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. 2014 Jan 2;5(1):63–67. doi: 10.1111/1759-7714.12060

Lack of epidermal growth factor receptor gene mutations in exons 19 and 21 in primary lymphoepithelioma-like carcinoma of the lung

Qianwen Liu 1,2,*, Guowei Ma 1,2,*, Haoxian Yang 1,2, Jing Wen 1,2, Mei Li 1,3, Hong Yang 1,2, Kongjia Luo 1,2, Yi Hu 1,2, Jianhua Fu 1,2,✉
PMCID: PMC4704286  PMID: 26766974

Abstract

Background

Primary lymphoepithelioma-like carcinoma (LELC) of the lung is uncommon in non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) targeted therapy has been applied in advanced common NSCLC. Whether EGFR-targeted therapy is also suitable for LELC of the lung remains unclear. As we know, EGFR gene mutation is a predictive factor. Therefore, EGFR gene mutations in exons 19 and 21 in Chinese patients with LELC of the lung were investigated.

Methods

Clinicopathological information was obtained by a retrospective review of the medical history recorded in the patients' charts. EGFR gene mutations in exons 19 and 21 were analyzed in 32 samples of LELC of the lung by TaqMan real-time polymerase chain reaction (RT-PCR).

Results

Eleven (34.4%) of the patients were male and 21 (65.6%) patients female. The mean age at diagnosis was 50.9 years (range, 25–71 years). Seven (21.9%) of the patients were smokers. In situ hybridization for Epstein-Barr virus-encoded small RNAs (EBERs) showed positive signals in all 32 patients. None of the tumors had mutations in exons 19 and 21. EGFR-targeted therapy was used in three patients with advanced disease and one patient with distant recurrence. However, no obvious therapeutic effect was found.

Conclusion

These data showed that LELC of the lung, a special histological type of lung cancer, lacked EGFR gene mutations in exons 19 and 21, which suggested that there was no opportunity for EGFR-targeted therapy for patients with LELC of the lung.

Keywords: EGFR, lung cancer, lymphoepithelioma-like carcinoma, mutation

Introduction

Primary lymphoepithelioma-like carcinoma (LELC) of the lung was first reported by Begin et al. in 1987.1 It is categorized as a subtype of large cell carcinoma according to World Health Organization (WHO) classification.2 It is histopathologically identical to the nasopharyngeal lymphoepithelioma, which is an undifferentiated carcinoma with predominant lymphocytic infiltration. Similar to nasopharyngeal carcinoma (NPC), LELC of the lung is associated with the Epstein-Barr virus (EBV).3,4 It is an uncommon primary carcinoma of the lung. There have been about 50 studies in the literature on LELC of the lung, involving about 200 patients, most of whom were from southern China,3,5,6 Hong Kong,4,7–12 and Taiwan.13–15 Patients with LELC of the lung have a better survival rate compared to those with non-LELC types of non-small cell lung cancer (NSCLC).5 The mainstay of treatment for early-stage disease is curative surgical resection, while multimodality treatment (surgery, chemotherapy, and radiotherapy) has been adopted in local advanced or metastatic diseases.9,16 It is unclear whether EGFR-targeted therapy, a novel modality possessing a promising clinical efficacy with less systemic toxicity, is a suitable treatment.

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (TK) expressed in NSCLC. The EGFR gene encompasses 118kb of sequence on the short arm of human chromosome 7 and consists of 28 exons, of which exons 18–21 encode the TK domain of EGFR, whose mutations correlate with clinical responses to tyrosine kinase inhibitors (TKIs).17–19 As 89% of mutations are small in-frame deletions in exon 19 and a point mutation (L858R) in exon 21, the so-called classical mutations, which confer dramatic sensitivity to TKIs gefitinib and erlotinib clinically,20,21 in clinical practice, detection of mutation status in exons 19 and 21 is adequate for selecting patients for EGFR-targeted therapy.

LELC of the lung belongs to a subtype of NSCLC. It is suitable to investigate EGFR gene mutation status when a patient with LELC of the lung develops advanced disease. Therefore, the aim of the present study was to detect the mutation status of EGFR in patients with primary LELC of the lung.

Materials and methods

Tumor cases

During the period from August 2009 to July 2012, 46 patients with primary LELC of the lung were diagnosed and treated at the Sun Yat-Sen University Cancer Center. Genetic analysis of the tumors was performed in 32 patients. Primary LELC of the lung was diagnosed according to the criteria set by WHO. Undifferentiated carcinomas without dense lymphoid infiltrates and negative Epstein-Barr virus-encoded small RNAs (EBERs) staining were excluded from the current study. Clinical information including gender, age at diagnosis, smoking history, stage, and treatment protocol was obtained by a retrospective review of the medical history recorded in patients' charts. Tumors were staged according to the International Union Against Cancer (UICC) Tumor Node Metastasis (TNM) classification of malignant tumors. The ethics committee of Sun Yat-sen University Cancer Center approved the study.

In situ hybridization (ISH) of Epstein-Barr virus-encoded small RNAs (EBERs)

EBERs were detected using the EBV Probe In Situ Hybridization Kit (DIG-AP, A300K.9901, PanPath Company, Amsterdam, Netherlands) as described in previous manuscripts.22 Briefly, the process included the following steps: (i) deparaffinization and dehydration of the paraffin sections using xylene and a series of graded ethanol; (ii) pretreatment with 0.4% pepsin for 10 minutes; (iii) hybridization with digoxigenin-conjugated EBV (EBERs) probe at 37°C for three hours; (iv) signal detection using peroxidase-conjugated anti-digoxigenin antibody and 3,3'-diaminobenzidine (DAB); and (v) counterstaining the sections with hematoxylin solution. The positive signals were brownish-yellow and localized within the nuclei.

DNA extraction and TaqMan real-time polymerase chain reaction (PCR) for epidermal growth factor receptor (EGFR) mutation analysis

Mutational analysis of the EGFR gene was carried out using TaqMan real-time polymerase chain reaction (PCR) as described in previous studies.23 Briefly, tissue sections of 10-μM thickness microdissected from formalin-fixed paraffin-embedded surgically resected tumor specimens were examined by microscopy after hematoxylin and eosin staining, and only tissue samples with greater than 80% tumor content were selected for the study. To obtain genomic DNA, the QIAamp DNA FFPE tissue kit (Qiagen, Hilden, Germany) was used according to the manufacturer's instructions. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) fragment was amplified as an internal control to ensure DNA integrity and for normalization. PCR was performed on a Peltier Thermal Cycler PCR system (Esco Technologies, Inc., St. Louis, MO) as described previously,24 and the PCR products were visualized on a 1.5% agarose gel. The EGFR mutations were analyzed using a Real Time PCR Detection Kit for the Analysis of EGFR Gene Mutations (GP Medical Technologies, Beijing, China), to detect two specific in-frame deletion mutations in exon 19 (A: E746-A750del B: L747-P753ins S del) and two point mutations in exon 21 (C: L858R D: L861Q) of the EGFR gene. The TaqMan PCR and genotyping analysis were performed on ABI7500 Real Time PCR System (Applied Biosystems, Life Technologies Corporation, Carlsbad, CA). Data were analyzed with SDS2.0.software (Applied Biosystems) according to the manufacturer's instructions.

Results

The clinicopathological characteristics of 32 patients with LELC are presented in Table 1. Eleven (34.4%) of the patients were male, and 21 (65.6%) female. The mean age at diagnosis was 50.9 years (range, 25–71 years). Seven (21.9%) patients were smokers. Nasopharyngoscopy with or without magnetic resonance imaging was examined in all patients, and showed normal findings in the nasopharynx. The stages of LELC were: nine (28.1%) in stage I, six (18.8%) in stage II, 12 (37.5%) in stage III, and five (15.6%) in stage IV. All specimens were from primary tumors. LELC of the lung was characterized by undifferentiated carcinoma cells with ill-defined cytoplasmic borders arranged in syncytial sheets and nests. The tumor cell nuclei were round, oval or elongated, with mildly irregular nuclear borders, vesicular chromatin and distinct nucleoli. The stromal tissue septa contained large numbers of reactive lymphoplasmacytic cells and other inflammatory cells. A few small lymphocytes percolated between tumor cells (Fig 1). In situ hybridization for EBERs showed positive signals in all 32 patients. None of the tumors had mutations in exons 19 and 21. Multimodality treatment was applied in most patients. It is necessary to point out that EGFR-targeted therapy was used in three patients with advanced disease and one patient with distant recurrence. However, no obvious therapeutic effect was found.

Table 1.

Patient characteristics

No. Gender/Age (years) Smoking history Overall TNM staging Treatment protocol
 1 F/53 No IB S + CT
 2 M/63 No IB S
 3 F/58 No IB S
 4 F/56 No IIIA S
 5 F/55 No IIIA S + CT
 6 F/35 No IB S + CT
 7 F/45 No IV S + CT
 8 M/42 Yes IIB S + CT
 9 M/44 No IIB S + CT + T
10 F/62 No IIIA S
11 M/48 Yes IIIA S + CT
12 F/60 No IA S
13 F/47 No IIIA S
14 F/63 No IIB S + CT
15 M/56 Yes IB S
16 F/54 No IIB S + CT
17 M/34 No IIIA S + CT
18 F/50 No IIIA S + CT
19 F/54 No IV CT
20 F/25 No IIIB CT + RT + T
21 M/52 Yes IIIB CT + T
22 F/37 No IV CT + T
23 M/48 Yes IV CT
24 M/71 No IIIA S + CT
25 F/49 No IB S + CT
26 F/59 No IB S + CT
27 M/46 Yes IV S
28 M/65 Yes IB S + CT
29 F/59 No IIIA S
30 F/44 No IIB S + CT
31 F/52 No IIB S + CT
32 F/42 No IIIA S + CT
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CT, Chemotherapy; F, Female; M, Male; RT, Radiotherapy; S, Surgery; T, EGFR-Targeted therapy; TNM, tumor node metastasis.

Figure 1.

Figure 1

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Histology of lymphoepithelioma-like carcinoma of the lung, showing islands of epithelial tumor cells surrounded by and partially infiltrated by lymphocytes (Hematoxylin and eosin × 100).

Discussion

LELC of the lung is an uncommon histological type of lung cancer. It has characteristic clinical and pathological features that are different from other histological types of lung cancer. More female patients with LELC of the lung were found in this series, in contrast to the male predominance of other histological types of lung cancer. The mean age was 50.9 years, which was consistent with a previous study.14 In that study, the mean age of patients was 10 years younger than that of other types of lung cancer. As we know, smoking is considered to be a major cause of lung cancer. Smoking history is usually more than 60% among patients with other histological lung cancers.14 In contrast, similar to a previous study,16 only 21.9% of patients had a smoking history, which suggested that smoking might not play an important role in the tumorigenesis.

In the first report on LELC of the lung, the relationship between this special form of carcinoma and EBV was suggested.1 Thereafter, many studies reported the association between EBV and LELC of the lung in Asian patients.3,5,14 However, a similar association has not been shown in Caucasians.25 There are several ways to prove the presence of EBV in LELC of the lung, such as PCR for EBV DNA, ISH for EBV DNA and RNA, and immunohistochemistry for EBV-associated proteins.11,26,27 Among these methods, ISH for EBERs detecting EBV within the tumor cells is considered to be the standard test. In the present study, all patients were EBERs positive, confirming the consistent association of EBV in Chinese patients with LELC of the lung, and suggesting a possible etiologic role of EBV.

EGFR has an intrinsic TK activity. Mutation in EGFR TK domain encoded by exons 18–21 is involved in both pathogenesis and progression of lung cancer. An earlier study on the prevalence of EGFR mutations among different histological types of lung cancer demonstrated a low prevalence (1 in 11) of EGFR mutations in LELC of the lung.28 Chang et al. found that EGFR mutations were identified in eight out of 46 tumors of LELC of the lung (17.4%), among which there were three tumors with mutations in exon 21, two with mutations in exon 20, one with mutation in exon 19, and one with mutation in exon 18. Another tumor had concurrent mutations in exons 19 and 20. However, only one tumor carried classical mutation in exon 19. The classical mutations, including in-frame deletion in exon 19 and L858R missense mutation in exon 21, are predominant mutations, and show a better response to TKIs than non-classical mutations.29,30 Liang et al.6 analyzed EGFR mutations in exons 18, 19, 20, and 21 in 11 patients with LELC of the lung by PCR, but wild-type EGFR was found. These results suggest that EGFR target therapy is not an encouraging treatment for patients with inoperable or advanced LELC of the lung.15

In order to clarify whether EGFR-targeted therapy is a suitable treatment for LELC of the lung, we also tested its EGFR mutation status. Because mutations in exons 19 and 21 account for most of the EGFR mutations in NSCLC, and are associated with a better response to TKIs, only mutation status in exons 19 and 21 was analysed in this study. However, similar to previous studies, no mutations in exons 19 and 21 were found. This indicated that pathogenesis and progression of LELC of the lung were not associated with EGFR mutations. In a study by Huang et al.,31 targeted therapy, including erlotinib and gefitinib, was used in six out of 21 patients with LELC of the lung as a salvage treatment. One patient had six months of stable disease, and two patients had three months of stable disease; however, three patients showed no treatment response. EGFR-targeted therapy was also used in four cases in our series, but it did not show an obvious therapeutic effect. It further suggested that EGFR-targeted therapy might not be favorable for LELC of the lung.

Conclusion

In conclusion, LELC of the lung is a special histological type of lung cancer. The lack of EGFR gene mutations in exons 19 and 21 suggested that there was no opportunity for EGFR-targeted therapy for patients with LELC of the lung.

Acknowledgments

The authors thank the pathologists from the Department of Pathology at the Sun Yat-sen University Cancer Center for their cooperation.

Disclosure

No authors report any conflict of interest.

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