Monoclonal antibodies recognizing CD5, CD10 and CD23 in formalin-fixed, paraffin-embedded tissue: production and assessment of their value in the diagnosis of small B-cell lymphoma

AIMS: Assessment of the expression of antigens CD5, CD10 and CD23 can be of value in the differential diagnosis of small B-cell lymphoma. Correct subclassification is important since optimal treatment regimes differ between the subtypes. The aim of this study was to generate monoclonal antibodies recognizing these antigens in paraffin-embedded tissue and to assess their efficacy using a panel of cases of small B-cell lymphoma of various subtypes. METHODS AND RESULTS: For each antibody synthetic recombinant protein and conventional murine hybridoma technology was employed. Monoclonal antibodies effective in formalin-fixed, paraffin-embedded tissue were successfully generated, designated NCL-CD5-4C7, NCL-CD10-270 and NCL-CD23-1B12, respectively. A series of 58 cases of small B-cell lymphoma including examples of each subtype (lymphocytic, follicle centre cell, mantle cell, marginal zone and lymphoplasmacytoid) was assembled and immunostaining for the respective antigens carried out using the monoclonal antibodies produced. Our results indicate that the antibodies are specific for their respective antigens and give the predicted phenotypic profile in the small B-cell lymphoma subtypes. CONCLUSIONS: These novel monoclonal antibodies may be of value in routine diagnostic practice.     

Activation and differentiation antigen expression in B-cell non-Hodgkin's lymphoma

In an attempt to establish whether extended immuno-phenotyping allows more accurate definition of subgroups of B-cell non-Hodgkin's lymphoma (NHL) we have stained a series of 145 cases with a large panel of monoclonal antibodies that recognize B-cell differentiation and activation antigens. No antigen was expressed by all cases. The B-cell histogenesis in many cases could be confirmed only by using a panel of immunoglobulin and pan B-cell markers. There was marked phenotypic heterogeneity within and between major groups of B-cell NHL as delineated by the Kiel classification although the differentiation antigens CD5 (lymphocytic and centrocytic NHL) and OKT10 (plasma cell tumours) were more often expressed by certain morphological groups. The activation antigens 4F2 and transferrin receptor were expressed more strongly and more often by high grade NHL but other activation antigens (CD23 and CD25) were not more frequently associated with these tumours. Extended phenotyping may be of value in improving the understanding of biological abnormalities and processes involved in B-cell NHL, but we conclude that a limited panel of markers (CD3, CD5, CD22, CD45, IgM, kappa, and lambda) should be sufficient for routine diagnosis and classification of most cases.     

Proliferation and apoptosis in primary gastric B-cell non-Hodgkin's lymphoma

To elucidate the role of cell turnover in primary gastric B-cell non-Hodgkin's lymphomas we studied tissue samples of 72 patients—26 small cell (25 MALT lymphomas) and 46 large cell (31 MALT lymphomas). Proliferation indices and apoptotic indices were measured by Mib-1 expression and terminal deoxynucleotidyl-transferase (TdT)-mediated nick end labelling, respectively. Furthermore, expression of the apoptosis related gene-products bcl-2 and p53 was studied. Large cell lymphomas showed significantly higher proliferation indices (59.1% vs. 15.4%) and apoptotic indices (3.2% vs. 0.7%) than small cell lymphomas. Proliferation and apoptotic indices were positively correlated ( r =0.371, P =0.03). Expression of the bcl-2 protein was significantly higher in the small cell lymphomas. Furthermore, cases with a high bcl-2 expression showed both a significantly decreased proliferation ( P <0.0001) and apoptotic index ( P =0.0096) compared to bcl-2 negative cases. Expression of the mutant p53 protein was present in 8.0% of small cell and in 18.2% of large cell lymphomas. p53 positive cases showed significantly higher proliferation indices, but showed no correlation with apoptotic index. These data suggest that impaired apoptosis by bcl-2 may be more prominent than proliferation in the genesis of small cell lymphomas, whereas a high cell turnover characterizes large cell primary gastric lymphomas.     

Automated analysis of fluorescence in situ hybridization on fixed, paraffin-embedded whole tissue sections in B-cell lymphoma.

Certain recurrent cytogenetic abnormalities are diagnostic of a specific neoplasm and may portend prognosis. As conventional cytogenetics may not reveal a neoplastic clone, and unfixed material for fluorescence in situ hybridization may be unavailable, performing fluorescence in situ hybridization on fixed tissues is diagnostically and prognostically valuable. Manual interpretation of fluorescence in situ hybridization signals may be difficult on paraffin-embedded tissue sections due to truncated nuclei. Therefore, we investigated the use of an automated image acquisition and analysis system (MetaSystems) for interpretation of fluorescence in situ hybridization signals in tissue sections from dual fusion translocation probes. Three probe sets were analyzed on archival specimens with a confirmed diagnosis of mantle cell lymphoma, follicular lymphoma or Burkitt lymphoma. 100% of mantle cell lymphomas (7/7) were positive for t(11;14), 91% of follicular lymphomas (10/11) for t(14;18) and 100% of Burkitt lymphomas (9/9) for t(8;14). Successful hybridization was achieved using various tissue fixatives and fluorescence in situ hybridization interpretation was blinded with respect to the underlying diagnosis. Based on these results, automated analysis of fluorescence in situ hybridization on fixed tissues is accurate and valuable in the evaluation of B-cell lymphoma, and may provide pertinent diagnostic and prognostic information.     

Secondary non-Hodgkin's B-cell lymphoma involving the breast: radiologic imaging

Secondary lymphomas involving the breast, although uncommon, represent the largest group of metastasic tumors to the breast. Primary non-Hodgkin lymphomas (NHLs) of the breast are also rare, accounting for 1.7% to 2.2% of extranodal NHL cases and 0.38 to 0.7% of all NHLs. Approximately 300 cases have been reported in the Medical literature. Mammographic appearances are described as round or oval areas of opacity. The breast mass may appear homogeneous or inhomogeneous. The lesion may be very well defined and may be mistaken for a benign process, most notable in patients younger than 35 years of age. Ultrasonographic appearance is described as a sharply defined mass with low or medium echoes. The posterior aspect of the mass is well defined (possibly with slight acoustic enhancement but with no associated posterior shadowing). In summary, the mammographic appearance of the breast lymphoma is nonspecific, but the diagnosis can possibly be excluded if calcifications or a desmoplastic reaction are present. Prominent lymph vessels in a patient with a breast mass should raise the suspicion of breast lymphoma. The most critical aspect in the workup of a breast mass is the tissue biopsy, since radiographically lymphomatous lesions are indeterminate. We present the case of a 79-year-old woman with abdominal pain and a palpable breast mass; we also make a brief summary of the clinical features and main imaging findings of NHL (plain radiograph, mammography, breast ultrasound and thorax tomography).     

Cytogenetic characterization of three cases of unusual B-cell non-Hodgkin's lymphoma

We report the cytogenetic results in three cases of B-cell non-Hodgkin's lymphomas with morphologic and immunophenotypic features compatible with a non-follicle center cell lymphoma. In all cases, a chromosome breakpoint at 14q32 and structural abnormalities of 1p were found. Increased copies of chromosome segment 12q and structural rearrangements of chromosome 6 were found in two cases. Translocation (14;18)(q32;q21) with rearrangement of bcl-2 was found in one case. These lymphomas have a perifollicular growth pattern and IgM+, IgD+, CD10-, and CD22+ immunophenotype features typical of non-follicle center cell lymphomas and probably belong to the follicle mantle lymphomas described recently. Little cytogenetic data about this group of lymphomas is available, possibly because in the Working Formulation for the Classification of Lymphomas they are not separated from follicle center cell lymphomas.     

Prognostic significance of activation and differentiation antigen expression in B-cell non-Hodgkin's lymphoma

Immunophenotyping shows heterogeneity of expression of activation and differentiation antigens in B-cell non-Hodgkin's lymphoma (NHL). To investigate whether antigen expression correlates with clinical behaviour we have studied the clinical presentation and follow-up of a series of 111 B-cell lymphomas previously phenotyped for a panel of antigens including CD groups 5, 9, 10, 21, 23, 25, 30, 38, 4F2 antigen, and transferrin receptor. CD antigens 5, 10, and 23 were expressed significantly more often by low grade lymphomas whereas CD38, 4F2 antigen, and transferrin receptor were more often expressed by high grade lymphomas. There was a significant correlation with survival and age, stage at presentation, histological grade, and expression of 4F2 antigen and transferrin receptor but not with the other antigens studied. 4F2 antigen and transferrin receptor may identify a poor prognostic group of cases in low grade lymphoma but we conclude that phenotyping B-cell NHL for many of the antigens expressed at various stages of B-cell differentiation and activation does not provide clinically useful information in addition to that obtained from standard histological classifications.     

Localization in situ of costimulatory molecules and cytokines in B-cell non-Hodgkin's lymphoma.

Costimulatory molecules are essential in cognate interactions between T and B lymphocytes. To study the prerequisites of functional interactions between malignant B cells and intermingled T cells in B-cell non-Hodgkin's lymphomas (B-NHL), we examined the expression of CD40, CD80 and CD86 and their ligands CD40 ligand (CD40L, CD154), CD28 and CTLA4 (CD152) using immunohistochemistry and confocal laser scanning microscopy. Almost all mucosa-associated lymphoid tissue (MALT) NHL were positive for CD40 and CD80 and in nine out of 14 cases were positive for CD86. The majority of follicle centre cell lymphomas (FCCL) expressed CD40, but were heterogeneous in their expression of CD80 and CD86. Most diffuse large cell lymphomas (DLCL) were CD80+, but lacked expression of CD86. These patterns reflect the differences in phenotype of normal marginal-zone B cells (as counterparts of MALT NHL) and germinal centre cells (as counterparts of FCCL and DLCL). Counter-receptors on T cells were detectable in 13 of 14 MALT NHL, 12 of 16 FCCL but only occasionally in DLCL (three of 12 cases). A subgroup of FCCL was identified with T-cell expression of CD40L, CD28 and CTLA4 simultaneously with strong expression of CD40 and CD86 on the tumour B cells. These results indicate that MALT NHL and a subset of FCCL are most optimally equipped for functional interactions with T cells. This may be supported by the demonstration of cytokine production - mainly in T cells - in MALT NHL [interleukin-2 (IL-2), interferon-gamma (IFN-gamma), IL-10] and FCCL (IL-2, IFN-gamma) and to a lesser extent in DLCL.     

PCR-based assays for the detection of monoclonality in non-Hodgkin's lymphoma: application to formalin-fixed,paraffin-embedded tissue and decalcified bone marrow samples

In crucial cases the diagnosis of non-Hodgkin's lymphoma (NHL) still represents a challenge to the pathologist since morphological criteria do not always help to distinguish between reactive and malignant lymphoproliferations. Clonality assays are a useful supplement since monoclonal cell proliferation is strong evidence for malignancy. The polymerase chain reaction (PCR) can be utilized to establish the clonal origin of B-or T-cell lymphocyte populations by amplification of rearranged immunoglobulin and T-cell receptor (TCR) genes. In the present study DNA was isolated from a variety of neoplastic and nonneoplastic formalin-fixed, paraffin-embedded lymph nodes (n=62), cutaneous tissue (n=9), samples of miscellaneous origin (n=11), and, reported here for the first time, decalcified bone marrow samples (n=35). These samples were submitted to PCR-based assays directed against the immunoglobulin heavy-chain (IgH), immunoglobulin light-chain (IgL), and TCR chain genes. The impact of various decalcifying agents on the ability to amplify DNA was investigated by PCR-based amplification of a single copy gene. Buffered and nonbuffered EDTA was found not to impede amplification of DNA fragments up to 300 bp in length. In lymph node and cutaneous specimens monoclonality was detected in 83% of B-NHL cases using a seminested PCR approach for the amplification of IgH, whereas the same approach gave rise to monoclonal bands in 80% of bone marrow samples. The subsequent amplification of IgL helped to raise the sensitivity of detection to 94%. Monoclonality was detected in seven of nine T-cell NHLs by amplification of TCR. Most of the false-negative results were related to DNA extracted from centroblastic-centrocytic lymphoma and lymphoplasmocytic immunocytoma (37% negative each). PCR-based rearrangement analysis of immunoglobulin and TCR chain genes should be used in diagnostic pathology for cases which are histopathologically and immunohistochemically questionable. The application of clonality assays to bone marrow samples previously decalcified with EDTA provides a new tool for the detection of minimal residual disease.Abbreviations BALT bronchus-associated lymphoid tissue - dNTP deoxynucleoside triphosphate - Ig immunoglobulin - IgH immunoglobulin heavy chain - IgL immunoglobulin light chain - MALT mucosa-associated lymphoid tissue - NHL non-Hodgkin's lymphoma - PCR polymerase chain reaction - TCR T-cell receptor     

Composite germ cell tumor and B-cell non-Hodgkin's lymphoma arising in the sella turcica

We report the case of a composite malignant neoplasm consisting of germ cell tumor and B-cell non-Hodgkin's lymphoma occurring in the sella turcica of a young girl who presented with hypopituitarism. Routine hematoxylin and eosin-stained sections of a resected suprasellar tumor demonstrated a neoplasm composed of 2 distinct morphologies. A panel of immunohistochemical markers was used to confirm the morphologic impression of germinoma (cytokeratin AE1/AE3-CAM 5.2, cytokeratin 7, neuron-specific enolase, and focally placental alkaline phosphatase positive) and mature B-cell lymphoma (CD20 positive; pancytokeratin, placental alkaline phosphatase, and terminal deoxynucleotidyl transferase negative). To the best of our knowledge, this is the first reported case of such a composite tumor in the central nervous system.     

Monoclonal antibodies reactive in routinely processed tissue sections of malignant lymphoma, with emphasis on T-cell lymphomas

The immunoreactivity of eight monoclonal antibodies was evaluated on 45 routinely processed lymphomas (22 T-cell lymphomas, 11 B-cell lymphomas, and 12 cases of Hodgkin's disease). Two antibodies reactive with leukocyte common (T200) antigens (PD7/26 and 2B11) stained most of the B- and T-cell lymphomas but did not stain the Reed-Sternberg cells and variants in Hodgkin's disease. Two antibodies known to stain B cells (LN-1 and LN-2) reacted with some of the B-cell lymphomas, but LN-2 also reacted with the neoplastic cells in six of 22 T-cell lymphomas and with the Reed-Sternberg variants in eight of 12 cases of Hodgkin's disease. The granulocyte antibody anti-Leu M1 reacted with most cases of Hodgkin's disease but also reacted with two of 11 B-cell non-Hodgkin's lymphomas. An antibody to epithelial membrane antigen (anti-EMA) stained some cases of T-cell lymphoma, B-cell lymphoma, and Hodgkin's disease. Leu 7 was expressed in one T-cell lymphoma and in one case of Hodgkin's disease. A novel antibody reactive with T cells (L60) stained all cases of T-cell lymphoma but also stained some cases of B-cell lymphoma and one case of Hodgkin's disease. We conclude that none of these antibodies, when used alone on routinely fixed paraffin-embedded material, is completely sensitive and specific for T-cell lymphoma, B-cell lymphoma, or Hodgkin's disease. However, a panel of antibodies is useful in distinguishing Hodgkin's disease from non-Hodgkin's lymphoma and in suggesting the B- or T-cell phenotype of non-Hodgkin's lymphomas.     

Monoclonal antibody phenotyping of B-cell non-Hodgkin's lymphomas. The Southeastern Cancer Study Group experience

This report describes the experience of the Southeastern Cancer Study Group (SECSG) with the frozen-section immunoperoxidase phenotyping of 162 cases of B-lineage non-Hodgkin's lymphomas. The authors used a panel of 13 different markers with varying degrees of specificity for B lymphocytes and B-cell neoplasms. All lymphomas were classified according to the International Working Formulation. Several antibodies, including anti-immunoglobulin, B1, Leu 12, and Leu 14 were B-cell-specific markers that were generally pan-reactive. Several other monoclonal antibodies, however, were selectively reactive with subpopulations of B-cell lymphomas. Three "selective-B" antigens (BA1, p24, CALLA) were found on about half of the B-cell lymphomas tested, while another three (HB31, transferrin receptor, C3d receptor) were found on about two-thirds of the lymphomas tested. Leu 1 reacted with 18% of the B-cell lymphomas, particularly the small lymphocytic lymphomas. When the reactivity of the monoclonal antibodies was compared with the histologic classification, two important points became apparent. First, with the large panel of antibodies, there was tremendous phenotypic diversity even among histologically similar tumors. Second, however, not all possible combinations of antibody phenotypes were encountered. That is, clusters of antigenic phenotypes were seen, and these phenotypes correlated to some degree with the histologic diagnosis of the tumor. Small lymphocytic and follicular lymphomas tended to be phenotypically distinct, although there was some overlap. Intermediate- and high-grade lymphomas were phenotypically more diverse. The more common phenotypes of lymphomas encountered could not be reconciled with any simple linear scheme of neoplastic B-cell differentiation.     

Immunophenotyping of non-Hodgkin's lymphomas in paraffin-embedded tissue sections. A comparison with genotypic analysis.

Several monoclonal antibodies (MoAbs) are now available for immunophenotyping non-Hodgkin's lymphomas (NHLs) in paraffin-embedded tissue sections. To determine the reliability of these reagents in predicting the genotype, 44 cases of NHL were studied with the alkaline phosphatase-anti-alkaline phosphatase technique with the use of the following MoAbs: leukocyte common antigen (CD45), Mac 387, L26, 4KB5, MB1, MB2, LN2, UCHL1, MT1, and MT2. The lineage of the neoplastic cells was determined in all cases by gene rearrangement studies for immunoglobulin heavy chain and for the T-cell receptor beta-chain. Genotypic results showed B-cell lineage in 33 cases (75%), T-cell lineage in 6 cases (14%), and mixed or undetermined lineage in 5 cases (11%). A concordance of lineage assignment by paraffin section immunophenotyping with gene rearrangement studies was observed in 37 of 39 (95%) lymphomas with an unequivocally defined genotype. MoAb L26 was the most sensitive in detecting B-cell genotype; MoAbs MT1 and UCHL1 were the most sensitive and specific, respectively, in detecting T-cell genotype. The authors conclude that lineage assignment of NHLs in paraffin sections is reflective of the corresponding genotype when an appropriate panel of MoAbs is used.