JOURNAL OF PATHOLOGY, VOL. 159: 87-90 (1 989)

SELECTED SUMMARIES

This section highlights some of the topical areas and papers published in 1988. It is by no means comprehensive but the intention is that it gives a brief overview to those who do not have a specialized interest in this area, and there is emphasis on topics which are of diagnostic value.

LYMPHORETICULAR PATHOLOGY

There has, as usual, been a large number of publi- cations on the subject of malignant lymphoma in the past year. A small selection will be summarized here. The areas to be highlighted comprise immunocyto- chemistry, the prognostic significance of immuno- phenotyping of lymphomas, and the cellular origin of nodular, lymphocyte predominant Hodgkin’s disease.

Immunocytochemistry on fixed and processed tissues is an expanding area in histopathology in general and lymphoreticular pathology in particu- lar. Several recent papers have dealt with potential problems in such work.

Antigen preservation in infarcted lymphoid tissue: a novel approach to the infarcted lymph node using monoclonal antibodies effective in routinely pro- cessed tissue. A. J. NORTON, A. D. RAMSAY and P. G. ISAACSON. Am J Surg Pathol 1988; 12 759-767.

Lymph node infarction is an uncommon event, but has a documented association with malignant lymphoma. It is therefore essential to gain the maximum information from such tissues. Tinctorial stains such as reticulin and trichromes may occasionally be of some help, but are of limited value. In this study the authors have made use of a range of monoclonal antibodies that react with fixa- tion-resistant epitopes to study infarcted lymph nodes.

Eleven cases of malignant lymphoma diagnosed concurrently with, or following, lymph node infarc- tion were studied. These comprised seven B-cell lymphomas, three T-cell lymphomas, and a single case of Hodgkin’s disease. A panel of antibodies was used including antibodies to leucocyte common

antigen (PD7/26), T-cell associated antigens (MT1, UCHL l), B-cell associated antigens(MB1, 4KB5, MT2, LNl, L26), CD15 (C3D-1), CD30 (BerH2), and HLA-DR (LN3, TALlB5). Staining with an antibody to leucocyte common antigen (CD45) and related antigens (CD45R) proved useful. Nine cases showed strong reactivity with PD7/26 in infarcted tissue; eight were strongly stained by the T-cell and B-cell restricted leucocyte common antibodies. In the case of Hodgkin’s disease, scattered T cells and fewer B cells were identified. T-cell clusters were noted around apparent empty spaces in the infarcted tissue, but when compared with viable tissue from the same case it was clear that these T cells were, in fact, clustered around Reed-Sternberg cells.

L26, a B-cell specific antibody, showed no stain- ing or diffuse non-specific staining in six cases. In three cases, some antigenicity was retained at the periphery of the infarcted tissue. In viable tissue, however, L26 reacted with all of the B-cell lympho- mas, highlighted groups of B cells in the T-cell lymphoma, and reacted with some of the Reed- Sternberg cells in the case of nodular sclerosing Hodgkin’s disease.

BerH2 (CD30) and immunoglobulin light chain antibodies gave rise to unstructured, diffuse, non- specific staining of infarcted tissues despite excellent results in the viable tissue. LNl produced strong cytoplasmic and surface staining in all the infarcted B-cell lymphomas. HLA-DR specific antibodies also showed strong membrane staining in infarcted tumour cells and in the case of Hodgkin’s disease they highlighted the Reed-Sternberg cells. LeuM 1 (CD15) reactivity was seen at the periphery of the infarcted tissue in the case of Hodgkin’s disease. In a CDl5-positive T-cell lymphoma, strong surface and cytoplasmic staining was evident in both the infarcted and the viable tissue.

0022-341 7/89/09008744 $05.00 0 1989 by John Wiley & Sons, Ltd.

88 SELECTED SUMMARIES

This study shows that diagnostically useful information can be gained by immunocytochemical examination of infarcted lymph nodes. Further- more, it may help in the assessment of infarcted tissue in general and may allow definitive diagnosis of lymphoma to be made even in the absence of viable tumour.

lmmunohistochemistry in bone marrow diagnosis. Value of a panel of monoclonal antibodies on rou- tinely processed bone marrow biopsies. P. VAN DER VALK, M. HULLINK, P. C. HUIGENS, T. M. TADEMA, W. Vos and C. J. L. M. MEIJER. Am J Surg Patholl989; 13: 97-106.

The authors investigated the reativity of a large number of antibodies in 73 bone marrow biopsies fixed in formol-sublimate, decalcified in an acetic acid/formaldehyde mixture, and embedded in paraffin. They were able to identify different cell lines in normal and diseased marrow.

Megakaryocytes were identified by Ulex euro- paeus lectin (UEA) and antibody to factor VIII related antigen. The myeloid series was stained by the antibodies MT1, LeuM1, LN2, LN3, and anti- elastase amongst others. In myeloproliferative con- ditions, the myeloblasts and promyelocytes stained for leucocyte common antigen. Erythroid cells, like megakaryocytes, stained with UEA and were also reactive with anti-glycophorin and LN1. Lympho- cytes marked for LCA and some reacted with LN2 and MB2. Plasma cells were readily identified when stained for immunoglobulin light chain. All the metastatic carcinomas studied reacted with MB2, and occasional cases reacted with other antibodies including LNI, LN2, MTI, and BerH2.

These results show that immunocytochemistry is feasible on routinely processed, decalcified bone marrow biopsies, and the authors recommend that a limited panel should be used. This would identify the cell lines found in the bone marrow and would comprise UEA, LeuM1 /MTl, anti-LCA, and immunoglobulin light chain antibodies. These are particularly suggested for the investigation of sus- pected bone marrow involvement by malignant lymphoma, plasma cell disorders, myelodysplastic syndromes, and the assessment of the proportions of various cell lines in the marrow. The authors comment, however, that they do not believe the panel would be of value in acute leukaemia.

An alternative to the use of antibodies on decalci- fied paraffin sections of bone marrow has also been suggested.

A simplified plastic embedding and immunohistologic technique for immunophenotypic analysis of human hematopoietic and lymphoid tissues. T. T. CASEY, J. B. COUSAR and R. D. COLLINS. Am J Pathol1988; 131; 183-189.

Biopsies of lymphoid and haematopoietic tissues were fixed overnight in acetone at -2O"C, infil- trated in glycol methacrylate (GMA) monomer with 5 per cent methylbenzoate, and embedded in cata- lysed GMA monomer using reagents available in a kit form. Two pm sections were cut, transferred to glass slides, and air-dried. Immunohistochemical examination was performed using the double conju- gated indirect immunoperoxidase technique with a large panel of antisera including B- and T-cell markers and antibodies to interleukin 2 receptors, HLA-DR, leucocyte common antigen, and CD15. The results showed that this particular embedding technique gave rise to comparable, or superior, antigen preservation to that observed in cryostat sections. Furthermore, morphological details were superior to both frozen sections and routine paraffin sections. Other detection methods such as avidin- biotin complex, alkaline phosphatase/anti-alkaline phosphatase, and immunogold silver staining were also investigated and produced satisfactory results.

The explanation for the retention of antigenicity is thought to lie in the inclusion of methylbenzoate in the embedding protocol. GMA usually masks antigens during its polymerization, apparently by binding to amino-terminal portions of protein mol- ecules. Methylbenzoate prevents this masking, although it is interesting to note that this occurs only in tissue fixed in acetone. Use of methanol as a fixa- tive does not achieve the desired effect.

The recommendation of this plastic embedding technique is particularly interesting in view of the recent papers regarding the more general use of plastic sections in diagnostic histopathology.

Is wax on the wane? G. I. MURRAY. J Pathol 1988; 156: 187-188; Plastic or paraffin? ANON. Lancet 1989; i: 139-140.

To the non-lymphoma specialist it appears that lymphoma pathologists apply ever-increasing numbers of antibodies to sections, perhaps in an attempt to confuse those who do not share their special interest. In the last year, two papers have examined the prognostic significance of immuno- phenotype in malignant lymphomas, a subject on which there are relatively few data available.

SELECTED SUMMARIES 89

Immunophenotyping of non-Hodgkin’s lymphoma, correlation with relapse free survival. H.-J. SCHUURMAN, W. HUPPES, L. F. VERDONCK, J. VAN BAARLEN and J. A. M. VAN UNNIK. Am J Patholl988; 131: 102-1 11.

This was a retrospective analysis of 92 patients with B-cell lymphoma using antibodies to the B-cell antigens CD9, CDIO, CD19-24, CD37, and CD38; the T-cell antigens CD5-7, HLA-DR, and CD25; and transferrin receptors. Staining for terminal deoxynucleotidyl transferase and peanut lectin was also performed. CD3 expression was the only marker to show a significant association with longer disease-free survival in the total patient group. Among high grade lymphomas, absence of CD38 or presence of CD24 was associated with a significant improvement in disease-free survival. An attempt was made to examine the stage of B-cell differen- tiation of the tumours by means of assessing patterns of CD9, CDIO, CD21, CD22, and CD23 reactivity. This showed that the more immuno- phenotypically primitive tumours had a shorter disease-free survival than the more mature cases, as one might predict. The authors conclude that detailed immunophenotyping of B-cell lymphomas is of limited value both in terms of confirmation and assessment of the histopathological classification and with regard to the prognosis. They do not, however, deny the potential value of immuno- phenotyping in cases where the histopathological classification is uncertain.

The aberrancy of immunophenotyping and immuno- globulin status as indicators of prognosis in B-cell diffuse large cell lymphoma. C. M. SPIER, T. M. GROGAN, S. M. LIPPMAN, D. J. SLYMEN, J. A. RYBSKI and T. P. MILLER. Am J PuthoE1988; 133 118-126.

In this study, 105 cases of diffuse large cell lym- phoma were studied using a panel of 40 monoclonal antibodies. Of these lymphomas, 83 were found to have a B-cell immunophenotype and these cases were further examined for prognostically significant markers. Absence of the pan-B antigens CD20 and CD22 was associated with a significant decrease in survival. Loss of expression of HLA-DR was also associated with poorer survival.

Immunoglobulin production was investigated; kappa light chain restriction was found to be a favourable feature when compared with lambda or to cases without demonstrable light chain. Heavy

chain phenotype had no significant relationship with survival. Multivariate analysis was performed and this included the presence or absence of B symp- toms, details of tumour bulk, and presence or absence of extra-nodal disease as well as the immu- nophenotypic data. This confirmed the independent prognostic significance of kappa light chain expres- sion, CD20 and 22 expression, and HLA-DR expression. B symptoms were also predictive of poor survival. The authors suggest that aberrant B-cell phenotype in diffuse large cell lymphoma is a predictor of poor prognosis. They point out, how- ever, that the number of cases they have examined is relatively small and confirmation is required in a large prospective study.

Another area of debate in the field of lympho- reticular pathology is the putative B-cell origin of nodular, lymphocyte predominant Hodgkin’s dis- ease. Two studies, one using immunocytochemistry and the other molecular biology and immunocyto- chemistry, have been reported recently.

Hodgkin’s disease, lymphocyte predominant type, nodular-further evidence for a B cell derivation. G. S. PINKUS and J. W. SAID. Am J Pathol 1988; 133: 21 1-217.

The monoclonal antibody L26 is a very effective B-cell marker which can be used on rountinely fixed and processed tissues. It was applied to 72 cases of Hodgkin’s disease covering all subtypes. In all cases of lymphocyte predominant disease, the large pleo- morphic cells typically seen in this condition exhibited strong membrane and weaker cytoplas- mic staining. Rare classical Reed-Sternberg cells showed similar reactivity. Varying numbers of small lymphocytes were also positive and these appeared most numerous in cases demonstrating the most distinct nodularity. In contrast to these findings, lacunar cells, classical Reed-Sternberg cells, and large pleomorphic cells in other types of Hodgkin’s disease showed only occasional L26 staining (CL5 per cent) in most cases. Eight cases, however, showed more extensive reactivity and in one mixed cellularity case more than 90 per cent of Reed- Sternberg cells were positive. The authors consider that the consistent reactivity of Reed-Sternberg cell variants in nodular, lymphocyte predominant Hodgkin’s disease with L26 provides further evi- dence for a B-cell derivation. The reactivity of Reed-Sternberg cells in other subtypes raises the possibility that in some of these cases they are also of B-cell origin. This observation raises a problem in

90 SELECTED SUMMARIES

diagnostic immunocytochemistry in cases where Hodgkin’s disease and non-Hodgkin’s lymphoma cannot be distinguished histologically. In order to avoid error the authors once again emphasize the need for the application of panels of antibodies for accurate lymphoma diagnosis.

Absence of B-cell or T-cell clonal expansion in nodular, lymphocyte predominant Hodgkin’s disease. M. D. LINDEN, A. J. FISHLEDER, W. E. KATZIN, and R. R. Tus~s. Hum PathoZl988; 19: 591-594.

Evidence for a B-cell origin of nodular, lympho- cyte predominant Hodgkin’s disease was sought by means of both immunocytochemistry and molecu- lar biology. DNA was extracted from the same frozen tissues used for cryostat section immuno- phenotyping. Only three cases were analysed and no evidence of light chain restriction was observed by

immunochemistry. No rearrangements of the immunoglobulin heavy and light chain genes or of the T-cell receptor B-gene were identified.

The authors conclude that the lack of detectable immunoglobulin gene rearrangement indicates that the predominant B-lymphocyte population in this form of Hodgkin’s disease is polyclonal. However, the possibility that the typical pleomorphic Reed- Sternberg cells are monoclonal remains since, because of their paucity, detection of rearrangement may be beyond the sensitivity of Southern blotting.

Further studies will no doubt follow using the polymerase chain reaction in order to assess the clonality of these enigmatic cells.

KEVIN WEST Department of Pathology

University of Leicester Leicester Royal Injirmary Leicester LEZ 7LX, U.K.