An immunohistochemical investigation of diagnostic biopsy material taken from short and long term survivors with small cell lung cancer.

An immunohistochemical study has been carried out on fibre optic-biopsy specimens from patients with small cell lung cancer (SCLC) who had either died within 3 months, or who had survived more than 2 years. Long term survivors (LTS) were identified from completed clinical trials at major UK centres and were matched for age and sex within the trial with short term survivors (STS). The panel of immunohistochemical markers included those previously reported to be associated with prognosis, and reagents representative of both neuroendocrine and epithelial differentiation. A preliminary screen of 17 antibodies identified 11 as consistently reactive on paraffin-embedded material using streptavadin-biotin immunoperoxidase. Of 186 identified patients, 110 biopsy samples were retrieved. Of these, 70 gave sufficient material for analysis. All sections were scored by three observers without knowledge of the prognosis. The analysis failed to identify any antigen whose expression was correlated with prognosis. We conclude that, in fibre-optic biopsy specimens, immunohistochemical analysis does not add prognostic information in SCLC.

Lack of expression of P-glycoprotein in 7 small cell lung cancer cell lines established both from untreated and from treated patients.

The establishment and characterisation of 7 small cell lung cancer cell lines is described. Four cell lines were established from biopsies taken from untreated patients and one of these was from primary tumour taken via the fibreoptic bronchoscope. The other three were from biopsies taken from patients who had relapsed after chemotherapy. All grow as non-adherent aggregates of cell and express a range of neuroendocrine and epithelial features characteristics of small cell lung cancer cell lines. No relationship was observed between the characteristics of the cell line in vitro and the treatment status of the patient from whom the biopsy was taken. Furthermore, none of the cell lines expressed p-glycoprotein even though 3 were derived from relapse biopsies where chemotherapy included drugs associated with the multidrug resistance phenotype.

Modulation of the cluster 1 and mucin antigens in human small cell lung cancer and other epithelial tumour cell lines after treatment with the differentiation inducing agent, sodium butyrate.

The expression of the human small cell lung cancer (SCLC) cluster 1 antigen and the human milk fat globule membrane (HMFG) antigen were studied in three SCLC, three lung adenocarcinoma, six ovarian adenocarcinoma and three colorectal adenocarcinoma cell lines before and after culture in the presence of the differentiation inducing agent, sodium butyrate. Before treatment, only SCLC and well differentiated ovarian cell lines expressed the cluster 1 antigen. After 4 days culture with sodium butyrate, expression of cluster 1 antigen was induced in poorly differentiated ovarian, colorectal and lung adenocarcinoma cell lines whereas existing antigen expression was enhanced in SCLC and well differentiated ovarian cell lines. The expression of the HMFG antigen was also modified. Induction of cluster 1 antigen correlated with increased levels of alkaline phosphatase in the treated cell lines, this enzyme being associated with a more differentiated state in colon and ovarian carcinoma cell lines. The pattern of induction of cluster 1 antigen expression suggests that several different epitopes of this antigen are recognised by the ten SCLC cluster 1 antibodies studied.

Establishment and characterisation of two new small cell lung cancer cell lines--one from a patient with previous familial retinoblastoma.

Two new small cell lung cancer (SCLC) cell lines have been established. The first, WX330, has been developed from a pleural effusion collected at presentation from a patient with extensive disease. The second, WX331, originated from a liver metastasis collected at post mortem from a patient who died of his SCLC disease. WX330 has been in culture for 4 years and WX331 for 11 months. Both cell lines have typical cytological features of SCLC, WX330 contains dense core granules on electron microscopic examination and both are tumourigenic in nude mice. Cell lines were analysed phenotypically using antibodies submitted to the Second International Workshop on SCLC Antigens by the immunoperoxidase method. WX330 is reactive with a wide range of SCLC and epithelial associated and WX331 with a more restricted group of antibodies of predominantly neuroendocrine and SCLC specificity. WX330 also reacts with an antibody against the multiple drug resistance-associated glycoprotein.

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 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 (MOV15), 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.

Characterisation and properties of a small cell lung cancer cell line and xenograft WX322 with marked sensitivity to alpha-interferon.

Controversy exists as to whether interferons usefully influence the growth of epithelial carcinomas. A small cell lung carcinoma (SCLC) cell line, WX322, has been derived which is greater than 1000-fold more sensitive to alpha-interferon (IFN) when grown in agar than other reported SCLC cell lines. The WX322 line has been characterised to prove its epithelial origin and its chemosensitivity compared with that of the NCI-H69 small cell line. The WX322 cell line expresses neuroendocrine and epithelial markers and possesses a morphology consistent with SCLC origin. A concentration of 5 IU ml-1 of IFN produced 50% inhibition of colony formation in agar in the WX322 line, whereas a concentration of greater than 10(5) IU ml-1 was required to produce a comparable effect with the NCI-H69 cell line. In contrast, WX322, possessed similar sensitivity to NCI-H69 cells when exposed to a range of cytotoxic agents. Analysis of the cell cycle indicated that IFN increased the percentage of cells in the G0/G1 phase for the WX322 cell line but increased the percentage in S phase for the NCI-H69 line. Growth of the xenograft, from which the cell line was derived, was also inhibited by IFN at doses greater than 10(5) IU/mouse/day. The WX322 cell line whether grown in agar or as a xenograft shows an unusually high sensitivity to IFN and provides an interesting model for studying mechanisms of IFN cytotoxicity to epithelial cells.

Immunocytological detection of residual marrow disease at clinical remission predicts metastatic relapse in small cell lung cancer.

A panel of monoclonal antibodies against neural and epithelial associated antigens was used to examine bone marrow from patients in clinical remission from small cell lung cancer (SCLC). A standard peroxidase-antiperoxidase technique and Ficoll-Hypaque enrichment were used to detect SCLC-like cells at the 1-2% level of contamination in 8 of 12 patients who were disease free by conventional criteria, including routine marrow cytology and histology and endobronchoscopic biopsy or cytology. Six of these patients ultimately relapsed, with metastatic sites found between 2 and 6 months after restaging. Furthermore, 6 patients had undergone chemointensification including autologous marrow rescue with radical irradiation to the primary lung tumor. Four of these 6 subsequently relapsed, also with metastatic sites. Of the 4 patients without bone marrow metastases at restaging using this technique, 2 relapsed, with cells found at the primary site, and 2 remained in complete remission. Serum free cell culture was attempted in 9 of 12 cases and SCLC-like cell colonies grew, in suspension, in 4. The SCLC-like nature of these cells has been confirmed by electron microscopy in 1 case and by repeat immunocytochemistry for small cell associated antigens in 3 cases. Bone marrow positivity using these techniques appears to predict a high risk of metastatic relapse regardless of further therapy.

Effect of sodium butyrate and other differentiation inducers on poorly differentiated human ovarian adenocarcinoma cell lines.

We have studied the effects of sodium butyrate, retinoic acid, and dimethyl sulfoxide on two human ovarian carcinoma cell lines PE04 and PE01. PE04 cells, after treatment with sodium butyrate at cytostatic doses (2-3 mM for 4 days), exhibited phenotypic changes including induction of alkaline phosphatase and determinants recognized by the monoclonal antibodies 123C3 and 123A8. These effects are not simply the result of cytostasis as they were not produced by dimethyl sulfoxide or retinoic acid. Other markers are also modified by sodium butyrate including lipid, acid mucin, and glycogen. Retinoic acid modulated expression of lipid and CA125, while dimethyl sulfoxide reduced expression of CA125. Other short chain fatty acids such as propionic acid and valeric acid (in addition to butyric acid) also induced alkaline phosphatase and the determinants recognized by 123C3 and 123A8 in PE04 cells. Other differentiation inducers and cytotoxic agents studied did not induce these markers at cytostatic concentrations. The effects of sodium butyrate (and related short chain fatty acids) thus appear to be relatively specific for this cell line.

Characterization and properties of nine human ovarian adenocarcinoma cell lines.

Four series of cell lines have been derived from patients with ovarian adenocarcinoma. Nine cell lines have been established at one from a solid metastasis. Six lines were derived from the ascites or pleural effusion of patients with poorly differentiated adenocarcinoma: PEO1, PEO4, and PEO6 from one patient, PEA1 and PEA2 from a second, and PEO16 from a third. Three lines (PEO14 and PEO23 from ascites and TO14 from a solid metastasis) were derived from a patient with a well-differentiated serous adenocarcinoma. Each set of cell lines was morphologically distinct. The five cell lines PEO1, PEO4, PEO6, PEA1, and PEA2 had cloning efficiencies on plastic of 1-2% and only a few cells in these lines expressed alkaline phosphatase or vimentin. Only a low percentage of these cells reacted with the monoclonal antibodies 123C3 and 123A8 but most reacted with OC125. Conversely the cell lines PEO14, TO14, PEO23, and PEO16 were characterized by low cloning efficiency values (less than 0.05%), marked expression of alkaline phosphatase and vimentin, and good reaction with 123C3 and 123A8 but not OC125. These four cell lines also exhibited dome formation. Four of the cell lines, PEO1, PEO4, PEO6, and PEO16, have been xenografted into immune-deprived mice and found to be tumorigenic.

Expression of human milk fat globule antigens HMFG1 and HMFG2 on ovarian tumours and lung tumours.

The patterns of reactivity of the monoclonal antibodies HMFG1 and HMFG2 were studied in epithelial ovarian tumours, small cell lung cancer, and non-small cell lung cancer. Twenty primary, paraffin embedded, ovarian tumours were analysed in addition to freshly obtained ascitic fluids (21), a node aspirate (1), solid metastatic tumours (3), and 6 established cell lines. SCLC tumours comprised paraffin embedded bronchial biopsies (31) and metastatic deposits (7) and fresh tissue from bronchial biopsies (3), pleural aspirates (4), bone marrows (5), node aspirates (6), solid metastatic tumour (3), and 4 cultured cell lines. Antigen expression was heterogeneous for all tumour types studied. However, significant differences in antigen expression were noted with the HMFG2 antibody between primary and metastatic lesions from both ovarian and small cell lung cancer groups.

Clinical applications of immunocytochemistry in the monitoring of the bone marrow in small cell lung cancer (SCLC).

A panel of monoclonal antibodies (MAbs) has been identified which may be useful in detecting SCLC tumour cells and which does not cross-react significantly with normal marrow elements. Compared with conventional techniques this MAb panel markedly increases detection of micrometastases in the bone marrow especially in limited disease and post-chemotherapy in responding patients. There is a close correlation between immunological positivity and the ability to grow SCLC-like cells for variable periods in serum-free culture. In 4 cases SCLC-like cells have been characterized by electron microscopy or by the establishment of SCLC cell lines in continuous culture. The prognostic importance of pre-treatment detection of marrow involvement is unknown. The implications of post-chemotherapy detection of micrometastases are also uncertain but there may be an association with early systemic relapse which may occur regardless of intervention, including high dose intensification with autologous marrow rescue with primary site and CNS prophylactic irradiation.

Prognosis in small cell carcinoma of the lung--relationship to human milk fat globule 2 (HMFG2) antigen and other small cell associated antigens.

Forty fixed tissue sections from patients with small cell lung carcinoma (SCCL) have been stained with a panel of 10 monoclonal antibodies using a peroxidase anti-peroxidase method and the incidence of staining has been compared to patient characteristics at presentation and to survival. An inverse association between HMFG2 staining and survival was found with median survival in HMFG2 negative patients 13 months compared to 8 months for HMFG2 positive patients. No such association was found with the other antibodies and no association was found between staining and disease extent or primary versus secondary deposits with this panel of antibodies. Epidermal growth factor receptor was detected in 3/38 presentation biopsies and in these 3 patients mean survival was only 5 months. Further prospective study of HMFG2 as a prognostic indicator in SCCL is suggested.

Protective effect of sodium-2-mercaptoethanesulfonate on the gastrointestinal toxicity and lethality of cis-diamminedichloroplatinum.

cis-Diamminedichloroplatinum (cis-platinum) is an effective and widely used antitumor drug. Patients receiving cis-platinum, however, experience very profound and long lasting gastrointestinal symptoms. The role of intestinal mucosal toxicity in the pathogenesis of these symptoms is unclear. In this study we have investigated the thiol-containing compound mesna (sodium-2-mercaptoethanesulfonate) as a potential antidote to cis-platinum-induced gastrointestinal tract damage. In mice, mesna caused a significant reduction in the gastrointestinal toxicity of cis-platinum assessed by electron microscopy, villus recovery rate, and by disaccharidase estimations. Mesna also significantly reduced serum creatinine levels following cis-platinum. Administration of mesna prior (or immediately following) a 67% lethal dose of cis-platinum protected 87-100% of the animals from the lethal effects. The antitumor efficacy of cis-platinum in L1210 leukemia bearing mice was not affected by coadministration of mesna indicating that the protective effect may be tissue specific. In addition this finding indicates that mesna has potential as an agent which may improve the therapeutic index of cis-platinum in clinical practice.