4th LTB W Abstracts/Lung Cancer 10 (1994) 347-373 353

Responsiveoess to multiple neuropeptides: acquisition during bronchial carcinogenesis and regulation in a small cell lung cancer (SCLC) cell line Frankel Aa, Tsao M-S’, Viallet J’*b. “Department ofMedicine, ‘Department of Oncology, ‘Department of Pathology, The Montreal General Hospital and McGill University, Montreal, Canada.

Previous work has demonstrated that human SCLC and NSCLC cell lines frequently show biological responsiveness to a broad variety of neuropeptides including bombesin (BOM), bradykinin (BRA), vasopressin (AVP), cholecystokinin (CCK) and neurotensin (NEU). We grew primary and immortalized cultures of normal human bronchial epithelial (NHBE) cells on coverslips and loaded them with the fluorescent calcium indica- tor Quin-2 in order to monitor for a transient increase in intra- cellular free calcium concentration ([Ca++]i) following exposure to the above neuropeptides. We observed responses at the following frequencies: BOM O/6; BRA 6/6; AVP O/6; CCK O/3; NEU l/5. These data indicate that mass cultures of NHBE cells have a limited responsiveness to neuropeptides. The fre- quent presence of such responses in lung cancer cell lines sug- gest that broad responsiveness to neuropeptides may be an important step in bronchial carcinogenesis.

Responses to neuropeptides are regulatable and may be amenable to pharmacological manipulation with consequences on lung cancer cell growth. The PKC activator PMA can acutely desensitize the BOM response in NCI-H345 SCLC cells and the recent description of the amino acid sequence of three BOM receptor genes shows the presence of potential PKC phosphorylation sites. These observations suggest that ligand stimulated activation of PKC may be involved in the homologous desensitization which is part of the prototypic re- sponse of SCLC cells to neuropeptides. We studied the regula- tion of the [Ca++]i response to BOM and AVP in NCI-H345 cells. Exposure of cells to increasing concentrations of PMA for 5 min leads to a progressive decrease in the amplitude of the [Ca++]i response to BOM and AVP with IC5Os of 13.7 f 10.6 nM and 1.3 f 0.6 nM, respectively; 100 nM PMA usually com- pletely abrogates the response. The PKC inhibitor staurosporine has no effects on these responses after a 5-min exposure to concentrations as great as 500 nM. However, as lit- tle as 10 nM staurosporine added for 5 min before exposure to 100 nM PMA leads to a partial recovery of the [Ca”]i responses to BOM and AVP. A dose-response to staurosporine was observed up to the highest concentration tested of 100 nM (0% vs. 74.1% of control for BOM and 0% vs 37.6% of control for AVP). In addition, incubation of cells for 48 h with 100 nM PMA led to an escape from the desensitizing effects of 100 nM PMA added for 5 min before the ligands (I 6.5% vs. 89% of con- trol for BOM; 3% vs. 69% of control for AVP). Neither acute pretreatment with staurosporine or prolonged exposure to PMA had any effect on the homologous desensitization to BOM or AVP despite the easily detectable effects on PMA in- duced desensitization. These data suggests that ligand induced PKC activation is not involved in homologous desensitization of neuropeptide responses in SCLC NCI-H345. Furthermore, there are quantitative differences in the regulation of the responses to BOM and AVP.

Immunopbenotypic changes associated with oeoplastic traosfor- mation of human respiratory tract epitbelium Franklin WA. Colorado Lung SPORE Tissue Bank Core Labo- ratory, University of Colorado Health Sciences Center, Denver, CO 80262.

Conventional histopathologic and cytological evaluation of specimens from patients at risk for lung cancer suggests that invasive tumor formation in the bronchial mucosa may be pre- ceded by a sequence of subtle morphologic abnormalities which occur over an extended period of time. Changes in the expres- sion of cell surface and nuclear macromolecules that may ac- company these morphological abnormalities have been the subject of increasing study but are still not well defined. The purpose of the present investigations has been to identify differ- ences in immunophenotype between invasive tumor and benign respiratory mucosa and to determine which of these differences are present in pre-invasive epithelium.

To accomplish this aim, we have employed a panel of mono- clonal antibodies against cell surface and nuclear antigens in a sensitive alkaline phosphatase-antialkaline phosphatase proce- dure to stain frozen sections of histologically normal respira- tory epithelium and in situ and invasive carcinomas of lung. The antibodies used in the panel are shown in Table 1 below.

Histologically normal respiratory mucosa. We have examined 39 samples of histologically normal bronchial mucosa and have found that specific epithelial cell populations within histologically normal respiratory mucosa have unique im- munophenotypes. For example, basilar cells strongly express EGFR but not RARa while ciliated and goblet cells are EGFR negative and RARo positive. In some instances, im- munophenotype is dependent on location within the respira- tory tract. In particular, epithelium at the level of the bronchiole strongly expresses FBP while this protein is focally unexpressed by proximal respiratory epithelium. Results of res- piratory epithelial immunophenotype analyses are summarized below. Neuroendocrine cells of normal respiratory epithelium are few in number and difficult to identify and for this reason their immunophenotype is incomplete at the present time.

invasive carcinoma. We have examined invasive lung car- cinomas of several histological types including four small cell lung carcinomas (SCLC) I5 squamous carcinomas (Sq Ca), 13 adenocarcinomas (Adeno Ca), and live poorly differentiated non-small cell carcinomas not easily classified which we have designated ‘Anaplastic’. Immunophenotypic changes in in- vasive tumors are both cell type specific and cell type nonspecific. Most tumors regardless of histological classitica- tion express increased amounts of Ki-67, TR, and RARo. All SCLC strongly express the NCAM cell surface marker but rare- ly express EGFR or FBP. Sq Ca strongly expresses EGFR but rarely express NCAM or FBP. A great majority of adenocar- cinemas strongly express FBP and many express EGFR. Most adenocarcinomas are NCAM negative. Finally, anaplastic non- small cell carcinomas focally express EGFP, NCAM and FBP. These last tumors thus exhibit immunophenotype evidence of mixed differentiation. The immunophenotypes of invasive tumors of various types are summarized in Table 2 below. The numbers of Ki-67 represent mean percentage of tumor cells positive. Numbers in the remaining columns represent propor-

354

tion of tumor which were immunoreactive in the series of Table 2, Immunophenotypic findings in non-neoplastic respir-

tumors reported here. atory mucosa During the course of these studies it was noted that uninvol-

ved peripheral bronchi from a few patients with invasive car- cinoma contain small clusters of cells unrecognizable in histological sections which are NCAM positive. These cells do not appear to represent fully developed neuroendocrine cells since staining of parallel sections with antichromogranin, a neurosecretory granule marker, fails to label these cells. They may represent partially differentiated neuroendocrine cells, the function and fate of which are unknown at the present time.

Cell type Ki-67 RARa TR EGFR NCAM FBP

Basilar <lo/o f - + - f Cells +

Ciliated/ <I% + - - - f goblet Cells +

Neuroen- < I% ? - ? + ? docrine Cells + (?)

Carcinoma in situ. In two cases of squamous carcinoma in situ (CIS) of the bronchus we have examined, im- munophenotypic changes intermediate between normal epithe- lium and invasive carcinoma are found (Table 3). These changes include increased expression of Ki-67, RARo and TR as well as increased expression of EGFR, reduced expression of FBP and absence of expression of NCAM.

Table 3. lmmunophenotype of invasive and in situ carcinoma

In summary, the cell surface and nuclear immunophenotype of invasive carcinoma of human lung differs distinctly from that of normal respiratory epithelium. These changes result in part from increased expression of cell cycle specific markers (i.e. Ki-67) transcription regulators (i.e. RARa) and growth factor receptors (i.e. EGFR, TR) and in part from variation in expression of differentiation antigens including FBP and NCAM. Expression of EGFR, NCAM and FBP generally cor- relates with histopathological classification of lung cancers. These preliminary studies suggest that immunophenotypic changes may occur early in neoplastic transformation of airway epithelium since these changes can be detected in CIS. Whether immunophenotypic differences described in these studies can be exploited for early detection and/or therapy is currently under investigation.

Histology Ki-67 RARo TR EGFR NCAM FBP

SCLC 86% 414 414 o/4 414 l/4 SqCa 45% 15115 15115 l4/15 3115 2115 Adeno Ca 26% 13/13 8113 II113 l/l3 l3/13 Anaplastic 60% 515 415 415 315 315 CIS 21% 212 212 212 o/2 o/2

Approaches to studying premelignant changes in the respiratory

epitbelium Gazdar AF. Simmons Cancer Cenrer and Department of Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX.

Lung cancers, both SCLC and NSCLC. are characterized by multiple genetic changes, including: (1) activation or over ex- pression of dominantly acting oncogenes; (2) loss or mutation of recessive growth regulatory genes (anti-oncogenes); (3) aneu- ploidy and multiple morphometric changes. Presently, there is little knowledge about the sequence of events in lung cancer. Newer approaches currently being used in our laboratory for the study of preneoplastic changes include (1) fluorescent in situ hybridization for interphase cytogenetics; microdissection of precisely identified small collections of cells; and ploidy and morphometric changes using a high resolution image analyzer.

Table I. Antibody panel

Antibody Antigen recognized Cellular position

Ki-67 (Dako) 9a (Gaubl

Rochette-Egly) Ber-T9 (Dako)

5G3,13A9,19C5 (Genetech)

NKH- I (Coulter)

FBP146,343,458, 7 IO (UCHSC)

Proliferation associated (Ki-67) Retinoic acid receptor a

(RARa) Transferrin receptor (TR)

Epidermal growth factor receptor (EGFR)

Neural cell adhesion molecule (NCAM)

Folate binding protein (FBP)

Nuclear Nuclear

Cell mem- brane Cell mem- brane Cell mem- brane Cell mem- brane

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

It is generally accepted that most adult epithelial cancers do not arise de novo, and that carcinogenesis is a multistep pro- cess. For centrally arising lung cancers (such as squamous cell and SCLC), the morphological steps include hyperplasia, metaplasia, dysplasia, carcinoma in situ (CIS), invasive car- cinoma and metastatic carcinoma. While the sequence for peripherally arising cancers (adeno- and large cell carcinomas) has not been established, they may be accompanied by hyperplastic and dsysplastic changes in peripheral airway cells.

The estimated number of mutations required for the develop- ment of invasive cancer is in double digits. How can so many lesions develop in a single cell? A possible explanation is the ‘field cancerization’ theory. The latter developed from observa- tions that the surface changes in head and neck cancers were ex- tensive, and that the incidence of synchronous, metachronous or second malignancies was very high, suggesting that much or all of the epithelium has undergone one or more genetic altera-