D. Y. Mason

JC70: a new monoclonal antibody that detects vascular endothelium associated antigen on routinely processed tissue sections.

A new monoclonal antibody, JC70, raised against a membrane preparation from a spleen affected by hairy cell leukaemia, recognises a membrane bound glycoprotein identical with that of the CD31 group of monoclonal antibodies. The antibody stains a fixation resistant epitope on endothelial cells in benign and malignant conditions in a wide variety of paraffin wax embedded tissue. JC70 stained malignant endothelial cells in 10 angiosarcomas with more consistency than monoclonal or polyclonal antibodies to factor VIII related antigen (FVIII-Rag). In four cases of Kaposis sarcoma the antibody stained malignant endothelial cells but not spindle cells. It is concluded that antibody JC70 is of value for studying benign and malignant human vascular disorders in routinely processed tissue.

MALIGNANT HISTIOCYTOSIS OF THE INTESTINE: A T-CELL LYMPHOMA

Malignant lymphoma complicating coeliac disease has been characterised on morphological and immunocytochemical grounds as malignant histiocytosis of the intestine (MHI). Fresh tissue from four cases of MHI was studied by means of a panel of monoclonal antibodies; in three cases tumour DNA was studied for immunoglobulin and T-cell receptor (TCR) gene rearrangement. Immunocytochemistry showed a T-cell phenotype in all four cases, confirmed by the demonstration of a rearranged TCR beta-chain gene in the three cases studied. Lymphoma complicating coeliac disease thus appears to be of T-cell, rather than histiocyte, origin.

Heterogeneity of vascular endothelial cells with relevance to diagnosis of vascular tumours.

AIMS: To determine the distribution of factor VIII related antigen, CD31, CD34 and CD36 in normal and malignant human vascular tissues using a panel of well characterised monoclonal antibodies. METHODS: Frozen and fixed material from a wide range of normal tissues and routinely processed material from 43 benign and malignant vascular tumours were examined. Single immunocytochemical labelling was performed using the APAAP technique. Double staining involved the sequential use of APAAP with the peroxidase method. RESULTS: Human vascular endothelium was antigenically heterogeneous. One of the most restricted markers was factor VIII related antigen, despite its having been widely used in diagnostic pathology as a marker of vascular endothelium and of the tumours which arise from it. Three antibodies against factor VIII related antigen, CD31 (JC70) and CD34 (QBend 10) were identified as immunostaining routinely processed, formalin fixed, paraffin wax sections. Each antibody gave different staining when tested on a range of vascular tumours, both benign and malignant. CONCLUSIONS: A small panel of three reagents (factor VIII related antigen, CD31 (JC70) and CD34 (QBend 10)) should be used by diagnostic pathologists who want to show the presence of cells of endothelial origin in routine material.

Cytokeratin expression in smooth muscle and smooth muscle tumours.

The expression of cytokeratin intermediate filaments by a tumour has been accepted as evidence of an epithelial origin. Although there have been anecdotal reports of Cytokeratin expression within tissues and neoplasms of non‐epithelial origin, particularly muscle, there have been no comprehensive studies of its frequency and distribution. In order to investigate this we have studied 51 cases of normal smooth muscle and benign and malignant smooth muscle tumours using a panel of monoclonal antibodies against a range of intermediate filaments (cytokeratins, desmin and vimentin). Cytokeratin expression was noted overall in 50% of normal, benign and malignant smooth muscle tissues. Such expression tended to have a focal or patchy distribution. No case expressed cytokeratins in the absence of both desmin and vimentin. The implication of these findings for diagnostic immunocytochemistry is that intermediate filaments alone are not completely reliable markers of tumour histogenesis and should be used as part of a larger panel of monoclonal antibodies.

REARRANGEMENT OF THE T-CELL-RECEPTOR β-CHAIN GENE IN THE DIAGNOSIS OF LYMPHOPROLIFERATIVE DISORDERS

The arrangement of the T-cell-receptor and immunoglobulin genes has been analysed in 77 cases of lymphoproliferative disorder. All 6 T-cell leukaemias and 16 of 19 T-cell lymphomas showed rearrangement of the gene coding for the beta chain of the T-cell receptor, associated in all cases with a germline arrangement of the immunoglobulin genes. All 36 B-cell leukaemias and all 16 B-cell lymphomas showed rearrangement of immunoglobulin genes; the T-cell-receptor gene was in the germline configuration in most of these cases but showed a rearranged pattern in 3 cases (2 chronic lymphatic leukaemias and 1 immunoblastic lymphoma). The combined use of T-cell-receptor and immunoglobulin gene probes promises to be a valuable means of identifying and classifying T-cell neoplasms.

Detection of T cells in paraffin wax embedded tissue using antibodies against a peptide sequence from the CD3 antigen.

Rabbit polyclonal antibodies were raised against a proline rich, peptide sequence, comprising 13 amino acids, in the cytoplasmic domain of the CD3 epsilon chain. Immunoprecipitation experiments showed that this antibody preparation recognised the CD3 antigen on human T lymphoblasts. The antibody stained normal T cells strongly in tissue sections which had been fixed in formalin or Bouins solution and embedded in paraffin wax. Its reactivity with T cell lymphoma, when evaluated on a series of 96 previously phenotyped cases, closely agreed with the results obtained on cryostat sections. These results indicate that the specific detection of T cells in routinely processed tissue biopsy specimens is now technically feasible on a wide scale in diagnostic laboratories using CD3 peptide antibodies, and they also suggest that in future the use of anti-peptide antibodies may detect other lineage specific antigenic markers in paraffin wax sections.

The immunohistological detection of platelets, megakaryocytes and thrombi in routinely processed specimens

The production and characterization of a new monoclonal antibody, Y2/51, against platelet glycoprotein IIIa is described. A useful feature of this antibody is its ability to recognize platelets and megakaryocytes in formalin‐fixed routinely processed material. It could also be used to reveal platelets both in thrombi in large vessels and in microthrombi too small to be readily apparent on conventional microscopic examination. For this purpose it was helpful to use the antibody in conjunction with a new monoclonal reagent (Ret40f) against red cell sialoglycoprotein β‐which detects red cells and their precursors in routinely processed tissue. The use of these antibodies should be valuable for the detection of thrombi in a variety of situations such as renal transplant rejection, coronary artery disease and vasculitis.

Genotypic analysis of large cell lymphomas which express the Ki‐1 antigen

The monoclonal antibody Ki‐1 reacts with Reed‐Sternberg cells in Hodgkins disease and with the tumour cells in a minority of large cell non‐Hodgkins lymphomas. This study describes the results of immunophenotypic and DNA analysis in 30 cases of non‐Hodgkins lymphoma, all of which expressed the Ki‐1 antigen. The genotypic analysis has been undertaken using both immunoglobulin and T‐cell receptor gene probes. Sixteen cases were shown by this method to be of monoclonal T‐cell origin, six of B‐cell origin, while in eight cases there was no evidence of either T‐ or B‐cell lineage. This confirms previous immunohistological data indicating that non‐Hodgkins lymphomas which express the Ki‐1 antigen may be of either T‐cell or B‐cell origin.

CD30 expression in non-Hodgkin's lymphoma

The CD30 antigen has been reported as the immunophenotypic hallmark of a recently described category of non‐Hodgkins lymphoma, termed anaplastic large cell lymphoma. From a series of approximately 500 lymphomas, 17 cases showing typical anaplastic features have been identified. They were strongly labelled by monoclonal antibodies recognizing CD30 (Ki‐1 or BerH2). However, 36 other lymphomas, mainly high‐grade, of non‐anaplastic cytology also expressed CD30, either diffusely or focally, with a staining pattern identical to that seen in anaplastic large cell lymphomas. This clearly suggests that such lymphomas cannot be identified solely on the basis of being high‐grade non‐Hodgkins lymphomas showing CD30 positivity. From the present results, the distinction between the anaplastic and non‐anaplastic types would be better made with antibodies to epithelial membrane antigen than to CD30. Clinical data, available for 48 of the patients (16 with anaplastic large cell lymphomas and 32 with non‐anaplastic) revealed no significant differences with regard to age at presentation, sex or clinical signs. A short‐term follow‐up study of 25 patients revealed that for the first 2 years after diagnosis there were no significant differences in patient survival between anaplastic large cell lymphoma, other CD30+ high‐grade lymphomas and all high‐grade non‐Hodgkins lymphomas considered together. These findings, which must be confirmed by larger studies, suggest that in a general lymphoma clinic there is probably little justification for differentiating anaplastic large cell lymphomas or CD30+ lymphomas from other high‐grade non‐Hodgkins lymphomas.

CLINICAL IMPORTANCE OF ANALYSING MALIGNANT TUMOURS OF UNCERTAIN ORIGIN WITH IMMUNOHISTOLOGICAL TECHNIQUES

The value of immunohistology in the histopathological diagnosis of malignant tumours was evaluated in a series of 120 consecutive routinely processed biopsy specimens, all of which had been referred by histopathologists because of diagnostic difficulties. The specimens were analysed by means of a panel of monoclonal and polyclonal antibodies reactive with antigens which resist routine tissue processing. The tumours had previously been considered unclassifiable (24 cases) or categorised as probable carcinomas (43 cases) or lymphomas (53 cases). All but 8 of the 120 cases could be classified with the immunohistological technique. Lymphoma was the commonest diagnosis (66%) and accounted for 29 of the 43 cases initially thought most likely to be anaplastic carcinomas. Immunohistological methods can now resolve the majority of difficulties arising over the histological diagnosis of malignant tumours, and these methods should therefore be used on a wide scale by diagnostic histopathology laboratories.