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p53 and epidermal growth factor receptor (EGFR) are common genes involved in the pathogenesis of lung cancer, but their roles in lymphoepithelioma-like carcinomas (LELC) are unclear. In this study, we investigate the roles of p53 and EGFR in LELC carcinogenesis. Forty-six pulmonary LELCs were identified to evaluate p53 and EGFR aberrations. p53 mutations were identified in three patients, which all occurred in exon 8. EGFR mutations were detected in 8 of 46 cases with a majority of exon 21 mutations but without L858R. The other cases harbored mutations in exons 20 and 18. Only one case gained a deletion in exon 19. Notably, EGFR mutation was more commonly observed in patients with tumor size ≤3 cm (P = 0.014). In addition, there was a trend of more common EGFR overexpression in female (22/30) than in male patients (7/16, P = 0.061). However, there was no correlation between p53/EGFR mutations and protein expressions, suggesting the presence of complex mechanisms. p53 and EGFR mutations are uncommon in LELCs, indicating that these genes are not the important events in carcinogenesis for this tumor subtype. The EGFR mutation in 35% patients with LELC tumors <3 cm in size suggests the potential benefits to EGFR tyrosine kinase inhibitors of inoperable LELCs. (Cancer Sci 2011; 102: 282–287)
The majority of tumors have evidence of mutational inactivation of tumor suppressor genes and activation of oncogenes. Mutation of one p53 allele and loss of the normal p53 allele occur in many tumors including lung cancers. These alterations apparently contribute to the development of cancer by interfering with the tumor suppressor activity of p53. In addition, overexpression of mutant p53 may cause cellular transformation, whereas transfection and expression of a normal p53 gene in cells having a mutated p53 can suppress cellular transformation and inhibit cell proliferation.(1) It has been reported that somatic mutations and increased expression of p53 are frequently found in approximately 23% and 65% of non-small-cell lung cancer (NSCLC), respectively.(2–4)
Epidermal growth factor receptor (EGFR), a transmembrane glycoprotein, is involved in promoting cell division, migration and angiogenesis, and inhibiting apoptosis.(5) Loss of control of EGFR because of deregulation, amplification, or mutations may result in malignant change of cells.(6) Gefitinib (Iressa, ZD1839; AstraZeneca, Wilmington, DE, USA), an inhibitor of EGFR tyrosine kinase, has a novel antitumor effect on NSCLC. The better treatment outcomes of tyrosine kinase inhibitor in NSCLC patients are strongly associated with mutations at the EGFR tyrosine kinase domain (exons 18–21) in EGFR of tumor tissues.(7,8) Depending on the tumor subtypes and different ethnicity, Caucasian NSCLC patients have an EGFR mutation frequency of <10% compared with a mutation rate of at least 30% in East Asian patients.(2,9,10)
Lymphoepithelioma-like carcinoma of the lung (LELC) was first reported in 1987.(11) It is a rare form of NSCLC predominantly affecting young non-smoking Asians(12–14) and has been described as being closely associated with Epstein–Barr virus (EBV) infection.(12,13) Lymphoepithelioma-like carcinoma of the lung shows a poorly differentiated epithelial component admixed with lymphocytic infiltrate(15) identical to undifferentiated nasopharyngeal carcinoma. It is also associated with a lower incidence of nodal disease or distant metastases and a better prognosis than other NSCLCs.(12,16,17) Due to its rarity, the treatment of advanced LELC is not only empirical, but controversial.(12,14)
In our previous study, we were unable to establish a significant role of p53 or bcl-2 in tumorigenesis of LELCs at the molecular level.(12) The aim of this study is to elucidate the mechanisms responsible for the development of LELCs by p53 and EGFR mutations, as well as p53 and EGFR expressions. Furthermore, the results of these gene expression statuses are also characterized for correlation with clinicopathologic features, including sex, smoking status, stage, tumor size, lymph node metastasis, and survival.
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Primary LELC of the lung is a rare and clinicopathologically distinctive neoplasm. In the past two decades, there have been just over 150 cases reported in published work.(12,21,22) In our study, 16 patients were male and 30 patients were female. The male:female ratio was different from other published reports of 2.2:1(23) and 6:5(16); the male:female ratio of NSCLC was 2.2:1 in our previous study.(24) The patients ranged in age from 40 to 85 years (mean, 57 years), similar to previous reports.(23,25) The mean age of the patients with LELC was 10 years younger than that of other histological types of NSCLCs.(12)
The etiologic and molecular events responsible for the occurrence of LELC are almost entirely unknown. The presence of EBV was indicated by in situ hybridization for EBV DNA and RNA in all of our cases, suggesting a relationship between pulmonary LELC and EBV. It has been well documented that specific genetic alterations and aberrant gene expressions in tumor cells may influence the biologic behaviors of a tumor. p53 is a tumor suppressor gene involved in cell cycle control and preservation of genomic integrity. Mutation in the p53 gene is relatively frequent in lung cancer,(26) and it is mutated in approximately 50% of NSCLCs.(2,27) To the best of our knowledge, this is the first time a study has revealed that a very low frequency of somatic aberration of p53, suggesting different somatic or inherited genetic mechanisms, could underlie cancer development in these patients. Our results also indicate that rare p53 mutations in these cases were closely related to favorable survival in pulmonary LELC.(12) This observation is different from ordinary NSCLC results.(24,27)
The low frequency of association with cigarette smoking in LELC of the lung (13% in the current series and 40% in cases in published reports),(22,28) in contrast to other lung cancers (smoking history usually more than 60%),(29) suggests that smoking probably does not play an etiologic role, further mandating separation of this tumor from other lung cancers.(21,22)
Considerable evidence indicates that the EGFR pathway also plays an important role in both the pathogenesis and the progression of lung cancer.(30) A detailed study of mutation patterns within the EGFR gene and their associations with clinicopathologic features of lung cancers suggested that lung cancer patients whose tumors have EGFR mutations respond better than those without such mutations to drugs that target the EGFR tyrosine kinase domain.(7,8,31) However, there are no data addressing the EFGR mutation status and EGFR tyrosine kinase inhibitor treatment response in LELC of the lung. In this series, we tested a larger number of primary pulmonary LELCs for EGFR mutations and explored the relationship between EGFR mutation status and multiple clinicopathologic parameters. In contrast to previous reports that 40–80% of patients of East Asian ethnicity have NSCLCs with EGFR mutations,(2,10,32) we found that only 17.4% of patients with LELCs carried EGFR mutations. This relatively low EGFR mutation frequency existing in LELCs, concurring with the results reported by Tam et al.,(33) is a proof that it is a distinct entity of special interest, especially in the Asia-Pacific region.
It has been previously indicated that mutations of a subset of NSCLCs are limited to the first four exons (exons 18–21) of the tyrosine kinase. Three types of mutations constitute 94% of the total: deletions in exon 19; duplications and/or insertions in exon 20; and a single-point mutation in exon 21.(10) Other investigators have also noted that the predominant mutations are single classical mutations (del-19 or L858R),(32,34) which all flank the ATP-binding pocket that is important for tyrosine kinase activity.(7,8) Among the patients with single classical mutations, a better response to gefitinib than those without the classical mutations has been noted.(35,36) In our study, we identified four mutations in exon 21, all of them non-classical mutations. Surprisingly, a relatively high prevalence of exon 20 mutations, without duplications or insertions, was detected in three cases. One of them coexisted with an exon 21 mutation. According to the published reports, there is an average 34% gefitinib response rate for the exon 20 mutation, which is much lower than that for classical mutations in exons 19 and 21.(37–39) These results suggest that EGFR target therapy is not an encouraging treatment for patients with inoperable LELCs. Our experience with the chemotherapy regimen, a combination of gemcitabine and cisplatin, showed a good partial response without accompanying adverse reactions. Based on the better prognosis, we would recommend future use of combination chemotherapy or neoadjuvant chemotherapy with additional surgical resection in advanced LELC of the lung.
It has also been noticed that EGFR mutations are statistically significantly more frequent in females and non-smokers in adenocarcinoma.(7,8,10,32,39) However, these characteristics are not present in patients with LELC. The meaning of this discrepancy is not clear.
As in the reports mentioned before, EGFR mutations are not associated with stage or patient survival, suggesting that EGFR mutations occur relatively early in the clinical course and are associated with pathogenesis of adenocarcinoma rather than progression.(2,10,32) Interestingly, the correlation between EGFR mutations and various clinicopathologic factors of LELCs reveals that EGFR mutations are tightly associated with tumor sizes of 3 cm or less. This observation suggests that EGFR mutations may be responsible for the relatively different clinical and biologic behaviors.
In this study, we did not find a concordance between p53/EGFR mutations and p53/EGFR protein expressions in LELCs, suggesting the presence of complex mechanisms. There was a tendency for more common EGFR overexpression in female patients. Notably, the frequency of the expression of p53 protein (19.6%) was much higher than the results in our previous study.(12) This was because the authors used bifunctional SkipDewax pretreatment solution (Insitus Biotechnologies, Albuquerque, NM, USA) as the antigen retrieval technique in this study instead of citrate buffer, as previously used. This is a product that combines the three pretreatment steps, deparaffinization, rehydration, and unmasking in immunohistochemistry stains, which generally results in a higher proportion of positive cells, greater staining intensity, and lower backgrounds than citrate solution, thereby producing more consistent, reliable results.(40)
In conclusion, this study shows for the first time that p53 and EGFR mutations are infrequent in LELC of the lung. The absence of association between classical EGFR mutations and female non-smokers, and the significant correlation between EGFR mutation and a tumor size of 3 cm or less imply that LELC is clearly a distinct entity of NSCLCs. The 35%EGFR mutation rate in tumors <3 cm suggests the potential benefits of EGFR tyrosine kinase inhibitors of inoperable LELCs.