lung cancer, 55 tumors from patients with squamous cell carcinoma (16), adenocarcinoma (29), or ..... Northern and Western blot anal yses were performed ... respiratory epithelial cells (1+), bronchial mucosal glands in the major airways (2+), ...
(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. Robinson Departments 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 Departments of 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 receptor and is postulated to be important in human carcinogenesis. To define the significance of the expression of this protein in human non-small cell lung cancer, 55 tumors from patients with squamous cell carcinoma (16), adenocarcinoma (29), or large cell carcinoma (10) of the lung were examined for plS5'"'" using ¡mmunohistological methods. Five of 16 squamous cell carcinomas and 10 of 29 adenocarcinomas were found to overexpress pl85"c" relative to levels of expression seen in uninvolved bronchiolar epithelium. For the adenocarcinomas, pl85nel1expression was associated with older age (66.6 ±10.1 versus 57.5 ±10.8 years) (P = 0.04) and shortened survival (83.7 ±94.1 versus 188.5 ±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 squamous cell carcinomas, pi85"'" expression was not correlated with any of our clinicopathological parameters. Our findings indicate that non-small cell lung cancers which express p 185'"'"do so at levels higher than that found in normal bronchiolar epithelium, and expression in adenocarcinomas of the lung is independently associated with diminished survival intervals.
INTRODUCTION Lung cancer is the leading cause of death from all malignan cies in men and women in the United States (1). In addition, it has the highest incidence of all tumors in men and the third highest incidence in women (1). During the past 10 years medical and surgical intervention has resulted in little change in the 5-year survival rate for lung cancer (1). Therefore, major efforts in research are being directed to identifying the relation ship between specific gene alterations and the clinical behavior of lung tumors. The outcome of such studies could provide more accurate and useful diagnostic tools and, eventually, more effective therapeutic options. In lung cancer, several genes and their protein products including 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 expres sion of p!85neu, the protein product of the HER2/neu protoon cogene (also known as c-erbB-2), occurred in approximately one-third of 22 cell lines derived from human non-small cell lung cancers and one-third of 17 biopsy specimens of human lung cancer (9). The HER2/neu protooncogene encodes a transmembrane protein that shows extensive homology to the recep tor for epidermal growth factor receptor, suggesting that the HER2/neu protooncogene protein product, pl85neu, is a mem brane-bound receptor with tyrosine kinase activity (10-15). However, a ligand for pl85"eu has not been identified. Ampli-
fication of the HER2/neu protooncogene has been found in 15-40% of primary breast carcinomas (16, 17) and 30% of ovarian carcinomas (17). Moreover, elevated levels of pl85neu expression in these tumors has been linked to shortened sur vival, shortened disease-free interval, the presence of lymph node metastasis, and a poor nuclear grade (16-19). Therefore, in this study we sought to determine the extent of pl85neu expression and the relationship between pl85"cu expression and prognosis in non-small cell human lung cancers. MATERIALS
AND METHODS
Study Population. We limited our study population to those individ uals who underwent resection or open biopsy procedures at the Univer sity of Iowa Hospitals and Clinics between 1982 and 1985. This approach was chosen so that we could precisely stage the lung tumor based on operative findings and allow adequate clinical follow-up time. Tumors were classified by morphological and immunohistochemical characteristics as adenocarcinomas, large cell carcinomas, or squamous cell carcinomas. No tumors were included if they contained elements of small cell carcinoma. Between 1982 and 1985, a total of 60 cases of non-small lung cancer were resected or underwent an open biopsy procedure and had tissue specimens available for analysis (Table 1). Those tumors having a mixed growth pattern (8 of 26) were classified according to the predominant pattern. Clinical parameters were ob tained by an chart review by a independent reviewer unaware of the results of the immunohistochemical analysis. The median follow-up time for all patients was 38 months (range, 0.5-89 months). Standard criteria as set forth by the International Staging System for Lung Cancer (21) were used to determine characteristics of the primary tumor, regional lymph nodes, métastases,and surgical stage. pl85™"-specific Antisera. DBW-2, a polyclonal anti-pl85ncu anti-
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 in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by grants from NIH HL01575, the American Lung Association, and in part by the University of Iowa Cancer Center. Dr. Weiner is supported by the Council for Tobacco Research. 2To whom requests for reprints should be addressed, at University of Iowa Department of Internal Medicine C33-A, General Hospital, Iowa City, IA 52242. 5184
serum, was prepared by immunizing rabbits with a synthetic peptide corresponding to amino acid residue 1240-1255 of the human pl85ne" sequence coupled to keyhole limpet hemocyanin (9). This antiserum specifically immunoprecipitates pl85"eu of rats, cats, and humans (9, 22,23) and has been used extensively by several laboratories for analysis of pi85™"in a wide range of applications documenting its specificity and tissue reaction pattern (9, 22-26). Cell Cultures. Human lung tumor cell lines were obtained from American Type Tissue Culture (Rockville, MD) (A-549, A-427, SKLU, CALU-1, CALU-3, CALU-6, SK-MES) and from Dr. A. Gazdar of the National Cancer Institutes (NCI-HI466 and NCI-H522). The cells were cultured in RPMI and 10% fetal bovine serum or Eagle's modified essential media and 10% fetal bovine serum and incubated at 37°Cin a humidified 5% CO2 atmosphere. The rat neuroblastoma cell line B104 was used as a positive control in Northern blot, Western blot, and immunohistochemical analyses. This cell line is known to express pi85""", contains DNA which when transfected into NIH 3T3 cells results in their transformation, and was the source of isolation of the neu cDNA3 (10, 14). Western Blot Analysis. Cells were disrupted in lysis buffer [20 mM Tris, pH 8-0.33 M sucrose-0.5 mM ethyleneglycol bis(/3-aminoethyl ether)-yV,Ar,A",./V'-tetraacetic acid-25 uM leupeptin-1.5 UMaprotinin-5 UM7V-ethylmaleimide-0.5% Triton X-100], and the protein content was determined by the Bradford method (27). Total protein, 100 Mg,from each cell line was resuspended in Laemmli buffer, heated at 100'C for 3The abbreviations used are: cDNA, complementary dodecyl sulfate; SSC, standard saline citrate.
DNA; SDS, sodium
P1851*" EXPRESSION
IN HUMAN LUNG CANCER
Table 1 Non-small cell lung cancers resected between 1982 and 1985 Adenocarcinomas" Squamous cell carcinomas Large cell carcinomas'*
Total 30 20 10
Studied 29* 16C 10
* Adenocarcinomas were further separated into acinar, bronchoalveolar, pap illary, and solid (positive mucicarmine staining) carcinomas with mucus produc tion (20). * One case was a metastasis from a nonpulmonary primary and therefore excluded. 'Four cases were métastasesfrom 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 electrophoresis analysis using a 5% stacking and 7.5% separating acrylamide gel with 0.192 M glycine-15 mM Tris, pH 8.3, upper and lower tank buffer. After completion of electrophoresis, the gel was equilibrated in transfer buffer (20% methanol-0.192 M glycine-15 mM Tris, pH 8.3) and the equilibrated gel was electrically transferred to a nitrocellulose mem brane with 30 V at 4°Cfor 16 h. Adequacy of transfer was determined by staining the gel after the transfer with 0.1% Coommassie blue. The nitrocellulose membrane was blocked in 4% nonfat dry milk, followed by the addition of the primary antibody at a 1:1000 dilution. After 16 h (4°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 -70°C. RNA Isolation and Northern Blot Analysis. Total cellular RNA was isolated by a guanidium isothiocyanate method with cesium chloride modification (28, 29). Equal amounts of RNA, as determined by A26o, were denatured in sample buffer and size fractionated by electrophoresis through a 1% agarose gel containing 6% formaldehyde and 2 Mg/dl ethidium bromide. Agarose gels were visualized by UV illumination to determine the position of 28S and 18S rRNA bands, to assess the integrity of RNA, and to verify that equal amounts of RNA had been loaded into all wells. Transfer of RNA to nylon membranes was accomplished by capillary blotting for 24 h using 2.5x sodium chloridesodium phosphate-EDTA (350 mM NaCl-80 mM NaH2PO„-7HIM EDTA, pH 7.4). After blotting, membranes were dried, baked at 80°C in vacuo, and prehybridized at 42°Cfor 16-24 h in prehybridization fluid (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 followed by hybridization for 24 h at 42°Cin the same solution with IO6 cpm/ lane of a heat-denatured "P-labeled plasmid DNA (30) with a full length HER2/ne« cDNA insert (31). Blots were washed twice in 2x SSC (ix SSC, 150 mM NaCl-15 mM sodium citrate, pH 7) and 0.1% SDS at 25°Cfor 30 min and twice in 0.1 % SSC and 0.1 % SDS at 50°C for 30 min and exposed at -70°C to Kodak XAR-5 film. To ensure equal loading and transfer of RNA in all lanes, radiolabeled probes were removed from the membranes by incubation with 0.2% SDS, 10 mM Tris at 85°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 Pathology at the University of Iowa Hospitals and Clinics. Sections (3-4 urn) were cut from the tissue blocks, placed on pretreated (Histostik) glass slides, dried at 37°Covernight followed by 60°Cfor 30 min, and exposed to xylene for 5 min (3 times). Tissues were rehydrated in decreasing concentrations of ethanol (100, 95, and 70%) for 2 min (twice) and rinsed in distilled water, and endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in methanol (30 min). Slides were washed in PBS (20 min), excess liquid was blotted off, and nonimmune goat serum (Vector Laboratories Ine, Burlingame, CA) was applied to reduce nonspecific background staining (20 min). The serum was drained and rabbit anti-pl85neu antiserum or a negative
Chemical Co., Milwaukee, WI) was added (30 min), and, again, the slides were rinsed with water and counterstained with Harris hematoxylin. After the counterstain, the tissue was dehydrated with increasing solutions of ethanol (70, 95, and 100%) for 2 min (twice) followed by clearing in xylene and mounting. Positive controls of formalin-fixed, paraffin-embedded human lung were used with each staining reaction. Negative controls consisted of tissue sections from the paraffin-embedded lung cancer block to be studied for p 185"'" expression treated with an irrelevant antibody (rabbit anti-mouse IgG) (Vector) or with pl85nel1antisera previously exposed to saturating amounts of immunizing peptide. All microscopic analyses were performed by the same pathologist (R. A. R.). A tissue specimen exhibiting membrane or membrane and cytoplasmic reactivity with DBW-2 antiserum throughout the tissue section was considered posi tive. No reactivity or cytoplasm without membrane reactivity was considered negative. Flow Cytometry Analysis. Flow cytometry of paraffin-embedded tis sue was accomplished by the techniques of Hedley et al. (33) and Vindelov et al. (34). A modified pepsinization technique of Hedley was used to obtain nuclei and the propidium iodide staining method of Vindelov was used to stain the DNA (35). Two to three SO-^m sections were cut from paraffin blocks that had > 1 cm2of tissue with a minimum of 50% tumor cells. The blocks were also chosen to include 5-10% of apparent normal tissue as an internal diploid standard. Hematoxylin and eosin sections were cut after the thick sections to confirm the presence of tumor in the sections being studied. The sections were deparaffinized by applying two 3-ml changes of xylene for 10 min at 20°Cafter hydration in decreasing alcohol concentrations. The tissue was then washed twice in distilled water and resuspended in 1 ml of 0.5% pepsin (pH 1.5) (Sigma Chemical Co., St. Louis, MO) for 90 min with intermittent vortexing. The nuclei were pelleted by centrifugation and the supernatant was aspirated. The nuclei were resuspended in 0.1 ml of citrate buffer (pH 7.6) and 9.9 ml of solution A (30 mg/liter trypsin, pH 7.6) (Sigma) was added. After 10 min, 0.75 ml of solution B (500 mg/liter trypsin inhibitor-100 mg/liter RNase A, pH 7.6) (Sigma) was added to the suspension. After an additional 10 min, 0.75 ml of solution C (420 mg/liter propidium iodide, pH 7.6) (Sigma) was added, and the suspension was filtered through a 30-^m nylon mesh filter. The DNA nuclear content was measured on a Becton Dickinson 440 flow cytometer (Becton Dickinson, Mountainview, CA). Nuclei from normal paraffin-embedded lymph nodes were examined as exter nal standards to set the diploid peak at channel 60. For each specimen, histograms of 10,000 events were recorded utilizing hardware gating on propidium iodide to eliminate debris material in the lower channels to the left of the G, peak. Integration procedures were performed to obtain phase fraction analyses. The coefficient of variance was deter mined by the full width peak method. Nuclear DNA ploidy patterns were categorized as either diploid, tetraploid, or aneuploid. Diploid tumors were defined as those histo grams having only one definable G! peak. Aneuploid tumors were identified as those histograms having a second peak that did not fall in the expected channels of the G, and the G2 + M peaks. Tetraploid tumors were defined as any histogram whose G2 + M peak was >3 SD above the G2 + M peak of normal lymphocyte nuclei. The nuclei in the G2 + M peak were sorted by flow cytometry and examined microscop ically to confirm that they were single nuclei and not doublets. Statistical Analysis. Nonparametric statistical tests (36) were used to evaluate all of our comparisons. Fisher's exact test was used to assess the relationship between categorical variables and the expression of pl85"eu, and the Mann-Whitney U test was used to calculate P values for continuous variables. The relationship between the cumulative probability of survival and a variety of clinical variables, including the expression of p 185"", was determined by using the log rank test, as
described by Kaplan and Meier (37). Censoring was performed for subjects who were either lost to follow-up or were followed for less than the longest survivor. The clinical factors that were significantly associ ated with the cumulative probability of survival were tested in a hier control applied (60 min). Slides were rinsed with PBS and drained, and a biotin-conjugated goat anti-rabbit antibody was added (Vector). After archical multivariate survival analysis, as described by Cox and Oakes 30 min, the slides were again rinsed with PBS, avidin was applied for (38). All interactions were tested among clinical factors that were found 30 min, the slides were rinsed in PBS, diaminobenzidine (Aldrich to be independently associated with survival. 5185
pl85~° EXPRESSION
IN HUMAN LUNG CANCER
RESULTS Immunohistological
Detection of pl85"eu Expression Corre
lates with Western and Northern Blot Analysis. In order to use the monospecific, polyclonal anti-pi85"°" antiserum DBW-2 to identify pl85"cu, we first studied the relationship between HER2/neu gene expression and DBW-2 antiserum-identified pl85neu protein expression. Total cellular RNA (28, 29) and protein (9) were isolated from 10 cell lines derived from human non-small cell lung cancers. Northern and Western blot anal yses 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 single transcript of 4.8 kilobases in expressing cell lines. By Western blot analysis (Fig. 1, lane B) DBW-2 antiserum recognized a single protein of M, 185,000. The Western and Northern blot autoradiogram signals representing pi85ncu protein and HER2/ neu mRNA expression from each cell line were quantitated by digital analysis and the degree of HER2/neu mRNA accumu lation was correlated with the degree of pl85neu protein expres sion. As shown in Fig. 1, gene expression and protein expres sion correlated significantly (r = 0.90, P = 0.0001). Therefore, we concluded that the level of pl85neu expression determined by Western analysis using DBW-2 antiserum is a good assess ment of HER2/neu gene expression. In these same cell lines, we next sought to determine the relationship between immunohistochemical identification of pl85ncu using DBW-2 antiserum and Western and Northern blot analyses. Cells from each cell line were fixed, pl85ncu
Correlation of HER2/neu Gene Expression and p185neuExpression
200 r = 0.90 p = 0.0001
O
100
A-427 kb
1
50
A-427 kDl 200.0-
f
i
50
100
82.5-
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 nonsmall cell lung cancers and analyzed for HER2/neu gene expression using a HER2/nfU cDNA probe. Total cell protein was isolated from the same cell lines and Western blot analysis performed using DBW-2 antiserum. A representative Northern blot (lane A ) with the single 4.8-kilobase (kb) HER2/neu mRNA transcript and 2.0-kilobase tf-actin control probe is shown. A representative Western blot (lane B) is also shown. Both protein and DNA were derived from the American Type Culture Collection cell line A-427. The autoradiogram hy bridization signals were quantitated by digital analysis and plotted, and a corre lation coefficient was derived. A total of 3 Northern blots and 5 Western blots were 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 to high) by an investigator unaware of the results of Western and Northern blot analyses. The immunohistochemical identifica tion of pl85"cu was found to be strongly related to levels defined by Western blot analysis (Kruskal-Wallis one-way analysis of variance, x2 = 11.0, /* = 0.03), as was the comparison of the level of HER2/neu gene expression defined by Northern blot analysis to the immunohistochemical identification of pl85neu by DBW-2 antiserum (x2 = 10.4, P = 0.03). Therefore, our results indicate that immunohistochemical identification and quantitation of pl85ncu with DBW-2 antiserum is strongly associated with either Northern blot analysis of HER2/neu expression or Western blot analysis of its protein product. From these studies we conclude that using DBW-2 antiserum for immunohistological analysis to determine pl85"eu expression is a valid technique. Expression of pl85"eu in the Adult Human Lung and in NonSmall Cell Lung Cancer. We next sought to immunohistologically determine pl85"eu expression in specimens of normal human lung and lung cancer. In sections of normal lung ob tained 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 ciliated respiratory epithelial cells (1+), bronchial mucosal glands in the major airways (2+), and cells with the appearance of type II pneumocytes (1+). By light microscopy it was difficult to determine whether staining was present in type I pneumocytes. Other normal cells from the lung, including fibroblasts, smooth muscle cells, and endothelial cells, did not react with the antiserum. Ten adenocarcinomas reacted with DBW-2 antiserum (Table 2). Staining was present in all areas of the tumor represented in the tissue section (Fig. 3). Reactivity was usually uniform throughout (4+) but occasionally ranged from 2-4+. Both membrane and cytoplasmic staining was present in all tumors examined, with membrane staining more intense, especially in the bronchioloalveolar subtypes. Interestingly, membrane stain ing was always most intense at the luminal border. Foci of clear cells showed less intense staining than non-clear cells, and tumor cells displaying more cytoplasm and showing a lower nuclear grade were more often positive. Five squamous cell carcinomas also reacted with DBW-2 antiserum (Table 2). Staining was uniform throughout the tumors but usually at lower levels than in adenocarcinomas (23+) (Fig. 4). Membrane staining was also less pronounced than in adenocarcinomas (2+). There was more intense staining of cells with large amounts of cytoplasm. Moreover, foci of welldifferentiated squamous cell carcinomas tended to be 4+ posi tive. None of the large cell carcinomas studied reacted with DBW2 antiserum (Table 2). In 5 cases (3 adenocarcinomas, 2 squa mous cell carcinomas), the immunohistochemical staining pat tern was indeterminate. These tumors had a single focus of DBW-2 antiserum reactivity as opposed to diffuse reactivity.
5186
Table 2 pl85"'" expression in human non-small cell lung cancer Expression of p 185"'" typeAdenocarcinomaSquamous Cell cellLarge cellTotalAbsent1691035Present105015%
Positive3836030
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 the antiserum and stronger luminal membrane reactivity (1+) (x 250). In /(, the same tissue block was stained with anti-pi85°*°-specificantiserum first exposed to saturating amounts of immunizing peptide. Note the loss of reactivity on the membrane and in the cytoplasm (x 250).
;
Fig. 4. Expression of pl85"*" in human lung squamous cell carcinomas. In A, human lung squamous cell carcinomas were stained with anti-plSS^-specific antiserum as described in "Materials and Methods." Note the uniform staining of p 185"'" throughout the tumor (2-3+) with some cell to cell variation (x 300). In lì, the same tissue block was stained with anti-pi85"'"-specific antiserum first exposed to saturating amounts of immunizing peptide. Note the loss of reactivity on the cell membrane and in the cytoplasm (x 300).
Since it could not be determined whether these tumors were pl85neu 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 at low levels throughout the respiratory tract in ciliated bronchiole epithelial cells (1+), bronchial mucosa! glands (2+), and type II pneumocytes (1+). In addition, expression of pl85"eu was found in 38% of adenocarcinomas and 36% of squamous cell carcinomas but not in large cell carcinomas. The level of pl85neu found in the expressing carcinomas was always higher than normal expression seen in uninvolved bronchiolar epithelium. Association Between p 185"'" Expression and CIinicopatho log ical Features. The expression of pl85neu was not associated with
B Fig. 3. Expression of plSS"" in a human lung adenocarcinoma. .1. section from a lung adenocarcinoma showing uniform p 185°"" reactivity (4+) throughout the tumor (x 400). B, same tissue block using anti-pi85™"-specific antiserum first exposed to saturating amounts of immunizing peptide. Note the loss of reactivity on the cell membrane and in the cytoplasm (x 400).
any unique clinical characteristics in patients with squamous cell lung cancer (Table 3). In particular, no relationship was found with poor prognostic indices, such as increased tumor stage (P = 0.35), older age (P = 0.25), and diminished survival (P = 0.50). Moreover, no consistent correlation existed between the 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 adenocar cinoma of the lung (Table 4). Adenocarcinomas expressing pl85neu were found to be derived from a significantly older
5187
pl85~" EXPRESSION
IN HUMAN LUNG CANCER
Table 3 Relationship between expression of the HER2/neu gene protein product p 185"" and clinicopathological variables for patients with squamous cell lung cancer plSS""1Categorical Expression of
Table 4 Relationship between expression of the HER2/neu gene protein product plS5*" and clinicopathological variables for patients with adenocarcinoma of the lung p!85°~Categorical Expression of
value"1.001.000.550.200.251.000.350.951.000.350.600.250.300.150.50 =10)68422127134355—415915—1682— value"0.700.601.001.000.5 (N=5)4433100412305022101463.4 (W variablesMaleSmokerCoughHemoptysisWeight
variablesMaleSmokerCoughHemoptysisWeight
lossChest painPrimary
lossChest painPrimary
tumor123Regional
tumor123Regional
nodes0I2Métastases01Surgical lymph
nodes012Métastases01Surgical lymph
stage1234OutcomeDeadAliveHistologyBronchioloalveolarPapillarySolidAcinarDNA
stage1234OutcomeDeadAliveContinuous
variablesAge 10.1*65.6 ± 4.550.0 ± (yr)Pack ±14.18.5 yrTumor 30.93.7 ± 1.7177.3± ± 6.3237.8+ ± (cm)Survival size 134.5P (wk)Absent(N=9)792152Ì518109071104558.6 129.7Present " 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 ±SD.
patient population (66.6 ±10.1 (SD) versus 57.5 ±10.8 years) (P = 0.04) with a significantly shorter survival (83.7 ±94.1 versus 188.5 ±120 weeks) (P = 0.01). Marginal, albeit statis tically insignificant, relationships were also observed in adenocarcinomas between the expression of pl85n
