High levels of the soluble form of CD30 molecule in rheumatoid arthritis (RA) are expression of CD30+ T cell involvement in the inflamed joints.

The CD30 is a surface molecule expressed by Th2-type lymphokine-producing T cells upon activation. CD30-expressing activated T cells release a soluble form of the molecule, which can be detectable both in vitro and in vivo. In the present study, high levels of soluble CD30 were found in peripheral blood and synovial fluid from patients with RA. However, CD30+ CD3+ cells, either CD4+ or CD8+, were significantly present in synovial fluid, but not in peripheral blood, of RA patients. Serum values of soluble CD30 were higher in active than inactive RA patients and directly correlated with rheumatoid factor serum titres. These data strongly support an involvement of CD30+ T cells in the immune processes of rheumatoid synovitis, and may suggest a relationship between Th2-type cytokine-secreting T cells and the pathological response in RA.     

Evidence for monoclonal T lymphocyte proliferation in angioimmunoblastic lymphadenopathy.

The arrangement of the T cell receptor and immunoglobulin genes was analysed in lymphoid tissue biopsy specimens from 25 cases of angioimmunoblastic lymphadenopathy. Nineteen cases showed a rearrangement of the gene coding for the beta chain of the T cell receptor, and in one case a clonal rearrangement of immunoglobulin genes was shown (in which the T cell receptor gene was in a germline configuration). These findings indicate that a monoclonal T cell proliferation is present in most cases of angioimmunoblastic lymphadenopathy, and they also correlate with the fact that some patients who present with this disorder subsequently develop a T cell lymphoma.     

Diversity of genomic breakpoints in TFG-ALK translocations in anaplastic large cell lymphomas: identification of a new TFG-ALK(XL) chimeric gene with transforming activity

Anaplastic large cell lymphomas are associated with chromosomal aberrations involving the anaplastic lymphoma kinase (ALK) gene at 2p23 that result in the expression of novel chimeric ALK proteins with transforming properties. In most of these tumors, the t(2;5)(p23;q35) generates the NPM-ALK fusion gene. However, several studies have now demonstrated that genes other than NPM may be fused to the ALK gene. We have recently described two different ALK rearrangements involving the TRK-fused gene (TFG) in which the same portion of ALK was fused to different length fragments of the 5' TFG region. These two rearrangements encoded chimeric proteins of 85 kd (TFG-ALK(S)) and 97 kd (TFG-ALK(L)), respectively. In this study, we have identified a new ALK rearrangement in which the catalytic domain of ALK was fused to a larger fragment of the TFG gene (TFG-ALK(XL)), encoding for a fusion protein of 113 kd. Genomic analysis of these three TFG-ALK rearrangements revealed that the TFG breakpoints occur at introns 3, 4, and 5, respectively, whereas the ALK breakpoints always occur in the same intron. No homologous regions or known recombination sequences were found in these regions. Transfection experiments using NIH-3T3 fibroblasts showed a similar transforming efficiency of TFG-ALK variants compared with NPM-ALK. In addition, in common with NPM-ALK, the TFG-ALK proteins formed stable complexes with the signaling proteins Grb2, Shc, and PLC-gamma. In conclusion, these findings indicate that the TFG may use a variety of intronic breakpoints in ALK rearrangements generating fusion proteins of different molecular weights, but with similar transforming potential than NPM-ALK.     

ALK Expression Defines a Distinct Group of T/Null Lymphomas (“ALK Lymphomas”) with a Wide Morphological Spectrum

The t(2;5)(p23;q35) translocation associated with CD30-positive anaplastic large cell lymphoma results in the production of a NPM-ALK chimeric protein, consisting of the N-terminal portion of the NPM protein joined to the entire cytoplasmic domain of the neural receptor tyrosine kinase ALK. The ALK gene products were identified in paraffin sections by using a new anti-ALK (cytoplasmic portion) monoclonal antibody (ALKc) that tends to react more strongly than a previously described ALK1 antibody with the nuclei of ALK-expressing tumor cells after microwave heating in 1 mmol/L ethylenediaminetetraacetic acid buffer, pH 8.0. The ALKc monoclonal antibody reacted selectively with 60% of anaplastic large cell lymphoma cases (60 of 100), which occurred mainly in the first three decades of life and consistently displayed a T/null phenotype. This group of ALK-positive tumors showed a wide morphological spectrum including cases with features of anaplastic large cell lymphoma “common” type (75%), “lymphohistiocytic” (10%), “small cell” (8.3%), “giant cell” (3.3%), and “Hodgkin’s like” (3.3%). CD30-positive large anaplastic cells expressing the ALK protein both in the cytoplasm and nucleus represented the dominant tumor population in the common, Hodgkin’s-like and giant cell types, but they were present at a smaller percentage (often with a perivascular distribution) also in cases with lymphohistiocytic and small cell features. In this study, the ALKc antibody also allowed us to identify small neoplastic cells (usually CD30 negative) with nucleus-restricted ALK positivity that were, by definition, more evident in the small cell variant but were also found in cases with lymphohistiocytic, common, and “Hodgkin’s-like” features. These findings, which have not been previously emphasized, strongly suggest that the neoplastic lesion (the NPM-ALK gene) must be present both in the large anaplastic and small tumor cells, and that ALK-positive lymphomas lie on a spectrum, their position being defined by the ratio of small to large neoplastic cells. Notably, about 15% of all ALK-positive lymphomas (usually of the common or giant cell variant) showed a cytoplasm-restricted ALK positivity, which suggests that the ALK gene may have fused with a partner(s) other than NPM. From a diagnostic point of view, detection of the ALK protein was useful in distinguishing anaplastic large cell lymphoma cases of lymphohistiocytic and small cell variants from reactive conditions and other peripheral T-cell lymphoma subtypes, as well as for detecting a small number of tumor cells in lymphohemopoietic tissues. In conclusion, ALK positivity appears to define a clinicopathological entity with a T/null phenotype (“ALK lymphomas”), but one that shows a wider spectrum of morphological patterns than has been appreciated in the past.     

PG-M1: A New Monoclonal Antibody Directed against a Fixative-Resistant Epitope on the Macrophage-Restricted Form of the CD68 Molecule

A new anti-macrophage monoclonal antibody (PG-M1) was produced by immunizing BALB/c mice with fresh spleen cells from a patient with Gaucher's disease. PG-M1 reacts strongly with a fixative-resistant epitope of an intracytoplasmic molecule, selectively expressed by virtually all macrophages of the human body. Although attempts to immunoprecipitate the molecule recognized by PG-M1 have failed so far, the reactivity of the antibody with COS-1 and WOP cells transfected with a human complementary DNA clone encoding for the CD68 antigen suggests that PG-M1 is a new member of the CD68 cluster. However, unlike other CD68 antibodies (KP1, EBM11, etc.), which react with both macrophages and myeloid cells, PG-M1 detects a fixative-resistant epitope on the macrophage-restricted form of the CD68 antigen. In 957 routinely fixed, paraffin-embedded samples, PG-M1 showed a more restricted reactivity with elements of the monocyte/macrophage lineage than the previously described monoclonal antibodies MAC-387 (anti-calgranulins), KP1 (CD68) and Ki-M1P. Among hematological malignancies, PG-M1 only labels acute leukemias of M4 and M5 type and rare examples of malignant histiocytosis/true histiocytic sarcoma. In contrast, acute leukemias of the M1, M2, M3, M6, M7, and L1-L3 types, non-Hodgkin's lymphomas, and Hodgkin and Reed-Sternberg cells of Hodgkin's disease are consistently PG-M1-negative. In the daily diagnostic practice, PG-M1 seems to be particularly valuable for the diagnosis of myelomonocytic or monocytic leukemia and neoplasms of true histiocytic origin in routine paraffin sections.     

Tumours of histiocytes and accessory dendritic cells: an immunohistochemical approach to classification from the International Lymphoma Study Group based on 61 cases

Neoplasms of histiocytes and dendritic cells are rare, and their phenotypic and biological definition is incomplete. Seeking to identify antigens detectable in paraffin-embedded sections that might allow a more complete, rational immunophenotypic classification of histiocytic/dendritic cell neoplasms, the International Lymphoma Study Group (ILSG) stained 61 tumours of suspected histiocytic/dendritic cell type with a panel of 15 antibodies including those reactive with histiocytes (CD68, lysozyme (LYS)), Langerhans cells (CD1a), follicular dendritic cells (FDC: CD21, CD35) and S100 protein. This analysis revealed that 57 cases (93%) fit into four major immunophenotypic groups (one histiocytic and three dendritic cell types) utilizing six markers: CD68, LYS, CD1a, S100, CD21, and CD35. The four (7%) unclassified cases were further classifiable into the above four groups using additional morphological and ultrastructural features. The four groups then included: (i) histiocytic sarcoma (n=18) with the following phenotype: CD68 (100%), LYS (94%), CD1a (0%), S100 (33%), CD21/35 (0%). The median age was 46 years. Presentation was predominantly extranodal (72%) with high mortality (58% dead of disease (DOD)). Three had systemic involvement consistent with 'malignant histiocytosis'; (ii) Langerhans cell tumour (LCT) (n=26) which expressed: CD68 (96%), LYS (42%), CD1a (100%), S100 (100%), CD21/35 (0%). There were two morphological variants: cytologically typical (n=17) designated LCT; and cytologically malignant (n=9) designated Langerhans cell sarcoma (LCS). The LCS were often not easily recognized morphologically as LC-derived, but were diagnosed based on CD1a staining. LCT and LCS differed in median age (33 versus 41 years), male:female ratio (3.7:1 versus 1:2), and death rate (31% versus 50% DOD). Four LCT patients had systemic involvement typical of Letterer-Siwe disease; (iii) follicular dendritic cell tumour/sarcoma (FDCT) (n=13) which expressed: CD68 (54%), LYS (8%), CD1a (0%), S100 (16%), FDC markers CD21/35 (100%), EMA (40%). These patients were adults (median age 65 years) with predominantly localized nodal disease (75%) and low mortality (9% DOD); (iv) interdigitating dendritic cell tumour/sarcoma (IDCT) (n=4) which expressed: CD68 (50%), LYS (25%), CD1a (0%), S100 (100%), CD21/35 (0%). The patients were adults (median 71 years) with localized nodal disease (75%) without mortality (0% DOD). In conclusion, definitive immunophenotypic classification of histiocytic and accessory cell neoplasms into four categories was possible in 93% of the cases using six antigens detected in paraffin-embedded sections. Exceptional cases (7%) were resolvable when added morphological and ultrastructural features were considered. We propose a classification combining immunophenotype and morphology with five categories, including Langerhans cell sarcoma. This simplified scheme is practical for everyday diagnostic use and should provide a framework for additional investigation of these unusual neoplasms.     

BCL-6 protein expression in AIDS-related non-Hodgkin's lymphomas: inverse relationship with Epstein-Barr virus-encoded latent membrane protein-1 expression.

This study aimed at investigating the expression of the BCL-6 protein in acquired immune deficiency syndrome (AIDS)-related non-Hodgkin's lymphomas (AIDS-NHLs) and at comparing the expression pattern with the presence of Epstein-Barr virus (EBV) infection of the tumor clone. A total of 34 AIDS-NHL biopsies, including 16 AIDS-related diffuse large-cell lymphomas (AIDS-DLCLs) and 18 AIDS-related small-noncleaved-cell lymphomas (AIDS-SNCCLs) as well as 7 AIDS-SNCCL cell lines were immunostained with a BCL-6-specific monoclonal antibody. Expression of BCL-6 protein was detected in 9 of 16 AIDS-DLCLs, 18 of 18 AIDS-SNCCLs, and 3 of 7 AIDS-SNCCL cell lines. Expression of BCL-6 among AIDS-NHLs occurred independently of the presence of molecular lesions of the bcl-6 gene. Notably, among EBV-positive AIDS-NHL biopsies and cell lines, expression of BCL-6 was mutually exclusive with the expression of EBV-encoded transforming antigen latent membrane protein-1. The mutual exclusion between BCL-6 and latent membrane protein-1 expression was further confirmed by in vitro superinfection experiments of an AIDS-SNCCL cell line originally devoid of EBV sequences. Overall, the frequent association between AIDS-NHLs and expression of BCL-6, a protein selectively expressed by germinal center B cells, suggests that these lymphomas may originate from the germinal center.     

Detection of terminal transferase in paraffin sections with the immunoperoxidase technique.

Terminal deoxynucleotidyl transferase (TdT) is an early T-cell differentiation marker in a number of species, including man. We have demonstrated TdT in the nuclei of cortical thymocytes in paraffin-embedded sections of calf, rat, and human thymus by indirect immunoperoxidase techniques. In addition, these techniques have been used to verify and extend enzyme assay results by detecting TdT in blast cells from 10 patients with convoluted T-cell lymphoma/leukemia, 1 patient with acute granulocytic leukemia, 1 patient with chronic granulocytic leukemia in blast crisis, and 1 patient with non-T non-B acute lymphocytic leukemia.     

ALK expression in extranodal anaplastic large cell lymphoma favours systemic disease with (primary) nodal involvement and a good prognosis and occurs before dissemination

AIMS: In anaplastic large cell lymphoma (ALCL), the site of origin has been described as an important prognostic factor. Recently, a fusion protein containing anaplastic lymphoma kinase (ALK) was described in systemic nodal ALCL, and shown to be associated with a good prognosis. The aims of this study were to investigate whether the presence of ALK protein differs between ALCL of different sites of origin; to determine whether ALK expression occurs before dissemination to other sites; and, finally, to investigate whether the site of origin remains a prognostic parameter in ALK negative ALCL. METHODS: ALK expression, as detected by immunohistochemistry using the monoclonal antibodies ALK1 and ALKc, was studied in 85 ALCLs from different sites of origin. In 22 patients, ALK expression was studied in multiple biopsies from different sites (including 13 skin, 16 lymph node, and nine other). Overall survival time was analysed using the Kaplan Meier method. RESULTS: ALK expression was found in 20 of 51 systemic ALCLs with (primary) nodal involvement. No ALK expression was found in 15 primary cutaneous, 14 gastrointestinal, and five nasal ALCLs. Multiple and subsequent biopsies of patients showed ALK expression to be identical to that seen in the primary diagnostic biopsy. Kaplan Meier survival curves showed that in ALK negative ALCLs originating from different sites, primary cutaneous cases are associated with an excellent overall survival, whereas the other cases show a comparable five years survival of less than 40%. CONCLUSIONS: If present, ALK expression favours systemic ALCL with (primary) nodal involvement, and can be used in differentiating between extranodal involvement of systemic (nodal) ALCL and primary extranodal ALCL. ALK is expressed consistently in multiple biopsies of a given patient, indicating that the chromosomal abnormality leading to aberrant ALK expression occurs before dissemination to other sites. Finally, in ALK negative non-cutaneous ALCLs, different sites of origin show comparable poor survival.