Br. J. Cancer

(1991), 63, Suppl. XIV,




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0 Macmillan Press Ltd., 1991 5-8©McilnPesLd,19

The expression of small cell lung cancer related antigens in foetal lung and kidney L.G. Bobrow, L. Happerfield & K. Patel Department of Histopathology, ICRF Human Tumour Immunology Group, 19 Riding House Street, University College and Middlesex School of Medicine, London, WIP 8BT, UK. Summary The presence and distribution of antigens recognised by the antibodies submitted to the Second International Workshop on Small Cell Lung Cancer Antigens has been mapped on human foetal lung and kidney using a streptavidin immunoperoxidase technique on frozen tissue sections. Thirteen of the antibodies showed no staining at all on these tissues and five showed staining of all structures in all tissues of equal intensity. The staining patterns of the remaining antibodies could be divided into several groups. All the cluster I antibodies recognising the neural cell adhesion molecule (NCAM) gave a distinctive staining pattern in both tissues. The cluster 2 antibodies and several groups of unclustered antibodies gave varying pure epithelial patterns of staining. The cluster w4 and 5 antibodies gave an interesting staining pattern which included some of the distinctive features of the cluster I pattern plus some epithelial expression. Various less distinctive patterns were also observed. Serial sections of foetal lung which had been stained with NCC-LU246 (cluster 1), anti-desmin, and anti-neurofilament antibodies were also studied to attempt to determine the nature of the cells expressing NCAM in the foetal lung.

In the first Small Cell Lung Cancer Antigen Workshop in 1987 the most significant antibody cluster to emerge was cluster 1 (Souhami et al., 1988). Subsequently it was shown that the cluster 1 antigen was neural cell adhesion molecule (NCAM) (Patel et al., 1989). This is a cell-to-cell adhesion molecule of molecular weight 120-180 kDa which is a member of the immunoglobulin superfamily (Cunningham et al., 1987). Several different isoforms of the molecule have been identified and these are all coded for by the NCAM gene on chromosome eleven (Linneman & Bock, 1989). The molecule has been demonstrated in foetal neural tissues and muscle (Kemshead, 1987) and in adult brain, peripheral nerves, endocrine tissues and striated muscle (Souhami et al., 1988). In the foetus, it is present in a highly sialilated form and is loosely adherent whereas in the adult it is poorly sialilated and much more tightly adherent (Rothbard et al., 1982). The molecule is also abundantly present in small cell lung cancer, neuroblastoma and a wide range of other neural tumours, Wilms tumour, hepatoblastoma and rhabdomyosarcoma (Patel et al., 1989; Souhami et al., 1988; Kemshead 1987). Since NCAM is a major antigen expressed in almost 100% of cells of all small cell lung cancer studied (Moss et al., 1986; Gatter et al., 1985) and since it does not appear to be expressed in adult human lung except within nerve fibres, we thought it would be interesting to ascertain whether NCAM is present foetal lung and if so to map its distribution there. Since NCAM has been previously reported to be present in foetal kidney (Van der Gugten et al., 1988; Roth et al., 1988) we decided to include foetal kidney in this study. Further, since this study was to be presented at the Second International Small Cell Lung Cancer Antigen Workshop, all the antibodies submitted to the workshop were included in the

study. Materials and methods Tissue from 14, 15 and 16 week foetuses was collected from routine surgical procedures. The tissues were dissected out and immediately put into liquid nitrogen following termination. They were then stored until needed at - 70°C. Four micron sections were cut and following brief acetone fixation were stained using a streptavidin-biotin peroxidase system.

Correspondence: L.G. Bobrow.

Primary antibodies comprised the Second International Small Cell Lung Cancer Antigen Workshop panel of antibodies numbered 1 to 98 which were applied according -to the workshop instructions; anti-desmin and anti-neurofilament antibodies supplied by Dako. In addition to the controls included within the panel, we carried out our own negative control by omitting the primary antibody step on a foetal lung and kidney section in each staining run performed. Known positive controls for desmin and neurofilament were included for the experiment using these antibodies. The staining intensity was evaluated as negative, weakly positive or strongly positive. Results Most of the antibodies could be divided into several distinct groups according to the staining pattern observed on the foetal lung and kidney. The single largest group was made up of antibodies which gave no specific staining at all. The antibodies which fell into this category were the following (listed by workshop numbers): 1, 2, 9, 10, 15, 22, 23, 24, 25, 29, 32, 33, 40, 51, 52, 57, 62, 63, 64, 65, 72, 84, 85, 88, 94. The 13 antibodies which clustered into cluster 1 on the dendrogram (numbers 4, 12, 21, 31, 41, 48, 58, 60, 61, 74, 77, 82) all gave a very distinctive staining pattern. In the lung, there was strong staining of a continuous sheath of cells around all small air spaces and air passages (Figure la). In addition, in the bronchi, there was strong staining of foci of epithelial cells. In the kidney, strong staining of the primitive blastema, ampullae and S tubes and weak staining of all the undifferentiated stromal cells was seen (Figure lb). Several groups of antibodies gave varying pure epithelial patterns of staining. All the antibodies which clustered in cluster 2 (11, 14, 16, 20, 53, 80, 90) plus 46 and 73 gave a distinct pattern. In the lung, all epithelial cells showed strong staining. In the kidney, there was uniform strong staining of all tubules except those within the glomeruli, glomerular capsular epithelium and pelvic epithelium. The cytokeratin controls (3, 28, 56, 71) all gave an identical pattern of staining. In the lung, all epithelial and all mesothelial cells stained strongly (Figure 2). In the kidney, there was strong staining of all tubular structures except the ampullae, the glomerular capsular epithelium and the pelvic epithelium. A group of antibodies including HMFG1 (17, 36, 43, 49, 50, 79, 87 and 93) showed a fairly consistent pattern. Thus in



Figure 2 Section of foetal lung stained with antibody no 3 (CAM 5.2 the cytokeratin control). Strong staining of all the epithelial cells lining the airspaces is seen. Calibration bar micron.

Figure la Section of foetal lung stained with antibody no 31 (cluster 1). Strong staining of the cuff of cells adjacent to the epithelium surrounding the air spaces is seen. b, Section of outer part of foetal kidney stained with antibody no 31 (cluster 1). Strong staining of the blastema (top) as well as the subjacent 1 micron. ampulla and S tube is seen. Calibration bar

the lung, there was staining of all epithelial cells which was often strongest along the luminal surface and, in the kidney, there was staining of all tubular epithelial cells and heterogenous staining of pelvic epithelium. These antibodies often gave high background staining. The two antibodies which were designated cluster w8 (35, 47) plus four other antibodies which were all close to this cluster on the dendrogram (6, 7, 91 and 92) gave a very distinctive staining pattern. In the lung, there was strong staining of occasional small foci of cells in the bronchial epithelium. In the kidney, strong staining was seen in individual cells and focal collections of cells in the epithelium lining the collecting tubules. Towards the pelvis, the foci of positive cells showed an increase in size and number to become continuous in the pelvis giving strong staining of the pelvic epithelium. Antibodies 81 and 95 both show some limited staining of epithelial cells. With 81 there is staining in the lung, but not the kidney. All except one of the antibodies clustered in clusters w4 and 5 (66, 67 and 69) plus 59, 70 and 75, all of which are very close to either of these clusters on the dendrogram,

showed a notably distinct pattern of staining. The pattern of staining seen with cluster 1 antibodies was seen with the additional staining of all epithelial cells in the lung and tubular epithelial cells in the kidney. Staining of epithelial structures in both organs plus uniform endothelial staining was seen with antibodies 27, 45, 54, 76 and 89. This group includes the w6 antibodies 45 and 54. Staining of smooth muscle only was seen with antibodies 13, 30, 97 and 98 the latter two antibodies constitute w7. Occasional dendritic stromal cells were stained with antibodies 38, 42 and 86. Uniform staining of all tissues was seen with antibodies 8, 39, 44, 68 and 83. Several antibodies gave one-off patterns of staining which are worthy of mention. Antibody number 5 stained stromal structures in lung which may represent developing nerves. Antibody number 21 stained all structures uniformly strongly except for endothelium and glomerular epithelium. Antibody number 26 stained all non-epithelial structures uniformly strongly. Antibody number 96 stained all undifferentiated stroma. Antibody number 78 stained all tissues except for the undifferentiated stroma. Sections stained with anti neurofilament antibody showed staining around bronchi which was more limited but nevertheless overlapping the distribution seen with cluster one antibodies. Sections stained with the anti-desmin antibody showed staining around bronchi and airspaces which also overlapped the cluster 1 antibody staining pattern to some extent. There was no overlap in staining pattern with either antibody seen in the kidney and staining of smooth muscle around blood vessels was also noted with the anti-desmin antibody. Discussion Cluster I pattern The staining patterns observed in lung and kidney in our study is similar to those observed by van der Gugten et al. (1988) in their study of a range of foetal tissues with some cluster 1 antibodies. Roth and colleagues (1988) in a study on foetal kidney and Wilm's tumour demonstrated a more limited distribution of NCAM in kidney, but they used a single polyclonal anti NCAM antibody in parallel with a monoclonal antibody to polysialic acid. Given this variation in pattern between the Roth study, and ours and van der Gugtens, the degree of consistency in staining between the 13 antibodies included in this group is remarkably striking. All


L.G. BOBROW et al.

the more so since several different isoforms of NCAM have been described and it might be supposed that some of the antibodies may recognise different isoforms of the molecule. The distribution of positive cells in the lung overlaps to some degree the distribution of the developing nerve plexuses in the walls of the bronchi and larger airspaces. This is demonstrated well by the serial section of lung stained with anti neurofilament antibody. The degree of staining around the smaller airspaces with cluster 1 antibodies is much more than can be accounted for by developing nerve. These positive cells probably represent developing smooth muscle or myoepithelial cells. This is to some extent supported by the pattern seen on the serial section stained with the anti-desmin antibody but desmin is lacking around smaller airspaces. We plan to extend these studies using an anti-actin antibody as this marker may identify myoepithelial cell precursors more


The cluster 1 positive cells in the developing kidney are not related to developing muscle or nerve. The NCAM molecule appears to have an important developmental role in the kidney which is unrelated to its function in neuromuscular tissues in the adult, since its distribution does not mirror that of these structures in the kidney.

Pure epithelial staining patterns Since cluster 2 antibodies recognise a membrane-associated glycoprotein which has so far only been identified in epithelial cells and epithelial derived tumours it is not surprising that a purely epithelial staining pattern is seen with them. The absence of staining of mesothelial cells is consistent with findings in adult human mesothelium. The staining pattern seen with the cytokeratin positive

controls is again consistent with the pattern of distribution of low molecular weight cytokeratins seen in adult human tissues. Likewise the antibodies grouped with HMFG1 showed a distribution in the foetal tissues which is consistent with the pattern of staining seen with HMFG1 and its homologues in adult human tissues. The pattern seen with the two antibodies in cluster w8 plus the four closely associated antibodies is very distinctive. We have little information on the nature of the antigen recognised by these antibodies except that antibody number 92 is reported by Hirohashi and colleagues (1985) to be against a group A determinant. Further studies on the nature of the antigens recognised by this group of reagents and their pattern of staining on adult human tissues and carcinomas is warranted. The staining pattern seen with the cluster w4 and associated antibodies is interesting since the cluster w4 antibodies have been reported to be positive in small cell and non-small cell lung cancer.

Epithelial plus endothelial staining pattern Antibodies giving this pattern included 45 and 54 which comprise cluster w6 plus 27 which is adjacent to w6 on the dendrogram. The antibodies in w6 are against the Y hapten. The same pattern of staining is also seen in with two antibodies which are far away on the dendrogram; antibody 89 which is against ICAM-1 and antibody 76 which is close to general anti-mucin antibodies. Smooth muscle pattern The four antibodies which stained smooth muscle only include 97 and 98 which comprise cluster w7 and are reported to be against high molecular weight mucins. The other two antibodies in this group are far distant from cluster w7 on the dendrogram and we have little information on the antigens they recognise.

Remaining patterns With three antibodies (38, 42 and 86) which were clustered in the negative cluster we saw staining of occasional single dendritic cells within the stroma of both organs studied. The nature of these cells is uncertain. Where we saw uniform staining of all cells in both tissues studied we interpreted the staining as non specific. Repeat staining with controlled furtheir dilutions of these antibodies is necessary to allow proper interpretation of the staining with these reagents (8, 39, 44, 68 and 83). The remaining antibodies gave varied patterns of mainly stromal type. We have tested all the antibodies submitted to the Second International Workshop on Small Cell Lung Cancer Antigens, on foetal lung and kidney tissue sections using an immunohistochemical system. These tissues were selected because we were particularly interested in the cellular distribution of NCAM in them. We have confirmed that the distribution of NCAM in them is quite specific. We have also demonstrated several other patterns of staining with groups of antibodies within the workshop. The distinctive pattern seen with the cluster w4 and 5 is of particular interest in the context of SCLC since SCLC expresses both epithelial and neural antigens and these are both seen by antibodies in cluster w4 and 5. We have also attempted to elucidate whether the cells expressing NCAM in these tissues are developing nerve or muscle. Further studies are under way as the findings of this part of the study were inconclusive. We would like to thank Professor Souhami and Dr Fiona Moss for their constructive comments on the ideas which led to the setting up of this project; Dr Amanda Fisher for supplying the foetal tissue; and Mrs Maureen Cohen for her valuable secretarial assistance.

References CUNNINGHAM, B.A., HEMPERLY, J.J., MURRAY, B.A., PREDIGER, E.A., BRACKENBURY, R. & EDELMAN, G.M. (1987). Neural cell adhesion molecule: structure, immunoglobulin like domains, cell surface modulation and alternative RNA splicing. Science, 236, 799-806. GATrER, K.C., DUNNILL, M.S., PULFORD, K.A., HERYAT, A. & MASON, D.Y. (1985). Human lung tumours. A correlation of antigenic type with histological type. Histopathology, 9, 805.


VAN DER VALK, M. (1988). The distribution of small cell lung cancer cluster I (SCLC-I) antigens in foetal tissue. Lung Cancer, 4 (suppl), A12. HIROHASHI, S., CLAUSEN, H., YAMADA, T., SHIMOSATO,Y. & HAKOMORI, S.-I. (1985). Blood group A cross-reacting epitope defined by monoclonal antibodies NCC-LU-35 and -81 expressed in cancer of blood group 0 or B individuals: Its identification as Tn antigen. Proc. Nati Acad. Sci. USA, 82, 7039.

KEMSHEAD, J.T. (ed) (1987). Neuroblastoma Antibody Workshop, pp. 1-70, WG Forbeck Foundation: Hilton Head Island, SC. LINNEMAN, D. & BOCK, E. (1989). Cell adhesion molecules in neural development. Dev. Neurosci., 11, 149. MOSS, F., BOBROW, L.G., SHEPPARD, M.N. & 5 others (1986). Expression of epithelial and neural antigens in small cell and non small cell lung carcinoma. J. Pathol., 149, 103. PATEL, K., MOORE, S.E., DICKSON, G. & 4 others (1989). Neural cell adhesion molecule in Wilm's tumour. Am. J. Pathol., 133, 227.

ROTHBARD, J.B., BRACKENBURY, R., CUNNINGHAM, B.A. & EDELMAN, G.M. (1982). Differences in the carbohydrate structures of

neural cell adhesion molecules from adult and embryonic chicken brains. J. Biol. Chem., 257, 11064. SOUHAMI, R.L., BEVERLEY, P.C.L. & BOBROW, L.G. (eds) (1988). Proceedings of the First International Workshop on Small Cell Carcinoma Antigens. Lung Cancer, 4 (suppl.), 1.

The expression of small cell lung cancer related antigens in foetal lung and kidney.

The presence and distribution of antigens recognised by the antibodies submitted to the Second International Workshop on Small Cell Lung Cancer Antige...
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