Lung Cancer 10 (1994) 347-373

LUNG CANCER

Abstracts

4th IASLC Lung Tumor Biology Workshop

Arile, Virginia, 12- 15 April 1993

Variant morphology small cell lung cancer (SCLC): laboratory, morphologic, or clinical reality? Aisner SC. Depariment of Pathology, University of Maryland School of Medicine, University of Maryland at Baltimore, MD.

SCLC has been shown to have unique clinicopathological features including kinetic behavior, early metastases, and sensi- tivity to both chemotherapy and radiotherapy. To further define prognostic importance, attempts to classify SCLC into different subtypes has been studied in depth over the past three decades. These attempts included the WHO-I, the WPL, the WHO-II and the IASLC classification schema. The subtypes of SCLC defined by both the WHO-I (lymphocyte-like, poly- gonal, intermediate, and fusiform) or the WHO-II (oat cell and intermediate) did not show any significant prognostic value for SCLC subclassification. The WPL classification raised the issue of mixed histologies, and a retrospective review of several clini- cal studies suggested that small cell/large cell (variant) mor- phology defined an adverse prognosis. In-vitro cultures of selected biopsies were also identified which showed some radia- tion resistance, and grew in a dimorphic pattern suggestive of a small cell/large cell histology. The similarity between the in vitro growth and biology appeared to translate to the clinical setting. This cell pattern was termed variant and thus the name variant morphology SCLC was applied clinically in the IASLC classification. The clinical significance of this subtype, how- ever, continues to be controversial.

The true clinical or histologic occurrence of variant morphol- ogy SCLC is not known. This subtype has accounted for 4-14s of patients entered onto small cell lung cancer studies. Six large series incorporating 1393 patients over a IO-year period have evaluated the significance of the variant morpho- logy SCLC. Three early studies suggested an adverse prognosis among patients with Variant Morphology SCLC subtype. Three recent studies including 914 of the 1393 patients, how- ever, contradicted this suggestion.

Several distinct problems arise in attempting to define the clinical significance. First, diagnostic reproducibility such as

inter-rater variability for this subtype is poor. Second, the biop- sies for SCLC tend to be very small. Third, the predictability of the clinical course among patients with variant morphology SCLC is difftcult because of the rarity of the subtype. In review- ing the available information, these problems and the lack of defined histologic criteria or prognostic impact suggest that Variant Morphology SCLC currently should not be used clinically. Should biologic criteria such as oncogene markers better define a prognostic subgroup, then the potential clinical utility could be re-evaluated.

Mammalian bombesin receptor signalling in health and disease Battey J, Fathi Z, Wada E, Way J, Dobrzanski D, Sausville E. Laboratory of Biological Chemistry, DTP. DCT, National Cancer Institute, Bethesda MD 20892.

Bombesin is a tetradecapeptide originally isolated from the skin of the European frog Bombina bombina. Two mammalian bombesin like peptides have been identified and characterized: gastrin-releasing peptide (GRP) and neuromedin B (NMB). Bombesin-like peptides are associated with numerous physio- logic effects, including regulation of secretion, neuromodula- tion, and growth. Analysis of the growth stimulatory properties of bombesin-like peptides in Swiss 3T3 fibroblasts has been used to great advantage as a model system, providing general insight into the biochemical and genetic changes leading to cell proliferation.

Several observations indicate that growth stimulation by bombesin-like peptides plays a significant role in the pathogen- esis and progression of human small cell lung cancer (SCLC). Many human SCLC cell lines synthesize and secrete GRP and NMB. The clonal growth of some human SCLC cell lines is increased by addition of bombesin-like peptides. A neutralizing monoclonal antibody directed against bombesin, as well as potent bombesin receptor antagonists, attenuate growth of some human SCLC cells both in vitro and in vivo. More re- cently, similar studies indicate that bombesin may be an impor-

0169-5002/94/$07.00 Q 1994 Elsevier Science Ireland Ltd. All rights reserved. SSDI 0169-5002(93)00324-3

348 4th LTB W Abstracts/Lung Cancer 10 (1994) 347-373

tant autocrine or paracrine growth factor in breast and prostate

cancer. Our research has focused on the molecular mechanisms that

mediate and regulate bombesin-dependent signalling. We and others have cloned and characterized three pharmacologically distinct human bombesin receptor subtypes: GRP-R, NMB-R, and BRS-3. All three receptors have a structure typical of receptors whose signal transduction is coupled to guanine nucleotide-binding proteins (G-proteins). GRP-R, NMB-R, and BRS-3 share a biochemically similar signal transduction pathway including association with a pertussis-toxin insensitive G-protein, which activates phospholipase C, resulting in the hydrolysis of inositol trisphosphate, elevation of intracellular calcium, and activation of diacylglycerol-dependent protein kinase C. Responses elicited from all three bombesin receptor subtypes undergo rapid and complete desensitization by an un- known mechanism. Curiously, persistent activation of GRP-R or NMB-R with saturating levels of agonist peptides results in activation of phospholipase A, and release of arachidonic acid, as well as a profound cytostatic growth response whose mechanism remains to be defined. We are currently exploring which receptor domains are critical for high affinity ligand binding, effector coupling, and desensitization to define these properties of bombesin receptor signalling.

At present, it is clear that bombesin signalling in different cellular contexts involves at least two distinct ligands, three dif- ferent receptors, and activation of several distinct signal trans- duction pathways. A definition of the molecular interactions between molecules important in receptor signalling will hope- fully help to explain the pleiotropic nature of bombesin responses in different cells.

Molecular features of tumor cells expressing multidrug resistance due to alterations in DNA topoisomerase IIa Beck WT. Danks MK, Suttle DP, Kim R, Wolverton JS. Sr. Jude Children’s Research Hospital, Memphis. TN 38101.

Using human leukemic CEM cells selected for resistance VM-26 (teniposide) and cross-resistance to VP-16-213 (etopo- side), a drug used in the treatment of both leukemia and small cell lung cancer (SCLC), we studied the biochemical and molec- ular lesions associated with a form of ‘natural product’ multi- drug resistance (MDR) that is characterized by alterations in DNA topoisomerase II (topo II) activity or amount (at-MDR). Such cells display cross-resistance to many anticancer drugs that interact with topo II, but are not altered in drug accumula- tion, do not overexpress P-glycoprotein (Pgp), and are unaf- fected by Pgp modulators such as verapamil.

Resistance in the CEM cells is associated with decreased topo II activity. We showed that the cDNA encoding topo Ha had a point mutation in an ATP binding sequence that con- verted Ark, - Gln. Recently, other cell lines with altered topo II have also been shown to harbor mutations in and around this region of the topo IIa gene. Since we do not yet know whether expression of any of these mutations confer at- MDR in mammalian cells, we have used single-strand confor- mational polymorphism (SSCP) analysis to screen many cell

lines with an at-MDR phenotype to determine whether they also harbor mutations in selected regions (3 ATP binding sequ- ences and a DNA binding region) of the topo IIa gene. We have identified mutations by this method (confirmed by se- quencing) in -25% of the ‘at-MDR-like’ cell lines analyzed. Our results suggest that mutations in these regions of the struc- tural gene are not uncommon.

In other studies, we have found that traditional anti-top0 II drugs such as VM-26 stimulate c-jun transcription in drug- sensitive cells and that this increased expression of c-jun is at- tenuated in at-MDR cells in rough proportion to their resis- tance. In at-MDR cells, the rate of c-jun transcription and the stability of its mRNA were both decreased, compared to the drug-sensitive cells. These data suggest a basis for the diminish- ed induction of c-jun mRNA in at-MDR cells after treatment with VM-26, and permit the speculation that transcription fac- tors and early response genes may be important in mediating the cytotoxicity of topo II inhibitors and may also play a role in at-MDR.

The studies described above constitute part of our efforts to develop microdetection assays for drug resistant tumor cells. Other efforts to do the same involve development of single-cell assays. One currently under investigation is based on topo II immunostaining differences in cells with functional topo II, and stems from our observation that the heterogeneous pattern of topo II immunostaining becomes homogeneous and intense in a subpopulation of cells after treatment with topo II inhibitors, Importantly, this change in immunostaining is attenuated in at- MDR cells. We speculate that the stabilization of DNA-top0 II complexes protects the enzyme from proteolytic degradation and permits more topo II to be detected. We believe that this effect of topo II inhibitors on topo II immunostaining can be used to distinguish among drug-sensitive and-resistant cells. and we are currently developing the conditions to apply the assays to the tumor cells of patients. Results of these studies will be reported, as will studies of at-MDR expression in SCLC cell lines. (Supported in part by research grants CA-30103, CA- 40570, and CA-47941, program project grant CA-23099, and CORE grant CA-21765, all from NCI. Bethesda, MD, and in part by ALSAC)

K-ras and ~53 mutations in rodent lung tumors Belinsky SA. Inhalation Toxicology Research Institute. Albu- querque. New Mexico 87185.

The majority of human lung cancers observed are associated with the use of tobacco products. However, only two in 10 habitual smokers actually develop lung cancer. These statistics indicate that other genetic and environmental factors must con- tribute to the initiation and development of a lung cancer. Peo- ple are exposed to carcinogens in their home and workplace. Establishing the role of these exposures in the genesis of human lung cancer may be facilitated by the use of animal models, where the molecular mechanisms underlying lung tumor fot?ha- tion from a single agent or a combination of agents can be evaluated. In addition, using animal models, one can readily determine the temporal relationship between tumor develop- ment and a specific gene alteration. Work in our laboratory