of 22 lines contained a single rearranged allele. With respect to specific restriction enzyme sites within the V lambda 1 gene, the active allele in any given clone was either diversified or nondiversified. In contrast, examination of the heavy-chain loci within these lines demonstrated that 16 of the 22 had both alleles rearranged. Further diversification of the V lambda 1 locus did not occur after prolonged in vitro passage of the cell lines. We propose that v-rel expression arrests diversification of the light-chain locus in these lymphoid cells, allowing the production of stable, clonal B-cell populations. The development of these and similar cell lines will make it possible to identify specific stages of avian lymphoid ontogeny and to study the mechanism of rearrangement and diversification in the avian B lymphocyte.     

B cell abnormalities induced by a mu Ig transgene extend to L chain isotype usage

We have analyzed the phenotype of B cell populations from mice transgenic for a rearranged Ig mu H chain gene. We find a decrease in the number of B cells in the spleens of these mice. Transgenic B cells have decreased surface levels of both IgM and IgD. The circulating IgM in these mice is 3- to 10-fold enriched in lambda L chains, compared with that in non-transgenic mice. Analysis of IgM-producing hybridomas, from transgenic mice that express the transgene at high levels, demonstrates that this higher lambda frequency is observed in transgene-nonexpressing as well as transgene-expressing hybridomas. A partial loss of L chain isotype exclusion is also noted in these hybridomas, and a significant proportion of primary B cells expressing both kappa and lambda L chains on their surface can be demonstrated. These findings suggest an ability of the transgenic Ig H chain to affect events in B cell ontogeny beyond the H chain locus. Our results support a quantitative model of exclusion for both the H chain alleles and the L chain isotypes.     

Evaluation of tissue preparation methods and paired immunofluorescence staining for immunocytochemistry of lymphomas

Eight cross-linking fixatives were tested for preservation of extracellular or intracellular IgG, IgA, IgM, IgD, kappa and lambda light chains, J chain and secretory component. Most of the selected fixatives have been used in recent immunohistochemical studies of lymphoproliferative processes and comprised routine formalin, glutaraldehyde(1%)-formalin, Baker's formalin-calcium, formalin-sublimate, acetic acid(2%)-formalin-saline, Bouin's fluid, Susa fixative, and carbodiimide. The results obtained in artificial test substrates with defined amounts of IgG or IgA and in biological substrates (colon mucosa, tonsils, and different types of lymphomas) were compared by immunofluorescence with the antigenic preservation afforded by fixation in cold 96% ethanol (with or without inclusion of a pre-fixation 48 h washing period). An antigen concentration at least an eight-fold higher was necessary for detection with most other fixatives. Bouin's and Susa fixatives were peculiar in that they required antigen concentration 150 times higher for detection of IgG but only 3-8 times higher for IgA. Light chains were relatively well preserved by all fixatives except glutaraldehyde. For all cross-linking fixatives, the extent of antigenic masking depended on the concentration of environmental proteins, and the efficiency of unmasking with pronase or trypsin, therefore, varied with the location in the tissue. The J chain was particularly vulnerable to degradation during proteolytic treatment. The extensive masking of extracellular immunoglobulin in formalin-fixed tissue afforded a relatively good signal-to-noise ratio for immunoglobulin-producing cells when kappa and lambda chains were traced. Thus, differentiation between polyclonal and monoclonal B-cell processes on the basis of cytoplasmic labelling was often better in undigested sections. However, the light-chain type of membrane immunoglobulin could usually not be determined in directly fixed tissue. Ethanol fixation preceded by washing in saline afforded such determination and also preserved certain T-cell and HLA-DR antigens as well as diffuse alpha-naphthylbutyrate esterase. Reactive and malignant macrophages could further be traced by their cytoplasmic expression of L1 antigen, both in formalin- and ethanol-fixed material.     

The effects of cytokines and adherent cells on the interleukin 4-mediated induction of Ia antigens on resting B cells

In this report we have extended our previous studies on interleukin 4 (IL-4) [previously termed B-cell stimulatory factor-1 (BSF-1)]. Our results demonstrate that 8 hr of exposure to IL-4 is sufficient to induce maximal expression of Ia antigens. This increase in expression of Ia antigens on resting B cells is due to the direct action of IL-4 on the B cells since adding or removing adherent cells or utilizing low density cultures of B cells at 50-100/culture had no effect on the IL-4-mediated increase in Ia. Monoclonal anti-IL-4 antibody completely abrogated the Ia-inducing activity of IL-4. A variety of other purified lymphokines including interleukin 2 (IL-2), interleukin 1 (IL-1), and a source of either B-cell differentiation factor for IgM (BCDF mu), or B-cell growth factor II (BCGF II), did not alter the expression of Ia antigens on resting B cells. However, interferon-gamma can partially inhibit the IL-4-mediated induction of Ia.     

Nanoscale mapping and organization analysis of target proteins on cancer cells from B-cell lymphoma patients

CD20, a membrane protein highly expressed on most B-cell lymphomas, is an effective target demonstrated in clinical practice for treating B-cell non-Hodgkin's lymphoma (NHL). Rituximab is a monoclonal antibody against CD20. In this work, we applied atomic force microscopy (AFM) to map the nanoscale distribution of CD20 molecules on the surface of cancer cells from clinical B-cell NHL patients under the assistance of ROR1 fluorescence recognition (ROR1 is a specific cell surface marker exclusively expressed on cancer cells). First, the ROR1 fluorescence labeling experiments showed that ROR1 was expressed on cancer cells from B-cell lymphoma patients, but not on normal cells from healthy volunteers. Next, under the guidance of ROR1 fluorescence, the rituximab-conjugated AFM tips were moved to cancer cells to image the cellular morphologies and detect the CD20-rituximab interactions on the cell surfaces. The distribution maps of CD20 on cancer cells were constructed by obtaining arrays of (16×16) force curves in local areas (500×500 nm²) on the cell surfaces. The experimental results provide a new approach to directly investigate the nanoscale distribution of target protein on single clinical cancer cells.     

IgM expressed by leukemic CD5(+) B cells binds mouse immunoglobulin light chain

Mouse immunoglobulin (Ig) molecules have previously been shown to bind to the surface of CD5(+) B cells from patients with B-cell chronic lymphocytic leukemia (B-CLL). The results indicated that surface IgM was involved in the interaction and suggested the phenomenon was an example of the polyreactive binding capacity of the surface Ig (sIg) expressed by these malignant cells. This article describes the further characterization of the interaction between human IgM and mouse Ig molecules and subunits. Mouse Ig molecules of both kappa and lambda light chain classes bound to the B-CLL cell surface. The dissociation constant for the interaction of mouse IgG1 (K121) with the B-CLL cell surface was 3.6 x 10(-7) M. To confirm the involvement of the human IgM expressed by the B-CLL cells in the interaction, the malignant cells were stimulated in vitro to induce secretion of human IgM. Enzyme immunoassay was used to show that secreted human IgM bound to intact mouse Ig, as occurred with the cell surface analysis. The mouse Ig epitope recognized by the purified secreted human IgM was shown by Western blot analysis to be located on the light chain of the mouse Ig molecule and to be conformationally dependent. K121 light chain was cloned and expressed in E. coli and the recombinant light chain bound to the surface of CLL B cells. The results confirm that human IgM is the reactive ligand in the interaction with mouse Ig and indicate that the interaction of polyreactive IgM with mouse IgG occurs via the light chain component of IgG.     

Long-term-cultured mouse B-lymphocyte line. I. Establishment and characterization of mouse B-lymphocyte line

Mouse B-cell line was established by culturing anti-Thy-1 antibody and complement-treated splenic B cells with the conditioned medium of concanavalin A-stimulated spleen cell-culture supernatant. At the eighth week of culture, it was revealed that 100% of the long-term-cultured cells had both cytoplasmic and surface immunoglobulin. These cells were then maintained in the conditioned medium together with T-cell-depleted splenic and then splenic adherent feeder cells. Flow cytometric studies of the B-cell line showed that they had surface mu, delta, and kappa but no gamma, lambda, Lyt-1, or Lyt-2. The growth of the B-cell line was dependent on the factor(s) derived from concanavalin A-stimulated conditioned medium. It was found that IL-2 was the major factor supporting the B-cell growth. The B-cell line did not secrete immunoglobulin spontaneously, but it could differentiate into antibody-forming cells through the stimulation of bacterial lipopolysaccharides. The technique for obtaining mouse B-cell lines are reproducible in our laboratory and one of those lines has been propagated and maintained for 16 months to the present.     

Restricted Ig variable region gene expression among Ly-1+ B cell lymphomas

The majority of the characterized Ly-1+ B cell lymphomas of B10.H-2aH-4bp/Wts origin (the CH series) bear surface Ig related by Ag specificity or idiotype or both. To determine the genetic basis for these structural similarities, we have sequenced the VH and VL region genes expressed by 10 CH lymphomas, and have compared their VH and V kappa gene rearrangements by Southern blot analysis to one another and to those of four other CH lymphomas. Sequence analysis identified only five different VH, and seven different VL genes, and indicated that these V genes are essentially unmutated. CH lymphomas which express the identical VH gene share at least one idiotope. Thus, the basis for shared idiotype and specificity is due in most cases to the use of the same V gene. This restriction in V gene expression is not due to the preferential use of V genes of any particular VH family or VL group, as the expressed V genes belong to four different VH families and four V kappa groups, and include V lambda 1 and V lambda 2. We hypothesize that Ag selection accounts for the restriction in V gene usage among CH lymphomas.     

Extinction of B-cell surface differentiation markers in hybrids between murine B-lymphoma and myeloma cells

Fusion between murine B-lymphoma cells bearing membrane IgM with either IgG or light chain secreting myeloma, resulted in cell hybrids synthesizing and secreting large quantities of IgM. In contrast, the hybrids did not secrete IgD even though it is also present on the surface of the B-lymphoma cells. B-Cell surface markers such as the IgM, IgD, Ia and the Fc receptor, which were present on the B-lymphoma cells, but not the myeloma cells were not expressed on the surface of the hybrids. Hybrids which secrete IgM and retain the B-cell membrane differentiation antigens were not detected, even when selection was done under conditions which favor the growth of the lymphoma parental cells.     

The poised B cell: lymphokines induce an Ia-increase and antigen-presenting function in B cells

Individual murine B cells express a wide range of Ia densities on the plasma membrane. Here we demonstrate that a dramatic increase in B-cell Ia could be induced by overnight exposure to an uncharacterized lymphokine (LK). Membrane I-A and I-E molecules were both increased after LK treatment, whereas membrane IgM remained unchanged. Two subpopulations of B cells were identified, based on their requirements for expressing maximal Ia; one subpopulation required only LK, the other required both LK and T cells in the overnight culture. Functional changes accompanied the Ia increase. The functional capacity to present antigens to T cells was lacking in normal resting B cells, but was acquired following LK treatment. We suggest that the LK-treated B cell has achieved a new differentiation state, one of preparation for interaction with T cells. We term this state the "poised" B cell, and propose that B cells in the poised state may significantly contribute to T-cell activation as antigen-presenting cells. Moreover, poised B cells may themselves find an advantage over normal B cells in successfully acquiring T-cell help.     

Ly1+ PRO-B lymphocyte clones. Phenotype, growth requirements and differentiation in vitro and in vivo.

Several clones obtained from the bone marrow of a BALB/c mouse were found to contain the heavy and light chain Ig genes in the germline configuration, to express Ly1 and to carry the B cell lineage markers B-220, Lyb8 and BP-1; these clones are Pgp-1+, LFA-1+, J11d+, Mac-1+ and Thy1-, Lyt2-, L3T4-, GM1.2- and Ia-. Three clones analyzed in detail (Lyd9, LyH7 and Lyb9) have receptors for interleukin (IL) 2 and IL3 as assessed with the 7D4 and CC11 monoclonal antibodies respectively. They grow in rIL3 but not in rIL2 or rIL1; both rIL4 and rIL5 also promote their proliferation, albeit to a much lesser extent than rIL3. None of the interleukins tested alone or in various combinations promoted the clones to differentiate in vitro along the B cell pathway. Treatment with 5-Azacytidine (5-Aza) induced cell surface Ia expression but not rearrangement or expression of Ig genes. However, 5-Aza-treated Lyd9, LyH7 and Lyb9 cells co-cultured with X-ray irradiated accessory cells and LPS gave rise to Ly1+, IgM+ B lymphocytes (range 14-51%) including mu + kappa + (78-93%), and mu + lambda + (9-25%) B lymphocytes. In vivo, the Lyd9, LyH7 and Lyb9 clones gave rise to IgM+ B lymphocytes (8.5-17%) including mu + kappa +, and mu + lambda +, but not to Lyt2+ or L3T4+ T lymphocytes after 4-6 weeks of transfer into Scid mice. Our results indicate that Ly1+ IgM+ cells comprise a subpopulation of B lymphocytes that is derived from IL3-responsive Ly1+ PRO-B lymphocytes.