Development of an immuno-wall device for the rapid and sensitive detection of EGFR mutations in tumor tissues resected from lung cancer patients.

Detecting molecular targets in specimens from patients with lung cancer is essential for targeted therapy. Recently, we developed a highly sensitive, rapid-detection device (an immuno-wall device) that utilizes photoreactive polyvinyl alcohol immobilized with antibodies against a target protein via a streptavidin-biotin interaction. To evaluate its performance, we assayed epidermal growth factor receptor (EGFR) mutations, such as E746_A750 deletion in exon 19 or L858R substitution in exon 21, both of which are common in non-small cell lung cancer and important predictors of the treatment efficacy of EGFR tyrosine kinase inhibitors. The results showed that in 20-min assays, the devices detected as few as 1% (E746_A750 deletion) and 0.1% (L858R substitution) of mutant cells. Subsequent evaluation of detection of the mutations in surgically resected lung cancer specimens from patients with or without EGFR mutations and previously diagnosed using commercially available, clinically approved genotyping assays revealed diagnostic sensitivities of the immuno-wall device for E746_A750 deletion and L858R substitution of 85.7% and 87.5%, respectively, with specificities of 100% for both mutations. These results suggest that the immuno-wall device represents a good candidate next-generation diagnostic tool, especially for screening of EGFR mutations.

Introduction

Lung cancer is the leading cause of cancer-related mortality worldwide [1], and ~85% of lung cancers are classified as non-small cell lung cancer (NSCLC) [2]. Epidermal growth factor receptor (EGFR) is a member of the ErbB receptor tyrosine kinase family, which plays an important role in NSCLC cell proliferation, motility, and differentiation [3]. Somatic EGFR mutations are detected in 10% to 16% of NSCLC patients in the United States and Europe [4] and 30% to 50% of those in Asia [5], with ~90% presenting as deletions in exon 19, most commonly the E746_A750 deletion, and an L858R substitution in exon 21. Several studies report that these mutations are associated with the sensitivity of NSCLC patients to EGFR tyrosine kinase inhibitors (TKIs) [6,7]; therefore, clinical testing for EGFR mutations has become a standard care for patients with NSCLC. In addition to EGFR mutations, other driver mutations have been examined, with assessment of anaplastic lymphoma kinase (ALK), c-ros oncogene 1 (ROS1), and B-Raf proto-oncogene, serine/threonine kinase (BRAF) currently part of routine molecular testing in Japan [8].

Direct sequencing of EGFR polymerase chain reaction (PCR) products is a common approach for EGFR-mutation testing [9]; however, its clinical usefulness is reduced by false-negative results due to the small proportion of cancer cells in collected samples available for DNA extraction. Other DNA-based analyses have been developed to detect EGFR mutations, including PCR-Invader [10] and peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp [11]. These methods show high sensitivity and can be used in patients with advanced NSCLC, even in those with low tumor-cell content [12]. However, routine testing using these methods is often limited by the associated high costs and technical complexity [9].

We previously developed various microfluidic immunoassay devices for rapid and highly sensitive molecular analyses. First, we proposed an immuno-pillar device [13] comprising antibody immobilized microbeads and a UV-curable polyethylene glycol-based resin cured using photolithography. Antibodies and antigens pass through pores in the cured resin to reach the microbeads, thereby allowing antibody–antigen reactions on the microbead surface. Utilizing sandwich-type immunoassays with accumulated three-dimensional (3D) fluorescence signals, immuno-pillar devices exhibit high biomarker detection sensitivity using human serum samples. However, floating substances, such as blood cells, cell debris, and fibrin, sometimes block the pores or persist near the microbeads and/or the cured resin after the immunoassay, resulting in false-negative or false-positive results. To solve this problem, we developed a new immunoassay device called an immuno-wall device from a non-porous photopolymer and that shows robust sensitivity, even in the presence of bodily fluids and lysed tumor tissue harboring large amounts of debris [14].

In the present study, we evaluated the ability of the immuno-wall device to specifically detect mutated EGFR proteins in surgically resected tissues from NSCLC patients and successfully performed rapid mutant EGFR detection in a small volume (1 μL) of lysed, debris-rich, surgically resected samples without the need for thorough pretreatments.

Materials and methods

Chemicals

We first prepared 1% (v/v) bovine serum albumin (BSA; Thermo Fisher Scientific, Waltham, MA, USA) in phosphate-buffered saline (PBS; Thermo Fisher Scientific) and PBS containing 1% (v/v) Tween-20 (PBS-T; Sigma-Aldrich, St. Louis, MO, USA). Washing buffer was prepared by mixing 1% BSA and 1% PBS-T (1:1; v/v). A photoreactive polyvinylalcohol (azido-unit pendant water-soluble photopolymer; AWP) for photo-immobilization was purchased from Toyo Gosei Co., Ltd. (Tokyo, Japan). Recombinant streptavidin was purchased from ProSpec (Cat. No. pro-791NJ; East Brunswick, NJ, USA). Plastic immuno-wall device substrates were acquired from Sumitomo Bakelite Co., Ltd. (Tokyo, Japan). A rabbit anti-human EGFR (L858R) biotinylated antibody (clone 43B2, Cat. No. 5354), rabbit anti-human EGFR (E746_A750 deletion mutant) biotinylated antibody (clone D6B6, Cat. No. 5747), and rabbit anti-human EGFR biotinylated antibody (clone D38B1, Cat. No. 6627) were purchased from Cell Signaling Technology (Danvers, MA, USA). These antibodies were diluted to 50 μg/mL in PBS and used as the capture antibody in our sandwich immunoassay. We dissolved 50 μg of goat anti-human EGFR antibody (Cat. No. AF231; R&D Systems. Minneapolis, MN, USA) in 1% BSA (1 mL) and used it as the detection antibody in our sandwich immunoassay. These antibodies were also used for western blot analysis and immunocytochemistry. Importantly, anti-EGFR [wild-type; WT] antibodies can capture both WT and mutant EGFR. DyLight 650-conjugated anti-goat IgG antibody was purchased from Abcam (Cat. No. ab102343; Cambridge, UK) and diluted to 50 μg/mL with 1% BSA before use. Mouse monoclonal anti-actin antibody (Sigma-Aldrich) was used as a loading control, and an anti-rabbit or anti-mouse antibody (GE Healthcare, Little Chalfont, UK) was used as the secondary antibody for western blot analyses. Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes; Invitrogen, Carlsbad, CA, USA) was also used as a secondary antibody for immunocytochemistry, and 4',6-diamino-2-phenylindole (DAPI) (DOJINDO LABORATORIES, Kumamoto, Japan) was used for nuclei staining. Pipettes were used to inject the samples and reagents into the microchannels (Research Plus; Eppendorf AG, Hamburg, Germany). Solutions were removed from the microchannels with an aspirator (VACUSIP; INTEGRA Biosciences AG., Zizers, Switzerland).

NSCLC cell lines and lysates

Human lung cancer cell lines H3255, and HCC827 were obtained from the Hamon Center Collection (University of Texas Southwestern Medical Center, Dallas, TX, USA) and H358, H1299, and PC9 were purchased from ATCC (Manassas, VA, USA). These cells were cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% FBS at 37°C in 5% CO2. Cells were lysed with lysis buffer (Cell Signaling Technology) supplemented with 1 mM phenylmethylsulfonyl fluoride. All cell lines were checked for mycoplasma contamination using MycoAlert Mycoplasma Detection Kit purchased from Lonza (Cat. No. LT07-118; Switzerland) and short tandem repeats profiling analysis has been submitted for authentication.

Resected tissues from NSCLC patients

Patients with pathologically confirmed NSCLC at Nagoya University Hospital between November 2010 and August 2015 were enrolled in this study. All participants provided written, informed consent, and the Ethics Review Committee of Nagoya University Graduate School of Medicine approved this study (No. 2014–0171). Surgically resected tumor tissues were preserved by snap-freezing in liquid nitrogen within 1 h of collection and stored at −80°C until use. Each tumor sample was divided into two pieces: one for the immuno-wall assay and the other for testing using a commercially available, clinically approved EGFR testing method (PNA-LNA PCR clamp or PCR-invader).

Preparation of tumor lysate

The frozen tumor tissue was lysed in 200 μL radioimmunoprecipitation assay buffer (Wako, Osaka, Japan) with protease inhibitor using a sample-grinding kit (GE Healthcare). The lysate was centrifuged at 12,000g for 10 min at 4°C, and supernatants were used for all assays.

Western blot analysis

Western blot analysis was performed as described previously [15]. Primary antibodies used for this analysis included the anti-human EGFR (L858R) antibody (clone 43B2, Cat. No. 3197) (1:2,000), rabbit anti-human EGFR (E746_A750 deletion mutant) (clone D6B6, Cat. No. 2085) (1:5,000), anti-human EGFR antibody (clone D38B1, Cat. No. 4267) (1:4,000), and mouse monoclonal anti-actin antibody (Cat. No. A2228) (1:20,000). Horseradish peroxidase-conjugated donkey anti-rabbit (1:2,000) or sheep anti-mouse (1:2,000) antibody was used as the secondary antibody. Actin was used as a loading control.

Immunocytochemistry

Lung cancer cells grown on plastic dishes were fixed and permeabilized for 30 min with 4% formaldehyde and 0.2% Triton X-100 in PBS. After blocking with 1% BSA for 1 h, cells were incubated with anti-human EGFR (L858R) antibody (clone 43B2, Cat. No. 3197) (1:1,000), rabbit anti-human EGFR (E746_A750 deletion mutant) (clone D6B6, Cat. No. 2085) (1:1,000), and anti-human EGFR antibody (clone D38B1, Cat. No. 4267) (1:1,000) in 1% BSA in PBS for 1 h at room temperature. Cells were then washed and incubated with Alexa Fluor 488 goat anti-rabbit secondary antibody (1:1,000) and DAPI (1:1,000) for 1 h at room temperature. Fluorescence images were obtained using an IX73 inverted microscope (Olympus, Tokyo, Japan) with a 10× objective lens.

Immuno-wall devices

Immuno-wall device schematics are shown in Fig 1. The plastic substrates were made with cyclic olefin polymer, with 40 microchannels formed in the substrate. The wall-like structure with immobilized antibodies at the center of the microchannel was constructed using standard photolithography techniques. Devices were fabricated as follows. Streptavidin diluted in PBS (10 mg/mL) was mixed with the same volume of AWP in a low-adhesion tube (PROTEOSAVE; Sumitomo Bakelite Co., Ltd., Tokyo, Japan) to prevent nonspecific streptavidin binding. The mixture was then introduced into the microchannels and irradiated with UV light (LA-410UV-5; Hayashi Watch-Works Co., Ltd., Tokyo, Japan) through a photomask. Streptavidin was also photo-immobilized onto the irradiated, cross-linked AWP, with uncured AWP containing free streptavidin removed using an aspirator. The AWP wall was constructed in the middle of the microchannels, which were washed and filled with washing buffer containing 0.5% BSA to prevent nonspecific analyte protein and antibody binding before use. The AWP wall extended from the microchannel floor to the roof. Therefore, the top and bottom surfaces of the AWP wall were not in direct contact with the loaded lysate sample or antibody solutions during the immunoassay.

Fig 1

Picture and schematic of the immuno-wall device.

To immobilize the capture antibody, 1 μL of biotinylated antibody in PBS was injected into the microchannel and incubated for 1 hour at room temperature. Most biotinylated antibodies were immobilized by streptavidin via a biotin–streptavidin interaction around the AWP wall, after which unbound antibodies were then removed. We named the antibody immobilized AWP wall structure the “immuno-wall.”

Immunoassay procedure

The immunoassay procedure was similar to that used for enzyme-linked immunosorbent assay (ELISA) using microtiter plates. Solutions were removed and injected using an aspirator or pipette, with the volume of the sample, antibody solution, and washing buffer at 1 μL/injection. First, we removed the washing buffer and injected the sample, and after incubation for 15 min, we removed the unreacted sample and washed by immersion (1 min) and sequential rinsing (15 times) with washing buffer. Cell debris remaining within the microchannel after sample incubation was removed by rinsing. The device was then immersed in detection antibody (30 s), washed by immersion (1 min) and 15 sequential rinses, and immersed in fluorescence-labeled anti-goat IgG antibody (30 s). The device was washed again and imaged using a fluorescence microscope (Nikon, Tokyo, Japan). The device was scanned by a fluorescence immunoassay reader (Hamamatsu Photonics K.K., Hamamatsu, Japan) according to manufacturer’s instructions.

Statistical analysis

Although no reference or ‘gold standard’ has been defined for EGFR mutation analysis, PCR-based methods are assumed to be the current gold standard in daily practice. Therefore, we calculated diagnostic sensitivity and specificity based on PCR-based results as described previously [16]. JMP pro 14 (SAS Institute Inc., Cary, NC, USA) was used for all statistical analyses in this study.

Results

Immuno-wall assays

To evaluate the immuno-wall devices, we analyzed mixed NSCLC cell lysates harboring mutant and WT EGFR. EGFR mutant cells (HCC827 for E746_A750 deletion and H3255 for L858R substitution) and WT (H358) were mixed with 0%, 0.1%, 0.5%, 1%, 5%, and 10% mutant cells. Mixed cell lines were lysed in 200 μL lysis buffer with a 1.5 mg/mL total protein concentration. Respective anti-EGFR antibodies were used as capture antibodies, and representative fluorescence images of the immuno-wall devices are shown in Fig 2. Fluorescence intensity increased along with increasing mutant EGFR cell proportions. All images, except for those for 0%, 0.1%, and 10% HCC827 cells, appeared as two bright lines, implying that the antigen–antibody reaction mainly occurred on the device side, likely due to the large amount of immobilized capture antibodies. Weak fluorescence observed in 0% H3255 samples indicated cross-reactivity of anti-EGFR (L858R) antibody with WT EGFR, which was consistent with immunocytochemistry and western blot results (S1 Fig).

Fig 2

Representative fluorescence images after immunoassays of lysates containing mixed populations of mutant EGFR cells.

Fluorescence intensity measured by a fluorescence immunoassay reader is indicated. Scale bars = 100 μm.

Fluorescence calibration curves of the devices generated using the mutant EGFR cells are shown in Fig 3. Each plot represents the average (mean ± standard error of the mean) fluorescence intensity of the immuno-wall. The limit of detection (LOD) was estimated at 1% and 0.1% for the E746_A750 and L858R mutant cell proportions, respectively, based on the threshold value calculated as three standard deviations above the signals observed in the analyses for lysate with 1.5 mg/mL protein concentration from the EGFR WT cell line (H358) that was applied for each immuno-wall. The background fluorescence for the H3255 cell line was slightly higher than that for the HCC827 cell line, possibly due to cross-reactivity of the anti-EGFR (L858R) antibody. To estimate potential cross-reactivity, we performed the immunoassay using lysates from only the EGFR WT cell line (H358) (S2A and S2B Fig), finding that the fluorescence intensities remained below the threshold. For L858R substitution antibody, western blot analysis revealed a weak band for L858R substitution in HCC827 cells, which also indicated cross-reactivity (S1 Fig). Therefore, immunoassay was performed using lysates from only HCC827 and observed that the fluorescence intensities remained below the threshold (S2C Fig). Additionally, we performed the immunoassay targeting only mutant EGFR cell lines (Fig 4), revealing LODs estimated at ~0.01 mg/mL for both mutations and resulting in signals at three standard deviations above the average for the EGFR WT cell line (H358). At high protein concentration (>1 mg/mL), the increased fluorescence signals overlapped.

Fig 3

Calibration curves derived from mutant EGFR cell lines.

Fluorescence intensities associated with (A) E746_A750 deletion and (B) L858R substitution versus background device fluorescence. The dashed lines indicate fluorescence values three standard deviations above the average for the EGFR WT cell line (H358) detected in each immuno-wall device.

Fig 4

Calibration curves derived from total protein concentration in mutant EGFR cell lines versus device fluorescence intensity.

The dashed lines indicate fluorescence values three standard deviations above the average for the EGFR WT cell line (H358) detected in each immuno-wall device.

During immuno-wall analysis, target proteins were identified after incubation for only 15 min. Because cell lysate contains several proteins that could potentially interfere with the antigen–antibody reaction, the capture-antibody density on the AWP wall might affect detection efficiency. We assumed that the biotinylated capture antibodies introduced into the microchannels would be immobilized at the AWP wall side surface when mixed with streptavidin. To evaluate immobilization efficiency, we compared direct and biotin–streptavidin immobilization to the AWP (S3 Fig). Naked or biotinylated goat IgG was injected into the device, and immobilization was detected by a fluorescence-labeled anti-goat IgG antibody. Representative fluorescence images showed increased fluorescence intensity in the biotin–streptavidin-binding device (S3A Fig), with fluorescence quantification showing a >10-fold higher fluorescence intensity in the device with biotin–streptavidin interactions (S3B Fig).

Clinical diagnostic application

The immuno-wall devices were capable of accurately analyzing small sample volumes (1 μL). The lysed samples from surgically resected tumors included a higher level of debris than that from cell-line samples (S4 Fig); however, pretreatments, such as thorough cell-debris removal and sample enrichment, were not required before the immunoassay, which allowed easy preparation and analysis of the clinical samples. The immuno-wall devices were then used to perform clinical diagnosis of surgically resected specimens from 22 NSCLC patients previously confirmed as harboring tumors with the E746_A750 deletion, L858R substitution, or EGFR WT according to PCR-based methods and the previously described threshold value (S1 Table). The results shown in Table 1 and Fig 5 indicated that immuno-wall analysis of the L858R substitution demonstrated weak fluorescence using mixtures of lysates containing both EGFR E746_A750 deletion mutants and EGFR WT, which was consistent with in vitro findings (Fig 2). However, the weak fluorescence was lower than the threshold, resulting in a negative diagnosis. For lysates harboring both E746_A750 deletion and L858R substitution, the immuno-wall returned a >85% diagnostic sensitivity, with one case of each mutation misdiagnosed as negative due to low fluorescence intensity. Samples including EGFR WT were all diagnosed as such according to the immuno-wall device.

Table 1

Genotype analysis of immuno-wall results.

Immuno-wall analysis	Genotype
	E746_A750 deletion (n = 7)	Other exon 19 deletion (n = 15)	L858R substitution (n = 8)	WT (n = 7)
E746_A750 deletion, n (%)	6 (85.7)	0	0	0
L858R substitution, n (%)	0	0	7 (87.5)	0
WT, n (%)	1 (14.3)	15 (100)	1 (12.5)	7 (100)

Fig 5

Representative fluorescence images from immunoassay of clinical samples.

Samples containing (A) the E746_A750 deletion mutation (patient No. 11) and (B) L858R substitution mutation (patient No. 18). (C) A sample with no EGFR mutations (patient No. 9). Fluorescence intensity is indicated. Scale bars = 100 μm.

We then analyzed specimens from 15 NSCLC patients harboring an exon 19 deletion other than the E746_A750 deletion and confirmed by PCR-based methods (S1 Table and Table 1). All samples with the exon 19 deletion showed low fluorescence intensity when targeting the anti-E746_A750 deletion mutation and were diagnosed as WT based on detection of only the WT variant, resulting in 100% diagnostic specificity for both mutations.

Discussion

Here, we describe fabrication of a new immunoassay device for highly sensitive and rapid detection of mutant EGFR variants in a small sample volume (1 μL) of lysed, surgically resected samples with containing high levels of cellular debris. For quantitative analysis, mixtures of lysed NSCLC cell lines (mutant and WT EGFR), resulting in an estimated LOD of 1% and 0.1% for the cell populations harboring the E746_A750 and L858R substitutions, respectively. Additionally, the LOD was estimated using a dilution series for each mutant EGFR cell line, revealing values as low as 0.01 mg/mL for both lines. Moreover, the diagnostic sensitivity and specificity of the immuno-wall device for the mutations were 85% and 100%, respectively. These results indicated that the device described here provided rapid and specific detection of EGFR mutations.

The immuno-wall device includes several features suitable for molecular assays, including employment of enhanced immobilization of capture antibodies using biotin–streptavidin binding (S3 Fig), thereby allowing a high probability of antigen capture. Compared with other immuno-assays, including western blot, the immuno-wall device employs a 3D reaction field at the side of the immuno-wall, where the fluorescence signals are integrated, which potentially increases the detection sensitivity of the device. Additionally, the wall-like structure enables easy removal of non-specifically bound molecules by injection of washing buffer. In general, specimens, including blood or lysed tissues, contain cell debris and fibrin (S4 Fig), which can disturb flow into and out of the microfluidic channel; therefore, removal of these items is important for microfluidic assays. However, the immuno-wall device is capable of accurately analyzing lysed tissue samples without the need for thorough pretreatment. Moreover, the limited area of the microchannel allowed rapid antigen–antibody interactions, resulting in shorter incubation times and rapid detection of target molecules. The results confirmed that the immunoassay procedure could be completed within 20 min, making this method suitable for clinical applications, including point-of-care diagnostics.

Recently, several studies examined the presence of EGFR mutations in lung cancer by immunohistochemistry (IHC) using the two mutation-specific antibodies employed in the present study and demonstrated sensitivity ranging from 24% to 100% and specificity ranging from 77% to 100% [16-23]. IHC is a well-established and cost-effective method routinely applied in lung cancer diagnosis; however, the results are sometimes affected by differences in assay procedures and scoring system [24]. In the present study, we established a fluorescence threshold based on the average fluorescence intensity of an EGFR WT cell line (H358) (Fig 3). Bellevicine et al. [25] demonstrated that IHC analysis using EGFR-mutant-specific antibody could detect 10% of mutated cells from a mixture containing cells harboring either WT or mutant EGFR. In the present study, our dilution series of each mutant EGFR cell line revealed an LOD estimated at between 0.1% and 1% (Fig 3), which meets the sensitivity requirement of CAP/IASLC/AMP guidelines promoting the use of more sensitive tests that can detect mutations in specimens with as few as 20% cancer cells [8]. Additionally, the immunoassay for lysates with high protein concentration from EGFR WT cell lines and specimens from NSCLC patients harboring EGFR WT showed fluorescence intensities below the threshold, suggesting a low probability of false positives, even at high protein concentrations. Moreover, the L858R substitution antibody might cross-react with WT EGFR or other EGFR mutations (S1 Fig). In previous studies where EGFR mutation was evaluated using IHC, false positive results were observed with the use of anti-EGFR (L858R) antibody [23,26]. To evaluate potential cross-reactivity, we performed the immunoassay using lysates from only HCC827, which were proven to be false positive by immunocytochemistry or western blotting (S1 Fig). We found that the fluorescence intensities remained below the threshold, indicating the robustness of the assay even in samples with confirmed cross-reactivity by the anti-EGFR (L858R) antibody. The importance of an absence of false positives in this situation is underscored by the ineffectiveness of EGFR TKIs in patients without EGFR mutations, which can result in early disease progression [27]. Although the sample size in the present study was small, the results demonstrated high diagnostic sensitivity and specificity for the device relative to those obtained by PCR-based assays.

The mutation-specific antibodies used in this study targeted only two types of representative EGFR mutations. Analysis of a sample harboring a different deletion mutation in exon 19 showed an extremely faint signal using the EGFR (E746_A750 deletion) antibody. Yu et al. [28] reported detection of the E746_T751 deletion mutation using an antibody targeting E746_A750 but not L747_ A750. Additionally, Kawahara et al. [23] reported that only two of seven minor deletion mutations in exon 19 were identified by the E746_A750 antibody, and Simonetti et al. [18] reported that 12 samples with minor deletions in exon 19 were undetectable by IHC using mutation-specific antibodies. These findings suggested that the clinical efficacy of some antibodies is reduced by the occurrence of certain similar mutations; however, because E746_A750 and L858R somatic mutations account for >70% of EGFR mutations [19], screening NSCLC patients using the device described in the present study could potentially increase diagnostic speed and accuracy and identification of candidates for EGFR TKI therapy. Further improvement of these mutation-specific antibodies is needed to detect other less frequent mutations in order to enhance the sensitivity of molecular diagnosis using our device as a tool for EGFR mutations screening.

Several factors may have been associated with the discordances between the PCR-based method and our procedure, even in the analysis of E746_A750 and L858R somatic mutations. Although the exact reasons were unknown, possible factors may have included variation in tumor cell content or tumor heterogeneity within samples, which were also observed in comparison studies for PCR-based testing [12,29-31].

This study has limitations. First, it was a retrospective, single-center study, in which mutation analysis was conducted using a limited number of surgical samples stored in the hospital. Therefore, selection bias might have affected the results. Second, direct comparison of IHC with the described method was not done due to the complexity of the staining procedure and the variability in scoring of staining intensity for IHC assays. Therefore, superiority over IHC was inconclusive. Additionally, direct comparison with ELISA was not conducted, because PCR is the current gold standard for EGFR-mutation analysis; therefore, we felt that this was the appropriate comparison. Moreover, ELISA generally requires several hours and higher sample volumes (≥100 μL). Third, overlap in the fluorescence signal occurred at high protein concentrations (especially at ≥1 mg/mL protein) (Fig 4), indicating loss of quantifiable accuracy. In cases of highly concentrated samples harboring EGFR mutation(s), signal variability can potentially occur due to various forms of cell debris, including blood cells and fibrin. However, given the importance to detect lower proportions and lower concentrations of mutant molecules for this type of testing, overlaps of these signals at higher concentrations might be acceptable. Furthermore, many NSCLC patients are diagnosed with advanced disease and not surgically treated. Because small biopsy or cytological samples are only collected for diagnosis and mutation testing for these patients, such specimens are not always stored for other purposes. Therefore, these samples were not examined in this study. Taken together, our findings need to be confirmed in a larger prospective multicentric study.

Conclusions

Here, we analyzed surgical samples from 37 NSCLC patients using a new immuno-wall device, revealing a diagnostic sensitivity and specificity of >85% and 100%, respectively. Moreover, samples could be prepared within 10 minutes, and the immunoassay procedure could be completed within 20 min, for a 30-min total assay time. These results suggest the immuno-wall device as a good candidate for next-generation diagnostics. Future studies will investigate the applicability of the device for small specimens or other molecular targets, such as ALK, ROS1, and BRAF.

Expression of EGFR mutations (E746_A750 deletion and L858R substitution) in NSCLC cell lines.

(A) IHC analysis of each NSCLC cell line for the indicated antibody (green) and nuclei (blue). Images were obtained using a fluorescence microscope with a 10× objective lens. Scale bars = 10 μm. (B) Western blot analyses of mutant EGFR protein in each NSCLC cell line. Actin was used as a loading control.

(TIF)

Click here for additional data file.

Calibration curves of total protein concentration of lysates of EGFR WT cells (H358) and EGFR mutation cells (HCC827) versus the fluorescence intensity for each EGFR mutant protein.

E746_A750 deletion for EGFR WT cell line (H358) (A), L858R substitution for EGFR WT cell line (H358) (B), and EGFR mutation cells (HCC827) (C). Dashed lines indicate three standard deviations above the average fluorescence determined in the EGFR WT cell line (H358) by each immuno-wall device.

(TIF)

Click here for additional data file.

Comparison of immobilization to the AWP.

Goat IgG in PBS (100 μg/mL) and streptavidin in PBS (10 mg/mL) were mixed with an equal volume of AWP, respectively, introduced into the microchannel, and irradiated with UV light through a photomask. Biotinylated goat IgG (50 μg/mL) was injected into the microchannel, mixed with streptavidin, and the device was incubated for 60 min at room temperature, followed by the washing procedure. To compare immobilization to the AWP, fluorescence-labeled anti-goat IgG antibody was introduced into the microchannels of each device and incubated for 5 min at room temperature. After washing, (A) fluorescence images of the immuno-wall were obtained using a fluorescence microscope with a 20× objective lens (exposure time: 0.25 s) and (B) scanned with a fluorescence reader. Scale bars = 100 μm.

(TIF)

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A representative microscopy image of cell lysates from NSCLC cell lines and tumor samples.

Scale bars = 100 μm.

(TIF)

Click here for additional data file.

Details of patients examined and immuno-wall analyses.

(DOCX)

Click here for additional data file.

25 Aug 2020

PONE-D-20-01970

Development of an immuno-wall device for the rapid and sensitive detection of EGFR mutations in tumor tissues resected from lung cancer patients

PLOS ONE

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'T. Hase received personal fees from Chugai Pharmaceutical Co. Ltd., Ono Pharmaceutical Co. Ltd., Bristol-Myers Squibb Co., and Boehringer Ingelheim, and research funding from Boehringer Ingelheim, and Taiho Pharmaceutical Co. Ltd., outside the submitted work. Y. Hasegawa received grants from Boehringer Ingelheim, AstraZeneca, Eli Lilly Japan K.K., Ono Pharmaceutical Co. Ltd., Bristol-Myers Squibb Co., Taiho Pharmaceutical Co. Ltd., Novartis Pharma K. K., and Chugai Pharmaceutical Co. Ltd., and personal fee from Boehringer Ingelheim, MSD K.K., AstraZeneca, Pfizer Inc., and Chugai Pharmaceutical Co. Ltd, outside the submitted work.'

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Please note that we cannot proceed with consideration of your article until this information has been declared.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: No

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: No

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: PLOS ONE

MS: PONE-D-20-01970

Development of immune-wall device for the rapid and sensitive detection of EGFR mutations in tumor tissues resected from lung cancer patients.

The research article by Yogo et al., 2020, developed a sandwich immunoassay based immuno-wall diagnostic tool to detect two prevalent EGFR mutants such as E746_A750 deletion in Exon 19 and L858R substation in exon 21. Both of these mutants are commonly observed in EGFR mutant NSCLCs and are good predictors of treatment efficacy upon EGFR TK inhibitors targeted therapy. Using both wildtype and mutant EGFR NSCLC cell lines and 37 lung cancer specimens, authors demonstrate that immuno-wall device detected the EGFR mutants E746_A750 and L858R mutants with sensitivities of 85.7% and 87.5% respectively.

Existing literature clearly reveal that immuno-wall device based detection of mutations in cancer has been published previously. An immuno-wall device for detection of IDH1-R132H mutation in low grade gliomas was described earlier (Yamamichi et al., 2016). Previously published article by Yogo et al., 2018 (same group as in the current paper) has already reported about the immuno-wall device for detecting Anaplastic lymphoma kinase 1 (ALK1) and C-ROS Oncogene 1(ROS1) fusion in lung cancer specimens. Thus, the development of the immuno-wall tool to detect EGFR mutants is relatively not novel and is merely an extension of the earlier research articles with replacement EGFR gene and its variants.

However, the scope of the study to identify the subset of NSCLC patients with EGFR mutations using immune-wall device have the potential to improve the therapeutic outcome and prognosis, as these mutations are associated with sensitivity to EGFR tyrosine kinase inhibitors.

Here are few concerns authors need to clarify.

Q1. In the figure S1B, lane 3, L858R substitution antibody picked up a less intense band in HCC827 (E746_A750del) cells but not in PC9 cells (E746_A750del). Whether HCC827 cells also carry L858R substitution. PC9 cells did not show any detectable band with L858R substitution antibody and thus, the PC9 cells should have been considered instead of HCC827.

Q2. The study tested very limited number of retrospective lung cancer specimens (N=37) for which DNA sequence for EGFR mutants were available. The test need to be performed on a large number of lung cancer specimens from multicentric studies and the revised sensitivity and specificity for each mutant should be considered as the diagnostic potential of this device. In the supplementary table 1, patient number # 14, (Female, age 51), the tumor lysate has the protein concentration of 6.32 µg/µl and carry E746_A750del as reported by DNA sequence. However, Immuno-wall device failed to detect the mutation in the specimen from the same patient (false negative). Did authors verify E746_A750del mutation in this patient specimen by western blot analysis.

Q3. In results section, please clearly explain and rewrite about estimation of limit of detection (LOD)

Reviewer #2: Reviewer’s Comments:

The present study by Yogo et al titled “Development of an immune-wall ………. Lung cancer patients” demonstrates an innovation comprises of a unique fluorescence-based immune-wall device, capably detecting a couple of frequently occurring EGFR mutations from both the cell line lysates and patient-derived samples in a very rapid, efficient and above all, cost-effective way; even from a very tiny amount of surgically resected and/or crude protein extracts. This is principally a biotin-streptavidin conjugated method to amplify fluorescence signals initiated by a basic epitope-based antigen-antibody interaction; which authors claim to be much better than the gold standard technic in the field, the conventional PCR-based detection procedure against these mutations. There are few pertinent questions this reviewer still possesses, which need better justification;

1. Though it is a rapid fluorescence-based technic, then how did authors claim this to be very unique; and above all, how it can be remarkably sensitive over conventional ICC/IHC methods?

2. During the assay, it looks like that the rinsing of cellular debris after the antigen-antibody reaction might be a critical step, failure of which can lead to false detection leading towards therapeutic mishaps. How do authors address this uncertainty?

3. The major problem of this entire study is the sample size; an overall 37 samples are not sufficient to prove their claim. This reviewer would have been satisfied if the patient sample size could have been at least 10-fold more with a multi-institutional level of verification via multiple hands.

4. Why H358 (EGFR WT) cells show a definite false positive signal utilizing the EGFR L858R antibody under Fig.S1A, in the lower panel? In such a scenario, how did authors claim their immune-wall detection methodology to be free from any significant errors; that’s why limiting sample size is always deceptive!

5. Why can’t authors patent their innovation before publishing it?

**********

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Reviewer #1: No

Reviewer #2: Yes: B. Saha, PhD

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9 Oct 2020

Response to Journal requirements and Reviewers’ Comments

We would like to thank the reviewers for their constructive comments. Our responses to all of the comments are provided below.

Journal requirements

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

Response: We have confirmed that this manuscript meets the style requirements of PLOS ONE.

2. Please provide additional information about each of the cell lines used in this work.

Specifically, please provide the specific source for each cell line used and any quality control testing procedures (authentication, characterization, and mycoplasma testing).

Response: We have revised the relevant sentences in the Methods section as suggested. (Page 7 line 115 to page 8 line 122)

Before: “Human lung cancer cell lines H358, H3255, H1299, PC9, and HCC827 were obtained from the Hamon Center Collection (University of Texas Southwestern Medical Center, Dallas, TX, USA) or purchased from ATCC (Manassas, VA, USA). These cells were cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% FBS at 37 °C in 5% CO2. Cells were lysed with lysis buffer (Cell Signaling Technology) supplemented with 1 mM phenylmethylsulfonyl fluoride.”

After: “Human lung cancer cell lines H3255, and HCC827 were obtained from the Hamon Center Collection (University of Texas Southwestern Medical Center, Dallas, TX, USA) and H358, H1299, and PC9 were purchased from ATCC (Manassas, VA, USA). These cells were cultured in Roswell Park Memorial Institute 1640 medium supplemented with 10% FBS at 37 °C in 5% CO2. Cells were lysed with lysis buffer (Cell Signaling Technology) supplemented with 1 mM phenylmethylsulfonyl fluoride. All cell lines were checked for mycoplasma contamination using MycoAlert Mycoplasma Detection Kit purchased from Lonza (Cat. No. LT07-118; Switzerland) and short tandem repeats profiling analysis has been submitted for authentication.”

3. Thank you for including your ethics statement:

'All participants provided written, informed consent, and the hospital institutional review board approved this study (No. 2014-0171)'.

a. Please amend your current ethics statement to include the full name of the ethics committee/institutional review board(s) that approved your specific study.

Response: We have revised the relevant sentences as follows:

Page 8 line 126 to line 128 (revised portion is in red font).

“All participants provided written, informed consent, and the Ethics Review Committee of Nagoya University Graduate School of Medicine approved this study (No. 2014-0171).”

b. Once you have amended this/these statement(s) in the Methods section of the manuscript, please add the same text to the “Ethics Statement” field of the submission form (via “Edit Submission”).

Response: As suggested, we have added the relevant sentence to the “Ethics Statement” field of the submission form.

4. At this time, we ask that you provide the product numbers and any lot numbers of the EGFR (L858R), EGFR (E746), EGFR and actin primary antibodies used in the Western blot analysis and immunocytochemistry analysis in this study.

Response: We have revised the relevant sentences as follows:

Page 9 line 139 to line 145 (revised portion is in red font).

“Primary antibodies used for this analysis included the anti-human EGFR (L858R) antibody (clone 43B2, Cat. No. 3197) (1:2,000), rabbit anti-human EGFR (E746_A750 deletion mutant) (clone D6B6, Cat. No. 2085) (1:5,000), anti-human EGFR antibody (clone D38B1, Cat. No. 4267) (1:4,000), and mouse monoclonal anti-actin antibody (Cat. No. A2228) (1:20,000). Horseradish peroxidase-conjugated donkey anti-rabbit (1:2,000) or sheep anti-mouse (1:2,000) antibody was used as the secondary antibody. Actin was used as a loading control.”

Page 9 line 149 to line 153 (revised portion is in red font).

“After blocking with 1% BSA for 1 h, cells were incubated with anti-human EGFR (L858R) antibody (clone 43B2, Cat. No. 3197) (1:1,000), rabbit anti-human EGFR (E746_A750 deletion mutant) (clone D6B6, Cat. No. 2085) (1:1,000), and anti-human EGFR antibody (clone D38B1, Cat. No. 4267) (1:1,000) in 1% BSA in PBS for 1 h at room temperature.”

5. To comply with PLOS ONE submission guidelines, in your Methods section, please provide additional information regarding your statistical analyses.

Response: We added statistics information under the Methods section.

Page 12 lines 193 to line 197 (revised portion is in red font).

“Statistical analysis

Although no reference or ‘gold standard’ has been defined for EGFR mutation analysis, PCR-based methods are assumed to be the current gold standard in daily practice. Therefore, we calculated diagnostic sensitivity and specificity based on PCR-based results as described previously [16]. JMP pro 14 (SAS Institute Inc., Cary, NC, USA) was used for all statistical analyses in this study.”

6. Thank you for stating the following in the Competing Interests section:

'T. Hase received personal fees from Chugai Pharmaceutical Co. Ltd., Ono Pharmaceutical Co. Ltd., Bristol-Myers Squibb Co., and Boehringer Ingelheim, and research funding from Boehringer Ingelheim, and Taiho Pharmaceutical Co. Ltd., outside the submitted work. Y. Hasegawa received grants from Boehringer Ingelheim, AstraZeneca, Eli Lilly Japan K.K., Ono Pharmaceutical Co. Ltd., Bristol-Myers Squibb Co., Taiho Pharmaceutical Co. Ltd., Novartis Pharma K. K., and Chugai Pharmaceutical Co. Ltd., and personal fee from Boehringer Ingelheim, MSD K.K., AstraZeneca, Pfizer Inc., and Chugai Pharmaceutical Co. Ltd, outside the submitted work.'

a. Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these.

Please note that we cannot proceed with consideration of your article until this information has been declared.

b. Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf.

Response: We have confirmed that the competing interests do not alter our adherence to all PLOS ONE policies on sharing data and materials. Additionally, we have included the updated Competing Interests statement to our cover letter as suggested.

Review Comments to the Author

Reviewer #1

Q1. In the figure S1B, lane 3, L858R substitution antibody picked up a less intense band in HCC827 (E746_A750del) cells but not in PC9 cells (E746_A750del). Whether HCC827 cells also carry L858R substitution. PC9 cells did not show any detectable band with L858R substitution antibody and thus, the PC9 cells should have been considered instead of HCC827.

Response: Thank you for your valuable comment. We have confirmed that HCC827 harbors E746_A750del, not L858R, by peptide nucleic acid-locked nucleic acid PCR clamp as shown below, indicating cross-reactivity of anti-EGFR (L858R) antibody. The cross-reactivity is an important factor in the immunoassay including the immuno-wall device. In previous studies evaluating EGFR mutation in IHC, false positive results were also observed with the use of anti-EGFR (L858R) antibody[1,2]. As suggested, PC9 should have been considered instead of HCC827; however, we believe that it might be important to show no false positive results even in samples with confirmed cross-reactivity in the immuno-wall assay. In S2 figure, we performed immunoassay using lysates from only the EGFR WT cell line (H358) to evaluate potential cross-reactivity. In addition, we also performed the immunoassay using lysates from only the HCC827 and found that the fluorescence intensities remained below the threshold, indicating the robustness of the assay even in samples with confirmed cross-reactivity by anti-EGFR (L858R) antibody.

Together with the addition of a new supplementary figure S2C, we have revised and included the following sentences as shown below.

Page 13 line 225 to page 13 line 229 (revised portion is in red font).

“For L858R substitution antibody, western blot analysis revealed a weak band for L858R substitution in HCC827 cells, which also indicated cross-reactivity (S1 Fig). Therefore, immunoassay was performed using lysates from only HCC827 and observed that the fluorescence intensities remained below the threshold (S2C Fig).”

Page 19 line 322 to line 329 (revised portion is in red font).

“Moreover, the L858R substitution antibody might cross-react with WT EGFR or other EGFR mutations (S1 Fig). In previous studies where EGFR mutation was evaluated using IHC, false positive results were observed with the use of anti-EGFR (L858R) antibody [23,26]. To evaluate potential cross-reactivity, we performed the immunoassay using lysates from only HCC827, which were proven to be false positive by immunocytochemistry or western blotting (S1 Fig). We found that the fluorescence intensities remained below the threshold, indicating the robustness of the assay even in samples with confirmed cross-reactivity by the anti-EGFR (L858R) antibody.”

Page 30 line 504 to line 509 (revised portion is in red font).

“S2 Fig. Calibration curves of total protein concentration of lysates of EGFR WT cells (H358) and EGFR mutation cells (HCC827) versus the fluorescence intensity for each EGFR mutant protein. E746_A750 deletion for EGFR WT cell line (H358) (A), L858R substitution for EGFR WT cell line (H358) (B), and EGFR mutation cells (HCC827) (C). Dashed lines indicate three standard deviations above the average fluorescence determined in the EGFR WT cell line (H358) by each immuno-wall device.”

Q2. The study tested very limited number of retrospective lung cancer specimens (N=37) for which DNA sequence for EGFR mutants were available. The test need to be performed on a large number of lung cancer specimens from multicentric studies and the revised sensitivity and specificity for each mutant should be considered as the diagnostic potential of this device. In the supplementary table 1, patient number # 14, (Female, age 51), the tumor lysate has the protein concentration of 6.32 µg/µl and carry E746_A750del as reported by DNA sequence. However, Immuno-wall device failed to detect the mutation in the specimen from the same patient (false negative). Did authors verify E746_A750del mutation in this patient specimen by western blot analysis.

Response: Thank you for your valuable comment. As suggested, this test must be evaluated with a larger number of lung cancer specimens in a multicentric manner; however, lung cancer specimens were not collected routinely, especially in community hospitals. Therefore, it was challenging to increase sample size in this retrospective study. Thus, a larger prospective multicenter study could be considered to confirm the result of the current study.

Regarding patient #14, although a western blot analysis was performed, we failed to show the presence of E746_A750del mutation protein as shown below. The exact reason for the discordance between the DNA sequences and our method was unknown; however, possible factors may have included variation in tumor cell content or tumor heterogeneity within samples, which were also observed in comparison studies for PCR-based testing[3-6].

We have revised the relevant sentences as shown below.

Page 22, line 369 to line 370 (revised portion is in red font).

“Taken together, our findings need to be confirmed in a larger prospective multicentric study.”

Page 20 line 348 to page 21 line 352 (revised portion is in red font).

“Several factors may have been associated with the discordances between the PCR-based method and our procedure, even in the analysis of E746_A750 and L858R somatic mutations. Although the exact reasons were unknown, possible factors may have included variation in tumor cell content or tumor heterogeneity within samples, which were also observed in comparison studies for PCR-based testing [12,29-31].”

Q3. In results section, please clearly explain and rewrite about estimation of limit of detection (LOD)

Response: Thank you for your valuable comment. The limit of detection (LOD) was estimated at 1% and 0.1% for the E746_A760 and L858R mutant cell proportions, respectively, based on the threshold value calculated as three standard deviations above the signals observed in the analyses for lysate with 1.5 mg/mL protein concentration from the EGFR WT cell line (H358) that was applied for each immuno-wall.

We have revised the relevant sentences as shown below.

Page 13 line 218 to line 221 (the newly added part is in red font).

“The limit of detection (LOD) was estimated at 1% and 0.1% for the E746_A760 and L858R mutant cell proportions, respectively, based on the threshold value calculated as three standard deviations above the signals observed in the analyses for lysate with 1.5 mg/mL protein concentration from the EGFR WT cell line (H358) that was applied for each immuno-wall. ”

Reviewer #2

1. Though it is a rapid fluorescence-based technic, then how did authors claim this to be very unique; and above all, how it can be remarkably sensitive over conventional ICC/IHC methods?

Response: Thank you for your valuable comment. ICC/IHC method is a well-established and cost-effective method routinely applied in lung cancer diagnosis; however, the results are sometimes affected by differences in assay procedures and scoring system[7]. Indeed, several studies have examined the presence of EGFR mutations in lung cancer by IHC using the two mutation-specific antibodies employed in the present study and demonstrated sensitivity ranging from 24% to 100% and specificity ranging from 77% to 100%[1,8-14]. In addition, the cross-reactivity of the antibodies employed had potential risk for false-positive results in ICC/IHC methods. In our study, although there was concern regarding cross-reactivity of the anti-EGFR (L858R) antibody, no false positive results were obtained in the L858R assay owing to the pre-specified threshold, indicating not only rapidity, but also the usefulness of our method.

2. During the assay, it looks like that the rinsing of cellular debris after the antigen-antibody reaction might be a critical step, failure of which can lead to false detection leading towards therapeutic mishaps. How do authors address this uncertainty?

Response: Thank you for your valuable comment. As mentioned, the rinsing of the debris following the antigen-antibody reaction was an important step in this assay. Compared with an immuno-pillar device, which was previously proposed in microfluidic immunoassays[15], an immuno-wall device utilizes a wall-like structure immobilized with antibodies at the center of the microchannel, which enabled easy removal of non-specifically bound molecules by the injection of washing buffer. These structural features might decrease the uncertainty, resulting in the diagnostic sensitivity and specificity of the immuno-wall device.

3. The major problem of this entire study is the sample size; an overall 37 samples are not sufficient to prove their claim. This reviewer would have been satisfied if the patient sample size could have been at least 10-fold more with a multi-institutional level of verification via multiple hands.

Response: Thank you for your valuable comment. We agree with your comment. As described in our response to the comment of reviewer 1, lung cancer specimens were not collected routinely, especially in community hospitals. Therefore, it was challenging to increase the sample size in this retrospective study. Thus, a larger prospective multicenter study could be considered to confirm the result of the current study.

We have revised the relevant sentences as shown below.

Page 22, line 369 to line 370 (revised portion is in red font).

“Taken together, our findings need to be confirmed in a larger prospective multicentric study.”

4. Why H358 (EGFR WT) cells show a definite false positive signal utilizing the EGFR L858R antibody under Fig.S1A, in the lower panel? In such a scenario, how did authors claim their immuno-wall detection methodology to be free from any significant errors; that’s why limiting sample size is always deceptive!

Response: Thank you for your valuable comment. As mentioned, H358 cells showed weak fluorescence under Fig.S1A, indicating cross-reactivity of anti-EGFR (L858R) antibody. Hence, to estimate potential cross-reactivity, we performed the immunoassay using lysates from only the EGFR WT cell line (H358) (S2 Fig) and found that the fluorescence intensities remained below the threshold. In addition, as commented by reviewer #1, HCC827 cells showed weak fluorescence (Fig.S1B). We also performed the immunoassay using lysates from only HCC827 and found that the fluorescence intensities remained below the threshold, indicating the robustness of the assay even in samples with confirmed cross-reactivity by the anti-EGFR (L858R) antibody. Regarding the limited sample size in this study, as you have mentioned, selection bias might have affected the results. Thus, a larger prospective multicenter study could be considered to confirm the result of the current study.

Together with the addition of a new supplementary figure S2C, we have revised and included the following sentences as shown below.

Page 13 line 225 to page 13 line 229 (revised portion is in red font).

“For L858R substitution antibody, western blot analysis revealed a weak band for L858R substitution in HCC827 cells, which also indicated cross-reactivity (S1 Fig). Therefore, immunoassay was performed using lysates from only HCC827 and observed that the fluorescence intensities remained below the threshold (S2C Fig).”

Page 19 line 322 to line 329 (revised portion is in red font).

“Moreover, the L858R substitution antibody might cross-react with WT EGFR or other EGFR mutations (S1 Fig). In previous studies where EGFR mutation was evaluated using IHC, false positive results were observed with the use of anti-EGFR (L858R) antibody [23,26]. To evaluate potential cross-reactivity, we performed the immunoassay using lysates from only HCC827, which were proven to be false positive by immunocytochemistry or western blotting (S1 Fig). We found that the fluorescence intensities remained below the threshold, indicating the robustness of the assay even in samples with confirmed cross-reactivity by the anti-EGFR (L858R) antibody.”

Page 30 line 504 to line 509 (revised portion is in red font).

“S2 Fig. Calibration curves of total protein concentration of lysates of EGFR WT cells (H358) and EGFR mutation cells (HCC827) versus the fluorescence intensity for each EGFR mutant protein. E746_A750 deletion for EGFR WT cell line (H358) (A), L858R substitution for EGFR WT cell line (H358) (B), and EGFR mutation cells (HCC827) (C). Dashed lines indicate three standard deviations above the average fluorescence determined in the EGFR WT cell line (H358) by each immuno-wall device.”

Page 22, line 369 to line 370 (revised portion is in red font).

“Taken together, our findings need to be confirmed in a larger prospective multicentric study.”

5. Why can’t authors patent their innovation before publishing it?

Response: Thank you for your valuable suggestion. We have already applied for a patent on our immunoassay.

Other changes:

#1. Due to substantial contributions to the experiments required for this revision, Dr Hirofumi Shibata, Dr Kazuki Komeda, Dr Nozomi Kawabe, and Dr Kohei Matsuoka have been included as co-authors.

References

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2. Seo AN, Park T-II, Jin Y, Sun P-LL, Kim H, Chang H, et al. Novel EGFR mutation-specific antibodies for lung adenocarcinoma: highly specific but not sensitive detection of an E746_A750 deletion in exon 19 and an L858R mutation in exon 21 by immunohistochemistry. Lung cancer (Amsterdam, Netherlands). 2014;83: 316–23. doi:10.1016/j.lungcan.2013.12.008

3. Eberhard DA, Giaccone G, Johnson BE, Group N-S-CLCW. Biomarkers of Response to Epidermal Growth Factor Receptor Inhibitors in Non–Small-Cell Lung Cancer Working Group: Standardization for Use in the Clinical Trial Setting. J Clin Oncol. 2008;26: 983–994. doi:10.1200/jco.2007.12.9858

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5. Oliner K, Juan T, Suggs S, Wolf M, Sarosi I, Freeman DJ, et al. A comparability study of 5 commercial KRAS tests. Diagn Pathol. 2010;5: 23. doi:10.1186/1746-1596-5-23

6. Goto K, Satouchi M, Ishii G, Nishio K, Hagiwara K, Mitsudomi T, et al. An evaluation study of EGFR mutation tests utilized for non-small-cell lung cancer in the diagnostic setting. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO. 2012;23: 2914–9. doi:10.1093/annonc/mds121

7. Xiong Y, Bai Y, Leong N, Laughlin TS, Rothberg PG, Xu H, et al. Immunohistochemical detection of mutations in the epidermal growth factor receptor gene in lung adenocarcinomas using mutation-specific antibodies. Diagnostic pathology. 2013;8: 27. doi:10.1186/1746-1596-8-27

8. Kato Y, Peled N, Wynes MW, Yoshida K, Pardo M, Mascaux C, et al. Novel epidermal growth factor receptor mutation-specific antibodies for non-small cell lung cancer: immunohistochemistry as a possible screening method for epidermal growth factor receptor mutations. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer. 2010;5: 1551–8. doi:10.1097/JTO.0b013e3181e9da60

9. Kitamura A, Hosoda W, Sasaki E, Mitsudomi T, Yatabe Y. Immunohistochemical Detection of EGFR Mutation Using Mutation-Specific Antibodies in Lung Cancer. Clinical Cancer Research. 2010;16: 3349–55. doi:10.1158/1078-0432.CCR-10-0129

10. Simonetti S, Molina M, Queralt C, Aguirre I de, Mayo C, Bertran-Alamillo J, et al. Detection of EGFR mutations with mutation-specific antibodies in stage IV non-small-cell lung cancer. Journal of Translational Medicine. 2010;8: 1–8. doi:10.1186/1479-5876-8-135

11. Brevet M, Arcila M, Ladanyi M. Assessment of EGFR mutation status in lung adenocarcinoma by immunohistochemistry using antibodies specific to the two major forms of mutant EGFR. The Journal of molecular diagnostics : JMD. 2010;12: 169–76. doi:10.2353/jmoldx.2010.090140

12. Wu S-GG, Chang Y-LL, Lin J-WW, Wu C-TT, Chen H-YY, Tsai M-FF, et al. Including total EGFR staining in scoring improves EGFR mutations detection by mutation-specific antibodies and EGFR TKIs response prediction. PloS one. 2011;6: e23303. doi:10.1371/journal.pone.0023303

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15 Oct 2020

Development of an immuno-wall device for the rapid and sensitive detection of EGFR mutations in tumor tissues resected from lung cancer patients

PONE-D-20-01970R1

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6 Nov 2020

PONE-D-20-01970R1

Development of an immuno-wall device for the rapid and sensitive detection of EGFR mutations in tumor tissues resected from lung cancer patients

Dear Dr. Hase:

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