Utility of Leu M1 monoclonal antibody in the differential diagnosis of Hodgkin's disease

We conducted a retrospective study of the utility of Leu M1 monoclonal antibody staining of paraffin-embedded tissue in the differential diagnosis of Hodgkin's disease (HD). Forty-two cases of HD of various histologic types and 33 cases of non-HD lymphomas and hyperplasias were stained with Leu M1 using the avidin-biotin-peroxidase complex technique. Varying numbers, but not all Reed-Sternberg (RS) and Hodgkin's cells in all 42 cases of HD, were Leu M1 positive. These cases included seven examples of interfollicular HD and nine cases of lymphocyte-predominance HD, eight of which were nodular. Four of five cases of immunologically proved T-cell lymphoma contained Leu M1-positive RS-like cells, and Leu M1-positive RS-like cells were noted in two of five cases of non-HD lymphoma that were not phenotyped but were morphologically consistent with T-cell lymphoma. We concluded that Leu M1 staining is an aid in the diagnosis of HD, but it cannot be used to differentiate HD from T-cell lymphoma containing RS-like cells.     

Leu M1 and S100 in Hodgkin's disease and non-Hodgkin's lymphomas

Leu M1 positivity of Reed-Sternberg (RS) cells has been reported. The authors studied the specificity and sensitivity of Leu M1 in Hodgkin's disease (HD) and non-Hodgkin's lymphoma (NHL). Within NHL, they particularly selected cases that were confused with HD. The authors also studied S100 antigen to determine the pattern of staining in HD and NHL. Paraffin-embedded sections of 23 HD cases (3 lymphocyte predominate, 10 nodular sclerosing, 10 mixed cellularity) and 22 NHL cases (13 diffuse large cell, 5 diffuse mixed small and large cell, 4 others) were studied using an ABC technic. In 20 of 23 HD cases, RS cells and variants were Leu M1+; most cases contained prominent paranuclear positivity; some had diffuse cytoplasmic staining; and some had apparent staining of the cell surface. Neutrophils were intensely positive for Leu M1 and occasional histiocytes also were labeled. In two of the three negative cases (MC), the neutrophils were only weakly positive, thus suggesting a problem with tissue preparation. Of 22 NHL cases, 15 were totally Leu M1 negative. In six cases, rare or occasional tumor cells contained Leu M1 positivity in either a weak punctate, granular, or surface pattern. In an additional case, extensive pleomorphic cell staining was seen indistinguishable from that observed in RS cells; this case was the fourth recurrence of a primary skin NHL which began two years earlier as a pure small cleaved cell NHL. A total of three cases had positive pleomorphic cells. Some carcinomas were also Leu M1 positive. Concerning S100 antigen, the authors found scattered non-neoplastic cells throughout both HD and NHL samples; no tumor cells stained with this antigen. The negative S100 reaction of RS cells fails to support the argument for a dendritic cell origin. In properly prepared tissue, Leu M1 staining is quite sensitive for RS cells and variants, displaying a characteristic pattern. However, occasional Leu M1 positivity identified in NHL raises doubt as to its complete specificity.     

Leu M1 and peanut agglutinin stain the neoplastic cells of Hodgkin's disease

It has been suggested that the malignant cells of Hodgkin's disease (HD), Reed-Sternberg cells, and their mononuclear variants may be related to cells of the monocyte-histiocyte system. To test this hypothesis, 20 cases of HD were tested with nine antibodies, monoclonal or polyclonal, that normally react with cells of monocytic/histiocytic/granulocytic lineages, as well as PNA, which binds to histiocytes directly. Only two reagents, Leu M1 and PNA bound to the neoplastic cells in 20/22 and 13/22 cases tested, respectively. Leu M1 was the most sensitive reagent and was negative in only two cases of lymphocyte predominant HD. Leu M1 also could be employed in routinely fixed and processed formalin or B5-fixed tissue. This antibody, which was negative in 27 cases of non-Hodgkin's lymphoma, including 12 peripheral T-cell lymphomas, should prove to be of value in differential diagnosis of HD and morphologically similar reactive and neoplastic lymphoid lesions.     

Enhanced staining for Leu M1 (CD15) in Hodgkin's disease using a secondary antibody specific for immunoglobulin M.

The utility of staining for Leu M1 (CD15) as a diagnostic aid in Hodgkin's disease has been questioned because of a relative lack of specificity and sensitivity. Furthermore, interpretation is often made difficult by staining that tends to be weak and focal. Because the murine monoclonal anti-Leu M1 antibody is of immunoglobulin M type, it is reasonable to wonder whether improved immunohistochemical staining might result from use of a secondary goat antibody specific for the mouse mu heavy chain instead of the traditional one against mouse immunoglobulin. The two methods were compared, using a biotin-avidin detection system, on paraffin sections from 15 cases of Hodgkin's disease: 9 nodular sclerosing, 1 mixed cellularity, and 5 of nodular lymphocytic and histiocytic (L&H) type. In the nodular sclerosing/mixed cellularity group, the mu-specific detection method resulted in a greater number of cases with reactive Hodgkin's cells (7 versus 5), stained an average of more than three times as many neoplastic cells in each case (49% versus 14%), and usually produced staining that was distinctly more intense, often in a membrane and paranuclear distribution characteristic of Leu M1 in Hodgkin's cells. In the noLeu M1 in Hodgkin's cells. In the nodular L&H group, 1 case showed weak, focal staining with the newer method. None of the L&H cases stained using the traditional technique. It is concluded that use of a second-stage antibody that is directed specifically against mu heavy chains results in an improvement in immunohistochemical staining for Leu M1 in paraffin sections, which is of practical significance.     

Expression of Leu M1 antigen on a monoclonal B cell line established from a patient with rheumatoid arthritis

The purpose of this study is to show that anti-Leu M1 antibody (anti-CD15), which has different staining characteristics in lymphoid and non-lymphoid cells, reacted against the surface antigen of a defined monoclonal B cell line. This antibody recognizes the sugar moiety, lacto-N-fucopentaose (LNF-III), which is linked to the cell membrane protein in several kinds of cells, but not in B cells. However, a human monoclonal B-cell line (TKS-1) which was established from the peripheral blood of a patient with rheumatoid arthritis, expressed the Leu M1 antigen spontaneously. The analysis of surface markers using a fluorescence-activated cell sorter (FACS) has revealed that the surface markers of TKS-1 were anti-mu, delta, kappa, HLA-DR, DQ, Leu 12 (CD19) and Leu M1 (CD15). TKS-1 cells were not reactive with any of the following antibodies: anti-OK M1 (CD11b), Leu M2, Leu M3 (CD14), Leu M4, Leu 1 (CD5), Leu 2 (CD8), Leu 3 (CD4), Leu 4 (CD3), Leu 7 and Leu 11 (CD16). In addition, TKS-1 was positive to Epstein-Barr nuclear antigen, weakly positive to non-specific esterase without staining inhibition by NaF, and negative to peroxidase. TKS-1 cells produced IgM in the culture supernatant and have kappa-light chain rearrangement in its DNA. As shown in other studies, distribution of Leu M1 is very wide. This antigen is not a specific immunodiagnostic marker to distinguish the cell type. We conclude that it is possible to express Leu M1 antigen on the membrane of a B-cell lineage cell.     

Differentiation of adenocarcinoma of the lung from mesothelioma. Periodic acid-Schiff, monoclonal antibodies B72.3, and Leu M1

The immunohistochemical reactivity of 38 mesotheliomas and 44 adeno-carcinomas or large cell carcinomas of the lung with monoclonal antibodies (MAb) B72.3 and Leu M1 was compared with their reactivity with the routine histochemic stains periodic acid-Schiff with diastase digestion (PAS-D) and alcian blue +/- hyaluronidase. Both MAbs reacted selectively with carcinomas when a positive test was set at greater than or equal to 10% reactive tumor cells. However, MAb B72.3 reacted with significantly more of the carcinomas (86%, chi-square test, P less than 0.01) and bound to a greater percentage of tumor cells (47 +/- 28%; mean +/- SD, t-test, P less than 0.001) than Leu M1 (57% and 25 +/- 28%, respectively). The similar reactivities of surgically resected tumor specimens and post mortem tissues with both antibodies confirmed antigen stability and suggested broad clinical utility. PAS-D stained 61% of the carcinomas. Using the markers for carcinomas (PAS-D, B72.3, and Leu M1), the tumors were classified into the correct group in 80 of 82 (98%) cases (95% confidence level: greater than 92% accuracy). The alcian blue stain was useful to confirm a diagnosis of dimorphic or epithelial mesothelioma (48% were positive).     

Value of monoclonal antibodies in the diagnosis of Hodgkin's disease in paraffin sections]

A comprehensive panel of monoclonal antibodies that mark R-S/H cell, T- and B-cell, monocyte/histocyte was tested in paraffin sections of 107 cases of Hodgkin's disease (HD) including 21 cases of lymphocyte predominance (LP) and 86 cases of Non-LP HD. Thirty cases each of peripheral T-cell lymphoma and B-cell lymphoma were also tested for comparison. R-S/H cells were not stained with T-cell marker (UCHL-1) or monocyte/histocyte marker (Mac387) in all of these cases of HD. The results showed presence of certain difference in the phenotype of LP from non-LP. The H+L type of R-S/H cells of LP often reacted with B-cell markers including L26, LN2, LN1 and MB2 (93.3%-100%), LCA (83.3%) and EMA (92.3%), but rarely with LeuM1,T mü 9, or BerH2. On the contrary, most of the R-S/H cells of non-LP reacted with LeuM1 (80%), T mu 9(84%), BerH2(65%) but not with B-cell markers, LCA or EMA. Our study suggests a B-cell (probably the follicular center cell) derivation for L+H type of R-S/H cells in LP. The fact that 1 case of LP in this group transformed to a large cell B-cell lymphoma also supports this consideration. PNA is a sensitive marker of R-S/H cells but is not a specific one, since PNA stains 43.3% of the peripheral T-cell lymphoma and 20% of the B-cell lymphomas. Our findings indicate that using a panel of antibodies which mark R-S/H cells, T- and B-cells in paraffin sections will be helpful in the diagnosis and subtyping of Hodgkin's disease.     

A biotin-labelled antigen radioimmunoassay (BILA) for antibodies to membrane antigens useful for monoclonal antibody screening

Viable cells or protein extracts were labelled with N-hydroxysuccinimidobiotin and used as target antigens in a biotin-labelled antigen radioimmunoassay (BILA). The binding of the biotinylated antigens to capture antibodies coated on the bottoms of 96-well plastic plates were measured using 125I-labelled streptavidin as the detection step. The assay circumvents some of the problems associated with solid-phase RIA and permits screening of antibodies to undefined protein antigens present in very small amounts in complex protein solutions.     

Species-specific monoclonal antibodies for a membrane antigen(s) in all developmental forms ofTrypanosoma cruzi

Two monoclonal antibodies (designated as TCF48 and TCF87) were raised againstTrypanosoma cruzi, strain Tulahuen. Both antibodies reacted with all developmental forms of several different strains ofTrypanosoma cruzi. The antibodies showed no detectable cross-reactivity with other species of Trypanosomatidae, so far examined. TCF48 and TCF87 were classified as immunoglobulin subclasses IgG1 and IgG2b, respectively. Apparent molecular weight of the corresponding antigen(s) to these monoclonal antibodies was 25,000 in amastigotes and epimastigotes, and 25,000 and 24,000 in trypomastigotes, as determined by the Western immunoblotting analysis. This antigen appeared to be located at the plasma membrane and the flagellum ofT. cruzi. However, no evidence supported the localization of the epitope(s) at the external surface of the live cell. Since this antigen reacted with the sera from the chronically infected mice, these monoclonal antibodies may be useful in the study of Chagas' disease.     

Histiocytosis X arising in Hodgkin's disease: immunophenotypic characterization with a panel of monoclonal antibodies

Summary This report describes the antigenic profile of the proliferating cells of pulmonary histiocytosis X (HX) in a patient treated with chemotherapy for Hodgkin's lymphoma; the association of pulmonary HX and Hodgkin's disease has rarely been described in the literature. The histopathological diagnosis of HX was confirmed with the aid of monoclonal antibodies (mAbs) to CD4, CD1a, and polyclonal serum anti S-100 protein. The phenotype of HX cells has been analysed using a panel of mAbs against HLA class I A, B, C monomorphic determinants, locus A and B,2-microglobulin, HLA class II distinct monomorphic determinants, DP, DQ, DR, intercellular adhesion molecule-1 (ICAM-1) and vitronectin receptors. Our results indicate that HX cells express HLA class I and II, including locus A, locus B and DP, DQ, DR, like their normal counterpart (represented by Langerhans cells) and detectable levels of ICAM-1 but not vitronectin receptors. We would like to stress the possibility of the association of HX and Hodgkin's lymphoma extending the immunophenotypic profile of HX cells.     

L&H variants of Reed-Sternberg cells express sialylated Leu M1 antigen.

Anti-Leu M1 generally does not stain the lymphocytic and histiocytic (L&H) variants of Reed-Sternberg cells in the lymphocyte predominant type of Hodgkin's disease. However, the authors found that after neuraminidase treatment for removal of sialic acid, the L&H cells in more than half of the cases studied could be stained by anti-Leu M1. This result strongly suggests that L&H cells differ from the Reed-Sternberg cells in other types of Hodgkin's disease in their unique capacity to sialylate the 150-kd Leu M1 antigen.     

Importance of antigen specificity for complement-mediated lysis by monoclonal antibodies

Lysis of human lymphocytes by autologous complement had been studied using a range of monoclonal antibodies against different antigens. Antigen specificity (and not antibody isotype) was the most important factor which influenced cell lysis and this could not be accounted for merely by differences in surface density between antigens. Three antigens with comparable surface density were studied in detail: CAMPATH-1 (lytic), major histocompatibility complex class I (lytic) and leukocyte common antigen (poorly lytic). C1q binding was roughly proportional to antibody binding and dependent on antibody isotype. However, the lytic antibodies were much better able to bind and activate whole C1 than the poorly lytic ones. This result would not have been predicted from traditional concepts of complement activation but can be interpreted in the light of models for C1 activation which involve Fc-Fc interactions, Fc-C1r2s2 interactions and a critical C1q stem-arm angle for C1 binding and activation.     

Hodgkin's disease,lymphocyte predominance type,nodular--a distinct entity? Unique staining profile for L&H variants of Reed-Sternberg cells defined by monoclonal antibodies to leukocyte common antigen,granulocyte-specific antigen,and B-cell-specific antigen.

Using monoclonal antibodies to leukocyte common antigen, granulocyte-related antigen, and B-cell specific antigens, L&H variants of Reed-Sternberg (R-S) cells in Hodgkin's disease, lymphocyte predominance type (nodular), exhibited a unique staining profile as compared with R-S cells of other histologic types. L&H variants were strongly immunoreactive for leukocyte common antigen, as defined by monoclonal antibodies PD6/27 and 2B11; whereas other types of R-S cells were negative or rarely positive. R-S cells and variants in 69 cases of Hodgkin's disease of nodular sclerosis (41), mixed cellularity (25) or lymphocyte depletion (3) types, were consistently strongly immunoreactive for Leu-M1, a granulocyte-related antigen, while L&H variants were uniformly nonreactive (4 cases). B-cell specific antigens, detected by three pan-B-cell monoclonal antibodies, were observed only for L&H variants. These observations suggest that L&H variants of R-S cells represent a distinct type of transformed cell, possibly of B-cell origin, and do not share a common lineage with other types of R-S cells. These studies provide further evidence that Hodgkin's disease, lymphocyte predominance type, nodular, may represent a distinct entity.     

Isolation and characterization of monoclonal antibodies specific for protein conformational epitopes present in prostate-specific membrane antigen (PSMA)

Prostate-specific membrane antigen (PSMA) is a 750-amino acid glycoprotein highly expressed in malignant prostate tissues. PSMA reacts with the murine monoclonal antibody 7E11.C5, whose binding epitope has been mapped to the N-terminal of the protein distributed on the cytoplasmic side of the plasma membrane. We have developed murine monoclonal antibodies specific for extracellular epitopes of PSMA. Three of these antibodies--1G9, 3C6, and 4D4--display distinct binding properties consistent with their recognition of conformational epitopes within native PSMA. Results indicate this panel of antibodies binds to native full-length PSMA, but not to fusion proteins containing portions of the linear sequence of the protein. Antibody binding is greatly reduced upon heat denaturation of native PSMA, and these antibodies do not detect PSMA by Western blot. Immunoprecipitation experiments demonstrate the ability of each to bind to full-length PSMA as well as PSM', a form of the protein missing the first 57 amino acids. These results indicate each antibody is specific for an epitope within the extracellular domain, a region spanning residues 44-750. Flow cytometric experiments indicate strong specific binding to live LNCaP cells. Antibody inhibition studies demonstrate that these antibodies recognize at least two distinct epitopes. Taken together, the results demonstrate that these antibodies are specific for native protein conformational epitopes within the extracellular domain. Their properties, in particular strong binding to live cancer cells, make them ideal candidates that are clearly superior to linear sequence epitope specific antibodies for in vivo applications.     

Two monoclonal antibodies specific for serotype 4 antigen ofNeisseria meningitidis

Two new monoclonal antibodies (MN₁₄G_21.17 and MN₁₄E_9.15) specific for the serotype 4 antigen of meningococci were isolated. The antibodies were raised against a previously non-typable serogroup B strain from the Netherlands and were shown to react with the serotype antigen of the prototype reference strains for serotype 4 and serogroup A as well as with that of the homologous strain. Further screening of 290 serogroup A and B case isolates with the monoclonal antibodies indicated that the serotype 4 epitope was present on all 100 serogroup A strains tested, including representative isolates from 28 epidemics, and on most isolates from a recent serogroup B epidemic (1981–1983) in Cuba. In addition, 68 isolates from recent sporadic cases (1980–1986) and/or clusters of cases of serogroup B disease in many parts of Europe, the USA, the USSR and Australia were also of this serotype.     

An evaluation of the utility of anti-granulocyte and anti-leukocyte monoclonal antibodies in the diagnosis of Hodgkin's disease.

The immunoreactivity of six different monoclonal antigranulocyte antibodies (Leu M1, TG1, 3C4, BY/87a, BY/37a, and 3CD1) has been evaluated in 23 cases of Hodgkin's disease (7 lymphocyte predominant, 12 nodular sclerosing, and 5 mixed cellularity); in a variety of non-Hodgkin's lymphomas and in a series of reactive and benign lesions of lymph nodes. Applying a monoclonal antibody (PD7/26) to leukocyte common antigen (T200), we have also investigated reports that the L&H variants in nodular lymphocyte predominant Hodgkin's disease are strongly immunoreactive for leukocyte common antigen in contrast to the lack of reactivity of Reed-Sternberg (RS) cells and variants thereof in other forms of Hodgkin's disease. All six monoclonal anti-granulocyte antibodies reacted against RS cells and "Hodgkin's cells" in the nodular sclerosing (NSHD) and mixed cellularity (MCHD) types, with strong cell membrane and juxtanuclear (Golgi) staining. In contrast an anti-leukocyte antibody PD7/26 was unreactive with RS cells and variants thereof in NSHD and MCHD. On the other hand, RS cells and L&H variants thereof in the nodular L&H form of Hodgkin's disease (nodular lymphocyte predominant type) showed reactivity with PD7/26 but not with the anti-granulocyte markers. Rare L&H cells in 2 cases of diffuse lymphocyte predominant type showed reactivity with some, but not all, of the anti-granulocyte antibodies. These findings provide further support for the concept that the nodular L&H type of Hodgkin's disease represents an entity distinct from other forms of this disorder. Our studies also demonstrate the usefulness of these immunoperoxidase techniques when applied to formalinfixed, paraffin-embedded tissues.     

Use of monoclonal antibodies in formol-paraffin sections

Summary Thirteen monoclonal antibodies (the VI-series) reactive with B- and T-lymphocytes, monocytes, granulocytes and erythrocytes were tested in sections fixed with formalin, formalin-sublimate or formalin-acetic acid. After fixation and embedding, most of the antigens were not detectable. However, VIE-G 4, a monoclonal antibody selective for glycophorin A, produced a strong reaction with erythroid cells in formalin fixed sections. The binding of the monoclonal antibodies VIM-D 5, VIM-2 and VIM-13 to myeloid, myelomonocytic and monocytic cells became either more intense or was inhibited by preincubation of the sections with pronase, trypsin, papain or neuraminidase. Following enzyme digestion, the monocyte-specific antibody VIM-13 reacted selectively with some of the germinal centre cells.