Identification of non-small-cell lung cancer with activating EGFR mutations in malignant effusion and cerebrospinal fluid: Rapid and sensitive detection of exon 19 deletion E746-A750 and exon 21 L858R mutation by immunocytochemistry

Lung Cancer 74 (2011) 35–40

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Identification of non-small-cell lung cancer with activating EGFR mutations in malignant effusion and cerebrospinal fluid: Rapid and sensitive detection of exon 19 deletion E746-A750 and exon 21 L858R mutation by immunocytochemistry Akihiko Kawahara a,∗ , Koichi Azuma b , Akiko Sumi a , Tomoki Taira a , Kazutaka Nakashima a , Emiko Aikawa a , Hideyuki Abe a , Tomohiko Yamaguchi a , Shinzo Takamori c , Jun Akiba d , Masayoshi Kage a a

Department of Diagnostic Pathology, Kurume University Hospital, 67 Asahi-machi, Kurume 830-0011, Japan Division of Respirology, Neurology, and Rheumatology, Department of Internal Medicine, Kurume University School of Medicine, Kurume 830-0011, Japan c Department of Surgery, Kurume University School of Medicine, Kurume 830-0011, Japan d Department of Pathology, Kurume University School of Medicine, Kurume 830-0011, Japan b

a r t i c l e

i n f o

Article history: Received 19 October 2010 Received in revised form 27 January 2011 Accepted 7 February 2011 Keywords: Activating EGFR mutations Mutation-specific antibody Immunocytochemistry Non-small-cell lung cancer Effusion cytology

a b s t r a c t Background: Recently, we have reported that EGFR mutation-specific antibodies performed well in immunohistochemical analysis, with good sensitivity. We investigated whether this method could detect non-small-cell lung cancer (NSCLC) carrying EGFR mutations in malignant effusions and cerebrospinal fluid (CSF), comparable to the peptide nucleic acid–locked nucleic acid (PNA–LNA) PCR clamp assay. Furthermore, we compared activating EGFR mutations between primary and recurrent NSCLC. Patients and methods: Twenty-four patients with NSCLC effusions and CSF were examined by immunocytochemistry using antibodies specific for the E746-A750 deletion mutation in exon 19 and the L858R point mutation in exon 21. The PNA–LNA PCR clamp assay was used to detect the E746-A750 deletion at exon 19, L858R mutation at exon 21, and T790M mutation at exon 20. Results: We were able to identify EGFR mutations in NSCLC effusion and CSF with a sensitivity of 100% (5/5) using the anti-delE746-A750 antibody and 100% (8/8) using the anti-L858R antibody. Furthermore, in samples without these EGFR mutations, immunocytochemistry with the two specific antibodies identified 91% (10/11) as negative for both the deletion and the point mutations in EGFR. Activating EGFR mutations decreased in recurrent NSCLC compared with primary NSCLC, and the T790M mutation was detected in recurrent NSCLC of patients receiving gefitinib treatment. Conclusions: Identification of EGFR mutations is important for patients with primary and recurrent NSCLC. Rapid and sensitive immunocytochemistry using mutation-specific antibodies to detect EGFR mutations will be useful for diagnosing responsiveness to EGFR-targeted drugs. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Lung cancer is the leading cause of cancer deaths worldwide. Non-small-cell lung cancer (NSCLC) is the major type of lung cancer and is classified into three histological types: adenocarcinoma, squamous cell carcinoma, and large cell carcinoma [1,2]. The majority of patients with NSCLC are in an advanced stage at the time of clinical and/or histological diagnosis, and less than 30% of patients undergo surgical resection [3]. Epidermal growth factor receptor (EGFR) is one of the members of the Erb-B family of receptors and is composed of an intracellular binding domain, a transmembrane

∗ Corresponding author. Tel.: +81 942 31 7651; fax: +81 942 31 7651. E-mail address: [email protected] (A. Kawahara). 0169-5002/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2011.02.002

segment and an intracellular tyrosine kinase domain. Since the introduction of the EGFR tyrosine kinase inhibitor gefitinib, and its approval for clinical use in the treatment of advanced NSCLC [4], subsequent studies have shown a significant association between the presence of EGFR-activating mutations in lung tumors and their sensitivity to both gefitinib and another EGFR tyrosine kinase inhibitor, erlotinib. Most of these mutations occur in exon 19, such as delE746-A750, and the L858R point mutation in exon 21, in the tyrosine kinase domain [5–7]. In contrast, several studies have reported the appearance of a threonine-to-methionine substitution at amino acid position 790 (T790M) in EGFR in NSCLC that acquires resistance to gefitinib after treatment, whereas almost no T790M mutations have been found in NSCLC before gefitinib treatment [8]. Cancer with effusions in the serosal (peritoneal, pleural, and pericardial) cavity is usually considered an incurable systemic

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disease with a high risk of distant metastasis. Malignant pleural effusions can be observed in patients with various types of neoplasm, but most frequently in lung adenocarcinoma [9]. Malignant pleural effusion is often present in patients with advanced disease, such as stage IV NSCLC, and these patients cannot undergo surgical resection of the primary NSCLC [10]. Therefore, approaches that examine molecular biological markers from non-surgical tissue biopsy samples or cytological diagnostic samples, such as bronchial brushing or effusions in NSCLC patients may identify clinically relevant signatures that will be helpful [11]. Several authors have demonstrated mutation-specific antibodies recognizing the delE746-A750 and L858R mutations, which can be used to identify the EGFR status of tumor samples and provide a simple immunohistochemical method for diagnosing EGFR mutations in human tissue [12–15]. We have previously also demonstrated the use of EGFR mutation-specific antibodies in immunohistochemistry, and their application to the diagnostic screening of NSCLC patients and their responsiveness to EGFR-targeted drugs [16]. Although mutations in p53, K-ras and EGFR have been detected in pleural effusion samples [17,18], immunostaining analysis of EGFR mutations has not yet been performed using cytological samples. In this study, we investigated the presence of EGFR exon 19 delE746-A750 and the exon 21 L858R point mutation in malignant effusion and cerebrospinal fluid (CSF) using EGFR mutation-specific antibodies, and examined the correlation between the EGFR mutation status by the peptide nucleic acid–locked nucleic acid (PNA–LNA) PCR clamp assay and the expression of EGFR mutationspecific antibodies. Furthermore, we compared the activating EGFR mutation status, including T790M mutation, between primary and recurrent NSCLC. 2. Methods 2.1. Patient selection We selected primary NSCLC carrying EGFR mutations, such as exon 19 delE746-A750 and the exon 21 L858R point mutation, from the EGFR mutation status records of the Department of Diagnostic Pathology, Kurume University Hospital, Kurume, Japan. These EGFR mutation status records had already been confirmed by DNA direct sequence or PNA–LNA PCR clamp assay. In these patients with NSCLC carrying an EGFR mutation, we searched for recurrent NSCLC patients with malignant effusion and CSF from cytology records. We finally selected 16 patients who had undergone surgical resection for NSCLC with recurrence detected by cytology, and 8 malignant effusions of NSCLC in patients who had not undergone surgical resection due to advanced stage, between 1998 and 2009 (Table 1). The age of the lung cancer patients ranged from 34 to 79 years (median, 62.9), 10 were men and 14 were women. Histological types were 22 adenocarcinomas and 2 squamous cell carcinomas. Cancer stages were 16.7% Stage I (IA + IB), 33.3% Stage II, 29.2% Stage III (IIIA + IIIB) and 20.8% Stage IV. The delE746-A750 mutation in exon 19 was present in 4 patients, and the L858R point mutation in exon 21 in 9 patients. No patients had received drugs before surgery, including neo-adjuvant chemotherapy. Four patients with NSCLC carrying EGFR mutations received gefitinib after lung surgery, and these NSCLC did not show T790M mutation prior to the gefitinib treatment (patients 1, 9, 11 and 13). 2.2. Cell preparation Cytological samples were received as fresh effusion, and were centrifuged for 5 min at 1500 rpm. Sediment smears were fixed in 95% alcohol and stained with Papanicolaou. The cytospin technique (Thermo Electron Corporation, Waltham, MA) was used to collect

cells from the CSF for 5 min at 900 rpm, and the specimens were fixed in 95% alcohol and stained with Papanicolaou. The effusion and CSF were considered malignant if malignant cells were found on cytologic examination. Immunocytochemical analysis was performed using Papanicolaou stained specimens. 2.3. Immunocytochemistry/histochemistry The cover glass of the Papanicolaou specimen was removed for immunocytochemical staining of EGFR mutation-specific antibodies. The primary antibodies used mutation-specific anti-EGFR antibodies recognizing the delE746-A750 mutation (6B6) in exon 19 and the L858R mutation (43B2) in exon 21 (Cell Signaling Technology, Inc., Danvers, MA). The specimens were boiled in a microwave for 30 min in 1 mmol/L EDTA, pH 9.0, target retrieval solution (DAKO, Glostrup, Denmark) to recover antigens. Intrinsic peroxidase activity was blocked by treatment with peroxidaseblocking reagent (DAKO, Glostrup, Denmark) for 5 min. After washing in Tris-buffered saline (TBS; DAKO, Glostrup, Denmark) for 5 min, primary antibodies were diluted 1:200 and applied to the cells. The cytological specimens were incubated at room temperature for 30 min, washed in TBS for 15 min, and incubated with labeled polymer-HRP secondary antibody (ChemMate ENVISION Kit; DAKO, Glostrup, Denmark) for 30 min at room temperature. After washing in TBS for 10 min, the slides were visualized using 3,3 -diaminobenzidine. We used the autostainer (DAKO, Glostrup, Denmark) for immunostaining. On immunohistochemistry, lung cancer tissue samples were described as in our previous studies [16]. In evaluating the expression of EGFR mutations as biomarkers, only cancer cells that showed a distinctive membrane and/or cytoplasm with a strong expression for EGFR mutation-specific antibodies were considered positive. 2.4. DNA extraction and PNA–LNA PCR clamp assay Mutations of the EGFR gene were examined in exons 19 (delE746-A750), 21 (L858R) and 20 (T790M) by PNA–LNA PCR clamp assay as described previously [19]. In brief, genomic DNA was purified from paraffin-embedded tissues using a QIAamp DNA Micro kit (QIAGEN, Valencia, CA). The PCR primers employed were synthesized by Invitrogen Inc. (Carlsbad, CA). PNA clamp primers and LNA mutant probes were purchased from FASMEC (Kanagawa, Japan) and IDT (Coralville, IA), respectively. PNA–LNA PCR clamp assay was performed using the SDS-7500 System (Applied Biosystems, Foster, CA). Exon sequences for EGFR kinase domain were amplified with a specific primer by nested PCR. 3. Results 3.1. Immunocytochemical analysis of EGFR mutation-specific antibodies in non-small-cell lung cancer effusion and CSF cytological samples Figs. 1 and 2 show representative immunostaining images of NSCLC carrying the delE746-A750 mutation or the L858R mutation. NSCLC carrying the delE746-A750 mutation was stained strongly by the anti-delE746-A750 antibody, but not with the anti-L858R antibody; that carrying the L858R mutation was stained strongly by the anti-L858R antibody, but not with the anti-delE746-A750 antibody. Each advanced stage patient with NSCLC carrying the delE746-A750 or L858R mutation (patients 17–19) showed strong positive staining with the anti-delE746-A750 or anti-L858R antibody, respectively (Fig. 2A and B). In contrast, advanced stage patients without EGFR mutations (patients 20–24) showed no strong positive staining with either the anti-delE746-A750 or the

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Table 1 Patient characteristics and EGFR mutations status in primary NSCLC samples. Patient no.

Age

Sex

Stage

1a 2 3 4 5 6 7 8 9a 10 11a 12 13a 14 15 16 17 18 19 20 21 22 23 24

68 74 58 67 63 49 53 60 70 69 41 69 65 60 68 66 68 76 59 62 55 73 34 58

F F F F F F F F F M F F F M M M F M F M M M M M

IV II II IA II IIIB II II IB IA II II II IIIA IA IV IIIB IV IV IIIB IIIB IIIB IIIB IV

Primary lung cancer tissue

Cytology

Histological types

Status (exon 19, 21)

T790M

Samples

Cell amount

AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD AD† AD† AD† SQ† AD† SQ† AD† AD†

E746-A750 E746-A750 E746-A750 E746-A750 L858R L858R L858R L858R L858R L858R L858R L858R L858R No mutation No mutation No mutation Not available Not available Not available Not available Not available Not available Not available Not available

–

PL PL PL PL PL PL PL PL PL PL AS CSF CSF PL PL PL PL PL PL PL PL PL PL PL

Abundant Abundant Abundant Abundant Abundant Abundant Small amount Abundant Abundant Abundant Small amount Abundant Abundant Abundant Abundant Abundant Abundant Abundant Small amount Abundant Abundant Abundant Abundant Abundant

– – –

AD, adenocarcinoma; SCC, squamous cell carcinoma; †, cytological diagnosis; PL, pleural effusion fluid; AS, ascites; CSF, cerebrospinal fluid. a Patient received gefitinib after lung surgery.

Fig. 1. Immunostaining of both histology and cytology preparations in NSCLC patients. Del E746-A750 antibody stains cancer cells only in samples with delE746-A750 mutation in exon 19 (A, patient no. 3), and anti-L858R antibody stains only cancer cells in samples with L858R mutations on exon 21 (B, patient no. 7) (histology ×200, cytology ×400).

L858R antibody (Fig. 2C). In the two CSF patients (patients 12, 13) with NSCLC carrying the L858R mutation, only one sample was strongly stained by the anti-L858R antibody (Fig. 3). The expression data in Table 2, for EGFR mutations identified by immunocytochemistry, are summarized in Table 3. We observed a very high correlation between the results from the PNA–LNA PCR clamp assay and immunocytochemistry in NSCLC effusion and CSF. EGFR mutation-specific antibodies detected delE746-A750 mutation in 100% (5/5) of patients identified by the PNA–LNA PCR clamp assay, and detected L858R mutation in 100% (8/8) of the patients. Furthermore, in NSCLC patients without EGFR mutations, immunocytochemistry with the two specific antibodies identified 92% (11/12) as negative for both the deletion and the point mutations in EGFR. There was only one misidentification, a positive result with the anti-delE746-A750 mutation antibody.

3.2. Alteration of activating EGFR mutations between primary and recurrent non-small-cell lung cancer NSCLC carrying the EGFR mutations of delE746-A750 or L858R was found in 4 (25.0%) and 9 (56.3%), respectively, of 16 patients who had undergone surgical resection, whereas these mutations were found in only 3 (18.8%) and 7 (43.8%), patients, respectively, in recurrent NSCLC effusion and CSF. In 4 patients who received gefitinib after lung surgery, 2 had the T790M mutation in recurrent NSCLC (50%) (Fig. 3). The T790M mutation was not detected in patients who were not administrated gefitinib (see Table 4). 4. Discussion We have investigated whether immunocytochemistry for EGFR mutation-specific antibodies detects NSCLC carrying EGFR muta-

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Fig. 2. Representative immunocytochemical findings in effusion samples of stage IV NSCLC patients. A sample with delE746-A750 mutation was stained with anti-delE746A750-specific antibody (A, patient no. 18) and a sample with L858R mutation was stained with L858R-specific antibody (B, patient no. 19). Samples without EGFR mutations were never stained with these two mutation-specific antibodies (C, patient no. 24) (Papanicolaou stain and immunocytochemistry; ×400).

tions in malignant effusions and CSF, comparable with the PNA–LNA PCR clamp assay, which is used to detect these mutations in NSCLC. We observed a very high correlation between the results from the PNA–LNA PCR clamp assay and immunocytochemistry in NSCLC effusion and CSF with a sensitivity of 100% using the antidelE746-A750 antibody and 100% using the anti-L858R antibody. Furthermore, in samples without these EGFR mutations, immunocytochemistry with the two specific antibodies identified 91% as negative for both the deletion and the point mutations in EGFR, suggesting that immunocytochemistry for EGFR mutation-specific antibodies could rapidly and sensitively detect NSCLC harboring EGFR mutations using cytological samples. Yu et al. generated antibodies specific for delE746-A750 and L858R mutations in EGFR and first reported that the sensitivity of immunohistochemical assays was 92% in 340 NSCLC lung cancer tissues compared with a sensitivity of 99% for DNA sequencing [12]. Recently, we have also demonstrated that EGFR mutation-specific antibodies performed well in immunohistochemical analysis, with good sensitivity [16]. Other authors have reported that paraffinembedded NSCLC tumor samples with these antibodies showed sensitivity and specificity of the immunohistochemistry assay of 47–92% and 96–99%, respectively [12–16]. The sensitivity of the anti-delE746-A750 mutation antibody is slightly inferior to that of the anti-L858R mutation antibody [13,15], which could be a result of the influence of formalin-fixed tissue, and the positive reaction to the anti-delE746-A750 mutation antibody differs from the molecular EGFR mutation [14], because rare exon 19 dele-

tion mutations, such as L747-T751 or L747-A750, do not react at all or have a weak reaction [16]. Kitamura et al. concluded that EGFR mutation-specific antibodies were relatively specific, whereas not all patients with EGFR mutations could be selected using EGFR mutation-specific antibodies [14]; therefore, we recommend the algorithm described by Brevet et al. for the possible use of these antibodies. On immunocytochemistry, the expression levels of these antibodies were very high and relatively regular, however, the number of samples used in the present study for EGFR mutation analysis of recurrent NSCLC was inadequate, and disseminated cancer cells in effusion or CSF form only a part of total lung cancer cells. Further study is necessary to understand how to develop personalized therapies in patients with NCSLC dissemination, including those with recurrence or drug resistance. In the present study there was only one error, a positive result with the anti-delE746-A750 mutation antibody (patient no. 16). This patient was EGFR mutation negative both in primary NSCLC tissue and in recurrent cytology by PNA–LNA PCR clamp assay, whereas immunocytochemistry in recurrent cytology was positive. Although a technical error, such as poor washing by TBS may be the cause of the overreaction, the actual cause is not known. It is well known that histological types with EGFR mutations are found more frequently in adenocarcinoma, and that squamous cell carcinomas with EGFR mutation are rare. We previously reported six cases of squamous cell carcinoma with EGFR mutation. [19] Although EGFR mutation was not detected in patients with squamous cell carcinoma (patient no. 20 and 22), identification of EGFR

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Fig. 3. Immunostaining analysis and detection of T790M using the PNA–LNA PCR clamp assay in NSCLC carrying L858R mutation. On immunohistochemistry, primary NSCLC tissue of patient no. 12 (A) is weakly stained focally, compared with that of patient no. 13 (B) by anti-L858R antibody. In contrast, this antibody stained only one out of two samples of recurrent CSF (B). T790M mutation was detected in patient no. 13 (B) who received gefitinib treatment, whereas patient no. 12 (A) who had never received gefitinib, did not show T790M mutation using PNA–LNA PCR clamp assay (histology ×200, cytology ×400).

mutation using cytological samples is important in patients who cannot undergo surgical resection due to advanced stage cancer. NSCLC carrying the delE746-A750 and L858R mutations decreased in recurrent NSCLC compared with primary lung tissue. In fact, the expression area and intensity levels differed by case in the

expression of mutation-specific antibodies of primary NSCLC tissue (Fig. 3), [13,15] suggesting, especially in patients no. 4 and 12, that cancer cells carrying EGFR mutations do not always recur. Acquired resistance to gefitinib after treatment is known, such as T790M mutation and loss of PTEN expression in NSCLC patients

Table 2 Results of immunocytochemistry using EGFR mutation-specific antibodies and EGFR mutations status in NSCLC malignant effusion and cerebrospinal fluid cytology. Patient no.

Malignant effusion and cerebrospinal fluid cytology EGFR mutation-specific antibodies

1a 2 3b 4 5b 6 7b 8 9a 10b 11a 12b 13a 14 15 16 17c 18c 19c 20 21 22 23 24 a b c

EGFR mutations detection by PNA–LNA PCR clamp assay

E746-A750

L858R

Exon 19del E746-A750 and exon21 L858R

T790M

Positive Positive Positive Negative Negative Negative Negative Negative Negative Negative Negative Not available Not available Negative Negative Positive Positive Positive Negative Negative Negative Negative Negative Negative

Negative Negative Negative Negative Positive Positive Positive Positive Positive Positive Negative Negative Positive Negative Negative Negative Negative Negative Positive Negative Negative Negative Negative Negative

E746-A750 E746-A750 E746-A750 No mutation L858R L858R L858R L858R L858R L858R No mutation No mutation L858R No mutation No mutation No mutation E746-A750 E746-A750 L858R No mutation No mutation No mutation No mutation No mutation

+

Patient received gefitinib after lung surgery. Patient received gefitinib after recurrence. Advanced non-small-cell lung cancer patient received gefitinib.

− − − − − − − +

− − −

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Table 3 Summary of immunocytochemistry and PNA–LNA PCR clamp assay in malignant effusion and cerebrospinal fluid cytology. Anti-del E746-A750 antibody

Positive Negative

PNA–LNA PCR clamp assay

Anti-L858R antibody

E746-A750 (n = 5)

L858R (n = 7)

No mutation (n = 10)

5 (100%) 0 (0%)

0 (0%) 7 (100%)

1 (10%) 9 (90%)

Table 4 Comparison of activating EGFR mutation status in primary lung cancer tissue and recurrent malignant effusion and cerebrospinal fluid cytology. EGFR mutations status

delE746-A750 L858R No mutation T790M Gefitinib-administrated patient after lung surgery Gefitinib-administrated patient after recurrence Gefitinib-administrated advanced non-small cell lung cancer patient

Non-small cell lung cancer Primary tissue

Cytology

4/16 (25.0%) 9/16 (56.3%) 3/16 (18.7%)

3/16 (18.8%) 7/16 (43.8%) 6/16 (37.5%)

0/4 (0%)

2/4 (50%)

Not available

0/5 (0%)

Not available

0/3 (0%)

[8,20]. In our present study, the T790M mutation was detected in recurrent NSCLC patients receiving gefitinib treatment, whereas it was not detected in patients who were not administrated gefitinib. Detection of EGFR mutations using cytology samples is important for evaluating responsiveness to EGFR-targeted drugs in recurrent NSCLC, while at the same time, the detection of T790M mutation may also be important if patients have received gefitinib treatment. Interestingly, EGFR mutations were not detected by PNA–LNA PCR clamp assay or by immunocytochemistry in recurrent cytology samples of patient no. 11, who had L858R mutation in primary NSCLC tissue, despite having received gefitinib treatment. It is not clear why EGFR mutations were not detected, and further study is needed to understand other gefitinib-resistance mechanisms in NCSLC. In summary, the therapeutic responses of NSCLC to EGFRtargeted drugs, such as gefitinib and erlotinib, are closely associated with EGFR mutations. Therefore, identification of EGFR mutation by immunocytochemistry using cytological samples is useful, especially for patients with recurrent NSCLC. Conflict of interest statement None declared. References [1] Devesa SS, Bray FI, Vizcaino AP, Parkin DM. International lung cancer trends by histologic type: male:female differences diminishing and adenocarcinoma rates rising. Int J Cancer 2005;117(2):294–9. [2] Parkin DM, Bray FI, Devasa SS. Cancer burden in the year 2000: the global picture. Eur J Cancer 2001;37:S4–66.

Positive Negative

PNA–LNA PCR clamp assay

E746-A750 (n = 5)

L858R (n = 8)

No mutation (n = 11)

0 (0%) 5 (100%)

8 (100%) 0 (0%)

0 (0%) 11 (100%)

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