Alterations of K-ras, p53, and erbb-2jneu in human lung adenocarcinomas

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1 lterations of K-ras, p53, and erbb-2jneu in human lung adenocarcinomas The development of human adenocarcinoma of the lung involves multiple genetic changes including activation of oncogenes and loss of tumor suppressor genes. Patients whose lung tumors contain K-ras oncogene mutation, accumulation of the protein product of the tumor suppressor gene p53, or erbb-2/neu oncoprotein overexpression have been shown to have a worse prognosis. We examined these three genetic indicators in 29 lung adenocarcinomas to determine whether these markers are present in the same tumors or if they represent molecular changes that define different subsets of patients. P53 nuclear protein accumulation and erbb-2/ neu protein overexpression were determined by immunohistochemical analysis of cryostat sections of tumor specimens and corresponding normal lung tissue. K-ras mutations were detected by radiolabeled oligonucleotide probes, specific for the various twelfth codon mutations, hybridized to exon 1 of K-ras, which was amplified by the polymerase chain reaction. Increased nuclear accumulation of p53 protein was found in 11 adenocarcinomas (38 %). ll of the p53 positive tumors were found to show high level staining and homogeneous expression of erbb-2/ neu protein. K-ras mutations were detected in seven tumors (24 %), all of which overexpressed erbb-2/neu. The presence of a K-ras mutation did not correlate with p53 accumulation. In total, 93% of the tumors were found to overexpress erbb-2/neu, the highest being in one tumor with erbb-2/neu gene amplification. The presence of K-ras twelfth codon mutation was associated with increased cigarette smoking. In conclusion, erbb-2/neu overexpression is a common event in lung adenocarcinomas. Furthermore, the presence of K-ras mutation and p53 protein accumulation define separate groups of patients. The mechanisms by which these genetic alterations interact or adversely affect prognosis is unknown. (J THoRc CRDIOVSC SURG 1994;107:590-5) Philip F. Bongiorno, MD (by invitation), Richard 1. Whyte, MD (by invitation), Eric J. Lesser (by invitation), Jason H. Moore, BS (by invitation), Mark B. Orringer, MD, FCS, and David G. Beer, PhD (by invitation), nn rbor, Mich. Investigation into the molecular biology of lung cancer has revealed multiple abnormalities in oncogenes and tumor suppressor genes that are consistent with a multistep process of carcinogenesis. I, 2 Numerous reports exist that implicate specific genetic changes as negative prognostic markers when occurring in lung adenocarcinoma. These markers may identify patients with early-stage lung cancer at risk for recurrence after resection. From the Section of Thoracic Surgery, University of Michigan Medical School, nn rbor, Mich Read at the Seventy-third nnual Meeting of The merican ssociation for Thoracic Surgery, Chicago, I11., pril 25-28, ddress for reprints: David G. Beer, PhD, B560 MSRBII, Box 0686, University of Michigan, nn rbor, MI by Mosby-Year Book, Inc /94 $ /6/51760 K-ras is a commonly activated oncogene in human lung cancer with alterations at the twelfth codon position accounting for 70% to 80% of the activating mutations. 3, 4 The function of K-ras in normal and lung cancer cells is not known, but the oncogene likely functions in growth signal transduction and cellular differentiation. 5, 6 Lung adenocarcinomas with K-ras mutations have been shown to have a significantly worse prognosis than K-ras-negative tumors.3, 7, 8 In one study, 18 patients with stage I, K-ras-positive tumors had a decreased 5-year survival of 53% compared with an 84% 5-year survival in patients with stage I, K-ras-negative tumors.3 The tumor suppressor gene p53 has also been shown to affect prognosis in lung adenocarcinoma. The normal p53 protein is involved in cell cycle regulation. 9 Mutation of p53 gene leads to a loss of function and to increased protein half-life resulting in nuclear accumulation of the 590

2 The Journal of Thoracic and Volume 107, Number 2 Bongiorno et at '.'" -.. '.,' " "., Fig. 1. Immunohistochemical staining for erbb-2/neu ( and B) and for p53 proteins (C and D) in frozen sections of lung adenocarcinomas. Homogeneous overexpression of the oncoprotein erbb-2/ neu is present on almost all cells within the tumor. denocarcinoma cells stain while adjacent normal lung cells (N) do not stain for erbb-2/neu (). P53 nuclear protein accumulation was seen to occur in two different staining patterns. Some tumors demonstrated p53 protein accumulation in all adenocarcinoma cells (C), whereas others demonstrated p53 protein accumulation in just a subset of the cells within the tumor section (D). Normal lung cells did not demonstrate p53 protein accu, mulation. mutant p53 protein.1 0 P53 mutation and protein accumulation are associated with a worse prognosis in lung adenocarcinoma. 11, 12 Patients with stage I and II adenocarcinomas demonstrating p53 protein accumulation were found to have a 5-year survival of only 15% whereas those without p53 accumulation had a 5-year survival of 35%.1 1 The protooncogene erbb-2jneu encodes a membrane bound, probable autocrine growth factor receptor. 13 Overexpression of erbb-2jneu protein is associated with decreased survival in several human cancers,14, 15 including lung adenocarcinoma. 16 ErbB-2jneu protein overexpression is associated with gene amplification in breast cancer,17 whereas gene amplification is rare in lung adenocarcinoma. 14,18 K-ras oncogene twelfth codon mutations, p53 tumor suppressor gene protein accumulation, and erbb-2jneu oncoprotein overexpression were examined in 29 patients who underwent resection for lung adenocarcinoma. The prevalence and coincidence of these three prognostic markers were examined to determine if they occur in the same tumors and thus identify the same patients or represent independent genetic events in carcinogenesis. Gene amplification as a mechanism for erbb-2jneu protein overexpression was examined. dditionally, correlations between the genetic alterations and age, sex, tumor stage, and smoking history were made. Materials and methods Tissue samples. nalyses were performed on specimens from 29 consecutive patients with the final diagnosis of primary lung adenocarcinoma, who were operated on at the University of Michigan Hospital between ugust 1991 and ugust Immediately after resection, normal lung and lung tumor tissues were divided into similar portions. One portion was frozen in liquid nitrogen for isolation of deoxyribonucleic acid (DN) and ribonucleic acid (RN); the other was embedded in OCT compound (Miles Inc., Elkhart, Ind.) and frozen in isopentane cooled to the temperature ofliquid nitrogen. Samples were then stored at C until analysis. Detection of K-ras twelfth codon mutation. Genomic DN was isolated from frozen tissue specimens by standard techniques. 19 Exon 1 of the K -ras gene was amplified by the polymerase chain reaction as described. 7 Polymerase chain reaction

3 5 9 2 Bongiorno et al. The Journal of Thoracic and February 1994 B SER c 0 E F G H B L c 0 E F G H B VL c 0 E F G H NTNTNTCC NTNTNTCC ,. SP CYS RG Table I. Summary of K-ras, p53, and erbb-2jneu TNM K-ras twelfth P53 ErbB-2/neu stage codon mutation accumulation overexpression 4/14 3/14 14/14 II 0/3 2/3 3/3 lila 3/11 6/11 10/11 IV...QLL...QLL...QLL Totals 7/29 11/29 27/29 (24%) (38%) (93%) mouse monoclonal antibody (b-2) (clone 9G6) (Oncogene Science), which reacts with an extracellular domain epitope. Detection of erhb-2/ neu gene amplification. Southern blot analysis was used to evaluate for erbb-2/ neu gene amplification in three tumors that highly overexpressed erb B-2 / neu protein as previously described. 2o DN 10 JIg was digested with the restriction enzyme Hind III. Probes used were a 1.6 Kb EcoRI fragment of human erbb-2/neu cdn and a 0.8 Kb Pvu II fragment of human MDR-I cdn as a single copy reference gene. Clinical correlations. Relations between p53 and K-ras alterations and age, sex, tumor stage, and smoking history were examined with either the x 2 or Fisher's exact test. Fig. 2. Composite of dot-blot membranes that were sequentially probed with radioactive oligonucleotides specific for the six possible twelfth codon mutations. Exon I of the K-ras gene was amplified by the polymerase chain reaction (PCR). The PCR products from 25 lung adenocarcinoma (2, 4, 6, and 8) and corresponding normal lung specimens (I, 3, 5, and 7) were blotted onto three nylon membranes. Control DNs with known serine and aspartic acid mutations were blotted at B7 and C7, respectively. One tumor sample was divided between C5 and C6 without a corresponding normal lung sample. Mutations were determined by positive autoradiographic signals that far exceeded background signals. In these 25 adenocarcinomas, one aspartic acid mutation, one alanine mutation, four cysteine mutations, and one valine mutation were detected. The dot-blots for the other four patients were prepared identically, with the same controls, and revealed no mutations (data not shown). products were then vacuum blotted onto nylon membranes (GeneScreen Plus, NEN, Wilmington, Del.) and probed with radiolabeled oligonucleotides (CIon-Tech, Palo lto, Calif.) homologous to the known twelfth codon mutations. Blots were then washed at high stringency and autoradiograms were prepared. Immunohistochemistry of p53 and erhb-2/neu. Overexpression of erbb-2 / neu protein and accumulation of p5 3 protein were detected by the avidin-biotin-peroxidase complex method on 5 JIm thick cryostat sections. 2o P53 protein was detected with a I: I 00 dilution of a mouse monoclonal primary antibody (b- 2) (clone Pb1801) (Oncogene Science, Inc., Uniondale, N.Y.), which reacts with the N-terminaldomain ofp53. ErbB- 2/neu was detected with a I :100 dilution of an erbb-2/neu Results Immunohistochemistry was used for the analysis of p53 protein accumulation and erbb-2jneu overexpression in 29 lung adenocarcinomas. P53 protein accumulation was detected in the nuclei of 11 of 29 tumors (38%). Tumors that either stained positively or negatively for p53 were found among all TNM stages (Table I). In six of 11 p53 positively staining adenocarcinomas all tumor cells demonstrated nuclear protein accumulation, whereas in the other five positive tumors only a subset of the cells demonstrated nuclear protein accumulation (Fig. 1). P53 protein accumulation was not observed in normal lung tissue. ErbB-2/neu protein expression was found in 27 tumors (93%) (Table I). The staining pattern was identical in all tumors, with every tumor cell showing expression of the erbb-2/neu protein. Three tumors expressed erbb-2/neu ata very high level, whereas erbb-2/neuwas either undetectable or expressed only at very low levels in normal lung tissue (Fig. O. K-ras twelfth codon mutations were detected in seven of 29 tumors with differential hybridization of radioactive oligonucleotide probes used to dot-blot membranes of polymerase chain reaction-amplified normal lung and tumor DN (Fig. 2). Wild-type K-ras twelfth codon was detected in all samples of normal lung and tumor DN (data not shown). Mutations were detected in four stage I tumors and in three stage IlIa tumors (Table I). No mutations were detected in normal lung.

4 The Journal of Thoracic and Volume 107, Number 2 Bongiorno et al lthough erbb-2/neu was detected in nearly all tumors, only two tumors were found to contain both K -ras mutation and p53 protein accumulation (Table II). The two tumors that did not show erbb-2/neu overexpression had neither p53 nor K-ras changes. Southern blot analysis was used to determine if erbb- 2/neu gene amplification was occurring in the three tumors that expressed erbb-2/neu protein at the highest levels. One tumor demonstrated low-level amplification, and the other two tumors had evidence for only a single copy of the erbb-2/neu gene (data not shown). No statistically significant differences between K-ras positive and negative or p53 positive and negative tumors were seen in regard to patient sex or age or tumor stage (p > 0.05). Patients with K-ras positive tumors had a longer smoking history-a mean of 71 pack-years compared with a mean of 41 pack-years for patients with K-ras negative tumors (p < 0.05). There were four nonsmokers in the cohort, none of whom had K-ras positive tumors. If the analysis is restricted to smokers, K-ras remains associated with increased smoking history with a x 2 p value of ccumulation of p53 nuclear protein was not found to be associated with smoking history. Discussion lterations in the structure or expression (or both) of K-ras,p53, and erbb-2/neu have all been correlated with worse than expected prognosis in lung adenocarcinoma. 3, 7, 8,11,12,16 The present study has examined these three markers in the same lung adenocarcinoma specimens. The finding of p53 and K-ras twelfth codon abnormalities in 38% and 24% of the tumors, respectively, is consistent with the results of previous studies. 7, 11 Interestingly, p53 and K-ras occurred together in only two tumors (7%), indicating that these changes may be independent events in lung carcinogenesis. Kishimoto and associates 21 reported a similar result when they analyzed for p53 alterations using single-strand conformational polymorphism analysis in a group of non-small-celliung cancer tumors, which had previously been analyzed for K-ras mutations?1 P53 and K-ras have also been found to be independent events in colon adenocarcinomas. 22 P53 and K-ras appear to define separate subsets of patients with lung cancer who have potentially decreased survival. nalysis of the groups of patients with either K -ras or p53 alterations revealed an increased smoking history in the patients with K-ras twelfth codon mutations. This relation remained even if the analysis included only those patients who smoked. Cigarette smoke is known to contain mutagens capable of forming DN adducts that can Table II. Summary of genetic changes in the 11 p53 positive tumors TNM No.o1P53 K-ras 12th ErbB-2/neu stage positive cells codon mutation overexpression all Cysteine Positive few WT* Positive few Valine Positive II all WT Positive II few WT Positive ilia all WT Strongly positive ilia all WT Positive ilia all WT Positive ilia all WT Positive IlIa few WT Positive ilia few WT Positive WT, Codes for wild-type amino acid glycine at twelfth codon position. result in K-ras mutations. These results are consistent with the results of Reynolds and colleagues,23 who found a high percentage of K-ras twelfth codon mutations in smokers. ErbB-2/neu protein was expressed in 93% of the tumors analyzed. This observation is consistent with the findings of Shi and coworkers 24 but appears in contrast with other reports of a much lower frequency of erbb-2/ neu overexpression. 16 These differences likely relate to conditions used during immunohistochemistry. Formalin fixation and paraffin embedding of tissue used by others 16 may alter erb B-2 / neu antigens, 14 whereas this study used frozen tissue sections. dditionally, we used a monoclonal antibody directed to the extracellular portion of the protein, whereas others have used either polyclonal or monoclonal antibodies directed toward different epitopes. 16,24 The high frequency of tumors showing overexpression of erbb-2/ neu protein suggests that erbb- 2/neu protein overexpression is a very common event in lung adenocarcinomas. We find similar expression of this protein on almost all adenocarcinoma cells within the tumor. This may indicate that an important selection process exists that maintains the expression of this protein in all tumor cells. ErbB-2/neu protein overexpression along with other molecular alterations such as K-ras or p53 gene mutations may playa key role in lung carcinogenesis. ErbB-2/neu protein overexpression is not likely to indicate prognosis in this sample of 29 lung adenocarcinomas inasmuch as it is present in most tumors. ErbB-2/neu overexpression is commonly associated with gene amplification in breast and ovarian cancer. 14, 17 ErbB-2/neu gene amplification is known to be rare in lung cancer, and erb B-2 / neu was reported to occur in only one of 60 non-smail-cell lung cancers. 14, 18 Southern blot

5 5 9 4 Bongiorno et al. The Journal of Thoracic and February 1994 analysis of the tumors that overexpressed erbb-2jneu to the greatest extent revealed only one tumor with gene amplification and two tumors with single copies of the erbb-2jneu gene. Other possible mechanisms for erbb- 2jneu protein overexpression exist and include increased gene transcription,increased messenger RN stability, and increased protein stability. They are currently being examined in this laboratory. Future improvements in lung cancer survival will likely require earlier detection of cancer, identifying patients at high risk for recurrence after resection, and the development of novel therapies. Patients at risk may be screened for specific molecular alterations, possibly through sputum samples, with the intent to detect malignancy at its earliest point. Patients with early-stage lung cancer may be identified as being at high risk for recurrence on the basis of these molecular markers and may be given further therapy in addition to resection. Novel therapies beyond standard chemotherapy and radiation therapy, directed at these molecular changes, may be developed. Monoclonal antibodies have been developed against the erbb-2jneu protein,25, 26 and there are ongoing trials using these antibodies in patients with breast cancer. Because of its abundant expression, erbb-2jneu protein may prove to be an effective target for monoclonal antibody therapy in lung adenocarcinoma. REFERENCES 1. S1ebos RJC, Rodenhuis S. The molecular genetics of human lung cancer. Eur Respir J 1989;2: Gazdar F. Molecular markers for the diagnosis and prognosis oflung cancer. Cancer 1992;69: Sugio K, Ishida T, Yokoyama H, Inoue T, Sugimachi K, Sasazuki, T. ras Gene mutations as a prognostic marker in adenocarcinoma of the human lung without lymph node metastasis. Cancer Res 1992;52: Lehman T, Bennett WP, Harris CC, etal. p53 Mutations, ras mutations, and P53-heat shock 70 protein complexes in human lung carcinoma cell lines. Cancer Res 1991 ;51: Barbacid M. Ras genes. nnu Rev Biochem 1987;56: Pan J, Roskelley CD, Luu-The Y, Rojiani M, uersperg N. Reversal of differentiation by ras oncogene mediated transformation. Cancer Res 1992;52: Slebos RJC, Kibbellelaar R, Rodenhuis S, et al. K-ras oncogene activation as a prognostic marker in adenocarcinoma of the lung. N Engl J Med 1990;323: Mitsudomi T, Steinberg SM, Gazdar F, et al. ras Gene mutations in non-small cell lung cancers are associated with shortened survival irrespective of treatment intent. Cancer Res 1991;51: Lane DP. P53 guardian of the genome. Nature 1992; 358: NigroJM, Collins FS, Yogelstein B, et al. Mutations in the p53 gene occurs in diverse human tumor types. Nature 1989;342: Quinlan DC, Davidson G, Summers CL, Wasrden HE, Doshi HM. ccumulation of p53 protein correlates with a poor prognosis in human lung cancer. Cancer Res 1992; 52: Horio Y, Takahashi T, Takahashi T, et al. Prognostic significance of p53 mutations and 3p deletions in primary resected non-small cell lung cancer. Cancer Res 1993;53: Holmes WE, Sliwkowski MX, Yandlen RL, et al. Identification of heregulin, a specific activator of p I 85erbB2. Science 1992;256: Siamon DJ, Godolphin W, Press MF, et al. Studies of the HER-2/neu protooncogene in human breast and ovarian cancer. Science 1989;244: Yonemura Y, Ninomiya t, Sasaki T, et al. Expression of c-erbb-2 oncoprotein in gastric carcinoma. Cancer 1991 ; 67: Kern J, Schwartz D, Robinson R, et al. Pl85 neu expression in human lung adenocarcinomas predicts shortened survival. Cancer Res 1990;50: Liu E, Thor, He M, Barcos M, Ljung B, Benz C. The HER2 (c-erbb-2) oncogene is frequently amplified in in situ carcinomas of the breast. Oncogene 1992;7: Schneider PM, Hung M, Roth J, et al. Differential expression of the c-erbb-2 gene in human small cell and non-small cell lung cancer. Cancer Res 1989;49: Maniatis T, Fritsch EF, Sambruck J. Molecular cloning: a laboratory manual. 2nd ed. New York: Cold Spring Harbor Laboratory, 1989;9: I-Kasspooles M, Moore JH, Orringer MB, Beer DG. mplification and overexpression of the EGFR and erbb-2 genes in human esophageal adenocarcinomas. Int J Cancer 1993;54: Kishimoto Y, Murakami Y, Shiraishi M, Hayashi K, Sekiya T. berrations of the p53 tumor suppressor gene in human non-small cell carcinomas of the lung. Cancer Res 1992;52: Bell SM, Scott N, Quirke P, et al. Prognostic value of p53 overexpression and c-ki-ras gene mutations in colorectal cancer. Gastroenterology 1993;104: Reynolds SH, nna CK, nderson MW, et al. ctivated protooncogenes in human lung tumors from smokers. Proc Natl cad Sci US 1991;88: Shi D, He G, Hung M, et al. Overexpression of c-erbb-2/ neu-encoded p185 protein in primary lung cancer. Mol Carcinog 1992;5: De Santes K, Slamon D, Press 0, et al. Radiolabeled antibody targeting ofthe HER -2/ neu oncoprotein. Cancer Res 1992;52: Hsieh-Ma ST, Eaton M, Shi T, Ring DB. In vitro cytotoxic targeting of human mononuclear cell and bispecific

6 The Journal of Thoracic and Volume 107, Number 2 Bongiorno et al antibody 2B 1, recognizing c-erhb-2 protooncogene product and Fc,), receptor III. Cancer Res 1992;52: Discussion Dr. Douglas J. Mathisen (Boston, Mass.). Did you look at the degree of histologic differentiation of these tumors and whether there is any correlation with your findings? Do you have any information on the survival of these patients? Dr. Whyte. We did not specifically evaluate the degree of differentiation, but we did look at the tumor stage and found that there was no significant relation between the genetic alterations and the tumor stage. Regarding patient outcome, most of these patients were operated on within the last 18 months and most had stage I tumors. The follow-up period is relatively short and I have not evaluated survival yet. Dr. Martin F. McKneaIly (Toronto, Ontario, Canada). Do you envision that these markers will be used to identify patients with stage I disease at poor risk for recurrence and to apply more intensive or more toxic adjuvant therapy? Dr. Whyte. That's exactly right. If we can identify patients who are at higher risk, they will be more inclined to get adjuvant therapy. s things are now, patients with stage I disease undergo surgery with no other treatment; if a group of patients at high risk for early recurrence can be identified, these patients can be offered adjuvant therapy.