Br. J. Cancer (1991), 63, 63, Suppl. Suppi. Br. J. Cancer (1991),

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46-48 XIV, XIV, 46 48

Macmillan Press Ltd., 1991

Macmillan

Expression of 'small cell carcinoma antigens' in primary small cell lung cancer and metastases: an immunohistochemical study B.G. Skovl, F.R. Hirsch"2, F.G. Hay3, J.E. Olsen' & L.G. Bobrow 'University Department of Oncology and Pathology, Rigshospitalet, DK-2100 Copenhagen; 2Department of Internal Medicine C,

Bispebjerg Hospital, Copenhagen, Denmark; 3Imperial Cancer Research Fund, University Department of Clinical Oncology, Western General Infirmary, Edinburgh EH4 2XU; 4Imperial Cancer Research Fund, Histopathology Unit, University College and Middlesex School of Medicine, London WIP 8BT, UK. Summary The presence of 'small cell lung cancer antigens' was evaluated in pretreatment biopsies of primary SCLC, liver metastases, and/or bone marrow metastases from 46 patients. The antigen expression was detected immunohistochemically by applying monoclonal antibodies to routinely formalin-fixed and paraffin embedded tissue. The antibodies applied were second workshop codes: 3(CAM 5.2), 45 (MOVI5), 54 (NCCST433), 75 (PE35), 81 (HMFG-1), 95 (LCA1/L38) and HMFG-2 123C3, F4 and MOC-21. High expression was observed for WS 3, 45, 75, 81 and HMFG-2, both in the primaries and metastases. A good correlation was observed between the presence of antigens in primary biopsies and metastases, but there was a general tendency toward a lower proportion of positive tumour cells in the metastases compared to the primaries. For WS 45, 54, 75 and 95 the difference between primaries and bone marrow was statistically significant, and this was also true for WS 45 and 81 in the liver. The clinical relevance is discussed.

The conventional classification of lung cancer based on haematoxylin-eosin and mucin stained slides subdivides it into squamous-, adeno-, large cell-, and small cell carcinoma (SCLC) (WHO, 1981). SCLC is the most aggressive of these types, frequently metastasising to liver and bone marrow. Indeed metastases are often present at these sites at the time of diagnosis. In the last few years the use of monoclonal antibodies (MAb), has suggested that this classification may be inadequate. Several studies have demonstrated that a subgroup of non-small cell carcinomas (non-SCLC) possess both epithelial and neuroendocrine features and that this subgroup may respond to chemotherapy (Graziano et al., 1989; Berendsen et al., 1989). On the other hand SCLC with epithelial but lacking neuroendocrine markers comprise a subgroup of tumours with SCLC morphology, but clinical features mimicking non-SCLC (Berendsen et al., 1987). Thus, MAbs may be useful in a clinically relevant classification of lung cancer. Furthermore, MAbs have been demonstrated to be useful in the diagnosis of metastases, despite their lack of restricted tumour specificity. Stahel et al. (1985) reported on the ability of MAb SM1 to detect bone marrow metastases unrevealed by conventional histology, and another study stressed the improved detection of SCLC microinfiltrates in the bone marrow by the use of a panel of MAbs (Moss et al., 1988). The purpose of the present study was to evaluate the reactivity of a panel of MAbs, selected from the First Workshop (Beverley et al., 1988) which were directed against antigens related to epithelial and neuroendocrine differentiation, on both primary and secondary tumours with SCLC morphology, and thus to assess whether some of the antibodies could predict the metastatic potential for the tumour cells.

SCLC was made at the referral hospital and confirmed before initiation of chemotherapy. The primary diagnosis of SCLC was made on tumour tissue obtained from bronchoscopy, mediastinoscopy or thoracotomy. As part of the routine staging all the patients underwent bilateral bone marrow examination (biopsy and aspiration) from the posterior iliac crests and ultrasound guided liver biopsy with a sure-cut needle. For the present study, the primary diagnostic materials was used together with pretreatment metastatic material from the bone marrow and liver. Four patients had no metastases in the liver or in the bone marrow, but they had metastases to lymph nodes outside the chest and these distant metastases were recorded together with the liver metastases. All the tissues were fixed in 10% formalin and embedded in paraffin, sectioned and stained with haematoxylin and eosin for conventional histologic examination. For immunohistochemistry, five micron sections were dewaxed and rehydrated, blocked for endogenous peroxidase activity by ethanol 99%-hydrogenperoxide 1% for 20min, pretreated with Pronase (Sigma, type 24, No. 8038) for 5 min, and were then stained using the following primary MAbs, partly selected from the First Workshop (the number referring to the antibody number in the Second Workshop); CAM 5.2 undiluted supernatant (3), MOV15 1:1000 (45), NCCST433 1:100 (54), PE35 1:100 (75), HMFG-1 undiluted supernatant (81), 123C3 1:1000, F4 1:10 and MOC21 diluted supernatant, see Table I. The primary antibodies were applied to the sections for 30min and a standard three step PAP (peroxidase-anti-peroxidase) technique was used (Sternberger, 1979). The peroxidase activity was detected by incubation in ethylcarbazole and a light counterstain was given with 3 min in haematoxylin. As negative controls the primary antibodies were omitted, but otherwise the same procedure was used.

Materials and methods The study material was taken from 218 consecutive patients with SCLC, who received chemotherapy according to ongoing protocols at the Finsen Institute during the period 1 January 1986 to 1 January 1989. The primary diagnosis of

Correspondence: B.G. Skov, Department of Pathology, 5444, Rigshospitalet, DK-2100 Copenhagen, Denmark.

Evaluation The immunostaining was assessed by one observer (BGS). Intensity of staining was scored as weak or strong and proportion of stained tumour cells in the whole of each seciton was estimated and put on a 0-4 scale with 0 representing no positive cells, one between 1 and 10%, two between 11 and 25%, three between 26 and 75%, and four more than 75% positive tumour cells. The primary and secondary tumours were evaluated blind. For statistical analysis a sign test was applied with a confidence limit of 95%.

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SMALL CELL LUNG CELL ANTIGENS Table I Antibodies used in the study Known reaction WS code Antibody 3 Cytokeratin 8-18-19 CAM 5.2 Pan epithelial 45 MOV15 54 Carbohydrate antigen NCCST433 Pan epithelial 75 PE35 81 Epithelial membrane antigen HMFG-1 Glycolipid 95 LCA1/L38 None Epithelial membrane antigen HMFG-2 None SCLC, brain, nerves (NCAM) 123C3 None Internal part of EGF F4 Neuroendocrine related antigen None MOC21 EGF - epidermal growth factor.

Results

Tissue from both primary tumours and metastases from liver and/or bone marrow was available for immunohistochemistry in 46 patients, and material from these patients was entered into the study. Twenty-six patients had metastases to the liver, eight patients had metastases to the bone marrow and 12 patients had metastases to both liver and bone marrow. Some of the biopsies were very small and tissue from these was not tested with the full panel of antibodies. The results of the investigation can be divided into (1) the expression of the antibodies in SCLC tumour tissue, (2) presence of antigens in primary vs the secondary tumours and the proportion of positive tumour cells in the primary vs the secondary tumours.

Expression of antibodies in SCLC tissue Antibodies recognising epithelial antigens (WS number 3, 45, 75 and 81 and HMFG-2) were positive in most of the tumours, often with a strong intensity, but varying in the proportion of stained cells. About 2/3 of the tumours were positive for WS 81, and about 3/4 were positive for HMFG2. With all these antibodies the staining was cytoplasmic or localised to the cell membrane. WS 54, recognising a sugar antigen, was also positive in the majority of the tumours. WS 95 was only detected in about half of the tumours, but often with intense staining. No tumours were positive for F4. With MOC21 some tumours were positive, often with nuclear staining, but many other non-neuroendocrine cells in the same section gave the same reaction. This staining was considered to be nonspecific and therefore not scored in any of the slides. Finally, 123C3 was weakly and unevently positive in a few tumours

Table II Proportion of staining cells by antibody Proportion of tumour cells staining NEG 1-10% 11-25% 26-75% >75% Total

WS 3 primary liver marrow WS 45 primary liver marrow WS 54 primary liver marrow WS 75

primary liver marrow WS 81 primary liver marrow WS 95

primary liver marrow HMFG-2 primary liver marrow

The antibodies recognising epithelial antigens stained a high proportion of both primary and secondary tumours (Tables II and III). In this group HMFG-1 (WS 81) and HMFG-2 stained a lower proportion of tumours (about 70% in both the primaries and the metastases) than WS 3, 45 or 75. WS 95 and 123C3 were expressed in a small number of cases in the primaries as well as the metastases. When paired primary and secondary tumours from the same patient were compared, many pairs contained the same proportion of positive tumour cells in both the primary and the metastasis (Table IV, column marked 'same'). When pairs were assigned different scores, the metastases scored lower more often (Table IV, column 'lower') than they scored higher than the primary (Table IV, column 'higher'). Thus, a tendency towards a lower expression in the metastases compared to the primaries was seen for all the epithelial antibodies. Among liver metastases significantly more tumours showed reduced antigen expression compared with that in the primary with antibodies WS 45 and 81. In the bone marrow deposits this was true for WS 45, 54, 75 and 95 (P

Expression of 'small cell carcinoma antigens' in primary small cell lung cancer and metastases: an immunohistochemical study.

The presence of 'small cell lung cancer antigens' was evaluated in pretreatment biopsies of primary SCLC, liver metastases, and/or bone marrow metasta...
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