The distribution of the c-myc oncogene product in malignant lymphomas and various normal tissues as demonstrated by immunocytochemistry

The expression of c-myc was studied in 51 malignant lymphomas and in a variety of normal tissues by immunocytochemistry using monoclonal antibodies raised to different synthetic peptides and reacting monospecifically with the c-myc product (p62C-Yc). The c-myc product was detected in only a minority of malignant lymphomas principally those containing cells with immunoblastic characteristics, and was predominantly localised to the cytoplasm. In normal lymphoid tissues only plasma cells and histiocytes were found to have immunoreactivity. In non-lymphoid normal tissues, however, the c-myc product was distributed widely. Marked differences in its intracellular distribution were apparent in different tissues. These findings suggest that the relationship of p62c-lYc expression to cell division may be more complex than previously suggested by in vitro studies, and raise the possibility that it may have other functions within the cell.

The human c-myc proto-oncogene is a cellular gene homologous with the 3' sequence of the transforming gene of the avian myelocytomatosis virus MC29 (Papas et al., 1984). It has been shown that in Burkitt lymphoma-derived cell lines the c-myc proto-oncogene is often involved in reciprocal translocations involving the immunoglobulin genes although the chromosomal break points may differ (Gellmann et al., 1983;Erikson et al., 1983;Hayday et al., 1984); in some circumstances this appears to be associated with mutation of c-myc . This type of rearrangement has also been described in murine plasmocytoma cell lines (Bernard et al., 1983). Rearrangement of c-myc has also been described in cell lines or fresh tissue from other lymphoid tumours, but these are less well characterised in terms of the precise genomic configuration (Rothberg et al., 1984;Dalla-Favera et al., 1983). Rearrangements of the c-myc proto-oncogene have been correlated with elevated expression but this does not appear to be invariably the case (Klein, 1981;. In some Burkitt cell lines levels of c-myc mRNA comparable to those seen in normal cells are found, but the untranslocated allele is silent (Taub et al., 1984). This raises the possibility that the translocation may result in expression at an inappropriate stage of cellular development. On the basis of this work, it has been suggested that J.C.-B abnormal expression of c-myc may be critically involved in development of lymphomas.
In contrast to the extensive study of Burkitt lymphoma derived cell lines there is little known about the expression of this gene in other types of lymphoid tumour or in normal lymphoid tissues. Elevated levels of c-myc mRNA have been found in some tumours (Rothberg et al., 1984;Roy-Burnam et al., 1983;Slamon et al., 1984), but the interpretation of these results is complicated by the extensive cellular heterogeneity which may be present in malignant lymphomas.
We therefore decided to investigate c-myc expression in lymphoid tumours using monoclonal antibodies to the p62c-mYc gene product. It was hoped that such an immunocytochemical study would give information about the cellular distribution of the c-myc product in both normal and malignant lymphoid tissue and the relationship of c-myc expression, if any, to cellular differentiation. It was hoped that it would be possible to comment on whether the gene was being aberrantly or constitutively expressed in tumour cells.

Tissue
Tissue was obtained from 51 lymphoid tumours including most histological types commonly seen and 20 examples of normal lymphoid tissue (lymph node, spleen, thymus, tonsil and bone marrow). With the exception of two cases originating in East Africa all were recent biopsy specimens from The Macmillan Press Ltd., 1986 Correspondence: A.S. Jack. Received 2 January 1986; and in revised form, 19 February 1986. Glasgow Royal Infirmary. All tissue was fixed in buffered formol saline and embedded in paraffin wax.
The diagnosis was made on the basis of routine histology and a wide variety of immunocytochemical tests. The diagnostic categories broadly follow those described in the Kiel classification or the Lukes-Butler classification of Hodgkin's disease (Wright & Isaacson, 1983;Lukes & Butler, 1966).
Monoclonal antibodies Two monoclonal antibodies were used. These were raised against synthetic peptides as previously described. The antibodies designated myc-I 6E10 and myc-I 9E10 have been shown to react with different regions of the p62c-mYc molecule and to be monospecific on immunoblotting with various cell lines .  in the form of a purified antibody (2mgml-1) and was used as a dilution of 1/500 in Tris buffer pH 7.6. Myc-l 6E10 was in the form of hybridoma supernatant and was diluted to 1/20. These concentrations were obtained after titration studies on normal and neoplastic tissue.

Immunocytochemistry
Tissue sections were dewaxed and hydrated. All sections were routinely trypsinised for 5-15 min. Endogenous peroxidase activity was blocked using acidified methanol containing hydrogen peroxide. After washing in Tris saline, pH 7.6, sections were incubated overnight with diluted primary antibody at 20°C. The sections were washed and incubated for 30 min with optimally diluted rabbit anti-mousehorseradish peroxidase conjugate (Dako). Antibody binding was demonstrated by immersion in diaminobenzidine and hydrogen peroxide for 10min. Sections were then washed, counterstained with haematoxylin and dehydrated. A control section was stained, for each specimen, omitting the primary antiserum.

Results
Antibody specificity An identical pattern of staining was obtained using both monoclonal antibodies. (Figure la,b). As these were raised to different parts of the p62c-mYc molecule it renders fortuitous cross reactions, commonly seen with monoclonal antibodies, unlikely.
Distribution of staining Normal lymphoid tissue All lymphoid cells seen in normal lymph node, spleen, tonsil and thymus were unstained with the exception of plasma cells (see Table I). (Figure 2). These cells showed marked and consistent cytoplasmic staining. In thymus strong granular cytoplasmic staining was present in the epithelial cells including Hassall's corpuscles. In tonsil granular cytoplasmic staining was present in crypt epithelium. In both spleen and lymph node rather faint cytoplasmic staining was seen in histiocytes. This was most marked in the tingeable body histiocytes associated with germinal centres.
Intense nuclear and cytoplasmic staining was present in the majority of haemopoietic precursor cells seen in normal bone marrow (Figure la, b). Weak cytoplasmic staining was sometimes seen in mature granulocytes.
Lymphoid tumours A summary of the staining patterns found in lymphoid tumours is given in Table II. A number of additional points should be noted. Two lymphoblastic tumours were included.    One of these was a typical example of African Burkitt lymphoma (BL), which showed minimal staining of tumour cells but a moderate degree of staining of the substantial component of reactive histiocytes (Figure 3). The other case which differed morphologically from BL showed weak cytoplasmic staining. Positive staining was seen in all Timmunoblastic malignant lymphomas although in some cases this was rather weak. Some cells in these tumours showed nuclear as well as cytoplasmic staining.
In addition, large cell elements, presumably immunoblasts, were found to be stained in two cases of pleomorphic peripheral T cell tumours (Figure 4), although most of the small or intermediate sized cells were unstained ( Figure 5). Two plasma cell tumours showed diffuse cytoplasmic and, occasionally nuclear staining of the type seen in normal plasma cells.
In the cases of Hodgkin's disease some Reed-Sternberg cells showed cytoplasmic and occasionally nuclear staining, although this was variable Normal non-lymphoid tissues Non-lymphoid cells were often present in biopsies of lymphoid tissue. As shown in Table III, a wide variety of cell types showed immunoreactivity. Some cells, such as connective tissue cells, showed diffuse cytoplasmic staining, similar to that seen in plasma cells. In other cells, notably hepatocytes and jejunal epithelium, (Figure 6) p62c-myc was localised to coarse, intensely staining cytoplasmic granules. The perinuclear distribution of these granules in enterocytes suggested an association with the Golgi apparatus.

Discussion
The aim of this study was to determine whether p62C-mYc was detectable in normal lymphoid tissues and tumours by immunocytochemical methods and whether this was related to cellular differentiation. Twenty-one of 51 cases of malignant lymphoma showed some evidence of immunocytochemical reactivity with the anti p62c-myc monoclonal antibodies. However, in the majority of cases, especially follicle centre cell tumours, the intensity of staining was very weak relative to that seen in some normal tissues. Nevertheless some tentative conclusions can be drawn about the relationship of anti p62c-mYc staining and cellular differentiation.
Positive staining was most consistent in T immunoblastic malignant lymphomas and this was further corroborated by the positive immunoreactivity in the immunoblast component of pleomorphic peripheral T cell tumours. It was of interest that many Reed-Sternberg cells in cases of Hodgkin's disease also stained positively; some authors hold the view that these cells are modified immunoblasts, in most cases of the T cell type (Stein et al., 1984). It was not possible to determine whether normal T-immunoblasts contained p62c-mYc since these cells are rarely seen in normal lymphoid tissue and suitable material from cases of reactive T immunoblastic hyperplasia such as infective mononucleosis was not available. Therefore it is impossible to comment on whether the tumour cells are aberrantly expressing the c-myc gene product.
Two cases of lymphoblastic malignant lymphoma were studied, one of which was a typical African BL. The latter case showed no definite evidence of tumour cell reactivity, but there was intense Granular cytoplasmic staining mainly in basal layer. Granular perinuclear staining in both crypts and villous epithelium. Granular cytoplasmic staining in hepatocytes and bile duct epithelium. Cytoplasmic staining in ducts. Salivary acini are negative. Granular cytoplasmic staining in thyroid acinar cells.
Cytoplasmic staining in both smooth and skeletal muscle. Weaker staining in other mesenchymal cells including adipocytes, fibroblasts and endothelium. Diffuse cytoplasmic staining of gastric antral epithelium. granular staining in the reactive histiocytes which are a prominent feature of this condition. The non-Burkett B-lymphoblastic malignant lymphoma showed weak cytoplasmic staining. Again no equivalent normal cell type could be studied as those cells are not identifiable in normal tissues.
In cultured lymphoid cells a transient increase in levels of c-myc mRNA is found after mitogen or growth factor stimulation at the GO/G1 transition (Kelly et al., 1984). As transcription in some systems does not appear to vary during the cell cycle, regulation of c-myc mRNA may be partly related to variable rates of degradation (Blanchard et al., 1985). In a wide range of other cells c-myc is induced in early G1 phase and persists during S and G2 phases of the cell cycle (Rabbitts et al., 1985). Our results are not consistent with a simple relationship of p62c-mYc expression and cell division. Positive immunoreactivity was seen in a wide range of cells, including terminally differentiated cells such as plasma cells and skeletal muscle cells as well as dividing cells such as haemopoietic marrow and Go arrested cells such as hepatocytes. Immunoreactive c-myc product has been described in normal testicular cells (Sikora et al., 1985). An imperfect correlation with cell division and c-myc mRNA expression has also been found using in situ hybridisation of human embryos (Pheifer-Ohlsonn et al., 1984). One possible explanation for the discrepancy between our results and the various in vitro studies could lie in the intracellular distribution of p62c-mYc. In cultured cells c-myc gene product is of short half life and localised to the nucleus (Rabbits et al., 1985). In this study a nuclear distribution was seen in the actively dividing cells of bone marrow and in some Tissue tumours. However, in most normal cells which show immunoreactivity the localisation of p62c-PyC was cytoplasmic. The pattern of staining of enterocytes and hepatocytes suggests that in these cells the protein was probably concentrated in the Golgi apparatus. This suggests that localisation of the protein, in vivo, may differ in cells which are dividing as opposed to those which are arrested or terminally differentiated.
In view of the unexpected results of this study the possibility of antibody cross-reactivity must obviously be considered. This is a serious problem when using single monoclonal antibodies. (Nigg et al., 1982). However, in this study antibodies to separate parts of the p62c-myc were used and taken with the detailed characterisation of these antibodies, cross-reactivity would seem to be a highly unlikely explanation. The second problem lies in the sensitivity of the methods used. Immunohistochemical methods are known to be very sensitive but nevertheless it cannot be definitively stated that very low levels of p62c-mYc may not be detected and that these could have biological significance. Tissues were fixed rapidly after removal from the patient which therefore makes it unlikely that c-myc protein would have been significantly catabolised before fixation. A further possibility is that the intracellular distribution of p62c-myc was altered by fixation, either by diffusion, or some other mechanism. However, it is difficult to envisage how the observed tissue-specific differences in distribution could be accounted for by this type of artefact. Furthermore, the intense granular localisation seen in some cells (Figures 5 and 6) would not be consistent with fixation associated diffusion.
In conclusion, our study provides little evidence to support the view that elevated expression of cmyc is a common or necessary feature of Ihuimian malignant lymphomas. Widespread distribution of this protein in normal tissues raises a number of interesting questions concerning its function. These concern the possible differing functions of nuclear and cytoplasmic p62c-myc since it may be that one is active and the other a storage form of the molecule. It would also be relevant to ask whether c-myc is involved solely in the control of cell division or whether it participates in other aspects of cellular metabolism. These questions clearly merit further investigation. Finally, it does not appear that the immunocytochemical demonstration of p62c-mYc is of value in the pathological diagnosis or subtyping of malignant lymphomas.