843
SCIENCE & PRACTICE
Biology of small-cell lung cancer DESMOND N. CARNEY
The incidence of lung cancer continues to rise worldwide ; in the European Community it accounts for 29% of cancer deaths and 21 % of all deaths among men. Despite major advances in our understanding of the clinical behaviour and prognostic factors for the various histological subtypes of lung cancer, the five-year survival for newly diagnosed patients remains between 5% and 10%. Smallcell lung cancer (SCLC) accounts for a quarter of all new cases and tends to progress very quickly; even in patients who receive systemic chemotherapy, with or without radiation therapy, the median survival is only 11months or so.
Since the early 1980s, there have been substantial advances in our understanding of the biological properties of lung cancer cells. Such advances have arisen through studies of panels of well characterised cell lines of both SCLC and non-small-cell lung cancer (NSCLC). We now know that heterogeneity exists within a given tumour type and that there is considerable overlap between the biological properties of SCLC and NSCLC cells. In this paper I review the recently recognised biological properties of SCLC cells and their relevance to clinical presentation and management.
Genetic factors
Although cigarette smoking
is the
primary
permanent cell lines of both SCLC and NSCLC.4 In general, SCLC grows as floating aggregates that may be tightly or loosely packed, whereas NSCLC grows in monolayers of adherent cells. Once established as permanent cultures, these cell lines can be maintained indefinitely; they form colonies in soft agarose and can be cryopreserved. When cultured cells are transplanted into nude mice, the resultant tumours are morphologically and histologically similar to the original human tumour. Permanent cell lines of SCLC and NSCLC allow studies that have both clinical and prognostic importance, and can be set up in about one-third of cases.5 Does culturability itself tell us anything about the tumour? In an early retrospective study, the median survival of 19 newly diagnosed and previously untreated SCLC patients from whom cell lines were established at diagnosis was 14 weeks; the median survival of 123 extensive-stage patients from whom cell lines were not established was 48 weeks.6 Prospective studies, however, suggest that for SCLC (in contrast to NSCLC) there is no relation between culturability and patient survival. The only clinical variable that seems to be related to the ability to establish such lines is the extent of metastatic disease.
Neuroendocrine SCLC exogenous
causal factor in lung cancer, genetic influences have an important role in the pathogenesis and development of these tumours.1 In a study of 337 families, each identified through a lung cancer proband, a mendelian codominant inheritance pattern was found, suggesting a rare autosomal gene that promotes early onset of disease.2 Among patients under 50, 27% of lung cancers were attributable to the mendelian gene alone. However, in patients aged 70 or over, only 9% of lung cancers could be attributed to such a gene (whose existence remains to be confirmed). Individuals who extensively metabolise debrisoquine are at significantly greater risk of lung cancer than those who are poor or intermediate metabolisers.3 Debrisoquine is broken down by P-45011D6; the gene coding for this enyme, CYP2D6, is located on chromosome 22. Current data suggest that the ability to metabolise debrisoquine is a marker for carcinogen metabolism and may be a major determinant of susceptibility to lung cancer in smokers. The feasibility of developing a screening test for this marker is now being evaluated.
SCLC
can be subdivided into two major categoriesclassic and variant.’,’,8 Classic cell lines, which namely, account for 70% of the total, are characterised by high expression of neuroendocrine markers such as L-dopa
decarboxylase (DDC), bombesin/gastrin-releasing peptide (GRP), neuron-specific enolase (NSE), and creatine kinaseBB (CK-BB). They have a long doubling time, a poor ability to be cloned in vitro, are radiosensitive, and have the typical morphological characteristics of the intermediate cell type of SCLC (fig 1). By contrast, variant cell lines have selective loss of some of these neuroendocrine markers including DDC and GRP, though they do continue to express increased amounts of NSE and CK-BB. Variant SCLC cell lines grow more vigorously in agarose, have a shorter doubling time in vitro, are radioresistant, and resemble large-cell undifferentiated carcinoma (fig 2). Many variant SCLC lines have substantial amplification of the c-myc gene.
These neuroendocrine properties may have important clinical correlates. In a retrospective study, patients from
CelI lines The development of hormone-supplemented serum-free medium has greatly enhanced our ability to culture
ADDRESS Mater Misericordiae Hospital, Dublin 7, Ireland. (D N. Carney, PhD).
Eccles
Street,
844
Fig 1-Classic SCLC cells
in vitro.
The typical appearances of intermediate SCLC cells are seen. scanty cytoplasm, nuclear moulding, and mconspicuous nucleoli. Phase contrast x
80
whom variant cell lines were established had a survival of 33 weeks compared with 53 weeks among those from whom non-variant classic cell lines were set Up.55 Similar endocrine markers are found in NSCLC; the two tumour types may therefore arise from a common stem cell in the bronchial tree. Furthermore, both in-vitro and in-vivo evidence suggests that classic SCLC tumours can evolve into a phenotype resembling NSCLC; oncogene interactions can promote direct transition of SCLC to NSCLC. Prospective studies must address the clinical importance of these neuroendocrine markers in
carcinogenesis. In-vitro
chemosensitivity
One possible use for permanent lines of lung cancer cells is in screening programmes for new cytotoxic drugs. Moreover, mechanisms of drug resistance can be investigated in such in-vitro systems. Both SCLC and NSCLC cell lines show a pattern of in-vitro chemosensitivity and radiation sensitivity similar to that of in-vivo results.9,10 For instance, in SCLC cell lines obtained from 25 patients, median drug concentrations required to produce a reduction of at least 50% in cell growth for both etoposide and cisplatin in patients who achieved a response to chemotherapy were 7-8-fold lower than those of in-vitro of Thus, non-responders. 11 patterns chemosensitivity of permanantly established cell lines can be predictive of in-vivo responses. Furthermore, the chemosensitivity of SCLC cell lines seems to be stable. Prospective clinical studies have shown that chemosensitivity testing is feasible for the initial selection of antitumour agents." In-vitro tests correlate well with clinical response. Selection of chemotherapy based on in-vitro testing may extend survival among patients with limited-disease SCLC.13 No survival benefit was found in patients with severe disease. Although most SCLC tumours are initially chemosensitive, multidrug resistance tends to develop after In other cancers, drug resistance is frequently associated with overexpression of the 170 kDa Pglycoprotein coded for by the MDR1 gene. Lung cancer cells show low expression of P-glycoprotein, with the exception of chemosensitive NSCLC cell lines expressing neuroendocrine markers and chemoresistant carcinoids. 14 Drug resistance in lung cancer cells may also be related to other mechanisms, such as P450 isoenzymes, glutathione
relapse.
Fig 2-Variant SCLC cells
in vitro.
The appearances are more those of a large-cell undifferentiated carcinoma: more cytoplasm and prominent nucleoli. Phase contrast x 80.
metabolism, or altered topoisomerase activity; for most lung cancers the mechanisms of cytotoxic resistance remain unidentified. The in-vitro selection of cell lines highly resistant to several cytotoxic agents may allow a greater understanding of this process. SCLC
antigen expression
Several monoclonal antibodies have been generated against lung-cancer-associated antigens. 36 monoclonals raised against SCLC antigens have been grouped into eight clusters according to their reactivity with lung tumours, cell lines, and normal tissues. is No antigen is specific for SCLC or universally present in all specimens examined. Antibodies belonging to the major cluster, cluster 1, were found to be directed against the neural-cell adhesion molecule (NCAM), while the nature of the other antigens remained unclear with the exception of cluster 6, which was probably the LY antigen.’6 Studies of both SCLC and NSCLC cell lines have shown that NCAM expression is associated with the neuroendocrine phenotype, irrespective of the histological type of lung cancer. Monoclonal antibodies might have therapeutic value if coupled with a radionuclide or a toxin such as ricin. In addition, radiolabelled antibodies can detect minimal disease in bone-marrow aspirates and biopsy specimens, and might be able to purge SCLC tumour cells from bone marrow. No prognostic value has been identified for any specific antibody or pattern of antibodies. A prospective study is now in progress to evaluate the role of monoclonal antibodies in the early detection of lung cancer. Such early detection may also allow tumour localisation and targeted cytotoxic therapy at earlier stages of disease. 17
Molecular genetics
Lung cancer cells show not only mutations that activate dominant cellular proto-oncogenes but also genetic mechanisms that inactivate recessive tumour suppressors.1s Cytogenetic studies suggest that the genetic events associated with the development of lung cancer are multiple. The most common abnormality is a deletion on the short arm of chromosome 3, which is found in over 90% of cases of SCLC and in about 50% of NSCLC cases. Several candidate recessive oncogenes exist on 3p: the &bgr;-retinoic acid receptor gene, the zinc-fmger containing genes, and the
845
protein tyrosine phosphatase (PTP)-gamma
gene. The
missing from cells of lung cancer for a tumour-suppressing enzyme that encodes and patients Whether cell genes such as this, mapping growth. regulates to the deleted area on chromosome 3, are involved in the pathogenesis of lung cancer remains to be proven. Other sites of loss of heterozygosity include lip (related to the Wilms tumour gene), 13q, and 17p. Mutations in the retinoblastoma and p53 genes represent 13q and 17p lesions, respectively. The retinoblastoma gene and its protein product seem to be altered in nearly all cases of SCLC. The gene encoding human p53, a 393 aminoacid phosphoprotein PTP-gamma
gene is
the nucleus and seems to be present on 17p, is mutant in almost 100% of SCLCs and 50% or more of primary NSCLCs. Mutations cluster within five highly conserved domains and lead to synthesis of a protein product that abnormally interacts with the ras oncoprotein, binds to heat-shock protein, and has a long half-life. Deletions of chromosome 9 have also been found in SCLC cell lines and correspond to the interferon gene cluster series.19 The dominant oncogene mutations in SCLC are among the myc family. Amplification of c-myc, N-myc, and L-myc have been noted in cell lines and fresh biopsy material from SCLC patients. These abnormalities are late events in the pathogenesis of SCLC; they are recognised mainly in patients who have relapsed after previous intensive chemotherapy. The most common myc family abnormality is overexpression without amplification. 18 that localises chromosome
to
Tumour
progression
in
Repeat biopsy patients with SCLC who later relapse systemic chemotherapy frequently reveals a phenotype more consistent with NSCLC. A similar after induction
evolution is sometimes observed in cultured SCLC cells. Cooperation between oncogenes may be important in this transition.2O-22 Expression of an exogenous c-myc gene in classic SCLC cells is associated with an increased growth rate and cloning efficiency, but only subtle phenotypic changes can be seen. Overexpression of a mutated Harvey (Ha)-ras gene in classic SCLC cells that do not normally overexpress the c-myc or N-myc genes caused no phenotypic changes. By contrast, insertion of a mutated Ha-ras gene in SCLC cells that overexpress either an endogenously amplified c-myc or N-myc gene, or a transfected human c-myc gene, causes transition to the large-cell undifferentiated cancer phenotype. Phenotypic changes include: alteration in the typical growth pattern of SCLC cells, which change from floating aggregates to monolayer culture more typical of NSCLC cells; variable decreases in expression of neuroendocrine markers typical of SCLC; development of ultrastructural features more typical of NSCLC; and acquisition of drug-resistant patterns typical of NSCLC. Finally, the transition to the large-cell undifferentiated carcinoma phenotype is associated with altered growth requirements for these tumour cells. The myc/ras-gene-induced transition of SCLC to the large-cell phenotype takes place in conjunction with a fall in cellular gastrin/bombesin-releasing peptide production; cells become much less dependent on this hormone for growth. These cells also express large quantities of epidermal growth factor receptor and begin to produce transforming growth factor-a. These genes and their protein products are not normally expressed in SCLC, but are found in NSCLC tumour lines. In-vitro studies show that SCLC cells can undergo transition to an NSCLC
phenotype and that such transition may be brought about by the effects of multiple oncogenes and their products. These molecular events could be of major importance to our understanding of mechanisms of SCLC carcinogenesis. Growth factors insulin-like growth important autocrine growth
Bombesin/gastrin-releasing peptide, factor-1, and transferrin
are
factors for SCLC. Recent studies have shown that other neuropeptides can stimulate the clonal growth of this tumour--eg, bradykinin, neurotensin, cholecystokinin, and vasopressin. These neuropeptides stimulate a rapid and transient increase in intercellular calcium concentration. The combined effects of calcium mobilisation and SCLC cell growth suggest that such growth may be sustained by several peptides functioning in both an autocrine and a paracrine fashion.23 Exogenous agents to inhibit or block effects of a single neuropeptide would be of limited value to interrupt tumour growth. Several studies have reported inhibition of lung tumour growth, both in vitro and in vivo, with analogues of bombesin. Bombesin/GRP has previously been shown to be a potent growth factor for SCLC. In addition, previous studies have shown that monoclonal antibodies that bind exogenous free bombesin could inhibit both the in-vitro and in-vivo growth of SCLC tumours. Mahmoud et al24 have shown that bombesin analogues that bind to the GRP receptor in SCLC cells, but which do not activate signal transduction, can disrupt both GRPstimulated growth of these tumour cells in vitro and growth of SCLC xenografts in athymic nude mice; these effects were dose dependent. Neuropeptide antagonists with a broad range of inhibitory function on SCLC cell growth may be of greater therapeutic benefit. Woll and Rozengurt found that analogues of substance P can inhibit SCLC growth in soft agarose and in liquid culture.25 The specific analogueArg,6 D- Trp,7,9 MePhe8-blocked receptor-mediated calcium mobilisation induced by a range of different peptides and thus offers the possibility of being able to disrupt effects of mutiple neuropeptides regulating SCLC growth. The characterisation of other autocrine growth factors for SCLC will help the design of new therapeutic
strategies. The effects of many biological response modifiers on lung cell growth have been tested extensively in vitro. With few exceptions, none of these have been shown to stimulate SCLC growth. However, the introduction of human granulocyte colony-stimulating factors to prevent myelosuppression in patients receiving chemotherapy may be of some concern. Granulocyte colony-stimulating factor receptors have been found on SCLC cells; some studies have shown that SCLC colony formation was enhanced by granulocyte colony-stimulating factor.26 The meaning of these studies remains unclear; few major adverse effects have been reported in clinical trials. cancer
Conclusion The past decade has seen major advances in our understanding of the biological features of both SCLC and NSCLC cells. Studies of tumour cell lines have revealed their biochemical properties, the influence of autocrine/ paracrine growth factors, the importance of antigen expression, and an understanding of the molecular genetics of lung cancer cells. Survival and cure of patients with SCLC will probably only come from such studies. The most
846
important issues will include an evaluation of the clinical importance of the neuroendocrine properties of lung cancer cells; the application of monoclonal antibodies to both diagnostic imaging and directed therapy; the characterisation of mechanisms of drug resistance; and how the effects of growth factors can be modified for therapeutic benefit. Finally, identification of the specific (tumour suppressor) genes that are deleted from chromosome 3 should allow opportunities to directly manipulate factors regulating SCLC growth. REFERENCES 1. Law MR. Genetic predisposition to lung cancer. Br J Cancer 1990; 61: 195-206. 2. Sellers TA, Bailey-Wilson JE, Elston RC, et al. Evidence for mendelian inheritance in the pathogenesis of lung cancer. J Natl Cancer Inst 1990;
82: 1272-79. 3.
Tucker MA, Hoover RN, et al. Lung cancer and the debrisoquine metabolic phenotype. J Natl Cancer Inst 1990; 82:
Caporaso NE, 1264-71.
De Leij L. Lung cancer biology. Semin Oncol 1988; 15: 199-214. 5. Stevenson HC, Gazdar AF, Linnoila RI, et al. Lack of relationship between in vitro tumour cell growth and prognosis in extensive-stage small-cell lung cancer. J Clin Oncol 1989; 113: 923-31. 6. Johnson BE, Ihde DC, Makuch RW, et al. Myc family oncogene amplification in tumour cell lines established from small cell lung cancer patients and its relationship to clinical status and outcome. J Clin Invest 1987; 79: 1629-32. 7. Camey DN, Gazdar AF, Belper G, et al. Establishment and identification of small cell lung cancer cell lines having classic and variant features. Cancer Res 1985; 45: 2913-23. 8. Gazdar AF, Carney DN, Nau M, et al. Characterization of variant sub-classes of cell lines derived from small cell lung cancer having distinctive biochemical, morphological and growth properties. Cancer Res 1985; 45: 2914-30. 9. Carney DN, Mitchell JR, Kinsella TJ. In vitro radiation and chemosensitivity of established cell lines of human small cell lung cancer and its large cell variants. Cancer Res 1983; 43: 2806-11. 10. Carmichael J, Mitchell JB, Degraff WG, et al. Chemosensitivity testing of human lung cancer cell lines using the MTT assay. Br J Cancer 1988; 57: 540-47. 11. Tsai CM, Ihde DC, Kadoyama C, et al. Correlation of in vitro sensitivity testing of long-term small cell lung cancer cell lines with response and survival. Eur J Cancer 1990; 26: 1148-52. 4.
Carney DN,
AF, Steinberg SM, Russell EK, et al. Correlation of in vitro drug-sensitivity results with response to chemotherapy and survival in extensive-stage small cell lung cancer: a prospective clinical trial. J Natl
12. Gazdar
Cancer Inst 1982; 82: 117-24.
Johnson BE, Salem C, Nesbitt J, et al. Limited stage small cell lung cancer (SCLC) treated with concurrent BID chest radiotherapy (RT) and etoposide/cisplatin (VP/PT) followed by chemotherapy (CT) selected by in vitro drug sensitivity testing (DST). Proc ASCO 1991; 10: 240. 14. Lai SL, Goldstein LJ, Gottesman MM, et al. MDR1 gene expression in lung cancer. J Natl Cancer Inst 1989; 81: 1114-50. 15. Souhami RL, Beverley PCL, Bobrow LG, et al. Antigens of lung cancer: results of the second international workshop on lung cancer antigens. J Natl Cancer Inst 1991; 83: 609-12. 16. Patel K, Moore SE, Dickson G, et al. Neural cell adhesion molecule (NCAM) is the antigen recognized by monoclonal antibodies of similar specificity in small-cell lung carcinoma and neuroblastoma. Int J 13.
Cancer 1989; 44: 573-78. 17. Mulshine J, Linnoila RI, Jensen S, et al. Application of monoclonal antibodies for use in the early detection and management of lung cancer. Second International Workshop on Small Cell Lung Cancer Antigens, London 1990. 18. Minna J, Maneckjee R, D’Amico D, et al. Mutations in dominant and recessive oncogenes, and the expression of opioid and nicotine receptors in the pathogenesis of lung cancer. Proc AACR 1991; 32: 445-46. 19. Oiopade OI, Buchagen DL, Minna JD, et al. Deletions of the short arm of chromosome 9 that include the alpha and beta interferon genes. Proc AACR 1991; 32: 1814. 20. Mabry M, Nelkin BD, Falco JP, et al. Transitions between lung cancer phenotypes—implications for tumour progression. Cancer Cells 1991; 3: 53-58. 21. Mabry M, Nakagawa T, Baylin S, et al. Insertion of the v-Ha-ras oncogene induces differentiation of calcitonin producing human small cell lung cancer. J Clin Invest 1990; 85: 1740-45. 22. Falco JP, Baylin SB, Lupu R, et al. V-ras induces non-small phenotype, with associated growth factors and receptors, in a small cell lung cancer cell line. J Clin Invest 1990; 85: 1740-45. 23. Bunn PA Jr, Dienhart DG, Chan D, et al. Neuropeptide stimulation of calcium flux in human lung cancer cells: delineation of alternative pathways. Proc Natl Acad Sci USA 1990; 87: 2162-66. 24. Mahmoud S, Staley J, Taylor J, et al. Inhibition of lung tumour growth by bombesin analogues. Cancer Res 1991; 51: 1798-802. 25. Woll PJ, Rozengurt E. A neuropeptide antagonist that inhibits the growth of small cell lung cancer in vitro. Cancer Res 1990; 50: 3968-73. 26. Vallenga E, Biesma B, Meyer C, et al. The effects of five haematopoietic growth factors in human small cell lung carcinoma cell lines: interleukin 3 enhances the proliferation in one of the given cell lines. Cancer Res
1991; 51: 73-76.
Management of small-cell cancer of the lung HEINE H. HANSEN
Striking changes have taken place in the treatment of patients with small-cell lung cancer (SCLC) during the past two decades. Before the 1970s, surgery and radiotherapy were the most common forms of treatment. Subsequent clinical studies showed that SCLC metastasises early and widely; moreover, this tumour was found to be sensitive to various cytotoxic agents. These observations provided the basis for introducing cytotoxic drugs into treatment regimens. Initial results with such agents, with a 4-5-fold increase in median survival, led to high hopes that there would be further improvements in clinical outcome similar
No standard treatment for SCLC exists. I shall discuss the present range of management options for patients with SCLC. To a large degree my conclusions will be based on the consensus reports from the Third Workshop on SCLC arranged by the International Association for the Study of Lung Cancer.12
those obtained with other chemosensitive tumours, such and testicular cancer malignant lymphoma. this did not Unfortunately, happen and the past decade has seen no change in the overall results of treatment. New developments in the biology of SCLC are described by Carney in the accompanying article. Whether these advances will translate into clinical benefit remains to be
(75-80%), but not always, because small-cell carcinoma is often characterised by submucosal growth. The identification of SCLC among the various types of lung to be reasonably straightforward for cancer seems
to as
seen.
Diagnosis The diagnosis of small-cell lung cancer is based on the presence of typical cytological or histological features. Cytological examination of sputum will usually be positive
ADDRESS: Department of Oncology, The Finsen Institute/ Rigshospitalet, DK-2100 Copenhagen, Denmark (Dr H. H. Hansen,
MD)
SCIENCE & PRACTICE
Biology of small-cell lung cancer DESMOND N. CARNEY
The incidence of lung cancer continues to rise worldwide ; in the European Community it accounts for 29% of cancer deaths and 21 % of all deaths among men. Despite major advances in our understanding of the clinical behaviour and prognostic factors for the various histological subtypes of lung cancer, the five-year survival for newly diagnosed patients remains between 5% and 10%. Smallcell lung cancer (SCLC) accounts for a quarter of all new cases and tends to progress very quickly; even in patients who receive systemic chemotherapy, with or without radiation therapy, the median survival is only 11months or so.
Since the early 1980s, there have been substantial advances in our understanding of the biological properties of lung cancer cells. Such advances have arisen through studies of panels of well characterised cell lines of both SCLC and non-small-cell lung cancer (NSCLC). We now know that heterogeneity exists within a given tumour type and that there is considerable overlap between the biological properties of SCLC and NSCLC cells. In this paper I review the recently recognised biological properties of SCLC cells and their relevance to clinical presentation and management.
Genetic factors
Although cigarette smoking
is the
primary
permanent cell lines of both SCLC and NSCLC.4 In general, SCLC grows as floating aggregates that may be tightly or loosely packed, whereas NSCLC grows in monolayers of adherent cells. Once established as permanent cultures, these cell lines can be maintained indefinitely; they form colonies in soft agarose and can be cryopreserved. When cultured cells are transplanted into nude mice, the resultant tumours are morphologically and histologically similar to the original human tumour. Permanent cell lines of SCLC and NSCLC allow studies that have both clinical and prognostic importance, and can be set up in about one-third of cases.5 Does culturability itself tell us anything about the tumour? In an early retrospective study, the median survival of 19 newly diagnosed and previously untreated SCLC patients from whom cell lines were established at diagnosis was 14 weeks; the median survival of 123 extensive-stage patients from whom cell lines were not established was 48 weeks.6 Prospective studies, however, suggest that for SCLC (in contrast to NSCLC) there is no relation between culturability and patient survival. The only clinical variable that seems to be related to the ability to establish such lines is the extent of metastatic disease.
Neuroendocrine SCLC exogenous
causal factor in lung cancer, genetic influences have an important role in the pathogenesis and development of these tumours.1 In a study of 337 families, each identified through a lung cancer proband, a mendelian codominant inheritance pattern was found, suggesting a rare autosomal gene that promotes early onset of disease.2 Among patients under 50, 27% of lung cancers were attributable to the mendelian gene alone. However, in patients aged 70 or over, only 9% of lung cancers could be attributed to such a gene (whose existence remains to be confirmed). Individuals who extensively metabolise debrisoquine are at significantly greater risk of lung cancer than those who are poor or intermediate metabolisers.3 Debrisoquine is broken down by P-45011D6; the gene coding for this enyme, CYP2D6, is located on chromosome 22. Current data suggest that the ability to metabolise debrisoquine is a marker for carcinogen metabolism and may be a major determinant of susceptibility to lung cancer in smokers. The feasibility of developing a screening test for this marker is now being evaluated.
SCLC
can be subdivided into two major categoriesclassic and variant.’,’,8 Classic cell lines, which namely, account for 70% of the total, are characterised by high expression of neuroendocrine markers such as L-dopa
decarboxylase (DDC), bombesin/gastrin-releasing peptide (GRP), neuron-specific enolase (NSE), and creatine kinaseBB (CK-BB). They have a long doubling time, a poor ability to be cloned in vitro, are radiosensitive, and have the typical morphological characteristics of the intermediate cell type of SCLC (fig 1). By contrast, variant cell lines have selective loss of some of these neuroendocrine markers including DDC and GRP, though they do continue to express increased amounts of NSE and CK-BB. Variant SCLC cell lines grow more vigorously in agarose, have a shorter doubling time in vitro, are radioresistant, and resemble large-cell undifferentiated carcinoma (fig 2). Many variant SCLC lines have substantial amplification of the c-myc gene.
These neuroendocrine properties may have important clinical correlates. In a retrospective study, patients from
CelI lines The development of hormone-supplemented serum-free medium has greatly enhanced our ability to culture
ADDRESS Mater Misericordiae Hospital, Dublin 7, Ireland. (D N. Carney, PhD).
Eccles
Street,
844
Fig 1-Classic SCLC cells
in vitro.
The typical appearances of intermediate SCLC cells are seen. scanty cytoplasm, nuclear moulding, and mconspicuous nucleoli. Phase contrast x
80
whom variant cell lines were established had a survival of 33 weeks compared with 53 weeks among those from whom non-variant classic cell lines were set Up.55 Similar endocrine markers are found in NSCLC; the two tumour types may therefore arise from a common stem cell in the bronchial tree. Furthermore, both in-vitro and in-vivo evidence suggests that classic SCLC tumours can evolve into a phenotype resembling NSCLC; oncogene interactions can promote direct transition of SCLC to NSCLC. Prospective studies must address the clinical importance of these neuroendocrine markers in
carcinogenesis. In-vitro
chemosensitivity
One possible use for permanent lines of lung cancer cells is in screening programmes for new cytotoxic drugs. Moreover, mechanisms of drug resistance can be investigated in such in-vitro systems. Both SCLC and NSCLC cell lines show a pattern of in-vitro chemosensitivity and radiation sensitivity similar to that of in-vivo results.9,10 For instance, in SCLC cell lines obtained from 25 patients, median drug concentrations required to produce a reduction of at least 50% in cell growth for both etoposide and cisplatin in patients who achieved a response to chemotherapy were 7-8-fold lower than those of in-vitro of Thus, non-responders. 11 patterns chemosensitivity of permanantly established cell lines can be predictive of in-vivo responses. Furthermore, the chemosensitivity of SCLC cell lines seems to be stable. Prospective clinical studies have shown that chemosensitivity testing is feasible for the initial selection of antitumour agents." In-vitro tests correlate well with clinical response. Selection of chemotherapy based on in-vitro testing may extend survival among patients with limited-disease SCLC.13 No survival benefit was found in patients with severe disease. Although most SCLC tumours are initially chemosensitive, multidrug resistance tends to develop after In other cancers, drug resistance is frequently associated with overexpression of the 170 kDa Pglycoprotein coded for by the MDR1 gene. Lung cancer cells show low expression of P-glycoprotein, with the exception of chemosensitive NSCLC cell lines expressing neuroendocrine markers and chemoresistant carcinoids. 14 Drug resistance in lung cancer cells may also be related to other mechanisms, such as P450 isoenzymes, glutathione
relapse.
Fig 2-Variant SCLC cells
in vitro.
The appearances are more those of a large-cell undifferentiated carcinoma: more cytoplasm and prominent nucleoli. Phase contrast x 80.
metabolism, or altered topoisomerase activity; for most lung cancers the mechanisms of cytotoxic resistance remain unidentified. The in-vitro selection of cell lines highly resistant to several cytotoxic agents may allow a greater understanding of this process. SCLC
antigen expression
Several monoclonal antibodies have been generated against lung-cancer-associated antigens. 36 monoclonals raised against SCLC antigens have been grouped into eight clusters according to their reactivity with lung tumours, cell lines, and normal tissues. is No antigen is specific for SCLC or universally present in all specimens examined. Antibodies belonging to the major cluster, cluster 1, were found to be directed against the neural-cell adhesion molecule (NCAM), while the nature of the other antigens remained unclear with the exception of cluster 6, which was probably the LY antigen.’6 Studies of both SCLC and NSCLC cell lines have shown that NCAM expression is associated with the neuroendocrine phenotype, irrespective of the histological type of lung cancer. Monoclonal antibodies might have therapeutic value if coupled with a radionuclide or a toxin such as ricin. In addition, radiolabelled antibodies can detect minimal disease in bone-marrow aspirates and biopsy specimens, and might be able to purge SCLC tumour cells from bone marrow. No prognostic value has been identified for any specific antibody or pattern of antibodies. A prospective study is now in progress to evaluate the role of monoclonal antibodies in the early detection of lung cancer. Such early detection may also allow tumour localisation and targeted cytotoxic therapy at earlier stages of disease. 17
Molecular genetics
Lung cancer cells show not only mutations that activate dominant cellular proto-oncogenes but also genetic mechanisms that inactivate recessive tumour suppressors.1s Cytogenetic studies suggest that the genetic events associated with the development of lung cancer are multiple. The most common abnormality is a deletion on the short arm of chromosome 3, which is found in over 90% of cases of SCLC and in about 50% of NSCLC cases. Several candidate recessive oncogenes exist on 3p: the &bgr;-retinoic acid receptor gene, the zinc-fmger containing genes, and the
845
protein tyrosine phosphatase (PTP)-gamma
gene. The
missing from cells of lung cancer for a tumour-suppressing enzyme that encodes and patients Whether cell genes such as this, mapping growth. regulates to the deleted area on chromosome 3, are involved in the pathogenesis of lung cancer remains to be proven. Other sites of loss of heterozygosity include lip (related to the Wilms tumour gene), 13q, and 17p. Mutations in the retinoblastoma and p53 genes represent 13q and 17p lesions, respectively. The retinoblastoma gene and its protein product seem to be altered in nearly all cases of SCLC. The gene encoding human p53, a 393 aminoacid phosphoprotein PTP-gamma
gene is
the nucleus and seems to be present on 17p, is mutant in almost 100% of SCLCs and 50% or more of primary NSCLCs. Mutations cluster within five highly conserved domains and lead to synthesis of a protein product that abnormally interacts with the ras oncoprotein, binds to heat-shock protein, and has a long half-life. Deletions of chromosome 9 have also been found in SCLC cell lines and correspond to the interferon gene cluster series.19 The dominant oncogene mutations in SCLC are among the myc family. Amplification of c-myc, N-myc, and L-myc have been noted in cell lines and fresh biopsy material from SCLC patients. These abnormalities are late events in the pathogenesis of SCLC; they are recognised mainly in patients who have relapsed after previous intensive chemotherapy. The most common myc family abnormality is overexpression without amplification. 18 that localises chromosome
to
Tumour
progression
in
Repeat biopsy patients with SCLC who later relapse systemic chemotherapy frequently reveals a phenotype more consistent with NSCLC. A similar after induction
evolution is sometimes observed in cultured SCLC cells. Cooperation between oncogenes may be important in this transition.2O-22 Expression of an exogenous c-myc gene in classic SCLC cells is associated with an increased growth rate and cloning efficiency, but only subtle phenotypic changes can be seen. Overexpression of a mutated Harvey (Ha)-ras gene in classic SCLC cells that do not normally overexpress the c-myc or N-myc genes caused no phenotypic changes. By contrast, insertion of a mutated Ha-ras gene in SCLC cells that overexpress either an endogenously amplified c-myc or N-myc gene, or a transfected human c-myc gene, causes transition to the large-cell undifferentiated cancer phenotype. Phenotypic changes include: alteration in the typical growth pattern of SCLC cells, which change from floating aggregates to monolayer culture more typical of NSCLC cells; variable decreases in expression of neuroendocrine markers typical of SCLC; development of ultrastructural features more typical of NSCLC; and acquisition of drug-resistant patterns typical of NSCLC. Finally, the transition to the large-cell undifferentiated carcinoma phenotype is associated with altered growth requirements for these tumour cells. The myc/ras-gene-induced transition of SCLC to the large-cell phenotype takes place in conjunction with a fall in cellular gastrin/bombesin-releasing peptide production; cells become much less dependent on this hormone for growth. These cells also express large quantities of epidermal growth factor receptor and begin to produce transforming growth factor-a. These genes and their protein products are not normally expressed in SCLC, but are found in NSCLC tumour lines. In-vitro studies show that SCLC cells can undergo transition to an NSCLC
phenotype and that such transition may be brought about by the effects of multiple oncogenes and their products. These molecular events could be of major importance to our understanding of mechanisms of SCLC carcinogenesis. Growth factors insulin-like growth important autocrine growth
Bombesin/gastrin-releasing peptide, factor-1, and transferrin
are
factors for SCLC. Recent studies have shown that other neuropeptides can stimulate the clonal growth of this tumour--eg, bradykinin, neurotensin, cholecystokinin, and vasopressin. These neuropeptides stimulate a rapid and transient increase in intercellular calcium concentration. The combined effects of calcium mobilisation and SCLC cell growth suggest that such growth may be sustained by several peptides functioning in both an autocrine and a paracrine fashion.23 Exogenous agents to inhibit or block effects of a single neuropeptide would be of limited value to interrupt tumour growth. Several studies have reported inhibition of lung tumour growth, both in vitro and in vivo, with analogues of bombesin. Bombesin/GRP has previously been shown to be a potent growth factor for SCLC. In addition, previous studies have shown that monoclonal antibodies that bind exogenous free bombesin could inhibit both the in-vitro and in-vivo growth of SCLC tumours. Mahmoud et al24 have shown that bombesin analogues that bind to the GRP receptor in SCLC cells, but which do not activate signal transduction, can disrupt both GRPstimulated growth of these tumour cells in vitro and growth of SCLC xenografts in athymic nude mice; these effects were dose dependent. Neuropeptide antagonists with a broad range of inhibitory function on SCLC cell growth may be of greater therapeutic benefit. Woll and Rozengurt found that analogues of substance P can inhibit SCLC growth in soft agarose and in liquid culture.25 The specific analogueArg,6 D- Trp,7,9 MePhe8-blocked receptor-mediated calcium mobilisation induced by a range of different peptides and thus offers the possibility of being able to disrupt effects of mutiple neuropeptides regulating SCLC growth. The characterisation of other autocrine growth factors for SCLC will help the design of new therapeutic
strategies. The effects of many biological response modifiers on lung cell growth have been tested extensively in vitro. With few exceptions, none of these have been shown to stimulate SCLC growth. However, the introduction of human granulocyte colony-stimulating factors to prevent myelosuppression in patients receiving chemotherapy may be of some concern. Granulocyte colony-stimulating factor receptors have been found on SCLC cells; some studies have shown that SCLC colony formation was enhanced by granulocyte colony-stimulating factor.26 The meaning of these studies remains unclear; few major adverse effects have been reported in clinical trials. cancer
Conclusion The past decade has seen major advances in our understanding of the biological features of both SCLC and NSCLC cells. Studies of tumour cell lines have revealed their biochemical properties, the influence of autocrine/ paracrine growth factors, the importance of antigen expression, and an understanding of the molecular genetics of lung cancer cells. Survival and cure of patients with SCLC will probably only come from such studies. The most
846
important issues will include an evaluation of the clinical importance of the neuroendocrine properties of lung cancer cells; the application of monoclonal antibodies to both diagnostic imaging and directed therapy; the characterisation of mechanisms of drug resistance; and how the effects of growth factors can be modified for therapeutic benefit. Finally, identification of the specific (tumour suppressor) genes that are deleted from chromosome 3 should allow opportunities to directly manipulate factors regulating SCLC growth. REFERENCES 1. Law MR. Genetic predisposition to lung cancer. Br J Cancer 1990; 61: 195-206. 2. Sellers TA, Bailey-Wilson JE, Elston RC, et al. Evidence for mendelian inheritance in the pathogenesis of lung cancer. J Natl Cancer Inst 1990;
82: 1272-79. 3.
Tucker MA, Hoover RN, et al. Lung cancer and the debrisoquine metabolic phenotype. J Natl Cancer Inst 1990; 82:
Caporaso NE, 1264-71.
De Leij L. Lung cancer biology. Semin Oncol 1988; 15: 199-214. 5. Stevenson HC, Gazdar AF, Linnoila RI, et al. Lack of relationship between in vitro tumour cell growth and prognosis in extensive-stage small-cell lung cancer. J Clin Oncol 1989; 113: 923-31. 6. Johnson BE, Ihde DC, Makuch RW, et al. Myc family oncogene amplification in tumour cell lines established from small cell lung cancer patients and its relationship to clinical status and outcome. J Clin Invest 1987; 79: 1629-32. 7. Camey DN, Gazdar AF, Belper G, et al. Establishment and identification of small cell lung cancer cell lines having classic and variant features. Cancer Res 1985; 45: 2913-23. 8. Gazdar AF, Carney DN, Nau M, et al. Characterization of variant sub-classes of cell lines derived from small cell lung cancer having distinctive biochemical, morphological and growth properties. Cancer Res 1985; 45: 2914-30. 9. Carney DN, Mitchell JR, Kinsella TJ. In vitro radiation and chemosensitivity of established cell lines of human small cell lung cancer and its large cell variants. Cancer Res 1983; 43: 2806-11. 10. Carmichael J, Mitchell JB, Degraff WG, et al. Chemosensitivity testing of human lung cancer cell lines using the MTT assay. Br J Cancer 1988; 57: 540-47. 11. Tsai CM, Ihde DC, Kadoyama C, et al. Correlation of in vitro sensitivity testing of long-term small cell lung cancer cell lines with response and survival. Eur J Cancer 1990; 26: 1148-52. 4.
Carney DN,
AF, Steinberg SM, Russell EK, et al. Correlation of in vitro drug-sensitivity results with response to chemotherapy and survival in extensive-stage small cell lung cancer: a prospective clinical trial. J Natl
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Johnson BE, Salem C, Nesbitt J, et al. Limited stage small cell lung cancer (SCLC) treated with concurrent BID chest radiotherapy (RT) and etoposide/cisplatin (VP/PT) followed by chemotherapy (CT) selected by in vitro drug sensitivity testing (DST). Proc ASCO 1991; 10: 240. 14. Lai SL, Goldstein LJ, Gottesman MM, et al. MDR1 gene expression in lung cancer. J Natl Cancer Inst 1989; 81: 1114-50. 15. Souhami RL, Beverley PCL, Bobrow LG, et al. Antigens of lung cancer: results of the second international workshop on lung cancer antigens. J Natl Cancer Inst 1991; 83: 609-12. 16. Patel K, Moore SE, Dickson G, et al. Neural cell adhesion molecule (NCAM) is the antigen recognized by monoclonal antibodies of similar specificity in small-cell lung carcinoma and neuroblastoma. Int J 13.
Cancer 1989; 44: 573-78. 17. Mulshine J, Linnoila RI, Jensen S, et al. Application of monoclonal antibodies for use in the early detection and management of lung cancer. Second International Workshop on Small Cell Lung Cancer Antigens, London 1990. 18. Minna J, Maneckjee R, D’Amico D, et al. Mutations in dominant and recessive oncogenes, and the expression of opioid and nicotine receptors in the pathogenesis of lung cancer. Proc AACR 1991; 32: 445-46. 19. Oiopade OI, Buchagen DL, Minna JD, et al. Deletions of the short arm of chromosome 9 that include the alpha and beta interferon genes. Proc AACR 1991; 32: 1814. 20. Mabry M, Nelkin BD, Falco JP, et al. Transitions between lung cancer phenotypes—implications for tumour progression. Cancer Cells 1991; 3: 53-58. 21. Mabry M, Nakagawa T, Baylin S, et al. Insertion of the v-Ha-ras oncogene induces differentiation of calcitonin producing human small cell lung cancer. J Clin Invest 1990; 85: 1740-45. 22. Falco JP, Baylin SB, Lupu R, et al. V-ras induces non-small phenotype, with associated growth factors and receptors, in a small cell lung cancer cell line. J Clin Invest 1990; 85: 1740-45. 23. Bunn PA Jr, Dienhart DG, Chan D, et al. Neuropeptide stimulation of calcium flux in human lung cancer cells: delineation of alternative pathways. Proc Natl Acad Sci USA 1990; 87: 2162-66. 24. Mahmoud S, Staley J, Taylor J, et al. Inhibition of lung tumour growth by bombesin analogues. Cancer Res 1991; 51: 1798-802. 25. Woll PJ, Rozengurt E. A neuropeptide antagonist that inhibits the growth of small cell lung cancer in vitro. Cancer Res 1990; 50: 3968-73. 26. Vallenga E, Biesma B, Meyer C, et al. The effects of five haematopoietic growth factors in human small cell lung carcinoma cell lines: interleukin 3 enhances the proliferation in one of the given cell lines. Cancer Res
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Management of small-cell cancer of the lung HEINE H. HANSEN
Striking changes have taken place in the treatment of patients with small-cell lung cancer (SCLC) during the past two decades. Before the 1970s, surgery and radiotherapy were the most common forms of treatment. Subsequent clinical studies showed that SCLC metastasises early and widely; moreover, this tumour was found to be sensitive to various cytotoxic agents. These observations provided the basis for introducing cytotoxic drugs into treatment regimens. Initial results with such agents, with a 4-5-fold increase in median survival, led to high hopes that there would be further improvements in clinical outcome similar
No standard treatment for SCLC exists. I shall discuss the present range of management options for patients with SCLC. To a large degree my conclusions will be based on the consensus reports from the Third Workshop on SCLC arranged by the International Association for the Study of Lung Cancer.12
those obtained with other chemosensitive tumours, such and testicular cancer malignant lymphoma. this did not Unfortunately, happen and the past decade has seen no change in the overall results of treatment. New developments in the biology of SCLC are described by Carney in the accompanying article. Whether these advances will translate into clinical benefit remains to be
(75-80%), but not always, because small-cell carcinoma is often characterised by submucosal growth. The identification of SCLC among the various types of lung to be reasonably straightforward for cancer seems
to as
seen.
Diagnosis The diagnosis of small-cell lung cancer is based on the presence of typical cytological or histological features. Cytological examination of sputum will usually be positive
ADDRESS: Department of Oncology, The Finsen Institute/ Rigshospitalet, DK-2100 Copenhagen, Denmark (Dr H. H. Hansen,
MD)
