p185neuExpression in Human Lung Adenocarcinomas ......P1851*" EXPRESSION IN HUMAN LUNG CANCER Table 1 Non-small cell lung cancers resected between 1982 and 1985 Total Studied Adenocarcinomas"

(CANCER RESEARCH 50. 5184-5191, August 15. 1990]

p185neuExpression in Human Lung Adenocarcinomas Predicts Shortened Survival1

Jeffrey A. Kern,2 David A. Schwartz, Joanne E. Nordberg, David B. Weiner, Mark I. Greene, Lisa Torney, and

Robert A. RobinsonDepartments of Internal Medicine [J. A. K., D. A. S., L. T.] and Pathology [R. A. R.J, University of Iowa College of Medicine, Iowa City, Iowa 52242, and Departmentsof Internal Medicine [J. E. A/., D. B. W.] and Pathology and Laboratory Medicine [D. B. W., M. I. G.J, University of Pennsylvania School of Medicine, Philadelphia,Pennsylvania 19104

ABSTRACT

plSS1"" is the protein product of the HER2/neu protooncogene. This

protein has characteristics of a tyrosine kinase growth factor receptorand is postulated to be important in human carcinogenesis. To define thesignificance of the expression of this protein in human non-small celllung cancer, 55 tumors from patients with squamous cell carcinoma (16),adenocarcinoma (29), or large cell carcinoma (10) of the lung wereexamined for plS5'"'" using Â¡mmunohistologicalmethods. Five of 16

squamous cell carcinomas and 10 of 29 adenocarcinomas were found tooverexpress pl85"c" relative to levels of expression seen in uninvolvedbronchiolar epithelium. For the adenocarcinomas, pl85nel1expression was

associated with older age (66.6 Â±10.1 versus 57.5 Â±10.8 years) (P =0.04) and shortened survival (83.7 Â±94.1 versus 188.5 Â±120 weeks) (P= 0.01). In this group, using Cox's multivariate survival analysis, pl85nel1

expression was found to be a significant determinant of survival (P =0.04) even after accounting for the effect of tumor stage. For the squamouscell carcinomas, pi85"'" expression was not correlated with any of ourclinicopathological parameters. Our findings indicate that non-small celllung cancers which express p 185'"'"do so at levels higher than that found

in normal bronchiolar epithelium, and expression in adenocarcinomas ofthe lung is independently associated with diminished survival intervals.

INTRODUCTION

Lung cancer is the leading cause of death from all malignancies in men and women in the United States (1). In addition, ithas the highest incidence of all tumors in men and the thirdhighest incidence in women (1). During the past 10 yearsmedical and surgical intervention has resulted in little changein the 5-year survival rate for lung cancer (1). Therefore, majorefforts in research are being directed to identifying the relationship between specific gene alterations and the clinical behaviorof lung tumors. The outcome of such studies could providemore accurate and useful diagnostic tools and, eventually, moreeffective therapeutic options.

In lung cancer, several genes and their protein productsincluding c-myc (2, 3), N-myc (3), L-myc (4), C-myb (5), K-ras(6), and c-erbB-l (7, 8) have been found to be amplified,rearranged, or overexpressed. We recently reported that expression of p!85neu, the protein product of the HER2/neu protooncogene (also known as c-erbB-2), occurred in approximatelyone-third of 22 cell lines derived from human non-small celllung cancers and one-third of 17 biopsy specimens of humanlung cancer (9). The HER2/neu protooncogene encodes a trans-membrane protein that shows extensive homology to the receptor for epidermal growth factor receptor, suggesting that theHER2/neu protooncogene protein product, pl85neu, is a membrane-bound receptor with tyrosine kinase activity (10-15).However, a ligand for pl85"eu has not been identified. Ampli-

Received 11/20/89; revised 4/20/90.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.

1This work was supported by grants from NIH HL01575, the American LungAssociation, and in part by the University of Iowa Cancer Center. Dr. Weiner issupported by the Council for Tobacco Research.

2To whom requests for reprints should be addressed, at University of IowaDepartment of Internal Medicine C33-A, General Hospital, Iowa City, IA 52242.

fication of the HER2/neu protooncogene has been found in15-40% of primary breast carcinomas (16, 17) and 30% ofovarian carcinomas (17). Moreover, elevated levels of pl85neu

expression in these tumors has been linked to shortened survival, shortened disease-free interval, the presence of lymphnode metastasis, and a poor nuclear grade (16-19). Therefore,in this study we sought to determine the extent of pl85neuexpression and the relationship between pl85"cu expression andprognosis in non-small cell human lung cancers.

MATERIALS AND METHODS

Study Population. We limited our study population to those individuals who underwent resection or open biopsy procedures at the University of Iowa Hospitals and Clinics between 1982 and 1985. Thisapproach was chosen so that we could precisely stage the lung tumorbased on operative findings and allow adequate clinical follow-up time.Tumors were classified by morphological and immunohistochemicalcharacteristics as adenocarcinomas, large cell carcinomas, or squamouscell carcinomas. No tumors were included if they contained elementsof small cell carcinoma. Between 1982 and 1985, a total of 60 cases ofnon-small lung cancer were resected or underwent an open biopsyprocedure and had tissue specimens available for analysis (Table 1).Those tumors having a mixed growth pattern (8 of 26) were classifiedaccording to the predominant pattern. Clinical parameters were obtained by an chart review by a independent reviewer unaware of theresults of the immunohistochemical analysis. The median follow-uptime for all patients was 38 months (range, 0.5-89 months). Standardcriteria as set forth by the International Staging System for LungCancer (21) were used to determine characteristics of the primarytumor, regional lymph nodes, mÃ©tastases,and surgical stage.

pl85â„¢"-specific Antisera. DBW-2, a polyclonal anti-pl85ncu anti-

serum, was prepared by immunizing rabbits with a synthetic peptidecorresponding to amino acid residue 1240-1255 of the human pl85ne"

sequence coupled to keyhole limpet hemocyanin (9). This antiserumspecifically immunoprecipitates pl85"eu of rats, cats, and humans (9,

22,23) and has been used extensively by several laboratories for analysisof pi85â„¢"in a wide range of applications documenting its specificityand tissue reaction pattern (9, 22-26).

Cell Cultures. Human lung tumor cell lines were obtained fromAmerican Type Tissue Culture (Rockville, MD) (A-549, A-427, SK-LU, CALU-1, CALU-3, CALU-6, SK-MES) and from Dr. A. Gazdarof the National Cancer Institutes (NCI-HI466 and NCI-H522). Thecells were cultured in RPMI and 10% fetal bovine serum or Eagle's

modified essential media and 10% fetal bovine serum and incubated at37Â°Cin a humidified 5% CO2 atmosphere. The rat neuroblastoma cell

line B104 was used as a positive control in Northern blot, Westernblot, and immunohistochemical analyses. This cell line is known toexpress pi85""", contains DNA which when transfected into NIH 3T3

cells results in their transformation, and was the source of isolation ofthe neu cDNA3 (10, 14).

Western Blot Analysis. Cells were disrupted in lysis buffer [20 mMTris, pH 8-0.33 M sucrose-0.5 mM ethyleneglycol bis(/3-aminoethylether)-yV,Ar,A",./V'-tetraacetic acid-25 uM leupeptin-1.5 UMaprotinin-5

UM7V-ethylmaleimide-0.5% Triton X-100], and the protein content wasdetermined by the Bradford method (27). Total protein, 100 Mg,fromeach cell line was resuspended in Laemmli buffer, heated at 100'C for

3The abbreviations used are: cDNA, complementary DNA; SDS, sodiumdodecyl sulfate; SSC, standard saline citrate.

5184

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P1851*" EXPRESSION IN HUMAN LUNG CANCER

Table 1 Non-small cell lung cancers resected between 1982 and 1985

Total StudiedAdenocarcinomas" 30 29*Squamous cell carcinomas 20 16CLarge cell carcinomas'* 10 10

* Adenocarcinomas were further separated into acinar, bronchoalveolar, papillary, and solid (positive mucicarmine staining) carcinomas with mucus production (20).

* One case was a metastasis from a nonpulmonary primary and therefore

excluded.'Four cases were mÃ©tastasesfrom nonpulmonary primaries and therefore

excluded.''All specimens were negative for mucicarmine staining and none exhibited

squamous differentiation.

10 min, and subjected to SDS-polyacrylaminde gel electrophoresisanalysis using a 5% stacking and 7.5% separating acrylamide gel with0.192 M glycine-15 mM Tris, pH 8.3, upper and lower tank buffer.After completion of electrophoresis, the gel was equilibrated in transferbuffer (20% methanol-0.192 M glycine-15 mM Tris, pH 8.3) and theequilibrated gel was electrically transferred to a nitrocellulose membrane with 30 V at 4Â°Cfor 16 h. Adequacy of transfer was determined

by staining the gel after the transfer with 0.1% Coommassie blue. Thenitrocellulose membrane was blocked in 4% nonfat dry milk, followedby the addition of the primary antibody at a 1:1000 dilution. After 16h (4Â°C),the membrane was washed in phosphate-buffered saline (15

min/wash, 4 washes), followed by a 1-h incubation with 100,000 cpm/ml of 125I-protein A. The membrane was washed with PBS (15 min/wash, 4 washes) and exposed to Kodak XAR-5 film at -70Â°C.

RNA Isolation and Northern Blot Analysis. Total cellular RNA wasisolated by a guanidium isothiocyanate method with cesium chloridemodification (28, 29). Equal amounts of RNA, as determined by A26o,were denatured in sample buffer and size fractionated by electrophoresisthrough a 1% agarose gel containing 6% formaldehyde and 2 Mg/dlethidium bromide. Agarose gels were visualized by UV illumination todetermine the position of 28S and 18S rRNA bands, to assess theintegrity of RNA, and to verify that equal amounts of RNA had beenloaded into all wells. Transfer of RNA to nylon membranes wasaccomplished by capillary blotting for 24 h using 2.5x sodium chloride-sodium phosphate-EDTA (350 mM NaCl-80 mM NaH2POâ€ž-7HIMEDTA, pH 7.4). After blotting, membranes were dried, baked at 80Â°Cin vacuo, and prehybridized at 42Â°Cfor 16-24 h in prehybridizationfluid (50% deionized formamide-lx Denhardt's solution-1% SDS-1

mM NaCl-5 mM Tris, pH 7.4-0.5 ng/ml heparin-250 ng/ml denatured,sheared salmon sperm DNA-10% dextran sulfate). This was followedby hybridization for 24 h at 42Â°Cin the same solution with IO6cpm/lane of a heat-denatured "P-labeled plasmid DNA (30) with a full

length HER2/neÂ« cDNA insert (31). Blots were washed twice in 2xSSC (ix SSC, 150 mM NaCl-15 mM sodium citrate, pH 7) and 0.1%SDS at 25Â°Cfor 30 min and twice in 0.1 % SSC and 0.1 % SDS at 50Â°Cfor 30 min and exposed at -70Â°C to Kodak XAR-5 film. To ensure

equal loading and transfer of RNA in all lanes, radiolabeled probeswere removed from the membranes by incubation with 0.2% SDS, 10mM Tris at 85Â°Cfor 60 min, and blots were reprobed as above using a

/3-actin (32) control probe.Immunoperoxidase Staining of Fixed Tissue. Paraffin-embedded, for

malin-fixed tissues were obtained from the Department of Pathologyat the University of Iowa Hospitals and Clinics. Sections (3-4 urn)were cut from the tissue blocks, placed on pretreated (Histostik) glassslides, dried at 37Â°Covernight followed by 60Â°Cfor 30 min, and

exposed to xylene for 5 min (3 times). Tissues were rehydrated indecreasing concentrations of ethanol (100, 95, and 70%) for 2 min(twice) and rinsed in distilled water, and endogenous peroxidase activitywas blocked with 0.3% hydrogen peroxide in methanol (30 min). Slideswere washed in PBS (20 min), excess liquid was blotted off, andnonimmune goat serum (Vector Laboratories Ine, Burlingame, CA)was applied to reduce nonspecific background staining (20 min). Theserum was drained and rabbit anti-pl85neu antiserum or a negative

control applied (60 min). Slides were rinsed with PBS and drained, anda biotin-conjugated goat anti-rabbit antibody was added (Vector). After30 min, the slides were again rinsed with PBS, avidin was applied for30 min, the slides were rinsed in PBS, diaminobenzidine (Aldrich

Chemical Co., Milwaukee, WI) was added (30 min), and, again, theslides were rinsed with water and counterstained with Harris hematox-ylin. After the counterstain, the tissue was dehydrated with increasingsolutions of ethanol (70, 95, and 100%) for 2 min (twice) followed byclearing in xylene and mounting.

Positive controls of formalin-fixed, paraffin-embedded human lungwere used with each staining reaction. Negative controls consisted oftissue sections from the paraffin-embedded lung cancer block to bestudied for p 185"'" expression treated with an irrelevant antibody (rabbitanti-mouse IgG) (Vector) or with pl85nel1antisera previously exposed

to saturating amounts of immunizing peptide. All microscopic analyseswere performed by the same pathologist (R. A. R.). A tissue specimenexhibiting membrane or membrane and cytoplasmic reactivity withDBW-2 antiserum throughout the tissue section was considered positive. No reactivity or cytoplasm without membrane reactivity wasconsidered negative.

Flow Cytometry Analysis. Flow cytometry of paraffin-embedded tissue was accomplished by the techniques of Hedley et al. (33) andVindelov et al. (34). A modified pepsinization technique of Hedley wasused to obtain nuclei and the propidium iodide staining method ofVindelov was used to stain the DNA (35). Two to three SO-^m sectionswere cut from paraffin blocks that had > 1 cm2of tissue with a minimumof 50% tumor cells. The blocks were also chosen to include 5-10% ofapparent normal tissue as an internal diploid standard. Hematoxylinand eosin sections were cut after the thick sections to confirm thepresence of tumor in the sections being studied. The sections weredeparaffinized by applying two 3-ml changes of xylene for 10 min at20Â°Cafter hydration in decreasing alcohol concentrations. The tissue

was then washed twice in distilled water and resuspended in 1 ml of0.5% pepsin (pH 1.5) (Sigma Chemical Co., St. Louis, MO) for 90 minwith intermittent vortexing. The nuclei were pelleted by centrifugationand the supernatant was aspirated. The nuclei were resuspended in 0.1ml of citrate buffer (pH 7.6) and 9.9 ml of solution A (30 mg/litertrypsin, pH 7.6) (Sigma) was added. After 10 min, 0.75 ml of solutionB (500 mg/liter trypsin inhibitor-100 mg/liter RNase A, pH 7.6)(Sigma) was added to the suspension. After an additional 10 min, 0.75ml of solution C (420 mg/liter propidium iodide, pH 7.6) (Sigma) wasadded, and the suspension was filtered through a 30-^m nylon meshfilter. The DNA nuclear content was measured on a Becton Dickinson440 flow cytometer (Becton Dickinson, Mountainview, CA). Nucleifrom normal paraffin-embedded lymph nodes were examined as external standards to set the diploid peak at channel 60. For each specimen,histograms of 10,000 events were recorded utilizing hardware gatingon propidium iodide to eliminate debris material in the lower channelsto the left of the G, peak. Integration procedures were performed toobtain phase fraction analyses. The coefficient of variance was determined by the full width peak method.

Nuclear DNA ploidy patterns were categorized as either diploid,tetraploid, or aneuploid. Diploid tumors were defined as those histograms having only one definable G! peak. Aneuploid tumors wereidentified as those histograms having a second peak that did not fall inthe expected channels of the G, and the G2 + M peaks. Tetraploidtumors were defined as any histogram whose G2 + M peak was >3 SDabove the G2 + M peak of normal lymphocyte nuclei. The nuclei in theG2 + M peak were sorted by flow cytometry and examined microscopically to confirm that they were single nuclei and not doublets.

Statistical Analysis. Nonparametric statistical tests (36) were used toevaluate all of our comparisons. Fisher's exact test was used to assess

the relationship between categorical variables and the expression ofpl85"eu, and the Mann-Whitney U test was used to calculate P values

for continuous variables. The relationship between the cumulativeprobability of survival and a variety of clinical variables, including theexpression of p 185"", was determined by using the log rank test, as

described by Kaplan and Meier (37). Censoring was performed forsubjects who were either lost to follow-up or were followed for less thanthe longest survivor. The clinical factors that were significantly associated with the cumulative probability of survival were tested in a hierarchical multivariate survival analysis, as described by Cox and Oakes(38). All interactions were tested among clinical factors that were foundto be independently associated with survival.

5185



pl85~Â° EXPRESSION IN HUMAN LUNG CANCER

RESULTS

Immunohistological Detection of pl85"eu Expression Corre

lates with Western and Northern Blot Analysis. In order to usethe monospecific, polyclonal anti-pi85"Â°"antiserum DBW-2 toidentify pl85"cu, we first studied the relationship betweenHER2/neu gene expression and DBW-2 antiserum-identifiedpl85neu protein expression. Total cellular RNA (28, 29) and

protein (9) were isolated from 10 cell lines derived from humannon-small cell lung cancers. Northern and Western blot analyses were performed on each cell line to identify the HER2/neu mRNA transcript and pl85neu. On Northern blot analysis

(Fig. IJaneA), the HER2/neu cDNA probe recognized a singletranscript of 4.8 kilobases in expressing cell lines. By Westernblot analysis (Fig. 1, lane B) DBW-2 antiserum recognized asingle protein of M, 185,000. The Western and Northern blotautoradiogram signals representing pi85ncu protein and HER2/

neu mRNA expression from each cell line were quantitated bydigital analysis and the degree of HER2/neu mRNA accumulation was correlated with the degree of pl85neu protein expres

sion. As shown in Fig. 1, gene expression and protein expression correlated significantly (r = 0.90, P = 0.0001). Therefore,we concluded that the level of pl85neu expression determinedby Western analysis using DBW-2 antiserum is a good assessment of HER2/neu gene expression.

In these same cell lines, we next sought to determine therelationship between immunohistochemical identification ofpl85ncu using DBW-2 antiserum and Western and Northernblot analyses. Cells from each cell line were fixed, pl85ncu

Correlation of HER2/neu Gene Expressionand p185neuExpression

200

O 100

1 50

r = 0.90p = 0.0001

A-427 A-427kb kDl

200.0-

f

i 82.5-

50 100 150

p185neu Expression

Fig. 1. Correlation of P185"'" protein level with HER2/neu gene expression.Total cellular RNA was isolated from 10 cell lines derived from human non-small cell lung cancers and analyzed for HER2/neu gene expression using aHER2/nfU cDNA probe. Total cell protein was isolated from the same cell linesand Western blot analysis performed using DBW-2 antiserum. A representativeNorthern blot (lane A ) with the single 4.8-kilobase (kb) HER2/neu mRNAtranscript and 2.0-kilobase tf-actin control probe is shown. A representativeWestern blot (lane B) is also shown. Both protein and DNA were derived fromthe American Type Culture Collection cell line A-427. The autoradiogram hybridization signals were quantitated by digital analysis and plotted, and a correlation coefficient was derived. A total of 3 Northern blots and 5 Western blotswere analyzed for each cell line. M,. in thousands.

immunohistochemically was identified with DBW-2 antiserum,and levels of expression were graded on a 0-4 scale (low tohigh) by an investigator unaware of the results of Western andNorthern blot analyses. The immunohistochemical identification of pl85"cu was found to be strongly related to levels definedby Western blot analysis (Kruskal-Wallis one-way analysis ofvariance, x2 = 11.0, /* = 0.03), as was the comparison of the

level of HER2/neu gene expression defined by Northern blotanalysis to the immunohistochemical identification of pl85neuby DBW-2 antiserum (x2 = 10.4, P = 0.03). Therefore, our

results indicate that immunohistochemical identification andquantitation of pl85ncu with DBW-2 antiserum is strongly

associated with either Northern blot analysis of HER2/neuexpression or Western blot analysis of its protein product. Fromthese studies we conclude that using DBW-2 antiserum forimmunohistological analysis to determine pl85"eu expression

is a valid technique.Expression of pl85"eu in the Adult Human Lung and in Non-

Small Cell Lung Cancer. We next sought to immunohistologi-cally determine pl85"eu expression in specimens of normal

human lung and lung cancer. In sections of normal lung obtained at autopsy from patients without lung cancer, pl85"eu

was found to be expressed throughout the respiratory tract (Fig.2). Specifically, DBW-2 antiserum reactivity was seen in ciliatedrespiratory epithelial cells (1+), bronchial mucosal glands inthe major airways (2+), and cells with the appearance of typeII pneumocytes (1+). By light microscopy it was difficult todetermine whether staining was present in type I pneumocytes.Other normal cells from the lung, including fibroblasts, smoothmuscle cells, and endothelial cells, did not react with the anti-

serum.Ten adenocarcinomas reacted with DBW-2 antiserum (Table

2). Staining was present in all areas of the tumor representedin the tissue section (Fig. 3). Reactivity was usually uniformthroughout (4+) but occasionally ranged from 2-4+. Both

membrane and cytoplasmic staining was present in all tumorsexamined, with membrane staining more intense, especially inthe bronchioloalveolar subtypes. Interestingly, membrane staining was always most intense at the luminal border. Foci of clearcells showed less intense staining than non-clear cells, andtumor cells displaying more cytoplasm and showing a lowernuclear grade were more often positive.

Five squamous cell carcinomas also reacted with DBW-2antiserum (Table 2). Staining was uniform throughout thetumors but usually at lower levels than in adenocarcinomas (2-3+) (Fig. 4). Membrane staining was also less pronounced thanin adenocarcinomas (2+). There was more intense staining ofcells with large amounts of cytoplasm. Moreover, foci of well-differentiated squamous cell carcinomas tended to be 4+ positive.

None of the large cell carcinomas studied reacted with DBW-2 antiserum (Table 2). In 5 cases (3 adenocarcinomas, 2 squamous cell carcinomas), the immunohistochemical staining pattern was indeterminate. These tumors had a single focus ofDBW-2 antiserum reactivity as opposed to diffuse reactivity.

Table 2 pl85"'" expression in human non-small cell lung cancer

Expression of p 185"'"

CelltypeAdenocarcinomaSquamous

cellLargecellTotalAbsent1691035Present105015%

Positive3836030

5186



pl85~" EXPRESSION IN HUMAN LUNG CANCER

Fig. 2. Normal expression of PI 85"*" in human bronchiole epithelium. In A,normal human lung sections obtained at autopsy were stained with anti-pi85"'"specific antiserum (DBW-2) as described in "Materials and Methods." Note the

weak cytoplasmic reactivity of normal bronchiole ciliated epithelial cells with theantiserum and stronger luminal membrane reactivity (1+) (x 250). In /(, the sametissue block was stained with anti-pi85Â°*Â°-specificantiserum first exposed tosaturating amounts of immunizing peptide. Note the loss of reactivity on themembrane and in the cytoplasm (x 250).

;

BFig. 3. Expression of plSS"" in a human lung adenocarcinoma. .1. section

from a lung adenocarcinoma showing uniform p 185Â°""reactivity (4+) throughoutthe tumor (x 400). B, same tissue block using anti-pi85â„¢"-specific antiserum first

exposed to saturating amounts of immunizing peptide. Note the loss of reactivityon the cell membrane and in the cytoplasm (x 400).

Fig. 4. Expression of pl85"*" in human lung squamous cell carcinomas. In A,human lung squamous cell carcinomas were stained with anti-plSS^-specificantiserum as described in "Materials and Methods." Note the uniform stainingof p 185"'" throughout the tumor (2-3+) with some cell to cell variation (x 300).In lÃ¬,the same tissue block was stained with anti-pi85"'"-specific antiserum first

exposed to saturating amounts of immunizing peptide. Note the loss of reactivityon the cell membrane and in the cytoplasm (x 300).

Since it could not be determined whether these tumors werepl85neu expressing or nonexpressing, they have not been in

cluded in any of our analyses.In summary, pl85neu was found by immunohistological tech

niques using DBW-2 antiserum to be normally expressed atlow levels throughout the respiratory tract in ciliated bronchioleepithelial cells (1+), bronchial mucosa! glands (2+), and typeII pneumocytes (1+). In addition, expression of pl85"eu was

found in 38% of adenocarcinomas and 36% of squamous cellcarcinomas but not in large cell carcinomas. The level of pl85neu

found in the expressing carcinomas was always higher thannormal expression seen in uninvolved bronchiolar epithelium.

Association Between p185"'" Expression and CIinicopathological Features. The expression of pl85neu was not associated with

any unique clinical characteristics in patients with squamouscell lung cancer (Table 3). In particular, no relationship wasfound with poor prognostic indices, such as increased tumorstage (P = 0.35), older age (P = 0.25), and diminished survival(P = 0.50). Moreover, no consistent correlation existed betweenthe expression of pl85neu and overall outcome.

In contrast, the expression of pl85neu was found to be asso

ciated with poor prognostic indices in patients with adenocarcinoma of the lung (Table 4). Adenocarcinomas expressingpl85neu were found to be derived from a significantly older

5187




Table 3 Relationship between expression of the HER2/neu gene protein productp 185"" and clinicopathological variables for patients with squamous cell lung

cancer

Table 4 Relationship between expression of the HER2/neu gene protein productplS5*" and clinicopathological variables for patients with adenocarcinoma of the

lungExpression ofplSS""1Categorical

variablesMaleSmokerCoughHemoptysisWeight

lossChestpainPrimary

tumor123Regional

lymphnodes0I2MÃ©tastases01Surgical

stage1234OutcomeDeadAliveContinuous

variablesAge(yr)PackyrTumor

size(cm)Survival(wk)Absent(N=9)792152ÃŒ518109071104558.6

Â±10.1*65.6

Â±30.93.7Â±1.7177.3Â±

129.7Present

(N=5)4433100412305022101463.4

Â±4.550.0Â±14.18.5

Â±6.3237.8+134.5P

value"1.001.000.550.200.251.000.350.951.000.350.600.250.300.150.50

" P values calculated using Fisher's exact test and Mann-Whitney U test. The/' value for survival was calculated using the log rank test as described by Kaplan

and Meier.4 Mean Â±SD.

patient population (66.6 Â±10.1 (SD) versus 57.5 Â±10.8 years)(P = 0.04) with a significantly shorter survival (83.7 Â±94.1versus 188.5 Â±120 weeks) (P = 0.01). Marginal, albeit statistically insignificant, relationships were also observed in adeno-carcinomas between the expression of pl85n


I.UTable

5 Relationship between survival and clinicopathological variablesforpatientswith adenocarcinoma of thelungCategorical

variables(n)GenderMale

(14)Female(12)Smoking

statusSmoker(22)Nonsmoker

(4)CoughNo

(16)Yes(9)HemoptysisNo

(20)Yes(3)Weight

lossNo(22)Yes

(3)Chest

painNo(20)Yes

(5)Primary

tumor1(8)2(17)3(1)Regional

lymphnodes0(11)1(8)2(6)MÃ©tastases0(17)1

(9)Surgical

stage1(6)2(8)

3(3)4(9)Histology

BronchioloalveolarNo(15)Yes

(11)SolidNo

(18)Yes(8)AcinarNo

(13)Yes(13)DNA

contentAneuploid(18)Diploid

(5)Tetraploid(1)Grade1(2)

2(15)3(9)plSS"*"

expression

AbsentPresentContinuous

variablesAge(yr)PackyrTumor

size (cm)Survival

(wk)130.3

Â±32.0*169.1

Â±35.5143.1

Â±25.9176.3Â±64.2143.0

Â±30.2157.4Â±44.5151.0

Â±28.6137.0Â±50.6164.0

Â±26.232.7Â±12.6143.1

Â±27.1168.6Â±64.2189.9

Â±52.4136.7Â±25.310.0155.5

Â±39.6180.1Â±41.386.8

Â±34.3198.8

Â±28.452.7Â±16.2244.7

Â±44.8184.1Â±46.7

146.0 Â±48.552.7 Â±16.2135.0Â±

30.2166.2Â±38.8133.4

Â±27.2181.5Â±47.0200.2

Â±33.796.2Â±27.3146.4

Â±27.7146.8Â±57.2

33.056.5

Â±23.5172.3Â±32.2128.4 Â±40.1185.5

Â±30.083.7Â±29.8-0.06

Â±0.032''0.01Â±0.01C-0.30Â±0.10CP

value"0.600.850.900.800.0040.900.00010.400.00010.00090.900.450.100.500.300.010.070.400.003Â°

P values for categorical variables were computed by the method of Kaplan

and Meier and comparison of statistics were calculated using the log rank test.For continuous variables a Cox's survival analysis was performed with thePvalues

for the coefficient estimatespresented.*Mean Â±SD.

' Estimated mean Â±SE.0.90.80.7CDf3_

0.5OX15n

Â°-4gC0.30.20.10.0iÃ¯

""U,LI\

111

1111111~~1\\

(1\\

\VI Stagf3 Stage1I\1

.LLLL,LStage

211Stage

4p

=0.00090

SO 100 150 200 250 300 350400Weeks

AfterDiagnosisFig.

5. Actuarial curve for survival of all patients with lungadenocarcinomaaccordingto surgical stage. The Kaplan-Meier method was used to estimate the

survival distribution for each subgroup. The log rank test was used to evaluatethe equality of the survivalcurves.man

non-small cell lung cancer and confirmed in anotherorgansystemthat expression of this protein, at least in lung adeno

carcinomas, is of potential prognostic importance. Ourstudieshaveshown that pl85neu is normally expressed in ciliated epi

thelial cells lining the respiratory tract, type IIpneumocytes,andbronchial mucosa! glands. Expression of this proteinoccursin

both squamous cell carcinomas (36%) andadenocarcinomas(38%)of the lung and is at levels higher than inuninvolvedbronchiolar

epithelium. Most important, inadenocarcinomasexpressionof pl85"cu is independently associated with a short

enedsurvival.Initialefforts in this study sought to determine thevalidityand

sensitivity of immunohistochemical analysis ofpl85ncuusingDBW-2 antiserum. Previous work has documentedthemonospecificity

of DBW-2 antiserum (9, 22-26). Bycorrelatingimmunohistochemical,Western blot, and Northern blot anal

yses performed on a series of cell lines derived fromhumannon-smallcell lung cancer, we found thatimmunohistochemicalstudies

of pl85ncu expression using DBW-2 antiserum signifi

cantly correlated with HER2/neu gene and proteinexpressionasdefined by Northern blot and Western blot analyses. There

fore, using DBW-2 antiserum in immunohistochemical techniques to determine pl85neu expression appears to be a valid

quantitative technique. In five cases we were unable to categorize the tissue p 185"euexpression status. A single focus of DBW-2

antiserum reactivity was present in these tumors whilethemajorityof the tissue specimen was nonreactive.Indeterminateimmunohistochemical

staining patterns have also been reported5189




1.0

0.9

0.8

0.7

_ 0.6

in"5

t!5S8Q.

0.5

0.4

0.3

02

0.1

0.0

No expression

p = 0.01

150 200 250 350 400

Weeks After DiagnosisFig. 6. Actuarial curve for survival of all patients with lung adenocarcinoma

according to plSS1"" expression. The Kaplan-Meier method was used to estimatethe survival distribution for each subgroup. The log rank test was used to evaluatethe equality of the sun Â¡valcurves.

Table 6 Multirariale analysis of survival for adenocarcinoma of the lung

VariableExpressionof plSS1""

SurgicalstageIntercept

Log likelihoodScaleEstimate

Â±SE-0.79Â±0.39

-0.74 Â±0.197.46

Â±0.62-31.39

0.81 Â±0.15x24.115.2146.7P

value0.04

0.00010.0001

in breast cancers (18). The biological significance of this expression pattern is unclear. It may represent cells in a differentiationor proliferation state distinct from the rest of the tumor orsimply a focus of cells with a slightly higher level of pl85neu

expression than surrounding tissue. Regardless, we thought itinappropriate to grade these specimens either positive or negative. Thus, these specimens were not included in any of dataanalyses reported.

In our patient population demographic and clinical characteristics reflect what has been previously reported for largerpopulations of patients with lung cancer (39). However, thesimilar survival that we observed for stage 2 and stage 3 diseaseindicates that our study population is somewhat different fromother patients with adenocarcinoma of the lung. Therefore,further studies are needed in larger, more representative populations to determine whether our findings are generalizable toall patients with non-small cell lung carcinoma.

pl85ncu is similar in structure to the epidermal growth factorreceptor (50% amino acid homology) (10, 13-15, 40) and is

presumed to be a membrane receptor, but the nature of itsnormal function(s) and ligand(s) remains unknown. Given theassociation of pl85ncu in lung adenocarcinomas to an older

population with a poorer prognosis, it is tempting to postulatea role for this protein as a component of tumorigenesis ratherthan simply a marker of lung adenocarcinoma. At least twoexperimental findings support such a role in tumorigenesis: (a)overexpression of pl85ncu in NIH 3T3 cells or epithelial cell

lines leads to transformation (41, 42) and (b) signaling througha chimeric receptor (epidermal growth factor receptor ligand-binding domain/pi85ncu tyrosine kinase domain) with epider

mal growth factor results in the transformation of receptorexpressing cell lines (43, 44).

Two alternate hypotheses may explain our clinical findingswith pl85neu expression in lung cancer and need to be considered. First, pl85ncu expression may occur at a late stage in the

natural history of all lung adenocarcinomas resulting in theidentification of an older patient population with a shortersurvival. However, thus far no primary lung tumor studied orits metastasis has been found to change pl85"eu expressionstatus. Second, expression of pl85neu may identify a specific

tumor subtype that occurs in older patients and has a rapidlyfatal course. Identification of histological subtypes of breastcancer by pl85ncu expression has been reported (45), but we

have not identified any correlation between histological featuresof the lung tumors studied and pl85neu expression.

The lack of any significant relationship between pl85"cu

expression and squamous cell carcinomas is difficult to understand. This may reflect our small study population, the differentlineage of adenocarcinomas and squamous cell carcinomas,differing mechanisms involved in tumorigenesis, or differentlevels of detectable pl85neu expression. Recent work by Slamon

et al. (17) indicates that fixation decreases the sensitivity ofimmunohistochemical detection of pl85"cu as compared to theuse of fresh frozen specimens. Therefore, the pl85"eu expression

reported here may actually underestimate the true incidenceand level of expression in these tumors. More sensitive techniques to detect and measure pl85ncu may result in additionalinformation regarding the expression of pl85ncu in squamous

cell carcinomas.The mechanism resulting in p 185"euexpression in lung cancer

is not known. Gene amplification, as has been described inbreast cancer (16-18, 45, 46), appears to be infrequent in lungcarcinoma (17), implying that transcriptional or posttranscrip-tional regulatory mechanisms are disordered. The detection ofconsistent biochemical or genetic alterations in human lungcancer should begin to provide insights into the mechanisms ofcell transformation and may carry prognostic information unrelated to clinical findings or histology. Our findings in thisstudy regarding survival and pl85neu expression in human lungadenocarcinomas is the first such example in non-small celllung carcinomas. Further prospective investigations are neededto assess the generalizability of our findings and to determinewhether more aggressive therapeutic modalities might be beneficial to patients whose tumors express pl85neu.

ACKNOWLEDGMENTS

The authors would like to acknowledge Dr. Adi Gazdar for supplyingthe NCI lung cancer cell lines, Scott Van Fossen for assistance incomputer programming, Helga Trabert for expert secretarial support,and Christine Bromley (H.T., A.S.C.P.) for excellent immunohistolog-

ical technique.

5190




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1990;50:5184-5191. Cancer Res Jeffrey A. Kern, David A. Schwartz, Joanne E. Nordberg, et al. Shortened Survival

Expression in Human Lung Adenocarcinomas Predictsneup185

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p185neuExpression in Human Lung Adenocarcinomas ......P1851*" EXPRESSION IN HUMAN LUNG CANCER Table 1 Non-small cell lung cancers resected between 1982 and 1985 Total Studied Adenocarcinomas"