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Clonality and Prognostic Implications of p53 and Epidermal Growth Factor Receptor Somatic Aberrations in Multiple Primary Lung Cancers

Authors' Affiliations: Departments of ¹ Pathology and ² Surgery and Traumatology, National Taiwan University Hospital and National Taiwan University College of Medicine; ³ Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan; and ⁴ Division of Biostatistics and Bioinformatics, National Health Research Institute, Miaoli, Taiwan

Requests for reprints: Yung-Chie Lee, Division of Thoracic Surgery, Department of Surgery, National Taiwan University Hospital and National Taiwan University College of Medicine, 6F-1, 99, Section 3, Roosevelt Road, Taipei 10646, Taiwan. Phone: 886-2-23625177; Fax: 886-2-23625176; E-mail: damu{at}ha.mc.ntu.edu.tw or Yuh-Shan Jou, Institute of Biomedical Sciences, Academia Sinica, 128, Yen-Chiu-Yuan Road, Taipei 11529, Taiwan. E-mail: jou{at}ibms.sinica.edu.tw.

	Abstract

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Purpose: For treatment decision and prognostic applications, we evaluated p53/epidermal growth factor receptor (EGFR) somatic aberrations in multiple primary lung cancers to differentiate multifocal tumors from intrapulmonary metastasis.

Experimental Design: Fifty-eight multiple primary lung cancers of 1,037 patients in a 10-year period were identified to investigate somatic mutations and altered expression of p53 and EGFR for clonality assessment. Genomic DNA was extracted from microdissected cells of paraffin-embedded multiple primary lung cancer tissues. Overexpression and somatic mutations in exons of p53 (exons 5-8) and tyrosine kinase domain of EGFR (exons 18-22) were examined by immunohistochemical staining and DNA sequencing, respectively.

Results: High frequency of somatic mutations in p53 (33 of 58, 56.9%) and/or EGFR (44 of 58, 75.9%) resulted in high discrimination rate of tumor clonality (50 of 58, 86.2%) of multiple primary lung cancers. Twenty-two cases (37.9%) were assessed as having the same clonality and 28 cases (48.3%) were determined as having different clonality, which further supported the carcinogenic theory of field cancerization. Notably, the occurrence of lymph node metastasis was more commonly observed in tumors with the same clonality (P = 0.045) and was associated with poor patient 5-year survival rate (P = 0.001). However, no correlation was found between tumor clonality and patient survival (P = 0.630). The EGFR somatic aberrations in 58 multiple primary lung cancers, including vascular invasion associated with EGFR overexpression (P = 0.012) and mutation (P = 0.025), further suggested the potential benefits of target therapy of inoperable multiple primary lung cancers.

Conclusions: Our results suggest that analysis of somatic alterations in p53 and EGFR can significantly improve the clonality assessment and impact management of multiple primary lung cancer patients.

The criteria proposed by Martini and Melamed (6) in 1975 are still commonly used for the diagnosis of synchronous multiple primary lung cancer for patient management. The diagnosis is primarily based on the histologic characteristics of tumors, such as morphology, location, presence or absence of carcinoma in situ, vascular invasion, metastasis, and other empirical features without biological and molecular basis. Therefore, molecular analysis of clonal relation between different lesions may solve the diagnostic riddle in these patients. In particular, PCR amplification of genetic markers on small amount of tumor DNA isolated from microdissection-purified cancer cells from the same patient allows detailed analysis and determination of tumor clonality.

Recent advances in molecular tumorigenesis indicated that accumulated genetic alterations of cancer genome during multistep tumor progression are potentially useful marker for clonality analysis. Several technologies detecting genetic alterations, including X-chromosome inactivation analysis, loss of heterozygosity analysis by using microsatellite markers, viral integration analysis, and detection of mutation in tumor-associated genes (7), have been widely used with limited success. To develop optimal genetic markers for clonality analysis, highly frequent and independent somatic aberrations that occurred in early stages and are maintained throughout the tumor progression are important for clonality assessment. For instance, the mutation analysis of the p53 tumor-suppressor gene is useful for clonality assessment in lung cancer due to highly frequent, stable, and well-distributed point mutations occurring in exons 5 to 8 (8, 9). Because coding mutations of p53 occur relatively early in the development of lung cancer and are potentially required for maintaining malignant phenotype, the acquired p53 mutations are preserved during tumor progression and metastatic spread (10–12). It is reported that somatic mutations and increased expression of p53 were frequently found in 45% and 65% of non–small cell lung carcinomas (NSCLC), respectively (13).

Because protein tyrosine kinases play important roles in the pathogenesis of many malignant tumors, development of selective tyrosine kinase inhibitors is currently one of the major efforts in cancer treatment (14, 15). The epidermal growth factor receptor (EGFR), the first receptor protein tyrosine kinase described, is detected with increase of expression by immunohistochemistry in 43% to 89% of NSCLC patients (16, 17). Two molecularly targeted agents, gefitinib (AstraZeneca, Wilmington, DE) and erlotinib (OSI Pharmaceuticals, Inc., Melville, NY), have been approved and have shown greater benefit for the treatment of advanced NSCLC. The better outcomes of tyrosine kinase inhibitor treatment in NSCLC patients are strongly associated with mutations of the tyrosine kinase domain (exons 18-22) in EGFR of tumor tissues (18, 19). Depending on the tumor subtypes and races, the Caucasian NSCLC patients have an EGFR mutation frequency of 10% compared with a mutation rate of at least 30% in Asian patients (20, 21).

Toward developing useful genetic markers for clonality assessments and patient management, we took advantage of highly frequent and early somatic mutations of p53 (5, 22–24) and EGFR (25) genes to define the clonal origins of tumors in 58 multiple primary lung cancers. The results of somatic mutations along with increase of protein expression of p53 and EGFR detected by immunohistochemistry were further characterized for correlation with the clinicopathologic features for potential diagnostic and prognostic applications.

	Materials and Methods

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Patient populations. Fifty-five synchronous and three metachronous multiple primary lung cancer specimens were obtained from 1,037 patients who underwent surgical resection at the National Taiwan University Hospital from August 1995 to December 2004. The research ethics committee of the hospital has approved this study. These patients were not treated with neoadjuvant chemotherapy and irradiation therapy.

Extraction of DNA from microdissected cells in paraffin-embedded tissues. Five-micrometer-thick paraffin sections were used for genomic DNA isolation. After deparaffinization with xylene, tissue sections were stained with hematoxylin, and areas were carefully microdissected with Laser Microdissection System (Leica LMD, Wetzlar, Germany) for obtaining >70% of neoplastic and adjacent normal-appearing cells. An estimated 1,000 microdissected cells were digested in 50 µL of buffer consisting of 20 mmol/L Tris-HCl (pH 8.0), 1 mmol/L EDTA (pH 8.0), 1% Tween 20, and 1 mg/mL proteinase K for 24 h at 56°C. The protease-treated DNA mixture was heat inactivated after incubation for 10 min at 95°C as described by Sugio et al. (26) and 1 µL of DNA mixture was used for each PCR reaction.

PCR sequencing and mutation detection of p53 and EGFR genes for clonality analysis. Exons 5 to 8 of p53 and exons 18 to 22 of EGFR with their short flanking intronic sequences were PCR amplified using specific primers in a 96-well format followed by nested PCR reactions as described by previous reports (27, 28). Purified PCR products were subjected to cycle sequencing. Multiple sequence alignments of exon sequences were conducted by using Sequencer 4.14 (Gene Codes, Ann Arbor, MI) for mutation identification. The diagnosis of different clonality was made when different mutations were found in two or more tumor clones from one patient. On the other hand, the same clonality was diagnosed when the tumors showed identical DNA mutation and lack of the same mutation in the adjacent normal lung tissue samples. When no mutation was detected in either p53 or EGFR gene in tumor samples from a patient, a diagnosis could not be made.

Immunohistochemical analysis of p53 and EGFR expression. For immunohistochemical analysis of p53 protein in the tumor tissue, deparaffinized 4-µm-thick sections were treated with 0.3% H₂O₂ in methanol, heated in a microwave oven for 20 min for antigen retrieval, and incubated with normal nonimmune goat serum. After blotting the excessive goat serum, the slides were incubated with a specific mouse anti-p53 protein antibody "p53 (Ab-6), pantropic" (diluted 1:50; Oncogene Science, Cambridge, MA) for 1 h at room temperature. After incubation with biotinylated goat anti-mouse antibody, the sections were incubated with peroxidase-conjugated streptavidin. 3,3'-Diaminobenzidine tetrahydrochloride (0.05%) was used as a chromogen.

The sections for immunohistochemical analysis of the EGFR protein expression were autoclaved in 0.01 mol/L phosphate citrate buffer (pH 6.0) at 121°C for 10 min and then treated with 3% H₂O₂-methanol, incubated with normal goat serum, and subsequently subjected to the primary antibody reaction. The antibody for EGFR protein (diluted 1:30; BioGenix, San Ramon, CA) was left to react with the sections overnight at room temperature. Detection of the immunoreactive staining was carried out by the avidin-biotin-peroxidase complex method according to the manufacturer's instructions (DAKO Corporation, Carpinteria, CA).

Immunostaining was classified in the following two groups according to both intensity and extent: (a) negative, when no staining or positive staining was detected in 50% of the cells; (b) positive, when immunostaining was present in >50% of the cells. Two independent pathologists (Y.-L.C. and C.-T.W.) were involved in the assessment of the expression.

Statistical analysis. The correlation between various clinicopathologic variables and the somatic aberrations of p53 and EGFR were analyzed by Fisher's exact test. Survival curves were estimated by using the Kaplan-Meier method. The log-rank test was used to compare survival curves. The Cox proportional hazards model was used to carry out the survival analysis without any adjustment or adjusted for age of 50 years, sex, and smoking status. Odds ratios (OR) and 95% confidence intervals (95% CI) were also calculated. The multiple logistic regression was used to assess the effect of the factor of somatic aberrations on lymph node metastasis without any adjustment or with adjustment for age of 50 years, sex, and smoking status. ORs of tumors with the same clonality versus tumors with different clonality and 95% CIs were also calculated. All tests were two-tailed, and P < 0.050 was considered significant. All statistical analyses were done using SAS statistical software (version 8.2, SAS Institute, Inc., Cary, NC).

	Results

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Patient characteristics. Of the 58 multiple primary lung cancer patients (Table 1 ), 33 cases with multiple tumors were located in the same lobe; 17 cases were unilaterally located in different lobes; and 8 cases were bilaterally located in different lobes. There were 36 cases with three or more tumors and 22 cases with less than three tumors in the same patient. The size of the largest tumor >3 cm in a patient was observed in 34 cases and 3 cm was detected in 24 cases. The histologic types of multiple tumors in each patient were the same in all cases, including 87.9% of adenocarcinomas (51 of 58), 5.2% of bronchioloalveolar carcinomas (3 of 58), 3.4% of squamous cell carcinomas (2 of 58), and 3.4% of adenosquamous carcinomas (2 of 58). Immunohistochemical staining indicated that 34.5% (20 of 58) and 29.3% (17 of 58) of cases showed up-regulation of p53 and EGFR proteins, respectively.

View larger version (21K): [in this window] [in a new window]   Fig. 1. Representative results of direct sequencing on exon 21 of EGFR gene and exon 5 of p53 gene in multiple primary lung cancers. Sequencing chromatograms of multiple primary lung cancers with different clonality (mutations of EGFR exon 21 in case 7 and p53 exon 5 in case 9) and with the same clonality (mutations of EGFR exon 21 in case 36 and p53 exon 5 in case 16). Arrows, positions of point mutations.

For clonality assessment with EGFR mutations, 19 (32.8%) patients were classified into the same clonality (group B); 25 (43.1%) patients belonged to either group A or C with conclusion of different clonality; and 14 patients (24.1%) without EGFR mutation (group D) were not classified according to clonality. Patients with EGFR mutations classified into the same clonality were also statistically associated with lymph node metastasis (P = 0.017; Table 2). The OR (Table 3) of tumors having the same clonality versus tumors having different clonality, with EGFR mutations for lymph node metastasis with adjustment, was 6.89 (95% CI, 1.56-30.45, P = 0.011).

Clonality analysis by using mutations in p53 and/or EGFR. Because 24% to 43% of patients show no detectable somatic mutations in either p53 or EGFR, we therefore combined mutations in both genes for clonality assessment. The tumors were classified into different clonality whenever either p53 or EGFR mutation in one patient belonged to either group A or C. In the rest of the patients, the tumors were categorized into the same clonality whenever either p53 or EGFR mutation in one patient belonged to group B. Indeed, our strategy increased the patient number for clonality analysis to 50 cases (50 of 58, 86.2%) with only eight patients showing no mutations. Among 50 cases accessible for clonality, 22 cases were classified as having the same clonality (37.9%) and 28 cases were categorized into different clonality (48.3%; Table 2). At least three advantages were immediately revealed by our strategy. First, there was no gender preference by combining mutation data from both genes for clonality analysis. Second, there was no conflict in the result of clonality assessment among 58 cases by using mutations that occurred in both genes. Finally, multiple primary lung cancers classified as the same clonality either by p53 or EGFR genes were correlated with lymph node metastasis (P = 0.005 and P = 0.017, respectively; Table 2). The multiple primary lung cancers that were classified as having the same clonality, determined by p53 and/or EGFR mutations, remained statistically associated with lymph node metastasis (P = 0.045). The OR (Table 3) of the tumors having the same clonality versus tumors having different clonality, with p53 and/or EGFR mutations for lymph node metastasis with adjustment, was 4.57 (95% CI, 1.30-16.01, P = 0.018).

Correlations of p53 and EGFR somatic aberrations with clinicopathologic features. Our results indicated that the overexpression of either p53 or EGFR occurred more frequently in the tumors of the same clonal origin (11 of 22, 50.0%) compared with tumors of different clonal origin (6 of 28; 21.4%; P = 0.042; Table 2) determined by p53 and/or EGFR mutations. The overexpression of p53 was marginally associated with poor survival adjusted for age of 50 years, sex, and smoking status (P = 0.066; Table 4 ). Unlike p53 mutations in exons 5 to 8 showing no correlation with protein expression in tumor tissues, EGFR overexpression was statistically associated with EGFR tyrosine kinase domain mutations (P = 0.046; Table 1). The vascular invasion of multiple primary lung cancers was also correlated with overexpression of EGFR (P = 0.012, data not shown). Although there was no significant correlation between clonality and survival (P = 0.630; Table 4, Fig. 2A ), the tumors having the same clonality were associated with lymph node metastasis and the 5-year survival rates in 44.2% and 83.0% of patients with and without lymph node metastasis, respectively (P = 0.001; Fig. 2B; Table 4). The OR (Table 4) for survival of lymph node metastasis over no lymph node metastasis with adjustment was 7.83 (95% CI, 2.29-26.72, P = 0.001).

View larger version (10K): [in this window] [in a new window]   Fig. 2. Survival curves of multiple primary lung cancer patients with relationships to tumor clonality and lymph node metastasis. A, no significant differences of probability of survival with tumor clonality (P = 0.630, log-rank test). B, multiple primary lung cancer patients with lymph node metastasis tend to have worse survival rate compared with patients without lymph node metastasis (P = 0.001, log-rank test).

	Discussion

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For the first time, we revealed that high frequent somatic aberrations of EGFR existed in multiple primary lung cancers are proven to be a good marker for distinguishing multifocal tumors from intrapulmonary metastatic cancers. In agreement with previous studies (29), EGFR protein expression in tissue specimen detected by immunohistochemical staining had good concordance in EGFR mutation, suggesting that EGFR may play an important role in tumorigenesis of NSCLCs and multiple primary lung cancers. The correlation between EGFR mutations and various clinicopathologic factors further revealed that EGFR gene mutations are tightly associated with vascular invasion and lymph node metastasis. Lymph node metastasis occurred more frequently in the tumors with the same clonal origin than in tumors with different clonal origin, suggesting that EGFR mutation might be a prognostic indicator for multiple primary lung cancer with highly invasive, metastatic potentials, and reduced patient survival (15, 30). Because the response rate of the EGFR tyrosine kinase inhibitors is high (69%) in EGFR mutation patients and the EGFR mutation status is strongly associated with the good response to gefitinib (18, 19), our current data suggest that gefitinib and other EGFR tyrosine kinase inhibitors may be particularly effective for treating the inoperable multiple primary lung cancer patients.

With the increase of clonality assessment rate to 86.2% (50 of 58), the revelation of different clonality of multiple primary lung cancers in our results supports the field cancerization concept (4) that multiple respiratory epithelia exposed to carcinogens and that undergo neoplastic transformation independently resulted in tumors with similar morphology but distinct clonality. Indeed, >70% of the second lung cancer was shown to have the same histologic subtype as observed in the first (6, 31). Our results implicated that carcinogenic insults affect many different susceptible cells in the respiratory tract and create various mutations in the same subtype of cancers. Our finding that p53 and/or EGFR mutations had higher incidence of lymph node metastasis in intrapulmonary metastatic cancers than that in carcinomas with different clonality further showed the clinical applications of using genetic markers in clonality assessment of multiple primary lung cancers.

In conclusion, this study is the first to show that the concurrent detection of p53 and EGFR mutations can increase the diagnostic usefulness in multiple primary lung cancers by direct sequencing of functional domains in limited exons. The current examination allowed not only clear diagnosis of multifocal lung cancers in the majority of patients despite similarities in histopathologic features but also genetically supports the independent field cancerization theory. Our results of no statistical association between tumor clonality to patient survival implicated that other factors, in addition to tumor clonality, should have contributed to poor patient outcomes. Nevertheless, the correlations between lymph node metastasis and intrapulmonary metastatic tumors, from the tumors with the same clonality, as well as poor patient survival support the clinical applications of clonality assessment in multiple primary lung cancers. In addition, our studies may provide the possibility for EGFR target therapy by using tyrosine kinase inhibitors in selected inoperable multiple primary lung cancer patients. The recent initiation of cancer genome sequencing projects of prevalent cancers are expected to further reveal additional frequently mutated cancer genes as therapeutic targets (32). Our strategy of combining frequent somatic mutations of newly discovered cancer genes in assessment of tumor clonality will be important to improve patient management of other multiple primary cancers.

	Acknowledgments

 
We thank Chih-Hsin Chen for her skillful technical support and Wen-Chen Wu for his help in preparation of the manuscript and artwork.

	Footnotes

 
Grant support: National Science Council, Republic of China, grants NSC 95-2314-B-002-326 and NSC 95-2320-B-002-110 (Y-L. Chang, C-T. Wu, and Y-C. Lee), and NHRI 93A1-NSCMM05-5 (S-C. Lin and Y-S. Jou).

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.

Note: Y-S. Jou and Y-C. Lee contributed equally to this work.

Received 7/16/06; revised 9/12/06; accepted 10/19/06.

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