4th LTBW Abstracts/Lung Cancer 10 (1994) 347-373 361

those patients with Stage 11 disease, the survival of the BGAA- negative group was shorter (P = 0.048), with a median survival of 11 months versus 53 months for the BGAA-positive group. Recently, Miyake et al. reported that expression of a blood- group antigen H-related carbohydrate antigen was an impor- tant prognostic factor in patients with NSCLC (N Engl N Med 1992; 327: 14). The 5-year survival rate was 20.9% in the antigen-positive group and 58.6% in the antigen-negative group. For the study population overall, antigen positivity was the most important prognostic factor, followed by N stage and T stage. These results are quite exciting, not only because the antibody they used (i.e., MIA-15-5 antibody) recognizes H/Ley/Leb antigens which accumulate in tumors negative for blood-group antigens A and B, indirectly confirming previous results, but also because this antibody has shown strong in- hibitory effects on cell motility and the potential for metastasis. These Endings clearly support the idea that cell surface carbo- hydrate structures play an important role in tumor progression and heterogeneity. Furthermore, this MIA-15-5 antibody was found to bind to a receptor for an autocrine motility factor, providing a clue to the biologic consequences of altered expres- sion of blood-group antigens in tumor cells. It would be of fur- ther interest to examine the BGAA-negative cell lines to better understand the underlying biologic mechanisms that may be useful for future design of therapy and prevention strategies.

Peripheral airway cell differentiation in neoplastic and non- neoplastic lung Linnoila RI. Biomarkers and Prevention Research Branch, National Cancer Institute, NIH, Rockville, MD 20850.

There has been an increase in the incidence of lung adenocar- cinemas in the United States. While these tumors are a heterogenous group, in our experience one half of all pulmo- nary adenocarcinomas demonstrate features suggesting an ori- gin from peripheral lung. They are thought to originate from metaplastic mucin producing cells or peripheral airway cells, including bronchiolar non-ciliated secretory cells (Clara cells), and alveolar Type II cells. Clara cells and Type II cells are pro- genitor cells also for the non-neoplastic pulmonary epithehum. We have used two well-defined proteins produced by these cells, the Clara cell IO-kDa protein (CCIO) and the major sur- factant associated protein A (SPA), to study the role of peripheral airway cell differentiation in human lung car- cinogenesis.

Immunohistochemistry (IHC) revealed CC10 reactivity in the cytoplasm of nonciliated secretory cells throughout the con- ducting airways. Epithelial basal cells, and alveolar cells were negative. The expression of mRNA paralled the expression of protein. Most abundant mRNA was seen in bronchioli. In the presence of atypia, CC10 expression decreased in bronchi, and became detectable in up to 20% of alveoli at both mRNA and protein level. Changes were minimal in bronchioh. The extent of the atypia, including fibrosis, squamous metaplasia, alveolar epithelialization, dysplasia, and hyperplasias of basal, goblet, and type II cells correlated with smoking history. By IHC SPA was detected in alveolar Type II cells, and macrophages. Stain- ing was cytoplasmic. SPA mRNA expression was detected in scattered alveolar Type 11 cells, and rare cells in the conducting

airways. In the presence of atypia, increased expression of SPA both at protein and mRNA level was seen in clusters of reactive Type II cells around alveoli. These cells were frequently posi- tive for markers of proliferating cells, overexpressed c-myc but lacked ~53 immunoreactivity.

By IHC, up to 40% of adenocarcinomas followed by large cell (25%) and squamous cell carcinomas (16%) were positive for CC10 and/or SPA. Staining was mostly focal, and less in- tense than in non-neoplastic lung. Focal expression of SPA mRNA was detected in 5119 and CC10 mRNA in l/19 tumors by in situ hybridization. Expression of SPA was associated with female gender and lighter smoking history; CC10 with younger age. In a subset of earlier stage patients, SPA and CC10 expres- sion was of prognostic significance.

We also examined a panel of well characterized NSCLC cell lines for the expression of peripheral airway cell markers. Twenty-eight out of 52 (54%) cell lines expressed mRNA for SPA and/or CClO, but there was a poor correlation with the presence of characteristic ultrastructural features such as lamellar bodies or electron dense granules. The expression, as analyzed by RNA-RNA in-situ hybridization, was often focal, and 13 cell lines were positive for both markers.

We conclude that the two markers are expressed at high cellular levels in non-neoplastic lung, and the expression is decreased in neoplastic lung. Atypia is associated with altered marker expression evidenced by deviations from expected dis- tribution patterns.

Small cell lung cancer differentiation: Oocogene modulation by all-trm-retinoic acid Mabry M, Kalemkerian G, Jasti R. The Johns Hopkins On- cology Center, 424 North Bond Street, Baltimore, IUD 21231.

The precise determinants of tumor progression and treat- ment resistance in small cell lung cancer (SCLC) are unknown. However, one component may be the movement of the SCLC phenotype along a differentiation continuum, linking SCLC to the non-small cell lung cancer (NSCLC) tumors [l]. Clinical support for this concept comes from studies which have demonstrated a 25-40% prevalence of conversion from SCLC to a NSCLC phenotype during therapy [2]. Based on this paradigm; we developed a laboratory model of SCLC progres- sion using genetic manipulations to induce transition from a SCLC to a NSCLC phenotype [3,4].

Our model features the genetic manipulation of SCLC cells so that they acquire features of NSCLC. This model involves the insertion of genes which appear, based on phenotypic segre- gation, to be candidates for driving the transition process. A mutated ras gene was chosen because many NSCLC, but not SCLC, tumors have ras mutations [5) and exhibit increased ex- pression of p21 la’ proteins [6]. Insertion of the mutant v-rasH gene into SCLC cells which overexpress the c-myc (NCI-H82) or N-myc (NCI-H249) gene product, induces acquisition of NSCLC features [3,4]. This transition from a SCLC to a NSCLC phenotype is associated with increased growth rate, loss of neuroendocrine marker expression, acquisition of epithelial marker expression, such as CEA, cytokeratin and desmosomes, and acquired resistance to polyamine depletion via difluloromethylornithine (DFMO) [3,4]. This directly con-

362 4th LTB W Absrructs / Lung Cancer 10 (I 994) 347-373

trasts with the findings in cells which overexpress the L-myc gene. When v-ras is inserted into SCLC cells which overexpress L-myc (NCI-H209), no alterations in growth or phenotype are observed. To directly test whether c-myc is required, v-rasH has been inserted into SCLC cells into which a c-myc gene had been inserted and overexpressed (NCI-H209 myc). These cells acquire NSCLC features, while the parent cell line (NCI-H209), with L-myc, but not c-myc, overexpression, shows no phenotypic change after v-rus insertion. In summary, it appears that interactions between c-myc or N-myc and the v-rus” gene are competent to induce a transition from the SCLC to the NSCLC phenotype. However, L-myc is not competent to cause ras-induced phenotypic alterations in our system.

prior to the induction of, or selection for, additional genetic derangements which result in tumor progression.

1

2

3

Upon completion of these studies, we began to look for clinically relevant agents which could inhibit the induced phenotypic transition by modulating myc-family gene expres- sion, with the ultimate goal of altering the clinical course of SCLC. This approach is supported by clinical evidence which shows that c-myc gene amplification and overexpression are found more frequently in tumor samples from SCLC patients who previously received chemotherapy than from untreated pa- tients, and correlates with shortened survival [7].

4

5

Mabry M, Nelkin BD, Falco JP et al. Transitions between lung cancer phenotypes - implications for tumor progres- sion. Cancer Cells 1991; 3: 53-58. Abeloff MD, Eggleston JC, Mendelsohn G et al. Changes in morphologic and biochemical characteristics of small cell lung carcinoma of the lung: a clinicopathologic study. Am J Med 1979; 66: 757-764. Mabry M, Nakagawa T, Nelkin BD et al. v-Ha-ras onco- gene insertion: a model for tumor progression of human small cell lung cancer. Proc Nat1 Acad Sci USA 1988; 85: 6523-6527. Falco JP, Baylin SB, Lupo R et al. v-rus” induces non- small cell phenotype, with associated growth factors and receptors, in a small cell lung cancer cell line. J Clin Invest 1990; 85: 1740-1745. Mitsudomi T, Viallet J, Mulshine JL et al. Mutations of ras genes distinguish a subset of non-small-cell lung cancer cell lines from small-cell lung cancer cell lines. Oncogene 1991; 6: 1353-1363.

6 Retinoids are vitamin A analogs which inhibit cellular

growth and induce differentiation in various human malignant cell types in vitro [8]. Gebert et al. reported that alterations of retinoic acid receptor(RAR)-fl transcripts are common in lung cancer 191. Doyle et al. treated SCLC cells with all-trans- retinoic acid (RA) and reported decreased growth, decreased c- myc gene expression, and increased sensitivity to etoposide [IO]. These data suggest that alterations of retinoid signalling pathways may influence the biologic behavior of lung cancer.

7

8 These findings were applied to our culture model of SCLC

progression. As mentioned above, insertion of the activated v- rasH oncogene, into a c-myc overexpressing SCLC cell line, NCI-H82, results in acquisition of NSCLC features, including increased growth rate and increased resistance to cytotoxic agents (31. Treatment of the NCI-H82 cells with 10m6 M RA resulted in growth inhibition and downregulation of steady- state c-myc mRNA expression, as previously reported by Doyle et al. [IO]. In addition, RA treatment resulted in upregulation of steady-state L-myc and RARfi mRNA expression. Since pre- vious data in our phenotypic transition model suggested that c- myc, but not L-myc, overexpression is competent to cooperate with v-ras “, we postulated that pre-treatment of NCI-H82 cells with RA would inhibit the rus-induced acquisition of NSCLC features. Indeed, treatment of the NCI-H82 cells with 10e6 M RA prior to V-rus” insertion blocked the acquisition of NSCLC features [ 111. However, treatment of NCI-H82 cells with RA after rus-induced acquisition of NSCLC features could not reverse these features. In this situation, RA had no effect on cellular phenotype, growth, or myc gene expression WI.

Kurzrock R, Gallick GE, Gutterman J. Differential ex- pression of pztras gene products among histological sub- types of fresh primary human lung tumors. Cancer Res 1986; 46: 1530-1534. Brennan J, O’Connor T, Makuch RW et al. Myc family DNA amplification in 107 tumors and tumor cell lines from patients with small cell lung cancer treated with dif- ferent combination chemotherapy regimens. Cancer Res 1991; 51: 1708-1712. Lippman SM, Kessler JF, Meyskens FL. Retinoids as pre- ventive and therapeutic anticancer agents (part I). Cancer Treat Rep 1987; 71: 391-405. Gebert JF, Moghal N, Frangioni JV et al. High frequency of retinoic acid receptor beta abnormalities in human lung cancer. Oncogene 1991; 6: 1859-1868. Doyle LA, Giangiulo D, Hussain A et al. Differentiation of human variant small cell lung cancer cell lines to a classic morphology by retinoic acid. Cancer Res 1989; 49: 6745-6751. Mabry M, Jasti R, Finzi E et al. Retinoic acid receptor y is upregulated in small cell lung cancer tumor progression models. Proc Am Assoc Cancer Res 1991; 32: 283.

Recent studies on bronchiole-alveolar tumors in mice Malkinson AM. Colorado Cancer Cenrer. School of Pharmacy, University of Colorado, Denver, Colorado 80206.

These data suggest that continuous exposure to RA can inhibit SCLC growth and progression to a more aggressive and therapy-resistant phenotype via modulation of myc-family gene expression. These data also predict that efficacy would be highest if RA was administered with initial chemotherapy,

Because of several similarities between primary lung tumors in mice and human adenocarcinoma (AC), the rodent system can provide relevant information not otherwise readily at- tainable. These similarities include the following: (I) Cells of origin: Both murine and a large proportion of human AC demonstrate surfactant apoprotein gene expression and have morphological features of alveolar type 2 and bronchiolar Clara cells. (2) Genetic predisposition: Inbred strains of mice vary at each stage of tumor progression in whether they will ad- vance to the next step (normal - initiated; initiated -