(CANCER RESEARCH 50. 6095-6099, September 15. 1990]

The Distinction of Small Cell and Non-Small Cell Lung Cancer by Growth inNative-State Histoculture1

R. A. Vescio, K. M. Connors, G. M. Bordin, J. A. Robb, T. Youngkin, J. N. Umbreit, and R. M. Hoffman2

AntiCancer Incorporated, San Diego, California 92110 [K. A. V., K. M. C., R. M. H.J; Laboratory of Cancer Biology, Department of Pediatrics, University of California,San Diego School of Medicine, M-009-F, La Jolla, California 92093 ¡R.M. H.j; Scripps Clinic and Research Foundation, La Jolla, California 92037 [G. M. B.,J. A. R.]; Mercy Hospital and Medical Center, San Diego, California 92103-2180 [T. Y.¡;and University of South Alabama Cancer Center, University of South Alabama,Mobile, Alabama 36688 fj. yV.U.J

ABSTRACT

Histological analysis remains the primary method of distinguishingbetween small cell (SCLC) and non-small cell lung cancer (NSCLC).This distinction has significant impact therapeutical!}' because of their

relative difference in chemoresponsiveness (J. D. Minna et al.. Principlesand Practice of Oncology, pp. 396-474, 1981). Yet for at least 10% oflung tumors, pathologists will disagree upon the classification (A. R.Feinstein et al.. Am. Rev. Respir. Dis., 101: 671-684, 1970). Furthermore, current neuroendocrine markers lack specificity for SCLC althoughthe presence of these markers may help predict chemosensitivity (S. L.Graziano et al., J. Clin. Oncol., 7: 1375-1376, 1989; S. B. Baylin, J.Clin. Oncol., 7:1375-1376, 1989; C. L. Berger et al., J. Clin. Endocrinol.Metab., 53:422-429, 1981; A. F. Gazdar et al., Cancer Res., 45: 2924-2930, 1985). In vitro growth characteristics may more accurately reflectbiological properties of aggressiveness and susceptibility to chemotherapy. In this study, 3-dimensional gel-histoculture was used to retrospectively distinguish between NSCLC and SCLC. Tumor expiants from 78patients with NSCLC and 13 patients with SCLC were grown in gel-supported histocultures with an overall success rate of 92%. These 2tumor types were distinguishable by their 3-dimensional in vitro tissuearchitecture. In addition, proliferation rates were measured by histológica! autoradiography after 4-day incorporation of |'11 |<l1lid. The percent

age of cells labeled in the most proliferatively active regions of theautoradiograms was termed the growth fraction index (A. F. Gazdar etal.. Cancer Res., 45: 2924-2930, 1985; R. A. Vescio et al., Proc. Nati.Acad. Sci. U. S. A., 84: 5029-5033, 1987; R. M. Hoffman et al., Proc.Nati. Acad. Sei. U. S. A., 86: 2013-2017, 1989). The mean growthfraction index for pure small cell lung cancer was 79 ±10%, differingmarkedly from that of 35 ±19% for mixed small cell/large cell tumors,adenocarcinoma (38 ±16%), large cell undifferentiated carcinoma (40 ±18%), and squamous cell carcinoma (33 ±15%) ( /' < 0.001 in eachcase). We therefore conclude that 3-dimensional gel-histoculture is auseful means of distinguishing pure SCLC from NSCLC, which mayimprove treatment decision making.

bility in classification occurs relatively frequently (3). Small celllung tumors have been identified by their neuroendocrine properties, yet significant overlap in the presence of these biologicalmarkers exists (4-8). In fact, a recent study demonstratedincreased chemoresponsiveness for NSCLC tumors possessingneuroendocrine markers (4). SCLC is further divided into 3subtypes, "pure" small cell, mixed small cell/large cell, and

combined small cell carcinoma (9). Recent studies have showna poorer response to chemotherapy and shorter survival inpatients with the mixed subtype (10). Clearly, biological andhistological markers that distinguish characteristics predictiveof general chemoresponsiveness would aid treatment decisionmaking.

With a goal of distinguishing biologically between small celland non-small cell lung carcinoma, we have utilized the nativestate histoculture system developed by us that successfullycultures human solid tumors at a high success rate (11-14). Inthe native-state system, tumor expiants are grown on 3-dimensional collagen-containing gels in histocultures whereby prolif-erative cells are labeled with ['H]dThd. Autoradiographs are

then made from tissue sections to determine cellular proliferation by grain formation due to [3H]dThd incorporation. The

percentage of labeled cells is then measured to determine theGFI. We have previously shown the correlation of GFI withstage and grade in breast and ovarian carcinomas (13).

In this report, we demonstrate that the growth characteristicsof small cell lung tumors differ markedly from those of non-small cell lung cancer. The GFI of small cell lung cancer ismarkedly elevated and can be used along with the histoculturegrowth patterns to reliably distinguish small cell from non-small cell lung cancer. We also demonstrate the ability to growlung tumors in vitro from 92% of the samples obtained.

INTRODUCTION

Lung carcinomas are clinically divided into two major types:SCLC3 and NSCLC (1). For patients with lung carcinoma, the

accurate determination of tumor type significantly influencestreatment decision making. In general, SCLC is much moreresponsive to chemotherapy and consequently this comprisesthe mainstay of treatment. This is in contrast to NSCLC, whichincludes adenocarcinomas, squamous cell carcinomas, and largecell undifferentiated carcinomas of the lung. NSCLC is relatively chemoresistant and thus primarily treated with surgicalresection for local disease (1, 2). Histological differences between these tumor types can be subtle and interobserver varia-

Received 1/18/90; accepted 6/6/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' This study was supported by National Cancer Institute Small Business

Innovation Research Grant R44 CA44 CA43411, National Cancer Institute GrantR01-CA27564. and American Cancer Society Grant PDT 330.

2To whom requests for reprints should be addressed.' The abbreviations used are: SCLC, small cell lung cancer; NSCLC, non-

small cell lung cancer; GFI. growth fraction index.

MATERIALS AND METHODS

Tumor Tissue Acquisition. Tumor samples were obtained from various hospitals in the San Diego region as part of a study to determinewhich in vitro biological parameters correlate with clinical data. Inaddition, specimens came from Alabama and Los Angeles via overnightdelivery systems.

Tumor Histoculture. Tumors were removed surgically and placed intoculture medium (Eagle's minimal essential medium containing Earle's

salts, glutamine, 10% fetal calf serum, nonessential amino acids, andgentamicin) by the pathologist and transported to the laboratory.

The tumors were minced into 1- to 2-mm-diameter pieces andplanted on previously hydrated collagen-containing gel matrices(Health Design Industries, Rochester, NY) within 48 h of removal fromthe patient. Six 1-mm' pieces of tissue, each representing a different

region of the tumor, were explanted atop one 1 x 2 cm gel to overcomesampling error due to tumor heterogeneity. After allowing tumor cellgrowth in culture for 10 days, [3H]dThd (4 MCi/ml; 1 Ci = 37 GBq)

was added to label replicating cells for 4 additional days.The cultures were then washed with phosphate-buffered saline,

placed in histology capsules, and fixed in 10% formalin overnight. Thehistocultures were consequently dehydrated and rinsed with paraffin.The gel was then placed in an embedding cassette, then embedded so

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SMALL CELL VS. NON-SMALL CELL LUNG CANCER—HISTOLOGICAL ANALYSIS

that all 6 pieces of tumor were at the front of the paraffin block. Theblock was then sectioned using a microtome so that a continuous ribbonof 5-¿imsections was made, each containing 6 representative pieces oftumor. Approximately 10-15 sections of the ribbon were placed oneach slide for autoradiographic processing and staining. Two slideswere made of each condition. The slides were deparaffinized and thencoated with Kodak NTB-2 emulsion in a dark room and allowed toexpose for 5 days at 4°Cbefore developing. After rinsing, the slides

were stained with hematoxylin and eosin.Determining GFI. The slides were then analyzed in a blinded fashion

with a microscope epi-illuminated with light polarized by an ICS cube(Nikon). Replicating cells were easily determined by the presence ofbright green-reflecting silver grains over the cell nuclei that had incorporated ('H)dThd. Cells were considered labeled if 10 or more grains

were present over the cell nucleus. Benign stromal cells were excludedfrom the measurement by their morphological appearance. Whencounting, each slide was scanned at x 40 or x 100 power to locate theareas of maximum label. Once these areas were ascertained, the percentage of cells undergoing DNA synthesis was determined in at least3 of the visual fields for each tumor piece at x 200. The GFI wascalculated by dividing the number of labeled tumor cells by the numberof total tumor cells.

Acquisition of Pathology Reports. The final pathology report wasused to determine stage, grade, and histological class. Tumors weredetermined unclassifiable if the histological class was equivocal or didnot fall into the following types (pure small cell, mixed small cell/largecell, squamous cell, large cell, or adenocarcinorna). Five bronchioalveo-lar cell tumors were cultured and were classified as adenocarcinomasfor this report.

Photomicrographs. The slides were reanalyzed at x 200 and x 400in a blinded fashion. Photographs were taken of at least 3 separateareas showing the greatest cell labeling and then grouped by tumornumber. Two observers were shown representative photographs of smallcell and non-small cell lung tumors that were taken at an earlier timeand not used later. They were then asked to group individual tumorsby patterns of cell labeling and tumor invasion into the gel, by reviewingthe photographs.

RESULTS

Differentiation of Small versus Non-Small Cell Tumors byGFI. Tumors were obtained from 9 patients with pure smallcell lung cancer. All specimens came from the primary tumorand all 9 tumors were successfully cultured in vitro. Large areasof tumor growth into the collagen gel were evident in each case,with a high percentage of cell labeling. The GFI was calculatedfor each small cell lung tumor specimen and ranged from 67%to 93% with a mean of 79% (SD ±10%) (Fig. 1).

Four additional tumors were identified as being of the mixedsmall cell/large cell subtype of SCLC by the criteria of theInternational Association for the Study of Lung Cancer (9).These tumors were analyzed separately and GFIs ranged from16% to 60% with a mean of 35% (SD ±19%).

Tumors from 71 patients with non-small cell lung cancerwere planted on collagen gels. Culture success rate was 91%.GFIs were calculated blindly with a mean of 37% (range = 8%-78%, SD ±16%) for the NSCLCs as a group. For squamouscell tumors the mean GFI was 33% (range = 8%-68%, SD ±15%) (Fig. 1). The mean GFI for large cell tumors was 40%(range = 17%-74%, SD ±18%), whereas the mean GFI foradenocarcinomas was 38% (range = 10%-78%, SD ±16%)(Fig. 1).

The mean GFIs between the 3 classes of non-small cell lungtumors were remarkably similar, ranging from 33% to 40%.This was in marked contrast to the mean GFI for small celltumors of 79% and was statistically significant (P< 0.001). Allnine small cell tumors had a GFI > 66%, whereas only 4 of 71

Lung Cancer100

Small Cell Mixed Small Large Cell Adenocarcinoma Squamous

Fig. 1. GFI were determined for 84 patients with lung cancer by histologicalautoradiography. Tumors were cultivated on collagen-containing gel matrices for10 days and then labeled with |'H]dThd for 4 days. Autoradiography was per

formed on sectioned histocultures. The percentage of labeled cells in the regionsof greatest labeling was determined and termed the growth fraction index. MeanGFI was 79 ±10% for 9 patients with the pure subtype of small cell lung cancer.Mean GFIs were significantly lower for: 4 patients with small cell lung cancer ofthe mixed small cell/large cell subtype (35 ±19%), 38 adenocarcinomas (38 ±16%), 12 large-cell undifferentiated carcinomas (40 ±18%), and 21 squamouscell carcinomas (33 ±15%)(P< 0.001 in all cases). See "Materials and Methods"

for further methodology.

non-small cell tumors had such a high GFI (Fig. 1). The mixedsmall cell/large cell tumors had GFIs similar to the NSCLCswith a significantly lower mean than that of the pure SCLCs(P< 0.001).

Differentiation of Small versus Non-Small Cell Tumors byGrowth Patterns. Fig. 2, a-c, was taken from 3 different smallcell tumors and is fairly representative of the growth patternobtained for this tumor type. The tumors were extremely cellular, frequently containing extensive regions of heavily labeledcells with infiltration into the gel matrix. The pattern of SCLCtumor growth was remarkably consistent in contrast to thewider phenotypic range of tumor growth present for the non-small cell lung tumors.

Most NSCLC expiants had large areas of central necrosiswith peripheral labeling in smaller clusters of cells. Althoughdifferentiation between the three non-small cell types was notpossible by the pattern of labeling and growth, certain characteristics of each tumor type seemed to be present. Large celltumors tended to grow into the gel matrix peripherally and hadthe greatest cellularity of the NSCLC types, with many cellshaving a high cytoplasm-to-nuclear ratio (Fig. 3, a-c). Adenocarcinomas frequently grew in duct-like patterns on the explantperiphery with moderate cellularity within the tumor and moderate gel invasion (Fig. 4, a-c). Finally, squamous cell tumorsgrew in swirls within the tumor expiant, which often containedlarge areas of necrosis (Fig. 5, a-c). Most of the cell labeling ofsquamous cell tumors occurred in scattered cells far out in thegel matrix. Some of the five bronchioalveolar cell tumors,interestingly, grew as a monocellular sheet that matched thepattern of growth in vivo (data not shown). The 4 mixed smallcell tumors had growth patterns more comparable to theNSCLCs. All 9 pure small cell tumors were correctly identifiable by photomicrographs alone by two separate blinded observers.

DISCUSSION

The ability to reproducibly culture human tumor expiants invitro has been a goal for many reasons. Prognostic information

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SMALL CELL VS. NON-SMALL CELL LUNG CANCER—HISTOLOGICAL ANALYSIS

Fig. 2. Representative histológica! autoradiograim of tumor expiants from 3different patients with small cell lung cancer (a. from patient 526; b, from patient743; c, from patient 701). Tumor expiants of 1 mm' in size were in culture for11 days followed by 4 days of labeling with |'H]dThd (4 nCi/ml). After fixation,

dehydration, and paraffin embedding, the histocultures were sectioned onto slidesand coated with emulsion. After a 5-day exposure, the slides were stained withhematoxylin and eosin. Epi-illumination polari/ation microscopy at x 400 wasused to identify replicating cells, depicted by bright green grains over cell nuclei.Small cell tumors were characterized by consistent high cellulari!) and celllabeling as demonstrated in a, h. and c. See "Materials and Methods" for further

methodology.

may be obtainable by measuring in vitro growth characteristicsthat would influence treatment aggressiveness. Individualizeddrug therapy may be obtainable by measuring in vitro drugsensitivities. New drug development may progress more rapidlyif an in vitro system can be developed that closely mimics in

Fig. 3. Representative histológica!autoradiograms of tumor expiants from 3different patients with large cell undiffercntiated carcinoma, (a, from patient 647;b, from patient 294; c, from patient 158). Large cell undifferentiatcd carcinomashad. in general, the most cellularity of the non-small cell lung tumors and tendedto grow by invasion into the gel matrix. Cell labeling in these areas of growth wasmuch less than in comparative regions in small cell lung tumors. See Fig. 2 and"Materials and Methods" for methodology.

vivo conditions. Finally, patterns of in vitro growth may be ableto distinguish different tumor classes.

We have developed an in vitro culturing system for humantumor expiants that maintains much of the 3-dimensional //;vivo tumor structure, is more rapid than nude mouse hetero-transplantation, and is highly successful in a variety of solidhuman tumor types (11-14). Tumor expiants are cultivated onfloating collagen-containing gels and then labeled with ['H]-

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SMALL CELL VS. NON-SMALL CELL LUNG CANCER—H1STOLOGICAL ANALYSIS

Fig. 4. Representative histológica! autoradiograms of tumor expiants from 3different patients with adenocarcinoma (a. from patient 159; ft. from patient 193;<•.from patient 496). Adenocarcinoma tumor expiants showed the greatest cellu-larity and cell labeling in polypoid outgrowths from the tumor edge or borderingducts within the tumor piece. See Fig. 2 and "Materials and Methods" for

methodology.

dThd to quantify cell proliferation by autoradiography.In a recent study, the ability to establish cell lines from small

cell lung tumors in vitro had a success rate of 31.3% (15). Inthe native-state histoculture system described here, the culturerate for a 2-week period was greater than 90%. Klein et al. (16)mechanically dissociated small cell and non-small cell lungcarcinomas onto feeder layers of a nonmalignant murineC3H10T1/2 cell line. Patterns of growth on the monolayer

Fig. 5. Representative histological autoradiograms of tumor expiants from 3different patients with squamous cell carcinomas, (a, from patient 1043: ft. frompatient 369; c. from patient 502). Tumor expiants from squamous cell carcinomasfrequently had large areas of central necrosis with low cellularity monolayersalong the tumor expiant edge. Scattered cells deep in the gel matrix were oftenpresent and usually labeled. A unique strand-like pattern of growth along thetumor periphery was present in one expiant as depicted in i See Fig. 2 and"Materials and Methods" for methodology.

were used to differentiate between the various small and non-small cell tumor types with a high degree of accuracy. Overallculture success rates matched those in our study. However, themethod of Klein et al. (16) is nonquantitative and the tumorcell growth pattern is not as representative of the in vivo state.

As mentioned above, there are frequently difficulties in distinguishing small cell and non-small cell lung tumors. This is

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SMALL CELL VS. NON-SMALL CELL LUNG CANCER—HISTOLOGICAL ANALYSIS

an important distinction clinically since the respective treatment protocols differ significantly. The presence of neuroendocrine markers is sometimes used to characterize SCLC tissues. Unfortunately, these markers are not yet specific withtheir expression in as many as 20% of NSCLC tumors (4-17).Indeed, the presence of these markers has been shown to predictgreater chemoresponsiveness in patients with NSCLC in vitroand in vivo (4, 18). These results suggest a common cell lineagebetween these tumor types (5, 7, 19, 20). Further evidencesupporting this theory came after insertion of v-Ha-ras genesin certain SCLC-derived cell lines was shown to induce cell-line characteristics more typical of large cell undifferentiatedlung carcinoma (21).

The presence of tumor heterogeneity also suggests a morphological continuum among lung cancer cell types. Many tumorshave isolated regions with characteristics of differing histolog-ical subtypes. This heterogeneity is further supported by autopsy studies. Five of 40 patients with biopsy-proven small cellcarcinoma had evidence of only NSCLC at autopsy (22). Thesefive patients had variable responses to chemotherapy and ingeneral had longer-than-expected survival, suggesting a lessaggressive tumor type. Interestingly, 4 of these 5 patients hadpresented with the intermediate subtype of SCLC, using theolder WHO classification scheme (23).

The mixed small cell/large cell subtype of SCLC seems to beless responsive to chemotherapy than the pure small cell subtype with a 16% versus 46% complete response rate in aNational Cancer Institute study (10). This also suggests thatthe mixed subtype of SCLC has diverse biological characteristics that may influence tumor aggressiveness and chemosensi-tivity. There were 4 cases of small cell lung tumors obtainedand cultured in our study that were classifiable as the mixedsmall cell/large cell subtype. These tumors had a variable GFIranging from 16% to 60% with a mean of 35%. All four ofthese tumors primarily had features of large cell carcinoma,and in 3 of the pathology reports the pathologist suggested thattreatment be based upon the large cell component. It is hopedthat the GFI represents a biological marker that could distinguish between the aggressive and indolent cases of the mixedsmall cell/large cell subtype of SCLC.

With the native-state histoculture technique used here, tumors classified as SCLC by histological analysis were successfully distinguished by both growth fraction index and growthpatterns in vitro from NSCLC tumors. This offers the opportunity to study the biology of each tumor type, particularly withregard to regulation of cell proliferation. Since this method ofcharacterization represents a "biological" assay based on cell

proliferation, it is hoped that a more accurate prediction ofchemoresponsiveness of both SCLC and NSCLC will be obtained. Identification of small cell and non-small cell lungcancers with high GFIs may be a means of distinguishing themost chemoresponsive tumors.

Furthermore, in vitro drug sensitivities can be obtained bythe addition of chemotherapeutic agents prior to tumor labeling. Thus, this in vitro system offers, in addition to the diagnostic distinction demonstrated in this study, the opportunityto evaluate new and standard agents against these tumor types.Further retrospective and prospective analysis will be necessaryto correlate in vivo tumor drug responsiveness with the parameters of in vitro growth patterns and GFI to determine the fullpotential of this methodology.

ACKNOWLEDGMENTS

The authors are grateful to Michael S. Vollmar for retrieving tumorspecimens and pathology reports.

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1990;50:6095-6099. Cancer Res   R. A. Vescio, K. M. Connors, G. M. Bordin, et al.   by Growth in Native-State HistocultureThe Distinction of Small Cell and Non-Small Cell Lung Cancer

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