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Usefulness of immunohistochemistry for the detection of the BRAF V600E mutation in Japanese lung adenocarcinoma
Lung Cancer 82 (2013) 51–54
Contents lists available at ScienceDirect
Lung Cancer journal homepage: www.elsevier.com/locate/lungcan
Usefulness of immunohistochemistry for the detection of the BRAF V600E mutation in Japanese lung adenocarcinoma Hidefumi Sasaki a,∗ , Shigeki Shimizu b , Yoichi Tani c , Masayuki Shitara a , Katsuhiro Okuda a , Yu Hikosaka a , Satoru Moriyama a , Motoki Yano a , Yoshitaka Fujii a a
Department of Oncology, Immunology and Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan Department of Pathology, National Hospital Organization, Kinki-chuo Chest Medical Center, Sakai, Japan c Dako Japan Company, Tokyo, Japan b
a r t i c l e
i n f o
Article history: Received 26 March 2013 Received in revised form 30 May 2013 Accepted 24 June 2013 Keywords: BRAF V600E expression Lung adenocarcinoma 599 insertion T IHC Linker EnVision
a b s t r a c t Purpose: Mutations in components of the mitogen-activated protein kinase (MAPK) cascade may be a new candidate for target for lung cancer. The usefulness of immunohistochemistry (IHC) as a new approach for the detection of BRAF V600E in cancer patients has been recently reported. Methods: To increase the sensitivity, we modified BRAF V600E expression detection assay by IHC using mutation specific antibody. From the screening step, we found a novel 599 insertion T BRAF mutation in lung adenocarcinoma. In this study included 26 surgically removed cases with EGFR, Kras, erbB2, EML4-ALK and KIF5B-RET wild-type (wt) lung adenocarcinomas, including 7 BRAF mutants (5 V600E, 1 N581I, and 1 novel 599 insertion T mutation) analyzed by DNA sequencing. Detection of the BRAF V600E mutation was carried out by the Dako EnVisionTM FLEX detection system using the VE1 clone antibody and compared with the results of direct sequencing. Results: The autostainer IHC VE1 assay was positive in 5 of 5 (100%) BRAF V600E-mutated tumors and negative in 20 of 21 (95.2%) BRAF non-V600E tumors, except for a novel 599 insertion T case. Conclusion: IHC using the VE1 clone and FLEX linker is a specific method for the detection BRAF V600E and may be an alternative to molecular biology for the detection of mutations in lung adenocarcinomas. This method might be useful for screening to use molecular target therapy for lung adenocarcinomas. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Lung cancer is a major cause of death from malignant diseases, due to its high incidence, malignant behavior and lack of major advancements in treatment strategy [1]. A great deal of progress has been made in the target therapy for non-small cell lung cancer (NSCLC), largely owing to the development of small-molecular inhibitors, such as epidermal growth factor receptor (EGFR) [2–5] and ALK [6] for lung adenocarcinomas. The pathway linking receptor tyrosine kinases to the Ras family to the Raf serine–threonine kinase to the MAP kinase cascade is critical for cell proliferation and is frequently activated in human cancers [7]. Activating mutations of the v-RAF murine sarcoma viral oncogene homolog B1 (BRAF) are found in a wide range of human cancers including lung adenocarcinomas [8–11]. According to the Catalogue of Somatic Mutations in Cancer, the vast majority of BRAF alterations are characterized
∗ Corresponding author at: Department of Oncology Immunology and Surgery, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuhocho, Mizuho-ku, Nagoya 467-8601, Japan. Tel.: +81 52 853 8231; fax: +81 52 853 6440. E-mail address: [email protected] (H. Sasaki). 0169-5002/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.lungcan.2013.06.014
by a missense substitution of valine by glutamic acid in a mutational hotspot at amino acid position 600. This exchange mimics the phosphorylation of amino acid residues T599 and S602 and induces a conformational change of the activation segment leading to a constitutive kinase activity of BRAF and consecutive phosphorylation of downstream targets [12]. The importance of recognizing this molecular subtype was highlighted by an inhibition using BRAF V600E inhibitors for clinical investigation [13]. Of these, the V600Especific inhibitor Vemurafenib has remarkable clinical activity in patients with BRAF V600E mutated metastasizing melanoma [14]. Therefore an accurate and practical assay is urgently needed to detect this molecular subset of cancer. Currently, the methods available for detecting the mutation are polymerase chain reaction (PCR) assay. PCR is a single detect test to detect the gene mutations, however, it generally requires multiple tissue sections, dissection for tumor cell enrichment and a multiplex system. Recently, it has been demonstrated that the mutation specific antibodies can reliably detect the exchange of single amino acids in routinely processed, formalin-fixed, and paraffin-embedded tumor tissue [15,16]. Our group has already screened the Japanese BRAF gene status using RT-PCR assay [17]. In this paper, to increase the sensitivity, we have modified BRAF V600E protein expression detection system
52
H. Sasaki et al. / Lung Cancer 82 (2013) 51–54
by immunohistochemistry (IHC) using specific monoclonal antibody, VE1 and Dako EnVisonTM FLEX, using linker. We have diluted the antibody for several concentrations for testing surgically treated lung adenocarcinoma samples. We have re-established the autostainer IHC method for detecting V600E mutation. From the screening step, we have found a novel 599 insertion T BRAF mutation in Japanese lung adenocarcinoma. 2. Material and methods 2.1. Patient samples The study group included lung cancer patients who had undergone surgery at the Department of Surgery, Nagoya City University Hospital. All tumor samples were immediately frozen and stored at −80 ◦ C until assayed. Because we have demonstrated that the BRAF V600E were in adenocarcinoma histology of lung cancer and exclusively with EGFR, Kras or erbB2 mutations [10,17], we mainly focused on wild type (wt) adenocarcinomas for the genes. The clinical and pathological characteristics of the 44 wt lung cancer patients for BRAF sequencing analyses were as follows; 30 (68.2%) were male 14 were female. 40 were diagnosed as adenocarcinomas, 2 were adenosquamous and 2 were pleomorphic carcinomas. 30 (68.2%) were smoker and 14 were non-smoker. 29 (65.9%) were pathological stage I. The clinical and pathological characteristics of the 26 lung cancer patients for BRAF V600E IHC analyses were as follows; 17 (65.4%) were male 9 were female. All were diagnosed as adenocarcinomas without EGFR, Kras codon12–13 [18], erbB2 [4,17], EML4-ALK or KIF5B-RET [19] mutations. 18 (69.2%) were smoker and 8 were non-smoker. 15 (57.7%) were pathological stage I. 2.2. PCR assay for BRAF gene Total RNA was extracted from fresh/snap-frozen lung cancer tissues using Isogen kit (Nippon gene, Tokyo, Japan) according to the manufactures’ instructions. RNA concentration was determined by Nano Drop ND-1000 Spectrophotometer (Nano Drop Technologies Inc., Rockland, DE, USA). RNA (1 g) was reverse transcribed by First strand cDNA synthesis kit with 0.5 g oligo (dT)16 (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufactures’ protocol. The reaction mixture was incubated at 25 ◦ C for 15 min, 42 ◦ C for 60 min, 99 ◦ C for 5 min and then at 4 ◦ C for 5 min. About 200 ng of each cDNA was used for polymerase chain reaction (PCR) analysis. The primer sequences for BRAF gene were as follows: the forward primer, 5-GACGGGACTCGAGTGATGAT-3 and the reverse primer, 5-GGGATCCCATCGTAAGGTTT-3 (594 bp). The cycling conditions were as follows: initial denaturation at 94 ◦ C for 5 min, followed by 40 cycles at 94 ◦ C for 40 s, 60 ◦ C for 40 s and 72 ◦ C for 45 s.
Fig. 1. Sequencing result for a novel BRAF insertion case. (Left) Nucleotides ACA (3-bp) were duplicated after 1797. The amino acid threonine (T) was duplicated after 599 T. (Right) Adjacent normal lung tissue samples showed BRAF wild type sequence, suggested that the mutation was somatic.
and counter staining with hematoxylin for 3 min. We selected 200× as an appropriate dilution and slides were scored semi quantitatively for cytoplasmic staining intensity and distribution. An IHC score was assigned to each case according to the following criteria showing the designated staining pattern; 3+, intense, granular cytoplasmic staining; 2+, moderately, smooth cytoplasmic staining; 1+, faint cytoplasmic staining; and no staining. The extent of the staining area was determined as 0–10%, 25%, 50%, 75%, and 90%. Tumors with intensity involving a minimum of 50% of the positive stained tumor cells were considered positive for BRAF V600E expression. These criteria were determined by the co-author with clinical data blind. 3. Results 3.1. BRAF gene alteration status in Japanese lung cancer patients We have sequenced for kinase domain of BRAF gene for 44 adeno histology samples. Of 44 patients, from direct sequencing using cDNA samples, we found a novel BRAF insertion case (Fig. 1). Nucleotides ACA (3-bp) were duplicated after 1797. The amino acid threonine (T) was duplicated after 599 T. This patient was female and non-smoker adenocarcinoma at stage I. Mixed subtype adenocarcinoma with acinar dominant (acinar 80% and papillary 20%). Adjacent normal lung tissue samples showed BRAF wild type sequence, suggested that the mutation was somatic. Other 43 patients did not have BRAF mutations within kinase domain. Previous our report demonstrated that from 133 wt lung cancers, 6 BRAF mutations were found. Totally, from 177 wt lung cancers, 7 (3.95%) BRAF mutation cases were found. 3.2. Immunohistochemistry for BRAF V600E
2.3. BRAF V600E protein immunohistochemistry 26 cases were immunostained by automated methods (Dako Japan Co.) for BRAF V600E expression using the mouse monoclonal BRAF V600E clone VE1 (Spring Bioscience, Pleasanton, CA). Unstained 4-m sections of FFPE tumor tissue were submitted to the analysis. The Dako EnVisionTM ELEX detection system included pretreatment with Dako PT LINK (pre-treatment module) and Target Retrieval solution, High pH (K8004, Dako Co., Tokyo, Japan) 97 ◦ C for 20 min, followed by incubation with 50×, 100× or 200× diluted mouse anti-BRAF V600E (clone VE1) with Antibody Diluent (K8006, Dako Co.) 4 ◦ C for overnight. Antibody incubation was followed by standard signal amplification including rabbit LINKER (K8019) at room temperature for 15 min, HRP conjugated EnVision TM FLEX at room temperature for 20 min, DAB reaction for 10 min
Immunohistochemical results are summarized in Table 1. V600E mutant adenocarcinomas showed different pattern of mixed subtypes, such as 2 acinar dominant, 1 papillary dominant, 1 solid dominant and 1 micropapillary dominant. Two cases in the V600E mutant cases were 3+, and one was 2+. These samples had more than 90% staining for VE1 (Fig. 2). In addition, two more V600E cases had the 1+ staining intensity, and more than 50% tumors had staining for both cases. The staining pattern was diffuse in all components of adenocarcinomas and cytoplasmic. We have additionally performed immunohistochemistry for nonV600E patients. The staining intensity was 1+ in one case, very weak (+−) in two cases, and no-stained in 18 cases. The positive case was 599 insertion T case. In this case, about 50% of tumor cells had cytoplasmic staining.
H. Sasaki et al. / Lung Cancer 82 (2013) 51–54
53
Table 1 Immunohistochemical results. No.
1. 2. 3. 4. 5. 6. 7.
BRAF mutant adenocarcinomas Pap
Lep
Acinar
80 30 50 10 20 20 20
10 20 30
10 50 20 20 45 30 80
Solid
70 30 10
Micropap
5 40
BRAF
V600EIHC
S581I V600E V600E V600E V600E V600E 599insT
– +50% +50% 3 + 90% 2 + 90% 3 + 90% +50%
Pap: papillary; Lep: lepidic growth; Micropap: micropapillary.
4. Discussion In this study, we have introduced modified methods for the detection of BRAF V600E by EnVison TM FLEX IHC, a sensitive method using linker, and the results showed an excellent concordance between RT-PCR assays. We have established optimal condition of IHC for BRAF V600E. Although BRAF V600E protein expression was not perfectly matched with the sequencing results, IHC analysis is an appropriate method for detecting BRAF V600E status in lung cancer. From the screening process, we found a novel 3-bp insertion in BRAF resulting in the duplication of threonine 599. The main focus of this study was to establish IHC methods for detecting V600E in routinely processed FFPE tissue and to increase the sensitivity. Original report demonstrated that clone VE1 positivity was perfect match with sequencing results in papillary thyroid carcinomas and melanoma metastases [15]. However, recent study has been reporting the 19 of 21 (90%) of V600E Caucasian lung adenocarcinoma were positive for IHC test [20]. In our study, one of non-V600E mutant sample was positive for VE1 IHC. Because the VE1 was made using peptides between 596 and 606 (GLATEKSRWSG) of V600E, it should not cross-react with a novel 599 insertion T case. If GLATEKSRWSG is similar to GLATTVKSRWSG in tertiary structure, the antibody may recognize the latter motif. Reduced VE1 immunostaining intensity of damaged or “prenecrotic” tissue parts has to be taken into account, as it may lead to false-negative results in cases with small or sub-optimally preserved tissue [15]. Tissue sections have to be freshly cut from FFPE blocks for optimal results, as previously observed a marked
decrease of VE1 immunostaining intensity in tissue sections stored for longer than approximately 2 weeks [16]. Although the mutation rate of V600E in Japanese lung cancers was lower than previous Caucasian report (9% of wt lung cancers) [17,20], a racial difference between the studies about mutant BRAF may be existed, like EGFR gene mutations [2–6]. Because the BRAF mutation statuses were examined by pyrosequencing or direct sequencing in Caucasian cohort [20], we cannot conclude that differences were owing to our methodology as our PCR and sequencing methods. However false IHC positive was not found in our wild type cases. Mutated BRAF V600E protein has recently emerged as a promising therapeutic target. Several specific small-molecule BRAF V600E inhibitors are currently under pre-clinical or clinical investigation [13]. Of these, the V600E-specific inhibitor PLX4032 (Vemurafenib) has remarkable clinical activity in patients with BRAF V600E mutated metastasizing melanoma [14]. The compound GSK 2118436 (Dabrafenib) also resulted in clinical response in metastasized melanoma and may also be effective in BRAF mutations [21]. Similar insertion mutation (1798 insertion TAC resulted in the insertion of one additional threonine residue at position 599) has been reported in pilocytic astrocytoma [22]. This mutant BRAF insertion T did display an in vitro kinase activity and cellular MEK/ERK activation potential comparable to those to BRAF V600E [22]. Replacement of threonines by valine residues has similar effects on BRAF activity, suggested that the distortion of the peptide backbone by additional amino acids rather than the insertion of additional, potential phosphorylation sites destabilizes the inactive conformation of the kinase domain [22]. Identification of a
Fig. 2. VE1 IHC results. (Left upper) V600E mutant case with 3+, and the sample had more than 90% staining for VE1. (Left lower) V600E mutant case with + and the sample had more than 50% staining for VE1. (Right upper) 599 insertion T case with + and the sample had more than 50% staining. (Right lower) Negative case.
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small fraction of lung cancers patients responsive to a molecular target therapy may have large clinical impact as highlighted by the recent FDA approval of crizotinib for ALK-rearrangement lung cancers [23]. This was based upon from the results of clinical trials for advanced lung cancers with ALK rearrangements, and early responses in ALK-rearrangement might be assessed by IHC [24,25]. Accurate and rapid screening of mutations is important in lung cancers, especially in determining their eligibility for molecular target therapy. Current standard technique to identify BRAF is PCR assay [10,17]. This assay requires extraction of RNA or DNA from clinical samples, is not routinely performed in many diagnostic pathology laboratories, and suffers from some important limitations. Another important limitation is the possibility of contamination with normal tissues and small sized biopsies obtained by limited invasive procedures, which might impair specificity and led to false-negative results. IHC enables the pathologist to perform a more reliable quantification of the genomic alteration. The use of an antibody to detect oncogenic mutations on tumor tissue sections allows simultaneous morphological examination, in particular an estimation of the percentage of tumor cells, as well as determination of the number and intensity of stained cells. The intensity of BRAF IHC may be important additional information in BRAF mutation screening. Approximately 40% of patients with BRAF mutant have resistance to Vemurafenib [14]. It should be tested whether cases with low level expression are less dependent on MAP pathway activation by BRAF V600E and may be resistant to target therapy. In summary, BRAF V600E mutation in Japanese lung cancers seemed to be rare, however, V600E specific IHC might be a role in detection and screening for molecular target therapy for lung cancer. Conflict of interest statement None declared. Acknowledgements The authors thank Mrs. Yuka Toda for her excellent technical assistances. This work was supported by Grants-in-Aid for Scientific Research, Japan Society for the Promotion of Science (JSPS) (Nos. 23659674, 24592097 and 25293303). References [1] Ginsberg RJ, Kris K, Armstrong G. Cancer of the lung. In: Principles and practice of oncology. 4th ed. Philadelphia: Lippincott; 1993. p. 673–82. [2] Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004;304:1497–500. [3] Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129–39.
[4] Sasaki H, Shimizu S, Endo K, Takada M, Kawahara M, Tanaka H, et al. EGFR and erbB2 mutation status in Japanese lung cancer patients. Int J Cancer 2006;118:180–4. [5] Fukuoka M, Wu Y-L, Thongprasert S, Sunpaweravong P, Leong S-S, Sriuranpong V, et al. Biomarker analyses and first overall survival results from a phase III, randomized, open-label, first-line study of gefitinib versus carboplatin/paclitaxel in clinically selected patients with advanced non-small-cell lung cancer in Asia (IPASS). J Clin Oncol 2011;29:2866–74. [6] Soda M, Choi YL, Enomoto M, Takada S, Yamashita Y, Ishikawa S, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature 2007;448:561–6. [7] Peyssonnaux C, Eychene A. The Raf/MEK/ERK pathway: new concepts of activation. Biol Cell 2001;93:53–62. [8] Brose MS, Voipe P, Feldman M, Kumar M, Rishi I, Gerrero R, et al. BRAF and RAS mutations in human lung cancer and melanoma. Cancer Res 2002;62:6997–7000. [9] Naoki K, Chen T-H, Richards WG, Sugarbaker DJ, Meyerson M. Missense mutations of the BRAF gene in human lung adenocarcinoma. Cancer Res 2002;62:7001–3. [10] Sasaki H, Kawano O, Endo K, Suzuki E, Haneda H, Yukiue H, et al. Uncommon V599E Braf mutations in Japanese patients with lung cancer. J Surg Res 2006;133:203–6. [11] Paik PK, Arcila MA, Fara M, Sima CS, Miller VA, Kris MG, et al. Clinical characteristics of patients with lung adenocarcinomas harboring BRAF mutations. J Clin Oncol 2011;29:2046–51. [12] Wan PT, Garnett MJ, Roe SM, Lee S, Niculescu-Duvaz D, Good VM, et al. Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell 2004;116:855–67. [13] Arkenau HT, Kefford R, Long GV. Targeting BRAF for patients with melanoma. Br J Cancer 2011;104:392–8. [14] Flaherty KT, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA, et al. Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med 2010;363:809–19. [15] Capper D, Preusser M, Habel A, Sahm F, Ackemann U, Schinder G, et al. Assessment of BRAF V600E mutation status by immunohistochemistry with a mutation-specific monoclonal antibody. Acta Neuropathol 2011;122:11–9. [16] Capper D, Berghoff AS, Magerle M, Ilhan A, Wohrer A, Hackl M, et al. Immunohistochemical testing of BRAF V600E status in 1120 tumor tissue samples of patients with brain metastases. Acta Neuropathol 2012;123:223–33. [17] Sasaki H, Shitara M, Yokota K, Okuda K, Hikosaka Y, Moriyama S, et al. Braf and erbB2 mutations correlate with smoking status in lung cancer patients. Exp Ther Med 2012;3:771–5. [18] Sasaki H, Okuda K, Kawano O, Endo K, Yukiue H, Yokoyama T, et al. Nras and Kras mutation in Japanese lung cancer patients: genotyping analysis using LightCycler. Oncol Rep 2007;18:623–8. [19] Yokota K, Sasaki H, Okuda K, Shimizu S, Shitara M, Hikosaka Y, et al. KIF5B/RET fusion gene in surgically-treated adenocarcinoma of the lung. Oncol Rep 2012;28:1187–92. [20] Ilie M, Long E, Hofman V, Dadone B, Marquette CH, Mouroux J, et al. Diagnostic value of immunohistochemistry for the detection of the BRAFV600E mutation in primary lung adenocarcinoma Caucasian patients. Ann Oncol 2013;24:742–8. [21] Hauschild A, Grob JJ, Demidov LV, Jouary T, Gutzmer R, Millward M, et al. Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomized controlled trial. Lancet 2012;380:358–65. [22] Eisenhardt AE, Olbrich H, Roring M, Janzarik W, Van Anh TN, Cin H, et al. Functional characterization of a BRAF insertion mutation associated with pilocytic astrocytoma. Int J Cancer 2011;129:2297–303. [23] Shaw AT, Yeap BY, Mino-Kenudson M, Digumarthy BR, Costa BR, Heist RS, et al. Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK. J Clin Oncol 2009;27:4247–53. [24] Takeuchi K, Choi YL, Togashi Y, Soda M, Hatano S, Inamura K, et al. KIF5B-ALK, a novel fusion oncokinase identified by an immunohistochemistry-based diagnostic system for ALK-positive lung cancer. Clin Cancer Res 2009;15:3143–9. [25] Mino-Kenudson M, Chirieac LR, Law K, Hornick JL, Lindeman N, Mark EJ, et al. A novel, highly sensitive antibody allows for the routine detection of ALKrearranged lung adenocarcinomas by standard immunohistochemistry. Clin Cancer Res 2010;16:1561–71.
Contents lists available at ScienceDirect
Lung Cancer journal homepage: www.elsevier.com/locate/lungcan
Usefulness of immunohistochemistry for the detection of the BRAF V600E mutation in Japanese lung adenocarcinoma Hidefumi Sasaki a,∗ , Shigeki Shimizu b , Yoichi Tani c , Masayuki Shitara a , Katsuhiro Okuda a , Yu Hikosaka a , Satoru Moriyama a , Motoki Yano a , Yoshitaka Fujii a a
Department of Oncology, Immunology and Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan Department of Pathology, National Hospital Organization, Kinki-chuo Chest Medical Center, Sakai, Japan c Dako Japan Company, Tokyo, Japan b
a r t i c l e
i n f o
Article history: Received 26 March 2013 Received in revised form 30 May 2013 Accepted 24 June 2013 Keywords: BRAF V600E expression Lung adenocarcinoma 599 insertion T IHC Linker EnVision
a b s t r a c t Purpose: Mutations in components of the mitogen-activated protein kinase (MAPK) cascade may be a new candidate for target for lung cancer. The usefulness of immunohistochemistry (IHC) as a new approach for the detection of BRAF V600E in cancer patients has been recently reported. Methods: To increase the sensitivity, we modified BRAF V600E expression detection assay by IHC using mutation specific antibody. From the screening step, we found a novel 599 insertion T BRAF mutation in lung adenocarcinoma. In this study included 26 surgically removed cases with EGFR, Kras, erbB2, EML4-ALK and KIF5B-RET wild-type (wt) lung adenocarcinomas, including 7 BRAF mutants (5 V600E, 1 N581I, and 1 novel 599 insertion T mutation) analyzed by DNA sequencing. Detection of the BRAF V600E mutation was carried out by the Dako EnVisionTM FLEX detection system using the VE1 clone antibody and compared with the results of direct sequencing. Results: The autostainer IHC VE1 assay was positive in 5 of 5 (100%) BRAF V600E-mutated tumors and negative in 20 of 21 (95.2%) BRAF non-V600E tumors, except for a novel 599 insertion T case. Conclusion: IHC using the VE1 clone and FLEX linker is a specific method for the detection BRAF V600E and may be an alternative to molecular biology for the detection of mutations in lung adenocarcinomas. This method might be useful for screening to use molecular target therapy for lung adenocarcinomas. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Lung cancer is a major cause of death from malignant diseases, due to its high incidence, malignant behavior and lack of major advancements in treatment strategy [1]. A great deal of progress has been made in the target therapy for non-small cell lung cancer (NSCLC), largely owing to the development of small-molecular inhibitors, such as epidermal growth factor receptor (EGFR) [2–5] and ALK [6] for lung adenocarcinomas. The pathway linking receptor tyrosine kinases to the Ras family to the Raf serine–threonine kinase to the MAP kinase cascade is critical for cell proliferation and is frequently activated in human cancers [7]. Activating mutations of the v-RAF murine sarcoma viral oncogene homolog B1 (BRAF) are found in a wide range of human cancers including lung adenocarcinomas [8–11]. According to the Catalogue of Somatic Mutations in Cancer, the vast majority of BRAF alterations are characterized
∗ Corresponding author at: Department of Oncology Immunology and Surgery, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuhocho, Mizuho-ku, Nagoya 467-8601, Japan. Tel.: +81 52 853 8231; fax: +81 52 853 6440. E-mail address: [email protected] (H. Sasaki). 0169-5002/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.lungcan.2013.06.014
by a missense substitution of valine by glutamic acid in a mutational hotspot at amino acid position 600. This exchange mimics the phosphorylation of amino acid residues T599 and S602 and induces a conformational change of the activation segment leading to a constitutive kinase activity of BRAF and consecutive phosphorylation of downstream targets [12]. The importance of recognizing this molecular subtype was highlighted by an inhibition using BRAF V600E inhibitors for clinical investigation [13]. Of these, the V600Especific inhibitor Vemurafenib has remarkable clinical activity in patients with BRAF V600E mutated metastasizing melanoma [14]. Therefore an accurate and practical assay is urgently needed to detect this molecular subset of cancer. Currently, the methods available for detecting the mutation are polymerase chain reaction (PCR) assay. PCR is a single detect test to detect the gene mutations, however, it generally requires multiple tissue sections, dissection for tumor cell enrichment and a multiplex system. Recently, it has been demonstrated that the mutation specific antibodies can reliably detect the exchange of single amino acids in routinely processed, formalin-fixed, and paraffin-embedded tumor tissue [15,16]. Our group has already screened the Japanese BRAF gene status using RT-PCR assay [17]. In this paper, to increase the sensitivity, we have modified BRAF V600E protein expression detection system
52
H. Sasaki et al. / Lung Cancer 82 (2013) 51–54
by immunohistochemistry (IHC) using specific monoclonal antibody, VE1 and Dako EnVisonTM FLEX, using linker. We have diluted the antibody for several concentrations for testing surgically treated lung adenocarcinoma samples. We have re-established the autostainer IHC method for detecting V600E mutation. From the screening step, we have found a novel 599 insertion T BRAF mutation in Japanese lung adenocarcinoma. 2. Material and methods 2.1. Patient samples The study group included lung cancer patients who had undergone surgery at the Department of Surgery, Nagoya City University Hospital. All tumor samples were immediately frozen and stored at −80 ◦ C until assayed. Because we have demonstrated that the BRAF V600E were in adenocarcinoma histology of lung cancer and exclusively with EGFR, Kras or erbB2 mutations [10,17], we mainly focused on wild type (wt) adenocarcinomas for the genes. The clinical and pathological characteristics of the 44 wt lung cancer patients for BRAF sequencing analyses were as follows; 30 (68.2%) were male 14 were female. 40 were diagnosed as adenocarcinomas, 2 were adenosquamous and 2 were pleomorphic carcinomas. 30 (68.2%) were smoker and 14 were non-smoker. 29 (65.9%) were pathological stage I. The clinical and pathological characteristics of the 26 lung cancer patients for BRAF V600E IHC analyses were as follows; 17 (65.4%) were male 9 were female. All were diagnosed as adenocarcinomas without EGFR, Kras codon12–13 [18], erbB2 [4,17], EML4-ALK or KIF5B-RET [19] mutations. 18 (69.2%) were smoker and 8 were non-smoker. 15 (57.7%) were pathological stage I. 2.2. PCR assay for BRAF gene Total RNA was extracted from fresh/snap-frozen lung cancer tissues using Isogen kit (Nippon gene, Tokyo, Japan) according to the manufactures’ instructions. RNA concentration was determined by Nano Drop ND-1000 Spectrophotometer (Nano Drop Technologies Inc., Rockland, DE, USA). RNA (1 g) was reverse transcribed by First strand cDNA synthesis kit with 0.5 g oligo (dT)16 (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufactures’ protocol. The reaction mixture was incubated at 25 ◦ C for 15 min, 42 ◦ C for 60 min, 99 ◦ C for 5 min and then at 4 ◦ C for 5 min. About 200 ng of each cDNA was used for polymerase chain reaction (PCR) analysis. The primer sequences for BRAF gene were as follows: the forward primer, 5-GACGGGACTCGAGTGATGAT-3 and the reverse primer, 5-GGGATCCCATCGTAAGGTTT-3 (594 bp). The cycling conditions were as follows: initial denaturation at 94 ◦ C for 5 min, followed by 40 cycles at 94 ◦ C for 40 s, 60 ◦ C for 40 s and 72 ◦ C for 45 s.
Fig. 1. Sequencing result for a novel BRAF insertion case. (Left) Nucleotides ACA (3-bp) were duplicated after 1797. The amino acid threonine (T) was duplicated after 599 T. (Right) Adjacent normal lung tissue samples showed BRAF wild type sequence, suggested that the mutation was somatic.
and counter staining with hematoxylin for 3 min. We selected 200× as an appropriate dilution and slides were scored semi quantitatively for cytoplasmic staining intensity and distribution. An IHC score was assigned to each case according to the following criteria showing the designated staining pattern; 3+, intense, granular cytoplasmic staining; 2+, moderately, smooth cytoplasmic staining; 1+, faint cytoplasmic staining; and no staining. The extent of the staining area was determined as 0–10%, 25%, 50%, 75%, and 90%. Tumors with intensity involving a minimum of 50% of the positive stained tumor cells were considered positive for BRAF V600E expression. These criteria were determined by the co-author with clinical data blind. 3. Results 3.1. BRAF gene alteration status in Japanese lung cancer patients We have sequenced for kinase domain of BRAF gene for 44 adeno histology samples. Of 44 patients, from direct sequencing using cDNA samples, we found a novel BRAF insertion case (Fig. 1). Nucleotides ACA (3-bp) were duplicated after 1797. The amino acid threonine (T) was duplicated after 599 T. This patient was female and non-smoker adenocarcinoma at stage I. Mixed subtype adenocarcinoma with acinar dominant (acinar 80% and papillary 20%). Adjacent normal lung tissue samples showed BRAF wild type sequence, suggested that the mutation was somatic. Other 43 patients did not have BRAF mutations within kinase domain. Previous our report demonstrated that from 133 wt lung cancers, 6 BRAF mutations were found. Totally, from 177 wt lung cancers, 7 (3.95%) BRAF mutation cases were found. 3.2. Immunohistochemistry for BRAF V600E
2.3. BRAF V600E protein immunohistochemistry 26 cases were immunostained by automated methods (Dako Japan Co.) for BRAF V600E expression using the mouse monoclonal BRAF V600E clone VE1 (Spring Bioscience, Pleasanton, CA). Unstained 4-m sections of FFPE tumor tissue were submitted to the analysis. The Dako EnVisionTM ELEX detection system included pretreatment with Dako PT LINK (pre-treatment module) and Target Retrieval solution, High pH (K8004, Dako Co., Tokyo, Japan) 97 ◦ C for 20 min, followed by incubation with 50×, 100× or 200× diluted mouse anti-BRAF V600E (clone VE1) with Antibody Diluent (K8006, Dako Co.) 4 ◦ C for overnight. Antibody incubation was followed by standard signal amplification including rabbit LINKER (K8019) at room temperature for 15 min, HRP conjugated EnVision TM FLEX at room temperature for 20 min, DAB reaction for 10 min
Immunohistochemical results are summarized in Table 1. V600E mutant adenocarcinomas showed different pattern of mixed subtypes, such as 2 acinar dominant, 1 papillary dominant, 1 solid dominant and 1 micropapillary dominant. Two cases in the V600E mutant cases were 3+, and one was 2+. These samples had more than 90% staining for VE1 (Fig. 2). In addition, two more V600E cases had the 1+ staining intensity, and more than 50% tumors had staining for both cases. The staining pattern was diffuse in all components of adenocarcinomas and cytoplasmic. We have additionally performed immunohistochemistry for nonV600E patients. The staining intensity was 1+ in one case, very weak (+−) in two cases, and no-stained in 18 cases. The positive case was 599 insertion T case. In this case, about 50% of tumor cells had cytoplasmic staining.
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Table 1 Immunohistochemical results. No.
1. 2. 3. 4. 5. 6. 7.
BRAF mutant adenocarcinomas Pap
Lep
Acinar
80 30 50 10 20 20 20
10 20 30
10 50 20 20 45 30 80
Solid
70 30 10
Micropap
5 40
BRAF
V600EIHC
S581I V600E V600E V600E V600E V600E 599insT
– +50% +50% 3 + 90% 2 + 90% 3 + 90% +50%
Pap: papillary; Lep: lepidic growth; Micropap: micropapillary.
4. Discussion In this study, we have introduced modified methods for the detection of BRAF V600E by EnVison TM FLEX IHC, a sensitive method using linker, and the results showed an excellent concordance between RT-PCR assays. We have established optimal condition of IHC for BRAF V600E. Although BRAF V600E protein expression was not perfectly matched with the sequencing results, IHC analysis is an appropriate method for detecting BRAF V600E status in lung cancer. From the screening process, we found a novel 3-bp insertion in BRAF resulting in the duplication of threonine 599. The main focus of this study was to establish IHC methods for detecting V600E in routinely processed FFPE tissue and to increase the sensitivity. Original report demonstrated that clone VE1 positivity was perfect match with sequencing results in papillary thyroid carcinomas and melanoma metastases [15]. However, recent study has been reporting the 19 of 21 (90%) of V600E Caucasian lung adenocarcinoma were positive for IHC test [20]. In our study, one of non-V600E mutant sample was positive for VE1 IHC. Because the VE1 was made using peptides between 596 and 606 (GLATEKSRWSG) of V600E, it should not cross-react with a novel 599 insertion T case. If GLATEKSRWSG is similar to GLATTVKSRWSG in tertiary structure, the antibody may recognize the latter motif. Reduced VE1 immunostaining intensity of damaged or “prenecrotic” tissue parts has to be taken into account, as it may lead to false-negative results in cases with small or sub-optimally preserved tissue [15]. Tissue sections have to be freshly cut from FFPE blocks for optimal results, as previously observed a marked
decrease of VE1 immunostaining intensity in tissue sections stored for longer than approximately 2 weeks [16]. Although the mutation rate of V600E in Japanese lung cancers was lower than previous Caucasian report (9% of wt lung cancers) [17,20], a racial difference between the studies about mutant BRAF may be existed, like EGFR gene mutations [2–6]. Because the BRAF mutation statuses were examined by pyrosequencing or direct sequencing in Caucasian cohort [20], we cannot conclude that differences were owing to our methodology as our PCR and sequencing methods. However false IHC positive was not found in our wild type cases. Mutated BRAF V600E protein has recently emerged as a promising therapeutic target. Several specific small-molecule BRAF V600E inhibitors are currently under pre-clinical or clinical investigation [13]. Of these, the V600E-specific inhibitor PLX4032 (Vemurafenib) has remarkable clinical activity in patients with BRAF V600E mutated metastasizing melanoma [14]. The compound GSK 2118436 (Dabrafenib) also resulted in clinical response in metastasized melanoma and may also be effective in BRAF mutations [21]. Similar insertion mutation (1798 insertion TAC resulted in the insertion of one additional threonine residue at position 599) has been reported in pilocytic astrocytoma [22]. This mutant BRAF insertion T did display an in vitro kinase activity and cellular MEK/ERK activation potential comparable to those to BRAF V600E [22]. Replacement of threonines by valine residues has similar effects on BRAF activity, suggested that the distortion of the peptide backbone by additional amino acids rather than the insertion of additional, potential phosphorylation sites destabilizes the inactive conformation of the kinase domain [22]. Identification of a
Fig. 2. VE1 IHC results. (Left upper) V600E mutant case with 3+, and the sample had more than 90% staining for VE1. (Left lower) V600E mutant case with + and the sample had more than 50% staining for VE1. (Right upper) 599 insertion T case with + and the sample had more than 50% staining. (Right lower) Negative case.
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small fraction of lung cancers patients responsive to a molecular target therapy may have large clinical impact as highlighted by the recent FDA approval of crizotinib for ALK-rearrangement lung cancers [23]. This was based upon from the results of clinical trials for advanced lung cancers with ALK rearrangements, and early responses in ALK-rearrangement might be assessed by IHC [24,25]. Accurate and rapid screening of mutations is important in lung cancers, especially in determining their eligibility for molecular target therapy. Current standard technique to identify BRAF is PCR assay [10,17]. This assay requires extraction of RNA or DNA from clinical samples, is not routinely performed in many diagnostic pathology laboratories, and suffers from some important limitations. Another important limitation is the possibility of contamination with normal tissues and small sized biopsies obtained by limited invasive procedures, which might impair specificity and led to false-negative results. IHC enables the pathologist to perform a more reliable quantification of the genomic alteration. The use of an antibody to detect oncogenic mutations on tumor tissue sections allows simultaneous morphological examination, in particular an estimation of the percentage of tumor cells, as well as determination of the number and intensity of stained cells. The intensity of BRAF IHC may be important additional information in BRAF mutation screening. Approximately 40% of patients with BRAF mutant have resistance to Vemurafenib [14]. It should be tested whether cases with low level expression are less dependent on MAP pathway activation by BRAF V600E and may be resistant to target therapy. In summary, BRAF V600E mutation in Japanese lung cancers seemed to be rare, however, V600E specific IHC might be a role in detection and screening for molecular target therapy for lung cancer. Conflict of interest statement None declared. Acknowledgements The authors thank Mrs. Yuka Toda for her excellent technical assistances. This work was supported by Grants-in-Aid for Scientific Research, Japan Society for the Promotion of Science (JSPS) (Nos. 23659674, 24592097 and 25293303). References [1] Ginsberg RJ, Kris K, Armstrong G. Cancer of the lung. In: Principles and practice of oncology. 4th ed. Philadelphia: Lippincott; 1993. p. 673–82. [2] Paez JG, Janne PA, Lee JC, Tracy S, Greulich H, Gabriel S, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 2004;304:1497–500. [3] Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med 2004;350:2129–39.
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