22 ANTIGENS

081

NSP GENE EXPRESSION AS A NOVEL MARKER OF THE NEUROENDOCRINE PHENOTYPE IN LUNG CARCINOMAS. H.J.K. van de Velde’, N.H.M. Sendens, A.J.M. Roebroek’, J.L.V. Broers*, F.C.S. Ramaekerss and W.J.M. Van de Ven’ (‘Lab. for Molecular Oncology, Center for Human Genetics, Univ. of Leuven, Belgium and *Dept. of Molecular Cell Biology and Genetics, Univ. of Limburg, the Netherlands).

We recently described the alternative transcripts of a novel human neuroendocrine-specific gene, designated NSP (Roebroek et al., J.Biol.Chem. 286,13439- 13447,1993). NSP transcription in neuroendocrine tissues results in 2 overlapping mRNA products, respectively of 3.4 kb and 1.8 kb. The function of the corresponding protein products is still unknown, but both proteins appear to be tightly attached to endoplasmic reticulum membranes through theircommoncarboxyterminalregion. Theirsubcellular localization and neuroendocrine-specific expression pattern raise the question as to whether the NSP proteins may constitute a novel class of neuroendocrine markers. As a first approach to resolve this question, we studied the NSP gene expression in lung cancer subtypes.

On Northern blot analysis, a vast majority (17/18) of classic and variant small cell lung carcinoma (SCLC) cell lines expressed either the 3.4 kb or the 1.8 kb NSP transcript or both. Remarkably, high levels of an additional 2.3 kb transcript were found in the variant NCI-H82 cell line. These transcripts were absent in the 11 tested squamous cell carcinoma and adenocarcinoma cell lines.

Immunohistochemical analysis of primary lung carcinomas was performed with a combination of monoclonal antibodies MON- 160, MON- 161 and MON-162, which recognize different epitopes uniquely present in the NSP protein encoded by the 3.4 kb transcript. A diffusely positive staining pattern was obtained in 3/3 bronchial carcinoid tumors and 5/12 SCLCs, whereas a more focal staining pattern was observed in 3/12 SCLCs. In non-small cell lung carcinomas focal staining was only observed in cases with known neuroendocrine features.

We conclude that NSP is specifically expressed in lung carcinomas with a neuroendocrine phenotype and that the NSP proteins may constitute useful additional neuroendocrine markers in lung cancer diagnosis.

083

ELASTOLYTIC ACTIVITY IN PRIMARY PULMONARY CARCINOMAS (PPC). S.J. Urbanski’, J. Luide?, M. Weave?, G.A.J. Gelfand:A. Maitla~&~ A. Hinekp Depts. of ‘Pathology, tiboratory Medicine, ISurgery, Foothills Hosp, Tom Baker Cancer Center, Calgary, AB;4Cardiovascular Research Unit, Hosp for Sick Children, Toronto, ON, CANADA.

Tumor invasion, phenotypic feature of malignancy is associated with local destruction of the extracellular mat& We have shown previously (Br. J. Cancer 1992;66:1188) that two gelatinases MMP-2 and -9, both of which have elastolytic activity (EA) are expressed in PFC at the mRNA level.

This analysis of EA in PPC was undertaken to correlate mRNA transcripts with functional proteins. We used primary PPC cultures where elastin degradation was analysed by ELISA when cells were grown on elastin membranes or free [‘HI was counted when [%I elastin was added to cells grown on plastic. Class specific inhibitors were used to identify class of pmteinases responsible for EA (EDTA and O- phenanthroline to block MMPs, Leupeptin - cysteine and PMSF - serine proteinases).

Substantial EA was shown in all 6 PPC and 1 metastatic carcinoma but not in metastatic sarcoma. EA was due to the action of serine and cysteine proteinases. MMPs inhibition showed drop in EA only in 1 case. Flow cytometric analysis ruled out a possibility that polymorphs are responsible for EA (no CD1 1 positivity).

PPC show uniform EA independent of the tumor histology. It results from the action of cysteine and serine proteinases. MMPs are of lesser significance in this process. We are at present investigating whether MMPs are uanslated but present in inactive form or whether their function is inhibited by overexpression of tissue inhibitors of MMPs - TIMP-1 and -2.

EVALUATION OF CK-BB MASS CONCENTRATION IN LUNG CANCER. P.Pi”so”, .J.Van Schoor, J.Delanghe, S.Mione, G..Joos, R.Pauwels. Univ.Hospital, Ghent, Belgium.

CK-BB was measured using a new two-site immunoenzymo- metric assay using a set of two monoclonal antibodies (Hybritech) directed against the B-subunit of the enzyme. Reference range of CK-BB in serum was O-2.8 "g/ml. Between- run CV of the method was 4.5 %.

Diagnostic performance of CK-BB was evaluated in a group of 124 patients with lung diseases : 50 lung turnours and 74 non-tumoral lung diseases. Upon diagnosis CK-BB values for small cell lung cancer (SCLC)(n=ZO) were 15.6+ 40.0 "g/ml (mean + SD) and for non-small cell lung cancer (NSCLC)(n=30) 2.75 3.1 "g/ml (SCLC vs NSCLC p < 0.05). In COPD patients (n=ZZ), CK-BB levels were comparable to the reference range : 2.320.9 "g/ml. Receiver-operating characteristics (ROC) analysis was performed and compared with the ROC-curve for neuron-specific enolase (NSE), soluble cytokeratin fragments (CYFRA 21-l) and carcino- embryonal antigen (CEA). At a cut-off level of 3.00 "g/ml, sensitivity and specificity for CK-BB were resp. 37 X and 96 X. For the whole group, ROC analysis showed an area under curve of 0.615 for CK-BB, which is comparable with the values for CEA (0.609), CYFRA 21-1 (0.641), but inferior to the value of NSE (0.873).

At diagnosis, none of the markers investigated were able to contribu'ce significantly to the prediction of prognosis in SCLC.

084

TIMP 1 GENE EXPRESSION IN RESECTED LUNG CANCER KM Fang- PV Zimmerman’, PJ Smith’. ‘Dept of Thoracic Medicine, The Prince Charles Hospital, ‘Queensland Cancer Fund Research Lab, University of Qld.. Brisbane, Australia

During the metastatic process, lung cancer invades through the basement membrane aided bycollagenase enzymes which degrade collagen. TlMPs (Tissue Inhibitor of Metalloproteinases) are the natural inhibitors of collagenases. TIMP 1 inhibits metastasis in cell and animal models, thus low levels would be expected in more invasive turnout-s.

We measured TIMP 1 gene activity using Northern Slot analysis of steady state total RNA levels in 46 resected non-small cell lung cancers (NSCLC). TIMP 1 RNA levels of each tumour was standardised for loading differences and calculated as a ratio compared to levels in adjacent uninvolved lung tissue. TIMP 1 expression was divided into low, normal and high groups as follows:-

TIMP 1 expression Ratio of normal luna No of cases low < 0.5 12 normal 0.5 - 1.5 21 high > 1.5 13

In the low expression group, there were 7 squamous cell carcinomas (SCCs), 2 adenocarcinomas (ACs) and in the high expression group, 3 SCCs, 8 ACs; a significant difference (p=O.O35, Fisher’s one-sided test). Although the low expression group had less early Tl stage (2/10) cases than the high expression group (6/13), this was not statistically significant. There was no significant difference in nodal involvement, TNM stage or survivti to a mean follow-up duration of 11 months between the low and high expression groups.

As ACs tend to be the most aggressive NSCLC subtype, the finding of more ACs in the high expression group may be due to less aggressive, high TIMP 1 tumours being selected for surgery. On the other hand, TIMP 1 gene expression levels alone did not correlate with tumour stage or progression. The balance between invasive factors and their inhibitors is likely to be more important in determining metastatic behaviour in individual turnours.